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Sexual in , and

Proceedings of the 8th international symposium on inseed plants, ferns andmosses 20-24August 1984, Wageningen, the Netherlands

M.T.M. Willemse and J.L. van Went (compilers)

-^ BIBUO FF (Stichting voor: gredeling) Droevendi INGEN

CPRO-DLO BIBLIOTHEEK Postbus1 6 6700A A Wageningen

Pudoc Wageningen 1985 CONTENTS

The 8th international symposium on sexual reproduction in seed plants, ferns and mosses 10

Congres "organization 11

PART 1

Microsporogenesis, , anther and in vitro 13 Brown, R.C. & B.E. Lemmon. Ultrastructure of in the Entodon seductrix 15 Chauhan, E. & M. Lai. Cytochemical and ultrastructural tudies on sporogenesis in the moss Physcomitrium cyathicarpum Mitt 16 Bednara, J. & B. Rodkiewicz. Distribution of and mitochondria during sporo­ genesis in hyemale 17 Keijzer, C.J. Anther development of Gasteria 20 Shridhar & D. Singh. Anther development and pollen formation in Cucurbitaceae 21 Souvré, A., L. Albertini, A.C. Dhar & M. Petitprez. and pollen grain forma­ tion in Rhoeo discolor Hance 24 Pacini, E., G.G. Franchi & L.M. Bellani. Pollen grain development in the (Olea europaea L.): ultrastructure and anomalies , 25 Sou^é, A., L. Albertini & A.C. Dhar. The effect of cold (4 °C) on H-proline and C-hydroxyproline incorporation in , arid connective of Rhoeo discolor Hance. A radioautographic study 28 Wiermann, R. Biochemistry of pollen pigmentation 32 Herich, R. & A. Lux. Degradation of tapetal cell nuclei during the formation of micro­ 33 Jensen, W.A. The effect of a chemical hybridiying agent on the development of wheat pollen 34 Habintore, E., J.P. Tilquin & A. Serrhini. Genetical approach of autosterility in Camélia sinensis L 35 Sawhney, V.K. Hormonal and temperature control of male-sterility in a mutant 36 Van Marrewijk, G.A.M. & L.C.J.M. Suurs. Characterization of cytoplasmic male sterility in Petunia x hybrida (Hook) Vilm 39 Bino, R.J., S.J. de Hoop & A. van der Neut. Cytochemical localization of cytochrome oxidase in anthers of cytoplasmic male sterile Petunia hybrida (Hook.) Vilm 44 Noher de Halac, I. Sterility of microspores in Oenothera hookeri Velans 47 Noher de Halac, I. Stages of tapetal cell cell development in sterile anthers of Oeno­ thera hookeri Velans 50 Tan, M.M.C., 3. de Bruin, G.A.M. van Marrewijk, A.J. Kool & H.J.J. Nijkamp. Isolation of Petunia hybrida cytoplasts 51 Schröder, M.B. & R. Hagemann. content of generative cells in 52 Hagemann, R. & M.B. Schröder. New results about the presence of plastids in generative and cells of Gramineae 53 Rowley, J.R. & B. Walles. The surface coating of microspores and microspore mother cells in Pinus sylvestris 56 Willemse, M.T.M. Light absorption of walls 57 Barnabas, B. Effect of water loss on germination ability of maize pollen 58 Van Lammeren, A.A.M. Microtubular configurations during and pollen develop­ ment in Zea and Gasteria as visualized by immunofluorescence 59 Derksen, J. & J.A. Traas. Growth of tobacco tubes in vitro; effects of drugs interfering with the cytoskeleton 64 Miki-Hirosige, H., S. Nakamura & Y. Yamada. Fine structural research on germinating lily pollen tubes in vitro and in vivo 68 Kroh, M. & B. Knuiman. Membrane-structure and events in non-plasmolyzed and plasmo- lyzed tobacco pollen tubes: a freeze-fracture study 71 Cresti, M., F. Ciampolini, D.L.M. Mulcahy & G. Mulcahy. Ultrastructure of Nicotiana alata and Petunia hybrida pollen tubes grown in semi-vitro conditions 72 Hough, T., P. Bernardt, R.B. Knox & E.G. Williams. Use of fluorochromes in pollen 73 Cadic, A., R.S. Sangwan & B.S. Sangwan-Norreel. Stress related structural modifications in the pollen of Datura 74 Dashek, W.V., C.S. Ridenour & R.R. Mills. longiflorum pollen germination and tube elongation as markers for solfur dioxide 75 Sangwan, R.S. & B.S, „Sangwan-Norreel. Early indicators of in vitro transformation from to in Datura 76 PART 2 , incompatibility and pollen germination in vivo 79 Ramm-Anderson, S. & R.B. Knox. Localization of pollen surface glycoproteins using monoclonal antibodies 81 Said, C, P. Zandonella, T. Gaude & C. Dumas. Cytochemistry of the stigma surface components using the print technique 83 Owens, S.J. Stigma structure and the pollen-stigma interaction in Caesalpinioideae - Leguminosae 84 Kenrick, 3., P. Bernardt, R. Marginson, G. Beresford & R.B. Knox. Acacia breeding systems 88 Kormutâk, A. Incompatibility between Pinus nigra Arn. and Pinus sylvestris L. and overcoming it with chemomutagenic substances 90 Wojciechowska, W. Immediate distinction between self- and cross-pollination and between self-compatibility and self-incompatibility in Papilionaceae - prelimina­ ry report 93 Serrhini, A., 3.P. Tilquin & E. Habintore. Cytological approac of sterility and incompati­ bility in Tea (Camélia sinensis) 96 Tilquin, J.P., A. Serrhini & E. Habintore. Incompatibility in Camélia sinensis; a general approach 97 Nakanishi, T. & M. Sawano. Pollen tube behavior by carbon dioxide in relation to over­ coming self-incompatibility in Brassica 99 O'Neill, P., M.B. Singh, T.F. Neales, R.B. Knox & E.G. Williams. Carbon dioxide blocks the stigma callose response following incompatible in Brassica .100 Gaude, T., A. Palloix, Y. Hervé & C. Dumas. Molecular interpretation of overcoming self-incompatibility in Brassica , 102 Dickinson, H.G., I.N. Roberts, C.J. Elleman, G. Harrocf, & M.I. Zuberi. Early events on the Brassica stigma following compatible and incompatible intraspecific matings 105 Hodgkin, T. & G.D. Lyon. Pollen germination inhibitors associated with the self-incom­ patible response of Brassica oleracea L 108 Kenrick, J., V. Kaul & R.B. Knox. Self-incompatibility in the nitrogen-fixing Le­ gume, Acacia retinodes: pre- or post-zygotic mechanism? Ill Larsen, K. Cell surface recognition glycoproteins and their possible role in angiosperm self-incompatibility 112 Rouse, J.L., R.B. Knox & E.G. Williams. Unilateral hybridization in Rhododendron 115 Kaul, V., J.L. Rouse, R.B. Knox & E.G. Williams. Early post-fertilization development in Rhododendron 116 Willemse, M.T.M. & M.A.W. Franssen-Verheijen. Pollen tube penetration in the style of Gasteria 118 Williams, E.G., P. O'Neill & T. Hough. The pollen • tube as an experimental growth system v 119 Gilissen, L.J.W. & F.A. Hoekstra. Style-controJled corolla wilting'in Petunia hybrida L 121 PART 3 Megasporogenesis, , sac development, fertilization and embryo and endo­ sperm development 123 Sheffield, E. & E.G. Cutter. Cryo scanning electron microscopy of reproduction in cryptogams 125 Tigerschiöld, E. Gametangia in Ceylonese species of the familie Thelypteridaceae 126 Llewellyn, G.C.,* J.D. Reynolds & W.V. Dashek. Water fern (Marsilea quadrifolia) repro­ duction as a bioassay for aflatoxin B. and T-2 Tricothecene toxin 127 Pennell, R.I. & P.R. Bell. Fertilization in Taxus baccata L 131 Polito,. V.S. & Nai Yan Li. Pistillate differentiation in Juglans regis: a develop­ mental basis for heterodichogamy 134 Went, J.L. van, N.M. van Beek & J.M. van Tuyl. development in relation to ovule position and flower development in Lilium 135 Sniezko, R. & C. Harte. Genetic basis of callose pattern, degeneration and polarity in the of Oenothera 136 Schulz, P. Ultrastructure and ultracytochemical localization of acid phosphatase in the dyad, tetrad and developing megaspore of Capseila bursa-pastoris 137 Cass, Û. & D. Peteya. Aspects of megagametogensis in Hordeum vulgare HO Wilms, H.J. Behaviour of spinach cells in pollen tubes prior to fertilization 143 Russell, S.D. Sperm specificity in Plumbago zeylanica 145 McConchie, Ç.A., S. Jobson & R.B. Knox. The structure of sperm cells in Brassica 147 Kadej, A. & F. Kadej. Sperm cells in the fertilization process in Lycopersicon esculentum 149 Czapik, R. Secondary nucleus in Rosoideae 150 Van Went, J.L., C.H. Theunis & A.P.M. den Nijs: Embryo sac ultrastructure before and after fertilization in Cucumis stivus 153 Bannikova, V.P. & T.A. Plyushch. Embryo sac ultrastructure in Peperomia blanda H.B. et K. before and after fertilization 155 Choisez-Givron, D. Sexual reproduction in the genus . Embryogenesis in F. Fulgens 156 Longly, B., T. Rabau & B.-P. Louant. A cytological method for detecting coexistence of sexual and diplosporic reproductive processes 1 157 Abeln, Y.S., H.J. Wilms & A.J.P. van Wijk. Initiation of apomictic seed production in Kentucky bluegrass, L 160 Nijs, A.P.M. den & P. Milotay. Pollination and fertilization in pickling and slicing cucumbers 165 Singh, M.B., R.B. Knox & E.G. Williams. competition in oil seed rape: effect of pollen - galactosidase deficiency on fertilization 166 Georgieva, I.D. Cytochemical investication of Lilium regale embryogenesis after pollina­ tion with -irradiated pollen 167 Erdelskâ, O. Some structural features of the embryo/endosperm interaction in angio- 168 Schel, J.H.N., A.A.M. van Lammeren & H. Kieft. Ultrastructural analysis of embryo- endosperm interactions in developing maize (Zea mays L.) 171 Shah, C.K. Reproductive physiology of Limnophyton obtusifolium (L.) Mig. A histochemi- cal analysis 172 Shah, C.K. Grass - A SEM and histochemical analysis 173 Viegi, L. & G.C. Renzoni. DNA measurements in the chalazal proliferating cells of Capsella bursa-pastoris (L.) Medic 176 Ryczkowski, M. Respiration rate of the embryo in its natural environment in vitro 177 Ryczkowski, M., A. Kowalska & W. Reczynski. Element concentrations in the s^p sur­ rounding the developing embryo 180 Borzan, Z. Hybridization between Pinus nigra and Pinus sylvestris and of 181 Pretovd, A. In Vitro embryogenesis of flax (Linum usitatissium) 182 Maheswaran, G. & E.G. Williams. Direct somatic embryogenesis for rapid genotype propagation 183 Batygina, T.B. Applied aspects of embryology 185 Chebotarus, A.A. To the morpho-functional status of higher 191 Willemse, M.T.M. Sexual reproduction of seed plants, ferns and mosses. A background 194

List of participants 200 Subject index 203 Plant index 205 Authors index .' 206 THE.8T H INTERNATIONAL SYMPOSIUM ON SEXUAL REPRODUCTION IN SEED PLANTS, FERNS AND MOSSES

The proceedings

These proceedings of the 81h International ted these practical aspects by visiting the Symposium on Sexual reproduction in Seed Uniyersity Department of Plant Breeding Plants, Ferns and Mosses links up the previous and the Institute of Horticultural Plant seven editions. Breeding. In 1968 Prof.Dr. M. Favre Duchartre took Compared with the preceeding symposia the initiative to organize a symposium in there was increasing interest on male sterility, Paris entitled: "Colloque sur les aspects incompatibilty and ; the number cytologiques de la reproduction sexuele des of presentations on mosses and ferns was plames ovulees". small. During the poster sessions the presenta­ This was the beginning of a long row of tion of specialized often adapted methods scientific symposia: Reims 1970, Siena 1972, was manifest. Structural studies combined Nijmegen 197*, Reims 1976, Lublin 1980, with histochémical, physiological or genetical High Tatra 1982, each with a growing interest data were very clarifying., from all countries of the world. Also in The programm covered the processes of Wageningen new colleagues were present sexual reproduction from sporogenesis to and many young scientists. seed formation, a broad but essential scope.

A symposium is a place of encounter, partly In this large field of research nearly all ordered in a programm, but for an important participants showed progress in iheir special part running during the day ••an d evening. field of interest by lectures, posters, discus­ Also at this symposium there were new sions and the exchange of reprints. Altogether and renewed acquintances. Both the Rector the presentations and conlacts resulted in Magnificus of the Agricultural University, a stimulus to continue this interesting and Prof.Dr. C.C. Oosterlee in his openingspeech, important field of scientific research and and Dr.Ir. H.N. Hasseloo at the reception applied aspects, not only by individual or of the Ministry of Agriculture and Fisheries, cooperative research, but also ihrough ihe mentioned the value of fundamental research next 9th symposium in 1986 at Reims, conve­ on sexual reproduction especially for plant nor Prof. Favre-Duchartre, and in Siena breeding and crop sciences. The location in 1988. of this symposium at Wageningen has promo­

10 Congress organizalion The panel of the poster discussion was composed of Prof.Dr. P.R. Bell, The scienlific program comprised three lec- Prof.Dr. R. Hagemann and Prof.Dr. lure sessions whh 29 ledures, ihree posier M.T.M. Willemse. sessions and posier discussions wilh 49 posiers and the film "Befruchtung und frühe Entwick- In this proceedings volume both lectures lung von Embryo und Endosperm beim Schnee­ and posters are compiled in three groups. glöckchen (Galanthus nivalis)" by D.O. Erdelska. I: microsporogenesis, microgametogenesis, anther and pollen germination in vitro; The lectures and posters were placed in II: stigma incompatibility and pollen germina­ three groups: tion in vivo; III: megasporogenesis, ovary, 1. Lectures: Microsporogenesis, anther embryosac development, fertilization, and and pollen development. This session embryo and endosperm development. was divided in three parts. Chairmen of these parts were respectively: Some contributions of colleagues, unable Dr. E.G. Williams, Dr.Ir. H.J. Wilms to come, are added. and Prof.Dr. J.L. van Went. 1. Posters: Microsporogenesis, pollen Members of the organizing committee were: development, progamic phase and A.C. van Aelst, F.M. Engels, C.J. Keijzer, incompatibility. The panel for the A.A.M. van Lammeren, J.H.N. Schel, J.L. poster discussion were composed of van Went, M.T.M. Willemse and H.J. Wilms. Dr. H.G. Dickinson, Dr. W.V. Dashek, Dr. F.M. Engels and Prof.Dr. H.F. Technical assistance was given by: I.B.A. Linskens. van Brakel, J. Cobben-Molenaar, J. Drijver, Ir. A.J.M. Groenenwegen, A.B. Haasdijk, 2. Megasporogenesis, ovary, embryo G.G. van de Hoef-van Espelo, E.J.L. Hotke- sac development, incompatibility, Siaal, P.A. van Snippenburg, E.P. van de and fertilization. This session was Wetering. divided in four parts. Chairmen of these parts were respectively: Dr. The following sponsoring organizations assisted S.D. Russell, Prof.Dr. W.A. Jensen, 1he symposium by making a major donation, Prof.Dr. L. Albertini and Dr. K. Larsen. which is gratefully acknowledged: II. Posters: megasporogenesis, embryo Ministry of Agriculture and Fisheries of sac development and embryogenesis. the Netherlands The panel of the poster discussion Agricultural University LH, Wageningen ws composed of Prof.Dr. D.D. Cass, International Agricultural Cenlre, I.A.C., Dr. J. Bednara and Dr. J.H.N. Schel. Wageningen Royal Netherlands Academy of Sciences, 3. Embryo and endosperm development. K.N.A.W. Apomixis and miscellaneous subjects. Royal Netherlands Botanical Society, K.N.B.V. This session was divided in two parts. Philips, Eindhoven, the Netherlands Chairmen of these parts were Prof.Dr. Algemene Bank Nederland, A.B.N., Wageningen E. Pacini and Prof.Dr. P.R. Bell. HI. Posters: reproduction in mosses and ferns, hybridization and applications.

11 This proceedings volume of the 81h meeling of planl embryologisls links up ihe previous editions, to begin with:

Paris 1968: Colloque sur les aspects cytologi- ques de la reproduction sexuée des plantes ovuies. Revue .de la,....Cytologie et Biologie Végétale 1969, 32: 1-404. »l«^ljSr.:'

followed by:

Reims 1970: Colloque sur les aspects cytologi- ques des gametogénèses femelles et mâles chez les Cormophytes. Ann. de 1' Université et de l'ARERS 1971, 9: 1-237. r," 31--:';

Siena 1972: From ovule to seed: ultrastructur­ al and biochemical aspects. Caryologia 1973, 25: 1-314. s*-••>• • ^»J

Nijmegen 1974: Fertilization in higher plants. H.F. Linskens, ed., North Holland Publ. Comp. 1974, p. 1-373. ib'A

Reims 1976: Cytobiologie de la reproduction sexuée des plantes ovulées. Bulletin de la Société Botanique de France 1978, 125: 1-299. ^5

Lublin 1980: Proceedings of the Vith Inter­ national Symposium Advances in plant cytoem- . Acta Societal is botanicorum Polo- niae 1981, 50: 1-356. l/n* Ï^HlbCj

High Tatra 1982: Fertilization and embryo- genesis in ovulated plants. O. Erdelska, H. Ciamporova, A. Lux, A. Petrova, I. Tupy, Centre of Biological and Ecological sciences. Institute of Experimental Biology and Ecology, Slovak Academy of Sciences, Bratislava, 1983:1-384. 'iH* 2. (lo^i, "•'-'/?'/;

12 PART I

MICROSPOROGENESIS, MICROGAMETOGENESIS ANTHER AND POLLEN GERMINATION IN VITRO

••*•••••••••*•••••*•••*••*••••••••••••••

13 Part I:Microsporogenesis ,microgametogenesis ,anthe ran dpolle ngerminatio n invitr o

Introduction opment. In that aspect also links with animal cells should be included. Also and microspore development, male the study of the normal process and the sterility, pollen tube formation in vitro exact estimation of the anomalies will and external influences on different be very helpful to understand the study developmental stages are the main topics of sporedevelopment . of this part. Mainly ultrastructural and biochemical methods are used, in On her way to Holland Dr. Williams from some cases a combination of structural Australia had a delay for 7 hours in and biochemical approach is offered. a KLM airplane. This situation stimulated Some more or less new methods are des­ her to produce a number of limerics about cribed as theus e of fluorochromes,immuno ­ KLM. During the symposium this poetical logical technics and quantitative measure­ attitude was still present and so she ment of pollen. For applications the shortly summarized thesituation : study of male sterility and the use of

hybridizing agents are important in plant The poster display it was fun. / breeding. ( As from poster to poster we run Sixtythree,sixtyfou r- During the poster session the complex andtwentyfiv emore ? question about the genetic order and But I still haven't seen number program arises related to the various one developmental processes.. Till now only few complete studies of anther and pollen Therefore the proceedings will permit development, including interaction the reader to run on his own way in his are available. However, gradually progress ownspeed . is made by comparison, and the impressionv arises of a genetic order.i n the major steps, as induction of meiosis, isolation of the meiotic cell by a special wall, the spore formation, formation of storage products and a resting period. When such stages appear to be common steps inmicro - and megasporogenesis, perhaps a common genetic base for the production of spores, pollen and seed as dispersal organs can be postulated. However, the development of spores, pollen and seed is also spe­ cies-specific and strongly dependend- ing on the surrounding tissues andenviron ­ ment. In such an approach a comparison between sporogenesis in mosses, ferns and higher plants is important too and can sustain a general view on sporedevel ­

14 ULTRASTRUCTURE OFMEIOSI S INTH EMOS SENTODO N SEDUCTRIX

R.C .Brow n and B.E .Lemmo n

University of Southwestern Louisiana,Lafayette ,L A 70504US A

Summary regular distribution ofplastids ,on et oeac h ofth efutur etelophas eI Ipoles ,i s Theai mo fthi sultrastructura l associated with anelaborat esyste mo f investigationo fmeiosi s inth emos sEntodo n microtubules. Typical preprophase bandso f seductrixwa st oprovid ea comprehensiv e microtubules areno t associatedwit hth e account ofnuclea ran dplasti d inheritance prediction of cleavage planes. Instead, and toprovid e informationo nth econtro lo f numerousvesicle sdevelo pi nth eplane so f divisionplane si nplan t sporogenesis. The cleavage furrows andther ei sreaso nt o eventual cleavageplane sar epredicte db y suspect thatmicrotubule s ofth eprophasi c aggregations ofvesicle stha tmar k thesite s system arenucleate d inthi sdivisio nsite . where,followin gmeiosis ,cel lplate swil l By lateprophase ,chromati n décondensesan d fusewit hth esporocyt ewall . Vesicle thediffus enucleu s isensheathe d bya syste m complexes and precocious infurrowingo fth e ofmicrotubule s that interconnectsth efou r cytoplasmpredic t thefutur eplan eo f plastids. Thecytoplas mha sbecom edeepl y division asaccuratel y asd opreprophas e lobedaroun d thetetrahedrall yarrange d bandso fmicrotubule s known frommitosi si n plastids and planeso fth efutur epost - parenchymatous plant tissues. Cytoplasmic meioticcytokinesi sar eclearl yestablishe d microtubules ofth eprophas e system arefirs t asdee p furrowsbetwee n the lobes. Itmus t associatedwit hvesicle so fth edivisio nsit e beremembere d that this'cytoplasmi c and latercompris ea nextensiv e system prepatterning anticipates thesecon dnuclea r associatedwit hprophasi c distribution ofth e division. InEntodon , asi nsom eothe r fourplastid8 ,on et oeac ho f thetetra d mosses (Brownan dLemmon , 1982d)a norganell e poles. bandincompletel y separatesth ecytoplas m intotw oportions . Followinga brie f Keywords:meiosis ,microtubules ,moss , inframeiotic interphase,th esecon d division polarity,sporogenesi s spindles,wit h axesa trigh tangle st oeac h other,for mbetwee npair so fplastid s located Introduction oneithe r sideo f theorganell e band. Cytokinesisalon gsecon ddivisio nequatoria l Previous studieso nmos ssporogenesi shav e planesprecede ssepta l formationi nth e emphasized thatth emarke d polarity exhibited persistantmeiosi sI organell eband .Cleavag e throughout sporogenesis stemsfro mevent so f alongplane spredetermine d inearl ymeioti c melotic prophase (Brown &Lemmon , 1982a,b , prophaseresult si nfou rtetrahedrall y c; 1983). Ofparticula r importance isth e arranged spores eachwit ha plasti dnea rth e presence of amechanis m for thequadri - distal surfacean d anucleu s proximal toit . partitioning ofth esingl eplasti d inth e premeioticsporocyt es otha t eachhaploi d sporereceive sa singl eplasti d inadditio n References toa nucleus .Migratio no f daughter plastids toth efutur e tetrad poles isa strikin g Brown,R .C .& B .E .Lemmon , 1982a. manifestation ofth eestablishmen t of Ultrastructureo fmeiosi s inth emos s polarity inmos s sporocytes. Fromou r Rhynchostegiumserrulatu m I.Prophasi c studiesi ti sclea r that themos s microtubules and spindledynamics . sporocyte approaches theproces s ofmeiosi s Protoplasma 110:23-33. asa singl eeven t rather thana stw o & ,1982b .Ultrastructura l aspects sequential divisions. Thethre epola raxe s ofmos smeiosis : Reviewo fnuclea ran d necessary forth etw onuclea r divisions are cytoplasmic events during prophase.J . established early inmeioti cprophase . HattorlBot .Lab .53:29-39 . & ,1982c .Ultrastructura l aspects Resultsan d discussion ofmos smeiosis :Cytokinesi san dorganell e apportionment inRhynchostegiu m serrulatum. Thenucleu smigrate sfro ma centra l J.Hattor i Bot.Lab .53:41-50 . positiont oa periphera l positioni nth e & ,1983 .Microtubul e organization synaptic stage and returns toa centra l andmorphogenesi s inyoun g sporeso fth e position inpachynema . Thesingl e plastid mossTetraphi 8pellucid aHedw .Protoplasm a undergoestw oconsecutiv edivision searl yi n 116:115-124. meioticprophas ean d thefou rresultin g plastidsmov e toth efutur etelophas eI I polesprio r tometaphas eI . Thispatter no f 15 CYTOCHEMICAL ÄND ULTRASTRUCTURAL STUDIES ON SPOROGENESIS IN THE MOSS PHYSCOMITRIUM CYA- THICARPUMMITT .

E.Chauha n & M.La i

Plant Cytochemistry and Ultrastructure Laboratory, Department of Botany, University of Delhi,Delh i- 110007 ,Indi a

Summary Resultsan d Discussion

Present investigations comprise a com­ Early stages of differentiation of prehensive cytochemical and ultrastruc­ the sporogonium are characterized by tural study of various stages of differen­ very active RNA and protein synthesis tiation in the moss Physcomitrium cya- in cells of the amphithecium and endothe- thicarpum Mitt., a member of Funariaceae. cium. There is a high content of these Presence of a special non-callosic PAS- till the spore mother cell stage. The positive sporocyte wall, monoplastidic mature SMC's show a single, elongated condition of the sporocytes, indications plastid around the nucleus (monoplasti- of intense RNA and protein synthesis dy). The SMC protoplast shows prominent in the archesporium, and marked histo- cytoplasmic protrusions. The SMC wall chemical events in the cells of the spore- is intensely PAS-positive and reveals sac corresponding to the development toluidine blue metachromasia. The wall of the sporogenous tissue are some of also stains with ruthenium red but the the interesting features recorded with callose is absent. Thus, it seems that the help of quantitative and qualitative the isolation of the spore-mother cells histochemicaltechniques . by callose walls may not be a stringent Electron microscopic studies have re­ prerequisite for normal sporogenesis vealed monoplastidic condition asa transi ­ (Chauhan, 1981; Lai & Chauhan, 1982). tory stage during sporogenesis. Also The SMC wall and the intersporal septum studied are some of important aspects are a product of the protoplast itself. of spore mother cell development, inter- The protoplast reveals abundance of sporal septum and spore wall formation, smooth ER and golgi along the SMC mem­ and the fine structural attributes of branes. the spore-sac which are parallel and Cytochemical and preliminary ultrastruc­ complimentary to the spore development. tural studies on the cells of the spore- Keywords: cytochemistry, moss, sporo­ sac suggest that these may be playing genesis,ultrastructure . a role in making available precursors for the deposition of the perine coat Material andmethod s on- the young spores as well as metabol­ ites for the development of the spores Capsules of various ages of P. eyathi- themselves. Fine structural attributes carpum were excised from the leafy plants of' cell s of the spore-sac are indicative and fixed according to conventional pro­ of their secretory nature, like those cedures for light and electron microscopy. of the secretory tapetum in higher Paraffin-embedded sections cut at 7 jim plants. The ornamentation on the spores were stained with PyroninY for thelocali ­ seems to be exogenous, possibly of the zation of RNA and with MBB for total tapetal (spore-sac)origin . proteins. Cytophotometric observations were carried out using a Reichert Micro- References photometer. Measurements were made at 550 nm (for RNA) and 610 nm (for total Chauhan, E., 1981. Ultrastructural, cyto­ proteins) peak transmission of the inter­ chemical and histoenzymological studies ference wedge filter. The mean absorption on some aspects of sporophyte develop­ values were substituted in the plug-method ment in the moss Physcomitrium cyathi- to determine the relative amounts of carpum Mitt. Ph.D. Thesis, Univ. of chromophore per cell. Sections of 1. 5 fjpa Delhi. thickness cut from the Glut-Osmium fixed/ Lai, M. & E. Chauhan, 1982. Cytochemical Durcupan embedded capsules were stained studies on sporogenesis in Physcomi­ with PAS and TBO reactions, for pectins, trium cyathicarpum Mitt. — Nature and for callose. Ultrathin sections, of the spore mother . Cryptoga- stained with uranyl acetate and lead mie,Bryol .Lichénol . 3:5.1-57 . citrate were viewed and photographed inPhilip sE M 300a t8 0kV .

1) Presentaddress :Universitä tUlm ,Abt .Allgemein e Botanik (Biol.II) ,Obere r Eselsberg, D-7900Ulm ,FR G 16 DISTRIBUTION OF PLASTIDS AND MITOCHONDRIA DURING SPOROGENESIS

INEQUISETU M HYEMALE

J. Bednara and B. Rodkiewicz

Institute of Biology, Maria Curie-SklodowskaUniversity , Akademicka 19 20-033 Lublin, Poland

Summary middle one of closely packed mito­ chondria and two others,mostl y of At themeioti c telophase Iplastid s plastids..(fig ,4) .Th e aggregation andmitochondri a of Equisetum aggre­ remains until the late telophase II, gate in an equatorial layer. Itper ­ when it changes its shape and sists until the telophase II, then it stretches between telophase nuclei. isreshape d and separates postmiotic Later a cell plate is set up along cells. After formation of cellplates themiddl e of the mitochondrial plastids take the position in a dis­ layer; plastids scatter in the cyto­ tal part of each spore whilemito ­ plasm, but aggregate again in the chondria are distributed randomly. distal part of the spore.Whe n the cell plate is completed, mitochondria Introduction are randomly distributed in the spore. Equatorial aggregation of organoids There are conflicting data on the has been observed inmeiosi s of a few behaviour of cytoplasmic organoids in plants - from mosses to Angiosperms sporogenesis of Equisetum (Lenoir, (for references see Rodkiewicz et al., 1934, Jüngers, 1934). Using an elec­ 1984), This type of arrangement,ac ­ tron microscope we followed localiza­ cording to some authors,ma y ensure tion of plastids and mitochondria in apportionment of organoids amongpost - meiosis and in spore differentiation meiotic cells. Itma y also play adif ­ of Equisetum hyemale. Plastids of ferent, not necessarily alternative, itsmeiocyte s contain grains role. All meiocytes with organoid visible in squash preparations of layers form tetrads after simulta­ sporangia after PAS-reaction . neous cytokinesis. Therefore it seems possible that the layers substitute Results and discussion cell plates and, at a later stage, are related to cell plate formation. During meiosis plastids andmito ­ Distal localization of plastids in chondria of Equisetum meiocytes young spores of Equisetum precedes change their localization in aregu ­ the division into rhizoidal andpro - lar pattern (fig. 1 a-p).I n the thalial cells. early prophase they are randomly dis­ persed throughout cytoplasm. Some­ References what later all plastids andmito ­ chondria aggregate in a large group Jüngers, V., 1934.Mitochondries , close to the nuclear envelope (fig. chromosomes et fuseau dans les 2). Later inprophas e they disperse sporocytes de 1'Equisetum limosum. again around the nucleus,bu t before Cellule 43:321-340. themetaphas e Ithe y form two groups Lenoir,M. , 1934.Etud e vitale de at the opposite sides of thenucleus . sporogénèse et des phénomènes d'ap­ At metaphase and early telophase parence électro-magnétique concomi­ plastids and mitochondria move to­ tants chez 1'Equisetum variegatum. wards the equator of the meiocyte Cellule 42:353-409. where they congregate in aring .A t Rodkiewicz, B.,K . Kudlicka &H . Sto- the late telophase Ial l these or­ biecka, 1984.Pattern s of amylo- ganoids occupy the equatorial plane plast localization duringmicro - (fig. 3)separatin g the cell into two sporogenesis inTradescantia , Impa­ parts. This aggregation of organoids tiens and Larix Acta Soc. Bot.Pol . differentiates into three layers:th e 53: (i n press).

Acknowledgements:V/ ethan k I.Gielwanowsk a M. Sc. for her assistance.

17 Fig. 1. Equisetum sporogenesis • -mitochondrion o-plastid

r ,o • o; > O O'O o \wi/;//n

Fig. 2 Light micrograph of pro­ phase Imeiocyte s after PAS reaction. Amyloplasts gathered in one dense group close to the nuclear envel­ ope. L Fig. 3 Electron micrograph of sporocyte in telophase I stage. All plastids and mitochondria arranged in a band at the equatorial region of the cell.

Fig. 4 Mitochondria closely packed and plastids inpos t telo­ phase band. ANTHERDEVELOPMEN T OFGASTERI A

C.J. Keijzer

Department ofPlan tCytolog y andMorphology ,AU ,Wageningen ,Th eNetherland s

The developing anther of Gasteria verru­ resistant structures, being the orbicules, cosa (Mill.) Haw. was investigated by the tapetal membranes and the exine (on means of light and electronmicroscopy. the microspores) arise gradually and syn­ Some aspects of the development and behav­ chronously. After the microspores are iour of living anthers in situ had been released from the callose wall, the tapetum followed earlier by means of micromanipula­ starts to degenerate. The plastids lose tion (Keijzer, 1983). These observations their starch forming lipid-like drops together lead to some functional relation­ and fuse to form a part of the pollenkitt. ships and can explain the development Next this substance fuses with the content ofth e livinganther . of the ER to form the pollenkitt. Shortly The two main goals of a developing before the tapetal cells consist anther are the formation and the exposure merely of pollenkitt and some cellremants , of mature pollen. The young Gasteria covered by the tapetal membranes with consists of an , an endothecium, theorbicules . one middle layer, a tapetum and amultilay - The first visible spare substance of ered sporogenous tissue. These strongly the future pollengrains is presumably differing cell layers appear to develop the meiotic callose wall, as the main on behalf of either the first or the second pollen storage product, starch, is formed goal,o r both.Moreove r interactions between immediately after the post-meiotic dissolu­ these tissues can be found besides the tion of this wall. After the first pollen onestha tar ealread yknown . the amount of cytoplasm in the The epidermis and the endothecium develop vegetative cell increases, by which the somewhat comparable. Both layers arealread y cell expands strongly. This expansion vacuolated before meiosis and contain is made possible by the stretching of chloroamyloplasts. After the first pollen the colporal region, less by the rest mitosis the endothecium cells lose most of the exine. Finally the swelling pollen­ of this starch and form their characteristic grains press themselves into the tapetum wall thickenings. At the same time the and the middle layer remnants, by which cells of both layers swell by vacuolation, the pollenkitt finds its way capillary causing the inward bending of the locule into the intercellulary spaces between wall, thanks to the inflexible innertangen ­ the pollengrains and even between the tial wall of the endothecium cells. This epidermis and the endothecium cells. In mechanism keeps the opened stomium closed this way the exines stick to each other and works opposite to thewel lknow noutwar d and to the orbicules and the tapetum mem­ directed bending later on, at"dehiscence. k branes. So the pollengrains stay on the It prevents the loss of immature pollen locule wall after dehiscence, waiting intoth e closedflowerbud . fo'ra . Shortly before the flower opens, the epidermis enlarges its cuticular surface Reference by the formation of ridges on its outer tangential wall. This enlargement enables Keijzer, C.J., 1983. Hydration changes a very fast evaporation from epidermis during anther development. In: D.L. andendothecium ,b ywhic hth eanthe r dehisces Mulcahy and E. Ottaviano (Eds.): Pollen, immediately afterth eflowe ropens . biology and implications for plant The function of the single middle layer breeding.Elsevier ,Ne wYork . is more difficult to explain. During anther development its cells are stretched in tangential direction by which they are flattened. Although this phenomenon is often described as disruption, degeneration takes place only just before anther de­ hiscence. Next the degenerating fatty cellconten tmixe swit hth etapeta lremnants , but keeps separated from the content of the bordering, swollen endothecium cells. The tapetum and the microspores interact to form three final products: the mature pollengrains,th epollenkit t andth eorbicul e covered inside of the locule wall. The amount of cytoplasm in the tapetum cells increases during meiosis. The acetolysis 20 ANTHER DEVELOPMENTAN DPOLLE N FORMATION INCUCURBITACEA E

Shridhar*an d DalbirSingh* *

*Dept .o fBotany ,Governmen t PostGraduat e College,Sriganganagar ,Raj ,Indi a **Dept .o f Botany,Universit y ofRajasthan ,Jaipur ,Rajasthan ,Indi a

Summary Dzevaltovsky et al (1973) and Dzeval- tovsky & Zhalalov 1976)hav e also reported Development of anther and formation 5-layered anther wall (including epidermis) of pollen grains were studied in 18 species in Cucumis anguria and Mukia scabrella, covering 14 genera and six tribes of Cucur- respectively. bitaceae. The number of anther lobes was The tapetum is secretary and the cells variable and they showed cohesion to vari­ become multinucleate (Castetter, 1926; ous degrees. The anther wall development Passmore, 1930; Asana & Sutaria, 1932). is of "dicotyledons type". The mature The multinucleate nature of the tapetal wall compresis epidermis, fiberous endothe- cells in Cucurbitaceae has attracted the cium, 2 ephemeral middle layers and the attention of many workers who tried to secretary tapetum. In Luffa cylindrica explain the mechanism by which this con­ alone tapetal cells formed periplasmo- ditionha sbee nattained . dicum in situ.Tapeta l cells showedenlarge ­ In India although a large number of ment, division of their nuclei and fusion investigations have appeared on the origin ofresultin gnuclei . and development of gametophyte Microsporogenesis is normal and the (Banerjee & Das, 1937; Chakravorty, 1947; microspores are arranged in tetrahedral Singh, 1970) which cover a good number and isobilateral fashion in tetrads.Usual ­ of plants from each tribe of the family, ly the microspore nucleus is uninucleolate studies on the development and structure but occasionally multinucleolate condition of anther and the male gametophyte are occurred inL .cylindric a and Mukiascabrel - scanty (Asana & Sutaria, 1932; Banerjee la• Enucleate cytoplasmic nodules were & Das, 1937). The present study was there­ observed in developing vacuolated pollen foreundertaken . grains of pepo, Trichosanthes bracteata and T. dioica, a feataure hither­ Materialsan dmethod s tounknow nt opolle ngrains . Pollen grains are 2 celled at shedding. of all stages, youngest to open Usually uninucleolate but rarely multi­ flower, of Momordica charantia, Citrullus nucleolate vegetative nucleus was observed colocynthis, C. lanatus, grandis, in Dactyliandra welwitschii, Edgaria dar- Lagenaria siceraria, Cucurbita pepo, Luffa jeelingensis, L. cylindrica and Tricho­ cylindrica, cyclanthera pedata, Sechium santhes spp. Mature pollen grains were edule, Trichosanthes anguina, T. bracteata found to be colpate, colporate and porate and T. dioica, T. lobata, Edgaria darjee- and exine surface varied from reticulate lingensis, Ctenolepis garcinii, Dactylian­ tospinate . dra welwitschii,Cucumi smel o var.utilissi - mus andMuki a scabrella were studied. Usual Keywords: dicotyledons, fibrous, ephemeral, microtome techniques were employed to secretary, multinucleolate, cytoplasmic prepare double stained slides for study. nodules. Observations Introduction Anther lobes in the members of Cucurbi­ Tropical family Cucurbitaceae with 110 taceae vary from 5 (Mukia scabrella) to genera and 640 species (Willis, 1973) 18 (Coccinia grandis). In Cyclanthera is characterized byman y distinctivemorpho ­ pedata, the whole androecium forms a cir­ logical features. Chakravary (1959) record­ cular disc with a central partition running ed 34 genera and 108 species from India. throughoutth edisc . The embryological work on the family has been summerised by Kirkwood (1904), Johan- Development and structure of anther wall sen (1950), Davis (1966) and Singh (1970). The literature on the development of anther The young anther comprises one to three and pollen is scanty. Kirkwood (1907) hypodermal male archesporial cells (Pig.1 ) reported, in addition to epidermis, three differentiate at each of the sites destined wall layers in anthers of Micrampelis to form the pollen sees. The archesporial and Cyclanthera and four in Cucumis and cell(s) divide periclinally forming an Fevillea. Heimlich (1927) observed four outer primary parietal layer and an inner parietal layers inCucmi ssativus . sporogenous layer (Fig. 1). The parietal

21 cells undergo periclinal as well as anti- isplosp l clinaldivisions forming outer and inner secondary parietal layers (Fig.2) . The latter enlarges and differentiates to form the tapetum (Figs 2, 3, 4) whereas the former redivides periclinally (Figs2 , 3). Of the two resulting layers the outer undergoes one more periclinal division (Fig. 4). Thus the parietal layer forms in all four anther wall layers. The outer­ most derivative functions as the endothe- cium, the innermost as the tapetum and the remaining two as the middle layers (Fig. 4). The development of the anther wall conforms to the "Dicotyledonous type" of Davis (1966) and is summarised in the schemegive nbelow :

jo.o.s.p..p.1 1._r~-|endotheciu . m (en) >.p.l. J l__Tiiiddlelaye r (ml) lo.s.p.1. _ tapetum (1)

The innermost wall layer forms the glan­ dular secretary tapetum (periplasmodium in L. cylindrical which is of dual origin. To begin with the tapetal cells are uni­ nucleate but later on as the microspore mother cells enter meiosis, these cells enlarge radially and become usually binuc- leate, rarely polynucleate in M. charantia (Fig. 5).

Microsporogenesis

The microsporogenesis follows a common pattern. The sporogenous tissue is usually 1-layered (Fig. 3). The cells undergo a few mitotic divisions and a mucileginous wall is secreted around each cell. The microspore mother cells undergo normalv meioticdivision .

Microgametogenesis

The young microspore grows in size and its nucleus is usually uninucleolate (Fig. i~S,9;10 SCf 7), rarely the multinucleolate condition is observed in L. cylindrica (Fig.9 ) Figs 1-11: Figs 1-5: Momordica charantia. andM .scabrella . Fig. 1: T.s. of anther portion showing An interesting phenomenon, hitherto two archesporial cells; Figs 2-4: same unreported' i n pollen grains, is the forma­ showing formation of wall layers and micro­ tion of enucleate cytoplasmic nodules spore mother cell; Fig. 5: T.s. part of observed in the pollen grains of Cucurbita anther. Note the degeneration of the inner pepo, T. bracteata and T. dioica. The middle layer and 2 or 3 nucleate tapetal nodules are observed as small balloon-like cells. Figs 6, 7, 8: Citrullus lanatus. outgrowths from the inner lining of the Fig. 6: T.S. part of mature anther wall; cytoplasm into the large central Figs 7, 8: one and two celled pollen grain (Fig. 11). respectively. Figs 9, 10:Luff a cylindrica, The nucleus of the microspore divides pollen grains showing multinucleate con­ to form a large vegetative and a small dition in microspore nucleus and vegetative lenticular generative cell. The vegetative nucleus, respectively. Fig. 11: Cucurbita nucleus which is usually uninucleolate pepo: uninucleate pollen grain showing (Fig. 8) becomes multinucleolate during enucleate cytoplasmicnodules . the maturation of pollen grains in L. cylindrica (Fig. 10) and in 4 other plants.

22 Chakravarty, H.L., 1959 . Monograph on Discussion Indian Cucurbitaceae. Rec. Bot. Survey The present study on 18 species of the India 17:1-234 . Cucurbitaceae gives the first comprehensive Chakravorti, A.K., 1947. The development account of the anther wall development of female gametophyte and seed of Coc- and structure in the family. The develop­ cinia indica. W. & A.J. Indian Bot. ment of the anther wall follows the "Dicot­ Soc. 26:95-104 . yledonous type" and the anther wall is Devis, G.L., 1966. Systematic Embryology of 4 layers. The inner middle layer de­ ofAngiosperms . generates soon after its formation. This Dzevaltovsky, A.K. et al., 1973. Some may account for the observation of only data on study of the male generative 3 wall layers in Cylanthera and Micram- spore in Cucumis anguria collected scien­ tific articles. Publ. House Kiev State pelis by Kirkwood (1907). The endothecium Univ. develops the characteristic fibrousthicken ­ ings. Dzevaltovsky, A.K. & G. Zhalalov, 1976. Periaswamy & Swamy (1966) have reported On embryology of Malothria scabra Naud. that the tapetum is of dual origin in Ukr.J . Bot.33 :287-290 . angiosperms. This is confiremd in the Heimlich, L.F., 1927. The development present study also. Initially the tapetal and anatomy of the staminata flower cells are uninucleate but they become of the cucumber. Am. J. Bot. 14:227-237 . binucleate in all the taxa investigated Johansen, D.A., 1950. Plant Embryology. presently. The tapetum is secretary and Waltham,Mass. ,U.S.A . in L. cylindrica alone it forms a peri- Kirkwood, J.E., 1904 . The comparative plasmodium which is - absorbed while in embryology of the Cucurbitaceae. Bull. situ. Turala (1958, 1963) has asserted N.Y.Bot .Gard . 3:313-402 . thatth etapetu m remainsuninucleat ethrough ­ Kirkwood, J.E., 1907. Some feature of out in 7 members studied by her and that pollen formation in the Cucurbitaceae. further differentiation is attained through Bull. TorreyBot .C1./34 :221-242 . endomitosis. This is in contrast to our Passmore, S., 1930. Microsporogenesis observations which have clearly shown in the Cucurbitaceae. Bot. Gaz. 990: that the cells become usually 2-nucleate 213-222. inal lth etaxa . Periaswamy,K . &B.G.L . Swamy, 1966.Morpho ­ logy of the anther tapetum of angio­ An interesting and hitherto unrecorded sperms.Curr .Sei . 35:427-430 . feature is the formation of enucleate cytoplasmic nodules in the uninucleate Singh, D., 1964. Cytoplasmic modules in pollen grains in C. pepo and Trichosanthes the endosperm of angiosperms. Bull. spp. The nodules arise from the inner TorreyBot .Cl . 91:86-94 . lining layer of the cytoplasm and buldge Singh, D., 1970. Cucurbitaceae: a review into the large central vacuole of the in comparative embryology of angios'perms. uninucleate pollen grains. Such cytoplasmic Bull. Indian Nat. Sei. Acad. 41:212-219 . nodules have been recorded in the endosperm Turala, K., 1958. Endomitosis in the tape­ of many plants belonging to dicotyledons tal cells of Cucurbita pepo. Acta Biol. and (see Singh, 1964). Cracov Bot. 1: 24-35 . The significance of the cytoplasmic nodules Turala, K., 1963. Studies in endomitotical even in endosperm development remains processes during the differentiation disputed. Singh (1964) consider it an of the tapetal layer of the Cucurbita­ active method of endosperm growth helping ceae. Acta Biol. Cracov. Bot. 6: 87-102. in the obliteration of the central vacuole. Willis, J.C., 1973. A dictionary of the Another interesting feature in pollen flowering plants and ferns. Cambridge grains is the multinucleolate condition Univ.Press . of the microspore nucleus and vegetative nucleus in someo f the investigated members.

References

Asana, J.J. & R.N. Sutaria, 1932. Micro­ sporogenesis in Luffa aegyptiaca Mill. J. Indian Bot.Soc . 11:181-187 . Banerjee, I. & M.C. Das, 1937. The devel­ opment of microspores in Trichosanthes dioica Roxb. Ind.J . Agr. Sei. 7:497-510 . Castetter, E.F., 1926. Cytological studies in the Cucurbitaceae: Microsporogenesis in Cucurbita maxima. Am. J. Bot. 13: 1-10.

23 MICROSPOREAN DPOLLE N GRAINFORMATIO N INRHOE ODISCOLO RHANC E

A. Souvré,L .Albertini ,A.C .Dha ran dM .Petitpre z

Laboratoired eCytologi e etPathologi eVégétales ,E.N.S .Agronomique ,14 5avenu ed eMuret , 31076Toulous e Cedex,Franc e

Introduction callose,cellulos e andpecti c compoundsbu t afterther ei sa progressiv e losso fth e Thepresen treport ,ultrastructural ,cyto - pecto-callosiccompound s. Whe nth edetachmen t chemicalan dradioautographi c studyo fmi ­ ofG Cfro mintin eproceed s ,th eV Can dth e crosporean dpolle ngrai ndevelopmen ti n GCar eseparate db y theirplasmalemma . Rhoeodiscolo r(Commelinaceae )i sa par to f Thenucleu s ofth eV Ci shighl yconvolute d ourtea mresearc hdevote d toth e studyo f andther ei sahig hdensit yo forganelle si n microsporocyte and tapetum(periplasmodial ) thevegetativ ecytoplas m ;particularely , developmentan dth einterrelatio no fthes e dictyosomes,mitochondri a and lipidicglo ­ twotissues . bules (inth eVC ,th eneutra l lipidsar e strongly stainedb ySuda nblac k andacidi c Observations lipidsb yNil eblue) .Afte r thepre-mitoti c amylolysis,amylogenesi soccur swhic hrenew s Microsporeprotoplas m thepolysaccharidi c granuleso famyloplasts . During theearl yperio d ofpollen ,G Ccon ­ After thelysi s ofth e specialcallosi c tainsth esam eorganelle s astha to fV Cbu t wallsurroundin g thetetrad ,th eyoun gmi ­ thedensit y islower .A tth ematur estag eo f crosporeswit ha centra lnucleu s (stage 1) thetwo-celle d pollengrains ,th egenerativ e areseparate d ;approximativel y 20% o fmi ­ nucleusi shorsesho e shaped and itoccupie s crospores arefertile .Durin g thematuratio n themajo rpar to fth eGC . ofmicrospores ,th enarro w zoneo fcytoplas m Aflash-labellin gwit h3u-uridin e(RNA )fo r which iscrushe dbetwee nplasmalemm a (close isolated pollengrain smark sfirstl y thege ­ toth e sporoderm)an dvacuola r tonoplast nerative andvegetativ enuclei .Fo rth elon g containsa nincreasin gdensit yo fribosome s period ofprecurso rsupply ,th eV Ci smor e andpolyribosomes .Th emitochondri a swell,th e labelledbot hi nit scytoplas man dnucleu s; RERprofile swit hnumerou s ribosomesattache d theincorporatio no f3n-uridin eint ovegeta ­ multiply and thediamete ro fth epolysaccha ­ tive cytoplasm canb eassociate dwit hth e ride granuleincrease s frommicrospor esta ­ multiplicationo fit sRE Rprofiles . ge 1(1- 2ym )t opremitoti cstag e(1,5 - Thevegetativ ecytoplas m stronglyincorpo ­ 3,5 Vim).But ,th enumbe ro fdictyosome sre ­ ratesth e3ji-gi Ucose,3n-acetat e (neutral mainspoor . lipids)an d 3H-choline (phospholipidswit h At thebeginnin g ofmicrospor ematuration, k choline),thes eactivitie s correspondt oth e itscytoplas mstain sdeepl ywit hFas tGree n amylqgenesis,th eincreas eo f lipidicglobu ­ FCF (basicproteins) .Durin g theperio do f le'san d thecytomembran emultiplication , maturation,ther ei sa slo wincorporatio no f respectively. severalprecursor s (3H-uridinean d3n-oroti c acid :RN A ;%-a rginin e :h istone s; Sporoderm 3H-leucine :tota lproteins )an da lo win ­ corporationfo rth e3H-tryptophan e (non­ Themai npar to fexin e isbuil tunde rth e histone proteins)int oth enucle ian dcyto ­ callosicwall .Th epresenc eo fwhite-line s plasmo fmicrospores .Th e cytoplasmiclabel ­ inth einne rpar t ofth eprotoexin eindica ­ lingwit h3n-prolin ean d '^C-hydroxyproline testh erol eo fplasmalemm a inth eexin ede ­ (theamino-acid softe n liedwit hglycopro ­ velopment.Afte r thebreakin g up ofth ete ­ teins)i smoderat e inth eyoun gmicrospore s trad,th eexin ereact spositivel y toth e and itdecrease sdurin gfurthe rdevelopment . lipid reagentsbu tonl y theexin eo fth e The3H-gl ucose ismainl y incorporated into fertilemicrospore s incorporates thelipi d the insolublepolysaccharides ,i nrelatio n precursors,particularel y the '^C-mevalonate toth eamylogenesi sa spreviousl y observed (carotenoidic lipids). byTEM . Intinewhic hi sdevoi do fcallose ,i spo ­ sitivefo rPA S andThier y tests.I tcontain s Pollenprotoplas m a loto facidi cpecti c compounds(whit hfre e -COOH)i nth eoute rlaye randpecto-cellulosi c Inth eyoun gpolle ngrain ,th ehemisphe - compounds inth e inner layer.Whe nth ethick ­ roidalwal ltha tseparatin g thevegetativ e nesso fintin e increases,th esporoder mo f cell (VC)an dth egenerativ e cell (GC)i s thefertil emicrospore s (fromstag e3 )an d attached tointin ean d iti sThiery-positive . pollengrain s incorporatesth e3H-myo - Atth ejuvenil e stagethi sfin ewal lcontain s inositol.

24 POLLENGRAI N DEVELOPMENT INTH EOLIV E (OLEA EUROPAEA L.):ULTRASTRUCTUR EAN DANOMALIES .

E. Pacini ,G.G.Franch i and L.M. Bellani

Department ofEnvironmenta l Biology,Botanica l Section,Sien aUniversity ,Ital y Department ofPharmaceutica l Chemistry andTechnologies ,Pharmaceutica l Botany Section, SienaUniversity ,Ital y

Summary Tapetum cooperates in the pollen grain The ultrastructure of pollen grain development producing: 1) nutritive development in the olive is described from substances, 2) control substances archesporium differentiation to pollen (callase), 3) sporopollenin precursors and shedding.A particula r emphasis isgive nt o orbicles,4 ) substances to be deposited on the relationships between tapetum and pollen grain surface e.g. tapetal prints, meiocytes/microspores/pollengrains ,an dt o sporophytic proteins and pollenkitt. some cytological events.Als o the presence Tapetal protopast volume (detected by of anomalies is described from the cross-sections) increases up to the early ultrastructural pointo fview . microspore stage, followed by tapetal Key words: Olea europaea, pollen grain degeneration in correspondence to pollen development,anthe rtapetum . first haploid mitosis. Also volume and number ofmitochondri a andundifferentiate d Introduction plastids (at last becoming elaioplasts) The ultrastructural features of some continuously increase up to pollen first aspects of pollen grain development in haploid mitosis, whilst free and ER olive (Olea europaea L.),i.e . the tapetum ribosomes, and ER cisterns, reach the development (Pacini & Juniper, 1979b; maximum atth emid-microspor estage ;the na Pacini & Casadoro, 1981), the formation of little decrease is detectable, before sporophytic proteins in correspondence to degeneration occurs.A sth e resulto fthei r the pores (Pacini & Juniper, 1979a), the degeneration,tapeta l cellsar etransforme d sporoderm pattern in some cvs. (Pacini & in round lipidic masses, which before Vosa,1979) ,th emai n anomalies atth eL.M . dehiscence will cover the grain surfaces (Pacini et al., 1978), have already been (pollenkitt). described. In this paper we intend to After archesporium differentiation, but complete the missing sections of the before the first meiotic prophase, mMC are ultrastructural description, together with connected to one another and to tapetal pollenanomalies . cells by plasmodesmata. During leptotene plasmodesmata between mMC and tapetum Materials andMethod s disappear, whilst some connecting mMC Olive anthers were collected, fixed, between themselves seal and someother sar e embedded, cut, stained and observed as transformed in cytomictic channels; these previously described (Pacini & Juniper, channelsclos edurin g interphase. 1979a). The callosic wall starts developing around meiocytes during zygotene, and is Resultsan d Discussion completed at diplotene; at the mean time Pollen grain development isth e resulto f mMC, firstly polyhedral, become round,an d an interaction between two tissues, which the locular fluid is formed. During the appear after archesporium differentiation, first and the second meiotic prophases, namely the microspore mother cells (mMC) some nucleoloids are formed: these are and the tapetum. This interaction is accessory nucleoli and are released in the explained in Fig. 1, which refers to the cytoplasm during the first and the second observations atth e E.M. level. In this karyokinesis (Dickinson & Heslop-Harri- figure all the steps leading to pollen son, 1977). They are detectable in the ripening are represented, starting from microspore cytoplasm up to the mid archesporium differentiation. This whole microspore stage, and probably originate processoccur si nabou t seventeen days. new ribosomes (Fig.2) .

25 LOCULtJS DOMAIN .4 . ARCHESPORIUM DIFFERENTIATION TAPBTUM DOMAIN"

iE [ y Icallosic wall formation Jpa >and prophase < JmMC roundishing Indivision interphase {a^divïsion

fcallosic walls formation TETRADS^ primexine formation [callosic walls dissolution-^.]

just released exine completion change of shape vacuolization/ new ribosomes foi h amylogenesis /amylolysis nucleus-cytoplasm interactions

'Ë Era 20-

s, V <ïy l HAPLOID MITOSIS . rprotoplas t volume and 'ejjeclsJlDEGENERATIONllsvmtomftjribosomes decrease, central /vacuolization/new ribosomes formation * (vacuole disappears 2celled amylogenesis/amylolysis GAMETO < PHYTl E PGdehydratio n # kripe PG

FIG.1 .Schemati c representation ofth emai n phenomena occurring inth etapeta l cellsan d loc\jlusdurin g pollen graindevelopment .

FIG.2.Microspor e during callosedissolution . C:callose ;P :primexine :N :nuçlcoloi dr e leasingpolyribosome s (arrow heads)i n the adjacent cytoplasm.

l"HM FIG.3 .Increas ei nth e equatorial diameters ofmicrospore s and pollen grains infunctio n ofdevelopmenta l stages.T :tetrads ;EM :ca £ lymicrospores ;MM :middl e microspores;LM : latemicrospores ;1s tIIM :firs t haploidmi _ FIG.4 .Twi n latemicrospores .Intin e (arrow tosis; SP:sheddin gpollen . heads)an d pores (P)ar ealread y formed.

26 Meiosis leads to the formation of moretha nthre epores ; tetrahedric tetrads and the callosic wall 4)polle n grainsgerminate d insideanthers . is formed centripetally after the second At the ultrastructural level,bot h micro meiotic division. As soon as microspores and macro-grains may be empty or full of are isolated from oneanother ,primexin ei s cytoplasm when shed, as they derive from laid down, showing all exine components, meiotic anomalies. It is remarkable that with the exception of endexine (Fig.2) . sometimes macro-grains contain two After thedissolutio n ofth ecallosi cwall , generative cells. These anomalies are the free microspores rapidly change their generally also linked to the presence of shapean d become round;a shar p increasei n irregularly placed pores,an d macro-grains the equatorial diameter occurs during the poresar eofte n fouro rmore . early and middle microspore stages (Fig. When pollen grains (two pollen grains, 3), followed by a slight increase in the and sometimes four) are connected by late microspore stage and the two celled cytoplasmic bridges (Fig. 4),the y might stage:whe n pollen isshed , grain diameter derive either from two adjacent tetrads, ismor eo r lessth edoubl e thantha to fth e where cytomictic channels between the mMC single microspore inside the tetrads (Fig. were not sealed off during the meiotic 3). Olea europaea microspores do not interphase,o r from two microspores of the increase very much ,i fcompare d withothe r sametetrad ,no tcompletel y separated after species (Heslop-Harrison, 1972). the second meiotic division (the callosic Exine is completed before the mid wall is incomplete and perforated - Pacini microspore stage, but the tapetum still & Cresti, 1977). In these twin grains produces sporopollenin precursors, up to cytoplasms are continuous, and plastids the first haploid mitosis, when coreless behave inth e same way,^hils t in adjacent orbiclesstar t toappear .Othe rcytologica l microspores/grains their development is events during the middle and the late slightly asynchronous in the amyloge- microspore stages are: microspore nesis/amylolysis. vacuolization,followe d byth eformatio no f Dehiscing anthers (in seven cvs. out of new cytoplasm and ribosomes; the first the forty-eight observed) often contain amylogenesis, with few small starch grains pre-germinated grains. The hypothetical per plastid (these grains are hydrolized causes and the ultrastructural features of justbefor e the first haploid mitosis);th e this anomaly have already been discussed formation of ER stacks from the nuclear (Pacini &Franchi ,1982) . membrane, which migrate towards the pores and,accordin g toPacin i& Junipe r (1979a), References are probably responsible for the Dickinson,H.G .& J . Heslop-Harrison,1977 . gametophytic proteins deposited into the Phil.Trans.R.Soc , Ser.B277:327-342 . tubuleso fpora lintine . Heslop-Harrison,J. , 1972.In :F.C .Stewar d The first haploid mitosis is (Ed.): :a treatise . asynchronous,lastin g about threedays ,an d Vol.V IC Physiolog y ofdevelopment :fro m isfollowe d by a new vacuolization, and by seeds tosexuality . Academic Press,Ne w a new wave of starch deposition in York.p.133-289. plastids. Starch in this second Pacini,E.& G . Casadoro,1981 .Protoplasm a amylogenesis is more abundant than in the 106: 289-296. first one, and it is cumulated in one Pacini,E.&M .Cresti ,1977.Plant a 137:1-4 . single big grain per plastid (often a Pacini,E.& G.G . Franchi,1982 . Caryologia composed grain). As reported by Pacini et 35:205-215 . al. (1978) in some cvs. this starch is Pacini,E.& B.E'.Juniper ,1979a .Ne wPhytol . hydrolized before pollen shedding, whilst 83:157-163. inothe r pollen isshe d starchy. Pacini,E . & B.E. Juniper,1979b . New As far as morphological anomalies are Phytol.83:165-174 . concerned they have beenclassifie d (Pacini Pacini,E.& C.G.Vosa ,1979 .Ann .Bot . 44: etal. , 1978),in fourgroups : 745-748. 1)macr o andmicro-grains ; Pacini,E.& M.Cresti,F.Ciampolin i& G.Bini , 2)polle n grainsno tseparate d butconnect ­ 1978. In:S.Sansavin i (Ed.): Laferti H edb ya cytoplasmi c bridge; ta nellepiant e dafrutto .Sociét éOrti - 3)polle n grains with irregular poreso r cola Italiana,Bologna ,p.643-654 .

27 THEEFFEC TO FCOL D (4°C)O N3 H-PROLINEAN D 1^C-HYDROXYPROLINEINCORPORATIO N INMICROSPORES , TAPETUMAN D CONNECTIVE OFRHOE ODISCOLO RHANCE .A RADIOAUTOGRAPHI C STUDY

A.Souvré ,L .Albertin i andA.C .Dha r

Laboratoired eCytologi e etPathologi eVégétales ,E.N.S .Agronomique , 145avenu ed eMuret , 31076Toulous e Cedex,Franc e

Summary 1970).Th eeffec t ofcol d (4°C)o namino - acid incorporation inth emicrosporocytes , The effect ofcol d (4°C)o nth e levelo f tapetum and connective ofanthe rha sbee n theincorporatio n of-'H-prolin ean dl^C - studied by comparing theincorporatio ndat a hydroxyproline intoth enuclear ,cytoplasmi c obtained from anther setstreate ddifferen ­ andwal lprotein s ofmicrosporocytes ,tape - tly :is tge t :2 4h labelling at22° C turnan d anther connective duringmicrosporo - (control 1); 2n dse t :2 4h labellinga t genesisha sbee n studied inRhoe odiscolo r 22°Cfollowe db y 48h incubatio nwithou t Hance,a plan t sensitive tochillin gstress . radiotracer at4° C ;3r d set :2 4h label - The cold treatmentwa s applied to theexci ­ lingat 22°C (14C-HYP) followedb y 48h incu ­ sed inflorescenceseithe rdurin g orafte r bationwithou t radiotracer at22° C (control labellingwherea s thecontrol swer e labelled 2)o r2 4h labelling at4° C (3H-PR0).Th e at22°C . anthers fixedb y ethanol-acetic acid3/1 , Keywords :col d treatment,-%-proline , thenembedde d inparaffin ,wer e sectionned -^H-hydroxyproline,radioautography ,micro - (8ym )an d treatedb yFicq' s (1961)radioau - sporogenesis,Rhoe odiscolor . tographic technique (Ilfordnuclea r emulsion inge lform ,K 2 typefo r 3{J-PRO an(j(5 5typ e Introduction for '^C-HYP ;tim eo fexposur e indar kroom : 1mont hfo r 3R-PR0 and 15day sfo r l^C-HYP). Rhoeodiscolo rHance ,a plan twhic hi s Beforemounting ,th e sectionswer estaine d characterized by ameiosi swit h aphenomeno n withmethy lgreen-pyronin . of catenationan db y aplasmodia ltapetum , Thetota lactivit y ofa give n cellular issensitiv et ochillin g stress.Recently , structurewa sdetermine d asbein gth enumbe r wehav estudie d byradioautoradiography ,th e of silver grains,counte d over thisstructure . effect of cold (4°C)o nprotei nsynthesi s in Eachresul t inTabl e 1i sth e averagevalu e microsporocytes and tapetum ofR . discolor obtained after counting thenumbe r of silver byusin g ^H-leucine (^H-LEU),3H-arginin e grains over 15-30nuclei ,cytoplasm s orwal l (3H-ARG)an d3n-tryptophan e (3H-TRP) portions ata give nstage . (Albertini and Souvré, 1983). Iti sknow ntha tpollen s containhig hle ­ Results (Tables 1an d 2) vels of freeprolin e and ofprotein s richi n proline (ZhangHong-qi ,Croe s andLinskens , a)2 4h labelling at22° C (control 1) 1982). Theglycoprotein s of-plan tcel lwall s containhydroxyprolin e (Lamport, 1977)pro ­ Pollenmothe r cells(PMC )an dmicrospores : ducedb y thehydroxylatio n ofprolin e (Chrispeels, 1970).Furthermore ,prolin eme ­ Thedetermination s carried oncontro l1 tabolismundergoe swid emodification s when corroborated earlier results obtainedwit h theplant sar echille d (Dashekan dErickson , 3H-LEU, 3H-ARG and 3H-TRP (Albertini,1970 , 1981). 1975 ;Albertin i and Souvré,1983 ) :th ein ­ Thepresen tpape r reports the influenceo f corporations of 3H-PR0an d l^c-TYPint oPM C cold onth e 3H-PRO and l^C-HYPincorporatio n nucleus and cytoplasmwer ehig h duringth e intoth eprotein s ofnucleu san d cytoplasm premeioticrestin g stage (PMR),the ndecrea ­ ofmicrosporocyte s and tapetum and intoth e sedmarkedl y during themeioti c prophasean d cellwal lprotein s ofconnectiv e duringmi - remained moderate to lowunti l theen do f crosporogenesis inR .discolor .Th epresen t meiosis and throughth e tetrad stagean dmi ­ dataar ediscusse dwit hsom eo fth eresult s crospore stages 1-4.Th enucleola r activity ofou rpreviou swor k and ofth eliterature . wasver yhig hdurin g thePMR ,the ndecrease d markedly (40% )a tmid-synizesi sbefor eva ­ Material andmethod s nishing at thebeginnin g of thezygoten e stage.Durin gPM Ran d synizesis,th eprimar y wallprotein s ofPM Cha sno t incorporated Thedifferen t stages ofth emicrosporoge - 3 14 nesis OfR . discolorHanc ehav ebee npre ­ H-PR0an d C-HYP but thePM Cwal lwa sla ­ viously described (Albertini, 1970).Th ein ­ belled from zygotene/pachytene untilth een d corporation of3 H-PR0an d l^C-HYPint oth e ofdivisio nI (during theformatio no fcallo - excised inflorescenceso fR .discolo rwa s sic special wall). The callosicwal lsur ­ monitored aspreviousl y described (Albertini, rounding thetetrad s didno t incorporate the

28 radiotracersbu t thepenetratio n ofthes e b)2 4h labelling at22° Cfollowe db y 48h radiotracers intoth etetra d cellsoccurre d incubationwithou t radiotracer at22° C asther ewer e silver grains found overth e (control 2; l^C-HY P :Tabl e2 ) cytoplasman dnucleus .Th eactivit y ofmi ­ crosporesporoder m issignifican t frommi ­ The levelo f '^C-HYPincorporatio nobser ­ crospore stage 1t ostag e4 . ved intoth edifferen t cellular structures wasmor eofte nequa lo reve nsuperio r (PMC Tapetum: nucleidurin gmeioti c prophase ;connectiv e cellwall )t oth e level of incorporationo f The incorporation of3 H-PROan d '^C-HYP thecontro l 1(2 4h labelling at 22°C). This intoth enucle i oftapetu ma tdifferen tsta ­ canb eexplaine db yth eprolonge d presence ges (meiosis,tetra d formation andmicrospo ­ ofth eradiotrace r inth eanthe r tissuesan d restage s 1-2)wa s relativelymoderat ewhe n more especially inth evascula r tissueo f compared toth eincorporatio n of 3H-LEU, connective. 3H-ARG,3 H-TRP (Albertini, 1970 ;Albertin i etal. , 1983). During thematuratio n ofmi ­ c) scol d treated at4° C crospores,th e incorporation ofprecursor s intotapetu mnucle iprogressivel y decreased When theinflorescence swer e treatedb y and finallywa s close tozer ojus tbefor e cold (4°Cfo r4 8h )afte rth eradioactiv e thedivisio n ofmicrospore .Th e cytoplasmic precursor supply,th eincorporatio n ofra ­ activity of thetapetu m (periplasmodium)in ­ diotracer intoth enucle ian d cytoplasmso f creased duringmeiosis ,becam ehig hbeyon d PMCan d tapetum and intoth e connective cell thepachyten e stage andreache d amaximu ma t wallmor eofte nmarkedl y decreasedwhe ncom ­ thetetra d stage (Tables 1an d2) . pared to thecontrol s 1(Table s 1an d 2)an d 2 (14c-HYP) ;Tabl e 2).Th e 3H-PRO labelling Cellwal lo fanthe r connective: ofconnectiv e cellwal lwa sver y sensitive tocol d aswel ldurin g themeiosi s asdurin g Frompremeiosi sunti l theen d ofmicrospo ­ themicrospor e development :th eobserve d restag e 2,th ecel lwal lan d thenarro wcy ­ reduction of labellingwa salway s above 73% toplasmic filmboun d toth ewal lincorpora ­ and evenreache d 100% a tmicrospor e stage2 tedmor e strongly the3 H-PR0 (104t o12 0 (Table 1).A marke d reductiono fwal llabel ­ silvergrain spe r 100p m length ofcel lwal l lingb y 3H-PR0 and ^C-HYPwa sals oobserve d section)tha nth e 14C-HYP inth eanthe rcon ­ inth ePM Cwal l (fromzygotene-pachyten eun ­ nective.Then ,th e incorporation ofthes e tilth een d ofdivisio n I)an d inth esporo ­ precursors intoth ecel lwal lprogressivel y dermo fmicrospore s during thestage s 1t o4 . decreased andbecam ezer oa tth e stageo f Wheninflorescence swer etreate d bycol d youngpolle ngrain . during theentir eperio d (24h )o f^H-PR O

Table 1.Distributio n ofradioautographi c silver grains overnucleu s and cytoplasm(i nbrac ­ kets)o fmicrosporocyte san d tapetuman dals oove rconnectiv ecel lwal l following-^H-prolin e (20yCi/ml )incorporatio n intoexcise d and chilled inflorescenceo fRhoe odiscolo rHance .

PMR Syn. D.M.) M2 M4

-K(2 4h a t22°C ) 59(31) 29(28) 10(8 ) 4 (8) 5(19) 2(16) PMC * (id.)the n4 8h°a t4° C 26(12) 10(5 ) 4 (8) 1 (2) 0 (6) 1 (0) _-*durin g 24h a t4° C 6 (8) 2 (2) 1 (1) 0 (2) 1 (2) 0 (3)

'-*(2 4h a t22°C ) 9(32) 15(62) 5 (5) 0 (0) Tapetum •*(id. )the n4 8h°a t4° C 5(22) 4(25) 0.(0 ) 0 (0) •ttdurin g 24h at4° C 1 (5) 2(18) 1 (5) 0 (2)

'*(2 4h a t22°C ) 115 - 104 120 60 Connective (100u mo f -X(id. )the n4 8h°a t4° C 11 - 27 0 11 cellwall ) -*durin g 24h a t4° C 16 - 17 17 5

•K= incorporation of radiotracer ;4 8h °= 4 8h incubationwithou t radiotracer. Abbreviations :PM C :microsporocytes ,PM R= premeioti crestin g stage,Syn .= synizesis , D.M.j= diakinesi s tometaphas e 1,T = Tetrad ,M 2 =microspor e stage2 ,M4 = microspor e stage4 .

29 Table2 .Distributio no fradioautographi c silver grains overnucleu san dcytoplas m (inbrac ­ kets)o fmicrosporocyte s andtapetu man dals o over connective cellwal l following l^C-hydro- xyproline (4uCi/ml )incorporatio n intoth eexcise dan dchille d (orno tchilled )inflores ­ cenceso fRhoe odiscolo rHance .

PMR Syn. D.M.] M2 M4

+++ ++ + + * (24h a t22°C ) (++) (++) (++) (+) +++ +++ +/++ +/++ PMC * (id.)the n4 8h°a t22° C +/++ (++) (+++) (++) (+/++) ++ + +/++ -/+ -*(id. )the n4 8h °a t 4°C ++/+ -/+ (++) (+) (+/++) (+)

"-X(2 4h a t22°C ) ++ ++ + (++++) (++++) (+++) (++) ++ ++ + Tapetum •*(id. )the n4 8h°a t22° C (+++) (++++) (+++) (+) + + + * (id.)the n4 8h °a t 4°C (++) (+++) (+/++

* (24h a t22°C ) +

Connective •*(id. )the n4 8h °a t22° C + +/+ + (cellwall )

* (id.)the n4 8h°a t 4°C -/+ -/+ -/+

* =incorporatio no fradiotrace r ;4 8h °= 4 8h incubatio nwithou t radiotracer. SeeTabl e 1fo rabbreviation s -= n olabelling ,-/ += ver ypoo r labelling (0t o2 grains) ,+ = poo r labelling (2t o1 0 grains),+ += moderat e labelling (10t o2 0grains) ,++ +averag e labelling (20t o4 0grains) , ++++= stron g labelling. The labellingo fM 2 andM 4 (PMC)correspon d toth ewhol emicrospores .

supply,th eleve lo fincorporatio n intoth e toth eyoun gmicrospor e stages :th ePM C la­ nucleian dcytoplasm so fPM Can dtapetu man d belling then'represent s 13t o2 6% o fth e intoth econnectiv e cellwal lwa smarkedl y PMCactivit ya tPMR) .A t22°C , 3H-PR0an d decreased with respectt oth econtro l 1(th e l^C-HYPwer e stillsignificantl yincorpora ­ activity lossmos t oftenrange d from7 0t o ted intoth eprotein so fcytoplas man dspo ­ 90 %). Theactivit yo fPM Cwal l (betweenth e rodermo fol dmicrospore s ;thi sresul ti s zygotenean dth een do fdivisio nI )an do f tob erelate dwit htw ofact s :1° / pollen sporodermo fmicrospore s atstage s 1t o4 proteins containPR O(Zhan gHong-q ie tal. , was lowo rzero . 1982),2° /tryphin ewhic h coatsth epolle n exine, contains antigenic proteins (Knox, Remarksan ddiscussio n 1971)tha tar eric hi nPR O(Underdow nan d Goodfriend, 1969).A t22°C ,th epatter no f Thepresen t resultsar et ob ecompare dwit h incorporationo f3 H-PRO andl^c-HY Pint oth e thedat ao fou rearlie r experimentso nth e increasing periplasmodium (fromth ezygoten e incorporation of 3H-LEU, 3H-ARGan d 3H-TRP toth etetra d stage)wa ssimila rt otha to f 3 3 (Albertinian dSouvré , 1983)an d3n- orotic H-ARGan d H-TRP (Albertini,1970 ,197 5; acid (Souvréan dAlbertini ,1978 )int oPM C Albertinie tal. , 1983);o nth eothe rhand , and tapetumo fRhoe odiscolo ran dt ob edis ­ duringth esenescenc e phase (afterth e te­ cussed inth eligh to fth edat aavailabl e trad stage)th eperiplasmodiu m incorporated fromliterature . lessactivel y 14c-HYPan despeciall y 3H-PR0 3 At 22°C,th egenera l trendo fincorpora ­ than H-LEU,3 H_ARG and 3H-TRP (Albertinie t tiono f3H-PR O and3 H-HYPint oPM Can dmi ­ al., 1983)int oit sproteins . crospores runsparalle lwit h thato f 3H-LEU Whenth ecol d treatment (4°C)wa sapplie d andmor e especiallywit h 3H-ARG (the3 H-PRO duringth e3 H-PRO supply,i nPM C(a tPM Ro r incorporation intoPM Ci smoderat ebu ti ti s meioticprophase )an di nexpandin go rfull y significant fromth ebeginnin go fzygoten e developed periplasmodium,onl ya smal lbu t

30 'significan t amount ofth eradiotrace r (7t o proline,glycosylatio n ofhydroxyprolin e 27% )o fth econtro l 1radioactivity )wa s and secretiono fth eglycoprotein .Bio - incorporated inth enuclea r and cytoplasmic chem.Biophys .Res .Commun .3 9 :p .732-737 . proteins (Table 1).Unde r similarconditions , noRN Asynthesi s couldb edetecte dwhe n 3H- Ficq,A. , 1961.Contributio n àl'étud ed u -oroticaci dwa suse d asa precurso r (Souvré métabolisme cellulaire aumoye nd e lamé ­ etal. , 1978). The latter tworesult ssug ­ thodeautoradiographique .Monographi e gest that theRNA spresen t inth ecel lbefo ­ n°9 ,Institu t Interuniversitairede s reth eactio no fcol d (particularly thelong - SciencesNucléaires ,Bruxelles . livedmRNA sdetecte d forexampl e inTrades - Kacperska-Palacz,A. ,Jasinska ,M. , cantiapolle nb yMascarenha s (1966))ar e Sobczyk,E.A . andWcizlinska ,B. ,1977 . ablet oinduc ea t4° Ca low ,bu t significant Physiologicalmechanism s offros ttoleran ­ proteinsynthesi s inth ePM Can dperiplasmo - ce :subcellula r localisation and some diumconsidered . physical-chemical propertieso fprotei n fractions accumulated under coldtreat ­ At 22°C,i nth eanthe r connective,th e ment.Biologi aPlantaru m 19 :p . 18-26. cellwal lan d thenarro w cytoplasmicfil m Knox,R.B. , 1971.Polle nwal l enzymes: bound toth ewal lhav e incorporated signifi­ taxonomiedistributio nan dphysica lloca ­ cantamoun t of3 H-PROan d 14C-HYP ;bu ta lization.I n :He slop-Harriso n (Ed.): reduction inth e levelo f incorporationwa s Pollen :developmen t andphysiology . observedwhe nth e inflorescenceswer eexpo ­ LondonButterworths .p . 171-173. sedt ocol d afterth eapplicatio no fth era ­ dioisotopes.Th ewal lprotein smigh thav e Lamport,D.T.A. ,1977 .Structure ,biosynthe ­ undergone theproces s ofproteolysis ,an dth e sisan d significance of cellwal lglyco ­ losso fprotein s inpartiall y compensatedb y proteins.I n :F.A . Loewusan dV.C .Rune - asubstitutio n synthesis as indicatedb yth e ckles (Ed.): Recen tAdvance s inPhyto - results concerning theeffec t of colddurin g chemistry 11.Plenu mPublish .Corp. , supply of3 H-PR0 (Table 1).Althoug h N.Y..p .79-115 . R.discolo r isa plan t sensitive tochillin g Mascarenhas,J.P. , 1966.Polle ntub egrowt h stress,th eresult spresente d here couldb e and ribonucleic acid synthesisb yvegeta ­ related tothos eo fKacperska-Palacz , tivean d generativenudle io fTradescan - Jasinska,Sobczy k andWcislinsk a (1977)wh o tia.Amer .J . Bot.5 3 :p .563-569 . noted,i nth ecold-toleran twinte r rape,a n Rochat,E .an dTherrien ,H.P. , 1976.Etude s increaseo ffre ePR Oresultin g fromth epro ­ desacide s aminése nrelatio nave c laré ­ teolysis induced bycol d and,mor eprecise ­ sistancea ufroi d chez lesblé s d'hiver ly,t othos eo fRocha t andTherrie n (1976); Kharkow etKent .Natur ,can .Helv .88 4: according tothes e twoauthors ,ther ewoul d p.138-163 . bea lysi so fth ewal lHYP-glycoprotein s Souvré,A . andAlbertini ,L. , 1978.Etud e withth ereleas eo fPR O asa resul t ofdehy - autoradiographique del'actio n dufroi d droxylationo fwal lHYP . sur lessynthèse snucléaire s duRN Ae tde s protéines dans lesmicrosporocyte s etl e tapis chez leRhoe odiscolo rHance .Soc . References Bot.Fr. ,Actualité sBot . 125 :p .33-37 . Underdown,B.J . andGoodfriend ,L. ,1969 . Albertini,L. , 1970.Le sacide snucléique s Isolationan d characterizationo f anal ­ etle sprotéine s aucour sd e lamicrospo - lergenfro m shortragwee d pollen.Bioche ­ rogénèse chezl eRhoe o discolorHance . mistry 8 :p .980-989 . Etudeautoradiographiqu e etcytophotométri - ZhangHong-qi ,Croes ,A.F .an dLinskens ,H.F. , que.Thès eDoct . d'Etat Sei.Nat. ,Toulouse . 1982.Protei n synthesis ingerminatin g Albertini,L ., . 1975.Etud eautoradiographi ­ 3 polleno fPetuni a :rol eo fproline . qued e l'incorporation dutryptophan e H Planta 154 :p .199-203 . dans lesmicrosporocyte s etdan s letapi s duRhoe odiscolo rHance .Caryologi a2 8 (4): p .445-458 . Albertini,L . and Souvré,A. , 1983.Th e effect ofcol d (4°C)o n3 H-leucine,3 H- arginine and3 H-tryptophaneincorporatio n inmicrosporocyte s and tapetum ofRhoe o discolorHance .A n autoradiographic study. In :Fertilizatio nan dembryogenesi si n ovulated plants,VEDA ,Bratislava , Czechoslovakia,p .349-385 . Dashek,W.V . andErickson ,S. , 1981.Isola ­ tion,Assay ,Biosynthesis ,Metabolism , Uptakean d Translocation,an dFunctio no f Proline inPlan t cellsan d tissues.Bot . Rev.4 7 (3): p .349-385 . Chrispeels,M.J. , 1970.Biosynthesi s ofcel l Wallprotei n :sequentia lhydroxylatio no f

31 BIOCHEMISTRY OFPOLLE N PIGMENTATION

R. Wiermann

Botanisches Institut, Münster, Federal Republic of Germany

Summary Experiment3 Pollen pigmentation is hi ghly variablean d The locationo fenzyme so f phenylpropanoid is generally duet oth eac e umulationo fcaro - metabolism wasstudie d using antibodiesa - tenoids and/or flavonoidss ucha schalcones , gainst chalcone synthase(CH S and phenyla- anthocyaninsan ddivers ef l avonolglycosides. lanineammonialyas e (PAL)i n immunohistoche- The anther tapetum playsa crucial rolei n mica l assays. the accumulation ofthes es ubstances. This After incubation ofcros ss ectionso ftu - was demonstrated byenzymo l ogical andimmuno - lip anthers with antibodiesd irected against histochemical studies. PALan dCH Sth emos t intense fluorescence Keywords: pollen pigmentati on, phenylpropa- was seen inth etapetu m cells (see Kehre!& noids, enzyme localization, anther tapetum. Wiermann,i n press). Introduction Table 1.Distributio n ofPA Lactivit yi n Pollen pigmentation,whic h ishighl y vari­ different anther tissues (1:whol e anther, able,i sgenerall y duet oth eaccumulatio no f 2: anther sections without pollen,3 :res t carotenoids and/or flavonoids sucha schal ­ ofth eanthe r after squeezing theloculu s cones,anthocyanin s anddivers e f1avonolgly­ material,5 :pollen ,6 :tapetu m cells. cosides. (Stanley& Linskens , 1974). As parto fa stud yo fth ebiochemistr yo f pollen pigmentation,th erol eo fth eanthe r PAL activity (ukat/ka protein) tapetum inth eaccumulatio n processo fpheny l- 1. 2. 3. 4. 5~ 6. propanoidswa sinvestigated . Ifth eaccumula ­ tiono fthes e substances isregulate d byth e• 33,6 34,7 31,1 9,7 - 755,5 tapetum cells, enzymes involved inthei r bio­ synthesis should belocate d inth etapetum .

Results anddiscussio n From these experiments oneca nconclude , thatth eenzym eo fphenylpropanoi d metabo­ lismar epredominatel y localized inth eta ­ Experiment1 petum cells.Th etapetu m thus playsa cru ­ cial rolei nthi s metabolism inth eIcculu s The contents ofanther s were,squeeze d out, . ofth eanthe ran ds oi nth epigmentatio no f ina buffer/sucros e mediuman dwer e seperated. pollen. by filtration into pollenan dtapetu m frac­ tions fordeterminatio n ofenzym e activities involved inphenylpropanoi d biosynthesis. The highest specific enzyme activities- References withth eexceptio n ofchalcone-flavanon e iso- merase- wer e obtained withth etapetu m frac­ Herdt, E.,R .SUtfel d &R .Wiermann , 1978. tion.Th epolle n fraction exhibited onlylo w The occurrenceo fenzyme s involvedi n activities (Herdte tal. , 1978). phenylpropanoid metabolism inth etapetu m fractiono fanthers . Cytobiologie17 : 433-441. Experiment2 Kehre!,B .& R .Wiermann , 1984.Immu ­ In ordert ostud yth edistributio n ofrele ­ nochemical localization ofphenylalanin e vant enzymes within theanthers , specifically ammonia-lyase (PAL)an dchalcon e synthase inth etapetu m cells,th etapetu m tissuewa s (CHS)i nanthers . Planta,i npress . isolated byth eus eo fhydrolyti c enzymesan d Rittscher,M .& R .Wiermann , 1983. was seperately analysed forcompariso n with Occurrenceo fphenylalanin e ammonia-lyase pollenan dth eothe r anther tissue. (PAL)i nisolate d tapetum cellso fTu lip a anthers. Protoplasma 118: 219-224. The results demonstrate thata hig h specific PAL activity islocate d inth etapetu m tissue. Stanley,R .G .& H .F ,Linskens , 1974. The pollenan dothe r anther tissues contain Pollen. Springer Verlag; Berlin,Heidelberg , only marginal activity (Table 1;Rittsche r& New York. Wiermann, 1983).

32 DEGRADATION OF TAPETAL CELL NUCLEI DURING THE FORMATION OF MICROSPORES R. Herich and A. Lux Department of Plant Physiology, Comenius University, Bratislava, Czechoslovakia

Degradation of tapetal cell nuclei or avacuole . Dilatation of inner and protoplast was followed in.diffe ­ membranes and vacuolar degradation rent species of family Liliaceae, of plastids occur. taking place during the last stages ofmicrospore s formation. It was The last stage of tapetal cell found, that the process is realized degradation in Lilium martagon L. indiffern t species by differing occurs without the presence ofva ­ ways . cuoles. Cytoplasm is filled with dark patches of reserve material. Nuclei are compact, globular, they In Lilium henryi Bak. a gradual does not form lobes. Chromatin is désintégration of nuclei occurs, condensed./Fig.3/. nuclei become lobed, karyotheca désintégrâtes, light vacuole-llke bodies appear in karyoplasm, passing to the cytoplasm /Fig.1/. A great amount of reserve material /lipids/ is formed in plastids. Plastids are filled with numerous plastoglobuli in later stages of ontogenesis, their envelope désintégrâtes. In Lilium davidii Duch. the nu­ cleus changing in shape becomes deeply lobed. It is of interest, that the process does not take place in all nuclei simultaneously, compact globular nuclei are present together with intensively lobed nuclei /Fig. 2/. In that time the number of plas­ tids is increasing, they are fre­ quently constricted in shape. Plas­ tids contain numerous small plasto­ globuli. A great number of parallel membranes of ER is present in the cytoplasm. Lobed nuclei are present in tape­ tal ceils of Tulipa kaufmanniana Regel var.Shakespeare. Plastids are cup-shaped in those cells in later stages cf ontogenesis. Their cava- tions can be filled with cytoplasm

33 THEEFFEC TO FA CHEMICA L HYBRIDIYING AGENTO NTH EDEVELOPMEN TO FWHEA TPOLLE N

WilliamA .Jense n

Department ofBotany ,Ohi o StateUniversity ,Columbus ,Ohio ,4321 0U.S.A .

Cultivated variation of wheat were treated with a chemical hybridiying agent develop­ ed by Rohm and Haas Chemical Corporation. This compound caused the pollen to abort and this study was undertaken to determine the mechanism of action of the chemical. Plants were treated with the compound early in flower development. Such treated plants had normal development except that the pollen wall formed was much thinner than in the control. When the stage of pollen development is reached where the cell goes from a highly vacuol­ ate stage to one where the cell is filled with cytoplasmic organelles, the cell collapse. Preliminary chemical studies indicate that carotene content of the anthers was normal or higher than in the control. Also, preliminary data in­ dicate that a Peroxydase with ap H optimum , of 7.2 is strongly inhibited in the treat- ( ed anthers. Thus, it appear that the mode of action of the compound is to prevent formation of the pollen wall by the inhibition of the deposition of the sporopollenlin.

34 GENETICAL APPROACH OF AUTOSTERILITY IN CAMELIA SINENSIS L.

E. Habintore, J.P. Tilquin & A. Serrhini

Faculty of Agricultural Sciences, Department of Crop improvement, Burundi University, BP 2940, Bujumbura, Burundi

Clone Seed Summary Unfertil. Aborted U L U L U L Thete aplan t hasa trilocula r ovarywit h twoovule spe r loculus.I na polyclona l seed Tri31/ 8 37 12 162 174 40 55 garden,th emajorit y of contentson e Tri31/1 1 44 19 145 173 51 66 seed,exceptionally ,five .A statistical BB3 5 44 17 157 211 32 38 studyo f thenumbe r of seed inuppe ran d IB17 4 66 9 86 113 80 73 lowerpositio n perclon e isgiven .Dialle l IB24 1 12 3 60 70 2 1 crossesafte rhan d emasculation andpollina ­ IB40 1 62 34 110 139 76 75 tionwer erealized .Th eresult s demonstrate IB10 8 73 20 97 135 67 84 amarke d difference between thereciproca l IB9 2 72 61 245 260 91 76 crosses,a tota lautosterilit y and amulti - SHAN 116 62 84 107 98 132 allelism. IB6 2 83 10 182 121 34 28

Introduction Experiment 2 :som eresult so fdialle l cros­ Clonal selectionwa s themor e important ses,% o f fruiting after6 strategy inte a improvement giving aunifor m months plantation and astabilit y of organoleptic characteristics.Bu t the seedage ofsynthe ­ Male Female % ticpopulation s isno twithou t interest. Tri31/ 8 Tri31/1 1 75 Synthetics are easily produced inbiclona l 16 seedgarden .Th evalu e of the syntheticca n Tri31/1 1 Tri31/ 8 Tri31/ 8 beestimate d by aprogeny-test ;al lprogeny - IB 174 65 testingprocedure s depend upon theestima ­ IB17 4 Tri31/ 8 20 tiono fcombinin g abilities (agenera lan d Tri31/ 8 IB10 8 20 aspecific) .Dialle l isa matin g patternfo r IB10 8 Tri31/ 8 0 estimationo f combining abilities and the relative importance of the twoan d so,autho ­ These results showa marke d differencebet ­ rizesa choic ebetwee n synthetic orclona l ween thereciproca l crosses,a grea t impor­ selection.Often ,th e twooccu r togethera s tanceo f thefemal e cytoplasm. inCacao ;man y clones areals oself-incompa ­ tiblean d synthetics showa "dramatic "inter - Reference populationheterosi s (Simmonds, 1979).A diallel isa nexcellen t toolt ounderstan d Simmonds,N.W. , 1979.Principle s ofcro p theincompatibilit y system. improvement.Longman ,Londo n &Ne wYork .

