Advances in Botanical Research, Volume 2

Advances in

BOTANICAL RESEARCH

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Advances in

BOTANICAL RESEARCH Edited bu

H. D. PRESTON The Astbury Deparlnaml of Biophp-ics The University, k d s , England

VOLUME 2

ACADEMIC PRESS, INC. (tiarcourt Brace lovanovic h. 'Publishers)

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98765432

CO-UTORS

TO VOLUME 2

M. B. DALE,Departmend of Botany, The Univeraity, Southampton, England (p. 35). DEREKT. A. LAMPORT, Now at the Michigan State University Atomic Energy Commi8eion Plant Research LuboraWy, RIAS, Baltimore, Maryland, U.S.A. (p. 151). JOHN LEVY,Botany Department, Imperial College, University of Londun, 8.W.7 (p. 323). I. MLCNTON, Botany Department, University of L e d , England (p. 1). P. MAHESHWARI,Department of Botany, Univereity of Ddhi, Delhi, India (p. 219). N. S. RILNOASWAMY, Department of Botany, Univerdy of Delhi, Delhi, India (p. 219). P. A. ROELOFSEN, Laboratory of General and Technical Biology, Technological Univeruity, Delft, Netherland8 (p. 69). W. T.W~LIAMB, Department of Botany. The UniuerSitgv.8wuthampton, England (p. 36).

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In preparing the first volume of this series we were well aware that, whatever eke we were doing and however valuable and rewarding this new series might prove to be, we were inevitably adding to the burden of reading which all professional saientists, and not leaat among them, the botanists, are called upon to bear. In the prefaoe to that volume we were therefore rather careful to point out what we hoped would be found to be an approach somewhat different from that of most other texts reviewing piecemeal a whole field of study. Though we ouraelves had faith in this new venture we neverthelees waited with more than a little nervousness for the reaations of readera mainly, though not entirely, voiced by reviewers in the learned journale. In the event all the reviewers were favourable and we pmoeed to the second volume, not so much perhaps with more assurance, as with relief both that we were not too far in error in assessing the need for and the merits of the approach we outlined and that the publisher, the authors, and the editor had together made a book which fulfilled theee aims. In presenting this second volume we have in mind the comment of one reviewer that we may 6nd difficulty in maintaining the standard set in the first volume, We are sure that this second volume will set his fears at rest and we are grateful to the distinguished authora in this volume that they have spared no effort in ensuring that t h b ehould be ao. It is naturally not pomible in two volumes to cover the whole field of botany, and an editor is therefore faced a t the outset with the task of selecting topics in such a way as to achieve a fair balance in each volume between the interests of the wide variety of readers at which it is aimed. The inclusion in this volume of two articles dealing with cell walls therefore calls for comment, particularly since this happens to he our own particular interesf. An editor is inevitably guided by one overriding principle. The topics he chooses must be in course of rapid development, must be significant and must be dealt with by an author who is, and is known to be, distinguished. This l e d to a deliberate choice of author rather than of topic. The editor must, however, be ready to accept an opportun$y if one unexpectedly comes his way. In choosing the growing cell wall as one of the more important topi08 of the day the only possible author was Prof. Roelofaen and we am Vii

viii

PREFACE

more than grateful to him for the warmth with which he accepted an onerous task. This was intended to be the only article on cell walls, for although Dr. Levy’s article on the recently discovered importance of soft rot fungi is involved with cell walls this is not its only, or even its major, importance. When, however, we saw the opportunity of a “stop preas” article on the moat recent cell-wall development we hsd no hesitation in pressing Dr. Lamport to write what in our opinion is an article complementary to that of Prof. Roelofsen but is also in itself a striking example of the sudden break-through which can occur in an old problem when an enquiring mind seizes upon a new clue. One of the most remarkable developments of our time in plant science has been the way in which hitherto purely observational regions are progressively becoming experimental or even mathematical. One case of the former is included here, in the remarkable developments in embryology a t the hands of Prof. Maheshwari and his colleagues and students; and one case of the latter is given in the mathematical approach to taxonomy represented here in the work of Prof. Williams and his associates, which is now giving a new look to this somewhat bewildering field. We do not need to emphasize the impact of electron microscopy in all fields of botanical enquiry; it is represented here in its position with regard to classification in which, as in other aspects of electron microscopy, Prof. Manton has played such a prominent part. We are again grateful to the authors in this volume for the time and care with which they have prepared the artioles and have thereby smoothed our path, and our thanks are due particularly to the publishers whose quiet efficiency we deeply appreciate. Finally we are indebted in this volume as we were in Volume 1 for secretarial assistance to Mrs. E. D. White (nee Lister), this time with the able assistance of Mrs. C. J. Parker.

R. D. PBESTON

beds, 1906

CONTENTS

......................................... v .......................................................... vii

CONTRIBUTORS TO VOLUME2

PREFA~E

Some Phyletic Implicatione of Flagellar Structure in Planta I . MANTON .. Introduction ................................................. Distribution of Flagella of the 8 +2 Type ......................... .. The Heterokont versus Isokont Condition ........................ Other Aspects of Flagellar Numbers and Relative Length .......... V . F l a ~ ~ a r.aS h..............................................

I I1 I11 IV

.

........................................ ........................................... ............................................ ............................................ . ............................................... . ............................................ . .................................................. x. summery ................................................... References., ..................................................

V I Flagellar Appendages A Flagellar Spines B Flagellar Hairs C FlagellarScales VII Flagellar Beeea VIII Flageller “Roots” IX Conclueions

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6 8 10 13

13 14

17 17 18 20 20 33

Fundamental Problem8 in Numerical Taxonomy w T WILLIAMS and M . B DALE

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I Introduction I1 The Nsture end Propertiea of Claseificetions A The Basic Axiom B Monothetic and Polythetic Claseifications C Maximization D Hierarchical and Non-hierarchicalClaaaificetiona E Probabilistic and Non-probabilistic Cleeeificationa 111 The Choice of Mathematic&%l Model A Introduction: Metrics B Metric Properties of Pair-functions C Intrinsicslly Non-metric Systems D Non-EuclidmnSyateme E Conclusions IV The Beeio Euclideen Model A . Duality: The R/Q F’roblem ................................. B. Adjustmenta to the Model C Heterogeneity v. StretegyofAnSlyais .......................................... A Simplification Methods ..................................... B. Pertition C Non-hierarchical Methods D Hierarchical Methods Achowledgementa References

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1

3

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36

37 37 37 38 42 43 48 48 49 51 62 63 64 64 66 56 69 59 61 61 62

67 07

X

CONTENTS

Ultrastructure of the Wall in Growing Celle and ita Relation to the Direction of the Growth P

. .

. A . ROELOFSEN

................................................. 09 ............................................. ................... 70 70 ........ 78 ........................ 82 ................. 85

I Introduction I1 Morphological Aspects of Constitution. Synthesis and Breakdown of theprimery WEll A Constitution and Morphology of Microfibrils B Constitution of the Amorphous Matrix in Primsry Wells C Some Aspecte of Microfibril Coherence D Site of Synthesis of Prim~ryWall Substances E Questionable Evidence of Breakdown in Primary Walle I11 Survey of the Microfibrillsr Arrangement in Different Typee of Growing Celle A Freely Growing more or less Isodiawetrio Cells B Freely Growing Tubular Cells or Perts of Cells C Tissue Cells with Isodiametric Growth D Tissue Cells with Predominant Growth in Length E Tissue Cells that Predominantly Widen., F Tips of Tissue Cells with Tip Growth I V Interreletion between Growth end Wall Ultrastructure A Effect of Growth on Wall Structure B Effect of Well Structure on the Direction of Growth C Theories on the Mechenism of Orientated Jnitiel Synthesis of Cellulose Microfibrils References

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......... 89 ................................................ 91 91 ................ ................ 98 ......................... 104 .............. 106 ..................... 112 113 ......................... ............ 114 .......................... 114

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128 139 145

The Protein Component of Primary Cell Walls DEREK T

. A . LAMPORT

. A. LJcopeandDefinitione ...................................... 161 151 B. HistoricalPerspective1888-1969 ............................ 152 I1. Experimental Methods end Materi~ls............................ 165 A. Cell Suepension Cultures....................................166 I Introduotion .................................................

............................................. 167 168 I11. The Hydroxyproline-rich Wall Protein: “Extensin” .............. 180 A . Intra-cellular Location of Hydroxyproline..................... 160 B. Chemical Cheracb&tion of 4-tmnu-hydroxy-~-pIine......... 167 C. The Amino Acid Composition of Primepy Cell Walle., .......... 168 D. Enzymic Degradation and Characterizefion of Wall Protein ..... 171 E. Disdphide B r i d e in Cell-Wdl P r ~ b i n....................... 172 F. Distribution of the Hydroxyproline-rich Wall Probin in the Plant Kingdom ................................................. 174 IV. The Biosynthesia of “Extensin” ................................ 177 . A . Uptake and Incorporation of W-Proline by Intact Cells........ 177 B. ProlineHydroxylation ..................................... 184 V . Veristion of Cell-wall Hydroxyproline Content ...................188 A . Walls Isolsted from Tissue Culturw .......................... 188 B. Wells Isolated from Plsnt Psrts ............................. 189 B. WholePlante

c. Ana~yticdTechniques......................................

CIONTENTS

xi

.................. 189 ...................................... 193 ...................................... 194 ........... 198

.

V I Degredation of the Sycamore P r b 8 r y Cell Well A ChemioelDegradation B Enzymic Degradation VII A Tentative Piature of “Extensin” in the Primsry Wall VIII The Contribution of “Extensin” to Wall Form end Teneile Strength I X EnzymiaW8llProtein A hoorbio Aoid Oxidaae B Hydrolyeeee C Other Wall-bound Enzymee D How does the Wall Rind Emymtw ? X The Role of “Extensin” Aoknowledgemente Referenoes

. . ... .. .. .

200 ......................................... . 204

...................................... 204 .............................................. 206 ................................. 206 ......................... 206 ....................................... 209 ........................................... 213 ................................................... 213

Embryology in Relation to Phyeiology and Genetice P MAHESHWARI and N 9 RANQA8WAMY

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................................................. 219 ....................................................... 221 . ........................................ 221 ...................................... 223 . ............................................... 227 ........................................ 281 . .................................... 232 . ..................................... 232 ....................... 234 ..................................................... 237

I Introduction 11 Pollen A Longevity of Pollen B. GerminationofPollen C Pollen Tube 111 Control of Fertilizetion A Treetmentoftheat igma B Treatment ofthe Spyle C . Intrmvarien and in vitro Fertilization IV Embryo A Growth of Vmbryo in Relation to Seed Development B Dependence of Embryo on Endoeperm C Bpeofioity in Nutrition of Embryo V Endoeperm . A Conetituent4Jof Endoeperm B Role of Endusperm in Seed Development C CultmofEndosperm VI Embryo culture A Cultural Conditions B GrowthMedia C Applications of Embryo Culture D Limitations of Embryo Culture M I. Cultureof0vulee M I. Culture of Ovsriea and Flowera M Perthenocsrpy X Polsembryony A Adventive Embryony B EmbryonslBudding XI P ~ h e n o g eeeis n XI1 . . .g.A n X I I I Antherculfure XIV Control of Sex Expression X V Conclueions Aoknowledgementa Refe~

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........... 237 ....................... 239 ........................... 289 .................................................. 240 ................................. 240 ..................... 242 ...................................... 243 .............................................. 246 ........................................ .............................................

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247 260 266 262 263 266 278 280 280 286 296 800 801 804 309 810 310

xii

CONTENTS

The Soft Rot Fungi: Their Mode of Action and Sicanee in the Degradation of Wood JOHN LEVY

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I Introduction 323 I1 Histology of Soft Rot 329 I11 A Technique for Studying Soft Rot Fungi........................ 337 IV Mode of Action of Soft Rot Fungi 339 A Peseive Penetration and Decay Penetration 339 B . Effect of Species of Wood on the Mode of Attack by the same Fungue 340 C Effect of Species of Fungus on the Mode of Attack in the same 344 Wood D Soft Rot Fungi on Posts in Ground Contact 348 V List of Fungi known to came Soft Rot 348 VI Dieoussion 349 Acknowledgements 366 356 Referenoes AUTHOSINDEX 369 SWBJEOTINDEX .................................................... 371

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Some Phyletic Implications of Flagellar Structure in Plants I. MANTON Botany Department, Univereily of Lee&, Enghnrl 1. Introduction..

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1

II. Distribution of FhgeUe of the 9 +Z Tspe. .................................. 3 5 III. The Hebmkont veraue Isokont condition.. ................................ Iv. Other depeote of Flegellsr Nnmbera and Relative Length.. .................. a

v.

BlEgdhrlSaape

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M. W q & u Appendzigw ........... .+ ...................................... A. F h g e k Spinen ..................................................... B. FbgellmHeire

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c. Flegellsr Scales .....................................................

10 13 13 14 17 I?

........................................................ ...................................................... ia .......................................................... 20 ..............................................................20

VII. FhgellarBBaee YIII. Flr4gellar “Roote” IX. ConolUBioM,. X. Summery

References.. ...........................................................

33

I. INTRODUCJTION Discovery of the essential similarity between cilia and flagella of both plant and animal kingdoms has exposed a linguistic contradiction which impinges too closely on taxonomy to be easily resolved. So long as protistologists must refer to major p u p s of organkms as Ciliates and Flagellates, recommendation to use the one word “cilium” for the filamentous appendages of both, leaving the other word “flagellum” for the very differently constructed filamentous appendages of bacteria, is a council of perfection that can scarcely be carried out. That inextricable confusion has not resulted from this linguistic impasse ie a tribute both to the resilience of biologists and to the clear-cut basic character of the organelles themselves. Illogical verbal usage at this level is indeed harmless in the sense that it rarely, in practice, leads to a fundamental misunderstanding of facts. I n contrast, the unforeeeen obstacles to mutual comprehension that have been raieed by reoent attempts to standardize nqmenclature a t a more intimate level ie a topic about which more will be said below. In the study of cilia anp flaplla of the non-bacterial sort by electron microscopy, three periods pan conveniently be distinguished. In the firat phaae, extending roughly from 1946 (Jakus and Hall) to 1964 (Fawcett and Porter), with the most intense period from 1980 to 1982 (Manton and Clarke), the ubiquity of the 9+2 fibre pattern waa B

2

I. MANTON

established. That each member of the peripheral ring is double and not a single strand was also established at the close of the same period (Manton and Clarke, 1962; Fawcett and Porter, 1964), though general agreement that the fibres of the central pair are not similarly constructed came later. A second phase of study, beginning with Af?elius (1969) and culminating in Gibbons and Grimetone’s classio paper of 1960 (see also Gibbons, 1961a), introduced better fixation, the use of new plastics and above all the successful applioation of new electron dense stains to thin sections, thereby opening up the study of additional structures which is still continuing. The new information on flagellar bases discussed below was made possible by thia work. Lastly a third phase, involving application of negative staining to dismembered flagella, is beginning to provide details of the fine structure within individual fibres (Andr6 and Thikry, 1963; Pease, 1963). It is not the intention to review here any of these phases as such, but only to discuss those aspects of flagellar structure which may have phyletic implications. Phase 3 so far has no phyletic implications and mention of it has been made only to bring the record up to date and to alert biologists who are not themselves electron microscopists to the existence of a very interesting new field of study. Phyletic implications, where they occur, are only rarely dependent on the micro-anatomical details associated with the 9+2. They are usually dependent on ancillary structures or on other details such as relative length and arrangement of flagella, some of which have long been known, in a general way, to light microscopists. The early history of knowledge of the existence of tile heterokont condition among algae (Thuret; 1861, Pringsheim, 1866; Thuret and Bornet, 1878; Luther, 1899) can be cited in illustration of this. Endorsement of the liiht microscopist’s view using the same type of evidence seen larger is not, however, the main function of the study of fine structure. The phyletic implications that I wish to discuss here are those that could not have been effectively worked out by light microscopy unaided and on wbich the electron microscope has supplied a considerable number of entirely new criteria and with them some new points of view. Phyletically significant new criteria have occasionally been sought by deliberate electron microscopical enquiry, but usually they have been encountered incidentally as unexpeatd additions to an investigation directed primarily towards other objectives. At the start of electron microscopy (phase 1mentioned above), when the main technical method was that of shadow-cast whole mounts, the great variety of external appendages borne by plant flagella in contrast to those of animah was an incidental h d h g of this kind. The reviews published by myself in 1962, 1964 and 1966 cover sufficiently this phase of the investigation

FLAUELLAR STRUOOTURE IN PLASTB

3

of plants. Subsequent developments have however been of two kinds. There is real progress when new methods have proved to be applioclble to new types of material, but there are also some unfortunate mistakes which, for one reason or another, have begun to enter the litemture. These, if accepted as facts by the inexperienced, which is all too easily done, can impede progress substantially. It is this last consideration as much as any other which has prompted selection of the present title at this time. Finality is not yet attainable in many matters on which knowledge is rapidly growing. A progress report based on &&-hand experience may nevertheless have positive value. There will necessarily be strong personal bias in the selection of topics for discussion, since an exhaustive treatment of literature alone is not the intention. Fortunately two recent publications can be used to supplement this report. There is Pitelka’aexcellent little book on the fine structure of protista both colourless and coloured (Pitelka, 1963) and a recent algological survey by Christensen (1962) which lends itaelf admirably to discussion of phylogeny. Since the text of the latter is in Danish (an English edition is promised but not yet available) it may perhaps be of value to Englieh readers if the giat of Christensen’sphyletic scheme is reproduced in the form of a list from his table of oontents (see p. a), supplemented by his diagram (Fig. l), translated where necessary, reproduced on p. 5. 11. DISTRIBUTION OF FLAGELLA OF TEE 9+2 TYPE An important biological concept not based on flagella but summing up much information on the micro-anatomy of protoplasmic structures of other kinds is that ofthe procaryotic versus the eucaryotic cell (expressed as Procaryota and Eucaryota in Christensen’s liat). Anyone unfamiliar with these terms can usefully consult the excellent short paper by Stanier and van Niel(1962). Procaryotic cells lack membranebounded internalorganelles though they do not lack internal membranes. If organs of locomotion exist, aa in bacteria, they are never of the 9+2 type. Eucaryotic cella (i.e. cells of all the main plant and animal groups other than bacteria and blue-green algae) possess membranebounded organelles of various kinds, e.g. nuclei, mitochondria, chloroplasts. Even when oell size is reduced to within the dimenaiom common in bacteria, as in the tiny green flagellate iKrmnOnae p u d h (Manton, 1969a), the eucaryotic nature of the main cell components remains sharply distinct. The introduction of the membrane-bounded space within which biochemical activities of some kinds can be carried on in relative isolation from biochemical aotivities of other (perhaps mutually incompatible)

4

I . MANTON

kinds is clearly an evolutionary advance of the greatest importance which must have preceded most of the phyletic diversity with which modern taxonomy is concerned. Membrane-bounded spaces in the sense of ccdouble-membranea”(for fiwther discussionof this terminology see Manton, 1961) occur conspicuously in cells of blue-green algrte, notably in their pigmented regions. It is membrane systema of this type, or still more elaborate variants of them (but not simple membranes), which delimit the organelles within eucaryotic cells. Such ceUe are also the only ones to possess oilia and flagella of the 9+2 type, not, however, in all eucaryotic groups. The absence of flagella of any kind from the red algae has long been known (of. Fritsch, 1936), though this fact has scarcely disturbed the prevailing view that, apart from blue-green algae, a flagellate anoeatry lies behind all the main algal groups. An early loss of flagella is of course not difficult to imagine and the prevailing tendency to & o w Rhodophyceae (Fritsch, 1936, or Rhodophyta in the terminology of Smith, 1951), late in the soheme of major algal groups has consciously

List of mujor algal groups according to ChriStemen, 1962 PROCARYOTA Cyanophyta Cyanophyeeae EUCARYOTA ACONTA Rhodophyta

RMphyme CONTOPHORA Chromophyta

Cryptophgceae Di?WphgWZ$ B h a p h i ~ ~ ~ Cb~OPkP= H*topkJCrw*h-

Bt7Cill&C?phyC4?4W Xadwphym

Chlorophyta

p*hY-

EWkVhYLxOPhYPrmhphyeeae Chlorqphyctw

FLAGELLAR S T R U C T U R E I N P L A N T S

6

or unconsciously led to a general feeling that they are late arrivals and intrinsically specialized. Christensen (1962) is most explicit another way : specialized they undoubtedly are in many ways (somatic structure, mode of growth, life histories), but this could be mainly sign of extreme antiquity within the eucaryotic scheme, an antiquity which could have resulted from an origin before the 9+2 flagellum had been evolved. The extreme simplicity of their plastid lamellations (cf. Bouck, 1962; a180 A. D. Greenwood, personal communication) is an important additional micro-anatomical character consistent with such a view. Christensen’s phyletic scheme (Fig. 1) thus places the Rhodophyta MI the loweet and most ancient eucaryotic group, preoeding in origin the

Fro. 1. Christensen’s Phyletic scheme of probable relationehip between the main group of algae and other main groups of living organisms. Algae are indieetad by thick lines and lettera, other organisms by thin lines and letters. After Christensen, 1982.

6

I. MANTON

simplest true flagellates and ante-dating the separation of the plant and animal kingdoms. The importance of the term Aconta as opposed to Contophora is thus greater, and the term itself more significant than the mere absence versus presence of flagella had previously suggested.

111. THE HETEROKONT VERSUS ISOKONT CONDITION There is now a good deal of evidence in favour of extreme antiquity for the heterokont condition. By this is meant the possession of flagella in pairs, the two members of each pair Wering in length, type of motion and presence or absence of external appendages, and wully with the basal bodies of a pair mutually attached at a wide angle. Among pigmented forms, at both the monad and algal levels, other features of cell structure accompanying the heterokont flagellation include absence of chlorophyll 6 , presence of a rather simple internal plastid structure devoid of grana (perhaps more correctly described as plastids with a different lamellar system from that of the higher green plants), a more intimate relation between chloroplasts and endoplasmic reticulum than in organisms possessing chlorophyll byand the possession of mitochondria with micro-tubules and not flattened cristae. It is significant that some of these characters, notably the tubular equivalents of cristae, are also encountered among Protozoa. It is therefore virtually certain that the latter antedate Metazoa phyletically and that the heterokont pigmented flagellates are likely to be ancestral not only to these but to the main groups of heterokont algae and fungi also. These views are adequately expressed in Christensen’s diagram (Fig. l), and are, on fhd whole not controversial. An important innovation in Christensen’s scheme is, however, the use of the term Chromophyta to cover all groups of Contophora lacking chlorophyll b. Such an arrangement may not last permanently, but in the present state of knowledge it brings clarity where confusion previously existed and is therefore to be welcomed. In addition to the basic heterokont groups (Chrysophyceae, Xanthophyceae, Yhaeophyceac) some new names are included on which comment will be added later (pp.12, I’letc.). Attention should however be directed here to the importance of the present position of Vauckeria. That t h h familiar classroom type is closely relevant to questions of origin of the water moulds is well known, but as long as it was treated a8 a member of the green algae, a total conflict of evidence was inevitable together with a tendency to over-estimate the antiquity and relative importance of Chlorophyta versus Chromophyta, which still adversely affects phyletic thinking. The heterokont condition of the Vuwkria spermatozoid

FLAGELLAR STRUUTURE I N PLANTS

7

waa however well known to Pringsheim (1866),though at that date the concept of Heterokontae (Luther, 1899) as a group did not exist. It took nearly a century before the chemistry of pigmentation (for litemture see Strain, 1961) plus supplementary details of flagellar struoture (Kooh, 1961) could counteract the seductive influenoe of its bright green colour and lead to the realization that its true afRnitiee are with the Chromophyta. Everything that has happened since (eee especially Greenwood et al., 1967;Greenwood, 1969)has confirmed thie conclueion and there can be no doubt that the tranafer of Vaw&& to Xanthophyceae (Perke, 1962)is correct. This, it may be said in passing, could in fad have been done half a century sooner had the value of flagellar characters as phyletic indicators been better understood. The Chlorophyta (containing chlorophyll b) include the familiar isokont group^ enumerated in Christensen’s liat under Chlorophyceae together with euglenoids and some additional new p u p a which are not iaokont in flagellation. The essential unity of all the major land plants (archegoniates,gymnosperms,angiosperms)with green algae is endorsed by rapidly accumulating information about salient details of fine structure of the cell as a whole which cannot be described here (see however Manton, 1964b,c,d). This unity neverthelees depends almost certainly on chlorophyll b (and its physiological and structural conaequences) rather thah on flagellation, and though the origin of chlorophyll b as such can scarcely be discussed here it ia important to notioe the conservative treatment of thia problem given by Chriabnaen. In placing Chromophyta and Chlorophyta on a level, he is in fact avoiding a question which he might have treated differently. There are many members of the Chromophyta containing chlorophyll a but no other chlorophyll, e.g. many Xanthophyceae and Chrgeophyoeas (Bogorad, 1962). A caae might therefore perhaps have been made out for greater antiquity of some groups of Chromophyta compared with Chlorophyta had Christensen wiahed to do 80. He has wisely avoided this question, but it ia perhaps uaeful to note that an argument in favour of the converaeposition,i.e. for greater antiquity of Chlorophyte, versus Chromophyte, could scarcely have been sustained at all. It ie therefore important that in the list (p. 4) the group to be mentioned last ie no longer the Rhodophym (Rhodophyta) aa in Fritsch (1936) and Smith (1961), but the Chlorophyta themselves. With this point of view I personally am in full agreement. The origin of iaokont flagellation aa such is not aa olosely linked with the origin of the Chlorophyta aa has sometimee been thought. It ia important here to realize that the tacit assumption eometimes made by algologiets that isokont flagella are in themaelves primitive in an aeeump tion baaed on indirect rather than positive evidence. We have in fsot

8

I. MANTON

no information regarding the nature of “the primitive flagellate’’ and it is probably important to realize that while many Chlorophyceae are isokont other members of the Chlorophyta (Euglenophyceae, Loxophyceae and Prasinophyceae on Christensen’s terminology) are not. Conversely some members of the Chromophyta, notably the group of genera sopnrated from the Chrysophyaeaeby Christensen under the title Haptophyceae, are isokont, e.g. leochryuiu (Parke, l949), Chy80chrmulinu (Parke et al., 1966,1962etc.), P q r n d u m (Manton and Leedale, 1963) and the Coccolithophoridae (Parke and Adams, 1900). The differences between these and the isokont Chlorophyceae are nevertheless profound and include general features of cell organization as well as details of flagellar bases, some of which will be discussed below. The possibility must be accepted that isokonty may have arisen more than once within the Contophora, though in all 0&888 from an essentially unknown type of ancestor, and its presence is thus in some ways less informative than is heterokonty when all the various ancillaries are fully developed.

IV. OTHERASPEUTB OF

B’LAGELLAR

NUMBEW AND RELATIVE LENGTH

The uniflagellate condition, encountered sporadically in many groups, is sometimes manifestly a secondary development by suppression of one member of a former (usually heterokont) pair. An extremely clear example of this is the brown alga Dictyota (Manton, 1969c)in which the uniflagellate spermatozoid retains a basal body for the hind member of a heterokont pair which fails to develop further. The single flagellum is clearly the of members of the Chrysophyceae such as Mdequivalent of the front flagellum of their heterokont relatives within the same group (Pihlka, 1949).It is possible that the single flagellum of fungi such as A l h y c m or OZpidium is aim&& the equivalent of t b hind flagellum of a former heterokont pair, but this is less certain. On the other hand, the nature of the single flagellum in green flagellah such as Pedinmnonas, Fig. 4, (Ettl and Manton, 1964), hficronumae (Manton and Parke, 1900),hfowmmtiz,Fig.6, etc.,is entirely uncertain. These organisms are small and apparently simple. They lack sexual processes and often lack true starch, though they posseas chlorophyll 6. The single flagellum they contain could be primitive. Whether this is in fact the case is however almost impossible fo prove. The special case of Euglena can be dismissed &om this category. While it is probable that all members of Euglenophyceae will eventually prove to be biflagellate (G. F. Leedale, personal communication), it is well established (Pdngsheim, 1966; Leedale, 1966) that all representatives, both aolourlessand coloured,previously described as hawing aaingle

FLAGELLAR STRUUTURE I N P L A N T S

9

flagellum bifurcate at the base, are biflagellate, though the moond flagellum if3 80 short that it fails to omerge from the oantll (formerly termed reservoir or gullet). Much elementary teaching could usefully be corrected on these points. Multiple flagella (beyond two) have certainly been achieved independently more than once and major phyletic conclusions based on this character have to be made with caution. The best known examples of multiple flagella are at present Oedoqonium (Hoffman and Manton, 1962, 1963) among algae and the gametes of many archegodate land plants (compareMsntonandClarke,1961a,withMmton, l952,1959b), all chlorophyll b-containing organism. The quadriflagellate condition is however a special case which should perhaps be mentioned first. It might have been expected that a group of four equal flagella would have been directly related (as a relatively simple precursor) to a large ring such a8 that of Oehqorjium (Fig. 10). This however is not 80. A recent study carridout in detail from this point of view (Manton, 1964a) has shown that at least in 8tigeoclonium and D r a v m U i a (members of the Chdophorales 8emu Christensen) the quafdrifiagellate condition is achieved by the grouping of two isokont flagellar pairs in a mirror image relation (Fig. 11A) and the same is almost certainly true for Ulothriz (Manton, 1962). Other quadriflagellate green cells, e.g. P y ~ a milnonas (Manton et at., 1963),Platymonas, 8 p ,may relate back to uniflagellate or biflagellate relatives in which flagellar number has increased in a different manner. Some information about these will be given below (pp. 9,11,17 etc.). It is enough to say here that the grouping of fourflagella seems to have occurred within the green algae many times and few, if any, are in a direct line towards either Oedogonium or a fern s*rmatozoid. While the origins of the special types of multifbgellate motile oelh characteristic of Oedogonium on the one hand and many land plants on the other are therefore still obscure, a positive finding relevant to the latter is perhaps important. The biflagellate condition of bryophyte spermatozoids is sometimes (e.g. 8phugnum, Manton and Clarke, 1962) undoubtedly the expression of two flagella of different lengths, attached at different points on the cell and therefore not a pair in either the isokont or ordinary heterokont mnse. Whether this oonditioa is primitive or specialized (i.e. due to reduction from a multiflagellate type) is unknown but it indioates that flagellar number is not in itself the moat significant character, but rather the overall symmetry of the cell. The spermatozoids of archegoniates are fundamentally aaymmetrical no matter whether they are bi- or multiflagellate. There is therefore no ease for attributing an isokont ancestry to them. The common textbook practice of treating C?damydummaa aa an unequivocally primitive

10

I. M A N T O N

type is thus valid, if at all, only for certain groups of green algae but not for the mainstream of phyletic change leading to the vegetation of the land. It is perhaps important to note that flagellar size as such is not directly connected with flagellar number, though minor phyleticallydetermined differences exist. In the large compound zoospores of Vaucheriu (Greenwood, et el., 1967) the average length of the shorter member of each flagellar pair was 9-8p and of the longer member 11.1 p. In Oedogonium, for which abundant numerioal data have ale0 been assembled (Hoffmann and Manton, 1962, 1963),an averago length of 174 p was encounterod both in epermatozoids with 30 flagella per cell and in Z O O E ~ O ~with ~ E over 120. The biflagehte gametes of bryophytea may give measurements of slightly greater length, e.g. 20p for both Marchantia (Manton, 1962) and 8phagnum (Manton and Clarke, 1962), but those of a fern (Dryoylleris) are only slightlylessthanthisat 18p (MantonandClarke, 196la;Manton, 1969b), though flagellar numbers in ferns may reach three figures. Uniflagellate cells are more varied, though lese so if the exceptional condition of the very reduced species Nicronumas puailb (see below and Fig. 6A) is excluded. P e a l i v rniw (Fig. 4A)with a cell only a few microns larger than N.puailb has a length for its single flagellum ranging from 10-17 p according to conditions. The extreme shortness of the hind flagellum in many heterokont members of the Chromophyta iR thus a sign of fundamental asymmetry of the cell as a whole, since it is in no way conditioned by flagellar number as such.

V. FLAGELLAR SHAPE The only feature of flagellar Rhape of importance in the present context is that of the tip. ThiR region, however, oannot be effectively studied by light microscopy uncontrolled by electron microscopy since flagellar tips are often the sites of structural breakdown either in vivo or after fixation, leading to artifacts of very peculiar kinds which need to be recognized for what they are before conclusions can be drawn. If distal breakdown ocour~without rupture of the flagellar membrane, osmotic swelling may lead to apparently spherica1 apices which have been desoribed aa such more than once by light microscopists (see for example Strasburger, 1880,for Vuucheria).Discussion of this artifact in relation to bryophyte epermatozoids will be found in Manton and Clarke (1962).On the other hand the flagellar membrane may beoome wholly or partially stripped offleading to an appearance aa of a long distal thin hair. This artifact is almost certainly in part the r w n for the special term “acronematic”, introduced by Deflandre (1934)for

FLAUELLAR S T R U C T U R E IN PLANTS

11

flagella lacking lateral hairs, since the early drawing8 of stained flagella (e.g. Fischer, 1894) show extreme developments of this kind whioh are mrtainly paztly artifacts in the light of subsequent eleotron mioroscopical investigations. The most extreme o w e of all, in which the whole flagellum is disrupted, displaying its fibrillar oompition, ie usually recognized for what it is (oompare for example Ballowitz, 1888, with Manton, 1960, and Manton and Clarke, 19/51), but even extreme disruption commonly starts at the flagellar tip and may not affect the base. Since the component fibrils of a disrupted flagellum are individually below the limit of effective resolution with the light microscope unless dried,suoh a flagellum in a stained preparation may appear to show a faint, splayed, distal extremity on a more densely staining narrower base. Another complictction that has to be remembered is that of occwional abnormalities which may simulate one or other of these conditions. An early example (in Fucw 8erratw) of an exceptional plant giving a long terminal “hair” as a genuinely structural addition to the front flagellum of its male gametes which other plants of the same species do not normally show, was illustrated in Manton and Clarke (1961~).When therefore a similar condition is described for the zoospores of Chordaria on the coast of Denmark (Petersen et al., 1968), such a description nee& to be followed up by a re-investigation of the same species in another locality before the suspicion of structural abnormality in the material can be excluded. Abnormalities and damage apart, there may be other difficulties in determining the true shape of flagellar apices in dried material examined optically or electron microscopically.The flagellum itself may be coated in some way and its tip not directly visible. The apparently te-1 tuft, of hairs described for M&mnnmua 8qu.amata (Manton and Parke, 1960), Pyrarnimmw, etc. (Manton et al., 1963), belongs to this category and further information about thew genera will be given below (see also Fig. 9). Knowledge of the internal structure is also probably more necessary to interpretation than has generally been recognized and this has rarely been provided for the flagellar apices in plants (see however Roth and Shigenaga, 1964, for some pertinent details for animals). The smoothly tapered cilia of ferns and some bryophytes seem likely to depend on the early termination of the two central strands followed by gradual petering out of the strands of the peripheral ring (evidencefrom dismembered speoimens, Manton and Clarke, 1961a, 1962). In other cases, notably AZicrowwncce pwilkc, Fig. 6A (Manton, 1969a), the central strancb are prolonged beyond those of the peripheral ring giving a long and slender but membranebounded terminal extremity, suggesting a hair-point when seen with

12

I. MANTON

the light microscope. One of the commonest types of flagellar ending8 is, however, shortly mucronate (Fig. 2B),but there is no information on the internal tip structure in any of the organisms with tips of this kind and it is therefore impossible to know whether a mucro L only a short “hair-point” or a genuinely separate morphological category. I personally suspect the latter, but morphology alone provides insufficient guidance in this’case. While therefore it is probable that phyletic information may ultimately be obtainable from apical shapes of more than one kind, there is only one context at present in which these seem likely to be important in the immediate futuk. Among small green flagellates in the group provisionally assigned to Loxophyceae by Christensen, the long “hairpoint” of Nicromoms pmilh has already been mentioned. It is roughly 2 p long on the tip of a flagellum only about 1p long. The hair-point is smoothly covered by the flagellar membrane and is unquestionably not an artifact (Fig. 6A). This peculiar little organism, the smallest known true flagellate, remained for several years in an anomalous taxonomic position (for furfher discussion, see Manton and Parke, 1960). Two other recently investigated genera of small green flagellates are nevertheless showing (Fig. 6) with four signs of bridging this isolation. In Sp-sir, flagella and Mononu;cstix with one flagellum (Fig. 6B), each flagellum is effectively bipartite ,possessing a long distal extremity conspicuously thinner than the almost equally long proximal part. This feature waa correctly recorded by light microscopy at leaat for Spemul-aie (Korschikoff, 1913) and ie undoubtedly a good morphological character. Since an affinity between these genera and M. pueilh can almoet certainly be justified on other grounds it is probable that these oharao. teristic flagellar apices may prove to be useful phyletio indicators within this group when more is known. However, it is precisely for this reason that the continued u88 of the word “acronematicJJfor any of these flagellar types, even those last discussed, is undesirable. The term suggests the presence of a terminal hair comparable to the lateral hairs which will be discussed in the next section, which is in fact never the caw. The word Peitechenge&8d (whiphh flagellum) is not inappropriate for the hind flagellum of a heterokont pair (Fig. 2A) a8 a contrast to F&mmrqek?e,! (hairy flagellum, Fig. 2B)for the front flagellum of the same pair. In all other cases ordinary botanical language is greatly to be preferred, e,g. mucronate, tapering or bipartite. For the most frequently needed distinction, that between hairless and hairy (with lateral hairs), latinized words such as glabrous and hirsute or their translated equivalents are available for use in languages such as French which lack a

PLAGELLAR STRUCTURE I N P L A N T S

13

simple vernacular word for “hairy”. My recommendation is therefore strongly that “acronematic” should be deleted from the vocabulary. Further comments on nomenclature will be found in the next motion.

VI. FLAGELLAR APPEXDAGES Information on flagellar appendages of the sort variously termed Flimmer or mastigonemes (see below) dates from the nineteenth century, more eapeoially Loeffler (1889) and Fisoher (1894). Qood reviewrt of the light microscopy will be found in Petersen (1929), Vlk (1931) and Deflandre (1934), with important personal observations by Petersen (1918) and Koch (1951). Apart from this brief list of excellent papers, the first fifty years after Loeffler were on the whole dominated by indifference if not frank disbelief by most botanists. The importance of electron microscopy in changing this picture has been reviewed several times, see especially Pitelka (1949, 1963) and also Manton (1952, 1954, 1956), and no attempt will be made to diacuas the whole field again. Consideration can nevertheless usefully be given t o topics not adequately covered by previous reviews and some of these are indeed to be found in relation to each of the more diverse categories of flagellar appendages encountered in plants, namely, spines, hairs of various kinds, and scales. A. FLAGELLAR SPINES

th el^^ spines are of special phyletic interest in relation fo certain more recent observations on the nature of the primitive Fucoid to be disoussedbelow (pp. 19-20). Thereareonlythreeknowncases,allmembeFs of the brown algae. There R i a Ringle spine on the front flagellum in Hinucntlralia (Manton el al., 1953) and another in a 8irnihr portition in Xiphqhura (Manton,1956), a Fucoid from the southern hemisphere. I n the third example, Diclyola (Manton et al., 1963), there GIa row of spines. A spine seem to be an excrescence caused by a lump of some unknown material borne on one of the peripheral fibm and therefore beneath the flagellar membrane. A phyletic interpretation is not available for Dictyota though the character waa of importance in determining the plane of “symmetry”* aa passing between but not through the two central strands (Manton, 1959). A phyletic interpretation

*

That strict bilateral symmetry does not exist in fbgella follows from the demonetration of spiral aqmmetry so clearly made for flagellar beses by Gibbone (1961). Gibbons and Grimetone (1960) and others. Nevertheleae the central strands per ae have 8 symmetrydoftheir own m d e fixity of position in relation to other attributes of e flagellum which it ie impo.tent to determine. Tbie ie therefore the sense in which the words “fhgeUar eymmetry” rn intended in the present context.

14

I. M A N T O N

supporting a common origin for X i p h p h m a and Himanthalia, on the other hand, is a matter of considerable btereet. Xip?uq~homdiffere from Himccnthalia in lacking the basal “button” and is therefore probably more primitive. It is otherwise not dissimilar since it mnsists essentially of dichotomous ribbon-like thalli with conoeptacles on both surfaces as in HimanthuZkz. An a w t y between these two genera in spite of their present wide geographical separation could be argued on general morphological grounds. The demonstration that they share a character as unusual as that of their flagehr spine nevertheless gives force to this comparison in a way which is perhaps important. B. FLAOELLARHAIRS

FZageZlar hair8 have recently become a serious source of confusion aa a result of three entirely different factors, namely, the nomenclature, the accidental introduction of errors of fact into the literature and genuine increases of knowledge. Nomenclature can at this point be relegated to a footnote” since my own strong recommendation, a8 already explained, is to discard for the time being all special terms except perhaps tho two oldest (Flirnmergei88d and Peit8chengei88d) limiting these however to the special case of the two differentiated members of a heterokont pair in the mnventional heterokont groups (Xanthophyceae, Chrysophyceae, Phaeophyceae and the water moulds). For the appendages themselves in these and other cases the word “hair”, at least to readers of English, is unambiguous and I propose to use it in the account which follows since it carries no overtones suggesting uniformity in all contexts which a single specially coined technical term such as “mastigoneme” inevitably does. The scepticism of biologists in accepting the concept of b i r a on flagella at all is connected with the obvious diffioulty in distinguishing fixation artifacts fiom real structures which are at or just below the threshold of visibility with the light microscope. A mistake of a Werent kind is exemplified by Thalaasomonae Butcher, a genus of small green flagellates, described (Butcher, 1969) as possessing full heterokont flagellation. However, this description has recently been shown (Parke and Rayns, 1964) to be due to confmion betwoen two *The more commonly encountered terms other than IrlhmrgeisseZ end Peitachengeiesel(Fiecher, 1894) were introduced by Deflandre (1934) end englicized by Pitelke (1049). who ale0 lists them in Pitelke (1963). On this terminology, the noun w t i g o n e m denotea 8 &gellar hair of any khd; the adjmtive pantonemtk denotes e f h g e h n which when Been in profile ehowa hsh on both sides; 8 t h h m ~ a C Aagellum with bira on one aide only; end amonematic 8 terminal hair (diecuseed on p. 12). Other terms, of which there are an increasing number, do not yet 8igniScSntly effect the general literetuw.

FLAQHILLAR STRUOTURID I N P L A N T S

15

different organism present together in the same culture, namely, a pigmented organiem identioal with Micromo7~cs8qtubmatQ (Menton end Parke, 1960) and a much larger colourless chrysophyoean predator. This naturally displayed hairs on its front flagellum of the normal heterokont type charaoteristic of Chrysophyceae, when examined electron microsoopioally by another observer whose miorographs wem reproduced. The genus Th.akceeomonas has thus to be rejected aa a nomen confusurn, synonymous otherwise with Xkwmunaa 8quu&, and all statements based on it, claiming the presence of true heterokont flagellation in a member of the green algae, should be deleted also. This does not, however, mean that hairy flagella as such are absent from members of the Chlorophyta and indeed there are hairs of more than one kind and in several different types of arrangement. For comparison Fig. 2 illustrates the type of hair present on a true Ff!irnwrgeissef! of a brown alga, in this case represented by the front flagellum of the zoospore of Scytoeiphon. The hairs are co&1ge,arranged in two rows (one on each side of the flagellum), but becoming sparaer towards the tip which is shortly mucronate. Each hair appears to be bipartite with a slender distal extremity emerging from a somewhat thicker proximal part. Whether these slender extremities are present in life or represent molecular threads ejected from the centres of tubes (the thicker bases) as a fixation artifact is unknown. F i p 4, on the other hand,illustrates the very different type of hair present on the single flagellum of the type-species of Pedinomrmc~e(Loxophyceae within the Chlorophyta8ewu Christensen).Thereare again two rowsof lateral hairs, but each hair is sothin aa to make the detachedbacterialflagellumpresent in the same field appear coarse in comparison. Moleculer threads are again suggested, though in this case the idea of a f h t b n a r t W i s m o r e difficultto introduce in view of their very regular arrangement. What effect, if any, their presence may have on the mechanics of swimming is impossible to determine, but a substantial mechanical effect seems excluded for threads as thin as this. In a somewhat similar caw, Chlorochitridim t u b e r c u b Vischer (Pedinomonacr tuberculata (Vischer) Cams), the hairs are more numerous and longer (Manton and Parke, 1960) but they are individually no thicker than in P, minor. Their exact arrangement in ChZorochitridion should be re-examined, since the “plume-like” appearance might in that o w have been caused by an all-over hairiness rather than by restriction of the hairs to two ranks. This possibility is excluded for P.m i w (rig. 4) and also for a true li”limmergei88d since the alignment of the point6 of origin of hairs, either singly or in tufts, w(t8 clearly demonstrated at an early date (Manton and Clarke, 1966;see also Manton et aZ., 1952). The special flagellar characteristicsof the euglenoids have often been

16

I. M A N T O N

illustrated (see especially Mainx, 1928; Manton, 1962; and may be taken as generallyknown. Thelimitation of the longest hairs to oneside of the flagellum (Fig. 3) was detected by Fischer (1894) and hae not subsequently been seriously disputed. Nevertheless to w e the name PZimmergeisseZ for this as well as for the front flagellum of a hetemkont alga is almost certainly ill-advised. Not only is the arrangement of hairs different but the individual hairs, not separately visible with the light microscope which can detect only hair tufts in a stained preparation, are very slender and, though longer and more copious than in the type species of Pdinommure, are definitely not comparable either in structure or in arrangement with the hairs of the heterokont algae and fungi. The presence of thick hairs of more than one kind has, however, also been recorded on unequivocal evidence within the Chlorophyta. Among members of the Volvocales the surface of the whole cell, both wall and flagellar membrane, is sometimes covered by a close tomentum, at least when seen in the iixed condition (Fig.7). The evidence of dividing ceUs (Fig. 7D) indicates that this apparent tomentum is almost certainly derived from material deposited upon the new daughter cells before liberation from the old wall of the mother cell. An extreme case (Fig. 8) is that of Haemtococcus pluvialis which has not yet been studied developmentally but which has a very conspicuous tomentum limited to the flagellttr surfaces only. The hairs in this c a m are very translucent when examined in whole mounts (Fig, 8A) though they were recorded as present as early as 1961 (Miihlpfordt and Peters). I n sections on the other hand they are extremely compicuouc(, Wme being apparently compound and curly. They cover the flagellar surface all over including the tip, but they are not an integral part of its structure since they are clearly related to a separate layer of material of unknown nature laid upon the surface. A different type of thick hair (Rg. 9) has been encountered among many genera of Christensen's new group Prasinophyceae (see List p. 4) and also in some colourless Cryptomonads, notably Chilomonae (Pitelkaand Schooley, 1966). Examples illustrated here are on the tip of the flagellum of illicrommras s q u a m t a (Fig. 9A), the surface of the flagellum in Heteromastix (Fig. 9B)and scattered fiee on the field near a group of flagella from which they have become detached in H&ephmra (Fig.9C).Such hairs are often slightly curved and tapered at both ends. They commonly show a pattern of transverse s t ~ t i o m when seen in section and they have recently been shown, in the special case of Heteromaetix (Manton et al., 1965), to be msnuftlcturod in vesicles within the body and to be depoeited thenos, together with scales, upon the surface.

FLAGELLAR STRUCTURE I N P L A N T S

17

C. FLAGELLAR SCALES

These last examples lead naturally to some of the most peculiar flagellar appendages so far discovered, namely, jlagellccr scales (Fig. 9). These were first encountered in Micromonas s q w w t a (Manton and Parke, 1960)as a single layer of fairly large scales covering the whole of the cell including the single flagellum (Fig. 9A). For some years this remained a unique occurrence, but now many other cases are known (Figs. 9B and 9C), with possible consequences to the taxonomy and nomenclature (includingthe naming of M . aqwmata itself) which have not yet been fully determined. Nephr08ehi8 gilva (Parke and R a p s , 1904) has a single layer of fairly large scales over body and flagella, though here the flagellar scales are not quite like those on the body, and the flagella themselves are two, somewhat unequal in length. Still more complex examples with scales in two layers (Fig. 11B) and with morphological differences affecting both layers according to location on the organism have been encountered in Pyramimonae, Hahaphaera, Pteromonas,Heteromastix,Prasimladw, Plutymonm to name only a few for which the facts have been published or are nearly ready for publication (Manton et aE., 1963; Manton et al., 1905; M . Parke and I. Manton, unpublished). It is possible that Christensen’snew group Prasinophyceae may eventually be used to oontain them all, though t o do so will involve bisecting the genus Micromonas since M . pwilkc, the t p species discussed on p. 12, has already been assigned to Loxophyceae. These problems need not, however, seriously affect a general botanist. The grouping of difficultgenera into larger units is a special taxonomic exercise which not many people will want to undertake. The important point in the present context is not the names but rather the undoubted fact that flagellar scales as well as flagellar hairs seem likely to provide some phyletic information of a new and interesting kind even though this has not yet been completely assimilated into the nomenclature of any existing system.

VII. FLAGELLAR BASES The use of flagellar bases as phyletic indicators is only just beginning. The absence of the central strands was the only significant observation before the introduction of0the improved methods mentioned under phase 2 (p. 2), but these, in the hands of Gibbons and UrimrJtone (1900), gave results of such dazzling clarity that they have remItined unchallenged. These authors showed among other things that, in the flagellar bases of certain colourless endoparasites, the peripheral strands became triplets instead of the doublets present in the 9+2 region. Each triplet group when seen in transverse section appeared tilted radially, C

18

I. MANTON

giving the type of spiral asymmetry referred to above, and within this region of tilted triplets or at least for a considerable part of it an elaborate central pattern, sometimes referred to as the “cartwheel pattern”, occupied the centre of the basal body. These observations have been substantially confirmed by other workers on several animals and plants and, until recently, major diversity was not bclieved to occur. Nevertheless there is diversity, at least among algae. That the flagellar bases of certain green algae are not identical in all respects with those of Gibbons and Grimstone’s material has been imperfectly shown in a general way several times. Some good transverse and longitudinal sections through the transition region in a species of Polytoma (colourless relative of Chlamydomonas) published by Lang ( 1 963), and two detailed studies by myself, carried out independently on several pigmented algae (Manton, 1964a,b), have clarified the factual position. There is no doubt that Gibbons and Grimstone’s triplet strands (Fig. 11G) and a form of the cartwheel pattern (Fig. 11H) are present in all these bases, the cartwheel pattern being characteristic of the extreme lower end of a flagellar base, but the transition region between base and flagellum proper shows a spectacular stellate pattern which was not observed by Gibbons and Grimstone. In the green algae, and indeed probably in most if not all chlorophyll b-containing organisms, the stellate pattern is so strongly developed as t.0 arrest the attention (Fig. l l D ) , but elsewhere it has to be looked for with care even when present and it is not yet known whether indeed it is always present outside the Chlorophyta and land plants. In a search for it among the Chromophyta it was found with difficulty (Fig. 11C) in Prymnesium parvum (formerly Chrysophyceae, now Haptophyceae sensu Christensen). The flagellar base of this organinm nevertheless proved to be so unlike those of both Gibbons and Brimstone’s material and the members of the green algae studied that a view of any one of three or four different levels would have differentiated it phyletically at a glance. Nevertheless even here the extreme base had triplet strands and a cartwheel pattern in no way different from that of almost any other plant or animal. The fixity of the 9+2 thus seems to be shared by some but not all the structural features of flagellar bases and it R i certain that these will repay closer study in a wider range of forms than have hitherto been examined.

VIII. FLAGELLAR “ROOTS” The phyletic signficance of the fibrous appendages internal to the cell, which radiate from the region of the flagellar barns, repreuents a new field which has only recently been effectively studied in plants.

FLAGELLAR STRUOTURE I N PLANTS

19

Only a limited number of examples are yet known in detail, but these are all in their various ways important. One of the simplest to describe though by no means the simplest t o explain is Oedogonium. The well-known crown of cilia in the zoospore and spermatozoids of this familiar green alga were studied in detail b y Hoffman and Manton (1962, 1963) who showed not only the fibrous substructure giving stability to the ring (Fig. 1OB) but the presence of radiating roots arising singly between each pair of ciliary bases and therefore equal in number to the cilia themselves (Fig. lOA). Each root proved to be bipartite (Fig. IOC) with a relatively short rod of crossbanded densematerial underlying a ribbon of three tubes or coated fibres running longitudinally beneath the plasmalemma for possibly the whole length of the body. No exactly similar case has yet been encountered elsewhere since in other green algae there are normally two kinds of roots arranged alternately no matter what the total number of cilia (flagella) may be, i.e. whether one (Pedinomonua), two anisokont ( H e t e r o w t i x ) , two isokont (Chaetomorpha) or four isokont (Stigeoclonium, Darprnaldia, Ulothrix). Roots of all these genera contain tubes or coated fibres in very definite numbers. The commonest fibre number ie two (Fig. 1lE), encountered in all these genera though in each case in only two of the four roots present. The other two roots, arranged alternately with these in tt cruciform manner, have been Rhown to possess five fibres (Fig. 1IF) in Stigeoclonium, Draparnaldia, Ulothrix (Manton, 1952; Manton et al., 1955; Manton, 1964s) and three fibres in Pedimmna.9 (Ettl and Manton,, 1964) and probably Chaelomorpha (Manton et al., 1955). This degree of numerical uniformity was unexpected and is difficult to explain except perhaps in terms of an ultimately monophyletic origin for very diverse chlorophyll 6-containing organisms now distributed in several different sections of Chlorophyceae, F’rasinophyoeae and Loxophyceae. This sqbject therefore deserves further attention with the expectation that the exact phyletic meaning may become better interpretable when more is known. The same may be said for flagellar roots in members of the Chromophyta, though here the pattern just outlined is not repeated. Instead we have some extremely peculiar but equally characteristic structures which have been studied most fully among brown algae. Thn need for doing so was determined by the existence of a special organ, the 00called proboscis attached to the flagellar apparatus of Fucuis (Manton and Clarke, 1951c), in a position apparently identical with that of an internal “root” detected later (Manton, 1959~)in the uniflagellate spermatozoid of Dictyota. This matter has recently been explored in greater detail (Manton, 1964c) with the discovery that the proboscis

20

I. M A N T O N

of Fucus is indeed homologous with a special type of internal root present generally in brown algal zoospores as well as in the male gametes of more primitive Fucoids, notably Halidrys and C‘ystoseira. The phyletic importance of this is considerable, since it indicntos among other things that Fueus itself is more specictlized thtin has sometimus been thought, while Cg~toseiruis Rubstantially more primitive.

IX. CONCLUSIONS

It is thus clear that flagellar characters have value as phyletic indicators in many more ways than could have been known even ten years ago. Emphasis can nevertheless usefully be laid here on two major sources of confusion which must at all costs be avoided if the full value of these somewhat unfamiliar criteria is to be utilized. One is the type of careless mistake leading to seriously erroneous records as discussed on p. 14 with respect to “Thahsomonae”, and the other is premature systematization of nomenclature which can obscure genuine and phyletically significant differences under the artificial unity of a system of Latin names, as discussed under flagellar apices (p. 12) and flagellar hairs (p. 14). The two most important attributes for work of this kind are meticulous care in recording and an open mind in interpreting. Given these, together of course with reasonable botanical knowledge and manipulative skill, accumulated information will almost automatically clarify interpretation provided that it is collated with other sources of evidence. It cannot be emphasized too strongly, however, that for phyletic interpretations there is no a priori guidance as to what characters will in themselves be of major and what of minor importance. Only when the work is finished will this become apparent. It is essential therefore to use information from fine-structure as additions to the pool of knowledge provided by general morphology, biochemistry, life histories, etc., so that phyletic conclusions when reached are based on all the evidence and conflict with none. Knowledge of very few plant groups is in this happy state and therefore it may assist a general reader without first-hand experience of the particular characters under discussion if a summary of what I personally regard as reasonably well authenticated conclusions in the present state of knowledge be appended.

X. SUMMARY (1) Absence of flagella in the case of Red Algae, combined with other facts of fine structure (notably the platid lamellations) in which members of this group seem primitive, is I believe correctly interpreted

FLAGELLAR STRUCTURE I N PLANTS

21

(Christensen, 1962) as indicating greater relative antiquity for this group than for any other type of pigmented algae after Blue-Green Algae. (2) On thesubject of isokont versus heterokont flagellation, the following points have been brought out. (a) Heterokonty in the strict sense is not found in Chlorophyta though it occurs in several groups of’ organisms lacking chlorophyll 6, collectively termed Chromophyta by Christensen (19f32),together with their colourless tlorivativen, notably the water moulds. The Chromophyta are tLt leant an d t l and could be oltlcr than the Chlorophyta. ( b ) lxokonty H i found in the Huptophyceae among Chromophyta and in the Chlorophyceao among Chlorophyta. There is no close affinity between these two groups. Anisokont members of the Chlorophyta occur in the Euglenophyceae, the Loxophyceae and the Prasinophyceae. The fundamental asymmetry of the motile cells of land plants points to an anisokont ancestry within the Chlorophyta, for which therefore Chlamydomonas is not on the direct line. ( c ) This means that the nature of the “primitive flagellate” within the Contophora is unknown, but it must not be assumed that it contained chlorophyll b and it need not have been isokont. (3) External flagellar characters of value as phyletic indicators include : (a) apical structure (especially in certain anisokont greens), ( b ) flagellar spines (brown algae), (c) flagellar hairs (different in Chlorophyta from Chromophyta but of value in different ways in both), ( d ) flagellar scales. (4) Internal flagellar chaructw of value an phyletic indimtore include : ( a ) flagellar bases (different in Chlorophyta and Chromophyta), ( 6 ) flagellar roots (different in Chlorophyta and Chromophyta). ( 5 ) A special case for which the sum of flagellar characters (including external morphology and the micro-anatomy of roots) has given important new insight is in the Fucales, where Cyatoseim now appears t o be more primitive and Fucua less so than has commonly been thought.

22

I. M A N T O N

LEGENDS OF FIQURES 2-11 FIG.2. Scytosiphon zoospore to show normal heterokont flagellation. A: showing part of the body with a romplete hind flagellum (Peitschengeissel) and part of the hairy front flagellum (Flimmergeissel); micrograph C5933, x 15,000. B: the tip of a front flagellum, C3706, x 20,000 (Bfter Manton 1964r). FIG.3. Euglena apirogyra flagellar tip x 20,000 (courtesy Dr. G. F. Leedalc).

FIG.4. Pedinomonae minor. A: complete cell showing the single flagellum, 5797 /21, ~3000. 13: flagellar tip showing very fine lateral hairs with an S-shaped detached bacterial flagellum lying awow the wpecimen; mirrograph C5006, ~ 3 0 , 0 0 0(after Ettl and Manton, 1964). B:

h a . 5. A : Micronionnr pueilh Hection of a complete re11 xE0,000 (after Manton, 1959). Yottomtinliz eczeca (courtesy 1)r. J. H. Belchor). Micrograph C5015, x 20.000,.

Pro. 6. l~lugellsrtips from R freshwatcr plankton sample (courteay 1)r. H. Ettl); two dagells from ~tL‘hlrrmydomonnn species below; four flagella with long “hair-pointa” from a Spermloroopin cell abovo. Micrograph C3028, X 15,000. PIG. 7. Chloropnium ronue (courtesy Dr. H. Ettl). A: transverse section of a flagellum ahowing the superficial tomentum; micrograph C3137, X 50,OOO. B: oblique longitudinal section of a flagellum near the surface of the subtending cell, both showing tomenturn; C3151, ~40,000.C: longitudinal section through a cell tip with emerging flagellum t o show distribution of tomentum and differing thicknesses of wall layers below; C3170, x40,OOO. I): part of two daughter cells within the mother cell wall to show probable origin of tomentum; C3156, X40,000. FIG.8. Haemutococcus pluvialis (courtesy Dr. H. Ettl). A: shadowcast flagellar tip showing very transparent tomentum (contrast with the detached spine of another organism top right), C7299, ~30,000.B: transverse section of a flagellum with tomentum attached to a separate layer of material outside the plasmalemma; C8208, ~60,000.C: oblique longitiidinal sertion of a flagellum with tomentum (N.B. the lower end of the profile is not a flagellar tip); C7439, x 40,000.

Fro. 9. Examples of flagella with hairH and wcalen. A: Micrornrmna eqmmulo, x30,CNN (after Parke & Rayna 1964). B: Hetrromu8lix rotunda, x30,WK) (after Manton el ul., 11965). C: Halosphnera sp. (courtesy Dr. M. Parkc), mic-rograph BW2r5, ~30,000. Fro. 10. Ocdogonium cardiaeum npermatozoicl (aftor Hoffman and Mariton, 1963). A: flagellar ring with bases and ‘ ‘ m O t H ” , X 16,000. B: section whowing the cartwheel p&ton, in flagellar bases and the various fibrow hands connecting them together, xf%,fw). (;: two adjacent ‘‘mots” cut transversely showing in each the nolid rod below and tho ribbon of three fibres or tubes above, Xm,oOo.

Frcr. 11. Flagellar bases and “roots” in variouH algae. A: Stigeoclonium Hertion of a tip of a settling zoospore ~30,000 (from Manton, 1964a). B: Heleromaslix dovclb wction o f a flagel. lum covered with two scale-layers (see also Pig. BB); micrograph C5552, x 100,CXw). C: the “stellate pattern” in Prymnenium parvum, X 100,OOO (after Manton. 1964h). L): the “st&ate pattern” in Stigeockmium, X 150,000 (after Manton. 1984b). E :Stigeoeloniurn, two-strandd root, ~ 5 0 , 0 0 0(after Manton, 1984a). F: Sligeoclonium, five-stranded root, x IOO,ooO (after Manton, 1964a). G: triplet strands from the middle region of&flagellar base of P r ~ w i ~ m p r v u m ; micrograph C4238, x 100,OOO. H: triplet strands and cartwheel pattern a t innemoet end of flagellar base of Prymnesium parvum; micrograph C4613, x 1OO.OOO.

Fro. 3. Euglena.

Fro. 3. Euglena.

FIG.5. Micrononae and Monomaetix.

FIG.6. 8permatozoopie and Chkzmydomnas.

Fio. 7. Chloroqniurn rome.

29

FXQ.8. Uaemaloeoccua.

FIG.10. Oedogonium.

FIG.11. FIagellar “roots” and flagellar bases.

B L A Q E L L A R STRUOTUR‘BI IN PLANTS

53

REPERENOES A d d , J. and Thi&y, J. P. (1963). J. M h ~ p i 2,s 71-80. Afzelius, B. (1969). J. Bwphya. Bioohem. Cytol. 5, 289-78. Bdlowitz, E. (1888).AT&. dk.A M . 82, 401-73. Bogorad, L. (1982). I n "Physiology and Bioohemistry of Algae” (R.A. h*, ed.). AccEdemio P m , New York. Bouok, (3. B. (1962).J . OSU BWl. lQ1,153-70. Butoher, R. W. (1969). An introduotory account of the smaller algae of Brithh O O M t d Wetere. Part I. Fkh. Invest. hd.8er. 4. Chriateneen, T. (1962). Botanik. Bind 11: Syetemtik Botamk, Nr. 2, A b r . 1-178. Munkegeerd, Copenhegen. Dei%uuh,0. (1934). C.R. A d . Rcd., Paria, 198, 497-8. Ettl, H. and Menton, I. (1964). Nowa Hedw@a. In the preae. Fawoett. D. W. end Pgrt-ar, K. R. (1964). J. Morph. 94,221-82. Fieoher, A. (1894). Pringa?&m’e Jahrb.f. +a. Bot. XirVI. 187-236. Frifech,F.E. (1936).“structureesldreproductionof the Algee.”Vol.I.Wbri+ Gibbons, I. R. (1961a). J. Biophya. Bwchern. Cytol. 11, 179-205. Gibbona, I. R. (1961b). N d w e , Lond. 19, 1128-9. 7, Gibbona, I. R. and Grimatone, A. V. (1960). J. Bhphye. Biochem. U@. 879-716. Greenwood, A. D. (1969).J. exp. Bot. 10, 56-88. Greenwood, A. D., Manton, I. and Clarke, B. (1967).J. exp. Bot. 8,71-86. H o ~L.,and Manton, I. (1962). J. EXP.Bot. 18, 443-9. Hoffmsn, L.and Menton, I. (1963).Amer. J. Bot. 60. Jakus, M. A. and Hall, C. E. (1948). BioZ. Bull. 91, 141-4. Koch, W. (1961).J . E k h a Mitohell eci. SOC.87, 123-31. Korechikoff, A. A. (1813).Ber. dtsch. bot. Qw. 81, 174. Lang, N. J. (1963).J. cell. Bwl. 19, 631-4. Leedale, G. F. (1964).Arch. Mikrobiol. In the preaa. Loeffler, F. (1889). Zbl. Bakt. 6, 209-24. Luther, A. (1899). K . Bvenska, Vetensk A M . L@ud. 24, 1-22. Maims, F. (1928). Arch. ProtLqtenk. IJL, p. 306. Manton, I. (1960).Demonstration of compound cilia in a fern aperm8tozoid by meene of the ultra-violet microscope. J. exp. Bot. I,89-70. Menton, 1.(1962).The fine structure of plant cilia. R p p . Roc. exp. Bwl. 8,300-19. Manton, I. (1954). Microsnstomy of cilia, flagella etc. 139. Recent work on the internal structure of plant cilia. Proc. Int. Confevmce on EZectron Micro-

-,

London. 694-9.

Manton. I. (1966).In ‘lCellulsr Mechanism in Differentiation mid Growth’’, @. Rudnick, ed.). Princeton, U.8.A. Manton, I. (19698). Electron microscopical observetione on 8 very amall fl8geht.e. The problem of C h r d i m gnceilla Butcher. J . Mar. biol. A M . U.K. 88. 319-33. Manton, I. (196Qb).Observations on the mi&oantatomy of the spermatozoid of the Bracken Fern.(Pteridiwnaqudinwn.)J . Bwphya. B i o c b . Cytol. 6,413-18. Manton, I. (19690).Obeervationa on.the internal structure of the spermatozoid of Ddclyola. J. exp. BOt. 10, 448-61. Manton, I. (1961). P l a t Cell 8tmcture, In “Contemporery Botanioal Thought” (MoLeod and Cobley, eds.) pp, 171-97. Oliver and Boyd, London. Manton, I. (19848). ~bsewationson the Ane structure of the zooapore and young gemling of Stigeoolonium.J . exp. Bot. 16, 399-411. Msnton, I. (1964b). The poeeible signi5usnce of aome detaile of flagellar baeee in plants. J . R.nric?.. 800. 111,82,279-80. Mmton, I. (1964~).A contribution towarda underatanding of “the primitive Fucoid”. New PWZ. 68, 244-64. Manton, I. (1964d). Obeervstions with the electron microeoope on the division cycle in the flagellate Prymneeium p a r u n . J. R. Mh.6oc. 88, 317-26.

34

I. MANTON

Manton, I. and Clarke, B. (1960). Electron miaroscopical observatione on the spermetozoid of FUGUE. Nature, Lo&. 160, 973. 8 fern Manton, I. and Clarke, B. (196la). Demonstration of compowld C i h eprmetozoid with the electron microscope. J. Wp. Bat. 2, 126-8. Manton, I. end Clarke, B. (1061b). Electron microscopical observations On the zoospores of Pylaielb and Lanrimrb. J. q. Bot. 2, 242-0. Manton, I. and Clarke, B. (10610). An eleatron miorowope etudy of the sp-tozoid of Fucw aewatw. Ann. Bot. N.B. 15, 461-71. Manton, I. end Clarke, B. (1962). An electron miommope study of the spennetozoid of19phgnum.J. exp. Bot. 8,206-75. Manton, I. and Parke, M. (1860). Further observetione on small green fl&lab with special reference to possible relatives of 0-m Mlla Butohm. J . MW. h i . AEE.U.H. a 9 , 2 7 ~ ~ Manton. I., Clarke, B. end Greenwood, A. D. (1953). Further observatiom With the electron miarwoope on spermatozoids in the Brown Algae. J. c ~ pBat. . 4, 310-29. b t o n , I., Clarke,B. and Greenwood, A. D. (1965). Obeervationswith the eleotron microscope on biciliate end quadriciliate zoospores in Green &m. J . q B O ~6,. 126-8. Manton, I., Oates, K. and Perke, M.(1963). Observations on the fine structure Of the Pyramimorure stege of Hahaphaera end preliminary observations on three species of Pyramimonae. J. Mar. bwl. Aae. U . K . 48, 225-38. Manton, I., Clarke, B., Greenwood, A. D. snd Flint, E. A. (1962). Further observations in the struoture of plant, cilie, by a combination of Visual and electron microscopy. J. exp. Bot. 3, 204-16. Manton, I., Raym, D. G., Ettl, H. and Parke, M. (1066). Further observations on green flagellates with scaly flagella: the genus Heterommtiz Korshikov. J. Mar. biol. Aas. U.K.45, 241-66. Muhlpfordt, H. and Peters, D. (1061). 2001.Anz. 16 (Suppl.) 163-0. Parke, M. (1949). J , Mar. Biol. Aaa. U.K. 28, 266-86. Parke, M. (1962). J. Mar. biol. Aaa. U.K.32, 407-620. Parke, M. and Adams, I. (1060). J. Mar. Biol. Aaa. U.K.89, 263-74. Parke, M. and R a p , D. a. (1964). Studies on Marine Flagellates. VII. Nephro. aebnie gilwa sp. nov. and some allied forma. J. Ma?.. biol. Am. U.K. In the press. Parke, M.,Lund, J. W. Q. end Manton, I. (1062). Arch. Mikrobiol. 42, 838-52. Perke, M., Manton, 1. end Clarke,B. (1066). J . Mar. Liiol. Rae. U.K. 84,579-608. Pea~e,D. C. (1063). J . cell. BWl. 18, 313-26. Petersen, B. (1918). k w k . Ndurh. For. 69, 346-7. Petereen, B. (1029). Bot. Ti&&. 40,373-89. Petersen. J. €3.. Caram, B. and Haneen, J. B. (1968). Bot. Tidssb. 64, 67-60. Pitelka, D. R. (1940). Uniw. C d q . Publ. 2002.68, 377-480. Piteh, D. R. (1963). “Electron-microscope etruature of Protom8.” Pergamon h, Oxford. Pitelka, D. R. end Schooley, C. N. (1966). Univ. Cdif. Publ. 2002.61, 79-128. Pringeheim, E. a. (1948). BWZ. Rev. 28, 46-61. Pringsheim, E. G. (1966). N o ~ Acts a Leop. CWOZ.18.1-188. m h e i m , N. (1866). M d . A M . Wku. Berlin. 133-66. Roth, L. E. and Shigenege, Y. (1064). J. cel2. Bwl. 20, 240-70. Bmith, 0.M. (1061). Menu81 of Phycology. N.8. 27, Ohmica Botunim 00. W d W , Maaa. Stanier, R. Y. and van Niel, C. B. (1062). Arch. Mikrobiol, 4a, 17-36. strain, H.H.(1061). In “Menual of Phyoology” ( a. 116. Bmith, ed.) p. 262. Cronioe Bot~nic& CO., W81tham, Maasaehueette. Strasburger, E. (1880). Znltbildzcngund 2-w. 3rd edition. Jena. Thuret, G. (1861). Recherches sur lea Zoospores dea Alguw. Ann. 8ei. W . (bot.) 38 10, 1-13. Thuret. a.md %ma+, Ti! ‘11S’711\ “F+.4- DL----l-A---- r t D-21

Fundamental Problems in Numerical Taxonomy

.

W. T. WILLIAMS and M B . DALE Department of Botany. The Univeraity. 8outhamptm. Bngland

.

............................................................. 86 .................................. 87 . ............................... ;..................... a7 B. Monothetio and Polythetic Clwi5~lltione.................................87 C. Maximizcltion ......................................................... 88 D. Hierarchical and Non-hierclrahical Clessiflmtione........................... 42 E. Probabilfstic and Non-probabllisttoClwifirntiom.......................... A3 I11. The Choice of Mathemstioel Model.......................................... 48 A . Introduction: Metrica .................................................. 48 B. Metric Properties of Pair-functions ...................................... 49 C. Intrinaidy Non-metrio Byatam ........................................61 D. Non-Euclidean Syntamn................................................. 52 E. Concluaionn ........................................................... 63 IV. The BM~C Euclidesn Model ................................................. 54 A . Duslity: The R/Q Problem .............................................. 64 B. Adjustments to the Model .............................................. 66 C. Heterogeneity......................................................... MI V. Strategyofhlysis ....................................................... 60 A. Simplification Methods ................................................. 69 B. Partition ............................................................. 61 C. Non-hierarchical Metbode ..............................................81 L). Hierarchical Methods .................................................. 82 Acknowledgements........................................................ 67 Referenw ..............................................................87 I

.

Introduction

I1 The Nature and Propertien of Clessiflcutione A TheBaeicAxiom

I. INTRODUCTION In any field of endeavour which transgrosoos the boundary between fundamental and applied disciplinefl there tend to be two alternative approaches: the user’s approach. “What do I wish to do. and how can it best be done?” and the more fundamental “What can most efficiently be done. and what can it be used for?” The approaches are more different than is commonly realized. and both are necessary. Them refleotions are prompted by the appearance of the firet major text-book devoted to numerical taxonomy. that due to Bokal and Sneath (1964). This will provide an admirable introduction for those botanists wishing to enter this rapidly developing field; and it is no denigration of this important work to suggest that the authors are less rigorous in their examination of the methods than they are in their Uee and htemreta-

W. T. WILLIAMS A N D M . B. D A L E

36

tion, for it is with these latter aspects that they are primarily concerned. The user’s interests in plant ecology are similarly met by Greig-Smith (1964) and, in a more limited context, by an article which to some extent complements our own (Lambert and Dale, 1964). Excellent bibliographies have been provided for taxonomy by Sokal and Sneath (1964) and for ecology by Goodall (1962) and Greig-Smith (1964). Our intention is different. The newcomer to this field is faced with a formidable diversity of methods, all apparently fullilling closely similar functions. It is nevertheless our contention that the number of fundamentally distinct methods is very small, and that criteria can be erected which will olarify the distinctions between them, and between their numerous variants. This is tho aim of this communication. We shall not be concerned with the problem of allocation to an existing classification, which is the province of discriminant analysis. Although all the methods we shall discuss are in principle applicable to botanical problems, few have yet been ao applied; our references will therefore of necessity be drawn from a wide variety of disciplines. Symbols used will be conventional; but in the 2 x 2 contingency table arising from the possession ( J , K )or lack (j,k)of two attributes J and K, two conventions now exist for the number of individuals in each class: the alternatives are set out below:

SJ

-J

SK

a

6

-K

C

d

+J

-J

(J)

Scheme (i)is older, and has long been used in elementary statistical texts; scheme (ii) is used by Sokal and Sneath. The latter is more informative, but is clumsy in algebraic expressionsand in our experience is easily misread. When such a table is at issue, we shall therefore adhere to the (a,b,c,d)convention.

FUNDAMENTAL PROBLEMS I N NUMERICAL TAXONOMS

37

TI. ‘I‘HIGNATUREAND PROPERTIES OF CLASSIFICATIONS A. THE BASIC AXIOMS

Most, ge~i(wtldixcurrsionn o t i c:lrcndicr~t h r two co~icc~iwd tto clc?fina. t’o (tistiiigiiirrli bctwcon, csistiirg typw of clrwnification; such , for examplc, are tlie discussions in Lawrence (IYGl), Beckner (196Y), Gilmour (1961)and Sokal and Srisatli (1964).It is important for our purposes, however, to establish the minimum requirements which all cliissifications must meet, and wc rcstnte tlie problem as follows. A population consist8 of elerncnts, each of which can be iridividually described by reference to a predetermined list of “relevant characteristics”. This population is subdivided into sets of elements. what requirements must be fulfilled by these sets for the subdivision to rank as a classification? We submit that the following axioms will suffice. iuid

(1) Within every many-membered set there must be, for every member of the set, at least one other membes with which it shares at least one relevant characteristic. (2) Membership of the set may not itself be a relevant Characteristic. (3) Every member of any one set must differ in at least one relevant characteristic from every member of every other set.

Axiom (1) introduces a concept of “likeness” and ensures that tin elenlent cannot be classified if nothing is known about it. Axiom (2) has two important ConsequenceH. First, division into groups defined solely as possessing a stated nuniber of membcrs (such a8 dividing a population into groups of ten, or dividing it equally into eight parts) is excluded ; secondly, all classifications mu& be open-ended-there may be no known members to add to a set, hut it must not be impowittlle by definition to add more. Axiom (3) not orily cnsurm that identicah cannot be distributed between different sctq, hit makes provision for the Mingle-membered set. Although these axioms will suffice to define a classification, they are not in general sufficient to define one which is useful. We therefore need to discover what additional constraints must be imposed to enable our classification to meet specific external requirements, and it is from this point of view that we now proceed to examine some of the basic problems in numerical taxonomy. B. MONOTHETIC AND POLSTHETIC CLASSIFICATIONS

These terms were introduced by Sneatll (1962) to replace Beckner’fi (1959)terms “monotypic” and “polytypic” (without changing Beckner’s definit.ions),since these terms have other meanings. The Nets in a mono-

38

W. T . WILLIAMS A N D M. B . DALB

thetic classification are completely defined by the presence or absence of specific characteristics. Since such classifications are always generated in practice by successive sub-division, it follows that there must always be at least one set all of whose members share at least one relevant charaoteristic. It is quite possible to oonstruct a population, classifiable by reference to the axioms, from which no such set can be extracted; in such a case monothetic classification is impossible. Monothetic classifications may nevertheless be useful. They have proved valuable in ecology, where the concept of “indicator species” has long been familiar; they may well be needed in criminology, in which a decision may have to be taken quickly and based on as few attributes as possible. They are normally unacceptable in taxonomy; in medical taxonomy, for instance, one does not wish a man to be treated for the wrong disease because he has one aberrant symptom. The occasional criticism that monothetic systems produce misclassification is, however, invalid, since the criticism automatically assumes that a polythetic system is desired, and the argument is circular. The real objection to manothetic classifications is that they assme a property of the population which it may not in fact possess. Polythetic classifications imply no properties beyond those invoIved in the basic axioms, and are therefore always possible. 0. MAXIMIZATION

1. principlee of maximization The basic axioms will serve to define a large number of alternative cla&kations, And a further constraint is needed to select from among these. The constraint universally required by users is that, in a sense yet to be defined, the members of any one set are to be as alike as possible and as unlike the members of other sets as possible. Differences within sets are to be minimized, differences between sets are to be maximized. Formal work in this field, usually loosely known a8 “maximization”, has been largely confined to discriminant situations, particularly in the field of pattern recognition (vide, e.g. Sebestye~, 1962); but the diverse methods of numerical taxonomy are simply variant methods of maximization. The method8 fall into two fundamentally distinot groups.

i. r%f-~tnLcturingmpdhodi~ (u)A function of the relevant characteristics ia defined between paim of elements. ( b ) An element may be either a member of a population or an entire set; if a set, then the set may be defined by one of its members, by all

BUNDAMENTAL PROBLIDMS I N NUMERIUAL TAXONOMY

39

of its members, or by an element constructed from all of its members. (c) Sets are to be construoted so that the funotion is minimum (or maximum) within them, maximum (or minimum) between them, or both.

ii. Derived-~tmturing?ne.t)wda (a) A function is defined between pairs of relevant oharaoteristios over a given set of members. (b) A characteristic, or a group of characteristics, is found for which the funotion, or a derivative of the function, is maximal. (c) Sets of members are defined in relation to the oharacteristic(e) so selected.

For certain purposes it is desirable that the analysis oan be “inverted”, in the sense that the elements and characteristias change plaaes. For this to be possible the data must fulfil certain oonditions which we explore later (Section IV A). The apparent diversity of methods in the literature largely concerns self-structuring methods, and in these the diversity is largely one of the function selected. Monothetic methods necessarily employ derived-structuring. 2. Intern1 and external ch&$cationa It is assumed in the foregoing paragraph that the members as defined by their relevant oharacteristics form a self-sufficient set within which maximization is to be effected; such systems, which comprise almost the wholo of existing literature in numerical taxonomy, we shall call “internal” classifications. It may nevertheless be desired to impose a restraint in the form of an external element or set of elements (selfstructuring) or an external characteristio or set of oharacterietics (derived-structuring). In such casm the maximization is entirely between the reference unit on the one hand and the internal eete on the other, the internql sets needing only to satbfy the besic olassifiwtory axioms. The process of maximization is, however, itself different from the all-internal case. The pkimary maximization is of the range of the selected function, in that the internal sets are to be as like or unlike as possible to the reference sef. The main use of these “external” classificatiom is likely to be predictive; if the population is heterogeneous in the sense we shall define in Section IV C, they will be more powerful than the classical regrmsions taken over the whole population. Their possible application t o problem in plant ecology is also under investigation. The only example known to us in the literature iu the derived-structure “predictive attribute ctnalysis” of Macnaughton-Smith (19SS), with whom we are currently collaborating in the development of more general S Y E ~ I W .

40

W. T. WILLIAMS A N D M , B. D A L E

3. Simultaneous alternative chsi$cations: clump Suppose the system be restricted by the requirements ( i ) that maximization shall extract only one sub-set from the population, and (ii) that this sub-set shall be subject to a specified constraint; the constraint normally imposed is that the subset must contain a specified element or group of elements which will act a8 its nucleus. Such a subset is normally termed a “clump” and the remainder of the population is without interest. Let this process be successivly repeated on the entire population by specifying a new constraint on each occasion ;the ultimate result is a set of clumps. This set is sometimes loosely termed an “overlapping olassification”, but such an extension of the term “classification” is not to be recommended; the clumps need not exhaust the population, and any one element can, and usually does, occur in more than one clump. Systems of this type are particularly associated with the work of Needham (vide,e.g. Needham, 1962; Needham and Jones, 1964) on linguistic data arising from problems in documentation and information retrieval ; but they have also found some application in anthropology and medicine (Bonner, 1964). They have been developed to meet circumRtances in which simplicity and speed of computation are more important than power, and they may well require re-examination before they can satisfy the more rigorous demands of plant taxonomy and ecology. A system of clumps can similarly be generated by the use of a changing external criterion as constraint. The groups delimited by the “deme” terminology (Gilmour and Heslop-Harrison, 1954) of plant taxonomy together form a system of precisely this nature, but it seems never to have been the subject of numerical study. We shall not be further concerned with clump systems in this article. 4. Weighting Sokal and Sneath (1964) accept the Adansonian postulate that “every character is of equal weight”. We need not so restrict ourselve8, and we shall first distinguish between a primi and a posteriori importance.

i. Importance a priori Classifications in, for example, modical or criminological corrtoxtn may be used as guides to action; in such cases particular characteristics may be of overriding importance. It might be regarded as undmirable to send epileptics to prison, no matter what their other characteristics suggested. Such cases do not disturb the systems we are considering, since they do not alter the classifications, but only the UBB that ie made of them. It has, however, frequently been suggested (wide, e.g.

FUNDAMENTAL PROBLEMS IN N U M E R I C A L T A X O N O M Y

41

Proctor and Kendrick, 1963)that characteristics should be assigned a differential importance from prior knowledge of the field ; as we have already pointed out (Williams et al., 1984) this destroys the objectivity which is the single most valuable feature of numerical taxonomy, and we cannot recommend it. ii. lmportunce a posteriori Derived-structuring methods maximize some function of the characteristics. After maximization, therefore, each characteristic will be associated with a numerioal value which reflects its contribution to the overall maximization, and which may therefore be regarded as tt measure of its importance. The application of this concept presents different problems in different systems, and the situation may best be explorcd by comideration of firstly, monothetio derived-struoture, and secondly, polythetio self-structure. (a) Mmthetic derived-etructure.If a population is such that it contains many shared charaoteristias and 80 can be defined as a set of final classes, a very large number of alternative monothetic classifications is possible. The characteristics used may be selected solely for external convenience, or even indiscriminately, and there is no internal maximization. Such are the “special claasifications” (into, e.g., food- or fibreplants) and the dichotomous keys in floras. These, which are in fact perfectly good external classifications, are commonly termed“artificia1”. It is therefore tempting to equate “artificial” with “absence of internal maximization”; but we defer to the views of Sneath (in Zitt.) to the effect that the terms “natural” and “artificial” have been so variously used that to provide them with new statistical definitions would confuse rather than clarify the situation. In contraat to these classifications, the method of Aseociation Analysis, whose properties are discussed in Section V D 2, ie a monothetio method whose defining CharacteriRtics have been obtained by a process of internal maximization. The characteristios now differ in a p8teriori importance, and this has by some workers been regarded an “weighting”. (b) Polythetic self-structure. Here again it is theoretically possible to effect classification without maximization, but since most real-life populations already themselves satisfy our Axiom (1) for a classification, the solution is usually trivial. A single maximization is therefore necessary in practice. All the “similarity” methods discussed in Sokal and Sneath (1964) are of this type : they use the least maximization which is in practice essential. However, the first step in such an analysb might be a derived-structure maximization, so that the characteristics were aa a first step provided with “importance” measures; a second

42

W.

T. WILLIAMS A N D X. B . DALE

maximization, using these weighted characteristics, would be necessary to complete the classification. The only such doubly-maximizedmethods known to us are those in whose development we have ourselves collaborated (Macnsughton-Smith et al., 1964; Williams et ul., 1964). 5. Unintentional weighting: “nui~ancecorrelations” The selection of attributes is not itself the concern of numerical taxonomy. Nevertheless, methods which employ derived-structure functions are prone to difficulties arising from so-called “nuisance correlations”-groups of attributes linked for reasons unconnected with the purpose of the analyeis. This problem does not arise in those ecological studies in whiah the attributes are plant species, since these are necessarily different things. The questionnaires of sociologiortl studies, however, normally contain much redundant information ; this is deliberate, since a question which may be avoided in one form may be answered readily in another. Some of the attributes are therefore logically linked, and these linked groups may dominate the subsequent analysis. It must be remembered that questionnaires have not normally been deaigned with modern numerical methods in mind, and the increasing use of these methods will doubtless in time iduence the design of questionnaires; but meanwhile the problem exists. The nature of the problem, however, has not always been clearly understood. The objection,to these links is simply that they can be known to be links without recourse to analysis ;if they could not be so known they would be of interest. It does not follow that they are in every caw easily recognized, and a preliminary numerical analysis may serve to este;blieh them. This is possible if the system is such that elements and chamteristics can change places, so that the characteristior, can be grouped into sets; if such a set inescapably suggests the hypothesis that the mombercl are linked for reasona-auch as intrinsic redundancy in a questionnaire -in which the investigator is not interested, the group can be replaced by one or more of its members or by a new attribute constructed from all of them. Despite statements to the contrary in tho literature, we submit that the objection to nuisance correlations does not lie in their logically necessary links; the sole criterion is the interest or otherwise of the wer. D. HIERARCHICAL AND NON-HIERILRCHICJAL CLASSIFICATIONS

Hierarchical classifications are of very real advantage to the taxonomiat, since they enable him to compare taxa at any desired level. This has probably contributed to the fact that the vast majority of existing numerical methods are hierarchical in nature. However, it

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43

may also generate a requirement that each level in division is aesoaiated with some measure which shall fall as the hierarohy deswnds. It is not always realized that this places an additional oonstraint on the ohoioe of maximizing function ; some functions (notably Euclidean distanoee and information statiatios) possess this property, whereas others (most of the derived-structure ooefficients and the “statistioal distance” coefficients) do not. The term “reticulate classification” seems .to include two quite different concepts. The f h t ia the unmaximized external classification with an embarrassing choice of alternatives, such aa arises in classifying books; this need not concern us. Truly reticulate claasifications arise out of an interest in inter-set relationships after division into sets has been complgted. If only the inter-set functions are required, a completely non-hierarchical method could be used; but, as shdl later point out, the choice of such methods is extremely restricted. In most caaes, therefore, both the hierarchy and the inter-set functions are of interest, and the problem is to generate either from the other. We ahall later demonstrate that maximization may, or may not, be uniform over the entire mathematical model in me. If it is uniform, as with unweightd Euclidean distances or information statiatios, no difficulty arises : inter-set functions and hierarohical divisions are everywhere oompatible. In those methods with which we ourselves have been associated, the maximization is deliberately non-uniform over the model; in these cases, which are hierarchical, no compatible inter-set function has yet been defined (vide Sections I11 D (i)(ii)).It is not permissiblo to define a completely new function,laincethe original hierarchical maximization may then fail; this is the cauae of the “recombination of aets” difficulty which Goodall (1953a) experienced in his pioneer studies in divisive methods. E. PROBABILISTIC AND NON-PROBABILISTIC CLASSIB+ICATIONE

This particular dichotomy has generated more confusion-and probably more rancour-than any other. It underlines the commonlyexpressed doubts a~ to whether these methods can, or cannot, be classed as statistics, and so has caused Greig-Smith (1964) to use the term “quantitative” and Sokal and Sneath (1964) and ourselves to fall back on “numerioel”. It underlies, too, the misgivings that authors frequently express concerning the “significance” of their results. The difficulty has been exacerbated by the fact that modern statistios is almost entirely concerned with estimates of probability, so that if well-known statistical parameters-x* or the correlation coefficient, for example-are uaed for maximizing, it is aaeumed that these am

44

W . T . W I L L I A M S A N D M . B. D A L E

estimates which should be associated with measures of probability. I n fact, the methods of numerical taxonomy are not, or need not be, probabilistic systems at all, but hypothesis-generating systems. We shall outline the two alternative approaches.

I . The non-probabilistic approach From this point of view, the methods of numerical taxonomy may be regarded as stemming from a branch of statistics of respectable antiquity-that concerned with finding mathematical formulations which will serve as a concise and economical description of an otherwise intractably oumbersome mass of data. Though superficially so dissimilar, their logical relatives are to be found among such projects as the fitting of Pearson curves to actuarial data (Elderton, 1938); the search for a flexible growth-curve (Richards, 1969; Nelder, 1961); and the application of contagious Poisson distributions to distributions of plants in the field (Archibeld, 1948).The fitting of regression lines is itself a member of the mme family, extended by the probabilistic concept of the significance of the parameters which the fitting requires. Now, these concise mathematical descriptions can with perfect validity be used to generate hypotheses concerning the nature of the data, but only if two conditions are rigidly satisfied. First, aa always, the hypotheses must be capable of being tested; secondly, any test must depend on new observations, and cannot again use the data from which the hypothesis was generated. Generation of the hypothesis may not be used as its own evidence; we forbear to cite examples of this practice, contenting ourselves by remarking that they can be found in biological literature. The precise statistical context of these methods can most clearly be demonstrated by comparing a vegetation survey in ecology with an agronomic experiment in, say, mineral nutrition. In the agronomic context the hypothesis is set from previous experience, and this determine8 the details of an experiment, which issues in a quantity of data; etatistical methods are applied to thaw data in order to test the hypotheHis-ueually in the form of the probability of obtaining a given deviation from a null hypothe6iB by chance alone. Tn tho ecological context, although experience may have informed its collection, the data is the starting-point ; function6 are elected and appropriately maximized in order to reduce the data to eimplor form; thin simpler form is used to generate a hypothesis-often in the form of “there is a change of some sort in this region”; and the hypothesis is tested by new, direct observations in the field. Examples of this type of hypothesisvalidation may be found in the work on Association Analysis (Williams and Lambert, 1960).

B U N D A M E N T A L YBOBL10MS 1N N U M E R I U A L T A X O N O M Y

46

Nor is validation difficult in applied taxonomy. In medicine, for example, the individuals classified may be diseme-producing organisms, or symptoms; in criminology they are normally delinquents. I n these cases the hypothesis takes the form of a suggestion for treatment. It may be remarked in passing that the power of the mathematical methods used is all-important in these fields, for although falsification of a hypothesis might gratify a dispassionate experimenter, it is apt to be disastrous if a human individual is concerned. The problem is more difficult in “classical” taxonomy. Here it is tempting to enunciate a phylogenetic hypothesis, normally based on inter-set functions, but fossil records are such that hypotheses of this type are rarely testable (vide, e.g. Sneath and Sokal, 1962). The basic requirement of taxonomy aemu stricto is stability, both of the membership of sets and of the pattern of characteristics that their members display within them. I n the first case (the membership of sets), addition of new characteristics followed by re-maximization should not ohange the membership of the sets. In the eecond (the pattern of characteristics), let a new element be disooverod whom chctracteristios are imperfectly known; if from the known chasacteristics it can be allocated unequivooally to an existing set, the pattern of its remaining characteristics, when these are examined, should conform to the pattern for the set. On this approach, therefore, the methods of numerical taxonomy are hypothesh-generating systems; and a hypothesis-generating system is neither valid nor invalid. Probability enters only, if indeed it enters at all, in the testing of the hypotheses that are generated. This approach exposes a possible danger, which we do not believe taxonometric writing has always avoided. This is that computer classifications might be regarded aa in some sense absolute-as “objective” and therefore “better”. They are not objective, since they depend on the user’s personal choice of maximizing function; and they are only better if they can be shown to fulfil a stated requirement more efficiently.

