Developments in Plant Genetics and Breeding, 4
Biology of Brassica Coenospecies
Developments in Plant Genetics and Breeding 1A ISOZYMES IN PLANT GENETICS AND BREEDING, PART A edited by S.D. Tanksley and T.J. Orton 1983 x +516 pp. 1B ISOZYMES IN PLANT GENETICS AND BREEDING, PART B edited by S.D. Tanksley and T.J. Orton 1983 viii +472 pp. 2A CHROMOSOME ENGINEERING IN PLANTS: GENETICS, BREEDING, EVOLUTION, PART A edited by P.K. Gupta and T. Tsuchiya 1991 xv + 639 pp. 2B CHROMOSOME ENGINEERING IN PLANTS: GENETICS, BREEDING, EVOLUTION, PART B edited by T. Tsuchiya and P.K. Gupta 1991 vi + 630 pp. 3 GENETICS IN SCOTS PINE edited by M. Giertych and Cs. M,~tyas 1991 280 pp. 4 BIOLOGY OF BRASSICA COENOSPECIES edited by C. G6mez-Campo 1999 x + 490 pp.
Developments in Plant Genetics and Breeding, 4
Biology of Brassica Coenospecies edited by
Cesar Gbmez- Campo Departamento de Biologia Vegetal Universidad Po/itecnica de Madrid E. T.S. Ingenieros Agronomos Ciudad Universitaria Madrid, Spain
1999 ELSEVIER Amsterdam
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FOREWORD
Brassica crop species a n d their allies (Raphanus, Sinapis, Eruca, etc.) are i m p o r t a n t s o u r c e s of edible roots, stems, leaves, b u d s a n d inflorescences, as well as of edible or i n d u s t r i a l oils, c o n d i m e n t s a n d forage. Many well k n o w n n a m e s of p l a n t s or p l a n t p r o d u c t s , s u c h as kale, cabbage, broccoli, cauliflower, B r u s s e l s s p r o u t s , kohl-rabi, Chinese cabbage, turnip, rape, r u t a b a g a , swede, colza or rapeseed, canola, m u s t a r d , rocket, etc. are directly a s s o c i a t e d to this botanical group. M a n y are also a s s o c i a t e d to o u r everyday life. Historically, m o s t cultivated forms a n d some of their wild relatives were u s e d in medicine as anti-escorbutic; a m o d e r n version of their medicinal value is the anti-carcinogenic effect of some of their c o n s t i t u e n t s . Some m e m bers of the g r o u p have also proved to be very effective in the control of soil n e m a t o d e s . O t h e r s have been positively tested for the c o n c e n t r a t i o n of heavy metals from c o n t a m i n a t e d soils. O t h e r s are a c t u a l or potential s o u r c e s of natural anti-fungic s u b s t a n c e s . In turn, m a n y wild relatives have traditionally been collected for different p u r p o s e s . In m a n y cases, t h e s e are an a c t u a l or potential s o u r c e of useful genes to t r a n s f e r to the cultivated forms. The development of highly productive lines for seed oil, where u n p l e a s a n t or u n h e a l t h y c o m p o n e n t s are completely removed, h a s rocketed the economic importance a n d the acreage of some Brassica crops. In some c o u n t r i e s of the n o r t h e r n t e m p e r a t e zone, for instance, r a p e s e e d occasionally displaces w h e a t from the first place, d e p e n d i n g u p o n the yearly prices. The scientific i n t e r e s t for this botanical g r o u p h a s r u n parallel to its economical i m p o r t a n c e , a n d r e s e a r c h a c h i e v e m e n t s in o u r d a y s would have certainly a p p e a r e d u n i m a g i n a b l e only two d e c a d e s ago. As the end of the c e n t u r y a p p r o a c h e d , entirely new fields (transformation, somatic fusion, etc.) have been a d d e d to the classical ones. T h u s , n o b o d y can d o u b t the o p p o r t u n e n e s s of this book, in which the m o s t recent a d v a n c e s in the biology of this group are compiled a n d p r e s e n t e d . The a u t h o r s a n d the editor only expect to have m a d e a useful w o r k which, at least partially, is in line with the above brilliant c i r c u m s t a n c e s . The editor w i s h e s to t h a n k Dr. S. P r a k a s h who first s u g g e s t e d the idea of p r e p a r i n g this book a n d collaborated in its initial planning. He is also very grateful to Miss E s p e r a n z a Garcia-Lanza, for her valuable help in the edition work. The editor
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vii
CONTENTS Chapter 1. TAXONOMY (C. G6mez-Campo) Cultivated B r a s s i c a species B. o l e r a c e a wild relatives The genus B r a s s i c a Other related genera The tribe B r a s s i c e a e References
3 4 12 14 16 19 23
Chapter 2. ORIGIN a n d DOMESTICATION (C. G6mez-Campo and S. Prakash) The phylogeny of B r a s s i c a and allied genera Domestication of cultivated brassicas and allies References
33 34 43 52
Chapter 3. CYTOGENETICS (by S. Prakash, Y. Takahata, P. B. Kirti and V. L. Chopra) T h e B r a s s i c a coenospecies Crop brassicas: cytogenetic architecture Genome manipulation Cytogenetics of wild allies: wide hybridizations Introgression of genes Cytoplasm divergence and genome homoeology Chromosome addition lines References
59 60 65 68 74 84 84 87 90
Chapter 4. SOMATIC HYBRIDIZATION (K. Glimelius) Protoplast technology Somatic hybrids produced between different B r a s s i c a species Intergeneric somatic hybrids within the tribe B r a s s i c e a e Limited gene transfer via protoplast fusion Cytological investigations using in s i t u hybridization The utilization of protoplast fusion to modify the cytoplasm Modification of cytoplasmic traits via protoplast fusion Conclusions References
107 108 110 116 123 124 126 132 135 136
Chapter 5. SELF-INCOMPATIBILITY (M. Watanabe and K. Hinata) Morphology and physiology Classical genetics and dominance relationships The S-multigene family Signal perception and signal transduction Molecular analysis of self-compatibility
149 149 150 151 160 164
viii Evolutionary aspects Related studies with future prospects References
165 166 168
Chapter 6. MALE STERILITY (R. Delourme and F. Budar) Genic male sterility Cytoplasmic male sterility Use for the production of commercial hybrids References 203
185 185 186 198
Chapter 7. GENOME STRUCTURE a n d MAPPING (C. F. Quiros) Linkage m a p s Structure of the B r a s s i c a genomes Cyclic amphiploidy and the origin and evolution of the B r a s s i c a species A r a b i d o p s i s as a model for a simpler genome Applications of the m a p s in breeding References
217 218 224
Chapter 8. IIAPLOIDY (C. E. Palmer and W. A. Keller) Historical overview Methodology Factors influencing microspore culture Developmental aspects of microspore embryogenesis Utilization of microspore-derived embryos of B r a s s i c a Conclusions and future prospects References
247 248 249 249 258 262 267 268
Chapter 9. G E ~ I C
Gene transfer methods Types of genes transferred Field tests of transgenic plants Legal issues Transgenic B r a s s i c a crops now being commercialized F u t u r e prospects References
287 287 288 290 293 300 300 302 303
Chapter I0. C,IIIt, M I C A I , COMPOSITION (E. A. S. Rosa) The importance of the B r a s s i c a and allies in h u m a n and animal diet The chemical composition of B r a s s i c a crops General components Secondary plant metabolites: the glucosinolates Other compounds References
315 318 319 319 323 342 346
ENGIgEERING (E. D. Earle and V. C. Knauf)
B r a s s i c a species transformed
232 234 234 236
ix C h a p t e r 11. PHYSIOLOGY {P. Hadley and S. Pearson) Germination Vegetative growth The transition from vegetative to reproductive development Hormonal control of flowering in Brassica Progress to crop m a t u r i t y Yield determining factors References
359 359 360 361 365 365 366 369
C h a p t e r 12. DISEASES (J. P. Tewari and R. F. Mithen) Blackspot or grey leaf c a u s e d by Altemaria brassicae a n d d a r k leaf spot caused by A. brassicicola Stem c a n k e r or blackleg caused by Leptosphaeria maculans Stem rot c a u s e d Sclerotinia sclerotiorum White r u s t a n d s t a g h e a d disease caused by Albugo candida Light leaf spot c a u s e d by Pyrenopeziza brassicae Downy mildew c a u s e d by Peronospora parasitica Verticillium wilt c a u s e d by Verticillium dahliae Clubroot c a u s e d by Plasmodiophora brassicae Other fungal diseases References
375 375 378 384 386 388 389 390 391 393 394
C h a p t e r 13. BREEDING: AN OVERVIEW (H. C. Becker, H. L6ptien a n d G. R6bbelen Breeding objectives Genetical resources Operational steps for breeding Breeding m e t h o d s Breeding results F u t u r e developments References
413 415 425 427 431 442 445 448
C h a p t e r 14. GENETIC RESOURCES (I. W. B o u k e m a a n d T. J. L. v a n Hintum) Strategies for conservation Availability S u m m a r i e s of Brassica genetic resources collections I m p o r t a n t collections Concluding r e m a r k s References
461 462 467 467 473 475 476
SUBJECT INDEX
481
Biology of Brassica Coenospecies
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Biology of Brassica Coenospecies C. G6mez-Campo (Editor) 91999 Elsevier Science B.V. All rights reserved.
TAXONOMY C6sar G 6 m e z - C a m p o
Dept. Biologia Vegetal, Universidad Polit~cnica de Madrid. 28040 - Madrid. Spain. Coenospecies is not a t a x o n o m i c category b u t a cytogenetical concept whose taxonomic c o u n t e r p a r t could c o n s i s t of a g r o u p of "allies" or "relatives" to a given taxon. Here, it is applied to the species a n d s u b s p e c i e s of the g e n u s Brassica, together with those of its closest related genera. Where s h o u l d the limits be placed? Special e m p h a s i s is p u t on g e n e r a Erucastrum,
Diptotaxis, Hirschfeldia, Eruca, Sinapis, Sinapidendron, Coincya, Raphanus and Trachystoma, together with Brassica itself. However, a brief i n c u r s i o n into the rest of the tribe Brassiceae will cover o t h e r possible wild relatives.
Some y e a r s ago, a book edited by T s u n o d a , H i n a t a a n d G 6 m e z - C a m p o (1980) was p u b l i s h e d with a similar scope to the p r e s e n t one. Almost two d e c a d e s later, trying to review new d e v e l o p m e n t s c o n c e r n i n g the t a x o n o m y of this group, p r o d u c e s a mixture of positive, negative a n d c o n f u s i n g feelings. On the one h a n d , a lot h a s been written on the s u b j e c t a n d references at the end of this article include only a part, t h a t t h o u g h t to be the m o s t significant, of all w h a t h a s been p r o d u c e d in this period. On the o t h e r h a n d , it is p e r h a p s too early to profit a d e q u a t e l y from the c o n t r i b u t i o n s t h a t cytogenetic a n d m o l e c u l a r m e t h o d s are m a k i n g to the knowledge of this botanical group as they do for m a n y others. In short, this book m a y be o p p o r t u n e as a whole, b u t a c h a p t e r on the t a x o n o m y of Brassica a n d allies would have benefited from being p o s t p o n e d p e r h a p s a decade or so to receive - a n d digest- f u r t h e r i n p u t s from o t h e r fast developing fields to become more useful a n d complete. A significant p a r t of recent w o r k on the t a x o n o m y of Brassica coenospecies is directly or indirectly related to a series of m o n o g r a p h i c t h e s e s which are worth listing together in advance" Baillargeon (1986) on Sinapis, Gladis (1989), E a s t w o o d (1996), Lann6r (1997) a n d Ls (1997) on Brassica, Leadlay (see Leadley a n d Heywood, 1990) on Coincya, Martinez-Laborde (1988a) on Diplotaxis, Pistrick (1987) on Raphanus, S a l m e e n (1979) on Brassica a n d S~nchez-Y61amo (1990) on the Diplotaxis-Erucastrum-Brassica complex. In addition to c u r r e n t literature a n d several new local floras, the publication of E u c a r p i a Cruciferae Newsletter h a s proved to be a positive factor in
s t i m u l a t i n g r e s e a r c h a n d c o m m u n i c a t i o n in this discipline a n d in m a n y ot h e r s related to Brassica. At the s a m e time, a n u m b e r of related w o r k s h o p s a n d meetings, periodic or not, at least in three c o n t i n e n t s , have also played a significant part. This c h a p t e r s t a r t s with a s h o r t reference to the already well known six cultivated species of the classical U-triangle (see c h a p t e r 3) to proceed to the wild relatives of Brassica oleracea, t h e n to the g e n u s Brassica as a whole, to its m o s t related g e n e r a and, finally, beyond the coenospecies limits, giving a few c o m m e n t s on the tribe Brassiceae as a whole. U p d a t e d s y n o p s e s are provided for the s u b g e n e r a , sections, species a n d s u b s p e c i e s of the ten genera m e n t i o n e d so far (Table 1.1) as well as for all the tribe g e n e r a (Table 1.2).
Cultivated B r a s s i c a species Regarding the infraspecific variability of B. oleracea, there is an unwritten a g r e e m e n t to describe this with t a x o n o m i c categories below the range of s u b s p e c i e s . This is r e a s o n a b l e b e c a u s e a l t h o u g h this variability is very high, it originated u n d e r d o m e s t i c a t i o n in a relatively s h o r t period of time. A detailed a c c o u n t is b e y o n d the scope of this c h a p t e r , b u t the r e a d e r is referred to Dickson a n d Wallace (1986) or to several articles grouped within the section "cole crops" in Kalloo a n d Berg (1993). A p r e l i m i n a r y information can be found in tables 10.1, 10.3 a n d 10.6 within the p r e s e n t book.
B. oleracea wild types are only truly wild along the E u r o p e a n Atlantic c o a s t s a n d t h e y have often been referred to var. sylvestris L. They are close to cultivated forms (Gustafsson a n d L a n n ~ r - H e r r e r a , 1997a) and almost morphologically i n d i s t i n g u i s h a b l e from m a n y kales. O t h e r so called "sylvestris" in other p a r t s of the world, are mere e s c a p e s from cultivation. Two o t h e r closely related t a x a originally c o n s i d e r e d as species, n a m e l y B. bourgeaui a n d B. alboglabra, s h o u l d p r o b a b l y merit an infraspecific s t a t u s within B. oleracea variation. B. bourgeaui, from the C a n a r y Islands, was included in the g e n u s Sinapidendron in early literature a n d t h o u g h t to be extinct for m a n y years. It was re-found by Borgen et al. (1979) who ascribed it to the n = 9 Brassica group. They only found two p l a n t s in La Palma Island. Today, m o s t opinions e m p h a s i z e its close relation to B. oleracea (Lann~r, 1998) t h o u g h , p e r h a p s , some possible differential c h a r a c t e r s in leaves a n d pod b e a k s s h o u l d be s t u d i e d more carefully. A second population h a s been found more recently (Marrero, 1989) in E1 Hierro Island. B. alboglabra, a n old cultivated form from China, h a s puzzled r e s e a r c h e r s for years bec a u s e of the p h y t o g e o g r a p h i c a l problem it poses. B o t h m e r et al. (1995) s u m marize the r e a s o n s w h y it s h o u l d be kept within B. oleracea. Although it h a d always been believed to have r e a c h e d C h i n a from the M e d i t e r r a n e a n region, conclusive evidence only a p p e a r e d w h e n H a m m e r et al. (1992) found it locally cultivated in S o u t h Italy. Flowers are n o r m a l l y white a l t h o u g h they m a y be yellow a n d they distinctively a p p e a r in early wintertime.
T a b l e 1 . 1 A t a x o n o m i c synopsis of B r a s s i c a and its allied genera with indication o f subgenera, sections, species and subspecies. B R A S S I C A L.
Subgen. Brassica Sect. Brassica B. oleracea L. B. montana Pourret B. incana Ten. subsp, incana B. i. subsp, cazzae (Ginz. and Teyb.) Trinajstic B. villosa Biv. subsp, villosa B. v. subsp, bivoniana (Mazzola and Raimondo) R.and M. B. v. subsp, drepanensis (Caruel) Raimondo and Mazzola B. v. subsp, tinei (Lojac.) Raimondo and Mazzola B. rupestris Rafin subsp, rupestris B. r. subsp, brevisiliqua Raimondo and Mazzola B. r. subsp, hispida Raimondo and Mazzola B. macrocarpa Guss. B. insularis Moris B. cretica Lam. subsp, cretica B. c. subsp, aegaea (Heldr. and Hal.) Snogerup et al. B. c. subsp, laconica Gustafsson and Snogerup B. botteri Vis. subsp, botteri B. b. subsp, mollis (Vis.) Trinajstic B. hilarionis Post. B. carinata Braun B. balearica Pets.
Sect. Rapa (Miller) Salmeen B. rapa L. subsp, rapa B. r. subsp, campestris (L.) Clapman B. r. subsp, chinensis (L.) Hanelt B. r. subsp, dichotoma (Roxb.) Hanelt B. r. subsp, narinosa (Bailey) Hanelt B. r. subsp, nipposinica (Bailey) Hanelt B. r. subsp, pekinensis (Lour.) Hanelt B. r. subsp, trilocularis (Roxb.) Hanelt B. napus L. B. juncea (L.) Czern.
Sect. Micropodium DC. B. fruticulosa Cyr. subsp, fruticulosa B. f subsp, djafarensis Blanco and Matarranz B . f subsp, dolichocarpa Emberger and Maire B . f subsp, glaberrima (Pomel) Maire
B. f subsp, mauritanica (Cosson) Maire B. f subsp, numidica Maire B. f subsp, pomeliana Maire B. f subsp, radicata (Desf.) Maire B. nigra (L.) Koch B. cossoniana Boiss. and Reuter B. spinescens Pomel B. maurorum Durieu B. procumbens (Poiret) O.E.Schulz B. cadmea O.E.Schulz B. desertii Danin and Hedge Sect. Brassicoides Boiss. B. deflexa Boiss.
Sect. Sinapistrum Willkomm B. barrelieri (L.) Janka B. oxyrrhina Coss. B. tournefortii Gouan
Sub~en. Brassicaria (Godr.) G6mez-Campo Sect. Brassicaria (Godr.) Cosson B. repanda (Willd.) DC. subsp, repanda B.r. subsp, africana (Maire) Greuter and Burdet B.r. subsp, almeriensis G6mez-Campo B.r. subsp, blancoana (Boiss.) Heywood B.r. subsp, cadevallii (Font Quer) Heywood B.r. subsp, cantabrica (Font Quer) Heywood B.r. subsp, confusa (Emb. and Maire) Heywood B.r. subsp, galissieri (Giraud) Heywood B.r. subsp, glabrescens (Poldini) G6mez-Campo B.r. subsp, gypsicola G6mez-Campo B.r. subsp, latisiliqua (Boiss. and Reut.) Heywood B.r. subsp, nudicaulis (Lag.) Heywood B.r. subsp, saxatilis (DC.) Heywood B.r. subsp, silenifolia (Emberger) Greuter and Burdet B. desnotesii Emb. and Maire B. gravinae Ten. B. jordanoffii O.E.Schulz B. loncholoma Pomel B. nivalis Boiss. and Heldr. B. elongata Ehrh. subsp, elongata B. e. subsp, integrifolia (Boiss.)Breistr. B. e. subsp, imdrahsiana Quezel B. e. subsp, pinnatifida (Smal'g) Greuter and Burdet B. e. subsp, subscaposa Maire and Weiller B. setulosa (Boiss. and Reuter) Cosson
B. somaliensis Hedge and Miller
Sect. Nasturtiops (Pomel) Salmeen B. souliei (Batt.) Batt. subsp, souliei B. s. subsp, amplexicaulis (Desf.) Greuter and Burdet B. dimorpha Coss. and Dur.
ERUCASTRUM C. Presl. E. gallicum O.E.Schulz E. nasturtiifolium (Poiret) O.E.Schulz subsp, nasturtiifolium E. n. subsp, sudrei Vivant E. leucanthum Coss. and Dur. E. palustre (Pir.) Vis. E. virgatum C.Presl. subsp, virgatum E. v. subsp, baeticum (Boiss.) G6mez-Campo E. v. subsp, brachycarpum (Rouy) G6mez-Campo E. v. subsp, pseudosinapis (Lange) G6mez-Campo E. varium Durieu subsp, varium E. v. subsp, mesatlanticum Maire and Wilczek E. v. subsp, subsiifolium Maire E. littoreum (Pau and F.Quer) Maire subsp, littoreum E. l. subsp, brachycarpum (Maire and Weiller)G6mez-Campo E. l. subsp, glabrum (Maire) G6mez-Campo E. rifanum (Emb. and Maire) G6mez-Campo E. elatum (Ball.) O.E.Sehulz E. brevirostre (Maire) G6mez-Campo E. canariense Webb and Berth. E. cardaminoides (Webb ex Christ.) O.E.Schulz E. ifniense G6mez-Campo E. arabicum Fisch. and Mey. E. elgonense Jonsell E. meruense Jonsell E. abyssinicum (A. Rich.) O.E.Schulz E. pachypodum (Chiov.) Jonsell E. rostratum (Balf. f.)G6mez-Campo E. strigosum (Thunb.) O.E.Schulz E. griquense (Brown) O.E.Schulz DIPL 0 TAXIS D C.
Subgen. Diplotaxis D. tenuifolia (L.)DC. subsp, tenuifolia D. t. subsp, cretacica (Kotov)Sobrino-Vesperinas D. muralis (L.) DC. subsp, muralis D. m. subsp, ceratophylla (Batt.) Mart.-Lab. D. viminea (L.) DC. D. simplex (Viv.) Spr.
Subgen. Hesperidium (DC.) Mart.-Lab. D. harra (Forsk.) Boiss. subsp, harra D. h. subsp, confusa Mart.-Lab. D. h. subsp, crassifolia (Rafin) Maire D. h. subsp, lagascana (DC.) O.Bol6s and J.Vigo D. antoniensis Rustan D. glauca (Schmidt) O.E.Schulz D. gorgadensis Rustan subsp, gorgadensis D. g. subsp, brochmanii Rustan D. gracilis (Webb) O.E.Schulz D. hirta (Chev.) Rustan and Borgen D. sundingii Rustan D. varia Rustan D. vogelii (Webb) Cout. D. pitardiana Maire D. acris (Forsk.) Boiss. D. villosa Boul. and Jail. D. griffithii (Hook.f. and Thorns.) Boiss. D. nepalensis Hara Subgen. Rh~nchocarpum (Prantl) Mart.-Lab. Sect. Rhynchocarpum Prantl D. assurgens (Del.) Gren. D. tenuisiliqua Del. subsp, tenuisiliqua D. t. subsp, rupestris (J. Ball.) Mart.-Lab. D. brachycarpa Godr. D. virgata (Cav.) DC. subsp, virgata D. v. subsp, australis Mart.-Lab. D. v. subsp, rivulorum (Br.-B1. and Maire) Mart.-Lab. D. v. subsp, sahariensis (Coss.) Mart.-Lab. D. berthautii Br.-B1. and Maire D. catholica (L.) DC. D. ollivieri Maire D. siifolia G. Kunze. subsp, siifolia D. s. subsp, bipinnatifida (Coss.) Mart.-Lab. D. s. subsp, vicentina (P.Cout.) Mart.-Lab. Sect. Heterocarpum Mart.-Lab. D. D. D. D.
ibicensis (Pau) G6mez-Campo siettiana Maire brevisiliqua (Coss.) Mart.-Lab. ilorcitana (Sennen) Aedo and Mart.-Lab.
Sect. Erucoides Mart.-Lab. D. erucoides (L.) DC. subsp, erucoides D. e. subsp, longisiliqua (Coss.)G6mez-Campo
SINAPIS L.
Sect. Sinapis S. alba L. subsp, alba S. a. subsp, mairei (H.Lindb.) Maire S. a. subsp, dissecta (Lag.) Bonnier S. flexuosa Poir.
Sect. Ceratosinapis DC. S. arvensis L. subsp, arvensis S. a. subsp, allioni (Jacq.) Baillargeon S. a. subsp, nilotica (O.E.Schulz) Baillargeon
Sect. Hebesinapis DC. S. pubescens L. subsp, pubescens S. p. subsp, virgata (Batt.) Baillargeon S. boivinii Baillargeon S. indurata Coss. S. aristidis Coss.
Sect. Chondrosinapis O.E.Schulz S. aucheri (Boiss.) O.E.Schulz ER UCA Mill. E. vesicaria (L.)Cav. subsp, vesicaria E. v. subsp, sativa (Miller) Thell. E. v. subsp, pinnatifida (Desf.) Emb.and Maire COINCYA Rouy C. richeri (Vill.) Greuter and Burdet C. wrightii (O.E.Schulz) Stace C. monensis (L.) Greuter and Burdet subsp, monensis C.m. subsp, cheiranthos (Vill.) Aedo, Leadlay and Mufioz-Garm. C.m. subsp, nevadensis (Willk.) Leadlay C.m. subsp, orophila (Franco) Aedo, Leadlay and Mufioz-Garm. C.m. subsp, puberula (Pau) Leadlay C. transtagana (Cout.) Clem.-Mufioz and Hern.-Bermejo C. longirostra (Boiss.) Greuter and Burdet C. rupestris Porta and Rigo subsp, rupestris C. r. subsp, leptocarpa (Gonz.-Albo) Leadlay RAPHANUS L. R. raphanistrum L. subsp, raphanistrum R. r. subsp, landra (DC.) Bonnier and Layens R. r. subsp, maritimus (Sm.) Thell. R. r. subsp, microcarpus (Lange) Thell.
10 R. r. subsp, rostratus (DC.) Thell. R. sativus L. HIRSCHFELDIA Moench
H. incana (L.) Lagr6ze-Fossat subsp, incana H. i. subsp, incrassata (Thell.) G6mez-Campo SINAPIDENDR ON Lowe
S. angustifolium (DC.) Lowe S. frutescens (Sol.) Lowe subsp.frutescens S. f subsp, succulentum (Lowe) Rustan S. gymnocalyx (Lowe) Rustan S. rupestre Lowe S. sempervivifolium Mnzs. TRACHYSTOMA O.E. Schulz
7". aphanoneurum (Maire and Weiller) M.and W. 7". balli O.E.Schulz T. labasii Maire
The s a m e r e a s o n to avoid u s i n g the s u b s p e c i e s category in B. oleraceaa r e c e n t history in cultivation - s h o u l d be logically applied to B. napus, since this is t h o u g h t to be a n amphidiploid derived from crosses B. oleracea x B. rapa which, m o s t likely, o c c u r r e d in cultivation. However, the s u b s p e c i e s category h a s b e e n traditionally applied to some g r o u p s of B. n a p u s cultivars s u c h as napobrassica, rapifera, pabularia, etc. (not included in Table 1.1). Infraspecific variability of B. rapa (syn. B. campestris) is given a different t r e a t m e n t , p e r h a p s in a c c o r d a n c e with its older history in domestication. For a time, the species category was widely applied (B. chinensis, B. p e k i n e n s i s , B. japonica, etc.) b u t this is now c o n s i d e r e d an excessive license. The s u b s p e c i e s r a n k is only r e c o m m e n d e d for the m o s t significant v a r i a n t s (Oost, 1985) t h o u g h a n intensification of the u s e of n a m e s according to the I n t e r n a t i o n a l Code of N o m e n c l a t u r e for Cultivated Plants (Trehane et al., 1995) would be desirable (Oost, p e r s o n a l c o m m u n i c a t i o n ) . The prioritary n a m e for the species is B. rapa (after the first c o m b i n a t i o n by Metzger in 1833) while the n a m e s u b s p , c a m p e s t r i s s h o u l d be reserved for non specialized semi-wild forms with slender root (Toxopeus et al., 1984). As a m a t t e r of fact, B. c a m p e s t r i s h a s been a very widely u s e d n a m e and, for many, it m i g h t take some effort to a d a p t to the new correct n o m e n c l a t u r e . In the Far East, d o m e s t i c a t i o n led to a wide variety of forms, parallel to those obtained in the M e d i t e r r a n e a n with B. oleracea (except cauliflower!). In the West, the i m p o r t a n c e of B. rapa h a s also been c o n s i d e r a b l e b u t more specialized final
11 forms developed d u e to the "competition" by a n extensive u s e of B. oleracea. The n u m b e r of e p i t h e t s u s e d , either from the E a s t or from the West is very high b u t Oost (1985) s u g g e s t s t h a t only 10-12 s h o u l d merit subspecific rank. Table 1.1 only includes the seven t h a t have been formally c o m b i n e d (Hanelt, 1986), b u t o t h e r epithets as dubiosa, japonica, perviridis, purpurar/a, etc. m i g h t p e r h a p s be equal c a n d i d a t e s for as m a n y s u b s p e c i e s . Upper a c u t e widely e m b r a c i n g leaves a n d long n a r r o w b e a k s in the pods are the best c h a r a c t e r s to d e t e c t B. rapa individuals (B. n a p u s is more g l a b r o u s a n d glaucous, its b e a k is thicker a n d its leaves are only loosely embrasing). An i n t e r n a t i o n a l effort to e s t a b l i s h a c c e p t a b l e criteria in the n o m e n c l a t u r e of Brassica crops s e e m s highly n e c e s s a r y .
B. juncea, a n a m p h i d i p l o i d B. rapa x B. nigra is a n i m p o r t a n t source of edible oil in S. Asia, of vegetables in C h i n a a n d of m u s t a r d c o n d i m e n t elsewhere in the World. Its variation h a s b e e n t h o r o u g h l y s t u d i e d over m a n y y e a r s a n d s o m e s u b s p e c i e s (juncea, crispifolia, foliosa, integrifolia, napiformis; not included in Table 1.1) have often been recognized. Some r e c e n t DNA a n a lyses are of relevance for its taxonomic differentiation (see below). The book by C h o p r a a n d P r a k a s h (1996) is mostly c e n t e r e d on this species a n d is a good source of l i t e r a t u r e references. B. carinata derives by a m p h i d i p l o i d y from one or several B. oleracea x B. nigra c r o s s e s a n d is cultivated locally in E t h i o p i a where it h a s a wide a r r a y of uses" s o u r c e of oils a n d spices, medicinal, vegetable, etc. The valid n a m e for this species is the widely u s e d B. carinata B r a u n a n d not its alternative B. integrifolia (West) Rupr., as it h a s been s o m e t i m e s p r o p o s e d (see Jonsell, 1982, p. 69). IBPGR, now IPGRI (International Plant Genetic R e s o u r c e s Institute) (1987, 1990) h a s p u b l i s h e d d e s c r i p t o r s for B. rapa a n d B. oleracea (+ R a p h a nus) respectively with the aim of helping p r o p e r c h a r a c t e r i z a t i o n of cultivars in this group of species. The six B r a s s i c a species of the U-triangle are u s u a l l y referred to as "crop b r a s s i c a s " , a f o r t u n a t e d e n o m i n a t i o n b e c a u s e B r a s s i c a species outside this g r o u p have a limited a g r i c u l t u r a l i m p o r t a n c e (only B. tournefortii does not belong to the U-triangle a n d is cultivated for oil in India) . However, it s h o u l d be n o t e d t h a t the correct Latin p l u r a l for B r a s s i c a is B r a s s i c a e (not "Brassicas"), so t h a t "brassicas" s h o u l d be only u s e d as a c o m m o n nonL i n n e a n n a m e in o t h e r l a n g u a g e s , w i t h o u t italics or a n initial capital. For f u r t h e r r e a d i n g on this group of species the review by P r a k a s h a n d H i n a t a (1980), is a u s e f u l d a t a source on its biology a n d use. F u r t h e r useful reviews in the s a m e line c a n be found in C h o p r a a n d P r a k a s h (1990, 1996). The infra-specific variability of these cultivated species h a s b e e n the object of diverse molecular, biochemical or morphologically b a s e d a n a l y s e s within the last decade either for B. oleracea (Kresowich et al., 1992; Dias et al., 1992; Dias et al., 1993; Arfls et al., 1987) for B. rapa (Lamboy et al., 1994) or for B. j u n c e a (Jain et al., 1994; B h a t i a et al., 1995).
12
B. o l e r a c e a
wild relatives
B. oleracea, a n Atlantic plant, h a s a n u m b e r of closely related species which grow a r o u n d the M e d i t e r r a n e a n basin. All of t h e m have n = 9 chrom o s o m e s a n d are m o r e or less interfertile a m o n g t h e m s e l v e s a n d with B. oleracea. Knowledge of these h a s been s t i m u l a t e d in the r e c e n t y e a r s as a c o n s e q u e n c e of active IBPGR s u p p o r t e d seed collecting activities (G6mezC a m p o a n d G u s t a f s s o n , 1991) which supplied not only living material b u t also a b u n d a n t geographical a n d ecological data. An i m p o r t a n t review by S n o g e r u p et al. (1990) describes with m u c h detail the t a x o n o m y a n d geograp h y of these taxa. These a u t h o r s propose some n o m e n c l a t u r a l readjustm e n t s , m a i n l y for B. cretica subspecies. The m a x i m u m morphological diversity of the group clearly o c c u r s in Sicily where at least four species - B. macrocarpa, B. villosa, B. rupestris a n d B. incana, from west to e a s t - are present. R a i m o n d o a n d Mazzola (1997), confer the r a n k of s u b s p e c i e s to some other Sicilian t a x a w h i c h are closely related to either B. rupestris or B. villosa (see Table 1.1).
The affinities a m o n g this group of species have been studied from different points of view. S t o r k et al. (1980) describe in detail a n u m b e r of seed c h a r a c t e r s a n d m i c r o - c h a r a c t e r s s u c h as t e s t a layers, surface s t r u c t u r e s a n d mucilage cells t h r o u g h o u t the species complex. G 6 m e z - C a m p o et al. (1999) c o m p a r e a n o t h e r m i c r o - c h a r a c t e r , the morphology of epicuticular wax c o l u m n s , a n d find a distinctive type for B. oleracea, B . a l b o g l a b r a a n d B. bourgaei a n d two o t h e r s for the other wild species. Mithen et al. (1987) analyzed eighteen p o p u l a t i o n s belonging to eleven specific or subspecific taxa for their c o n t e n t s of nine glucosinolates. Some taxonomically meaningful differences were found for wild vs. cultivated B. oleracea, as well as a m o n g wild Sicilian species, a n d also for S a r d i n i a n vs. T u n i s i a n p o p u l a t i o n s of B. insular/s. Aguinagalde et al. (1992) u s e d flavonoids, seed proteins a n d five isozyme s y s t e m s a n d found a comparatively high p h y t o c h e m i c a l diversity in B. cretica, even h i g h e r t h a n for the Sicilian group of species. No differences were found between wild a n d cultivated B. oleracea. In t u r n , B. bourgeaui closely r e s e m b l e s the g r o u p formed by B. oleracea a n d B. m o n t a n a (all lacking isor h a m n e t i n ) , while B. oleracea a n d B. alboglabra, considered co-specific by m o s t a u t h o r s , c a n be d i s t i n g u i s h e d by their flavonoid p a t t e r n s . S t u d i e s with RAPDs a n d isozymes on twenty-two p o p u l a t i o n s belonging to fifteen specific a n d sub-specific t a x a (Ls a n d Aguinagalde, 1998 a,b) clearly d i s t i n g u i s h e s a group with the Sicilian species except B. incana, a second W e s t e r n g r o u p including B. oleracea, B. m o n t a n a a n d also B. i n c a n a a n d B. b o u r g e a u i and, finally, a third group including B. alboglabra (suggesting some gene influx) together with B. cretica a n d B. hilarionis. O t h e r importa n t s t u d i e s c o m p a r i n g either species or p o p u l a t i o n s are those by H o s a k a et al. (1990), G u s t a f s s o n a n d L a n n ~ r - H e r r e r a (1997a) a n d Lann~r (1998). Evaluations of interfertility a m o n g all these taxa (Snogerup, 1996) show t h a t r e d u c e d fertility m e a s u r e d by pollen stainability often occurs, b u t is only loosely correlated to morphological differences.
13 Other works focus on single species. Aguinagalde et al. (1991) study eight populations belonging to all the three existing subspecies of B r a s s i c a cretica. By analyzing seed storage proteins and five isoenzyme systems the a u t h o r s find again a high inter-population diversity. The only exception is provided by subsp, taconica (Gustafsson and Snogerup, 1983) whose populations seem to be more homogeneous. Maselli et al. (1996) st udy five enzyme systems on seven populations of B. insularis from Tunisia, Sardinia and Corsica. Though it does not appear too clearly in their dendrogram, a clinal variation from S o u t h to North is evident if the p a t h followed by the clustering process is closely observed. This agrees with the expectations derived from morphological analyses. A few years before, Widler and Bocquet (1979) had recognized four differentiated varieties of this species in Corsica alone. G u s t a f s s o n a n d Lann~r-Herrera (1997b) compare wild B. oleracea populations u s i n g different criteria while Lann~r-Herrera et al. (1996) perform a similar c o m p a r i s o n on eighteen popul at i ons u s i n g isozyme and RAPDs analyses. Though interesting results were obtained on the intra- and inter-populational variation, no evident correlation with the geographical origin (Spain, France and Great Britain) was found. The truly wild s t a t u s of B. oleracea populations in Noth Spain has been s u p p o r t e d by Fern~ndez-Prieto and Herrera-Gall~stegui (1992) on phytosociological grounds. Regarding B. m o n t a n a , a good detailed a c c o u n t of the French populations with morphological analyses and a study of several enzymatic s y s t e m s was produced by Cauwet-Marc et al. (1993). The investigations by Rac a n d Lovric (1991) and by Eastwood (1996) help considerably to u n d e r s t a n d some imperfectly known variability roughly related to B. incana existing in the Adriatic coasts and islands. Trinajsic and Dubravec (1986) recognized four different taxa with the range of subspecies, one part ascribed to B. i n c a n a itself and a n o t h e r part to B. botteri, a differentiated form which is recognized to merit specific rank. A critical additional species B. balearica, from Majorca Island, was first considered a small-sized m e m b e r of this group a n d later excluded from it. In fact, it is an d it is not. Snogerup and Persson (1983) studied it cytologically and found t h a t the n = 9 genome is present as part of its 2n = 32 chromosome complement. They suggest t h a t an ancient hybridization involving B. insularis gave rise to this species by amphidiploidy. G6mez-Campo (1993a) suggests t h a t the other p a r e n t might have been a m e m b e r of Subgen. Brassicaria (see below). Seed and seedling morphology, the general a p p e a r a n c e of the plant a n d some phytogeographical considerations are in favor of this hypothesis a l t h o u g h it is necessary to a c c o u n t for some c h r o m o s o m e losses. In Table 1.1, B. balearica is included in Sect. B r a s s i c a , together with a n o t h e r hybrid species, B. carinata, for which one of the p a r e n t s belongs to the n = 9 genome group. Though B. n a p u s is a similar case, it is placed in Sect. R a p a due to its capacity to form t u b e r o u s roots.
14
The genus Brassica D i s t r i b u t i o n s into sections a n d lists of Brassica species have been proposed in very s e p a r a t e times (Schulz, 1936; Salmeen, 1979) a n d with very different a m p l i t u d e - the former a u t h o r a d m i t s three sections a n d the second nine. These w o r k s have been u s e d as b a c k g r o u n d for the s h o r t analysis which follows. In t u r n , Table 1.1 provides a n u p d a t e d version where m o s t r e c e n t r e s e a r c h r e s u l t s have been incorporated. U-triangle species a n d their r e l a t i v e s - here all included in Subgen. Brassica - s h a r e a few c o m m o n c h a r a c t e r s s u c h as the p r e s e n c e of seeds in their pod b e a k s (or at least the potentiality for it), spherical or quasi spherical seeds, n o t c h e d cotyledons a n d well defined l y r a t e - p i n n a t i p a r t i t e b a s a l a n d m e d i a n leaves, where s i n u s e s r e a c h the mid-nerve at least toward the leaf base. D o u b t s in the i n t e r p r e t a t i o n s o m e t i m e s arise b e c a u s e some of the above c h a r a c t e r s m a y a p p e a r in a regressive form a n d b e c o m e difficult to detect. For i n s t a n c e , a p a t i e n t observation of m a n y pods of B. oleracea m a y be n e e d e d to find only a few seeded beaks. It m a y be even more difficult with the slender long b e a k s of B. rapa, t h o u g h at least some seed primordia can be discovered w h e n green pods are observed. In B. nigra, the p r o n o u n c e d s h o r t e n i n g of the whole fruit m i g h t r e n d e r the s a m e t a s k impossible. However, all o t h e r c h a r a c t e r s agree with those of the group. In o t h e r taxa, leaves m i g h t show a r e d u c t i o n in the n u m b e r of their s e c o n d a r y s e g m e n t s so t h a t w h e n these are a b s e n t (as in B. carinata) the r e s u l t a n t simple silhouette can h a r d l y be a s s o c i a t e d with a lyrate leaf. Five sections have been recognized in Subgen. Brassica (Table 1.1). Sect Brassica h a s a l r e a d y been d i s c u s s e d above. While S a l m e e n (1979) considers t h a t B. rapa alone deserves a Sect. Rapa, the a m p h i d i p l o i d s B. napus a n d B. juncea, have b e e n a d d e d to this, since all three have the capacity to form t u b e r o u s roots even if this c h a r a c t e r is not always expressed. However, it is recognized t h a t Sect. Rapa a n d Sect. Brassica are akin. The B. f r u t i c u l o s a / s p i n e s c e n s / maurorum complex, all with n = 8, together with B. procumbens from Algeria a n d B. cadmea clearly s h a r e the s a m e c h a r a c t e r s mentioned above for b e a k s , seeds, cotyledons a n d leaves. They m i g h t r e p r e s e n t the closest relatives of B. nigra (Truco a n d Quir6s, 1995) a n d they are all p u t together in Sect. Micropodium. B. dej~exa deserves a special section b a s e d on its deflexed siliques, some cytological c o n s i d e r a t i o n s (unique Brassica species with n = 7) a n d a n i n d u m e n t u m with very long p a t e n t h a i r s which is s o m e h o w atypical. B. assyriaca is c o n s i d e r e d to fall within the variability of B. dej~exa. The trio B. tournefortii / B. barrelieri / B. oxyrrhina also shows spherical seeds, seeded b e a k a n d lyrate leaves, a l t h o u g h b e c a u s e of their t e n d e n c y to form c o m p a c t basal rosettes the leaves of B. barrelieri a n d B. oxyrrhina are b e t t e r described as r u n c i n a t e . In Table 1.1 they have been conservatively g r o u p e d together u n d e r Sect. Sinapistrum a l t h o u g h some recent r e s e a r c h s u g g e s t s t h a t the trio is not h o m o g e n e o u s (Horn a n d V a u g h a n , 1983; P r a d h a n et al. 1992). Their similitudes a n d differences can be well d e s c r i b e d by c o n s i d e r i n g all possible pairs of species v e r s u s the r e m a i n i n g
15 third species. T h u s , B. barrelieri a n d B. oxyrrhina are identical in the vegetative stage by their a d p r e s s e d r o s e t t e s a n d can be well d i s t i n g u i s h e d from B. tournefortii. B. tournefortii a n d B. oxyrrhina s h a r e small sized flowers probably a s s o c i a t e d with a u t o g a m y while B. barrelieri flowers are bigger. In t u r n , B. barrelieri a n d B. tournefortii b e a k s are s h o r t e r t h a n t h o s e of B. oxyrrhina. The last species is also u n i q u e in s h o w i n g white (against yellow) flowers. Let u s finally a d d a n o n - t a x o n o m i c a l fact: B. tournefortii is often cultivated in India as a s o u r c e of oil while the two o t h e r s are only wild. A n o t h e r quite different g r o u p - Subgen. Brassicaria -exemplified by B. repanda (n = 10) always s h o w s a seedless beak, their seeds are flattened to a greater or lesser extent, a n d their leaves c a n rarely be described as properly lyrate b u t r a t h e r as s i n u a t e or pinnatifid (sinuses never or very rarely reach the mid-nerve). Their t e n d e n c y to be s c a p o s e a n d / o r to show s h o r t s u b t e r r a n e a n s t e m s with leaf s c a r s (as if they were small b u r i e d b u s h e s ) is also characteristic. B. repanda h a s a l m o s t fifteen, often poorly defined, s u b s p e c i e s from the M a r o c c a n Atlas to the E u r o p e a n Alps. Galland (1988) found highly polyploid forms in the Great Atlas, a u n i q u e case for the g e n u s Brassica. B. glabrescens from N. Italy s h o u l d be c o n s i d e r e d as an additional s u b s p e c i e s of B. repanda. Significantly, B. repanda is a b s e n t from the Balearic Islands. F u r t h e r to the E a s t in Europe, B. jordanoffii a n d B. nivalis s h a r e the s a m e set of c h a r a c t e r s a n d are very local in Bulgaria a n d Greece, respectively. Akeroyd a n d Leadlay (1991) s u g g e s t e d t h a t b o t h s h o u l d be only s e p a r a t e d at the subspecific level, b u t they s h o w sufficient morphological differences a n d different c h r o m o s o m e n u m b e r s (n = 11 a n d n= 10, respectively). In t u r n , B. elongata is very c o m m o n from the B a l k a n s to I r a n a n d it occasionally o c c u r s to the West (Roux a n d G u e n d e , 1995). In fact, two s u b s p e cies are local in Morocco. It is not always completely s c a p o s e a n d c o n t a i n s an n = 11 genome. In North Africa, there are other m e m b e r s of the group s u c h as B. gravinae, B. setulosa and B. loncholoma from Algeria a n d B. desnottesii local in Morocco, the last three close to B. repanda. Also, the original description of B. somaliensis strongly a d v o c a t e s for it to be included in Subgen. Brassicaria. Most r e c e n t r e s u l t s t e n d to confirm t h a t m e m b e r s of Subgen. Brassicarla are very s e p a r a t e from Subgen. Brassica. For i n s t a n c e , Clemente-Mufioz a n d H e r n ~ n d e z - B e r m e j o (1980) o b t a i n seven d e n d r o g r a m g r o u p i n g s in Brassica, in w h i c h all s e e d l e s s - b e a k e d species fall into the s a m e group, separate from all o t h e r m e m b e r s of the genus. Curiously, they did not s t u d y fruits at all b u t only the m o r p h o l o g y of the sterile p a r t s of the flower, sepals, petals, nectaries, which is considered to be very c o n s t a n t t h r o u g h o u t the whole Brassicaceae family. F u r t h e r s u p p o r t for the s a m e idea can be found in m a n y r e c e n t morphologically or biochemically b a s e d w o r k s ( T a k a h a t a a n d Hinata, 1986; Horn a n d V a u g h a n , 1983, etc.). However, some of these d a t a seem to s u g g e s t a s e p a r a t e section for B. elongata.
16
B. souliei from NW Africa a n d Sicily a n d B. dimorpha from Algeria do not easily fit in a n y of the above sections. Both are morphologically closer to Subgen. Brassicaria b u t they c o n s t a n t l y a p p e a r very s e p a r a t e from other Brassica t a x a in m o s t recently p r o d u c e d m o l e c u l a r - b a s e d d e n d r o g r a m s . Two d e c a d e s ago, S a l m e e n (1979) placed t h e m in Sect. Nasturtiops which is here recognized a n d located within Subgen. Brassicaria.
Other related genera Erucastrum a n d Diplotaxis have traditionally b e e n considered as very close relatives of Brassica, a n d several r e c e n t investigations s t u d y the relative positions of the t h r e e genera. S~nchez-Y~lamo (1990) a n d S~nchez-Y~lamo et al. (1992) s u c c e s s f u l l y differentiate t h e m on the b a s i s of seed proteins. S~nchez-Y~lamo a n d Aguinagalde (1996) i n c o r p o r a t e d s t u d i e s with flavonoids. M a n y o t h e r c o m p a r i s o n s a m o n g these three g e n e r a have been carried out within s t u d i e s on larger g r o u p s of t a x a (below). Some specific a s p e c t s of the t a x o n o m y of Erucastrum have been studied by G 6 m e z - C a m p o (1982, 1983, 1984). F o r m e r t a x a k n o w n u n d e r E. laevigaturn are d i s t r i b u t e d b e t w e e n E. littoreum a n d E. virgatum. A new species, E. ifniense, is newly d e s c r i b e d as a c o n t i n e n t a l v i c a r i a n t of the M a c a r o n e s i a n group (E. canariense a n d E. cardaminoides). A n o t h e r controversial taxon from Morocco formerly k n o w n u n d e r several generic d e n o m i n a t i o n s is t r a n s ferred into this g e n u s as E. rifanum. Hirschfeldia incana a n d Erucastrum littoreum are p r o p o s e d as p a r e n t s of the amphidiploid species E. elatum, a hypothesis later confirmed by S~nchez-Y~lamo (1992) b a s e d on isoenzyme a n a lysis. The recognition of sections within Erucastrum r e m a i n s an open possibility. If we restrict ourselves to the c i r c u m - m e d i t e r r a n e a n taxa, E. gallicum, E. leucanthum a n d E. nasturtiifolium show c h a r a c t e r s closer to Diplotaxis (thinner pods, smaller s e e d s a n d shallowly n o t c h e d cotyledons) while E. virgatum, E. littoreum or E. varium are s o m e h o w closer to Brassica. The species of E a s t a n d S o u t h Africa are a p p a r e n t l y closer molecularly to the Diplotaxislike trio so t h a t f u r t h e r detailed morphological or caryological c o m p a r i s o n s between the species of each biogeographic a r e a would be convenient to clarify the possibility of e s t a b l i s h i n g sections. J o n s e l l (1982) h a s described two new species, E. elgonense in U g a n d a a n d E. m e r u e n s e in Tanzania.
Hirschfeldia is very similar to Erucastrum b u t its a m p l i t u d e as a g e n u s h a s been differently interpreted. The r e a s o n lies in its definition by a combin a t i o n of c h a r a c t e r s w h i c h are often feeble a n d do n o t always occur together even in the type species H. incana. In o u r opinion, H. incana should p e r h a p s r e m a i n as s u c h b u t H. rostrata from S o k o t r a Island s h o u l d be ascribed to Erucastrum. H. incana s u b s p , incrassata is in m a n y r e s p e c t s intermediate between Hirschfeldia a n d Erucastrum. Diplotaxis h a s a t t r a c t e d m u c h interest, as j u d g e d by the n u m b e r of papers p r o d u c e d . G 6 m e z - C a m p o (1981a) gives specific s t a t u s to D. ibicensis
17 a n d ascribes a frequently confused yellow f o w e r e d taxon from Algeria to D. erucoides- as s u b s p , longisiliqua. The work by Martinez-Laborde (1988ab, 1989, 1991a, 1991b, 1991c, 1992 a n d 1993) is i m p o r t a n t b e c a u s e it not only brings to light a n d gives a proper specific s t a t u s to some o b s c u r e taxa s u c h as D. ilorcitana, D. brevisiliqua, D. brachycarpa, etc. a n d p r o p o s e s several infraspecific taxa within some variable species s u c h as D.virgata, D. harra and D.siifotia, b u t also p r e s e n t s an u p d a t e d s y s t e m for the genus. This system is f u r t h e r developed by the s a m e a u t h o r within a recent p a p e r by G6m e z - C a m p o a n d Martinez-Laborde (1998) a n d is fully reflected in Table 1.1. In turn, B r o c h m a n n et al., (1997) exhaustively refer to the frequently neglected taxa of Cape Verde Islands (also included in Table 1.1). S~nchezY~lamo (1994) s t u d i e s flavonoid c o n t e n t of m a n y Diplotaxis species w i t h o u t clearly d i s c r i m i n a t i n g the s u b g e n e r a , while the work by S~nchez-Y~lamo a n d Martinez-Laborde (1991) on the isozymes of Subgen. Diplotaxis s h e d s light on the p a r e n t s of the amphidiploid species D. muralis. S o b r i n o - V e s p e r i n a s (1985, 1993, 1996) refers to some p u n c t u a l p r o b l e m s of this genus. In the work by Warwick et al. (1992) based on chloroplast DNA, a lack of congruity between m o l e c u l a r a n d morphological d a t a is reported. However, it is noteworthy (Table 1.1) that, at least within the s u b g e n e r a a n d sections of Diplotaxis, there is a high degree of congruity between morphological a n d molecular results. In Diplotaxis, leaves are always pinnatifid or p i n n a t i s e c t (sinuses never reach the mid-nerve) a n d seeds are always small a n d more or less ovoid or elliptic. Two of the s u b g e n e r a (Diplotaxis a n d Hesperidium) c o n s t a n t l y include species with seedless beak. Together with Brassica, Diplotaxis is u n i q u e in containing b o t h k i n d s of taxa - with seedless a n d seeded beaks. It is to be noted t h a t the presence of seeded b e a k s (hetero-arthrocarpic fruits) is an exclusive d e v e l o p m e n t of the tribe Brassiceae, p r e s e n t only in p a r t of the genera. It is believed t h a t m u c h of the general variability p r e s e n t in Brassica a n d Diplotaxis including molecular heterogeneity is, in fact, a s s o c i a t e d to this b e a k duality. Exclusively b a s e d on floral m e a s u r e m e n t s , Hern~ndezBermejo a n d Clemente (1986) found a m a x i m u m variability in Brassica, followed by Diplotaxis a n d t h e n by Erucastrum, well above t h a t of sixteen other related genera. The s a m e three genera p r e s e n t the h i g h e s t variability at the molecular level (Warwick a n d Black, 1991, 1993). However, Erucastrum does not show a n y convincing signs of b e a k duality.
Raphanus is treated in an extensive morphological study, by Pistrick (1987). Two species are recognized viz. Raphanus sativus a n d R. raphanistrum, the latter including several subspecies. The origin a n d the infraspecific variability of Raphanus sativus h a s merited special a t t e n t i o n a n d a m o n g s t the m o s t recent p a p e r s on this species, those by Crisp (1995) a n d Rabbani et al. (1998) s h o u l d be m e n t i o n e d . The i n d e h i s c e n t fruit of Raphanus is i n t e r p r e t e d as a fully developed b e a k i.e. w i t h o u t valvar portion. However, at least in R. raphanistrum, a stereomicroscope can often help to
18
detect two small scales at the base of the fruit vestigially representing the missing valves.
Sinapis is the object of a detailed thesis by Baillargeon (1986). This author distinguishes four sections (Table 1.1) and introduces some interesting novelties s u c h as placing all cultivated forms of S. alba u n d e r subsp, alba and all wild ones u n d e r subsp, mairei. He also describes or recombines some other taxa by s u b o r d i n a t i n g them u n d e r S. arvensis or S. pubescens (Baillargeon, 1983). The morphological and molecular heterogeneity present in genus Sinapis can easily be correlated with the existence of two basic chromosome n u m b e r s , n = 9 and n = 12. All the species are hetero-arthrocarpic. Coincya h a s been studied in a n o t h e r thesis (see Leadlay and Heywood, 1990) soon after u p d a t e d for Spanish material by Leadlay (1993). Both works constitute a significant step toward u n d e r s t a n d i n g this difficult heteroarthrocarpic genus. The t r e a t m e n t is synthetic and gets rid of a n u m b e r of superfluous n a m e s t h a t had produced m u c h confusion, mostly in the Iberian P e n in s u la where the genus reaches its m a x i m u m variability. SobrinoVesperinas (1991) carried out hybridizations with material from Sierra Morena (S.C. Spain) to s t u d y the inheritance of fruit shape. An extensive phytochemical s t u d y of the lipids by Vioque-Pefia (1992) h a s no significant systematic implications. Molecular studies (Warwick and Black, 1993) show a distinct and f u n d a m e n t a l l y hom ogeneous genus. Trachystoma, with three species, has been the object of some intergeneric crosses (see c h a p t e r s 3 and 4 of this book) and also of anatomic studies on its strongly hetero-arthrocarpic fruit (Giberti, 1984). It is known (Maire and S a m u e l s s o n , 1937) to hybridize s p o n t a n e o u s l y with Ceratocnemum. In a way, this challenges the presently defined limits for the ~coenospecies" and invites an exploration of several other genera with shortened fruits. Eruca h a s become a monotypic genus after two poorly known former Eruca species (setulosa and loncholoma) from North Africa are ascribed to Brassica Subgen. Brassicaria (Table 1.1). The uni que species E. vesicaria is an a n n u a l n o n -het er o- ar t hr ocar pi c plant, one of whose subspecies (subsp. sativa) h as reached c i r c u m - m e d i t e r r a n e a n distribution and is cultivated in m a n y other parts of the World. The difficulty to distinguish the three (or perh a p s four) subspecies of E. vesicaria using pressed material has been noted by G6mez-Campo (1993b), but it is expected t hat further characterization work would clarify this. Sobrino-Vesperinas (1995) observed partial sterility between subsp, sativa and subsp, vesicaria. Sinapidendron is a n o t h e r apparently seedless-beaked genus with five species plus one subspecies restricted to Madeira Island (Hansen and Sunding, 1993). Former S. palmense should be referred to Sinapis pubescens (Rustan, 1980). Sinapidendron is woody and the only non-heteroarthrocarpic genus which belongs to the so called ~nigra" molecular lineage (see next ch a p ter or Warwick and Black 1991; for B. nigra itself see above). Testing an obvious possible interpretation, the a u t h o r of this chapter stu-
19 died several h u n d r e d b e a k s in s e a r c h for vestigial seed primordia, so far w i t h o u t success.
The tribe Brassiceae Table 1.2 tries to u p d a t e previous lists with all the g e n e r a of the tribe Brassiceae t o g e t h e r with their e s t i m a t e d n u m b e r of species a n d the type of fruit exhibited. S u b g e n e r a for Brassica a n d Diplotaxis are also included. Novelties c o n s i s t of Quidproquo a n d Dolichorhynchus, while Reboudia is det a c h e d after its u n i q u e species was placed u n d e r Erucaria by G r e u t e r et al. (1986). We have only tentatively re-included Calepina, very distinct, b u t of doubtful position elsewhere in the family Brassicaceae. It is obvious t h a t not all these 54 g e n e r a (with r o u g h l y 240 species) have received the s a m e attention in the p a s t two decades. Only those not t r e a t e d above b u t considered in recent s t u d i e s will be briefly c o m m e n t e d , s o m e t i m e s with still briefer references to s o m e of their m o s t relevant relatives.
Moricandia is often considered to belong to the Brassica coenospecies on the basis of morphological a n d cytogenetical similarities. The g e n u s pres e n t s its m a x i m u m variability in N. Africa (where M. suffruticosa, M. spinosa, M. foleyii a n d M. nitens are endemic) a n d in the Iberian P e n i n s u l a (where M. moricandioides a n d M. foetida are endemic). S o b r i n o - V e s p e r i n a s (1983) suggests t h a t so called M. arvensis var. robusta Batt. from the C o n s t a n t i n e a r e a in Algeria is exactly the s a m e form which once e s c a p e d a n d h a s now invaded the M e d i t e r r a n e a n b a s i n as a weed. The s a m e a u t h o r p o s t u l a t e s the identity of M. sinaica (W. Asia) with M. arvensis var. g a r a m a n t u m Maire from the Hoggar m a s s i f (S. Algeria). The m o s t e a s t e r n m e m b e r of the tribe Brassiceae, Douepia is very similar to Moricandia except for its capitate stigma a n d its s a b a n a - o r i e n t e d fenology. A m m o s p e r m a a n d Pseuderucaria are a d a p t e d to s a n d y s u b d e s e r t i c h a b i t a t s a n d are very similar to each other. Quezeliantha was described u n d e r Quezelia by Scholz (1966) b u t the original generic n a m e was later a d j u s t e d (1982) by the s a m e a u t h o r . This grows in Tibesti massif. Vella w a s revised by G 6 m e z - C a m p o (198 lb). Crespo (1992) describes a new distinct species V. lucentina in SE Spain, not far from Alicante. More recently, Crespo et al. (1999) have p r o d u c e d a c o m b i n e d a n a l y s i s of the subtribe Vetlinae involving ITS ribosomal s e q u e n c e s a n d morphological data. In t u r n , Ponce-Diaz (1998) h a s s t u d i e d the s a m e g r o u p with i s o e n z y m e s a n d ISSR m a r k e r s . Warwick a n d A1-Shehbaz (1998) propose to include Boleum a n d Euzomodendron within Vella. However, the three g e n e r a exhibit very distinct sets of adaptive c h a r a c t e r s in a c c o r d a n c e with their respective mec h a n i s m s for seed dispersal. Quidproquo is a new g e n u s p r o p o s e d by G r e u t e r a n d B u r d e t (1983) for material from L e b a n o n a n d Israel t h a t h a d been e r r o n e o u s l y included in Sinapis aucheri. A s t u d y by Baillargeon (1985) h a s d i s s i p a t e d some previous d o u b t s on its t a x o n o m i c s t a t u s a n d geographical distribution. Its fruit s h o w s
20 a well developed seeded beak. Dolichorhynchus was described by Hedge and Kit (1987) from W e s t e r n Arabia. It is a desert a d a p t e d p l a n t resembling Moricandia b u t exhibiting seeded beaks.
Crambe h a s often a t t r a c t e d the a t t e n t i o n of b o t a n i s t s b e c a u s e of its evolved simplified fruits ( n u c a m e n t a c e o u s , derived from one-seeded beaks) a n d the a n t i q u i t y s u g g e s t e d by the E-W geographic d i s j u n c t i o n it presents. Using flavonoids, Aguinagalde a n d G 6 m e z - C a m p o (1984) reflect s u c h disj u n c t i o n at the biochemical level. Khalilov (1991) r e a d j u s t s the sections of this g e n u s after i n c l u d i n g two new species. More recently Warwick a n d Black (1997) s t u d y c h l o r o p l a s t DNA a n d F r a n c i s c o - O r t e g a et al. (1999) ribosomal ITS s e q u e n c e s with similar results. Crambe s e e m s to be a well defined g e n u s with clearcut limits a n d no evident close relatives. Division of the tribe Brassiceae into s u b t r i b e s h a s been an unresolved issue for a long time (Schulz, 1919; J a n c h e n , 1942) mainly b e c a u s e it is difficult to e s t a b l i s h a d e q u a t e criteria. Today, m o l e c u l a r m e t h o d s can be very useful at providing a rich source of h y p o t h e s e s (Yanagino et al. 1987; Song et al., 1990; Warwick a n d Black, 1991, 1994, 1997). Clustering m e t h o d s a r o u n d r e p r e s e n t a t i v e genera might be the way to define indubitable n a t u r a l groups, b u t as morphological, biochemical or m o l e c u l a r phenetic distances become greater, the b o u n d a r i e s of these defined g r o u p s become more and more diffuse. The case of the Vellinae is a good example of this situation (Warwick a n d Black, 1994; Crespo et al., 1999 a n d Ponce-Diaz, 1998). It s e e m s obvious t h a t Boleum, Euzornodendron a n d Carrichtera are the closest allies to Vella, followed by Succowia a n d Psichyne in this order. On the contrary, Schouwia, s h o w i n g a winged fruit similar to t h a t of Psichyne, is normally rejected as a relative of this group. All these genera show seedless b e a k a n d u s u a l l y s h o r t e n e d fruits a n d perennial habit, being a d a p t e d to either semiarid or m e s o p h y t i c s t e p p e s or m o u n t a i n s . Thus, the subtribe Vellinae s e e m s to c o r r e s p o n d to a n a t u r a l a n d c o h e r e n t group where Euzomodendron might r e p r e s e n t the long-fruited a r c h e t y p e a n d Succowia an evolved a n n u a l representative. It is u n f o r t u n a t e t h a t Kremeriella was not t a k e n into a c c o u n t b e c a u s e its a s y m m e t r i c u n i l o c u l a r fruit might well have derived from a bilocular one by lateral abortion. A fusion of s u b t r i b e s VeUinae a n d Savignynae was proposed by the a u t h o r of this article on the basis of their seed wing (sometimes vestigial) b u t molecular d a t a (Ponce-Diaz, 1998) suggest t h a t they s h o u l d be kept separate. Henophyton (perennial) a n d Savignya (annual), are not too d i s t a n t from each o t h e r a n d are b o t h a d a p t e d to more arid e n v i r o n m e n t s in North Africa. Quezeliantha, from Tibesti, might be also ascribed to the subtribe Savignynae. Therefore, this subtribe is small a n d its limits are not well defined. The s a m e o c c u r s with Cakilinae, where the s u p p r e s s i o n of Reboudia h a s left the pair Erucaria a n d Cakile alone. The isolated position of Crambe a n d its evolved strongly hetero-arthrocarpic fruit m i g h t well deserve a monotypic s u b t r i b e (Crambinae). The E-W
21 T a b l e 1.2 Genera and subgenera of the tribe Brassiceae (Cruciferae).
+ H H H H H H H H + H
H H H H H ?
Ammosperma (1) Boleum (1) Brassica Subgen. Brassica (27) Subgen. Brassicaria (11) Cakile (7) Calepina (1) Carrichtera (1) Ceratocnemum (1) Chalcanthus (1) Coincya (6) Conringia (6) Cordylocarpus (1) Crambe (35) Crambella (1) Didesmus (2) Diplotaxis Subgen. Diplotaxis (4) Subgen. Hesperidium (14) Subgen. Rhynchocarpum (13) Dolichorrhynchus (1) Douepia (1) Enarthrocarpus (5) Eremophyton (1) Eruca (1) Erucaria (6) Erucastrum (21) Euzomodendron (1) Fezia (1) Foleyola (1)
In parentheses: estimated number of species. H = heteroarthrocarpic genera or subgenera. + = genera with species of both types. ? = fruit status doubtful.
H H H H ? H H H H
H H H H
H H
?
Fortuynia (2) Guiraoa (1) Hemicrambe (1) Henophyton (2) Hirschfeldia (1) Kremeriella (1) Moricandia (7) Morisia (1) Muricaria (1) Orychophragmus (1) Otocarpus (1) Physorrhynchus (2) Pseuderucaria (3) Pseudofortuynia (1) Psychine (1) Quezeliantha (1) Quidproquo (1) Raffenaldia (2) Raphanus (2) Rapistrum (2) Rytidocarpus (1) Savignya (2) Schouwia (2) Sinapidendron (5) Sinapis (8) Succowia (1) Trachystoma (3) Vella (5) Zilla (2)
22 M e d i t e r r a n e a n geographic d i s j u n c t i o n it p r e s e n t s is i n t e r p r e t e d as an indication of antiquity. At first view, Crambella or Muricaria fruits are similar to those of Crambe b u t m o l e c u l a r r e s u l t s (Warwick a n d Black, 1997; FranciscoO r t e g a et al., 1999) do not s u p p o r t the existence of a close relationship. A similar s i t u a t i o n o c c u r s with Hemicrambe. F u r t h e r s t u d i e s are n e c e s s a r y to find a suitable position for t h e s e genera. Also, m a n y a u t h o r s have found similitude between Crambe a n d Calepina b a s e d on their n u c a m e n t a c e o u s fruits. In o u r opinion, the u n i q u e p e n d e n t seed found in Calepina fruit m e a n s t h a t a d r a m a t i c r e d u c t i o n a n d loss of d e h i s c e n c e of the valvar portion h a s t a k e n place while the u n i q u e erect seed of Crambe fruit m e a n s t h a t we are faced with a r o u n d e d o n e - s e e d e d beak. In o t h e r words, it is a case of mere convergence w h e r e the fruit of Calepina is derived from the valves a n d t h a t of Crambe from the beak. On the other h a n d , it is d o u b t f u l t h a t Calepina a m e m b e r of the tribe Brassiceae. Were it ever fully a c c e p t e d in the tribe, it would merit a n o t h e r monotypic s u b t r i b e for itself. All m e m b e r s of the s u b t r i b e ZiUinae (Zilla, Foleyola, Physorrhynchus a n d Fortuynia) s h a r e c o m m o n d e s e r t - a d a p t i v e morphological traits (Schulz, 1936) a n d are a p p a r e n t l y close molecularly (Warwick a n d Black, 1994, Crespo, pers. comm.}. F r u i t s in the E a s t e r n g e n e r a (Physorrhynchus a n d Fortuynia) are clearly h e t e r o - a r t h r o c a r p i c . However, seeds in the w e s t e r n genera (Zilla, Foleyola) are p e n d e n t , strongly s u g g e s t i n g t h a t their simplified fruits might not c o r r e s p o n d to b e a k s b u t to i n d e h i s c e n t valvar portions. If this is confirmed t h r o u g h a n a t o m i c a l studies, the s t r u c t u r e of this s u b t r i b e s h o u l d p e r h a p s be t h o r o u g h l y reconsidered. For the r e m a i n i n g s u b t r i b e s Brassicinae, Raphaninae a n d Moricandinae, it is p r e m a t u r e to try to modify the p r e s e n t s t a r e s b e c a u s e their limits are u n d e r c o n s t a n t a s s a u l t by the r e s u l t s of new r e s e a r c h a n d interpretations. B e a k d e v e l o p m e n t in size or s h a p e a n d / o r fruit s h o r t e n i n g are agile evolutionary t r e n d s w h o s e variation c a n often be observed within a single g e n u s (for i n s t a n c e Coincya, Trachystoma, etc.) while n a t u r a l h y b r i d s are frequently found between long fruited g e n e r a - T r a c h y s t o m a a n d Cordylocarpus- a n d s h o r t fruited ones s u c h as Ceratocnemum a n d Rapistrum respectively. Thus, s u b - t r i b e Raphaninae h a s p r o b a b l y derived polyphyletically from different m e m b e r s of the s u b t r i b e Brassicinae a n d a c l e a r - c u t limit between both cannot really be e s t a b l i s h e d . The Moricandinae are also close to the Brassicinae in m a n y respects, their differences being mostly a s s o c i a t e d with a d a p t a t i o n of the former to more xerophytic h a b i t a t s . We s h o u l d note t h a t while subtribe Moricandinae only c o n t a i n s n o n - h e t e r o - a r t h r o c a r p i c g e n e r a (with seedless beaks) a n d s u b t r i b e Raphaninae only c o n t a i n s h e t e r o - a r t h r o c a r p i c gen e r a (with seeded beaks), s u b t r i b e Brassicinae c o n t a i n s both types of genera. F u r t h e r m o r e , both t r e n d s are p r e s e n t within Diplotaxis a n d Brassica. If this m o r p h o g e n e t i c c h a r a c t e r is eventually recognized a higher relative taxonomic i m p o r t a n c e with r e g a r d to other merely adaptive c h a r a c t e r s (see next chapter) this would m e a n a split not only for s u b t r i b e Brassicinae, b u t also for the g e n e r a Diplotaxis a n d Brassica. If this idea is t a k e n further, this could
23 lead to the recognition of only two subtribes (genera with seedless beak and genera with seeded beak) but this seems somewhat exaggerated. However, giving an excessive or u n b a l a n c e d emphasis to molecular data might equally lead to similar exaggerated situations. Future developments should be based on the intelligent integration of the new information which is continuously arriving from different directions.
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32 Warwick, S. I. and Black, L. D. 1997. Phylogenetic implications of chloroplast DNA restriction site variation in subtribes R a p h a n i n a e and Cakilinae (Brassicaceae, tribe Brassiceae). Can. J. Bot. 75,960-973. Warwick, S. I., Black, L. D. and Aguinagalde, I. 1992. Molecular systematics of Brassica and allied genera (subtribe Brassicinae, Brassiceae) chloroplast DNA variation in the genus Diplotaxis. Theor. Appl. Genet. 83, 839-850. Widler, B. E. and Bocquet, G. 1979. Brassica insularis Moris: Beispiel eines messinischen Verbreitunsmusters. Candollea 34, 133-151. Yanagino, T., Takahata, Y. and Hinata, K. 1987. Chloroplast DNA variation among diploid species in Brassica and allied genera" J a p a n J. Genet. 62, 119-125.
Biology of Brassica Coenospecies C. Gdmez-Campo (Editor) 91999 Elsevier Science B.V. All rights reserved.
33
2 ORIGIN
and
DOMESTICATION
C~sar G 6 m e z - C a m p o (1) a n d S h y a m P r a k a s h (2)
(1) Dept. Biologia Vegetal, Universidad Potit~cnica de Madrid. 2 8 0 4 0 Madrid. Spain. (2) National R e s e a r c h Centre on Plant Biotechnology. Indian Agricultural Research Institute. N e w Delhi - 110012. India. Inferences from antiquity, excavated p l a n t parts, references in a n c i e n t literature of v a r i o u s civilizations, c o m p a r a t i v e t a x o n o m y , n a t u r a l distribution, cytogenetical a n d biochemical evidence and, in r e c e n t years, m o l e c u l a r m a r k e r s have greatly helped in r e c o n s t r u c t i n g the p a s t events of origin a n d evolution of Brassica a n d its allied genera. However, the origin of a n y cultivated p l a n t involves two s e p a r a t e aspects. On the one h a n d , the origin of the taxon itself before d o m e s t i c a t i o n , as a result of the evolutionary process in the wild. On the other, its origin in cultivation or, in o t h e r words, the h i s t o r y of its d o m e s t i c a t i o n a n d s u b s e q u e n t u s e a n d diversification. For wild allies - w h o s e c o n s i d e r a t i o n is of great i m p o r t a n c e n o w a d a y s - the first case would always a n d u n i q u e l y apply. B u t conversely, some p l a n t species were p r e s u m a b l y b o r n in cultivation - s u c h as Brassica napus, R a p h a n u s sativus, etc. - a n d lack previous evolutionary history. Evolution o c c u r s in b o t h cases, b u t a p a r t from m u t a t i o n it is the r e s u l t of n a t u r a l selection in the first case a n d of h u m a n influence a n d artificial selection in the second. H u m a n influence h a s also been exerted t h r o u g h the practice of p l a n t collection not only before b u t also d u r i n g the practice of agriculture, b u t its evaluation is extremely difficult a p a r t from a highly p r o b a b l e selection a g a i n s t the m o s t edible forms. M a n y p l a n t s are also a d a p t e d to m a n w i t h o u t being c u l t i v a t e d - s u c h as weeds, for instance. The r e l a t i o n s h i p between crops a n d weeds is not only one of competition. Adaptation to d i s t u r b e d h a b i t a t s a n d vigorous h a b i t have m a d e m a n y weeds s o m e h o w p r e - a d a p t e d to d o m e s t i c a t i o n (for instance, weedy a n d cultivated Daucus carota are the s a m e species). On the contrary, m a n y c o n t e m p o r a r y weeds are often crop p l a n t s e s c a p e d from cultivation (Medicago sativa). There are r e a s o n s to believe (see below) that, chronologically, the first d o m e s t i c a t e d Brassica species was the diploid B. rapa (turnip rape). Also the u s e s of B. nigra (black m u s t a r d ) s e e m to be very a n t i q u e a n d the s a m e can
34 p r o b a b l y be said of B. j u n c e a (Indian m u s t a r d ) an a m p h i d i p l o i d originated by c r o s s e s between b o t h species. In t u r n , B. oleracea (kale, cabbage) seems to have entered cultivation later b e c a u s e its n a t u r a l a r e a (Atlantic coasts) was too far from the i m p o r t a n t c e n t e r s of domestication. Therefore, amphidiploid species in which B. oleracea intervenes as a parent, n a m e l y B. n a p u s (rape) a n d B. carinata (Ethiopian m u s t a r d ) , s h o u l d have been the last b r a s s i c a s to be i n c o r p o r a t e d into agriculture. We will first c o m m e n t on the evolutionary origin of the m e m b e r s of B r a s s i c a coenospecies a n d will follow the above chronological order in o u r s u b s e q u e n t a c c o u n t on domestication.
The p h y l o g e n y o f B r a s s i c a and allied g e n e r a . Morphologically b a s e d c o n c e p t s a n d / o r morphologically based numerical t a x o n o m y s t u d i e s of relevance for the phylogenetic relations between B r a s s i c a species a n d its wild allies, are relatively f r e q u e n t in the literature of the p a s t two d e c a d e s (G6mez-Campo, 1980, 1999; T a k a h a t a and Hinata, 1980, 1986.). Many o t h e r biochemically b a s e d p u b l i c a t i o n s relevant to their phylogeny have already b e e n m e n t i o n e d in c h a p t e r 1 of this book and they roughly s u p p o r t ideas t h a t could also be derived from morphology. Incongruities of m o r p h o l o g y with reproductive or cytogenetic d a t a have often been detected in the past, b u t they were not paid m u c h attention. After all, sexual compatibility, for i n s t a n c e , is of less taxonomic or phylogenetic importance in p l a n t s t h a n in a n i m a l s b e c a u s e p l a n t evolution h a s often occurred with the protection of geographic barriers. In an extreme bizarre situation, nobody d o u b t s t h a t self-incompatibility does not imply the existence of two species in a single individual! Equally, different c h r o m o s o m e n u m b e r is not e n o u g h to s e p a r a t e two species w h e n all other visible c h a r a c t e r s are homogeneous: autopolyploidy or disploidy are j u s t a first step toward a possible speciation b u t they do not m e a n speciation by themselves. T a x o n o m y h a s been s u p p o s ed to follow p h y l o g e n y as m u c h as possible b u t it h a s also been expected to be p r u d e n t w h e n p h y l o g e n y is not well e s t a b l i s h e d - as o c c u r s in so m a n y cases. B u t above all, t a x o n o m y s h o u l d be pragmatic, practical and easy to use.
The a d v e n t of m o l e c u l a r biology h a s resulted in the addition of massive a p p a r e n t incongruities with e s t a b l i s h e d taxonomy, so t h a t frequent interm i x t u r e s of taxa in newly p r o d u c e d d e n d r o g r a m s raises a certain discomfort to a n y taxonomist. However, at least with o u r material, we can see t h a t incongruities are not so w o r r i s o m e and, on the other h a n d , there can be m a n y e x p l a n a t i o n s for the i n t e r m i x t u r e of taxa. S a m p l e d genes are evidently different for each case. While m o l e c u l a r m e t h o d s have aimed to s t u d y a few genes localized in organelles or a larger r a n d o m sample of the n u c l e a r genome where a large p r o p o r t i o n of genes are r e d u n d a n t or encode for primary metabolic p r o c e s s e s - classical t a x o n o m y h a s r a t h e r been b a s e d on a handful of specific genes w h o s e effects are externally visible.
35 In Brassica, m o l e c u l a r biology s t a r t e d with exploration of the origin of amphidiploid species via cpDNA (Palmer et al. 1983; E r i k s s o n et al. 1983; I c h i k a w a a n d Hirai, 1983). Shortly after, Yanagino et al. (1987) c o m p a r e d for the first time cpDNA of a set of eleven species a n d found clear incongruities with t a x o n o m y , since a group of four Brassica species a p p e a r e d split a n d intermixed in the d e n d r o g r a m with other five allied genera. The correlation with c h r o m o s o m e pairing (Mizushima, 1980) was very high, while t h a t with some n u m e r i c a l t a x o n o m y r e s u l t s ( T a k a h a t a a n d Hinata, 1986) w a s low or not significant. Soon after, Song. et al. (1990) s t u d i e d n u c l e a r RFLPs of thirty-eight a c c e s s i o n s belonging to fifteen species of Brassica a n d of three additional g e n e r a a n d found similar incongruities. Warwick a n d Black (1991, 1993, 1994, 1997) a n d Warwick et al. (1992) with cpDNA a n d P r a d h a n et al. (1992) with cp a n d mtDNA extend this type of s t u d y to m a n y other m e m b e r s of the tribe Brassiceae with c o m p a r a t i v e l y similar results. According to t h e s e a u t h o r s , at least in the a r e a of the phylogenetic tree where Brassica a n d its closest allies are found, two lineages c a n be distinguished: '~nigra" a n d " r a p a / o l e r a c e a " lineages - a c c o r d i n g to the n o m e n c l a ture u s e d by W a r w i c k a n d Black. M e m b e r s of large g e n e r a viz. Brassica, Diplotaxis, Erucastrum a n d Sinapis are r e p r e s e n t e d in both the lineages while monotypic or smaller g e n e r a are only r e p r e s e n t e d in one of these lineages. Cp a n d m t DNA RFLP information is therefore in favor of recognizing a polyphyletic origin, at least for these genera. C o m p a r a t i v e r e s u l t s by three aut h o r s r e g a r d i n g the d i s t r i b u t i o n of several species in t h e s e two lineages are s h o w n in Table 2.1. Let u s p u t forward t h a t s u b g e n e r a a n d sections recognized in Brassica, Diplotaxis a n d Sinapis on strictly morphological g r o u n d s (Schulz, 1936; Salmeen, 1979; G 6 m e z - C a m p o , in this book) a l r e a d y provide a basis on which s u c h p o l y p h y l e t i s m c a n be envisaged a n d u n d e r s t o o d . In general, m o l e c u l a r d a t a s u p p o r t the existence of s u c h large t a x o n o m i c divisions a n d act as a g e n e r a t o r of h y p o t h e s e s on how the evolution of p a r t i c u l a r t a x a or g r o u p s of t a x a might have occurred.
The "nigra" and "rapa" lineages Within Brassica Subgen. Brassica, the ~nigra" lineage involves a group of species i. e. r o u g h l y those included in Sect. Micropodium, w h o s e affinities with the g e n e r a Sinapis a n d Hirschfeldia a n d with some Erucastrum or Diplotaxis species are h i g h e r t h a n with the o t h e r m e m b e r s of Brassica belonging to sections Brassica, Rapa or Brassicoides). Sinapis species are all i n c l u d e d in ~nigra" lineage except Sect. Chondrosinapis w h o s e u n i q u e m e m b e r is S. aucheri, a t a x o n w h i c h h a s always b e e n c o n s i d e r e d to be very s e p a r a t e from the others. Within Diplotaxis, only the m e m b e r s of Subgen. Rhynchocarpum (except Sect. Erucoides) s e e m to belong to this lineage. In the case of Erucastrum w h e r e no sections have been recognized, m o s t species fall within this lineage (as it does Hirschfeldia, very close to Erucastrum) a n d only a few
36
Table 2.1 A select list of genera and species of the tribe Brassiceae comprising the two lineages - Brassica (Rapa) and Sinapis (Nigra) - as reported by various authors. Compiled by M. Lakshmikumaran. Song & aL 1990 TAG 79:
Warwick & Black 1991 TAG
Pradhan & al. 1992 TAG
497-526 (nuclear RFLP)
82:81-92 (cp RFLP) and 1992
85:331-340 (cp RFLP)
TAG 83:839-850 (cp RFLP) Sinapis lineage
Brassica lineage
Sinapis lineage
Brass&a
lineage
lineage
Sinapis lineage
Brassica rapa
B.futiculosa
B. rapa
B. Higra
B. rapa
B. Fligra
B.oleracea
B.nigra
B.oleracea
Xarvensis
B.juncea
B.carinata
Diplotaxis erucoides
S.arvens&
D.erucoides
S.alba
B.oleracea
S.arvensis
B.tournefortii
S.alba
B.deflexa
S.flexuosa
B.napus
S.flexuosa
Eruca sativa
S.aucheri
D.siettiana
D.erucoides
S.alba
Hirschfeldia incana
Raphanus sativus
B.fruticulosa
R.sativus
D.siettiana
B.oxyrrhina
S.pubescens
S.aucheri
D.siifolia
B.barrelieri
B.tournefortii
B.gravinae
S.pubescens
D.harra
H. incana
D.muralis
H. incana
E.sativa
D.siifolia
B.oxyrrhina
B.maurorum
B.gravinae
E.sativa
B.spinescens
D.muralis
D.harra
B.fruticulosa
Erucastrum abyssinicum
B.tournefortii
Moricandia arvensis
Erucastrum. varium
Brassica
B.souliei
37 (see below) are apart. The g e n u s Trachystoma a n d two s t u d i e d Sinapidendron species also belong to this lineage. Coincya is closer to "nigra" t h a n to "rapa" lineages b u t occupies a s e p a r a t e position, p e r h a p s s u g g e s t i n g a lineage of its own. The "rapa" lineage (short for "rapa/oleracea") will therefore contain all m e m b e r s of Sect. Brassica, Sect. Rapa a n d Sect. Brassicoides. Within Sect. Sinapistrum, B. barrelieri a n d B. oxyrrhina s h o u l d also be included. In t u r n , B. tournefortii h a s offered c o n t r a d i c t o r y r e s u l t s w h e n n u c l e a r or chloroplast DNA were u s e d , as d i s c u s s e d below. Significantly, "rapa" lineage also contains m o s t t a x a with seedless beak: not only m e m b e r s of Brassica s u b g e n u s Brassicaria b u t also all Diplotaxis species of the Diplotaxis a n d Hesperidium s u b g e n e r a as well as the g e n u s Eruca. As for Erucastrum, the "rapa" lineage c o n t a i n s the g r o u p gallicum \ nasturtiifolium \ l e u c a n t h u m - all morphologically close to e a c h other as indicated in c h a p t e r 1 - as well as some e a s t e r n a n d S. African species. G e n u s R a p h a n u s also falls within this lineage. We can see t h a t m o l e c u l a r d a t a are not as i n c o n g r u o u s with t a x o n o m y as initially t h o u g h t , since at least the larger t a x o n o m i c g r o u p s s h o w no difficulties in being m a t c h e d to DNA lineages. In t u r n , lineages offer a new view of evolution where not only m o r p h o g e n e t i c genes with visible p h e n o t y p i c effect are involved b u t s a m p l e s of a m u c h larger a n d / o r different sets of genes are manifested. B e c a u s e of its low c h r o m o s o m e n u m b e r , Hirschfeldia incana (n = 7) h a s been p r o p o s e d as an a n c e s t o r for the "nigra" lineage. However, Hirschfeldia could be seen as a n Erucastrum with specialized fruit, so t h a t o t h e r less specialized species of Erucastrum with n = 7 as E. virgatum or E. varium might better play t h a t role. Nonetheless, as the "nigra" lineage goes b a c k to the Sect. Rhynchocarpum of Diplotaxis a n d f u r t h e r b a c k into the g e n u s Sinapidendron - this case showing primitive seedless b e a k - p e r h a p s the real archaeotype for the "nigra" lineage s h o u l d be traced t h a t far.
S. arvensis h a s been r e g a r d e d as primitive within its genus, a n d its closeness to B. nigra is s u p p o r t e d by s t r o n g evidence. Both species show high homology in their n u c l e a r DNA (Song et al., 1988ab; P o u l s e n et al., 1994; Kapila et al., 1996), chloroplast DNA (Warwick a n d Black, 1991; Pradh a n et al., 1992), fraction I protein (Uchmiya a n d W i l d m a n 1978) a n d seed proteins ( V a u g h a n a n d Denford, 1968). Both species have 3 pairs of satellited c h r o m o s o m e s (Cheng a n d Heneen, 1995). The c h r o m o s o m e s of these two species p o s s e s s similar genic s e q u e n c e s as e x p r e s s e d in the high degree of pairing in the inter-generic hybrid B. nigra x S. arvensis (up to 8 bivalents, Mizushima, 1950). F u r t h e r evolution of Sinapis s e e m s to have o c c u r r e d in two directions, one line toward S. p u b e s c e n s a n d its relatives a n d a n o t h e r tow a r d S. alba a n d S. j~exuosa. The last two are very similar morphologically, cytologically a n d molecularly. On the o t h e r h a n d , S. aucheri m i g h t recognize a different origin.
3g Molecular d a t a also s u p p o r t a high degree of coherence for Sect. Microp o d i u m of Brassica. A close relationship exists between B. nigra and B. fruticulosa (n = 8) in regard to n u c l e a r DNA (Song et al., 1990), cp DNA (Warwick a n d Black, 1991; P r a d h a n et al., 1992), fraction I protein (Uchimiya a n d Wildman, 1978), a n d c h r o m o s o m e pairing (up to 7 bivalents in the hybrid. B. nigra x B. fruticulosa (Takahata a n d Hinata, 1983). Another three species viz. B. fruticulosa, B. maurorum a n d B. s p i n e s c e n s are ascribed to the same B. fruticulosa cytodeme (Harberd, 1976; T a k a h a t a a n d Hinata, 1983). Hybrids between t h e m are o b t a i n e d with m u c h ease (Takahata a n d Hinata, 1983) alt h o u g h their c h r o m o s o m e behavior reveals meiotic irregularities suggesting these species are u n d e r differentiation. Among them, B. maurorum is an unspecialized weed, while B. fruticulosa (with several subspecies) a n d B. spin e s c e n s have developed morphological c h a r a c t e r s of a d a p t a t i o n to coastal habitats. B. cossoniana w a s proposed to be an a u t o t e t r a p l o i d of B. fruticulosa (Harberd, 1972) b u t it s e e m s to show closer similarities in cp a n d m t DNA with B. maurorum ( P r a d h a n et al., 1992). B. p r o c u m b e n s is morphologically similar to other m e m b e r s of this group b u t it h a s recently been shown to have n = 9 c h r o m o s o m e s ( Baldini, 1998). Its lineage r e m a i n s u n k n o w n . For the rapa/oleracea lineage, Diplotaxis erucoides (n = 7) h a s been p r o p o s e d to be the closest ancestor, b a s e d on cp DNA (Warwick a n d Black, 1991; P r a d h a n et al., 1992). High homologies of repeat s e q u e n c e s between D. erucoides a n d B. rapa a n d B. oleracea (96 a n d 94% homology respectively, H a r b i n d e r a n d L a k s h m i k u m a r a n , 1990) s u b s t a n t i a t e this view. According to this, stocks r e p r e s e n t e d by a part of the genera Diplotaxis, Erucastrum and Brassica, plus the complete genera Eruca a n d R a p h a n u s evolved from this species. However, we would like to m a k e again o u r point to this scheme. It is obvious t h a t "rapa" lineage is well rooted in the group of taxa with a s p e r m beak, since those from Brassica s u b g e n u s Brassicaria, Diplotaxis s u b g e n u s Diplotaxis a n d s u b g e n u s Hesperidium a n d also the g e n u s Eruca belong to it. This m e a n s t h a t s u c h primitive a s p e r m - b e a k stock could never have derived from an a n c e s t o r similar to D. e r u c o i d e s - m u c h more evolved - b u t from more primitive a n c e s t o r s . These might tentatively be identified with Cape Verde Diplotaxis of Subgen. Hesperidium. S o m e c a s e s for f u r t h e r s t u d y
B. tournefortii (n = 10) belongs to the "nigra" lineage. Although Song et al. (1990) held the view t h a t this species evolved from a "rapa/oleracea" a n c e s t o r with n u c l e a r introgression from B. nigra, studies by Warwick and Black (1991) a n d P r a d h a n et al. (1992) on cp DNA suggest the opposite: it a p p e a r s t h a t B. tournefortii evolved from a B. fruticulosa-like a n c e s t o r with strong n u c l e a r a n d cytoplasmic introgression from B. r a p a / R a p h a n u s material (both s y m p a t r i c in their distribution) as reflected in the high a m o u n t of c h r o m o s o m e pairing p r e s e n t in the h y b r i d s (Mizushima, 1968).
39 Sinapis aucheri (n = 7) origin a n d evolution have been related to t h a t of R a p h a n u s (n = 9) b a s e d on their strongly h e t e r o a r t h r o c a r p i c fruits a n d their cp DNA similarity (Warwick a n d Black, 1991; P r a d h a n et al., 1992). However, we believe t h a t fruit similarity is a mere morphological convergence while cp DNA similarity is j u s t t h a t showed by two m e m b e r s of the "rapa" lineage. C h r o m o s o m e n u m b e r , leaf i n d u m e n t u m a n d shape, geographic distribution, etc. all advocate for a relation between Sinapis Sect. C h o n d r o s i n a p i s a n d B r a s s i c a Sect. Brassicoides. In other simpler words, Sinapis aucheri m i g h t be to B r a s s i c a dej~exa w h a t R a p h a n u s s a t i v u s is to B r a s s i c a rapa or B. oleracea. They both r e p r e s e n t the evolution of long seminiferous b e a k s from a n c e s t o r s with m o d e r a t e l y developed ones. Cultivated taxa
From i n t e r p r e t a t i o n of cytogenetic evidence, diploid B r a s s i c a species B. nigra, B. oleracea a n d B. r a p a - were t h o u g h t to have evolved in a n ascending order from a c o m m o n a r c h e t y p e (R6bbelen, 1960; P r a k a s h a n d Hinata, 1980). However, recent m o l e c u l a r investigations clearly d i s c a r d a monophyletic origin a n d s u g g e s t t h a t B. rapa a n d B. oleracea evolved from one progenitor while B. nigra evolved from another. In t u r n , several sets of evidence point to a c o m m o n origin for B. oleracea a n d B. rapa. These include: close similarities in their c y t o p l a s m s (Palmer et al., 1983; E r i c k s o n et al., 1983; Yanagino et al., 1987; Warwick a n d Black, 1983; P r a d h a n et al., 1992); high c h r o m o s o m e pairing in the interspecific hybrid B. rapa x B. oleracea (up to 9 bivalents, Olsson, 1960a; Namai, 1976); serological similarities in seed p r o t e i n s (Vaughan et at., 1966); n u c l e a r DNA RFLPs (Song et al., 1988a) a n d the presence of one satellite c h r o m o s o m e in b o t h g e n o m e s (Wang et al., 1989; C h e n g et al., 1995). The r e l a t i o n s h i p s of B. nigra within the "nigra" lineage h a s already been d i s c u s s e d . According to R6bbelen (1960) the B. o l e r a c e a / B , rapa real a r c h e t y p e is now extinct a n d h a d six pairs of c h r o m o s o m e s . Evidence in s u p p o r t of this view comes from s e c o n d a r y association of bivalents, c h r o m o s o m e pairing in haploids (see P r a k a s h a n d Hinata, 1980) a n d the presence of d u p l i c a t e d loci for rDNA genes (Quiros et al., 1985, 1987). Diploid species evolved from this prototype by two m e c h a n i s m s (i) selective c h r o m o s o m e d o u b l i n g i.e. secondary polyploidy, a n d (ii) c h r o m o s o m e re-patterning. RFLP d a t a s u b s t a n t i a t e this view of c h r o m o s o m e duplication a n d also s u g g e s t c h r o m o s o m e re-organization t h r o u g h localized sequence duplication a n d s e q u e n c e t r a n s p o s i t i o n (Song et al., 1991; Chyi et al., 1992; S n o w d o n et al., 1997). Recent r e s e a r c h on genome m a p p i n g (see c h a p t e r 7) h a s a d d e d m u c h information a n d new views to this subject. It s h o u l d be a d d e d t h a t a n y c o n s i d e r a t i o n on the origin of B. oleracea c a n n o t be s e p a r a t e d from the origin of its n = 9 relatives, since B. oleracea itself s h o u l d be considered as having evolved in close relation to t h a t group. As the m a x i m u m diversity of the whole group o c c u r s in Sicily, this Island a p p e a r s to be the m o s t probable c a n d i d a t e for its center of origin.
40
Additionally, Sicilian B. villosa and B. i n c a n a show several primitive characters such as hairy leaves and beaks with 1-3 seeds (primitive only for this material!). From there, five lines of evolution involving hair loss and wax development gave rise to the other taxa, after extending geographically through the Mediterranean and Atlantic coasts and islands (G6mez-Campo and Gustafsson, 1991). Alloploid species - B. carinata, B. j u n c e a and B. n a p u s - originated following multiple natural interspecific hybridizations (Olsson, 1960ab; Prakash, 1973, 1974; Song and Osborn, 1992). Natural hybridizations were always unidirectional as revealed by the studies on Fraction-I protein (Uchimiya and Wildman, 1978) and cp DNA restriction patterns, which conclusively established that B. nigra and B. rapa are the cytoplasmic donors of B. c a r i n a t a and B. j u n c e a respectively (Erickson et al., 1983; Ichikawa and Hirai, 1983; Palmer et al., 1983; Warwick and Black, 1991; Pradhan et al., 1992), while in B. n a p u s there is a slightly altered B. oleracea cytoplasm (Palmer et al., 1983). Similarities in cp genomes of diploids and their derived alloploids indicated that cp genomes have been well conserved in B. j u n c e a and B. c a r i n a t a since their origin (Erickson et al., 1983) while in B. n a p u s it has diverged slightly from the maternal B. oleracea cp genome. These studies also revealed that mt and cp genomes were co-inherited during their evolution. The close similarity in cp DNA of diploids and alloploids suggests that alloploids are of recent origin. A survey of rDNA of the allotetraploid species led Quiros et al. (1985) to propose that B. j u n c e a was the first to evolve and B. n a p u s and B. c a r i n a t a originated later. This agrees with the delayed entrance of B. oleracea in the agricultural world postulated in another part of this chapter. Amount of DNA in tetraploids has not changed significantly since their origin though there has been a reduction in nuclear size, probably due to higher DNA density resulting from greater condensation of chromosome material (Verma and Rees, 1974). Nuclear DNA composition of alloploid species is more closely related to the maternal cytoplasmic donors than to the male parents (Song et al., 1988a). Nuclear genomes of male progenitors in B. j u n c e a and B. carinata have undergone extensive changes while the maternal parents have preserved genomic integrity during evolutionary history (Song et al., 1988a). This suggests co-evolution of nuclear and cytoplasmic genomes. Only in B. n a p u s are these changes in nuclear genomes minimal (Parkin and Lydiate, 1997). It is enigmatic that each monogenomic species has not contributed cytoplasm to more than one alloploid species in spite of the fact that alloploids are of polyphyletic origin involving several interspecific hybridization events. Song et al. (1993) suggested that low seed fertility might be the cause for the elimination in nature of alloploids having opposite cytoplasm. The presence of different types of cytoplasm in various accessions of B. n a p u s strongly supports the concept of multiple origins of B. n a p u s (Song and Osborn, 1992).
41
Raphanus p r o b a b l y s h a r e s a c o m m o n a n c e s t r y with B. rapa/oleracea s t o c k s as indicated by the close c h r o m o s o m e homology between B. oleracea a n d Raphanus g e n o m e s - u p to 7 bivalents in R. sativus x B. oleracea (2n = 18) a n d u p to 7 bivalents in R. sativus x B. rapa (2n = 19) (Richharia, 1937). Many other similarities exist a m o n g Raphanus, B. oleracea a n d B. rapa. They can easily form intergeneric h y b r i d s a n d it is n o t e w o r t h y t h a t a m o n g the diploid species of Brassica coenospecies, only two - Raphanus a n d B. rapa s u b s p , rapa (turnip) - form t u b e r o u s roots. R. sativus is t h o u g h t to have derived from an a n c i e n t d o m e s t i c a t i o n of R. raphanistrum. Similarities in n u clear DNA RFLPs a n d morphological c h a r a c t e r s s u c h as flower size between Eruca a n d Raphanus led Song et at. (1990) to s u g g e s t t h a t b o t h h a d evolved from a c o m m o n ancestor. However, P r a d h a n et al. (1992) d i s c o u n t e d this in view of large differences in their cp DNA. Eruca h a s cp DNA similarities with Diplotaxis tenuifolia a n d D. pitardiana (all with n = 11 a n d a s p e r m beaks) while Raphanus h a s n = 9 a n d strongly h e t e r o a r t h r o c a r p i c fruit. The " i s t h m u s " c o n c e p t . I n t r o d u c e d by G 6 m e z - C a m p o (1999) it a t t e m p t s to help a better u n d e r s t a n d i n g of the evolution of the tribe Brassiceae as a whole. It c o n s i s t s of giving an a n g u l a r i m p o r t a n c e to the p r e s e n c e of seeds within the stylar cavity in m a n y of its m e m b e r s as a s i n g u l a r a c h i e v e m e n t t h a t can only be f o u n d within this tribe, while it is a b s e n t in all other Crucifer tribes. Within the tribe Brassiceae, r o u g h l y one half of the g e n e r a do not s h o w s u c h develo p m e n t , so they resemble o t h e r Crucifers at this respect. The other half s h o w seeded stylar portions (heteroarthrocarpy), a n d they often develop this t r e n d into bizarre types of seeded beaks. Two successive evolutionary r a d i a t i o n s are p o s t u l a t e d (Figure 2.1). The first involves t h o s e g e n e r a where the fruit b e a k keeps its primitive seedless condition ( n o n - h e t e r o a r t h r o c a r p i c fruit) s u c h as Sinapidendron, Eruca, Euzo-
modendron, Vella, Boleum, Carrichtera, Succowia, Moricandia, Rytidocarpus, Douepia, Conringia, Chalcanthus, Pseudofortuynia, Ammosperma, Pseuderucarla, Savignya, Henophyton, Quezeliantha, etc. as well as to the s u b g e n e r a Diplotaxis a n d Hesperidium of Diplotaxis a n d s u b g e n u s Brassicaria of Brassica. An a r c h e t y p e of the tribe m i g h t have r e s e m b l e d p r e s e n t M a d e i r a n Sinapidendron (but with biseriate i n s t e a d of u n i s e r i a t e seeds) or p r e s e n t Cape Verde Diplotaxis of subgen. Hesperidium (but with p e r h a p s woodier stems). The a c h i e v e m e n t of h e t e r o a r t h r o c a r p i c fruits (with seeded beaks) m i g h t have o c c u r r e d with the a d v e n t of s u b g e n u s Rhynchocarpum of Diplotaxis, so t h a t Diplotaxis s t a y s at b o t h sides of the i s t h m u s or bridge between b o t h radiations. F r o m here, a second evolutionary r a d i a t i o n originated Erucastrum, Hirschfetdia, Brassica (Subgen. Brassica), Sinapis, Coincya, Erucaria,
Trachystoma, Raphanus, Enarthrocarpus, Cakile, Ceratocnemum, Cordylocarpus, Crambe, Didesmus, Eremophyton, Fortuynia, Guiraoa, Hemicrambe, Muricaria, Otocarpus, Physorrhynchus, Rapistrum, Fezia, etc. All of t h e m s h o w
Guiraoa / Otocarpus / Ceratocnemum I
-0
Morisia
Brassica - Sinapis / Trachystoma / Coincya /
Sinapidendron / Brassicaria (Archetype)
I
< -I
rn
Fezia
v)
Erucastrum / Hirschfeldia / Cordylocarpus - Rapistrum ===
B)
Moricandia - Douepia Rytidocarpus / Conringia Pseudofortuynia Ammosperma - Pseuderucaria
Rhynchocarpum
Dolichorhynchus
-
-
-
-
-
X
rn ;[I
0 Erucaria - Cakile Didesmus / Eremophyton
Henophyton / Quezeliantha Savignya /Schouwia Foleyola - Zilla?
v)
Raphanus / Enarthrocarpus / Raffenaldia / Quidproquo
Eruca
Hesperidiurn ---------- Diplotaxis
rn 0
Psychine Succowia / Kremeriella? Vella - Boleum Euzomodendron / Carrichtera
A)
E
Hemicrambe / Crambe Crambella, Muricaria
Calepina?
-0
I
< --I
rn
cn Physorrhynchus - Fortuynia
Figure 2.1 The “isthmus” concept is exemplified by tentatively placing fifty genera (and five subgenera, underlined) into two separate evolutionary branches: A) genera with seedless beak (non-hetero-arthrocarpic), and B) genera with seeded beak (heteroarthrocarpic). Within each of these radiations, taxa are roughly grouped by their morphological or ecological affinities.
i3
43 h e t e r o - a r t h r o c a r p i c fruits with very different degrees of b e a k development, a c c o m p a n i e d or not with overall fruit reduction. It is believed t h a t m a n y previous c o n f u s i o n s derived from the joint consideration of b o t h r a d i a t i o n s a n d from the a b u n d a n t c a s e s of adaptive convergence involved. The i s t h m u s c o n c e p t provides a n u m b e r of h y p o t h e s e s to be either confirmed or rejected. Its c o n g r u i t y with p r e s e n t t a x o n o m y is high. While the c o n g r u i t y between morphological a n d m o l e c u l a r d a t a is not always high, it is believed t h a t this c o n c e p t m i g h t help to a b e t t e r i n t e r p r e t a t i o n of the existing divergences. For i n s t a n c e , the duality of lineages found by molec u l a r m e t h o d s in Diplotaxis a n d B r a s s i c a largely c o r r e s p o n d s with the d i s t r i b u t i o n of their s u b g e n e r a a n d sections on both sides of the i s t h m u s . However, it is obvious t h a t the s e c o n d r a d i a t i o n m a y not be completely monophyletic since both lineages seem to go a c r o s s the i s t h m u s .
Domestication Brassica
rapa
of cultivated brassicas and allies L.
B r a s s i c a rapa L. (syn. B. campestris) s e e m s to have grown n a t u r a l l y from the West M e d i t e r r a n e a n region to Central Asia, a n d is still p r e s e n t t h r o u g h o u t this area, in general a s s o c i a t e d to weedy h a b i t a t s . Its wide availability m a d e it p r o b a b l y the first d o m e s t i c a t e d Brassica, p e r h a p s several millenia ago, as a m u l t i p u r p o s e crop (roots in turnip, leaves in Chinese cabbage, y o u n g flowering s h o o t s in Galician "grelos", seeds in original colza or r a p e s e e d , etc.) a n d it h a s b e e n widely u s e d by all civilizations developed in t h a t ample region.
Nonetheless, t r u e a n t i q u i t y of B. rapa is not well d o c u m e n t e d . The earliest reference to a B. rapa ecotype is of yellow s a r s o n in S a n s k r i t literat u r e U p a n i s a d a s a n d B r a h a m a n a s (c. 1500 BC) w h e r e it w a s referred to as 'Siddhartha' (Prakash, 1961; Watt, 1989). Some B. rapa s e e d s have b e e n recovered from the s t o m a c h of Tollund m a n (Renfrow, 1973). Burkill (1930) c o n s i d e r e d E u r o p e as the place w h e r e B. rapa w a s first d o m e s t i c a t e d as a biennial p l a n t from w h i c h a n n u a l forms arose t h r o u g h selection. A c o m p a r ative morphological s t u d y led S u n (1946) to propose the existence of two lines: Western, w h i c h i n c l u d e s oilseed forms a n d t u r n i p , a n d are d i s t r i b u t e d t h r o u g h o u t E u r o p e , Central Asia a n d the I n d i a n s u b - c o n t i n e n t ; a n d E a s t e r n , c o m p r i s i n g E a s t Asian vegetable forms. Isozyme d i s t r i b u t i o n p a t t e r n s (Denford a n d V a u g h a n , 1977) a n d RFLP a n a l y s i s (Song et al., 1988b) s u p p o r t S u n ' s views. Evidence from morphology, geographic d i s t r i b u t i o n , isozymes a n d n u c l e a r RFLPs indicate t h a t t h e s e g r o u p s r e p r e s e n t two i n d e p e n d e n t c e n t e r s of origin. E u r o p e c o n s t i t u t e s the p r i m a r y c e n t e r for oleiferous forms a n d turnip. E a s t e r n forms evolved in the n o r t h - w e s t of I n d i a in the oleiferous direction, while Chinese forms differentiated as leafy vegetables in S o u t h China.
44 T u r n i p (B. rapa s u b s p , rapa) is believed to have evolved in Europe. Carbonized t u r n i p s have been recovered from Neolithic sites (Hyams, 1971). DeCandolle (1886) p r o p o s e d its cultivation in Europe a r o u n d 2 5 0 0 - 2 0 0 0 BC a n d its s p r e a d to Asia after 1000 BC. The Chinese book 'Shih-ching'by Confuceous (551-479 BC) m e n t i o n e d t u r n i p (Keng, 1974). T h e o p h r a s t u s (370285 BC) m e n t i o n e d t u r n i p in 'Enquiry into Plants'. He also m e n t i o n e d a wild p o p u l a t i o n with long n a r r o w roots having small hairy leaves. Similarly, Rom a n Cato (234-149 BC) referred to t u r n i p in 'On Agriculture' as a vegetable. Columella (42 AD) a n d recorded its uses. Plinius (23-79 AD) wrote extensively a b o u t t u r n i p in 'Natural History' a n d referred to it as rapa and napus. He stated t h a t Greeks d i s t i n g u i s h 3 k i n d s of turnip: a fiat one, a r o u n d one a n d a wild form with very long root. Columella (c. 60 AD), a u t h o r of one of the best R o m a n h a n d b o o k s on agriculture gave significant information on t u r n i p s . He m e n t i o n e d long 'Roman', 'Round' from Spain, the 'Syrian', the 'White' a n d the 'Egyptian'. The existence of m a n y Semitic, Greek a n d Slavic n a m e s for t u r n i p is significant a n d indicative of the antiquity of its domestication. In Middle English, napus b e c a m e nep a n d this together with turn (made round) gave the n a m e turnip (Boswell, 1949) which a p p e a r e d only after 1400 AD. Leafy forms are believed to have differentiated in C h i n a from oilseed forms of B. rapa after its i n t r o d u c t i o n t h r o u g h w e s t e r n Asia or Mongolia in the 1st c e n t u r y AD. Pak-choi (subsp. chinensis) with a n a r r o w or wide greenwhite petioles was the first to evolve in central C h i n a (Li, 1982). Its antiquity is s u g g e s t e d by a vast range of morphological diversity (Li, 1982) a n d high levels of DNA p o l y m o r p h i s m (Figdore et al., 1988; Song et al., 1988b). This was also the m o s t primitive form of East-Asian group from which s u b s p parachinensis developed in central China. The history a n d origin of Chinese cabbage - B. r. s u b s p , p e k i n e n s i s - is well d o c u m e n t e d (Li, 1982). The primitive loose-leaved form a p p e a r e d in the 10th c e n t u r y as a hybrid between p a k - c h o i (subsp. chinensis) a n d t u r n i p (subsp. rapa) in the city of YoungChow. This information is based on a reference in the book Ben-Cao-Tou-Jing (The Classics of Illustrated Medical Herbs). Its hybrid origin is also s u p p o r t e d by RFLP analysis (Song et al., 1988b). This primitive form is still grown in s o u t h e r n China. The h e a d i n g form with thick petioles a p p e a r e d for the first time in n o r t h e r n C h i n a a n d is recorded in 12th c e n t u r y literature. Better agronomy, irrigation a n d n u t r i e n t s u p p l y helped the a p p e a r a n c e of semih e a d i n g forms. S u b s e q u e n t l y , solid h e a d forms evolved t h r o u g h selection. A 14th c e n t u r y book Shua-Pu-Tsa-Su (Miscellanea of Gardening) mentioned s u c h forms. These forms were f u r t h e r improved by g a r d e n e r s a n d solid h e a d s with fluffy tops or fully solid h e a d s were developed. These types were described in Shuin-Tian-Fu-Tse (Local Records of Shuin 7~an FU) in the 17th century. Oleiferous B. rapa is d i s t r i b u t e d from Europe to China. It is believed t h a t E u r o p e a n forms developed in the M e d i t e r r a n e a n area (Sinskaia, 1928). On the other h a n d , Asian forms originated in the region comprising Central
45 Asia, A f g h a n i s t a n a n d adjoining n o r t h - w e s t India. In the I n d i a n s u b c o n t i n e n t there are 3 ecotypes of oleiferous B. rapa: b r o w n s a r s o n , toria a n d yellow sarson. Of t h e s e brown s a r s o n a p p e a r s to be the oldest (Singh, 1958). Two views r e g a r d i n g its origin exist: a) it evolved in the n o r t h - w e s t of the I n d i a n s u b - c o n t i n e n t from the original B. rapa stock (Sinskaia, 1928) a n d b) it r e a c h e d n o r t h - w e s t of India t h r o u g h Iran a l r e a d y in the s u b - d i f f e r e n t i a t e d s t a t e (Alam, 1945), from w h e r e it m i g r a t e d e a s t w a r d s a n d differentiated into o t h e r ecotypes. In t u r n , Toria is a n early m a t u r i n g crop very similar to b r o w n s a r s o n in m o r p h o l o g y except for the growing period a n d the size of the plant. It is believed to have been selected from a brown s a r s o n p o p u l a t i o n in the s u b - m o u n t a n e o u s t r a c t of the H i m a l a y a s . Yellow s a r s o n is c h a r a c t e r i z e d by yellow colored seeds a n d self-compatibility. M a n y of the cultivars have 3-4 valved siliquae a n d for this r e a s o n it w a s once n a m e d B. trilocularis (Roxburg, 1832). However, this c h a r a c t e r does not s e e m intrinsic to it as forms with bilocular fruits also o c c u r in n a t u r e . It is believed to have evolved from brown s a r s o n as a m u t a n t a n d have survived b e c a u s e of its self-compatible n a t u r e . It m i g h t have b e e n selected by f a r m e r s for its a t t r a c t i v e yellow seed color a n d a bigger seed size. It w a s first m e n t i o n e d as Siddhartha in S a n s krit l i t e r a t u r e from c. 1 0 0 0 - 8 0 0 BC i n d i c a t i n g t h a t it w a s well e s t a b l i s h e d by t h a t time. H i n a t a a n d P r a k a s h (1984) s u g g e s t e d c. 1200 BC a n d n o r t h - w e s t of India as the tentative date a n d place of origin.
B r a s s i c a nigra (L.) K o c h . This species w a s m e n t i o n e d by H i p p o c r a t e s (480 BC) for its m e d i c i n a l value. The New T e s t a m e n t m e n t i o n s ~ m u s t a r d " as a '~plant growing fast a n d high u p from a small seed, b r a n c h i n g a n d allowing birds to n e s t on its b r a n ches" in a c o m p a r i s o n with the growth of the Kingdom of God. This m i g h t n o t be too e x a g g e r a t e d since in good c o n d i t i o n s this a n n u a l species c a n easily b e c o m e t h r e e to four m e t e r s high in only a few weeks. Its n a t u r a l a r e a is c i r c u m - m e d i t e r r a n e a n e x t e n d i n g into C e n t r a l Asia a n d the Middle East, so it w a s easily available for d o m e s t i c a t i o n by m a n y Old World civilizations. PreC o l u m b i a n M e d i t e r r a n e a n civilizations were p a r t i c u l a r l y k e e n for spices to m a k e food m o r e tasteful, a n d c o n d i m e n t s b a s e d on Cruciferae, Labiatae or Umbelliferae were very i m p o r t a n t . The s t r o n g t a s t e of Brassica nigra (black m u s t a r d ) or Sinapis alba (white m u s t a r d ) seed m e a l s w a s therefore m u c h a p p r e c i a t e d . E i t h e r cultivated or s i m p l y collected from the wild, t h e s e two species have also been the object of m e d i c i n a l u s e s (as in s i n a p i s m s ) since very a n c i e n t times. The word m u s t a r d is t h o u g h t to come from Latin ~mustus ardens", b u r n i n g juice in English. Nowadays, m u s t a r d u s e s of B. nigra (black m u s t a r d ) have d i m i n i s h e d in favor of B. juncea (Indian m u s t a r d ) or in a growing scale B. carinata (Ethiopian m u s t a r d ) . F u r t h e r m o r e , B. nigra h a s also benefited m a n k i n d by p a r t i c i p a t i n g as a p a r e n t in t h e s e two a m p h i diploids.