Resultsan d discussion

Experiment1

Inorde r tounderstan d thenatur e ofth e encountered sterility, swer ecollecte d atrando m ina nopenpollinate d seed garden (polyclonal).Fruit swer e carefullydissec ­ ted.Thre ecategorie s are found :seed ,un ­ fertilized and aborted ovules (table 1).A distinction ismad e between theuppe r (style) and lower ()positio n ofovules . A.2analysi s showa significantl y difference between thenumbe r of seed in loweran duppe r Positionwit h all theclone s andn odiffer ­ encewit h theaborte d ovules.A compariso n °fth emean s of seed inuppe r and lower posi­ tionsho wa significantl ydifference .

35 HORMONAL AND TEMPERATURE CONTROL OF MALE-STERILITY IN A TOMATO MUTANT

V.K . S awhney

Department ofBiology ,Universit y ofSaskatchewan ,Saskatoon ,Saskatchewan ,Canada ,S7 N0W 0

Summary conditions and also provide information on the protein patterns associated with male- The male-sterile, single , stamenless- s terility / fertility. 2 (SI2/SI2) mutant of tomato can be induced to form normal and viable pollen either by the application of gibberellic acid (GA3) or by growing plants under low A line C line temperatures. IAA treatment or high temp­ Male-sterile Fertile eratures resulted in the production of ms ms Ms Ms 1 carpel-like stamens. The mutant stamens (9 paren t (d parent ) contained lower levels of proteins and different banding pattern in SDS Polyacryla­ mide gels than the normal (+/+) stamens. The reversion of mutant stamens to normal Hormonal by GA3 or low temperatures partly restored or the differences in the protein content and Environmental- the banding pattern. induced revertant Fertile Keywords: male-sterility, tomato mutant, ms ms GA3, IAA, temperature, proteins. / (maintainer) Introduction

Male-sterility in plants is extremely valuable for the production of hybrid seeds and for increasing the yield of crops. ms ms Generally, cytoplasmic male-sterility (CMS) B line is used in producing hybrids, since CMS can be readily maintained in the F^ generation. Alternatively, nuclear or genie male-sterile lines can also be useful for hybrid produc­ tion, but the maintenance of such lines is Fig. 1. A proposal for the maintenance of tedious and involves the process of roguing genie male-sterile line by the experimental (Frankel and Galun, 1977). -" ." induction of revertants and the production k of mutant and hybrid lines (Adapted from We propose that if male-sterile mutant Frankel &Galun , 1977 ) plants can be reverted by chemical or environmental treatments to produce normal stamens, such pollen can be used to polli­ nate mutant and the seeds thus pro­ Materials and Methods duced will generate all mutant plants. This approach can therefore be useful in main­ Seeds of the stamenless-2 mutant were taining the pure male-sterile lines (Fig. 1) . originally obtained from Dr. C.D. Clayberg of the Connecticut Agricultural Station, New In tomato (Lycopersicon esculentum Mill.) Haven, CT, U.S.A. Initially, mutant plants CMS is not commonly known, but several were maintained through cuttings; later the single gene male-sterile mutants exist which gibberellic acid-reverted flowers were used exhibit a varying degree of male-sterility for selfing and pure homozygous seeds were (Rick, 1953, 1960, 1962; Hafen and Stevenson, obtained. The normal line used was 1955) . These mutants have been shown to homozygous dominant (+/+). serve as female parents in hybrid seed production (Hafen and Stevenson, 1958; Hormonal and temperature experiments were Lapushner and Frankel, 1967). As proposed performed on clones of plants obtained through cuttings. Gibberellic acid (GA3, in Fig. 1, if the fertility of these mutants 3 - can be restored by hormonal or environmental 10~ M) and indole-ace tic-acid (IAA, 10 %) means, such mutants can be very useful in was applied with a micropipette (10 or 25 ul) hybrid seed production. to the axil of the youngest immediately before floral initiation. For temperature In this article we report on the regula­ experiments, plants were grown in growth tion of male-sterility in a male-sterile, chambers maintained in one of the following single gene, stamenless-2 (SI2/SI2) mutant of three regimes: Low - 18°C day/15°C night, tomato by plant hormones and temperature

36 intermediate - 23°C/18°C night, and high - 28°C day/23°C night. Table 1. Effects of GA3 (2 X 10 pi of 10"%) and IAA (2 x 25 yl of 10 M) on Che development of stamens of the Proteins were extracted by homogenizing stamenless-2 (sl2/sl2) mutant of tomato. Y.P. = yellow Che tissue in 10 mMTris-HC l buffer (pH 7.5) and pubescent (normal), c.s. = carpel-like stamens. with mercaptoethanol. The homogenate was Values presented are the mean, n = 15. centrifuged at 10,000 x g for 10 min. and the supernatant was used for protein determina­ tions and electrophoresis. Protein determi­ Treatment No. of Y.P. No. of c.s. Starne n Staminal stamens/ stamens/ length cone (% nations were performed according to the flower flower (mm) flowers) method of Bradford (1976) . The reaction

mixture was read at 595 nm in Beekman DU-7 a a a spectr cphotometer. GA3 6.60 0.0 9.30 100.0 b b b For electrophoresis, the supernatant of IAA 0.25 • 4.25 6.50 0.0

plant extract was mixed with equal volume of sl /sl e a C 2 2 2.25 0.5 7.80 a buffer containing 10% glycerol, 5% g- (control) 0.0

mercaptoethanol, 2.3% SDS (sodium dodecyl- a a a +/+ 6.36 o.o 9.80 100.0 sulfate) and 0.0625 M tris-HCl (pH 6.8). The (control) mixture was dipped in boiling water for 3 minutes and the cooled sample was loaded on a Note: Means followed by the same letter in a vertical 12% Polyacrylamide slab gel in the Bio-Rad not different at 5% level. Protean Cell. The gels were run at a constant electric current of 20 mA for each (Data from Sawhney and Greyson, 1973b). gel and were stained with coomassie blue (R-250). pollen and viable seeds were produced Results and Discussion following GA3 treatment, it is clear that this treatment can be used to maintain the The stamenless-2 mutant produced abnormal male-sterile line, i.e.^he B-line (Fig. 1) . stamens which were pale yellow in color, bore Mutant plants grown under different naked external ovules (E.0.) on the adaxial temperature regimes also showed somewhat surface, were laterally free, produced non­ similar results. Plants grown under low viable pollen and were shorter in length than temperatures produced normal stamens the normal stamens (Sawhney and Greyson, which were yellow and pubescent, were as 1973a) . The normal stamens were - long as +/+ stamens and were fused to form yellow in colour and pubescent (Y.P.), and a staminal cone (Table 2, see also Sawhney, were laterally fused to form a staminal cone. 1983a ) . The pollen produced in these Gibberellic acid at the concentration of stamens was normal and viable and if used 10~JM induced the formation of normal stamens to pollinate the mutant flowers, resulted in in flowers which were initiated after the fruits with seeds which were all SI2/SI2. treatment. The stamens were yellow and The high temperature conditions on the pubescent, produced normal pollen, formed a other hand caused the production of carpel- stamina l cone, and were as long as the normal stamens (Table 1; see also Sawhney and Greyson, 1973b). The pollen produced were viable and when used to pollinate the sl2/slg Table 2. Temperature effects on the development of flowers formed fruits with normal seeds, the stamenless-2 (SI2/SI2) mutant of tomato. Temperature which upon germination produced all mutant regimes: LTR = low, ITR - intermediate, HTR - high. Y.P. - plants . yellow and pubescent (normal), c.s. = carpel-like stamens. Values presented are the mean, n = 30. Indole-acetic-acid induced the production of carpel-like stamens (C.S.). The C.S. showed no signs of microsporogenesis and Genotype/ No. of Y.P. No. of c.s. Stamen Staminal possessed a basal ovary-like region which Temperature stamens/ stamens/ length cone (% contained ovules inside (see Sawhney and regime^ flower flower (mm) flowers) Greyson, 1973b). The stamen length of such 3 a a stamens was smaller than that of the GA3- sl2/sl2 (LTR) 6.16 0.13 9.92 66.6 treated sl2/sl2-.and +/+ flowers, and the stamens did not fuse to form a staminal cone " (ITR.)- 0.50b 1.30b 7.61b 0.0 b (Table 1). More importantly, such stamens " (HTR) o.oo 5.46C 5.72e 0.0 were functionally male-sterile. a a a These observations showed that the male- +/+ (ITR) 6.70 o.oo 9.42 100.0 fertility of the sl2/sl2 mutant can be either restored by GA3 treatment or the stamens can Note: Means followed by the same letter in a column not e induced to form a completely sterile different at 5% level. structure by IAA. The timing of hormonal For details on different temperature regimes see the text. treatment was, however, a critical factor (Sawhney and Greyson, 1979). Since normal (Data from Sawhney, 1983a).

37 like stamens (Table 2) in which mlcro- breeding. Springer-Verlag, New York. sporogenesis did not take place and Hafen, L. & E.C. Stevenson, 1955. New male- instead ovules were produced at the basal sterile and stamenless mutants. Tomato end (See Sawhney, 1983a). Genet. Coop. Rep. 5: 17. Thus the male-sterility of SI2/SI2 mutant Hafen, L. &E.C . Stevenson, 1958. Stamen- can also be controlled by growing mutant less mutants as female parents in the plants under different temperature regimes. production of F^ hybrid seed. Tomato This can therefore be an alternative Genet. Coop. Rev. 8: 18. approach for the maintenance of pure sterile Lapushner, D. &R . Frankel, 1967. Practical lines (Fig. 1). That temperature effects aspects and the use of male sterility in may be mediated through changes in the production of hybrid tomato seed. endogenous hormones was discussed elsewhere Euphytica 16: 300-310. (Sawhney, 1983b) . Rick, CM., 1953. New male-sterile mutants. A protein analysis showed that the normal Tomato Genet. Coop. Rep. 3: 19-21. stamens contained nearly twice as much Rick, CM., 1960. New male-sterile mutants. protein as the mutant stamens under the Tomato Genet. Coop. Rep. 10: 35-37. intermediate temperatures (12.26 for SI2/SI2 Rick, CM., 1962. New male-sterile mutants. and 24.34 for +/+, yg/mg fresh wt. of Tomato Genet. Coop. Rep. 12: 39-41. protein). The SI2/SI2 stamens reverted to Sawhney, V.K. , 1983a. Temperature control normal by low temperatures (15.86 yg/mg) or of male sterility in a tomato mutant. J. GA3 (18.44 yg/mg) contained greater protein Hered. 74: 51-54. than the untreated SI2/SI2 stamens, but Sawhney, V.K. , 1983b. The role of temp­ less than the +/+ stamens. The carpel-like erature and its relationship with stamens produced under high temperatures gibberellic acid in the development of contained lowest levels of protein (10.1 yg/ floral organs of tomato (Lycopersicon mg) than all the other conditions. esculentum) . Can. J. Bot. 61: 1258-1265. The lower level of protein in the mutant Sawhney, V.K. &R.I . Greyson, 1973a. vs normal stamens, may be primarily related Morphogenesis of the stamenless-2 mutant to the proteins associated with pollen £n tomato. I. Comparative description of development; the reversion to normal stamens the flowers and ontogeny of stamens in the and pollen by GA3 and low temperatures normal and mutant plants. Am. J. Bot. 60: restored some of these proteins. 514-523. An SDS-polyacryalamide gel analysis showed Sawhney, V.K. &R.I . Greyson, 1973b. that mutant stamens contained 3 bands of Morphogenesis of the stamenless-2 mutant approx. 33, 28 and 23 k daltons which were in tomato. II. Modifications of either faint or absent in the +/+ stamens. organs in the mutant and normal flowers . The low temperature reverted mutant stamens Can. J. Bot. 51: 2473-2479. showed the absence of these bands, but Sawhney, V.K. &R.I . Greyson, 1979 . they were present in the high temperature Interpretations of determination and stamens. The +/+ stamens had a zone of canalisation of stamen development in a protein bands ca. 35-45 k daltons which tomato mutant. Can. J. Bot. 57: 2471-2477. were also more distinct in the low tempera­ ture reverted mutant stamens, than in the intermediate or high temperature mutant stamens. In the GA3- treated stamens also protein bands found in the mutant stamens were much fainter and the zone of protein bands found in +/+ stamens was much more dis tine t. It appears therefore that the differences in the protein levels or protein bands in the mutant and normal stamens were partly recovered by the restoration of normality in the mutant stamens either by low temperature or by GA3. The nature of such proteins, however, remains to be resolved.

References Bradford, M-.M. , 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. Frankel, R., E. Galun, 1977. Pollination mechanisms, reproduction and plant

38 CHARACTERIZATION OFCYTOPLASMI CMAL ESTERILIT Y INPETUNI AX HYBRID A (HOOK)VILM .

G.A.M.va nMarrewij k& L.C.J.M . Suurs

Dept.o fPlan tBreeding ,AU ,Wageningen ,Netherland s

Summary consisting ofonl y 50%mal esteril eplants . Soth ebreede r iscompelle d torogu ehal fo f Cytoplasmicmal e sterility (CMS)i nman y thesee dlin eplant st opreven tundesire d speciesi saccompanie d bya numbe r ofcyto - sibbing.Usuall y thisi stechnicall yan d logicalan dbiochemica l deviations inth e economicallyunfeasible . malese xorgan .A ninventor y ofal labnorma ­ CMS, onth eothe rhand ,lend s itselfver y litiesi nCM SPetuni ax hybrid ai sunderway . well tohybri d breeding.Crossin gbetwee na Callaseactivit y appearst ob estrongl y CMS-plantan da mal e fertilemaintaine rlin e reduced inCM Stype scompare d tonorma lmal e results ina completel y malesteril eprogeny , fertileplants .Als odeviation shav ebee n sinceth eplasmagene sresponsibl e forsteri ­ observed inth eesteras ean d acidphosphatas e lityar etransferre d fromth emothe rt oal l iso-enzymepatterns ,wherea speroxidas ecom ­ theoffsprin gvi ath eeg gcells . positiondi dno tsho wconspicuou sdifferences . CMSca nb erestore d by the introductiono f Keywords:cytoplasmi cmal e sterility (CMS), fertility restoringgene s (Rf-).Th ewa y Petuniax hybrida ,callase ,isoelectri c inwhic h thegen eproduct so fth esterilizin g focussing,iso-enzym epattern . cytoplasm andnuclea rRf-gene sinterac tha s notye tbee nunravelled . Malesterilit y Thecausa lagen to fCM S/ Malesterilit y (MS)ca nb edefine d asth e incapacity ofplant st oproduc e ort oreleas e Several investigatorshav epreoccupie d them­ functionalpolle n (VanMarrewijk , 1979).Mal e selveswit hth equestio nabou tth ecausativ e sterility occurs inman y forms:absenc eo f factoro fCMS .I nth enineteen-sixtie s itwa s themal ese xorgan ,atroph y ofanthers ,reduc ­ predominantly thoughttha tthi sagen to fCM S tiono fth estamen s intostaminodi ao rconver ­ had aviroi d orepisomi cnatur e (Atanasoff, siono fth emal ese xorga n intocarpel so r 1964). Thishypothesi swa scorroborate db y .Th emos tcommo n type,however ,i s theobservatio n thatCM Scoul db etransferre d pollensterility . Inthi scas eo fM Sth ean ­ toa norma l fertileplan tb ymean so fgraf ­ thersdevelo pnormall y butirregularitie si n ting,suc ha swit hpetuni a (Frankel,1956) , thedevelopmen to fth emicrospore scaus e sugarbeet (Leonova,1974 )an dsunflowe r abortiono fth epolle ngrains .Polle nsteri ­ (Pimakhin& Agashev , 1978).O nth estrengt h lity iswidel y spread throughout thePlan t ofman ynegativ eresult swit hth esam ecrop s Kingdom. Itsometime soccur s spontaneously aswel la swit hothers ,th eaccurac y ofthes e underextrem e environmental conditions,bu t observations,however ,ha sbee nquestione d isusuall y controlled byhereditar yfactors . (seei.a .Va nMarrewijk , 1970).Recen tstudie s Inmos tcase spolle nsterilit y isgoverne db y havereveale d aninvolvemen to fth emitochon ­ onenuclea rgene :geni emal e sterility,GMS . drialgenom e inth emechanis m ofCMS ,espe ­ Somespecie s (tomato,barley )involv elarg e cially inmaiz e (Levings& Pring ,1979) , numberso fGM Sloci . sorghum (Levings,1983 )an dmos tprobabl y Ina limite dnumbe ro fcase spolle nsteri ­ petunia (Koole tal ,1982) . lity isbrough tabou tb y extranuclearhere ­ ditaryfactor so rb yth ejoin tactio no fbot h Expressiono fCM S nucleargene san d extrachromosomalfactors : cytoplasmic malesterility :CMS . Cytoplasmic malesterilit y expressesitsel f primarily by deterioration ofth esporogenou s Significance ofmal e sterility tissue.Thetim eo fcollaps erange sfro mpre - meiosisu pt oth ematurin gpolle nstage .On e Malesterilit y hasbee n extensively adopted particularCM Styp eca ncaus edegradatio na t inplan tbreeding . Itsmai nus e isfoun di n differentstage s indifferen tgeneti cback ­ theproductio n ofhybri d varieties.Introduc ­ groundso reve n inth esam eplan t (Chauhan& tiono fM S inon eo fth eparenta l linesena ­ Singh,1966) . Generally CMS isaccompanie db y blesth ebreede r toproduc epur ehybri dseed . otherhistologica l andbiochemica labnormali ­ Acomplicatin g factor isth e inheritanceo f tiesi nth ese xorgan . malesterility .Nearl y allcase so fGM Sinhe ­ Schreur (1978)inventorizin g the literature ritmonofactoriall yrecessive .Suc himplie s onCM Sexpressio n distinguishessi xmai n thatth emal esteril eplant shav et ob emain ­ categorieso fdeviation s from thenormal e tainedb ycrossin gwit ha heterozygou s fertile fertilesituation : isogenic counterpart,resultin g ina progen y

39 1.Deviation si nth edevelopmen to fth etape - Allclone sar egrow n inclimati cchamber s tallayer . understandar d conditionso filluminatio n 2.Abnormalitie s inth evascula r systemo f anddaylengt h ata temperatur erang eo f13° - thefilamen tand/o rconnectiv etissue . 170-21°-25°C.No tal lcondition sno ral l 3.Substantiall y modified freeamin oaci d planttype sar euse d ineac hexperiment . composition. Sinceth ecompositio nan dactivit y ofenzyme s 4.Decrease d contento fnuclei c acidsan d andothe rcomponent s isdependen to fth e theirderivatives . stageo fdevelopmen t ofth eanther ,al lde ­ 5.Change d activities ofenzymes ,especiall y terminations areexecute d in8 developmenta l ofredo xsystems . stages.Th eprope r stagei sestablishe d 6.Deregulate d callaseactivity . cytologically aftersquashin gon eo fth ean ­ Alsoagriculturall y important characteristics therso rwit hth eai do fa nempiricall y mayb eaffected ,bu ti ti softe ndifficul tt o determined relationbetwee n flowerbu dsiz e saywhethe rthi sresult sfro mpleiotropi cgen e and sporogenic stages.Th elas tmetho di s actiono rfro m closely linkedgenes . especially used forCM Stype s inwhic hth e Thedat ai nth eliterature ,however ,caus e sporogenoustissu edeteriorate salread y confusion,becaus eth edifferen t aspectshav e duringmeiosis . beeninvestigate d separately indifferen t Insevera lenzym eactivit y assessmentsus e 'stageso fdevelopmen t fordivers e cropsunde r ismad eo fultrathin-laye risoelectri cfocus ­ varyingcondition san db ymean so fnon-stan ­ sing (IEF)i nacrylamid egels .Th efocussin g dardizedmethods . isdon ea ta temperatur e of4° C ina nLK B Ultrophor flat-bed container connected toa Motivationo fth eresarc h subject powersuppl y (PharmaciaECP S3000/15 0wit h Volthour integratorVH-1 )a ta constan t Inth eprecedin gpar tth eimportanc eo fCM S inputo f3 Watt .Sample sar eobtaine db y forhybri d seedproductio nha sbee nmentioned . extractiono f1 0m ggroun d anthertissu ei n Practical application,however ,i slimite d 20(i lHO , centrifugation (10.000g )fo r1 0 byth efac ttha tCM Sonl y occurs ina limite d minan ddialyzatio no fth esupernatan t numbero fspecies .M Sinductio nb ychemica l (2*5n D against2 1 HO. pollenkiller so rgametocide sha soffere ds o Thecompositio n ofth e10 0ur nge li saccor ­ farn oreliabl e alternative (VanMarrewljk , dingt oRadol a (1980)addin g 10%glycero lt o 1979). Thepresen tresearc h isaime da t preventdesiccation . obtaining insight inth emechanis m ofCM S andt odiscove rwher e theproces so fth e Callase activity deregulation inth esporogenou s development takesa start . Callaseactivit y playsa nimportan tpar ti n Thewor k isdon ewit hwell-describe dmaterial , thedevelopmen t ofth emicrospore .Prope r grownunde rstandar d conditions.Thi smate ­ timingo fth ebuild-u p anddegradatio no f rial willb eexamine d foral lpossibl edif ­ callaseappear sessentia l forth eproductio n ferencesbetwee nmal efertil ean dcytoplas ­ ofvita lpollen .Franke l eta l (1969)stu ­ micmal e sterileplants .I ti shope d thatth e dying isogenicmal e fertile andcm sline so f dataobtaine d inthi swa y canb ejoined-a s* ' Petuniahybrid acv .Ros yMor nobserve d are ­ pieceso fa jigsa wpuzzl e andma y lead toa markable difference incallas e activity.I n fathomingo fth eCMS-system .B y imitating fertileplant scallas eactivit y wasni lbe ­ thismechanism , itwil l thenb e triedt o tweenprophas e and tetradformation andin ­ inducemal esterilit y infertil eplants . creased sharply during thereleas eo fth e Someresult sobtaine d sofa rwil lb edis ­ microspores.Steril e antherso nth econtrar y cussedbriefl ybelow .I ti sintende dt o displayed astron g activity ofcallas ebe ­ publishth eresult swit h theseparat esub ­ tweenprophas e and thesecon dmeioti cdivi ­ jects elsewhere inmor edetail . sion.Afte rtha tth eactivit y decreased. Theauthor sconclude d 'thatth eprecociou s Materialsan dmethod s riseo fcallas e activity inCM Santher s isa causerathe rtha na neffec to fth ebreakdow n Theplan tmateria l consistso fa numbe ro f ofmicrosporocytes' .I na late rarticl e cloneso fPetuni ahybrida ,namely : Izhar& Franke l (1971)demonstrat e therela ­ 1.Tw oclone so fcytoplasmi c malesteril e tionshipbetwee n callaseactivit y andp H idiotypeso fcvs .Snowbal l (SBS),Blu e level. Bedder (BBS)an dRos yMor n (RMS)an dth e Wehav e investigated whetherw ecoul dveri ­ matchingmal efertil emaintainer s(SBF , fyth eresult so fFranke lc.s .throug hou r BBF, RMF). material.Thi sha sbee ndon e (i)b ymean so f 2.Tw oclone so frestore d fertile idiotypes thesemiquantitativ e bioassay usedb yFranke l (R1.-2,R2-2) . c.s.an d (ii)wit ha quantitativ ebiochemica l Thismateria lwa sdescribe d earlierb y approach,th elaminari ndiges ttest . VanMarrewij k (1969). 3.F -progenie s from crossesbetwee nCMS - andR-idiotypes .

40 Thesemi-quantitativ e lacmoidtes t anthertissue .Th elaminari n isconverte db y thecallas e inth eextrac t intoD-glucose , Thelacmoi d testi sa semi-quantitativ e which init stur n isoxidize d togluconi c bioassay.Th eessentia lpar to fthi stes ti s acidb yglucos eoxidase .Th erelease dhydro ­ thatextract sar emad eo fanther sa tdifferen t genperoxid ereact swit h indicatorreagent s developmental stages.Thes e extractsar e (4-aminophenazon ,sodiu mhydroxybenzoate , subsequently added toa standar d substrate andperoxidase )t obecom ea stabl ered - whichconsist so fyoun g tetradso ffertil e stained quinoncomplex.Th e intensity ofth e anthers.Th elatte r containsa grea tamoun t stainingo fthi scomple x isassesse dspecto - ofcallose ,whic h canb edigeste d byth e photometrically at51 0n m and isdirectl y callaseo fth eextract .Afte r incubationa t proportionate toth eD-glucos e concentration 38°Clacmoi d (resorcinblu e inabsolut e inth esample .Suc hassessmen trequire slarg e ethanol)i sadde d tosto pth ereactio nan d samples (300m gpe rassessment )an d isthere ­ tostai nth e indigestedcallose .Th ecallas e foreperforme dwit honl yon enorma lfertil e activity inth eextrac t isinversel ypropor ­ (BBF),on emal e sterile (BBS)an don eresto ­ tionatet oth estainin g intensity ofth e red fertile (R2-2)clone . substrate.Th emetho d isdescribe d inFranke l Theresult so fth e laminarin testar e eta l (1969)an dha sbee nadopte db yu safte r given inFig .2 . afe wmodifications . Fig.2 .Callas eactivit y inrelatio nt o Allfertil e idiotypes showed thesam etren d flowerbu d sizei nmal e fertile (BBF1)an d inactivity :lo wcallas eactivit y frompre - cytoplasmicmal e sterile (BBS1)Petuni a meiosisu pt oth eyoun g tetrad stagean da hybridacv .Blu eBedde ran d ina restore r suddenoutburs to factivit y inth elat ete ­ line (R2-2)determine d withth elaminarin - tradstag ejus tbefor eth ereleas eo fth e digesttest . microspores (Fig.1) . Fig.1 .Callas eactivit y inrelatio nt o microsporogenesisi nmal e fertile (RMF1)an d cytoplasmicmal e sterile (RMS1)Petuni a 40 hybridacv .Ros yMorn ,determine d withth e • I' •i \ lacmoidstainin gtest . 35 1 >• '-BBF-1 /"' 1 » »-BBS-1 Callase 9 30 // • ..R 2-2 activity '/ / \ * 4 ; / ~--t] \ \ b25 ; . \ ""A "•c

Ji A < , A- — . . . ^ i.— " XQ-- n-— --a-- —J

PREMEI PRO DIV. DIV TETRAD RELEASEÏÜUN G OLD POLLEN OSIS PHASE I I STAGE MICROSR MICR. MICR.

] RMFa 04 08 12 16 20 24 28 32 36 4.0 4.4 4.8 5.2 5.6 6.0 64 68 size cm (Frankel et aI. ,1969) " "(Own experiments) . RMSi CMSidiotype swer echaracterize db ya nequa l orslightl yhighe rcallas eactivit y compared Thefigur eshow stha tth efertil eanther s toth efertil ecounterpart s inth eearl y havever y little callaseactivit y inth e developmental stagesan d ales scomplet ean d firstphase so fdevelopment .A tth eflower - inconsistent increaseo factivit y inlate r bud sizeo f2. 3 mm,whic h correspondendswit h stages.Ther e isn oclea rpoin to ftur nan d thesecon dmeioti c division,a rapi d increase theleve lo factivit y isconsiderabl y lower inactivit y occursreachin g itsmaximu ma t thani nth efertil eplants . flowerbu d sizesbetwee n3. 4 and3. 8m m Restored fertileplant sac ti ncomplet e (=microspore release).Steril eanther sappea r accordancewit hth epatter n followedb y tohav ea ver y lowcallas eactivit y atal l normalfertil eplants . stageso fdevelopment ,wherea srestore dfer ­ tileanther ssho walmos tth esam etren da s Thelaminarin-diges ttes t observed innorma l fertileones . Althoughth eresult so fbot htest sar e Thelaminarin-diges t testmake s itpossibl e certainly not identical,the yd odemonstrat e toquantitativel y assesscallas eactivity . aclea rsimilarity . Inan ycase,neithe ro f Laminarini sa commercia l preparationwhos e theapproache s lendssuppor tt oth ehypothe ­ primary structure completely correspondswit h siso fFranke l c.s.tha tth esporogenou s callose. cellsdegenerat ebecaus eo fa to oearl y cal­ Inthi stes tlaminari n isadde d asa sub ­ laseactivity . stratet ocarbohydrate-fre e extractso f

41 Iso-enzymepattern s thana qualitativ enature . Esterase Acid phosphatase Esterasesar easpecifi c enzymeso fcommo n Acidphosphatas e isdetecte d afterstainin g occurrencewhos e function iti st ohydroliz e thege l in a-naphtylaci dphosphat e andFas t esters.Esteras e iso-enzymepattern sar ei n GarmetGB C atp H4.8 . usea sa too l forth e identificationo f With thisenzym e thenumbe ro fiso-enzym e cultivarso fa crop . bandsan d theirdensit y increasebetwee nth e AfterIE Ftreatmen t esterasewa sstaine d premeioticstag ean dth etetra d stage,bu t withth eai do f a-naphtaleneacetat ean d toa considerabl y lesshig hdegre etha nwit h FastBlu eR Ra tp H7.2 . esterases.Thi sproces soccur s inth eferti ­ Inmal e fertileplant sa gradua l increase lea swel l asi nth eCM Stypes .A mos tcon ­ both innumbe ran dactivit y ofesteras e iso­ spicuous thinghoweve r istha tth ezymogram ­ enzymeswa sobserved ,maximu m intensity meso fBB F andBB Sdiffe rqualitatively .BB S beingreache d atth eearl y tetradstage . showsthre eclea rband stha tar eabsen ti n CMS idiotypes,i ncontrast ,showe d lowenzy ­ BBF,wherea sBB Fha sa fe w indistinctband s meactivit y and fewband s inal ldevelop ­ whichar eabsen ti nBBS . mentalstages . Incv .Ros yMorn ,though ,thes edifference s Nodifference sbetwee n cultivarso nbot h haveno tbee nfound . thefertil ean d theCM S levelwer ediscerned . Therestore r idiotypesproduce dzymogramme s whichwer enearl y identical tothos eo fth e References malefertil eplants .Bot h thenumbe ro f bandsan d thedensit y ofth eparticula r Atanasoff,D. , 1964.Viruse san d cytoplasmic bandswer emuc halik e those inth efertil e heredity. Z.Pflanzenz .51:197-214 . zymogrammes. Chauhan,S.V.S .& S.P . Singh,1966 .Polle n abortion inmal e sterilehexaploi d wheat Wema y concludetha tCM Saffect sth eiso ­ (/Norin1)havin gAegilop sovat aL .cyto ­ enzymepatter nbot h qualitatively andquan ­ plasm.Cro p Sei.6 :532-535 ~ titatively.Also ,th eeffect sar ei ntur n cancelledb yth eintroductio no fRf-genes . Frankel,R. , 1956.Graf t induced transmission toprogen y ofcytoplasmi cmal esterilit y Peroxidase inPetunia .Scienc e124:684-685 . Peroxidasesoccu r inal lplan ttissues . Frankel,R. , S.Izha r& J .Nitsan ,1969 . Theiractivit y and iso-enzymenumbe rin ­ Timingo fcallas eactivit y andcytoplasmi c creaseswit h theag eo fth eplant .Accordin g malesterilit y inPetunia .Bioch.Genet .3 : toParis h (1968)i ti sa nindicato r forth e 451-455. physiological situation ofa plant .Peroxi ­ Izhar,S .& R .Frankel ,1971 .Mechanism so f dasei sa ver y stable enzymewit ha hig h malesterilit y inPetunia .Th erelation ­ water-solubility. shipbetwee npH ,callas eactivit y inth e anthersan d thebreakdow n ofth emicro - Itdoe sno tdenaturat e whentissue sar e sporogenesis.TA G41:104-108 . stored indeep-froze n conditions.Th ebio ­ chemical function ofperoxidas e isunclear:* ' Kool,A.J. ,J.H . deHaa s& G.A.M ,va nMarre - onerecen thypothesi s istha ti tha st ob e wijk,1982 .Analysi so fmitochondria l and considered asa degradatio nproduc to f 'chloroplas tDN A from fertile andcytoplas ­ catalase.Bot hanther san d leaveswer ein ­ micmale-steril ePetuni ahybrida .In : vestigated forperoxidas epatterns . C.Broertje s (Ed.). Induced variability in Peroxidaseswer e detectedb y incubatingth e plantbreeding .PUDOC ,Wageningen:127 . gelafte rIEF-treatmen ti na stainin g Leonova,N.S. ,1974 .Th enatur eo fmal este ­ solution according toSha w& Prasa d (1970). rility transmitted bygraftin g andappea ­ ring inth esee dprogen y ofscion si nsuga r Itappeare d that iso-enzyme patternsar e beet.Genetic s (USSR)10:13-17 . -specific.Th ezymogramm e oflea fsam ­ pleswa s identical foral l idiotypesinvol ­ Levings,CS. ,1983 .Th eplan tmitochondria l ved.Sligh tdifference s couldb ediscerne d genome and itsmutants .Cell.32:659-661 . inth eperoxidas e composition ofanther s Levings,C.S .& D.R . Pring.Molecula rbase s between developmental stages,varietie san d ofcytoplasmi c malesterilit y inmaize . fertility types.So ,a fe wband swhic hoccur ­ In:J.E .Scandalio s (Ed.). Physiological red inBB F couldno tb etrace d inth esteril e Genetics.Ac.Press ,Ne wYork ,etc. :171-193 , versionan dvic eversa .Ther eals oseeme dt o Marrewijk,G.A.M .van ,1969 .Cytoplasmi cmal e bea ninfluenc eo fth etemperatur e inwhic h sterility inpetunia .I .Restoratio no f theplants.ha dbee ngrown .A fe wband swhic h fertility with specialreferenc e toth e wereclearl yvisibl e inth e17° CBB Fsampl e influenceo fenvironment .Euphytic a18 : disappeared orwer e lessclea ra t25°C ,bu t 1-20. alsorevers e situations occurred. Marrewijk,G.A.M .van ,1970 .Cytoplasmi cmal e sterility inpetunia .II .A discussio no n Differencesbetwee n varietieswer ehardl y male sterility transmission bymean so f moreconspicuou s thanthos ebetwee nfertili ­ .Euphytic a19:25-32 . tytypes .I twa s concluded thatth eobserve d differenceswer eo fa quantitativ erathe r

42 Marrewijk,G.A.M ,van ,1979 .Mal esterilit y forhybri dproduction .In :Plan tBreedin g perspectives.PUDOC ,Wageningen :120-134 . Parish,R.W. , 1968.Studie so nsenescin g tobacco leafdisc swit h specialreferenc e toperoxidase .I .th eeffect so fcuttin g ando finhibitio n ofnuclei c acidsan d proteinsynthesis .Plant a82 :1-13 . Pimakhin,V.F .& O.V . Agashev, 1978•.Trans ­ missiono fcytoplasmi cmal esterilit y (cms)b ygraftin g insunflower .PB A49 : 2997. Radola,B.J. , 1980.Ultrathin-laye riso ­ electric focussing in50-10 0(i mPolyacry ­ lamidegel so nsilanize d glassplate so r polyester films.Electrophoresi s 1:43-56 . Schreur,E. ,1978 .Biochemisch e achtergronden vand emannelijk e steriliteit.Ir-thesi s PlantBreeding ,AU ,Wageningen :3 9pp . Shaw,CR . &R .Prasad ,1970 .Starc hge l electrophoresis-A compilatio n ofrecipes . Bioch.Genet .4 :297-320 .

43 CYTOCHEMICAL LOCALIZATION OF CYTOCHROME OXIDASE IN ANTHERS OF CYTOPLASMIC MALE STERILE PETUNIAHYBRID A (HOOK.)VILM .

R.J.Bino ,S.J . deHoo pan dA .va nde rNeu t

Department ofPlan tCytolog y andMorphology ,Wageningen ,Th eNetherland s

Summary (Watson et al., 1977). Throughout sporoge- nesis, sterile anthers showed fewer iso­ In Petunia it was demonstrated that zymes than their fertile counterparts. changes inmitochondria l DNA are associated In addition, the enzymatic activity of with cytoplasmic male sterility (Kool cytochrome oxidase appeared to be differ­ et al., in preparation). Investigations ent. Sterile anthers of various types of translational products from isolated of cms maize plants, exhibited a signifi­ mitochondria have revealed several unique cantly lower cytochrome oxidase activity polypeptides. This distinct polypeptide than fertile ones (Watson et al., 1977; composition possibly influences sterile Ohm.asa et al., 1976). During fertile microsporogenesis and may ultimately meiosis, cytochrome oxidase activity result in abortion of the tapetal and increased rapidly, a phenomenon not found sporogenous cells. In the present study, in sterile sporogenesis (Ohmasa et al., cytochrome oxidase is localized at the 1976). However, since the differences ultrastructural level, using the diamino- in enzyme activity occured rather late benzidine method. Cytochrome oxidase in sporogenesis, they may be evoked by activity is demonstrated in the cristae the preceding process of degeneration. and at the membranes of the mitochondria. Cytochrome oxidase activity assayed We could not detect any difference in from leaf tissue, was similar for fertile staining specificity, correlated with and sterileplants . sterility. In Petunia, Kool et al. (in prepara­ Keywords: cytoplasmic male sterility, tion), demonstrated that changes in mito­ Petunia, cytochrome oxidase, cytochemical chondrial DNA and its translational pro­ localization. ducts are correlated with sterility. Investigations of translational products Introduction from isolated mitochondria have revealed several unique polypeptides associated Cytoplasmic male sterility (cms) is with male sterile cytoplasms. Recently, a maternally inherited trait which inter­ specific sequence analysis of mitochon­ feres with pollen production and is used drial DNA affirmed changes in the genetic to produce F1 hybrids by preventing self code for subunit II of cytochrome coxida ­ fertilization of the seed parent -(fo r se, possibly related with cms (Hanson more details the reader is kindly referred at *al., 1984). In the present study, to the contribution of Van Marrewijk). we attempt to establish whether these Substantial evidence, especially in alterations in mitochondrial DNA do have maize and Sorghum, suggests that the implications for enzymatic activity in trait is encoded by the mitochondrial the anther at the ultrastructural level. genome (Levings, 1983). A number of pro­ The results demonstrate the localization teins are produced within themitochondria . of cytochrome oxidase in the cristae For example, three subunits of cytochrome and at the membranes of the mitochondria. c oxidase, two subunits of the cytochrome We could not detect any difference in be complex and mitochondrial ATPase are staining specifity correlated withsterili ­ synthesized within this organelle (Dillon, ty- 1981). Deviations in the synthesis of one of these proteins may ultimately Materials andmethod s result in abortion of the tapetal and sporogenous cells. In several studies, Two isogenic types of Petunia hybrida sterility appears to be associated with (Hook.) Vilm. were used in this study, alterations in cytochrome oxidases. Mito­ i.e. male fertile cv. Blue Bedder (BBF), chondria from a cms Sorghum, synthesize and the cms Blue Bedder (BBS), described a cytochrome c oxidase subunit I, 4000 byVa nMarrewij k (1968). daltons larger than the form found in The localization of cytochrome oxidase fertile cytoplasms (Dixon and Leaver, was based on the methods developed by 1982). In maize, differences in the number Seligman et al. (1968) and Anderson et of isozymes of cytochrome oxidase were al. (1975). The medium contained 2.5 observed during meiosis in the anther mM 3,3'-diaminobenzidine (DAB) in 0.05 MNa-phosphat ebuffe ra tp H7.2 .

44 Fig. 1. Part of a microspore of BBF, Fig. 3. Distribution of the reaction stained for cytochrome oxidase activity. product of DAB within mitochondria of Enzyme reaction product appears in the a microspore of BBF. A few cristae and mitochondria. the outer surface of the inner membrane Tetrad stage,x 15,000. ofth eenvelop ear e stained. Tetrad stage,x 52,500.

*. "t^t

Pig. 2.Par t of a meiocyte of BBS,staine d Fig. 4. Part of a microspore of BBF. for cytochrome oxidase activity. Enzyme Staining for cytochrome oxidase is inhib­ reaction product appears in the mitochon­ ited with KCN. No formation of reaction dria. product isobserved . Prophase I,x 15,000. Tetrad stage,x 15,000.

45 Resultsan d discussion activity between fertile and sterile anthers. Hence, the situation in maize The electron transport system regulates appears to be not representable for cms the flow of electrons during oxidative speciesi ngeneral . phosphorylation and is an integral part of the inner mitochondrial membrane (Dil­ References lon, 1981). Cytochrome oxidase, which catalyzes the oxidation of cytochrome Anderson, W.A., G. Bara & A.M. Seligman, c, is an essential component of this 1975. Ultrastructural localization system and consists of a complex of several of cytochrome oxidase via cytochrome transmembrane polypeptides that are in­ c. tegrated into the inner membrane. Using J. Histochem. Cytochem. 23: 13-20. DAB as an electron acceptor, the consistent Dillon, L.S., 1981. Ultrastructure, macro- morphologic visualization of an oxidase molecules and evolution. Plenum Press, system can be demonstrated on the outer NewYork ,London . surface of the inner membrane (Seligman Dixon, L.K. & C.J. Leaver, 1982.Mitochon ­ et al., 1968). The initial step in the drial gene expression and cytoplasmic mitochondrial oxidation of DAB relies malesterilit y inSorghum . upon cytochrome c, acting as the electron Plant.Mol . Biol. 1:89-102 . acceptor. The next step is the reoxidation Hanson, M., S. Izhar, M. Boeshore, E. of DAB by cytochrome oxidase. The oxidated Clark, M. Rothenberg, C. Snyder, I. DAB forms a nondroplet, amorphous reaction Lifshitz & H. Nivison, 1984. Organelle product at the enzyme site.Th e precipitate genomes in cytoplasmic male sterile is osmiophilic and insoluble in the dehy­ and fertile somatic hybrid Petunia dration and embedding agents used in plants. In: Plant mitochondrial DNA. specimen preparation. If the reaction EMBO workshop, Melrose, Scotland, is conducted under controlled conditions, p.35 . the intensity of the reaction product KjSol, A.J., J.M. de Haas, J.N.M. Mol affords a dependable criterion for indi­ & G.A.M, van Marrewijk, in preparation. cating cytochrome oxidase activity (Perotti Isolation and physiocochemical charac­ etal. , 1983). terization of mitochondrial DNA from In Petunia, the DAB reaction product cultured cells of Petunia hybrida. is located in the cristae and at the Levings, C.S., 1983 . The plant mitochon­ mitochondrial envelope (Figs. 1, 2 and drialgenom ean d itsmutants . 3). Cytochrome oxidase reaction ispresume d Cell. 32:659-661 . because formation of the reaction product Marrewijk, G.A.M. van, 1968. Cytoplasmic is inhibited by short prefixation in male sterility and restoring fertility glutaraldehyde and by cyanide (Pig. 4). in the garden petunia.Doctorat ethesis , Mitochondria from fertile and sterile AU, Wageningen, The Netherlands (in sporogenous cells comprise only a small Dutch,wit h English summary). number of reacting cristae. The distribu­ Ohmasa, M., Y . Watanabe & M. Nobuo, 1976. tion of the reaction product is not 'in­ A biochemical study of cytoplasmic fluenced by the developmental stage. , 'malesterilit y ofcorn . Staining specificity of the meiocytes JapanJ . Breed.26 :40-50 . of BBS, and themicrospore s ofBB Fappeare d öpik, H., 1975. The reaction of mitochon­ tob esimilar . dria in the coleoptiles of rice with We could not detect any difference diaminobenzidine. in enzyme localization correlated with J. Cell. Sei. 17:43-55 . sterility. The localization of the reaction Perotti, M.E., W.A. Anderson & H. Swift, product in fertile and sterile mitochon­ 1983. Quantitative cytochemistry of dria, suggests a similar cytochrome oxidase the diaminobenzidine oxidase reaction activity. Nevertheless, it cannot be product in mitochondria of cardiac ruled out thatdifference s inmitochondria l muscle andpancreas . enzyme activity play a role in sterility. J. Histochem. Cytochem. 31: 351-365. Viz• , the specificity of the reaction Seligman, A.M., M.J. Karnovsky, H.L. for cytochrome oxidase is not completely Wasserkrug &J.S .Hanker , 1968.Nondrop - certain. In plant tissues, other cytochro­ let ultrastructural demonstration mes may participate in the oxidation of cytochrome oxidase activity with of DAB (Opik, 1975). Comparison of tissue a polymerizing osmiophilic reagent, specific localization of cytochrome oxidase diaminobenzidine. and total biochemical activity should J. Cell. Biol. 38:1-14 . reveal whether the altered mitochondrial Watson, C.V., J. Nath & D. Nanda, 1977. DNA correlates with differences in enzyme Possible mitochondrial involvement activity in the anther.However ,prelimina ­ in mechanism of cytoplasmic male steri­ ry results could not establish any bioche­ lity inmaize . mical difference in cytochrome oxidase Biochem.Gen . 15:1113-1124 .

46 r.STERILIT Y OF MICROSPORES IN OENOTHERA HOOKERI.VELAMS I.Nohe r de Halac Centro tieMicroscopi a Electronica, Universidad Católica de Córdoba, Cordoba, Argentina.

Summary 2. Vacuolate microspores; 3. Two-celled vac­ uolate pollen nrains;4 . Two-celled pollen During the non vacuolate stage dense mate­ grains with starch. These are coincident with rial accumulates outside the precursor layer those generally described by Laser & Lersten of the ektexine. Later the microspores vacu­ (1972). olate but no paracrystalline structure is After release from the tetrad the microspo­ formed in the ektexine. Enzyme digestion of res are covered by an external fibrillar lay­ theendexin e is followed by dissolution of erwhic h can be considered the ektexine pre­ the abnormal dense deposits of the microspore cursor layer. An internal, dark, homogeneous surface. Lamellations are the last remains of layer can be identified as the endexine. The theexin e on the external surface of the apertural chamber has fibrillar contents and intine which comes in direct contact with the shows at the base lamelles which look like pollen sac environment. After the microspores compressed vésicules (Fig.1 a).Late r the collapse without suffering mitosis, lamellar paracrystalline structure of the ektexine ap­ material aswel l as dense deposits and cyto­ and the beaded viscin threads become plasm remains are finally contained in the their final structure. Dark sporopollenine denenerating anthers. Tapetal cells show grains appear at the base of the apertural concomitant modifications to these changes, chamber. At this stage the tapetal cells affecting mainly the transpher phase, the reach the transpher phase. E.R. and the plastids and mitochondria. The vacuolization of the microspores is concomitant to the deposition of the intine Introduction (Fig. 1 b).Th e tapetal cells at this stage are characterized by plastids which contain The aim of this work is to compare the de­ vésicules of light material surrounded by a velopmental stages in normal and sterile an­ dark halo. Deposits of dark material are seen thers after the release of the microspores between the mitochondrial membranes and the from the tetrad. It seems important to state ammount of REP.decreases . the developmental stages of microspores and After the first mitosis of the microspore pollen nrains using electron microscopy meth­ is completed, the sporoderm arrives at the ods. typical final structure. The anther tapetum Some work on pollen sterility in the genus shows some cells with degenerating cytoplasm Oenothera was first published using methods still "in situ" but abundant cell remains are of light microscopy (Oehlkers, 1927). After swimming in the pollen sac. this the work of Harte 1 Bissinger (1952)re ­ Shortly before anther dehiscence, the pol­ ported on the changes occuringin 0. hookeri. len grains reach the dormancy stage which is velan s using paraffin-embedded cuttings of marked by the loading of abundant plastids normal and sterile anthers. The same hybrid with starch and at the same time the disap­ is the material used in this work,whic h was pearance of the big central vacuole (Fig.1 kindly supplied by Prof. C. Harte of the Uni­ c). No tapetal cells remain "in situ". versity of Cologne. At electron microscopy An accurate description of the modifica­ level only the pollen sterility of 0. ery- tions occuring in sterile anthers during de­ trosepala BORBAS (lamarckiana DE VRÏTS)wa s velopment will be published elsewhere. Only studied before (Jean, 1971). Normal develop­ the main differences with normal development ment is partial!y know n through work done are pointed out in this symposium book. using electron microscopy (Afzelius, 1956; As Harte *iBissinge r stated (1952), pollen Lepousé s.Romain , 1967;'Skvarl a et al., 1978; sterility is monogenic. The ster gene in ho- Skvarla, 1975; Skvarla et al., 1976). No work mozigous plants determines pollen sterility, was done before with a developmental point of causing a very deep modification of theme ­ view. tabolism in the flower. The maturation se­ quence of the anthers in the flowers of the Results and discussion inflorescence is altered, as well as the syn­ chronism of developmental stages in the pol­ Four developmental stages were recognized len sacs of the same anther. after release of the microspores from the Regarding the microspores no differences tetrad (Fig. 1):1 . Non vacuolate microspores; between normal and sterile development could

47 Fig. 1. Stages of normal development in 0. hookeri. velans , a, vacuolate microspore, b,Two - celled vacuolate pollen grain, c, Two-celled pollen grain with starch.Symbols : | ektexine precursor; ektexine;M endexine;™ intine; B| apertural chamber; wmlamelle s Qplas- tids with starch.

be verified during meiosis.Thi s is coinci­ during sporoderm development. This is also dent with findings in 0. erytrosepala (Jean, supported by the fact that they fail tode ­ 1971), where at a very late stage pollen die velop normally in this hybrid. The tapetal leaving empty grains with almost normal cell mitochondria also look different,whic h sporoderms. is remarkable. In 0. hookeri. velans differences between the dissolution of the endexine and later normal and sterile pollen development appear oA the digestion of the dark deposits on the as the final consequence of deep metabolic microspore surface can be a secondary con­ alterations of the last stages of sporoderm sequence of the metabolic disturbance in the morphogenesis,whic h fail to develop the pollen sac. Enzymes could be responsible for paracrystalline structure of the ektexine the initiation of digestion of the endexine. (Fig. 2a) . These enzymes are acting from the inside of Two main changes could be stated in sterile the layer towards the outside. Once the anthers (Fig. 2 a-c^: endexine becomes discontinuous the dark ma­ 1. Dark material deposited on the outer sur­ terial of themicrospor e surface is also di­ face of the microspores is the first evidence gested. In barley,esterase s are shown to ofmodification s during sterile development. digest the exine (Ahorkas, 1976). Otherwise It is produced in vésicules of the tapetal the endexine is not a continuous layer,a s cytoplasm, passing on to the ppllen sac and, has been demonstrated in Epi1ob iur n angusti- accumulating on the outer surface of the * folium L. (Rowley, 1976). The endexine chan­ ektexine precursor without,originating the nel« could be the sites of location of the paracrystalline structure. inactive enzymes.Th e beginning of enzyme 2. Later on themateria l secreted by the digestion ismarke d by suitable conditions -tapetal cells becomes lamellated. Lamella- arriving in the environment. This occurs -tions of the exine seem to be a common fact either during germination on the stigmatic inmodifie d pollen grains.The y were observed surface after pollen dormancy in normal in aborted pollen of several species (Rowlev compatible pollinations,o r still in the pol­ & Flynn, 1969). len sac before pollen grains arrive at their On the base of my observations I assume maturity and enter dormancy in sterile an­ that dark material accumulated in tapetal thers. vésicules and.o nth e microspore surface is probably a kind of lipid self-assembly References (Sitte,1981 )o f colourless sporopollenin precursors produced in the tapetal cells Afzelius,B.M. ,1956 . Electron Microscope during the transpher phase,-whic h fail to investigations into exine stratification. organize as definitive sporopollenin mole­ Grana Palyn. 1: 22-37. cule in sterile anthers. Production of col­ Ahorkas, H., 1976. Evidence of a Pollen ourless precursors of sporopollenin wasas ­ esterase capable of hydrolizing sporopol­ sumed before (Heslop-Harrison, 1968). Three lenin. Experientia 32: 175-176. alternative pathways can be postulated for Harte. C. & B. Bissinger, 1952. Entwcklungs- •thes e precursors: they originate either geschichtliche Untersuchung der durch die sporopollenin or Bcarotenoid s in normal Faktoren fr. und ster Bedingten Pollen­ anthers and anorphous lipids in sterile pol­ sterilität bei Oenothera. Z. Vererbungsleh­ len sacs. Plastid morphology in tapetal cells re 84:251-269 . allow the assumption that they play a role Heslop-Harrison, J., 1968.Anthe r carotenoids

48 9:403-413. Oehlkers.F., 1927. Entwicklungsgeschichte der Pollensterilitat einiger Oenotheren. Z. Vererbungslehre 43:265-348. Rowley,J.R. , 1976. Dynamic changes in pollen wall morphology. In: I.K. Ferguson &Mulle r (Ed.): The evolutionary significance of the exine. Academic Press,London , p.39-66 . Rowley,J.R . &J . Flynn, 1969. Membranes pressure and exine Form. A reinterpretation ofexin e formation on aborted pollen grains. Pollen et Spores 1:169-180. Sitte,P., 1981. Role of lipid self-assembly in subcellular morphogenesis. In: 0. Kier- mayer (Ed.): Cytomorphogenesis inPlants . Springer Verlag,Wien,Ne w York. p.401-421 . Skvarla, J.J. et al., 1978.A n ultrastructu­ ral study of viscin threads in pollen. Pollen et Spores 20:5-143. Skvarla,J.J. , P.H. Raven &J . Praglowski, 1975. The evolution ofpolle n tetrads in Onagraceae. Am. J. Bot.62:6-35 . Skvarla,J.J. , P.H. Raven &J . Praglowski, 1976. Ultrastructural survey of Onagraceae pollen. In: I.K. Ferguson &J . Muller (Ed.): The evolutionary significance of theexine . Academic Press,London , p.447-479 .

Fig. 2. Stages of sterile development in0_ . hookeri. velans , a, vacuolate microspore with dense deposits on the ektexine precursor, b, beginning of the endexine dissolution, c, last stages of exine dissolution, symbols: as Fig. 1.

and the synthesis of sporopollenin. Nature 220:605. Jean, R., 1971. La paroi du pollen d'Oenothe- ra lamarckiana. Bull. Soc. Bot. du Nord de la France24:93-102 . Laser, K.D. & N.R. Lersten, 1972.Anatom y and cytology ofmicrosporogenesi s in cytoplasmic male sterile Angiosperms. Bot. Rev.38:425-454 . Lepouse,J. &M.F . Romain, 1967.Etud e de 1' ultrastructure des enveloppes polunique s chez Oenothera biennis. Pollen et Spores

49 STAGESO FTAPETA L CELL CELL DEVELOPMENT INSTERIL EANTHER S OFOENOTHER AHOOKERI .VELAN S

I.Nohe r deHala c

Centro deMicroscopï aElectrónica ,Universida d Católicad eCordoba ,Cordoba ,Argentin a

Summary

Thetapeta l cellstage swer e identified and theirultrastructur e related tomicro ­ sporemorphology . During sterile development they show changes in theirplastid s andER . Vesicleswit h dense contents andparamura l bodies appear asmai nmodifications .

Introduction

O.hookeri.velans isa hybri dwit hsporo - phytically controlledmicrospor e sterility b * (Noher deHalac,1984).Developmenta lstage s Fig.l.Autolyti c tapetalcel l s.a ,Tapeta l of tapetal cellsha dno tbee n recognized layerwit h lamellarmateria l at thelocula r before atelectro nmicroscop y level,eithe r face.Scale:25um.b ,Lamella r materialan d innorma lo ri n degenerating anthers,i n darkdroplet s inth e autolyti c cytoplasm. spiteo f the fact thatpolle n sterilityha s Sc4le:2 urn . beenknow n tob epresen t inth e genusOenothe ­ c rafo ra lon gtime . velans adee p disturbanceo f lipidmetabolis m in tapetal cells conducesi n theektexin et o Results and discussion the failureo f theparacrist a llineorganiza - tiono fsporopollenin . Itha s been assumed After release from tetrads themicrospore s that thetapetu mplay s arol e inth esynthe - are at theno nvacuolat e stage.Th e secretory sis ofinformationa lmacromo l ecules (Ragha- tapetal cells reach the transfer phasewhic h van,1981). Furtherwor kshou l dprovid emor e is characterized by thedigitation s ofth e evidence on thekin d ofmacr o molecules invol- plasmalemmawit h amitochondrio n at thebas e vedi nlipi dmetabolis mwhic h couldb emodi - of each digitation..A tth evacuolat e stage fied in thesehybrids . ofmicrospores ,th e tapetal cells gradually loose the digitations of theplasmalemm aan d References the amount of RER decreaseswhil e theplas-* - tidsbecom e fullo fligh t vésiculessimila r Dickinson,H .G 197 3Th eI Dl eo fplastid s inth e to thoseobserve d inRaphanu s (Dickinson, ' formation ofpolle n grain coatings.Cytobios 1973 ),Antirrhinu mmaiu s (Lombardo &Carraro, 8:25-40. 1976 )an dPle aeuropae a (Pacini&Casadoro , Lombardo,G.&I.Carraro.1976.Tapeta lultrastruc ­ 1981). Vacuolation of tapetal cells follows tural changes during pollen development.I. and the cellremain s partially migrate into Caryologia29:114-125 . thepolle n sac.Tapeta l cellscompletell y Noher deHalac,I.1984.Thi s symposiumbook . desappearwhe n pollen grains get charged Pacini,E.&G.Casadoro.1981.Tapetumplastid s with starch aftermitosis . ofPle aeuropae aL .Protoplasm a 106:289-96. Raghavan,V.1981.A transient accumulationo f During sterile development,tapeta lcell s poly(A)-containing RNA in the tapetumo f show alterations in the transferphase .Late r Hyoscyamusnige r duringmicrosporogenesis . onn oplastid s containing lightvesicle sde ­ Developmental Biol.81:342-348 . velop. Instead,a hypertrophy a of theE Ri s Reznikowa,S.A.&T.M.Willemse.1981 .Electron - seen and darkmaterial s accumulate firsti n Microscopic andhistochemica l investigation vesicles and then asparamura lbodie soppo ­ of tissue of thedevelopin g lily anther site the locular face of the cells(Fig.1). in connectionwit h metabolism ofreserv e This demonstrates the relationship between nutrient substances.FiziologiyaRastenii . thetransfe rphas eo f tapetal cells andth e 28:1181-1189. development of theparacristallin e structure inth eektexine . Themai n functional activity of thetape - turni sassociate dwit h synthesis oflipid s and carotenoids.Thi swa s assumed forth e developing lily anther (Reznikova &Willemse , 1981). In thesteril e anthers ofO.hookeri .

50 ISOLATIONO FPETUNI AHYBRID A CYTOPLASTS

M.M.C.Tan ,J . deBruin ,G.A.M ,va nMarrewijk* ,A.J .Koo lan d H.J.J. Nijkamp

Dept.o fGenetics ,Vrij eUniversiteit ,D eBoelelaa n 1087,108 1 HVAmsterdam , TheNetherland s •Instituteo f Plant Breeding,Agricultura l University,Wageningen ,Th eNetherland s

Somatic cell fusion of protoplasts with cytoplasts (enucleated protoplasts) rep­ resents an ideal tool for transfer of specific cytoplasmic genetic information from one plant into another, without changing the nuclear background of the acceptorprotoplast . By using a simple method of Percoll centrifugation, we have developed aproce ­ dure for cytoplast isolation of Petunia hybrida protoplasts, aiming at the trans­ fer of cytoplasmic male sterility from P.hybrid a toLycopersico nspecies . Freshly isolated Petunia protoplasts were centrifugea (50,000g for one hour at 12°C ) on top of a discontinuous Per­ coll gradient. Fractions of protoplasts (A), cytoplasts (B) and nuclear fragments (C) were collected and analyzed micros­ copically after staining with 1% aceto- carmine.

--* \ * £iW

.A B

51 Plastidconten t ofgenerativ ecell si nLiliacea e

M.B.Schröder andR.Hageman n Department ofGenetics ,Martin-Luther-University ,Domplat z1 DDR-4o2oHalle/S. ,Germa nDem .Rep .

Summary thefirs tpolle nmitosis .Durin gth e maturationo fth egenerativ ecel l Theplasti d content ofgenerativ e theseplastid ssee mt odegenerate . cellsi nspecie so fth eLiliacea eha s Onlymitochondria ,dictyosomes ,ribo - beenstudie dusin g electronmicrosco ­ somes,endoplasmi creticulu man d py.Matur egenerativ e'cell swithou t myelin-likestructure scoul d befoun d plastidsar eforme d eitherb yth eun ­ withinth egenerativ ecytoplas ma t equaldistributio n ofplastid sdurin g anthesis.Th elac k ofplastid si n firstpolle nmitosi s (G.verrucosa& maturegenerativ e cellslead s toa F.meleagris)o rb yth edegeneratio n maternalinheritanc eo fplastid si n ofplastid sdurin g thematuratio no f thesespecies . generative cells (F.imperialis& H . 3.Gasteri averrucos aan dFritillari a ventricosa).Th egeneti c consequence meleagris:Th eyoun ggenerativ ecel l isa materna linheritanc eo fplastids . aswel la sth ematur egenerativ ecel l Generativecell so fLiliu mmartago n dono t containplastids .Plastid sar e containplastid sdurin g thewhol e excluded fromth egenerativ ecel l pollendevelopment .Th emod eo fth e during thefirs t pollenmitosis . plastid inheritance inthi sspecie s 1^.Gasteri averrucos aw ecoul dob ­ dependso nth efat eo fplastid sdur ­ servea polarizatio no fplastid sfa r ingsper mcel lformatio nan dfertil ­ fromth edividin gmicrospor enucleu s ization;thi sha st ob eanalyze di n startinga tth elat eprophas ean d futureinvestigations . endinga tth eearl y two-cellular stage.Thi splasti darrangemen t pre­ Introduction venting theplastid sfro mbein g transmitted intoth egenerativ ecel l Themod eo fplasti d inheritancede ­ mayb econtrolle db ya biochemica lo r pendso nth emod eo fplasti ddistri ­ abiophysica lgradien t (Schröder butiondurin gth edevelopmen t ofth e 1984).Th egeneti c consequence ofth e malegametophyt ean dth efertlizatio n absence ofplastid si nmatur egenera ­ inangiosperms .Accordingly ,fou r tivecell si sa materna linheritanc e typeso fplasti d transmissionhav e ofplastids . beendescribe d (Hagemann,1983) *Th e underlying cytologicalmechanism sar e .References notye t fullyunderstood .Furthe rin ­ vestigations onman y speciesar e Hagemann,R.,1983 .Th eformatio no f necessary. generativean dsper mcell swithou t plastidsi nangiosperm san dth e Resultsan ddiscussio n underlyingmechanisms .I nErdelska , 0.,ed.Fertil.Embryogen.Ovul.Plant s Fivespecie so fLiliacea ehav ebee n VEDA,Bratislava,97-99 . investigated using electronmicrosco ­ Schröder,M.B.,1984 .Ultrastructura l py.Thre etype so fplant shav ebee n studieso nplastid so fgenerativ e found: andvegetativ ecell si nLiliaceae . 1.Liliu mmartagon :Th eyoun gmicro ­ 3.Plasti ddistributio ndurin gth e sporea swel la sth egenerativ ean d pollendevelopmen t inGasteri a thevegetativ ecel lcontai nplastids . verrucosa (Mill.)Duval . Degenerating plastidshav eno tbee n Protoplasma.Inpress . observed.Therefore ,w eassum etha t thetransmissio no fplastid sint oth e spermcell san dlate rint oth ezygot e mayb epossible .Furthe rstudie so n theplasti d distributiondurin gth e progamic phasean d fertilizationhav e tob eperformed . 2.Fritillari aimperiali san dHost a ventricosa:Th eyoun g generativecel l containssom eplastid sa sa resul to f

52 ISWRESULT SABOU TTH EPRESENC EO PPLASTIE S INGENERATIV EAN DSPER MCELL S OFGRAMINEA E

R.Hageman n andII.B .Schrode r Department ofGenetics ,Martin-Luther-University ,Domplat z1 DDR-4020Halle/S. ,Germa nDem .Rep .

Summary Datai nth eliteratur e seemedt o datasee mt omak e arevisio nneces ­ indicate astrikin gheterogeneit y of sary:Th ecereal ssee mt ob emuc h thecereal sregardin gth eplasti d morehomogeneou sregardin gth eplas ­ content ofthei rgenerativ eand .sper m tidconten t ofthei rgenerativ ean d cellsa swel l asth etransmissio no f spermcell stha npreviousl y thought. maleplastid sint oth e eggcell . However,electro nmicroscopi cstudie s Resultsan ddiscussio n inou rla b andcritica lanalyse so f recentliteratur e datamak e arevi ­ Hordeumvulgar e: sionnecessary :Th eGraminea esee m tob emor e homogeneous thanprevious ­ Cass& Kara a (1975)reporte dtha t lythought .Al l cereals,studie d inbarle y 'therear en orecognizabl e intensively,d ocontai n plastidsi n plastids inth egenerativ ecell' . thegenerativ e andsper mcell so f Miodzianowaki (person,coram .1982 ) theirpolle ngrain san d tubesreap . reported theobservatio n ofplastid s Mosto fthe mbelon gt oth eTriticu m ingenerativ e cells.Mogense n (1982) type:Presenc e ofplastid s ingener ­ statedtha tth esper mcytoplas mo f ativean dsper mcells ,bu tthei r barley 'containsnumerou sorganelles , strippingof fth esper mnucleu s mosto fwhic happea rt ob emitochon ­ duringth efertilizatio nprocess . dria,bu t someo fwhic hma y bepro - Carefulgeneti c studies areneces ­ plastids'.- V/ ehave ,thus ,t otak e sary toanalys e thesituatio n inSe - intoaccoun t thepresenc eo f(a t calewhic hma y showa nexceptiona l leastsome )plastid si ngenerativ e behaviour. andsper mcells . Thegeneti c datao fArnaso n etal . Introduction (1946)sho wa uniparenta lmaterna l inheritance ofchlorophyl ldeficien t Onth epreviou sSymposiu mi nth e mutant plastidsi nbarley . HighTatr a thefirs t authorha s characterized 4.typeso fangiosper - Zeamays : mousspecie sregardin gth epresenc e orabsenc eo fplastid si ngenerativ e Larson (1965)reporte d theobserv ­ andsper m cellso fth epolle nan d ation ofsper m cellswithou t starch thetransmissio n ofmal eplastid s suggesting anabsenc e ofplastid3 . intoth e eggcel l (Hagemann 1983): Butth epape ro fChebotar u (1981) Lycopersicontype , Solanumtyp e clearly showsth epresenc e ofplastid s 'Jriticumtyp e and Pelargoniumtype . insper mcells . Genetic studies demonstrate auni ­ Datai nth e (older)literatur ele d parentalmaterna l inheritanceo f toth eopinio n thatth eGraminea e mutantplastid s (Shumway andBauman , (= )sho wa strikin ghetero ­ 1967). geneity intha trespect ;different - speciesseeme d tobelon gt oal l4 Oryzasativa : types.Ther ear epaper sreportin g thatHordeum ,Agropyro n and Zeabe ­ Vaughn etal . (1980)discusse dric e longt oth eLycopersico ntype , asa nexampl efo rthei rhypothesi s Oryzat oth eSolanu m type,Triticu m that 'chloropiastsma y bephysicall y andTritical e toth eTriticu mtype , altered inth eproces s ofaicrosporo - andSecal et oth ePelargoniu mtype . genesis,preventin gthe mfro mbeing - transmitted'int oth eeg gcell ,bu t Electronmicroscopi c studies inou r haveno tdocumente d thisstatemen t laboratory anddetaile d criticalana ­ byelectro nmicrographs . lyseso fsevera lrecen t literature Incrosse sPa lan dRamanuja m (1941)

53 found auniparenta lmaterna linherit ­ Secalecereale : anceo fplasti dmutations . Electronmicroscopi c studiesi nou r Triticum aestivum: labprove d thepresenc eo fplastid s ingenerativ ean dsper mcell so fry e In ourla b inHall e thepolle nde ­ (Laub,Schröder ;Pig.3) . velopment ofwhea twa s studied.Th e generative andsper mcell sregularl y Incontras tt oth efinding sabou t contain plastids (Schröder,Risch ; thespecies ,mentione d above,ther e Fig.1). Geneticstudie s showeda uni ­ isa pape rb yFros te tal.(1970 )re ­ parentalmaterna l inheritance ofmu ­ portinga biparenta l inheritanceo f tantplastid s (Briggle, 1966).Th e anextranuclea rchlorophyl ldeficien ­ uniparentalinheritanc e ofplasti d cy.Thi si sth eonl y report abouta characterswa s alsodemonstrate db y biparental plastid inheritance ina restriction analyses aftercrosse s monocotspecies . (Vedele tal .1981) .Pro m thesefact s wedre w (1983)th econclusio ntha t However,th eanalysi so frestric ­ wheat represents ane w type:I nth e tionpattern so forganell eDBA so f Triticum typeo fplasti d distribution eightTritical eline swit hdifferen t bothgenerativ e andsper mcell sd o wheat cytoplasms showed exclusively contain plastids,bu tdurin gth epro ­ thepresenc eo fth ematerna lplastid s cesso ffertilizatio nth eplastid s andmitochondria ,thu sgivin gn ohin t arestrippe d offth esper mnucleu s ata transmissio n ofpaterna l (Secale andar eno t transmitted intoth eeg g organelles intoth ehybri d cell. (Vedele tal . 1981).- Studie so fth e Themitochondri a ofwhea t seemt o restriction patternso fplasti dDÎJ A behavei nth esam eway :purel ymater ­ ofprimar y triticales inou rla b(Sie - naltransmissio n (asdoe s cytoplasmic mfenroth)als oreveale d only thepre ­ male sterility). senceo fwhea tplastids ,an dn oin ­ dicationfo ra transmissio n ofmal e Triticale-: ryeplastids . Therefore iti shighl y desirable Ourstudie swit hTritical edemon ­ toperfor m reciprocal crosses inry e stratedals o thepresenc eo fplastid s withregar d togeneti c plastiddiffe ­ ingenerativ e and spermcell s(Schro ­ rencesi norde rt oconfir m ort odis ­ der,1983 ;Fig.2) . proveth erepor to fFros t etal . Sofa rgeneti cresult sabou tth e transmission ofplastid si nTritical e Concludingremark s areno tavailable . Thestud y ofth edistributio no f Common characteristics of the specifes, plastids andmitochondri a during dealt with in the preceding paragraphs: ,malegametogenesi s andth eelucid ­ ation,whethe rmal eplastid san dmi ­ Summarizingth emai nresult so fou r tochondria aretransmitte d intoth e studies andth ecritica l analysiso f (andthu sth enex tgeneration ) theliteratur e dataregardin gbarley , areno tonl y interesting questionso f maize,rice ,whea t andtriticale ,w e thefertilizatio nbiolog y ofhighe r comet oth econclusion : plants.Thi sproble m isals oo fgrea t - Inal lthes especie s thegenerativ e practicalvalu efo rplan tbreeders , andsper mcell scontai n plastids because theywis ht okno wwit hwha t (Thesituatio n inric ei sno tclea r typeso fcytoplasmi c organellesa inth emoment. ) hybridnucleu si scombined ,an d - Thegeneti c resultsindicat e auni ­ whetherothe rplastid so rmitochon ­ parentalmaterna l inheritanceo fplas ­ driamigh tsho wa bette rinteractio n tids.Thu si ti st ob econcluded , witha hybri d nucleuswit hregar dt o thatth eplastids ,stil lpresen ti n hybridvigou ran dplan tproduction . thesper mcells ,ar estrippe d offth e spermnucleu si nth eproces so ffer ­ tilization andar eno t transmitted References intoth eeg gcell . -Tha ti st osay ,tha t thespecies , Arnason,T.J. , J.B.Harringto n &H.A . mentioned above,al lbelon gt oth e Friesen,1946 .Canad .Journ .Res.24 : Triticumtyp eo fplasti ddistribution , 145-157. described by Hagemann (1983). Briggle,L.ÏÏ. ,1966 .Crop .Sei .6 : 43-45 Cass,D.D .& I .Karas ,1975 .Canad . Journ.Bot .53 :1051-1062 .

54 Chebotaru,A.A. , 1981.Act aSoc .Bot . Polon.50 :265-268 . Frost,S. ,L .Vaivar s &G .Carlbom , 1970.Heredita s 65:251-260 . Hagemann,R. , 1979.Stadle rGenetic s Ssmpos. (Columbia,Miss .USA )11 : 91-115. Ilagemann,R. , 1983.In :Erdelska ,0 . etal . (Ed.):Fertilizatio nan d Embryogenesis inOvulate dPlants . Bratislava Slov.Acad .Sei .Brati ­ slava,p .97-99 . Larson,D. ,1965 .Amer .Journ .Bot . 52:139-154 . Mogensen,H.L. ,1982 .Carlsber gRes . Comm.47 :313-354 . Pal,B.P . &Ramanujam ,S .1941 .In ­ dianJourn.Agr.Sei .11 :170-176 . Schi-öder,M.B. , 1983.In :Erdelska , 0.e tal . (Ed.):Fertilizatio n and Smbryogenesisi n OvulatedPlants . Bratislava SLov.Acad .Sei .Brati ­ slava,p .101-104 . Shumway,l.K .& L.F .Bauman ,1967 . Genetics 55:33-38 . Vaughn,K.C. ,L.R . DeBonte ,K.G . Wilson& G.W .Schaffer ,1980 . Pig.2.Triticale .Generativ ecel l Science208 :196-197 . containinga proplasti d (P)an da Vedel,P. ,P .Quetier ,Y .Cauderon , mitochondrion (Mj.Generativ enucle ­ P.Posb a& G .Doussinault ,1981 . us (GIJ).Electro nmicrograph .Ba r Theor.Appl . Genet.59 :239-245 . represents0. 5 /urn.

,^^>¥\.#

i?X.

Pig.1.Triticu maestivum .Sper mcel l Pig.3.Secal ecereale .Generativ e containing aproplasti d (P),a mito ­ cell containing aproplasti d (P) chondrion (1,1)an dothe rcytoplasmi c anda mitochondrio n (K). Generative organelles.Electro nmicrograp hS . nucleus (Glï).Electro nmicrograp h Risch.Ba rrepresent s0. 5 /um. II.Oldenburg .Ba rrepresent s 0.5 um.

55 THESURFAC E COATINGO FMICROSPORE S ANDMICROSPOR EMOTHE RCELL SI NPINU S SYLVESTRIS

J.R .Rowle y andB .Walle s

BotanyDepartment ,Universit y ofStockholm ,S-10 69 1 Stockholm,Swede n

Summary and carotenoid esters)bu tals o surface receptors,according toth e findingso f Components of themicrospor e surface Atkinson et al. (1972)wit h algalcells . coating are structurally distinctive soon We assume that the structureo f thesporo ­ after cytokinesis of the tetrad ofmicro ­ polleninous coating isinfluence d byth e spores andbefor e arecognizabl e preexinous specific chemical composition ofth e pattern isdiscernible .Th edistinctiv e receptors. structures includea syste mo frod-lik e subunits andprocesses .Thi s appears tob e References duet ospecifi c glycocalyx receptorso n which sporopolleninaccumulates . Atkinson,A.W. ,B.E.S .Gunnin g &P.C.L .John , Keywords:exine ,pollen ,glycocaly x 1972.Sporopolleni n inth ecel lwal lo f receptors,sporopollenin . Chlorella and other .Plant a107:1-32 .

Introduction

Surface coatings ofmicrospor emothe r cells and ofmicrospore sbot h forma splasm a membrane glycocalyces.Bot har epresen twhe n a sporopolleninous exine isinitiate d so thatmonomer s arepresumabl y availablet o both coatings.A s iswel lknown ,a nexin e forms only on themicrospor e — themicro ­ sporemothe r cell coating eventually vanishes.Thi s situation offers anopportuni ­ tyfo r investigating systemshavin g orno t having thecapabilit y for sporopollenin polymerization.

Results and discussion

Themicrospor emothe r cell surface coating isstructurall y distinct from'thesurface». ' coating ofmicrospores .Th emothe r cell coatingha s theappearanc eo fa n irregular filamentousmes h (Fig. 1)wherea s themicro ­ spore surface coatingha scomple x structures which include rod-like subunits ca3 0n mi n diameter (Fig.2) .Rodlet s appear tob e linked and associated withprocesse s that arec a 100n m inwidth .Thes eprocesse s areprominen t inth e "airbladder "region s butar eals opresen t ingenerall y radial orientation onal lbu t theapertura l areao f microspores.The yma yb econsidere d asunit s ofa nexin e template since theoute r portion Figs. 1-3.Karnovsk y fixativeplu s ruthenium of the exine forms directly onthem .Bot h red.Staining :U ,Pb .Bars : 100nm . when iti sinitiate d andagai n laterwhe ni t Fig. 1.Microspor emothe r cell surfacecoat ­ isthickene d the exine surface isroug hbe ­ ing (M).— Fig.2 .Microspor e surfacecoat ­ causeo f thepresenc e of the rodlet surface ingbetwee nplasm amembran e (P)an d callosic coating (Fig.3) .Th eprocesse s are smoothed envelope (C).Dar k rodlets (arrows)occu r out asthe y increase inthicknes s due,pre ­ between and onprocesse s of future exine(S) . sumably,t o theadditio n of sporopollenin. — Fig.3 .Exinou sprocesse s (S)an d tectum The distinctive plasmamembran e surface (R)ar e studdedwit h components of themicro ­ components areals oobserve d ontapeta lsur ­ sporesurfac e coatingwhic h arec a3 0n mi n faceswhic h likewise accumulate sporopollenin. diameter and appear tob erod-shaped ;en d Sporopollenin accumulation not onlyrequir ­ views aremarke d by arrowhead s androd - esth e availability ofmonomer s (carotenoids -shaped sideview sb yarrows .

56 LIGHTABSORPTIO NO FPOLLE NTUB EWALL S Measuringmatur epolle no fdifferen tspecie s ANDPOLLE NWALL S three types ofspectra l absorption curves can be distinguiwhed: amaximu mpresen tbetwee n M.T.M.Willems e 400-450n m (Delphinium elatumhyb. ,Digitali s purpurea,Campanul abononiensis ,Phyostegi a Department of Plant Cytology and Morphology, virginiana);a maximu mpresen tbetwee n450 - AU, Wageningen, The Netherlands 550n m (Chrysanthemumsegetum ,Potentill a pyrenaica,Lupinu smirabilis ,Gasteri averru ­ cosa)an dn omaximu mpresen t atal l (Cedrus Summary atlantica,Colchicu m autumnale,Hibiscu ssyria - Iti sfeasibl eusin g amicroscop e todeter ­ cus,Liliu m c.v. Enchantment). mineth e lightabsorptio n of aselecte d small Thenatur e and concentration ofpigment s areao fa pollengrai n orpolle n tube.Ligh t such asdelphidin e and carotenoids probably absorption ofdevelopin g pollen,differen tpol ­ determine absorptionbetwee n400-45 0nm .Flavo ­ lenspecie s and thepolle n tube canb erelate d noids absorb ingenera lbetwee n 200-400nm .Th e toth echange s inwal l thickness andcomposi ­ specific absorption spectrumo feac hpolle ni s tionan d the cytoplasmicvolume . determinedb y their specific cellwal lcompo ­ nents includingpigment s and its cellcontents . Methods 2.Polle n tubean dpolle n tubetip . Thepolle ngrai ni ssuspende d inwate ro n amicroscop e slidean d theabsorptio ndeter ­ Thepolle n tubean d theti po f thepolle n minedo fa know n fixedare ausin ga 40 xobjec ­ tubeo fImpatien swallerian a showdistinc t tive.A 75Wxeno n lampan d anautomaticall y absorption curves.Th e ratio inabsorptio n drivenIvon-Jobi nmonochromato r provides acon ­ from45 0n mbetwee npolle n tube and itsti p tinuous spectrum from400-75 0nm .Transmitte d is about 1:0,75. See fig.2 ,measure d area lightwa smeasure d using aRC Aphotomultiplie r 10x10y ma t 1170V . I0a t50 0n m= 6, 4m V= suppliedwit h aselecte d voltage.Compare d 100%, witha contro lwithou t specimen (= 100%),i t From 400-450n mth epdlle ntub eshow sa waspossibl e todetermin e theoptica l density strong decrease inabsorption .Th e tipha sn o ofa sampl e inmV ,whic hwa s expressed asA% . slope and alowe rpercentag e ofabsorption . Ifno totherwis ementione d thevarianc e inth e The differencebetwee n theti pan d theothe r meanvalu e of3 measurement s is7% . parts of the tube canb e related toth ecompo ­ sitiono f thetub ewall ,th epresenc e ofcal - Results and conclusions losean d the cytoplasm,a t the tip richi n vesicles,i nth e tubea swit h starchgrains . 1.Developin g andmatur epollen .

During thedevelopmen t ofGasteri averrucos a pollen absorption remainsnearl y constantdurin g theearl y stages.Increase s atth ematur e stages aremainl y due tochange s inth ecom ­ positionan d augumentation of theplasma .Ab ­ sorptionfro m 700-750n mi sprimaril y duet o thecytoplasmi c content of thepollen . Absorptionb y pollenwal l inaborte d empty polleni shig h inth eyoun gmicrospor e duet o thecontinuin gwal l formation.Thereafte r the 0_400 50Ö 600 700 nm wallstreche sbecaus e of theincreasin g cell When stainedwit h calcofluorwhite ,mainl y volume. See figure 1,measure d volumewa s forpolysaccharides ,th eintensit y of ameasur ­ 20x20x10ur na t 1070V ; I0 at50 0n m= 4, 5m V ed areao f 10x10y m for theti p is 24 Î 2m V 100%. (n=5)an d for thetub ei s 16! 1m V (n=7)a t Comparable results inLiliu man dPhysios - 450n man d 1000V .Thi s suggests a 1.5xhighe r tegiasuggest s thatespeciall y thethicknes s levelo f cellwal l components inth etip , andcompositio n of thepolle nwal l andth e partlypresen t inth evesicle s inth ecyto ­ volumeo f thecytoplas m influence therat eo f plasm. absorption. When stainedwit hwate rblue ,a stai nmain ­ ly for callose,th eintensit y of ameasure d •——• young GASTERIA sp. areao f 10x10y m for theti p is2 0± 6 m V (n (n •—• middle • • mature =7)an d for thetub e3 0 ± 8m V (n=5)a t50 0 C"V ,—. wallyoung —- waif middle nman d 1170V (Willemse, 1981). Thereexsist s ; V, C---0 wall mature a0. 6 x lower callose concentration inth etip . •-.. \\ -^^® - Preference --:q--- =--.,_..--!;*"' '"•••.. ,-—-.--"""'"""^~NI Willemse,M.T.M. , 1981.In :D.C .Robinso n& ~°-=-=-:va,:; \ - H.Quade r (Eds.). In:Proc . IICel lWal l Meeting.Wiss .Verla g Gesell.MBH ,Stuttgart , p. 242-250. 57 EFFECT OF WATER LOSS ON GERMINATION ABILITY OF MAIZE POLLEN

B. Barnabas

Agricultural Research Institute of the Hungarian Academy of Sciences, Martonvâsâr, Hungary

Summary With regard to the length of the pollen tubes, it was observed that The correlation b etween the water pollen grains containing less than content and viabili ty of maize pol­ 25 per cent water developed len grains was stud led on the basis considerably shorter pollen tubes os the germination ability of pollen during the period 60-120 min after from a single cross hybrid. There pollination than those with higher was found to be a c lose correlation water contents /Fig.1./. between viability o f the grains and their tolerance to desiccation. Key words: maize, p ollen viability, dehydration, pollen treatment. Fig.1 . 12rmax .lenqt ho fpolle ntuba sm m177 1 water content Introduction

Knowlton /1922/ has ascribed the I 40-55 I rapid loss of viability in cereal pollen to the desiccation of the grains. According to Shivanna E 25-40 I Heslop-Harrison /1981 / structural changes take place in the membranes I 10-25 I of the pollen grains during dehyd­ ration, and the permeability of the plasma membranes alters. No records- W < 10 Z appear yet to have been published h with respect to the effect of various, degrees of water loss on the viability of maize pollen. _0S=_ ÉÊL III 15 30 120 mm Materials and methods

Pollen was collected from a single It is reasonable to assume that is cross maize hybrid and was then dried the most tolerant grains which sur­ to various water conte nts'. 15-20 mm vive the experimental treatment pieces of silk were po llinated with /drying/ without loss of their pollen under examinati on and were normal function. incubated for 15, 30, 60 and 120 min at 25 °C in petri dish es. After these periods the numb ers of germinated pollen grai ns were counted References and the maximum length of the pollen tubes was measured. Knowlton, H.E., 1922. Studies in pollen with special reference to Results and discussion longevity. Agric. Exp. Sta. Mem. 52:751-793. • A comparison of the germin ation of pollen grains w ith various'w ater Shivanna, K.R. Heslop-Harrison, J. content showed that grains w ith high 1981 . Membrane state and pollen /40-55 per cent / water conte nt viability. Annals of Botany. adhered to the stigma and ge rminated hi: 759-770. most rapidly an d in greater numbers. Almost 60 min w ere required for ger- mination of the pollen grain s with low /10-25 per cent/ water c ontents.