2. The probabilistic approach It is, &B we shall show, easy to conceive of probahilietio clamificatione in theory; but we are here concerned to defend tho thesitc that auch classifications are usually both impracticable and unprofitable. Fimt, it should be noted that a probabiliRtic classification requiree a null hypothesis; this will normally take the form of stating that the pairfunctions available for maximization in a given population or set could have been generated by a random process. The null hypothesis cannot, in fact, be independent of the function selected for maximization,

46

W. T. WILLTAMS A N D M. B. DALHl

i. DifJicultiea inherent in null hypothea Since the null hypothesis depends on the maximizing function, it will be convenient to select two well-known cases for consideration. (a) Multivariate nomnal popubtiom. In this case the null hypothesis would state that the observed variation in characteristics could have been generated by a set of independent normal variates, usually the characteristics themselves. The function available for test would probably be the correlation matrix. Now, Bartlett’s (1950) test for the roots is not available if the matrix is singular, and experience suggests that it is sensitive to departures from normality. To demand that all the coefficients be individually significant is nomally regarded aa too stringent ; and Goodall (19G3a) has in effect suggested that the ooefficients be themselves treated as normal deviatos, so that the proportion of them which exceeds the individual signifioanoe level be regarded &B a test of significanoe of the whole matrix. There is, in fact, no simple, unequivocal and robmt test available. ( 6 ) Qualitative populations. The function used (though others are available) is often related to the Euclidean diRtance between elements (or set centroids) plotted in an n-dimensional space where the j t h co-ordinate for an element is 1 if it possesses the j t h attribute and 0 if it lacks it. The problem now is to state a null hy&hesis at all. Use of the binomial expansion would imply that possession of all characteristics was equally likely; and the solution obtained by Rohlf (1962) for even n makes assumptions as to the distribution of the frequencies. If we assume, however, that the hypothesis should not involve the frequencies, an obvious solution would be to retain the frequency totals and to construct from them the entirely dissociated class-frequencies; that is, the numbers of individuals that would be required in all possible sub-classes if, without change in the total numbers possessing each attribute, all pairs of attributes were to have zero association. It is straightforward, though tedious, so to calculate the probabilities (for 0,1, d2)in the two-characteristic case; but the resulting algebraio expressions are extremely cumbersome, and lend little hopo of extension. In any case, construction of the general null population may present formidable difficulties. If we write (A) for the number of individuals possessing attribute A, (AB) for the number posmsing both A and B, and so on, then in the oompletely dissociated population W C .

N

*.

.)

( 4 * ( B )((7... .

=x

“3

Unfortunately, for more than two characteriafics, this relationship is necessary but not s d c i e n t (vide, e.g. Yule and Kendall, 1960), and oannot therefore be UBePx M a generating function.

F UNDAM INT A L PROBLIMS I N N U Y I R I O A L TAXONOMY

47

It has been suggested to UB (Macnaughton-Smith, in W.) that information statistics might provide a solution of the qualitative problem, in view of their remarkable additive propertiea and their relationship to x*. Let a p u p of n individuals be specified by the possession or lack of p attributes, and let the number possessing the j t h attribute be a,; we have made preliminary ObservationA, using ecological data, on the behaviaur of the statistic: I = Iyn log It-

E [u, log a,+(n-aj)

log (n-tzj)l*

j-1

Ecological data not uncommonly contain groups of identical or nearidentical individuala, and these groups may vary greatly in size; the data will have the properties of a stratified, rather than of a random, sample. Unfortunately, we find that the stati8tio above is seneitive to this particular form of non-randomnew, and is therefom unduly senaitive to set size--sets tend to be fused if they contain comparable numbers of membera. This is incompatible with our second classificatory axiom, since it implies that a function of the set may determine the allocation of one of its members. This difficulty may be removed by normalizing for group size, though, in some forma of analysis, a t the expense of replacing it by the generation of an “ambiguity” problem related to that arising from unweighted Euclidean diatanwa (Section V D 3 (i)). Nevertheless,these statistics have many desirable properties and would repay further investigation. (c) GoodaZl’8 coe&ien$. Very recently Goodall (1964) has proposed a probabilistic similarity index. For every pair of individuals, the probability that the two are as similar as in fact they are is calculated for each attribute separately , and the attribute-probabilities then combined. The method is cumbersome for qualitative b t a , but it is the only method known to us which ia in principle applicable to mixed data i.e. data in which the attributes are 80 unlike that any common scaling would be unrealietic. No example of it8 use has yet been published. ii. AelicaliOn Of $WObabili8tic Ch8$C&iO?l% Suppose an appropriate criterion of significance, and therefore an appropriate null hypothesis, to be available; and s u p p a a popubtion to have been divided by maximization into two eete whose dididididididididididididididididkincfio fails to reach signihance. It still does not follow that the division should not be effected. For the population may be so intractably large that the best possible sub-diviaion, though non-significant,may be mom uaeful than none at all. However, although the overall characbristicpattern may not define a significantdifference, sub-seta of charmbh-

48

W . T . WILLIAMS A N D M. B . D A L E

tics may exhibit stability (this phenomenon may occur if the population exhibits n o h , which are briefly discussed in Section I V C 4). I n either case, it is the usefulness of the division which will be of importance; the division will therefore in any case be subjected by the user to a second, pragmatic, test which will override the fist, probabilistic, test. We are therefore not convinced that any useful purpose is served by the probabilistic test, quite apart from its inherent difficulties. 111, THECHOICEOF MATHEMATICAL MODEL A. INTRODUOTION : METRIOS

The ultimate test of a numerical method is whether the u8er fin& it useful. However, all mothods are of some we to the usor; and if he ie to bear the sole responsibility of deciding between them, he will be faced with an immense amount of empirical work,still with no assuranm that the method may not fail under extreme conditiona-as, we believe, some existing “similarity” methods have s h a d y failed. The literature contains many despondent remarks on the paucity of available information relating to comparison of methods. This is particularly true of the pair-functions themselves, often loosely classed as “similarity coefficients”. The best-known have been reviewed by Goodman and Kruskal (1954, 1959), Dagnelie (1960) and Sokal and Sneath (1904); but it k doubtful whether even these extensive collections are complete. The problems would be relatively unimportant if all such functions were jointly monotonic, in the sense that, if element-pairs are so ordered that one function forms a monotonic series (i.e. a series which either increases or decreases over the whole of its length), the remainder will also be monotonic, To take only three well-known functions, 2a/(%+b+c), (a+d)/(a+b+c+d), and the correlation coefficient, it ie easily shown that no one of these is jointly monotonic with either of the other$. A choice is therefore necesmy; and the testing diffioulty oan be overcome, at lea& in part, if the methods and function8 are required to fulfil appropriate mathematical conditions. We consider it essential that any meamre used for maximization should define a model, and, if possible, a model in Euclidean space. The advantages of such Rystems are threefold. First, many simple, robust and powerful methods are available in Euclidean systems that are not available outside them. Secondly, as mentioned in Section I1 D, they have hierarchical advantages. Thirdly, and perhaps most important, our daily experience gives us an intuitive perception of Euclidean systems, and thereby enables us to grasp their properties and to predict these properties in extreme cases. If the function is such that it is not known

FUNDAMENTAL PROBLEMS I N N U Y E R I U A L TAXONOMY

49

to be associated with any particular probabilistic or spatial model (models which me neither probabilistic nor spatial are possible, but we know of no published work on them) we propose that it must, aa a minimum requirement, be a metric; it will then necessarily define a spaoe whose properties oan be explored. We deal in this motion with the general problem of metrios, and it will be oonvenient Brat to etate the conventionaldefinitione. The subject is fully dieoueaedin geometrioal texts; the formulation we use ia substantially that of Kelley (1965). De$dtion. A numerical function d(z,y) of pairs of points of a eet E is said to be a metric for E if it satisfies these oonditions: (1) d(z,y) = 4Y,430 (symmetry) (2) d(z,z)
where true we shall call qua&-metric. In the oontext of numerical taxonomy, metrio properties may fail for two remons : the characteristics may be intrinsically metric but the pair-function selected is not; or only a pair-function exists, and this is not metric. We deal with these cases in order. B. METRIU PROPERTIES OF PAIR-FUNUTIONS

It would be unprofitable to examine the properties of all the many functions in the literature, and we shall largely conhe our observations to the three moat familiar: (a+d)/(a+b+c+d); 2a/(%+b+c); and the correlation ooeffiaient.

I . ia+d)/(a+b+c+d)

This is the coefficient normally used by Sneath. Using the model mentioned in Section I1 E 2(i)b, and following Soh1 and Sneath in writing d for the Euclidean distance between the two points, the ooefficient is equal to (1-A*/N). It is baaed on a Euclidean metrio and satisfies the requirements we have suggested. 2. 2a/(2a +b +c) This coefficient is probably among the oldest in the literature; it specifies the ratio between the number of charaderiefics common to two elements and the arithmetic mean of the numbers possessed by eaoh. It is monotonic with the coefficient a/(a+b+c), used by Sneath for the purpose of exoluding double-negative matches; the intention Ic

50

W. T. WILLIAMS AND 116. B. D A L l

here, in the context of our model, is to prevent points being gmupd solely beoause they are near the origin. Enumeration of the I)-oh&raoteristic case for either coefficientwill immediately demonstrate that the ooeffioients are quasi-metric ; they are also necessarily semi-metrio, and thus do not Ratisfy our requirements on either oount. It is of intent& to note that Sokal and Sneath (1964) no longer recommend the exolwion of double negativea. The earlier coefficient can be ueed to generate a different model. If, in the ordinary 2 x2 table, we have (2a+b+c)#N, there must be Ia-dl double-positive or double-negative matches ; there may be more but there cannot be less. If the coefficient is written in the form:

it may be regarded as derived from a distance whose dimensions am scaled to remove all logically-necessary matches. The dimensions of the model now change from place to place, so that the model is nonEuclidean; it could be topologically embedded in a Euclidean space of not more than 2N dimensions, but we are not ourselves competent to explore the utility of this approach.

3. The correhtion coeficient Several alternative models have been suggested for this funotion, only one of whioh fulfils our requirements. First, it should be noted that of the four requirements for a metric, simple unsigned derivativea of this coefficient (such as (1-r)) fail t o satisfy requirements (2)and (3), and cannot therefore be handled in this way (its semi-metric properties are of value if “shape” coefficients are r e q u i r e d 4 Rohlf and Sokal, 1963). A model commonly used in factor analysis, however (vide, e.g. Cattell, 1962), supposes the points to be rigidly attached to their ,co-ordinate axes by extensible perpendichrs. If the axes are now rotated about the origin until all correlations are zero, the final angles between them will be the inverse cosines of the original coefficiente. These angles between pairs of lines now serve to define a Euolidean space with oblique axes ;providing this model is in me, our requiremente tm therefore satisfied.

4. Aeymmetric fU%i?b?&? Goodall (1963b), in the c o w of an emmination into the phytosociological concept of “fidelity” , has suggested that twymmetrio funotions would be of value in this mntext. Several euoh funotions am in fact on record in the literature, although only Goodall appem to have appreciated their nature and possible applioation. 80 far aa we 5m

FUNDAMENTAL PROBLEMS IN N U M I R I O A L T A X O N O M Y

61

aware, no practicable strategy for the maximisetion of aaymmebrio functions has ever been suggested or even sought; unldl this ia done, further investigation of their properties will remain unproilttable. U. INTItINSIOALLY NON-METRIO SYSTEMS

In the previous section it has been assumed that all characteristics are, or can be regarded as being, measurable; failure of metricity ia due only to the calculation of a measure which is not a metric. We are here concerned with two problems of greater fundamental difficulty; firat, the case in which individual characteriatioa, though they exist, cannot be provided with a simple measure; and secondly,the caw in which characteristics do not exist, though a pair-function between elements does. 1. I n d i ~ d dCharWteTi8tiGB This situation arises when all that can be measured in respect of a given characteristic is some cornparison between two elementacommonly in the form of a difference or a ratio. It is then neceaaary to operate on these comparisons in such a way m to generate a metric which will uniquely order the elements along that ahmaateriatic considered a8 a dimension. This problem, usually known as “scaling”, is of great importance in psychometric work, and hae given rise to 4n extensive literature ; the reoent communiwtion by Phillips (1903) will aerve as an introduction to the field. We h o w of no botanical work of this type; but since comparative measures are not unknown in taxonomic descriptions,the methods may yet prove applicable, and botanists should be aware of their existence. 2. I80laled pair-functim Consider a sociological study in which has been recorded, for the members of each pair of individuab in a group, the number of times they met each other in a given period; all that is available for analysis is a pair-funotion. Functions of this type are often semi-metrio (some pairs of individuals never meet) and are almost alwaye quaai-metric. The problem is to generate a Euclidean system of hypothetical characteristice such that the distances between elements shall be related to the original pair-functions. A solution itl provided by the “proximity analyais” of Shepard (1962tt,b).This generates a system of coo-ordinates, of the lowest order which will permit a unique solution, such that the Euclidean distances between the elements are monotonic with the original pair-funotions. The solution is iterative, and a oomputer

62

W . T . WILLIAMS A N D M . B . D A L E

program exists. Again, we know of no published botenical applioations, but the method might conceivably be of interest in competition studies where the records took the form of the number of times pairs of species were in contact.

D. NON-EUCLIDEAN SYSTEMS

1.

I~TdUCtiOn

A Euclidean space is necesmrily metric, but the converse is not true. It will be convenient to begin with a conventional definition : A Euclidean space of order n is the set of all n-tuples (q. . , xi . z,,) where all xi are real numbers and where the distance between two points

.

n is given by [d(z,y)]Z = Z(xj-yi)z. It can be shown that such i =1 distances are metrics. There are three obvious ways in which Euclidean properties may be lost. Firstly, n itself may not be constant, so that the dimensions vary locally; this is the situation for the coefficient discussed in Section I11 B 2 above. Secondly,the n-tuples may be constrained in some way, e.g. to the surface of a sphere ;we know of no application in numerical taxonomy. Thirdly, the distance function may fail; in the cmes we shall consider, the distance-function holds within sets, but fails between some or all of them. The space defined is thus locally Euclidean, and hence (if varying continuously) Riemannian; and no difficulty arises unless inter-set functions are required. We shall disouse briefly three rnethode in which this type of problem arises.

2. Examples (i) Attempts to we “8tati8tical distance" The group of statistia of which the Mahalanobis Dzis the bestknown is essentially probabilistic in concept; it relates inter-& distance to a common within-set function, and involves the postulation of a common dispersion matrix for the two sets. If the sets are manifestly unlike, this is an unrealistic assumption; end if such a matrix ie artificially constructed, it may well be singular. It is not uncommon (cf. Harberd, 1962) to postulate that the common dispersion matrix is an identity matrix, and to regard the Euclidean distance so calculated as a derivative of the Mahalanobis statistic; we ourselves believe this fo represent an unrealistic model, and consider that, as suggesw by Kendall(1957), a Riemannian metric is needed.

FUNDAMENTAL PROBLEMS IN NUM’IORIOAL T A X O N O M Y

63

ii. weighted polfietic & d a r n This method (Maonaughton-Smithet al., 1964) employs a Euclidem model with axes scaled by a p t e r i o r i importance me&Bures.As in the previous case, the scaling depends on the dispersion (or cornlation) matrix, but it is the individual axes which are affected. New scales &FB calculated before each sub-division; as a reault, any two sets derived by sub-division of a single set share the same metric, but this is not true of set-pairs in general. The final model resembles a Riemannian system in being locally Euclidean; bu6 the space is now divided into blocks with the local metric changing abruptly at the boundaries, and may best be described aa a “disjoint metric space”. Although models of this general type have received some attention from topologists, we have been unable to trace any work on the metrization of such a space. The diiliculty is exacerbated by the fact that the space remains undefined between sets. iii. Aeeociution A?ldg&8 Since this is a puro derived-structure method, no measure .)f intor-sot distance arises naturally from the maximization. Again, however, the axes vary in a poakriom‘ importance from set to set, and a Euclidecm metric would be unrealistic. E. CONOLUSIONS

We may now conveniently classify the acceptable ooefficients under three headings : 1. Information stat&8tica These can be maximized over the whole model; as a result, they automatically provide inter-set functions and progressively-falling hierarchy measures. Their relationship with x* permits them to be used in a probabilistic context. If our misgivings (Section I1 E 2(i)b) as to their dependence on set-size prove to be unfounded, or can be overcome, they will be very attractive; but more work is needed. 2.’ E‘uclihn CEialancecr These, too, can be maximized over tho whole model, and provide inter-set functions and hierarchical measures with the desired properties. They aeem likely to be probabilistically intractable, but we have given reaaons (Section I1 E 2(ii))for regarding this as relatively unimportant. Compared with the doubly-maximized coeffioienta they appear to lack power, especially in populations defined by small numbers of characteristica.

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3. Riemannh and diejoint-ape fundions It is our opinioii that these provide the moet Fealistio models mid the most powerful methods for olassifioation;but work on inbr-aet funotioris is badly needed, #or the biologist (inoluding the p m n t authom) the mathematioe required for suoh work is out of reaoh; but the d i f l i o u i t i e ~ are not entirely mathematical. In the probabilistio o w , since the dispersion matrices are known to be different, what is the null hypothesis which is fo be tested? In methods using sub-division with changing weights, what properties is an inter-set funotion required to possees? Before the geometers oan be expected to collaborate, the users must be prepared to consider theae questions.

IV. T m BASIOEUULIDEAN MODEL A. DUALITY: THE R/& PROBLEM We have already given reasons for our preference of a Euolidean model; this is not incompatible with essentially Riemannian or disjoint models, shoe the region of space undergoing maximization is always locally Euolidean. We therefore now oonsider the problems that arise in setting up suoh a model. We begin with n elements (which we shall henceforth uall idvidmh) specifled by p uharacteristics (whioh we shall henceforth call attributes, a term we use in an extended sense to inolude variables and variates). Provided all attributes can be given values, either inherently or by the methods of generation outlined in Section I11 C, the sy~temis symmetrioal; the data-matrix can be transposed so that the individuals and attributes exohange status. Two models immediately present themselvee: a set of rc points in a p-spsce, or a set of p points in an n-space. This duality haa given rise to the symboh R and &. Unfortunately, two mutally incompatible traditions aa to the definition of these symbols exist side-by-side in the literature; and this oonfusion-to which we have ourselves contributed-must now be resolved. The early workers in factor analysis oommonly refer to a model in which the individuals am points imagined as in a spaae apeofled by co-ordinate axes representing attributes: this is our n points in a p-space that we shall for the moment mll an attribute-space. The arithmetical operations were oarried out on a matrix of correlations (or oooaeionally 00variances) between attributes. Suoh a method ww oalled an R-method. Later, the entire prome WM transposed for oertain purpoees; the model is now p points in an n-space that we ehall for the moment ortll an individual-spaoe, and the arith6tical operatiom were octrried out on a matrix of correlations between individuals. This waa a 0-method.

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This now is the problem: do R and Q refer to the model or the matrix? In the early work there was no obvious ambiguity; but oonaider a matrix of Euolidean distances between individuale. Shoe it is a matrix between individuals, it hi Q; but since it is bmed on a model in the attribute-space, it is R. Sokal and Sneath (1964) define the symbols unequivooally by the matrix, and the individual-diatanoe matrix is, for them, Q; but in all publications from our laboratory we have d e h e d the symbols by the model, and the individual-distance matrix has been, for us, R. We have now decided that the Sokal-Sneath definition should prevail, for two rewons. First, the widespread circulation that their book will deservedly attain will ensure that many taxonomists previously unfamiliar with the symbols will first meet them in the Sokal-Sneath sense; and to attempt to assert a rival definition would cause unjustifiable confusion. Secondly, there is some historical precedent. Most early work obtained approximate solutions for principal axes by the “centroid” method. Although this opemtes arithmetically on an attribute-combtion matrix, it is based on a model in the individual-space;but it hw always been known as R, though by the Southampton definition it would be &. We suggest, then, that R and & refer to the matrix; but it w i l l still be oonvenient to have symbols for the model, and we suggest the symbols A (for a model in the attribute-space) and I (for a model in the individual-spaoe). It seem likely that the indecision frequently expressed concerning the relative merits of R and Q-methods stems partly from inadequate understanding of the models implied, and we believe that the introduction of the new symbols will olarify the situation. A matrix of interindividual distances and an inter-individua! oomlation matrix are both &; but the former implies relationships between points in an A-space, the latter between angles in an I-space. An attribute-oorrelation matrix is R,and an individual-distancemtrk ie Q; but both am A - s p w models, the first ooncerned with angles and the second with points. In fact, two @methods may require models which differ from each other more fundamentally than do some It/& pairs. B. ADJUSTMENTS TO THE: MODEL

V i r t d l y all numerical methods involve diEcultiss oonwrned with the dimemione of physioal quantities. Only in truly qwlitative data do these difS.Oultiesnot arise; whatever the nature of the quantities which have been diohotomized, addition of either rows or mlumns of the datamatrix is interpretable in terms either of the number of individuclls possessing an attribute or of the number of attribute p o d by an

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individual. If the data is quantitative and not all in the same units, addition of attributes across a single individual is not teohnioally possible. This difficulty arises immediately in Euclidean distances or principal components in the A-space, and in correlation coefficients (“betweenpersons”) in the I-space. It is discernible,too, in the tendenoy to regard principal components as “taking out” a proportion of a variance constructed by the illegitimate addition of sepmate variances. It is the invariable, and inevitable, convention in numerical taxonomy to regard all attributes as dimensionless, and hence available for ethmetioal manipulation; but the highly autocratic nature of this convention must be clearly realized. Methods which involve the addition of different attributes are not, in general, invariant under changes in scale of the co-ordinate axes. The initial scaling of axes is thus irrevocable, and will in a sense determine the results of the analysis. Euolidean distanoes are very sensitive to the scales of the axes, but independent of the position of the origin ;principal oomponents are very sensitive to both. In the o&ae of Euolidean distances we have, we believe (Maanaughton-Smith et al., 1964; Williams et al., 1964), turned this sensitivity to sale to advantage, though at the expense of ending the analysis with a disjointspace model. There tend to be two schools of thought concerning principal components, those who leave the variances unohanged and those who standardize them all to unity; one advantage of such standardization is that it renders the variates genuinely dimensionless. It would be equally permissible to standardize the variates by “importance” measuros as in the case of our scaled distances; this might conceivably increase the power of the method, but it haa never been tried. In factor analysis it is usual to rescale the factors to unit variance after their extraction. The position of the origin is a far mom intractable problem, since in highly heterogeneous data there is no obvious “best” place for its location. It is traditional to take the decision appropriate to the multivariate normal distribution and locate the origin at the common mean; we ourselves have no better solution to offer. U. HETEROOENRITY

1. Introduction If material is presented for claesification, it must be suspected of being heterogeneous in aome way. In the context of our model, this heterogeneity may take two forms. In the h t , all attributes may be meaningful for, and measurable on, all individuals; but attributes, either singly or in linked groups, may be markedly polymodal. I n the

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A-epaoe model, the points form discrete galaxies (we d h o w the possibility of non-galactic heterogeneity below). In the aeoond, $he attributes may, again either singly or in groups, beoome &em.If only the zero or non-zero nature is at stake (qualitative dab) them ia no difficulty; the diffloulty arisee when measurable attributee am sometimes zero. For the ooncept of “zero” embraces two quite dietinot concepts-that whioh happens to be zero and that which must be zero: the number of haira on the third pair of legs be zero in an inseot, but it muat be zero in man. In many cases the pattern of zeros and nonzeros is itself of primary importance.

2. The data-Ch88&3 Since data may be homogeneous, or heterogeneous in either or both of two ways, we find it convenient to distinguish fom classes of data. C h 8 1. &-ordinates measurable on all axes; no sub-populations everywhere zero on sub-sets of axes; dietributions substantially unimodal on all axes. Claas 2. Co-ordinates measurable on all axes ;no sub-populations everywhere zero on sub-sets of ams; polymodal on at least some axes, the points forming galaxies in the A-space. Class 3. Qualitative data, the oo-ordinatestaking only the values 0 or 1; sub-populations exist which are everywhere zero on eub-set8 of ares. C h 8 4. &-ordinates measurable on all axes; sub-populations exht which are everywhere zero on sub-sets of axes; diatributionson the non-zero axea may be polymodal.

CLaSs 1, of courae, approaches the multivariate norma1 distribution, and is of no interest in classificatory problems. CIaSs 2 data is the raw material of taxonomy, so long as the individuale are known to be oloeely similar. Bince random sampling of individuals of widely disparate nature would be pointless for taxonomic purposes, this requirement is normally fulfilled. Class 3 is the familiar “presence-or-absend’ data of the ecologist ;perhaps because of the modesty of ita mathematical demands, it has been extensively studied by “biological” biometrioians. Moreover, owing to the rebtive ease with which such data can be analysed, it is frequently generated from Class 4 data by dichotomizing the variatee. It has, however, been pointed out to us by MaonaughtonSmith (in Zitl.) that this raisee a new difficulty. In ecology the 1/0 situation is truly asymmetrical, in that only the preeencee carry useful information, but this is not true in sociology; if 1 is taken to repremnt drunkenness, 0 represents sobriety, and both am meaningful. It

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remains only to say that Class 4 data are normal in sociology, and in ecology if a measured attribute (such &B peroentage cover) is d.

3. Trampotition of data-chases The data-classes are not necessarily invariant under trctnspodtion. Classes 1 and 2 may become inter-converted under A / I tranepoaition; so may Clasees 9 and 4, with certain limitations. The nature of the

data may thua appear to change markedly when transposed. We believe that thia is one c a w of the prevailing uncertainty regarding B/Q differenma : an R/Q difference is inherently likely to be greater if it dso involves an A / I difFerenoe.

4. Noda The term “nodum” was apparently introduced by Poore (1966) in the context of phytosociology; its numerical implications have so far been examined only in the cam of monothetio olassifioations of Clase 8 data (Williams and Lambert, 19618; Lambert and William,1962). This concept is, however, most easily illuatrated in Class 2. Consider points in a 3-spac13,disposed within two elliptioal cylindera whose long axes are parallel to the Z-axis. The projection on the (X,Y)-plane will show two sharply-definedgalaxies; projections on the other two planee will show no strikingly galactic structure, and may not even separate the two cylindem. A nodum, in our dehition, is an enumeration both of a set of points and of the set of &xesin which the pointa constitute a galaxy or “cluster”. In Class 3 data, a nodum consists of an enumeration of a set of individnala and of a set of attributes for which they are substantially all non-zero. Noda may be regarded as foci around which the population is varying; they are potentially of great value as a basia for sheddingperipheral information. Unfortunately, no general method of extracting them ie yet known. We now incline to the view that the solution provided by Willi~msand Lambert (1961a) is open to objeotiona; it is in any oase applicable only to monothetic situatione. One of UB (Dale, 1904) has carried out a preliminary inveatigation into the oharaoteriaation and combinatorid properties of noda, but the problem is a6 yeb far from solution. The d%culty is more fundamental than may appear at firet sight, The existing method involves aetting up the fwo modela (n pointa in p-spaoe and p pointa in n-space) and cohting the reaulte; but the mnoept of a nodum &B “central” infomtion intrinsically requiree that the individuals and attributes be manipulated simulteneously. This is impossible 80 long &B either ia regarded &B a set of pointa in a apace defined by GO-ordinate axes of the other. Deepite our advocaoy

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of a Euolidean model, and despite its inoontesbble power, the searoh for nodd teobniquea may yet force us to abasdon spatial models and seek methods of maximhing some funotion of a da.b-matrix whioh ehall be symmetrioal ES regards rows and oolumne.

5. N o n - g ~ t i Cheterogewity In Class 2 data, the pinla need not olustm into galexiee; they might, for example, be dispereed along intatwined filaments, or on the surfam of oonoentrio sphem. We me not aware that m y suoh data have been reported (except, of oourse, in eoologioal situations where pattern on the ground is at issue); but if it were to be suspeoted a psrtiouhr stirategy of analysis is indimtecl (vide Beotion V D I(%) below).

V. STRATEGY OF ANALYSIS A. BIIIIPLIFIUATION METHODS

By “simplifiwtion” we intand Borne means of r e d u h g the dimensions of the original Euolidean model, so that the data (ULIL be dhphyed in a small number of dimemions with the minimum loss of information. The p r o m may fl any of three quite distinot funotions, though these are seldom olwrly distinguished in the literature. 1. 8ubjeetive clmaa$cation of c o m ( p b data A taxonomist may legitimatelynot wish to invoke objeotive numerid methode, preferring for some specifio purpose to utilize his own knowledge and experienoe to delimit taxonomioally intrautable m~terial. The data may nevertheless be speoified by too m y attributes for the iaxonomist fo handle oonfidently; the requirement is to find transkmations of the original attributm whioh o m be graphed in two or three dimeneions. Prinaipal oomponent analysis ia oommonly uded for this purpose, but ie intrimically liable to prodm a dilemma. If fhe clispereion matrix is d, the data is in no way dbtorbd; but if any of the attributes have appmaiably higher variance th&nthe remainder, these attributes will dominate the analysis, 80 providing information which oould have been more simply obtained by univariate inapeofion. If, on the other hand, the oorrehtion matrix ie usedit normally isthe data being o b i f i e d is not the original data. The Hotelling solution oommonly given in test-booksinvolves two suodaeeivesh-tions to unit vtwhnce-kt of the attributes, then ot the oompnenband so atill further distorte the originel dab. Feotor analysis h w oomsionally been u88d for the =me purpose, but the elementa being so olsaified are further removed again from t h

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W . T. WILLIAMS A N D Y. B . D A L B

specified by the original data. However, the cme reported by Pettett (1960)~of a population of Viola spp. whioh showed marked disoontinuity on the first factor but not on the fist component, iS potentially of great interest, and would merit further investigation. Relatively crude approximations to such methods exist in the literature ; the method of Curtis (1969), for example, can be regarded aa an approximation to a component analysis with the origin outside the population, at the point of intersection of the tangent-planes perpendicular to the axes of the hyper-ellipsoid. The coefficient used is the quantitative counterpart of 2a/(2a+b +c) and is thus non-metric. Such methods were unavoidable while computing facilities were limited ;now that fast programmes exist for the calculation of large dispersion or correlation matrices, and for the extraction of their roots and vectors, there is little point even in such approximations as the centroid solution.

2. Preliminary evaluation of data A non-probablistic approach in practice necessarily assumes that there ie heterogeneity to be found ;but it may well be desired to explore the general configuration of this heterogeneity, whether or no it is everywhere sharply-dehed, and whether or no it is galactio. Unlws the data is exceptionally aomplex, the first two or three principal components will normally provide the information required.

3. Generation of “underlyingfactor” hypothesw It may be desired to erect hypotheses more far-reaching than those [Section I1 E I] which are purely classificatory; such hypothesee normally take the form of postulating the existence of a small number of underlying “factors” which would be sufficient to generate the interrelationships within the entire numerical system under study. It is natural to explore the simplest possible hypotheses-i.e. those that can be contained in the smallest number of postulated factors-with due regard to Kendall’s (1967) warning that “this seems to w u m e on Nature’s part a much more indulgent behaviour than we have my right to expect”. If classical methods are in use-aa distinct from those maximum likelihood methods (Lawley and Maxwell, 1963) which do not require rotation-the appropriate solution is iterated communalities, double standardization and rotation to simple structure. Rotation normally aims at providing the simplest possible relationship between old and new axes; but it may instead be required to seek the simplest possible relationship between individuals and new axes-the solutione are not necessarily identical. In cases of extreme heterogeneity, Dale (1964) has shown that there may be no factor-analytic solution : there may be no real values of the communalitiw which will substantially

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reduce the order of the matrix, and the oentroid iteration of communalities may fail to oonverge, These methods are ourrently out of favour, probably as a result of the inoautioua olaizps whioh have in the past been made for them. They do not “rev8&1” or “demonstrate” any struoture in the data, and we deprecatethe tendency to “identify” the factors which are extracted. If they me regarded purely ae hypothesis-generatingsystems fheir use is unexoeptionable, and they are potentially of great power. B. PARTITION

Attributes may be suoh that not only are they present or absent (and the pattern of presences or absences important) but they may also, if pment, be meaeurable. Buch situations are more oommon then is usually realized. In partioular, the data of plant eoology are of this type if a mwure suoh as percentage cover is in use. They arise in pure taxonomy if, for given t p of specimens (suchas herbarium speoimens), some attributes oamot be observed; and they arise in sociology if parts of a questionnaire are not answered. Essentially, the data is of Class 4, and the primary need is to ascertain whether the major heterogeneity is qualitative or quantitative. We have suggested elsewhere (Williams and Dale, 1962) a method by which this may be effected, but the computation is heavy and no computer programme exists at present. The method consists essentially of a partition into qualitative and quantitative elements; it can be extended without M c u l t y to the threefold system (known/unknown): (if known, presentlabsent): (if known and present, then measured). We incline to the opinion that Class 4 data should normally be partitioned-i.e. eeparated into Class 2 and clese 3 elements-before numerical analysis; but the methods of subsequent analysis will require modification from their normal forms, and no investigation of this kind has yet been attempted. C. NON-MERAROHIUAL METHODS

The most familiar non-hierarchical method is that whioh u888 canonical variates (h, 1952) for the comparison of ~ O U P Sof individuals. Recent examples are mainly zoological, though the method haa been wed successfullyon Populus, Betulcc and UZmu.9 spp. by J.M.R. Jeffers (personal oommuuioation)andhiscobborators. Like all methods relate3 to the Mahahnobis D*strttistic,it is not applicable to i n d i v i d d or to groups not known a pimi to be sufficiently aimilar to sham a common within-group dispersion matrix, and its defailed aoneideration is therefore oufside the scope of this article.

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Component analysis and factor analysis me non-hierarohiwl, but are normally made the basis of subjeotive daasification: completely objeotive non-hierarohioal methob, suoh as the “multi-dimemdona1 group analysis” under development by R.Janoey (pereon4oopmunication), seem to be extremely rare. We have a3ready pointed out (8eotion I1 D) that hierarchical ohwilloations are oommonly regarded aa deairable by the users,and it ia preambly for this reason that they dominate the literature. Despite their intrinsio theoretioal intereet, we inoline to the View that non-hiermchicalmethods are of limited value in numerical taxonomy. A n exception should perhaps be made for the method assooiated with Tanimoto : but this, though non-hierarohical, is closely related to oertain hierarchical systems, and it will be more convenient to defer its consideration to the section which follows. D. EXERARUHIOAL METHODS

1. General CmLeiderations

i. Pairs Hierarchical methods are oompletely dominated by the concept of all possible pairs of points or of axes. There is no difficulty in conceiving methods bamd on all possible triangles or tetrahedra of points, or all possible solid angles. We know of no work of this type. It would involve far more computation than do the pair-system, and until it is oertak.1 that a11 possible power has been extracted from such systems, it is doubtful whether more oomplex methods are worth pursuing.

ii. Direction The analysis m y either begin with the entire population and progressively break it down (divisive methods), or begin with the individuals and progressively fuse them (aggIomerative methods). The relative idvantages and disadvantagee are best disonssed in connexion with speoific systems; it is only necessary here to point out that, if a hiemrohical classification is required, monothetio methods cannot (except in a trivial sense)be agglomerative ;after the fbt groups have been formed there may be no attribut6 by whioh they ortn be fused. iii. Sorti?q The problem here may be reduced to that of defhing a distance between a point and a set of points, and is parficuhrly relevant to the agglomerative methode. Three methods are in u88. In the first, the distance is defined as that between the point and the neeseet member of the set (“neareat-neighbow” sorting). 8inoe this u888 only 8 small

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part of the available information concerning the set, the method is normally regarded as lacking in power. It is, however, computationdy very economical, requiring the calculation of @(a-1) diabnoes, and it is the only form of sorting which will eluoidate non-gahotio heferogeneity. In the second method, the distance is defined &B the average of all the distttnces between the point and the individual membera of the set. It requires the ca.louhtion of (1c-1)~dietanoes or averagea of distances, but demands complex sorting procedures to use the oalouhtions economically. It is nevertheless the only method whioh has regard to set density. h t l y , the set may be represented by the ooordinates of its centroid. This also requires (a-l)a oaloulations, but the computational strategy is very simple; it is our opinion that the simplicity and elegance of strategy that this method allows conclusively justifiee its use. 2. Momthetic eub-cEivieiue: “iwaociationa d y & ” For detailed accounts of the uae of the method, see Williams and Lance (1968), Willi~msand Lambert (1969, 1960, 198lb). It uses derived-structure maximization; zajkis calculated between every pair of attributes j and k (in terms of the number of individuals possessing

or lacking them singly or jointly) and the B U ~ .%bis formed of all k#J the 2 9 whioh involve a particular attribute j.Sub-division is on the attribute for which

$jxa,k ie maximum.Sincefor the 2 x 2 case%a = Nra,

’

. T ~ , ~in ; this form the method ie kZ3 possibly applicable to quantitatively-specified data (vide Dele, 1964, for a method of subdivision on a quantitative variable) but no work of this type has yet been undertaken. The original investigations in

the parameter may be regarded as

fact used Ir,&laa sub-didion-parameter ;but private oommunicationa k#j from H. Shin (usingamultiple-regressionmodel)and from P. MacnaughtonSmith (using an information-theory model) have independently demonstrated that Zra is the efficient parameter if the greateat reduotion of residual variance is required. Lawley (aZit&) haa pointed out that, aa originally suggested, Z(rJmay be regarded as a crude approximation to a factor analysis (using averoid communalifies), and thue may perhaps be treated aa a monothetic approximation to an essentially polythetic system. Empirical trials on ecological d ~ t ah v e suggested it is less sensitive that Zlrl has in fact cercain advantages:in p&i&r to the presence of “outlying” individuals, whose innate similarities it may preeel.ve. The more effioientBY*tend8 to split off outl*g indivi-

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W . T . WILLIAM8 A N D M. B . D A L E

duals as single-membered sets, thus hgmenting the analysis. Further comparative tests on different types of data are desirable. The method has now been used in variety of contexts and appears robust in that it is not unduly sensitive to occasional errom in transcription of data. It is, however, extremely sensitive to “nuisance correlations” as defined in Section I1 C 4 ; because of the large contributions that such correlations can make to Zr*, they are intrinsically liable to dominate the analysis. The largest individual x k has been u a d as a memure of “rank” for each successive sub-division, and Williams and Lambert (1900) give reasons for not using more sophisticated parameters. The measure is nevertheless unsatisfactory, since it does not neoessarily fall with the hierarchy; this is particularly troublesome at the lower levels of subdivision. We have some reason to believe that

kz-;xajk

would be a better

measure, and we propose to subject this possibilityto empirical test.

3. Polythetic ccggherative: “aimihrity” amty8ea Most of the published accounts of such methods use parameters which we consider unsatisfactory for reasons given in Section I11 B, often combined with inevitable but relatively inefficient hand-sorting; these methods need no critical examination. We shall also exclude information statistics and the Goodall probablistic coefficient, since no fully developed methods are yet in use. With these provisos, there are currently only three genuinely distinct methods, associated respectivelywith Sneath, with Tanimoto and with ourselves. We consider these in turn.

i. Sneuth: unweighted methods References: Sneath (1967); Sneath and Cowan (1968); Sokal tLnd Sneath (1904). The earlier work used the quaeimetria aoeffioient a/(a+b+c), though in his more recent writing heath, like ourselves, inclinea towards the fully metric (a+d)/(a+b+c+d); the earlier work also used nearest-neighbour sorting as a computational convenience, though here also Sneath concedes the greater power of group-sorting techniques when computational facilities are available. The important feature of his methods is the strict adherence to the Adaneonian postulate that, unless there is some special rewon for so doing, all attributes are equal and should not be weighted. A ditIioulty immediately arises if only few attributes are available or if many of the attributes are lacked or possessed by nearly ad the individuals: the intrinsic information content per individual is 60 low that it is impossible to specify the “best” fusion at any stage. Sneath has always made it

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66

clear that his methods are not applicable to such data, and stresees that the number of attributes used should not be less than about 40. Despite the undoubted sucoesses that his methods have achieved with suitable data, we believe that this limitation ia a severe and undesirable restriction on the wide application of the method. It is clear that this restriction can be overcome if further information can be imported into the system at the individual level, which will necessarily involve some form of weighting. On the assumption that a piori importance measures are undesirable, the only remaining s o m of information ia contained in such a posteriori measures as can be obtained from the population as a whole. The remaining two methods offer Werent solutions to this problem. ii. Tanimto: weighted individmla Tanimoto (1968); Rogers and Tanimoto (1960). This method in fact uses a quasimetric coefficient, but this is not important in the present context. The coefficients are summed for all individuals, thus providing an a po.deriori importance measure for each individual; the individuals with the highest values are used as “apices” for beginning the aggomerative process. Unfortunately, the existing sorting process is non-hierarohical and involves decisions on the part of the operator, and, aa Sokal and Sneath (1964) point out, the increasing tendency to separate operator from computer renders “steered” programmes undesirable. h p i t e the early successes of the method, its sorting strategy requires revision ; if its concept of information-importing can be combined with the use of a fully-metric coefficient and a mechanioal (and preferably hierarchical) sorting system, the method, already of great intrinsio interest, might be a widely applicable strategy of coneiderable power.

iii. W i l l i a m et al. : weighted attributa Williams, Dale and Macnaughton-Smith (1964). This method ~ 8 8 8 Euclidean distances in an A-space with axes permanently scaled by the a pogteriori importance measure of Association Analysia, i.e. k+j& z Its succeeeful classification of a 6-attribute population demonstrates that it is free from the attribute limitations of Sneath’s unweighted method. Its chief demerit is the use of invariant weighta: the a n a l p h is necessarily dominated by what may loosely be regarded aa “firstfactor” relationships. 4. Polythetic divi8ive Several.authors (wide, e.g., Regcigno and Maccaccaro, 1960; C o c h n P

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and Hopkins, 1961;Macnaughton-Smithetal., 1964) have considered the general problem of finding what is in some sense the beat of all p s i b l e alternative sub-divisions; but the only practical method known fo US is that of Edwards (1963). This calculates between/within partitions of Euclidean distances for all possible sub-diviaiona into two p u p s . Since there are 2n-1-1 such sub-divisions, the method is neoesedy limited to a small number of individuals; and shoe the distanaes unweighted, some diffidties due to ambiguity may be expot6d at low levels of diviaion. If the number of individuals is to be increaeed to reahtic proportions, some form of “directed search’, is inevitable. The Macnaughton-Smith et al. “dissimilarityanalysis” finds,in accordanoe with a stated criterion, the individual least representative of the pophtion aa a whole; this individual is then used aa the basis for a sub-population, and the remaining individuals allocated sequentially to this sub-population or to the remainder of the population. The “objectively-weighted Euclidean distance” of Section V D 3(iii)above has been used as criterion in the preliminary trials. The method has two advantages. Firat, the computation required, though still considerable, is considerably less than iS required for an ‘MI possible sub-divisions” method. Secondly, the weights for the axes can be recalculated for each successive sub-division, thus removing the “first-fact~r~’ dependence of the corresponding agglomerative method. The present criterion has the disadvantage of defining a disjoint-space model. 5. Genera2 conclwione If a monothetic classification is desired, association analysis is olearly indicated; if monothetic clasRification is acceptable, and if p 4 IZ (as is often the case), the computation required is less than for othor methods, and association analysis is again indicated. If a polythetic classification is essential, a sub-divisive method which will provide the major discontinuities at the beginning of analysis is obvioualy preferable; we can only say that “dissimilarity analysis” ahowe considerable promise, though further development and experience is necessary before it can be uIuc888Nedly recommended. It is in the agglomerative field that we find ourselves most at vazianoe with current practice. We believe that the completely unweighted methods lack power, and are only suitable where very sharply defined heterogeneities exist ; we suspect that the “cloudy clusters’7 stigmatizsd in a recent h l i b discussion (1962, p. 268) as “a criticism not of the method, but of the material” may yet be found to be due to uaing a. method of insufEcient power. Furthermore, we oonaider that weights calculated internally from the data contravene only the letter, and not

FUNDAMENTAL PROBLEMS IN NUMERIOAL T A X O N O M Y

07

the spirit, of the Adansonian postulatea: Admaon oould hardly have foreseen the possibility of internal weighting. It will not have esoaped notice that we inoline towcmls the u88 of methods in whose developments we have o d v m been oonoerned. This is inevitable, sinoe had we not been dissatisfied with exiating methods we should not have been led to devise our own. It does not follow that we are right :when all the programmes am freely available, the mre will decide.

h K N 0WLEDQMENTS

We are indebted to many mathematicians and statisticians for their patient assistanoe, among whom we pmtioularly wish to aokuowledge the help of M i . P. Maonaughton-Smith, Dr. F. Rhodes and Mrs. N. Wilson;but these must not be held responsible for any heretical views we may have expressed. We are also greatly indebted to Dr.P. H. A. Sneath for enabling the senior author to read Lsokal and Sneath’s book in proof. The arfiole inoorporates material arising from work &d out by one of UB (M.B.D.)during the tenure of a D.S.I.R. Studentahip.

REPBIRENUES h h i b e l d , E. E.A. (1948). Ann. Bot. N.S. IS, 221. ABLIB Conference on Cleseification (1962). AaZib Proo. 14, 216. Bartlett, Y. 8. (1960). B.J.P. SWkt. 8, 77. Beckner. M. (1969). “The Biologid WSY of Thought”. Columbia Universify Prees, New York. Bonuer, R E. (1984). I.B.M. J . 8,22. Cattell, R.B. (1962). “Factor Andpis”. Harper & Bros., New York. Cochran, G.and Hopfrine, C. E. (1981). BiometriOe 17, 10. Curtis,J.T.(1969). “The Vegetationof Wisconsin”.University of Wisconsin Press. Dagnelie, P. (1980). Bull. Hem. Can%phytogeogr. B 6, 7. Dale, M. B. (1964). Ph.D. The&, University of Southampton. Edwards, A. W. F. (1903). 5th I d . Cwj. BiomStrh (Cambridge, 1963). Elderton, W. P.(1938). ‘‘FrequencyCurvee and Correlation”, 3rd ed. Cambridge. cfiknow, J. 8. L. (1961). Nature, Loncl. 168. 400. Oihow, J. 8. L.end HaIop-Befiieon, J. (1964). U e n d a 87, 147. aoodsll, D. W.(1@638).A d . J . Bot. I,89. Goodall, D.W. (1963b). Auet. J . Bot. I,434. ooodall,D.W. (1982). Ebot. B 4; 10. Goodall, D. W.’(1984).N&re, M .203. 1098. Uoodmsn, L.A. and Krusksl, W. H.(1964).J . Amer. aldiat. A@#.40,732. Goodman, L.A. and h k d , W. H.(1969). J . A m r . slabist. AM. 04,128. Om&-Smith, P. (1964). “QwmtitmtivePlant Ecology”. Butterworthe, Landon. Wberd, D. J. (1962). J . E d . 60, 1.

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Kelley, J. L. (1966).“General Topology”. Van N o s t w d , New York. Kendall, M. G. (1967).“A Course in Multivariate Analyaid’. G m , London. Lambert, J. M. and Dale, M. B. (1964). “Advences in Ecologictll Reeeeroh,” Vol. 2. Academic Preas, London. Lambert, J. M. and Williams,W. T. (1962).J. EwZ. 80, 776. Lawley, D. N. and Maxwell, E. A. (1963). “Fsctor A d y s i a 88 6 Btetisticsl Method”. Butterworth, London. Lawrence, G. H. M. (1961). “Taxonomy of Vaeculer Plants”. Mmmillsn, New York. Macnaughton-Smith, P. (1963).Biornetrka 19,364. Mmnaughton-Smith, P., Williams, W. T., Dale, M. B. and Mockett, L. 0. ( 1964). Nuhre, Lond. Needham, R. M. (1962).In “Information Processing 1962”,p. 284.North Holland Publishing Co. Amsterdam. Needham, R. M. and Jones, K. 8. (1964).J . Document. 20,6. Nelder, J. A. (1961).B i m & 17, 89. Pettett, A. (1960). Ph.D. Thesis, Univcrsity of Southampton. Phillip, J. P. N. (1963).Nature, Lond. 200, 1347. Poore, M. E.D. (1966).J. Ewl. 48,226, 246, 606. Proctor, J. R. and Kendrick, W. B. (1903).Ntbtwe, Lond. 187, 716. R m , C. R. (1962).“Advanced Statistical Methods in Biometric h e a r c h . ’ ’ W h y , New York. Rescigno, A. and Meooaccaro, G. A. (1960). The Information Content of Biological ClaesScations. I n “Symposium on Information Theory”. Butterworth, London. Richards, F. J. (1969).J . ezp. Bot. 10, 290. Rogers, D.J. and Tanimoto, T. T. (1960).Science 182, 1116. Rohlf, F.J. (1962).Ph.D. Thesis, University of Kanses. Rohlf, F. J. and Sokal, R. R. (1963).Kana. Univ. 815.BuU. Sebestyen, G. 8. (1962).‘‘Daision-making Processea in Pattern h o g n i t i o n ” . Mmmillan, New York. P q c h t r i k a 27, 126. Shepard, R. N. (1962~). Shepesd, R.N. (1962b).P8ychomelrika. 27, 219. Sneath, P.H.A. (1967).J . g m . Microfiol. 17,201. Sneath, P.H.A. (1962).Symp. SOC.gen. Microbiol. 12, 283. Sneath, P.H.A. and Cowan, S. T. (1969).J. gem Mii7robiOZ. 19,651. Sneath, P.H.A. and Sokal, R. R. (1962).Nature, Land. 198, 865. Sokal, R.R. and Sneath, P. H. A. (1964). “Numerical Taxonomy”. W.H.Freeman, San Francisco and London. Tanimoto, T. T. (1968).“An Elementary Matherrmtical Theory of Claurifioclti4n end Prediction”. I.B.M.Corp., New Ymk. Williams, W. T. and Dale, M. B. (1962).Nahre, Lmcd. 198, 602. William, W. T. and Lambert,J. M. (1969).J . E d , 47, 83. Williams, W. T. and Lambert, J. M. (1960).J. E d . 48,689. Williams, W. T. and Lambert, J. M. (1961a).Nature, Lond. 191,202. William, W. T. and Lambert, J. M. (1961b). J . Ecol. 49,717. Williems, W. T. and Lance, G. N. (1968).Nature, h n d . 182, 1766. William, W. T., Dale, M. B. and Maonaughton-Smith, P. (1964).Nature, Laul. 201, 426.

Yule, 0. U. and Kendell, M. G. (1960). “An Introduction to the Theorg of Ststistics”. Grifh, London.

Ultrastructure of the Wall in Growing Cells and its Relation to the Direction of the Growth * P. A. ROELOFSEN

Laboratory of Gmml and Technical Biology,Tech-cal Delft, Netherlad

Univerdty,

I. Introduction ............................................................. 69 11. Morphological Aspects of Constitution, Synthesis and Breekdown of the Primary

Well .................................................................. 70 A. Constitution and Morphology of Microfibrile., ............................. 70 B. Comtitution of the Amorphoue Matrix in Primary Walls 78 C. &me Aepeots of Microfibril Cbherence .................................... 82 D. Site of Byntheeb of Primary Wall Substances. ............................. 68 E. Qnwtionable Evidence of Breakdown in Primary W& 80 III. Burvey of the Mim5brlllm Arrangement in Different Types of Qrowing Cells.. 91 A. Freely Qrowing more or less Iaodiametric Celle 91 B. Freely Qmwing Tubular Cells or Parts of Cells.. ........................... 98 C. Tieaue Cells with Iaodiemetrio Urowth ................................... .lo4 D. Tissue Cells with Predominant Growth in Length ..........................lo5 E. Tiesue Cells that Predominsntly Widen.. ................................ .112 F. T i p of "issue Cells with Tip Urowth .................................... .113 IV. hterreletion between Urowth and Wall Ultrastructure ....................... .114 A. Effeut of Omwth on Wall Structure ..................................... .114 B. Effect of Well Structure on the Direction of Urowth.. ..................... .I28 C. Theories on the Meahanism of Orient&tedInitial Synthesis of Cellulose Microfibrilel30 References .............................................................. .146

....................

..................... ... .............................

I. INTRODUOTION This review deals with the present knowledge of the submicrosmpia st;ructure of growing Geus of Thallophyta and Cormophyta and the interrelation between this and the direction of surface growth. Also morphological aspects of constitution, synthesis and breakdown of cell wall substances will be discussed. As 8 rule only literature more recent than 1969 will be mentioned. For older literature and for electron micrographic documentation of structures, the reader is referred to the monographs of Frey-Wyssling (1959) and Roelofaen (1959), and to the reviews of Setterfield and Bayley (1961), Wardrop (1962) and Northcote (1963). The review is restricted to a consideration of the primary w t l l , which is defined as the part of the wall that is produced by a cell or by its parent cell while growing in surface. The s d 9 y wall is defined as the part produced after growth has ceased and is more compact. The growing cell may sometimes, or locally, produce more wall The survey of literature p r t a i i g to this review was concluded in June 1 W .

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material than is needed to compensate for wall extension, which results in thickening. Hence thickened primary w& do exist, so that thickening cannot be adopted as a criterion for the definition of a secondary wall. However, there still remain transitions between primary and secondary wall layera as defined above, since growth may cease slowly. Then an intermediate layer may occur, called the transition layer (Uebergangslamelle), which has the more compact structure. typical of the secondary wall but shows signs of extension, such as wide spacings between microfibrillar bundles and crossed interwoven bands. Probably the low extensibility of this layer is aofually the cause of the cessation in growth. Naturally, the wall of a freely growing cell and the outer wall of an epidermal cell consist of one primary wall, whereas walls in tissues consist of two primaries. Their outermost parts are fused to form the middZe lamella, easily recognizable by its non-birefringence due to the absence of cellulose, and by microchemical reactions, etc. The partition wall between daughter cells embodies the cell plate, but in growing cell walls,e.g. in the side walls in the files of daughter cells in primary meristems, the cell plate has disappeared completely. *

11. MORPHOLOGICALASPEOTS OF

CONSTFlWTION, SYNTHESIS AND

BREAKDOWN OF TEE PRIMARY W u

All growing plant cell walls consist of a f r a m m k of microjibrile of either cellulose or chitin or, more rarely, another plysaccharide, embedded in a continuow matrix of usually amorphous and highly swollen substances. The microfibrils are necessary to bear the stress in the wall due to turgor pressure. This has been extensively treated by Frey-Wyssling (1952,1969, p. 306). For instance in wheat mots with cells of 16 p diameter and turgor pressures of 3 atm, wall stresses of 30-60 kg/cmz occur. I n a V u h i u cell of 10 mm diameter at only 1 atm, however, it is a formidable 20,000 kg/cm*, since the stress is proportional to the diameter. Therefore the wall stress in ba&& is at the most 1 kg/cm,. This permits the absence of any fibrihr component in bacterial walls ; with those species which do produce cellulose, this arises in the culture medium. A. CONSTITUTION AND MORPHOLOGY OB MICROFIBRILS

7. Celluloee micro$br&Ee

In the primary walls of all Cormophyta, of all algae of which the ultrastructure will be discussed here and of the Ollrnyoetes, cellulose

U L T R A S T R U O T U R E O F THE WALL I N G R O W I N G UBILLS

71

ie the sole miorofibrillar component. Its amount varies between 20 and 30% (see below). Since i&e chemical and physical properties of oeuulose have been treated in detail in the monographs cited above (ale0 in Treiber, 1967; Ruhland, 1958; Honeymctn, 1969), it may suffim to mention here only

properties relevant from a morphological point of view.

mElem6/lary fibril'

X)x70h

Fro. 1. Diagram of the otruoture of oellulcme microfiW. A and B: generally ocoepted itrnotrve and dimensions. C: with "elementary fibrils" t~coordiagto Frey-Wpling. Now oelluloric polyas&uidea locsted d i n g to preeton (1962).

Ae is shown diagrammatically in Fig. 1, the miorofibrile are very long with respect to their diameter, at least several p, unbranched and somewhat flattened. Their width hae been estimrcted by most authors at 100-300 A, their thickness at 60-1OOA. The microfibrils consist of a bundle of parallel, equally unbranched, cellulose (&1-4 glucan) chain molecules, with lengths varying between about a half and several p. In tramverse section there are about three of these chains per square mp.

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Some two-thirds of each microfibril may be considered c r y s t a k e since it produces X-ray interferences. The crystal structure is of aa called cellulose I, which occurs only in nature and is probably the only crystalline form occurring in nature. The remaining third is less well, or not at all, ordered (no regular hydrogen bonds between adjacent chains) and is called semi- or paracrystalline. A small part of this is probably localized in very small regions throughout the crystal lattice (no crystal is 100% perfect). The main part, however, occurs firstly in short segments at intervals alon the length of the microfibrils, thus separating crystallites 300-600 long, and secondly, at the surface (see Fig. 1). Despite this crystallinity the microfibrils can make curves down to 1400 A in dirtmeter (Muhlethaler, 1960) due to their thinness and, probably, to the paracrystalline regions. Frey-Wyssling’s concept (1969, pp. 17, 114; by-Wyssling and Miihlethaler, 1963) of microfibril structure is somewhat Werent, see Fig. 1(C). He envisages “elementaryfibrils” of 35 x36 A (30 x70 Ain the original concept), each having thin paracrystalline sheaths, which would either occur freely, or be aggregated, thus forming the thicker microfibrils usually found. In our view the evidence supports the first concept. Firstly, the thickness of 36 A as determined by Miihlethaler (1960, cf. also Ohad et a,?.,1962), might, according to Colvin (1963), be a considerable underestimate. Also data from X-ray and electron diffrcldion diagrams have often permitted the conclusion that the crystallites were much thicker (e.g. Preston, 1962). I n that case, however, the fibrils would no longer be “elementary”, but just common micro5brils, and there would be little difference between the two concepts, ~ince it is commonly aocepted that microfibrils tend to aggregate into bundles. Secondly, in the range of 36-200 A all thicknesses seem to occur (cf. Giinther, 1960) and not only multiples of 36 A. The occurrence of smoothly tapering ends in growing microfibrils with a thickness of more than 100 A (see below) also seems a t variance with the concept. Further, cellulose, which is attacked chemically or enzymrttically, disintagrates in pieces of much more than 35 A thick (see, for example, Norkrans and R&nby, 1966). Since paracrystalline regions may be supposed to be primary points of attaok, crystallites of 36 A thickness are to be expected in them caws. The non-crystalline condition of the surface of the microfibrile is of great importance for their coherence. Within the crystallites all OH-groups, three in each glucose-anhydride monomer, are involved in hydrogen bonds which interconnect the cellulose chain molecules. At the surface, however, many of them become available. These may bind to the paracrystalline surfaces of other microfibrib. The great strength

x

U L T R A S T R U U T U R E O F T H E W A L L IN G R O W I N G OELLS

73

of cellulose fibres, both wet and dry, demonstrates the extent of this bonding. I n most secondary and in all primary walls, however, hemicelluloses and pectic substances are associated with the microfibril surface, as indicated in Fig. 1. Part of these are held so tenaciously that it is notoriously difficult to obtain pure cellulose from such cell walls (Adams and Bishop, 1955). These substances are more hydrophilic than cellulose; this means that more hydroxyl groups are involved in hydrogen bonds with water molecules and fewer will be available for hydrogen bonding between microfibril surfaces. Therefore, in the presence of water the microfibril coherence is reduced as compared with pure cellulose (in the dry condition coherence is increased, but this does not occur in living cell walls).

FIG. 2. Growing extrscellular cellulose microfibrils in a culture of Acelolmcter Zylinum. Note the tapering ends along about 0-6 p and the blunt points. A: from Millman and Colvh (1961); B: from Colvin and Dennis (1964).

For the biochemistry of cellulose synthesis we refer to reviews of Setterfield and Bayley (1961) and Neufeld and Hassid (1963; cf. also Elbein et al., 1964). The morphological aspects of microfibril formation have mainly been studied in young cultures of Aeebbacter xylinum, which produces cellulose extracellularly. It waa started by Miihlethaler (cf. Frey-Wyssling, 1959, p. 123) and carried on mainly by Colvin and co-workers (e.g. Colvin and Beer, 1960; MiUmaa and Colvin, 1901; Colvin, 1963, 1964a).Around clean bacteria under favourable conditione microfibrils less than 0.6 ,uin length appear within half a minute. These elongate constantly, but except at the two ends, the final diameter Qf 150-200 A, is very soon attained. The microfibrils are often twisted, and both ends taper for about 0.5 p, but not indefinitely, 90 that the very points are blunt (see Fig. 2). At 34°C the growth velocity of each microfibril is on the average 2 ,u per min, but there are faster growing ones, since after 1.5 m h microfibrils of 8 ,u are to be found. At 25OC the growth velocity is about F*

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P. A . ROELOFSEN

half of this. Whether one or both tips grow is unknown, but the similar morphology would suggest the latter. Some authors (originally Miihlethaler, and more recently Ohad el al., 1962) reported that the microfibrils arose from slimelike or granular prestages that were supposed to be, at least partly, amorphous cellulose molecules. This was denied by Colvin and co-workers (most recently Colvin, 1963) who observed the formation of microfibrils remote from amorphous materials which they considered to be excreted proteins, etc, Using labelled glucose they failed t o find the postulated intermediate cellodextrins and, moreover, demonstrated the formation of microfibrils from an excreted soluble low molecular precursor by a similarly excreted bacterial enzymatic system (Khan and Colvin, 1961 ; Colvin, 1964a). Colvin and co-workers therefore favour the hypothesis of tipbiosynthesis of the microfibrils, the addition of glucose monomers to the ends of the cellulose chains, exposed a t the tapered microfibril ends. The crystalline ordering of the chains would then follow tbe growing molecules. This hypothesis was originally proposed by the author (1968) on the following considerations. (a)Microfibril formation from preformed cellulose molecules should, just as in the production of synthetic cellulose fibres, give rise f a interconnected microfibrils with a crystal structure (cellulose 11) different from that found in nature. (b) The “unusual” tip-biosynthesis would make the formation of the unusual crystal lattice and the formation of unbranched microfibrils more comprehensible. Since enzymatic synthesis of polysaccharides is as yet known to occur only at the non-reducing molecule ends, the hypothesis suggested that the molecule ends at the growing tips would all be non-reducing ones and this, in its turn, would require that the commonly accepted crystal structure with so-called an tiparallel molecule chain orientation should be rejected and replaced by one with parallel chain orientation. The latter suggestion had in fact been proposed long ago, but had been rejected on insufficient grounds (cf. discussion in the paper by Preston, 1964).

We thought that it also implied that the microfibrils should @OW at one tip only, the non-reducing one (“reducing” on p. 188 of our monograph is an error). However, while writing this article it occumd to us that a microfibril might equally start with chain8 growing in opposite directions. While moving away from the “tail to tail” mid& part, both ends will soon become non-reducing ;at least to a large extent, since antiparallel molecules of 0.5 ,u length at most (the tapering ends) might be incorporated in the growing microfibril. (A small number of

ULTRASTRUCTURE O F THE WALL IN GROWING CELLS

78

the cellulose molecules are actually about this length, cf. Marx-Figini and Schulz, 1963.) Colvin (1964b) has determined the isotope-labelling and reducing activity of microfibril ends, and concludes that all tips are similar. As discuesed, this does not solve the parallel-antiparallel, nor the one or two enzymes problem.

Fro. 3. The inner cell wall surface of a plasmolyzed cell of C h e t o m o t p b melagmiurn. Note tapering ends of microfibrils covering microfibrils running in main direction and ends covered with files of granular bodies (from Prei and Preston, lO6la).

How cellulose microfibrils in plants are formed is unknown, but the occurrence of soluble precursors (Colvin, 1961; Webb and Colvin , 1963; Elbein et a,!., 1964) and, ae is apparent in Figs. 3 and 4, of similar tapering ends with blunt points, suggest a similar mechanism. It certainly does not suggest that in plants, microfibrils would be formed in a way any different from that in bacteria, e.g. be modelled within or along microtubules (see pp. 85, 141).Tip growth ie also suggested by the intertwining of the microfibrils in plant cell walls as e.g. the one depicted in Fig. 3. The twisted microfibrillar bundles frequently observed also suggest tip growth, but the twisting might have occurred during drying (Millman and Colvin, 1961). Finally, the striking alignment of microfibrils in many cell walls would be quite comprehensible

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on the basis of growth of tips through pre-existing interfibrillar spaces. Such template action of pre-existing microfibrils was also observed in Acetobacter cellulose formed on agar (Millman and Colvin, 1961). However, this template phenomenon, if occurring in plant cell walls, is evidentlysubordinate to some other orienting mechanism (see p. 139). Growth of microfibrils in thickness not only at the tapering ends, but all along their length, has been postulated several times (cf. Roelofsen, 1959, p. 29), but most emphatically by Gunther (1960), who in the protonema of Funaria found thicker microfibrils on the outer wall surface (as compared with the inside) and in old protonemata (as

FIG.4. Cellulose microfibrile with tapering end regions and blunt points at the edge of B perforation, formed to reloase swarmem in a cell of Chm!mwrpha melagmiurn. CrScLe in microfibrils are artefacts (from Frei and Preston. 198111).

compared with young ones). These remlts do not, in our view, prove the point, but certainly merit attention. 2. Chitin microjibrib Chitin is found in all fungi, except Obmycetes and lome exceptional cases (cf. Roelofsen, 1959, p. 40; Northcote, 1963). It doe# not occur in algae, as is still asserted in recent encyclopaedicworks and probably never occurs together with cellulose. (The presence of cellulose in the chitin containing fungys Rhizidimyces was reported by Fuller (1960) and Fuller and Barshad (1960), but this is based only on X-ray ring diagrams, which have more than once led to misinterpretation.) Chemical and physical properties of chitin are discussed in the monographs already mentioned and by Foster and Webber (1960). Recent literature on biosynthesis is mentioned by Jawomki et a,?.

ULTRASTRUCTURE

O F THE W A L L IN Q R O W I N Q CELLS

77

(19631, on crystal structure by Dweltz (1960) and Rudall (1963). From our point of view it is relevant that chitin microfibrils are usually very similar to those of cellulose in length, diameter, probable flatness, chemical resistance and crystallinity. Here too, the microfibril surface will probably be paracrystalline and will more readily bind polymers from the non-fibrillar matrix. However, in cell walls which contain small quantities of chitin, like those of many yewts, the chitin is not fibrillar but granular (Houwink and Kreger, 1953). In such walls another fibrillar component often occurs, viz. glucan (see below). Very long and rather thicker are the curious so-called “mycofibrila” discovered by Liese and Schmid (1962, 1963). They are formed extracellularly by wood-rotting fungi, both when grown on wood and on agar. However, their chemical constitution is BR yet unknown. The chitin content of fungal walls varies greatly. One yeast (Schizoeacchromyc,cee)contains no chitin, baker’s yeast only about O-lyo(Korn and Northcote, 1960), Phycomycea sporangiophores 30%, Allornycee mycelium as much as 60% (Aronson and Machlis, 1959). This applies to full-grown cell walls; growing ones have not been analysed. They probably differ greatly, since in young mycelium of Allontycae, which still would have contained non-growing walls, considerably less chitin was found than in old mycelium. 3. Micro$brils of other materiala

In Cormophyta the sole non-cellulosic microfibrillar cell wall substance known is the mannan in the endosperm of palm seeds (Meier, 1958; also Roelofsen, 1959, p. 56). These cells will not be discussed in this review since they do not grow and, moreover, have not been studied sufficiently. Furthermore in certain collenchyma cells pectin has been found to occur in the form of axially orientated crystallites. In the “pectin skeletons”, obtained after extraction of cellulose and hemicellulose, short aggregated microfibrils occurred. However, these might have arisen after the chemical treatment (Roelofsen and Kreger, 1951; Roelofsen, 1959, p. ‘72) and their original presence is questionable. In nearly all green, red and brown algae studied so far, cellulow is the sole microfibrillar component. In some green algae (Bryopie, Caulerpa, Penicillua, Udoteu, Halimeda) and in two red algae (Pmphyra and Banqia) the microfibrilsconsist of B- 1,3-linkedxylan ;while in some other green algae (Codium, Amtabularia, Dmyccla&us,Ba&pa.ura) and in the “cuticle” of Porphyra the crystalline wall component is /3-1,4linked mannan (identical with that found in palm seeds) but no microfibrils have been detected ( h iand Preston, 1961c, 1964). Furthermore in the green algae Hydrodictyon and Halicyetie the microfibrile probably consist of a partly crystallized mixture of cellulose, and mannan and

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P. A . ROELOFSEN

xylan respectively (Roelofsen, 1959, p. 228; Kreger, 1960; Northcote et al., 1960).* Until recently it was accepted that alginic acid, universally found in brown algae as well as cellulose,occurred in orientated (birefringent)but amorphous state. Recently, however, Frei and Preston (1962) showed that one component of it, polyguluronic acid, partly occurred in the native walls in orientated crystallites. As far as is known at present, however, these are not incorporated into microfibrils. The situation seems more or less comparable with that of the related pectin in collenchyma cell walls. Finally we have to mention the glucan microfibrils demonstrated in the walls of yeasts. They seem to be very short, nearly granular, at least in the untreated cell walls (Houwink and Kreger, 1953; Roelofsen, 1959 p. 304). In the chemically treated walls conspicuous aggregation occurs. There is about 30% glucan in yeast cell walls, so it certainly is an important structural component, unlike the chitin, of which often only traces occur. Yeast glucan is probably a linear polymer with both /?-1,3 and /?-l,S links; like cellulose it tenaciously associates with other substances, viz. mannan and protein (see below). It seems relevant that all these non-cellulosic and non-chitinous microfibrils differ in appearance from cellulose and chitin microfibrils. The latter are clearly individual, easily separable, somewhat stiff threads. The former appear to be more supple, are often short and seem sometimes to be interconnected. Torn edges of cell walls never show the fringe of loose microfibrils, which are typical of cellulosic walls. B. CONSTITUTION OF THE AMORPHOUS MATRIX IN PRIMARY WALL8

The matrix in which the microfibrillar framework i H embedded consists of a highly hydrated molecular network of a number of polymeric Substances, viz. polysaccharides and polyuronides, intermixed with a minor amount of proteins and lipids. Meristematic cell walls have often been analysed. Unfortunately the available analytical methods, even for separating such main groups a8 cellulose, hemicelluloses and pectic substances, lack specificity, mainly since these substances associate readily and since each group contains molecules varying greatly in molecular weight, the hemicellulosefl also varying in chemical constitution. The separation of these hemicellulose components is even more difficult. Since glucose oocurs both in cellulose and in some hemicelluloses and uronic acids occur both in the latter and in pectic substances, the analysis cannot be based upon the quantitative constitution of the hydrolysate of the whole cell wall. This * Xylan microfibrils show the exceptional feature of negative birefringence (Frei and Preston, 1%).

U L T R A S T R U C T U R E O F T H E W A L L I N Q R O W I N Q CELLS

79

unsatisfactory situation explains why the results of analyses of the same object by different investigators using different methods usually differ considerably, often more than the analyses of different objecta by the same investigator. Roelofsen (1959, pp. 2, 127) and Setterfield and Bayley (1961) gave summaries of quantitative analyses. Some more recent analyses have been compiled in Table I. Regarding these data the following pointa TABLEI Quantitative Chemical Analyaee of Growing Cell Walk Publisherl Bince 1960 (Valuearepreaent yo of total dry wall) Number of HemiPedic reference. Cellulose eelluloee aubatance Protein Lipid Lignin

Object --

h’adaonia (yeaat)

>6l

3-6

46 33 46 63 40 26 39 13 40 mixed 46 with 80

26 17 16

26

Avena coleoptile Avena coleoptilo Allium. root Pieurn, stem Acer pepl., cambium Nitella opm NiteUa tranalucena Cham auatralia Hydrodictyon afric.

10

4

30 30 29 37 17

9

2.8

<30 26 28

4

+

4 11 4

7

-

8

11

9

*Reference numbers (1) Bishop et al. (1968),pectic substances corrected according to King and Bayley (1063).(2) Ray (1062).(3) CRlCdRtedfkOm Jeneen and Ashton (1060). (4) Matchett and Nance (1982). (6) Thornber and Northcote (1961, 1062). (6) Probine and Preston (1081). (7) Anderson and King (1961). (8) Northcote el d.(1960). (9) Dyke (1984).

should be noticed. The corrected pectin content of Avenu ooleoptiles is more in line with the earlier results of Alberaheim and Bonner (1969) and of Jansen et al. (1960); and with the recent results of Ray and Rottenberg (1964), who found 4% galacturonic acid incorporated in pectin and 2% glucuronic acid in xylan-hemicelluloses. The finding of lignin in cambium and its high cellulose content indicates the admixture of secondary walls. This probably applies even more to the walls of Nitella translucens and Chara; the “lignin” in these cases must have been some other substance. I n these algae the pectic substances would be wholly westerified, which the authors relate to their auxin insensitivity. In Hydrodictyon the hemicellulose is mannan and pectin is less than 6% in amount. The hemicellulose of Nadeonia contains equel quantities of glucose and mannose, probably occurring aa microfibrillar

80

P . A . ROELOFSEN

glucan and amorphous mannan, as in other yeasts. Chitin in this caae is at most 0.4y0. If both recent and older tmalyses are considered and any extreme values are neglected, it can be stated that, usually, primary walls of Cormophyta contain 20-30% cellulose, 3 5 5 0 % hemicelluloses, 10-20y0 pectin substances, 3-10% proteins and 2-7% lipids. Avena coleoptiles are exceptional in having very low pectin and very high hemicellulose contents. Collenchyma walls are exceptional in containing more than 40% pectin. In recent years a number of investigations were concerned with the changes in wall components during growth and induced by auxin (Galston and Purves, 1960; Setterfield and Bayley, 1961 ; Thimann, 1963; Ray, 1962; Probine and Preston, 1962; King and Bayley, 1963). In some objects an increme of hot water soluble substances, especially pectic substances, and an increase of methylation of the pectin, was found; however, in others it was not. Other cell-wall components did not change significantly in amount and constitution ; neither did the amount and the distribution of calcium. This does not, however, disprove the eventual role of these substances in plasticization of the wall for growth, since the physical properties of polymers may be profoundly changed by slight, so far undetected, chemical changes. Since the amorphous matrix is involved in the coherence of the microfibrils, the chemical constitution may be briefly mentioned. The hemicelldoses are a mixture of polysaccharides, some of which contain glucuronic acid. They may involve straight chain homopolymers, e.g. mannan, galactan, glucan, xylan and araban, or these may bear other monomers, usually in short side-chains but sometimes also in the main chain, so that heteropolymers arise, e .g. glucomannan, galactomaman, arabinoxylan and arabinogalactan. Glucuronic acid or its methylester may be attached to some heteropolymers as side chains, e.g. glucuronoxylan and arabinoglucuronoxylan. In marine algae sugar sulphates are usually incorporated in the hemicelluloses. Each of these polymers varies greatly in degree of polymerization (DP) and hence in solubility. On the average the DP is 100-300, corresponding to a maximum length of 500-1500 A, which is short as compared with t h e microfibril dimensions. The pectic substances of the Cormophyta are straight chain polymers of galacturonic acid and of its methylester. The solubility in neutral water is less in fractions with higher DP, lower methyl and higher calcium content. For chemical details of the amorphous polysaccharides the reader is referred to the reviews of Aspinal (1959, 1962), Kreger (1962, algae

U L T R A S T R U U T U R E O F THE W A L L IN G R O W I N G UELLS

81

only) and Northcote (1963,algae and fungi), and for papers on the biosynthesis to Setterfield and Bayley (1961)and Neufeld and H W (1963). Special attention has been given in recent years to cell wall proteh, whose presence has now been conclusively proved (see Roelofaen, 1969, p. 77; Newoomb, 1963; Dyke, 1964). The embodiment of proteins in walls also follows from the occurrence in the wall of enzymes (Newcomb, 1963). In some cases the enzymes were shown to be present only in cell wall fractions isolated from tissue homogenatee. Since adsorption of proteins certainly occurs, the evidence in such cmes can never, in our opinion, be conclusive. More convincing is the demonstration of the occurrence of metabolic turnover within the cell wall, e,g. by Matchett and Nance (1962).Also the secretion of enxymes into the culture medium by both tissue cultures and intact plant roots (e.g. Constabel, 1963; Chang and Bandurski, 1964)involves the presence of proteins, at least temporarily, in the wall. With yeasts and other fungi, many exocelluhr enzymes and enzymes attached to the wall have, of course, been known for a long time. Possibly significant with regard to cell wall plasticization is a cell wall protein, probably non-enzymatic, that wm found in the cell walls of both plant tissue cultures and, to a lesser estent, normally grown plants. Its discovery was due to an extraordinary high content of the unusual amino acid hydroxyproline (Lyndon and Steward, 1963; Newcomb, 1963;Steward and Chang, 1963).It has been supposed that this protein is involved in the control of growth by forming labile cross linkages between the cellulose (Brown, 1963;Lamport, 1963,see p.212 of this book); also, that it would cement the cells together in the middle lamella (Ginzburg, 1961).The latter supposition does not c o r n pond with the possibility that the cells of many tissues can be separated with substances that do not react with proteins, like calcium binding agents (Letham, 1962)and purified pectinase preparations (Zaitlin and Coltrin, 1964). The protein of yeaat cell wall was also studied recently (Nickereon et! al., 1961; Northcote, 1962; Dyke, 1964). With careful extraction glucan-protein and glucan-mannan-protein complexes are obtained. Glucosrcmine would act as a binding agent in them. The protein dnot contain hydroxyproline. Although its cystein costent is small it would, according to Nickerson and Falcone (1956),be involved in the strengthening of the coherence of the glucan microfibrils in the cell wall by forming disulpbide bridges. Breaking these bonds by a reducing enzymrttic system would weaken the wall fabric in the s p t where a bud is to be formed. Brown (1963)has postulated that in higher plants the breaking of protein disulphide bonds is also involved in growth.

82

P. A . R O E L O F S E N 11. SOME ASPEOTS OF MIUROFZBRIL OOXERENOB

Growth is the yielding of the wall to the stress generated by the turgor pressure. The stress is born by the microfibrils and by the coherence between these. The coherence will be highest between microfibrils that are in direct contact. It will probably decrease sharply with an increase of the interfibrillar distance shoe it then becomes dependent on the strength of the bonding of hemicelluloses, pectic substances rtnd possibly proteins. Since the molecular chain length of hemicelluloeee, for example, varies between 600 and 1500& smallinterfibrillar distanms will require the participation of two of these to establish one link, and this number would increase proportionately with distance. The binding of hemicelluloses to the paracrystalline microfibril surface is ratber strong, as is seen by the dWcdty in purifying oellulose, but in all probability the points of contact between two hemicellulose moleoules in the amorphous interfibrillar material are too limited to establish strong links. In electron micrographs the “cellulose skeletons” obtained after extraction of most other substances look very compact, but this is due both to shrinkage as a result of extraction and desiccation, and to a comparativelygreat depth of focus in the electron microscope. In order to estimate the true volume percentage of oelluloee in the untreated wall, one has to take into consideration the highly hydrated condition of the intersbrillar substances as compared with cellulose. In 1892 Cohn found 61-72% water in collenchyma. Corn coleoptile walls contain about 60% water (Roelofsen, 1969, p. 127, the water content of 90%, often quoted, is without any experimental basis). In contact with water, pure cellulose contains only about 16% water, which is wholly restricted to the paracrystalline regions. Accepting a total water content of 60% and 30% cellulose on dry weight, the interfibrillar substances would contain about 67% watm and would oompy about 90% of the total volume. The cellulose would occupy about lo%, which implies that the mean distance between microfibril s u r f a c ~ ~ would be about four times their diameter, that ie 600-1000 A. This is an average of the whole wall; the middle lamelb does not contain cellulose. The outer parts of the primary wall contain much less than the inner ones, as is apparent from electron micrographs of cell wall sections (cf. Setterfield and Bayley, 1968). If the innem& layer would contain 76% cellulose on a dry weight basis, this would occupy about half of the volume. The mean intedibrillar diatmm would be less than their diameter; there would be considertcble direot contact, with many interfibrillar links involving only one mole& of hemicellulose. In the middle and outer layers many more weaker linka,

ULTRASTRUUTURE O F T H E W A L L IN GROWING UELLS

83

involving more than one molecule per link, will occur (see Fig. 5). Besides the amount of amorphous substances, the degree of alignment of the microfibrils naturally will also be very important with regard to their coherence. A network of microfibrils orientated at random will, like a piece of felt, be equally extensible in all directions. With two crossed microfibrillar directions the network will, like textile fabric, be least extensible in these directions and most extensible (more than a random network) in the directions bisecting the angles between the microfibrillar directions. Finally, a wall with more or less parallel microfibrils only will extend with difficulty parallel to these and with

Meon dislonce lW&-------

Cellulose content of: 75 1.%, 50 vol. %

------30w.'L,K)vDI.%

------15w.%,5wl.%

Fra. 6. Diagram of the role of interfibrillary eubatances in the coherence of mimofibrilr, at varying callulose oontent.

w e at right angles. We will see that all these possibilities are actually realized in growing walls. The only bonds possible betwean polysaccharides are hydrogen bonds between two hydroxyl groups (I)or between one hydroxyl and the 0-atom of either pyrmose or furanose-rings or of the glycosidic bridge (11). In chitin there might, in addition, exist hydrogen bonde between two NH-CO-groups of the acetylglucosamine monomers (111) or involving one of these. The carboxyl groups of uronides or of uronic acids in hemialldosea may bind hydroxyl groups, both when ionized (IV) or non-ionized (V). Relatively strong double bonds may occur between two non-ionized

P. A . R O E L O F S E N

84

(- )OOC

Hb

\

CH,

\N-CH,-CH,-N

/\

/\

CH,

\

W++)

/ \

COO(-)

(-)OOC

(VW

\

/ C

/

bH

,’

p

I

I (VIII)

carboxyl groups (VI), but only in an acid medium. Since proteins have both peptide-groups and several kinds of H-donating and H-accepting groups in side chains, they may form complexeswith all polysaccharides. Not only protons (H-ions) are attracted by the free electron pairs of electronegative atoms like 0 and N, but also metal-atoms and metalions that may incorporate such pairs in the outer electron shell. Many metals can take up four, six or even more of such electron pa&. In this way metal-complexes, called chelates, may be formed with molecules having two or more electron-donating atoms. Calcium and magnesium, the main metals in plant cell walls, are known to decrease the plasticity of primary cell walls and it has been supposed that they are involved in the growth-promoting action of auxin. This would induce the production of some chelating agent that would bind the atoms that previously connected two uronic acid groups in pectic substances or in acid hemicelluloses (see BurstrUm, 1963). Usually the possibility of the binding of acid groups only ia considered. It may be remembered, however, that by binding an anionio group like carboxyl, the a f i i t y of the metal for hydroxyl oxygen or for nitrogen increases, as is demowtrated by the Ca-EDTA complex (VII), the complexing of calcium by lactic acid, gluconic acid, etc. Therefore it is likely that in cell walls calcium and magnesium a t o m form metal bridges not only between uronic acid or otheranionicpups,

UL T RAS T RU C T U R E O F THE W A L L I N G R O W I N G CELLS

I

86

but also between these and hydroxyl groups of polysacoharidee, including cellulose (VIII). The co-ordinative binding properties of many other bivalent and trivalent metals, e.g. copper, zinc, aluminium, iron and silver are much greater. These may bind OH-groups in a natural medium without the help of carboxyl groups. Belford e l al. (1959) showed that heavy metals are taken up in a more or less crystalline arrangement on the surface of cellulose fibres in amounts that parallel their content of xyhn. Of these metals, however, only traces are found in natural cell walls. D. SITE OF SYNTHESIS OF PRIMARY W A L L SUBSTANCES

It will appear below that cellulose microfibrils are, in all probability, synthesized partly between plasmalemma and the inner wall surface (strict apposition) and partly within the wall veryneartheinnersurfbce. Considered submicroscopically the latter process is intussusception, but considered on a microscopic scale it still is apposition. In the following discussion we will use the term apposition in the latter sense except where specifically stated otherwise. Amorphous cell wall polysaccharides are probably synthesized outside the plasmalemma. In at least one instance ( N i W ) this process occurs, as in the caw of cellulose, at or near the inner wall surface, but in higher phnta probably throughout the growing wall. The grounds leading to these conclusions are as follows. (a) If cellulose microfibrils were synthesized in the cytoplasm, free microfibrils should have been observed regularly in the electron micrographs of section through growing cells as for instance those of Bayley and Setterfield and co-workers (cf. Roelofsen, 1959, pp. 142, 150, leO), which is not the cam. Moreover, the strict apposition this would mply does not correspond with the considerable interwining of the microfibrils, nor with the covering of microfibrile that very probably were the youngest ones, see Fig. 3. Thia Ruggests that microfibrils are formed on the spot by tip growth as in the formation of bacterial cellulose. This interwining comprises several microfibrih. Therefore a thickness of say 600 A of the innermost part of the wall must be able to synthesize them throughout (Frei and Preston, 19618; Preston, 1964), at least, in this particular case. In yeaet, intimate contact between glucan microfibrils and particles at the outer surface of the plasmalemma has been reported (Moor and Miihlethaler, 1963). The microtubles discovered recently by Ledbetter and Porter (1963) near growing walls and by Hepler and Newcomb (1964) near secondaq thickenings probably do not synthesize cellulose, but are related only to the orientation of the growth of cellulose microfibrils w i t h the

86

P. A . ROELOFSEN

wall. One of the electron micrographs of the first-mentioned paper is reproduced in Fig. 28. This point will be discussed in Section IV C. (b) The molecules of both cellulose and other wall plysaccharides are insoluble, so that they cannot diffuse. Thus, if they would be synthesized endoplasmatically, this would imply strict apposition, unless (i) there would either occur numerow tiny cytoplasmh protrusions into the wall, or (ii) there would occur outward flow of the interfibdar substances through the pores of the microfibrillar network. Possibility (i) must be rejected. Electron microscopy has revealed a distinct, continuous plasmalemma, which only enfers the wall at plasmodesmata (of. Buvat, 1963; Newcomb, 1963). The tiny threada between wall and plasmolyf ed protoplast, originally described by Hecht, always correspond with plasmodesmata (Sitte, 1963). Also the plasmic “invaginations”, reported by Frey-Wyssling (1962) and celled plasmic papillae, are, in our view, curved entrances of plasmodeernrcta. They were demonstrated only in oblique sections of very young, shrunken walls, which explains their abnormal shape. Similar Str’UCtUreS occur in the micrographs of, for example, Strugger (1967), who apparently also considered them to be plasmodesmata. Plrtsmodesrrmfa are numerous, but nevertheless there are far too few to explain wall growth by intussusception. Moreover, they are grouped in a limited number of primary pit fields, which are not centres of growth but “centres of stagnancy”, as will appear later (p. 117). The wall grows between these (cf. Roelofsen, 1969 p. 133). Finally there are growing walls without any plasmodesmata, e.g. those of tips showing intrusive growth, of fungi, and of green algae. Possibility (ii)seem very unlikely, since there is no pressure gradient normal to the wall (for consideration of tangential gradients see p. 116 and Fig. 18). Consequently, cytophmic synthesis implies strict apposition. However, as will appear below, the btter ie unlikely and, henoe, exophmatic synthesie is more probable. The considerable activity of cytoplasmic organelles like the endoplasmic reticulum, the dictyosomes and their vesicles, probably pertaim to the production and the transport to the walI of precursore of cell wall substances, of co-enzymes and of eynthmizing enzymes, needed for synthesis outside the plasmalemma. (c) In Acelobacter xylinum, ceilulose microfibrils arise extracellularly. Cellulose molecules naturally are too insoluble to pass the wall and in fact enzymatic synthesis in the culture medium, from an excrefed soluble precursor and from a similar precursor extraoted from green Plants, been reported (see p. 74). This does not prove anything

ULTRASTRUCTURE O F T H E WALL IN G R O W I N G CELLS

87

about the site of synthesis in higher plants, but is at least consistent with exoplasmatic synthesis. (d) The investigations of Ordin et al. (1967) and Jansen et a2. (1960) on pectin methylation, of Margerie and €'6aud-Lenoel(I 961) on cellulose and of Matchett and Nance (1962) on cellulose, hemicellulose and pectic substances, indicate metabolic turnover of these cell wall substances. This clearly suggests synthesis outside the plasmalemma, although in our view it is not strictly proved. There might be breakdown of existing wall substances by hydrolysing enzymes, accompanied by apposition of new wall substances that has been synthesized in the cytoplasm. (e) Setterfield and Bayley (1959) followed the site of cell wall synthesis during growth by means of autoradiographs of sections of Avenu coleoptile segments that had been incubated in tritium-labelled sucrose for several hours. Labelled cell wall material appeared not only

FIG.6. Autoradiographs of transverse sertions of the epiderml wall of Avena coleoptiles, grown for 21 h on tritiated sucrose. A : unextracted wall, containing 76"/n non-cellulorce.

B: wall extracted with acid and alkali, containing about 40% non-cellulose (from h t t e d e l d and Bayley, 1969). 1

at and in the inner part of the wall, but also elsewhere throughout the depth of the thick outer epidermal walls. This is apparent in Fig. 6A. Figure 6 B is of a section that was treated to remove mogt of the noncellulose, but still contained 40%. Since the non-cellulose molecules cannot diffuse into the wall and probably do not flow through it either, synthesis everywhere within the wall is clearly indicated. This does not, in our view, apply to cellulose since in the section of Fig. 6B a large amount of non-cellulosic material was present. Moreover Bahmer (1958) concluded from autoradiographs of parenchyma cells of coleoptiles, subjected to a similar treatment, that there wm no cellulose synthesis within the wall rJince the edge thickeningfi (see Fig. 14) lagged behind in labelling (as also Heen in the autoradiographn published by Setterfield and Bayley, 1957, 1959). (f) Green (1968a) exposed Nitella cells for short periods to tritiumcontaining water and, by comparing the radio-activity on the outer

88

P. A . ROELOFSEN

and the inner sides of the cleaned walls, showed that new wall material, apparently both cellulose and non-cellulose, must, have been added to the wall at or very near the inner surface. This observation is consistent with both apposition of materials synthesized in the cytoplasm and synthesis immediately outside the plasmalemma. Evidently, the site of synthesiH of at least non-cellulosic material is different in Nitella as compared with the Avena coleoptile. (9) Both the so-called multinet structure found in tubular c e b (Figs. 12 and 14)involving a difference in microfibrillar orientation between inner and outer lamellae in crossed polylamellate walls (Figs. 10, 13 and 15), and the peeling off of outer lamellae in many algae, are only comprehensible on the basis of the apposition of the microfibrils, either in the strict sense or in the sense of being formed both on and within the wall near the inner surface. See Section IV A 5. ( h ) On the other hand, the retention of the thicknew of the middle lamella during growth ie only comprehcneihle on the haUiH of either outward migration of its constituents through the interfibrillar spaces, which Beems unlikely, or synthetiis in or near the middle lamella. This is discussed on p. 117. (i) If intuwusception of cellulose microfibrils were to occur at a greater distance from the inner surface, then one would expect the occurrence of cellulose in the middle lamella of all tissue cells and the non-existence of walls with separate cellulose lamellae. What would occur if cellulose microfibrils were to be formed throughout the wall is demonstrated by the completely interwoven structure of the cellulose films of Acetobacter xylinum. A microfibril in this felt-like structure cannot be ascribed to one particular bacterium, whereas in plant tissues, for each microfibril a cell can be indicated to which it belongs. It should be mentioned here that Bayley et al. (1957), studying the outer epidermal wall of the Avena coleoptile, and Beer and Setterfield (1958), studying collenchyma cells, concluded that intumusception of cellulose within these thick polylamellate walls seemed likely on certain morphological grounds. Their conclusion regarding the epidermal wall wan, however, criticized by Bolliger (1969) and in our view the origin of the structure of collenchyma cells is more readily comprehensible without accepting intussusception (see p. 111). The previouN discussion mainly concerned polysaccharide~.Chitin microfibrils are probably also formed at or near the inner wall nurfam, but thiH is based only on the multinet structure of Y h y m y c e s sporangiophores. Wall proteins are presumably synthenized in the cytoplasm, nince the ribosomes and RNA needed do not, as far as is known, occur in cell walls. The hydroxyproline, however, seems to be formed by oxidation

ULTRASTRUCTURE O F THE W A L L IN G R O W I N G CELLS

89

of proline that was incorporated in wall protein (Lamport, 1963). Nothing is known of the site of synthesis of wall lipids. E. QUESTIONABLE EVIDENCE OF BREAKDOWN IN PRIMARY WALLS

Evidently, full-grown walls may occasionally be dissolved physiologically, e.g. in cross walls of wood vessels, lysigenous oil cavities, the flesh of ripening berries, endosperm of seeds, eta. (see Kiister, 1966, pp. 139, 726; Frey-Wyssling, 1969, p. 77). Recently Wartenberg (1963) reported cell wall attack in starving epidermal cells. To our knowledge, none of these cases have been studied electron-optically, so that we do not know the morphological changes of the cellulose microfibrils. When attacked by fungal cellulase they disintegrate into pieces 300 A long and 150 A thick (Meier, 1965; Norkrans and Ranby, 1966). We do not know these changes in chitin microfibrils either. The diHsolution of the partition wall in zygotes of P h y m y c a was studied by SaRsen (1962), but the morphology of the attack of the chitin microfibrils it!jnot revealed. Breakdown of cell wall substance in primary walls, going beyond the plasticization of the amorphous interfibrillar substances, has, in our view, not been proven to exist. Until a few years ago it was often considered that in meristematic cells plasmodesmata aroee by dissolution of at least the amorphous matrix and by either a pushing aside, or else a dissolution, of the microfibrils (see e.g. Frey-Wysslhg, 1959, p. 68). However, in the partition walls between daughter cells, cytoplasmic connections have now been shown to persist during the formation of the cell plate, thus giving rise to the plasmodesmata (of. Buvat, 1963). Also in the side walls of apical meristem cells, for example, plasmodesmata very probably do not arise by dissolution of cell wall substance. Up to 1967 they were either thought to be absent in the youngest stages, or to exist as invisibly thin plasmic threads (Meeuse, 1957). Later they were also found here (Strugger, 1957; Frey-Wyssling, 1962; other literature in Krull, 1960; Newcomb, 1963; Buvat, 1963). As will appear later (p. 117) they ure formed it1 the dividing parent cell cell by the cleaving of existing plltsmodcHrrit~Lti that were widened as a result of wall exteneion. One case of dissolution of wall material in the formation of I ~ u H ~ ~ ( J desmata is evident, viz. of the callow platelet8 at both sides of the pores in the sieve plates of growing sieve vcfisel elements (Esau et al., 1962). Cellulose and other wall mdwtanoeN, originally prerjcnt at thcnc pore sites, need not have been dissolved (see p. 120). Two cases of perforation of growing walh of lower plants huw: ttccn studied electron-optically. Firstly, the perforation of ChcLetorwy~hu cells for the release of swarmers. According to Frei and Prefiton (196la) the cytoplasm protrudes into the wall by pufihing microfibrils anide and

90

P . A . ROELOFSEN

apparently by digestion of the matrix substanoes and the last few wall larnellae only are burst through. Meanwhile the deposition of wall lamellae goes on. but there are two differences with normal lamelhe. Firstly, the microfibrils are oriented at random, as compared with the strictly parallel arrangement of normal IameUaa (Fig. 13). Secondly, they end at the perforation, as is clearly demonstsated in the electron micrograph of Fig. 4. Note the tapering ends of about 0.6 p in length similar to those found with growing microfibrils, both of C - p b (Fig. 3) and of Acetobacter (Fig. 2). This indicates that they were being synthesized. If they were being attacked, disintegration in pieces would probably occur. (The ruptures visible in the miorofibrils in Fig. 4 are not signs of' breakdown. According to Preston, they are arbifacts caused by the electron beam.) Secondly, we may mention the perforation of the wall of yeaeta in the formation of buds. It has been mentioned already (p. 81) that, at the site where the bud is to be formed, the wall would (according to Nickerson and Falcone, 1960) be plasticized by a reducing enzymatic system. The wall yields abruptly and a naked protoplasmic sphere ie extruded. According to the authors the glucan microfibrils are displaced into a circular rim at the base of the sphere. The latter fonns a wd, expands and is thrown off after a separating wall htw been laid down. The circular r i m s remain visible like a bud scar in the parent cell and a birth scar in the bud, see Fig. 9. If the perforation of a growing wall is to be considered the result of an extreme local extension identical in principle with growth, and if even this does not aeem to require true breakdown of cell wall substances, then it seem unlikely that normal growth would do so. cells may in fact elongate so fast that there can be no question of ohemical change; for instance, when the cellulose parts of the cells of Tr&cizntia stamina1 hairs attain a threefold length in half a minute, or when stamina1 filaments of grasses elongate sixfold in several minufee (eee p. 114).A1so the fact that the chemical compoeition of a rapidly growing wall often does not differ from that of a dower growing one tiow not s ~ p p ~the r t idea of chemical change. It does not disprove it either, for synthesis might just compensate breakdown in both slow and rapidly growing walk Matchett and Nance (1962) demonstrated metabolic turnover of 0eU wall substances, including cellulose, by a decrease of radioactivity during growth. This wm stimulated by auxin. It waa interpreti3d as indicating increased rates of breakdown and of resynthesis of wall components. However, in our view, this still does not prove the occurrence of true breakdown, since it was not demonstrated that the turnover could not be ascribed to mere exchange of monomers between

ULTRASTRUOTURE OF T H E W A L L I N GROWING U ELLS

91

the cell-wall substances and their precursors for synthesis, oatdysed, of course, by the synthesizing enzymes present in the wall. Enzymetic synthesizing reactions often are reversible. Hydrolytio reactions are w a rule practically irreVereible and if it were demonstrated, therefore, that e.g. c e l l h e , chithaw, h e m i d u lases Or pectinme were present and active in the well, breakdown would have to be accepted. As far a~ we know, of all these enzymes only c e l l b h w been demonstrated in growing tissues (Ruhland, 1968; Gascoigne and Gaacoigne, 1960; Maclachlan and Perrault, 1964). However, whether it occurs in the cell wall and is aotive them, is unknown. Mrtclachlan and.Young (1962) postulated the breakdown of cell wall in growing pea stems, but the evidence 1388108 quesfionable. Moreover, in view of the slownees of the attack of insoluble celluloee by known cellulases it seems hard to understand a degradation of microfibrils that is rapid enough to be effective in plasticization of primary walls. Also, disintegrated microfibrils have not been observed. A limited depolymerization or side-chain removal of matrix materiala aeems more likely as the basis of plasticization.