46
Brassicajuncea (L.) C z e r n . Prain (1898) held t h a t B. juncea originated in China, a view also supported by Sinskaia (1928) who believed t h a t E a s t - E u r o p e a n B. juncea is also of Chinese origin from where it migrated naturally t h r o u g h the Kirgiz steppes. In evidence, she stated t hat it grows wild along this route. She further proposed t h a t forms with lyrate-pinnatisect leaves are the most primitive from which evolution occurred in three directions" bipinnate leaves dissected into th r ead like s e g m e n t s (in the E a s t Asian forms), crisp leaves (in the Chinese forms) and non-divided leaves (comprising the Central Asian and Indian forms). However, Burkill (1930) and Sun (1970) discard its Chinese origin placing it in the Middle East. S u n (1970) argued t h a t since the parental species are not found naturally in China, B. juncea had to be introduced from outside. Vavilov (1949) proposed Afghanistan and adjoining regions as the primary center of its origin and favored Central and Western China, Eastern India and Asia Minor t h r o u g h Iran as the secondary centers. However, the cytogenetical, biochemical and, in recent years, molecular evidence, point to a polyphyletic origin (Olsson, 1960b; Prakash, 1973a; Vaughan, 1977; Song et al., 1988a) at m a n y places where the parental species have a sympatric distribution. Due to the occurrence of wild forms of B. rapa and B. nigra, the region of the Middle East has strongly been favored as its original place of origin (Olsson, 1960b; Mizushima and Tsunoda, 1967). Wild forms of B. juncea still grow in this region particularly in the plateau of Asia Minor a n d s o u t h e r n Iran (Tsunoda and Nishi, 1968). Regions of southwestern China and north-west India constitute two secondary centers showing e n o r m o u s variations. Biochemical studies of V a u g h a n et al. (1963) and V a u g h a n an d Gordon (1973) provided strong evidence for the existence of two geographical races viz. Chinese, where the seeds have a marked mucilagenous epidermis and produce allylisothiocynates, and Indian which produce 3-butenyl isothiocynate. RFLP studies (Song et al., 1988a) also give s u p p o r t to two main centers of origin: a) Middle East-Indian region where p r e d o m i n a n t l y oil forms evolved; a n d b) China, where evolution occurred mainly towards leafy forms. China h a s a long history of B. juncea cultivation (Wen, 1980; Chen, 1982). Its first mention is in literature from the Chou Dynasty (1122-247 BC). Its use as a flavouring agent is recorded during the West Han Dynasty (206 BC-24AD). Dai in his work Liji (The book of Rites) referred to a "sliced j a m of fish with mustard". Uses for its seeds and leaves are also mentioned in C h i a - s s u - h s i c h ' s book Himin-yao-shu of late 5th or early 6th century (Li, 1969). F r e q u en t references are found in Su's (10-61 AD) work Tu-Jin-Bin-Cao (Illustrated Book of Medicinal Herbs) indicating that m u s t a r d was a popular crop of the time. Wang (1576-1588) mentioned root forms in his work 'Gua Guo Shz~ (Explanations of Cucurbits and Vegetable Crops). The famous work by Li (1578) described m a n y forms which were used as leaves or shoots during Ming dynasty.
47 A fascinating a c c o u n t r e g a r d i n g the origin of v a r i a t i o n s in Chinese B.
juncea h a s b e e n p r e s e n t e d by Wen (1980). Description in a n c i e n t literature
from the 5 t h c e n t u r y AD indicate t h a t the primitive type w a s a small a n n u a l p l a n t with poor leaf growth cultivated for its p u n g e n t seeds. S u b s e q u e n t variations of leaf s h a p e , size a n d colour, petiole width, h e a d i n g type a n d s p r e a d of leaves evolved a n d were selected. F o r m s with l u x u r i o u s b r o a d leaves were developed in the Tang D y n a s t y (618-907 AD) a n d u s e d as greens in t e m p e r a t e a n d h u m i d s o u t h China. A form with deeply dissected leaves a d a p t e d to arid e n v i r o n m e n t s was developed in n o r t h e r n China. This, in t u r n , p r o d u c e d a tillering form which was more productive, b r a n c h e d early d u r i n g vegetative growth a n d was good for pickles. F o r m s with broad, thick mid-ribs a n d petioles were developed d u r i n g the Chin d y n a s t y (1644-1911 AD). Later, h e a d e d forms with leaves with fleshy m i d r i b s a n d petioles evolved. Types with swollen s t e m were also bred. F l e s h y root forms evolved i n d e p e n d e n t l y from b r o a d leaved forms p r e s u m a b l y after the 12th century.
B. juncea w a s also a c o m p o n e n t of the a g r i c u l t u r e of the I n d u s Valley civilization w h i c h flourished a r o u n d 2 3 0 0 - 1 7 5 0 BC. The a r t of e x t r a c t i n g oil w a s k n o w n to this civilization. In fact, seeds of B. juncea have been excavaed from C h a n h u d a r o , a site of this civilization (Allchin, 1969: Mackey, 1943). W h e n the A r y a n s c a m e to India c. 1500 BC, they a d o p t e d B. juncea oil as a preservative. Its u s e w a s later e x t e n d e d to cooking a n d m a s s a g e p u r p o s e s . A r o u n d 1000 BC, it s p r e a d e a s t w a r d s with m i g r a t i n g people. Reports of Chinese travelers H u e n T s a n g (c. 640 AD) a n d Itsing (c. 690 AD) reveal t h a t it was e s t a b l i s h e d as a n oil crop in the I n d o - G a n g e t i c Plains by 700 AD. T h o u g h conflicting views have been e x p r e s s e d r e g a r d i n g the route of e n t r y of B. juncea into India, it s e e m s t h a t B. juncea r e a c h e d n o r t h - w e s t India from the Middle East, its place of origin, t h r o u g h A f g h a n i s t a n b e t w e e n 5 5 0 0 - 2 3 0 0 BC (Hinata a n d P r a k a s h , 1984). Intensive differentiation of m a n y agro-ecotypes was later c o m p l e m e n t e d by new h y b r i d i z a t i o n s b e t w e e n the constit u e n t p a r e n t s in n o r t h - w e s t India. Brassica
o l e r a c e a L.
B. oleracea L., grows wild in the Atlantic c o a s t s of Europe, where it m i g h t have b e e n cultivated by Celts in its primitive form (kales). W h e n it was eventually b r o u g h t to the E a s t M e d i t e r r a n e a n region (estimatedly by the limit between the first a n d second millenia BC) it b e c a m e fully d o m e s t i c a t e d a n d s t a r t e d an explosive diversification giving rise to a n e n o r m o u s r a n g e of cultivated forms. The m o s t widely k n o w n forms or g r o u p s of forms are: 1) kales w h i c h develop a s t r o n g m a i n s t e m a n d are u s e d for their edible foliage. These are old cultivated forms a n d include green curly kales, n a r r o w s t e m kale, a n d giant J e r s e y kale. Land r a c e s of these kales are widely scattered.
48
2) cabbages are charaterized by formation of h e a d s of tightly packed leaves and are represented by head cabbage, savoy cabbage and Brussels sprouts. 3) Kohlrabi is grown for its thickened stem. 4) Inflorescence kales are u s e d for their edible inflorescences. Major forms are cauliflower, broccoli and calabrese. 5) Chinese albogtabra kale, u s e d for its leaves. Earlier, it was widely held t h a t wild oleracea kales which are found along the Mediterranean coasts, were progenitors of the cultivated forms (see P r a k a s h an d Hinata, 1980), b u t the pr es ent concept is the opposite: Mediterr a n e a n kales are mere escapes from early cultivations. More recently, a polyphyletic origin by incorporation of genes into the B. oleracea genome from different wild Mediterranean species (Gustafsson, 1979; Snogerup, 1980; Mithen et al., 1987) was suggested. The species preferently considered in this respect were B. cretica, B. rupestris, B. insularis, and B. montana. However, Hosaka et al. (1990) do not find molecular evidence to suggest specific wild ancestors for the different B. oleracea types. Today, the tendency is to minimize the possible introgression by other species, t h u s returning again to the Atlantic B r a s s i c a oleracea the main role in the development of cultivated forms b u t admitting t h a t introgression of genes from wild species has probably been responsible for increasing the variability and adaptability of cultivated B. oleracea. The earliest cultivated B. oleracea was most likely a leafy kale which gave rise to a wide variety of kales along the coasts of the Medit e r r a n e a n an d Atlantic from Greece to Wales (Song et al., 1990). Diverse forms were developed in different areas primarily due to selection in different climates, n a t u r a l hybridization and gene introgression. Macro- and microm u t a t i o n a l events and c h r o m o s o m a l changes also played a substantial role (Chiang an d Grant, 1975; Kianian and Quiros, 1992). Song et al. (1988b, 1990) considered Chinese kale (var. alboglabra) to be very close to the primitive type which spread to the center of the East Mediterranean and eventually reached China. The extent to which Celts domesticated B. oleracea in western and n o r t h w e s t e r n Europe, as believed by DeCandolle (1886), is an open question, since it is also likely t h a t invading Celts a r o u n d the VI-VIII centuries BC found cabbage already domesticated by aborigines and adopted it from them. Another open question is that of how a wild plant from the Atlantic coasts could reach the centers of civilization, at that time Egypt and Mesopotamia. A slow diffusion t h r o u g h France by 1000 BC seems improbable. However, a rapid way could have been provided by the tin route, linking the ~Casiteride" Islands (British Islands) with the East Mediterranean by sea (G6mez-Campo and Gustafsson, 1991). Tartessians, from SW Spain, were exploiting the tin mines of Cornualles a n d selling the ore to the Phoenicians in Gades (now Cadiz). The Phoenician maritime commercial network then at its peak in the Mediterranean coasts did the rest. The early nam e of B. oleracea (~krambe")
49 u s e d by the Greeks, w a s probably of Phoenician origin. Brassica itself h a s been linked to Celt n a m e s , b u t it p r o b a b l y comes from "prasikein", vegetable in Greek. Greek T h e o p h r a s t u s (370-285 BC) a n d R o m a n Cato (234-149 BC) a n d Plinus (23-79 AD) already described s t e m k a l e s a n d h e a d e d cabbages. It is believed t h a t an a r r a y of cultivated coles was available to R o m a n s at the time of Christ. Cauliflower a n d broccoli evolved in the e a s t e r n M e d i t e r r a n e a n (Hyams, 1971; S n o g e r u p , 1980). T h o u g h s p r o u t i n g forms of c a b b a g e were m e n t i o n e d by early G r e e k s a n d R o m a n s a distinction was not a p p a r e n t between cauliflower a n d broccoli, indicating t h a t the differences did not exist or t h a t the two were c o n s i d e r e d as v a r i a n t s of the s a m e form. A S p a n i s h - A r a b i c a u t h o r , Ibn-al-Awam, (c.1140) m a d e the first clear distinction between h e a d i n g a n d s p r o u t i n g forms in his book Kitab-al-Falaha, wherein he devoted a s e p a r a t e c h a p t e r to cauliflower (Hyams, 1971). He u s e d the n a m e 'quarnabit', the present day Arabic word for cauliflower. Herbalist D o d o e n s (1578) referred to cauliflower as B. cypria indicating its origin in Cyprus. H y a m s (1971), b a s e d on the o b s e r v a t i o n s of Ibn-al-Awam who referred to it as Syrian or Mosul cabbage, c o n s i d e r e d Syria as the place of its origin. Cauliflowers are generally r e g a r d e d as derived from broccolies (Crisp, 1982; Gray, 1982). Crisp (1982), b a s e d on his w o r k on hybridization between broccoli a n d cauliflower, c o n c l u d e d t h a t a single m a j o r gene m u t a t i o n in broccoli gave rise to cauliflower. Broccoli is an Italian word derived from the Latin '~brachium" which m e a n s a n a r m or b r a n c h (Boswell, 1949). It includes h e a d i n g forms with a single large t e r m i n a l inflorescence. Another form is s p r o u t i n g broccoli, a b r a n c h e d type in which the y o u n g edible inflorescences are referred to as sprouts. Broccoli p r o b a b l y originated between 4 0 0 - 6 0 0 y e a r s BC w h e n the a n c e s t r a l forms of m o d e r n varieties were selected (Schery, 1972). Dalec h a m p ' s (1586) description in the 16th c e n t u r y h e r b a l Historia Generalis Plantarum c o n s t i t u t e s the first d o c u m e n t e d report. Broccolis were introd u c e d into Italy from the e a s t e r n M e d i t e r r a n e a n where diversification took place a n d m a n y forms, including h e a d i n g a n d s p r o u t i n g ones arose. The regular h a n d s o m e inflorescence of recently commercialized '~Romanesco" broccoli is often cited by the s t u d e n t s of fractal geometry. B r u s s e l s s p r o u t s were developed n e a r B r u s s e l s in Belgium d u r i n g the 14th century. A kale r e s e m b l i n g B r u s s e l s s p r o u t s , b u t with finely dissected leaves a n d n u m e r o u s b u d s , is d e s c r i b e d by Gerarde (1597) as ~persil cabbage" (Henslow, 1908). These s p r o u t s were reportedly served at a wedding feast in 1481 (Hyams, 1971). Brassica
napus
L.
Brassica napus is not k n o w n to occur truly wild in n a t u r e t h o u g h it often o c c u r s as an escape. The first reference to r a p e s e e d (B. napus s u b s p . oleifera) was by D o d o e n s (1578) in 'Cruydt Boeck' wherein he referred to Slo-
50 o r e n being grown for oilseeds (Toxopeus, 1979). This was probably an early form of winter rape. As a crop, it a p p e a r e d a r o u n d the y e a r 1600. Its oil was k n o w n as "raepolie" a n d was u s e d in l a m p s for lighting, as a food a n d in soap making. S u m m e r rape developed a r o u n d the y e a r 1700 and is cultivated in places where winter is not too severe. Some older records have been o b s c u r e d by its similarity to B. rapa.
S i n s k a i a (1928) a n d S c h i e m a n n (1932) p r o p o s e d t h a t it originated in the M e d i t e r r e n e a n region of s o u t h - w e s t E u r o p e where the two contributing p a r e n t s , B. o l e r a c e a a n d B. rapa, overlap in their n a t u r a l distribution (Olsson, 1960b; T s u n o d a , 1980). However, s u c h overlapping did not probably exist. Wild B. r a p a is very poorly r e p r e s e n t e d today in S p a i n (only in Girona, the n o r t h e a s t e r n extreme) a n d it is difficult to envisage it a p p r o a c h i n g the Atlantic m a r i t i m e cliffs where B. o l e r a c e a was growing wild. Thus, we believe t h a t it m a y have originated elsewhere, even outside the Mediterranean region, b u t obviously in a n agricultural environment. In fact, the B. o l e r a c e a • B. r a p a cross h a s o c c u r r e d several times a n d in both directions. Palmer et al. (1983) explored the m a t e r n a l lines in the U s c h e m e t h r o u g h chloroplast analysis. C o n t r a d i c t o r y phylogenies for two lines of B. n a p u s might indicate either f u r t h e r introgressive hybridization or multiple origins. Ar~s, Baladr6n a n d Ord~s (1987) u s e d isozymes to analyze B. n a p u s a n d their parents. Aguinagalde (1988) detected additive profiles of flavonoids not only for B. n a p u s b u t for all the three hybrid species. An alternative view on the origin of B. n a p u s h a s been provided by Song a n d O s b o r n (1992) b a s e d on chloroplast a n a l y s e s of several B. n a p u s accessions. His r e s u l t s suggest t h a t B. nap u s cDNA is closer to t h a t of B. m o n t a n a t h a n t h a t of B. oleracea. R u t a b a g a s or swedes (B. n a p u s s u b s p , rapifera) are also believed to be of recent origin. According to Boswell (1949), the h e r b a l i s t B a u h i n mentioned swede for the first time t h o u g h no a c c u r a t e reference is provided. Zwinger (1696) described a form S t e c k r u e b e n - k o h l which in all probability r e p r e s e n t s a n early form of r u t a b a g a . It b e c a m e p o p u l a r in Scandinavia a n d later s p r e a d to E n g l a n d in the late 18th c e n t u r y (McNaughton and Thow, 1972). Cultivated r a p e s e e d or colza started with oleiferous varieties of B. r a p a (which are still in use), b u t B. n a p u s h a s progressively t a k e n the s u p r e m a c y in this role. This h a s o c c u r r e d to a point t h a t B. n a p u s , only 400 years old as a k n o w n species, h a s now climbed to the second or third places in economic i m p o r t a n c e a m o n g edible crops in several c o u n t r i e s s u c h as C a n a d a a n d a few in Central Europe. D e p e n d i n g u p o n the yearly prices, it sometimes surp a s s e s w h e a t in relative importance. Double-zero varieties (without erucic acid a n d w i t h o u t glucosinolates) have given a new e n o r m o u s impulse to its use. For f u r t h e r information a b o u t recent scientific a n d applied developm e n t s involving B. n a p u s , the reader is referred to o t h e r c h a p t e r s of this book, especially to n u m b e r 13.
51 B. c a r i n a t a Braun
An a m p h i d i p l o i d derived from c r o s s e s b e t w e e n B. oleracea a n d B. nigra, it h a s b e e n g r o w n for c e n t u r i e s locally in Ethiopia. M i z u s h i m a a n d T s u n o d a (1967) failed to locate a n y wild t y p e s in t h e E t h i o p i a n p l a t e a u , b u t t h e y often o b s e r v e d t h e p a r e n t s growing close to e a c h o t h e r in c u l t i v a t i o n or a s escapes. The u s e s of t h i s p l a n t in E t h i o p i a are m u l t i p l e (Astley, 1982; Riley a n d B e l a y n e h , 1982): oil is e x t r a c t e d , the c a k e is u s e d a s a m e d i c i n e , c r u s h e d s e e d s are c o n s u m e d in s o u p s or as spice, leaves are boiled a n d e a t e n , etc. Brassica carinata is n o w b e c o m i n g m o r e a n d m o r e p o p u l a r in o t h e r p a r t s of the World as a p r o m i s i n g oilcrop a n d s o m e i m p o r t a n t b r e e d i n g p r o g r a m s a r e u n d e r w a y (Rakow a n d Getinet, 1998).
O t h e r g e n e r a o f agricultural s i g n i f i c a n c e
R a p h a n u s sativus L. is n o t k n o w n in the wild s t a t u s ( t h o u g h it h a s bec o m e w e e d y in m a n y a r e a s , e s p e c i a l l y in t e m p e r a t e S o u t h America). It is t h o u g h t to h a v e o r i g i n a t e d by d o m e s t i c a t i o n a n d selection of t h e wild s p e c i e s R a p h a n u s raphanistrum. Mainly u s e d for its root, t h i s h a s r e a c h e d a h i g h m u l t i p l i c i t y of s h a p e s , sizes a n d colors. B u t leaf forage v a r i e t i e s are also c u l t i v a t e d a n d t h e long fruits of var. caudatus are often a n object of h u m a n c o n s u m p t i o n in s o m e p a r t s of Asia. Eruca vesicaria (L.) Cav., t h e rocket, h a s four c o n s p i c u o u s s u b s p e c i e s , t h r e e r e s t r i c t e d to the W e s t M e d i t e r r a n e a n region. The fourth, s u b s p , sativa ( c o m m o n l y referred to a s E. sativa) s h o w s a c i r c u m - m e d i t e r r a n e a n a r e a w h i c h e x t e n d s (in its wild form) into I r a n i a n plains. C u l t i v a t i o n h a s f u r t h e r e x t e n d e d t h i s a r e a into the I n d i a n s u b c o n t i n e n t , C h i n a a n d N. a n d S. America. C u l t i v a t e d f o r m s are easily d i s t i n g u i s h e d b e c a u s e t h e i r p o d s are c o m p a r a t i v e l y m o r e r o b u s t a n d t h e i r s e e d s are bigger. S a i n t Isidoro of Seville (VIII c e n t u r y ) a n d Ibn a l - A w w a m (XII c e n t u r y ) m e n t i o n its c u l t i v a t i o n in Spain, b u t t h i s is t o d a y c o m p l e t e l y m a r g i n a l . O t h e r w i s e , r o c k e t is very p o p u lar in Italy a s a s o u r c e of p u n g e n t s a l a d , or in T u r k e y a n d Egypt, w h e r e n o n p u n g e n t v a r i e t i e s are a s a p p r e c i a t e d a s l e t t u c e b u t c h e a p e r . In India, '~taramira" s e e d s are a c o m m o n s o u r c e of oil for b u r n i n g a n d also for h u m a n consumption. O t h e r Eruca vesicaria s u b s p e c i e s are n o t c u l t i v a t e d b u t t h e y s h o w som e p r o m i s i n g traits. For i n s t a n c e , s u b s p , vesicaria from S p a i n is h i g h l y p u n gent a n d its odor c a n even be d e t e c t e d in the field from a few m e t e r s away. S u b s p . pinnatifida grows in b a r r e n a r e a s close to NW African d e s e r t s , showing a h i g h r e s i s t a n c e to d r o u g h t c o n d i t i o n s a n d a very s h o r t life cycle. S o m e Diplotaxis s p e c i e s are also c u l t i v a t e d , m o s t l y in Italy, for u s e s sim i l a r to t h o s e of Eruca. T h e s e are a l m o s t exclusively D. tenuifolia a n d D. muralis, t h o u g h the g e n u s Diplotaxis c o n t a i n s m a n y o t h e r species, m a i n l y w i t h i n t h e s u b g e n u s Diplotaxis t h a t c o u l d be e q u a l l y u s e d to provide p u n gency to s a l a d s a n d o t h e r d i s h e s .
52
Acknowledgement The a u t h o r s are indebted to Dr M. L a k s h m i k u m a r a n for her permit to reproduce the content of Table 2.1.
References Aguinagalde, I. 1988. Flavonoids in Brassica nigra (L.) Koch, B. oleracea L., B. campestris L. and their n a t u r a l amphidiploids. Bot. Mag. Tokyo, 101, 55-60. Alam, Z. 1945. Nomenclature of oleiferous brassicas cultivated in Punjab. Indian J. Agr. Sci. 15, 173-181. Allchin, F. R. 1969. Early cultivated plants in India and Pakistan. In: Ueko, P.J. and Dimbleby G.W. (eds.). The domestication and exploitation ofplants and animals. Duckworth, London, pp. 323-328. Arfls, P., Baladrdn, J . J . and Ord/ts, A. 1987. Species identification of cultivated b r a s s i cas with isozyme electroforesis. Cruciferae Newsl. 12, 26. Astley, D. 1982. Collecting in Ethiopia. Cruciferae Newsl. 7, 3-4. Baldini, R. M. 1998. Rediscovery of Brassica procumbens (Poir.) O.E.Schulz (Cruciferae) in Italy with some systematic and distributional observations. Webbia 53, 57-68. Boswell, V. R. 1949. Our vegetable travellers. Nat. Geogr. Magaz. 96, 134217. Burkill, I. H. 1930. The Chinese m u s t a r d s in the Malay Peninsula. Gad's
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Chen, S. R. 1982. The origin and differentiation of m u s t a r d varieties in China. Cruciferae Newsl. 7, 7-10. Cheng, B. F. and Heneen, W. K. 1995. Satellited c h r o m o s o m e nucleolus organizer regions and nucleoli of Brassica campestris L., B. nigra (L.) Koch. and Sinapis arvensis L. Hereditas 122, 113-118. Cheng, B. F., Heneen, W. K. and Pedersen, C. 1995. Ribosomal RNA gene loci and their nucleolar activity in Brassica alboglabra Bailey. Hereditas 123, 169-173. Chiang, B.Y. and Grant, W. F. 1975. A putative heterozygous interchange in the cabbage (Brassica oleracea var capitata} cultivar ~Badger Shipper'. Euphytica 24, 581-584. Chyi, Y.S., Hoenecke, M. E. and Sernyk, J. L. 1992. A genetic linkage map of restriction fragment length polymorphism loci for Brassica rapa (syn. campestris). Genome 35, 746-757. Crisp, P. 1982. The use of an evolutionary scheme for cauliflowers in the screening of genetic resources. Euphytica 3 1 , 7 2 5 - 7 3 4 .
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57 Song, K. M., Suzuki, J. Y., Slocum, M. K., Williams, P. H. and Osborn, T. C. 1991. A linkage map of Brassica rapa (syn. campestris) based on restriction fragment length polymorphism loci. Theor. Appl. Genet. 82, 296-304. Song, K. M. and Osborn, T.C. 1992. Polyphyletic origins of Brassica napus: new evidence based on organelle and nuclear RFLP analyses. Genome 35, 992-1001. Song, K. M., Tang, K. and Osborn, T. C. 1993. Development of synthetic Brassica amphidiploids by reciprocal hybridization and comparison to natural amphidiploids. Theor. Appl. Genet. 86, 811-821. Sun, V. G. 1946. The evaluation of taxonomic characters of cultivated Brassica with a key to species and varieties. I. The characters. Bull. Torrey Bot. Cl. 73, 244-281. Sun, V. G. 1970. Breeding plants of Brassica. J. Agr. Assoc. China, 71, 4152. Takahata, Y. and Hinata, K. 1980. A variation study of subtribe Brassicinae by principal component analysis. In: Tsunoda, S., Hinata, K. and G6mez-Campo, C. (eds.), Brassica crops and wild allies. Biology and breeding, J a p a n Scientific Societies Press, Tokyo, pp. 33-49. Takahata, Y. and Hinata, K. 1983. Studies on cytodemes in the subtribe Brassicineae. Tohoku, J. Agri. Res. 33, 111-124. Takahata, Y. and Hinata, K. 1986. A consideration of the species relationships in subtribe Brassicinae (Cruciferae) in view of cluster analysis of morphological characters. Pl. Sp. Biol. 1, 79-88. Toxopeus, H. 1979. The domestication of Brassica crops. Proc. Eucarpia Conference on the breeding of Cruciferous crops., 47-56. Tsunoda, S. 1980. Ecophysiology of wild and cultivated forms in Brassica and allied genera. In: Tsunoda, S., Hinata, K. and G6mez-Campo, C. (eds.), Brassica crops and wild allies. Biology and breeding, Japan Scientific Societies Press, Tokyo, pp. 109-120. Tsunoda, S. and Nishi, S. 1968. Origin, differentiation and breeding of cultivated Brassica. Proc. XII Int. Congr. Genet. 2, 77-88. Uchimiya, H. and Wildman, S. G. 1978. Evolution of fraction I protein in relation to origin of amphidiploid Brassica species and other member of Cruciferae. J. Heredity 69, 299-303. Vaughan, J. G. 1977. A multidisciplinary study of the taxonomy and origin of Brassica crops. Bio. Sci. 27, 35-40. Vaughan, J. G. and Denford, K. E. 1968. An acrylamide gel electrophoretic study of the seed proteins of Brassica and Sinapis species, with special reference to their taxonomic value. J. Exp. Bot. 19, 724732.
58 V a u g h a n , J. G. a n d Gordon, E. I. 1973. A taxonomic s t u d y of Brassica juncea u s i n g the t e c h n i q u e s of electrophoresis, gas-liquid chromatog r a p h y a n d serology. Ann. Bot., 37, 167-184. V a u g h a n , J. G., Hemingway, J. S. a n d Schofield, H. J. 1963. Contributions to a s t u d y of variations in Brassica juncea Coss et. Czern. Bot. Journal of the Linnean Society 58, 435-447. V a u g h a n , J. G., Waite, A. Boulter, D. a n d Waiters, S. 1966. Comparative s t u d i e s of the seed proteins of Brassica campestris, B. oleracea a n d B. nigra. J. Exp. Bot., 17, 332-343. Vavilov, N. I. 1949. The origin, variation, i m m u n i t y a n d breeding of cultivated plants. Chron. Bot. 13, 1-364. Verma, S. C. a n d Rees, H. 1974. Nuclear DNA a n d the evolution of allotetraploid Brassicae. Heredity 33, 61-68. Wang, X. H., Luo, P. a n d Shu, J. J. 1989. Giemsa N - b a n d i n g pattern in Cabbage a n d Chinese kale. Euphytica 41, 17-21. Warwick, S. I. a n d Black, L. D. 1991. Molecular s y s t e m a t i c s of Brassica a n d allied genera (Subtribe Brassicinae Brassicae) - chloroplast genome a n d cytodeme congruence. Theor. Appl. Genet. 82, 81-92. Warwick, S. I. a n d Black, L. D. 1993. Molecular r e l a t i o n s h i p s in subtribe Brassicinae (Cruciferae, tribe Brassiceae). Can. J. Bot. 7 1 , 9 0 6 - 9 1 8 . Warwick, S. I. a n d Black, L. D. 1994. Evaluation of the s u b t r i b e s Moricandiinae, Savignynae, Vellinae a n d Zillinae (Brassicaceae, Brassiceae) u s i n g chloroplast DNA restriction site variation. Can. J. Bot. 72, 1692-1701. Warwick, S. I. a n d Black, L. D. 1997. Phylogenetic implications of chloroplast DNA restriction site variation in s u b t r i b e s Raphaninae a n d Cakilinae (Brassicaceae, tribe Brassiceae). Can. J. Bot. 75, 960973. Warwick, S. I., Black, L. D. a n d Aguinagalde, I. 1992. Molecular systematics of Brassica a n d allied genera (subtribe Brassicinae, Brassiceae) c h l o r o p l a s t DNA variation in the g e n u s Diplotaxis. Theor. Appl. Genet. 83, 839-850. Watt, G. 1989. Brassica. In: Dictionary of Economic Products of India. I., Calcutta, pp. 520-534. Wen, L. C. 1980. Classification a n d evolution of m u s t a r d crops (Brassicajuncea) in China. Cruciferae Newsl. 5, 33-35. Yanagino, T., T a k a h a t a , Y. a n d Hinata, K. 1987. Chloroplast DNA variations a m o n g diploid species in Brassica a n d allied genera. Japan J. Genet. 62, 119-125. Zwinger, F. 1696. Theatricum Botanicum. Basel.
Biology of Brassica Coenospecies C. G6mez-Campo(Editor) 91999 Elsevier Science B.V. All rights reserved.
59
3 CYTOGENETICS S h y a m P r a k a s h (1}, Yoshihito T a k a h a t a (2), P u l u g u r t h a B. Kirti (1) a n d Virender L. C h o p r a (1) (1) National R e s e a r c h Centre on Plant Biotechnology. I n d i a n Agricultural R e s e a r c h Institute. N e w Delhi - 11 O012. India. (2) Faculty o f Agriculture. I w a t e University. Morioka 020. J a p a n
D e t e r m i n a t i o n of c h r o m o s o m e n u m b e r for B r a s s i c a rapa (syn. B. campestris) by a J a p a n e s e r e s e a r c h e r T a k a m i n e in 1916 w a s the beginning of cytogenetical r e s e a r c h in Brassica, a l t h o u g h interspecific a n d intergeneric h y b r i d s were reported m u c h earlier ( R a p h a n u s s a t i v u s x B. oleracea, Sageret, 1826; B. n a p u s x B. rapa, Herbert, 1847). Hybridizations between different species a n d s t u d y of their meiotic behavior led Morinaga (1928-1934) to u n r a v e l the genetic a r c h i t e c t u r e of crop brassicas. A r o u n d t h a t time, Manton (1932) carried o u t a n extensive g e n o - s y s t e m a t i c a l survey of Cruciferae a n d d e t e r m i n e d the c h r o m o s o m e n u m b e r s for a n u m b e r of taxa - a s t u p e n d o u s pioneering t a s k indeed. During the 1970s, wild g e r m p l a s m of B r a s s i c a a n d related genera was extensively collected a n d cytogenetical s t u d i e s were initiated. While the t h e m e of r e s e a r c h in the early p h a s e centered a r o u n d polyploid breeding, later the priorities shifted to exploitation of wild allies for introgression of n u c l e a r genes for desirable agronomic traits, cytoplasmic s u b s t i t u t i o n s a n d c o n s t r u c t i o n of c h r o m o s o m e m a p s , t a k i n g a d v a n t a g e of d e v e l o p m e n t in the a r e a s of somatic cell a n d m o l e c u l a r genetics in recent years. Use of m o l e c u l a r t e c h n i q u e s h a s considerably helped in c o n s t r u c t i n g linkage m a p s by applying restriction f r a g m e n t length p o l y m o r p h i s m (RFLP), r a n d o m amplified polymorphic DNA (RAPD) a n d DNA fingerprinting. These investigations have yielded i m p o r t a n t information on g e n o m e organization, extent of gene duplications, c h r o m o s o m e s t r u c t u r a l c h a n g e s a n d intergenomic gene introgression. Recent years have seen a s p e c t a c u l a r d e v e l o p m e n t in in vitro t e c h n i q u e s s u c h as ovary a n d embryo culture, a n d somatic hybridization. Somatic hybridization n o t only overcomes reproductive barriers b u t also generates cytoplasmic variability which is n o t possible t h r o u g h the conventional m e t h o d of sexual hybridization. T h o u g h some of these recent d e v e l o p m e n t s are more t h r o u g h l y d i s c u s s e d in other p a r t s of this book, we
60 will m a k e some occasional reference to t h e m from the cytogenetical point of view.
The Brassica
coenospecies
The Brassica coenospecies e n c o m p a s s e s those genera a n d species which are genetically related to crop b r a s s i c a s a n d are capable of exchanging genetic material with t h e m (Harberd, 1976). T a x o n o m i s t s in the last 200 y e a r s have collected a n d described m a n y species a n d have often classified the s a m e species u n d e r different n a m e s resulting in large-scale synonymy. A c o m p r e h e n s i v e t a x o n o m i c investigation was a t t e m p t e d by Schulz (1919, 1936). H a r b e r d (1976) for the first time classified this g e r m p l a s m biologically into c y t o d e m e s or c r o s s i n g g r o u p s taking into c o n s i d e r a t i o n c h r o m o s o m e n u m b e r a n d pairing, a n d extent of fertility in the hybrids. He included 91 species belonging to 9 g e n e r a of the s u b t r i b e Brassicinae of Schulz (1919) viz. Brassica, Diplotaxis, Erucastrum, Eruca, Sinapis, Coincya (syn. Hutera), Hirschfeldia, Trachystoma, a n d Sinapidendron b u t excluded the g e n u s Reboudia. Two more g e n e r a from the related subtribe Raphaninae viz. Raphanus a n d Enarthrocarpus were also included. H a r b e r d recognized 44 cytodem e s including the crop species. Six c y t o d e m e s have two species each a n d a large n u m b e r c o n t a i n only one single species. Two c y t o d e m e s include more t h a n six species each - these are Coincya (syn. Hutera a n d Rhynchosinapis), a n d B. oleracea. T a k a h a t a a n d H i n a t a (1983) f u r t h e r extended this s t u d y a n d a d d e d a few more c y t o d e m e s in recent years. S y s t e m a t i s t s have investigated the t a x o n o m i c s t a t u s a n d r e l a t i o n s h i p s of a wider g e r m p l a s m beyond the b o u n d a r i e s of the s u b t r i b e Brassicinae employing m o l e c u l a r techniques which include nuclear, m i t o c h o n d r i a l a n d chloroplast DNA restriction fragm e n t length p o l y m o r p h i s m . This r e s e a r c h h a s s u b s t a n t i a t e d the earlier proposed t a x o n o m i c s t a t u s a n d cytogenetical r e l a t i o n s h i p s of m a n y taxa and h a s also s u g g e s t e d new r e l a t i o n s h i p s between g e n e r a a n d species. These investigations (Warwick a n d Black, 1994) proposed, for instance, the close relationship of three more genera viz. Moricandia, Rytidocarpus a n d Pseuderucaria from the related s u b t r i b e Moricandiinae with crop b r a s s i c a s suggesting their inclusion in the Brassica coenospecies. T h u s , Figure 3.1 and Table 3.1 refer to the coenospecies in its b r o a d e s t sense. The lowest gametic c h r o m o s o m e n u m b e r in the group is n = 7 and is c h a r a c t e r i s t i c of seven cytodemes. H a r b e r d (1976) was of the view t h a t all c y t o d e m e s of n = 13 or less are diploids (only one, t h a t of Diplotaxis harra is n = 13), while c y t o d e m e s with n = 14 or higher c h r o m o s o m e s are polyploids. A total of 43 c y t o d e m e s are diploids where every n u m b e r from n = 7 to n = 13 is r e p r e s e n t e d . A r o u n d 50% of these c y t o d e m e s have gametic chromosome n u m b e r n = 9 a n d n = 10. Polyploidy is not u n c o m m o n a n d is characteristic of 20 additional c y t o d e m e s c o m p r i s i n g both a u t o a n d allopolyploids (Table 3.2). Level of ploidy is k n o w n to exceed tetraploidy only in Moricandia (6x, 8x; S o b r i n o - V e s p e r i n a s , 1980) a n d Brassica repanda (Galland, 1988).
61
Family:
Tribe
Subtribe:
Genus
Cruciferae
Brassiceae
9
Brassicinae
I
: Brassica (20)
Diplotaxis (13) Erucastrum (11)
Raphaninae
Moricandiinae
Raphanus (1)
Moricandia (4)
Enarthrocarpus (1)
Rytidocarpus ( 1)
I
I
Pseuderucaria (1)
Eruca (1) Hirschfeldia (1) Sinapis (5) Coincya (2) Trachystoma (1) Sinapidendron (1)
Figure 3.1 Architecture of Brassica coenospecies (number of cytodemes in brackets)
62 T a b l e 3. 1 Cytodemes in Brassica coenospecies sensu lato (originally based
In Harberd with modifications by Takahata and Hinata (1983), W a r w i c k and Black (1994) and G 6 m e z - C a m p o (pers. comm.).
n
Principal species
1
7
2
7
Brassica deflexa Boiss. Diplotaxis erucoides (L.) DC.
3
7
Erucastrum virgatum C. Presl.
4
7
Erucastrum varium Durieu
5
7
Sinapis aucheri (Boiss.) O.E. Schulz
6
7
Hirschfeldia incana (L.) Lagreze-Fossat
7
7
Pseuderucaria spp. O.E. Schulz
8
8
Brassica nigra (L.) Koch
9
8
Brassica fruticulosa Cyr. (+ maurorum + spinescens)
10
8
Diplotaxis siettiana Maire
11
8
Erucastrum abyssinicum (A. Rich.) O.E. Schulz
12
8
E. nasturtiifolium (Poiret) O.E. Schulz (+ leucanthum)
13
8
Erucastrum strigosum (Thunb.) O.E. Schulz
14
8
Trachystoma spp.
15
9
Brassica oleracea L. and 8 wild Mediterranean allied species
16
9
Brassica oxyrrhina Coss.
17
9
Diplotaxis assurgens (Del.) Gren.
18
9
Diplotaxis catholica (L.) DC.
19
9
Diplotaxis tenuisiliqua Del.
20
9
Diplotaxis virgata (Cav.) DC.
21
9
Diplotaxis berthautii Braun-Blanq. & Maire
22
9
Erucastrum cardaminoides Webb & Berth. (+ canariense+ ifniense)
23
9
Raphanus L. all species and subspecies
24
9
Sinapis arvensis L. (+ allioni)
25
9
Sinapis pubescens L.
26
10
Brassica tournefortii Gouan.
27
10
Brassica barrelieri (L.) Janka
28
10
Brassica gravinae Ten.
29
10
Brassica repanda (Willd.)DC. (+ desnottesii)
63 30
10
Brassica rapa L. (+ many cultivated subspecies)
31
10
Diplotaxis siifolia G. Kunze
32
10
33
10
Diplotaxis viminea (L.) DC Enarthrocarpus spp.
34
10
35
11
Sinapidendron spp. Brassica souliei Batt.
36
11
37
11
Diplotaxis acris (Forsk.) Boiss. Brassica elongata Ehrh.
38
11
Diplotaxis tennuifolia (L.) DC. (+ pitardiana)
39
11
40
12
Eruca spp. Mill. Coincya spp. (syn. Hutera and Rhynchosinapis)
41
12
Sinapis alba L.
42
12
43
13
44
14
Sinapis flexuosa Poir. Diplotaxis harra (Forsk.) Boiss. (+ several subsps.) Erucastrum virgatum C. Presl. (subsp. pseudosinapis)
45
14
Moricandia arvensis (L.) DC.
46
14
Moricandia moricandioides (Boiss.) Heywood
47
14
Rytidocarpus moricandioides Coss.
48
15
49
15
Erucastrum gallicum (Willd.) O.E. Schulz Erucastrum elatum (Ball.) O.E. Schulz
50
16
51
16
Brassicafruticulosa (Boiss.& Reut.) N. African subpecies (+ B. cossoniana) Brassica balearica Pers.
52
16
Erucastrum nasturtiifolium (Poiret) O.E. Schulz (4x)
53
16
Erucastrum abyssinicum (A. Rich.) O.E. Schulz (4x)
54
17
Brassica carinata A. Braun
55
18
Brassicajuncea (L.) Czem & Coss.
56
19
Brassica napus L.
57
20
58
21
Brassica gravinae Ten. (4x) Diplotaxis muralis (L.) DC.
59
22
Brassica dimorpha Coss. & Dur.
60
24
Coincya spp. (4x)
61
28
62
42
63
80?
Moricandia suffruticosa (Desf.) Coss. & Dur. Moricandia spinosa Pomel Brassica repanda (Willd.) DC. (High Atlas)
64 Table 3.2
Polyploid cytodemes in B r a s s i c a coenospecies
Alloploids
Diploid progenitors
Reference
Brassica carinata, n = l 7
B. nigra, B. oleracea
U, 1935
Brassica juncea, n = 18
B. rapa, B. nigra
U, 1935
Brassica napus, n = 19
B. oleracea, B. rapa
U, 1935
Diplotaxis muralis, n=21
D. tenuifolia, D. viminea
Harberd and McArthur, 1972
Erucastrum gallicum, n=15
E. leucanthum x sp?
Harberd,1976
Erucastrum elatum, n = 15
Hirschfeldia incana x Erucastrum sp.
G6mez-Campo, 1983
Brassica balearica, n = 16
B. oleracea group x
Snogerup and Persson,1983
another species
Tentative autopoll:ploids
Diploid homologue
Reference
Moricandia arvensis, n = 14
unknown
Harberd, 1976
Moricandia moricandioides, n =14
unknown
Harberd, 1976
Rytidocarpus moricandioides, n=14 unknown
Harberd, 1976
Erucastrum virgatum, n = 14 (subsp. pseudosinapis) Brassica cossoniana, n - 16
E. virgatum, n = 7
Harberd, 1976
B. maurorum, n--8
Pradhan et al., 1992
Erucastrum abyssinicum, n--16
E. abyssinicum, n=8
Harberd,1976
Erucastrum nasturtiifolium, n=16
E. nasturtiifolium, n=8
Harberd,1976
Brassica gravinae, n = 20
B. gravinae, n= l O
Takahata and Hinata, 1983
Brassica dimorpha, n=22
B. souliei, n=l l
G6mez-Campo, 1980
Coincya spp., n=24
Coincya, n=12
Harberd, 1976
Moricandia suffruticosa, n - 2 8
Moricandia sp., n = 14
Sobrino-Vesperinas,1980
Moricandia spinosa, n=42
Moricandia sp., n=14
Sobrino-Vesperinas,1980
Brassica repanda n - 8 0
B. repanda, n=l 0
Galland, 1988
65
Crop brassicas: cytogenetic architecture Morinaga (1928-1934) pioneered genome analysis and unravelled the cytogenetic structure of cultivated B r a s s i c a species. These investigations established that crop brassicas consist of six species. Of these, B. nigra (n = 8), B. oteracea (n = 9) and B. rapa (n = 10) are diploid monogenomic elementary species. The other three viz. B. carinata (n = 17), B. j u n c e a (n = 18) and B. n a p u s (n = 19) are high chromosome digenomic species which evolved in n a t u r e through convergent alloploid evolution following hybridization between any two of the diploid species. This proposal was subsequently verified by experimental synthesis of B. n a p u s by U (1935). The relationship amongst crop species was presented in a diagram which is commonly referred to as U's triangle (Figure 3.2). The diploid species represent an ascending aneuploid series (Manton, 1932) and are regarded as secondary balanced polyploids. The earlier view held that they evolved from a common prototype with x = 6 (Mizushima, 1950; R6bbelen, 1960). However, recent investigation on nuclear, mitochondrial and chloroplast DNA restriction fragment length polymorphism established their evolution from two prototypes: B. nigra from one prototype and B. oleracea and B. rapa from the other. The evolutionary diversion is also reflected in their cytoplasm (Palmer, 1988; Warwick and Black, 1991; P r a d h a n et al., 1992). B. oleracea and B. rapa cytoplasms are closer to each other than either is to B. nigra (Palmer, 1988). Although variations were observed in the cytoplasms of B. rapa and B. oleracea in cp and mt DNA patterns (Song and Osborn, 1992), the mitochondrial genome of B. rapa was more variable t h a n that of B. oleracea. Pachytene chromosome analysis by R6bbelen (1960) and Venkateswarlu and Kamala (1973) revealed that diploids have 6 basic types of chromosomes. The B. rapa genome is represented by AABCDDEFFF (tetrasomic for chromosomes A and D; and hexasomic for chromosome F), B. oleracea by ABBCCDEEF (tetrasomic for chromosome B C and E) and B. nigra by AABCDDEF (tetrasomic for chromosomes A and D). Chromosomes of each type have lost homology due to structural a n d / o r genic alterations during their long evolutionary history (see chapter 7 in this book). However, all the three genomes are partially homologous as revealed by cytogenetic (Mizushima, 1950; Atria and R6bbelen, 1986; P r a k a s h and Hinata, 1980), and molecular evidence (Hosaka et al., 1990; Teutonico and Osborn, 1994). It is also held that the genetic information in all the three genomes is similar, only its organization and distribution on the chromosomes is different (Truco et al., 1996). Chromosomal duplications and translocations have played a pivotal role in differentiating the chromosomes (Quiros et al., 1988; Hosaka et al., 1990; McGrath et al., 1990; Truco and Quiros, 1994). Deletions may also have contributed in repatterning the chromosomes (Hu and Quiros, 1991). Moreover, because of the secondary balanced n a t u r e of B r a s s i c a genomes, these changes were tolerated and adjusted (Kianian and Quiros, 1992a).
66 The high c h r o m o s o m e species B. carinata, B. j u n c e a a n d B. n a p u s are chromosomally balanced with a regular bivalent forming regime devoid of any homoeologous pairing. Their alloploid n a t u r e has been confirmed by m a n y reports on their artificial synthesis (see P r a k a s h and Chopra, 1991), nuclear DNA content (Verma a n d Rees, 1974), r RNA genes (Quiros et al., 1987), nuclear DNA m a r k e r s (Song et al., 1988; H osaka et al., 1990; Demeke et al., 1992) an d genomic in situ hybridization (Snowdown et at., 1997b). Research involving Fraction-1 protein (Uchimiya and Wildman, 1978) and chloroplast a n d mitochondrial DNA analysis (Palmer et al., 1983; Erickson et al., 1983; Warwick and Black, 1991; P r a d h a n et al., 1992) has revealed the directionality of n a t u r a l hybridizations. These studies established B. nigra and B. rapa as the cytoplasmic donors of B. carinata and B. j u n c e a respectively. Chloroplast DNA restriction patterns for B. n a p u s were different from both B. rapa and B. oleracea b u t were more similar to B. oleracea. (Palmer et al., 1983; Erickson et al., 1983). A detailed s t u d y by Song and Osborn (1992) based on the RFLP pattern of cp an d mt DNAs provided strong evidence for multiple origins of B. n a p u s and suggested t h a t a closely related ancestral species of B. rapa and B. oleracea identical to B. m o n t a n a was the cytoplasmic donor of most of the B. n a p u s accessions. The cp genomes have been conserved in B. carinata and B. juncea, however it is slightly altered in B. n a p u s (Palmer et al., 1983). There are also indications of coinheritance of m t a n d cp genomes in all the 3 species (Palmer, 1988). It is also observed t h a t when the parental diploid species of the amphidiploid had highly differentiated cytoplasm as in B. j u n c e a and B. carinata, the nuclear genomes of the alloploids contributed by the male parents were altered considerably compared to the nuclear genomes of the female p a r e n t s (Song et al., 1988; 1995). Extensive s t r u c t u r a l r e a r r a n g e m e n t s of c h r o m o s o m e s have occurred since the evolution of these alloploid species (Slocum et al., 1990; Song et al., 1991; Kianian a n d Quiros, 1992b; Harrison and Heslop-Harrison, 1995). However, the average c h r o m o s o m e size remained u n c h a n g e d (Parkin and Lydiate, 1997). N u c l e a r DNA
Nuclear DNA content in crop species has been estimated by several investigators (Yamaguchi and Tsunoda, 1969; Verma and Rees, 1974; Arumug u n a t h a n an d Earle, 1991, Figure 3.2). An appreciable reduction in DNA content and nuclear volume in autotetraploids of B. oleracea and natural B. n a p u s led Yamaguchi and T s u n o d a (1969) to believe that nuclear DNA had been lost with evolution. Verma and Rees (1974) on the other h a n d were of the view t h a t there is a diminution in DNA content in allotetraploid subseq u e n t to their formation and lower DNA values in tetraploids were associated
67
III
(Solid and broken lines represent female and male parents, respectively, DNA values Mbpc/IC)
Figure 3.2 Cytogenetic relationships of crop brassicas (U, 193 5)
68 to reduced nuclear volume. Higher DNA density of nuclei in natural alloploids might be due to chromosome condensation at interphase because of switching off the r e d u n d a n t gene copies in tetraploids.
S a t e l l i t e c h r o m o s o m e s and rDNA loci Among the diploid species, B. r a p a has been reported to have one pair of chromosomes showing secondary constriction and satellites (Olin-Fatih and Heneen, 1992; Cheng et al., 1995). B. oleracea also has one pair of satellited chromosomes (Wang et al., 1989; Cheng et al., 1995) while B. nigra has three pairs (Cheng and Heneen, 1995). This is not in accordance with previous observations by Lan et al. (1991) who reported two satellited chromosome pairs. In situ hybridization with r DNA probes, however, revealed five pairs of loci in B. r a p a (Maluzynska and Heslop-Harrison, 1993; Fukui et al., 1998). Cheng and Heneen (1995) were of the opinion that four pairs are inactive in nucleolus formation and since it has 2 nucleolar organizing region bearing chromosomes, it represents one pair of active rDNA loci. B. nigra has two pairs of rDNA loci (Maluzynska and Heslop-Harrison, 1993) supporting the earlier observations of R6bbelen (1960) who found 2 pairs of chromosomes associated with nucleoli at pachytene. Variations in n u m b e r of rRNA gene loci have been observed in B. oteracea. T w o major and one minor pairs of loci were observed by Maluzynska and Heslop-Harrison (1993), and McGrath et al. (1990) indentifying two synteny groups linked to strong rDNA signals and one synteny group linked to weak signals. Similar observations were found by Kianian and Quiros (1992 ab) reporting three pairs of rRNA gene loci. However, Cheng et al. (1995) found two major and two minor rRNA gene loci located on satellited chromosome pair 9 and non-satellited pair 8 respectively and concluded that only two pairs of chromosomes are involved in nucleoli formation in agreement with earlier observations of R6bbelen (1960) and Kamala (1976) who found two bivalents associated with nucleoli at meiosis in B. oleracea and also of Delseny et al. (1990) who reported two pairs of rRNA gene loci. The alloploids have fewer rDNA loci than the s u m of their parents. B. j u n c e a has five major pairs and one minor pair, one less than those of the parental sum. B. n a p u s possess six major rDNA loci while B. carinata has four loci. Maluzynska and Heslop-Harrison (1993) and Snowdon et al. (1997
a) believed that the n u m b e r of loci has been reduced during evolution of these alloploid species and might be ascribed to chromosome rearrangments leading to loss of rDNA site.
Genome
manipulation
Genome manipulation is one of the ways to achieve breeding goals. It supplements nature's role in crop evolution by designed synthesis of the alloploid species through selectively choosing the donor progenitors. Such
69 experimental synthesis of a B r a s s i c a alloploid species dates back to 1935 when U d e m o n s t r a t e d the successful artificial synthesis of B. n a p u s . Subsequent literature during the last 60 years a b o u n d s in reports on synthesis not only on the naturally occurring alloploid species B. n a p u s , B. j u n c e a a n d B. c a r i n a t a b u t also on new alloploids combining entirely new genomes using a variety of methods. The early syntheses were entirely academic in n a t u r e aiming at verifying the proposals of Morinaga (1934). However, in the 1960s the priorities shifted to comprehensive programs of designed e n h a n c e m e n t of genetic variability of breeding value. Extensive variability in highly polymorphic B. r a p a and B. o l e r a c e a h a s been exploited in the synthesis. The objectives in these syntheses varied and included: development of productive B. j u n c e a forms (Olsson, 1960a; Prakash, 1973a); early a n d productive B. c a r i n a t a (Prakash et al., 1984); high seed yielding B. n a p u s (Olsson, 1960b); early developing B. n a p u s (Prakash, 1980; P r a k a s h a n d Raut, 1983; Akbar, 1987); synthesizing fodder forms of B. n a p u s (Namai a n d Hosoda, 1967, 1968; Ellerstrom and Sjodin, 1973); synthesis of root forming r u t a b a g a s or swedes (Olsson et al., 1955; Namai a n d Hosoda, 1968; Kato et al., 1968); a new head forming type (Shinohara and Kanno, 1961) a n d developing yellow seeded B. n a p u s (Chen et al., 1988). Synthesis Synthetic alloploids have been obtained t h r o u g h conventional sexual hybridizations from reciprocal cross (Table 3.3). It h a s been generally accepted that B. j u n c e a a n d B. c a r i n a t a are easier to synthesize t h a n B. n a p u s . In general, the cross B. o l e r a c e a • B. r a p a is very difficult to obtain followed by B. nigra x B. r a p a due to the operation of strong post-fertilization barriers. Failure of normal e n d o s p e r m development is the major c a u s e of interspecific hybrid embryo abortion (Inomata, 1975; Wojciechowski, 1985). Various embryo rescue techniques viz. ovary, ovule and embryo culture have been developed in recent years leading to the obtention of a large n u m b e r of hybrids (see Inomata, 1993; Shivanna, 1996). Cytoplasm type plays a crucial role in the success of producing synthetic alloploids. Synthetic B. j u n c e a with B. nigra cytoplasm a n d B. n a p u s with B. o l e r a c e a cytoplasm are very difficult to obtain. It is reported t h a t crosses with the same cytoplasm donor as the natural alloploids yield more hybrids t h a n the others (Song et al., 1993). A few reports indicated e n h a n c e d crossability when hybridization was carried out between tetraploids (Olsson, 1960b). In recent years alloploid species have been synthesized following protoplast fusion which not only helped in nuclear g e r m p l a s m e n h a n c e m e n t b u t also generated novel cytoplasmic organelle combinations. The first a t t e m p t was by S c h e n c k a n d R6bbelen (1982) on B. n a p u s . Somatic hybrid plants were intermediate between the p a r e n t s in general morphology a n d growth pattern and comparable to n a t u r a l accessions. However, some floral abnormalities were observed. This subject is throughly treated in c h a p t e r 4 of this
70
Table 3.3 Major investigations on artificial synthesis of natural alloploid species B. c a r i n a t a , B. j u n c e a and B. n a p u s through sexual hybridization Species B. carinata B. nigra x B. oleracea
and reciprocals
Frandsen, 1974; Mizushima, 1950b; Pearson, 1972; Prakash et al., 1984; Song et al., 1993
B. juncea B.rapa x B. nigra
and reciprocals
Frandsen, 1943; Ramanujam and Srinivasachar, 1943; Olsson, 1960a; Prakash, 1973a,b; Campbell et al., 1991; Song et al., 1993
B. napus B. rapa x B. oleracea
and reciprocals Oil rape
U, 1935; Karpechenko and Bogdanova, 1937; Frandsen, 1947; Rudorf, 1950; Hoffmann and Peters, 1958; Olsson, 1960b; Gland, 1982; Prakash and Raut, 1983; Akbar, 1987; Chen et al., 1988; Mithen and Magrath, 1992; Song et al., 1993
Forage rape
Hosoda, 1950, 1953, 1961; Hosoda et al., 1969; Sarashima 1967, 1973; Nishi, et al., 1970
Rutabaga
Olsson et al., 1955; Olsson, 1960b, Hosoda et al., 1963, 1969; Namai and Hosoda, 1967, 1968; Kato et al., 1968
Heading form
Shinohara and Kanno, 1961 ; Takada, 1986
71 book, so t h a t we will restrict ourselves to c o m m e n t some cytogenetical aspects.
Meiosis and fertility Meiosis is generally disturbed in sexually obtained synthetic taxa in early generations due to the occurrence of multivalents a n d univalents (Table 3.4). With advancing generations these abnormalities decrease and complete meiotic stabilization is achieved by AT-As generations. Multivalents (upto 4 quadrivalents) were frequently observed in B. c a r i n a t a (Mizushima, 1950b) and B. n a p u s (upto 2 IV + 1 III; S a r a s h i m a , 1973; P r a k a s h and Raut, 1983). Interestingly, higher associates were a b s e n t or rare in B. j u n c e a (Olsson, 1960a; P r a kas h, 1973a). Occurrence of univalents was of c o m m o n observance in all the synthetics in early generations. Synthetics invariably show reduced pollen and seed fertility in early generations (Table 3.4). Selections resulted in a considerable improvement accompanied by stabilization of c h r o m o s o m e pairing. Synthetic plants generally achieve complete fertility by As-A6 generations. C h r o m o s o m e analysis of somatic hybrid pl ant s revealed t h a t besides forms with n o r m a l c h r o m o s o m e s , hypo a n d hyperdiploids were also obtained in B. n a p u s (Schenck and R6bbelen, 1982; Terada et al., 1987; S u n d b e r g et al., 1987). High c h r o m o s o m e plants probably resulted from multiple fusions. Meiosis was by and large regular with formation of 17 bivalents in A1 generation plants of B. c a r i n a t a (Narasimhulu et al., 1992) and 19 bivalents in B. n a p u s (Schenck a n d R6bbelen, 1982; Rosen, et al., 1988). However, multivalents were also noticed - up to 3 quadrivalents in B. c a r i n a t a (Narasimhulu et al., 1992) a n d in B. n a p u s (Schenck and R6bbelen, 1982). Somatic hybrids had varied fertility. Pollen and seed fertility in B. carin a t a obtained by N a r a s i m h u l u et al. (1992), ranged from 36-87% and 18% respectively while those obtained by J o u r d a n a n d Salazar (1993) had 4-98% fertile pollen. However, these a u t h o r s reported t h a t pollen was ineffective in producing seed on selfing and on crossing to n a t u r a l B. c a r i n a t a seed set was very poor. In B. n a p u s , Schenck and R6bbelen (1982) reported low fertility, while Rosen et al. (1988) observed very low or no seed set. But pollination with n a t u r a l B. n a p u s gave a high seed set. Although S u n d b e r g et at. (1987) observed 38-70% pollen fertility, seed set was very poor. Similar resuits were also obtained by Heath and Earle (1996, 1997). In spite of regular meiosis, the pl ant s were highly sterile, the r e a s o n s for which are unknown. Analysis of cytoplasmic organelles indicated t h a t besides chloroplasts and mitochondrial combinations similar to n a t u r a l forms, other situations s u c h as recombined mitochondria and novel c o m b i n a t i o n s of chloroplasts and mitochondria were obtained. For example B. c a r i n a t a with B. o l e r a c e a cp and B. nigra m t (Narasimhulu et al., 1992; J o u r d a n and Salazar, 1993) and
Table 3.4 Chromosome pairing and seed fertility in synthetic Brussicu alloploids in different alloploid generations. Alloploid
B. curinata
Chromosome pairing
Seed fertility
Al
A2
17II-I6II+2I
-
17II-4IV+9II
171I-4IV+91I Mostly 1711 Poor
A3
Al
A2
Very poor Very low 2 1.3
A3
A4
A5
A6
27-53
References Frandsen, 1947
47-62
87
92
Mizushima and Katsuo, 1953 Prakash (unpub.)
B. juncea
B. napus
18I1-1611+41
1811 mostly
1811
6.2
26.6
3 1-64
43-92
Fully fertile
Fully fertile
Olsson, 1960a
18II-I4II+8I
1611+41-1811
1811 mostly
26.1
45.8
64
96
Fully fertile
Fully fertile
Prakash, 1973a
1911 occasional univalents
1911
1911
7.9
Highly fertile Highly fertile Highly fertile Highly fertile Normal fertile Olsson, 1960b
21V+2111+711
21V+1511-1911 1911 mostly
8.8
24
36
59
31V+9II+8I
1911 mostly
7-29
19-52
58-92
Highly fertile Highly fertile Highly fertile
1911 mostly
79
Sarashima, 1973 Prakash and
73 B. n a p u s with B. oleracea cp and B. rapa mt were reported (Sundberg, 1987; Rosen et al., 1988).
Agronomic potential of synthetic alloploids A survey of synthetic alloploids reveals t hat research with B. j u n c e a is concentrated in India whereas with B. carinata and B. n a p u s research takes place in several countries, primarily Europe and C a n a d a and also India. Synthetics in general are inferior to n a t u r a l cultivars in productivity. Since they r ep res ent new genetic variability, these have been u s e d to advantage as genetic stocks in breeding p r o g r a m m e s and as bridging material for introgressing disease resistance. As a result, several cultivars have been developed. Synthetic B. j u n c e a forms originating from leafy B. rapa have vigorous vegetative growth with quick growing large sized leaves which m ake t hem suitable for fodder. In contrast, plants involving oily forms of B. rapa have their leaves of small size b u t are productive and rich in oil content (Prakash, 1973a). Oil rape B. n a p u s h a s attracted considerable attention in Europe and of late in India while fodder rape and swedes have been exploited in Europe and J a p a n . Several promising oil rape forms were released in Sweden (Olsson, 1986). These include Svalof Panter, m a r k e t e d for its higher oil yield and comparatively rapid growth at low t em per atures; Svalof Norde which possess high seed a n d oil yield and considerable resistance to Peronospora and Verticillium; Brink and J u p i t e r for their high seed yields and very low erucic acid content. Early and productive selections of oil seed B. n a p u s have been found to be very promising in India (Prakash and Raut, 1983) a n d Bangla Desh (Zaman, 1989). Synthetic B. n a p u s derived from clubroot r e s i s t a n t B. rapa a n d B. oleracea p o s s e s s e d very effective resistance to clubroot (Diederichsen a n d Sacristan, 1996). Synthetic forms developed from B. rapa x wild B. oleracea were resistant to blackleg disease caus ed by L e p t o s p h a e r i a m a c u l a n s . Resistance was ascribed to a high level of alkenyl glucosinolates in the leaves (Mithen and Magrath, 1992). Heath a n d Earle (1996) recovered somatic hybrid B. n a p u s plants which were dwarf with n o n - s h a t t e r i n g pods and bold seeds. They also possessed a large a m o u n t of erucic acid. Fodder rape forms have been bred from synthetics originated from leafy and root forms of B. rapa viz. subsp, chinensis, narinosa, nipposinica, p e k i n e n s i s an d rapa. Hosoda (1953) bred a fodder rape-CO which was cultivated in J a p a n for its vigorous growth and winter hardiness. A synthetic head forming type (which does not exist in nature) developed from the cross B. oleracea var. capitata • B. rapa subsp, p e k i n e n s i s was m a r k e t e d in J a p a n
74 u n d e r the n a m e H a k u r a n in 1968. It h a s soft leaves with less fibres, tastes like h e a d lettuce a n d p o s s e s s e s a high degree of r e s i s t a n c e to soft rot (Takada, 1986).
C y t o g e n e t i c s of wild allies: wide hybridizations. Genetic e n r i c h m e n t of crop species from their wild relatives is currently one of the major a p p r o a c h e s in crop i m p r o v e m e n t p r o g r a m s . Extensive genetic diversity occurs in the wild a n d weedy g e r m p l a s m of B r a s s i c a allies for n u c l e a r a n d cytoplasmic genes. This g e r m p l a s m occurs in n a t u r e in a vast stretch from the w e s t e r n M e d i t e r r a n e a n area to the e a s t e r n end of the Sahara desert in the n o r t h - w e s t of India. It occupies very diverse ecological habits s u c h as coastal d u n e s , slopes of coastal volcanos, stony p a s t u r e s and arid to semi-arid regions (Tsunoda, 1980). Wild allies p o s s e s s a n u m b e r of agronomically useful traits which include n u c l e a r gene encoded resistance to diseases a n d insects; i n t e r m e d i a t e C3-C4 p h o t o s y n t h e t i c activity, tolerance to cold, salt a n d heat; a n d m i t o c h o n d r i a l genes for i n d u c i n g cytoplasmic male sterility. B r a s s i c a crops h o s t an array of p a t h o g e n s a n d p e s t s a n d since wild taxa are potentially capable of exchanging genetic material with them, cytogeneticists a n d b r e e d e r s are t u r n i n g more a n d more t o w a r d s this variability for incorporating desirable genes into existing crops. As we have already mentioned, M a n t o n (1932) d e t e r m i n e d the c h r o m o s o m e n u m b e r for m a n y of the species which were s u p p l e m e n t e d s u b s e q u e n t l y (see G 6 m e z - C a m p o and Hinata, 1980). R e s e a r c h with wild g e r m p l a s m was initiated by Mizushima in the 1950s (Mizushima, 1950a, 1968) a n d consisted of hybridizations, studying c h r o m o s o m e pairing in hybrids a n d interpreting g e n o m e homologies. A major step was the collection expeditions a r o u n d the Mediterranean particularly in Spain, Portugal, Morocco a n d Algeria by S p a n i s h and J a p a n e s e scientists from 1968 o n w a r d s . Harberd (1972, 1976) carried o u t an extensive cytotaxonomic survey a n d grouped this g e r m p l a s m into cytodemes (crossing groups). This s t u d y w a s further extended by T a k a h a t a a n d Hinata (1983), a n d Warwick a n d Black ( 1991, 1993, 1994) as already d i s c u s s e d .
Hybridization between wild a n d crop b r a s s i c a s is a pre-requisite for their exploitation in a n y breeding program. As the wild g e r m p l a s m belong to the s e c o n d a r y a n d tertiary gene pool, several kinds of hybridization barriers operate. The hybridization process is a complex p h e n o m e n o n culminating into seed formation. A close coordination between pollen a n d pistil during pollen-pistil interaction, a n d developing embryo a n d e n d o s p e r m after fertilization is essential. A b r e a k at a n y level results in incompatibility. Barriers operate either at the time of fertilization or after fertilization depending u p o n the extent of reproductive isolation - the wider the distance, earlier is the stage of operation of barriers. Pre-fertilization barriers include pollen germination, pollen t u b e entry in the stigma, a n d growth of the pollen tube t h r o u g h the style. Many crosses show unilateral incompatibility i.e. pollina-
75 tion is effective only in one direction while the reciprocal c r o s s e s show strong pre-fertilization barriers (see S h i v a n n a , 1996). In general, it is observed t h a t in the majority of crosses, s u c c e s s is greater w h e n wild species are female parents. Post-fertilization barriers c a u s e embryo abortion leading to formation of shrivelled or r u d i m e n t a r y seeds, w i t h o u t embryo. Several forms of m a n i p u l a t i o n s have been carried o u t to obtain sexual h y b r i d s s u c h as grafting, mixed pollinations, b u d pollinations a n d s t u m p pollinations. In recent years in vitro fertilization h a s effectively been u s e d to raise several intergeneric h y b r i d s (Zenkteller, 1990). E m b r y o rescue h a s been the m o s t effective technique, pioneered by J a p a n e s e scientists, to overcome post-fertilization barriers a n d is very widely u s e d to raise hybrids. This p h e n o m e n o n covers all the t e c h n i q u e s which are u s e d to p r o m o t e the growth of hybrid e m b r y o a n d includes ovary culture, ovule culture a n d sequential culture (see S h i v a n n a , 1996). As a result, a large n u m b e r of h y b r i d s have been obtained a n d extensively studied for their meiotic c h r o m o s o m e behavior (Table 3.5). One of the characteristic features of meiosis of these wide h y b r i d s is the occurrence of a low to very low level of c h r o m o s o m e pairing. Bivalents wherever they occur, are in general, rod s h a p e d m o n o c h i a s m a t e s . Multivalents in h y b r i d s between diploid species are either a b s e n t or occur rarely. However, a high n u m b e r of bivalents a n d frequent t r i v a l e n t / q u a d r i v a l e n t are formed in triploid (tetraploid x diploid) or tetraploid (tetraploid x tetraploid) hybrids. Simply b a s e d on o c c u r r e n c e of bivalents in diploid hybrids, it is difficult to arrive at the extent of auto a n d allosyndesis. We have very limited information on a u t o s y n d e s i s b a s e d on pairing in the h a p l o i d s which m a y help in inferring homologies between the different genomes. In previous years, M i z u s h i m a (1950a, 1968, 1980) considered c h r o m o some pairing in the h y b r i d s as an index of genome homoeology a n d p r o p o s e d a partially h o m o l o g o u s relationship between g e n o m e s belonging to Brassica, Sinapis, Diplotaxis, Eruca, Hirschfeldia (syn. B. adpressa) a n d Raphanus. This relationship c a n now be e x t e n d e d to other genera of coenospecies as Diplotaxis, Erucastrum, Enarthrocarpus, Sinapidendron, etc. Many of the wild species are genetically very d i s t a n t a n d sexually incompatible with the crop species, t h u s m a k i n g the genes of wild t a x a inaccessible. Somatic hybridization overcomes the incompatibility barriers in s u c h situations. Cell fusion also allows the generation of extensive cytoplasmic heterogeneity t h r o u g h organelle reorganization a n d r e c o m b i n a t i o n . Many somatic h y b r i d s have been o b t a i n e d c o m b i n i n g wild a n d crop species r e p r e s e n t i n g interspecific intergeneric a n d intertribe c o m b i n a t i o n s (Tables 4.3 a n d 4.4 in the next chapter). T h o u g h initial s y n t h e s e s were for d e m o n s tration, s u b s e q u e n t l y the objectives shifted t o w a r d s their practical utilization to introduce n u c l e a r a n d cytoplasmic genes. The desirable traits for introgression include the Ca-C4 p h o t o s y n t h e t i c s y s t e m (Moricandia arvensis), high nervonic acid c o n t e n t (Thtaspi perfoliatum), club root r e s i s t a n c e (Raphanus
Table 3.5 Wide hybrids in Brussica coenospecies and their meiotic behaviour Hybrids Diplotaris erucoides (n=7) x Hirschfeldia incana (n=7) x Brassica nigra (n=8) x Sinapis pubescens (n=9) x Brassica oleracea (n=9) x Brassica rapa (n= 10) x Brassica juncea (n= 18) x
Brassica napus (n=19)
Erucastrum varium (n=7) x Brassica nigra (n=8) Erucastrum virgatum (n=7) x Sinapis pubescens (n=9) Hirschfeldia incana (n=7) x Brassica nigra (n=8) x Brassica napus (n= 19) Brassicafruticulosa (n=8) x Brassica nigra (n=8) x
x x x
Erucastrum littoreurn (n=8) ssp. glabrum Erucastrum cardaminoides (n=8) Brassica barrelieri (n=10) Brassica rapa (n=l 0 )
Brassica maurorum (n=8) x Erucastrum varium (n=7)
2n
Chromosome pairing at M1 of meiosis
Reference
14 15 16 25 17 25 25 45
141-611 + 21 151-711 + 11 1 6 1 4 1 1 + 81 7II+ 111 171-1111 + 311 + 81 111 + 231-1IV + 611 + 91 311 + 191-511 + 151 1111+ 1911 + 41 1IV + 1111 + 5-1311 + 6 2 3 1
Quiros etal., 1988 Quiros etal., 1988 Harberd and McArthur, 1980 Vyas et al., 1995 Vyas etal., 1995 Vyas etal., 1995 Inomata, N., 1998 Vyas etal., 1995 Delourme et al., 1989
15
151-511
16
1 6 1 4 1 1 + 81
+ 51
Takahata and Hinata, 1983 Harberd and McArthur, 1980 Mattsson, 1988 Kerlan et al.. 1993
15 26 161-711 + 21 1 6 1 4 1 1 + 81 711 + 21-811
Mizushima, 1968 Truco and Quiros, 1991 Takahata and Hinata, 1983
17 18 18
171-311 + 111 8 1 4 1 1 + 101 81-711 + 41 81-511 + 81
Takahata and Hinata, 1983 Harberd and McArthur, 1980 Mizushima, 1968 Takahata and Hinata, 1983 Nanda-Kumar et al., 1988
15
51-411 + 71
16 16
Takahata and Hinata, 1983
4
m
+ 21
x
Brassica nigra (n=S)
16
161-711
x
Sinapis arvensis (n=9) Brassica barrelieri (n=10) Brassica rapa (n=lO) Raphanus sativus (n=9)
17 18 18 17
171-311 + 111 181-211 + 141 181-511 + 81 17&6II+SI
15 15 16
111 + 1 3 1 4 1 1 + 71 15I--IIII+5II + 21 161-211+ 121
16 16 17 17
11 + 151-811
16 18
-
16 18
-
16 16
161- 411 + 81 611 + 41-161
18 21 21
181-511 + 81 211 + 171-711 + 71 111 + 191-511 + 111
x x x
Brassica nigra (n=8) x Erucastrurn virguturn (n=7) x Hirschfeldia incana (n=7) x Brassicafruticulosa (n=8)
x
x x
x
Brassica rnaurorum (n=8) Brassica spinescens (n=8) Raphanus sativus (n=9) Sinapis arvensis (n=9)
Brassica spinescens (n=8) x Brassica nigra (n=8) x Brassica rupa (n= 10) Diplotaxis siettiana (n=8) x Brassica nigra (n=8) x Brassica rapa (n= 10) Brassica oleracea (n=9) x Diplotaxis erucoides (n=7) x Hirschfeldia incana (n=7) x Raphnanus sativus (n=9) x Sinapis arvensis (n=9) x Coincya monensis (n=12) x Sinapis alba (n=12) x Moricandia arvensis (n=14)
+ 11
167-411 + 81 1 6 1 4 1 1 + 41
-
Takahata and Hinata, 1983 Truco and Quiros, 1991 Truco and Quiros, 1991 Takahata and Hinata, 1983 Takahata and Hinata, 1983 Bang et al., 1997 Harberd and McArthur, 1980 Quiros etal., 1988 Harberd and McArthur, 1980 Prakash et aL, 1982; Mattson, 1988 Prakash etal., 1982 Prakash etal., 1982 Matsuzawa and Sarashima, 1986 Mizushima, 1950a Harberd and McArthur, 1980 Bing el al., 1991, Mattson, 1988 Truco and Quiros, 1991 Takahata and Hinata, 1983 Nanda-Kumar and Shivanna, 1993 Mizushima, 1980 Quiros et aL, 1988 Sarashima et aL, 1980 Mizushima, 1950a Harberd and McArthur, 1980 Harberd and McArthur, 1980 Ape1 et al., 1984
J.
4
x Diplotaxis muralis (n=2 1) Brassica oxyrrhina (n=9) x Brassica nigra (n=8) x Brassica oleracea (n=9) x Sinapis pubescens (n=9) x Raphanus sativus (n=9) x
x x
Brassica barrelieri (n= 10 ) Brassica rapa (n=10) Brassica tournefortii (n=lO)
Diplotaxis catholica (n=9) x Brassica rapa (n=10) x Brassica juncea (n=l 8) Diplotaxis virgata (n=9) x Brassica rapa (n= 10) x Brassica juncea (n=l 8) Erucastrum canariense (n=9) x Brassica oleracea (n=9) Erucastrum cardaminioides (n=9) x Brassica oleracea (n=9) Raphanus sativus (n=9) x Brassica nigra (n=8) x Brassica oleracea (n=9) x Brassica rapa (n= 10) Sinapis arvensis (n=9) x Brassica nigra (n=8) x Raphanus sativus (n=9) x Brassica napus (n= 19) Brassica barrelieri (n=lO) x Brassica fruticulosa (n=8) x Brassica nigra (n=8) x Brassica oxyrrhina (n=9)
30
111 + 281-611 + 181
Harberd and McArthur, 1980
17 18 18 18
-
Prakash et al., 1982 Harberd and McArthur, 1980 Harberd and McArthur, 1980 Kaneda and Kato, 1997 Bang et al., 1997 Mattsson, 1988 Prakash and Chopra, 1990 Mattsson. 1988
211 + 141-611 + 61 111+ 161-411 + 101 181- 311 + 121 181- 111 + 611 + 61 -
19
191411 + 111
19 27
211 + 151-1 111 + 311 + 101 131+ 711-2111 + 711 + 71
Mohanty, 1996 Mohanty, 1996
19 27
191411 + 71 111+ 251-811 + 111
Takahata and Hinata, 1983 Harberd and McArthur, 1980
18
1I1 + 161-811
+ 21
Harberd and McArthur, 1980
-
18
1111+111-91-11V+1111+111+91
Mohanty, 1996
-
-
18 19
181-1111 + 611 + 31 1I1 + 171-711 + 51
Matsuzava and Sarashima, 1986 McNaughton, 1973 Mizushima, 1950a
-
Bing et al., 1991 Mizushima, 1950a Mathias, 1991
181411 + 101 181-411 + 101
Takahata and Hinata, 1983 Takahata and Hinata, 1983
181-311 18 18
+ 121
Sinapis pubescens (n=9) Brassica rapa (n=10) Brassica rapa (n= 10) X Hirschfeldia incana (n=7) X Brassicafiuticulosa (n=8) x
x
X
X X
X X X
X X X
X
Brassica spinescens (n=8) Erucastrum leucanthum (n=8) Brassica atlantica (n=9) Brassica bourgeaui (n=9) Brassica cretica (n=9) Raphanus sativus (n=9) Sinapis arvensis (n=9) Brassica barrelieri (n= 10) Brassica tournefortii (n=l 0 ) Eruca sativa (n=l 1)
X Moricandia arvensis (n= 14) Brassica tournefortii (n=lO) x Brassica fruticulosa (n=8) x Brassica nigra (n=8) x Brassica oleracea (n=9)
19 20
191-4511+ 71 201-4511 + 81
Harberd and McArthur, 1980 Takahata and Hinata. 1983
17 18
171-511 181-711
+ 71
18 18 19 19 19 19
-
19
191-511
Mizushima, 1968 Mizushima, 1968 Harberd and McArthur, 1980; Nanda-Kumar et al., 1988; Prakash et al., 1982 Prakash et al., 1982 Harberd and McArthur, 1980 Mithen and Herron, 1991 Inomata, 1986 Inomata, 1986 Mizushima, 1950a Harberd and McArthur, 1980 Mizushima, 1950a Mattsson, 1988 Prakash and Narain, 1971 Mizushima, 1950a Harberd and McArthur, 1980 Takahata and Takeda, 1990
+ 41
111+ 161-511
+ 81
191- 0-511 + 9
-
+ 91
20 21
2 0 1 4 1 1 + 121 211-811 + 51
24
241-511
18 18 19
-
181-311 191-311
+ 141 + 121
+ 131
x x
Brassica oxyrrhina (n=9) Raphanus sativus (n=9)
19 19
-
191-711
+ 51
x
Sinapis arvensis (n=9)
19
191-511
+ 91
20
201-511
+ 101
x Brassica rapa (n=l 0 ) Diplotaxis siifolia (n=lO)
Prakash et al., 1982 Narain and Prakash, 972 Mizushima, 1968 Narain and Prakash, 972 Mattsson, 1988 Mizushima, 1968 Harberd and McArthur, 1980 Mizushima, 1968 Harberd and McArthur, 1980 Sikka, 1940, Mizushima, 1968 I . \o
Brassica rapa (n= 10) Brassica juncea (n=l8) x Brassica napus (n= 19) Diplotaxis viminea (n=lO) x Diplotaris tenufolia (n=l 1) x Brassica carinata (n= 17) x Brassica napus (n=19) Enarthrocarpus lyratus (n=10) x Brassica oleracea (n=9) x Brassica rapa (n=lO) x Raphanus sativus (n=9) x Erucastrum abysinicum (n=16) x Brassica carinata (n=17) x Brassica napus (n=19) Sinapidendronfi-utescens (n= 10) x Brassicafruticulosa (n=8) x Sinapis pubescens (n-8) x Brassica juncea (n= 18) Diplotaxis tennuifolia (n=l 1) x Erucastrum virgatum (n=7) x Hirschfeldia incana (n=7) x Brassica nigra (n=8) x Brassica oleracea (n=9) x Brassica rapa (n=lO) x Brassica elongata (n=l 1) x Coincya leptocarpa (n= 12) x Brassica juncea (n=l8) Eruca sativa (n=l 1) x Brassica oleracea (n=9) x x
20 28 29
-
Batra et al., 1990 Batra et al., 1990 Batra et al., 1990
21 27 29
211-411 + 131 3111 + 411 + 101 21V + 1111 + 211 + 141
Harberd and McArthur, 1980 Mohanty, 1996 Mohanty, 1996
19 20 25 26 37 29
191-1111 +4II + 81 201-2111 + 411 + 61 211 + 2 11-911 + 71 611 + 141-1011 + 61 1111 + 1311 + 81 291-1 IV + 1111 + 611 + 101
Gundimeda et al., 1992 Gundimeda et al., 1992 Bang et al., 1997 Harberd and McArthur, 1980 Gundimeda et al., 1992 Gundimeda et al., 1992
18 19 28
311 + 121-911 111 + 171-611 + 71 211 + 2 4 1 4 1 1 + 121
Harberd and McArthur, 1980 Harberd and McArthur, 1980 Harberd and McArthur, 1980
18 18 18 20 21 22 23 29
181-711 + 41 1 8 1 4 1 1 + 61
Harberd and McArthur, Harberd and McArthur, Salisbury, 1989 Harberd and McArthur, Salisbury, 1989 Harberd and McArthur, Harberd and McArthur, Salisbury, 1989
20
x
Raphanus sativus (n=9)
20
x
Brassica rapa (n= 10)
21
281
-
201-211
+ 161
-
221-411 231-511
+ 141 + 131
-
201-311
+ 141
00
0
1980 1980 1980 1980 1980
Matsuzawa and Sarashima, 1986 U etal., 1937 Dayal, 1987 Matsuzawa and Sarashima, 1986 Agnihotri et al., 1988
x Diplotaxis tennuifolia (n=l 1) Sinapis alba (n=l2) x Brassica oleracea (n=9) x Brassica napus (n=l9) Eurcastrum laevigatum (n=14) x Hirschfeldia incana (n=7) Moricandia arvensis (n= 14) x Brassica nigra (n=8) x Brassica oleracea (n=9) x Raphanus sativus (n=9)
Brassica rapa (n=10) x Brassica juncea (n=l 8) x Brassica napus ( ~ 1 9 ) Moricandia moricandioides (n=l4) x Brassica juncea (n=l8) Erucastrum gallicum (n=l5) x Diplotaxis erucoides (n=7) x Erucastrum virgatum (n=7) x Hirschfeldia incana (n=7) x E. nasturtiifolium (n=8) x
Raphanus sativus (n=9) Sinapis arvensis (n=9) x Sinapis pubescens (n=9) x Brassica barrelieri (n=10) x Enarthrocarpus lyratus (n=10) x Sinapidendronfrutescens (n=lO) x Brassica juncea (n=18) x Brassica napus (n=l9) Brassica balearica (n=16) x Brassica oleracea (n=9) var. alboglabra x
x
22 21
221-511
+ 121
211
U etal., 1937 Ripley and Arnison, 1990
-
21
311 + 151-911
22 23 23 23 24 32 33
221--1III+ 5II+91 231-2111 + 611 + 51 1111 + 0-511 + 13-231
+ 31
-
241-511
+ 141
32 22 22 22 23 24 24 24 25 25 25 33 38 25
Takahata and Hinata, 1983
Harberd and McArthur, 1980 Takahata and Takeda, 1990 Takahata, 1990 Bang etal., 1995 Takahata et al., 1993 Takahata and Takeda, 1990 Takahata et al., 1993 Takahata et al.. 1993 Takahata et al., 1993
211 + 181-811 + 61 111 + 201-711 + 81 111 + 201-711 + 81 511 + 131-1011 + 31 711 + 91-811 + 71 311 + 181-811 + 81 211 + 201-911 + 61 411 + 161-911 + 61 311 + 191-811 + 91 311 + 171- 811 + 91 311 + 171-911 + 71 3111 + 1I1 + 221 3111 + 611 + 171
Harberd and McArthur, 1980 Harberd and McArthur, 1980 Harberd and McArthur, 1980 Harberd and McArthur, 1980 Takahata and Hinata, 1983 Harberd and McArthur, 1980 Harberd and McArthur, 1980 Harberd and McArthur, 1980 Harberd and McArthur, 1980 Harberd and McArthur, 1980 Harberd and McArthur, 1980 Batra etal., 1989 Batra etal., 1989 Snogeroup and Persoon, 1983
2
Brassica oleracea (n=9) var. insularis Brassica cossoniana (n=l6) x Brassica napus (n=19) Erucastrum abyssinicum (n=16) x Erucastrum virgatum (n=7) x E. leucanthum (n=8) x E. nasturtiifolium (n=8) x Brassica oleracea (n=9)
25
911 + 71
35
1311 + 91-1611
23 24 24 25
711 + 91-911 + 511 511 + 141-811 + 81 511 + 141-1011 + 41 111 + 231-911 + 71
Brassica rapa (n=l 0 ) Brassica carinata (n= 17) Brassica juncea (n= 1 8) Brassica carinata (n=l7) x Brassicafruticulosa (n=8) x Diplotaxis assurgens (n=9) x Diplotaxis tenuisiliqua (n=9) x Diplotaxis virgata (n=9) x Raphanus sativus (n=9) x Sinapis arvensis (n=9)
26 33 34
-
25 26 26 26 26 26
311 + I ~ I - - ~ I I171 + 311 + 201-1011 + 61 1I1 + 241-1 011 + 61 411 + 181-1 1I1 + 41 211 + 221-911 + 81 261 + 811 + 101
26
3-711
x
x x x
x
Sinapis pubescens (n=9)
Orychophragmus violaceus (n=12) 34 Erucastrum gallicum (n=15) 32 Brassica juncea (n=18) x Diplotaxis virgata (n=9) 27
Snogeroup and Persoon, 1983
711 +I21-1011+
+ 31
61
Harberd and McArthur, Harberd and McArthur, Harberd and McArthur, Harberd and McArthur, Rao et al., 1994 Harberd and McArthur, Rao et al., 1994 Rao et al., 1994
1980 1980 1980 1980 1980
5-1 211 + 8-221
Harberd and McArthur, 1980 Harberd and McArthur, 1980 Harberd and McArthur, 1980 Harberd and McArthur, 1980 Harberd and McArthur, 1980 Mizushima, 1950 Bing etal., 1991 Harberd and McArthur, 1980 Bing eral., 1991 Li et al., 1998 Harberd and McArthur, 1980
0 - 1 IV + %-2III+ 7 -1211 +
Inomata, 1994
+ 12-201
x x
Harberd and McArthur, 1980
12-201 27 27 27
-
x
Raphanus sativus (n=9) Sinapis arvensis (n=9) Sinapis arvensis (n=9)
x
Sinapispubescens (n=9)
27
0 4 1 1 + 15-271
x
x
7-1011
+ 7-131
Fukushima, 1945 Bing et al., 1991 Harberd and McArthur, 1980 Mizushima, 1950 Inomata, 1991
Brassica gravinae (n= 10) Brassica tournefortii (n=10) x Enarthrocarpus lyratus (n=10) x Sinapis alba (n=l2) x Orychophragmus violaceus (n=12) x Crambe abyssinica ( ~ 4 5 ) Brassica napus (n= 19) x Diplotaxis erucoides (n=7) x Hirschfeldia incana (n=7) x Raphanus raphanistrum (n=9) x Raphanus sativus (n=9) x Sinapis arvensis (n=9) x
x
Sinapis pubescens (n=9) Brassica gravinae (n=10) x Eruca sativa (n= 1 1) x Orychophragmus violaceus (n=12) x Brassica cossoniana (n=l6) Diplotaxis muralis (n=2 1) x Brassica rapa (n= 10) x Sinapidendronfrutescens (n=lO) x Diplotaxis harra (n=13) x Erucastrum gallicum (n=l5) x Brassica napus (n=19) x
x
38 38 28 30 24-36
281-1811
+ 21
Nanda-Kumar et al., 1989 Yadav etal., 1991 Gundimenda et al., 1992 Bajaj, 1990 Li et al., 1998 Wang, 1997
-
281-711
+ 141
-
__
-
26 26 28
3-1011 + 6-201 0-1IV + 1-711 + 10-241 0-1IV + 4-1011 + 8-201
28
0-2111
28 39 30 31 35
0-1111 + 2-1 111 + 5-241 391-1011 + 191
--
31 31 34 36 40
+ 0-1011 + 6-281
-
12-1611
+ 3-1
1I
311-511 + 211 1I1 + 291-511 + 2 1I 341-4511+ 221 311 + 301-1011 + 161 -
Harberd and Mc Arthur, 1980 Kerlan et al., 1993 Kerlan et al., 1993 Takeshita et al., 1980 Kerlan et al., 1993 Mizushima, 1950a Inomata, 1994 Nanda-Kumar et al., 1989 Bijral and Sharma, 1996 Li and Luo, 1993 Harberd and McArthur. 1980 Harberd and McArthur, Harberd and McArthur, Harberd and McArthur, Harberd and McArthur, Ringdhal et al., 1987 Fan etal., 1985
1980 1980 1980 1980
00 W
84 sativus), alternaria leaf spot resistance (Sinapis alba, Camelina sativa), nematode resistance (Sinapis alba) and cytoplasmic genes for inducing male sterility.
Introgression of g e n e s Several fungal diseases cause heavy losses to seed yield in Brassica crops. The major ones are white rust caused by Albugo candida, alternaria leaf spot (Alternaria spp.), blackleg or stem canker (Leptosphaeria maculans), and sclerotinia stem rot (Sclerotinia sclerotiorum). Nuclear genes conferring resistance to these diseases (see chapter 12) and for other desirable agronomic traits have been incorporated from related sources exploiting non-homologous recombination following sexual / somatic hybridization and also by generating alien chromosome addition lines. Successful introgression of genes following sexual hybridization are black leg resistance from B. juncea to B. napus (Roy, 1984) and from B. nigra to B. napus (Chevre et al., 1996, 1997); clubroot resistance from B. napus to cabbage (Chiang et al., 1977); self-incompatibility alleles from B. rapa to forage rape, earliness to oil rape leading to release of cultivars like Norin 16 and Asahi-Natane in J a p a n (Shiga, 1970; Namai et al., 1980); resistance to pod shattering from B. juncea to B. napus (Prakash and Chopra, 1990a); chlorosis corrections and fertility restoring genes from Raphanus sativus to CMS B. napus (Paulmann and R6bbelen, 1988) and resistance against Phoma lingam from B. juncea to B. napus (SacristAn and Gerdemann, 1986) and from B. nigra to B. napus (Struss et al., 1996). Examples of introgression through somatic hybridization include beet cyst nematode resistance (Heterodera schachtiz) from Sinapis alba and R a p h a n u s sativus to B. napus (Lelivelt et al., 1993; Lelivelt and Krens, 1992); alternaria leaf spot resistance from S. alba to B. napus (Primard et al., 1988); clubroot resistance (Plasmodiophora brassicae) from Rap h a n u s sativus to B. oleracea var. botrytis (Hagimori et al., 1992), blackleg resistance from B. nigra to B. napus (Gerdemann et al., 1994) and fertility restoring genes from Raphanus to B. napus (Sakai et al., 1996) and from Trachystoma baUii and Moricandia arvensis to B. juncea (Kirti et al., 1997; Prakash et al., 1998b).
Cytoplasm divergence and g e n o m e h o m o e o l o g y The taxa in Brassica coenospecies are classified into 2 distinct lineages based on their chloroplast DNA RFLPs (Warwick and Black, 1991; Pradhan et al., 1992). Each lineage is further separated into groups and four major groups have been recognized within each lineage. Five genera viz. Brassica, Diplotaxis, Erucastrum, Sinapis and Trachystoma are represented in both the lineages, t h u s indicating the incongruence between cp DNA data and the morphology based taxonomy. An interesting observation is that the taxa of these genera show more cp DNA homologies among them than to cogeneric
85 taxa. The lowest level of divergence was observed between taxa belonging to the s a m e cytodeme, e . g . B , oleracea, B. fruticulosa, E r u c a s t r u m virgatum a n d Coincya. On the contrary, divergence was h i g h e s t for B. rapa - B. oleracea v e r s u s B. nigra; B. rapa v e r s u s S i n a p i s arvensis, S. p u b e s c e n s a n d Coincya. Chloroplast g e n o m e similarity was h i g h e s t between B. nigra a n d S. arvensis. B. rapa a n d B. oleracea are also very close. This close affinity is also reflected in c h r o m o s o m e pairing in their h y b r i d s (B. nigra x S. arvensis, 2n = 17, 1-8 II, Mizushima, 1950a; H a r b e r d a n d McArthur, 1980; B. rapa x B. oleracea, 2n = 19; 9II + 1I, Olsson, 1960b). However, c h l o r o p l a s t genome divergence is not always c o n s i s t e n t with n u c l e a r genome divergence as the c h r o m o s o m e pairing between the taxa within a lineage is not always higher t h a n a c r o s s the lineage. At times, chrom o s o m e pairing could be as high or even higher in h y b r i d s a c r o s s the lineage. This high extent of pairing m a y be ascribed to f r e q u e n t hybridization leading to gene introgression, since a large n u m b e r of t h e m are s y m p a t r i c in their distribution. A few s u c h e x a m p l e s are the interlineage hybrids: Diplotaxis erucoides ( r a p a / o l e r a c e a lineage) x B. nigra (nigra lineage), 2n = 15, 7II (Quiros et al., 1988); B. f r u t i c u l o s a x B. rapa 2n = 18, 7II + 4I, (Mizushima, 1968); E r u c a s t r u m c a n a r i e n s e • B. oleracea, 2n = 18, 811 + 2I, (Harberd a n d McArthur, 1980); B. tournefortii • R a p h a n u s s a t i v u s 2n = 19, 7II + 5I, (Harberd a n d McArthur, 1980); Diplotaxis tennuifolia x E r u c a s t r u m virgatum 2n = 18, 7II + 4I, (Harberd a n d McArthur, 1980). On the o t h e r h a n d , some of the h y b r i d s between the t a x a within the lineage show little c h r o m o s o m e pairing indicating a high degree of genetic differentiation b e c a u s e of repatterning. E x a m p l e s include B. f r u t i c u l o s a • E r u c a s t r u m c a r d a m i n o i d e s , 2n = 17, 3II+11 I, (Harberd a n d McArthur, 1980); B. tournefortii • B. nigra, 2n = 18, 31I + 12I, (Narain a n d P r a k a s h , 1972); Dip l o t a x i s tenuifolia • B. oleracea, 2n = 20, 2II + 16I, (Harberd a n d McArthur, 1980). An interesting feature of these h y b r i d s was the o c c u r r e n c e of a high degree of c h r o m o s o m e pairing in the intertribe h y b r i d s involving Moricandia a r v e n s i s a n d B r a s s i c a spp. 1III + 5II in M. a r v e n s i s • B. nigra, 2n = 22 a n d 2III + 6II in M. a r v e n s i s • B. oleracea, 2n = 23 ( T a k a h a t a a n d Takeda, 1990; T a k a h a t a , 1990). Similarly, the somatic hybrid M. a r v e n s i s + B. j u n c e a (2n = 64) exhibited u p t o 1 q u a d r i v a l e n t a n d 3 trivalents (Kirti et al., 1992). These higher a s s o c i a t i o n s m i g h t be an e x p r e s s i o n of h o m o e o l o g y between chromos o m e s of the two species. This c l o s e n e s s is also reflected in their chloroplast g e n o m e s as Moricandia h a s affinities with B. rapa a n d B. oleracea. (Warwick a n d Black, 1994). These o b s e r v a t i o n s led these a u t h o r s to s u g g e s t the inclusion of Moricandia in B r a s s i c a coenospecies a n d also indicate t h a t recognition of Moricandiinae as a s e p a r a t e s u b t r i b e from s u b t r i b e B r a s s i c i n a e m a y be artificial. At the morphological level they b o t h s h o w elongated or siliquose d e h i s c e n t fruits.
86 Cytoplasmic substitutions Maternally inherited pollen sterility is of major importance as a pollination control m e c h a n i s m in developing hybrid cultivars. This trait - the cytoplasmic male sterility (CMS) - is encoded in mitochondrial genome and arises either as a mutation in mt DNA or can be engineered by placing the crop nucleus in alien cytoplasm -the alloplasmics (see chapter 6). Wild germplasm in B r a s s s i c a coenospecies is a rich repository of diverse cytoplasms as revealed by cp and mt DNA RFLPs (Warwick and Black, 1991, 1994; Pradhan et al., 1992). By combining their cytoplasms, alloplasmics of crop species have been synthesized exhibiting stable pollen sterility. These were obtained following sexual a n d / o r somatic hybridization. A major difference is that in the former the cytoplasm is exclusively contributed by the female parent and is unaltered while in the latter, organelle a s s o r t m e n t and mitochondrial DNA r e a r r a n g e m e n t s are of frequent occurrence. The first case of CMS in Brassica was reported in a wild population of R a p h a n u s (Ogura, 1968). This sterility inducing cytoplasm was later introduced to several B r a s s i c a spp. (Bannerot, 1974; Kirti et al., 1995a). Pearson (1972) developed a CMS system in broccoli (B. oleracea var. italica) by placing its nucleus in B. nigra cytoplasm. A little later, Hinata and Konno (1979) combined the cytoplasm of a wild species Diplotaxis muralis with B. rapa subsp, c h i n e n s i s nucleus (a leafy type). Subsequently, a n u m b e r of CMS systems have been developed in Brassica spp. carrying the cytoplasms of related wild species as B r a s s i c a oxyrrhina (Prakash and Chopra, 1990b) or Diplotaxis siifolia (Rao et al., 1994), Trachystoma ballii (Kirti et al., 1995b) and Moricandia a r v e n s i s (Prakash et al., 1998b). Somatic hybridization has added m a n y other possibilities (see Prak a s h et al., 1998a and chapter 4).
C y t o g e n e t i c c o n s t r u c t i o n of fertility restorers Fertility restorer lines for some of the CMS systems have been developed through chromosome manipulations. Restorer genes for ogu system were located in synthetic R a p h a n o b r a s s i c a (R. s a t i v u s x B. napus) and R. s a t i v u s x B. oteracea progeny by Heyn (1976) and Rouselle and Dosba (1985) respectively. Heyn (1976, 1979) introgressed these genes into B. napus, however, the fertility of restored plants decreased considerably. Pellan-Delourme and Renard (1988) observed that although restored B. n a p u s plants had the normal 38 chromosomes, 0-3 quadrivalents, 0-3 trivalents and upto 8 univalents per cell formed frequently. Decrease in fertility was attributed to high degree of ovule abortion. However, intensive selection led to developing restored lines with normal fertility. P a u l m a n n and R6bbelen (1988) also introgressed such genes for Ogu CMS in B. napus. Their method consisted of synthesis of hexaploid (ogu) R a p h a n u s s a t i v u s • B. n a p u s (2n = 56, RRAACC) and backcrossing it to B. rapa thus allowing recombination between the A and C genome chromosomes in BC1 hybrid AACR and s u b s e q u e n t introgression of restorer genes into B. napus. Stiewe et al. (1995) introduced such
87 genes in CMS (tournefortiz) B. n a p u s from B. tournefortii usi ng the synthetic alloploid B. tournefortii • B. rapa as a bridge species. Similarly, restorer genes for CMS (Moricandia) B. j u n c e a were transferred from Moricandia a r v e n s i s (Prakash et al., 1998b). Individual c h r o m o s o m e of M. arvensis was added to CMS B. j u n c e a and introgression was achieved t h r o u g h homoeologous chrom o s o m e recombination. In a n o t h e r study, fertility restorers were constructed for CMS (Trachystoma) B. j u n c e a by allowing intergenomic recombination between T. baUii a n d B. j u n c e a c h r o m o s o m e s in the backcross progeny of somatic hybrid T. ballii + B. j u n c e a (Kirti et al., 1997).
Chromosome
addition lines
Generation of c h r o m o s o m e addition lines have opened up the possibility of studying genome organization and evolution, identifying gene linkage groups and assigning species specific char act ers to a particular c h r o m o s o m e and comparing gene synteny between related species. They also have considerable value in transferring desirable cha ract ers of agronomic value from alien species to crop cultivars. All the three basic diploid genomes viz. B. nigra, B. oleracea a n d B. rap a have been dissected and a series of m on osom i c and disomic addition lines have been generated. As B r a s s i c a c h r o m o s o m e s are very small and lack morphological a n d cytological l a n d m a r k s , they have been characterized t h r o u g h genome specific m a r k e r s s u c h as isozymes, rDNA, RFLP and RAPDs. A general observation on these addition lines was that, unlike other genera, they did not show characteristic morphological features specific for a particular c h r o m o s o m e and they were rarely distinguishable from each other. It may well be t h a t the b a c k g r o u n d genome m a s k s the effect of the alien chromosome. Or the homoeologous chromosome, because of secondary polyploid nature, may nullify the effect of the alien chromosome. Another significant observation was t h a t high t r a n s m i s s i o n of alien c h r o m o s o m e t h r o u g h ovules and pollen was observed in the b a c k g r o u n d of amphidiploid species in c o n t r a s t to alien lines developed in a diploid backg ro u n d where t r a n s m i s s i o n occurs mostly t h r o u g h ovules. B. nigra addition lines B. nigra c h r o m o s o m e c o m p l e m e n t was dissected and m o n o s o m i c / d i s o mic addition lines were generated in the b a c k g r o u n d of the B. n a p u s genome following the r e c u r r e n t backcrossing of the trigenomic AB hybrid with B. nap u s (Jahier et al., 1989; Chevre et al., 1991; S t r u s s et al., 1991). Disomic addition lines were developed either on selfing or following a n t h e r culture and characterized by isozymes, fatty acid and RFLP m a r k e r s (Jahier et al., 1989). Six enzymes viz. 6-PGDH, GOT, TPI, PGM, PGI and ADH were able to discriminate B. n a p u s and B. nigra genomes. Therefore, loci coding for these
88
e n z y m e s were u s e d for identification of B. nigra c h r o m o s o m e s . The results s u g g e s t e d t h a t 6 PGDH-2 a n d GOT-5 formed p a r t of the s a m e s y n t e n y group. A similar s i t u a t i o n exists for TPI-1 a n d PGM-3. S y n t e n y g r o u p 1 displayed high levels of linoleic a n d linolenic acids in the seeds of B. nigra p a r e n t s ; s y n t e n y group 3 a c c u m u l a t e d higher levels of eicosenoic a n d erucic acid t h a n B. nigra. RFLP m a r k e r s also confirmed the six s y n t e n y groups. The p r e s e n c e of multiple f r a g m e n t s for several probes a n d a different d i s t r i b u t i o n in two c h r o m o s o m e s s u g g e s t the existence of duplicated c h r o m o s o m e s e g m e n t s in B. nigra. Meiosis revealed the formation of 19II+ 1I in m o n o s o m i c addition (Struss et al., 1991) a n d 19II+ 1II in disomic additions (Jahier et al., 1989). Transm i s s i o n of alien c h r o m o s o m e w a s high both t h r o u g h ovules a n d pollen ranging from 50 to 100 per cent. Very little c h r o m o s o m e pairing w a s noticed between B. nigra a n d B. n a p u s c h r o m o s o m e s t h u s confirming the earlier views t h a t B. nigra g e n o m e is genetically d i s t a n t from A a n d C genomes. B. oleracea addition lines B. oleracea m o n o s o m i c a n d disomic c h r o m o s o m e addition lines were g e n e r a t e d in the b a c k g r o u n d of B. rapa (Quiros et al., 1987; McGrath et al., 1990; C h e n et al., 1992) by r e c u r r e n t b a c k c r o s s i n g of n a t u r a l or synthetic B. n a p u s with B. rapa. They were c h a r a c t e r i z e d by genome specific m a r k e r s . F o u r e n z y m e s y s t e m s viz. 6-PGD, PGI, LAP a n d PGM, rDNA genes a n d RFLP m a k e r s were u s e d to identify individual c h r o m o s o m e s . T h e s e m a r k e r s revealed the existence of a high level of gene duplication in B. oleracea genome (McGrath et al., 1990) s u p p o r t i n g the earlier h y p o t h e s i s of its s e c o n d a r y polyploid origin. Addition lines were, in general, morphologically undisting u i s h a b l e from e a c h other. Only one morphological c h a r a c t e r , rugose or p u c k e r e d leaf w a s c h a r a c t e r i z e d for a B. oleracea c h r o m o s o m e which was always a s s o c i a t e d with a n isozyme m a r k e r 6-PGD-1. No o t h e r c h a r a c t e r s could be a s s o c i a t e d with other B. oleracea c h r o m o s o m e s . C h e n et al. (1992) developed five B. oleracea var. alboglabra c h r o m o s o m e addition lines. One of these was r e p o r t e d to have 3 loci viz. EC, WC a n d L a p - l C controlling the b i o s y n t h e s i s of erucic acid, white flower color, a n d faster migrating b a n d of leucine a m i n o p e p t i d a s e respectively on s a m e c h r o m o s o m e . The alien chrom o s o m e mostly r e m a i n s u n i v a l e n t b u t also u n d e r g o e s pairing with B. rapa c h r o m o s o m e s forming a trivalent (Chen et al., 1992). K a n e k o et al. (1987) developed B. oleracea var. a c e p h a l a m o n o s o m i c c h r o m o s o m e addition lines in the b a c k g r o u n d of R a p h a n u s s a t i v u s by r e c u r r e n t b a c k c r o s s i n g of synthetic R a p h a n o b r a s s i c a (2n=36 RRCC) with R. sativus. Seven addition lines could be d i s t i n g u i s h e d by morphological a n d physiological c h a r a c t e r s s u c h as seedling, leaf, root a n d pod traits a n d growth habit. C h r o m o s o m e configuration of 9II + 1I w a s p r e d o m i n a n t in these addition lines, a n d formation of a trivalent or a q u a d r i v a l e n t w a s rarely observed. Since one of these addition lines showed r e s i s t a n c e to t u r n i p mosaic virus, the r e s i s t a n c e gene is consi-
89 dered to locate on the added c h r o m o s o m e of B. oleracea (Kaneko et al., 1996).
B. ox.yrrhina addition lines Seven different B. oxyrrhina c h r o m o s o m e a d d i t i o n s were identified with reliable morphological m a r k e r s in the b a c k g r o u n d of B. rapa s u b s p , oleifera (Srinivasan et al., 1998). Novel p h e n o t y p e s s u c h as c u p p e d leaves a n d crinckled petals were observed which p r e s u m a b l y r e s u l t e d from intergenomic interactions between both genomes. However, no deleterious effect on floral morphology or seed fertility was appreciated. Since these lines were developed on alien cytoplasm, they were all pollen sterile except s y n t e n y group 6 which showed 12-16 % pollen fertility. Four m o n o s o m i c additions showed trivalent formation suggesting allosyndetic c h r o m o s o m e associations. The m a t e r n a l t r a n s m i s s i o n frequency a m o n g the a d d i t i o n s indicated r e d u c t i o n in the ovule t r a n s m i s s i o n frequency a g a i n s t theoretical expectations. RAPD analysis of these a d d i t i o n s revealed u n i q u e B. oxyrrhina specific b a n d s r e p r e s e n t i n g each a d d i t i o n s except for s y n t e n y group 6. S a m e sized PCR p r o d u c t s from a single primer, r e p r e s e n t i n g different B. oxyrrhina c h r o m o s o m e s indicated the possibility of i n t r a g e n o m i c r e c o m b i n a t i o n s a m o n g B. oxyrrhina c h r o m o s o m e s .
Concluding remarks Brassica a n d allied genera form a gene pool which can be easily manip u l a t e d genetically a n d chromosomally. As these are very a m e n a b l e to in vitro t e c h n i q u e s as d e m o n s t r a t e d by m a n y studies, new a n d exciting develo p m e n t s can be expected in developing s u p e r i o r a g r o n o m i c types. Some of the s u g g e s t i o n s in this regard are: A major limitation in a breeding p r o g r a m is a lack of a d e q u a t e variability. It is widely recognized t h a t genetically a n d geographically d i s t a n t genotypes p r o d u c e a m u c h superior progeny. P r o d u c t i o n of synthetic s t r a i n s of B. juncea, B. n a p u s a n d B. carinata exploiting large variations of the const i t u e n t p a r e n t s will r e s u l t in useful variability. Since n a t u r a l h y b r i d s were always unidirectional, s y n t h e t i c s with new c y t o p l a s m s , opposite to n a t u r a l ones a n d somatic h y b r i d s with new c o m b i n a t i o n s of cytoplasmic organelles will give more variations. New variability can also be o b t a i n e d exploiting nonh o m o l o g o u s r e c o m b i n a t i o n in F1 interspecific h y b r i d s a n d s u b s e q u e n t functioning of duplication-deficiency g a m e t e s (Prakash, 1973b). Wild g e r m p l a s m will be of major i m p o r t a n c e in a n y crop i m p r o v e m e n t p r o g r a m in the future. They not only p o s s e s s n u c l e a r gene controlled desirable agronomic traits, b u t also their c y t o p l a s m s also carry DNA s e q u e n c e s which control c h a r a c t e r s of significance. These include m a t e r n a l l y inherited male sterility, herbicide r e s i s t a n c e a n d p h o t o s y n t h e t i c activity. We are fortunate in having a large gene pool of wild taxa p o s s e s s i n g e n o r m o u s varia-
90 bility in c h l o r o p l a s t a n d m i t o c h o n d r i a l g e n o m e s (Warwick a n d Black, 1991) a n d where n u c l e a r genes for useful c h a r a c t e r s are liberally distributed. A s p e c t r u m of male steriles can be p r o d u c e d for developing heterotic hybrids b a s e d on these wild taxa. With the a d v a n c e m e n t of in vitro techniques, it is even possible to obtain intertribe hybrids. Wide hybrids of every possible c o m b i n a t i o n s h o u l d be obtained for c h r o m o s o m e m a n i p u l a t i o n . S u s t a i n e d efforts s h o u l d be directed t o w a r d s developing alien chromosome addition lines particularly from the g e n o m e s of wild allied g e r m p l a s m to locate the genes of i m p o r t a n c e on a specific c h r o m o s o m e . These will be of i m m e n s e value in introgressing c h a r a c t e r s of agronomic value t h r o u g h cytogenetic m a n i p u l a t i o n s a n d also for the application of genetic engineering t e c h n i q u e s for crop improvement. Brassica c h r o m o s o m e s are small and lack cytogenetic l a n d m a r k s . However, in situ hybridization techniques, RFLPs a n d RAPDs provide the m e a n s to identify the specific c h r o m o s o m e as these allow a large n u m b e r of molecular m a r k e r s to be associated with them. Efforts in this direction are very e n c o u r a g i n g since molecular m a p s for B. nigra, B. oleracea, B. j u n c e a a n d B. n a p u s are now available (see c h a p t e r 7).
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Biology of Brassica Coenospecies C. G6mez-Campo(Editor) 91999Elsevier Science B.V. All rights reserved.
107
4 SOMATIC HYBRIDIZATION Kristina Glimelius
Department of Plant Breeding Research, The Swedish University of Agricultural Sciences, Box 7003, 750 07 Uppsala, Sweden Many efforts have been made to improve Brassica crops by sexual crosses designed to enrich their g e r m p l a s m t h r o u g h artificial resynthesis and wide hybridization of naturally existing amphidiploids (for reviews see Olsson and Ellerstr6m, 1980; Prakash, 1980; Namai et al., 1980; Downey a n d R6bbelen, 1989; Buzza,1995). However, even though there is a large tolerance for interspecific hybridization between the Brassica crop species a n d several of the wild species related to Brassica species, successful use of sexual hybridization is limited. A n u m b e r of pre- and post-fertilization barriers limit the diversity of different genomic combinations. F u r t h e r m o r e , in sexual hybrids, the haploid gametes a n d t h u s the haploid genomes of each p a r e n t are combined, which usually results in problems of pairing of non-homologous c h r o m o s o m e s during meiosis. Poor pairing results in the presence of large n u m b e r s of univalent chromosomes, a n d even some tri- a n d quadrivalent c h r o m o s o m e s (Chopra et al., 1996). Besides the reduction in fertility c a u s e d by d i s t u r b a n c e s in chromosome pairing, m a t e r n a l inheritance of organelles can result in cytoplasmic incompatibility barriers which further restrict the possibilities for hybridization. Dysfunctional n u c l e a r - c h o n d r i o m e a n d / o r unclear-plastom combinations may be expressed in s u c h defects as cytoplasmic male-sterility (CMS; Edwardson, 1970), leaf chlorosis (Bannerot et al., 1977), floral abnormalities (Beillard et al., 1978; Bonnett et al., 1991), poorly developed nectaries (Bannerot et al., 1977) and reduced female fertility (Rawat and Anand, 1979). Methods to overcome these restrictions have been developed, viz interspecific hybrids t h r o u g h embryo, ovule, a n d ovary culture. At present, sexual hybridization is limited to combinations between certain species a n d genotypes representing closely related genera s u c h as Bras-
sica, Diplotaxis, Eruca, Erucastrum, Enarthrocarpus, Hirschfeldia, Coincya, Raphanus, Sinapis, Sinapidendron a n d Trachystoma (for a review see Chopra et al., 1996). Protoplast fusion can circumvent the limitations on sexual hybridization, so t h a t more taxonomically divergent species may be employed in breeding programs. Fusing protoplasts can b y p a s s problems with non-
108 homologous chromosome pairing, as well as create new nuclear-cytoplasmic combinations. In this review, a survey is presented of the results from using protoplasts as artificial gametes for the production of somatic hybrids and cybrids in the family B r a s s i c a c e a e .
Protoplast technology Isolation of protoplasts from the genus B r a s s i c a was first reported by Wenzel (1973). One year later a presentation on callus formation and plant regeneration from B. n a p u s was published by Kartha et al. (1974). However, it was not until the beginning of the 1980's that reproducible protocols for plant regeneration from isolated B r a s s i c a protoplasts were fully developed (Schenck and R6bbelen, 1982; Pelletier et al., 1983; Glimelius, 1984). These protocols expanded the possibilities of protoplast technology for plant breeding purposes. Reports have continued to be published on successful culture methods of different B r a s s i c a species (see Wamling and Glimelius, 1990). Efficient m a s s fusions of protoplasts isolated from different sources of tissues make it possible to combine genetic material independently of species and sexual compatibility (Wallin et al., 1974; Kao and Michayluck, 1974). The main procedure used for protoplast fusion is the polyethylene glycol (PEG) method, first described by Wallin et al. (1974) and Kao and Michayluck (1974) and adapted for B r a s s i c a species by, for example, Pelletier et al. (1983), Sundberg and Glimelius (1986), Morgan and Maliga (1987) and Robertson et al. (1987). High frequencies of heterodimeric fusion products can be obtained following PEG treatment. Successful utilization of electric stimulation has also been reported to promote fusion (Zimmermann, 1982). However, selection of the desired heterokaryons from the mass population of protoplasts after fusion is still a bottleneck. Several different techniques are used to enrich and select fused protoplasts. The most efficient method is flow cytometry combined with cell sorting (Glimelius et al., 1986). However, the most commonly used method is the donor-recipient fusion method, originally developed by Sidorov et al. (1981), in which one of the parental protoplasts is pretreated with iodoacetate or iodoacetamide (IOA) and the other with irradiation. A method in which one parent exhibiting poor growth and regeneration capacity is fused with a pretreated parent has also been used successfully in several instances (Terada et al., 1987; Gerdemann-Kn6rck et al., 1994; Waiters and Earle, 1993; O'Neill et al., 1996). Other methods include m a n u a l selection of fusion products using the micromanipulator (Sundberg and Glimelius, 1986; Lelivelt et at., 1993) or simply utilising special growth requirements for the protoplasts, which allow for sustained growth and development of the hybrid but not the parental protoplasts (Schenck and R6bbelen, 1982; Ros6n et al., 1988). To verify the hybrid nature of the regenerated plants, isoenzyme analysis has proven to be an easy and efficient method (Sundberg and Glimelius, 1986). RFLP-analysis using nuclear probes is even more efficient. Hybridi-
109
f spe ies B
Ng
speciesA
//
~~
!!