58 MICROTUBULAR CONFIGURATIONS DURING ENDOSPERM AND POLLEN DEVELOPMENT IN ZEA AND GASTERIA AS VISUALIZED BY IMMUNOFLUORESCENCE

A.A.M. van Lammeren

Department of Plant Cytology and Morphology, Wageningen, The Netherlands

Summary ment the callose cell wall and later on the sporopollenin cell wall impeded IgGs The arrangement of microtubulàr (MT) •to penetrate protoplasts. This problem configurations was studied in differenti­ was initially overcome by enzymic digestion ating cells to elucidate the role of the of the cell walls (Van Lammeren, 1982). cytoskeleton during cytomorphogenesis. The subcellular organization, however, Endosperm from Zea mays and anthers from changes during cell wall digestion because Gasteria verrucosa were fixed with aldehy­ protoplasts round up and protease activity des and embedded in polyethylene glycol may interfere (Wick &Duniec , 1983). (PEG) at various stages of development. To circumvent the problem, Wolosewick Semi-thin sections were mounted on slides and De Mey (1982) embedded animal tissue and processed for immunocytochemistry. in polyethylene glycol (PEG). This paper The specificity of antisera was confirmed sets out to describe a modification of byprope r controlexperiments . their method leading to a successful appli­ In the developing maize endosperm a cation for plant material. Microtubular variety of MT arrangements was detected. arrangements will be shown using this During cell division microtubules were technique in the study of developing maize conspicuously labeled in spindles and endosperm and developing Gasteria pollen. . At interphase, however, they were preferentially oriented along Materials andmethod s the cell membrane. In isodiametric cells the microtubules appeared in a criss-cross Young caryopses of Zea mays L., strain texture while in the elongated cells of A-188 (kindly provided by Dr. CE. Green, the epidermal layer they were highly or­ Minnesota) were dissected in the greenhouse dered into bundles arranged parallel to at various developmental stages. Sagittal each other along the anticlinal cellwalls . sections of endosperm were fixed in a Throughout the process of pollendevelop ­ mixture of 3% depolymerised paraformaldehy­ ment in Gasteria MTs were demonstrated de and 0.25% glutardialdehyde in phosphate in various concentrations and configura­ buffered saline (PBS: 135 mM NaCl; 25 tions. Their location at meiosis and pollen mM KCl; 1.5 mM KHP O; mM »aHPO, , pH mitosis was well established. The shape 7.2) for 2 hours at room-temperâture. of the generative cell proved to be deter­ Anthers from immature flowers of the mined and maintained by bundles of MTs. inflorescence of Gasteria verrucosa (Mill. H. Duval) were dissected and to improve Introduction penetration of the same fixative into the loculi the distal ends of the anthers In recent years immunocytochemistry were cut prior to fixation. The developmen­ has been used frequently to study the tal stages of the cells in the loculi arrangements and functions of microtubules were determined for each flower by Nomarski (MTs) in various (for surveys, optics. see Osborn & Weber, 1982; Lloyd, 1982). After fixation tissues were subsequently Besides functioning during cell division rinsed in PBS for 30 minutes, treated in the formation of the spindle (Mcintosh, with 15 mM NaBH„ and 0.1 M NH„C1, both 1984) their functioning in cell motility 4 4 was stressed (Hyams, 1982). During inter­ in PBS, for 15 minutes each and rinsed phase their main function has proved to again in PBS for 30 minutes. Samples were be the forming of a cytoskeleton estab­ washed in water and processed through lishing and maintaining cell shape, both a graded series of ethanol. Temperature in animal and plant cells (see Brinkley was raised up to 55°C when ethanol 100% e t al., 1980;Marchant , 1982). was replaced by a 1:1 mixture of ethanol Plant tissues, however, often cause : PEG 1500/4000 (w/w PEG 1500 : PEG 4000 specific difficulties during preparation = 2:1). After two hours of infiltration, for immunocytochemistry as was discussed the mixture was replaced by pure PEG 1500/- by Knox et al. (1980). Ensuring access 4000. After one hour samples were transfer­ °f antibody to cellular sites of antigen red into silicone moulds or gelatine cap­ seems to be the main problem. Indeed in sules containing pure PEG 1500/4000 at maize endosperm the cell wall 55°C. This temperature was maintained aPpeared to be a barrier to immunoglobulins during another two hours. Then the heater '!gGs ) and also during pollen develop­ was switched off to allow the PEG to cool down slowly. Semi-thin sections (5 jum) were prepared with a Kulzer Histoknife 59 (Kulzer, Friedrichsdorf) mounted on a sity of their labeling as was concluded LKB Ultrotome V. Serial sections were from data obtained from unembedded FITC- picked up with hanging drops of 40% PEG labeledtissues . 6000 in water to permit wrinkles to disap­ The composition of the mixture of PEG slowly. Then the sections were care­ with various molecular weights was deter­ fully tipped onto poly-L-lysine coated mined experimentally and the combination slides (poly-L-lysine, Sigma, MW >70,000 , used gave the best results during section­ 0.1% w/v in distilled water, pH 8.6) for ing. A microtome was used to obtain repro­ adhesion. Slides were rinsed twice in ducible section thickness which proved PBS to remove the PEG prior to the applica­ tob e of particular importance forquantita ­ tiono fantisera . tive analysis and photography. Semi-thin For immunocytochemistry, the first sections did adhere well at poly-L-lysine antiserum was raised in rabbit against coated slides and the cut single cells tubulin purified from calf brain essential­ in the loculi of the anthers remained ly according to Shelanski et al. (1973). localized when PEG was washed away and The IgG fraction was isolated with column slides were further processed. It should chromatography (DEAE Affi-gel Blue, Bio- be taken into account that direct micro­ rad). Sections were incubated at 37°C scopic observation of the fluorescent with 20 /ul diluted antiserum (1:15 in cytoskeleton revealed far more details PBS) or 20 jUl IgG solution for one hour, than can be shown on prints or slides subsequently rinsed in PBS and exposed because of the small depth of field and to the secondary fluorochrome-labelle d the limited resolving power of the photo- antibody (goat anti-rabbit IgG-FITC, Nor­ graphicalemulsion . dic, Tilburg) diluted 1:40 in PBS for one hour. All steps were at 37°C. Slides Microtu.bu.lar configurations in the endosperm. were rinsed in PBS for 1hou r and sections Approximately nineday safte rpollinatio n(DAP ) were embedded in Mowiol" 4-88 (20% Mowiol several regions could be distinguished in 4-88, Hoechst, Frankfurt am Main, in 0.1 the developing maize endosperm. At the MTris-HCl ,p H8.5) . base of the endosperm, near the , Sections were observed with epifluores- cells are elongated and densely cytoplas­ cence on a Leitz Ortholux microscope with mic. The central region of the endosperm Xenon 150 or HBO 100W high-pressure mercu­ consists of isodiametric cells which often ry lamp illumination. The objectives used' have large central . The outer were Nikon Plan Achromate 50x oil (N.A. region of the endosperm consists of an 0.50-0.85), Leitz Fluor 95x oil (N.A. epidermal layer with small, densely cyto­ 1.10-1.32) or Leitz 100x oil (N.A. 1.25). plasmiccells . Black and white images were recorded Both in the epidermal layer and in on Agfapan 400. Color slides were made the central region of the young endosperm, on Kodac Ektachrome 400 and processed cell divisions were detected at low fre­ commercially. quency. Mitotic spindles were observed and during cytokinesis various stages Results of development were seen. Irt interphase cells of the central region Technical aspects. Both the fixation and PEG MTs were detected preferentially surround­ embedding proceduresappeare dt o be appropri­ ing the nucleus and running criss-cross ate to detect MTs at various stages of along the cellmembran e (Fig.1a) . the cell cycle. This not only holds for When the was about 15 DAP the endosperm and the developing pollen cells of the central region of the endo­ tested but for the embryo and carpels sperm contained only few vacuoles but of maize and for tips of maize, numerous plastids. Microtubules were still and radish as well (Van Lammeren, unpub­ found running along the cell membrane lished). With respect to immunolabeling but also throughout the central part of both the first antiserum and the IgG frac­ the cytoplasm. Interphase cells of the tion gave excellent visualization of MTs. epidermis had a strikingly different confi­ However, serum dilutions less than 1:25 guration. Here, MTs were highly ordered coincided with increasing aspecific la­ into bundles which ran along the anticlinal beling of the background. Two control cell walls parallel to the surface of experiments in which either the first the endosperm (Fig. 1b). In the nucellus antiserum was omitted or pre-immune serum tissue also two cell types were observed. was applied resulted in but a faint as­ The isodiametric cells showed a cortical pecific labeling of the cytoplasm while mesh-work of MTs. The epidermal cells no MTs were to be detected. The first exhibited bundles of MTs as can be seen antiserum was stored for more than 2 years inFig .1c . at -60°C without an apparent loss of bind­ ing capacity. The high temperature of Microtubular configurations during pollen devel­ 55°C during PEG embedding influenced nei­ opment. At the pre-meiotic stage of pollen ther the distribution of MTs nor the inten­ development, cells of the sporogenous tissue

60 and young microspore mother cells ofGaste - ria are polyhedral and about isodiame- tric. The large nucleus occupies the cen­ tral region and only few small vacuoles appear in the cytoplasm. Starting at this stage MTs were detected in various, but stage specific configurations. As is shown in Fig. 2a, microtubules in microspore mother cells are arranged in a criss-cross texture throughout the whole cytoplasm. At meiosis MTs were visualized in spindle and phragmoplast configurations (Figs. 2ban d c). Cell wall synthesis of pollen starts from early tetrad stage; when microspores are set free in the loculus they have a bilateral symmetry. Then microspores are elongated and the nucleus is situated near the cell wall opposite the colpus (Fig. 2d). Strands of MTs preferentially appear in between the nucleus and the colpus. Cytoplasmic MTs disappear at pollen mitosis. The position of the fluorescent spindle and phragmoplast indicate the asymmetric plane of division (Fig. 2e). The newly formed generative cell is firstly Fig. 1. Microtubular configurations in attached to the intine,bu t as it differen­ Zeamays . / tiates, it becomes completely surrounded a.Endosper m cells at the central region by the cytoplasm of the vegetative cell at interphase. Note the criss-cross and subsequently elongates. Eventually textureo f fluorescentMTs . the spindle-like cell merely consists b.Epiderma l endosperm cells with cortical of a nucleus surrounded by a thin layer bundles of MTs (arrows) at interphase. of cytoplasm, which is enveloped by the Epidermal cells of the nucellus (Nc) plasma membrane. In the cytoplasm of the with MTs along the anticlinal cell.- vegetative cell only a diffuse fluorescence walls. The MTs are oriented parallel was observed and MTs were rarely seen. to each other and perpendicular to However, in the generative cell striking thesurfac eo f theepidermis . fluorescent cables running from pole to pole indicate a highly ordered arrangement The cytoplasmic MTs of the interphase ofMT s (Fig. 2f). cells of maize endosperm appear to be organized in two different configurations, Discussion depending on the location of the cells. In the small epidermal cells they are Sectioning plant tissues embedded in mainly arranged into bundles which align a mixture of polyethylene glycol proved the anticlinal cell wallswherea s inisodia - to be useful in overcoming the problem metric cells a cortical criss-cross texture of the walls forming a barrier of MTs appears to be most frequent. These to igGs in their penetration of the proto­ findings are in agreement with the now plasm. The antigenicity of tubulin struc­ widely accepted view that the asymmetric tures appeared not to be altered compared shape of plant cells is largely the result to hand made sections. Moreover it was of the orientation in which cellulose quite easy to study serial sections. Ini­ microfibrils are deposited within their tial experiments directed towards the cell walls. During the establishment of addition of PEG with a lower molecular the mature shape and size of cells, MTs weight which strongly improved the section appear to influence the pattern of cellu­ quality. In pure PEG 4000 as used by Wolo- lose deposition and thus MTs control indi­ sewick and De Mey (1982) sections were rectly the developing shape (Marchant, too brittle. The use of the solidifying 1982; Traas et al., 1984). Spindle and Polyvinylalcohol Mowiol 4-88 (see Osborn phragmoplast MTs are not likely to interfe­ & Weber, 1982) enabled us to manipulate re with the ultimate cell shape. The plane the slides without risk of damaging the of division, however, is determined at sections. mitosis. The position of the cell plate It is beyond the scope of this paper of epidermal endosperm cells ispredominant ­ to describe and interpret the various ly perpendicular to the outer surface, MT configurations extensively. However, while the orientation is random in the some striking features will be discussed. centralregion .

61 proposed that the MTs play part inorienta ­ tion of the nucleus and in transport of materials to the cell surface. In our observations your microspores of Gasteria have a length axis while they are still encapsulated in the callose wall and the MTs that run throughout the cytoplasm sometimes have a pole-to-pole orientation. The same pole-to-pole orientation is most striking after pollen mitosis in the spin­ dle-shaped generative cell in which several bundles of MTs appear. The morphogenetic function of these MT bundles was already stressed by Sanger and Jackson (1971) who reported that the elongation of genera­ tive cells of Haemanthus katherinae is accompanied by the development of a peri­ pheral array of MTs which are oriented parallel to the long axis of the cell. When they destroyed MTs generative cells partiallyreverte d to their former spheroi­ dal shape. Elongated generative cells with bundles of MTs were shown in avariet y of plant species (Cresti et al., 1984) and indeed for Gasteria their presence was also recorded by electron microscopy (Keijzer, this volume). In free microspores which have a colpus and a pollen wall, MTs were not equally distributed throughout the cytoplasm but merely restricted to the colpus region (Fig. 2d).Thi s probably indicates the conservation of the cell shapea tth ecolpu s zoneb y theMTs . The data obtained indicate a coincidence of a proper MT configuration with a certain Fig. 2. Microtubular configurations in cell shape. We suggest that MTs influence Gasteria verrucosa. the cell shape as follows: MTs in a criss­ a.Microspor e mother cells with fluorescent cross texture only permit cells to differ­ MTs in a criss-cross texture throughout entiate isodiametrically while MTs in the cytoplasm. parallel arrays direct towards longitudinal b.Diad e stage with MTs radiating from cell growth or shape. In all it can be the nuclei and forming the phragmopiaat'. stated that the PEG embedding procedure c.Tétrad e stage. Note MTs radiating from as t modified by us was suited to study the nuclei towards newly formed cell MT configurations and their function in walls. the endosperm cells ofmaiz e and indevelop ­ d.Microspore s showing MTs preferentially ing pollen of Gasteria. More experimental in between the nucleus (N) and the conditions interfering MT arrangements colpus(C) . willb euse d infurthe rstudies . e.Firs t pollen mitosis at metaphase with spindle MTs indicating the asymmetric Acknowledgements plane of celldivisio n (arrows). f.Polle n grains with generative cells The author thanks Miss W.M.J. Poelma (G) in transverse section. Bundles and H. Kieft for technical assistance of cytoplasmic MTs surround the unstain­ and Prof. M.T.M. Wiilemse and Dr. J.H.N. ed nucleus of the generative cell. Schelfo rreadin g themanuscript .

In Gasteria only the isodiametric micro­ References spore mother cells exhibited the criss­ cross texture throughout the cytoplasm. Brinkley, B.R., S.H. Fistel, J.M. Marcum All other cell types showed altered MT & R.L. Pardue, 1980. Microtubules in configurations. cultured cells; Indirect immunofluores- In the diade (Fig. 2b) and tetrad (Fig. cent staining with tubulin antibody. 2c) stage MTs were observed extending Int.Rev .Cyt .63 :59-95 . from the nuclear envelope to the plasma Cresti, M., F. Ciampolini & R.N. Kapil, membrane as was also reported recently 1984. Generative cells of some angio- for very young microspores in Lilium by sperms with particular emphasis on Dickinson and Sheldon (1984). These authors theirmicrotubules . J. Submicrosc. Cytol. 16: 317-326. 62 Dickinson, H.G. & J.M. Sheldon, 1984. A radial system of microtubules extend­ ing between the nuclear envelope and the plasma membrane during early male haplophase inflowerin g plants. Planta 161:86-90 . Hyams, J.S., 1982. Microtubules. In: C.W. Lloyd (Ed.): The cytoskeleton in plant growth and development. Academic Press, London,p .31-53 . Knox, R.B., H.I.M.V. Vithanage & B.J. Howlett, 1980. Botanical immunocytoche- mistry: a review with special reference topolle n antigens andallergens . Histochem.J . 12:247-272 . Lloyd, C.W., 1982. The cytoskeleton in plant growth and development. Academic Press,London . Marchant, H.J., 1982. The establishment and maintenance of plant cell shape by microtubules. In: C.W. Lloyd (Ed.): The cytoskeleton in plant growth and development. Academic Press, London, p.295-321 . Mcintosh, J.R., 1984. Mechanisms of mito­ sis. Trends Bioch. Sei. 9: 195-198. Osborn, M. & K. Weber, 1982. Immunofluores­ cence and immunocytochemical procedures with affinity purified antibodies: Tubulin-containing structures. In: L. Wilson (Ed.): Methods in cell biology vol. 2, part A. Academic Press,London , p.97-132 . Sanger, J.M. & W.T. Jackson, 1971. Fine structure study of pollen development in Haemanthus katherinae Baker. II. Microtubules and elongation of the generativecells . J. CellSei . 8:303-315 . Shelanski, M.L., F. Gaskin & C.R. Cantor, 1973. Microtubule assembly in the absen­ ceo fadde dnucleotides . Proc. Nat. Acad. Sei. 70: 765-768. Traas, J.A., P. Braat & J.W. Derksen, 1984. Changes in microtubule arrays during the differentiation of cortical rootcell so fRaphanu ssativus . Eur. J. Cell Biol.34 :229-238 . Van Lammeren, A.A.M., 1982. Detection of cytoplasmic and spindle microtubules in plant cells by indirect immunofluores­ cence. Proc. 23rd Dutch Fed. Meeting, Amsterdam,p .256 . Wick,S.M . &J . Duniec, 1983.Immunofluores ­ cence microscopy of tubulin and microtu­ bule arrays in plant cells. I. Prepro- phase band development and concomitant appearance of nuclear envelope-associat­ edtubulin . J. Cell Biol.97 :235-243 . Wolosewick, J. & J. De Mey, 1982.Localiza ­ tion of tubulin and actin inpolyethyle ­ ne glycol embedded seminiferous epithe­ lium. Biol.Cel l 44:85-88 . GROWTHO FTOBACC OPOLLE NTUBE SI NVITRO ;EFFECT SO FDRUG SINTERFERIN GWIT HTH ECYTOSKELETO N

JanDerkse nan dJa nA .Traa s

Department ofBotany ,KUN ,Toernooiveld ,652 5E D Nijmegen,Th eNetherland s

Abstract Materialan dmethod s

Thepresenc eo fth e drugs colchicine,deu ­ Culture conditions teriumoxide ,cytochalasi n Ban dphalloïdi n inhibitedgerminatio n andtub e growtho f Pollen fromNicotian atabacu m (L)C VSam - Nicotianatabacur npolle n invitro .Th epol ­ sunwer e collectedan dstore d asdescribe d lentube s didno tloos ethei rpolarit yi n byKro han dKnuima n (1982). thepresenc eo fth edrugs .Th eeffec t onth e Pollen (10mg )wer e germinated andgrow n microtubule skeletonwa s examinedusin gim ­ insucrose/borat emediu m (2ml )fo rsevera l munofluorescence.I nth epresenc eo fcolchi ­ hoursunde r standardization conditions (Kroh cinen oo ralmos tn omicrotubul e skeletons andKnuiman , 1982).Sample swer etake na t couldb eobserved -I fdeuteriu m oxide,phal ­ 2an d3 hour safte r starto fth eincubation . loïdino rcytochalasi nB wer epresen t inth e Representative area'swer ephotographed . culturemedium ,microtubul e skeletons,thoug h Germination andtub elengt hwer edetermine d distorted,remaine dpresen t frommicrograph swit h suitablemagnification ; Germinationwa s scored atleas t 1000polle n Introduction grains.Polle ntub elengt hwa s determined froma tleas t 200germinate d pollenusin ga Pollentube sar eabl et odirec tthei r Kontronvideopla n computer.Drug swer eadde d growth,probabl y guidedb yenvironmenta lfac ­ toth emediu mi nvariou s concentrations tors. ^priort oth e incubation.Al lexperiment swer e Theyar egrowin ga tthei rti ponl yan dthu s carriedou ti nduplo . showa distinc tpolarit y (Sieversan dSchnepf , 1981).Therefor epolle ntube smus thav ea t Immunofluorescence their disposal agrowt h directing systemtha t isprobabl y located ino rnea rth etip .Th e Immunofluorescencewa s carriedou tprincip ­ main structuralelemen to fplan t cells,th e allya sdescribe db yWic k etal .(1981 )wit h cellwall ,canno tb e considered sucha syste m somemodification s (Derksene t al.manus ­ sinceth ewal l isa passiv e elementonly . cript inprep.) . Theprimar yantibod ywa sa Thepre-eminen t morphogenetic elementi n ratmonoclona lanti-tubuli n (Mas077 ;Ser a plant cells isprobably ,lik e inanima lcells , labs),th e second antibodywa sa rabbi tFit c the cytoskeleton,a coheren tnetwor ko f labelled anti-rat Igg (Nordic BV).Preprara - various elements,mainl ymicrotubule san d tionswer eexamine d ina Leit zorthopla n microfilaments (forreview s seea.o. :Lloyd * microscope equipped forimmunofluorescence , I98I+eCh dRobards ,1983) .Th epresenc e'o f usinga Leit z 100xwate r immersionlens . these elements inpolle ntube sha sbee nshow n Photographswer emad ewit h aLeit zvario - already in 1972b yFrank e andcoworkers . orthom*combinatio n oneithe rFujichrom e*t0 0 Microfilaments (Pictonan dSteer ,1982 )and , orAgfapa n ^00professiona lfilm . duet othei rpresumptiv e role incel lwal l deposition,especiall ymicrotubules(Lloyd , Results 198U)hav ebee n supposedt ob e involvedi n plant cellmorphogenesis .Variou sdrug sar e Germination knownt o interferewit hth ecytoskeleton . Weuse dphalloïdi nan d cytochalasinB , both Inpreliminar y experimentsth e curvesre ­ knownt o interactwit hmicrofilament s andt o presenting germinationwer e sigmoidwit ha inhibitplasm astreamin g andcolchicin ean d linearrang ebetwee n 1an d 3hour safte r deuterium oxide (DO)respectivel y depolymeri- starto fth eincubation .Thoug h experiments zingan d stabilizingmicrotubule s (reviews: carried out indupl o showed almost identical Robards,1983 ,Sat oe t al., 1982).Her ew e results,large r differences occuredbetwee n presentno tonl yth eeffect so ngerminatio n independent experiments. Therefore,experi ­ andpolle n-tub egrowt ho fthes e drugs,bu t mentswer eno tpooled .Th e effect ofvariou s alsothei reffec to nmicrotubul e skeletons concentrations ofcolchicine ,DO , cytocha­ using immunofluorescence (Wicke t al., 198l), lasinB an dphalloïdi n ofa representativ e recently adaptedt opolle ntube s (Derksene t experiment isshow n infig .1 . al.manuscrip t inprep.) .

64 pigure 1.Inhibitio no fgerminatio nb ycolchichine ,DO , cytoehalasin Ban dphalloxdin . explanation:se etext .

Germination (%) 100 L COLCHICINE B20 i •—• 2 hrs. .—• 2 hrs. x x 3 hrs. x x 3 hrs.

\

3 10"' 5x10-' 10" M l 20 50 % PHALL0ÏDIH ^-x \ .—. 2 hrs. x x 3 hrs. \ \

h- \ -•

3 0,006 0,01 OJBjjg/ml 10"' 2x10"' 1Û" M Concentration

Figure2 .Inhibitio no fpolle ntub e growthb ycolchicine ,DpO ,cytoehalasi nB an dphalloîdin . explanation:se etext .

Length (p x 200)

0,0050,0 1 0,05pg/ml 10'' 2x10"' 10"J M Concentration

65 Theeffec t ofcolchicin e showna ta concen ­ Figure 3.Immunofluorescenc e ofmicrotubule ; tration of 10"-'Mi shardl y stronger atin ­ creasing concentrations.D O stronglyinhibi ­ tedgerminatio n andalmos t completeinhibi ­ tion isreache da tconcentration s somewhat highertha n 50%.Bot hphalloîdi n andcytocha - lasina tlo wconcentration s didno t inhibit germinationan deve nshowe d slightstimulation . However,thi s effectwa sno t consistent and probablyma yno tb e significant.Highe rcon ­ centrations ofthes e drugswer e clearly inhi­ biting.I ncontro lexperiment sth egermina ­ tionpercentag ewa s almost 100%afte r 3hours . Ifa ninhibitor y effect ofa dru gwa srecor ­ dedals oth emaximu m germination percentage becamelowe rtha n 100%.Th emaximu mgermina ­ tionpercentag ewa s stillreache dthe nafte r about 3hours .

Pollentub egrowt h

Inrepresentin g ourresult sw echose dt o useth emea nvalu eo fth epolle ntub elength s asa narbitrar ymeasure .Al ldrug sclearl y inhibitedpolle ntub e growth (fig.2) . Inhibitiono fpolle ntub e growthb y colchicine andD O showed asimila r course asth einhi ­ bitiono fgerminatio n (compare figs 1an d2) . The effectso fpolle ntub e growth ofeyto - chalasinan dphalloîdi noccurre d atlowe r concentrations thanth eeffect so npolle n germination (compare figs 1an d 2). Theeffec t ofth edrug so ntub e growth seemt ob emor e pronounced thano ngermination .

Effectso fdrug so nmicrotubul eskeleton .

Inimmunofluoresenc epreparation sparalla l strandso fmicrotubule swer evisibl ethrough ­ outth e cellwit h mainly axialorientation s (fig. 3Aan dtabl e 1J ,bu t alsohelice swit h arigh t turn (Shelix )occurred . • Thepresenc eo fth edrug s causedmarke ddiffe ­ rences inmicrotubul e organization (fig.3 ) andar edescribe dbelo wfo rth edifferen t drugs.Th econcentration s usedwere : 10 M colchicine,50 %DO , 10 Mphalloîdi nan d Fig.3 .Immunofluorescenc e ofmicrotubules . 0.002ugr/m lcytochalasi nB . A:untreated ;B :colchicine ,C :D„0 ,D :pha l Inth epresenc eo fcolchicin e pollentube s loîdin,an dE :cytochalasi ntreated .Pointe r generally didno t shownumber so fparelle l microtubule's.Al lmagnifications :300 0x . strandso fmicrotubule s anymore.Onl y fewin ­ dividual strands,mainl y axially oriented Themai norientation s remained axialbu tmo r werepresen t (fig. 3B),bu t occasionally andsteepe rhelice soccurre d (seetabl e1) . arrayso fmicrotubule s couldb eobserved .Th e Near theti po fth etub e feweran dles s tubes showed ahig hbackground .Th estrand s strictly orientedmicrotubule s strandswer e thatremaine dwer eles s strictlyoriente dan d visible.Organizatio n ofcytoplas m andplas m less straighttha n inth econtrol s (compare streamingwer edisturbed .Th ediamete ro ft h figs 1Aan d B). Thediameter s ofth epolle n tubeswa s irregular,mostl y largertha ni n tubeswer e irregular andlarge rtha n inth e thecontrol san dendknob soccurre dregularl y controlcells ,sometime s akno b atth eti po f Often,abou t 50%,th epolle ntube swer e thetube.occurred .A sjudge dfro mphas econ ­ twisted ina righ ttur n (Shelix ;se efig s trastmicroscopy ,th eorganizatio n ofth e 3Aan dC) . plasmaan dth eplasm astreamin gwa sdisturbed . Phalloîdin didno t clearly effectth enum ­ DO cuased changes inth emicrotubul eskele ­ bero fmicrotubul e strands,bu tthe yofte n ton:mor ean dshorte rstrand s thatwer eles s appearedt ooccu r inbundles .Th estrand swe : strict oriented andles s straight thani nth e lessstrictl y oriented,especiall ynea rth e control cells (figs 1Aan dC an dtabl e1) . tip,bu tthe yremaine d straight (fig.3D) .

66 Crossing strands were observed (fig. 3D). colchicinetreate dpolle ntube si scause db y More and steeper helices occurred (see: numerous small,non-cortica lmicrotubule stha t table 1). Organization of the cytoplasm and persist inth e cell.I nvariou stype so f plasma streaming seemed to be normal. cellsgrowin g atthei rti pcolchicin e causes The effects of cytochalasin were similar to losso fpolarit y (fordiscussio n seea.o. : those of phalloîdin but much more pronounced Mizukamian dWada , 1983).However ,fo rpolle n (fig 3E and table 1). Clear wavelike patterns tubesi twa sconclude d (Frankee tal. , 1972) with many crossing strands (fig. 3E) were thatmicrotubule swoul dpla yonl ya mino r seen. Also bundles of non wavelike strands rolei nmorphogenesis .I nou rmateria ln o and spirals were observed. The density of the losso fpolarit ywa s observed. microtubule strands is obviously higher than Increasedmicrotubul e densities aftercyto ­ in the control cells. Near the tip the chalasintreatment ,a sobserve dher ehav e microtubule skeleton is less well organized alsobee nreporte d forroo thair s (Seagull and sometimes the helices in the tip are andHeath ,198o )an dassociation sbetwee nmi ­ steeper than in the rest of the cell. crotubules andmicrofilament sar eregularl y Occasionally, pollen tubes appeared to be observed (seeals o forrefs. :Traa s et al., twisted in a S.helix. No obvious disturbances manuscript submitted).Suc hobservation sin ­ in the organization of the cytoplasm could be dicate anarro wrelationshi pbetwee nth emi - observed out plasma streaming seemed to be crotubular andmicrofilamenta lsystem .Thu s irregular. itmigh twel lb etha tbot h systemsac tto ­ None of the drugs caused great changes in the gether,on e systemtakin gove rwhe nth eothe r microtubule strands of the generative cell. system fails.Thu scell smigh t stillb eabl e However, colchicine, but especially D_0 and tomaintai npolarit ywhe non eo fth e systems cytochalasin B caused a somewhat wavelike isnon-operational .A spropose d recentlyb y pattern in the microtubule skeleton of the Lloyd (198I*)th eoccurrenc e ofsteepe rheli ­ generative cells. cesafte rinhibitio nma yb erelate dt oa lowe r growthrate . Table 1. Main direction of the microtubules, Literature range mode mean N Franke,W.W. ,W .Herth ,W.J .va nde rWoud e control 0°-l+5° 0°{60%) h° 95(-0 andD.J . Morré,1972 . ^o U ,o D20 0-85 0 (20^) 2-T 118(-17) Planta 105:317-3^1 . Kroh,M .an dB .Knuiman ,1982 . cytochalasin 0°-70° 0°(32#) 1U° 121(-16) Planta 151*:2*11-250 . phalloîdin 0°-T5° 0°(23$) 12° 129(—11 ) Lloyd,C.W. , 1898!* Int. Rev.Cytol .86 :1-51 . Table 1.Th eangl eo fmicrotubul e skeletons Mizukami,M . andS .Wada ,1983 . withth ecel laxi smeasure d atinterval so f Protoplasma 11It :151-162 . 5 .Betwee nparenthesi s incolum n2 :percen ­ Picton,J.L . andM.W .Steer ,1982 . tageo fmicrotubule•skeleto nwit h anangl e J.Theor .Biol .98 :15-20 . withinth emode .N :numbe ro fcell smeasured ; Robards,A.W. ,1983 . numbero fcell s fromwhic h nomai norienta ­ In: H.Ellenber g (ed):Progres s inBotany , tioncoul db edetermine d inparenthesis . vol. 1*5:1-17 .Springer-Verlag ,Berlin , Heidelberg. Discussion Sato,H. ,T .Kato ,T.C .Takahash i andT .Ito , 1982. Alldrug s showed significant effectso nger ­ In: H.Sika ian dH .Mohr i (eds):Biologica l mination,growt h andmicrotubul e organization functionso fmicrotubule s andrelate dfunc ­ ofpollen .Cell s growing inth epresenc eo f tions.Acad .Press ,Ne wYork .p .211-226 . phalloîdin orcytochalasi n stillshowe d Seagull,R.W . andJ.B .Heath ,1980 . plasmastreaming ,indicatin g aresistanc e of Protoplasma 103:205-229 . themicrofilament s ora failur eo fth ecel l Sievers,A .an dE .Schnepf ,1981 . totak eu pthes e drugs.Th e inhibitory effect In: 0.Kiermaye r (ed):Cytomorphogenesi si n ofD O isstrikin g andi sprobabl y relatedt o plants.Springe r Verlag,Wien ,Ne wYork . its*freezing 'effect .However ,th edistur ­ Wick,S.M. , R.W. Seagull,M .Osborn ,K .Webe r banceo fgrowt han dplasm aorganizatio n indi­ andB.E.S .Genning ,1981 . catesdifferen t effectso nmorphogenesi s J. CellBiol .89 :685-690 . leavingthei r interpretation uncertain (seefo rdiscussion :Sat oe t al., 1982). Acknowledgement Thoughcolchicin e inhibited germinationan d growth,stil lnumerou s pollen couldgro wi n Wethan kDr sM.M.A .Sasse nan dM .Kro hfo r itspresence .Thes epolle ntube shardl yshow ­ various support.W e alsothan kJ .Broekman s edan ycoheren tmicrotubul e skeletons,ail - fortypin gth emanuscript . thoughmicrotubule s remained alwayspresent , layb eprotecte db yMAPs .I t cannotb eex ­ cludedtha tth e fluorescencebackgroun di n

67 FINESTRUCTURA LRESEARC HO NGERMINATIN G LILYPOLLE NTUBE SI NVITR OAN D INVIV O

H.MIKI-HIROSIGE*,S.NAKAMURA*an dY.YAMADA* * »BiologicalLab..Kanagaw aDenta lCollege,Yokosuka,**Biol.Lab..Gunrn aUniversity ,Maebashi , Japan

Summary For the experiments in vitro, 5 mg of In vitro, the percentage of germination pollengrain swer eshake na t28 Cfo r3 hi n and tube length of the pollen grains in a small Erlenmeyer's flask with 1 ml of Lilium longiflorum are best when thegrain s the medium, which was composed of dis­ are cultured in a medium containing boron. tilledwater ,10 %sucrose ,10 %sucros ean d Thethicknes s ofth etub ewal lo fth epolle n 0.03% Ca(N0 ), 10 %sucros e and 0.001%H grainsculture d ina sucros emediu mcontain ­ B0 , 0.03% Ca(N0 ) and 0.001% HBO in ing calcium is the greatest, and they are 10% sucrose, Dickinson's medium(ï968) thinest in a medium containing boron. The contained 0.03%Ca(N 0 ), 0.001 %H B 0 and pollen tube walls in compatible and incom­ 0.01%KN0 in 0.322Mpentaerythritol , res­ patible styles are thin, while those in pectively.I naddition ,polle ngrain swer e incompatible stylar exudate are very thick. germinated in the compatible and incom­ Structural differences are observed on the patible exudates. Calcium ion(Picton & surface of the stylar canal cells between Steer,1983) and boric acid(Stanley & compatible and incompatible styles. The Lichtenberg,1963; Vasil,1964) concent­ cytoplasm of the pollen tubes in the rations are optimum. The elongated pollen incompatible style at 96h after pollination tubeswer ecollecte d from thecana lo fth e degenerates. compatible or incompatible styles 48,72 , 96 hours after pollination, and the Keywords: pollen tube, compatible, incom­ germinated pollengrain sculture d invitr o patible,boron ,calcium . ^ in the media described above, werefixe d with 3% glutaraldehyde in cacodylate Introduction buffer(pH 7.0) for 3h, postfixed with 1% osmium tetroxide for 3h,dehydrate d ina n Pollen germination and pollen tube elon­ ethanol series at 4 C, embedded in low gationar egreatl y stimulated bycalciu mand . viscosity epoxy resin, sectioned with a boron contained in the culture mediaused . diamond knife on an LKB-Ultratome and The presence of boron has been reported to observed with a JEM-100B TEM. Uranyl help the translocation of (Gauch & acetate and Raynolds' lead citrate(1963) Dugger,1953) , and play a definite role in were used for staining. For scanning pectic synthesis in germinating pollen- electronmicroscopy ,pollinate d compatible (Stanley & Loewus,„1964) . In addition,th e and incompatible styleswer efixe dwit h3 % requirement for Ca ions during membrane glutaraldehyde for 3h, rinsed and post- fusion and the release of the secretory fixed with 1% osmium tetroxide for 3h, product is well established(Morris>.' & dehydrated with alcohol, critical-point Northcote,1977 ;Kote re tal. ,1978 ;Baydou n *dried , coated with carbon and gold and & Northcote,1980). examined ina JEM-1 5SE Ma t15kV . We report here the fine structural di­ fferences of the germinated pollen tubesi n Lilium longiflorum, which have different Resultsan d discussion physiological conditions depending on the presenceo rabsenc eo fsucros ei nth emediu m Thepolle ngrain so fLiliu m longiflorum and depending on the presence orabsenc eo f cultivated inth emedi awhic har edescribe d calcium and/or boron. In the incompatible inTabl e1 ,absor bwate rfro mth ecultur e lily style, pollen tube elongation ceases medium andgerminate .Th epercentag e ofger ­ aftergrowt hha soccure d forhal fth elengt h mination,polle n tubelengt han dth ethick ­ of the style(Ascher & Peloquin,1966). A nesso fth etub ewal lo fgerminate d pollen comparison of the fine structure ofpolle n grainsi neac hcultur emediu m areshow ni n tubes and conductive tissues in the style Table1 . between compatible and incompatible condi­ The percentage of germination in the tions was also made by thetransmissio nan d sucrose medium and in sucrose medium plus scanningelectro nmicroscopes . calcium and/or boron, is higher than that For materials, pollen grains and pistils obtained in Dickinson's medium. The tube ofLiliu m longiflorum Georgiawer eused .Th e length in the sucrose medium plusboron ,i s compatible pollen grains were obtained from longer than that in the medium lacking another variety, and they were tested for boron, and is similar to thati nth eexuda ­ their fertilitly on the flowers of Lilium te. In general, the percentage of ger­ longiflorum Georgia. For the test ofincom ­ mination and tube length of the pollen patibility, a pistil, pollen grains and grains in the medium containning boron are exudatewer eobtaine d from thesam eflower .

68 thebes ti nvitr oconditions . Iti ssuppose d thatth etissue so fth estyl e All of the pollen tube walls showed in offer protection to the developing tube so Table1 hav edoubl e layers.Th ethicknes so f that thick tube walls are notforme d andi n the pollen tube wall in the sucrose plus addition ahig hrat eo felongatio nmitigate s calcium medium is the greatest among those againstthickness . inth eartificia lmedia . Onth eothe rhand , Ultrastructural observation of thepolle n the tube wall is thin in the mediaconta ­ grain cultured in 10%sucros e medium, show ining boron. It was suggested that calcium thatth eGolg ibodie sproduc enumerou sGolg i combined with pectin substances made cell vesicles, the number of amyloplasts in­ wallcomponent s(It o& Fujiwara,1968)- ,promo ­ creases, and starch grains in amyloplasts ted pollen tube growth by stimulatingvesi ­ and lipid bodies are converted to poly­ clefusio n at the tip(Herth,1978), andtha t saccharide particles(p-particles,Mikl- boron helped the translocation of sugaran d Hirosige & Nakamura,1982) which sometimes pectic synthesis. From our results, it is enclose fibrous substances. The situations assumed that calcium promotes the vesicle of the cell organelles in the pollen tubes fusiona tth eti pmor etha nboron . cultured in the used media except water In stigmatic exudates, germination is medium,ar ealmos tth esame .Th eoute rlaye r better in the incompatible stylarmaterial , of the tube wall is formed at the pollen and growth is better in the compatible tube tip region by Golgi vesicles,an d the material.Whil e the thickness of thepolle n inner layer is formed at the lateral tube tubewal l in the compatible stylarexudate , wallregio nfro mp-particles(Miki-Hirosig e & is not great, and resembles the results Nakamura,1982). Although uniform large obtained in 10 % sucrose medium, the tube vesicles(ca.400 nm in diameter) which make wall is very thick in the incompatible thepolle ntub ewall ,ca nb esee ni nth eti p stylarexudate .I ti ssuppose d thatth ewal l region of the pollen tubes cultured in formation is affected by some aspecto fth e distilled water, small vesicles(ca. 150 nm incompatiblity. There were no ultrastructu­ indiameter )derive d fromth eGolg ibod yan d ral difference among the cell organellesi n large vesicles(ca. 500-1000n m in diameter) thepolle ntube si neithe rth ecompatibl eo r which are made by the fusion of small incompatible exudates,excep tfo rth ethick ­ vesicles(thesevesicle smak eth epolle ntub e ness of the tube wall. On the otherhand , wall) , are seen in tubesculture d inothe r the thickness of the tube wall in the media. The number, size and distributiono f style(in vivo) is very thin; the thickness vesicles in the pollen tubes in the medium ofth ewall si ncompatibl e stylesi ssimila r containning calcium and those in themediu m to the results obtained in the medium con­ containning boron,appeare d to be thesame . taining boron, and the thickness of the Amyloplasts and lipid bodies which are the walls in incompatible styles is similar to reserve substances in the pollen grain,ar e the results obtained in Dickinson's medium. scarce in the pollen tube cultured inwate r

Table1 .Percentag e ofpolle ngermination ,tub elength ,an dtub ewal lthicknes so fth e pollengrai no fLiliu m longiflorumi nartificia l culturemedia ,exudate san dstyles .

%o f Tube Tubewal l CULTUREMEDIU M germination length(mm)thickness(nm ) Water 33.4-41.3 1.23+0.42 550

10%sucros e 76.7-81.7 7.79+2.43 180

10%sucros e 0.03%Ca(N 0) 65.7-71.6 8.48+1.64 600

10%sucros e 0.03%Ca(N 0 ) 0.001%HgBO g 71.8-77.6 12.04+1.98 150

10%sucros e 0.001%H B 0 82.9-87.8 19.04+2.04 110

0.322M 0.03%Ca(N 0 , 0.001%H B 0 0.01%KN 0 49.4-57.9 8.38+1.97 190 3.2 ..,_..._.,à^j d pentaerythrito l(Dickinso n's mediu m,1968 '

E Compatible 87.1-91.2 17.06+2.13 170 XD UA T Incompatible 93.8-97.1 12.76+1.80 880 E afterpollinatio n 48h 96h In compatiblestyl e 110 120 In incompatible style 120 250

69 and Dickinson's medium, but they arenume ­ 32:433-440. rous in those cultured in media containing Cresti,M.,F.Ciampolini& G.Sarfatti ,1980 . sucrose. In the pollen tube cultured in Ultrastructural investigationso n water, very thick tube wall, numerousroug h Lycopersicumperuvianu m pollenactivatio n ERs(rERs)an d fewGolg ibodie sca nb eseen . andpolle n tubeorganizatio n aferself - In the pollen tubes which develop inth e andcross-pollination .Planta,150:211-217 . compatible style,numerou svesicle s andrER s Dickinson,D.B.,1968 .Rapi d starch are seen. Cresti et al(1979)mad e thesam e synthesisassociate dwit hincrease d observation in Prunus avium. In ourexperi ­ respiration ingerminatin glil y ment,i ti sobserve d thatth enumber so fth e pollen. PlantPhysiol .43:1-8 . vesicleswhic hcontai nfibrou smaterial san d Dickinson,H.G.& J.Lawson , 1975.Polle n form thepolle ntub ewall ,an drER sar emuc h tubegrowt h inth estigm ao fOenother a greater in the pollen tubes grown in the organensisfollowin g compatiblean d compatible style than those found in tubes incompatible intraspecificpollinations . grown in the artificial media andexudates . Proc.R.Soc.Lond.B.188:327-344 . Many rERs are associated with manyvesicle s Gauch.H.G.& W.M.Dugger,J R 1953.Th erol e in the in vivo situation, but it is not ofboro ni nth etranslocatio n ofsucrose . clear that the rERs are makingth evesicle s PlantPhysiol .28:457-466 . or secreting something into them. The Herth.W.1978 .Ionophor eA 2318 7stop s cytoplasmic fine structure of the pollen tipgrowth ,bu tno tcytoplasmi c tubes growing in incompatible styles is streaming,i npolle n tubeso fLiliu m normal until 48h after pollination, but at longiflorum.Protoplasma ,96:275-282 . 96h and after, degeneration occurs. Cresti Ito.A.& A.Fujiwara ,1968 .Th erelatio n etal.(1980 )als oreporte d thatn odifferen ­ betweencalciu m andcel lwal l ingrowin g ces were observed in vivo between pollen riceleaf .Plan t& Cel lPhysiol .9:433-439 . tubeso f Lycopersicumperuvianu mgrowin gi n Koter,M.,B.DeKruijf f& L.L.M.Va nDeenen , compatible and incompatible styles during 1978.Calcium-induce d aggregationan d activationan dorganization ,unti l4 han d3 0 ''fusiono fmixe d phosphatidylcholine- min after pollination, while Dickinson & 'phosphatidi c acidvesicle sa sstudie s Lawson(1975) reported that differences be­ by PNMR .Biochim.Biophys.Acta . come evident from a very early stage in 514:255-263. Oenotheraorganensis .I nth estyle ,secrete d Miki-Hirosige,H.& S.Nakamura ,1982 .Proces s materials from the stylar canal cells are < ofmetabolis m duringpolle n tubewal l observed on the surface of the conductive formation.J.Electro nMicrosc .31:51-62 . canal tissue. The amount of the secreted Morris,M.R.& D.H.Northcote ,1977 . materials from the incompatible tissue is Influenceo fcation sa tth eplasm a less than those from the compatible one. membranei ncontrollin g polysaccharide Presumably the pollen tubes grown in the secretionfro mSycamor e suspension incompatible style are affected with some cells.Biochem.J .166:603-618 . substances secreted from thecana l tissueo f Picton.J.M.& M.W.Steer ,1983 .Evidenc e thestyle ,an ddegenerate .Mos tprobabl yth e forth erol eo fC a ions inti p entirepolle ntub ei nth eincompatibl estyle ' extension inpolle ntubes .Protoplasm a is damaged uniformaly. Furthermore,a new ^15:11-17. layer(Miki-Hirosige & Nàkamura,1982) has Reynolds,E.,1963 .Th eus eo flea d citrate been found to form between the cytoplasmic athig hp Ha sa nelectron-opaqu e stain membrane and the intine layer 30mi n after inelectro nmicroscopy .J.Cel lBiol .17 : water absorption. This layer protrudes from 208-212. the aperture and develops into the pollen tubewall ,therefore ,w ewis ht opropos eth e Stanley,R.G.& E.A.Lichtenberg ,1963 . term "germinative layer"fo rthi sne wlayer . Theeffec to fvariou sboro ncompound s oni nvitr ogerminatio no fpollen . Physiol.Plant.16:337-346 . References Stanley,R.G.& F.A.Loewus ,1964 .Boro n andmyo-inosito l inpolle npecti n Ascher,P.D.& S.J.Peloquin ,1966 .Effec t biosynthesis.in:H.F.Linsken s (Ed.):Pollen offlora lagin go nth egrowt ho f physiology and fertilization.North - compatible and incompatiblepolle ntube si n Holland Pub.Co..Amsterdam,p.128-136 . Lilium longiflorum.Amer.J.Bot .53:99-102 . Vasil.I.K.,1964 .Effec to fboro no n Baydoun.E.A.H.& D.H.Northcote ,1980 . pollengerminatio nan dpolle ntub e Measurement and characteristics offusio n growth,in:H.F.Linsken s (Ed.):Pollen ofisolate dmembran efraction sfro mmaiz e physiologyan d fertilization.North - root tips.J.Cel lSei .45:169-186 . Holland Pub.Co..Amsterdam,p.107-119 . Cresti,M.,F.Ciampolini,E.Pacini,G.Sarfatt i & B.Donini,1979 .Ultrastructura l features ofPrunu saviu mL .polle n tubesi nvivo . 1.Th ecompatibl epolle n tube.Caryologia ,

70 MEMBRANE-STRUCTUREM D EVENTSI NNON-PLASMOLYZE DAN DPLASMOLYZE D TOBACCOPOLLE NTUBES : AFREEZE-FRACTUR ESTUD Y

M.Kro han dB .Krmima n

Department ofBotany ,Universit y ofNijmegen ,Th eNetherland s

Introduction areth e result of distortions ofth eplasm a membranecause d e.g.b y alos s inturgidit y Theoute rlaye ro fth etobacc opolle ntub e ofth etube . wall (primarywall )i sforme db yth egrowin g pollentub etip ,wherea sth e innerwal l Reductiono fplasm amembran e layer (secondarywall )an dth eplug sar e depositedb yth enon-growin g parto fth e Duringth eformatio no fth e secondarywal l tube.Th e secondarywal lan dth eplug s andth eplug s innon-plasmolyze d pollentube s containcallos e inadditio nt opectin ,hemi - ando fth ewal l capi nplasmolyze dtube sa cellulosean dcellulos e (Kroh &Knuiman , reductioni nsurfac eo fth eplasm amembran e 1982). occurs.Thi s reduction isapparentl y achieved Plasmolyzed tobaccopolle ntube s forma byvesiculatio n andofte nals ob ystron g capo fwal lmateria l (withcellulos emicro ­ invaginations ofth eplasm amembran ewit ha fibrils)betwee nth eretracte dprotoplas t subsequent outwarddetachmen t ofvesicle s andth etub eti p (Kudlickae t al., 1981). andmembran epieces . Membrane-boundvesicle s andirregula rmem ­ branesystem swer eobserve djus toutsid eth e References plasmamembran ebu tals owithi nth eca p / material.Th emembraneou s structureswer e Kroh,M .& B .Knuiman , 1982.Ultrastructur e assumedt opla ya rol ei nexocytosi san d ofcel lwal lan dplug so ftobacc opolle n reductiono fth eplasm amembran e (Krohe t tubes after chemicalextractio no fpoly ­ al., 1983). Inorde rt otes tthi s assumption saccharides.Plant a 15^:2^1-250 . wecarrie dou ta freeze-fractur e studyo n Kroh,M. ,B .Knuima n &K .Kudlicka ,1983 . non-plasmolyzedan dplasmolyze dtobacc o Effectso freduce dturgo rpressur eo nto ­ pollentubes . baccopolle ntubes .In :0 .Erdelsk a (Ed.): Fertilization andEmbryogenesi s inOvulate d Resultsan d conclusions Plants,VEDA ,Bratislava ,Czechoslovakia , p. 121-121*. Exocytosis Kudlicka,K. ,M .Kro h &M.M.A .Sassen ,1981 . Cellwal lregeneratio n inplasmolyze dpol ­ Polysaccharides forth ematri xmateria lo f lentube so flil yan dtobacco .Act aBot . tubewall ,plug s andwal lca pma yb eextru ­ Neerl.30:316-317 - ded A.b y fusiono fsecretor yvesicle swit hth e plasmamembran e andincorporatio no fth e vesiclemembran e intoth e extending plasmamembrane , B.b ymembrane-boun d vesiclesvi ainvagina ­ tionso fth eplasm amembrane ;n oexten ­ siono fth eplasm amembran e occursthen . Theformatio no fth eprimar ywal lma y occurpredominantl y inth e firstmanner .Th e formationo fth e secondarywall ,th eplug s andth ewal l capo fplasmolyze dpolle ntube s possibly occur inth e secondway .

Hexagonalarray so fparticle s

Hexagonalarray so fparticle swer eobserve d m theplasmati c fracture faceso fmor etha n 50%o fbot hnon-plasmolyzed/polle ntubes .I n fewcase so fnon-plasmolyze d pollentube s smallhexagona larray so fparticle swer e observedtogethe rwit himprint s ofmicrofi ­ brilso fth etub ewall .Fo rthes e casesi t canb e excludedtha tth ehexagona lpattern s andplasmolyze d

71 ULTRASTRUCTURE OF NICOTIANA ALATA AND PETUNIA HYBRIDA POLLEN TUBES GROWN IN SEM1-VITRO CONDITIONS.

M.Cresti ,F . Ciampolini,D.L.M .Mulcah y and G.Mulcah y Department ofEnvironmenta l Biology,Universit y of Siena,5310 0Siena ,Ital y * Departmento fBotany ,Universit y ofMassachusetts ,Amherst ,U.S.A .

Research on pollination and fertilization granule were frequent. In comparison to "in vitro" could be important for plant what was described "in vivo" (Cresti et breeding as it could allow the functional al., 1979), thecallosi c tubewal l in"sem i characterization and selection of various vitro"i sslightl y undulated. types of pollen (Tilton & Russell, 1984). Similarly pollen tube growth in References "semi-vitro" conditions could be used for Brewbaker,J.L.,Kwack,B.H. ,1969 :In :H.F . the same purposes with the advantage of Linskens (Ed.):Polle n physiology andfe £ quick results and the possibility of tilization.N.H.Elsevier ,Amsterdam ,N.Y . relating tube growth rate and Cresti, M., et al., 1979: J. Submicr. ultrastructural features. In order to test Cytol.11 :209-219 . thishypothesi sw ehav estarte d anumbe ro f Cresti,M.,e tal.,1984:Amer.J.Bot.(i n rev.) experiments especially inorde r tofin dth e Tilton, V.R., Rüssel, S.H., 1984: Bio- mostsuitabl etechniques . sciences34 :239-242 . Styleso fN^ _ alata and P_^hybrid a (self and cross-pollination) were cut in two equal parts1 8 h after pollination and the samples with the stigmas were transferred toth e culture medium (Brewbaker & Kowach, 1964)unde r controlled conditions. After compatible pollination the pollen tubes of N^_alat a and P^hybrid a grow out ofth e style and successively developed in thecultur emedium .Afte r self-incompatible pollination nopolle n tubegrow sou to fth e style.Bot h inNL _alat a and P. hybrida the same cytological zonation was observed as already described for pollen tubes of, Nicotiana grown "in vitro" (Cresti etal.' , 1984)an d Petunia grown "in vivo" (Cresti et al., 1979). Nicotiana alata (Fig. 1):i n comparison toth etube sgrow n "in vitro"th e tubes in "semi vitro" show the presence, especially inth esub-apica l zonean d nuclear zone,o f many lipidic bodies and concentric configurations of rough endoplasmic reticulum (CER); sometimes autophagic vacuoles are present and surround the lipidic bodies.Thes emorphologica l aspects may be interpreted as cessation of protein synthesis (CER) or as the beginning of degeneration (lipid formation) and thereby arresto ftub egrowth . InPetuni a hybrida pollen tubes (Fig. 2) the cytoplasm shows the presence of numerous lipid bodiesalso ,bu tRE R con­ centric configurations were not observed while plastids containing a single starch

72 USEO FFLUOROCHROME SI NPOLLE N BIOLOGY T.Hough ,P .Bernhardt ,R.B .Kno x and E.G.William s PlantCel lBiolog y Research Centre,Schoo lo fBotany ,Universit y ofMelbourne ,Parkville , Victoria 3052, Australia

sucroseo rH3325 82 0jug/m li n 18%sucrose . The fluorescent DNA probes ethidium Atvariou stime safte r pollintionpistil s bromide or Hoechst 33258, when used in were fixed,cleare d and stained insiro ­ conjunction with the aniline blue fluor (0.25mg/ml) . fluorochrome (sirofluor) which stains the Observationswer emad ewit hZeis s callose component of pollen tube walls, epifluorescence opticso nmaterial smounte d allowlocatio n ofnucle iwithi n pollen tubes instai no rwater . Specimens stained with and permit discrimination between the tube EBwer eviewe d usingblue-viole t nuclei and those of the background pistil excitation (FITCsystem )an d specimens tissue. stained withH3325 8wer e viewedwit hU V excitation. Introduction Resultsan d discussion Fertilization dependso nprecis ebehaviou r andmigratio n ofpolle n nuclei inth epolle n Double stainingwit hth ecallos estain , tubea si ttraverse sth epistil . Single- sirofluor,i ncombinatio n witheithe ro f staining techniques used to locate nuclei thefluorescen tDN Aprobes ,ethidiu mbromid e have been of limited use for the study of (EB)o rHoechs t 33258 (H33258),allowe d pollination,owin g toth elengt h andnarro w visualisation ofal lpolle no rpolle ntub e diameter of the tubes,togethe r with both nuclei anddiscriminatio n notonl ybetwee n theobscurin g density of the grain andtub e vegetative and generativeo r spermnuclei , wallsan d the similarity of pollen tubest o butals obetwee n nucleiwithi n thepolle n narrow, elongate cells in the transmitting tubean d thoseo fth episti l tissueafte r tract of many types of pistil. Here we pollination. Under blue-violet excitation report on techniques for visualising pollen with EBplu ssirofluor ,orang enucle iwer e tube nuclei, and for selective double enclosed by yellow green tube walls.Unde r staining to contrast nuclei against a UV excitation with H33258 plus sirofluor, differentially stained pollen tube wall blue nuclei were contrasted against light withinth epistil . yellow tubewalls . For ungerminated pollen grainsan d i£vitr o grown tubes,definitio n Stainingprocedure s of nuclei was better after vital staining than after fixation. Amajo r advantage of Ethidium bromide (EB)an d Hoechst 33258 sirofluor over decolorised aniline blue is (H33258) were used as vital stains or that it will stain atneutra l pH and hence applied after fixation with 1:3aceti cacid : can be used for vital staining. Pre- ethanol. They were employed singly orwit h pollination vital stainingwit h EBo rH3325 8 sirofluor (purified fluorochrome from followed by sirofluor staining after pistil aniline blue stain. Evans & Hoyne 1982. fixation, allowed visualisation of pollen Commercial release pending). Pollen and in tube nuclei against unstained pistil tissue vitro grown tubes were treated with stains at least in the early stageso ftub egrowt h 5-10 min. on microscope slides and whole within thepistil . pistils for 1-7 hours in small vials. Thetechnique sdescribe d arepotentiall y Osmotic adjustment of stains wasmad e only suitable for monitoring nuclear behaviour for vital prestaining of pollen before duringpolle n tubegrowth ,an d possibly pollination,wher e a substantial period of during thefertilizatio n processitsel fwit h viability was required before observation. further development ofpre-pollinatio nvita l (Forfurthe r details seeHoug h eta_l .1984) . stainingprocedures . Forsingl e stainingwit hE Bth estai nwa s applied asa 0.01 %aqueou ssolution . For References doublestainin gwit h sirofluor thestain s wereapplie d together asa naqueou smixtur e Evans,N.A. ,Hoyne ,P.A . 1982 Afluoro ­ of3 part s0.01 %EB : 1par t sirofluor chrome fromanilin e blue: structure, (0.25mg/ml) . For single stainingwit h synthesisan d fluorescenceproperties . H33258th estai nwa sapplie d inaqueou s Aust. J. Chem. 35:2571-5 . solution (20jug/ml). Fordoubl e staining Hough,T. ,Bernhardt ,P. ,Knox ,R.B . withsirofluo r thestain swer eapplie d asa Williams,E.G . 1984 Applicationso f mixture of 1par tH3325 8 (40ug/ml ): 1 fluorochromesi npolle nbiolog yII . Partsirofluo r (0.25mg/ml) . Forvita lpre ­ TheDN A probesethidiu mbromid ean d staining for in_viv opollinatio nstudies , Hoechst 33258i nconjunctio nwit hth e freshpolle nwa shydrate d 5minute sbefor e callose-specific anilineblu efluoro ­ Pollination ineithe r EB0.01 %i n18 % chrome.Stai nTechnol .(i n press).

73 STRESS RELATED STRUCTURAL MODIFICATIONS IN THE POLLEN OF DATURA

Cadic, A. , Sangwan, R . S. and Sangwan-N orreel, B. S.

Present address: Université de Picardie, Androgenèse et Biotechnologie, 33 rue Saint Leu, 80039 Amiens Cedex, FRANCE; Université de Paris VI, Cytologie expérimentale et Morphoge­ nese végétale, Bât. N2, 4 place Jussieu, 75005 Paris, FRANCE (The described experiments have been done in the laboratories of Cytologie expérimentale et Morphogenèse végétale, Université de Paris VI and of Biologie de la reproduction des végétaux supérieurs, Université de Paris VII)

Under certain in vitro conditions , the micro­ and spl it endexine . spores of a number of Angîosperm species can - a rapid disorganization of the tapetum. be induced to an embryogénie developmental This give rise to a mass of cytoplasm which pathway to produce . The key to infiltrated between the microspores. the formation of a greater number of microspo- - the formation of periodic structures in va­ re-derived sporophytes therefore lies in the cuoles and apertural intine of some pollen means of modifying the normal microspore de­ grains. velopment so that embryos or calli differentia­ - in somatic tissues of the anthers, many te into plants. A number of factors controlling cells seem damaged and starch grains in the the deviation from a gametophytic to a sporo - plastids begin to dissappear or to reduce. phytic pathway have already been elucidate: among these , cold treatment and centrifuga- /These results suggest that the efficiency of tion have been shown to increase androgenic

Dead pollen grains and young embryos were counted in the anthers after IT' days of cu-ltn/1 References re. Statistical studies of the results show that the treatments did not increase the percentage 1 -Cadic, A. and Sangwan-N orreel, B.S., of androgenic microspores when applied 1-2 1983. Effet du refroidissement et de la days before the first haploid mitosis. However centrifugation sur I'ultrastructure des when applied 1-2 days after this division the microspores du Datura innoxia Mill. Ann. same treatment greatly enhanced the embryo- Sei. Nat. (in press' genie potentialities of the pollen grains.After 2 -Camefort, H. and Sangwan, R.S.,1979. mitosis chilling flower buds increased the num-* ber of androgenic pollen grains five fold; cen- Action d'un choc thermique sur certaines trifugating the anthers for 5 min at 150 g ultrastructures des grains de pollen embry- before culture increased six fold the number ogènes du Datura metel L. C. R. Acad. of embryogénie pollen grains; but the best re­ Sei. Paris, D, 2887T383-1386. sult was obtained by the association of the 2 3 -Sangwan, R.S., 1984.Cold treatment treatments which increased 12 folds the yield related structural modifications in anthers of embryos (8.6 % androgenic pollen grains). of Datura. Cytologia (in press). 4 -Sangwan-N orreel, B.S., 1977. Androge­ nic stimulating factors in the anther and Structural changes.. isolated pollen grain cuhure of Datura The general pattern of ultrastructural chan­ innoxia Mill. J. exp. Bot., 28: 843-852. ges by the above treatments are: - a modification in the pollen wall. The midified exine shows sole slighly crumbled

74 jjJTM LONGIFLORUMPOLLE N 2 30 T-Test (Mendenhall &Ott , 1972). GERMINATION ANDTUB E ELONGATION AS MARKERS FOR SULFUR DIOXIDE Results *+ * W. V. Dashek , C. S. Ridenour and Germination- Fumigatio no fpolle n R. R. Mills**, Depts. Biology, uponth eanther swit h2. 5pp mSO , '*West Virginia Univ., Morgantown, followedb yin vitroG resulte di n1. 5 W 26506, +Atlanta Univ. Atlanta, and1.2-fol delevation si n% G > th e GA 30314 and **Virginia Commonwealth controla t2 an d4 hr ,respectively . Univ., Richmond, VA 23284, USA At1 0ppm ,th eenhancement swer e3. 4 and2.7-fold s (Fig. 1). Summary. Tube elongation- Neithe r2. 5no r 10.0pp mSO ,significantl y (p=0.05) Atmospheric sulfur dioxide (SO,) affectedT Ea teithe r2 o r4 h r (Fig. is an air-pollutant requiring surveil­ 2). lance as it impairs human health and promotes crop damage at certain levels.

This surveillance has included attempts FIG.2 >»' to utilize both pollen germination (G) and tube elongation (TE) as air-pollu­ r |—1 tant indicators. Here, we report SO,- induced alterations of LUium. pollen G f and TE.

Introduction

While a variety of plants has been surveyed for their assay of air-pollu­ tants (Kellerher &Feder , 1978), few investigations have dealt with the Discussion possibility that pollen G and/or TE could serve to bioassay SO,. Here, we The datawithi nFigs .1 an d2 demon ­ report SO, -induced alterations in strate thatonl yG coul db ea possibl e Lilian pollen G and TE. monitor sinceneithe r2. 5no r10. 0pp mSO , significantly affectedT Ea teithe r2 o r3 feteria1 s and Methods hr. These results differ markedly from investigationswit h otherpollens . Fumigation conditions - Whole anthers (stored 4 C, 1-2 months) with Acknowlegements adhering Lilium lonaiflorum, cv. 'Ace' pollen were fumigated with either 2.5 We thankMs .B .Boggs ,Ms . S.Schel l (4 hr) or 10.0 (10 hr) ppm SO, within a andMs .C .Capell efo rtechnica lassis ­ Plexiglass chamber and the SO, quanti­ tance,Dr .S .Erickso nfo rfumigation san d fied as in Erickson & Dashek T1982). Ms. L.Le efo rclerica l assistance. Germination conditions - Twenty mg fr wt lots of both fumigated and un- References fumigated pollen were sown in sterile Petri dishes containing 10 ml each of Dickinson,D .B. ,1965 . Germinationo f sterile Dickinson's (1965) medium lily pollen: Respirationan dtub e except for tetracycline. Pollen was growth. Science,N Y150:1818-1819 . germinated either 2 or 4 hr at 24 ± 2 C Erickson,S .S .& W .V .Dashek ,1982 . in constant darkness. At either time Accumulationo ffolia r soluble proline medium aliquots were withdrawn to shell in sulphur dioxide-stressed Glycine vials containing 100 ,ul 40% formalde­ max. cv.'Essex 'an dHordeu m hyde. The experiment was replicated vulgarecvs. ,'Proctor 'an d'Excel ­ sior' seedlings. Environ. Pollut., three times. y Quantification of both % G and TE Ser. A.28:89-108 . ~ Germinated pollen was removed from Kellerher,T .J .& W .A .Feder , 1978. the vials and transferred to microscope Phytotoxic concentrationo fozon eo n slides for scoring of both % G and TE. Nantucket Island;lon grang e transport Sixteen hundred grains and tubes were fromth emiddl eAtlanti cState sove r scored for both the control and each theope nocea nconfirme db ybioassa y treatment. Tube lengths were measured withozone-sensitiv eplants . Environ. with a calibrated ocular micrometer. Pollut.17:187-194 . Significant differences in either Mendenhall,W .& L .Ott ,1972 . Under­ % Go r TE between fumigated and non- standing statistics. DuxburyPress , fumigated pollen were assessed by a n Belmont,CA . 75 EARLY INDICATORSO F INVITR OTRANSFORMATIO N FROMGAMETOPHYT E TOSPOROPHYT E INDATUR A

R. S.Sangwa nan dB .S .Sangwan-Norree l

Université dePicardie ,Androgenès e etBiotechnologie ,3 3ru eSain tLeu ,.8003 9Amien sCede x FRANCE -Universit é dePari sVII ,Membrane sBiologiques ,tou r5 42 ° et., 75551 ParisFRANC E

Summary find aspecifi c cytologicalmarke r inorde r todistinguis h embryogénie fromno nembryogé ­ At theverg eo fth efirs thaploi dmitosis , niemicrospore s ata nearl y stageo fandroge ­ someo f themicrospores/polle n grainso fDa ­ nesis. turama yb e induced tofollo wa sporophytT c development.A t this stage,th emicrospor e Material andmethod s beforecultur e invitro ,for mthre edistinct s size typeshavin gmea n diameter of32 ,39 ,4 5 Anthers,fo rfixin gdirectl y and forcultu - umrespectively .A t thematur eanthe rstage , ring,wer e takenfro m theplant so fDatur a only twomajo r typesar eeviden twit h amea n innoxiagrow ni n thePhytotro n (Gifsu rYvet ­ diametero f4 8u man do f5 8um . te)accordin g to themethod spreviousl y des­ Invitro ,th efirs tmanifestatio n ofth e cribed (Sangwanan d Camefort, 1982;Sangwan , androgenic induction isth edevelopmen to f 1983). acoatin go rdeposi to n the tonoplast,onl y Forexaminatio no f theandrogeni c(uninu ­ in theembryogéni emicrospores .Cytochemica l cleate orearl ybinucleate)an dmatur epolle n tests indicated thepresenc e of tanninsi n grainstages ,5 anther so f each floralbud s thedeposits .Th eearl yembry o globularstag e werepresse d gently in2 m lo fdistilledwate r isals o characterized by theformatio no fri - with2 % sucrose .Th epolle nsuspension sthu s bosomalbodies .Thes ebodie spersist -onl ya t obtainedwer eobserve d directly underth e theglobula r stagean d disorganizewhe nth e lightmicroscope .A minimu mo f 1000grain s embryo enter theheart-shape d stage.Simila r werecounte d foreac hstag e bodies areals oobserve d in theyoun g zygotic embryoso fDatura .Th epresenc eo f theabov e Results and discussions indicators,i nrelatio n toi nvitr o androge- • nesis,ar ediscussed . a)I nviv o characterization ofdifferen t Keywords:i nvitr o culture,androgenesis , pollenpopulation s pollen,embryo ,Datura ,haploidy . Although allstage so fpolle n development Introduction havebee n studied,her eonl y twoparticula r stages (i.e.androgeni c andmatur epolle n Since Guhaan dMaheshwar i (1964)obtaine d stages)ar edescribed .Polle n ofandrogeni c haploid plants fromculture d anthers,work s stagei scharacteriz eb ya larg ecentra lva ­ inou r laboratoryhav ebee naime d tounder ­ cuole, thinlaye r cytoplasm and periphral stand themechanis mo f invitr o androgenic nucleus/nuclei.A s iseviden t from theTabl e inductioni nvie w toincreas e theyiel do f I,Thre eprincipal s typeso fpolle ncoul db e haploid plants.W ehav ebee n successfult o increase theyieldsb yusin g differenttrau ­ Table I.Percentage s ofdifferen t typeso f matic shocksbefor e theanther sar eculture d pollengrain safte rspore-diamete ra tandro ­ (seeSangwan-Norreel , 1980).But ,i ti sno w genic/uninucleate andmatur epolle nstag ei n evident thafth epercentage s ofmicrospore s Datura.MP:mea n diameter,A :androgeni c stage forming embryosca nno tb eincrease dbehon d maturepolle nstage . certain level (approxymately5 % i nDatur a Stage Floral Type1 Type 2 Type3 evenb yapplyin g thebes tcultur e conditions buds MD: 32um MD: 39um MD:45u m (stress,media ,hormone s etc.). Theabov e facts suggest thatsom especie s e.g.Solana - ceaean d Graminae familyma yhav epolle n grainswhic h aregeneticall y predisposed to 27.3 52.8 19.9 develop inculture .Therefore ,i ti sno tth e 30.4 56.6 13 normalpolle n grainswhic har edeviate d from 22.6 63 14.4 theirgametophyti c pathway invitro .I nfac t pollendimorphis mhav ebee n observed inman y Smaller Larger types species (Sunderland, 1980;Maheshwar i eta l type 1983).Ou raim s in this investigationhav e MD: 45u mMD :5 8ur nMD :7 0u m been: 1)t ocatalogue ,dependin g on thedia ­ meter, themai n typeso fpolle na t theandro ­ genic aswel l asa t thematur epolle nstage , 46.10 45.65 8.38 2)t oevaluat e theearl y cytologicalbeha ­ 42.15 50.98 6.85 viouro fi nvitr opolle npopulation ,3 )t o 33.55 60 6.44

76 recognized.Typ e 1wit h amea n diametero f ninswer eobserve d inth ematur eno nandroge ­ 32u mwhic h represent about 30%o f thetota l nicpolle ngrain s asdiscret ebodie so rglo - pollenpopulation .Typ e2 wit h amea ndiame ­ ules in theremaider s of thecentra lvacuol e tero f3 9u mwit hmor e than5 0% an d type3 rather thana sa coating .Th eorigi nan d the witha mea ndiamete r of45u mwhic h represent roleo f this invitr o deposits areno tclear , alo wpercentag e of pollenpopulatio n atth e although thereseem s tob e acorrelatio nbet ­ androgenicstage . ween itspresenc e and theembryogéni epathwa y Although themajorit y of grains,i nmatur e of themicrospor e invitro . antherswer ebicellular , therewer e twodis ­ tinguish types:smalle r (meandiamete r 48um ) - Ribosomalbodie s formation. andlarge r (withmostl y amea n diametero f Themos t importantcytoplasmi c featureo f 58u man da fe wwi h 70 um), (Table I).Th e theearl yglobula r embryos (4t o 10cells ) frequencies of theabov e typesvar y largely was theformatio no f condensed pyroninophilic fromon eflora lbu d toothe ran d evenfro m structures afterUnn astainin gwit h positive oneanthe r toanothe r in thesam e floralbud . Brächettes tfo rRNA .Abou t 3-5 suc hstruc ­ Suchtype so fvariation s inth epolle npopu - turescoul dbe 'see npe rcel l section,situa ­ lationhas frquentlybee nobserve d inman y tedaroun do rver ynea r thenucleus .The scy ­ species (Maheshwari et al, 1983).Whe nstai ­ toplasmic structures,calle d ribosomalbodie s nedwit hacetocarmine ,n oclea rcu tdifferen ­ (Fig.3 ;RB )laste d onlydurin g theglobula r cescoul db eobserve dwithi n these threety ­ stagean d disorganized progressively asth e pesa tth eandrogeni c stage.However ,a tth e embryoentere d inth eheart-shape d stage. matureanthe r stage,polle n grainswit h small Thesebodie sconsi ste do fdens emasse s ofri - diameter (48um) )wer eweakl y stained ascom ­ bosomes and aroug hendoplasmi c reticulum. pared with thelarge rone s (58ur no r7 0 um). Similarbodie swer eals o observed inth ezy ­ Thelarge rsiz eseem s tocorrespon d toregu ­ gotic embryoso fDatura .Th eR B are therefore larnorma lmatur epolle ngrain s as iseviden t characteristic of theearl y embryogenesis in after thefluorescin ediacetat e staining. whatever form.The y areno tobserve d during pollenmaturatio n invivo .Thei rrol ean d b)I nvitr o observations functionar eno tclea ralthoug h inanimal s theyar esuggeste d tob ea nimportan t siteo f Theearl y evolutiono f themicrospore s to­ enzyme formationnecessar y for inductionan d wardsembryogenesi s couldb e traced fromFeul - developmento fembryo s (Vande nBeggelaar , gen-squashes ofmateria l fixed atregula rin ­ 1976). tervalso f48 h for 16day s (Sangwan, 1983). Approxymately 3t o5 % o f themicrospore s Conclusions formedembryos . -Tonoplas t feature during invitr o culture 1-Thtit ear eno t two,a sobserve d inothe r Beforecultur e thevacuolat e androgenicmi ­ speciesbu t three typeso fpolle n grainsi n crosporeha s athi n tonoplast (Fig. 1).Onl y Daturaafte r sporesize . about5 0% o f themicrospore s remained viable 2-N oclea rcu trelationshi p couldb eobser ­ after48 h invitr o and couldb e distinguished vedbetwee n these typesan d invitr oandroge - byelectro nmicroscop y into three types:1 ) nesis. i.e.whic h typewil lonl yform sembry o microsporeswit h densely contrasted/coated instead offollowin g thenorma lgametophyti c tonoplast (Fig.2 ;4 to6 %), 2)microspore s development.Th e smallerpolle n grains inth e inwhic h tonoplast dono t change (15t o2 0% ) mature anther stageseem s tocorrespon d to 3)microspore swhic h develop intomatur epol ­ theabnorma lpolle npopulation .I ti squit e lena sa resul to fwhic h tonoplastruptur e possible that this typema ydevelop sembry o andfinall ydisapear . whencultured .However ,th epercentage so f After8 day si ncultur e around 94% o fth e thesegrain s islargel y superior to theper ­ microsporeswer e dead, 1% becam ematur e pol­ centages ofpolle n grains undergoing irivitr o lenan d theres tdevelo p intoembryos .Thes e androgenesis. percentages agreewit h our lightmicroscopi c 3-Tannin-coate d tonoplastwa s found tob e observations on theevolutio n ofmicrospore s specific formicrospoe s formingembryos . .lnvitr oan d theorigi no fdifferen tpathway s 4-Ribosoma lbodie swer eonl ypresen ti n ofembry o formation (Sangwanan dCamefort , theearl yembryogenesi s inDatura . •982;Sangwan , 1983) All themicrospore-derive d embryos showed thecoate d tonoplast.Thi s coatingpersiste d References °nlyi n theglobula r stage.B ymean s ofcyto - chemical testes (Sangwanan d Camefort, 1983) Guha,S .an dS.C .Maheshwari , 1964.Natur e wehav econfirme d thepresenc eo f tanninsi n 204:497 . thecoatin go fvacuola rmembrane/tonoplast . Maheshwari,S.C , A.Rashi d andA.K .Tyagi , Theform san d dimensions of thedeposits/coa ­ 1983.I.A.P.T.C .Newslette r 41:2 -9 . tingswer eusuall y uniform at thebeginnin g Sangwan,R.S ..1983.IN :D.L .Mulcah y andOtta - °fth eembryogenesi s although itwa soccasio ­ viano (Eds.),Pollen :Biolog yan d implica­ nallymor e irregular insom emicrospores .Tan ­ tions forplan tbreeding .Elsevie rScienc e

77 publishing co.:28 7- 293 . Explanation offigure s Sangwan,R.S .an dH . Camelort, 1982.Experi - Fig. 1- Electro nmicrograp h ofa nuninucl e ëntia,38 :39 5- 397 . temicrospor ebefor e culture.Not etono - Sangwan,R.S .an dH .Camefort ,1983 .Histo ­ plast (t)withou t deposit.C :cytoplasm , chemistry,78 :47 3- 480 . Ex:exine ,N :nucleus ,nu :nucleolus ,V : Sangwan-Norreel,B.S. , 1980.Bull .Soc .Bot . parto f the largecentra lvacuole .Arrow s Fr., 126:41 3- 443 . indicate the tonoplast (x6 100)-, Sunderland,N. , 1980.In :D.R ;Davie s and D.A. Fig.2 - Microspor eafte r4 8h o fi nvitr o Hopewood (Eds)I V thJoh n Innes Symposium. culture.Not e thedensel y contrasted tono Theplan tgenome ,Joh nInne sCharity ,North - plast (arrows)an d thetoo-like nucle i, wick, :17 1- 183 . (x4 950) . Vande nBiggelaar ,J.A.M. , 1976.Proc .K .ned . Fig.3 - Ribosomi c amas (RB)i na cel lo fa Akad.Wet .Sect .C ,79 :42 1 -430 . youngpollen-embryo , (x7 200) .

x3^ ï'^4.^'0^\^

78 PART II

STIGMA, INCOMPATIBILITY AND POLLEN GERMINATION IN VITRO

• ••••••••••••••••••••••••••••••••••it****

79 Part II:Stigma ,incompatibilit y andpolle ngerminatio n inviv o

Introduction Also the development of specific cytochemi- cal methods is very important. Recogni­ The stigma surface, incompatibility and tion is a very important mechanism, working pollen germination in vivo are the topics on different moments from pollination of this part. Ultrastructural and biochemi­ till after fertilization, between sporophy- cal methods are used and combined. A recent te or gametophyte and gametophyte. This new technic is the thin layer chromatogra­ interaction is very dependent on timing phy - bio assay and the pollen tube test and environmental factors. Constant compar­ system. For application the study of incom­ able conditions of the flower and its patibility is of high importance in plant way of acting in acceptation or rejection breeding. should be well known before conclusions The sporophytic incompatibility gets atten­ canb emade . tion as an interaction which occurs at Because of the complexity in recognition, the place of contact between pollen coat it is perhaps not by accident that Dr. and stigmaticsurface . Williams omits a word as "Incompatibility" One of the major questions is still the or male sterility in the following verse: way of recognition and nature of the signal / A poem you asked me for - please or signals during this first contact. with a few words of "technicalese" I said "nott oworry " Different possibilities are proposed offer­ Butno w Imus thurr y ed, but to fit them in the process of Toge ta sman yword slike : apomixis incompatibility is still difficult. The gametophyte complexity of the pollination stimulus and sporogenesis into the last line as I can possibly is also demonstrated in the wilting of squeeze '. the flower. Several other aspects of the To avoid all negative effects of squeezing flower behaviour, such as colour shift, in recognition systems a male sterile changes in position, rate of opening or life form seems to be a simple solution, its general morphology could be studifed suojn as Lord Bommel, probably a male ster­ in relation to pollination. Also the study ile boar, a well known cartoon hero (see of the differences in stigma morphology fig. 1a, b). So he has to live in his can offer more data about the conditions own world of fairy tales, a situation and tasks present in pollen or stigma. of no squeezing at all (see fig. 1c).

This figure"suggest s an intensive meeting between individuals (a, b, c).I n respect to a possible interbreeding, a lot of differences may be expected. However, contacts by hands and eyes show some incompatibility. Probably this also becomes clear in fig. c, shown by one person on which male sterility can be observed. Note the flower in c too. (From 'Den AndereWereld' .Marte n Toonder. Druk:Va n Boekhoven Bosch B.V. Utrecht, 1982.Wit h permission ofMarte n Toonder/De Bezige Bij,Amsterdam .No tpermitte d tocop y the figure). 80 LOCALIZATION OF POLLEN SURFACEGLYCOPROTEIN SUSIN GMONOCLONA L ANTIBODIES

S.Ramm-Anderso nan d R.B.Kno x

PlantCel lBiolog y Research Centre,Schoo lo fBotany ,Universit y ofMelbourne ,Parkville , 3052 Australia.

Summary Table 1. Approximatemolecula rweigh tan d Thecellula r siteso fallergeni cglyco ­ specificities ofFMC- A serieso fhybridom a proteins inmatur e ryegrasshav ebee n antibodies (Smarte t al. 1983). located by immunoelectronmicroscopy using monoclonal antibodies. Several chemical Antibody approx.m w specificity fixationprocedure s and tworesin s (Spurr's (x1000daltons ) (S)an dLondo n resinwhit e (LRW)wer e evaluated forthei r suitability inpre ­ FMC-A1 35 GroupI servingbot h structure and antigenicityi n FMC-A5 16 Group IIo rII I maturepollen . Allergenic glycoproteinswer e FMC-A7 30,28 GroupI localised inth epolle n graincytoplasm , FMC-A9 71,87,49,47,18 GroupI V walllayer san d onth e grain surfacean d antherorbicules . Negative Control: Supernatant from cultureso fth emous emyelom a lineX-6 3whic h Introduction secretesa nIg Gmolecule .

Inryegras spollen ,fou r groupso f allergens (groupsI-IV )hav ebee n described Thebindin g ofal lfou rmonoclona lanti ­ (Marsh, 1975). Theseallergen shav en o bodies inth ecytoplas m ofth epolle ngrai n knownrol e inth epolle n grain,an d thusth e alwaysoccurre d near ERmembrane so rmem ­ precise localization ofthes e proteinsan d branesaroun d vesiclesan d P- glycoproteinsan d their siteso fsynthesi s (polysaccharide)particles . Inth eintine , areessentia l fora nunderstandin g ofthei r thegol d particlesboun dmainl y toth e function. tubular structureso fth ematrix . Inth e Recently,monoclona l antibodiest o exine thegol d particleswer e localised in allergenic components ofryegras spolle n thearcades ,th epore san d theexin elayers . havebee ndevelope d (Smarte t al., 1983)an d Although thedifferen tmonoclona l anti­ madeavailabl e tous . Inth epresen tinvesti ­ bodies showdifferen t densitieso fbindin g gation,indirec t post-embedding immunogold inth epolle n grain sections,bindin g always methodshav ebee n used togetherwit h occurred insimila r regions (Table2) . different specificmonoclona l antibodiest o Further information concerning theprecis e precisely localise someo fth eallergens . siteso fsynthesi so fth eallergen swil l depend onmor edetaile d characterization of Resultsan d Discussion themonoclona l antibodiesused .

Immunogold localization ofpolle n allergens Conclusions Overall,th eperiodate-lysine-para - formaldehyde (PLP)fixativ e (McLeanan d Monoclonal antibodiesbin d tothi nsec ­ Nakane,1974 )produce d superior structural tions,an d mayb edetecte d withimmunogol d preservation even though thepolle nwal l labelling. Three interesting featureso f layerswer ewel lpreserve d usingglutaral - pollen allergensemerge : dehydean d cetylpyridinium chloride allth emonoclonal sbin d toa simila r (GCC)fixativ e (Grotee t al., 1983). rangeo fcellula r sites inth epolle ngrains . Pollengrain sembedde d inSpurr' sresi nwer e - differencesi nnumbe ro fgol d particles lessdistorte d and sectionscoul d beviewe d boundma y notreflec tdifference si n forlonge r periodsunde r theelectro n glycoprotein concentration sinceth evariou s microscope than those inLondo n ResinWhite . antibodiesha d different titres,an dth e However,Londo n ResinWhit e iseasie r tous e surface receptorsma yno talway sb e and requires lessinfiltratio n time. The accessible forantibod ybinding . distribution ofcolloida l gold particles - antibody binding inth epolle n cytoplasm (10n mdiameter )i ndifferen t regionso fth e isassociate d with ERan d vesicles,an d Pollen grain showed considerable variation P-particles,suggestin g thatthes e dependingo nth especifi cmonoclona lantibod y organellesma yb eassociate d withth e used (Table1) . extracellularsecretio n ofth eglycoprotein s intoth epolle nwall . Table2 . Semi-quantitative distribution ofgol d label onthi n sectionso fryegras s pollen grainsafte rdifferen t fixationan d embeddingprotocols .

Fixation FMC- Pollen PollenWal l Pollen & Resin Anti-Cyto - Intine ExineSurfac e body plasm Arc- & GAM ardes Probe

PLP/S A1 ++ + + - GCC/S A1 ++ + + + GCC/LRW A1 + + + +

PLP/S A5 ++ + + + GCC/S A5 + + + + GCC/LRW A5 + + + +

PLP/S A7 + + + + GCC/S A7 - - - - GCC/LRW A7 ++ + ++ +

PLP/S A9 +++ ++ ++ ++ GCC/S A9 +++ +++ ++ ++ GCC/LRW A9 +++ ++ ++ ++ PLP/S ) GCC/S ) X-63 - - - - GCC/LRW )

PLP/S ) GCC/S ) GAM - - - - GCC/LRW )

- : nolabelling ;+ :< 1 0grains/section ; ++: < 100grains/section ;+++ : > 100grains / section; GAM: colloidal goldgoat-anti - mouse IgG.

References

Grote,M. ,Fromme ,H.G .& Sinclair ,N.J . 1983. The useo f cetylpyridinium chloride topreserv e water-soluble surfacemateria l inpolle n forelectro n microscopy. Micron 14:29-32 . McLean,I.W .& Nakane ,P.K . 1974 Periodate- lysine-paraformaldehyde fixative ane w fixative for immunoelectronmicroscopy. J.Histoch .Cytoch . 22:1077-83 . Marsh,D.G. , 1975. Allergens and the geneticso fallergy . InSela ,M . (Ed.): TheAntigens . Academic Press,London . 3:271-359 . Smart,I.J. , Heddle,R.J. , Zola,H .& Bradley,J . 1983. Developmento f monoclonalmous e antibodies specific for allergenic components inryegras s (Loliumperenne )pollen . Int.Arch s Allergy appl. Immun. 72:243-48 .

82 CYTOCHEMISTRY OFTH E STIGMA SURFACE COMPONENTSUSIN G THE PRINT TECHNIQUE

C.SAID ,P .ZANDONELLA , T.GAUDE ,C . S

UniversitéCl .Bernard-Lyo n I,4 3B d du 11Nov .1918 .696Z 2Villeurbann e Cedex.FRANC E

Summary Fixation Cytochemical test Dryan dwe tstigma sposses ssurfac ecompo ­ none CTC SB PAS CB EA nents,easil y diffusible.Stigma-prin ttech ­ niqueallow s tovisualiz e and identify these none + nt surfaceproducts ,wit h light and scanning 0s04 + nt electronmicroscopy ,withou t any inclusion Glutaral­ procedure.W e demonstrated thatpolysaccha ­ dehyde rides,proteins ,lipid s ,enzymati c activity andminera l ions,especiall y Ca canb e * CTC:chlorotetracyclin e for Ca ,SB :Suda n revealed bythi smethod .Dr ystigma s (Brassicaolerace aL. ,Populu s albaL. )o r Black forlipids ,PAS :PA Sreactio n forpoly ­ wetstigma swit h smallexudat e (Forsythia saccharides,CB :Coomassi eBlu e forproteins , intermediaZab. )gav e bestresult s thanwe t EA:esteras e activity (review inKnox , 1984). stigmaswit h copiousexudat e (Petuniahybri - da L.). Petuniahybrid adoe sno tprovid e aclea r Keywords:cytochemistry ,stigma.print ,prin t print;jus tgenerall y remainso nth eslid ea technique. diffuse lipophilic exudate.Th eshap e ofth e stigmacell scanno tb eidentified . Introduction Discussion Inorde r togai n abette r understanding ofpollen-pisti l interactions inAngiosperms , Hydrophilic and adhesive compounds ofth e iti snecessar y todetermin e thenatur eo f stigmasurfac e havebee n demonstrated byth e theinteractin g surface components.Thes e print technique.However ,i fth etim e ofcon ­ componentsdiffus eeasil y ina naqueou s tacto fth estigm awit h theslid e ismor e medium:thi spropert y hasbee n used bysom e than 30min. ,protoplasmi c components mayb e authorst odevelo p the print method (Heslüp- released fromth estigm a cells inducing false HarniSùrte tal. ,1973 ,1975) .W ehav eadap ­ reactions. tedan d improved theprin tmetho d forbot h Stigma printsar ebette r afterglutaralde ­ lightan d scanning electron microscopy. hyde vapour fixation thanafte r osmicvapour . Theresult sdiffe rwit h thespecie stested : Materialan d method drystigm atype so rthos ewit h asmal lamoun t ofexudat e give the bestresults . Thestud y wasperforme d onsevera lspecie s Inconclusion ,th e stigmaprin tmetho d pro­ withdr ytyp estigmas :Brassic anapu sL. , vides asimpl emetho d forth e characterization Pogulusalb aL .an dwe t typestigmas : cell surface components which may beinvol ­ Forsythia intermedia Zab.,an d Petuniahybri - ved inpollen-pisti l recognition. daL .Print swer e realized onglas sslide s and fixed withglutaraldehyd e orOsO .vapou r References ina hermeticall y sealedbox . For scanning electron microscopy,print s Heslop-Harrison,J. , Heslop-Harrison,Y. , werecoate d with gold palladium and observed Knox,R.B .& B .Hewlett ,1973 .Polle nwal l ina Cambridg e 600,a t1 5kv . proteins:'gametophytic' and 'sporophytic' For lightmicroscopy ,differen tcytoche ­ fractions inth epolle nwall so fth eMalva ­ micaltest ssummarize d inth e following ceae.Ann .Bot.37 :403-412 . tablehav e beenused . Heslop-Harrison, J., Heslop-Harrison,Y .& J . Barber, 1975.Th estigm asurfac e inincom e Results patibility responses.Proc .R . Soc.Lond . B. 188:287-297 . Knox,R.B. ,1984 .Pollen-pisti l interactions. Cytochemicaldat a from light andU.V .micro ­ PlantPhysiol .17 :508-609 . scopy forBrassic a napus,Populu s albaan d Forsythiaintermedia . (+= positiv e reaction,- = negativ e reaction, i =wea kreaction ,n t= no t tested).