111. SURVBY OF THE MIUROFIBRILWLR ARMGEXENT IN DIFFERENT TYPESOF GROWINGCELLS In our monograph (Roelofsen, 1959,pp. 134-160,273-305)the microfibrillar structure of growing cells of lower and higher plants has been described in detail and documented with electron miorographs. Here we will try to summarize this knowledge by giving schematic drawings of the structural types known so far. Since many lower plants and mamy types of tissue cells of higher plants have not been studied, or not sufficiently, this review will necessarily be very inwmplefe. In 80 fiGr as the diagrams are baaed on recent papers, these will be cited; for older papers, our monograph might be consulted. Freely growing cslls and tissue cells are treated separately since the former, not being infiuenoed by adjacent cells, are structurally simpler. Miarofibrils alwaye lie mom or less parallel to the plane of the w d . With random or isotropic orientation there is an abeenoe of preference for any direction within this plane. It will be undersfood further that e.g. “random layer”, “orientated layer”, etc., are corruptionsof: “ b p r being constituted of microfibrile orientated at random”, an #‘parallel in a certain direction”, respectively. A. FREELY GROWING MORE OR LESS ISODIAMBTBIU OELLB

This group includes yeasts, some algae and young stegm of other algae and fungi. There are four structural types. Type lA, Fig. 7, is the most simple type conceivable. There a m (3

92

P . A . ROELOFSEN

A

FXQ. 7. Structural type 1 of isndiametric cells. A: more or I ~ k lE y growingecelisof algae and fungi; B: t h e cells.

randomly orientated microfibrils all around the cell end through the whole depth of the wall. Probably the meah Will be wider at the outer surface as a result of extension combined with apposition, but thie has not been observed. The outer surface is covered by e cuticls-lh amorphous granular material that disappears on treatment with alkali. In some cases the cells are truly free, in other cams they are fixed at one or both ends. Probably the walls of a great many Unicellular algae and fungal stages have this microfibrillar structure, but only very few have been studied, viz. Chlorelh pyremidosa (Northcote and Godding, 1958), the youngest sporeling stage of some algae ( V h h , Dictgmphiwia, Chaedmwrpha, Cludupha, 8pongvnwrpb and A m W M , me Nicalai and Preston, 1959) and young stages of the fungi Wwp&&m (&onson and Preston, 196Ob) and A w c m (Aronson and Preston, 19604. In C h h e l h , r 5 m m p h and A c r o e i p h h pmumably young and old cell w a b have the same structure notwithstendingthat the twolatter ones elongate (see p. 101). In the other algae, however, radically different lamellar appear very soon (types 4 and 7) but if they are plaamolymi, the retracted protoplasts again produce this efrudmal type of wall (Frei and Preston, 1962a). In Hydrodictym ufricunwn, young cells are tubular (w p. NO),but older cells become more and more spherical and, mrreaponding)y, acquire the isotropic wall atructure of t p 1A (Breen, 1963b).

U L T R A S T R U C T U R E OR T H E W A L L I N G R O W I N G C E L L S

93

Sometimes the structures of different parts of one cell belong to different types. In young P h y m p + ssporangiophores, the tip is blown up to a spherical SporCLngium with, again, ieotropio structure, whereas the growth-zone below it had and will keep quit6 a different strudmm (seep. 100).

/Ifit

-

l@A

Birefringence in Holicystis

Fro. 8. 8tructural type 2 ie a8 lA, but lamellab. Found in Ha&&, induced globoid celle of Nit&.

red algae, colchioin-

Type 2, Fig. 8 is similar to type IA, but there am many isotropio lamellae separated by amorphous material. This is typioal of all spherical or barrel-shaped cells of fhmentoue and also thalloid red algae (Myers and Preston, l969b;Dawes d al., 1961),of which in particular Uri~thaia$oeculoea(Hyers et d.,1966), Rhim?ymenia palmata (Myers and Preston, 1959s)and € € e b n i & M caZifomica (Dawes et al., 1961)have been studied. 8trictly, the c d b of thrtlloid red algae should not be mentioned here but in &&ion IIC. Apparently, the growth in tissue ha8 no significant struotural consequences,except that pits &re present (see Myers et d., 1959). Experimentrtlly this structure was obtained with internodal cells of Nitella (otherwisetype 6) by cultivation in water with 0.35~0colohicide (Green,1962, 196%). The cells of Halicyeti8 ovalie are probably built in this way, but ody the outer and the inner surfaces have been observed (Roelofeen, 1959, p. 282).These walle differ in being built up of three polyhmellatelayers, of which the middle one ia exceptional in exhibiting negative double refiaotion, see Fig. 8. Except for cell walls containing wax, this eeemed to ooour in one other case only, viz. in CauZerp (see e.g. Dippel, 1898, p. 240).Its cause was obscure, but now it is evident that it is due to the negative birefringence of xylan microfibrib, as WLW found and expleined recently by Frei and Preston (1964), see also Bryopk etc., p. 77.

94

P . A . ROELOBSEN

Type 3 (Fig. 9) represents the wqll structure of non-myceliumforming yeasts (literature cf. Roelofsen, 1969, p. 304; Hagedorn, 1964; MacClary, 1964). In chemically untreated cell walls, the short randomly orientated, possibly interconnected glucan microfibrils, are visible only on the inside of the wall. The outer side is covered by a grmuhr, lipid-rich material that may be removed by treatment with alkali. The wall is usU&uytwo-layered, the inner one being rich in gluoan, the outer in mannan. Each cell has one birth scar (always at one of the ends of the egg-shaped cells) and a varying number (up to about 100) of bud scars. As far as known, young cells without bud scars and fullgrown cells with bud scars have the same cell wall structure.

6 t h scar

Fro. 9. Structural type 3 is

as l A , but with birth scar8 and

mycelium-forming yeasts.

bud

BCU~.

repreeenting uou.

In the scars a concentric orientation of microfibrib ia Vieible, especially in the rims; in the centre is a little dent. Such a ~otlrlike structure was also observed in hyphas of the fungus PoZy&a!w (eee p. 98). The perforation of the wall prior to budding haa been discussed on p. 90. Type 4, Fig. 10, represents the big (up to 4 cm in diameter) coenocytic (multinuclear) cells of several Valonia s p e c k and the cells (about 0.5 mm) constituting the thallus of D i d y m p M famhaa. Very probably a similar structure occurs in iaodiametrio cells of related genera. There is a gradual transition to tubular wlb with the wme crosswise polylamehte walls described under type 7, Fig. 13. The diagram is based on literature mentioned in our monograph (1959, p. 274). The aplanospore has a cellulose-free wall with a, granular ~ d a 0 8 . The finst cellulose lamella deposited on its inside is felt of randomly arranged microfibrils embedded in an amorphous matrix. This stage was mentioned under type 1, p. 92. Unlike waUs of some other algae

U L T R A S T R U O T U R E O F THE W A L L IN OROWING CELLS

96

Amorphous wall aplanospore

I

Successive larnellae deposited with increasing parollelism

Lamelloe 4,7,10 etc., less dense, sometimes missing

[

Effect of growth on successive lomelloe

FIG.10. Btructurd type 4, found in eella of V a h h spec. div., tbllue mlla of LXc@qhu&a and related gonera.

and of higher plants, this cannot extend very much, 80 that it is torn soon by the extension of the cell. Before this occurs, a lamella of amorphous material followed by a second cellulose lamella (2 in Fig. 10) has been deposited, again on the inside. In the latter the cellulose ie more or less orientated in a flat left-handed (S)helix around the cell. This direction is marked A in Fig. 10. Pollowing this, amorphoue material end then lamella No. 3 is deposited. Its direction, B, is more or less along the meridians from the base of the cell converging to s

96

P . A . ROELOFSEN

“pole” at or near the top. The sharp angle a between A and B varied between 67” and 89” in one cell that waa studied in detail. On to the inner side of lamella No. 3, amorphow material and then lamella No. 4 is (at least may be, see below) deposited, b v h g microfibrillar direction X. Thk intersocte the obtuse angle between A and B and hence is in a right-handed (Z) helix around the cell, also oonverging at the pole. Then again a lamella with direction A is deposited md 80 on ABX ABX, etc. The sets A and B are alwaye well developed and always present, but the X-lamella are thin and sometimes they may even be missing, so that the sequence may be an irregular one, e.g. A B A B X, etc. The degree of regularity varies in different s p e c b and even in cells of the same species. Also the helix angles vary considerably. In growing and full-grown cells there are 20-40 cellulose lamellae present. Each is about 0.4 p thick. The first couple of lamelbe a m laid down with an increasing d e b e of fibrillar alignment. AU are separated by thin lamellae of amorphous hemicellulosicand polyuronide material, so that they can be separated easily after mild maceration. As a result of more or less isodiametric externion of the cell, lamella No. 7 is torn soon to isolated patches that are thrown off. The orientated lamellae are also torn, but initially only along lines parallel to their main orientation, so that broad fissures arise (see Fig. 10).Later, teare normal to these also appear so that pieces are formed that peel off. In Dictyo8phueGa there is a very regular three-lamella repeat and all lamellae are equally developed. Taken strictly, thew cells am not freely growing and should be mentioned in Section 111E. It ehould be noted that no orientated streaming of protoplasm could be observed in the living cells mentioned in this section. ’

B. FREELY UROWlNU TUBULAR CELLS OR PARTS OF UEUB

A tubular cell may arise as a result of either tip growth, or growth in the aide wall only, or both. 1.

Tubular celle with tipgrowth only

Pollen tubes and root hairs of land plants certainly grow in this way and probably also hyphae of fungi, rhizoids of aIgae and fungi and the protonema of mosses. Recent papers confirming this are of &rmack et al. (1963, root hair), Rosen e.t al. (1964, p l l e n tube) and 8trnnk (1983, hyphae). For other literature we refer to our monograph (1969 p. 188) and that by Frey-Wysshg (1969, p. 83). The structure of the tip may, as shown diagrammatically in Fig. 11 under type 6, vary in detail. After a cutiole-like amorphous maa has been removed, the outer surface usually shows randomly orientated microfibrilcl (X), but sometimes more or less a d y orientated ones (Y).

U L T R A S T R U C T U R E OF T H E W A L L IN OROWING UIELLS

87

The inner side of the tip has in no case been observed electron-optioally but, as judged from the birefringence of the whole wall, the microfibrils here are usuaJly also orientated isotropically (A), sometimeg, however, transversely (B) Since the tubular part does not grow, the outer surface is the same as at the tip, either X or Y. Underneath, the secondary thickening

.

& A : FUIIY isotropic or

Cuticle

1’ May be lomallole

Fro. 11. Btructural type 6, found in tip of frcely growing w l l ~or of per& of cell8 and of tieaue wlle with intrusive growth.

is often visible, better, of course, when observed from the inner side. Its orientation is usually axial, sometimes croeswiae or transverse. In exceptional cmes no secondary thickening is deposited. We will now discuss the structure of the various objects as far aa it has been studied. Concerning root hairs of land plants some recent publications have been added to the ones mentioned in our monograph (1969, pp. 140,160) :Belford and Preston (1961, on 8imyie),Dawes and Bowler (1969, on Raphanue) and Scott el al. (1963a, on Raphanw, Bramica, Triticum and Cucurbita). In nearly a11 oakw a combination of the structures X with either A or B wag found in the tip. In R a p h w one author found type Y,others X. Different shapes of the tip may explain this (me p. 127). The root hair of Tritznea bogotenai8 wa8 studied by Roelofaen and %lorn6 (1980). Here again the combination XB waa found. Bince the random structure is retained in the tubular part, it can be upp posed that, as with land plants, this part does not grow. The inner surfiwe, presumably a secondary thickening, is irregular but predominently transverse in orientation, which is exceptional. AB is well known, the protoplasm streams lengthwise. The protonema of Funaria hygrometrica wm studied electron-optically by Gunther (1960). The combination XA waa found both in the tip

98

P. A . ROELOFSEN

and in the tubular part. Extension here is presumably by tip growth only, though Giinther herself does not report about the site of growth. Pollen tubes have, as far as is known (cf. Roelofsen, 1969, p. 168; Sassen, 1964),also the combination XA. This may occur in the tubular part, but also type 6A (p. 100).Probably,themisnosecondarythickening. Tips of fungal hyphae show at their outer surface a random orientation (Roelofsen 1969, Fig. P210; Aronson and Preston, 1960a,b; Strunk, 1963). In some tips of hyphae of PoEystidue, Strunk (1983) found a concentric structure with a pore-like centre, that would be centre of growth. These structures also occurred in aide walls. They look like bud scars in yeasts and this raises the question whether they are scars of thrown-off branches and whether tips showing it were actually growing. Rhizoids of Alhkycm have structure Y at their tips (Aronson and Preston, 1960a).Structure XA was also found in the tips of rhizoids of sporelings of Chaetmorphct and Chdophora and, as already mentioned on p. 89, in the bulges formed in full-grown oeh prior to the perfor&tion of these spots for the release of Bwarmera. Probably isotropic structure also occurs in the hemispherical apical cell of Nitella, but this is only deduced from its isotropy between crossed nicols (Green, 1968b). The red algae present a problem in that we do not know if the tubular cells occurring in many species have been formed by tip growth only, or if there also was elongation in side walls. All wall lamelhe were reported to be isotropic (Myers and Preston, 1969a,b; Dawea et d., 1961). If we accept this (the micrographs published by the h t mentioned authors do show orientation), it suggests restriction of the growth to the tip of the apical cells, since pasaive axial reorientation would presumably occur if the sidewalls would elongate (see Section I V A 5). Lack of knowledge concerning the localization of growth also hampers classification of the structure of the green algae A C T W $ ~ ~ U and Spongmpha, which is like that of the red algae (Nimbi and Preston, 1959). They are therefore also proviBionally classified under tip growth. Also unknown is the eitc of growth in elongated celh of brown aka%. According to Dawes et al. (1961)(for older literature cf. Roelofsen, 1969, p. 297) a thin outer layer is isotropic, the rest of the wall is axially oriented. Perhaps the latter is “secondary wall”. 2 . Tubular cells growing in sidewalls only Tbere are two structural types in these cells, depending on the initkl orientation of the microfibrils deposited at the inside.

U L T R A S T R U U T U R E O F THM W A L L IN Q R O W I N G O l L l i S

90

(a) New microfibrile &po&ted with t m w e ~ s eOtientatiOn. AU freely growing tubular cells that grow in the tubular part and in which new microfibrils are deposited in transverse orientation have the structural type 6. It is visible in the electron micrograph of Pig. 23 and shown schematically in Fig. 12.

Cu tide

- ---May

tM:

or intermediate

Intermediate

May be lomellale

(N/!c//u)

Fro. 12. Structural type 6, called multinet ntructcvo. k”ount1 iu all frwly

growing

oelb

that elongate in lltteral walle cmd in which the initial cclluloso orientation is cunetantly traneverse.

On the inner wall surface the structure is comparatively compact and the microfibrils are orientated more or lees transvemely (if there is spiral growth, the mean orientation is according to a flat helix, me p. 132). After removing the cuticle, either a very widely meshed mimfibrillar network R i visible, with the microfibrils running more or lee8

100

P. A . ROELOFSP1N

lengthwise (A) or a less widely meshed network with microfibrils running in all directions (B), or a structure transitional between them. The intermediate part of the wall always ahows a gradml transition between the inner and the outer surface. I n some oases regulasly crossed structures as in C appear as a transitioml stage, or on the inner surface when the elongation ends, aee p. 128. This is known as multinet structure (Roelofsen and Houwink, 1963) (although the walls represented by Figs. 10, 13 and 16 would be more entitled to that name). This term was given since the wall may be imagined to consist of a number of nets with varying wideness of mesh and varying orientation of microfibrils. Usually, however, there are no separate nets, but one three-dimensional network. As will be disoussed in Section IV A 5 (b), the multinet structure arises aa a result of p d v e reorientation of the transversely orientated microfibrils deposited at the inner surface (see Fig. 23). Free cells of higher plants that grow in this way and that have been cltudied electronoptically are restricted to two, viz. the ceb of the stamina1 hairs of Tradeecuntia virginica and the arms of the stellate aerenchyma cells in the medulla of young leaves of Jumw eJu8ua (of. Roelofsen, 1969,p. 146-149). In Juncwr (Rg. 22)the outer surface waa according to type A ; in Tradacantia, type B waa seen on very young cells, and type A on full-grown cells that also had extended through endosmosis (see p. 132). Among fungal cells, the growing zone of the sporangiophom of Phywmyces blakes1eeacnu.a in the stage with sporangium belonge to thie type (cf. Roelofsen, 1959,p. 298).The outer surfaoe is aa B in the middle part of the growth zone and more like A after full extension further down. Below the growth zone (more than 2 mm below the sporangium) the axially oriented secondary wall is deposited on the inner side. Of the algae, the cells of Nitella (Green, 1968b, 1960a;Probine and Preston, 1961;Limaye et al., 1962)and 8pirogyra (Limye ei! aZ., 1962) can be mentioned. In both, the outer surface is, &B A. The Nit& wall is lamellate and the central lamellae have in fact structures intermediate between the innermost and outermost ones (Probine and %ton, 1961). Transverse orientation was also observed in ToZypelb by Fridvalgky (1968),but in To~ypdlopsisand in two species of C h a , thirn only occurred in the outer part of the wall, the inner one showing axial or steep helical orientation (Fridvaleky, 1967, 1960). Thh, in our view, suggests secondary thickening, which, &R far &B we know, waa not reported 80 far in Algae. Hydrodiclyon is provisionally classified here since the cells are also tubular, negatively birefringent and without the striations typical for the crossed lamellate walh of type 7. However, unpublished eledton

U L T R A S T R U C T U R E O F T H E WALL I N U R O W I N U (3ELLS 101

micrographs (prepared by B. J. Spit) only showed a microfibrillar reticulum with, at least in young cells, a low degree of orientation. Cross walls have only beon studied in Tradacantia and Nilella. Both have random orientation. There are many phmodesmata in the former, but they seem to be lacking in the latter caae (eee a h hiohart, 1962). As far as has been observed, protoplasm streaming in all these ceb was either more or leas lengthwise, or irregular, never transverse. (b) New microfibrib depo8ited in larnellae with al&mting helical micro$brillar miendation. When tissue celh are exoluded (see p. 104) only algal cells fall into this group, namely C w h , C w p h u and probably also the related R?&oclonium and other genera of the Siphonocladales with tubular cells. These are arranged in files, forming filaments, which are unbranched in Chaetmwrph and branched in CZudoplwra. All cells are multinucleate, and so may divide and grow. I n C w l w r a a major part of the growth is located in the tip of the apical cell, which usually falls off in Chaetmwtpha. The submicroscopic structure has been studied exclusively by Nicolai and Preston and co-workers (cf. Roelofeen, 1969, p. 284; Nicolai and Preston, 1969; Frei and Preston, 1961a,b). As mentioned on p. 94 the swarmem, after settling, first acquire a cellulose lamella with random orientation, as in F’ig. 7. Soon, however, the polarity which the cell apparently develops is also expressed in the wall. A lamella is laid down with a flat helical microfibrillar orientation and having a “pole” somewhere near the top of the cell. This stage is quite similar to that of a young sporeling of V a h i a (Wg. 10) in which only the lamellae nos. 1 and 2 are present. Then, however, the sporelings of C h a d w p h a and CWphora elongate and acquire a wall that is, at least in detaib, different. It is represented in Fig. 13 rn structural type 7. The wall is built up of cellulose-rich layers separated by thin amorphous layers. The former consist of several lamellae (total number of lamellae is several tenths). In each lamella the microfibrils are parallelly orientated in an oblique direction with respect to the cell axis, thus running in helices around the cell. As in Valania, there are always two p directions and, in some major sets A and B, in flat and s t ~ oblique species of both genera, a third direction, X, that b b o t a the o b t w angle between A and B. In most species A and X form S-helicteR; B, a Z-helix, ie as drawn in Fig. 13, but in C l a d q h a polVera A iR Z and B and X are S. Since new Iamellae are laid down on the inner surfaoe only, the outer lamellae in any cell are extended more than the inner ones. The extension is in an oblique direction, in most species Z-helical, with an average a~ is indicated in Fig. 13. This causes the cell and the whole

102

P. A . KOELOFSEN Birefringence

Rotation

\,

fe

2

.Cellulose ond

~

Amorphous layers

1

I

!I

Steepness

Outer

+4 -4 +4

Alignment-

Spacing+

+ + +

I

Middle

Inner

/

Average oricnlotion of upper growlh wall spiral of cellin

\

Y

Effects of elongation on structure of lornelloe (Outer compored with inner)

Fro. 13. Structural type 7, found in C'hdophora, C'haelomorpha and other Siphonocladales with tubular cells and alternating initial cellulose orientation.

filament to rotate around its axis and it aho cawes the clrsngm in orientation and in alignment in the cellulose lamellae &R indicated. This evidently requires some slipping past one another of adjacent lamellae. Outermost lamellae may be 80 torn that they peel off. In rapidly growing cells the A lamellae always have flatter, and the B lamellae steeper, microfibrillar orientation aa compared with slower growing cells. When growth stops in the autumn, all lamellae are similar to the outer lamellae of growing celh. By then the cells have also become more or less barrel-shaped so that they might a h be classified with ceUs of F'ulonia in type 4. During the stage of widening the initial orientation of the A lamellae becomes steeper and that of the

U L T R A S T R U C T U R E O F T H E W A L L I N Q R O W IN O C ELLS

103

B-lamellae flatter, just contraxy to the difference observed when a cell increases its growth rate. Whether this also occure in the la,mellae while being displaced outwardly, is questionable. The sign of rotation during widening has not been reported. As in V&ntizc, there is no orientated protoplasm streaming. 3. Tubular cells growing both in the tip and in sidemlla

These naturally are merely combinations of type 5 (Fig. 1 1 ) with either 6 (Fig. 12) or 7 (Fig. 13). The cotton hair shows a combination of 5 XB with 6 A. I n full-grown hairs the crosswise structure 6 C has been observed at the inner surface. The struoture of the tip is demonstrated by Figs. 24 and 26. The hairs of Ceiba pentandra and A8ckp’a.s cornudi exhibit in the tubular part, like cotton, the structural type 6 A. Their tips have not been studied ;nor has the tip of the apical cell of Traderrcantiastamina1 hairs. Immature sporangiophores of Phycmym (with no sporangium yet), also grow both at the tip and in the tapering part ,down to 2 mm below the tip (Castle, 1968; cf. Roelofsen, 1969, p. 298). Electron-optioally, structure 6 B was observed; the inner surface waa sometimes aa 6 C. The tip was not studied by means of this method, but since it is negatively birefringent its structure probably R i according to type 5 XB. In the apical cell of Cbdophora filaments, a struoture as 6 XA waa fourid. ‘I’his merges into the crossed polylamellate structure of type 7 (Frei and Preston, 1961a). Rhizoids of this alga, apical celh of red algae and the protonema of Funaria were provisionally chsified in the group with tip growth only, but in reality it is not known whether these do not grow below the tip. The green alga Bryopsis (a large coenocytic call,Rhaped like a feather) was, until recently, studied with the polarization and the interference microscopes only (Kuster, 1933; Green, 1960b), so that its structural pattern is only partly known. The results indicate that the tubular parts have a structure as in type 6 A and that the tips of the main axis and of the laterals are as in type 5 B. Green’s (1963b) revised Rtructural model of Rryop.6~does not hold, since Kiister (1933) had observed a reverml of the double refraction on stretching and releasing the flaccid cells. This is evidence of traneverse orientation. The condition in Byopsits has been resolved by Frei and Preston (1964) who show that the negative birefringence of the xylan microfibrils is overcompensated by the positive birefringence of glucan chains which lie parallel to them. Electron micrographs show

104

P. A . R O E L O F S E N

that the microfibrils lie in fact almoRt at random with only a slight tendency towards transverse orientation. It s e e m unknown whether growth is confined to the tips in B ~ g q e i e cells, or occurs in the tubular parts as well. The multinet-like structure points to the latter possibility. Green (1902, 1903a) has induced Nitella internodal cells to produce lateral protuberances by surrounding them with a tight jacket provided with a hole. These induced laterals are negatively birefringent throughout with respect to their axis. This suggests a combination of types 0 A and 5 B. U. TISSUE UELLS WITH 180DIAMETRIU GROWTH

Although more or less isodininotric cells oocur very frequently, the submicroecopic structure of both the growing and the full-grown walls has received little attention. Since tho cell faces are isotropic in surface view, it has tacitly been accepted that the microfibrile are randomly orientated both in primary and in secondary walk. Thus the stru&ural type 1 A (Fig. 7) would apply, only with pits and with thickening at the ribs of the polygonal cells. However, such ideal random orientation has been observed in only one case, viz. at the outer surface of the central spherical part of the stellate medullary cells of Juncua (Houwink and Roelofsen, 1954). This surface is contiguous to intercellular spaces. The outer surface of macerated parenchyma cells of the apple And the potato (Wardrop, 1954a, 1956) and of the avocado (Scott et a?., 1963b) showed either some orientation or a random arfangement of broad bands and areas, consisting of more or less parallel microfibrils. A similar structure was seen in parenchyma cells of the D u M h tuber, but this micrograph might be of the inner surface and hence of secondary thickening (Wardrop, 1954b). Czaja (1903) has reported that, if the birefringence iR enhanced with chlor-zinc-iodine,the walls of more or less isodiametric parenchyma cells of fleshy f d t s are no longer isotropic as was thought previowly. The present author confumed this, using the truly isodiametric and growing cells obtained by maceration of young tubers of A s m v aprengeri. In our view this phenomenon does not prove that the tot81 wall is anisotropic, since presumably the reagent will enhance the birefringence especially of exposed and of the beet obaned wall layers, so t h t the anisotropism might be artificial. However, it confirme the electron-optical observations that parenchyrm walh need not have randomly orientated microfibrile, but may have randomly orientated overcrossing bands of more or less aligned microfibrils. This concept has been schematized in Fig. 1B.

U L T R A S T R U C T U R E O F THE W A L L IN GROWING

CELLS

106

Tissue cells in suspension culture become truely spherical except when they divide or form clumps. Since, as far aa known, their cell wall was not studied, it is unknown whether they belong t o type IA or IB. As far as can be judged h m the photographs in Lamport (1.964),however, they lack rib thickenings. Although they do not grow truly free, the isodiametric thallus cells of red algae were mentioned in IIIA. They have the structural type 2, Fig. 8. D. TISSUE OELW WITH PREDOMINANT GROWTR IN LBNQTII

1. With M without thin axial rib thickenings

To this group belong parenchyma cells of apical merhteme, fusiform cambium initials and their derivatives, cortical fibres, perivaacular fibres and latex vessels in the stage of lengthening. They all have a relatively simple multinet structure in their growing walla, at least at the initial stages. (a) Parewhymu celk of alyicul nzeristem. Since them have been studied most of all, their structure, which is schematized aa type 8 in Fig. 14, will be discussed in most detail. The main objeots of study were the Avenu coleoptile and the onion root, which gave ementially similar results (cf. Roelofsen, 1969, p. 142). After division the cells are 15-20 ,u high. They are intermediate between isodiametric polyhedral and prismatic (A in Fig. 14). Phmodesmata are grouped in pit fields, occurring both in orom walls and, very crowded, in side walls, At junctions with two adjacent cells there are slightly thickened ribs. Exoept those at the cross walls, these were probably “inherited” from the parent cell. The microfibrils are randomly orientated in the crom walls and more or less transversely in the side walls, including the inner surface of the edge thickenings. The ler3;ter are restricted to the outer surface and consist of a band of microfibrils aligned parallel to the direction of the edge. Many microfibrils at the border of bands are partly lying loosely at the outer surface of the inter-rib regions. These young cella first grow both in length and in girth (cell B), then only in length (cell C). Growth must be distributed equally all along the cell since the labelling of the cell wall by eupplying W labelled sugars is regular, and since the spacings between the pit fielda increase eqully all along the cell (of. Roelohn, 1969 p. 133). During widening, the pits in the side walla are drawn out transversely and ale0 the tramverse orientation of the microfibrils becomes more pronouncsd. In more elongated cells (C) the transversely orientated microfibrib are restricted to a compact inner layer which is continuous along the

P. A . R O E L O F S E N

106

C

A s seen In

- -section -- - - As seen in

surface vtew

FIG.14. Structural type 8, found in tubular tissue cella with thin axial rib thickening. M occur generally in Cormophyta. Special type of multinet structure.

whole inner surface. On the outer surface of the inter-rib regions a loose mesh of microfibrils with random orientation occurs. Later, more and more axially orientated microfibrih appear here with randomly orientated ones underneath. Thus essentiallythe same multinet strudiure as in type 6 appears in these regions. Here too, a crossed f i b f i r structure may occur. The vertical edge thickenings, usually six, grow in width and in

ULTRASTRUCTURE OF T H E W A L L IN G R O W I N G UELLS 107

thickness. The microfibrils in them are axially orientated. Aa a result, narrow cells may at the end of the elongation acquire positive double refraction with respect to the cell axis. I n transverae sections the outer part of the thickenings appears less donse than the inner part adjacent t o the continuous wall. The collulose network in the pit fields is very thin. It is perforated by 10-50 plasmodesmata. Thick microfibril bundles encircle and strengthen the fields borders and may subdivide the fields. The number of pit fields per cell does not increase during growth and seems in some caaes even to decrease (cf. Frey-Wyssling, 1969 p. 69). As discussed in Section I V A 3, the pit fields themselves do not grow in size during extension growth, but during division growth they very probably do so as well MI dividing. The cross walls thicken, but retain the interwoven mesh structure. At the cross-wall edges, microfibrilsof the vertical parent edge thickenings may, in young cells, run through before being disrupted in older ones. (6) Fwiform mmbium ircitiale. Of these much less is known (cf. Roelofsen, 1969 p. 162). Structures similar to stage C of the parenchyma cells (Fig. 14) have been found. There are similar edge thickenings, only thinner ones and, presumably, only four of them. At the outer side of the inter-rib regions there are again axially or randomly orientated microfibrib, either loose or in a network with wide meshes. Underneath, a, more oompact structure with a predominant transverse orientation, that may be crosswise helical, is detectable. At the inner surface a transverse structure occurs all around the cell. However, too few electron microgmphs have been published to be Sthat the outer surfaces of the four cell faces are actually similar in details. Differences are to be expected, since the direction and the degree of their extension vary greatly and this will affect the microfibril,k orientation (SectionI V A 5).The radial cell faces ariae by widening of the cella and the tangential ones from cell pbtes (data on the increase in area of the various cell faces are given by Wilson, 1963). (c) Vmcular elem&. Only the phloem cells and tracheary elements in coleoptilea of Avena and maize have been studied t o any extent (Miihlethaler, 1950; cf. Roelofaen, 1959, p. 158). Initially, there is no essential difference between these and parenchyma cells. Tranaverae orientation dominates also here, but the axial thickenings are less conepiauous and later even disappear. Loose, more or less axially orientated, microfibrils occur on the outer face. In somewhat older cells, that have both widened and lengthened, crowd helical structures appeaz regularly. The sieve elements differentiate into sieve oelle, with tapering ends and with many large pit fields in Hide w a b and sieve K

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P . A . ROELOFSEN

tube members, with obtuse ends provided with sieve plates. The tracheary elements acquire annular or helical thickenings in which the microfibrils are aligned. Also in primary xylem of Ricinw, axial edge thickenings and cell faces with transverse and random structures were seen (Scott el d., 1960).

(a) Fibrw of cortex, phloem arul wood. As far as can be deduced from the limited amount of work devoted to these c e b , multinet structure, often with thin longitudinal edge thickenings aa in Fig. 14(C), seema to prevail also here. It occurs in phloem and wood fibres of Fmxinw (Bosshard, 1962), in cortical fibres of (Sterling and Spit, 1967), in perivascular fibres of Secule (Roelofaen and Spit, 1960) and very probably in the growing tubular part somewhat below the tip of the wood fibre of Papuodendron (Wardrop, 1964). I n somewhat older fibres of Aspamgw that have both lengthened and widened, and in the central part of the Papuodkndron fibres, again crossed helical, at least oblique, structures have been found. We will return to this point when discussing widening tubular cells (p. 128). ( e ) Latex weasels during lengthening. Only latex vessels of Ewpllotbia splendens were studied (Moor, 1959). Three developmental stages may be discerned. In the embryonic tissue, some initials grow by intrusive tip growth into the intercellular spaces and form a highly branched cell. In the second stage, the different tubular parts of this cell elongate together with the growing surrounding tissue. Finally, the vessel widens considerably in all parts. Here only the second stage is considered (see p.128). As sketched in Fig. 16, the tips are covered with a randomlyorientated mesh of microfibrils (A). As a result of extension this becomes axiclug orientated, while underneath a more nearly transverse, usually croeswiee, structure has appeared (B). This is called the transition l a m e b by Moor. In our view, it might equally be considered as part of the primerg wall, since it extends considerably; ale0 there s e e m 8 to exiat a gradual transition with the outer lamella, as in typical multinet structure, There are no edge thickenings and no pit fields so that there is a closer analogy with the pattern of freely growing cells (Fig. 12) than with parenchyma cells (Fig.14). 2. Tubular cella with thick axial thickening8 (a) Epidermal eel& of apical rneristew. Mowt attention hae given to the epidermis of the Aeenu coleoptile, less to that of the onion root. There is no essential difference in structure between these. Other epidermal walls have only been studied occasionally. For the a p p o priate literature see Roelofsen (1969 p. 160) and B6hmer (1968).

ULTRAS TRUUTURE O F T H E W A L L I N O R O W I N O UELLS

+

c

\

I

is

109

:ondai'Y wall lamellas

k n i n g

50- 100 lamellae

FIG.15. Structures found in latex vesvela during the initial intrusive tip growth (A), lengthening (B)End widening (C). The eroaswk polylamellate wall at C ia celled etructural typs 9.

The epidermal cells elongate along their whole length, as was proved with both the autoradiographic method nnd hy mcam of mark8 at the outer Burface of Avenu coleoptiloll. 'J'troy ~ ( J Wto a higher extent than the parenchyma celh in the garnu ohjcct, rcnching in Avena 160 t i m a and in the onion root 30 timeN the initial length. As drawn schemtLtically in Fig. 16, there are two similar, thin radial walls and two thicker tangential ones, of which the outer ie very thick. The pits are tramversely &-like in the radial wall8 and more rounded in the inter tangential wall. In the outer wall of onion root epidermis ectodesmats (ending dead under the cuticle) were reported. The microfibrillar pattern of both the radial and the inner tangential walls is similar to that of parenchyma cell8 and is typically multinet. There are also two axial edge thickenings. The radial walls differ from

110

P. A . ROELOFSEN

the tangential inner one in being thinner and having fewer longitudinally oriented microfibrils a t the outer surface. The outer epidermal wall is polylamellate in Avem coleoptile, onion root and all other epidermal walls studied so far. I n young Awna coleoptiles 10-15 lamellae occur in a wall about 1 p thick, in old ones about 26 lamollae in a wall 2-3 p thick, In onion root cells and in very

1'1

In older cells

Inner rwioce (young utllst

Fro. 18. fitructural type 10, found in tubular epidermel oelln of apical meristama.

young cells of Avena coleoptiles this wall also shows typical multinet structure. The overall biregringence in such celle is negative, but the axially orientated outer part is already present in greater amount than in normal walls. The outermost lamellae of this part are certainly leas dense than the inner ones, but whether they are ale0 more perfectly aligned is questionable. Regarding older walls in Avena, opinions differ with respect to the orientation of the innermost lamella. According to Setterfield and Bayley this has acquired axial orientation, but according to Mtihlethaler and to Buhmer it is still tramverse, as in young celh and &a in the other cell faces of older cells. Definite settling of this point by electron-microscopy of the inside in surface view seem worthwhile, since if the first-mentioned view is correct, this would be the first o w of a polar cell with dissimilar initial orientation of microfibril ayntherris in the different side walls. This might be important with mpct to the mechanism of orientated synthesis (see Section IV C).

U L T R A S T R U O T U R E OF T E E W A L L IN G R O W I N G CELLS 111

( 6 ) Coltenchyma celZs. Our knowledge of the ultrastructure of these cells is based on their study in petioles of Apium, Heruckurn and Petkles (cf, Roelofsen, 1969 pp. 140, 160, 248) and on an occasional electron micrograph from the pen &em (Setterfiold, 1961). Ae is well known, all transitions occur between cortical parenchyma cells with slightly thickened axial ribs (Fig. 14) via collenchyma cells with moderate edge thickenings (Fig. 17(A)) to those with heavy thickenings (Fig. 17(B)).As in the outer opidormal wall, the thickenings in collenchyma cells are polylamellate, but they differ in having thicker non-cellulosic lamelluo between tho cellulose lltmellae. Pectic substances are the main constituents of these (up to 4WY0 of the wall).

l h . 17. Eitruotural typen 11, found in collonc.hym wlb.

Very young collenchyma cells do not differ much from parenchyma cells. The walls are isotropic between crossed nicols, evidently since the transverse continuous wall and the axially orientated thickenings compensate each other. Soon they become positively birefringent m a result of the appearance of more axially orientated lamellae in the edges. In type A, all thickening lamellae are situated at the outer side of a lamella which is continuous around the cell. Thie lamella is, in all probability, transversely orientated. In collenchyma of celery petioles this wa8 observed in c e b of all lengths and ages only in so-called “mature” heads, which are resting stages prepared for growth in the next season of this biennial plant. It was not found in cells of young growing plants, but with the method used (transverse sediions) thin lamellae might have escaped detection. It is a similar inconclusive H*

112

P . A . ROELOFSEN

situation as with the inner surface of‘ the outer epidermal wall (see p. 110). In collenchyma of growing Heruckurn and Pelasites petioles, at least, the innermost layer ie always less axially orientated than the rest of the wall, but whether it is aotually tranevem, is again questionable. In the edgo thickenings, the outer lamelhe are conspicuously less dense than the inner ones and in Petmites collenchyma cells, outor lamebe also appear better aligned. In type B collenchyma cells, there are, at least in celery petioles, alao edge thickenings on the inner side of the continuous lamella. Thus, there are outer and inner ribs. Also in the latter, cellulose orientation is axial and probably including the innermost lamella. Whether, in “mature” heads, cells with inner ribs also acquire a transversely orientated continuous layer is another quecution that requires investigation. Czaja (1961) reported that if collenchyma cells in sections of petioles of various plants are observed between crossed Ncols in nearly orthogonal position, bright and dark transversely orientated bands, 0.5-1 p wide, appear all along the length of the cell. We can confirm this; but not, however, the explanation which in our view is: that as a reault of axial contraction, the cellulose lamellae become undulated transversely and normal to the plane of the wall. When observed parallel to the plane of the wall, this produces alternating left and right oblique orientations, which consequently extinguish right and left of the orthogonal positions. This follows firstly, from the fact that the bright bands become dark and dark ones bright at the other side of the orthogonal position and, secondly, from the disappearance of all these bands on stretching the cells and the reappearance on releasing them, This is a striking demonstration of the well-known elmticity of collenchyma celh and, moreover, it shows that the collenchyma tissue bears axial stress generated by other tissues in the petiole or in the stem in question (see p, 130). E. TISSUE CELLS THAT PREDOMINANTLY WIDEN

The third and final growth stage of a latex vessel is, as mentioned on p. 109, one of the predominant widening, m is indicated in Fig. 16(C). In this stage the microfibrils in the pre-existing lamellae naturally acquire a more nearly transverse orientation and become more widely spaced. Meanwhile, more lamellae, finally up to 100 of them, each about 0.1 u , thick, are deposited on the inner mrface. The microfibfi am well aligned in each lamella and the orientation alternates, 80 that left- and right-handed helical structures appear. Their pitch R i initially about 48’ but as a result of the widening, they flatten in older lame1la.e which

U L T R A S T R U C T U R E O F T H E W A L L I N O R O W I N Q OELLS

113

explains the overall negative birefringence of the wall. As in similar polylamellate crossed walls of algae (cf. p. 101 and Figs. 10 and la), the changes in lamellar orientation must involve slipping between adjacent lamellae. AE waa mentioned already when discussing wood and phloem fibres (p. 108), crossed helicsl structureR wero also found in somewhat older stages or older parts of these colb and Irrey-Wyesling (1962) and Wardrop (1964) have related this to the simulttlneoua occurrenoe of lengthening and widening in these cases, just &B in latex oeb. Ale0 in parenchyma cells, cambium initials and espeoially vaecular elements, crosswise helical structures, although less conspicuous ones, have often been observed (pp. 106,107). These too may be related to simult,sneous widening and lengthening. I n cells that only widen, this structure does not seem to occur; for instance, neither in widening wood vessel cells of Frminw, (Bosshard, 1962) nor in the anticlinal walls of disk-shaped epidermal cells. The latter invariably have a predominant radial microfibrilla,r arrangement (normal to the periclinal walls), but the occurrence of multinet structure withtangentially orientated outermost microfibrils is to be anticipated when these walls come to be studied electronoptioally. Just as in the case of the cross walls of other widening cells, the periclinal walls of disk-shaped epidermal cells grow in all directions and would presumably also have random microfibril orientation. In fact, they usually are optically isotropic in surface view. Therefore, the conspicuous orientation and multinet etruoture in the outer wall of PhiMe&m leaf cells, found by Bolligor (1969), is a surprise, and at the same time an invitation to further research. F. TIPS OF TISSUE CELLS WITIF TIP GROWTH

To these belong vascular elements, tracheids, fibres in wood, in phloem and in cortex, and latex vessels in the firat growth stage (cf. Roelofsen, 1959, p. 134; Schoch-Bodmer, 1960). Either one or both tips show intrusive growth. In front of the advancing tip, the adjacent cells become separated in the middle lamella,. As a result their plasmodesmata are ruptured. Later, new ones may be formed with the intruding cell. Some accept that growth is restricted to the extreme tip, so that the new wall is apposed along the outer surfaces of the celh with which new contacts are established. Others accept that there is also growth below the tip, which, at least when the adjacent cells do not grow or grow more slowly, involves some gliding of the new wall awes existing wtllla. Only very few electron micrographs have been publhhed revealing the Rtructural pattern at the outer surface of the tip. These are phloem

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P. A. ROELOFSEN

and tracheary elements of Avena coleoptile. a tracheid of Pinus, a wood fibre of Eucalyptuq a phloem fibre of Praxinw, a cortical fibre of Asparagus (for all these see Roelofsen, 1969, pp. 157-160), latex vessels of Euphorbia (Moor, 1959) and a wood fibre of Papuodendron (Wardrop, 1964). A network with predominant axial orientation was found in the tips of most of these. In those of phloem cells this was scanty and a transverse structure underneath was dominant. Finally, in tips of latex vessels and of Papuodendrm wood fibres, random structure occurred. Evidently there is, just as in tips of freely growing cells, Fig. 11, a considerable variation of the structural pattern of different objects. This was confirmed recently by Wtirclrop (1962 p. 272) with respect to tips of one object, viz. Buculyptus wood fibres. Variations in the shape of the tips may perhaps explain this (p. 127).

IV. INTERRELATION BETWEEN GROWTHAND WALL

ULTR ASTRUOTURE

In this section we will discuss three points: the effect of growth on the wall structure, the effect of wall structure on the direction of growth and, finally, the possible cause of the initial oriented synthesis of the microfibrils. A. EFFEUT O F GROWTH ON WALL STRUCTURE

I . Displacement of microfibrils (a)Radial displacement is the inevitable result of apposition in (the non-strict sense), which is the niiiin if not the sole way microfibrils are added t o the wall (see Section IT D). ( b ) Increase in microjibrillar spocinr~,or tangential displacement. This is likewifie the inevitablc result of apposition in combination with the spreading out of the wall over a greater area. In case8 of exceptionally rapid extension the whole wall io thinned out, because there is little or no wall synthefiis, e.g. in the weta of Pellia, in &mind filaments of grasses and in Tradescantia stamina1 hairs subject to endosmosis (p. 90). In other cases there may be only a temporary thinning of the wall, as in Helianthus hypocotyls and in NiteZla (Green, 1958b). Usually, however, wall synthesis just about compensates thinning out, 60 that a steady state of synthesis, outward displacement, wall thickness and wall extension is reached, as was demonstrated, for example, in onion roots (Jensen and Ashton, 1960) and Auenrc coleoptiles (Ray, 1962). Probine and Preston (1961) calculated the distribution of the integrated extension along the depth of a cell wall, on the basis of a tenfold cell lengthening and with an apposition that just compensatm

ULTRASTRUCTURE O F T H E W A L L I N GROWING OELLS

115

the thinning out. This resulted in the instructive graph of Fig. 23C which shows that the total expansion is heavy especially in the outermost part of the primary wall, If the amount or the physical properties of the i n t e r j i b r i b material do not allow extensive sliding of microfibrih past one another, fissures and tears must appear, as in Vulrmia and some other algae (Figs. 10 and 13). If the microfibrils do not cohere so strongly, their mutual spacing just increases, rn has evidently happened in outer parts of walls with multinet structure (Figs. 12, 14, 15B. This a h occurs in the outermost lamella of the axial rib thickenings of collenchyma cells, Fig. 17, and of the outer epidermal wall (Fig. l0), which are conspicuously less dense than the inner ones. Jn widening latex vessels (Fig. lac)the thinning-out effect is likewise obvious. (c) Possible origin of rib thickenings. The author’s hypothesis (1958,1969,p. 178)of a migration of plasticized amorphous substances of the middle lamella and of the outer parts of adjacent primary walls, from the cell faces towards the cell edges is not more than a possibility. With turgid cells therq will be a pressure gradient in this direction as a result of a difference in cell curvature, see Fig. 18. This effect is even more apparent if there is an air-fUed intercellular space, or even an outer space at the cell edge. In foams, the liquid between the polyhedral air bubbles is pressed from the faces towards the edges and runs off through the latter. Admittedly, the amorphous cell wall substances are not’liquid, but since they are plastic and since pressure differences of several kg/oma along distances of the order of only 20 p may be involved, migration does not seem impossible. Moreover, wall extension already keeps the amorphous substances moving and these might be somewhat thixotropic. If such tangential displacement does occur, it might drag lome microfibrils with it from the outer cell faceH of the primary walls towards the edges and thus give rise to edge rib thickcningn. ThiH would explain why edge thickenings are always present in polyhedral or prismatic tissue cells but not in spherical or truly cylindrical tiwue cells (thallus cells of Algae, wide vessels), nor in freely growing cells of either lower plants orprobably,seep. 105,of higher plants in suspension culture. Obviously, plasmodesmata will impede such displacements of microfibrils and, especially in young cells with crowded pit fields, displacement will be restricted to the regions near the edges. The course of microfibrils will be restricted to the regions near the edges. The c o m e of microfibrils both around pit fields and at the borders of the edge thickenings certainly do support the migration hypothesis, but of course cannot prove it. Perhaps thin edge thickenings, as occur in parenchyma cells, vascular

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P . A . ItOELOFSEN

elements, cambium initials and cortical fibres (Fig. 14) might arise principally in this way, but this seems improbable with respect to the thick axial thickenings of epidermal cells (Fig. 16) and of collenchyma cells (Fig. 17). Here, locally increased cell wall formation is evident, especially in collenchyma cells with inner edge thickenings (Fig. 17B) I n the latter case the migration hypothesis does not even apply. It seem logical to suppose, therefore, that with thin edge thickenings &I)

Fxo. IS. Diagram illustrating the concept of Roelofsen (1956, 1969, p. 17) of migration of plasticized amorphous wall substances with loose microfibrilsfrom cell faces towctnls cell edgea as a result of pressures being lower in the latter.

well an increased cell wall synthesia in the edges contributes to the formation of the thickenings. Non-cellulosic aubstances are synthesized throughout the wall, but opinions differ regarding the site of cellulose synthesis in all these thickenings. Setterfield and Bayley (1968, 1959, 1961), Bayley et al. (1957) and Beer and Setterfield (1958) accept the formation of microfibrils within the edge thickenings and within the outer epidermal wall. As was discussed in detail in Section I1 D, we consider the formation of microfibrils at some distance from the plasmalemma. althou~h

U L T R A S T R U C T U R E O F T H E W A L L IN G R O W I N G CELLS 117

possible in principle; to Ee unlikely for several remons and so far it has not been proved in any one case. With respect to the thick axial thickenings, the esdent thinning out of outer lamellae in both epidermal walls and collenchyma thickenings, and the reorientation observed in some cases, e.g. in young epidermal walls, do support the concept that there was simply an increased apposition of cellulose at the inner surface. A speoial sort of rib thickening is the circular rim which arises during the budding of yeast cells as a result of a displacement of the glucan microfibrils. They are pushed away by the protruding naked protoplast (p. 94, Fig. 9). 2. Neceegity of sgnthesis of middle lamella constituew within the wall

Since the middle lamella is not renewed by apposition as is the primary wall, it will be extended most. Nevertheless, electron micrographs do not show any sign of thinning out of the middle lamella in growing walls. This means that its constituents must either be synthesized on the spot or, if synthesized like the microfibrils at or near the inner surface of the wall, they must have migrated outward through the pores in the microfibrillar network. Since we cannot see any reason for the existence of a radial pressure gradient that might cause such flow normal to the wall surface, Rynthesis in or near the middle lamella seems a necessity. As discussed on p. 87 and demonst'rated by Fig. 6, this was in fact proved by autoradiography and the apparent demonstration of metabolic turnover in the wall supports it. 3. Multiplication of plasmodesmata and of primary pit jklds

It has already been remarked (p. 107) that even the youngest stages of meristematic cells me provided with the full number of primary pit fields in their side wa&. During the phase of growth by extension these become wider spaced, but they do not increase in number nor significantly in size. Apparently, the microfibrils within the pit fields cohere so strongly that they do not slip past one another aa occurs elsewhere in the extending wall. The pit fields are not islands of wall synthesis, and growth as was once thought, but may be termed islanda of stagnancy, spots of statm quo, at least during the main growth phase. Since both the number of pit fields per cell in a file of daughter cells in an apical meristem and, apparently, the number of plasmodesmata per pit field remain fairly constant, doubling of both must occur in the parent cell before or during division. There are convincing grounds to accept that this occurs as a result of a cleaving of the pit fields and of the plasmodesmata. Firstly, Scott and Lewirc (1963), ZiegenApeck+

* Sea also K. Wilson (1HG8). Ann. Bol. N.S. 22,44e458.

P . A . ROELOFSEN

118

(1953) and KrulI (1960) have described differences in the number, the shape, the structure and the size of primary pit fields in dividing meristematic cells, that induced them to conclude that the fields were being cleaved. Secondly, K r d (1960) found branched plasmodesmata which had also been found earlier by others whom she mentions, and she explained their origin by accepting that the original plasmodesmata had been widened by wall extension and had subsequently been partly cleaved y the apposition of younger wall lamellae, a process which is illustrated diagrammatically in Fig. 19.

4

A Section

=

o n n o

\

\

initial wall n

0 0 Surface view

0 FIO.19. Diagram of the mechanism of cleaving of plaernodeam;rta in growhg walls according to Krull (1980).

The photograph of branched plasmodesmrtta published by Krull, and also the branched plasmodesmata described prcviowly, apply to secondarily thickened wall. Since this, in our view, did not prove the point, she kindly provided us with another one which is reproduced in Fig. 20A. This is of a section of a pit field in an undoubtedly growing, very young wall and shows several highly branched plasmodesmata. These also occur in other objects. One of the photographs in each of the papers of Buvat (1957, 1960) and that of Frey-Wyssling (1962, Fig. 6), seem to show it and Buvat supplied us with another unpublished photograph, which is reproduced in Fig. 20B. The general occurrence of such branches is also indicated by the fact that in electron micrographs of oblique and tangential wall sections in these and other

IJLTRASTRUCTURE O F T H E W A L L I N Q R O W I N Q CELLS

119

papers, intersected plasmodesrna tubules occur conspicuously often in groups. I n addition, inany electron micrographs of the “cellulose skeletons”

FIG.20. Branched plsnmodesmstt& in tranwerse section8 of primary pit fields of: A, the sidewall of a prtrenchymii roll in a 4 mm long, growing shoot of Viacum album (unpublished micrograph of Krull. 1960); B, tho sidewall of rt parenchyma cell of the conducting ntrmda of a growing shoot of Cueurbitn p e p (unpublished micrograph of S u v a t , 1960).

of very young parenchyma cells, e.g. those published by Stecher (1952), are evidently crowded with pit fields, many of them in pairs and with a loose niicrofibrillar net work. Actually, these loose spots were recogH**

120

P. A . R O D L O P S E N

nized as growing spots by him, but since they were not recognized as widening and dividing pit fields, they gave rise to the concept of mosaic growth, which had later to be rejected. Presumably, in sonic unknown wtty the cytoplasm regulates the coherence between the 1nicrofil)rilx in the pit field and in this way determines whether the extending wall will draw them apart, as occurs in the dividing parent cell and perhaps also in the very young daughter cells, or not, as applies to the wall in the main growth phase. 4. Origin of pores and perforations Obviously, if a plasmodesma has been widened as discussed in the

previous section, without subsequent deposition of cleaving bundles of microfibrils, it will not multiply but widen into bigger pores, that may become microscopically visible. I n co-operation with the local synthesizing and plasticizing activities of the cytoplasm, probably the pores arise in this way in the margin of bordered pits in the radial walls of young conifer tracheids (cf. Roelofsen, 1959, p. 157). Also the disappearance of microfibrils in the formation of the big pores of sieve plates (see p. 89) may thus be understood. Neither pushing apart by cytoplasm (E’rey-Wyssling, 1959, 3). 70) nor dissolution of cell wall material necessarily seem8 to be involved, except of callose in sieve plates (see p. 89). Pushing apart by the cytoplasm, or at least by cell contents, presumably does occur when the growing wall is perforated, as in the budding of yeasts and prior to the release of swarmera by algal cells (pp. 89, 90). 5. Reorientation of microjihrils, milltinet growth

( n ) Role of iiirgor pressure in retaining transverse orientation in growing walls of fwhitinr cells. If an extensible material, containing r a n d o d y oriented elongated particles, is extended equally in all directions, the particles will stay random, but with unidirectional extension they will become orientated. This in principle also occurs in primarywalh with the inicrofibrils. In 1935 Bonner stretched plasmolysed Avena coleoptiles and with only 80/, extension he observed a change from negative to positive birefringence, that is from a mean transverse to a mean axial orientation. Equally slight mechanical extensions reverse, in the author’s experience, the sign of hirefringenue in punctured Z’ruhclcntiu stamina1 hairs, in young cotton liair~i d in the growth zone of Phycomyce? (cf. Roelofsen, 1959). It may undoubtedly be observed in all young cells and tissdes that are stretched provided, to be ~ u r ethat , they are in non-turgescent condition. The reorientation of the microfibrils is shown diagrammatically in Fig. 21B. In a turgid cell, however, the cell wall appears to behave radically

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differently. This was already deduced about thirty years ago by Van Iterson from the following observations (cf, Roelofsen, 1959, p. 139). The seta of PeZZia grows so fast, namely 20- to 40-fold (some cells 60-fold) in two days, that the wall is thinned out to one-tenth, but still it remains negatively birefringent (cf. Diehl el al., 1939, p. 785). One might object that this irJ not pure extension, since meanwhile new (transversely orientated) cell wall niaterial was undoubtedly deposited on the wall and this ,might have obscured the reorientation of the pre-

A With turgor pressure

T

Without turgor pressure

B

FIG.21. l k g r n m s of: A, displacement without reorientation of microfibrils in the inner wall lemellae of an rlongating turgid tubular cell; €3, displarement with reorientation on mtclinnictll stretching of the sitme cell in non-tnrgid condition.

existing wall. This objection is not valid in the following cases. The stamina1 filaments of Secale elongate sixfold in several minutes, but stay negatively birefringent all the time. If stamina1 hairs from wilted flowers of TrnlZesca.nlia virginica are mounted in water, the cuticle is disrupted and, rapidly taking up water by endosmosis, the cells may, without widening to any extent, elongate threefold within half a minute. The negative birefringence, however, ix retained. Van Iterson realized that, as in any tube with internal pressure, the transverse stress in the wall ia twice the axial one and that i t muot have been this transverse stress that prevented the microfibrils from being reorientated by the axial extension. He considered the microfibrils

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P. A . R O E L O F S E N

to 1)chavcas if they malie part of circular pieces of thread that are tied around an inflated. rubber tube. In case there is an increase in pressure these prevent the tube from widening, but they do not prevent its lengthening and mean\vhile setain t'heir transverse orientation. If, however, the tube had not boen inflated and had been lengthened by pulling, such loops would be reorientated very soon. Thus Van Iterson arrived at the important conclusion that the turgor pressure provides for a rectifying mechanism that ensures transverse orientation of the wall structural elements, not,withstanding considerable lengthening. ( 6 ) MultirLet growth t A e ~ r y .Tliis concept has been used and elaborated in our inultinet growth theory (1958, 1959, p. 170), which explains how the multinet structure :wises, that was found in both the quickly extending Tradwcantia hairs ant1 in all tubular cells except those with helically crossed polylninellate wnlh tind those in which only the tip grows. 111wdls with niultinet stxucture the inner part is a sheath, consisting of bui~llcsof' tmnnvcrwly orien tzaLtctlinirrofibrils, see Figr;. 12 and 22. Tliis is the part which apparently is inimuiie for reorientation by even nianifolti axial extension, provided the bundles are kept taut by transverse stress and strain due to turgor pressure. During extension these buiidles must have split in several thinner ones and any obliquely oriented microfibril8 that overcrossed them, must have slipped along them without causing reorientation to any extent. This is indicated tliagrammatically in Fig. 21A. However, on being shifted outwardly by the apposition (non-strict sense, see p. 85) of new wall material, a point of time arrives for any wall lamella at which it acquires random and, eventually, axial orientation. It follows that this must have been brought about at a time when the transverse stress had become too small to prevent reorientation. Since interfibrillary materials are synthesized and possibly broken down within t'he wall (p. as), it Reems probable that thiH l o s ~of stress has been the reclult of a decrease of coherence between the microfibrils, effected either by an increase of, or by a chemical change in, the interfibrillar substances in that part of the wall. Here, the ringlike microfibrillar bundle8 yield to the trarwverno stress and are widened. The stress will bc taken over by a younger part of the wall. Once freed, reorientation is rapid, a8 is apparent from the immediate reversal of the birefringence on stretching flaccid cells. This concept implies, therefore, t h a t there is not only a steady state in wall thickness as a result of a balance in apposition and thinning out, but also a steady state in the distribution of the transverse and of &he axial stress across the depth of the wall. The transverse stress is

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123

I k . 22. Toni nrni of stollato medullur cell of' n growing Juncue leaf basis, showing typical multinet structure, HCU I4g. 1% (from Hoiiwink iunl Itoelofaen, 1954).

evidently borne mainly by the inner part, the axial stress probably by central and outer parts (in Trade.scantia stamina1 hair8 apparently by the cnticle). In Fig. 23A this multiriet growth theory is represented diagrammatically, accepting three separate lamellae for drawing facilities. I n fact a gr'ltdual transition must he accepted (Fig. 12), except in lamellate walls as with Nitella. In Fig. 23B the hypothetical distribution of the transverse and the axial stress across the depth of the wall is shown. This might be different in different places in the wall of the same cell. For instance, a t the edges of tubular cells and in the outer walls of epidermal cells, the transverse stress might be reduced sooner, causing earlier reorientation. In Fig. 23C the distribution of the integrated strain across the depth of the wall is prefiented as calculated by Probine and Preston (1961) on the basis of a tenfold wnll extenfiion and of a conutant thicknefis brought about by apposition. It has become apparent that the tiiff'erence in mechanical properties between walls of turgid and of flaccid cells is the bash for the mrrltinet

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P . A . ROELOFSEN

growth theory. Recently Kamiya et al. (1963)determined this difference quantitatively. They found that in Nitella internodal cells (unfortunately not growing ones) the longitudinal extension brought about by

B

fl ../I ,’

c:

-w 0

/’

/*

/I0*

/‘ Inside Outside Possible distribution o f : Transverse stressAxial s?ress------

Inside

5p

Distance within wall

Distribution of strain

PIG.23. Diagrams related to t.he multinet growth concept. A: thinning out and microfibril reorientation in the side wall of an elongsting tubular cell; B: Supposed radial distribution of transverse and axial stress in euch a wsll; C: Calculated radial dietribution of the integrated etrain in a wal1,after a tenfold elongation and while the wall thickness is maintained at 4 by apposition (from Pmolie and Prcston, 1901).

turgor pressure was only a third to

a

quarter of the extenHion by an

equivalent axial stress in a flaccid cell. They considered that in the

randomly or crosswise orientated parts of the wall the transverse stress in a turgid cell generates a component force which induces axial contraction. This is analogous t o the vertical contracting tendency to be noted in scissors-gates as they extend horizontally. It will naturally reduce the axial exten~ibilityin turgid cells as compared with flaccid

ULTRASTHGCTURE OF T H E WALL I N QEaWINQ OELLS

125

FIQ.24. Tip of A growing cotton hair showing reorientation of the outermost microfibrile cit the trttmition of the hemiepherird tip t o the tubular part (from Houwink and Roelofeen, 1951).

cells or wall strips. Green ( 1 W3b) has suggested a similar mechanism to explain low transverse extensibility in other cells. Another experimental support of the rnultinet growth theory may be seen in the following results of (ireen and Chen (1960) and Green (1963a). An increase of the axial stress and strain in Nilella enhanced reorientation in outer wall p r t s 80 that the negative birefringence was reduced or even reversed although transverse orientation wafi retained

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P . A . ROYLOFSEN

at the inner wall surface. Thus “super-multinet structure” was obtained. Furthermore, Green showed that no axial, orientation arose in the absence of axial strain. Transverse orientation was retained in all wall lamellae of young Nitella cells that were jacketed so that there was no axial or transverse strain at all, or were pressed between glass plates so that there was only transverse drain. ( c ) Reorientation in growing tips. When we first postulated multinet growth (1960 p. 98), at least in principle (the role of stress distribution within the wall and the necessity of appoHition were not realized), most cell wall electron-microwopists considered reorientation unlikely, since the microfibrillar network seemed too dense and the microfibrils too intertwined. Therefore, the first proof of reorientation may be revived here, particularly since it is still the most convincing one and illustrates at the same time the complications involved in tip growth. Figure 24 shows tl part of the outer surface of the hemispherical tip of a growing cotton hair. This tip tias the structural type 5 XB (Fig. 11) and accordingly the micrograph shows random orientation at the outer surface. Immediately below the hemisphere of the tip, about 1 ,LA below the randomly orientated area, however, the outermost microfibrils appear to be more or less axially orientated. This cannot have been achieved by synthesis of new axially orientated microfibrils on top of the randomly orientated ones. If synthesis juHt below the cuticle would occur the microfibrils should have been short ones. Reorientation is therefore evident. A similar reorientation of outermost microfibrils was observed recently by Wardrop (1964) in the tip of a wood fibre of Papuodendron. In addition, there are many other electron-optical observations that support reorientation, but since either different cells, or inner and outer lamellae, have to be compared, these are lese convincing. Figure 25 is a diagram of the microfibrillar orientation at the outer and

Fio. 85. Microfibrillar arrangomerlt at the outer and thc inner surface of LL growing cotton hsir (from Houwitik and ItoolofHen, 1064).

U L T R A S T R U C T U R E O F T H E W A L L IN G R O W I N G CELLS

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at the inner surface of the cotton hair tip. All the microfibrils occurring at the outer surface of the hemisphere were, in all probability deposited in random orientation on the inner surface at the top of the hemisphere. This area is first extended nearly equally in all directions so that the microfibrils retain their random orientation. Where the hemisphere merges into the tubular part, however, axial extension prevails greatly and reorientation takes place. The microfibrils that are deposited on the inner surface of the hemisphere below its very top, are initially more or less transversely orientated (negative double refraction). These will probably arrive at the outer surface further down in the tubular part. Meanwhile they will have been reorientated somewhere within the wall as shown in Pig. 23A. It will be clear that it is mainly the shape of a tip that determines whether extension on the tip surface is about equal in all directions as with the cotton hair tip, or unequal as in a more elongated, ellipsoidal tip. This might explain the varying superficial microfibril orientations found in various growing tips. Castle (I968) has determined the longitudinal and ciroumferential relative elemental growth along the tapering tips of young P h y m y m sporangiophores. In the apical 1 mm of the tip axial growth predominates, in the distal 0.5 mm widening. The characteristic shape of the tip is a result of a steady d a t e in the distribution of the ratio of the two relative elemental growth rates along the length of the growth zone. De Wolff and Houwink (1984) tlreatled reorientation during tip growth mathematically, but the preHent author is of opinion that their assumptions do not apply to cell walls that grow a8 a result of apposition. ( d ) Remientation in some special cases (a) Axial thickeninge. In thin edge thickenings and in young epidermal walls of onion root and of Avena coleoptiles. the structural differences between the inner and the outer parts clearly indicate reorientation. Of the epidermal wall of older coleoptiles and of the edge thickenings of collenchyma cells, the structures are still not sufficiently known to either support or disprove reorientation in them (p. 111). As discussed on p. 112 these ce& are mbject to “passive growth”, due to considerable additional axial stress and strain, so that we anticipate “super-multinet structure”, as obtained experimentally under comparable conditions in Nitelk (p. 126). (ii) Widening of cells. Obviously, no reorientation will occur widening cells that already have transverse orientation. This applies to radial walls of cambium initials (p. 107) and to Nitella internodal cells pressed between glass plates (Green, 1963a). Anticlinal walls of disk-shaped hodiametric epidermal c e b of leaves,

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P . A . RoELOFSEN

etc., should (like those of tubular epidermal cells), exhibit multhet structure as a result of reorientation from normal to parallel with respect to the leaf surface, but these walls were not studied electronoptically so far. As discussed earlier (p. loti), latex vessels first mainly lengthen then both lengthen and widen, and &ally mainly widen. As a result, the outermost lamella and the so-called transition lamella, both originally isotropic, are first reorientated lengthwise and later transversely, see Fig. 15. At certain stages this transition lamella acquires a crosswise helical structure. This also often occur8, though not always, in other tubular cells, that both widen and lengthen (p. 113). It has not been explained so far how this structure arises and why only in some cases, whereas in other ones randomly orientated structures that extend similarly retain their random orientation. (iii) Reorientation in crossed polyhmellrcte wa&. I n these the crosswise structure does not arise as a result of wall extension but has been deposited as such. In Fig. 15, representing latex vessels, these lamellas are numbered. During widening a decrease in the pitch of the helical orientations in the lamellae occum. As is to be expected, the decrease is higher in older lamellae, since of these the initial diameter waa smaller and thus the increase in circumference higher. Changes in the steepness of helical structures as a result of growth have also been observed in the outer wall lamellae of algae with crossed polylamellate walls by Frei and Preston (1961a,b), see Fig. 13. The changes differ in the three sets of lamellae in conformity with the spiral growth occurring in these cells. B. EFFECT OF WALL STRUCTURE ON "HE DIRECTION OF QROWTH

1. General correlatiow, pamive growth by tissue tension It seems now to be generally accepted that the wall grows becaw it yields to a more or less constant turgor pressure. By means of a mechanism which is unknown and, moreover, outside the scope of this review, the cytoplasm determines when and to what extent the wall is plasticized and thus, when and how fast the wall grows. Accordingly, a proportionality between wall extensibility and growth velocity and similar effects of auxin on both have been found in many instances (cf. Setterfield and Bayley, 1961; Probine and Preston, 1962). Proportionality is, however, not obligatory. In the growth zone of P h y m y c m sporangiophores, for example, the highest extensibility is found just below the sporangium, but growth is fastest 0.2 mm further down (Roelofsen, 1950).

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129

Furthermore, the cytoplasm determines whether the wall of the whole cell will grow or only a special part of it as, for example, in the formation of root hairs, perforations, branches, sporangia, buds in yeast, a r m in stellate cells, etc. Sovereignty of the cytoplasm in this respect is strikingly demonstrated when, in a young sporangiophore of Phycmnyce.9, lengthening is stopped, the tip is blown up like a balloon to produce a sporangium and the lengthening is resumed. The cytoplmm also finally determines whether the wall will yield multidirectionally or unidirectionally and if 60, in what direction. However, as far as the author knows, most if not all investigators of the structure of growing cells now agree that the cytoplrtsm performs this directional e#e.ct through the intermediary of the structure of the wall it produces. Although relative data were obtained only recently (Pmbine and Preston, 1962), it was generally realized more than thirty years ago, that an isotropic wall will extend multidirectionally, whereas an anisotropic one will be most extensible normal to the predominant orientation of structure. This general rule will have become apparent in Chapter 2 and in Section I V A 6. The structural type of a cell, especially of the freely growing ones, appears to be interrelated completely with the direction of growth. We always find isotropic wall structure associated with isotropic growth, either of the whole cell andleading to isodiametric cells (Section 111A and C, Figs. 7, 8 and 9) or of a tip, leading to a tubular cell with a hemispherical growing tip (Section I11 B 7 and 3 and F, Fig. 11). When orientated structures occur in isodiametric cella these are either in randomly orientated bandH (Fig. I(B)), or the wall is C ~ O S S W polylamellate ~ ~ ~ and may still be consideredetatistically isotropic Section (111 A, Fig. 10). Freely growing tubular cells, with elongating side walls, alway8 have predominantly transverse structure, either simply and leading to multinet structure (Section I11 B 2u and 3, Fig. 12) or statistically transverse, but with crossed lamellae (Section I11 B 26 and 3, Fig. 13). As wm noted, in tubes the transverse stress in the wall is about twice the axial one. Hence for inducing a tubular cell to lengthen, the transverse structural predominance must be so considerable that the transverse extensibility becomes less than half the axial one. If it is more than half the axial one, the cell will mainly widen. This applies to the extensibilities aa measured in turgid cells; those in the flaccid cell and in wall strips may be different (see p. 120). The correlation between structure and direction of growth, or anticipated direction of growth, is likewise apparent in most tissue c e h . Also here, isodiametric cells have isotropic or etatisctically isotropic

130

P . A . ROELOFSEN

walls, at least at the cell faces (Section I11 C), as also have multidirectionally growing parts of cells, like cross-walls in widening cells and tangential walls in disk-shaped epidermal cells. Similarly, tubular tissue cells usually have predominantly transverse microfibrillar arrangements (Section I11 D I , Fig. 14), as have unidirectionally expanding cell faces like anticlinal walls of tubular epidermal cells). If oriented structures occur in more or less isodiametric cells, these are often prepared for unidirectional growth, such as the youngest cells in apical meristems (Fig. 14A, the disk-shaped cells in young setae e of Pellia and young palisade parenchyma cells of K u ~ ~ c h uleaves (Von Witsch, 1941). Such anticipation of future growth direction probably occurs rather generally. It demonstrates that the initial cellulose orientation is not determined by the cellshape (see SectionIV C). However, there are also deviations from these simple correlations. One of these seems to be the widening latex vessel (Fig. lSC), of which the wall is predominantly transversely orientated. However, considering the small predominance, the transverse extensibility will not be less than half the axial one and hence the predominant widening is quite as expected. Another seemingly exceptional case is the widening young wood vessel cell that, at least in Fruxinw, has a more or less isotropic wall. However, here too bhe transverse extensibility will be more than half the axial one and thus widening is comprehensible. Also tubular cells with thin axial rib thickenings are on close consideration not at all exceptional since the thickenings do not change the over-all transverse structural predominance. True deviations are epidermal cells of apical meristems (p. 108, Fig. 16) and collenchyma cells (p. 111, Fig. 17). In these, the axial thickenings predominate heavily and the problem arises why these cells simply lengthen. If free, a collenchyma cell would, if its turgor pressure would be high enough to cause growth at all, probably widen. The epidermal cell in question would likewise not lengthen, but would probably become U-shaped with the outer wall at the concave side (cf. Van ItorRon in Dichl eta,?.,1939, p. 781) (see Fig. 26). Undoubtedly the reason for this morphologically surpri8ing direction of growth in so-called passive growilb (Kiister, 1956, p. 626), a type of growth that was quite acknowledged by classic plant anatomists, but neglected in the last few decades. The epidermal and collenchyma cells am subject to very high additional axial stress, generated by other cells in the tissue. This is demonstrated by the well-known curvature to be observed when growing stem apexes, or petioles with collenchyma, are split lengthwise. Also by the appearance of undulations both in epidermal walls in tangential sections of Helianlhw hypocotyls and of

-

ULTRASTRUOTURE 01’ T H E W A L L I N GROWING OELLB 131

Epidsrmol call

/

,free 7

Passive growth

Collenchymo

--Hi4

I

cell

Cambium initiol

Fro. 26. Diegram of ex~mp1esof pmeive growth.

Avenu coleoptiles and in collenchyma cells (p. 111). Taking a coleoptile as a whole, transverse structure still dominates sufficiently to explain the axial growth. In this connection, it might be noted that the epidermis in the Avena coleoptile in fact limits the growth rate (a fact which seems not always to be fully realized, e.g. by those who in the study of growth-wall relations analyse the wall of whole coleoptiles). Cells may also acquire quite abnormal shapes as a result of passive growth as e.g. the star-shaped cella in the pith of Juncus leavm and the retort-shaped cells in aerial roots (cf. Kuster, 1956). If cells do not grow fast enough, tissue tension may even tear them aa in the well-known examples of primary tracheary elements with annular or helical thickenings and as in the stamina1 filaments of Anthomnthum and the seta of Pellia in which the central cells are torn so that these organs become hollow and filled with ah. A less specfacular example of passive growth in a structurally unexpected direction ie possibly the radial wall of the cambium initial, which widens although

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P. A . R O E L O F S E N

the structure is transverse. It may be passive widening, but in principle it may also be a cme of impossibility of lengthening, so that the wall only yields in the direction of least extensibility (see Fig. 26). Similarly questionable is the cause of the widening of young apical meristematic cells (Fig. 14A).These problematic cases are only mentioned to point out that tissue cells may acquire “abnormal” shapes a i a~ result of both passive growth and imposed stagnancy.

2. Spiral growth in cells with multinet .structure If in the wall of a tubular cell the mean microfibrillar arrangement is neither axial nor transverse but oblique, both lengthening and widening will be accompanied by a rotation of the cell around its axis, a twist. One may also Ray that the extension is along a spiral (although helix is more correct). Since these cases demonstrateeven more strikingly the effect of wall structure on the direction of growth, we will discuss them in more detail, at least in so far as they were studied (there are many ca8es which so far have received no attention). A right-handed or dextral helix, as occurs in a woodscrew, is called a Z-helix; a left-handed, or sinistral one, an S-helix; since in the upper parts the direction is similar to the middle part of Z or S. (In old botanical literature Z-helices were called left-handed.) S-Helical growth corresponds with clockwise rotation of the tip as seen from above, which therefore is called a sinistral rotation. ( a ) Tradescantia stamina1 hairs. During the rapid elongation of the cuticle-free stamina1 hair cells of Tradescantia (p. loo), a twist is to be observed and in fact there is a flat helical structure in the wall (cf. Roelofsen, 1959,p. 187).Twist during normal growth has not been studied, but is very likely in view of both the well-known helical striations in the cuticle and the helical protoplmm streaming. Both Z-helical and S-helical structures occur. (b) Phycomyces sporangiophwea. More research has been done on spiral growth and wall structure of sporangiophores of P h y m y m (cf, Roelofsen, 1969,pp. 184,298)Growth, both in length and in girth, is restricted to the apical 2 mm, which, as long as there is no sporangium, includes the tip. Before and after sporangium formation, the tip (or the sporangium) usually rotates clockwise as seen from above, both during growth and on squeezing, either by hand or in an “ironlung” device by increasing the air pressure (Roelofsen, 1960). Thus, growth is usually in the direction of a steep S-helix that intersects the cell-axis under 6” on the average (angle a in Fig. 27). Extension by pressure induces about two-thirds of the rotation per mm lengthening that was observed in the same sporangiophore during the preceding growth. This shows a dominating role of the physical

U L T R A S T R U U T U R E O F T H E W A L L I N O R O W I N Q CELLS 133

wall properties in determining the direction of growth. The author once (1950) believed that some direct influence of the cytoplasm on the direction of the yield of the wall, called “active intussusception”, had to be accepted to account for the missing third of the rotation. However, the evidence was inconclusive, since there are apparent differences in the two extension processes (unlike pressure-extension, growth is slow, at a constant pressure and without disturbance of the cytoplmm). The wall ultrastructure is typically multinet, Fig. 12, with a mean chitin microfibril orientation along a flat Z-helix. This could be demonstrated in several sporangiophores by unwinding strips of wall. Model experiments with cellophane tubes showed that an S-helical twist is aotually to be expected on increasing the pressure in tubes with a flat Z-helical structure. However, a corresponding Z-helical major extinction position ( m a p . angle y in Fig. 27) between crossed nicols could only sometimes be observed and moreover only after chemical treatments and after enhancement of the birefringence with congo red. I n most cases the extinction was practically transverse and in any case several degrees lower than the 6’ in Z-helical direction that would occur if the average microfibril orientation would be normal to the direction of growth. Hence, p is always less than a. We will return to this point in paragraph ( d ) . Another fact indicating the major role of wall structure in directing the growth is that during some time after the resumption of growth following the formation of the sporangium, both growth and pressureextension are accompanied by dextral (anti-clockwise) rotation. Explanations that were suggested for this ephemeric reversal of rotation are : the effect of the steep S-helical inner secondary wall and of the outer wall part that probably was pulled out to a steep 8-helix during previous normal growth. Steep structural S-helices would induce widening accompanied by dextral rotation (Fig. 27). Widening actually occurs in the growth-zone of both young and mature sporangiophores (p. 127). It may be remembered that twisting in sporangiophoresof Phycumyw was not discovered during their lengthening but during the widening (with concomitant small lengthening) of the sporangiophores of the variety piloboloida. In 1916 Burgeff observed in these a flattening in the angle of protoplasm streaming and a strong S-helicalrotation of the sporangium (which in our view sugge8ts that a Z-helix with values of y greater than 46’ was the dominant structural helix in this o w ) . Flattening of the streaming helix has also been observed in sporangiophores of the normal variety, that widened as a reoult of maltreatments (cf. Roelofsen, 1969, p. 301). It may be noted that in the growth zone

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P. A . ROELOFSEN

the protoplasm does not stream. Streaming is only seen below this zone, where secondary wall is deposited. However, the directions of streaming, of the m.e.p. of the secondary wall and of previous growth do coincide. (c) Nitellrt internodal cells. Another classical example of spiral growth, that was intensively studied during the last ten years, is the internodal cell of N i t ~ Z h( ( h e n , 1958a,b, 1959, 1960a, 1962, 1963a,b; Green and Chen, 1960; Probine and Preston, 1968, 1961, 1962; Probine, 1963; Kamiya et al., 1963; older literatlure: Roelofsen, 1969, pp. 187, 290). The wall structure is typical multinet and in this cam polylamellate, Fig. 12. The hemispherical apical cell has the pattern of type 5 XA, Fig. 11. The internodal cell grows along its whole length from 0.03 to 30-100 mm and at the same time widens from 0:l to 0-5-0.75 mm. The percentage increase in length is constantly about 4.5 (3-5-6 in different cells) times the percentage increase in breadth. So there is a constant inequality of wall yield, which produces a tubular cell (Green, 1963b). In N . axiZlaris (object of Green, presumably also N. opaca, object of Probine and Preston), cells up to 0.3 mm length have no spiral growth. Then growth becomes accompanied by a rapid dextral rotation, so that in cells of only 0.6 mm length, both the helical files of chloroplmts, the two chloroplast-free striations and the streaming direction of the protoplasm, have become Z-helical. The angle 6, in the figure below, is about 40". I n this case, both species follow the peculiar, changing helical growth pattern as indicated by the data in Table 11. Up to a length of about 10 inm the direction of growth (angle a) stays Z-helical. Nevertheless the streaming angle 6 becomes steeper ; this means that a must have become less than 0. The helices of streaming and of growth me by no means identical, as is sometimes assumed. The former is merely the integrated state of twist, the summation of the differentiated growth events to which the cell was subjected previously. The actual helix of growth at a given time has not been deterniined in Nilella (the data of Green, 1954, would allow it). While elongating further, the growth (a) becomes S-helical, corresponding with a sinistral rotation. This, of come, untwists the streaming angle even more, but at the end a net Z-helical orientation, with an angle 6 of a few degrees, is still left. If a cell is forced to grow between pressed glass plates, so that it does not lengthen or twist, but only widens, the streaming helix is flattened (Green, 1963s). With N. opaca, the twist due to an incrercse of turgor pressure was studied (Probine, 1963). Its sign always corresponds with the growth (a) at that time, but in how far the consistency is quantitative remains

U L T R A S T R U U T U R E O F T E E W A L L I N O R O W IN O C ELLS 186

TABLEI1 Dda on Spiml Growth of Nitella Internodal Cl'elle Diameter (mm) Length (mm) Direotion of growth, a (andpreseure twist ' with N. qpaea) Major extinction position (m.e.p.) q o =ria stmclmingangle e (inhgpae b b of twist)

} {NN:

0.13 0.6

0.2-0.6 10

dextral, 2..change over 10-13"s 3vz

0.4-0*7 30-100

.. s i n i ~ h lS,

m

06°S-10Z

14'2

2-4"2

nil to lees then 4"s

Cell axis

Upper cell wall

to be determined (in PhyGmycee it is two-thirds, see above). Whether N. axilkcris shows pressure twist is unknown (P. B. Green, personal communication). It is certain that there is helical structure in the wall of both species. Helical unwinding of strips in torn walls has been observed in both (Probine, 1963 ; P. B. Green, personal communication). Birefringence and obliquity of structure are much higher in N. opaca than in N.asiZZuri.3 in which the major extinction position (y)is nearly transverse (sometimes S-helical obliquity is suggested, but it could never have been more than 4'). In N. 02~;cathe m.e.p. also appears to be S-helical, except in the final growth stage when transverse and very low Z-helical orientations were found. Two facts merit special mention. Although, unfortunately, not the angle of growth ( a ) but only the integrated state of twist (0) is known, it is evident, that, as waa observed in Phycomycm, the overall structural obliquity indicated by y must be very much less than the obliquity of growth incidated by a. In very young cells, 0 and a cannot differ much and here y is about ono-third of 8. A possible explanation will be discussed in the next section. hcondly, it should be noted, that in N. o m the reversal of twist (during growth and on increase of pressure) from Z to S does not coincide with the change in the overall structural helix y from S to Z, which occurs much later. Hence during a certain period both a and y are S-helical. Opposite signs are usual, and, as will appear in the next

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section, coniprehensible. An explanation of this deviation waa not advanced. As a possibility we see a similar one as suggested for the temporary reversal of rotation in Phycomyces (p. 133), viz. an effect of the outer wall part, that very probably has during previous growth acquired a steep Z-helical structure (angle 0). (d) Mechanism of spiral growth in walls with multinet structure. Theories on the mechanism of spiral growth in plant cells have been discussed e.g. by Castle (1963) and the author (1969, p. 186). They mainly centred around the spiral growth of P h y m y c a sporangiophores Rince this had been the sole experimental object. As mentioned previously, all investigators of the structure of growing walls now seem to agree that the cytoplasm determines the direction of yield of the wall through the intermediary of the microfibrillar structure of the wall. Therefore theories on spiral growth involving direct effects of the cytoplasm need not be considered further; nor need structural theories that have become incompatible with present knowledge of the ultrastructure of growing walls. As far as walls with simple multinet structure are concerned, there remains only one basic concept. It originated, rather self-evidently, from the finding some thirty years ago that, as discussed in Section IV €3 I , a wall yields the most at right angles to the predominant microfibrillar orientation (see Fig. 21A). At that time, steep S-helical yield to pressure was observed in Pitycornyeea growing S-helically, and since also a flat Z-helical m.e.p. was found, at least in some cases, the spiral growth was simply taken as being due to wall extension at right angles to this structure. This is shown diagrammatically in Fig. 27A). It should be noted that this implies that the angles a and p in this figure must be equal. However, as mentioned above, it appeared later that both in P h y m y c e s and in Nitellcc y is invariably much less than a. A mechanism that might explain this discrepanoy waa postulated by the author (1958) and is shown schematically in Fig. 27B. In harmony with the observed very low obliquity of p, nearly all microfibrils of the dominant inner part of the wall are supposedto be orientated transversely, or nearly so, and to make part of microfibrillar bundles encircling the tubular cell. A small number of the microfibrils in this part of the wall are supposed to be obliqueIy orientated whereas the majority of the latter are orientated in the Z-helical direction, for example. Only the effect of this numerically small majority need be considered further. Evidently, these microfibrils will have points of contact and coherence with several transversely orientated microfibril bundles. On elongation of the cell, these 2-helically orientated microfibrile will induce mutual shifts between the transverse bundles that are being separated axially. As a result, the transverse bundles will tend to move a little pest one

ULTRAS TRUCTURE O F T H E W A L L I N G R O W I N Q UELLS 137

&\ T /-----?A i& A

T

------

f

E’ro. 27. Diagram of the mechanism of helical growth and rotation. A and B during lengthening of a tubular cell with flat 2-helicalwall structure. A: original concept; B: aocordbg to h l o f s e n (1D68), to explain emall structural angle w aa compared with growth angle a; C :during widening.

another in the directions indicated by arrows. The summation of a huge number of such minute, but similarly directed, movements might well result in an appreciable rotation of the cell. As a result of this mechanism the angle a of the resulting spiral growth might become considerably greater than y. In principle this mechanism is related to the “scissor-gate” effect postulated later by Kamiya et al. (1963) and Green (1963b) to explain other effects (see p. 124). The origin of spiral growth on the basic concept mentioned was treated mathematically by Probine (1963). In this treatment the possibility of difference between a and y is included and it is not at variance with the mechanism proposed by us. A semiquantitative treatmeqt WM also given by Preston and his co-workers (Preston, 1948) some years ago. It must be admitted, however, that the concept certaidy oversimplifies the point, since it disregards the presence of multinet structure and, therefore, of wall parts transitional between nearly transverse and nearly axial. Its sole merit is to make the discrepancy between the obliquities of structure and of extension comprehensible.

138

P. A . R O E L O F S B N

As was observed in Phycomyccs sporangiophores, and as is selfevident, helical structures will also cause rotation in widening c e b . Presumably such cells will have steep helical structures with a pitch of more than 45". Otherwise they would not predominantly widen. As was apparent from unpublished model experiments with cellophane tubes carried out by w, the direction of extension and of rotation may also be expected to be opposite in sign to the structural helix here. (This is shown schematically in Fig. 27C.) It seems difficult to imagine a process analogous to B in this cam. 3. Spiral growth in cells with helically cros8ed lamellate walls Recently, spiral growth has also been detected and studied in Clauhphora and Chadomwpha by Prei and Preston (1961b). The filaments rotate when the cells elongate or widen and therefore often become entangled when cultivated in undisturbed water. The rotation per mm lengthening remains constant for any one filament for weeks, but it varies from 170"to 600' in different filaments and is about inversely proportional to the mean width of the cells. The rotation increases in hypotonic solutions temporarily reverses in hypertonic solution (while shortening) and then returns to the normal course, but at a slower rate and in harmony with a reduced elongation. I n sign, the growth spiral is always opposite to the flat structural spiral A in Fig. 13 and similar, but less steep than B. Its angle with the axis (a)varied in the filaments studied between 44 and 28, and was on an average 38". This explains why the B lamellae, on passing outwards in the wall, become less steep whereas the A lamellae become steeper. The rotation per mm lengthening that waa observed was quantitatively consistent with the observed changes in pitch of both structural helices. It suggests that the direction of growth is completely determined by the wall structure. The sign of rotation during lengthening indicates that in this process t.he A lamellae are mechanically dominating. I n accordance with the situation in Nitella and Phpmnyca, we see that the angle of growth (a)exceeds the obliquity of the A lamellae. I n late sewon the cells widen. Then both the initial orientation of the A lamellae and their orientation throughout the wall become steeper, and those of the B lamellae become flatter. The sign of rotation during widening is not known, but the changes in lamellar obliquities suggest in our view that the sign was the same i w during lengthening, and thus opposite to that of the structure in the A lamellae. This, in its turn, would suggest that the A lamellae are also mechanically predominant during widening.

ULTRASTRUOTURE O F T H E W A L L I N Q R O W I N G OELLS 139

C. THEORIES ON THE MEUHANISM OF ORIENTATED INITIAL SYNTHESIS OF CELLXJLOSE MIUROFIBRILS

I n Sections I1 A 7 and D it appeared that very probably the miorofibrils arise as a result of polar or bipolar tip growth both on the inner surface of the wall and within it very near this surface. Here we will discuss theories on the mechanism which determines their initial orientation.