~ ~ ~-~
@
~
~
[-//-] laserlight ~ ........ detector, _ ~ +§ computer o 4- ~ o% droplet ~( o ~) deflector I~ ~ . B " selectionby dfl~ s~ wast~e ! 0
0r
protoplasts
droplet chargesignal
cuitu~plate " (~sel~t!o.n ~ ~ ~ oy,,sning .....CMturepiate
~
Analyses; fertility, seedset
'""'"~
"'
~ ~else;tned ~7~ products
~ ;~I'
division
isoenzyme& molecular markers ABHHP RFLP, chloroplast & mitochondrial _ _ = = DNA
~
~~~~
, ~
callus
~ ~
DNA-cont. measurements chromosome counting
nuclei p~
"
~
shoot regeneration
~
root tip
in situ hybridization squash
Figure 4.1 Schematic drawing of the different steps used for production of somatic hybrids and for analysis of the obtained plants.
110 zation of probes which are either species-specific or giving species-specific patterns is useful for hybrid confirmation. Repetitive species-specific sequences which assay for large parts of the parental genome have proven to be very useful (Fahleson, 1993; Fahleson et al., 1994a) and can even be used for a quantitative estimate of the proportion of genomic material from each parent in asymmetric hybrids (Imamura et al., 1987; Piastuch and Bates, 1990). RFLP analysis is also used to determine the organellar composition of hybrid plants (Beillard et al., 1978; Aviv et al., 1980; Pelletier et al., 1983). Steps used for the production and analysis of somatic hybrids are presented in Figure 4.1.
Somatic hybrids produced between different B r a s s i c a species Resynthesis of B r a s s i c a n a p u s Cytological experiments have shown that B r a s s i c a n a p u s is a hybrid between B. rapa (syn. B. c a m p e s t r i s , AA genome) and B. oleracea (CC genome) (U, 1935). These results prompted researchers and breeders to re-synthesize rapeseed by sexual hybridization from the progenitor species (Namai et al., 1980). For breeding purposes, the main goal of producing B. n a p u s hybrids is to broaden genetic diversity of the cultivated crop. However, hybrid production often requires that B. rapa is used as the female parent, and sexual hybridization between some genotypes never results in hybrid production (Namai et al., 1980). Thus, the restrictions on sexual hybridization have stimulated researchers to resynthesize rapeseed by somatic hybridization. A substantial number of investigations have described production of somatic hybrids between B. oleracea and B. rapa (Table 4.1). Several of the regenerated hybrids had the expected chromosome n u m b e r of 38, which will result from fusing somatic ceils with the diploid chromosome number from B. rapa (2n = 20) and B. oleracea (2n = 18). However, deviations from the expected chromosome n u m b e r were also recorded in several hybrids. This could be due to triple fusions involving one B. rapa protoplast with two B. oleracea protoplasts, or vice versa, resulting in hexaploid plants containing either the genome composition AAAACC or AACCCC, as discussed by Terada et al. (1987) and Heath and Earle (1996a). Aneuploid n u m b e r s were also obtained in several cases, which most likely reflected chromosome elimination during regeneration and development (Sundberg et at., 1987; Terada et al., 1987). Nevertheless, most of the plants could set seed, either after selfing or crossing to an established rapeseed cultivar (Table 4.1). Rapeseed hybrids from protoplast fusion generally exhibited lower self fertility than hybrids obtained by sexual hybridization (Schenck and R6bbelen, 1982; Sundberg et al., 1987; Ros~n et al., 1988; Ozminkowski and Jourdan, 1993). Reduction in self fertility could be attributed to chromosomal alterations induced by the in vitro culture or from genetic factors such as incompatibility (Sundberg et al., 1987; Heath and Earle, 1996a). Another factor which could affect the
Table 4.1 A summary of the results obtained from resynthesis of Brassica napus via somatic hybridization of B. rapa (+) B. oleracea where regeneration of hybrid plants per number of calli, chromosome number, traits and fertility are presented. Hybrid plants 12
Calli
Chromosome number
Trait
Fertility
Reference
139
38, 36,54, 18
ND*
F
Schenck and Rlibbelen 1982
4
ND
38
vegetable properties
F
Taguchi and Kameya 1986.1987
10
ND
23
38, 33,36,49, 56, 57
ND*
F, MS
Terada et al. 1987
2300
38,50-60
ND*
F, MS
Sundberg et al. 1987
1
1089
36-38
CMS
ND
Robertson et al. 1987
5
1980
38
earliness, winter hardiness
F
Rosen et al. 1988
34
1136
38,58, aneuploidy
atrazinR CMS
F, MS
Jourdan et al. 1989
72
ND
38,58
fertility, SI-genes organelle segregation
F
Ozminkowski and Jourdan 1993; 1994 a; b
109
3 80
38
erucic acid, shattering and lodging resistance; seed size.
F
Heath and Earle 1995
39
110
38, 56, 5 8
CMS, cold tolerance
F, MS
Heath and Earle 1996a
F = fertile,
MS = male sterility,
ND = not determined,
CMS = cytoplasmic male sterility,
* basic investigations of hybrid features and fertility,
112 fertility of the hybrids is the expression of self-incompatibility (SI) genes from the parental genomes (Ozminkowski and J o u r d a n , 1993).
Comparison of resynthesized rapeseed produced by somatic and sexual hybridization. In a thorough investigation Ozminkowski a n d J o u r d a n (1993, 1994ab) have compared the results obtained from resynthesis of rapeseed by interspecific somatic a n d sexual hybridization. The same parental material was u s e d for hybrid production in both experiments. They found that more hybrids were obtained by somatic hybridization. In addition, when combining different genotypes of the parental species successful results were more likely via somatic hybridization. Only one of the chosen B. rapa genotypes gave progeny after sexual hybridization. Furthermore, the time required to obtain somatic hybrids was shorter (7 months) t h a n for sexual hybrids obtained by embryo rescue (usually a r o u n d 1 year). Time to flowering was shorter for the somatic hybrids t h a n for the sexual hybrids. Some of the latter required vernalization to flower, while the somatic hybrids did not. Hybrids derived from protoplast fusions are not uniform and they often differ in ploidy level, morphology, fertility a n d cytoplasmic composition (Schenck and R6bbelen, 1982; S u n d b e r g et al., 1987; Terada et al., 1987). Nevertheless, Ozminkowski a n d J o u r d a n could establish t h a t within each fusion experiment, it was possible to obtain some hybrid plants with the expected and balanced c h r o m o s o m e n u m b e r of n = 19, utilizing protoplasts from one genotype of each parent. These plants displayed uniformity in morphology and fertility, were expected to contain all the aUelic combinations present in the p a r e n t s and, thus, were considered clones for the unclear-encoded characteristics. However, additional investigations, such as characterization of hybrids utilizing RFLP m a r k e r s would be needed before drawing further conclusions. In c o n t r a s t to nuclear traits, the organelle composition differed a m o n g the somatic hybrids, whereas the sexual hybridization resulted in m a t e r n a l inheritance of the organelles. Heath a n d Earle (1996a) have also m a d e a comparison of resynthesized rapeseed produced from the same parental material by sexual and somatic hybridization. Their investigation included a comparison between somatic hybrids a n d sexual hybrids produced by s p o n t a n e o u s seed development as well as by embryo rescue. The hybrids compared in their study differed in several characters, s u c h as leaf morphology, flower color, fertility, DNA content and organelle composition. Their investigations revealed more variability in the somatic hybrids than in the sexual hybrids, especially for leaf morphology, b u t also for flower color. When analysing the sexual hybrids they found less segregation in the different traits in the spontaneously obtained hybrids t h a n in the hybrids obtained via embryo rescue. Their conclusion was t h a t the s p o n t a n e o u s l y obtained hybrids may have been derived from u n r e d u c e d gametes rather t h a n via hybridization. Another difference was
113 that the somatic hybrids displayed a higher DNA cont ent t h a n the expected DNA co n te n t in a hybrid, possibly due to a three-way fusion involving one B. c h i n e n s e s a n d two B. o l e r a c e a protoplasts. All the sexually produced hybrids had a DNA c o n t e n t similar to the amphidiploid DNA content in rapeseed. Fertility investigations of the material revealed t h a t the pollen viability was high in the sexually produced hybrids, s o m e w h a t lower in the putative hexaploid somatic hybrids, while it was low for all the other somatic hybrids having aneuploid or multiple genome compositions. Only the s p o n t a n e o u s sexually derived hybrids set seeds after selfing while the hybrids obtained via embryo rescue were self and sib-incompatible. However, they were able to set seeds when crossed with anot her B. n a p u s cultivar. None of the somatic hybrids could be selfed, b u t two of the putative hexaploid hybrids were able to set seeds after elimination of the B. c h i n e n s i s genome. Regarding the chloroplast composition in the somatic hybrids a strong bias towards the B. chin e n s i s chloroplasts was found. This might have been due to the fact t h a t most hybrids were hexaploid and contained two AA genomes. From these investigations it can be concluded t h a t somatic hybridization can be beneficial for hybrid production of some cultivars whereas for others sexual hybridization is better. The reasons for the variations in somatic hybrid plants may depend on the cultivars combined or the m e t h o d s u s e d for hybridization and regeneration. Nevertheless, new breeding materials of potential importance can be p r oduced via somatic hybridization. Several of the somatic rapeseed hybrids are being evaluated in field trials for agronomic traits such as earliness, winter hardiness, erucic acid content, lodging and shattering resistance, CMS, atrazine resistance and cold tolerance (Table 4.1). Furthermore, the vegetable "Hakuran" or r u t a b a ga, has also been produced by somatic hybridization of cabbage with Chinese cabbage (Taguchi and Kameya, 1986). An area where somatic hybridization might be of practical importance is to modify the fatty acid composition in rapeseed. As an example, efforts have been mad e to resynthesize B. n a p u s with a high content of erucic acid. Fatty acids consisting of long carbon chains with 20 or more carbon atoms, like erucic acid, are of great importance in the industrial applications for production of polyethylene films, polymers a n d nylon p r o d u c t s (Sonntag, 1995). Thus, experiments were conducted fusing a B. o l e r a c e a cultivar exhibiting high levels of erucic acid (C22:1) with a B. r a p a cultivar also with high erucic acid content. This resulted in a hybrid line exhibiting as m u c h as 57.4% erucic acid (Heath and Earle, 1995). A c o n t e n t of 56.6% was recorded a m o n g the progeny obtained after selfing and culture in field conditions. However, to be of real practical importance levels higher t h a n 66% should be obtained. This could be the case if erucic acid is esterified to all 3 positions in the glycerol molecule (Uppstr6m, 1995). Thus, a transfer of the genes responsible for esterification of erucic acid to all 3 carbon positions in the glycerol molecule would result in the m o s t optimal combination (Taylor et al., 1994).
114
Combinations of other Brassica
g e n o m e s into s o m a t i c hybrids
Besides the resynthesis of rapeseed a large n u m b e r of somatic hybrids have been produced by combination of the other Brassica genomes (Figure 4.2, Table 4,2). Somatic hybridization of the different Brassica progenitor species within the triangle of U (1935) have resulted in the production of all three amphidiploid species, B. napus, B. carinata (Jourdan and Salazar, 1993; N a r a s h i m h u l u et al., 1994) and B. juncea (Campbell et al., 1990). Furthermore, the combination of all three Brassica genomes, A, B, C, has been performed by the production of B. naponigra (Sj6din and Glimelius, 1989a). Several of the combinations have been produced to improve disease resistance in the cultivated species B. napus, B. juncea or B. oleracea (Table 4. 2). Numerous efforts have been made to breed for resistance to blackleg caused by the pathogen Phoma lingam with the perfect stage Leptoshaeria maculans (Desm). This fungus causes blackleg disease in all cruciferous crops and is a severe problem in Europe, Australia and Canada. Resistance to the pathogen has been found in several different germplasms of B. carina-ta, B. nigra and B. juncea (Sj6din and Glimelius, 1988), but also in B. tournefortii (Salisbury, 1991) and Sinapis alba (syn. B. hirta) (Pl~mper and Sacristan, 1995). Disease-resistant hybrid plants have been obtained from most fusion experiments (see review by Dixelius and Glimelius, 1995). Analysis of some hybrid progenies obtained after backcrossing to rapeseed have confirmed presence of resistant lines after 10 to 12 generations, indicating stable inheritance of the resistance genes (Axelsson and Dixelius, 1994). Other diseases of Brassica crops include beet cyst nematode (Heterodera schachtil), clubroot (Plasmodiophora brassiceae), black spot (Alternaria brassiceae), white rust (Albugo candida) and black rot (Xanthomonas campestris pv. campestris). High levels of resistance have been found in the primary hybrids produced between rapeseed and the resistant Sinapis alba (syn. B. hirta) for beet cyst nematode (Lelivelt et al., 1993). Clubroot resistance was derived from B. nigra (Gerdemann-Kn6rck et al., 1994), black spot resistance from the donor Sinapis alba (Chevre et al., 1994) and black rot resistance was transferred from B. napus to B. oleracea (Hansen and Earle, 1995). The primary hybrids have been utilized as bridges for transferring the genes for resistance to the cultivated crop by backcrossing. In several cases, for example clubroot, black spot and black rot, BC1 progenies have been obtained displaying disease resistance. This confirms that transfer and inheritance of the resistance genes can be established via protoplast fusion.
C o m p a r i s o n of s e x u a l v e r s u s sion of alien genes~
s o m a t i c hybridization for introgres-
Chevre et al. (1994) performed an investigation comparing the efficiency of protoplast fusion to sexual crosses as a means to introduce new traits in a crop. Cytogenetic and molecular characterizations of somatic hybrids produced between B. napus (AACC) and Sinapis alba (Sal) were compared with
115
Genus Brassica
B.oleracea
Figure 4.2 Somatic hybrids produced between different
Brassica species. The somatic hybrids are represented by grey circles. Arrows indicate which species were combined. The number of circles represents the number of genomes in the species and hybrids. The Brassica species are arranged according to the triangle of U (U, 1935) with solid black lines showing the phylogenetic relation between the allotetraploid and diploid species.
116 sexually produced hybrids from the same species. The molecular studies revealed t h a t all the somatic hybrids except one had the complete genomes of both parents. This was confirmed by chr om osom e n u m b e r analysis. However, the c h r o m o s o m e n u m b e r varied from 42 to 54 after backcrossing to rapeseed. Thus, instead of obtaining the expected c h r o m o s o m e n u m b e r 50, which would be the result from the combination AACCSal, a variation was observed. This might be due to d i s t u r b a n c e s during meiosis. Nevertheless, in the b a c k c r o s s e d plants, meiotic abnormalities were found, s u c h as trivalents a n d quadrivalents, indicating homologies which enabled c h r o m o s o m e s from the two p a r e n t s to pair, and possibly to recombine. From the meiotic analysis it was confirmed t h a t c h r o m o s o m e r e a r r a n g e m e n t s occurred more frequently in the somatic t h a n the sexual hybrids. The conclusion from these results is t h a t r e a r r a n g e m e n t s induced by protoplast fusion might facilitate introgression of new traits from taxonomically d i s t a n t species to a crop. This seems to have occurred t hr ough recombination rat her t h a n translocation.
I n t e r g e n e r i c s o m a t i c hybrids w i t h i n t h e tribe B r a s s i c e a e Intergeneric hybrids have been produced between all the different Brassica species in the U triangle and species belonging to the genera Eruca, Sinapis, Raphanus, Moricandia, Diplotaxis and Trachystoma (Figure 4.3, Table 4.3). All intergeneric combinations resulted in hybrid plants. Some of these hybrids h a d c h r o m o s o m e n u m b e r s representing the s u m of the parental species, while large variations, ranging from c h r o m o s o m e n u m b e r s slightly higher t h a n the c h r o m o s o m e n u m b e r of the Brassica species to multiples of the expected s u m of the two species were found. According to isozyme and RFLP analysis hybrid plants lacking c h r o m o s o m e s also lacked some of the genetic markers. However, even t h o u g h some of the genetic material from the "donor" parental line was missing, partial hybridity was confirmed by presence of some m a r k e r s from both species. Thus, in the hybrids with fewer c h r o m o s o m e s t h a n expected, and even in the plants with a chromosome n u m b e r equal to the Brassica species (regarded as the "acceptor"), alien DNA was present. In a s t u d y performed by Sun dberg and Glimelius (1991a) the correlation between the frequency of somatic hybrids with eliminated chrom o s o m e s and the genetic distance between the species in each combination was investigated. They concluded t h a t a larger genetic distance between the two species resulted in a greater degree of c h r o m o s o m e elimination. This was significantly greater after fusing species with different ploidy levels.
Intertribal somatic hybrids The great and u n i q u e potential of protoplast fusion technology is that it produces species combinations t h a t could never be obtained via sexual hybridizations, even when using the embryo rescue and in vitro fertilization techniques. The very first somatic hybrids produced between different spe-
Table 4.2 A summary of the somatic hybrids produced between different Brassica species within the tribe Brassiceae. The traits of interest to modify with the somatic hybridization and fertility have been listed. Somatic hybrid
Trait
Fertility
References
Brassica napus
Brassica nigra
Phoma lingamR
F
Sjodin and Glimelius 1989 a; b
Brassica napus Brassica napus
Brassica nigra Brassica nigra
?
Sacristan et al. 1989 Gerdemann-Knorck et al. 1995
Brassica napus Brassica napus
Brassicajuncea Brassica carinata
Phoma lingamR Phoma lingam' Plasmodiophora brassiceae' Phoma lingamR Phoma lingamR
Brassica napus
Sinapis alba (syn. B. hirta)
Brassica napus
Brassica oleracea
Brassica napus Brassica napus
Brassica oleracea Brassica tournefortii
Brassica napus Brassica oleracea
Brassica tournefortii Brassica nigra
Brassica oleracea
Brassica napus
Brassica juncea
Brassica spinescens
F = fertility,
MS = male sterility,
Alternaria brassiceae' Heterodera shachtii' Drought tolerance CMS triazinR, seedling chlorosis ND* Phoma lingam' CMS CMS Albugo candid2 A lternaria brassiceaeR Xanthomonas campestriis pv campestrisR,CMS, atrazinR Albugo candid2 salt tolerance
CMS = cytoplasmic male sterility.
S, F(few) F F F S F MS, FF
Sjodin and Glimelius 1989 b Sjiidin and Glimelius 1989 b Plumper and Sacristan 1995 Primard et al. 1988 Lelivelt et al. 1993 Chevre et al. 1994 Kao et al. 1992
F F MS F, MS F FF, MS F
Stiewe and Riibbelen 1994 Narasimhulu et al. 1992 Jourdan and Salazar 1993 Hansen and Earle 1995
FF, MS
Kirti et al. 1991
Sundberg et al. 199 1 Liu et al. 1995
*basic investigation of chromosome elimination and chloroplast segregation
118
Tribe Brassiceae
Genus Moricandia
Genus Eruca
Moricandia arvensis Brassica
B. juncea
B. oleracea
muralis Diplotaxis catholica
Smapls
Figure 4.3. Intergeneric somatic hybrids produced within the tribe Brassiceae. Hybrids, the number of genomes and the phylogenetic relationships are marked as in Figure 4.2.
Table 4.3 A summary of the somatic hybrids produced between species from different genera from the tribe Brassiceae where the desired traits and fertility are listed. Somatic hybrid
Trait
Fertility
Reference
Brassica napus
Diplotaxis muralis
CMS
?
McLellan et al. 1987
Brassica napus
Diplotaxis harra
CMS
S
Klimazewska and Keller 1988
Brassica napus
Eruca saliva
Drought tolerance, AphidR, erucic acid
F
Fahleson et al. 1988,1997
Brassica napus
Raphanus sativus
CMS
MS
Sundberg and Glimelius 1991b
Brassica napus
Raphanus sativus
Heterodera schachtiiR
S
Lelivelt and Krens 1992
Brassica napus
Moricandia arvensis
Alternaria brassiceaeR, Phyllotreta crucijereaeR,, Plasmidiophora brassiceaeR
F
O’Neill et al. 1996
Brassica juncea
Eruca saliva
Drought tolerance
F
Sikdar et al. 1990
Brassica juncea
Diplotaxis muralis
ND*
F
Chatterjee et al. 1988
Brassica juncea
Diplotaxis catholica
AphidR,Alternaria brassiceaeR
F
Kirti et al. 1995c
Brassica juncea
Moricandia arvensis
Photorespiration
MS
Kirti et al. 1992a
Brassica juncea
Trachystoma ballii
Alternaria brassiceaeR, hard pods, CMS
S
Kirti et al. 1992b
Brassica oleracea
Sinapis turgida
ND
ND
Toriyama et al. 1987b
Brassica oleracea
Moricandia arvensis
Photorespiration, Albugo candid2
?
Toriyama et al. 1987a,1988
Brassica nigra
Sinapis turgida
ND
ND
Toriyama et al. 1987b
Brassica oleracea
Raphanus sativus
Plasmodiophora brassiceaceR
F
Hagimori et al. 1992
CMS = cytoplasmic male sterility,
MS = male sterility,
ND = not determined,
S = sterile,
F = fertile
*basic cytogenetic investigations
120 cies in the family of B r a s s i c a c e a e were made between B. rapa (syn. B. campestris) and A r a b i d o p s i s thaliana, two species from different tribes of the family (Gleba and Hoffman, 1979, 1980). Their success in obtaining intertribal somatic hybrids with genetic material from both parental species, encouraged other researchers to try other species from different genera and tribes. As presented in Figure 4.4 and Table 4.4, intertribal combinations between species derived from five different tribes have been produced. In all the experiments reported, hybrid or partial hybrid plants have been obtained according to molecular m a r k e r analysis of the regenerated plant material. However, evaluations of chromosome n u m b e r s , isoenzyme content, and RFLPpatterns revealed that a higher degree of asymmetric hybrids were obtained among the intertribal hybrids than among the intrageneric and intergeneric hybrids. Surprisingly, no differences in the frequency of hybrid fusion products and hybrid shoots were recorded, when comparing the intertribal hybrids with inter or intrageneric hybrid. Only after attempts to culture and establish the hybrid material in the greenhouse was a clear difference noted. The intertribal combinations were, in general, more difficult to root and culture to m a t u r e plants outside in vitro conditions (Fahleson et al., 1994a). These difficulties were especially pronounced in fusions between B. n a p u s and B a r b a r e a vulgaris, which, in spite of leading to hybrid plants that could be cultured in vitro, never resulted in plants that could grow under ordinary greenhouse conditions. According to Oikairinen and Ry6ppy (1992), fertile plants were obtained after fusing B. rapa with B a r b a r e a vulgaris. However, clear evidence that these plants were real hybrids has not yet been reported. The problem of establishing somatic hybrids between species from different tribes was also encountered by other researchers. Hybrids between B. n a p u s (+) A. thaliana (Bauer-Weston et al. 1993) and between B. n a p u s (+) Lesquerella f e n d l e r i (Skarzinskaya et al., 1996) were difficult to root and culture in the greenhouse. Similar problems were observed in the hybrids produced between B. rapa and A. thaliana by Gleba and Hoffmann (1979, 1980). Success in obtaining hybrids that could be rooted and established in soil in the greenhouse was increased by partially eliminating DNA from the wild species using irradiation (Bauer-Weston et al., 1993; Skarzhinskaya et al., 1996). Furthermore, the irradiation improved fertility of the hybrid plants. For example, all L. fendleri (+) B. n a p u s hybrids obtained from symmetric fusions were self-sterile, while 38% of the asymmetric hybrids could be selfed (Skarzinskaya et al., 1996). A clear significant positive correlation between degree of a s y m m e t r y and seed set after selfing was obtained in asymmetric hybrids produced between B. n a p u s (+) A. thaliana (Forsberg et at., 1998b). Even though no positive effect of irradiation on fertility was found in hybrids between Thlaspi perfoliatum (+) B. napus, it improved the efficiency of hybrid production (Fahleson et al., 1994a). It is also interesting to note that some of the somatic hybrids produced between B. n a p u s (+) A. thaliana developed to fully differentiated plants that could be cultured in the greenhouse as well as set seeds even after selfing (Forsberg et al., 1994). The reasons for these
121
Tribe)
Lepideae ,
Family
Brassicaceae
Tribe
Sisymbrieae Ara~is thaliana
Brassiceae
Arabideae
Figure 4.4 Intertribal somatic hybrids within the family Brassicaceae. Hybrids are illustrated by the grey circles and the different species combined are marked with arrows.
Table 4.4 Intertribal somatic hybrids produced within the family Brassicaceae Somatic hybrid Parent A (tribe Brassiceae) Parent B
Tribe
Brassica rapa
Arabidopsis thaliana
Brassica napus
Trait of interest
Fertility
Reference
Sisymbrieae
ND
ND
Gleba and H o h a n n 1979; I980
Arabidopsis thaliana
Sisymbrieae
acetolactate-synthase
MS
Bauer-Weston et al. 1993
Brassica napus
Arabidopsis thaliana
Sisymbrieae
Phoma lingamR
F
Forsberg et al. 1994
Brassica carinata
Camelina sativa
Sisymbrieae
Alternaria brassiceaeR
ND
Narasimhulu et al. 1994
Brassica rapa
Barbarea stricta
Arabideae
cold tolerance
MS
Oikarinen and Ryoppy 1992
Brassica rapa
Barbarea vulgaris
Arabideae
cold tolerance
MS
Oikarinen and Ryoppy 1992
Brassica napus
Barbarea vulgaris
Arabideae
cold tolerance
S
Fahleson ei al. 1994b
Brassica napus
Thlaspi perfoliatum
Lepideae
nervonic acid
MS
Fahleson ei al. 1994a
Brassica napus
Lesquerella fendleri
Drabeae
lesquerolic acid
F
Skarzhinskaya et al. 1996
F = fertile,
MS = male sterility,
ND = not determined,
S = sterile
123 differences are unclear, but could be the result of the genotypes combined, as well as differences in the fusion and culture conditions.
Modification of fatty acid c o m p o s i t i o n by intertribal hybridization The practical importance of utilizing somatic hybridization between species from different tribes would be to modify the composition of the fatty acids in rapeseed. Several wild and distantly related species within the Brassicaceae exhibit fatty acids in their seeds which would be valuable to utilize for production of technical oils and replace the use of mineral oil. Thus, efforts to modify the fatty acid composition in rapeseed in order to obtain high enough quantities to be of economic importance is of interest to investigate. With that purpose in mind Fahleson et al. (1994a) performed hybridization experiments between rapeseed and Thlaspi perfoliatum. Seeds of T. perfoliatum have high levels of nervonic acid (C24:1), which is an oil of value as a lubricant. Some of the somatic hybrids were able to set seeds when backcrossed with rapeseed pollen. Seeds from the backcrossed progeny had an increased level of nervonic acid compared to rapeseed, reaching 4.9%. However, this content is still m u c h lower than the 20% level in T. perfoliaturn. Nevertheless, the results from these investigations clearly show that gene(s) coding for synthesis of nervonic acid have been transferred via protoplast fusion and are expressed during seed development. The relatively low level of nervonic acid in hybrid plants indicates that additional genes may be responsible for the elongation of C22 to C24 fatty acids, or that expression of the genes is modified in the hybrid material. The somatic hybrids produced between Lesquerella fendleri (+) B. napus were also made with the aim to tranfer genes coding for valuable fatty acids {Skarzinskaya et al., 1996). In Lesquerella the presence of high levels of hyd r o x y - u n s a t u r a t e d fatty acids has been recorded (Muuse et al., 1992). Since castor oil is the main source for hydroxylated derivates of oleic acid (C 18:1OH), breeders have sought other crops containing hydroxy fatty acids which are possible to cultivate in temperate regions. Actually, several attempts to domesticate L. fendleri have been made. Another approach would be to identify and transfer the genes coding for the elongation and hydroxilation steps into rapeseed, using protoplast fusion. Progeny from somatic hybrids obtained between L. fendleri and rapeseed have been investigated for their fatty acid composition (Schr6der-Pontoppidan et al., 1998) and material producing high levels (64%) of very long chain fatty acids (VLCFA) have been obtained.
Limited gene transfer via protoplast
fusion
Induced a s y m m e t r y in combination with a selection pressure To facilitate the transfer of specific traits after somatic hybridization, the use of a selection pressure for the trait of interest is a suitable way to succeed. In the case of improving resistance to Phoma lingam in B. napus a che-
124 mically characterized toxin, sirodesmin, isolated from culture filtrates of in vitro fungal cultures, was u s e d as a selective agent. Analysis of resistant and susceptible Brassica species revealed the selective properties of the toxin (Sj6din et al., 1988). To promote transfer of the resistance trait, donor materials of r e s i s t a n t B. nigra, B. juncea or B. carinata were irradiated prior to fusion (Sj6din and Glimelius, 1989b). The small cell colonies developed from the fusion p r o d u c t s were exposed to concentrations of the toxin in which only the r es is tan t het er okar yon cells could divide and differentiate into plants. Most of the hybrid pl ant s exhibited resistance to Phoma lingam, indicating t h at transfer of the resistance genes had occurred. According to chromosome analysis, isoenzyme a n d RFLP patterns, parts of the alien donor genome were p r e s en t in the material (Sj6din and Glimelius, 1989b). By backcrossing the asymmetric hybrids to rapeseed and screening for resistance to pycnospores from Phoma lingam, resistant lines have been selected exhibiting both cotyledon a n d adult leaf resistance (Wahlberg and Dixelius, 1997). These lines have been investigated in greenhouse and field conditions and represent valuable material both for studying the molecular regulation of P. lingain resistance and for breeding resistant rapeseed for agricultural use.
Selection of desired traits by using marker genes Another a p p r o a c h to select for desirable traits in asymmetric hybrids is the utilization of selectable m a r k e r genes t hat have been transferred to the donor species via transformation. This approach was first utilized by MUller and Schieder (1987) who showed that m a r k e r genes introduced by Agrobacterium tumefaciens t r a n s f o r m a t i o n were transferred from protoplasts of an inactivated donor species into recipient protoplasts. Besides the T-DNA, other genetic material of the donor genome was also transferred. This approach h a s later been u s e d by Sacristan et al. (1989) and GerdemannKn6rck et al. (1994, 1995). As donor material in the fusion experiments they u s ed a B. nigra line t h a t was resistant to Phoma lingam and Plasmodiophora brassiceae (Sacristan et al., 1989). This line also contained the gene for hygromycin p h o s p h o t r a n s f e r a s e (HPT), which was inserted into the genome by t r a n s f o r matio n with Agrobacterium tumefaciens. By screening for tolerance to hygromycin, asymmetric hybrids could be selected after fusion of irradiated B. nigra lines with B. napus. Besides presence of the m a r k e r gene in the hybrids, the genes coding for resistance to Plasmodiophora brassicae and Phoma lingam were also present, even t hough the genes were not coupled. However, to be certain to transfer the desired traits of agronomic importance, the m a r k e r gene and agronomically valuable genes have to be closely linked.
Cytological investigations using in situ hybridization Somatic hybridization between distantly related species is mainly performed to obtain a somatic hybrid which can serve as a bridge between the
125 alien gene d o n o r and the cultivated crop. Thus, it is of interest to confirm the transfer by cytological investigations, since the desired result is transfer and stable inheritance of specific genes. The combination of two alien chromosome sets in the same cell, obtained after protoplast fusion, might result in the production of a new species, like R a p h a n o b r a s s i c a (Olsson and Ellerstr6m, 1980), or in partial hybrids in which c h r o m o s o m e s and traits of interest are either p r e s e n t or eliminated (Glimelius et al., 1991). To follow the chromosome constitution and inheritance of specific c h r o m o s o m e s after somatic hybridization, genetic studies utilizing in situ hybridization have been performed on two different somatic hybrid c o m b i n a t i o n s in our laboratory. Progeny from the intergeneric hybrids between Eruca sativa (+) B. n a p u s (Fahleson et al., 1997), a n d the hybrids between Lesquerella f e n d l e r i (+) B. n a p u s (Skarzinskaya et al., 1998) have been studied. To confirm the presence of alien c h r o m o s o m e s , species specific repetitive DNA sequences and total DNA have been u s e d as probes for genomic in situ hybridization (GISH). In addition, the c h r o m o s o m e constitution was analysed by morphological studies. GISH analysis of B. n a p u s c h r o m o s o m e s revealed t h a t total DNA from B. n a p u s hybridized preferentially to the centromeric regions (Fahleson et al., 1997). Other species investigated with the GISH technique have shown more uniform labelling of c h r o m o s o m e s (Parokonny et al., 1992; A n a m t h a w a t - J 6 n son et al., 1995; Hoan et al., 1989). The difference might be due to the generally low c o n t e n t of dispersed repeats and high cont ent of t a n d e m l y organized centromeric repeats found in B r a s s i c a species. In contrast, GISH analysis of E. s a t i v a (Fahleson et al., 1997) a n d L. f e n d l e r i c h r o m o s o m e spreads revealed uniform labelling of all c h r o m o s o m e s . Thus, we were able to analyse the c h r o m o s o m e constitution in some of the hybrids a n d their progeny. From the studies of progeny of the E. sativa (+) B. n a p u s hybridization we found the presence of one or two extra E. sativa c h r o m o s o m e s . These plants m ost likely r e p r e s e n t addition lines in which the genetic material from E. sativa will probably not be stably inherited. In contrast, in the L. f e n d l e r i (+) B. n a p u s hybrids, we were able to confirm the presence of c h r o m o s o m e s made up of c h r o m o s o m e s egm ent s from both parents, t hus, constituting recombined a n d t r a n s l o c a t e d chromosomes. In the L. fendleri (+) B. n a p u s results, asymmetric hybrids were produced after irradiation of the L. fendleri genome, while the E. sativa (+) B. n a p u s hybrids were produced w i t hout irradiation. These results indicate t h a t it might be beneficial to induce chromosome fragmentation by irradiation in order to introgress alien genetic material into the acceptor genome. Whether the translocated c h r o m o s o m e s will persist, achieving stable introgression of alien DNA r e m a i n s to be investigated. Another m e t h o d to determine w h e t h e r introgression h a s t aken place in somatic hybrids is to compare their RFLP p a t t e r n s with the p a t t e r n s found in m a p p e d genomes s u c h as B. n a p u s , B. nigra, B. j u n c e a and A. thaliana. We have b e g u n a s t u d y in our laboratory of the asym m et ri c B. n a p u s (+) A. thaliana somatic hybrids (Forsberg a n d Glimelius, 1995), and of the B. nap u s (+) B. nigra, B. n a p u s (+) B. carinata, and B. n a p u s (+) B. j u n c e a hybrids
126 (Sj6din an d Glimelius, 1989b). The pur pose of the analysis is to follow the inheritance of the alien B r a s s i c a and A. t h a l i a n a DNA in the somatic hybrids. An additional p u r p o s e in the study involving the B. n a p u s (+) A. t h a l i a n a hybrids is to investigate w het her different p r e t r e a t m e n t s of the donor material with X-ray, UV a n d restriction enzymes will result in specific breakpoints in the genome, and increase transfer of specific c h r o m o s o m e fragments. The results from these investigations have clearly shown t h a t analyses of RFLPm a r k e r s spread over the c h r o m o s o m e s are very suitable for studying presence of alien DNA and c h r o m o s o m e segments in the somatic hybrids (Forsberg et al., 1998a). The investigations revealed t h a t U V - and X - irradiation proved to be efficient m e a n s of inducing asymmetry, while the treatment with restriction enzymes did not result in any significant elimination of DNA from the donor protoplasts. Furthermore, besides fragmentation and elimination of donor c h r o m o s o m e s intergenomic translocations were most probably obtained according to presence of RFLP-band deviating from those in the A. t h a l i a n a p a r e n t (Forsberg et al., 1998b).
The utilization of protoplast fusion to modify the cytoplasm Sexual hybridization prevents the mixing and exchange of cytoplasmic genetic material, since the chloroplast and mitochondrial genomes are generally maternally inherited. In contrast, somatic hybridization allows mixing and exchange, leading to unique combinations of organelles and even to organelles with recombined genetic material (Nagy et al., 1981; Boeshore et al., 1983; Pelletier, 1986). Since plant organelles encode several traits of agronomic importance including CMS, herbicide resistance, nectar production, resistance to fungal toxins and cold tolerance, m a n i p u l a t i o n of the cytoplasmic genomes is of interest for plant breeding purposes.
Production of cybrids Fusion of protoplasts of different parental species always results in a mixing of the cyt opl as m s into a single cell. However, nucl ear fusion does not always follow cell fusion, so t hat cells may form with the nucl eus from only one p a r e n t in a hybrid cytoplasm. Such a cell is called a cybrid. A more successful way to obtain cybrids is to eliminate the n u c l e u s from one of the fusion partners. Several m e t h o d s have been applied with that purpose. Zelcer et al. (1978) u s e d X-rays to destroy the nuclei of the protoplast from one parent. The irradiated nuclei are unable to divide b u t the cell can still contribute organelles to the fusion product. A combination of X-ray treatm e n t of one p a r t n e r with t r e a t m e n t of the other fusion partner with the metabolic inhibitor, IOA, increases the possibility t h a t the nucleus from the IOA pretreated p a r e n t will be combined with the organelles from the irradiated parent. An even more sophisticated way to obtain cybrids is to enucleate the protoplasts from one fusion par t ner before fusion, for example, by centri-
127 fugation a n d / o r chemical treatment (Wallin et al., 1978; L6rz, 1985), or to enrich for spontaneously formed or plasmolytically induced cytoplasts, i.e., protoplasts without nuclei (Sundberg and Glimelius, 1991 b).
Organelle segregation and recombination after protoplast fusion. From the initial mixture of organelles obtained by protoplast fusion, segregation and sorting out of the organelles will take place during cell division and plant differentiation. Usually the chloroplasts sort out during subsequent mitoses, resulting in an establishment of one or the other parental type (Pelletier 1986). In rare cases a mixture of the two chloroplast types has been found in the hybrid plants and their progeny. It has not been confirmed that this mixture will persist in s u b s e q u e n t generations. Even more rare is recombination between combined chloroplast genomes. Nevertheless, Medgeyesy et al., (1985) demonstrated, by using two Nicotiana m u t a n t lines, that recombination had taken place after fusion and selection for the traits coded by m a r k e r genes present in the chloroplast genomes in the m u t a n t lines. In the investigation of different cybrids and hybrids produced between species in the family of B r a s s i c a c e a e it is difficult to draw conclusions and generalize about factors influencing segregation of chloroplasts. However, in a systematic investigation performed by Sundberg and Glimelius (1991a), the influence of genetic divergence and ploidy level on chloroplast segregation was studied. In this investigation the hybrids were produced with uniform and reproducible methods, with no pretreatment of the cells or directed selection. This study was extended by investigating hybrids from B. n a p u s (+) T. perfoliatum (Fahleson et al., 1994a), B. n a p u s (+) A. thaliana (Forsberg et al., 1994) and B. n a p u s (+) L. f e n d l e r i (Skarzhinskaya et al., 1996). The resuits are reported in Table 4.5. The methods to produce and enrich the fusion products, as well as culture and regenerate the plants were the same in all these studies. Chloroplasts from rapeseed were favored in almost all of the somatic hybrids. The most r a n d o m segregation of chloroplasts appeared in fusions between B. oleracea (+) B. rapa. These species represent closely related species, and the hybrid nuclei contained the complete nuclear genome of both parents. In all the other combinations the nuclear genome of the hybrids was dominated by the B. n a p u s genome, since a varying n u m b e r of chromosomes from the diploid species B. rapa, B. oleracea, B. nigra, R. sativus, E. sativa, T. perfoliatum, A. thaliana and L. f e n d l e r i had been preferentially eliminated. The biased segregation might be due to genetic divergence between the nuclear genomes of the species resulting in chromosome elimination of one species and t h u s incompatability reactions between nuclei and chloroplasts. The bias towards B. n a p u s chloroplasts could also reflect ploidy level differences between the species combined. A higher ploidy level of a species usually leads to larger n u m b e r s of chloroplasts per cell (Butterfass, 1989). Thus, the amphidiploid rapeseed protoplasts might provide more chloroplasts to the fusion products t h a n the protoplasts from the diploid
Table 4.5 Organelle composition in somatic hybrids produced between different species within the family of Brassicaceae Hybrid combination
No of hybrids
Parent B
Parent A
Frequency (YO) of hybrids with chloroplasts mitochondria from Parent A Cp Mt
Parent B CP Mt
Mix recom. Cp Mt
References
Intrageneric hybrids
B. napus"
(+)
B. oleracea
B. rapa B. napus" B. napus" B. napus"
(+) (+) (+) (+)
B. oleracea B. nigra B. juncea (tour) B. juncea (rap)
58
72
ND
38
ND
0
ND
II
45 92 ND 100
10
0 22 ND
55 8 ND 0
0 5 17 ND
0 0 ND 0
90 95 61 ND
91 88
33 56
9 12
8 0
0 0
59 44
94
50 57 26
6 0 21
0 0 9
0 0
50 43 65
24
18 6
Sundberg and Glimelius 1991a
Intergeneric hybrids
B. napus" B. napus"
(+) (+)
R. sativus (Ogura) E. sativa
12
A . thaliana T. perfoliatum L. fendleri
16 7 34
17
Intertribal hybrids B. napus"
B. napus" B. napus"
a
(+) (+) (+)
The B. napus cultivar contained B.rapa cytoplasm,
100
16
ND = not determined
3
Forsberg et a/. 1994 Fahleson et a[. 1994a Skarzhinskaya et al. 1996
129 species. In a n o t h e r s t udy performed by S u n d b e r g et al. (1991) the effects of cell type on chloroplast segregation was investigated. No significant correlation between the type of tissue u s e d for isolating protoplasts and segregation was found. However, even t h o u g h the segregation and sorting out of chloroplasts is u s u a l l y biased the r e a s o n s for this n o n - r a n d o m segregation have not been elucidated. This indicates t hat it is very difficult to control which chloroplast type will be established in the hybrid plants. The fate of the mitochondrial genome after protoplast fusion differs from t h at of the chloroplast. Besides segregation and sorting out t h a t takes place during cell division and development, the heteroplasmic state often results in intergenomic recombination between the mitochondrial genomes. The hybrid mitochondrial genome contains DNA fragments characteristic of both parents, as well as novel and u n i q u e fragments not found in the parental mt DNA (Beillard et al., 1979). Segregation of the m i t ochondri a in the somatic hybrids produced in our laboratory was biased in all hybrid c o m b i n a t i o n s (Table 4.5) an d favored the m i t ochondr i a from rapeseed (Landgren and Glimelius, 1994). This c o n t r a s t s to the segregation of chloroplasts, which was generally more r a n d o m when closely related species were hybridized. This bias to rapeseed (B. rapa) m i t o c h o n d r i a was proposed to result from i nherent mitochondrial d e t e r m i n a n t s since no clear effects of differences in genetic divergence or ploidy level were found on mitochondrial segregation (Landgren and Glimelius, 1994; Landgren et al., 1994). As for the chloroplast segregation the cell type u s e d for protoplast isolation did not influence the segregation of m i t o c h o n d r i a (Landgren et al., 1994).
The effect of p r e t r e a t m e n t of protoplasts on organelle segregation Since the organelles encode traits of agricultural importance, scientists desire to control the tranfer of these traits by somatic hybridization. Therefore, several studies have been u n d e r t a k e n to influence the sorting out and segregation of the organelles after fusion. These investigations have focused on the effects of IOA t r e a t m e n t combined with irradiation. The m o s t substantial studies on B r a s s i c a materials have been performed by Morgan and Maliga (1987) a n d Waiters and Earle (1993). In these studies, protoplast fusions were induced between a male-fertile and a CMS line of B. n a p u s (Morgan and Maliga, 1987) and a male-fertile a n d CMS line of B. oleracea (Waiters a n d Earle, 1993). In the latter investigation protoplasts isolated from different tissues were fused with protoplasts subjected to different pret r e a t m e n t s , in a variety of combinations. The results from both investigations, in which a large n u m b e r of calli was analysed, revealed t h a t segregation of chloroplasts was biased in favor of the fertile parent. According to Morgan an d Maliga (1987) segregation of chloroplasts was complete after 19 to 22 cell divisions, so t h a t no calli were found to have a mixture of chloroplasts. On the contrary, Waiters a n d Earle (1993) found both types of chloroplasts in calli, indicating a mixed cytoplasm. It c a n n o t be excluded, how-
Table 4.6 A presentation of selected cybrids with the desired features produced via protoplast fusion within the Brassicaceae family including their organelle composition and modified features. ~~~
N. of selected
~
Nuclear donor
Cytoplasm donor
Cytoplasmic trait
plants/ regenerated
Organelle composition Cp Mt
B. napus
B. napus CMS
Ogura-CMS AtrazinR
71131
Bn
Ogu-rec
4
B. napus (cam) atrR
B. napus
1/85
Bc
Ogu-rec
1
B. napus (cam)
B. napus CMS
16/36
Pol Pol Bc Bc
Pol Bc Bc Pol
21 5 10
Ogu
ow
2
Bn
ogu
2 2
B. napus
B. napus
1987
MF
CMS
B. napus
B. napus
MF
CMS
B.rapa ATR~
B. oleracea
MF
CMS
Polima-CMS
Ogura-CMS
411
Number of plants
Reference Pelletier et al. 1983 Chetrit et al. 1985
Barsby et al. 1987
0
Ogura-CMS
2/87
Bn
OP
AtrazinR Ogura-CMS
114 10134
Bc
Ogu-rec
1 10
Morgan and Maliga
Jar1 and Bornman 1988, Jar1 et al. 1989
Robertson et al. 1987 Jourdan et al. 1989 Temple et al. 1992
B. n a p
B. napus
ATR
AtrazinR Polima-CMS
11261
Bn
Pol-rec
1
Chuong el al. 1988
CMS
B. napus
R. sativus
Kosena-CMS
10117
ND
Kos-res
1
Sakai and Imamura
ND ND
rec Bn
3 6
Bn
Ogu-rec
3
Kao et al. 1992
Br Br
2 1
Earle and Dickson 1994
rec
1990
CMS B. oleracea
B. napus
Ogura-CMS
419
CMS B. oleracea CMS
B. rapa atrR
:Y-CMS3/62
e W
0
Ogura-CMS
B. oleracea Dicksson 1994 MF
B. oleraca
B. napus MF amR
B. oleracea CMS
Ogura-CMS
I ix
Bo
Ogu
1
B. oleracea
B. napus CMS
Polima-CMS
313
ND
rec
3
Wang et al. 1995a
B. juncea MF
B. napus CMS
Ogura-CMS
41123
Bj
rec
4
Kirti el al. 1995a
B. juncea MF
Trachvsfoma ballii
CMS
1/10
rec(?)
rec
1
Kirti et al. 1995b
313
Bo
Ogu 3 Earle and
CMS
MF B. napus (cam) MF
B. tournefortii MF
Tour-CMS
611674
Bc
Bt
4
Stiewe and Robb. 1994
B. napus MF
B. tournefortii
Tour-CMS
7/25
Bt Bn Bt
Bt Bt rec
4
Liu et al. 1996
1
2
B. oleracea Ppt
B. juncea HygR CMS MF
Tour-CMS
B. rapa MF atrR
B. oleracea CMS
Ogura-CMS
2 1/53
Br
Ogu
21
B. oleracea
B . rapa
Tour-CMS
17141
Bo Bo Bo Bt Bt
Bo Bt rec Bo Bt
1 1 1
CMS
39/78
Bo Bo
Bt rec
25 14
Arumugam et al. 1996
Heath and Earle 1996b Cardi and Earle 1997
0
6
atrR= Atrazin resistant chloroplasts, Ppt'= Phosphinotricin resistance, Bn = B. napus, Br = B. rapa, Bc = B. rapa subsp. campestris, ole-racea, Bj = B. juncea, Bt = B. tournefortii, rec = recombined mitochondria1 DNA, ND = not determined.
BO= B.
132 ever, t h a t the calli were chimeric. They did not find a n y correlation to age or size of the callus. Nevertheless, the r e g e n e r a t e d p l a n t s c o n t a i n e d either the Brassica or the R a p h a n u s c h l o r o p l a s t s revealing t h a t the segregation a n d sorting o u t p r o c e s s e s were complete in the differentiated p l a n t a n d can occur both d u r i n g the first cell divisions a n d formation of the callus as well as during the early s t a g e s of differentiation of a shoot p r i m o r d i u m . Since, neither the p r o t o p l a s t source, cell type, nor the p r e t r e a t m e n t by itself affected chlor o p l a s t segregation the b i a s e d segregation a g a i n s t the R a p h a n u s chloroplasts w a s s u g g e s t e d as a n effect of the n u c l e a r - p l a s t i d incompatibility. Analysis of the m i t o c h o n d r i a l composition in the calli revealed t h a t the mtDNA was r e c o m b i n a n t a n d t h a t the segregation was t o w a r d s the Raphan u s m i t o c h o n d r i a . F r o m the s t u d i e s where the effects of cell type on segregation w a s investigated in correlation with p r e t r e a t m e n t of the protoplasts with IOA it w a s found t h a t the p r e t r e a t m e n t of hypocotyl p r o t o p l a s t s resulted in a r e d u c e d m i t o c h o n d r i a l c o n t r i b u t i o n to the fusion products. This r e s u l t w a s also found by H e a t h a n d Earle (1996b) a n d Liu et al. (1996). However, in the e x p e r i m e n t s performed by O z m i n k o w s k i a n d J o u r d a n (1994b) utilizing m e s o p h y l l p r o t o p l a s t s from both p a r e n t s in the fusions, biased segregation of m i t o c h o n d r i a t o w a r d s the IOA t r e a t e d material w a s not found. The c o n c l u s i o n s from t h e s e investigations are t h a t chloroplasts a n d m i t o c h o n d r i a segregate, after fusion a n d r e g e n e r a t i o n of calli a n d plants, in a n i n d e p e n d e n t , b u t biased way. F u r t h e r m o r e , the segregation of chlorop l a s t s was not affected by IOA p r e t r e a t m e n t . In the case of mitochondria, the r e s u l t s s u g g e s t t h a t IOA h a s the effect of c a u s i n g a r e d u c t i o n of the mitoc h o n d r i a from the p r e t r e a t e d hypocotyl p r o t o p l a s t s in the hybrids. The generality a n d significance of t h e s e r e s u l t s r e m a i n to be proven, however. With r e s p e c t to irradiation of the p r o t o p l a s t s before fusion, no significant effects on segregation a n d e s t a b l i s h m e n t of the organelles have been recorded.
M o d i f i c a t i o n o f c y t o p l a s m i c traits via p r o t o p l a s t fusion Cytoplasmic male sterility (CMS) Protoplast fusion provides a n excellent tool to create novel combinations of organelles, w h i c h modify cytoplasmic traits. In spite of the rapidity with which t r a n s f o r m a t i o n technology h a s been developed, the practical a n d most suitable way to genetically modify the organelle c o m b i n a t i o n s a n d organellar DNA so far is to utilize p r o t o p l a s t fusion. Unique c o m b i n a t i o n s of cytoplasmic traits m a y arise as a r e s u l t of novel c o m b i n a t i o n s of chloroplasts a n d m i t o c h o n d r i a a n d of r e c o m b i n a t i o n s of organellar DNA (Pelletier, 1986). An elegant d e m o n s t r a t i o n of s u c h a modification is the i m p r o v e m e n t of the CMS B. n a p u s lines c o n t a i n i n g the "Ogura" c y t o p l a s m from R a p h a n u s sativus. Even t h o u g h this male sterility h a s been found to be highly stable in B. n a p u s g e n o t y p e s it suffers from chlorophyll deficiency, which is expressed especially at low t e m p e r a t u r e s . Moreover, the male-sterile plants have
133 low nectar production, which causes the flowers to be less attractive to honey bees (Pelletier et al., 1988). By producing hybrids between the B. n a p u s cultivar and a rapeseed line with the "Ogura" cytoplasm, an exchange of chloroplasts was obtained in some lines resulting in normal chlorophyll production, while retaining the male-sterile trait. Furthermore, a b u n d a n t nectar production was recovered in some cybrids (Pelletier et al., 1986). By this procedure a most valuable material was obtained, which has been used for further breeding to improve existing CMS cultivars (Renard et al., 1992). The material has also been very valuable for molecular analysis of malesterility (Bonhomme et al., 1992; Grelon et al., 1994). Similar experiments intended to restore chlorophyll levels and nectar production in cytoplasmic male-sterile B. n a p u s lines were carried out by Jarl and B o r n m a n (1988). These experiments have been followed by several other investigations, successfully transfering the '~Ogura" mitochondria coding for the cytoplasmic male-sterility and replacing the "Ogura" chloroplasts with the chloroplasts from the nuclear donor line in, for example, different cultivars of B. napus, B. j u n c e a and B. oleracea (for references see Table 4.6). Other cytoplasms known to code for cytoplasmic male sterility in Brassica species are the "Polima" (Fang and Mc Vetby, 1989) and '~CMS juncea" ("Anand", "Tour") (Rawat and Anand, 1979). Both these cytoplasms have been transferred via protoplast fusions to a nuclear acceptor line for which the CMS trait was desired (for references see Table 4.6). Also, in these studies material was produced which is utilized for commercial production of CMS cultivars. The '~CMS juncea" cytoplasm is reported to be derived from B. toumefortii (Pradhan et al., 1991; Szasz et al., 1991). To investigate whether the '~CMS juncea" was of alloplasmic origin and whether cytoplasm from fertile lines of B. toumefortii could induce male sterility in B. n a p u s , asymmetric fusion experiments between B. n a p u s and B. toumefortii have been carried out (Stiewe and R6bbelen, 1994: Liu et al., 1996). From these experiments lines of B. n a p u s were recovered, exhibiting the CMS trait and containing the B. toumefortii mitochondria, or mitochondria with recombined mt-DNA from the two parental genomes (Stiewe and R6bbelen, 1994; Liu et al., 1996). Phenotypically, flower morphology closely resembled the malesterile B. n a p u s plants obtained after sexual transfer of the cytoplasm of the original CMS B. j u n c e a line (Sodhi et al., 1994). Besides the male-sterile cybrids exhibiting floral abnormalities like narrow petals, swollen pistils, and abnormal or degenerated anthers, male-sterile cybrids with a more normal flower morphology was also obtained. In the cybrids displaying a more normal flower morphology the mt-DNA was recombined (Liu et al., 1996). A r u m u g a m et al., (1996) synthesized a hexaploid (AABBCC) somatic hybrid between B. j u n c e a (AABB), carrying the cytoplasm of B. toumefortii, and B. oleracea (CC), in order to produce a bridging material. From these fusions both male-sterile and fertile plants have been obtained with different compositions of organelles, including recombined mt-genomes. The hybrids are also represented by male-sterile lines that besides the male-sterility are free
134 from other floral abnormalities. These results are in agreement with the findings of Liu et al. (1996), indicating t h a t mt-DNA recombinations can rectify the a b n o r m a l floral features. Similarly, Cardi and Earle (1997) have produced a new CMS-B. oleracea by transferring the "CMS-tour" cytoplasm from B. rapa via protoplast fusion. A new alloplasmic CMS line of putative importance was produced via protoplast fusion between B. j u n c e a and T r a c h y s t o m a ballii (Kirti et al., 1992b). Backcrossing with a B. j u n c e a cultivar as the male parent, stable inheritance of the CMS trait was achieved (Kirti et al., 1995b). Molecular analysis revealed t h a t the CMS-line had r e c o m b i n a n t mitochondrial mt-DNA and suggested t h a t the cp-DNA was also recombinant, although more detailed investigations are required (Kirti et al., 1995b). Nevertheless, a new sterilityinducing cytoplasm was transferred to B. j u n c e a by protoplast fusion, which will be evaluated as a putative CMS-system for B. napus. A s y m m e t r i c h y b r i d s as a t o o l to t r a n s f e r n u c l e a r f e r t i l i t y r e s t o r e r g e n e s to CMS l i n e s
Even t h o u g h production of cybrids is performed mainly to create novel nuclear-cytoplasmic combinations, the nuclear genome can also be modified. If the cytoplasm donor cells are treated with ionizing radiations (X or gamma) prior to fusion, fragmentation of c h r o m o s o m e s is promoted, resulting in a limited transfer of a nuclear DNA (Dudits, 1987). This can be of practical importance aiming at producing cytoplasmic lines with nuclear restorer genes. Sakai a n d I m a m u r a (1990, 1992) have reported on the production of CMS lines of B. n a p u s obtained by introducing CMS from radish ( R a p h a n u s s a t i v u s cv. Kosena). This line differs from the R a p h a n u s lines containing the "Ogura" cytoplasm with respect to the CMS-inducing genes, and might therefore require different nuclear genes to restore the male sterility (Sakai et al., 1996). A stable CMS-line was used as a source of protoplasts to fuse with a restorer line of radish by the donor-recipient fusion m e t h o d (Sakai et al., 1996). Male-fertile lines were obtained from asymmetric hybrids, in spite of the presence of the CMS-inducing Kosena cytoplasm. Genetic analysis was performed on one of the lines, which gave progeny segregating to male-fertile and male-sterile plants after back-crossing. Several successive backcrosses to rapeseed were performed and the monogenic inheritance of fertility in the BC2S1 plants was interpreted to result from an integration of the restorer gene from radish in the nuclear genome of rapeseed. F u r t h e r studies will be expectedly performed to clarify the genomic organization and m ap the locus of the putatively integrated restorer gene in the acceptor rapeseed line. Similarly, in the studies performed by Liu et al. (1996) in which the alloplasmic CMS-lines of rapeseed were produced by introducing the mitochondria from B. tournefortii, the asymmetric hybrids segregated into fertile and male-sterile lines. The fertile lines are being u s e d as pollinators to establish w h eth er they can restore rapeseed lines containing the CMS-inducing cyto-
135 p l a s m of B. tournefortii. Besides, this m a t e r i a l is of clear i n t e r e s t for genetic a n d m o l e c u l a r s t u d i e s of C M S - i n d u c i n g a n d restoring genes. H e r b i c i d e r e s i s t a n c e or t o l e r a n c e
A n o t h e r trait of i n t e r e s t in b r e e d i n g of B r a s s i c a crops is triazine or a t r a zine tolerance. An atrazine t o l e r a n t B. rapa line w a s discovered in C a n a d a ( S o u z a - M a c h a d o et al., 1978) a n d explained by a m u t a t i o n in the c h l o r o p l a s t 32 KD QB protein (Hirschberg et al., 1984). Since b r o a d leaf w e e d s in c a n o l a fields are a s e r i o u s problem, a n extensive s e a r c h for h e r b i c i d e s to control t h e s e weeds h a s b e e n made. The c h l o r o p l a s t coded trait of a t r a z i n e tolerance c a n only be c o m b i n e d with the m i t o c h o n d r i a l coded CMS trait via p r o t o p l a s t fusion. In Table 4.6, different c o m b i n a t i o n s , bringing together a t r a z i n e toler a n t c h l o r o p l a s t s with CMS i n d u c i n g m i t o c h o n d r i a , are listed. As s h o w n in the table, a large n u m b e r of a t r a z i n e t o l e r a n t CMS lines have b e e n created. It m i g h t be a p p r o p r i a t e to a d d some c o m m e n t s a b o u t the trait of atrazine tolerance in this context. Triazine herbicides are not c o n s i d e r e d environm e n t a l l y a p p r o p r i a t e due to p r o b l e m s with the d e g r a d a t i o n of the herbicide. Their u s e will be limited in the future, a n d t h e y are a l r e a d y strictly regulated in several countries. F u r t h e r m o r e , a t r a z i n e t o l e r a n t c h l o r o p l a s t s in B. nap u s c a u s e a yield p e n a l t y (Reboud a n d Till-Bottraud, 1991) a l t h o u g h no yield p e n a l t y h a s b e e n found in B. oleracea vegetables (Christey a n d Earle, 1991). However, as d i s c u s s e d by Earle a n d Dickson (1994), a t r a z i n e r e s i s t a n t lines m i g h t be benefitial w h e n p l a n t i n g in soil with high levels of triazine residues. Conclusions
F r o m this overview of the l i t e r a t u r e r e g a r d i n g utilization of p r o t o p l a s t technology to improve the crops within the family of B r a s s i c a c e a e , it is clear t h a t s u b s t a n t i a l p r o g r e s s h a s been m a d e d u r i n g the last 15 years. Although the first r e p o r t s of p r o t o p l a s t isolation were from the b e g i n n i n g of the 1970's, it w a s not until the middle of the 80's t h a t real efforts were m a d e to utilize the p r o t o p l a s t technology to improve crops. It is obvious t h a t p r o t o p l a s t fusion c a n be u s e d as a m e t h o d to b y p a s s b a r r i e r s restricting s e x u a l hybridization of different species. Although h y b r i d p l a n t s c a n be o b t a i n e d in m o s t of the somatic hybridization e x p e r i m e n t s performed, low fertility severely restricts the likelihood of seed p r o d u c t i o n u p o n self-fertilization. However, seeds c a n u s u a l l y be o b t a i n e d by pollinating the h y b r i d s with pollen from one of the p a r e n t s . T h u s , the i m p o r t a n c e of the m e t h o d of somatic hybridization is not so m u c h to p r o d u c e a m p h i d i p l o i d s , c o m b i n i n g the complete g e n o m e s from the two species, as to utilize the somatic h y b r i d as a bridge for t r a n s f e r of desirable traits between the alien gene d o n o r a n d the crop. The i n h e r i t a n c e of certain desired traits, t r a n s f e r r e d to different B r a s sica crops, h a s b e e n followed in several investigations. In o r d e r to improve B r a s s i c a crops, traits like disease r e s i s t a n c e to different p a t h o g e n s , modifi-
136 cations of fatty acid composition, cytoplasmic male sterility, herbicide tolerance and repair of seedling chlorosis or sensitivity to cold have been modified and stably inherited over several consecutive generations of back-crosses. Thus, several lines have been derived from somatic hybrids, that are of great potential value for further breeding. However, more detailed investigations of the inheritance of the alien DNA carrying the desired genes and how to enhance the introgression of the alien DNA is also of importance to study. An introgression of DNA might occur via intergenomic translocations, recombination between homologous chromosomes, or even between nonhomologous chromosomes. Tools to detect intergenomic translocations have been developed; GISH and Prince (Koch et al., 1993) analyses will most probably become powerful techniques to elucidate configuration and constitution of chromosomes in the somatic hybrids. Besides these methods RFLP analysis of the linkage groups of the genomes in the somatic hybrids and the parents will most probably reveal whether recombinations a n d / o r translocations between the genomes have occurred. Of even greater importance would be the possibility to promote introgression of certain alien genes in a directed way. However, production of useful breeding material and new cultivars derived from somatic hybrids will be the final proof of the importance of the technique, as is the case for the improved Ogura-CMS-lines produced.
Acknowledgements. Thanks are due to all the research partners and students in my group performing the work with production of somatic hybrids and for discussions and suggestions of improvements of the manuscript. Special acknowledgements are devoted to Dr. E. Sundberg for allowing me to modify and utilize figures from her thesis. Thanks are also due to the Swedish Research Council for Forestry and Agricultural Research for support to this research.
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Biology of Brassica Coenospecies C. G6mez-Campo (Editor) 91999 Elsevier Science B.V. All rights reserved.
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Masao Watanabe (1) and Kokichi Hinata
(2)
(1) Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka 020-8550, Japan (2) Faculty of Agriculture, Tohoku University, Sendai 981-8555, Japan Self-incompatibility is an elaborated breeding system for securing outcrossing and m a x i m u m recombination in the angiosperms. It is classified into heteromorphic and homomorphic types with respect to flower morphology. The homomorphic type is comprised of gametophytic and sporophytic types according to the phase (gametophyte or sporophyte) of S gene expression. The self-incompatibility in the Brassicaceae is classified into the homomorphic sporophytic type. According to Bateman's survey (1955), out of 182 species in the Brassicaceae, 80 species express self-incompatibility. In Brassica species and its closest allies, 50 species were self-incompatible out of 57 species examined (Hinata et al., 1994). The self-incompatibility system has probably played an important role in the differentiation and adaptation of species in this group. A n u m b e r of economically important vegetables are included in the Brassiceae (Brassica oleracea, B. rapa (syn. B. campestris), Rap h a n u s sativus etc.) and hybrid variety seeds made by using self-incompatibility are highly evaluated. Recently, self-incompatibility-aided hybrid breeding has been conducted in oleiferous B. napus (Pink, 1993). Investigations on the mechanism of self vs. non-self recognition and of resulting pollen-tube rejection may greatly contribute to the understanding of reproduction in plants, as well as to the further development of breeding techniques. A few short recently published review articles on this subject (de Nettancourt, 1997; Nasrallah, 1997; Suzuki et al., 1997d; Charlesworth and Awadalla, 1998) may be useful for general understanding of this field.
Morphology and physiology The stigma of Brassicaceae belongs to the so-called "dry stigma" group and is covered with one layer of papilla cells on its surface. The papilla is a typical secretory cell, in which endoplasmic reticulum and Golgi a p p a r a t u s are developed (Kishi-Nishizawa et al., 1990). The cell wall of papilla cell is
150 composed of an inner cellulose layer and an outer cuticle layer, on which waxes are deposited. Yellow colored pollen grains are generally transferred by bees. They have three germ slits, and the exine is covered with a lipoidal coating, which is called a pollen kit, tryphine or pollen coat (Dickinson and Lewis, 1973; Roggen, 1974). The coating is believed to include some other substances that may provide the signal of pollen identity. In cross-pollination, pollen grains absorb water from papilla cells, resulting in germination, and the pollen tubes, penetrating the outer cuticle layer, grow in the cellulose layer toward the stylar tissue. In self-pollination, absorption of water and consequently germination are disturbed (reviewed in Dickinson, 1995). When ambient humidity is sufficiently high, the pollen grains can germinate and the pollen tubes penetrate the cuticle layer, but they cannot grow into the cellulose layer. Therefore, self-incompatibility in this family is considered to derive from an interaction between a pollen grain (or pollen tube) and the cell wall of papilla (Kanno and Hinata, 1969). Upon self-pollination, callose deposition is observable at the plasmalemma of papilla. The site is limited to the attached portion with pollen and pollen tubes. Callose is also recognizable at the tips of pollen tubes. It has not yet been determined whether callose deposition is a cause or a result of pollen tube arrest (reviewed in Hinata et al., 1993).
Classical g e n e t i c s and d o m i n a n c e relationships Bateman (1952, 1954, 1955) first proposed that self-incompatibility in
Iberis a m a r a was controlled by a sporophytic multiple allelic system at a single locus, S, and the general applicability of this proposal to B r a s s i c a
species was also pointed out. In this sense, self-incompatibility is equivalent to self vs. non-self recognition between S alleles. Since then, a number of studies have supported this scheme (Thompson, 1957; Sampson, 1964; Thompson and Taylor, 1966; Okazaki and Hinata, 1984; Nou et al., 1991, 1993a, b), although there are different opinions as to two-locus systems with multiple alleles (Verma et al., 1977; Lewis 1977) and the joint expression of both sporophytic and gametophytic systems (Zuberi and Lewis, 1988; Lewis et al., 1988). In the sporophytic system, the behavior of pollen tubes is determined by the phenotype of the sporophyte that produced the pollen. Therefore, the phenotype of the pollen and the stigma of heterozygous plants depends upon the outcome of complex dominant / recessive allelic interactions (Thompson and Taylor, 1966). Dominance relationships between S alleles have been investigated for several species in the family B r a s s i c a c e a e : I. a m a r a (Bateman, 1954, 1955), B. o l e r a c e a and R. s a t i v u s (Haruta, 1962), B. oleracea (Thompson and Taylor, 1966; Ockendon, 1975; Visser et al., 1982; Wallace, 1979), R. r a p h a n i s t r u m (Sampson, 1964, 1967), S i n a p i s a r v e n s i s (Stevens and Kay, 1989) and recently, in B. r a p a (Hatakeyama et al., 1998a). Among the cha-
151 racteristic features of d o m i n a n c e r e l a t i o n s h i p s in these species are that: (a) c o d o m i n a n t r e l a t i o n s h i p s occur more frequently t h a n d o m i n a n t / recessive ones; (b) d o m i n a n t / recessive r e l a t i o n s h i p s occur more frequently in the pollen t h a n in the stigma; (c) the d o m i n a n t / recessive r e l a t i o n s h i p s are not identical for S alleles between stigma a n d pollen; a n d (d) n o n - l i n e a r domin a n c e r e l a t i o n s h i p s are observed more frequently in the s t i g m a t h a n in the pollen. N u m b e r s of S alleles were e s t i m a t e d by several s t u d i e s (Sampson, 1967; O c k e n d o n , 1974, 1980; Ford a n d Kay, 1985, Stevens a n d Kay, 1988, 1989; Karron et al., 1990; Nou et al., 1991, 1993a, b). Generally speaking, a b o u t 30 or more S alleles have been found in a population. Nou et al., (1993a) e s t i m a t e d t h a t there are more t h a n one h u n d r e d S alleles in B. rapa t h r o u g h o u t the world.
The S-multigene family Since the discovery of SLG, several genes whose s e q u e n c e s are similar to S L G have been isolated a n d characterized. They are c o n s i d e r e d to form an S-multigene family. A brief e x p l a n a t i o n for t h e m in Table 5.1 m a y provide an overview. SLG
(S-locus glycoprotein)
Detection a n d protein c h e m i s t r y of S L G The p r e s e n c e of S-specific a n t i g e n s in s t i g m a w a s first detected by immunological m e t h o d s (Nasrallah a n d Wallace, 1967; H i n a t a et al., 1982). Isoelectric focusing (IEF) a n a l y s i s of s t i g m a proteins revealed t h a t the Sspecific glycoproteins h a d different pI values c o r r e s p o n d i n g to respective S alleles (Nishio a n d Hinata, 1977). The c o n t e n t of the c o r r e s p o n d i n g S-glycoproteins in the stigma of S-heterozygotes was a b o u t half t h a t of the stigma of S-homozygotes (Hinata a n d Nishio, 1978). These S-glycoproteins (SLGs) cosegregated with S alleles w i t h o u t exception (Hinata a n d Nishio, 1978; Nou et al., 1991, 1993a). The S-glycoproteins were p r o d u c e d in s t i g m a s a few days before flower a n t h e s i s a n d the expression was coincident with the expression of self-incompatibility (Nishio a n d Hinata, 1977). This line of evidence s u p p o r t e d the idea t h a t the S-glycoproteins were the p r o d u c t s of S genes a n d they were c o n s i d e r e d to be the m o s t likely s u b s t a n c e to be involved in self vs. non-self recognition of self-incompatibility. Three S L G s were isolated from the s t i g m a s of S 8, S 9 a n d S12-homozy gotes of B. r a p a (Takayama et al., 1987; Isogai et al., 1987). Ninety-eight percent of the a m i n o acid s e q u e n c e of SLG s (B.r.) w a s d e t e r m i n e d . The three SLGs (B.r.) (SLG s, S L G 9 a n d S L G lz) h a d two k i n d s (A type a n d B type) of Nlinked oligosaccharide c h a i n s in c o m m o n . The ratios of the A type to the B were very similar, a n d no discernible differences were found in the N-glycosidic c a r b o h y d r a t e c h a i n s a m o n g these three SLGs ( T a k a y a m a et al., 1989).
152
Table 5.1
Brief explanation
of t h e m e m b e r s
of S - m u l t i g e n e family.
SLG (S-locus glycoprotein)
A secreting glycoprotein in the m a t u r e papilla cells, co-segregates with S allele. This w a s called S-protein by Nasrallah a n d Wallace (1967), a n d Sglycoprotein by Nishio a n d Hinata (1977). This protein group is highly variable; each h a s different pI value c o r r e s p o n d i n g to respective S allele a n d possibly involved in the self vs. non-self recognition of self-incompatibility. The n u m b e r of s u p e r s c r i p t s denotes the protein or gene t h a t is isolated from the S homozygote having its n u m b e r . S alleles have been d e n o t e d by s u b s c r i p t s widely, b u t in this report they are s h o w n by s u p e r s c r i p t s to clear t h a t they are controlled by allelic genes.
SRK (S-receptor kinase) A t r a n s m e m b r a n e protein k i n a s e ( s e r i n e / t h r e o n i n e type), whose receptor d o m a i n is highly h o m o l o g o u s with SLG, existing in the m a t u r e papilla cells. The S L G a n d S R K of the same S allele are considered to play a recognition role as a haplotype. SLR1 (S-locus related gene 1)
This was first described as NS glycoprotein in the m a t u r e papilla cells by Isogai et al. (1988), a n d the cDNA cloned as SLR1 by Lalonde et al. (1989) is c o n s i d e r e d to c o r r e s p o n d to the NS glycoprotein. This protein is not so variable as SLG; only 4 types being found in B. rapa. It does not participate in the recognition reaction, so far as the d a t a are concerned. C h r o m o s o m a l location of this gene is i n d e p e n d e n t of S allele. This was cited as SRA by H i n a t a et al. (1993). S L R 2 (S-locus related ~ene 2)
This was cloned to be linked with SLR1 by Boyes et al. (1991). Its DNA s e q u e n c e is highly h o m o l o g o u s with the Class II SLG. This was cited as S R B by H i n a t a et al. (1993). S L R 3 (S-locus related gene 3)
This gene cloned by Cock et al. (1995) was not linked to the S locus. The putative S L R 3 protein lacks a few cysteine r e s i d u e s t h a t are conserved in the other proteins of the S-multigene family. The expression of this was found in petals, sepals, a n d vegetative apices, in addition to stigmas a n d anthers.
153 Therefore, the specificity is considered to be determined by the protein portions of SLGs, a l t hough there is a possibility t hat the variation of the N-glycosylation sites might have significance for S-specificity (Takayama et al., 1987; Isogai et al., 1987). SLC~ protein was produced by a transgenic tobacco cell line with the c o n s t r u c t encoding S L G driven by duplicated CaMV 35S promoter. The characteristics of the SLC~ protein produced by this transgenic tobacco were very similar to those from B r a s s i c a stigmas in molecular m a s s and pI value (Perl-Treves et al., 1993).
Cloning of S L G cDNA clones of S L G were isolated from eight S-homozygotes of B. olerac e a (Nasrallah et al., 1985, 1987; Lalonde et al., 1989; Trick and Flavell, 1989; Chen a n d Nasrallah, 1990; Scutt and Croy 1992; Delorme et al., 1995 a), seven S-homozygotes of B. r a p a (Yamakawa et al., 1994; W a t a n a b e et al., 1994; M a t s u s h i t a et al., 1996; H a t a k e y a m a et al., 1998b; H a t a k e y a m a et al., 1998c) and two S-homozygotes of B. n a p u s (Goring et al., 1992a, 1992b), which contained d o m i n a n t and recessive alleles. These different S L G s so far cloned were classified into two groups. The first group c o n t a i n s pollen-domin a n t SLGs (Class I SLG) and the second contains pollen-recessive S L G s (Class II SLG). The amino acid sequence homology of S L G s within each class is a b o u t 78-90%, but t h a t between classes is a b o u t 65%. Several genomic clones derived from d o m i n a n t S alleles of B. r a p a and B. o l e r a c e a had no intron (Nasrallah et al., 1988; Dwyer et al., 1991; Suzuki et al., 1995; Delorme et al., 1995a). Pollen-recessive S L G 2 (Class II SLG) of B. o l e r a c e a had a small intron. This gene produced two transcripts: a secreted glycoprotein a n d a m e m b r a n e - a n c h o r e d protein. Transcripts of the m e m b r a n e - a n c h o r e d type were not detected in the S L G s of the pol l en-dom i nant group (Tantikanj a n a et al., 1993). On the other hand, three genes of the Class II S L G in B. r a p a so far cloned contained an intron b u t encoded only secreted glycoproreins (Hatakeyama et al., 1998b). The m e m b r a n e - a n c h o r e d form of S L G is not necessary for the pollen-recessive n a t u r e in B r a s s i c a species. Each sequence contains twelve conserved cysteine residues at the carboxyl terminal. These residues may be involved in the e s t a b l i s h m e n t of the tertiary configuration of this protein (Isogai et al., 1987; Nasrallah et al., 1987). The p a r t s having relatively different amino acid sequences a m o n g S L G s are located in 180-200, 250-280 residues (Yamakawa et al., 1994). These parts are relatively highly hydrophilic and m ay be responsible for the specificity of S L G s (Isogai et al., 1987; Nasrallah et al., 1987). The sequence of EP1 (embryonic protein 1), whose cDNA was isolated from non-embryonic carrot s u s p e n s i o n cells, h a d a region of high homology with SLG. Beside sequence homology, EP1 s har ed other characteristics with SLGs: all were 5060 kDa in size, they contained several glycosylation sites in the N-terminal two-thirds of the protein, and the C-terminal domain was cysteine-rich (van
154 Engelen et al., 1993). In the plant kingdom, there seem to be several genes or DNA clones whose sequence is similar to SLG. Expression and localization of S L G The mRNA t r a n s c r i p t s and protein products of SLG were not detected in the early p h as e of stigma development, b u t were expressed in m a t u r e papilla cells of the stigma surface (Nasrallah et al., 1988; Mariac et al., 1992; Cappadocia et al., 1993). A very small a m o u n t of SLG protein has also been identified in the style of B. oleracea (Kleman-Mariac et al., 1995), although the papilla cell is the recognition site for self-incompatibility. Electron microscopic observation using a n t i - S L G a n t i s e r u m has revealed t h a t S L G a c c u m u l a t e s in the m a t u r e papilla cell wall, where inhibition of self-pollen tube development occurs (Kandasamy et al., 1989; Kishi-Nishizawa et al., 1990). SLG was secreted via the route t h a t involved the r u m e n of ER, Golgi bodies and the small vesicles (Kishi-Nishizawa et al., 1990; Kandas a m y et al., 1991). Promoter analysis of SLG A chimeric toxic gene consisting of the diphtheria toxin A chain gene fused to 3.65kb SLG 13 promoter was us ed to detect expression in transgenic A r a b i d o p s i s and B. n a p u s . In the transgenic A r a b i d o p s i s , the papilla cells were s t u n t e d and became biologically inactive. Anther development was also impaired by toxic gene expression. The combined defects of pistil and ant her rendered the t r a n s f o r m a n t s self-sterile. However, the t r a n s f o r m a n t s were cross-fertile with u n t r a n s f o r m e d plants: the viable pollen of ablated plants was rescued by the wild-type stigmas, and the ablated papilla cells allowed the growth of wild-type pollen (Thorsness et al., 1993). In the transgenic B. n a p u s , flower morphology was normal except for a b e r r a n t papilla cell develo p m e n t and partial pollen sterility. Papilla cells lost their ability to elongate, to synthesize cell-specific proteins, and to s u p p o r t pollen germination after self- and cross-pollination (Kandasamy et al., 1993). A r a b i d o p s i s was transformed with a chimeric gene consisting of the 3.65-kb promoter region of an SLG 13 (B.o.) fused to the reporter GUS (~-glucronidase) gene. GUS activity was found in a different pattern between stigm a s an d anthers. In stigmas, the time and distribution of GUS activity was similar to t h a t described for SLG gene expression in B r a s s i c a . In anthers, however, the expression of GUS activity was detected in the sporophytic t a p e t u m tissues at an earlier stage of flower development (Toriyama et al., 199 lb). Another transgenic plant, B. oleracea, with SLG 13 (B.o.)-GUS showed a slightly different expression p a t t e r n than the transgenic Arabidopsis. Histochemical and fluorometric a s s a y s revealed that, in addition to its primary site of expression in the stigmatic papilla, this gene was expressed in the t r a n s m i t t i n g tissue of the stigma, style and ovary in pistils. Furthermore,
155 in anthers, the SLG-promoter was active not only in the t a p e t u m cells, with sporophytic expression, but also in the haploid microspores (Sato et al., 1991). For analysis of the promoter region, tobacco was transformed with truncated versions of the S L G 13 (B.o) promoter fused to the GUS reporter gene. The promoter had a modular organization and consisted of separable DNA elements that independently specify the gene expression in pistil and pollen. A 196-bp region was sufficient to confer stigma and style specificity to the marker gene. Two distinct, but functionally r e d u n d a n t domains allowed specific expression of the gene in pollen. The functional domains identified within the S L G 13 promoter contained several sequence elements (Box I to Box V) that were highly conserved in different alleles of the S L G (Dzelzkalns et al., 1993). In Sg-homozygote of B. rapa, the promoter regions of S L G 9 and S R I ~ were completely identical for 1,407 bp u p s t r e a m from their respective initiaion codons. The five sequence elements were also found in these clones, although one conserved element (Box III) lacked 7 of 11 bp. The transgenic tobacco plants (Nicotiana t a b a c u m ) transformed with S L G 9 and S R I ~ separately expressed their transcripts in the pistil tissues. This indicates that box III is not always necessary for their expression in pistil (Suzuki et al., 1995, 1996, 1997a). Transformation of S L G In order to analyze the function of SLG, gain of function and loss of function would be powerful strategies. In a transgenic B. r a p a with antisense S L G driven by the S L G promoter, the transcripts of S L G and S R K decreased, and the transformant became self-compatible (Shiba et al., 1995). A similar p h e n o m e n o n was also observed in different materials, but the progeny recovered its self-incompatibility (Takasaki et al., 1998). These experiments clearly show that S L G a n d / o r S R K function in self / non-self recognition reaction. Another interesting point is whether the S-specificity of recipient plants can be changed by the introduction of the S L G gene. Transgenic B. o t e r a c e a with the SLC~ (B.r.) altered their pollen-stigma interaction and became fully self-compatible. Reciprocal pollinations between transgenic and u n t r a n s formed plants showed that the stigma reaction changed in one recipient strain, whereas the pollen reaction was altered in the others (Toriyama et al., 199 l a). Self-compatible B. n a p u s , which is an amphidiploid between B. r a p a and B. oleracea, was transformed with four different SLGs. In these transgenic plants, the newly produced SLGs had pI values and molecular weights similar to those of donor plants. However, the production of S L G in the transgenic B. n a p u s was lower in quantity t h a n that of donor self-incompatible plants, and introduction of the S L G to the B. n a p u s cv. Westar did not alter the self-incompatibility phenotype (Nishio et al., 1992).
156 Takasaki et al., (1998) transformed a self-incompatible B. rapa with SLC~ and S L G 9 t h a t were isolated from the same species. The expression of one S allele in the host plant was c o - s u p p r e s s e d by the introduced SLGs. However, this c o - s u p p r e s s i o n was not observed in the progeny. An S L G gene of self-incompatible B. r a p a u n d e r control of a tapetumspecific promoter was introduced into self-compatible B. n a p u s . A pollination test indicated t h a t the pollen of the transgenic B. n a p u s did not gain the self-incompatibility phenotype (Sasaki et al., 1998). Simple identification m e t h o d s of S allele by S L G in breedinR programs Identification of S genotypes was d e m o n s t r a t e d by polymerase chain reaction (PCR) of genomic S L G followed by restriction analysis. Primers homologous to the conserved regions near the 5' and 3' ends of the SLG coding sequence were u s e d to amplify the SLGs. The S genotypes of the plants t h u s determined agreed well with the results based on pollen tube growth tests (Brace et al., 1993). When applying this m e t h o d to 50 different S-homozygotes in B. oleracea, RFLP using six restriction enzymes showed a u niq u e p a tter n for each S-allele with two exceptions (Brace et al., 1994). In a n o t h e r case, SLG-specific primers permitted amplification of SLG without any c o n t a m i n a t i o n of S L R 1 , SLR2, and other m e m b e r s of the S multigene family. This was applied to several cultivars and 27 different S-homozygote tester lines in B. rapa. The use of combinations of specific primers and restriction enzymes is r e c o m m e n d e d for registering S alleles (Nishio et al., 1994, 1996). Application of this system for R a p h a n u s species and some o r n a m e n t a l plants in the B r a s s i c a c e a e suggested t hat the diversification of the S L G alleles predates generic differentiation (Sakamoto et al., 1998). Another strategy for the identification of the S genotypes is the observation of single-strand conformation polymorphism in the PCR products of SLG. This technique distinguishes the PCR products derived from different S-homozygotes by electrophoretic mobility (Delorme et al., 1995b).
SRK (S-receptor kinase) Identification and cloning of S R K Walker an d Z hang (1990) pointed out t hat the amino acid sequence of the extracellular dom ai n of a serine / threonine type putative t ransm em brane protein kinase from maize had high homology to t hat of SLG. This finding suggested t hat a t r a n s m e m b r a n e protein kinase may play an import a nt role in self-incompatibility, since m a n y kinds of protein kinase are involved in signal t r a n s d u c t i o n in animals (Pawson, 1991; Trewavas and Gilroy, 1991). Stein et al. (1991) cloned S R K 6 from the genome of B. oleracea. Its extracellular domain, showing 89% homology to SLG ~, was connected via a single-pass t r a n s m e m b r a n e domain to a protein kinase catalytic center. This S R K gene comprised seven exons and had two stop codons, one in the first
157 intron at the e n d of the extracellular d o m a i n a n d one at the end of the seventh exon. Two S-locus linked genes, SLUr 2A a n d SLUr2B, w e r e isolated from the S2-homozygote, a n d this SLC_r2B w a s c o n s i d e r e d to be a n S R K 2 (Chen a n d Nasrallah, 1990; Stein et al., 1991). B e c a u s e b o t h S L G a n d S R K are considered to p a r t i c i p a t e in the recognition reaction a n d m a p to the S locus, a n S allele is referred to as a n S haplotype (Nasrallah a n d Nasrallah, 1993). Since t h e n , cDNA or genomic DNA clones of S R K have been isolated u n der different cloning strategies from two S-homozygotes of B. o l e r a c e a (Kum a r a n d Trick, 1994; Delorme et al., 1995a), five S-homozygotes of B. r a p a (Watanabe et al., 1994; Y a m a k a w a et al., 1995; S u z u k i et al., 1995; H a t a k e y a m a et al., 1998b; H a t a k e y a m a et al., 1998c), a n d two S-homozygotes of B. n a p u s (Goring a n d Rothsetin, 1992; Glavin et al., 1994). In e a c h case, S L G a n d S R K were found to be tightly linked to e a c h other, a n d they were also linked with S g e n o t y p e s d e t e r m i n e d by pollen t u b e behavior (Stein et al., 1991; W a t a n a b e et al., 1994; Delorme et al., 1995a). The positions of i n t r o n s were well c o n s e r v e d in e a c h S R K gene, t h o u g h the length of the first intron varied (Stein et al., 1991; K u m a r a n d Trick, 1994; Delorme et at., 1995a; Suzuki et al., 1997a; H a t a k e y a m a et al., 1998b). The homology between S L G a n d the S d o m a i n e n c o d e d by S R K varied from 85 to 90% in m a n y cases. A rapid identification s y s t e m for the diversity of the S R K genes w a s d e m o n s t r a t e d recently by PCR-RFLP by u s i n g a set of specific p r i m e r s (Nishio et al., 1997). E x p r e s s i o n a n d localization of S R K The t r a n s c r i p t of S R K w a s m a i n l y detected in s t i g m a tissue. Even in s t i g m a tissue, the e x p r e s s i o n of S R K was extremely low relative to S L G (Stein et al., 1991; W a t a n a b e et al., 1994; Glavin et al., 1994; Delorme et al., 1995a). The time of the e x p r e s s i o n of S R K w a s similar to t h a t of S L G (Glavin et al., 1994; Stein et al., 1996). B e c a u s e the S R K genomic clones h a d a n inframe stop codon, S R K s e e m e d to direct the s y n t h e s i s of several t r a n s c r i p t s (Stein et al., 1991; K u m a r a n d Trick, 1994; S u z u k i et at., 1995, 1996; Delorme et al., 1995a). These t r a n s c r i p t s were a p p a r e n t l y g e n e r a t e d by a c o m b i n a t i o n of alternative splicing a n d the u s e of alternative p o l y a d e n y l a t i o n signals (Stein et al., 1991; S u z u k i et al., 1996; G i r a n t o n et al., 1995). The t r u n c a t e d S R K w h i c h were derived from the S d o m a i n were detected as a protein a n d as a n RT-PCR p r o d u c t (Giranton et al., 1995). Recently, several n a t u r a l a n t i s e n s e t r a n s c r i p t s of S R K were identified in B. oleracea. W h e n different RNase protection p r o b e s were u s e d , regions of the promoter, exon I a n d intron I of SRK, were t r a n s c r i b e d in a n a n t i s e n s e direction. These antisense t r a n s c r i p t s would be correlated with a lower level e x p r e s s i o n of S R K t h a n t h a t of S L G (Cock et al., 1997). An e l e c t r o p h o r e s i s e x p e r i m e n t with s t i g m a p r o t e i n s s h o w e d t h a t S R K was a glycoprotein targeted to the p l a s m a m e m b r a n e (Delorme et al., 1995a). The s a m e r e s u l t w a s d e m o n s t r a t e d u s i n g t r a n s g e n i c tobacco p l a n t s with
158 S R K ~ (Stein et in E. coli, was late on serine and Nasrallah,
al., 1996). The product of SRK, expressed as a fusion protein a functional protein kinase, and was able to autophosphoryand threonine residues (Goring and Rothstein, 1992, Stein 1993).
Transformation of S R K A genomic clone of S R K 6 (B.o.) was introduced into S2-homozygotes. Although S R K was expressed in both stigmas and a n t h e r s of the transgenic plants, the self-incompatibility phenotype was not altered (Stein et al., 1991). Further transgenic approaches were u n d e r t a k e n using several chimeric genes. The transgenes led to a dramatic reduction in the expression of the endogenous S locus and related genes. The homology-dependent silencing of endogenous genes was associated, in at least some cases, with increased cytosine methylation. The silencing of SLG a n d / o r S R K genes in self-incompatible host plants results in the breakdown of self-incompatibility, whereas a reduction in SLG a n d / o r S R K gene transcripts in anthers does not affect pollen phenotype (Conner et al., 1997). A self-incompatible B. n a p u s was transformed with an inactive copy of the SRK gene. The transformants became self-compatible because of co-suppression and dominant-negative effects. The change of the S phenotype was only observed in stigma, but not in pollen (Stahl et al., 1998). Molecular characterization of S locus
The long genomic regions spanning the S locus were analyzed in some S haplotypes. These analyses have revealed that several genes exist on the flanking region of S L G and S R K genes. In self-incompatible B. n a p u s , two genes, SLL1 (S-locus linked gene 1) and SLL2 (S-locus liked gene 2), were located between the S L G and S R K genes, and expressed in the anthers. However, SLL1 did not have allelic specificity and SLL2 was also expressed in stigmas, indicating that these genes might not be the pollen S gene (Yu et al., 1996). In S 8 haplotype of B. rapa, two nonpolymorphic and vegetatively expressed sequences, 2 9 8 and 299, were located at the 3'-flanking region of the SLG 8. It was found that the 2 9 9 encoded SLL2 gene, and the 2 9 8 encoded ClpP (Clp protease) homologue (Boyes et al., 1997; Conner et al., 1998; Letham and Nasrallah, 1998). Using P 1-derived artificial chromosome (PAC) vector, Suzuki et al. (1997 c) directly cloned an 80-kb M/ul genomic fragment containing both SLG and S R K genes of S 9 haplotype in B. rapa. They have found more than ten genes are located in this genomic region, and this is currently being studied more throughly. The physical localization of the S locus (SLG and SRIO in the chromosome of B. rapa was visualized by multi-color fluorescent in situ hybridization. The S L G gene is localized at the interstitial region close to the end of the chromosome (lwano et al., 1998).
159 For the S 8 haplotype of B. rapa, a 100-kb region s p a n n i n g the S locus was m a p p e d with several cDNA and genomic DNA clones of A r a b i d o p s i s . Comparative m a p p i n g between the S locus region of B r a s s i c a and its homologous region in A r a b i d o p s i s revealed t h a t no sequences similar to S locus in B r a s s i c a were detected in the A r a b i d o p s i s genome (Conner et al., 1998).
Other members of S-multigene family S o u t h e r n blot analysis of B r a s s i c a genome with the S L G cDNA probe exhibited S haplotype-associated restriction site p o l y m o r p h i s m (Nasrallah et al., 1985; Nasrallah et al., 1988; Nou et al., 1993b). A part of these hybridized b a n d s have been isolated a n d characterized as S L G a n d SRK. In addition to these, self-incompatible B r a s s i c a species possess S related genes called SLR1 (Isogai et al., 1988, 1991; Lalonde et al., 1989; Trick a n d Flavell, 1989; Trick, 1990; Y am akaw a et al., 1993; W a t a n a b e et al., 1998), S L R 2 (Scutt et al., 1990; Boyes et al., 1991; T a n t i k a n j a n a et al., 1996), a n d S L R 3 (Cock et al., 1995). These genes are not linked to the S locus (Lalonde et al., 1989; Boyes et al., 1991; W a t a n a b e et al., 1992; Cock et al., 1995). Considerable evidence suggests t h a t the SLR1 gene does not participate in the selfvs. non-self recognition events, at least not directly (Franklin et al., 1996). Anyhow, together these genes form a large S multigene family (Dwyer et al., 1989). Observation of the SLR1 a n t i s e n s e transgenic B. n a p u s revealed t h a t it reduced the a d h e s i o n between pollen a n d stigma, and indicated t h a t SLR1 is one of the factors involved in the pollen-stigma adhesi on (Luu et al., 1997). When the region encoding the S R K catalytic d o m a i n was u s e d as a probe, m a n y genomic clones hybridized a n d some of t h e m were cloned (Kum a r and Trick,1993). One clone was identified as a pseudogene, b u t some others encoded functional protein kinases. Three clones each of 5 functional protein k in a s es cross-hybridized with an SLCr 29 cDNA probe, indicating the presence of u p s t r e a m receptor d o m a i n s t h a t m ay be homologous to the S L G gene. The previously reported S sequence complexity m ay be ascribed to a large gene family of receptor kinase (Kumar and Trick, 1993). In some of these clones, the t r a n s c r i p t s were detected in several t i ssues by RT-PCR analysis. RFLP analysis revealed t h a t one or two clones co-segregated with the S locus (Kumar and Trick, 1994; Oldknow and Trick, 1995). In self-incompatible B. rapa, 12 groups of genomic clones were isolated to obtain SLG-homologous regions. Of these groups, two corresponded to S L G and SRK, respectively. Of the remaining ten groups, four were SLG-like clones and six S R K - l i k e clones. Two clones with two S R K - l i k e sequences and one with an SLG-like sequence co-segregated with the S locus (Suzuki et al., 1995; Suzuki et al., 1997b). A pulse-field gel electrophoresis s t u d y in conjunction with DNA blot analysis d e m o n s t r a t e d t h a t S L G and S R K were separat ed by a m a x i m u m distance of 2 2 0 k b to 350kb. C o m p a r i s o n of the d a t a from the two genotypes, S 2 an d S~, revealed t h a t a high level of p o l y m o r p h i s m existed across
160 the entire S locus (Boyes a n d Nasrallah, 1993). Kianian a n d Quiros (1992) showed t h a t the S m u l t i g e n e family w a s organized in a linkage group of three loci by RFLP analysis. The p r e s e n c e of three linked loci indicates t h a t the self-incompatibility reaction m a y be the r e s u l t of the concerted action or interaction of several loci. F u r t h e r m o l e c u l a r a n a l y s i s will be n e c e s s a r y to d e t e r m i n e w h e t h e r all three m e m b e r s of the S m u l t i g e n e family are expressed a n d w h e t h e r t h e y have r e s u l t e d from the t a n d e m duplication of an ancestral locus (Kianian a n d Quiros, 1992). S u z u k i et al., (1997a) showed t h a t the p r o m o t e r s e q u e n c e s of S L G 9 a n d S R K 9 are completely identical to each other. This identity m a y suggest the r e c e n t o c c u r r e n c e of gene conversion in this locus.
Signal perception and signal transduction signal of pollen The recognition reaction of self-incompatibility is ascribed to the identity of S genes in pollen a n d stigma. Therefore, a s u b s t a n c e which correlates with S genes is expected to be p r e s e n t in pollen. The first possibility is t h a t the p r o d u c t of S L G or S R K functions as a n S gene d e t e r m i n a n t in pollen. Another possibility is t h a t the p r o d u c t of a gene t h a t is tightly linked to SLG a n d S R K f u n c t i o n s as a n S gene d e t e r m i n a n t in pollen. In e a c h case, genes m u s t m a p to the S locus b e c a u s e the self vs. non-self recognition reaction is r e g u l a t e d by the S locus. F u r t h e r m o r e , in the case of sporophytic self-incompatibility, this s u b s t a n c e would be p r o d u c e d before meiosis in pollen m o t h e r cells, or, if later, in the sporophytic a n t h e r wall from which it is t r a n s f e r r e d to pollen. The t r a n s c r i p t s of a recessive S L G were detected not only in the stigma b u t also in the a n t h e r , b u t the levels of t r a n s c r i p t i o n were different between s t i g m a a n d a n t h e r ( T a n t i k a n j a n a et al., 1993). Fully spliced a n d unspliced S R K 6 (B.o.) t r a n s c r i p t s were m a x i m a l l y e x p r e s s e d in a n t h e r s at the binucleate pollen stage (Stein et al., 1991). In the S 2 haplotype of B. oleracea, a gene, S L A 2 located in the 3' flanking region of S L G 2 gene, t r a n s c r i b e d two c o m p l e m e n t a r y anther-specific t r a n s c r i p t s by two promoters. One was spliced a n d the o t h e r unspliced. They a c c u m u l a t e d in a n antiparallel m a n n e r in developing m i c r o s p o r e s a n d a n t h e r s . S e q u e n c e s related to S L A (S locus anther gene) were not detected in either DNA or RNA in other p l a n t s carrying S h a p l o t y p e s o t h e r t h a n S 2 (Boyes a n d Nasrallah, 1995). In f u r t h e r studies the p r e s e n c e of a f u n c t i o n - d i s r u p t e d S L A gene by a large insertion was identified in a n S h a p l o t y p e of self-incompatible B. o l e r a c e a (Pastuglia et al., 1997b). Therefore, this gene does not seem to be required for the self-incompatibility response. In a t r a n s g e n i c A r a b i d o p s i s t h a t carried a r e p o r t e r G U S gene with the p r o m o t e r of S L G 13 (B.o.) G U S activity was found in a n t h e r tapetal cells at the u n i n u c l e a t e m i c r o s p o r e stage (Toriyama et al., 199 l b). In the t r a n s g e n i c B.
161
oleracea with the s a m e reporter gene (SLG 13 (B.o.)-GUS), e x p r e s s i o n of GUS was detected in the t a p e t a l cells a n d in the pollen g r a i n s at the bi- a n d trinucleate pollen s t a g e s (Sato et al., 1991). These o b s e r v a t i o n s s u p p o r t the presence of an SLG-derived t r a n s c r i p t in a n t h e r s . The p r e s e n c e in y o u n g a n t h e r s of the t r a n s c r i p t h o m o l o g o u s with SLG was s u g g e s t e d by the aid of PCR m e t h o d with c o m b i n a t i o n s of primers. Several t r a n s c r i p t s differing in size were detected in s e x u a l as well as in vegetative t i s s u e s (Guilluy et al., 1991). The p r e s e n c e of a n a n t h e r - s u b s t a n c e t h a t c r o s s - r e a c t e d with anti-SLGS(B.r.) a n t i s e r u m w a s r e p o r t e d by W a t a n a b e et al., (1991). This s u b s t a n c e , SAP (S-glycoprotein-like a n t h e r protein), generated a single distinct b a n d at pl 5.0 on a n I E F - i m m u n o b l o t . SAP w a s found in a n t h e r walls at the uni- a n d b i n u c l e a t e stages r a t h e r t h a n in pollen. This observation m a y also s u g g e s t t h a t SLG-like p r o d u c t s are p r o d u c e d on the ant h e r side. However, correlation between t h e s e s u b s t a n c e s a n d S genotypes h a s not yet b e e n d e m o n s t r a t e d . Several pollen coat proteins (PCI~ have b e e n looked for a n d isolated. W h e n a m i x t u r e of s t i g m a e x t r a c t s with pollen coat p r o t e i n s w a s applied to isoelectric focusing analysis, one b a n d was newly detected; this w a s considered to be a p r o d u c t formed by the i n t e r a c t i o n b e t w e e n a certain pollen coat protein a n d SLGs. This pollen s u b s t a n c e , d e s i g n a t e d PCPT, w a s a 7 - k D a nonglycosylated peptide (Doughty et al., 1993). In a n o t h e r e x p e r i m e n t , PCP1 was isolated from a n t h e r mRNA u s i n g RT-PCR. PCP1 c o n t a i n e d a single intron a n d e n c o d e d a small, basic peptide c o m p o s e d of 83 a m i n o acids containing a h y d r o p h o b i c signal peptide s e q u e n c e . Eight cysteine residues, which have high homology to a n u m b e r of o t h e r anther-specific genes, were found in the c e n t r a l p a r t a n d C - t e r m i n a l region. T r a n s c r i p t s of PCP1 were detected in the c y t o p l a s m of the t r i n u c l e a t e pollen grain, b u t not in the t a p e t u m . The PCP g e n e s formed a large m u l t i g e n e family, c o m p o s e d of 30 to 40 copies per g e n o m e of B. oleracea, b u t s h o w e d no b a n d linked to the S locus (Stanchev et al., 1996). The cDNA clones h o m o l o g o u s to PCP1 were also isolated from a cDNA library of i m m a t u r e a n t h e r s by u s i n g polyclonal a n t i s e r u m raised a g a i n s t the e x t r a c e l l u a r pollen p r o t e i n s (Toriyama et al., 1998). Recently, by u s i n g a b i o a s s a y s y s t e m , addition of PCP-A {renamed from PCP7) w a s e x a m i n e d on s t i g m a surface. W h e n "self" PCP-A fraction was u s e d , the s u c c e s s of compatible cross-pollination w a s prevented, while a "cross" PCP-A fraction could i n d u c e the g e r m i n a t i o n a n d growth of selfpollen. This s u g g e s t s a possibility t h a t a m e m b e r of the PCP-A protein family could be a d e t e r m i n a n t at the pollen side in the B r a s s i c a self-incompatibility s y s t e m ( S t e p h e n s o n et al., 1997; D o u g h t y et al., 1998).
Signal transduction cascade via protein phosphorylation By analogy with a n i m a l growth factors, one could imagine t h a t S R K accepts a signal from pollen a n d t r a n s d u c e s the signal into papilla cells via a protein p h o s p h o r y l a t i o n c a s c a d e (Figure 5.1).
162 Using a yeast two-hybrid system, proteins interacting with SRK-910 kinase domain were screened. Two different kinds of cDNA clones were isolated and characterized. One of them included two thioredoxin-h-like clones, THL-1 and THL-2. These clones specifically interacted with the kinase domain of SRK-910. THL-1 was expressed in a variety of tissues, but THL-2 preferentially expressed in floral tissues. Thioredoxin may possibly be one of the effector molecules in the signal cascade of self-incompatibility (Bower et al., 1996). Another cDNA clone was ARC1 (Arm Repeat Containing) gene. The ARC 1 specifically interacted with the kinase domain of SRK, but not with the kinase domains taken from different kind of Arabidopsis receptor-like kinases. The interaction was phosphorylation dependent (Gu et al., 1998). Two independent experiments have been reported on phosphorylation using inhibitors of serine / threonine protein phosphatase. One showed that treatment with okadaic acid via transpiration stream against the newly opened flower bud was sufficient to overcome self-incompatibility, though the magnitude of this effect was S genotype dependent. At the higher concentrations used, pollen tube growth was arrested before the pollen tubes reached the ovary, b u t this effect was also observed in cross-pollinated styles treated in the same m a n n e r (Scutt et al., 1993). In contrast, the treatment of mature flowers with p h o s p h a t a s e inhibitors, okadaic acid and microcystin, had no effect on the self-incompatibility response of four different S genotypes, although the treatment of flower b u d s j u s t prior to anthesis allowed self-pollen tube invasion of papilla cells (Rundle et al., 1993). A cDNA clone encoding type 1 serine / threonine protein phosphatase (BoPP1) was isolated from B. oleracea stigmas. It was suggested that the arrest of self-pollen development might result from activation of the SRK and the phosphorylation of specific protein substrates, whereas to allow the growth of compatible pollen tube, protein p h o s p h a t a s e activity might be required to dephosphorylate these substrates (Rundle and Nasrallah, 1992). A new A r a b i d o p s i s m u t a n t (pop1) gives us an insight into the pollen tube arrest in self-pollination. Stigma cells that had been in contact with the m u t a n t pollen produced callose, a It-1,3-glucan. The m u t a n t pollen failed to germinate because it did not absorb water from the stigma; yet it germinated in vitro, indicating it was viable (Preuss et al., 1993). In these points, the similarity between the pop1 mutation and the self-incompatibility studied in Brassica species is striking. Both regulate pollen germination at the stigma surface, often through control of pollen hydration. In addition, there are m a n y examples of callose production in stigma cells that are in direct contact with incompatible self-pollen. Singh et al. (1989) showed that self-pollen had higher levels of chlorotetracycline fluorescence and higher calcium content than cross-pollen in energy-dispersive analysis of X-rays. Callose deposition was found to be a calcium dependent process in pollination, by using a calcium channel antagonist and a calcium ionophore. However, pretreatment of pistils with 2-
163
Figure 5.1 A hypothetical picture drawing the recognition reaction. A pollen specific substance (unknown) is transferred onto the stigma papilla cell as a ligand. The ligand is accepted by SLG and/or the receptor domain of SRK at the cell wall of papilla. The accepted signal is transferred to the cytoplasm of the papilla cell by SRK, and then through protein phosphorylation cascade the incompatibility reaction would be controlled.
164 deoxy-D-glucose abolished the callose formation in self-pollination, whereas self-pollen remained inhibited and cross-pollen grew normally. This suggests that callose formation in the stigma is not an essential factor preventing the growth of pollen tubes in the self-incompatibility (Singh and Paolillo, 1990). Furthermore, transgenic plants with the [t-1, 3-glucanase gene were raised. In the plants, little or no callose was detected in the papilla cells, though the self-incompatibility system appeared to be unaffected. This result significantly indicates that callose deposition is not required for the rejection of incompatible pollen (Sulaman et al., 1997).
Molecular analysis of self-compatibility A self-compatible (Sc) line of B. oleracea possessed SLG sc which had a similar spatial and temporal expression to that of self-incompatible B r a s s i c a plants. Immunological analysis of an F2 population revealed that the SLG sc segregated with the self-compatibility phenotype, suggesting that the changes in SLG were responsible for the self-compatibility character. The deduced amino acid sequence of an SLG sc cDNA clone showed a high level of homology with that of pollen-recessive SLC~ (Gaude et al., 1993). From selfcompatible B. n a p u s cv. Westar, two different stigma-specific cDNA clones homologous to S L G were isolated. One of these sequences, S L G - w s l , showed high homology to Class I SLG, whereas the other, S L G - w s 2 , to Class II SLG. Both genes were expressed at high level in stigmas following a developmental pattern typical to SLG in the self-incompatible diploids, B. rapa and B. oleracea (Robert et al., 1994). A single s u p p r e s s o r gene caused the reduction of SLG content (Nasrallah and Nasrallah, 1989). This self-compatibility trait is not tightly linked to the S locus, and the genetic data suggested action of a single recessive gene. The genetic locus defined by the self-compatible mutation was designated as s c f l (stigma compatibility factor 1). The s c f l mutation affected the RNA expression level of secreted type glycoproteins, SLG, SLR1 and SLR2, but not the receptor protein kinase, S R K and, therefore, it is considered that this disrupted a regulatory gene which possibly encodes a trans-acting factor required for high-level expression of the secreted-type glycoproteins in stigma (Nasrallah et al., 1992). In one self-compatible B. oleracea, transcripts of S R K were not detected, though SLG was normally expressed. The analysis of S R K genomic clones demonstrated that the first and the second exons were deleted (Nasrallah et al., 1994). On the other hand, in spontaneous self-compatible B. rapa var. y e l l o w s a r s o n (C636), self-compatibility was mainly explained by a recessive epistatic gene, m. The transcript of SLG was less a b u n d a n t than in self-incompatible strains, though SLR1 and S L R 2 were normally expressed. Furthermore, the transcript of S R K was not detected. This down-regulation of SLG and S R K may be related to this self-compatibility (Watanabe et al., 1997). Similar trends were observed in different strains of y e l l o w s a r s o n
165 (Lalonde et al., 1989, Nasrallah et al., 1994). Recently, the self-compatible rood (renamed from m) locus was dissected with molecular techniques. The phenotype was associated with the absence of t r a n s c r i p t s encoded by an aquaporin-related gene. This may suggest t h a t a water c h a n n e l is required for the self-incompatibility response of B r a s s i c a species (Ikeda et al., 1997). Besides, the importance of pollen coat lipids for pollen germination was also stressed (Wolter-Arts et al., 1998). An isolated S R K from self-compatible B. n a p u s h a d a 1-bp deletion toward the 3' end of the S domain. This deletion would a p p e a r to lead to the p r e m a t u r e t e r m i n a t i o n of translation and the production of a t r u n c a t e d SRK. An active S R K might be required for the expression of self-incompatibility (Goring et al., 1993). Genes homologous with SLR1 were cloned from several self-compatible species (Oldknow et al., 1995, Lalonde et al., 1989). So far, self-compatibility seems to be connected with the down regulation of SLG and SRK, t h o u g h the main p a t h w a y involved h a s not yet been determined.
Evolutionary aspects The role of self-incompatibility in plants is considered to promote outbreeding, t h r o u g h which plants maximize genetic recombination and maintain the genetic heterogeneity. Uyenoyama (1988) proposed t h a t the evolutionary function of self-incompatibility s yst em s is to serve as a eugenic mec h a n i s m for the improvement of offspring quality. Production of offspring t h a t are heterozygous at specific antigen loci is adaptive if the expected viability or fertility of those offspring is greater. U y e n o y a m a (1989) considered conditions for the origin of partial sporophytic self-incompatibility, a s s u m i n g two quantitative models, which differ with respect to the determination of offspring viability. In both cases, the origin of self-incompatibility requires t h a t the relative change in the n u m b e r s of offspring derived by the two reproductive m o d e s c o m p e n s a t e for the twofold cost of outcrossing. Sporophytic self-incompatibility systems could arise in response to identity disequilib r iu m between modifiers of incompatibility a n d a locus subject to overdom i n a n t viability selection. Recent a d v a n c e s of studies on SLG, S R K and related genes have made it possible to d i s c u s s the variation of S alleles in view of evolutionary trends. Hinata et al. (1995) compared s y n o n y m o u s and n o n s y n o n y m o u s base substitutions in the S dom ai n and kinase d o m a i n separately between SRKs. Acc u m u l a t i o n of s y n o n y m o u s and n o n s y n o n y m o u s base s u b s t i t u t i o n s per site in the S d o m a i n was mostly comparable with those of SLGs, respectively. In the kinase domain, however, the level of n o n s y n o n y m o u s base s u b s t i t u t i o n was as low as half of those of the S domain, while the level of s y n o n y m o u s base s u b s t i t u t i o n was mostly comparable between these two domains. This may indicate t h a t the n o n s y n o n y m o u s base s u b s t i t u t i o n of the kinase domain is c o n s t r a i n e d because this d o m a i n should keep its kinase activity,
166 while the S d o m a i n could diversify b e c a u s e the c h a n g e of s t r u c t u r e would be recognized as different S alleles. Analysis of nucleotide s e q u e n c e s on S L G a n d related genes h a s been c o n d u c t e d by Trick a n d H e i z m a n n (1992), U y e n o y a m a (1995), Hinata et al. (1995) a n d King a n d Lynn (1995). It was s u g g e s t e d t h a t the age of the sporophytic self-incompatibility s y s t e m expressed in B r a s s i c a exceeds species divergence within the g e n u s by four to five folds. The extraordinarily high levels of s e q u e n c e diversity exhibited by S alleles a p p e a r s to reflect their ancient derivation, with the alternative h y p o t h e s i s of h y p e r m u t a b i l i t y rejected by the a n a l y s i s (Uyenoyama, 1995). According to d e n d r o g r a m s c o n s t r u c t e d on the basis of s y n o n y m o u s a n d n o n s y n o n y m o u s s u b s t i t u t i o n s , it was considered t h a t SLR1 differentiated first, followed by S L R 2 (Figure 5.2). The differentiation of S L G a n d the S d o m a i n of SRK, b o t h of which occurred coincidentaUy, is one of the prerequisite factors for the e s t a b l i s h m e n t of self-incompatibility. The allelic differentiation was e s t i m a t e d to have occurred more t h a n tens of million y e a r s ago (Hinata et al., 1995). Phylogenetic analysis of m a n y S L G a n d S R K alleles suggested t h a t intragenic r e c o m b i n a t i o n was involved in the evolution of S L G a n d t h a t gene conversion might have often occurred between S L G a n d SRK. The generation of a large n u m b e r of SLG a n d S R K alleles m a y have been c a u s e d not only by point m u t a t i o n b u t also by i n t r a / i n t e r genic recombination. Since S L G a n d S R K alleles are classified into Class I a n d Class II b a s e d on their s e q u e n c e similarity, it is possible t h a t self-incompatibility was first expressed in a two-haplotype system with a d o m i n a n t Class I a n d a recessive Class II haplotype (Kusaba et al., 1997). The c u r r e n t p a r a d i g m for the origin of self-incompatibility p o s t u l a t e s multiple episodes of r e c r u i t m e n t a n d modification of pre-existing genes in each major m e c h a n i s m of self-incompatibility. U y e n o y a m a (1997) a n d S c h i e r u p et al. (1997, 1998) have d i s c u s s e d evolutionary d y n a m i c s a n d the allelic genealogies t h r o u g h stochastic simulation.
R e l a t e d s t u d i e s with future p r o s p e c t s In higher plants, self vs. non-self recognition reactions are only known in the pollination system, involving self- a n d interspecific incompatibility. Besides, gene-to-gene recognition reaction is k n o w n in p l a n t - p a t h o g e n interactions. Hodgkin et al. (1988) have c o m p a r e d B r a s s i c a self-incompatibility a n d p l a n t - p a t h o g e n interactions. Both reactions have m a n y features in common: both c o n c e r n cell surface c o m p o n e n t s s u c h as glycoproteins a n d both are active p r o c e s s e s requiring enzyme synthesis. The m o s t obvious differences between these two s y s t e m s are t h a t self-incompatibility in B r a s s i c a is m e d i a t e d t h r o u g h the s u p p l y of water a n d does not exhibit cross-protection reactions. A characteristic feature of self-incompatibility is a pre-programmed process in p l a n t s r a t h e r t h a n a n i n d u c e d r e s p o n s e to external events.
167 SLG 13 Bo SLG 8 Bc SLG 29 Bo
47
, SLG 14 Bo
4 98
SLG 6 Bo , SRK 6 Bo(RD) r S L G Bc loo
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b: Nonsynonymous substitution per site
Figure 5.2 Dendrograms of 17 OTUs constructed by neighbour-joining method based on synsynonymous and non-synonymous base substitution per site. The SLR1 differentiated first, followed by SLR2. The differentiation of SLG as well as the receptor domain of SRK is one of the prerequisite factors for the establishment of self-incompatibility in the Brassicaceae. (From Hinata et al. (1995); reproduced with permission from the Genetic Society of America).
168 Recently, several resistance genes against p a t h o g e n s were isolated (reviewed in Bent, 1996). Two of them, PTO and X a 2 1 , were encoded membrane b o u n d serine / threonine protein kinase (Martin et al., 1993, Song et al., 1995). In this respect, both self-incompatibility a n d pathogen-plant interaction are regulated by similar m e c h a n i s m s via phosphorylation cascade in the signal transduction. Another different protein kinase had an important role in plant morphogenesis. Erecta (er) m u t a n t of A r a b i d o p s i s Landsberg showed a compact inflorescence, blunt fruits, and short petioles. The er gene was encoded by a receptor type serine / threonine protein kinase (Torii et al., 1996). Several receptor type protein kinase genes were isolated and characterized, although their biological functions remain u n k n o w n (Chang et al., 1992; Kohorn et al., 1992; Tobias et al., 1992; Dwyer et al., 1994; Walker, 1992; Zhang a n d Walker, 1993; Wang et al., 1996; Pastuglia et al., 1997a). The elucidation of signal t r a n s d u c t i o n in these physiological traits would contribute to the u n d e r s t a n d i n g of the m e c h a n i s m of signal transduction of self / non-self recognition in self-incompatibility.