83 STIGMASTRUCTUR EAN DTH EPOLLEN-STIGM A INTERACTION INCAESALPINIOIDEA E -LEGUMINOSA E

S.J. Owens

Jodrell Laboratory,Roya lBotani c Gardens,Kew ,Richmond ,Surre y TW93AB ,Englan d

Summary collection ofth eKe wherbarium . Thesefor m thebasi so fanothe r publication (Owens& Stigmatic surfaceso f6 gener a inth e McGrath inpreparation) . Caesalpinioideae arebriefl y described.The y can bedivide d into2 groups ,W Pan d WN, SEM which showmorphologica l affinities toth e moreadvance d sub-families Papilionoideae Stigmaswer eremove d directly fromflowers , (WP)an d Mimosoideae (WN).Th egerminatio n stuck toa stu busin gconductin g 'Dotite' responseo fsel fpolle nappear s furthert o paint,examine d and photographed fresha t endorse thesedifferences . 10K Vi na Jeo lS35 . Cassiafloribund a (WN)i sexamine di n detailan d datao fself ,interspecifi can d LightMicroscop y intergeneric pollinations presented. Stigmatic exudatesi nW Ntyp estigma sar e Morphology considered tob eefficien tgerminatio nmedi a and thestigm a tolac kspecifi c recognition Observationso n freshan d spiritmateria l molecules. wereals omad eusin gdissectin gmicroscope s andmicroscope sfitte dwit hepi-objectives . Introduction Fartherexaminatio no fstigm a structurewa s made from semi-thin (1*im)sections ,fixed , Thefamil yLeguminosa e isgenerall y dehydrated and embedded using conventional considered tocompris e 3sub-families ;th e TEMtechniques . more primitivegener a and speciesar e classified inth eCaesalpinioideae .15 2 Non-specific Esterase generaar erecognise d (seeth etaxonomi e analysis inPolhil l &Rave n 1981)an d these Esterasewa slocalise d on stigmasusin g canb eassigne d to5 tribes . themetho d ofPears e (1972). Datao nstigmati c surfacesar eavailabl e fromth esurve yo fHeslop-Harriso n &Shivann a Cutin (1977). Twogener ao fth eCaesalpinioideae , Browneaan d Cassia,wer eexamine d andwer e Thepresenc eo fcuticl eo n thestigmati c classified intoth eW Pcategory ,th esam e»• ' surfacewa steste d onfres hstigma susin g category asal lpapillonoi d genera examined. Aur^imine0 (Heslop-Harrison 1977).Examinatio n Thebreedin g systemsan d pollination wasmad ewit ha Vicker sPhotopla nusin g biology haverecentl y beenreviewe db y transmitted UVillumination . Arroyo (1981).Te nspecie sou to fsixtee n examined inth eCaesalpinioidea e werefoun d Pollenviabilit y tob eself-incompatible ;dioec yan d andromonoecy weremor ewidesprea d thani nth e Pollenviabilit ywa sestimate d foral l other sub-familiesan d apomixiswa swel l species inthi sstud yusin g thetechniqu eo f documented inth eAustralia n Cassiaserie s Shivanna &Heslop-Harriso n (1981).Wit hth e Subverrucosae ofsectio n Psilorhegma.A wid e exception ofCassi aobtus a (13%)an d Cercis spectrum ofpollinatin gagent swa sals o species (25-80%),al lpollen swer e>80 % recorded including bees,wasps ,butterflies , viable. birdsan d bats. Pollentub egrowt h Materialsan d Methods Pollentube swer eexamine d intransmitte d Representatives ofal l tribesi nth e UVilluminatio n (asabov efo r Auramine0 ) Caesalpinioideae aregrow na tKe walthoug h aftermountin g freshly dissected stigmasint o atpresen tonl y specieso f3 tribe sflowe r anilineblu e (O'Brien &McCull y 1981)o na regularly.6 gener a (Cassia,Caesalpinia , slide. Cercis,Bauhinia ,Brownea ,Gleditsia )an d 16specie shav ebee nuse d insom epar to r Pollinations allo fthi sstudy . Afurthe r 45gener aan d approximately 92specie swer eavailabl efo r Allpollination swer ecarrie d outi nth e studyo fstigmati cmorpholog y inth espiri t laboratory onvirgi n stigmas. Laboratory

84 temperaturesrange d from21-2 7 Can d appearst oincreas e inviscosit y onexposur e relativehumidit y between 36-56%. toth eatmosphere . Wholeindividua l flowerswer euse di n pollinationso fBauhinia ,Browne a (these 2.Th epollen-stigm a interaction;polle n wereals oemasculate d beforeremova lfro m germination theinflorescence )an d Caesalpiniaan d eitherindividua l flowerso rwhol einflor ­ A General escencesi nCassi aan d Cercis.Polle nwa s removeddirectl y from freshlydehisce d Thetw ostigm a typesdiffere d inthei r anthersusin geithe ra singl epaintbrus h requirements forsuccessfu l experimental hairo ra custom-mad eglas smicro-needle . pollination.Al lstigma so fth eW Ptyp e Grainswer eplace d onth estigmati c surface needed pollent omak egoo d contactwit hth e andi nsolid ,papillat e stigmaswer epushe d surfacebefor ether ewa san yresponse .I n downt omak egoo d contactusin ga custom - Bauhiniaan d Brownea speciesth epolle n mademicro-scalpel .Fo r Cassia stigmas grainsha d tob epushe d downont oth esurfac e pollenwa spushe d intoth estigm apor eusin g betweenth estigm apapilla ean d inbot h theglas smicro-needl e and largenumber so f specieson epolle ngrai nwa sno t enought o Cassiapolle ngrain scoul d beintroduce d provokea response .I nBrowne a 5grain swer e intoth epor e ina singl epollinatio n theminimu muse d and ofthes ea maximu m of4 attempt.Tw oo rthre egrain s (Bauhiniaan d hydrated.Th eesteras etes tdi d notappea r Caesalpinia)wer euse da teac hattemp ti n toindicat etha tther ewer ean y specific intergeneric crossesbecaus eo fgreate r receptive siteso nth epapill a surface,how ­ pollensize . eversinc e theBrowne a stigma iscoloure d red-brown localisation ofth ereactio n Results produced isdifficult . ForW Ntyp estigma scontac twit hth e 1.Stigm a structure secretionwa senoug ht oensur egerminatio n evenwhe nonl y asingl egrai nwa sused . Speciesca nb eseparate d into2 group s based onstigm astructure .Th e classification ofHeslop-Harriso n (1981)i sused . Table 1.Th eminimu m timet oobserve d WP Thestigm a issolid ,papillat ean d germination after self-pollination. appearswe ta tmaturity .Secretion ,whic hi s generated by thepapilla ean d cellsbeneat h thepapilla e (Owens& McGrat h inprep.) ,i s Species Minimum time morecopiou s in Cercisstigma sprio rt o toobservabl e pollination than inBauhinia ,Gleditsi ao r pollengerminatio n Brownea. (Mins.) WN Thestigm a iseithe r funnel-shaped and theinne rreceptiv e surface rugose WP (Caesalpinia)o r flask-shaped with anarro w Bauhiniatomentos a 60(16) entrance porean dno tpapillat e (Cassia). Cercissiliquastru m 55(25) Bothtype so fstigm aar ewet ,th esecretio n beingproduce d byth ecel l layers liningth e WN receptive surface.Amon gman y morphological Caesalpinia gilliesii 30(7) differenceswhic hexis tbetwee nstigma so f C.pulcherrim a 43(3) Caesalpinia and Cassiaa majo r distinction Cassia floribunda 20(40) isth elac ko fa cuticl eove r thestigmati c surfaceo fCaesalpinia .Th estigm ao f Caesalpinia appearst olac ka nepiderma l ()numbe ro fstigma steste d surfacelayer ,leavin gth estyla rtransmit ­ tingtissu ecell sa sth elaye ront owhic h pollenlands . Alarg esecretio ndrople twhic hcover sth e B Cassia whole stigmatic surfacea treceptivit yan d Becauseo fit sabundan t flowerproductio n intowhic h thepolle n lands isregularl y amor edetaile d examination could bemad eo f produced byth estigma so fCassi amarylandica , thepollen-stigm a interaction inCassi a _Cmimosoides ,C .nemophil aan d Caesalpinia floribunda.Tabl e2 show stha tth ereceptiv e pulcherrima.I ti spresen t irregularly onth e stigmai swe teve nthoug h stigmasrarel y stigma in Cassiaobtus aan dapparentl yver y producea secretio n droplet.Examinatio n of rarei nCassi afloribunda .I ti sno tproduce d hand cut sectionso fth estigm aclearl y atal lb y Caesalpiniagilliesi iwhic hdoe s showed thatth epolle ni sreceive d intoa havea copiou ssecretio n onth esurfac eo f largechambe r (itca nhol da tleas t 100 thestigm abu ti ti sno t enough togenerat e grains).Th echambe r isline d witha cuticl e adroplet .Th estigm ao fC .gilliesi ii s whichma y rupturebefor epollinatio n orma y larger than C.pulcherrima .Th esecretio ni s berupture d followingpollination .Ruptur e a clear,colourless ,water y liquid which

85 releases thesecretio nan d thisi softe n 3.Self-incompatibilit y accompanied byth ereleas e intoth e secretion ofhundred so fth ecell slinin g Preliminary datao n self-incompatibility thechamber .Th erespons e toth eesteras e suggeststha tth etw oBrowne a hybrids (both test (Pearse 1972)i svariable ,a positiv e highly pollen fertile)ar e self-incompatible reactiongenerall y occurringa ta sit ejus t while Cassia floribunda,Caesalpini agillies i belowth eporat eentranc et oth echambe ri n and C.pulcherrim a areself-compatible . intactstigmas .Polle ngerminatio n takes placebetwee n 20-30minute safte r pollination Discussion formos t self-an d cross-(intraspecific) pollinations. Thestigma so fCaesalpinioi d legumesca n beeasil y classified intotw otype swhic h possesssimila r characteristics toeithe r stigmaso fth ePapilionoidea e orth e Table2 .Th erespons eo fpolle ngrain so n Mimosoideae.A goo d exampleappear st ob e stigmaso fdifferen t states (weto rdry ) thesimila rmorpholog y ofstigma so f after eitherself -o r cross-(intraspecific) Caesalpinia (WN)an d those inAcaci aan d pollination in Cassiafloribunda . Mimoseae (Kenrick &Kno x 1981, 1982). Similaritiesbetwee n the Papilionoid areles seas y whencomparison sar emad ewit h Stigma state Pollen (individual Trifolium (Heslop-Harrison &Heslop-Harriso n response flowers) 1983)an d Phaseolus (Heslop-Harrison & Germination Nogerminatio n Heslop-Harrison 1984)bu trecen tunpublishe d work inou r laboratory on theprimitiv e Wet 31 19 Papilionoid genera Sophoraan d Swartzia Dry 21 Suggesta close rrelationshi p withthos e 'Caesalpinioideae inth eW Pgroup .Cuticl e rupturing,a characteristi c feature ofman y Papilionoideae (Heslop-Harrison &Heslop - Harrison 1983,Owen sunpub.) , appearst ob e Theresult so finterspecifi can dinter - required forth esuccessfu l pollinationo f generic crossesar e shown inTabl e 3.Al l ' Bauhinia andBrowne a (WP). However,i nth e pollensgerminate d although theresponse s Caesalpiniodeae,a swit h theprimitiv e were slower orth esam ea sfo rself - Papilionoideae thisi sno tassociate d witha pollination.Th eon enotabl eexceptio nwa s sophisticated trippingmechanism . inth ecros sinvolvin g Bauhinia pollenwher e amuc hmor erapi d responsewa sobserved . Itwoul d beinappropriat ea t thisstag et o overgeneralizeabou t theevolutio no fstigm a typeswithi n thefamil y fori ti scertai n Table 3.Th erespons eo fforeig n (inter­ thata substantia l number ofmorphologica l specific &intergeneric )polle no nth e typesexis thithert oundiscovered .Howeve r stigmaso fCassi a floribunda, C.obtus aan d therear eclea r indications (Owens& McGrat h Caesalpiniagilliesii . unpub.)tha tbot h inmorpholog y andstructur e stigmasi nth e Caesalpinioideae canb e divided intogroup swhic happea r tolin k Cross Min. timet o closely witheithe ron eo rothe ro fth etwo , germination moreadvanced ,sub-families . (Mins.) Thestigma so fal lspecie sar ewe t (see C.obtus a however thediscussio n ofHeslop-Harriso n & x C.floribund a 35(9) Heslop-Harrison 1983)an d contactwit h secretion isa necessar y pre-requisitet o C.obtus a germination.Th epresenc e ofsecretio ni n X Caesalpinia pulcherrima 45(9) Cassiafloribund a isno tstimulate db y pollination.Manua l pollinationma yruptur e C floribunda theintac t cuticleo fsom e stigmasan dthu s X Caesalpinia pulcherrima 45(10) bring thepolle n intocontac twit hth e secretion butpollinatio n itselfdoe sno t C floribunda appear toinfluenc eth egeneratio n of X Caesalpinia gilliesii 45(19) secretion. Thehig hrat eo fgerminatio n ofa wid e Caesalpiniagilliesi i selectiono fpolle n inth estigmati c x C.floribund a 30(4) secretiono fth etw o Cassia speciesuse dan d ofCassi apolle no nth e Caesalpinia stigma C.floribund a mayb erelate d toth eevolutio n ofcontrolli i 30(8) xBauhini a tomentosa systemsi nth estyl erathe r thana tth e stigmatic surface i.e. self-incompatibility inLeguminosa e isapparentl y astyla r ()numbe r offlower steste d reaction (DeNettancour t 1977). Itappear s Heslop-Harrison,J . &Heslop-Harrison ,Y. , thatth eexudate so fCassi aan d Caesalpinia 1984. Stigmaorganizatio nan d thecontro l stigmasar e justefficien tgerminatio n offertilizatio n inPhaseolus .In : media.Thi si sals ointerestin g inrelatio n Reimann-Phillip,R . (Ed.): Proc.Eucarpi a toth enon-specifi c exudation responseo f meeting,Hamburg ,Jul y 1983.p -88-96 . Acaciastigma st ovariou spollens ,a Heslop-Harrison,Y. , 1977.Th epollen-stigm a situationwhic hwa sconsidere d tob ea interaction:polle ntub epenetratio ni n generalpollinatio n signalinitiate d atth e .Ann .Bot .41 :913-922 . commencemento fpollen-stigm a interactions Heslop-Harrison,Y- , 1981.Stigm a (Kenrick& Kno x 1981). characteristics andangiosper m . Nord.J .Bot .1 :401-420 . Heslop-Harrison,Y .& Shivanna ,K.R. ,1977 . Thereceptiv esurfac eo fth eangiosper m Acknowledgements stigma.Ann .Bot .41 :1233-1258 . Kenrick,J . &Knox ,R.B. , 1981.Structur e Gratefulthank sar eowe d toM sS .McGrat h andhistochemistr y ofth estigm aan d style andK rL .Fo x fortechnica lassistance . ofsom eAustralia n specieso fAcacia . Aust.J .Bot .29 :733-745 . Kenrick,J .& Knox ,R.B. ,1982 .Functio no f thepolya d inreproductio n ofAcacia . References Ann. Bot.50 :721-727 . Nettancourt,D .de ,1977 .Incompatibilit y in Arroyo,M.T.K. , 1981. Breeding systemsan d Angiosperms.Monograph s on Theoreticalan d pollinationb iology inLeguminosae .In : Applied Genetics,3 .Springer-Verlag , Polhill,R.M . & Raven,P.H . (Ed.): Berlin. Advancesi nL egumesystematics ,par t2 . O'Brien,T.P .& McCully ,M.E. ,1981 .Th e CrownAgents , London,p .723-769 . study ofplan t structure^.Principle san d Polhill,R.M .& Raven,P.H. , (Ed.),1981 . selectedmethods .Termarcarphi ,Melbourne . Advancesi nL egumesystematics ,par t1 . Pearse,A.G.E. ,1972 .Histochemistry , CrownAgents , London,p .57-142 . Theoretical and Applied,Vol .2 . Heslop-Harrison ,J . &Heslop-Harrison ,Y. , Churchill,London . 1983. Pollen- stigmainteractio ni nth e Shivanna,K.R .& Heslop-Harrison ,J. , 1981. Leguminosae: theorganizatio n ofth e Membranestat ean d pollenviability . stigma inTr i folium pratenseL . Ann. Bot.47 :759-770 . Ann. Bot.5 1: 571-583.

87 ACACIABREEDIN G SYSTEMS

J.Kenrick ,P .Bernhardt ,R .Marginson ,G .Beresfor d and R.B.Kno x

Plant CellBiolog y Research Centre,Schoo lo fBotany ,Universit y ofMelbourne ,Parkville , Victoria 3052, Australia.

Summary

Breeding experimentshav e showntha t severalAustralia n speciesar esel f ^•'. -'t 777 A.pycnantha incompatible: A_^_pycnantha ,A .retinodes , 7•"-:• • ~- - A.terminalis ;partiall y selfcompatible : - IMLîîO I A.retinodes A,,myrtifolia ;an d selfcompatible : : 4 : "7 A.ulicifolia . Many ,especiall y ••$•-•-< 1 1 1 } Aterminal s 5 familieso fbees ,forag e forAcaci a ;7*.s,jv;7 polyadsan d servea spolle nvectors . 'fr]:'l ' Two genera arewide-spectru m , while theother ssho wnarrowe r - 1 11 1 [ „ 1 Z 2 specificities. Acacia terminalis isals o A ulicifolia visited bysmal lbird stha t foragefo r , ... /(— . thehexose-ric h inextra- l 0-2 10 INDEX OF SELF INCOMPATIBILITY nectaries thatappea r tob ea nadaptatio n forbir d pollination. Fig. 1. SIrelationship s infiv especie s Introduction ofAcacia . Open circle:mea nISI ; horizontalline :rang eo fIS Ivalues ; Acacia isa nitrogen-fixin g treegenu s arrows: individual parentvalues ;number s ofconsiderabl e economic importancean d 'beneath arrowssho wth enumbe ro fparent s there isevidenc e for self-incompatibility with indicated ISIvalue . (SI). Experiments atth eWattl e Research Institute inSout hAfric a showed that selfingresult s inreduce d seed seti n Inmos t ofthes especies ,polle n transfer A.decurren s and A^mearnsii . Wepresen t fromon etre et oanothe r isessentia lfo r resultso fou rbreedin gexperiment si n seed set. natural populations insouth-easter n Australia ofa summer-flowerin g species, Insectswer eattracte d toth enectarles s A.retinodes ;a nautumn-flowerin gspecies , flowersb ya combinatio n ofth emassiv e A.terminalis ;a winter-flowerin gspecies , yellowdispla y and strong fragrancestha t A.ulicifolia ;an d two spring-flowering varied between fouro fthes e species species,i Lmyrtifoli a and A^pycnantha . (A.ulicifoli a notye t studied). Beetles Wehav e alsoinvestigate d the-pollen ,.' and fliesat eth epolyads . Solitary bees, tl vectorso fthes especies ,wit ha vie wt o most commonly collected insects,gathere d establishing themode so fpolle n transfer polyadst ofee d tothei r larvae. Wehav e betweentrees . added twobe e families (Colletidaean d Megachilidae)t oth ethre efamilie sprevious ] knownt opollinat eAustralia n Acacia. The Resultsan d Discussion most constant pollinators,regardles so f seasono rhabitat ,wer eLeioproctu sspp . These specieso fAcaci a areprotogynous . (Colletidae)an d Lasioglossum spp. The stigma cup isjus tlarg eenoug ht o (Halictidae),whic har ewidel ydistribute d accepta sinlg epolya d and 80%o f and pollinated allfou rAcaci aspp . pollinationsar eo fthi skin d in Conversely,som ebee s showed narrowspecif ­ A.retinodes . Theuniqu e 16grai npolyad s icitiest oon eo rtw oAcaci a spp. For provide asyste m forpolle n transferi n example,specie so fEuhesma ,Hylaeu s which allth emeioti c products ofa nanthe r (Colletidae),Megachil e (Megachilae)an d loculusar eretaine d asa singl eunit . fiveou to fsi xHomalictu sspp . (Halictidae) Thismean stha t inmos tpollination sal lth e wererestricte d toA ^retinodes . seedsi na po dma yhav e the samefather . InA_ ^terminali sbee sshare d pollination SIha sbee n analysed interm so ffrui t dutieswit h smallbird s likethornbill s set,usin g the Index ofSel f (Acanthiza),silvereye s (Zosterops)an d Incompatibility (ISI)ie .yiel d offrui t spinebills (Acanthorhynchus). A.terminali s following self/crosspollinatio n (see bearslarge ,scarle t extrafloral nectaries Bernhardt etal. , 1984). Theresult swit h on itslea fpetioles . At floweringtim e 5 specieso fAcaci a showa rang eo fS I thesegland ssecret edrop so fnecta r rich levels (Fig.1) . inthre e sugarsan d 18amin oacid s (collaborationwit h DrsI .an d H.G.Baker , U.o fCalifornia ,Berkeley) . The flowering branchesar e strategically located arising inpair sfro mth enodes . Asbird ssearc h forth eglands ,an ddrin kth enectar ,thei r bodiescontac tth e flowering branches transferring polyadst othei rplumage . Observationso fth ebirds ,an d analyseso f thepolle n loadso fbird san d bees, confirmed thatbot hma y acta s pollenvector si nthi sspecie so fAcacia .

Reference

Bernhardt,P. , J.Kenrick ,an d R.B.Kno x 1984. Pollinationbiolog y andth e breeding systemo fAcaci a retinodes (Leguminosae:Mimosoideae) . Ann. MissouriBot .Gard . 71:1 7- 29 .

89 INCOMPATIBILITY BETWEENPINU SNIGR AARN .AN DPINU S SYLVESTRIS L.AN D OVERCOMING ITWIT H CHEMOMUTAGENIC SUBSTANCES

A.Kormut'â k

x • 1 Arboretum Mlynany-Institute of Dendrobiology CBES SAS, Vieska n. Zitavou, Czechoslovakia

Summary pollenbud s ofSect spin e seems tob emor e efficient in thisrespec ta s indicate the Using thepolle n grains ofScot s pine results of our investigation presented in /P.sylvestrisL. /mutagenicall y treated thispaper . with the two derivatives ofnitrosoure a and sodium azide itwa s possible to obtain Material and methods thehybri d seeds of interspecific combina­ tionP.nigr ax P.sylvestri swhic hunde r Twoattempt s with overcoming incompatible normal conditions is incompatible. barrier between theEuropea nblac k pine/P . nigra Arn./an d the Scots pine/P.sylvest ­ Introduction risL. /hav e beenmad e in theyear s 1978 and 1981.I n the first case one individual Hybridological status ofEuropea nblac k of theEuropea nblac k pine was used asfe ­ pine /P.nigraArn. /an d Scots pine /P.syl­ male parent,whil e in theexperimen tperfor ­ vestris L./ha s in the pastbee n thesub ­ med in198 1 three individuals of the same ject of certaincontrovers y which tosom e species were involved. The Scotspin e tes­ extent persists until no?/.Th e spontaneous ted asa mal e specieswa srepresente d by origino fP.nigr ax P.sylvestrishybrid s the two individuals separately used inbot h firstpostulate d in the year 1867i nAus ­ t&eabov e experiments. tria/Vidakovic , 1974/an d laterassume d ' Asa preparator y step to the artificial by Wettstein/1951 /an dVidakovi c /1958/ hybridization, the application ofN-nitro - wasprimaril y based onth e finding ofseve ­ so-N-methylurea/NMU/ ,N-nitroso-N-ethyl - ral individuals of intermediate morphology. urea/NEU /an d sodium azide /HaU /o npol ­ However,accordin g to the same author /Vi-< lenbud s ofScot s pine was performed inth e dakovic,1977/ , thehybri d nature of these yearprecedin g pollination.Th emixture so f putative hybrids hasneve r beenconvincib - thesemutagen s prepared witha vaselin e pas­ ly demonstrated.Also ,th e attempts tohyb ­ tehav e beenapplie d separately at theba ­ ridizeEuropea nblac kpin e withScot s pine ses ofbud s during their initiation inth e artificially,thoughpreviousl y reported as firsthal f ofJuly . In theyea r 1977onl y successful /Johnson, 1939;'//righ tan dGab ­ 25an d 50ni Mconcentration s ofNE U were tes­ riel, 1958/,hav e notbee n confirmed by the ted,wherea s the treatments in198 0wer e extensive experiments carried outb y VidaT extended of thevariant swrit h10 ,3 0an d5 0 kovic and Borzan/1973 / over a span of'1 ^ mM concentrations ofN aN as wella swit h years. ,10 »an d 50m M concentrations ofbot hNM Uan d NEU/Tabl e l/. Onaverag e 300bud s ofeac h Cytological examinationreveale d thatin ­ variantwer e treated inthi sway .A s acon ­ hibition of thepolle n tube growth inth e trolserve d the non-trea,ted buds ofth esa ­ nucellar tissue of the ovules is theprima ­ me tree. ry cause of theirabortiv e development/Vi ­ dakovic,Jurkovic-Bevilacqua , 1970/. Inor ­ Viability of thepolle ngrain s collected der to overcome thishybridizatio nbarrier , in the spring of the nextyea rwa s tested theapplicatio n ofirradiate d polleno f by their germination invitro .Th e pollen Scots pine aswel l as themento r pollen originating from theabov e treatmentsha s technique are recommended /Vidakovic et al., been separately used inartificia l pollina­ 1975/. In theirattempt s with disturbing tion of the ovulate strobili ofEuropea n themechanis m of incompatibility ofthi s blackpine .Th e conventional technique of crossing, theauthor s treated thepolle no f artificial pollinationwa sapplie d inwhic h Scots pine aswel l as the conelets ofEuro ­ the isolatingbag san d pollinator of own peanblac k pine with seven types ofchemi ­ constructionwer e used.Totall y 100ovulat e cal compounds involvinghormones ,growt h strobili were pollinated in 1978,whil e the substances and .Aqueou s solutiono f amount ofstrobil ipollinate d in.198 1reach ­ yeastwa suse d aswell .Non e of these tre­ ed the number 183.Th e exactproportion s of atments hashoweve r beenprove d todistur b the ovulate strobili pollinated by thepol ­ this-mechanism . Ourapproac hbase d onap ­ lengrain s treated by the individual types plication ofnitrosomethyl- ,nitrosoethyl - ofmutagen s are giveni n table 1.Th e qua­ urea and sodium azide on the developing lity ofmatur e seeds obtained was examined

1.Presen t addresss 9515 2 Slepcany, okr.Nitra , Czechoslovakia

90 byth eX-ra y radiography /Simak, 1980/.

Results and discussion

The results ofviabilit y test indicate thatapplie d chemomutagensha d differenti­ ally affected the ability ofmatur e pollen togerminat e but ingenera ladversel y in­ fluenced the growth ofpolle n tubes.A si t indicatesFig .1 , the lowest proportion of germinating pollengrain s nasregistere d in thesample s treated by NMJ. Theex ­ tento fgerminatin g pollengrain sascertai ­ nedi nit svariant s with 10an d 50m M con­ centrations hasa s arul e beenmaintaine d atth eleve l of4 4 and 47 %wha t represents thefigur e only a little worse thana con ­ trolwit h the 49% fraction ofgerminatin g pollenrevealed . The two other variants of treatment,i.e .wit hNE U and SaJ have on the otherhan d yielded the pollen samples 20 60 100 140 which consistently differed from a control sollen tube length,J> incontainin g the larger populations ofger ­ minatingpolle ngrains .Th e corresponding Fig.1 .Germinatio npercentag e and pollen parameters ranged between 54an d 59 i n % tube length inpolle ngrain s ofScot s pine the former case and between 57 and 65% in affected by 10m M/2 /an d 50m M/3 /NMU ,1 0 thelatte rone . mM /4/an d 50m M/5 /NEU , 10m M /6/, 30m M Sith the outlined variationpatter nha s /!/ and 50m M /8/l aH ; L-control onlypartiall y correlated thepolle ntub e intraspecific level,wherea s the species growth.I ncompariso nwit h a control,-achie ­ vingunde r experimental conditions themea n combinationP.nigr ax P.sylvestris-control lengtho f10 3 microns,wa s the growth of serves asa control variant at the inter­ pollen tubes inal l the variants ofmutage ­ specific level.I t isapparen t from the tab­ nictreatmen t retarded. The degree of inhi­ le thatbot h these controls differ strikin­ bitionvarie d considerably, depending on gly inqualit y of the seed progenies produ­ theconcentratio n of the applied mutagens ced.Th e intraspecific crossings ofP.nigr a whichha d generally reduced thepolle ntu ­ involving self-pollinations of thematerna l belengt h to the level of76,1-93, 9 microns treesan d cross-pollination ofdifferen t whati nrelatio n tocontro l represents sta­ individuals of the species have asa rul e tistically highly significant deviation. yielded the viable seed progenies.Th eef ­ However,i nspit e ofsuc h awid e variation ficiency ofselfin g differed annually but inlengt ho fpolle n tubes,th eresult sob ­ the presence ofsoun d seedswa s a constant tained from the individual types oftreat ­ feature of itsprogenie s inbot hyear s tes­ mentswer e comparable,irrespectiv e ofth e ted.Particularl y remarkable was theredu ­ chemical nature of themutageni c compounds ced amount ofsoun d seeds in this category utilized.Remarkabl e was the tendency dis­ ofprogen y harvested inth e year 1981wit h playedb y the derivatives ofnitrosourea , only 24 %o f fully developed seeds as com­ i.e.NM U and NEU whichbein g tested intw o pared with the 84% proportion of seeds of concentrations exerted their strongestre ­ the same category resulting from across - tardanteffect s in the variants with 10m M pollinationa swel l aswit h the 91,5 % of concentrations differing thus from the tre­ filled seeds obtained from selfing in1978 . atmentswit h the corresponding mutagens in With the above data contrasted the results 50m M concentrations.N o such tendency has of interspecific hybridization ofP.nigr ax onth e other hand been observed in the case P.sylvestris inwhic h the non-treated pollen ofJ aJ . ofScot s pine vas used /control/.A s it fol­ lows from table 1,i n the 1978experimen t The pollen grains ofScot s pine origina­ this combinationyielde d 0,39 %o f filled tingfro m the above described treatments seedswha t represents only four fully deve­ nave subsequently beenutilize d inartifi ­ loped seeds of the totalpopulatio n of102 3 cialpollinatio n of theEuropea nblac kpin e seeds obtained,whil e inth e year 1981n o Performed in theyea r 1981.Th e results of filled seedswer e revealed atall .Take n thisattemp t are summarized in the lower together, these data support incompatible part of table 1,whil e those referring to nature ofP.nigr ax P.sylvestri scombina ­ thehybridizatio n experiment in197 8ar e tion. tabularized in the upper part of the table. T he self-an d cross-pollinations of thema ­ Ina nattemp t toovercom e incompatibili­ ternal stand for the control atth e ty of this crossing, thepollinatio n of fe-

91 Table 1.Th e results ofattempt s with overcoming the incompatible barrier betweenPinu s nigra Arn.an dPinu s sylvestrisL . species Combinations tested Number of' Number of Number of Total Number of Percentage pollinate äcollected cones witï number sound of H male parents female mature full of seeds seeds sound CD CH strobili cones seeds obtained seeds P.nigra-selfing 24 16 16 983 899 91,50 P.sylvestris-oontrol 34 22 4 1023 4 0,39 CO P.sylvestris-25 mM NEU 42 20 11 793 24 3,00 P.sylvestris-50 mM NEU 80 42 19 1507 43 2,90 ho P.nigra-selfing 8 6 6 129 31 24,00 •H P.nigra-cross-pollination 42 26 26 1096 920 84,00 d P.sylvestris-control 17 13 - 269 - - ra P.sylvestris-10 mMN a N 18 4 1 149 1 0,60 3 P.sylvestris-30 mM Na^N 8 4 2 173 2 1,10 0 P.sylvestris-50 mMS a J 5 4 2 121 3 2,40 •H P.sylvestris-10 mM NMU 14 4 1 177 1 0,50 AH P.sylvestris-50 mM NMU 12 3 - 69 - - P.sylvestris-10 mM NEU 41 26 4 739 11 1,40 P.sylvestris-50 mM NEU 18 12 3 146 3 2,00 male strobili of thematerna l specieswa s nera.Canad . Jour.Res .C .17:411-444 . carried outusin gmutagenicall y treated Simak,M. , 1980.X-ra y radiography inre ­ pollen ofScot s pine.Particularl y efficient searchan d testing offores t treeseeds . was in thisrespec t the pollination in197 8 ^Rapporter Nr.3.Th e Swedish University of duringwhic h thepolle ngrain s originating / Agricult.Sciences ,Depart .Silvicult. , from treatments with2 5 and 50m MNE Uwer e Umea. used. Insuc ha wa y itwa s possible torai ­ Vidakovic,M. , 1958.Investigatio n onth e se thepercentag e of fully developed hybrid intermediate typebetwee n theAustria n seeds to3 an d 2,9 %, respectively. Onth e and the Scots pine.Silva e Genet.,7:12 - otherhand , theattemp t repeated in198 1 19. nad based on the involvement of twoaddi ­ Vidakovic,M. , 1974.Genetic s ofEuropea n tionalmutagen s /NaN an d NMU/»/a sles s blackpin e/Pinu s nigraArn./ .Annale s successful.Thehighes t proportion of filled Forestales 6/3:57-86. hybrid seeds resulted from the pollination Vidakovic,M. , 1977.Som e morphological with the polleninfluence d by 50m MN a N characteristics ofPinu s x nigrosylvis and reached the value of2, 4 %. Comparable /P.nigrax P.sylvestris/ .Annale sFores - resultsha dals obee nachieve d by the pol­ tales 8/2:15-27. lengrain s treated with 50mM REU»/her e2,J é Vidakovic,M .an d Jurkovic-Bevilacqua,B. , offille d seedswer e obtained.The variant 19^0. Observations onth e ovule development with application ofpolle ngrain sderive d following cross-pollinationbetwee nAus ­ from the treatmentwit h NMUha s onth e trianan d Scots pines using irradiated otherhan dbee nles s efficient indistur ­ and non-irradiated pollen.Proc .Sexua l bing themechanis m ofincompatibilit y bet­ Repr.For.Trees,Vol.3 ,Varparanta ,8pp . weenbot h the pine species.Amon g 177seed s Vidakovic,M .an d Borzan,2. ,1973 .Contri ­ of this class only 1see d was revealed to bution to the investigations ofincompa ­ be fully developed /0,5 %/. tibility by crossing Scots pine andEuro ­ Summarizingv/ eca nstat e thatamon g2.30 0 peanblac kpine .Intern .Symp . Gen.Scot s seeds collected from the interspecific cros­ pine. IUFR0Divis .2 ,Workin g Party S2 . singP.nigr ax P.sylvestri si n1978 ,6 7 0.3.5., Warszawa-Kornik ,1973 . seedswer e detected tob e filled,wherea s Vidakovic,M. ,Borzan ,Z .an d Jurkovic-Bevi­ the experiment repeated in198 1resulte d in lacqua,B ., 1975 .Breedin g ofEuropea nblac k 21hybri d seeds.Expresse d inth e relative pine and Scots pine using irradiated pol­ values, these data represent 2,91 and 1,33 len.Act a Biol.,/Zagreb /7/4:309-317 . %o f filled seeds,respectively , obtained Wettstein,S .von ,1951 .Übe r BastardePi ­ with ahel p ofmutagenicall y treated pollen nus nigra var.austriac a x silvestris. grains ofScot spine . Ztschr.Pflanzenzuchtun g30:473-477 . Wright,J . W.an d Gabriel,W .J. , 1958.Spe ­ References cieshybridizatio n in thehar d pines,se ­ ries Sylvestres.Silva e Genet.,7:109-115 . Johnson,L .P .V. , 1939.A descriptive list ofnatura lan d artificial interspecific hybrids inNort hAmerica n forest-tree ge-

92 IMMEDIATE DISTINCTION BETWEEN SELF-POLLINATION AND CBOSS*POLLINATIONAN D BETWEEN SELF-COMPATIBILITY AND SELF-INCOMPATIBILITY IN PAPILIONACEAE - PRELIMINARY BEPOBT

W.Wojciechowak a Institute of Plant Genetics,Polis h Academy ofScience ,Poznan , Poland

Summary fore they are pollinated rather rarely or in case of cleistogamy areno t pollinated Suitabilityo f "now"morphologica l tra­ by insects at all.Nearl yever y flower is itsa s indices of pollination mode was pollinated inthes e plants,becaus e own tested onsevera l species of Papilionaceae pollen always fits to own stigma and it Keywords:Papilionaceae , self-pollination, alwaysmature s inth emos t suitable time. cross-pollination, self-compatibility, Poorly blooming causesther e are rather self-incompatibility. enough available resources tomatur e every pollinated flower into fruit Aable 1/, Introduction Themod e of pollination has the funda­ Table 1.Fructificatio n in "typically mental influence on the next generations. self-pollinated plants". Progenies of full self-pollinated plants are genetically uniform while the proge­ nies of full cross-pollinated plants are mode of Per CO n ta ge genetically very different.Ful l self-po­ Species polli­ 0 f llination appears in case of cloistogamy nation podsi »et seeds set and- contrary to it- the absolutely cross-pollination appears inmonoalinon* , Vicia angu- bisexual plants population in case of full stifolia L. open 91.9 87.7 self-incompatibility. But we exactly know there areman ymode s of pollination bet­ Vicia hir­ open 95.7 90.0 ween full self-pollination and full cross- sute/L. / pollination.I tmus t be also noted that S.F .Gra y isola­ themod e of pollination may vary depending ted 80. 4 95.0 on external conditions and- like other traits- undergoes evolutional changes. Ornithopus isola­ Therefore certain adaptations to cross-po­ sativus L. ted 88.7 82.5 llinationma y be noted inactuall y self- pollinated plants- e.g. , brush mechanism in - and vice versa.Thu s at least So such plants,a sw e can see on the several easily recognized traits have to Table 1, in sufficient environmental con­ be considered in plants populations for ditions can produce in infruetescence the accurate determination of the pollina­ nearly suchman y pods as there were the tionmode . Inman y botanical and agricultu­ flowersan d nearly suchman y seeds in pods ralmanual s there are the pieces of advi­ as there was the number of ovules.I hav e ceho w to recognize amod e of pollination also assumed that inthes e typically self- e. g., such well-known books aswritte n by pollinated plants the strong tendency sho­ Allard or Elliot.T o recognize more pre­ uld appear to form short styles of pistil. cisely amod e of pollinational l authors But why do Ihop e to see such a tendency? advise to do the special experiments.No w The style of pistil - ingametophyt e sys­ I want to show that "new"morphologica l tem of self-incompatibility in Papiliona­ traitswhic h have not been used till now ceae - is like a "strainer"whic h inhibits for this purpose help ustha t inman y ca­ the inadeqate pollen tubes from the pro­ ses these experiments could be omitted. cess of growing.O f course,suc h a strai­ ner in the plants which have mostly "deci­ Besults and discussion ded" for self-pollination is unnecessary. There ison e more tendency in typically For easier presentation of the results, self-pollinated plants.I hav e noticed investigated plants have been divided into that the petals of these plants come out the groups according to theirmode s of slowly from the calyces and therefore, in florescence,pollinatio n and fructifica­ adult flowers the calyces are rather big. tion. "Typically self-pollinated plants" /W.Wojciechowsk a 1983/.Th e petalso f the have been included into the first group. typically self-pollinated flowers dono t It consists of such self-pollinated plants have to growver y fast because they do not which often bloom rather poorly and there­ need toattrac t the insects.Slo w growing of petals whichar etogethe rwit h genera­ usually more plentifulan dcontrar yt oth e tiveorgan s deeply hiddeni sth ecalyx , second groupi tmus tb epollinate db yin ­ isusefu li nthi s case becausei tprotect s sects.Th epercentag eo fth epod sse ti s these organs from damaging, diseasesan d distinctly lower similarlyt oth esecon d soon .Whe nw ese eth eplant s withth e afo­ group thanth e numbero fflower san dth e resaid traitsw enigh t assume that there percentageo fseed si npod s isals o high is typically self-pollinated population. as inth epreviou s group /Tabla3/ . Inmos t "typically self-pollinated plants" examinedb ym eth eshor t stylesan dlon g calyces have been foundbu tmor e research Table3 .Fructificatio ni nentomophilou s works shouldb emad e /Figs. 1-7/. but self-compatible plants.

Mode of Percentage Species polli- of nation podsse t seedsse t

open 42.9 82.6 Astragalus isola- glycyphyl- ted 1.5 los L. artifi­ cial 47.5 77.3

lupinus °Pen 25'° 80.5 JT^Idl1- artifi- inflorescencesse t * cial pods with seeds

Figs. 1-7.Pistil s from "typically"self , pollinated plantswit h short styles- accordingt om esuc ha goo d seedsse ti n 1.Vici aangustifolia ,2 .V ,tetrasperma , podsi nentomophilou s plant« indicates 3.7 .hlrsuta ,4 .V .sativa ,5 .Tetragona - their self-compatibility. Self-compatibi­ lobus purpureus,6 .Gllcyh e hipsida, lity likea sa self-pollinatio n Increases 7* Ornithopus sativusx 5,3 . the chanceo fpollinatio nan dfertilisa ­ tion.A sw eca nse eo nth etabl e3 , the The second group consistso fsuc h self- legitymacyo fthi s assumptionha sbee n pollinated plants which bloom rather plen­ proveda ttw ospecies ,bu to fcours ei ti s tifullyan dtherefor e therear eals o often not sufficient.I forese e that very good pollinatedb yinsects .The y produce,in * seedsse ti nentomophilou s speciesca nb e contraryt oth efirs t group, distinctly as*a rul e connected with their self-compa­ less pods there were flowers.Bu tth enum ­ tibility.O nth eothe rhan d poorse t need bero fseed si npod si snearl yth esam ea s not always Indicate self-incompatibility. thenumbe ro fovule s similarlyt o the Asw eknow , besides self-incompatibility first group.I ti s called "self-pollinated therear eothe rmechanism sa s- e . g., and partly entomophilous plants"/Tabl e2 / dichogamy which prevent from self-polli ­ nation. Iti sjus t impossible,havin g onlyth e Table2 .Fructificatio ni nself-pollinate d table data,t odistinguis h self-pollina­ and partly entomophilous plants. tedbu tpartl y entomophilous plants from entomophilousbu tself-compatibl e ones. Mode of Percentage Accordingt om yhypothesi s both aforesaid Species polli- o f groups couldb eeasil y distinguishedb y nation podsse t seedsse t accurate observationo fa tleas t several infructescences.Th emai n factor whichli ­ mitsth epod sse ti nth esecon d groupi s Lupinus availabilityo fresources .Beside si tthe ­ luteusL . open 27.8 83.6 rei sals o another important factori nth e third group whichca nfor mth einfructes - Lupinus cence.Thi si sth e"disposition "o fin ­ albus I>. open 52.3 96.2 sects.Thei r "indisposition"cause db yth e bad weather conditions during blooming bringsabou tth egap s betweenth epods .O f "Entomophilousbu tself-compatibl e coursea sth e"indisposition "o finsect s plants"belon gt oth ethir d group.I ti s isa rando m event,a tleas t several infruc similart oth esecon d groupbu ti tbloom s tescencesmus tb elooke d through because

94 itnee d not be any gap ineach .Phot o1 these cases some viable seeds could be set showsth e appearance of entomophilousan d after self-pollination.Suc h events are autogamous lupins infructescence /Photo 1/ very well documented especially in Polish literature /Barcikowska 1966,ltlynie c1962 , Spies 1972,Wojciechowsk a I963an d many others/.O f course insuc h cases a percen­ tage of seeds set should be higher than in full self-incompatible plantsbu t lower than in self-compatible ones. At the end Ipresen t shortly the"new* * traits or tendencies which should be exa­ mined usingmor e species: 1.Th e length of the style of pistil - in typically self-pollinated plants we ex­ pect the tendency to form the short styles. 2.Th e sise of the calyx in the ratio to the petals- intypicall y self-pollinated plants we expect the tendency to form the long calyces,bu t these traits are only used in cases when the fundamental unit which attracts the insects isa singl e flower. 3.Th e position of pods in infructes­ cence - the gaps between the pods indicate Photo 1.Appearanc e of lupins infructes­ cross-pollination. cence- I- entomophilou s specieswit h the 4.Th e very good seeds set in entomophi­ gapsbetwee n the pods!a - Lupinus polyp- lous plants indicates their self-compati­ hyllus,b - L.autabilia , II- autogamous bility. / specieswithou t the gaps:e - i. Intens, d- l.albus . Table 4 presents fructification in ento­ Acknowledgment mophilous full self-incompatible plants. I wish to express my thanks to Professor Stanislaw Sulinowskl forvaluabl e discus­ Table 4.Fructificatio n in self-incompa­ siono f the results. tible plants. Mode of Percentage References Species polli­ o f nation pods set seeds set Allard,B .W. , 1960.Principle s of plant breeding. John Willey and Sonc. Inc., open 34.5 32.5 New York.p . 31-42. Coronilla Barcikowska B., 1966.Self-fertilit y and varia L. isola­ inbreeding depression inwhit e and ted 0.0 0.0 yellow sweetclover /Melilotus albus Desr. and Melilotus officinalis A./ Desr. open 62.5 34.5 Gen.Pol .? : /I/ 1-H. Elliot,F . C, 1958.Plan t breeding and Lathyrus isola­ cytogenetics.M c Graw-Hill Book Company, praten­ ted 0.0 0.0 Inc. NewYork ,Toronto , London,p . 1-55. sis L. Mlyniec W., 1962.Th emechanism s ofpolli ­ artifi­ 3.3 5.0 nation and generative reproduction in cial aphides Vicia villosâ.Gen .Pol .3 :/3 /285-299 . Spiss L., I972.Wply w geitonogamicznego zapylenia naosadzani e nasion u roélin motylkowatych drobnonasiennych.Hodowl a The attention canb e put to the lowper ­ roslin inasiennictw o - biuletyn branzo- centage of seeds set in these plants.Thi s wy.4 5 /37/15-18 . percentage should be always lower than in Wojciechowska B., 1963.Embryologica l stu­ the previous groups but itma y differ de­ dies inth e genus Lotus.Par t I.Ferti ­ pending on the external and internal con­ lization and seed development following ditions from year to year and from popula­ open- and self-pollination of Lotus tion to population.Beside s enthomophilous corniculatus L.Gen .Pol .1 :/4 /53-63 . full self-incompatible plants there are Wojciechowska W., 1983.Th e development only partly self-incompatible ones.I n rhytm of the flower-bud in some Papilio- such plants the mechanisms of self-incom­ naceae species.III .Acta Soc.Bot .Pol . patibility areno t quite efficient.I n 52:/! /9-21 .

95 CYTOLOGICALAPPROAC HO F STERILITYAN D INCOMPATIBILITY INTE A (CAMELIA SINENSIS)

A.Serrhini ,J.P .Tilqui n& E .HABINTOR E

Cytogenetical Laboratory,Carno y Institut,Catholi cUniversit y ofLouvain ,4 Plac ed el aCroi x duSud ,B-134 8Louvain—la-Neuve ,Belgiu m

Summary

Microscope observations ofself-incompati ­ pollen tubes reaching theovule s isdistinc ­ bility and sterility inth e teaplan t show tlylower . that thetim enecessar y toreac h theovule s istwic ea slon gafte r self-pollination than 2.Observation s onsterilit y after crossing.A lowproportio n ofth e embryo-sacs faildurin g thedifferentiatio n Theovar y istrilocula rwit h 4ovule spe r of thegametophyt e or showsom eirregulari ­ loculus andaxil eplacentation .Th eovul ei s ties.Th e seed sterility ofth ete aoccur s anatrope,th e embryo-sac isbispori c ( ato rafte r fertilizationo f theovule san d type)an d embryo iso f theSolanu mtype . couldb eattribute d togeni eo rphysiologi ­ Ovules showing abnormal development areno t calfactors . frequent.Abou t1 1 % fai lbefor e thematura ­ tiono fembryo-sac .Som eembryo-sa c (9% ) Introduction containon eo rsevera l supernumerarynuclei , differentiated ornot .Th emos t frequentcas e Tea ispropagate d bycuttin g inorde rt o isa duplicatio n of thewhol econten t ofth e secure theuniformit y of thecultures .Th e enforyo-sac.On eovul e showed 24nuclei .Some ­ shortcoming of thatfor mo fpropagatio n isa times,onl y the sexualapparatu s isduplica ­ lacko fgeneti c diversity inth epopulatio n ted.W ehav eobserve d pollen tubesreachin g that couldhav e serious consequences inth e themicropyl e ofovule swit h duplicated em­ caseo fepidemi c diseaseo rdrought .See d bryo-sacs,bu t theultimat e fateo fthes e multiplication couldb e favourable tonow , abnormal embryo-sacs isno tye tknown . sexual reproduction of the teaplan tha s Theabortio n ratebefor e thematurit yo f littlebee n investigated.Th e sterilityan d embryo-sacs isno t sufficient toexplai nth e self-incompatibility maymak edifficul t the actual levelo f seed sterility.Tha t level crossing andbreedin g of somecultivars . ishig h and couldb edu et ogeni eo rphysio ­ Thepresen twor k isa cytologica l studyo f logicalphenomeno noccurin g atth efertili ­ thecause s of sterility and incompatibility zation timeo rsoo nafte r inth ete aplan t afterartifica lpollination .

Results »'

1.Observation s on incompatibility

1.1.I nvitr o germinationo fpolle n

Pollengrai n isbinucleat e and showsthre e pores. Invitr o germinationha sbee nachie ­ ved ona cultur emediu m containing only 8% sucrose and 1% agar ,a tp H= 5 .A fewminu ­ tesafte r sowing thepolle ngerminate san d nearly 90% o fnormall y differentiatedpolle n grainshav eproduce d atub eafte rabou t3 hours;8 5% o f thepolle n isapparentl yfer ­ tile.

1.2.I nviv opolle n germination andpolle n tubegrowt h

Onehou r afterpollination ,th egermina ­ tiono f theself-polle no nth estigm a isa s good asafte rcross-pollination ,bu tth e elongationo f thepolle n tubes isslower . The levelo f theovule s isreache d after Squasho f style showingpolle n tubesan dthei i about 40hour s forcrossin g and 72hour sfo r calloseplug s (fluorescence). selfing.I ntha t latercase ,th enumbe ro f

96 INCOMPATIBILITY INCAMELI A SINENSIS;A GENERA LAPPROAC H

J.P.Tilquin ,A . Serrhini& E .Habintor e

Faculty ofAgricultura l Sciences,Departmen t ofCro p improvement,Burund i University,B P 2940,Bujumbura ,Burund i

Summary

Inspit eo f itseconomi c importance,th e carryingmal e gameteswhic h are incompatible teaplan tha s littlebee n investigated on with thefemal eplan t arearreste d inthei r itsreproductiv ebiology ; thecro pproduc ­ growth inth e stylean d so,fai l toreac h tioni svegetativ e and clonal selectioni s theembryo-sac .Th e reactiono f thepolle n themos t important ofbreedin gstrategies . towards thediploi d tissuesma y insom eca ­ However,a goo dknowledg e of itsincompati ­ sesb edetermine db y thehaploi d genotype bility system isno twithou t interest;sinc e of individual pollengrains ,i nsuc hcase , stumpfro m seed garden (biclonal)i sneces ­ the incompatibility reaction issai d tob e sary insom edr y areas,furthermore ,atten ­ gametophytic.I nothe r instances,th epol ­ tioni sfocuse d onth ehybridizatio n ofma ­ len'sreactio n isdetermine d by thediploi d terialso fwidel y different geographical genotype,the yal lbehav ephenotypicall ysi ­ provenance forhybri d vigour and anove l milarly towards the style,suc hcase sar e objective isappeare d :oi l extractionfro m said tob esporophytic . seeds.Th esterilit y ishigh ;one ,tw oo r Theobromacaca o isa nanomal y inth eincom ­ threeseed s for 12ovule spe r fruit andfrui ­ patibility world;th e incompatibility system tingrat e is20-3 0% .Afte r severalinvesti ­ isno t established inal l thetree s (Cope, gations sucha sdialle l crosses,polle nger ­ 1967-1958;Bouharmont , 1960).Th eincompati ­ mination,cytologica l examinations,i tseem s bility reaction isdelaye d untilmal ean d tob ea ne w incompatibility system.A lik e female gameteshav eme t inth eembryo-sa c or inth etw omajo r systems,autosterilit y is insom ecases ,durin g theearl y development therule ,bu t thepolle n tubes inhibition of theembryogenesi s (Bouharmont, 1960). doesno t occur inth e styleno r inth eovary . Oncepenetrated ,th eovule s areno trecepti ­ Alik e inTheobrom acaca owhic h ispartl y ve towards any otherpolle n tubes,s otha t autofertile,incompatibilit y reactioni s anenormu swast eo f femalemateria l isrea ­ localized inth eembryo-sa c aftergametophy - lizedwhe n sometree sar eselfed ,5 0% non - ticfusion ;th e system isthu sprimitive ; fusiono rabortio n appear inth eovary ,du e otherspecie swhic h discriminate against toencounte r of carrying thesam e incompatible pollen tubes inth e stylehav e allele.Th econtro l isdoubl y gametophytic theadvantag eo fconservin g their female (Bouharmont, 1960). Only theheterozygou s gametesunti l such timea scompatibl epolle n combination ofS-gene s isviable . arises.Suc ha syste mwher e autopolleni s Knight and Rogers (1953-1955 )suggeste d that always thefirs t on the stigmacoul d explain a single locuswa s concerned which showed high sterility encountered ina polyclona l multiple allelomorphism,wit h dominanceo r seedgarden . equality relationshipsbetwee nalleles .Fo r Keywords :tea ,incompatibility ,sterility . Cope (Cope, 1957-1958),tw oothe r lociar e required besides theS-locus ,thes ehav e Introduction beencalle d Aan d Ban d are thought tob e concerned with theproductio n ofa non-spe ­ Therear e severalkind s ofself-incompati ­ cific incompatibility precursorupo nwic hth e bility systems inplan t kingdom,som echa ­ S-allelesac t togiv e specific incompatibili­ racterized bymorphologica ldifferentiatio n tyreactions .Thi s system isprimitiv ean d ofth ematin g types (heteromorphic sucha s seems tob ealik e that found insom eOenothe ­ inCinchona) , someno t (homomorphic). Inth e rawher e themaintenanc eo fheterozygosit y majorityo f thereporte d cases ofincompati ­ isassure d by abalance d lethal systema t bility,a reactio n of thepolle no rpolle n zygotic levelwhic h involves a5 0% reductio n tubewit h the stigmao r the style isinvol ­ insee d set.Fo r Cleland (1972), inthes e ved.I nincompatibl e pollinations,th eger ­ Oenothera,th enon-fusio n or theabortio no f minationo f thepollen ,whe napplie d toth e fertilized ovules isno t duet oa gene ,bu t stigma,ma yb e totally inhibited;o r ifger ­ toth eabsenc e ofnecessar y genes togamete s minationdoe s takeplace ,th epolle ntube s fusiono rcoordinate d development ofembry o

1-Cytogenetica l Laboratory,Carno y Institut,Catholi cUniversit y ofLouvain ,4 plac ed el a Croixd uSud , 1348Louvain-la-Neuve ,Belgiu m and endosperm.A complementatio n betweenth e several pointsmus tb e investigated;th e male and female gametes secures thefertili ­ growth ofmixe d auto-allopollen tubes inth e zationproces so rth edevelopmen t ofth e style,th enatur eo fth eabortio n inth ede ­ seed andmaintain s theheterozygosity . veloping ovules.

Results and discussion Acknowledgements

Onecompatibl efécondatio nassure sth e Theauthor s thank Prof.J . Bouharmont,th e fruitdevelopment . generalDirecto r ofISAB U (Institut des Sci­ Theembryo-sa c isbispori c (Alliumtype ) encesAgronomique s duBurundi )an dhi scolla ­ andno t asPolygonu m type (Simuraan dOoso - borator,M rJ . Flémal,responsibl e ofth e na, 1956).Th ebispori c natureo f theembryo - teaprogramme . sacma ycomplicat e theunderstandin g ofth e incompatibility system. Insuc ha case ,th e References reactiona tth ezygoti c and endospermicle ­ velwil lb edifferen t andma y explainth e Bouharmont,J. , 1960.Recherche s cytologiques abortion ofovule s after someday so fdeve ­ sur lastérilit é et l'incompatibilité chez lopment. Theobromacacao .Publication s I.N.E.A.C., Abnormal gametophytogenesis may explain série scientifique n°89 . apartia l sterility; 11% o fth eembryo-sa c Cleland,R.E. ,1972 .Oenothera ,cytogenetic s aredegenerate d atanthesi s and some9 % and evolution.Acad .Press ,Londo nand 'Ne w contain surnumerical nuclei.Thi sovula r York. sterility isprobabl y variable fromclon e Cope,F.W. , 1957-1958.Incompatibilit y in toclone ;i nannua l crops produced by seed, Theobroma cacao.A repor t onCaca oResearc h therei sintensiv e selection forreproduc ­ I.C.T.A.,Trinidad :7-17 . tivenormalit y but inclona lcrops ,a degre e Knight,R . &H.H .Rogers ,1953 .Sterilit y in ofreproductiv e derangement ispresen tan d Theobromacacao .Nature ,London ,172:164 . sometimes sohighl y developed ast oforbi d Knight,R .& H.H .Rogers ,1955 .Incompatibi ­ normal sexual reproduction (Tilquinan d al, lity inTheobrom a cacao•Heredit y 9:69-72. 1983). Simura,T . &K .Oosone , 1956.Studie s onth e Theautopolle n growsmor e slowly thanal - fertilizationo f teaplant .J . J.Breedin g lopollenwhe napplie d separately onth e 6:114. stigmabu tw ekno wnothin g about thegrowt h Tilquin,J.P. ,K .d eBrouwe r &M .Calier , of themixe d pollen.Th etw o typesreac hth e 1983.Effect s ofvegecultur e onsexua lre ­ embryo-sac.Th erat eo f seed issignifican ­ production.I nFertilizatio n andembryo - tlydifferen tbetwee n thebasa l- an duppe r genesis inovulate d plants,VEDA ,Bratis ­ ovuleswhil e theabortio ndurin g thedeve ­ lava,Czechoslovakia . lopment isa trandom ,suggestin g thatauto - pollen isth efirs t tovisi t thebasa lovu ­ les. From thedialle l crosses,som e features mustb epointe d out: - atota lautosterility ;th e incompatibility mechanism isthu sdifferen t fromth ecaca o system. Itmus tb e sporophytic.Th emarke d differencebetwee n reciprocal crosses argues inthi smeaning .A strong effect of thefe ­ male cytoplasm (Tri31/ 8x Tr i31/1 1 -7 5% offruitin gan d thereciprocal ,Tr i31/1 1 x Tri31/8,1 6% - a sibcross) .A greatdele ­ tion ismor edecisiv e inth eembryo-sa c than inth emal egamete . Someobservation s showdominanc erelation ­ ships,fo rexample ,i nth ebiclona l seed garden ofKayanza ,onl y oneclon e (IB92 ) setfruit ,th eothe r (Tri31/11 )i stotall y sterile.Thi s latter isfertil e inth epoly ­ clonal seed gardeno fNyakagez i (seeabove) . Thedominanc e relationships couldb eexplai ­ nedb y theimportanc e of thedelectio ncar ­ riedb y femaleo rmal egametes ;th eabsenc e ofon egen e isrecessiv e toth eabsenc eo f twogene s and soon . The incompatibility system inCaméli asi ­ nensis isunknow n inplan tkingdom ;t oge t thefundamenta lknowledg e onth ebreedin g systeman d seed production of the teaplant ,

98 POLLENTUB EBEHAVIO RB YCARBO NDIOXID E INRELATIO NT OOVERCOMIN G SELF-INCOMPATIBILITY IN BRASSICA

T.Nakanishi& M.Sawan o

Department ofAgricultur e andHorticulture ,Kob euniversity ,Kobe ,Japa n

Summary Resultsan d discussion

Studieso f selfed-pollen behavior following Most ofth eself-incompatibl epolle ngrain s applicationwit hhig hconcentratio n of-C0 „ produced snaked orradiall y coiledpolle n gas,whic hha sbee nnote d toovercom eth e tubesont oth estigmati cpapill acells . self-incompatibility reaction inCruciferou s These tubeswer eno tunifor m inwidth . speciesreveale d that significant changesi n Pollen tubestreate d by5 % o f C0„gre w thepolle n tubegrowt ho fgerminatin g grain fat, whether thetube s spread overth e onstigm a occurred prior toit spenetratio n papilla cello rpenetrate d intothem . The intoth epapill acell .Remarkabl e increasei n thickest regiono f thepolle n tubegrow ni n pollentub ebreadt hwa sfoun d todepen do n highcone ,o fC0 „ga swa sapproximatel y 10ja m theconcentration s of C0„whic henable sth e ascompare d to5 ja mi nth econtrol . pollent ogerminat e andprotrud eth etub e Callosedepositio nwhic h looked likelens e intoth estigmati cpapilla eagains t theself - alwaysappeare d onth eti po fpapill a cells incompatible response.Observatio n ofthes e where self-incompatiblereactio noccurred . pollentube susin g SEMals oreveale d that Thesympto m of thesecallosi c reactionstil l mosto f thepolle n tubes enlarged atthei r appeared onth epapilla ,whe nself-incompati ­ tipso fconnectin g region toth epapill a cell. bility had beenbroke ndow nb yC O gas. Significance ofth erespons e ofpolle n tubes Pollen tubebehavio rwa sals o studied in tohig hC0 „concentratio n isdiscusse d in response to1,3.1 0an d 20% o fC O inair . relation tothei r effectupo nsubsequen t Thedistributio n of thepolle n tubewidt h penetrationan d overcoming self-incompati­ showed that thepolle n tubegrowt hwa saffect ­ bility. edb y theconcentration s of C0„gas .Abou t 30% o fth etube s treated with 3% C0 „wa s Introduction over 3time s thicker than thecontrol . Pollen tubespenetrate d into thepapill acell s Carbondioxid eapplicatio n toself-incom ­ whenth ega sconcentration swer e3 t o1 0% . patibleBrassic a speciesha sa grea tadvan ­ Pollen tubebehavio r onstigm awer eals o tagei nproducin g selfed seedswhic har e observed by theus eo fSEM .Th epolle ntub e necessary toobtai n inbred lines forF seed whichwa senlarge d byC0 „showe d simillar production (Nakanishi& Hinat a 1974). behavior totha to f self-incompatible ones. Previouswor k (Nakanishie t al. 1969)showe d Thetube softe n severely ridged botha tth e thathig hC O ranging from3 t o5 % i nai r contact regionan d theinne rwal lo fth e enables theself-incompatibl e pollent o papillacell . penetrate into thestigmati c papillacell . Thepresen t study indicated thatincrease d Further studies revealed theeffectiv etim e atmospheric C0„ga st oovercom eself-incompa ­ ofC0 „applicatio n for self-pollination tibility could haveaffecte d pollenb ychang ­ (Nakanishi &Hinat a 1973). Theobjectiv eo f ingth emetaboli c pathway of thetub egrowth . thisstud ywa s todemonstrat e thepolle n behavior onstigm afollowin g C0„treatment . References

Materials &method s Nakanishi,T. ,Y .Esash i &K .Hinata ,1969 . Controlo f self-incompatibility byC0 „ga s Self-incompatible strainso fB.oleracea , inBrassica .Plan t &Cel lPhysiol .10:925 - D.ercoides andB.campestri swer eused . 927. Flowerswer epollinate d and thentreate dwit h Nakanishi,T .& K .Hinata ,1973 .A neffectiv e 1-20 %o fC0 „usin g air-tight flask,fo r 6h r timefo rC O gastreatmen t inovercomin g at23°C .Stigma swer e collected and stained self-incompatibility inBrassica .Plan t& withwate r blue forfluorescen t observation. CellPhysiol .14 :873-879 . Eachpolle n tubegrowt ho nstigm awa sevalu ­ Nakanishi,T .& K .Hinata ,1974 .Self-see d ated bymeasurin g thewidt h of itsnarrow , productionb y C0_ gastreatmen t inself - median andwid eportion . incompatible cabbage.Euphytic a24:117-120 . Stigmaswer e alsosubmitte d toth escannin g electronmicroscop y toexamin e theeffec to f C0„applicatio n onth eprofil eo fpolle n tubesadherin g toth epapill acells .

99 CARBON DIOXIDE BLOCKSTH ESTIGM A CALLOSE RESPONSEFOLLOWIN G INCOMPATIBLE POLLINATIONS INBRASSIC A

P.O'Neill ,M.B .Singh ,T.F .Neales ,R.B .Kno x and E.G.William s

Plant CellBiolog y Research Centre,Schoo l ofBotany ,Universit y ofMelbourne ,Parkville , Victoria 3052,Australi a

pure CO to a concentration of3 % (v/v)an d Summary fed into container B. Both containersA (ambient air)an d B (3ÏC0 ?)wer eplace d in Carbon dioxide enrichment is known to the light at 25°C After 22 hours the overcome sporophytic self-incompatibility in -1 pistils were fixed in acetic acid:ethano l Brassica. Elevated C02 (30mmo l C02 mol (1:3), washed in 70? (v/v) ethanol and air), supplied via a flow*-through gas stained with decolorised aniline blue. system,wa s shown to block the formationo f Fixed pistilswer e thenlightl y squashedan d rejection callose in the surface stigmatic observed by incident fluorescence papillae of Brassica campestris following microscopy. self-pollination. Possible mechanisms by which COp may affect callose formation are Resultsan d Discussion discussed. Unpollinated pistilsfro mbot hambien tai r Keywords: Brassica campestris, callose, and 3% C0? treatments showed no fluorescence self-incompatibility, carbondioxide . in stigmatic papillae, although a strong callose rejection response was observed in Introduction aelf-pollinated stigmas exposed to ambient air. On self-pollinated stigmas,anilin e The induction ofsel fsee d setwit hcarbo n blue fluorescent material could be seen in dioxide inself-incompatibl e plantsha sbee n stigmatic papillae and a fewpolle n grains investigated extensively in Brassica. The had germinated but no penetration of the C0_ response was observed cytologically as stigmaha d occurred (Fig.1) . the extent of pollen tube growth (Nakanishi However, in the 3% CO treatment most et al., 1969)o r by seed-set following CO pollen grains had germinated and many had treatment of self-pollinated Brassica penetrated the stigma. This bundle of pistils (Nakanishi and Hinata 1975). pollen tubes could be traced down thestyl e Callose, a1-3-/ 3- linke d glucan,ha sbee n toth eovules . Therewa sn oaccumulatio no f shown to accumulate in the papillae of rejection callose in stigmatic papillae Brassica stigmas in response to self- other than that in the wallso fpenetratin g incompatible pollinations (Heslop-Harrison,, pollen tubes. The appearance of these C0? et al., 1974). Our studywa sundertake n tö treated stigmaswa ssimila r totha tobserve d observe the effect of CO on this callose 'in compatible crosspollinations . rejectionresponse . The primary action of C0„ in breaking incompatibility may be to directly inhibit Materials and Methods theformatio no fcallos ean d thusremov eth e physical barrier to self pollen entry. Mature flower buds of Brassica campestris Alternatively, C0? may disturb molecular var. T15 were emasculated and branches recognition between pollen and stigmatic bearing these buds were placed indistille d papillae such that the callose reaction is water overnight. The following morning, not induced against self pollen. The open flowers were excised and supported in Brassica pollen-stigma interaction offersa n agar. Pollen contaminated stigmas were attractive system for further explorationo f discarded. Clean stigmas were the role of C0„ self-pollinated, and several replicates in breaking including unpollinated controls were placed self-incompatibility. in each of two identical perspex containers Aan d B (1.5dm 3volume )a t 75%R.H . Two different gas mixtures were prepared callose by drawing ambient air from the laboratory roof. Thisai rwa scleane d through afilte r of activated charcoal and humidified. Half of this air was pumped directly into container A. The restwa s bubbled through KOH and passed through two soda limetower s before thisno w CO-fre e air wasmixe dwit h Self Cross Self+C02

100 References

Heslop-Harrison,J. , Knox,R.B .an d Heslop-Harrison,Y . (1974) Pollen-wall proteins:exine-hel d fractionsassociate d withth e incompatibility responsei n Cruciferae.Theoret .Appl .Genet .44 : 133-137. Nakanishi,T. ,Esahi ,Y .& Hinata ,K .196 9 Controlo fself-incompatibilit y byC O gasi nBrassica . Plant CellPhysiol . 10:925-927 . Nakanishi,T .& Hinata ,K . 1975 Self-seed productionb yC0 „ga streatmen t inself - incompatible cabbage. Euphytica24 : 117-120.

101 MOLECULAR INTERPRETATION OFOVERCOMIN G SELF-INCOMPATIBILITY INBRASSIC A

12 3 1 T.Gaude ,A .Palloix ,Y .Herv é andC .Duma s 1-Universit é Cl.Bernard-Lyo n 1,4 3B dd u1 1Nov .1918 .6962 2Villeurbann eCedex . FRANCE 2-INRA ,Statio nd'Amélioratio n desPlantes ,Avignon ,FRANC E 3-INRA ,Statio nd'Amélioratio n desPlantes ,Rennes ,FRANC E

Summary thepseudo-compatibilit y appearsrelate dt o the immaturestag eo fstigm awher erecogni ­ Inplan tbreeding ,severa lmethod s have tionfactor scontaine d inth epellicl e beendevelope d totemporar yovercom eself - wouldb eabsen t (Shivannae t al., 1978).Thi s incompatibility inflowerin g plants inorde r method iscertainl y themor e oftenuse db y toobtai nparenta l pure lines.Suc hmethod s plantbreeder s tomultipl y highlyself-incom ­ arebase do nth eus eo ftreatment swhic har e patible lines.However ,bud-selfin g istime - applicated eithero npolle no rstigm aalone , consuming andver y expensive becauseo fth e either onpollinate d flowers.I nthi spaper , hand labour involved.Then ,mor epractica l wereviewe d thesedifferen t techniquesan d methodshav ebee ninvestigated ,base d onth e givesom ene wdat aobtaine d inBrassic aolera - useo ftreatment s applicated either onpolle n cea.Treatment swit hcontrolle dconcentration s orstigm aalone ,eithe ro npollinate dflowers , ofcarbo ndioxid ean d controlled relative Self-incompatiblepolle ngrain sca ntur nin ­ humidity (rH)hav eeffect so npolle nquality , tocompatibl ewhe nmixe dwit hkille dcompati ­ pollenadhesio nan dgermination ,stigm a cal- blepolle no rthei rextrac t (mentor effect). loserespons e and pollen tubeentranc e inth e Théwashin g ofself-polle nwit horgani csol ­ style.A t theligh to fthes eresults ,a mole ­ ventsha sals obee nshow nt oovercom e incom­ cular interpretation ofovercomin g self-incom­ patibility inBrassic a (Roggen,1974) .Th e patibility inBrassic ai sproposed . efficiency ofthes e treatments seemst ocom e Keywords:Self-incompatibility ,carbo ndiox ­ fromdiffusio no fsubstance s fromcompatibl e ide,Brassic aoleracea . pollen.Thes ecompound swoul d acteithe rdi ­ rectlyo nth estigm asurfac eeithe ro nth e Introduction polleno rpolle n tube inalterin g theirreco ­ gnition potentialities.Thoug hth eprecis e Insporophyti cself-incompatibilit ysystems , natureo fthes esubstance s isstil lunknown , therecognitio nreactio nbetwee nmal ean dfe ­ Howlette tal . (l975)foun d thatsom epuri ­ male partnersoccur s atth estigm a level(se e fied proteinso f thepolle nwal l inCosmo s Heslop-Harrison et al., 1975).A characteris ­ bipinnatuswer eabl et oovercom eself-incom ­ tico fthi s incompatibilitysyste m isth eoc ­ patibility. curenceo fcallos e lenticules instigmati cpa ­ Onmatur e stigma,physica l andchemica l pillae cells inrespons e toself-polle no r treatments havebee n triedwit hsucces si n pollentubes .Th especificit yo fthi scal ­ Crucifers:mechanica ldamage ,hig htempera ­ loserejectio nrespons eha sbee nrecentl yexa ­ ture,loca lheating ,electri c shocks,organi c mined inBrassic a (Kerhoase t al., 1983).Th e solvents,hig hrelativ ehumidit yan dhig hcon ­ callosereactio n isthough tt ob edu et ospe ­ centrations ofcarbo ndioxide.. .(se eOcken - cificmolecule s associated withexpressio no f don,1978 ,fo rexample) .Suc h treatmentsin ­ S-alleles.Th eexistenc e ofself-incompatibi ­ ducea partia lbreakdow n ofth eself-incompa ­ lityconstitute s anobstacl e inproducin g in­ tibilitywhic h ismor eo rles spronounce dac ­ bredline suse dt omak eF 1 hybrid cultivarsi n cording toth especie so rth e linetested . breeding programs.However ,severa lmethod s Theeffect so fthes evariou s treatments onth e havebee ndevelope d toovercom e thesebarrier s incompatibilityreactio nremai n difficultt o toselfe dsee dproduction .I nthi spaper ,w e determine since thecellula r ormolecula ral ­ brieflyreviewe d thesemethod s anddiscus s terations brought toth e stigmaar eno tpreci ­ somene wdat aobtaine d incauliflowe rb yth e selyknown .Nevertheless ,thes edat ademons ­ use ofC O treatments associatedwit hhig hre ­ tratetha t theefficienc yo fth estigmati c lativehumidit y (Palloixe t al., 1984). barrier dependso n thephysica l andchemica l integrityo fth estigm asurface . Classicalmethod suse d toovercom e self-incom­ The effecto fhig hconcentration s ofC O patibility inovercomin g self-incompatibility inBrassic a hasbee nextensivel y investigated sinceth e Theflowe rag eha searl ybee nreporte dt o firstwor ko fNakanish ie t al. (1969).Thei r affectth epartia lbreakdow n ofself-incompa ­ methodconsiste d inincubatin g self-pollinated tibility inBrassica .Selfe d seedca nb eob ­ flowerswit h3 t o5 %C O levelsfo r6 hours . tainedafte rself-pollinatio n ofage dflower s Recently,O'Neil l etal . (1984)foun d thatth e (Kakizaki,1930 )o rafte rbu d self-pollination stigmacallos erespons e inBrassic açampestri s (Kakizaxi& Kasai , 1933).I nbu d pollination, wasblocke d byexposur et oa 3 %leve lo fC O.

102 Inorde r toclarif yth eeffect so fcarbo n afterpollinatio n seemsdirectl ycorrelate d to dioxideo npollen-pisti l interactioni n theacceptanc eo rrejectio n ofth egrain .Th e Brassica,a stud ywa sattempte d inou rlabo ­ facttha thig hrelativ ehumidit yma yovercom e ratorybase d ona n invitr o systemo fpollina ­ self-incompatibility confirms the importance tion(Palloi x etal. , 1984).Som eo fthes e ofth ehydratio nste p inth erecognitio nme ­ resultsar eno wdiscussed . chanism. Theseobservation s ledu s torecentl y pro­ Effectso fincreasin gconcentration so fC O posea molecula rmode l toexplai npollen - ona self-incompatibl ematin g inBrassic a stigmarecognitio n insporophyti cself-incompa ­ oleraceavar .Botryti s tibility systems (seeDuma se tal. , 1984). Thismode l isbase do nth econtro l ofwate r Thevariou sC0 „ treatmentswer e applicated flowfro m stigmat opolle nb y thereorganiza ­ toexcise dflower swhic hwer e placedo naga r tiono fcel lsurfac ecomponent so fbot hinter ­ medium ina hermeti cglas scontainer .A tth e acting partners .I na compatibl ematin g the endo ftreatment ,4 eventso f fertilization specific interaction between therecognitio n processwer eexamined :th eadhesion ,th eger ­ factors (S-products)woul d permitth earrange ­ mination,th epenetratio n ofpolle n tubesi n mento flipid san dprotein s ina macromolecu - thestyl ean d thecallos erejectio nrespons e laredific ewhic hpresen tfacilitate dway sfo r ofstigma .Th e following conclusions havebee n water flowan d thus,woul d ensurea ful l pol­ reachedfo rth e3 differen thig hself-incompa ­ lenhydratio n (Fig.a) .I na nincompatibl e tibleline stested : mating,th eS-produc t interactionwoul d induce 1-A sfirstl ydemonstrate d incabbag eb y theformatio no fa hydrophobi cmacromolecula r ïïakanishie tal . (1969),lo w concentrations structurewhic hwoul d constitute abarrie rt o ofC O applieddurin g theprogami c phasewil l efficienthydratio no fpolle ngrai n (Fig. b). overcome theself-incompatibilit y mechanism. Themos tefficien tconcentration s (4t o 6%) aresimila r tothos ereporte d forB .napu sb y Dhaliwale t al. (1979)an dfo rB .campestri s byDhaliwa l etal .(1981 )an dO'Neil l et al. (1984).However ,a longe rperio d ofapplica ­ tion (8,1 6o r24h )prove d toproduc e themos t effectiveresul t incauliflower .

2-Highe r concentrations ofC0 p (above 8%) haveapparentl y atoxi ceffect . 3-A sfoun d inB .campestri s (OWeill et al. 1984),C0 „ levelsefficien t inovercomin g in­ compatibility block the stigmacallos erespon ­ s&e\, sefollowin g self-incompatiblepollinations . 4-r Htreatmen t alonedi dno tovercom eth e 0,0: L incompatibilityreactio n incauliflower . 5-C O actsa tth epollen-stigm a interface Àfi :P rathertha no nth epolle no rstigm aseparatel y sincen oeffec t onth e self-incompatibility responsewa sobserve dwhe nC O was applied priort opollination . Fig.a& b :Mode lo fmolecula r interaction bet­ 6-C O actsearl y inth einteractio nsinc e weenpolle nan dstigm asurfac ecomponent s in thefirs teven t infertilization ,th eadhesio n Brassica (fromDuma se tal. , 1984).L=lipids , step, ismodified . P=proteins,arrow s indicatewate r flowsfro m Ifth eeffect so fC O applicationshav ebee n stigmat opollen . described,ho wca nac tth ega sa ta molecula r levelt omodif y the incompatibility reaction? Thismode lfo rpollen-stigm a interactioni s inagreemen twit hth evariou s techniquesuse d Interpretation ofth eCO ^ effects inoverco ­ toovercom e self-incompatibility.Treatments , ming self-incompatibility:molecula rmode lo f inmodifyin g either thepolle nsurfac eo rth e pollen-stigma interaction stigmapellicl ewoul d preventth eformatio no f thehydrophobi cedific eo ralter eit shydro ­ Recently,w eobserve d bytransmissio nelec ­ phobiccharacter . tronmicroscop y thatth efirs tcontac to f' Theactio no fCO .ma yb et odistur bth e pollengrai no nstigm a papillaewa sfollowe d dialoguebetwee nmal e andfemal esurface sa t bya reorganizatio no fstigm asurfac ecompo ­ themomen to fpollination .Th e gasma yac to n nents (Gaude& Dwnas , inpreparation) .Thi s thevariou scomponent s ofth e pollen-stigma eventma ypla ya preponderan trol e inpolle n interfaceo rspecificall y onth e interaction adhesionan drecognitio n processes inassurin g between putative stigmaan d -gen e control ofth ewate r flowfro mstigm at opol- , products.Th eprecis emechanis m ofthi sactio n len.A sshow nb y thewor k ofRobert s etal . ofC O isno tye tclear . (1980)i nBrassica ,th epolle nhydratio nleve l Carbondioxid eha sbee nshow nt ob ea meta -

103 bolieregulato ri nanima lan dplan tcell s oleracea.Theor .Appl .Genet .52 :113-117 . (reviewo fMitz,1979) .CO .mor e particularly O'Neill,P. , Singh,M.B. ,Neales, T.F. ,Knox , affectsprocesse ssuc ha smembran epermeabi ­ R.B.& Williams ,E.G. ,1984 .Carbo ndioxid e lityan dmolecula r interactions.A tth e blocksth estigm acallos erespons efollowin g membrane level,carbo ndioxid einterfere sa s incompatible pollinations inBrassica .Plan t wella so nprotein s thato nth elipi dbilayer . CellEnviron .7 :i npress . CO.interact swit huncharge damino-group so f Palloix,A. ,Hervé ,Y. ,Knox ,R.B .& Dumas ,C . proteinst ofor mcarbamate swhic hcoul dlea d 1984.Effec to fcarbo ndioxid ean drelativ e toconformationa l changes inth emolecule .O n humidityo nself-incompatibilit y incauli ­ thelipi dbilayer,th ega sappear s tochang e flower,Brassic aoleracea .J .Cel lSei . thephysica lstat eo flipid s inincreasin g (inpress) . solubility. Roberts,I.N. , Stead,A.D. ,Ockendon ,D.J . & During pollen-stigma interaction,i tseem s Dickinson,H.G. , 1980.Pollen-stigm ainter ­ possibletha tconformationa l changes inpro ­ action inBrassic aoleracea .Theor .Appl . teinsan dmodificatio n ofth einterfac elipi d Genet.58 :241-246 . fluiditycoul d affect theS-produc t interac­ Roggen,H. ,1974 .Polle nwashin g influences tion.Thi swoul dresul t ina mor epermeable - (incompatibilit y inBrassic aolerace avari e interfacewhic hwoul dallo wful lhydratio no f ties.In :H.F .Linsken s (Ed.):Fertilizatio r self-pollen grainsan dgrowt ho fself-polle n inhighe r plants.Nort hHollan d Publ.Co. , tubes inth estyle,'i nth epresenc eo felevate d Amsterdam,p .273-278 . C0„levels . Shivanna,K.R. ,Heslop-Harrison ,Y .& Heslop - Harrison,J. , 1978.Th epollen-stigm ainter ­ action:bu dpollinatio n inth eCruciferae . References ActaBot .Neerl .27 :107-119 .

Dhaliwal,A.S. ,Malik ,C.P .& Singh ,M.B. , 1979.Som eexperimenta l studieso nth eself - incompatibilityo fBrassic a species.1.Th e effecto fC O onincompatibilit y inBrassic a napus.Incompatibilit yNewslette r 11:84-87 « Dhaliwal,A.S. ,Malik ,C.P .& Singh ,M.B. , 1981.Overcomin g incompatibility inBrassica , campestrisL .b ycarbo ndioxid ean ddar kfixa ­ tiono fth ega sb ysel fan dcros spollinate d pistils.Ann .Bot .48 :227-233 . Dumas,C , Knox,R.B .& Gaude ,T. ,1984 .Pol ­ len-pistilrecognition :Ne wconcept sfro m electronmicroscop y andcytochemistry .Int . Rev.Cytol .90 :239-272 . Heslop-Harrison,J. , Heslop-Harrison,Y . & Barber,J. , 1975.Th estigm asurfac e in». " incompatibilityresponses .Proc.R.Soc.Lond . B,188 :287-297 . Howlett,B.J. ,Knox ,R.B. ,Paxton ,J.D . & Heslop-Harrison,J. , 1975.Polle nwal lpro­ teins:physicochemica lcharacterizatio nan d role inself-incompatibilit y inCosmo s bipinnatus.Proc.R.Soc.Lond.B ,188 :167-182 . Kakizaki,Y. , 1930.Studie s onth e genetics andphysiolog yo fself-incompatibilit y in thecommo ncabbage .Jap .J .Bot .5 :134-208 . Kakizaki,Y .& Kasai ,T. ,1933 .Bu dpollina ­ tion incabbag ean dradish .Som eexample s ofconspicuou s "pseudo-fertility" innormal ­ lyself-incompatibl eplants .J .Hered .24 : 359-360. Kerhoas,C , Knox,R.B .& Dumas ,C , 1983. Specificity ofth ecallos erespons ei n stigmaso fBrassica .Ann .Bot .52 :597-602 . Mitz,M.A. ,1979 .C O biodynamics:A ne w concepto fcellula r control.J .Theor .Biol . 80:537-551 . Nakanishi,T. ,Esahi ,Y .& Hinata ,K. ,1969 . Control ofself-incompatibilit y byCO .ga s inBrassica .Plan tCel l Physiol.10 :925-92 7 Ockendon,D.J. , 1978.Effec to fhexan ean d humidityo nself-incompatibilit y inBrassic a

104 EARLYEVENT SO NTH EBRASSIC A STIGMAFOLLOWIN GCOMPATIBL EAN D INCOMPATIBLE INTRASPECIFIC MATINGS

H.G.Dickinson ,I.N . Roberts,C.J . Elleman,G .Harro dan dM.I .Zuber i

Departmento fBotany ,Plan tScienc eLaboratories ,Universit y ofReading ,Whiteknight s Reading,RG 62A S

Summary system.

Acombinatio n ofne welectro nmicroscopi c Thesurfac e ofth estigmati cpapilla e (EM)technique san d simplephysiologica lex ­ perimentshav e identified asuperficia l layer Theonl y evidence that self-incompatibility lyingo nth epolle ngrai ncoating .Th ebeha ­ factorsar econtaine d inth epellicl e ispro ­ viouro fthi s layerafte rcompatibl e andin ­ vided byth eadhesio nassay so fStea d etal . compatiblemating sstrongl y suggeststha ti t 1980whic hdemonstrate d notonl y thatadhe ­ playsa nimportan tpar t inth econtro lo f siono fpolle n toth e stigmawa srelate dt o pollendevelopment .Thes e findingsar edis ­ SI,bu tals otha tenzymi ctreatmen t ofth e cussed inth eligh to fmodel s currentlypro ­ pellicle reduced 'compatible'adhesio n toa n posed forth eoperatio no fth eself-incompat ­ 'incompatible'level .Interestingly ,th e ibility (SI)syste m inBrassica . sourceo fth emolecule s implicated inS Iwa s shownt ob eth estigmati c cytoplasm,an d full Introduction adhesivecapabilit ywa s restored toth estig ­ matic surfacewithi n 2h,indicatin g eithera Fromth e firstmicroscopica l investigations very rapid repair system/o rtha tmolecule s itbecam e evidenttha tth e sporophytically- are 'turnedover' .A clu et oth enatur eo f controlled SIsyste m ofBrassic a operateda t thesefactor swa sprovide d byth eobservatio n thesurfac eo fth estigm a for,i fi tgermina ­ thatacquisitio no fth eS Isyste mb ybud swa s teda tall ,incompatibl epolle n formedcon ­ accompanied byth esynthesi so fa glycoprotei n torted tubesapparentl y incapableo fpenetra ­ (pi5.6 ) present inth e stigmaticpapillae . tingth epapillae .Althoug hth eus eo fth eE M Subsequent elegantexperiment shav edemonstra ­ (Kannoan dHinata ,1969 ) confirmed thattube s ted thisglycoprotei n tob eS-gen especifi c from self-pollen failed toente rth estigm a (Nishioan dHinat a 1979)an d involved incon ­ cuticle,an dMattsso n etal.(1973 )reporte d trolling pollendevelopmen t onth estigm a thepresenc eo fa 'pellicle'o nth esurfac e (Ferrari etal . 1981).Whil e iti spossibl e ofth epapillae ,recen t investigations into tous eth e stigma surfacea sa prob e to bin d theS Imechanis m inBrassic ahav e indicated molecules extracted fromth epollen-grai n itt ob ea highl y complexproces san dno t coating,an dvic eversa ,thi sbindin gha s analagous,fo rexample ,wit hth eantigen - provedvariabl e andno tS-gen especifi ci n antibody systemo fanimals .Further ,th edev ­ the aqueousenvironment s used.Mos trecentl y elopmento f self-pollenma y rangefro mlac k anautoradiographi c techniqueha sconfirme d ofgerminatio n toproductio n ofa lon gtub e thatcyclin g ofprotein sdoe s indeed occuri n which,o nattemptin g toente rth efemal e the stigmaticwall ,an dmayb e eveni nth e papillae,ofte nappear st ostimulat e thepro ­ pellicle (Roberts,e tal . 1984a)an dtest s ductiono fcallose .Thi srang eo fdevelopmen t with inhibitors indicate thatmaintenanc eo f isthu sno teasil y reconciledwit ha simpl e protein synthesis isrequire d forth econtin ­ interactionbetwee n S(incompatibility)gen e uedoperatio no fth eS Isyste m (Robertse t productso n initial contactbetwee n thespor - al. 1984b).Thu sa pictur eemerge so fth e ophytically-synthesisedpolle ngrai ncoatin g pellicle asa dynami c fluidmosai c containing andstigm asurface . atleas ton ecyclin gprotei n involved inth e Investigation intoth eS Imechanis moperat ­ SIsystem . ingi nBrassic aha sals obee nhindere db yit s extremegeneti c andphysiologica l lability. Thepolle ngrai nan d itscoatin g Anumbe ro fBrassic a S-genotypes areavailabl e ranging fromver y 'weak't over y 'strong', There isconsiderabl e circumstantial evi­ andi tha s longbee nknow nt oplan tbreeder s dencetha tth epolle ngrai ncoating ,o rtry - thatth eS Isyste m canb eoverridde n either phine,i sactivel y involved inth e selfin ­ byraisin ghumidit y (Carteran dMcNeill y1975 ) compatibility response.Extract s frompolle n orb yincreasin g atmospheric CO .Whil ere ­ coatingshav ebee nshow nt ostimulat eth e centwor k has succeeded inidentifyin g some productiono fcallos e in 'incompatible' ofth efemal emolecule s involved (Roberts,e t stigmatic papillae (Dickinsonan dLewi s1975 ) al. 1979,Ferrar i etal . 1981)an d hasunder ­ whileexchang eo fcoating sbetwee npolle n lined thedynami c natureo fth e pellicle grainsca nalte rthei rbehaviou r onself - (Roberts,e tal . 1984b),n odetail sar eavail ­ stigmas (Dickinson,H.G.»unpublished) . ableo fth epollen-hel d components ofthi s

105 Neverthelessth epresenc eo fS-specifi c mol­ some structure.Th e structurevisibl ema y eculeswithi n thepolle n coating remainst o vary from simple electron-lucent lineso f beproven . membrane-like dimension,t oprofile sreminis ­ Themai nbarrie r toprogres s inthi sfiel d cento fvesicle s and organelles (Fig.2) . istha texperiment s arenormall y carriedou t ina naqueou s environment,whil e the surface ofth estigm a iscomparativel y dry.Th epol ­ lenitsel f iss osensitiv e tomoistur e that itca nb eshow nt oadsor bmoistur eimmediate ­ lyfollowin g anthesis.Similarly ,attempt s toexamin e thestructur eo fgerminatin g pol­ lenar efraugh tb yth e facttha tth egrain s willhydrat edurin g the fixationprocess .W e Fig.2Exin e (E)hel d pollencoating,'conver ­ haveno wdevelope d arang eo ffixatio ntech ­ ted'followin g acros spollination . niqueswhic hd ono tinvolv eaqueou ssolution s Scalebar =lji m (Elleman,e tal.1984) . Further,sinc efixa ­ tion isnearl y instantaneous,i tha sprove d While thischang e isfirs tdetectabl e after possiblet ochar tth edehydratio n ofth epol ­ 30s,i ttake sa matte r of2-3mi nfo ral lth e leni nth eanthe ran d itsrehydratio n onth e coatingbetwee n thegrai nan d theare ao fcon ­ stigma surface ona minute-by-minut ebasis . tactt obecom e converted.Th ephysico-chemica l Inth eanthe rth epolle n coating assumes basiso fthi s coatconversio n isno tknown , itsfina l form justprio r toth edehydratio n buti tma ywel lreflec ta chang e froma pri ­ ofth egrai n itself.Instea d ofth eelectron - marily covalentt oa nioni c environment in­ opaque imageoffere db ynorma l fixitives, ducedb yth e flowo fwate r intoth ecoat . thecoatin gno wappear smottle d andelectron - Oncecomplete ,coatin g conversion isfol ­ lucentbounde d bya 'membrane'.Thi s 'mem­ lowedb ya chang e inth eorganisatio n ofth e brane' ,whic h issom elOn mi ndept h invests pollenprotoplas t fromtha tcharacteristi c of bothcoatin g and theexin e (Fig.1) . deWydrationt oa stratifie d condition(Fig.3).