1. Protoplasm-streaming theory The old hypothesis, that streaming protoplasm determines initial orientation either directly or indirectly, must clearly be rejected. So far, in growing cella no streaming parallel to the initial cellulose-orientation has ever been observed. In many caaes, there is no orientated streaming at all, although there is highly orientated synthesis, e.g. in Phgmyces, Spirogyra, Valonia, Chaetonwrpha and C h d o p b a . I n other caaes them is orientated streaming, but in a direction nearly at right angles to that of the microfibrils, e.g. in Tradacantia stamina1 hairs, Nit&, T r k m root hairs and, probably, cotton hairs (Denham, cf. Roelofson, 1969, p. 230). However, in secondary walls a coincidence in directions often, though not always, occurs (cf. Roelofsen, 1969, p. 262) and protoplasm streaming has widely been held responaible for cellulose orientation in this cam. If there actually is a causative relation then the orientatingeffect of the cytoplasm must be indirect. A direct effect, e.g. on loose ends of microfibrila, is excluded, since the outermost cytoplasm is stationary and, moreover, since the microfibrils very probable arise outside the plasmalemma. With reference to the spiral structures of cell walls, Thiele (1964) described physico-chemical model experiments, in which spiral structures were produced as a result of streaming in combination with precipitation of polymers by a gradient in ionic concentration. These me interesting, but have little to do with botany. 2. Wall-8treastheory Some thirty years ago, Van Iterson suggested that the predominant transverse stress in tubular cells would not only be responsible for the retahment of transverse orientation during elongations (see p. 106), but also for the initial deposition in transverse direction. This theory was supported by the present author (1968, 1969, pp. 170, 266). Initially, the strain caused by the predominant stress would orientate the microfibrila transversely in a young tubular cell. Microfibrila formed subsequently would be similarly orientated, either by template action I

140

P. A . R O E L O P S E N

or as a result of facilitated intussusception into the transversely orientated meshes that naturally arose as a result of the axial extension, or by both mechanisms, The change in initial orientation on deposition of the secondary wall wm suggested to be due to reorientation in the plasmalemma, as soon &B its growth would no longer keep pace with the extension. It waa realized that at lea& one objection could be made, viz. that the theory failed completely with alternating crossed lamellate walls. Other objections resulted from a series of elegant and significant experiments with NilpZZa by (heen (1962, 1963a) and by Green and Chen (1960) of which the main o n w will now be mentioned. When grown in 0-35(;/, colchicine solution, NiteUa cells start to deposit isotropic wall lamellae on the pre-existing transverse ones and with further growth finally become spherical. On removal of the alkaloid, transverse order and lengthening are restored again. This proves that there is no (or at least not decisive) template action of the pre-existent wall. This is also apparent from the covering by isotropic spots in the wall of orientated lamellae in the pre-swarming stage of Cktorncnpha cellR (p. 90). Further, if all stress and strain, both axial and trwverse, were eliminated by constraining Nit& cells in a tight jacket, deposition of transverse inner wall lamellae still continued. Hence, no stress or strain was needed for preserving orientated synthesis. This was not merely template action since jacketed colchicine cells restored transverse order on removal of the drug, and while still being constrained in the jacket. Here, transvereely orientated microfibrils were deposited on an isotropic waU, in which no stress occurred. Finally, Nitella cells were cultivated in 10% Carbowax solution to reduce turgor pressure, and then they were stretched by a submerged cork or via a pulley by weights, so that the predominant stress in the wall became axial. This only induced a greater part of the wall to be axially orientated (“super-multinet” growth, p. 126), but the inner lamella continued to be deposited transversely. This experiment also suggests that transverse stresses or strains are not causes of traneveme synthesis, but unlike the previous one, does not, in our view, furnish conclusive proof, since transverse stress might still have predominated in the innermost lamellae. Taken as a whole, these experiments clearly disprove that stress in the wall or the.strain it causes would determine the initial orientalion of microjibm’l 8ynthm.8. We wish to emphasize first, that they do not in any way disprove that transverse stress is necessary in elongating ceb to retain transverse orientation, and secondly, that they support the occurrence of passive reorientation by axial strain. These points do not

U L T R A S T R U C T U R E O F T H E W A L L I N CSROWIKQ C E L L S

141

coiicern tjhe initial orientat,ion and have been discussed in Section I V A 5.

3. C:ytoplasmicelement theories Green (1962, 1963ti) postulated a new theory, vis. that ux:iuZZy orientated, laterally h i d e d , cytoplasmic threads would occur in NiblZu and would induce the niicrofihrik in the adjacent inner wall lamella to grow at right, iknglen to the threads’ i L x i N .

FIG.28. Olilicluc nrrtion though re11 wall ( c n ) and outer cytoplksma of a growing cell in n rout of Phlrua pmlenee. The mic.rotuliuleH (mt) run parallrl to the microfibrils in the adjacent wit11 t ~ r i t lprohal)ly ctetcrminr their initin1 oricmtation (from Ledbetter and Porter, 1963).

However, since he nniendetl this theory (19G3b) in the face of the discovery of .bam?wme/y orientatcd microtubules in growing tubular root cells by Ledbetter and Porter (1!)83, 19fi4), t h k latter important finding may be discussed first. A s is visible in Fig. 28, numerous tubules of about 250 A diameter and at least several /I, long were foirntl lying in the outer cytoplasm

142

1’.

A . EOELOE’SEN

bet ween ent toplmniatic reticulum and plasmalemma of growing root cortical cells. Some may he attached t o the plasmalemma. Each consists o f a cylinder of thirteen elementary tubules. They are identical in morphology to those found in flagellae and in mitotic spindles of plant and animal cells, except that the latter are a little thinner and consist of somewhat fewer elementary tubules. Especially significant from our point of’ view is their orientatkm, which is parallel to the microfibrils a t the inner surface of the adjacent wall. Thus they are arranged tmnsvcrsely near side w a h of tubular cells and at random near their crose walls. The discoverere suggested that they might undulate and in this way generate protoplasm streamirig parallcl t o their long axk, which would induce thc calluloee orientation, Also, that they might be cut off and act as primers for microfibril synthesis, but such separation was not observed. (It appears t o us that the fibrillar bodies observed by Frei and Preston, 1961a, Fig. 52, at the inner wall surface of Chaetomorph may be similar microtubules.) Hepler and Newcomb (1964) found identical tubules lying parallel t o the cellulose microfibrils in developing bands of secondary thickening in parenchyma cells redifferentiating into xylem cells. I n addition, clusters of similar but thinner (130 A) osniiophilic fibrils with less dense cores were found in cisternae of the adjacent granular endoplasmatic reticulum lying deeper in the cytoplasm. These authors conjectured that the “tubules” might determine the deposition and orientation of the microfibrils and that the “fibrils” might be associated with polysaccharides or developmental stages of microfibrils. I n view of‘these observations (heen (1963b) supported the suggestion that the microtubules determine the orientation of the microfihril synthesis. He expressed the view that they may repreuent the “taut transverse bands” in the cytoplasm which he had found necessary t o explain the stability of the transverse microfihrillar synthesis in Nitella. His finding that colchicine, known to disorganize the mitotic spindle, induces the formation of isotropic walls in Nitella confirms similar observations of Gorter (1945) with root hairs, although the evidence that colchiciiie actually disorganizes the ectoplasmic tubules is not a t hand. Colchicine was already known t o produce more or lem spherical cells in (!ham (Delay, 1957) and in apical nieristems, leaves and protonernata (Biinning, 1957). Another argument in favour of this role of the microtubules, in our view is that a t last one mechanism in both primary and secondary cell wall forination may be held responsible for the orientation of cellulose synthesis. Previous theories applied to only one of these and thus two were needed. As mentioned, microtubules were found aligned along

UL T RAB T RU O T U R E O F T H E W A L L I N U R O W I N G U E L L S 143

bands of secondary thickening and it may be remembered that colchicine does, in fact, prevent the deposition of thickenings in &+urn leaves (Biinning, 1957). However, the additional suggestion of Ledbetter and Porter (1963) that the microtubules might act aa primers for microfibril synthesis, i unlikely in our view, since there either before or after being cut off, A probably is no strict apposition (see Section I1 D) and since quite similar microfibrila are produced by Aeetobacter xylinum without microtubules (see p. 86). Their suggestion (1963, 1964) that protoplasm streaming generated by the microtubules might be involved seems even less likely (p. 139). Instead, some physical condition or some force must, in our view, be generated by the microtubules, that can direct cellu2oee aynthesia at 80me dktance. For instance, the growing microfibril tips might be polarized through induction by an electric current running along the microtubules, possibly generated by the flow through or along the tubules of ions or eleotrons. Perhaps one may try whether cells with oriented cellulose synthesis can orient the synthesis extracellularly when mounted in a synthesizing cell-free system obtained from Acetobarcter qZinum or from meristems. However, the supposed role of the microtubules is at variance with theories accepting simultaneous cellulose synthesis in two different directions, e.g. transverse on the inner face and axial on edge thickenings deeper in the wall.* If the microtubules direct the initial microfibril orientation, the next question is what directs the microtubules ? Green (1963b) suggests that in elongating tubular cells they would retain their tramverse position in the cytoplasm by virtue of their tautness that would be the result of the simultaneous increase in girth. Thus a similar “loop around inflated tube rectifying mechanism” would occur as, according to the multinet growth theory, would be responsible for the retainment of transverse microfibril orientation in the growing wall. As an explanation of the apparent reorientation of the tubules when Nitella cells are induced to produce lateral protnrsions, he suggests that the second type of protein fibrils, found by Hepler and Newcomb (1964), and situated deeper in the cytoplasm, might be involved. I n our view this microtubule-stress rectifying mechanism cannot determine the initial microtubule orientation but might indeed play a part in retaining transverse orientation. However, it is apparent that

* A mechanism has already been suggested for simultaneous deposition of cslluloee microfibrils in two or three directions simultaneouely (Preeton, 1964) baaed on obaergetions on elgal cell walls (Frei and Preston. 1961s). “his ViaUeliEsll grandee about 300-800 A diameter in cubic close packing et the cell amtiace. As mentioned on p. 85 such grsnnlae were in fact o k e d in contact with gluoan &rofibrils in yeaat.

144

P. A . R O E L O F S E N

if this occum it is quite subordinate to some other orientating capacity in the cytoplasm that on it8 own can preserve transverse orientation, that further can change the orientation at will, and that also haa directed the initial microtubule orientation. We believe that two of Green’s own experiments that disproved the wall-stress theory indicate this subordinate role of an eventual microtubule-stress rectifying mechanism namely, the retainment of transverse orientation in jacketed cells (no widening, and hence no stress) and the re-establishment of transverse orientation on removal of the alkaloid in jacketed (no stress) colchicine-treated celh (no pre-existent order). Sovereignty of an unknown cytoplasmic factor also appears from the observation of Setterfield (1961) that in a horizontally grown pea stem, the cells in the upper part develop axially orientated thickenings, while in the lower part of the stem, transverse orientation is retained. Rotation of the stem through 180” also reverses the direction of deposition. This hierarchy is also indicated by the occurrence of changes in initial cellulose orientation in the apparant absence of changes in the orientation of ectoplasmic strain. For instance, in the crossed lamellate wallsof somealgae (Figs. 10 and 13), in latexvessels (Fig. 15), in the preswarming stage of Chaetomorpha (p. lo]), and in the development of poles in the wall structure of settled swarmers of Cladophorales (p. 95). A study of the microtubule orientation in these cases would be extremely interesting. Like the rectifying mechanism, template action must also, if it occurs, be assigned a subordinate role. That it might occur to some extent is indicated in oultures of Acetobacter xylinum on agar (Millman and Colvin, 1961). The expression “polarity” for this cytoplasmic orientating mechanism at the heirarchic top imposes itself, but has unfortunately, at present, little factual meaning (cf. Biinning, 1957). It seems that the explanation why in some cases “polarity” causes transverse initial cellulosesynthesis, in other cases constant helical or changing helical orientations, why random orientation occurs in cross walls and in tips of polar cells, etc., must evidently await elucidation of the essence of “polarity” itself.

ULTRASTRUOTURE,OF THE WALL I N UROWINU UELLS

146

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The Protein Component of Primary Cell Walls

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DEREK T A LAMPORT*

RIAB. Baltimore. Marylud. U.S.A. 161 I. Introduction ............................................................ A SoopeandDefinitions ................................................ 161 B Hi~toriaalPempeotive 1888-1969 ....................................... 162 166 I1* Experimental Methods &ad lldotel’bh ....................................... A. Cell Suspension Cultures ............................................... 165 B Whole Plants 157 C Amlytioal Teohniquea 158 180 I11. The IIydroxyproline-richWall Protein: “Extonsin” A Intre-oe~d&w Looation of Hydroxyproline 180 B Chemioel OhtmoteriCetionof 4-trad1ydroxy-~-pmIine 167 C. The Amino Aoid Compoclitionof Primary Gll-wella 168 D Emymio DegradationandCharaotarizationof Wall Protein 171 E Dinulphide Bridp in Cell-wall Protein 172 F Distribution of the Hydroxyproline-rioh Wall Protein in the Plant Kingdom ...174 177 I V . The BioeJmthesis of “Extemin” ........................................... A . Uptake and Incorporation of 14C-Prolineby Intact Cells 177 B. Proline Hydroxylation ............................................... 164 188 V . V d t i o n of Cell-wallHydroxyprolineContent A. Walls Iaolated fmm TiesueCulturea ..................................... 188 B Wells bohted from Plant P&s 189 183 VI Degradation of the Sycamore Primary Cell Wall A Chemiod h p d t b t i o n ................................................ 193 B Emymio Degradation 194 198 VII. A Tentative Pioture of “Externin” in the Primary Wall VIII. The Contribution of “Externin” to Wall Form and TensileStrength .............200 204 IX EncymioWallF’rotem ................................................... A Aworbio Aoid Oxidsae ................................................ 204 B Hydrolyeeee ......................................................... 206 C. Other Wd1-bound Enzymes ........................................... 205 D How doea the Well Bind Enzymes ? ...................................... 206 208 X . The Role of “Extamin” ................................................... Acknowledgements ...................................................... 213 Referencea .............................................................. 21:1

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I . INTRODIJUTION A

. SCOPE AND

DEFMITIONS

It is the aim of this review topresent data and discuss oell-wall prolain from several points of view ranging from the purely enzymio aspects (oell-wall enzymes) to the structural hydroxyproline-rich wall proteins Although the subject of wall protein is hardly a new field it does begin to look as though the demonstration of wall-bound hydroxyprohe

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*Now at the moJ&8n state University Atomic Energy Commhion Plsnt b r & Laboratory

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has imparted a-revivifying stimulus to an old problem-this is more evident from work known to be in progress than from the amount of published material, Therefore, material has been included which is as yet unpublished or published only in abstract form. “Primary wall” has been used throughout as defined by Kerr and Bailey (1934), on the basis of phylogenetic, physiological, chemical and physical criteria: “The term primary wall . . should be used solely in designating the cambial wall and its homologue8 in other tissues . . .” where the cambial wall or homologous* structure they defined physiologically as a “. . . wall capable of expansion and of increase in 8urface area . . .” with a corollary that “. . . layers of secondary thickening are not deposited until the cell has attained its mature diameter”. Physically and chemically they defined it by writing: ‘ I . . . the anisotropic cambial walls contain both cellulose and polyuronides”. Electron microscopic studies (see Roelofsen, 1959) show that we must recognize the more or less randomly interwoven cellulose microfibrila as another definitive characteristic of primary cell walls.

.

B. HISTORICAL PERSPECTIVE 1888-1959

Do cell walls contain protein? This simple question excited controversial discussions almost as soon as early investigators made a distinction between cell walls and cytoplasm, As early tw 1888 Julius Wiessner replied to a barrage of criticism with positive statements-that the growing cell wall contained protein, and that the growing cell wall waa a living structure : . . die Zellwiinde sind, zum mindesten so h g e sie wachsen, eiweisshaltig, . . . das Wachsthum der Zellhaut ist ein actives, und dieser uberhaupt bis zu einer gewissen Grenze ihres Daseins ein lebendes Gebilde”. I n the following years opinion oscillated violently, as a result of differences in experimental materials and methods. At one extreme were Tupper-Carey and Priestley (1924). They were the first to isolate cell walls from meristematic tissues ( Vicia faba). They fractionated the isolated walls, analysed the fractions (protein, cellulose, pectin, etc.), and concluded that the meristematic cells had walls containing protein. At the other extreme, Wood (1926) questioned Tupper-Carey and Priestley’s results and concluded from histochemical data that “. . . more than 0.001% of protein does not occur in the cellulose cell walls of any of the plants examined . . . if indeed any is present”. I n between the extremes most other workers found protein associated with isolated cell walls (Tupper-Carey and Priestley, 1924; Thimrtnn and Bonner, 1933; Christiansen and Thimann, 1950; Tripp et al., 1951; I‘.

* For a recent discussion of homology, Bee Grimstone (1969).

THE P R O T E I N COMPONENT O F P R I M A R Y UELL WALLS 163

Bishop et d.,1968; Dieckert and Snowden, 1960; King and Bayley, 1963), but were unable to rule out the poesibility of cytoplasmic contamination. This last possibility was emphasized by the view that, during wall biosynthesia, cytoplaam becomes inextricably mixed up with the wall, although it is not apparent why the secondary wall should contain so much leas protein than the primary wall. Other more general considerations led several workers to consider the possibility of cell-wall protein. From a consideration of how cells grow, Tupper-Carey and Prieatley (1924) considered that new walls “. . . commencing as interfaces in a protein-containing medium may be regarded as composed at first mainly of protein”. Later, h s t o n (1956) ooncluded from 8 study of microfibril deposition that “the inevitable conclwion seems to be that in these growing walls the cytoplasm interpenetrates the wall . . . that the wall is not & dead envelope but marks instead the outer limits of the living cytoplasm”. Frey-Wyssling (1969) emphasized the difficulty of the problem: “Am schwerigsten ist der Eiweissgehalt der wachsenden Zellwiinde zu beurteilen.” The difficulty of “estimating” cell-wall protein arose from lack of oriteria, but once these were formulated the problem became less formidable. The criteria for cell fractionation formulated by Schneider and Hogeboom, applied t o the problem of cell-wall protein, gradually led to the appearance of methods for distinguishing specific wall protein from oytophsmic contamination. “Although the purification of enzymes is not a primary objective of cell fractionation, purification of subcellular structures is” (Schneider, 1967). This is the ruieon d’bre of cell fractionation. The very first step, then, is to explore methods for isolating the subcellular structure of interest-here the primary cell wall-bearing in mind that some experimental materials are more amenable than others. Theoretically there are four or more choices for isolation of the cell wall :

I

(i) selective (chemical or enzymic) extraction ; (ii) autolysis ; (iii) osmotic lysis ; (iv) cell rupture and differential centrifugation of the released subcellular structures.

In practice the fourth alternative presents least difficulties, and its use will be disoerned to a greater or less extent throughout this review. To complete this brief historical survey and to orientate the reader towards a more critical appraisal of the highly speculative conclusions of this review it is worth while even at this stage to jump ahead of the

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data and map out quite a different line of reasoning which not only points to the presence of cell-wall protein but also raises the possibility of the direct involvement of wall protein in cell extension. From his classical studies of “The Mechanism of Cell Stretching” Heyn (1931) concluded that wall plasticity was auxin dependent. Here it is important to note that, by Kerr and Bailey’s (1934) definitions, auxin dependent changes in plaaticity are restricted to the primclry wall. Bonner (1935) suggested that changes in wall plasticity depended upon labile cross-linkages (Haftpunkte) between cellulose micelles. The chemistry of these cross-linkages was quite unknown (hence the noncommittal term Hafpunkte), but in 1937 Ruge proposed that wall p k ticity arose from changes in the pectic substances. This propom1 stimulated further study of pectin metabolism, and in its most refined form appeared as Bennet-Clark’s (1966) hypothesis that wall plasticity was enhanced when the carboxyl groups of pectin were methylated. Conversely wall plasticity would be low following demethylation and calcium bridge formation. This hypothesis found support in the work of Glasziou (1969) which pointed to cell wall pectin methylesterase as controlling pectin estorification. Sat0 (1966), Ordin et al. (1966) and Albersheim and Bonner (1969) also viewed pectin esterification as perhaps the chemical basis for changeR in wall plasticity. They showed that auxin enhanced the incorporation of W-methyl groups from methionine into pectin. But Jansen et al. (1960a) could not detect an effect of auxin on the net cell-wall methyl ester content. Jansen et al. (1960b) also disagreed with Glasziou’s conclusion that the effect of auxin in promoting the elongation of cells was mediated through an effect on pectin methylesterase. Finally Cleland (1963a) has given what may well be the final blow to the Bennet-Clark hypothesis by showing that in coleoptiles the early effects of auxin on cell-wall methylation and elongation arc indvpended. Furthermore, Cleland (1963b) could find no significant auxin-induced increase in pectin methylation in three dicotyledonous tissues. He concluded that “the restricted occurrence of auxin-induced pectin methylation makes it unlikely that this process plays an important role in the growth and development of cells. It seems more likely that the enhanced methylation occurs simply because the methylating system in certain tissues is sensitive to one of the biochemical changes which occur in tissue8 treated with auxin.’’ The great merit of Bennet-Clark’s hypothesis waa to focus attention on cell-wall metabolism. But the question rem~im,which aspect of wall metabolism is responsible for plasticity? It is still profitable to return to Heyn’s (1931) work. He suggested two clear alternatives: “An increme in elasticity or plasticity of the wall could arise through altera-

T H E PROTEIN UOMPONENT O F PRIMARY OELL W A L L S 156

tion of the amount of wall substance available for cell expansion, or through a change in the quality of the cell wall.” Jackeon’s (1960) work supports the second alternative; the rate of root-hair elongation was enhanced significantly within a very few minutes after application of 10-ISM auxin. Ray and Ruesink (1962) report that auxin increases the growth rate of coleoptile cylinders after a lag of about ten minutes. Such rapid changes rule out the direct involvement of RNA, protein, and polysaccharide biosynthesis as the auxin dependent changes of wall plasticity; instead, changes of plasticity must result from the alteration of a pre-existing wall component. Buratrom (1969)reached a similar conclusion after he found that large growth responses were not accompanied even by small changes in cell wall composition. He concluded that “the regulators should act upon the structural organization of the cell walls, not specifically on the aynthesis of cell wall matter”. But, if changes occur within the wall, surely protein must be involved. If protein in the wall is involved in cell extension, how is this protein to be studied? Which systems are best? Which analytical techniques must be used? Wbioh criteria must be applied? I n this review an attempt will be made to amwer these questions and show how they lead inevitably to a reconsideration of one of the great problems of plant growth-the auxin question.

11. EXPERIMENTAL METHODSAND MATERULS A. UELL SUSPENSION UlJLTURES

7. Origin, maintenance, and ~pecialpropertiecr

There are three main advantages of using cell suspension cultures aa a source of primary cell walls. First, the rapid growth of a cell suspension culture generally eliminates secondary thickening or lignification ; secondly, such cella are readily broken by a variety of techniques, and the wall fraction is easily separated and washed by repeated centrifugations. Thirdly, gram amounts of cell walls can be obtained readily. In a d i t i o n to these advantages it is worth pointing out that fre8hly ieolated cell-wall suspensions can be pipetted accurately; thus, Rtandard wall suspensions can be prepared and the concentration expressed aa mg dry wall/ml. After freezing and thawing, cell-wall suspensions tend to become rather c l m p y and difficult to pipette-these clumpy wall swpemiona, however, provide better flow characterietica than normal cell-wall suspensione, for cell-wall columns desoribed later (Section I X D).

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D E R E K T. A . L A M P O R T

A t present, the origin and isolation of cell suspension cultures is at best an empirical process (cf. Lamport, 1964a), although there is no doubt that continued selection from an initially clumpy suspension culture sooner or later yields better cell suspensions (Scott et al., 1964). Once a suitable strain of cells in suspension has been isolated, reserve stock cultures can be grown slowly on agar elants. A further insurance against loss of a valuable culture arises from the convenience of cell suspensions as an experimental tool in several fields. Consequently, cell suspension cultures tend to become wideepread among laboratories. Three examples of fairly widespread cultures are Paul's Scarlet Rose (Nickel1and Tulecke, 1900; Weinstein et aZ., 1962), the pollen-derived Ginkgo (Tulecke et al., 1962) and a Sycamore (Acer pseurloplatanw) culture which was isolated by the author in November 1968. 2. Isolation of the cell-wall fraction

The cells of suspension cultures are, unlike many plant tissues, readily broken by the use of several different techniques (Table I). Among the most useful of these, at least for the subsequent isolation of the cellwall fraction, are those methods involving the use of glass beads (2 mm diameter for plant cells) in some sort of vibrating cell mill (cf. Lamport and Northcote, 1900a,b).Zillig and Hslzel's (1968) cell mill* has special advantages. It is able to accommodate either very small or large amounts of material in the different sizes of breakage cups, around which iced water can be pumped continuously. Routine microscopic examination gives an approximate idea of the degree of cell breakage. Generally no intact cells remain after one minute in the cell mill. The homogenate is decanted from the glass beads and the walls are obtained, by gentle centrifugation (e.g. 30 sec at 800 x 8). If a fairly dilute (e.g. about 6% packed cell volume) cell suspension has been broken, most of the cell contents will have already been removed and five or six water washes by centrdugation yield optically and electron microscopically fairly clean walls. If breakage conditiom are too mild, some nuclei survive intact and these tend to contaminate the wall fraction slightly. The isolated wall fraction can be cleaned further by washing with sodium chloride solutions increasing stepwiae in concentration (e.g. the author regularly uses, 0.1, 0.2, 0.6,and 1.0 M NaCl) followed by water washes to remove the salt. Depending upon the experiments in mind, the wall fraction can be used immediately, stored cold, frozen, or dried from the frozen state. Obtained from Edmund Buhler Company, Postfach 23, Tiibhgen, Gemany.

T H E P R O T E I N C O M P O N E N T O F P R I M A R Y UELL W A L L S

167

TABLEI Methods of Breakuge for Sycuwre Cell Suapenabn.9 Apparutue and principle Cell mill (Shockman et al., 1967). vertical vibra- lOOyo (1 min) tion et 26 c/s with glass beads 2 mm diameter Cell mill (Zillig and H(ilze1, 1968). Vertical vibra- 100% ( 1 min) tions at 60 c/s with glass beads 2 mm diameter Ultrasonic generator giving powerful oscillations 100% (1 min) at 26 kc/s (of. Dbvies, 1969) High-pressure extrusion (French and Mihier, 1966) 100% High-pressure shearing (Hughes, 1961) 100% Homogenizer (Dounce et al., 1966). Tight plunger 26% ball in cylinder Potter’s homogenizer. Rotating plunger in cylinder 6% Osmotic shock (a)by transferring cells from their growth medium 0% to distilled water* (b) by trensferring colls from a glycerol plasmoly6% ticum to diRtilled water* Freezing and thawing