Acknowledgements We t h a n k Dr. M. K. Uyenoyama, Duke University, U.S.A., Dr. E. Newbigin, University of Melbourne, Australia, and Dr. A. Isogai, Nara Institute of Science a n d Technology, J a p a n , for helpful c o m m e n t s and improving the manuscript. This work was supported in part by Grants-in-Aid for Special Research on Priority Areas (nos. 07281102 a n d 07281103; Genetic Dissection of Sexual Differentiation and Pollination Process in Higher Plants) from the Ministry of Education, Science, Culture and Sports, J a p a n .
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177 quired for the self-incompatibility response in Brassica oleracea. Plant Cell 9, 2065-2076. Pawson, T. 1991. Signal transduction in the control of cell growth and development. Trend. Genet. 7, 343-345. Perl-Treves, R., Howlett, B. and Nasrallah, M. E. 1993. Self-incompatibility related glycoproteins of Brassica are produced and secreted by transgenic tobacco cell cultures. Plant Sci. 92, 99-110. Pink, D. A. C. 1993. Swede and turnip Brassica napus L. var. napobrassica, B. rapa L. var. glabra. In: Kalloo, G. and Bergh, B. O. (eds), Genetic improvement of vegetable crops, pp. 511-519, Pergamon Press. Preuss, D., Lemieux, B., Yen, G. and Davis, R. W. 1993. A conditional sterile mutation eliminates surface components from Arabidopsis pollen and disrupts cell signaling during fertilization. Genes Dev. 7, 974985. Robert, L. S., Allard, S., Franklin, T. M. and Trick, M. 1994. Sequence and expression of endogenous S-locus glycoprotein genes in self-incompatible Brassica napus. Mol. Gen. Genet. 242, 209-216. Roggen, H. 1974. Pollen washing influences (in)compatibility in Brassica oleracea varieties. In: Linskens, H.F. (ed.) Fertilization in higher plants, pp 273-278, North-Holland. Rundle, S. J. and Nasrallah, J. B. 1992. Molecular characterization of type 1 serine/threonine p h o s p h a t a s e s from Brassica oleracea. Plant Mol. Biol. 20, 367-375. Rundle, S. J., Nasrallah, M. E. and Nasrallah, J. B. 1993. Effects of inhibitors of protein serine / threonine p h o s p h a t a s e s on pollination in Brassica. Plant Physiol. 103, 1165-1171. Sakamoto, K., Kusaba, M. and Nishio, T. 1998. Polymorphism of the S-locus glycoprotein gene (SLG) and the S-locus related gene (SLR1) in Rap h a n u s sativus L. and self-incompatible ornamental plants in the Brassicaceae. Mol. Gen. Genet. 258, 397-403. Sampson, D. R. 1964. A one-locus self-incompatibility system in R a p h a n u s raphanistrum. Can. J. Genet. Cytol. 6, 435-445. Sampson, D. R. 1967. Frequency and distribution of self-incompatibility alleles in R a p h a n u s raphanistrum. Genetics 56, 241- 251. Sasaki, Y., Iwano, M., Matsuda, N., Suzuki, G., Watanbe, M., Isogai, A. and Toriyama, K. 1998. Localization of an SLG protein expressed u n d e r the regulation of a tapetum-specific promoter in a n t h e r s of transgenic Brassica napus. Sex. Plant Reprod. 1 1 , 2 4 5 - 2 5 0 . Sato, T., Thorsness, M. K., Kandasamy, M. K., Nishio, T., Hirai, M., Nasrallah, J. B. and Nasrallah, M. E. 1991. Activity of an S locus gene
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179 Stein, J. C. a n d Nasrallah, J. B. 1993. A plant receptor-like gene, the S-locus receptor kinase of Brassica oleracea L., encodes a functional serine / threonine kinase. Plant Physiol. 101, 1103-1106. Stein, J. C., Howlett, B., Boyes, D. C., Nasrallah, M. E. a n d Nasrallah, J. B. 1991. Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea. Proc. Natl. Acad. Sci. USA, 88, 8816-8820. Stein, J. C., Dixit, R., Nasrallah, M. E. and Nasrallah, J. B. 1996. SRK, the stigma-specific S locus receptor kinase of Brassica, is targeted to the p l a s m a m e m b r a n e in transgenic tobacco. Plant Cell 8, 429-445. Stephenson, A. G., Doughty, J., Dixon, S., Elleman, C., Hiscock, S. a n d Dickinson, H. G. 1997. The male d e t e r m i n a n t of self-incompatibility in Brassica oleracea is located in the pollen coating. Plant J. 12, 1351-1359. Stevens, J. P. a n d Kay, Q.O.N. 1988. The n u m b e r of loci controlling the sporophytic self-incompatibility system in Sinapis arvensis. Heredity
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180 Suzuki, G., Watanabe, M. and Hinata, K. 1997d. Molecular genetics of selfincompatibility in Brassica. Recent Res. Dev. Agri. Biol. Chem., 1, 235-242. Takasaki, T., H a t a k e y a m a , K., Ojima, K., Watanabe, M., Toriyama, K. and Hinata, K. 1998. Alteration of self-incompatibility phenotype by sense and antisense t r a n s g e n e of S-locus glycoprotein in Brassica rapa L. (in preparation). Takayama, S., Isogai, A., Tsukamoto, C., Ueda, Y., Hinata, K., Okazaki, K. a n d Suzuki, A. 1987. Sequences of S-glycoproteins, products of the Brassica campestris self-incompatibility locus. Nature 326, 102104. Takayama, S., Isogai, A., Tsukamoto, C., Shiozawa, H., Ueda, Y., Hinata, K., Okazaki, K., Koseki, K. a n d Suzuki, A. 1989. Structure of N-glycosidic saccharide chains in S-glycoproteins, products of S-genes associated with self-incompatibility in Brassica campestris. Agric. Biol. Chem. 53, 713-722. Tantikanj ana, T., Nasrallah, M. E., Stein, J. C., Chen, C-H. and Nasrallah, J. B. 1993. An alternative transcript of the S-locus glycoprotein gene in a class II pollen-recessive self-incompatibility haplotype of Brassica oleracea encodes a m e m b r a n e - a n c h o r e d protein. Plant Cell 5, 657-666. Tantikanjana, T., Nasrallah, M. E. a n d Nasrallah, J. B. 1996. The Brassica S gene family: molecular characterization of the SLR2 gene. Sex. Plant Reprod. 9, 1O- 116. Thompson, K. F. 1957. Self-incompatibility in n a r r o w - s t e m kale, Brassica oleracea var. acephala. I. demonstration of a sporophytic system. J. Genet. 55, 45-60. Thompson, K. F. a n d Taylor, J. P. 1966. Non-linear dominance relationships between S alleles. Heredity 2 1 , 3 4 5 - 3 6 2 . Thorsness, M. K., Kandasamy, M. K., Nasrallah, M. E. and Nasrallah, J. B. 1993. Genetic ablation of floral cells in Arabidopsis. Plant Cell. 5, 253-261. Tobias, C. M., Howlett, B. and Nasrallah, J. B. 1992. An Arabidopsis thaliana gene with sequence similarity to the S-locus receptor kinase of Brassica oleracea: Sequence and expression. Plant Physiol. 99, 284 -290. Torii, K. U., Mitsukawa, N., Oosumi, T., Matsuura, Y., Yokoyama, R., Whittier, R. F. and Komeda, Y. 1996. The Arabidopsis erecta gene encodes a putative receptor protein kinase with extracellular leucinerich repeat. Plant Cell 8, 735-746.
181 Toriyama, K., Stein, J. C., Nasrallah, M. E. and Nasrallah, J. B. 1991a. Transformation of B r a s s i c a oleracea with an S-locus gene from B. c a m p e s t r i s changes the self-incompatibility phenotype. Theor. Appl. Genet. 8 1 , 7 6 9 - 7 7 6 . Toriyama, K., Thorsness, M. K., Nasrallah, J. B. and Nasrallah, M. E. 1991 b. A B r a s s i c a S-locus gene promoter directs sporophytic expression in the a n t h e r t a p e t u m of transgenic Arabidopsis. Dev. Biol. 143, 427431. Toriyama, K., Hanaoka, K., Okada, T. and Watanabe, M. 1998. Molecular cloning of a cDNA encoding a pollen extracellular protein as a potential source of a pollen allergen in B r a s s i c a rapa. F E B S Lett. 424, 234-238. Trewavas, A. and Gilroy, S. 1991. Signal transduction in plant cells. Trend. Genet. 7, 356-361. Trick, M. 1990. Genomic sequence of a B r a s s i c a S-locus-related gene. Plant Mol. Biol. 15, 203-205. Trick, M. and Flavell, R. B. 1989. A homologous S genotype of B r a s s i c a oleracea expresses two S-like genes. Mol. Gen. Genet. 218, 112-117. Trick, M. and Heizmann, P. 1992. Sporophytic self-incompatibility systems: B r a s s i c a S gene family. Inter. Rev. Cytol. 140, 485524. Uyenoyama, M. K. 1988. On the evolution of genetic incompatibility systems: Incompatibility as a m e c h a n i s m for the regulation of outcrossing distance. In Michod, R. E. and Levin, B. R. (eds.) The evolution o f sex: A n e x a m i n a t i o n o f current ideas, pp 212-232, Sinauer, Sunderland, MA. Uyenoyama, M. K. 1989. On the evolution of genetic incompatibility systems. V. Origin of sporophytic self-incompatibility in response to overdominance in viability. Theor. Pop. Biol. 36:339-365. Uyenoyama, M. K. 1995. A general least-squares estimate for the origin of sporophytic self-incompatibility. Genetics 139, 975-992. Uyenoyama, M. K. 1997. Genealogical structure among alleles regulating self-incompatibility in natural populations of flowering plants. Genetics 147, 1389-1400. Van Engelen, F. A., Hartog, M. V., Thomas, T. L., Sturm, A., Van Kammen, A. and De Vries, S. C. 1993. The carrot secreted glycoprotein gene EP1 is expressed in the epidermis and has sequence homology to B r a s s i c a S-locus glycoproteins. Plant J. 4, 855- 862. Verma, S. C., Malik, R. and Dhir, I. 1977. Genetics of the incompatibility systems in the crucifer Eruca sativa. Proc. R. Soc. Lond. B., 196, 131159.
182 Visser, D. L., Hal, J. G. Van. and Verhoeven, W. 1982. Classification of Salleles by their activity in S-heterozygotes of Brussels sprouts (Brassica oleracea var. gemmifera (DC.) Schultz). Eupytica 3 1 , 6 0 3 611. Walker, J. C. 1992. Receptor-like protein kinase genes of Arabidopsis thaliana. Plant J. 3, 451-456. Walker, J. C. and Zhang, R. 1990. Relationship of a putative receptor protein kinase from maize to the S-locus glycoproteins of Brassica. Nature 345, 743-746. Wallace, D. H. 1979. Interactions of S-alleles in sporophytically contorolled self-incompatibility of Brassica. Theor. Appl. Genet. 54, 193-201. Wang, X., Zafian, P., Choudhary, M. and Lawton, M. 1996. The PR5K receptor protein kinase from Arabidopsis thaliana is structurally related to a family of plant defense proteins. Proc. Natl. Acad. Sci. USA, 93, 2598-2602. Watanabe, M., Shiozawa, H., Isogai, A., Suzuki, A., Takeuchi, T. and Hinata, K. 1991. Existence of S-glycoprotein-like proteins in anthers of self-incompatible Brassica species. Plant Cell Physiol. 32, 10391047. Watanabe, M., Nou, I. S., Takayama, S., Yamakawa, S., Isogai, A., Suzuki, A., Takeuchi, T. and Hinata, K. 1992. Variations in and inheritance of NS-glycoprotein in self-incompatible Brassica campestris L. Plant Cell Physiol. 33, 343-351. Watanabe, M., Takasaki, T., Toriyama, K., Yamakawa, S., Isogai, A., Suzuki, A. and Hinata, K. 1994. A high degree of homology exists between the protein encoded by SLG and the S receptor domain encoded by SRK in self-incompatible Brassica campestris L. Plant Cell. Physiol. 35, 1221-1229. Watanabe, M., Ono, T., Hatakeyama, K., Takayama, S., Isogai, A. and Hinata, K. 1997. Molecular characterization of SLG and S-related genes in a self-compatible Brassica campestris L. var. yellow sarson. Sex. Plant Reprod. 10, 332-340. Watanabe, M., Watanabe, M., Suzuki, G., Shiba, H., Takayama, S., Isogai, A. and Hinata, K. 1998. Seqeunce comparison of four SLR1 alleles in Brassica campestris (syn. B. rapa). Sex. Plant Reprod. 11,295-296. Wolters-Arts, M., Lush, W. M. and Mariani, C. 1998. Lipids are required for directional pollen-tube growth. Nature 392, 818-821. Yamakawa, S., Watanabe, M., Isogai, A., Takayama, S., Satoh, S., Hinata, K. and Suzuki, A. 1993. The cDNA sequence of NS3-glycoprotein from Brassica campestris and its homology to related proteins. Plant Cell Physiol. 34, 173-175.
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Biology of Brassica Coenospecies C. G6mez-Campo (Editor) 91999 Elsevier Science B.V. All rights reserved.
185
6 MALE STERILITY R6gine D e l o u r m e (1) a n d Fran~oise B u d a r (2)
(1) INRA, Station d'Am~lioration d e s Plantes, BP 29, 3 5 6 5 3 Le Rheu Cedex, France (2) INRA, Station de G~n~tique et d'Am~lioration d e s Plantes, Route de St-Cyr, 7 8 0 2 6 Versailles Cedex, France The value of h y b r i d s in the breeding of Brassica crops h a s s t i m u l a t e d interest in r e s e a r c h on male sterility. This h a s r e s u l t e d in a large a m o u n t of literature c o n c e r n i n g all a s p e c t s of male sterility (cytological a n d physiological analysis, m o l e c u l a r analysis, interspecific transfer). These s t u d i e s have d r a w n benefit from f e a t u r e s of the Brassicaceae family (some of which are covered in o t h e r c h a p t e r s of this publication) including the relative ease with which it c a n be m a n i p u l a t e d in a b r o a d r a n g e of genetic a n d biotechnological e x p e r i m e n t s , s u c h as i n t e r s p e c i f i c / i n t e r g e n e r i c crosses, in vitro culture, somatic hybridization, a n d genetic engineering. The fact t h a t m i t o c h o n d r i a l gen o m e s of Brassica are a m o n g the s m a l l e s t a n d the s i m p l e s t k n o w n a m o n g Angiosperms, h a s u n d o u b t e d l y facilitated m o l e c u l a r a n a l y s e s of cytoplasmic male sterility. Some good reviews on male sterility in Brassica have b e e n p u b l i s h e d in the p a s t y e a r s (Kaul, 1988; Shiga, 1980; Stiewe et al., 1995b). In this c h a p ter we will specifically focus on s t u d i e s on male sterility or its application in r e c e n t years.
Genic male sterility The first genic male sterility d e s c r i b e d in Brassica species w a s found in green s p r o u t i n g broccoli (Anstey a n d Moore, 1954). It h a s b e e n later described in m o s t of the cultivated b r a s s i c a s (Table 6.1) a n d in the majority of c a s e s it is i n h e r i t e d as a monogenic recessive c h a r a c t e r . Only four monogenic d o m i n a n t male sterilities have b e e n reported, two in B. oleracea (Dunem a n n a n d G r u n e w a l d t , 1991; Ruffio-Chable et al., 1993), one in B. rapa (Van der Meer, 1987) a n d one in B. n a p u s (Mathias, 1985a). A digenic male sterility s y s t e m h a s been identified in C h i n a (Li et al., 1988): one d o m i n a n t gene (Ms) i n d u c e s male sterility a n d male fertility c a n be r e s t o r e d by a n o t h e r
186 dominant gene (Rf). Other cases of genic male sterility reported in China are controlled by two or three recessive genes (Fu and Yang, 1995). The male sterile plants have mainly arisen as spontaneous mutants. However, one recessive male sterility in B. n a p u s (Takagi, 1970) and one dominant male sterility in B. o l e r a c e a (Dunemann and Grunewaldt, 1991) were obtained through mutagenic treatment. More recently, Plant Genetic Systems has developed a genic male sterility system by genetic engineering. Mariani et al. (1990) fused the tapetum-specific promoter T A 2 9 from tobacco (Goldberg, 1988) to the coding regions of two genes encoding RNases: barna s e from B a c i l l u s a m y l o l i q u e f a c i e n s and R N a s e T1 from A s p e r g i l l u s oryzae. Male sterile B. n a p u s plants were produced by transferring these chimaeric genes into the plant genome via A g r o b a c t e r i u m t u m e f a c i e n s . The male sterile plants with the b a r n a s e construction can be restored to fertility by crossing to restorer lines carrying the b a r s t a r gene, encoding a specific inhibitor for b a r n a s e (Mariani et al., 1992). The T A 2 9 - b a r n a s e construct was also introduced into B. oleracea var botrytis and male sterile transformants were obtained (Reynaerts et al., 1993). In most histologically described male sterilities, microsporogenesis breaks down at the tetrad stage and sometimes at the uninucleate vacuolate microspore stage (Table 6.1). These two stages of degeneration are observed in the d o m i n a n t male sterility of Mathias (1985a) and Ruffio-Chable et al. (1993) depending on the temperature conditions (Theis and R6bbelen, 1990). The digenic male sterility of Li et al. (1988) shows a specific degeneration process: the two meiotic divisions and the s u b s e q u e n t cytodieresis are disturbed (micro-nuclei formation) or stopped (Zhou, 1990). When male sterility is complete, the meiosis is completely stopped and the disturbed nuclear envelope does not perfectly separate the nuclear chromatin from the surrounding cytoplasm. In the engineered male sterility of Mariani et al. (1990), the disruption of microsporogenesis is correlated with the activity of the chimeric T A 2 9 - R n a s e or b a r n a s e genes (De Block and Debrouwer, 1993; Denis et al., 1993). Degeneration of the tapetal RNA takes place after microspore release from the tetrads and is immediately followed by the disappearance of the RNA from the microspores, after which they collapse. This confirms the intimate relationship of the tapetum and microspores during their development.
Cytoplasmic male sterility Many cytoplasmic male sterility (CMS) systems have been elaborated in the cultivated B r a s s i c a species. Table 6.2 summarizes the extensive research work that has been developed in this field mainly during the last 25 years. As cytoplasmic male sterility is the result of specific nuclear/mitochondrial interactions, the association of cytoplasm and nucleus from different species often results in total or partial male sterility, sometimes associated with other striking effects on floral morphology. In this section we will describe how the most popular CMS systems were obtained, their morphology, and
Table 6.1 Genic male sterilities in the Brassiceae (Cruciferae) Species
References
Inheritance
Origin
Anstey and Moore (1954); Cole (1959)
1 recessive gene ms-1
spontaneous
Dickson (1970)
1 recessive gene ms-6
spontaneous
Dunemann and Grunewaldt (1991)
1 dominant gene
mutagenic treatment
Nieuwhof (1961)
1 recessive gene ms-5
spontaneous
Borchers (1 966)
1 recessive gene ms-4
spontaneous
Chatterjee and Swarup (1972)
1 recessive gene
spontaneous
Ruffo-Chable et al. (1993)
1 dominant gene
spontaneous
Reynaerts er al. (1993)
1 dominant gene
genetic engineering
Nishi and Hiraoka (1958)
1 recessive gene
spontaneous
Rundfeldt (1960)
1 recessive gene
spontaneous
Johnson (1958)
1 recessive gene ms-2
spontaneous
Nieuwhof ( 1961; 1968)
1 recessive gene
spontaneous
Das and Pandey ( 1961)
1 recessive gene
spontaneous
Chowdhury and Das (1967; 1968)
recessive genes
spontaneous
Microsporogenesis breakdown stage
Brassica oleracea
var italica
var botrytis
var capitata
var gemmifera
after microspore release
tetrad - microspore *
tetrad
tetrad
Brassica rapa
brown sarson
tetrad tetrad / anther indehiscence
yellow sarson
Chowdhury and Das (1966; 1968)
1 recessive gene
spontaneous
var pekinensis
Van der Meer (1987)
1 dominant gene
spontaneous
Takahata ei al. (1996)
1 recessive gene
spontaneous
uninucleate microspore
Brassica juncea
Banga and Labana (1 983)
I recessive gene
spontaneous
vacuolate microspore
Brassica napus
Takagi (1 970)
2 recessive genes
mutagenic treatment
Heyn (1973)
2 recessive genes
spontaneous
Mathias (1985a)
1 dominant gene
spontaneous
Li et al. (1988)
digenic
spontaneous
Mariani ei a/. ( 1 990; 1992)
digenic
genetic engineering
Theis and Robbelen (1990)
1 recessive gene
N P Z system
vacuolate microspore
Raphanus salivus
Tokumasu (1951; 1957)
1 recessive gene
spontaneous
late prophase I of meiosis
* Ruffo-Chable, 1994
** Theis and Robbelen (1990)
*** Zhou,
**** Denis eial.. 1993
1990
tetrad
tetrad
**
tetrad or vacuolate microspore meiosis
**
***
vacuolate microspore
** ** **
189 nuclear restoration of fertility. We will go on to describe the recently acquired knowledge on the molecular basis of three of these systems. Establishment
of CMS
Most of these CMS s ys t em s have been obtained after transfer of the n u c l e u s of the studied species into the cytoplasm of an alien species, and t h u s result from alloplasmy. Only in a few cases, CMS h a s arisen spontaneously within the species as in Chinese cabbage (Okhawa and Shiga, 1981) a n d in rapeseed i.e. 'Polima' system (Fu, 1981) and ' S h a a n 2A' system (Li, 1986 in Fu an d Yang, 1995). The male sterility system first described by Ogura (1968) in an open pollinated radish cultivar h a s been recently reported to be widespread in wild J a p a n e s e populations of radish and in some specimens of wild R a p h a n u s raphanistrum (Yamagishi and Terachi, 1997). This probably corresponds to a s p o n t a n e o u s male sterility in this species. Another CMS system occurred s p o n t a n e o u s l y in a field trial of B. j u n c e a in India (Rawat and Anand, 1979) but, recently, molecular analyses of the cytoplasm showed t h a t this system probably results from s p o n t a n e o u s intergeneric hybridization between B. tournefortii and B. j u n c e a (Pradhan et al., 1991). In a few other cases, CMS h a s been obtained after intraspecific crosses. The first reports of CMS in rapeseed originated from intraspecific crosses using 'Bronowski' (Thompson, 1972) or 'Hokuriku 23' (Shiga and Baba, 1973) as male parents. This CMS is know n as the nap system. In the same way, O k h a w a (1984) identified a CMS in B. rapa close to this nap system. All the other reported CMS s y s t e m s result from interspecific or intergeneric crosses. In some cases they have been obtained by transferring a previously identified male sterility-inducing cytoplasm to the desired species. Thus, the male sterility-inducing cytoplasm of radish originally found by Ogura (1968) was successfully transferred to B. oleracea, B. n a p u s (Bannerot et al., 1974) a n d B. j u n c e a (Labana and Banga, 1989) and induced CMS in all these species. CMS p la n ts of B. n a p u s were also produced by crossing a male sterile radish line to B. n a p u s (Paulmann and R6bbelen, 1988). In radish, this male sterility was decribed as genic male sterility (Tokumasu, 1951). Its transfer to B. n a p u s resulted in cytoplasmic male sterility. Two h y p o t h e s e s might be drawn: either it was a non identified CMS in radish or the resulting CMS is due to the created alloplasmic situation. CMS s y s t e m s can also arise after crossing two male fertile species and can then be transferred to a n o t h e r species. The CMS obtained in B. oleracea with the cytoplasm of B. nigra (Pearson, 1972), in B. rapa with the cytoplasm of Diplotaxis muralis (Hinata and Konno, 1979) or in B. j u n c e a with the cytop l a s m s of B. tournefortii (Rawat and Anand, 1979) and Diplotaxis siifolia (Rao et al., 1994) were then transferred to B. n a p u s (Mathias, 1985b; Pellan-Delourme et al., 1987; Rao and Shivanna, 1996). These interspecific crosses were performed t h r o u g h sexual hybridization. More recently, protoplast fu-
190 sion between more or less related species was used by Kameya et al. (1989) in B. oleracea, Sakai and I m a m u r a (1990) in B. napus, Mukhopadhyay et al. (1994) in B. carinata, and by Kirti et al. (1995a) and Prakash et al. (1995, 1998a) in B. j u n c e a to acquire new CMS systems. Protoplast fusion was used by Yarrow et al. (1990) to transfer the 'Polima' cytoplasm from B. n a p u s to B. oleracea and by Cardi and Earle (1997) to transfer the B. tournefortii cytoplasm from B. rapa to B. oleracea. Protoplast fusion was also extensively used to induce new combinations of cytoplasmic traits. Male sterility has often been obtained by introducing the genome of one species into an alien cytoplasm and results from mitoc h o n d r i a / n u c l e u s interactions, but at the same time some defects may appear from chloroplast/nucleus or m i t o c h o n d r i a / n u c l e u s incompatibilities. When the male sterility-inducing cytoplasm of radish (Ogura, 1968) was introduced into B. oleracea, B. n a p u s , B. rapa and B. juncea, male sterile lines were obtained, b u t these showed a severe chlorophyll deficiency and low nectar secretion. This CMS system was first improved in B. n a p u s by protoplast fusion (Pelletier et al., 1983). Male sterile cybrids with normal photosynthesis and improved nectar secretion were obtained through chloroplast exchange and mitochondrial recombination. One of these cybrid cytoplasms was introduced to B. rapa and B. j u n c e a to produce male sterile lines (Delourme et al., 1994b). Then, similar experiments were performed on 'Ogura' CMS in B. n a p u s (Jarl and Bornman, 1988; Jarl et al. 1989; Menczel et al., 1987) as well as in B. oleracea (Kao et al., 1992; Pelletier et al., 1989; Waiters et al., 1992; Waiters and Earle, 1993) and in B. j u n c e a (Kirti et al., 1995a). Improved 'Ogura' cytoplasms were then transferred to vegetable B. rapa (Heath et al., 1994) and cabbage (Sigareva and Earle, 1997). Likewise, male sterile plants of B. rapa and B. j u n c e a carrying B. oxyrrhina cytoplasm have delayed flowering and chlorotic leaves (Prakash and Chopra, 1990). Chlorosis correction in B. j u n c e a was achieved through protoplast fusion (Kirti et al., 1993). Stiewe and R6bbelen (1994) and Liu et at. (1996) fused B. n a p u s and X-ray treated B. tournefortii protoplasts and obtained cybrids with B. n a p u s chloroplasts and B. tournefortii or recombined mitochondria. Development of improved CMS lines through protoplast fusion is also in progress in B. j u n c e a for B. tournefortii and B. oxyrrhina CMS (Pradhan et al., 1995). Protoplast fusion was also an efficient way of combining atrazine resistant chloroplasts of B. rapa with the CMS trait of B. nigra cytoplasm in B. oleracea (Christey et al., 1991) and with nap cytoplasm (Yarrow et al., 1986), 'Polima' cytoplasm (Barsby et al., 1987) or 'Ogura' cytoplasm (Jourdan et al., 1989) in B. napus.
Flower morphology and microsporogenesis Two main stages of microsporogenesis inhibition have been observed. For the nap, 'Polima' and tournefortii CMS, a premeiotic inhibition of microspore division and cell differentiation is already observed in the archespore
191 tissue when male sterility is complete (Theis and R6bbelen, 1990; Mishra and Anand, 1985; Gourret and Delourme, u n p u b l i s h e d results). In these systems, male sterile flowers are also characterized by narrow petals, and the stability of the male sterility is largely dependent on the environment or on maintainer lines. Fertile pollen grains may be produced at temperatures higher than 25-30~ in nap and 'Polima' systems (Burns et al., 1991; Fan and Stefansson, 1986) or at low temperatures depending on the maintainer lines in 'Polima' system (Fu et al., 1990). After protoplast fusion, Stiewe and R6bbelen (1994) obtained a cybrid line whose male sterility was more stable at high temperature than that of plants with the original B. tournefortii cytoplasm, but its petals were also narrower. For the B. oxyrrhina system, flowers have a normal appearance but short and u n d e h i s c e n t anthers; microsporogenesis breaks down after the tetrad stage (Prakash and Chopra, 1990). In the CMS system induced by 'Tokumasu' cytoplasm in B. n a p u s , degeneration also begins after the tetrad stage leading to empty exins and t a p e t u m walls in the a n t h e r s (Theis and R6bbelen, 1990). Gourret et al. (1992) compared the expression of 'Ogura' CMS in cybrids of B. n a p u s and in male sterile lines of B. n a p u s with the original 'Ogura' cytoplasm (from radish, R a p h a n u s sativus). Their results confirmed the observations made by Ogura (1968) in radish i.e. an excessive vacuolization of the tapetal cells leading to their degeneration prior to the sudden collapse of the microspores at the uninucleate stage. This expression of male sterility was attributed to the 'Ogura' male sterility already present in radish. In addition, reduction in n u m b e r and size of the microsporangia and feminization of the androecium (carpelloidy) were observed in the B. n a p u s plants with 'Ogura' cytoplasm and in some cybrids (Gourret et al., 1992). Polowick and Sawhney (1987; 1990; 1991) have shown that low or moderate temperature during bud development favors feminization of the androecium of B. n a p u s with 'Ogura' cytoplasm. This feminization was related to the expression of a second independent male sterility due to alloplasmy (Gourret et al., 1992). These two CMS determinants i.e. 'Ogura' male sterility and alloplasmic male sterility have been hypothetized after the study of the different cybrids produced (Pelletier et al., 1987; Primard et al., 1992). Other alloplasmic male sterilities also show an abortive pattern that includes feminization of the androecium e.g. Diplotaxis muralis CMS in B. n a p u s (PellanDelourme and Renard, 1987), B. tournefortii CMS in B. j u n c e a (Mishra and Anand, 1985) and in B. n a p u s (Gourret and Delourme, u n p u b l i s h e d data) or B. nigra CMS in B. n a p u s (Pellan-Delourme et al., 1987). In this latter CMS, petaloid s t a m e n s were also observed in cabbage B. oleracea (Pearson, 1972) and in rape B. n a p u s (Pellan-Delourme et al., 1987). Some male sterile plants with petaloid s t a m e n s were also produced in B. rapa by Mekinayon et al. (1994, 1995) with the cytoplasm of rocket (Eruca vesicaria subsp, sativa or in B r a s s i c a j u n c e a by Kirti et al. (1995b) with the cytoplasm of wild Trachystom a ballii.
192
Male fertility r e s t o r a t i o n The genetics of male fertility r e s t o r a t i o n is indicated in Table 6.2. When r e s t o r e r genes have b e e n identified, they always act as d o m i n a n t genes. The d e t e r m i n i s m is m o n o g e n i c or oligogenic according to the male sterility-inducing cytoplasm. For the nap CMS system, various n u m b e r s of r e s t o r e r genes have been found according to the cultivars s c r e e n e d (Rousselle a n d Renard, 1982; Shiga, 1976). The genetics of the 'Polima' s y s t e m is m u c h more simple since it is monogenic. Restorer genes have been found in a s u m m e r oilseed rape variety 'Italy', in the line 'UM2353' (Fang a n d McVetty, 1989) a n d in B. j u n c e a 'Zem' variety (Fan a n d S t e f a n s s o n , 1986). In C h i n a a n d India, several restoring lines were selected in B. n a p u s species (Banga a n d Gurjeet, 1994; Yang a n d Fu, 1990). F a n g a n d McVetty (1989) reported t h a t the two r e s t o r e r genes originating from 'Italy' a n d 'UM2353' were non allelic a n d unlinked. Recently, J e a n et al. (1997) identified DNA m a r k e r s linked to these two genes (named R f p l a n d Rfp2, respectively) a n d to a restorer gene of the nap s y s t e m (Rfn) (Jean, 1995). One of t h e s e m a r k e r s co-segregated perfectly with R f p l , Rfp2 a n d Rfn, indicating t h a t the three genes m u s t be at least tightly linked to one a n o t h e r a n d m a y reside at the s a m e locus. The b a s i s of the d i s c r e p a n c y between these r e s u l t s a n d those of McVetty et al. (1989) is still not clear. If the three genes reside at the s a m e locus, the allele of Rfn capable of restoring the nap c y t o p l a s m would be a m a i n t a i n e r allele for the 'Polima' CMS (Jean, 1995). For the B. tournefortii system, genotypes partially restoring male fertility have been found in B. nigra a n d B. rapa for male sterile lines of B. j u n c e a (Anand et al., 1986). These restorer genes have been b r o u g h t together in the B. j u n c e a g e n o m e a n d completely restoring g e n o t y p e s have been obtained (Angadi a n d A n a n d , 1988). In B. napus, genotypes partially restoring the male fertility of this CMS have been identified, s u c h as Asian genotypes 'Mokae' a n d 'Yudar (Delourme et al., u n p u b l i s h e d data) or a fodder winter variety 'Arvor' ( B a r t k o w i a k - B r o d a et al., 1991). The genetic d e t e r m i n i s m seems to be oligogenic. In the r e s t o r e r lines isolated by B a n g a et al. (1994), one or two r e s t o r e r genes are hypothezized. Sodhi et al. (1994) also identified restorer genes in 'Yudar as well as in a n o t h e r Asian variety 'Mangun', a n d concluded t h a t the i n h e r i t a n c e w a s monogenic. With these latter two genotypes, Stiewe et al. (1995a) o b t a i n e d restored p l a n t s with i n t e r m e d i a t e fertility a n d the det e r m i n i s m did not s e e m to be simple. These genotypes also partially restored the male sterile cybrid line developed by Stiewe a n d R6bbelen (1994). Stiewe et al. (1995a) a n d Pental et al. (1995) also a t t e m p t e d to t r a n s f e r restorer genes from B. tournefortii to B. n a p u s or to B. j u n c e a via B. tournefortii / B. rapa h y b r i d s or via somatic hybridization. Plants with nearly n o r m a l fertility were found in the BC 1 generation (Stiewe et al., 1995a).
193 For the 'Ogura' CMS, no restorer has been found in any of the Brassica species (Rousselle, 1982). Restorer genes were identified in E u r o p e a n radish varieties (Bonnet, 1975). They were introduced into rapeseed from a R a p h a nobrassica (Heyn, 1976). Segregation studies with the derived B. n a p u s restorers showed t h a t the original 'Ogura' cytoplasm needed several restorer genes to be restored to fertility b u t subsequently, protoplast fusion t h r o u g h mitochondrial recombination m ade it possible to simplify the genetics of the CMS system. Fully restored plants with only one d o m i n a n t restorer allele (Rfo) could be selected on the best cybrid cytoplasms (Pellan-Delourme, 1986; Pelletier et al., 1987). Those cybrids have been shown to bear the det e r m i n a n t inducing sterility in the original 'Ogura' radish, whereas a more complex genetic system for restoration is e n c o u n t e r e d w hen this 'Ogura' det e r m i n a n t is combined with the alloplasmic sterility due to the association of a B r a s s i c a n u c l e u s with radish type mitochondria (Pelletier et al., 1987; Prim a r d et al., 1992). The introduction of the Rfo gene into CMS lines of B. rapa and B. j u n c e a (with 'Ogura' cybrid cytoplasm) is in progress (Delourme et al., 1994b). An isozyme (Pgi-2) gene (Delourme and Eber, 1992) a n d DNA markers (Delourme et at., 1994a) were found to be perfectly linked to the Rfo gene. Such m a r k e r s can be us ed for m a r k e r assisted selection of restorer lines (Hansen et al., 1997). Recently the radish introgression carrying the Rfo gene was characterized (Delourme et al., 1998). This Rfo gene h a s been found to be widely distributed in the wild radish populations which the Ogura cytop l as m is s u p p o s e d to originate from (Yamagishi, 1998). In the other CMS B. n a p u s systems derived from radish, restorer gene(s) were also introduced from radish. P a u l m a n n and R6bbelen (1988) introduced restorer genes t h r o u g h intergeneric crosses for the 'Tokumasu' cytoplasm. Sakai et al. (1996) transferred a restorer gene by protoplast fusion for the 'Kosena' CMS system. More studies are needed to know w het her this restorer gene is the same. For m o s t of the other CMS systems, the genetics of restoration is u n k n o w n or no restorers are yet available, especially for those derived from wide interspecific or intergeneric crosses. Nevertheless, restorer genes could recently be introgressed into B. j u n c e a or B. n a p u s from the cytoplasm donor species i.e. T r a c h y s t o m a baltii (Kirti et al., 1997), Moricandia arvensis (Prak a s h et al., 1998b) and B. oxyrrhina (Banga and Banga, 1998). Molecular features of some cytoplasmic
male sterilities
The 'O~ura' a n d 'Kosena' systems. Although the mitochondrial genome of the 'Ogura' radish has been extensively analysed and compared to the normal fertile radish one (Makaroff et al., 1989; 1990; 1991; Makaroff and Palmer, 1988) the identification of the mitochondrial d e t e r m i n a n t for this CMS has been possible only after Brassica cybrids were obtained via the protoplast fusion experiments described above (Pelletier et al., 1983).
Table 6.2 Cytoplasmic male sterilities in the Brussicaceue Transfer method
Fertility restoration
B. rapa
lnterspecific cross
2 dominant genes
Pearson (1972) Christey et a/. (1991)
B nigra
lnterspecific cross Protoplast fusion
2 dominant genes
Chiang and Crete ( 1 987)
B. napus
lnterspecific cross
unknown **
Species
References
Brassica aleracea
Nishi and Hiraoka (1958)
Yarrow et a/. (1 990) McCollum (1981) Bannerot et al. (1974) Pelletier ef al. (1989) Kao el 01. (1992) Walters et al. (1992) Kameya ef al. (1989) Brassica rapa
B.napus (Polima) R.sativus ('Early Scarlet Globe') R. sofivus (Ogura)
R. safivus ('Shougoin')
-*
Protoplast fusion Intergeneric cross Intergeneric cross Protoplast fusion
unknown no restorer
Protoplast fusion
Okhawa and Shiga (1981)
B. napus
spontaneous
2 dominant genes
Okhawa (1984)
Brapa '14'
lntraspecific cross
unknown
B. oxyrrhina
Interspecific cross
Prakash and Chopra ( 1990) Delourme et a/. (1994)
Brassica juncea
Cytoplasm origin
modified 'Ogura' in B. napus
lnterspecific cross
1 restorer gene unknown
Hinata and Konno (1979)
Diplofaris muralis
Intergeneric cross
Mekiyanon eta/. (1994; 1995)
Eruca sativa
Intergeneric cross
Rawat and h a n d (1979) h a n d et al. (1986)
B. fournefortii
Alloplasmy
Prakash and Chopra (1990) Kirti ef a/. ( I 993)
B. oxyrrhina
lnterspecific cross Protoplast fusion
Labana and Banga (1989) Kirti et al. (1995a) Delourme et al. (1994)
R. sativus (Ogura)
Intergeneric cross Protoplast fusion Interspecific cross
no restorer
Intergeneric cross
unkonwn
Rao et al. (1994)
modified 'Ogura' in B. napus Diplotaxis siifolia
4 dominant genes
I restorer gene
Brassica juncea
Brassica napus
Kirti et al. (1995b)
Trachystoma ballii
Protoplast fusion
no restorer
Prakash et al. (1995)
Moricandia arvensis
Protoplast fusion
no restorer
Prakash et al. (1995)
Diplotaxis catholica
Protoplast fusion
no restorer
Prakash et al. (1995)
Sinapis alba
Protoplast fusion
no restorer
Thompson (1 972) Rousselle and Renard (1982) Shiga and Baba(1973); Shiga(1976)
B. napus
Intraspecific cross
1 to 2 genes
Fu (1981); Yang and Fu (1990) Barshy et al. (1987)
Polima
spontaneous Protoplast fusion
1 dominant gene
Li(1986)inFuandYang(1995)
Shaan 2A
spontaneous
unknown
Pellan-Delourme et al. (1987)
B.nigra
Interspecific cross
unknown
Mathias (1985b) Battkowiak-Broda et al. (1991) Sodhi et al. (1 994) Stiewe and Robbelen (1994) Liu et al. ( I 996)
B.tournefortii
Interspecific cross
Bannerot et al. (1974) Pelletier el al. (1983; 1987) Jar1 et al. ( 1988) Jourdan et al. (1989) Paulmann and Rdbbelen (1988) Sakai and Imamura (1990); Sakai et al. (1 996)
Brassica carinata
1 to 4 genes
oligogenic I dominant gene Protoplast fusion Protoplast fusion R.sativus (Ogura)
Intergeneric cross Protoplast fusion
oligogenic 1 dominant gene
R.sativus (Tokumasu) R.sativus (Kosena)
Intergeneric cross Protoplast fusion
unknown 1 dominant gene
Pellan-Delourme and Renard (1987)
Diplotaxis muralis
Intergeneric cross
unknown
Rao and Shivanna (1996)
Diplotaxis siijolia
Interspecific cross
no restorer
h a n d (1987)
B. tournefortii
Interspecific cross Protoplast fusion
unknown unknown
Rmtivus (Ogura)
spontaneous
1 dominant gene
Mukhopadhyay et al. (1994) Raphanus sativus
Ogura (1968)
* not studied
** restorer genes have been identified but the genetics was not determined
196 These fusion e x p e r i m e n t s not only gave the B. n a p u s a n d B. oleracea cytotypes now m o s t extensively u s e d for the c o m m e r c i a l p r o d u c t i o n of hybrid seeds, b u t also offered the best m a t e r i a l for the m o l e c u l a r c h a r a c t e r i z a t i o n of the sterility d e t e r m i n a n t . B o n h o m m e et al. (1991; 1992) identified a mitoc h o n d r i a l gene, orf138, originally found in the 'Ogura' r a d i s h mitochondrial genome, a n d w h o s e e x p r e s s i o n at the RNA level is strictly correlated with the sterile p h e n o t y p e of B. n a p u s a n d B. oleracea cybrids (in the a b s e n c e of a n y r e s t o r e r gene). E x p r e s s i o n of this m i t o c h o n d r i a l gene h a s since been associated with the sterile p h e n o t y p e of 'Ogura' r a d i s h ( K r i s h n a s a m y and Makaroff, 1993). The orf138 gene is e x p r e s s e d in r a d i s h a n d m o s t B. napus cybrids on a bicistronic RNA also encoding orfB. The orf138 gene p r o d u c t is a m e m b r a n e b o u n d protein which is p r o b a b l y a s s o c i a t e d in oligomers in the m i t o c h o n d r i a l m e m b r a n e (Grelon et al., 1994; K r i s h n a s a m y a n d Makaroff, 1994). The p l a n t s carrying the orf138 gene in their m i t o c h o n d r i a l genome exhibit the O R F 1 3 8 protein in all the organs. However, in male-sterile radish, the O R F 1 3 8 protein h a s been r e p o r t e d to be highly a c c u m u l a t e d in roots relative to the a l f a - s u b u n i t of m i t o c h o n d r i a l ATPase ( K r i s h n a s a m y a n d Makaroff, 1994) w h e r e a s in B. n a p u s cybrids, its a c c u m u l a t i o n seems to follow the a b u n d a n c e of n o r m a l m i t o c h o n d r i a l proteins in the different o r g a n s of the p l a n t (Bellaoui et al., in preparation). The r e s t o r a t i o n of fertility by n u c l e a r r e s t o r e r s h a s a d r a m a t i c effect on the a c c u m u l a t i o n of the ORF138 protein in b u d s a n d leaves in r a d i s h ( K r i s h n a s a m y a n d Makaroff, 1994). In B. napus, r e s t o r a t i o n is a c c o m p a n i e d with the abolition of ORF138 a c c u m u lation in a n t h e r s (Bellaoui et al., in preparation). In both species, the restoration of fertility does not affect at all the level of a c c u m u l a t i o n of the mitochondrial m e s s e n g e r RNA of the orf138 gene, indicating t h a t the m e c h a n i s m of r e s t o r a t i o n is acting either on the t r a n s l a t i o n efficiency of the m e s s e n g e r or on the stability of the ORF138 protein ( K r i s n a s a m y a n d Makaroff, 1994; Bellaoui et al., in preparation). Most if not all of m i t o c h o n d r i a l male sterility a s s o c i a t e d genes described so far p r e s e n t a c h i m a e r i c s t r u c t u r e where p a r t s of n o r m a l mitochondrial genes c a n still be recognized, strongly suggesting t h a t they occured by genetic r e c o m b i n a t i o n inside the m i t o c h o n d r i a l genome, even if some of their s e q u e n c e is of u n k n o w n origin (Braun et al., 1992; H a n s o n , 1991). The orf 138 gene h a s long been entirely of " u n k n o w n origin", since its sequence does not significantly r e s e m b l e a n y t h i n g in the d a t a b a s e s (Bonhomme et al., 1992). The complete s e q u e n c i n g of the m i t o c h o n d r i a l genome of Arabidopsis thaliana recently allowed the identification of the end of the coding sequence a n d 3' flanking region of the orf138 gene as identical (70 nucleotides including the last 12 codons) to the 3' u n t r a n s l a t e d region of Arabidopsis thaliana orf557 p r o b a b l y encoding an NADH d e h y d r o g e n a s e s u b u n i t (Bellaoui et al., 1998). Recently, the B. n a p u s orf577 gene, homologous to the bacterial ccll gene h a s been described by H a n d a et al. (1996) a n d M e n a s s a et al., (1997). This gene is very similar to the A. thaliana orf557 a n d also p r e s e n t s in its 3' u n t r a n s l a t e d region the s a m e s h o r t a n d perfect homology to orf138. So the orf138 gene m i g h t also be a r e s u l t of m i t o c h o n d r i a l genetic recombination.
197 The O R F 1 3 8 protein p o s s e s s e s a h y d r o p h o b i c N-terminal region, probably c o m p r i s i n g a m e m b r a n e s p a n n i n g domain, a n d a highly hydrophilic Ct e r m i n a l region s t r u c t u r e d in three repetitions of 13 a m i n o - a c i d s . The 'Kosena' cultivar of radish, as well as some wild s a m p l e s exhibiting O g u r a - t y p e m i t o c h o n d r i a , p o s s e s s a deleted orf138 gene where one repetition of the hydrophilic region of the protein h a s been lost (Sakai et al., 1995; Yamagishi a n d Terachi, 1996). However, it is not clearly e s t a b l i s h e d w h e t h e r this t r u n c a t e d ORF138 is responsible for the male sterility p h e n o t y p e in the 'Kosena' cultivar. The 'Polima' system. The pol m i t o c h o n d r i a l genome shows some r e a r r a n g e m e n t s c o m p a r e d to the n o r m a l B. napus (nap) one (Handa a n d Nakajima, 1992; Singh a n d Brown, 1991; Witt et al., 1991). Differences in the t r a n s c r i p t p a t t e r n of the atp6 gene b e t w e e n sterile, fertile a n d r e s t o r e d p l a n t s were detected a n d directed i n t e r e s t t o w a r d s this region of the genome as the possible determ i n i n g locus of 'Polima' sterility. In the pol m i t o c h o n d r i a l genome, the atp6 gene is c o t r a n s c r i b e d with (and d o w n s t r e a m of) a new gene c a p a b l e of encoding a 224 a m i n o a c i d protein, orf224. The first 58 codons of the orf224 gene are highly similar to the a m i n o - t e r m i n a l coding region of the orfB gene, followed by 43 bp of homology with the last exon 1 of rps3, the r e s t of the seq u e n c e r e m a i n i n g of u n k n o w n origin. Therefore, this C M S - a s s o c i a t e d gene h a s probably been g e n e r a t e d by m i t o c h o n d r i a l genetic r e c o m b i n a t i o n s . The putative protein p r o d u c t of the orf224 gene h a s not been detected in sterile p l a n t s so far. In the p r o g e n y of crosses segregating sterile a n d r e s t o r e d p l a n t s with the pol cytoplasm, the bi-cistronic t r a n s c r i p t orf224-atp6 h a s b e e n correlated with the sterile p h e n o t y p e of the plants, w h e r e a s in fertile r e s t o r e d plants, a m o n o c i s t r o n i c atp6 t r a n s c r i p t p r o d u c e d by RNA p r o c e s s i n g is a c c u m u l a t e d . Singh et al. (1996} have elegantly d e m o n s t r a t e d t h a t the r e s t o r e r gene of 'Polima' c y t o p l a s m is a n allele of a locus influencing RNA p r o c e s s i n g of some m i t o c h o n d r i a l t r a n s c r i p t s in B. napus.It Mapping e x p e r i m e n t s on the n o r m a l B. napus c y t o p l a s m s u g g e s t e d t h a t s e q u e n c e s similar to orf224 are also pres e n t in these m i t o c h o n d r i a , b u t not a s s o c i a t e d with the atp6 gene (L'Homme a n d Brown, 1993}. Hence, the e m e r g e n c e of the sterile p h e n o t y p e could be d u e to the a s s o c i a t i o n of the orf224 coding s e q u e n c e with e x p r e s s i o n signals of the atp6 gene Recently, the e x p r e s s i o n of a new gene, orf522, a s s o c i a t e d with the nad 5c region a n d potentially encoding a peptide which s h a r e s 79% s e q u e n c e similarity to the predicted p r o d u c t of orf224 h a s b e e n a s s o c i a t e d with nap CMS in B. napus (L'Homme et al., 1997). Even more interesting, it a p p e a r s t h a t the locus previously a s s o c i a t e d with 'Polima' fertility r e s t o r a t i o n a n d involved in m i t o c h o n d r i a l t r a n s c r i p t m a t u r a t i o n is also allelic to the r e s t o r e r for nap CMS (Li et al., 1998}. Hence, this is the first e x a m p l e of two CMS
198 s y s t e m s being r e s t o r e d by two different alleles of the s a m e locus a n d associated with m i t o c h o n d r i a l genes which are probably evolutionarily related. The B. tournefortii system. The m o l e c u l a r b a s i s of this s y s t e m h a s been explored by a survey of the e x p r e s s i o n of m i t o c h o n d r i a l genes in p l a n t s with the s a m e B. tournefortii c y t o p l a s m b u t in different n u c l e a r b a c k g r o u n d s , where the sterility is either e x p r e s s e d (B. juncea, B. napus) or not (B. tournefortii, B. napus restored). L a n d g r e n et al. (1996) t h u s showed t h a t the e x p r e s s i o n of the mitochondrial B. tournefortii g e n o m e is extensively affected by the n u c l e a r b a c k g r o u n d . They have correlated alterations of the atp6 t r a n s c r i p t p a t t e r n with the e x p r e s s i o n of the male sterile p h e n o t y p e in the plants, the B. napus a n d B. juncea sterile alloplasmic lines exhibiting a larger RNA molecule revealed with atp6 probe in addition to the n o r m a l m o n o c i s t r o n i c atp6 t r a n s c r i p t found in all the plants. This larger RNA is actually a bi-cistronic m e s s e n g e r which carries a n open reading frame, orf263, d o w n s t r e a m of atp6. The orf 263 gene is, therefore, p r e s e n t b u t not e x p r e s s e d in n o r m a l B. tournefortii plants. It potentially e n c o d e s a 2 9 k D a protein. About one third of the orf263 s e q u e n c e s h o w s homology to a p a r t of the nad5 gene. T h u s , orf263 also s e e m s to be a chimaeric m i t o c h o n d r i a l gene. In organello s y n t h e s e s of proteins revealed a 3 2 k D a polypeptide w h o s e expression s e e m s to be correlated with the sterile p h e n o t y p e of the plants. However, no evidence h a s yet been o b t a i n e d t h a t this polypeptide is indeed the p r o d u c t of the orf263 gene. Analysis of r e s t o r e d B. napus progeny segregating male sterile a n d male fertile p l a n t s showed t h a t the p r e s e n c e of the 32 k D a protein in in organello s y n t h e s e s is correlated with the sterile phenotype, b u t the bicistronic RNA atp6-orf263 is p r e s e n t in all the progeny (Landgren et al., 1996). F u r t h e r e x p e r i m e n t s are now n e c e s s a r y to e n s u r e t h a t the 3 2 k D a protein is the protein e n c o d e d by the orf263 gene. In t h a t case, the r e s t o r a t i o n of fertility in the s t u d i e d B. napus progeny would be effective by a control on protein accum u l a t i o n w i t h o u t affecting the RNA level, which is r e m i n i s c e n t of the situation in the 'Ogura' system. However, one can a s s u m e t h a t n o r m a l B. tournefortii p l a n t s do not exhibit sterility b e c a u s e they can efficiently process the bicistronic m e s s e n g e r atp6-orf263 a n d only a c c u m u l a t e the monocistronic atp6 RNA. In this case, we can predict t h a t B. tournefortii p o s s e s s e s restorer gene(s) which act on the p r o c e s s i n g of the atp6 m e s s e n g e r , r e m i n i s c e n t of the s i t u a t i o n in the 'Polima' system.
Use for t h e p r o d u c t i o n o f c o m m e r c i a l hybrids As we have seen above, a large n u m b e r of male sterility s y s t e m s have been p r o p o s e d for the p r o d u c t i o n of hybrid Brassica seeds. However, m a n y of these s y s t e m s have been s h o w n to p o s s e s s i n h e r e n t d i s a d v a n t a g e s when u s e d in breeding p r o g r a m s .
199
Genic male sterility The u s e of genic male sterility is limited b e c a u s e in m a n y c a s e s the male sterility p h e n o t y p e is u n s t a b l e a n d b e c a u s e male fertile p l a n t s m u s t be d i s c a r d e d j u s t before flowering in h y b r i d seed p r o d u c t i o n fields. However, in B. oleracea var botrytis, the selected male sterile p l a n t s c a n be vegetatively p r o p a g a t e d (in vivo or in vitro). T h u s , it h a s b e e n possible to u s e recessive male sterility for the c o m m e r c i a l p r o d u c t i o n of F1 h y b r i d s a n d a d o m i n a n t male sterility is now u s e d in the s a m e way (Ruffio-Chable, 1994). The digenic male sterility identified by Li et al. (1988) h a s been applied in C h i n a to p r o d u c e 100% male fertile F1 h y b r i d r a p e s e e d varieties from a 100% male sterile line (Figure 6.1). This implied, however, the m a n u a l elimin a t i o n of the male fertile p l a n t s in the female strips d u r i n g m a i n t e n a n c e a n d multiplication of the A line. These h y b r i d varieties have b e e n cultivated on 4 0 , 0 0 0 h a over the last five y e a r s (Li S.L., pers. comm.). O t h e r h y b r i d s b a s e d on different genic male sterilities are registered a n d u s e d for p r o d u c t i o n in C h i n a (Fu a n d Yang, 1995). In the e n g i n e e r e d digenic male sterility of Mariani et al. (1990; 1992), a herbicide r e s i s t a n c e gene is linked to the Ms gene. The male sterile p l a n t s c a n t h e n be selected in a l t e r n a t i n g strip seed p r o d u c t i o n fields by s p r a y i n g the herbicide. Elite Ms a n d Rf alleles have been selected in s u m m e r B. n a p u s after trials p e r f o r m e d in different locations a n d y e a r s a n d have been t r a n s ferred to w i n t e r B. n a p u s a n d also to B. rapa a n d B. j u n c e a (De Both, 1995). Two s u m m e r B. n a p u s F1 h y b r i d s were a c c e p t e d for r e g i s t r a t i o n a n d commercial scale p r o d u c t i o n in F e b r u a r y 1996 in C a n a d a (Joos, 1997). Breeding w i n t e r B. n a p u s a n d B. j u n c e a F1 h y b r i d s is in progress. The d e v e l o p m e n t of s u c h t r a n s g e n i c varieties will d e p e n d on their r e g u l a t o r y approval.
Cytoplasmic male sterility The a c h i e v e m e n t of a n effective CMS s y s t e m h a s often been slowed down or i m p e d e d by various difficulties s u c h as the instability of the male sterility, the a b s e n c e of one of the c o m p o n e n t s of the s y s t e m i.e. the a b s e n c e of m a i n t a i n e r or r e s t o r e r lines, or negative effects of the male sterility-inducing cytoplasm. Instability of male sterility e x p r e s s i o n u n d e r v a r i o u s e n v i r o n m e n t a l conditions is the r e a s o n w h y the nap s y s t e m is not u s e d in B. n a p u s a n d w h y m u c h work h a d to be done on the 'Polima' s y s t e m to obtain good m a i n t a i n e r genotypes. The selection of very few m a i n t a i n e r lines could be achieved by s c r e e n i n g a great n u m b e r of lines in different e n v i r o n m e n t a l conditions ( B a r t k o w i a k - B r o d a et al., 1991) or u s i n g haplodiploidization (BartkowiakB r o d a et al., 1995). In some s y s t e m s s u c h as B. nigra CMS or D. muralis CMS in B. napus, the a b s e n c e or the too rare o c c u r r e n c e of m a i n t a i n e r lines have
200 p r e v e n t e d their u s e for F1 h y b r i d breeding (Pellan-Delourme et al., 1987; P e l l a n - D e l o u r m e a n d Renard, 1987). The male sterility-inducing c y t o p l a s m m a y have negative effects on chlorophyll content, n e c t a r secretion, flower morphology a n d yield. For the 'Ogura' s y s t e m , the chlorophyll deficiency was corrected by obtaining cybrid cytop l a s m s in B. n a p u s a n d B. oleracea. Male sterile cybrid r a p e s e e d plants also s h o w e d improved n e c t a r secretion (Mesquida et al., 1991). T h u s , high yielding male sterile lines c a n be u s e d efficiently in F1 hybrid seed production (Renard et al., 1991, 1992). In the B. tournefortii CMS system, the cybrids p r o d u c e d in B. n a p u s m a y also be useful to correct the light-green color of the leaves of the male sterile p l a n t s with the original B. tournefortii cytoplasm. B. tournefortii c y t o p l a s m h a s been s h o w n to i n d u c e negative effects on some yield c o m p o n e n t s in B. j u n c e a b u t heterotic c o m b i n a t i o n s can be found (Sheikh a n d Singh, 1996). The 'Polima' c y t o p l a s m was also reported to i n d u c e yield p e n a l t y in B. n a p u s (McVetty et al., 1990). Nevertheless, it is possible to get h y b r i d s which exhibit a superior p e r f o r m a n c e c o m p a r e d to the c o n v e n t i o n a l varieties ( B a r t k o w i a k - B r o d a et al., 1995; McVetty et al., 1990). Some male sterile p l a n t s have flowers with small a n d n a r r o w petals e . g . B , n a p u s p l a n t s with 'Polima' or B. tournefortii cytoplasm. This h a s to be t a k e n into c o n s i d e r a t i o n for pollination efficiency since these floral morphology c h a n g e s greatly increase the n u m b e r of "sideworking" bees. Studies carried o u t by McVetty et al. (1989) revealed t h a t this did not lead to a decrease of yield on the male sterile lines b u t this r e s u l t could be due to the design u s e d (alternating A- a n d B-lines) a n d to the flooding of the trials with leaf c u t t e r bees, e n s u r i n g n u m e r o u s visits to each flower. However, these floral morphology c h a n g e s m a k e pollination more u n c e r t a i n in the seed production d e s i g n s generally used, which d e c r e a s e s F1 hybrid seed yield. This led McVetty et al. (1995) to propose a 3A:3R row ratio design a n d 50,000 leaf c u t t e r b e e s . h a -1 for efficient F1 h y b r i d seed p r o d u c t i o n in W e s t e r n Canada. The last r e q u i r e m e n t which s h o u l d be met for oilseed B r a s s i c a crops is the availability of efficient restorer lines. As decribed above, restorer genes have been identified in B. n a p u s for 'Polima' c y t o p l a s m a n d can be easily i n t r o d u c e d in elite lines b e c a u s e of a monogenic inheritance. For B. tournefortii CMS, the genetic d e t e r m i n i s m is not so clear b u t it s e e m s t h a t good r e s t o r e r lines of B. j u n c e a a n d B. n a p u s could be selected (Angadi a n d Anand, 1988; B a n g a et al., 1995). The restoration b r o u g h t by the genotypes 'Mangun', 'Mokae' or 'Yudal' is not complete a n d its t r a n s f e r in double low genetic b a c k g r o u n d s is not very easy. The transfer of a r e s t o r e r gene from B. tournefortii to B. n a p u s is in p r o g r e s s a n d the crosses are being followed by RAPD a n a l y s i s to find selectable m a r k e r s for r e s t o r a t i o n a n d to identify p l a n t s c o n t a i n i n g the m i n i m u m a m o u n t of T genome b a c k g r o u n d (Stiewe et al., 1995a). In 'Ogura' CMS, one d o m i n a n t r e s t o r e r gene is available for the cybrids selected by Pelletier et al. (1983). However, the i n t r o d u c t i o n of this restorer gene from r a d i s h to r a p e s e e d was a c c o m p a n i e d by a d e c r e a s e in seed set
201
Step 1"
Maintenance of the A line
A
I ~s~s~ 50% F
A
I
I ~s~s~, I
I MsMsRfrf I 50% F
50% S
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discarded
MsMsrfrf I
Ms MsRfrf I 50% F
Step 2:
50% S
Multiplication of the A line
A
A
B
msmsffff I 50% F
50% S
discarded
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Step 3:
Production of F1 hybrid seeds A
R
[ Msms rfrf I
X
[ ms ms RfRfl
lOO% s
/
lOO% F
F1 hybrid Ms ms Rf rf ms ms Rf rf 100% F
Figure 6.1 Seed multiplication scheme using a digenic male sterility system.
50% S
202 (Pellan-Delourme and Renard, 1988). It was a s s u m e d t hat restored plants had retained too m u c h radish genetic information a r o u n d the restorer gene or elsewhere in the genome. Improvement of the restorer material was achieved t h r o u g h b ackcr os s and pedigree breeding a n d restorer lines with good female fertility have been obtained (Delourme et al., 1991). However, the length of the introgressed radish segment and the fact t h a t a linkage was found between this introgression and glucosinolate content have slowed down the use of these restorer lines in double low F1 hybrid breeding (Delourme et al., 1995). This led the breeders to propose varietal associations which do not use male fertility restoration (Renard et al., 1992). Recently, double low restored F1 hybrids were obtained (Renard et al., 1998). For m o s t of the other CMS systems, no restorers are yet available. Thus, until now, commercial F1 hybrid production based on CMS has been achieved in B. oleracea us i ng the improved 'Ogura' cytoplasm obtained by Pelletier et al. (1989). F1 hybrids of various B. oleracea types (cauliflower, s a u e r k r a u t cabbage, garden cabbage and Savoy cabbage) have recently been registered (Leviel, 1998). Other improved 'Ogura' cytoplasms are available in B. oleracea (broccoli, cauliflower and cabbage) as well as in vegetable B. rapa (Chinese cabbage and pak choi) and are being tested for use in commercial hybrid production in seed companies worldwide (Earle and Dickson, 1995). In B. n a p u s , s u m m e r F1 hybrid varieties have been registered in Canada (Downey, 1994), in France (Pinochet, 1995), in Australia, in China and in India using the 'Polima' system. In China, the most cultivated F1 hybrid 'Qinyou 2' was developed with the 'Shaan 2A' CMS system. A hybrid based on B. tournefortii CMS was released for general cultivation in Punjab during 1994 (Banga et al., 1995). Hybrid and line composite varieties of winter and spring B. n a p u s based on the improved 'Ogura' CMS (named Ogu-INRA CMS) have been registered and cultivated in Europe since 1994 and, more recently, restored F1 hybrids have been also registered (Renard et al., 1998). In B. j u n c e a , development of the first hybrid on a commercial scale was achieved in India with B. tournefortii CMS (Angadi and Anand, 1988). In the years to come, the n u m b e r of commercial hybrid varieties in B r a s s i c a crops will certainly increase significantly due to the intensive work made in this field all a r o u n d the world. New systems are always appearing, like the rapeseed F1 hybrids recently registered in Europe, which are based on a system ('NPZ' system) developed in a seed company. Development of new engineered male sterilities are also in prospect.
Aknowledgements We t h a n k F. Eber, M. Renard, V. Ruffio and I. Small for helpful criticism of the m a n u s c r i p t . We are grateful to B.S. Landry and T. Sakai for providing their m a n u s c r i p t s prior to publication.
203
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212 Pelletier, G., Primard, C., Vedel, F., Chetrit, P., Renard, M.-D., R and Mesquida, J.1987. Molecular, phenotypic and genetic characterization of mitochondrial recombinants in rapeseed. Proc. 7th Int. Rapeseed Conference, Poznan, Poland, pp. 113-118. Pelletier, G., Ferrault, M., Lancelin, D. and Boulidard, L. 1989. CMS Brassica oleracea cybrids and their potential for hybrid seed production. 12th Eucarpia Congress, G~ttingen 11(7), 15. Pental, D., Pradhan, A. K., S o d h i , Y. S., Arumugam, M. and Mukhopadhyay, A. 1995. Heterosis breeding in m u s t a r d (Brassica juncea) and rapeseed (B. napus) by a combination of molecular and conventional methods. Proc. 9th Int. Rapeseed Congress, Cambridge, UK, pp. 122-124. Pinochet, X. 1995. Arriv~e de materiel de types hybrides en France. GCIRC Bulletin. 11, 32-37. Polowick, P. L. and Sawhney, V. K. 1987. A scanning electron microscopic study on the influence of temperature on the expression of cytoplasmic male sterility in Brassica napus. Can. J. Bot. 65, 807-814. Polowick, P. L. and Sawhney, V. K. 1990. Microsporogenesis in a normal line and in the ogu cytoplasmic male-sterile line of Brassica napus. I The influence of high temperature. Sex Plant Reprod. 3, 263-276. Polowick, P. L. and Sawhney, V. K. 1991. Microsporogenesis in a normal line and the ogu cytoplasmic male-sterile line of Brassica napus. II. The influence of intermediate and low temperatures. Sex Plant Reprod. 4, 22-27. Pr~adhan, A. K., Mukhopadhyay, A. and Pental, D. 1991. Identification of putative cytoplasmic donor of CMS system in Brassica juncea. Plant Breeding 106, 204-208. Pradhan, A. K, Arumugam, N., Mukhopadhyay, A., Gupta, B. S, Yadav, J. K, Verma, J. K. and Pental, D. 1995. Development of improved cytoplasmic male sterile lines in Brassica through somatic cell hybridization. In: Proc. 9th Int. Rapeseed Congress, Cambridge, UK, pp. 52-54. Prakash, S. and Chopra, V. L. 1990. Male sterility caused by cytoplasm of Brassica oxyrrhina in B. campestris and B. juncea. Theor. Appl. Genet. 79, 285-287. Prakash, S., Kirti, P. B. and Chopra, V. L. 1995. Cytoplasmic male sterility (CMS) systems other than ogu and Polima in Brassicae: current status. Proc. 9th Int. Rapeseed Congress, Cambridge, UK, pp. 44-48. Prakash, S., Kirti, P. B. and Chopra, V. L. 1998a. Development of cytoplasmic male sterility fertility restoration systems of variable origin in m u s t a r d - Brassica juncea. In: Thomas, G. and Monteiro, A. (eds.)
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Brassica 97, International symposium on Brassicas, Rennes, Acta Hort. 4 5 9 , 299-304. Prakash, S., Kirti, P. B., Bhat, S. R., Gaikwad, K., Kumar, V. D. a n d Chopra, V. L. 1998b. A Moricandia arvensis- based cytoplasmic male sterility a n d fertility restoration system in Brassica juncea. Theor. Appl. Genet. 97, 488-492. Primard, C., Delourme, R., Eber, F., Lancelin, D. a n d Pelletier, G. 1992. Identification of three types of CMS after somatic fusion between a fertile a n d a CMS B. napus. XIIIth EUCARPIA Congress, Angers, France, pp. 113-114. Rao, G. U. a n d Shivanna, K.R. 1996. Development of a new alloplasmic CMS Brassica napus in the cytoplasmic b a c k g r o u n d of Diplotaxis siifolia. Cruciferae Newsl. 18, 68-69. Rao, G. U., Batra Sarup, V., Prakash, S. a n d Shivanna, K. R. 1994. Development of a new cytoplasmic male-sterility system in Brassica juncea t h r o u g h wide hybridization. Plant Breeding 112, 171-174. Rawat, D. S. a n d Anand, I. J. 1979. Male sterility in Indian m u s t a r d . Indian J. Genet. 39, 412-415. Renard, M., Mesquida, J., Delourme, R. a n d Vallee, P. 1991. Les contraintes de la production de semences hybrides de colza. Bulletin Semences 117, 63-65. Renard, M., Delourme, R., Mesquida, J., Pelletier, G., Primard, C., Boulidard, L., Dore, C., Ruffio, V., Herve, Y. and Morice, J. 1992. Male sterility and F1 hybrids in Brassica. XIIIth EUCARPIA Congress:, Reproductive Biology a n d Plant Breeding, Angers, France, pp. 107-119. Renard, M., Delourme, R., Vallee, P. a n d Pierre, J. 1998. Hybrid rapeseed breeding a n d production. In: Thomas, G. a n d Monteiro, A. (eds.) Brassica 97, International s y m p o s i u m on Brassicas, Rennes, Acta Hort. 4 5 9 , 291-298. Reynaerts, A., Vandewiele, H., Desutter, G. a n d J a n s s e n s , J. 1993. Engineered genes for fertility a n d their application in hybrid seed production. Scientia Hort. 55, 125-139. Rousselle, P. 1982. Premiers r6sultats d'un p r o g r a m m e d'introduction de l'androst6rilit6 "Ogura" du radis chez le colza. Agronomie 2, 859864. Rousselle, P. a n d Renard, M. 1982. Int6r6t du cultivar "Bronowski" pour l'obtention de plantes m~le-st6riles cytoplasmiques chez le colza (Brassica napus L.). Agronomie 2, 951-956. Ruffio-Chable, V. 1994. Les s y s t ~ m e s d'hybridations chez le chou-fleur (Brassica oleracea L. van botrytis L.). Application ~t l'am~lioration g~n~tique., Thesis, ENSA de Rennes.
214 Ruffio-Chable, V., Bellis, H. a n d Herve, Y. 1993. A d o m i n a n t gene for male sterility in cauliflower (Brassica oleracea var. botrytis). Phenotype expression, inheritance a n d use in F1 hybrid production. Euphytica 67, 9-17. Rundfeldt, H. 1960. U n t e r s u c h u n g e n zur Z ~ c h t u n g des Kopfkohls (B. oleracea L. var capitata). Z. Pflanzenzficht. 44, 30-62. Sakai, T. a n d I m a m u r a , J. 1990. Intergeneric transfer of cytoplasmic male sterility between Raphanus sativus (CMS line) a n d Brassica napus t h r o u g h cytoplast-protoplast fusion. Theor. Appl. Genet. 80, 421427. Sakai, T., Iwabuchi, M., Kohno-Murase, J., Liu, H. J. and I m a m u r a , J. 1995. Transfer of radish CMS-restorer gene into Brassica napus by intergeneric protoplast fusion. Proc. 9th Int. Rapeseed Congress, Cambridge, UK, pp. 3-5. Sakai, T., Liu, H. J., Iwabuchi, M., Kohno-Murase, J. and I m a m u r a , J. 1996. Introduction of a gene from fertility restored radish (Raphanus sativus) into Brassica napus by fusion of X-irradiated protoplasts from a radish restorer line and iodacetoamide-treated protoplasts from a cytoplasmic male-sterile cybrid of B. napus. Theor. Appl. Genet. 9;t, 373-379. Sheikh, I. A. a n d Singh, J. N. 1996. Exploitation of male sterility in Indian m u s t a r d . Cruciferae Newsl. 18, 70-71. Shiga, T. 1976. Cytoplasmic male sterility a n d its utilization for heterosis breeding in rapeseed, Brassica napus L. Japan Agric. Res. Quarterly 10, 178-182. Shiga, T. 1980. Male sterility a n d cytoplasmic differenciation. In: Tsunoda, S., Hinata, K. and G6mez-Campo, C., (eds.), Brassica crops and wild allies. Biology and breeding, J a p a n Scientific Societies Press, Tokyo, pp. 205-221. Shiga, T. a n d Baba, S. 1973. Cytoplasmic male sterility in oilseed rape (Brassica napus L.), a n d its utilization to breeding. Japan. J. Breed. 23, 187-197. Sigareva, M. A. a n d Earle, E. D. 1997. Direct transfer of a cold-tolerant Ogura male sterile cytoplasm into cabbage (Brassica oleracea ssp. capitata) via protoplast fusion. Theor. Appl. Genet. 94, 213-220. Singh, M. a n d Brown, G.G. 1991. Suppression of cytoplasmic male sterility by n u c l e a r genes alters expression of a novel mitochondrial gene region. The Plant Cell 3, 1349-1362. Singh, M., Hamel, N., Menassa, R., Li, X.-Q., Young, B., J e a n , M., Landry, B. S. a n d Brown, G.G. 1996. Nuclear genes associated with a single
215
Brassica CMS restorer locus influence transcripts of three different mitochondrial gene regions. Genet. 143, 505-516. Sodhi, Y. S., Pradhan, A. K., Verma, J. K., Arumugam, N., Mukhopadhyay, A. and Pental, D. 1994. Identification and inheritance of fertility restorer genes for 'tour' CMS in rapeseed (Brassica napus L.). Plant Breeding 112, 223-227. Stiewe, G. and R6bbelen, G. 1994. Establishing cytoplasmic male sterility in Brassica napus by mitochondrial recombination with B. tournefortii. Plant Breeding 113, 294-304. Stiewe, G., Sodhi, Y. S. and R6bbelen, G. 1995a. E s t a b l i s h m e n t of a new CMS-system in Brassica napus. Proc. 9th Int. Rapeseed Congress, Cambridge, UK, pp. 49-51. Stiewe, G., Witt, U., Hansen, S., Theis, R., Abel, W. O. and R6bbelen, G. 1995b. Natural and experimental evolution of CMS for rapeseed breeding. Genetic m e c h a n i s m s for hybrid breeding. Adv. in Plant Breeding 18, 59-76. Takagi, Y. 1970. Monogenic recessive male sterility in oilseed rape (Brassica napus L.) induced by g a m m a irradiation. Z. Pflanzenzfichtg. 64, 242-247. Takahata, Y., Nagasaka, M., Kondo, H. and Kaizuma, N. 1996. Genic male sterility in Brassica campestris L. In: Dias, J. S., Crute, I. and Monteiro, A.A. (eds.), Proc. Int. Syrup. on Brassicas, Ninth Crucifer Genetics Workshop. Acta Hort. 407, 147-150. Theis, R. a n d R6bbelen, G. 1990. Anther and microspore development in different male sterile lines of oilseed rape (Brassica napus L.). A n g e w a n d t e Botanik 64, 419-434. Thompson, K. F. 1972. Cytoplasmic male sterility in oilseed rape. Heredity 29, 253-257. Tokumasu, S. 1951. Male sterility in J a p a n e s e radish (Raphanus sativus L.). Sci. Bull Fac. Agric. Kyushu Univ. 13, 83-89. Tokumasu, S. 1957. Histological studies on pollen degeneration in male sterile J a p a n e s e radish (Raphanus sativus L.). Japan J. Breed. 6, 4550. Van Der Meer, Q. P. 1987. Chromosomal monogenic d o m i n a n t male sterility in chinese cabbage (Brassica rapa subsp, pekinensis (Lour.) Hanelt). Euphytica 36, 927-931. Waiters, T. W and Earle, E. D. 1993. Organellar segregation, r e a r r a n g e m e n t and recombination in protoplast fusion-derived Brassica oleracea calli. Theor. Appl. Genet. 85, 761-769.
216 Walters, T. W., Mutschler, M. A. and Earle, E. D. 1992. Protoplast fusionderived Ogura male sterile cauliflower with cold tolerance. Plant Cell Rep. 10, 624-628. Witt, U., Hansen, S., Albaum, M. and Abel, W. O. 1991. Molecular analyses of the CMS-inducing 'Polima' cytoplasm in Brassica napus L. Curr. Genet. 19, 323-327. Yamagishi, H. 1998. Distribution and allelism of restorer genes for Ogura cytoplasmic male sterility in wild and cultivated radishes. Genes and Genetic Systems 73, 79-83. Yamagishi, H. a n d Terachi, T. 1996. Molecular a n d biological studies on male-sterile cytoplasm in the Cruciferae. III. Distribution of Oguratype cytoplasm among j a p a n e s e wild r a d i s h e s and Asian radish cultivars. Theor. Appl. Genet. 93, 325-332. Yamagishi, H. a n d Terachi, T. 1997. Molecular a n d biological studies on male sterile cytoplasm in the Cruciferae. IV. Ogura-type cytoplasm found in the wild radish, Raphanus raphanistrum. Plant Breeding 116, 323-339. Yang, G. a n d Fu, T. 1990. The inheritance of polima cytoplasmic male sterility in Brassica napus L. Plant Breeding 104, 121-124. Yarrow, S. A., Wu, S. C., Barsby, T. L., Kemble, R. J. a n d Shepard, J. F. 1986. The introduction of CMS mitochondria to triazine tolerant Brassica napus L., var."Regent", by micromanipulation of individual heterokaryons. Plant Cell Rep. 5, 415-418. Yarrow, S. A., Burnett, L. A., Wildeman, R. D. and Kemble, R. J. 1990. The transfer of Polima cytoplasmic male sterility from oilseed rape (Brassica napus) to broccoli (B. oleracea) by protoplast fusion. Plant Cell Rep. 9, 185-188. Zhou, X. 1990. La st~rilit~ mole dig~nique dominante du colza (Brassica napus). Recherche du g~ne restaurateur R f et ~tude cytologique en microscopie ~lectronique. M6moire D.E.A., Labo. de Biologie Cellulaire. Universit6 Rennes I.
Biology of Brassica Coenospecies C. G6mez-Campo (Editor) 91999 Elsevier Science B.V. All rights reserved.
217
7 GENOME STRUCTURE AND MAPPING Carlos F. Quiros
Department of Vegetable Crops. University of California. Davis, CA 95616, U.S.A. Most of the m a p p i n g work in Brassica h a s t a k e n place d u r i n g the p a s t ten years. This activity h a s been focused mostly on r a p e s e e d B. napus a n d on all three diploid cultivated species, B. nigra, B. oleracea a n d B. rapa. More recently, m a p p i n g h a s been e x p a n d e d to include B. juncea. The m a p s p r o d u c e d in Brassica crops are b a s e d mainly on F2 progenies developed by various laboratories, which will require in the future their integration for a more efficient use. In addition to linkage maps, a few synteny m a p s based on alien addition lines have been created, allowing chromosome assignment of some of the existing linkage groups. The m a r k e r m a p s are being u s e d to locate genes d e t e r m i n i n g traits of economic interest, including quantitative trait loci. Another i m p o r t a n t application of the maps, which is quite active at the p r e s e n t time, is on the s t u d y of the structure, origin a n d evolution of the Brassica genomes. Recent reviews of m a p p i n g activity in Brassica are those of Quiros et al. (1994), Altenbach et al. (1995) a n d Paterson (1997). Relatedness between the three basic Brassica genomes, A (n = 10), B (n = 8) a n d C (n = 9), h a s been cytogenetically investigated by u s i n g digenomic diploids from interspecific hybrids (Attia a n d R6bbelen, 1986), from amphidiploids through haploidy (Morinaga a n d F u k u s h i m a , 1933; Olsson a n d Hagberg, 1955), a n d digenomic triploids from crosses between amphidiploids a n d diploids (Morinaga, 1929, 1934; Attia et al., 1987). In general, the three genomes are considered to be mutually a n d partially homologous and p r e s u m a b l y derived from a common ancestral genome (Mizushima, 1950). The advent of modern molecular techniques is playing an i m p o r t a n t role in u n d e r s t a n d i n g the organization a n d relationships of the Brassica genomes. Results from these studies not only confirmed the origin of the amphidiploid species, b u t also suggested t h a t the A a n d C genomes originated from a single lineage, whereas the B genome is genetically dist a n t to both A a n d C genomes forming a separate lineage (Song et al., 1990; Warwick a n d Black, 1991). A c o m m o n a s s u m p t i o n is t h a t the n = 8, 9 a n d 10 cultivated species have evolved in a n a s c e n d i n g diploid series from a c o m m o n
218 primitive genome, "Urgenome' (Haga, 1938). Although there are no known B r a s sica species in n a t u r e with genomes of less t h a n n = 7 chromosomes, Catcheside (1934), Sikka (1940) a n d R6bbelen (1960) postulated t h a t the ancestral genome for these species consisted of five or six basic c h r o m o s o m e s , which through polysomy originated the p r e s e n t day cultivated genomes ranging from n = 8 to n = 10 chromosomes. T h u s , as a corollary of these hypotheses, the cultivated diploids can be considered s e c o n d a r y polyploids (Prakash a n d Hinata, 1980). Genetic m a p s in B r a s s i c a serve a double purpose" a) u n d e r s t a n d i n g the relationship a m o n g the genomes of the B r a s s i c a cultivated diploid species, and b) utilization in applied genetics and breeding of the n u m e r o u s B r a s s i c a crops.
Linkage m a p s B. o l e r a c e a maps Several m a p s have been developed independently for this species involving crosses between different crops. Slocum et al. (1990) reported an extensive RFLP m a p of 258 m a r k e r s covering 820 recombination u n i t s in nine linkage groups, with average intervals of 3.5 units. This is a proprietary m a p developed from a broccoli • cabbage cross. Landry et al. (1992) c o n s t r u c t e d a m a p consisting of 201 RFLP m a r k e r s distributed on nine major linkage g r o u p s coveting 1112cM. The F2 progeny u s e d to c o n s t r u c t this m a p was developed by crossing a cabbage line r e s i s t a n t to clubroot to a rapid cycling stock (Figure 7.1). The cabbage line was derived from an interspecific cross involving B. o l e r a c e a a n d B. n a p u s , followed by a series of b a c k c r o s s e s to B. oleracea. According to the authors, it is likely t h a t the resulting cabbage line h a d two c h r o m o s o m e segments of the A genome carrying the disease resistant genes. These h a d been introgressed or s u b s t i t u t e d in the C genome of the cabbage line. Kianian a n d Quiros (1992) developed a m a p comprising 108 markers, spread in 11 linkage groups and coveting 747 cM. This m a p was b a s e d on three intra-specific a n d one inter-specific F2 populations, namely: collard • cauliflower, collard • broccoli, kale • cauliflower and kohlrabi • B. insularis. The majority of the markers in the map are RFLP loci, with a few morphological a n d isozyme markers. Later, a few RAPD markers were added a n d the m a p was r e d r a w n based only on the three intra-specific crosses using JOINMAP (Stare, 1993). It consists of 82 markers distributed on eight major linkage g r o u p s covering 431 cM (Quiros et al., 1994). R e c o m b i n a n t inbreds by single seed d e s c e n t are u n d e r development for the collard by cauliflower and collard by broccoli progenies. C a m a r g o et al. (1997) recently reported a m a p developed in an F2 population of c a b b a g e • broccoli. It includes 112 RFLP a n d 47 RAPD loci and a selfincompatibility locus on nine m a i n linkage groups. Kearsey et al. (1996) and R a m s a y et al. (1996) c o n s t r u c t e d a linkage m a p based on backcross progenies obtained from double haploid lines of broccoli a n d B. alboglabra. Recombination of m a r k e r s was higher in the BC2 t h a n in the BCI generation. These differences were a t t r i b u t e d to differential c h i a s m a frequency in female a n d male meiosis.
LG 2
LG 1 WG6M
E m % WG2E12 wMB2b AAlOD W G W . AAlSC
EcIDll A834 WwD7 WGEASb'
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51 21 14
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17
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MlW ABlbE AUB ACIC' TG.?f2 l
Figure 7.1 B. oleruceu linkage map produced by an F2 from a cross cabbage x broccoli. It includes 112 RRFLP and 47 RAPD loci and a self-incompatibility locus on 9 main linkage groups. Letters in parenthesis after some of the markers indicate recessive alleles originating from cabbage (A) or broccoli parent (B). Asterisks indicate loci with significant segregation distortion (Cmargo et al., 1997).
220 B o u h o n et al. (1996) u s e d this m a p for aligning the C genome linkage groups of B. oleracea a n d B. n a p u s . Lagercrantz a n d Lydiate (1995) also observed sex dep e n d e n t recombination rates in B. nigra. In this species, however, recombination rate in each sex varied for some c h r o m o s o m e segments. If differential recombination rates in male a n d female gametes proves to be a widespread phenomenon, it posses a n o t h e r complexity to consider when applying linkage information to breeding problems. We have aligned m o s t of the linkage groups of four of these m a p s (Hu et al., 1998). For this purpose, a linkage m a p was c o n s t r u c t e d from an F2 population of 69 individuals with s e q u e n c e s previously m a p p e d i n d e p e n d e n t l y in three linkage m a p s of this species. These were the m a p s p u b l i s h e d by Kianian and Quiros (1992), Landry et al. (1992) a n d C a m a r g o et al. (1997). The base m a p developed in this s t u d y consisted of 167 RFLP loci in nine linkage groups, plus eight m a r k e r s in four linkage pairs, covering 1738 cM. Linkage group alignment was also possible with a fourth m a p p u b l i s h e d by R a m s a y et al. (1996), containing c o m m o n loci with the m a p of Camargo et al. (1997). In general, cons i s t e n t linear order a m o n g m a r k e r s were m a i n t a i n e d , a l t h o u g h often the dist a n c e s between m a r k e r s varied from m a p to map. A linkage group in Landry's m a p carrying a clubroot resistance QTL was found to be rearranged, consisting of m a r k e r s from two other linkage groups. This was not surprising considering t h a t the resistance gene was introgressed from B r a s s i c a n a p u s . The extensively duplicated n a t u r e of the C genome w a s revealed by 19 s e q u e n c e s detecting duplicated loci within c h r o m o s o m e s a n d 17 sequences detecting duplicated loci between c h r o m o s o m e s . The variation in m a p p i n g distances between linked loci pairs on different c h r o m o s o m e s d e m o n s t r a t e d t h a t sequence r e - a r r a n g e m e n t is a distinct feature of this genome. Although the consolidation of all linkage groups in the four B. oleracea m a p s compared was not possible, a high n u m b e r of m a r k e r s to c o r r e s p o n d i n g linkage g r o u p s were added. Some c h r o m o s o m e s e g m e n t s were enriched with m a n y m a r k e r s which m a y be useful for future r e s e a r c h in gene tagging or cloning. The a s s i g n m e n t B. oleracea linkage groups to their respective chromosomes h a s been partially accomplished. This t a s k h a s been based on the development of alien addition lines allowing the construction of synteny maps. We developed two synteny m a p s for the nine C genome chromosomes including isozyme, RFLP and RAPD markers. One m a p has approximately 194 markers and was constructed using a set of alien addition lines B. rapa-oleracea extracted from artificial B. n a p u s "Hakuran' (McGrath and Quiros 1990; McGrath et al., 1990). The second m a p h a s approximately 103 m a r k e r s a n d was assembled from a set of addition lines B. rapa-oleracea extracted from natural B. n a p u s (Quiros et al., 1987). Chen et al. (1992) a n d Cheng et al. (1994ab, 1995) also developed B. rapa-oleracea {alboglabra) alien addition lines, allowing the identification of the chromosome carrying genes for seed a n d flower color. Later, various c h r o m o s o m e s of these lines were characterized by molecular m a r k e r s (Jorgensen et al., 1996). Although it was possible in most cases to physically assign linkage groups to chromosomes using these sets of lines, two major complications arising from this activity
221 deserve comment. The first one is the frequent lack of p o l y m o r p h i s m of inter specific m a r k e r s of the alien c h r o m o s o m e s in the intraspecific crosses u s e d to develop the linkage maps. Low coincidence of p o l y m o r p h i s m between these two sets of materials m a k e s c h r o m o s o m e a s s i g n m e n t tedious a n d time consuming. The second complication is the instability of the alien c h r o m o s o m e s . We have addressed this problem by following groups of syntenic m a r k e r s in the progeny of monosomic addition lines of B. rapa-oleracea (Hu a n d Quiros 1991). Data have been obtained from two progenies of approximately 100 plants each, derived from two monosomic addition lines of B. rapa-oleracea for chromosomes C4 and C5 (2n = 21). After following several m a r k e r s located on each c h r o m o s o m e , the alien c h r o m o s o m e s were found not to be always stable. All the expected m a r k e r s were recovered in approximately 50% of the plants carrying the alien chromosome. The remaining 2n = 21 plants h a d one or more m a r k e r s missing. These observations were extended to other six c h r o m o s o m e s of the C genome in the "Hakuran' derived lines, with a larger n u m b e r of m a r k e r s (Hu and Quiros, unpublished). The study d e m o n s t r a t e d that this is widespread in the C genome a n d seems to occur to all chromosomes. Most of the deletions were terminal, with a few exceptions. The deletions were found useful to physically individual m a r k e r s of linkage segments to specific c h r o m o s o m e a r m s (Hu and Quiros 1991). Another anomaly of the alien c h r o m o s o m e s is their ability to undergo n o n - h o m o l o g o u s recombination at a low frequency when present in the same cytoplasm in double or triple monosomic condition or forming part of similar hyperploids. This type of situation provides the opportunity for association of homoeologous segments resulting from duplicated segments. Although we were u n a b l e to quantify this type of recombination precisely, it is estimated to occur at a frequency ranging between 5 to 10% (Hu a n d Quiros, u n p u b l i s h e d ; S t r u s s et al., 1996). B. r a p a
maps
The two m o s t extensive m a p s created for this species are proprietary. The first one, developed by Chyi et al. (1992) resulted by crossing s a r s o n by canola. It includes 360 loci on 10 linkage groups covering 1876 recombination units. The average distance between m a r k e r s is 5.2 m a p units. The second one developed by Slocum (1989) a n d Song et al. (1991) was obtained by crossing Chinese cabbage x spring broccoli. This m a p includes 273 loci covering 1455 recombination u n i t s in 10 linkage groups with average intervals of 5 units. An u p d a t e d version was used to locate genes involving 28 phenotypic traits (Song et al., 1995a). Kole et al. (1996) created a m a p involving a white r u s t resistance line for the purpose of mapping the resistance gene. Other m a p s have been developed by Schilling and Bernatzky (Schilling, 1991) including 58 RFLP loci covering approximately 700 cM after crossing the oilseed cultivar "Candle' by a rapid cycling strain. McGrath and Quiros (1991) developed another m a p from an F2 of turnip x Pak Choi containing 49 m a r k e r s in eight linkage groups a n d covering a total of 262 cM. Both m a p s have been consolidated by exchanging probes a n d DNA from the m a p p i n g populations (Hu, Vernatzky a n d Quiros, unpublished).
222 B. n i g r a m a p
Truco and Quiros (1994) developed a map for this species based on a single F2 population of 83 plants, involving two parental individuals from geographically divergent populations. The map constructed with the program Mapmaker (Lander et al., 1988), has 67 markers arranged in eight major linkage groups which may correspond to the eight B. nigra chromosomes, plus two small groups. The markers include RFLP's, RAPD's and a few isozymes. The RFLPs are based on EcoRI digestions. The map covers 561 cM with average intervals of 8.4 cM (Figure 7.2). Lagercrantz and Lydiate (1995) developed a RFLP map in a backcross population of B. nigra, consisting of 288 loci covering a length of 855 cM. A trend of higher recombination rates was observed in male gametes for the distal portions of the linkage groups. Also, recombination rates tended to higher in the proximal regions of some of the linkage groups. This map was recently expanded by Lagercrantz (1998) who incorporated 284 additional loci based on A. thaliana probes. It has been possible to assign only four linkage groups of the first map to their respective chromosomes by synteny mapping based on alien addition lines (Chevre et al., 1991). Different species combinations for developing these lines have been used. These include a B. oleracea-nigra series extracted from B. carinata (Quiros et al., 1986) for five of the eight B genome chromosomes. The second set consists of Diplotaxis erucoides-B, nigra covering seven of the eight chromosomes in the B genome (Quiros et a/., 1988; This eta/., 1990). The third and fourth sets are in tetraploid background, B. napus-nigra lines, and were developed independently in France (jahier et al., 1989; Chevre et al., 1991) and Germany (Struss et al., 1991, 1996). Chevre's set covers at least seven of the eight B genome chromosomes. The alien chromosomes are in disomic condition and contain RFLP, RAPD and isozyme markers. Also in this set the chromosomes carrying genes for fatty acid chain elongation and desaturation and possibly a chromosome carrying resistance to blackleg have been identified. The addition lines developed by Struss et al. (1991) include B genome chromosomes extracted from B. nigra, B. carinata and B. juncea. Struss et al. (I 996) constructed synteny map for most of these B genome alien with isozyme, RAPD and RFLP and phenotypic markers. Although they found extensive intra- and intergenomic recombination in these chromosomes, it was possible to construct a consensus synteny map for the B genomes extracted from all three sources. In this map, chromosomes carrying genes for erucic acid, sinigrin and possible blackleg resistance were identified. B. n a p u s m a p
Most of the mapping activity in Brassica has been focused to this species because of its economic importance. This has resulted in the construction of at least six independent maps. Several maps for this amphidiploid species have been reported by various laboratories. The first map was developed by Landry et al. (1991), and resulted from crossing two rapeseed cultivars, "Westar' and "Topaz', based on single-enzyme digestions by four enzymes, BamHI, EcoRI, EcoRV and HindIII. It included 120 loci arranged in 19 linkage groups covering 1413 recom-
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Figure 7.3 (continued from the previous page) Linkage map of B. napus resulting from double haploid lines of the F1 resulting from crossing the winter rapeseed cv "Mansholt' with cv. Samourai". Mapped genes and QTLs are underlined: Pale (pale yellow flower), ACP (acyl-carrier-protein), KAS (beta-ketoacyl-ACP synthase) I, gls (see glucosinolate content QTLs) (after Uzunova et al., 1995). bination units. Hoenecke a n d Chyi, (1991) reported the s a m e year at the International Congress of Rapeseed a proprietary m a p developed by crossing two breeding lines BN0011 • BN0031 based on EcoRI digestions. This m a p consisted of 125 m a r k e r s a r r a n g e d in 19 linkage g r o u p s covering 1350 m a p units. In the p a s t three years, four other m a p s have been developed b a s e d on F2 progenies of double haploid lines. Ferreira et al. (1994) c o n s t r u c t e d a m a p by crossing the a n n u a l canola cultivar ' S t e l l a r ' b y the biennial cultivar 'Major'. Double haploid lines generated from the resulting F1 served to construct a m a p of 132 loci covering 1016 cM in 22 linkage groups. A partial m a p was also c o n s t r u c t e d from the F2 progeny of the two p a r e n t a l lines. For the c h r o m o s o m e s e g m e n t s compared, no significant differences on linkage associations were observed between the two maps. Uzunova et al. (1995) constructed a m a p based on double haploid F1 lines by crossing the two winter rapeseed varieties, ~lVlansholt's H a m b u r g e r Raps' a n d 'Samourai'. It consisted in 204 RFLP a n d two RAPD m a r k e r s distributed in 19 linkage g r o u p s covering 1441 cM (Figure 7.3). S h a r p e et al. (1995) reported an integrated m a p based on two populations, one of which included a resynthesized B. n a p u s line crossed to a rapeseed cultivar. The c h r o m o s o m e instability of the resynthesized line a d d e d to the complexity of these maps. Parkin et al. (1995)
226 published also a map based only on this synthetic population, consisting of 399 RFLP markers. The integration of the maps by Sharpe et al. (1995), based in the combination of the two populations resulted in a reduction of over 100 loci. Most recently, Foisset et al. (1996) constructed a map based on RFLP, RAPD and isozyme markers developed from a double haploid progeny of the F1 generated by crossing a dwarf rapeseed isogenic line 'Darmor-bzh' of cultivar 'Darmor' and ~Yudal', a spring Korean line. The map based on 153 double haploid lines had 254 markers on 19 linkage groups coveting 1765 cM. Several economically important genes were located on it.
B. j u n c e a map The activity in this species is quite recent. Sharma et al. (1994) developed a small map based on a F2 mapping population resulting of crossing 5/aruna', a brown seeded cultivar and BEC 144, a yellow seeded accession. This map has only 25 markers on nine linkage groups covering 243 cM. A more extensive map developed by Cheung et al. (1996) is based on a double haploid progeny from a canola quality line by a high oil content line. The map was developed with probes from B. n a p u s , consisting of 343 loci on 18 main linkage groups covering 2073 cM.
Structure of the Brassica genomes General attributes A great deal of information has been gathered during the past few years on the genomic structure of all three cultivated genomes. Diverse analysis of synteny maps for the B and C genomes (McGrath et al. 1990; Struss et al., 1996), and the linkage maps for the A, B and C genomes (Slocum et al., 1990; Song et al., 1991; McGrath and Quiros 1991; Kianian and Quiros, 1992, Truco and Quiros, 1994) reveal extensive sequence duplication. For example, Slocum et al. (1990) reported for B. oleracea that 35% of the genomic clones produced more than one locus, and 56% besides disclosing single locus segregations often produced other monomorphic fragments that may represent duplications. Kianian and Quiros (1992) also in B. oleracea found 50% of their cDNA sequences mapping to more than one locus. McGrath et al. (1990) reported over 40% sequence duplications in B. oleracea chromosomes represented in a series of addition lines. Song et al. (1991) reported in B. rapa that 36% of their genomic clones produced segregating RFLPs at more than one locus and 41% detected sequences segregating as single locus but also revealed additional monomorphic fragments. Truco and Quiros (1994) found approximately 40% of RFLP loci duplicated in B. nigra. This finding was confirmed by Lagercrantz and Lydiate (1995) who reported that "essentially every chromosomal region [is present] in three copies" in this species. Thus, these reports demonstrate that close to 50% of the loci in all three cultivated genomes are duplicated, supporting the hypothesis that the B r a s s i c a diploid species are secondary polyploids (Prakash and Hinata, 1980; Quiros et al., 1994). In general, these duplications are distributed on more than one chromosome. Some of the
227 linkage and synteny groups have sequences present in more than one group. When linkage arrangement is conserved, the distances are usually changed. Rerrangements of linkage groups may be explained by chromosomal translocations due in part to homoeologous recombination. Translocations are of common occurrence in B r a s s i c a and have been reported by independent investigators as a widespread event in various species (Songerup, 1980; Quiros et al., 1988, Kianian and Quiros, 1992). In addition to duplications and linkage rearrangements, deletions seem to be another important molding force of the B r a s s i c a genomes. The four independent F2 linkage studies cited above have detected a large n u m b e r of loci containing dominant markers, where one allele is apparently null, which may be due to deletions. This phenomenon, however, may be also due to the masking of common alleles in duplicated loci (Uzunova et al., 1995). Sometimes these loci are assembled in linkage blocks implying large deletions in some chromosomes (McGrath and Quiros, 1991; Song et al., 1991). The presence of deletions has been demonstrated cytologically for the C genome in alien addition lines (Hu and Quiros, 1991). Finally, inversions have also been observed in the F2 linkage maps of B. o l e r a c e a by Song et al. (1991) and Kianian and Quiros (1992). Similar levels of genome duplication have been reported in B. n a p u s . Landry et al. (1991) found that 88% of the probes disclosed more than one locus. B. n a p u s being an amphidiploid, these duplications are expected to correspond to both intra-genomic and inter-genomic sequences. This indeed seems to be the case, because some of the probes disclosed three or four segregating loci located on different chromosomes (Landry and al., 1991; Hoenecke and Chyi, 1991). The same trend was observed by Uzunova et al. (1995), detecting ten different clusters of two to four duplicated loci in 11 linkage groups. Ferreira et al. (1994) also detected a similar level of duplicated loci in B. n a p u s . Another interesting attribute of the B r a s s i c a maps in general is the relatively large number of loci deviating from Mendelian segregation, which often cluster in linkage blocks (McGrath and Quiros 1991; Figdore et al., 1993; Landry et al., 1991; Kianian and Quiros 1992; Chry et al., 1992; Ferreira et al., 1994; Uzunova et al., 1995; Foisset et al., 1996). The deviations in some cases may be due to genomic divergence between the parents involved in the crosses (McGrath and Quiros 1991). This conjecture is supported by the fact that the number of deviating loci increases with the level of divergence of the parents (Kianian and Quiros, 1992). In androgenic plant material such as double haploid lines, often used in B. n a p u s for mapping purposes, loci with biased segregation ratios can reach up to 35% (Ferreira et al., 1994; Foisset et al., 1996). Inclusion of loci with distorted segregation ratios in linkage maps is risky, since caution is necessary to avoid disclosing pseudolinkages due to biased statistical tests (Foisset et al., 1996).
Plasticity of the B r a s s i c a g e n o m e s The highly duplicated nature of the genomes have important implications in structural changes of the chromosomes. The homoeologous regions arising by duplications u n d e r certain situations, such as those imposed by hybridization,
228 will facilitate intra-genomic and inter-genomic recombination events. This is especially true in Brassica, where hybridization often results in aneuploidy and amphiploidy. Because of their plasticity, the B r a s s i c a genomes are prone to frequent structural changes.
Intragenomic homoeologus recombination This type of recombination has been detected mostly in aneuploids, such as alien addition lines and newly synthesized amphidiploids. Evidence from the C genome derived addition lines (McGrath et al., 1990) indicates that non-homologous recombination may take place in the B. oleracea genome when two or more of these chromosomes are present in single copies as alien additions. Furthermore, in addition to the nine expected synteny groups resolved from the B. n a p u s C genome, four recombinant groups including markers from different chromosomes were recovered (Quiros et al., 1994). The chromosomes most often involved in these recombination events were 4C, 5C and 6C, indicating that some chromosomes are more recombinogenic than others. Comparison of chromosomes of addition lines derived from natural and synthetic B. n a p u s "Hakuran' dissecting the C genome (McGrath et al., 1990) disclosed also possible syntenic differences. Although chromosomes 3, 4, 5 and 6 aligned well in both sets, chromosomes 1, 2 and 7 displayed rearrangements of the markers. The synteny changes observed in the alien addition lines occurred in 12% of the plants carrying more t han one alien chromosome. Variation in synteny has also been observed for the B genome chromosomes in addition lines from independent origins (This et al., 1990; Chevre et al., 1991; Struss et al., 1996). Extensive intergenomic recombination was detected for addition lines originating from trigenomic hybrids of constitution ABC. In this type of hybrid the chromosomes of the three genomes have maximum opportunity for auto and allosyndetic pairing resulting in intra and inter-genomic recombination events (Struss et al., 1996). Other instances of probable intergenomic recombination was disclosed by comparative mapping in B. rapa and B. n a p u s (Hoenecke and Chyi, 1991). They found significant linkage arrangement differences between the A genomes from the diploid and amphidiploid species. However, it was still possible to identify major conserved linkage groups. The structural changes observed in the three cultivated genomes indicate that this p h e n o m e n o n is widespread, not exclusive of a single genome. Although amphiploidy and interspecific aneuploidy may serve to induce these genomic changes, some rearrangements might take place in the diploid environment.
Intergenomic homoeologous recombination Occasionally a few of the diploid individuals derived from B. rapa-oleracea monosomic addition plants were found to carry a few C-genome-specific markers present in the alien chromosomes of the parental plant, indicating that inter-
229 genomic recombination had taken place. Earlier during the development of these lines, we detected intergenomic recombination between the A and C genome chromosomes for the isozyme locus Pgi-2 (Quiros et al., 1987). Recently we have observed intergenomic recombinants for rDNA sequences, where B. r a p a individuals display EcoRI fragments typical of B. oleracea (Hu et al., unpublished). Another line of evidence for this type of recombination has been obtained by Sharpe et al. (1995) who followed segregation of genome specific RFLP markers in B. n a p u s progenies. Parkin et al. (1995), detected non-homoeologous recombination, resulting in non reciprocal homoeologous translocations in B. n a p u s cultivars at approximately 0.3%. In resynthezided B. n a p u s recombination of this type was approximately 10%. Early generations of synthetic B r a s s i c a amphiploids after several cycles of selfing displayed genomic changes. Some of these changes could be attributed to inter genomic recombination (Song et al., 1995b). Other instances of intergenomic recombination have been reported (Struss et al., 1996; Plieske et al., (1998) in B. n a p u s - n i g r a addition lines. AACC euploids derived from some of the lines carried genes for resistance to balckleg, erucic acid and sinigrin content from the B genome. It is unknown whether the B genome chromosome segments translocated to either the A or C genome or both. A similar situation was reported by Landry et al. (1991), where resistance genes from B. rapa were transferred to the C genome by crossing and backcrossing B. n a p u s to B. oleracea.
Genomic relationships Comparative mapping of the genomes extracted from diploid and amphidiploid species is j u s t beginning. Hoenecke and Chry (1991) comparing linkage groups for the A genome of B. rapa and B. n a p u s found mostly conserved loci associations. This allowed the identification of the A genome linkage groups in the amphidiploid species. However, it was also possible to detect linkage arrangement differences between the A genomes from the diploid and amphidiploid species. Teutonico and Osborn (1994) found good coincidence for most of the linkage groups of the A and C genomes from B. rapa and B. oleracea, respectively, with those of B. n a p u s . Because of the small n u m b e r of loci used in the comparative study, it was no possible to trace the linkage groups of B. n a p u s to the respective parental diploid species. In contrast, Lydiate et al. (1993) and Parkin et al. (1995) reported that the A and C genomes of B. rapa a n d B. oleracea, respectively, conserve virtually the same loci arrangement as in natural amphidiploid B. n a p u s . However, in a parallel study from the same laboratory. Sharpe et al. (1995) reported altered linkage arrangement at low frequencies due to nonreciprocal homoeologous translocations. Comparative mapping for all three basic genomes from the cultivated diploid species is at its infancy in B r a s s i c a . Previous work on this subject was limited to comparison of the A and C genomes (Slocum 1989; McGrath and Quiros 1991, Camargo 1994). Only recently comparative mapping for the A, B and C genomes has been reported, allowing to draw inferences on the origin of the three genomes based on their relationships (Lagercrantz and Lydiate 1996; and Truco
230 et al., 1996). All three Brassica species share regions of homology in their genomes (Figure 7.4). Often a single linkage group showed regions of homology with more than one group of the other species. This is in agreement with a comparative study between m aps of B. rapa and B. oleraeea by Slocum (1989), who found that in some cases it was possible to align linkage groups from one to the other species. Some of the linkage groups, however, also shared homologous regions that were separated into more t h a n one group in the other species. It is evident that extensive gene reordering has taken place during the evolution of Brassica species, even though there is considerable conservation among certain chromosome regions within and among the three genomes. This results in complex intra and intergenomic chromosomal relationships where co-linearity is maintained for some segments, but broken up for other chromosomal regions. The extensive map of B. rapa produced by Chyi et al. (1992) illustrates this p h e n o m e n o n quite well for intragenomic homology, where six of the ten chromosomes, representing approximately half of the genome, associate in this fashion. Similarly, in B. oleracea we found seven of the nine chromosomes showing homologous segments, covering also approximately 40% of the genome. On the basis of marker arrangement conservation, we have drawn phylogenetic relationships among the chromosomes of the three genomes. This allowed u s to postulate the possible n u m b e r of chromosomes in the hypothetical ancestral genome originating the A, B and C genomes u n d e r the two following assumptions: 1) The A genome is related to the C genome and possibly derived from it. 2) Two main lineages originated the diploid cultivated Brassica species, the B. rapa/B, oleracea and B. nigra lineage. These a s s u m p t i o n s are supported by taxonomic studies based on chloroplast (Warwick and Black 1991; Pradhan eta/., 1992) and nuclear DNA (Song et al., 1990). Based on the above a s s u m p t i o n s , no more t h a n seven ancestral chromosomes can be postulated to explain the existing linkage groups and their homologous relationships (Figure 7.5). However, analysis of the comparative mapping data of Lagercrantz and Lydiate (1996) in a similar fashion suggests that this n u m b e r could be as low as four. Although hypothetical, chromosomal relationships derived in this fashion serve as a framework to characterize the Brassica chromosomes and determine the m e c h a n i s m s leading to their origin. For example, the higher level of intergenomic homology between the A and C genomes supports the conjecture t h at the former derived later on from an already established C genome. More recently Gale and Devos (1998) based mostly on Lagercrantz and Lydiate (1996) presented an hypothetical alignment of all Brassica chromosomes of the A, B and C genomes. However, the a m o u n t of mapping data available at this time for the three diploid cultivated Brassica species is not sufficient to support this scheme.
On the origin o f the g e n o m e s Based on the mapping data disclosing extensive loci duplication, it is clear that the three diploid Brassica cultivated species are indeed ancient polyploids as suggested earlier by Prakash and Hinata (1980). The reiteration of chromosomes within the genomes certainly agrees with the hypotheses that the existing Brassica genomes derive from a smaller ancestral genome. The n u m b e r of chromo-
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232 somes of this ancestral genome in u n k n o w n , b u t it is reasonable to a s s u m e that it h a d four or five, as explained in the previous section. Since the Cruciferae genera Physaria a n d Stenopetalum contain species of n = 4 (Rollins, 1966), the existence of an ancestral c h r o m o s o m e n u m b e r of n = 4 is a clear possibility. In any case, it is unlikely t h a t the Brassica genomes originated by polysomy or duplication of whole chromosomes. Therefore, the complexity of the existing chrom o s o m a l relationships discards the possibility that auto-polyploidy explains the higher c h r o m o s o m e n u m b e r s now observed in the cultivated genomes. Further, Brassica genomes cannot be simply described by formulae including a few founder c h r o m o s o m e s reiterated 3 or 4 times (R6bbelen, 1960). Lydiate et al. (1995), Lagercrantz a n d Lydiate (1996) and Lagercrantz (1998) concluded that an hexaploid ancestral species originated all three Brassica cultivated genomes. This conclusion was based on the observation t h a t a fraction of the duplicated loci detected by the RFLP probes used were triplicated, in spite of the fact that these probes detected on the average 1.9 loci. Another factor leading the a u t h o r s to this hypothesis is the fact that the Brassica genomes contain approximately three times the DNA found in the Arabidopsis genome (Arumuganat h a n and Earle 1991). Unfortunately the complexity of the Brassica genomes containing a large proportion of repetitive DNA would argue against this possibility.
Cyclic amphiploidy and the origin and evolution of the B r a s s i c a species Sikka (1940) mentioned two main forces molding the Brassica genomes departing from an ancestral genome of n = 5. The first one is hybridization and the second one is c h r o m o s o m a l s t r u c t u r a l changes. S p o n t a n e o u s interspecific hybridization in Brassica is well d o c u m e n t e d by the existence of natural amphidiploid species, driven mainly by u n r e d u c e d gametes (Harlan a n d deWet 1975). Tolerance of c h a n g e s in genomic n u m b e r s h a s been experimentally demonstrated by the development of alien addition lines (Quiros et al., 1994). During the formation of amphiploids, crosses of sesquidiploid individuals back to their diploid parents likely have produced in nature changes in genomic numbers. Additionally, newly formed amphidiploids m a y have crossed to r e m n a n t sexqui-diploids. These crossing possibilities will produce a plethora of aneuploids, some of which will have multiple copies for some c h r o m o s o m e segments. Previous to the hybridization events, the ancestral Brassica genome may have originated derived cytotypes of x = 4 to 5 chromosomes. These would arise as a result of c h r o m o s o m a l s t r u c t u r a l modifications due to differential evolutionary forces c a u s e d by spatial isolation of the species containing the ancestral genome. Most likely mainly reciprocal translocations, which are quite common in Brassica species (see above) have been responsible to these structural modifications. Further, translocations are known to generate not only duplications and deletions b u t also aneuploidy, opening also the possibility of limited changes in c h r o m o s o m e n u m b e r s previous to hybridization. Thus, following Sikka's insight,
233
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Figure 7. 5 Hypothetical ancestral genome of six chromosomes (Wl to W6) originating specific A, B and C genome chromosomes deduced by homoeologous relationships. Bx and Cx are intermediate chromosomes. Broken lines indicate tentative homologies (after Truco et al., 1996). based on common events observed in B r a s s i c a species, it is proposed that the A, B and C diploid genomes are actually partial amphiploids derived by hybridization. Their DNA content have remained practically constant in spite of changes in chromosome n u m b e r and structure, 507-516 Mbp (million base pairs) for B. rapa, 468 Mbp for B. nigra and 599-662 Mbp for B. oleracea (Arumuganathan and Earle 1991). Thus similar amounts of genetic information exist in each of these genomes but are packed in a different n u m b e r of chromosomes. Because of their ancestral common origin, these three genomes have conserved chromosome segments and extensive duplications, which will cause further modification by homoeologous recombination. After genomic stabilization these have generated by another cycle of hybridization the cultivated allotetraploid species we know today. Therefore, cyclic amphiploidization would explain the origin of diploid and amphidiploid
234 Brassica species. The separation of the A a n d C genomes diploid species in a separate lineage from B. nigra (B genome) (Warwick a n d Black, 1991; Song et al., 1990), would indicate t h a t the first two shared at least one ancestral cytotype during their initial amphiploid synthesis. On the contrary, the B genome might originate from different cytotypes due to colonization of different geographical niches.
A r a b i d o p s i s a s a m o d e l for a s i m p l e r g e n o m e Taking advantage of the fact that A. thaliana (tribe Arabideae) (Price et al., 1994 ) is a related crucifer to Brassica species (tribe Brassiceae), comparative mapping between the two genera is taking place at an increasing pace (for review see Paterson, 1997). A. thaliana h a s a simple genome of only 145 Mbp (Arumugan a t h a n a n d Earle, 1991) a n d n = 5 chromosomes, a n d approximately 12- 17% duplicated loci (McGrath et al., 1993; Kowalski et al., 1994). Kowalski et al. (1994) found extensive r e a r r a n g e m e n t s between RFLP m a p s of A. thaliana and B. oleracea, however, islands of conserved gene organization were identified. Sadowski et al. (1996) exploited the genetic m a p of A. thaliana (Hauge et al., 1993) to probe the Brassica genomes with A. thaliana gene complex carrying five genes within a 20 Kb span (Gaubier et al., 1993). This complex comprises a well characterized Emlike protein-coding gene and other four flanking genes on chromosome 3. The five gene complex array from A. thaliana was conserved on a single chromosome of each genome, b u t additional copies for most of the genes were found in one or two other c h r o m o s o m e s . A similar situation was observed for a six gene complex on A. thaliana chromosome 4, including the disease resistance gene RPS2 (Sadowski and Quiros, 1998). In this case, besides the conserved array in one Brassica chromosome, four other c h r o m o s o m e s contained copies for some of the genes. Lagercrantz et al. (1996), in a similar study found mostly triplicate copies for each A. thaliana gene in the B. nigra genome. This finding prompted them to hypothesize that B. nigra derived from an hexaploid ancestor. The conservation of the A. thaliana array in one c h r o m o s o m e of all three Brassica genomes indicates that the gene cluster arrays predates the separation of the genera. Interestingly enough, although the genetic distances between genes were similar in A. thaliana and Brassica, the physical distances, based on pulse field electrophoresis, tended to be m u c h larger in Brassica (Sadowski et al., 1996). However, Conner et al. (1998) comparing a 250 kb Arabidopsis fragment in B. rapa found that spacer size varied for each pair of genes. In some cases their sizes were similar in both species, in other cases, spacer size was bigger or smaller in Arabidopsis. Therefore, it is not possible to generalize t h a t spacing between most genes, is equivalent in both Brassica species and A. thaliana, as proposed by Lagercrantz et al. (1996).