Fig.1. Superficial layer (L)boundin gth e Fig.3 'Stratified'polle n cytoplasm.Not eth e pollen coating (C)an d exine(E) . coating (C),exin e (E),vesicula r layer (V) Scaleba r= 0.1(i m andmitochondrio n (M). Scal eba r= l\im

Sinceth ecoatin g ishel d tob e lipidic in* ' These changesi nth epolle ncytoplas m arecon ­ nature,thi s 'membrane'clearl y cannotb e sidered indetai l elsewhere (Elleman,e tal . regarded asa tru ebiologica lmembran ean d 1984). Between 5mi nan dl hafte rpollinatio n weshal l therefore refer toi ta sth ecoat ­ the stratified aspect islos tan d thepolle n ing superficial layer (CSD. I ti salread y cytoplasm reorganises ina nassymetrica l knowntha tth eCS Lha s somemembrane-lik e manner,concentratin g largeaccumulation so f properties, fori twil l notonl y exclude vesicles atth egermina lpor e fromwhic hth e lanthanum,bu t canals obin dit . tubewil l eventually emerge.Furthe rdevelop ­ Inth efina l stageso fpolle n dehydration mento fth etub e isdescribe d inDickinso n& inth eanthe r theperipher y ofth ecel lass ­ Lewis1973 . umesa nelectron-opaqu e aspect,containin g Development isver ydifferen t following only small fibrillarsphere s some20 0n mi n selfing for fusiondoe sno t immediatelyoccu r diameter. Itwoul d also seemtha t eveni n betweenth eCS Lan d thepellicle .Indeed , thismos tdehydrate d forma plasm amembran e material sampled upt o5 mi nafte rpollinatio n doesinves t thepolle nprotoplast . showstha tth eCS L remainsintact . Events overth enex t 45mi n suggesttha tcontac ti s Postpollinatio n developmentan d itsmodifi ­ established betweenpolle nan d stigma,fo r cationb yth eS Isyste m somecoatin g conversiondoe soccur,albei ta t amuc h reduced pace andonl y onth e 'stigmatic' The firstcontac tbetwee n compatiblepolle n sideo fth e layer.Developmen t continueswit h and the stigmatic surface involves fusiono f achang e toth estratifie d typeo fcytoplas ­ theCS Lwit h thepellicle .Thi soccur s ina micorganisatio n in some,bu tno tal lgrains . matter of secondsan d isfollowe d by acon ­ Inth epresenc e ofa 'strong'S-gene ,polle n spicuousreactio no fth epolle n coatingadja ­ development ceasesa tthi sjuncture . centt oth eregio no fcontact .Here ,th emot ­ Although thedifferenc e in thebehaviou r tled appearance ofth ecoa t isreplace d bya n ofth eCS Lgoe s somewa y toexplai n thead ­ electron-opaque aspectan d theemergenc eo f hesion resultso fstea d eta l (1980), itdoe s

106 notindicat e amechanis m bywhic hdevelop ­ Acknowledgements mento f incompatible pollenma yb earrested . Thereis ,however ,a larg ebod y ofevidenc e Wethan k theAFR Co fth eU K forfinancia l pointing toth einvolvemen t of inhibitors supportove rth ecours eo fthi swork . ina tleas t somepar to fth eS Irespons e References (e.g.Hodgki n sLyo n1984 ). Allth edat acurrentl y available indicate CARTER,A.L .S T .McNEILL Y1975 . Euphytica thatth eS Isyste moperate sdurin g theear ­ 24,805-813 . liest stageso fpolle ndevelopmen t and iti s thusno tclea rwh y someincompatibl e grains DICKINSON,H.G . SD .LEWI S 1973. Proc.R.Soc. developtubes ,an dwh y thesetube sapparent ­ Lond.(B) 183,21-38 . lyar earreste d byth edepositio n ofcallose . Thefirs tquestio n isprobabl y the easiert o DICKINSON,H.G . &D .LEWI S1975 . Biol.J.Lin. answeri ntha tatmospheri cwate reithe rby ­ Soc.7 ,Suppl.1 ,165-177 . passesan ybarrie r toflo wbetwee npolle n andstigm aor ,dependin g uponth ehypothesi s ELLEMAN,CE. ,G .HARRO D SH .G .DICKINSO N198 4 infavour ,dilute sth e levelo finhibito rt o (inth epress) . belowa threshol d necessary forgrai ndevel ­ opment.Th e formationo fstigmati ccallos e FERRARI,T.E. ,D . BRUNSS D.H .WALLAC E1981 . isno t sosimpl y explained,i ti spossibl e PlantPhysiol ,67 ,270-277 . thatsporophyti c coatingmateria laccompan ­ iesth etub e tipan d engenders areactio n onceth ecuticl eha sbee nerode d(Dickinso ns HODGKIN,T .S G.D . LYON 1984.Ne wPhyto l (in Lewis 1975), but,equally ,i tma yb etha tth e thepress) . callosic response isstimulate d bya second , gametophytically-controlledgene ,no wknow n KANNO,T .s K .HINAT A 1969.PI.Cel lPhysiol . tob eactiv e inthes eplants .Nevertheless , Tokyo 10,213-216 . sinceth eformatio no fcallos ema y alsob e / stimulatedb ymechanica ldamag e and fungal MATTSSON,O. ,R.B.KNOX ,J.HESLOP-HARRISO NS infection,i tremain spossibl etha ta dis ­ Y.HESLOP-HARRISO N 1973.Natur e (Lond)247 , ruption inpolle n tubeorganisatio n byth e 298-300. activitieso fth eS-gene sdurin g thegermin ­ ation stages sodisorganise s thepolle ntubes , NISHIO,T .& K .HINAT A 1979.Japa nJ.Genetic s thatth estigm a 'recognises'the ma s for­ 54,307-311 . eignorganisms .Suc ha nhypothesi s isno t easily tested,bu tth eturgo ro fgrowin gcom ­ ROBERTS, I.N., G.HARRO D and H.G.DICKINSON patible tubesca nb e raised by supplying 1984a. J.CellSei .66 ,241-253 . highlevel so fatmospheri cwater ,an d iti s very striking thatthi streatmen t isaccom ­ ROBERTS, I.N., G.HARRO Dan dH.G .DICKINSO N paniedb yth edevelopmen t oflong ,tortuou s 1984b. J. Cell Sei.66 ,255-264 . tubesan d theformatio no f stigmaticcallose . ROBERTS, I.N., A.D.STEAD ,H.G .DICKINSO N& Inconclusion ,wha t canw eno w sayo fth e D.OCKENDO N 1979. Planta,146 ,179-183 . SIsyste m operating inBrassica ?Wit hth e pellicle,th eCS L clearly emerges asplayin g STEAD,A.D. ,I.N . ROBERTS SH.G .DICKINSO N acentra l rôle inth econtro lo fadhesio n 1980. J. Cell Sei. 42,417-423 . andth e subsequent development ofpollen . Further,th elac ko f full fusion inincompat ­ iblepollinatio n seemst oresul ti nslo w coat-conversion,an d thepolle n cytoplasm taking longer toassum ea stratifie dorganis ­ ation.Whil e the suppressiono fpolle ndev ­ elopment canb eexplaine d sofa ri nterm so f theavailabilit y ofwater ,ther ei ssom eev ­ idencethat ,i nth e stratified condition,th e pollenprotoplas t secretesprotei n intoth e intineand ,perhaps ,th ecoating . Itremain s possible thatthi s interaction forms thebasi s ofa secon dphas e ofth eS Isyste m -a phas e involving theproductio n of inhibitors,wheth ­ erS-gen eactio n isdirectl y orindirectl y responsible forcallos e synthesis following incompatible tubegrowt h seemsunclea rbut ,i n viewo fou r increasing knowledge offactor s stimulating callose formation inplants ,th e latteralternativ e appearsth emor eattractive .

107 POLLEN GERMINATION INHIBITORSASSOCIATE DWIT H THESELF-INCOMPATIBL E RESPONSE OFBRASSIC A OLERACEA L.

THodgki nan d GD Lyo n

Scottish Crop Research Institute,Invergowrie ,Dunde e DD25DA ,Scotlan d

Summary thendeveloped ,dried ,spraye d with Petunia hybrida orLiliu m lankongense polleni n Pollengerminatio n inhibitors inBrassic a appropriategerminatio n mediaan d incubated oleracea L.tissu e extractswer edetecte d overnight topermi tpolle ngermination . To usinga thin-laye r chromatographic bioassay. obtaingerminatio n ofB .olerace a pollenth e Extracts ofleaf ,seeds ,polle nan d styles procedurewa smodifie d by soaking TLCplate s allcontaine d inhibitory compounds,an d for0.7 5 hi npH 8TAP S (Sigma Ltd)prio rt o stigma extractsha d thegreates t amountsan d loading theextract san dusin g thegermin ­ largestnumbe r ofinhibitors .Som einhibitor s ationmediu m ofHodgki n (1983). Zoneso f werecommo n toextract s ofunpollinated , inhibition werereveale d by sprayingth e self-pollinated and cross-pollinated stigmas incubated platewit hanilin eblu et ostai n whilst otherswer eonl ydetecte d instigm a germinated pollenan d examining underU V extractsobtaine d after incompatibleself - lighto rb y incident light fluorescence pollinations. Theinhibitor s detected microscopy. The effect of incompatible uniquely from self-pollinationswer ea t pollination onth einhibitor s inth estigm a their highest concentration approx.2 h wasexamine d using extractsprepare d from after pollination.Th esignificanc e ofthes e stigmascollecte d 2,4 o r2 4h afte r self- findingswit h respect topossibl e parallels o/ cross-pollination orafte r being leftun ­ between pollen-stigma interactionsan d pollinated for2 4h (Hodgkinan d Lyon,1984) . plant-pathogen recognition isdiscussed . Keywords: Brassica oleracea,polle ngermin ­ Results ation,thi n layerchromatography ,germin ­ ation inhibitors,self-incompatibility , Fig. 1summarise s theresult sobtaine d for bioassay,phytoalexin . varioustissu e extractsusin g P.hybrid ao r

Introduction Fig. 1. Diagram ofth emajo r zoneso fP . hybrida pollengerminatio n inhibition onTL C Thesporophyti c self-incompatibility system platesfollowin g chromatography ofextract s ofBrassic a oleracea (Thompson 1957)provide s ofdifferen tB .olerace a tissues:A ,seed ; a usefulmode l forstudie so nth enatur eo f B, Leaf;C ,pollen ;D ,style . Zoneswit ha pollen-stigma recognitionan d formor egen-* ' variable degreeo finhibitio n arestippled . eralinvestigation s ofrecognitio n reactions inflowerin g plants (Roberts etal. , 1984). RecentlyHodgki nan d Lyon (T9797,Bushnei l (1979), Lewis (1980)an dKee n (1981)hav e drawnattentio n toth e similarities between the interactions ofplan tan d pathogenan d stigmaan d pollen. Wehav edevelope d abio ­ assay thatca nb euse d toinvestigat e some aspects ofth estigma' srespons e following anincompatibl e pollination inB .olerace a (Hodgkinan d Lyon, 1983; 1984). Weher e summariseth eresult sobtaine d usingthi s bioassay and discuss theirsignificanc ei n relation topossibl eparallel sbetwee nth e reaction ofplan tan d pathogen and stigma andpollen .

Materialsan dMethod s

Inhibitors ofpolle ngerminatio nwer e detected inB .olerace a tissue extracts usingth etechnique s described byHodgki n and Lyon (1983). Ethylacetat e soluble extractswer eprepare d fromth erequire d plant tissuesan d loaded ont ocleane d thin B D layer chromatography (TLC)plate swhic hwer e

108 L.lankongens epolle n todetec t inhibitory thedevelopmen t ofimprove d quantitative zones. Onezon eo finhibitio n (Rf0.5 )wa s techniques. common toal l tissuesteste d although leaf B.olerace a pollengerminate d lesswel l and pollenextract s oftengav eles sinhib ­ than P.hybrid a orL .lankongens e polleno n itioni nthi szon etha nothe r tissue TLCplates . Percentagegerminatio n of50% - extracts. Pollenextract sgav ea zon eo f 65%wer e obtained and tubegrowt hwa spoo r inhibition (Rf0.25 )no tfoun d inothe r (20-50urn) . In contrast,B .olerace apolle n extractsan d extracts ofunpollinate d stigma inhangin gdrop sgav e 80-90%germinatio nan d tissuegav eth elarges tnumbe r (upt o5 )o f tubelength so fu pt o 1mm . Inth ebioassa y inhibitory zonesan d theseha d larger B.olerace apolle ngerminatio n wasinhibite d dimensions than those fromothe rextracts . inclearl y defined zonesalthoug h theyha d When extractso fstigm a tissue collected2 , different Rfvalue s fromthos eobtaine d 4o r2 4h afte r self-o r cross-pollination usingP .hybrid apollen . Zoneso finhib ­ werebioassaye d with P.hybrid apollen , itionwer edetecte d forunpollinate d stigma zoneso f inhibition were detected fromth e2 extractsa t Rf0 (zonediamete r 3mm )R f hself-pollinate d extractwhic hwer eno t 0.11 (diameter 10mm )an d Rf0.2 5 (diameter detected from thecross-pollinate d orun ­ 10mm) . Noothe r zoneso fcomplet einhib ­ pollinated stigma extracts (Fig.2) . The itionwer edetecte d although severalarea s sizeo fthes eadditiona l zoneswa sreduce d ofpartia l inhibition wereseen . Extracts in4 h extract san d theywer evirtuall y from stigmasself -o rcross-pollinate d for absent from 24h extracts . The inhibitory 2, 4o r2 4h possesse d thesam e3 zone sbu t zonesfro m extractso f self-pollinated additional zoneso fpartia linhibition , stigmastende d tob e larger thanthos efro m which haveye tt ob efull y characterised, cross-pollinated orunpollinate d extracts. wereals odetected . Differencesi nnumbe r and Rfvalue so fth einhibitor y zonesma y Fig. 2. Diagram ofth emajo r zoneso fP . result fromth eTL C platepretreatmen tan d hybridapolle ngerminatio n inhibitiono nTL C highp Hneede d forB .olerace apolle n platesfollowin g chromatography ofextract s germination. When individual extractswer e ofB .olerace a stigmas: A,unpollinated ;B , separated byHPLC ,t oavoi d thenee d tous e cross-pollinated for2 h ;C , self-pollinated pretreated platesfo rchromatography ,th e for2 h ; D,self-pollinate d for4 h ;E ,self - numbers,R fvalue san d sizeso fth einhib ­ pollinated for 24h . itory zoneswer esimila r to P.hybrid aan d B.olerace a pollen (Hodgkin andLyon , unpublished). -f Discussion

Severalworker shav edraw nattentio n to similarities between thepollen-stigm a interactions in interspecific and intra- specific incompatibility and host-pathogen interactions inplan t disease.I nparti ­ cular,the yhav enote dgeneti c parallels (Bushneil, 1979;Hogenboom , 1983),draw n attention toth esignificanc e ofcallos e deposition (Bushneil,1979 ;Lewis ,1980 ) and suggested acommo n rolefo rcel lwal l components,particularl y glycoproteins,i n both recognition systems (Keen,1981 ; Roberts, 1984). However,muc h ofth edis ­ cussion onth esimilaritie sha sbee n speculative,reflectin g keygap si nou r © knowledge. Thisi sparticularl y trueo fth e eventstha t follow theprimar y pollen-stigma O recognition reaction and result inth e failure ofincompatibl e pollen topenetrat e thestigm asurface . B D Wehav edescribe d results froma bioassa y techniqueanalogou s tothos euse d toinvest ­ igatehost-pathoge n interactions (Smith, Atleas t twoo fth e zones (Rf0.12 , 0.67) 1982)an d haveshow ntha tstigm aextract s detected from thebioassa y of2 h self - areparticularl y rich inpolle ngerminatio n pollinated stigmaswer enote d in2 4h seif ­ inhibitors thatar eeffectiv e invitr o and cross-pollinated stigmaso fdifferen t againstpolle nfro mdifferen t species. The inbred lines indicating thatquantitativ e compounds involved would appear tob epoten t differences may exist between plants inth e germination inhibitorsbecaus ea namoun to f amountso fth einhibitor spresent . The theextrac tcorrespondin g to 10-15stigma s clarification ofsuc h differencesmus tawai t

109 gave inhibitory zonesu pt o2 0m m india ­ hasshow nho wtechnique sdevelope d for meter. Furthermore,followin g incompatible investigating oneclas so frecognitio n self-pollinations,th eamoun t ofpre-existin g systemsca nassis t ininvestigatin gothers . inhibitors increased and additionalinhib ­ Itha sals o opened upne wavenue s tob e itorswer eproduce d inth estigm atha twer e explored inpollen-stigm a interactionsan d notfoun d incross-pollinate d stigmas. The enabled ust odetec ta possibl ene wclas so f chemical identity ofth einhibitor si s biologically activecompounds . unknown butthei r solubility inorgani c solventsan d mobility onTL Cplate sindicat e References thatthe yhav ea lo wmolecula r weight.Thei r presenceonl y 2h afte r self-pollination Bushnell,W.R. , 1979. Thenatur eo fbasi c shows that they areproduce d rapidlyfollow ­ compatibility: comparisons betweenpistil - ingth eappropriat e induction and their pollenan d host-parasite interaction. In: declineafte r 4h indicate sa rapi d break­ J.M.Dal y &I .Uritan i (Ed.): Recognition down. and Specificity inPlan t Host-Parasite Phytoalexins arelo wmolecula rweigh t Interactions. Jap.Sei .Soc .Press ,Toky o compoundscapabl e ofinhibitin g fungalgrowt h and Univ.Par k Press,Baltimore ,p .211 - and areproduce d byplant si nrespons et o 227. challengeb yincompatibl e fungi (Keen, 1981). Hodgkin,T. , 1977. Sibnumber s inindivid ­ Wehav e suggested (Hodgkinan d Lyon, 1979) ualBrussel s sprout siliquae. Cruciferae thatcompound sanalogou st o phytoalexins NewsletterNo .2 :25-26 . might playa par ti npollen-tub e inhibition Hodgkin,T. , 1983. Amediu m forgerminatin g inincompatibl e pollinations. Thecompound s brassicapolle ni nvitro . Cruciferae detected byou rbioassa y have someo fth e Newsletter No.8 :63 . properties ofsuc h inhibitorsan d someo f Hodgkin,T .& G.D .Lyon , 1979. Inhibition themma y thereforeb e involved inpollen - ofSolanu m pollengerminatio n invitr ob y stigma interactions invivo . /he phytoalexin rishitin. Ann.Bot .4 4: However,severa l factorsmus tb eborn ei n 253-255. mind when examining thishypothesis .Pollen - Hodgkin,T .& G.D .Lyon ,1983 . Detectiono f stigma recognition and responsear epart so f pollengerminatio n inhibitorsi nBrassic a a complex series ofevent swhic h involve oleracea tissue extracts. Ann.Bot .5 2T extrememorphologica l specialisationan d 781-789. ensure successful fusion ofmal ean d female Hodgkin,T .& G.D .Lyon , 1984. Pollen gametes. The intraspecific incompatibility germination inhibitors in extractso f responseinvolve sinhibitio n of"self" ,i n Brassica oleracea L.stigmas . NewPhytol . contrast tohos tpathoge n interactions (or 96 :293-298 T animal systems)wher e "non-self"i srejected . Hogenboom,N.G. , 1983. Bridging aga p Incompatible pollinationsma yb erecognise d between related fields ofresearch : rapidly (10-15minute safte r pollination)an d Pistil-pollen relationshipsan d the iti sno tclea rwhethe r inhibitorso fth e distinction betweenincompatibilit yan d kind detected could bemobilise d soquickly* : incongruity innonfunctionin g host Current theories ofth einductio nan d ipéfrasit erelationships . Phytopath.73 : operation ofphytoalexin s involve thetrans ­ 381-383. mission ofa 'stimulus'b ya secondar y Keen,N.T. ,1981 . Evaluation ofth erol eo f messengert oth ecell' snucleu san d toneigh ­ phytoalexins. In:R.C .Staple s& G.H . bouring cells followed byd enov osynthesi s Toennesen (Ed.): Plantdiseas econtrol : ofprotein . Infact ,th eabilit y ofneigh ­ Resistancean d Susceptibility. J.Wile y bouringcell st orespon d iscentra l those & Sons,Ne w York.p .155-177 . theorieswherea s eachpolle n grain-stigma Lewis,D.H. , 1980. Arethe r inter­ papilla cell interaction isusuall yconsidere d relationsbetwee n themetaboli c roleo f tob ea n independent event (butse eHodgkin , boron,synthesi s ofphenoli c phytoalexins 1977, forevidenc e toth econtrary) . and thegerminatio n ofpollen . New Allrecognitio n reactions involvedetect ­ Phytol.8 4 :261-70 . ion, discrimination and response. Atleas t Roberts, IN., G.Harro d &H.G .Dickinson , oneo fth eparticipatin g organisms orcell s 1984, Pollen stigma interactionsi n must detect theothe ran d theremus t thenb e Brassica oleracea.II .Th efat eo fstigm a aproces so fdiscriminatio n followed bya surfaceprotein sfollowin g pollinationan d discernible responsedependen t onth eresul s their rolei nth e self-incompatibility ofth ediscriminatio n processes. Host- response. J. Cell Sei.6 6 :255-264 . pathogen and pollen-stigma interactionsma y Smith,D.A. , 1982. Toxicity of Phytoalexins shareothe r features intha t cellsurfac e In: J.A.Baile y &J.W . Mansfield (Ed.): glycoproteinsappea r tob einvolve d inth e Phytoalexins,Blackie ,Glasgow .p218-25 2. initial recognition events (detectionan d Thompson,K.F. , 1957. Self-incompatibility discrimination)an d lowmolecula r weight inmarro wste mkale ,Brassic a oleracea growth inhibitorsca n bedetecte d inrespons e var. acephela• 1 .Demonstratio n ofa toincompatibl e recognition inbot hsystems . sporophytic system. J.Genet .5 5 :45-60 . Theexten tan d significance ofthes ecommo n featuresha sye tt ob edefine d butou rwor k

110 SELF-INCOMPATIBILITY INTH ENITROGEN-FIXIN G TREELEGUME ,ACACI ARETINODES : PRE-O RPOST-ZYGOTI CMECHANISM ?

J.Kenriok ,V .Kau lan d R.B.Kno x PlantCel lBiolog y Research Centre,Schoo l ofBotany ,Universit y ofMelbourne ,Parkville , Victoria 3052, Australia

Summary synergids, with its filiform apparatus, nucleus and cytoplasm breaks down during Thecytolog y of pollen tube growthha sbee n pollentub eentry ; followed in the self incompatible species — cell and nuclear size of the zygote are Acaciaretinodes . Inbot h selfan d cross larger than thato fth eunfertilize d egg; pollinations,th e pollen tubes grow through — in the central cell, one large fusion thestyl et oth eovule sa tapproximatel y the nucleus is present after fertilization of same rate reaching the ovules within 11 the embryo sacwhic h may have one or three hours. Though pollen tubesadhere d toth e nucleoli present compared with two smaller nucellus,ther ewa sonl y 1.5%o fovule s polar nuclei in unfertilized sacs. Pollen fertilized after self pollination compared tubespenetratin g thenucellu sar edifficul t with 14.1%afte r crosspollination . Apre - todetect ,a sthe ydi dno tsho wABF . zygoticmechanis m ofsel f incompatibility is suggested. Based on these criteria, the number of fertilized ovuleswer ecompare d incros san d self pollinations, 72 hours after Introduction pollination (Table2) .

Inou r studieso nnatura l populationso f Table 2. Incidence of fertilization in Acacia retinodes theplant swer e found tob e ovuleso ftw o parents ofA ^retinode sfixe d highly self-incompatible (SI) (Bernhardt et 72 hours after self or reciprocal cross al., 1984). Here, we present a brief pollination. Data obtained from 75 to 160 account of the cytology of SI in A. ovulespe rtreatment . retinodes. FemaleParen t % fertilized Resultsan d Discussion ovules Usingth eanilin eblu e fluorescence (ABF) SELF POLLINATION method,w efin d thatther e isn oinhibitio n 1 1 .6 of self pollen tube growth on the stigma 1 surfaceo r inth e style. Pollen tubesente r 2 3 ovuleswithi n 11 to2 4h afte rpollination . CROSSPOLLINATIO N There is no significant difference in the 14 rate of pollen tube growth in the style 1 .7 12 between selfan d cross pollinations 2 8 (Table 1). During the first 6 h, tube Inth e selfpollinate d group,ther ewer e growth rates were higher than during the 1.5% of fertilizedovules and 14.1% after final5 h . cross pollination between the same two parents. The difference is highly Table 1. Rate of growth of pollen tubes significant (P < 0.001). The Index of through the style ofth e same femaleparen t Self-Incompatibility (ISI), computed onth e ofAcaci aretinodes . basiso fyiel d ofzygote sfollowin gself / cross pollination, 0.10, is in good Pollination Leading PollenTub e agreement with the ISI computed from fruit Growth Rateum/mi n setdat a (0.08). (a)0- 6 h (b)6-1 1 h We can conclude that the site of pollen tubearres t isi nth eovary ,probabl y inth e Self 3.3 1.2 nucellus and is likely to bea pre -zygoti c mechanism. The question ofwhethe r SIi sa Cross 4.5 2.0 result ofpolle n tube/ovule or gamete/gamete interactions isye tt ob edetermined . Differences < 10%wer e detected in the pollen tube growth rate between different pollenparents . Unpollinated and most self pollinated Reference flowers underwent abscision 6 to 8 days after pollination. We examined the Bernhardt,P. , J.Kenrick ,an d R.B.Knox . cytological events inth eovule sdurin g this 1984 Pollination biology and thebreedin g critical period. Fertilized and systemo fAcaci aretinode s (Leguminosae: unfertilized embryo sacs differed in the Mimosoideae)Ann .Missour i Bot.Gard . appearance ofke ystructures . 71: 17- 29 . — in fertilized embryo sacs oneo fth etw o

111 CELL SURFACE RECOGNITION GLYCOPROTEINS ANDTHEI R POSSIBLE ROLEI NANG'IOSPER M SELF-INCOMPATIBILITY

Knud Larsen Departmento fGenetics ,Roya l Veterinary andAgricultura l University, Copenhagen,Denmar k

Summary Skurray, 1980). This specific recognition triggers adhesion andsubsequen t DNAtransfe r Ability todistinguis h "self"o r"kin "fro m from donort orecipien t cell. "not self"o r"no tkin "i sa prerequisit efo r proper contact,co-operatio n oreve n fusion In haploid yeast cellso fopposit e mating between cells. Examples ofcel l surface recog­ types,fusio n ismediate d bycomplementar y nition glycoproteins span from mere adhesion sex-specific glycoproteins inth ecel l walls, systems toth eutmos t ingenious major histo­ through recognition/agglutination. The two compatibility complex (MHC)o fhighe r verte­ glycoproteinsar eo fdifferen t size:A smalle i brates.I propos e that angiosperm self-incom­ one withM Wbetwee n 23,000an d60,000 ,an da patibility iscause d bya mechanis m similart o largeron ebetwee n 130,000an d500,000 ,depen ^ the MHC-restricted antigen recognition bycy ­ dento nth especie s (Tohoyamae tal. , 1979; totoxic T-lymphocytes: Pistil/pollen isrecog ­ Pierce& Ballou , 1983;Burk ee tal. , 1980; nized through identical S-glycoproteinsi n Mendonca-Previatoe tal. , 1982). Thelarge ro- association with complementary sex-specific these glyproteins iscompose d ofmonovalen t glycoproteins totrigge ra n active inhibition subunits attached toa cor e molecule through of self pollen tube growth.I furthe r propose disulfide links. thatth eextrem e S-gene polymorphism iscause d Amongth eascomycetes ,th ehermaphroditi c and maintained by gene conversion, utilizing Neurospora crassa hason elocu sfo rit s two silent S-genes asreservoir so fdiversity . mating types,A an da .Thes e determine the ho' This also explains thegeneratio n ofne wS - • mogenic sexual incompatibility,i.e . onlyga ­ alleles occasionally seen after inbreeding. meteso fopposit e mating types will fuse(Ca r 1ile& Gooday , 1978). This iscalle d hetero- Keywords: Self-incompatibility, recognition, thallism. glycoprotein,cel l surface, fertilization, pollen,pistil ,histocompatibility , gene poly­ Inth eunicellula r green alga chlamydomonas morphism,gen e conversion,matin g type. gametes from opposite mating types (+an d- ) recognize each otherb ythei r flagella tips. Introduction The flagellarmembrane s adhere toeac h other, triggering wall lysisan dsubsequen t fusion Early inevolution ,cel l discrimination must (Mitsudae tal. , 1982). Sex-specific glycopro have becomea nessentia l condition forcel l- 'teinso fhig hM Whav e been detected in c. eu- cell interactions. Certain cell surface mole­ gametos (Musgravee tal. , 1981)an dc. P.ein- cules evolved intomonitors ,triggerin g co­ hardi (Coopere tal , 1983). -Homa ne tal . operativeo rdefensiv e responses,dependen to n (1982) found indications thata tleas tth e(- the signal received. Speciation mustb efol ­ agglutination factor isexternall y anchored b lowed (orpreceded )b ydifferentiatio n ofsuc h an intrinsic membrane glycoprotein (arecep ­ recognition molecules toestablis h stable bar­ tor), whicho nagglutinatio n forwardsa n in­ rierst osexua l and/or somatic intermixingo f tracellular signal. species. Cell surface glycoproteins seemt o Ascidians (sea-squirts)ar ehermaphrodite s be involved inmos to fthes e mechanisms. and usually self-sterile. Inth esimpl e asci- dian Ciona intestinalis, theglycoprotei neg g Sexual recognition systems envelope "chorion" prevents self-fertilizati­ on, polyspermy andheterospecifi c fertiliza­ Itha sbee n suggested that cell recognition tion (Dalee tal. , 1978). systems originated with theappearanc eo fsex ­ uality (Monroy& Rosati , 1979), thus giving Oka& Watanab e (1957)observe d that fertili riset oothe r cellular communication systems zation inth ecompoun d (colonial)ascidia nBo inth eevolvin g multicellular organisms. tryllus primigenus isonl y possible between different colonies.Scofiel d etal . (1982)i n In bacteria, "sexual" conjugation isbes t Botryllus schlosseri found this quality tob e known from Escherichia coli. Here,th eF(o r governed byon egen e locus with multiple alle F-like)plasmi do fF cells codesfo rpilin , les. Thehaploi d sperm isincompatibl e with a glycoprotein witha molecula r weight (MW)o f the diploid,maternall y derived eggenvelope s 11,000-12,000 daltons,polymerizin g intoth e ifit sallel e inthi s locus ismatche d byth e sex-pili (extracellular filaments)whic hat ­ latter.Thi s self-incompatibility isclosel y tach toreceptor so nF "cell s (Willetts& similart oth e"gametophytic " typeo fangio -

112 sperm self-incompatibility. reaction between Petunia pollenan dstyle . Kinsey& Lenhar z (1981)foun da neg gsurfa ­ Further,i n196 0h eprovide d evidenceo f like ceglycoprotei n fraction fromth ese aurchi n S-specific antigens inpolle nan dstyl e from Avbacia punotulata that interfered with both the same S-genotype of Petunia hybrida. sperm bindingan dfertilization ,bu ti na non-specific manner.Thi sle dthe mt othin k Somatic recognition systems thatbindin g andspecies-specificit y needno t reside neither inth esam e siteno ri n the Somatically, intercellular recognition is as samemolecule ,contrar yt oprevailin g ideas essential forprope r cell interactionsa si n regarding fertilization specificity.- Lop o the sexual mechanisms above.- A fe wexample s & Vacquier (1980) purified anantiseru ma - should bementioned : gainst sperm surfaceo fth ese aurchi n stron- In Neurospora crassa, somatic fusion iscon ­ gyloaentrotus purpuratus ,an d found itt o trolledb ygene s inabou tte nloci ,al lmedia ­ cross-react with sperm from all 28teste d spe­ ting heterogenic incompatibility.On eo fthes e cies from7 phyla ,includin g amphibia,bird s isth ematin g locus mentioned above. I.e.,A andmammals . fuses somatically only withA ,s oth ereactio n is completely reversed comparedt oth egamete s Themammalia neg gi ssorrounde d byth e gly- (Carlile& Gooday,1978 ) coprotein layer "zona pellucida" (producedb y theoocyt e itself)whic h isresponsibl efo r Also in Botryllus sehlosseri, theabov emen ­ species-specific fertilizationan dsubsequen t tioned ascidian,th elocu sfo rsexua l self-in­ blockt opolyspermy .On eo fth eglycoproteins , compatibilityi sinvolve d insomati c incompati­ "ZP3" (MW83,000 )i sidentifie da sth especi ­ bility.Scofiel de tal . concluded thismulti - ficsper m receptor (Blei1 & Wassarman , 1983). allelic locust ob esolel y responsiblefo r Likewise,a sper m glycoprotein seemst ob e in­ both phenomenons,agai n determining homogenic volved inth esperm-eg g recognition (Moore& incompatiblity between gametes,contrar yt o Bedford, 1983). heterogenic discrimination between somatic cells (Scofield etal. , Î982). Angiosperm self-incompatibility (SI,o rS , when useda sa prefix )mus t result from recog­ In plants,host-pathoge n andsymbioti c inter­ nition events,bu tthoug h thoroughly investi­ actions with microorganisms attract atten­ gatedfo rdecades ,definitiv e conclusionsa s tion. Though thoroughly investigated,th ede ­ toth ebiochemica l basisha sno tbee n achie­ tailed mechanism ofcompatibilit y remains elu­ ved.Recently ,i tha seve n been suggested sive.I tha sbee n discussed whetherth ebasi c that SI,a tleas tth egametophyti c type (pol­ reactionsar esimila rt othos eo fself -(o r lenbehaviou r governed byth epolle n S-geno- interspecific) incompatibilityo rno t (e.g. type)migh tno tb edu et oan yqualitativ eme ­ Hogenboom, 1982). Itmigh t suffice heret o chanism,rathe r beingth equantitativ e result mention that therear eno wreport so f agly ­ ofdeleteriou s récessives (Mulcahy& Mulcahy , coprotein receptor (MW40,000 )o nlegum e 1983).Th eauthor s enumerate several examples, for Rhizobium cells (Apte& Modi , 1983),an d someo fwhic hma ywel l beplausibl y explained that Phytophthora glycoproteins elicit soybean bythi s alternative hypothesis.However ,th e productiono fphytoalexi n (Keen& Legrand , evidenceo f qualitative SImechanisms ,thoug h 1980). -Phytoalexin sar eplant-produced , non­ somewit h additional quantitative traits (e.g. specific antibiotics,whic har eals o reported Beta vulgaris, Larsen 1983)see ms ooverwhelm ­ tob eautophytotoxi c (Boydstone tal. , 1983). ing,an dconcordan t with other recognitionsy ­ stems,tha t purelyquantitativ e modelsma ya t Themajo r histocompatibility complex the highest applyt oexception s fromth erule . In higher vertebrates,histocompatibilit yi s There isno wgrowin g evidenceo f glycopro­ governedb ya comple xo fclosel y linked loci teins asbein gth especifi c productso fangio ­ (MHC),whic h codesfo rtransmembraneou s glyco­ sperm S-genes: Inth estigm ao f Brassica ole- proteins (MW44,000 )(fo ra brie f review,se e racea (sporophytic SI,i.e .polle n behaviour e.g. Kleine tal. , 1981). Itdoe sno tappea r governed maternally),severa l authors have immediately logical forsuc h organismst o found S-genotype-specific glycoproteins with maintain acomplicate d mechanism forrejectio n MWbetwee n 54,oooan d66,00 0 (Nishio& Hinata , of foreign tissue.Ther ear especulation s that 1982;Nasralla h âNasrallah , 1984;Ferrar ie t its primaryai mi ssurveillanc e ofmodificati ­ aI• , 1981). -Fro m styleso f Prunus avium(ga ­ onso fcel l surfacemolecule s withinth eindi ­ metophytic SI),Ma ue tal . (1982) isolated vidual,and ,a squote d earlier,th eancien to - twoglycoproteins ,M W37,oo oan d39,ooo ,asso ­ riginma yhav e beenth eris eo fsexuality .I n ciated withth egenotyp e S,S.. 1974, Zinkernagel &Dohert y revealedth ephe ­ nomenon nowknow na s"MH Crestriction "o fcel ­ Glycoproteins arecommo n angiosperm pollen lular immunity: Cytotoxic T-cells only killin ­ constituents (e.g.Vithanag ee tal. , 1982), fected cellsi fthe y exhibita specificit yco ­ outu pt oth epresen ta clea r association with dedb yth eindividual' sow nMHC-genotype .I n Siha sno tbee n demonstrated. -However , al­ otherwords ,t ob eeffective ,th eT-cel l must readyi n1954 ,Linsken swa sabl et odetec t associatively recognize "identity +differen ­ Slycoproteinsa sproduct so fmutua l ce"(MHC-mo lecu !e + foreig n antigen).

113 Another peculiarityo fth eMH Ci sit sextre ­ sevier/North Holland, Amsterdam, p.219-265, me gene polymorphism, possibly only surpassed Burke,D., L.Mendonca-Previato, C.E.Ballou, by thato fth eangiosper m S-genes. Thisvari ­ 1980. Proc. Natl .Acad. Sei. USA 77: 318-22, ability,whic h asa rul e involves blockso f Cooper,J.B., W.S.Adair, R.P.Mecham, J.E.Heus­ nucleotides,rathe r than single base substi­ er, U.W.Goodenough, 1983. Proc. Natl. Acad, tutions (asexpecte d ifgenerate d though point Sei. USA 80: 5898-5901. )no wseem s prooved tob ecause db y Dale,B., S.De ni s-Dom 'ni , R.deSantis, A.Monroj gene conversion, i.e. non-reciprocal transfer F.Rosati, V.Taglietti, 1978. Biol. Cell. 3! of information froma dono r (presumablya 129-133. pseudogene)t oa recipien tDN Aduple x (Weiss Ferrari, T.E., D.Bruns, D.H.Wallace, 1981. et al., 1983). Plant Physiol. 67: 270-277. Hogenboom, N.G. 1983. Phytopathol. 73:381-38- Conclusion Homan, W.L., P.G.Hodenpijl, A.Musgrave, H.var den Ende, 1982. Planta 155: 529-535. The examples above were deliberately select­ Keen, N.T. & M.Legrand, 1980. Physiol. Plant edt oillustrat e whatt oth eautho r appearo - Pathol. 17: 175-192. verwhelmingly indicativeo falmos t universal Kinsey, W.H. & W.J.Lenharz, 1981. J. Cell. principles inintercellula r recognition,du e Biol. 91: 325-331. to common origin. Particularly,i ti sinfer ­ Klein,J., A.Juretic, C.N.Baxevanis, Z.A.Nagy, red thatS Ii nangiosperm s (aswel l asi n Bo- 1981. Nature 291:455-460 . tryllus, Neurospora andothers )ma yb ecause d Larsen, K.,1983 . In: Mulcahy,D.L .& E.Otta - bya mechanis m parallel toth eMH Crestrictio n viano (ed.): Pollen:Biolog yan dimplicati ­ phenomenon:Associativ e recognitiono fidentit y onsfo rplan t breeding. Elsevier,N.Y .p . (S-glycoprotein)an ddifferenc e (?versu s i 205-210. glycoproteins),triggerin g an active rejection Linskens,H.F.,1954 . Proc.8 Congr . Int. Bot. response,possibl y through releaseo fphyto - .(Paris)Sect .10 : 146-147. alexin (orphytoalexin-lik e substance],strict ­ L/nskens,H.F.,1950 . Z.f.Bot. 48:126-135 . ly localized onth eindividua l pollen tube Matsuda.Y..S.Kazuko,K.Naoko ,T.Mizuochi ,Y . surface,o rwithi n it.Considerin g therelati ­ Tsubo,A.Kokata , 1982.Arch .Microbiol . ons in Neurospora and Botryllus, this appar­ 131: 87-90. ently doesno texclud eth epossibilit y that Lopo,A.C .& V.D.Vacquier , 1980.Natur e288 : (some of)th eS-gene s also have functionsi n 397-399. interspecific, heterogenic incompatibility. Mau, S.L., J.Raff,A.E.Clarke ,1982 .Plant a Finally,i ti spropose d thatth eextrem eS - 156: 505-516. gene polymorphism inangiosperm s iscause dan d Mendonca-Previato,L., D.Burke, C.E.Ballou, maintained by gene conversion. This alsoex ­ 1982. J.Cell .Biochem .19 : 171-178. plains several reportso fhigh-frequenc y gene­ Monroy.A.& F.Rosati, 1979.Natur e 278: 165-6 rationo fne w(o rknown )S-allele s following Moore,H.D.M.& J.M.Bedford , 1983.In : Hartman inbreeding (reviewed byd eNettancourt , 1977). J.F. (ed.): Mechanisman dcontro l ofanima l Silent S-genes (pseudogenes)ma yserv ea sRe ­ fertilization. Acad.Press,N.Y . p.453-497. servoirso fdiversit y (and/or sometimesb e Mulcahy,D.L. &G.B.Mulcahy , 1983.Scienc e called into action in extenso). • «20: 1247-1251. Looked upon inthi s way,angiosper m SIi s no Musgrave.A.,E.va n Eijk,R.teWelscher ,R.Bro ­ more thana specia l caseo fa genera l cell- ekman,P.Lens ,W.Homan ,H.va nde nEnde , cell recognition mechanism. Accordingly,i t 1981. Planta 153:362-369 . may risean dfal l from timet otime ,withou t Nasrallah,J.B .& M.E.Nasrallah , 1984.Expe ­ affecting itsbasi c elements:Ubiquitou s cel­ rientia 40:279-281 . lular recognition glycoproteins. Nettancourt,D.de , 1977. Incompatibilityi n Angiosperms.Springer-Verlag , Berlin.230p p Acknowledgments NishioJ.& K.Hinata,1982.Genetic s 100: 641-7 Oka, H.& H.Watanabe , 1957.Proc .Jap .Acad . The present study hasi npar t been supported 33: 657-659. by Kobmand Sven Hanseno ghustr u InaHansen s Pierce,M.& C.E.Ballou , 1983.J .Biol .Chem . Fond. Ia mmuc h indebted toProf .E .Andrese n 258: 3576-3582. for animating inspiration bycallin gm yatten ­ Scofield.V.L.,J.M .Schlumpberger ,L.A.West , tiont oth eMH Crestrictio n phenomenon. I.L.Weisman, 1982.Natur e 295:499-502 . Tohoyama,H.,M.Hagiya ,K.Yoshida , N.Yanagishi References ma, 1979.Mol .Gen .Genet . 174:269-280 . Vithanage,H.I.M. ,B.J.Howlett ,S.Jobson ,R . Apte.R.& V.V . Modi, 1983.Experienti a39 : B. Knox, 1982.Histochem .J .14 :949-966 . 509-510. Weiss,E.,L.Golden ,R.Zakut ,A.Mellor , K.Fahr Bleil,J.D . & P.M.Wassarman, 1983.Dev .Biol . ner, S.Kvist, R.A.Flavell, 1983.EMB 0J .2 : 95: 317-324. 453-462. Boydston.R.,J.D.Paxton , D.E.Koeppe, 1983. Willetts,N.& R.Skurray , 1980.Ann . Rev. Ge­ Plant Physiol. 72: 151-155. net. 14:41-76 . Carlile, M.J. & G.W.Gooday, 1978. In:Poste,G. Zinkernagel,R.M .& P.C.Doherty , 1974.Natur e & G.L.Nicholson (ed.): Membrane fusion. El­ 248: 701-702.

114 UNILATERAL HYBRIDIZATION IN RHODODENDRON J.L. Rouse2, R.B. Knox1 and E.G. Williams1 'plant Cell Biology Research Centre, School of Botany, and ^School of Physics, University of Melbourne, Parkville, Victoria 3052, Australia

Summary Hybridizationbetwee nVirey a Rhododendrons and species in the Azalea compex is seed was obtained. Inal l but two crosses unilateral, occurring only with Vireya pollen tubeswer e arrested within thestyl e species as female parents. Hybridity has or at the style base. In the two been confirmed by the, intermediate exceptional crossespolle n tubesentere d the morphology of leaf hairs on hybrid ovarybu tdi d notente r theovules . seedlings. Bymonitorin g pollentub egrowt h In pollinations with Azalea pollen on mehav e also determined the arrest points Vireya pistilsovule swer e entered bypolle n associated with incompatibility of Vireya tubes foral lexcep t thoseinvolvin g pistils polleno nAzale apistils . of jU_kawakami i in which arrest occurred within the ovary or at the style base. Introduction Viable intersubgeneric seed was produced on Vireya seed parents in a total of eight To our knowledge no previous confirmed crosses involving pollenparent s insection s successful crosses have been recorded Tsutsusi, Tsutsusiopsis, Pentanthera and betweenVirey a Rhododendronswhic hhav efla t Choniastrum: (R^laetu m xR_; _aurigeranum )x scales on their first true , and R. indicum, R. retusum x R^ simsii, (R. species in the Azalea complex which have intranervatum x R. 'Souvenir de J.H. simple or glandular hairs. As part ofa n Mangles') x R^ periclymenoides, (R. extensive survey of interspecific intranervatum x R. 'Souvenir de J.H. compatibility in the genus Rhododendron we Mangles') x R_;_japonicum , (R. 'Dr Hermann have made a number of controlled hand Sleumer' x R. herzogii) x R^_bakeri , R. pollinations between these groups and here retusum x R_^periclymenoides , R. retusumx report on the occurrence of a general R. ellipticum, and [(_R_imacgregoria e x R. unilateral compatibility, and the lochae)x R_ ^macgregoriae ]x Rj_tashiroi . recognition of hybrid seedlings by Hairs on the first true leaves of morphology ofth e juvenile indumentum. surviving hybrid seedlingswer e intermediate in morphology between the Vireya flat Materials andmethod s multicellular scales on a short stalk and the longer simpler hairs of the Azalea Pollinations involved the following parents. Typically theywer e stalked witha materials: bulbous glandular tip. The morphological Subgenus Rhododendron, section Vireya, differences in the juvenile indumentum of subsection Euvireya,a number ofspecie san d parent species and hybrids are suficiently fertile hybrids including Rj_sessilifolium , marked to be used as an indicator of (jj.laetum x R_i aurigeranum), (R. hybridity. Many of the intersubgeneric intranervatum x R. 'Souvenir de J.H. seedlingswer eweak ,a numbe r havedied ,an d Mangles'), (R. 'Dr Hermann Sleumer' x R. ofth e survivorsnon ehav eye tflowered . herzoRii)& [(R_;_macgregoria ex R_; _lochae )x AllVirey a Rhododendrons behaved similarly ii macgregoriae]; subsection Pseudovireya, incrosse so nAzale a pistils,showin g arrest iiretusum , R. quadrasianum (tropical)an d of pollen tubes before entry to ovules. j_.kawakami i (temperate). Azalea complex, However,amon g Vireya Rhododendrons used as subgenusPentanther a (deciduous Azaleas),R . seed parents, only Rj_kawakami i showed P_griciymenoides,, R . japonicum, R. bakeri, rejection of Azalea pollen before entry to subgenus Tsutsusi, section Tsutsusi ovules. We therefore conclude that the (evergreen Azaleas), R. indicum, R. simsii, barrier to interbreeding of Vireya and section Tsusiopsis, R_^tashiroi ; subgenus Azalea Rhododendrons is,i n general terms, Azaleastrum, section Choniastrum, R. unilateral,bein g more easily overcomewhe n gjj-ipticum. Vireya Rhododendrons are used as female Pollen tube arrest points in incompatible parents. crosses were determined by fluorescence microscopy of pistil squashes stained with decolorised aniline blue (Williams e_tal . References 1982). Williams,E.G. ,Knox ,R.B. ,Rouse ,J.L . 1982 Resultsan d discussion Pollination sub-systemsdistinguishe d by pollen tubearres tafte r incompatible In pollinations with Vireya pollen on interspecific crossesi nRhododendro n Azalea pistils no capsules developed andn o (Ericaceae)J .Cel l Sei.53 :255-77 . 115 EARLY POST-FERTILIZATION DEVELOPMENT IN RHODODENDRON

V.Kaul ,J.L . Rouse,R.B .Kno x and E.G.William s

Plant CellBiolog y Research Centre,Schoo lo fBotany ,Universit y ofMelbourne ,Parkville , Victoria 3052,Australi a

Summary blue fluorescence-positive callosic walli s initiated adjacent to the degenerating Early post-fertilization development has synergid,extend st ocove r theentir ezygot e been studied in several species of surface, and remains visible as the zygote Rhododendron (Ericaceae). Fertilization is elongates prior to the first proembryonal marked by rapid development of a callose division. This division is delayed 10-15 wall around the zygote. The first days or more after fertilization. (Wehav e proembryonal division isdelaye d for 10day s observed undivided zygotes at 60day safte r ormor eafte rfertilization ,bu tth eprimar y pollination in R^_konor i developing at endosperm nucleus begins development winter temperatures inMelbourne) . Priort o immediately after triple fusion. The the first proembryonal division the zygote endosperm is cellular from the first elongates at the end distant from the division. Apparent self-incompatibility in micropyle,an d the first division separates three species has been shown tob e apost - a small cap cell from a larger basal cell. zygotic mechanism. Interspecific The distal portion of the embryosac ,whic h incompatibility may also, in certain is lined with a single endothelial layer, crosses,b e expressed after entry ofpolle n enlarges and becomes filled with cellular tubesint oth eovules . .endosperm before the first division ofth e zygote. Introduction Inovul oexpressio n of incompatibility In an extensive study of pollen-pistil interactions and interspecific pollinations Interspecific incomptibility in inRhododendro n wehav eobserve d anumbe ro f Rhododendron may be expressed atan yon eo f instanceswher e foreigno roccasionall y self at least 7 different levels in thepistil , pollen tubes penetrate the ovules but from the stigma surface to the ovules produce little or no viable seed. (Williams et al_. 1982a,b). In species Accordingly,w ehav e setou tt odescrib eth e combinationswher eovule sar eentere d butn o course of early post-fertilization events viable seeds produced we have observed two leading to normal seed development,an d to basicoutcomes : Ina fe winstance s (e.g.R . observe the deviations from this patter^i• kawakamii x Kalmia latifolia (Ericaceae) which result in interspecific incompat­ normal fertilization is not achieved, and ibility or apparent self-incompatibility. tftere is an abnormal coiled overgrowth of Observationshav ebee nmad e either bybrigh t the undischarged pollen tube within the field microscopy of semi-thin plastic embryo sac. More commonly,achievemen t of sections of ovaries stained with PAS fertilization is marked by formation ofa (periodic acid/Schiff)- toluidin e blue,o r normal zygote callose wall and the fluorescence microscopy of whole pistil initiation of endosperm development, with squashes or ovary sections stained with incompatibility being expressed as failure decolorised aniline blue (Williams et al. of zygote or early proembryonal divisions 1982a,b, 1984a,b). together withendosper m degeneration (eg. Rj_kawakami i x R^ retusum and R. Compatible Development retusum xR^ _kawakamii) . Somecrosse s (eg.R ^retusu m xR ^santapaui ,R .retusu mx Ovule development has been studied in R.ovatu m andR_ ^retusu m xR_ ^tashiroi )sho w compatible hand-pollinations of seven later abortion,producin gmature d seedswit h Rhododendron species (Williams et al. partially developed, inviable embryos,o r 1984a). Pollen tubes reach the ovules in small seeds giving rise to weak, abortive about 4-7 days after pollination and seedlings. discharge their contents into one synergid Self-incompatibility inR ^ellipticum ,R . cell. The endosperm begins development championaean dj U amamiense iscontrolle d by immediately after fertilization, and is a post-zygotic barrier to development of cellular from the first division. The selfed embryos (Williams et al., 1984b). zygote shrinks slightly relative to the Fertilization is apparently normal. The unfertilized egg, and lays down a special zygote contracts slightly and lays downa callose wall in the first two days after callose wall as seen after compatible fertilization. The PAS-postive, aniline pollination. Some pre-divisional zygote elongation occursbu tn odivision shav ebee n observed. The endosperm begins development 116 normally, but within two weeks after fertilization degeneration becomesapparen t (eg. at the 12-16 celled stage in R. amamiense). Collapse ofth e embryosa can d ^vïiïë wall follow rapidly. Although endosperm degeneration isth e first visible signo fabnormality ,thi si sno tnecessaril y causal to embryo abortion, and may be a parallel expression of the same underlying factors. Self-incompatibility ispresumabl y controlled by a system of multiple lethal genes functioning to produce severe "inbreedingdepression "i nearl y embryogeny.

References

Williams,E.G. ,Knox ,R.B .& Rouse ,J.L . 1982a Pollination subsystems distinguished bypolle n tubearres t afterincompatibl e interspecific crosses inRhododendro n (Ericaceae).J .Cel lSei . 53:255-77 . Williams,E.G. ,Knox ,R.B .& Rouse ,J.L . 1982b Pollen-pistil interactionsan dth e controlo fpollination . Phytomorphology 31: 148-57. Williams,E.G. ,Knox ,R.B. ,Kaul ,V .& Rouse,J.L . 1984a Post-pollination callose development inovule so f Rhododendron and Ledum (Ericaceae): Zygote specialwall . J. CellSei . (inpress) . Williams,E.G. ,Kaul ,V. ,Rouse ,J.L .& Knox,R.B . 1984b Apparentself - incompatibility inRhododendro n ellipticum,R .championa e and R. amamiense: Apost-zygoti c mechanism. Incompatibility Newsletter (inpress) .

117 POLLENTUB EPENETRATIO N INTH E STYLEO FGASTERI A

M.T.M.Willems e andM.A.W .Franssen-Verheije n

Department ofPlan tCytolog y andMorphology , AU,Wageningen ,Th eNetherland s

Summary

The centralpar t of thestyl e isfille d with amateria l ofchangin g consistency.I n thefirs tpar tbeyon d the stigma themateria l issolid ,i nth edirectio n of theovul ei ti s liquid. The courseo f thepolle n tube through the styleseem s tog othroug hmateria lmainl y consisting ofmono -an doligosaccharides .

Introduction

As inothe rLiliaceae ,Gasteri averrucos a Afterpollinatio n ofa receptiv e stigmath e has ahollo w style (Sears,1937 )wit h awe t growingpolle n tubes (pt)penetrat e thethic k stigma (Heslop-Harrison &Shivanna , 1977). parto f thepapillä rcel lwal l andgro wint o However asubmorphologica l study revealsa thesoli d centralpar to f thestyle .A par t special typeo fa styla r canal inGasteria , of thepolle n tubes areflatene d (fig.2) . especially atth e tipo f thestyle . THepolle n tubewal l (w)consist s ofa thi n finefibrilla r layer,whic h shows awea k Methods fluorescence after stainingwit haniline-blu e

Parts ofmatur e styles ofGasteri averru ­ cosawer e fixed in3 %glutaraldehyd e inphos -' phatebuffe r0. 1 M at7. 2 pHfo r 18hr sa t 4C .Afte rwashin g the specimenswer e stained in 1%OSO 4i nbuffe r during 2hr s at20°C . After dehydration andembeddin g inEpon ,sec ­ tionswer epoststaine dwit h leadcitrate and uranylacetate,an dexamine d ina Philip sE M 301.

Results *'

The stigmao fGasteri aha spapillä rcell s ofmediu m length.Th e cellsar eelongate d and surroundedb y awal l consisting ofa , x40PQ0 small layer around the cellan d athic klaye r which isbordere db y anundulatin g cuticle. Beyond thestigm ath ethic kpar to fth epapil ­ Conclusion lärcel lwal l iscontinuou swit h thecentra l part of thestyl e (cw).I nthi spar tremnant s Among thedifferen t stylar types,Gasteri a of thecuticl e (c)o f the three carpels,whic h shows acentra l stylarpar twit h changing border threever y small flat canals,ar e consistency and astar-shape d canaltapere d present.Th e centralpar tbeyon d thestigm a off tothre epoint snea r the stigma.Th epol ­ contains elongated cells.A t the corticalpar t lentube spenetrat e thepapillä rcel lwall , of the style the cells laymor e closetogether . and followa wa y rich incarbohydrates .Be ­ See fig.1 . causeo f thehig h osmotic rate and theim - Themateria l of thethic kwal l layer ofth e bition capacity of thesurroundin gmateria l papillär cells and its continuation inth e inth estyl ebeyon d the stigmaprobabl y the centralpar t of thestyl e iselectro ntrans ­ pollen tubesbecom e flattened. parent.Histochemica l testso n thismateria l forpectine ,cellulos e and callose arenega ­ References tive.Stainin g of the liquidwit hnaphtol - H2SO4i spositiv e and suggests thepresenc e Sears, 1937.Cytologica lphenomen aconnecte d of carbohydrates asmono - (glucose teststrip s with self sterility inth e flowering plants arepositive )an doligosaccharides . Genetics 22:130-181 . Heslop-Harrison,Y . &K.R . Shivanna,1977 . Thereceptiv e surfaceo f theangiosper m stigma.Ann .Bot .41 :1233-1258 . llf THEPOLLE NTUB EA SA NEXPERIMENTA LGROWT HSYSTE M

E.G.Williams ,P .O'Neil l and T.Houg h

PlantCel lBiolog y Research Centre,Schoo lo fBotany ,Universit y ofMelbourne ,Parkville , Victoria 3052,Australi a

temperature and plant genotype (Williams & Knox 1982,William s et al., 1982b). Summary Qualitative invitr obioassa y The effects of various factors on pollen tube growth jjiviv o can be quantified by Pollen is applied with the edge of a counting numbers of pollen tubes passing coverslipa sa thi nlin eacros sa microscop e throughoptica l transectso f stylesa tfixe d slide coated with an agarose-solidified intervalsafte r pollination. Pollentub e germination medium. Two 2mm wide filter growth ni vitro can be quantified by paper strips containing a test substancei n measurement of pollen tube lengths in solution are then placed 2 mm out oneac h hanging drop cultures or cultures on the side of the line of pollen (Williamse tal . surface of a semi-solid medium. These two 1982c), or a single test strip directly in vitro techniques have been developed as above the pollen (Williams et al. 1982b). bioassays for the effects on pollen tube The slide isplace d onmois t filter paperi n growth of microgram amounts of test a closed petri dish,incubate d 18hour sa t substances. 20-23°C, and then fixed with formaldehyde vapour by adding 0.5 ml formaldehyde Introduction solution to the filter paper and reclosing the dish for 10 min. Mean pollen tube To study the control of pollen-pistil length for each slide isdetermine d usinga interactions,polle n tubes are grown either Carl Zeiss Videoplan Computerized Image in association with the living pistil in Analysis system attached to a compound vivo, or separately in.vitro . The i]iviv o microscope. Thismetho d has been used to system istypicall y used to examine effects survey stylar proteins for fractions on the interaction, of factors such as affecting pollen tube growth (Williams et temperature,flowe rage ,plan tgenotyp eetc . al. 1982c). Themetho d cannotb e used for However,t o identify signalmolecule swhic h precise quantification of the effects of pass between the pistil and pollen tube,a these molecules since the diffusion gap system is required with a less complex between filter paper test stripsan d pollen metabolism than thato fth ewhol epollinate d tubesvariabl y reducesth e timeo ftreatmen t pistil. The obvious simplification ist o and the concentration of test molecule remove the pistil and grow pollen tubes in reachingth etubes . vitro on a simple defined medium. Pistil fractions or other known molecules canthe n Quantification of growth invitr o be introduced intoth eculture s todetermin e their effects. Here we reporta metho d for Quantification of the effects of quantification of pollen tube growth in substances on pollen tube growth in vitro vivo, and two techniques for ^n vitro can be achieved using multiple 200ju l bioassay ofmicrogra m amountso f substances hanging drop cultures in the wells of a affectingpolle n tubegrowth . 96-well PVC disposable microtitre tray (Hough et al. 1984). Cultures are setu p Quantification ofgrowt h inviv o using aconstan t amount ofpolle n suspended in germination medium, and a varying Pollinated pistilsar e fixed after aknow n concentration of the test substance, The interval and squashed indecolorise d aniline tray is carefully inverted so that the blue for visualisation of pollen tubesb y cultures formhangin gdrop ssuspende d inth e fluorescence microscopy (Williams et;al . wellsb y surfacetension . The inverted tray 1982a, Willams & Knox 1982). Counts are isthe n laid across two shallowsupport si n made of numbers of pollen tubes passing a closed container lined withmois t filter througha serieso foptica l transectsacros s papers, and incubated at 20-23°c. For the style and pollen tube numbers are then sampling thetra y isreverte d and a 15-25/U l plotted against distance down the style. aliquot withdrawn from each well. Each This technique has been used for sample is placed on a microscope slide, Lycopersicon peruvianum, to determine rates fixed by addition of a small drop of of compatible pollen tube growth, and to formaldehyde solution, and seated under a examine the effects on self-incompatible coverslip for microscopic measurement of tube arrest of flower age,nutrien tstatus , pollen tube lengths as in the previous method above. After sampling the tray can be reinverted and incubation continued.

119 This method has been used to quantify the effectso f inhibitor and promoter molecules on pollen tube growth (Hough et al. 1984, Hewitte tal . 1984).

Conclusion

Quantification of pollen tube growth in thepisti lca n providedat a on pollen-pistil interactions, particularly the gametophytic self-incompatibility response. _In vitro pollen tubeculture sca nb euse d toidentif y stylar molecules which may be active in pollen-pistil interactions, and to investigate the effects on pollen tubeso f defined molecules. In the future, these methodsma y also be useful for selectiono f superior pollen sourcesan d screening pollen forphysiologica lmutants .

References

Hewitt,F.R. ,Sasse ,J.M . &Rowan ,K.S . 1984 Theeffect so fbrassinolid e onpolle n germination and tubegrowth . Aust.J . PlantPhysiol ,(submitted) . / Hough,T. ,O'Neill ,P. , &Williams ,E.G . 1984 Multiplehangin gdro passa yfo r substancesaffectin g pollentub egrowt h invitro : Effecto fcycloheximid eo n Prunusaviu m pollen tubes. Aust.J .Plan t Physiol, (submitted). Williams,E.G. ,& Knox ,R.B . 1982 Quantitative analysiso fpolle ntub e growth inLycopersieo nperuvianum ,J . Palynol. 18:65-74 . Williams,E.G. ,Knox ,R.B. ,& Rouse , J.L. 1982a Pollination sub-systems distinguished bypolle n tubearres tafter , incomptible interspecific crossesi n Rhododendron (Ericaceae). J. Cell Sei. 53:255-77 . Williams,E.G. ,O'Neill ,P .& Hough ,T . 1982b Thepolle n tubea sa n experimental system. In:"Pollinatio n '82"(ed. ) Williams,E.G. ,Knox ,R.B. ,Gilbert ,J.H . & Bernhardt,P . Plant CellBiolog y Research Centre,Universit y ofMelbourne , pp. 15-26. Williams,E.G. ,Kamm-Anderson ,S. ,Dumas , C, Mau,S.-L. ,& Clarke ,A.E .1982 c The effect ofisolate d componentso f Prunusaviu mL .style so nij ivitr ogrowt h ofpolle ntubes . Planta 156:517-9 .

120 STYLE-CONTROLLED COROLLAWILTIN G INPETUNI AHYBRID AL .

LuudJ.W . Gilissenan dFolker tA .Hoekstr a

ResearchInstitut e ITAL,P.O . Box48 ,670 0A A Wageningen, theNetherland s (L.J.W.G.); andDepartmen t ofPlan tPhysiology ,Agricultura lUniversity ,Arboretumlaa n 4,670 3B D Wageningen,th eNetherland s (F.A.H.)

Summary throughth estyl et oth ecoroll aoccurre d within4 h afterpollinatio n orwoundin go f Inthi sposte r arevie w isgive no fth e thestigma .Ethylen e evolutionals o resultso fresearc ho n style-controlled increased immediately upon thesetreatments . corollawiltin g inPetuni ahybrid aL . Because ofth esimilarit y ineffect so f Pollinationfollowe d bypolle ntub e pollentub epenetratio n and stigmawoundin g penetration intoth estigma ,o r injuryo f oncoroll awilting ,a simila rmod eo factio n thestigm ao r style led toth e generationo f ofth ewilting-inducin g factor isproposed . awilting-induein g factoran d itstransfe r Eluates collected fromth eovaria nend so f .t oth ecoroll awithi n 4h .Th ewiltin g styles contained awilting-inducin g factor, inducingfacto r isdifferen t from1-amino - particularly inth ecas eo fpreviou s cyclopropane-1-carboxylic acid (ACC). pollinationo rwounding .Th econten t ofth e Keywords:pollination ,stigm awounding , ethyleneprecurso rAC C inth eeluate swa s corollawilting ,ethylene ,1-aminocyclo - belowdetectio n (Gilissen &Hoekstra , 1984). propane-1-carboxylic acid (ACC).

Introduction References

Insom eplan t species corollawiltin go r Gilissen,L.J.W. , 1976.Th erol eo fth e abscission is initiated by pollination style asa sense-orga ni nrelatio nt o (orchids,Dianthus ,Petunia , , wilting ofth e flower.Plant a131 : Digitalis etc.). InDigitali s the stimulus 201-202. foraccelerate d abscissionreache sth e Gilissen,L.J.W. , 1977.Style-controlle d corollabasi swel lbefor epolle ntube shav e wilting ofth e flower.Plant a133 : traversed the style (Stead andMoore , 1979). 275-280. Ethylenebiosynthesi s isgenerall y involved Gilissen,L.J.W . &F.A . Hoekstra,1984 . inthes epost-pollinatio nphenomena . Pollination-induced corollawiltin gi n Thisposte r reportso nth eeffect so f Petuniahybrida :Rapi d transfer through pollination andwoundin g of the stigmai n the styleo f awilting-inducin g substance. Petuniaflower san do na possibl e roleo f PlantPhysiol .75 :496-498 . 1-aminocyclopropane-1-carboxylicaci d (ACC) Stead,A.D . &K.G .Moore , 1979.Studie so n orethylen e inth etransfe r of thewilting - flower longevity inDigitali s: -inducing factor from the stigmat oth e Pollination-induced corolla abscissioni n corolla. Digitalis flowers.Plant a 146:409-414 .

Results and conclusions

The timerequire d for50 %o f theflower s ofP .hybrid a towil t (W50)wa s considerably reduced afterpolle n tubepenetratio n into thestigma .Pollinatio nwithou t tube penetrationdi dno t affect corolla longevity.Th eW50 ,du e topolle ntub e penetration,wa shighe r afterself-polli ­ nationtha nafte rcross-pollination .Wiltin g wasaccelerate d inproportio n toth enumbe r ofpenetratin g tubes.Injur y of thestigm a orstyl eals o strongly acceleratedwilting . Style-induced acceleratedwiltin gwa s prevented by excisiono f theentir e style (Gilissen, 1976; 1977).Althoug hthi s treatment caused some decreaseo fW5 0a s comparedwit h untreated flowers,i tallowe d forkineti c analyseso f the transfer ofth e wilting-inducing factor.Thi s transfer

121 PART III

MEGASPORGENESIS, OVARY, EMBRYO SAC DEVELOPMENT EMBRYO AND ENDOSPERM DEVELOPMENT

• ••••••••••••••••••••••••••••••••••••••y»-

123 Part III: Megasporogenesis, ovary, embryo sac development, fertilization and embryo an endosperm development.

Introduction this problem with an idea for research Megasporogenesis, sperm cells, embryo onsexua lreproduction :

sac structure, fertilization, apomixis Well, Prof. Willemse, I declare, and embryogenesis are the main topics Is that the Delta Project there? They tell us that the Dutchmen try in this part, but to begin with some data To keep their little country dry. on ferns. Most studies are done using Butsurel y thati shar dt odo , And costs a lot of guilders too. the electron microscope, some combined Why fight to keep the water out - with quantitative data and biochemistry. If it came in, you could farm trout. And we could study something new, The use of image analysis systems and Like algal sex andgamete stoo . ultracryotomy is new. For application And scubadivin gw ecoul dg o Totak eou r samples frombelow . in breeding the study of apomixis, the Toanalys eth esubmarin e in vitro embryoid formation and the data Would open up our research scene. Sow einvit eyou ,i fyo ulike , on sperm cell composition are of interest. To pull your finger from the dyke.

Ultrastructural studies "on plant reproduc­ tion today become more detailed because the general phenomena are well known. In spite of the studies on the developing embryo sac and fertilization untill now' too little attention has been payed to ultrastructural studies on embryogenesis and seed coat formation. The resolving of problems in keeping seed qualities can use some submorphological information. More comparisons could be made with somatic embryogenesis.

In experiments with ovules, but also in general on sexual reproduction, the repro­ As is illustrated by Noud Schel and can ductive calendar of the plant under study be find on the end of• thi s volume, Prof. is very important, such as in relation Willemse has followed this advise and to possible aberrations as apomixis. pulled his finger from the dyke. He is liberated with all his fingers free in In this part different aspects of the the sea. From this position he isstimulat ­ reproductive processes from,megasporogene ­ ed to think how sexual reproduction started sis to embryogenesis are considered. Com­ in the sea. Plants using the power of pared with studies on pollen more attention sexual reproduction have ultimatelycoloniz ­ should be payed to the very complicated ed land. In this way, pulling a finger process of ovular development. Many con­ from a dyke , itwoul d stimulate the study tacts were made during the congress. The of sexual reproduction in the sea, and excursions focussed on the problem how it would again ultimately lead up to the to keep Holland dry. This stimulated Dr. study of seed plants on land, especially Williams again to compile a poem linking theflower .

124 CRYOSCANNIN GELECTRO NMICROSCOP Y OFREPRODUCTIO N INCRYPTOGAM S

E.Sheffield ,E.G .Cutte r

Department ofBotany ,Universit y ofManchester ,Manchester ,M1 3 9PL,U.K .

Thescannin gelectro nmicroscop e (SEM) Preparativeprocedur e studyo freproductio n inpteridophyte san d bryophytesha spreviousl y reliedupo n 1. Generateplant s inasepti c culture preparative techniques employing fixation, wherepossible . thencritical-point-dryin g orfreeze-drying . 2. Removematerial ,attac ht oSE Mstu b Thesetechniques ,however ,yiel dvariabl e with silverpaint . resultsan d frequently causedistortion , 3. Plunge stubint oslushe d liquid shrinkageand/o r collapseo fth efragil e nitrogen. structureswhic heffec treproductio ni n 4. Evacuatechamber ,transfe rstu b cryptogams.Th ene wtechniqu eo fcry oSE M (undervacuum )t oSEM . avoidsartefact s and damage attributable 5. Observespecime ni nSE Mo ncry ostag e tofixatio nan ddehydratio n sinceth etissu e (Hexland)a t-190°C . issimpl y frozenan d observeddirectly . 6. Sublimean y superficialic eb y raising Themetho d istherefor emuc hquicke rt o to-80°C . performtha nconventiona lprocedure san d 7. Withdraw specimen topre-chambe r canyiel d specimenso fvastl y superior (-190°C),sputte rcoat . appearance (henceinformationa lcontent) , 8. Return tomicroscop e (Cambridge S150 ) asillustrate d bythes emicrographs . forobservatio n at-190°C .

Antheridiao fPteridiu m aquilinum,eac h hastw oring-lik ejacke t cellsan da singl e opercular cell. Bar= 50u m

Marchantiaalpestris.Par t ofa narchegon - iophore,bisecte d prior tofreezing ,showin g Antheridiao fEquisetu m arvense,mos t maturependulou s archegonia.Thes e sito na havefou ropercula r cells. Bar= lOOu m cup-shapedpedestal . Bar= lOOu m

125 GAMETANGIAI NCEYLONES E SPECIES OFTH EFER NFAMIL Y THELYPTERIDACEAE

E.Tigerschiöl d

Department ofBotany ,Universit y of Stockholm,S-10 69 1 Stockholm,Swede n

Summary layer sotha t itlook s like thearchegoniu m isstandin g ona widene d foundation.I nmos t Variation ingametophyt eanatom y in species thefou r cellrow s terminate ina Thelypteridaceae isunde r investigationan d triangular cell onto p of thearchegonium , includes studies of the structure ofse x where there consequently arefou r triangular organs.I ngenera l archegonia consistextern ­ cellsmeeting .Sometime s only threecell s ally of fourrow so f cells and onero wha s meeto n the topo f thearchegonium ,th e enlarged cells forming acurve d "chord". fourth rowendin gbelow .I nMacrothelypteri s Variation isfoun d innumbe r and sizeo fto p torresiana the top cellsar eno t triangular cells.Antheridi ausuall y consist of three but differ fromeac hothe r inshap ean dsize . cells surrounding the spermatozoids.Differ ­ Viewed from theoutsid e theantheridiu m entmechanism s ofopenin goccur . (Fig2 )generall y consists of threecells , onebasa l cell,on erin gcel l that iswide r Introduction thanth ebasa l cellan d anundivide d capcel l InMetathelypteri s flaccida thebasa lcel li s Thepreviou s studies of gametophyte wider than therin g cell.I nsom e speciesth e anatomy inThelypteridacea e havebee nmad e c^pcel l isoccasionall y divided into two,o f with lightmicroscope .Antheridi a andarche ­ jfhichon e isluneat e andpartl y surroundsth e gonia are located onth ecentra l cushioni n other circular cell.Ver y often theopenin g fully developed prothallia.Dehiscen so f of theantheridiu m isb ydetachmen t andlift ­ antheridia isreporte d togenerall y occur ingo f theca p cell. Sometimes there isals o bya por e (Atkinson &Stoke y 1973,Atkinso n apor e inth eoute rcel lwal l of theca pcel l 1975)bu t inon e species theca pcel li s when iti slifte d off.I nmos t cases theca p lifted off (Atkinson &Stoke y 1973).Th e cell istotall y lost.Th e conditionwit ha archegonial neck iselongated ,4- 6 cells pore inth eca p cellwhic h remained attached long and curved away from thenotc hregio n toth eantheridiu mwa s alsofound . (Nayar &Kau r 1971,Atkinso n 1975). As theknowledg e about gametophytemorpho ­ References logy inThelypteridacea e isscarc e itseeme d important toundertak e adetaile d study Atkinson,L.R . &Stokey ,A .Th egametophyt e makingus eo f theresolutio n powero f the, of someJamaica n thelypteroid ferns.Bot .J SEM.Th eresult s about sexorgan s reported Linn.Soc .66:23-36 . here ispar to f amor e extensive investiga­ Atkinson,L.R . 1975a.Th egametophyt e offiv e tiono f thegametophyt e generation in OldWorl d thelypteroid ferns.Phytomorpho - Thelypteridaceae. logy25:38-54 . Nayar,B.K .& Kaur ,S . 1971.Gametophyte so f Material andmethod s homosporous ferns.Bot .Rev .37:295-396 .

Fertile sporophytes of 27specie so fth e familyThelypteridacea e werecollecte d in Ceylon.Plant s originating fromthi smateria l arekep t incultivation .Gametophyte sfo r structural investigationwer e grownfro m spores insteril e culture onagar .Th ere ­ searchmateria lwa s critical point dryedan d studied ina Cambridg e stereoscan600 .

Results

Archegonia (Fig 1)wer e found toconsis t of fourrow so fcell s that are 5-6 cells high.I nal l species studied thematur e archegonia areben t away fromth enotc han d Fig.1 .Amauropeltahakgalensis.44 0 leaning somewhat toon e side.On eo fth e Fig.2Antheridium.Christell ahispidula.560 x four rowso fcell s thatbuil du p thearche - goniumar eenlarge d forming acurve d "chord. Inon e species,Amauropelt ahakgalensis , therear emor e than fourcell s inth ebasa l

126 WATERFER N (MftRSILEA OUADRIEOLIA) Sporocarps- Fo rgermination ,th etip so f ^PRODUCTION AS A BIOASSAY POR AFLATOXIN B, Marsileaquadrifoli aL . sporocarps (Carolina AND T-2 TRICOTHECENE TOXIN Biological Supply Inc.,Burlington ,NC )wer e excisedan dthe nplace dint oa Petr i dish containing1 0m lo fsteril edistille dILO . G.C. Llewellyn, J.D. Reynolds and W.V. One sporocarpwa suse dpe r treatmentwhic h Dashek + ,* occurredwithi na nenvironmenta l chambera t 14h rdayligh t (6:00a mt o8:0 0pm) ,2 1C , Depts.Biology, Virginia Commonwealth Univ., 550 lumensfluorescen t light. At8:3 0a m Richmond, VA 23284, Atlanta Univ.+, Atlanta, one sporocarpwa sprepare da sabov ean db y GA 30314, and West Virginia Univ.*, 1:00p nmegaspore sisolate dan db y2:0 0p m Morgantown, W, 26506, USA treated. - A tmegaspor ereleas efro mth e sorus,eac hmegaspor edisplaye da smal l papillaprojectin g fromon eend . This Summary indicatedth emegaspor e nucleuswhic h underwent division resultingi narchegoniu m Bothaflatoxi nB . (AFB,),a secretio no f formation. Thelatte r consistso fbot hnec k Aspergillus spp.,an dT- 2 trichothecene cellsan da singl eeg gcell , external toxin,a by-produc to fFusariu m spp.,ar e evidencesfo rarchegonia l formation subjectso fintens e research interests. (Bierhorst,1971) . Approximately10 0 Thisstem sfro mth ecarcinogenic , mutagenic megasporeswer eproduce db yon esporocar p andteratogeni cpropertie so fAFB ,an dth e germination. Followingthei r collectionvi a hemorrhagicattribute so fT- 2toxin . The a5 0id .capillar y pipet,3- 5megaspore spe r latterha sbee nuse d (allegedly)a sa wellwer eadde dt oa tissu e culture warfareagen t therebymandatin gth e multi-well plate (LinbroDivisio n Flow developmento fquantitative , rapid, reliable Laboratories, Inc.,Hamden ,CT). Each andsimpl eassay sfo rthes emycotoxins . megasporewa s developedt oth epapillar y Here,w erepor tth epossibl eus eo fwate r stagewithi n coveredwel lplate san dth e fernreproductio na sa mycotoxi nbioassay . environmental chamber. Eachwel l contained 2m lsterile ,distille dBLO . Archegonial Introduction formation,fertilizatio nan dresultan t sporophyte developmentwer e observeda t Whereasaflatoxin s (APTs)ar esecretion s leasttwic e dailyfo r5- 7day sa t7-30 X ofAspergillu saavu san dA . parasiticus magnification. (Goldblatt,1969) , trichothecenesar e T-2toxi ntreatmen to fmegaspore s- Usin g productso f1 7Fusariu m species (Joffe, a5 0il lcapillar ypipet ,th eH, 0withi neac h 1965). Onderprope r environmental wellwa sremove dvi aa nautomati cvacuu m conditions,thes e fungica ngro w upon crops pipettor connectedt oa serie so ftraps . (Mirocha sLsH., 1976;Dashe k& Llewellyn , TheT- 2toxi nwithi n0. 5m lIL O(Calbiochem , 1982)thereb y posinga healt h riskt obot h SanDiego ,CA )wa sadde dt oeac hwell . For manan dhi sdomesticate danimal ssinc eAFT s toxinstoc kconstruction ,1 m laceton ewa s arecarcinogenic , teratogenican dmutageni c addedt o500 0p gT- 2toxi nwit hth e (Vfogan,1965 )an dtrichothecene sar e dissolved toxinbein gadde dt o19 8m lo f hemorrahgic (Schoentale tal. . 1979). sterile,distille dH-O . Asecon dm lo f Recently,evidenc eha saccumulate d forth e acetonewashe dou tan yresidua lvia l allegedus eo fcertai ntrichothecene sa s toxin. Thestock ,2 5y gtoxin/ml ,wa s chemical/biologicalwarfar eagent s (Rosen& heatedwit h stirringfo r3 h rt obot hmi x Rosen,1982 ;Miroch a.§ £âl. , 1983). anddriv eof fth eacetone . Following Furthermore,AFT scoul d servea ssuc h cooling,0. 5m laliquot swer eadde dt owell s agents. Thus,i ti simportan tt odevelo p followinga 3 0se caeratio n involving rapid,accurate , reliablean dsimpl e vigorous shakingo f20 0m lwithi na flask . quantitative assaysfo rth etw otoxins . The Theacetone-wate r control (AC)receive d2 m l currently-available, highly-sensitive ofaceton ei n19 8m lo fwate r concurrently analyticalmethodologie sare : withth eT- 2toxi n stock. Another control non-transportablet oth efield , expensive (C)withou t either toxino raceton ewa suse d andrequir ehighly-traine d personnel. Thus, also. Toxin (T-2),A Can dC solution swer e wear egeneratin g bioassaysfo rbot hAFB . maintaineda t2 2C prio rt oadditio nt oth e andT- 2toxin . wellscontainin gth emegaspores . Here,w erepor twhethe rwate r fern Megasporeswer e treatedwit h0. 5m lo fT- 2 reproductionca nb ea bioassa yfo rbot h toxin,A Co rC solution shavin g3- 5 toxins. megasporespe rwell .Th eplate' sfirs t eight wellscontaine dT- 2toxin ,th enex teigh tA C .Materialsan dMethod s andth elas teigh tC . Megasporeswer e treatedfo ron eh rwhe n solutionswer e T-2Toxi nbioassa y withdrawnusin gth eautomati cpipette san d traps. Anew ,steril e5 0ja lpipe twa suse d

127 for eachevacuate dtreatmen tgroup . 16.0 ppm but was reduced to 57.14% at 0.5 Immediatelyafte r removalo fth esolution s ppm. Whereas the 1.0 ppm-treated sporocarps fre mth ewells ,tw om lo fsteril eH~ 0wa s exhibited a germination of 85.71%, the 20.0 addeda sth efirs twash . Therewer ethre e ppm-treated group showed a percentage of washeswhic h requiredabou t1 0min . 71.43. Generally,th etota lmegaspor e "n"numbe r foreac htreatmen twa s>9 0wit ha nattemp t Fertilization tohav e100 . TheT- 2toxi n concentrations werea si nTabl e1 . AF&, - A 50% fertilization level for the Microsporeadditio nt otreate dmegaspore s sporocarps that had germinated was observed inth ewell s- Followin gwashes ,treate d for control cultures. In contrast, megasporeswer emaintaine di n2 m lsteril e fertilization ranged from 8.0% for the 0.5 H-O. Toeac hwel lwer eadde dnumerou s ppm replicas to 71.43% in the 2 ppm gelatinousclump so fmicrospore st oensur e cultures. The highest AFB, concentration anexces so fsper mfo rfertilization . Prom (20.0 ppm) possessed a germination thistim emicroscopi cexamination swer e percentage of 17% > the controls. performed routinelya tleas ttwic edaily . Sporophyte production AFB, bioassay T-2 toxin - Table 1 summarizes sporophyte Marsilea sporocarpswer e scarifiedprio r development for T-2 toxin-treated megaspores tothei rplacemen t intovariou sAFB , five days after treatment. Percent concentrations. A2 0.ug/m lAFB ,stoc kwa s megaspores developing normal sporophytes was preparedb ycombinin g1 0m gAFB , (Lotno . linear between 0.314 and 0.039 .ug/ml. 387032,Calbiochem ,LaJolla ,CA Twit h44 0m l Further refinement of this potential of sterileBU Oan d6 0m lo facetone . From .bioassay might be accomplished by increasing thisstock ,SFB ,dilution so f16.0 ,12.0 , /the concentrations between the extremes. 8.0,4.0 ,2.0 ,1. 0an d0. 5pg/mL were AFB, - Both the control and 20.0 ppm prepared. treatment shewed sporophyte occurrences of Sevenreplicate scontainin g2 0m lo feac h 100%. For sporophyte production originating dilutionwer eprepare dan dplace dint o from a single sporocarp, means of 81 sterilePetr idishes . Sevencontrol s sporophytes for controls and 87 for 20.0 ppt (sterileH, 0an dacetone )wer emad ealso . A were observed. Sporophyte production in singlesporocar pwa splace dint oeac hdis h seven of eicht remaining toxin levels was < whichwer e sealedwit hpetroleu m jellyt o that of both control and 20.0 ppm groups. retardevaporation . Disheswer e stacked1 4 highwithi na nenvironmenta lchambe rwit ha Discussion photoperiodo f1 2h rligh tan d1 2h rdar ka t 23+ 2 C . Cultureswer eexamine ddail yfo r Comparisons of Marsilea T-2 and AFB. toxin sorophoreappearance ,microspor erelease » responses with those for other systems spermrelease ,fertilization ,sporophyt e L(Tables 3, 4 and 5* the latter two productionan dsporophyt eoccurrence . in Llewellyn si al. (1982)) T-2 toxin - When the data within Tables 1 Essults and 3 are compared, it is apparent that except for tobacco callus, water fern Tables 1 &2 summarize T-2 toxin (1) and reproduction appears to be the most T-2 AFB, (2) data related to sporocarp toxin sensitive plant system thus far germination percentage, fertilization examined yielding a linear dose-response percentage for those germinating, sporophyte curve (Fig. 1). production for those fertilized and AFB, - The data of Table 2 do not lend sporophyte occurrence for those replica themselves to construction of dose-response shewing fertilization. curves and thus prohibit the utilization of water fern reproduction as an AFB, bioassay. Sorophore appearance, microspore and sperm Perhaps 'fine-tuning' of the concentrations releases between 0.000 and 0.625 ,ug/ml may yield a curve. AFB, - Sorophore appearance and both Comparison of the responses of M^TSÙl^a microspore and sperm releases were not sporocarp germination and sporophyte inhibited by any AFB, level. Megaspore production (Table 2) with the germination of release was reduced to 66.67% at 1 ppm and various seeds and pollen (Table 4 ) and the 85.71% at 2 ppm, a response not observed at elongation of plant parts (Table 5 ) reveals either higher or lower concentrations. Marsilea reproduction to be the mostAFB. , sensitive (0.5 jug/ml) system employed to Sporocarp production date as most are affected by 100 and even 1000jug/ml . Of interest is OnocJ.ea AFB, - Germination was increased from sensibilis where a 43.8% inhibition of spore 85.71 (C) to 100% at 2.0, 4.0, 8.0, 12.0 and germination was obtained at 3.90 ;ug/ml.

128 Inconclusion ,fern sappea rt ob equit e Llewellyn,G.C ,C.L .Gentry ,E.S . Mayo sensitivet omycotoxin san dthu swarran t &W.V .Dashek ,1982 . Aflatoxin further investigationa spossibl ebioassa y effectso nspic e seed germinationan d tools. rootelongation , z.Lebens .Unters . Forsh.174:18-22 . Mirocha,C.J. ,S.V . Pathre,B . Schauerhamer& CM . Christensen, theAFB , resultswer e completedb yM . 1976. Naturaloccurrenc eo fFusariu m Meredith"an dG . Alexander Stephenson. toxinsi n foodstuff. Appl.Environ . Assaysfo rABB ,wer eb yThoma sEadie , Microbiol. 32:553-556. VirginiaDivisio no fConsolidate d Laboratory Mirocha,C.J. , R.A.Pawlosky ,K . Service,Richmond ,VA . Wethan kMs .Mar y Chatterjee ,S .Watso n& w .Hayes , Wesleyfo rthoughtfu l clericalassistance . 1983. Analysiso fFusariu mtoxin si n various samples implicatedi nbiologica l warfarei nSoutheas tAsia . J.Assoc . Off.Ana l Chem.66:1485-1499 . Bierhorst,D.W. , 1971. Morphology of Rosen,R.T .& J.D . Rosen,1982 . Presence vascularplants . Hie MacMillianCo. , of fourFusariu mmycotoxin san d NY. syntheticmateria li n"yello wrain" . Dashek,W.V .& G.C .Llewellyn , 1982. Evidencefo rth eus eo fchemica l weapons Aflatoxinsan dplants . Postepy inLaos . Biomed.Mass .Spectrom . Microbiologii21:65-84 . 9:443-450. Goldblatt,L.A. ,1969 . Aflatoxin: Schoental,R. ,A.Z . Joffe& B .Yagen . Scientific background,contro l and 1979. Cardiovascular lesionsan d implications. Academic Press, NY. varioustumor sfoun di nrat sgive n Joffe,A.Z. ,1965 . Toxin-productionb y T-2toxin ,a trichothecen emetabolit e cereal fungicausin g toxicalimentar y of Fusarium. Cancer Res.39:2179-2189 . aleukiai nman . In:G.N .Woga n(Ed. ) Wogan,G.N . 1965.Mycotoxin si n Mycotoxinsi nFoodstuff . MIT Press, foodstuffs. MITPress ,Cambridge , MA. Cambridge,MA , p.77-84 .

Table1 . SporophyteDevelopmen t ForT- 2Toxin-Treate d MarsileaFiv e Days After.Treatment *

T-2Toxi n Percent ofMegaspore s Percento fMegaspore s Concentration TotalMegaspore s Showing Potential Actually Attaining Normal Wg/m? Tested For Development Sporophyte Status

1.250 69 10.3 + 17.9 0.0 + 0.0 0.625 63 17.3 + 30.0 0.0 + 0.0 0.314 51 11.0 + 15.5 0.0 + 0.0 0.156 59 66.0 + 33.7 31.7 + 38.1 0.078 55 72.3 + 32.4 72.3 + 34.2 0.039 51 85.3 + 25.4 83.3 + 28.9 0.020 66 95.3 + 4.5 82.7 + 22.7 0.009 63 87.6 + 6.9 81.0 ± 2.6 0.005 64 68.3 + 8.3 63.0 + 14.1 0.002 60 78.3 + 6.7 71.3 ± 8.5 0,001 66 83.0 + 9.8 69.3 ± 14.4 0.000 70 84.7 + 3.8 71.7 ± 13.8

*Dataar emean san dstandar d deviations forthre e replicate experiments.

129 Table 2. Responseo fMarsile aGermination ,Fertilizatio n andSporophyt eProductio nt o AflatoxinB , Treatments

Germination Fertilization SporophyteProductio n SporophyteOccurenc e Percentage PercentageFo r ForThos eFertilize d ForThos eReplic a ThoseGerminatin g (MeanNo./Sporocarp ) Showing Fertilization {% o fcultures )

0.0 85.71 50.00 81 100.0 (3/3) T.2TOXI NDOSE-RESPONS EC 0.5 57. 14 0.00 <_l 111' -T- 1.0 85.71 33.33 56 100.0 S r FIG cc 100 (2/2) o 2.0 100.00 71.43 89 100.0 (5/5) h- z 8 4.0 100.00 28.57 50 100.0 5 UJ fN« r=C a. (2/2) ÜJ O 60 _J 8.0 100.00 42.86 58 66.7 >- Ul I > (2/3) a. UJ a 40 12.0 100.00 57.14 68 100.0 o (4/4) o- \ 20 16.0 100.00 57.14 69 100.0 (4/4) 20.0 71.43 66.67 87 100.0 0.1 0.2 0.3 (4/4) >ig/ml T-2 TOXIN

Data are means for seven rep!

Table 3. Summary ofSom ePlan tSystem sUse da sPossibl eBioassay so fTrichothecene s

System Concentration Effect Investigator

Lesion development 20t o 100m g Inhibits infection Bawdenan d upontobacc onecrosi s trichothecin/1 wbjehspraye dove r Freeman (1952) viruso nbean s leaveso rdfc yafte r theyhav ebee nin ­ cubatedwit hviruses , but ineffectivewhe n applied 2day sbefor e

Pea seedgerminatio n 0.5ugT- 2 Germination reduced Burmeister Toxine/ml bymor e than50 % andHesseltin e (1970)

Rhodotorularubi a 4.0 ygT-2 / Growth retardation Burmeisteran d NRRLY-722 2an d toxinassa y Hesseltine Pénicilliumdigitatu m disk (1970) NRRL 1202

Tobacco callusupo n 6.0 X 10 uM 50%growt h Helgeson agar T-2 toxin inhibitor etall.(1974) ; Stahle tal.(1973 )

Fullreferecnec e citations canb efoun di nProtectio n againsttrichothecen e mycotoxins, 1983.Nationa lAcadem yo fSciences ,Washington ,DC ,USA .