O%t

~

Very good

very good Very good

Good Poor Good Good

Nil. Poor

Nil

~~~

The releaae of leucoanthocysnin having a high sbeorbency st 280 mp W M used 88 a sensitive indicator of cell rupture (cf. Goldstein el al., 1962). t The cell wall was not obviously ruptured, but judging from the release of leucmnthocyanin, permeability barriera were broken down completely.

B. WHOLE PLANTS

I . Isolation of the cell wall fraction Isolation of a clean waU fraction from various parts of whole plants is much more difficult than from suspension cultures. An important point is that the primary wall is often contaminated with secondary wall. This increases the difficulty of cell breakage and removal of the cell contents. Cell mills and glass beads are of little help in isolating these wall preparations. The simplest and often most effective method is to w e a pestle and mortar-sometimes this gives sufficient maceration ; often a little sand can be added (sand then tende to contaminate this type of wall preparation), or the material can be frozen in solid carbon dioxide and ground to a powder in a cold mortar. Water can be added finally and the wall fraction isolated and cleaned by centrifugation as for cell suspensions. Tupper-Carey and Priestley (1924) adopted an esaentially similar procedure. In a mortar they ground dry plumules and radicles obtained h

168

D E R E K T . A . LAMPORT

from ungcrminsted seeds of the bean Vicia faba, and extracted the powdered maherial with various reagents. Thimann and Bonner (1933) ground coleoptiles and extracted them with cold water. More recent workers, notably Kivilaan et al. (1969, 196l), homogenized coleoptiles in 80% glycerol, a medium which they described as I ‘ . . . essentially nonaqueous . “ . . chosen to minimize elution of protein from the wall fragments” ; a somewhat hfortunate choice, in the light of Haurowitz’s (1950) statement that “if the concentration of glycerol does not exceed 85% a large portion of tho soluble protein pastlea into the glycerol extract”. Bean and Ordin (1961) ground tissues frozen in liquid nitrogen and examined the effect of different methods of isolation on the wall composition. They concluded that ‘‘. . . the cell walls isolated by the three procedures, cold water extraction, glycerol extraction, or glycerolglycol extraction, might be comparable in nature and composition”.

. .”

.

C . ANALYTICAT, TECHNIQUES

I . Hydrotysis No single technique suffices for the simultaneous hydrolysis of both polysaccharides and proteins. Thus, while dissolution in 72% sulphuric acid (Saeman et al., 1964), followed by dilution to 2.5 N and autoclaving for 1-2 hours, is sufficient for the hydrolysis of most polysaccharides, this procedure only leads to partial protein hydrolysis (cf. Table XV). On the other hand, suitable standard conditions for complete hydrolysis of protein (6 N HCl, 18 hr at 106’ in a sealed tube) lead to losses of carbohydrate, humin formation (reduced by porforming the hydrolysis in a large excess of acid, Tristram and Smith, 1963) and the loss of eulphur amino acids, unless the material has first been oxidized with performic acid to convert unstable cystine or cyateine residues into cysteic acid (Schram et al., 1964). Total nitrogen determinations (e.g. Chibnall ei! al., 1943) are an important adjunct to the above hydrolyses. The various methods of enzymic hydrolysis of cell walls will be discussed as they occur.

2. El+?clrophore& and chromatography Table I1 summarizes the electrophoretic and chromatographic techniques which the author has found most useful for qualitative and quantitative analysis of cell-wall hydrolytic products. We still lack a well-tested rapid and accurate method for sugar separation and estimation (but see Sweely et al., 1963; Perry, 1964). The reagents described by Trevelyan, et aZ. (1950), Partridge (1949), Wibon (1969), and Frahn and Mills (1969) are useful especially for sugar identification. A nin-

T H E P R O T E I N OOMPONENT O F P R I M A R Y UELL WALLS 159

hydrin (002% in acetone &lo% acetic acid) dip is the beat general reagent for amino acids, with a few exceptions such as proline and hydroxyproline which give low colour (yellow/brown) yields and are deteoted best by the specific reagents of Acher et d.(1960). Hydroxyproline is estimated best by the Neuman and Logan (1960) method or a modification (e.g. Hutterer and Singer, 1960). Very often this estimetion can be performed directly on a small sample of the acid hydrolysate (with appropriate blanks), but where there is little hydroxyproline or much interfering material, the hydroxyproline must be estimated after an initial separation (e.g. paper electrophoresis pH 1.9) and elution. Similarly, Chinard’s (1962) specific method allows a good estimation of proline. TABLEI1 Methoda for Beparation of W d l Hydrolytic Prod&

Paper electrophorecrie with kerosene coo&nt pH 1.9 buffor: wetic acid 2.6% v/v formic acid 8.7% v/v water

pH 9.2 buffer: 0 . 1 ~sodium tetraborate Paper c h m r n d o p p h y ethyl acetate, pyridine, water (8 : 2 :1 v/v/v) (Timoll, 1900)

tertiary amyl alcohol plus buffer: 1 : 1 v/v buffer: 6% v/v pyridho -I 043% N ethyl morpholino adjusted to pH 8.2 with acetic acid (essential to tiso Whatmen No. 4 or nimilar Paper) Ion mchange chromatography Dowex 60 H+ resin with 1~ HCl aa eluent Moore and Stein columns Udfuhation Sephedex G26 with 0.01~1 acetic acid aa eluant.

AYpecicllapplieatiora Separation of hydroxyproline isomers and proline (cf. Sheehsn and Whitney, 1963). Qualitative after direct reaction of paper. Quantitative after elution of separated material, e.g. after autoradiography. Also useful for peptide sepamtion Qualitative sugar separation

Separates, in order of increeshg R,, g a h h e , glucose, mannose, arabinose, xylose, rhamnose. The sugars can be estimated by the method of Wilson (1969) Conibinod with pH 1.Qeloctrophorosk(1st dimension), this method (2nd dimension) is extromely useful for quantitstivo amino acid eetimation hy the method of Hoilmann et al. (1967)

For initial soparation of liydroxyprolintt isomers and proline For complete qualitative amino acid estimation

Peptide separation

160

D E R E K T . A . LAMPORT

111. THEHYDROXYPROLINE-RIUH WALLPROTEIN :‘

r

E

~

~

~

A. LNTRA-CELLULAR LOCATION O F IIYDROXYPROLINE

Early workers who isolated cell walls from young tissues always found appreciable amounts of organically bound nitrogen (Table 111). TABLEI11 Nitrogen Content of Isolated Primary Cell Walla ~~

~

Tkeue Cotton fibres

~~

~~

Nitrogen content of dried wall Method wed for iaohting wall aa percentage of dry weight

Maize colooptile

0*4-0*8

Artichoke tubor

0.7

Oat coleoptile

1.5

Oat coleoptile

1.9

Pea stem

Cotton fibres boaten in a waring blender (Tripp et aZ., 1961) Homogenized in Servall omnimixer followed by filtrat&i through a bed of g h beeo;a (Kivilaan et al., 1969) Homogenized in Servall omnimixer followed by filtration through a bed of glese beads (King and Bayley, 1963) Grinding in mortar (Bishop eL al., 1958) Grinding in mortar (Thimm and Bonnor, 1933) Grinding in mortar (Christiansen and Thimann, 1960)

2.2

0.55

Culture 899 897 895 902 901

1.7 1.2 1.6 1a 8 1’55

90 1

1 .B

I

Sycamore cell* mapelteion culture0 Zillig and Holzel’s (1958) Cell mill + water %illigmd Holzol’~(1068) Ch11 mill -1 40y, sucrom ~~~~

* Nitrogen

~-

content of dricd whole eyramore cells in ntationary phase ie approximately 2.4% (Zillig and Holzcl, 1968).

Lack of criteria for dist,inguishing cytoplaHmic contamination from a genuine wall protein component severely hindered theRe early studies of wall protein. Tripp et al. (1951) made the first chemical investigation of wall-bound nitrogen and after acid hydrolysis of the isolated primary cell wall identified serine, aspartic acid and glycine. The problem of finding suitable criteria of cell-wall protein remained until 1960 when Dougall and Shimbayashi (1960) and Lamport and Northcote (19608, b)

~

~

T H E PROTEIN COMPONENT O F PRIMARY CELL WALLS

161

carefully examined the amino acid composition of cell walls isolated from tissue cultures. From this work emerged the rather startling discovery that hyroxyproline was a major component of the wall hydrolysate, and a very minor component of other hydrolysed cell fractions. Here it seemed at one fortunate stroke Nature had provided us with a way in toward further studies of a specijic cell-wall protein fraction. Before accepting this conclusion several legitimate objections must be met. For example, the hydroxyproline might be associated with the outer cell membrane (plasmalemma), remaining firmly bound to the wall even after cell breakage ; or a cytoplasmic hydroxyproline component rnight be adsorbed by t h e wall during cell breakage; or perhaps a particular cytoplasmic hydroxyprolinc-ricll Mystem is anchored on to the cell wall. It iw convenient to deal with each of thene possibilities in turn. Sycamore cells grown in suspension culture readily’ plasmoly8e in hypertonic sucrose or salt solutions. The outer cell mehbrane remains with the shrunken protoplast (Figs. 1 and 2) and there is no significant difference between the hydroxyproline content of ways isolated from normal cells and plasmolysed cells (Table VIII). This is the clearest experimental evidence for concluding that the outer cell membrane does not contribute to wall hydroxyproline. Both Dougall and Shinibayashi (1 960) and Lamport and Northcote (1960 a, b) very seriously considered that during cell breakage the wall might adsorb the cytoplasmic hydroxyproline-rich material more or less completely. Both groups therefore examined the wall hydroxyproline content after isolating the walls under conditions ranging from extremes of pH (2-10) to extremes of tonicity (distilled water to 40% sucrose). But the wall hydroxyproline content remained the same within the limits of experimental error. Finally, one must consider the possibility of‘ a wall-anchored but mainly cytoplasmic hydroxyproline-rich material, becaune of the presence of Hecht’s threads (Hecht, 1912) in plasmolysed cells (including sycamore cells in suspension culture), and in view of the recent demonstration by Ledbetter and Porter (1964) of microtubules oriented in the direction of the cellulose microfibrils. Electron microscopical studies combined with careful hydroxyproline estimations reasonably exclude these possibilities. Although electron micrographs of plasmolyfiecl sycamore cells (Figs. 1 and 2) certainly show probable Hecht’s threads, especially in the regions of new wall formation, neither these nor other niembraneous materials are nppreciably present in electron micrographs of sectioned isolated sycamore walls (Fig. 3). The microtubules remain exclusively with the protoplast during plasmolysis (E. H. Newcomb, private communication). Yet another general Consideration which could conceivably fall T<

*

162

D E R E K 1‘. A . L A M P O R T

Fro. 1. A. Low power electron micrograph of plasmolyned sycamore cells (incollaboration with 0. Srhidlovskg). Exponentially growing syramore cells were plaemolysd in 0-4 M NaCl, fixed in permanganate, embedded and sectioned. AP = amyloplast; DCW = newly-formed daughter re11 wall; MCW = mother re11~ ~ 1N1 : ; nucleus; V = vacuole; I = incipient intercellular spare. Note that plaamodewmnta are rare or absent in cells grown in suspension culture. Note also old mothcr re11 walls with torn end&,probably resulting from cell expansion rupturing the older mother cell walls (rf. Lamport, 19fXa). Note incipient “intercellular s p c e formation nt “I”which must rupture the old mother cell wall and extend along the daughter cell wall cleavage plane before the new daughter cells can separate (cf. Martens, 1937). These electron micrographs provide no evidenre for a “middle lamella” defined (classically from optiral microscopical observstions) its the intracellular cellulose-frac cement substance.

THE PROTEIN COMPONENT O F PRIMARY CELL WALLS

163

PIQ.1. R. Electron micrograph of plnsrnolysetl sycamore cells, (in collaboration with a. Schidlovsky). Similar to A but viewtd st a grcatcr mltgnification. Symbola w in A with the following additions: GI3 = golgi hody; HT Hecht’s threads ( 1 ) ; M = mitochondria; 1’ = plasma membrane; T = tonoplant. A

164

D E R E K 1’. A . L A M P O R T

Fio. 2. High power elcrtron microgrnph of plnnmolynud nyremore re11 (in oollahoration with G. Schidlovsky). The experimental rnatrrinl was the same aw shown in Pig. 1. Thu micrograph shows n region having thr leaat cytopftwrn. Symtroh as in rig. 1, with thr following trdditions: ER :-cntloplanmic reticulum; M’ .-w d l .

THE PROTEIN COMPONENT O F PRIMARY CELL WALLS

165

within the category of a cytoplasmic system anchored to the wall is the possibility that wall biosynthesis requires a very special type of structural protein. This may well be, but it does not explain the great variations in wall hydroxyproline content.between different species or within species (Table VIII). Thus, if hydroxyproline is localized exclusively in the wall (i.0. ignoring traces of cytoplasmic hydroxyproline) a simple relation allows one to calculate, from the hydroxyproline figures, the percentage of the tissue (on a dry weight basis) which exists as cell wall:

(yo cell wall

=

hydroxyproline in cells or tissue x 100. % hydroxyproline in isolated wells

Applying this relation to higher plants (Tables V I I I and IX) grown either as tissue cultures or normally, always gives entirely reasonable figures for the percentage of cell wall, regardless of the actual amounts of hydroxyproline involved. The figure for sycamore agrees very well with that obtained by other methods (Table 1V).

~~

* .I figure of 3 9 . 4 WIM olitained

~ _ _

by direct recovery of cell walls from i t bomogenate.

A t this stage one can be reasonably confident that at least one point is now firmly established, viz. that the bulk of cellular hydroxyproline is firmly bound t o the cell wall. Other workers (Olson et al., 1962; Brown 1963; Edelman and Hall, 1964), have experimentally established this point for themselves and concur. Most recently Oleon (1964) reporb the results of preliminary auto-radiographic experiments which ah0 confirm that hydroxyproline-rich protein is associated with the cell wall. There is unanimity with one exception, namely Steward et al.

166

D E R E K T. A . L A M P O R T

Fro. 3. Electron mirrograph of thc i H o l i i t i v l H y i v i r n r w wrrll (in wIla1~oraliiinwith 1’. F. Ihkcr). Walls isolatril from syrimorc ~ i i s l ~ i ~ t i n i oI Wi i I ~ U ~ C Hwere fixed in pc*rrn;rnganatc,em. bedded, and sectioned. The mic*rogrn1rh HIIOWL( ;I typirnl nample. Non-wall membranous material WEE present in lens than 10% of the t w t i o n H rxaniined. Note that the outer surface (08) of the i~olatetlwdl tlhowa the vhttrwtmistw hixzy apptwanre, also seen in sections of intact cell^ (cf. Fig. 1).

T H E P R O T E I N C O M P O N E N T O F P R I M A R Y CELL W A L L S

167

(1951) who firet drew attention to the existence of hydroxyproline in plants, which was released by acid hydrolysis, and later concluded (Pollard and Steward, 1959) that the hydroxyproline-rich material waa present mainly in the cytoplasm rather than in its particulate inclusions. The fact that these investigtttors discarded the cell-wall fraction (J.K. Pollard, private communication) explains the disagreement in part only, for recently Steward and Chang (1963) concluded, from experiments with 14C-prolineand tissue cultures, that : “. . . the W-proline and Whydroxyproline-containing protein eventually and progressively accumulates in the bulk of the insoluble protein which at this point can only be designated as the main protein fraction which constitutes the organization (i.e. the cytoplasm and its organelles) of the cell, which centrifuges at low speed and is insoluble in a buffer solution at pH 8.3”. This section would hardly be complete without mentioning the brilliantly intuitive (1959) suggestion of Preston (published in 1961), who wrote : “Here, then, is a protein of which at least the proline- and hydroxyproline-containing portion is guarded from the metabolic machinery of the plant. One possibility is that t h i s is tho protein which is closely associated with the developing cell wall.” B. CHEMICAL CHARACTERIZATION O F 4-THANS-HYDROXY-LPROLlNE

The Iiyciroxyl group can occur at carbon 3 or 4, and can then be either cia or lrur~sto the casboxyl group, giving four possible isomers each of which can exist in the D or L form giving a final total of eight possible isomers of hydroxyproline. The 5-hydroxyproline isomera are probably not found because of the greater stability of the equivalent structure glutainic eemi-aldehyde. Five of the possible hydroxyproline isomers have been found in nature, viz. 4-tram?-hydroxy-L-proline(most common), 4-cis-hydroxy-~-prolinine (Wieland and Witkop, 1940; Radhakrishnan and Giri, 1964), 4-ciahydroxy-D-proline (Sheehan et ul., 1958) ; 3-truns-hydroxy-~-proline (Ogle et al., 1962), and 3-cis-hydroxy-~-proline(Irreverre et al., 1963). Only 4-truns-hytlroxy-~-proline and 3-tmrr~-)iydroxy-~-proI~e occur as protein constituents. Today, therefore, one can characterize a hydroxyproline isomer by comparing it with the known isotricre which have previoudy been chgracterized by other workers. The 4-hydroxyproline isolated from collagen by Fischer (1902) was fir& characterized using rigorous chemical methods by Neuberger (1945) and Hudson and Neuberger (1950) as 4-truns-hydroxy-~-proline.

TanrE

v

Amino Acid Composition of Cell Walls (residues

HYPm &P Thre Ser Glu PrO GlY Ala Val Met Ileu Leu

TYr cbala

Lys His Arg Cya

Met SO,

I81 49 54 92 38 61 48 40 65 4 20

33 29 14 91 23 21 n.d. -

142 50 51 93 61 60 85 39 61 4 39 41 18 20 81 1s 11 n.d.

-

182 59 35 94 65 57 51 46 49

154

77 49 92 65 54 62 52 50

2

3

26 44 15 24 77 21 18 10 10

30 61 15 27 55 20 20

12 10

114 74 38 108 64 46 56 50

63 12 33 51 31 30 102 25 22 -

85

61 51 82 a5 60 S8 67 62 15

42 73 28 37 66 17 47 -

-

55 104 38

90

-11

80 66 45 83 9 31 57

24 24 86 19

25

-

lo5 g protein)

64 71 51 93 86 69 112 68 61 trace

44 71 11 26 66 10 16

-

-

71 106 56 119 92 66 85 55 64

47

63 8 18 62 trace 12 12 12

47 66

64 66 108 53

81 74 73 trace 47 75 22 33 77 22 26

-

-

102 81 54 86 84 61

95 63 44

45 73 15 34 69 9 11 3 11

52

50

i0

87 50 51

64 i4

102 85 105 6.5

Go 9 36 89 12 19 89 9 16

-

fA 42 146 66 64 trace 39 61 23 37 32 26 14

-

-

43 69 69 78 116 54 50

90 70 20 41 74 tmce 28 72 29 24 I

T H E P R O T E I N C O M P O N E N T O F P R I M A R Y C E L L W A L L S 169

The presence of a hydroxyproline isomer in an acid hydrolysate can be detected following an electrophoretic or chromatographic separation by treating with ninhydrin at 106O, or with the more sensitive and specific reagents of Acher et al. (1960). When only small amounts of material are available the characterization can be pursued even further by reactions on paper. For example, the hydroxyproline isolated from sycamore cell walls migrated identically during eleotrophoresis (pH 1.9) with 4-trans-hydroxyproline. These conditions separate 4-tranahydroxyproline from the other epimers. Furthermore, neither hydroxyreacted proline from sycamore cell walls nor 4-truw-hydroxy-~-proline with D amino acid oxidase under conditions similar to those described by Wagner (1960), and which are known (e.g. Corrigan et al., 1963) to oxidize hydroxy-D-prolineisomers. Van Etten et al. (1961) have characterized the bound hydroxyproline of plants more rigorously. From an mid hydrolysate of the seed coat of Iris germanica they obtained 120 mg of crystalline material. They found that its elementary analysis, optical rotation, and X-ray pattern, were identical with those of an authentic sample of 4-traw-hydroxy-~-proline. C. THE AMINO ACID COMPOSITION OF PRIMARY CELL WATLS

All the common acid-stable amino acids which occur in protein are present in hyrolysates of primary cell walls. Table V summarizes the available data. The most outstnnding feature of these analyses, besides' the presence of hydroxyproline, is the wide range found in the (Notes to Table V on facing page) (a) From Dougall and Shimbeyashi (1960). (W The walls were isolated from suspension vultured material and h ~ h l y e e d .Amlno acids were estimated after electrophoreticand chromatographicaepratiion on peper (cf. Table

11).

(c) The w e b were oxidized with performic acid and thcn hydrolyd. The amino acid analpie wm performed by thc Awlytica Corporation of New York Inc. (a) The walls were treated with cellulaae. The insolubleresidue WM oxidized with perfode acid and then hydrolyaed. The amino acid analysis was performed by the Analytica Cerpomtion of New York Inc. ( 8 ) Taken from Lamport find Northcote (leaOa). (0An originally tetraploid strain WM grown in suspension culture. Dr.R. P. Ambler verg kindly analymd the wall hydrolysate on a Spinco automatic amino acid analyser. (0) The walls were isolated from a suspension culture and hydrolysed. EB. Betty Brown very kindly performed the amino acid analysis manually on Moore aod Stein columns. (h) Walls were isolated by grinding three-day-old roots or bypocotyls in a mortar wish liquid nitrogen. The washed walls were bydrolysed without further t m t m e n t (aolumn l),or effer performic acid oxidation (column 2). Amino acids were estimated after electrophoretic and chromatographic separation on paper. (1) Walls were isolated from a callus culture by grinding in water in a mortar, and hydroI@. Amino acids were estimated after electrophoretic and chromatographic separation on poper.

170

D E R E K T. A . LAMPORT

value of any particular amino acid when the amino acid composition of one wall is compared with another. The range is sufficiently wide to make one consider the possibility that there is perhaps other bulk protein firmly bound to the wall in addition to the hydroxypmline-rich protein. This problem will probably best be resolved by a comparison of different wall protein peptides by methods involving peptide fingerprinting and subsequent amino acid sequenoe determination. Until these data have been gathered, it is best to take the simple view, that the walls contain only one hydroxyproline-rich protein, in view of the large amount of hydroxyproline present ;and that this protein accounts for the bulk of the protein in the cell wall. This simple view still leaves unexplained why wall protein should show such wide variations in composition from species to species. Certainly this variation may have some taxonomic value. Table V does indeed show a striking similarity between the amino acid composition of Nicotkm tabacunt and Lympersicon esculentum cell walls; both species being members of the Solanaceae. Presumably the composition of the protein relates to its role in cell-wall organization, If it is a structural protein its role will be a priori morphogenetic ! Differences in composition might then rank high nmong the crucial determinants of cell size and shape. That all the hydroxyproline is present as polyhydroxyproline can be ruled out by data presented in the next section (111 D). For reasons which will become apparent later in this discussion, the presence of sulphur amino acids, especially cystine or cysteine in the wall protein, is of especial interest. But the sulphur amino acids undergo extensive degradation. during acid hydrolysis, especially so in the presence of carbohydrate-at present an unavoidable drawback to studies of wall protein. This, of course, explains the lack of figures for cystine or cysteine in the earlier published analyses of wall protein. But the sulphur amino acids can be measured after converHion to a more stable form. For example, after performic acidbxidation and acid hydrolysis of a number of cell-wall fractions from different species, the author was able to detect and measure cysteic acid (Table VI) by the method of Heilmann et al. (1957), after an initial electrophoretic (pH 1.9) separation. Aasuming for the Rake of argument at this point that the amino acid composition irJ that of one main protein, the inevitable comparison with collagen shows differences rather than similarities. Thus wall protein predominates in polar amino acid residues (especially hydroxyatnino acids); has an average amount of glycine, tyrosine, and cystine; and lacks hydroxylysine. Collagen, on the other hand, predominates in non-polar amino acid residues ; consists of approximately onethird glycine residues ; has hydroxylysine, little tyrosine, and lacks cystine.

T E E PROTEIN COMPONPNT O F PRIMARY CELL WALLS

171

TABLEVI Hydroqproline/U@&c Add Molar Ratio8 in P e r f m i c Acid Oxidized Cell-waUPrepardione"

A m peeudoplatcmoce Solunum tuberoaum (2n) Rosa (Paul's Scarlet) P k m 8ativum (root) Nicoticmo tabacu7n var. Xanthi Nieotianu tabaeum var. Turkish P k m Bativlcrn (epiootyl) Centaurea cyanua Ginkgo &ba ( n )

17 12 12 11 10 7.0 7.0 7.0

3.0 1.2

orym 8aliua

Exoept whem stated, the walls were isolated from suspension cultures.

At the moment the value of theue amino acid analyses lies in the fact that they show all the acid-stable amino acids commonly found in proteins also occur in wid hydrolysates of cell walls. It is therefore reasonable to conclude that we are more likely to be dealing with protein than with mucopeptide-likestructures fourid in bacterial cdl w a b . D. ENZYMIU DEGRADATION AND CHARACTERIZATION OF WALL

PROTEIN

TABLEVII The Effect of Proteasea on Sycamore Cell-wdl Protein" Proteme -

Percentage of total c m n h rdeutwd

~~

pH 7.8 pH 7-8 pH 5.0 pH 7.8 p~ 7.8 pH 7.8 pH 2-2 pH 7-8 ( + CaCI,) Bromelin pH 7.8 Muremideee pH 7.8 ( 0 - 0 5 HC1 ~ 24 hr at 105" Chymotrypsin Pronaao Papain Subtilisin Elasfaso Trypain Pepsin Collqpnese

40 38 35 33 28 26

21 10

10 3 -5 70)

* w& were ieolated from a culture grown in a medium containing I ) mixture of *~.IPb&d emino aoids derived from an algal hydrolysate.Thin gives wells with only the protein hcrbelled Two ml cell wedl nuepension, containing approximately 1 mg dry weight, waa inoubet& 18 born in the appropriate buffer et 97". with exces8 proteae (1 mg). Release of mdioa&ide wan oalculated from meantbe radioactivity in samples from the incubation mixtm k fore and after centrifugation.

172

D E R E K T . A . LAMPORT

The effect of various proteolytic enzymes on isolated sycamore cell walls (Table VII) provides additional evidence for the existence of hydroxyprolirw in peptide linkage with other amino acids. Especially noteworthy is the effect of trypsin, in view of its specificity for lysyl or arginyl bonds. Trypsin released one-quarter of the 1 ‘ W label from walls labelled either with a complete mixture of amino acids or with p r o h e and hydroxyproline. Furthermore, an unlabelled sycamore wall preparation initially containing 1.2% hydroxyproline (dry weight) contained only 0.8% hydroxyproline after incubation with excess trypsin at pH 8.0. This corresponds to a loss of approximately one-quarter of the wall hydroxyproline and justifies the conclusion that each of the three different methods described above does in fact measure the same thing-release of hydroxyproline-rich peptides from the cell wall. The trypsin-resistant protein remaining with the sycamore wall probably corresponds to Olson’s (1964) “residual wall protein” which remains after extraction with 1 N NaOH. Wall protein is exceptionally resistant to proteolytic attack-probably a result of its unusually high imino acid content, its close association with carbohydrate and presumably its insolubility. To date “pronase”, a powerful proteolytic preparation isolated from Streptomycea q~iaeu.9 (Nomoto et al., 1960; Hiramatsu and Ouchi, 1963), removes the greatest amount of wall protein from sycamore-approximately 60% after 00 hours’ treatment at 37”. Pronase degradation of wall protein yields mainly free amino acids and small peptides. Therefore, despite tho good yield of soluble material, pronase is of little help in further structural studies of wall protein. At the moment, among the proteases chymotrypsin offers the greatest promise as a structural probe of wall protein. Chymotrypsin releases a reasonable amount of sycamore wall protein as both high and low molecular weight material (Fig. 4) which is currently under study in the author’s laboratory. E. DISULPHIDE BRIDGES IN CELL-WALL PROTEIN

One very important reason for pursuing the rather difficult enzymic degradation of cell-wall protein is that we are now at least able to begin an experimental investigation of what has hitherto been an almost completely speculative field. In particular, there i s the problem of wall “cross-linkages” and their possible significance in extension growth. Many workers, for example Ginzburg (1958, 1961), Cleland (1960), Ray (1961), and Brown (1963), have in fact suggested that protein is the basis for wall cross-linkages. Nickerson and Falcone (1969) showed the presence of a disulphide-rich (pseudokeratin) protein in yeast ceU walls. These workers also found that the disulphide bridge8 could be opened

THE P R O T E I N C O M I ~ O N E N TO F P R I M A R Y C E L L W A L L S 173

by cnzyniic reduction and. therefore, suggested that the disulphide-rich protein was the inolecular basis for the localized areas of increased wall plltxt,icity required during budding (Nickerson, 1963). Although sycamore cell-wall protein is less amenable to analysis, i i s it cannot, unlike yeast' wall protein, be removed from the wall, the present results clearly point towards the presence of disulphide bridges in the wall: first tlhereis the tlenionstnLtion of cysteic acid in all perfor-

Tube number

Yiu. 4. Sephatlcx frccrtionation of 11 rhymotryptir hydrolysatc of *~S-lahIledsycamore cell VSIIN. Syramore rcllw were grown for 3 wceks in IL nrilphur-deficit.nt medium containing 1 mC asS80,. The wall fraction wtcs isoletcd and washed with water and salt eolutions as described in Section I1 A 2, and s~tq'c.iitledin a toticl volume of Id ml 0 . 0 2 ~ tris-HC1 pH 7.7 buffer. After rrddiirg rhymotrypsin (1 m g in 0.1 nil 0 . 0 1 ~ HCI), thc mixture was incubated a t 37" and sampled periodictllly to dctermine the tunount of radioactive matorial released from the walls. Relecise of radioactivity had practically rranrcl after 6.5 hours' incubation. The mixture wan filtered. The filtrate was dried from the frozen ntate, rodissolved in 1 m l 0 . 1 ~ acetic acid, and applicd to the tnp of a 110 rm x 1 cm diamctcr sephadex G I 5 cohmn. The column was then eluted with 0 . 0 1 licetic ~ acid; S rul frnrtions were collected; 2.5 p1 aliquot8 were monitored for rndioartivity in a Nurlear-Chicugo willtillation ronntcr. Tubcs were pooled corresponding to p k s I, 11, and 111, nnd analyard furthcr.

inic ticid oxidized wall preparations examined so far (Table VT). Secondly there are the results of experiments in which sycamore cells were grown in a low sulphate medium containing "SO4. IJnder those conditions sycamore cellx rapidly incorporate 3aS in to protein. After isolation and incubation of the labelled W ~ with H c.tiymotrypHin,sephadex frrtotionation of the hyclrolysate shows three main 36S-lnhelledpeptide o l a s ~ e ~ (Fig. 4). Peaks I1 and 111 then received the Brown and Hartlcy (1963) t
174

D E R E K T . A . LAMPORT

chrornut,ographic and electrophoretic amino acid separation. Both the arrowed peptides of Fig. 5 yielded cysteic acid and other amino acids, but hydroxyproline was not detected. At the moment then there is only incomplete evidence for regarding these cysteic acid peptides as part of the intact hydroxyproline-rich protein.

FIG.5. IJctcction of ryntiiiu-iJridgtd prlitirlo~in nywmrtrc: trrll w~LJJH. Thc nwfhod involven two-dimensional c~cctrophormin. Tho 1’11 remains thc nctmc. for crach dimannion. After the first dimenflion, the paper in dried and cxpotwtl to performiu acid vaporir, which oxitlieen oach cyntine peptidc to a “twin” puir of cyRtcic, wid pcptiden. Thcne migrate in tho Hecond dimcnnion according t o their new properties. Thus, t u in puir8 of cyntc*icacid (Cya)peptides fall off the diagonal occupied by tho othrr unaltered prptitlcfi. The nutoradiogram shove showa twin cysteic acid peptidea originnting from (I pirti(111y fractionid nycamorc cell wall chymotryptic hydrolymte. F. DISTRIBUTION OF THE HYDROSYPROLINE-RICH WALL PROTEIN IN THE PLAKT KINGDOM

Despite the small number of different species so far surveyed (Tables V I I I and IX) it is clear that wall-bound hyroxyproline is widespread and probably ubiquitous at least among the higher plants (Sperinatophyta). It seems reasonable, on the basis of experimental

T H E PROTEIN OOYPONENT O F PRIMARY UELL W A L L S 176

TABLEV I I I Hychxyproline DiStrd6uth in Tieme Czltturea Percentage dry weight h@vxprolim Wholetkeue C e l l d

specie8 Phaeeolw d g a & LycopmOn aculentwm S o l a n m tuberomm (2n) Wego 080inalie A p h m gravwlem Nicotiana tdmcuna var. Turkish Nicotiana tabacurn var. Xanthi Acer peudvplatanua Daww OarOtcL tXdqo bi2oba (2n)

*wum

0.42 n.d. 1.42 0.97 0.74

69 60

49

-

1.3

-

0.6 0.6 0.6 0.66

1.25 1-1 1. I 0.96

41 46 64

n.d, n.d. n.d. n.d. n.d.

0.8

eatiacum

Uamellia Sane& Centaurea oyanus Roaa ( P d ' s Bcerlet) 07Q(Zae&Diua Ginkgo bildm (n)

16

2.7 2-6 2-4 1.6 1.6 1.6

n.d. n.d.

% of cell exidingascetlwaw

69

-

0-22-0d 0.28 0.2 0.07

-

* "hie figure hne deorenaed very slightly since the original isolation of the culture in 1968. No significant difference hea been found in the bydroxyproline content of wdln ieoleted from cell8 in log phaee or etationmy pbee, plaamolymd or unpleemolyeed. TABLEIX H@mqyproline Dietributh in Va&w Plant Paria

SpeCia

Part

Pieurn aatiwm

Root Epicotyl Cotyledon Cotyledon cellua Hypocotyl HypocotYl Cambial tissuea Pericerp Hypocotyl Leaf Coleoptile Coleoptiluand leaf Pericarp

Percentage dry weight hydroxyproline % of cell Whole Cell existing ad tieme walle cell wall n.d. n.d.

1.1 0.68

0.2 0.8

n.J. n.d.

0.36 0.08 0.06

0.03 0.02

0.88

0.17 n.d. 0.14 0.1

0.03 0.02

0.06 0.06 0.06

0.01

0.04

0.02

~

L

40

49

26 20 41 80

41 30

170

D E R E K T. A . TIAMPORT

results obtained with sycamore and other species, to consider that the wall-bound hydroxyproline of other species not examined in detail does, nevertheless, represent specific wall-bound protein. Again, judging from the amino acid analyses of cell walls isolated from different species (Table V), the amount of hydroxyproline can be taken as an approximate indication of the amount of wall protein present. Hydroxyproline occurs free only rarely in plants ; for example, 4-cia-hydroxy-~prolinc in Santalunt album (Radhakrishnan and Diri, 1954). Therefore, the nppcarance of hydroxyproline in hydrolysates of plant material can be talcen more often than not as a measure of cell-wall protein. At one extreme, where a particular tissue consists largely of primary cell wall and this wall contains a large amount of hyroxyproline (e.g. most tissue cultures), the overall impression, of course, will be of a tissue rich in hydroxyproline. At the other extreme, a particular tissue (e.g. storage tissues and meristems) may contain very little cell wall and this cell wall may contain little cell-wall protein. Under these conditions the “dilution” of cell-wallprotein by cytoplasmic proteins may be sufficient to lower the hydroxyproline concentration in the total tissue hydrolysate below the level for accurate measurement. All this adds up to the reasonably obvious rule that when hydroxyproline levels in a tissue hydrolysate are high they can be taken fairly safely as a measure of wall protein. But when hydroxyproline levels in the tissue hydrolysate fall below the level of detection one cannot conclude that, in this particular tissue, hydroxyproline is of no significance, before carefully examining the isolated wall fraction. Two examples will illustrate this point. Consider first the pollen-derived haploid Ginkgo cultures isolated by Tulecke (1957). Tulecke et al. (1902) analysed the alcoholinsoluble residues of thc Qinlcgo culturc nnd found no hydroxyproline. However, isolation and hydndy~ixof thc wall yielded Nmall amountN of hydroxyproline (Table VIII). Another example, but not quitc so clear, is to be found in data presented by Wetmore (1954) who determined thc amino acid composition of the total protein fraction of different organs at different stages of growth in Lupinus albw. Rearranging these data (Table X) and comparing “young” organs with “old” organa (e.g. young roots with old roots, or unexpanded leaves with expanded leaves) shows that as the plant ages, hydroxyproline accounts for an increasing proportion of the total nitrogen. As older organs tend to decrease in total nitrogen on a dry weight biL(SiR,this apparent enrichment in hydroxyproline in any given organ may be a reflection of the decreasing cytoplasm /primary cell-wall ratio.

THE PROTElN COMPONENT O F PRIMARY UELL WALLS

177

TABLEX HydmxyproZim and Pmline Uontent of Lupinm albus*

HYPOWtYQ

Stem7

Young roots?

44 4.0 1.1

6-3

3.8

Hypro Pro HYPwPro

4.2 1.26

6.6 0.84

Old roots$

-

6.6 11, 9.3 0.71

* Theee data are quoted from Wetmore’e (19G4) figuws ebowing the amino acid compoeition of the total protein fraction, the nitrogen of each amino acid being expressed BB a percentage of the total nitrogen determined. t From plmb 14 &YE old. $ From plmb 30 daya old with wtyledone removed at 23 daye.

IV. THE BIOSYNTHESIS OF “EXTENSIN” A. UPTAKE AND INUORPORATXON OF 1*C-P130LINE BY INTACT CELLS

I , Preliminary data Because more than 90% of the cellular hydroxyproline is in wall protein, hydroxyproline itself is the most convenient indicator of wall protein and, as a working hypothesie, one can assume that looking at hydroxyproline is the same thing as looking at the hydroxyprolinerich wall protein. It follows that one should be able to amwer a number of questions about wall protein even before it can be isolated and purified. Such questions for instance as the conditions necessary for synthesis-nergy availability, substrate requirements-the rate of synthesis, the inhibition of synthesis, and the variou~reactions and reaction mechanismsinvolved in the synthesis. All these questions have a bearing on the oentral problem-the preciEie role of wall protein. Stetten (1949) showed that in animal systems the best precursor for hydroxypmline was proline. From her lsN-proline feeding experiments she concluded that : “The hydroxyproline of the proteins is not derived to any appreciable extent from dietary hydroxyproline but rather from the oxidation of proline which is already bound, p m m abty in peptide linkage” (my italics). Steward and P o h r d (1968) showed that a similar eituation existed

178

DEREK T. A . L A M P O R T

in carrot tissue cultures ; 1%-hydroxyproline entered the carrot cells

but was not incorporated into the alcohol-insoluble “protein” fraction. On the other hand, radioactivity from 1%-proline rapidly entered the alcohol-imolublefraction and appeared as 1%-prolineand 1%‘-hydroxy proline in a ratio of about 4 : 1. This ratio decreased with time, showing that proline incorporated into the alcohol-insoluble fraction was progressively converted to hydroxyproline. From such data these workers concluded that proline in peptide linkage in a protein was oxidized to hydroxyproline. They did not consider the alternative explanation (discussedin Section IV A 3), viz. that during the biosynthesis of a hydroxyproline-rich protein (if it “turned over” slowly or not at all) this protein would progressively capture radioactivity released from proteins which did turn over. Thus, the final W-hydroxyproline/ W-proline ratio might simply reflect the overall ratio of hydroxyproline to proline in the most stable proteins.

Hr

Fro. 6. Sycamore cell euepensions: uptake, incorporation, and redistribution of W-proline

with time. Approximately 6.6 pg W-proline (0.5 pC) was added to 2 ml eamplea (7mg/ml dry weight) of rapidly powing cella suspended in their own growth medium. After incubation a t 27”for variow times, the cells were washed rapidly with water and homogenized in the 6 ml pot of Zillig and Hokel’s (1958) cell mill. The homogenate waa recovered quantitatively. Cell fractions were prepared by centrifugation and &re designated by Alexander’s (1956) notation. For example, the 10.P.20fraction ie the pellet obtained after centrifugingat 10,OoO g for twenty minutes. The wall fraction is designated a8 1.P.i.Total radioactivity in each fraction is expressed as a percentage of the total radioactivity meaenreed in the complete (&ctionated) homogenate. The “pool” waa estimated aa the difference between the diBlyaed and ~ n d i a l p dlOO.S.30fraction.

T H E P R O T E I N C O M P O N E N T O F PRIMARY OELL W A L L S

179

Like the carrot cultures mentioned above, sycamore cells also incorporated lV-L-proline into alcohol-insoluble material. Sycamore cells also concentrated l'C-trans-4-hydroxy-~-proline (a gift from Dr. N. M. Green), but it did not enter protein and was extracted completely by ethanol or 8% trichloracetic acid. In exponentially growing sycamore cell cultures the uptake process for W-proline had properties summarized as follows. (i) A net uptake of proline against a concentration gradient was possible, since free intra-cellular proline waa greater in amount than free extra-cellular proline, for a short period following a pulse of W-proline (Fig. 6). (ii) Substantial uptake did not occur at 1' or in the presence of 2 X 10% cyanide at 27". (iii) The maximum rate of proline uptake was 2.02 pg proline/mg dry weight cells/hr/27' (Fig. 7).

Fro. 7. Uptake and incorporation of 1%-proline. Saturation curve for rapidly growing syckmore cells. Rapidly growing cells wero washed three times with a pH 5.6 wlt solution of the mme compositionas the growth medium except for the omieeion of sumlie and coconut water (cf. Lamport. 1964a). One ml samples of weclhed d 8 were agitated for 1 hr a t 27" with 0.1 pC '%-proline (11.3 mC/mmole). Tho mmplee contained incroaeingamounts of carrier proline. Uptake WEE stopped by the addition of 0-lrOemer proline and the cells were then rapidly wsehed with water three times, t r a n s f e d to weighed planchettee, dried and monitored for radioactivity, lrom which ' ~ ~Oelauleted t l the actual amount of proline taken up. The a~?,nel emount of proline inoorpor~tedWEE obtained by recounting the planchettee after exhaustive washing in ethanol containing oarrier proline.

D E R E K T. A . L A M P O R T

180

(iv) The extra-cellular proline concentration wt~sabout 7 X 1 0 “ ~ at half the maximum rate of proline uptake (Fig. 7). (v) The process showed stereospecificity (Fig. 8).

I

2

I

10

I

I

8

6

I

I

4

2

I

I

I

0 2 4 Uptake of I4C-proline ( c t s h i n X lo3)

I

6

I

8

I

10

FIG. 8. Uptakc of W-proline into sycamore cells: effect of proline andogues. One ml sample8 of rapidly growing cells (4 mg dry wcight) were either pretreated for 1 hr at 27” with a given imino acid analogue or incubated simultaneously with the amlogue and 0.1 pC 143proline (11.3 mC/mmolo) for 1 hr at 27”.After each inoubationthe cell8 were washed rapidly in water. Finally the cell6 were tmnaforrccl to planchettes and the radioactivity measured.

The incorporation process phowed properties as follows. (i) The intracellular (protein precursor) proline pool was minute: a small “pulse” of 14C-prolinegave an “immediately” linear rate of incorporation into the ethanol-insoluble fraction (Fig. 9); and when the pool was artificially expanded, it contracted at a rate comparable with that of growth (Fig. 6). (ii) The maximum rate of proline incorporation was 1.2 pg/mg dry weight cells/hr/27” (Fig. 7). (iii) The extra-cellular proline concentration wm about 7.8 x 10% at haIf the maximum rate of proline incorporation (Fig. 7).

2. Inhibition by proline analogues Steward et al. (1968) showed that a number of substances, which could be regarded as proline or h ydroxyproline analogues, inhibited

T H E PROTEIN C O M P O N E N T O B P R I M A l t Y C E L L W A L L S 181

the growth of plant tiasue cultures. Especially potent were traneL4hydroxy-L-prohe, cie-4-hydroxy-~-proline,tmne-4-hydroxy-~-prolylglycine, 0-acetylhydroxy-L-prolineand azetidine-2-carboxylic acid ; all were competitive inhibitors of proline. Fowden and Richmond (1963) have recently shown that azetidine-2-carboxylic acid and dehydroproline (Fowden et al., 1983)are incorporated into protein and replace some of the proline.

'@OOt/

Y -

0

I

2

*

8

4

I

1

6

*

8

I

*

1

@

*

0

Mlnutea

Pro. 9. Time courae of incorporation of W-prolino into exponentiallygrowing sycamore calla during a small pulse of 1%-proline.Twentyfivo ml containing 16 mg/ml (dry weight) rapidly growing cells were inoubated in their own growth medium. At zero time 0.6 pC W-proline (10 mC/mmole) was added and I ml samples taken at two-minuteintervals were pipetted into 3 ml ethsnol containing carrier proline. The ethanol-insolublecell residuea were washed four t h e e with ethanol, then transferred to planchettes and the radioactivity was measured.

I

Proline analogues alao inhibit the growth of sycamore cell suspeneion cultures. The high proportion of imino acids in wall protein might make its biosynthesis unusually sensitive to one or more proline analogues. The way would then be open for a study of the effoct of modified wall protein on cellular properties. So far, however, the proline analogues wed seem to inhibit sycamore cells indiacriminately. For example, the analogues studied inhibit both growth and proline transport (Fig. 8) in the sycamore cultures. However, Cleland (19630,1964)reporta that M hydroxyproline inhibits auxin-induced cell elongation in oat coleoptiles within 3 hr of application.

3. Change%of diatfibution with time (a) The pool. The pool must be defined on the basis of experimental results as ". . . the total quantity of low molecular weight compounds which may be extracted from the cell under conditions such that the macromolecules are not degraded into low molecular weight subunits

182

D E R E K T. A . L A M P O R T

. , .” (Britten and McClure, 1962). However, one must remember that pool defined in this way includes at least two cellular compartments in cultured cells, viz. cytoplasm and vacuole. I n exponentially growing sycamore cells the rapid loss of 1W-proline from the pool (Fig. 6) and the absence of kinetic delays in the incorporation of W-proline (Fig. 9) both indicate that there was only a smll pool of imino acid precursors to protein, and that at any particular instant there was less than 60 sec supply of free proline available for protein synthesis. (b) Turnover of soluble protein and redistribution of radioactivity. Sycamore cells rapidly take up a small 1% pulse and incorporate it into various alcohol-insoluble cell fractions. The label content of the soluble proteins rose sharply when the free proline pool was expanded, and fell drastically after the free pool became depleted (Fig. 6). I n contrast with the soluble proteins, the wall fraction showed a continued increase in total label throughout the experimental period. Therefore, the walls were capturing label from the soluble proteins, either in the form of intact protein synthesized while the pool was expanded, or as the degradation products of cytoplasmic protein which had re-entered the amino acid pool before resynthesis arid transfer to cell wall protein; or perhaps in both these ways. Olson (1964) recently reported the results of similar experiments performed with tobacco suspension cultures, with the important addition of a large proline “chase” 0.6 hr after the proline pulse began. He found that even as long as 10 hr after the chase began W-proline was steadily being lost from the cytoplasmic fraction and was reappearing in the wall fraction both as W-proline and lW-hydroxyproline. Olson concluded that during the chase free proline could not be the source of the wall label, which was more likely derived from a cytoplasmic protein or polypeptide wherein proline was hydroxylated before or during deposition in the cell wall. The significance of this conclusion is discussed in Section I V B 3. ( c ) 8ite of synthesis and transfer of the hydroxyproliw-rich protein. Experimental results discussed so far leave little room for doubting that the hydroxyproline-rich protein appears fbst in the c y f o p k m before being transferred to the wall. This conclusion finds additional support in the observation that in sycamore cells (Table XI) and in tobacco cells (Olson, 1964) the specific activity of the soluble protein hydroxyproline is significantly higher than the specific activity of the soluble protein proline during the 1%-proline pulse. These data point to the exiRtence of a small cytoplasmic pool of hydroxyproline-rich protein which, unlike wall protein, gains and loses radioactivity with ease. This dynamic state of cytophsmic hydroxyproline-fich protein

THE P R O T E I N OOMPONENT O F P R I M A R Y CELL WALLS

183

could arise from either its continual degradation or its transfer to another cell fraction. Continual degradation of a hydroxyproline-rich protein can be ruled out because these cultured ceUs can neither utilize free hydroxyproline nor do they accumulate significant amounts of free hydroxyproline. Therefore, in the absence of degradation, cytoplasmic hydroxyproline must be transferred and accumulate somewhere. Evidently, it is the precursor to wall hydroxyproline! Reinforcing this conclusion are data derived from attempts to deal with wall protein itself, I n experiments with sycamore cells (Lamport, 1962a) it wm possible to show that after a pulse of W-proline, followed by isolation of the cell-wall fraction (washed with distilled water only) and packing it as a column,it was possible to obtain a small amount of high spec& activity, triohloracetic acid-precipitable, hydroxyproline-rich material by eluting the cell-wall column with a linearly-increasing sodium chloride gradient (Fig. 19). This represents ionically-bound wall protein precursor and corresponds partly at least to Olson’s (1964) hydroxyproline-rich protein (“wall extracted fraction”) extracted from the wall by NaOH, KCl, or urea. The kinetics of Olson’s “wall extracted fraction” also show characteristics typical of a component in transit (Olson, 1964). TABLEXI 8pecifio Actidiea of Protein-Bound Proline and Hydroxyproline in afferent Cell Fmtkma of 8 y m m e Cell Suepem‘ons at Varbua Tinzee after a “C-Proline P&e*

Hours after %“proline p&e Th~uanndaof CPM/ymole

Fraction 100.5.30 hydroxyproline proline 1O.P.20 proline 1 .P.t hydroxyproline (wall) proline

0.6

-

11.6 12.6

2

6

12

86

96 102 121

69

57 63 42.6

30.5

63 107

66

-

A2

-

24

-

48

-

-

92

73

66.6

66

84

40

* Cells were incubatedand frectionated as described in Fig. 6. Specific ectivitien of hydroxyproline and proline were determined after electrophoresis (pH 1.8); autoradiopphj ; and elution of tbe labelled proline and hydroxyproline.hmples of thew imino acids woro chocked end found to be radiochemicallypure after eloctrophoreeie at pH 6.8. One can conclude that in all probability, after ribosome-mediated synthesis of hydroxyproline-rich protein, it is released aa a Boluble protein into the cytoplasm from whence, by processes unknown, it makes its way to the wall, appearing finally a8 firmly bound insoluble wall protein.

184

D E R E K T . A . LAMPORT B. PROLINE HYDROXYLATION

1. Oxygen jixation

Hydroxyproline biosynthesis is field of its own yet drawing together workers from many disciplines. The peculiar properties of hydroxyproline-rich proteins add significance to these studies. For example, both animals and plants produce wound tissue containing hydroxyproline-rich protein. How cells synthesize these proteins is understood in small part only. Outstanding is the problem of the preaise stage at which proline becomes hydroxylated and whether ascorbic acid plays a significant part in the reaction. Recent workers inquired into the origin of the hydroxyproline hydroxyl group. The answer may illuminate the problems above. Fujimoto and Tamiya (1962) and Prockop et al. (1962) reported that 1*0, was incorporated into collagen hydroxyproline hydroxyl oxygen. Similarly it was possible to show that in sycamore cell suspension cultures the hydroxyproline hydroxyl oxygen was derived exclusively by the fixation of atmospheric oxygen (Lamport, 1963a). In a preliminary communicat$ionStout and Fritz (1964) report that etiolated soybean and maize seedlings also incorporate molecular oxygen into hydroxyproline. These results lead inevitably to a consideration of the effect of oxygen tension on wall protein synthesis, bearing in mind the possibility of dissociating these effects from the cytochrome system. Summarizing the recent experimental results obtained with sycamore (Table XII) it seems that these cultured cells grow equally well at oxygen tensions of from 6-40% vlv. Tensions above 80% are toxic. Tensions below 6% remain to be explored in more detail; however, preliminary rosults indicate that cultures grown in oxygen tensions lower than 6% yield a higher proportion of giant cells. TABLE XI1

Effectof Oxygen Tension on the Yield of Sycamore Cell Suepensions

Yield after 14 ohye’ growth Approximate 0, tr?isioir* Packed cell volume Dry weight mg/nJ ~

6% v/v 10 40 80 100

48 50 48 33 4

~

~

11.6

12.3 11.7 7-7 1.6

* &lle were grown in the closed system &E previously described (Lemport, 1963~).The nitial inoatdurn was spproxh~tely1 mg/ml cells (dry weight).

T H E P R O T E I N C O M P O N E N T O F PRIMARY ClLL W A L L S

186

2. Hydrogen lo88 In pursuit of the proline hydroxylation mechanism, several workers (Prockop d al., 1962, 1964; Stone and Meister, 1962; Meister d al., 1902) used a specifically tritkted 3-4 'H-proline and drew various conflicting conclusions depending on the amount of tritium that was lost from proline during the hydroxylation. Part of the confusion may be traced to the view that the tritium at C3 and C4 was distributed randomly, whereas in fact there is some evidence for considering that the tritium at C3 and C4 is tram to the carboxyl group (Lamport, 1936b? 1964b). If thiR is so then, knowing that 3-4 SH-prolineloses 60% of its tritium on oonversion to hydroxyproline in sycamore c e b , one can conclude that the mechanism of proline hydroxylation is analogous to some steroid hydroxylationa (Hayano et al., 1958). Additional experiments in which sycamore cella grew in the presence of 4-*H glutamate or in *H,O containing a "swamp" of proline, also indicated that during hydroxylation proline lost only one hydrogen from C4 (Lamport, 1964b). Bearing in mind the tritium results which indicate that the hydrogen lost from C4 is tram to the carboxyl group, one can oonclude that the reaction mechanism probably involves a single proton displacement with retention of configurcLtion by the incoming substituent. If nothing else this work at least shows that the judicious me of isotopes (*H, 8H, l*O, W) can lead to the detailed considerations of a !reaction mechanism in the intact cell.

3. The immediate precursw to hydroxyproline The immediate precursor to hydroxyproline remains rather a mystery. The important question still unresolved, despite earlier claims (e.g. Steward and Pollard, 1968), is whether proline is hydroxylated hcfore Or after it enters into peptide linkage. Unfortunately, there is no lack of inconclusive evidenoe for both views distributed fairly evenly on both sides of the fence. Peterkovsky and Udenfriend (1901, 1963) obtained a cell-free chick embryo system which converted W-proline to peptide-bound "(3-hydroxyproline. This conversion apparently continued when ribonucleaae or puromycin inhibited further IF-proline incorporation into peptide linkage. They concluded that the source of peptide-bound 1'6-hydroxyproline under these inhibitory conditions was peptide-bound lPC-proline, which must, therefore, be hydroxylated in 8itu. Olson (1964) arrived at a similar conclusion as a result of his observation that in tobaooo suspension cultures a pulse of l'C-proline gave rise to bound 1'C-proline which nevertheless continued to give rise to bound14 C-hydroxyproline even during the proline cliase. These results above are difficult to reconcile '

DEREK T . A . LAMPORT

186

with reports of Coronado et al. (1963), Manner and Gould (1963), and Jackson e.i aZ. (1964), who claim to have isolated hydroxyprolylsRNA from in vitro systems. In addition, Manner and Gould (1963) found that puromycin severely inhibited the incorporation of Whydroxyproline. An unusual feature of the chick embryo system is that free hydroxyproline is normally present. TABLE XIII

I8ohtion of Putative Hydroxyprolyl-8I1" f r m Syoam0l.e Cell Suapc%%km CUh4TM

A. Results of treating W-prolino-labelled RNA with '

hydroxylamine

Counta above paper backprmnd Origin Hydroxyproline hydroxamic acid Proline hydroxamic acid

43 27 60

B. Results of treating W-proline-labelled RNA with ammonium hydroxide (pH 11.6)

~ o u above h paper backpou?ld Origin

Hydmxyproline Proline

274

61 42

Ezperimenlal metihod. Three litres of expnentially-growing cells (10% packed cell volume) were hervcsted rapidly on a 600 ml sintered glam funnel. About 260 ml packed cells were resuspendcd in their own growth medium (total volume WO ml) incubated a t 27" and aerated by rotary agitation. Ten pC W-proline (specific activity 11-3 mC/mMole) were added, and 3.5 min later the whole suepeneion wm dumped on t o crushed ice. The cells werc reharvested on 8 sintored funnel (in the oold mom at 4') and broken batchwiee in Zillig and H6lzel's (I@.%) cell mill. The homogenate was centrifuged and nucleic acid was extracted from the 100.5.80 fraction by Kirby's (1966) method. The freeze-driedcrude nucleic acid W 8 8 incubated for 3 hr a t 26"with anhydrous hydroxylamine,and 26 pg each of the hydroxamic acide of proline and hydroxyprolinewere added a8 carrier. (Thesehydmxemic acids were 8 kind gif't from Dr. N.M. Green.) The incubation mixture was diluted with 2 ml water; dried from the frozen state; resuepended irr 0.6 ml 70% ethanol, and centrifqed. The ethanolic supernstant wau then electrophoresed for 2 hr at pH 6.5 (A0 v/cm) on Whatman number 62 paper, which wau then dipped in an acetone solution containing 1% ferric chloride buffered to pH 0.9 with triohloracetic acid. The pink-coloured areas corresponding to the hydroxamic acid ferric complexes of proline and hydroxyproline were eluted with water, on to planchettes, dried, and the radioactivity measured (A). The hydmxylamine-treated ethanol-extracted nucleic acid fraction wm incubated for a further 3 hr a t 37" with ammonium hydroxide to strip the ERNAof imino acids. Material soluble in 70% ethanol wm eleotrophoresed a t pH 2.1 with markers; and eluted with water, and the radioactivity measured (B).

THE PROTEIN C O M P O N E N T OF PRIMARY CELL WALLS

187

Experiments with cultured sycamore cells also indicate (Table XIII) that these cells contain small amounts of hydroxyprolyl-sRNA. Whether or not all these data can be reconciled remains for future experiments. If it turns out that hydroxyprolyl-sRNA i8 the precursor to peptidebound hydroxyproline, then one should observe no restrictions on hydroxyproline peptide sequences in wall protein. Alternatively, if hydroxylation of proline occurs in the protein one ahould observe a regularity of the hydroxylation pattern-only certain proline residues will be recognized by the hydroxylase-and the hydroxyproline peptide sequences will therefore be restricted. Unlike wall protein, one-third of the collagen residues is glycine, and this fact alone imposes severe restrictions on possible sequences (see Harrington and von Hippel, 1961; Ramachandran, 1963). Another important structural protein, elastin, has little hydroxyproline but has about the same number of glycine residues as colhgen. Partridge (1958) suggested that the three chain helix structure of collagen might also he possible for obstin. If the secondary and tertiary structure8 of collagen and elastin woro similar this would raise even more difficultiw for an in situ type of hydroxylation. 1f hydroxyproline - peptido Roquonces aro, like other amino tukl wquences, determined by a nucleic acid code, tho reHtrictionn dinappcar and the range of structures containing hydroxyproline becomcn more flexible. In this situation adenyl proline is likely to be the immediate precursor to hydroxyproline. Prolyl-sRNA can almost be ruled Pro --+AMP-Pro

+sRNA-Pro

-

Protein

Ascorbote

FIG.10. Hypothetical scheme for prolino hydroxylation. Tho scheme illu8tratex how a m r bic acid might be a necessary part of the hydroxylation roection, by acting aR the two dootron donor which reducos enzyme-boundperferry1 ion (E-FcO,++) to enzymeboundforryl ion (E-FeO++)which could be the actual oxygen donor tanproline (rf. diecussion by Lnprahum, 1962, p. 68).

188

D E R E K T . A . LAMPORT

out as the immediate precursor, because the sequence-determining mechanism only recognizes the nucleic acid part of the sRNA-amino acid complex (Chapeville et al., 1962). However, Manner and Gould (1963) found that although sRNA from Escherichia coli markedly increased the incorporation of 1%-proline in a chick embryo system, the E. coli sRNA was (unlike chick embryo sRNA) completely ineffective in stimulating the formation of hydroxyproline. They concluded that one should consider the possibility of two sRNA acceptors for proline, only one of which can be hydroxylated. Figure 10 is an attempt to summarize all the available data as a reasonable scheme which can be tested further experimentally. The scheme includes ascorbic acid on the bwis of suggestions by Robertaon and Schwartz (1963), Gould (1968), and Stone and Meister (1962), that ascorbic acid may be directly involved in proline hydroxylation in animals. A similar role for ascorbic acid in plants would also be consistent with Bonner and Bonner’s (1938) demonstration of an ascorbate requirement in plants.

V. VARIATION OF CELL-WALLHYDROXYPROLINE CONTENT A. WALLS ISOLATED FROM TISSUE UULTURES

Table VIII summarizes the author’s results of estimating hydroxyproline in walls isolated from various tissue cultures. The walls most abundant in hydroxyproline contain nearly forty times more hydroxyproline than walls containing least hydroxyproline. The average hydroxyproline content of primary cell walls isolated from tissue cultures is 1.2% on a dry weight basis, perhaps better expressed as 92 pmoles hydroxyproline/g dry cell wall. Thus, the hydroxyproline content of the primary wall is clearly dependent on the plant Rpecies from which the culture originated, and even continues at the variety level as in tobacco (Table VIII). It would be especially interesting to know if the cell-wall hydroxyproline content varied, even in those cultures which were isohted fmm diferent regions of the 8ame p b n t (ornear olonal material). The answer to this question is y e s md no. For example, the hydroxyproline content of Phaseolua VuZgaris listed in Table VIII actually refers to identical results obtainqd from two cultures, one isolated from hypocotyl (Lamport and Northcote, 1960a), the other isolated from the cut surfact of the cotyledon. Here there is obviously no effect of position on wall hydroxyproline. However, in a more recent and extreme example the author finds that whereas Tulecke’s (1967) haploid pollen-derived Birckgo culture coiitains little wall hydroxyproline, a rather poorly

T H E PROTEIN U O M P O N E N T O F P R I M A R Y CJELLWALLS 189

growing callus culture isolatod from Ginkgo cambial tissues yielded a wall fraction relatively rich in hydroxyproline (Table VIII). Because pollen tubes themselves contain little hydroxyproline (cf. !Necke ed al., 1982, and various authors in Linskens, 1984), one can speculate that the low hydroxyproline content of Tulecko’s culture is not a result of haploidy per se (if it were, polyploidy should reverse the situation !), but is a result of differentiation-the haploid culture is suggested by the author to retain some important attributes of pollen (e.g. low wall hydroxyproline) and is in that aenae differentiated. LaRue, who pioneered the isolation of “pollen cultures”* (see Tulecke, 1969), also considered that these pollen-derived cultures retained some of the characteriatics of pollen ;for he regarded these cultures as representing extensions of the vegetative tissue of the male gametophyte (Straua and LaRue, 1964). As other pollen cultures become available it will be interesting to see how far this generalization can be extended. Finally there is the possibility, which has probably been in the reader’s mind throughout this section, that perhaps the primary wall hydroxyproline content can be altered or influenced by purely cultural conditions. If so, these conditions have yet to be found. The nearest approach to date was obtained with Centaurea cyanw (Table VIII) which after one month in suspension culture yielded a wall fraction aontaining only 0.22% hydroxyproline. But after six months of continued growth by subculture every two weeks, the isolated wall fraction contained 0.6% hydroxyproline. Correlated with this dramatic increase in wall hydroxyproline was a concomitant decrease in the number of secondarily thickened cells. These were abundant in the early cultures when up to 80% of the cells showed prominent spiral, reticulate or scalarifom thickening, but were rare in later cultures. The apparent increase in wall hydroxyproline was therefore in this instance probably a reflection of an increasing proportion of primary cell wall. The wall hydroxyproline content of sycamore suspension cultures has remained constant within experimental error, and independent of the various experimental conditions imposed on these cells, over the last six years! The dissociation of hydroxyproline biosyntheeis from wall biosynthesis in cultured cells is therefore a problem for the future. B. WALLB ISOLATED FROM PLANT PARTS

Unlike most cultured material, the variable presence of the secondary cell wall accentuates the problem of comparing the hydroxyproline

* Workers who looaely refer to pollen cultures and oambisl cultures, and draw conolush

based on the assumption that them are pollen cultum and cambial cultures per 6e. ahodd refer to Huiley (1943) for enlightenment !

D E R E K T. A . L A M P O R T

190

content of primary waUs isolated from various plant parts. I n fact, secondary wall is invariably present to a greater or lesser extent. Ordinary optical and polarization microscopy gives a very rough idea how much secondary wall contaminates a primary wall preparation. A somewhat more quantitative rule firat suggested by Sultze (1967) seems to be emerging from a comparison of arabinose/xylosemolar ratios in cell wall hydrolysates. Work with tissue cultures (Table X N ) shows that the arabinose/xyloae ratio is always greater than unity in primary walls. This rule also seems to be applicable to walls isolated from the TABLEXIV

Primary Cell Wall Sugar Molar Ratwe Galaotose Glucose Mannoee Arabinoee Xyloae Acer pseudoplutandb) 1960 A 1960 B

0.4 1.8

1 1

2.4

1

0.8 Camellia sinemid") 2.4 Ginkgo biloba (n)@) 0.3 Ginkgo biloba (2n)(O) 0.7

1

0.9 1.4

0.3

R h m e

0.1

trace trace trace trace n.d. trace

3.0 0.6 2.0 0.6

0.3 2.8

n.d.

0.4

n.d.

trace

0.8

0.36

1.4 M d w qlveetridd) Nicotiana tabaoum(") 0.4 1.3 oryza e a t i v a ( ~ )

1 1

0.3

1

1

n.d. n.d.

2.7 0.1 1.3

Populue Iremula(''

1

0-OG

6.6 0.26 1-6 0.66

n.d. 0.38 0.1 n.d. n.d.

1960 C 1964

(8)

0.7

I I

0.6

n.d.

0.G

0.4 0.1

0.6

Wells isolated from suspension culture.

(b) Wdls isolated from callus cultures showing some secondary thickening. (c)

(d) (e) (f)

Wells isolated from callus culture with no obvious secondary thickening. W d l isohted ~ fromfleahy fruit. Tobacco pith cell walls analyead by Wilson (1969). Sultze (1967).

intact plant (Sultze, 1957; Thornber and Northcote, 1961; Ray, 1903) where the advent of secondary thickening corresponded to a. rapid decrease in the arabinoselxylose ratio. The secondary wall problem can, of course, be minimized by judiciously selecting material containing little or no seoondary thickening, for example, young rapidly growing tissues and the soft flesh of fruits. Table IX lists the hydroxyproline content of walk isolated from several different tissues. The average, 0.3%, is considerably lower than for tissue cultures, and the general rule seem to be that primary walls from tiasue cultures are richer in hydroxyproline than primary walls from plant parts. Nature does not apply this rule rigorously. For emmple, primary walls isolated from young pea roots contain more hydroxyproline than primary walls

THE P R O T E I N C O M P O N E N T O F P R I M A R Y CELL W A L L S 191

isolated from some tissue cultures ; conversely primary walla isolated froin rose, rice, and haploid #inkgo contain less hydroxyproline than walls isolated from various plant parts (Tables VIII and IX). Unlike tissue cultures, the intact plant seems able t o control hydroxyproline content of the primary wall. For example, pea root walls contain roughly twice as much hydroxyproline as do pea epicotyl walls (Table IX).Also hydroxyproline-poor material such as sycamore cambial tisme gives rise to tissue cultures with hydroxyproline-rich walls. It almost looks as though the hydroxyproline-rich tissue culture represents a disturbed situation resulting from the removal of cells from the controls presumably present in the organized plant. A study of the effect on the entire plant of disturbing hydroxyproline biosynthesis could be most illuminating. D. S. Mathan and A. C. Olson (unpublished data, private communication)have recently discovered such a disturbed situation, namely, a mutant tomato, in which there is a correlation between wall hydroxyproline content and leaf shape. They find that the homozygous lanceolate leaf shape mutant produces at most a seedling with very small simple leaves (Mathan and Jenkins, 1962). These leaves contain 100 times more wall-bound hydroxyproline than do leaves of the normal seedling. Even the heterozygous lanceolate seedlings contain 36 times the normal amount of wall-bound hydroxyproline. The implication that wall-bound hydroxyproline directly influences leaf morphogenesis is too strong to be ignored and makes one await impatiently the results of future work with the tomato mutants. The work of Van Etten ed al. (1961, 1963) can also be interpreted as supporting the suggestion made above, that intact plants exert a strict control over the wall hydroxyproline, the different amounts in the same plant presumably being determined by the m e r e n t needs for wall protain in different tissues, regions, or organs. Van Etten and colleagues determined the amino acid composition of hydrolysates from 200 different seed meals. Hydroxyproline occurred often, sometimes in unusually large amounts, localized mainly in the pericarp or testa rather than in seed tissue itself. The hydroxyproline was insoluble and remained firmly bound to the original tissue, so it seems reasonable in view of all the data presented so far to conclude that it was bound to the cell wall. The variation in amount of this hydroxyproline seems too hrge to be explained as a result of variations in the proportion of cell wall and cytoplasm in the material examined. More probably the variation reflects the type of primary cell wall present. Why aome should be so rich in hydroxyproline is yet another aspect of the more general unsolved problem of wall protein. One cannot ignore the possibility that wall protein might confer the necessary tensile or elmtic properties on cell walls of tissues involved in the dehiscence mechanism. M

D E R E K T. A . L A M P O R T

192

The relatively high hydroxyproline content of some young dicotyledonous herbaceous stems such as peas, beans, etc., contrasts strongly with the relatively low hydroxyproline content of the Gramheae examined so far (Tables VIII and IX). These differences in hydroxyproline content are brought out even more clearly by E. R. Stout and G.J. Fritz's recent obHervation (private communication) that, unlike maize, the soybean seedling hydroxyproline content per gram fresh weight increases during the two-week period studied (Fig. 11). On a fresh weight basis both plants initially contained about the same amount of hydroxyproline, but after two weeks the soybean seedlings contained five times as much hydroxyproline as the maize.

- 250 e

f 200t

. I -

4

0

l

*

2

'

4

6

8 10 Age (days)

~

~

12

14

~

I

Fro. 11. Tho hydroxyproline content of etiolated soybean and maim seedlings dwing germinetion and growth (E. It. Stout and G. J. Fritz, personal communication).The hydroxyproline content was measured in seedlings minua endonperm and eeed coat. The figure above shows the results expressed on a fresh weight basis.

It seems logical to look for 11, correlation between hydroxyproline content and general growth form. In this respect it is worth recalling the significant contributions of Schwendener (1874), who first snalysed the mechanical tissues of stems and leaves in terms aimilar to those used by engineers when analysing the arrangement of girdem. Among herbs, the monocotyledons exemplify the most advanced types of mechanical construction based on the distribution of sclerenchym, collenchyma, and xylem. Herbaceous dicotyledons rely for mechanical support rather more on turgidity. It would be surprisingif this difference were not reflected in the composition of the wall. Continuing this line of reasoning, it seeins worth aaking whether the fact that young pea root walls are richer in protein than young pea epicotyl walls is, like the arrangement of root vascuIar tissue, a reflection of the longitudinal pul-

T H E P R O T E I N C O M P O N E N T O F P R I M A R Y C ELL W A L L S

193

ling stresses and strains to which roots are subjected, in contrast with the mainly transverse bending strains in stems. Another aspect worth mentioning is that some of the more exotic structures produced during the reproductive phase must involve a sufficiently high degree of control over cell extension, even for the precise determination of the shape of a single oell. If wall protein is involved in this process (cf. Section VIII) it seems inevitable that one form of control at the cellular level would involve a non-uniform but predetermined distribution of wall protein within the cell wall. Examples might be found among the six-rayed stellate parenchyma pith cells of Jur~cwror the multibranched unicellular hairs of many plants.

VI. DEGEADATION OF THE SYCAMORE PRIMARY CELL WALL A. CHEMICAL DEGRADATION

Before further attempts to answer questions about the role of wall protein it is necessary to know something about the other components of an isolated primary cell wall. The sugar profile obtained by estimating the sugar molar ratios in a total acid hydrolys&teis a simple, rapid and crudely quantitative way of comparing isolated cell walls. Preliminary results of such a crude comparison (Table XIV) show similarities rather than differences; for example, this mcthod does not distinguish a @inkgo primary wall from a sycamore primary wall; hardly surprising in view of the surmised common properties of primary cell walls. There is, therefore, some reassurance that the necessarily detailed chemical work on one primary wall will reveal details which u're essentially similar for many other primary walls. The ease of isolating the wall fraction from a good (i.e. pipettable) cell suspension culture dictated the choice of sycamore. Figures 12 and 13 show the results of a typical experiment in which the walls were successively extracted by the classical procedures involving somewhat harsh conditions. This type of extraction reveals several noteworthy features : first, appreciable protein is only removed by reagents known to lead to some peptide bond cleavage, but even under these conditions a small amount of protein remaim insoluble; second, the protein content of the a-cellulose can be reduced further (to about one-tenth of the value shown in Fig. 12), but still incompletely, by longer treatment with strong KOH. Third, the a-cellulose contains other sugars additional to glucose. After calculating the theoretical amount of pure microfibrillar 1,4-/?-glucanfrom the actual glucose content of the a-cellulose, one findR that the primary wall contains nearly as much protein as microfibrillar cellulose !

D E l t E K T. A . L A M P O R T

194

B. ENZYMIC DEGRADATION

Despite the difficulty of obtaining pure carbohydrases, specifically catalysing known reactions, even crude enzymic methods of analysing cell wall structure complement the chemical methods of extraction. At the present moment both chemical and enzymic methods are crude, but enzymic methods offer the greater hope of drastic improvement. Table XV summarizes the effect of various crude commercial carbohydrnses on the cornpodion of the isolated sycamore cell walls.

Pectic aubstonces 36.5

I

I

I

4% H3BQ5-------

Lipid Protein

Polysocchoride

0 Glucan

PIG. 12. Chemical fractionation of aycamorc ccll Walls. The results shown in Fig. 12 repreeent the results from one complete sequence of cxtraotiona of w d h bohkd from stationary phase sycamore cells. During stationary phase the cells contain loast starch which otherarise ten& to contaminate the wall fiaction. The sequenoe of extractiona proceeds from left to right acmes the figure beginning with ethaqol and ether refluxes, and ending with a 3 d q extraction under nitrogen with a 24% KOH/4% H,BO, mixture. Material aolubilized by potessium hydroxide WBB neutralized with acetic acid, then dial@ against distilled water, and h l l y precipitated by addition of alcohol to a h a 1 concentration of 70% v/v. The pectic subetancee were obtained by refluxing the walls with water for 24 hr. This released the mme amount of m d o acid residues Mdid treatment with ethylamine diamine tetra acetic acid. Uronic acid residues were eetimated by the method of McCrermdy and McComb (1962). protein wan calculated from the hydroxyproline content of the insoluble wall residue remaining af'ter each fraction, on the assumption that hydroxyproline represented approximately 14% by weight of the probin present. In the light of more recent results this may be a slight over-estimation.

T H E P R O T E I N C O M P O N E N T O F P R I M A R Y CELL W A L L S

196

Although three types of crude enzymic preparations are available, viz. “pectinase”, “hemicelluI&8e”, and “cellulase”, the names give little guide to their specific actions, which must therefore be determined experimentally and re-checked for each batch of identically named enzyme when the “lot number” changes. For example, the crude pectinase preparations which the author has examined remove rather more than polygalacturonides from the wall but have negligible cellulase activity. Some commercial preparations labelled “hemicellulase” behaved very much like the crude “pectinme”, while others showed an additional powerful cellulolytic activity. Finally, crude “cellulase” preparations generally showed pectinase hemicellulase cellulase and proteam activities. Thus, to save time one could omit the “pectinase” step and immediately treat the walls with cellulase. ______~

15

Hemicellulose II

a-Cellulose

10

5

0

Gal G Ar Xy M Rh

Gal G Ar Xy

M

Rh

Gal

G

Ar Xy

M Rh Gal G Ar Xy M

F

Fro. 1s. Sugar profiles of sycamore cell wall fractions. Each fraction was hydmlyaed in 72yu w/w bulphuric acid for 12-36 hr. The solution was then diluted to 2 . 6 and ~ heated in an autoclave for 1 hr at 16 lb/in*. The solutions were ncutralizcd by addition of IRQB (HCO,form) resin, filtered, evaporated and chromatographod 24 hr in Timoll’s (1960) ethyl acetate pyridine, water (8 :2 : 1 v/v/v) solvent. Nugars wcrc then estimated. by Wilnon’n (1959) method. The profile for each wall fraction expreswe thc sugar molar ratios relstive to glucose which han been arbitrarily set at 10. These figures are uncorrected for the small amount of pugar decomposition during hydrolysis. The u-cellulosc of the experiment shown ~ b v eone tained an amount of mannose and hydroxyprolinr which could be reduced 90% by prolonging the extraotion procedure.

The most obvious result of dograding sycamore walls this way was to produce a carbohydrate-protein complex (approximately one-third protein), which more or less retained the wall shape and was considerably enriched in hydroxyproline and arabinose, when compared with the original wall (Lamport, 19640). This enrichment of hydroxyproline and arabinose occurred iilso in other cellulase-treated w a b

D E R E K T. A . L A M P O R T

196

TABLEXV Enzymic I)egrtctlation of Sycurnore Cell Walk

yo 1088 in ___-

weight for each extruct i o u

--____-

Intact .wall

-__

C‘unaulative lose i n Ir~(+#~l - ___ __---

yo bypro found 1.2

(1

60

60

2.6

Carbohydkte20 protein m p Z e x ( ’) 24% KOH/4% HaBO, 3 days at 26” undor

70

4.2

94

n.d.

+ “Cellulme”(b)

aal Q

M

Ar S y

+-

0.5 0.3

1 _ 1 1 -

0

+ “Poctinme”(’)

Sugar projlWd)

0.8

1

3.0 1 0.28 9.6 1.0

+

nitrogen “Limit -cellulose”

24

1.0

1

-

2-6 1.0

(a) Incubated for 24 hr a t 37 ’ tLt pH 6 in a 10% pectinam solution prepared from a dry powder (catalogue number 908) obtained from Mann Remarch Laboratories Tnc., New York. (b) Incubated for 24 hr a t 37” a t pH 6 in a 10% technical cellulase solution prepared fiom a dry powder (lot number 49643) obtained from General Biochemicals, Chagrin Falle, Ohio. ( 0 ) When the carbohydrate-protein complex was hydrolysed in 72% w/w sulphuric wid for 86 hours a t 26” and followed by dilution to 2 . 6 ~and autoclaved for 1 hr at 16 Ib/in*, only 62% of the material was solubilized. The sulplruric acid-insoluble raidue was dried; hydrolysed in 6N HC1 (106” 18 hr) and hydroxyproline was estimated. The material which rerrmined undissolved in 72% sulphuric acid contained 2.3% hydroxyproline on a dry weight bash! One can only agrec with Tupper-hroy trnd I’rieRtley (1924) who a180 observed that B portion of the wall was resistant to sulpliuric acid, and concluded: “On the whole, a combination of protein with the cellulose, scorn8 the most probable explanation of this resistance to sulphullc acid.” (d) Molar ratios. Gal. : gulticto8e; G gluro~c;M = manno8e: Ar nrabinoae; Xy = sylose. 7

-:

provided that these walls were initially hydroxyproline-rich* (Table XVI).The hydroxyproline-poor walls isolated from the haploid Ginkgo culture dissolved completely in a crude cellulase solution and the hydroxyproline-poor walls of rose dissolved almost completely. Cellulase removed the walls from intact osmotically stabilized haploid Ginkgo cells sufficiently rapidly to aUow the isolation of metabolically

* From one p i n t of view the hydroxyproline-richcarbohydrate-probh complex reeemftlea humus, which is in part produced by accumulation of the more resistant residua of plants. The nitrogen content of humus aruouiits to as much as 6%, is much more resttant to d c m . bial attack and is also largely resistant to chemiral extraction (cf. Tabla XV)and hydrolyais (Whitehead and Tinnley, 1983; rf. Srharpmeel, 1962).

THE PROTEIN COMPONENT O F P R I M A R Y CELL WALLS

197

TABLEXVI Effect of Crude C e l l h e on thc Cmpm‘tion of Cell

yo hydroxyproline after trentvnent Ccnta;tcrea:cyarrue*

Cenhurea cyanus? Nicoticcna labacum. vmr. xanthi* v m . turkiah* Aeer pseudoplatccnuat Acer pseudoplata;nua* Lywpareicon* caoJcntim

0.6 0.69 2.0 2.45 4.2 4.6 4. I

UaC 2

0.6 4.6

6.0

3.0 4.6

W&

Sugar molar ratws Q M Ar X y 1 1

1 1 1 1

1 1.2

1-8

2

6 1-6

8.0 18.0 0.26 9.6

3.0 1.0

0-2622.0

0-4

-

1.0

n.d.

The 0011wnlls of all tliv nbovo n p v i c n worn obtained from suspenNion aulturos. All the orude commercial carbohytlrasesRO far oxamined also show protoolytic activity and this may explain why some walls are not onrii:hd in hydroxyprolino in proportion to tho amount of cell wall eolubilized. The results shown for Ccnfazcrca Cyonud w m obtained using walls ieolated within one month of obtaining a Cenhurea cyanzla culture. The wslle of Ccnlaurur cpntrst were isolated after about 6 months in suspension culture. Indicates material remaining ineolublo after trcstment with a 10% Bolution of General Biochemiml’s “Hemicellulase” for 24 hr at 37”. This preparation showed poworful cellulaae activity. It is no longer commerciallyavailable. t Indicates material remaining imoluble after treatment with a 10% solution of General Biochemical’s technioal cellulnae (lot number 49453) for 24 hr a t 37”.

active protoplasts. This difference in cellulase solubilization between hydroxyproline-rich and hydroxyproline-poor walls is direct evidence for believing that the hydroxyproline-rich proteifr plays some part in holding the wall together, a suggestion made earlier on a purely theoretical basis (Lamport, 1962a,b). Judging from the low glucose content and lack of reaction with Schdze’s chlor-zinc-iodide specific cellulose reagent, the insoluble oarbohydrab-protein complex, obtdned by ccUulase treatment of the sycamore wall, contains little or no cellulose. A t this stage i t was useful to supplement the enzymic methods by a further treatment with a strong potassium hydroxide-boric acid mixture which is known to be very effective in removing non-cellulosic polysaccharides (Jones and Painter, 1957). After this treatment the h a 1 insoluble residue gave a positive reaction with Schulze’s cellulose reagent. Furthermore, it showed 8 considerable enrichment in glucose (Table XV), although etill far from being pure cellulose. This result raiws the old problem of accounting for the traces of non-glucose sugaw invariably found in acelluloge preparations (Saeman el al., 1954; Dennis and Preston, 1961). Treating sycamore walls sequentially with cellulase and KOH has

198

DEREK T . A . LAMPORT

produced a “cellulose” containing a remarkably large amount of nonglucose sugars. It seems also significant that reversing the sequenoe to KOH treatment followod by cellulase, which amounts to incubating O a glucm oonsidorably onriohed in a-collulose with cellulaso, C ~ S yields non-glucose sugnrR (Tablo XVLT). TABLEXVII Thr. effect of K O H m d Cellulaee on S y m m r e Walls

Sugar molar ra&a Treatment eequmce 1. “Cellulase”* 2. 24y0KOH 4% H W S 1. 24% KOH 4% HSUOS 2. “CtlllUlAse”t

%, L O U 8

Cumulative loas

of ineoluble r d w Gal G M Ar X y 0.25 0-6 1 2.5 1

70 24

70 94

3 1

75

75

0.08

1

0.05 0.01 0.08

17

92

0.3

1

0.5

1 1

0.6

0.3

Walls* or a-cellulose t wero incubated in (I 10% solution of techniral cellulase (General Biochomicals lot number 49453) for 24 hr at pH 5 and 37“.

VLI. A TENTATIVE PICTURE OF “EXTENSIN” IN THE PRIMARY WALC “If therefore some may bo apt to think that I have sometimes too far indulged in Conjecture, in the Inferences I have drawn from tho Events of some experiments; they ought to consider that it is from these kind of Conjectures that fresh Discoveries first take their Rise ; for tho’ some of them may prove false, yet they may often lead to further and new Discoveries.” Stephen Hale, “staticalE88ay8” (1733)

As outlined here, the results of chemical and enzymic degradation of the sycamore wall might be taken to suggest that the carbohydrateprotein complex merely represents resistant components of the wall held together by their mutual insolubility. But cmnplete solubilitation of hydroxyproline-poor walls by cellulase argues strongly a g a h t this possibility. What then does the hydroxyproline-rich carbohydrateprotein complex signify? Two main lines of evidence point to a covalent link between the carbohydrate and the protein. h t , it is impossible to separate protein from the intact wall unless hydrolytic methods are employed (Dougall and Shimbayashi, 1960; Lamport and Northcote, 1900a; Olson, 1964). Even then mild methods of hydrolysis lead to incomplete removal of protein (Tablo VII). Secondly, “ceUuL&se” removes about 70% of the sycamore wall, leaving a wall-shaped struc-

THl P R O T E I N COMPONENT O F P R I M A R Y C E L L W A L L S 199

ture oontaining 8040% of protein initially present, and oonsiderably enriohed in plrabinose and gdaatose. The oellulase-treatedw a b of three different tissue cultured species examined so far (Table XVI)yielded R carbohydrate-protein complex with an arabinose Igdaotose ratio of 3 : 1. This indioatea the presenae of a galacto-araban.The small amounts of cellulose remaining in the syca-

FIG.14. Possible arrangement of “extensin” in the primary cell wall. One can imeghe the Mockd regions &a more or lees preserving the integrity of the wall during enzymic (“wlluI-”) attack.

200

D E R E K T . A . LAMPORT

more carbohyhte-protein complex probably represent those portions of wall cellulose which, being most intimately associated with protein and galrtcto-araban, are masked and therefore unavailable to the action of cellulase. Figure 14representsthisschematically ;it also shows gdactoaraban the link (adaptor?) between cellulose and protein and the possible arrangement of diaulphide bridges (of. Section X).

VIII. THECONTRIBUTION OF “EXTENSIN” TO WALL FORM AND TENSILE STRENUTH It is true that the schome (Fig. 14) for the arrangement of “extensin” in the wall represents a considerable extrapolation from the experimental data. Viewed purely as a working hypothesis the extrapolation justifies itself: not only are the actual suggestions open to direct experimental attack, but they lead to further predictions about the properties of hydroxyproline-rich primary walls. One such prediction is that if protein forms cross-links between wall polysaccharides, it follows that the protein would contribute to wall tensile strength. The corollary, of course, is that loss of wall protein would decrease the wall tensile strength. It therefore becomes necessary to compare the tensile strength of walls containing protein, with walls lacking protein. But the direct approach, involving enzymic removal of the protein, is aa yet unsatisfactory mainly because most of the wall protein is resistant to enzymic attack (Table VII). Olson et a2. (1964) found that although 6 6pronase” removed over 900,(o of the protein from oat coleoptiles, a small amount of protein (less than 10% of the total) remained. This resistant protein contained more than 60% of the hydroxyproline in the whole coleoptile. Its presence might explain why these workers found no significant difference between the stress-atrain measurements of pronase-treated and untreated coleoptiles. Unfortunately, the oat coleoptile contains an unusually small amount of hydroxyproline (Table IX) and may not be the best test object for this type of system. Therefore, if the stress-strain type of experiment were to be repeated using a hydroxyproline-rich system such as bean hypocotyls or pea roots, the results might be more significant. However, a study of cell suspension cultures may throw some light on this problem despite the fact that it is neither possible to effect removal of wall protein directly, nor a simple matter to measure the wall tensile strength of singlecells from suspensioncultures. Both can be accomplishedindirectly. The force required to rupture the wall of a spherical cell is, aa b y Wyssling (1957) pointed out, a strict function of wall thicknees, cell size, turgor pressure, and tensile strength of the wall components. Therefore, the osmotic fragility of celL in suspension culture would

T H E PROTEIN U O M P O N E N T O F P R I M A R Y CELL WALLS 201

reflect the wall tewile strength, But hydroxyproline-rich cell suspension cultures are not osmotically fragile. For example, even when sycamore cell suspensions grow in media containing up to loo/, mcrose (Table XVIII) (of. Straw and LaRue, 1964), and the internal osmotic pressure rises to about 30 atm, suddenly transferring these cells to dwtlled water does not buret them I Tulecke’s haploid Ginkgo suspension culture TABLEXVIII Urowth Yield of Sycamwe Cella Grown in fiffereni Sucrose Cmentrdione+

Initial Bucro8e concentrdiora w/v

Yield Packed o&f oalume mg &/dry weight

2

13

3

7

11 10 9 9 8.6

9 10

6.6 6.6 6.6

4 6 6

a

6.2

6.3 4.7 4.7 4.9 4. a 4.6 4-6 4.6

% dry weight 4.0 4.8 4-7 6.2 6.4 6.6 7.1

7-2 9.0

* The other coinponents of the growth medinm were &B previoiouely deecribed (Lemport, 1Wa). The celle were grown for 14 days in 280 ml Erlenmyer ffaslte. Each flssh oontsined a total volume of 100 ml medium and was sheken on a New Brunawick Gyrotory ehaker. circumvents the problem of removing wall protein because the Ginkgo wall contains so little protein to begin with (Table VIII). Furthermore, some of the Ginkgo cells are osmotically fragile even after growth in the normal medium which contains only 2y0 sucrose. Increasing the sucrose concentration increases the osmotic fragility of the cells. All the cells are osmotically fragile after growth in a medium containing 10% sucrok. Preliminary results of wall analyses indicate that the primary wall of haploid Ginkgo cells has a sugar composition much the same aa other primary walls (Table XIV). The wall does not appear excessively thin in the optical microscope, but further judgment of this aspect must be deferred until after an electron microscopic examination. Another hydroxyproline-poor system gave similar results. Gaminating pollen contains mere traces of bound hydroxyproline (Tulecke et al., 1902; of. Linskens, 1964). Table XIX shows that increasing the sucrose concentration increases the osmotic fragility of germinating pine pollen. The paucity of experimental data restricts one at the preeent time to

D E R E K T.

202

A . LAMPORT

TABLEXIX The Effectof Sucroec Concentration on the Oarnotic Fragility of Q e r n h a t i n g Pine Pollen* Sucroee concantration w/v in tile germimtion medium. 0 2 4 7 10 16 20

Per cent tubes burat in dietilled water

Approximate NaCl malady for 50%

plasmolysia

U

n.d.

0 0 40

nsl.

74 95 100

0.5

04 0.7 0.8

0.83

* Pine pollen was mllrwted in mid-May 1961, and wits allowed to germinate in a weak solution of salts identical to that used as tho inorganic components of the medium supporting growth of sycamore suspension rulturos.Tho appropriate amount of Ru(me.ewas added together with 100 pg/ml “mysteclin” (Squibh I’lmrmacnuticals. A wide epeetrum bactcrio8bt and fungicide.) The pollen was allowed to germinate 44 hr hefore testing the osmotic fragility. the tentative conclusion that walls containing little . hydroxyproline have a lower tensile strength than walls rich in hydroxyproline. But is osmotic fragility related to lack of hydroxyproline Po& hoc ergo propter hoc? The answer lies in future work which will show whether or not one can say as predicted that the increase in tensile strength of hydroxyproline-rich walls is due directly to the presence of the hydroxyproline-rich protein. The most direct evidence for involving wall protein in wall form iR, as stated earlier (Section VI B), the retention of wall form by the carbohydrate-protein complex remaining after cellulase digestion of the sycamore walls. These and other data therefore support Tupper-Carey and Priestley’s (1 924) conclusion that some growing cell walls contained a cellulose protein complex. Essentially in agreement also is the work of Chayen (1952) who, like Tupper-Carey and Priestley (1924), used Viciufuba as the experimental material, but adopted a combined enzymic and microchemical approach.

“It was found, however, that after prolonged treatment with the fungal pectinaRe preparation, which may also contain celluloBe enzymes, a well-marked cell envelope remained about the cell and apart from the membrane which bounded the cytoplafim. ThiH envelope did not absorb appreciably at 2650 A. but stained with the tetrazotized dianisidine reaction for proteins. The wall could be plaRmoiysod, or contracted by other methods, away from this

THE: P R O T E I N C O M P O N E N T O F P R I M A R Y C ELL W A LLS 203

envelope, indicating that the stained material was not residual cytoplasm. It could also be Been in cells in which the cytoplasm lay close to the envelope. It would seem, therefore, that besides the polysaccharide components, there is also some protein in the wall of meristem cells, probably ns n coniplete envelope, but possibly only as part of the wall.” (Chaycn, 1952.) One can only attempt to guess the possible way in which “extensin” might be involved in controlled changes of cell shape. By analogy with the highly orderod way in which pollen cells lay down their complex walls, it seems quite possible that ‘there could be a simila,r highly ordered non-uniform distribution of wall protein within the primary wall of a single cell. On this basis the morphogenetic potential of the wall would be built into the wall during primary wall formation. Again, the took and techniques are available for a thorough-going experimental investigation of this point. If, as suggested earlier in thio mction, “extensin” contributes to wall tensile strength by cross-linking the polysaccharides,it is also reasonable to consider those aspects of “extensin” which might contribute to its structural stability. Here, despite the differences, comparison with collagen might be useful. The high imino acid content (proline and hydroxyproline) rules out an a-helical structure for “extensin”, but the low glycine content of “extensin” also rules out the structure favoured for collagen (viz. collagen 11, cf. Harrington and von Hippel, 1961). Quite possibly “extensin”, unlike the triply-stranded collagen, exists in situ aa separate polypeptide chains. One can suggest that the polysaccharide environment enhances the structural stabilization of “extensin”. A more definite stabilizing factor is the high imino acid content which, in collagen, is clearly correlated with the shrinkage temperature (TB) or denaturation temperature (T,) cf. Josse and Herrington, 1964). collagens range in imino acid content from about 100 to 240 residues/ lOag moles protein. The shrinkage temperature of various collagens varies from below 35’ to above 60’. Table V shows the wide range in imino acid content of wall protein, As yet there are no experimental data relating the imino acid content of cell walls to a particular physical characteristic, but the work of Ginzburg (1961) may be relevant. Ginzburg showed that a heat pretreatment of pea root tips subsequently treated with a chelating agent enhanced separation of the cells. This heat pretreatment waa effective only above 60”, and increase in cell separation occurred rapidly over a narrow temperature range, Ginzburg (1961) regarded these and other data as evidence for a protein gel structure in the intercellular cement of plant tissue. Thb may be the first observation of the effect of heat on “extensin” denaturation.

204

DPREK T. A . LAMPORT

IX. ENZYMIU WALLPROTEIN Since tho time of Wiessner (1888) various workers have emphasized the peculiar propertieH of the wall surrounding a growing cell, by describing the wall as living ! Bonner (1935), for example, wrote “Die jugoiidliche Zellwand ist hiernach also nicht nur ala sine passive Haut, tjondern als ein fur sich lebendes organ zu betrachtcn.” Heyn (1940) also considered that “the wall must be considered more as a living organ than as a dead structure of the cell”. And Picken (1960) pointed out that “the practice of discriminating between a ‘wall’ usually thought of as an inert, rigid covering, and an active ‘living’ cell surface, has led to a false notion of the independence of the cell wall from the underlying cytoplasm”. If it is at all correct to describe the primary cell wall as part of the active living cell surface, some useful predictions should emerge. Of course, the expression “living cell wall” is hardly an explicit definition and must be regarded more as a signpost pointing to the direction our thoughts must take. It follows that the first question to be answered is whether or not the primary wall is metabolically active. Secondly, do the metabolic activities of wall and cytoplasm interact reciprocally? To answer the h t question one must determine the enzymes present in the isolated primary wall preparation, bearing Hogeboom and Schneider’s (Schneider, 1957) criteria in mind. A . ASCORBIC ACID OXIDASE

Tho first systematic study of a wall-bound enzyme was Newcomb’s (1951) work with the ancorbic acid oxidase of auxin-treated tobacco pith cells. This important work established that most of the ascorbic acid oxidase activity was localized in the wall fraction; non-dividing pith cells increaaed in volume under the influence of auxin; during this rapid cell expansion the wall-bound ascorbic acid oxidase activity increased about tenfold. Numerous other workers (Honda, 1966; Butt el al., 1958; Butt, 1969; Mertz, 1961,1964) confirmed Newcomb’s general conclusion that high ascorbic acid oxidase activity of the wall fraction was characterisitic of actively growing (i.e. expanding) plant tissues. These workers also found that (a)a large percentage of the total ascorbic acid oxidam of the homogenate was recovered in the wall fiwtion; (b) the specific activity of the wall fraction (activity/mg nitrogen) wm several times as great as that of the homogenate; and (c) the specific activity of the wall fraction remained constant upon repeated washing and was hardly affected by preparing walk from plmmolysed cells or by salt washes. The 8ame is true for walls isolated from tissue cul-

T H E P R O T E I N UOMPONENT O F PRIMARY C E L L WALLS 206

tures (Table XX), with the intriguing exception of haploid ainkyo cella which show no wdl-bound ascorbic acid oxidam activity. T u r n XX Aecorbic Acid O x h e Activity of W& pension U&uree+

Iaolded fm Sue-

p?nolea oxygen up&kejdnute 10 mg cell svdls (dry w i g h t )

Acor psPudoplatanm

0.1

Solanurn tuberoeurn (2n)

0.33

Nicotiana labcrcrsln var. Turkieh var. Xanthi oryza edim Centatma cyanua

1.0 2.2 2.2 2.6

* The wall suspensions were incubated in citrete/phosphate buffer pH 6.8 at 27". A total volume of 7 ml containing about 1 mg wall suspension was magnetically stirred. Exoeea buffered sodium ctscorhte (c. 2 mg) wae added and the oxygen uptake was mwiured by means of a Clark oxygen electrode. B. HYDROLASES

Three main groups of wall-bound enzymes catalysing hydrolysis are common in rapidly growing cells. K i v h n et aZ. (1961), Straw (1962), Straus and Campbell (1963), and Edelman and Hall (1964) reported the widespread distribution of invertase firmly bound to the wall fraction and not eluted by salt solutions. Gbziou (1969) presented evidence for wall-bound pectin methylesterarre which could be partly removed by elution with dilute salt solutions. Variow wallbound phosphatase activities are a180 widespread (Lamport and Northoote, 1960b; Kivilaan et &., 1961). One should note that none of thew wall-bound enzymes attains the ubiquity of wall-bound ascorbic acid oxidase. C. OTHER WALL-BOUND ENZYMES

K i v h a n el al. (1961) investigated the enzymic activities of isolated walls in view of the possibility that enzymes involved in wall bioaynthesis might be localized in the wall. These workera were able to demonstrate uridine diphosphoglucose pyrophosphoryhe activity in isolated maize coleoptile walls. The enzyme was of highest specificactivity (on a nitrogen baaia) in the wall fraction and there was no evidence that this was due to non-specific adsorption during cell breakage.

206

D E R E K T. A . L A M P O B T D. HOW DOES THE WALL BIND ENZYMES?

Cell walls bind enzymes either ionically or covalently. The ionic properties of cell walls are especially liable to create the illusion of specific binding of enzymes. Jansen d al. (1960b,c) showed that Avena coleoptile cell walls would bind large amounts of pepsin and peroxidase as well as chymotrypsin and its di-isopropyl fluorophosphate inhibition product. They also showed that salt solutions eluted these enzymes. Salt solutions also completely removed the wall pectinesterase activity which they therefore concluded was only ionically bound.

11.5

F I ~ 16. . Acid phosphatase of sycamore cell walls: Km determination by the method of Lineweaver and Burk (1934), and demonstration of competitive inhibition by inorganic phosphate. Cell walls (1 mg/ml dry weight) were suspended in 4.9 ml pH 5.8 20 m~ sodium acetate/ acetic Reid buffer and magnetically stirred a t 27". Buffered p-nitrophenyl phosphate waa added (0.1 ml). After one minute two drop 10% w/v tnchloracetic acid were added to stop the reaction. The reaction mixture was centrifuged two minutes a t Soap. The supernate was decantad and adjusted to pH 10-11 by the addition of two drop molar Na,C03. The optical density of the motion mixture at 400 my was read against a blank consisting of the reaction mixture minus the substrate. The optical density reading was converted to pg p-nitrophenol released, by referring to a standard curve made from an aqueous pH lop-nitrophenol solution. 9 pg pnitropheny1 phoaphate/5 ml; u = total ymoles p-nitrophenol releaeed/minuw in 6 ml reaction mixture. Open circles represent substrate only. Closed circlc8 represent eubntrate containing additional inorganic phosphate (6.5 x IO-' M).

-

T H E P R O T E I N C O M P O N E N T O F P R I M A R Y C ELL W A L L S

207

Many of the workers quoted so far found that other wall activities were not solubiked by treatment with salt solutions, and yet others found that salt solutions solubilize only trace amounts of wall-bound

enzymes. Sycamore cell suspensions, for example, aontain an active acid phosphatase firmly bound to the cell wall (Lamport and Northcote, 1960b). It readily hydrolysed several phosphate esters including p-nitrophenyl phosphate ( K , 6.6 x lo%, Fig. 16) and was competiM, Fig. 16). The tively inhibited by inorganic phosphate (K, 4 x

Inorganic phosphate concenlration X

M

FIQ.16. Arid phosphataae of eycamore cell walla: determination of the K,for i n o ~ n i c phosphate, by Dixon'e (1963) method. Tho general methods used were as described in Fig. 16. Open oirclee show rates obtained using a p-nitrophenyl phosphate concentration of 1.67 X lo-' Y while varying the inorganic phosphate concentration.Closed circles show the rates wben p-nitrophenyl phoephatc concentration was 0.835 x 10-4 M while varying the inorganic phosphate concentration.

wall phosphatase accounted for 60-80%, of the total activity of u dialys 5% of the sed homogenate. Salt solutions (up to IM) removed l e ~ than wall-bound phosphataee ;apparently only Rmall amounts were ionimlly bound. Gradient elution of a small column of walls illustrated this rather dramatically (Fig. 17). These small amounts of phosphatase may represent precursor to the more firmly (covalently?) bound wall enzyme. Some of the elutable peaks of pliospliatase activity may represent traces of cytoplasmic contamination, for peaks I, T I , and I11 did not appear when a column of intact cellR was eluted (Fig. 18). N

208

D E R E K T. A . L A M P O R T

In contrast to the well-bound phosphatam of sycamore suspension cultures is the behaviour of the soluble cytoplasmic enzymes which are not appreciably adsorbed on to the cell walls when the cells are homogenized. For example, less than 1% of the glutamine synthetctee activity of a sycamore homogenate was found in the water-wctshed cell wall fraction (J.E. Varner, private communioation). The fact that salt does not elute the bulk of some wall-bound enzymes is evidence for considering these enzymes as covalently bound. As yet there is no more direct evidence than that. It is worth recalling,

FIG.17. NaCI gradient elution of sycamore cell walls: acid phosphatase activity of fractions. Cell walls were isolated and washed with water ten timer. The walls were then packed a8 a 1 cm diameter x 6 cm long column which was then eluted with a linearly-increasinggradient of NaCl in pH 7.06 20 mM tria-HC1 bu5er. Two-ml fraction6 were collected and aaseyed for phosphataee activity by incubating 1 ml samples for ten minutes with 220 pjij p-nitrophenyl phosphate in 2 ml sodium acetate pH G.8 buffer at room tcmpersture (0.20").The reaction wan stopped by adding three &opn of 1 M sodium carbonnto. Tbe optical density a t 4UO mp was read. Left-hand scale repremntn optical density a t 400 my, and phosphatnm activity is shown as a continuous line. 1tight.hnnil wale rrprcsentN NuCI rnolnrity w11ic.h ilc showii an u darhtul line.

however, that the most widespread of these wall enzymes ie ascorbic acidoxidme, and Stark and Dawson (1903) showed that a Aoluble homogeneous ascorbic acid oxidase preparation from summer crookneck squash contained ten hexosamine residues /mole enzyme. They suggested that the enzyme might"be (t covalently-bonded carbohydrate-protein complex. This might provide the structural baais for its inclusion in the wall, covalently bound.

THE P R O T E I N C O M P O N E N T O F PILIMARY C E L L W A L L S 209

In the light of d a b presented in this section one must oonaider the possibility that the hydroxyproline-rich wall protein accounts for some or all of the wall enzymio activities. This possibility seem unlikely for several reasons: (a) allowing 10% or more of the wall to exist aa emymic protein would surely be an enihiirrassmont of riches ; ( 6 ) hydroxyproline is, as far as we know, exclusively restricted to structural proteins; (c) a purified preparation of ascorbic aoid oxidaae contains no hydroxyproline (Stark and Dawson, 1962); (d) there wm no correlation between wall hydroxyproline content and ascorbic acid activity

3

06

-

0.4

-

8ti d

Fraction number

Pro. 18. NaCI grdiunt elution of intact sycamore cells. Acid phosphateiee sctivity of h c tions. Bycamore collswore washed with 20 mM pH 7-06tris-HC1 buffer and pcked &B a column, 1 om diameter x 6 cm high. This column was then eluted with a linearly-increeainggredient of NaCl in pH 7.06 mM tris-HC1 buffor, fractions collectod and msayed &B described in Fig, 17. Left-hand scale represents optioal density at 400 mv and phospheteaa activity ie shown M B continuous line. Right-hand scale represents NaCl molarity which is shown 8 s a dashed line.

(Tables VIII and XX); and (e) sycamore cell walls, from cells exposed to a pulse of 8H-proline,eluted with a buffered sodium chloridegradient, yielded fractions rich in phosphataae activity which did qwt coinoide with the labelled hydroxyproline-rich fractions (Fig. 19).

X. THE ROLEOF EXTENSIN “Because of the stimulating character of hypothesis, it is not to be suppreased, however daring. It may be objected that there is danger in giving wider currency to hypotheses still insufiiciently sharply formulated; but this is the way in which science b s always developed. Time sifts. There is danger to no one, and those only will be disooncerted who believe that mience in Absolute Truth.” Laurence Picken (1960).

210

D E R E K T. A . LAMPORT

The experimental evidence supports a simple argument. The primary cell wall plays a unique role in cell extension, and localized therein is a unique hydroxyproline-rich protein. I n biological systems hydroxyproline is located exclusively in structural proteins. Therefore, the hydroxyproline-rich primary cell wall protein plays a structural role in the wall. Accoptance of this argument has wider implicationa. If this protein plays a structural role in the wall it inevitably playR some part in cell extension since all the wall components play B O M ~part in cell extension. The problem is to pinpoint components responsible for the auxin-dependent wall plasticity changes. Protcim initiate dynamic: biocltcmical changer,; thorefore, wall proteii, is a priori involvcd in call-wall pInHtic:it,y.RoolofHcii (1959) likens the wall to R multihyered net; when the ocll oxpic1H under the influence of turgor p e e -

LPhosphatase

l

Fraction number

FIG. 19. NaC1-gradient elution of *H-luhlIed sycamore cell walls. Exponentially growing sycamore cclls (c. 100 mg dry weight) wcru exporred for 6 hr (about one-eighth of the mean generation time) to 100 pC *H-prolinc(141 mC/m mole). The walls wcre ircolated, washed ten times with water and then packed aw a coliimn. and eluted. Fractiona were aRHay4 for phmphatase activity as described for lcig. 17. A 1 ml. sample from each fraction waw ulm mixed with 1 ml 10% w/v trichloracctic acid ant1 filtered through a 2 cm membrane filter (Oxoid. grade AP). The filters were waelied three times with 2 mlh% w/v trichloracetic acid containing 1 mg/ml carrier proline, twice with 2 nil 1% acetic acid, anddriecl. Radioactivity retained by the membrane filters was then mcarrurcd in a liquid scintillation counter. Left-hand scale represents thousands of counts per minute retained by the membrane filters. Radio activity of fractions is shown RS R. dotted line. Right-hand scale represents N&l molarity and optical density at 400 mp. PhoRphatase activity is shown as a contiiuoua line. NaCl molanty is shown as a dashed line. sure, the cellulose microfibrils of the multi-layered net are forced to dide over one another. The author suggests the cell controls this process by providing the multi-layered net with protein “knots” which must be loosened before extension call occur. These knots correspond to Bonner’s “Haftpunkte” (Bonner, 1935).

T H E P R O T E I N C O M P O N E N T O F P R I M A R Y C E L L W A LLS 211