Applications
of the maps in breeding
The Brassica linkage maps are now extensively applied to tag genes of interest, including quantitative trait loci (QTL) of economic importance.
235 Vernalization requirement for flower induction: Two genomic regions determining biennial habit in B. rapa have been identified, by crossing biennial to annual types. These regions associate to two linkage groups in B. n a p u s carrying genes related to flowering time. (Teutonico and Osborn 1995; Ferreira et al., 1995a). CO gene homologues determining flowering time in A. t h a l i a n a were detected in B. nigra by Lagercrantz et al. (1996). Camargo and Osborn (1996) peformed QTL analysis of flowering time in F3 populations B. oleracea obtained by crossing cabbage and broccoli. A total of five QTLs were detected. One of these was linked to the S locus locus slg6. Another QTL was associated to petiole length. No conservation for flowering time QTLs were detected between B. oleracea and B. n a p u s a n d B. rapa. Freezing tolerance and winter survival in B. n a p u s and B. rapa: QTL analysis of these traits have been carried out in both species. In the former species, no QTLs were detected. In B. rapa, however, four QTLs were observed. Two of these linked to A. t h a l i a n a RFLPs for cold induced (COR6.6a) and stress related (DHS2) cDNAs (Teutonico et al., 1995). Linolenic acid content in B. n a p u s : Markers for this trait have been identified by several laboratories. One of the four markers detected by T a n h u a n p a a et al. (1995a), corresponded to one m ar ker detected by Hu et al. (1995), which accounted for 37% of the variation for this trait. The markers disclosed by Jourdren et al. (1996a,b) and T h o r m a n n et al. (1996) were related the f a d 3 (omega 3 desaturase) gene of A. t h a l i a n a (Arondel et al., 1992). Oleic acid content in B. rapa: A QTL in a linkage group of six markers associated to oleic acid content was detected by T a n h u a n p a a et al. (1996). OPH- 17, a codominant RAPD m ar ker was the best for selection of this trait. It was converted to a SCAR m ar ker for better reproducibility. The oleic acid QTL also affected content of palmitic and linoleic acid content, which indicates that it controls either chain elongation or desaturation steps. Palmitic acid content in B. rapa: T a n h u a n p a a et al. (1995b) detected a RAPD marker associated to this trait in the same linkage group associated to oleic acid content. Erucic acid content in B. n a p u s : All variation for this trait is explained by genomic regions (Thormann et al., 1996; Ecke et al., 1996). These correspond to two of three QTLs detected for seed oil content. Markers for loci determining this trait were reported by J o u r d r e n et al. (1996c). Glucosinolate content in B. napus: Four (Uzunova et al., 1995 ) to five (Toroser et al., 1995) QTLs have been detected for this trait, two of which were major. De Quiroz a n d Mithen (1996) integrated both studies revealing RFLPs for low (GLS-1 marker) and high (GLS-2 marker) content after screening varieties with contrasting a m o u n t s of this compound. Varieties with intermediate content displayed both markers. Clubfoot (Plasmodiophora brassicae) resistance in B. oleracea: RAPD markkers associated to this trait were detected by Grandclement et al. (1996). Pre-
236 vious work in B. n a p u s by Figdore et al. (1993) had identified three QTLs for this trait. Landry et al. (1992) found two QTLs associated to resistance to this disease in B. oleracea. Blackleg resistance (Leptosphaeria maculans) in B. napus: For cotyledon and stem resistance, two to three c o m m o n QTLs detected. For field resistance two other u n r e l a t e d were observed (Ferreira et al., 1995b). Dion et al. (1995) also detected two QTLs for field resistance to this disease, one of which seems to correspond to a major resistance gene n a m e d LmF~I.This gene was flanked by two RFLP m a r k e r s at 5% recombination on each side. White r u s t (Albugo candida race 2) in B. napus: A single gene responsible for resistance to this disease was m a p p e d (Ferreira et al., 1995c). The locus responsible, for the resistance, n a m e d ACA1, h a s been located on linkage group 4, flanked by two m a r k e r s at approximately 5 cM on each side (Kole et al., 1996). Black rot ( X a n t h o m o n a s campestris) in B. oleracea: Two QTLs were found associated to this trait by Camargo et al. (1995). One of this was also associated to petiole length. Fertility restorer gene for cytoplasmic male sterility in B. napus: An isozyme a n d a RAPD m a r k e r were found associated to the fertility restorer gene for the O g u r a r a d i s h system by Delourme et al. (1994). Morphological traits: Genes or m a r k e r s for 28 traits, some of which were associated to as m a n y as five QTLs were determined in a B. rapa progeny of Chinese cabbage ~Vlichihili x Spring broccoli (Song et al., 1995b). The same type of s t u d y was done by Kennard et al. (1994) for 22 traits in an F2 progeny B. oleracea, resulting of crossing broccoli by cabbage. U p a d h y a y et al. (1996) found a m a r k e r for yellow seed coat color in B. juncea. Markers were also detected for six quantitative traits. A RFLP for seed coat content was detected by Teutonico and Osborn (1994) a n d van Deynze et al. (1995) in B. napus.
Acknowledgments Part of this work was funded by USDA Competitive G r a n t s # 9201568 and 9600835.
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Biology of Brassica Coenospecies C. G6mez-Campo (Editor) 91999 Elsevier Science B.V. All rights reserved.
247
8 HAPLOIDY C o n s t a n t i n e E. P a l m e r (1) a n d Wilfred A. Keller (2)
(1) Department of Plant Science, University of Manitoba, Winnipeg, MB R3T 2N2, Canada. (2) NRC-Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, SK S7N owg, Canada. The d e v e l o p m e n t of h a p l o i d s nized as the m o s t r a p i d r o u t e to d u c t i o n of p u r e lines. T h e y o c c u r angiosperms and gymnosperms. c a n be achieved.
in a n u m b e r of p l a n t s p e c i e s is now recogthe a c h i e v e m e n t of h o m o z y g o s i t y a n d proin a n u m b e r of families a n d g e n e r a of b o t h T h e r e are several w a y s in w h i c h h a p l o i d y
(a) S p o n t a n e o u s o c c u r r e n c e in p l a n t p o p u l a t i o n s w h e r e t h e y are recognized by s m a l l n a r r o w leaves, m a l e sterility a n d a b n o r m a l p h y s i c a l f e a t u r e s . S u c h h a p l o i d s o c c u r a t very low f r e q u e n c y a n d m a y be of m a t e r n a l or p a t e r nal origin. (b) A n t h e r a n d m i c r o s p o r e c u l t u r e w h e r e t h e m a l e g a m e t e develops into e m b r y o by a p r o c e s s called a n d r o g e n e s i s . (c) The c u l t u r e of unfertilized ovules a n d ovaries l e a d i n g to e m b r y o dev e l o p m e n t from one or m o r e of the h a p l o i d cells w i t h i n t h e unfertilized e m bryo sac. This p r o c e s s is called g y n o g e n e s i s . (d) C h r o m o s o m e e l i m i n a t i o n as a c o n s e q u e n c e of wide c r o s s e s a s is the c a s e with the m a i z e - m e d i a t e d s y s t e m of h a p l o i d y in w h e a t (Laurie a n d Bennett, 1988). To be u s e f u l in crop i m p r o v e m e n t h a p l o i d s m u s t be p r o d u c e d efficiently in a wide r a n g e of species. With a n t h e r a n d m i c r o s p o r e c u l t u r e the p o t e n t i a l exists for the r e c o v e r y of large n u m b e r s of h a p l o i d p l a n t s b e c a u s e of the large n u m b e r of m i c r o s p o r e s / p o l l e n g r a i n s in e a c h a n t h e r . As a c o n s e q u e n ce, this is t h e m e t h o d of choice for h a p l o i d p r o d u c t i o n in m o n o c o t s a n d dicots. In Brassica a n d r e l a t e d s p e c i e s t h i s is the m o s t widely u s e d m e t h o d of h a p l o i d p l a n t p r o d u c t i o n a n d a l t h o u g h t h e r e are v a r i a t i o n s , s u c h p l a n t s c a n be p r o d u c e d in a wide variety of g e n o t y p e s s o m e of w h i c h are of c o m m e r c i a l i m p o r t a n c e . W i t h t h e available m e t h o d s for c h r o m o s o m e d o u b l i n g , d o u b l e d h a p l o i d p l a n t s c a n be efficiently r e c o v e r e d in m o s t c a s e s .
248 In this review the cur r ent s t a t u s of haploidy in B r a s s i c a will be presented with special e m p h a s i s on the culture of isolated microspores and the recovery of doubled haploid plants. The u s e s of these haploids in crop improvement, m u t a t i o n and selection and in basic studies of storage produced biosynthesis an d storage will also be examined.
Historical overview Although the process of microsporogenesis follows a precise pathway leading to the differentiation of the male gametophyte and haploid sperm cells, it has long been recognized t hat environmental d i s t u r b a n c e s such as high t e m p e r a t u r e can alter this pat hw ay resulting in a sporophytic pattern of development and the emergence of pollen embryos (Stow, 1930; La Cour, 1949). This process is referred to as androgenesis and occurs naturally, but at low frequencies, in some species where the male gamete develops into an embryo in the embryo sac (see reviews by Kimber and Riley, 1963; Pandey, 1973). In Brassica, s p o n t a n e o u s haploids occur at a low frequency (Thompson 1974) and can be produced t h r o u g h interspecific crossing (Prakash, 1973, 1974). However, there is no evidence that such haploids are of pollen origin. With the recognition that homozygous plants could be recovered from these haploids t h r o u g h chromosome doubling, their value in plant breeding was a p p a r e n t and research was directed towards the development of experimental protocols for the production of such plants by a n t h e r culture. Even t h o u g h mo s t n a t u r a l haploids are probably of maternal origin, the limited n u m b e r of haploid cells in the embryo sac is a barrier to large scale production of haploids. In contrast, a n t h e r s usually contain an a b u n d a n c e of fairly uniform haploid cells with the potential to produce large n u m b e r s of embryos. The first definitive report of haploid embryogenesis from microspores was by G u h a and Maheshwari (1964). This was followed by other reports in which haploids were obtained by a n t h e r culture in a n u m b e r of plant species including those of B r a s s i c a (Thomas and Wenzel, 1975; Nitsch, 1972; Kameya and Hinata, 1970). With the e s t a b l i s h m e n t of procedures for successful a n t h e r culture it became a p p a r e n t t hat the confines of the a n t h e r walls might qualitatively and quantitatively limit haploid embryogenesis (Hoffmann et al., 1982) as only a small n u m b e r of embryos were produced, compared to the large number of microspores within each anther. As a consequence, m e t h o d s were developed for the culture of isolated microspores and in tobacco, Nicotiana tabacum, the frequency of embryogenesis increased several folds with microspore culture, compared to a n t h e r culture (Nitsch 1974ab). In brassicas, an isolated microspore culture protocol was first reported by Lichter (1982) and, with modifications, it is now possible to recover ha-
249 ploid embryos from isolated microspores of a n u m b e r of B r a s s i c a species. There are a n u m b e r of critical factors affecting the efficiency of microspore embryogenesis in B r a s s i c a and manipulation of these factors is required to improve microspore embryogenesis in some species. The most extensive studies have been conducted with B r a s s i c a n a p u s where the frequency of embryogenesis can be as high as 50% (Pechan and Keller, 1988; Swanson, 1990). The response for B r a s s i c a r a p a (syn. B. c a m p e s t r i s ) is somewhat less although embryogenesis has been reported in a n u m b e r of genotypes (Ferrie and Keller, 1995). As a consequence, this technique is now employed as a means to produce homozygous lines for improvement of cultivated brassicas (Charne and Beversdorf, 1988; Hoffmann et al., 1982). Table 8.1 shows the n u m b e r of species in which microspore-derived embryos have been produced from isolated microspore culture. Embryos are produced either through anther or isolated microspore culture with the latter being the more efficient (Lichter, 1989; Siebel and Pauls, 1989).
Methodology For successful culture of microspore floral buds at the appropriate stage of development are homogenized and microspores separated by filtration and centrifugation (Polsoni et al., 1988; Coventry et al., 1988; Swanson, 1990; Baillie et al., 1992; Ferrie and Keller, 1995). Gradient centrifugation is seldom necessary as a purification step but can be used to enrich the preparation (Fan et at. 1988). Microspores are usually cultured in liquid medium or agarose overlayed with liquid, in dark for 21-28 days (Keller et al., 1987; Lichter, 1982; 1989, Gland et al., 1988; Mathias, 1988). Once embryos develop to the torpedo or cotyledonary stage, further development is normally achieved in light on reduced levels of sucrose.
F a c t o r s i n f l u e n c i n g m i c r o s p o r e culture Donor plant genotype For the efficient recovery of haploid embryos from B r a s s i c a species one of the most important controlling factors is the genetic m a k e - u p of the donor plant (Dunwell et al., 1985; Baillie et al., 1992; Ferrie and Keller, 1995). This is evident in the frequency of embryogenesis, the quality of embryos and the mode of plant regeneration (Arnison and Keller, 1990; Chuong et al., 1988; Duijs et al., 1992; Kieffer et al., 1993; Ferrie et al., 1995a). Breeding experiments reveal that the traits responsible for responsiveness to culture can be transferred from highly responsive genotypes to low or nonresponsive ones (Cardy, 1986; Dunwell et al., 1985; Wenzel, 1980; Ockendon and Sutherland, 1987). Within a genotype there may be plant to plant variation (Ockendon, 1985; Phippen and Ockendon, 1990; Seguin-Swartz et al., 1983). Even
250 though genetics is a major factor, environmental interactions do modify this effect (Dunwell et al., 1985; Roulund et al., 1990; Thurling and Chay, 1984). To underscore this observation, field grown plants of B. oleracea convar c a p i t a t a (L.) Alef. were generally more responsive t h a n greenhouse grown plants (Roulund et al., 1990). It is commonly observed that the more responsive the genotype the less stringent are the donor plant growth conditions for microspore embryogenesis, with the converse being true for non-responsive genotypes (Ferrie et al., 1995b). There are probably a few genes controlling microspore culture responsiveness in B r a s s i c a (see review by Palmer et al., 1996) and chromosomal regions involved in microspore culture responsiveness have been proposed (Cloutier et al., 1995). While knowledge of the biochemical and physiological basis for genotypic responsiveness is limited it may involve endogenous growth regulators (Arnison et al., 1990; Biddington et al., 1988; Biddington, 1992; Gland et at., 1988).
Donor plant physiology For successful microspore culture there is general agreement in the literature that donor plant growth conditions are decisive factors (Dunwell et al., 1985; Keller and Stringham, 1978; Thurling and Chay, 1984; Ferrie et al., 1995). For most B r a s s i c a species, growth at low temperatures appears to be required. The extent of such low temperature requirement may be genotype or species dependent as highly responsive microspores were obtained from B. n a p u s plant growth at 10~176 day-night temperature (Keller et al., 1987) while for some genotypes of B. j u n c e a a temperature regime of 20~176 daynight was required (Thiagarajah and Stringham, 1990). Reports indicate that microspores from winter type rape may be more responsive to low temperatures compared to s u m m e r type rape (Dunwell et al., 1985). With B. oleracea, some genotypes responded to low growth temperatures in a range of 5~ to 20~ (Duijs et al., 1992; Arnison and Keller, 1990; Roulund et al., 1990; Yang et al., 1991). In some cases there may be seasonal variations in microspore culture response and in B. rapa, the best responses were obtained in spring and a u t u m n (Sorvari, 1985). Even though low growth temperatures are conducive to a high embryogenic frequency in B r a s s i c a , responsive microspores have been obtained from greenhouse and field grown plants (Jain et al., 1989; Sorvari, 1985). The basic a s s u m p t i o n is that low temperatures modify the physiology of the donor plant and this is reflected in the responsiveness of the microspores. There is little definitive evidence to support this assumption. However, it has been observed that donor plant growth conditions affect the distribution of microspores of a particular nuclear stage (Duijs et al., 1992). In B. n a p u s developmental characteristics of the donor plant were altered by varying the growth conditions and the highest embryo yields were correlated with growth conditions which delayed flowering and increased
251
T a b l e 8. I B r a s s i c a s p e c i e s a n d a l l i e s i n w h i c h e m b r y o s h a v e been reported through microspore culture.*
Species
Reference
B. carinata
Chuong a n d Beversdorf 1986
B. r a p a / c a m p e s t r i s
Lichter 1989
_
Baillie et al., 1992 B u r n e t t et al., 1992 Guo a n d Pulli, 1996 B. rapa subsp, p e k i n e n s i s
Sato et at., 1989
B. rapa subsp, chinensis
Cao et al., 1994
B. rapa subsp, p a r a c h i n e n s i s
Wong et al., 1996
B. j u n c e a
Thiagarajah a n d Stringham, 1990,1993 Bhojwani a n d Agarwal, 1996
B. n a p u s oleifera
Swanson et al., 1987 Lichter, 1982, Mathias, 1988 Gland et al., 1988
B. n a p u s rapifera
H a n s e n a n d Svinnset, 1993
B. nigra
Lichter, 1989
B. oleracea
Duijs et al., 1992, T a k a h a t a a n d Keller, 1991 Lichter, 1989
* This listing is not intended to be exhaustive b u t r a t h e r to indicate species of Brassica where embryos developed t h r o u g h microspore culture. For a a more complete listing the reader is referred to a review by Palmer et al., 1996.
252 plant height (Lo and Pauls, 1992). Thus, low temperatures may alter the endogenous levels of hormones and metabolites in the anthers and modify the culture behavior of the microspores (Lo and Pauls, 1992). Cytological analysis of microspores from donor plants grown at different temperature regimes revealed that embryogenic microspores were cytologically different from nonembryogenic ones (Lo and Pauls, 1992). The importance of both high and low temperatures in the regulation of pollen development has been recognized for sometime. In T r a d e s c a n t i a b r a c t e a t a short-term exposure to high and low temperatures inhibited gametophytic development and resulted in anomalous cell divisions of the vegetative cell (Sax, 1935,1937; LaCour, 1949). With B r a s s i c a n a p u s , plants grown at high temperatures yielded microspores with a high frequency of symmetric cell division, compared to those grown at normal temperatures (Telmer et al., 1993). Also, in the culture of isolated microspores temperature controls the pattern of development (Custers et al., 1994). There has been no systematic analysis of qualitative and quantitative changes in hormones and metabolites in donor plants of B r a s s i c a grown at low temperatures. However, such temperatures may induce changes in hormones, specific amino acids and proteins which are known to respond to stress conditions. These may account for the embryogenic potential of the microspores. Other factors such as light intensity, mineral nutrient status and freedom from disease and pests affect donor plant physiology and the responsiveness of microspores. At least in B. n a p u s plant age was shown to be a critical factor. Higher frequencies of embryogenesis were obtained with older inflorescences compared to younger ones when young plants were used, while with older ones new inflorescence produced more embryogenic microspores (Takahata et al., 1991). In another study, as inflorescence age increased the frequency of embryogenesis declined (Chuong et al., 1988). This may be an important aspect of microspore culture as the physiology of the microspor e s / a n t h e r will probably vary with age. Growth temperature of 18~ and continuous light was superior to higher temperatures or 18~ light period for B r a s s i c a n a p u s compared to B. r a p a where 24~ with continuous light the most effective (Aslam et al., 1990).
Stage of microspore development In B r a s s i c a species microspore embryogenesis is restricted to a specific stage of microspore development, the uninucleate to early binucleate stage (Fan et al., 1988; Kott et al., 1988a; Telmer et al., 1993). With B. n a p u s , the highest frequency of embryogenesis occurred with microspores at the late uninucleate stage (Kott et al., 1988a). In the highly embryogenic cultivar Topas, the late uninucleate to the early binucleate stage was the most responsive (Fan et al., 1988; Hansen and Svinnset, 1993; Pechan and Keller,
253 1988; Telmer et al., 1992). For other B r a s s i c a species this stage was also optimal (Baillie et al., 1992; Duijs et al., 1992; H a m a o k a et al., 1991; Kieffer et al., 1993; Yang et al., 1992). There are only a few instances in which embryogenesis have been recorded in microspores outside this nuclear stage (Cao et al., 1990; Kameya and Hinata, 1970). It is unresolved whether microspores outside the late u n i n u c l e a t e to early binucleate stages have not acquired, or have lost competence for embryogenesis. The late uninucleate to early binucleate stages are essentially mitotic an d these cells are, therefore, a c c u m u l a t i n g or utilizing gene products associated with mitosis. Culture conditions such as t e m p e r a t u r e shock and a n t i m i c r o t u b u l a r agents s u c h as colchicine are able to stimulate cell division and embryogenesis in microspores at the above m ent i oned unclear stage (Simmonds, 1991; Zaki and Dickinson, 1991). This does not explain the specificity for a restricted nuclear stage of development. It is, however, evident that u n d e r permissive culture conditions microspores at this stage of development undergo sporophytic development leading to the elaboration of an embryo instead of the gametophytic p a t t e r n of development destined for the production of the gametes. The n u m b e r of microspores which develop into embryos is a small fraction of the total population and the question arises as to w h e t h e r all microspores are c o m p e t e n t for embryogenesis or w h e t h e r there is a distinct s u b p o p u l a t i o n of embryogenic microspores (Heberle-Bors, 1989). The existence of p o l l e n / m i c r o s p o r e dimorp h i s m h a s been reported in some species, b u t h a s not been defined in B r a s sica species (Horner and Street, 1978). If such a population exists t hen it is reasonable to infer t h a t conditions which favor microspore embryogenesis e n h a n c e the occurrence of embryogenic microspores. Pretreatments
o f i s o l a t e d b u d s or m i c r o s p o r e s
P r e t r e a t m e n t of isolated microspores, whole inflorescences or excised b u d s can e n h a n c e the frequency of embryogenesis. With B. n a p u s , the frequency of microspore-derived embryos was e n h a n c e d by i n c u b a t i n g flower b u d s at 4~ before microspore isolation (Lichter, 1982). Similarly, with B. oleracea b u d s kept at 4~ for 40h showed e n h a n c e d microspore embryogenesis in a g en o ty pe- dependent m a n n e r (Osolnik et al., 1993). P r e t r e a t m e n t of a n t h e r s of S i n a p i s alba (syn. B r a s s i c a hirta) with reduced a t m o s p h e r i c p res s u r e e n h a n c e d microspore embryogenesis (Klimaszewska and Keller, 1983). These t r e a t m e n t s are probably of little benefit if donor plants are grown u n d e r o p t i m u m environmental conditions. Exposure of isolated microspores to low level irradiation, ethanol, modified a t m o s p h e r e or antimicrot u b u l a r agents s uch as colchicine e n h a n c e d embryogenesis in a n u m b e r of experiments (Pechan and Keller, 1989; MacDonald et al., 1988ab; Klimaszewska an d Keller, 1983; Zaki and Dickinson, 1991; Iqbal et al., 1994; Zhao et al., 1996). However, these t r e a t m e n t s are not routinely u s e d even t h o u g h
254 the u s e of a n t i m i c r o t u b u l a r a g e n t s have the a d d e d a d v a n t a g e of c h r o m o s o m e doubling a n d the direct recovery of d o u b l e d - h a p l o i d embryos. Culture conditions
M e d i u m composition Media f o r m u l a t i o n s are designed to s u p p l y m i n e r a l salts, vitamins and c a r b o n required for e m b r y o development. The liquid NLN c u l t u r e m e d i u m originally developed by Lichter (1982) is now the m o s t c o m m o n l y u s e d with various modifications (Gland et al., 1988; H u a n g a n d Keller, 1989; Lichter, 1989; Ferrie a n d Keller, 1995). Variations in m i n e r a l composition m a y enh a n c e e m b r y o g e n e s i s b u t the m o s t critical c o m p o n e n t of the m e d i u m is the c a r b o h y d r a t e type a n d a m o u n t .
Carbohydrates The m o s t c o m m o n l y u s e d c a r b o h y d r a t e for B r a s s i c a microspore embryogenesis is sucrose. The o p t i m u m c o n c e n t r a t i o n varies from 10 to 14 per cent ( M a t s u b a y a s h i a n d K u r a n a k i , 1970; Gland et al., 1988; Keller a n d Armstrong, 1978; R o u l u n d et al., 1991; Duijs et al., 1992; Lichter, 1989). S u c h high levels provide both a c a r b o n source a n d an o s m o t i c u m for the ind u c t i o n process. In some c a s e s higher levels of s u c r o s e are required for ind u c t i o n of e m b r y o g e n e s i s t h a n for e m b r y o development. Dunwell and Thurling (1985) u s i n g 4 cultivars of B. n a p u s reported t h a t a n initial culture period at 20 per c e n t s u c r o s e followed by lower levels gave the highest embryo p r o d u c t i o n from c u l t u r e d a n t h e r s . These a u t h o r s indicated t h a t the osmotic potential of the a n t h e r h o m o g e n a t e was equal to t h a t of 17 per cent sucrose a n d c o n s e q u e n t l y , s u c r o s e levels in this r a n g e were more favorable to e m b r y o induction. Elevated s u c r o s e levels in the c u l t u r e m e d i u m removed some r e s p o n s e differences between cultivars even u n d e r poor donor plant growth conditions (Dunwell a n d Thurling, 1985). This two-step sucrose regime was also effectively employed in microspore embryo i n d u c t i o n of a n u m ber of B. r a p a (syn. B. c a m p e s t r i s ) genotypes (Baillie et al., 1992). There is no e x p l a n a t i o n for the a p p a r e n t specific r e q u i r e m e n t for sucrose in B r a s s i c a m i c r o s p o r e e m b r y o g e n e s i s b u t it is clear t h a t other carbohyd r a t e s s u c h as glucose, fructose a n d maltose are less effective (Baillie et al., 1992; Gland et al., 1988; Yang et al., 1992). S u c r o s e is a c o m m o n t r a n s p o r t c a r b o h y d r a t e in higher p l a n t s a n d a c c u m u l a t e s d u r i n g low t e m p e r a t u r e s t r e s s (Guy et al., 1992). It m a y have a protective function d u r i n g cell desiccation (Hoekstra et al., 1991). It m a y also serve a similar function during high t e m p e r a t u r e stress. This p h e n o m e n o n is observed in a s t u d y by Hamoa k a et al. (1991) which d e m o n s t r a t e d a specific r e q u i r e m e n t for sucrose during microspore cell division in a n t h e r s of B. r a p a c u l t u r e d at high temperature. However, no definitive information is available on s u c r o s e utilization
255 during microspore embryo induction and it would be useful to distinguish between its osmotic role and that of a carbon source. Nitrogen supply Organic nitrogen is a beneficial component of the microspore culture medium and L-glutamine and L-serine or casein hydrolysate are generally included in formulations (Gland et al., 1988). However, there is no indication that they are specifically required either for isolated microspores or anther culture. Moreover, the main nitrogen source in most cases is nitrate which suggests that its utilization is not a limitation to embryo induction. However, the a m o u n t of total nitrogen, ratio of NO3:NH4 and ratio of inorganic to organic nitrogen influenced microspore embryogenesis in barley (Mordhorst and Lorz, 1993) and manipulation of these components may improve the efficiency of embryogenesis in Brassica. Plant growth regulators There are two aspects of plant growth regulator response in microspore embryogenesis, the exogenous requirement, or lack thereof, and the production of endogenous growth regulators which influence embryogenesis. The requirement for exogenous growth regulators has not been rigorously established and in some cases embryogenesis occurs without exogenous auxin or cytokinin (Keller et al., 1987; Swanson et al., 1987). Studies with B. n a p u s microspores demonstrated an e n h a n c e m e n t of embryogenesis with benzylaminopurine (BAP) (Charne and Beversdorf, 1988). This response may be species specific as cytokinins suppressed embryogenesis in a n t h e r cultures of cauliflower (B. oleracea var. botrytis) (Yang et al., 1992) while the response was genotype dependent with broccoli a n t h e r cultures (Arnison et al., 1990). Low levels of auxin enhanced microspore embryogenesis in B. oleracea while high levels were inhibitory (Ockendon and McClenaghan, 1993; Yang et al., 1992). These responses were obtained with a n t h e r cultures and it is possible that the a n t h e r tissues may modify the response to exogenous growth regulators. Callusing of a n t h e r tissues appears to increase with high levels of auxins and this was correlated with reduced microspore embryogenesis (Yang et al., 1992). Although the requirement for auxins and cytokinin in microspore culture is undefined they are usually included at low levels in the culture medium (Gland et al., 1988; Lichter, 1982,1989; Yang et al., 1992). Studies on the relationship of ethylene and abscisic acid to microspore embryogenesis have been performed with B. oleracea a n t h e r cultures. Exogenous ethylene and conditions which enhance endogenous ethylene production are inhibitory to pollen embryogenesis (Biddington and Robinson, 1990,1991). Also B. oleracea genotypes which are unresponsive to culture showed e n h a n c e d ethylene production (Biddington and Robinson, 1990;
256 Biddington et al., 1992,1993). The conclusion is t h a t endogenous ethylene production is a limiting factor in B. o l e r a c e a microspore embryogenesis and a n t h e r filament and wall tissues are rich sources of endogenous ethylene (Biddington, 1992). Exogenous abscisic acid inhibited embryogenesis in a n t h e r cultures of B. o l e r a c e a by stimulating ethylene production (Biddington et al., 1992). However, its role in embryo induction of isolated microspores remains to be defined. While there may not be an obligatory requirement for plant growth regulators in the embryo induction process, development of the embryo is undoubtedly regulated by auxins as is the case with development of zygotic and somatic embryos (Liu et al., 1993). Microspores are a rich source of auxin (Stead, 1992) which may be available for embryo development. The developing embryo may also acquire competence for auxin production making exogenous supply u n n e c e s s a r y . It is generally accepted that ABA plays a significant role in embryogenesis where it controls normal m a t u r a t i o n and the a c c u m u l a t i o n of storage products. This is probably the same for microspore-derived embryos as the low osmotic potential of the culture m e d i u m is conducive to ABA biosynthesis. Therefore, even t h o u g h it inhibits embryo induction (Biddington et al., 1992) it is certain to exert a positive effect on embryo development. Other factors A n u m b e r of other factors such as pH, influence the frequency of embryogenesis in B r a s s i c a species (Baillie et al., 1992; Gland et al., 1988; Arnison et al., 1990). The use of activated charcoal or frequent media changes a p p e a r s to remove toxic s u b s t a n c e s and improved embryogenesis (Gland et al., 1988; Kott et al., 1988b; Hansen and Svinnset, 1993). Temp e r atu r e The culture t e m p e r a t u r e is a critical factor for successful microspore culture in brassicas. In most cases embryogenesis is initiated by exposure to t e m p e r a t u r e s of 30 to 35~ for 24h to 72h and embryos can then develop at lower t e m p e r a t u r e s (Arnison et al., 1990; Gland et al., 1988; Dunwell et al., 1985; Baillie et al., 1992; Duijs et al., 1992; Yang et al., 1992; Keller and Armstrong, 1979). Within this t e m p e r a t u r e range, embryogenic response varies with genotype and species. For B. o l e r a c e a 32 to 35~ was optimal (Arnison a n d Keller, 1990; Duijs et al., 1992; Yang et al., 1992). For several genotypes of B. r a p a 32~ for 48h to 72h gave the best results (Baillie et al., 1992). This need for high t e m p e r a t u r e may not be obligatory as colchicine t r e a t m e n t was shown to replace this requirement (Zhao et al., 1996). A minim u m period of 6-18h at elevated t e m p e r a t u r e s is required soon after micro-
257 spore isolation to induce embryogenesis (Pechan et al., 1991; Duijs et al., 1992; Ockendon, 1984). The physiological and biochemical bases for this high temperature response are u n d e t e r m i n e d but it is clear that high culture temperature is a decisive factor in the transition from gametophytic to sporophytic development in microspores. When B. n a p u s microspores were cultured at low and high temperatures, gametophytic development predominated at low temperature while at high temperature sporophytic development occurred at high frequency (Custers et al., 1994). Qualitative analysis of soluble proteins indicated a close similarity between microspores cultured at low temperature and isolated pollen, but not with those cultured at high temperature. In these studies the synthesis of certain heat-shock proteins in response to elevated t e m p e r a t u r e s appears to be related to embryo induction (Cordewener et al., 1995). Elevated culture temperatures increased the frequency of symmetric cell division of microspores (Hause et al., 1993; Telmer et al., 1992,1993) and promoted division of the vegetative nucleus in binucleate pollen (Custers et al., 1994). These all favored sporophytic development and embryo organization. Starch metabolism and the expression of specific genes may also be related to high temperature treatment (Pechan et al., 1991; Telmer et al., 1993,1995). Ethylene metabolism may also be a factor as in B. oleracea recalcitrant genotypes produced higher levels at elevated temperatures compared to low levels by responsive genotypes (Biddington et al., 1988; Biddington and Robinson, 1990). Ethylene inhibitors, AVG and AgNo3 e n h a n c e d embryogenesis in non-responsive genotypes but a high temperature treatment was still required (Biddington et al., 1988). This suggests that other factors besides ethylene are involved in the high temperature response. At least in B. rapa, the high temperature response is dependent on elevated levels of sucrose (Hamoaka et al., 1991). Whether or not this specific carbohydrate requirement is nutritional, osmotic or protective remains to be determined. It can be a s s u m e d t h a t in brassicas, high temperature provides an inductive stress factor, since in Nicotiana t a b a c u m pollen stress resulting from nutrient starvation elicited the switch from gametophytic to sporophytic development (Zarsky et al., 1992). Light Microspore-derived embryos are generally initiated in d a r k n e s s but there is no definitive information on the importance of light quality and quantity to the frequency of embryogenesis. In B. oleracea a n t h e r cultures, incubation in the dark was more favorable to embryogenesis t h a n a light intensity of 10 w / m 2 for 16h (Yang et al., 1992). Once embryos are formed further development and maturation occurs in light of relatively low intensity (100 ~mol m -2 S -1) until fully green (Baillie et al., 1992).
258 Microspore culture density Culture densities range from 4x104 cells per ml to l x l 0 5 cells per ml (Fan et al., 1988; Kott et al., 1988b; Polsoni et al., 1988; Baillie et al., 1992). In highly responding genotypes of B. n a p u s a density of 3x10 a cells per ml was the m i n i m u m for embryogenesis (Huang et al., 1990). The optimum density is required to provide the putative conditioning factor and to prevent over crowding and the a c c u m u l a t i o n of toxic metabolites (Huang et al., 1990; Kott et al., 1988b). The positive effect of nurse cultures on embryogenesis at low culture density may be due to conditioning factors (Simmonds et al., 1991).
Developmental aspects of microspore embryogenesis Studies with B. n a p u s microspores have revealed morphological and cytological differences between potentially embryogenic and nonembryogenic microspores (Kott et al., 1988a; Lo and Pauls, 1992; Telmer et al., 1993; Zaki and Dickinson, 1990). These differences include changes in the a b u n d a n c e of starch grains a n d the volume of the vacuole (Telmer et al., 1993). The u n i n u c l e a t e to the early binucleate stages of microspore development are the most favorable for embryogenesis (Fan et al., 1988; Kott et al., 1988a; Telmer et al., 1992; Baillie et al., 1992). It is reasonable to a s s u m e that conditions which favor a high proportion of microspores at these stages, in the general microspore population, will favor a high frequency of embryogenesis. Also, the population of embryogenic microspores can be enriched by bud selection, gradient centrifugation a n d flow cytometric techniques (Fan et al., 1988; DesLauriers et al., 1991; Pechan et al., 1988). Under optimal culture conditions, embryogenic microspores divide symmetrically instead of the asymmetric division characteristic of nonembryogenic microspores (Fan et al., 1988; Telmer et al., 1993; Zaki and Dickinson, 1991; Custers et al., 1994). Figure 8.1 indicates the p a t h w a y s to embryogenesis from the u n i n u c l e a t e microspore. High culture t e m p e r a t u r e during the early stages increases the frequency of symmetric cell divisions where as at low culture t e m p e r a t u r e s cell division is asymmetric a n d microspores mature as pollen like s t r u c t u r e s (Hause et al., 1993; Telmer et al., 1993; Custers et al., 1994). The high t e m p e r a t u r e response was evident with isolated microspores and with those from intact plants exposed to high temperature (Telmer et al., 1993). The a r r a n g e m e n t of microtubules and the cytoskeleton is important for symmetric cell division and embryogenesis (Hause et al., 1993; Zaki and Dickinson, 1991; Iqbal et al., 1994). C o m m i t m e n t to the embryogenic pathway of development requires the synthesis of specific proteins associated with embryogenesis. Culture temperature to a certain extent controls this process as the soluble protein profile
259
Pathways to Haploid Embryos from Microspores or Pollen in Brassica
.•,
Asymmetric / cel'l divisi~///
Uninucleate
microspore
\ Symmetric x ~ ell divisiOn
Binucleate pollen
/
1
Gametophyte development
l
Globular embryo
Multiplication of vegetative cell and degeneration of generative nucleus Cotyledonary embryo Figure 8.1 The illustration shows both symmetric and asymmetric cell division from the uninucleate microspore. This would be favoured if donor plants are subjected to high temperatures (Telmer eta/., 1993). Under culture conditions both uninucleate microspores and binucleate pollen may divide to produce embryos without gametophyte development.
260 of microspores cultured at elevated t e m p e r a t u r e s closely resembled that of the embryo while the same microspores cultured at low non-inductive temp e r a t u r e s h a d a soluble protein profile resembling t hat of pollen (Custers et al., 1994; Cordewener et al., 1994). Some of these proteins are specifically associated with early embryo induction and are probably related to heat shock or s t r e s s - i n d u c e d proteins (Cordewener et al., 1994,1995; Boutilier et al., 1994). The involvement of heat shock proteins in pollen embryogenesis h a s been suggested (Zarsky et al., 1995; Cordewener et al., 1995). In B r a s s i c a microspore cultures high t e m p e r a t u r e t r e a t m e n t elicited the a p p ear an c e of a n u m b e r of mRNAs and proteins in embryogenic microspores (Pechan et al., 1991). These were less a b u n d a n t or a b s e n t in microspores ma in ta in ed u n d e r non-inductive conditions. These proteins have not been characterized in B r a s s i c a but in Nicotiana t a b a c u m a n u m b e r of phosphoproteins were identified in embryogenic microspores (Kyo and Harada, 1990). In this species it is also evident t h a t n u t r i e n t starvation activates DNA replication in the vegetative n u c l e u s of binucleate pollen and transcription of specific genes occurs (Zarsky et al., 1992; Garrido et al., 1993). In these experiments the biochemical events associated with embryogenesis are triggered by n u t r i e n t stress and, while a similar situation h a s not been described for B r a s s i c a microspores, it is evident t hat elevated t e m p e r a t u r e s (stress) can elicit s u s t a i n e d cell division in the vegetative cell of a binucleate pollen followed by embryo development (Custers et al., 1994). Recent studies of nuclear DNA synthesis in isolated microspores or pollen of B r a s s i c a n a p u s revealed th at the vegetative cell re-entered the cell cycle resulting in embryogenic development, when cultured at elevated temperature, while at lower t e m p e r a t u r e s DNA synthesis was restricted to the generative nucleus and development was gametophytic (Binarova et al., 1993). These authors concluded that microspores can enter the embryogenic p a t h w a y at the G1 to the G2 stages of the cell cycle, while entry into this pat hw ay by binucleate pollen is restricted to the G1 stage. An early event in microspore embryogenesis is the e s t a b l i s h m e n t of polarity. This is u n d o u b t e d l y related to the p h y t o h o r m o n e auxin which is responsible for polarity during early zygotic embryogenesis (Liu et al., 1993). During the early stages of culture, cell divisions of the microspores occur within the confines of the microspore cell wall resulting in a m a s s of small cells (Hause et al., 1994). Polarity is probably established at the proembryo stage of development and may involve calcium, starch metabolism and endogenous h o r m o n e s (Timmers and Schel, 1991; Hause et al., 1994).
Embryo maturation Even t h o u g h microspore-derived embryos of B r a s s i c a are developmentally similar to the zygotic embryo, there are physiological and biochemical differences. This largely reflects the environment in which the two types of embryos develop. The zygotic embryo develops in the confines of the cotyle-
261 don and the seed coat. Throughout development this embryo is influenced by the physiological and biochemical events of the intact plant. In contrast, microspore-derived embryos have no endosperm or seed coat and are in direct contact with the closed system of the culture medium environment. These embryos do not undergo the normal maturation process seen in intact zygotic embryos. The critical hormone regulating this process is abscisic acid (Finkelstein and Sommerville, 1989; Finkelstein et al., 1985; Maquoi et al., 1993). With microspore-derived embryos, there is very little ABA synthesized (Mandel, 1991) and consequently the normal maturation process does not occur. Instead, embryos germinate precociously and culture manipulations are required to allow normal embryo maturation. The application of ABA and lowering the osmotic potential of the culture medium e n h a n c e d normal maturation of microspore-derived embryos and resulted in the accumulation of storage products similar to those of the zygotic embryo (Eikenberry et al., 1991; Gruber and R6bbelen, 1991; Senaratna et al., 1991; Brown et al., 1993; Pomeroy et al., 1994). In B r a s s i c a , the acquisition of desiccation tolerance and biosynthetic competence, by microspore-derived embryos required ABA treatment and best results were obtained when these were treated at the late torpedo or cotyledonary stage (Brown et al., 1993; Pomeroy et al., 1994; Taylor et al., 1990b; Wilen et al., 1990).
Plant regeneration and doubled haploid production Under optimal conditions fully developed embryos at the cotyledonary stage can be obtained after 21 d in culture for most B r a s s i c a microspore cultures and green embryos after an additional 14 days in light. The quality of embryos can be improved by low temperature treatment, partial desiccation treatments and culture agitation (Brown et al., 1993; Coventry et al., 1988; Gland et al., 1988; Kott and Beversdorf, 1990; Lichter, 1989; Mathias, 1988; Polsoni et al., 1988). Fully m a t u r e cotyledonary embryos often germinate directly into plantlets but there may be plant regeneration through secondary shoot formation from the cotyledonary areas (Duijs et al., 1992; Siebel and Pauls, 1989). The latter tendency may be genotype dependent (Duijs et al., 1992). There may also be extensive secondary and tertiary embryo development from the initial embryo (Loh and Ingram, 1982). Plant regeneration frequencies are often low and variable. With B. nap u s frequencies ranged from 1 to 47 percent (Chuong et al., 1988; Kott and Beversdorf, 1990) and 5 to 20 percent for B. r a p a (Baillie et al., 1992; Burnett et al., 1992). With B. rapa var. p e k i n e n s i s , plant recovery was 5 to 10 percent of the embryos produced (Sato et al., 1989) while in B. oleracea, genotype had a pronounced effect on plant recovery which varied from 35 to 70 percent (Duijs et al., 1992).
262 The stage at which embryos are transferred to a solid, hormone-free medium is critical for succesful plant recovery. Embryos transferred at the cotyledonary stage were found to give the highest plant regeneration frequency (Burnett et al., 1991; Coventry et al., 1988; Polsoni et al., 1988; Zhang et al., 1991). Histological analysis of cotyledonary embryos of B. n a p u s indicated that abnormal vascular connection between the shoot axis and the root axis was a factor in low plant conversion (Rahman, 1993). One important aspect of microspore technology consists of obtaining an easy generation of homozygous plants for use in breeding programs. This is usually achieved with the use of antimicrotubule agents such as colchicine (Coventry et al., 1988; Lichter et al., 1988; Swanson et al., 1988; Polsoni et al., 1988). Treatment with colchicine is frequently done at the plant stage by immersing rooted plants in a 0.1 to 0.2 percent solution of colchicine for 5 to 6 hours (Swanson et al., 1988; Coventry et al., 1988). Alternatively, colchicine can be injected into secondary buds of intact plants or into young plantlets treated in vitro prior to their transfer to soil (Lichter et al., 1988; Mathias and R6bbelen, 1991). In either case a high degree of diploidization can be achieved. Chromosome doubling at high frequency can also be obtained by exposing microspores to antimicrotubule agents during the early stages of culture (M611ers et al., 1994b; Chen et al., 1993; Eikenberry, 1993; Zhao and Simmonds, 1995; Zhao et al., 1996; Hansen and Andersen, 1996). The major limitation to the widespread use of this technique appears to be regulation of the concentration and time of exposure to these doubling agents to maximize diploidization and minimize aberrations. With B. n a p u s , microspores exposed to 50 mg.1-1 colchicine for 24 h showed 80-90% diploidization (M611ers et al., 1994b). In a few instances spontaneous diploids have been obtained, perhaps through endoreduplication, and fertile homozygotes have been recovered (Charne et al., 1988; Lichter et al., 1988; Foisset et al., 1997). Following cryopreservation of isolated microspores of B. n a p u s a very high percentage of spontaneous diploids was obtained in s u b s e q u e n t cultures (Chen and Beversdorf, 1992a). This response was unexpected and the reason for it is unexplained. So far, it has not been reported neither in other B r a s s i c a species nor in other related taxa.
Utilization of microspore-derived embryos of Brassica Genetics and breeding One advantage of microspore culture technology in B r a s s i c a is the rapid production of homozygous lines from haploid embryos through chromosome doubling compared to conventional backcrossing. With doubled haploids all loci are homozygous and all functional genes are expressed.
263 The value of this technology is evident in outcrossing or self-incompatible species such as B r a s s i c a oleracea and B r a s s i c a r a p a where it is a reliable method of rapid homozygous plant production (Duijs et al., 1992). With male sterile genotypes microspore culture is an advantageous method of achieving homozygosity where there is microspore development to the responsive stage (Sodhi et al., 1995). As a consequence of these attributes, microspore culture technology is now a part of several B r a s s i c a breeding programs (Keller et al., 1987; Wenzel et al., 1977; Hoffmann et al., 1982; Scarth et al., 1991; Chen and Beversdorf, 1990; Stringham and Thiagarajah, 1991; Keller et al., 1982; Dewan et al., 1995). With microspore-derived plants there is no dominance and recessive interaction and this makes selection easier as characteristics are more defined (Choo et al., 1985). Also population sizes for selection are smaller than those required for conventional breeding (Siebel and Pauls, 1989ab; Thiagarajah and Stringham, 1993). The value of doubled haploids for breeding depends on the regenerants representing a random array of gametic recombinations and there should be no preferential regeneration from selected microspores. This appears to be largely the case (Charne and Beversdorf, 1991; Chen and Beversdorf, 1990; Thiagarajah a n d Stringham, 1993). However, segregation distortion patterns have been reported in microspore-derived embryos of B. n a p u s and this may be due to genes regulating embryogenesis (Foisset et al., 1993, 1997). Mutation and selection
In brassicas, the haploid system has been used for the selection of herbicide tolerant m u t a n t s (Swanson et al., 1988,1989; Beversdorf and Kott, 1987; Polsoni et al., 1988; Saxena et al., 1990; Ahmad et al., 1991a; Kott et al., 1996). Stable m u t a n t s resistant to chlorsulfuron and imidazolinone herbicides have been obtained (Swanson et al., 1988,1989 ). Selection pressure by the herbicides resulted in the recovery of resistant embryos and plants. Thus, mutagenesis may not be required to recover stable mutations. Mutants for altered fatty acid composition were isolated following mutagenesis of microspores (Turner and Facciotti, 1990; Huang et al., 1991). A major advantage of the haploid embryogenic system for the isolation of storage product m u t a n t s is that analysis can be performed and one of the two cotyledons and the rest of the embryo retained for plant regeneration. One of the first attempts at exploitation of the haploid embryogenic system for m u t a n t analysis in B r a s s i c a was the selection for disease resistance using fungal culture filtrates (Sacrist&n and Hoffman, 1979; Sacrist&n, 1982, 1985; MacDonald and Ingram, 1986; Newsholme et al., 1989; MacDonald et al., 1989; Ahmad et al., 1991b). Although resistant cells were selected using culture filtrates of A t t e m a r i a brassicicola and L e p t o s p h a e r i a m a c u l a n s , no resistant plants have been reported. This may be due to the non-specificity of
264 the culture filtrates. However, the use of haploid plants in the selection for blackleg tolerance may result in heritable resistance (Stringham et al., 1993). With the identification of a specific toxin in the culture filtrate of Leptosp h a e r a m a c u l a n s (Pedras and Seguin-Swartz, 1992; Pedras et al., 1993) it should now be possible to isolate resistant plants. The use of chemical and physical m u t a g e n s should a u g m e n t this system (SacristAn, 1982; MacDonald et al., 1989; Ahmad et al., 1991b). In Brassica oleracea, doubled haploid plants were screened and resistance to clubroot disease identified (Voorrips and Visser, 1990). This was identified without in vitro selection pressure and represented gametoclonal variation which existed in the culture (Morrison and Evans, 1988). Gene transfer
A n u m b e r of techniques have been used for the introduction of foreign genes into haploid B r a s s i c a cells. Direct microinjection of DNA into cells of microspore-derived embryos of B. n a p u s resulted in transgenic plants (Neuh a u s et al., 1987). Such a technique is obviously advantageous for DNA delivery, but is technically demanding and it is not widely used. Particle b o m b a r d m e n t combined with desiccation and imbibition of DNA resulted in a high frequency of transformants in B. n a p u s microsporederived embryos (Chen and Beversdorf, 1994) with the recovery of transgenic plants. Transient GUS expression was achieved in microspores of B. n a p u s follow-ing DNA uptake by electroporation (Jardinuad et al., 1993). These are poten-tially valuable techniques for haploid cell transformation especially with the emergence of reliable protocols for secondary embryogenesis from cultured embryos (Nehlin et al., 1995). Agrobacterium t u m e f a c i e n s - m e d i a t e d gene transfer is the most commonly used for haploid cell transformation in Brassica (Swanson and Erickson, 1989; Oelck et al., 1991; Huang, 1992). Transgenic plants of B. napus, resistant to antibiotics and the herbicide L-PPT have been recovered following cocultivation of haploid embryo segments with Agrobacterium (Swanson and Erickson, 1989; Oelck et al., 1991). While this method is attractive because of the high regenerative potential of these embryos, it has not been fully exploited. Perhaps the most attractive method is direct co-cultivation of microspores with Agrobacterium. In early studies, successful co-cultivation was reported but no plants were regenerated (Pechan, 1989). Other reports indicate the successful recovery of transgenics following co-culture of Agrobacterium and freshly isolated microspores (Dormann et al., 1995,1996). The response appears to be related to the timing of co-cultivation and media manipulation to remove bacterial cells following co-culture. This is a potentially useful approach as individual transformed cells give use to haploid embryos and the introduced trait can be readily fixed.
265
P r o t o p l a s t c u l t u r e and fusion The major advantage to the use of haploid cells for protoplast fusion is that complete diploids can be recovered with the potential for increased fertility. In contrast with diploid fusions, even where the nucleus of one partner is inactivated, there can be chromosomal aberration and low fertility. Haploid-haploid fusion was employed in B r a s s i c a n a p u s to recover CMS plus herbicide tolerant plants (Chuong et al., 1988, 1989). The resulting diploids were completely fertile. There are a n u m b e r of reports of protoplast isolation, culture and plant recovery (Thomas et al., 1976; Kohlenbach et al., 1982; Chuong et al., 1987; Li and Kohlenbach, 1982). This system is of value for mutagenesis and the selection of naturally occurring variants and is particularly useful where plant regeneration is efficient as is the case with secondary embryogenesis from haploid primary embryos of some B r a s s i c a species (Ingram et al., 1984).
B i o c h e m i c a l and p h y s i o l o g i c a l s t u d i e s Evidence indicates a close developmental similarity between microspore embryogenesis and zygotic embryogenesis (Pomeroy et al., 1991a; Crouch, 1982). This system is, therefore, a convenient tool for studies of embryogenesis and the physiological and biochemical aspects of storage product accumulation. All stages of embryogenesis are easily identified with microspore derived embryos, and the influence of hormonal, media and environmental factors on embryo development can be readily determined (M611ers and Albrecht, 1994; Albrecht et al., 1994; M611ers et al., 1994a). For these and other reasons, microspore derived embryos are now used extensively for a n u m b e r of physiological and biochemical studies. These embryos are a rich source of enzymes involved in lipid biosynthesis (Taylor et al., 1990a) and they have been used to define the enzyme systems involved in the biosynthesis of long chained fatty acids (Taylor et al., 1990b,1992; Holbrook et al., 1992). The triacylglycerol (TAG) profiles of both microspore-derived and zygotic embryos are similar (Chen and Beversdorf, 1991; Pomeroy et al., 1991a) and the seed specific fatty acid, erucic acid, occurs in both types of embryos (de la Roche and Keller, 1977). In some cases culture conditions can be manipulated to allow accumulation of triacylglycerols by microspore-derived embryos, at rates greater t h a n those of zygotic embryos (Wiberg et al., 1991). This system is, therefore, very suitable for rapid screening for oil quality (Wiberg et al., 1991; Albrecht et al., 1994; M61lers and Albrecht, 1994). Among the advantages outlined by Wiberg et al., (1991) are reduction in labor requirement for screening, ability to evaluate recessive characteristics and nondestructive analysis, where single cotyledons can be analyzed and plants regenerated from the remainder of the em-
266 bryo. Also, with any purposeful modification of the haploid embryo for oil quality or quantity, screening can be done at the embryo level (Wiberg et al., 1991). Storage protein accumulation, napin and cruciferin, occurs in microspore-derived embryos (Crouch, 1982; Taylor et al., 1990a; Wilen et al., 1990; Van Rooijen et al., 1992; Pomeroy et al., 1994). Some physiological aspects of protein accumulation have been examined using these embryos, and the similarity to protein accumulation in zygotic embryos outlined (Pomeroy et al., 1994; Finkelstein et al., 1985; Holbrook et al., 1992). There are a n u m b e r of other uses of microspore-derived embryos of B r a s s i c a , including studies of chilling and freezing tolerance (Johnson-Flanagan et al., 1986; Orr et al., 1990; Cloutier, 1990; Pauls, 1990). Microspore-derived embryos have been used in studies of chlorophyll metabolism and the degreening process (Johnson-Flanagan et al., 1992; Johnson-Flanagan and Singh, 1991,1993) and in studies of glucosinolate metabolism (McClellan et al., 1993; Iqbal et al., 1995).
Germplasm storage and artifical s e e d t e c h n o l o g y The embryos arising from microspore culture represent a range of genetic recombinations and some of these may be useful in crop improvement. The system can, therefore, be viewed as a source of germplasm. Because of the large n u m b e r s of embryos generated from highly responding species and genotypes of brassicas, a convenient and reliable method of storage is required. Any storage method should allow recovery of viable microspores or embryos. There are two general methods, cryopreservation and desiccation storage. Isolated microspores have been cryopreserved without loss of embryogenic potential (Charne et al., 1988; Chen, 1991; Chen and Beversdorf, 1992b; Skladal et al., 1992). An apparent advantage of low temperature storage of B. n a p u s microspores is the recovery of a high frequency of spontaneous diploid plants following embryogenesis (Chen and Beversdorf, 1992a). Microspore-derived embryos have been cryopreserved and plants successfully recovered (Bajaj, 1983,1987). Desiccation storage is simple and inexpensive provided viable embryos can be recovered. Embryos have been desiccated, stored at room temperature and plants recovered at high frequency (Brown et al., 1993; Takahata et al., 1993; S e n a r a t n a et al., 1991; Eikinberry et al., 1991). Important factors for successful embryo desiccation were, the stage of embryo development and the use of exogenous abscisic acid. Once desiccated, microspore derived embryos can be handled as artificial seeds. This technology has been applied to somatic embryos from a n u m b e r of species (Redenbaugh, 1993; Janick, 1989) and has been recently examined with microspore-derived embryos of B r a s s i c a species (Takahata et
267 al., 1992,1993). E m br yos could be desiccated to 10 percent water content without loss of viability (Takahata et al., 1993). Where embryos of desirable genotypes can be generated at high frequency artificial seed technology can be exploited. An advantage of this system is the possible recovery of homozygous artificial seed t h r o u g h s p o n t a n e o u s or chemical induced c h r o m o s o m e doubling during the initial stages of embryo development.
C o n c l u s i o n s and future p r o s p e c t s In brassicas, isolated microspore culture is the m o s t efficient m et hod for doubled haploid production and the value of these plants in crop improve-ment is now firmly established. In almost all B r a s s i c a species it is now pos-sible to generate haploid embryos and doubled haploid plants t h r o u g h mi-crospore culture. When compared to other crop species, the use of doubled haploids in breeding is a m o n g the m ost successful and cultivars have been developed us i ng this technology (Thompson, 1975; Agric. C a n a d a Variety Description, 1993). However, effective utilization of this technology requires microspore culture an d plant recovery techniques t h a t are applicable to a wide range of B r a s s i c a species. Key factors governing microspore culture response have been identified a n d this has u n d o u b t e d l y advanced the use of this technology. However, m e t h o d s are still largely empirical a n d an u n d e r s t a n d i n g of the influence of factors s u c h as genotype, culture t e m p e r a t u r e , carbohydrate requirement a n d the influence of plant growth conditions, at the physiological an d biochemical level is still lacking. There are suggestions t h a t u n d e r the appropriate culture conditions microspores from any B r a s s i c a species or genotype can be responsive (Lichter, 1989). While this m ay be so, it is still difficult to reconcile the complete lack of response in some genotypes with the presence or absence of media components. For almost all B r a s s i c a species so far examined the m o s t responsive stages of microspore development are the late u n i n u c l e a t e to early binucleate stages. It would be useful to allow microspores to a c c u m u l a t e to these stages by some temporary blockage in development. It is not clear why microspores outside this window of development are generally unresponsive. A potentially fruitful area of research is examination of the dedifferentiation of the fully binucleate pollen. The expectation is t h a t advances will be m ade in this area as biochemical studies on t e m p e r a t u r e regulation of gametophytic and sporophytic development of cultured microspores are now being reported (Pec h a n et al., 1991; Binarova et al., 1993; Custers et al., 1994). The influence of high and low t e m p e r a t u r e on a n o m a l o u s pollen development in plants was observed more t h a n 6 decades ago (Sax, 1933), yet it is only recently t h a t there h a s been any a t t e m p t at u n d e r s t a n d i n g its overriding role in microspore embryogenesis.
268 There have been advances in the physiology and biochemistry of microspore-derived embryo maturation and it is now recognized that ABA plays a key role in this process (Pomeroy et al., 1994; Brown et al., 1993). However, conditions should be optimized for the recovery of normal embryos at high frequencies. The recovery of doubled haploid plants appears to be mostly through colchicine treatment of plantlets. However, in m a n y instances attempts are being made to recover doubled haploid embryos by chromosome doubling at the early stages of microspore culture. Such treatments do not appear to reduce the efficiency of embryogenesis and may even enhance it (Iqbal et al., 1994). In oilseed brassicas microspore embryos have been successfully exploited for studies of lipid and protein metabolism and this system is well suited for selection for oil quality and quantity (Wiberg et al., 1991; Albrecht et al., 1994). There is an enormous potential for the use of haploid microspore culture systems for mutation studies, selection and gene transfer and basic studies of plant embryogenesis. One particular advantage of the haploid culture system is due to the random array of genetic recombinations which microspores represent. Where embryos are generated at high frequencies they become a rich source of genetic variation. Culture conditions and chemical-induced chromosome doubling may also contribute to such variations. Since microspore derived embryos can be produced in large n u m b e r s and are more uniform, compared to somatic embryos, there is the potential to apply artificial seed technology to such embryos (Takahata et al., 1992). This is particularly attractive if diploidization can be achieved during the early stages of embryo culture so that doubled haploid embryos can be recovered. These embryos could be a valuable source of germplasm.
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Biology of Brassica Coenospecies C. G6mez-Campo(Editor) 91999 Elsevier Science B.V. All rights reserved.
287
9 GENETIC ENGINEERING Elizabeth D. Earle (1) a n d Vic C. Knauf (2)
(1) Department of Plant Breeding, Cornell University, Ithaca, NY14853-1901 U.S.A. (2) Calgene, Inc., 1920 Fifth Street, Davis, CA 95616 U.S.A. The m o s t dr am at i c scientific development affecting Brassica crops in the p a s t two decades is the ability to introduce genes from any source (viruses, bacteria, m a m m a l s , etc. as well as plants whose genes c a n n o t be accessed by sexual crosses). Transformation technology h a s already greatly expanded the genetic diversity of Brassica crops. It is providing improved forms of current oilseed a n d vegetable crops and m ay also permit new agricultural u s e s for these materials. In addition, rapeseed is a good test syst em for basic studies in plant genetics. This c h a p t e r reviews the t r a n s f o r m a t i o n techniques for Brassica materials, the types of genes already transferred, issues a b o u t the pl ant s recovered, a n d prospects for the future. The c u r r e n t s t a t u s of the first commercially i m p o r t a n t transgenic rapeseed lines is also presented.
Brassica species transformed Transgenic pl ant s have been recovered from all six species in the triangle of U (Tables 9.1 and 9.2), b u t far more work h a s been done with B. napus t h a n with any of the others. The spring canola line "Westar" is especially widely used. B. oleracea t r a n s f o r m a n t s include all the major vegetables (broccoli, cauliflower, cabbage, kale, b r u s s e l s sprouts), as well as rapid cycling lines. In B. rapa (syn. B. campestris), both oilseed and Chinese cabbage (subsp. pekinensis) forms have been transformed. A few reports dealing with B. nigra, B. juncea, and B. carinata have also appeared. There is little published work on other Brassica species or related crucifers, except for Sinapis alba. Considerable work on regeneration a n d gene transfer s y s t e m s for crucifers (often involving the model crucifer Arabidopsis thaliana) is in progress t h r o u g h o u t the world. The list of transformed m e m b e r s of the tribe Brassiceae is therefore likely to increase in the near future, a l t h o u g h successful plant regeneration does not necessarily lead to high efficiency transformation.
288
Gene transfer methods Delivery of DNA into Brassica materials has been accomplished by many different methods (Tables 9.1 and 9.2; Thomzik, 1993; Earle et al., 1996; Poulsen, 1996; P u d d e p h a t et al., 1996). At present the most common approach uses the crown gall bacterium, Agrobacterium tumefaciens, as a biological vector for transfer of DNA into multicellular explants. The wild type bacterium produces plant tumors by inserting DNA containing genes for synthesis of auxin and cytokinin (T-DNA from the bacterial Ti plasmid) into plant chromosomes. Removal of the hormone genes ("disarming of the TDNA"), together with introduction of reporter genes, selectable markers, and genes of interest, has converted A. tumefaciens into an excellent general system for gene transfer. Explants for which an efficient regeneration system has been developed are co-cultivated with the bacteria for a brief period (usually several days) and then cultured u n d e r conditions that kill the bacteria and favor the growth and regeneration of transformed cells. In work with Brassica the explants are usually pieces of young seedlings grown in vitro, especially hypocotyls and cotyledonary petioles. Other targets of Brassica transformation include portions of larger plants, such as slices or thin cell layers from flowering stalks, and isolated protoplasts (Table 9.1).
Agrobacterium rhizogenes is an alternate biological vector for DNA transfer, especially for B. oleracea (Tables 9.1 and 9.2). Explants incubated with wild-type A. rhizogenes can integrate and express T-DNA from its Ri (rootinducing) plasmid, leading to formation of hairy roots. Such roots, which show rapid, highly branched, and plagiotropic growth, are of interest for root-related p h e n o m e n a such as formation of secondary metabolites and other types of metabolic studies (Downs et al., 1994ab). Hairy roots can also serve as a starting point for recovery of transformed plants, via spontaneous or hormone-induced plant regeneration from transformed hairy root cultures (Christey, 1997). The resulting plants very often have an abnormal phenotype that is due to integration of the rol loci from the TL-DNA (e.g., reduced apical dominance, shortened internodes, wrinkled leaves, plagiotropic roots, late flowering and reduced fertility; Tepfer, 1989). The extent of these symptoms can vary considerably from plant to plant, and some morphologically normal plants have been recovered. Genes of interest have also been transferred to vegetable, oilseed and forage brassicas via separate binary plasmids introduced into A. rhizogenes. Plants regenerated from transgenic hairy roots show a relatively wide range of Ri phenotypes due to differences in expression of the rol loci. In addition, some normal plants carrying the gene of interest can later be obtained in progeny since the two T-DNA regions may be integrated into different chromosomes and segregate at meiosis (Boulter et al., 1990).
289 Table 9.1 Methods and target tissues used in transformation of B. n a p u s *
METHOD
TARGET TISSUES/CELLS
REFERENCE
Biolol~ical Vectors
Agrobacterium tumefaciens
Seedling explants - cotyledonary petioles - hypocotyls Stem explants Thin cell layers Microspores or microsporederived embryoids Hypocotyl protoplasts
A. rhizogenes
Internodes Cotyledon nodes Hypocotyls Seedling leaves; cotyledons
Moloney et al. 1989 Radke et al. 1988 De Block et al. 1989 Fry et aL 1987 Pua et al. 1987 Charest et al. 1988 Pechan 1989 Swanson and Erickson 1989 Thomzik and Hain 1990 Guerche et al. 1987b Boulter et at 1990 Damgaard and Rasmussen 1991 Christey and Sinclair 1992
Direct DNA Transfer
Electroporation PEG-mediated uptake Microinjection Bombardment + desiccation /DNA imbibition
Leaf protoplasts Leaf protoplasts Microspore-derived embryoids Hypocotyls of microspore embryoids
Guerche et at 1987a Golz et at 1990 Neuhaus et al. 1987 Chen and Beversdorf 1994
* For widely used methods (e.g., A. tumefaciens and seedling explants), only initial eferences focused on methodology are cited. Isolated DNA h a s sometimes been transferred directly into B r a s s i c a materials, thereby avoiding the need to deal with A g r o b a c t e r i a (Tables 9.1 a n d 9.2). Uptake of DNA by protoplasts can be induced by t r e a t m e n t with polyethylene glycol (PEG) or electroporation. This a p p r o a c h should produce nonchimeral transgenic plants, b u t regeneration from protoplasts is more difficult and genotype-specific t h a n regeneration from multicellular explants.
290 Particle b o m b a r d m e n t (biolistics, gene gun), an alternate method for direct DNA delivery, is particularly convenient for comparison of promoter strength or specificity. When different promoter sequences are linked to the bacterial B-glucuronidase ( g u s ) reporter gene and bombarded into cell cultures or plant tissues, a blue color representing expression of the gene can be scored in within a few days. To date, b o m b a r d m e n t has only rarely been employed for recovery of transformed B r a s s i c a plants. The combined use of bombardment and desiccation/DNA imbibition was more effective than either method alone for recovery of transgenic B. n a p u s t r a n s f o r m a n t s (Chen and Beversdorf, 1994). Microinjection is a another possible route for direct transfer of DNA. Transgenic rapeseed plants were obtained after microinjection into 12-cell microspore-derived embryoids (Neuhaus et al., 1987), but not when uninucleate microspores were the targets (Jones-Villeneuve et al., 1995). Improved transformation efficiency is an important goal because large n u m b e r s of t r a n s f o r m a n t s m u s t usually be screened in order to find some with the expression, inheritance, and agronomic characters required for practical use. Efficiency is influenced by m a n y factors, including cultivar, type of explant, conditions for plant regeneration and selection, and DNA construct used. Intra- and inter-lab variation in success is often noted even using the same protocol, indicating that not all relevant variables have been identified. In highly successful experiments with A. t u m e f a c i e n s , over 50% of rapeseed cotyledonary petioles produced at least one transgenic plant (Moloney et al., 1989); however, rates of 10% or less are more typical. This is substantially lower t h a n the rates routinely achieved in tobacco transformation experiments. B r a s s i c a materials tend to be more sensitive to Agrobacterium than solanaceous crops, and browning of explants can be a problem. Kanamycin or hygromycin are the u s u a l selective agents, although some others, such as methotrexate and phosphinotricin herbicides, have also been tested (Mukhopadhyay et al., 1991). Because B r a s s i c a is quite sensitive to kanamycin, the concentrations used for selection are low, usually about 15-25 mg/l vs. 300 mg/l for tobacco. To avoid inhibition of regeneration by kanamycin, selection is often delayed until several days to weeks after co-cultivation. Addition of silver nitrate to the nutrient medium for control of ethylene levels has greatly e n h a n c e d recovery of transformants in some studies (DeBlock et al., 1989; M u k h o p a d h y a y et al., 1992 ), but not in others (Radke et al., 1992; Metz et al., 1995a).
T y p e s o f g e n e s transferred Many studies with species other t h a n B. n a p u s have been limited to establishing and optimizing the transformation system, generally using the g u s reporter gene or selectable marker genes, although a few genes of interest have been tested (Table 9.2). In contrast, a variety of genes affecting
291 T a b l e 9.2 T r a n s f o r m a t i o n o f B r a s s i c a species (other than B. n a p u s ) *
SPECIES
VARIETY
METHOD
GENE (other than markers or Agrobacterium genes)
REFERENCE
A. t.
S-locus genes
Toriyama et al.
or subsp. B. oleracea
italica
1991; Conner et aL 1997 A.t.
Bt [crylA(c)]
Metz et al. 1995 a,b Hosoki et al. 1991
A.r
A.r botrytis
anti-sense ACC
Henzi et al. 1998
A.r.
Christey et al. 1997
A. r. (onc.)
David and Temp6
A. t. (onc.)
Srivastava et al.
1988 1988 A.t.
bar
DeBlock et al. 1989
Direct, to PP
bar
Mukhopadhay et al. 1991
CaMV genes
Passel6gue and
A.r.
Anti-sense EFE
Christey et al. 1997
A.t
Bt [crylA(r
Bai et al. 1993;
A.t. (onc. +
Kerlan 1996
disarmed) capitata
Metz et al. 1995b Rapid-cycling Rapid-cy-
A. r~
Christey et al. 1997
A.t. (onc. +
Berthomieu et al. 1994
disarmed)
Berthomieu and
A.r
Jouanin 1992
cling acephala
Sato et al. 1991
A.t.
A.r.
ALS
Christey and Sinclair 1992
A. t. andA. r
Hosoki et al. 1989, 1994
292
gemmifera
Hamada et al. 1989"
A. r.
Christey et al. 1997 Rapid cy-
A.t.
Millam et al. 1994
A.t.
Mukhopadhyay
cling B. rapa
oleifera
et al. 1992;
Radke et al. 1992 pekinensis
A.t.
TMV coat protein
Jun et al. 1995 He et al. 1995;
A.r.
Christey et al. 1997 rapifera
A.r.
ALS
Christey and
A.t.
Bt (crylA)
Li et al. 1995
Sinclair 1992 B. napobrassica
B. nigra
A.t. or direct to PP
Gupta et al. 1993
B. juncea
A.t.
Matthews et al. 1990 Barfield and Pua
A.t.
1991 Pental et al. 1993
h,t.
A.t. B. carinata
Anti-sense ACC
Pua and Lee, 1995 Narasimhulu et al.
A.t.
1992 A.t.
Hirudin+oleosin
Chaudhary et al. 1998
Sinapis alba
A.r.
Yadav et al. 1996
A.t.
Hadfi and Barschauer 1994
* This table lists only work in which transformed plants were recovered. A.t.: Agrobacterium tumefaciens, disarmed except when noted as oncogenic (onc.); A.r.: A. rhizogenes;
PP: protoplasts; S-locus genes: Brassica genes related to self-incompatibility;
Bt: 6 -endotoxin gene from Bacillus thuringiensis: ACC: ACC oxidase; bar: confers resistance to glufosinate (phosphinotricin) herbicides; EFE: ethylene forming enzyme; ALS: confers resist-ance to sulfonylurea herbicides.
293 agriculturally important traits have been transferred into B. n a p u s (Table 9.3). These include ones designed to improve performance of the crop in the field (resistance to herbicides, viruses, fungi, insects, heavy metal), modify the composition of oils or proteins in seeds, alter the levels of secondary metabolites, or manipulate male or female fertility. It is now possible to create "designed oils" containing fatty acids (FA) of chain lengths not normally seen in rapeseed, novel ratios of the u s u a l FA, and alterations in the degree of saturation and loading of FA onto specific positions of triacylglycerides (Kinney, 1996). Many of these changes have been achieved by transfer of genes for relevant enzymes isolated from other plants. Amino acid levels in seeds have been altered in several different ways: 1) via introduction of genes for proteins produced in other plants such as Brazil n u t (e.g., Altenbach et al., 1992); 2) via antisense versions of native genes for storage proteins (e.g. Kohno-Murase, 1994, 1995); 3) via bacterial genes encoding enzymes with altered feedback inhibition (e.g., Falco et al., 1995). A few products of medical or industrial interest have also been produced in seeds of B. n a p u s (Vanderkerckhove et at., 1989; Poirier et al., 1995). Recovery of valuable recombinant proteins may be facilitated by targeting them to oil bodies in seeds. The feasibility of this approach has been demonstrated using the g u s gene fused to an A r a b i d o p s i s oleosin gene (Van Rooijen and Moloney, 1995) as well as with the medicinal compound hirudin (Parmenter et al., 1996) and the industrial enzyme xylanase (Liu et at., 1997). The level of gene expression achieved in the studies listed in Tables 9.2 and 9.3 varies substantially. In some cases, c o - s u p p r e s s i o n / g e n e silencing p h e n o m e n a make it difficult to achieve the phenotype sought by transfer of plant genes. An example is the difficulty encountered in modifying self-compatibility by introduction of a B r a s s i c a S-locus gene (Conner et al., 1997). Nevertheless some transgenic B r a s s i c a plants recovered are already in commercial use (see below).
Field t e s t s of t r a n s g e n i c plants Insertion, expression, and inheritance of a transgene are prerequisites for a genetically engineered crop, but m a n y additional steps are necessary as well, including selection of lines with good agronomic or horticultural characters. This requires evaluation u n d e r field conditions in a variety of environments. By December 1995, 665 field tests of transgenic canola had been conducted worldwide, primarily in Canada, the United States and European Union countries but also in Australia, J a p a n , China, Argentina, Mexico, Hungary, and South Africa (James and Krattiger, 1996). The traits most frequently evaluated were herbicide tolerance, quality characters, and male sterility. In 1998, 2.4 million hectares of transgenic canola were grown world-wide, twice the area grown in 1997 (James, 1998). A few field trials of B. oleracea vegetables have also been conducted.
N
\o
P
Table 9.3 Genes (other than markers) transferred to B. nupus plants TRAIT AFFECTED
GENE
SOURCE
REFERENCE
mutant acetolactate synthase (target of herbicide)
Arabidopsis thaliana
AroA gene for altered EPSPS; glshosate degradation gene (COX) bar gene (for enzyme that inactivates herbicide)
Agrobacterium sp. strain CP4; Achromobacter sp. strain LBAA
Miki et al. 1990; Christ. and Sincl. 1992 Blackshaw er al. 1994 Bany et al. 1992
Streptomyces
DeBlock et al. 1989
chitinase
bean
chitinase
tomato + tobacco
Broglie et al. I991 Benhamou et al. 1993 Grison et ul. 1996
Stilbene synthase
Arachis hypogaea
Thomzik 1993
sense or antisense non-coding region
TYMV
Zaccomer et al. 1993
Resistances Tolerance to herbicides Sulfonylureas
Glyphosate
Glufosinate (phosphinotricin)
Tolerance to fungal Dathogens Rhizoctonia solani Cylindrosporium concentricum Phoma lingam, Sclerotinia sclerotiorum ? Control via synthesis of phytoalexin (resveratrol)
Tolerance to viral Dathogens Turnip yellow mosaic virus
Tolerance to heaw metal (cadmium)
Metallothionein
human
Misra and Gedamu 1989
C8:0, C1O:O fatty acids
acyl-ACP thioesterase
Cuphea hookeriana
Dehesh et al. 1995
High laurate (12:O)
I2:O-ACP thioesterase
Umbellularia californica (Califor-
Voelker et al. 1992, 1996
Storage Products Seed oils
nia bay) C12:O in sn-2 position of triacylglycerols
lysophosphatidic acid acyltransferase
COCOS nucifera (coconut)
Knutzon et al. 1995
High palmitate (16:O)
palmitoyl-ACP thioesterase
Cuphea hookeriana
Jones el al. 1995
Increased stearate (1 8:0)
anti-sense stearoyl-ACP desaturase
B. rapa
Knutzon et al. 1992
Increased oleate (1 8: 1)
ad-desaturase (for cosuppression of native gene)
B. napus
Reiter et al. 1994
Altered C 18 ratios
3-ketoacyl-ACP synthase
E. coli
Venvoert et al. 1995
Increase in fatty acids with > I 8C (20:1,22:1,24:1)
beta-ketocyl-CoA synthase
Simmmonhia chinensis (jojoba)
Lassner et al. 1996
Trierucin (22: 1) triacylglycerols
lysophosphatidic acid acyltransferase
Limnanthes alba (meadowfoam)
Lassner et al. 1995
I -acyl-sn-glycerol-3-phosphate acyltransferase
L. douglasii
Brough et al. 1996
ketoacyl-CoA synthase
Lunaria
Lassner, pers. c o r n .
napin
B. napus
Radke el al. 1988
increased cruciferin
anti-sense napin
B. napus
Kohno-Murase el al. 1994
increased napin
anti-sense cruciferin
B. napus
Kohno-Murase et al. 1995
increased methionine
2 s albumin
Bertholletia excelsa (Brazil nut)
increased lysine
dapA (for lysine-insensitive dihydroxy-picolinic acid synthase)
Corynebacterium
Guerche et al. 1990 Altenbach et al. 1992 Denis et al. 1995 Falco el al. 1995
tryptophan decarboxylase
Catharanthus roseus
Chavedej et al. 1994
Induction of male sterility
barnase
Bacillus amyloliquefaciens
Mariani et al. 1990 Denis et al. 1993
Restoration of male fertility
barstar
B. amyloliquefaciens
Mariani et al. 1992
No effect on male fertility
antisense tapetum-specific A9 gene
B. napus
Turgut el al. 1994
24:1 fatty acid
Seed Proteins
Secondary Metabolites Decreased indole glucosinolates
Reproductive Characters
Self-incompatibility
S-locus genes
B. oleracea, B. campestris
Nishio et al. 1992
Receotivitv to pollination
toxin, A chain
Coynebacterium diphtheriae
Kandasamy et al. 1993
Medical
enkephalin (neuropeptide)
Plastics
phaB, phaC
coding sequence added to Arabidop- Vanderkerckhove et al. 1989 sis 2s albumin Afcafigeneseufrophus cited in Poirier ef al. 1995
Taraetina proteins to oil bodies
oleosin
Zea mays;
Lee et 01. 1991;
hirudin + oleosin
Arabidopsis
Van Rooijen and Moloney
xylanase + oleosin
Hirudo medicinalis
Parmenter et al. 1996
Neocallimastix patriciarum
Liu et al. 1997
oleosin gene
Arabidopsis
Plant el al. 1994
low temperature responsive gene
B. napus
White et al. 1994
S-locus glycoprotein gene
Brassica
Sat0 ef al. 1991
Medical or industrial products
Regulation of Promoters
1995
- Source ofpromoter -
h)
W J.
298 M o v e m e n t of t r a n s g e n i c Brassica m a t e r i a l s from the laboratory a n d g r e e n h o u s e into the field h a s been a c c o m p a n i e d by c o n c e r n s a b o u t i m p a c t s on h e a l t h a n d the e n v i r o n m e n t a n d the a p p r o p r i a t e levels of regulatory oversight. Most of the i s s u e s raised are similar to those raised with other t r a n s genic crops, b u t some are p a r t i c u l a r l y relevant to Brassica: 1) Are the t r a n s g e n i c p l a n t s directly d a n g e r o u s to h u m a n s , animals, or the e n v i r o n m e n t ? This is a p p r o p r i a t e l y studied on a case by case basis, d e p e n d i n g on the specific t r a n s g e n e a n d crop use. For example, e x p r e s s i o n of the Brazil n u t 2S a l b u m i n protein in s o y b e a n m a y c a u s e allergic r e a c t i o n s in people who are allergic to Brazil n u t s (Goldberg, 1994). The s a m e gene h a s been introduced into r a p e s e e d seed proteins to i n c r e a s e m e t h i o n i n e levels in animal feed (Altenbach et al., 1992; Denis et al., 1995). 2) C a n the t r a n s g e n i c p l a n t s c a u s e indirect u n d e s i r a b l e e n v i r o n m e n t a l effects? This also m u s t be a s s e s s e d with a t t e n t i o n to the t r a n s g e n e a n d the cropping s y s t e m used. Availability of herbicide t o l e r a n t t r a n s g e n i c p l a n t s could lead to either i n c r e a s e d or d e c r e a s e d u s e of herbicides or to shifts toward more e n v i r o n m e n t a l l y benign chemicals. W i d e s p r e a d d e p l o y m e n t of t r a n s g e n i c crops c a r r y i n g Bacillus thuringiensis endotoxin genes for insect control h a s the potential to accelerate d e v e l o p m e n t of r e s i s t a n t insects, t h e r e b y m a k i n g these crops ineffective a n d also h a m p e r i n g the u s e of Bt s p r a y s (McGaughey a n d Whalon, 1992). This issue is of i m m e d i a t e concern for crops like cotton a n d corn for which large a c r e a g e s of Bt-transgenic p l a n t s are a l r e a d y being grown in the U.S., yet little relevant experimental information is available. B t - t r a n s g e n i c broccoli p l a n t s are being u s e d in g r e e n h o u s e a n d field e x p e r i m e n t s to test r e s i s t a n t m a n a g e m e n t strategies involving refuges a n d seed m i x t u r e s (Metz et al., 1995b). The insect u s e d is the d i a m o n d b a c k m o t h (PtuteUa xylosteUa), a crucifer p e s t from which resist a n t s t r a i n s have a l r e a d y been recovered following e x p o s u r e to Bt sprays. 3) Are the t r a n s g e n i c p l a n t s t h e m s e l v e s likely to become weeds? This h a s been e x a m i n e d in a variety of s t u d i e s c o m p a r i n g s u c h plants either for c o m p e t i t i v e n e s s t h r o u g h o u t the whole life cycle or at specific stages, s u c h as seedling e m e r g e n c e (e.g., F r e d s h a v n et al., 1995). British trials have s h o w n t h a t t r a n s g e n i c r a p e s e e d carrying tolerance to k a n a m y c i n or the herbicide BASTA is not more invasive or p e r s i s t e n t t h a n controls (Crawley et al., 1993). Generally similar r e s u l t s have been o b t a i n e d in o t h e r studies on d o r m a n c y , survival, emergence, or growth of seedlings from transgenic plants, including ones modified to have high l a u r a t e or high s t e a r a t e levels in the seeds (Linder a n d Schmitt, 1995). Results of s u c h oilseed mo-
299 difications are of p a r t i c u l a r interest since they seem more likely to alter seedling p e r f o r m a n c e t h a n m a n y other types of t r a n s g e n e s . Some differences were seen in tests of the s a m e lines in different geographic areas, California a n d Georgia (Linder a n d Schmitt, 1995), indicating t h a t e v a l u a t i o n s s h o u l d be done at multiple locations e n c o m p a s s i n g the areas in which the crops are to be grown. 4) Will t r a n s g e n i c p l a n t s t r a n s f e r ~enes to wild relatives, leadin~ to new weed p r o b l e m s ? C o n c e r n s are often raised a b o u t possible m o v e m e n t of t r a n s g e n e s from the crops to related plants. This is particularly relevant to B. napus b e c a u s e of its k n o w n potential for genetic e x c h a n g e with wild or weedy relatives. M u c h of the earlier work in this area dealt with m a n u a l pollination, sometimes followed by embryo rescue, or somatic hybridization. In more recent studies, s p o n t a n e o u s hybridization between r a p e s e e d a n d wild relatives s u c h as B. rapa s u b s p , campestris (syn. B. campestris), R a p h a n u s raphanistrum or Hirschfeldia incana h a s been m o n i t o r e d in field tests, u s i n g either t r a n s g e n e m a r k e r s or o t h e r t e c h n i q u e s to recognize h y b r i d s (Baranger et al., 1993; Leckie et al., 1993; Darmency, 1994; Eber et al., 1994; J ~ r g e n s o n a n d Andersen, 1994; Scheffler et al., 1994). It is clear t h a t s p o n t a n e o u s hybridization can in any case occur, b u t the rates vary considerably d e p e n d i n g b o t h on the specific lines of r a p e s e e d or wild species a n d on the test conditions used. Although fertility of the inter-specific h y b r i d s is u s u a l l y low, seeds c a n often be recovered from them. Competitiveness a n d persistence of s u c h seeds t h e n b e c o m e s the e x p e r i m e n t a l key question. While m u c h of this work is motivated by regulatory i s s u e s a b o u t a p p r o p r i a t e u s e of t r a n s g e n i c r a p e s e e d crops, there is clearly potential for productive interaction of ecologists a n d molecular biologists to a d d r e s s more general biological a n d ecological questionso Various strategies to decrease gene flow from t r a n s g e n i c crops to wild relatives have been tested or proposed. One is u s e of b o r d e r a r e a s t h a t lack vegetation, c o n t a i n p l a n t s t h a t are n o t pollinated by insects or t h a t will be destroyed, or c o m b i n a t i o n s of the two p r o c e d u r e s (Morris et al., 1994; Scheffler et al., 1995). Genetic strategies include u s e of male sterile p l a n t s t h a t produce no pollen or i n t r o d u c t i o n of t r a n s g e n e s into c h l o r o p l a s t DNA r a t h e r t h a n into the n u c l e a r genome (McBride et al., 1995). The latter app r o a c h s h o u l d prevent pollen t r a n s m i s s i o n b e c a u s e pollen does not c o n t a i n chloroplasts. To date, chloroplast t r a n s f o r m a t i o n h a s been achieved only in tobacco (Nicotiana tabacum) b e c a u s e special c o n s t r u c t s a n d selection proc e d u r e s are required, b u t work is in p r o g r e s s on d e v e l o p m e n t of the proc e d u r e s required for both Arabidopsis a n d Brassica (P. Maliga, p e r s o n a l communication).
300
Legal i s s u e s M a n y a s p e c t s of gene t r a n s f e r technology have been p a t e n t e d , including Agrobacterium tumefac~ens s y s t e m s , m a n y of the widely u s e d promoters a n d selectable m a r k e r s , genes of interest, c o n c e p t s (e.g., a n t i s e n s e technology, u s e of coat protein or replicase a p p r o a c h e s to virus resistance), etc. Calgene holds a p a t e n t on Agrobacterium tumefaciens-mediated t r a n s f o r m a tion of Brassica (United States P a t e n t No. 5,188,958, Feb. 23, 1993). Permission to u s e p a t e n t e d or p r o p r i e t a r y m a t e r i a l s for r e s e a r c h p u r p o s e s can often be obtained; however, the licensing a n d financial a r r a n g e m e n t s required for c o m m e r c i a l u s e m a y delay or p r e v e n t the a g r i c u l t u r a l u s e of t r a n s f o r m e d p l a n t s p r o d u c e d in a c a d e m i c r e s e a r c h p r o g r a m s or small companies.
Transgenic Brassica crops now being commercialized The first c o m m e r c i a l planting of a t r a n s g e n i c Brassica crop took place in the S o u t h e r n United S t a t e s in the A u t u m n of 1994. Approximately 2.5 million p o u n d s of seed from a n e n g i n e e r e d B. napus t e r m e d "Laurate Canola" were h a r v e s t e d , c r u s h e d by the following s u m m e r , a n d the resulting oil delivered to a n u n - n a m e d c o m p a n y for i n d u s t r i a l p u r p o s e s . L a u r a t e Canola was developed by Calgene (Voelker et al., 1996) by Agrobacterium-mediated transfer of a gene c o n s t r u c t e d with a cDNA cloned from seed of the California bay tree, Umbellularia californica. This chimeric gene c o n s t r u c t conifers the trait of lauric acid a c c u m u l a t i o n in the seed oil. The oil composition trait in L a u r a t e Canola is specific to the seed storage lipid as e x p r e s s i o n of the t r a n s g e n e is driven by a p r o m o t e r element derived from a highly e x p r e s s e d n a p i n storage protein gene from B. rapa (Kridl et al., 1993). To limit t r a n s g e n e effects specifically to the seed of oilseed b r a s s i c a s , n a p i n p r o m o t e r s are often u s e d a l t h o u g h p r o m o t e r elements derived from cruciferin (Sjodahl et al., 1995), oleosin (Lee et al., 1991), a n d o t h e r embryo-specific genes have also been employed. L a u r a t e Canola is physically i n d i s t i n g u i s h a b l e from r e g u l a r canola in the field. Within a few cycles of selection u s i n g haploid b r e e d i n g t e c h n i q u e s , crop yields have r e a c h e d e s s e n t i a l parity with top open pollinated varieties in C a n a d i a n field trials. L a u r a t e C a n o l a is a notable e x a m p l e of the u s e of t r a n s g e n e s to enrich
Brassica g e r m p l a s m , since l a u r a t e molecule is n o r m a l l y p r e s e n t only in trace
a m o u n t s in crucifer seed oils. Years of screening for v a r i a n t s a n d / o r mut a n t s of oilseed b r a s s i c a s t h a t m i g h t p r o d u c e lauric acid have not been fruitful. Moreover, the pre-existing c o m m e r c i a l s o u r c e s of l a u r a t e oils, coconut a n d p a l m kernel, c o n t a i n oil triacylglycerols (TAG) with lauric acid in a n y of the three positions of the lipid molecule (as does U. californica). Laurate C a n o l a t e n d s to exclude lauric a n d other s a t u r a t e d fatty acids from the second position of the TAG molecule (Del Vecchio, 1996). T h u s its oil is chemically different from c o c o n u t or p a l m kernel oils, a n d L a u r a t e Canola oil t u r n s out to be u n i q u e l y a n d significantly better for certain high value food applications~
301 Other transgenic B. n a p u s with a modified oil composition a n d in advanced development include lines in which fatty acid d e s a t u r a s e enzymes are s u p p r e s s e d in seed via antisense (Knutzon et al., 1992) or co-suppression m e t h o d s (Reiter et al., 1994). Down-regulation of the stearoyl-ACP desaturase is an example where m u t a t i o n breeding in oilseed rape (and also A r a b i d o p s i s thaliana) failed to identify lesions in the stearoyl-ACP desaturase gene or the expected corresponding increase in levels of stearic acid. This may be because there are multiple genes for this enzyme, and knocking out any one of t h e m will have no phenotypic effect. Alternatively, lesions in a locus significantly affecting stearoyl-ACP d e s a t u r a s e activity may be lethal. However, if multiple stearoyl-ACP d e s a t u r a s e genes share e n o u g h DNA homology a n d ant i s ens e control is limited to the p h a s e of seed development during storage lipid biosynthesis, it m a y be possible to engineer higher levels of stearic acid in transgenic canola oil. This is indeed the case (Knutzon et al., 1992), a l t h o u g h it r e m a i n s to be seen if the trait will be stable over m a n y generations in multiple environments. Regarding the d e s a t u r a s e activities t h a t convert oleic acid to linoleic acid and linoleic acid to linolenic acid ("D 12" and "D 15", respectively), mutation breeding h a s succeeded in developing lines with lowered polyunsaturated fatty acids. Perhaps the m o s t interesting involves increasing oleic acid by s u p p r e s s i n g activity of the D 12 d e s a t u r a s e . High oleic lines with seed oil containing a b o u t 75% oleic acid have shown good agronomic properties; however, efforts to raise oleic levels above 80% by combining two non-allelic high oleic m u t a t i o n s have resulted in yield penalties and poor agronomic characters. This a p p e a r s to be due to D 12 d e s a t u r a s e activity deficiencies in leaf and other parts of the plant. The DuPont group (Reiter et al., 1994) has addressed this problem by transgenic s u p p r e s s i o n of D 12 activity specifically in the seed. Field trials are currently u n d e r w a y with materials having seed oils considerably above 80% oleic. The second transgenic rapeseed p r o d u c t to be commercialized was the sale of seed from a variety of oilseed rape developed by Agrevo to be resistant to the herbicide glufosinate. The t r a n s g e n e basis underlying the herbicide resistance derives from observations (DeBlock et al., 1989) t h a t a Strept o m y c e s gene can be engineered to express in B. n a p u s and B. oleracea an enzyme t h a t degrades glufosinate. G l y p h o s a t e - r e s i s t a n t oilseed rape developed by Mo n s a nt o d e p e n d s partly on a bacterial gene coding for an enzyme active in the degradation of glyphosate (Barry et al., 1992). These transgenic approaches were able to take advantage of procaryotic genetic elements selected in bacterial environments. Another transgenic development t h a t highlights a synergistic contribution to both B r a s s i c a g e r m p l a s m and more conventional breeding approaches is engineered nuclear male-sterility (Mariani et al., 1990, 1992). Plant Genetic S y s t e m s scientists and breeders are close to commercializing hybrid canola seed in C a n a d a made possible with a male-sterile female p a r e n t t h a t can be directly selected after pollination. Selection against the male p a r e n t
302 during hybrid seed production is accomplished with the herbicide glufosinate; the above m e n t i o n e d herbicide resistance t r a n s g e n e is directly linked to the t r a n s g e n e encoding the nuclear male-sterility trait. The female parent of the hybrid cross is male-sterile due to a transgenic RNAse (again from a bacterial source) engineered to express specifically in the t a p e m m tissue. When expressed, the RNAse kills the hos t cell and t h u s blocks normal pollen development. Fertility can be restored with a transgene encoding an effective inhibitor of the RNAse enzyme; contribution of this gene from the male p a r en t allows efficient seed set by plants grown from F1 hybrid seed. Whereas both nuclear a n d cytoplasmic male-sterile g e r m p l a s m is available in m a n y Brassica species, the transgene system promises to be more efficient with no likely yield penalty as observed in some other systems. In addition, the transgene system m a y be easily transferred to plant species within and outside the crucifers, wherever transformation syst em s are available. Other examples of commercially interesting t r a n s g e n e s in the crucifers (Table 9.3) include an altered acetolactate s y n t h a s e enzyme conferring resistance to sulfonyl u r e a herbicides; Bacillus thurigiensis toxin genes conferring insect feeding resistance; a short chain acyl-ACP thioesterase from Cuphea hookeriana t h a t allows synthesis of C8:0 and C 10:0 fatty acids; a palmitoylACP thioesterase from C. hookeriana t h a t allows synt hesi s of high levels of palmitic acid; a lysophosphatidic acid acyltransferase cDNA from meadowfoam t h a t allows s y n t h e s i s of trierucin triacylglycerols; a ketoacyl-CoA synthase cDNA from Lunaria t hat allows B. napus to m a k e a C24"1 fatty acid; and a castor bean hydroxylase cDNA t h a t allows synt hesi s of ricinoleic acid by Arabidopsis. These examples and still others to come underscore the potentially wide impact of us i ng transgenes in Brassica a n d related genera.
Future p r o s p e c t s Work in progress with the model crucifer Arabidopsis thaliana has major implications for genetic engineering of Brassica crops. Many additional genes for tests in Brassica will become available t h r o u g h complete sequencing of the Arabidopsis genome, analysis of m u t a n t s , and gene isolation efforts. Genes t h a t e n h a n c e resistance to crucifer diseases will be of particular interest. The impact of genetic engineering is likely to be greatest with the oilseed b r a s s ic as because of the large acreages grown a n d their high commercial value. Resistance to herbicides, pathogens, and pests can enhance performance in the field a n d reduce costs to growers. Alteration of oil or protein quality permits production in rapeseed of improved p r o d u c t s or ones previously obtained in other ways. This may, of course, result in loss of revenue for the former sources of such pr oduct s (e.g., producers of tropical oils). Production of n o n - p l a n t products within seeds of B. napus ("molecular farming") could expand the us e of the crop still further.
303 The impact of transgenic Brassica vegetables is harder to predict. Because the vegetables are so diverse, less support is available for work on any given one. Obtaining the data required for regulatory and legal approval is onerous and expensive, especially for minor crops. On the other hand, transgenic resistance to herbicides, diseases or insects could have important environmental benefits for vegetable production by allowing use of modern herbicides and reduction in application of insecticides. Such changes could be especially beneficial in developing countries where applications of chemicals are often high and poorly regulated. Opportunities for use of other genes already identified in other systems include control of pollination, alteration of growth habits, or extension of shelf life (provided cost comparisons to other approaches are favorable). Current attention to Brassica vegetables as anticarcinogens m a k e s manipulation of nutrient and flavor components another attractive target for genetic manipulation. Quite apart from commercial applications, current studies of crucifer genomes combined with the ability to produce genetically altered plants provide m a n y opportunities to gain basic information about developmental biology, physiology, biochemistry, molecular biology, population genetics, and other aspects of Brassica species.
Acknowledgment The a u t h o r s t h a n k Dr. M.C. Christey, New Zealand Institute for Crop and Food Research, Ltd., for helpful comments about Agrobacterium rhizogenes-mediated transformation and for access to papers in press.
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311 Nishio, T., Toriyama, K., Sato, T., Kandasamy, M. K., Paolillo, D. J., Nasrallah, J. B. and Nasrallah, M. E. 1992. Expression of S-locus glycoprotein genes from Brassica oleracea a n d B. campestris in transgenic plants of self-compatible B. napus cv Westar. Sex. Plant Reprod. 5, 101-109. Parmenter, D. L., Boothe, J. G., Rooijen, G.J.H. van, Yeung, E. C. a n d Moloney, M. M. 1996. Production of biologically active hirudin in plant seeds using oleosin partitioning. Plant Molec. Biol. 29, 1167-1180. Passel6gue, E. a n d Kerlan, C. 1996. Transformation of cauliflower (Brassica oleracea var. botrytis) by transfer of cauliflower mosaic virus genes t h r o u g h combined cocultivation with virulent and avirulent strains of Agrobacterium. Plant Science 113, 79-89. Pechan, P. M. 1989. Successful cocultivation of Brassica napus microspores a n d proembryos with Agrobacterium. Plant Cell Rep. 8, 387-390. Pental, D., Pradhan, A. K., Sodhi, Y. S. a n d Mukhopadhyay, A. 1993. Variation a m o n g s t Brassica juncea cultivars for regeneration from hypocotyl explants and optimization of conditions for Agrobacterium-mediated genetic transformation. Plant Cell Rep. 12, 462-467. Plant, A. L., Van Rooijen, G.J.H., Anderson, C. P. a n d Moloney, M.M. 1994. Regulation of an Arabidopsis oleosin gene promoter in transgenic Brassica napus. Plant Molec. Biol. 25, 193-205. Poirier, Y., Nawrath, C. a n d Somerville, C. 1995. Production of polyhydroxyalkanoates, a family of biodegradable plastics a n d elastomers, in bacteria a n d plants. Bio/Technology 13, 142-150. Poulsen, G. B. 1996. Genetic transformation of Brassica. Plant Breeding 115, 209-225. Pua, E. C. a n d Lee, J. E. E. 1995. E n h a n c e d de novo shoot morphogenesis in vitro by expression of a n t i s e n s e 1-aminocyclopropane-l-carboxylate oxidase gene in transgenic m u s t a r d plants. Planta 196, 69-76. Pua, E. C., Mehra Palta, A., Nagy, F. a n d Chua, N. H. 1987. Transgenic plants of Brassica napus L. Bio/ Technology 5, 815-817. Puddephat, I. J., Riggs, T. J. a n d Fenning, T. M. 1996. Transformation of Brassica oleracea L.: a critical review. Molecular Breeding 2, 185210. Radke, S. E., Andrews, B. M., Moloney, M. M., Crouch, M. L., Kridl, J. C. and Knauf, V. C. 1988. Transformation of Brassica napus u s i n g Agrobacterium tumefaciens: developmentally regulated expression of reintroduced napin gene. Theor. Appl. Genet. 75, 685-694. Radke, S. E., Turner, J. C. a n d Facciotti, D. 1992. Transformation a n d regeneration of Brassica rapa u s i n g Agrobacterium tumefaciens. Plant Cell Rep. 1 1 , 4 9 9 - 5 0 5 .
312 Reiter, R. S., Mauvais, C. J., Ripp, K. G., Yadav, N. S., Kinney, A. J., Chen, Z., Debonte, L. R. and Hitz, W. D. 1994. Transgenic modification of seed lipid desaturation in Brassica napus. 4th International Congress of Plant Molecular Biology. Amsterdam, Abstract 1469. Sato, T., Thorsness, M. K., Kandasamy, M. K., Nishio, T., Hirai, M., Nasrallah, J. B. and Nasrallah, M. E. 1991. Activity of an S locus gene promoter in pistils and anthers of transgenic Brassica. The Plant Cell 3, 867-876. Scheffler, J. A., Parkinson, R. and Dale, P. J. 1995. Evaluating the effectiveness of isolation distances for field plots of oilseed rape (Brassica napus) using a herbicide-resistance transgene as a selectable marker. Z. Pflanzenzucht. 114, 317-321. Sjodahl, S., Gustavsson, H. O., Rodin, J. and Rask, L. 1995. Deletion analysis of the Brassica napus cruciferin gene crul promoter in transformed tobacco. Planta 197, 264-271. Srivastava,V., Reddy, A. S. and Guha-Mukherjee, S. 1988. Transformation and regeneration of Brassica oleracea mediated by an oncogenic Agrobacterium tumefaciens. Plant Cell Rep. 7, 504-507 Stewart, C. N. Jr., Adang, M. J., All, J. N., Raymer, P. L., Ramachandran, S. and Parrott, W. A. 1996. Insect control and dosage effects in transgenic canola containing a synthetic Bacillus thuringiensis crylAc gene. Plant Physiol. 112, 115-120. Swanson, E. B. and Erickson, L. R. 1989. Haploid transformation in Brassica napus using an octopine-producing strain of Agrobacterium turnefaciens. Theor. Appl. Genet. 78, 831-85. Tepfer, D. 1989. Ri T-DNA from Agrobacterium rhizogenes: a source of genes having applications in rhizospere biology and plant development, ecology and evolution. In: Kosuge, T. and Nester, E. W. (eds.), Plant-Microbe Interactions. Molecular and Genetic Perspectives, Vol. 3, McGraw-Hill, Inc. pp. 294-342. Thomzik, J. E. 1993. Transformation in oilseed rape (Brassica napus L.). In: Bajaj, Y. P. S. (ed.), Biotechnology in Forestry and Agriculture, Vol. 23, Plant Protoplasts and Genetic Engineering I V , Springer-Verlag, pp. 170-182. Thomzik, J. E. and Hain, R. 1990. Transgenic Brassica napus plants obtained by cocultivation of protoplasts with Agrobacterium tumefaciens. Plant Cell Rep. 9, 233-236 Toriyama, K., Stein, J. C., Nasrallah, M. E. and Nasrallah, J. B. 1991. Transformation of Brassica oleracea with an S-locus gene from B. campestris changes the self-incompatibility phenotype. Theor. Appl. Genet. 8 1 , 7 6 9 - 7 7 6 .
313 Turgut, K., Barsby, T., Craze, M., Freeman, J., Hodge, R., Paul, W. and Scott, R. 1994. The highly expressed tapetum-specific A9 gene is not re-quired for male fertility in Brassica napus. Plant Molec. Biol. 24, 97-104. Vanderkerckhove, J, Damme, J. Van, Lijsebettens, M. Van, Bottermanm J., Block, M. De, Vandewiele, M, Clercq, A. de, Leemans, J., Montagu, M. Van and Krebbers, E. 1989. Enkephalins produced in transgenic plants using modified 2S seed storage proteins. Bio/Technology 7, 929-932. Van Rooijen, G. J. H.,Van and Moloney, M. M. 1995. Plant seed oil-bodies as carriers for foreign proteins. Bio/Technology 13, 72-77. Verwoert, I.I.G.S., Linden, K. H. Van Der., Walsh, M. C., Nijkamp, H.J.J. and Stuitje A. R. 1995. Modification of Brassica napus seed oil by expression of Escherichia coli fabH gene, encoding 3-ketoacyl-acyl carrier protein synthase III. Plant Molec. Biol. 27, 875-886. Voelker, T. A., Worrell, A. C. anderson, L., Bleibaum, J., Fan, C., Hawkins, D. J., Radke, S. E. and Davies, H. M. 1992. Fatty acid biosynthesis redirected to medium chains in transgenic oilseed plants. Science 257, 72-74. Voelker, T. A., Hayes, T. R., Cranmer, A. C., Turner, J. C. and Davies, H. M. 1996. Genetic engineering of a quantitative trait: metabolic and genetic parameters the accumulation of laurate in rapeseed. The Plant Journal 9, 229-241. White, T.C, Simmonds, D., Donaldson, P. and Singh J. 1994. Regulation of B N l l 5 , a low-temperature-responsive gene from winter Brassica napus. Plant Physiol. 106, 917-928. Yadav, R. C., Konishi, H., Kamada, H. and Kikuchi, F. 1996. Transformation of Brassica carinata A. Braun using Agrobacterium rhizogenes. Cruciferae Newsl. 18, 44-45. Zaccomer, B., Cellier, F., Boyer, J. C., Haenni, A. L. and Tepfer, M. 1993. Transgenic plants that express genes including the 3' untranslated region of the turnip yellow mosaic virus (TYMV) genome are partially protected against TYMV infection. Gene 136, 87-94.
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Biology of Brassica Coenospecies C. G6mez-Campo (Editor) 91999 Elsevier Science B.V. All rights reserved.
315
10 CHEMICAL COMPOSITION E d u a r d o A. S. Rosa Horticulture Section. Universidade de Tras os Montes e Alto Douro. 5 0 0 1 - 9 0 9 Vila Real, Portugal.
Several species of Brassica a n d ally g e n e r a w h i c h are given a wide range of uses. A p a r t from direct h u m a n a n d a n i m a l c o n s u m p t i o n i n d u s t r i a l u s e s include the m a n u f a c t u r e of r a p e s e e d oil a n d the p r e p a r a t i o n of m u s t a r d products. R a p e s e e d oil r a n k s third b e h i n d s o y b e a n a n d oil palm, showing the i m p o r t a n c e of this product. After extraction of the oil from r a p e s e e d (Brassica n a p u s a n d B. rapa), the r e s i d u a l m e a l w h i c h is high in protein, is u s e d in a n i m a l feeds. Those w h i c h are u s e d for h u m a n c o n s u m p t i o n are often subjected to p r o c e s s e s s u c h as: blanching, freezing, freeze-drying, boiling or fermentation. D e p e n d i n g on the species c o n c e r n e d , a l m o s t a n y p a r t of the p l a n t p a r t including the roots, s t e m s a n d petioles, leaves, inflorescences a n d b u d flowers c a n be u s e d (Table 10.1). The p e r capita c o n s u m p t i o n of vegetables r a n k s in third place worldwide after cereals a n d o t h e r n o n - p l a n t sources (Table 10.2). Within the vegetables, Brassica crops r a n k third a m o n g the major vegetable p r o d u c i n g botanical g r o u p s in developed c o u n t r i e s after potatoes a n d t o m a t o e s , r a n k i n g also in third place in c o n s u m p t i o n (FAO, 1992). P r o d u c t i o n a n d c o n s u m p t i o n in developing c o u n t r i e s could be h i g h e r b u t take s e c o n d place to high calorie grain crops. In m o s t Brassica species, after the h a r v e s t of the m a i n p l a n t part, the r e m a i n i n g b i o m a s s m a y be u s e d for fodder. For i n s t a n c e , in cauliflower, broccoli a n d B r u s s e l s s p r o u t s , the p a r t s which are u s e d for h u m a n c o n s u m p t i o n r e p r e s e n t only a small portion of the total b i o m a s s production. T h u s , a c h e m i c a l c h a r a c t e r i z a t i o n of t h e s e "residual" p a r t s is i m p o r t a n t in order to evaluate their e v e n t u a l u s e as fodder. C o m p r e h e n s i v e information on the c h e m i c a l c o m p o s i t i o n of Brassica crops is also r e q u i r e d w h e n o t h e r u s e s are envisaged s u c h as medicinal applications a n d in the p r o d u c t i o n of a r o m a t i c s , c o s m e t i c s a n d additives a n d more recently for the control of soil-borne d i s e a s e s a n d n e m a t o d e s . With the d e v e l o p m e n t of analytical c h e m i s t r y , new m e t h o d s have been i n t r o d u c e d t h a t p e r m i t a more c o m p r e h e n s i v e knowledge of the chemical composition of the plants. In the past, food c o m p o s i t i o n t a b l e s were consi-
316 T a b l e 10. 1 Brassica species and their potential uses.
Species
Human consumption
Fodder
White cabbage (B. oleracea var. capitata)
Heads
Leaves and petioles
Savoy cabbage (B. oleracea var. bulata)
Heads
Leaves and petioles
Caulifower
Inflorescences
Leaves and petioles
Broccoli (B. oleracea var. italica)
Inflorescences
Leaves and petioles
Leaves
Leaves and petioles
Vegetative buds
Leaves and petioles
Roots
Roots, leaves and petioles
Loose heads
Leaves and petioles
Roots
Roots, leaves and petioles
(B. oleracea var. botrytis)
Kale, collards (B. oleracea var. acephala) Brussels sprouts (B. oleracea var. gemmifera)
Turnips (B. napus) Portuguese cabbage (B. oleracea var. tronchuda) Swede (B. napus var rapifera)
dered to s u p p l y e n o u g h information a b o u t the quality of a p a r t i c u l a r vegetable. Today, however, information on s e c o n d a r y p l a n t metabolites is often required. I n t e r e s t e x t e n d s beyond basic information a b o u t the levels of protein, fat, c a r b o h y d r a t e s , v i t a m i n s a n d minerals, a n d the s e c o n d a r y plant metabolites are a group of s u b s t a n c e s t h a t can explain some of the effects of vegetables in h u m a n s a n d a n i m a l s which in m a n y c a s e s h a d been already d e s c r i b e d by the a n c i e n t R o m a n a n d Greek writers b a s e d on empirical knowledge t h r o u g h the c e n t u r i e s . For i n s t a n c e , Plinius s t a t e d t h a t "Cruciferae are s u p e r i o r to o t h e r vegetables in their n a t u r a l properties". E u r o p e a n policy h a s focused on the quality of vegetables to the d e t r i m e n t of high yields a n d al-
317 T a b l e 10. 2
World food sources and average annual per capita food consumption, and its daily calorie, protein and fat contribution (Rubatzky and Yamaguchi, 1996).
Food source
Annual volume
Protein
Fat
(kg)
(cal/day)
(cal/day)
(cal/day)
Cereals
188
1371
33.4
5.7
62
141
2.0
0.3
Vegetables
69
46
2.5
0.4
Sugar
25
237
0.1
0.0
Pulses
6
58
3.6
0.4
Nuts and oils
7
50
2.4
3.6
Fruits
53
64
0.8
0.4
188
710
25.6
55.5
Starchy roots and tubers
Meats and non-plant sources
m o s t every c o u n t r y is c o n c e r n e d with diet a n d its effect on h u m a n h e a l t h with the objective of r e d u c i n g costs in h e a l t h care services. For instance, it is expected t h a t the h i g h e s t c a u s e of d e a t h in the world, h e a r t disease, which is responsible for 7.2 millions every year m i g h t be r e d u c e d with the improvem e n t of diet which s h o u l d include greater a m o u n t s of fruit a n d vegetables (50 th HWO Assembly, 1998). Nutrition is b e c o m i n g a n increasingly i m p o r t a n t factor in the choice of food p r o d u c t s and, as a c o n s e q u e n c e , a c h a n g e in eating habits, to e n s u r e a more h e a l t h y diet, h a s been n o t e d in m a n y countries. For i n s t a n c e Americans are m a k i n g food choices b a s e d on fat c o n t e n t ( J o h n s t o n , 1998). More c o m p r e h e n s i v e food labeling, indicating the major c o n s t i t u e n t s of the food together with the m o s t i m p o r t a n t beneficial effects is probably the next step. In fact, other c o n c e p t s have been recently i n t r o d u c e d which in the future migh also apply to B r a s s i c a p r o d u c t s , including "functional foods", "novel foods", "probiotics", "prebiotics", a n d "design foods". In this chapter, the chemical c o m p o s i t i o n of the m a i n B r a s s i c a crops will be a d d r e s s e d focusing on the information available from food composi-
318 tion tables. There will be special e m p h a s i s on s e c o n d a r y p l a n t metabolites, particularly the glucosinolates, a m a j o r group of c o m p o u n d s p r e s e n t in the family Brassicaceae (=Cruciferae) which have been the subject of several c o m p r e h e n s i v e reviews.
T h e i m p o r t a n c e o f t h e B r a s s i c a a n d a l l i e s in h u m a n a n d animal diet This group of p l a n t s are well a d a p t e d to a wide range of climatic conditions which p e r m i t s t h e m to grow in different latitudes t h r o u g h o u t the world. In a p a r t i c u l a r site u s i n g different species a n d primitive cultivars it is a l m o s t possible to grow Brassica p l a n t s t h r o u g h o u t the year. Good examples are kale a n d collards which might be available as fresh green vegetables the whole year a r o u n d even at higher n o r t h e r n latitudes, a situation that can be u s e d to a d v a n t a g e in the p r o d u c t i o n of fodder kales, a crop with a high u n tritive value within the Brassica group, as seen in Table 10.1. Brassica crops in general, could play a n even more i m p o r t a n t role in the nutritional quality of diets with better d i s s e m i n a t i o n of information a b o u t their nutritive value together with c h a n g e s in eating h a b i t s t h a t will benefit people, especially those of m a r g i n a l diets. However, in some countries, due to limited food availability, the t e n d e n c y is to meet caloric d e m a n d s first, regardless of other n u t r i t i o n a l n e e d s leading generally to u n d e r n o u r i s h m e n t or malnutrition. Dietary fibre (either soluble a n d insoluble), a low caloric value, the vitamin a n d m i n e r a l c o n t e n t s a n d the protein quality are some of the a r g u m e n t s to increase Brassica c o n s u m p t i o n . O t h e r nutritional factors p r e s e n t in this group of plants, which will be d i s c u s s e d later, also justify the recommendation for their increased c o n s u m p t i o n .
Brassica crops have also been grown as catch crops for forage (Sheldrick et al., 1981; Rosa a n d Heaney, 1996). By definition, a catch crop occupies the land for only p a r t of the growing season. They are utilized in the aut u m n a n d early winter as grazing either for sheep or, occasionally, for dairy cows. The dry m a t t e r c o n t e n t of b r a s s i c a s d e p e n d s on the crop species and growing season, varying between 2.0 % a n d 12.0 % a n d is considered to be highly n u t r i t i o u s (Kay 1971). Cruciferous crops, a l t h o u g h m u c h bulkier t h a n cereals, are capable of supplying high yields or energy per hectare with digestibility values of a r o u n d 0.7 a n d N c o n c e n t r a t i o n s between 20.0 to 39.0 g.kg -1 dry m a t t e r (Sheldrick et al., 1981). The a m i n o acids, lysine, methionine, cystein a n d t r y p t o p h a n , which are likely to limit growth in y o u n g animals are found in greater a m o u n t s in the green t h a n in the root crops (either Brassica napus, B. rapa or even s o m e t i m e s B. j u n c e a a n d also R a p h a n u s sativus). The crude protein c o n t e n t of root crops (10-13%) is also lower t h a n in the green crops (13-16%), being largely degradable in the r u m e n suggesting s u p p l e m e n t a t i o n with a protein which is only slowly degradable (Kay et al., no date).
319
The c h e m i c a l c o m p o s i t i o n o f B r a s s i c a crops During this c e n t u r y several food composition tables have been developed to supply information on the m os t diverse products, facilitating an improvement in diets and generally for a healthier population with less health risks. The information on vegetables is now comprehensive enough to take advantage of their characteristics to improve the diet, a l t h o u g h in m a n y countries due to social-economic c o n s t r a i n t s the food composition tables are not used e n o u g h or even at all. The B r a s s i c a c e a e is a major family of plants generally u s e d worldwide, including m e m b e r s which in m a n y situations are seen as having a low food value. In the following pages the compositional characteristics of this group of plants will be treated with e m p h a s i s in the genus Brassica.