130 FERTILIZATION IN TAXUS 8ACCATA L.

R.I. Pennell & P.R. Bell

Department of Botany & Microbiology, University College London, UK

Summary of instances, in Pinus (Haupt, 1941; Came- fort, 1969) karyogamy takes place after The development and nature of the gam­ the sperm nucleus has entered a cup-shaped etes of the English Yew, Taxus baccata depression in the surface of the egg nucleus L., have been studied in depth. Mitosis and the envelopes of the pair become closely of the body (spermatogenous) cell within parallel. The formation of local adhesions the pollen tube is rapidly followed by the and pores at the sites of contact then ocurs dissolution of the boundary cell, degenera­ in a manner essentially similar to that which tion of the cytoplasm, and liberation of takes place between sperm cell and synergid the sperm nuclei in to the tube lumen. in Gossypium (Jensen & Fisher, 1967). The The sperms lack any identifiable motile inner membranes of. each envelope fuse apparatus. in turn. Similar events also take place during The mature egg measures 20-30 x 50 karyogamy in Plumbago (Russell, 1983), but Urn, the micropylar surface of the nucleus in Gossypium nuclear contact comes about lying 10-15 um below the neck cells. Insemi­ not by direct adhesion, but via the fusion nation is brought about shortly after the of cisternae of endoplasmic reticulum mid­ pollen tube penetrates the archegonium way between the two surfaces (Jensen, 1964; and the wall is lysed at its tip. The cause Jensen & Fisher, 1967; ^Schultz & Jensen, of the movement of a single sperm nucleus 1977). When several such contacts have towards the egg nucleus is not evident. been established the length of each cisterna The egg nucleus becomes cup-shaped as decreases and the interposing membranes the sperm draws near, and the male gamete of the envelopes fuse over their entire sur­ becomes partially enclosed by the depression faces. so formed. Karyogamy then takes place as a number of local adhesions are formed Materials and Methods between the parallel surfaces of the two gametic nuclei. Pore formation at these Thin median slices of well-pollinated ovules sites is followed by the enlargement of were fixed for 2-3 hours at room tempera­ the areas of fusion and the complete co- ture in 2.5% glutaraldehyde (Taab, Reading) mingling of the genomes. in 0.05 M Sorensen's phosphate buffer. After Keywords: fertilization, Taxus. washing in buffer the slices were post-fixed for 2 hours in 2% aqueous osmium tetroxide Introduction and dehydrated in acetone. Embedding was in Epon 812 (Taab, Reading). 4 urn sections Fertilization in the so-called siphon- were searched for evidence of gametogenesis ogamous higher vascular plants is brought and fertilization. Sections containing appro­ about by non-motile sperms. After the priate structures were remounted according formation by lysis of a small pore near to the technique of Woodcock & Bell (1967) the tip of the pollen the two sperms are and resectioned in the thickness range 50- liberated either into a single or adjacent 80 nm. Post-staining in uranyl acetate and archegonium (Singh, 1978), or into an lead citrate was followed by critical exami­ embryosac (Jensen & Fisher, 1968; Russell, nation in a Siemens Elmiskop 102 Trans­ 1982). In genera in which the male gametes mission Electron microscope, running at are cells, discharge of the sperms is follo­ 60 kV. wed by the apposition of the plasmalemma of the sperm to that of either the synergid Results and discussion (Jensen & Fisher, 1968) or the egg (Russell, 1983). Many local adhesions then develop Although it has been widely reported that between the two membranes and, at the two sperms of Taxus are entire cells (Stras­ site of each of these, a pore develops. burger, 1879; Rohr, 1973; Gianordoli, 1974), The subsequent enlargement and fusion this study demonstrates that the cytoplasm °f the pores ultimately removes the barrier is lost as they mature (Fig. 1). Sister sperms between the two gametes. The means by w appear to differ from one another only in hich the male nucleus subsequently moves respect of the extent to which the chromatin through the egg cytoplasm are not clear is condensed. The mis-identification of the however. The final events in the sexual body cell and stalk nucleus (Gianordoli, Process have been observed in a number 1974) is in part responsible for the belief

131 that the sperms are cells of different sizes. The microtubules within each group appear to originate from the nuclear envelope. Microtubules are also conspicuous features Fig. 1 The sperms shortly after liberation of the cytoplasm of the young zygote of into the tube appear to differ from one Pinus (Camefort, 1969). another only in the degree of condensation Insemination of the archegonium takes of chromatin. place following the penetration of the tip Scale: 1 um. of the pollen tube between the archegonial neck cells and the lysis of its wall. In the v. : «w electron microscope lysis is seen to be corre­ lated with the passage of vesicles from the surface of the plasmalemma into the wall. This may indicate the secretion of hydrolases into the wall, leading to weaken­ ing and ultimately dissolution. One or both sperms, sometimes accompa­ nied by the stalk nucleus and some pollen tube cytoplasm, then move from the pollen tube into the egg. It is not clear how the sperm penetrates the cytoplasm, but in view of the known behaviour of the pollen tubes of some angiosperms (e.g. Cass & Jensen, 1970), it is possible that ??% the plasmalemma of the female gamete is interrupted by the tube itself. In Taxus the eccentric position of the body The cause of the movement of the sperm cell nucleus, and the consequent lateral nucleus within the egg is not evident. Micro­ displacement of the cell plate following tubules within each of the groups that arise division of this nucleus, have also given from the surface of the egg nucleus extend rise to the notion that the sperms differ for distances of up to 20 um into the cyto­ in size. However, following degeneration plasm. Were they to attach to the envelope of the boundary of the body cell, and of of the sperm they could, by means of sliding the cytoplasm, the nuclei alone are liberated against one another, as is known in ciliary into the tube. The situation is probably motion (Gibbons, 1981), draw the male similar in Athrotaxis (Brennan & Doyle, gamete towards the nucleus of the egg. 1956), although in this instance the later Indeed, microtubules are sometimes evident stages of were not observed. in the narrow region of cytoplasm which The sperms lack any visible motile appara­ subsequently separates the two apposed gam­ tus, but bodies which may be relict blepharo- etic nuclei. Other proteins, possibly contrac­ plasts (Pennell, 1984) are conspicuous within tile, may be involved in the movement, the cytoplasm of the body cell, and ere and these are currently being searched for often associated with microtubules. by immunocytochemical means. Conversely, The egg cell occupies the whole archegon- the formation of the cytoplasm of the micro- ial chamber (Fig. 2). The nucleus of the pylar region of the egg into radially elonga­ egg lies deep within the protoplast, its micro- ted channels may suggest that cytoplasmic pylar surface approximately 15 um from streaming of the 'reverse-fountain' type the neck cells. Groups of microtubules are is responsible. This has been suggested for occasionally seen radiating from egg nuclei. flowering plants (Jensen, 1974). The egg nucleus becomes cup-shaped as the sperm approaches. The sperm itself enters the depression so formed and becomes Fig. 2 The mature egg cell. The micro- partially enclosed by the egg nucleus (Fig. pylar pole of the egg is to the right. 3a). The envelopes of the two gametic nuclei Scale: 10 um. come to lie parallel to each other separated by a region of cytoplasm no more than 2 um wide. At this stage, the adjacent membranes develop undulations, those of the egg nucleus being in register with those of the sperm nucleus (Fig. 3b). The outer membranes of the undulations ultimately make contact (Fig. 3c) and fuse. This fusion of the inner membranes then gives rise to pores which provide contact between ÜW*^Ï/»^** -'* Vi :&-, «'•^ % the two genomes. As each pore widens it merges with others so that the parallel envelopes become wholly fused. Fusion events v-5v-~?-

132 of this kind are well described features References of fertilization in both (e.g. Camefort, 1969) and angiosperms (e.g. Brennan, S.J. & J. Doyle, 1956. The gameto- Russell, 1982). phytes and embryology of Athrotaxis. Sei. Proc. Roy. Dublin Soc. 27: 193-257. Cass, D.D. & W.A. Jensen, 1970. Fertilization Fig. 3. S, sperm; EN; egg nucleus. in barley. Amer. J. Bot. 57: 62-70. a. Nuclear apposition at the time of kary- Camefort, H. 1969. Fécondation et proembryo­ ogamy. Scale: 10 urn. genèse chez les Abietacee (notion de neo- b. Local adhesions (arrow) of the parallel cytoplasme). Rev. Cytol. et Biol. Veg. 32: nuclei are visible in the electron micro­ 253-271. scope. Scale: 1 um. Gianordoli, M. 1974. Ultrastructure des sperma- c. Membrane adhesion is followed by the atozoides de trois gymnospermes, Cephalo- dissolution of the interposing inner mem­ taxus drupacea, Sciadopitys verticillata, branes of the nuclear envelopes (arrow). Taxus baccata. Cr. hebd. Seanc. Acad. Sei. Scale: 0.5 urn. Paris, Ser. D. 278: 2637-2640. Haupt, A.W. 1941. and fertilization in Pinus lambertiana and P. monophylla. Bot. Gaz. 102: 482-489. Jensen, W.A. 1964. Observations on the fusion of nuclei in paints. J. Cell Biol. 23: 669-672. Jensen, W.A. 1974. Reproduction in flowering plants. In Dynamic Aspects of Plant Ultra- structure (ed. A.W. Robards) pp. 481-503. McGraw Hill, Maidenhead. Jensen, W.A. & D.B. Fisher, 1967. Cotton embryogenesis: . Phyto- morphology 17: 261-26». Jensen, W.A. & D.B. Fisher, 1968. Cotton embryogenesis: the entrance and discharge i.'iV ••..»ƒ-..,• of the pollen tube in the embryo sac. f- Planta 78: 151-183. ^ .\»Vv •: • *- ' - . '-A A 'Ik**.."* -1 Penneil, R.I. 1984. Ph.D. Thesis, University of London. r -, Rohr, R. 1973. Ultrastructure des spermato­ -. ifcr * »f. zoïdes de Taxus baccata L. obtenus à partir \* .- .4./*V de culture aseptiques de microspores sur un milieu artificiel. C.r. hebd. Seanc. Acad. Sei. Paris. Ser. D. 277: 1869-1871. v - 1, &N. i- ••V Russell, S.D. 1982. Fertilization in Plumbago ,f zeylanica: entry and discharge of the pollen tube in the embryo sac. Can. J. Bot. 60: 2219-2230. Russell, S.D. 1983. Fertilization in Plumbago

r zeylanica: gametic discharge and fate of the ÏÎ. "'"" »* ?' male cytoplasm. Amer. J. Bot. 70: 416-434. Schulz, R. & W.A. Jensen, 1977. Cotton emb­ ryogenesis: the early development of the free nuclear endosperm. Amer. J. Bot. 64: 384-394. Singh, H. 1978. Embryology of Gymnosperms. Handbuch der Pflanzenanatomie, vol. 10(2). Gebrüder Borntraeger, Berlin & Stuttgart. Strasburger, E. 1879. Die Angiospermen und die Gymnospermen. G. Fischer, Jena. Woodcock, C.L.F. & P.R. Bell, 1967. A method for remounting 4 ym resin sections routinely for ultrathin sectioning. J. r. microsc. Soc. 87: 485-487.

133 PISTILLATE IN JUGLANS REGIA: A DEVELOPMENTAL BASIS FOR HETERODICHOGAMY

Vit o S. Polito & Nai Yan Li Department of Pomology, University of California, Davis CA 95616, U.S.A.

Walnuts (Juglans spp.) have a hetero- sinks as nutritional requirements of devel­ dichogamous with some individ­ oping fruits increase. uals being protandrous and others proto- This abrupt termination of pistillate gynous (Forde & Griggs, 1975; Gleeson, flower organogenesis as the growing fruits 1982). We investigated the phenology of attained full size was noted in each of the pistillate flower differentiation in several clones examined, regardless of the mode of protogynous and protandrous clones of J. dichogamy characteristic for that clone. regia in order to determine the relationship However, differences were evident in the between the timing of floral organogenesis extent of organ initiation that had occurred and the mode of dichogamy. at the time organogenesis stopped. In each Because previous work on walnut pistil­ of the six protogynous clones pri- late flower development (Lin et al, 1977) mordia were initiated before the cessation provided data on protandrous , of organ initiation in the spring. In pro­ which are more common, we chose to emphasize tandrous clones did not form until protogynous individuals and examined each of growth resumed following the winter dormancy the six protogynous clones available to us. period. Thus, protogynous individuals enter These included early ('Chico', 'Sharkey'), the next growing season with pistillate midseason ('Amigo', 63-378) and late ('Mey- flowers having attained a developmental lan', 'XXX Mayette') individuals. Three stage that allows gynoecial differentiation protandrous cultivars ('Ashley', 'Chandler', to proceed immediately. By contrast, the 'Howard') not previously examined were in­ protandrous cultivars require more organo- cluded for comparison. genetic activity during the weeks prior to Buds were dissected to reveal floral pri- anthesis as perianth parts must be initiated mordia and prepared for optical or scanning in'addition to the . As the time electron microscopy using standard methods. required for this additional developmental Results indicated that initiation of' activity is sufficient to account for the pistillate flowers in J. regia begins within relative differences in pistillate bloom six to eight weeks following pistillate times between protandrous and protogynous anthesis in spring. By eight to eleven walnuts, it appears that a fundamental basis weeks organogenesis stops and does not re­ for heterodichogamy in walnut is in the sume until shortly before bloom the extent of pistillate flower differentiation following spring. Cessation of organogene­ that occurs during the post-anthesis period. sis corresponds to the time that the growing fruits attain their full size and undergo References marked changes in development and in their requirements for photosynthate (Pinney & Forde, H. I. & W. H. Griggs. 1975. Pollin­ Polito, 1983). Prior to this time there is ation and blooming habits of walnuts. rapid growth in fruit size characterized by Univ. Calif. Div. Ag. Sei. Lft. 2753. increases mainly in endosperm and involucral. Berkeley, CA. tissues. As the fruits reach full size they Gleeson, S. K. 1982. Heterodichogamy in begin a series of developmental events that »ralnuts: Inheritance and stable ratios. include shell lignification, rapid embryo growth and, 'ultimately, accumulation of Evolution 36: 892-902. sugars and lipids in the embryo. This se­ Lin, J., B. Shabany & D. Ramos. 1977. Pis­ quence suggests that nutrient partitioning tillate flower development and fruit phenomena may be an important factor growth in some English walnut cultivars. leading to the cessation of organ initiation J. Amer. Soc. Hort. Sei. 102: 702-705. in the primordial flowers with the develop­ Pinney, K. & V. S. Polito. 1983. English ing pistillate flowers becoming less favored walnut fruit growth and development. Sei. Hort. 21:19-28.

Pistillate flower differentiation in (A) J. regia cv. Ashley (protandrous), 13 March, approximately one month prior to pistillate anthesis, and (a) j^ regia cv. XXX Mayette (protogynous), 26 October. B, ; I, involucre; P, perianth.

134 OVULEDEVELOPMEN T INRELATIO N TOOVUL EPOSITIO NAN DFLOWE RDEVELOPMEN T INLILIU M j.L.va nWent* ,N.M .va nBeek* ,J.M . vanTuyl* *

* Department ofPlan tCytolog y andMorphology ,Wageningen ,Th eNetherland s **Institut e forHorticultura l PlantBreeding ,Wageningen ,Th eNetherland s

Summary % mature ovules per ovary seg­ ment in relation to flower Lilium ovules does not develop synchronously. development in three different Ovule development is quickest in segment 2 and cultivars. 3 (central half of ovule), slower in segment 1 (apical quarter), and slowest in segment 4 (basal quarter). The used cultivars show considerable variation in development, which may partly result from differences in growth conditions. At one day before anthesis no ovule has reached maturity. At anthesis approximately 50% of ovules of culti­ vars 'WhiteEurope 'an d 'WhiteAmerican 'ha sreache d maturity. In 'Gelria' at anthesis only 20%o fovule sha sreache d maturity. Keywords: lily, Lilium longiflorum, ovule develop­ ment.

Introduction

For breeding purposes information on ovule

development is essential to establish the optimum cultivar "White American pollination time, the possible seedsetting, and the effect of manipulation as e.g. bud pollination. Therefore a quick and reliable method for the study of sporogenesis and gametogenesis was devel­ oped, based on staining with haemalum and clearing withmethy l salicylate.

Resultsan ddiscussio n

For the study of megasporogenesis and gametoge­ nesis a method developed by D. Stelly, Dept. of Genetics, Wisconsin University, U.S.A., and E. Jongendijk, Dept. of Plant Breeding, Agricultural University, Wageningen, The Netherlands, was modi­ fied. The method eventually employed comprises fixa­ tion in a mixture of formaldehyde, propionic acid, and alcohol 50 % (5,5,90 v/v);stainin g in haemalum (Sass' modification of Mayer's haemalum); and clearing inmethy l salicylate. Three cultivars of Lilium longiflorum has been studied: 'White Europe', 'White American', and 'Gelria'. Ovule development appears to be not synchronous.

The ovules of the apical and basal quarters of the ovary are considerably slower in development (segment 1 and 4). At one day before anthesis no ovule has reached maturity. At anthesis approxi­ Conclusions mately 50% of the ovules of 'White Europe' and 'White American', and 20%o f the ovules of 'Gelria' The results indicate that have reached maturity. In all cultivars ultimately in Lilium breeding experiments 80-90%o f theovule sreache smaturity . the seedsetting can be influ­ The observed differences in development of enced by the time and method the various cultivars may partly be due to differ­ of pollination and the stage encesi ngrowt hconditions . of development of the ovary. 135 GENETIC BASIS OF CALLOSE PATTERN,MEGASPOR E DEGENERATION AND POLARITY IN THE OVULES OP OENOTHERA

Renata Sniezko and Cornelia Harte Instytut biologii UMCS,Lublin ,Poland ; Institut für Entwicklungsphysiologie, Köln,FRG .

Summary Oe.suaveolens forms hetero- and equipolar tetrads ofmegaspores .Degen ­ The independence of the polarity in eration can start from various posi­ the ovule and the callose pattern of tions inth e tetrad. The embryo sac the meiotic cells in our material develops mainly from the micropylar contradicts the assumption of agen ­ megaspore,bu t can develop from the eral causal relation between the two chalazal spore as well. phenomena. The segregation for different types F^hybrids of the course ofmegasporogenesi s in the Fj-generationgive s proof for a Similar types ofmegaspor e tetrad genetic basis ofmor e than onebu t polarity and embryo sac development as not many genes for each of the charac­ in Oe. suaveolens are observed. The ters include«i n this investigation. quantitative analysis indicates,tha t the plasmon and the genom effect the Introduction polarity in the hybrid ovules. In Oenothera a causal relationship Fp-hybrids between the deviations from mendelian segregation ratios and development of A considerable variability ofpheno - chemegaspor e -cetrad Is supposed,A S types and embryological processes are polarity phenomena in the ovule are observed, including the hookeri-type. important,th e relation between the The pattern of callose deposits inth e development and degeneration ofth e walls ofmeioti c cells is variable and megaspores and the formation of callo­ independent from the type of megaspore se deposits in the ovule has tob e degeneration in the later stages.A investigated. correlation between the pattern of In spite of recent investigations embryo sac development and other concerning the relation between the phenotypic characters of Fp-plants is megaspores competing in the ovule and established. polarity ofth e tetrad it is still open, The pattern of callose formation in if on aquantitativ e basis an * the walls ofmeioti c cells and the agreement between development ofth e ordgr of degeneration of the megaspores megaspore tetrad in Oenothera hybrids are variable between the ovules ofan y and genetic data from their progenies ovary in the F1- and Fp-hybrids. canbejestablished . The differences between theF g-plant£ Two species of Oenothera were used are quantitative rather than qualitat­ asparents . They differ inth e tetrad ive. Differences are seen in the types polarity and competition between the of callose pattern and the polarity megaspores. The pattern of the callose of themegaspor e tetrad. deposits in thewall s ofth e meiotic In the ovules of Fp-plants with cells is different in the twospecies . equipolar tetrads two embryo sacs can If there is ageneti c basis for these develop.The y differ in the genom of phenomena,tha n inth e Fg-generation Their nuclei and compete for becoming ofhybrid s a segregation for the type the functioning embryo sac.Thi s is of the embryo sac development canb e the morphological basis for the gene­ expected. tic RENNER-effect.

Results and discussion References Sniezko,R . and C. Harte,198 4 Species of Oenothera Polarity and competition between megaspores in the ovule ofOenothe - Oe. hookeri shows a stable pattern ra-hybrids. of polarity in the ovules. Inth e Plant Syst.Evol. 144,83-97 , megaspore tetrad the degeneration - - ,198 4 starts inth e chalazal part cfth e Callose pattern and polarization tetrad. The embryo sac develops always phenomena in the ovules inth eFp - from the micropylar megaspore. hybrids. PI.Syst.Evol. 144,i n print. 136 ULTRASTRUCTURE AND ULTRACYTOCHEMICAL LOCALIZATION OF ACID PHOSPHATASE IN THE DYAD, TETRAD AND DEVELOPING MEGASPORE OF CAPSELLA BURSA-PASTORIS p. Schulz Department of Biology, University of San Francisco, San Francisco,C A 94117

and ultracytochemical changes that occur during female meiosis in Cap­ Ovules of Capsella bursa-pastoris sella and describes the dyad, the (monosporic Polygonum type ofdevel ­ tetrad, the developing megaspore and opment)a t the dyad and tetrad stages the 2-nucleate gametophyte. ofdevelopmen t and at the megaspore and 2-nucleate stages of thegarneto - Materials and methods phyte were fixed routinely or incu­ bated ina modified Gomori medium for Field grown plants of Capsellabur ­ acid phosphatase localization using sa-pastoris (L.)Medic , wereharves ­ beta-glycerophosphate as asubstrate . ted at the University of California Organelle distribution and develop­ Botanical Garden. The procedures for mentwer e studied aswel l as the fate the preparation of tissues forelect ­ ofautophagi c vacuoles that localize ron microscopy and fluorescencemic4 > acid phosphatase inherited from the roscopy and for acid phosphatase lo­ megaspore mother cell. calization have been previously des­ Some plastids and mitochondria are cribed (Schulz and Jehsen, 1981). destroyed and absorbed into the large Tissue prepared for histochemical lo­ vacuoles of the megaspore and young calizations was fixed in glutaralde- gametophyte. Other plastids andmito ­ hyde, embedded in Epon and sectioned chondria survive meiosisan d give rise at 1% pm. The procedure for theper ­ toplastid s and mitochondria of the iodic acid-Schiff (PAS)reactio n for gametophyte generation. There isn o insoluble carbohydrates vas taken evidence for the de novo origin of from Jensen(1962) and for the aniline these organelles. The rapidly grow­ blue black stain for general protein ing functional megaspore synthesizes from Fisher (1968). large numbers of ribosomes and pro­ ducesmicrovillus-lik e extensions into Results the cell wall. The megaspore and 2- nucleate gametophyte maintain plasmo- The dyad. A transverse wall divides desmataa t the chalazal endo f the the meiocyte into two unequal cells. cell. The chalazal cell receives the great­ Keywords: ultrastructure , acid phos­ er share of cytoplasm but themicro - phatase,ovule ,megasporogenesis , pylar cell gets a proportionate share Capsella. of all types of organelles,present in the meiocyte. The transverse wall Introduction contains membranous vesiclesan d shows strong aniline blue fluorescence. Both Theultrastructur e and ultracytochem- cellshav e autophagic vacuoles (AVs) ical localization of acid phosphatase that contain ribosomes andorganelles . inth emegasporocyt e of Capsella have Many plastids in the micropylar cell been described (Schulz and Jensen, are enclosed inAVs,whil e those in the 1981). Dramatic changes occur in the chalazal cell usually are not.Mito ­ meiocyte at the beginning of meiosis chondria in both cells look like those that include the formation of acid in the meiocyte. Both cells contain phosphatase-rich autophagic vacuoles microbooies, small vacuoles and long, that encapsulate portionso f cyto­ single RER cisternae that are closely, plasm containing ribosomes and organ­ associated with the transverse wall. elles, the blebbing of double-membrane The tetrad. Meiosis II produces a bound vesicles from thenuclea r envel- linear tetrad of haploid megaspores °pe, distinctive changes in plastids a with the chalazal most cell being some­ nd mitochondria, and the appearance what larger. The wall separating the °fanilin e blue fluorescent substances micropylar two cells of the tetrad is ifith e cell wall. This is acontin ­ often incomplete or irregular. The uation of the study of ultrastructural transverse walls show strong aniline

137 blue fluorescence. The chalazal end chalazal end of the cell. Plastids wall and the lateral walls of the andmitochondri a in the cytoplasm ap­ functional megaspore contain plasmo- pear to behealth y and dividing. Long desmata but there are no - single RER cisternae begin to develop ta in the non-functional megaspores. and ribosome density remainshigh . Both the functional megaspore and the non-functional megaspores inherit AVs Discussion containing ribosomes and organelles that are rich in acid phosphatase. This study shows thatAV s that lo­ In the functional megaspore some AVs calize acid phosphatase play a major appear tob e moving into the large role in the destruction ofribosomes , micropylar vacuole of the cell. organelles and other cytoplasmic con­ Many plastids and mitochondria in stituents during megasporogenesis in the degenerating megaspores,bu t not Capsella. Many of theAV s that seg­ in the functional megaspore, localize regate into the functional megaspore acid phosphatase. Theplastid s of the are absorbed into the enlarging vac­ functional megaspore lose their in­ uoles of the maturing megaspore and ternal membranes and some show a dis­ 2-nucleate gametophyte. Concentric solved plastid envelope. Plastids membrane-bound organelles similar to in the functional megaspore do not the AVs in Capsellahav e been obser­ accumulate starch but nucellar cells ved and assigned various functions do. Mitochondria begin to enlarge during female meiosis in several spe­ and lose electron density in the func­ cieso f LiHu m (Rodkiewicz andIlikul - tional megaspore. Thenucleolu s is ska,1965 ;Dickinso n andAndrews , small and granular and ribosomes are 1977; De-Boer-De-Jeu, 1978). The pre­ dense in the cell. sent study which shows the specific The developing megaspore. The ex­ localization of acid phosphatase in panding functional megaspore crushes these structures and follows their the nucellus surrounding it while the fate during meiosis and early gameto­ degenerating megaspores are crushed phyte development confirms that in by encroaching nucellar cells. The Capsella they function asAV s that nucleolus of the functional megaspore mediate the turnover ofmacromolecule s becomes very large and the cytoplasm and organelles of the sporophyte gen­ of the cell becomes extremely elect­ eration during the transition to the ron dense with ribosomes. Twopromi ­ gametophyte phase of the life cycle. nent vacuoles fill with debris. AVs, A decrease in the concentration of plastids and lipid globulesar e seen cytoplasmic ribosomes has been obser­ in the vacuoles. Other plastids in ved during female meiosis in Pisum the cytoplasm appear to be healthy sativum (Medina et al.,1981), during and developing and possess a fewthy. - female and male meiosis in LiHu m lon- lakoids and in some cases starch. giflorum (Dickinson and Andrews,1977 , Some mitochondria appear to be destroyed 'Dickinson and Heslop-Harrison , 1977), at this stage while others appear to and in other species. In Capsella be quite healthy. Some plastids and this reduction in ribosomes iscorrel ­ mitochondria look like they are div­ atedwit h their destruction inside of iding and dictyosomes are active. AVs. While the synthesis of newri ­ The ceilwal l is thickened and bosomes isprobabl y initiated in the shows strong aniline blue fluorescence functional megaspore of the tetrad but doesno t stain with PAS or with there is a dramatic increase in the aniline blue black. Plasmodesmata size of the nucleolus and amassiv e occur in this wall and microvillus- outpouring of new ribosomes in the like projections at the megaspore enlarging megaspore. Ribosome res­ surface extend into thiswal l toin ­ toration in the tetrad and maturing crease the absorptive surface area megaspore has also been reported in of the rapidly expanding cell. Pisu m sativum (Medina et al., 1981), The 2-nucleate gametophyte. This Zeamay s (Russell, 1979)an d LiHu m cell isver y broad at the micropylar longiflorum (Dickinson and Fotter, end. The two haploid nuclei are sep­ 1978; Dickinson and Heslop-Harrison, arated by a large central vacuole 1977). which contains cellular debris includ­ The fate of plastids and mitochon­ ing degenerating plastids andmito ­ dria during female meiosis is diffi^ chondria and AVs that localize acid cult to follow. Some plastids inher­ phosphatase. The thickened cell wall ited by the functional megaspore are and themicrovillus-lik e projections encased inAV s and are eventually ab­ of the megaspore have disappeared but sorbed into the vacuoles of the grow­ plasmodesmata are still present at the ingmegaspor e and 2-nucleate garneto-

138 phyte. Other plastids in the cytosol then degenerate in the 4-nucleate gam­ appear to dedifferentiate by losing etophyte. internal membranes and in some cases theplasti d envelope. Plastids never References disappear altogether and some appar- antly survive to proliferate in the Da-Boer-de-Jeu, M.J., 1978.Ultrastruc ­ enlarging megaspore. There isn o evi­ tural aspects of megasporogenesis dence from this study that plastids and initiation ofmegagametogenesi s are regenerated de_nov o from another in Lilium. Bull. Soc. bot.Fr .Actu - membrane system in the cell. Plastid alitsbotanique s 125:175-181. dedifferentiation and redifferentia- - Dickinson,H.G . & L.Andrews ,1977 . tion during male and female meiosis The role ofmembrane-boun d cytoplas­ has been reported in LiHu m longiflor ­ mic inclusions during gametogenesis um (Dickinson and Potter, 1978;Dick ­ in Lilium longiflorum. Planta134 : inson and Heslop-Harrison, 1977)an d 229-240. during female meiosis in Pisum sativ­ & J. Heslop-Harrison, 1977.Ribo-. . um (Medina et al., 1981). A sizable somes and organelles during meiosis portion of the plastid population is in angiosperms. Phil. Trans.R . Soc. thought to be eliminated in LiHu m Lond. B277:327-342 . (Dickinson and Potter, 1978). & U. Potter, 1978.Cytoplasmi c chan­ Themitochondria l cycle during fe­ gesaccompanyin g the female meiosis male meiosis in Capsella is also com­ in Lilium longiflorum Thunb. J. Cell plex. The functional megaspore inher­ Sei. 29:147-169. itsdense ,contracte d mitochondria Hill,R.A. , 1977.Th e ultrastructure characteristic of the megasporocyte of the synergids of Gossypiurnhir - (Schulz and Jensen, 1981). Some of sutum: From anthesis through pollen these organelles localize acid phos­ tube discharge. Ph.D. Dissertation, phatase. It is not known if these University of California, Berkeley. are destined to degenerate or are only Medina, F.J., M.C.Risuen o &M.I .Rod - undergoing molecular reorganization. riguez-Garcia, 1981.Evolutio n of Hill (1977)observe d the localization cytoplasmic organelles during fe­ of significant levels of acid phos­ male meiosis in Pisum sativum L. phatase in mitochondria ofpost-anthe - Planta 151:215-225. sis synergids of cotton ovules just Rodkiewicz, B. &E .Mikulska ,1965 . prior to their degeneration. Some of The development of cytoplasmic stru­ the mitochondria of the functional ctures in the embryo sac of Lilium megaspore of Capsella enlarge and be­ candidum asobserve d with the elec­ gin toproliferate . Then a large num­ tron microscope. Planta67:297-304 . ber ofmitochondri a appear to be des­ Russell, S.D., 1979.Fin e structure troyed and absorbed by the large vac­ ofmegagametopyt e development in uoles of the maturing megaspore and Zeamays . Can. J. Bot.57:1093-1110 . the 2-nucleate gametophyte. Surviv­ Schulz, P & VI.A. Jensen,1981 .Pre - ingmitochondri a proliferate in the fertilization ovule development in 2- and 4-nucleate gametophyte stages. Capsella:ultrastructur e and ultra- Aswit h plastids there isn o evidence cytochemical localization of acid from this study that at any time all phosphatase in the meiocyte.Proto ­ ofth e mitochondria of the dyad, func­ plasma 107:27-45. tional megaspore or 2-nucleate gam­ etophyte are destroyed or formed de Woodcock, C.L.F. &P.R . Bell,1968 . novo. Mitochondria are reported to Features of the ultrastructure of go through stages of dedifferentiation the female gametophyte of Myosurus and redifferentiation during female minimus. J. Ultrastruct. Res. meiosis in Myosurus minimum (V/oodcock 22:546-563. and Bell, 1968), Lilium longiflorum (Dickinson and Heslop-Harrison, 1977) and Pisum sativum (Medina et al., 1981), Aportio n of the mitochondrial pop­ ulation in Lilium is thought to be e- liminated (Dickinson and Potter, 1978). Russell (1979)observe d apostmeioti c ^differentiation and subsequent des­ truction of mitochondria in Zea mays. InZe amitochondri a apparantly dedif­ ferentiate during meiosis,increas e in complexity in the functional mega­ spore and 2-nucleate gametophyte and

139 ASPECTS OFMEGAGAMETOGENESI S INHORDEU MVULGAR E

D.Cas san dD .Petey a

Department ofBotany ,Universit y ofAlberta ,Edmonton ,Canad a

B.Robertso n

BotanyDepartment ,Universit y ofPor tElizabeth ,Por tElizabeth ,Republi c of SouthAfric a

Summary Megagametogenesis

Megagametogenesis inHordeu mvulgar e Barley ismonosporl cwit h degeneration (barley)ha sbee n studied using 1.5u m occurring inth e 3micropyla rmegaspores . sectionsphotographe d withNomarski - The functionalmegaspor e isa larg ecel l interference opticsan d thenre-embedde d with itslon gaxi soriente d toward the for transmission electronmicroscopy . ovularmicropyle . Itscytoplas mha sa Stages inmegagametogenesi s tob econsidere d distinctly polar distribution ofplastids ; willb e fromth e 1-nucleatemegaspor estag e themicropyla r portiono f thefunctiona l toth eearl y8-nucleate ,7-celle dembry osac . megasporeha smor eplastid s thanth e Byusin g agynoecia l staging technique chalazalportion . Thisplasti d distribution modified afterWaddingto n et al. (1983)w e difference ismaintaine d throughth e were able toobtai n afairl ycomplet e 4-nucleate stagedurin gwhic hmajo rvacuole s developmental sequence,particularl ywit h b^gint oappear . Anadditiona lmitosi s respect tostage s inth eproces so fembry o occurs inbot h themicropyla r and chalazal saccellularization . pairo fnucle ia tth e4-nucleat e stageprio r tocel lwal lformation . Introduction Wallformatio n amongbot hmicropyla ran d The studyo fmegagametogenesi s inflowerin g chalazalquartet so fnucle i isaccompanie d plants isdifficult ,particularl y inth e by changes incytoplasmi ccomponents , cellularization phase,becaus eo f theabsenc e specifically dictyosomes,endoplasmi c ofa reliabl eexterna lmorphologica lmarke r reticulum andmicrotubules . Dictyosome forstagin g and theapparen t rapidityo f activity during thewal lformatio nproces s changesoccurrin g oncemitosi s iscomplete . results inth eproductio n ofbot hdens ean d Inthi spape rw ewil lpresen t someobserva ­ rather clearvesicles . Theendoplasmi c tionso nmegagametogenesi s and oncel lwal j reticulum duringwal lformatio nha sdilate d formationparticularl ywit hrespec t toth e cisternae and isdistribute d both ina eggapparatu s ofbarle y using astagin g 'branched fashionan d incircula rarrays . techniquemodifie d afterWaddingto n et al. Inman y casesgrowin gwall san d thebranche d (1983). endoplasmic reticulum arenearl y congruous (Figs. 1& 2) . Microtubules arecommo nnea r Results and discussion thefre eend so fgrowin gwall san d inarea s ofdiscontinuitie s (Figs.1 & 2) . Manysuc h Staging technique microtubules areparalle l toeac hothe ran d perpendicular toth egrowin g cellwal l(Figs . Waddington et al. (1983)assigne d numbers 1 & 2). of4 t o 10t ogynoecia ldevelopmen t stages fromappearanc e of thepisti lprimordiu m Afterwal l formation in theeg gapparatu s (4)t opollinatio n (10). We found aver y isnearl y complete,th e3 cell sexhibi t rough correlationbetwee n theirnumbere d rather flatprofile swit hbeade d cellwall s stagesan dmegasporogenesi so rmegagameto ­ (Fig.1) . Somewhat later cellso f theeg g genesisstage s inwhic hw ewer e interested. apparatus developmor erounde d profilesbu t Their stage 7.5,fo rexample ,produce d retain thefairl yunifor mwal lbeadin g megasporocytes forus . Stage8. 5 gaveu s visible earlier. Cellwal lbeadin gbecome s 2-o r4-nucleat eembry o sacs. Stage 8.75 lesspronounce d during chalazalexpansio no f gaveu scellularizatio nwit h lownumber so f theeg gapparatus . This isapparen t inFig . embryo saccells . Stage 9gav eu shig h 3 aswel la sa marke d thinning ofth e chala­ numberso fembry o saccell s (barleyha su pt o zalcel lwall . Thecel l inFig .3 i sa 100antipoda l cells). Although thisstagin g synergid,identifiabl eb y theappearanc eo f techniquewa sno tprecise ,i tallowe d ust o wall ingrowths of thefilifor m apparatusa t makea numbe r ofobservation s onthi s itsbase . Theultrastructura l appearanceo f process. thefilifor mapparatu s issimila r totha to f transfer cellwal l ingrowths. Theadjacen t

140 Fig. 1.Tw oeg gapparatu s cellsan dportio n (X34,000). Fig.3 . Aportio n ofcentra l ofcentra l cell (CC)fro mearl y cellular cell (CC),portio n ofeg g (E),an d 1o f2 barley embryo sac. Theeg gapparatu scell s synergids fromolde rembry o sac. Synergid havelo wprofile san d arepartiall y separated differentiationi sindicate d byappearanc e bya 'T-shaped',beaded ,discontinuou swall . ofwal l ingrowths of filiform apparatus Microtubule clusters (Mt)mar k 2discontinu ­ (FA)an db ythinnin go fit swal l(small , ities. Small,unlabelle d arrowsdenot e unlabelled arrows)a s itexpand schalazally . vertical andhorizonta lmember s of 'T';a (X5,500). Insert(* ) showsportio no f large,unlabelle d arrowindicate sinter ­ filiformapparatu s (X14,500). sectiono f 'T'. Dilated endoplasmicreticu ­ lum (ER)show nadjacen t tovertica lwal l member. Part of thenucleu s (Nu)o f 1eg g apparatus cella tlowe rrigh t (X17,000). Mlcropyle isdownwar d inal l3 figures . Fig.2 . Growingen d (large,unlabelle d arrow)o fanothe r sectiono fvertica lwal l fromFig . 1. Amicrotubul e clusteri s indicated (Mt)a swel la sendoplasmi c reticulum (ER)adjacen t togrowin gwal l

141 eggapparatu s celli sth eeg gbase d on References identificationo fth esecon d synergid cell inlate rsections . Thisi sth eearlies t Bajer,A.S .& J .Mole-Bajer ,1972 . The stagea twhic hw ehav e observed morphological courseo fmitosis . In:Bourne ,G.H .& differentiation ofeg g apparatus cells;thi s J.F.Daniell i (Editors): Spindledynamic s embryo sacha d about 10cell s (3eg gappara ­ and chromosomemovements . International tuscells ,centra lcell ,abou t 6antipoda l Reviewo fCytology ,Supplemen t 3,Academi c cells)an dwoul d fitwithi n stage8.7 5 of Press,Ne wYork ,p .34-97 . Waddingtone t al. (1983). Transfercel l Fineran,B.A. ,D.J.C .Wil d &M .Ingerfeld , wallingrowth s alsoappeare d inantipoda l 1982. Initialwal lformatio n inth e cells immediatelyborderin g theembry osa c endosperm ofwheat ,Triticu maestivum :a walla t thisstage . réévaluation. Can.J .Bot .60 :1776-1795 . Morrison,I.N . &T.P .O'Brien ,1976 . Discussion Cytokinesis inth edevelopin gwhea tgrain ; divisionwit h andwithou t aphragmoplast . Initialwal ldevelopmen t inbarle yembry o Planta 130:57-67 . sacsi si n2 stages . First isa beade dwal l Waddinton,S.R. , P.M. Cartwright &P.C . (Figs. 1& 2 )see n inal lincomplet ewall s Wall, 1983. A quantitative scaleo fspik e and inyoun g completewalls . Second isa initialan dpisti ldevelopmen t inbarle y completewal l (Fig.3 )wit h amor eregula r andwheat . Ann.Bot .51 :119-130 . appearancebu twhic hma yvar y inthickness . Thebeade d nature of incompletewall s suggests that their initialgrowt h isb y vesicleaccretion . Vesicles similari n appearance toth ebead sbu t ofvariabl e sizes havebee nsee nnea rgrowin gwalls .Activ e dictyosomes occurring neargrowin gwall shav e vesicleswit h similarmorpholog y totha to f thewal lbeads . Microtubule clusters (Fig. 1 &2 )occu r atwal lend san d discontinuities but thesemicrotubule s dono tappea r tob e consistently associated with anyorganelle . TheE Rma y contribute toth e transformation ofyoun gbeade dwall s tomor eunifor mwalls . Dilated ER (Figs.1 & 2 )i sclosel y appressed tobeade dwall swherea sregula rwall sar e associatedwit h slender ERsegment swit honl y approximate alignment typicalo fmatur ewalls .

Ourobservation s ofmicrotubul ecluster s inclos eassociatio nwit hfre eend so fgrow ­ ingwall ssegment san dwit h intercalarywal l discontinuities suggests the involvemento f phragmoplasts inth eproces so fwal lformatio n inth ebarle y embryo sac. The questiono f whether thesephragmoplast s arisefro m mitotic spindlesa swoul d be suggestedb y therecen twor k ofFinera ne tal . (1982)o n wheat endosperm ordj snovo ,a nalternativ e originpropose d byBaje r& Mole-Baje r (1972), must await furtherwork .

Theoccurrenc e of transfer cellwal l ingrowths inantipoda l cellso fbarle ya t thesam e stage that similar ingrowthsar e forming insynergid s suggests thatbot hpole s of theembry o sacma yb e involvedwit h nutrient influx. Sucha ninflu x ofnutrient s would becompatibl ewit h theprobabl ehig h metabolic demands of theembry o saca tthi s time.

Weacknowledg e theNatura l Sciencesan d Engineering Research Council ofCanad afo r itssuppor t toD .Cas s (grantA6103) .

142 BEHAVIOURO FSPINAC H SPERMCELL SI NPOLLE NTUBE SPRIO RT OFERTILIZATIO N

H.J.Wilm s

Departmento fPlan tCytolog y andMorphology , AU,Wageningen ,Th eNetherland s Summary of this nucleus starts (fig. 6). The sperm cells with the long slender runners Cytological sperm cell changes are follow the vegetative nucleus through investigated during pollen tube growth the pllen tube. In the stylar tissue in vivo, starting at the pollen germina­ these runners are still in close vicinity tion and ending at the tube penetration in the degenerating synergid. The number of organelles and the amount of male cytoplasm in the sperm cells gradually decrease, while the contact between the two sperm cells remains intact. Keywords: spinach, Spinacia oleracea, pollentub egrowth ,sper m cell(s).

Introduction

In order to investigate the fusion of male and female gametes and the in­ fluence of the male cytoplasm in the inheritance, the sperm cytology is impor­ tant to know. Not only at the moment of fusion but already from the moment of formation of the sperm cells in the pollen grain. Priviously the situation in the mature pollen grain has been described (Wilms & Van Aelst, 1983). A consistent association of the sperm cells and the vegetative nucleus suggests a structurally connection. How this connection behaves during pollen tube growth is presented in this report.

Resultsan ddiscussio n

In the mature pollen grain stage the sperm cells are linked with each other (fig. 1). Their long, slender projections are found around the irregular shaped vegetative nucleus. The sperm

Fig. 1. Sperm cells-vegetative nucleus Figs. 2,3. Irregular shaped vegetative association in the pollen grain..Schemati c nucleus (VN) and sperm cell (S1) in reconstruction from serial sections. pollentub ei nstyla rtissue , S1, S2 = sperm cell; SN = sperm nucleus; of the vegetative nucleus. In the sperm VN= vegetativ enucleus . cell cytoplasm mitochondria, dictyosomes, cells - vegetative nucleus association rough endoplasmic reticulum next to becomes less evident in the style (figs. free ribosomes can be observed (fig. 2,3). Later on the shape ofth e vegetative 3). Later degeneration of mitochondria nucleus becomes more elongated and less is clearly present (fig. 5).Th e number irregular (fig. 6); also degeneration of organelles and the amount of male

143 cytoplasm in the sperm cells gradually References decrease. The sperm cell runners also decrease in length, while the contact Wilms, H.J., 1981. Pollen tube penetra­ between the two sperm cells remains tion and fertilization in spinach. intact (figs. 4,5,7). The process of ActaBot .Neerl . 30:101-122 . deminishing of the total amount of sperm Wilms, H.J. & A.C. van Aelst, 1983. cell cytoplasm is not clear, but it Ultrastructure of spinach sperm cells finally leads to almost naked sperm in mature pollen. In: 0. Erdelska nuclei which were observed earlier in e.a. (Eds.): Fertilization and embryo- close vicinity of the female gametes genesis in ovulated plants. VEDA, (Wilms, 1981). Bratislava,p .105-112 .

144 SPERMSPECIFICIT Y INPLUMBAG O ZEYLANICA

ScottD .Russel l

Department ofBotan y andMicrobiology ,Universit y ofOklahoma ,Norman ,Oklahoma ,U.S.A . 73019

Summary at 18t o23° Cwit h 16hou rday si ngrowt h chambers atth eUniversit y ofOklahoma . Inth eangiosper m Plumbago zeylanlcath e Ovarieswer e collected 8-1/4 to9 hour safte r roleo findividua l spermcell sdurin g gametic pollination,dissecte d and fixed atroo m fusionca nb edetermine d using paternal temperature in3 %glutaraldehyd e in0.15 M organellesobserve d withinmaterna l cytoplasm phosphatebuffe r (pH6.8 ) for6 t o8 -hours . totrac epattern s offusio nafte rfertili ­ Tissuewa srinse d briefly,fixe d in2 % zation.Th e two spermcell s aredistinguishe d buffered osmium tetroxide,dehydrate d in bysignifican t differences inconten to f ethanol,an dembedde d inlo wviscosit y resin. mitochondriaan d plastids.I n>94 % ofth e For furtherdetail so fpreparatio n see casesobserved ,th eplastid-ric h spermcel l Russell (1983). unassociated withth evegetativ e nucleus fuseswit h theeg g tofor mth ezygote .I n Results and discussion onlyon e instancedi d theopposit e pattern occur.Sinc e thepossibilit y ofthi sresul t The tricellularmicrogametophyt eo fP . occurring asth econsequenc e ofchanc ealon e zeylanica contains twodimorphi c spermcell s isles s than 1i n7,000 ,thi s represents whichhav ebee ndesignate d Svnan d Suaan d strong evidence forth epresenc eo fa fina l aredistinguishe d bywhethe ro rno t thesper m putativerecognitio n event occurring atth e celli sphysicall y associated withth e gameticlevel . vegetativenucleu s (Russell,1984) .Th esper m cell that isno t associated withth e Introduction vegetativenucleu s isdesignate d Sua;th e other sperm cell,whic h isphysicall y Gametic fusion isth ecellula r processb y associated with thevegetativ enucleus ,i s whichth esper mnucleu senter s thefemal e designated Svn.Th e twosper m cellsdiffe r gamete,permittin g nuclear fusion and the significantly inorganell econtent .Th eSu a formationo f theembryo .Presumabl y themean s hasth emajorit y of theplastids ,a naverag e bywhic hgameti c fusionoccur sentail s of24.1 8plastid s and fewmitochondri a cellular fusion,an d consequently atleas t (average:39.81 ;Russell ,1984) . Incontrast , somesurroundin gmal e cytoplasmma yb e theSv ncontain s anaverag eo f0.4 5 plastids transmitted inth eproces s (Russell,1983) . and themajorit y ofmitochondria ,a naverag e Sincemal e cytoplasmic organellesar e of>25 6 (Russell,1984) .Thus ,organell e inherited ina numbe r ofangiosperm s (Kirk differences alonema y alsob euse dt o andTilney-Bassett ,1978) , therecen t report distinguish the twosper mcells . ofdimorphis m insper m cellorganizatio nan d Interestingly,th eSv nusuall y doesno t organelle content (Russell,1984 )raise s contain anyplastid s (Russell,1984) . questions aboutwhethe r the spermcells ,s o produced,ar eequall y capable of fertilizing Paternalplastid s canb euse d totrac e theegg ,o rwhethe r preferential patternso f which of thetw omal egamete s fuseswit hth e fusionwil loccur . eggan d centralcel lsinc ethei r ultrastructure differs from thato fmaterna l Inth eangiosper mPlumbag o zeylanicaL. , plastids.Assumin g that spermcytoplas m is thetw osper m cellsdiffe rsignificantl yi n transmitted,a nassumptio n thatha sbee n bothmitochondrio n and plastid content verified inP . zeylanica (Russell,1983) ,th e (Russell, 1984). Furthermore,i tha sbee n presence and location ofpaterna lplastid s in demonstrated that thepaterna l andmaterna l cells of thefemal egametophyt eshoul d organellesma y beobserve d and distinguished indicatewhic ho f the twomal egamete sfuse d byultrastructura l differences inbot hth e with theegg .Provide d thatnumerou spaterna l zygotean d endosperm of thisplan t following plastids areseen ,th efat eo fth e gametic fusion (Russell,1980 ,1982 ,1983) . plastid-rich Suadurin g fertilization canb e Thepresen t studyuse s these characteristics clearly deduced. asth ebasi s forexaminin g thefat eo f individualsper mcell sdurin g gameticfusion . Theprobabilit y oferro r inidentifyin g whether aparticula r sperm celltyp e .Materialsan dMethod s fertilized theeg g reduceswit h eachpaterna l plastid seeni na give n femalegametophyte . Plantso fPlumbag o zeylanicaL .wer egrow n Since thestatistica l probability of theSv n

145 containing asman y asthre eplastie si sles s Therecognitio no f sperm cellsb yth e than 1%,th epresenc eo f threeo rmor e femalereproductiv e cellsi spresumabl y paternalplastie si nth ematerna l cytoplasm mediated by thesam emechanism s influencing ofeithe r thezygot eo rendosper m should recognitionevent s inothe r systems (Clarke clearly establish thefat eo fth e andKnox ,1978) ;however ,th eidentit yan d plastid-richsper mcel love r 99% ofth etime . modeo factio no fan yspecifi crecepto ri s The actualmargi no ferro r isprobabl y presently undetermined.Th epresenc eo f significantly lesstha n 1%,i fon econsider s unfused spermcytoplasmi c bodies(Russell , howlikel y iti stha t allpaterna lplastid s 1983)suggest s thata threshol d effect willb eobserve d without serially occurs,whereb y only themai n spermcel lbod y reconstructing thefemal egametophyte . caneffec t gameticfusion .Difference si n sperm cellvolume ,whic hma y approacha rati o Inth eovule suse d inthi sstudy ,media n of2: 1 (Russell,1984) ,ma y alsob ea n sectionswer eprepare d throughbot hth e importantfacto r indiscriminatin gbetwee n zygotean dprimar y endospermnucleu s inorde r thedelivere d malegametes ,whethe rsuc h toexamin e theperinuclea r cytoplasm inwhic h recognitioninvolve s aspecifi c receptor,th e plastidspreferentiall yappear .Thi sapproac h cooperationo fmultipl ereceptors ,o rwhethe r permits thefat eo feac hsper m cellt ob e differences inmembran echarg ean dsurfac e confirmed inbot hfemal ereproductiv ecells . areaalon emigh tprovid e themechanis mfo r Insom e cases,numerou smitochondri a similar discrimination arecentra l tofurthe r insiz et othos enormall y observed inth e elucidationo f thebasi s ofsper mspecificit y spermcel l (Russellan dCass ,1981 ;Russell , duringdoubl efertilization . 1983)wer e observed inon eo f thetw ofemal e reproductive cells.I neac hcircumstance , numerouspaterna lplastids .wer eobserve di n References theopposit efemal ereproductiv e cell. /

146 THESTRUCTUR EO FSPER M CELLS INBRASSIC A

C.A.McConchie ,S .Jobso nan d R.B.Kno x

PlantCel lBiolog y ReseachCentre ,Schoo l ofBotany ,Universit y ofMelbourne ,Parkville , Victoria 3052,Australi a

Summary nections. Within thesper m cells,ther e isa nuneve n Inmatur e trioellular polleno fBrassic a distribution ofmitochondri a and noplastids . campestrisL. ,th epai r ofsper m cellsar e Thevegetativ enucleu si sremarkabl ycon ­ held together within thecommo n plasmamem ­ voluted. Thecytoplas m inth eembayment s braneo fth evegetativ e cell. Thesper m containsprominen tcircula r profileso fE R cellmos t closely associated withth eveg ­ which may or may not be coated with etativenucleus ,beside scontainin gmos to f ribosomes. These appear to originate from themitochondri a inth e sperm cells,ha sbee n the outer membrane of the nuclear envelope. shownt oposses sa lon gtail ,> 10u mi n Also present are portions ofth e tail ofS C length,tha tpenetrate sthroug h apassag e 1, the sperm cell most closely associated inth ehighl y convoluted vegetativenucleus . with the vegetative nucleus. This sperm In all grains examined, this long tail contains more mitochondria than the other contained a forked array of microtubules sperm cell (SC 2),an d possessesa lon gtai l linked toarray saligne d within theridge so f that penetrates through a passage in the thesper mcell . Plastidswer eabsen t inbot h highly convoluted vegetativenucleus . spermcells . Several different arrays of microtubules have been detected inth e cytoplasm adjacent Introduction to the plasma memebrane^of the spermcells . In SC1, an array is present ineac h ofth e Sperm cell associationshav ebee npre ­ tworidge so fth etail . Towardsth epoin to f viouslyreporte d inthre etricellula r entry into the nucleus,th e arrays fusean d pollen systems. The first isPlumbag o continue as a single array intoth etip ,bu t zeylanica,wher ea nassociatio nbetwee n end prior to the branches. Each array spermcell san d vegetative nucleuswa sre ­ appears as agrou p of linearly arranged rows ported in the mature pollen and pollen tube of microtubules. In SC2, several arrays (Russell and Cass, 1981). Subsequently, extend along the major ridges to the end similar associations were found in Brassica adjacent to SC1 where they terminate. The oleracea (Dumas et al. 1984), and s.pinach, arrays of microtubules presumably provide a Spinacia oleracea (Wilms and van Aelst, cytoskeleton for the sperm cell, giving the 1983). Here, we report the finding of a tailth e strength tomaintai n theassociatio n spermcel lassociatio n inBrassic a campestris with thevegetativ enucleus . and have demonstrated its 3 dimensional Our quantitative data support the concept structure. of the male germ unit recently developed for the sperm cell association in B^_olerace a Resultsan d Discussion (Dumase tal. , 1984). The importanceo fthi s conceptlie si nit srelationshi p todoubl e Serial thin sectionswer e cuto fentir e fertilization: spermcel lassociation so f3 polle ngrains , — all the DNA ofheredity ,bot h cytoplasmic and 3-dimensionalcoordinat e informationob ­ and nuclear, is held together in a single tained bydigitizatio n using theZeis sVideo - unit; planImag eAnalysi s system. Precise lengths — there isdimorphis m inbot hth e shapean d and placement of the sperm cells and mitochondrial content of the pair of sperm vegetative nucleus in three dimensions were cells (see Russell and Cass, 1983)an d some computed and steresscopic images generated. evidenceo fthei r non-random Thesewer e confirmed bymanuall y constructed participation in the two fertilization 3-dimensionalmodels . events. The sperm cellassociatio n issuspende d While iti saccepte d thatthes e conclusions centrally inth ecytoplas m ofth evegetativ e apply in only some tricellular pollen cell. The sperm cells,lik e thoseo fB . systems,take n together,thes eresul ssugges t oleracea (Dumase tal. , 1984)ar esurrounde d thatfertilizatio nma yb etargeted . Themal e bothb ythei r ownplasm amembranes ,an d the germ unit therefore appears to provide a continuous innerplasm amembran eo fth e means by which the male gametes are vegetative cell.Th etw oplasm amembrane sar e transported toth e female reproductivecells . separated bya nelectro n lucent zone,whic h raaycontai n varioustype so fvesicles ,o r evenwha tappea rt ob ecytoplasmi ccon ­

147 References

Duraas,C , Knox,R.B. ,& Gaude ,T .1984 b Pollen-pistil interactions:ne wconcept s fromelectro nmicroscop y and cytochemistry. Int.Rev .Cytol . (in press). Russell,S.D . and Cass,D.D . 1981 Ultrastructure ofth e spermso fPlumbag o zeylanica. I.Cytolog y and association withth evegetativ enucleus . Protoplasma 107:8 5- 107 . Russell,S.D .& Cass ,D.D . 1983 Unequal distribution ofplastid san dmitochondir a during spermcel l formation inPlumbag o zeylanica. In:"Pollen :Biolog yan d Implications forPlan t Breeding ".(ed. ) Mulchay,D .& Ottaviano ,E . Elsevier SciencePublishin gC , N.Y.pp .135-140 . Wilms,H.J .& va n Aelst,A.C . 1983 Ultra- structureo fspinac h sperm cellsi nmatur e pollen. In: "Fertilization andEmbryo - genesis inOvulate d Plants,1983" . Veda, Bratislava, pp.105-111 .

/ * SPERMCELL S INTH EFERTILIZATIO N PROCESS INLYCOPERSICO NESCULENTU M

A.Kadej ,F .Kade j

Institute of Biology of the M. Curie-Sklodowska University, Lublin, Poland

References Summary In tomato, just before merging with Cass, D.D., 1981. Structural relationship the egg- and central cell, the sperm among central cell and egg apparatus cells show conspicuous reduction in size cells of barley as related to trans­ andi namoun to forganelles . mission of male gametes. Acta Bot. Soc.Pol .50 ,nr . 1-2:177-180 . Introduction Kadej, A. & F. Kadej, 1983. The ultra- structure of the egg cell in Lycopersi- In few cases e.g. in barley (Cass, con esculentum (Miller) at the moment 1981) sperm nuclei and other organelles of fertilization. In: Fertilization take part in the fertilization process. and Embryogenesis in Ovulated Plants. In Spinacia (Wilms, 1981) the sperm nuclei VedaBratislava . have no cytoplasm before they merge with Wilms, H.a., 1981. In: Reproduction in the cytoplasm of the egg- and central Spinaciaolerace aL. ,Wageningen . cell.

Resultsan ddiscussio n

Electronograms present sections from tomato ovules fixed at 34 h after pollina­ tion with the method reported by Kadej & Kadej (1983). These sections come from the same egg apparatus from three levels of cutting. They have been intrepreted as follow: the sperm cells in the tomato before leaving the pollen tube continue their transformation. These are mainly visible in the rejection of the outer cytoplasmic layer with the majority of mitochondria (M), dictyosomes (D) and - endoplasmic reticulum (ER) in them (Fig. *^>V * 3). Plastids were not observed. The male gametes so formed cover themselves with V. .-:;•? a new cell membrane containing pollen tube material (Fig. 3, arrows). The pre­ j&'•vi served remnants of the sperm cell wall $ft 4' I (Fig. 2, arrows) and a considerable cavity in the pollen tube as well as the nature à '-*$?'£$*>• of the cytoplasmic structures in it point 54-^ •>' .-A ' to the fact that the whole reduced sperm cellget s intoth eeg g cell. The mechanism of the fusion of the male gametes with the egg- and central cell would be a simple fusion of two cells. This fusion is preceded by resorb- ing of the cell wall which would cause theopenin g of twoareas . The role of the cytoplasm and the organ­ elles of the sperm cells could be however insignificant. The may soon undergo com­ pletedegeneration .

Presentaddress :Institut eo f Biology,Akademick a 19,20-03 3Lublin ,Polan d

149 SECONDARY NUCLEUS IM fiOSOIDEAE

R. Czapik

Department ofPlan t Cytology and Embryology Institute of Botany, Jagellonian University, Krakow, Poland

Summary where the published data for particular genera are fragmentary or completly In 16 examined species from 10 genera of lacking. Accidental observations are the subfamily Rosoideae the three types mostly insufficient and the studies on of polar nuclei behaviour were found.Po ­ this character must be carefully planned lar nuclei fused: 1. long or 2. shortly to get the exact view when polar nuclei before fertilization, and 3- simultaneous fuse and what kind of variation of their triple fusion occurred(Table 1).I n the behaviour may be expected. last case no secondary nuclei were formed, The problem was investigated by the or they were rare, in unfertilized embryo authoress inRubu s( Czapi k 1983 ai b). sacs and the degenerating polar nuclei The four examined species of the sub­ lay attached one to another. The results genera Eubatus,Cyclacti s and Chamae- confirmed the view that the behaviour of morus differed inrespec t of thischarac ­ polar nuclei is one of the embryological ter and the following three types ofbe ­ characters of the species and it may vary haviour of the polar nuclei were found. within the genus. ^ 1 chamaemorus type: polar Keywords: polar nuclei, secondary nucleus, nuclei fused long before fertilization, Rosoideae. the secondary nucleus was always formed. 2 Rubus saxatilis type: polar nuclei Introduction fused shortly before fertilization, simultaneous triple fusion was noted in Schnarf (1931)an d Davis (1966)empha ­ rare cases. In this type the secondary sized the importance of the behaviour of nucleus was present in old embryo sacs of polar nuclei in mature embryo sac as an unpollinated flowers. embryological character which may be of 3 Rubus caesius type: simultaneous taxonomie value. Davis, however, stressed triple fusion occurred. In old unfertil­ that there was little exact information ized embryo sacs no secondary nuclei were connected with this process. found, or they were very rare. The data for families of Angiospermae The influence of some external factors have been lately compiled by Poddubnaya- did not seem to contribute to the above Arnoldi (1982). According to the cyto- typ*eso fibehaviou r of polar nuclei.JMo r embryological characteristics given in was it possible tocorrelat e these her monograph, in ca 80%o f investigated differences, for certain, with any of the families polar nuclei fuse forming ase ­ internal factors taken into consideration condary nucleus which may be fertilized. taxonomie relationship of the species, Among the remaining familis the most in­ mode ofreproductio n or level of teresting are those in which variation of examined plants. the behaviour of polar nuclei was noted. The aim of the present studies was to One of them are . Poddubnaya-Ar- check whether the types of behaviour of noldi(1982)write s that in this family polar nuclei described for Rubus occur in polar nuclei fuse either long before fer­ other genera of the subfamily Rosoideae. tilization or at fertilization. On the basis of this description of the Material and methods character one can presume that in some taxa of Rosaceae, or under some special 16 species from 10 genera of the sub- conditions, secondary nucleus is not for­ family Rosoideae were exami ned. They were med. Such reports were published for the members of the four tribes Filipenduleae, polyploid Rubus species investigated by Rubeae,Potentilleae , Sangu isorbeae and Christen( 1950) , Berger (1953), Markarian Dryadeae( 'J-abl e1 ) . et al. (1959) and Dowrick (1966), while In each species at least two plants Thomas (1940)an d Virdi et al.(1969 ) from two distant localities in Poland observed early formation of the secondary were examined, except Duche snea indica nucleus in some diploids( ref . Czapik and Waldsteinia geoides tak en from the 1983 b). collection of the Krakow Bo tanic Garden, The observations on polar nuclei in The numbers of specimens Hubus were comparatively more frequently were established( Tabl e1) . The examined performed than in other taxa ofRosaceae , plants were potted and cult Ivated in the

150 experimental field in Modlnica where werevisible .However ,i nRubu ssaxatilis , emasculation, isolation and controlled representingthi styp eo fpola rnucle i pollination were performed. The ovaries behaviour,th esimultaneou s triplefusio n of various age as well as of pollinated wasobserve d sporadically.I tma yb eth e and unpollinated flowers were fixed in resulteithe r ofsom edela yo fth epola r acetic alcohol 1:3, 5 to 48 hrs or 4 to 6 nucleifusio no ro fth equicke rgrowt ho f days after anthesis. Mictotome sections thepolle ntubes .I ti shighl ypossibl e 8 - 12/i-m thic were stained with Heiden- thatsimila rproces sma yhav eoccurre d hain's haemathoxylin, alone or combined alsoi nothe rplant swit hRubu ssaxatili s with PAS or ruthenic red. The Feulgen typeo fpola rnucle ibehaviour . method was used for some embryo sacs fixed at the time of fertilization. Table1 .Behaviou r ofpola rnucle ii n examinedspecie so fRosoideae . Results Species 2n Tribe Innormall ydevelopin gflower so fth ee examinedplant sembry osac swer efull y (1)Rubu schamaemoru s type -pola r organized atth etim eo fexpansio no f nucleifus elon gbefor efertilizatio n •flowerbud san djus tbefor eanthesis .I n gynoeceawit hman ypistill s someyounge r Rubuschamaemoru sL . 56Rubea e ovulesmigh thav eoccurre d attha tstag e Duchesnea indicaFock e 42Potentillea e butth eyoun gembry o sacswer eeas yt o Sibbaldiaprocumben sL .1 4Dryadea e recognize. Wald,geoide sWilld . 14Dryadea e Thebehaviou r ofpola rnucle i inexa ­ Dryasoctopetal aL . 18Dryadea e mined taxafollowe d oneo fth ethre e patternsestablishe d forRubu s( Tabl e 1) . (2)Rubu s saxatilis type- pola rnucle i Infact ,th espea d ofth efusio nwa sth e fuseshortl ybefor efertilizatio n point inwhic hth emai ntype so fth e Rubussaxatili sL . 28Rubea e polarnucle ibehaviou rdiffered .Th enu ­ Filip.hexasepalaGilib .1 4Filipendulea e cleiapproache d eachothe ran dla yi n Pot.arenari aBorkh . 28Potentillea e closeproximit ydurin grelativel ylon g Comartunpalustr eL . 42Potentillea e orshor tperio do ftime . Fragariavesc aL . 14Potentillea e Inspecie sassigne d totyp e( 1 } Rubu s Geumurbanu mL . 42Dryadea e chamaemorus,secondar ynucleu swa sforme d Sang,officinali sL . 28Sanguisorbea e mostlywithi na close dflowe r budbefor e anthesisan dpollination .I ntyp e(2 ) (3)Rubu scaesiu styp e- simultaneou s Rubussaxatili sfusio no fpola rnucle i triplefusio n tookplac ea tth etim eo fpollination , A.i nunfertilize d embryo sacspola r sometimes justbefor efertilization .I n nucleii npairs ,secondar ynucle irar e thethir dtyp esimultaneou s triplefusio n occurredan dn osecondar ynucleu swa s Rubuscaesiu sL . 28Rubea e formed. Pot.puberul aKraSa n 42Potentillea e Thegener awer erepresente d byon e specieseac hexcep tRubu s studiedpre ­ B. inunfertilize d embryosac sal l viously,an dPotentilla .Th especie so f stageso ffusio no fpola rnucle i thelas ttw ogener adiffere d inrespec t RubusBellardi iWeih e 28Rubea e ofth eexamine dcharacter .Th edifferen ­ cesi nRubu sle dt oth erecognitio no f C.i nunfertilize d embryosac spair s thethre emode lgroup so fpola rnucle i ofunfuse dpola rnucle i behaviour. Intetraploi dPotentill aare ­ nariasecondar ynucleu swa sforme dshortl y Agrimoniaeupatori aL . 28Sanguisorbea e beforefertilization ,whil e inhexaploi d P.puberul asecondar ynucle iwer erar ei n Wald.= Waldsteinia ,Filip .= Filipendul a unfertilized embryo sacs.Suc hembry o Pot.- Potentilla ,Sang .= Sanguisorb a sacscontaine dunfuse dpola rnucle ilay ­ ingclosel yattache d toon eanother . Thistyp ewa srepresente d byon especie s Thefusio no fpola rnucle ibefor e fromeac htribe ,fro mPotentillea e- b y anthesisan dpollinatio n type(1)wa s threespecies .Th eplant swer ediploi d noted infiv especies :thre ediploid s (Filipendulahexasepala ,Fragari avesca ) fromth etrib eDryadea e( Sibbaldi apro - orpolyploi d( Rubu ssaxatilis ,Potentill a cumbens,Waldsteini a geoides,Drya socto - arenaria,Comaru mpalustre ,Sanguisorb a petala)an d twopolyploid s fromth etribe s officinalis,Geu murbanum/ . Rubeaean dPotentillea e( Rubu schamaemoru s Thethir dtyp eo fpola rnucle ibehaviour , andDuchesnea .indica) . i.e.simultaneou s triplefusion ,seeme d Thepola rnucle ifuse dshortl ybefor e tob ea rul e inth ethre etetraploids : fertilization in7 species ,i nthei r Rubuscaesius ,R .Bellardi i(Rubeae )an d unfertilized embryosac ssecondar ynucle i ( Sanguisorbeae)a s 151 well as in one hexaploid - Potentilla pu* fusion was observed, while the formation berula (Potentilleae) . In Agrimonia eupa- of secondary nuclei was arule . toria secondary nuclei were not formed in The influence of external factors on unfertilized embyro sacs till the sixth such a process as fusion of nuclei cannot day after anthesis,whe n the degeneration be excluded. Some unknown factors caused started. Such state was observed in Three that 1,7% of central cells ofunfertil ­ specimens from two distant populations, ized embryo sacs contained unfused polar in one of them during two following nuclei inRanunculu s investigated by seasons. InRubu s caesius,R . Bellardii Rutishauser (1954). Ormrod et al.(1967 ) and Potentilla puberula secondary nuclei found inPhaseolu s vulgaris that the either were not formed or they were very number of embryo sacs with fused polar rare in unfertilized embryo sacs. nuclei was modified by temperature. How­ There were some similarities between ever, nothing is known about the pheno- species of type(3 )Rubu s caesius and typic control and very little about the plants with multiple embryo sacs from the degree of interspecific variation of the group(2 )Rubu s saxatilis.Whethe r they examined character inRosoideae . were essential or casual it was not A separate problem for the variation obvious. Fragaria vesca,Waldsteini a geo- studies is offered by plants with multi­ ides and Fillpendula hexasepala developed ple embryo sacs where the main embryo sac 1-4 mature embryo sacs per ovule. The contains a secondary nucleus and the additional embryo sacs sometimes did not additional ones have unfused polar nuclei. form secondary nuclei in unfertilized The hypothesis of the correlation between conditions.In some ovules the discrepancy apomixis and the behaviour of polar nuclei could be explained by a somewhat younger requires examination of the developmental age of the embryo sac with unfused polar processes in ovules of the plants with nuclei. There were, however, embryo sacs multiple embryo sacs as well as of those for which such an interpretation seemed wi/thsingl e sacs, in which secondary to be doubtful. In Waldsteinia geoides nuclei are not formed in unpollinated the longest, probably strongest, embryo flowers. sac contained always a secondary nucleus. The present report on the behaviour of The embryo sacs with unfused polar nuclei polar nuclei inRosoidea e may beconsi ­ were shorter, often with converted polar­ dered as the first step of further stud­ ity and situated in chalaza. Many of them ies on one of the embryological charac­ seemed to have unreduced chromosome num­ ters of Angiospermae which has seldom ber(Czapik , in press). The hypothetical been investigated in detail. correlation between the apomictic nature of embryo sacs and the lack of secondary References nucleus or the delay of its formation in unfertilized embryo sacs was considered Czapik R., 1983 a. Embryological problems previously( Czapi k 1983 bj. There is, inRubu s L. Proc. Vll Intern. Cytoembr. however, no certain evidence which could», help to verify that assumption. Symp. Bratislava. p. 373 - 379« CzapikR. i 1983 b. The secondary nucleus in four species of the genus Rubus. Discussion Acta Biol. Cracov. ser. Bot. 25: The observations showed that the beha­ 179 -188 . viour of polar nuclei varies within the Davis G., 1966. Systematic Embryology of subfamily Rosoideae. It may be considered theAngiosperms . J. Willey, New Kork. as one of the embryological characters of Ormrod D.P., C. J. Woolea, G. W. Eaton, a species,a t least in genera Rubus and E. H. Stobbe, 1967.Effec t of tempera­ Potentilla, where more than one species ture on embryo sac development in was examined and in which differences of Phaseolus vulgaris I. Canad. J. Bot. the behaviour of polar nuclei were stated. 45: 948 - 950. There are,however , several questions Poddubnaya-Arnoldi V.A. , 1982.Harakte - left unanswered. The first is-why in some ristika semejstv pokrytosemennyh ra- species, such as Agrimonia eupatoria, or stenij po citoembriologicheskim pri- in some embryo sacs of Rubus caesius and znakam. Nauka Moskva. R. Bellardii, the polar nuclei were un­ Rutishauser A., 1954. Die Entwicklungs­ able to fuse without fertilization, or at erregung des bei pseudo- least why their fusion was delayed. gamen -Arten. Mitt. Katurf. The second question refers to the range Ges. Schaffhausen. 25: 1-45 . of variability of the character both at Schnarf K., 1931. Vergleichende Embryo­ the intra- and interspecific levels. The logie der Angiospermen. Gebr.Born - subtypes of type(3 ) Rubus caesius are an traeger, Berlin. example of the interspecific variation. The intraspecific variation was noted in Rubus saxatilis. In a few embryo sacs of the the examined plants simultaneous triple

152 EMBRYOSA CULTRASTRUCTUR EBEFOR EAN DAFTE RFERTILIZATIO N INCUCUMI SSATIVU S

J.L.va nWent' ,C.H . Theunis',an dA.P.M .de nNijs' 1

'Departmento fPlan tCytolog y andMorphology ,AU ,Wageningen ,Th eNetherland s "Institutefo rHorticultura l PlantBreeding ,Wageningen ,Th eNetherland s

Summary

The composition and development of ovule and embryo sac of Cucumis sativus, from anthesis up to fertilization has been studied. The mature ovule of Cucumis is anatropous, bitegmic and crassinucellate, and the embryo sac is of the Polygonum type. Development, composition and ultra- structure of the embryo sac generally resemble the pattern as already described for many other angiosperm species. The .polle n tube grows through the micropylar nucellar tissue towards the embryo sac and strongly proliferates. After discharge Fig. 1.Developmen to fembry osa c of part of its contents into one of the A:immature ;B :mature ; synergids, the pollen tube is disconnected C: justfertilized ;D :j u from thedegenerate d synergid by thedeposi ­ before firstzygot edivis i tiono fa plug .

Introduction

As basis for a breeding project on thepartia l transfer ofmal e geneticmateri ­ al by pollination with irradiated pollen, an investigation on thenorma l fertilization in Cucumis has been started. This part describes and relates the ultrastructure of the different cell types of the embryo sac in some stages, from their formation until some time after fertilization. Special attention is paid to the cells which are directly related to the process of fertili­ zation.

Material andmetho d

Unpollinated (just before anthesis) and pollinated (respectively 25, 45 and 70hr safte r pollination) flowerso f Cucumis sativus L.var.Hardwickii ,wer e taken from a greenhouse culture. The ovaries were sectioned and fixed in 3.5% glutardialdehyde for 10 hrs and post fixed in 1% OsO in 0.1 m sodium cacodylate buffer (pH 7.2) for 12 hrs at room temperature. Then they were dehydrated and embedded in epon.

Resultsan d discussion

The mature ovule of Cucumis sativus is anatropous,bitegmi c and crassinucellate, and the embryo sac is of the Polygonum type (Fig. 1).Th e ovule strongly enlarges during development and becomes flask shaped, with a long and narrow micropylar part of the nucellus (Fig. 3).Th e egg apparatus strongly enlarges within 30 hrs after anthesis, while the antipods disappear before maturity of the embryo sac. The Fig. 2Compositio n and ultrastructure cytoplasm of the various cells of the ofmatur eembry o sac 153 •\

••••'

Fig. 3Compositio n of nucellus after fertilization, showinç porliferationo fpolle ntube . Pig.4 Uïtrastructur e of zygote and blocked pollen tube Fig. 5Ultrastr.ucliur e of.persisten tan d degenerated synergic egg apparatus increases. The total volume the degenerated synergid (Fig. 5).Afte r of the embryo sac does not increase very this discharge, the remaining pollen tube much, nor does the volume of the central is disconnected from the embryo sac by cell (Fig. 1). Simultaneously the cell the deposition of a plug (callose), just types of the embryo sac each develop a outside the embryo sac (Fig. 4).Th eremain ­ specific morphology and ultrastructure. ing cytoplasm in the pollen tube and pollen The mature egg cell is pear shaped and tube proliferation shows very active mito­ appear active with large RER accumulations, chondria, many ER and dictyosomes, which many mitochondria and dictyosomes (Fig. indicates a high metabolic activity. Even 2). Development , composition and ultrastruc­ after fertilization,th epolle n tubecontin ­ ture of the embryo sac generally resembles ues to increase in volume, on account the pattern as already described for many of the surrounding nucellar tissue (Fig. other angiosperm species. The filiform 3). This growth implies haustorial activity apparatus is well developed and highly (Tilquin, 1983). Thispolle n tubeprolifera ­ convoluted (Fig. 2). Shortly before the tion may also form a blockage for other pollen tube enters the embryo sac one pollentube st openetrat e theovule . of the synergids starts to degenerate. The pollen tube grows through themicropyla r References nucellar tissue towards the embryo sac and strongly proliferates in this nucellar Tilquin, J.P., K. de Brouwer, Ä. Mathieu tissue (Fig. 3). The pollen tube enters & M. Calier, 1983. Haustorial pollen theembry o sacthroug hth efilifor m apparatus tubes in Fuchsia boliviana. Annals and discharges part of its contents into ofBotan y 52:425-428 . 154 EMBRYO SAC ULTRASTRUCTURE IN PEPEROMIA BLANDA H.B.ET K. BEFORE AND AFTER FERTILIZATION.

V.P.Bannikova, T.A.Plyushch N.G.Kholodny Institute of Botany,Ukr.S.S.R.Academy of Sciences.

Summary. central cellan dmoderat ei nth eeg g Inth epape rth eultrastructur e apparatus cells.Othe r characterso f ofal ltype so fcell s ofth ePepero - theultrastructura l organizationo f miabland aembry osa ci sdescribed . cells aresimilar .O nth eearl ysta ­ ge,nucle iwit heve nprofil e ofth e Introduction. envelope andmainl ydiffus e chroma­ Species ofth egenu sPeperomi a tinar etypical .Th edensit yo fri - 'arecharacterize d byth etetrasporic , bosomedistributio ni s low.E R cis­ sixteen-nucleate,polypola rembry o ternsar eno tnumerous . Duringth e saco fth ePeperomi atyp erathe rra ­ embryo sacdifferentiatio nth equan ­ refo rangiosperms .Th enumbe ro f tity ofribosome srise st osom eex ­ central cellnucle i andtha to fla ­ tent,an dalmos t completereductio n teral cells,homologou s toantipods , ofE Ri ncell so fal ltype soccurs . isvaried .Problem s concerningth e Inth e egg cell,aswel la si ncent ­ numbero fsynergid s andfunctiona l ralan d lateral cells,th enucle i specializationo feg gapparatu s become lobar.I nsynergids ,th enuc ­ cellsar ediscusse d inliterature . leipreserv eth eeve nenvelop e con­ Thedat ao nultrastructur e ofth e tour.I nth ebot hsynergids ,befor e Peperomiatyp eembry o sacar eno t thepolle ntub eenter sth eembry o reported. sac,betwee nmembrane s oforganelle s envelopes osmiophilicsubstance sar e Results. accumulated. Thepolle ntub eenter s All cellso fth eP.bland aembry o theembry osa cthroug ha synergid . sacbefor e andsom e timeafte rfer ­ Thefusio no fgamet e nucleiproceed s tilizationar esurrounde d onlyb y slowly.Durin gthi sproces s thecon ­ plasmalemma,wherea s polysaccharide tent ofribosome s andorganelle si n wallsar e absent.Th eultrastructur e alltype so fcell sincreases .E R(ci ­ ofeac htyp eo f cellsi sspecific . sternalan dtubular )i sdeveloped . Aconsiderabl e quantity ofplastid s Cisterns ofE Rar emos tgreatl yde ­ istypica lo fth eeg gapparatu s veloped inth e central cell.Th etu ­ cells,thenumbe ro fwhic h constantly bularreticulu mi sth emos tabundan t equals 5«Therei sa smal lamoun t (re­ inzygote .I nal l cellsth enucle i lative toth e cellvolume )o fplas ­ have lobarshape,diffus echromatin . tidsi nth e central cell.I nth ela ­ Thepolysacharid e cellwall sar e teral cellsplastid s arerathe rra ­ formedi nth efina lperio do fnucle i relyobserved .Mitochondri aar eth e fusioni nzygote . mostabundan ti nlatera l cellsan d their quantityi sals ohig hi nth e

155

_ SEXUALREPRODUCTIO N INTH EGENU SFUCHSIA . EMBRYOGENESIS INP.FULGENS .

D.Choisez-Givron .

Cytogenetical Laboratory,Carno y Institut,Catholi c Universityo fLouvain , 4Plac ed el a Croixd uSud ,B-134 8Louvain-la-Neuve ,Belgiu m

Summary 2.Embryogenesis .

Fertilization andembry odevelopmen ti n Researchha sbee nmad eo nmicrotom e slides F.fulgens are investigatedpreliminarl y in cuts stainedwit hhaematoxyline-fast-green . order toperfor m the fertilization in-vitro. Thepollinatio n dayi srepresente d byDo . Thepolle n tubesgro wbetwee nth ecell so f The fertilization occurso nD o+4 .Th eembry o thetransmittin g tissue andreac h thelocu - ishidde nb ydegenerate d synergids.Th epri ­ luso fth eovar y threeday safte rpollina ­ mary endospermic nucleusundergoe sth efirs t tion.The yente r theovul ethroug h themicro - division immediately.Th e inneran doute r pylean dpenetrat e oneo fth e synergidsbe ­ integumentshav e respectively 2an d 4cell - fore toreac hth eegg-cell .Th ediploi dzy ­ layers.The ywill ,ver y early,g othroug h gotegrow su pi na coenocyti c endospermwhic h changestha twil lmak e thespermoder m outo f becomes cellularafte r the lothday .Th e them.Som emitose s canstil lb eobserve di n mature seed isexalbuminate . theoute r integument,whils tth ecell so f theinne r integument are loadedwit hdark - Introduction brown coloured substances.Mitose soccu rin ­ sideth enucellus .A tD o+ 7 ,th ealbume nha s TheFuchsi a speciesbelong s toth efamil y about 10nuclei .Th enucellu s cellsbecom e ofth eOnagraceae .Th eresearc hconducte do n vacuolated whichpushe sth enucleu sagains t the fertilization andembryogenesi scome s thecel lwall .N omitos e canb efoun dany ­ intoth e frameo fth e selectiono fne wva ­ more.Th epolle n tubepersist s longafte r rieties inhorticulture .Man y Fuchsialover s fertilization andprobabl y assumesa hausto - keep searching forne whybrids .Th edevelop ­ rialfunctio n (Tilquine tal. , 1983). mento fhybrid s isi nsom ecase shampere db y Thepolle n tubecytoplas m ishighl yconcen ­ barriers ofincompatibility ,b e itbefor eo r trated.Th eoute r integuments differentiate: after fertilization; thesebarrier smigh tb e theoute ran dinne rlayer sge tflat ,whils t removed through test-tube fertilizationo r themiddl e layercell sge tempt yo fthei r throughin-vitr o cultivating abortingem ­ content.Th ecell sar eloade dwit hdar kgra ­ bryos.Th e studyo fembryogenesi s inF.ful ­ nulesan d thecell-wall sge tthicker . gens (sectionEllobium ) isa preliminar yt o AtD o+10 ,th eembry oha sgo tri do fth e thisresearch . * micropylar dark stainan d carries 1t o6 ,cel#ls.The"albume n iscoenocyti cwit h scores ofnuclei' .A tD o+ 17,th eembry o isglobu ­ Resultsan ddiscussio n larwit h 45t o 140cells .Th ealbume ni s cellularised around theembryo .Som enucellu s 1.Fro mpollinatio n tofertilization . cells stillcontai na nucleus ,whic hdoe sno t divideanymore .A tD o+20 ,th eembry o isco - Thegerminatio n ofpolle no n thestigma , tyledonary and countsove ra hundre dcells , aswel l asth ecours e ofth epolle ntube s insideo fa celle d albumenwit h severalhun ­ inth e styleunti lenterin g inth eovar yar e dred cells.A tth echalazae l endo fth eovule , followed thankst osmear smad eafte r 1,2 thealbume n remains coenocytic.Fro mD o+ 30 , 3day s (stainedwit h anilin-blue;fluore ­ upt oth ematurit y ofth efrui t (Do+50) , scencemicroscope) .Abou t 3day sar eneces ­ theembry odevelops ,progressivel y fillsi n sary forth epolle n tubest oente r theovar y allth ecavit yo fth e seed,rebsorb sth e and fertilize thefirs tovules .I ttake s2 4 albumenan d scratchesnucellu swhic hdisap ­ hoursmor e foral lovule so fth eovar yt o pearscompletely .A tmaturity ,th esee di s be fertilized, i.e.a maximu m of 85%.Th e exalbuminate. entranceo fth epolle n tube intoth eovul e occursthroug h themicropyle ; itcrosse s thenucellu s and entersth eembry osa c Reference througha synergi dwher e itsconten t isdis ­ charged.Th esynergi d becomesopaque .Th e Tilquin,J.P. ,d eBrouwer ,K. ,Mathieu ,A . second synergid degeneratesa t once. andCalier ,M. , 1983.Haüstoria lpolle n tubes inF.boliviana .Ann .o fBot .52 ,425 - 428.

156 ACYTOLOGICA LMETHO DFO RDETECTIN G COEXISTENCE OF SEXUALAN DDIPLOSPORI C REPRODUCTIVE PROCESSES1

B.Longly , T.Raba u &B.-P .Louan t

Laboratoire dePhytotechni eTropical e etSubtropicale , Place Croixd u Sud3 , Sc.15D , B-1348Louvain-la-Neuve , Belgium

Summa ry reference inth elittératur et oth e sizeand / orth edifférenciatio no f theovul e (forex ­ A study realized inhermaphroditi c plants ample: Esau , 1946), more rarely ofth e by linking up theconcomitan t stages offe ­ ovaryo r theflowe r (Pacini &Sarfatti , 1978). male sporogenesis andgametogenesis , onth e However,w e found ina Compositae , Cichorium onehand , ando fmal e sporogenesis andga ­ intybusL. , littlean dunderdeveloppe dovu ­ metogenesis, onth eothe rhand , allowedt o les inol d ovaries, preventing anyreferenc e reveal differences ingametophytogenesise s toflowe r components.Sometime s too, itwa s dynamics. used astric t time referencebut ,followin g Each dynamicsca nb ecorrelate dwit ha Favre-Duchartre et al.(1979), estimateso f definitereproductiv eproces s onbasi so f angiospermoogenesise s durations, inabsolut e cytological observations.So , itwa stha t value, areextremel y rare.I ti sindee dver y thecoexistenc e of sexual and apomictic insecure toevaluat e thespee do f aphysio ­ reproductive processes couldb ebrough t to logical process ona chronologica l basis thefor e inBrussel' schicor y (Cichorium because ofth einfluenc eo nth e development intybus L,), theter m "apomictic "coverin g ofenvironmenta l factors (temperature,light , here diplospory sensu lato.Ther e issom e hygrometry...)an d ofgerieti cfactor s res­ confirmation of the difference indynamic s ponsible for interplantvariations . between sexuality and diplosporywithi na So, we thought tous ea mor e intrinsic samegenu s (Eragrostis). factoro f theflowe r development, i.e.th e In somecases , thismetho d must allowt o malegametophytogenesis .W e established then bring toth efor efacultativ e apomixisi n a "reproductiv e calendar"wher eth estage s species thought tob eentirel y sexualo r offemal e sporogenesis and gametogenesis obligate apomicts.Moreover , itmake spos ­ werecorrelate d with theconcomitan t stages sible toestimat e the degree ofapomixi s in ofmal e sporogenesis andgametogenesi s in plantswher e thisreproductiv e processi s the sameflowe r (Louant &Longly , 1981a; appearingnaturall y orb yhybridization . Longly &Louant , 1982). Inthi sview , theyoun g stages ofmega - Introduction gametophytogenesis, corresponding toth edi ­ vision of thearchesporia l cell, areth emos t Eachgametophytogenesi sproces s (meiotic, important.I nmonospori c species,th e "key apomeiotic, mitotic...)seem s tohav eit s stage"wil lb eth etetra d one.However , older owndynamics .Thi sdynamic s dependso nth e stages canb euseful , forexampl ewhe n sexual initiation andprogres s of thefirs t divi­ and diplosporic embryo sacs aremorphologi ­ siono f the archespore.Indeed , anydistur ­ callydiscernible . bance1i nth emeiosi s leading tounreduce d (mitotisationo f themeiosi s: Application of thereproductiv e calendar Stebbins, 1941)give s rise tochange si n method gametophytogenesisprogress .O nanothe rhand , apurel ymitoti c division generally gives a)Withi n aspecie s rise tochange s ingametophytogenesi s ini­ tiation.Apparently , themor e themitotisa ­ So, inCichoriu m intybusL. , sucha cyto - tioni simportant , themor e there isa dela y chronological detailed studyha sbee nunder ­ inth efirs t division of the archesporial taken.A t thesam etime , itwa s seen there cell (Bergman, 1941). were several types of structures inth e ovules of this species (Louant &Longly , Conceiving of the reproductivecalenda r 1981aan d b;Longly , 1984). These structures produce generally the same typeo fgameto - Toevaluat eth edegre eo f sucha delay , it phyte.Mos t ofthe mca nb ecorrelate dwit h isnecessar y tohav e areference .Wit hre ­ aknow nmod e ofreproduction .Beside sth e gard toth edynamic s of thegametophytoge ­ normal progress ofmeiosis ,w e found several nesis inth eovules , itwa smor e oftenmad e forms ofapomeiosi s andmeioti c irregula-

1.Researc h subsidizedb y IRSIA (Institu tbelg epou rl'encouragemen t del aRecherch e Scientifique dans l'Industrie etl'Agricultur e) .