In the chemistry of knot loosolling, labile disulphide bridges are probably important, especially in view of the well-known correlation between growth, and reduced glutathione and ascorbate in the cytoplasm. Moreover, there is direct chemical evidence for disulphide bridges in sycamore cell walls (Section I11 D). Now the auxin problem devolves upon explaining how auxin affects the lability of diaulphide bridges of “extensin”. In general terms it is not difficult to imagine that the intra-cellular -SHlevel influences the opening and closing of wall disulphide bridges. But, does auxin control the intracellular -SH level, and if so, how! Marre and his colleagues (cf. Marre and Arrigoni, 1957; Tonzig and Marre, 1961; Galston and PurveR, 1900) huve shown a clear correlation between the intra-cellular -SH level and auxin-induced growth. Cytoplaamic -SH compound8 arise from the pentose phosphate cycle, which generates the TPNH, specifically required to reduce glutathione. This surmise, that the pentose phosphate cycle is connected with wall plasticity via the production of TPNH, is consistent with the correlation between plant tissue age and cycle activity (Gibbs and Beevera, 1966; Yamamoto, 1963). Furthermore, auxin enhances the activity of the pentose phosphate cycle per 8e (Shaw et al., 1958; Humphreys and Dugger, 1959) and TPNH production (Marre and Bianchetti, 1961). Now one muat ask how auxin might control the pentose phosphate cycle. Marre and Forti (1958) showed a rapid auxin-dependent increase in the ATPlADP level which may provide an answer. Glycolysia and the pentose phosphate cycle are probably substrate limited, and Garfinkel and HesR (1964) concluded that the adenine nucleotides were the most important controlling factors in their computer model glycolytic system. Thus auxin-enhanced ATP production could increase the level of glucoso-6-phosphate available for the pentose phosphate cycle. In considering how auxin might mediate changes in the ATP/ADP ratio, it is worth recalling the suggestion of Bonnor and Uundurrrki (1952) that since auxin increases renpiration in a Ayetem whose rate limiting step is the phosphorylating one, then auxin must in Bome manner affect this phosphorylative process. Rhodes and h h w o r t h (1962) suggested that auxin itself became phosphorylated, and Koves and Sirokm&n (1963), in a preliminary report, provide evidence for a phosphorylated auxin. Szent-Gyorgyi and Isenberg (1960) report that auxin can form charge transfer complexes. This might allow auxin to act as a specific coupling agent for niitochondrial phosphorylation. Figure 20 summarizes the preceding arguments as a hypothetical sequence of auxin-dependent events starting with enhanced mitochondrial phosphorylation and ending with wall extension. It depicts

212

D E R E K T . A . LAMPORT

reductive disulphide-bond cleavage in the wall as the ma,jor auxindependent event. It seems likely that the rate of disulphide-bond turnover via a disulphide sulphydryl exchange type of reaotion (of. Jensen, 1969) would be just as important as the actual disulphide-mlphydryl ratio at any particular instant. On this basis wall-bound ascorbic acid oxidase might facilitato the disulphide turnover via the production of dehydroascorbate or monodehydroascorbate whose function would be to oxidize sulphydryl groups of “extensin”. Even if this scheme does not stand the test of further experimentation, the hydroxyproline-rich protein remains a contender for a role in cell extension. To summarize this hypothetical aspect concisely the

&

CYTOPLASM

Plasma membrane

WALL

FIQ.20. Hypothctical schemc for wall cxtension. Tho scheme depicts thc possible sequence of metabolic events which might lcad to an increase in the reductive cleavage of “extanain” disulphide bridges. Auxin (IAA) incrcascs the ATP level, perhap by acting as a coupling factor for mitochondria1phosphorylation in reaction (1). The increased ATP level increases the level of glucose 6-phosphate (2). which provides an increased level of substrate for glucose 0-phosphate dehydrogenaae (3) thereby leading to an increase in the level of TPNH. This leads to a geneml incream in the level of reduced sulphydryl compounds, one or more of which may reductively cIeave “extonsin” disulphide bridge& Ascorbic acid oxidase might complete the control mechanism cycle by gcnerating dchydroasoorbic (or monodehydroascorbic) acid which would rc-oxidizo the sulphydryl groups of “extonsin” back to the disulphide bridge. Abbreviations: ADP = odenosine diphosphate; ATP = adenosine triphoaphate; A8c = ascorbic acid; DHA = dchydmascorbic acid; 0.6.P = glucose 6-phosphate; (3 = glucoee; IAA = indole acetic acid; TPN = triphosphopyridine nucltmtide; 6.PG = B-phosphoglutanic acid.

T H E P B O T E I N C O M P O N E N T O F P R I M A R Y O E L L W A L L S ‘113

author has given the hydroxyproline-rich primary cell-wall protein the provisional name “extensin”. It can, so named respectably join the ranks of other structural proteins, such as collagen and elastin. The name is, if nothing else, at least euphonious !

AOKNOWLEDGEMENTS

I am grateful to Dr. n. H. Northcote for advice and support from 1958-61, and the Agricultural Research Council for financial support during that time. From 1961 to the present I am grateful to Dr. J. E. Varner for support which has enabled me to continue studies of cellwall protein. I am also grateful to Miss Nancy Joseph for her help, especially with the sugar and amino acid analyses. The more recent work was also supported by generous financial support from the U.S. National Institutes of Health (NIH grant A57081 and the US.Atomic Energy Commission (Contract AT(30-1)-3150). I am much indebted to Dr. J. E. Varner and Dr. M.H. Proctor for thoir helpful criticism of the manuscript.

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Embryology in Relation to Phygiology and Genetics*

.

..

P MAHESHWARI and N S RANGASWAMY Department of Botany. Univerdy of Delhi. Delhi. India

.

I Introduction ...........................................................

I1. Pollen

219

................................................................. 221 A. Longevity of Pollen ...........!k ..................................... 221 B . Uermination of Pollen ............................................... 223 C. Pollen Tube ........................................................ 227 231 111. Control of Fertilization ................................................. A. Trentment of the Stigma .............................................232 B. Treatment of the Style ............................................... 232 C. Intraovarian and in Vitro Fertilization ................................. 234 237 IV. Embryo ............................................................... A. Urowth of Embryo in Relation to Sccd hvclopment .................... 237 B . Dependence of Embryo on Endosperm ................................ 239 C. Specificity in Nutrition of Embryo ..................................... 230 240 V . Endoaperm ............................................................ A . Constituents of Endosperm ........................................... 240 B. Role of Endosperm in Seed Development., ............................. 242 C. Culture of Endonperm ................................................ 843 248 VI. Embryo Culture ....................................................... A . Cultural Conditions. ................................................. 241 B. QrowthMedia ...................................................... 260 C. Applloatione of Embryo Culture ....................................... 256 D. Limitetiom of Embryo Culture ....................................... 262 263 VII. Culture of Ovules ...................................................... 266 VIII. Culture of Ovariee and Flowers .......................................... 273 IX. Parthenocarpy ......................................................... X . Polyembryony ......................................................... 280 A . Advontivo Embryony ................................................ 280 B. Embryonal Budding ................................................. 28f1 2M XI. Parthenogenes ie ........................................................ 300 XI1. Androgeneeis ...:...................................................... XI11. Anther Culture .......................................................... 301 304 XIV. Control of Sex Expression ............................................... xv . Concluaions............................................................ 309 Acknowledgements ...................................................... 310 References ............................................................ 310 \

1. INTRODUCTION During the years i8&1-1830 Alnici made some fundamental discoveries when he observed that pollen grains germinate on the stigma. and the pollen tubes grow into the style and ovary. and Snally enter the ovules Schleiden (1837) confirmed the observations of Amid but erroneously thought that the tip of the pollen tube ifself became transformed into the embryo . In 1849 Hofmeister reported the presence of germinal vesicles (i.e. constituents of the egg apparatus) in embryo saw and * The main eurvoy of the litcrkturc WHB complctcd by thc end of 1963.

.

220

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M A I I E S H W A H I A N D N. Y. R A N Q A S W A M Y

emphasized that it was ond of these germinal vesicles that gave rise to the embryo and not the tip of the pollen tube. He believed that the fluid discharged by the tube activated the germinal vesicle to develop. Strasburger (1884) observed the sperm nuclei in the pollen tube and their discharge into the embryo sac. He demonstrated the fusion of a sperm with the egg and showed that it is the product of the union of the gametes that gives rise to the embryo. Nawaachin (1898) and Guignard (1899) completed our knowledge of the basic facts of fertilization in flowering plants by showing that while one out of the two male nuclei brought in by the pollen tube unites with the egg to produce an embryo as already found by Strasburger (1884), the other fuses with the secondary nucleus to give rise to a tissue called the endosperm. Whereas the embryo is diploid and is the progenitor of the next generation, the endosperm is normally triploid and acts 88 a nurse, tissue for the embryo. With the year 1900 came the rediscovery of Mendel's laws followed by an unprecedented activity in the hybridization of varieties, species and genera in order to produce newer and more useful types. Plant breeders put the pollen upon the stigma and looked for results in the ovary, often succeeding but not always. Their failures to obtain the desired hybrids stimulated more intensive research into the morphology and physiology of the changes that follow pollination and ultimately lead to fertilization and then the formation of the seed and the f i t . These aspects have gradually been recognized aa constituting a new discipline, namely experimental plant embryology. The beginnings of experimental embryology may be said to have been laid when Hannig (1904) grew the young embryos of certain plants in an artificial medium. Several others followed him and in 1925 Laibach gave a valuable hint to plant breeders that if a cross does not result in a fertile hybrid and the sterility is due to the death of the resultant embryo it may be useful to excise the embryo at an early stage and grow it in a nutrient medium. This in Witro culture of embryos has now become a common practice in plant breeding and n more detailed account of it is given in a later section of this essay (see p. 246). Although an exckion of the young embryo and its culture in an artificial medium may sometimefl save it from premature death and thus prove helpful, there are other and still more primary barriers to successful hybridization. The chief among them are: (a) the relatively short life of the pollen, (6) the inability of the pollen to germinate on the stigma, (c) the slow growth of the pollen tubes 60 that they do not reach the embryo sac in time, (df the failure of the sperm to fuse with the egg, and (e) the diafunction of the zygote, or endooperm, or both.

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AND G E N E T I C S

221

Some of these impediments are indicators of a sexual incompatibility which manifests itself as a physiological imbalanoe between the male gametophyte and #e carpellary tisaues or between the produots resulting from syngamy and triple fusion. We shall, therefore, disouae both the pre- and post-fertilization events. The former deal with the role of the pollen in relation to the pistil, and the latter with the endosperm, embryo, and other tissues of the seed.

11. POLLEN A. LONGEVITY OF POLLEN

In natture pollination is aohieved through wind, water, insects or other agenoies. Sometimes pollination occurs even in the bud when the anther and the stigma come in direct contact or lie so close to each other that any little movement of the flower bud causes the pollen to land on the stigma. Most pollens lose their viability soon after shedding from the anther. The pollen of grasses is especially short-lived. Bennett (1969) reported that in Pmpalum dilatatum it loses its capacity to germinate within 30 minutm after shedding. Only in a few plants does the pollen remain viable for more than 2 or 3 days, and a period of a few weeks or months is rather rare although it?has been observed in several fruit trees and gymnosperms. Methods of prolonging the vitality of pollen are, therefore, of considerablevalue for they would enable the transport of pollen in plants wbioh are geographioally isolated. They would also help in bridging over the undesirable interval, if any, between the blooming periods of the two parents. The longevity of the pollen iu governed by a number of factors such as temperature, relativo humidity, light, and the time of blooming. Of special significance are temperature and relative humidity and their effects are interdependent. Sub-freezing temperatures (-6 to -10°C) and 25-60% relative humidity (RH) are conduoive t o a satisfactory storage of most pollens. The pollen of certain species of Niwtiam shred for a year at -6°C and 60% RH germinated aa easily aa fresh pollen (Daniel, 1965). Similarly, the pollen of apple stored at -16OC for 9 months showed 95% germination (Visser, 1966). King (1969) h u reported a successful freeze-drying of the pollen of Pinus although that of P e e ~ & ~ w was u affected adversely (Livingston et al., 1962). Aocording to Whitehead (1963) freeze-drying greatly increased the longevity of the pollen of Coc0.3 nuciferu; pollen stored at room temperatures over sulphuric acid remained viable for 3 week, that at low temperature over silica gel generally retained its longevity for 2-3 months, and pollen stored over damp calcium ahloride remained alive

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even up to 18 months. Hansoii (1961) has demonstrated that subfreezing of the pollen of alfalfa can prolong its vitality from 8-15 to 183 days. Ordinarily the pollen of mango remains viable for 8 days only, but at a temperature of 4.5-9"C and at 10, 26, or 60% RH it retained its longevity for 5 month8 and at -23°C and 0% R H for 14 months (Siiigh, 1962). Temperatures as low as -190°C obtained by using liquid air or liquid oxygen, have proved still better in enhancing the longevity of pollen. For example, Bredemann et al. (1947) stored the pollen of Lupinus polyphyllus at -190°C for 3 months without loss of vitality, whereas storage at 0°C proved unsatisfactory. The pollen of apple stored at -190OC was as effective as fresh pollen even after 2 years (Visser, 1966). Like low temperatures, a low relative humidity also prolongs the life span of most pollens. The pollens of apple, apricot, peach, pear, plum, sour cherry (Nebel, 1939), grape (Olmo, 1942), and pistachio (Stone ei al., 1943) remained viable for one year or even longer (6.5 years in sour cherry) at 10-60% RH and a temperature of 0-10°C. The pollen of Datum remained viable for only 2 weeks when air-dried, but when refrigerated over calcium chloride it kept alive for almost a year (Blakeslee, 1945). The pollens of Arachig hypogaeu, BrassiCa; nigra, Solanum melongem and S. tuberosum showed the highest viability at 31-40% RH (Vasil, 1962). Singh (1962) reports that the pollen of Marqiferu indicu remained viable not only for a longer period (20 days) but also showed a higher percentage of germination when stored at 0% RH than at 10, 25 or 60% RH. On the other hand, a low relative humidity is harmful to the pollens of members of the Gramineae which can be stored somewhat better, although still only for a short period, at a relatively high R H (70-100%). For example, Sartoris (1942) observed that in Sacchrum and Zeu the pollen remained viable for 10 days at 90-1000/, R H and a temperature of 4°C. Daniel (1955) reported that temperatures lower than 7°C and a relative humidity below 50% were detrimental to the pollen of Zea mays. Pennisetum typhoideum is an exception as its pollen can be stored for 186 days at 16-35°C and 0% RH, but a R H of 60% or above limited the viability to 5 days (Vasil, 1962). To minimize deterioration during storage, particularly duo to desiccation, the pollen ix often mixed with certain pulverized, anhydrow materials called diluents. Out of over 30 mbstances, lycopodium powder, egg albumen, casein and talc are the most acceptable diluents. However, they are useful with granular pollen only; with sticky pollen they cause agglutination and a decrease in viability, Of interest is also the, report that hand-collected pollen of Prunw amygdalus stored in a home freezer at about -18°C remained alive

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for 801 days and bee-collected pollen for 1130 days (Griggs et al., 1953). The maximum longevity so far recorded is 9 years for the pollen of some rosaceous fruit trees (Ushirozawa and Shibukawa, 1961). Although no conclusive physiological interpretations can be offered, the practical value of prolonging the life of pollen is recognized by all . . plant breeders. B. GERMINATION OF POLLEN

Whereas the life span of the pollen of many species can be extended by artificial devices, this should not be taken to imply that stored pollen would always retain its capacity for germination and subsequent potency for fertilization to the same degree as f m h pollen. Some probable explanations for this are: loss of moisture, depletion of food reserves, and a decrease and/or inactivation of the enzymes or other key substances. The logical corollary is that stored pollen may require for its germination a higher relative humidity and a higher level of nutrient material than fresh pollen. This ha6 been confirmed by some investigators. Pfeiffer (1944) observed that on providing a favourable humidity and temperature the pollen of Cinchona showed some recovery of its capacity for germination. The pollen of Pinw freshly collected in June yielded optimum germination in 2% sucrose, while a sample stored until December required as much as 20% sucrose (Kiihlwein and Anhaeusser, 1961). Similarly, the comeeponding requirements of Aradiie hypogaea and of the varieties T.65 I.C.1472 and T.25 of Pennisetum typhoideum were 10, 12.5 and 26% sucrose for fresh pollen; and 12.5, I5 and 27.6% for stored pollen (Vasil, 1962). For tomato Gorobec (1968) observed that 4-day-old pollen stored in the laboratory yielded fruits of normal size while older pollen gave only small fruits. At the other extreme it is recorded that the pollen of apple gave some germination even 9 years after storage (Ushirozitwa and Shibukawa, 1951). Nielwn (1956) observed that the content of pantothenic acid (chief constituent of coenayme A) decreases Hubstantially in the pollenn of Pinw and Alnua after storage for one year. This imbalance in coenzyme A upsets the general metabolism which in turn shortens the life of the pollen. It is of interest to note that stored pollen which fails to germinate in vitro may nevertheless cause a satisfactory seed set. The investigations of Olmo (1942) on grape, of Stone et al. (1943) on pistachio, of Hagiaya (1949) on tobacco and of Visser (1966) on tomato seme as examples. The stored pollen of these plants showed only 6% gemina0

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tion in vitro but proved quite effective for field use. Similarly, the pollen of potato stored at -344OC for 7-13 months gave almost no germination in culture but effected R good fruit set when used on the stigma (King, 1988). The pollen of cacao stored over calcium chloride at -20 to -30°C for 1-4 weeks showed only a low germination on an agar-dextrose medium, but field pollinations gave 40 - 50% fruit set (Soria and Denys, 1961). This is readily understandable because the stigmatic and stylar tissues help to make up some of the deficiencies imminent to storage and provide a natural environment for the germination of the pollen and the growth of the pollen tube. Thus, the failure of germination of stored pollen in cultures does not necessarily mean that the pollen is dead or useless. Similarly, the mere germination of stored pollen in w h o is no assurance that it wiU effect fertilization. For example, the pollen of wheat showed &lo% gennination on an agar medium but proved unsatisfactory for pollination in nature (KovkEik and Holienka, 1962). This implies that there are other factors which also control the germination of pollen. The pollen requires an adequate supply of moisture, inorganic elements, and a source of energy-usually a sugar. Some of these requirements may be met from the reserves of the pollen itself, but very often one or more of these act as limiting factors. The ambient humidity is critical for the germination of pollen. As substrates the lower surfaoe of fresh leaves of aquatic plants and sometimes even moist parchment paper have proved adequate. I n some instances good germination has also been obtained by merely placing the pollen near a hanging drop of water in a microchamber. Excessive moisture is deleterious to the pollen, and it is a common experience of plant breeders that pollinations carried out soon after rain or dew are frequently infructuoua. Schmucker (1933)reported that the pollen of Nymphueu germinated in a solution of glucose only when mixed with the stigmatic extract of the plant. Later, he detected boron in the extract and, therefore, replaced it'by traces of boric acid. This proved succeissful and quantitative estimations revealed that the pollen required almost the same concentration of boron aa was present in the stigma. Following Schmucker, many 0th- have identified boron in the stigma and style and have confirmed its favourable effect on the germination of pollen. It was believed that boron is related, in some unknown way, to the incorporation of carbohydrates in the pollen tube membrane. However, some studies indicate that it is essential to the spthe8ia of peotine in the middle lamella of newly formed calls (Spurr, 1967) and in the pollen tubes (Raghavan and Baruah, 1969). Stanley (1964) and Stanley and Luewus (1964) suggested that myo-inositol is probably an inter-

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mediate in the conversion of hexose sugar to pectin. On using tritiated myo-inositol with two conmtrations of boron (0.75 and 7.5 pg/ml) in cultures of the pollen of Pyrua communis they found that (a)boron combined with a speoific enzyme enabling it to bind and react with inositol, (6) myo-inositol waa readily converted into pectin, and (c) pectin synthesis inoreaaed at higher levels of boron. Theee observations suggest that boron plays a significant role in peotin synthesis in germinating pollen. . Like boron, calcium has been shown to enhance the germination of pollen aa well as the growth of the pollen tubes, and especially the latter. Its role is d i s o u s ~under h t i o n I1 C. Low conoentrations (0~001-0~0001 M) of dicarboxyb acids, such aa succinic, fumaric and adipic, have also been reported to stiIn&b the germination of pollen (Petrochenko, 1962). The pollens of many members of the family Ma1vaoea.e germinate only with some difficulty, but Bronckern (1961) obtained 7 0 4 2 % germination of the pollen of cotton iti artificial media in the preaence of amnaphthene. It is well known that during germination the pollen grains exercise a m m effeot (Brink, 1924). The ancient Arabs did not discard their stocks of old pollen of date palm but mixed it with the fresh lot and uaed the mixture quite liberally. It is possible that the old pollen, although inviable, contributed some chemical subatanoes to the mixture. Savelli and Caruso (1940) also mentioned a “mutual stimulation effect” in Nicotianu. When a large sample of pollen of one species wm added to a small population of pollen of another species both pollens were stimulated and showed better growth. Several investigators have also reported that in culturea a denser sowing of the pollen grains results in better germination than when only a few grains are wed

Fro.

1. Qerminetion of pollen showing masa effect; note good &tion pollon is dense (after Iwensmi, 1959).

w h v e r the

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(Fig. 1). Tigin (1962) found that tho addition of pollen of Hibiscus, Malva neglecta and Qossypium arboreum to that of cotton 108F, promoted the germination of pollen and the development of bolls as compared to selfing and pollination with limited pollen. Similarly, kedrina (1962) observed that supplementary pollinations as well as mixtures of pollen from more than one variety of maize increased the frequency of fertilization from 71*8-97*4y0 and fertilization was accomplished in 24-28 hours. On the contrary, selfing and pollinations with a restricted number of pollen grains caused a delay in fertilization and a decrease in the amount of food reserves in the grain. Likewise, VoZda (1962) has reported that mixed pollinations in maize resulted in an increased weight of the ears but the other characters remained unaltered. According to Bardier (1960) in wheat the grain set was considerably improved if rye pollen was used either before or a few hours after pollination by wheat pollen. Such a “mentor” effect of foreign pollen has also been reported for sugar beet (Kovarskij and Guzan, 1960), rye (Sulima, 1960) and sorghum (Zajceva, 1961), but all these observations need confirmation on the basis of more critical data. The receptivity of the stigma is another link in the chain of events leading to fertilization, although it is often rather difficult to control. Generally the receptivity of the stigma is determined by the age of the flower, and the ambient humidity and temperature. Jones and Newel1 (1948) have reported that under favourable conditions the stigma remained receptive for 19 days in Ruchloe dactyloides and for 24 days in Zea mays. According to Eghiazarjan (1962) stigmas of Nicotiana, less than a day old after anthesicc, possessed greater receptivity than those of unopened or older flowers, and mature pistils preferred self pollen to foreign pollen. The effect of the stigma is well known in certain phiits in which the pollen fails to germinato after self-pollinations (see Brewbaker, 1957). Jost (1907) attributed this self-sterility to a retarded growth of the pollen tubes. On the bad, of hi6 work on Corydalis cava, Lilium bzilbiferum, Secale cereale and other plants, he propounded the concept of “Individualstoffe”, meaning that many plants have a characteristic substance in the pistil which inhibits the growth of their own pollen tubes. Correns (1912) noted, however, that while individuals of the same clone or pure line were cross-sterile, they could hybridize freely with individuals of other clones or pure lines. He therefore postulated the presence of “Linienstoffe” as the underlying cause of self-sterility. I n other words, each pure line is charncterized by a substance which is specific to all of its individuals and is inhibitory to their pollen. This thcory opened the genetical approach to wlf-sterility, and Compton

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(1913) attempted to explain it in Mendelian terms. He itssumed that substances are formed in the pistil which stimulate or retard the growth of the pollen tube. He also drew an analogy between selfsterility and the growth of fungous hyphae into the host tissue end compared the mechanism of self-sterility with that of immunity against a pathogen. In some self-sterile plants successful self-pollinations can be made in the bud stage of the flower. Bmsica oleracea (Attia, 1960), Trifo2ium hybridum (Williams, 1961) and Nicotianu a h (Pandey, 1963) are some examples. In Muau the frequency of fertilization is often discouragingly low, even if abundant pollen is supplied. However, several clones show an increased fertility if bud pollination8 are m*de (Shepherd, 1954, 1960). No conclusive explanation is available for this phenomenon but it is postulated that some factors, which would inhibit the germination of pollen soon after natural self-pollination, might be absent or ineffective in the immature stigma. At the same time it may be noted that bud pollination is not always satisfactory and may lead to the formation of weaklings, heteroploids and sterile individuals (Iizuka, 1960). Liko bud pollination, bee pollination has also been reported to be beneficial. In certain intravarietal crowma of cotton, bee pollination resulted in an increased number of bolls, seod set, and the quality and yield of the fibre (Arutjunova and Skrebcov, 1962).

In the Cruciferae the incompatibility reaction is localized in the stigma and after self-pollinations the percentage of germination of pollen is negligible, Sometimes, this can be improved by increasing the ambient humidity. El Murabaa (1987) has reported that high temperatures (26°C) proved more favourable in overcoming self-incompatibility in Ruphanua sutivzcs than lower temperatures (17°C) which promoted crose-pollination. Sexual incompatibility is common in interspecific and intergeneric crosses. Nevertheless, several examples can bo mentioned of successful interspecific and intergeneric crosses, especially among the orchids, where the stigma has little or no deleterious effect on the germination of foreign pollen. C. POLLEN TUBE

The germination of pollen may not be NO diffigult as sustenmce of the growth of the pollen tube. A knowledge of tho extent of growth of pollen tubes in the pistil is tliereforo a pre-requieite in devising techniques to circumvent the bai~iersto crowability. Dissections, whole mounts and phase contrast microscopy are usually employed for this. In Rome studies on interspecific hylwiclizations in Nicotiana, Swnmina-

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than and Murty (1967a) demonstrated that autoradiography serves as a rapid and reliable meane for estimating the degree of growth of pollen tubes. Recently, Dempaey (1962) suggested the application of ultraviolet fluorescence for assessing the growth of pollen tubea in styles. Little direct evidence is available of the nature of the factors influencing the growth of the pollen tubes in the pistil. Some correlations have been suggested between the structure of the style and the growth of pollen tubes in it. Haeckel (1961) reported that (a) solid styles (i.e. where a transmitting tissue is present) generally hrcve a low starch oontent and show a very high phosphatase activity during the growth

A

Stigma /

Style

FIG.2. Growth of pollen tubes in Lilium lonqiflorYm. A. Part of style from which a pime. waa removed end thbn rephoed in inveree position and glued with gelatin. When pollen waa sown at the top, all the pollen tubes grew downward in the etyle. B and C. Growth of pollen t u b in horizontally orienhtwl etylee. From the region marked X in B Bome of the styhr tiesue wm ecooped out. In C an incision waa made in the style. &me pollen tuber grew toward the ovary end and others in the oppoeite direation. D. Excised stigma plsoed on M agar medium along with pollen ehowing growth of pollen tubes along ita inner wall (after Iwenami, 1959).

of pollen tubes, and (b) hollow styles (i.e. those which possess a canal) are rich in starch and show a high amylase activity. According to Brewbaker (1957), in incompatible plants having a solid style the growth of the tubes becomes arrested in the style itself, but where the style is hollow (LiZium and Lotus) the inhibition occurs in the ovary. Iwanami (1959) investigated the growth of the pollen tubes in LiZium longiJEormnt. In one aet of experiments he cut off a part of the style, replaced it upside down and glued it with gelatin (Fig. 2A). When the

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pollen was sown at the top almost rtll the pollen tubes were observed to grow downward in the style. In another experiment the style was placed horizontally and a portion of it was scooped out or an incision waa made in it, On sowing the pollen into the operated region some of the pollen t u l m grew toward the stigma and others in the opposite direction (Fig. 2B and C). I n still other experiments the stigma was cut at its baae and placed on an agar medium (Fig.2D). If the pollen waa sown around it, the tubes grew toward and along the inner w d of the stigma. On the basis of these observations Iwanami (1969) concluded that an “inductive substance” is present in the pistil and it serves to draw the pollen tubes which are initiated at the surface of the stigma first into the stigmatic tissue and then into the style. Once the pollen tubes enter the style they grow of their own acoord in the direction of the ovary. Among the physical factors influencing the growth of the pollen tubes, temperature is the most important. With an increase in tempemture, the rate of growth is appreciably enhanced, a maximum being reached at between 20 and 30°C. However, in many illegitimate selfpollinations the optimal temperature favouring the growth of pollen tubes is lower than in the corresponding legitimate cross-pollinations. As the temperature rises the growth rate of the incompatible tubes diminishes. Lewis (1949) attributes this to an inhibitory reaction which proaeeda faster at higher temperatures. A good deal of information has accumulated on the iniluenoe of several kinds of chemicals i n d extracts of tissues from h o s t all parts of the plant including even the root. Among the substances which have been reported to have a stirnulatory effect on the growth of pollen tubes are alcohols, amino acids, auxins, carotenoids, colchicine, gibberellic acid, kinetin, metallic ions, purines, sugars, vitamins and yeast extract (see Chandler, 1957; Sawada, 1958; Raghavan and Baruah, 1969; Takami, 1969; John and Vasil, 1961; Singh and Randhawa, 1961; Prasad and Mehrotra, 1963). Further investigatiom are required to determine whether these substances, particularly the carotenoids, also possess any chemotropic effect (see Rosen, 1962). The physiology of sexual incompatibility is not fully understood and moet of the explanations are only speculative. East (1929) propounded the immunity theory suggesting an antigen-antibody mction between the pollen and the pistil. Preformed substances in the style are thought to react with antigens from the pollen, and the reaction products are believed to cause an inhibition of the pollen tubes, On the basis of his experiments with Petunia,Straub (1947) believed that the pollen tubee produce a substance necessary for their growth through the style, but in illegitimate pollinations this substance is rapidly used up and its

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shortage terminates the further growth of the tubes. Later, Straub (1964) explained that in illegitimate self-pollinations certain inhibitory substances are formed which prevent the normal growth of the pollen tubes. The recent studies of Tomkovti (1969), Linskens et al. (1960), and Brewbaker and Majumder (1961) support this hypothesis. Tomkov6 demonstrated the presence of an inhibitory principle in the Rtyles of self-pollinated flowers of Nicotianu a h h . On the other hand, in the styles of cross-pollinatedflowers it was detected only in negligible amounts. In a self-incompatible Rpecies of Petunia, Linnkens et al. (1960) X-irradiated mature st'yles and elf-pollinated them immediately. Nearly 60y0of the treated flowers set seed. When flowers were selferl 20 hr prior to or 24 hr after irradiation, there was no seed set. It is explained that once the incompatibility reaction has begun to operate in the style it cannot be destroyed by irradiation, although it can be arrested by X-irradiating the unpollinated style at a stipulated dose and time. However, such an inhibition is reversible because the irradiation merely causes some biochemical changes in the immunological mechanisni and not any mutation. As already mentioned a mass effect occurs in artificial cultures of pollen (see Fig. 1). Whereas small populations of pollen germinate but poorly, large populations show good germination and an excellent growth of pollen tubes. Brewbaker and Majumder (1961) demonstrated that the population effect is due to the action of a stable, water-soluble and highly diffusible factor which was designated as the pollen growth factor (PGF). They suggested that in cultures of small populations of pollen the quantity of the PGF is deficient. Recently, Brewbaker and Kwack (1963) adduced experimental evidence to show that in artificial cultures the PGF leaches into the external medium and therefore the germination and growth of pollen tubes are negligible. They added cell-free extracts of anthers to the medium on which small populations of pollen were cultured. With incretwing concentrations of the anther extract (number of anthers extracted in 2 ml medium) the percentage of germination also increased. The optimum lev& of germination were obtained with the use of 100 anthers. The population effect could be replaced by a growth factor obtained from aqueous extracts of many plant tissues, even including the root. UHing pollenash as the starting material they identified this factor as the calcium ion. In further experiments Brewbaker and Kwack cultured the pollen in sucroseboric acid medium supplemented with different cations and observed that satisfactory germination and growth of pollen tubes occurred on a medium which included calcium, boron, magnesium and potassium. When these elements were used individually no germ-

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ination occurred. By an elimination technique, they conclude that all the four ions (calcium, boron, magnesium and potassium) me essential for germination and growth of pollen tubes. In the Cruciferas the incompatibility reaction is manifested right on the surface of the stigmatic papillae which contain an inhibitory substance (Kroh, 1966). Heinen and Linskens (1961), and Linskens and Heinen (1962) observed that in Brassica nigra the pollen tubes formed after s e h g are unable to penetrate the cuticle of the papillae, but this barrier is easily overcome by treating the stigma with a cutinase obtained from moulds. On the basis of his work on apple and tobacco, Tupg (1959) found that in compatible crosses the pollen tubes grow rapidly and the available glucose is utilized to build the wall material with hardly any midue left in the form of callose. In incompatible matings, on the other hand, tho pollen tubes grow very slowly. The utilization of glucose is appreciably restricted; and large quantities of callose accumulate and hinder the further growth of the pollen tubes. These observations are supported by the cytochemical investigations of Schlasser (1961) on Petunia. On the basis of tracer studies, Tupj (1961a,b) has further concluded that in incompatible pollinations the arrest in the growth of pollen tubes is not due to a deficiency of the respiratory substrate but to the inhibition of respiration. This explanation is based on the following observations: (a) during the growth of the pollen tubes in the style there is an increased inflow of carbohydrates; (b) in contrast with compatible crosses, in incompatible pollinations the utilization of the carbohydrates is limited and there is a relative deficiency of y-aminobutyric acid and alanine; and (c) such a deficiency of these two amino acids is due to lack of a-ketoglutaric acid which is involved in their transamination.

111. CONTROLOF FERTILIZATION The basic facts about fertilization have already been outlined in the introduction. This section deals with some of the barriers to fertilization and their control. One or both of the participants in the act of fertilization may mmetimes pose problems which eventually lead to the lack of embryo formation. Sometimev the failure of fertiha'tion is merely due to an early abmission of the flower. I n such instances i t may be possible to prolong the life of the flower by the application, of hormones (see Crane and Marks, 1952; Brock, 1954).

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It is also feasible to apply to the stigma an artificial medium which is more suited for tho germination of the particular pollen than the secretions of the stigma. For some fruit trees boric acid sprays have been reported to increase the germination of the pollen (Batjer and Thompson, 1949). I n interspecific crosses of Trifolium Evans and Denward (1956) found that the application of NOA to the stigma resulted in a successful germination of pollen. I n some intervarietal crosses of potato a better germination of pollen and improved seed set have been obtained by treating the stigma with an extract of the anthers of a freely crossing variety of potato (Frimmel, 1966). I n self-sterile plants such a8 Petunia and Niwtiana the stigma has heen reported t o be inhibitory to Relf pollen. Takfishima (1964) demonstrated that in Petunia a washing of the mature stigma followed by smearing it with the secretion from a compatible strain removed the barrier to self-sterility. Such a disguised stigma did not exhibit any antagonism to self pollen and allowed it to germinate normally. In the self-incompatible Nicotianu ahta seed sot was increased more than 200-fold by an application of the stigmatic secretion of a mature stigma of the female parent to a dry stigma of the recipient flower bud (Pandey, 1963).

n. TREATMENT OF THE STYLE More often, however, the cause for the failure of fertilization lies in the style. Many experiments have, therefore, been directed to an understanding of the physiological role of the style in the control of fertilization. Some of these go back to the year 1907, when Jost demonstrated that pollen tubes can grow through the styles of two species placed end to end. Later investigators improved this technique and successfully overcame many instances of incompatibility (see Maheshwari, 1960). Their work has shown that the incompatibility response operates at any level either in the stigma or in the style. Employihg R new technique of stylar grafting Hecht (1960, 1963) demonstrated that in Oenothra the incompatibility reaction k stronger in the stigmatic region than in the style. Prom a oompatible strain of 0. organensis he excised the style 16 mm below the stigma and grafted it on to the lower part of a similarly cut style of an incompatible strain. The stock and the scion were superposed by a “splint” made of 6% lactose in 10% gelatin and a piece of lens paper. The “grafts” were maintained at 27°C for 15 hr in petri dishes containing moist filter paper. Under these conditions the self pollen tubes grew through the

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compatible scion into the incompatible stock up to an average length of 22.1 mm. I n another set of experiments on 0.w p i t o 8 a , 0.orgamm&9 and 0. rhombipdab Hecht (1963) made grafts (a)botween n compatible scion and a stigmtio lobe (stock) which WILH RIAO compntible, and (b) between a oompntible mion and an incompatible stigma lobe. I n the first type the pollen tubes grew readily from the cut end of the scion into the stock. I n the second where an incompatible stigma lobe formed the stock the pollen tubes were completely halted at the stigmatic surface. To determine the degree of the incompatibility reaction in the stigma, and style Bali (1963) made similar grafts in 0.rhmnbipetala. In the pontrol the pollen tubes grew through the scion, then through the stigmatic lobe of the stock and finally up to the baw of the stook style. In the incompatible grafts, on the other hand, the pollen tubes growing through the scion failed to penetrate the stigmatic lobe of the’ stock. These experiments suggest that the incompatibility reaction is complete a t the stigma and the self pollen tubes are debarred from entering into an incompatible stigma even if they had previously passed through a compatible region. Whether these techniques of stylar grafting can be applied to overcome incompatibility in fertilization is yet to be ascertained. Hybridization of a long-styled female parent with a short-styled male parent is usually unsuccessful whereas the reciprocal cross between a short-styled pistillate and a long-styled pollen parent presents no special diffculty. This is in accordance with the observation that the longer the style posses~edby a species, the greater is the growth potency of its pollen tubes. For example, the pollen tubes of Nicotiana rustica (10 mm long style) fail to reach the ovary of N. pniculatac (20-30 mm long style) while the reciprocal cross is eaaily achieved, Mention may also be made of the experiments of Gardella (1950) on Datura inrwxia, D. quercifolia and D . ferox whose styles me 130-170, 30-40 and 25-36 mm long respectively. When I ) . innoxia waN used as the female parent and the other two species as male parents she observed that the seed set was negligible. To improve it G a r d e b performed two kinds of experiments. Jn the fir&the upper portion of the style of the female parent was cut away and substituted by the stigma-bearing stylar portion of the male parent so that the total length of the “graft” was reduced to about 20 mm. This permitted the pollen tubes to grow as far as possible in thoir own stylar environment. In the second method, called RtyIe insertion, the Htyle of the female parent was completely replaced by that of the pollen parent. The situation in Lathyrus odoratus (10 mm long style) xL. hirsutug (4 mm long style) is similar. Davies (1967) amputated the style of L. odoratw and pollinated the cut end. Swaminathan (1956) mcom-

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mended the use of an artifioid stigma for some interspecific crosses in tlie Mexican solanuma. By applying an agar-sucrosegelatin medium 011 the stub and covering it with moist cotton wool after pollination, he obtained the otherwise incompatible croeses between Solanurn pinnutisecturn xS. bulbocastanum, and 8.pinnutisecturn x S . h n c i f m e . A similar procedure was tried with success in the cross Nicotiana tabacum x N . r?mticu (Swaminathan and Murty, 1957b). When trisomic plants of Datura stramonium are used as ,-3 parents, fertilization by sperms of the ( n + l ) complement is only a chance occurrence owing to tho slow growth of the pollen tubes. Buchholz et al. (1932) showed that at tho time when the pollen tubes of the n type had advanced to the base of the style, those of the ( n + l ) type had grown to only about lialf the length of the style. If the lower part of the style was now cut away to elirninatc the n pollen tubes and the upper portion containing the ( n + l ) pollen tubes affixed to the ovary, fertilization was achieved by the ( n + l ) sperms. When a diploid plant is treated with pollen from a tetraploid there is usually a high degree of incompatibility. Generally the pollen grains fail to germinate and even if they do tlie pollen tubes burst in the diploid styles. In Datura it is explained that the epidermal layer which lines the interior of the stigma and style in the diploid female parent exercises an antagonistic effect on the diploid pollen tubes derived from a tetraploid male parent. To enable such a cross Satina (see Avery et al., 1959)troated the female parent with colchicine and induced the formation of a periclinal chimaera which now had a tetraploid epidermal luycr. although it was otherwise diploid. The pollen tubes grew in such a style and fertilized the emlwyo sace.

C. INTRAOVARIAN AND I N VITRO FERTILIZATIO

A direct introduction of pollen into the ovary was thought of aa early as 1886 by Strasburger wlio tried to make such int'rwvarian pollinations in orchids. Kusano (1915) repeated Strasburger's experiments with another oschid, Gmtrodia ehta. For introducing the pollinium, he severed tlie upper portion of the ovary or made an opening on it. A satisfactory germination of the pollinium occurred followed by fruit formation. Several ovules received the pollen tubes and the seeds showed a normal development of the embryos. When polliriia of Bletia hyacinthinu were used, germination still occitrred and stimulated the growth of the ovitrios of Gastrodia but the seeds had no embryos. The experiments of Dahlgren (1926) on CorEonopsi~ ovata, of Cappelletti (1937) on Digitalis purpurea and of Bosio (1940)

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on species of He11ebortu arid Paeouici fiirther indicated the feasibility of this technique. In Yaeonia some viiLble seeds were also obtained. More recontly Mahediwari i ~ n dKttntci (1961, 1962), and Kanta and Maheshwari ( 1963a) have tfasrrihetl the results of their experiments with Payaver rhoaa8, P.BO?NniJorrr?ti, fitwh.ucholzl:acali,fornicu,ArcJemone mexirana anti A . ochruleiccu. A pollen suHpenrrion was prepriretl in (I

Fro. 3. Intruovlrrinn pollinntion in I’upuver ~ o ) ~ i i i ~ e rA. t ~ Ovary )a. at t h e time of injection of jwllen suspension in I00 ppni boric: twill aolution; wrow shown point of injection. 13. Vertical half of ovary shown in A . C. T.S. Iiortion of ovirry 3 iluyw nftrr injct:tion of pollon nunpcnHirm; showing 4-(!1dli!iJ iiriw~nt~ryt~; note gcrminntion of p11i.n. 1). Id.niicmpylar portion ol‘ WIIIC notc pmiatcnt p l l c n tubc. 14:. 47-l)ny-oltl fruit. ol~titinidttirouyh intrtwvclritin pollinrrtion itnci fcitilization. 10. Vt?rticd lirrlf of C.ILPNII~I! sliown in E; whitc: Imlion are tlic tiitlJle HOC~JH. C. Ncrdling ruiard from ficld-borne nt*rtl. FI Swtlling rc:;iretl from secd obtained thr~iugh int,mov:iriun pollination niid fcrtiliziit~ion(nfter Kantrb and Miiliunliwari, 1963a).

.

2 1111 solution of O.Ol‘ll, boric acid in sterile, double dilltilled water. The flowers were eninsculated iind the surfitue of the ovary wiped with cotton soaked in ethyl alcohol. T w o punctures were made 011 the ovnry--ime to dlow the air to escnpe mid the other to inject the

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suspension of pollen (Fig. 3-4and 13). After the injection the punctures were se;tled with petroleum jelly. T n the treated ovaries germination of t,he pollen, entry of pollen tubes into embryo sacs, and double fertilization occurred as in nature (Fig. 3C and n). The ovaries

PIG.4. In oilro fertilimtion in P ~ J ~ J ~oirin,ijertlm.. IIVP~ A. Culture of I d l e n wit1 ~ V U ~ Con H Nit,srh’s iigiir medium. 1%i i r i t l ( ’ . 7-lhiy-oldd t o r e u ; tlic white bodies are the ilcvcloping rrcedri. 1). Whole mount of ovule frnm 3-iIriydI cdtiire showing sever&] germinating pollen grains. E. 1)isnc.ction from n 5-day-nit1 culture slinwinp n 4-uc.lletl proembryo imheddod in the endoqicrrn. I: rind G. (:loliul;ir c1llIJr;vO i d fully hrmrd embryo excised from 9-day-old and 2!?-dny-oltlW Y I ~o l ~ t , ~ i nini ~viilturc: l (after l i t m t t t e6 (I!,., 1962).

grew nonniilly aiid developed into capsules bearing viable seeds (Pig. 3E t o H ) . The nl~ovemetiiod lins becn used to hybridize A . mexicana (an = 28) and A . ochroleucn (2n -- 56). ‘L‘hey rarely cross in nature. Stigmatic pollinations to raise 11yhrids have been discouraging especially when A . och:sobuca is used its the fomaIe parent. Kanta and Maheshwari

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(1963a) applied the technique of intruovarian pollination and found that it proved markedly efficacious in tho cross A . ochrole~%X A . mexicuna. In the reciprocal crow also intraovnrian pollinations oonsiderably improved the seed set and the seeds were fully viable. These results suggest that intraovarian pollinations may be specially uaeful in instances where the zone of incompatibility lies in the stigma and the style. A still more promising method is to excise the ovules and rear them together with the pollen in an artificial medium, thus eliminating the gynoecial tissues altogether. Of much interest in this connection are the recent papers by Kanta et al. (1962), Kanta and Maheshwari (1963b), and Maheshwari and Kanta (1964). They excised the unfertilized ovules o f P . eomniferum, A . mexiccana, E . alifornim and Nicotiana tabacum just after anthesis and cultured them on an agar nutrient medium along with pollen grains collected from ripe anthers. All the stages from the germination of pollen to double fertilization were observed and mature seeds containing viable embryos were obtained in culture (Fig. 4). On germination the seeds gave rise to normal seedlings. The technique of test-tube fertilization involves two major stepsovule culture and gernlination of pollen. Whereas it is not too difficult to achieve a satiefactory germination of pollen, the chief concern is the rearing of unfertilized ovuletj to victble seeds through fertilization. With further refinements thiv technique, like that of embryo culture, would prove a boon in wide hydridizntiori in that it is possibly the best approach to overthrow the various incompatibility barriers instituted by the gynoecial tissues. Even when fertilization can be accomplished with reasonable success, there are other problems like the abortion of the young embryo. The control of these post-fertilization barriers demands a n understanding of the development of the embryo and its relationships with the endosperm and the maternal tissues of the seed.

IV. EMBRYO A. GROWTH OB EMBRYO IN RELATION TO

SEED DEVELOPMENT

The embryo is the young fiporophyto of the new generation. For its normal growth and difforentiation, it is dependont on it8 immediate environment, namely the endosperm. The entloHperm in turn draws upon the somatic tissues of the plant via the placenta and funicle. This implies that the parent sporophyte exercises control, at least in part, over the development of the seed by the nature and quantity of the material which it supplies to tho seed. Conversely, the seed in

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turn affecta the growth of the fruit. Thus, fruits with many seeds are usually larger than those with fewer seeds (see Nitsch, 1952; Luckwill, 1969). Gustafson (1939) demonstrated that in immature fruits of the tomato the seeds, placentae, septa and pericarp contain decreasing concentrations of auxin. This suggests that auxin is synthesized in the me& and transported to the other parts of the fruit along a decreming gradient. That auxins play a role in the growth of the fruit is also confirmed by other data. One indicator is that an application of synthetic auxins may stimulate the development of the fruit even in the absence of fertilization. The spraying of auxins to improve fruit set has now become a standard prtwtice in many countries for certain varieties of tomato, apricots, figs, grapes and oranges. I n the blackberry and strawberry there is often partial sterility or inadequate pollination leading to tho formation of fewer Reeds and a subnormal growth of the fruits. In uuah instanceiJ applications of auxins have proved beneficial in increasing the size of the fruits. Dionne (1968) observed a negligible seed set in some interspecific crosses of Sohnum. He attributed it to a poor development of the fruit which in turn was correlated with the presence of too small n number of seeds to stimulate the optimum growth of the ovary. A treatment of the ovaries, 24 hr after pollination, with 2,4-D (1 drop of a 3-6 ppm solution) promoted the development of the pericarp which in turn stimulated the formation of seeds. Similarly, in the self-incompatbile Lilium longifEOrum Emsweller et al. (1962) obtained a good seed set through the use of naphthalene acetamide and potassium gibberellate. At the time of pollination a lanolin paste of 1% naphthalene acetamide was applied to the wound caused by tievering one of the petals. Potassium gibberellate was used as an aqueous spray (0.01-0.2% plus 0.1% Tween 20). Alternate flower8 on tho inflorescence were left untreated. Observations made from the twelfth to the twenty-sixth day after pollination revealed that naphthalene acetamide prevented the senescence of the ovaries and ovules, and favoured their development into normal fruits and seeds. In the untreated controls, on the other hand, the ovules showed a progressive degeneration and collapse of the ovaries. Potassium gibberellate proved less efficacious than naphthalene acetamide. For raising hybrids between Bramica oleracea var. m p h a l a and Raphnw Honma and Otto (1962) used Nrn-tolyphthalamic acid. In this case a piece of cotton soaked in a 100 ppm solution was applied to the pedicel at the time of pollination. Following the treatment, several pods were formed, although the number of viable seeds ww discouraging. These experiments suggest that the development of the fruit and the formation of the seeds proceed in harmony and that both the phenomena are influenced by growth regulators.

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B. DEPENDENCE OF EMBRYO ON ENDOSPERM

In general the endosperm is regarded as a nurse tissue for the embryo and there are several circumstantial observations which support this view. 1. At the time of fertilization the embryo R ~ has C very little nutritive material. As the endosperm advmces in development it stores enough food substances to insure an adequate supply for the developing embryo. 2. I n the majority of flowering plants the zygote segments only after the endosperm has reached a reasonable stage of development. Even in suoh instances where the division of the zygote precedes or occurs simultaneously with that of the endosperm, the latter soon surpasses the embryo in growth. 3. Generally, the embryo develop8 only when the endosperm is properly organized. If the endosperm aborts, as in many incompatible matings, the growth of the embryo is adversely affected. 4. In the absence of the endosperm (as in Podostemaceae, Trapaceae and Orchidaceae) special provisions exist to ensub the nutrition of the developing embryo (see Subramanyam, 1960; Johri, 1962). I n the Podostemaceae a pseudoembryo sac ig formed as a substitute for the endosperm. T r a p has a large suspensor haustorium. I n the orchids also, the suspensor often develops into a haustorial organ of considerable dimensions. The hauRtorial branches seem to function as absorptive structures which enable the embryo to grow even when it is not accompanied by the endosperm. Large and hamtorial suspensors are no doubt recorded in many other plants which also have a functional endosperm (for examples see Maheshwari, 1950), but here they probably serve as accessory structures which convey food materials to the main body of the endosperm which in turn relearres them for the embryo. 6. During its growth the embryo depletes the surrounding cells of the endosperm of their contents. In many plants, such as members of the Leguminosae, Cucurhitaceae and Compositae, the embryo consumes nearly the whole of the endosperm. I n the Graminem, Euphorbiaceae and Solanaceae, on the other hand, the endosperm stores food uuhstances which are utilized during the germination of the seed. ln wch plants only those emdosperm cells are digested during the development of the seed which lie in immediutc proximity to tho embryo. C. SPECIFTCITY IN NUTRITION O F EMBRYO

Two questions may arise regarding the function of the endoHperm aa a nurse tissue: (a) what is the nature of the material which t h e

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endosperm provides to tlio embryo, and (b) how specific is this substance. In other words, can the endosperm of a species serve to nurse the embryo of another species? For convenience the second question is discussed first. The available information on the specificity of the endosperm is based chiefly on experiments with mature or nearly mature endosperm whose food reserves are normally utilized during the germination of the seed and not during the earlier development of the embryo. This is particularly true of heterologous transplantations, where embryos of one species are exciwd and cultured on tho endosperm of another species. Sting1 (1907) observed that embryos of Triticum grown on the endosperm of Secale frequently gave better seedlings than when the embryo was reared on the endosperm of its own species. Similar experiments have been made by Grekoff (1940), Ciimara (1943), Yamasaki (1947, 1960), David and SOchet (1948), Hall (1964, 1966) and Strun (1966). Clmara made interspecific embryo-endosperm transplantations in Aqibp8, Avena, Hordezim, ISecale and Trilicum at the “milk” stage of the endosperm and noted differences in some features of the seed produced in Fl plant8. Also, the embryo of Triticum vdgare grew better on the endosperm of T.duruni than on its own endosperm or that of T.turgidurn. Several Russian investigators have claimed heritable changes in the plants raised by this method. However, Mathon (1952) has explained that such genetic diversities arise by a modification in the rate of growth and differentiation of the embryo due to the altered environmental conditions. More critical observations are necessary before these conclusions can be accepted. Studies on cultured embryos of related taxa suggest that in earlier stages also the embryo does not show an absolute spocificity for the endosperm. At the hcart-shaped stage the embryos differ only slightly in their requirements for inorganic elements, sugars and nitrogen compounds, arid the range of their tolerance to nutrient materials is fairly large (see Rappaport, 1954). Furthermore, even embryos of plants belonging to diverbe fanlilies can grow in almost identical nutrient media.

V. ENDOSPERM A. CONSTITUENTS OF ENDOSPERM

As already mentioned, the available information largely concerns the nutritional requirements of older embryos while the cature of nutrients supplied by the endosperm to the embryo during its early growth is less understood. A knowledge of the Binds and quantities of food materials and other substances which are stored and /or synthesized

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in the endosperm during its development is therefore fundamental to an understanding of its role in the growth of the embryo. Van Overbeek et a,?. (1941, 1942) found that the milk of young coconuts had a stirnulatory effect on isolated embryos of Datura stramonium. The “embryo factor’’ in coconut milk is now known to be a complex of potent substances. According to Steward (1963) this is probably related to the formation of proteins, and its effect stems from a complex of substances each of which is individually unable to control growth to the same extent as the entire complex (see also Steward and Mohan Ram, 1981). Growth-promoting substances also occur in the liquid endosperm of Zeu m y 8 (Steward and Caplin, 1952), Allanblackia parvi,fora (Nitsch, 1963), Aesculus hippocastmum (Shaiitz and Steward, 1955), in the banana, in the feniale gametophytc of Ginkgo biloba (Steward and Shatnz, 1969), and in the endosperm of Sechium edule, Camellia japonica and Thea sinensie (Tto, 1961). Extracts of these tissues have the property of inducing the multiplication and enlargement of cells in explants. Some of the substances which have already been isolated and identified are : 1,3-diphenyl urca; indoleacetie acid; xanthine, chlorogenic acid, hexitola, purine-type conipounds and leucoanthocyanins. Once it is known what subrrtsnces are provided by the endosperm for the growth of the embryo, thcir specific actions can be studied. At present, however, we have only a partial knowledge of these. Earlier, Haagen-Smit et al. (1946)had reported that in the immature endosperm of corn only 9% of the auxin is accounted for by indoleacetic acid, thereby suggesting the presence of other substances of similar activity. Evidence is accumulating for the rather widespread occurrence in seeds of substances with gibberellin-like properties and they have already been recognized in the endosperms of Aesculus californica, Echinocystis mcrocarp, Persea amerimna, Prunw amygdalw, P . armeniaca and P . domestica (Phinney et al., 1957),Pyrus m l w r (Nitsch, 1968), Cocos nucifera (Radley and Dear, 1958), and in the seeds of Phaseolwr, Pisum and Zea muys (Radley, 1958). The endosperm thus contains a variety of growth-regulating substances which, at least partly, control the growth and differontiation of the embryo. The investigations of Avery et al. (1942)on developing maize kernels suggest that at the time of pollination their free auxin content is extremely low, but immediately after pollination it rises very sharply and a peak is reached within 1-3 weeks. No correlation could be established between the vigour of hybrids or polyploids and the amount of auxin stored in their kernels. Kernels with B sugary endosperm wem consistently richer in total auxin than those with a starchy endosperm. It is also suggested that the amount of auxin in the seed is nearly P

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proportional to the quantity of cellular endosperm present. The concentration of auxin in the endosperm is considerably higher than that in the embryo (see Luckwill, 1957). Duviuk (1962) has analysed the endosperm of four varieties of maize at different periods of development and reported the presence of nine amino acids and two amides. He found that most of the proteins of the glutein complex (alkali-soluble) are synthesized during the first half, and most of those of the zein complex (alcohol-soluble) during the second half of maturation of the grain. For Datura stramonium it has been shown that 2-3 weeks aftor fertilization the concentrations of amides and free amino acidx reach a maximum, but when the see& are mature the concentrations drop off considerably, and only asparagine and aspartic acid show a steady increase (Rietsema and Blondel, 1959).

Chemical analyses of the ertdoaperm like the above have, however, so far yielded only meagre data on the role of the endosperm in the growth of the embryo. Tho main source of information on this mpect is the culturo of isolated embryos, whose requirements may be expected to correspond largely with the materials received by them from the endosperm. However, the favourable effect of a substance on the growth of excised embryos in aseptic culture does not necessarily imply that it is also an active factor in the seed. Thus, in the seeds of Datura stramonium the embryos have much oil but no starch, whereas excised embryos grown on a medium containing sucrose accumulate starch instead of oil. Further, the endosperm is not a homogeneous tissue; often the cells frQm its different regions vary not only in size, shape and staining capacity but also in function. For example, the cells adjacent to the embryo become “dissolved” while those away from it continue to store reserve foods. The findings of embryo culture must, therefore, be interpreted with caution in explaining the role of the endosperm for the growth of the embryo. B. ROLE OF ENDOSPERM IN SEED DEVELOPMENT

It is more probable that the function of the endouperm is related in some ways to the quantity and proportions of the growth substances present in it. Any disturbance in tbia equilibrium leads to the abortion of the seeds. For example, the content of free amino acids in tho ~eeds of Datura innoxia after selfing and after crosfiing with D. d i a w h (d) varies considerably and the cross with D . discolor is incompatible because the ovules abort after fertilization (Rietsema and Satina, 1959).

The presence of growth substances in the seed becomes manifest immediately after fertilization. Murneek and Wittwer (1942) and

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Wittwer (1943) found that fertilization has a stirnulatory effect on the metabolism of the whole plant. In corn, McLane and Murneek (1962) reported a substance which they termed as syngnmin. According to them its concentration reached the peak 5 days after fertilization and it greatly stimulated tho growth of excised embryos of corn. MEIT* and Murneek (1963) stated that the hormones produced in the seed have an important role in regulating the movement and accumulation of carbohydrates and nitrogenous substances towards the developing fruits. In naturally pollinated ears of corn as well as in unpollinated ears treated with NAA or the ethyl ester of IAA they observed (a) a distinct increase in the content of sbiI3rch,( b ) a decrease in the content of sucrose chiefly during the first three days after treatment, (c) an increasing Concentration of reducing sugars, and (d) a moderate but significant increase of hexose phosphates. On the contrary, no significant changes occurred in the carbohydrate content of unpollinated controls. h r t h e r , the similar respon,ses of both t h e kernel and the cob to treatment suggest that hormones influence the carbohydrate metabolism not merely in the fruit and the seed but may also have analogous effects on the neighbouring tissues. The above discussion has indicated the iniportanco of the endosperm in the development of the seed. Many exampla are known in which the aborting seeds show a breakdown of the endosperm after a certain number of cell divisions. In incompatible crossos there often develop empty seeds which are much s~rinllerthan tho seods formed &B the result of successful pollinations. In other words, a degenerating endosperm may lead to a poor development of the seed itself (Rietsema et al., 1966). The formation of viable seeds is therefore a function of the balance between the embryo and the endosperm (Shepherd, 1960). This necessitates an examination of the factors which govern the growth of the two partners independently of each other. C. CULTURE OF ENDOSPERM

It is well known that of the two sperms delivered by a poIlen tube, one fertilizes the egg and the other tho secondary nucleus. The zygote organizes itself into the embryo and the triple fusion nucleus produces the endoaperm. What directs this differential behaviour is not understood. That it is not the difference in the chromosome numbers of the two tissues which is significant is indicated by the condition in the Oenotheraceae where the embryo and the endosperm are both diploid. A logical approach to the problem is to grow the endosperm and the embryo of the same species in vitro and to experiment with the tissues obtained from them.

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La Rue (1947) obtained a successful culture of the endosperm of Zecc m y 8 on a medium contnining tomato juice, or yeast extract, or the extract of unripe kernels of maize itself. Subsequently, Straus and La Rue (1954) cultured the endosperms of the sugary, starchy ,and waxy varieties of Zaa. Only the sugary endosperm readily formed a callus tissue. Tomato juice brought about an erratic growth but yeast extract was quite satisfactory, the Seitz-filtered solution being twice &B effective as the autoclaved one. Sternheimer (1954), and Tamaoki and Ullstrup (1968), also succeeded in rearing the endosperms of the sugary, waxy and starchy varieties of 8. m y 8 on a medium supplementod with yeast extract. Coconut milk did not prove beneficial to the cultures of the endosperm. In tosting the effects of several carbohydrates Straus and Ln Rue found Rucrose to be the most effective in supporting tho growth of the endosperm while glucose was the least. Some other sugar8 (arabinoso,galactose, glycerol, lactose and rhamnose) did not enhance the growth of the tissue at all; instead they caused a decrease in its weight. The endosperm tissue grown in vitro often showed a number of chromosomal aberrations leading to the formation of polyploid cclls. Since the endosperm grew in cultures only when yeast extract waa supplied, Straus (1960) studied the effects of some amino acids and amides present in the extract. Aspartic acid, glutamic acid, aaparagine and glutamina all supported the formation of a callus, but only asparagine could suitably replace yeast extract. The endosperms which had a pigmentod aleurone layer produced callus tissues which were purple due to the presence of anthocyanins. Straus (1959) reported that aspartic acid and cywteine strongly promoted the synthesis of anthocyanins in endosperm tissue cultures. Norstog (1956) excimd the endosperm of Lolium perenne at variow stages of development and cultured it on White's medium eupplemented with yeast extract or coconut milk. It proliferated vigorously FIQ.6 (See facing p a g e ) .

FIQ.6. Culturc of cmbryo and endosperm of Santalum albwm (hyp, hypocotyl; ra, radicle). A. 3-week-old culture of a seed on a modified White's basal medium; the seed enlarged slightly but failed to germinate. B. Culture of liimilar age on the basal medium supplemented with coconut milk (20%)+ caacin hydrolysate (400 ppm): the primarg root and hypocotyl are well developed; plumule and cotyledons arc still partially enclosed in the degenerated endosperm. C. 6-Week-old culture as in B; a normal plantlet has formed. D. 3-Week-old culture of seed on the basal medium+yeast extract (0.25%)+ kinetin (6 ppm)+P,CD (2 ppm). The endosperm hea proliferated along the sides and the embryo has just emcrged from it. E. A similar culture BB in D. from which the embryo waa excised. The endosperm continued ite proliferation and in 3 weeks formed an extensivc mass of callus tissue. F. 2-Week-oldsubculture of a fiagmcnt of the endosperm callus (after Rangaswamy and Rao, 1983).

1’

*

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and mitie of the cells developed into starch storing cells. Many showed c:hroniosomnl aberrations similar t o those in maize endosperm. Ntiktijimu (1902) excisctd the endosperm of Cucumia sativw 10 days d t e r pollination and cultured it with considerable success on White’s medium supplemented with yeast extract. A mixture of IAA, 1,3diphenylurea and casein hydrolysate could effectively replace yeast extract. According to Nakajinia the successful growth of the endosperm depends on the presence of an auxin, a kinin and an organic substance rich in nitrogen. Recently, Rnngaswaniy and Rao (1963) have successfully grown the endoRperin of Santdwn album. When the seeds were cultured on Wliite’n mediuni, there WIW no npprecinbla response except for an ovordl enlargenient of tho Heed (Fig. 6A). If the ba~ttlmedium was supplemented with casein hydrolysate (400 ppm) and coconut milk (‘LOO/,)the embryos gerininated, conmiming the endosperm. I n about G weeks normal seedlings were formed (Fig. 6B and C). If the seede were cultured on a medium supplemented with yeast extract (0-25y0), kinetin (5 ppm) and 2,4-I)(2 pprn), the seeds enlarged many times their initial size in 2-3 weeks. In t;0-70% of the cultures they grew into a large mass of callus tissue (Fig. SD). Both microtomepreparations and dissections showed that only the endosperm proliferated while the embryo merely elongated slightly. If the embryo was excised from the proliferating endosperm nnd grown separately it made no growth. The endosperm continued its proliferation and formed an extensive mass of callus tissue from which small masses of tissue broke away (Fig. SE). Fragments of the endosperm callus were subcultured and the cultures have been maintained for more than 12 months through four passages (Fig. BF). These investigations suggest a similarity of the growth requirements of the endosperms ofwidely aeparated taxa like Zen, Lolium, Cucumit~ and Santalum. For a satisfactory proliferation readily lending to subculture the endospermfi probably require an auxin and kinin (2,4-D and kinetin for Santalum) in conjunction with yeast extract. The establishment in tissue cultures of endosperm is of significance in understanding the physiological implications concerning embryo growth . I

VI. EMBRYO CULTURE In 1904 Hannig grew the embryos of a few plants in an artificial medium. Several others followed him, but the most significant contribution was that of Laibach (1925). In crosses between Linum perenne x L. austriacum he found that the seeds and embryos were inferior to

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those of the parents in size as well as weight. Further, in the reciprocal cross the fruits shed prematurely, and the seeds were incapable of germination. Lttibach excised the embryos from the young seeds and reared them t o maturity in a solution of 10-1570 sucrose. Prompted by his succew in obtaining the hybrid seedlings, Laibach suggested that in all cases of difficulty in obtaining viable seeds of hybrids it niight be appropriate to try t o excise their embryos and grow them in an artificial nutrient medium. Laibach's work gave a treniendous impetus to the technique of embryo culture. In the growth of the embryos of angiosperms there are three periods : ( a ) it period of slow growth following fertilization, (6) a phase of rapid growth. and findly (c) a period of physiological niaturation of the cmhryo. 'L'hc Hucccssfril culture of embryos, thercfore, comprises two niiLjor phawc": prcgorminal ant1 postgerminal (Rijven, 1952). The first is the proniotiori of embryotittl growth during which differentiation continues ; tlio second is trhegermination of embryo and the formation of a nornird needling. A. ClILTURAL C?ONDITIONS

The younger the embryo, the more difficult it is to excise it under aseptic conditions. Secondly, young embryos are extremely susceptible t o osmotic shock. In cultures of embryos of Hordeurn wu2gare the addition of casein hydrolybcate increiised the osmotic value and thus favoured the pregerminal growth (Ziebur et al., 1950). Rietsema et al. ( 1963) also reported that osmotic concentration influenced the growth of embryos of Dalzwa. Using maniiitol to obtain the desired osmotic values, they demonstrated that the younger the embryo the greater was its affinity for a medium of high osniotic concentration. According t o Rijveii (1958) a high concentration of phosphate buffer also had a similar influencc on the embryos of Capella bursa-pastoris. Contrarily, Raghavan and Torrey (19634 have reported that the osmotic value of the culture medium is of relatively little importance in the morphogenesis of cultured embryos. Early heart-shaped embryos (81-406 L,A in length, Fig. 6A) of C. bursa-paetoris grew normally in an agar nutrient medium containing mineral salts, three H-vitamins and only 2yo sucrose. Three weeks after culture the root and shoot primordia had already formed and the cotyledonary lobes were well marked (Fig. OB and C). The primordia of the first pair of leaves developed in 4-6 weeks (Fig. (ID). Globular embryos (<80 p in length, Fig. 6E) cultured on the basal medium failed to develop even after a long time. An increase in the concentration of the vitamins five or ten times over the initial quantity, variations in the p H of the medium, and a high

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oxygen tension failed to improve the growth of the globular embryos in vitro. However, a successful development of the globular embryos (80-80p in length) wns obtained, if the bnsnl medium was supplemented with indoleacetic acid, kinetiti and /or ittlenine aii1phiLte. A combinntion of d l tho thrcs ctdjiivttrrta providoti tho host wrz'liau for growth. Ihiririg the first 7-10 ckya, the bnll of ernbr.yonrd cells continuoti to atltl to its t t u further bulk by cell divieions and cell enliirgement (Fig. el!'). development was siiniltlr to that of older embryos (Fig. 6G). Thus, in C . bur.~a-pastoristhe younger embryos did not show a n absolute requirement for n high osmotic: concentration although a high percentage (12 or 18) of sucrose or a high quantity of mineral salts (ten times that in the basal medium) partly replaced the effect of the three growth substances in inducing their development. On the basis of these observations Raghavan and Torrey (1963a) suggest that the growth and differentiation of excised embryos in cultures are controlled not so much by the osmotic value of the culture medium but by the availability of specific growth factors. For the embryos of Bo,~,~ypium Mauney (1961) used White's medium with all the ingredients a t five times the usual concentration, and supplemented it by 0.7% sodium chloride and certain growth substances. This had the useful effect of retarding the enlargement as well as elongation of cells, which ia typical of germination, and of favouring the division of cells which is otisentiel for differentiation. When the embryos grew older, they were tratiafctml to ;L medium oontuining only 0-3(% sodium chloride. l'he lowered osmotic pres~ureof the medium improved the postgcrminal tieveloptncnb of the embryo. A low osmotic concentration, which i H easily achioved by using decreasing amounts of sucrose in the medium, promotes the germination of embryos (Ziebur and Brink, 1951; Rijven, 1952; Honma, 1955; Iwanowskaya, 1962). Recently, Ryczkowski (1962b) Btudied the changes in the osmotic values of' the embryo and endosperm in the

Fro. 8 (see fnring pxrge). FIG.0. I n rilro rultliro of rmbryos of (!fip3t?lhbur8ri-pmlori8 (cot, cotyledon; hyp, hyporotyl: s, uuspenaor).A. Early heart-sliaptl rmbryo (81 in lengt,hexclusivc of the Buspensor). 13. 2-\Vwk-old rulture on nil agiw nutrient, medium incubnted in dark; note formation of rotylrctons. C. 3-U'oek-old culture grown in light ; tho cotylcdon8 and the plumule are well developed. 1). Ax in C'; 5-h'eok-okl emtxyo ~howitigthe formntion of the first pair of leaves. P. Globular embryo (51 p in loiigt,h inclunive of tho aimpensor). P. 10-Day-old culture of n globular ambryo, rxriwd nt Htiigc wliown in E. on tho 1)nHuI mcclium supplmnmtcxl with J A A (0.2 ppm)+ kinetin (0.001 ppm)+ ibclfminu nolpliittc: ( O W 1 ppm). Ct. fLWt!t!k-olfl culturt! ahowing the differentintion of mot, hypowt~yluntl i.ot.yk!iltni~(ufbr Httfihttvun nr~d'rorruy, 196311).

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clevelopirig seeds of soine representiitives of both monocotyledonous and dicotyledonus plants. In both, tho embryo generally showed a higher osmotic value than the endosperm. However, the osmotic value of the embryo increased with its growth only in monocotyledonous plants. Ryczkowski Iias, therefore, suggested that a knowledge of the osmotic values of t'lie e m h y o tliiring its growth ant1 development in the o v i h would be Iiclpful iii tlonigniiig a propor nirtricnt medium for its growth irb ailro. I,ike tho oxinotic virlu~,the cwnwntratioii of the oxygen in the rne~liunialso intluenccs t l w growth of the embryo in culture. White (1939) reported that a tlecrcnsc in the content of the oxygen in the surrounding nietlium is essentid for enabling the embryo to follow the normal course of differentiatioii. "hi8 can be achieved by lowering the enibryos into the nutrient medium. Embryos of some gymnosperms, like Ginkgo hiloba and I'inw lambwtiana, showed their best growth only when their cotyledons were imhedded in the medium (Bulard, 1952; Brown and (:ifford, 1958). Sinii1:irly. embryos of Dafuru (Van Overbeek et at., 1944) and Cydamm pwsicum ((hrter, 1955) grew best only when they were implanted below the surface of the agar medium. However, other reports are contrary to this. Submerged embryos of Pinus hrnbertiuna failed to grow normitlly hut developed tumorous growths whereas those of Horderrm vulgaw did not grow a t all (Norstog, 1961). U. UROW'I'H M RI)IA

Aliot,lier wninioii t?xl)crieticcis t l l i b t t i nutritive medium suitable for older er11l)ryos is oftcn quite iiiisiiit;d)le for youngcr ern bryow. Also, yoling embryos usually grow prwociously in culture and produao inalformed seedlings. Thiw is vci*y i~il(lc!sirihlespecially when tho aim is t o raise plants from otherwisp iLborth embryos. Mu& of tho investigations on embryo culture arc. therefore, concerned in formulating the conditions and media w1iic.h would promote the normal growth of t,he eiiibryo in its pregcrminal and postgcmiinal phases. Sugars not only alter the osmotic value of the medium but also play it nutritional role. High osmotic values and therefore high concentrations of carbohydrates (as inuch as 12-18';;> mcrose) favour the pregerminal phase ; low osmotic values achieved by lower concentrations of sugars (2';; and below) promote the postgerminal phase (see Rappaport, 1954; Norstog, 1961). Sucrose has been found to be by far the best source of carbohydrate for embryo culturea. Van Overbeek el al. (1941) showed that unautoclavecl coconut milk induced normal, pregerminal growth of young embryos of Uaturu. Since then coconut milk has h e n used with success on embryos of several plants including Cocoa ncccifera.

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Merry (1942) excised embryos of Hordeurn salivum 7-28 days after fertilization and cultured them on an agar nutrient medium containing 2yh sucrose. The youngest embryos (7- and 8-day-old) lay quiescent even up to 8 weeks, whereas 9- to Il-day-old embryos initially showed some indications of growth. Only the 12-day-old or still older embryos grew normally in culture. Recently, Norstog (1961) cultured embryos of H. ndgare, ranging from fio to 1500p in length, on White’s medium Rupplemented with none or one or more of the following subtitanceti: danine, nspnragine, glutnmine, rnixtureti of amiiio acids, and coconut milk. On White’s iiictliiitii t ~ l o i i oembryos lotin than 500 p in length failed t o survive!, wliilc t,tiow 500 14 in length tihowed only a limited growth (Fig. 7A-C). It iH only those twyontl 50Op that attained a size coniparnblo to that of rtnihryos iiL r h o . However, no differentiation W ~ observable H in any of them. Mere ninino acitln were of little henefit, but coconut milk (10, 20 or YO)(,) iiiducetl a marked growth even in embryos which were smaller than 500 p. I n 2 weeks after culture roots and additional leaf primordia developed in 500 p long embryos (Fig. 7D and E). I n combination with glutamine (400 ppm) or with a mixture containing 585 ppm of a total of 14 amino acids, coconut milk caused excellent growth and differentiation of embryos of all ages. I n a few instances even 6Op long embryos produced masses of tissue which developed leaf and shoot priniordin (sometimes more than one) (Fig. 7F and (2). Nevertheless, the embryos grown in culture did not develop scutelluni. (’hang (1963) illtio reported that embryos of barley grown in. iiitro allowed tieveral deviations in their morphology and failed t o reach the mature stage. T n an attempt to discover a suitnhle sutwtitute for coconut milk, Matsubara ( 1968) tested the effects of (:wein hytlrolysdn, dried brewer’s yeast, skimmed milk, diffiinnteti froin the endotipcrtns of Ginkgo and froin the neetln of t ~ t imaiiy an iiinc rmgiofqmrns on yoling embryos. He observed that the alcoholic difFutiltten from young Heedn of Lupinus lutezu rLnd from old seeds of 8rchium edde were as effective as coconut milk in promoting the growth of embryos of Datura tutula which were only 0.15-0-55 mm in lengbh. Embryo factor activity, similar to that of coconut milk, has also been found in extracts of almond inenl, banana, Datura ovules, nondormant apple buds, wheat germ (Van Overbeek, 1942; Van Overbeek et nl., 1942), malt extract (Solomon, 1950), and peat and yeast extracts (Gorter, 1955). Like coconut milk, casein hydrolysate (a complex of several amino acids) is yet another substance which has been widely used in embryo culture. I n their experinients on Hord~urnuirlgare, Kent and Brink (1947) tried casein hydrolysate, Noditiin nucleate, and tomato juice

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and discovored that these substances too can promote the growth of young embryos t o maturity. Sanders and Burkholder (1948) observed that, unlike casein hydrolysate, individual amino acids or mixtures of a few of them did not favour t,he growth of embryos of Da.tirm

C

E

D

-

F

White’s medium i 20% coconut milk t 4 0 0 p p m glutornine OW 130 doys) (C.80cells)

Shoot 1 Shoot2 Shoot 3-

.

Shoot 5

Root

G

FIQ.7. Culture of young embryos and procmbryos of ffordeum vulyare. A. Embryo (0.5 mm in length) a t thc time of culture. U. 14-lhy-old culturc: on Whit& medium. C. Embryo from B in longisection; on White’s medium the growth was negligible. D and E. Embryos (0.5 mm in length) grown for 14 days on Whito’u mcdiurn+coconut milk (20%);note primordie, of additional roots and leaves and comprrc with I3 and C. F. Proembryo (60 p in length). G . 30-Uay-old culture of p ~ ~ l l l l J ~in y OWhite’s medium+ coconut milk (20%)+ glutamine (400 ppm). Tho embryo prodticwl n muss of tistme from which five shoots differentiated (nfter Norstog, 1961).

innoxia and D. stramoniurn. However, a mixture of 20 amino acids, or of cysteine and tryptophan i n concentrationu proportional to their

quantity in casein hydrolyrrate, proved favourable.

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According to Ziebur et al. (1950) tho amino acid atid sodium chloride components of casein hydrolysitte prevent the precocious germination of embryos, ant1 that the iitnino ;ic*ids together with the phosphate complex serve as nutrients in p m o t i n g the normal growth of embryos. Amino ttcids by themselves ftdetl. to diiplicate the effeclt of orwein hydrolysate. Therefore, Ziel)ur et ul. (l!)50) concluded that tho growth of immature embryos of barley, ohtiiined i n vitro by using casein hydrolysate, “may he the result of ;In interplay of both nutritional and physical factors”. The works of Lofland (1950) and Mauney (1961) on Cossypium, of Rijven (1952) on C’apscEb hursa-pasforis, and of Nakajima (1958) on Cucurbita maxirna confirm that casein hydrolysate strongly supports the growth of young embryo8. Tn his work on Cucurbita maxima Nakajima investigated the effects of casein hydrolysate, 1,3-diphenylurea (a component of coconut milk), kinetin, IAA, malt extract, and a n extract of the endosperm of its own species on young torpedoshaped embryos. The most marked promotion of embryonic growth and formation of seedlings occurred when casein hydrolysate and 1,3diphenylurea were used in combination. For the growth of proembryos of Citrus microcarp aluo, casein hydrolysate proved beneficial (Rangaswamy, 1961). Very young globular proembryos (14-28 x 14-28 p ) cdlapaed within 3 days of culturing on n modified Whitbe’s mccliunt (Fig. 8A). An addition of 3, 5 or even 10‘,~{~siicrose did not promote the growth of proembryos to any appreciable degree but merely prolonged their survival t o 2CJ days. With 10f;G sucrose the embryos enltirged t o twice their original size. The proembryoe, cultured on White’s medium supplementetl with casein hydrolysate (400 ppni), faithfully continued their pregerminal development through d l the entbryological utageu. I n 3-4 weeks fully organized embryos were formed in 8V1,,of the cultures (Fig. 8B and C). This is one of the few instsnc+esof successful growth of very young proenibryov (<18 p in diameter) in vifro. The fostering of the normal growth of young embryos 1)v sii1)stiincex x u d i as coconut milk and casein hydrolysate is iniportmt for rearing non-aberrant seedlings from cultures of proeinbryos. In addition t o rarboliydmteu, untitio acids and plant tissue extracts, a large number of other organic. coinpounds belonging to the class of vitainins, plant hormones, kinins, xterols, steroids and steroidal sapotiins have been screened with R view to discovering substances beneficial for the growth of ewisetl embryos (see Sanders ant1 Ziebur, 1963). Among these. gibberellitis i ~ n dkinetin have reooived increaning recognition :ts clieniioals c.apiLl)le of rcgiilatitig the growth untl tlovclopmerit of embryos.

of dvena fnliiu (Ni~yIorntitl Sitiil)son, 1961). Later, Simpson and Naylor (1962) tlcmonstrtttcvl that w i twyo tlormancy in A . f d u a in due to a critictil t):ilnrtce in tlic ; a m o u n t of' rnrrltiaso i r i the grrtinn. I f tho xynttlenis of fllibIttlS(? is hlockctl Or i t s c:orltcrlt is low, the! hydIY~lynis of stnrdi in prevwtetl i m 1 the oni t)ryo p s w into u state of'rlormancy. Naturally, in ciiltiirvs of' embryos exrisetl from dormant caryopnen a high conccntration of' exogenous nngar, or ntaltase, or gihttorellic. acid (50 ppni) W R S Iiclpf'iil hi overcoining the dormancy. That gihberellic acid is effective i n 1)rwtking tl(~~tiliL11~y tmtl promoting germination

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is explained as due to its capacity to stimulate the activity of maltme. I n embryo cultures of Hor&*um z~tilgarealso Paleg et al. (1962) found that GA, activated the mobilization of food reserves from the endosperm. Veen (1963)reports that gibberellic acid did not improve the growth rate of globular and heart-shaped embryos of Capselk bursap t o r d s . However, in cultures of torpedo-shaped embryos GA, increased the growth rate considerably by stimulating the activity of the root meristem as well as the development of root hairs, and induced epinastic curvatures of the cotyledons. Since these responses normally occur during germination Veen considered gibberellic acid to be a promoter of this process. Nickell's (1958) dbservation that gibberellin-like substances appear in very old tissue cultures of the cotyledons of Phaseolw uuZgaris is of interest, and suggests that GA-like substances occur in the embryo. Corcoran and Phinney (19W2) and Ogawa (1963a,b)have demonstrated a oorrelation between the growth of the embryos and the quantity of gibberellin-like substances present in the soeds. Like gibberellic acid, kinetin has also been reported to affect the growth of the embryo. I n Veen's experiments it showed no significant effect on the torpedo-shaped embryou of Capsella bur8a-pastm*8,but in to 10-0 g/ml it increased the growth rate of concentrations of globular as well US heart-shaped embryos. Nevertheless, this kinetininduced growth resulted only in malformed embryos. C. APPLICATIONS OF EMBRYO CULTURE

I n addition to these fundamental investigations a large number of papers have appeared on the applications of the embryo culture method, Since the time of Laibach (1926) the technique has been extensively used in agriculture and horticulture where the failure to obtain viable seeds is due to one of the two causes: (a) the embryo of the first generation hybrid aborts in the seed, or (b) the F, hybrid produces seeds which are incapable of supporting the development of the embryo to the germinable stage. In several instances of intergeneric and interspecific matings, which had been declared ineffectual or discouraging because of only a low yield of adult progeny, embryo culture has proved a boon. Some such achievements with cereal, pulses, fruit and vegetable crops and ornamentals etc. are dincumxi below. Species of EZymw are characterized by (a) thc development of a deep-growing and strong root system which enctbles the plantn to resist drought, and (6) the production of a large number of ueeds per ear. When Tritimm was crossed with Elpmw no hybridrc were obtained

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unless the embryos were cultured in artificial media. Iwanowskaya (1946) crossed T. durum x E . arenariua, excised the hybrid embryos, and grew them in culture. Later, Iwanowskaya (1962) raised several other hybrids and observed that the viability of the embryos of T. d u r u m x E . arenarius was greater than that of the embryos of T. durum x E. giganteus and of T . uulqare x E . arenariua. Many of these hybrids are said to be perennials having a height of 1.5 m and bearing abundant foliage and large ears. Uttaman (1949a-0) used tho technique of embryo culture to test the viability of young embryos of Zea mays. Three-week-old cobs were refrigerated immediately rtftor harvont and ten kernels were removed from them daily. The embryos excised from 3-week-old cold-stored cobs retained viability for about 46 days. Embryos excised directly from 4- to 7-day-old kernels (which were not cold-stored) and grown at 31&1°C showed only a slight growth and even this ceased five days after culture. The addition of n few drops of cold-preserved coconut milk (expressed from mature nuts) to 3- or 4-day-old cultures had a depressing effect, particularly on the root. If coconut milk medium was used to culture embryos excised from 2-week-old kernels, germination was suppressed; but good growth resulted when it was added on the second day after culture. Only embryos which were at least 16-day-old could readily develop into plants. Hordeum vulgare is completely self-fertile but susceptible to cold and to the powdery mildew, Erysiphe graminis f. h.uraei, whereas H . bulbosum, although loss self-fertile, is appreciably winter hardy and resistant to mildew. Hybrid progenies between the two species have always been desired, but in practical hybridization the resulting caryopses are frequently devoid of an endosperm so that the embryo dies prematurely. Thvim (1960) applied the tochnique of embryo culture to raim interspecific hybrids between H . vulgare, H . bulbosum and €?.californicurn. He performed cross-pollinations in nine combinations, excised the embryos 14-28 days after pollination, and cultured them on a modified Randolph-Cox medium. Seedlings capable of successful transplantation were obtained from the cross H . vdqare ( 2 n ) x H . bulbosum (4n). Seedlings were also obtained in other combinations but the transplantations were more dificult. Unlike H . vulgare, the wild H. hexupodium is resistant to Hdminthosporium sativum. When the two were crossed the endosperm usually failed to develop in the hybrid caryopses and the embryos were rendered inviable. To circumvent this difficulty, Schboler (1962) made cultures of excised embryos and successfully raised the Fl and also some subsequent generations. Similarly, in crossing Hordeum jubatum and Secale cereale many of

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the hybrid seeds oollapsed in 6-13 days after fertilization. At maturity even the largest seeds were malformed and failed to germinate. Histological examination showed that the embryos made considerable growth but were arrested because of an incompatible endosperm. To overcome this Brink et al. (1944) excised tho embryos from the hybrid seeds and cultured them on White’s medium. Out of 81 cultures, the majority showed undifferentiated growth, but one embryo differentiated normally and developed into a seedling. In interspecific crosws in O y z a it is generally observed that although fertilization occurs the caryopses fail t o produce viable embryos. I n crosses between 0. minuta and 0.sativa Nakajima and Morishima (1958) noted the formation of only imperfect grains containing only malformed embryos. When 0. sativa (diploid) was crossed with 0. mint& (tetraploid) the caryopses produced were viable but those obtained in the cross 0. sativa (tetraploid) x 0 . minuta were inviable. By culturing them it was possible not only to raise seedlings of this otherwise unsuccessful cross but also to improve the performance of the hybrids of the diploid 0. saliva x 0 . minula. Bouharmont (1961) has reared hybrids between 0. saliva ( 9 ) and three other species (0.schweinfurthiana, 0. perennea and 0. latifolia) which were used &B pollen parents. He cultured 7-day-old embryos on a nutrient medium containing dextrose and obeerved that the endosperm adherent to the embryos had no inhibitory influence on the formation of seedlings. However, according to another report a n extract of the endosperm of 0. sativa retarded the initial stages of the germination of embryos in culture (Sircar and Lahiri, 1956). Sirice indole-3-aceticacid duplicated the retarding effect, Sircar and Luhiri concluded that the endosperm contains a substance similar to IAA. The legumes are yet another group of plants to which embryo culture has been extensively applied, and many new hybrids have been obtained which show a blending of several desirable features. Of special interest are the investigations on Melilotw. M. oficinalis is more drought resistant and better adapted to grow in the phhs (in the USA) than M.dba;but it htts a high content of coumarin (harmfd to cattle) unlike M.dentata and some lines of M. alba. Webster (1955) reported that efforts to introduce genes for low coumarin from ill. dentata into M. oficinalis were a failure. Reciprocal croxses were then attempted between M . oficinalis and the low coumarin lines of M. alba. In these, the embryos grew for some time but failed to mature becaum the ovules aborted 8-10 days after pollination. Only through embryo culture was it possible to rear two hybrid individuah from the cross bl. OfiCiW& x H.a&. Almost identical rwults were obtained by SchlosserSzigat (1962) who has also raised a hybrid of M.oflcinalio x M . alba.

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According to Webster (1966) “other species crosses, previously thought to be impossible, should now be reconsidered”. I n Medicago sutiva (alfalfa) an abortion of o d e s is quite frequent after self-pollinations and in interspecific crosses. I n self-pollinated plants Fridriksson and Bolton (1963) excised the embryos when they were 21-day-old and nurtured them into seedlings. A few interspecific hybrids of L a t h y w cornicktw, L. tenub and L. uliginoszls have also been made possible through embryo culture (Davies, 1961). It is known that only “non-viable” or (‘physiologically sterile” seeds result from tho cross Phmeolus wulgnris x P. acutifoliw. However, when embryos were excised 14-24 days after pollination and cultured on White’s medium satisfactory seedlings were obtained (Honma, 1966). I n this case the embryos were initially cultured in a liquid medium and then gradually transferred to media containing decreasing concentrations of sucrose ranging from 4 to 0%. Interspecific crosses in the genus Prunw have been &,tempted quite frequently. Tukey (1938) applied the embryo culture technique and reported success with embryos of apple, peach, pear and plum, which often abort before completing their full development. Skirm (1942) germinated 414 embryos derived from 16 different interspecific crosses of Prunw. Lammerts (1942) demonstrated that embryo culture helps to shorten the breeding cycle of deciduous fruit trees such as apricot, nectarine end peach, and to hasten the germination of hybrid seeds. Nectarine and double-flowering peach hybrids reared by this method grew large enough to be used as female parents within two years and proved superior to the plants raised from seeds subjected to stratification (treatment with moisture followed by low temperature). Gilmore (1960) studied the growth of embryos of peach excised from seeds treated with various disinfectants. He also investigated the influence of light, temperature, moisture, and the composition and age of the medium on the growth of embryos in witro, and etandardized a technique for culturing the embryos of peach. Many raaceous fruit trees are characterized by a long period between the ripening of the seeds and the maturation of the embryos. The requirements for germination of embryos excisod from after-ripened seeds and of the embryos isolated from non-after-ripened seeds am variable (Tukey, 1944). Lesley and Bonner (1962) reported that it is helpful to store the fruits of Prunus at 2°C for 7 weekR before excising the embryos from them. Hesse and Kester (1966) and Kester and Hesse (1966) cultured the embryos of almond, plum, peach and cherry. They observed certain correlations between the ability of excised embryos to germinate in vitro and their developmental status at harvest,

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the method and length of storage of seeds or fruits before the excision of embryos for culture, and the nature of the medium. Embryos of the early ripening s p i e s were favoured by the presence of sugar in the storage solutions as well as in the culture media, whereas those of the intermediate ripening varieties readily germinated in a non-sucrose medium irrespective of a previous storage in sugar or non-sugar solutions. Embryos of late maturing varieties responded best when grown in a sucrose-deficient culture medium. Zagaja (1962) studied the after-ripening requirements of immature embryos of apple, apricot, pear, plum, sour and sweet cherry. He raised embryo cultures and subjocted them to low ,temperatures. Cold treatments significantly stimulated the germination of embryos. Zagaja reported (a) that the after-ripening requirements of immature as well as mature embryos were somewhat similar, and (b) that in immature bmbryos the cold treatment stimulated a 'better translocation of materials from the ootyledons to the embryo axes and in turn increased the capacity for cell divisions. Some wild species of Lywpersicum-L. peruvianum and L.glanduloeum--are resistant to viruses. Several attempts have been made to transfer this character to the cultivated tomato. However, the cross L. peruvianum x L . emulenturn proved infructuous and even in the reciprocal cross only collapsed seeds were formed (Smith, 1944; Choudhury, 1966). From his enibryologicalstudies Smith found that the endosperm collapsed first and then the embryo within 30-40 days after pollination. He excised the embryos prior to their undergoing necrosis, oultured them for about 10 days and obtained hybrid seedlings. Similarly, Choudhury (1966)excised the embryos nearly a month after fertilization when they were still turgid, and cultured them on a medium supplemented with 50% coconut milk. A week later when the seedlings were formed they were transferred to a medium without coconut milk, These hybrid seedlings grew rigorously and also produced flowers. Alexander (1950) reported that nearly 9% of the fruits resulting from the cross L. esculentum x L . peruvianum bore seeds from which the embryos could be cultured into hybrid plants. The embryo culture method is also useful for overcoming dormancy in seeds. To give an example, the seeds of Iri.9 hihve a variable period of dormancy. To reduce the long interval between the formation of the seed and the flowering of the plants Randolph and Cox (1943) employed the embryo culture method and thereby shortened the life cycle from two or three years to less than a year, Like many other ornamental plants the rose normally takes a whole year to come into flowering. However, by embryo culture it has been possible to produce two generations a year resulting in a shortening of Q

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its breeding cycle (Lamnierts, 1946; Asen, 1948). Dormancy of seeds and a slow growth of the seedlings are undesirable features in many other horticultural plants. Thus, in the crab apple (MaZuesp.) 3-4 years are required to obtain the F, progeny. Nickell (1951) tried to ourtail this period. The excised embryos germinated within 24-48 hr of culturing and in 3-4 weeks transplantable seedlings were formed. In soil the seeds germinated only 9 months after Eowing. In recent years other reports have appeared. In hybridizations between diploid and tetraploid Chrysanthemum boreale (9) and C. paci$cum, Kaneko (1967) observed that although fertilization occurred the embryos collapsed early during the development. Hybride were obtained by excising the embryos and culturing them on a nutrient medium. The brtyding of lilies has been greatly facilitated by embryo culture. All the attempts to cross Lilium hen@ with L,regak. were unsuccessful until the embryo culture technique wm adopted (Skirm, 1942). When the varieties “Album” and “Rubrum” of L. spe&mm were treated with pollen from L. auratum, the resultant seeds were largely non-viable and contained embryos at various stages of degeneration. With the embryo culture technique Emsweller et al. (1962) successfully raised over 1000 seedlings of L. speciomm album"^ L. auratum and 100 seedlings of L. speciosum “Rubrum” x L. auratum. The F, hybrids, named L. p r k n i i , bloomed normally but were df-sterile. Interpollinations among them and back-crosses with their parents have resulted in a wide range of cultivars such as “Allegra”, “Aurora” and “Advance”. Among other economic plants whose improvement has been sought through embryo culture technique are the fibre-yielding crops such as C o r c k s (jute) and Qossypium (cotton). Corchorus q s u l a r i a yields the “white” jute of commerce. T t is fairly resistant to drought and flood, and can be cultivated on low lands. However, it is susceptible to certain pests and diseases, and although the fibre is white it is less strong than that of C. olitoriw. This species generally grows on high lands and produces a red but strong fibre. Plant breeders have long wished to evolve an interspecific hybrid combining the desirable features of both species. However, the croes 0.olituriw xC. txpw.hri8 gives a very poor fruit set without any viable a d s , and the reciprocal cross gives entirely negative regults owing to the abscission of the flowers shortly after pollination. Ganesan et al. (1967) investigated the causes for the failure of seed formation in the cross C. olitoriwrx C. capsularis and found that the hybrid embryo usually aborts before reaching the heart-shaped stage. Sulbha and Swaminathan (1969) adopted such techniques as smearing the ovary with hormonea and using the reciprocally grafted plants as parents. The b a t mentioned

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technique considerably increased the percentage of fruit set not only in the cross C. olitoriua xC. mpsulam's but also in C. cupaulat$sx C. olitoriwr. Islam and Raehid (1961) improved the fruit set by applying IAA to the pedicels of flowers soon after pollination, and obtained a few viable F, seeds in the cross C. ol~tom'usxC.cupsularis. More recently, Islam (1964) coupled the application of hormone and the technique of embryo culture to rear hybrid seedlings of the reciprocal crosa, C. capeulariip xC. olikdwr. A medium supplemented with 0.1% yeast extract and 0.06 ppm each of kinetin and IAA proved best for the germination of the hybrid embryos which grew into tramplantable seedlings. In Gbesypiunz repeated attempts have been made to obtain hybrids between the species of the New World and those of the Old World. According to Beasley (1940) in the cross B. arhmeurn (2n = 26) XU. h~rsutum (2n = 62) the endosperm disorganized in 15 days after pollination without reaching the cellular stage and the embryo aborted. J. B. Weaver (1968) excised the embryos from capsules varying in age from 20 days after pollination to maturity and oultured them on White's medium. Those which were excised from 30-day-old fruita grew satisfactorily and one normal seedling was obtained. I n the reciprocal cross, B. hireutum XU. arboreurn, the hybrid embryos grew normally for about 10 days after pollination. Thereafter, the growth was retarded and within 15 days after pollination the fruits became abscissed. In the few capsules which remained on the mother plant one or more ovules showed a cellular endosperm but there were no embryos. Out of 600 pollinations only one embryo grew to a stage that waa advanced enough for excision and culturing. This waa reared in vitro (J.B. Weaver, 1957). Among other investigations on embryo culture may be mentioned those of McLean (1946), Wall (1964) and Nishi el al. (1969). McLoan raised interspecific hybrids of D.ceratocaula with nine other herbaceous species of Datura. Wall (1964) successfully hybridized Cucurbitu p e p with C . moechata, and Nishi et al. (1959) obtained a hybrid Chinose cabbage. The work on the parasitic Caaytha jitiforrnie (Rangaswamy and Rmgan, 1963) haa shown that normal germination and the subsequent growth of the seedling can be obtained in culture without the intervention of any host plant. This confirms the earlier observations on other pamites, namely Cwcutu rejiexa, Dendrophthoe falcala and 8aWlum album. These have been discussed elsewhere in this Damr.

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D. LIMITATIONS O F EMBRYO CULTURE

Despite mtiny years of experimentation and its application in practical biology and fundamental research the problem of the culturing of embryos cannot be regarded as solved. The embryo undergoes a gradual transition from a stage of dependence in the beginning t o that of relative autonomy. Consequently, its requirements for growth also change with age. Of interest in this connection is the work of Raghavan and Torrey (1963b) on the embryos of an intervarietal hybrid of Cattlep, They observed that with progressive ontogenetic development the embryo acquired a capacity for the activation of certain enzyme systems, especially those concerned with the utilization of nitrogen compounds. The ability to assimilate nitrate nitrogen corresponded with the appearance of the enzyme nitrate reductase in the embryo. In the sced the embryo is nourished by the tissue adjacent to it, namely the endosperm (the Podostemaceae, Orchidaceae and Trctpaceae are the only exceptions in which little or no endosperm is formed). It is, therefore, logical t o presume that as a rule the changes in the contents of the endosperm largely correspond to the requirements of the embryo. In their attempts to provide the embryos with natural nutrients, many investigators have added extracts of endosperms, ovules, or fruits of the same species to the culture medium. Van Overbeek et al. (1944) reported that an extract of the ovules of Datura favoured the growth of the young embryos. Likewise, Ziebur and Brink (1951) observed that the endosperm of Hordeum vulgare had a high stimulatory effect on the growth of its embryos. Endospenns and embryos of similar as well as of different stages were tested. The endosperms were excised and placed around the embryos either directly on the surface of the agar medium, or in a box made of filter paper or cellophane. It was found that the older endosperms wer0 more effective in improving the growth of young cmbryoa. Further, freshly excised, entire living endosperms were superior to (a) overnight-frozen endosperms, ( b ) a crude extract of' endospernis, and (c) riutoclavod endosperm. i to nurse t h o omtryo That the main function of the eticlosperm N is cleitrly demonstrated by the work of Pieczur (1962) on Zea may8. Previously established tiwue cultures of its endosperm served as beneficial substrata for the growth of excised embryos of the parent species. It has ttlso beon reported that the addition of the excised endosperm to the cultme medium c a w s an increase in the protein content of the embryos of corn (Oaks and Beevem, 1901). Similarly, the best growth of the embryos of Cocos nucifera was obtained in a