General c o m p o n e n t s The major food composition aspects of B r a s s i c a oleracea plants m o s t commonly u s e d for h u m a n c o n s u m p t i o n are show n in Table 10.3. The high water content and the low fat a n d carbohydrate values are relevant features, being responsible for the low caloric value, a characteristic of these products. When comparing B r a s s i c a vegetables with other of high water content, the levels of fibre and protein are relatively high. Protein levels in Brussels s p r o u t s and kale are high each having over 4 % of fresh edible portion. B r a s s i c a are also a good source of minerals particularly, p o t a s s i u m a n d calcium, the latter being a b u n d a n t in kales and broccoli. These two brassicas together with Brussels s p r o u t s also have i m p o r t a n t levels of vitamins, particularly the two carotenoid anti-oxidants and vitamin C. In several cultivars of Chinese cabbage, vitamin C c o n t e n t was between 22 and 97 m g / 1 0 0 g of fresh weight whilst the carotenoid content (expressed as gcaroten) varied between 31 and 219 lug/100g fresh weight (Bajaj et al., 1991). Broccoli and B r u s s e l s s p r o u t s are an i m p o r t a n t source of lutein (Table 10.4). Increased bioavailability of carotenoids to h u m a n s was reported after the typical "in home" cooking (Table 10.4) (Scott a n d Hart, 1995). Kale an d Br us s el s s p r o u t s have the highest amino acid content followed by broccoli. Special attention should to be paid to the nine essential amino acids which a n i m a l s are strictly d e p e n d e n t on. Among these, methionine permits the entry of s u l p h u r in animal cells a n d is involved in the synthesis of cysteine a n d other s u l p h u r c o m p o n e n t s with major functions for cell metabolism (protein biosynthesis, catalytic activities, detoxification processes, etc.) (Gaki~re et al., 1999).Of the two s u l p h u r amino acids, methionine and cysteine, the first is c o m m o n to all B. oleracea forms, whilst cysteine was the only one reported in white and red cabbages a n d in kale which has more t h a n twice the a m o u n t pr es ent in the other two varieties (Table 10.3). The other major group of crops within the g e n u s B r a s s i c a are the oilseed species (B. n a p u s , B. rapa and B. juncea) generally k n o w n as rapeseed.
Table 10.3 a - Major constituents of the most commonly used Brussicu oleruceu types for human consumption. Brassica crops
Water
(“w
Protein Fat Carbohydrates Fibre
Na
K
Mg
Ca
P
Fe
Se
(mg) (mg) (mg) (mg) (mg) (mg) (pg)
1
B
(w) (m)
Mn
Cu
Zn
(vg)
(pg)
(
(8)
(g)
(g)
89.70 3.30 89.20 3.60 88.20 4.40
0.20 0.35 0.90
2.51 5.70 1.80
3.00 1.40 2.60
19.0 23.0 8.0
373.0 389.0 370.0
24.0 105.0 23.0 101.0 22.0 56.0
91.60 2.46 91.10 2.60 88.40 3.60
0.28 0.22 0.90
2.34 5.20 3.00
2.92 0.88 1.80
16.0 16.0 9.0
328.0 328.0 380.0
17.0 20.0 17.0
20.0 25.0 21.0
54.0 0.63 0.94 0.64 150.0 170.0 - 57.0 1.00 - 300.0 64.0 0.70 Trace Trace
41.92
257.0
30.00
600.0
85.00 4.45 84.80 4.20 84.30 3.50
0.34 0.40 1.40
3.29 8.60 4.10
4.40 1.60 4.10
9.6 22.0 6.0
387.0 404.0 450.0
22.0 26.0 8.0
31.0 61.0 26.0
83.6 0.60 40.00 0.70 270.0 260.0 - 80.0 1.50 77.0 0.70 - 200.0 ND 1.00
64.86
590.0
20.00
500.0
86.30 4.30 85.00 4.00 2.93 -
0.90 0.80
2.54 6.80
4.20 1.40
42.0 55.0
31.0 212.0 35.0 177.0 21.4 117.1
87.0 1.90 71.0 2.00 78.4 1.07
55.60
330.0
-
490.0 383.0 357.0
(g)
W h)
0
Font
broccoli [ B. oleracea
convar. bottytis var. italica ]
82.0 77.0 87.0
1.30 0.70 15.00 160.0 260.0 126.00 605.0 - 1.20 - 1.70 Trace 2.00 - 200.0 20.00 600.0
1
2 3
cauliflower [ B. oleracea
convar. bottytis var. botrytis ]
-
-
I 2 3
Brussels sprouts [ B. oleracea
convar. oleracea var. gemmi$ra ]
-
-
I 2 3
kale [ B. oleracea
convar. acephala ]
-
-
-
1.37 12.00 240.0 550.0
-
-
- 495.0
-
- 1470
Portuguese kale [ B. oleracea var. tronchuda 0..costata
- _ - 855.0 -
840.0
42.36
239.0
-
-
-
-
310.5
31.1 486.0
69.0
1.70
-
91.80 1.50 0.18 91.70 1.60 0.22
3.54 6.10
2.50 1.00
4.0 17.0
266.0 257.0
18.0 35.0 17.0 43.0
30.0 0.92 36.0 0.70
100.0
90.49 1.37 0.20 92.50 1.40 0.22 90.70 1.40 0.20
4.16 5.10 5.00
2.96 0.09 2.10
11.7 17.0 7.0
208.0 272.0 240.0
23.0 16.0 6.0
46.0 49.0 49.0
27.5 2.51 37.00 1.93 600.0 100.0 - 29.0 0.50 - 200.0 29.0 0.50 Trace 2.00
33.31
90.00 2.95 90.50 2.50
2.41 4.90
2.57 0.60
9.0 20.0
252.0 262.0
12.0 20.0
47.0 54.0
55.6 1.23 22.00 51.0 0.80
34.68
-
4.00
I 2 4
4
red cabbage [ B. oleracea
convar. capitate var. rubra ]
-
5.20 250.0
-
-
100.0
-
-
-
1
2
white cabbage [ B. oleracea convar. capitata var alba ]
224.0
1
10.00 200.0
2 3
-
-
Savoy cabbage [ B. oleracea
convar. capitate var. sabauda I
0.38 0.24
Units- d l 0 0 g edible portion, I-Adaptedfrom Souci et a l ( l 9 8 4 ) 2-Adaptedfrom Rubatzky and Yamaguchi ( I 996) 3-Adaptedfrom 4-Adaptedfrom E.Rosa and R.Heaney (1996)
5Ih
-
300.0 200.0
-
-
-
262.0
-
suppl. t o McCance and Widdowson ‘s (1991)
1
2
Table 10.3 b - Major constituents of the most commonly used Brassica oleracea types for human consumption.
broccoli [ B. oleracea convar. botrytis var. italica 1
cauliflower [ B. oleracea
convar. botrytis var. botrytis ]
Brussel sprouts [ B. oleracea convar. oleracea var. gemmiferal
kale
[ B. oleracea
convar. acephala ]
red cabbage [ B. oleracea convar. capitate var. rubra 1
white cabbage [ B. oleracea
convar. capitate var alba ]
Savoycabbage [ B. oleracea
convar. capitate var. sabauda 1
846.0 -
575.0
10.4
174.0
98.5
- 100.0 -
-
110.0 100.0
50.0
-
-
447.0
275.0
126.0
-
215.0 5170.0
-
167.0
-
1.00
-
- _
100.0 100.0
0.60
-
- _
I .29
280.0 0.50
_
-
-
140.0
ND
1.01
200.0
1.50
-
-
280.0 1.50
-
-
- - - - -
- - - - -
- I
- - 2
- - 3
-
I .oo
370.0 0.40
100.0 110.0
250.0 210.0
2.10
-
250.0 0.50
-
187.0 105.0 300.0 69.0 240.0 140.0 250.0 52.0 140.0 130.0 64.0 230.0 180.0 69.0 I - 119.0 - - - - - - - - - - - - 2
0.32
150.0 2.00
35.0
-
-
71.8
79.5
48.6 60.0
37.3 50.0
0.32
0.26
-
-
-
59.2 60.0
-
-
-
0.43
-
-
336.0 0.40
50.0 50.0
39.0
140.0 110.0 170.0 48.0 77.0 110.0 34.0 150.0 35.0
-
68.0 80.0
40.0
66.0
73.0 110.0 49.0 75.043.0 - -
-
-
_
125.0
_
0.67
24.5
_
-
0.60
_
15.0
_
111.0 115.0 190.0 63.0 150.0 130.0 160.0 50.0 120.0 120.0 37.0 170.0 - - I 109.0- - - - - - - - - 2 90.0 87.0 - - - - - - - - - - - - 3
_
134.0 150.0
- 110.0 817.0
178.0 200.0
-
- _
63.8 60.0
-
_
-
-
-
-
-
174.0 3.08
182.0 112.0 280.0 110.0 250.0 210.0 230.0 40.0 150.0 160.0 50.0 240.0 98.0- - - - 135.0 115.0 - - - - - - - - - - - -
_
-
31.0
- - _
0.21
180.0 0.10
34.0
0.33
0.21
156.0 0.10
90.0
-
-
-
-
-
- - _ _ _ _
50.0 110.0 27.0 58.0 - -
71.0 -
-
45.2 100.0 25.0 50.035.0 -
65.0
39.0
49.4 150.0 47.0 47.0-
92.0
-
-
43.0
-
-
-
-
-
-
56.0 13.0 30.0 38.0 - 42.0
- -
-
- - _
- 27.0
- _ - _ _ -
-
-
_
-
-
100.0 190.0 27.0 120.0 110.0 32.0 140.0 -
2 3
- 30.0 I - - 2
61.0 14.0 32.0 42.0 12.0 46.0
-
- I
_ _ _
-
I 2
3
- - I - - 2
Units- @I00 g edible portion. I - Adaptedfrom Souci et al(l984) 2 - Adaptedfrom Rubatzky and Yamaguchi (1996) 3 - Adaptedfrom 5Ihsuppl to McCance and Widdowson's (1991)
322
Table 10.4 Carotenoid content (pg/100g wet weight, as eaten) of some Brassica oleracea varieties (Scott and Hart, 1995)
Vegetable
Type
Lutein
Brussels sprouts
Raw
610
441
112
Brussels sprouts
Cooked
621
411
144
Broccoli-fresh
Raw
1614
800
119
Broccoli-fresh
Cooked
1949
1125
256
White cabbage
Raw
80
51
8
White cabbage
Cooked
111
65
6
Savoy cabbage
Raw
103
50
nd
Savoy cabbage
Cooked
341
240
33
Savoy cabbage
Outside 14457
10020
1829
Leaves
Zeax 13-cryp Lyco m-car 13-car cis 0-car
Cauliflower
Raw
Tr
nd
nd
Cauliflower
Cooked
Tr
nd
nd
The seed of these three species contain 40 to 50% of oil which was originally u s ed for lighting and a lubricant, and now, after refining mainly for edible purposes. The fatty acid composition of seed oils affects their chemical, physical and nutritive characteristics (Rudloff and Wehling, 1998). One of the major fatty acids in oilseed, erucic acid (C22:1), responsible in part for the antinutritive properties of rapeseed oil, has been reduced by selective breeding from 45% to less t h a n one percent in the so called "double low" cultivars of rapeseed (low in erucic acid and glucosinolates). The fatty acid composition of these cultivars h a s higher (55-65% and could be up to 80%) oleic acid (C18:1) an d lower (20-23% and even down to 3%) linoleic acid (C18:2) contents t h a n m o s t other vegetable oils whilst the linolenic acid (C 18:3) content is higher (8-12%) t h a n in sunflower, olive or poppy oils and similar to soybean (8%) (Krzymafiski, 1998; Rudloff and Wehling, 1998). The quality of the oil can be increased by new techniques involving the fusion of protein / p e p t i d e s rich in essential amino acids, enzymes or t h e r a p e u t i c / p h a r m a c e u tical proteins to oleosins (structural proteins closely associated with the oil body, the n a t u r a l oil storage organelle of the plant seed) (van Rooijen et al., 1998). After oil extraction the residual rapeseed meal const i t ut es 52-58% of the original seed weight. This meal has a protein content of 38 to 46% (levels
323 in B. j u n c e a being slightly higher (45.9%) than B. n a p u s and B. rapa (44.6 and 43.1%, respectively)) (Newkirk et al., 1997), whilst levels of the amino acids lysine, methionine, cysteine, threonine and tryptophan compared favorably to other oil meals and cereals (Krzymafiski, 1998). The seed protein content of either B. n a p u s and B. j u n c e a might be increased by nitrogen and sulphur fertilizations (Trivedi and Sharma, 1997; Wang et al., 1997; Aulakh et al., 1995). The fibre content (11%) of the seed, mainly localized in the hulls and accounting for 27 to 30% of the rapeseed meal, is higher t h a n that of soybean seed (7%). The meals from B. j u n c e a contained less (27%) total dietary fibre t h a n B. n a p u s and B. rapa (29.5 and 29.7%, respectively) (Newkirk et al., 1997). The utilization of these high-quality products is more or less reduced due to oil mill processing but the aqueous enzymatic extraction procedure h a s been further improved to allow more optimal utilization of the various high-quality products in oilseed rape, resulting in a "Green Chemistry" biorefining process (Bagger et al., 1998). Another important aspect of rapeseed chemical composition is the chlorophyll content of the seed. The presence of as little as 2% of green seeds resuits in a downgrading of the crop (Morissette et al., 1998). Thus, seeds of B. rapa and B. n a p u s have been submitted to breeding programmes and genetic transformation in order to reduce chlorophyl content, seed coat and levels of polyphenols resulting in reduced off-flavors and odors and improved shelf life of the oil (Morissette et al., 1998). Other species such as Indian (B. juncea) and Ethiopian (B. carinata) mustards, with reported levels of erucic of between 0 to 50% of the total fatty acid content, might be used as oilseeds for h u m a n and industrial purposes u n d e r Mediterranean or semi-arid conditions, due to their resistance to drought, to pod shattering and to a wide range of diseases and pests (Velasco et al., 1998; Getinet et al., 1996).
S e c o n d a r y plant m e t a b o l i t e s : t h e g l u c o s i n o l a t e s Occurrence and distribution When considering the secondary metabolites of Crucifers, there can be little doubt t h a t the glucosinolates, a class of sulphur-containing glucosides are by far the most important. Although more than 100 such compounds are known, only a r o u n d 15-16 occur in significant a m o u n t s t h r o u g h o u t the brassicas a n d within any particular species the n u m b e r is usually m u c h lower. Glucosinolates are present to some extent in the seeds, roots, stems, leaves and flowers of all B r a s s i c a species in which they co-exist with an enzyme called myrosinase (thioglucoside glucohydrolase, E.C. 3.2.3.1.) which mediates their breakdown to a range of physiologically active compounds. The presence of high levels of such c o m p o u n d s in rapeseed meal, a by-product of the rape-oil industry, rendered the meal unsuitable for use in m a n y
324 potential a n i m a l feeding applications t h u s s t i m u l a t i n g i n t e r n a t i o n a l r e s e a r c h p r o g r a m s a i m e d at r e d u c i n g the levels of these c o m p o u n d s . F u r t h e r r e s e a r c h i m p e t u s was g e n e r a t e d w h e n s u l p h u r c o m p o u n d s derived from g l u c o s i n o l a t e s were shown to be partly r e s p o n s i b l e for the flavor of B r a s s i c a vegetables a n d t h a t selection of varieties for breeding p u r p o s e s , b a s e d on glucosinolate criteria might afford a m e a n s of d e t e r m i n i n g this imp o r t a n t a t t r i b u t e . R e s e a r c h was f u r t h e r s t i m u l a t e d w h e n studies showed t h a t glucosinolates are closely involved in the prevention of insect a n d fungal a t t a c k in p l a n t s a n d m o r e recently, t h a t t h e y m a y have a n i m p o r t a n t role in the control of biological p r o c e s s e s a s s o c i a t e d with the initiation and progression of carcinogenic p r o c e s s e s in m a n .
Chemistry In general, glucosinolates conform to the basic s t r u c t u r e shown in Figure 10.1 (Fenwick et al., 1983a). The s t r u c t u r a l diversity of this large group of c o m p o u n d s is due a l m o s t entirely to the different s u b s t i t u e n t s possible at the s i d e c h a i n position R, a l t h o u g h some exceptions have been reported (Sor e n s e n , 1990), n o t a b l y esterification of the s u g a r moiety with sinapic, caffeic or malic acids.
R - C - S - 13 - D - Glucose II N " OSO3
Figure 10.1 General s t r u c t u r e of glucosinolates. The R s u b s t i t u e n t m a y be a n alkyl or alkenyl side c h a i n which itself m a y c o n t a i n s u b s t i t u e n t hydroxyl g r o u p s or s u l p h u r in various oxidation states. Alternatively, the R s u b s t i t u e n t m a y be an a r o m a t i c or a hetero-aromatic group, the possible hydroxy or m e t h o x y s u b s t i t u e n t s which f u r t h e r complicate the picture. As will be s h o w n below, this variability in the sidec h a i n at R is d u e to the biosynthetic p a t h w a y s involved in the formation of the glucosinolate a n d as a c o n s e q u e n c e of these v a r i a t i o n s a great n u m b e r of b r e a k d o w n p r o d u c t s are possible. The s t r u c t u r e s a n d n a m e s of the glucosinolates m o s t c o m m o n l y found in b r a s s i c a s (particularly in B. oleracea cultivars, B. rapa a n d in the a m p h i diploid B. napus) are given in Table 10.5 (Bjerg a n d S o r e n s e n , 1987). In addition to t h e s e m a j o r c o m p o u n d s , a f u r t h e r 15-16 glucosinolates are commonly detected b u t t h e s e are u s u a l l y p r e s e n t only in m i n o r a m o u n t s (< 10 p m o l / 1 0 0 g fresh weight).
325
Table 10. 5 Structure of the side chain R of the major glucosinolates occurring in the Brassicaceae (from Bjerg and Sorensen, 1987). Chemical name
Trivial name
CH2=CH-CH2 -
2-propenyl- or allyl glucosinolate
Sinigrin
CH2=CH-CH2-CH2 -
but-3-enyl glucosinolate
Gluconapin
CH2=CH-CH2-CH2-CH2 -
pent-4-enyl glucosinolate
Glucobrassicanapin
CH2=CH-CH-CH2 -
2-hydroxybut-3-enyl glucosinolate
Progoitrin
CH2=CH-CH2-CH-CH2 I OH
2-hydroxypent-4-enyl glucosinolate
Gluconapoleiferin
CH3-SO-CH2-CH2-CH 2-
3-methylsulfinylpropyl glucosinolate
Glucoiberin
CH3-SO-CH2-CH2-CH2-CH2-
4-methylsulfinylbutyl glucosinolate
Glucoraphanin
2-phenethyl glucosinolate
Gluconasturtiin
indol-3-ylmethyl glucosinolate (R1 = R4 = H)
Glucobrassicin
1-methoxyindol-3-ylmethyl glucosinolate (R1 = OCH3 ;R4 = H)
Neoglucobrassicin
4-hydroxyindol-3-ylmethyl glucosinolate (R1 = H ; R4 = OH)
4-Hydroxyglucobrassicin
4-methoxyindol-3-ylmethyl glucosinolate (R1 = H ; R4 = OCH3)
4-Methoxyglucobrassicin
Structure of R
Aliphatic glucosinolates
i
OH
Aromatic glucosinolates
Indole glucosinolates
326
I
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327
Biosynthesis, degradation and breakdown Although the bi os ynt hes es of glucosinolates and a n o t h e r group of n a t u ral toxicants, the cyanogenic glycosides both follow the sam e initial stages (Halkier et al., 1991; Koch et al., 1992; Larsen 1981) the enzyme syst em s involved are different and the two classes of c o m p o u n d have seldom been reported to occur together with only the glucosinolates occurring in brassicas. The similarity between glucosinolates a n d side c h a i n s of some amino acids was an early indicator t h a t glucosinolates may be derived from n a t u r a l amino acids (Ettlinger and Kjaer, 1968), s upport i ng earlier observations (Kut~cek et al., 1962) t h a t t r y p t o p h a n was converted to indol-3-yl-methyl glucosinolate and t h a t benzyl glucosinolate was biosynthesized from phenylalanine (Underhill et al., 1962). The first stage of the biosynthesis of glucosinolates (Figure 10.2) involves the modification of an amino acid (or a derivative thereof) with the formation of an aldoxime intermediate (Rosa et al., 1997). S u c h amino acids may be c o m m o n protein amino acids s u c h as valine and iso-leucine (giving rise to isopropyl and sec-butyl glucosinolates), phenylalanine a n d tyrosine (benzyla n d hydroxybenzyl glucosinolates) or tryptophan, the p r e c u r s o r of indol-3ylmethyl glucosinolate. Alternatively, chain elongation of amino acids s u c h as methionine m a y be followed by oxidation of the s u l p h u r group to produce sulphoxides or sulphones. Insertion of a s u l p h u r a t o m into the aldoxime to produce a thiohydroxamic acid is t hen followed by glycosylation and sulphation (see Rosa et al., 1997). None of these latter conjugation reactions is affected by the s t r u c t u r e of the side chain (GrootWassink et al., 1987, 1990, 1994). F u r t h e r side-chain modifications m ay occur after the formation of the glucosinolate skeleton. Glucosinolates occur in the plant in conjunction with an enzyme called myrosinase which mediates the hydrolytic b r e a k d o w n of glucosinolates. Myrosinase (more correctly described as thioglucoside glucohydrolase E.C.3.2. 3.1.) h a s been found in seeds, leaves, stems a n d roots a n d occurs within the myrosin cells a n d is t h u s separated from the glucosinolates (Thangstad et al., 1990). It h a s been suggested t h a t myrosin cells are formed at an early stage of leaf development, after which no new cells are p r o d u c e d (Pocock et al., 1987). Young plant tissue generally shows a higher m yrosi nase activity. Only when the plant cells are di s r upt ed by physical m e a n s or w hen autolysis occurs within the plant, does the enzyme come into cont act with its substrate glucosinolates, resulting in a rapid conversion to a range of different c o m p o u n d s . It is these b r e a k d o w n c o m p o u n d s r a t h e r t h a n glucosinolates p e r s e which are responsible for the m a n y and varied physiological properties associated with this class of c o m p o u n d s . C r u s h i n g of oilseeds (B. n a p u s , B. rapa, B. j u n c e a ) in order to extract the oil, results in a seedmeal with intact glucosinolates a n d enzymes and the introduction of s u c h material into animal feeds resulted in an in vivo breakdown of the glucosinolates to a range of p r o d u c t s harmful to animal health.
328 Similarly, the cutting, shredding or c r u s h i n g of B r a s s i c a vegetative material results in an immediate loss of glucosinolates, t h u s creating problems for the analytical chemist. The n a t u r e of the m y r o s i n a s e - m e d i a t e d breakdown p r o d u c t s is determined by a n u m b e r of factors, the m o s t i m p o r t a n t of which is the structure of the side chain R. Other influences include the pH a n d / o r the presence of certain co-factors s u c h as ferrous iron.(MacLeod and Rossiter, 1987; Uda et al., 1986; Bones et al., 1994) (Figure 10.3)
At pH 5-7 aliphatic side chains tend to produce isothiocyanates, whereas at more acidic pH, nitriles r a t h e r t h a n isothiocyanates are formed. Since isothiocyanates have more significant organoleptic, biological and plant protective roles it is clear that the pH at which hydrolysis occurs is important. Glucosinolates with an aliphatic side chain s u b s t i t u t e d at carbon 2 with a hydroxyl group produce u n s t a b l e isothiocyanates which cyclize spontaneously to form oxazolidine-2-thiones, c o m p o u n d s with irreversible goitrogenic or antithyroid activity. C o m p o u n d s with indolic or s u b s t i t u t e d indolic side chains also produce u n s t a b l e isothiocyanates which in t u r n give rise to the corresponding indole-3-carbinol (which may react further to give diindolylmethane) and thiocyanate ion (Searle et al., 1984). Under acidic conditions the production of nitriles is favored and during autolysis nitriles tend to be produced even when pH appears to be unfavorable, probably due to the inhibitory effect of ferrous iron on isothiocyanate formation (Uda et al., 1986). Similarly, when the R side chain h a s a terminal u n s a t u r a t e d group, the presence of ferrous iron together with epithiospecifier protein, results in the formation of episulphides (MacLeod and Rossiter, 1985; Petroski and Kwolek,
1985) Analysis As shown above, sampling of material for s u b s e q u e n t extraction and analysis is fraught with problems and should be reserved for the skilled analyst. Glucosinolate levels can differ widely even within a single plant and may be further affected by a n u m b e r of factors (see below). Preparation of extracts m u s t be carried out in a m a n n e r s u c h that losses of glucosinolates due to myrosinase activity are negligible. This usually m e a n s careful freeze drying or freezing the plant material in liquid nitrogen followed by extraction of glucosinolates us i ng boiling a q u e o u s methanol. The choice of a suitable analytical method is complex and may be determined by the need for quantitative or qualitative information, speed, accuracy, or a combination of any of these factors. The need for rapid methods and for accurate quantitative m e t h o d s for the regulation of glucosinolate levels in oilseed rape p r o m pt ed the E u r o p e a n Commission and national governments to s u p p o r t their development with the result t hat several s u c h methods are now available to meet the needs of analysts in other areas of glucosinolate research.
(D
o
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329
330 Methods have been described for the analysis of total glucosinolate content based on the stoichiometric release of glucose and bisulphate by myrosinase (McGregor et al., 1983; Heaney and Fenwick, 1993). In common with methods which quantify individual glucosinolates, the preparation of the enzyme in a pure state is an essential prerequisite. Such methods worked well for rapeseed and for m a n y other brassicas, but the possible presence of glucose esters of (for example) sinapic acid, render methods based on glucose unsuitable for some applications (Sorensen, 1990). Furthermore, methods based on glucose or bisulphate release, furnish no information about the nature and levels of individual glucosinolates. As will be shown below, this information is vital in studies of the biological and physiological effects of glucosinolates and their derived compounds. The development of methods which yield quantitative information about each constituent glucosinolate have been hampered by a lack of compounds suitable for use as internal s t a n d a r d s and reference markers, the preparation of which from natural sources was both time-consuming and expensive. The early use of gas chromatographic (GC) methods for the separation and quantitation of glucosinolates was limited by the failure of the methods to accurately quantify those compounds with indolic side chains. The ubiquity of these compounds was only appreciated with the development of high performance liquid chromatography (HPLC) methods. Using HPLC in reverse-phase together with ion-pairing agents, glucosinolate extracts could be analyzed without further enzymatic steps (Helboe et al., 1980; Betz and Page, 1990). However, later HPLC methods involved the adsorption of glucosinolates onto small columns of anion-exchange material and washing with water followed by treatment with the enzyme sulphatase (Spinks et al., 1984). This approach afforded a highly specific method in which all of the glucosinolates occurring in the Crucifer crops could be resolved with good baseline separation and was adopted as an official method of the European Commission (EC, 1990). Mass spectrometric (MS) methods have provided m u c h useful information and in the absence of suitable reference compounds, MS coupled to GC or HPLC permits the identification of individual peaks. More recently, the use of capillary electrophoresis has provided the basis for rapid and inexpensive methods with good separation of both glucosinolates and desulphoglucosinolates (Feldl et al., 1994; Michaelsen et al., 1992).
Levels o f g l u c o s i n o l a t e s in B r a s s i c a The importance of glucosinolates in the diets of both h u m a n s and animals has prompted a large n u m b e r of studies resulting in a wealth of data showing wide variations in levels both between and within different parts of the plant In general, seeds contain the highest levels, a fact which is of particular importance in the oilseed rape and m u s t a r d industries. Regardless of genetic origin, any B r a s s i c a subspecies or variety tends to contain the same
331 spectrum of glucosinolates and, although there may be several glucosinolates present in small amounts, usually only three or four are present in relatively high concentrations. There are m a n y other factors which have a bearing on glucosinolate levels and these are given in detail in the next section. Although the type and a m o u n t of glucosinolates vary according the part of the plant analyzed, most reported values for B r a s s i c a vegetables are for plants which have been harvested at the optimum time for sale on the market and refer to the part normally consumed. Early methods of analysis failed to differentiate between the different indolyl glucosinolates which were assessed by measuring released thiocyanate and recorded as total indole glucosinolates. Table 10.6 summarizes the findings of some of the studies carried out in the USA and more recently in the UK. In cabbages and Brussels sprouts, glucosinolate levels reported by American workers tend to be m u c h lower t h a n those found in the UK studies. The reasons for this are not clear but may be due to differences in the genetic base of the material or possibly the effect of site, growing season or sampiing time. The major glucosinolates of white cabbage and Savoy cabbage are prop2-enyl- and 3-methylsulphinyl glucosinolates together with the indolyl glucosinolate, glucobrassicin. These three c o m p o u n d s are also major components of Brussels sprouts in addition to but-3-enyl- and 2-hydroxybut-3-enyl glucosinolates, a spectrum similar to that shown by kale. In red cabbage varieties 4-methylsulphinylbutyl glucosinolate is a major component. Apart from glucobrassicin, the major components of broccoli are 4-methylsulphinylbutyl glucosinolate and 3-methylsulphinylpropyl glucosinolate (found in purpleheaded varieties). The glucosinolate content of cauliflower was both quantitatively and qualitatively similar to that of cabbage although some varieties were found to contain high levels of 4-methylsulphinylbutyl glucosinolate. The compounds mentioned above and shown in Table 10.6 represent the major constituents, in addition to which several other glucosinolates may be present in minor amounts. In addition to these major crops, the glucosinolate contents of a n u m b e r of commercially less important brassicas have been reported. The major glucosinolates of swedes were reported to be 2-hydroxybut-3-enyl-, 2-phenethyl- and 4-methoxyindol-3-yl glucosinolates. Kohlrabi contained 4-methylthiobutyl- 3-methylthiopropyl- and 2-phenethyl glucosinolates. The main components of r u t a b a g a were hydroxybut-3-enyl- and 4-methylthiobutyl glucosinolates. Chinese cabbage contained up to 465 ~moles of total glucosinolates per 100g of fresh weight {Bajaj et al., 1991). Glucosinolate levels in seed and vegetative parts of wild B r a s s i c a species have also been reported (Mithen et al., 1987; Horn and Vaughan, 1983). The pattern and a m o u n t of glucosinolates in B r a s s i c a plants are subject to wide genetic variation and other factors including the stage of the plant's development, growing environment, cultural practices, pests and diseases,
332
Table 10.6 Levels of the principal glucosinolates occurring in the main Brassica oleracea varieties and in rapeseed (B. napus and B. rapa) Concentration (lamoles 100 g-1 of fresh weight) Species
Giucosinolate
Average
Range
Reference
White cabbage (B. oleracea L., capitata group) 2-propenyl-
36.3 26.4 57.2 66.2
4.3-147.4 8.8-148.6 18.6-104.3 18.6-162.7
VanEtten et al. (1976) VanEtten et al. (1980) Sones et al. (1984a) Sones et al. (1984c) z
3-methylsulphinylpropyl-
34.7 28.3 72.7 97.6
13.0-70.9
5.0-193.1 5.0-279.8
VanEtten et al. (1976) VanEtten et al. (1980) Sones et al. (1984a) Sones et al. (1984c) z
indole-glucosinolates
49.4 31.2
28.0-106.4 10.5-104.9
VanEtten et al. (1976)Y VanEtten et al. (1980)Y
indol-3-ylmethyl-
39.3 60.7
9.3-129.8 9.3-200.0
Sones et al. (1984a) Sones et al. (1984c) z
143.8 117.3 68.6 200.9 238.3
66.4-236.7 57.5-234.5 17.7-112.8 93.8-348.2 78.8-602.6
VanEtten et al. (1976) VanEtten et al. (1980) Mullin and Sahasrabudhe (1977) Sones et al. (1984a) x Sones et al. (1984c) z
14.2 93.2
35.8 0.1-39.7 31.5-162.7
VanEtten et al. (1976) VanEtten et al. (1980) Sones et al. (1984a)
46.7 169.8
100.7 15.2-91.1 72.5-279.8
VanEtten et al. (1976) VanEtten et al. (1980) Sones et al. (1984a)
80.5
111.3 61.7-108.2
VanEtten et al. (1976)Y VanEtten et al. (1980)Y
123.0
70.2-199.8
Sones et al. (1984a)
Total
10.0-58.6
Savoy cabbage (B. oleracea L., sabauda group) 2-propenyl .
.
.
.
3-methylsulphinylpropyl-
indole-glucosinolates
indol-3-ylmethyl2-hydroxybut-3-enyl-
0.5
1.6 0.0-1.3
VanEtten et al. (1976) VanEtten et al. (1980)
333
13.8
Total 164.5 461.3
5.6-29.5 275.6 100.4-265.0 267.1-653.4
Sones et aL (1984a) VanEtten et aL (1976) VanEtten et al. (1980) Sones et al. (1984a) x
Red cabbage (B. oleracea L., capitata group) et et et et
al. al. al. aL
12.6 10.5 16.1 14.5
11.1-14.1 1.5-25.7 12.4-19.7 4.8-31.0
VanEtten VanEtten VanEtten VanEtten
4-methylsulphinylbutyl-
56.8 52.3
46.7-66.9 31.6-82.1
VanEtten et al. (1976) VanEtten et al. (1980)
indole-glucosinolates
72.8
42.6-102.9 31.9-67.9
VanEtten et al. (1976)Y VanEtten et aL (1980)Y
2-propenyl3-methylsulphinylpropyl-
.
-
-
(1976) (1980) (1976) (1980)
but-3-enyl-
15.1 9.9
13.9-16.3 4.6-15.6
VanEtten et al. (1976) VanEtten et al. (1980)
2-hydroxybut-3-enyl-
12.2 8.3
10.1-14.3 4.4-5.5
VanEtten et al. (1976) VanEtten et al. (1980)
Total
150.5-258.1 88.2-234.4 34.4-98.9
VanEtten et al. (1976) VanEtten et al. (1980) Mullin and Sahasrabudhe (1977)
136.0 10.7 112.1
27.7-392.9 3.9-22.7 4.0-280.6
Heaney and Fenwick (1980 Carlson et al. (1987a) Sones et al. (1984c)
3-methylsulphinylpropyl-
76.6 11.8
0.0-154.2 2.4-18.9
Sones et aL (1984c) Carlson et aL (1987a)
4-methylsulphinylbutyl-
8.2
0.4-22.6
Carlson et al. (1987a)
204.3 163.4 68.8
Brussels sprouts (B. oleracea L., gemmifera group) 2-propenyl-
indol-3-ylmethyl-
1-methoxyindol-3-ylmethylbut-3-eny|-
2-hydroxybut-3-enyl-
113.2
Heaney and Fenwick (1980) Sones et al. (1984c) Carlson et al. (1987a)Y
128.4 391.8
45.3-228.4 54.3-326.3 327.8-469.4
21.3
1.9-34.3
36.5 61.3 4.2
7.3-121.7 6.1-221.2 0.5-12.2
Heaney and Fenwick (1980) Sones et al. (1984c) Carlson et al. (1987a)
67.9 111.9 8.3
93.7-231.9 29.3-303.5 1.0-25.4
Heaney and Fenwick (1980) Sones et al. (1984c) Carlson et al. (1987a)
Sones et aL (1984c)
334
Total
367.2 461.9 495.0 553.0
330.3-406.5 138.6-900.7 318.4-861.9 465.6-600.6
2-propenyl
20.7
12.6-28.7
Carlson et al. (1987a)
3-methylsulphinylpropyl-
38.6
8.4-69.3
Carlson et al. (1987a)
indol-3-ylmethyl-
55.5 47.3
44.2-69.5 ---
Carlson et al. (1987a)Y VanEtten and Tookey (1979)
220.4
64.4-306.7
Carlson et al. (1987a) w
2-propenyl-
97.0
62.5-197.3
Carlson et al. (1987a)
3-methylsulphinylpropyl-
11.7
0.0-49.9
Carlson et al. (1987a)
107.5
67.2-165.3
but-3-enyl-
21.3
5.8-38.1
Carlson et al. (1987a)
2-hydroxybut-3-enyl-
70.1
16.8-130.3
Carlson et al. (1987a)
439.1
316.1-600.0
Mullin and Sahasrabudhe (1977) Heaney and Fenwick (1980) Sones et al. (1984c) Carlson et al. (1987a) w
Collards (B. oleracea L., acephala group)
Total Kale (B. oleracea L., acephala group)
indol-3-ylmethyl-
Total
Carlson et al. (1987a)Y
Carlson et al. (1987a) w
Broccoli (B. oleracea L., italica group) 2-hydroxybut-3-enyl3-methylsulphinylpropyl4-methylthiobutyl4-methylsulphinylbutyl-
74.1
8.0-161.0
Hansen et al. (1997)
0-327.2 17.0-78.0
Lewis et al. (1991 ) Hansen et al. (1997) Fahey et al. (1997) p
541.0 63.9 97.5 1660.0 108.0
-
-
-
28.9-88.3 54.0-190.2 152.0-384.0 -
-
.
53.0-128.0 pent-4-enyl-
4-hydroxyindol-3-yl methyl-
Hansen et al. (1997)
6.0
but-3-enyl
5.0-14.0 71.0
Carlson et al. (1987a) Lewis et al. (1991 ) Hansen et al. (1997) Fahey et al. (1997) p Fahey et al. (1997) Rosa and Rodrigues (unpub)
Hansen et al. (1997) Fahey et al. (1997) p
335
indol-3-ylmethyl-
59.4 56.0
42.2-71.7 22.8-101.0 107.0-334.0
Carlson et al. (1987a)Y Lewis et al. (1991 ) Hansen et al. (1997)
8.6
2.4-18.4 26.0-199.0
Lewis et al. (1991 ) Hansen et al. (1997)
7.0-20.0
Hansen et al. (1997) Fahey et al. (1997)
1-methoxyindol-3-yl methyl-
4-methoxyindol-3-yl methyl62.0
Total
161.9 188.2 248.4 .
.
.
98.5-323.9 102.2-262.7 152.2-448.6 134.0-351.0
Mullin and Sahasrabudhe (1977) Carlson et al. (1987a) w Lewis et al. (1991) Rosa and Rodrigues (unpub)
Cauliflower (B. oleracea L., botrytis group) 2-propenyl-
37.8 35.8 10.0
1.3-157.9 1.3-157.9 2.9-16.5
Sones et al. (1984b) Sones et al. (1984c) Carlson et al. (1987a)
4-methylsulphinylbutyl-
63.8
1.8-190.1
Lewis et al. (1991)
3-methylsulphinylpropyl-
41.0 37.5 5.2 51.0
0.0-90.9 1.3-90.9 0.0-22.8 0.0-327.2
Sones et al. (1984b) Sones et al. (1984c) Carlson et al. (1987a) Lewis et al. (1991)
indol-3-ylmethyl-
50.0 46.7 60.6 42.1
14.8-162.3 13.6-162.3 18.8-104.7 21.0-101.0
Sones et al. (1984b) Sones et al. (1984c) Carlson et al. (1987a)Y Lewis et al. (1991)
10.0 9.3 7.3
1.1-32.0 1.2-32.0 2.3-17.4
105.0 161.9 135.7 94.6 178.2
59.1-180.6 30.2-520.4 30.2-455.8 41.1-160.6 57.1-448.6
1-Methoxyindol-3-ylmethyl-
Total
Sones et al. (1984b) Sones et al. (1984c) Lewis et al. (1991) Mullin and Sahasrabudhe (1977) Sones et al. (1984b) Sones et al. (1984c) Carlson et al. (1987a) w Lewis et al. (1991)
Turnip tops (B. rapa subsp, rapa and subsp, campestris groups) but-3-enyl .
.
.
. 103.0
294.0 38.0-181.0
Carlson et al. (1981) Carlson et al. (1987b)
336
pent-4-enyl-
Total
--58.0
151.0 20.0-112.0
Carlson et at (1981) Carlson et al. (1987b)
--186.0
586.0 80.0-292.0
Carlson et at (1981)w Carlson et al. (1987b)w
Rapeseed (B. napus L.) 3187
Fenwick et at (1983a)
10937
Fenwick et at (1983a)
pent-4-enyl-
824
Fenwick et at (1983a)
2-hydroxypent-4-enyl-
522
Fenwick et at (1983a)
but-3-enyl-
2-hydroxybut-3-enyl-
Total (Summer rape) (Spring rape)
8031 --2175
8425-17002 8140-12582 1000-2700
Fenwick et at (1983a) Fenwick et al. (1983a) Sang and Salisbury (1988)
13455 3863 14207 23450
10706-16107 1302-10281 11960-15698 ---
Fenwick et aL (1983a) Sang and Salisbury (1988) Sang and Salisbury (1988) Davis et al. (1991)
Rapeseed (B. rapa L.) but-3-enyl-
2-hydroxybut-3-enyl-
1836 209 250
1050-2387 0.0-520 ---
Sang and Salisbury (1988) Sang and Salisbury (1988) Davis et al. (1991)
pent-4-enyl-
1704 240
1092-2941 0-334
Sang and Salisbury (1988) Sang and Salisbury (1988)
322 161 396 261
234-385 0-334 294-475 0-474
Sang and Salisbury (1988) Sang and Salisbury (1988) Sang and Salisbury (1988) Sang and Salisbury (1988)
2-hydroxypent-4-enyl4-hydroxyindol-3-ylmethyl-
y Indole glucosinolates content based on thiocyanate levels x Total glucosinolates by GC method w Total glucosinolates calculated by the glucose released method (average molecular weight 457) p Broccoli sprouts etc. c a n f u r t h e r c o m p l i c a t e t h e p i c t u r e ( R o s a e t a l . , 1 9 9 7 ) . C l e a r l y s u c h v a r i a bility a f f e c t s t h e b i o l o g i c a l p r o p e r t i e s of t h e p l a n t a n d h a s i m p o r t a n t i m p l i c a t i o n s for s a m p l i n g m a t e r i a l for a n a l y s i s . G l u c o s i n o l a t e c o n t e n t is g r e a t l y a f f e c t e d b y t h e p l a n t p a r t a n d l e v e l s i n t h e s t e m s a n d p e t i o l e s h a v e b e e n f o u n d to b e l o w e r t h a n l e v e l s in r o o t s a n d
337 h e a d s of kale, broccoli, cauliflower, r u t a b a g a a n d savoy cabbage. Although roots tend to show higher levels t h a n other parts, exceptions include kale in which root levels were lower t h a n in the l am i na (Sang et al., 1984). In a study of the glucosinolate content of different part s of the cabbage it was found t h at the stem cambial cortex contained a b o u t twice the level found in the pith or leaves (VanEtten et al., 1979). A 'dilution' effect due to rapid expansion of leaves h a s been postulated to explain the lower glucosinolate levels of the more m a t u r e outer leaves of cabbage h e a d s com pared to the inner core material (Pocock et al., 1987). A similar difference has been noted between the inner and outer parts of Brussels s p r o u t 'buttons'. Despite differences in levels between different plant parts, glucosinolate p a t t e r n s of p a r t i cul ar plant parts have been u s e d to characterize different cultivars. Wild B r a s s i c a populations have been reported to have higher glucosinolate c o n t e n t t h a n the cultivars and the analysis of glucosinolates supports the h y p o thes i s of a multiphyletic origin of cultivated forms of B r a s s i c a oleracea from a n u m b e r of wild species whilst wild populations of B. r a p a had very similar glucosinolate content (Mithen et al., 1987). However, whereas the glucosinolate profile of seeds h a s been u s e d to predict the glucosinolate pattern of cabbage heads, a t t e m p t s to use levels in seedlings to forecast the glucosinolate content of the seeds of new oilseed rape varieties were less successful (Tookey et al., 1980). Variation in glucosinolate levels in the seeds of different cultivars of rapeseed, whilst levels in foliage remained similar, indicates th at levels are controlled by different genetic and physiological mec h a n i s m s (Inglis et al., 1992). The genetic regulation of the side-chain of aliphatic glucosinolates has been the subject of several studies (Magrath et al., 1994; Mithen et al., 1995; Parkin et al., 1994). E n v i r o n m e n t is i m p o r t a n t in determining the glucosinolate content. Soil type, temperature, daylight and water are all determining factors and seasonal effects would therefore be expected. In line with this b r a s s i c a s grown in a u t u m n / w i n t e r seem to have lower glucosinolate cont ent due, p r e s u m a b l y to shorter days, lower t e m p e r a t u r e s and more rainfall (Rosa a n d Heaney, 1996). Year to year variation in the glucosinolate levels of cultivars of several B r a s s i c a species have been observed and u n d e r controlled conditions, individual glucosinolate levels in young cabbage plants changed markedly during the course of a single day (Rosa and Rodrigues, 1998). Some cultural practices have been shown to affect glucosinolate levels Increasing plant density was found to increase levels of glucosinolates and other s u l p h u r c o m p o u n d s , although this could be due to smaller head size equating with less leaf expansion and a reduced 'dilution' effect as described above. A similar result with Brussels s p r o u t s was attributed to stress conditions which are k n o w n to favor increased levels of low molecular weight flavor p r e c u r s o r s (Rosa et al., 1997). There is little evidence t h a t i n p u t s of pesticides or herbicides significantly affect glucosinolate levels.
338 Soil type a p p e a r s to affect the glucosinolate content of some brassicas, with organic soils increasing levels in r u t a b a g a a n d turnip, probably due to higher s u l p h u r levels. S u l p h u r has been recognized as an i m p o r t a n t nutrient for o p t i m u m growth a n d development of plants, particularly for a group of high s u l p h u r d e m a n d p l a n t s - the family B r a s s i c a c e a e . A deficiency generally results in decreased yield and quality, and S:N imbalances c a u s e d by insufficient S-supply are responsible for fluctuations of both ion a n d amino acid pools (Hawkesford, 1999). Although stored reserves of sulphate, normally localized in the vacuole might be re-mobilized (Hawkesford, 1999), studies with w heat plants have shown that little remobilization of S might occur when the S reserves are low (Blakekalff et al., 1999). Glucosinolates have been suggested to be a sink of S (Schnug, 1990), b u t it is not yet k n o w n if the contribution of S from glucosinolates is sufficient to co u n ter the effect of S starvation, because the concentration in vegetative tissues is less t h a n 8% of total S (Fieldsend a n d Milford, 1994) and decreases during leaf expansion (Porter et al., 1991). Experiments with oilseed rape showed t h a t S application is fundamental to improve the efficient use of nitrogen. When no s u l p h u r was applied, N fertilization alone led to a total pod abortion of spring oilseed rape, showing t h at the metabolism of N is disrupted when N is in excess over S (Lassere et al., 1999). These a u t h o r s further suggest t hat the best N:S ratio value was of 6, whilst for a ratio value of 15 the s u l p h u r transferred to the seeds dramatically decreased. On the other hand, u n d e r excess availability of sulphate, uptake is repressed by cellular regulation (Hawkesford, 1999). Although early studies indicated a negative correlation between soil nitrogen levels an d glucosinolate levels in the plant, more recent work has suggested t h a t increased glucosinolate levels are likely provided the s u l p h u r supply is ad e q u at e (Zhao et al., 1993). Soil m i c r o n u t r i e n t s are important in determining the glucosinolate content of brassicas. Low levels of boron result in increased a m o u n t s of glucosinolates whereas zinc has a positive effect. Post-harvest t r e a t m e n t s affect the glucosinolate content of and in particular any process involving a loss of cellular integrity will result in myrosin a s e - i n d u c e d glucosinolate breakdown. These processes clearly include cutting or shredding as in the preparation of coleslaw. Myrosinase activity increases at t e m p e r a t u r e s up to 60 ~ and heating at 100 ~ is necessary to effect d e n a t u r a t i o n (Bj6rkman and L6nnerdal, 1973). Thus, an inadequate blanching prior to freezing will result in loss of glucosinolates on thawing. Correctly blanched material will store well at t e m p e r a t u r e s below 18 ~ but long-term storage of fresh material at chill t e m p e r a t u r e s results in lower levels of glucosinolates. Cooking B r a s s i c a vegetables in water reduces glucosinolate content partly due to the effect of heat and as a consequence of leaching into the cooking water.
339
Biological effects Although interest in the role of glucosinolates as flavor precursors prompted m a n y early studies, the m a i n t h r u s t of glucosinolate research s t e m m e d from the negative effect these c o m p o u n d s and their derivatives had on animals with a diet t h a t included high-glucosinolate rapeseed meal. The development of analytical methodology together with the isolation of a range of naturally occurring glucosinolates was an essential prerequisite for such studies and facilitated research into other areas, the m o s t i m p o r t a n t of which are the effect of glucosinolates on plant pests a n d diseases and the role of these c o m p o u n d s in the prevention of cancer. The flavor of B r a s s i c a vegetables is complex and a l t h o u g h glucosinolates p e r s e contribute little to flavor, there is no d o u b t t h a t their hydrolytic breakdown p r o d u c t s have an i m p o r t a n t role together with other s u l p h u r containing c o m p o u n d s . Glucosinolate-derived isothiocyanates have important flavor characteristics having been described as p u n g e n t , acrid, garliclike and horseradish-like (Fenwick et al., 1983b). Other descriptions include lachrymatory a n d bitter a n d t h u s it is p e r h a p s surprising t h a t the right balance of these properties contributes to the valued overall flavor of B r a s s i ca vegetables. In fact when levels of isothiocyanates are low, a flat and flavorless p r o d u c t results. However, excessive bitterness is undesirable and may result from the b r e a k d o w n of 2-hydroxybut-3-enyl glucosinolate to the goitrogen, 5-vinyloxazolidine-2-thione (goitrin). The bitterness score of Brussels s p r o u ts correlated well with the goitrin content and careful selection of breeding lines could result in cultivars containing reduced levels of this comp o u n d (Fenwick et al., 1983b). Extraction of the oil from high glucosinolate rapeseed results in a seed meal which c o n t a i n s all of the original glucosinolates (some of which may be partly degraded) a n d m o s t of the original m yrosi nase activity, factors which may be i m p o r t a n t in determining the acceptability of animal diets in which it h a s been included. More i m p o r t a n t however, are other physiological consequences of diets containing high glucosinolate rapeseed meal, problems which led eventually to the development of very low glucosinolate varieties of rapeseed. C o m p a r i s o n s of the m a n y studies on the nutritional value of rapeseed meal have been complicated by m a n y variables, not least the species, sex and age of the ani m al s studied (Mawson et al., 1993, 1994a, 1994b; Bell 1993). Nutrient levels and energy content of diets have often been insufficiently described and rapeseed processing conditions were variable. However, feeding trials comparing the use of either high or low glucosinolate meal show t h a t the problems encount er ed as a result of the inclusion of high glucosinolate meal include reduced feed intake, enlarged thyroids, reduced levels of circulating thyroid hormones, abnormalities in the liver and kidneys and reduced growth and reproductive performance. Studies have shown t h a t some of these problems can be attributed directly to the glucosinolates or
340 their breakdown products, several of which are goitrogenic. The antithyroid effects of oxazolidinethiones and nitriles are irreversible and have been demonstrated in experimental animals, in pigs and in poultry. Ruminants appear to be less affected, possibly due to microbial breakdown of precursor compounds in the gut. Liver enlargement in rats fed on B r a s s i c a based diets is reduced when glucosinolates are removed. Glucosinolates and their breakdown products have been found to have a profound effect on the susceptibility of the family B r a s s i c a c e a e to attacks by insects, mites, microorganisms, viruses and fungal pathogens (see Rosa et al., 1997). Although glucosinolate-derived compounds are considered to be part of the plant's defensive m e c h a n i s m against generalized consumers, including mammals, birds, insects, bacteria and fungi, they are not absolute in their effectiveness as a defense against all nonadapted feeders and they also stimulate feeding and oviposition in adapted insects (see Rosa et al., 1997). Their biological activity is dependant upon the n a t u r e of the side-chain, the concentration of the glucosinolate and the type of insect. A recent review addresses such differences (Louda and Mole, 1991). Crucifers have been suggested to interfere with fungal, bacteria and nematode populations, a property that has been related to the pattern and level of glucosinolates. The antifungal and antibacterial activity of glucosinolates and their breakdown products, particularly the isothiocyanates, has been reviewed by Rosa et al. (1997). Isothiocyanates, such as allyl- and p-hydroxybenzyl isothiocyantes, seem to exert a good control over plant infection due to certain types of microorganisms, fungi and plant viruses but the particular involvement of glucosinolates in plant resistance to diseases needs further clarification. Soil-borne diseases and nematode populations might be decreased by a m e n d m e n t s with the green tissue of some B r a s s i c a species or by certain glucosinolate derivatives, particularly allyl- and 2-phenylethyl isothiocyanates (see Rosa and Rodrigues, in press; Pinto et al., 1998; Kirkegaard et at., 1998; Potter et al., 1998). To achieve a better soil fumigant effect, B r a s s i c a crops with high concentration of fungicidal volatile compounds, m u s t be used particularly those high in allyl isothiocyanate, which has been reported to be as toxic to fungi as the commercially available methyl isothiocyanate (Lewis and Papavizas, 1971; Vaughn et at., 1993; Mayton et at., 1996). During the last decade there has been increased interest in the potential use of B r a s s i c a c e a e as bioherbicides due to the release of volatile allelochemicals (Brown and Morra, 1995; Vaughn and Boydston, 1997). Epidemiological evidence has led to the recommendation that, in order to limit the risk of cancer, people should eat several portions of fruit and vegetables each day and particularly B r a s s i c a (Verhoeven et al., 1996) The benefits of increased dietary fibre intake are well known and recent studies have provided strong evidence that brassicas in general and glucosinolates
341 in particular have a further role in the prevention of cancer, due to the modification of endogenous detoxification processes (see Rosa et a. 1997). The foundations for this i m p o r t a n t area of study were laid by Wattenberg and coworkers who, in an early study showed e n h a n c e d benzo(a)pyrene activity in rats fed diets containing B r a s s i c a vegetables (Wattenberg, 1993). These workers postulated a classification of anticarcinogens to include c o m p o u n d s which act either by preventing the formation of carcinogens from their precursors or by blocking the action of a carcinogen a n d s u p p r e s s i n g agents which prevent the progression of initiated cells to fully transformed cells. Metabolic detoxification of carcinogens h a s been described as a three stage process which may involve activation of the carcinogen by oxidation (phase I) followed by conjugation to a more polar state (phase II) t h u s facilitating eventual excretion a n d finally, t r a n s p o r t out of the cell (phase III). B r a s s i c a diets have been shown to increase the oxidative metabolism (phase I) of phenacetin a n d antipyrine in h u m a n s a n d later to facilitate glucuronide conjugation (phase II) of paracetamol (Johnson et al., 1995). A procedure h a s been developed for screening plant extracts and pure c o m p o u n d s for their ability to induce p h a s e II enzymes (Prochaska et al., 1992). The test m e a s u r e s increased activity of quinone r e d u c t a s e (an enzyme which is coordinately induced with other phase II enzymes) in m o u s e hepatoma cells a n d the use of microtitre plates permits the screening of large n u m b e r s of plant extracts. B r a s s i c a plants and mainly broccoli, were found to be effective inducers a n d in particular, s u l p h o r a p h a n e , a sulphinyl-containing isothiocyanate was identified as a major inducer of quinone reductase and glutathione S-transferase activity in m o u s e cells (Zhang et al., 1992, 1994). Broccoli hybrids have been produced with higher levels of the p a r e n t glucosinolate (4-methylsulphynilbutyl) (Faulkner et al., 1998). Subsequent studies have shown other B r a s s i c a extracts to be effective inducers in this assay (Tawfiq et al., 1994) as well as h u m a n diets including Brussels sprouts (Nijhoff et al., 1995). A reduction in the incidence, multiplicity and rate of m a m m a r y t u m o r s in rats confirms the beneficial effects of broccoli extracts in the diets (Fahey et al., 1997). Glucosinolate-derived indole c o m p o u n d s (particularly indole-3-carbinol) have been found to be very potent inducers of hepatic a n d intestinal enzymes in rats (see Rosa et al., 1997). N u m e r o u s studies have d e m o n s t r a t e d the inhibitory effect of indole c o m p o u n d s on t u m o r formation in animals challenged with various carcinogens (see Rosa et al., 1997). However, despite the a p p a r e n t benefits afforded by dietary indole c o m p o u n d s , it a p p e a r s that timing is important. Indole-3-carbinol administered to rainbow trout after challenge with aflatoxin B 1, actually e n h a n c e d carcinogenesis w h e n compared to indole-3-carbinol given before or during exposure to the carcinogen. Similarly, the inclusion of indole-3-carbinol in a wheat b r a n diet containing cholesterol a n d tallow, e n h a n c e d the tumorigenic effect of dimethylhydrazine, indicating the possible synergistic effects of dietary components.
342 Crucifer isothiocyanates have been described as exerting an anticarcinogenic effect via a detoxification process involving induction of phase I and p h a s e II enzymes (Benson and Barretto, 1985; Zhang et al., 1992; Zhang and Talalay, 19994). However, in other studies the same c o m p o u n d s appeared to have a contradictory effect (Musk a n d J o h n s o n , 1993). F u r t h e r studies are needed to clarify these aspects. In the h u m a n diet brassicas are eaten fresh, cooked or used as condim e n t s (Brassica or Sinapis m u s t a r d s a n d horseradish, Armoracia) on the basis of their organoleptic properties. Isothiocyanates are responsible for the "hot" flavor of brassicas and have been described as pungent, lachrymatory, acrid, garlic-like, horseradish-like, a n d bitter (Fenwick et al., 1983b), the allyl-2-hydroxybut-3-enyl-, but-3-enyl- a n d 4-methylthiobut-3-enyl- iso-thiocyanates being predominant. Thus, plant glucosinolate pattern and concentration are responsible, probably together with other compounds, for the organoleptic properties of this group of plants and for their potential effects. Browning in most Brassica p r o d u c t s is a major problem, particularly after shredding. Allyl isothiocyanate h a s been shown to be closely involved in the s u p p r e s s i o n of the induction of enzymes responsible for this problem as well as for ethylene synthesis triggered by wounding (Nagata 1996).
Other c o m p o u n d s Flavonoids Plant flavonoids a p a r t from being i m p o r t a n t as taxonomic and phylo-genetic m a r k e r s which help to clarify systematic and evolutionary problems (Aguinagalde, 1988; Aguinagalde et al., 1992; Aguinagalde and G6mez-Campo, 1984) are also part of the h u m a n diet and are considered to have positive health effects (Bridle a n d Timberlake, 1997). Epidemiological studies have shown a correlation between c o n s u m p t i o n of high levels of flavonoid-containing fruits a n d vegetables and reduced risks for coronary heart disease (Nielsen et al., 1997). Flavonoids are part of the chemical composition of Brassicaceae a n d a total of 21 have been identified in 14 Crambe species (Aguinagalde a n d G6mez-Campo, 1984) whilst a group of 19 of these compounds, some acylated, were reported for B. nigra, B. oleracea and B. rapa (Aguinagalde, 1988). Flavonoid composition h a s also been reported for several other wild species (B. incana, B. rupestris, B. montana, B. drepanensis, B. bourgeaui, B. alboglabra, B. macrocarpa, a n d B. cretica) (Aguinagalde et al., 1992) and for other Brassica, Erucastrum a n d Diplotaxis species (S&nchez-Y61amo, 1992, 1994). Table 10.7 s u m m a r i z e s these investigations. In turn, Broccoli h a s been reported as a good source of the anti-carcinogenics kaempferol and quercetin with an average of 2.5 a n d 1.8 mg per 100 g fresh weight, respectively (Nielsen et al., 1997). Since these two c o m p o u n d s appeared to have a different metabolism in h u m a n microsomes, further studies are required to elucidate this process.
343 Table 10.7 Flavonoids identified in the genera Brassica, Erucastrum and Diplotaxis (Aguinagalde, 1988; Aguinagalde et al., 1992; S~inchezY61amo, 1992, 1994). Isorhamnetin 3- sophoroside
Kaempferol 7-diglucoside
Isorhamnetin 3- sophoroside 7-glucoside
Kaempferol 7-glucoside
Isorhamnetin 3, 7-diglucosid
Kaempferol 7-galactoside
Isorhamnetin 3-glucoside
Kaempferol 7-galactoside 3-digalactoside
Isorhamnetin 3-galactoside 7- rhamnoside
Quercetin 3- sophoroside
Isorhamnetin triglycoside (rhamnose
Quercetin 3- sophoroside 7-glucoside
+ galactose)
Quercetin 3, 7-diglucoside
Isorhamnetin 3-digalactoside
Quercetin 3-gentiobioside
Kaempferol 3-digalactoside
Quercetin 3-glucoside
Kaempferol 3-digalactoside 7-rhamnoside
Quercetin 3-diglucoside
Kaempferol 3- gentiobioside
Quercetin 3-glucoside 7-rhamnoside
Kaempferol 3, 7-diglucoside
Quercetin 3-digalactoside
Kaempferol 3-feruloyl- sophoroside
Quercetin 7-glucoside
Kaempferol 3-glucoside
Quercetin 7-galactoside
Kaempferol 3-sinapoyl- sophoroside
Quercetin 3-sinapoyl- sophoroside
Kaempferol 3-sophoroside
Quercetin 7-diglucoside
Kaempferol 3-sophoroside 7-glucoside
Quercetin 7-glucoside
Anthocyanins are a group of flavonoids which have lately received renewed interest for natural pigment extraction due to public concern on the safety of synthetic colorants. In Brassica oleracea more than 15 anthocyanins have been separated and four fully characterized (Idaka, 1987, 1988 cited by Bridle and Timberlake, 1997). These are based on Cy 3,5-diglucoside, and Cy 3-diglucoside-5-glucoside acylated at the 3- position with ferulic, p-coumaric and sinapic acids. Within this botanical group, red cabbage has been commercially used as one of the major sources of anthocyanin food colorants (Bridle and Timberlake, 1997). Flavonoids also play a role in hostplant interactions as observed with the monarch butterfly (Danaus plexippus) (Baur et al., 1998) and red cabbage would be expected to stimulate oviposition more than other Brassica varieties. The flavonoid composition might
344 be changed by environmental factors such as high UV radiation (290-320 nm) as recently shown in rape (B. napus) (Greenberg et al., 1996).
S-methylcysteine sulfoxide (SMCSO) Another class of o r g a n o s u l p h u r compounds is the non-protein m-amino acid (+)-S-methyl-L-cysteine sulphoxide (SMCSO) and its enzymatic by-product, methyl methanethiosulphinate (MMTSO). The SMCSO was discovered to occur naturally in B r a s s i c a in 1956 by two independent research groups (Synge and Wood, 1956; Morris and Thompson, 1956). After plant tissue disruption, the SMCSO is hydrolyzed by cystine lyase (EC 4.4.1.8), an enzyme with a similar behaviour to that of another present in garlic (alliinase- EC 4.4.1.4) to produce pyruvate, a m m o n i a and suspected allyl thiosulphinates. Marks et al. (1992) showed, u n d e r natural conditions, the enzymatic production of methyl methanethiosulphinate as well as dimethyl trisulphide from SMCSO. Production of the first compound was later confirmed by Nakamura et al. (1996) who report a marked influence of pH on the a m o u n t produced. SMCSO can be found in concentrations ranging between 1 and 2% on a dry weight basis with considerable intraseasonal variations (Mae et al., 1971; Whittle et al., 1976; Maw, 1982; Griffiths and Macfarlane Smith, 1989). According to the last authors no significant differences can be found between the SMCSO levels of leaves and stems of forage rape (B. napus). Brussels sprouts were found to have the highest levels (68.0 mg / 1 0 0 g fresh weight) when compared to broccoli, cabbage, and cauliflower which have 19.1, 18.5 and 14.3 m g / 1 0 0 g fresh weight, respectively (Marks et al., 1992). Apart from being involved in the aroma and flavor of these vegetables SMCSO appears to be able to inhibit carcinogenesis (Marks et al., 1992). In garlic this compound exhibited a weaker andiabetic effect (Sheela et al., 1995; Kumari and Augusti, 1995). In forage B r a s s i c a the metabolites, methanethiol and dimethyl disulphide are generally produced by ruminal fermentation, and are implicated in the reduction of food intake and in the m e c h a n i s m s of oxidation, denaturation and precipitation of haemoglobin, with the destruction of erythrocytes and the formation of Heinz bodies (Prache, 1994; Helclova, 1996). Apart from a rise in Heinz bodies, no other significant effects were observed in sheep receiving diets to which dimethyl disulphide had been added and, on the contrary, a reduction was noted in the depression of feed intake caused by allyl cyanide, a breakdown product from sinigrin (Duncan and Milne, 1993).
Phytates and others Phytic acid, myo-inositol 1,2,3,4,5,6-hexakis-dihydrogen phosphate is the primary phytic compound, but it also occurs as mono-, di-, tri-, tetra-, and p e n t a p h o s p h a t e s of inositol. The phytic acid content in whole rapeseed is between 2.0 and 4.0% whilst in the defatted meal it tends to be slightly
345 h i g h e r (2.0-5.0%) a n d even h i g h e r in the protein c o n c e n t r a t e s (5.0 a n d 7.5%) (Thompson, 1990). A d e c r e a s e in phytic acid in the seeds m i g h t be achieved by imbibition for a 2 4 h period a n d by g e r m i n a t i o n d u e to a n i n c r e a s e in the e n z y m e p h y t a s e ( m y o i n o s i t o l - h e x a p h o s p h a t e p h o s p h o h y d r o l a s e , EC 3.1.3.8.) levels a n d activity (Mahajan a n d Dua, 1997). In r a p e s e e d meal it is found in free form or a s s o c i a t e d with proteins a n d p o l y m e r s i n d u c i n g a r e d u c t i o n in protein digestability (Bjergegaard et al., 1998). It h a s b e e n c o n s i d e r e d as a n a n t i n u t r i o n a l c o m p o u n d d u e to its ability to bind divalent c a t i o n s b u t also due to the low bioavailability of the p h o s p h o r o u s p r e s e n t in the c o m p o u n d (Rickard a n d T h o m p s o n , 1997). A mixed salt of phytic acid a n d cations is formed w h e n several cations complex within the s a m e phytic acid molecule. The p r e s e n c e of more t h a n one cation m a y i n c r e a s e synergistically the precipitation of the p h y t a t e salts as h a s been observed in vitro with Zn 2§ a n d Ca 2+ or Zn 2+ a n d Cu 2+. According to Rickard a n d T h o m p s o n (1997), high doses of phytic acid m a y be tolerated by h u m a n s who c o n s u m e excess calories a n d n u t r i e n t s , t h u s benefiting from its hypoglycemic, hypolipidemic a n d a n t i c a n cer effects Proteinase inhibitors, are also found in B r a s s i c a c e a e a l t h o u g h in lower a m o u n t s t h a n in o t h e r families (Poaceae, F a b a c e a e a n d Solanaceae). However, Nielsen et al. (1996) reported levels of t r y p s i n inhibitors in r a p e s e e d to be similar to t h o s e found in peas. Aromatic choline e s t e r s are a g r o u p of c o m p o u n d s w h i c h belong to the t a n n i n pool, i.e. related to gallic acid. S i n a p i n e is the d o m i n a n t c o m p o u n d in r a p e s e e d being r e s p o n s i b l e for the off flavor in m e a t from bulls (Fenwick et al., 1983a). Phenols have been referred as m i n o r a n t i n u t r i t i o n a l c o m p o u n d s in the Crucifers (Mahajan a n d Dua, 1997). Levels in seeds of r a p e s e e d (Brassica rapa s u b s p , c a m p e s t r i s var. oleifera) were u n d e r 1%, being r e d u c e d by seed imbibition a n d germination. For i n s t a n c e the c i n n a m i c acid derivatives associated to the dietary fibre fraction in the seed of B. n a p u s were b e t w e e n 390 a n d 1190 n m o l e s / g being u p to 2 0 0 0 n m o l e s / g in the protein rich meal (Anderson et al., 1998). Levels in the r a p e s e e d hulls were b e t w e e n 850 a n d 1270 n m o l e s / g whilst in the lipoprotein fraction t h e y were b e t w e e n 160 a n d 1240 n m o l e s / g . F u r t h e r a n a l y s i s revealed the p r e s e n c e of v a r i o u s sinapoyl derivatives as e. g. sinapoyl e s t e r s of choline, m a l a t e a n d c a r b o h y d r a t e s (And e r s o n et al., 1998). Several B r a s s i c a species are c h a r a c t e r i z e d by a relatively thick epicutic u l a r wax the composition of which h a s been s h o w n to vary b e t w e e n species as well as b e t w e e n varieties a n d g e n o t y p e s of the s a m e species ( S h e p h e r d et al., 1995). These a u t h o r s r e p o r t e d the wax m o r p h o l o g y a n d c o m p o s i t i o n to be d e p e n d e n t on the e n v i r o n m e n t a l conditions in addition to a role for the h o s t - p l a n t recognition for certain insects a n d the r e s i s t a n c e to d r o u g h t a n d diseases.
346
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357 G.R. (ed.). Food a n d cancer prevention: chemical a n d biological aspects, Royal Soc. Chem. Cambridge. pp. 12-23. Whittle, P. J., Smith, R. H. and Mclntosh, A. 1976. Estimation of S-methylL-cysteine sulfoxide (kale anaemia factor) and its distribution among Brassica forage and root crops. J. Sci. Food Agric. 27, 633642. Zhang, Y. and Talalay, P. 1994. Anticarcinogenic activities of organic isothiocyanates: chemistry and mechanisms. Cancer Res. 54 (Suppl.), pp. 197-198. Zhang, Y., Talalay, P., Cho, C.-G. and Posner, G. H. 1992. A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure. Proc. Nat. Acad. Sci. USA 89, 2399-2403. Zhang, Y., Kensler, T. W., Cho, C. G., Posner, G. H. and Talalay, P. 1994. Anticarcinogenic activities of sulforaphane and structurally related synthetic norbornyl isothiocyanates. Proc. Nat. Acad. Sci. USA 91, 3147-3150. Zhao, F., Evans, E. J., Bilsborrow, P. E. and Syers, J. K. 1993 Influence of sulphur and nitrogen on seed yield and quality of low glucosinolate oilseed rape (Brassica n a p u s L.). J. Sci. Food Agr. 63, 29-37.
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Biology of Brassica Coenospecies C. G6mez-Campo (Editor) 91999 Elsevier Science B.V. All rights reserved.
359
II PHYSIOLOGY Paul Hadley a n d Simon P e a r s o n
Department of Horticultural and Landscape. The University of Reading. Reading RG6 2AS. United Kingdom. To review the whole a r e a of the physiology of the Brassica coenospecies - e v e n of the g e n u s B r a s s i c a - would be b e y o n d the scope of a single chapter. In this c h a p t e r , we therefore have limited ourselves to reviewing o u r p r e s e n t knowledge of the physiology of Brassica crops with e m p h a s i s being m a d e on the effects of e n v i r o n m e n t on vegetative a n d reproductive d e v e l o p m e n t in particular. M u c h of this c h a p t e r will c o n c e n t r a t e on the physiology of Brassica oleracea, b u t where a p p r o p r i a t e o t h e r Brassica species will be d i s c u s s e d . F u r t h e r r e a d i n g on o t h e r species c a n be found in B h a r g a v a (1996). Particularly for Brassica crops of h o r t i c u l t u r a l i m p o r t a n c e , there is a n i n c r e a s e d r e q u i r e m e n t to s c h e d u l e crops a c c u r a t e l y to m e e t the i n c r e a s i n g d e m a n d s of the fresh m a r k e t for specific v o l u m e s of p r o d u c e at p a r t i c u l a r times. The time from sowing to h a r v e s t d e p e n d s to a large e x t e n t on the variation in t e m p e r a t u r e a n d also on light conditions. In addition, crop d u r a tion in m a n y Brassica is d e p e n d e n t on the t r a n s i t i o n from vegetative to reproductive development, a n d this is p a r t i c u l a r l y sensitive to variation in temp e r a t u r e . Moreover, the s e a s o n for growing m a n y Brassica c r o p s is being e x t e n d e d so t h a t , for example, crops are often grown u n d e r conditions where the incidence of bolting is large. A clear u n d e r s t a n d i n g of the effects of envir o n m e n t on vegetative a n d reproductive growth is therefore e s s e n t i a l if these crops are to be p r o d u c e d reliably a n d predictably. The growth of B. oleracea p r o g r e s s e s t h r o u g h a series of well defined dev e l o p m e n t a l s t a g e s b e g i n n i n g with g e r m i n a t i o n , vegetative growth, r e p r o d u c tive growth a n d finally crop m a t u r i t y . We will e x a m i n e e a c h of t h e s e stages highlighting the m a i n e n v i r o n m e n t a l f e a t u r e s t h a t d e t e r m i n e p l a n t response.
Germination G e r m i n a t i o n in Brassica is epigeal a n d the time from sowing to emergence is largely d e t e r m i n e d by t e m p e r a t u r e so that, for e x a m p l e for cauliflower, a c c o r d i n g to Wagenvort et al. (1974) the rate of p r o g r e s s to germina-
360 tion i n c r e a s e s linearly with t e m p e r a t u r e from a b a s e t e m p e r a t u r e of approximately 4.50C (reflecting the t e m p e r a t e origin of this species) u p to an optim u m a n d t h e n p r e s u m a b l y declines with f u r t h e r i n c r e a s e s in t e m p e r a t u r e , a l t h o u g h this h a s not been clearly d e t e r m i n e d for b r a s s i c a s . S u c h a linear r e l a t i o n s h i p indicates t h a t g e r m i n a t i o n occurs after the a c c u m u l a t i o n of a c o n s t a n t t h e r m a l time (Hadley et al., 1983). D a t a p r e s e n t e d by Wagenvort et al. (1974) indicate t h a t g e r m i n a t i o n in cauliflower o c c u r s after the a c c u m u lation of a p p r o x i m a t e l y 1000 degree h o u r s above a base t e m p e r a t u r e of 4.5 ~ C a n d requires a f u r t h e r 500 degree h o u r s for 90% of the seed to germinate. T h u s at 5.5 ~ C g e r m i n a t i o n will begin after 40 d a y s a n d 90% germination will be achieved after a f u r t h e r 20 days, w h e r e a s at 10oC this is reduced to a time from sowing to g e r m i n a t i o n of 9 d a y s a n d a s p r e a d of germination of 4 days. This c o n t r a s t s with the g e r m i n a t i o n of crop species within the family Umbelliferae where g e r m i n a t i o n a p p e a r s to be m u c h slower, for example, g e r m i n a t i o n in celery requires a t h e r m a l time a c c u m u l a t i o n of 2500 degree h o u r s above a base t e m p e r a t u r e of 2.5 - 4 ~ C a n d 50% g e r m i n a t i o n is achieved after the a c c u m u l a t i o n of 5 0 0 0 degree h o u r s . More recently Vigil et al. (1997) indicated t h a t emergence of spring canola (Brassica napus) occurred after the a c c u m u l a t i o n of 1 5 6 0 - 1 9 4 0 degree h o u r s a n d winter canola after 1 6 0 0 - 2 8 0 0 h o u r s b o t h above a b a s e t e m p e r a t u r e of 0.4 - 1.2oC. Squire (in press) indicates a n o n - l i n e a r r e s p o n s e in the rate of g e r m i n a t i o n of oil seed rape s u c h t h a t e m e r g e n c e at low t e m p e r a t u r e s was faster t h a n would be predicted from a linear r e s p o n s e suggesting a lower base t e m p e r a t u r e of a b o u t -loC. Later g e r m i n a t i n g seeds of some rape cultivars c a n enter second a r y d o r m a n c y u n d e r cool c o n d i t i o n s (Squire, in press). This condition is also s t i m u l a t e d by w a t e r stress , d a r k n e s s a n d far-red light (Pekrun et al., 1996; L 6 p e z - G r a n a d o s a n d L u t m a n , 1998). This i n d u c e d d o r m a n c y can be b r o k e n by e x p o s u r e to higher t e m p e r a t u r e s or large d i u r n a l variation in t e m p e r a t u r e . T h u s , early cohorts of a u t u m n sown rape will germinate quickly whilst later c o h o r t s m a y e n t e r s e c o n d a r y d o r m a n c y a n d t h e n not germinate until spring t e m p e r a t u r e s rise sufficiently to allow g e r m i n a t i o n to occur leading to a second flush of seedlings.
Vegetative growth D u r i n g the vegetative phase, the plant is c h a r a c t e r i s e d by a small apex s u r r o u n d e d by small leaf p r i m o r d i a which arise acropetally in a spiral succession a r o u n d the s h o o t apex. J u v e n i l e leaves are, in general, simpler in contour. For cauliflower, leaf p r o d u c t i o n rate i n c r e a s e s a p p r o x i m a t e l y linearly with i n c r e a s e d t e m p e r a t u r e with a n o p t i m u m in excess of 20 ~ C and falling to zero at a p p r o x i m a t e l y 6 ~ C (Wiebe, 1972). T h u s , leaf p r o d u c t i o n inc r e a s e s by 0.05 leaves per day per "C above 6 ~ C. A similar s t u d y on s u m m e r rape (Morrison a n d McValty, 1999) also showed a linear relationship between leaf p r o d u c t i o n a n d t e m p e r a t u r e so t h a t leaf p r o d u c t i o n increases by 0.02 leaves per day per "C.
361 Leaf p h o t o s y n t h e s i s in B r a s s i c a species a p p e a r s to be typical of m o s t o t h e r C3 species. Paul et al. (1990) s t u d y i n g p h o t o s y n t h e t i c activity in oilseed rape (B. n a p u s L.) showed t h a t w h e n p l a n t s were grown at 13 ~ C m a x i m a l values of leaf p h o t o s y n t h e t i c rate were a p p r o x i m a t e l y 29 ~moles CO2 m -2 s -1 a n d o c c u r r e d at a t e m p e r a t u r e of 22 ~ C. Plants grown at 30 ~ C h a d lower r a t e s of p h o t o s y n t h e s i s , whilst o p t i m u m r a t e s were recorded at t e m p e r a t u r e s of a p p r o x i m a t e l y 31" C. J e n s e n et al. (1996) showed t h a t in oil seed r a p e during the early growth stages light s a t u r a t e d p h o t o s y n t h e t i c rate w a s 3 5 - 4 5 ~moles CO2 m -2 s -1 a n d s t o m a t a l r e s i s t a n c e w a s 1-1.5 moles m -2 s -1 a n d both varied linearly with leaf nitrogen content. P h o t o s y n t h e t i c rate a p p e a r e d to vary significantly a m o n g s t 39 B r a s s i c a t a x a a n d related species of the coenospecies as s t u d i e d by S u r a c h et al. (1997). A m o n g s t t h e s e species there w a s a significant negative correlation b e t w e e n net p h o t o s y n t h e t i c rate a n d s t o m a t a l r e s i s t a n c e b u t a significant positive correlation b e t w e e n net photos y n t h e s i s a n d chlorophyll c o n t e n t a n d specific leaf weight. The latter suggests t h a t t h e r e is good potential within the g e n u s to select for high phot o s y n t h e t i c potential. Yields of m o s t B r a s s i c a species are i n c r e a s e d w h e n p h o t o s y n t h e t i c rate is i n c r e a s e d artificially by growing u n d e r elevated CO2 c o n c e n t r a t i o n s This fact indicates the potential benefit of i n c r e a s e d phot o s y n t h e s i s by this method.
The t r a n s i t i o n from v e g e t a t i v e to reproductive development Flower initiation is a critical stage d u r i n g the d e v e l o p m e n t of m o s t commercial B r a s s i c a crops. In the case of c a l a b r e s e a n d cauliflower, flower initiation m a r k s the beginning of c u r d / s p e a r development. Variation in c u r d / s p e a r d e v e l o p m e n t will u l t i m a t e l y lead to similar v a r i a t i o n s in h a r v e s t time. However, for c a b b a g e a n d B r u s s e l s s p r o u t s , flower initiation m a r k s the o n s e t of bolting, a condition w h i c h r e n d e r s the crop u n m a r k e t a b l e . Flower initiation is also critical to oilleed r a p e (B. n a p u s or B. rapa) w h e r e early flowering m a y lead to injury of reproductive o r g a n s by low t e m p e r a t u r e s a n d conseq u e n t i a l fruit loss. Flower initiation is a c c o m p a n i e d by a g r a d u a l b r o a d e n i n g of the apex to form a clearly d o m e d s t r u c t u r e a p p r o x i m a t e l y 0.6 m m in diameter. In c a u liflower, w h e r e apical d e v e l o p m e n t h a s b e e n s t u d i e d in detail, apical enlargem e n t o c c u r s rapidly at t e m p e r a t u r e s of a p p r o x i m a t e l y 11 ~C b u t this d e c r e a s es with i n c r e a s e s in t e m p e r a t u r e so t h a t above 22 ~ C the width of the apex r e m a i n s c o n s t a n t or even s h r i n k s (Wiebe, 1972c; N o w b u t h a n d Pearson, 1998). Flowering will only take place after the p l a n t h a s p a s s e d t h r o u g h a juvenile phase. The length of the juvenile period a p p e a r s to vary greatly b e t w e e n different B r a s s i c a types a n d b e t w e e n cultivars of similar B r a s s i c a types. Indeed, a l t h o u g h Stokes a n d V e r k e r k (1951) a n d T h o m a s (1980) in B r u s s e l s s p r o u t s a n d Wiebe (1972) in k o h l r a b i found t h a t g e r m i n a t i n g seeds could
362 n o t be v e r n a l i s e d , i n d i c a t i n g a j u v e n i l e period, N a k a r w r a a n d Hattori (1961) in c a b b a g e a n d F u j i m e a n d Hirose (1979) in cauliflower i n d i c a t e t h a t vernalising the seed c a n lead to a r e d u c t i o n in leaf n u m b e r s u g g e s t i n g t h a t a juvenile period m a y n o t exist. The l e n g t h of t h e j u v e n i l e p h a s e c a n be e v a l u a t e d by t r a n s f e r r i n g p l a n t s from w a r m (less inductive) to cool (inductive) t e m p e r a t u r e s a t intervals before r e t u r n i n g t h e m a g a i n to w a r m t e m p e r a t u r e s (Stokes a n d Verkerk, 1951; Sadik, 1967; H a n d a n d A t h e r t o n , 1987). If the p l a n t h a s p a s s e d t h r o u g h the j u v e n i l e p h a s e , leaf n u m b e r below t h e a p e x d e c l i n e s on e x p o s u r e to cool t e m p e r a t u r e c o n d i t i o n s . However, p l a n t s still in the j u v e n i l e p h a s e flower a t the s a m e leaf n u m b e r a s p l a n t s g r o w n c o n t i n u o u s l y in w a r m conditions. Bec a u s e cool t e m p e r a t u r e s r e d u c e t h e r a t e of d e v e l o p m e n t , u s e of leaf n u m b e r s a s a m e a s u r e of p l a n t d e v e l o p m e n t h a s b e e n c o n s i d e r e d to be m o r e satisfactory t h a n the u s e of c h r o n o l o g i c a l time. Following this a p p r o a c h , H a n d a n d A t h e r t o n (1987) e x p o s e d cauliflowers to a four w e e k period at 5 ~ C a t v a r i o u s p l a n t a g e s followed by r e t u r n i n g the p l a n t s to a w a r m g l a s s h o u s e (20 ~ C). U n d e r w a r m c o n d i t i o n s p l a n t s flowered after i n i t i a t i n g 43 leaves, b u t flowered a t a m i n i m u m of 17 leaves w h e n e x p o s e d to cool t e m p e r a t u r e c o n d i t i o n s s u g g e s t i n g t h a t the e n d of the juvenile period is a c h i e v e d after the p l a n t s h a v e formed 17 leaves so t h a t the opt i m u m t e m p e r a t u r e for flowering w a s close to 5 ~ C. However, Wiebe (1972) p r e s e n t s t h e r e s u l t s of a controlled e n v i r o n m e n t s t u d y , w h i c h clearly s h o w s t h a t the m i n i m u m t i m e to c u r d initiation o c c u r s a t a p p r o x i m a t e l y 16 ~ C. W u r r et al. (1996) also s h o w on o p t i m u m t e m p e r a t u r e of 14 ~ C for c a l a b r e s e . This a p p a r e n t d i s a g r e e m e n t in the l i t e r a t u r e m a y be d u e to a p o t e n t i a l flaw in t h e e x p e r i m e n t a l a p p r o a c h u s e d to d e t e r m i n e t h e e n d of the juvenile period. The d a t a of Wiebe (1972) s u g g e s t s t h a t the o p t i m u m t e m p e r a t u r e for the f a s t e s t time to c u r d initiation is a p p r o x i m a t e l y 14 to 16 ~ C, w h i c h is closer to the ' n o n - i n d u c t i v e ' t r e a t m e n t e m p l o y e d by H a n d a n d A t h e r t o n (1987) s t u d y . T h e y also m e a s u r e d the l e n g t h of the j u v e n i l e period in t e r m s of leaf n u m b e r below t h e curd. The r a t e of leaf initiation is also k n o w n to be a f u n c t i o n of t e m p e r a t u r e so t h a t c h a n g e s in leaf n u m b e r c a n be a t t r i b u t e d to e i t h e r a c h a n g e in t h e d u r a t i o n to c u r d initiation, w h e n leaf p r o d u c t i o n c e a s e s , or to the effect of t e m p e r a t u r e on the r a t e of leaf initiation. In their s t u d y , a low o p t i m u m t e m p e r a t u r e for c u r d initiation is likely to have b e e n b e c a u s e t h e r a t e of leaf initiation w a s m u c h slower t h a n the r a t e of p r o g r e s s to c u r d initiation a t 5 ~ C giving a lower n u m b e r of leaves initiated below the floral a p e x even t h o u g h p r o g r e s s to c u r d initiation w a s m u c h f a s t e r at w a r m er t e m p e r a t u r e s . The d a t a of Wiebe (1972) s h o w t h i s very clearly, since a l t h o u g h t h e m o s t r a p i d c u r d initiation o c c u r r e d a t 16 ~ C leaf n u m b e r below the a p e x r e a c h e d a m i n i m u m a t 7 ~ C a s leaf p r o d u c t i o n r a t e declined to zero a t a p p r o x i m a t e l y 6 ~ (Figure 11.1). The effects of t e m p e r a t u r e on p r o g r e s s to flower initiation c a n only be q u a n t i f i e d a c c u r a t e l y w h e n time to flower initiation is m e a s u r e d directly at a r a n g e of t e m p e r a t u r e s a n d n o t b a s e d on leaf n u m b e r s alone.
363
120
60
100 c o
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.E
,D
E
, , m
60-
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-2o
40a
200
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5
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15 20 Temperature
25
Figure 11.1 The effect of temperature on the time to curd initiation (e) and final leaf number (o) in cauliflower cv. Aristocrat, after Wiebe (1972).
A l t h o u g h t h e effects of t e m p e r a t u r e on c u r d i n i t i a t i o n a n d g r o w t h in s u m m e r a n d a u t u m n cauliflowers h a s b e e n s t u d i e d in s o m e detail, w i n t e r cauliflower h a s received less a t t e n t i o n . H a i n e (1959) s h o w e d t h a t w i n t e r c a u liflowers m a i n t a i n e d above 15.6 ~ C r e m a i n e d vegetative a n d t h a t cool conditions p r o g r e s s i v e l y r e d u c e d the t i m e to h e a d i n g . More recently, W u r r a n d Fellows (1998) h a v e s h o w n t h a t p r o g r e s s to c u r d i n i t i a t i o n a p p e a r e d to o c c u r m o s t r a p i d l y b e t w e e n 12 ~ a n d 14 ~ C a n d d e c l i n e d a t t e m p e r a t u r e s b o t h above a n d below this. T h i s is s i m i l a r to s u m m e r / a u t u m n cauliflower, w h e r e p r o g r e s s to c u r d i n i t i a t i o n is k n o w n to be q u i c k e s t b e t w e e n 9 ~ a n d 14 ~ C (Pearson et al., 1994, G r e v s e n a n d O l s s e n , 1994). The g e n u s B r a s s i c a a p p e a r s to h a v e e i t h e r a long d a y or d a y n e u t r a l flowering r e s p o n s e to p h o t o p e r i o d a n d a m o n g s t t h e s e c a b b a g e , B r u s s e l s s p r o u t s a n d k o h l - r a b i h a v e a d a y n e u t r a l r e s p o n s e (Friend, 1985). In cauliflower, S a d i k (1967) a n d Wiebe (1981) i n d i c a t e t h a t it is d a y n e u t r a l after its cool t e m p e r a t u r e r e q u i r e m e n t h a s b e e n satisfied. A more. r e c e n t s t u d y by T h a p a (1994) also s h o w e d no evidence for p h o t o p e r i o d s e n s i t i v i t y in t h r e e c o m m e r c i a l cauliflower varieties. However, closer i n s p e c t i o n of d a t a p r e s e n t -
364 ed by P a r k i n s o n (1952) indicates t h a t long days a d v a n c e d cutting date by 10 to 30 days d e p e n d i n g on cultivar. Therefore a l t h o u g h m o s t cultivars a p p e a r to be day n e u t r a l , there is still a q u e s t i o n w h e t h e r certain genotypes p o s s e s s some sensitivity to photoperiod. Low light also a p p e a r s to delay floral induction. Wiebe (1972a) showed t h a t cauliflower grown u n d e r low light conditions initiated more leaves before the curd t h a n p l a n t s grown u n d e r high light conditions. H a n d (1988) also observed a delay in cauliflower curd initiation with lower light levels, b u t only at relatively w a r m t e m p e r a t u r e s (20 ~ ). This h a s been confirmed in a more recent s t u d y by N o w b u t h a n d P e a r s o n (1998) where lower light levels progressively delayed the o n s e t of curd initiation, particularly u n d e r less inductive t e m p e r a t u r e conditions. T o m m e y a n d E v a n s (1991) s t u d i e d the effects of low t e m p e r a t u r e a n d d a y l e n g t h s on flower initiation in winter oilseed rape (Brassica napus L.) u n der controlled e n v i r o n m e n t conditions. Flower initiation occurred m o s t rapidly at t e m p e r a t u r e s of 6 ~ or 9 ~ C; t e m p e r a t u r e s higher a n d lower then lead to slower r a t e s of progress to flower initiation. At 12 ~ C, s h o r t days partially s u b s t i t u t e d for the cold requirement. However, photoperiodic induction appeared to be less i m p o r t a n t t h a n the influence of low t e m p e r a t u r e . Although flowering is p r o m o t e d by exposure to low t e m p e r a t u r e s d u r i n g the pre-floral growth p h a s e , e x p o s u r e to high t e m p e r a t u r e (30 ~ C) before or after exposure to low t e m p e r a t u r e s delay flowering in spring rape (B. n a p u s var. a n n u a L.) resulting in increased node n u m b e r to flowering ( D a h a n a y a k e and Galway, 1998). Similar delays or prevention of flower induction by exposure before or after the application of a period of low t e m p e r a t u r e which would otherwise promote flowering have been d e m o n s t r a t e d in Chinese cabbage (Brassica rapa s u b s p , pekinensis). (Eles a n d Wiebe, 1984) a n d Brassica oleracea var. gongylodes L. (Wiebe et al., 1992). The t e r m vernalisation a p p e a r s to be u s e d r a t h e r loosely in the published literature on flowering in Brassica species. In particular, the more recent genetic literature on Brassica often refer to m a p p i n g loci controlling vernalisation r e q u i r e m e n t in Brassica species (eg. Teutonio a n d Osham, 1995; O s h u m et al., 1997), w h e n physiologically it a p p e a r s t h a t there is no requirem e n t for cold t e m p e r a t u r e s to induce flowering a n d t h a t m a n y species simply have a cool t e m p e r a t u r e o p t i m u m for flower iniation. The classic definition of vernalisation refers to an inductive p h e n o m e n o n where floral development can only o c c u r after an initial exposure to cold t e m p e r a t u r e s in the order of 6 ~ C b u t t h a t initiation only occurs after the plants are exposed to a second e n v i r o n m e n t a l s t i m u l u s , often long days, implying an obligate response to cold t e m p e r a t u r e s . More specifically t h a t flower initiation does not occur u n til the p l a n t s are transferred to s e c o n d a r y inductive conditions. Although looser definitions of vernalisation exist (Bernier et al., 1981), for m a n y Brassica species, s u b s p e c i e s or varieties, the o p t i m u m t e m p e r a t u r e for flower induction a p p e a r s to be m u c h higher t h a n those normally associated with vernalisation r e s p o n s e s .
365
H o r m o n a l c o n t r o l o f flowering in B r a s s i c a There h a s been considerable interest in the fact that floral development in brassicas is controlled via endogenous growth hormones. Thomas et al. (1972) showed that the level of gibberellin-like s u b s t a n c e s increased about 2 weeks before curd initiation in cauliflower. This peak in hormone levels was increased when plants were exposed to cold, confirming a similar study by Kato (1965) in cauliflower and by Tortes et al. (1970) in broccoli. Exogenous application of a mixture of GA4 and GA7 applied at the end of the juvenile stage reduced the n u m b e r of leaves initiated before curd development indicating an a d v a n c e m e n t in the progress to curd initiation (Booij, 1989). This has recently been confirmed by Fernandez et al. (1997), who showed that u n d e r a weak curd-inducing temperature of 22 ~ C application of gibberellins reduced the n u m b e r of initiated leaves by 30% and curd initiation occurred one week earlier. Under strongly inducing conditions (10 ~ C) gibberellin application did not advance curd initiation, suggesting that gibberellin application advances curd initiation only u n d e r sub-optimal inducing conditions. Gibberellins have been implicated in the flowering process in other B r a s s i c a species. For example, Zanewich and Riod (1995) investigated the role of gibberellins in vernalised and non-vernalised winter canola ( B r a s s i c a napus). Following exposure to low temperatures, endogenous GA1, GAa, GAs, GA19 and GA20 levels were 3.1, 2.3, 7.8, 12 and 24.5 fold higher respectively than in unvernalised plants, demonstrating that exposure to low temperature inducing conditions influence GA content, with GAs serving as probable regulatory intermediaries between chilling treatment and s u b s e q u e n t development.
Progress to crop m a t u r i t y Curd growth following the initiation of the floral apex in both cauliflower and calabrese is strongly dependent on prevailing temperature. Salter(1969) showed a close linear relationship between the logarithm of curd size and thermal time indicating that the time to curd maturity is determined by accumulated temperature. The close relationship between curd growth and thermal time has subsequently been confirmed in other studies on cauliflower, for example Wurr et al. (1990) and Pearson et al. (1994), although the response over the full growth period appears to be curvilinear (Figure 11.2). Similar relationships have also been shown for calabrese (Pearson and Hadley, 1988; Wurr et al., 1991) although in this crop radiation had also been shown to modulate the rate of progress towards maturity (Marshall and Thompson, 1987; Wurr et al., 1991). A more detailed anlysis of curd growth has been provided by Pearson et al. (1994). From data collected u n d e r field conditions, there is a linear fall in the potential relative growth rate of the curd with thermal time from curd initiation so t h a t the m a x i m u m rate of curd growth occurred after curd initiation falling to zero at curd maturity. The second component describes the
366 i n s t a n t a n e o u s effect of t e m p e r a t u r e on curd growth rate in which relative curd growth rate increased linearly u p to an o p t i m u m of 16 ~ , 2 lo or 25 ~ C depending on cultivar a n d declined thereafter. The resulting model provided good a g r e e m e n t with a wide range of i n d e p e n d e n t dat a indicating that it could provide a basis for crop scheduling. For both cauliflower and calabrese, it is still difficult to predict flower initiation from sowing or planting with great accuracy. Therefore, current systems to predict m a t u r i t y in these crops are based on m e a s u r e m e n t s of curd size at a stage prior to maturity, and then forecasting maturity using the relationships described above in combination with long-term meteorological data. When u s e d commercially, if such s y s t e m s are also provided with information on planting areas of specific varieties and their planting dates they are capable of predicting yield of a complete enterprise over an extended period. For single growers and grower cooperatives, such analyses can provide i m p o r t a n t information on peaks and t roughs in production. This allows rapid changes in m a r k e t i n g and labour plans. Gluts and t roughs can be considered in s u p e r m a r k e t promotions, ens ur ing supplies m a t c h demand. On a regional basis, the s ys t em could be scaled up to include a n u m b e r of major growers. F u r t h e r m o r e , supply problems can be identified well in advance and coordinated by grower associations, avoiding the large crop losses which have dogged this sector of the industry.
Yield d e t e r m i n i n g factors Yield determining factors u n d e r the control of the grower include plant density and planting date. Everaarts et al. (1998) studied the effects of three planting dates and three planting densities on yield determining factors in Brussels sprouts. Dry m at t er a c c u m u l a t i o n after planting was higher after planting late in the season but the final dry m a t t e r a c c u m u l a t i o n was reduced by late planting. Plant density did not influence the final dry matter acc u m u l a t i o n or significantly influence radiation use efficiency which had a m e a n value of 2.2 gMd -1. Planting late in the season decreased the n u m b e r of b u d s per plant a n d decreased bud dry m a t t e r content. However, a decrease in the n u m b e r of b u d s per plant due to increased plant density was more t h a n c o m p e n s a t e d for by the increase in n u m b e r of plants per hectare. Harvest index, of 30 - 45%, was not affected by the treatment. They concluded th at high yields would be achieved by planting as early as field conditions allow. Ha (1997) us ed a sensitivity analysis within a crop growth model to study options for increasing seed yield in winter oilseed rape. The most promising crop type for high seed yield combined late m a t u r i t y with early flowering, with a m a x i m u m LAI of about 3 for m a x i m u m light absorption and erect clustered pods for source improvement. Increased source activity has to be combined with increased sink capacity t h r o u g h a high rate of seed set, increased potential growth rate of seed and apetalous flowers.
367
_
9
o
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I
I
I
200
400
600
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Day d e g r e e s
Figure 11.2 The relationship between thermal time and the logarithm of curd growth for crops of cv. Revito grown in the field, data from Pearson et al. (1994). Yield of calabrese is strongly influenced by plant density so t h a t yield per unit area increases with higher density u p to a plateau (Cutliffe, 1975; T h o m p s o n a n d Taylor, 1976; Chung, 1982; Salter et al., 1984). However, increased density reduced head diameter a n d weight in calabrese a n d led to greater variability at higher densities (Warr et al., 1992). In cauliflower increased density can lead to increased yields b u t also leads to a decrease in curd size (Salter a n d J a m e s , 1975; T h o m p s o n a n d Taylor, 1973). However, in contrast to calabrese, uniformity was increased at closer spacings (Salter and J a m e s , 1975). In cabbage, head weight a n d variability declined with increasing density. Moreover, crop m a t u r i t y was delayed as plant spacing increased (Stoff a n d Fleming, 1990). During heading, approximately 40% of carbon assimilated is translocated to the growing head (Hara a n d Sonoda, 1981). However, this is reduced by reduced light a n d high nitrogen. In Brussels sprouts, increasing density results in a decrease of plant size a n d a reduction in the growth rate of individual s p r o u t s (Thompson a n d Taylor, 1973; Fischer a n d Melbourne, 1974; Abuzeid a n d Wil, 1989). Otherwise, increases in plant density do not affect the n u m b e r of s p r o u t s per plant b u t reduce m e a n s p r o u t size at harvest so t h a t yield is only increased when
368 the growing season is long enough so that these reach a marketable size (Kirk, 1982). As density increases so does the uniformity of the size and distribution of sprouts down the stem permitting machine harvesting, whereas wide spacing, where the lowest sprouts m a t u r e first, is more suitable for h a n d picking over a more extended period (Fischer, 1974). Greater sprout uniformity is also achieved commercially by 'stopping' the plants by removing the apical region of the plant when the first bud is approximately 1012 m m in diameter. The consequent removal of apical dominance increases the growth of sprouts, particularly the immature sprouts in the upper regions of the stem. S h a r m a and Ghildiyal (1992) studied the contribution of leaf and pod photosynthesis to seed yield in Brassica rapa (syn. B. campestris). Shading pods decreased seed yield by 95% over u n s h a d e d controls by decreasing seed n u m b e r per pod and seed size. In contrast, defoliation decreased seed yield by decreasing pod n u m b e r and seed size, indicating the complementary role of leaf and pod photosynthesis in determining seed yield in mustard. In a similar defoliation study on four Brassica species (B. rapa, B. juncea, B. nigra and B. carinata) (Ramana and Ghildiyal, 1997) defoliating B. rapa and B. j u n c e a decreased seed yield by decreasing pod n u m b e r but not seed n u m b e r per pod and seed weight. Defoliation also decreased seed weight in B. nigra and seed n u m b e r per pod in B. rapa. Thus, in B. rapa and B. juncea, leaf photosynthesis appears mainly restricted to the formation of pods whereas, in B. nigra and B. carinata leaf photosynthesis also contributed towards seed development. Also in B. n a p u s seed growth mainly depends on h u s k CO2 assimilation. Gammelind et al. (1996) showed that the predominant effect of increasing N application in oil seed rape u n d e r conditions without water deficiency was enhanced expansion of photosynthetically active leaf or silique. Photosynthetic rates of both leaves and siliques increased linearly with increased N content up to about 2gm -2. At higher tissue N contents, photosynthetic rate responded less to changes in N status. Expressed per unit N, light saturated photosynthetic rate was three times higher than in silique valves, indicating more efficient photosynthetic N utilization in leaves then in silique. Two weeks after flowering and onwards, total net CO2 fixation in silique valves exceeded that in leaves because siliques received much higher radiation intensities t h a n leaves and because the leaf area declines during the reproductive phase of growth. In conclusion, considerable challenges remain to fully exploit the yield potential of brassicas. Key areas of further research required include studies on yield potential and carbon partitioning and further elucidation of the genetic basis of m a n y of the important responses to environment. Of particular importance in the future will be studies on how environment controls and regulates eating or oil quality. Such information will help underpin the future production and crop improvement the of Brassica crop species and relatives.
369
References Abuzeid, A. E. a n d Wilcockson, S. J. 1989. Effects of sowing date, plant density and year on growth and yield of Brussel s s p r o u t s (Brassica oleracea var. buUata subvar, gemmifera). J. Agric. Sci. l l i , 359375. Bhargava, S. C. 1996. Physiology.
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371 Nowbuth, R. D. and Pearson, S. 1998. The effect of t e m p e r a t u r e and shade on curd initiation in temperate and tropical cauliflower. Acta Hort. 4 5 9 , 79 - 87 Osborn, T. C., Kole, C., Parkin, I.A.P., Sharpe, A. G., Kuiper, M., Lydiate, D. J. and Trick, M. 1997. Comparison of flowering time genes in Brassica rapa, B. napus and Arabidopsis thaliana. Genetics 1 4 6 , 11231129. Parkinson, A. H. 1952. Experiments on vegetative and reproductive growth of cauliflower. Annual Report, National Vegetable Research Station, pp.38-51. Pearson, S. a n d Hadley, P. 1988. Planning calabrese production. Grower 100(9), 2 1 - 2 2 Pearson, S., Hadley, P. and Wheldon, A. E. 1994. A model of the effects of t e m p e r a t u r e on the growth and development of cauliflower (Brassica oleracea L. botrytis). Sci. Hort. 59, 91-106. Pekrun, C., Lutman, C. and Lopez-Granados, F 1996. Population dynamics of volunteer rape and possible m e a n s of control. Proc. 2 n d Intl. Weed Control Congress, Copenhagen, pp. 122-1227. R a man a , S. and Ghildiyal, M. C. 1997. Contribution of leaf p h o t o s y n - t h e s i s towards seed yield in Brassica species. Zeitschrift f u r Acker und Pflanzenbau, 178, 185-187. Sadik, S. 1967. Factors involved in curd and flower formation in cauliflower. Proc. Am. Soc.Hort. Sci. 90, 252-259. Salter, P. J. 1969. Studies on crop m a t u r i t y in cauliflower: I. Relationship between times of curd initiation and curd m a t u r i t y of plants within a cauliflower crop. J. Hort. Sci. 44, 129-140. Salter, P. J., Andrews, D. J. and Akehurst, J. M. 1984. The effects of plant density, spacial a r r a n g e m e n t and sowing date on yield a n d head characteristics of a new form of broccoli. J. Hort. Sci. 5 9 , 79-85. Salter, P. J. and J a m e s , J. M. 1974. F u r t h e r studies on the effects of cold t r e a t m e n t of t r a n s p l a n t s on crop m a t u r i t y characteristics of cauliflower. J. Hort. Sci. 49, 329-342. S h a r m a , P. an d Ghildiyal, M. C. 1992. Contribution of leaf and pod photosynthesis to seed yield in m u s t a r d . Photosynthetica 2 6 , 91-94. Squire, G. R. 1999. T em per at ur e and heterogeneity of emergence time in oilseed rape. Annals Appl. Biology (in press) Stokes, P. and Verkerk. 1951. Flower formation in Brussel sprouts. Mededlingenen van de L andbouw Hogeschool te Wageningen 50(9), 143160.
372 Stoffella, P. J. a n d Fleming, M. F. 1990. Plant p o p u l a t i o n influences yield variability of cabbage. J. Am. Soc. Hort. Sci. 115, 708-711. S u r e s h , K., Rao. K.L.N. a n d Nair, T.V.R. 1997. Genetic variability in photos y n t h e s i c rate a n d leaf c h a r a c t e r s in Brassica cenospecies. Photosynthetica 33, 173-178. Teutonico, R. A. a n d Osborn, T. C. 1995. Mapping loci controlling vernalization r e q u i r e m e n t in Brassica rapa. Theor. Appl. Genet. 91, 12791283. Thapa, M. P. 1994. The effect of photoperiod on curd initiation of cauliflower (Brassica oleracea van botrytis). MSc Thesis, University of Reading. U.K. T h o m a s , T. H., Lester, J. N. a n d Salter, P. J. 1972. H o r m o n a l changes in the s t e m apex of the cauliflower p l a n t in relation to c u r d development. J. Hort. Sci. 47, 449-455. T h o m p s o n , R. a n d Taylor, H. 1973. The effects of p o p u l a t i o n and harvest date on the yields a n d size grading of an F1 hybrid a n d open pollinated B r u s s e l s s p r o u t s cultivar. J. Hort. Sci. 48, 2 2 5 - 2 4 6 T h o m p s o n , R. a n d Taylor, H. 1976. Plant competition a n d its implications for c u l t u r a l m e t h o d s in calabrese. J. Hort. Sci. 5 1 , 2 3 0 - 2 3 1 . Tommey, A. M. a n d Evans, E. J. 1991. T e m p e r a t u r e a n d daylength control of flower initiation in winter oilseed rape (Brassica napus L.) Annals Appl. Biology 118, 2 0 1 - 2 0 8 Vigil, M. F., Anderson, R. I. a n d Beard, W. E. 1997. Base t e m p e r a t u r e a n d growing-degree-hour r e q u i r e m e n t s for the emergence of canola. Crop Science 37, 8 4 4 - 8 4 9 Wagenvoort, W. A., Boot, A. a n d Bierhuizen, J. F. 1974. O p t i m u m temper a t u r e range for g e r m i n a t i o n of vegetable seed. Gartenbauwissenschaft 46, 97 101. Wiebe, H. J. 1972a. Wirking yon t e m p e r a t u r u n d Licht a u f W a c h s t u m u n d E n t w i c k l u n g yon B l u m e n k o h l I. D a u e r der J u n g e n d p h a s e fur Vernalisation. Gartenbauwissenschaft 37, 165-178. Wiebe, H. J. 1972b. Wirking von t e m p e r a t u r u n d Licht a u f W a c h s t u m u n d E n t w i c k l u n g von B l u m e n k o h l II. Optimale Vernalisations-temper a t u r u n d Vernalisationdauer. Gartenbauwissenschaft 37, 293303. Wiebe, H. J. 1972c. Wirking von t e m p e r a t u r u n d Licht a u f W a c h s t u m u n d E n t w i c k l u n g von B l u m e n k o h l III. Vegetative phase. Gartenbauw i s s e n s c h a f t 37, 455-469. Wiebe, H. J. 1981. Influence of t r a n p l a n t characteristics a n d growing conditions on curd size (buttoning) of cauliflower. Acta Hort. 122, 99105.
373 Wiebe, H. J., Habegger, R. a n d Lubig, H. P, 1992. Q u a n t i f i c a t i o n of vernalization a n d d e v e r n a l i s a t i o n effects for kohlrabi (Brassica oleracea convar, acephala var. gongyloides L.) Sci. Hort. 50, 11-20. Wurr, D.C.E. 1990. Prediction of the time of m a t u r i t y in cauliflower. Acta Hort. 2 6 7 , 3 9 7 - 3 9 4 . Wurr, D.C.E., Fellows, J. R. a n d H a m b r i d g e A. J. 1991. The influence of field e n v i r o n m e n t a l conditions on c a l a b r e s e growth a n d development. J. Hort. Sci. 66, 4 9 5 - 5 0 4 . Wurr, D.C.E., Fellows, J. R., Phelps, K. a n d Reader, R.J. 1994. Testing a vern a l i s a t i o n model on field grown crops of four cauliflower cultivars. J. Hort. Sci. 69, 251-255. Wurr, D.C.E., Fellows, J. R. a n d Phelps, K. 1996. Investigating t r e n d s in vegetable crop r e s p o n s e to i n c r e a s i n g t e m p e r a t u r e a s s o c i a t e d with climate change. Sci. Hort. 66, 255-263. Wurr, D.C.E. a n d F e l l o w s , J. R. 1998. Leaf p r o d u c t i o n a n d c u r d initiation of w i n t e r cauliflower in r e s p o n s e to t e m p e r a t u r e . J. Hort. Sci. Biotech. 73, 6 9 1 - 6 9 7 . Zanewich, K. P. a n d Rood, S. B. 1995. Vernalization a n d gibberellin physiology of w i n t e r c a n o l a - e n d o g e n o u s gibberellin (GA) c o n t e n t a n d m e t a b o l i s m of GA1 a n d GA20. Plant Physiology 108, 6 1 5 - 6 2 1 .
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Biology of Brassica Coenospecies C. G6mez-Campo (Editor) 91999 Elsevier Science B.V. All rights reserved.
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12 DISEASES J a l p a P. Tewari (1) a n d Richard F. Mithen (2)
(1) Department of Agricultural, Food and Nutritional Science. Faculty of Agriculture, Forestry and Home Economics. University of Alberta. Edmonton, T6G 2P5 Canada (2) Department of Brassica and Oilseeds Research. John Innes Centre. Colney. Norwich, NR4 7UH. U.K. In this c h a p t e r we review the m a j o r d i s e a s e s of Brassica c a u s e d by fungal p a t h o g e n s . The i n c r e a s e in economic i m p o r t a n c e of Brassica crops, a n d in p a r t i c u l a r of oilseed rape, h a s led to a n i n c r e a s e in r e s e a r c h on h o s t - p a t h ogen interactions. For the majority of t h e s e interactions, notable a d v a n c e s have b e e n m a d e p a r t i c u l a r l y in the u s e of m o l e c u l a r m e t h o d s for a s s e s s i n g genetic variation in the p a t h o g e n a n d developing diagnostic t e c h n i q u e s , a n d in locating the position of r e s i s t a n c e genes on genetic m a p s of Brassica. The w i d e s p r e a d a d o p t i o n of Arabidopsis thaliana (L.) Heynh. as a model p l a n t s y s t e m for m o l e c u l a r genetics h a s also led to a n i n c r e a s e in r e s e a r c h on crucifer p a t h o g e n s , b u t p a r t i c u l a r l y on Peronospora parasitica (Pers. ex Fr.) Fr. a n d Albugo candida (Pers.) Kuntze.
Blackspot or grey leaf c a u s e d by A l t e r n a r i a b r a s s i c a e (Berk.) Sacc. and dark leaf spot c a u s e d by A. b r a s s i c i c o l a (Schw.) Wiltshire F o u r species of Alternaria are p a t h o g e n i c on Brassica spp. ( H u m p h e r s o n - J o n e s , 1992; Paul a n d Rawlinson, 1992; S i m m o n s , 1992, 1995; Tewari, 1985; V a a r t n o u a n d Tewari, 1972; V e r m a a n d S a h a r a n , 1994): A. alternata (Fr.) Keissler, A. brassicae, A. brassicicola a n d A. japonica Yoshii. Of these species, A. brassicae a n d A. brassicicola are w i d e s p r e a d a n d m o s t i m p o r t a n t economically. Both p a t h o g e n s are found a r o u n d the world especially where crucifers are c o m m e r c i a l l y grown. Alternaria brassicae is c o m m o n in m a n y t e m p e r a t e p a r t s of the world d u r i n g the s u m m e r a n d in m a n y s u b t r o p i c a l a n d tropical p a r t s d u r i n g the winter. Alternaria brassicicola r e q u i r e s w a r m e r conditions t h a n A. brassicae. This r e s t r i c t s its d i s t r i b u t i o n in more n o r t h e r l y a n d cooler a r e a s as c o m p a r e d to A. brassicae ( D e g e n h a r d t et al., 1982; H u m p h e r s o n - J o n e s a n d Phelps, 1989; H u m p h e r s o n - J o n e s , 1992).
376 The host range of both species includes m a n y oleiferous, vegetable, ornamental, and wild crucifers, and also some non-cruciferous plants. However, species identity and pathology of at least some records on non-cruciferous plants need to be re-examined. Both species may occur on the same crop but A. b r a s s i c a e is most frequently associated with the oleiferous B. j u n c e a (L.) Cosson, B. n a p u s L., and B. rapa L. and A. brassicicola with the vegetable B. oleracea L. (Humpherson-Jones, 1992). Alternaria brassicae and A. brassicicola exhibit some cultural, morphological, pathogenic and genetic variation (Verma and Saharan, 1994; S h a r m a and Tewari 1995, 1997). However, variation is perhaps not as profound as in some other pathogens. Both A. b r a s s i c a e and A. brassicicola cause conspicuous spotting of the aerial plant parts and their symptoms overlap (Figures 12.1 and 12.2). The lesions caused by A. b r a s s i c a e range from grey to almost black while those caused by A. brassicicola are almost sooty black. These necrotic lesions are solid or concentrically zonate and are s u r r o u n d e d by chlorotic borders (Conn et al., 1990; Verma and Saharan, 1994). Lesioning of leaves reduces photosynthetic area and causes accelerated senescence. Alternaria b r a s s i c a e produces abscisic acid, N-methyl-2,5-dimethyl-N'-cinnamoyl-piperazine, and 3-carboxy-2-methylene-4-pentenyl-4-butenolide, all of which are senescence promoting c o m p o u n d s (Dahiya et al., 1988; Dahiya and Tewari, 1991). Lesions caused by A. b r a s s i c a e and A. brassicicola often exhibit an incipient green island effect. This may be due to production of cytokinins by these fungi (Suri and Mandahar, 1984, 1985; Dahiya and Tewari, 1991). Lesioning of siliques and inflorescent stems is a common feature of infection when the weather is conducive. This has a significant effect on seed yield as the siliques themselves produce photosynthates necessary for their own increase in size and weight (Allen et al., 1971). Lesions on siliques cause prem a t u r e ripening, increased fruit shattering, direct infection of developing seeds, and an increase in dockage (Conn and Tewari, 1990; Conn et al., 1990; H u m p h e r s o n - J o n e s , 1992; Seidle et al., 1995). Alternaria brassicae can increase green seed in B. rapa and these seeds are often situated close to deep-seated lesions in siliques of this species (Rude et al., 1994, Seidle et al., 1995). Green seed may be caused by production of cytokinins by the pathogen or by killing of funiculus preventing the passage of water and photosynthates between seed and silique, and is an important factor in downgrading commercial canola seed (Suri and Mandahar, 1985: Dahiya and Tewari, 1991; Rude et al., 1994; Seidle et al., 1995). The pathogens are also reported to affect seed germination and the quality and quantity of oil (Humpherson-Jones, 1992; Verma and Saharan, 1994; Seidle et al., 1995). Rainy weather during silique maturation promotes lesioning of fruits and may result in substantial yield losses (Tewari, 1993). Alternaria diseases cause seed losses of up to about 70% in countries such as Canada, France, Germany, India, Poland, and Britain (Kolte, 1985; Kolte et al., 1987, Tewari and Conn, 1988; Conn and Tewari, 1990; H u m p h e r s o n - J o n e s , 1992; Verma and Saharan, 1994; Seidle et al., 1995).