157 rities. gyny shouldmak e ineffectual thecalendar . Theform s ofapomeiosi s showa mor eo rles s -Detection ofapomicti c reproductive pro­ important delay of thefirs t divisiono fth e cessb y thiswa y applies only incas eo f archespore comparedwit hth emeiosis .Th e diplospory sensulato .Somati c apospory is moreth edeviatio n isstartin g soon, themor e anotherproblem .I tconcern s othercell s thedela y isimportant .Thi si si naccor d thanth earchespore .Ou rcalendar ,only , withBergma n (I.e., cfr introduction).Al l allows tocompar edifferen t dynamics ofth e theseprocesse s give rise tounreduce d game- samecell . tophytes. -Whencalendar shav e tob e compared, ina Themeioti c irregularities, on theothe r samespecie s orbetwee n different ones, the hand, giveris e totetrad s orpolyad swit h microgametophytogenesis processesmus tb e thesam e "timin g" asi ncas e ofregula r identical or,i nan y case, superimposable .Ho ­ meiosis.Th eresultin g gametophytesar eher e wever, a nabnormalit y doesn't changeine ­ reduced and generally aneuploîd. vitably theprogres s of amicrogametophyto ­ Here, only thereproductiv e calendarallow ­ genesis. edt orevea l caseso f apomixisno tstructu ­ rally different of sexuality but showinga Fieldso fus e slightdelay . -Thecalenda r isappropriat ewhe n iti s b)Withi n agenu s difficult orno tpossibl e todiscer n sexual and apomictic ripegametophytes . Themetho d of reproductive calendarwa s -Themicrogametophytogenesi s referencebe ­ alsoapplie d to,comparetw o specieso fth e comes indispensablewhe n iti s impossiblet o genusEragrosti sWol f (Graminae): E. tef utilizequantitativ echaracter s sucha sth e (Zucc.)Trotte r andjï .curvul a (Schrad.)Nees . inversiono f theovul e incas e ofanatropy , E. tef follows sexualmod eo f reproduction theovule san d ovaries size, thefillin go f (monosporicbipola r embryo sac of the "Poly ­ the/ovariancavity.. .t oestimat e thepro ­ gonum"typ e: Mengesha , 1964). gresso f femalegametophytogenesises . Inth e Relating to E. curvula, cytomorphological opposite situation, itca nserv ea sa comple ­ studieso f themegagametophytogenesi ssug ­ ment. gested (Brown &Emery , 1958)an d thencon ­ -Thecalenda rprevent s fromlookin gfo r firmed (Streetman, 1963)reproductio nb y division figures inth earchesporia lcells . diplospory. These are indeed seldomdu et othei rquick ­ Since themicrogametogenesise ssee mt ob e ness.I tallow s totak eint oaccoun tcyto ­ identical inbot ho f these species,w eca n morphological consequences of thesedivision s get themt ocoincid e ina nonl y reference todefin e areproductio nmode . scale.Th eelaborate d reproductive calendar -Innatura l orhybri d facultativeapomicts , enables togiv eprominenc e ort osituat e theus eo fa calenda rallow s torealis ea cytochronological and cytomorphological dif­ screening apomixisversu s sexualitywithi n ferencesbetwee n apomixis and sexuality in a same inflorescence, asam eplan t ora popu ­ both species.I nthi sway ,megaspore s tetrads' lation, tospecif y therelativ erat eo fapo ­ ofE .te f arefoun d atth e samemal e stagea s mixis.Th e stamplesca nb eanalyze d either archespores in .E.curvula .Thes e archespores bymean s ofparaffi n sections orclearin g start todivide , atth eearliest , at thesam e squashes. timea sth emitoti c division of thefunctio ­ nalmegaspor e in E. tef.Th eresultin g game­ Conclusion andprospec t tophytes (sexual andapomictic )ar eher edif ­ ferent.Initially , thefirs t onesar eocto - Aprogen y test combinedwit h acytologica l nucleatewhil e thesecon d ones, tetranucle- study ofmegasporogenesi san d embryo sacde ­ ate. velopment aregenerall y required toconfir m At the timeo f reproductive calendaresta ­ apomixispresenc e and toidentif y hismecha ­ blishment,w e discovered, inE_ .curvula,. , nism (Bashaw, 1980). Nevertheless, according symptoms of sexuality besides apomictic toStebbin s (1950), irrefutable evidenceo f structures. So,.,a tetra d ofmegaspore san d eitherpresenc e orabsenc e ofapomixi sca n anembry o sacwit h sexual appearencewer e onlyb eprovide d by cytomorphological obser­ observed atth e samemal e stages asthos e vations.Th e reproductive calendarreplie s corresponding toidentica l female development tothi srequirement . inE .tef .Thi sfact s confirm thefacultativ e With thecalenda rmethod , it isabov eal l apomixis concept for thespecie s (Voigt& possible todetec t coexistence of sexuality Bashaw, 1973;Brix , 1974). andapomixi s ina sam e species ori na sam e genus onbasi so f thenon-equivalenc e ofth e Limits ofus e dynamics of thereproductiv emodes . Inspecie s interesting from theevolutiv e Nevertheless, therear elimit s toth eus e or agronomic point ofview , thesystemati c of thereproductiv e calendarmethod . applicationo fou rmetho d would permit to -The flowershav e tob ehermaphrodite . bringu p todat e theunsuspecte d diversityo f Moreover ato opronounce d protandry orproto - reproductionmode swithi n somuc h asam espe -

158 ciesa sa sam egenus .Thi sdiversit y isi n Streetman,L.J. , 1963.Reproductio n ofth e connectionwit h the thought ofAske r (1979) Lovegrasses, thegenu sEragrostis— I: E . accordingwic h only facultative apomixis chloromelasSteud. , E_.curvul a (Schrad.) withvariou s degrees occursi nspecie scon ­ Nees, E.lehmannian aNee s andE .superb a sidered asobligator y apomicts. Peyr.Wrighti a3 : 41-51 . ~ Voigt, P.W. &E.C .Bashaw , 1973.Facultativ e References apomixis inEragrosti s curvula.Agron .Ab ­ stract.6 5: 16 . Asker, S., 1979.Progres s inapomixi sre ­ search.Heredita s 91: 231-240 . Bashaw, E.C., 1980.Apomixi s and itsappli ­ cation incro p improvement.I n: W.R .Feh r & H.Hadle y (Ed.): Hybridization ofcro p plants.Madison ,p .45-63 . Bergman, B., 1941.Studie s onth eembry osa c mothercel l andit sdevelopmen t inHiera - ciumsubg .Archieracium .Svens k Bot.Tid - skr.35(1 ): 1-41. Brix,K. , 1974.Sexua lreproductio n inEra - grostiscurvul a (Schrad.,)Nees .Z .Pflan - zenzuecht 71: 25-32 . Brown, W.V.& W.H.P .Emery , 1958.Apomixi s inth eGraminea e: Panicoideae .Ann .Jörn . Bot.45(4 ): 253-263 . Esau,K. , 1946.Morpholog y of reproduction inGuayul e andcertai nothe r specieso f Parthenium.Hilgardi a 17(2): 61-120 . Favre-Duchartre,M. , B.-P.Louan t &J.P . Tilquin, 1979.Duré ed evi ee torganisa ­ tiond usa cembryonnair e ausei n del'ovu ­ led eCichoriu m intybusL . (Compositae)e t théorie taxalienne.CR . Acad. Se, Série D, 288: 331-334 . Longly, B., 1984.Tétrade s irrégulières,dé ­ veloppements apoméiotiques et aposporie dansle sovule s deCichoriu m intybusL . (Chicorée deBruxelles) .Can .J . Bot.6 2: 635-646. Longly, B.& B.-P .Louant , 1982.Cytochrono - logied el amégagamétogénès eche zCicho ­ rium intybusL .Ann .Univ .Arer s 18: 17 - 20. Louant, B.-P.& B .Longly , 1981a.Correspon ­ danceschronologique s entre lessporogé - nèses etgamétogénèse smâle s etfemelle s chez Cichoriumintybu sL . (Chicoréed e Bruxelles).Rev.Cytol.Biol.Vég.4:187-201 . Louant, B.-P.& B .Longly , 1981b.Symptôme s d'aposporieobservé s aucour s del asporo - génèse etd el agamétogénès eche zCicho ­ rium intybusL.(Chicoré e deBruxelles ). Rev.Cytol .Biol .Vég .4 : 173-186 . Mengesha,M.H. , 1964.Eragrosti s tef (Zucc.) Trotter;it s:embry osa c development,gene ­ ticvariabilit y andbreedin g behavior.Ph . D.Dissert .Purdu eUniv .Dissert .Abstr . 25: 4338-4339 . Pacini, E.& G .Sarfatti , 1978.Th erepro ­ ductivecalenda ro fLycopersicu mperuvia - numMill. . Soc.Bot .Fr. ,Actualité sBo ­ taniques 1-2 :295-299 . Stebbins, G.L., 1941.Apomixi s inth eAngio - sperms.Bot .Rev.Vol .VI I 10: 507-542 . Stebbins, G.L., 1950.Variatio n andevolu ­ tion inplants .Columbi aBiologica l Series XVI. ColumbiaUniv .Press ,New-Yor k and London.

159 INITIATIONO FASEXUA L SEEDPRODUCTIO N INKENTUCK YBLUEGRASS ,PO APRATENSI SL .

Y.S. Abeln,H.J .Wilm san dA.J.P .va nWijk *

Departmento fPlan tCytolog yan dMorphology ,AU ,Wageningen ,Th eNetherland s *Vande rHav eC oPlan t Breeding Station,Rilland ,Th eNetherland s

Summary finds two developing embryo sac producing cells: A so called aposporous cell situated The sexual embryo sac development of beside a haploid sexual cell (Rütishauser, Poa pratensis follows the Polygonum type, 1967). Both cells develop into embryo while the apomictic embryo sac development sacs. 'Sexual cell' indicates any stage follows the aposporous way as in . of development from megaspore mother cell Initiation of the asposporous cell occurs to mature embryo sac, while 'aposporous next to the chalazal part of the sexual cell' indicates any stage of development cell. This initiation takes place at some from aposporous initial cell to mature stage of the development from megaspore aposporous embryo sac. The egg cell of to mature embryo sac. While developing, the embryo sac derived from the aposporous the aposporous cell enlarges in the direc­ cell' (diploid ) can eventually undergo tion of the micropyle. In the more sexual , probably after fertiliza­ experimental variety of Poa pratensis tion of its central cell nucleus (Akerberg, L. in a short period of embryo sacdevelop ­ 1942). Therefore one can expect toorigina ­ ment a huge complex of antipodals develops. te from such Poa pratensis flowers seed It is possible that this complex restricts with two diploid embryos, one of sexual initiation and development of aposporous origin, the other of asexual origin (twins). cells. The results of this study indicate Engelbert (1941) came across this phenome­ that it ispossibl e topredic tth ereproduc ­ non in up to 5% of studied Poa pratensis tive character of Poa pratensis plants plants. In 0,5% of the germinated seeds by observing their very first flowers he found twins,bot h of which showed matro- when the embryo sac development has reached clinic character. Hence these twins both the four-nucleatestage . have been of aposporous origin. The sexual Keywords: Kentucky bluegrass, Poa pratensis embryo sac (cell)mus t have stoppeddevelop ­ h., apomixis, apospory, embryo sac develop­ ing at a certain time and it probably ment. disappeared. In Poa pratensis varieties listed as obligate apomicts, none or very Introduction few plants deviate phenotypically from the female parent. In these varieties Kentucky bluegrass, Poa pratensis L., the sexual embryo sac development apparent­ is a grass species used for lawns and, ly stops very regularly. At what stage sportfields. It can produce seed in à exactly the sexual cell aborts and is sexual way (amphimictic), and/or in an •replaced,,,b y the developing aposporous asexual way (apomictic) (Winkler 1905). sac is 'unknown (Bashaw, 1980). The same Plant breeders utilize this phenomenon: holds for the causes of this abortion the sexual way is used when creating varia­ and replacement (Grazi et al., 1961; Ny- bility through intra-specific hybridi­ gren, 1967;Va n Dijk, 1971). While studying zation and then only those F plants which these problems it became apparent that deviate phenotypically from the female it is difficult to distinguish the sexual parent are selected for the assessment cell from the aposporous one, especially of turf performance and seed production; in the more advanced stages of the gameto- the asexual way is used when maintaining genesis (Bashaw, 1969, 1980). varieties in an uniform and stable way. Up till now in Poa pratensis the degree Materialan dmethod s of apomixis is determined afterwards in field experiments by counting the deviating Panicles were taken from plants of Poa plants,whic h isa time consumingprocedure . pratensis L. varieties Parade and Enwarto, Therefore plant breeders have asked for as representatives with a high degree a method to determine the reproductive of apomixis,an d of amor e sexualexperimen ­ character. a s soon as possible, preferably tal variety. The plants had not yet flow­ with the help of the very first flower. ered. The samples were preserved informal ­ This •reproductive character is determined dehyde acetic acid aethanol (FAA). 8 to by the way in which embryo sac development 10 spikelets were taken from a panicle: proceeds. So what one should look for the 4 or 5 uppermost spikelets of the are the crucial moments inthi sdevelopment . panicle spindle and 4 or 5 spikelets of According to the literature in ovules the lowest positioned side spindle (Fig. of apomictic Poa pratensis varieties one 1).

160 Results

Sexualembry o sacdevelopmen t

Megaspore mother cells (mmc) were found in young hemitropous ovules of approximate­ ly 0,16 mm width (Fig. 3a).Th e megaspore mother cell stretches towards the chalaza. There is one layer of nucellus cells be­ tween the megaspore mother cell and the micropyle. The mmc nucleus is notably big, with a large nucleolus. The nucleus undergoes a meiotic division sagittaly (Fig. 3b). The division of the chalazal dyad cell is completed sooner than that of the micropylar one (Fig. 3c). The four developed megaspores are cylindrical to round (Fig. 3d).Th e size of each of them is roughly the same as that of the young megaspore mother cell. Their contours are clearly visible, presumably because of callose deposits. The chalazal megaspore

Fig. 1. Panicle of Poa pratensis-variety Parade. Arrows point at used spikelets.

A certain number of dissected ovaries was transferred to a clearing fluid (Herr, 1971). After clearing, the ovary tissue surrounding the ovule was removed and the ovules (5-15) were placed in clearing fluid on a slide (Fig. 2).The y were exam­ ined with a Nomarski light microscope.

••• • ••• • ••• •

Fig. 2. Slide with one coverglass on each side of objects. Third coverglass on top ofobject san dothe rglasses . Icallose - deposits Another number of ovaries was processed II'ca p for embedding in plastic: the varieties IIIantipoda l Parade (cleared in Herr fluid) and Enwarto IVpola rnuc l (fixed in FAA) were treated for 3x24 hrs V eggcel l in 100% aethanol and 3 hrs in 1% OsO VI synergid in aethanol. The variety Enwarto (FAA) Fig. 3. Development of sexual embryo sac 3.5 hrs in 1% OsO in phosphate buffer. and surrounding nucellus tissue of Poa The fixed material was then washed in pratensis varieties Parade and Enwarto. ethanol or buffer, dehydrated in graded Vacuole has been shaded, nucellus layers 'ethanol' series and embedded in Epon. have been dotted. The ovaries surrounding Sections (4 jjm) were placed consecutively these ovules have to be pictured with on a slide and examined with a phase con­ their styles upwards while their points trastmicroscope . ofattachmen t area tth ebottom . 161 grows, whereas the others degenerate (Figs. stage the antipodals disappear or become 3e, f).Conspicuou s then becomes the 'cap' extra large having nuclei with numerous between the megaspores and the one layered very dark nucleoli. This antipodal complex nucellus near the micropyle (Figs. 3e, was especially found in the more sexual f, g, h). The functional megaspore can experimental variety of Poa pratensis. become twice as big while growing in the It was remarkable that in this variety, direction of the micropyle. The nucleus in comparison with the other two varieties, contains one or two nucleoli and issituat ­ more embryo sacs were found in a late ed in the middle of the cell. A first stage. The central cell with two polar cytoplasmic vacuole becomes visible. The nuclei occupies two thirds of the embryo nucleus undergoes a mitotic division sagit- sac. Fused polar nuclei have not been taly. The two nucleated cell is pear shaped found. The egg apparatus consists of an and grows considerably (up to 6x itsorigi ­ egg cell and two synergids. The egg cell nal size) (Figs. 3g, h, i). The vacuole is bell-shaped and contains a vacuole. enlarges as well. Its position is in be- The nucleus is about as large as a polar tween the two nuclei. A smaller vacuole nucleus and at an early stage it issituat ­ is visible at the chalazal end. Through ed at the micropylar side, at maturity its enlargement, the cell gets closer in the centre of the cell. The two syner­ to the micropyle (Fig. 3i). The two nuclei gids are oblong pyramidal. Their nuclei divide: the chalazal one sagitally, at are in the centre. During the embryo sac right angles to the micropylar nucleus development the ovule changes from a hori­ (Fig. 3j). The four nucleated cell is zontal to a vertical position in the ovary now separated from the micropyle by only (Fig.3) . one nucellar cell layer (Figs. 3k, 4). Aposporous embryo sacdevelopmen t

•Jhe aposporous initial cell is very characteristic (Fig. 5). Initially its shape is round, in a later stage oblong. Some small vacuoles are visible.Th einitia ­ tion of this aposporous cell takes place in the nucellus near the chalazal part of the developing sexual embryo sac cell. In the studied material ovules have been found with more than one aposporous initial cell. Sometimes the initial cell was found growing into the sexual cell. The apospo­ rous embryo sac development is similar to the sexual embryo sac development des­ cribed above. Only the eight nucleated aposporous cell has not been found. From the^w o nucleated stage on the cell grows towards the micropyle through nucellus tissue, passing the developing sexual Fig. 4.A four nucleated cell ofPo apraten ­ cell. In one occassion the cell wall of sisvariet y Parade (Herr clearing). the aposporous embryo sac was observed to have grown into that of the sexual The nuclei are spherical with one, two embryo sac, in between the polar nuclei or three nucleoli. The vacuoles have en­ and the egg-apparatus. In a later stage larged as well. When dividing for the the aposporous embryo sac reaches the third time mitotically, the cell grows micropylar opening. Moving towards the broadwise. The direction of the division micropylar opening it sometimes grows of the two outer nuclei is at right angles through one or two layers of the inner to the line micropyle chalaza. At the integument. same time the direction of the division The aposporous and the sexual cell have of one of the nuclei is at right angles been found next to each other in the same to the direction of the division of the and/or different stages of development. other. The two inner nuclei divide sagital­ Data on these observations are given in ly (Fig. 31). The vacuoles in the eight Fig.6 . nucleated cell resemble the vacuoles in preceding stages (Fig. 3m). Eventually Discussion the cell differentiates into an eight nucleated, seven celled embryo sac (Fig. According to Rütishauser (1969) the 3n). There are three antipodals at the sexual embryo sac development of Poapraten ­ chalazal side. In a later stage their sis follows the Polygonum type, the apospo­ cytoplasm is darker. They are V-shaped, rous embryo sac development follows the with their nuclei in the bend of the V. Hieracium type. If one has to decide wheth­ They do not have vacuoles. In a later er one deals with a sexual or an aposporous

162 T3 JD rO -o aj T3 a) 0) E •U e- . c O o ,Q cell eu U P 'o 3 o ^ e megaspores

one-nucleated sexualcel l @ ê two-nucleated sexualcel l © Ô four-nucleated sexualcel l eight-nucleated sexualcel l differentiated sexualembry osa c differentiated sexualembry osa c withantipodal - complex Fig. 6. Numbers of observed sexual and aposporous embryo sac development stages in Poa pratensis. The aposporous cells areshaded .

development, three characteristic stages oneha st okee p inmind : In the one- and two-nucleated stage: the aposporous cell contains less cytoplasm and more vacuolar volume than the sexual cell; in the sexual development one can expect to find degeneration rests of the threemicropyla rmegaspores . In the two-, four- and eight-nucleated stage: while stretching towards the micro- pyle the aposporous cell bends round the sexual cell towards the micropyle and pushes nucellus and integument tissue aside. In the mature embryo sac stage: in the aposporous embryo sac one can expect to find an embryo (without fertilization having taken place); in the sexual embryo sac one can expect to find an antipodal complex, which covers the largest part of the embryo sac and has pushed out nucel­ lustissue . Observations of various authors confirm the characteristic distinctions given above: publications by Tinney (1940), Grazi et al. (1961) and Nygren (1967). Nygren shows drawings of the aposporous initial cell which correspond to the pres­ ent observations, and drawings by Nielsen (1945) show degeneration rests of mega­ spores. From Fig. 6 it can be seen that initiation of the aposporous cell was Pig. 5. Embryo sac of Poa pratensis-variety found to occur next to a developing sexual Enwarto with synergids (s), egg cell (e), one-nucleated or older cell, but it appears polar nuclei (p), antipodal complex (ac) that up to and including the four nucleated and aposporous initial cell (ai) (section­ stage most of the aposporous initial cells ed). have come into existence. Next to an embryo

163 sac with an antipodal complex there were Hereditas 47:489-541 . only few aposporous initial cells found. Herr, J.M. Jr., 1971.A new clearing-squash It is possible that the antipodal complex technique for the study of ovuledevelop ­ restricts initiation and development of ment in angiosperms. Am. J. Bot. 58: the aposporous cells, since in the experi­ 785-790. mental variety relatively more sexuality Nielsen, E.L., 1945. Cytology and breeding isfoun d inth eF plants. behavior of selected plants of Poa From Fig. 6 xt can also be seen that pratensis. Bot. Gaz. 106: 357-382. above all aposporous cells develop into Nygren, A., 1967. Apomixis in the angio­ embryo sacs when had been initiated in sperms. Handb. Pflanzenphysiol. 18: an early stage of development. When the 551-596. sexual cell has reached the eight nucleated Rütishauser, A., 1969. Embryologie und stage of development it fills up most Fortpflanzungsbiologie der Angiospermen. of the space in the ovule. This leaves Eine Einführung. Springer-Verlag, Wien. little room for aposporous embryo sac Tinney, F.W., 1940. Cytology of parthenoge­ development. The latter only has a chance nesis in Poa pratensis. J. of Agric. to develop next to the sexual cell when Res. 60:351-360 . this sexual one is not yet fully grown. Winkler, H., 1908. Ueber Parthenogenesis Occasionally the aposporous cell might und Apogamie in Pflanzenreiche. Verlag even take the place of the sexual one. vonGusta vFischer ,Jena .

The results of this study indicate that it ispossibl e topredic t thereproduc ­ tive character of Poa pratensis plants by observing their very first flowers when the embryo sac development has reached the four nucleated stage or beforehand. If there is an aposporous cell at these stages, the plants will, in all probabili­ ty, be aposporous. If there is not an aposporous cell, the plants will, in all probability,reproduc esexual .

Acknowledgements

It is a pleasure to thank Prof.dr. J.L. van Went for critical reading the manuscript, Mrs J. Cobben-Molenaar and Mrs Drs. J.S.G. Brefeld for their assist­ ancesi npreparin g themanuscript . *•

References

Akerberg, E, 1942. Cytogenetic studies in Poa pratensis and its hybrid with Poaalpina .Heredita s 28:1-126 . Bashaw, E.C., 1969. Unpublished Annual Report, Grass and Turf Investigations, U.S. Dept. Agric. ARS, College Station, Texas. Bashaw, E.C., 1980. Apomixis and its ap­ plication in crop improvement. In: W.R. Fehr & H.H. Hadley (Eds.): Hybridi­ zation of Crop Plants. Am. Soc. Agron. and Crop Sei. Soc. Am., Madison,Wiscon ­ sin,p .45-63 . Dijk, G.E. van, 1971. Breeding of apomictic grasses. 1st international course on plant breeding. Int. Agric. Centre, Wageningen. Engelbert, V, 1941. The development of twin embryo sacs, embryos and endosperm in Poa artica R Br.. Can. J. Res. 19:135-144 . Grazi, F., M. Umaerus & E. Xkerberg, 1961. Observations on the mode of reproduction and the embryology of Poa pratensis.

164 POLLINATION ANDFERTILIZATIO N INPICKLIN GAN D SLICINGCUCUMBER S 1 2 A.P.M.de nNlj s ,P .Milota y Inst.Hort .Plan t Breeding,Wageningen ,th eNetherland s 2 VegetableCrop sResearc h Inst.,Keckskemet , Hungary

the1- 5longes tpolle ntube swa smeasure d as Summary wella stha t ofth emai nfron to f Pollination ofparthenocarpi cDutc hslicin g pollentube s (atleas t10-2 0tubes) . cucumbers oftenyield s lownumbe ro fseeds , Ovulean d seed development ofparthenocar ­ becauseonl y theproxima lsegmen t ofth efrui t pican d pollinated fruitso fcv sG 6 ,Mara m containsplum pseeds .T oclarif y thereaso n andNane twer eexamine d atanthesi san d after forthi s inadequate seed set,i nviv opolle n 4,8an d 12days . tubegrowt h and ovulean dsee d development Fruitswer edivide d infiv esegment sa t werestudie d insi x cucumber cvswhic hdiffe r maturity and ovulesan dseed swer ecounte d forfrui t lengthan d parthenocarpy.Polle n persegment . parentsha d nosignifican t effecto npolle n Resultsan ddiscussio n tubegrowt h orsee d set.Polle n tubesneve r reached thebasa lfrui tsegment so flon g Atpollinatio ntim ether ewer en osiz ediffer - fruited types,becaus eth efrui telongatio n rencesbetwee n ovulesthroughou tth elengt h proceeded morerapidl y thanpolle ntub e ofth eovar y inal lcvs .Enlargemen t ofovu ­ growth.Therefore ,rathe rtha nparthenocarpy , lesbecam eeviden t after k days.Fo rG 6 an d thelon govar yan d rapid elongation offruit s Vestervang thisoccurre d inth eblosso men d isresponsibl efo rth e lowsee d seti nlon g ofth efrui tonly . fruited cucumbertypes . Inviv opolle ngerminatio n and tubegrowt h wasno taffecte d by pollenparent ,ther e Introduction weren odifference sbetwee nsel fan dcros s pollinations.A t k daysafte r pollination Pollination ofteninduce sa typica lmal ­ small,bu tsignifican t differences inpol ­ formation ofth efrui t inDutc hslicin gcu ­ lentub egrowt h existed betweenmaterna l cumbers:th eblosso men d ofth efrui ten ­ parents,c vMara m ovariesha d the longest largeswhil eth eres t ofth efrui tremain s pollentubes . slender.Thi sthic kfrui tsegmen t contains theseeds .I ti scalle d 'seedhead' . Fruit lengtha tA -day safte rpollinatio n Pollinated fruitso fAmerica n salad cucum­ differed greatly.Th eovarie so fG 6 wer e bersan d pickling cucumbers grow outevenl y already longa tpollinatio ntime ,an dtri ­ and contain seedsthroughou t thefruit .See d pled their length in h days.Th eshor tova ­ yield perfrui t ofsuc htype s ismuc hhighe r rieso fth epickl ean dbeth-alph acv selon ­ thanca nb eobtaine d with Dutchslicers . gatedmor eslowly . Whenth ereaso n forth elo wsee d yieldo f Fruitso fth e longfruite d cvscontaine d Dutchslicin g types isknown ,i tma ybecom e very many ovulesi nthei rfruits ,bu tonl y possiblet oimprov eth eefficienc y ofsee d veryfe wha dgrow nou tint oplum pseeds . production. Fruitso fth eshor tfruite d cvscontaine d Dutch slicingtyp ecv sar echaracterize d lessovules ,bu thig hpercentage s ofseeds . bygeneti cparthenocarp y and largefrui t Inth e longfruite d cvsonl y thefirs ttw o length.Parthenocarp y generally reducessee d fruit segmentscontaine d seeds -th e 'seed yieldspe rpollination ,bu t iti sno twel l head'. knownhow .I nviv ostudie so fth egrowt ho f pollentube sthroug hth estyl ean d ovaryan d Seed setwa sno tinfluence d bypolle nparen t> ofth efertilizatio n processma yhel pt o therewer en odifference sbetwee n selfan d clarify theobserve d differencesi nsee dset . crosspollinations .See dse twa sstrongl yin ­ fluenced bymaterna lparent . Experiments Conclusion Sixdifferen t cucumber cvswer eused :G 6 'Seed-heads'an dlo wsee dyiel d ofDutc h (slicingtype ,parthenocarpic) ;Vestervan g slicingcvs ;ar eno tcause db ygeneti cparthe ­ (slicing type,no n parth.);Mara mF l (beth nocarpy,bu tmos t likelyb ythei r largeovar y alphatype ,parth.) ;Bet hAlph a(no nparth.) ; sizean d very fast initialfrui telongation . NanetF l (pickling type,parth. )an dUnicurb a Pollentube so f longfruite d typeshav esi ­ El (pickling type,no n parth.).Al lwer esel f milar growth capacity asthos e ofshor tfrui ­ pollinated and crosspollinate d withtw otes ­ tedtypes .The yar eunabl et oreac hth eovu ­ tercv s (Nanetan d Vestervang).Glasshous e lespas tth eproxima l onethir d ofth eovary . temperature ranged from 20°C (night)t o32° C Theovule s inth eres to fth e ovary-althoug h (day).Fou rday safte rpollinatio n sixfruit s they seemno tt ob edifferent -remai nunused . percros swer e removed,fixed ,macerate d and Selectionfo rfaste ran d longerpolle ntub e stained forUV-microscopi c study ofpolle n growthcoul d improveth efertilizatio nsuc ­ tubegrowt h and fertilization.Th e lengtho f cessan d increasesee dyields . 165 GAMETE COMPETITION INOI LSEE DRAPE : EFFECTO FPOLLE N S- GALACTOSIDAS E DEFICIENCYO N FERTILIZATION

M.B.Singh ,R.B .Kno x and E.G.William s

Plant Cell Biology Research Centre,Schoo l ofBotany ,Universit y ofMelbourne ,Parkville , Victoria 3052,Australi a

Summary Table 1. Segregation of progeny ofGal/ga l crossed reciprocally to wild type Gal/Gal, Wehav e recently reported thedetectio n of (expected ratio:1 Gal/Ga l :1 Gal/gal ) a new haplophase in crop plants thataffect sth eexpressio n ofth eenzym e CROSS OFFSPRING /5-galactosidase in pollen of oilseed rape, Gal/Gal Gal/gal ChiSquar e Brassica campestris. Plants heterozygous df=1 for the enzyme deficiency, Gal/gal shed pollen grains, 50% of which are enzyme Gal/galo x deficient. We have compared male gamete Gal/Gal à transmission abilities ofnorma l (Gal)an d galactosidase deficient (gal)polle nan d ß - Observed 14 0.04 N.S. report that mutant pollen shows reduced 13 Expected 13.5 competitive ability infertilization . 13.5 Ga^/Galo x Keywords: oil seed rape, Brassica GsuL/galo » campestris, ß -galactosidase, fertilization. Observed 57 35 5.30 p0.0 1 Expected 46 46 Introduction

In higher plants, there are very few The results show that there is normal molecular geneticmarker s which can beuse d transmission of gal through the egg cell. to study the physiological and biochemical However, gal pollen grains show reduced basis of male gametophyte competition. competitive ability in fertilization. This Until now only three segregating loci were isth e first example in angiosperms ofmal e known, including waxy (wx) , alcohol gamete competition related to enzyme dehydrogenase (Adh)i nmaiz e pollen,an dno w deficiency. Experiments are in progresst o /)-galactosidase (gal) in oil seed rape t determinewhethe r the selective advantaget o pollen. Adh deficiency had no delaterious GaJ»polle n is associated with the initial effect (Schwartz 1969), while wx pollen events of pollen adhesion, hydration and showed only a slight reduction in male germination or later stages of pollen tube gamete transmission (Sprague 1933). We penetration and growth through thepistil . report here the results of experiments on the effect of pollen ß -galactosidase deficiency onfertilization . References Materials and Methods Schwartz,D . 1969 Anexampl eo fgen e fixation resulting from selective Pollen quality was determined by the advantage insuboptima lconditions . Am. fluorochromatic reaction (FCR)test . The Nat. 103:479-481 . ß-galactosidas e activity in pollen was Singh,M.B .an dKnox ,R.B . 1984 detected cytochemically byusin g 5-bromo- Quantitative cytochemisty of/3 - 4-chloro-3-indoxyl- /i -galactoside as galactosidase innorma l (Gal)an d substrate (Singhan d Knox 1984). deficient (gal)polle n ofBrassic a campestris: applicationo fth e Resultsan d Discussion indigogenic method. Histochem.J . (inpress) . WhenGal/Ga l isuse d asfemal ean dGal/ga l asmale ,th e progeny should be 1Gal/Ga l :1 Sprague,G.F . 1933 Pollentub e Gal/gal. Thelatte r showspolle n establishment and thedeficienc y of segregation for the enzyme deficiency and waxy seeds incertai nmaiz ecrosses . thus can easily be detected by the pollen Proc.Natl .Acac .Sei . 19:838-841 . staining test. Such a plant can only be obtained if gal pollen fertilized the egg cell. This criterion was used todetermin e percentage of progeny originating from enzymedeficien t galpolle n (Table1) . m CÏTOCHEMICALINVESTIGATIO N OFLILIU MREGAL EEMBRYOGENESI SAFTE R POLLINATIONWIT Hy-IRRADIATE D POLLEN

I.D. Georgieva

Instituteo fGenetics ,Sofi a 1113,Bulgari a

Summary

SevenD Hinvolved ,i ndifferen tmetaboli c anddy eabou t2 0d afte rpollination . pathwayswer einvestigate d cytochemicallyi n Inou rpreviou swor k (Georgieva,1983 )wa s theembryosa c ofL.regal eafte r pollination foundtha tdurin gth e fertilization andearl y withy-irradiate d pollen.Abnormalitie s in embryogenesis theenzym s ofcitrat e cycle theembryogenesi s andmetabolis mwer e observed. (IDH,MDH),o f PPP(G6PDH,6PGDH), ofalcoholi c The activities of IDH,MDH,G6PDH,6PGDH and LDH fermentation (ADH),o fglycolysi s (LDH)a s inth eembryosa cwer eno t changed ascompare d wella sGD Hbecam e activated inth e embryosac with controls,bu tAD H andGD Hwer e completely ofL.regale .I nth epresen t study itwa s shown inactivated. that althoughth e embryogenesisha dbee n des­ Keywords:y-irradiate d pollen,dehydrogenases , troyed afterpollinatio nwit h y-irradiated cytochemistry,abnorma lembryogenesis . pollenth e degradation ofcarbohydrate svi a thecitrat ecycl ean dPP Pa swel la sth egly ­ Introduction colysiswer eno taffected .Th erat eo fanaero ­ bicbreakdow no fcarbohydrate sb yAD Hhoweve r Marked changes inth elil y embryogenyan d drasticallydecreasedwit hth e increasingy-ra y morphology ofth e seedstak eplac e afterpol ­ dose.I nth esam etim eth e connectionbetwee n linationwit hy -an dX-irradiate d pollen themetabolis m ofcarbohydrate s andamin oacid s (Vassileva-Dryanovska,1°ó6;Priee ,1957 ; mediatedb yth eke y enzymeGD Hi sdisrupte d Poolee t al.,1978).A sth ephysiologica l because ofth eGD Hinactivation .A sth eimpor ­ peculiarities andth ebehaviou r ofth ecell s tantmetaboli c pathways i.e. citrate cycle,PP P inth eembryosa c duringearl y embryogenesis andglycolisi swer e notaffecte d inth eembryo ­ canb e distinguished only cytochemically, sac, theearlies t stages ofembryogenesi swer e weundertoo k thecytochemica linvestigatio n characterizedb y enchanced growth.I tca nb e of7 DH ,involve d indifferen t pathwayst o admittedtha tth ebreak-u po fsom emetaboli c revealsom emetaboli c disturbances following chains (e.g.thos e inwhic hAD H andGD Har e pollinationwit h y-irradiated pollen. involved)probabl y completely destroyth e metabolismo fth e embryosac andprovok eth e Materialan dmethod s seedfailur e

Flowers fromLiliu mregal eW wer epollinate d References withy-irradiate dpolle n (10,25an d5 0krad) . Freshpistil s 1-30d afterpollinatio nwer e Georgieva,I.D.,1983.Histochemica llocalizatio n used.Th e cytochemical localization ofD H ofsom edehydrogenase s inth epistil s of (IDH,MDH,G6PDH,6PGDH,LDH,ADH and GDH) was Liliumregal edurin gth eembryogenesis . carriedou tb yth emetho do ftetrazoliumreduc - Fertilization ansembryogenesi s inovulate d tases (Lojdae t al.,1979). plants.VEDA ,Bratislav a(Czechoslovakia) , p.273-277. Results anddiscussio n Lojda,Z.,R.Goffrau& T.Schiebler,1979 . Enzymehistochemistry .Springer ,Berlin . Itwa s establishedtha tth e 3dose sunde r Price,P.,1957.Reproductio n andgrowt hi n studystimulate d fertilization and celldi ­ various lily species followingX -an dy-ir - vision inth e embryosacan daffecte dth e radiationo fpolle n andX-irradiatio n of appearance ofsimila rcytoembryologica lab ­ seeds.Diss.Abstr . 17(8): 165O-I65L normalities,whic hwer ebette r expresseda t Poole,J.E.P.,D.T.Morga n &R.D.Rappleye,1978 . higher doses.Th epredominat e oneswa sth e Embryologicalan dflora leffect s attributable formationo fembry o asa nirregula rmas so f todeficiencie s following crosseswit h cellsan datypica l freenuclea r endosperm X-rayedpolle no fLiliu mregal eW.Cytologi a with smallnuclei . U3:689-69!* . Thiskin d ofembryosa c almost didn't grow

Abbreviations: DH -dehydrogenases ,IDH - isocitrate dehydrogenase,MDH -malat edehydrogenase , G6PDH-glueose-6-phosphat e dehydrogenase,6PGDH -6-phosphogluconat edehydrogenase ,LDH -lactat e dehydrogenase,ADH -alcoho ldehydrogenase ,GDH -glutamat e dehydrogenase,PPP -pentos e phosphate pathway

167 SOME STRUCTURAL FEATURES OF THE EMBRYO/ENDOSPERM INTERACTION IN ANGIOSPERMS

O.Erdelskâ Institute of Experimental Biology and Ecology CBES, SAS,Dübravsk ä 14, 814 34 Bratislava, Czechoslovakia

Summary tase the reaction withoC - naphtylpho- sphate and FAST RED TRwa s used and By the underlying examination of to prove non-specific esterase the the early embryogenesis of selected reaction with tO - naphtylacetate and species of angiosperms, threecorre ­ the FAST BLUE Bwa s used. lation types were marked off inemb ­ On the individual sampling days the ryo and endosperm development.The y number of cells /nuclei/o f both en­ areboun d to a definite typeo fendo ­ dosperm and embryo was counted and sperm,t o aduratio n of endospermal the structural changes traced that function, to the dynamics of embryo reffered to the spatial and functio­ growth and to the onset of thediges ­ nal interaction of embryo andendos ­ tive or digestion stimulating func­ perm on the level of optical micros­ tion of the embryo. copy. Keywords: embryo, endosperm, correla­ tion types. Results Introduction The course of the early embryogene­ sis,in selected species of angiosperms Correlation in the development of ha%allowe d to characterize 3type s both embryo and endosperm is one of of correlation in embryo and endos­ the basic supposition of normal seed perm development. development.Th e type of correlation Type Ii s characterized by amarke d ischaracteristi c of the individual advance inendosper m development in taxa of angiosperms. In various spe­ the first phases of embryogenesis cies of angiosperms both embryo and /Fig. 1/. endosperm are developing with diffe­ rent absolute and relative speed. Our work aimed at ascertaining thedi ­ fferences in the relative speed of their development that characterize the type of correlation. The type of correlation appears to point atpossir - bledifference s in the function of the endosperm as well as at differences in the growthan d regulation activity of the embryo in the first phases of embryogenesis.

Material and methods

The early embryogenesis was studied of selected species of angiosperms / somniferum, Nicotiana taba- cum, Jasione montana, Linumusitatis - simum, Capsella bursa pastoris,Rap - hanus sativus,Pisu m sativum, Phaseo- lusvulgaris/ .Youn g seeds of these species were sampled from the field in 1- 2day s intervals, starting with the day of pollination up toth e 7th- -12th day of seed development.The y were embedded in paraffin, sectioned Fig.l Globular embryo of Papaver som­ and stained with haematoxiline accor­ niferum in the advanced endosperm. ding to Heidenhain or by the PASrea ­ ction.A part of the seedswa s fixed In this period there gradually fall according to Baker, embedded inpara ­ to one embryo cell from more than ten ffin and sections were analysed by cells /e.g.wit h Ni'cotiana tabacum, histochemical methods to prove hydro- Jasione montana/t o several hundreds lytic enzymes.T o prove acid phospha­ of cells / e.g. with Papaver somnife- rum//Tabl e 1/.Th e 1st type ofcorre ­ stored into the cotyledons.A small lation is usually bound to the cellu­ portion of the endosperm, however, lar endosperm or to the nuclear en­ may be preserved until seed maturity. dosperm that cellularizes relatively early, and to the albuminous type of seed.A t the time when the endosperm reaches the final number of cells the '• • f embryo is in the globular phase or à" ' passes from theglobula rt o transitio­ nal or to heartshaped phase of its development.A t the same time itpa ­ sses from the suspensoral to the sur­ facenutritio n inwhic h it obtains supply material from the disintegra­ ting cells of the endosperm. The em­ bryo acquires,a t a relatively early phase of itsdevelopment , a digestive or digestion stimulating function.

Table 1 Embryo and endosperm cells number /in some selected days/

embryo endosperm Papaver/I / 16 4 034 7 days Papaver 169 16 722x 12 days Fig.2 The embryo and endosperm ofLi ­ num usitatissimum in the parallel de­ Nicotiana/I / 36 558 velopment. 9 days Nicotiana 304 2 522x Correlation type III occurs with 11 days species exhibiting markedly exalbumi- nous seed.Afte r an initial short- Linum /II/ 5 059 1504 5 -term rise in the number of endosper­ 7 days mal nuclei, it is the embryo that Pisum/III / 24 533 19 357 acquires dominance in the development 7 days /Fig.3/. x Data represent roughly the final or nearly final cell number.

The development of the embryo and endosperm in type II is the parallel one.I t usee,t o be bound to a nuclear type of endosperm /Linum usitatissi- mum, Capsella bursa pastoris,Rapha - nus sativus/. In this type ofcorre ­ lation the number of endosperm cells falling to 1embry o cell stabilizes soon after the starto f embryogenesis and the embryo proliferates and diffe­ rentiates parallel with the prolifera­ tion of the endosperm. /Fig.2/ Embryo and endosperm jointly fill the inner space of the embryo sac.Thei r spatial interaction,manifeste d also by the désintégrationo f endospermal cells round theembryo , sets in only at amor e advanced stage of embryoge­ nesis, at a time when the embryo is already in its late heartshaped or tor­ pedo phase of development. In this ty­ peo f correlation most of the supply Fig.3Th e advance of embryo development material needed for germination is in the seed of Pisum sativum

!69 The number of embryo cells soonexce ­ and others/,ha s allowed to characte­ eds the number of endosperm cellsma ­ rize three types of embryo and endos­ ny times over.Th e endosperm develop­ perm correlation.Th e facts referred ment is very limited and all supply to in thepape r show that embryos of substances needed for germination are various species pass over from the stored into the cotyledons.Th e wea­ suspensory to the endospermal nutri­ kly developed endosperm layer désin­ tion invariou s phases of their deve­ tégrâtes as soon as it gets into spa­ lopment. Some already in the globular tial contactwit h the developing em­ or early heartshaped phase /correla­ bryo, thus in themicropyla r area of tion type I/,otherone s in the late the embryo sac soonner and in thecha - heartshaped phase or later /correla­ lazal area later.Thi s type ofcorre ­ tion types IIan d III/.No t only the lation isknown , for example, in the character and duration of thenutriti ­ family Viciaceae, and bound to the ve function of endosperm but also the nuclear endosperm. character of the growth and digestive The portion of endosperm to embryo or digestion stimulating activity of cells number during early embryogene- the embryo are connected with this. sis in the species typical for all The question of the character of em­ correlation types is indicated on the bryo activity /digestive or digestion Fig.4 . stimulating/ during embryogenesis re­ mains open /Jones 1974,Olso n 1981 andothers/ .

16 References /"-I. Davis, G.L., 1966.Systemati c embryo­ 14 logyo f the angiosperms. John Wiley / i and Sons, Inc.,Ne w York. Jones, R.L., 1974.Th e structure of 12 / the lettuce endosperm. Planta 121: 133 -146 . §• 10- Olson, R., 1981.Embry o and endosperm -O cr S development in ovules of Papaver nu- •Nicotian a dicaule after in vitro placental pollination, Canad.J.Bot. 59:1738 - K3» -1948. Poddubnaja-Arnoïdi,V.A. , 1982.Cyto - embryological characteristics of angiosperm families.Nauka ,Moskva .

4-

*-—«-- .&. 2 Pisum 1+ oy I r- 0 1 2 3 4 5 6 7 8 9 10 11 « tJ 14 fdaysj *•

In type II and III,embryo s veryof ­ ten contain during embryo- genesis.

Discussion Theknowledg e and completion ofre ­ sults obtained by examining the early embryogenesis of selected species of angiospermous plants by literary data on the embryogenesis of other species /Davis 1966,Poddubnaja-Arnold i 1982

170 ULTRASTRUCTURAL ANALYSIS OF EMBRYO-ENDOSPERM INTERACTIONS IN DEVELOPING MAIZE SEEDS (ZEA MAYS L.)

J.H.N. Schel, A.A.M, van Lammeren & H. Kieft

Department of Plant Cytology and Morphology, Wageningen, The Netherlands

Summary Results and conclusion

The fine structure of developing caryopses During the first week of seed development from maize (Zea mays L.) was investigated. the young embryo is surrounded by a region Samples were taken from 4 to 11 days of small, plasma-rich cells (Fig. 1, arrows). after pollination. It is concluded that a Electron microscopy of this "modified endo­ strong interaction between embryo and sperm" bordering the embryo suspensor endosperm exists and that nutrient supply at the end of the first week showed a high of the embryo occurs by various pathways. level of Golgi and ER activity, while mito­ The cytological evidence leading to this chondria were located near the plasma view is presented. •membrane adjacent to the embryo (Fig. 2). After the first week the large, highly vacuol­ Introduction ated endosperm cells near embryo axis and scutelium degenerate (Fig. 3). There is a lack of studies dealing with It is concluded that in the first week embryo development in relation to the neigh­ food supply of the immature embryo occurs bouring tissues, as was pointed out recently mainly by the modified endosperm bordering by Smart & O'Brien (1983). Although embryo the suspensor. Amino acids and proteins formation in maize has been studied extens­ might be main constituents. After that ively, an integrated study examining func­ period, also food uptake by the embryo tional embryo-endosperm relationships at axis and the scutelium takes place. Lipids an ultrastructural level was not yet carried and carbohydrates (cell wall remnants) out. We therefore studied the early develop­ seem to be the main nutrients present in mental stages of maize caryopses by electron that region. microscopy, putting emphasis on cytological details indicating important functions of References the endosperm surrounding the embryo in relation to nutrient synthesis and/or trans­ Smart, M.G. & T.P. O'Brien, 1983. The port. development of wheat embryo in relation It was thought that - while in vitro culture to the neighbouring tissues. Protoplasma methods using immature embryos of maize 114: 1-13. are becoming increasingly important (see Vasil, V., Vasil, I.K. & C. Lu, 1984. Somatic Vasil et al., 1984) - more knowledge of embryogenesis in long-term cultures of the in vivo nutrient supply of these immature Zea mays L. (Gramineae). Am. 3. Bot. embryos is strongly required. 71: 158-161-

Fig. 1. Light micrograph; about 4 days after pollination. The modified endosperm (arrows) borders the embryo. Fig. 2. Electron micrograph of the modified endosperm at 7 d.a.p. Fig. 3. Degenerated endosperm near the scutelium (11 d.a.p.). Note the presence of lipid droplets (arrows).

171 REPRODUCTIVE PHYSIOLOGY OF LIMNOPHYTOÎÏ OBÏUSIFOLIUM (L) Miq.-A HISTOCII-ïïlCAL AÎÏALYSIS CK. Shah

Botany department, School of Sciences, Gujarat University, Ahmedabad-9 The embryo sac fellows a bisporic , (ii) On such a spherical Scilla pattern. All the eight fema­ interior first axial and afterwards le nuclei have different diameters appendicular proeambial systems but identical extinction values for embark at the same time, (iii) Coty- DNA. The endosperm is helobial. Its ledonary initial divides rapidly in polyploid nuclei have multiples of a plane at right angles to the DNA G values. The DNA content per hypocotyledonary initial, one forms tapetl nucleus remains amplified the cotyledonary mound while the because of fusions but after meiosis other designs the embryo axis. Thus in microspore mother cells it decre­ one half of the embryo consists of ases dramatically. -SH proteins many large cells, less basiometric showed peak values during periods of as compared to the hypocotylar half meiosis and mitosis indicating their which has smaller cells but richly functional role. The female gamete pyroninophilic. The first histoche- showed a distinct staining for mical departure appears with the Feulgen reaction confined only to formation of the first indentation the nucleus and even during matura­ demarcating the germinal and abger- tion of the egg, DNA did not reveal min-al portions on the globular embryo. qualitative or quantitative changes. Thé intensity of ascorbic acid (AA), Acidic proteins (NHGP) detection by RNA and -SH proteins becomes more toluidine blue 0 or by fast green pronounced on hypocotylar face than FCP at acidic pH of 0.5 left firm the cotylar one. It initiates the unspeciflc binding with cell-walls • change of symmetry. A SEM, ontogenic and cytoplasm of ovular tissues. as well as histochemical profiles The cell çb remains unicellular and from the first dipression to mature ever enlarging while only ça gives embryo fabric manifest three diffe­ rise to the organs of the embryo. rent extensions of the single lateral The course of divisions thus depicts cotyledon viz. sheath, middle piece the Aponogetonad pattern. An intense and the haustorium. At the end the reducing condition prevails in the embryo has one lateral cotyleuon and hypertrophied çb. In fact ascorbic an axis bearing two growing poles- acid and -SH proteins ascend unifor-* radicle and plumule. Surprisingly in mly. The RNA and protein show vigo­ the,matur,e seed, various tissues of rous fluctuations, çb multiplies Llmnophyton yield extremely variable its genome 16 times resulting in a quantitative levels of metabolites. large polyploid 32C nucleus. However, Anther tapetal cells, meiocytes, the acidic nuclear proteins (NHCP) suspensor, (çb), endosperm, vegetative content increases multifold followed cell and male gametes in the polen by a sharp decline. The synthetic grain, antipodals, seed coat cells performance of the suspensor cell and perianth tissues show varying was at full swing in the quadrant ( 2n = 22, h-h, 52, 66) chromosome and octant stages of embryo develop­ set up in numbers on metaphase plates. ment but reached its peak value at Surprisingly in spite of the huge the globular embryo where cells differences in the volume and chroma­ determine their developmental pathway tin make up in these nuclei, the The various tiers of proembryo do not hydrolysis curves (6o°C 1 N HCl) for show differential distribution of the above diverse cell nuclei showed the metabolites indicating their even a similar plateau value as with the physiological state. At the end it vegetative apical . is the globular embryo which starts This if the virtual optimal period revealing a dispersed reaction of of hydrolysis. To arrest Feulgen metabolites followed by the differen­ hydrolysis at its peak, the prepara­ tiation of specific precursor cells. tions are dipped in cold 1N HCl for Three critical histogenic events 1 minute and then rinsed with disti­ appear in the interior of the globul­ lled water for 2 minutes. This step ar embryo: (i) Two loci or initials replaces 2-3 peaks and produces a of presumptive sites for the embryo­ flat curve. Meiocytes show two/three nal regions viz. hypocotyl and peak periods of Feulgen hydrolysis.

172 GKASS EMBKYO - A SEM AND HISTOCHEMICAL ANALYSIS C.K.SHAH

Botany department, School of Sciences, Gujarat University, Ahmedabad-9

Summar y both are unit extensions and form a single lateral cotyledon. This Differentiation of the grass results further in organised develop­ embryo proceeds by two primary inde­ ment of the embryo and the formation ntations on the face of the globular of tubeliKe coleoptiles-coleorhiza embryo and several ones on the which emerge from the central region surface of the scutellum. All embry­ of the cup-shaped scutellum. The onal cells maintain equal but high cotyledonary mound enlarges and metabolic potential till the embryo ridges and furrows appear an the assumes a radially symmetrical surface of the scutellum radiating form. Basically the embryonal form from the surface of tti e scutellar of the grasses is identical with node towards its periphery. In this the rest of the monocotyledons. Ouïs way the peripheral part of the is an attempt to integrate the scutellum becomes lobed, deeply histochemical turnovers which seem divided and nodular and forms 3 or to condition or control the unfold­ k outgrowths. Coleoptile is scutellar ing of the axis (hypocotyl) as well in origin. The origin of leaf like structures is not at all associated as the appendage (cotyledon) in the / grass embryo by localising differe­ with a shoot . Future presum­ nt metabolites or substances during ptive sites of these outgrowths are different stages of embryo develop­ traced to subepidermal initials of ment and correlate them with morpho­ the scutellum. Thus the grass embryo logical changes. has two apertures one is guarded by auricles and scales and the other by the coleoptile. The ridges of tissues Observations and conclusion are cotyledonary or scutellar. Auri­ cles and scales appear in ontogeny In an attempt to settle the wide and develop after the covering of gulf of the grass embryo, it heral­ plumule by the coleoptile and radicle ds the basic plan that it has an by the coleorhiza. The region of axis known as the hypocotyl. On its adnation between the scutellar node one terminal lies the shoot apex and the coleoptilar node is the on the other end the radicle. It mesocotyl. It is the fusion product bears a solitary senescing lateral of the hypocotyl and the scutellum. cotyledon. Basically the embryonal The attachment or the point of topography of the grasses is iden­ divergence between the two is an tical with the rest of monocotyle­ expanse running from 0.12 mm to 1 cm. dons but its single cotyledon is Coleoptile develops from abgerminal functionally divergent like its sector and makes its appearencs as a leaf. The compact scutellum shields circular ring before the shoot apex; the embryonal axis leaving only the it becomes a part of the cotyledon. ends of the coleoptile and coleorhi- Early appearance of coleoptile before za visible. The embryonal axis shoot apex formation helps to protect shows leaf primordia enclosed with­ embryonal shoot apex. Histochemical in the coleoptile and a primary tum overs and statistical analyses root surrounded by the coleorhiza. for RNA, total proteins and total In SEM, it has a boat or shoe-shap­ nucleic acids in maize reveal that ed scutellum wrapping the embryo the developing latency of all the axis leaving a broad mouth towards constituent cells of octant (m and the radicle end only. This open £) tiers is nearly the same and the plateau is guarded by an epiblast, fascimile of all the cell derivatives a pair of auricles and ventral of ça remain exactly alike till the scales. These appendages even ensh- globular embryo. The origin of the eath the inner coleoptile-coleor- organs cannot be assigned to specific hiza cylinder. Thus embryo axis of tiers in the young embryo. The extin­ the hypocotyl is protected by two ction values of five metabolites in concentric covers or veils-one of four different cells of 1 and 1' the coleoptile-coleorhiza and the tiers show the coefficient of other of the scutellum. Neverthless

173 variation of ?.00 for polysacchari­ plumule and radicle have higher des, 10.08 for -SH proteins, 6.38 concentrations of RNA, DNA and for RNA, 3-65 for histones and 5.39 histones as compared to that of the for DNA. Thus the cells have not cotyledonary regions indicating a been summoned for different behavi­ high metabolic turnover in these ours either at quadrant or octant growing apiees. While scutellum, stage. As the development proceeds eoleoptile, coleorhiza and mesocotyl the stain intensity (e. values) for possess higher content per cell/ DNAan d his tones in the derivatives nucleus of DNA, histon e and RNA. The of ça rises and reaches its peak, mesocotyl occupies an intermediary values. At the elongated and inden­ position between the cotyledon and ted embryo, the extinction values embryo axis. Extinction values for decline. The quantitative evidence DNA are higher in the scutellum of from the extinction values, content 2ea as well as Sorghum as compared per cell and concentration per unit to the rest of the regions probably area of DNA and histones, profile indicating higher ploidy level in of the procambial strands, origin this storage sink of the cotyledon. of the organelles of the cotyledon E. values for histones are also point to a positive correlation higher in this region in both Zea (r = + 0.68). DNA/histone ratio and Sorghum suggesting the senescing remains fairly constant. Its steep fate of the scutellum. The concen­ rise runs parallel and an intense trations of DNA in the plumule and reducing condition prevails in the radicle are 2-8 times higher than suspensor cells till the embryo is the cotyledonary region in 2ea and globular. In fact ascorbic acid and 1 to 3 times higher in Sorghum. -SH proteins are maximum at this However content per cell shows" a stage. The globular stage does not reverse trend the cotyledonary regi­ inherit either any basic cytoplasmic ons portraying 9-10 times higher variance or ultra-structural diver­ values in Zea and 3-5 times more in sity to merit uneven potential of Sorghum. There is a gradual diminution all the cells. In this light absence in the free and bound (ASG) ascorbic of differential detection of genetic acid (AA) from 2h to 72 hr in all as well as labile cellular molecules the regions of the embryo. There is imprint equal but high physiological a decrease in the AAU from 2^ to ^8 potency till the first depression hr in the coleorhiza, radicle and appears. It separates the axial plumule while it increases in the hypocotylar unit from the lateral scutellum and eoleoptile. RNA,proteis, cotyledonary unit. Two loci of the ascorbic acid and -SH and -SS contai­ growing regions viz. hypocotyl and ning proteins exhibit wide fluctuati­ cotyledon lie in the interior of ons in embryonal tissues embarking the radially symmetrical embryo and on differentiation. Histon e level not at its distal end. Estimations remains higher in the cotyledonary of ascorbic acid, -SH proteins ana regions than in the radicle and total nucleic acids reach its peak plumule. RNA, protein and total values at the globular embryo in nucleic acids increase manifold in rice, ragi and oat and subsequently the plumule and radicle than the decline at first indentation or cotyledonary regions depicting high inlet. Globular embryo occupies a metabolic turn over. The estimation transitory but conclusive place bet­ of coefficients of correlations ween pro- and adult embryonic stages. elucidate that proteins have signi­ It shows two faces -1 germinal or ficant and positive correlations axial and 2. abgerminal or cotylar. with significant and positive corre­ The level of ascorbic acid and non­ lations with eight metabolites out histone chromosomal .proteins (NHCP) of the nine studied such as DNA remain low on the abgerminal face (r = + 0.3^) RNA (r = + 0."+7), total than the germinal one. Differentiati­ nucleic acids (r = + 0-5'+)> basic on in the globular embryo is express­ (r = + 0.29), acidic (r + 0.^7), ed by two primary and several secon­ -SH and -SS containing proteins dary successive indentations. Cyto- ( = + 0.73) and insoluble polysaccha­ photometrlc analysis of ENA, histones rides (r = + 0.^3), indicating its and DNA in 6 embryonal regions of intense role in growth and differen­ mature embryos is Sorghum bicolor tiation of the germinating embryo. and Zea mays shows that scutellum, Only ascorbic acid did not reveal mesocotyl, eoleoptile and coleorhiza any significant correlation with possess higher cell area and nuclear proteins (r = + 0.05)- Proteins volume as compared to that of plumule containing -SH and -SS groups are and radicle. In both the plants

174 highly significant and show positive with total proteins (r = 0.77) correlations with DNA (r = + 0.59), total nucleic acids (r = O.69), RNA (r = + O.52), total nucleic HNA (r = 0.72) and -SH and -SS acids (r = + O.63), acidic proteins proteins (r = 0.56) all significant (r = + 0.55) and total proteins above 99 %• However, there is no (r = + O.73)• Ascorbic acid displa­ correlation found between MA and ys significant positive correlation acidic proteins indicating that the with DNA (r = + 0.V/) and insoluble synthesis of acidic proteins is not polysaccharides (r = + 0.4-0) but a consonant with that of DNA upto negative correlation (r = - 0.26) the end of etnbryogeny, the plumule with acidic proteins. The colsest radicle poles have a high level of correlations of RNA are with total ascorbic acid. But as soon as the nucleic acids (r = + 0.92), total axis is organised there is a surge proteins (r = + O.h'/), acidic of AA towards the scutellum. proba­ proteins (r = + 0.72) and -SH and bly this depletion of AA from the -SS groups (r = + 0.52). Thus it axis and its transfer to scutellum is the total proteins followed by and pile of the basic proteins are total nucleic acids having maximum responsible acts leading to dormancy correlations with most of the while the reverse is the behavior macromolecules followed by -SH and of hi ston es and ascorbic acid in -SS groups, acidic proteins, RNA Rhizophora - a viviparous plant. and insoluble polysaccharides, Differentiation of the hypocotyl is which occupy the next positions. marked by the first and second While DNA and ascorbic acid are indentations earring its distal and highly selective in their correlati­ proximal terminals respectively ons with the rest of the metabolit­ albng with a part of the cotyledon. es. The presumptive site of'the cotyledon brings aoout a change in the symmetry Changes in the levels of MA and of the embryo from radial to bilate­ proteins indicate that cells of the ral. As a result two organogenic apical me^istems and leaf primordia centres are discerned. A longisecti- have a high concentration of meta­ on passing through these two sectors bolites as compared to those of imprint certain histochemical diffe­ scutellar extensions. Moreover the rences too. One half of the globular mature embryo of monocotyledons the embryo eonsists of many large cells, embryonal axis shows a profound less basiochromatic as compared to staining intensity of MA, DNA, the plumular half which are smaller proteins but they lack starch grains in area but richly pyroninophylic. while scutellum and its annexures Further initially cells of the evince a feeble staining reaction cotylaconar part are more active Dut their cells are studded with with respect to mitotic divisions, starch grains. The scutellum and whe^e as its complementary half shows its organells however, contain the little mitotic activity. The early major guantum of these metabolites. divisions in the plumular half are Large starch grain in plenty a few anticlinal where as the cotyledonary fairly conspicuous fat droplets and cells divide in irregular planes to many small peripherally positioned form a massive cotyledon. Thus the protenaceous granules constitute above data does not support either the major storage products. The Hansteinian-Soueges nor Swamy- amount of DNA and histon es points Haccius Schools on the organography to a positive correlation (r - + of the embryo in monocotyledons. 0.86) in the expance of the cotyle­ Histochernical staining reactions don. Acidic proteins in Sorghum manifest the ontogenetic homogeneity cotyledonary regions showed signi­ among different extensions of the ficant and positive correlations scutellum. extinction values of substances in four fertile cells of the grass octant embryo

DNA Histones RNA Total proteins -SH and SS protei ?irst cell of the tier 1' 0.066 0.266 0.097 0.0706 0.095 Second cell of the tier 1' 0.06+3 0.28 0.098 J.0810 0.092 Third cell of 1 0.0703 0.^3 0.097 0.076 0.080 Fourth cell of 1 0.073 0.29 0.0 8+ 0.064 0.07+ Standard deviation +0.0037 +0.0103 +O.OO6 +0.004-9 +0.0086 Coefficient of variation 5.39 3-65 6.38 6.30 10.07 all the four cells have an identical developmental potential 175 DNAMEASUREMENT S INTH ECHALAZA LPROLIFERATIN G CELLSO FCAPSELL ABURSA-PASTORI S (L.)MEDIC{* )

L.Viegi ,G .Cel aRenzon i

Department ofBotani cSciences ,University ,Pisa ,Ital y

Summary Fig. 1.DN Acontent s (arbitrary units)i n different zoneso f thechalaza lprolifera ­ DNA content inchalaza lproliferatin g cells tingtissue ,a tvariou s embryostages . ofCapsell aha sbee n studied.Cytophotometri c measurements showed somedegre eo fendopoly - ploidy inal lembry o developmentalstages . Ahypothetica l role forthi s tissue iscon ­ sidered inrelatio nt oembry onutrition .It s probable function asa substitut e forth e antipodals isdiscussed .

Introduction

Previous studieso fchalaza lproliferatin g tissue inCapsell a (Pollock &Jensen ,1967 ; Schulz &Jensen , 1971)hav e clarified its origin anddevelopment .I norde r tofin d outmor eexaclt ywha t role isplaye d bythi s 24 a 12 4 8 12 16 20 24 2 16 20 24 tissue,w ehav emeasure d cytophotometrically theDN A content inth enucle io f thecells , alreadyhypotheticall y attributed polyploid.

Results and discussion

Chalazal cellular populations examined in theglobula r (G),earl yheart-shape d (Ci)an d heart-shaped (C)embry o stagesar ethu sdi ­ stributed inth efollowin g zones,wit hdiffe ­ rentDN Avalue s (fig.1) : A,dista l (withrespec t toth e embryo-sac): Thever y limited synthetic activity ofth e 2-12cinG and Ci;2-8 c inC antipodals inthi s species and theirbrie f B, intermediate:4-24 ci nG an d Ci;2-16 c inC duration strongly support theteor y thatch a C,proximal :variabl eDN Avalue s lazalproliferatin g cellsma yb ea substitut e Thedegre eo fploid y inth ecell sha sbee n structure. calculated inrelatio nt ovalue s 2can d 4co f theembry ocells . References Endopolyploidy,particularl y evident inB , confirms theactivit y of thechalaza lproli ­ Pollock,E.G .& W.A .Jensen ,1967 .Ontogen y ferating tissue inth e earliest stages ofem ­ andcytochemistr y ofth echalaza lprolife ­ bryodevelopmen t and itsrapi d degeneration. rating cellso fCapsell abursa-pastori s Asdiscusse d byPolloc k &Jense n (1967), (L.)Medic . Schulz &Jense n (1971)thi sstructur ema y NewPhytol .66 :413-417 . hypothetically beattribute d arol e inth e Schulz, P.& W.A .Jensen , 1971.Capsell a differentiationo fth ecentra l cell anddeve ­ embryogenesis:th echalaza l proliferating loping endosperm,wit hbot ho fwhic h itha s tissue. aclos eplasmati c relationship. J. Cell.Sei . 8:201-227 .

(*) Supported by ItalianMinistr y ofPubli c Instruction

176 RESPIRATIONRAT EO PTH EEMBRY O INIT SNATURA LENVIRONMEN TI NVITR O

M.Ryczkowsk i Institute ofMolecula rBiology ,Cracow ,Polan d

Summary tween8 an d9 a.m..Th enumbe ro fday s countedfro mth eda yth eperiant hwil ­ Respirationrat efcjOg )o fth eembry o tedt oth eda yo fsamplin gan dth e (HaemanthusKatharinae ,Aesculu shyb - dimensionso fembryo sfH.Katharinae )o r rida)insideth eintac tovule ,an di n elseth edimension so fembryo s(A.hyb ­ thecentra lvacuol esa prespectivel y rida)onl ywer eth eadopte dcriteri a isstrongl yrestricte d invitr oan d ofthei rdevelopment .I tshoul db eno ­ invivo .I tha sbee nsuggeste dtha t ticedtha tplant so fH.Katharina epro ­ mainfunctio ni nth eQO 2o fth eem ­ duceda smal lnumbe ro fovule so fnor ­ bryoan d endosperm inintac tovul ei n maldimension sbu twithou tembryos . vitroan di nviv oi saccomplishe db y Theprocedur eo fmeasurin gth edi ­ 'pentosecycle .Afte rpreparatio nth e mensionso fembryo swa sdescribe di n coat,embry oan d endospermtissu e paper(Ryczkowski ,1962b),an dth ete ­ fromth eovul eth eglicoliti csequenc e chniqueo fth esa pcollectio ni npape r pathwayan dtricarboxyli caci dcycl e (Ryczkowski,1962a) .Befor eus eth e ismainl yuse db ythes etissue si n collected sapwa sfiltere d througha vitro. Schott11G 3filter . Keywords:A .hybrida ,H .Katharinae , Q02 ofth eovule san dembryo swa s embryo,ovule ,respiratio nrat e« QO 2 determinedb ymean so fWarbur gappar ­ centralvacuol esa p« . atusa t25 °* 0,2 °C i ndarkness .Q0 2 measurementso fH.Katharina eovule s Introduction orembryo swer eperforme di nmois t atmosphere(Ryczkowski,1976) . Therear eonl yfe wpapers(Cutter ,e t Measurementso fQO 2o fA.hybrid aem ­ al.,195 2;Stanley,1957;Forman ,Jensen , bryoswer ecarrie d outi ntw odiffer ­ 1965;Johri ,Maheshwari ,1966 ;Rycz ­ entenvironments :a.mois tatmosphere - kowski,1976 ;Ryczkowski ,Szewczyk , maincompartmen to fth eroanometric 1978)containin gth eresult so nQO 2 vesseltyp e- W A011 0containe d 10m l ofth edevelopin gembryo ,endosperm , ofdistille dwater ,an d itssid ear m coatan dovul ei nhighe rplant si n 0,5m lo f20 %KOH ,embryo swer eplace d vitro. ona plasti cperforate dplat eabov e Theseresult san d conclusionscon ­ thewater ,b.sa p- mai ncompartmen t cerningth ebiochemica lan denergeti c ofth emanometri cvesse lcontaine d 10 aspecto fth eembryo ,endosper man d mlo fth esa pan dembryos ,an dit s ovulei nvitr oQO 2ar eno tconcordant . sidear m0, 5m lo f2 Cf)i KOH. Themai nai m ofth eperforme dmeas - Thevessel swer eequilibrate dfo r urmentso fth eembry oQO 2wa st oge t •30min.,an dpressur e changesrecorde d somecomplementar ydat aconcernin g every5 o r1 0min .dependen to nembry o itsQO 2insid eth eovul ei nvitr oan d dimensionsfo ron ehour . indirectly invivo .Thi spape rpre ­ AfterQO 2measurement sth eovule s sentsth eresult sconcerning :1.QO 2 andembryo swer eweighte dt odeter ­ ofa novul econtainin gth eembryo,an d minethei rfres hweight .Befor etha t anovul ewithou ti to fth esam eag e A.hibridaembryo swer ewashe d indis ­ asth epreviou sone ,2.QO 2o fa no - tilledwate ran ddrye dwit ha pic eo f vulecontainin gth eembryo,an dth eem ­ -wool. bryoo fth esam eag ea sth eovul ewit h Theresult spresente d inTable s 1,2 theembryo ,3.QO 2o fth eembry oplace d and3 ar emea nvalue so f2- 3meas ­ inth ecentra lvacuol esa pi.e .i n urementso fQO2 ,an dar eexpresse di n itsnatura lenvironmen t invitro . mgC02/h/ovul erespect ,embryo ,an d inm gC02/h/ gfres hwt . Materialan dmethod s Results Ovuleso fHaemanthu sKatharina eBak . fofth eag e132-15 0day safte rth epe ­ H.Katharinae.Q0 2(mgCOo/h/ovule )o f rianthwilted ,stationar yphas eo f theovul ewit han dwithou tembry oa t growth),and embryoso fAesculu shyb ­ age13 5- 15 0day-ol d laywithi nth e ridaD.C .(A.octandr ax A.pavia ,expo ­ limitsV0,0179- 0,032 9m g C02respect . nentialphas eo fgrowth )wer euse da s (Table1 ).Th eQ02Jdifferenc eBetwee n experimentalmateria lcollecte dbe ­ theseovule s(o fth esam eage )wer e Vo,0206- 0,036 6an d 177 Table 1. H.Katharinae. QO2 of the ovule with embryo, and the ovule without it (the same age)in moist atmosphere; + higher Q02 the ovule with embryo; - higher QO2 the ovule without embryo. Age Fresh wt. g QO2 in mg CO2/I1 days 1 ovule per ovule per g fresh wt. with without with without difference with without difference embryo embryo embryo embryo embryo embryo 135 0,4372< 0,4583 0,0206>0,0179 0,0027+ 0,0462>-0,0391 0,0071+ 135 0,6440> 0,6432 0,0366>0,0329 0,0037+ 0,0569>0,0511 0,0058+ 150 0,4875> 0,4007 0,0297>0,0270 0,0027+ 0.0610-C0.0674 0,0064-

Table 2. H.Katharinae. Q02 of the ovule with embryo, and embryo (the same age as the ovule with embryo) in moist atmosphere.

Age Dimensions Fresh wt. g QO2 in mg C02/h days of embryos ovule embryo perovul e perembry o perg fres hwt . in mm ovule embryo 132 9,9x2,8 0,49620,034 3 0,0215 0,0054 0,0432 0,1587 135 9,8x2,6 0,51660,032 1 0,0211 0,0058 0,0408 0,1832 142 9,6x2,6 0,47610,030 8 0,0186 0,0046 0,0390 0,1465

withinth erang e0,0027-0,003 7m g C02. Table3 .A .hybrida .Q0 2 ofth edeve ­ Q02(mgC0 2/h/gfres hwt. )o fth e0 - lopingembry oi nth ecentra lvacuol e vulewit hembry oa tth eag e135-15 0 sapan di nmois tatmosphere . day-old laywithi nth elimit s0,0462 - 0,0610m gCO2 ,an dthi svalu eo fth e ovulewithou tembry owa swithi nth e 'Dimensions Fresh QO2i nm gC0 2/h range0,0391-0,067 4m g C02 (Table1 ). ofembryo s wt.g1 per embryo Differencesi nth eQ0 ?betwee nbot h inm m embryo sap moistatm . typeso fovule sla ywithi nth elimit s 0,0058-0,0071 mgC0 /h/gfres hwt . 12,7x5,5 0,059 0,0285-CO,051 4 2 17,9x5,6 0,060 0,0389-CO ,047 6 TheQ0 2(mgC0 2/h/embryo)o f132-14 2 day-old embryoswa swithi nth elimit s 20,9x5,8 0,159 0,0492<0,095 7 0,0046-0,0058m gCO? ,an dconstitute d 20,1x8,0 0,201 0,0687-CO ,121 0 onefourt ho fth eQO oo fth eovule s • 25,1x8,6 0,272 0,0454-CO ,163 1 withembryo s (Table 28,3x10,7 0,566 0,0744-CO,247 7 2). 30,"te10,7 0,509 0,0732-CO,116 1 Q02 ofth eembryo(m gCOo/h/ gfres h wt.)waswithi nth erang e0,1465 - 34,8x14,6 1,129 0,1310-CO,468 0 0,1832m g C02an dwa s3,7-4, 5time s highertha nthi svalu eestablishe d forth eovul ewit hembry o(Tabl e2) , 1.Differencebetwee nth eQO oo fth e A.hybrida.Q0 2 (mgC02/h/embryo)o f ovulewit hembry oan dQO 2o fth eovul e thedevelopin g embryoplace d inth e without it(H.Katharinae,independen t sapo rmois tatmospher eincrease d onthei rage ,an dsmal ldifferenc ei n irreglarlyfro m0,028 5t o0,131 0an d theirweight)lie swithi nth erang eo f from0.051 4t o0,468 0m g C02respect ­ biologicalvariabilit yan dmethodica l ively [Table 3). errors.2.Th eQ0 2 ofth eembryo(H . QO2o fth eembryo(m gC02/h/ gfres h Katharinae)constitute sabou ton e wt.)placedi nth esa po ri nth emois t fourtho fth eQ0 2 ofth eovul ewit h atmospheredecrease d from0,481 4t o embryoi nvitro ,i nth emois tatmos ­ 0,1160,an dfro m0,868 4t o0,414 6m g phere.3.Th eQO 2o fth eembryos(bot h COorespectivel y (resultsno tplace d species,mois tatmosphere )i nvitr oi s inTabl e3) . muchhighe rtha ntha to fthes eembryo s inthei rnatura lenvironment si n Discussion vitro(Table1,2.3)i.e .i nth eovul e ori nth esap .4.Th eQ0 2 ofth eA . Basingupo nth eobtaine dresults , hybridaembryo sar e1,8-3, 6time slow ­ concerningth eQO oo fth eembry oo f eri nth esa ptha ni nmois tatmos ­ differentag ei nit snatura lenviron ­ phere( xable3) .Hence ,i tresult s menti.e .insid eth eovul eo ri nth e thatQO 2o fth eembryo s(bot hspecies ) sap,an di nmois tatmosphere ,th e isstrongl yrestricte d inth eovul e followingfact swe restablishe d: invitr oan di nvivo .Thi sconclusio n

178 isi nagreemen twit hth ever ylo wox - Theendogenou soxyge nuptak eo ftis ­ gentensio ninsid eth eovul e(Ryczkow ­ suesi nth edevelopin gfrui to fCo - ski, 1973),an dunpublishe ddat a and cosnucifera .Amer.J.Bot.39:51-56 . itsanatom y(Netolitzky ,1926) . Porman,M.,W.A.Jensen,1965.Respiration Onth ebasi so fliteratur edat a andembryogenesi s incotton .Plan t (Forman,Jensen ,1965 ;Johri ,Mahesh - Physiol.40:765-769 . wari,1966 ;Kolloffel ,1970 )an dth e Gibbs,M.,H.Beevers,1955 .Glucos edis ­ author'spreviou sresult s(Ryczkowski , similationi nth ehighe rplant . 1976; Ryczkowski,Szewczyk ,1978) , Effecto fag eo ftissue .Plan tPhy ­ andpresen t investigationso nth eQ0 £ siol.30:343-347 . ofth eovule ,coat ,endosper mtissu e Johri,M.M.,S.C.Maheshwari,1966.Grow ­ andembry oi tshoul db etake nint o th,developmen tan drespiratio ni n accounttha tthes etissue sar epotent ­ theovule so fZephyranthe slancaste - iallycapabl eo frespirin gby :a .gli - ria tdifferen tstage so fmaturation . coliticsequenc epathwa yan dtricarbo ­ Plantan d CellPhysiol .7:49-58 . xylicaci d cycle,b .direc toxydatio n Kolloffel,C,1970 .Oxydativ ean dpho - ofsugar s- pentos e cycle,an d c.gli - sphorylatlveactivit y ofmitochon ­ coliticsequenc ean danaerobi crespi ­ driafro mpe acotyledon sdurin gma ­ ration. turationo fseed .(Plant aBerl.) .91 : Itseem stha tth emai nfunctio ni n 321-328. therespiratio nrat eo fth eendosper m Netolitzky,P.,1926.Di eAnatomi ede r tissuean d embryo inintac tovule ,i n Angiospermen,Hb.Pflanzenanatomie , vivoan di nvitr oi saccomplishe d hersg.v.K.Linsbauer ,Berlin . byth epentos e cycle(Barne te tal. , Ryczkowski,M.,1962a.Change si nth e 1953; Gibbs,Beevers ,1955 ;Wager , osmoticvalu eo fth ecentra lvacuol ­ 1983).Mos tprobabl y itoccur si nbi g arsa pi ndevelopin govules .Bull . ovules(H.Katharinae ,A.hibrida" )i n Acad.Polon.Sei.,Ser.Sei.Biol.10 : whichoxyge npenetratio ninsid eth e 371-374. ovulei sstrongl yrestricted(Ryczkow ­ Ryczkowski,M.,1962b.Change si nth e ski, 1973;Ryczkowski ,Szewczyk ,1978) . osmoticvalu eo fth esa pfro membry ­ Onth eothe rhan dafte risolaitin g os,th e centralvacuol ean dcellula r thecoat ,endosper m tissue,an dembry o endospermdurin gdevelopmen to fth e fromth eovule ,an di ndependenc eo n ovules.Ibid .10 :375-380 . itsdimension san danathomy ,thes e Ryczkowski,M.,1973.Change so fth e tissues,organ srespir ei na naerobi c oxygentensio npO oi nth edevelop ­ way -glicoliti c sequencepathwa yan d ingovul edurin ginhibitio nan dex ­ tricarboxylicaci dcycle(Stanley,1957 ; ponentialphas eo fembry ogrowth . Porman,Jensen ,1965 ;Johri ,Mahesh - Ibid.21:303-308 . wari, 1966).Thi ssuggestio ni sals o Ryczkowski,M.,E.Szewczyk,1975.Furth ­ confirmed bya distinc tincres eo f erresearc ho nth ephotosynthesi s QOgo fthes etissue safte rthei rpre ­ inth egree ndevelopin g embryosdi ­ parationi ncompariso nwit hth eQO , cotyledonousplants .Z.Pflanzen - ofa nintac tovul e (Ryczkowski,1976 ; physiol.75:175-180 . Ryczkowski,Szewczyk , 1978). Ryczkowski,M.,1976.Respiratio nrat e Inth ecas eo fth eovul ewit hgree n ofth eovule ,coat ,endosper mtissu e embryoscapabl eo fphotosynthesi si n andembry odurin gthei rdevelopment . vitro(Ryczkowski ,Szewczyk ,1975)an d Bull.Acad.Polon.Sei.,Ser.Sei.Biol. probably inviv otoo ,th eoxyge nfro m 24:237-242 . thisprocce scoul db euse d insit ui n Ryczkowski,M.,E.Szewczyk,1978.Respir ­ theprocce so fembry oan dendosper m ationrat eQ0 2 ofth eovule ,coa t tissuerespiration .Thi sfac tmake s andendosper m indifferen tenviron ­ theproble m ofrespiratio no fthes e ments invitro .Ibid .26:415-420 . tissuesi nintac tovul estil lmor e Stanley,R.,1957.Kreb scycl eactivit y obscure. ofmitochondri afro m endospermo f Anaerobicrespiratio no fth eembry o sugarpin esee dPinu sLambertan a and endosperm tissuei nintac tovul e Dougl.Plan tPhysiol .32:409-412 . inviv oan di nvitr oseem simprobabl e Wager,H.G.,1983.Th elabelin go fglu - butno texclud ea ta determine ddeve ­ cose-6-phosphate inpe aslice ssup ­ lopmentalstage . pliedwit hC u glucose,an dth eas ­ sessmento fth erespirator ypath ­ waysi nair ,i n 10%CO 2an d in 2% References 02 inN .J.exp.Bot .34:211-220 . Barnett.R.C,H.P.Stafford,E.E.Conn , B.Vennesland,1953 .Phosphogluconi c dehydrogenase inhighe rplants . PlantPhysiol .28:115-122 . 1952- Cutter,V.M.Jr.,K.S.Wilson,J.P.Dube X 179 ELEMENTCONCENTRATION S INTH ESA PSURROUNDIN GTH EDEVELOPIN GEMBRY O

M.Ryczkowski ,A .Kowalsk aan dW .Reczynsk i Instituteo fMolecula rBiology ,Cracow ,Polan d 1 Laboratory ofPhysicochemica lAnalysi san dStructura lResearch ,Cracow ,Polan d

Introduction Inrecen tliteratur ether ei sa lac k tionther ewa sa smal lincreas ei n ofquantitativ e investigations onele ­ theseconcentration ,an ddurin gfurth ­ mentsi nth eovul edurin gembryogenes - erdevelopmen tthes econcentration s is(Maheshwari,1963 ;Wardlaw ,1965) . decreased.Thes echange sar ei nagree ­ Thepresen tabstrac tcontain sth e mentwit hchange so fosmoti cvalu eo f resultso felemen tdetermina-tion si n thesa p(Ryczkowski ,1962a) .d.Th eele ­ thecentra lvacuol esa p(= sap)carrie d mentsoccurrin gi nth esa pwer euse d outwit hth eai mt oestablish :a .th e upb yth edevelopin gembryo .Thi scon ­ numbero felement soccurrin gi nth e clusioni sconcordan twit htw ofacts : sap,b .thei rconcentration san d decreaseo fthes eelement si nth esap , changesdurin gembryogenesis . andoccurrenc eo fth esam eelement s inth eembry o(preliminar yinvestigat ­ Materialan dmetho d ions). Theoccurrenc eo felement s inth e Ovuleso fAesculu shybrid aD.C.(A . sap± si naggreemen twit hliteratur e octandrax A .pavia)wer euse da sex ­ data"(Tammes ,1959) . perimentalmaterial .Th edimension s ofembryo swer eth eadopte d criteria References ofthei rage .Th eprocedur e ofmeasur ­ Maheshwari,P.(Ed. ):Recen tadvance si n ingembryo swa sdescribe d inpreviou s theembryolog yo fangiosperms .In ­ paper(Ryczkowski ,1962b)an dth etech ­ tern.Soc.PlantMorphol .Delhi.1963 . niqueo fth esa pcollectio ni npape r Ryczkowski,M.,1962a.Change si nth e (Ryczkowski,1962a) . osmoticvalu eo fth ecentra lvacuol ­ SpectrophotometerPerkin-Elme rMode l arsa pi ndevelopin govules .Bull . 503(AA S- method )wa suse dfo rdetermi ­ Acad.Polon.Sci.,Ser.Sci.Biol.10: nationo fK,Ca,Mg,Mn,]?e ,Z ni nair - 371-374. acetyleneflam e(standar d condition), Ryczkowski,M.,1962b.Change si nth e andHGA7 4Graphit eFurnanc efo rCu . osmoticvalu eo fth esa pfro membry ­ ». os,th ecentra lvacuol ean dcellula r Resultsan ddiscussio n endospermdurin gdevelopmen to fth e ovules.Ibid .10 :375-380 . Itwa sestablished :a.Concentration s Tammes,P.M.L.,Nutrientsi nth egian t ofK,C aan dM gwer edistincl yhighe r embryosac-vacuoleo fth ecoconut . inth esa ptha nconcentration so fFe , ActaBot.Neerl .8 :493-496 .1959 . Mn,Znan d Cu(Table ). b.Th elowes tcon ­ centrationwa scharacteristi c ofC u Wardlaw,C.W.,1965.Physiolog y ofem ­ (Table),c.Tota lconcentratio no fK+C a bryonicdevelopmen t incormophytes . andMg+Fe+Mn+Zn+C urespectively , In:W.Ruhlan d(E dJ : Encyclopedi ao f changeddurin gth eembry odevelopment . plantphysiology.v .15/ 1Berlin , Atth ebeginin go fembry odifferentia ­ Heidelberg,Ne wYork .p.844-965 .

Table.A.hybrida .Elemen tconcentration s inth esa psurroundin gth eembryo . Embryom m K Ca EX Ca Mg Fe Mn Cu Zn 2M gF eM n Dimensions ^mg/cm-1 ,ug/cm5 CuZ n 0,87x0,54 3,20 1,27 4,47 113,00 8,17 3,40 0,080 3,35 128,00 1,23x0,53 3,20 1,35 4,55 125,00 7,40 3,15 0,150 3,36 139,06 3,80x2,00 3,50 1,43 4,93 125,00 6,00 2,40 0,050 2,90 136,35 4,70x2,10 3,20 1,33 4,53 108,00 7,75 2,90 0,160 3,43 122,24 16,10x5,40 3,00 1,15 4,15 88,00 7,65 2,53 0,150 2,53 100,86 25,30x9,70 3,20 1,10 4,30 115,00 4,40 1,90 0,125 2,30 123,73 28,10x14,0 2,45 1,00 3,45 80,00 6,45 2,63 0,095 1,55 90,73 39,90x19,2 1,50 1,10 2,60 100,00 3,20 1,60 0,080 0,90 105,78 S.D. 0,30 0,28 11,48 0,15 0,75 0,008 0,55

180 HYBRIDIZATION BETWEEN PINU S NIGRA ANDPINU S SYLVESTRIS ANDMORPHOLOG Y OF CHROMOSOMES 2. Borzan Faculty of , Department of Forest Genetics and , Zagreb, Yugoslavia

Summary thout irradiation,mento ro rothe r pollentreat ­ Thekaryotype so fparen ttree sPinu snigr aArn , ments,fo rtheP^ .nigra xP .sylvestri s combination. (treen i22 1) an dIP .sylvestrisL .(tre e sy77 ) Figure 1.Th eidiogra m ofth ehybri d tree nisy410 . havebee ncompare dwit hth ekaryotypeo f hybrid treeP .x nigrosy lvi sVid . (treenlsy410) .Th e differences ofchromosom etota lan dar mlengths ,

aswel la sth ecentrome ri cpositio nbetwee npa ­ 66 64 renttree sar eles sexpresse d thanth edifferen ­ 56 65 ces ofth esam e paterns between eachof th e S = 47 parent trees when compared toth ehybrid . Introduction Oneof th esi xverifie d hybridtree sP .x nigro - sylvis (thetre en is y4 10) ,grow nsinc e1968 ,wa s L = 53 51 57 used toge tth ekaryotyp efo rth ecomparisonw it h 60 58 66 the karyotypesof paren t treesP' .nigr a(tre e ni 221)andP .sylvestri s(tre esy77) .Borza npre ­ sentedkaryotype sof theseparen ttree si n1981 . I II III IV V VI VII VIII IX X XI XII Work method, results and|discussio n 18 suitable cells oftheendosper m indeve ­ Onth econtrar ythe£ .sylvestris xP .nigr acros - lopmentwer euse d forworkin gou tth ekaryotyp e sin gseem st ob ecom p1ete'l yincompa ti ble .Bac k ofth ehybri d treenisy410 .A l1o fth eee lI shav e crosseso fth eP_ .x nigrosy lvi shybrids ,wit hbot h originated fromoneovuleonly ,i.e .th esam e parentspecies,ar eeas yt odo, ,an dth enumbe rof slidemad e byFeulgensquashmetho d (Darlington fertil eseed spe rcon ei sth esam ea safte r intra- SLaCour, 1962).I n1981 , Borzan described ka­ specifi chybridization .Fro mthi spoin to fview , ryotype analysis anddat a processing.Th ecom ­ theresu ltso fkaryotyp ean aly si so findividua l parisono fnumerica l karyotypevalue so ftree s treesn i221 ,s y77an d theirprogen ynis y41 0ar e ni 221,s y7 7an dnis y41 0wa sdon eusin gt-tes t interesting. Inconclusion , thedifference so f ona computer. Table 2.Result so fteste d differencesbetwee n investigated trees. Table 1.Hybri dtreenis yMI Onumerica lkaryotyp evalues . Tested dif- Total Short Long Arm Centromeric ferences be- chromosome arm arm ratios indexes •Relative chromosome lengths Arm Centromeric tween trees lengths lengths lengths S/L 100S/S+L Shortar mLon gar m index ratio ni22 1 Nosignif . Nosign . x* No sign. No signif. S L S/L sTTT differencesdiffer .• differ. differences 125.26 59.87 65.39 .917 47.80 nisy41 0 Nosignif . * ** * „<• (7.97) (4.24) (4.73) (.056) (1.58) m 221 differencesv •X I x •" 116.32 55.30 61.02 .911 47.59 nis.syy41' 0 A ftft* ft * ft ftft* fti (5.16) (2.26) (4.57) (.070) (1.99) ^77 VII XI V ; XI V ;XI V 111.91* 52.51 59.43 .889 46.95 (3.62) (2.60) (4.20) (.087) (2.57) Significant differencesa t5 %level ; 1% level; 0.1% level. 108.40 51.60 56.80 .911 47.59 (3.05) (2.94) (2.70) (.078) (2.22) chromosomemorpholog ypattern sbetwee nparen t 105."t7 50.93 54.54 .935 48.30 trees areles sexpresse d thanth edifference so f (2.81) (1.56) (1.97) (.037) (1.01) thesam epattern sbetwee neac hof th eparen ttree s 102.26 49.19 53.07 .929 48.12 whencompare dt oth ehybrid.l ti sinterestin gtha t (2.51) (1.87) (2.39) (.061) (1.70) thechromosom eX Iof th eprogen ynis y41 0i sno t 99.03 46.67 52.36 .897 47.13 (2.00) (3.21) (3.29) (.104) (3.10) submetacentric,accordin gt oSaylor' s(1961 )de - 45.27 51.45 .885 46.81 finition.Thi s iswh yth edifference sbetwee ntha t (2.90) (3.28) (.100) (2.83) chromosomecompare dt oth echromosom eX Io fth e 93.50 43.92 49.59 46.97 parent treesar esig ni ficant .T ounderstan dthes e (3.19) (2.16) (2.44) (1.80) resultsi ti swort hstressin g theheterozygosity 87.83 40.79 47.04 .867 46.33 (4.98) (4.14) (1.75) (.087) (2.67) existing inboth parentalpin etrees ,larg epos ­ 81.U7 38.05 43.42 .882 46.74 sibleva riab i1 i t yo fth egarnet sformin gth enis y (5.39) (2.93) (3.88) (.087) (2.55) 410zygote ,a swe l1 a sth efac t thatth eendosper - 71.80 28.44 43.36 ,663 39.51 mal nisy4l0tissue,use dtowor kou tth ekaryotype , (6.01) (4.64) (4.38) (.134) (4.83) hasbee ndevelope dafte rth emeios istoo k place. From thestandpoin to f incompatibi1 i ty ,i t i s References interesting thatP • nigr axP .s yIve str ishybrid s have been produced,bu tno tviceversa .W it h2 0 Borzan,2. ,1981 .Karyotyp ean aly si sfro mth een ­ yearexperienc ei ncrossin gthes etw ospecies ,i t dospermo fEuropea nblac kp in ean dScot s pine. seems thatdifferen tmethod s inovercommingth e Ann. Forest. 10/1, 1-42. incompatibi1 i t yca nonl y increaseth enumbe rof Darlington,CD .6 L.F .L aCour , 1962. The seeds,tha tcan ,i nsmal lnumber sb eproduce dwi ­ handling of chromosomes.G ,A lle nÊUnwi nLtd . Saylor, L.C.,I96 L Silvae Genetica 10,77-84. 181 INVITR OEMBRYOGENESI S OFFLA X (LINU MUSITATISSIMU M ) A.Pretov â Institute ofExperimenta lBiolog y and Ecology ofSlova kAcadem y ofSciences , Bratislava,Czechoslovaki a

Summary teststimulatio n effectwa sobserve d atth econcentratio no f5.1 0 molo f Embryoso ffla xca nb ecultivate d kinetin.Kineti ni nconcentratio no f invitr o beginingwit h theirglobu ­ 5.10 molcause d unorganised growth larstag e (th e firstcritica lsta ­ and callus formation.Kineti n ,_gaini ge ).Afte rinitia lenlargemen to f lyi nth econcentratio no f5.1 0 mol, bothlengt han d width,cotyledon s speeded upreachin go fth esecon d started todifferentiate .Widt hi s criticalstag e (i na naverag e 2-3 more increasing thanth elength .Th e daysearlie r thanth e controlembryos ) globularembry oreache s thesecon d invitro . critical stage on the8% h- 10t hda y ofcultivation .Th ewidene d globular Kinetinwa sals oincreasin gth e stage isreachin g the secondcriti ­ percentage ofembryo scompletin g calpoin to n6t h- 8t hda yan dth e theirembryona l development.A tth e heartstag e embryoattain s thesecon d globularstag ei twa sincrease d from criticalstag e already on2n d -6t h 20 % to 35- 4 0 %.A t theexcise d day,o rth e embryoimmediatel y passes more proceeded embryos thispercenta ­ into theexponentia lgrowth .Kineti n ge increased from6 0 %t o8 5 %. addeô into thecultivatio nmediu m The/stimulativ e effecto fkineti n promotes the transitiono fth eemb ­ on'ithedevelopmen t ofexcise demb ­ ryosint o thesecon d criticalstage . ryos isperhap sdu e toth eabilit y ofkineti n tostimulat e cytokinesis ( K611ere tal . 1962,Ioffe &Zhukova , Introduction I.965).Thi sca n beals oi na connec ­ tionwit hth econfirme d stimulation Themetho d ofembry o cultivation ofproteosynthesi s .(Klämbt ,1976) . invitr o became aver yeffectiv e Itseem s thatkinetin ,i na sui ­ method forplan t breeding.Thi spa ­ tableconcentration ,ca nb euse d for perstudie s completingo fembryoge ­ speedingu p the invitr o embryogene­ nesi si nvitro ,mainl y under theef ­ siso ffla xembryos . fecto fkinetin .