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medium containing filter-sterilized milk from tender coconuts. The older cellular endosperm, heat-sterilized milk, and milk from mature coconuts markedly. inhibited the growth of these embryos in dtro (Cutter and Wilson, 1964). It is curious that sometimes “foreign” endosperms support the growth of embryos better than the endosperm of the same species. This is amply elucidated by the stimulative effect whioh the endosperm of coaonut has on embryos of widely separated species. The picture which emerges from this observation and other investigations carried out during the last six decades is that underlying the development of the zygote into the embryo there is an interplay of many nutrients, growth stimulants and inhibitors, whose exact duplication in artificial cultures is by no means easy. Consequently, when freed from the confines of the ovule, the young embryo often shows an unrestrained behaviour but also manifests growth responses of a most varied nature.

VII. CULTUREOF OWLES In orchids the interval between fertilization and formation of mature seeds lasts from several days to a few weeks. This lag is rather annoying, especially in epecies of ornamental value. The embryo is too tiny to be dissected out, but the fertilized ovules can be cultured as such and seedlings raised from them. Working with the ovules of Epidendrum cochlmtum, E . tarnpense and of a hybrid of Cattleya OctavexC. nws&e, Withner (1942, 1943) was able to shorten the duration between the pollination of the ovary and the maturation of the seeds, and thus hasten the production of seedlings. Curtis (1947) raised normal seedlings of Van& tricolor through ovule culture. On adding barbiturates to the medium the embryos showed a copious proliferation. Later, Spoerl (1948) tested the usefulness of different sources of nitrogen, and found arginine to be most satisfactory for supporting the growth of the unripe seeds and aspartic acid for that of the mature seeds. Vacin and Went (1949) obtained a better differentiation and germination of embryos in cultures made on a medium fortified with tomato juice or prominogen (a protein hydrolysate). It0 (1961) etates that the germination of the seeds and the growth of the protocorm of orchids do not take place satisfactorily in the absence of peptone in the medium. However, Rao (1963) was able to germinate the seeds of an interspecific hybrid of Van& on an agar nutrient mediuni without any special supplement. He observed that some of the seeds directly produced seedlings wheresu others developed into a callus which later differentiated into new plants. Similarly, Raghavan and Torrey (19638) obtained ready germination of weds and plantlet8 of an intervarietal hybrid of Cuttlega on a medium containing

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ammonium nitrate as the sole source of nitrogen. Other ammonium salts such as sulphate, chloride, acetate and oxalate were as effective as ammonium nitrate, whereas the nitrites and nitrates of sodium, potassium and calcium were poor sources of nitrogen for the growth of the embryos. Neither kinetin nor ascorbic acid proved useful. Professional orchid growers are well acquainted with several media and growth supplements which enable the raising of plants through seed culture. Vacin and Went’s medium, Knudson’s medium, RandolphCox medium, Difco orchid agar, and fish emulsion are some of them (see Withner, 1969). One of the advantages of ovule culture is that it serves as a tool (a) to support the growth of such embryos that fail to develop after isolation, and (b) to study the behaviour of embryos which are difficult to excise aseptically, Of interest in this connection is the work of N. Maheshwari (1968) on Papaver emnifetmm. She raised ovules containing a zygote (or 2-celled proembryo) and a few endosperm nuclei into viable seeds on Nitsch’s medium supplemenfed with 0.4 ppm kinetin. Subsequently, Maheshwari and La1 (196lb) reported that kinetin accelerated the growth and differentiation of the proembryos in the ovulos. They observed that in kinetin-treated ovules, 10 days after culture, the embryos had grown to a length of 450 p exceeding that attained by embryos in wivo. The cotyledons and the stem tip were also well developed. This initial rate of growth was, however, not maintained, so that the final length of the mature embryos (640 p ) in cultured ovules was less than that of the embryos which grew in the field controls (660 p ) . Using a 10% solution of sucrose PoddubnayaArnoldi (1960) successfully grew the ovules from pollinated ovariea of several orchids (Calunthe veitchii, Cypripedium insigne, DencErobium nobile, and Phaluenopsis scldleriana). I n such material she was able to trace the events commencing from the entry of the pollen tube up to the development of the embryo and also described the histochemical changes accompanying these stages. Work on the culture of the ovules and seeds of angioepermic parwittw is comparatively recent. It is generally believed that in obligafe root parasites such as Striga and Orobanche the formation of seedlings is dependent on some stimulants from the host root. Experimental verification of this point can be best carried out only through seed cultures. In dtrigu wiaticu, kinetin, certain other 6-substituted aminopurinee, scopoletin and 4-hydroxycoumarin (Worsham et al., 1969, 1962) and gibberellic’ acid (Williams, 1961) are said to replace the germination stimulant excreted by host roots. However, a aupplemontary treatment with kinetin is needed for the development of the

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shoots. Woraham e.t ad. (1962) also consider that although scopoletin and 4-hydroxycoumarin promote the extension of the radicle, the enlargement of the cotyledons as well us the growth of the shoot may nevertheless depend on a eupply of kinetin-like substances. In some experiments at the University of Delhi seed8 of Strigct euphwioick.8 were sown on White's medium supplemented with kinetin (10 ppm). In a few days they ruptured and released the embryoR, but instead of germinating into seedlings these proliferated and produced a mass of callus from the cotyledons (T. S. Rangan, unpublished data). Privat (1960) hm demonstrated that in Orobunche hederae a contact with the root of the host (ivy) is indispensable'for the initiation of the shoot. However, recent work on 0. uqyptima (Rangaswamy, 1963) has demonstrated that the differentiation of shoots can be induced in the absence of any stimulus from the host root (Fig. 9). Chopra and Sabharwal (1963) have recorded a beneficial effect of the placental tissue on the growth and maturation of the seeds in Qymndropeis gynandra but not in Impatiens balsamina. I n Opuntia dillenii also Sachar and Iyer (1959) observed that the ovules showed no growth on any of the media even when they were cultured along with the placenta. However, on a medium containing both IM and kinetin, the placenta sometimes proliferated to form a callus of limited growth. According to Niimoto and Sagawa (1961) the formation of a zygote is a prerequisite for the further growth of the ovules in vitro. In Dendrobz'umphalaenopsie, for example, they observed that fertilization did not occur oven when the placenta was excised with its ovules and pollen tubes and then planted on the culture medium. In this connection the experiments of Kanta el al. (1962) on in vilro fertilization are quite significant (see p. 235). Kazimieraki (1963) has reported that unlike Lupinus luteus, L. rothrrutleri produces much smaller seeds and is characterized by several other desirable qualities. In attempts to transfer these features into L.lulew,he observed that no well-developed seedu were obtained in the croae L. luteus xL. rothmleri whereas in the reciprocal cross ~ome viable seeds were formed although their number was negligible. The hybrids produced only about 20y0 fertile pollen. The high sterility waa mainly due to the failure of the ovules to form functioiial embryo mca. Ovule culture may possibly help in solving such problems. It may be worth while t o excise the ovules or the entire placenta bearing the primordia of ovules and nurture them up to the stage of the mature embryo sac and then to bring about fertilization in uitro. Ovule culture may also prove useful in the artificiai induction of parthenogenesis. A direct handling of' the eggs of angiosperms is by

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no means easy because of the problems involved in removing them without injury. However, there is no such bar to the use of unfertilized ovules. While ovules excised at the zygote or 2- to C-Celled proembryo stage can be reared in zrilro (see p. 268), unpollinated and unfertilized ovules have not proved amenable. According to Ryczkowski (1962a), a knowledge of the changes in the osmotic value of the o d e during its growth and development may prove helpful in understanding the requirements of excised ovules in cultures. He found that while in early stages the osmotic concentration of the central vacuolar sap of the ovule showed an increase, with the further growth of the embryo and especially the elongation of the cotyledons the value decreased considerably.

VZTI. CULTURE OF OVARIES AND FLOWERS La Rue (1942) waR the first to initiate the technique of culture of ovaries and observed root formation from the pedioels. Jansen and Bonner (1949) grew the ovaries of Lycopersicum pimpinellifolium on a medium supplemented with casein hydrolysate, IAA and a mixture of some B-vitamins. Ovaries with an initial diameter of 1 mm enlarged to a diameter of 4-8 mm and developed the pigment lycopene. However, no viable seeds were obtained. Nitsch (1949, 1951) cultured the ovaries of Lycopereicum mulenturn, Cucumis anguria (gherkin), Phseolue piulgaris (bean), Fragaria sp. (strawberry), and Nicotiana tubacum, excised two or more days after pollination. On media supplemented with 2,4-D or 2,4,5-Tor NOA even unpollinated ovaries of Lycopersicum esculentum formed smrtll but seedless fruits. Nitsch, concluded that certain growth substances can replace the stimuli of pollination and seed development on the growth of ovaries into fruits. Since the development of fruits in dtro followed a normal pattern he suggested that the technique of ovary culture could be used a tool for studies on fruit growth. Fro. 9 (See facing page). FIG. 9. In oilro growth of emhryoR of Orobonck aeqyptiuca (0, callus; end, endosperm; p, proliferation; t , testa). A. Whole mount of embryo excised from a seed grown for 2 weeks on a modified White’s medium+ caeein hydrolysate (a00ppm.). The radicle end shows some proliferation and the remainii portion has taken a deep stain with cotton blue in lactophenol. B. L.S. seed from a 3-week-old culture showing further proliferation of radicle end of the embryo. Some of the cells along the margin8 and plumular end appear necrosed. C. L.S. shoot apex differentiated on callus. Note apical dome, scale leaves, leaf primordia and proveacular strand; there is no indication of a root. D. 2-Month-oldsubculture of embryo callns on White’s medium+ casein hydrolysate (400 ppm)+ coconut milk (16%). E.Microtome preparation of part of callus showing “internal” divisions (after Rangaswamy, 1963).

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With a view to studying the effect of chemicals on the pattern of development of the embryo and endosperm Rau (1956) excised the ovaries of Phlox drummondii one day after pollination and cultured them on Nitsch’s medium. Both embryo and endosperm showed a normal development. If the cultures were made on a medium containing colchicine (0.1576) the endosperm nuclei showed aberrant divisions. Nuclear fusions and aggregations were common and eventually the endosperm degenerated. The embryo also showed certain cytological abnokmalities, although the general pattern of its development was normal and its size was also larger than that of the embryos in ovaries grown on the basal medium, If the ovaries were retained on the colcliieine niedium for more than 12-14 days, the seeds became malformed and the embryos aborted. Since young proembryos are l e s ~amenable to excision and their nutritional requirement8 are also not well understood, RBdei and RBdei (1955) cultured whole ovaries, flowers and spikelets of Triticum aestimm and T.apelta, excised 4-43 days after pollination, on a nutrient niedium containing yeast extract. If ovaries deprived of all the floral envelopes were cultured, the embryo8 seldom continued to grow. When ovaries enveloped by two paleae were cultured the embryo furthered its normal development. The development of the embryo as well as the caryopsis was better when both rachillas and paleae were retained. After growing the ovaries for 8-12 days, R6dei and RBdei excised the etnbryos, which hnd sufficiently progressed in growth by now, and cultured them on a medium supplemented with casein hydrolysate (0.50/,). The embryos continued their development and eventually germinated to form nornial seedlings. The experiments of R6dei and RBdei suggest that the floral bracts and rachillae have an iniportnnt role in fruit development. That the floral envelopes are not unessential organN but play a significant role in fruit development is aLo borne out by several other investigations. Chopra (1968, 1962) studied the effects of IAA, IBA, kinetin and GA8 on both unpllinater! tLnd poliinated ovaries of AZthuecc rmea cultured on Nitsch’s medium. On a medium supplemented with IRA (20 ppm) the unpollinated ovaries developed into parthenocarpic fruits comparable in size with those which developed in nature although the seeds were devoid of any endosperm or embryo. The addition of kinetin (0.5 ppm) or IAA ( 5 ppm) had no pronounced effect. However, when used in conjunction kifietin and IAA had a synergistic effect in inducing parthenocarpy. It is important to note that even the growth of the pollinated ovaries W&B considerably affected by the calyx. If the calyx was severed before implanting the ovaries in culture, the fruits p w to a diameter of only 12 mm, the endosperm remained free nuclear, and

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the embryo reached only the heart-shaped stage. On the other hand, when the calyx was left intact the fruits enlarged to a diameter of 19 mm, the endosperm became cellular. and the embryo showed its full development. Maheshwari and La1 (1958, 1961a) cultured the flowers of lhericr a m r a on Nitsch’s medium supplementod with the B-vitaminll. Flowers cultured one day after pollination produced normal fruits and the embryos grew to maturity. However, this was possible only when the calyx was not removed before implanting the flower hi the medium. If the calyx was removed, it was necessary to add 5% sucrose in order to obtain a satisfactory growth of the ovary, but even then the embryo did not grow as well as when the calyx was left intact. However, when the flowers were collected more than 8 days after pollination, the removal of the calyx did not affect the growth of the ovaries in vitro (Fig. 10). La Croix et al. (1968) found that in barley the development of the proembryos proceeded normally in florets whose lemma and palea were kept intact. If these were detached the development was impaired. In cultures of excised spikes the growth of the proembryos was normal even when all the florets had been dehulled provided a single leaf was retained on the tlpike. La Croix et al. concluded that a “hull factor” is necessary for a proper development of’the embryo and it ig supplied by the tissues external to the ovary. I n the absence of the hull faotor the cells of the proembryo merely enlarged and even showed a duplication of the DNA (twice the level of the diploid prophase), but failed to undergo mitoses. Kinetin could not replace the hull factor, It may be concluded that ovary, flower and inflorescence cultures are additional means for tackling difficult situations in rearing the embryos to maturity . That developing seeds are rich in auxin and control the growth of the fruit has been confirmed by the observations on ovary cultures. Sachar and Kanta (1958) studied the effects of certain growth substanaes on the ovaries of Tropueolum majus. Ovaries excised two days after pollination were cultured on Nitsch’s medium supplemented with three B-vitamins and glycine. Although initially the growth rate of the cultured ovaries matched with that of others grown in the field, eventually the test-tube fruits were smaller even when the medium was supplemented with one or more of the following substances : biotin, colchicine, casein hydrolysate, kinetin, 2,4-D, CA,, IAA, TBA, tomato juice and yeast extract. Sachar and Kanta (1958) also studied the development of the embryo and endosperm in the ovules of ovaries grown on the basal medium. At the time of btarting the cultures the ovules contained a young pro-

Fro. 1 0 . Culture of ovwien of l b r r h ( m u r # . A. Ovsry grown an Nitnch’n I J I L H ~medium (9-wwk-oId). B. Similsr culture on Nitwcli’n medium+ vitamins+ IAA (5 pprn). C and I). OvHrieH grown on Nitwh’n rnetliuni+ vitiirninfi-t kinetin (0.5 ppm)+IAA (5 ppm). E and P. OvwieR grown on Nitnrli’s mctliurn+ kinetin (04 ppm)+ I A A (10 ppm). G. Culture showing growth of fruit in Nitwh’a medium 1 vitamins+ kinetin (0.R ppm)+IAA (5 ppm). The pedicel i# hypertropltietl; compare with fruit Hhown in E ;&ndF. H. Culture Rhowing growth of fruit in Nitach’s medium+ vitamins+ I,inctiii (0.5 ppm)+ titleniitr (10 ppm); only rut end of pedicel in hypcrtropliirtl (after Miihl.rliwiiri snd 1.1~1, 19fillt).

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embryo and a nuclear endosperm. Although both embryo and endosperm began to develop normally, signs of degeneration were evident by about the third week. The influence of growth substances has also been studied on fruit and seed development in Linaria ntaroccaru~(Sachar and Baldev, 1958). Pollinated ovaries cultured on Nitsch’s medium developed into fruits, but these were smaller than the field-grown controls. The addition of kinctin, or TAA, or IBA, or 2,4-D, or adenine improved the growth only slightly. However, if yeast extract was added to the medium the fruits not only attained the natural size but also reached maturity at a much earlier stage (15-17 days) than in nature (21-23 days). A few ovule8 also developed into seeds containing viable embryos. With a view to determining the nutritional requirements of developing fruits Guha (1962) cultured the flowers of AZZium c e p excised two days after pollination. On the basal medium there was only a 6 7 % seed set. The addition of IAA or GA, increased this to 20%, and if tryptophan was also supplied seed set increased to 30% (see also Johri and Guha, 1963).

Ovary culture has also been useful in inducing polyembryonic tendencies in Ranunculm sceleratua (Sachar and Guha, 1962), Anethum gravedens (Johri and Sehgttl, 196%) and Foeniculum vulgare (Sehgal, 1964) which otherwise bear monoembryonate seeds. A reference t o these is made in the section on polyembryony (p. 280). Dulieu (1963, 1965) grew unpollinated pistils of Nicotiana tabacum on a nutrient medium and 24 hr later applied pollen to the stigmas. It was found that the number of the ovules which were fertilized depended on the quantity of pollen applied. Under the best cultural conditions nearly 25% of the ovules were fertilized and 50 seeds developed per ovary. Such a low seed set was due to the degeneration of a large number of ovules both before and after fertilization. Some of the seeds germinated while still in the ovary and produced normal seedlings. To increase the percentage of seed set Dulieu excised the ovules 5 days after fertilization and cultured them on a fresh medium. While the endosperm grew normally, the embryo attained only the heart-shaped stage even after 45 days, It is well known that double fertilization stimulates not only the formation of the embryo and endoeperm but alxo the development of the fruit. Even in most apomicts pollination stimulafes the growth of the ovary and seed although there is no fertilization. The culture of the pistils of apomictic plants may, therefore, help to elucidate the nature of the stimulus provided by pollination. So far the only cultural experiments of this kind are on Aerva tomentom (family Amaranthaceae) which is an obligate apomict. Since there are no stctminate

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plants at Delhi, seed formation is independent of the stimulus of pollination. Puri (1963)studied the effects of IAA, casein hydrolysate, coconut milk and yeast extract on the growth of the embryos in cultures of ovaries, flowers, and portions of the spike. I n ovary cultures only 7% of the ovules developed into seeds and even these contained only poorly differentiated embryos. In flower cultures raised on the basal medium 15% of the ovules bore mature embryos. Yeast extract and casein hydrolysate increased the seed set to 20%’ and with coconut milk 26% of the ovules matured into seeds. The best response was elicited when portions of the spike were cultured instead of individual flowers. I n the presenoe of IAA and yeast extract the seed set was comparable to that in nature. Except for slight variations in size, the growth of the embryo and endosperm waH quite normal. In Zephyrantha which is also an apomict, Kapoor (1959) observed that ovaries, excised two days after pollination, developed into normal fruits on the basal medium itself. In the confinements of the culture vial ovaries often fail to grow into full-size fruits. To overcome this Ito (1961) devised a “partial sterile culture method” in which, instead of implanting the entire pistil, only the flower stalk is inserted into the aseptic nutrient medium through an opening in the stopper of the culture vial, and the ovary is left free to grow outside the vial. Using this technique Ito studied the growth requirements of the ovaries of Dendrobium nobile. The ovaries developed well on Nitsch’s medium containing only inorganic salts and a sugar. Generally, the disaccharides were superior to the monosaccharides, and among the former maltose and lactose were better than sucrose. Organic supplements were not indispensable, but vitamins B, and B, stimulated ovary growth, and tocopherol acetate (vitamin E) increased seed fertility. The seed set was best when all the three vitamins were used together. Coconut milk as well as peptone favoured fruit growth and also increased seed fertility. The partial sterile culture method is useful for studying the effects of variouR physical agencies on the growth and development of fruits, especially in plants bearing pedicellate flowers and in which the mature fruit is many times larger than the ovary. The floral meristem has been intorpretetl as a system of reactions which pass through several phases that correspond with the initiation of the floral organs (Wardlaw, 1957; Heslop-Hamison, 1969). To test this, Tepfer et al. (1963)used the culture technique. Flower buds of Aquilegia fwnwaa ranging from the pre-sepal t o the young carpel stage were excised and cultured on White’s medium supplemented with coconut milk, ten water-soluble vitamins, IAA, kinetin and GA,. When the buds were cultured a t the ‘%tar-carpel&age” the carpels

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surpaased both the stamens ailti the atamiiiodirt in growth (Fig. 11A and B). Nearly 26 days after culture the carpels had reached the maximum stage. The stamens and staminodia were poorly differentiated and later aborted. If the buds were cultured at “dimpled-carpel stage” the carpel8 were very well developed cbnd tho petals grew to about the normal oize for t h i H carpel stago, but the androeoium aborted (Fig. 11 C and U).When the buds were cultured at the “grooved-carpel stage” in 19 days after culture they reached the “erect-carpel stage” (Fig. 11 E and F). However, the stamens and staminodia aborted. In many cultures the carpel reached a length of 10-16 mm but showed no ovules. Fewer carpels differentiated in vitro than in buds on the control plants. The filaments and anthers differentiated but %hesporogenous tissue developed only up t o the pre-meiotic stage. The sepals developed in a manner similar to those in buds of intact plants. Tepfer d al. (1963)also observed that if the sepals enclosed the floral apex the inner floral whorls ceased to develop and when the sepals were removed the cther members resumed their growth.

TX. PARTHENOCARPY That fruits can develop without the act of fertilization (parthenocarpy) was first recognized as early as 1849 by Giirtner who obtained seedless fruits of certain cucurbits by “pollinating” the ovaries with the spores of Lywpodium. Massart (1902)placed dead pollinia on the stigma of some orchids and observed n r~wellingof the ovary. That some chemical substances might be involvud in the development of parthenocarpic fruits was inferred from the observations of Fitting (1909). He injeoted an aqueow extract of the pollinia into the ovaries of certain orchids and found that it not only prevented the abmission of the flowers but also caused a swelling of the ovaries. Kusano (1915) confirmed the observations of Massart and Fitting and also performed several other experiments 011 tho orchid Gastrodia ehta. Yasuda (1930, 1940) succeeded in producing pathernocarpic fruits of nearly normal size in several plants, especially those belonging to the Solanaceae and Cucurbitaceae, by treating the flowers with extracts of pollen. After Thimann’s (1934; see also Fukui el al., 1958) demonstration of the presence of indoleacetic acid in pollen, this and several cther growth substances belonging to the indole and the naphthalene group and the gibberellins have been utilized to induce parthenocarpy in a number of plants (see Guetafson, 1942; Maheshwari, 1960; Leopold, 1956). There are also reports of the natural o c c m n c e of auxins, gibberellins, and other substances of R similar nature in ovules, seeds, ovaries and fruits (Teubner, 1953; Luckwill, 1957; Von Bargen, 1960; Corcopn and Phinney, 1962).

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Many varieties of pear grown under low but not freezing temperatures produce an abundant blossom and yet fail to give a satisfactory fruit set. This is generally attributed to inadequate pollination and/or a failure of fertilization. Osborne and Wain (1961) reported parthenocarpic fruit formation after treatment with naphthoxypropionic acid. Ratjer and Uota (1951) and GriggA et al. (1961) considerably increaRed the fruit set in the variety “Bartlett” by treatments with 2,4,6-trichlorophenoxyacetic acid (2,4,S-T). However, similar attempts with other varieties were unsuccessful. Luckwill (1960) and Griggs and Iwakiri (1961) reported that GA, increased the fruit set in pears. The fruits resulting from gibberellin treatments had a more elongated form than the pollinated controls. Thompson (1963) used GA, and certain other growth substances on five varieties of pear. At full bloom, lanolin emulsions of GA,, IAA, IBA, and 2,4,5-T were injected, individually or in combinations, into the calycine cup. Applications of lanolin emulsions only (without any test chemical) resulted in an early nbscission of the flowera which could not be remedied by IBA. On the contrary, 2,4,5-T usually delayed abscission; whereas GA, promoted the retention of the flowers as well a8 the parthenocarpic development of the fruits. Crane (1963) investigated the effects of GA, on the development of fruits of peach. Aqueous solutions of a potassium salt of GA, (1000 ppm) were sprayed a t different stages after emasculating the flowers. Fruits which were obtained from treatments given after the petals had fallen matched very well with fruits developed in t h e openpollinated controls. With apples also the gibberellins have proved superior to other substances. Davison (1980) obtained seedless fruits in some four varieties by treating the flowers with gibberellic acid. After removing the petals, stamens and style a lanolin paste of 1% GA, was smeared on the cut surface as well as on the inner region of the receptacle. Thin induced the development of parthenocarpic fruits which compared well with naturally formed fruits in quality, colour and the time of ripening. FIG.11 (See facing page).

FIG.11. In vitro culture of flornl buds of Aquilegia fwmosn (cl-c5. carpels; st, staminode). A. C and E. Top view of buds from which sepals wcre removed before photography. B, D and F. Same buds after their growth on White’s medium+ IAA (O*6ppm)+QA, (2 ppm)+ kinetin (0.6 ppm) +coconut milk+ 10 vitamina. A. Bud at “star-carpelstage”; note the five carpels arrsnged in a pentarch manner; the crescent-shaped structures immediately next to the carpels arc the staminodes; and the remaining outcr whorls represent the stamens. B. Bud shown in A photographed 19 days after culture; all tho floral organ8 ere well developed. C. Bud at “dimpled-carpelstage’’ showing the dimpled carpels, stamene and etaminodis. D. Bud shown in C grown for 27 days in culture; only two petals and part of one sepal were retained for photography. E and P. ‘‘Croovc4cq-A RtUge’’ of bud beforo and 19 days afbr culture (after Tepfw el nl., 1988).

Fro. 11. Soe Icgcntl facing page.

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Dennis and Edgerton (1962) induced parthenocarpy in the McIntosh apple by applying a lanolin paste of potassium gibberellate to the emasculated flowers, Bukovac (1963) applied lanolin smeara of gibberellin A, to the cut surface of the style and the adjacent regions of the receptacle resulting in the development of parthenocarpic fruits. The gibberellins have elicited a similar response in several other rosaceous fruit crops belonging to the genus Pr?inzs.Crane et al. (1960) obtained a successful induction of prthenocarpy by spraying aqueous solutions of 50, 260 and 600 ppni of potassium gihberellate a t full I)loom or eight h y s after anthesis. With ttlmoncl arid npricot two applicatioiis of tho gibl)erellnte, ; L t ill1 intervid of eight tlitys, induced the fornitition of I)artlieiiocarl)i(.fruits. ‘l’lie peach rexp)ntletl best to all the colicelitrations of GA, irrespective of the nuiiiber of applications aiid the partlietiorarpic. fruits iiintc*lietl those formed naturally. However. the tretttnieiitq were ineficctive on cherry and plum. Rebeiz and (‘rane (19fil) sprayed tLqueous solutioiis of (;A3 alone or niethioniiie (2.4-DM) suppleniented with 2,S-dic~liloro~~Iienoxytloetyl on “Bing” cherry. However. the pnrtlienocnrpic fruits were smaller than the pollinated cwitrols. For strawberries Zielinski aiitl (hrren (19.38) reported that NAA (80 ppni) caused a 301:, increase in fruit set if sprayed two weeks after pollination when the production of auxin in the achenes reaches a peak. Lord t ~ n dWhite (1902) obtnined seedless strawberries by treating the emasculated flowers of five varieties with lanolin miears, solution smears, cqueous sprays. or eniulsions of naphthalene acetamide and IBA, iisetl indivictually or i n mixtures. However, the parthenocarpic fruits were not superior to the control berries. r i l h c genus Hosii c*oinprisesiii)otnictic*ILS well ILH noii-ai)otiiic:ticHpecieH. I’artheiioc,nri)y is i i iioriiinl feature in tlic fornicr hut not of the nonitponiicts. tJii(’kS0II tint1 1’1~sser( 1950) ctttcti1l)tetl to intlucc pnrthenociirpy iii the iio!i-iLl)oriiic‘tic.qx.icx : ii. uriarutiH, K.r?iVoeu nntl H.spinosiesima. Lanolin piistes of N A A , NAI) iLll(1 2,4,6-‘I’ were applied to emasculated flowers of field-grown plants. All treatments gave parthenocarpic fruits in H. ncqosu. However, 2. spinosiaeim was less responsive tliaii II. riigovo antl the best fruit set (W;:,)waH obtained with NAA-treatment. R . urvenvis wns the least responsive to any of the treatments. If’ the greenhouse plants of II. spinosiasima were treated with NAD, or UA,. or a mixture of both, parthenocarpic fruits resulted. Both NAD and (;A, gave W-70”0 fruit set, and a mixture of the two gave a fruit set of 9-j+:;,. Zatyk6 (19fi2a,b, 1963) induced parthenocarpy in GrossuZaria reclinata (cultivated gooseberry), in Hibes nibrum and R. niyrum (red and black currant) antl in some t w o interspecific hybrids of Ribee

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throiigh treatments with GA, and IAA. A pronounced synergistic effect of the two chemicals was observed in the gooseberry. Schroeder and Spector (1967) demonstrated a similar effect in the growth of pericarp tissues in vitro. Tn temperate countries serious lo~sesoccur in the glass-house pmduction of tomatoes during winter owing to a paucity of good pollen. To mitigate such losses tomato growers have resorted to the use of TAA, NAA and 4-chlorophenoxyctcetic acid all of which have been found very effective. Wain (1950) nhowod that a mixture of NAA and 4-chlorophenoxyacotic acid N i alro very useful. Pollination is not excluded in tomatoes ; the application of chemicals suppleme& the work of pollination and improves the fruit set. McGuire (1952) and later Dempsey (1962) showed that old pollen of tomato induced the formation of berries without participating in fertilization. Sell el aE. (1963) observed that rnethylated and ethylated esters and chlorinated derivatives of IAA wore more effective than IAA itself in inducing parthenocarpy in tomato. Some of the gibberellins have also been reported to be 500 times more potent than IAA in inducing parthenocarpy (Wittwer el al., 1957). Bukovac and Wittwer (1958) found that even very low concentration (3 x lo-%) of GA,, GA,, and GA, was inore effective than IAA. Weaver and Williams (1950, 1951, 1952) found that spraying of 4-chlorophenoxyacetic acid considerably improved the fruit set in the seedless varieties of Vitis viniferu. Stewart et al. (1958) reported that if GA, wns sprnyod eithcr at nnthesis or shortly thereafter it incremed the fruit set aB well as the size of the berrien. Similar results were alno obtained by R. J. Weaver (1968), Weaver nnd McCuno (1959), and Wittwer and Bukovac (1958). Pratt and ShaulL (1961) intlucecl parthenocarpy in grapes by tho application of gibberellic acid. In tho absence of pollination a B well as under conditions of inadoquato pollination GA, induced the dovelopment of oarly maturing, parthenocarpic berries. Among citrus fruitx the iiavol orange is naturally parthenocarpic, but Furusato and Suzuki (1955) were able to induce parthenocarpy in Citrus nataudaidai by a treatment with 2,4,5-T or NAA. Tn fig culture the quality of the fruits is dependent on caprification. Stowart and Condit (1949) found that spraying6 of 2,4-D and 2,4,5-T could induce the formation of seedless figs which were comparable to the caprified fruits in size and in the content of sugar. Blondeau and Crane (1950) induced parthenocarpy in the Calimyrns fig using aqueous or oil emulsion sprays of IBA, NAA, 2,4,5-T and para-chlorophenoxyacetic acid. IBA was effective at 200 ppm; NAA was increasingly so at 26-250 ppm; and 2,4-T (10 ppm) not only induced parthenocarpic

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developnmlt but also accelerated the maturation of fruits. The treatments with para-chlorophenoxyaceticacid (40, 60, 80 ppm) resulted in parthenocarpic fruits which matured normally and were comparable to the caprified controls in size, colour and taste. Balasubramanyam and Rangaswami (1959) observed that the artificially pollinated ovaries of some varieties of Psidium quajma developed into profusely seeded fruits whereas those of “Allahabad Round” yielded parthenocarpic and seedless fruits. They attributed the development of the parthenocarpic fruits t o the action of a “pollen hormone”. On applying an aqueous extract of the pollen to the emasculated flowers nearly all the ovarie6 developed into seedless fruits which ripened earlier than those formed through natural pollinations. On making chromatographic analyses of extracts of the pollen and the ovaries of “Allahabad Round”, Rangaswami and Kaliaqerumal (1960) detected traces of an indole-like compound in the extracts prepared immediately after antheah and much larger quantities of the name substanco in 5-day-old ovaries of selfed flowers. Tho cornpound could not be detected in 20-day-old ovaries a8 well as in the seeded varieties. However, exogenous applications of neither IAA nor JBA were effective in inducing parthenocarpy . Instead, parthenocarpic development was obtained by treatments with N U , NOA and 2,4-D. Teaotia et al. (1961) and Shanmugavelu (1962) were able to induce the formation of parthenocarpic fruits through the use of gibberellic acid. Such fruits showed a granular pulp and contained more ascorbic acid than the normal fruits. The induction of parthenocarpy is of considerable value in the production of greenhouse cucumbers, since it helps in bypassing tho laborious process of hand pollination. Wong (1941) obtained parthenocarpic fruits in several members of the Cucurbitaceae and Solanaceae through treatments with NAA, IBA, potassium naphthalene acetate, colchicine, acenaphthene, sulphanilamicle and trimothylamino. Dzevaltovsky (1962) reported that while IAA, gikhorellins, and tho sodium salt of 2,4,5-T had little or no effect 2,4-1) (0-002”/) readily induced prirthenocarpic development of the ovaries of Homo mom how of the Cucurhiticeae. Yakar-Olgun (1962) induced parthetioc:arpy in Viciafaba by the application of a 100 ppm aqueoun nolution of (;Aa. The parthenocarpic fruits were larger than those which developed from the pollinated controls. Sachar (1962) studied the eReect of gibberellin6 and certain indole compounds on the self-incompatible Pereskia aculeata (family Cactaceae). Sprays of IAA (50-500 ppm) delayed abscission of the ovaries, but. failed to induce partbenocarpy. IBA (50-500 ppm) induced 15% fruit set ; with GA, (100-500 ppm) it was loo(:/, .

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Britten (1947, 1950) sprayed the dehusked ovarieH of Zaa vliays with an emulsion of 0.1% NAA. Trentnients given 24 hr before pollination stimulated the ovaries to develop part1:enocar~)ically. If the ovaries were treated after pollination tho incidence of parthenocarpy was less pronounced. Sachar and Kapoor (1959) found that both GA, and IAA were effective in inducing parthenocarpy in Zephyranthes. The parthenocarpic fruita obtained by gibberellin treatment excelled the untreated controls in size. Kinetin, as well as mixtures of kinetin and IAA, were ineffective. According to Nitsch (1952, 1963) parthenocarpy may be of three types ; genetical, environmental, and chemical or induced. The navel orange, several varieties of cucumbers and the cultivated banana may be cited as examples of the first kind. Sometimes environmental factors such as temperature can also cause a parthenocarpic development of fruits, Lewis (1942) obtained parthenocarpic pears by exposing the flowers to freezing temperatures. Yasuda (1934) made similar observations on Solunum mehgenu and Nicotianu tabacum. Many instances of induced parthenocarpy have already been cited above. According to Gustafson (1939) plants which have a high content of auxin in the ovary show a tendency to be naturally parthenocarpic. Muir (1942) reported that soon after pollination the auxin content in the ovary increases considerably (see also Lund, 1966). For the successful development of the fruit a supply of auxin is essential. In naturally parthenocarpic fruits this is met partly by the pollen and partly by the seeds, whereas in induced parthenocarpy the auxin is supplied exogenously. Thus, pollen has two chief functions: (a) participation in fertilization and (b) an aid in fruit development. This is s u p p o h d by the experiments of Nitsch (1960) on strawberry. If the fertilized achenes were removed the growth of the receptacle ceased. A removal of the achenes as late as 21 days after pollination also elicited a similar response. If the achenes were removed and lanolin pastes of NOA or IBA were applied in their place, fruits of normal size and shape developed. The investigations carried out during the last decade emphasize the importance of the gibberellins in the development of parthenocarpic fruits. Recent reports of the occurrence of gibberellins A,, A,, A, and A, in the seeds of Phaseolus muEtiJorus (MacMillan and Suter, 1968; MacMillan et al., 1961) and gibberellin-like substances in the ondonperm of apple (Nitsch, 1958) indicate that theBe substances play an important role in the growth of fruits. Of interest also is the report of the occurrence of a kinetin-like substance in apple (Goldacre and Bottomley, 1969). However, further studies are needed on the upecific role of the gibberellins and kinins in the growth of the fruit.

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x. 1’OLYEMURYONY As early as 1719 Leeuweiihoelr noted that orange seeds contained more than one embryo. Since then many other plants have also been found t o produce polyembryonilto seeds. Stradmrgor (1878) Rhowed that the additional eiiibryos usually originate from the maternal tissues like the nucellus or the integument (adventive embryony) and later protrude into the embryo sac, Subsequently, other sources of accessory embryos were discovered. Among these are a cleaving or budding of the zygotic embryo (cleavage polyembryony), an3 a development of the synergids or antipodals either parthenogenetically or after fertilization by a male gamete. A. ADVENTIVE EMBRYONY

Hatierlandt (1921, 1922) assumed that the degenerating cells of the ovule secrete some substances (“necrohormone”) which stimulate the neighbouririg cells of the nucellus to develop into embryos. He attempted to induce tho formation of adventive ombryos in Oencl-thera by pricking the ovules or by syuceziilg the ovary HO as to cmse a Blight damage t o some cells. I n one of the treated ovulecl he observed two embryos and considered one of them to be of nucellar origin. Bedemaim (1931) followed Haberlandt’s technique and obtained D 2-celled embryo in Mirabilis unijlora, but whether it originated from a nucellar cell or the egg wa8 not ascertained. Similar efforts by NBmec (19351, Doak (1937) and Ivanov (1938) on other plants were unsuccessful. Despite his careful and intensive experiments Beth (1938) was unable to induce adventive embryony even in Oenotheru lurnarkiunu which was the object of Haberlandt’s studies. According to some workers adventive embryos are the products of a “somatic fertilization” of nucellar cells (see Glushtchenko, 1956). They believe that when two or more pollen tubes enter an ovule, the male gametes are sometimes discharged into the nucellar celh and also fertilize them. However, this observation remains unconfirmed (see Maheshwari end Rangaswamy, 1958). The adventive embryos are of considerable importance to the horticulturist as they are genetically uniform and roproduce the genotype of the seed parent without inheriting the variations kJrOUght about by the chromosomal segregations during q~orogcnminor by the gene recombinations in fertilization. Adventive emhryon are, therefore, used for propagating selected varieties of some horticultural CrOIJS and methods of artificial induction of adventive embryony have acquired special significance. Van Overbeek et al. (1941) injected several chemical suhstances

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into the ovaries of Datura stramonium. A treatment with O*l‘:/b NAA or IBA produced in some ovules a multicellular, tumoroid structure from the endothelium. Similar results were also obtained by Chopra and Sachar (1957) with D. fastuosa. However, the tumoroid structures did not differentiate into embryos. Fagerlind (1 946) reported that if II 1 (;(, lanolin paste of heteroauxin was applied to unpllinated pistils of Hosta they produced young adventive embryos. However, owing t o the lack of an endosperm the embryos failed to develop further. In many species of Citrus nucellar embryos are common. Furusato (1953) studied the possibility of controlling their number in C. natsudaidai and C . unshu. When a solution of maleic hydrazide was injected into young fruits the seeds showed a reduced number of embryos, sometimes only one per seed. More critical studies are, however, necessary to determine whether the treatments had any tjelective effect on the zygotic embryo or the embryos arising from the nucellus. In recent years the technique of tissue and organ culture has been used as a tool in studies on polyembryony. Rangaswamy (1961) excised the nucellar tissue from the fertilized ovules of C. microcarpa and cultured it on a modified White’s medium supplemented with casein hydrolysate. The nucelli proliferated and formed an exuberant callus. The callus differentiated into embryo-like regenerants (designated as “pseudobulbils”) which eventually developed into plants (Fig. 12). Similar observations have also been made on a few other species of Citrw (Sabharwal, 1963). These investigations demonstrate that if the nucellar tissue is freed from the confinements of the ovule and grown on an appropriate nutrient medium it can be activated t o unlimited growth and induced to yield a continuous supply of nucellar embryos. As yet, this has been found to be true of the nucelli of only such Citrus species that are naturally polyembryonic. The nurturing of the nucelli of monoembryonic species on the nucellar callu8 of a polyemhryonic Citrus is a line for future investigation. The zygote is a unique cell since it carries the genetic c:omplcment necessary to build a new individual. Recent rexearch ha8 s h o w n that the capacity to produce a new plant hody may well he manifcxtcci hy alniost any living cell of the organism. Somatic c c h rdc~s(!dfrorn differentiated tissues such as the vascular parenchyma of‘rootn,rncclullary parenchyma of stems, leaf parenchyma, and even thc ccllx c ~ fthe endosperm (unpublished observations on Sunhlurn ulhum) have heen observed to form enibryo-like structures. Steward et uE. ( I $ % ) and Steward (1963) showed that full-grown plants of BUUCUB wrnto can be raised from its phloem cells. Iliscs of phloem were excised from the root and cultured on a nutrient solution supplemented with coconut r\

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milk. I n 80 days the explants increased 80-fold in weight. On trcbnsfer to shake cultures the tissue i i i n ~ sdiusorittted to form t~ suspciision of single cells. The individual cells developed into cell aggregates which typically resembled the early stages of growth of the embryo. When transferred to an agar medium they established a root-shoot axis. On transplanting into the soil they gave rise to new and normal plants of carrot. Almost similar observations were made by Reinert (1959, 1963), Butenko and Yakovleva (1962), Kato and Takeuchi (1963), and Wetherell and Halperin (1963). Reinert (1959) found that on a medium containing coconut milk the callus tissue derived from cwrot root formed only roots. The differentiation of the root and the shoot could be controlled by changing the composition of the medium. In the presence of auxins (IAA or 2,4-D) root formation was inhibited, whereas in their absence the shoots and buds were inhibited. The roots generally arose as typical monopolar organs while the shoots originated from bipolar adventive embryos which differentiated in the callus. Butenko and Yakovleva (1962) observed that the growth and differentiation of the carrot root explants occurred in two phasee. On a modified White’s medium supplemented with the four RNA nucleosides, casein hydrolysate, coconut milk and 2,4-D, the explants proliferated vigorously and also showed the formation of buds. The second phase of development, namely the differentiation of roots and an active growth of the buds, occurred only when nucleosides and amino acids were both excluded from the medium. A transition to the second phase was stimulated by introducing into the medium antimetabolites of nucleic acid, of protein, as well as of auxin metabolism. Kato and Takeuchi (1963) reared callus tissues from excised root discs of carrot cultured on an agar medium containing yeast extract ( O . l * a ) and IAA. The callus showed two patterns of differentiation into embryos and their development into seedlings. Initially the callus was orange yellow. In one pattern a colourless tissue emerged along its periphery (40 days after culturing) and showed several nodule-like growth points (Fig. 13A and B). Some of them already exhibited a root primordium (Fig. 13C). In another 20 days a shoot primordium also originated in the nodules and completed the root-shoot axis (Fig. 131)). In the second pattern of differentiation the friahle callus rliseociatcd into single cells and small cell aggregates in about four months after culture. Through successive stages of differentiation resembling those of normal embryogenesis the cell colonies established ~~lantletrl (Fig. 13E-J). When a single cell was cultured in complete isolation

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on a fresh medium it failed to manifest the totipotency, but on a medium which had previously supported the growth of the callus tissue or when several of them were nurtured in a mass, differentiation occurred suggesting the normal pattern of embryogenesis (Fig. 13E-I),

Explant from tap root

Seedling

formation

\/A Torpedo- shoped stage

Production of roots

/

t Heart-shaped sloge

Formotion of shoot; also isolation j f single cells

Globular stage

Late globular stage

FIG.13. Formation of seedling^ from root disc8 of Daucu.9 awolr~culturctl on Wltito'n agu medium flupplemcnted with yeaat cxtrtwt (0.20/,)mil JAA ( I or 1 0 pprn). A,I$,~;,IJ und J represent one pnttarn of devehpmcnt of Iihntlatn. 1G.J iridii:8ktf>tho nc:c:ortd ptterrt in which embryo8 are organixc!rl prior t o formation of pliintlotn (after K i i t ~ ,and 'Ikkr:ur+ti, 1 !)fcjI.

ThurJ, multicellular units (nodules) as well as siriglc cells U'CI'C h J t h capable of producing plantlets (see also Mackawa ct al., 1968). Wetherell and Halperin (1963) cultured the root tissue of' thc wild carrot on an agar medium of semi-solid consistency. 'J'ho tissou p r Y J -

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duced a callus capable of continued growth. Portions of this were transferred to liquid cultures which were put on a rotator. Ten days after transfer 50-100 embryo-like structures developed in some of the cultures. These ranged from globular embryos (0.2 nun in diameter) to torpedo-shaped embryos. Subsequently, they devclopcd typical roots and green cotyledons. On transfer to a moist agar substratum containing only the mineral components of the ciilture medium, the plantlets developed into adult plants. Bearing close relevance to the above reports of the totipotency of carrot root cells are the recent observations of Bergmann (1959, 1960)

FIG. 14. Formation of embryo-like Ntructurw olinervcd in cell culturoa tlerivcd from pith pnrenehyma of Nicofiana h h ~ c u m(hfter Ibrgmann, 1851)).

on Nicotiana tabaczim and of Wadhi and Mohan Ram (1964) on Kalanchoe pinnata. Bergmann raised callus tissues from portions of the stem of Nicotiana cultured on White’x medium xupplemented with 7% coconut milk and 0.5 ppm 2,4-D. When the callus wax grown in liquid shake cultures it dissociated into small clusters and xingle cells. Suspensions of these were plated on an agar nutrient medium in petri dishes. Several cells showed active growth and gave ri8e to Glamentous structures some of which were organized like proembryos (Fig. 14). The leaves of Kalanchoe pinnata bear the so-called “foliar embryos” in their marginal notches, With a view to ascertaining their morphogenetic behaviour, Wadhi and Mohan Ram excised the leaf notches and cultured them on a modified White’s medium containing coconut milk and 2,4-D. The explants produced a callus tiswe which

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eventually differentiated into structures reminiscent of proembryos. Whether these can estnblish themselves into plnntlets st’illremains t o be investigated. A reference may also be made here to the investigations of Ito (1960) on the protonemata and prothalli of Pteris vittata, and of Schroeder et al. (1962) on the pericarp of Persea gratissima (avocado). By pricking with a fine glass needle Ito killed all but one cell in some mono-layered portions of the gametophytes of Pteris vittata and cultured them on Knop’s agar medium. Each cell isolated in this manner promptly regenerated into i t normal gametophyte. Schroeder et al. (1962) observed that in explants of the pericarp of avocado cultured on White’s medium the callus tissue developed typical roots. Undoubtedly “the apparently unlimited growth in culture of a tissue definitely limited in its natural growth” is of much interest t o morphogenesis. That an already organized tissue like the vascular parenchyma of roots, or the nucellus, or the leaf parenchyma, or the medullary parenchyma of stems, or the fruit parenchyma can be made t o proliferate and then again differentiate in culture completes the cycle of differentiation--,dedifferentiation=redifferentiation. This confers on the somatic tissue a capacity simulating that o f the zygote. Being a potential and continuous source of adventive embryos such tissue “banks” acquire a special interest for physiologists and geneticists (see Maheshwari and Rangaswamy, 1963). Whereas the artificial induction of adventive emhryos has undoubted advantages, at times i t is necessary to eliminate them. For example, there are obvious difficulties and uncertainties if the breeder has t o deal with a mixed population of seedlings, and hybridization experiments would indeed be rendered unfruitful if the nucellar emhryos suppress the zygotic embryos. If methods could he devised for a selective elimination of either one or the other kind of emhryos, it would be an important contribution to our techniques of plant improvement. B. EMBRYONAL BUDDINU

Like adventive embryos, identical twins and Nuper-twins resulting from a cleavage of the zygote are also of great interest to geneticists and plant breeders. Such cleavage polyembryony is common in gymnosperms but is much less frequent in angiosperms (see Maheshwari and Sachar, 1963). A plant which is of interest in this connection is Erardhis hiemlis (family Ranunculaceae). At the time of shedding (April-May) the seeds contain an undifferentiated embryo which is pear-shaped and possesses

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a long suspensor (Figs. 15 and 17). The radicle as well as the cotyledons differentiate several weeks later while the seed is in soil. Haccius (1955, 1957, 1963) investigated the effects of certain antimitotic substances on undifferentiated embryos. The seeds were soaked in solutions of the test chemicals for 24-96 hr imtnediately after shedding. In treatments with a O.loA, solution of 2,4-D, or 2,4,6-trichlorophenoxyacetic acid, or NAA, Haccius (1955) obtained many abnormalities such as syncotyly and pleiocotyly in nearly lOOY, of the embryos and twinning in 3-80,; of them (Fig. 16 A-C). Occasionally,the embryos

Fra. 16. Time schedule showing development of embryo of Emnlhis hipmulis after shedding of seeds. H+R, hj’pocotyl and radicle; L, lamina of cotyledons; S, cotyledonary sheath; and V, plumule. At the timo of shedding (May) the embryo is 0.1 mm. in length and undifferentiated. Further devaiopment takes place after shedding (after HacciuR, 1963).

showed a one-sided regeneration (Fig. 16D). It is explained that owing to the treatments ttie plumule uas damaged and the cells which were normally destined to produce the cotyledonn gave rise to ittldithrtl stem tips. In another set of experiments the frexhly harvested xeetls were treated with isopropyl-N-phenylcarhamate (0-2‘%,)and maleic hydrazide (O.l”/b). Four treatments administered within 24 hr caused a necrosis of the embryo yhich now appeared like a brown s p t . After a couple of months new regenerants originated from the suspenwr end of the necrosed embryd. If the same treatments were given 3 days after the shedding of the seeds, the development of the original ernhr.yo was arrested but there was no regeneration. Recently, Haccius (19639 has reported that the original embryo died if the test solutions were acidic. Xo effect was observed between

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pH

5 and 7 , but 5004 of the embryos treated a t pH 3.5-4.5 showed a marked basipetal necrosis and after 2-3 weeks only the suspensor cells remained alive (Fig. 17A-C). Tn due course these were re-embryonized and gave rise to embryoids. Initially the suspensor enlarged and became club-shaped due to initotic activity in its uppermost cells, and its connection with the necrosed embryo became severed (Fig. 17D). Eventually, a new bipolar embryo was reconstituted which showed

E

F

C

G

FIG.17 (see frrciny page). FIG. 17. Eranlhis hiemstlie (I), bud; dc, degenerating embryo; e, embryo proper; re. regenerating embryo; s, suspensor; v, plumule). A. L.S.part of secd at time of shedding showing undifferentiated embryo and elongated suspcnsor. B. L.S. seed 18 dayn after acid treatment; the embryo is necrosed but the cells of the suspensor immediately below i t have been activated. C. As in B, 22 days after treatment; the suspensor ha8 heCIJme club-ehaped owing t o the enlargement of the cells a t its upper end and the degenerated embryo has heen pushed aside. D. L.S. seed 6 weeks after acid-treatment showing prominent activity of the suspensor. E. As in D, 10 weeks after treatment. The new embryo is well developed and also shows a lateral bud (b). The remnants of the parent embryo have been completely pushed aside (after Hacrius, 1963).

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normal development (Fig. 17E). However, twins and nialformed embryos were also seen occasionally (Fig. 16E-H). Eunus (1955) X-irradiated the developing caryopses of Hordeurn vulgure and studied its effect on the growth anti development of the embryos. Generally, young embryos were more sensitive to the treatment than old embryos. When the caryopses were subjected to high doses of X-rays (400-800r) the embryo showed certain regions of deeply stained meristeniatic cells. These gave rise to numerous proliferations. Their frequency was highest in embryos irradiated with 800 r. Further, the proliferations developed in larger numbers near the haw of tho c:otylcdon r ~ n din regions directly oppowite or close to the plumulc. Sonictimes thc prolifcrtLtioiirr ronernhletl tho plrrmulc. itwlf. In studies on hybritlizations of U. ccratowulu with nine ottior herbaceous species of Dulura McLean (1946) observed thiht the hyhritl embryos became arrested during their growth. They varied from a completely undifferentiated mass to a differentiated but often malformed structure. With a view to raising F, plants McLean (1946) excised the undifferentiated embryos obtained in the crosses between the male parent D . ceratocuulu and three female parents D.discolor, D. innoxia and D. leichhrdtii, and in the cross D. cerutocaula (9)x D . metel. The excised embryos were cultured on an agar nutrient medium containing malt extract (0.5%). They frequently formed an undifferentiated mass and produced several buds (“multiple growth”). In one instance (D. cerutocuulu xD. metel) as many as 106 buds were produced. Eventually the buds severed from the parent mass and developed into normal seedlings. The orchids have proved specially favourable for studies on embryonal budding. Curtis (1947) cultured the seeds of Vundu tricolor. On the basal medium they prcdocctl normal Heetllinp, tjut if the medium was supplemented with harhituric acid or its sodiurn sdtn the embryos proliferated into a large numher of c.hkmq)hyllous tistme masses which readily lent themselvew to su t)culturing. Enihryon o f n

FIQ.18 (see facing p a g e ) . FIG.18. Culture ofembryos of Czrsculu rejnezn on a modified White’s medium (AE, accessory embryo; C, callus; NS. normal shoot). A-C. Stagm in germination of mature embryo. In home cultures the radiculsr end of the embryo callused and accessory embryos differentiated from it. D. Culture showing further growth of callus; arrow indicates an accessory emhryo. E. T.S. portion of cultured embryo shoring the development of accessory emtJryoH. F. Culture of young embryo ahowing formation of callus and its differentiation into awcsnory rmbryos. G. A few accessory embryos; the one on the extreme right show8 the formation of hhoot. H. 10-Week-old culture of accemory embryo; note repetition of cyc le of formation CJf Hiipernumerary embryos (after Maheshwari and Baldev, 1962).

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hybrid Cymbidium also developed rdlus on a medirun supplemented wit11 0-l0i, peptone. Curtis and Nichol (1948) studied the further growth of the original explants of both the orchids. TWOpatterns of growth were observed in Van&. Explants of Cymbidium gave rise to five patterns which differed in the size of the individual lobes in the callus and the degree of development of the rhizoids. I n their morphology and anatomy the proliferating structures resembled the protocorms produced during normal germination of seeds. Further, they showed a capacity for. continued growth, and germination was generally inhibited. Occasionally, however, true seedlings originated in some cultures. Recently, Rao (1963) reported embryo proliferation and differentiation in an interspecific hybrid of Vanda. It may be noted that in some orchids the embryo possesses a natural capacity to cleave into additional embryos and this can be augmented under artificial conditions. As in orchids, in many angiospermic parasites also the embryo shows a remarkable capacity for proliferation and Hubsequent differenrqflvza tiation. Work done a t the IJniversity of Delhi on L'W~CU~U (Maheshwnri and Snltlcv, I M a ) , llendrophthoc. Ji~Zcatu (,Johri uiid Bnjaj, 1963) and Orohurbche uell.l/r,tiacu (Rangaswemy, 1963) are some examples. ~ were When young embryos (0.5-1-0 mm in length) of C U S C Ureflem cultured on an agar nutrient medium containing casein hydrolysate and IAA,they became greatly swollen and ultimately produced a mass of callus from the radical end. Within six weeks after culturing the resultant callus differentiated into small whitish or yellowish-green bodies resembling normal embryos (Fig. 18F-H). If larger embryos (1.0-2.5 mm in length) were cultured, they developed into wedlings in about a month (Fig. 18A-C). However, some of them formed a maus of tissue a t the radical end which produced supernumerary e m h r p similar to those originating on the callus from younger embryOS (Fig. 18D and E). On transfer to fresh medium the accessory embryos formed normal shoots, but if they were retained on the ' ' ~ l d "medium FIG.19 ( S e e facing page). FIQ. 19. C'ultllre O f Cmbrj'OS (If Ilerrrlrophlhos j f 4 h i 1 l l ( I L O , l&l!l'l:HHi)ry(!Ill IJQ'~J; ill, III'I'I~UMJ~~ leaf; c, d u n ; h, holdfant; pl, pluinular J i d ; r, rudir:ulrrr ond; N, nunrx!riHiw). A. 31atuw crritirycl. B. 10-Week-oldcmbryo grown on N'hitc'H rnetliurn+ I A A (1 ppm)+ ymxt extrnr+ (500 pprn); the cotylcdons and the rudiculiir cnd hrrw prolifcrutetl. C. 20-Wcck-old ~:ILIJIIH c*ultiv:rted on White's medium t IAA (0-5 ppm)+ casein hydrolysatu (500 ppm) Nhowirig w w r a l pupillate structures. D. 20-Week-old seedling showing one plurnular and mverul ncccxaory leaven, and a massive holdfast. E. Globular proembryo. F mid G . 3- and 6-week-old cultures nhowing the differentiation of accessory embryos from the tLalll:s.H. 10-Week-old pdyernhryona1 muss (after Johri and Bajaj, 1903).

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they gave rise to a tissue which was capable of repeating the cycle of polyembryony. Somewhat similar results have been obtained with Dendrophlhoe falmtu (Johri and Bajaj, 1962, 1963). Differentiated embryos (4-8 mm in length) usually formed normal seedlings, but occasionally the radical end or the cotyledons produced a callus or buds of limited growth (Fig. 19A-D). If globular embryos (0.8-1*5 mm in length )were cultured on a medium supplemented with casein hydrolysate, they proliferated abundantly and the callus gave rise to several accessory embryos (Fig, 19E-H). On subculturing, these produced normal seedlings. Like the embryos of Cuscuta and Dendrophthoe (stem parasites), those of Orobanche aegyptiaca (a root parasite) have also shown a considerable capacity for proliferation (Rangaswamy, 1983). Tn seed cultures the ovoid, untiifferentjatetl embryos pro(lucct1 a massive ~t~llris ay)able of continuou8 growth. Several shoot tips arose from this, but there was no evidence of root formation (see Fig. 9). These investigations suggest that under cultural conditions the embryos of orchids and parasitic angiosperniu have a remarkable capacity to produce tissue masses of unlimited growth which can, however, be made to yield embryonal buds and seedlings. Recently, Steward et al. (1963, 1964) have reported that the cells of immature embryos of a wild carrot (“Queen Anne’s Lace”) express their totipotency more readily than do the mature phloem cells of the root. The excised embryos were grown on a liquid medium containing coconut niilk. Here they proliferated and formed a dense suspension of cells. When this was spread on an agar medium in petri dishes the resulting growth comprised some large, vacuolated undifferentiated cells and a larger number of embryo-like forma termed as “emhryoirls”. These passed through the globular, heart-shaped, torpedo and cotyledonary stages of’ embryogeny and eventually produced mature plants (Fig. 20). In some instances polyembryony has been induced in cultures of ovaries. In Ranunculus scelerutus the ovaries were cultured to study the growth of embryos. The embryo usually developed r~ormallyo r 1 the basal medium (Sachar and Guha, 1962), but on a medium containing casein hydrolysate it showed a tendency to produce additional emhryolike structures from the hypocotyl (Fig. 21). More striking results have been obtained with Anethum pi:eolene (Johri and Sehgal, 1963a,b). When the ovaries were cultured thrce rlayH after pollination on a medium supplemented with casein hydrolysate, IAA and yeast extract, in 7-21?;, of the cultures the zygote underwent cleavage and budding to form 15-52 embryo-like regenerants which showed varying degrees of cotyledonary abnormalities. The Hwollen

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polyembryonal mass projected outside the niericarp and produced multiple shoots. If the regenerants were isolated and cultured they grew into normal plants. Recently, Sehgal (1964) has also obtained similar responses from cultures of‘ ovclriee of Foeniculwn uulgarp. Whereas the production of a callus has been reported from cultured embryos of many other plants, only those instances where differentiation has been achieved are of interest here in that they recall the cycle of differentiation, dedifferentiation and redifferentiation. They may

in suspension ,cultured in medium DIUS

Storage root

/2 md phloem

/

@

explants

Transverse section of the root

t 2 \

\

\

Embryoid from cultured

\

cells from embrvo

-@

\

FIG.30. Prom individual cells of carrot to mature plant: diagrammatic representation of the cycle of differentiation in culture. Cells from tho phloem explants of the root as well 88 those from immature embryos pass through a basici~llysimilar course of development to form embryoida, plantlets and mature plants (after Steward et al., 1984).

also serve as a possible means of abundant clonal propagation and may turn out to be of special value in such inetance8 where ordinarily vegetative multiplication is not possible. XI. PARTHEPI’OOENESIN In angiosperms the first cell of theembryo, i.c. the zygote, i~ a product of fertilization. However, t h i g in not true of nucellar emhryon ]lor ~f the haploid embryos. Sometimes the haploidn arc a consequenr:c of fertilization of the secondary nucleus without an accompenying fertilization of the egg. Or, the zygote and one of the synergide mny both develop resulting in diploid-haploid twins. Since the discovery of the first haploid in Datura etramonium in 1921 (see Avery et al., 1959) Huch occurrences have been reported in more than 80 species belonging to

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as ninny AS 18 families (see Kiinber i w i d Rilcy, 1963; Milgooil tin([ Khanna, 1963). In ciicumber Aalders (1958) noted that embryos measuring 4-6 mm in length were likely to be haploids; these were reared in witro for further observations. Haploid sporophytes are of special interest to the geneticist because by a mere doubling of the chromosomes a haploid organism can be made to yield a homozygous diploid. In this way, plants homozygous for self-incompatibility alleles have been obtained without resorting to a lengthy and difficult programme of inbreeding. Owing to the great theoretical and applied value of haploids, many methods have been tried to induce their occurrence. Although considerable success has been achieved in this direction in animals, particularly the sea urchins and some amphibians, in angiosperms the occurrence of haploid sporophytes is still a rare and uncontrolled phenomenon.* Various techniques, such as treatment with abortive or irradiated pollen, delayed pollination, distant hybridization, and physical and chemical treatments, have been employed to raise haploids. Although abortive pollen is useless for fertilization, it may sometimes stimulate the egg to develop into a parthenogenetic embryo and might also induce an autonomous formation of the endosperm. Honic examples of this kind are Pharbitis nil (U, 1932), Nicotianu ylutinosa (Webher, 1933), and Oryza spp. (Nakamura, 1933). X-Ray treatment has been often reported to result in the forniation of haploid embryos. Ehrensberger (1948) pollinated normal plants of Antirrhinum nmjm with irradiated pollen and found some haploids in the progeny. Natarajan and Swaminathan (1958) treated the seeds, seedlings and inflorescences of Triticum aestivum var. N.P. 809 with Among algae Hiroe and Inoh (1954) obtaincd chemical intliiction of parthc*nr)gc*ncninin 8arga5r~tnpilulijwum. La1 (1963) induced the formation of haploid (apogamouq) rrImroljhytcw

in cultures of callus tissues raised from the protonema of the mom Phy8wm~triumcwqetzse. The young prothalli of Pterie cillnla grown in cultures in dark proliferated into callus tiwirs which showed a capacity t o produce apogamous aporophyteb (Kato, 1963).

FIQ.21 (see facing p z g e ) . FIQ.21. Rearing of ovarie8 of h h u n e u l u s acckralus. A. Owrion excisctl ti (hysafter r ~ t ~ I l i i ~ ; i tion and grown for 14 weeks on Sitsch’s medium+ c:nsi-iri hytlmlynntc (I000 prim); notc: formation of aeedhgs. !A mirropylar portion of :r~~honc! ro:ircsl for 2 wc!i!kn oil Sitwli’s ~ ; medium+ casein hydrolysatc ( M H J ppm); ttjc IJrcJf!mbryrJ drown txginniny of ;I ( ~ I V I I wljili? the endosperm has aborted. C. L.S. micropylar portion of achcmc! rwmd fijr 2 w w k H fJflSitnr+i’a medium+kinetin (0.5.ppm)+ 1A.a (5 prim); thc: niinpmsor is tiy~x:rt.r~~ptiicsl icrirl thr. c.nclrj. sperm has degenerated. b and E. \Vhole mountn of cmtJryon oxcisc:ri from l l ~ ! h f ~ f rcarctl l~H on ;L medium rontaining casein hydrolysatc ( 3 J f J ppm); noto the fiwiittionn. 1’. I,.S. (mi bryo dissected from achene reared on Sitsrh’s medium +casein hydrolysate (5fJO ppm) stmwirlg formation of supernumerary rotyledons and budding (after Sachnr and Gutla, I!&?).

u.

Fio. 91. See legend facing mxe.

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different doses of X-rays and obtained a few haploid embryos in such cases where the inflorescences had been subjected to 5200r for 2 or 3 days prior to anthesis. Like X-rays, radioisotopes have also been utilized in producing haploid sporophytes. Using P32Arnason et al. (1952) discovered a few haploid individuals in Trilicum wdgure. Natartjajtjan and Swaminathan (1958) reported that was effective in inducing haploidy in wheat when the treatment was given just before the onset of microsporogenesis. Pai and Swaminathan ( 1 959) observed a nulli-haploid with 20 chromosomes in the progeny of an S36-treatedplant of T . aestiwum (2n = 42). Delayed pollination has also been observed to enhance the frequency of haploids in certain crop plants. Kihara (1940) states that none of the 41 individuals of T. monococcum which were pollinated 3-5 days after anthesis produced any haploids, but three haploids were obtained from 8 individuals pollinated 9 days after emasculation. Pollinations made in the intervening periods gave a frequency ranging from 0-37". Smith (1946) confirmed Kihara's findingH ; in his experiments the frequency of the haploids increased from 0.1-20% by delaying pollination up to about 12 days after emasculation. In some distant hybridizations, although the pollen may not effect fertilization, it can often stimulate the parthenogenetic development of the egg into an embryo. The investigations of Chase (1952) on Zea mays indicated that the freqnency of haploids increased 20-fold by the use of certain male parents. Coe (1959) isolated a stock of maize which produced about 3% haploids and showed that the pollen of this stock was effective in inducing haploids in other lines also. Tiihara and Tsunewaki (1962) described the production of haploids in TriticaZe and in the cross Aegilops mudata x Triticum aestivum. Yasuda (1940) reported that by injecting an aqueous solution of belvitan (probably indoleacetic acid) into ovaries of Petunia violacea, the egg cells were stimulated to a few cell divisions resulting in small proembryos. A treatment with colchicine was reported to prwluce haploids in Nicotiuna langsdqfii (Smith, 1943) and I j o h v t ~ l y a r i x (Levan, 1945). Deanon (1957, see Magoon and Ktianrin, 1 Y M ) tre;Lf(:
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only. Huskins (1948) found that in roots of Allium cepa, which developed from bulbs grown in a solution of sodium nucleate (14%) for a period of 3-36 hr, reduction divisions and haploid nuclei occurred frequently. Similar investigations were made by Wilson and Cheng (1949) on Trillium erecturn, T. ovaturn and T. sessile, and by Patau and Patil (1961) on R b e o discolor. Haploid c e b have also been reported to occur occcisionally in root meristems of seedlings of Hordeurn vulgave and Triticum mnmoccurn raised fmm X-irradiated seeds (Swaminathan and Singh, 1968). During their studies on the effect of irradiation on sex expression in Citrullw vulgaria (2n = 22) Swaminathan and Singh (1958) found that a whole branch of a plant raised from an irradiated seed (48,000r) showed only the haploid number of chromosomes (2n = 11). This branch produced only one pistillate but many staminate flowers. Swaminathan and Singh presumed that the X-irradiation probably caused somatic reduction in the meristem from which the haploid branch originated. Somatic reduction has also been reported to occur in callus tissue cultures of certain plants such as H q l o p p p w (Mitrtr, et at., 1960). Since somatic reduction occurs in cultured c e b and because single cells are known to exhibit totipotency, there is reason to hope that this may be a way of obtaining haploid plants and then a homozygous diploid progeny. Little is understood of the causal factors responsible for the development of the unfertilized egg. That agencies like X-rays, colchicine* and foreign pollen are effective only in certain but not in all instances suggests that the role of the genotype is also significant. Further, the frequency of haploidy is variable in different species and sometimes even within clones of the same species. This increws the importance of selection of desirable genotypes suitable for a high frequency of haploids. However, the paucity of a technique for easily producing gametic chromosome constitutions a t will has been the greatest limitation t o the wide use of haploids. Even granting that a parthenogenetic development of the egg can be induced by artificial methods, there is still a serious handicap if the caretaker of the embryo, namely the endosperm, does not develop in the embryo sac. I n other words, a successful induction of parthenogenesis would involve two steps: (a) normal cell divisions in the egg, and (6) formation of a nutritive tissue, whether it is endosperm or an aawptable substitute. A recent report on the culture of the unfertilized eggs of HorcEeum aativum (Walker and Dietrich, 1963) is of much interest. The eggs were excised and cultured in micro-chambers by the *~Cblobioinehas been generally effective in inducing chromoeomal duplication, but the reporta of Smith (1943) and Levsn (1945) that its application resultad in the formation of heploidn are intemting. S

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hanging drop technique, and the effects of kinetin and adenosine triphosphate (ATP) studied under the phase contrast microscope. ATP caused a rapid vacuolization whereas kinetin induced cell divisions. XII. ANDROGENESIS The origin of a haploid embryo is generally traced to the parthenogenetic development of the egg or some other haploid component of the embryo sac. However, a few examples are known of haploid sporophytes which reproduce the characters of the male parent only. I n such instances the embryo is believed to be formed by the male gamete and the sporophyte is described as androgenic.*? Among earlier records of androgenic haploids are those by Clausen and Lammerts (1929) for Nicotiana digluta (2n = 7 2 )x N . tabacum (2n = 48), by Kostoff (1929) for N . tabucum (3n = 7 2 ) x N . langadbrJii (2n = 18), by Kostoff (1934) for N . tabacum aylvestria hybrid x N . sylve8tria, and by Gerassimova (1936) for Crepia tectorum. In all these instances the paternal haploids were the result of interspecific crosses and /or experimental treatments. Androgenic haploidy has also been induced in Antiwhinum mju.8 by pollinating irradiated flowers with normal pollen (Ehrensberger, 1948), and in N . glutinosa (2n = 24) crossed with N . repanda (2n = 48) (Kehr, 1951). From crosses between the natural tetraploid Hordeum bulbosum (2n = 28) and the artificially induced tetraploid H . mdgare (2% = 28) three F, plants resembled the male parent, H. VuZqare, in several features suggesting an androgenic origin (Davies, 1958). Haustein (1961)has reported an androgen of Oenotkra ambra. Campos and Morgan (1968) obtained an androgenic haploid in Capaicum frutemena in an attempt to cross two varieties differing in the charactem of their foliage’and fruit. Since the Fl showed feature8 of the male parent only it was concluded that a sperm had given riw to the sporophyte. It is well known’ that during syngamy the male and the female gametes unite, but only the female gamete transmits its cytoplasm to the progeny. In many instances of androgenic haploidH it is presumed that the male gamete continues to develop in the milieu of the cytoplasm of the egg cell. This has considerable appJication in plant breeding. For example, Goodsell (1961) reported that androgenesis in maize is useful in transferring the genotypes of inbred lines into cytoplasm that causes male sterility. For example, “Texas-sterile” is a male sterile strain and “Nebraska 6 (N,)” is an inbred line of Zm

* For haploid spomphytas originating from sperms, several term8 have been used in the literature : androgens, androgenic haploids, androgenetic haploida and paternal haploids. t Haploid sporophytea are not known among gymnosperms. In CepJialOtanu, drupaaa, however, Favre-Duchartre (1966. 1967) observed supernumerary divisions of one of the nperms inside the archegonium and interpreted it a8 the initiation of an androgenic haploid.

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m a p When Texas-sterile was used as the cytoplasmic donor (female parent) for the homozygous N,, it resulted in the production of a few androgenic haploids. Reoently, Chase (1963) demonstrated that an appreciable proportion of androgenic diploids originated when the cytoplaemic donor or female partner was a tetraploid. A method of producing androgenic haploids is very much to be desired but so far no specific technique is known. Of interest in this connection &rethe haploid tissues derived from cultures of pollens of Ginkgo biZoba, T m (see Tulecke, 1961),Torreya nucifera (Tulecke and Sehgal, 1963; 688 also Tulecke, 1964) and EpWra fo2icbtQ: (Konar, 1963). In E . fo2iata about-to-dehisce microsporangia bearing 4-Or &nucleate pollen grains were cultured on a modified Reinert’s medium supplemented with coconut milk (15%) and 2,4-dichlorophenoxyacetic acid (1-5 ppm). In 10-12 days tiny mames of tissue were obtained in 57% of the cultures. Histological observations revealed two patterns of growth: (a) the pollen grains first underwent transverse diviRions to form uniseriate filaments which later assumed a multiseriate form ; or (b) the pollen grains showed an overall enlargement followed by cell divisions leading to the formation of discoid multicellular masses. I n about 90 days after culture the pollen tissue increased nearly 614 times its original mass. This pollen tissue is haploid and undifferentiated and haa shown considerable capacity for continued growth. If haploid pollen tissues could be made to differentiate into embryos, it would open a new area of research. No angiosperm pollen has, however, been known to yield a tissue. Yamada e l al. (1963)cultured stamens of Tradescartl&&rejieza, excised from very young buds, on White’s medium supplemented with a-naphthaleneacetic acid (0.8 ppm). A callus capable of subculture was formed and cytological obaervations showed it to be haploid (n = 12). Several cells of the callus also showed pollen tube-like protmions. According to Yamada et al. thia haploid callus tissue probably originated from the pollen mother cells.

XIII. ANTHER CULTURE During the last few decades many attempts have been made to culture excised sporogenous tissues and anthers. Shimakura (1934) grew the microspore mother cells of Trudemzntia at metaphase I in a solution of sucrose and found that 2 6 4 0 % of them formed tetrah. Gregory (1940) excised the anthers of Datura stramonium, Lywpereicurn emdenturn and LiZium longi-rn at various stagee of development and cultured them in nutrient solutions. Those of Datum and Lywper&um failed to continue their growth. The young anthers of LiZiurn excised

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at the archesporial or sporogenous tissue stage at fist showed similar growth rates both in culture and in vivo. However, the growth rate in vitro rapidly decreased at the time when meioses were to be initiated; the sporogenous ceUs lost their characteristic appearance and became elongated and vacuolated. If the anthers were cultured when the diplotene stage had been already initiated in the microspore mother cells the meiotic divisions were completed. Nevertheless, the microspore tetrads degenerated, and Gregory concluded that the initiation of meiosis is not vested in the mother cells themselves but is probably governed by an interplay of one or more materials transported to the anther. Taylor (1 950) studied the differentiation of sporogenous tissue and the formation of microspores in Tradescantia paludo8a. Anthers were excised at stages ranging from early sporogenous tissue (5-8 days prior to leptotene) to the completion of meiosis in the microspore mother cells. The nutrient media were supplemented with B-vitamins, coconut milk and IAA. He observed that younger anthers required a longer period to undergo differentiation in culture. Those excised during the formation of the sporogenous tissues showed a gradational development but failed to initiate meiosis. Anthers excised at preleptotene and leptotene grew only to a limited extent whereas those cultured at the zygotene-pachytene stage regularly completed meiosis. However, the development did not continue beyond the tetrad stage. When the anthers were excised at the tetrad stage the microspore nucleus divided, but even so the pollen grains failed to mature. In Trillium erectum Sparrow et al. (1955) excised the anthers at stages ranging from pachytene to diakinesis and cultured them on Taylor’s medium supplemented with coconut milk. An average of 76% of the anthers attained the stage of division of the microspore nucleus. Media supplemented with casein hydrolysate, yeast extract and glutamic acid were less satisfactory and in these an average of only 27% of the anthers progressed to the division of the microspore nucleus. An average of 22% of the anthers cultured at the pachytenc stage survived through microsporogenesis. If excined a t n Rlightly later stage, the anthers showed a much higher degree of Hurvival; at diplotenediakinesis it was 670/,. Vasil (1967, 1959) excised the anthers of Allium wpu and Rhow discoEo?.at the leptotene-zygotene and diplotendinkinenis ntagen. In Allium the davelopment progressed up to the one-celled microRpore stage on media supplemented with GA, and kinetin. In rndith Hupplemented with RNA almost all the microspore mother cells formed tetrads in Allium cepa as well as in R h e o discolor. However, in either case the development did not proceed beyond the one-celled microspore

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stage. Only when the medium was supplemented with the four nucleotides of RNA did the microspores develop into two-celled pollen grains (Vmil, 1963). Walker (1957) studied the effect of colchicine on microsporogenesis in the excised anthers of Tradescantia paludosa. In anthers cultured a t the pachytene-diplotene stage colohicine (0.01”/o) promoted the formation of dyads and tetrads. If the anthers were cultured at metaphase I in a medium containing colchicine (0.1%) this resulted in the formation of monads. Beatty and Beatty (1953) reported that a period of 7 days elapses between the initiation and completion of meiosis in T . plu&osa. Walker and Dietrich (1961a) showed that this period could be curtailed to less than 2 days if the anthers were excised at mid-diplotene and cultured in a sucrose medium supplemented with kinetin (0.26ppm). I n it sucrose-deficient medium there was a cessation of kinetin activity and a “meiotic stasis”, but this could be overcome by the addition of citrate. In a test involving a comparison between the two lobeA of the anther, Walker and Dietrich observed that the lobe treated with kinetin markedly surpaesed the untreated lobe in an acceleration of the prophase beyond pachytene. Walker and Dietrich (1961b) also cultured the anthers on a lactose medium. If cultured at the mid-diplotene stage for a period of 48 hours, the anthers went through the meiotic divisions but cytokinesis did not occur. An addition of kinetin did not improve the response; instead it arrested meiosis 11. When sucrose was tlubstituted for lactose the kinetin-induced stasis was overcome and wall formation occurred normally. Citric acid, uronic acids and amino-sugars also proved helpful. There is another question that arises from the experiments on anther culture. It is known that at least on some occasions the egg can develop pathenogenetically into an embryo. Is it possible to obtain something similar from pollen grains? Of special interest are the pollen-embryo Bacs sometimes observed in Hyacinthus orientalis (Stow, 1934) and Ornithogalum nulam (Geitler, 1941). Stow (1934) found that when the pollen-embryo sacs of Hyacinthus were placed on an agar medium together with some normal pollen grains, the pollen tubes formed by the latter coiled around the former. I n one instance a male gamete was observed entering the pollen-embryo sac and in another the pollen-embryo sac showed 10 nuclei, prefiumahly the products of diviaion of a triple fusion nucleus. Stow concluded that all pollen grains are potentially capable of antluming either the male or the female form and that whereas normally the “male potency” i R dominant, sometimes the “female potency” get8 the upper hand owing to A release of necrohormones. Naithani (1937) also ntudied plant8 of 8*

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H. orientalis which were treated for early flowering and confirmed that some of the microspores developed into pollen-embryosacs. He believed that temperature is the decisive factor in the formation of such structures. It would be of interest to repeak the experiments of Stow and Naithani by exposing aseptically cultured anthers to various physical and chemical agencies to see if in other plants also pollen grains can be made to form embryo sacs which can later be fertilized by male gametes from normal pollen grains. A reference may be made here to the observntions of Ram (1959) on Leptomeria billardierii. No normal pollen grains were observed in the anthers; instcad some of the sporogenous cells enlarged, became vacuolated and underwent three nuclear divisions to produce eight nuclei. These organized into embryo sac-like structures (Fig. 2'2). Such embryo sac-like structures originating directly from the sporogenous cells have not been reported in any other plant. Whether they can be induced artificially still remains to be investigated. Yet another problem which may be approached through anther culture concerns the Cyperaceae. TTnlike the majority of angiosperms in the Cyperaceae three of the four microspore nuclei of the tetrad degenerate and only one functions. Whether all the microspores of a tetrad can be made to develop into pollen grains in vitro remains to be investigated.

XIV. CONTROLOF SEX EXPRESSION One of the less understood facctn in plant biology is the expression of sex in flowers. From time to time several suggestiorm have been put forth to explain it, and recently Borne investigationn have been directod to assess the role of growth substances in sex expression. A few of these are discussed here. Mehndrium dioicum is a dioecious member of the family Caryophyllaceae. When the male flowers are infected with Uslilugo violacea, the anthers become filled with the spores of the smut and the formation of pollen is prevented. However, if the female plant is attacked, the flowers produce functional stamens. Love and Lave (1945) were able to duplicate the effects of the smut by treatments with animal hormones. When lanolin pastes of oestrone and oestradiole (female hormones) were applied to leaf axils of staminate plants, the stamens were partially suppressed and a pistil was formed in the flowers. Similarly, in the pistillate plants the male hormone testosterone suppressed the expression of femaleness and promoted the formation of stamens. That plant hormones control sex determination in flowers i8 a relatively new concept. Laibach and Kribben (19BO), Laibach (1952),

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and Nitsch et aZ. (1962) believe that the sex of the flower may be determined by the relative quantity of native auxin present at the time of the initiation of the flower. They made the interesting observation that in Cwurbita pep0 a treatment with auxin increased the number of pistillate flowers and decreased that of the staminate flowers. A pwte or an aqueous spray of IAA, NAA and 2,4-D was applied to the cut ends of the stalks of the first two leave8 of 16- to 18-day-old plants. There was a diminution in the total number of flowers but a definite

FIU.22. “Pollen-embryoeace” of LspsOrnerM billardierii. A. L.S. anther lobe showing two binucleata pollen-embryo eats developed from microepore mother oells. B and C. Squash preparation from anther ehowing organized pollen-embryoa c e (after Ram, lB59).

increase in the production of female floworu. Nitsch et al. (1952) also showed that if the plants were spraycd at the two-leaf stage with a 100 ppm solution of N U the first female flower buds were initiated even at the ninth node, whereas in the controh the Ihtillato flowcrn did not originate until 20 or mom nodes developed. Wittwer and Hillyer (1954) investigated the oxpreuHion of HCX in Cucumis sativus and Cucurbitu p ~ p oWhen . a solution of 100 ppm NAA, or 25 ppm 2,3,5-TIBA was sprayed at the two- or three-leaf utagc: the ratio of male to female flowers went down from 23: 1 to 8 : 1, and from 14: 1 to 2 : 1. In Cucurbitupep0 an application of NAA (100 ppm) af‘ter the unfolding of the first leaf resulted in a decrease in the ratio of male : female flowers from 1.47: 1 to 0.4: 1; and sometimes no staminate flowers were produced a t all. If N M was applied after the formation of four or five leaves, the treated plants bore only pistillate flowers for 8 days. Ito m d Saito (1957) also induced sex reversal in cucumber. The apical bud was removed and the cut end treated with 2,3,6-TIBA. Pistillate flowers arose at the lower nodes in place of the usual staminate flowers. Heslop-Harrison (1956) subjected plants of ~‘unnabissativa (dioec-

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ious) to a period of minimal photoperiodic induction (ten short days) and then studied the influence of NAA on sexuality. Immediately after the short day treatment, a lanolin paste of NAA (0.5%) was applied to the lower surface of the central lobe of one of the trifoliate leaves at the third node. Five days later similar quantities of NAA were smeared on both leaves at the fourth node. For interpreting the results all the NAA-treated plants were induced to flower 70 days after the beginning of the experiment. It was observed that in plants which were genetically male only female flowers had developed in sites which should have been occupied by male flowers. According to HeslopHarrison (1957) the differentiation of sex is controlled by the endogenous level of auxin in the regions adjacent to the floral primordium. The formation of the pistil may be favoured by high level of auxin in the vicinity of the differentiating primordium, and that of the stamens hy lower levels. Setyanarayana arid Rengab:wttmi ( 1959) inventigated the effect of 2,4-D, NAA and p-chlorophenoxyacetic acid (CLPA) on Luffa acutangula. The plants were sprayed at regular intervals from thc time of germination of the seed to the appearance of the first flower. CLPA induced an S070 reduction in the number of male flowers with a corresponding increase in the percentage of the female flowers. Yet another chemical which has been extensively used in studies on sex expression is gibberellic acid. Laibach and Kribben (1950) demonstrated that in the monoecious inbreds of Cucurnis sativus an application of NAA increased the female tendency whereas GA, decreased it. Wittwer and Bukovac (1957, 1968) reported that in cucumber gibberellins delay the formation of pistillate flowers but speed up that of the staminate flowers. Similarly, Peterson and Anhder (1960) demonstrated that GA, is effective in inducing staminate flowers on plants which otherwise bear mostly female flowers. Bukovac and Wittwer (1961) found gibberellin A, to be more effective than A,, A, and A, in inducing the formation of staminate flowers. Wittwer and Bukovac (1962) tested the effect of gibberellin8 A, to A, on exclwively pistillate plants.of cucumber. The chemicals were applied to tho fist plumular leaf at its emergencc and once again after a week. Tho number of plants that produced staminate flowers, the number of male flowers per plant, and the number of nodeb: bearing male flowern were taken as criteria for judging the effect of the test chemical. Gibbcrellin A, was the most active and gibberellin A, the lead in inducing rna1enosr;l. Gibberellins A,, A,, A, and A, had an intermediate effect in decreasing order. Since gibberellins A,, A,, A, and A, are structurally similar, Wittwer and Bukovac suggest that there is some relationship between the structure and degree of the biological activity of the gibberellins.

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In recent years the technique of tissue and organ culture has also been employed in investigations on sex expression. Petrt and htovsk? (1957) germinated the caryopses of some varieties of Zea mays on an agar medium. Prom 7-day-old seedlings the stem tips were excised and cultured on an,agar medium. They regenerated into new plants which were transferred to pots and grown in glasshouses, Tlicy produced only female infloreocences. In Cucumis sativuo gibberellins favoured the formation of staminate flowers (Galun, 1959). To investigate whether the growth substances have any direct control on sex expression Galun et al. (1962) made in vitro cultures of young floral buds (0-7 mm in diameter) of C. m t i m . Buds of potentially staminate flowers (borne on the eighth node) were excised from 20-day-old seedlings of a monoecious line and cultured on a modfied White’s medium. When these were grown for 20 days on a medium supplemented with IAA (0.1 ppm) the stamens showed early degeneration but there was a reasonably good development of the ovary. The effect of IAA was, however, reduced when GA, (0.33.0 ppm) was also added to the medium and in buds cultured on a medium containing both JAA and GA, the stamens continued their development. These results suggest that IAA and CIA, do have a direct effect on the differentiation of the ovaries and stamens and they can exert their influence on floral buds without any obligatory interference h m the leaves or other organs of the plant. It remains to be seen whether such female flowers could be made to grow into fruits through pollination and fertilization in vitro. In recent years various chemicals called “gametocides” have been reported to induce male sterility without seriously affecting female fertility. Maleic hydrazide (MH), 2,3-dichloroisobutyricacid (FW-460), end 2,2-dichloropropionic acid (dalapon) are some of them. Moore (1950) and Naylor and Davies (1960) reported that maleic hydrazide (MH)is effective in inducing male sterility in maize. Wittwer and HilIyer (1964) also obtained a similar response in some members of the Cucurbitaceae. A repeated dipping or spraying of the plants at intervals of 6-7 days with a solution of 100 ppm MH suppressed the development of male flower buds whereas the female flowers developed normally and were fertile. Rehm (1962) described similar results in Citrullus vulgaris and Lycopersicum ecrcukntum. Recently, much attention has been given to FW-450 and dalapon. Eaton (1957)induced male sterility in “Empire cotton” by thc applicntion of FW-450. Moore (1959) obtained total male sterility in tomato by spraying a 0.3% solution of a sodium salt of FW-460 at anthoeis. Similar results were obtained by Hensz and Mohr (1969) in Citrullw, vulgaris by using dalapon.

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Recently, Starnes and Hadley (196%)applied FW-450 (0-2000 ppm) as a foliar spray on soybeans. This resulted in the inviability of pollen and a failure of dehiscence of the anthers. At 750 ppm FW-450 effected maximum male sterility, the highest incidence (93%) of pollen abortion being observed in the variety “Dunfield”. Kho and De Bruyn (1962) tested the gametocidal activity of FW-450 on Antirrhinum wjw.Aqueous sprays of its sodium salt (0*6y0)soon after the appearance of first flower buds caused male sterility. More recently Nasrallah and Hopp (1963) reported induction of pollen sterility in Solanum rpelongena by the application of a sodium salt of FW-450. Aqueous sprayings of a 0.2% solution 2-3 weeks before anthesi8 or when the buds were 60-80 mm in diameter proved effective. Among other subetances carbon monoxido, NAA, 2,4-D, and GA, have been reported t o have a gametocidal action in some instances. Genetically monoecious plants of Mercurialis ambigwt produce exclusively male flowers a t the first one or two nodes, but at subsequent nodes they bear both kinds of flowers. Heslop-Harrison (1957) inveutigated the effect of carbon monoxide in inducing male sterility h.such plants, A treatment with carbon monoxide reduced the ratio of male to female flowers from 27: 1 in the controls to 6: 1 in the treated plants. The output of pollen waR relatively low in the treated plants but the female flowers were unaffected. Heslop-Harrison and HeslopHarrison (1958) obtained auxin-induced male-sterility in Silene pendukz (family Caryophyllaceae). In the male-sterile flowers the gynoecium showed a remarkably precocious development. The styles were frequently exserted and the stigmas became receptive even before the unfolding of the corolla. In a few instances a treatment of the leaves with auxins resulted in parthenocarpic fruits without pollination. Hensz and Mohr (1959) reported that a treatment with GA, (2000 ppm) induced male Sterility in Cucumia vulgari8. Choudliury and George (1962) induced male uterility in Solanurn rnelongenu by applications of MH (600 ppin), NAA (50 p~mi),and 2,4-L) (20 ppm), the percentage of pollen sterility ranging from 90 to 100. The application of NAA also caused sex reversal by converting the &amen8 into miniature pistils. Although the intricate phenomenon of floral organogeneuis is only imperfectly understood, there is some evidence t o show that sex expression in flowering plants can be modified through the application of growth substances notably auxins, gibberellins and gametocides. In this the technique of tissue and organ culture, as employed by Galun et at!. (1962), offers a promising field for further investigations. While the induction of inale sterility has an obvious value in plant

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breeding, much research is necessary on the effects these chemical6 produce on the ovules.

XV. CONCLUSIONS Ever since the discovery of double fertilization in 1898 tho etudy of the embryology of angiosperms has made rapid progress. Apart from its role in taxonomy (Maheshwari, 1904) embryology 1iaR a close relation to physiology and genetics. The in &ro oulture of embryos, endosperm, ovules, ovaries, flowers and anthers has considerably advanced our understanding of the physiology of these organs. The test-tube fertilization of ovules followed by their full development into seeds is another notable achievement. Nevertheless, thore in still a great paucity of dotailed information on many aspects of fertilization. Although one can pinpoint the site of incompatibility in the reproductive system of flowering plants, a knowledge of the factors whioh cause it remains a matter of theories and conjecture. The most rewarding approach to the study of control of fertilization would be through co-ordinated embryological, physiological and biochemical investigations on the pollen tube, stigma, style and ovary, coupled with genetical research on sexual incompatibilities. The artificial culture of embryos has an obvious application in plant breeding and in overcoming dormancy. From the viewpoint of physiology and morphogenesis a study of the embryos of the orchids, insectivorous plants and certain phanerogamic parafiites and naprophytos is likely to prove very rewarding. For the horticulturist tho artificial induction of polycmhryony is of great value. Its induction in those plant8 in which it doen not occur in nature and its control in thofie in which it exifitn an a normal fcuturo will both need much experimental work. The recent HtudieH on tho cultivation of single c e b have an important implication in t h i n context. These have clearly demonstrated the totipotency of vegctative cclk, in that they too, like the zygote, can build up a new organism (Steward, 1963).

Although the zygote and the endosperm are both products of fertilization, their morphogenetic destinies are so divergent that the former develops into an embryonic sporophyte while the latter remains merely as an attendant on it. Plants of the family Oenotheraceae, where both embryo and endosperm have the same chromosomal complement, are worthy objects for studying the nature of the factors which bring about such a functional diversity. Although the pollen grain and thc egg arc both haploid ntructuree, they behave quite differently. On some occctrjions the unfertilized egg

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may develop directly into a well-organized group of cells, namely the embryo, but the obtaining of a tissue from the pollen is far more difficult. In fact, even the initiation of meiosis and the maturation of the pollen gl‘ains in escised anthers have not yet provcii feasible. A parthenogenetic development of the ovum has becn achieved in many animals and a few cryptogams, but the angiosperms have so far defied all attempts in this direction. Haploids do occur in nature sometimes but all methods to induce the development of the unfertilized eggs of angiosperms have proved virtually infructuous. It is possible that excised ovules cultured in vitro may be more easily amenable to physical and chemical stimulations than those borne on the plant in nature. The control of sex expression has far-reaching applications and there is increasing evidence of the role of growth substances in this. Further research has to be directed towards an understanding of its physiological basis and for finding methods for a selective control of the two kinds of gametes. These problems raise several fundamental questions the solution of which c a b for a further strengthening of the bonds between embryology, physiology and genetics.

ACKNOWLEDGEMENTS We are grateful to our pupils, Messrs. P. S. Rao, T. S. Rangan, and K. R. Shivanna, and Miss S. V. Usha, for their diligent help and cooperation in the preparation of the manuscript.

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The Soft Rot Fungi : Their Mode of Action and Significance in the Degradation of Wood JOHN LEVY Botany Department, Imperial College, London I. Introduction.. ........................................................... U. Hiatologg of So& Rot .................................................... III. A Technique for studying &ft Rot Fungi .................................

323 329 337 339 IV. Mode of Action of &ft Rot Fungi .......................................... 339 A. Pmeive Penetretion end D a y Penetration .............................. B. Effect of species of Wood on the Mode of Attack by the name Fuhgus. ........ 340 C. Effect of Species of Fungue on the Mode of Attack in the esme Wood. 344 348 D. Boft Rot Fungi on Po& in Ground Contact .............................. 348 V. Liat of Fungi known to came Soft Rot.. .................................... 349 VI. Disouaaion.. ............................................................. Acknowledgemente ....................................................... 355 355 Referencea ..............................................................

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I. INTRODUCTION I n the natural forest, the wood rotting fungi serve a w f u l purpose in breaking down the complex materials manufactured by the living tree into simpler substances which are ultimately returned to the soil and the atmosphere and 80 become available for the nutrition of other trees. Thus the natural cycle of a tree consists of a building-up phase which may last a very long time and a breaking-down phase which is usually very much shorter. The secondary xylem of the tree possesses a natural strength and resilience in order to perform the two functions of keeping the tree erect whilst supporting the weight of the crown and of providing a plumbing system capable of conducting water from the roots to the crown. This natural strength has cawed it to be utilized by man as a structural material. It is in this context that the fungal decay of wooden structures and materials becomes undesirable and often a hazard. The decay of wood has been observed from the earliest timeH, but it was not until the end of the eighteenth and the bdginning of the nineteenth centuries that it came to be considered sufficiently serious to be regarded as a matter of national importance. This was mainly due to the rapid expansion of the British Navy at this time. The decay of existing vessels and the difficulty of finding timber of suflicient quality to w e for repairs and new building gave rise to considerable concern. Bryant (1942) quotes figures of the ships in commission and building T

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for the Navy in 1793 as 268 of which 135 were line of battleships; by 1801 this figure had risen to 479 of which 202 were ships of the line. Anyone who has seen H.M.S. Victory a t Portsmouth can imagine what a ship of this size meant in terms of suitable timber and forest acreage. Ramsbottom (1937) has given an interesting account of the damage to ships caused by wood rotting fungi which, in one case, involved the complete rebuilding of a new battleship and its ultimate scrapping before it was commissioned, due to the ravages of fungal decay. By the middle of the nineteenth century the development of the railways and telephone and telegraph services increased the use of wood as sleepers and poles, and the need to take some action to combat decay soon became obvious. Many workers became involved in the subsequent study of the reasons for this decay. The association between fungi and decay in timber had been noticed from antiquity, but it was not until the second half of the last century that Robert Hartig (1878) established the fact that the fungi were responsible for the decay. His published work has become a classic and has influenced subsequent workers in this field ever since. Cartwright and Findlay (1958) give a concise account of the development of this work up to the present day. Following Hartig’s work it became generally recognized that a group of the Basidiomycetes and a small group of Ascomycetes were responsible for the fungal decay of wood. Of these the Basidiomycetes, the group thought to be of considerably the greater economic importance, could themselves be divided into two groups, the brown rots and white rots, according to the way in which the cell wall materials of wood were broken down (Cartwright et al., 1931). The idea thus became prevalent that decay of timber was normally due to one or other of these two types of organism. This state of affairs existed for the first half of the twentieth century in spite of a series of observations made between 1850 and 1950, until Findlay and Savory (1950) established that the deterioration of timbers in water-cooling towers, thought to be brought about solely by chemical means, was in fact caused by a particular type of fungal decay. This type of decay was subsequently called “wft rot”. Schacht (1850) was the first to obxerve the preHence of cavitiw with pointed ends within the wall of woody celk. In a later paper (1803) he was able to show that these cavities were c a ~ c dby the longitudinal penetration of cell walls by fungal hyphae and figured uuch cavities in the walls of isolated cells of Anona laevigata, Dracaena draco, Hernarulia s o m a and Caryota wens. His drawing of the latter specieu shows several features whose significance ha8 hitherto been overlooked, but which have been reported by recent workers (Corbett and Levy, 1963) and will be dealt with at length later in this paper. Dippel (1898)

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observed cavities in Fagua sylvatica (beech), Anona laeuiyala, Sabal umbracilifera and Taxodium distichurn. This method of fungal attack was first studied in detail by Bailey and Vestal (1937) who confirmed the existence of these cylindrical cavities and noted that the conical ends formed in the secondary cell walls had a remarkably constant appearance in a variety of timbers. They suggested that the enzyme action of the fungus in the cavity must take place along two predetermined sets of planes. They did not identify the fungi responsible, but suggested that they might be members of the Pyrenomycetes. Tamblyn (1937) observed similar penetration in Jarrah but was not able to arrive a t any more definite conclusions. Barghoorn and Linder (1944) found a similar type of attack in wood that had been submerged in the sea and they went further by the isolation of a number of Pyrenomycetes and Fungi Imperfecti from the decaying timber, thus begining the study of marine fungi. At this stage these observations were little more than interesting curiosities and hardly considered important as regards timber decay a8 a whole. It was left to Findlay and Savory (1950) to observe this type of decay in wood removed from water-cooling towers, and to isolate a fungus, Chaetomium globosum, with which they were able to produce this type of decay in beech in pure culture in the laboratory. Savory (1954a, b, 1955) described the effect on the wood produced by this fungus and coined the term “soft rot” to describe it on account of the softening produced in the surface layers by the action of the fungus. He described the widespread occurrence of soft rot in wood, but claimed that it was masked by the quicker acting Basidiomycetes where they were present. Findlay and Savory (1954), reviewing the work they had carried out on soft rot, suggested that this type of decay could have important economic significance under those conditions that would retard or inhibit the growth of Basidiomycetes. The term “soft rot” is now used to designate any instance of the characteristic penetration and growth of hyphae within the secondary cell walls of wood as described by these workers, whether or not softening of the surface is evident. It is caused by a number of micro-fungi, i.e. members of the Ascomycetes and some Fungi Imperfecti. Soft rot appears to occur in all old timber which has been expoNed to mokt conditions. A number of papers published in the 1950’s described fungi associatedwith wood in a way that can now be interpreted as being soft rot attack (Gandy, 1965 ; Johnson, 1956 ; Warrelmann, 1956 ; Wilson, 1956 ; Verrall, 1959). Both Johnson and Wilson were concerned with marine fungi, and described or figured soft rot attack. It is more commonly found in timber from broad-leaved trees than in coniferous woods, but no timber appears immune from attack. In water-cooling