377 Both A. b r a s s i c a e and A. brassicicola produce phytotoxic c o m p o u n d s which may be i m p o r t a n t in pathogenesis by these fungi (MacDonald and Ingram, 1985; Bains et al., 1993). The phytotoxic c o m p o u n d s produced by A. b r a s s i c a e have been chemically characterised a n d consist of destruxin B, h o m o d e s t r u x i n B, a n d destruxin B2. Another related compound, desmethyldestruxin B, is also produced by A. b r a s s i c a e b u t its phytotoxic activity, if any, h a s so far not been demonstrated. Destruxins are u n i q u e c o m p o u n d s as they act as both phytotoxins a n d mycotoxins. Destruxin B is also produced by M e t a r h i z i u m anisopliae (Metschn.) Sorokin, a fungus parasitic on insects (Gupta et al., 1989). Crucifers susceptible and some t h a t are resista n t to A. b r a s s i c a e are sensitive to destruxin B (Conn et al., 1991). Rapid a c c u m u l a t i o n of phytoalexins in some plants could a c c o u n t for pathogenresistance of destruxin B-sensitive crucifers (Conn et al., 1991; Jejelowo et al., 1991; Browne et al., 1991). Destruxins are produced by M. anisopliae and two species of Alternaria, namely A. b r a s s i c a e and A. linicola Groves and Skolko (Evans et al., 1995). All these attributes of destruxin B indicate that it should be placed in a new group of phytotoxins. Recently, a 35 kDa hostspecific toxin (probably a protein) produced by A. brassicicola h a s been purified (Otani et al., 1998). All commercial brassicas are essentially susceptible to A. b r a s s i c a e a n d A. brassicicola. However, there are some differences in their degrees of susceptibility (Jasalavich et al., 1993). Crucifers s u c h as B. n a p u s a n d B. carin a t a A.Br. are less susceptible to A. b r a s s i c a e t h a n B. rapa a n d B. j u n c e a (Skoropad a n d Tewari, 1977; Bhowmik and Munde, 1987; Conn a n d Tewari, 1989; Katiyar a n d Chamola, 1994). Epicuticular wax crystals providing a hydrophobic surface are primarily responsible for this difference (Skoropad and Tewari, 1977; Conn and Tewari, 1989). Some somaclones of B. j u n c e a have high degrees of field resistance to A. b r a s s i c a e (Katiyar a n d Chopra, 1990; S h a r m a and Singh, 1995). Little is known regarding genetics of resistance to A. b r a s s i c a e in B r a s s i c a species. Resistance in the Indian cultivar RC781 is governed by a single d o m i n a n t gene (Tripathi et al., 1980) while horizontal resistance h a s been reported in some other genotypes (Saharan a n d Kadian, 1983). Some cultivars of S i n a p i s alba L. are also appreciably r e s i s t a n t to A. b r a s s i c a e (Dueck a n d Degenhardt, 1975; B r u n et al., 1987). E r u c a vesicaria (L.) Cav., in general is susceptible to A. brassicae. However, one accession of this species showed a hypersensitive reaction to this pathogen (Conn and Tewari, 1986). Certain wild crucifers have high degrees of resistance to A. brassicae (Tewari, 1991; Tewari a n d Conn, 1993; Tewari, 1993; G u p t a et al., 1995) and a t t e m p t s are being m a d e to transfer this resistance to cultivated B r a s s i c a species (Shivanna a n d Sawhney, 1993; N a r a s i m h u l u et al., 1994). Resistance to A. b r a s s i c a e in crucifers is layered a n d m u l t i c o m p o n e n t (Tewari, 1991, 1993). Some wild crucifers are highly r e s i s t a n t to A. b r a s s i c a e and elicit phytoalexins when challenged by this pathogen (Conn et al., 1988). Phytoalexins elicited in C a m e l i n a s a t i v a (L.) Crantz consist of camalexin (CllHsN2S) a n d 6-methoxycamalexin (C12H10N2SO) (Browne et al., 1991).
378 They appear to be the first reported naturally occurring thiazoyl-substituted indole phytoalexins which contain a 2-substituted thiazole ring. Camalexin is also produced by A. thaliana when challenged by P s e u d o m o n a s syringae pv. s y r i n g a e Van Hall (Tsuji et al., 1992). Thiabendazole, a well known synthetic fungicide, is a 4-substituted thiazole and is closely related to camalexin. However, unlike camalexin thiabendazole is not toxic to A. brassicae (Maude et al., 1984; Conn et al., 1988). This raises some interesting chemical structure-function relationships. In a recent study, activities of some compounds related to camalexin were compared and the natural chemistry of camalexin was found to be most toxic to A. brassicae and A. brassicicola (Dzurilla et al. 1998). Capsella bursa-pastoris (L.) Medic. elicits camalexin, 6methoxycamalexin, and another phytoalexin, N-methylcamalexin (C12H10N2S) upon challenge by A. b r a s s i c a e (Jimenez et al., 1997). The phytotoxin, destruxin B also elicits phytoalexin response in Sinapis alba (Pedras and Smith, 1998) and may be involved in resistance of this crucifer to A. brassicae. In the absence of high degrees of resistance in cultivated brassicas to both these pathogens, practical control m e a s u r e s have to include cultural and chemical methods (Humperson-Jones, 1992; Verma and Saharan, 1994; K h a r b a n d a and Tewari, 1996). Little work has so far been done towards developing integrated pest m a n a g e m e n t and early warning systems (Eastburn, 1989; Fontem et al., 1991; H u m p h e r s o n - J o n e s , 1991; Hong et al., 1996). A non-radioactive diagnostic DNA probe has been developed for A. brassicae (Sharma and Tewari, 1998). S t e m c a n k e r or b l a c k l e g c a u s e d by Leptosphaeria maculans (Desm.) Ces. a n d de Not. L e p t o s p h a e r i a m a c u l a n s (anomorph P h o m a lingarn (Tode ex Fr.) Desm.) is the most important pathogen of oilseed B r a s s i c a crops, causing stem canker or blackleg disease. It is also an important pathogen of horticultural B r a s s i c a species. Pycnidia and pseudothecia are found on decaying Brassica tissue which release splash dispersed pycnidiospores and air borne ascospores which colonise healthy tissue. The fungus initially grows intercellularly without causing cell death. The length of this latent period is variable, and may be partially determined by the host genotype. Latent or symptom-
Figure 12.1 Figure 12.2 Figure 1 2 . 3 Figure 1 2 . 4
Blackspot on a leaf of B r a s s i c a sp. caused by Alternaria sp. in Portugal. Blackspot on the fruits of B r a s s i c a rapa caused by Alternaria b r a s s i c a e in C a n a d a (Courtesy Dr. I. R. Evans). Leaf lesions caused by L e p t o s p h a e r i a m a c u l a n s in Germany. Stem canker on B r a s s i c a sp. caused by L e p t o s p h a e r i a maculans in Canada.
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381 less infection of crops m a y be very extensive (Petrie et al., 1995). Following h o s t cell d e a t h greyish leaf lesions develop (Figure 12.3). These often p r o d u c e pycnidia which m a y r e s u l t in s e c o n d a r y infection. The lesions are r a t h e r variable in a p p e a r a n c e , a n d m a y be a s s o c i a t e d with a r e a s of chlorosis in surr o u n d i n g leaf tissue. The f u n g u s frequently b e c o m e s systemic a n d colonises the v a s c u l a r tissue in the petiole a n d stem ( H a m m o n d a n d Lewis, 1987). Stem c a n k e r s (Figure 12.4), which often arise from this systemic infection, u s u a l l y develop after flowering. C a n k e r s y m p t o m s are variable, a n d range from r a t h e r superficial s t e m lesions to large 'crown' c a n k e r s in which extensive regions of dry corky tissue develop a r o u n d the base of the s t e m leading to lodging. The f u n g u s will c o n t i n u e to colonise s t u b b l e after the p l a n t h a s died. P s e u d o t h e c i a develop on stubble which release a s c o p s o r e s for new infections. The factors which stimulate p s e u d o t h e c i a d e v e l o p m e n t are poorly u n d e r s t o o d , b u t are likely to be involved with c h a n g e s in the n u t r i t i o n a l stat u s of the p l a n t tissue. The f u n g u s can persist on s t u b b l e for several years. A detailed description of the epidemiology of Leptosphaeria m a c u l a n s is given by Hall (1992). Isolates of L. m a c u l a n s can be divided into two m a j o r classes. One class is often t e r m e d highly virulent or aggressive a n d is c h a r a c t e r i s e d by being strongly p a t h o g e n i c a n d having a slow rate of growth in c u l t u r e in which it p r o d u c e s a s i r o d e s m i n toxin, while the other class is of low aggressiveness a n d is avirulent on m a n y cultivars. The low aggressive isolates grow rapidly in culture in which they p r o d u c e a b r o w n pigment, b u t do not p r o d u c e toxins. The two classes of isolates can be d i s t i n g u i s h e d by m a n y m o l e c u l a r m e t h o d s , s u c h as isozyme variation, m o n o c l o n a l antibodies, RFLPs, RAPDs, karyotypes from pulse-gel electrophoresis, analysis of rDNA s e q u e n c e a n d occurrence of repetitive DNA e l e m e n t s a n d linear p l a s m i d s (Taylor a n d Borgman, 1994; S t a c e s m i t h et al., 1993; Lim a n d Howlett, 1994, P l u m m e r et al., 1994; Gall et al., 1995; Sippell a n d Hall, 1995). There is no evidence of regular gene flow between these two classes of isolates, which p r o b a b l y r e p r e s e n t distinct biological species. However, there is some evidence t h a t the genetic transfer of a repetitive DNA element from the high aggressive class to the low aggressive class h a s o c c u r r e d (Taylor a n d B o r g m a n , 1994). Within each of these major classes there is also considerable variation. In general, there is
Figure 12.5 Stem rot on Brassica n a p u s c a u s e d by Sclerotinia sclerotiorum in C a n a d a .
Figure 1 2 . 6 White r u s t on a leaf of Brassica j u n c e a in India. Figure 1 2 . 7 S t a g h e a d on the fruiting axis of Brassica rapa c a u s e d by Albu-
go candida in C a n a d a . Note the mealy growth of Peronospora parasitica on the s t a g h e a d surface. Figure 1 2 . 8 Downy mildew on a leaf of Brassica alboglabra c a u s e d by Peronospora parasitica in India.
382 less variation observed in the high aggressive class of isolates (Koch et al., 1991). O t h e r forms of L. m a c u l a n s have b e e n isolated from cruciferous weeds which m a y also r e p r e s e n t distinct biological species (Rouxel et al., 1994). The relative f r e q u e n c y of the low a n d high aggressive classes of isolates vary. In C a n a d a a n d Europe, the low aggressive isolates are relatively frequent, while in A u s t r a l i a they are relatively rare a n d m a y be a b s e n t from some of the r a p e s e e d growing areas. M a h u k u et al. (1996a) investigated the interaction b e t w e e n t h e s e classes of isolates in planta. They showed t h a t low aggressive isolates c a n i n d u c e r e s i s t a n c e to high aggressive isolates. It is not k n o w n w h e t h e r this i n d u c t i o n of r e s i s t a n c e operates in a field situation, b u t it m a y be crucial to u n d e r s t a n d i n g the a p p a r e n t virulence of L . m a c u l a n s in Australia a n d c h a n g e s in the incidence of s t e m c a n k e r observed in Europe. Various diagnostic t e c h n i q u e s for a s s e s s i n g frequencies of the two classes of p a t h o g e n s m a y be of p a r t i c u l a r value in this type of s t u d i e s ( M a h u k u et al., 1996b). D u r i n g the 1960's, 1970's a n d the early 1980s there were severe epidemics of s t e m c a n k e r disease in Europe. In the mid eighties, susceptible cultivars s u c h as Primor a n d R a p p o r a were replaced with the r e s i s t a n t F r e n c h cultivar J e t Neuf which soon d o m i n a t e d the r a p e s e e d growing areas. This cultivar h a s b e e n u s e d as a source of r e s i s t a n c e in the majority of breeding p r o g r a m m e s in all r a p e s e e d growing a r e a s . Since the mid eighties, the levels of s t e m c a n k e r in E u r o p e have fluctuated. Generally there was relatively little c a n k e r in the late 1980's a n d early 1990's which m a d e it difficult for b r e e d e r s to select for r e s i s t a n c e in field n u r s e r i e s . The r e a s o n s for this lack of c a n k e r are not clear, it m a y have b e e n d u e to a s u c c e s s i o n of dry a u t u m n a n d winters, or possibly the effectiveness of r e s i s t a n c e genes derived from J e t Neuf. Since the mid 1990's there h a s been a n i n c r e a s e in c a n k e r levels. This m a y be d u e to the loss of r e s i s t a n c e genes, or to c h a n g e s in the p a t h o g e n p o p u l a t i o n a n d w e a t h e r conditions. The m o s t i n t e n s e disease p r e s s u r e o c c u r s in Australia, where susceptible cultivars are killed e a c h y e a r by a highly virulent a n d aggressive pathogen population. In the early 1980's blackleg epidemics c a u s e d devastation over the majority of the c a n o l a growing a r e a a n d m a n y farmers stopped growing c a n o l a as a result. Breeding p r o g r a m m e s successfully u s e d J a p a nese spring cultivars a n d F r e n c h winter cultivars to p r o d u c e a series of relatively r e s i s t a n t cultivars which h a s led to a r e s u r g e n c e of the rapeseed industry. A review of blackleg in A u s t r a l i a h a s recently been published (Salisbury et al., 1995). The low aggressive strain of L. m a c u l a n s is k n o w n in C a n a d a since the early 1960's. Isolates of the high aggressive strain were first detected in 1975 in central S a s k a t c h e w a n . The disease h a s since s p r e a d to other r a p e s e e d growing a r e a s of C a n a d a . Disease p r e s s u r e is m u c h less t h a n in A u s t r a l i a a n d a lower level of r e s i s t a n c e is required in successful cultivars. The disease is of little i m p o r t a n c e in C h i n a despite its large oilseed rape acreage. It h a s been s u g g e s t e d t h a t this m a y be d u e to rapid decomposition of s t u b b l e following flooding of fields (Rimmer a n d Buchwaldt, 1995).
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Crop rotation a n d good s a n i t a t i o n are the m o s t effective m e a n s of control in the a b s e n c e of highly r e s i s t a n t cultivars. Infected s t u b b l e can contin u e to p r o d u c e a s c o s p o r e s for several years, a n d these m a y travel several kilometres. The p a t h o g e n c a n also infect the seed coat. In the oilseed crops, seed infection is p r o b a b l y only significant in i n t r o d u c i n g the disease into new areas, b u t it m a y be of more i m p o r t a n c e in the h o r t i c u l t u r a l i n d u s t r y . Chemical control is p r a c t i s e d on winter oilseed crops in Europe, in which prochloraz is often applied in late A u t u m n after the a p p e a r a n c e of leaf spots. The role of toxins in the d e v e l o p m e n t of disease by this p a t h o g e n h a s been investigated by several r e s e a r c h e r s . Ferezou et al. (1977) first described the p r o d u c t i o n of the toxin s i r o d e s m i n PL in liquid c u l t u r e s of L. m a c u l a n s . Pedras et al. (1988, 1989) s u b s e q u e n t l y reported the o c c u r r e n c e of other s t r u c t u r a l l y related toxins. The ratio of these toxins vary between isolates, b u t none, or very low a m o u n t s , are p r e s e n t in low aggressive isolates. These toxins are h o s t non-specific, t h a t is, they c a u s e cell d e a t h on both r e s i s t a n t a n d susceptible B r a s s i c a h o s t s as well as n o n - h o s t p l a n t s s u c h as wheat. J u s t as the role of toxins in the infection p r o c e s s is not u n d e r s t o o d , the role of p h y t o a l e x i n s p r o d u c e d by B r a s s i c a as a r e s p o n s e to L. m a c u l a n s infection r e m a i n s to be clarified. Several indole p h y t o a l e x i n s have been purified. While the level a n d type of p h y t o a l e x i n s which are i n d u c e d following either infection or abiotic elicitation varies between genotypes, there is no evidence of an association with r e s i s t a n c e (Rouxel et al., 1991). The m e t a b o l i s m of certain phytoalexins by L. m a c u l a n s h a s been described by P e d r a s a n d K h a n (1996). There is c o n s i d e r a b l e variation in r e s i s t a n c e to s t e m c a n k e r disease a m o n g s t B. n a p u s cultivars. The n a t u r e of 'Jet Neuf r e s i s t a n c e is not fully u n d e r s t o o d . It is clearly not a typical h y p e r s e n s i t i v e r e s i s t a n c e , a n d is likely to be regulated by several genes. Apparently, r e s i s t a n t p l a n t s m a y in fact have extensive s y m p t o m l e s s infections by L. m a c u l a n s . There have been several s t u d i e s on the i n h e r i t a n c e of resistance. These vary c o n s i d e r a b l y in the methodology a d o p t e d which m a k e direct c o m p a r i s o n s between s t u d i e s difficult. Several scientists have employed cotyledon tests a n d have described a series of Mendelian r e s i s t a n c e genes which c o r r e s p o n d to avirulence genes in the pathogen. G l a s s h o u s e tests are also u n d e r t a k e n on y o u n g p l a n t s by eit h e r leaf inoculation of direct infection of the s t e m or petiole. It is not clear, however, how t h e s e seedling tests relate to field resistance. B a n s a l et al. (1994) c o m p a r e d g l a s s h o u s e a n d field t e s t s for r e s i s t a n c e a n d found a good correlation in r e s i s t a n c e ratings. However, several o t h e r scientists have reported r a t h e r poor correlations between seedling a n d a d u l t p l a n t r e s i s t a n c e (Ballinger a n d Salisbury, 1996; Ferreira et al., 1995a; C r o u c h et al., 1994). T h u s , while single d o m i n a n t r e s i s t a n c e genes have been d e s c r i b e d in seedlings, it is not clear w h e t h e r these c o n t r i b u t e to a d u l t p l a n t resistance. An informative s t u d y seeking to resolve some of t h e s e i s s u e s was reported by Ferreira et al. (1995a). A p o p u l a t i o n of 105 d o u b l e d haploid recomb i n a n t lines derived from a cross between the cultivars Stella (susceptible) a n d Major (resistant) were evaluated for r e s i s t a n c e by several different me-
384 thods. A single m a j o r locus for r e s i s t a n c e was positioned on linkage group 6 b a s e d on qualitative scores on the interaction p h e n o t y p e with seedlings. This locus also a p p e a r e d to be involved in q u a n t i t a t i v e a s s e s m e n t s of resistance following g l a s s h o u s e inoculations of stems. However, w h e n field evaluated p l a n t s were a s s e s s e d , variation at this locus w a s not i m p o r t a n t , a n d variation at two o t h e r q u a n t i t a t i v e trait loci on linkage g r o u p s 12 a n d 21 were described. In addition to exploiting r e s i s t a n c e genes within existing B. napus cultivars, several p r o g r a m m e s have s o u g h t to i n t r o d u c e novel genes into breeding lines. The m o s t f r e q u e n t source of r e s i s t a n c e genes h a s been the Brassica B genome, r e p r e s e n t e d by either B. nigra or B. juncea. The first studies were those u n d e r t a k e n by Roy (1978) who described a monogenic control of res i s t a n c e in B. j u n c e a • B. n a p u s crosses. Several o t h e r studies involving the d e v e l o p m e n t of addition a n d s u b s t i t u t i o n lines have s u g g e s t e d a more complex mode of i n h e r i t a n c e of r e s i s t a n c e involving u p to 3 loci (Zhu et al., 1993; Pang a n d Halloran., 1996). The u s e of r e s i s t a n c e genes from B. j u n c e a a n d B. nigra in b r e e d i n g p r o g r a m m e s a n d the d e v e l o p m e n t of canola quality B. juncea cultivars h a s s t i m u l a t e d r e s e a r c h into possible virulence of L. maculans on B. juncea. C h e n et al. (1996) have d e s c r i b e d A u s t r a l i a n isolates of the f u n g u s which c a n infect the B. j u n c e a cultivars Stoke a n d Zaria. This virulence p h e n o t y p e is r e g u l a t e d by a n allele at a single locus in crosses with n o n B. j u n c e a a t t a c k i n g isolates. C r o u c h et al. (1994) described a similar type of r e s i s t a n c e occurring in a wild form of B.rapa from Sicily. This r e s i s t a n c e w a s successfully transferred to oilseed rape b r e e d i n g lines via the d e v e l o p m e n t of synthetic amphidiploids. F u r t h e r s o u r c e s of r e s i s t a n c e m a y o c c u r in other related crucifers s u c h as Eruca sativa a n d Brassica tournefortii (Liu et al., 1995). Transfer of r e s i s t a n c e from t h e s e species will involve m a x i m i s i n g the c h a n c e s of homoeologous c h r o m o s o m e r e c o m b i n a t i o n between the Brassica n a p u s a n d alien genomes.
S t e m rot c a u s e d S c l e r o t i n i a s c l e r o t i o r u m (Lib.) de Bary Sclerotinia s t e m rot or cottony soft rot is c o m m o n a r o u n d the world in the t e m p e r a t e regions (Mordue a n d Holliday, 1976; Kolte, 1985; Thomas, 1984; Tewari, 1985; Paul a n d Rawlinson, 1992; Howard et al., 1994). The disease is also serious d u r i n g storage of cruciferious vegetables. Sclerotinia sclerotiorum h a s a very broad h o s t range c o n s i s t i n g of 42 s u b s p e c i e s or varieties, 408 species, 278 genera, a n d 75 families of p l a n t s (Boland a n d Hall, 1994). This i n c l u d e s 48 m e m b e r s of B r a s s i c a c e a e consisting of oleiferous, vegetable, o r n a m e n t a l a n d wild types. In oleiferous b r a s s i c a s , infection is u s u a l l y seen s t a r t i n g from the early flowering stage. Sclerotia from the previous year(s) u n d e r g o carpogenic g e r m i n a t i o n (Dillard et al., 1995; H u a n g a n d Kozub, 1993) a n d a s c o s p o r e s are forcibly liberated from the apothecia. The a s c o s p o r e s b e c o m e w i n d b o r n e a n d are deposited on various plant parts,
385 especially leaf axils and points of b r a n c h i n g of stems. Leaves of B. rapa and B. n a p u s are sessile and auricled. The auricle is an excellent infection court as it collects ascospores, senescent petals, pollen, and water. Infection of stems is facilitated t h r o u g h pre-colonization of petals and pollen. Stem lesions are soft, watery, and whitish (Figure 12.5). Sometimes they are light brownish a n d zonate. Older lesions show shredding of the outer fibrous elements. Some sclerotia undergo myceliogenic germination (Huang, 1991) and this results in infection at the g r o u n d level. In some areas infection of seedlings is also seen (Paul and Rawlinson, 1992). Infected plants undergo p r e m a t u r e ripening and can be easily spotted a m o n g green plants in a field. Sclerotia develop inside a n d / o r on the outside of infected stems. Yield loss in oleiferous brassicas varies considerably based on stage of plant when infection took place, m a x i m u m loss taking place when the plants get infected d u r i n g early bloom stage. On average, yield losses approximate 0.4 to 0.5 times the percentage of infection (Thomas, 1984). Disease forecasting systems based on weather-, host-, a n d pathogenrelated criteria have been developed in m a n y countries (Thomas, 1984; Turkington et al., 1991; Twengstrom et at., 1998). A s s e s s m e n t of petal infestation plays a major role in one of these s y s t e m s (Turkington and Morrall, 1993). An i m m u n o f l u o r e s c e n c e technique to s t u d y the distribution of ascospores of S. sclerotiorum on petals of B r a s s i c a spp. h a s been developed (Lefol a n d Morrail, 1996). Oxalic acid is the pathogenicity d e t e r m i n a n t for S. sclerotiorum a n d oxalic acid m i n u s m u t a n t s are non- pat hogeni c (Godoy et al., 1990). There is no association between pathogenicity and pectolytic enzymes. Role of oxalic acid in the pathogenicity of S. sclerotiorum h a s been confirmed by u s i n g Arabidopsis t h a l i a n a (L.) Heynh. as a model s ys t em (Dickman and Mitra, 1992). Bacterial strains capable of degrading oxalic acid have been identified. They have shown prevention of infection by S. sclerotiorum in A. t h a l i a n a in bioassay tests, an d will serve as sources of genes for oxalic acid degradation in transgenic i m p r o v e m e n t of crop plants for resistance to S. sclerotiorum (Dickman and Mitra, 1992). In view of the importance of oxalic acid, control strategies based on foliar application of calcium-containing c o m p o u n d s to sequester pathogen-secreted oxalic acid in the infection court are being developed (Conn an d Tewari, 1993). Calcium n u t r i t i o n of crucifers is likely to be import a nt in reaction to S. sclerotiorum as the susceptibilities of p u m p k i n and sunflower seedlings have been shown to be inversely proportional to calcium concentration in the n u t r i e n t solution (Chrominski et al., 1987). This may have implications towards m a n a g e m e n t of soft rot of crucifers. Appreciable degrees of resistance to S. sclerotiorum in cultivated crucifers are non-existent. Disease control, therefore, h a s to rely largely on cultural an d chemical control (Thomas, 1984; K h a r b a n d a and Tewari, 1996). Development of petalless cultivars is one promising line of research (Fu, 1990). Selection for resistance to S. sclerotiorum may be possible t h r o u g h use of
386 oxalic acid directly on plants (Rowe, 1993) or in callus cultures (Wu and Liu, 1991). In another novel approach, a gene coding for oxalate oxidase from barley was transferred into B r a s s i c a sp. and was successfully expressed in the transformants (Thomson et al., 1993). Other promising disease control m e a s u r e s include use of biocontrol agents such as Coniothyrium m i n u t a n s (Sandys-Winsch et al., 1993) and hypovirulence (Boland et al., 1993). Multiple disease resistance to S. sclerotiorum, viruses, and Peronospora parasitica (Pers.: FR.) Fr., and tolerance to low temperatures h a s been reported in a swede rape cultivar from China (He et al., 1987). Sclerotinia sclerotiorum is able to form stable heterokaryons and has field populations that are genetically heterogeneous (Kohn et al., 1991; Ford et al., 1995). This should be recognised in developing genotypes for resistance to this pathogen. White rust a n d s t a g h e a d d i s e a s e c a u s e d by Albugo candida Numerous members of B r a s s i c a c e a e and some other families are hosts to A. c a n d i d a (Biga, 1995; Singh, 1989; S a h a r a n and Verma, 1992; Choi and Priest, 1995). Among the crucifers, the host list includes m a n y oleiferous, vegetable, ornamental, and wild types. The disease has been reviewed in detail in many publications (Thomas, 1984; Kolte, 1985; Tewari, 1985; Paul and Rawlinson, 1992; S a h a r a n and Verma, 1992; Howard et al., 1994). The disease has two distinct phases. The white rust stage (Figure 12.6) is the repeating s u m m e r stage and produces zoosporangia. Later during the season, the affected parts, especially portions of inflorescence, undergo hypertrophy and form galls called stagheads (Figure 12.7). Oospores which are the sexual resting spores develop in large n u m b e r s in the stagheads. Some work has been done on the physiological and biochemical changes in tissues infected with A. c a n d i d a (Saxena, 1985; Aldesuquy and Baka, 1992; Saharan and Verma, 1992; Yadav et al., 1994; Khangura and Sokhi, 1995). Plant tissues infected by A. c a n d i d a tend to have lower level of indole-3-acetic acid and higher levels of IAA-oxidase, gibberellic acid, zeatin and abscisic acid. Substantial yield losses in B r a s s i c a crops have been reported as a result of A. c a n d i d a infection (Singh et al., 1990; S a h a r a n and Verma, 1992; Bisht et al., 1994). The disease is of special concern on R a p h a n u s s a t i v u s L., B. j u n c e a and B. rapa in seed production (Ferreira et al., 1995b). Most North American and European cultivars of B. n a p u s are resistant to A. c a n d i d a whereas the Asian cultivars are susceptible (Fan et al., 1983; Petrie, 1988; Ferreira et al., 1995b). The staghead phase accounts for most of the yield losses. Harper and Pittman (1974) derived a yield loss equation (yield loss = 0.952 X % stems infected systemically) for use u n d e r field conditions. Maxim u m staghead formation is obtained when differentiating flower buds of 26day-old (growth stage 3.1) B. j u n c e a plants are inoculated with a zoospore suspension of A. c a n d i d a (Goyal et al., 1996). Exposing the apical meristem of flower buds with forceps is conducive to staghead formation. This should prove to be a useful technique for screening germplasm for resistance to
387 s t a g h e a d formation. M a n y s t u d i e s have reported t h a t P. p a r a s i t i c a preferentially colonises p l a n t t i s s u e s infected with A. c a n d i d a ( S a h a r a n a n d Verma, 1992; Achar, 1993). The p r e s e n c e of some n u t r i t i o n a l factors in t i s s u e s galled by A. c a n d i d a a n d their a b s e n c e in the h e a l t h y t i s s u e s is s u g g e s t e d to be the probable c a u s e of this p a r a s i t i s m by P. parasitica. Both are, however, restricted to living t i s s u e s being obligate parasites. Peronospora p a r a s i t i c a on its own c a u s e s little h y p e r t r o p h y a n d s t a g h e a d s with only A. c a n d i d a are c o m m o n in m a n y crucifers especially d u r i n g earlier stages of p l a n t growth. Many n e c r o t r o p h i c s e c o n d a r y - i n v a d i n g fungi are also f o u n d growing on p l a n t t i s s u e s infected by A. c a n d i d a (Petrie a n d Vanterpool, 1974). P s e u d o m o n a s s y r i n g a e is also a s s o c i a t e d in the field with s t a g h e a d s in B. n a p u s a n d B. rap a incited by A. c a n d i d a a n d a r r e s t s the d e v e l o p m e n t of o o s p o r e s in early stages of gall d e v e l o p m e n t (Tewari, J. P., u n p u b l i s h e d data). It is t e m p t i n g to consider u s i n g this b a c t e r i u m for biological control of A. c a n d i d a oospore d e v e l o p m e n t b u t this b a c t e r i u m alone also c a u s e s lesioning of the various plant p a r t s of several crucifers. B a s e d on specificities of different crucifer h o s t species, at least 10 physiological races of A. c a n d i d a have so far been identified (Pound a n d Williams, 1963; Hill et al., 1988; S a h a r a n a n d Verma, 1992; Liu et al., 1996). The pathogenicity of a race of A. c a n d i d a is not restricted to a p a r t i c u l a r h o s t species, i n s t e a d it also e x t e n d s to some genotypes of related B r a s s i c a species which are the h e t e r o l o g o u s h o s t s (Liu et al., 1996). The h o s t range of race extends especially to B r a s s i c a species which s h a r e g e n o m e s with the h o m o l o g o u s h o s t s from which it was originally collected (Liu et al., 1996). It s e e m s t h a t races of A. c a n d i d a are in a state of d y n a m i c flux as two "new" races 2V a n d 7V from B. j u n c e a a n d B. rapa cultivars, respectively, have recently been reported (Petrie, 1994). Many w o r k e r s have s t u d i e d the i n h e r i t a n c e of r e s i s t a n c e to A. c a n d i d a in B. carinata, B. j u n c e a , B. n a p u s , B. nigra, B. rapa a n d R. s a t i v u s a n d resistance h a s generally b e e n found to be conditioned by one, two or three d o m i n a n t genes (Williams a n d Pound, 1963; Delwiche a n d Williams, 1981; Fan et al., 1983; Tiwari et al., 1988; V e r m a a n d Blowmik, 1989; Liu a n d Rimmer, 1991; Paladhi et al., 1993; Rao a n d Raut, 1994; S u b u d h i a n d Raut, 1994; J a m b h u l k a r a n d Raut, 1995; Liu et al., 1996). A rapid-cycling population of B. rapa is r e p o r t e d to have polygenic control for r e s i s t a n c e to A. cand i d a (Edward a n d Williams, 1987). Eruca vesicaria is also a useful source of r e s i s t a n c e to A. c a n d i d a as it is not far phylogenetically to B r a s s i c a spp. In a recent study, all wild a n d cultivated a c c e s s i o n s of this crucifer were r e s i s t a n t to race 2 of A. c a n d i d a which infects B. j u n c e a . All wild a c c e s s i o n s of this crucifer were also r e s i s t a n t to race 7 which infects B. rapa. However, m o s t cultivated a c c e s s i o n s of E. vesicaria were susceptible to race 7 (Bansal et al. 1997). M a n y of t h e s e a c c e s s i o n s are also r e s i s t a n t to L e p t o s p h a e r i a m a c u l a n s a n d m a y provide s o u r c e s of multiple disease r e s i s t a n c e (Tewari et al., 1996). B r a s s i c a tournefortii G o u a n is also a good s o u r c e of r e s i s t a n c e to A. c a n d i d a a n d a t t e m p t s have been m a d e to hybridise it with B. alboglabra
388 Bailey an d B. j u n c e a (Yadav et al., 1991; Ljungberg et al., 1993). Some sources of resistance have recently been identified in the genus Diplotaxis (Gupta et al., 1995). Sources of resistance have been generated through somaclonal v a r ia nt s (Katiyar and Chopra, 1990; S h a r m a and Singh, 1995) and protoplast fusion of B. j u n c e a with B. s p i n e s c e n s (Kirti et al., 1991). Some investigations have focussed on multiple resistance for diseases such as those c a u s e d by A. candida, Alternaria spp., L. m a c u l a n s , and P. parasitica (Hill and Williams, 1983; Yadav et al., 1991, Ljungberg et al., 1993; MitchellOlds et al., 1995). Several workers have studied the histology of infection in susceptible a n d r es i s t ant reactions (Williams and Pound, 1963; Verma et al., 1975; Liu a n d Rimmer, 1987; Liu et al. 1989). Molecular mapping of loci controlling resistance to A. c a n d i d a h a s been done in A. thaliana, B. n a p u s and B. rapa (Crute et al., 1993; Holub et al., 1995; Ferreira et al., 1995b; Kole et al., 1996). Besides h os t resistance, cultural and chemical control is also effective in reducing the severity of disease c a u s e d by A. c a n d i d a (Saharan and Verma, 1992; K h a r b a n d a and Tewari, 1996).
Light l e a f s p o t c a u s e d by P y r e n o p e z i z a brassicae S u t t o n and Rawlinson P y r e n o p e z i z a b r a s s i c a e (anamorph Cylindrosporium concentricum Grev.) has been k n o w n as a pathogen of horticultural B r a s s i c a crops since 1822. Although widespread, it did little damage to commercial B r a s s i c a crops until the expansion of oilseed rape occurred in the 1970's. Since then there have been several major epidemics on oilseed rape crops which h a s resulted in severe yield losses. There has also been an increase in its importance on horticultural brassicas, p r e s u m a b l y due to greater inoculum levels (Yarham and Giltrap, 1989). It is now regarded as a major pathogen of oilseed B r a s s i c a crops in the United Kingdom and parts of France and Germany. The perfect stage was described in 1978 (Rawlinson et al., 1978). The pathogen is also i m p o r t a n t on B r a s s i c a crops in New Zealand (Cheah and Hartill, 1985). Several aspects of the epidemiology still require to be resolved. Primary infections on a u t u m n sown crops may arise t hrough wind dispersed ascospores released from stubble and infected volunteer seedlings, splash dispersed conidia (which can become airborne in small water droplets) and possibly infected seed (Lacey et al., 1987; C heah et al., 1982; McCartney and Lacey, 1990). The infection process has been described in detail by Rawlinson et al. (1978). Following infection, there may be a long symptomless latent phase during which the fungus produces a m a t of mycelium u n d e r n e a t h the leaf epidermis, b u t with relatively little penetration into the mesophyll. Acervuli erupt t h r o u g h the epidermis and produce large n u m b e r s of conidia which are s p las h dispersed to produce secondary infection. Secondary infections may also arise t h r o u g h airborne ascospores produced on decaying leaf tissue. The extent of systemic infection is not known, b u t it is likely that the
389 fungus can grow t h r o u g h o u t the plant prior to sporulation. Severe infection can kill y o u n g plants. Infection can spread to flower b u d s a n d pods where it can cause large decreases in yield. Grey lesions a p p e a r on stems where small apothecia m ay be found. It is likely t h a t winter oilseed rape plants become infected in A ut um n. Frequently no s y m p t o m s are observed for several weeks. The first s y m p t o m s are often distortion of leaves and some bleaching, which can be m i s t a k e n for frost damage. The long latent period has reduced the effectiveness of chemical control, as fungicides are often applied after extensive infection. Acervuli frequently e r u p t t h r o u g h the epidermis following a s u d d e n cold weather. Recent studies in the UK have helped to clarify aspects of the epidemiology and have highlighted the importance of t e m p e r a t u r e and leaf w et ness in establishing infection and the length of the latent period (Figueroa et al., 1994, 1995a, 1995b), and timing of fungicide applications (Jeffery et al., 1994). The f u n g u s is heterothallic with two mating types which can be readily crossed in culture (Ilot and Ingram, 1984; Coutrice and Ingram, 1987). This fungus h a s been u s e d for more f u n d a m e n t a l studies on the control of sexual reproduction of fungi (Chamberlain et al., 1995). The relative importance of sexual reproduction in the field as a m e a n s of generating variation is not known. Non pathogenic m u t a n t s of the fungus have also been produced which have provided evidence for the involvement extracellular protease in pathogenicity (Ball et al., 1991). There is variation in the degree of resistance within existing oilseed rape cultivars. However, the resistance ratings of cultivars change quickly with time, and widely grown cultivars which have been r e s i s t a n t for one or two s e as o n s have become susceptible. This is likely to be due to changes in the frequency of virulence genes within the pathogen population. Studies by Majer (1997) u s i n g molecular m e t h o d s have d e m o n s t r a t e d genetic differences between populations of P. b r a s s i c a e from different parts of the UK and continental Europe. Breeding lines with high levels of resistance, derived from wild B r a s s i c a species have been developed (Majer, 1997). There is some evidence of differential h o s t - p a t h o g e n interactions (Maddock et al., 1981; Sim o n s and Skidmore, 1988; Majer, 1997) b u t well defined resistance genes a n d physiological races have yet to be adequately described. Unlike m a n y other crucifer pathogens, P . b r a s s i c a e appea rs to have a h o s t range limited to B r a s s i c a (Maddock and Ingram, 1981), suggesting t h a t it m a y be possible to introduce resistance genes into B r a s s i c a crops from related genera.
Downy mildew caused by Peronospora parasitica P e r o n o s p o r a p a r a s i t i c a is distributed almost all over the world (Verma et al., 1994) in parallel with A. candida. Various details of the disease are described by T h o m a s (1984), Kolte (1985), Tewari (1985), Lucas (1988), Paul and Rawlinson (1992) and Howard et al. (1994). An extensive bibliography is given by Verma et al. (1994).
390 Infection c a u s e d by P. parasitica c a n be localised or systemic (Kolte, 1985). All p l a n t stages are susceptible to infection. The localised lesions on leaves have greyish to whitish d o w n y growth (Figure 12.8), whereas, s u c h growth is p r e s e n t all over on the m a l f o r m e d p l a n t parts. This growth consists of c o n i d i o p h o r e s p r o d u c i n g conidia. The oospores are p r o d u c e d e m b e d d e d in the h o s t tissue. Isolates of P. parasitica are either homothallic or heterothallic (Sherriff a n d Lucas, 1989). B r a s s i c a n a p u s isolates are p r e d o m i n a n t l y homothallic. C o n t r i b u t i o n of P. p a r a s i t i c a to c a u s a t i o n of s y m p t o m s of disease is h a r d to a s s e s s , as often A. c a n d i d a is also p r e s e n t c a u s i n g a complex infection especially on the h y p e r t r o p h i e d inflorescent stems. The p a t h o g e n h a s a b r o a d h o s t r a n g e in the family Brassicaceae. Economically it is i m p o r t a n t on the oleiferous a n d vegetable crucifers especially r a d i s h where seed loss of u p to 58% h a s been reported (Achar, 1992). It is r e g a r d e d as one of the m o s t d a m a g i n g diseases of oilseed r a p e in Sweden a n d Poland (Paul a n d Rawlinson, 1992). G e r m a n y a n d Britain also experience severe a t t a c k s d u r i n g certain y e a r s (Paul a n d Rawlinson, 1992). The disease is also economically i m p o r t a n t on B. j u n c e a in India. It is considered relatively u n i m p o r t a n t in C a n a d a . However, its c o n t r i b u t i o n to yield losses is h a r d to a s s e s s as often A. c a n d i d a is a s s o c i a t e d with this disease syndrome. C o n s i d e r a b l e r e s e a r c h is being done towards elucidating the m e c h a n i s m of p a t h o g e n e s i s / r e s i s t a n c e a n d finding s o u r c e s of r e s i s t a n c e to P. parasitica in crucifers (Dickson a n d Petzoldt, 1993; M a t s u m o t o , 1994; N a s h a a t a n d Rawlinson, 1994; W a n g et al., 1994; G u p t a et al., 1995; N a s h a a t a n d Awasthi, 1995). T r e a t m e n t of A. thaliana with 2,6-dichloroisonicotinic acid (INA) r e s u l t s in r e s i s t a n c e to P. parasitica a n d e x p r e s s i o n of genes corresp o n d i n g to three p r o t e i n s (Uknes et al., 1992). P a t h o g e n infection a n d salicylic acid (SA), the latter being a putative e n d o g e n o u s signal for acquired resistance, also c a u s e e x p r e s s i o n of these genes. It h a s been s h o w n t h a t INAa n d SA- i n d u c e d systemic a c q u i r e d r e s i s t a n c e (SAR) is not an ethylene-dep e n d e n t p r o c e s s (Lawton et al., 1994). SAR is also activated in the other rosette leaves of a n ecotype of A. thaliana u p o n d e v e l o p m e n t of a limited lesion r e s p o n s e c a u s e d by F u s a r i u m o x y s p o r u m in the lower leaves (MauchMani a n d S l u s a r e n s k o , 1994). Molecular m a p p i n g of some r e s i s t a n c e loci in P. p a r a s i t i c a h a s been done (Crute et al., 1993; P a r k e r et al., 1993; Tor et al., 1994). Positive correlation h a s been found between r e s i s t a n c e to P. parasitica a n d L. m a c u l a n s in B. rapa (Mitchell-Olds et al., 1995). C u l t u r a l a n d chemical control of P. parasitica h a s been developed (Howard et al., 1994). V e r t i c i l l i u m wilt c a u s e d by V e r t i c i l l i u m d a h l i a e Kleb This p a t h o g e n h a s a world wide distribution on m a n y p l a n t s (Schnathorst, 1981; S u b b a r a o a n d H u b b a r d , 1996). Verticillium wilt or stem rot of crucifers is i m p o r t a n t in c o u n t r i e s s u c h as Sweden, G e r m a n y and U.S.A. (Paul a n d Rawlinson, 1992; Koike et al., 1994). Long periods of rape cultivation or rape being f r e q u e n t in r o t a t i o n s m a y r e s u l t in yield losses of u p to
391 50% (Paul a n d Rawlinson, 1992). Various a s p e c t s of the d i s e a s e are described in Paul a n d Rawlinson (1992). The leaves t u r n chlorotic a n d bronze on one side. The s t e m s h o w s b r o w n i s h d i s c o l o u r a t i o n in the form of s t r e a k s on one side. This u n i l a t e r a l colonization m a y be due to c o m p a r t m e n t a l i z a t i o n of the p a t h o g e n (Baayen et al., 1996). N u m e r o u s microsclerotia are p r o d u c e d at the b a s e of s t e m s a n d roots. Although m u c h work h a s been d o n e on Verticillium d i s e a s e s of p l a n t s s u c h as cotton a n d potato, little r e s e a r c h h a s been done on the disease on crucifers. There is little or no h o s t specificity a m o n g the isolates of V. d a h l i a e (Chang a n d E a s t b u r n , 1994; Ligoxigakis a n d Vakal o u n a k i s , 1994; S u b b a r a o et al., 1995). Molecular a n a l y s i s of V. d a h l i a e isolates h a s been done by Koike et al. (1995). Also, a PCR-mediated a s s a y of V. d a h l i a e directly from soil h a s been developed (Vollossiouk et al., 1995). G r u n z e l m a n n et al. (1991) developed a diagnostic ELIZA t e c h n i q u e u s i n g commercially available a n t i b o d i e s a g a i n s t V. dahliae. V. d a h t i a e microsclerotia c a n survive in the soil for u p to 10 y e a r s a n d are crucial in c a u s i n g infection of p l a n t s ( S u b b a r a o a n d H u b b a r d , 1996). Disease control is a c c o m p l i s h e d by a c o m b i n a t i o n of chemical a n d c u l t u r a l m e t h o d s ( S u b b a r a o a n d H u b b a r d , 1996). Verticillium wilt does n o t o c c u r on commercial broccoli crops a n d microsclerotia do not form on its roots (Subb a r a o et al., 1995; S u b b a r a o a n d H u b b a r d , 1996). I n c o r p o r a t i o n of broccoli residue in soil r e s u l t s in a r e d u c t i o n in n u m b e r of microsclerotia a n d s u b s e q u e n t lower wilt incidence in cauliflower ( S u b b a r a o a n d H u b b a r d , 1996).
C l u b r o o t c a u s e d by P l a s m o d i o p h o r a b r a s s i c a e Wor. P l a s m o d i o p h o r a brassicae, a n d o t h e r m e m b e r s of the P l a s m o d i o p h o r a les, are of u n c e r t a i n phylogenic affinities. They are conventionally placed within the m y x o m y c e t e s , largely due to the p l a s m o d i a l p h a s e of their life history. O t h e r a s p e c t s of their biology s u c h as distinctive type of n u c l e a r division a n d probable holozoic mode of n u t r i t i o n are more typical of protozoa (Buczacki, 1983). While this p a t h o g e n was described more t h a n a h u n d r e d y e a r s ago, a n d c a u s e s significant a m o u n t of d a m a g e on crucifer crops in all t e m p e r a t e areas, several a s p e c t s of its life history still require elucidation. The m o s t widely accepted life history is t h a t p r o p o s e d by I n g r a m a n d Tomm e r u p (1972). A haploid resting spore g e r m i n a t e s to p r o d u c e a motile zoospore which e n c y s t s on a root h a i r or e p i d e r m a l cell of a potential host, a n d injects a u n i n u c l e a t e p l a s m o d i u m . A m u l t i n u c l e a t e p l a s m o d i u m develops in the h o s t cell by meiotic division, followed by cleavage into a m a s s of zoospor a n g i a which p r o d u c e f u r t h e r zoospores. These s e c o n d a r y z o o s p o r e s migrate to the base of the root hair a n d p e n e t r a t e f u r t h e r into the root tissue. They m a y also be released b a c k into the soil to infect other e p i d e r m a l root cells. Within the root, large m u l t i n u c l e a t e p l a s m o d i a develop a n d root galls are p r o d u c e d as a r e s u l t of h o s t cell e n l a r g e m e n t a n d division. K a r y o g a m y m a y occur d u r i n g this p h a s e , to be followed by meiotic division a n d the cleavage
392 of the p l a s m o d i u m to p r o d u c e resting spores which are liberated into the soil following root decay. A modification of this life cycle h a s b e e n suggested by Mithen a n d M a g r a t h (1992); t h o u g h basically similar, it gives more e m p h a s i s to an amoeboid p h a s e of the p a t h o g e n which actively moves t h r o u g h the cortical root cells prior to the d e v e l o p m e n t of s e c o n d a r y plasmodia. Some aspects of the life history have been described in great detail; for example the initial p e n e t r a t i o n of root h a i r s cells by p r i m a r y zoospores (Aist a n d Williams, 1971), a n d the cruciform mitotic divisions (Garber a n d Aist, 1979). P l a s m o d i o p h o r a b r a s s i c a e infects the majority of c r u c i f e r o u s species. It is i m p o r t a n t in h o r t i c u l t u r a l B r a s s i c a production, a n d the majority of commercial cultivars of B. o l e r a c e a a n d B. r a p a are highly susceptible. It c a u s e s severe losses on crops s u c h as cauliflower a n d chinese cabbage. It is import a n t on oilseed b r a s s i c a s in c e r t a i n areas, s u c h as S c a n d i n a v i a a n d p a r t s of C a n a d a . R e s i s t a n c e o c c u r s in B. rapa, B. o l e r a c e a a n d B. n a p u s b u t it h a s been difficult to t r a n s f e r to commercially s u c c e s s f u l cultivars. The presence of r e s i s t a n c e genes h a s led to two s y s t e m s of p a t h o g e n classification. Williams (1966) developed a s y s t e m for the d e t e r m i n a t i o n of r a c e s of P. brass i c a e which h a s been widely a d o p t e d in North America, while Buczacki et al. (1975) developed a set of five a c c e s s i o n s each of B. rapa, B. n a p u s a n d B. oler a c e a which have become k n o w n as the E u r o p e a n Clubroot Differential set (ECD), a n d devised a n u m e r i c a l s y s t e m for c h a r a c t e r i s i n g P. b r a s s i c a e pathotypes. Field isolates of P. b r a s s i c a e (i.e. spores isolated from infected field grown plants) have been s h o w n to consist of several p a t h o t y p e s . This h a s m o s t effectively b e e n d e m o n s t r a t e d t h r o u g h the d e v e l o p m e n t of single spore isolates by artificially inoculating a p l a n t with a single zoospore (Buczacki, 1977; Haji Tinggal a n d Webster, 1981; J o n e s et al., 1982; Voorrips, 1996). Single spore isolates are being u s e d for the m o l e c u l a r c h a r a c t e r i s a t i o n of genetic variation in P. b r a s s i c a e with RAPD a n d other m o l e c u l a r m a r k e r s (Buhariwalla et al., 1995). Molecular m e t h o d s a n d polyclonal antibodies are being investigated in order to develop m e t h o d s of detection of resting spores in soil ( W a k e h a m a n d White, 1996).
R e s i s t a n c e in B. r a p a is d e t e r m i n e d by a series of single d o m i n a n t genes. Certain s t u b b l e t u r n i p lines, s u c h as ECD04, p r o b a b l y contain three s e p a r a t e r e s i s t a n c e genes which provide effective r e s i s t a n c e a g a i n s t the majority of p a t h o t y p e s (Wit, 1964). The interaction between P. b r a s s i c a e a n d B. r a p a m a y be of the classic gene-for-gene n a t u r e , b u t so far it h a s not been possible to genetically a n a l y s e virulence factors in the p a t h o g e n . Resistance in B. o l e r a c e a h a s traditionally been considered to be non-differential a n d det e r m i n e d by a series of recessive r e s i s t a n c e genes (Chiang a n d Crete, 1976; H a n s e n , 1989). Some genotypes of B. oleracea, s u c h as ECD 15 a n d Bohmerwaldkohl c o n t a i n very high levels of resistance. Interestingly, isolates which c a n overcome the ECD04 r e s i s t a n c e of B. r a p a are a v i r u l e n t on these lines (Crute et al., 1983). B. n a p u s c o n t a i n s both types of r e s i s t a n c e , although c o m m e r c i a l cultivars lack the high levels of r e s i s t a n c e which c a n be found in both diploid progenitors. G u s t a f s s o n a n d Falt (1985) have s h o w n t h a t there
393 are likely to be four r e s i s t a n c e genes in B. napus. Synthetic lines of B. n a p u s have been developed c o m b i n i n g the m o s t effective s o u r c e s of r e s i s t a n c e from B. oleracea a n d B. rapa a n d this r e s i s t a n c e h a s been t r a n s f e r r e d into oilseed rape breeding lines (Diederichsen et al., 1995). R e s i s t a n c e genes in the respective B r a s s i c a g e n o m e s are being m a p p e d t h r o u g h the u s e of m o l e c u l a r m a r k e r s which m a y help with their i n t r o g r e s s i o n into agronomically acceptable genotypes (Figdore et al., 1993; L a n d r y et al., 1992; G r a n d c l e m e n t a n d T h o m a s , 1996). A r e s i s t a n c e gene h a s also been described in the model crucifer A r a b i d o p s i s thaliana which m a y be a m e n a b l e to cloning (Fuchs a n d Sacristan, 1996). In the a b s e n c e of r e s i s t a n c e cultivars, crop rotation, application of lime to raise pH levels, good drainage a n d s a n i t a t i o n are r e c o m m e n d e d to control clubroot. Boron is k n o w n to be effective at r e d u c i n g clubroot s y m p t o m s , possibly by inhibiting s p o r o g e n e s i s in infected cells (Dixon, 1996). O t h e r chemical control m e t h o d s are also partially effective i n c l u d i n g the u s e of the nitrogen fertiliser c a l c i u m c y a n a m i d e (Klasse, 1996). It h a s also been s u g g e s t e d t h a t trap crops of r e s i s t a n t B r a s s i c a cultivars c a n be u s e d as a control m e a s ure. These s t i m u l a t e the g e r m i n a t i o n of the resisting spores b u t do not allow the p a t h o g e n to complete its life cycle.
Other fungal diseases M e m b e r s of B r a s s i c a c e a e suffer from m a n y other fungal d i a s e a s e s , m o s t of which are described in Kolte (1985), Williams (1985) a n d Paul a n d Rawlinson (1992). Some of these d i s e a s e s s u c h as the white leaf spot a n d gray stem c a u s e d by Mycosphaerella capsellae are w i d e s p r e a d a n d c a u s e severe d a m a g e b u t there is little information on their effect on yield (Paul a n d Rawlinson, 1992). The white leaf spot p h a s e is often c o n f u s e d with Alternaria blackspot. In w e s t e r n C a n a d a , the gray s t e m p h a s e develops too late in the s e a s o n to c a u s e a n y appreciable yield loss. D a m p i n g off a n d root rot d i s e a s e s c a u s e d by Rhizoctonia solani K ~ h n are w i d e s p r e a d in w e s t e r n Can a d a especially in the n o r t h e r n prairies a n d are u s u a l l y c a u s e d by a n a s t o m o s i s group AG 2-1 (Sippell et al., 1985; Tewari et al., 1987; Yitbarek et al., 1987). The d i s e a s e also a p p e a r s to be of some i m p o r t a n c e in G e r m a n y (Paul a n d Rawlinson, 1992). Powdery mildew c a u s e d by E r y s i p h e polygoni DC. is c o m m o n in some p a r t s of the world, b u t u s u a l l y does not c a u s e a n y m a j o r d a m a g e (Kolte, 1985; Paul a n d Rawlinson, 1992). In w e s t e r n C a n a d a , this p a t h o g e n infects canola late d u r i n g the s e a s o n a n d is not damaging.
Acknowledgement A p a r t of this w o r k was s u p p o r t e d by a n o p e r a t i n g g r a n t (OGP 0491) from the Natural Sciences a n d Engineering R e s e a r c h Council of C a n a d a to J. P. Tewari.
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411 Wu, C. R. a n d Liu, H. L. 1991. Selection of rape (Brassica napus L). callus c u l t u r e s r e s i s t a n t to oxalic acid. Cruciferae Newsl. pp. 14-15 a n d 80-81. Yadav, O. P., Yadav, T. P., K u m a r , P. a n d G u p t a , S. K. 1994. I n h e r i t a n c e of r e d u c i n g s u g a r s in relation to white r u s t r e s i s t a n c e in I n d i a n m u s t a r d . Indian Phytopathol. 47, 56-59. Yadav, R. C., Sareen, P. K. a n d C h o w d h u r y , J. B. 1991. Interspecific hybridization in Brassica juncea x B. tournefortii u s i n g ovary culture. Cruciferae Newsl. 14-15, 84. Y a r h a m , D. J. a n d Giltrap, N. J. 1989 Crop d i s e a s e s in a c h a n g i n g agriculture: a r a b l e crops in the UK.: a review. Plant Path. 38, 4 5 9 - 4 7 7 . Yitbarek, S. M., Verma, pathogenecity seedlings a n d chewan. Can.
P. R. a n d Morrall, R.A.A. 1987. A n a s t o m o s i s groups, a n d specificity of Rhizoctonia solani isolates from a d u l t r a p e s e e d / c a n o l a p l a n t s a n d soils in S a s k a t -
J. Plant Path. 9, 6-13.
Zhu, J. S., S t r u s s , D a n d R6bbelen, G. 1993. S t u d i e s on r e s i s t a n c e to Phoma lingam in Brassica napus-Brassica nigra addition lines. Plant Breeding 111, 102-197.
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Biology of Brassica Coenospecies C. G6mez-Campo (Editor) 91999 Elsevier Science B.V. All rights reserved.
413
13 B R E E D I N G : AN O V E R V I E W Heiko C. Becker (1), H a r m L6ptien (2) a n d G e r h a r d R6bbelen (1)
(1) Institut ffir Pflanzenbau und Pflanzenzfichtung, Georg August Universitdt, G6ttingen (2) Marner GZG Saaten AG, K6nigstrasse 47, D-25709 Marne, Germany Plant b r e e d i n g is a technology b a s e d on n u m e r o u s empirical skills a n d e s t a b l i s h e d practices, b u t also on critical scientific knowledge s u c h as t h a t described in the p r e c e d i n g c h a p t e r s . The sole aim of p l a n t breeding is the d e v e l o p m e n t of p l a n t cultivars with high p e r f o r m a n c e in farm p r o d u c t i o n s a n d the i m p r o v e m e n t of those p r e s e n t l y grown. F r o m its s t a r t to this very end every p l a n t breeding p r o g r a m requires more t h a n a decade of intensive research. Therefore, the financial i n v e s t m e n t r e q u i r e d t o d a y for a single new cultivar of a m a j o r crop species m a y easily exceed a few million US dollars. B e c a u s e of this long period a n d high financial i n v e s t m e n t the economic success of a p l a n t breeding p r o g r a m is highly d e p e n d e n t on the effectiveness of the s e p a r a t e o p e r a t i o n s employed as well as on a n optimized efficient succession of these individual steps within the general outline (Schnell, 1982a). Convincing evidence h a s been p r e s e n t e d above on the i m p o r t a n t p r o g r e s s which h a s been m a d e due to an i n c r e a s e d the u n d e r s t a n d i n g of the p l a n t genome a n d the t e c h n i q u e s of its m a n i p u l a t i o n at a m o l e c u l a r level d u r i n g the last one or two decades. High e x p e c t a t i o n s have b e e n p u t forward regarding their benefits in p l a n t breeding applications. However, so far only a few of these h o p e s have been proven a n d m o s t of the new cultivars p r o d u c e d until now still r e s u l t from r a t h e r traditional p r o c e d u r e s . With this backg r o u n d in mind, p r e s e n t c h a p t e r a i m s to elaborate on the p r e s e n t s t a t u s a n d future potential of p l a n t breeding within the Brassica coenospecies. The g e n u s Brassica c o m p r i s e s a considerable n u m b e r of crops of greatly diverging biological characteristics. The extent of this diversity clearly relates to their u s e s as well as to the history of their d o m e s t i c a t i o n (see c h a p t e r 2 a n d also P r a k a s h a n d Hinata, 1980). The h i g h e s t variability in p l a n t morphology is exhibited w h e r e the p l a n t s are c o n s u m e d as vegetables with root, stems, leaves, a n d t e r m i n a l or axillary b u d s s o m e t i m e s drastically modified to a t t a i n high nutritive qualities. This is p a r t i c u l a r l y true for Brassica oleracea, which h a s a long tradition of c o n s u m p t i o n in Europe. As early as in the 4th c e n t u r y B.C., T h e o p h r a s t o s described the u s e of two different types of
414 cabbage in Greece. Plinius described the cultivation of even six distinct Brassica cultivars in the Roman empire including types which resembled varieties of cabbage, kohlrabi, curly kale, or broccoli of today (K6rber-Grohne, 1987). During the distribution of these novel cultivated forms, new genetic characters were generated also p r e s u m a b l y by genetic introgression through hybridization with other related wild B r a s s i c a species (Gustafsson, 1981). Most interestingly, a parallel evolution occurred in China with B. rapa and B. j u n c e a . In these species, too, ideotypes were developed by m e a n s of selective cultivation, which formed modified plant organs with particular qualities for vegetable uses. For example, as in the E u r o p e a n B. oleracea, varieties of B. rapa and B. j u n c e a evolved which exhibited head forming terminal buds, or thickened shoot stems, or com pr es s ed and enlarged inflorescences (Wen, 1980; Toxopeus et al., 1987). Much less diversity in phenotype is evident from species cultivated for foraging p u r p o ses , where the growers were mainly interested in the total m a s s of green m a t t e r of the above-ground stands. Plants in which seed is the only harvest, s u c h as the condiment m u s t a r d or in seedoil uses, plant phenotypes r e main ed even more uniform, and they differ from their ancestral weeds in little more t h a n seed retention and seed size. However, two oilseed forms have evolved in the temperate climate of Central Europe within B. nap u s and B. rapa, one for prewinter sowing and one for spring sowing. These do not only differ in their tolerance to cold b u t also in their photoperiodical adaptation, constituting a n o t h e r i m p o r t a n t agronomical distinction. Recently, the oilseed producing brassicas have attracted considerable attention in the world m a r k e t after their seedoil u n d e r w e n t f u n d a m e n t a l quality improvements by genetic selection, which primed rapeseed oil to become one of the world's m o s t valuable health food commodities. This change-over was started 25 years ago by the amphidiploid B. n a p u s , b u t dragged other "rapeseed" species along, s u c h as B. rapa, B. j u n c e a and B. carinata. Notwithstanding all this morphological diversity, the activity of the breeder is still more affected by differences in reproductive systems of the plants. Most of the diploid B r a s s i c a species represent self incompatible, outcrossing forms, while the amphidiploids are predominantly self compatible and inbreeding. The latter, of course, are the phylogenetically younger species (see c h a p t e r 3). Correspondingly, B. n a p u s is still subject to insufficient shattering resistance, while because of their longer history of cultivation B. j u n c e a and B. c a r i n a t a contain more s h a t t e r resistant types. However, the greatest diversity resides within the B. oleracea and B. rapa group, where the diploid n a t u r e and the n a t u r a l cross fertilization coincide with the most ancient cultivation history allowing for wide ecological dispersal and variable crop characteristics. Again, 'Yellow Sarson', an Indian oilseed subspecies of the diploid B. rapa, not only attained large seeds and s h a t t e r resistant siliques, b u t also self compatibility. In this way, crop development is contin u o u s l y s u p e r i m p o s e d by the effects of (unconscious or conscious) selection a n d corresponding genetic changes.
415
Breeding objectives Vegetable brassicas Since each Brassica vegetable is considered typical for its special characteristics, individual breeding p r o g r a m s have to observe r a t h e r different goals and priorities. Here, for r eas ons of limited space the situation is mainly presented for the case of breeding white cabbage. This B. oleracea form can roughly be grouped according to the u s e s of its harvested heads. If cultivars are to be grown for industrial processing, highest possible yields, i.e. high head weights, d e m a n d first priority. However, the reverse is also followed in cabbage breeding, i.e. varieties with relatively small h e a d s and low n o n - w r a p p e r leaf size allow for dense planting. This cabbage is to be delivered fresh to the vegetable m a r k e t s and greengroceries, and in this trade low head weights are w ant ed convenient for the small sized family of today. This m a r k e t sector again requires two plant types, one of which is sold directly from the field, while the other needs to tolerate longer storage in a cold depot; the latter type is u s u a l l y distinguished by high dry m a t t e r content as well as by a tight waxy layer. This waxiness of the leaves additionally provides effective protection against biotic and abiotic stress not only during storage, b u t also in the field. For instance, a positive correlation was found to exist between the degree of leaf waxiness and resistance a g a i n s t infestation with Albugo candida and Alternaria ssp. (Subudhi and Raut, 1994). In designing the cabbage ideotype, the breeder h a s to consider t h a t m a r k e t d e m a n d s can be highly divergent in different countries. In Central Europe, a c o m p a c t head is requested for industrial processing, while in the former Yugoslavia or in Turkey more loose leafed h e a d s are preferred. Many m a r k e t s see special value in r o u n d heads, b u t others desire pronouncedl y fiat and even pointed headed cabbages. The situation is similar in Chinese cabbage, B. rapa subsp, pekinensis: more ovate and more cylindrical heads are favored in different geographical regions. With regards the main production characters, white cabbage is known to exhibit a wide variation, e.g., in harvest date. For cultivation in Central Europe varieties are available which are ready for harvest from 55 to 150 days after transplanting. A similar s p e c t r u m of cultivars is offered for the Chinese cabbage with m a t u r i t y dates between 55 and 110 days after sowing (Opena et al., 1988). For some forms, for instance curly kale or the late ripening savoy cabbages, winter h a r d i n e s s is of considerable relevance. Here, FI hybrid varieties between white and the m o s t h a r d y Savoy cabbages have been developed specifically for English productions. Based on their frost tolerance these intermediate types with attractive head qualities can be marketed in England all t h r o u g h the winter u p to March directly from the field. F u r t h e r e m i n e n t breeding aims consist in resistance against bolting and splitting of the heads. Good s t a n d i n g ability is a n o t h e r i m p o r t a n t condition for adequate plant health and easy mechanical harvesting. For r e a s o n s of
416 high p r o d u c t quality, p a r t i c u l a r a t t e n t i o n m u s t be paid to physiologically conditioned d i s e a s e s s u c h as t i p b u r n c a u s e d by local calcium deficiency in the leaf tissue. Finally, it goes w i t h o u t saying t h a t genetic p a t h o g e n resist a n c e (see c h a p t e r 12) is a n essential r e q u i r e m e n t for all types of vegetables, since c h e m i c a l m e a s u r e s of p l a n t protection are as u n d e s i r e d today as for example m y c o t o x i n s derived from fungal infestations. For obvious r e a s o n s , the b r e e d e r m u s t pay careful a t t e n t i o n to the chemical composition of the m a r k e t e d vegetable. For i n s t a n c e , a high c o n t e n t of vitamin C gives rise to a n improved n u t r i t i o n a l value a n d also prevents greyish discoloration of the " s a u e r k r a u t " , the pickled white cabbage (K~nsch et al., 1989) on i n d u s t r i a l processing. Another role is a s s i g n e d to the glucosinolates, which for long were considered to be noxious a l t h o u g h the positive value of some of t h e s e c o m p o u n d s as antimicrobial or a n t i c a n c e r o g e n i c factors is now well recognized. Glucosinolates are also e s s e n t i a l flavor c o m p o n e n t s a n d it is r e c o m m e n d e d to reduce the c o n t e n t s of sinigrin a n d progoitrin, which both g e n e r a t e b r e a k d o w n p r o d u c t s with a bitter taste in favor of other glucosinolate c o n s t i t u e n t s (Olsson, 1993). Oilseed brassicas
The r a p e s e e d h a r v e s t from the respective B r a s s i c a species is processed to p r o d u c e two m a i n products: oil a n d meal. The oil is u s e d for h u m a n cons u m p t i o n , as salad oil or in the form of m a r g a r i n e , as well as for technical p u r p o s e s , s u c h as l u b r i c a n t s a n d h y d r a u l i c oils, as base chemicals for the o l e o c h e m i s t r y or biodiesel fuel (Shahidi, 1990; Kimber a n d McGregor, 1995). The meal which r e m a i n s after the oil extraction is high in nutritionally firstclass proteins. However, today b e c a u s e of the c r u d e traditional "oil-milling"process, the defatted meal or oilcakes are largely only a s s i g n e d to a n i m a l feed m i x t u r e s or even to fertilizer uses. Recently, the valuable proteins of the r a p e s e e d received new attention, including for u s e in h u m a n c o n s u m p t i o n , a n d gentle oil e x t r a c t i o n t e c h n i q u e s b a s e d on "green chemistry" u s i n g enzyme m a c e r a t i o n a n d avoiding organic solvents are being developed (Jensen et al., 1990). These m a y finally allow f u r t h e r c o - p r o d u c t s , s u c h as lecithins, tocopherols (i.e. vitamin E), hulls a n d fibres to be o b t a i n e d as well as glucosinolates with a h i g h e r quality t h a n ever before (Bagger a n d Sorensen, 1996). T h u s , t a k i n g all potential p r o d u c t s into a c c o u n t , s u c h new concepts will promise economic feasibility a n d greatly add value to even the high-valued r a p e s e e d c o m m o d i t y of today.
Yield p e r f o r m a n c e In the b r e e d i n g of rapeseed, the first a n d ever i m p o r t a n t t a s k is to increase the seed h a r v e s t s per u n i t l a n d surface (see Table 13.1, a n d reviews by A n d e r s s o n a n d Olsson, 1961; Downey a n d R6bbelen, 1989; Downey a n d
417 Rimmer, 1993; Buzza, 1995). This trait, however, is n e i t h e r simple nor indep e n d e n t of e n v i r o n m e n t a l influences a n d agronomic practices. Primarily, the seed yield is c o m p o s e d of three d e t e r m i n i n g c o m p o n e n t s , i.e. the n u m b e r of siliques per u n i t area (determined as n u m b e r of siliques per p l a n t + the n u m b e r of p l a n t s per u n i t area), the n u m b e r of seeds per silique, a n d the individual seed weight. Since these traits are negatively correlated with each other, yield i n c r e a s e s of cultivars u s u a l l y r e s u l t from small i m p r o v e m e n t s r a t h e r t h a n from a m a r k e d step u p w a r d s in one of these comp o n e n t s . Principally all seed m a t t e r is derived from p h o t o s y n t h e s i s in the green leaves a n d s h o o t surfaces a n d the s u b s e q u e n t allocation of these prim a r y a s s i m i l a t e s into the developing seed. This is why the breeder aims for an o p t i m u m h a r v e s t index, i.e. a high seed weight h a r v e s t e d as p e r c e n t of the total p l a n t dry m a t t e r produced. This index averages a b o u t 35% for the best E u r o p e a n w i n t e r - r a p e s e e d cultivars, b u t m a y reach values above 40% by c o n t i n u e d selection. However, for e x p e r i m e n t a l r e a s o n s the breeder rarely if ever d e t e r m i n e s the dry m a t t e r p r o d u c t i o n directly, b u t i n s t e a d selects for a n imagined p l a n t ideotype, which he a s s u m e s to secure a sufficient productivity in the vegetative p h a s e a n d a high t r a n s l o c a t i o n efficiency after flowering (Thurling, 1991). This ideotype conception is b a s e d on his long p e r s o n a l experience with high yielding genotypes at his e x p e r i m e n t station. It not only includes morphological p l a n t c h a r a c t e r s , s u c h as total p l a n t height, internode p a t t e r n , b r a n c h i n g s t r u c t u r e , or leaf a r e a index, b u t also implies developm e n t a l traits, s u c h as seedling vigor, leaf rosette formation, flowering initiation, or length of the seed filling period. Some of the morphological c h a r a c t e r s , in p a r t i c u l a r p l a n t height or basal s h o o t diameter, also c o n t r i b u t e to lodging resistance, which is essential for the final low-loss m e c h a n i c a l h a r v e s t of the seed produced. In addition, early lodging of the green p l a n t greatly i m p a i r s assimilation a n d translocation p r o c e s s e s a n d t h e r e b y m a y reduce seed yield a n d quality considerably. In the same way, a n y other d a m a g e to the green p l a n t surface is d e t r i m e n t a l to yield, w h e t h e r it is yellowing or withering u n d e r salt, d r o u g h t , or h e a t s t r e s s or p l a n t decay from frost d a m a g e . Genetic differences in tolerance to these abiotic s t r e s s e s are well k n o w n between the different oilseed B r a s s i c a species. B. n a p u s , particularly its winter form, is best a d a p t e d to the coastal or at least cooler n o r t h e r n areas. In the c o n t i n e n t a l or dryer a n d w a r m e r regions, B. j u n c e a or some spring types of B. rapa, a n d in the tropical highl a n d s of Ethiopia, B. c a r i n a t a forms are the m o s t productive B r a s s i c a oilseed crops. In order to extend the growing a r e a of the oilseed crop "canola", extensive efforts have recently been directed in C a n a d a to the exploration a n d breeding of these "other" B r a s s i c a species (Rakow, 1995, 1997). In the future, it m a y be possible to transfer s t r e s s tolerance from these species to B. nap u s by interspecific hybridization. In all cases, however, screening for perform a n c e a n d a d a p t a t i o n h a s been of little success, if b a s e d on morphological or physiological traits. Breeders, therefore, still select for the best lines in the field u n d e r h a r s h n a t u r a l conditions. As w e a t h e r c h a n g e s from year to year,
418 T a b l e 13.1 Main target characters for yield improvement in oilseed brassicas.
Component traits:
Number of siliques per unit area Number of seeds per silique Seed weight
Securing traits:
Tolerance to late sowing Winter hardiness Effective regeneration for damage repair Fertiliser use efficiency Tolerance against heat, drought, or salt stress Lodging resistance Uniform seed ripening Early maturity Shattering resistance Disease and pest resistance
T a b l e 13.2 Main diseases of oilseed rape (Brassica napus) in Europe and the prospects of breeding for resistance. Scores range from 1 - low, to 9 = high (after R6bbelen, 1994, modified).
Pathogen
Occurrence
Damage potential
Resistance derived from
Breeding potential
Phoma lingam Sclerotinia sclerotiorum Plasmodiophora brassicae Verticillium dahliae Alternaria brassicae Cylindrosporium concentricum Botrytis cinerea Peronospora parasitica Erysiphe cruciferarum
7 7 4 6 7 5 6 5 2
8 7 7 6 4 4 3 3 1
7 (nap, jun, nig) 1 6 (ole, rap, nig) 5 (napus) 5 (S. alba) 7 (napus) 3 (napus) 2 (napus)
9 1 6 7 3 7 3 2 1
Virus (TuYV)
7
3
7 (rapa)
5
Heterodera spec.
6
2
5 (Sin, Raph)
5
419 stress resistance is a difficult trait to breed for. The same holds true for nutrient use efficiency, which is a m oder n d e m a n d advanced for r e a s o n s of lowering agricultural i n p u t s and minimizing loads to the environment. In particular, ground water pollution by mineral nitrogen fertilizer is u n d e r public debate, since rapeseed with its high nitrogen r e q u i r e m e n t for o p t i m u m yields and low nitrogen export by the harvested seed, m ay leave unfavorably high a m o u n t s of nitrogen in the harvested fields (Yau and Thurling, 1987 a,b). This draws attention to the last, b u t not the least i m p o r t a n t character for o p t i m u m yields, i.e. the agronomic suitability of the crop within the agricultural production system and its ecological a d a p t a t i o n to the given climatic conditions. In Central Europe, for example, winter forms of rapeseed are essential to m a t c h with the winter cereals in the same rotation. However, not the winter h a r d i n e s s of the rapeseed cultivars, b u t r a t h e r the missing rainfalls in August at sowing are the main obstacle to a satisfactory prewinter e s t a b l i s h m e n t of the stands. In these not infrequent situations, the p r o n o u n ced intolerance of B. n a p u s to late sowing (or late field emergence) may cause severe growth r e t a r d a t i o n s u n d e r the diminishing daylengths and temperatures. This photo- or thermoperiodic sensitivity is expressed to a m u c h greater extent in winter forms of B. n a p u s t h a n of B. rapa. Improved genetic neutrality in this respect might also help to escape late frosts by retarding precocious growth in early spring. But vice versa, every favorable day not utilized for dry m a t t e r production does m e a n a loss to yield potential (Diepenbrock and Grosse, 1995). Because of these complex relations, breeding of high performance cultivars will remain a regional business. Disease an d pest resistance In B r a s s i c a breeding, a great effort is always devoted to improve plant resistance against diseases and pests (see Table 13.2, and reviews by Davies, 1986; Paul an d Rawlinson, 1992; Rimmer a nd Buchwaldt, 1995). As in every other crop, several diseases cause problems in m o s t growing areas, others are of more local importance, while also individual species m ay suffer from the a ttack of specialized pathogens. Separate breeding p r o g r a m s towards disease or pest resistance will be rewarding only for the major cultivated species and against the m o s t h a z a r d o u s pathogens, irrespective of possible local calamities. When promising, the breeder will first try to establish effective ways of screening for r e s i s t a n t genotypes and to find sources of disease resistance, which t hen have to be transferred into a useful cultivar. Some principles of this "resistance breeding" are described here for three of the p a th o g en s which affect brassicas. In any rape growing area, particularly at coastal, h u m i d and wind-protected sites, epidemic infections by Sclerotinia s c l e r o t i o r u m can cause premature ripening, deficient seed filling, and extensive shat t eri ng in oilseed stands. Since the f u n g u s covers a wide host range, it is r a t h e r unlikely t h a t durable resistance can be established in cultivars, a l t h o u g h moderate tole-
420 r a n c e h a s b e e n reported to o c c u r in some B r a s s i c a lines (Kolte, 1985; S e d u n et al., 1989). Therefore, a p e t a l o u s m u t a n t s have been p r o p o s e d to provide a n alternative m e a n s of r e d u c i n g Sclerotina a t t a c k s . Usually the s e n e s c e n t petals, w h i c h fall to the leaves or are lodged in the leaf axils after flowering, serve as a n ideal m e d i u m for the a s c o s p o r e s to g e r m i n a t e a n d the mycelium to p e n e t r a t e into the stems. This route is blocked in a p e t a l o u s forms; b u t productive cultivars of this type are still to be developed (Buzza, 1983; Fu et al., 1990; Thurling, 1991). O t h e r m e a n s of protection m a y be provided by gene technology. For example, w h e n the f u n g u s invades the plant, p r o d u c e s oxalic acid as a phytotoxin, which c a u s e s necrosis in the h o s t tissue. After i n t r o d u c t i o n of a gene isolated from w h e a t , which codes for oxalate oxidase, the extent of disease, i.e. the surface of leaf necrosis, was significantly smaller t h a n in n o n - t r a n s g e n i c isolines (Thompson et al., 1995; F r e y s s i n e t et al., 1995). Nowadays, m a n y similar strategies interfering with the p a t h w a y of pat h o g e n e s i s are being extensively studied by biotechnologists (see c h a p t e r 12). Worldwide of u t m o s t i m p o r t a n c e (except in China) is the 'phoma' disease, n a m e d after the a s e x u a l stage of the f u n g u s , Phoma lingam, belonging to the a s c o m y c e t e L e p t o s p h a e r i a maculans. This "blackleg" infection can proceed from b o t h p y c n o s p o r e s a n d a s c o s p o r e s , a n d the r e s u l t i n g mycelia after p e n e t r a t i o n c a n completely girdle the s t e m b a s e ("stem canker") and kill the plant. C h e m i c a l control is not economical p a r t i c u l a r l y in r a p e s e e d winter forms b e c a u s e of the long e x p o s u r e period to infection. B u t breeding h a s been p r o m i s i n g since genetic r e s i s t a n c e w a s first identified a n d widely u s e d in the F r e n c h cultivar 'Jet Neuf (Anonymous, 1982). Selections have been p u r s u e d in field n u r s e r i e s after n a t u r a l or artificially enforced (using diseased stubbles) infections. Meanwhile, g r e e n h o u s e tests by inoculation at the s t e m base have also been successfully applied. New s o u r c e s of resistance are now available in B. napus, being derived from species with the C-genome of B. nigra (Rimmer a n d van den Berg, 1992; Chevre et al. 1996) or from wild forms, e . g . B , rapa s u b s p , sylvestris (Crouch et al., 1994). It is, however, imp o r t a n t to observe t h a t for s u c h genetic testing isolates of L. m a c u l a n s from geographically d i s t a n t s o u r c e s are not t r a n s p o r t e d negligently from region to region on s e e d s or p l a n t residues, b e c a u s e genetic r e c o m b i n a t i o n between t h e m m a y r e s u l t in new i n c r e a s e d virulence (Petrie a n d Lewis, 1985). White r u s t from Albugo c a n d i d a infection m a y c a u s e s u b s t a n t i a l losses of seed yield w h e n it a t t a c k s a n d deforms the floral parts. However, serious d a m a g e is only k n o w n for B. rapa (with race 7 of the fungus) a n d B. j u n c e a (with race 2), while m o s t cultivars of B. n a p u s a p p e a r to be r e s i s t a n t to the p r e v a l e n t r a c e s (Fan et al., 1983). B u t for B. rapa, a high proportion of plants r e s i s t a n t to race 7 were found in the C a n a d i a n cultivar 'Tobin', a n d m a n y yellow seeded oriental m u s t a r d a c c e s s i o n s of B. j u n c e a were r e s i s t a n t to race 2 (Tiwari et al., 1988). M a n y i n s e c t s feed on the l u s h B r a s s i c a p l a n t s a n d those c a u s i n g serious d a m a g e vary with the region a n d p l a n t d e v e l o p m e n t (for review see Lamb, 1989; E k b o m , 1995). Most insect p e s t s of the oilseed b r a s s i c a s are
421 crucifer specialists, particularly a d a p t e d to the glucosinolates which are u b i q u i t o u s in this family. The same s e c o n d a r y c o m p o u n d s which act as feeding d e t e r r e n t s or toxins to livestock a n i m a l s , are a t t r a c t a n t s , feeding stimuli, or oviposition stimuli for the major insect p e s t s of rapeseed. So far, all these can be controlled by insecticides w i t h o u t risk of r e s i d u e s in the processed products; b u t the risk is always p e n d i n g t h a t insect p o p u l a t i o n s m a y develop pesticide resistance. So far, breeding for genetic h o s t p l a n t r e s i s t a n ce to insects h a s yielded very little success. Only selection for rapid seedling development or s h o r t e r flowering period h a s c o n t r i b u t e d to reduce insect damage. In c o m p a r a t i v e trials differences in insect a t t r a c t i o n have been demostrated: e.g., pod midges d a m a g e B. j u n c e a less t h a n B. n a p u s , or seedlings of Sinapis alba are less preferred by flea beetles. Transfer of these characters into B. n a p u s a n d B. rapa m a y be possible; b u t all screening, u s i n g mobile insect agents, is highly laborious indoors a n d unreliable outdoors.
Oilseed quality _
One of the m o s t s p e c t a c u l a r a d v a n c e s m a d e in recent p l a n t breeding h a s been the i m p r o v e m e n t in n u t r i t i o n a l quality of the traditional r a p e s e e d cultivars. The c h a n g e s from high to low erucic acid c o n t e n t of the oil a n d from high to low c o n t e n t of glucosinolates in the meal to p r o d u c e -the so-called "canola"- or 0 0 - r a p e s e e d cultivars have o p e n e d a l m o s t u n l i m i t e d a v e n u e s into the food a n d feed m a r k e t s . This s u c c e s s achieved with traditional breeding m e t h o d s h a s been frequently reviewed (e.g. Downey et at., 1975; Kramer et al., 1983; Downey a n d Rimmer, 1993; Scarth, 1995; Rakow, 1995; Uppstr6m, 1995). Today, r a p e s e e d quality is in the top class c o m p a r e d to other major oilseeds, so at this point the focus m a y be directed on the c h a r a c t e r s still left for f u r t h e r i m p r o v e m e n t (Figure 13.1). Oil: The oil is the m o s t valuable fraction of the seed. Within e s t a b l i s h e d cultivars grown on a large scale, the oil c o n t e n t in the air dry B r a s s i c a seed varies between 40 a n d 47 % for B. n a p u s , 36 a n d 44% for B. rapa, a n d 28 a n d 34% for B. j u n c e a . Within the first two species, winter forms definitely s u r p a s s the seedoil c o n t e n t of the c o r r e s p o n d i n g spring sown forms. Seed of 'Yellow Sarson' (see above), however, p r o d u c e s oil c o n c e n t r a t i o n s m u c h higher t h a n the u s u a l I n d i a n forms of B. rapa, reflecting the s u c c e s s of the long traditional selection of local farmers t o w a r d s large seeded a n d yellow coated, high-oil t u r n i p rape. Altogether, sufficient genetic variability is at h a n d for the breeder, to still secure a slow b u t s t e a d y increase of the oil c o n t e n t in new cultivars, in p a r t i c u l a r b e c a u s e of a d v a n t a g e s from a relatively high heritability (as c o m p a r e d to yield) a n d the i n t r o d u c t i o n of new efficient screening m e t h o d s , i.e. n u c l e a r magnetic r e s o n a n c e (NMR, Madson, 1976) a n d n e a r infrared reflectance s p e c t r o s c o p y (NIRS; T k a c h u k , 1981) for its analytical d e t e r m i n a t i o n . Breeding for oil quality s t a r t e d with the identification of p l a n t s with essentially no erucic acid in their seedoil in B. n a p u s (Stefansson et al., 1961)
422
22.5
Jlllllllllllllllllll Crude protein
IIIIIIIIIIIIIIIIIIII Vitamines, Cholin ~
Minerals Glucose Saccharose
Starch-
(1) o0 tl)
(1) .Q
38.3
m
~
1.1 0.7
E
n
5.0 7.7 ~2.5 4.9
c-.
t-
Hemicellulose 13.0
(D J=
Oligosaccharidea-
Phe~olicsothers-
Phytates Sinapovl esters
4.7
tim
L _ ,
Lignin
m
>
2.5
2.23.2
,
m
t~ (o ~
100 %
C
. . m
43.5
Cr Jde fat !
i
100 % Figure 13.1 Chemical composition of the seed of a 00-cultivar of oilseed rape, Brassica napus (after Thies, 1994).
423 and B. rapa (Downey, 1964), followed by "zero erucic" B. j u n c e a (Kirk and Oram, 1981) and B. carinata (Alonso et al., 1991). This change in erucic acid content led to c o n c o m i t a n t shifts of all other fatty acids c o m p o n e n t s for purely calculatory r e a s o n s (see Table 13.3). While the relative increase in linoleic acid (vitamin F) was welcomed by h u m a n nutritionists, the technological disadvantage of the relatively high linolenic acid cont ent induced extensive breeding efforts (R6bbelen and Thies, 1980a; Scarth et al., 1995). These yielded promising lines with no more t h a n 3% linolenic acid (Rficker a n d R6bbelen, 1996), b u t none of these h a s yet been established at larger scales in agricultural productions. F u r t h e r d e m a n d s of the food i n d u s t r y were met by the breeders after m ut ageni c seed t r e a t m e n t a n d extensive screening, with B. n a p u s forms containing high oleic acid a n d others (B. rapa) with elevated palmitic acid c o n t e n t s (Table 13.3). For oleochemical purposes, where rapeseed oils with high erucic acid contents are desired, B. n a p u s lines with up to 60% erucic acid have been selected from interspecific crosses with preselected parents, i . e . B , oleracea cony. botrytis x B. rapa subsp, trilocularis 'Yellow Sarson' (Lfihs and Friedt, 1994). But m u c h beyond any n a t u r a l variability, molecular t r ans f or m at i on today offers the potential to produce transgenic varieties with u n u s u a l fatty acid profiles or even principally new c o n s t i t u e n t s not synthesized in the conventional B r a s s i c a seed till now (Table 13.4; for review see Murphy, 1994; Voelker, 1997). Evidently, the composition of storage c o m p o u n d s in the B r a s s i c a oilseeds is r e m a r k a b l y open to change, a n d breeding is highly promising provided efforts are sufficiently c o n t i n u o u s an d intense. Meal: The reduction of glucosinolates in the traditional rapeseed to less t h a n 10 % of their original contents (see reviews cited above) was a n o t h e r dramatic d e m o n s t r a t i o n of the potential of conventional breeding (R6bbelen and Thies, 1980 b). Oilseed meal from the new 00-rapeseed cultivars now allows full exploitation of the valuable protein in animal feed mixtures. However, the net protein utilization of rapeseed meal is still m u c h below soybean meal and the total of u n d e s i r e d c o m p o n e n t s still a m o u n t s to more t h a n 25% of the dry m a t t e r (Figure 13.1; for review see Bell, 1993; Uppstr6m, 1995). Hemicellulose with a b o u t 13 % of the dry m a t t e r const i t ut es a major share of the detrimental meal constituents. Youngs (1967; see also Downey et al., 1975) was the first to draw attention to the thin seed coat of yellowseeded B r a s s i c a forms. Since t hen breeders have wished to reduce the total fiber co n te n t of the meal by introducing this easy-to-screen c h a r a c t e r into their cultivars. However, the a p p r o a c h was more complicated t h a n expected and the first phenotypically stable lines of B. n a p u s were obtained only recently (Stringham et al., 1974; Chen and Heneen, 1992; Rashid et al., 1994; Tang et al., 1997). In the dehulled meal fraction, on the other h a n d , soluble oligosaccharides are the major u n d e s i r e d carbohydrates, i.e. raffinose (0,3 %, Gal-Glu-Fru) and stachyose (2,5 %, Gal-Gal-Glu-Fru), which are undigestible for swine (and h u m a n s ; see also Slominski et al., 1995); they a c c u m u -
424 T a b l e 13.3 Fatty acid c o m p o s i t i o n o f seed oils from B r a s s i c a species (abridged after U p p s t r 6 m , 1995); ~A u l d e t a l . , 1992; 2 including 4% 16" 1. Cultivar/type (a)
Fatty acid content (%) (b) 16:0
18:0
18:1
18:2
18:3
20:1
22:1
Traditional types
n
Victor (winter)
3.0
0.8
9.9
13.5
9.8
6.3
52.3
n
Target (spring)
3.0
1.5
20.9
13.9
9.1
12.2
38.6
r
Duro (winter)
2.0
1.0
12.9
13.4
9.1
8.9
49.0
r
Echo (spring)
4.5
1.3
33.3
20.4
7.6
9.4
23.0
r
Yellow Sarson
1.8
0.9
13.1
12.0
8.2
6.2
55.5
j
Indian origin
2.5
1.2
8.0
16.4
11.4
6.4
46.2
c
Ethiopian mustard
3.2
0.9
9.8
16.2
13.9
7.5
41.6
Zero erucic types
n
Low 22:1
4
2
62
20
9
2
<1
n
Low 18:3
4
1
59
29
3
1
<1
n
High 18:1 1
4
2
80
4
5
2
<1
r
High 16:0
14 2
1
51
19
13
1
<1
a: n = B r a s s i c a n a p u s ; r = B. rapa; j = B. j u n c e a ; c = B. c a r i n a t a b: Fatty acids represented by carbon chain length and number of double bonds.
late in s e e d d e v e l o p m e n t a n d their c o n t e n t is highly v a r i a b l e with climatic c o n d i t i o n s , w h i c h m a k e s t h e m difficult to select for. Utilization of the seed a n d m e a l is f u r t h e r limited by phenolic acids not only with r e g a r d to a n i m a l feed, b u t also to u s e s of food-grade protein. Their c o n t e n t s in B r a s s i c a oilseeds e x c e e d s t h a t of cotton s e e d a n d p e a n u t by a factor of 10 a n d t h a t of s o y b e a n by 30. Similarly, p h y t a t e s exert a n t i n u t r i t i o nal p r o p e r t i e s in swine feeding, while s i n a p i c acid e s t e r s c a u s e the fishy taste of eggs laid by c e r t a i n h e n races. The l a t t e r two g r o u p s t o g e t h e r m a y r e a c h 10 % of the total seed d r y m a t t e r . B r e e d i n g for r e d u c e d c o n c e n t r a t i o n s h a s n o t yet b e e n s u c c e s s f u l . B u t sufficient genetic v a r i a t i o n is a p p a r e n t at l e a s t from o t h e r c r u c i f e r o u s s p e c i e s a n d q u i c k p h o t o m e t r i c m e t h o d s have b e e n e l a b o r a t e d r e c e n t l y for efficient a n a l y t i c a l s c r e e n i n g (Thies, 1991, 1994).
425 T a b l e 13.4 Varieties of oilseed rape with novel chemical compositions of their seed (Carruthers, 1995).
Non-transgenic varieties (1)
Transgenic varieties (2)
Available now
Zero-erucic acid ('double-low', 50-60% oleic acid) Medium-erucic acid (15-30%) High-erucic acid (40-55%)(HEAR)
Laurie acid (30-40%) Stearic acid (30-40%)
Anticipated in 5-15 years
Very-high-oleic acid (85%) Palmitic acid (10-20%) Very-low
Very-high-erucic acid (60-80%) Ricinoleic acid Petroselinic acid ~,-linolenic acid Epoxy fatty acids Wax esters Polyhydroxybutyrate Pharmac. peptides and proteins
1 Using conventional plant breeding and non-transformation based techniques 2 Using transformation based techniques
Genetical
resources
Because of the m a n y centuries of cultivation and their worldwide distribution, B r a s s i c a crops, - in fact vegetables more t h a n oilseed forms, - passed through an extensive differentiation into an a b u n d a n c e of morpho-, physioand chemotypes. Although regional centers originated during cultivation with specifically adapted landraces in popular use. For example, in Germany close to the city of Stuttgart a landrace of white cabbage, called 'Filderkraut', was grown in the fields until recently, which was conspicuous by its strong growth and its peculiar pointed head. Overall, such high diversity is the genetic base of all our present cultivars, and although no longer grown p e r se, these materials are still of great importance for every modern plant breeding programme; they are the primary resource for essential genes required for the development of new cultivars. Therefore, collections such as those in gene banks, but also individual genotypes existing in remote regions or in the h a n d s of hobby-gardeners will hopefully be conserved and publicly held available for future breeding exercises.
426 Records on the evolution of B. n a p u s as winter oilseed rape are particularly well d o c u m e n t e d in Central E u r o p e (for review see B a u r , 1944), where breeding s t a r t e d from a n u m b e r of l a n d r a c e s locally cultivated in the various rape growing regions. One of these, from which the y o u n g H a n s Lembke selected his ' L e m b k e s W i n t e r r a p s ' at the b e g i n n i n g of this c e n t u r y showed a n e x t r a o r d i n a r y potential for high crop p e r f o r m a n c e , since this landrace h a d evolved on the productive soils a n d in the suitable coastal s e a s o n s at the Baltic Sea, b u t also u n d e r frequent frost s t r e s s in the n o r t h e r n winters a n d d r o u g h t in the early s u m m e r s . A similar local l a n d r a c e from Oberschlesien provided the b a s e for ' J a n e t z k i ' s S c h l e s i s c h e r Winterraps', while other landraces also u s e d in Central E u r o p e were less successful in further cultivar evolution. Different winter rape m a t e r i a l s were derived in the first few deca d e s of this c e n t u r y from E a s t - E u r o p e a n , e.g., Polish l a n d r a c e s which were typically lower in height a n d less vigorous b u t highly frost tolerant, while W e s t - E u r o p e a n , e.g., F r e n c h origins were r a t h e r l u x u r i o u s vegetatively, b u t lacked w i n t e r h a r d i n e s s . Similar l a n d r a c e s are k n o w n for the other Brassica oilseed species a n d their c h a r a c t e r i s t i c s c a n be d e d u c e d from the local e d a p h i c a n d climatic local conditions of their respective c e n t e r s of origin a n d traditional d i s t r i b u t i o n (see c h a p t e r 2). On the other h a n d , long distance seed t r a n s f e r of the Old World B r a s s i c a oilseeds h a p p e n e d early. 'Mansholt's H a m b u r g e r Raps' was i n t r o d u c e d to J a p a n at the t u r n of the 20th century. D u r i n g World War II B r a s s i c a oilseeds were i n t r o d u c e d to C a n a d a , where they are still called 'Argentine' type (B. napus), or 'Polish' type (B. rapa) (Downey et al., 1975). W h e n ongoing available breeding l a n d r a c e s no longer offered sufficient variability to profit from selection, j u s t before the middle of this c e n t u r y b r e e d e r s began to s y s t e m a t i c a l l y compose the desired genetic variations in their basic breeding stocks. Infraspecific crossing between selected p a r e n t s of different l a n d r a c e s or p r e s e n t cultivars was the first a p p r o a c h to be applied. Next c a m e the knowledge t h a t a l t h o u g h spring forms of B. n a p u s exhibit a lower genetic variability, they often c o n s i d e r a b l y i n c r e a s e d selection gain in c r o s s e s with winter forms (Baur, 1944). Obviously, the gene pool of the spring forms is sufficiently different from t h a t of the winter types to provide positive c o m b i n i n g ability (Diers a n d Osborn, 1994; Knaak, 1996). Altogether, the existing high performing cultivars are the m o s t valuable initial breeding m a t e r i a l s a n d so far c o n s t i t u t e a n e s t i m a t e d 80 - 90 % of all genetic r e s o u r c e s applied in r a p e s e e d breeding p r o g r a m s . If r e q u i r e d for specific c h a r a c t e r s not available in the p r e s e n t cultivars, other more or less closely related B r a s s i c a species provide a "secondary gene pool" (Harlan a n d de Wet, 1971) to tap. After U (1935) h a d first synthesized B. n a p u s by interspecific crossing of B. rapa a n d B. oleracea (see c h a p t e r 3), r e s y n t h e s i s of the amphidiploid r a p e s e e d h a s repeatedly been reported in scientific p u b l i c a t i o n s (Andersson a n d Olsson, 1961). B u t the s t a t e m e n t of the last a u t h o r s in 1961 (p. 20): "The possibility of i n c r e a s i n g crop variation is of wide application to p r e s e n t a g r i c u l t u r a l practice" was definitely not true. The t e c h n i c a l difficulties of p r o d u c i n g interspecific h y b r i d s even between more d i s t a n t l y related g e n e r a in the B r a s s i c e a e today are minimized by mo-
427 dern in vitro techniques (see chapter 4). Nevertheless, relatively little use is made of experimentally resynthesized rapeseed in regular cultivar development. This m a y be so because, e.g., G e r m a n breeders clearly r e m e m b e r t hat for breeding the first zero erucic rapeseed cultivar, the semisynthetic cultivar 'Rapol' d e m o n s t r a t e d promising agronomic potential in its cross progenies, b u t had carried t h r o u g h u n n o t i c e d a high P h o m a sensitivity from its B. olerac e a p a r en t producing a devastating epidemic in the first year of commercial 0-rape production in Germany. Of course, these different related B r a s s i c a species contain m a n y useful genes (see the example given above on P h o m a resistance derived from the B. nigra C-genome). But again, allocating these genes into a well performing rapeseed cultivar is a serious m a t t e r of concern, the more alien and extended the transferred genetic s e g m e n t s are. For this reason, B r a s s i c a breeders have widely u s e d ionizing irradiation and chemical seed t r e a t m e n t to induce m u t a t i o n s and screen for desired traits in large progenies (e.g. Rakow, 1973; Velasco et al., 1995; R~cker and R6bbelen, 1996, 1997). This procedure is based on the a s s u m p t i o n t h a t except at the m u t a t e d site the rest of the genotype of the m u t a n t s represents the original high performing cultivar, which is, however, far from being confirmed. This expectation of genetic identity in the b a c k g r o u n d genotype is certainly more valid if molecular gene transfer is applied (see c h a p t e r 9). On the other h a n d , a new transgenic line does not m e a n a comprehensive improvement on the recipient cultivar. Except a single gene t r a n s p o s e d , it still represents the earlier genotype, which in its general characteristics of performance m a y soon be s u r p a s s e d by new cultivars as these c o n t i n u o u s l y emerge from breeders' activities. In this way, m u t a n t s a n d transgenics will finally only contribute some new genes to the c o m m o n pool of the species.
Operational s t e p s for breeding In order to create cultivars better t h a n existing ones and in compliance with specific breeding objectives, every breeding program is divided into three p h a s e s according to Schnell (1982a): 1) procuring appropriate initial variation; 2) establishing candi dat es for potential cultivars, a n d 3) selecting final cultivars via performance tests. Each of the p h a s e s involve different operations a n d it is the efficiency of these steps p e r s e as well as their effective positioning in the overall breeding method, which determines the pace of progress in breeding.
Procuring variation The first phase of breeding refers to the "genetic resources", i.e. to procure the initial breeding materials, which m u s t include the genetic variation
428 required for the p l a n n e d cultivar. In the breeding history of every crop, the origin a n d n a t u r e of t h e s e base m a t e r i a l s follow a typical s u c c e s s i o n starting with n a t u r a l l y o c c u r r i n g l a n d r a c e s of the species in q u e s t i o n a n d ending with highly e x p e r i m e n t a l genotypic p o p u l a t i o n s p r o d u c e d by sophisticated breeding p r o c e d u r e s . B r a s s i c a b r e e d e r s are u s u a l l y offered a wealth of genetic variations as was a l r e a d y described above. Often, for various r e a s o n s the desired c h a r a c teristics are c o n t a i n e d in a practically u s e l e s s genetic environment. Methods to trim s u c h initial genetic variation for first inclusion in a breeding prog r a m r a n g e from simple to r a t h e r s o p h i s t i c a t e d ones. Cultivation u n d e r appropriate n a t u r a l conditions with, e.g., high disease or p e s t incidence, d u r i n g cold w i n t e r s or u n d e r d r o u g h t or salt s t r e s s m a y preselect in given populations at low cost for desired genetic c o m b i n a t i o n s . In the case of in-breeding m a t e r i a l s desired r e c o m b i n a t i o n s m a y be a u g m e n t e d by a n introduction of male sterility s y s t e m s favoring cross-pollination (Rticker a n d Kr/iling, 1996). In directed crosses, b a c k c r o s s principles will often be applied. If the gene donor p a r e n t is a low yielding "exotic" genotype, the a d v a n c e d recipient parent will a g a i n be u s e d for a s u b s e q u e n t cross in order to increase its genetic proportion in the r e s u l t i n g progeny. A similar a d v a n t a g e m a y come from a "three way cross", where the first h y b r i d is again c r o s s e d with a n o t h e r high performing cultivar with expectedly good combining ability. The more distantly related the cross p a r e n t s are, the more s u c h p r e p a r a t o r y operations are r e q u i r e d to provide for a n initial v a r i a n t useful in f u r t h e r cultivar development. For example, from p r i m a r y a m p h i d i p l o i d s between a B r a s s i c a crop a n d a n alien species, u s u a l l y several b a c k c r o s s e s with the crop genotype a n d s u b s e q u e n t selections are required to remove m o s t of the exotic genotype, to be left with little more t h a n the desired gene or c h r o m o s o m e segment in a r e c o m b i n a n t or t r a n s l o c a t e d fashion within the productive genome. The potentially m o s t p r o m i s i n g genetic material, which is not p r e - a d a p t e d in a n y of these ways to the genotypes prevalent in a practical breeding program, m a y be lost even d u r i n g the first selection cycle b e c a u s e of destructive weakness. B a c k c r o s s i n g c a n be significantly enforced in effect by m a r k e r assisted selection for the genetic b a c k g r o u n d of the r e c u r r e n t p a r e n t , a n d n e a r isogenic lines, which m a y be e s s e n t i a l for the introgression of t r a n s g e n i c traits, can be p r o d u c e d in a more direct way (see c h a p t e r 9).
A highly direct a p p r o a c h of e s t a b l i s h i n g cultivar c a n d i d a t e s is molecular t r a n s f o r m a t i o n (for review see Poulsen, 1996). However, it s h o u l d be well recognized t h a t not every t r a n s f o r m a n t is a l r e a d y a p r o m i s i n g candidate. Apart from the more or less s o p h i s t i c a t e d m o l e c u l a r t e c h n i q u e s to verify the successful t r a n s f o r m a t i o n , copy n u m b e r a n d site of DNA insertion further inform a t i o n is required to identify relevant c a n d i d a t e s . At least twenty or even more i n d e p e n d e n t t r a n s f o r m a t i o n s m i g h t be n e c e s s a r y to s e c u r e the establ i s h m e n t of one p a r t i c u l a r t r a n s g e n i c line for potential application in breeding.
429
Establishing candidates Different types of cultivars can be distinguished by the last reproductive process taking place in the propagation of a cultivar (Schnell, 1982a): Self pollination will lead to line cultivars, panmictic cross pollination to population cultivars, and artificial crossing between seed and pollen parents to hybrid cultivars. Hence the methods to establish candidates are highly dependent on the n a t u r a l reproductive system of a crop. The diploid B r a s s i c a species B. rapa, B. o t e r a c e a and B. nigra are normally strictly cross pollinated due to their self incompatibility system (see chapter 5). However, exceptions exist like the self fertile B. r a p a subsp, trilocularis ('Yellow Sarson'). The amphidiploid B r a s s i c a species generally shows a mixed mating system with predominantly self pollination. The outcrossing rate is about 30 %, but depends on genotype and environmental conditions and may vary in B. n a p u s from 5 % to 70% (Rudloff and Schweiger, 1984; Rakow and Woods, 1987; Becker et al., 1991). Breeding of partially allogamous crops is very difficult and, therefore, in
B. n a p u s breeding artificial selfing is widely used in early generations. In this way, B. n a p u s is converted to a self pollinating crop, and s t a n d a r d methods
like pedigree selection or single seed descent can be applied. Without selfing, at least in the early generations, an efficient improvement of quality characters is hardly possible. Moreover, in most countries the high d e m a n d s for uniformity, distinctness and stability of cultivars strongly favor pure lines or F1 hybrids as the desired type of cultivar. An alternative to artificial selfing in s u b s e q u e n t generations is the development of doubled haploid lines. B r a s s i c a crops are among the pioneer ones to use this technique in plant breeding (Chen et al., 1994; Gland-Zwerger, 1995). Already in 1975, the cultivar 'Haplona' was released in the UK, which originated from a spontaneous haploid plant selected in 'Oro' (Thompson, 1979). Today, microspore culture techniques, which have been successfully promoted particularly in B. n a p u s to high effectiveness with almost every plant genotype (see chapter 8), produce the desired homozygosity at once, fully replacing earlier methods with similar effects, e.g. single seed descent. They also work with B. j u n c e a , and even B. rapa, although this self incompatible, fully cross breeding species usually suffers from inbreeding depression, and useful dihaploids are rare (Dewan et al., 1995; Ferrie and Keller, 1995).
Selecting cultivars As soon as individual plants have been obtained, which represent the genotype of the desired cultivar, the third phase commences in the breeding program with intensive testing for final cultivar selection. This is the phase in which modern biotechnologies have produced m a x i m u m benefit and a rapid progress in selection at both the phenotypic and the genotypic levels.
430 At phenotypic levels, yield is still and will continue to be determined largely in field tests over several years and locations. Here, nursery machinery during the last two decades h a s made remarkable progress. Plot seeders, e.g. Oyjord system, or single seed planters guarant ee the possible best positioning of the seed in the seedbed, and plot combines the desire for lowloss, clean seed harvest with highly computerized yield dat a assessment. For prescreening of disease resistance, greenhouse tests with artificial inoculation of y o u n g seedlings u n d e r well controlled conditions yield useful results for, e.g., Verticillium dahliae, Alternaria brassicae, Sclerotinia sclerotiorum or even P h o m a lingam. Last but not least the revolutionary success of seed quality improvement is d e p e n d e n t on the development of quick, cheap and sufficiently reliable qualitative analytical tests as well as s u b s e q u e n t accurate quantitative ones described in detail by R6bbelen and Thies (1980a, b). Meanwhile, new physical e q u i p m e n t h a s been developed and n o w - in addition to NMR for the estimation of oil content - NIR spectroscopy allows for a s i m u l t a n e o u s determination of seed humidity, oil, protein and glucosinolate c o n te n ts as well as t hat of oleic acid and possibly other fatty acids, each within s o m e w h a t different but mostly satisfying accuracy (Reinhardt, 1992; Tillmann, 1997; Velasco and Becker, 1998). Taken together accounts for an e n o r m o u s l y increased selection intensity in B r a s s i c a oilseed breeding. At genotypic levels, DNA p o l y m o r p h i s m s today offer m a r k e r s to assist in selection for i m p o r t a n t traits as well as in the identification and differentiation of genetic lines a n d cultivars. For example, using DNA-markers virus resistance can be identified without employment of infective insect vectors and prior to the expression of phenotypic symptoms, e.g. in breeding against the turnip yellow virus of rapeseed, B. n a p u s (Graichen, 1994). Restriction fragment length polymorphism (RFLP) m a p s have been produced by several groups of researchers, after the first m a p was published by Landry et al. (1991) for spring rape, B. n a p u s (Chyi et al., 1992; Uzunova et al., 1995). The u s ef u ln es s of these molecular m a p s increases quickly with the density of linked loci as well as with the n u m b e r of important agronomic traits positioned on the map. For this purpose, cDNA probes may be as valuable to associate particular probes with agronomic traits of interest as detailed phenotypic information from the segregating population is u s e d for molecular mapping. With these data, positions may also be m arked for quantitative trait loci (QTL) which are of particular interest in breeding (Weissleder et al., 1996). Like with isozymes, RFLPs and also non-radioactive RAPD-markers permit the introgression of genes, which are difficult to m e a s u r e in their phenotype to be followed. They also offer the chance of pyramiding genes, e.g., for fatty acid synthesis or disease resistance, where phenotypically one gene m a s k s the effect of a n o t h e r (for example see Dion et al., 1995; J o u r d r e n et al., 1996; Kuginuki et al., 1997; Chevre et al., 1997; Voorrips et at., 1997; J e a n et al., 1997). Ultimately, molecular m a r k e r s may provide additional characters for the identification of cultivars for registration and prosecution of property rights (e.g., Mailer et al., 1994; Lee et al., 1995).
431 One aspect rarely considered when describing breeding operations, t h ou g h increasingly important, is the "logistics" of a breeding program (Buzza, 1995). While for winter B. n a p u s in Europe the evaluation of breeders' lines takes u p almost the whole year from sowing in A u g u s t to harvest in following July, spring type and other Brassica species, or more favorable areas where the growing season is b r o u g h t to an earlier end, m a y allow for an extra generation outside the normal season. This can occur in growth rooms or greenhouses, b u t also sometimes m o s t efficiently in "off-season" or "contra-season" (other hemisphere) nurseries.
Breeding m e t h o d s The general usage of the term breeding m e t h o d refers to the overall working program which the breeder p u r s u e s from the initial identification of appropriate source materials until the ultimate release of the new cultivar (Schnell, 1982a). The strategy employed thereby is strongly determined by the mode of reproduction of the species in question and the type of variety to be bred accordingly. In the outbreeding B. oleracea, new "population cultivars" of, e.g., cabbage which were previously raised by m a s s selection, were generated by m e a n s of single plant selection and consecutive progeny testing. The gain in selection could be increased by vegetative m a i n t e n a n c e of the female stock plants, and clones of these were planted together in an outcrossing plot as soon as sufficient performance d a t a h a d been raised for their effective selection (Rundfeldt, 1960). On the other h a n d , al t hough the amphidiploid species B. n a p u s a n d B. j u n c e a u n d e r field conditions always undergo some outcrossing, pedigree breeding has been m o s t widely employed to develop "line cultivars". The procedure is similar to t h a t in the self pollinating cereals; b u t B. n a p u s a n d B. j u n c e a differ from these in two import a n t aspects: Firstly, they have a m u c h higher multiplication rate per generation (ca. 1000 : 1) and secondly, they have a p l a n t - t o - p l a n t outcrossing rate ranging from as low as 5 % to m u c h beyond 30 %. Therefore, replicated progeny testing can begin early in the F3 and certain levels of heterosis can be captured from the initial cross and retained in s u b s e q u e n t generations. From this, breeding of improved populations as "synthetic cultivars" was considered b u t little use h a s been made of this m e t h o d in the alloploid species. However, in the self incompatible diploids, mainly B. rapa, population improvement by r e c u r r e n t selection has been an efficient way to develop high yield cultivars (Downey and Rakow, 1987), and early progress by this m e a n s h a s been reported in vegetable breeding of cabbage cultivars (Rundfeldt, 1960). Of course, full exploitation of heterosis always requires a complete fertilization control as u s e d in the breeding of conventional "hybrid cultivars"; B. n a p u s cultivars of the latter type are j u s t now (1997) entering seed markets.
432
Breeding of line cultivars Although there are m a n y variations according to the genetic materials to work with, the type a n d inheritance mode of the c h a r a c t e r s to be improved, the available technical a n d financial inputs, or j u s t to the personal preference of the breeder, the m e t h o d of pedigree breeding for B. n a p u s oilseed cultivars essentially proceeds as follows (Figure 13.2): 1. Select p a r e n t a l p l a n t s (P1 a n d P2) with the view to combine the desirable traits from each. 2. Grow F1 p l a n t s in the g r e e n h o u s e u n d e r selfing (to save one year) a n d vernalize artificially (if winter form). 3. Raise spaced p l a n t s of F2 in the field a n d h a r v e s t vigorous, healthy single p l a n t s in a sufficient n u m b e r (to satisfy the expected recombination rate). 4. S u p p l e m e n t preceding p l a n t selection d u r i n g the season in the F2 plot with selection tests d u r i n g winter for quality a n d resistance p a r a m e ters of F3 seeds a n d seedlings, respectively. 5. Raise A-lines from selected F3 plants, bag for self-pollination (where needed) a n d confirm the selection traits. 6. Again raise A-lines from selected F4 plants, bag for selfing a n d after h a r v e s t of the selfed, b u l k all rest p l a n t s per plot. 7. C o n t i n u e A-line sowing a n d single plant selection for field performance within a n d between plots t h r o u g h Fs a n d F6 generations. 8. C o n d u c t replicated p e r f o r m a n c e trials with the F4 bulked seed at several different locations for two successive years. 9. Considering these results, b u l k h a r v e s t the best type-identical, hom o g e n o u s F6 plots belonging to the s a m e F4 single p l a n t descent to obtain a first lot of breeder's seed of the new candidate cultivar. 10. Finally, increase this F7 seed u n d e r isolation to produce the foundation seed, of which part is u s e d for the official trials aimed to secure Plant Breeder's Rights as well as to entitle to seed commercialization. The r e m a i n i n g seed is held in reserve for future cultivar m a i n t e n a n c e a n d multiplication needs. Alternatives to this pedigree m e t h o d are the d e v e l o p m e n t of doubled haploid (DH) lines or the single seed d e s c e n d (SSD) m e t h o d . From a genetical point of view these two m e t h o d s are very similar: both are rapid a p p r o a c h e s to reach homozygosity w i t h o u t a n y selection. In experimental comparisons, DH a n d SSD p o p u l a t i o n s from the s a m e crosses were similar in m e a n a n d variance of the lines (Charne a n d Beversdorf, 1991; S t r i n g h a m and Thiagarajah, 1991). Application of microspore culture saves time, b u t a more intensive selection is required in later g e n e r a t i o n s and, therefore, the time saving effect c a n be smaller t h a n s o m e t i m e s expected. In winter oilseed rape, the
433
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Figure 13.2 Breeding of line cultivars using pedigree selection of oilseed winter rape, Brassica napus, as applied in Europe (after Downey and R6bbelen, 1989).
434 u s e of d o u b l e d h a p l o i d s m a y r e d u c e the time to develop a new cultivar by one or two y e a r s ( P a u l m a n n a n d F r a u e n , 1991; F r a u e n , 1994). However, the additional y e a r s required for pedigree selection can be u s e d for a better evaluation of the y e a r to year variation of the material. Today double haploids are u s e d in m a n y breeding p r o g r a m s , b u t systematic r e s e a r c h on their efficiency including relative costs c o m p a r e d to o t h e r m e t h o d s is rare. Theoretically, the u s e of DH lines s h o u l d be less efficient in crosses, where one of the p a r e n t s is not a d a p t e d or lacks required quality traits. In s u c h material pedigree selection in early segregating gene r a t i o n s is relatively easy, w h e r e a s u n s e l e c t e d DH lines would contain a large p r o p o r t i o n of u n a c c e p t a b l e types. If, on the contrary, crosses a m o n g relatively n a r r o w elite material are performed, visual selection is m u c h easier a m o n g u n i f o r m DH lines t h a n a m o n g segregating progenies in a pedigree program. There is still some d e b a t e on the q u e s t i o n of which generation s h o u l d be u s e d for the p r o d u c t i o n of DH lines. In m o s t c a s e s F1 derived DH lines are u s e d to get a m a x i m a l gain in time, b u t DH p r o d u c t i o n from F2 or F3 p l a n t s will allow for more r e c o m b i n a t i o n a n d for some preselection of the material as well.
Breeding of population cultivars In the cross pollinated B r a s s i c a species v a r i o u s m e t h o d s of population i m p r o v e m e n t are used. In m a n y c a s e s m a s s selection h a s been successful, b u t this simple m e t h o d is more a n d more replaced by a d v a n c e d types of rec u r r e n t selection (Downey a n d Rakow, 1987). In B. rapa, where self incompatibility e n s u r e s a high degree of heterozygosity u n d e r open pollination, s y n t h e t i c s can be p r o d u c e d by mixing two or at m o s t three p a r e n t a l c o m p o n e n t s . The ' S y n - l ' seed borne from a two c o m p o n e n t synthetic will be c o m p o s e d 25 % of each of the p a r e n t a l s and 50 % hybrids, which provides for a n equal c h a n c e of cross between a n d within lines or h y b r i d s a n d t h u s g u a r a n t e e s stable gene frequencies. This procedure h a s been successfully applied in the first B. r a p a s y n t h e t i c s (Hysyn 100 and H y s y n 110), which were registered in C a n a d a in 1994. Even in p r e d o m i n a n t l y self pollinated species, the breeding of population cultivars was s u g g e s t e d to m a k e at least partial use of the heterosis. Already in the 1970s, before efficient s y s t e m s of fertilization control became available for hybrid breeding in B. n a p u s , breeding of synthetic populations (Figure 13.3; Schnell, 1982b) h a d been proposed. S c h u s t e r (1982) had discovered t h a t if two or more lines or cultivars h a d been grown in a mixed s t a n d (Syn-0), the next generation (Syn-1) will exhibit some of the heterosis of seed yield expected from F1 hybrids. This ' S y n - l ' p o p u l a t i o n will be composed of the c o m p o n e n t p a r e n t a l lines a n d all possible F1 h y b r i d s between them. Leon (1987, 1991) f u r t h e r d e t e r m i n e d t h a t the recovered yield increases over the calculated p a r e n t a l m e a n s were c a u s e d by heterozygosity a n d heterogeneity of this mixture as well a n d t h a t not only yield height b u t also
435 0
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Initial population Preselection of parents Test crosses
Topcross
GCA yield test
f
t
Combining cross (Syn-0) Syn-1 Syn-2
Figure 13.3 Breeding of synthetic cultivars of oilseed rape using inbred lines (Becker, 1993).
436 yield stability increased over years and locations (Schnell and Becker, 1986). The process can be c o n t i n u e d by sowing 'Syn-1' seed to produce 'Syn-2' seed and so on. But the genetic composition of the consecutive generations will change with the n u m b e r of parental lines used and the outcrossing rates between t h e m until the population has reached its equilibrium (Geiger, 1982). This led to debates a b o u t how synthetic cultivars should be constructed (Becker, 1982) a nd m a i n t a i n e d (Geiger, 1982) and which synthetic generation should be u s e d for commercial oilseed p r o d u c t i o n s (Becker, 1989) in order to secure a sufficient degree of heterosis. The proportion of hybrids might be increased by selecting parental lines with a high level of self sterility (Hackenberg and K6hler, 1996) or by initiating the synthetic with h a n d crossed F1 seed. So far, m os t of the efforts on utilizing heterosis in oilseed rape, B. n a p u s , are d~rected worldwide to establishing true hybrid cultivars. But this can only be achieved at a great expense and application of the m u c h simpler a p p r o a c h of developing synthetic cultivars should be considered in those cases where the production size and therewith the ret urn of economic investment will be restricted, such as in certain oleochemical or specialized technical oil m a r k e t s (Becker et al., 1998).
Breeding of hybrid cultivars For breeding of hybrid cultivars, three principal requirements have to be fulfilled: (1) there m u s t be a sufficient a m o u n t of heterosis; (2) a system for large scale production of hybrid seed m u s t be available; and (3) an efficient method to identify hybrids with high combining ability. A n u m b e r of studies have shown t h a t there is a considerable heterosis for yield in the oilseed B r a s s i c a species (Table 13.5; Becker, 1987; McVetty, 1995). However, in practical breeding programs, m a n y interesting lines are related by pedigree. In order to maximize heterosis it will be necessary in the long r u n to develop different gene pools. In B. n a p u s it has been suggested to use the diversity between spring and winter type (Diers and Osborne, 1994; Knaak, 1996) or to use resynthesized rapeseed (Becker and Engqvist, 1995). The problem for al m os t two decades has been to find a reliable system for fertilization control, which allows for the production of hybrid seed. Finally, extensive worldwide research and development have led to several different s y s tems which are presently being introduced with first F1 hybrid cultivars in the leading rape growing countries and now have to prove their suitability (also see Downey and Rimmer, 1993; Stiewe et al., 1995; and chapter 6). In principle, three different genetic m e c h a n i s m s exist to produce hybrid seed: (1) self incompatibility (SI), (2) nuclear male sterility (NMS), and (3) cytoplasmic male sterility (CMS). B r a s s i c a is probably the only crop eliminated. Inbreds m a y be obained from in vitro anther- or microspore culture as doubled haploids (Keller and Armstrong, 1983) or by selfing of about five generations. In the latter case, stigmas are h a n d pollinated either several (5-10) days prior to anthesis, when the self incompatibility barrier in the
437 stigma has not yet been developed (Tatebe, 1951), or after previous spraying of the open flowers with a solution of 3% NaC1 (Monteiro et al., 1988). After e s t a b l i s h m e n t of the SI lines, their m a s s propagation is usual l y performed by in vitro microcloning (Clare and Collin, 1973) or by cultivation of the flowering self pollinated plants at an increased concentration (ca. 5 %) of CO2 gas (Nakanishi et al., 1969; Palloix et al., 1985), which reduces the SI reaction to such an extent t h a t self pollen can attain fertilization and seed set. Another method for m a s s propagation in inbred lines is based on the use of near isogenic sublines which only differ with regard to their S-allele constitution, i.e. $1S1 v e r s u s $2S2 and $3S3 v e r s u s 8484 ( Figure 13.4a). These sublines are easily crossed by insect pollination giving rise to very uniform and incompatible S1S2 a n d $3S4 parental lines which are directly u s e d for hybrid seed production (Kuckuck, 1979). Occasionally, high t e m p e r a t u r e s , high air h u m i d i t y or similar environmental anomalies may d i s r u p t SI functions. Therefore, seed lots harvested for F1 plant cultivation are routinely tested for purity of their hybrid character. All seeds, which may have formed within the female by selfing or intracrossing will generate weak inbred plants, which of course are undesired. The proportion of these seeds is determined by use of morphological seedling m a r k e r s or by biochemical markers, s u c h as isozymes or PCR-markers (Nijenhuis, 1971). From experience, the off-types are mainly contained in the small seed fraction. Meanwhile, the combination of breeding procedures and seed production techniques in cabbage regularly produce almost 100 % hybridity in the commercial seed of m o s t cultivars. Hybrid development t h r o u g h SI h a s also been a t t e m p t e d in oilseed breeding with B. n a p u s (McVetty, 1995; O d e n b a c h a n d Beschorner, 1995), although this species is normally self compatible. But SI alleles can be selected within given rapeseed populations or be introduced from B. o l e r a c e a or B. r a p a a n d applied to produce hybrids. Both d o m i n a n t or recessive SI can be u s e d (Ekuere et al., 1995; Kott, 1995). Nuclear male sterility (NMS) is c o m m o n in all plants, b u t its use in breeding is normally restricted by the fact t h a t NMS lines can not be maintained. They have to be pollinated by a fertile line and the progeny will segregate 1:1 for sterile and fertile plants. In China, NMS hybrids are produced after removing the fertile plants by h a n d (Fu et al., 1997). Alternatively, a two-gene s y s t e m is also in use in China, which allows the production of completely male sterile lines (Renard et al., 1997). By transgenic m eans, the Belgian c o m p a n y Plant Genetic Systems (PGS) h a s developed the "Seedlink" system (de Both, 1995), where a nuclear sterility gene is linked to a gene for herbicide tolerance; hence, the fertile plants can easily be removed from the propagation fields by spraying with the respective herbicide (see chapt er 6). Cytoplasmic male steriliW (CMS) is a m o s t elegant genetic system of fertilization control. It has been found to occur whenever a species was under intensive research (K~ck and Wricke, 1995). The system consists of three
438 c o m p o n e n t s : the A-line, i.e. the male sterile seed p a r e n t carrying a cytoplasmic (mitochondrial) genome which codes for a dysfunction of pollen d e v e l o p m e n t (Stiewe et al., 1995), the fertile B-line to m a i n t a i n the A-line by pollination, a n d the R-line to act as the fertility restorer a n d heterotic partner to the sterile A-line (Figure 13.4b). The respective genic c o m p o n e n t s , i.e. the pollen sterility allele(s) a n d the fertility restorer allele(s) as well as the c o r r e s p o n d i n g c y t o p l a s m are i n t r o d u c e d into existing a d a p t e d cultivars a n d high yielding breeder's lines by m e a n s of backcrossing. In m a n y cases, restorer alleles exist in the actual breeding materials a n d good combining ability m a y easily be selectable within the broad general gene pool. Once the s y s t e m is established, f u r t h e r p r o g r e s s in new hybrid cultivars d e p e n d s on a n a p p r o p r i a t e genetic i m p r o v e m e n t of the hybrid partners, i.e. the A- a n d the R-lines. The breeding of R-lines m a y essentially occur by pedigree breeding as outlined above. Lines of this gene pool with satisfying p e r f o r m a n c e will be exposed to additional tests of c o m b i n i n g ability with corr e s p o n d i n g A-lines. On the other h a n d , A-line i m p r o v e m e n t in a CMS system is a m u c h more t e d i o u s exercise, not only b e c a u s e the first A-line u s u a l l y h a d been j u s t one r a n d o m genotype, b u t also b e c a u s e the development of this pool involves the parallel t r e a t m e n t of both the sterile seed p a r e n t a n d the c o r r e s p o n d e n t fertile m a i n t a i n e r lines (Figure 13.5). Recently estimates of c o m b i n i n g ability have been raised t h r o u g h m o l e c u l a r analysis of genomic (DNA) r e l a t i o n s h i p s between the c a n d i d a t e cultivars or breeding lines by principle c o m p o n e n t a n a l y s i s or by d e n d r o g r a m s . S u c h d a t a m a y provide the breeder with useful e s t i m a t e s for g r o u p i n g his gene pool into heterotic groups for u s e in h y b r i d breeding (Knaak a n d Ecke, 1995; Becker a n d Engqvist, 1995; Voss et al., 1996). In oilseed spring rape, hybrid cultivars have been available since the end of the 1980s m a i n l y based on the 'Polima' CMS s y s t e m (McVetty et al., 1995). In winter type rapeseed, however, it is more difficult to obtain stable CMS. In addition to the 'Polima' s y s t e m (Bartkowiak-Broda, 1995) the 'oguINRA' s y s t e m a n d several other s y s t e m s (Stiewe et al., 1995; P r a k a s h et al., 1995) are in use. In the 'ogu-INRA' system, all presently available restorer lines generate a too high glucosinolate c o n t e n t due to linkage of the restorer gene with a gene for glucosinolate b i o s y n t h e s i s (Delourme et al., 1995). But in cabbage breeding, where the F1 hybrid is h a r v e s t e d as a vegetative produce a n d its p r o d u c t i o n is not d e p e n d e n t on fertility restoration, the oguCMS is well accepted a n d u s e d as a valuable hybridizing system (ClauseSemences, 1994; de Melo et al., 1994). To avoid the p r o b l e m s of high glucosinolate c o n t e n t in oilseed rape production, u n r e s t o r e d 'ogu-INRA' F1 h y b r i d s are mixed with a certain percentage of fertile pollinator plants. S u c h 'composite h y b r i d s ' have been employed in E u r o p e for several years, b u t with i n c o n s i s t e n t results. Under favorable conditions d u r i n g flowering they m a y have an additional advantage due to a positive "sterility effect" b e c a u s e of the energy saved for not producing pollen; b u t in a n u n f a v o r a b l e e n v i r o n m e n t s o m e t i m e s poor seed set can be ob-
439
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Figure 13.4 a) above: Self incompatibility (SI) based hybrid seed production of cabbage (Kuckuck, 1979). b) below: Cytoplasmic male sterility (CMS) based hybrid seed production of oilseed rape (Buzza, 1995).
440
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441 served ( F r a u e n a n d Baer, 1996). Alternatively, t h r e e - w a y h y b r i d s c a n be prod u c e d b e t w e e n a CMS m o t h e r line a n d a n F1 as the male c o m p o n e n t being heterozygote for the r e s t o r e r gene. S u c h t h r e e - w a y h y b r i d s segregate into u n r e s t o r e d p l a n t s with low glucosinolate a n d r e s t o r e d p l a n t s with h i g h e r glucosinolate in a 1:1 ratio. Another male sterility system, n a m e d MSL (Male Sterility Lembke), h a s been developed by the private G e r m a n b r e e d i n g c o m p a n y NPZ-Lembke. This s y s t e m h a s its origin in a sterile p l a n t selected as s p o n t a n e o u s m u t a n t in NPZ n u r s e r i e s in 1983. In a large s c r e e n i n g p r o g r a m m a i n t a i n e r a n d r e s t o r e r genes could be selected. The MSL s y s t e m c a n be c h a r a c t e r i z e d as follows ( P a u l m a n n a n d F r a u e n , 1997): -
-
-
M a i n t a i n e r genes m u s t be selected carefully in a u n i q u e c y t o p l a s m in the a p p r o p r i a t e genetic b a c k g r o u n d . This m a i n t a i n e r m a t e r i a l is the p r o p e r t y of NPZ-Lembke only. Restorer genes are of f r e q u e n t o c c u r r e n c e ; are f u n c t i o n a l restorers.
m o s t released cultivars
Selection for modifier genes is n e c e s s a r y to obtain a n e n v i r o n m e n t a l ly stable male sterility.
The m a i n a d v a n t a g e of the MSL s y s t e m is t h a t r e s t o r a t i o n b e c o m e s only a s e c o n d a r y p r o b l e m since all breeding m a t e r i a l s c a n be directly u s e d as restorers. The v a r i o u s hybridization s y s t e m s differ in their practicability to p r o d u ce hybrid seed. A practical a d v a n t a g e of SI s y s t e m s is t h a t the h y b r i d seed c a n be p r o d u c e d by simply growing a m i x t u r e of the two c o m p o n e n t s . Using male sterility the two c o m p o n e n t s m u s t be grown in stripes a n d only the male sterile c o m p o n e n t harvested. This seed p r o d u c t i o n s y s t e m at h a r v e s t requires a d d i t i o n a l a t t e n t i o n a n d labor a n d , moreover, only t h o s e p a r t s of the field p r o d u c e h y b r i d seed, where the A-lines are raised. Therefore, it is u n d e r d i s c u s s i o n w h e t h e r the male sterile c o m p o n e n t s h o u l d be grown in a m i x t u r e with a small p e r c e n t a g e of the p a r e n t a l c o m p o n e n t a n d all p l a n t s harvested. In this case, the h y b r i d seed would c o n t a i n a certain a m o u n t of the p a r e n t a l genotype, b u t seed p r o d u c t i o n would be m u c h facilitated. S u c h a s y s t e m is s u c c e s s f u l l y u s e d in E u r o p e for h y b r i d rye a n d is u n d e r investigation for c a n o l a in C a n a d a (Renard et al., 1997). Cultivars p r o d u c e d in this way are a p o p u l a t i o n consisting m a i n l y of h y b r i d s with a small p e r c e n t a g e of the male p a r e n t a l line. This type of h y b r i d h a s some similarities with the above m e n t i o n e d composite hybrid cultivars. However, for composite h y b r i d s in a first step 100 % n o n - r e s t o r e d male sterile F1 h y b r i d s are p r o d u c e d , a n d these in a s e c o n d step, are artificially mixed with a n y pollinator, w h i c h m u s t not be genetically related to the hybrid. So far, r e s e a r c h in hybrid b r e e d i n g h a s b e e n very m u c h focused on the d e v e l o p m e n t of a reliable fertilization control. After this s y s t e m is available, it is n e c e s s a r y to optimize all steps of the m e t h o d to identify the b e s t c o m b i n a -
442 tion. Selection of the b e s t cross c o m b i n a t i o n c a n be m a d e in three steps: (1) selection a m o n g the c o m p o n e n t s d u e to their p e r s e performance, (2) selection a m o n g test c r o s s e s for high general c o m b i n i n g ability, a n d (3) selection a m o n g h y b r i d s for b o t h general a n d specific c o m b i n i n g ability. Selection for line p e r s e p e r f o r m a n c e is more i m p o r t a n t in r a p e s e e d t h a n in completely o u t c r o s s i n g crops, like rye a n d maize, b e c a u s e of its relatively lower level of heterosis (Geiger, 1990). If only a limited n u m b e r of sterile testers is available, as is u s u a l in the beginning of h y b r i d breeding p r o g r a m s , it m a y be a r e a s o n a b l y short term strategy to c o n c e n t r a t e on selection of superior pollinators; the seed compon e n t of the hybrid c a n t h e n be directly u s e d as the male sterile tester. But in the long r u n it would be more efficient to u s e a s y s t e m a t i c a p p r o a c h to improve both gene pools for both GCA a n d SCA in two steps like in maize breeding (Figure 13.5).
Breeding results The p r o d u c t i o n of cabbage, B. oleracea, h a s profited considerably from hybrid breeding. The benefits not only consist in i n c r e a s e d yields a n d better targeted c h a r a c t e r c o m b i n a t i o n s , b u t p a r t i c u l a r l y in a better uniformity of the p l a n t s a n d their p r o d u c t s . O p t i m u m m a r k e t qualities as required today can only be o b t a i n e d from F1 hybrid cultivars, which, therefore, by now are the only ones p r o d u c e d in cabbage by all major seed c o m p a n i e s . The application of SI in c a b b a g e breeding h a s been pioneered a n d led to first economic s u c c e s s in J a p a n . Already by 1951 J a p a n e s e h y b r i d s h a d been licenced a n d sold in the seed m a r k e t , with two self incompatible genotypes as p a r e n t a l lines (Yoshikawa, 1993). Similar a p p r o a c h e s are employed in cabbage breeding worldwide. But also in oilseed rape, the first commercial hybrids b a s e d on the SI s y s t e m are m a r k e t e d in C a n a d a a n d China. Worldwide, h y b r i d oilseed rape cultivars m a i n l y rely on the Polima CMS system. The c o u n t r y pioneering in hybrid r a p e s e e d p r o d u c t i o n is China, where in 1996 a b o u t 1.5 million h a were sown with hybrids, which is a b o u t 22 % of the total acreage. In C a n a d a in 1997, the p e r c e n t a g e of hybrids was still less t h a n 10 % of the area. In E u r o p e in 1997, a b o u t 2 2 0 . 0 0 0 composite h y b r i d s b a s e d on the o g u - I N R A s y s t e m were grown, mainly in F r a n c e (Ren a r d et al., 1997). The first spring r a p e s e e d cultivars u s i n g the transgenic "Seedlink" s y s t e m from the Belgian PGS c o m p a n y were released in 1997 in C a n a d a (Renard et al., 1997). However, only with the MSL s y s t e m of CMS, the first fully r e s t o r e d winter r a p e s e e d hybrid cultivars, i.e. 'Joker' a n d 'Pronto', were released in G e r m a n y in 1995, a n d in 1 9 9 6 / 9 7 a b o u t 30 000 h a were p l a n t e d with t h e s e h y b r i d s (Frauen a n d Baer, 1996). Oilseed rape b r e e d i n g since the early 1960s h a d been s h a p e d by the need to improve the quality of the oil a n d meal. The first low erucic acid spring rape cultivar 'Oro' (1966) was developed in C a n a d a from a cross be-
443 tween 'Nugget', a n Argentinian selection, a n d a low erucic selection from the G e r m a n accession 'Liho' (Limburger Hof, BASF), a n d seed of 'Oro' was generously d i s t r i b u t e d to b r e e d e r s all over the world to s t a r t domestic quality breeding p r o g r a m s . Similarly, 'Span' (1971) was developed in C a n a d a by selection for low erucic acid c o n t e n t in B. rapa from the initial Polish accession a n d the cultivar 'Arlo'. Low glucosinolate p l a n t s were first selected from 'Bronowski', a cultivar from Poland i n t e r m e d i a t e between spring a n d winter type, which interestingly e n o u g h belonged to the s a m e gene pool as 'Liho', the d o n o r of the zero erucic character. Worldwide all of the 0 0 - r a p e s e e d cultivars were developed from this origin. 'Erglu' in G e r m a n y a n d 'Tower' in C a n a d a were the first spring 00-types released in 1973, a n d 'Ledos' was the first winter 00-type licensed in G e r m a n y 1976; b u t only with 'Librador' (1981) first p r o d u c t i o n scales were r e a c h e d in 0 0 - w i n t e r rape. Despite of the u n d e n i a b l e s u c c e s s of breeding r a p e s e e d with 00-quality s t a n d a r d , it m u s t be noticed t h a t with each quality change, the first cultivars released were lower in seed a n d oil yields t h a n the earlier low quality forms, a n d it always took m u c h effort a n d several y e a r s to r e a c h a n d later s u r p a s s the former level of p e r f o r m a n c e (see Figure 13.6; alike for C a n a d i a n cultivars see Downey a n d R6bbelen, 1989). This lagging of the quality forms occurred for several r e a s o n s , the m o s t i m p o r t a n t being the drain of selection intensity from yield to quality c h a r a c t e r s ( S a u e r m a n n , 1988; R6bbelen 1989), reflecting breeding priorities a n d capacity limits r a t h e r t h a n physiological constraints. On the contrary, the increasing a t t e n t i o n which r a p e s e e d breeding received from the s e n s a t i o n a l conversion of seed quality, s u p p o r t e d the worldwide s e a r c h a n d exchange of g e r m p l a s m conditioning new, more productive plant ideotypes with higher h a r v e s t index a n d better suitability for m e c h a n i c a l h a r v e s t i n g (Leitzke, 1975; R6bbelen a n d Leitzke, 1974). However, with the e n o r m o u s e x p a n s i o n of acreage, s t r e s s factors increased in n u m b e r a n d intensity. Therefore, after e s t a b l i s h i n g the 00-quality level in winter rape in the 1970s in Europe, the prime of selection w a s aimed at p l a n t resistance. Considerable progress was a t t a i n e d in r e s i s t a n c e a g a i n s t fungal diseases. In p a r t i c u l a r Phoma resistance, which h a d allowed for the u n u s u a l l y wide d i s t r i b u t i o n of the F r e n c h cultivar 'Jet Neuf in the early 1980s, was successfully i n t r o d u c e d into m o s t of the i m p o r t a n t winter rapeseed cultivars. I m p r o v e m e n t s in resistance a g a i n s t Cylindrosporium, Alternar/a a n d Sclerotinia are perceptible, too, in the recent official list of r a p e s e e d cultivars in G e r m a n y (Anonymous, 1996). Breeding p r o g r e s s is also reflected by a strong increase in the n u m b e r of new breeders' lines e n t e r i n g official cultivar tests as in G e r m a n y each year. In winter rape for seed production, there were only 2 c a n d i d a t e s in 1968, b u t there were 47 in 1982, a n d a l m o s t a double n u m b e r is now s u b m i t t e d by the breeders. Correspondingly, the cultivar s p e c t r u m c h a n g e s quickly with a life time of a b o u t 4-6 y e a r s of economic i m p o r t a n c e for m a n y of t h e m (R6bbelen, 1994). For these r e a s o n s , for the individual breeder an a p p r o p r i a t e re-
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445 t u r n of his financial i n v e s t m e n t is only g u a r a n t e e d by the p r e s e n t l y large total r a p e s e e d acreage in the relevant regions, which his cultivars m a y share. Today, the m a i n activities in r a p e s e e d breeding worldwide c o n c e r n the e s t a b l i s h m e n t of F1 hybrid cultivars (Renard et al., 1997). The increase of yield expected from the additional full u s e of heterosis gives promise to produce the a t t a i n e d high p r o d u c t quality of r a p e s e e d in a c o n s i d e r a b l y higher quantity, which will a d d to the superiority of this vegetable oil production. Although only initial c o m b i n a t i o n s are available for commercial production, hybrid cultivars like 'Joker' a n d 'Pronto' have confirmed their yield benefit from heterosis in farmers' fields in 1996 (Table 13.5). While at first there was only one application for concession of license a n d release of F1 hybrid cultivars in G e r m a n y in 1993 a n d three in 1994, far more t h a n half of all cultivar c a n d i d a t e s for the G e r m a n official tests in 1996 were of F1 hybrid type. This alone d e m o n s t r a t e s the powerful a d v e n t of hybrid cultivars in rapeseed, as takes place today in all rape growing c o u n t r i e s of the world.
Future d e v e l o p m e n t s From their experience a n d s u c c e s s a t t a i n e d to date, p l a n t b r e e d e r s expect o p p o r t u n i t i e s for c o n t i n u e d genetic i m p r o v e m e n t of B r a s s i c a crops by straight forward selection to be a l m o s t as p r o m i s i n g today as w h e n the quality i m p r o v e m e n t p r o g r a m was first initiated three d e c a d e s ago. Most of the breeding objectives will be the s a m e as in those early days. However, with the r e m a r k a b l e e x p a n s i o n of breeding tools, in p a r t i c u l a r those derived from biotechnology a n d m o l e c u l a r genetics, the breeder will be able to optimize his classical p r o c e d u r e s considerably as is displayed t h r o u g h o u t this monog r a p h a n d roughly s u m m a r i z e d in Table 13.6. Seed yield, p r o d u c t i o n stability, a n d p r o d u c t quality will r e m a i n the pillars of the r a p e s e e d industry. In the details, however, new objectives will be m a d e accessible to breeding a n d p r o d u c t i o n of B r a s s i c a . Herbicide tolerance as well as disease a n d pest resistance p r o d u c e d by alien gene t r a n s f e r will have a n economic i m p a c t on all B r a s s i c a crops including vegetable cultivars. Tayloring seedoil composition for new technical a n d oleochemical u s e s will be as attractive as "molecular farming" of high value materials s u c h as p o l y h y d r o x y b u t y r a t e , drugs, or horm o n e s (see Table 13.4). This overall confidence in future breeding progress h a s general as well as specific reasons. In general, the rapidly e x p a n d i n g world p o p u l a t i o n will heavily d e p e n d on increased supplies of p l a n t p r o d u c t s . Life on e a r t h is driven by p l a n t phot o s y n t h e s i s which provides the p r i m a r y energy a n d raw m a t e r i a l s for all biological s y n t h e s e s . Man, like all a n i m a l s , is absolutely d e p e n d e n t on a sufficient global level of p l a n t productivity in order to satisfy his n e e d s for food, for direct c o n s u m p t i o n or after refinement by a n i m a l h u s b a n d r y , as well as for all renewable supplies of i n d u s t r i a l raw m a t e r i a l s a n d even organic energy sources. D e m a n d s rapidly multiply with growing world p o p u l a t i o n a n d p r e t e n t i o n s of prosperity. On the other h a n d , crop p r o d u c t i o n increase is
446 T a b l e 13.5 Performance of different types of cultivars of winter rapeseed, Brassica napus; selected data from the official tests in Germany in 1995
and 1996 from a total of 23 locations (*only 9 in 1996 and ** 12 in 1995, respectively) for grain and oil yield relative to the standard cultivars Falcon and Lirajet (= 100). Cultivar
Released
Grain
Oil
Line cultivars
Falcon Liraj et Express Zenith
1989 1989 1993 1997
98 102 103 109
98 102 109 115
1996 1996
103 * 120
104" 124
1995 1995 1996
107" 119"* 119
109" 122"* 119
40,6
16,8
Composite hybrid eultivars
Synergy Life Restored hybrid cuitivars
Joker Pronto Panther Absolute yield (in dt/ha = 100)
where all three m e c h a n i s m s are successfully used to produce commercial cultivars. Self incompatibility (SI) exists in the diploid B r a s s i c a species and is commercially used to produce hybrid cultivars in B. oleracea. In all cruciferous species, SI is u n d e r sporophytic control (see chapter 5), i.e. both Salleles of the male determine inability of the principally fertile pollen grains to penetrate the stigma and reach the egg cell of the female receptor plant carrying one of these S-alleles in common (Hinata et al., 1994) The first step of an SI based hybrid breeding programme consists in the production of inbred lines with stable SI expression u n d e r all relevant environments. Lines with pseudocompatibility and offspring including self fertile individuals, which occur rather frequently in cauliflower and early red cabbage, m u s t be
447 T a b l e 13.6 Breeding goals for future oilseed rape cultivars.
Preferably used procedures: CS MMGT IC QA-
Classical selection by phenotype Molecular marker use for DNA analysis Gene transfer and gene construction In vitro cell and tissue culture Instrumental quality analytics
1. Increase of grain yield
Broadening of the genetic base by, e.g., interspecific crosses (IC, CS) Improved precision of selection by using - doubled haploid lines (IC, CS), and - molecular markers for selection by genotype, i.e. at the DNA level, rather than by phenotype (MM) Hybrid parents with better combining ability by determination of genetic distances (MM), and establishment of complementary gene pools (MM, CS) 2. Improvement of yield stability
Improved disease resistance (MM, IC, CS, GT) Improved winter hardiness, shattering and lodging resistence (CS, GT) Protection against insect damage (GT) Herbicide tolerance (GT) Improved nutrient efficiency (CS, IC, GT) 3. Improved seed quality
Higher oil content (QA, CS, MM) Higher protein content (QA, CS, MM) Altered fatty acids profile (GT, CS, MM) Synthesis of new fatty acids (GT) Synthesis of new storage proteins (GT)
448 limited by worldwide decline of available arable l a n d a n d by enlarging recognition of the b u r d e n w h i c h intensive a g r i c u l t u r a l land u s e s impose on n a t u ral e c o s y s t e m s . Genetic i m p r o v e m e n t is the only m e a n s to a t t a i n more effective p l a n t p r o d u c t i o n not b a s e d on additional e x t e r n a l i n p u t s b u t on a more efficient t r a n s f o r m a t i o n of solar irradiation a n d i n t e r n a l regulation of metabolic f u n c t i o n s in the crop p l a n t s p e r se. This is the crucial c h a n c e for coming h u m a n g e n e r a t i o n s to s u s t a i n the essential conditions for their life in the future. After the i n d u s t r i a l revolution by c h e m i s t r y a n d physics the 21 st will be the "century of biology" a n d p l a n t breeding will be a major application of all forthcoming scientific a n d technical d e v e l o p m e n t s in this field. In the s a m e context, Brassica crops offer exceptional progress by furt h e r breeding. Brassica oilseeds have gained worldwide a t t e n t i o n in recent decades, a n d a highly potential p l a n t breeding i n d u s t r y h a s been established in m u l t i n a t i o n a l as well as in m e d i u m sized private c o m p a n i e s in all rape growing countries. T h e s e are able to actively utilize m o d e r n biotechnological knowledge a n d discoveries, which h a s been a special d o m a i n of the brassicas. Brassica species have pioneered in the d e v e l o p m e n t of in vitro culture t e c h n i q u e s for m a s s propagation, for microspore c u l t u r e to p r o d u c e doubled haploid lines, a n d for wide hybridization by p r o t o p l a s t fusion. Various gene t r a n s f e r m e t h o d s are highly effective in Brassica a n d information on u s a b l e genes is rapidly e x p a n d i n g s u p p o r t e d by early scientific cooperation worldwide as well as by r e c e n t benefit of extensive r e s e a r c h into the genetic model p l a n t Arabidopsis thaliana, which, b a s e d on genetic s y n t e n y paves the way for even more innovative genetic t r e a t m e n t of the related b r a s s i c a s . There is ample a c c e s s to genetic diversity by classical m e t h o d s , too, a n d m u c h rem a i n s to be exploited in both e s t a b l i s h e d Brassica crops a n d in m a n y allied wild species. Last, b u t not least, m a r k e t s are r e a d y to a b s o r b not only large quantities, b u t also a wide palette of very different quality p r o d u c t s derived from Brassica species, s u c h as vegetables, seedoils as well as seed meals for food, feed, a n d non-food uses, the latter e x t e n d i n g as far as to biodiesel fuels. In all i n s t a n c e s , p l a n t breeding will be pivotal. The preceding c h a p t e r h a s hopefully been convincing t h a t its s u c c e s s is already well programed.
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458 Sedun, F. S., Seguin-Swartz, G. and Rakow, G.F.W. 1989. Genetic variation in reaction to sclerotinia stem rot in Brassica species. Canad. J. Plant Sci. 69, 229-232. Shahidi, F. (ed.), 1990. Canola and rapeseed. Production, chemistry, nutrition and processing technology. Van Nostrand, pp. 1- 355. Slominski, B. A., Simbaya, J., Campbell, L. D. and Guenter, W. 1995. Untritive profile of yellow seeded canola rapeseed. Proc. 9th Int. Rapeseed Congress, Cambridge, U.K. 1, 148-150. Stefansson, B. R., Hougen, F. W. and Downey, R. K. 1961. Note on the isolation of rape plants with seed oil free from erucic acid. Canad. J. Plant Sci. 41, 218-219. Stiewe, G., Witt, U., Hansen, S., Theis, R., Abel, W.O. and R6bbelen, G. 1995. Natural and experimental evolution of CMS for rapeseed breeding. Adv. Plant Breeding 18, 89-110. Stringham, G. R., McGregor, D. I. and Pawlowski, S. H. 1974. Chemical and morphological characteristics associated with seed coat color in rapeseed. Proc. 4th Int. R a p e s e e d Congress, Giessen, Germany, pp. 99-108. Stringham, G. R. and Thiagarajah, M. R. 1991. Effectiveness of selection for early flowering in F-2 populations of Brassica napus L. - A comparison of doubled haploid and single seed descent methods. In: McGregor, D. I. (ed.), Proc. 8th Int. R a p e s e e d Congress, Saska-toon, Canada, 1, 70-75. Subudhi, P. K. and Raut, R. N. 1994. White r u s t resistance and its association with parental species type and leaf waxiness in Brassica juncea L. Czern and Coss x Brassica napus L. crosses u n d e r the action of EDTA and gamma-rays. Euphytica 74, 1-7. Tang, Z. L., Li, J. N., Zhang, X. K., Chen, L. and Wang, R. 1997. Genetic variation of yellow-seeded rapeseed lines (Brassica napus L.) from different genetic sources. Plant Breeding 116, 471-474. Tatebe, T. 1951. Studies on the behaviour of incompatible pollen in Brassica. IV. Brassica oleracea L. var. capitata and var. botrytis L. J. Hort. As. J a p a n 20, 19-26. Thies, W. 1991. Determination of the phytic acid esters in seeds of rapeseed and selection of genotypes with reduced concentrations of these c o m p o u n d s . Fat Sci. Technol., 93, 49-52. Thies, W. 1994. Die w e r t b e s t i m m e n d e n Komponenten des Rapsschrotes. Vortr. PflanzenzfAchtg. 30, 89-97. Thompson, C., Dunwell, J. M., J o h n s t o n e , C. E., Lay, V., Ray, J., Schmitt, M., Watson, H. and Nisbet, G. 1995. Degradation of oxalic acid by
459 transgenic oilseed rape plants expressing oxalate oxidase. Euphytica 85, 169-172. Thompson, K. F. 1979. Superior performance of two homozygous diploid lines from naturally occurring polyhaploids in oilseed rape (Brassica napus). Euphytica 85, 127-135. Thurling, N. 1991. Application of the ideotype concept in breeding for higher yield in the oilseed brassicas. Field Crops Res. 26, 201-219. Tillmann, P. 1997. Recent experiences with NIRS-analysis of rapeseed. GCIRC Bulletin 13, 84-87. Tiwari, A. S., Petrie, G. A. and Downey, R. K. 1988. Inheritance of resistance to Albugo candida Race 2 in m u s t a r d (Brassica juncea (L.) Czern.). Canad. J. Plant Sci. 68, 297-300. Tkachuk, R. 1981. Oil and protein analysis of whole rapeseed kernels by near infrared reflectance spectroscopy. J. Am. Oil Chem. Soc., 58, 819-822. Toxopeus, H., Yamagishi, H. and Oost, E.H. 1987. A cultivar group classification of Brassica rapa L., update 1987. Cruciferae Newsl. 12, 5-6. U, N. 1935. Genome analysis in Brassica with special reference to the experimental formation of B. napus and peculiar mode of fertilization. Japan. J. Bot. 7, 389-452. Uppstr6m, B. 1995. Seed chemistry. In: Kimber, D. and McGregor, D. I. (eds.), Brassica oilseeds. Production and utilization, CAB International, Wallingford, Oxon, U.K, pp. 217-242. Uzunova, M., Ecke, W., Weissleder, K. and R6bbelen G. 1995. Mapping the genome of rapeseed (Brassica napus L.). I. Construction of a RFLP linkage map and localization of QTLs for seed glucosinolate content. Theor. Appl. Genet. 90, 194-204. Velasco, L. and Becker, H. C. 1998. Estimation of fatty acid composition of the oil in intact-seed rapeseed (Brassica napus L.) by near-infrared reflectance spectroscopy. Euphytica 101, 221-230. Velasco, L., Fern~ndez-Martinez, J. and De Haro, A. 1995. Isolation of induced m u t a n t s in Ethiopian m u s t a r d (Brassica carinata Braun) with low levels of erucic acid. Plant Breeding 114, 454-456. Voelker, T. 1997. Genetic engineering of oil crops. Proc. 5th Syrup. Nachw. Rohstoffe, Berlin. Schr.reihe BMELF Bonn, Reihe A, Angew. Wiss. K611en Druck, Bonn, pp. 39-49. Voorrips, R. E., Jongerius, M. C. and Kanne, H. J. 1997. Mapping of two genes for resistance to clubroot (Plasmodiophora brassicae) in a population of doubled haploid lines of Brassica oleracea by m e a n s of RFLP and AFLP markers. Theor. Appl. Genet. 94, 75-82.
460 Voss, A., Ltihs, W., Friedt, W. and Ordon, F. 1996. Erste Ergebnisse zur Sch~itzung genetischer Distanzen beim Raps mittels RAPDs. Vortr. Pflanzenzfichtg. 32, 91-93. Weissleder, K., Uzunova, M. and Ecke, W. 1996. Genetische Markierung von Loci fur ph~notypische Merkmale bei Winterraps (Brassica napus L.). Vortr. Pflanzenzfichtg. 32, 88-90. Wen, L. C. 1980. Classification and evolution of m u s t a r d crops (Brassica juncea) in China. Cruciferae Newsl. 5, 33-35. Yau, S. K. and Thurling, N. 1987a. Variation in nitrogen response among spring rape (Brassica napus) cultivars and its relationship to nitrogen u p t a k e a n d utilization. Field Crops Res. 16, 133-155. Yau, S. K. a n d Thurling, N. 1987b. Genetic variation in nitrogen uptake and utilization in spring rape (Brassica napus) and its exploitation t h r o u g h selection. Plant Breeding 98, 330-338. Yoshikawa, H. 1993. Vegetable breeding in J a p a n . Chronica Horticult. 33, 34. Youngs, C. G. 1967. A m o u n t and composition of hull in rapeseed and m ustard. In: Fats and oils in Canada; a semiannual review, Dept. of Industry, Trade a n d Commerce, Ottawa, pp. 39.
Biology of Brassica Coenospecies C. G6mez-Campo (Editor) 91999 Elsevier Science B.V. All rights reserved.
461
14 GENETIC RESOURCES Ietje W. B o u k e m a a n d Theo J. L. v a n H i n t u m
Centre for Plant Breeding and Reproduction Research (CPRO-DLO}, Centre for Genetic Resources. The Netherlands (CGN), P.O. Box 16, 6700 AA Wageningen, The Netherlands. Plant genetic r e s o u r c e s are the b a s e for the i m p r o v e m e n t of crop varieties a n d therefore n e c e s s a r y to m e e t food d e m a n d s of g r o w i n g p o p u l a t i o n s , c h a n g i n g c o n s u m e r d e m a n d s a n d c h a n g i n g c u l t u r e m e t h o d s . Most c r o p s are widely d i s t r i b u t e d a n d far from t h e i r c e n t e r of origin w h i c h implies t h a t no c o u n t r y is self sufficient for t h e i r genetic r e s o u r c e s . Availability of the diversity of a crop in g e r m p l a s m collections is e s s e n t i a l for b r e e d i n g a n d o t h e r research. Genetic r e s o u r c e s collections of Brassica are m a i n t a i n e d like m o s t collections of o t h e r c r o p s by v a r i o u s o r g a n i z a t i o n s , s u c h as n a t i o n a l or regional g e n e b a n k s , d e p a r t m e n t s of u n i v e r s i t i e s , s t a t e o w n e d or p r i v a t e i n s t i t u t i o n s a n d b r e e d i n g c o m p a n i e s . Brassica is a wide g e n u s with s o m e very i m p o r t a n t c u l t i v a t e d s p e c i e s (B. carinata, B. juncea, B. napus, B. nigra, B. oleracea, B. rapa). The g e n u s r e p r e s e n t s a wide r a n g e of c r o p s i n c l u d i n g oilseed, m a n y vegetables a n d fodder crops. In s o m e species this whole r a n g e of u s e s is present. The g e n u s Brassica also c o n t a i n s m a n y wild species (see c h a p t e r 1). M a n y collections at b r e e d i n g i n s t i t u t e s were i n t e n d e d as w o r k i n g collections a n d c o n t a i n g e r m p l a s m n e c e s s a r y for s h o r t t e r m b r e e d i n g r e s e a r c h . The wide r a n g e of crops, species a n d a p p l i c a t i o n s c a u s e Brassica genetic r e s o u r ces to be even m o r e s c a t t e r e d over collections t h a n o t h e r crops; no single collection h o l d s the c o m p l e t e genetic diversity of Brassica. For a n i n t e g r a t e d a p p r o a c h of c o n s e r v i n g genetic r e s o u r c e s collections, n e t w o r k s on species or c r o p s are e s s e n t i a l . By c o l l a b o r a t i o n in n e t w o r k s the t a s k s of long t e r m c o n s e r v a t i o n of the genetic diversity a n d m a k i n g the genetic diversity available to u s e r s c a n be m o r e s t r u c t u r e d , d u p l i c a t i o n of w o r k c a n be avoided a n d g a p s in collections c a n be traced. For several c r o p s t h e r e are i n t e r n a t i o n a l n e t w o r k s c o o r d i n a t e d by the I n t e r n a t i o n a l P l a n t Genetic R e s o u r c e s I n s t i t u t e (IPGRI). For Brassica s u c h a n e t w o r k d o e s n o t (yet) exist. Large c o u n t r i e s like the USA a n d C h i n a have t h e i r o w n n a t i o n a l netw o r k s . In t h e USA several Regional P l a n t I n t r o d u c t i o n S t a t i o n s hold collec-
462 tions, a m o n g s t t h e m Brassica. Data from these collections are included in the d a t a b a s e for G e r m p l a s m Resources Information Network (GRIN) at the Beltsville Agricultural Research Center. Duplicates of these collections are stored in the long-term storage of the National Seed Storage Laboratory at Fort Collins (ARS-GRIN, 1997). In China a b o u t 20 regional crop germplasm g e n e b a n k s have been established in the last 15 years. The long-term storage of all germplasm from the entire country (350000 accessions) is the responsibility of the Institute of Crop G e r m p l a s m Resources of the CAAS (Chinese Academy of Agricultural Sciences, J i a n g Chaoyo and Liu Xu, 1992). Data of all the Chinese collections are being stored in the Chinese Genetic Resources Information System (CGRIS). In Europe, where m a n y national collections of different size exist, IPGRI co-ordinates the E u r o p e a n Cooperative Program for Crop Genetic Resources Networks (ECP/GR), which was founded in 1980. In this program, crop working groups were established. A B r a s s i c a Working Group was established in 1989 and the first meeting took place in 1991 (IBPGR, 1993). In the Working Group meetings activities regarding collecting, regeneration, safety duplication, descriptors lists, in situ conservation, research and international collaboration are discussed (Gass et al., 1995; Maggioni et al., 1997; Maggioni, 1998). An i m p o r t a n t activity of the working groups is the establishment of E u r o p e a n databases. The European B r a s s i c a d a t a b a s e (BrasEDB) was created by the CGN after a decision in the 1991 Working Group meeting and is cont i nuous l y being u p d a t e d (Hintum and Boukema, 1993; B o u k e m a and Hintum, 1998). Although no world wide network of B r a s s i c a exist, for overviews of the world collections of B r a s s i c a the inventory made by FAO of all world collections (FAO, 1997a) can be used.
S t r a t e g i e s for c o n s e r v a t i o n Some i m p o r t a n t ger m pl as m collections have a very long history, such as t h at of the N.I. Vavilov Research Institute of Plant I n d u s t r y (VIR) in St. Petersburg, Russia. However, most g e n e b a n k s have started in the last three decades, when it became increasingly clear that without an activity directed towards the conservation of genetic resources, they would be lost for future generations. In the 1980's the International Board for Plant Genetic Resources (IBPGR) and several other institutions from different regions realized that genetic erosion in cruciferous crops was increasingly severe and had to be prevented. IBPGR asked several experts to s u b m i t s t a t u s reports and organised a consultation in Rome (IBPGR, 1981). At this consultation recomendations were given on planning collection missions, procedures for regeneration, storage conditions and descriptor lists. As a result several collecting missions started. In Europe, being the centre of origin and diversity for Bras-
463
Figure 14. 1 Partial view of the seedstore of the Centre for Genetic Resources (CPRODLO) of the Netherlands, mostly devoted to crop species Figure 14. 2 Seed bank devoted to wild Crucifers in the Universidad Polit6cnica de Madrid (UPM), Spain.
464 sica oleracea, l a n d r a c e s of cruciferous crops were collected (Meer et al., 1984) in the f r a m e w o r k of a project s u p p o r t e d by the E u r o p e a n Community. Wild species related to B r a s s i c a oleracea were collected in Mediterranean c o u n - t r i e s s p o n s o r e d by IBPGR (G6mez-Campo a n d G u s t a f s s o n , 1991). In C h i n a a large scale nation-wide collection effort was u n d e r t a k e n by the Chinese g o - v e r n m e n t in the early 1950s yielding a b o u t 2 0 0 0 0 0 accessions, a m o n g s t which B r a s s i c a could be found (Jiang a n d Liu, 1992).
In general, two types of collections can be distinguished: collections m e a n t for breeding, variety testing, taxonomic or other types of research, a n d collections m e a n t for conservation of crop genetic diversity. These types are not always clearly distinguishable; m a n y 'conservation collections' started as 'research collections', s u c h as the previously m e n t i o n e d VIR collections. Most 'research collections' are m a i n t a i n e d by g o v e r n m e n t a l research a n d breeding i n s t i t u t e s a n d private breeding companies. Most 'conservation collections' can be found in specialised g e n e b a n k s in i n t e r n a t i o n a l institutes s u c h as those of the Consultative G r o u p on I n t e r n a t i o n a l Agricultural Res e a r c h (CGIAR), b u t also in national g e n e b a n k s or genetic r e s o u r c e s departm e n t s of breeding or r e s e a r c h institutes.
Maintenance of seeds Most B r a s s i c a collections are conserved as seeds. The collections in g e n e b a n k s are in general conserved u n d e r long term storage conditions to m a i n t a i n seed viability for ten to h u n d r e d years or even longer (Figures 14.1 a n d 14.2). Optimal storage conditions for the long term storage are -18~ with a 3-7% seed m o i s t u r e c o n t e n t (FAO/IPGRI, 1994). However, very often g e r m p l a s m collections, especially 'research collections', are m a i n t a i n e d u n der s u b optimal conditions. For safety r e a s o n s collections need to be duplicated in a n o t h e r genebank. Different actions are being u n d e r t a k e n to achieve this safety storage. In the USA seeds of the active regional collections are duplicated in the longterm storage of the National Seed Storage Laboratory at Fort Collins. In Chin a all material from the regional g e n e b a n k s is also being preserved in the long term storage of the Institute of Crop G e r m p l a s m Resources of the CAAS. In Europe the E C P / G R e m p h a s i z e d t h a t safety duplication is a n integral part of the long term conservation a n d t h a t the g e n e b a n k s holding national base collections are responsible for the safety duplication of these collections. They promote the 'black box' a r r a n g e m e n t , w h e r e b y the g e n e b a n k of origin h a s the r e s p o n s a b i l i t y for the quality of the stored material a n d its regeneration w h e n required (Maggione et al., 1997).
Regeneration Although it is possible to keep the seed viable for a very long time, regen e r a t i o n of the s a m p l e becomes n e c e s s a r y if the germinability starts to de-
465 crease, or if there is a shortage of seed. Maintaining the genetic integrity of the samples in a g e n e b a n k is of the highest importance. Since m o s t B r a s s i c a crops are outbreeding, it is necessary to genetically isolate the populations during regeneration to avoid c o n t a m i n a t i o n with foreign pollen. To avoid genetic drift due to small population size, a m i n i m u m n u m b e r of 100 plants should be u s e d for regeneration (IBPGR, 1981). Furt herm ore, m o s t B r a s s i c a crops are biennial. These facts m ake regeneration very costly. Knowing t h a t some change in the genetic m a k e - u p can not be avoided completely since some selection, n a t u r a l or unintentional, is b o u n d to occur, the necessity for good storage conditions to reduce the regeneration frequency as m u c h as possible, becomes obvious. Some B r a s s i c a crops such as perennial kale (B. oleracea L. var. r a m o s a DC), which, in some cases, was propagated vegetatively for centuries, have lost their ability to flower and can only be propagated vegetatively (Zeven et al., 1996). In s i t u
approaches
In situ conservation is a strategy c o m p l e m e n t a r y to the e x situ a p p r o a c h of g e n e b a n k s , in which g e r m p l a s m is conserved in its norm al habitat, i.e. in n a t u r e reserves or 'on farm'. Though in situ m e t h o d s are generally u n s u i t a b l e for conserving cultivated material it is particularly r e c o m m e n d e d for conserving wild species. One of the r e a s o n s for this is t h a t m o s t wild species are very difficult to regenerate e x situ. Wild species are mainly included in e x situ collections to make t h e m readily available to the users. A strategy for in situ conservation of wild species related to B r a s s i c a oteracea was proposed by G u s t a f s s o n (1995), which was elaborated later (Maggioni et al., 1997; Maggioni, 1998).
Description For efficient m a n a g e m e n t a n d us e of g e r m p l a s m collections, it is necessary to have good information a b o u t the identity, b a c k g r o u n d , and, above all, the genetic traits of the material. In order to standardize the description of g e r m p l a s m collections, the International Plant Genetic Resources Institute (IPGRI, formerly k n o w n as IBPGR) prepared descriptor lists for m a n y crops, in close collaboration with experts in g e n e b a n k s and universities. For B r a s sica two s u c h descriptor lists were published, one for B r a s s i c a a n d R a p h a n u s (IBPGR, 1990) a n d one for B. c a m p e s t r i s (a s y n o n y m for B. rapa, IBPGR, 1987). The latter was developed in collaboration with the Asian Vegetable Research a n d Development Centre (AVRDC) in Taiwan. Because these lists try to be complete, it is not possible to describe all c h a r a c t e r s listed. Therefore, the E C P / G R B r a s s i c a Working Group created m i n i m u m descriptor lists for the different B r a s s i c a crops (Baert, 1995; Bart kow i ak-Broda a n d Herv6, 1995; B o u k e m a a n d Astley, 1995; G u s t a f s s o n a n d G6mez-Campo, 1995).
466
Documentation Having good information a b o u t the collections is of little u s e if this information is not accessible. For this r e a s o n each g e r m p l a s m collection generally h a s its own d o c u m e n t a t i o n system. These s y s t e m s are becoming more a n d more u s e r friendly. An i n c r e a s i n g n u m b e r of s y s t e m s are accessible via the Internet, allowing direct access to the information to a n y u s e r with an I n t e r n e t connection. To avoid the n e c e s s i t y of a p p r o a c h i n g each s y s t e m individually, central crop d a t a b a s e s are being developed. The E u r o p e a n Brassica d a t a b a s e (BrasEDB) is an example of s u c h a d a t a b a s e c o m b i n i n g d a t a on all E u r o p e a n Brassica collections ( B o u k e m a a n d Hintum, 1998). In compiling this database the lack of s t a n d a r d i z a t i o n between the d a t a b a s e s of the contributors was one of the m a j o r obstacles. Especially taxonomic n o m e n c l a t u r e proved to be different in each collection. For example broccoli, the taxon called B. oleracea botrytis italica in the Bras-EDB, c o m p r i s e s material received u n d e r the n a m e s of B. botrytis italica, B. oleracea botrytis cymosa, B. oleracea botrytis italica, B. oleracea convar, botrytis var. itatica, B. oleracea italica, B. oleracea var. italica to varying extents completed with a u t h o r n a m e s . The datab a s e is available on I n t e r n e t via the ' E u r o p e a n Information Platform on Crop Genetic R e s o u r c e s ' (www.cgiar.org/ecprg/platform).
Duplication Having good a n d accessible information on the material in the collections allows the tracing of duplication within a n d between collections. Since m o s t collections have a considerable history of m u t u a l e x c h a n g e of material, m u c h material is d u p l i c a t e d in m a n y collections. Tracing this duplication is a r a t h e r complicated p r o c e s s since the transfer of information between syst e m s was done in the p a s t by m a n u a l l y entering it in the computer. This often resulted in m a n y errors s u c h as omission of i m p o r t a n t information, wrong t r a n s l a t i o n or i n c o n s i s t e n t t r a n s c r i p t i o n / t r a n s l i t e r a t i o n rules or plain typing e r r o r s - ( H i n t u m a n d Kn~pffer, 1995). After probable d u p l i c a t e s have been traced it is not at all certain t h a t these are a c t u a l duplicates. The genetic m a k e - u p of the probable duplicates m i g h t have c h a n g e d c o n s i d e r a b l y due to a variety of r e a s o n s including hum a n or n a t u r a l selection, drift, c o n t a m i n a t i o n , etc. (Hintum and Visser, 1995). B u t given the costs of m a i n t a i n i n g s a m p l e s of an o u t b r e e d i n g crop s u c h as Brassica it is of u t m o s t i m p o r t a n c e to reduce or avoid u n n e c e s s a r y duplication w h e n e v e r possible (Hintum et al., 1996).
Core c o l l e c t i o n s To m a k e large genepools more accessible for evaluation a n d use, core collections can be created. F r a n k e l (1984) i n t r o d u c e d the c o n c e p t of a core
467 collection w h i c h r e p r e s e n t s the genetic diversity of a crop species a n d its relatives with a m i n i m u m of repetitiveness. Since t h e n the c o n c e p t h a s been d i s c u s s e d intensively a n d m e t h o d s how to create t h e m have b e e n developed (Brown, 1994). Core collections c a n never replace g e r m p l a s m collections b u t are c r e a t e d to impyove their accessibility. The Barley Core Collection (Kntipffer a n d H i n t u m , 1994) is a n e x a m p l e of a n i n t e r n a t i o n a l effort in this direction. In B r a s s i c a a core collection of B. o l e r a c e a w a s c r e a t e d for a n E u r o p e a n Union project to s c r e e n E u r o p e a n g e n e b a n k collections on p e s t a n d d i s e a s e r e s i s t a n c e ( B o u k e m a et al., 1997).
Availability Genetic r e s o u r c e s held in g e n e b a n k s a n d public r e s e a r c h i n s t i t u t e s in i n d u s t r i a l i z e d a n d developing c o u n t r i e s have, traditionally, a l w a y s b e e n freely accessible, u n l i k e the m a t e r i a l in collections held by private i n d u s t r i e s . However, t h i s free a c c e s s will no longer be g u a r a n t e e d in the f u t u r e d u e to a n u m b e r of d e v e l o p m e n t s . First of all, there h a s been a t r e n d in r e c e n t y e a r s to privatize b r e e d i n g a n d r e s e a r c h i n s t i t u t e s . As a result, g e r m p l a s m is c o n s i d e r e d a n a s s e t and, therefore, a c c e s s to third p a r t i e s is no longer obvious in all cases. Secondly there is a n ongoing d i s c u s s i o n on a n i n t e r n a t i o n a l level concerning a c c e s s to p l a n t genetic r e s o u r c e s . The C o n v e n t i o n on Biological Diversity (CDB) gave n a t i o n s the j u r i s d i c t i o n over their biological diversity. However, t h e i m p l i c a t i o n s to crop genetic r e s o u r c e s c o n s e r v a t i o n p r o g r a m s were n o t clear. D e b a t e s on this m a t t e r are going on at different i n t e r n a t i o n a l platforms s u c h as the Conference of P a r t i e s of the CBD (COP) a n d the FAO C o m m i s s i o n on Genetic R e s o u r c e s for Food a n d Agriculture w h i c h are negotiating a h a r m o n i z e d I n t e r n a t i o n a l U n d e r t a k i n g on P l a n t Genetic R e s o u r c e s . The o u t c o m e of t h e s e d i s c u s s i o n s are, however, still u n c e r t a i n . Finally the influence of Intellectual Property protection legislation (as formalized in the WTO TRIPS-agreement) on a c c e s s to p l a n t genetic r e s o u r ces is i n c r e a s i n g . The effects of this d e v e l o p m e n t are also still not clear.
Summaries of Brassica genetic resources collections The m o s t i m p o r t a n t s o u r c e of i n f o r m a t i o n on w h i c h the overviews of the B r a s s i c a collections given here w a s b a s e d w a s the World I n f o r m a t i o n a n d Early W a r n i n g S y s t e m (WlEWS) c r e a t e d a n d m a i n t a i n e d by the FAO (FAO, 1997b). This i n v e n t o r y w a s m a d e in 1997. For s o m e collections the d a t a were c o r r e c t e d with i n f o r m a t i o n found in several sites on the I n t e r n e t , inform a t i o n provided in c o r r e s p o n d e n c e with collection h o l d e r s a n d finally inform a t i o n from the E u r o p e a n B r a s s i c a d a t a b a s e (Bras-EDB). The n u m b e r s of a c c e s s i o n s given s h o u l d be c o n s i d e r e d as m e r e l y indications. Since the FAO g a t h e r s its i n f o r m a t i o n from m a n y s o u r c e s s u c h as directories, q u e s t i o n -
468 n a i r e s a n d w o r k s h o p reports, it c a n h a p p e n t h a t some collections are counted twice a n d o t h e r s are n o t a c c u r a t e a n y m o r e . F u r t h e r m o r e , it is difficult to d e t e r m i n e the availability a n d quality of the collections in the FAO surveys. For o u r overviews, species n a m e s u s e d by Warwick (1993) are u s e d without authority.
Regions and countries Total n u m b e r of k n o w n conserved B r a s s i c a a c c e s s i o n s totals 74,041. F r o m these a c c e s s i o n s very large a m o u n t s are conserved in the E a s t a n d Far E a s t a n d in E u r o p e (each 41%, Table 14.1). Lower n u m b e r s are conserved in the Americas (12%), O c e a n i a (in this case Australia, 3%), Africa (2%) a n d in the Middle E a s t (1%). In E u r o p e the collections are s p r e a d over m a n y countries. The United Kingdom holds 10% of the total n u m b e r of accessions, G e r m a n y 8%, France, Spain, R u s s i a a n d B u l g a r i a e a c h a b o u t 3%, the Czech Republic a n d The N e t h e r l a n d s e a c h 2% a n d Portugal, the Ukraine, Poland a n d Italy a r o u n d 1% a n d several o t h e r c o u n t r i e s less t h a n 1%. In the E a s t a n d F a r E a s t some c o u n t r i e s hold very extensive collections like C h i n a (17%) a n d India (15%). Also c o n s i d e r a b l e n u m b e r s are held by Korea (3%), J a p a n a n d Taiwan (each a b o u t 2%) a n d the Philippines (1%). F r o m the collections in the America's the USA (9%), C a n a d a (2%) a n d Brazil (1%) are the m o s t important. Australia (here classified u n d e r Oceania) holds 3% of the world B r a s s i c a accessions. In Africa Ethiopia's collection is the only significant one (about 2%). In the Middle E a s t there are few collections. Only Israel holds a collection which a m o u n t s to n e a r l y 1% of the total.
Species and crops Regarding the species in the B r a s s i c a collections (Table 14.2), B. oleracea is r e p r e s e n t e d with the highest n u m b e r of a c c e s s i o n s (27%) followed by B. rapa (25%). B. n a p u s a n d B. j u n c e a are e a c h r e p r e s e n t e d with 18%, B. carinata a n d B. nigra are m u c h less r e p r e s e n t e d with 2% a n d 1% respectively. The wild species are r e p r e s e n t e d with less t h a n 1%. In a b o u t 7% the species n a m e is not k n o w n or is difficult to interpret. For B. oleracea a f u r t h e r classification is m a d e a c c o r d i n g to well k n o w n varietal g r o u p n a m e s (Table 14.3). To simplify the overview, the available d a t a are g r o u p e d a c c o r d i n g to this level a n d no further. B. oleracea capitata (cabbage) is r e p r e s e n t e d with the h i g h e s t n u m b e r of a c c e s s i o n s (37%), followed by B. oleracea botrytis (20%), c o m p r i s i n g cauliflower (the main part) a n d broccoli. B. oleracea g e m m i f e r a (Brussels sprouts) a n d B. oleracea acep h a l a (kale) are r e p r e s e n t e d in smaller a m o u n t s , 7% a n d 6% respectively. F r o m B. oleracea g o n g y l o d e s ( k o h l r a b i ) a n d B. oleracea alboglabra (Chinese kale) only few s a m p l e s are conserved (2% a n d 1%). In 28% no infraspecific n a m e is provided.
469 T a b l e 14. 1 N u m b e r and percentage o f conserved B r a s s i c a accessions in the world per region Region
N. of accessions
%
Europe
30145
41
East & Far East
30258
41
America
8894
12
Oceania
2426
3
Africa
1649
2
669
1
Middle East
T a b l e 14. 2 N u m b e r and percentage o f conserved B r a s s i c a accessions in the world per species. Species
N. of accessions
%
1486
2
B. juncea
13549
18
B. napus
13543
18
B. nigra
1146
2
B. oleracea
20106
27
B. rapa
18224
25
412
1
5575
7
B. carinata
Brassica wild species
B. unknown or other species
470
T a b l e 14. 3
N u m b e r and percentage o f conserved Brassica oleracea accessions in the world per varietal groups.
Varietal groups
B. oleracea acephala
N. of accessions
%
1156
6
152
1
B. oleracea botrytis
4016
20
B. oleracea capitata
7487
37
B. oleracea gemmifera
1417
7
B. oleracea gongylodes
377
2
5501
27
B. oleracea alboglabra
B. oleracea unknown or wild
T a b l e 14. 4 N u m b e r and percentage o f conserved Brassica napus accessions in the world per type o f end use.
End use
N. of accessions
%
food/fodder leaf type
2468
18
food/fodder root type
464
4
oilseed
2434
18
unknown type
8177
60
471 T a b l e 14. 5 N u m b e r and percentage of conserved B r a s s i c a rapa accessions
in the world per type of end use. End use
food/fodder leaf type food/fodder root type food/fodder flower type oilseed unknown type
N. of accessions
%
4919 1166 14 3546 8579
27 6 <1 19 47
For B. n a p u s a n d B. rapa a f u r t h e r classification is m a d e according to types of end u s e (Tables 14.4 a n d 14.5). For b o t h species the end u s e of large p a r t s is not k n o w n (60% a n d 47% respectively), b u t considerable p a r t s are of the f e e d / f o d d e r type (18% a n d 34%) of which the leaf types are the m o s t i m p o r t a n t . Both species also c o n t a i n i m p o r t a n t a m o u n t s of oilseed types (18% a n d 19%). Wild species
The k n o w n a m o u n t of c o n s e r v e d wild species are given in table 6. They are g r o u p e d a c c o r d i n g to their genomic s t a t u s after Warwick (1993). Most of the wild species belong to the g r o u p with the oleracea g e n o m e (80%). Of these, B. cretica is r e p r e s e n t e d in relatively high n u m b e r s followed by B. i n c a n a a n d B. m o n t a n a . Wild B. oleracea a c c e s s i o n s c a n n o t be given in table 14.6, b e c a u s e from the available d a t a it is not clear which of the u n k n o w n B. oleracea a c c e s s i o n s (Table 14.3) are wild. The g r o u p s with the f r u t i c u l o s a a n d r e p a n d a g e n o m e are m u c h less r e p r e s e n t e d (3% a n d 2% respectively) a n d the other species comprise 15%. End use
Regarding the end u s e of all conserved B r a s s i c a material a r o u g h estim a t e s h o w s t h a t a b o u t 40% is u s e d as feed or fodder, 30% as oilseed a n d in 30% the u s e is n o t k n o w n (Table 14.7). For t h e s e e s t i m a t e s all B. oleracea material, m a i n l y u s e d as a vegetable b u t s o m e t i m e s for feeding animals, is classified in the f e e d / f o d d e r group. F r o m the f e e d / f o d d e r g r o u p s of B. n a p u s a n d B. rapa (Tables 14.4 a n d 14.5) the classification is clear. Although end
472 T a b l e 14. 6 N u m b e r a n d p e r c e n t a g e o f B r a s s i c a w i l d species g r o u p e d acc o r d i n g to their g e n o m e status (after W a r w i c k 1993).
Genome status/species
N. of accessions
%
oleracea genome:* Brassica Brassica Brassica Brassica Brassica Brassica Brassica Brassica Brassica
bourgeaui cretica hilarionis incana insularis macrocarpa montana rupestris villosa
fruticulosa genome: Brassica fruticulosa Brassica maurorum Brassica spinescens repanda genome: Brassica desnottesii Brassica repanda
6 116 4 46 28 17 45 32 36
1 28 1 11 7 4 11 8 9
330
80
10 2 2
2 <1 <1
14
3
1 7
<1 2
8
2
4 6 3 11 4 2 1 5 24
1 1 1 3 1 <1 <1 1 6
60 412
15 1O0
own genome: Brassica balearica Brassica barrelieri Brassica deflexa Brassica elongata Brassica gravinae Brassica oxyrrhina Brassica procumbens Brassica souliei Brassica tournefortii
total
* wild B. oleracea are omitted because of insufficient data to discriminate between wild and cultivated.
473 T a b l e 14.7 Estimated n u m b e r and percentage o f conserved Brassica acces-
sions in the world per end use. .
.
.
.
End use
N. of accessions
%
food/fodder
29137
40
oilseed
22161
30
unknown or other
22743
30
u s e of B. carinata, B. nigra a n d B. j u n c e a is n o t solely oilseed a n d especially B. juncea w h i c h also i n c l u d e s vegetable types, t h e s e species were classified u n d e r oilseed. Of the B. juncea m a t e r i a l c o n s e r v e d by the CAAS in C h i n a (+3000 accessions) p a r t will be of the vegetable type, b u t from the available d a t a the end u s e is difficult to trace. Important
collections
Since i n f o r m a t i o n on 134 collections in 56 c o u n t r i e s w a s u s e d in the overviews given, only a r a t h e r r a n d o m selection of large or otherwise import a n t collections will be d e s c r i b e d below.
East and Far East 9 In C h i n a the I n s t i t u t e of Crop G e r m p l a s m R e s o u r c e s of the CAAS in Beijing holds very large collections of B. rapa (>5000), B. j u n c e a (+3000) a n d B. napus (1500) a n d some s m a l l e r of B. oleracea (+500). 9 In India very extensive collections of oilseed b r a s s i c a s are m a i n tained. The National B u r e a u of P l a n t Genetic R e s o u r c e s at New Delhi holds a large collection of m a i n l y B. juncea (+ 1170) a n d B. rapa (+ 1040). The All India C o o r d i n a t e d Project on Rape a n d M u s t a r d at the H a r y a n a Agricultural University, Hisar holds large a m o u n t s of B. juncea (>5400) a n d B. rapa (+1700), a n d the Division of Genetics of the I n d i a n Agricult u r a l R e s e a r c h I n s t i t u t e m a i n t a i n s a b o u t 1200 B. juncea a c c e s s i o n s . 9 In J a p a n the National I n s t i t u t e of Agrobiological R e s o u r c e s at T s u k u b a holds a r o u n d 700 B. rapa (vegetables) a n d a r o u n d 100 B. oleracea a c c e s s i o n s .
474 9 In Korea the G e r m p l a s m M a n a g e m e n t Office of the Crop Experim e n t S t a t i o n a t Y o n g d a n g h o l d s a large oilseed collection of B. n a p u s
(_+2200). Europe 9 In R u s s i a the N.I. Vavilov R e s e a r c h I n s t i t u t e for P l a n t I n d u s t r y , St. P e t e r s b u r g h o l d s a b o u t 1800 B. oleracea, 170 B. rapa a n d 30 B. carinata. 9 In t h e U n i t e d K i n g d o m t h e H o r t i c u l t u r e R e s e a r c h I n t e r n a t i o n a l (HRI), a t W e l l e s b o u r n e h o l d s m o r e t h a n 2 0 0 0 B. oleracea, 3 6 0 B. rapa, 3 0 0 B. n a p u s a n d 45 B. j u n c e a a c c e s s i o n s . 9 In G e r m a n y the G e n e b a n k of the I n s t i t u t e for P l a n t G e n e t i c s a n d Crop P l a n t R e s e a r c h (IPK) at G a t e r s l e b e n h o l d s a l m o s t 1000 a c c e s s i o n s of B. oleracea, a r o u n d 4 5 0 of B. rapa, 200 of B. napus, 130 of B. juncea, besides a b o u t 45 B. nigra, 40 B. carinata a n d 65 a c c e s s i o n s of wild Brassica species. The B r a u n s c h w e i g P l a n t Genetic R e s o u r c e s Collection (BGRC) of the F e d e r a l C e n t r e for B r e e d i n g of C u l t i v a t e d P l a n t s (BAZ) a t B r a u n s c h weig h o l d s a b o u t 390 B. oleracea a c c e s s i o n s , 2 6 0 of B. napus, 195 of B. rapa, 115 of B. carinata a n d 95 e a c h of B. nigra a n d B. juncea. 9 In t h e N e t h e r l a n d s the C e n t r e for G e n e t i c R e s o u r c e s the Netherl a n d s (CGN) h o l d s a b o u t 590 B. oleracea a c c e s s i o n s , 100 of B. napus, 335 of B. rapa, 100 of B. carinata a n d 20 e a c h of B. nigra a n d B. juncea. 9 In F r a n c e the P l a n t B r e e d i n g S t a t i o n of the National Agricultural R e s e a r c h I n s t i t u t e (INRA) in Le R h e u m a i n t a i n s a b o u t 525 l a n d r a c e s of B. oleracea, 80 of B. n a p u s a n d 30 B. rapa. 9 One of the l a r g e s t collections of wild n = 9 Brassica species (240 a c c e s s i o n s ) a n d of o t h e r Brassica species a n d allied c r u c i f e r o u s g e n e r a (up to 6 0 0 a c c e s s i o n s e x c l u d i n g the a l r e a d y m e n t i o n e d with n = 9) is held by the U n i v e r s i d a d Polit~cnica in Madrid, S p a i n ( G 6 m e z - C a m p o , 1990). It s t a r t e d in 1966.
America 9 In the USA the N o r t h - e a s t e r n Regional P l a n t I n t r o d u c t i o n Station at the New York S t a t e A g r i c u l t u r a l E x p e r i m e n t Station, G e n e v a holds a large n u m b e r of B. oleracea (1600), s o m e 5 0 0 B. rapa, a b o u t 75 wild species a n d of B. juncea, B. nigra a n d B. n a p u s e a c h a r o u n d 50 a c c e s s i o n s . The North C e n t r a l Regional P l a n t I n t r o d u c t i o n S t a t i o n a t t h e Iowa State University, A m e s h o l d s n e a r l y 3 0 0 0 a c c e s s i o n s of oilseeds of w h i c h a r o u n d 1250 are B. rapa, 1030 B. j u n c e a a n d 5 6 0 B. napus. 9 In C a n a d a the C r u c i f e r a e G e r m p l a s m Node at the AAFC R e s e a r c h C e n t r e , S a s k a t o o n , p a r t of t h e P l a n t G e n e R e s o u r c e s C a n a d a , holds
475 a b o u t 240 B. n a p u s , 200 B. r a p a , 70 B. j u n c e a a n d 50 B. oleracea. This collection is expected to be e x t e n d e d with c o n s i d e r a b l e m a t e r i a l c u r r e n t l y m a i n t a i n e d in o t h e r collections in C a n a d a .
Oceania 9 In A u s t r a l i a the A u s t r a l i a n T e m p e r a t e Field Crops Collection at the Victorian Crops R e s e a r c h Institute, H o r s h a m holds a collection of 1900 B r a s s i c a accessions. Africa 9 In E t h i o p i a the Biodiversity I n s t i t u t e in Addis A b a b a holds a large n u m b e r of B. c a r i n a t a (+ 1100) besides B. nigra (+ 120).
Concluding remarks Although the n u m b e r s given m i g h t be o v e r e s t i m a t e d , high n u m b e r s of B r a s s i c a a c c e s s i o n s are conserved in collections all over the world. The high a m o u n t s of B. o l e r a c e a in E u r o p e a n collections, B. j u n c e a in collections in India a n d China, B. c a r i n a t a in Ethiopia, B. r a p a in collections in E u r o p e
a n d in the E a s t a n d F a r E a s t reflect the c e n t e r s of origin a n d diversity of these species (IBPGR, 1981). For B. n a p u s this relation does n o t exist t h a t clearly. F r o m the available information s o u r c e s it is difficult to d e t e r m i n e which p a r t of the m a t e r i a l listed is actually alive a n d available for exchange, nor is it possible to d e t e r m i n e how m u c h of the m a t e r i a l c o n s i s t s of duplicates. Since this m i g h t strongly differ b e t w e e n species or origin regions, it is also difficult to trace possible o m i s s i o n s in the world g e r m p l a s m holding, a n d t h u s to prioritize f u t u r e collection efforts. R e g e n e r a t i o n of B r a s s i c a is very costly. Therefore good storage conditions in order to m a i n t a i n the seed viability are essential. It is also very imp o r t a n t to avoid u n n e c e s s a r y duplicates. C o n s e q u e n t l y good a n d accessible information on the m a t e r i a l in the collections is n e e d e d to trace t h e s e duplicates. An i n t e r n a t i o n a l central crop d a t a b a s e with high quality a n d highly accessible d a t a would be of great help. This would also e n c o u r a g e a more efficient u s e of the material. For the wild B r a s s i c a species in situ c o n s e r v a t i o n is of high i m p o r t a n c e ; the e x situ collections help to m a k e a small p a r t of the diversity readily accessible for utilization. In order to g u a r a n t e e availability of B r a s s i c a g e r m p l a s m to f u t u r e generations, f u n d i n g agencies, s u c h as n a t i o n a l g o v e r n m e n t s , n e e d to c o m m i t t h e m s e l v e s to s u p p o r t c o h e r e n t c o n s e r v a t i o n p r o g r a m s . These s h o u l d com-
476 prise of both ex situ strategies for the conservation of cultivated material, and in situ strategies, for the wild material. Genebanks and the other entities involved in the conservation of Brassica have to coordinate their activities better, to avoid and reduce u n n e c e s s a r y duplication of efforts and set targeted priorities.
Acknowledgements We would like to t h a n k Dr. Jerzy Serwinski from the FAO, Rome, Italy for the B r a s s i c a data of the WlEWS, which formed the main source for our overviews and Dr. Seifu Ketema from the Biodiversity Institute, Addis Abeba, Ethiopia, Dr. Richard Gugel from the Cruciferae Germplasm Node, AAFC Research Centre, Saskatoon, C a n a d a and Dr. Richard L u h m a n from North Central Regional Plant Introduction Station, Ames, USA for the valuable information on their collections.
References ARS-GRIN. 1997. Internet site: Germplasm resources information network, Na-tional Genetic Resources Program of the USDA's Agricultural Re-search Service, URL: http://www.ars-grin.gov/ Baert, J. 1995. Minimum descriptor list for forage Brassica. In: Gass, T., Gustafsson, M., Astley, D. and Frison, E. A., compilers, Report of a working group on Brassica. 2nd meeting, 13-15 November 1994, Lisbon, Portugal, European Cooperative Programme for Crop Genetic Resources Networks, International Plant Genetic Resources Institute, Rome, Italy, pp. 16-17. Bartkowiak-Broda, I. and Herv6, Y. 1995. Minimum descriptor list for oilseed Brassica. In: Gass, T., Gustafsson, M., Astley, D. and Frison, E. A., compilers, Report o f a working group on Brassica. 2nd meeting, 1315 November 1994, Lisbon, Portugal. European Cooperative Programme for Crop Genetic Resources Networks, International Plant Genetic Resources Institute, Rome. Italy, pp. 17-18. Boukema, I. W. and Astley, D. 1995. Minimum descriptor list for vegetable Brassica. In: Gass, T., Gustafsson, M., Astley, D. and Frison, E. A., compilers, Report of a working group on Brassica. 2nd meeting, 1315 November 1994, Lisbon, Portugal. European Cooperative Programme for Crop Genetic Resources Networks, International Plant Genetic Resources Institute, Rome, Italy, pp. 15-16. Boukema, I. W. and van Hintum, Th. J. L. 1998. The European Brassica database. In: Thomas, G. and Monteiro, A. (eds.), Proc. of an Intermational S y m p o s i u m on Brassicas, ISHS. Acta Horticultura 459, 249254.
477 Boukema, I. W., van Hintum, Th. J. L. and Astley, D. 1997. Creation and composition of the Brassica oleracea core collection. Plant Genetic Resources Newsl. 111, 29-32. Brown, A. H. D. 1994. The core collection at the crossroads. In: Hodgkin, T., Brown, A. H. D., van Hintum Th. J. L. and Morales, E. V. A. (eds.), Core collections of Plant Genetic Resources. J o h n Wiley and Sons, U.K., pp. 3-19. FAO, 1997a. Internet site: World Information s y s t e m on ex situ collections, The FAO's World Information Database on Plant Genetic Resources. URL: http://www.icppgr.fao.org/WIEWS/wiews.html FAO, 1997b. Report of the technical meeting on the methodology of the world Information and early warning s y s t e m on plant genetic resources, in Radzikow, Poland. Food and Agriculture Organization of the United Nations (FAO), Rome, Italy, 59 pp. FAO/IPGRI, 1994. Genebank standards. Food and Agriculture Organization of the United Nations, International Plant Genetics Resources Institute, Rome. Italy, 13 pp. Frankel, O. H. 1984. Genetic perspectives of germplasm conservation. In: Arber, W., Llimensee, K., Peacock, W. J. and Starlinger, P. (eds.), Genetic manipulation: impact on man and society. Cambridge University Press. Cambridge. pp. 161-170. Gass, T., Gustafsson, M., Astley, D. and Frison, E. A., compilers. 1995. Report of a working group on Brassica. 2nd meeting, 13-15 November 1994, Lisbon, Portugal. European Cooperative Programme for Crop Genetic Resources Networks (ECP/GR), International Plant Genetic Resources Institute, Rome, Italy. 87 pp. G6mez-Campo, C. 1990. A germplasm collection of crucifers. Colecc. Cat~logos I.N.I.A., no 22, Instituto Nacional de Investigaciones Agrarias, Madrid. 53 pp. G6mez-Campo, C. and Gustafsson, M. 1991. Germplasm of wild n=9 Medit e r r a n e a n species of Brassica. Botanika Chronika 10, 429-434. Gustafsson, M. 1995. A strategy for in situ conservation of wild Brassica. In: Gass, T., Gustafsson, M., Astley, D. and Frison, E. A., compilers. Report of a working group on Brassica. 2nd meeting, 13-15 November 1994, Lisbon, Portugal. European Cooperative Programme for Crop Genetic Resources Networks, International Plant Genetic Resources Institute, Rome, Italy, pp. 75-76. Gustafsson, M. and G6mez-Campo, C. 1995. Minimum descriptor list for wild Brassica. In: Gass, T., Gustafsson, M., Astley, D. and Frison, E. A., compilers. Report of a working group on Brassica. 2nd meeting, 13-15 November 1994, Lisbon, Portugal. E u r o p e a n Coopera-
478 tive P r o g r a m m e for Crop Genetic R e s o u r c e s Networks, International Plant Genetic R e s o u r c e s Institute, Rome, Italy, pp. 18. H i n t u m , Th. J. L. van, a n d B o u k e m a , I. W. 1993. The e s t a b l i s h m e n t of the E u r o p e a n D a t a b a s e for Brassica. Plant Genetic Resources Newsl.
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H i n t u m , Th. J. L. van, a n d Kn~pffer, H. 1995. Duplication within a n d between g e r m p l a s m collections. I. Identifying duplication on the basis of p a s s p o r t data. Genetic Resources and Crop Evolution 42, 127133. H i n t u m , Th. J. L. van, a n d Visser, D. L. 1995. Duplication within a n d between g e r m p l a s m collections. II Duplication in four E u r o p e a n barley collections. Genetic Resources and Crop Evolution 42, 135-145. H i n t u m , Th. J. L. van, B o u k e m a , I. W. a n d Visser, D. L. 1996. Reduction of duplication in a Brassica oleracea g e r m p l a s m collection. Genetic Resources and Crop Evolution 43, 343-349. IBPGR, 1981. Genetic resources of cruciferous crops. IBPGR S e c r e t a r i a t Cons u l t a t i o n on the Genetic R e s o u r c e s of Cruciferous Crops, 17-19 November 1980. I n t e r n a t i o n a l Board for Plant Genetic Resources, Rome, Italy, 47 pp. IBPGR, 1987. Descriptors for Brassica campestris L. I n t e r n a t i o n a l Board for Plant Genetic Resources, Rome, Italy, 27 pp. IBPGR, 1990. Descriptors for Brassica and Raphanus. I n t e r n a t i o n a l Board for Plant Genetic Resources, Rome, Italy, 51 pp. IBPGR, 1993. Report of the first meeting of the ECP/GR Brassica Working Group. E u r o p e a n Cooperative P r o g r a m m e for Crop Genetic Resources Networks. I n t e r n a t i o n a l Board for Plant Genetic Resources, Rome, Italy, 52 pp. J i a n g C h a o y u a n d Liu Xu, 1992. The progress of r e s e a r c h work on crop g e r m p l a s m r e s o u r c e s in China. Genetic Resources and Crop Evolution 39, 55-58. Kntipffer, H. a n d van H i n t u m , Th. J. L. 1994. The barley core c o l l e c t i o n - an i n t e r n a t i o n a l effort. In: Hodgkin, T., Brown, A.H.D., van H i n t u m Th. J. L. a n d Morales, E. V. A. (eds.), Core collections of Plant Genetic Resources. J o h n Wiley a n d Sons, U.K. pp. 171-178. Maggioini, L. 1998. Activities a n d a c h i e v e m e n t s of the E C P / G R B r a s s i c a Working Group. In: T h o m a s , G. a n d Monteiro, A. (eds.), Proc. of an Intermational Symposium on Brassicas, ISHS. Acta Horticultura 459, 243-248. Maggioni, L., Astley, D., G u s t a f s s o n , M., Gass, T. a n d Lipman, E., compilers. 1997. Report of a working group on Brassica. Third Meeting,
479 27-29 November 1996, I n t e r n a t i o n a l Institute, Rome, Italy, 92 pp.
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Meer, Q. P. van der, Toxopeus, H. Crisp, Roelofsen, H. a n d Astley, D. 1984. The collection of land-races of Cruciferous crops in the EC countries. Final report of the EC r e s e a r c h p r o g r a m m e 0890. IVT, Wageningen. 209 pp. Warwick, S. I. 1993. Guide to the wild g e r m p l a s m of Brassica a n d allied crops I. T a x o n o m y of the g e n o m e s t a t u s in the tribe B r a s s i c e a e (Cruciferae). Agriculture C a n a d a Research B r a n c h Technical Bulletin 1993 -14E. 33 pp. Zeven A. C., S u u r s L. C. J. M. a n d Waning, J. 1996. Diversity for enzymes, flowering b e h a v i o u r a n d purple p l a n t colour of perennial kale (Brassica oleracea L. var. ramosa DC.) in the Netherlands. In: Dias, J. S., Crute, I. a n d Monteiro, A. A. (eds.), Proc. of an International S y m p o s i u m on Brassicas. ISHS. Acta Horticultura 4 0 7 , 61-66.
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481
SUBJECT INDEX Abscisic acid (ABA), 256. Afghanistan, 45,46. Agrobacterium rhizogenes, 288,289,292. Agrobacterium tumefaciens, 124,186,264,265,288 -290,292. Albugo candida, 117,236,381,382,386-388,418,420 resistance, 387. Algeria, 14-17,19,74. alloploids, 40,41,64,66,68,69,73,88. Alps, 15. Alternaria, 375-378,418. resistance, 84,117,119,122,377. Alternaria brassicae, 375-378. Alternaria brassicicola, 375-377. aminoacids, 319-321. Ammosperma, 19,21,41,42. anthocyanins, 343. antibacterial effects, 340. anticarcinogenic effects, 303,341,342. antifungal effects, 340. aphid resistance, 119. Arabia, 20. Arabidopsis thaliana, 116,120,122,125,126,154, 160-162,168,196,234,287,302,375,393,448. Armoracia, 342. artificial seed technology, 266. Asia, 11,19,43,44,45,51. Asia Minor, 46. asymmetric hybrids, 134. Atlas mountains., 15. Australia, 382,468,474. autopolyploids, 64. auxin, 255. Bacillus thuringiensis, 292. Barbarea stricta, 120-122. Barbarea vulgaris, 120-122. barnase genes, 186. barstar gene, 186. beet cyst nematode (Heterodera). see nematodes. bioherbicides, 340. biolistics, 290. biotin, 321. black leg, see Leptosphaeria/Phoma. black rot, see Xanthomonas. Boleum, 19,20,21,41,42. Brassica, 3,14,21,41,42,60,61. A,B,C genomes, 67,217,224-231. allies, 3,18. cultivated species, 4,11,39,40. database, 462. genome plasticity, 225,227. genome structure, 65,224-230. genomic relationships, 229.
germplasm, 469. leaf shape, 14,15. molecular lineages, 35-37. n=9 genome, 12,13. origin of the genomes, 230-234. phylogeny, 34-39,42,232-234. plural of the name, 11. polyphyletism, 35. sections, 5-7,14-16. somatic hybrids, 115-119. subgenera, 5,6,14,21. working group, 462,465. Brassica adpressa see Hirschfeldia. Brassica alboglabra, 4,12,48,81,342. Brassica amplexicaulis, see B. souliei. Brassica balearica, 5,13,63,64,81. Brassica barrelierL 6,14,15,37,62. wide sexual hybridizations, 76-79. Brassica botteri, 5,13. Brassica bourgaei, 4,11,79,342. Brassica cadmea, 6,14. Brassica campestris, see B. rapa. subsp, rapifera see B. rapa subsp, rapa. Brassica carinata, 5,11,13,67. cytodeme, 63,64. cytogenetic architecture, 65. domestication, 34,51. haploids, 251. male sterility, 195. molecular lineage, 36. nuclear DNA, 66-68. origin, 40. somatic hybridizations, 120-122. synthesis, 69-72. transformation, 292. wide sexual hybridizations, 80,82. Brassica chinensis, see B. rapa. Brassica cossoniana, 6,36. cytodemes, 63,64. Brassica cretica, 5,12,13,342. subspecies, 5,13. Brassica crops, 315,317,339. economic importance, 315,317. nutritional value, 339. Brassica cypria, see cauliflower, 49. Brassica deflexa, 6,14,36,62. Brassica desertiL 6. Brassica desnotesii, 6,15. Brassica dichotoma see B. rapa. Brassica dimorpha, 7,16,63,64. Brassica elongata, 6.15,80. cytodeme, 63. subspecies, 6. Brassica fruticulosa, 5,14.
482
cytodemes, 62,63. molecular lineage, 36,38. subspecies, 5,6. wide sexual hybridizations, 76-83. Brassica gravinae, 6,15,36,83. cytodemes, 62-64. Brassica hilarionis, 5,12. Brassica hirta, see Sinapis alba. Brassica incana, 5,12,13,40,342. Brassica insularis, 5,13. Brassica jordanoffii, 6,15. Brassicajuncea, 5,11,67. CMS cytoplasm, 132,133. cybrids, 131. cytodeme, 63,64. cytogenetic architecture, 65. domestication, 34,46,47. haploids, 251. linkage map, 224. male sterility, 188,194,195,202. molecular lineage, 36. nuclear DNA, 66-68. origin, 40,46. somatic hybridization, 117,119. synthesis, 69-72. transformation, 292. wide sexual hybridizations, 76-83. Brassicajuncea subsp, crispifolia, 11. Brassicajuncea subsp, foliosa, 11. Brassicajuncea subsp, integrifolia, 11. Brassicajuncea subsp, napiformis, 11. Brassica kaber, see Sinapis arvensis. Brassica loncholoma, 6,15. Brassica macrocarpa, 5,12,342. Brassica maurorum, 6,14,36,38. wide sexual hybridizations, 76,77. Brassica montana, 5,12,342. Brassica napus, 5,13,33,67. cybrids, 130,131. cytodeme, 63,64. cytogenetic architecture, 65. diseases, 418. domestication, 34,49,50. germplasm, 470. haploids, 251. linkage map, 222,224,225,226. male sterility, 188,195,202. molecular lineage, 36. nuclear DNA, 66-68. origin, 40. seed chemical composition, 422. somatic hybridizations, 1 ! 7,119,128. synthesis, 69-72. synthesis by protoplast fusion, 110-113. transformation, 292,294-297,300-302. wide sexual hybridizations, 76-83. Brassica napobrassica, see B. napus.
Brassica napus subsp, napobrassica, 10. Brassica napus subsp, pabularia, 10. Brassica napus subsp, rapifera, 10,50. Brassica narinosa see B. rapa. Brassica nigra, 6,14,67. addition lines, 87,88. cytodeme, 62. cytogenetic architecture, 65. domestication, 33,45. haploids, 251. linkage map, 222,223. molecular lineage, 36. nuclear DNA, 66-68. somatic hybridizations, 117,119. transformation, 292. wide sexual hybridizations, 76-80. Brassica nipposinica, see B. rapa. Brassica nivalis, 6,15. Brassica oleracea, 5,12,14,60,67. addition lines, 88,89. chemical composition, 320,321. cybrids, 130,131. cytodeme, 62. cytogenetic architecture, 65. domestication, 34,47-49. germplasm, 470. glucosinolates, 332,336. haploids, 251. linkage maps, 218-221. male sterility, 187,194,202. market qualities, 415,416. molecular lineage, 36. nuclear DNA, 66-68. origin, 39,40. rapid cycling, 291-292. satellite chromosomes, 68. somatic hybridizations, 117,119. transformation, 291-292. varieties, 4, 316,320,321,332. var. sylvestris, 4. wide sexual hybridizations, 76-83. wild relatives, 12. wild types, 4. Brassica oxyrrhina, 6,14,15. addition lines, 89. cytodeme, 62. male sterility, 190,191,193. molecular lineage, 36,37. wide sexual hybridizations, 78,79. Brassica pekinensis, see B. rapa. Brassica procumbens, 6,14. position, 38. Brassica rapa, 5,10,11,14. cybrids, 130,131. cytodeme, 63. cytogenetic architecture, 65. domestication, 33,43,44.
483
haploids, 251. infraspecific variability, 10,11. leafy forms, 44. linkage maps, 221. male sterility, 187,188,194. molecular lineage, 36. name priority, 10. nuclear DNA, 66-68. oleiferous, 44. origin, 39. satellite chromosomes, 68. somatic hybridizations, 128. transformation, 292. wide sexual hybridizations, 76-83. Brasstca rapa subsp, campestris, 5. Brasslca rapa subsp, chinensis, 5,10,73,113. Brasstca rapa subsp, dichotoma, 5. Brasslca rapa subsp, narinosa, 5,73. Brasslca rapa subsp, nipposinica, 5. Brasslca rapa subsp, pekinensis, 5,10,44,73,113, 415. Brassica rapa subsp, rapifera = B. rapa subsp.
somatic hybrids, 117. wide sexual hybridizations, 77,79. Brassica Subgen. Brassica, 5,14,21,41,42. Brassica Subgen. Brassicaria, 6,7,13,21,38,41,42.
disease resistance, 419,420. future goals, 447,448. methods, 431. oilseed quality, 421. of hybrid cultivars, 436-442. of line cultivars, 432-434. of population cultivars, 434-436. operational steps, 427,431. pest resistance, 420,421. results, 442-446. British Islands, 48. broccoli,218. origin, 49. chemical composition, 315,316,322,334. brown sarson, see B. rapa. Brussels sprouts. chemical composition, 315,316,320-322,333. origin, 49. Bulgaria, 15,468. C3-C4 photosynthesis. cabbages, 34,44,48-50,58,84,113,202. chemical composition, 316,320-322,332,333. head quality, 415. cadmium tolerance, 294. Cakile, 20,21,41,42. calcium, 320. Calepina, 19,21,22,41,42. Camelina sativa, 84,377. Canada, 50,73,417,468,474. Canary Islands, 4. cancer, 303,341,342. canola, 300,417,421,441. Cape Verde Islands, 17. carbohydrates, 319,254,320. carbon dioxide effects, 361. carotene, 321. Carrichtera, 20,21,41,42. catch crops, 318. Cato, 44,49. cauliflower, 218,362. composition, 315,316,320-322,334. curd formation, 363,367. origin, 49. Ceratocnemum, 18,21,22,41,42.
cytodeme, 62. cytoplasm, 133,132,200. male sterility, 189,190,192,198. molecular lineage, 36-38. somatic hybridizations, 117. wide sexual hybridizations, 78,79,83. Brassica trilocularis, see B. rapa, 45. Brassica villosa, 5,12. position, 40. subspecies, 5. Brazil, 468. breeding objectives, 415-417,419-424.
chemical composition, 315-345. China, 4,43,46,47,461,462,473. Chinese cabbage, 113,287. chloroplast genome, 40,4 l, 129. chloroplast (cp) DNA, 17,20,35,37-41,50,60,65, 66,71,73,84-86,162. choline esters, 345. chromosome addition lines, 87-89. chromosome numbers, 62-64,74. chromosome pairing, 39. clubroot, see Plasmodiophora. CMS see cytoplasmic male sterility.
rapa. Brassica rapa subsp, trilocularis, 5. Brassica repanda, 6,15,60.
cytodeme, 62,63. subspecies, 6. Brassica rupestris, 5,12. subspecies, 5. Brassica Sect. Brassica, 5,14. Brassica Sect. Brassicaria, 6,7,13,15,21,38,41,42. Brassica Sect. Brassicoides, 6,14. Brassica Sect. Micropodium, 5,14. Brassica Sect. Nasturtiops, 7,16. Brassica Sect. Rapa, 5,14. Brassica Sect. Sinapistrum, 6,14. Brassica setulosa, 6,15. Brassica somaliensis, 7,15. Brassica soulieL 7,16,36,63. Brassica spinescens, 6,14,36,38.
Brassica tournefortii, 6,14,15.
Chalcanthus, 21.
484
coenospecies, 3,18,60-62. Coincya. 3,9,18,21,22,36,41,42,60,61,107. cytodemes, 63,64.
Coincya longirostra, 9. Coincya monensis, 9,77. susbpecies, 9.
Coincya richeri, 9. Coincya rupestris, 9,80. Coincya transtagana, 9. Coincya wrightii, 9. colchicine, 262. cold tolerance, 122. collards, 218,334. collecting expeditions, 12,74. Columella, 44. colza, 50. compatibility see self incompatibility. Confuceous, 44. Conringia, 21,41,42. conservation strategies, 462,464. Convention on biological diversity, 467. Cordylocarpus, 21,22,41,42. core collections, 466. Corsica, 13. Crambe, 20,21,22,41,42,83. Crambella, 21,22,41,42. cybrids, 126,130,131,191,193. cyclic amphiploidy, 232-234. Cylindrospermum concentricum, see Pyrenope-
ziza.
cytodemes, 60-64. cytoplasm substitutions, 86. modified by protoplast fusion, 132. cytoplasmic male sterility, 119,132,133,186-202, 437,438. establishment, 189,190. in somatic hybrids, 117,119. use in the production of hybrids, 199-202. Dai, 46. dark leaf spot, see Alternaria. dextruxins, 377. Didesmus, 21,41,42. Diplotaxis, 3,7,21,41,42,60,61. cultivated species, 51. flavonoids, 342-343. molecular lineages, 35-37. sections, 8. subgenera, 7,8,17,21. Diplotaxis acris, 8,63.
Diplotaxis Diplotaxis Diplotaxis Diplotaxis Diplotaxis Diplotaxis
antoniensis, 8. assurgens, 8,62 berthautiL 8,62. brachycarpa, 8,16. brevisiliqua, 8,16. catholica, 8,62,78.
somatic hybridizations, 119.
Diplotaxis erucoides, 8,16.
cytodeme, 62. molecular lineage, 36,38. wide sexual hybridizations, 76,77,81,83.
Diplotaxis glauca, 8. Diplotaxis gorgadensis, 8. Diplotaxis gracilis, 8. Diplotaxis griffithii, 8. Diplotaxis harra, 8,16,36,60.
cytodeme, 63. somatic hybridizations, 119. subspecies, 8.
Diplotaxis Diplotaxis Diplotaxis Diplotaxis
hirta, 8. ibicensis, 8,16. ilorcitana, 8,16. muralis, 7,16,36.
cytodeme, 63,64. cytoplasm, 189,199. somatic hybridizations, 119. wide sexual hybridizations, 78,83.
Diplotaxis nepalensis, 8. Diplotaxis ollivieri, 8. Diplotaxis pitardiana, 8. Diplotaxis Sect. Erucoides, 8. Diplotaxis Sect. Heterocarpum, 8. Diplotaxis Sect. Rhynchocarpum, 8. Diplotaxis siettiana, 8,36,62,77. Diplotaxis siifolia, 8,16,63,79,80. cytoplasm, 189.
Diplotaxis simplex, 7. Diplotaxis Subgen. Diplotaxis, 7,17,21,41,42. Diplotaxis Subgen. Hesperidium, 8,17,21,41,42. Diplotaxis Subgen. Rhynchocarpum, 8,21,41,42. Diplotaxis sundingii, 8. Diplotaxis tenuifolia, 7,63. wide sexual hybridizations, 80-82.
Diplotaxis tenuisiliqua, 8,62. Diplotaxis varia, 8. Diplotaxis villosa, 8. Diplotaxis viminea, 7,63,80. Diplotaxis virgata, 8,16,78,82. cytodeme, 62. subspecies, 8.
Diplotaxis vogelii, 8. direct DNA transfer, 299. diseases, 375-393. DNA markers, see markers. Dolichorrhynchus, 19,20,21,41,42. double haploids, 261,262,433,434. double-zero varieties, 50,421,423,443. Douepia, 19,21,41,42. downy mildew, see Peronospora. drought tolerance, l 17,119. Egypt, 5 I. electroporation, 289. embryo culture, 258-261.
485 Enarthrocarpus, 21,41,42,60,61,63. wide sexual hybridizations, 81-83. epicuticular waxes, 12,345. Eremophyton, 21,41,42. Eruca, 3,9,18,21,41,42,60,61. cytodeme, 63. Eruca vesicaria, 9,18. Eruca vesicaria subsp, sativa, 9. domestication, 51. molecular lineage, 36. origin, 41. resistance to white rust, 387. somatic hybridizations, 119. wide sexual hybridizations, 79-81,83. Erucaria, 20,21,41,42. Erucastrum, 3,7,16,17,21,41,42. molecular lineages, 35-37. Erucastrum abyssinicum, 7,36. cytodemes, 62-64. flavonoids, 342,343. wide sexual hybridizations, 80,82. Erucastrum arabicum, 7. Erucastrum brevirostre, 7. Erucastrum canariense, 7,16,78. Erucastrum cardaminoides, 7,16,62,76,78. Erucastrum elatum, 7,63,64. Erucastrum elgonense, 7,16. Erucastrum gallicum, 7,16,63,64. wide sexual hybridizations, 81. Erucastrum griquense, 7. Erucastrum ifniense, 7,16. Erucastrum laevigatum, Europe see E. virgatum. Erucastrum laevigatum, Africa see E. littoreum. Erucastrum leucanthum, 7,16,37. Erucastrum littoreum, 7,16,37. subspecies, 7. wide sexual hybridizations, 76-83. Erucastrum meruense, 7,16. Erucastrum nasturtiifolium, 7,16. cytodemes, 62-64. Erucastrum pachypodum, 7. Erucastrum palustre, 7. Erucastrum rifanum, 7,16. Erucastrum rostratum, 7,16. Erucastrum strigosum, 7. Erucastrum varium, 7,16,37,62. subspecies, 7. wide sexual hybridizations, 76. Erucastrum virgatum, 7,16,37. cytodemes, 62-64. subspecies, 7. wide sexual hybridizations, 76,77,80,81. erucic acid, 73,113,235,322,323. Erysiphe polygonL 393. Ethiopia, 11,51,475. Ethiopian mustard, 34. Eucarpia, 3.
Europe, 43,382. European Atlantic coasts, 4,12,40,47. European Brassica database, 462. Euzomodendron, 19,20,21,41,42. fat content, 320,322. fatty acids, 123,321,322. composition, 424. fertility restorer genes, 86,134,236. Fezia, 21,41,42. fibre, 318,320. flavonoids, 342,343. flavor, 339. flower initiation, 361-362. flowering, 361-365. hormonal control, 365. fodder rape, 70,73. Foleyola, 21,22,41,42. folic acid, 321. Fortuynia, 21,22,41,42. France, 13,48,468,474. freezing tolerance, 235,266. genebanks, see germplasm. gene gun, 290. gene transfer, 84,123,124. after microspore culture, 264. by protoplast fusion, 123,124. by wide hybridization, 84. in transformation, 288-290. genetic engeneering, 287-303, see transformation. maps, see linkage maps. resources, 461-475,425-427. genic male sterility, 185-188. use, 199,437. genome divergences, 84,85. manipulation, 69. mapping, 39,217-224. structure, 224-232. Germany, 468,474. germination, 359-360. germplasm, 461-475. availability, 467. conservation, 462-464. description, 465. documentation, 466. duplication, 464,466. end use, 471,473. in vitro, 266,267. regeneration, 464,465. of wild species, 471,472. gibberelins, 365~ GISIq analysis, 124. glucobrassicanapin, 325. glucobrassicin, 325. glucoiberin, 325. gluconapin, 325.
486
gluconapoleiferin, 325. gluconasturtiin, 325. glucoraphanin, 325. glucosinolates, 12,50,73,323,325. analysis, 328-330. biological effects, 339-342. biosynthesis, 326,327. breakdown, 327-329. chemical structure, 324,325. content, 235,296. levels in Brassica, 330-338. side chain, 325. goitrogenic effects, 340. gray leaf spot, see Alternaria. Greece, 15. Guiraoa, 21,41,42. Hakuran, 74,220,221,228. haploidy, 247-268. see microspore culture. hemicellulose, 423. Hemicrambe, 21,22,41,42. Henophyton, 20,21,41,42. herbicide resistance/tolerance, 89,135,263,294, 301,302. hetero-arthrocarpic genera, 21,41,42. Heterodera, see nematodes. heterosis, 436,445. hexaploid AABBCC, 133. Himalayas, 45. Hippocrates, 45 Hirschfeldia, 3,10,16,41,42,60,61,107. Hirschfeldia incana, 10,36,37,62. wide sexual hybridizations, 76-83. Hoggar massif, 19. homoeologous recombination intragenomic, 228,231. intergenomic, 228,229,231. horseradish, 342. human diets, 318. Hutera, see Coincya. hybridization barriers, 74. hydroxy-glucobrassicin, 325. Iberian Peninsula, 18. Iberis amara, 150. Ibn-el-Awwam, 49,5 I. IBPGR, see IPGRI. in situ conservation, 465. in situ hybridization, 124,125. incompatibility see self incompatibility. India, 15,43,51,73,74,473. industrial products, 297. insects, 303. intergeneric hybrids naturally occurring, 22. by protoplast fusion, 116,118,119. by sexual crosses, 75-85.
intertribal somatic hybrids, 116. introgression of genes, 84,114,116. IPGRI, 11,461,462,464,465. Iran, 15,51. iron, 320. isorhamnetin flavonoids, 12,343. isothiocyanates, 342. isozymes, 12,13,17,43,50,87,88,108. Israel, 19,468. isthmus concept, 41,42. Italy, 4,15,49,51,468. Japan, 473. juvenile period, 362. kaempferol flavonoids, 343. kales, 4,34.47-49,218,316,320,321,465. kohl-rabi, 48,218. Korea, 474. Kosena cytoplasm, 130,134,193,196. Kremeriella, 20,21,41,42. laurate canola, 300. leaf spot, see Alternaria. Lebanon, 19. Leptosphaeria maculans, 378-384,418,420. resistance, 73,84,114,122,236,294,383,384. Lesquerella fendleri, 120-122,125. lesquerolic acid, 122. light effects, 364. in vitro, 257. light leaf spot, see Pyrenopeziza. linkage maps, 218-224. linolenic acid, 235,322. Lunaria, 302. Madeira Island, 18. magnesium, 320. Majorca Island, 13. male fertility restoration, 86,192. by transformation, 295. male sterility, 185-202. see cytoplasmic,/genic. marker genes, 155,192,193,200,218,220-224,228, 230,235,236,320. maturity, 365. meal composition, 423. medical products, 297. Mediterranean region, 4,12,19,22,40,43-45,47-51, 74. methoxy-glucobrassicin, 325. microinjection, 289,290. microspore culture developmental aspects, 258-262. doubled haploid production, 261,262. embryo maturation, 261. influencing factors, 249,250,258. methods, 247,249,250-257,259. plant regeneration, 261,262. applied aspects, 263-267. microsporogenesis, 191.
487
mineral elements, 320. mitochondrial (mt) genome, 129. mitochondrial (mt) DNA, 35,38,60,65,66,73, 84-86,90,132. molecular biology, 35. molecular farming, 303. molecular lineages, 35-38,84. morphological traits, 236. Moricandia, 19,21,36,41,42,60. cytodemes, 63,64. somatic hybridizations, 119. species, 19,63. wide sexual hybridizations, 81. Morisia, 21,41,42. Morocco, 15,74. Muricaria, 21,22,41,42. mustards, 11,33,34,45,46. mutants, 427. recovered from microspore culture, 263,264. Mycosphaerella capsellae, 393. myrosinase, 327,328. national seed collections, 461,462,474. nematodes effects of glucosinolates, 340. resistance to, 84,86,114, 117,119. neoglucobrassicin, 325. nervonic acid, 122,123. Netherlands, 468,474. Nicotiana, 127,155. nigra lineage, 35-37. nitriles, 328. nitrogen, 255, 368. non-hetero-arthrocarpic genera, 21,41,42. numerical taxonomy, 34,35. nutritional value, 339. "Ogura" cytoplasm, 130-134,190,191,193,196, 200,202. oil quality, 125,300,301,322,323,421,422,425. oleic acid content, 235. oligoelements, 320. 00-varieties, see double-zero. orfmitochondrial genes, 196,197,198. organelles, 35,71,75,89,107,126,127-129,132. see chloroplasts. see mitochondria. Orychophragmus, 21,83. Otocarpus, 21,41,42. pak-choi, 44. palmitic acid content, 235. panthotenic acid, 321. Peronospora parasitica, 381,387,389,390,418. phenols, 345,424.
Phoma lingam, see Leptoptosphaeria. photoperiod, 363,364. phosphorilation. in self incompatibility, 162. phosphorous, 320.
photorespiration, 119. photosynthesis, 361,368. in the pod, 368. Physaria, 232. Physiology, 359-368. Physorrhynchus, 21,22,41,42. phytates, 344,345,424. phytoalexins, 377,378. Plasmodiophora brassicae, 391-393,418. resistance, 73,75,84,119,124,235,236,392, 393. Plinius, 44,316. Poland, 468. Polima cytoplasm, 130,131,133,197,438. pollen germination, 150. signal, 160. recognition reaction, 163. polyethylene glycol, 108. polyploid cytodemes, 64. Portugal, 74,468. Portuguese cabbage, 316. potassium, 320. powdery mildew, see Erysiphe. progoitrin, 325,416. proteinase inhibitors, 345. protoplast 129. fusion, 123,124,190,265. pretreatment, 129. technology, 108-110. Pseuderucaria, 19,41,42,60-62. Pseudofortuynia, 21,41,42. Psychine, 20,41,42. Pyrenopeziza brassicae, 388,389. QTL, see quantitative trait loci. quantitative trait loci, 234-236. quercetin flavonoids, 343. Quezelia, see Quezeliantha, 19. Quezeliantha, 19,20,41,42. Quidproquo, 19,41,42. Raffenaldia, 21,41,42. rapa/oleracea lineages, 35-39. rape, 336. rapeseed, 49,50,336. Raphanobrassica, 88. Raphanus, 3,9,17,21,41,42,60,61. cytodeme, 62. fruit, 17. molecular lineage, 36,37. origin, 41. Raphanus raphanistrum, 9,10,17,51,189. subspecies, 9,10.
Raphanus sativus, 1O,17,41,51.
cytoplasm, 134. domestication, 51. male sterility, 188, 195. somatic hybridizations, 119.
488
wide sexual hybridizations, 77-83. RAPDs, 12,13,59,90,218,220,222,224,430. Rapistrum, 21,22,41,42. Reboudia, see Erucaria, 19,60. RFLPs, 35,36,39,44,46,59,60,65,66,90, 218,220, 222,224,430. Rhizoctonia solani, 393. Rhynchosinapis, see Coincya. rocket, 51. Russia, 468,474. rutabagas, 50,69,70. Rytidocarpus, 21,41,42,60,61. cytodeme, 63-64. S-multigene family, 151 - 160. Sahara desert, 74. Saint Isidoro, 51. salt tolerance, 117. Sardinia, 13. Savignya, 21,41,42. Savoy cabbage, 202,316,320-322,332,333. Schouwia, 21,41,42. Sclerotinia sclerotiorum, 381,384-386,418,419. tolerance, 294,385. seed availability, 467. banks, 463. characters, 12. dormancy, 360. duplication, 464,466. germination, 359,360. meal, 423,424. oils, 295,296. proteins, 12,296. quality, 447. regeneration, 464,465. storage, 464,475. self incompatibility, 149-168. classical genetics, 150. evolutionary aspects, 165,166. in breeding, 436,437. induced by transformation, 296. molecular analysis, 164. molecular characterization of S-locus, 158. S-locus glycoprotein, 151-156. S-locus related genes, 152,159,160. S-multigene family, 151-160. S-receptor kinase, 152,156-158. signal of pollen, 160. signal transduction, 161 - 162. types, 149,150. sexual compatibility, 34. sexual hybridization limitations, 107. vs. somatic, 114,116. shattering resistance, 414,418. Sicily, 12,39,40. Sinapidendron, 3,4,10,18,21,41,42,60,61,107.
cytodeme, 63. molecular lineage, 36,37. wide sexual hybridizations, 80,81,83.
Sinapidendron angustifolium, 10. Sinapidendron frutescens, 10. Sinapidendron gymnocalyx, 10. Sinapidendron rupestre, 10. Sinapidendron sempervivifolium, 10. sinapine, 345.
Sinapis, 3,9,18,21,41,42,60,61. Sinapis alba, 9,18,36,81. cytodeme, 63. resistance to blackspot, 377. somatic hybridizations, 117. transformation, 292.
Sinapis aristidis, 9. Sinapis arvensis, 9,18,36,37.
cytodeme, 62. somatic hybridizations, 119. wide sexual hybridizations, 77-79,82.
Sinapis aucheri, 9,19.
cytodeme, 62. molecular lineage, 36,37,39.
Sinapis boivinii, 9. Sinapis flexuosa, 9,36,63. Sinapis indurata, 9. Sinapis pubescens, 9,18,36,62. Sinapis turgida, see S. arvensis. Sinapis Sect. Ceratosinapis, 9. Sinapis Sect. Chondrosinapis, 9. position, 39.
Sinapis Sect. Hebesinapis, 9. Sinapis Sect. Sinapis, 9. sinigrin, 222,325,416. sirodesmin, 123. sodium, 320. SMCSO, 344. soft rot resistance, 74. Sokotra Island, 16. somatic hybridization, 59,107,109. somatic hybrids cytological analysis, 124-126. inter-specific, 113,115,117. inter-generic, 116,118,119. inter-tribal, 116,119-122. Spain, 13,18,19,44,48,50,51,74,474. staghead disease, see Albugo. stem canker, see Leptosphaeria. stem rot, see Sclerotinia. Stenopetalum, 232. Su, 46. subtribe Brassicinae, 22,60,61. subtribe Cakilinae, 20. subtribe Crambinae, 20. subtribe Moricandinae, 22,60,61. subtribe Raphaninae, 22,60,61. subtribe Savignynae, 20.
489 subtribe Vellinae, 20. subtribe Zillinae, 22. Succowia, 20,21,41,42. sulphur, 338. Sweden, 73. swedes, 50,69,73,316. synteny, 68,87-89,220-222,227,228. synthetic alloploids. by sexual hybridization, 69-73,89. by somatic hybridization, 114. taramira, 51. taxonomic synopsis, 5. taxonomy, 3. temperature effects, 362,363,367. in vitro, 256,257. testa layers, 12. Theophrastus, 44,49. thermal time, 360,367. theses, 3. Thlaspi perfoliatum, 75, 120-122. Tibesti massif, 19. toria, see B. rapa. Tour CMS, 131,133. Trachystoma, 3,10,18,22,37,41,42,60,61,107. cytodeme, 62. Trachystoma aphanoneurum, 10. Trachystoma balli, 10. somatic hybridizations, 119. Trachystoma labasii, 10. transgenic plants, 293,298. being commercialized, 300. environmental effects, 298,299. field tests, 293,299 legal issues, 300. possible dangers, 298,299. questions raised, 298,299. transformation, 288-303. methods, 288-290. types of genes transferred, 290-292. tribe Brassiceae, 19,61. evolution, 34-43. fruit beak, 14,15. subtribes, 20. Tunisia, 12,13. Turkey, 51. turnip (B. rapa), 33,41,43,44. chemical composition, 316,334. turnip yellow mosaic virus. tolerance, 294. Ukraine, 468. United Kingdom, 468,474. United States, 474. U-triangle, 4,11,67. Vella, 19,20,21,41,42. vegetative growth, 360-361. vernalization, 235,364. verticillum wilt, 390-391.
Verticillum dahliae, 390,391,418. vitamins, 321. Wales, 48. water content, 319,320. waxes, 12,345. white leaf spot, see Mycosphaerella. white rust, see Albugo. wide sexual hybridizations, 74-85. techniques, 75. wide sexual hybrids, 75-85. chromosome pairing, 85. cp-DNA, 84. cytoplasm divergence, 84. fertilization barriers, 74. genome homoeology, 84. introgression of genes, 84. meiosis, 75. wild germplasm, 75,89. Xanthomonas, 117,236. yellow sarson, see B. rapa, 164. yield factors, 366-368. improvement, 418, 419. performance, 416,419. target characters, 418. Zilla, 21,22,41,42.