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towers, however, the conditions appear to be exceptionally favourable for soft rot organisms, for softwood slats suffer serious decay. Since degradation is usually confined to the superficial layers of the wood, soft rot may cause the greatest amount of damage in structures in which the surface/volume ratio is high (e.g. plywood or hulls of small boats (Becker and Kohlmeyer, 1958)). Savory (1955) noted that the treated zone of timber impregnated with preservatives does not remain protected from attack by micro-fungi. Liese has published a number of papers including a general review of soft rot (1959). He produced evidence of bacterial attack on wood (1965); was one of the first to show that timber treated with a wood preservative could be decayed by soft rot organisms (1960); and has shown that there is a range of natural durability amongst woody species (1961). In laboratory tests Duncan (1960) has observed that many fungi capable of producing soft rot have greater variation in tolerances to different wood preservatives than the common wood-destroying Basidiomyctes. Moreover, direct attack of the surface of treated wood has been noted in poles and piling (Duncan, 1960), and such superficial attack, though not in itself causing severe loss of strength, is believed to facilitate the entry of destructive Basidiomycetes. On the other hand, soft rot may be of considerable importance in situations where the softened surface is eroded away as with the wooden slats in water-cooling towers, on wooden marine piling and on the undersurface of boats. Several authors have shown that marine fungi can cause soft rot (Johnson, Ferchau and Gold, 1959; Meyers and Reynolds, 1959a, b, 1960; Siepmann and Johnson, 1960; Jones, 1962a, 1963; Oliver, 1962). There is also a considerable literature on the occurrence of soft rot in watercooling towers (Dost, 1959; Baechler, Blew and Duncan, 1961; Goff and Excell, 1961; Price, 1961; Walters, 1961; Jones, 1962b). The prohlems of finding effective preservatives and of retaining a toxic level of preservative in timber in situations such an those just demribed requires further investigation. A recent review has set out the latest developments (1964, For. Abstr. 25, (2) xxviii-xxix). I n laboratory experiments, the presence of certain chemicals appears to favour soft rot attack. Using a mycelial-mat wood-block technique (after Abrams, 1948), Savory (in Findlay and Cartwright, 1958) showed that the decomposition of beech wood by Chaetomiurn globosum may be accelerated by the addition of nitrogen and phocJphates to the culture medium and that within certain limits the decay is directly proportional to the concentration of these nutrients. Duncan (1960), and Savory and Farmer (1969) have shown that the normally more resistant coniferous woods are more susceptible to soft rot attack after prior degradation of the wood by treatment with chlorinated water under acid conditions.

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The latter workers pointed out that typical soft rot occurs where there is no possibility that chemical degradation of the wood took place before the penetration of fungi and that the light chlorine treatment under alkaline conditions, which is found in water-cooling towers, may not have an effect on the wood comparable with that obtninetl in the laboratory. Later work* with low concentrations of chlorirrc yielded a result that was in direct opposition to that of Duncan and ~howetlno increase in soft rot attack on blocks of Sequoia sempervirens. The discovery that fungi other than Basidiomycetes were capable of degrading wood, especially under conditions which normally do not favour attack by Basidiomycetes, has aroused considerable interest and has led to comparisons of the properties of soft rot with the two characteristic Basidiomycetg rots-“white rot” and “brown rot”. Particular attention has been given to the possible chemical nature of the degradation process (Meier, 1958; Savory and Pinion, 1958) and to the meahanical properties of wood undergoing fungal attack (Armstrong and Savory, 1959). Brown rot fungi (e.g. Coniophora cerebella, Poria monticola) preferentially attack the carbohydrate constituents of wood (celluloseand pentosans) and cause little depletion of lignin. A considerable proportion of lignin may be left when the carbohydrates are completely decomposed. White rot fungi (e.g.Polgsticlus versicolor), on the other hand, decompose both cellulose and lignin. Chemical analysis of beech wood undergoing attack by Chaetomium globosum (Savory and Pinion, 1958) has shown that there is a marked ce1lulor;e depletion from the early stages of attack and that when the wood in decayed to 82 per cent weight loss three-quarter8 of the residue is lignin. In t h i s respect soft rot resembles brown rot. The alkali solubility of the decaying wood has heen contkJcrec1 to he one of the chief chemical methods of distinguishing hetween white and brown rots (Campbell, 1952). The alkali solubility in regartlccl HS ti measure of the amount of degradation products, in excenn of‘ th(JHC not immediately utilized by fungus, which are formed during the enzymatic breakdown of wood constitutents by the attacking fungun. A rapid increase in alkali solubility in the early stages of decay in characteristic of brown rot, whereas in a white rot there is a gradual increase an decay progresses. Thus, in terms of alkali solubility the action of 6’.@oboeurn resembles a white rot. The effect of the decay of beech by 6’.globosum on the strength and toughness of beech has also been compared with that caused by white and brown rot fungi (Armstrong and Savory, 1969). These results suggest that soft rot of beech by C‘haetomium globoaum is more closely comparable to a white rot than R 1)rown rot i n its effect upon the impact resistance of WCJOd. Unpuhli~hctlrqiort in thv I:Cwcwt T*

I’rwlucts I h w w t ~I ( c w , r t l H ,

I d o i ~ ~ I f ~ Ir !lI,~ $ I ) .

328

JOHN LEVY

Microscopically, soft rot is generally quite different from either n white rot or n brown rot. In wood attacked by a fiingus M liicli causes a brown rot. hyphae ramify through the cell lumina and penetrate the walls a t right angles to the long axis, forming bore holes which become much wider than the diameter of the hyphae (Sachs, 1962). I n white rots there is a general thinning of the cell walls as well as the forniation of bore holes, since both the lignin and the cellulose of the wills are attacked by thehe fungi. Soft rot, 011 the other hand. has been recognized by t h e presence of' distinct elongated cavities within the middle layer of the secondary wall of fibres, tracheids and vessels. I n addition, the slow rate of decay compared with the Basidiomycete rots and the characteristic macroscopic appearance distinguish soft rot from either white or brown rot and the causal organisins must be placed in a separate class of' wood-rotting fungi. One interesting point which arose from the anatomical (Meier, 1955) and chemical (Savory and Pinion, 1958) studies referred to above is that of the extent t o which fungi, particularly soft rotting microfungi, can attack lignin. Some workers (13asi1, 1 948) claim that C'haelomium sp. and s/ac?hybotr?/s sp. tmak down lignin in jute fihre. Recent workers disagree on the extent to which lignin as found in wood is attacked, but this appears to be due chiefly to a lack of detailed microchemical information about the composition of the secondary cell wall. Meier (1955) attributed the lower attack by C'haetomium globoszim of coniferous u-oods in comparison with hardwoods to the higher lignin content of the former and suggested that lignin was largely absent froni the central layer of cell wiills of the hardwood (hirch) that he used. Savory and Pinion (1 958) claim that the complete dissolution of the central layer of the secondary cell \Val1 of Scots pine (soft\+oocl)hy Chaetomirrm globoszrin n hich they observed suggestr that t h i s f'ungus is capable of destroying lignin. The true answer is probably a combination of several factors, not the least of which appears to he the anatomical characteristics of the timber, which, as the present article will attempt to show. has some bearing 011 the ease with which the fungal hyphae can penetrate through the wood and reach the middle layer (S,)of the secondary wall. The work of Courtois (1963) already siihstantiatcs this point of' view. Working with two hardwood and five soft ~ v o o t lspecies hc: h s ttrlillysed soft rot attack on these timbers and tlescrihctl the type of t.;tvity i r l fitlrcq, tracheicls, parenchyma cells and versels. Hc sugg,,uststhat, ;LH 11cr:ay proceeds,thedestruction oftlic cell wall can he scparatetl into fburt1istinc:t stages. These are an initial stage, a developed stage, a late stage, and finally, as the last stage, the "destruction zone" occurs. The cavities in the cell wall could be differentiated into fourteen groups. His final

T H E SOFT HOT FUNGI

329

conclusioii is that “the shape and orientation of infection patterns as well as the differences between the wood species are mainly influenced by the fine structure of the cell wall and its topochemical composition”. Levi (1964a) states that the differences that exist in the effects of various wood-destroying fungi are of both a physical and chemical nature, with enzynles playing a very big part. He suggests that future work should be directed towards the complete analysis of decayed wood and to a study of the enzynies involved. In a recent, detailed, interesting and instructive account of an attempt to do this (Levi, 1964b), he describes the results from experiments on the progressive degrade of beech veneers by Cimetorniurn (nlobosurn. Chemical analysis of the degraded wood has been cornbined with optical and electron microscopic obeervations and attempted isoltitions of the enzyme systems responsible for tho degrade. The reactions of t’hese isolates on both the wood itself and its individual components is described. The results incidentally confirm Corbett’s observations. whilst the chemical analyses are striking in their own right. They indicate, amongst other things, an initial increase in the high polymer cellulose fraction and suggest that the lignin content of the wall is only partially altered and not, as was previously thought, completely removed. The implications of these results are considerable and provide the basis for opening up new Iines of thought and speculation on both the nature of the cell walls and the mode of action of the fungi degrading them.

11. HISTOLOGY OF SOFTROT The characteristic appearance of soft rot attack is shown in Figs. 1 and 2. The cavities as seen in transverse section are formed round fungal hyphae in the middle layer (Sz)of t h e secondary w d l . Whcre enzymic action has been considerahle the cavitieH havc c:ortlenc:etl forming a large cavity which contains more than ono hyph;i. ‘I’hc! ultimate enlargement and coalescence of these cavitiw rcmovon t t i c w tioh! of the S, layer. As seen in longitudinal section, the cavities are elongated with sharply pointed ends. I n three dimensions, therefore, they form cylindrical canals ending in sharply conical points. A chain of cavitieH may be seen frequently to follow the supposed line of the orientation of the microfibrils in the S, layer, each cavity being separated from the next by a very short distance. It is often possible to see a final hyphal projection from the end cavity, designated by Corbett (1963) as thc proboscis hypha. Figures 3 and 1 show examples. Courtois (1963) has described clearly and in considerable detail the ways in which the hyphae penetrate through the cell wall, the sequence T**

330

JOHN LEVY

F I ~1.. Transverse section of Pinu8 r8ylwslri8 showing hyphae of HOft rot fungi in the s, layer ' of the secondary vki11 (('orlJett,1963). ( x 1520.)

T H E S O F T RO'T F U N G I

FIG.

33 1

2. Transverne section of F q u s aylaa/;ca with Hoft rot hypheo in thc: Sa layer of a fibre showing cavity formation (Corhctt, 1'563). ( x3OWJ.)

332

JOHN LEVY

FIQ. 3. Tangential longitudinal mction of I % y u ~wybc&o H h l J W i I l g chrrrc.ti.rint i v HOft rot cavities with pointcd W I ~ I H .( / 1520.)

T H E S O F T R O T V'UNGI

FIG.

333

4. Longitudinal section of Pinus ayluedrb nhowing characteristic point,erl (:ndn f J f tho soft rot cavity. ( x 1520.)

334

JOHN L E V Y

of decomposition of the wall layers and the differences that can be seen in different cell types and between softwoods and hardwoods. Using a technique which involved observations on isolated macerated cells as well as an examination of microscopic sections, he has described fourteen different groups which can be recognized on the basis of shape, size, position and orientation of the decomposed regions (Fig. 6). In tracheids and $fibres he observed that three different initial stages can occur in the cell wall attacked by fungal hyphae. Thus the decomposition begins ( a )directly from the cell lumen, ( b ) in the middle of the secondary wall, and ( c ) in the outer secondary/primary wall region. (a) Beginning from the cell lumen the HecontInry wall in infected after the preceding local decomposition of the tertiary wall. The patterns are parallel or almost parallel to the longitudinal axix of the cell. Three form-groups were observed. Geometric Figures Group 1: Right-angular, rhomboid or trapezoidal eroded regions in the cell wall; these are not cavities but are partially similar to them. Irregularly Developed Forms Group 2: Smooth-edged, irregular cell wall erosion of variable size. Group 3: Irregularly indented notches of the cell wall. ( b ) In the case of an infection beginning within the secondary wall, only this wall is decomposed. The tertiary wall is decomposed a t a later stage. The cavities differ in their shape and angular form. Irregularly shaped patterns with irregularly developed forms were not observed. Qeometric Figures Group 4: Very narrow, more or less long cylindrical cavities with or without conical ends. They can be considered as the initial formx of the final cavities and lie in a "Z-helix" with angles between 3"-14", bctween 15'-32' and more rarely between 40"-66" to cell lengths. Group 5: Short and long, narrow and wide cylindrical cavitien with conical ends. They occur singly and show either a t one end or at both ends thin hyphal channels as continuation. In relation to the cclI longitudinal axis they can be orientated in the same angleu LLU thoue of group 4. Group 6: Longer and shorter cylindrical cavities with conical ends, generally lying one after the other like a row of dropletn. Frequently they are orientated parallel to the longitudinal axis of the cell, but alHo in flat angle as a "Z-helix". Group 7: Short rhombic or rhomboid cavities lying one after the other in series mostly parallel or almost parallel to the longitudinal axis of the cell. Group 8: Shgly-occurring cylindrical cavities, conical at both ends,

T H E SOFT R O T F U N U I

( 0 ) From

335

the cell lumen

2

Oecoy type 1

Lc

3

(b) Of the centrol secondary wall 38 ..14' 159..34 40?..50° ~

40

4b

4c

5b

50

-

-

\-,

4d

4e

5c

(c) Of the outer secondary WOW primory wall

90 9 b

IOa

Ilb

IOb

IlC

Examples of attock in the cell wall of vessels

Example of attack in the cell Wall of wood vorenchymo cells

13

14

&a. 5. Examplee of atfb& produced by soft rot fungi in the cell wall8 of tracheids, wood flbres, veclsels and wood parenchyma cells (Courtoi~,1983).

336

J O H N LEVY

changing into inore narrow cylinders and then becoiniiig spiiidleshaped. They are orientated either almost parallel or as a "Z-helix" at a small angle to the longitudinal axis of the cell. ( c ) Beginning from the outer secondarglprimary wall region, the S, layer is decomposed in either geometrically or irregularly developed forms; the cavities can lie a t large angles to cell length in the S, layer. Geometric Figures Urowp 9: Longer, unilaterally trapezoidal patterns in the outer cell wall IHyerH, orientafetl in the longitiidind direction to the cell axis ;triangular extractions w e less freqnciit. Irregularly Devdoped Forms Group 10: lrregular indents of the cell wall, smooth-edged, slightly indented, proceeding from the outer cell wall limits, partially in connection with cell wall penetrations. Group 11: Long, tube-shaped patterns which branch off, occurring in the outer cell wall layers. They are orientated in flat Z- or S-helices (e.g. 60') or more or less a t right angles t o the longitudinal axis of the cell. The vessel walk exhibit only one form-group. Group 12: Longer or shorter, generally narrow but also wide cavities follow the microfibrils in a Z-helix with an angle of between 36"-60° to cell length, or they surround the pits concentrically. Their edge i q smooth or slightly indented. Tliey ciln deviate froni their direction with a short bend and then, after another deviation, return to the former direction. Constrictions frequently subdivide the patterns of infection or lead over t o n thin hyphal channel, from the end. The ends of the cavities are either irregulnrly round or indistirictly coriicid. I n pareneh?ymtz cells only two form-groups occur. Group 13: Narrow, tube-shaped cavities, which widen a s t h c y grow older, are orientated as Z-helices with angles of between 39"-77" to the longitudinal axis of the cell and occur frequently. Group 14: Cylindrical cavities with conical ends, parallel to the longitudinal axis of the cell; they occur rarely. These groups were based on the following criteria : (1) According to the kind of cell in which the cavities or areas of decomposition occurred. Tracheids and fibres have the greatest ahundance and variety of groups, whilst parenchyma cells only show two group patterns and vessels only one. (2) According t o the shape and form of the cavity pattern. T h e shape, length, diameter and age of the cavities arc of importance, untl they generally lie one after another in the cell wall freyuently fieprLrrLtcd by short regions that show no decomposition. (3) According t o the position of the fungal hyphlt. Frequently

THE SOFT HOT F U N G I

337

decomposition begins in the 8, layer after the hypha has penetrated into the cell wall, and the S, layer (soinetinies called the tertiary wall) shows little sign of being infected. Sometimes, however, the wall appears to have been decomposed prcferentiidly from the Iiinien, whilst on ot#liero(wnions it, is t8hooiit.c!i*l i i , y c t r s (11' t I I C scwoiitlii.iy w i i l l i m ( 1 t h i r primary will1 \VIIWCt>hc?iLt'ttt('I
111. A TECHNIQVE FOX STUDYIKG SOFTROT FLWI Levy and Lloyd (1960) compiled a list of the fungi isolated from a disused copper mine in Cornwall. They found soft rot to bo widespread in the timber underground, which was predominantly softwood species, and compared their isolates with those listed by other workers. This was clearly not good enough and they indicated that ti tscliniquc suitable for screening the fungi isolated for their wft rotting potenti-

338

JOHN LEVY

alities was being sought. Lloyd (1960) surveyed the conditions under which soft rot was found in nature and came to the conclusion that the water content of the wood was usually high and that, where the attack waa of economic importance, it was frequently made in contact with water in movement (e.g. the fill of water-cooling towers ; underground mining timbers ; river kind marine timbers ; ;wid t,inihers in ground contact subjected to the lci~(:hiiigaction of r h ) . l~urican (1960) had reported that, whereas hardwoods could be attacked readily under ordinary laboratory conditions, softwoods were much more tlificirlt to Ii;~ntllcant1 a strong leaohiiig treatment of tlicsc timber WII.S r s of‘ton ii(x:esstiniplos prior. t,o iiioculat>ioiiwith i L soft rotting f ~ ~ n g ~ snry t II”! sy t II 1)tA )I ti s ( ) f t,tic: (I(!c:rLy (1 0 V(! lo]I(:( I . I,lo,ytl ( I !)(iO) slrgg(!st.(:(lt~tiiLt!ijlic iriovcrri(!rit ( 1 1 wiLi,(!l*i ~ t t f ’ ~ J I l ttic ~~1 soft,wootl tini her W;LH necessary c i t h r t,o r(!rnovc ti11 i i i t i i t)it,or. prcserit in the tiniI)cr or formed :LS a hy-product of the fungal iitt,tk(:l(,or even in order to introduce oxygen into t,he wood once the fiingus had penetrated the surface layers. She, anti later (lorbett (1963),described unsuccessful experiments to establish this ascertion and the latter decided that the only reliable means of determining the presence of soft rot attack in its early stages was t o cut sections of the timber and examine them under the microscope. It was soon observed that, there appeared to he differences in the early stages of attack by Chaetomium globosurn in beech (Fagus sylvatica) and in Scots pine (Pinus sylvestris), and a technique was worked out to attempt to examine these details more closely and to see what correlation, if any, there might he with the great difference in the rate of attack of these two species (as measured by loss in weight) when exposed to the fungus in a petri dish. The technique evolved ( b h ? t t ,1:JtX) was to use small c u t m of wood, hetween 0 . 7 cm and 1.0 c:rn in cr1gu clirtiension. ‘hw: tiloc:ks W(!IC w r c fully cut SO that t w o fikcos worc i r i thc: i,r;lrlsVCrHC l ) l i L r t c : , I,wo i t 1 tjll(! rJ1,fli;t,i longitudinal plane and ttio rciri;iirtirig !,WO i r i ttic! i,rirtgc:tti,iriI I ~ i t ~ ~ i f ~ i r ~ plane. Four faces of catch hloc:k w(!r(! swhd with “Ar:rl~lit,c:” (11 I W f J prietary formulation of a n epoxy rcsiri ;ulhesivo), HO t , h i ~ tttic c:rit.ry of the fungal hyphae was restricted to two ftices only, arid these were always opposite faces of the cube and therefore in the same plane. In this may it was possible to present a particular orientation of cells to the active growth of fungus on an agar medium, so that it might be possible to observe the way in which the fungal hyphae penetrated through the block. The size of the block was a convenient one for mounting directly on t o a sledge microtome so that sections could be cut in any plane without having to re-shape t.he block. Using this technique, experiments were set up with the sapwood of Scots pine (Pinus sylvestris) and beech (Fugue sylvuticu). Three blocks

I i ~ i d

T H E S O F T R O T E 'IrN 0 1

439

with a different pair of faces exposed in each case were placed on a mycelial mat of the fungus Chaeforniwm globosum growing on either a modified Abrams medium or on 1.5 per cent malt agar. With both species, considerable differences were observed in the rate in which the fungal hyphae penetrated through the block from the various faces and emerged on the upper unexposed face. With both species the transverse face provided the quicker entry and the hyphae emerged on the upper face sooner than with the tangential faces exposed and much sooner than the blocks with the radial faces exposed. In the case of the transverse faces, profuse sporulation occurred arid completely covered the upper face ; sparse sporulation occurred on the tangential face and little or no sporulation occurred on the radial face. With both species it appeared that the fungus was following the line of least resistance and W ;Lpvrt urc thtL1, rriiglit o w u r to I);LSS taking ;Ltlv;Lnt;qc of' iLny S ~ ) ~ Lor t t IP t I y 1)t i ;LC (1 11i(* k I y t I t roug t I t I I(! Id( k . 'I' I 10 1)cn(:trn t i o r 1 frc ) 111 tmr t s vcrsc f i ~ c cto trtLrlsvcwc! fiwe t ~ p p e ; ~ rto ( ~IrrLvc. l oc.c.irrrct1 prir11iLrily through the ~ I r i tho ( m e of' the tangentiul lririieii of trwlieids, vessels ~ L I Iti hex. faces the quickest route seemed to 1r;tve been d o n g tho length of' tlie medullary rays and the contents of' these cells may well have assisted in the nutrition of the fungus. With the radial faces, however, there appeared to be no straightforward path unless the fungal hyphae could penetrate readily from cell to cell through the bordered pits of the tracheids and simple pits in the fibres. Microscopic observation confirmed that this penetration did, in fact, occur.

Iv. MODE OF ACTIONOF

SOFT

ROT PUNGJ

A. PASSIVE PENETRATION A S D I I E C A Y I'PNIFTltAl'ION

In the case of Scots pine the rays were quickly colonized h y the fiirrgus, which appeared to penetrate fairly rodily throrigh thc: sirtilll(: [)it,H between parenchyma cells o f ttic r;Ly ; ~ r i ( I t h vcr1,ic:nl t,rii(*h(:iilsol' 1,tw wood. However, although h y p h e wcro s w r t f'rorn l,irri(? ~ J J 1 i r r i c : tlf) penetrate thrcmgh the bortlcred pits froni om trti(:ttttid t,(J t1h:rtr:xt,, this was by no means common in the early stiLgcs of infec:t,icm i ~ r r ( 1whiLt Corbett (1963) describes as the passive pcnetration of the furtgus apr)wm to proceed from ray t o tracheid and tracheid to ray more easily than directly from tracheid to tracheid. In the case of beech, the main path of passive penetration from cell t o cell was through the simple pits. These pits in beech are of small diameter and frequent occurrence and it seemed highly probable that one of the reasons for t,he rapid loss of weight of beech in the laboratory

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JOHN LEVY

by these cultural methods in comparison with the niuch slower loss in weight, of Scots pine was due primarily to the speed with which the fungus could make a pasfiive penetration through the former species and so with a greater volunie of timber in contact with the actively growing fungal hypliae t’he suhsequent decay penetration into the tva,ll was likely t>oo(:cur o i l i L greater nrinilier of occasions. It, seems iiiiporttuit therefore to tlistinguish hetween (a)tlie gross penetration of ,the fungiis into itnd through the hlock, ; ~ n d( b ) the pllct,riLt,iotiof i n d i v i d ~ i i f1111jii~1 ~l hyl)t~aeinto the cell wall C ~ L I I S ~ breakJI~ tlowti o f t h t w d l . ( lorl)et,t,(1!163) uiitlcrlitit!n this tlistiric:tioii a i i t l suggests t IIO t,(:r.liis “l)ilsfiivc! I)(!ii(!tr;st,ioii”il11(1 “tl~c:;~yi)(!ti(!t”iLt,ioli”t,o c o v w ttliosc! t,w(J li)i-tiis of l)rogrwsioti of i tic I’itiigys. I’ii.ssivc! l~ciict,riit,iotii r r I i c d i Wi1.s i~iiic:lic!r tjlirrii iii Si:ot>s1)itic.

R. EFFECT OF SPECIES O F WOOD O N THE MODE OF ATTACK BY THE SA3TE FUNGIJS

Corbett and Levy (1963) described the initiation of decay penetration of Chaetomirim ylohoswvn into Scots pine. The hyphae enter the lumina of the tracheids via the simple pits between t,he tracheid and t,he ray parenchyma cells. The hyphae t,hen align themselves parallel to the long axis of the trnclieitl and usudly contiguous with its wall. Lateral branches develo
* Recent work (Levi, 1963b; 11. Ci. S t w m s , pc~monalcomrrlciriic.ilt,iolI) tItLs ehown branching in tho S, layer of t11o first wall.

T H E S O F T HOT F I - S G I

341

branch in the opposite direction to that of the new growth. The two arms of the T thus formed grow at approsimate!,y the saiiie rate within the fia layer. Subsequently the rate of hyplial pelletration decreases and

a cavity forms round the hypliae and is en!argetl laterally. .It tliis stage the tips of the cavity begin to taper (Fig. fio), giving riw tit this point to the sharply defined conical ends c.tlar:wteristic: of tlw htcr stages of the rot (Fig. (id). This type of attack ~ V H Sfig~~rcbcl\r.ithout

342

JOHN L E V Y

comment by Schaclit in 1883 in Gnryota uren8. Courtois (1963) also figures it, but does not include it, in his fourt,een groups of attack patterns. The dist,ance from tjhe point of vertical brancliing to the conical ends of the cavity, when t.liey first appear, is variable and would appear t,o be related rather to the diameter of the cavity than length of the hyplin. Further cavities are initiated by lateral hyphal 1)r;tnclies froiii miLtJurcoiivities or hy fine Iiy1)liitC froni the tips of existing cavities (:IiLinln tIiiLi8 c:liiLins o f liclicidly dignatl , i!M:i) (Pig. 7 ) . (!art) ;LIT: li)imiotl wiihiii t l i I;lay(!L.1,)' i h ! 1IL r I )r()(*(!ssi1.1I( I t'lI ibt,, ;&,lioiigli ~ l : l i ( ! l ~ i L l tlissoliittion of t,lic! S, IiLyw (11' tho tjruc:lic:itl w d I s OC(!IWS in the iLdviLiic:eti stiLgtts of'rot, the S:,layer appears to tx sii t)st;ttititilly unattitoketl. She suggests that in summer wood tracheitls the cavities tend t,o he sliort stid wide and the chains of cavities to he almost parallel to the long ibxis of tlie tracheitl. I r i spring wood tracheids however, cavities arc gcnernlly longer antl finer antl arc set at a wider angle to t,lie long axis. In beech, tlie fibres undergoing at,trZck and the fibres just in advance of those showing at>tackare characterized by the presence of darkly stained hyphae in the cell luniina and ia pits connecting adjacent fibres. Because of the thickness of the fibre wall and the small size of the lumen, observation in longitudinal sections of the initiation of attack presents much more difficulty tlinn wibli Scots pine and in most, of the material examined the most common evidence of t'he rot was mature cavities wit11 conical ends having darkly stained hyphal contents and generally lying almost p t d l e l to the long axis of the fibre. Transverse hyphar piwxiiig ;Ldjac:ent fihre \\riLIIs were rilrcly seen. I I I scveribl iiistiLiiws ;I firic? I i ~ y p h iW;LN ~ sttl:Il in tlie f i l ) r ~\ ~ i l l I (x)liI1
,

THE SOFT HOT PUNOI

343

FIG.7. Hetrla sp. longitudinal noction from ti fwiw I J I J S ~sho\ving prot)oHcifi hyphw giving r i w to chains of cnvitirs of tiirninkhiiig tliritnvtcr (C'orl)ctt, 1963). ( Y Ill#).)

341

JOHN LEVY

occurred in the S, layer of the a d j w m t fibre traclieid wall. followed by ciivit,y forniiit,ion i t s found in Scots pine. I n the majorit,y of cases, however. i m l partioularly wlien transverse or tangential fares of the test blocks were exposed to the fungus. it' w a s found t,liat the Iiyplinc in t,he lumen proceeded to erode the wall IvIicrever it came into rolititct with t,he wall surfiice. Figure 8 s1~on.sthe characteristic appearance of the tiniber destroyed in this way. In some institiices i i lurninal hyplia formed a very short lateral t)rttnc:h which hegan to crode the wall at, ttio p i n t of i~ st>dnctl.'I'his iriitiiil erosion cwntw:t iLIi(\ w a s found to b o c o ~ densely af)l)ciLrl>(l iLs it stiiirI)-Sitlctl V-slii1~lte~l nick in the OCII wiilt wllen viewed in longitutli~li~1 scotion, c:onsitlcrtLttl,y I;trger a~icimore sl1iirl)l,y tlefiiied t.hm t h i k t o1)served 1j.y I'ro(:t,or (I!U1) witti t)ro\\.n t*ot.i i t l ( l 1 ' 1 ~ j 1 1 ~ 1 i t ~ l y cxtcn(1itig ILX fw iLs t1ho 1tri111iir,yWiLIl. Sonictinit:s i L liit,(?riil t1,y1)tIi11 t)r;m(:ti pssitig iiit,o,t,ll(: wit11 ;i,t, SII(:II it, t i i d ( t)(!(;ii,lll(! v(!ry litic! ii,ritI t8r;ivc!rsc(Ithis will1 i t r l ( l t,llc! w;LII of' t IIO i L ( l j i ~ ( ~ ( ~(:ell l l t ~ ill; right ;I.IIKICS to t,hcir Ioiig iwis ~ ~ n limiiccl tl i L l l o t l l C r V-SlliiItc(1i i i o k iit t,tle otlicr siirfii~x~. 'I'his 1)ror:i?ssof destruction let1 to nlirrketl scU1J)tUriIlgof the wit11 when viewed in longitudinal section. I n trimsverse sections, however, the cavities were almost always found t.o have rounded edges. Cavities within the thickness of the wall, usually forming a series of spiralling chains?of cavities, were found only at the extreme edges, beneath a sealed face, of t'hose blocks where transverse or tangential faces were exposed: Other parts of' these blocks showed only erosion of the wall from the lumen. In blocks where the radial longitudinal faces were exposed, however, decay penetration was restricted t o the cells nearer the exposed €ace and was mainly in the form of helically directed chains of cavit'ies in the S, layer and only rarely by erosion from the lumen. The initiation of decay penetration in the three tirnhers n1iL.y t h i i s tw seen to follow quite different paths. The reasons for these diflccrenr;es are not entirely obvious, but must he related to the fine structure and chemistry of the cell wall; they are tliscusscd later in this p a p r . F(*rthe present kt it suffice to say thtit the SiLnic fungus, ~~hudorrtiurn, yhhwrr~,, does not hchavc i n tho SiLnlC w;iy in e i ~ ( : Hhp C i C H o f woo(l. C. EFFECT O F SPECIES O F FI'S(;I.S

OX THE MODIC OF ATTACK

I?J THE SrlMlE WOOD

Corbett (1963) tested several species of fungus in culture against bloclrs of birch (BetitZa sp.) which had all faces unsealed iind one tar]gential face presented to the fungus. The fungi were L'mnim/hyriurn f i i c l i e l i i . Sphaeronema sp.. Stysmztis sfpmonilis, all of which had actively attacked the wood after three n,esks' exposure, Camarosporizrm amhiens

THE SOFT ROT FVNGI

315

T H E SOFT R O T F U N G I

347

adjacent cell; the erosion of the wall by hyphae in the lumen, forming V-shaped nicks; and cavities with conical ends within the S, layer, were all observed. Destruction of the wall was severe, even in cells with a low density of hyphal growth. Corbett (1963) figures four electron-micrographs taken by Preston and Levi (1963) using this material, one of which is reproduced as Fig. 9. This shows R hypha lying in a mature cavity within the wall with a portion of the finer lateral hyphn from which the cavity was initiated shown crossing the middle lamella and primary wall region. This is a very remarkable picture indeed. Sphaeronema sp. The exposed (tangential) face of the block was severely rotted after three weeks’ exposure. Hyphae were confined chiefly to the S, layer, and of relatively large diameter in comparison with wall thickness. Erosion from the lumen was rarely observed. Stysanw &emonnitis. The edges of the block and patches of tissue associated with vessels were severely rotted after three weeks’ exposure. Hyphae did not erode the wall from the lumen, but penetrated the wall and formed discrete vertical chains of helically arranged cavities within the S, layer. The hyphal walls stained quite strongly with picro-aniline blue compared with the hyphal contents which showed only a pale coloration. Rotted cells retained their form even though the hyphae ramified completely through the wall. I n longitudinal sections rotted tissue had a lacy appearance, because only the primary walls and middle lamellae remained and the hyphae filled the whole space between the undigested framework. Hyphal diameter increased with the development of the cavities. There was an extremely sharp differentiation between rotted and unrottecl zonen and it was more difficult to identify any early rstagea of penetration of the wall. Camarospwhm ambiem. Slight wulpturing of tho wall kJy liirnerid hyphae was observed in fibre-tracheidn on the e x p m d edge of tho block, but only when dense “ropes” of hyphac were present in the lumen. Cephubsporium sp. After three weeks the walls of some fibre-tracheids on the extreme edge of the exposed face were undergoing crouion hy luminal hyphae and there was also evidence of cavities within the wall. Cross-penetrartion hyphae were extremely common. The pattern of attack by all f i v ~fungi is similar in nature to that already observed for Chetomium globosum. Although differences in detail occur, the similarities are striking. It would be interesting to test these organisms on other timbers and see whether the effect of the dpecies of timber is similar in all species of fungus.

348

JOHN LEVY D. SOFT ROT FUNGI ON POSTS IN GROUND CONTACT

Corbett (1963) made some st,riliing observations on tlie occurrerice of soft rot in birch and Scots pine fence posts on three different sites. Soft rot cavities were observed to be present in birch posts a t the ground-line to a depth of 30-45 cells from the surface eleven months after the posts had been driven into the ground. After eighteen months’ exposure, soft rot cavities were also observed in the top of the birch posts. A similar pattern was observed in Scots pine, but the depth of penetration was less and showed considerable variation with the site conditions. Birch and Scots pine posts of a similar type t o those examined had previously been observed to fail a t the ground-line due to attack by Basidiomycete fungi, after three years’ exposure. This a t once lays open the possibility that the soft rot fungi may play a part in a succession of fungi colonizing these fence posts and could well be an important precursor of attack by Basidiomycetes. More work t o elucidate this point would be of very considerable interest and value.

V. LIST OF FUNGIKNOWN

TO CAUSE

SOFTROT

Corbett (1963) compiled a list of all the fungi which have been recorded as having the ability to induce soft rot in hardwoods or softwoods in pure culture. Trichoderma viride was not included in this list since there have been conflicting reports as to the ability of this species of fungus to produce soft rot. The list is by no means complete, since it omits marine fungi (0.g. Jones, 1963), and some other species, but is still a useful compilation, in spite of the difficulties with identification that often occur. Her list is as follows. ASCOMYCETES Chaetomium cochliodes C . elatum C . funicola C. globosum Ophiostoma coerulesccns 0 . piceue 0 . pini Xylariu sp.

FUNGIIMPERFECTI Acremoniella sppp. Acremonium Alternariu sp. A . humicola

Reference* 2, 4 4

2, 5 1, 2, 4, 7 3 6 3, fi 2

c

1

2 2 6

349

THE S O F T ROT FUNGI

Biapora effuaa B. pwilla Biaporomycea sp. Bobordeniellrt sp. Canta7osporiu7n ambiena Cephaloeporium sp. Ceratocyetis pilqera CWTOPaiS sp. Conwthyrium sp. C . fuckelii cyto8porelEa Rp. Dendryphium sp. Diplococcium sp. Diacula pinocolu v. mamnwaa Haplochulara sp. Hel&coaporiurnaurcum Hormkcium sp. Nematogenium sp. Orbicuh sp. 0 . porietinu Pestaloztia sp. Phialophora richardsiae P . faatigiata Phoma sp. Pullularia sp. Sclerotium sp. Sphaetonema sp. Sperocybe sp. Styannus sp. S . stemnitis Torula sp. Trichoapwium heteromorphuin Tridhurua terrophilua

* 1. Corbett, 1963. 2. Duncan, 1960. (Identifications stated t o be tentative.) 3. Krapivina, 1960.

2

5 2 2 1 1, 2

2 2 2, 4 1 2 2

2 3 2 2 2, 3 2 2

2 2 2 2 2

2

a 1 2 2, 4 1 2

3 2, 4

4. Savory, 1954. 5. Snvory, 19548.

6. Tumanyan, quoted by Krapivina, 1960. 7. Z)a Costa and Kerruixh, 1963.

VI. Drsccssioiv The differencesobserved between the initiation of the decay penetration of Chaetomitcm ylobosum into beech, birch and Scots pine can reasonably be explained only on a basis of differences in the habitat provided by the three species of wood. These differences, as Corbett (1963) and Courtois (1963) have pointed out, can be found in variations of the gross anatomy of the timbers; in variations of tho fine structure of the wall of the cells; and in chemical differences between the wall components.

350

JOHN LEVY

Scots pine. Passive penetration depends very largely on the existence of free space for the growing hyphae to develop. This may well involve the utilization of cell contents, but does not include enzymic breakdown of cell wall material nor the mechanical intrusion into the wall structure such as might be envisaged by the development of appressoria. In Scots pine there appears to be no difficulty in colonizing the rays, where proliferation of hyphae takes place, and the hyphae appear to pass freely through the large simple pits between ray parenchyma cells and vertical tracheids. Penetration of hyphae from tracheid lumen to tracheid lumen through the bordered pits is not as regular or as frequent as might be expected and would seem to suggest a barrier, probably associated with aspiration of the pits. The resistance to passi ve penetration in ICScots pine, therefore, must depend primarily on the ease with which growth occurs in the ray parenchyma cells with subsequent movement of the hyphae through the Bimple pits into the traclieici lumina. Corbett (1965) gives a photograph which show8 perithecial and mycelial development in Scots pine blocks, twelve days after exposure (Fig. lo), which suggests different rates of penetration. In reporting observations on such blocks exposed to Chaetmium globosum for only three days, she notes that the hyphae were observed, in those blocks with tangential faces exposed, preferentially to enter the ray parenchyma cells and to pass into the tracheids through the pits between the two cells. Little hyphal development was noted in the tracheids. In blocks with exposed radial faces, the hyphae were observed entering the tracheids and passing from tracheid to tracheid via bordered pits, a type of penetration which appears to occur to a greater estent in the spring wood than in the summer wood. Since the ohwrved rate of growth on the block was slower, the penetration through 1mrderc:d pits must therefore be a slower process and one can infer that cithcr the hordered pits form a barrier to progrcssion or else ti. build-iij~of flYpJILL1 material is necessary in the my prenchyrna to riU[)[JlY ttla tiritlgotwid and necessary logistics for an attack to develop. It is axiomatic so far as soft rot fungi are aoncernorl that unless tho hyphae reach the cells, the walls will not be decayed. It therefore follows that the rate of decay of a piece of timber must depend in the first instance on the speed with which passive penetration is effected. If this is slow, then subsequent decay will be slow and the chief reason for the difficulty experienced in using Scots pine as a laboratory test species could well be the slow passive penetration. Duncan's results with the leaching technique could well be due to the leaching action opening up the bordered pit apertures sufficiently to permit easy passage of growing fungal hyphae.

T H E SOFT ROT FUNGI

35 1

Decay penetration in Scots pine is less emy bo explain. The ptLtt,ern of development of first the lateral bmnch, then its penetration t'lwough the first wall and vertical branching developing in the S , layer of the adjacent wall is strange, to say the least. Why should vertical branching occur more frequently in the S2layer of one ccll and not in the other,

FIQ.10. Block8 of Pinus eyloestris 12 days after exposure to the fungus Chnefomirm globosum (Corbett, 1963) ( ~ 2 . )

when both have apparently equal chances of being attacked? Why does the lateral branch never enlarge in size or produce a cavity round i t ? The answers must lie in the physical and chemical nature of the cell wall. One question that may well be pertinent concerns plasmotlosmata. These exist in the primary wall between two young cell^, a8 cytoplasmic

352

JOHX LEVY

connectlions between the cells,.Do they persist i i s t,lw ~ c c ~ o ~ i t l nlayers iy of the wall nre laid c1o\vn? If t,liey do. their prexcn(:c ~\oul(lprovide n very fttcile esplaiint,ion of the observed fiwts. OIIOcould sugpcst, t,li;it t,hc initiathi of the developnient of the hteral brniicli f’rom Iiyphite in t.he lumen of the cell is in response to a stiniulus from possible cytoplasmic or protein residues remaining in the plasmadesma. The branch, through fine, may then produce sufficient enzymes to break down t.he protein residues when the growth of tthe hyplia would then follow the path of enzymic dissolution. ‘J’liose hyphac that penetrate complntely through t,he wall would be growing i n a plasmatlemia that reniains coruplete in spite of the disturbance caused hy the production of the secondary wa,ll. This, however, i m y not be true of all such plasmadesniata and where an unconformity occurs the hyphae can 1jenetrat.eno longer and extension growth is suppressed. Meanwhile, the fine hypha has heen producing ina all imounte of cellulolytic enzynies which, when extension growth ceases, accmnniulate near the tip of the hyphae. Roelofsen (1959) suggests that flow is likely t o be easier along the microfibrils than across t.liem clue to the encrustiitk)ns of lignin and so dissolution may be init,iiLt,cdl).v t,lic flow or tliflusion of’ cnxyincs a l ~ i i gthe easiest path, iw it, is il(!cll~~iiIliLt(!d ;it tlic I ~ y p l i t ~tip. l ‘J’liis would ;~c:c~oirnt for the ‘I’-xhapc?d vc!rt,ic:id hr;~iwIiingof’tliu liypl~~io. r 1 I his i s ~1)(:~:11l~ttiot1 t)ilsctl 0 1 1 i l l ] irriknowri fii(:tor, ~ t r i f lif it is ~ t r o ~ t l t l ( ~not, mist i n the sacoriclory wr~11, tlic WIIOIC of t h t , ~~li~sti~;~tlestiiiiti~ the foregoing pragtxph hccoiiics ~o ~iiuc:l~iionser~sc. H(JLV~\.W: it explains so many of t,he observed facts that further investigations ~ tt proportion of the ultrashould lie nlikde. Who kiiows? it inay I Jthat microscopic: cnvit,ies mentionetl by many ;uit,hors an! scctions of plasrnadenmat,tL which Iiuve heen l~rol<enup (luring the find strlgcs of‘ formation of the secondary \mil. Beech. Passive penetration is effected rapidly hecausc of’ tlie erne with which tlie fungal hyphae can penetrate from cell to cell via the simple pits. Tlie pits are relativeiy abundant in the fihre walls i111t1 so the fungus has inany spaces through tvhich i t rnay pass from cell to cell. The lumen of t,he fibres often appears to be filled by a liirgc hyjhri, from which branches pass through the pits, completely filling the pit apertures. There is thus a close contact between the hyphal material and the wall, which may amount to a higher percentage of the total hyphal surface area than with the other species of WCJOd cx~miirifxl.‘I‘hin may be a reason for the speed with which rleoiiy mcirrs i r i t w d i , clac:ay penetration 1)cginning as ;I t)r;iric:h frorn IL t~,yptiai r i ;L pit, t l p W t , l l ~ ( ? going straight into the w d . ‘I’hc siiiitll clir~rnctorof’ tlic: f i l m s nltulc observation difficult from longitudinal sections, h u t thu (:losoj)roxirnity of hyphae starting to form cavities in the S, layer with latcraf tryphac

:

THE SOFT ROT FUNGI

363

penetrating through the Pits seems to provide at least circumstantial evidence of this. Birch. Passive penetration is effected, as with the other two species, wherever there is space for the hyphae to grow, and there is considerable colonization of the rays. Decay penetration is effected in one of two ways, either lateral penetration and T-shaped branching in the wall, or by erosion of the wall from the lumen. This latter attack is effected by the formation of V-shaped nicks in the wall which may expand considerably but which invariably show a characteristically angled end to the erosion pattern (Fig. 8). This is similar to the angles observed and discussed by Roelofsen (1956) and Frey-Wyssling (1966) at the ends of cavities in the S, layer of the wall. The orosion of tho cell wiills from the lumen in birch is tt Htriking feature and must be related to the nature of the cell wall. Wardrop arid Dadswell (1967) suggest that the S, layer is absent in birch, slthough Meier (1965) shows illustrations in which it appears to be present. Courtoia (1963) quotes Meier (1957) in suggesting that the S, layer of birch fibres contains little lignin, which is not true of softwood tracheids. On the other hand he further suggests that differences in erosion may be due to differences in the mannan and xylan content of this layer and quotes Meier and Yllnev (1956), Liese (1963) and Sachs, Clark and Pew (1963) in support of this assertion. More work is obviously needed before this point can be elucidated, but it should be a relatively easy matter to discover how soft rot fungi react to mannan and xylan in culture and to observe the decay penetration into other timber species with a similar composition to the S, layer, or into reaction wood. From these observed differences in reaction to the three species of wood it does become apparent that the soft rot fungi may well prove a useful tool in the elucidation of the nature of the plant cell wall. Further work, with or without fungi, could well be designed to to& the validity of some of these epeculations. A t thc mme time, thwe in ti n:al need to follow up the work already done with other npcciw of tirntwr and with other species of fungue; not only soft rot fungi, but I m w n nJt, white rot and staining fungi as well. The same fungus, Chaetomium globoeum, can, in one spooies of wood, show afhities with the characteristic habit of staining fungi on the one hand and of Basidiomycete attack on the other. Cartwright and Findlay (1958) describe staining fungi as making a lateral penetration of two adjacent cell walls from a hypha in the lumen of one cell to a hypha in the lumen of the other. The hypha penetrating the wall R i very fine by comparison with the lurnenal hyphae. Chaetomiumglobosum can do this in both birch and Scots pine. This at once raises the question that, if the soft rot fungi pan behave like staining fungi, can the latter r

384

JOHN LEVY

type of fungus produce cavities in the wall like the former? Krapivina (1960) claims that this is so and illustrates typical early stages of cavity formation. More observatioii is required with stnining fungi nnd work is needed to throw light on the nature of the penetration ttlirough the wall in this particular case. Do plasmadesmata play a part here, or is the penetration purely mechanical, by means of appressoria? The V-shaped nicks observed by Corbett (1963) during erosion of the wall of fibres of birch by hyphae of Chuetomiumgloboaum are larger and more sharply defined, yet nevertheless show remarkable similarity to the V-shaped nicks observed by Proctor (1941) during brown rot attack. It would be interesting to discover the conditions which give rise to this effect. What factors determine the way in which the soft rot fungus will behave ? There is a very real need to combine morphological and histological studies, such as are described here, with biochemical studies. What do these observations mean in biochemical terms? How far are chemical differences in the wall materials suppressing or stimulating one enzyme or enzyme system at the expense of others? The amount of enzyme produced by the fungus must be an important factor in the way in which the wall is attacked. Those fungi which produce much extracellular enzyme are able to react with the wall materials at some distance from the hyphal tip, by the simple diffusion of the enzyme ahead of the fungus, such as occurs with the brown rot fungi. Does this mean that the soft rot fungi produce only small amounts of extracellular enzyme? Does the size or shape of the enzyme molecule prove a limiting factor to any extent? Why is the dissolution of the S, layer such a characteristic feature of this type of fungal attack? TH there a correlation between the T-shaped branching prior to cavity formation in the S, layer of the adjacent wall and the fact that the hypha has just penetrated the middle lamella and primary wall? Could this be the reason why no branching occurs in the first wall? If the reason for t h e decay of the S, layer depends on the high cellulose and low lignin content of that layer, how do these fungi compare with the fungi and bacteria known to attack cotton and other cellulose fahrics? Can bacteria cause soft rot, or do they act as a precursor of the soft rotting organisms? Wood preservatives provide another series of investigations that should be combined with these histological studies. Preston [1959) showed that waterborne mixtures of inorganic salts penetrated into the cell wall. How far is this true of other fungistatic and fungicidal materials used as wood preservatives? Is it possible to find a material that has a preference for the S, layer in softwoods and could therefore be used specifically against the soft rot fungi? Alternatively, i t might

T H E S O F T ROT FL‘NOL

355

well be that a material that penetrated only the S, layer could be more effective against soft rot attack in birch. Madhosingh (1961) has shown that a soil fungus, Fwrarium oxysporum, may well reduce the toxicity of certain wood preservatives to the entry and spread of a wood-rotting Basidiomycete. How far is this true of the soft rot fungi and their apparent tolerance of wood preservatives present in wood in conmntrations sufficient to inhibit the development of wood-rotting Basidiomycetes1 The main conclusion to be drawn from this is that much remains to be done to understand the behaviour of wood rotting fungi in wood. Work along these lines could well throw light on the nature of the cell wall and on new ways of arresting the decay penetration of fungal hyphae into the cell wall. The soft rot fungi &re an interesting group and rewarding to work with. They are of economic importance only where insufficient or excess moisture or low dosage of preservative treatment inhibits the growth of the more vigorous Basidiomycetes. They may also turn out to be a stage in the colonization of wood in ground contact and precursor of attack by wood rotting Basidiomyoeta fungi.

ACKNOWLEDGEMENTS The writer wishes to express his thanks to two research assistants, Mrs. Flora Deverall (nbe Lloyd) and Dr. Nanette Corbett, who carried out much of the work described in this article, and who listened patiently to much speculation on their observations. Thanks are also expressed to Prof. R. D. Preston, F.R.S., for his many kindnesses and discussions. Mr. J. G. Savory, earns specis1 thanks for making his list of references and reprints available and giving ungudgingly of hifi time to discuss the subject. Mr. A. Horne, who took most of the photomicrographs, is another to whom special thanks are due.

REFERENCES Abrams, E. (1948). C‘irc. US.Bur. Stand. 188. Armstrong, F. H. and Savory, J. G. (1069). Holzforschung 13 (3), 84-9. Baechler, R. H., Blew, J. 0. and Duncan, C. 0.(19fJl).Americnr~,So&i?~ ./ Mechanical Engineere, P8Per No. 61-PET-5. Bailey, I. W. and Vestal, M. R. (1937). J . Arnold Arbor. 18, 106-208. Barghoorn, E. S. and Linder, D. H. (1944). ParlowuA 1, 396-467. Barn, S. N. (1948). J . Tert. Id., 39, 232. Becker, GI. and Kohlmeyer, T. (1968). A T C ~fiir ~ V~ i 8 C h ? z k & ~ 8 ~ CQ h(1). f$ 2940.

Bryant, Sir A. (1942). “The Years of Endurance 1793-1802”, p. 332. Coiihe, London.

356

,JOHN L E V Y

Campbell, W. G. (1952).“Wood Chemistry” (L. E. Wise and E. C. Jahn), Vol. 2, pp. 1061-1116. Reinhold, New York. Cartmight, K. St. G. and Findlay, W. P. K. (1958).“Decay of Timber and its Prevention”. 2nd ed., H.M.S.O., London. Cartwright, K. St. G., Findlay, W. P. K., Chaplin, C. J. and Campbell, W. G. (1931).Bulletin. Forest Products Research, London, NO. 11. Corbett, N. H. (1963). “Anatomical, Ecological and Physiological Studies on Microfungi associated with Decaying Wood”. Ph.D. Thesis, University of London. Corbett, N. H. and Levy, J. F. (1963).Nature, Lond. 198 (4887). 1322-3. Courtois, H.(1963).Holzforschung und Holzverzuertung 15 (a),88-101. Da Costa, E.W. B. and Kerruish, R. M. (1963).Holzforschung 17, 12. Dippel, L. (1898).Dae Mickroskop Pt. 2, 116-19. Dost, W.A. (1959).Proceedings ofthe 20th Annual Water Conference, Pittsburgh. Pennsylvania. Duncan, C. G. (1960). U.S. Forest Products Laboratory, Madison, Report No. 2173. Findlay, W. P. K. and Savory, J. G. (1950).Proceedings of tho V I I International Botanical Congress, I. 3 15. Findlay, W. P. K. and Savory, d. (4. (1954).Holz Rub-u. Werkst. 12, 293-96. Frey-Wyssling, A. (1956).HoZz u Roh-u. Wwkst. 14 (6).210. Gandy, D. G. (1955).MOA Bull. 64, 551-2. Goff, J. It. and Exeell, J. S. (1961).American Society of Mochanical Engineers 61 (8). Hartig, It. (1878). “Die Zersetzungsersclicinungon des Holzcs der Nadelholzbliume und der Eiche”. Berlin. Johnson, T. W. (1956).M?jcoZogia 48, 841-76. Johnson, T. W., Fercliau, 11. A. and Gold, H. 8. (1959).Phyfon 12 (l),65-80. Jones, E.53. G. (19625).Trans. Brit.mycol. SOC.44, 93-114. Jones, E. B. G. (1962b). British Wood Preserving Association Convention Record, 41-3. Jones, E. 1%. G. (1963).J . Inst. Wood A%. No. 1 I , 14-23. Krapivinrt, I. G. (1960).Lesnoi Zhurnal 3 (l), 130-33. (C>.S.I.I<.O.Translation 5329 ,M.Slade, 1961). Levi, 31. P. (1964a).J . Insf. Wood S c i . No. 12, (5646) Levi, M. P. (1964b).“A Biochemicill Study of the Actiori of th4: Soft 1Cot l?urigi, Chaeioviizo,i Globosum on Fugiis Sylvaficrs. Ph. D. Thcris, University of Leeds. Levy, J. F. and Lloyd, F. J. (1960).J . 1 s t . Wood S c i . No. 8 , 14-24. Liese, W. (1955).British Wood Proservativc Association Corivcntion Hocord, 159-60. Liese, W. (1959).Nuturw. Idddh. 11, 419-26. Lieso, W.(1960.fffJkfor.?Ch?L?kg und Holzvrrwertuuy 12 (41, f i 1 - 4 . Liese, W. (1961).Mitt. dtsch. C.J. If. 48, 18-28. Licse, W. (1963).J . Polym. .%1 1%.C., No. 2, 213-29. Lloyd, F. J. (lg6O). “Studios in Tirnbcr Mycology”, D.I.C. Thesis, Imprial College, London. Madhosingh, C. (1961).For. Prod. J., 11, 20-22. Meier, H.(1955).HoZz a. Roh-u. Werkst. 13 (9),323-38. PuIeier, H.(1957).“Die Chemie der Pflanzenzellwmd” (E. Treiber), Berlin. Meier, H. and Yllnev. S. (1956).Svemk Papp-Tidn. 59, 395-401. bIeyers, S. P. and Reynold$ E. S. (19598).Can. J . Microbiol., 5, 493-602.

(I.

THE SOFT ROT FUNGI

3.57

Meyers, S. P. and Reynolds, E. S. (1969b).Bull. Mar. Sci. Gulf & Caribbean 9, 441-66. Meyers, S. P. and Reynolds, E. S. (1960).Can. J . Bot. 38,217-26. Oliver, A. C. (1962).J . Imt. Wood Sci., No. 9, 32-92. Preston, R. D. (1969).Brit. Wood Preserving Association Convention Record. 31-72. Preston, R. D. and Levi, M. (1963).Personal communication. Price, E. A. S. (1961). Wood.26, 56-6 and 99-100. Proctor, P. (1941). Yale Sch. For. Bull. 47. Ramsbottom, J. (1937).E88ex Nut. 25, 231-67. Roelofsen, P. A. (1956).Holz a Roh-u. Werkst. 14 (6),208-10. Roelofsen, P. A. (1969).‘The Plant Cell Wall”, Gebruder Borntraoger, BerlinNikolassee. Sachs, I. 13. (1962).U.S.Forest Products Laboratory, Madison, Report No. 2256. Sache, I. I%.,Clark, 1. and Pew, J. (1903).J. Polym. Sci. Pt. C, No. 2, 203-11. Savory, J. (1. (19544.Ann. appl. Biol. 41, 330-47. Savory, J. C. (I954b). J . app!. Buct., 17, 213-18. Savory, J. G. ( 1966). British Wood Prcwrving A~~ociation Convention Rocorcl. 3-20. Savory, J. 0.and Farmer, R. H. (1969). Forest Products Research Records, London. Report. Misc. I, 216. Savory, J. G. and Pinion, L. C. (1968).Holzfor8cl~ung12,99-103. Schacht, H.(1860).Bot. Ztg. 8 (39),697-713. Schacht, H.(1863).Jb. &8. Bot. 3, 443-83. Siepmann, R. and Johnson, T. W. (1960).J. Elieha Mitc?u% mi. SOC.,76 (I), 160-54. Tamblyn, N. (1937).Am&. For. 2, 6-13. Verrall, A. F. (1959).FOT.Prod. J . 9 (l),1-22. Waltere, N. E. M. (1961). Forest Products News Letter C.S.I.R.O. Aust. No. 273. Wardrop, A. B. and Dadswel1,H. E. (1957).Ho2zfor8chu~,I1 (2), 33-41. Warrelmann, E. (1956).E~eclriz~tatsw~rlsc~af~ 65 (23),869-76. Wilson, 1. M. (1960). Tram. Brit. mycol. SOC.88 (4),401-16.

This Page Intentionally Left Blank

Author Index Page numbers in ordinary figures are text references; page numbera in italic figureri are bibliographical referencea.

A

Aalders, L. E., 296, 310 Abrams, E.,326, 355 Acher, R.,169, 168, 213 Adams, G. A.. 73,145 Adams, I., 8, 34 Afzelius, B., 2, 33 Albersheim, P.,79, 145, 154, 2€3, 215 Alexander, L. J., 269, 310 Alexander, M.,178, 213 Alleraheim, P.,87, 146 Allende, J. E., 186, 214 Amici, G. B., 219, 310 Anderson, D.M. W., 79, 145 Andr6, J., 2, 33 Anhaaueser, H.,223, 315 Anhder. L.D., 306, 317 Archibald, E.E. A., 44, 67 Arbghaus, R.B., 167, 216 Armstrong, F.H., 327, 355 Arneson, T.J., 298, 310 Aronson, J. M.,77, 92, 98, 145 Arrigoni, O.,211, 215 Arutjunova, L. G., 227, 310 Asen, S., 238, 260, 310, 312 Ashton, M., 79, 114, 146 Ashworth, R. de B., 211, 216 Aspinal, G. 0.. 80,145 Attia. M. S., 227, 310 Aushermann, L. E., 257, 311 Avery. A. G., 234, 241, 310 Avery, G.S., Jr., 296, 310 ~

Barghoorn, E. S., 325, 355 Barker, C . A., 73, 76, 146 Barrollier, J., 159, 169, 214 Bershad, J., 76, 146 Bartlett, M. 5.. 46, 67 Baruah, H. K., 224, 229, 317 Basu, 5. N., 328, 355 Batjer, L. P., 232, 274, 311 Hayley, S. T., 69, 73, 79, 80, 81, 82, 87, 88, 116, 128, 145, 146, 148, 163, 160, 213, 215 Beaman, T.C., 158, 160, 205, 215 Bean, R. C., 168, 213 HeaRloy, J. O.,261, 311 Ueatty, A. V., 303, 311 Boatty, J. W., 303, 311 Becker, G.,326, 355 Beckner, M.,37, 67 Beer, M.,73, 88, 116, 145 Beevers, H., 211, 214, 262, 317 Belford. B. S., 85, 97, 145 Bonnet-Clark, T.A., 164, 213 Bennett, H.W. 221, 31Z Bontloy, R.,158, 217 Benzer, S., 188, 213 Berger, J., 295, 310 BOrgmm, La,285, 3 1 1 Beth, K., 280, 321 Bianchetti, R.,21 1, 215 Bigwood, E.J., 168, 217 Bishop, C. T., 73, 79, 145, 163, 160, 213

Blakely, L. M.,294, 319 B Blakeslee, A. F., 222, 234. 241, 243, Baechler, R. H., 326, 355 247,250, 251,280, 311, 318, 320 Bailey, I. W., 152, 164, 189, 213, 215, Mew, J. 0.. 326, 355 326, 355 JJlontfoau,R.,277, 311 Bajaj, Y. P. S., 292, 294, 314 13londo1, B.. 242, 243, 31H Belasubramanyam, V. R., 278, 310 Bogorad, L., 7, 33 Baldev, B.,271, 290, 292, 315, 318 Hohmer, H..87, 108, 146 Bali, P. N., 233, 310 Bolliger, R.,88, 113, 145 Ballowitz, E.,11, 33 Rolton, J. L., 258. 312 Bandurski,R. S.,81,145,158, 160,206. Bonner, D.,188, 213 211, 213, 214 Bonner, J., 79, 87, 120, 135, 1#6, 147, Bardier, N. O., 226, 311 152, 154, 158, 180, 165, 188, 200,

360

AUTHOR INDEX

Bonnor, J., corrt. 204, 206, 210, 211, 213, 215, 216, 21 7, 258, 266, 314 Bonner, R. E., 40, 67 Bornet, E . 2, 34 Bosio, M . G., 234, 311 Bosshard, H . H . 108, 113,145 Bottomley, W., 279, 313 Bouck, G. B., 5, 33 Bouharmont, J., 257, 311 Bowler, E., 93, 97, 98, 104, 108, 14.5,

148 Uredornttnn, G., 222, 311 Brewbakrr, J . L., 226, 228, 230, 311 Brink, R. A., 224, 247, 249, 261, 253, 257, 262, 311, 314, 321 Britten, E. J., 279, 311 Britten, R. J., 182, 213 Brock, R. D., 231, 311 Bronckrrs, F., 224, 311 Brown, A. I>.,81, 145 Brown, C. L., 250, 311 Brown, J . R., 173, 213 Brown, R., 165, 172, 213 Bryant, Sir A., 324, 355 Buchholz, J . T., 234, 311 Bukovac, M . J., 276,277,306, 311,321 Bulard, C., 250, 311 Biinning, E., 142, 143, 144, 145 Burgeff, H . 133, 145 Burk, D., 208, 215 Burkholder, P. R., 252, 318 Burstrom, H., 84, 145, 156, 213 Butcher, R. W., 14, 33 Butenko, R. G., 283, 311 Butt, V . S., 204, 213 Buvat, R., 86, 89, 118, 119, 14.5 Bystrom, B. G . , 97, 104, 148

C Calvin, J. R., 88, 116, 145 Ctmara, A,, 240, 311 Campbell, R. C., 276, 312 Campbell, W . A., 206, 217 Campbell, W . G., 324, 327, 956 Campos, F. F., 300, 311 C a p h , S. M., 241, 319 Cappelletti, C., 234, 311 Caram, B., 11, 34 Cartwright, K . St. G., 324, 326, 353, 356 Caruso, C., 225, 318

Castle, S., 103, 127, 136, 145 Catell, R. B., 50, 67 Chandler, C., 229, 311 Chang, c. w., 145, 251, 311 Cheng, L. O., 81, 148, 167, 217 Chapeville, F., 188, 213 Chaplin, C. J., 324, 356 Chase, S. S., 298, 301, 311 Chayen, J., 202, 203, 213 Cheadle, U . I., 89, 146 Chon, J. C. W., 125, 134, 140, 146 Chong, K. C., 299, 321 Chibnall, A. C., 158, 214 Ctiiiiwd, F. P., 159, 214 Ching, P.T., 277, 319 Ching, K . K., 221, 315 Ching, T . M., 221, 315 C'hopra, R. N., 265, 268, 281, 311 ('houclhury, I%.,259, 308, 311 C'Iiri&rtrmn, G. S.,152. 160, 214 L'liristonson, T., 3, 4, 5, 21, 33 Clarkc,U., 1,2, 7 , 9, 10, 11, 13, 15, 19, 33, 34 Clark, I., 353, 357 Clauson, R. E., 300, 312 Cleland, R., 87, 147, 154, 172, 181, 214, 216 Cochran, G., 65, 67 Coe, E. H., Jr., 298, 312 Coltrin, D., 81, 149 Colvin, J . R., 73, 14, 75, 76, 144, 145, 146, 147, 149 Compton, It. J., 226, 312 Condit, I . J., 277, 319 h n k l i r i , M. K., 241, 25(4 251, 2x0, J2I) Conxtth:l, P.,HI, 1.16

(,'tJOlJf'r, u. c., 257, ,311 ('oopar, F. P., 88, 116, f 4 5 Corbett, N. H., 324, 32!), 330, 331, 337, 338, 34(J, 341, 342, 343, 344, 345, 347, 348, 349, 350, 351, 354, .J56 Corc:oran, M. R., 25.5, 273, 512 Cormack, R. G. H., 96, 145 Coronado, A., 186, 214 Corrcns, C., 226, 312 Corrigan, J . J., 168. 214 Cottone, M . A., 157, 214 Courtois, H., 328, 329, 335, 342, 349, 353, 356 Cowan, S. T., 64, 68 Cox, L. G., 258, 317 Craig, W . R., 272, 273, 274, 320

Crane, J. C . , 231, 274, 276, 277, 311, 312, 31 7 Curtis, J. T., 60, 67, 263, 290, 292, 312 Cutter, V. M., 263, 312 Czaje, A. Th., 104, 112, 145

D Du, Costa, E. W. €3.. 349, 356 Dadswell, H . I<., 353, 357 Dagnelie, P., 48, 67 Dahlgren, K . V. O., 234, 312 Dale, M . B., 36, 41, 42, 63, 66, 60, 61, 63, 65, 66, 67, 68 Dandliker, W. B., 241. 313 Daniel, L., 221, 222, 312 Danon, D., 72, 74, 147 Dashek, W . V., 96, 148 David, R., 240, 312 Davies, A. J . S., 233, 312 Davies, D. R., 300, 312 Davies, E. A., 307, 316 Davies, R., 167, 214 Davies, W . E., 256, 268, 312 Davis, L. H., 286, 307, 318 Davison, R. M., 274, 312 Dawes, C. J., 93, 97, 98, 145 Dawson, C. R., 208, 209, 21 7 Deanon, J . R., Jr., 298, 312 Dear, E., 241, 317 De Bruyn, J . W., 308, 314 Deflandre, C., 10, 13, 14, 33 Delay, C., 142, 145 Dempsey, W . H., 228, 277, 312 Dennis, D. T., 73,145, 197, 214 Dennis, F. G., 276, 312 Denward, T., 232, 312 Denys, G., 224, 319 De Tar, J . E., 274, 313 De WoIff, P. M., 127, 145 Dieckert, J. W.,153, 214 Diehl, M . J., 121, 130, 145 Dietrich, J. F., 299, 303, 320, 321 Dionne, L. A., 238, 312 Dippel, L., 93, 145, 325, 36G Dixon, M., 207, 214 Doak, C. C., 234, 280, 311, 312 Donk, van M., 230, 315 Dorfman, R. I., 185, 214 Dost, W . A., 326, 356 Dougell, D. K . 160, 161, 169, 198, 214 DOUCQ,A. L., 167, 214 Dugger, W . M., 211, 215

Dulieu, H. L., 271, 312 Duncan, C. G., 326, 338, 349, 355, 356 Dure, L. S., 254, 312 Duvick, D. N., 242, 312 Dweltz, N . E., 77, 145 Dyko, K . G. H., 79, 81, 145 Dzevaltovsky, A. K., 278, 31%

E Earlo, F. R., 168, 191, 218 East, E. M., 229, 312 Eaton, F. M., 307, 312 Ebcrt, P. S., 185, 216 Edelman, J., 165, 205, 214 Edgerton, L. J., 276, 312 Edwards, A. W . F., 66, 67 Eghiazarjan, D., 226, 312 Ehrensberger, R., 296, 300, 312 von Ehrenstein, G., 188, 213 Elbein, A. D., 73, 76, 146 Elderton, W . P., 44, G7 El Murabaa, A. I. M., 227, 312 Eniswollor, S. L., 238, 260, .3I2 J':sau, K., 89, 146 Ettl, H.. 8 , 16, 17, 19, 22, 33, 34 Eunus, A. M., 290, 312 Evans, A. M., 232, 312 Excoll, J . S., 326, 356

F Fnprlintl, F., 28 1, 312 Falcono, G., 81, 90, 117, 172, 216 Farmer, R. H., 326. 357 Favre-Duchartre, M., 300, 313 Fauxett, D. W., 1, 2, 36 Fcrchau, H . A., 326, 366 Findlay, W. P. K., 324, 325, 326, 353, 356 Fischer, A., 11, 13, 14, 16, 33 Fiseher, E., 167, 214 Fitting, H., 273, 312 Flint, E. A , , 15, 34 Forti, G., 211, 215 Fostor, A. IS., 76, 14G Fowtlon, l.., 181, 214 Fratin, J. L,168, 214 Vroi, E., 75, 76, 77, 78, 85, 89, 92, 93, 101, 103, 128, 138, 142, 143,146

French, C. S., 157, 214 Frey-Wyssling, A., 69, 70, 72, 73, 86, 89, 96, 1CJ7, 113, 118, 120, 146, 153. 200, 214. 363. 350

362

AUTHOR INDEX

Fridriksson, S., 258, 312 Fridvalsky, F., 100, 146 Frimmel, G., 232, 312 Fritsch, F. E., 4, 7, 33 Fritz, G., 184, 192, 217 Fromageot, C., 159, 168, 213 Fujimoto, D., 184, 214 Fukui, H. N., 273,312 Fuller, M. S., 76. 146 Furusato, K., 277, 281. 312

G Galston, A. W., 211, 214 Galston, W. W., 80, 146 Galun, E., 307, 308, 312 Gandy, D. U., 325, 356 Ganesan, A. T . , 260, 312 Garber, K., 222, 311 Gardella, C., 233, 313 Garfinkle, D., 211, 214 Garren, R. G., Jr., 276, 321 Gartner, K. F., 273, 313 Gascoigne, J. A., 91, 146 Gascoigne, M. M., 91, 146 Gawlik, S. R., 96, 148 Geitler, L., 303, a13 George, P. V., 308, 311 Gerassimova, H., 300, 313 Gibbons, I. R., 2, 13, 17, 18, 3 3 Gibbs, M., 211, 214 Gifford, E. M., Jr., 250, 311 Gilmoro, A. R., 258, 513 Gilmour, J. S. L., 37, 40, G7 Ginzburg, I3. I., 81,146 Ginzburg, B. Z., 172, 203, 214 Giri, K. V., 167, 176, 216 Glasziou, K. T., 154, 205, 214 Glushtchenko, I. E., 280, 313 Goff, J. R., 326, 356 Gold, H. S., 326, 356 Goldacre, P. L., 279, 313 Goldstein, J. L., 157, 216 Goodall, D. W., 36, 43, 46, 47, 50, 67 Goodman, L. A,, 48, 67 Goodsell, S. F., 300, 313 Gorobec, A. M., 223, 313 Gorter, C. J., 121, 130, 142, 145, 146, 250, 251, 313 Gould, B. S., 186, 188, 214, 215 Goulding, K . J., 78, 79, 92, 147 Graf, L. H., 247, 253, 321 Green, P. B., 87, 92, 93, 98, 100, 103,

104, 114, 126, 127, 134, 136, 137, 140, 141, 142. 143, 1.10' Gremiwood, A. I>., 6, 7, 10, 13, 15, 19,

33, 34 Gregory, W. C., 301, 313 Greig-Smith, P., 36, 43, 67 Grekoff, P. I., 240, 313 Greyson, R. I., 272,273, 274,320 Griggs, W. H., 223, 274, 313 Grumstone, A. V.. 2, 13, 17, 18, 33, 152, 214 Guha, S., 271, 294, 296, 313, 314, 318 Guignartl, L., 220, 313 Giinthcr, I., 72, 76, 97, 146 Custafuon, I?. G., 238. 273, 279, 313 Gut, M., 185, 214 Guzan, I. I., 226, 314

H Haagen-Smit, A. J., 241, 250, 262, 313, 320 Haberlandt, G., 280, 313 Haccius, B., 287, 288, 313 Hadley, H. H., 308, 319 Haeckel, A., 228, 313 Hagedorn, H., 94,146 Hagiaya, K., 223, 313 Hall, C. E., 1, 33 Hall, M. A., 165, 205, 214 Hall, 0. L., 240, 313 Hallaway, M., 204, 213 Halperin, W., 284, 321 Halsoy, I)., 277, 311) Hancock, V. J. I?., 204, 213 Hannig, E., 220, 240, 313 Hansen, J. B., 11, 34 Hamon, C. H., 222, 3 1 3 Harberd, D. .J., 52, 67 Harrington, W. P.,187, 203, 214, 215 Harrison, J . S., 158, 217 Harteck, P., 222, 311 Hartig, R., 324, 356 Hartloy, B. S., 173, 213 Hassid, W. Z., 13, 75, 81, 146, 147 Haurowitz, F., 158, 214 Haustein, E., 300, 313 Hayano, M., 186, 214 Hecht, A., 232, 233, 313 Hecht, K., 161, 214 Hedemann, E., 280, 313 Hcilmann, J., 156, 169, 214 Heinen, W., 231, 513, 516

Hendricks, R. H., 166,217 Hensz, R. A., 307, 308, 313 Hepler, P. K., 86, 142, 143, 146 Heslop-Harrison, J., 40, 67, 272, 306, 300, 308, 313

Healop-Hamison, Y., 308,313 Regs, B., 211, 214 Heme, C. O., 268, 313, 314 Hestrin, S., 72, 74, 147 Heyn, A. N. J., 164, 204, 214, 215 Hillyer, I. O., 306, 307, 321 Hindman, J. L., 272, 273, 274, 320 von Hippel, P. H., 187, 203,214 Hirarnatsu, A., 172, 215 Hiroe, M., 296, 313 Hoffman, L., 9, 10, 19, 22, 33 Hofmeister, W., 219, 313 Holionka, J . , 224, 314 Holsten, R. D., 204, 298, 319 H(ilze1, H., 150, 167, 100, 178, 186, 218 Honda, S. I., 204, 215 Honoyman, J., 71, 146 Honma, S.,238, 249, 268, 313 Hopkins, C. E., 66, 67 Hopp, R. J., 308, 316 Horne, R. W., 78, 79,147 Houwink, A. L., 77, 78, 100, 104, 123, 126, 126, 127,145,146,148 Hudson, C. S., 167, 215 Hughes, D. E., 167, 215 Humphreys, T. E., 211, 215 Huskins, C. L., 299, 313 Hussey, H., 160, 217 Hutterer, F., 169, 215

I Iizuka, M., 227. 314 Ingraham, L.L.,187, 216 Inoh, S., 296, 313 Irreverre, F., 167, 215 Isenberg, I., 211, 217 Islam, A. S., 261, 314 Ito, H., 263, 272, 314 Ito, I., 306, 314 Ito,M., 286, 314 Ivanov, M. A., 280, 314 Iwakiri, B. T., 223, 274, 313 Iwanami, Y.,226, 228, 229, 314 Iwanowskaya, E. V., 249, 266, 314 Iyer, R. D., 206, 318

J

Jackson, D. S., 186, 215

Jackson, G. A. D., 276, 314 Jackson, W. T., 166, 215 Jakus, M. A., 1, 33 Jancey, R., 62 Jang, R., 79, 87, 146, 164, 206, 215 Jansen, E. F., 79, 87,146, 164, 200, 215 Jansen, L. L., 260, 314 Jaworski, E., 70, 146 Jeffers, J. M. R., 61 Jenkins, J. A., 191, 215 Jennings, A., 266, 317 Jensen, E. V., 212, 216 Jenuen, W. A., 79, 114,146, 264,312 Johnson, T. W., 326, 326, 356, 357 Johri, J3.M. 229,230,271,292,294,314 Jones, E. 13. a,,320, 348, 356 J O ~ O SJ., K. N., 197, 21li Joncu, K. S., 40, 68 Jonas, L. I<., 222, 223, 319 Jonau, M. I)., 220, 314 .Jones, Q . , 168, 191, 2Z8 J O E J J~.,, 203, 216 JORt, L., 226, 232, 314 Jung, Y., 307, 308, 312 Jiitisz, M., 169, 168, 213

K Kaliaperumal, T. T., 278, 317 Kamiya, N., 124, 134, 137, 146 Kaneko, K., 260, 314 Kanta, Kusum, 236, 230, 237, 266, 269, 314, 316, 318

Kaplan, A., 184, 185, 216 Kapoor, Manjii, 272, 279, 314, 318 Kato, H., 283, 284, 314, 315 Knto, Y., 2!M, d l 4 Kuwata, J 2f1I , 31(i KUY.E., 286. 318 KazimiorHki, T.,268, 818 Kehr, A. E., 300, 314 Kelley, J. L., 49, 67 Kendall, M. G., 40, 62. 00, 67 Kendrick, W. B., 41, 08 Kent, A. JL, 294, 296, 819 Kent, N., 261, 314 Kurr, ,.'l 162, 164, 215 Kerruidi, 14. M., 340, 3.50 Kesslcr, G., 81, 147 Kester, D. E., 268, 313, 314 Khan, A. W., 74,146 Klianna, K. R.. 296, 298, 315 Klio, Y. O., 308, 314

.,

364

AUTHOR J N D E S

Kihara, H., 298, 324 Kimber, G., 296, 314 King, J. R., 221, 224, 314 King, N. J., 79, 80, 145, 146, 153, 1601, 215

Kirby, K. S., 186, 215 Kivilaan, A., 158, 100, 205. 216 Kl(kiliooi~t,(-, A., 121, 130, I 4 5 Kliiigiiitiii, ( :. ( '., 264. 266, 321 Koch, W., 7, 13, 33 Kohlmeyer, T., 326, 355 Kojan, S., 302, 319 Kolb, J. J., 157, 217 Konar, R. N., 301, 314 Korn, E. D., 77, 147 Korschikoff, A. A., 12, 33 Kostoff, D., 300, 314 Kostoff, R., 300, 314 KovdEik, A., 224, 314 Kovsrskjj, A . E., 226, 314 Kovcs, E., 21 1 , 216 Krapivina, I. G., 349, 3.54, 356 Kregor, D. R., 77, 78, 80, 146, 147, 14b Kribbcn, F. J., 304, 300, 315 Kroh, M., 231, 316 Krull, R., 89, 117, 118, 119, 147 Kruskal, W. H., 48, 67 Kiihlwein, H., 223, 315 Kurtz, E. TJ., 305, 316 Kusano, S., 234, 273, 315 Kiister, E., 89, 103, 130, 131, 147 Kwack, B. H., 230, 311

L La Croix, L. J., 269, 315 Lahiri, A. Pi., 257, 319 Laibach, F., 220,246,255,304,306,315 Lal, M., 264, 269, 250, 296, 315 Lambert, J. M., 36, 44, 58, 63, 64, 67 Lammerts, W.E., 258, 260, 300, 312,

Laurencot, H. J., 156, 176, 189, 201, 21 7, 228 Lawley, D. N., 60, 63, 68 Lawrence, G. H. M., 37, 68 Lawson, W. H., 167, 217 Ledbottor, M. C., 85. 141, 143, 147, 161, 215

F,,8,

I A Y ! d d ( . , (>.

*7J

IAt~cuwctril~oo1~, van A., 280, 315 Lemay, P., 96,146 Leopold, A. C., 273, 315 Lesley, J. W., 258, 315 Letham, D. S., 81, 147 Lcvan, A., 298, 299, 315 Levi, M. P., 329,340,346,347,356,357 Levy, J. F., 324, 337, 340, 341, 356 Lewis, D., 229, 279, 315 Lcwis, M., 117, 148 Liese, W., 77, 147, 353, 356 Limayc, W. J., 100, I 4 7 Lintlcr, D. H., 328, 356 Lint-weaver, H., 206, 216 Liriskcns, H. F., 201, 215, 230, 231, 31.7, 315 Lipmann, F., 188, 213 Liverman, J. L., 305, 316 Livingston, G. K., 221, 315 Lloyd, F. J., 337, 338, 356 Loefflcr, F., 13, 33 Loewus, F. A., 224, 319 Lofland, H. B., Jr., 253, 315 Logan, M. A., 159, 167, 216 Lord, W.J., 276, 315 Love, A., 304, 315 Lvve, D., 304, 315 Luckwill, L. C., 238, 242, 273, 274, 315 Lund, H. A., 279, 315 Luther, A., 2, 7, 33 Lyndon, 1%.F., 81, 147

315

Lamport, D. T. A., 81, 89, 105, 147, 156, 160, 161, 162, 169, 179, 183, 184, 185, 188, 195, 197, 198, 205, 207, 215 Lance, G. N., 03, 68 Lang, A., 307, 308, 312 Lang, N. J., 18, 3 3 Larter, E. N., 269, 315 LaRue, C. D., 189, 201, 217, 244, 206, 315, 319

AllcLarw,S.R., 243, 31.5

AUTHOR INDEX

McLean, S. W., 261, 290, 315 MacMillan, J., 279, 315 Macmughton-Smith, P., 39, 41, 42, 47, 63, 66, 67, 63, 65, 66, 68

Madhosingh, C., 366, 356 Maekawa, F., 284, 315 Magoon, M. L., 296, 298, 315 Maheshwari, P.. 232, 236, 236, 237, 239, 264, 266, 209, 270, 273, 280, 280,290,292, 309,314, 315, 316 Mainx, F.. 16, 33 Majumder, S. K., 230, 311 Makita, M., 158, 217 Manner, G., 186, 188, 215 Manning, J . M., 185. 216 Manton. I., 1, 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 16, 10, 17, 18, 19, 22, 3 3 , d l Mapos, M. 0.. 281, 294, 295, 299, 316,

319 Marco, G., 76, 146 Mardones, E., 186, 214 Margerie, C., 87, 147 Marks, E., 231, 312 Mar& E., 211, 215, 217, 243, 316 Martens, P., 163, 215 Martin-Smith, C. A., 87, 145 Marx-Figini, M., 75, 147 hesart, J., 273, 316 Matchett, W . H., 79, 81. 87, 90, 147 Mathan, D. S., 191, 215 Mathon, C. C., 240, 316 Mathur, R. S., 278, 320 Matsubera, S., 251, 316 Mauney, J . R., 249, 253, 254, 316 Maxwell, E. A., 60, 68 Mears, K., 281, 319 Meeuse, A. D. J., 89, 147 Mehrota, N., 229, 317 Meier, H., 77, 89, 147, 327, 3211, 383, 356 Meister. A., 168, 186, 188, 214, 216, 217 Merry, J . 215, 316 Mertz, D., 204, 216 Meyers, S. P., 326, 356, 357 Miller, E. K., 165, 217 Miller, R. W., 168, 191, 218 MiUett, M. A., 168, 197, 217 Millman, B., 73, 75, 76, 144, 147 Mills, J. A., 168, 214 m e r , H . W., 167, 214 Mitchell, R. L., 168, 197, 217 Mitra, J., 299, 316

365

Mockett. L. G., 42, 83, 56, 66, 68 Mohan Ram, H. Y., 241, 285, 319, 321 Mohr, H . C., 307, 308, 313 Monty, T<. J., 187, 214 Moor, H., 85, 108, 114, 147 Moore, A. T., 160, 317 Mooro, R. H., 307. 316 Moore, S., 158, 217 Moore, W . E., 162, 168, 197, 217 Moreland, D. E., 264, 266, 321 Morgan, D. T., Jr., 300, 311 Morishima, H., 267,316 Morita, K., 167, 215 Morrb, D. J., 165, 200, 216 Miihlothalor, K., 72.85, 107, 146, 247 Miihlpfortlt, H., 16, 34 M i l k , 14. M., 279, 316 Murakemi, M., 172, 816 Murneok, A. E., 241, 242, 243, 31.3, 315, 316 Murty, B. R., 228, 234,319,320 Myom, A. 85, 93, 98, 145, 147

N Naithani, 8. P., 303, 316 Nnkajima, T., 246, 253, 257, 316 Nakamura, S., 296, 316 Nance, J . F., 79, 81, 87, 90, 147 Narohashi, Y., 172, 216 Nasrallah, M. E., 308, 316 Natarajan, A. T., 296, 298, 316 Xeweschin, S. G., 220, 316 Naylor, A. W., 307, 316 Naylor, J . N., 254, 269, 298, 310, 31& 316, 318 Ncalo, S., 181, 214 Nobel, 13. R., 222, 316 Nocttlharn, It. M., 40, RY N~tctly,1’. M., 241. :J17 Ncldw, J. A., 44, 68 Ntirnoc. U., 280, 316 Nciihwgcr, A., 107. 213, 2L6 Ncufc:ld, R. F.,73, 81, 117 Sournen, It. E., 169, 216 Nowcomb, E. H., 81, 85, Hfi, U!#, 142, 143, 146, 247, 161, 204, 216 Xewoll, L. C., 226, 314 Nichol, M. A., 292, 312 Nickcll, L. G., 166, 216, 255, 260, 316 Nickerson, W . J., 81, 90, 147, 172, 173, 216 Nicolai, E., 92, 98, 101, 147

366

AUTHOR I N D E X

Niel, C. B. van, 3, 34 Nielsen, N., 223, 316 Niimoto, D. H., 265, 316 Nishi, S., 261, 316 Nitsch, J. P., 238, 241, 266, 279, 305, 316 Nomoto, M., 172, 216 Norkrane, B., 72, 89, 147 Norstog, K. J., 244, 250, 251, 252, 317 Northcote, D. H., 69, 76, 77, 78, 79, 81, 92, 147, 148, 156. 160, 161, 169, 188, 190, 198, 205, 207, 215,217

0

Oaks, A., 211, 217, 262, 317 Oaks, K., 9, 11, 17, 34 Ogawa, Y., 255, 317 Ohad, J., 72, 74, 147 Ogle, J. D., 167, 216 Oliver, A. C., 326, 357 Olmo, H. P., 222, 223, 317 Olson, A. C., 166, 172, 182, 183, 185, 191, 198, 200, 216 Ordin, L., 87,147, 154, 158, 213,216 Osborne, D. J., 274, 317 Otto, H., 238, 313 Ouchi, T., 172, 215

P rai, It. A., 298, 317 I'aintor, T. J . , 197, 215 Paleg, L. G., 255, 317 Pandey, I. C., 278, 320 Pandey, K. K., 227, 232, 31 7 Parke, M., 7, 8, 9, 11, 12, 14, 15, 16, 17, 28, 34 Partridge, 8. M., 158, 187, 216 Patau, K., 299, 317 Patchett, A. A., 180, 217 Pate, S., 157, 214 Patil, R. K., 299, 317 Pease, D. C., 2, 34 PBaud-Lenoel, C., 87, 147 Perrault, J., 91, 147 Perry, M. B., 158, 216 Person, C. O., 298, 310 Peterkovsky, B., 185, 216 Peters, D., 16, 34 Petersen, B., 13, 34 Petersen, J. B., 11, 34

Peterson, D. H., 185, 214 Peterson, E . E., 306, 317 Petrochenko, U. A., 225, 31 7 Pet& E., 307, 317 Pettett, A., 60, 68 Pew, J., 353, 357 Pfeiffer, N. E., 223, 317 Phillips, J. P. N., 51, 68 Phimey, R. O., 241, 255, 273, 312, 317 Picken, L., 204, 209, 216 Pieczur, E. A., 282, 317 Pinon, L. C., 327, 328, 357 Pitelka, D. I%.,3, 8, 13, 14, 16, 34 Poddubnaya-Arnoldi, V. A., 264, 317 Pollard, J. K., 167, 177, 180, 185, 216, 217 Pond, V., 302, 319 Poore, M. E. D., 68, 68 Porter, C. A., 156, 218 Porter, K. R., 1, 2, 33, 85, 141, 143, 147, 161, 215 Prasad, A., 229, 317 Pratt, C., 277, 317 Preston, R. D., 71, 72, 74, i 5 , 76, 77, 78, 79, 80, 85, 89, 92, 93, 97, 98, 100, 101, 103, 114, 123, 124, 128, 129, 134, 137, 138, 142, 143, 145, 146, 147, 148, 153, 167, 197, 214, 216, 347, 354,357 Price, E. A. S., 326, 357 Priostley, J. H., 152, 153, 157, 196, 202, 218 Primttv, 1'. E., 278, 312 Pringshcirn, E. G., 8 , 34 Pringshcirn, N . , 2, 7, 34 Privat, G., 265, 617 Probine, M. C., 79, 80, I(JfJ, I 14, 123, 124, 128, 129, 134, 135, 137, 148 Prockop, D. J., 184, 185. 216 Procter, D. P., 158, 217 Proctor, J. R., 41, 68 Proctor, P., 344, 354. 357 Prosser, M. V., 278, 314 Puri, P., 272, 317 Purves, W. K., 80, 146, 211, 214

R Radhakrishnan, A. W., 167, 176, 216 Radley, M., 241, ,317 Raghavan, V., 224, 220, 247, 249, 262, 263, 317

367

AUTHOR I N D E X

Ram, M., 304, 306, 317 Ramctchsndrrtn, G . N.. 187, 216 Ramsbottom, J., 324, 357 Ranby, B. G., 72, 89,147 kdhaW8,

s. s.,

229, 318

Randolph, L. F., 268, 317 Rangan, T. S., 261, 266, 317 Rangsswami. G., 278, 306, 310, 317, 318

Rangsswamy, N. S., 236, 237, 244, 246, 263, 264, 261, 266, 266, 280. 281,282,280,292,294,314, 316,317 REO,A. N., 263, 292, 317 Rao, c'. R., 61, 68 W, P. S., 244, 246, 317 Rappaport, J., 240, 260, 327 Rmhid, A., 261. 314 Ran, M. A., 268, 317 Ray, P. M., 79, 80, 114, 148, 172, 190, 216 Ray, W. J., 166, 188, 213 R a p , D. G., 14, 16, 17, 22, 34 Rebeiz, C. A., 276, 317 Rebstock, T. L., 277, 318 RBdei, G., 268, 31 7 Redemann, C. T., 277, 318 Rees, M. W., 168, 214 Rehm, S., 307, 31 7 Reichart, G., 101, 148 Reinert, J., 283, 317 I$migno, A., 66, 68 Retovskf, R., 307, 317 Reynolds, E. S., 326, 556, 357 Rhodes, A,, 211, 216 Richards, F. J.. 44, 68 Richmond, & H.. I.181, 214 Rietsema, J . , 234, 241, 242, 243, 310, 318 Rijven, A. H. G . C., 247, 249, 263, 318 Riley, R., 296, 314 Ripley, G . W., 93, 147 Risley, E. B., 89, 146 Ritzel, M., 241, 317 Robertson, A. V., 167, 215 Robertson, W. van B., 188, 216 Roelofsen, P. A., 69, 74, 76, 77. 78, 79, 81, 82, 85, 86, 91, 93, 94, 96, 97, 98, 100, 101, 103, 104, 106, 107, 108, 111, 113, 114, 116, 116. 120, 121, 122, 123, 125, 126, 128, 132, 133, 134, 136, 137, 139, 146. 147, 148, 152, 210, $16, 362, 363, 357

Rogers, D. J., 66, 68 Rohlf, F. J., 46, 60, 68 Rollins, M. L., 162, 160, 217 Rosen, W. G., 96, 148, 229, 318 Roth, L. E., 11, 34 Rottenberg, D. A., 79, 148 Riidall, I<. M., 77, 148 Ruosink, A. W., 156,216 Ruge, U., 154, 216 Ruhland, W., 71, 91, 148 Rutnor, A., 166, 176, 189, 201, 217 RycekowHki, M., 249. 206, 318

S

Sabharwal, P. S., 266, 281, 311, 318 Snchar, R. C., 286, 209, 271, 278. 279, 281.286,294,296,311,310.318

Sachs, I. B., 328, 363, 357 Saeman, J. F., 168, 197, 217 Sagawa, Y., 266, 316 Saito, T., 306, 314 Sfblorn6,M. M., 97, 148 Samborski, D. J., 211, 217 Sanders, M. E., 262, 263, 318 Sartoris, G . B., 222, 318 Sassen, M. M. A., 89, 98,148 Sstina, S., 234,241, 242,243, 247.310, 318

Sato, C. S., 164, 217 Satyanarayana, G . , 306, 318 Savelli, R., 225, 318 Savory, .J. G., 324, 326, 326. 327, 328, 349, 35.5, 358, 6.57 fjawada, Y., 22U, 318 Sfiodrina, It. N., 228, 318 S c h d l t , H., 324, 342, 367 Scharpenscml, 11. W., 166, 217 Schleiden, M. .I ., 2 19, 318 Schliisser, K., 231, 318 Schliiswr-Szigat,G., 267, 318 Schmid, R., 77, 1 4 1 Schmuckor, T., 224, 318 Schneider, W. C., 163, 204, 217 Schoch-Bodmer,H., 113, 148 Schooler, A. B., 264, 266, 318 Schooley, C. N., 16. 34 Schram, E., 168, 217 Schrauwen, J. A. M., 230, 315 Schroeder, C. A., 277, 286, 318 Schulz, G. V., 75, 247 Schwartz, B.. 188, 218

368

AUTHOR INDEX

Schwendener, S., 192, 217 Scott, F. M., 93, 97, 98, 104, 108, 117, 145,148 Scott, T. A., 156, 217 Seaton, J. C., 279, 315 Sebok, 0. K., 185, 214 Sobcstyon, G . S., 38, 68 SBchet, J., 240, 312 Sehgal, C. lJ., 271, 294, 314, 318 Sehgal, Nanda, 296, 301, 320 Sell, H. &I., 273, 277, 312, 318, 321 Sotterfidd, G., 69, 73, 79, 80, 81, 82, 87, 88, 111, 116, 128, 144, 145, 148, 153, 160, 213 Shah, S. S., 260, 312 Shalucha, B., 295, 310 Shanmugavelu, K. G., 278, 318 Shentz, E. M., 241, 318, 319 Ghapiro, B. M., 185, 216 Shaulis, N. J., 277, 317 Shaw, M., 211, 217 Sheehan, J. C., 159, 167, 21 7 Shepard, R. N., 61, 68 Shepherd, K., 227, 243, 318 Shibukawrt, J., 223, 320 Shigenaga, Y., 11, 34 Shiinakura, K., 301, 318 Shimbayaxhi, R.,160, 161, 169, 198, 214 Shockman, G. D., 157, 217 Shoji, T.,301, 321 Sirgosmuntl, K. A., 96, 148 Siepmann, R., 326, 357 Simpson, G. M., 254, 316, 318 Singer, E. J., 159, 215 Singh, J. P., 229, 318 Singh, M. P., 299, 320 Singh, S. N., 222, 318 Sinoto, Y., 301, 321 Sircar, S. Ri., 257, 319 Sirokma'n, F., 211, 215 Sitte, P., 86, 148 Siu, R., 260, 262, 320 Sjaholm, V., 108,148 Skelton, C., 156, 217 Skirm, G. W., 258, 260, 319 Skrebcov, M. F., 227, 310 Smith, G. M., 4, 7, 34 Smith, H. H., 298, 299, 319 Smith, L.,298, 319 Smith, P. G., 259, 319 Smith, R. H., 168, 217

Sneath, P. H. A., 35, 36, 37, 40, 41, 43, 45, 48, 49, 50, 55, 64, 65, 68 Snowden, J. E., Jr., 153, 216 Sokal, R. R., 35, 36, 37, 40, 41, 43, 45, 48. 49, 60, 55, 64, 65. 68 Solomon, 13., 261, 319 Soria, V. J., 224, 319 Sparrow, A. H., 302, ,319 Sparrow, D. H. B., 255, 317 Spector. C., 277, 318 Spit, B. J., 100, 108, 147, 148 Bpoerl, E., 263, 319 Spurr, A. R., 224, 319 Stahman, M. A., 247, 253, 321 Staniur, It. Y., 3, 34 Stanley, R. G., 224, 319 Stark, 0.R., 208, 209, 217 Starnes, W. J., 308, 319 Stecher, H., 119, 148 Stein, H., 63 Sterling, C., 108, 148 Stenheimer, E. P., 244, 319 Stetten, M. R., 177, 217 Stevens, M. G., 340 Steward, F. C., 81, 147, 148, 165, 167, 177, 180, 185, 216, 217, 241, 281, 294,295,299,309,316,318,319 Stewart, W. S., 277, 319 Stingl, G., 240, 319 Stono, C. L., 222, 223, 319 Storic, N., 186, 188, 216, 217 Stout, E., 184, 1!+2, 217 Stow, I., 303, 319 Strain, H. H., 7, 34 Strasburger, PI., 10, 34, 221, 234, 280, 319 Straub, J., 229, 230, 319 Strnus, J., 189, 201, 205, 217, 244, 319 Strugger, S., 86. 89, 1111 Strun, hi., 240, 319 Strunk, C., 96, 98, 148 Subramanyam, K., 239, 319 Suhr. K1. A., 222, 311 Sulbha, K., 260, 319 Sulima, Ju. G., 226, Ql9 Sultze, R. F., 190, 217 Suter, P. J., 279, 31.5 Suzuki, E., 277, 312 Swain, T., 167, 224 Swarninathan, M. S., 227, 233. 234, 260. 296, 298, 299, 312, 316, 317, 319, 520

AUTHOR INDEX

Sweeley, C. C., 158, 217 Szent-Gyorgyi, A., 211, 217

T Takami, W., 229, 320 Takaehima, S., 232, 320 Takata, T., 124, 134, 137, 146 Takeuohi, M., 283, 284, 314, 315 Tamwki, T., 244, 320 Tamblyn, N., 326, 357 Tamiya, N.. 184, 214 Tanimoto, T. T., 65, 68 Taylor, J. H., 302, 320 Tazawa, M., 124, 134, 137, 146 Teaotia, S. S., 278, 320 Tepfer, S. S., 272, 273, 274, 320 Teubner, F. C., 273, 312, 320 Thiele, T., 139, 148 ThiBry, J. P., 2, 33 Thimarm,K. V., 80,148, 162,168, 160, 214, 217,273, 320 Thomas, M. D., 165, 217 Thompson, A. H., 232, 311 Thompson, J. F., 165, 217 Thompson, P. A., 274, 320 Thornber, J. P., 79,148, 190, 217 Thuret. G., 2, 3.4 Timell, T. E.. 169, 195, 217 Tinsley, J., 196, 218 Tiitin, A. I., 226, 320 Tjhio, K. H., 167, 214 Toda, M., 261, 316 Toennies, G., 157, 217 TomkovB, J., 230, 320 Tonzig, S., 211, 217 Torrey. J. G., 247,249,262,263,317 Treiber, E., 71, 148 TreveIyan, W. E., 158, 2!7 Tripp, V. W., 152, 160, 217 Tristram, H., 168, 181, 214 Tsunewaki, K., 298,314 Tukey, H. B., 258, 320 Tulecke, W., 156, 176, 188, 189, 201, 216, 217, 301, 320 Tupper-Carey, R. nf., 162, 153, 157. 196, 202, 218 TupJi,J., 231, 320

U,N., 296, 320

U

Udenfriend, S., 184, 186, 210 Uhring, J., 238, 260, 312 X

369

Ullstrup, A. J., 244, 320 Uota, M., 274, 311 Ushirozawa, K., 223, 320 Uttaman, P., 256,320

V

Vacin, E. F., 263, 320 Van Etten, C. H., 168, 191, 218 Van Iterson, (I. 121, , 130, 145 Van Overbeek, J., 241, 260, 261, 202, 280, 320 Vansell, G. H., 223, 313 Varner, J. E.. 208 Vasil, I. K., 222, 223, 229, 302, 303, 314, 320 Veen, H., 255, 320 Verall, A. F., 325, 357 Vestal, M. R., 325, 355 Visser, T., 221, 222, 320 Vlk, W., 13, 34 Von Barrgen, G., 273, 320 Von Witsch, H., 130, 149 Vozda. J., 226, 320

W Wadhi, Mridul, 286, 320 Wagnor, M., 108, 218 Wain, R. L., 274, 277, .?17, 320. Walkor, Q. W. R., 299, 303, 320, 321 Wall, J. R., 261, 321 Welters, N. E. M., 326, 357 Wang, L., 70,146 Wardlaw, C. W., 272, 321 Wardrop, A. 13.. 09, 104, I O X , 113, 114, 126, 149, 363, 357 Warrelmann, E., 325, 357 Wartenberg, A., 89, 149 Watkins. D.. 186, 216 Watzke, E., 169, 169, 214 Weaver, J. B., Jr., 261, 321 Weaver, R. J., 277, 321 Webb, T.E.. 75, 149 Webber, J. M., 76, 146, 296, 321 Webster, G. T., 257, 258, 321 Weinstein, L. H., 150, 176, 189, 201, 217, 218

Woisblurn, 13., 188, 213 Wollor, 1,. 15., 277, 621 Wellnor, D., 108, 214 WCllH, w. w., 168, 217 Went, F. W., 2fi3, SOG, 316, 880 Wost, C. A., 241. .?I7

370

AUTHOR INDEX

Wetherell, D. F., 284, 321 Wetmore, R. H., 176, 177, 218 White, D. G., 276, 315 White, P. R., 250, 321 Whitehead, D. C., 196, 218 Whitehead, R. A., 221, 321 Whitehouse, W . E., 222, 223. 319 Whitney, J. G., 169, 21 7 Wieland, H., 167, 218 Wiessner, J., 162, 204, 218 Williams, C. N., 264, 321 Williams, S. E. F., 158, 224 Williams, W. O., 227, 277, 322 Williams, W. T., 41, 42. 44, 53, 66, 58, 61, 63, 64, 65, 66, 68 Wilson, B. F., 107, 149 Wilson, C. M., 158, 159, 190, 195, 218 Wilson, G. B., 299, 321 Wilson, I. M., 325, 357 Wilson, K . S., 263, 312 Winkler, A., 186, 216 W i t h e r , C. L., 263, 264, 321 Witkop, B., 167, 180, 215, 217, 218 Witter, R. F., 157, 214 Wittwer, S. H., 241, 242, 243, 273, 211, 305, 306, 307, 311, 312. 313, 316, 318, 321

Wolff, I. A., 168, 191, 216 Wong, C. Y., 278, 321 Wood, F. M., 152, 218 Worsham, A. D., 264, 265, 321

Y Yakar-Olgun. N., 278, 321 Yakovleva, S. M.. 283. 311 Yamadu, T., 301, 321 Yamamoto, Y., 211, 218 Yctmasaki, Y., 240, 321 Yaauda, S.,273, 279, 29H, 321 Yllnev, S., 353, 356 Young, M., 91, 147 Yule, G. U., 46, 68

z Zachau, H. G., 167, 217 Zagaje, S. W., 258, 321 Zaitlin, M., 81, 149 Zajceva, Ju. F., 226, 321 Zatyk6, J. M., 276, 321 Ziebur, N . K., 247, 249, 253, 262, 318, 321 Ziegenspeck, H., 117, 1.19 Zielinski, Q. B., 276, 321 Zillig, W., 156, 167, 160, 178, 186, 218

Subject Index A flower sex, 305-308 fruit development, 209 Aconte, 0 Algae, blue-green 8ee ccleo individucclly inhibition of root formation, 283 ovary culture, 266 mdcalgae Christensen’s survey, 3-6 parthenocarpy, 279, 308 diversity of flagellar bases, 18 pith cells, 204 growth, 98 sex expression, 305-308 synthesis in seeds, 238 list of, 4 membrane system, 4 wall plestioity, 164, 210 microfibrillar arrangement, 92,98, Avena coleoptiles, cell ultrastructure, 79, 87, 106, 107-110,114, 127 103 microfibrillar componenh, 77 unity with major land plmtn, 7, 0 B Algao, brown ace ale0 individunlly B e t h , 61 named algae Brooding cycle of fruit tmon, 258 flagdar ~ p h e s 13 , growth, 08 microfibrillar arrangemont, 98, Cell 103, 105 membrane systems, 4 microfibrillar components, 77 uniflagellate spermatozoid, 8 middle lamella syntheais, 117 migration of wall substences, 116 Algae, red see also individually named origin of pores and perfofstions, algae 120 absence of flagelle, 4, 20 pit fields, 105, 117 growth, 98 plasmodesmata, 89, 105, 106, 113, list of, 4 117 microfibrillar arrangement, 98, suspension cultures, 155 103, 106 Cell growth Bee ale0 Mdcrof&&? (which microfibrillar components, 77 include8 referencea to 8peCke8) Allium, chemical content of cell walls, auxins, 164 79 collenchyma celh, 111 Androgonesis, 300 Anther culture, 301 cytoplasmic element theoriacl, I41 displrtoernont of microfibrilH, I I4 Allium cepa, 302 W u m stmmonium, 301 offect on wall Btructure, 114 effect of chemicals, 303, 304 ctpidermal colh of apical mnrigrowth media, 302-304 Htern.9, 108 Lilium longiflorum, 301 growth volocity. 73, 90, 121, 12n Lywperakum eemclentum, 301 isodiamotric cells, 9 I, 104 male and female potency of latex v w o h , 108. 113, 114, 148, pollen grain^, 303 130 m e W , initiaOion of, 302 lengthening, 106 Rhoeo diecolar, 302 middle lamells synthewis, 1 17 Tmdeecanticc, 301, 302, 303 multinet growth, 122, 132 Trillium eredum, 302 multiplicstion of pltwmadesmata AUXh and primary pit fields, 117 anther culture. 302 parenchyme cells of apical meriendosperm, 241, 244 stems, 106

c

372

SUBJECT INDEX

Nicotinia tabacuni, 197 Crll growth coiit. perforation of Chnetomorpliu cells, passive growth by tissue tension, 89 128, 130 physiological dissolution, 89 pectin metnbolism, 154 primary walls, A!) perforation of growing walls, 90 sycnmore, 196, 198 pores and perforations, origin, 120 yonsts, 90 protein, role of, 153 zygotes of Phyomyces, 89 protoplasm streaming theory, 134, 139 Cell wall reorientation of microfibrils, 120 amino acid composition, 168-17 1 spiral growth in cells with heliccolluloso content per cent, 82 ally crossed lamellate walls, 138 definitions, 69, 152 spiral growth in cells with multienzymes, 81, 171, 194, 204-208 net structure, 132, 136 form, role of protein, 202 tearing in conditions of rapid fractions, isolation, 156, 157 growth, 131 metabolism, 81, 204 tip growth, 74,96, 103, 113, 126 nitrogen content, 160 tubular cells growing both in tip protein, 81, 88, 151 and sidewalls, 103 for detailed entries see Protein tubular cells growing in sidewalls site of synthesis of primary wall only, 98 substances, 85, 88, 182 tubular cells with tipgrowth only, tensile strength, 200 96 transition layer (Uebergangslaultrastructure see Microfibrils melle), 70 wall extensibility, 83 water contcrit pcr cent, 82 wall strem, 70, 139 Coll-wall plasticity wi$ening, 112, 127 auxin8, 154, 204, 210 Cell, intorrelation botwoen growth and rncthgl ostor coritont, 154 wall ultrastructure pectin, 164 offect of growth on wid1 striictiirt!, ('ell wall, primury, conHtitutiori and 114 morphology of rriicrolihrils see ulso offect of wall etructuro on clirocMirrofi bri Is tion of growth, 128 cdlulose rnicrofitjrih, 70 mechanism of orientated initial phitin microfibrils, 76 synthesis of cellulose rnicromicrofibrils of ottror materials, 77 fibrils, 139 Cell wall, pr~mary, constitution . of Cellulose amorphous matrix see ulso Hydromicrofibrils, 50, 139 xyproline, Protein synthesis, 73, 85, 116, 139 analysis 78 Cell-wall analysis see Protein meristematic cell walls, 78 Cell -wall breakdown and degradation protein component, 81, 88, 151 see also Fungi Cell wall, secondary, 69 Acer pseudoplatanus, 197 attack by fungal cellulase, 89 Cell-wall ultrastructure see abo MicroCentaurea cyanwr, 197 fibrils chemical degradation, 193 interrelation between growth and enzymic degradation, 171, 194, wall structure, 114 200 microfibrillar arrangomant in difformation of plaamoclesmata, 89 feI'Wlt tyJ)(# O f KrCJWirlK CC*llh, 0 1 Gingko. I96 rriorphologicul m p w t y of C f J r l d . Lycopersicon esculenturn, 197 lion, xynthoHiH and tirc:z~kclowri meristematic cells, 89 of' growing wall, 70, 177, 193

SUBJECT INDEX

Chars a w t d i a , chemical content of cell walls, 79 Christensen’s Phyletic scheme, 5 Classification ~ e eaLo Mathematical models in numerical taxonomy basic axiom, 37 hierarchical and non-hierarchical clmsifications, 42, 01, 62 maximization, 38 monothetic and polythetic clmsifications, 37, 03, 04 numerical taxonomy, 36 phyletic implications of flagellar structure in plante, 1 probabalistic and non-probabalistic classifications, 43 “similarity” analysw, 64 subjective classification of com. plex data, 59 “underlying factor” hypotheses, 00 unmeaaured attributes, 01 Cormophyta cell wall substances, 77 structure of growing cell^. 09, 100

D Degradation of wood aee Fungi Dormancy, 254, 259

E Embryo dependence on tho endosperm. 289, 262 embryo-endosperm transplantations, 240 “embryo factor” in coconut milk, 241

373

hybridization, 220, 238, 266 et 8eq. incompatibility with endosperm, 257

limitations of ombryo culture, 202 osmotic shock, 247 oxygen concentration, 260 post-germinal development, 250 progerminal development, 247, 250

specific growth factoru, 249 tumorous growths, 260 1Smbryology in relation to physiology and genntics, 219 aee also detuiled individual entriea, P.B. Anther, PolEen, etc. androgenesis, 300 anther culture, 301 embryo, 237 embryo culture, 246 endosperm, 240 fertilization, 231 ovules, culture, 263 ovaries and flowers, culture, 206 parthenocarpy, 273 polyembryony, 280 parthenogenesis, 296 pollen, 221 sex expression, control of, 304 Endosperm abortion of seeds, 242 constituents, 240 chemical analysos, 242 culturc, 243 tlcgenuration, 243 dCpCndoIlC0 Of tho ( ! IIlt J r y ~J , 239, 262

cmbryo-ondortprm trtmqplarltrrtiom, 240 “ombryo-fsc.tor” in coconut milk,

growth in relation to seed development, 237 24 1 growth-regulating substances, 241 growth-robwletingsubstances, 241, specificity of the endosperm, 239 242, 244 substitutes for the endosperm, hormonal influence on plant meta289, 246, 202 bolism, 243 Embryo culture role in seed development, 242, 262 applications of embryo culture, 255 Enzymes breeding cycle, 268-280 degradation of cell walls, 171,194, cultural conditiona, 247 200 dormancy, breaking of, 254, 269 role in soft rot, 329, 352. 364 excision, 247 wall-bound enzymes, 81, 204, 206 growth media, 247, 250, 268, 259, Eucaryota, 3 262 Extenein see Hydroypolinc, Protein

374

SUBJECT INDEX

Cystoseira, 20 F Dictyota, 8 , 13, 19 Fertilization, control of h p m l d i a , 9, 19 diploid/tetraploid incompatibility, Dryopteria. 10 234 euglenoids, 7 , 8, 16, 21, 23 hybrids, 233, 236 Fucwr serratua, 11, 19, 20, 2 I intraovarian fertilization, 234 Haematococcua pluvialis, 16. 28 in witro fertilization, 237 Halidrye. 20 treatment of the stigma, 232 Halonphuera, 16, 17, 29 treatrnunt of tho style, 232 Haptophycoae, 8, IS, 21 Flagella Heterornaatiz, 16, 17, 19, 29, 31 abnormalitiut+,1 1 Himanthalia, 13, 14 acroncmatic, 10, 12, 14 Isochrysie, 8 artifacts, 10, 14 Loxophyceae, 8, 12, 15, 17, 19, basen, 17 21 distribution of 9 +2 type, 3 Mallomonae, 8 E’limmergeissel, 12, 14-16 Marchanticr, 10 “hairs”, 10, 14 Mioromonaa pudla, 3, 8, 10, 12, heterokont and isokont conditions 17, 25 tions, 6 M i c r o m o m squumata, 11, 15, 16, mastigoneme, 14 17, 29 multiple, 9 Monmruwtix, 8, 12, 25 pantonematic, 14 Nephroselmis gilva, 17 Peitschengeissel, 12, 14 Oedogonium, 9, 10, 19, 30 phylectic implications, 1 Olpidium, 8 roots, 19 Pedinmnonae, 8 , 10, 15. 19, 24 scales, 17 Phaeophycecte, 6, 14 shape, 10 Phtymonas, 9 spines, 13 Polytome, 18 stichonematic, 14 Prasinophyceae, 8, 16, 17, 19, 21 structure, 1 Prosinoclaele p l a t y n z m , 17 symmetry, 13 f’rymne&um pamum. 18. 31 tornentuni, 1 (i unifiagellatiori, 8 I’termnan, I7 Flagellate cells l’yr~m~imoru~s, U, I I , I 7 Allomycea, 8 Prymnmim, 8 C‘huetomorpha,19 , Y c y t o ~ i p h 16, , 22 C‘hiJomwws, 16 Aphupum, 9, I 0 Chlamydomonaa, 9, 21, 26 dyper?n&lzflfJfYh, 9, 12, 2fi Chlorochitridion tuberczhta Vi4A’tiyeoclonium, 9, 19, 3 1 cher, 15 Thahs-, 14, 15 Chlorogonium roaae, 27 Ulothrix, 9, 19 Chlorophyceae, 7, 8, 19, 21 Vaucheriu, 6, 7, 10 Chlorophyta, 6, 7, 8, 15, 16, 21 Xanthophyceae, 6, 7, 14 Chordaria, 11 Xiphophora, 13, 14 Chromophyta, 6,7, 10, 18, 19, 21 Floral e n v e l o p and fruit developChrysophyceae, 18 ment, 268 Cryptomonads, 16 Fruit set Chrysochromulinu, 8 auxins, 238, 273-279 Chrysophyceae, 6, 7, 8, 14 chemical agents, 273-279 Cladophorrtles, 9 parthmocarpy, 273-279 Coccolithophoridae, 8 Fungi ., Contophora, 6,8, 21 otiitiri, 76, 77

37s

SUBJECT I N D E X

Fungi conf. flegella, 8 microfibrillar arrangement, 91, 98 Fungi, soft rot alkali uolubilit,y of deaaying wood, 327

antltoniical charac,,kristics of timber, 328, 344, 349 Anona laet+ata, 324,326 Ascomycetes, 324, 326 atteok, chemicala favouring, 326 attack, rate of, 328, 329, 340, 360, 362

attack, variations accgrding to speciea of fungus, 344 attack, variation in mode of according to 'species of wood, 340

13a&liomyc:otuu, 324, $26, 348, 363, 366

bnech, 326, 326, 327, 338, 339, 340, 342, 349, 352

birch, 342, 344, 348, 363 brown rots, 324, 327, 364 Camarosporiurnambiena, 344,347 Caryota urens, 324, 342 cellulose, 327, 329 CepWspOtium sp., 346, 341 Cblmnitlm globosum, 326. 326, 327. 328, 329, 338, 339, 340, 342, 344,347,349,360,363,364

chemical analysis of degraded wood, 329 chemical nature of degradation process, 327, 354 coniferous woode, 326, 326, 328, 338

Coniophora cerebella, 327 Coniothyriumfuckelii, 344, 346 definition, 326 Ih.acaena dram, 324 enzymes, role of, 329, 362, 364 Epiwwum nipurn, 340 Fungi Imperfecti, 326 hardwoods, 328, 338 Hernandicr s o m , 324

histology, 326 history, 323 lectohing, 338, 360 lignin, 327, 328, 329, 363 marine fungi, 325, 326 microscopic exambetion, 328

mode of action, 330 natural durability of timber, 326 penetration, decay. 339, 349, 361 penetration of medullary rayu, 339, 360, 363

penetration, pawive, 330, 342, 360 pcntotians, 327 Phoma sp., 346 Pinua aylvealris, 338, 339, 340, 348, 360, 351, 363

Poria montkh, 327 posts in ground contact, 348 Pyrenomycetes, 326 Sabal umbmcilifm, 325 Scots pine, 338-340, 348, 350, 361, 363

Sequoiu sempervirena, 327 npocius, list, 348 Rpeciw, variation in attwk. 344 Sphaeronema sp., 344,347 Stackybotrys sp., 328 Stysanus stemonitia, 344, 347 Taxodium dktiehum, 326 technique for study, 337 water cooling towers, 324-326 white rots, 324, 327 wood-preserving substances, 326, 354

Fungi, soft rot, histology, 329 decomposition stages, 333-337 fibres, 333, 337 geometric figures, 333, 336 hyphae penetration of cell wall, 329. 337

irregularly developed formR, 333, 337

parenchyma celk, 336 tracheid~,333, 337 vowel walls, 338

c Genetic# and ernhrydogy, 210 Germination see also Fertilization,Polien ambient humidity, 224-227 dicarboxylic acids, 226 maw effect. 226, 230 metals, 224, 231 stored pollen, 223

376

SUBJECT INDEX

Cassytha J l i f o r ~ v ~ i26 s, 1 H Cattleya, 262, 263 Haploids Chrysanthemum, 260 Aegilops cauduta X Triticum nestiCorchorus, 260 vum, 298 cotton, 260 androgenesis, 300 Cucurbita, 261 angiosperms, 296 Cuscuta re$cxa, 261 Antirrhinurn majus, 296, 300 Datura, 233, 234, 242, 261, 290 Beh vulgar&, 298 Dendrophthoe falcata, 26 1 CapSiCUm fT?AtESCEnS,300 Elymus/Triticum, 255 CitruUw udgark, 299 G'ossypium, 200 Crepis t e c t m m , 300 ffordeum hexapodium/Helrninlhocucumber, 296 aporium sativum, 256 Datura atramoniuin, 295 Hordcum jubutum/Scwde cereole, Ephedra folkta, 301 250 CXinko biloba, 301 tiordeum vulyui-e/lCrysiphe yraHaplopappa, 299 minis, 256 H O T ~ Esp., U ~299, 300 Iris, 259 induction by abortivo pollon, 206 jute, 260 induction by chomical troatment, Lathyrus, 233, 258 298 logumes, 257 induction by delayed pollination, Lilium. 260 298 Linum, 246, 247 induction by radioisotopes, 298 L u p i n w , 265 induction by X-ray troatmcnt, 296, 299 Lycopersicum, 259 in roots, 299 Nicothna, 233, 234 Oryzu, 257 maize, 298, 300 ovule culture, 265 male sterility, avoidance, 300 N i c o t h m , 296, 298, 300 Pheolua, 258 pollen, germination, 223 Oenothera sabra, 300 pollon, longevity, 221 O y z a sp., 296 P e t u n h violacea, 298 pollen tube, growth, 227 potato, 232 Pharbitis nil, 296 Prunus, 25s Rhoeo discolor, 299 role of genotype, 299 Sunhdum cillmm, 2ti I somatic reduction, 298, 2'31) Sohnum, 234 Tazus, 301 Htylo, Jeriyth or, 233 Torrcya nucifera, 301 l'ryolium, 232 Tradcacantia ~ ~ J E x u , 801 Vululu, 203 Trillium sp., 299 Hylrrorlictyon, chomiaal ccmtorrt, of Tritiuzk, 298 coll walls, 71) Triticum sp., 296, 298, 299 Hydroxyproiino SCE alao Protoin wheat, 298 chemical charcLctwieation, 1li7 Zea mays, 298, 300 distribution in thc: plarit kingfjorn, Heterokont flagellation, 6, 21 174 Hybridization, 255 in cell walls, 151 androgenesis, 300 variation of cell-wall content, 188 Argamone ~ E X ~ T U 230, Z , 237 APgarnone ochroleuca, 236, 237 barriers to, 220 BrassicaJRaphanus, 238 I by embryo culture, 220, 238, 265 Isokont flagellation, 6, 21

SUBJECT INDEX

377

Ceiba pcntccndra, 103 Chaetomorph, 92, 98, 101, 102, Mathematical models in numerical 138, 139, 140, 142, 144 taxonomy C h r a , 100, 142 choice of model, 48 chitin microfibrils, 76 Euclidean modol, 64 ChlorcJh pyrenoidom, 92 motrica, 48 C‘krtlophorfl, 02, !#1& 0 1 , I102, , 103, inotric i)roporl,iwof I)i~ir-fii~ic!Lioi,n. 13H. 130 49 wIic~roiic*o, 72. HO, H I , 82, 120 non-Euclidoan systoms, 62 collonchyma culls, 1 1 I , 1 1 2, 116, non-metric systems, 61 130 Meiosis, 302 constitution and morphology, 70 Metazoa, 6 cortical fibres, 116 cotton hair, 126, 126, 139 Microfibrils crystallinity, 72, 77 Acetobacter xylinum, 143, 144 Cucurbita, 97 Acroeiphonb, 92 cytoplasmic element theories, 141 algae, brown, 98 Dahlia tuber, parenchyma cells, aee aluo individually named algae 104 algae, green, 92, 93, 98, 103 Dictyosphaeriu, 92, 94, 95 eee also individually named algae displacement by growth, 114, 116 algae, red, 93, 9A, 103, 105 elementary fibrils, 72 eee alao individually named algae epidermal cells, 108-110, 114-116, Allomycea, 92, 98 127, 130, 131 apical meristems, epidermal cells, Euphorbia, latex vessels, 108, 114 108, 130 fibres of cortex, phloem and wood, apical merktems, parenchyma 107, 108, 113 cells, 106 k’raxinua,p h h m and wood fibros, apical morintems, pit fields, 117 108, 113, 114, 130 Apium ptiolw, collenchyma Colin, lf’unarh h y p ~ n c t r i m ,97, 103 111 fiingal Iiyphao, !J8 applo, parenchyma colls, 104 &@lh& jloaeuloea, 83 arrangament in different types of growth in thicknons, 76 growing cells, 91 growth vclocity, 73, 90, I2 I , 1 2 X Aeclepku c m u t i , 103 Halieysticr, 03 Aaparagw, cortical fibres, 108 Helianthzce hypocotyk, 114, 130 Aaparagua, pmenohyma cells, 104 Helminthocludia californiccc, 93 Aepamgw, wood fibre tips, 114 Heracleum petioles. collenchyma Avena coleoptile, parenchyma cells. 111, 112 cells, 106, 109 Auem coleoptile, phloem cells, 107, Hydrodictyon africnnum, 92, 100 in tissue cells that predominantly 114 Avena coleoptile, tracheary elewiden, 112 ments, 107, 114 in tissue cells with predominant Auena coleoptile, epidermal cells, growth in length, 106 108-110, 114, 127, 130, 131 in tissue cells with kodiametric Avene coleoptile, older walls, 110 growth, 104 avocado, parenchyma cells, 104 in freely-growing tubular cells or parts of cells, 90 Bmseica, 97 breakdown, 89 in frody-growing moro or low Bryopsi.9, 93. 103, 104 isodiarnutric colls, 0 I cambium initials, 107, 1 1 3, I 1 6 &ncw /ffwuu,100, 104. 123, 1 3 1 Caulerpa, 93 Kulhnchoii Intlvon, I 3 0

M

378

SUBJECT l N D E S

Microfibrils c o d . latex vessels during lengthening, 108, 113, 114, 128. 130 maize, phloem cells and tracheary elements, 107 microfibrils of sundry materials, 77 microtubules, role in microfibril synthesis, 142 morphology, 70 mdtinet structure, 100, 104, 106, 108, 109, 110, 113,122, 129,132 NiteZla, 93, 98, 100, 101, 104, 114, 123-127, 134-143 onion root, epidermal cells, 109, 110, 127 onion root, parenchyma cells, 105 orientation, 83, 90-144 Papuodendron, wmd fibre, 108, 114,126 parenchyma cnlls. 104, 105, 100, 115 Pellia, ueta, 114, 121, 130 Petasitea, collenchyma COIIH, I 1 I , 112 Philodendron leaf cells, 11 3 phloem cells, 107, 114 Phycomyces sporangiophores, 93, 100, 103, 127. 128, 129, 132, 133, 135, 136, 138, 139 Pinus, tracheid, 114 pollen tubes, 98 P0&8tiCtW, 94, 98 potato, parenchyma cells, 104 protoplasm streaming theory, 139 R a p h n w , 97 Rhizoclonium, 101 Rhizophydium, 92 Rhodymenia palrnata, 93 rib thickenings, 115, 117 Ricinwr, primary xylem, 107 root hairs, 97 Secale, perivwcdar fibres, 108 Secale, stamina1 filaments, 121 sinapscs, 97 Siphonocladales, 101 spiral orientation, 132 Spirogyra, 100, 139 Spongomrpha, 92 synthesis, 86, 139 tip-growth, 74, 96, 103, 113, 126 Tolypellu, 100 Tollypellopsia, 100

Tradescnntia virginiea, 100, 101, 103, 114. 121, 122, 132, 139 Trianea bogotensk, 97, 139 Triticu?n, 97 turgor pmsure and orientation, 120, 140 Valonia, 92, 94, 95, 101, 102, 115, 139 vascular elements, 107, 113, 115 wall stress theory, 139 yemts, 91, 94, 117

N N&onia 8ee Yeast Nitellu cell-wall synthesis 86, 87 chemical content of cell walls, 79 microfibrillar arrangements, 93, 98, 100, 101, 104, 114, 123-127, 134-143

0 Oiirnyccttw, 70, 78

Ovary and flowor culture in artificial modia, 266 Allium cepu, 271 Anethum graveolene, 27 1 rapomictic plants, 27 1 Aquilegia f m s a , 272 barley, 269 Cucumia anguria, 266 Dendrobrium nobile, 272 Fragaria sp., 266 growth media, 288 et aeq. growth promoters, 271 Iberis amara, 269 Linaria maroccam, 27 1 Lycopersicurn eeculentum, 266 N k o t k n a , 256, 271 partial Rterilo culturn mathocl, 272 Phascolus vulyarie, 260 Phlox drummondii, 268 polyembryony, 27 I , 294 Itanunculw acelomlue, 27 1 Tritkum, 268 Tropaeolum m j w , 269 Oviilo culturo, 283 angionpermic parwibu, 264 artificial induction of parttrogeniuis, 256 C'attleya, 263 I)endrobium pha.!wnopa.ia, 265 Epindrum. 263

SUBJECT I N D E X

Ovule culture cont. growth medie. 263 growth promotera, 264 aY?tan&rop& gpandra, 265 hybrids. 266 I m p a t h a balaamim. 266 Opuntia dillenii, 266 orchids, 263, 264 Orobanche, 264, 266 Papaver mn$wurn, 264 Striga, 264, 266 Vavnnda tricolor. 203

P Parthenocarpy, 273 auxin, 279, 308 Cwtaceaa 278 chemicallyinducedpartlienocarpy, 273 et 8eq.

cherry, 276 citrua, 277 Cucurbitaceae, 273, 278 c-ts, red- and blwk- 276 environmental parthenocarpy, 279 ~ g s ,278 genetical parthenocmpy, 279 gooseberry, 276 Nieotbm, 279 orchi&, 273 pollen extracts, 273 Prunls, 276 Psidium guajava, 278 Row, 276 Solanaceae, 273, 278, 279 strawberries, 276 tomato, 277 Vicia faba, 278 V i t k vinqera, 277 ??U3y8,279 Zephyrantha, 279 Parthenogenesis, 295 &o Haploids abortive pollen, 296 androgenesia, 300 delayed pollination, 298 haploid sporophytea, 296 haploids, induction of, 296-299 radiokotopee, 298 role of genotype, 299 somatic reduction, 298, 299 X-ray treatment, 296, 299

Bee

379

Phylectic implications of flagellar structure in plants bases, 17 distribution of the 9 +2 type, 3 hairs, 14 heterokont, kokont conditions, 6 multiple condition, 9 roota, 19 scales. 17 shapu, 10 spines, 13 uniflagellation, 8 Physiology and embryology, 2 19 P k m , chomical content of cell walls, 79

Plasmodesmata formation of, 89 in parenchyma cell8 of npical meristems, 106, 106, 117 in tip cells, 113 Pollen androgenic haploidy, 300 anther culture, 301 bud pollinations, 227 formation of embryo sacs, 303, 304

germination, ambient humidity, 224, 226, 227

germination, boron, calcium, magnesium and potassium, 224,231 germination, dicarboxylic acids, 225

germination, maw effect, 226, 230 gcmnination. ntorod pollm, 223 induction of Hhrility, 308 irradiatctl pollon, 2118, 300 longwity, 221 male and femalu poterioy. 303 mutho& of prolonging vitality, 22 1 mutual stimulatiori of poll or^^, 226 pmtothunic wid ((YJ(3rMyIJJO A) content, 223 parthenogenoHi8, 296, 298, 299 Paapalum dilatatum, 221 pectin synthesis, 226 pollen growth factor, 230 self-sterility, 226, 230 sexual incompatibility, 229 storage, humidity, 221,222, 223 storage, nutrients, 223 storage, temperature, 221

stored pollen, field use, 223

380

SUBJECT I N D E X

Pollen tubes, growth influence of chemirals and natiiml abstracts, 229, 231 influence of temperature, 229 inhibition of, 229-231 mode of, 96, 98 relation to structure, 228 Pollen tubes, microfibrils, 98 Polyembryony adventive embryony, 280 Anethum graveolena, 271, 294 angiospermic parasites, 292 avocado, 286 carrot, 283, 294 chemicalpromoters, 280 et 804.- 287 Citrua, 281 cultivation of excised nucellnr tissue, 281 Cuacuta r e . e m , 292 Cymbidium. 292 Dutura, 290 Daucua carota, 281 Dendropthoe falcata, 294 embryonal budding, 286 Eranthis hicmalk, 286 Foeniculum vulgare, 271, 295 Hordeum vulgare, 290 irradiation of developing raryopses, 290 Kalanchoe pin&, 285 Niwtkna, 285 orchids, 290, 292 Orobanche aegy&wa; 294 ovary and flower culturp, 271, 294 Persea grattisima, 286 pseudobulbils, 281 Pteris vittata, 286 Ranuncuhs scleratua, 271,294 somatic fertilization, 280 somatic tissues, differentiationdedifferentiation - rediffercntiu tion, 286 totipotency of carrot root collx, 283-285, 294 Vunrh tricolor, 290 Procaryota, 3 Protein, analysiR of primary cell wall ascorbic acid oxidase, 204 cellulase, 195 enzymic degradation, 194 Ginkgo, 196 hemicellulase, 195

hydrol&q.se, 205 poctiiimo, 196 sugar molar ratios, 193, 196 sycamore, 196 uridine diphosphoglucose pyrophosphorylme, 205 Protein-carbohydrate link, 198 Protoin component of cell walls, 151 Acerpeeuhplatanua, 175, 190 amino acids, 168-171 analytical techniques, 158 Apium gravwlena, 175 ascorbic acid oxidase, 204 Avena sativa, 175 biosynthesis, 177 Camellia einensi.8, 175, 190 cell extension, 193, 210 cell suspension cultures, 155 Centauria cyanus, 175, 189 chemical tlogrdatiori, 193 chromatography, 168 Citrua paradiai, 175 collagen, 169 cystoino, 159 Uaucua carota, 175 dogradation of sycamoro primary cell wall, 171, 193 disulphide bridges, 172, 21 1 electrophoresis, 158 enzymic degradation and charmterization, 171,194,200 enzymic wall protein, 204 Eruca sativa, 175 experimental matttrials and methods, 155 Ir’ectucu sp., 176 Gulega o e i n a l i a , 175 Ginkgo biloba, 176, 176, 188, IQO, I91 Helknthw annuus, 175 history, 152 Iiydrolyasrrs, 205 hytlroly~is,158 hydroxyprolinc:, 100. 172, 174, 177 Lcpicliurn mtivum, 1 76 L U Y I ~ ~l ~bU 176, ~ ,177 Lycoper&icon caculenlum, 175 MdW 8YkXcltTi8, 190 Nicoticlrca tabacurn, 175, 1‘30 OTyZcl BUtiVU, 175, 190 I ’ h w 3 l w uulgari8, I 75, 188 I’isum sativum, 175

38 1

SUBJECT INDEX

Protein component of cell walls cont. P@ua tremdu, 190 proline hydroxylation, 184 . W-proline, uptake end incorpora. tion, 177 Pyrua cmmunis, 176 resistance to proteolytic attaok, 172 Rosa, 175 Santalum album, 176 Solanum tuberosum, 176 sulphur amino acids, 169 sycamore cells, 157, 171, 173, 174,178,179,191,193,196,198 tensile strength, 200 variations in composition betWMn K p ) C i O K , 168 wall-hunt1 onzymw, 204-208 wall form, 208 Zea mu&, 176 Protein in cell wrrlls, biosynthtwk, 88, 177 changes of 1.C distribution with time, 181 chick-embryo system, 186 oollagen, 187 elastin, 187 inhibition by proline analogues, 180 oxygen fixation, 184 oxygen tension, effect of, 184 proline hydroxylation, 184 1%-proline, uptake and incorporation by intact cells, 177 site of synthesis, 88, I82 sycamore cell cultures, 178 Protein in cell walla, variation of content, 188 arabinose/galactoae ratio, 109 arabinose/xylose ratio, 190 cambial cultures, 189 control by the plant, 191 control over cell extension, 103, 210 correlation with general growth form, 192 oultural conditions, influence of, 189 effect of disturbed bioaynthosis, 191 effect on leaf morphogenosis, 191 Gramineae, 182

in different regions of tho same plant, 188 Juncua, 193 list of specios, 176 maize, 192 peas, 191, 192 pollen cultures, 189 secondary wall, contamination by, 190 sceds, 191 soybean, 192 sugar moler ratios, 190 sycamore cambial tissues, 191 Protozoa, 6

It

lttlotlopllyttlL,5 , 7 Itnot l i n i r ~motlo , of growth, !If;, !I7 Rot 8c(! E'ungi

8 8cx expresuion, control of, 304 Antirrhinum majua, 308 auxins, 305, 306, 307, 308 Cuntuzbis aativa, 305 chemical control, 306 et scq. Citrullua vulgaris, 307 cotton, 307 Cucurnie, 305, 306, 307, 308 Cucurbita pepo, 305 folirtr epray, 308 gametocides, 307 growth suhstancm, role of, 304 el 8eq.

Luffu uculu,qula, 306 Lycoprnicurn esculenlurn, 307 maizct, 307 malo sterility, iriiliiotiori of', 3 0 7 , 30H

i%!!ehndriurn dioiciurri, 304 hf.lercuruilie umbiguu, 308 ptiotoporiodic control, 3Ofi plant hormones, 304 pollen sterility, induction, 308 sox reversal, 305, 308 &lime psnrluh, 308 Solanurn rnetonpna, 308 soybeans, 308 tissue and organ culture, 307 tomato, 307 Soft rot see Fungi

382

SUBJECT INDEX

T Taxonomy, 1,35, 168 Taxonomy, numencd 8e.e &o Mathematical modela in numerical taxonomy CleSsScatione, nature and propertiee of, 37 Eiiclidean model, 64 mathomatical modal, choico of 48 statistical approach, 36 MtI'8bm O f 8IldJ'E& 69 Thallophyta, structure of growing cells, 69

U Uniflagellation. 8

Volvocales, 16

V W

Wood degrsdation 8e.e Fungi

Y Yeaf3t coll wall brucrkdown, 90 chemical content of cell WdlH, 77, 79 glucan microfibrils, 78 microfibrillar arrangement, 91, 04 protein content of cell wall, 82