Materialan dMethod s » References Porou rexperiment s embryoso ffla x Ioffe',I.D. , Zhukova,Ja.,1965 .Kul - ( Linumusitatissimu mL .cv .Vier a) turaizzolirovanyk hzarodyshe jpo - were used.Th emetho d wasdescribe d krytosemjannykhrasteni jn aIss - indetail si nou rpreviou spape r kustvennojsrede .Bot .Zhurnal.50 : ( Pretova,198 3 ).T o the basicMon - 1157- 1182 . nier' smediu m (Monnier ,197 8 )w e Klämbt,D.,1976.Cytokini neffect so n added.kinetinineoncentrationsa f 4 proteinsynthesi s ofi nvitr o sys­ 5.10 mol,5.1 0 molan d 5.10°mol . temso fhighe r plants.Plan tan d CellPhysiol .17::73--77 . Resultsan dDiscussio n Koller,D.,Mayer,A.M. ,Poljakof f- -Mayber,A. ,Klein,S. ,1962 .See d In theproces s offla xembryogene - germination.Ann .Rev .o fPlan t sisi nvitro ,som e differenceswer e Physiol.13 :43 7- 465 . observed inembry odevelopmen ti n Monnier,M.,1978.Cultur e ofzygoti c dependence onth estag e inwhic hth e embryos.In :(Frontier s ofPlan t embryoswer eexcise d (Pretfova .1983) . ïissueCulturerjl978) .Proc .o fth e Whenkineti nwa sadde d toth eculti ­ 4thInternat..Cong ,o fPlan tTis e vationmediu mi nconcentration sdesc ­ suean d CellCulture ,Univ .Cal ­ ribed above,a promotio no fth eemb ­ gary,Alberta,Canada,Augus t2 0-25 , ryodevelopment ,o fgrowt ho femb ­ 1978. ryosan d of the finishingo fthei r Pretfova,A.1983.Cultivatio no fglobu ­ embryogenesiswa sobserved .Postemb ­ laran d heart stage flaxembryos . ryonaldevelopmen t -th e germination In: (Proc.o f7t hInternat .Cyto - ofembryo san d thegrowt ho fseed ­ embryologicalSymposium ,Hig hTat ­ lingswer e stimulated,too .Th egrea ­ ra,Jun e 14-17 ,1982) ,Bratislav a

182 DIRECTSOMATI C EMBRYOGENESISFO R RAPIDGENOTYP E PROPAGATION

G.Maheswara n and E.G.William s

Plant Cell Biology Research Centre,Schoo lo fBotany ,Universit y ofMelbourne ,Parkville , Victoria 3052,Australi a

Summary a potentially convenient technique for multiplying selected genotypes of Bydirec t somaticembryogenesi s «1vitr oa outbreeding species and bypassing sexual clone of aseptic plantlets can be raised reproduction with its attendant genetic from a single immature zygotic embryo of recombination. Since cellular Trifolium repens (white clover)withi n about destabilization (Meins 1983) in callus and six weeks from pollination. Somatic suspension cultures is now well known to embryoids arise directly from superficial result in genetic and cytological cells of the hypocotyl with no visible alterations ("somaclonal" variation,Larki n callus phase. Similar systems for direct etal . 1984),pathway so fmorphogenesi ssuc h primary somaticembryogenesi shav eals obee n asdirec t somatic embryogenesisan dmultipl e developed for T^ pratense (red clover),T . shoot induction which show no gross resupinatum (Persian or annual de-differentiation, aremor e likely togiv e clover), Medicago sativa (alfalfa), Lotus true clonal régénérants. Herew erepor to n corniculatus (birdsfoot trefoil) and systems for somatic embryogenesis from Brassica campestris (oilseed rape). In zygoticembryos . addition,secondar y cycleso fdirec t somatic embryogenesis on subcultured embryoids have Resultsan d Discussion ' also been achieved for T\_repen s and B. campestris. Immature zygotic embryos were dissected from surface-sterilized pods at the Introduction "torpedo" stage (cotyledons initiated, embryo filling 1/2 -2/ 3 embryo sac). This We are developing methods for propagation stage showed amarke d proliferative response by direct somatic embryogenesis or direct to appropriate hormone treatments. Embryos multiple shoot formation on immature zygotic were placed on nutrient medium in sealed embryos,wit h theaims : Petri dishes and incubated at 22°c in 1. tominimis e culture-induced variationb y continuouslight . avoiding cellular destabilization; Primary somatic embryogenesiswa sachieve d 2. to develop simple, rapid, space- onth emedia : efficient propagation techniques which can T.repen s ) EC6 (Maheswaran &William s be applied as early in the life cycle as T.pratens e ) 1984)wit h lgl"1 possible; T_;_resupinatu m ) yeast extract plus 0.05 3. to develop continuous embryogénie M^_sativ a ) mgl-1 BAP cultures with potential for mechanized U_corniculatu s EC6 (withoutYE )plu s propagation andharvesting . 1-2mgl" 1BA P ( B5 (Gamborge tal . 1968) In outbreeding self-incompatible species ( plus0.0 5 mgl"1 BAP;o r such as Trifolium repens and Brassica ( modified SH campestris,natura l populationsar enormall y B.campestri s ((Bhattachary a &Se n 1980) highly heterozygous and variable. ( pluslgl" 1YE ,0.02 2 Consequently, individual seeds, even from ( mgl-1 2,4-D and0.0021 6 the same plant, tend to vary widely in (mgl-1 kinetin genotype. Although many perennial species such as Tj_ repens can be propagated by Secondary direct embryogenesis from cuttings, this procedure is slow and cannot subcultured primary embryoids was achieved provide aseptic material equivalent to onth emedia : seedlings. For annualssuc ha sa numbe ro f T.repen s EC6wit h0.25-1. 0gl" 1Y E B. campestris varieties, propagation by plus2 mgl -1 BAP cuttings may be extremely difficult. B.campestri s B5wit h2 %sucrose ,n o Furthermore, when self-incompatible annual hormones. plants are artificially self-pollinated to Primary and secondary embryoids of all generate homozygous inbred stocks, these specieswer ereadil y separated and rooted on lines must normally be maintained by the corresponding media without hormones, artificial manipulations such as bud beforetransfe r tosoil . pollination. In T. repens the origin of primary Rapid propagation intissu e cultureoffer s embryoids was examined by scanning electron microscopy and lightmicroscop y ofsectione d

183 material. Embryoidsarose>superficiall yb y proliferation of one or a few hypocotyl epidermal cells,withou t attachment to the vascular system ofth eparen tembryo . The uniformity of régénérantsha s notye t been confirmed. However, if thisprove st o be satisfactory, direct somatic embryogenesis from zygotic embryos may provide not only amean s for propagation, but also "seedling" clones for multiple screening inhost/pathoge n orhost/Rhizobiu m studies, or experiments where breeding is combined with destructive sampling. Also, since embryoids appear to arise fromon eo r a few cells, direct somatic embrogenesis after mutagen treatment of immature embryos in vitro may improve recovery rates of whole-plant mutations and reduce chimerism inmutatio nbreeding .

References

Bhattacharya,N.M .& Sen ,S.K .198 0 Production ofplantlet sthroug h somaticembryogenesi s inBrassic a campestris. Z.Pflanzenphysiol .99 : ^ 357-65. • Gamborg,O.L. ,Miller ,R.A. , &Ojima ,K 1968 Nutrient requirementso f suspension cultureso fsoybea nroo tcells . Exp.Cel l Res.50 :151-8 . Larkin,P.J. , Ryan,S.A. , Bretten, R.I.S.& Scowcroft,W.R . 1984 Heritable somaclonal variation inwheat . Theor.Appl .Genet . 67: 443-55 Maheswaran,G. , &Williams ,E.G . 1984 Direct somaticembryoi d formationo n immature embryoso fTrifoliu mrepens , T.pratens ean d Medicago sativa,an d rapid clonalpropagatio n ofT\ _repens . *• Ann.Bot .54 :201-11 . Meins,F .Jr .198 3 Heritablevariatio ni n plant cellculture . Ann.Rev .Plan t Physiol 34:327-46 .

184 APPLIEDASPiJCT SO FEMBRïOLOG r

T.B.Batygina KomarovBotanica lInstitute » Leningrad,USS R Introduction authorsdat amad epossibl et oestab ­ Twomai ntrend sca ntoe distinguish ­ lisha serie so fcommo nregularitie s edi nmoder n embryology.Thefirsi ti s ofdevelopmen tcommo nfo rtn eproces ­ the establishing ofregularitie so f sestakin gplac e indistan thybridi ­ theevolutionar yproces san dsolutio n zation.Incompatibilit y indistan t? ofquestion s ofphylogen y andsystema - crossesca nb eexpresse di nthre e tics.Thebasi cmetho do fthes einvesti ­ stages:1interrelationship ofth epol ­ gationsstil lremain sth ecomparative - lenan dpolle ntub ewit hth etissue s descriptivemethod.Th esecon dlin eo f ofth epistil.2)Th eproces s offerti ­ investigation isth eclearin g outth e lization(syngam yan dtripl efusion) . lawso findividua ldevelopmen tconcer ­ 3)Thedevelopmen t ofth eembry oan d ningplan treproductio n (sexual,asex ­ endosperm. ual,agamic)whic hha swid eapplicatio n Themanifestatio no fincompatibi ­ inpractice.Th emetho do finvestigati ­ litya tth efollowin g stagei sno t oni sbot h comparative-descriptive alwaysconnecte dwit hth epictur eo f andexperimental.Bot hthes etrend s thecours eo fprocesse si nth eprevi ­ areclosel yconnecte dwit heac hother . ousstage.Fo rexample ,a goo dgrowt h Stillmor e significance hasacquire d ofth epolle ntub edoe sno talway s atpresen tth eexperimenta lline.On e secureth esucces so ffertilization . ofth etask so fexperimenta lembryolo ­ Comparisono fth echaracte ro fdis ­ gyi st oattai nsom epossibilitie so f turbances ofembryologica lprocesse s controlling separatestage so fontoge ­ indistan thybridizatio nwit hth e nesis.Plantembryolog yi sa necessar y normalcours eo fdevelopmen t enables basisfo rpractica l investigations ust odistinguis htw omai ntype so f concerningth equestion so fplan tre ­ disturbances:converte dan dnon-con ­ production.Atpresen tw eca nconcrete ­ verted.Toconverte d disturbancesbe ­ lydetermin e sometrend si nwork so n longsuc hdeviation swhic hd ono t selectionan dplan tgenetic swhic hur ­ disturbhomeostasi s inth esyste m gentlydeman dknowledg e ofembryolog y pollen-pollentube-eg gcell-zygote - andapplicatio n ofsom ecytoembryolo - endosperm-surroundingmaterna ltissue s gicalmethods.Som edirection san dme ­ ofth e ovule-theovary.The yar emain ­ thodswil lb e considered indetail . lyconnecte dwit hslowin gdow nth e tempo ofdevelopmen t andseldo m- Distanthybridizatio n withth eacceleratio n ofth ewhol e correlative intergratedsystem . Thedevelopmen to finvestigation s Toth enon-converte dbelon gth e inth efiel d of'comparativ ean dexpe ­ disturbanceswhic hchang ecorrelati ­ rimentalembryolog y ofwil dan dcultu ­ onsi nth edevelopmen t ofsom eo r redplant smad e itpossibl e toconsi -' , other structures.Thesedeviation s deri na ne w lightth epossibilitie s passth elimit so fself-regulation . ofcontro lan dregulatio n ofprocesse s Non-converted disturbances,i nthei r ofdistan thybridizatio nan d establish turn,ca nb edivide d intosevera l some.cause so fnon-crossability.Ther e groups.1.Disturbancesconnecte dwit h ison eexample.Selectionist sattemp t thegerminatio n ofth epolle nan dth e to obtainhybrid swit hth eparticipa ­ growth ofpolle ntubes.2.Disturbance s tiono fdiploi dT.monococcu mL.,beca ­ connected witha nanomalou seffusio n usethi sspecie so fwhea ti sdistin ­ ofth epolle ntub econtent sint oth e guishedb y itshig hresistabilit yt o embryo saccavit y andanomalie scon ­ manydiseases.However ,an yattempt st o nectedwit hth edisturbance so fth e obtaina cros s ofone-grai nwhea twit h mechanism ofdivergenc e ofsperms : otherspecie so fwhea thav ebeen^sel ­ forexample ,on eo rsevera lsperm s domsuccesful.Investigatio no fnorma l areretarde d inth esynergid ,wher e embryology ofwhea ti ntypica lrepresen ­ theygraduall y undergolysi san dth e tatives ofth epolyploi dseries(T.mo - endosperm isno tformed.Al lth esperm s nococcum,T.dicóccumScrank,T.aestivu m arei nth ecentra lcell.Th eeg gcel l L.)(Batygina 1959-1974)wa scarrie dout . isno tfertilize d- th eembry oi sno t Thisenable d ust ostud yal lembryolo - formed.3.Disturbanceso fcorrelation s gicalprocesse s indistan thybridiza ­ atsyngam y andtripl efusion ,th ede ­ tioni nth egenu sTriticum . velopment ofth eembry oan dendosperm . Theanalysi sof'literatur ean dth e Thecharacte ran dth edegre eo fexpre - Presentaddress :Laborator y ofEmbryology ,Komaro vBotanica lInstitute ,Le ­ ningrad,USS R 185 ssiono fanomalie sar edifferen ta t Thecultur eo fembryo sa tearl y different stageso fembry oan dendo ­ stageso fembryogenesi si son eo fth e sperm.Thati swh ywhil eworkin g out mostcomplicated ,a s»vei la smos tim ­ themethod s ofgettin gviabl eseed s inincompatibl ecrosse si ti simpor ­ portantproble mo fembryology . tantt otak ei nmin dno tonl yth echa ­ Since197 0th eLaborator y ofembry ­ racter ofdisturbances ,bu tals oth e ology ofth eBotanica lInstitut eo f time ofthei r expression.Inth ereci ­ theAcadem y ofScience sha scarrie d procalcrossin g ofone-grain(T.mono - onwor ko nstudyin gth ebehaviou ro f coccum)wit hhexaploi dspecie s(T.ae - isolated embryosi ndifferen t species stivümvar.Diamant )th estag e ofbla - offlowerin gplants.V/ ehav eworke d stomerizationi nth eembry otake splac e outa syste mapproach ,permittin gal ­ withoutan y specialdisturbances.How ­ readytoday ,t omanag e somemorphoge - ever» indirec tcrossin gth eembry o neticprocesses . stopsit sdevelopmen t atth emomen t Theproble mo findependenc e ofth e ofturnin gfro mMastomerizatio nt o embryowa sdiscussedii nliterature . organogenesis.Inreversed(back )cros ­ However,th eauthors ' solutionha s singth eformatio no fal lorgan si s beenone-sided.Th edependenc e ofth e usually observed insingularl yforme d embryofro mth ematerna lorganism ,i n embryos» andthei rdevelopmen tpasse s particular,fro mth eendosperm,wa s withoutanomalie swhic har eme twit h consideredi nconnectio nwit hsom eas ­ atdifferen t stageso fdevelopment . pects ofphylogen y andsystematics , andonl yth ecorrelatio n ofsize so f Indistan thybridizatio nreciproca l theembry oan dth eendosper mwa scon ­ crossingsar ealmos talway s"effective " sidered (Tachtajan1966).I twa sals o inon edirection.I nth ecombination so f discussedfro mth eviewpoin t ofphy ­ crossingdiscussed » mostsuccessfu l siologicalindependenc e (metabolicin ­ ill seemst ob eth ebac kcrossing » that dependence)an dabilit yt ogerminatio n is,whe nhexaploi dwhea tT.aestivu m (Raghavan1976).Th eprogres sachieve d isuse da sth ematerna lorganism ,a s inth efiel do fexperimenta lscienc e relatively fullgrain swer e obtained. enableu st osolv ethi sproble mi n Thus, embryologicalinvestigatio n âne wway . hasshow ntua tincompatibilit y inre ­ V/ehav eestablishe da ne wfeature - ciprocalcrossin gmanifest s itselfi n the embryo autonomy. Au­ allth ethre e stagesdiscusse dabove , tonomy isa specia lstructural-functi ­ butth echaracte r ofdisturbance san d onalstat eo fth eembryo ,whic hreflect ) thetim eo fthei rmanifestatio ni n itsindependenc e fromsurroundin g tis­ directan dbac kcrossing sar edifferen t suesan di smanifeste d init sabilit y Wehav ediscusse d onlyth emorpholo ­ tocomplet enorma l embryogenesisout ­ gicalmanifestatio no fvariou sdis ­ sideth ematerna l ,an ddeve ­ turbances.One ofth emos tpossibl erea ­ lopint oa norma lplan t(Batygina,Vasi - sonsfo rth edisturbance si nthes e lieva*1977-1984-).Thi si sth emos tim ­ processes,a sca nb esee nfro mvas t portantphas eo fembryogenesis ,becaus e literature (Linskens1959-1983 )ar e justwit hi tbegin sa relativel yinde ­ thepeculiaritie s ofth eimmunologi ­ pendent (autotrophic)developmen t of calrelationship sbetwee nth etissue s the embryo.Thestag eo fautonom yca n ofth ematerna lplant ,polle nan dth e be establishedb y themetho do fcul ­ pollentubes ,th ehybri dembry oan d ture invitro.I tshoul db especiall y theendosperm ,whic hlead st oth edis ­ notedtha tth edevelopmen t ofth eplan t turbances ofregulatio no fdevelop ­ inembryocultur e inth especie sstu ­ mentbot ho fa separat eorga nan dth e diedbegin swit hth estag ewhe nth e wholesyste m ofstructure sa sa whole . embryoacquire sindependenc efro mexo ­ Thisgive spossibilit yt ogeneticist s genichormones.l tmean stha tb ythi s andselectionist st owor kou tcertai n timei tcontain sa certai namoun to f approachesan dus edifferen tmethods . endogenous stimulants,physiologicall y activesubstance swhic hsuppl yth e Thecultur e ofembryos .Autonom y normalfurthe rdifferentiatio n ofth e embryoan dit sgermination.Th estag e Oneo fth emethod sfo rovercomin g ofautonom y isspecies-specifi c for non-crossability invitr oi sth ecul - eachtaxon.Probabl yw e canspea ko f turingo fth e embryo invitro.I nth e progressiveautonomizatio no fontoge ­ majority ofwork sdevote dt oembry o nesisan dconside r iti ncertai ntaxon s culture,ar euse dmainl yth eembryo s asa directe dproces s ofevolutio no f isolateda trelativel y latestage so f livingsystems . them developmentwhic hhav epasse d Thewide-sprea d opinionabou tth e inth ematerna lorganis mmos tpar t facttha tth estag efro mwhic hth eem ­ ofthei rdevelopment . bryobecome sindependen t ofth emater -

186 rialorganis m issimila rfo ral lan - andit sorgan san d otherorgan so f giospermsan di scharacterize db ydif ­ plants,especiall y aftervariuo sex ­ ferentiationo fcotyledon san dth e posures(fo rexample ,irradiation) , point ofgrowth ,ha sno tbee nconfir ­ canb euse da son eo fth epossibl e medb yus . methodso fobtainin gne wform so f Thestudie so fal lstage so fembryo - plants,i nparticular ,ric e(Davoya n genesis» and» firsto fall ,th estag e 1979.Davoyan,Batygin a1982,1984-,Da ­ ofautonomy ,i sa necessar ytheoreti ­ voyan 1983). calpreconditio nfo rapplie dinvesti ­ Complete autonomy gationssuc ha sovercomin g ofincom ­ isa specia lstructural-functiona l patibility indistan thybridization , state ofth eembry oreflectin g itsin ­ obtaining ofhaploproduction ,shorten ­ dependencefro msurroundin gtissue s ingo fth eperio d ofres to fseeds , andshow nb yit sabilit yt ocomplet e choice ofoptima ltim eo fcrops ,an d normal embryogenesisoutsid eth ema ­ someothers . ternalorganis man ddevelopjint oa nor ­ Ourdat ao nth eautonom y ofth eem ­ malplant.Autonom yo fth eembry oi s bryoar eals ouse da sa basi sfo rothe r determinedmainl yno tb ycondition s works,an dfin dthei rconfirmatio ni n ofgrowin gi ncultur ei nvitro .Spea ­ them(Davoyan,Batygin a1982,Lukyaniu k kingabou tth eautonom y ofth eembry o 1982,1983an dothers )an dca nb econ ­ andsom eothe rstructure si ti sneces ­ sidereda son eo fne wapproache so f sary,fo rconvinience ,t ointroduc e untraditionaltechnolog y ofselectio n theter m"th edegre e ofautonomy" . Aperspectiv e lineo finvestigatio n Naturally,i nnatur ether ear en ostruc ­ inselectio n isth eus eo fdihaploi d tureswhic hwoul ddevelo p incomplet e linesobtaine db ymean so fa haplo - autonomyfro ma comple xo fdifferen t producer- Hordeu mbulbosum(Ho,Jone s factors(physiological ,gravitativ e, 1980, Simpson,Snape1981,Lukjanuk , electric,etc.)B yrelativ eautonom y Ignatova 1980,1982).Bu tKash a(1975 ) ofth eembry omus tb eunderstoo dth e hasshow ncytologicall y thati ncros ­ ability ofth eembryo st odevelo pin ­ singH.vulgar ewit hH.bulbosu mther e toa whol eplan t incultur ei nvitr o takesplac e elimination ofchromoso ­ ondifferen tmedia . mesi nth eembry osac ,whic hresult s Naturally,th estag e ofisolatio n inhaploi d embryoso fH.vulgare.Afte r willb eabsolutel y differentan dwil l thisthey.ar ediploidized.Th ediffic u bedetermine dmainl yb yth econdition s ltyo fthi swor k isth eperishin go f ofculturin g specificfo reac htaxon . embryosa tearl y stageso fdevelopmen t becausethei r endospermdegenerate s Culturingo fanthe r invitro .Haploid s inth efirs tday so fdevelopment(Jen - Togetherwit hth ecultur eo fiso - sen1977,Lukyanu k1982) .Workin gou t latedembryos ,stil lmor eseriou sat ­ ofth etim eo fthei risolatio ni sne ­ tentionfo rpractica l aimsshoul db e cessary.S .P .Lukyanu kha scarrie d outa givent oth eworkin g outo fth etheo ­ adetaile dresearc ho fstudyin gauto ­ reticalfoundation s ofcultur e invitr o nomy inhaploi dan ddiploi d embryos ofbot hvegetativ e andgenerativ e ofbarle ywhic hwer eobtaine dwit h structuressuc ha sth eanthei' ,th eova ­ thehel p ofhaploproduce r (Hordeum ry, the ovule,th e embryosac ,th e bulbosum)an dha sgo ta numbe ro fpre ­ endosperman dth eembryo.Th eantne r ciousforms.I twa sestablishe dtha t being acomple xintergrate d system, thediploi d embryobecome sautonomi c fromtu emethodica lviewpoint , byth e13-14-t hda yafte rpollinatio n doubtlessly isa simple rmo ­ whenit ssiz ei smor etha n2, 0mm . delfo r investigation insitu ,i nvi ­ Atthis ,a sw ehav eshown ,th efor ­ voan di nvitro ,becaus e 01a. iti ti s mation ofit sautonom ytake splac e easiert oobserv ean d establishsepa ­ duringtw oday s(tha ti s11-12t hday s ratemorphogeneti cprocesse san dthe ­ afterpollinatio n inculture).I twa s ircorrelations.Po ra mor esuccesfu l necessaryfo rth ehapl^id so fsuc h studyo fth eanthe ra sa mode lsyste m sizet ogerminate ,t ochoos eth eme ­ iti snecessary ,firs t ofall ,t o diawit hadditio n ofdifferen tphy ­ work outa methodica lbasi so fstudies : siologically activesubstances.l t unification ofterminolog y ofstage s meanstha tthe ywer eonl yrelativel y ofdevelopment ,workin g outo fth e autonomic. mainproblem sinvolve d inth einvesti ­ Iti snecessar yt omentio nher e gation,unifie dstudie so fth eway so f thatth eselectio nproces sinclude s morphogenesis ofth eanthe ri ncultur e severalstages ,an don eo fth emos t invitr oi ndifferen t specieso fangio - important isth ecreatio n ofne w ini­ sperms,elaboratio n ofquestion so f tialmaterial•Th ecallu s jointstudie so fal lth estructure s obtainedfro m embryos ofth estamen ,th eanthe ran dth efe -

JS7 malereproductiv e sphere ofplants . toobtai nonl y equaldivisio n(Norrel l Becauseo fth efac ttha tfo rth e 1973an doth )an da 10 0% régénérants . purposeso fselectio ni ti snecessa ­ Probably,th echoic e ofth estag e ryt oobtai na mas squantit y ofhap - ofth eanthe ran dpolle ndevelopmen t loids,i nliteratur e iswidel ydis ­ forputtin gthe mint ocultur ei nvit ­ cussed,fo rexample » theus efo rcul - roca nb e exactlydetermine donl yb y turingi nvitr o ofstage so fdevelop ­ successivedetaile dstudyin go fth e mento fsporogeni ctissue ,o fmicro ­ stameno fth einvestigate d objecton ­ spores,o fmicrospores ,pollen ,th e lyfro mth eviewpoin to fsyste mapp ­ stateo fdono rplants .Ther ei sgive n roach.Thi sstag ei ndifferen tspecie s evidenceo fth einfluenc eo fvariou s willb ealway sdetermine db ydiffe ­ physiologicalfactor so nmorphogene ­ rentcomple xo ffactor san di ncerta ­ sis.Th edifferen tway so fformin g insens ei sspecies-specific.Fo rth e embryoidsar edescribed.However ,i n managing ofth eproces s ofgettin go f themajorit y ofcases ,i tca nno tb e haploidsar enecessar y additional saidfo rcertai n- wha twil lb eth e theoreticalfindings . resulto frégénérants ,i nwhic hwa y Generativestructure san dfactor so f ofmorphogenesi swil lg othei rdeve ­ envirdment( microelement s) . lopment,wh yth eway so fmorphogene ­ sisar ediffBren ti ndifferen tspe ­ Aric hexperimenta lmateria lha s cies.Thes equestions ,firs t ofall , beencollecte do nth ereactio nt oth e needa detaile dknowledg e ofth eob ­ factorso fenvironmen t atdifferen t jectinvestigate dwhic hw eshoul dad ­ periods ofontogenesis .Man ydat awit ­ viset ostud yfro mth eviewpoin to f nesst oth eimportan trol eo fmicro ­ systemapproac h elaboratedb y usan d elementsi nth egrai ncrop,i nparti ­ secondlyth estud yo fmorphogenesi s cular,boron.It sshortag edurin gth e wayso nth esyste mo freproductio ni n growtho fwhea tresult si nth edis ­ naturalcondition san di ncultur ei n turbance ofth eprocesse so fea rfor ­ vitro,thatis ,th emetho d ofdifferen ­ mation,thei routle tfro mth etubes , tiationo fvariou smorphologica l andals olea fformatio n(Troitskaya , structures. Batygina197 0).Th e detailedembryo - .logicalinvestigatio n ofthei rdeve ­ Nowadays,i nspit eo fth eeffecti ­ lopmenti nwhea ti nth ecas eo flac k venesso fsuc happroache sa shaploidy , ofboron ,ar epracticall yabsent . parasexualhybridizatio nan dinducti ­ Ourexperiment s(Batygina,Troitskay a ono frégénérant sfro msomati ccells , Alimova1966 )hav eshow ntha tth este ­ nophysiologis t orbiochemis tdealin g rility ofth espik ei nth elac ko f withthes eprocesse sknow sjus tho w boroni sth eresul t ofdisturbance s therégénérant sar eforme dfro mth e occuringi nth eanther .Anomalie s pollen.Itha sbee nknow ntheoretical ­ werefoun di never yanther.The yaffec ­ lytha tgenerativ ean dvegetativ e ». tedbot hsporogeni ctissu ean dpolle n cellsma yparticipat e inthis ,eithe r (mitosisan dmeiosis) ,a swel la sth e oneo fthem ,o rth emicrospor e (Sun ­ wallo fth emicrosporangia .I nal l derlandan doth .) .However ,nobod y ovariesther ewer edevelope dnorma l hasstudie dste pb yste pwit hth e embryosac swhich ,becaus eo fth e helpo fa ceitrafe rth esuccessiv esta ­ lacko fpollen ,degenerated .I narti ­ geso fdifferentiatio no frégénérant s ficialpollinatio no fthes eovarie s fromth epollen.Dee pan dwid eanaly ­ withth epolle nfro mplant sgrow nwit h siso fliteratur eo nmorphogenesi s boron,a 10 0 %formatio no fgrain s in culturei nvitr oan dou rpersona l wasobserved.I nconnectio nwit hth e datasho wtha t iti sno tclear :fro m introductiono fTritical e intocro p whatpolle nhaploid sar eformed :nor ­ industrywer ecarrie d outall-biolo ­ malo rabnormal ,a certai npe rcen t gicalinvestigatio no fth eperspecti ­ ofwhic hi salway spresen t inth e veexample-A D537 .Manufacturin gtest s anther. haveshow ntha ti twa sa remarkabl e Brilliantwork so fIndia nscientist s silosculture .However ,it sdrawbac k andother swh ohav ediscovere dthi s iswea kproductio n ofgrain san dal ­ phenomenonhav eestablishe dtha t a ternationo fgrains.I twa sfoun dtha t certainpercen to fhaploid si nDatu ­ alarg equantit y ofsteril epolle ni s rai sconnecte dwit ha certai nper ­ developed inth eanther s- 51, 1% cento fanomalies .Sto w( 193 6)]&av e ( 2,8- i nrye , 7,7 -i nwhea t ).A11 discoveredth echange so fsexualiza - thislead st oth elac ko fsufficien t tionan dformin go fth eembry osac s amounto fnorma lpolle nnecessar yfo r inth eanther ,thoug hi ti sknow n fertilization.I nconnectio nwit hth e thatwit hth ehel p ofdifferen tfac ­ above-mentioned,experiment so faddi ­ tors(fo rexample ,temperature )th e tiono fboro nt oA D53 7wer econduc ­ microsporema yb echanged ,tha tis , tedfo rman yyears ,a tth estag eo f formingleaves ,tha ti swhe nth efor ­ 9.Useo fnucella rpolyembryony , mationan ddevelopmen to ffemal ean d forexample ,fo robtainin gwilding s malegenerativ estructure stoo kplace , andstock s resistantt odraught , asa resul tw emanage dt oincreas e diseasesetc . theproductivit y ofth espike .Th e 10.Elaborationo fmethod so facce - preliminaryresult ssho wtha tth eamo ­ laratedreproduction :microclona l unto fth efertil epolle nha sincrea ­ reproductiono fseparat e genera­ seda tth eexpens eo fdecreas eo f tivean dvegetativ estructure si n asynchronity ofth eproces so fit s culturei nvitr o(anthers ,ovules , development.O nth esam eline swer e etc.) . carriedou twork so nth estud yo fapo ­ 11.Creationo fne wform so fplant s mixisi ndifferen tspecie so fPo a fromcallu s(includin gth einfluenc e (Batygina1977 »Batygina ,Freiber g ofdifferen tfactors )obtaine dfro m 1977,Batygina,Mametiev a1979) .I n differentreproductiv e structures Poapratensi sal ltype so fapomixi s ( inflorescence,flowe r) . havebee ndiscovere dfo rth efirs t 12.Shorteningan deliminatio no f timejändtw omethod s offormatio no f therestin gperio di nseeds . nucellarembryo s( embryoid sfro mon e 13.Creationo fth eban ko fplan t nucellarcell ,simila rt oth ezygotic , tissueso fvaluabl especie so fplant s sexualan dthrou gth eembryoni ccel l (fromdifferen tgenerativ ean dvege ­ complex (EO C).Th e analysiso flite ­ tativestructures) . ratureo nth eproble mo fmorphogene ­ 14.Findingou to fth eway so fmor ­ sisi nth ereproductio n systemo fflo ­ phogenesisi nth esyste m ofreproduc ­ weringplant si nnatura lcondition s tion.I tisjêspeciall yimportan tfo r andi ncultur ei nvitr o (Batygina themanagin g ofmorphogeneti cproces ­ 1976-1984)mad ei tpossibl et opu t sesoccurin gi ncultivatio no fgene ­ forward sometheoretica lan dpracti ­ rativean dvegetativ e structuresan d calidea sconcernin greproductio nan d organs. closelyconnecte dwxt hth emos tcomp ­ 15.Establishingo fth estage so f licatedproble m ofdifferentiatio n antherdevelopmen t indifferen tspe ­ of'variou s embryonic structures cieso fflowerin gplants ,o fpolle n ( fig- 1) . forth eeffectiv eobtainin g ofrégé ­ Besidesth easpect sdiscussed , nérants. whereknowledg e ofembryolog y isab ­ Allenumerate dproblems ,approa ­ solutelynecessary ,w eca nnot eth e chesan dmethod si nsom edegre ear e following: connectedwit hgenetics-selectio n 1.The determinationo fviabilit y works.A tpresen ti ti sdifficul tt o ofth eovule ,stigmas ,eg gcells , imaginetha tth eproble m ofreproduc ­ pollenfo restablishin g thetim eo f tioncoul db esolve dwithou t embryo- artificialpollinatio nan dpossibl e logists.However ,al lthes equestion s borderso fwork so nartificia lpolli ­ areall-biological .Thi si swh yfo r nation. theaim so fconsciou s managingo f 2.Elaborationo fmethod so fpre ­ separatestage so fontogenesi swid e servationo fviabilit y ofpollen . theoreticalgeneralization sar e 3.Cytoplasmicmal esterilit y necessarywhic hca nb edon eonl yi n ( CMS) . complexwit hothe rscience s(selec ­ 4.Elaborationo fbiologica lmethod s tion,genetics ,morphology ,cytology , ofstruggl ewit hparasiti cplants . physiology,physics ,chemistry,etc.) . 5.Obtainingo fparthenocarpi c fruitsan dsuppressio no fthi spro ­ cess. Abbraviatedreference s 6.Applicationo fth emetho do f artificialpollinatio nan dfertiliza ­ BatyginaT.B. ,1974 .Embryolog yo f tioninjectio no fsuspensio nint o wheat."Kolos" ,20 6p . ovary,combine dcultivatio no fpolle n BatyginaT.B. ,Butenk oE.G. ,1981 . andth eovules . Morphogeneticpotencie so fth e 7.Déterminâtiono fth etyp eo f embryoso fangiosper mplant s(a t apomixisfo rpossibl econservatio no f the exampleo fth egener aPaeoni a necessaryvaluabl efeatures . L.» Paeoniaceae).Bot.zh .66,11 . 8.Determinationo fth etyp eo f BatyginaT.B. ,V.E.Vasilieva ,1983 . sterility andelaboratio no fth eme ­ Systemapproac ht oth eproble mo f thodso fit sovercomin g(becrosses , embryodifferentiatio no fangio ­ obtaining ofamphiploids ,microclona l spermplants .Ontogenesis.14,3 : reproduction,condition s ofgrowin g 304-311. etc. Ho K.M., K.J.Kasha, 1975. Genetic technical bull. Ail-Union Selec­ control of chromosome elimination tion Genetic Inst., Odessa. during haploid formation on bar­ Lukjanuk S.P., 1983. The working out ley. Genetics. 81» 1o. of devices in vitro for getting Lukjanuk S.F., 1982. Early étapes of haploids of barley and triticale. growth and development of isola­ Thesis of candidate diss. ted embryos of barley. Science-

THE UNiVERSALiTy OF THE PATHWAYS OF MORPHOCENESiS IN FLOWERING PLANTS iN NATURAL CONDITIONS AND iN CULTURE iN Pathways of morphogenesis I. Embryogenesis II. Embryoidogenesis IN VITRO JN SJTU JN VlVO III. Organogenesis - gemmorhizogenesis

H - gemmogenesis m rhyzogenesis IV \IV. Histogenesis ^ m i ii m IV i II m IV

II I

II III

IV

l. - from the zygote: in the most of the flowering plants II. - from the flower: Allium odorum (Hegelmaier, 1897; Modilevski, I925, 1930), Erythronium americanum (Jeffrey, 1895), Eulophea epidendraea (Swamy, 1943) and other. - from the stem: Linum usitatissimum (Poddurnaya-Arnoldi, 1976). - from the leaves: ? - from the roots: ? HI. - from the flower: Poligonum viviparum, Poa, , Eringiu*n viviparum (Braun, 1956) and other. - from the stem and the roots: Bryophyllum (Ossenbeck, 1927), Pogonia oplfioglos- soides (Carlson, 1938, 1950), Lilium tigrinum, domestica, vitis vinipera, Prunus, Aristolochia (Sinnot, I960) and other. - from the leaves: Linaria (Van Tieghem, 1887), Cardamine pratensis (Goebel, 1908), rex (Prevot, 1938, 1939), BryophylJum calycinum (Berge, 1877; Yarbrough, 1932), Kalanchoe rotundifolia (Sinnot, 1960) and other. IV.- from the flower: ? - from the stem: in different woody plants. - from the leaves: ? - from the roots: Salix (Sinnot, 1960).

I. - from the flower: Hurdeum (Norstog, 1961), Paeonia (Batygina, Butenko, 1981), , Dactylorhiza maculata (Batygina, Vasilyeva, 1980 and other, II. - from the flower: Nicotiana tabacum (Tanaka, 1968), Ranunculus scelèratum (Norrell, 1973), Paeonia (Batygina, Butenko, 1981) and other, - from the stem: Ranunculus sceleratum, Foeniculum vulgare (Konar, Nataraja, 1965), Petunia hybrida (Rao et al., 1973) and other, - 'rom the leaves: Daucus carota, Coffea robusta (Norrell, 1973), Petunia hybrida (Rao et al., 1973) and other, - from the- roots: Daucus carota (Reinert et al., 1970, " 1973), Petroselinum (Vasil,. Hildebrandt, 1966), Panax ginseng (Butenko et al., 1968) and other. III. - from the flower: Cuscuta reflexa (Maheswari, 1961), Paeonia, Dactylorhiza maculata (Batygina, Vasilyeva, 1979, 1982) and other, - from the stem: Asparagus (Steward, , 1971), Petunia hybrida (Rao et al., 1973) and other, - from the leaves: Macleaya cordata (Kohlenbach, 1965). - from the roots: Panax ginseng (Butenko et al., 1968). IV. - from the flower: Daucus carota (Hildebrandt, 1970), Paeonia (Batygina, Butenko, 1981) and other. - from the stem: ? - from the leaves: ? - from the roots: Daucus carota and other (Hildebrandt, 1970).

190 TO THE MORPHO-FUNCTIONAL GAMETOGENESIS STATUS OP HIGHER PLANTS

A.A.Chebotaru Botanical Garden (Institute)o fth eAcadem y ofScience s ofth eMoldavia n SSR,Kishinev ,USS R

Summary relationwithi nth esystem : maternal organism -*»th esporogenou s In this communicationa nattemp t tissue ->th esexua l cells— »th e wasmad e tosummariz e theaccumula ­ zygote— *th eembry o- >th eseed . teddat aabou tgametogenesi s andt o Itwa sshow n thatth eformatio nan d indicate somepecularitie so fgamet e thedifferentiatio n ofsporogenou s formationprinciples .Bein gi na andparticularl y thehapioi dstruc ­ fullagreemen twit h theopinio n turesoccur s toa grea texten tdu e about therol eo fth ewhol ei nth e toth especifi cbac klink so fth e formationo fa particula rpar t sexual cellswit h thematerna lorga ­ (sexual cells)w ewan tals o tothro w nism (see thearrow sa tth escheme) . someligh to nth eembryona ldifferen ­ tiationproblem . sporophytej—»jgametophyt e}-^ > Introduction Atth etim eo fgamet eformatio n theserelation sar emanifeste d inth e Thegametogenesi s occupiesth e buildingo fgrowthactivate dsubstan ­ centralplac ei nth esexua lreproduc ­ ceslik eauxins ,hormone s etc.,sti ­ tiono fhighe rplants .It' sa prere ­ mulating thegrowt ho fpericarp ,th e quisite toth efertilizatio nan dt o formationo fparthenocarpi c orapo - somedegre ei tcanalise s thesubse ­ mictic fruits (seeds),th estrengthe ­ quentevent so fth eembryona ldeter ­ ningo ftrophica lan dphysiologica l mination.Gametogenesi shide si nit ­ reconstruction ofmaterna lorganis m selfimportan t ontogenesisiniti ­ inth ewhole . ations,individua l development,it s Theothe rwidesprea d events,con ­ stability andadaptability .Thes e comitant thesexua l cellsformatio n- questionsar econcerne d directlyth e thisi sth edegeneratio n ofth e problem ofadaptiv ereactio nnorm e surroundingsporophyt e cells (that's (Shmalgausen,1940) . Gametogenesis ofth ematerna l organism).Whil e is theech o ofdarwinia nselection . studuinggametogenesi s onei ssurprise d Theretrospectiv e analysiso f byth econstanc y ofth emeioti cdi ­ available information concerningth e visionnumbe rwit hstrictl yrepeatin g gametogenesis biology ofdifferen t chromosomal andbiochemica lrearran ­ taxai sfa rfro mfullness .V.A.Pod - gements observed atal lhighe rplant s dubnaja-Arnoldi (1982)ha sshow ntha t rangs.An d thoughgametogenesi si s of43 8familie s ofangiosperm s14 0 fora lon g timeconsidere d asa mai n onesar efa rfro m thecomplet e sourceo finitia lmateria l forth e embryologicalinvestigatio n oraren' t darwiniannatura lselectio nit sunder ­ investigated atall . standingi srathe rcontradictory . Atth esporo-gametogenesi s (beginning Resultsan ddiscussio n from thearchesporia l cella nendin g with thebuildin go fsexua l cells) Inth epreviou sstudie swit hth e therei ssee na constantl yrepeate d lightan d electronmicroscope s (Che- separating ofth ecommo nint ouneve n botaru,I960 ,1972 )th estructura l orpotentiall yuneve nformation s andfunctiona lpecularitie so fmal e thatstrength s thespectr eo fshed - andfemal e differentiationo fgameto - ding-elimination ofa grea t narto f phytesporogenou s cells areanalysed . apparentlylittl eadapte dhaploi d Theattentio nwa sdraw nt oth einter ­ structures.Th egeterogenou sstructur e

I.Presen taddress : Botanical Garden ofth eMoldavia nAcadem y ofSciences , Kishinev,USS R 191 -2 - formationa tth emicro- ,macrosporo - Thiswa yo fse xformatio nappearin g andgametogenesi si ndifferen tsyste ­ toa grea texten t "unstable"pro ­ maticphyl ai sregularl yaccompanie d videsnevertheles sa stead ygeno ­ by thedroppin gou t (elimination)o f typicalrenewa lwhic hi scanalise d thegeneticall y defectivehaploi d bypanmixis . structures calledb yu s a)th eprin ­ S.Gr.ïïavashin(1950 )ha sreveale d cipleo feliminatio n ofgeneticall y theenantiomorphica lnatur e ofth e defectivestructure swhic hexhibit s sisterspermcells .Thi spermitte d atth ephylo-ontogenesi sa sa pecu ­ tounderstan d theirbiologica l acti­ liarevolutionar ysiev eo fth enatu ­ vity.Apparentl y thesam ei soccu - ralselectio no nth ecel llevel .I n ringi nth efemal egametophyte , thisproces sa geneticall y fixedwa y though thereth edifferentiatio no f ofeliminatio n ofth e"exhauste d eggan dcentra l celli sa nindividua l slags" (inGoldshmidt' sunderstan ­ andver yspecifi c event.Amphimixi s ding)an d theprovidin go fth egete - ofenantiomorphica l structuresha s rogenouspotentia l bfsexua lcell s lefta genera lbiologica l imprinto n from onegeneratio nt oanothe ri s allth eflowerin gplant sari di ti s alsoseen .Pro m thispoin to fvie w formulated byu sa sc )th epricipl e thetendenc y ofgamet evariabilit y ofsynergis m ofenantiomorphica l within theecotype ,population ,phy - formations.I ti sth esynergis mo f tocenosishavin gspecifi camplitude s enantiomorphical ofmal e andfemal e ofth ereactio nnorme si squit eclear . formations thatresult si narisin g Itarise sa sa resul t ofth ereali * ofracemica l labilegenom esystems * sationo fdarwinia nselectio npres ­ (ontogenesis)whic har ebase d onth e sureo nth epas to fth epreviou s morphogeneticalhomeostasis .Th e parentalbasi san d thisi nit stur n latteri nadditio n canalisesth e causesa nadditiona l eliminationo f adaptivevariabilit y ofsporo-game - thegamete sthemselves ,th eembry o tophytesa tth eonto-phylogenesis . abortiono rappearanc eo fa steril e progeny. Gametogenesis isa ninstanc ewher e Iti snecessar y topoin tou tth e iti spossibl e tofollo w thesource s facttha tgametogenesi s thati sth e oforgani c (bioecological)advisabi ­ haploid structurebuildin goccur s lity,th ebeginnin g ofadaptiv ereac ­ onlyi nth estrictl ypolarise d tionnorm e (afterShmalgausen )an d coenocyteformations .Outsid eth e otherver yimportan tregularities . coenocyte thesexua l cellsar eno t Ingametogenesi s theactio no fdar ­ differentiating.Consequentl ymicro - winianselectio n isfollowe dan dth e andmacrospore sbefor e takingth e manifestation of theVavilov' sla w roado fsexua l cellformatio nar e ofth ehomologica l rows.I ngameto ­ subjected tocomple xstructura l and *• genesisi toccur sa har dselectio n physiological rearrangements ofth e ontl^ eleve l of meiosis,fertili ­ coenocytepattern .Ultrastructura l zationan dzygotogenesis ,Ther eth e analysiso fth emal ean dfemal ega ­ compatibilityo fpaternal(previous ) meteso fth edifferen tspecie so f genomesi sfitted'(i n themanne ro f floweringplant s (Ghebotaru,1972 , azipper . 1976;Willemse ,1972 )ha sreveale d In thegametogenesi sBaer' sla w astrikin gsimilarit y ofmal egametes . ofintegrate ddeterminatio nfind s Thusi tsuggest s thesecon dimportan t itsconfirmation .Throug hgametoge ­ conclusion thatth esexua lcell sar e nesis therei sth ewa yo fbuildin g arisinga selement so fintegrate d ofspecie sdiversity ,withou twhic h evolutionary-fixed phylogenetical theevolutio ni sunthinkable .There ­ systems.Th esmal ldifference s forei ti simpossibl e toagre ewit h between themal ean dfemal egamete s theopinio n thatth esexua lreproduc ­ (Paolilo,196 9Î Chebotaru ,197 6e tal. ) tiona sa suc hbear sonl yth epoten ­ within thespecies ,populatio nar e tialpossibilitie s ofth especie s ratherelement swhic hsupplemen t renewal (Levina,1982) .Th ehighe r eachother .Al l thispermitt st o plantsgametogenesi sha s tob econ ­ formulate thefollowin grul e- sidered"a s anorigina lye tunknow n b)th eprincipl eo f equipotentiality toth een d alphabetb ywhic hth e ofhaploi d formations (9ando*) . evolutionarywa yo fbiogeochemica l Thisprincipl ei smanifeste di nth e pastdevelopmen t (theplan tworld ) buildingo fful lgenotypica lspeci ­ iswritten .An di fth emountai n ficityi nth ecourc e ofgametogenesis . formations areremindin g thepas to f Theequiootentia lfeature s ofth e ourplane t bythei rhardene dint o gametes arerealise di nth ecourc e stonesilence ,the n theflowerin g offertilization . plantsmak e thisb ythei rconstan t

192 ontogenetiealrenewal .Th egenome s NavashinS.G. , 1950.Opi tstructur - oncearise d inth egeologica lpas t nogoizobragenij asvoist vpolovic h arerepeate d in thenumerou sgene ­ kletok.Izbr .trudi ,t.I ,p.326-36 1 rations throughgametogenesis . PaoliloD.J. , 1969.Ultrastructura l and developmental aspects ofplan t sperms.J.Deü.Bot.Univ .o fIII , References USA.Abstract sX IIBS ,USA . Poddubnaja-ArnoldiV.A. ,1982 .Charac ­ ChebotaruA.A .I960 .0 macrosporoge - teristics,semeist vpocritosemennic h nesei razviti i zarodishevogo rasteniip ocytoembryologicheski m meshka cucuruzl (macrosporogenez- priznakam.Moskva ,"Nauka" ,25 3p . osnovaranneg oontogenesa) .Trud i ShmalgausenI.I. ,1940 .Izmenchivost i institutabiologi iM PA NSSSR . ismen aadaptivnic hfor mv process e Chebotaru A.A.1972 .Embryologi a evolucii.J .Obshche ibiol. ,t.I , cucuruzi.Kishinev ,"Shtiintsa" , F 4. p.384. ChebotaruA.A. ,1978 .Som echaracte ­ WillemseM.T.M . ,1972 .Morphologica l ristics ofvegetative ,generativ e and quantitativechange s inth e andspermcel l cytoplasm ofmaiz e populationo fcel lorganelle s (Zeamai sasp .saccharat a Sturt.). duringmicrosporogenesi si n Soc.Bot .Prance .Actualité sbota ­ Gasteriaverrucosa .Act abot . niques,J N1-2 ,Reims ,Prance . Neerl.,v.21 ,K I,Dp.17-31 . LevinaR.E. ,1981 .Reproductivnaj a biologiasemennic hrastenii . Moskva,"Nauka ,9 6n .

193 SEXUAL REPRODUCTION OF SEEDPLANTS, FERNS AND MOSSES. A BACKGROUND

M.T.M. Willemse

Department of Plant Cytology and Morphology, AU, Wageningen, The Netherlands The flower The sporophytic nature of the plant, diploidy, prevails and also gene exchange The flower, the organ of reproduction, by sexual reproduction. The preference shows during its development many functions, for cross fertilization develops further. ultimately intended to produce a renewed Vegetative propagation decreases strongly. individual with good changes of survival. The flower is a continuously developing Mosses, ferns and seedferns organ which, during a long period of time and at the cost of much energy, will produce The earliest small archegoniates on land, viable seeds. Each plant species has its ferns, but also the mosses need, besides own characteristic in sexual reproduction, light, very humid conditions to survive. being marked by variations within a series Both for sexual reproduction and the excre­ of different steps. These steps comprise: tion of attractive substances, dispersed induction of meiosis, meiosis itself, cell in all directions to promote fusion of dissimi­ isolation by a callosic wall, attraction and lar gametes, water is necessary. Plant disper­ recognition, cell fusion and a resting period. sal proceeds with spores relying on abiotic These are the standard events of sexual factors such as wind and high or low humid­ reproduction in green plants. ity. The germination of the spore remains dependent on water. That characteristic, From water to land bein^ adapted partly to a wet and partly to 'a dry biotoop during the course of their The biotope for unicellular algae is water. life, is demonstrated by mosses. Spore germi­ In water the gametes are attracted by very nation, protonema , mossplant and especially specific substances produced by one partner. sexual reproduction, are dependent on water. Such substances are also operative during Dispersal is a function of the sporophyte the recognition processes. After attraction and occurs in dry conditions. Ferns also and recognition gamete cell fusion will show this ability to survive in wet and dry follow. biotoops, with spore germination and sexual In the course of life, algae as well as reproduction depending on water and spore fungi show the reproductive characteris­ dispersal being adapted to land life. tics of induction of meiosis, meiosis itself, Within the mosses and ferns there is cell isolation, attraction and recognition, a great variety in constitution and form. cell fusion, and a resting period. Also alter­ Their starts to develop in two nation in ploidy of individuals within a genera­ different ways, preparing the heterospory. tion exists. From unicellular algae to multi­ In same mosses and even more ferns a differ­ cellular algae few new elements are added ence in sexuality in the spores is expressed, to sexual reproduction. Only the organisation promoting the possibility of interbreeding. of gamete producing organs became more The number of spores produced in ferns specialized. change. The number of microspores, with The role of the sexual game in thallic their dependence of dispersal by wind, re­ and complex algae becomes more specialized mains numerous. The shape of the megaspore within the basic characteristics of sexual enlarges and their number is greatly reduced, reproduction. Multicellular or thallic algae in special cases to one. The dimension limits have extend their strategies by a higher their dispersal in the vicinity of the plant. level of differentiation, partly due to their This means that the task of sexual crossing diploid level and the possibility to use each is mainly restricted to the microspore, unicellular phase within their course of the megaspore determines the rate of dispers­ life as a starting point for a new organism al. The problem for the gametes to meet With these characteristics the land biotope each other is beautifully demonstrated by can be conquered. The possibility to change the appearence of appendages on the spores from a haploid to a diploid level enlarges to promote a collective spore dispersal. the chance of occupying more areas with Horsetails and waterferns show haptera different environmental conditions. The and anchor mechanisms. The interbreeding, first land plants inherited the possibility however, is not obvious from the point to alternate from ploidy level, oogamy, of view of one individual. The heterospory attraction and recognition mechanisms, culminates in the seedferns were the mega­ monoecy and and the spore as the spore is reduced to one, receives its nutrient unit for their dispersal. The conquest begins supply from the sporophyt and remains with the use of the ability of sexual reproduc­ there till after 'pollination'. In this respect tion and during the colonization the diversity also some of the waterferns show seedfern in land plants increases. characteristics. In seedferns diploid tissues

194 appears around the megaspore and the separa­ of the food supply in a relative short period tion of the megaspore is dependent upon of time to form a seed within one growing abscission of sporophytic tissues. Within this season as occurs in most angiosperms. This tissue the megaspore and megagametophyte initiation of embryogenesis is triggered by develops in isolation of the surrounding tissues. pollination and by double fertilization, which In the transition from diploid to haploid during starts the embryo and also the' endosperm. the meiosis, this isolation is preceeded by In gymnosperms this endosperm development the formation of a callose wall. is started after pollination during the genesis Within the mosses and ferns, self-fertiliza­ of the megagametophyte. tion as a survival strategy remains clearly The reason for the high differentiation possible, along with cross-fertilization. Incomp­ grade of the megagametophyte is its function atibility mechanisms should not be excluded in the formation of the seed. In relation to although incompatibility is scarcely reported dispersal of the plant, seed development in mosses and ferns. shows a lot of differences partly related to the biotic or abiotic factors, partly to the expected different germinating conditions. From seedfern to seedplant In that respect the seedferns develop a strate­ gy to colonize the land by the formation The longer retention and feeding of the of a presemen. In seed ferns also sporophytic megaspore on the sporophyte permits a further elements become involved in the . development and employment of the gameto- phyte and sporophyte for sexual reproduction. This longer stay on the sporophyte and the The sporophytic development for the use presence of specialized surrounding tissue of sexual reproduction of seedplants permits the micro- and megaspore to develop into a highly specialized gametophyte in which The abiotic and biotic way of transport cells get a high level of differentiation. of the microgametophyte be-comes an important This differentiation means for the micro- fact of wich the sporophyte plays a role and gametophyte the preparation for an abiotic will adapt. or, in the case of angiosperms also a biotic The pollen of gymnosperms is prepared transport, the possibility to rest, thereafter for wind transport over, in general, a long to germinate and to produce sperm cells. distance and during a long period. Ultimate­ It becomes manifest that mature pollen grains ly sucked in by a pollination droplet, the of different plants strongly differ in their pollen will reach the upper part of nucellar level of development when anthesis occurs tissue. Recognition reactions occurs between especially in relation to transport, recog­ sporophytic nucellar tissue with its exudate nition and germination. and the microgametophyte, surrounded in From the megagametophyte in gymnosperms general by an uncovered pollen wall. This a relative large prothallium develops with mechanism of recognition is unclear. one to several very specialized archegonia In angiosperms the pollen lands on the surrounded by cells, or there is a stigma. The pollen wall is covered with sporo­ simple egg cell. An egg cell receives one phytic remnants containing recognition sub­ or two sperm cells, depending on the level stances. The first pollen-stigma contact occurs of differentiation of this egg cell and on in fact between sporophyte and sporophyte. the way of transport of the sperm cells. The pathway of the pollen tube permits to In angiosperms the size of the megagameto­ add recognition between the sporophytic stylar phyte is reduced, but its consituting cells tissue and substances originating from the are highly specialized. In fact, to speak of pollen tube, a recognition between sporophyte 'the reduction of the gametophyte' draws and gametophyte. A recognition between attention to only a minor aspect in compara­ pollentube and nucellar tissue as in gymno­ tive gametophytogenesis. An egg cell contains sperms, occurs also in some angiosperms. but little storage substances and will receive In seedplants the classic attraction between one sperm cell. The egg cell is surrounded sperm cell and egg cell seems mainly to be by two synergids and by the central cell, shifted to an attraction after recognition commonly soon diploid. A degenerating synergid between pollen tube and egg cell in gymno­ will receive the two sometimes connected sperms and probably the synergids in angio­ sperm cells. Then the other synergid and sperms. In angiosperms not only the recogni­ usually the antipodals degenerate. After fertili­ tion but also aspects of the attraction are zation all haploid cells dissappear. Only the mainly taken over by the sporophyte. This ploidy level of the embryo and that of develop­ coincides with the development of the pistil ing endosperm differs. This difference in in relation to promotion of cross pollination. ploidy level after fertilization is common The mixture of different relationships between in seed plants. Probably this difference between sporophytic-gametophytic recognition to sporo­ embryo and the nutritive surroundings, haploid phytic recognition systems and the effort in gymnosperms, tri- or polyploid in angio­ to avoid self-pollination mainly by appealing sperms, is partly a result of a difference on biotic pollination, makes this system very in the start of the development of the new complicated. plant. The sporophyte provides a great deal The primitive flower construction devel-

195 ops as a nutrition source for different kinds angiosperms probably some attraction features of animals. Pollen, nectar, and the complex are taken oven by the synergids and extend flower shape are involved in the life cycle to the central cell as well. The pathways of different animals, mostly insects. Colour, of the pollen tubes are partly fixed by the odour, shape and position of the flower may sporophytic tissues in stigma and style. The attract animals in different ways. , arrival of two sperm cells is actual in some petals, stamens and pistil all can be involved gymnosperms but is resolved by a selective in this function. In this way the whole flower degeneration of one of the sperm cells in is pre-emintly an organ of communication the egg cell. Otherwise the ability to make functional in cross pollination. Within these a barrier around the egg cell after penetration plant- animal relationships the diversity in of one sperm cell seems to be lost in seed- flowers increases strongly. The shape of the plants. flower, its expression in signalling, its nutritive A rare feature of the embryo sac is the value, the way of recognition, all vary to post-zygotic abortion which occurs as a characterize their individuality. result of ovular incompatibility In ferns and mosses a kind of zygotic abortion seems The gametophytic development for the use to be unknown. of sexual reproduction of seed plants The origin of the ovule and anthers from sporangia is basically comparable and similar, Also within the structures and mechanisms especially in their functions. Heat shock of haploid tissues directly involved in sexual and breeding experiments, however, show reproduction of gametophytic origin a range that the ovule can produce pollen and the of changements occur. anther embryo sacs, the opposite type of Pollen development, as a process from gametophyte. mother cell to sperm cell, is a programmed Both mega- and microgametophytes deter­ process and prepares the future functions mine the very specific individuality of the of the pollen. However, the mature pollen sexual reproduction of each distinct species. grain shows several variable characteristics when its transport starts, depending on the The flower, organ of communication species involved. This is not only expressed in the number of cells present, but principally In the foregoing paragraphs the sexual in the possibility to germinate directly after reproduction is put forward as a developmen­ maturity or at a later point of time, its dry­ tal process during the course of time, strongly ness and the amount of Pollenkitt present. evolved in relation to the biotope, abiotic The sculpture and composition of the pollen and biotic, in order to develop diversity wall are highly specific and sustain the palyno- and to renew the individual species. On land logy. Although the relation is not yet known the flower develops, above all to avoid self- between the shape of the pollen and the sculp­ pollination. The gametophytes developing ture of the pollen wall on the one side and within the flower progressively attain a the receiving stigma surface on the other. higher level of differentiation. In conquering The more or less physically determined stickirtg landlife, the flower, in its overall functioning, forces may not be the only true factor. Also suqih as pollination and seed dispersal, became in the nature and place of recognition many involved in both abiotic and biotic surround­ possibilities are well known and yet the know­ ings, which evolved too. ledge about the basal mechanism in recognition During this conquest the plant uses the is not unambiguous , partly because of the inherited steps in sexual reproduction, ac­ diversity in interaction patterns. quired in the biotope, water. These possibil­ Embryo sac development too is a process ities are amongst otherones the use of each of several steps, which can be reduced to unicelluar step during the life cycle to form the number of mitotic divisions versus the an organism, the alternation within a gener­ formation of cell walls, the position of the ation, from a diploid to a haploid level and spindle and nuclei and the nuclear migration the other way around, which enables the or its degeneration. In fact a mixture of plant to live in different circumstances various conditions maybe present in the and which leads to a highly differentiated embryo sacs of ovules within the same ovary. sporophyte and gametophyte. The recognition Thereby possibilities to escape from sexual and attraction mechanism shifts for the reproduction within the embryo sac or the greater part from the gametes to the sporo­ ovular tissue are well known as apomixis. phyte and to the gametophyte. The prologa- In the submorphology of the embryo sac tion of the retention of the female gameto­ many diversifications can be noted at the phytes on the sporophyte ultimately results moment of its maturation. in a more simply constructed egg cell and Of the original functions connected with the formation of a seed. New and mani­ the egg cell, such as attraction, recogni­ fest is the construction of the flower, a tion and prevention of polyspermy, several product of the sporophyte which reflects alternations can be observed. Recognition the relation mainly with the biotic environ­ occurs on, or by means, of sporophytic tissues: ment. In this respect the flower is the organ attraction can be a function of the egg cell of communication, representative of a high in a number of gymnosperms; whereas in level of specialization. 196 Communication is an very important basic in as yet. The realization of the existence characteristic of life. In this context the of a reproductive calendar, the characteristics gamete fusion is the first step, the function of development, the external circumstances, of the flower a manifest accentuation. the course of the normal process and the The communication leads to a further diversi­ multiple function of the flower, are some fication and to a higher level of differenti­ of the points to keep in mind during experi­ ation. mental research. Budding, flowering and fruiting all are Reproduction research closely related to sexual reproduction and seed dispersal. Research on sexual reproduc­ Reproduction research studies the flower tion, a fascinating complex study, is flowering, in its development, complexity and diversity. and will be fruiting at both the fundamental Some of the experimental results can already and applied levels. be incorporated in our cognizance of reproduc­ tion, but owing to our insufficient in sight The author thanks Prof.Dr. A.D.J. Meeuse into the normal complicated development for the corrections and the critical reading of sexual processes some cannot be fitted of the manuscript.

197 J 8thINTERNATIONA L SYMPOSIUMO NSEXUA LREPRODUCTIO N INSEE DPLANTS ,FERN SAN DMOSSE S IW IK 20-24Augus t 1984,Wageningen ,th eNetherlands .

198 1. E.3.L. Hotke-Staal 2. J.L. van Went 3. S.D. Russell n. R.I. Pennell 5. G.A.M, van Marrewijk 6. W. Lange 7. S.3. Owens 8. T. Gaude 9. 3.H.N. Schel 10. C.3. Keijzer 11. H.G. Dickinson 12. B. Larsen 13. C. Nitsch 14. A. Souvré 15. F.M. Engels 16. N.M. van Beek 17. K. Theunis 18. R. Wiermann 19. L. Albertini 20. 3. Bednara 21. D.D. Cass 22. M. Kroh 23. A. Kadej 24. 3. Kenrick 25. 3.P. Tilquin 26. R.J. Bino 27. O. Erdelska 28. B. Walles 29. 3. Greilhuber 30. M. Ryckzkowski 31. T. Visser 32. E. Pacini 33. M. Crest i 3

199 LISTO F PARTICIPANTS Czapik, R. Dept."'ó f Plant Cytology and Embryology, Jagellonian University, Grod- Abeln, Y. Dept. of Plant Cytology and zak 52,31-04 4Krakow ,Poland . Morphology, Agricultural University, Arboretumlaan 4, 6703 BD Wageningen, Daaleman-Boersma, M. Dept. of Plant Cyto­ TheNetherlands . logy and Morphology, Agricultural Uni­ Aelst, A.C. van. Dept. of Plant Cytology versity, Arboretumlaan 4, 6703 BD Wagen­ and Morphology, Agricultural university, ingen,Th eNetherlands . Arboretumlaan 4, 6703 BD Wageningen, Dashek, W.V. Department of Biology, At­ TheNetherlands . lanta University, 223 Chestnut Street Albertini, L. Laboratoire de Cytologie S.W., Atlanta, Georgia 30314, U.S.A. et de Pathologie Végétales,E.N.S .Agrono ­ Derksen, J.W.M. Dept. of Botany, KU Nij­ mique, 145, Avenue de Muret, 31076 Tou­ megen, Toernooiveld, 6525 ED Nijmegen, louseCedex ,France . TheNetherlands . Barnabas, B. Agricultural Research Insti­ Dickinson, H.G. University of Reading, tute of the Hungarian Academy of Scien­ Dept. Botany, University of Reading, ces, 2462Martonvâsâr ,Hungary . Whiteknights, Reading, RG 6 2AS ,England . Bednara, J. Institute of Biology, Maria- Engels, F.M. Dept. of Plant Cytology Curie Sklodowska University, Ul. Akade- and Morphology, Agricultural University, micka 19,20-03 3Lublin ,Polen . Arboretumlaan 4, 6703 BD Wageningen, Beek, N.M. van. Dept. of Plant Cytology TheNetherlands . and Morphology, Agricultural Universi­ Erdelska, 0. Institute of Exp. biology ty, Arboretumlaan 4, 6703 BD Wageningen, and Ecology CBES, Dubravska 14, 81434 TheNetherlands . Bratislava,Czechoslovakia . Bell, P.R. University College, London, Dept. of Botany and Microbiology, Gower- Frans/en-Verheijen, M.A.W. Dept. of Plant street,Londo nWC1 EGB6 ,England . Cytology and Morphology, Agricultural Berkmortel, L. van de. Bruinsma Seed University, Arboretumlaan 4, 6703 BD Co., P.O. Box 24, 2670 AA Naaldwijk, Wageningen,Th eNetherlands . TheNetherlands . Gaude, T. Université de Lyon 1, 43, BD Bino, R.J. Department of Plant Cytology du 11 Novembre 1918, 69622 Villeurbanne- and Morphology, Agricultural University, Cedex,France . Arboretumlaan 4, 6703 BD Wageningen, Gilissen, L.J.W. Research Institute ITAL, TheNetherlands . P.O. Box 48, 6700 AA Wageningen, The Borzan, 2. Faculty of Forestry, Dept. Netherlands. of Forestry Genetics, P.O. Box 178,4100 1 Zagreb,Yugoslavia . Givron, D. Unité de Cytogenetique, Uni- t. versité Catholique de Louvain, 4 Place Briarty, L.G. Botany,Dept. , Nottingham Croixd uSud ,Ba tCarnoy ,Belgique . University, University Park, Nottingham Greilhuber, J. Institute of Botany, Uni­ NGT 2rd.England . versity of Vienna, Rennweg 14, A-1030 Brown, R.C. Dept. of Biology, University Wien,Austria . of Southwestern Louisiana, Lafayette, LA 70504,U.S.A . Hagemann, R. Dept. of Genetics, Martin- Luther-University, Domplatz 1, DDR-4020 Cass, D.D. Dept. of Botany, University Halle/S.,Germa nDem .Rep . of Alberta, Edmonton, Alberta T6G 2E9, Canada. Harte, C. Institut für Entwicklungsphys­ iologie, Universität zu Köln, Gyrhof- Cela Renzoni,G . Dept.o f BotanicalScien ­ strasse 17, D-5000 Köln 41, Germany. ces, University of Pisa, Via Luca Ghini 5,5610 0Pisa ,Italy . Hodgkin, T. Scottish Crop Research Insti­ tute, Invergowrie, Dundee DD2 5DA, Scot­ Ciampolini, F. Dept. of Environmental land,Unite dKingdom . Biology, Via P.A. Mattioli 4, 53100 Hoekstra, F. Dept. of Plant Physiology, Siena,Italy . Arboretumlaan 4, 6703 BD Wageningen, Costers, E. Unité de Cytogenetiqe, Uni­ TheNetherlands . versité Catholique de Louvain, 4 Place Janse, J. Dept. of Genetics, Agricultural Croixd uSud ,Ba tCarnoy ,Belgique . University, Gen. Poulkesweg 53, 6703 Cresti, M. Dept. of Environmental Biolo­ BMWageningen ,Th eNetherlands . gy, Via P.A. Mattioli 4, 53100 Siena, Jensen, W.A. Dept. of Botany, University Italy. of California, Berkeley, CA 94720,U.S.A . J.B.M. Custers, Institute for Horticul­ Kadej, A. Central Laboratory, Maria tural Plant Breeding, P.O. Box 16, 6700 Curie-Sklodowska University, ul. Akade- AAWageningen ,Th eNetherlands . micka 19,20-03 3Lublin ,Poland .

200 Kenrick, J. Plant Cell Biology Research Nijs, A.P.M. den. Instituut voor de Ver­ Centre, School of Botany, University edeling van Tuinbouwgewassen (IVT), P.O. of Melbourne, Parkville 3052, Victoria, Box 16, 6700 AA Wageningen, The Nether­ Australia. lands. Keijzer, C.J. Dept. of Plant Cytology Owens, S.J. Jodrell Laboratory, Royal and Morphology, Agricultural University, botanical Gardens, Kwe, Richmond, Surrey Arboretumlaan 4, 6703 BD Wageningen, TW9 3DS,Unite d Kingdom. TheNetherlands . Pacini, E. Dept. of Environmental Biolo­ Rieft, H. Dept. of Plant Cytology and gy, Via P.A. Mattioli 4, 53100 Siena, Morphology, Agricultural University, Italy. Arboretumlaan 4, 6703 BD Wageningen, TheNetherlands . Paré, J. Résidence 'Les Primeéeres' , 1 Rue A. d'Aubigne, 80000 Amiens, Prance. Kroh, M. Dept. of Botany, University of Nijmegen, Toernooiveld, 6525 EDNijme ­ Pennell, R.I. Dept. of Botany and Micro­ gen,Th eNetherlands . biology. University College London, Gower Street, London WC1E 6BT, United Kingdom. Lammeren, A.A.M. van. Dept. of Plant Cytology and Morphology, Agricultural Polito, V.S. Dept. of Pomology, Universi­ University, Arboretumlaan 4, 6703 BD ty of California, Davis,C A 95616, U.S.A. Wageningen,Th eNetherlands . Pretovâ, A. Institute of Experimental Larsen, K. Dept. of Genetics, The Royal Biology and Ecology of SAS, Dûbravska Veterinary and Agricultural University, cesta 14, 814 34 Bratislava, Czechoslova­ Bülowsvej 13, 1870 Copenhagen V, Denmark. kia. Leferink-ten Klooster, H.B. Dept. of Raamsdonk, L. van. Institute for Horti­ cultural Plant Breeding, P.O. Box 16, Plant Cytology and Morphology, Agricul­ A tural University, Arboretumlaan 4, 6703 6700 AA Wageningen, Th e Netherlands. BDWageningen ,Th eNetherlands . Reinders, M.C. Dept. of Plant Cytology Lemmon, B.E. Dept. of Biology, University and Morphology, Agricultural University, of Southwestern Louisiana, Lafayette, Arbóretumlaan 4, 6703 BD Wageningen, LA 70504,U.S.A . TheNetherlands . Linskens, H.F. Dept. of Botany, Toernooi­ Rowley, J.R. Dept. of Botany, University veld, 6525 ED Nijmegen, The Netherlands. of Stockholm, 106 91 Stockholm, Sweden. Longly, B. Université Catholique de Lou- Russell, S.D. Dept. of Botany &Microbio ­ vain, Labatoire Phytotechnie Tropicale logy, University of Oklahoma, Norman, et Subtropicale, Place Croix du Sud 3, OK 73019,U.S.A . SC 15 D, B-1348 Louvain-la-Neuve, Bel­ Ryczkowski, M. Institute of Molecular gique. Biology, Jagiellonian University, Al. Louant, B.P. Phytotechnie Tropicale et Mickiewicza 3, 31-120 Krakow, Poland. Subtropicale. Université Catholique de Sangwan, R.S. Lab. des Membranes Biologi­ Louvain, Place Croix du Sud 3, 1348 Lou­ ques, Université Paris VII, Tour 54-53, vain-la-Neuve,Belgique . 2 étage, 2 Place Jussieu, 75251 Paris, Prance. Lux, A. Dept.o f PlantPhysiology ,Comeni - us University, Mlynskâ Dol. B-2, 84215 Sangwan-Norrell, B.X. Lab. de Membranes Bratislava,Czechoslovakia . Biologiques, Université Paris VII, Tour 54-53, 2 étage, 2 Place Jussieu, 75251 Marrewijk, G.A.M. van. Instituut voor Paris,France . Plantenveredeling, Lawickse Allee 166, Wageningen,Th eNetherlands . Sawhney, V.K. Dept. of Biology, Universi­ ty of Saskatchewasn, Saskatoon,Saskatche ­ Miki-Hirosige, H. Biological Laboratory, wan,S7 N 0W0Canada . Kanagawa Dental College, Inaokacho 82, YokosukaCity ,Japan . Schel, J.H.N. Dept. of Plant Cytology and Morphology, Agricultural University, Munting, A.J. Dept. of Plant Cytology Arboretumlaan 4, 6703 BD Wageningen, and Morphology, Agricultural University, TheNetherlands . Arboretumlaan 4, 6703 BD Wageningen, TheNetherlands . Schulz, P.J. Dept. of Biology, University of San Francisco, 2130 Fulton Street, Nakanishi, T. Dept. of Agriculture & SanFrancisco ,C A 94117,U.S.A . , Kobe University, Kobe 657, Sheffield, E. Dept. of Botany, University Japan. of Manchester, Oxford Road, Manchester Neut, A. van der. Dept. of Plant Cytology M13 9PL,Unite d Kingdom. and Morphology, Agricultural University, Arboretumlaan 4, 6703 BD Wageningen, TheNetherlands . 201 Sniezko, R. Institute of Biology, UI. Akademicka 19, 20-033 Lublin, Poland. Souvré, A. Laboratoire de Cytologie et de Pathologie Végétales, 145 Avenue de Muret, 31076 Toulouse Cedex, France. Theunis, K. Dept. of Plant Cytology and Morphology, Agricultural University, Arboretumlaan 4, 6703 BD Wageningen, TheNetherlands . Tigerschiöld, E. Dept. of Botany, Univer­ sity of Stockholm, 106 91 Stockholm,1 Sweden. Tilquin, J.P. Dept. d'Amélioration des Plantes, University of Burundi, P.O. Box 2940,Bujumbura ,Burundi . Tuyl, J.M. van. IVT, Mansholtlaan 15, Wageningen,Th eNetherlands . Viegi, L. Dept. of Botanical Sciences, Via Luca Ghini 5, 56100 Pisa, Italy. Visser, T. Institute for Horticultural Plant Breeding, P.O. Box 16, 6700 AA Wageningen,Th eNetherlands . / Walles, B. Dept. of Botany, University / of Stockholm, S 106 91 Stockholm, Sweden. Went, J.L. van. Dept. of Plant Cytology and Morphology, Agricultural University, Arboretumlaan 4, 6703 BD Wageningen, TheNetherlands . Wiermann, R. Botanisches Institut der Universität, Schlossgarten 3, 4400 Mün­ ster/West f. ,Germany . Willemse, M.T.M. Dept. of Plant Cytology and Morphology, Agricultural University, Arboretumlaan 4, 6703 BD Wageningen, TheNetherlands . •• Williams, E.G. Plant Cell Biology Re- * search Centre, University of Melbour­ ne, Parkville, Victoria 3052, Australia. Wilms, H.J. Dept. of Plant Cytology and Morphology, Agricultural University, Arboretumlaan 4, 6703 BD Wageningen, TheNetherlands . Wojciechowska, W. Institute of Plant Genetics, Strzeszyhska 30-36, 60-479 Poznan,Poland .

202 SUB3ECT INDEX

A Abortion 96, ,111 Heterodichogamy 134 Androgenesis 76 Histochemistry 137,167,172,173 Anther development 20,21 Hybridiying agent 34 cytoplasmic nodules 21 Hybridization 115,181,185 enzyme localization 32 1 tissue interaction 20,24,25 Incompatibility 86,88,93,111,112,185 Vitro culture 76,187 bioassay 109 Apomixis embryo sac 97 apospory 160,162 intraspecific 90 detecting method 157 mechanisms 112 diplospory 157 model 102,105 parthenocarpy 165 overcoming 90,102 Autoradiography 24,28 overcoming bij CO_ 99,100,103 Autosterility 35 in ovule 116 B pollen tube behaviour 68,96,99 Breeding systems pre/post zygotic mechanism 111 S genes 114 C Callose 25,100 3 pattern 136 K zygote 116 Karyotypes 181 Connectivum 28 Cytoplasmic nodules 21 L

D M Diplospory 157 Male sterility Dimorphism 145,147 callase 40 E cytochrome oxydase 44 Embryo cytoplasmic male sterility 39,44 development 153,155,161 ektexine 48 endosperm interaction 168,171 hormones 37 nutrient supply 171 isoenzymes 42 Embryo sac mitochondria 44 abortion 96,98,111 proteins 36 egg cell 142,155 tapetal cell ultrastructure 50 ultrastructure 140,153,155 temperature 36 Embryogenesis transfer of cytoplasm 51 abnormal 167 ultrastructural changes 47 correlation types 168 vacuolation 47 differentiation 156,191 Megagametogenesis DNA chalazal cells 176 egg apparatus 142 elements in vacuolar sap 180 synergid 140 histochemistry 167, 168,172,173 ultrastructure 140 in vitro 182,183,186 Megaspore respiration rate 177 degeneration 136 somatic 183 enzymes 137 Embryoids 183,188 histochemistry 137 Endothecium 21 polarity 136 Endosperm ultrastructure 137 development 115,168 Meiosis element analysis 180 microtubules 15 microtubules 59 polarity 15 vacuolar sap 177 ultrastructure 15,16 Enzymes 32,40,42,44,137 163,166,167 Microspore glycocalix receptors 56 storage 20 Fertilization 111,115,116,131,149,153,156,166 vitro embryogenesis 76 Flower wall development 56 differentiation 134 Microsporogenesis organ of communication 194 anomalies 25 G autoradiography 24,28 Gametangia in ferns 126 callose wall 25 Gametogenesis 192 cold treatment 28 Generative cell microtubules 59,66 plastids 52,53 ultrastructure . 24 203 Microtubules 15,59,64,147 Pollination 5,165 Mitochondria 15,44 distinction cross/self 58,93 N with irradiated pollen 167 Print techniques 83 O Ovule development 135,137 R pollen tube entry 115,153 Recognition position 135 glycoproteins 102,112 respiration 177 somatic 112 sterility 95 post zygotic barrier 116 P Reproduction Phenylpropanoids 32 background 194 Phylogeny 191,194 bioassay 127 Phytoalexine 110 calendar 157 Plastids 17,52,53,146,152 physiology 172 Polarity 15,17,136 process 156 Polar nuclei SEM 125 position 150 S fusion 150 Scanning electron microscopy 125,173 Pollen Seed setting 165,166 allergens 81 Sperm cell centrifugation 74 association 143,147 cold treatment 74 composition 132,143,147 galactosidase 166 dimorphism 145,147 phenylpropanoids 32 fertilization 148,185 pigmentation 32,56 plastids 53,145 plasm 24 structure 147 surface proteins 81 Sporogenesis ultrastructure 74 mitochondria 17 viability 58 mosses 15,16 Pollen germination 165 plastids 17 inhibitors 108 polarity 17 semi vivo 72 Sterility 96,97 vitro 64,68,91 Stigma water loss 58 callose responce 100 Pollen-pistil interaction 84,112,121 events after mating 105 Pollen-stigma interaction 102,105,112 pellicle 105 contact 85 post pollination development 106 molecular model 103, proteins 105 Pollen tube surface components 83 compatible 68 surface structure 105 cytoskeletation 64 structure 84 collapse 118 Style drugs 66 canal 118 duration 96 control in wilting 121 exocytosis 71 length 95 growth 64,91,115,153 growth system 118,119 T growth quantification 119 Tapetum fluorochromes 73 cold treatment 24,28 inhibition 105,107 degeneration 33 light absorption 56 enzyme localization 32 membrane structure 71 function 20,21,25 minerals 68 Tissue interaction 20,24,25,168,171 microtubules 66 Toxin bioassay 127 semi vitro 72 U sulfurdioxide 75 Ultrastructure 15,16,24,47,50, ultrastructure 72 72,74,137,140,153,155 wall 71 Pollen wall V coating 81,106 Vacuole 47,177,180 ectexine 48 Vitrocultur e 76,182,185 exine 24,27,56 W glycocalix receptors 56 Wilting 121 light absorption 57 sporopollenin 34,56 X Y Z

204 PLANT INDEX

Bryophytes Lagenaria 21 Entodon 15 Lathyrus 95 Marchantia 125 Lilium 33,52,57,68,75,135,167 Physcomitrum 16 Limnophyton 172 Linum 168, 182 Lotus 183 Amauropelta 126 Luffa 21 Christeüa 126 Lupinus 94 Equisetum 17, 125 Lycopersicon 36,119,149 Macrothelypteris 126 Marsilea 127 Medicago 183 Pteridium 125 Momordica 21 Mukia 21 Gymnospermae Pinus 56,90, 181 Nicotiana 64,71,72, 168 Taxus 131 Oenothera 47,50,136 Angiospermae Olea 25 Acacia 88 111 Ornithopus 93 Aesculus 177 180 Oryza 53,187 Astralagus 94 Papaver 168 Bauhinia 8t Petunia 39,44,51,72,83,113,121 Brassica 83,99,100,102,105,IC (»7,166,183 Peperomia 155 Brownea m Phaseolus 168 Physiostegia 57 Caesalpinia 84 Pisum 16S Camelia 35,96,97 Plumbago 145 Capsella 7,168, 176 Poa 160,187 Cassia 84 Populus 83 Cercis 84 Potentilla 150 Cichorium 157 Prunus 113 Citrullus 21 Coccinea 21 Raphanus Comarum 150 Rhododendron 115,116 Coronilla 95 Rhoeo 24,28 Ctenolepis 21 Rubus 150 Cucumis 1,153, 165 Cucurbita 21 Sanquisorba 150 Cyclanthera 21 Seeale 54, 81 Sechium 21 Dactyiiandra 21 Sibbaldia 150 Datura 74,76 Spinacea 143 Dryas 150 Sorgum 174 Duchesnea [50 Trichosanthes 21 Edgaria 21 Trifolium 183 Eragrostis 158 Triticale 54 Triticum 34,54,185 Fillipendula 150 Tulipa 32 Forsythia 83 Fragaria 150 Vicia 93 52 Fuchsia 156 Waldsteinia 150

Gasteria 20,52,57,59,118 Zea 53,58,59,171,174 Geum 150 Gleditsia 84

Haemanthus 177 Hordeum 53,140, 187 Hosta 52

Jasione 168 Juglans 134

205 AUTHORS INDEX

3 R A Jensen, W.A. 34 Rabau, T. 157 Abeln, Y.S. 160 24,28 Jobson, S. 147 Ramm-Anderson, S. 81 Albertini, L. Reczynski, W. 180 K Renzoni, G.C. 176 B Kadej', A. 149 Reynolds, J.D. 127 Bannikova, V.P. 155 Kadej, F. 149 Ridenour, C.S. 75 Barnabas, B. 58 Kaul, V. 111,116 Roberts, I.N. 105 Batygina, T.B. 185 Kenrick, J. 88,111 Rodkiewicz, B. 17 Bednara, J. 17 Keijzer, C.J. 20 Rouse, J.L. 115,116 Beek, N.M. van 135 Kieft, H. 171 Rowley, J.R. 56 Bell, P.R. 131 73,81,88 Russell, S.D. 145 Bellani, L.M. 25 Knox, R.B. 100,111,115,116,147,166 Ryckzkowski, M. 177,180 Bereslord, G. 88 Bernardt, P. 73,88 Knuiman, B. 71 Bino, R.J. 44 Kormutâk, A. 90 S 180 Said, C. 83 Borzan, Z. 181 Kowalska, A. 71 Sangwan, R.S. 74,76 Brown, R.C. 15 Kroh, M. Sangwan-Norreel, B.S. 74,76 Bruin, J. de 51 L Sawano, M. 99 Lal, M. 16 Sawhney, V.K. 36 C Lammeren, A.A.M. van 59,171 Schel, J.H.N. 171 Cadic, A. 74 Larsen, K. 112 Schröder, M.B. 52,53 Cass, D. 140 Lemmon, B.E. 15 Schulz, P. 137 Chauhan, E. 16 191 Llewellyn, G.C. 127 Serrhini, A. 35,96,97 Chebotaru, A.A. 157 Shah, C.K'. 172,173 Choisez-Givron, D. 156 Longly, B. 157 Sheffield, E. 125 Ciampolini, F. 72 Louant, B.-P. 33 Shridhar 21 Cresti, M. 72 Lux, A. 108 Singh, D. 21 Cutter, E.G. 125 Lyon, G.C. Singh, M.B. 100,166 Czapik, R. 150 M Sniezko, R. 136 Maheswaran, G. 183 Souvré, A. 24,28 D Marginson, R. 88 Suurs, L.C.J.M. 39 Dashek, W.V. 75,127 Derksen, J. 64 Marrewijk, G.A.M. van 39,51 McConchi, C.A. 147 T Dhar, A.C. 24,28 Miki-Hirosige, H. 68 Tan, M.M.C. 51 Dickinson, H.G. 105 75 Theunis, C.H. 153 Dumas, C. 83,102 Mills, R.R. Milotay, P. 165 Tigerschiöld, E. 126 Mulcahy, D.L.M. 72 Tilquin, J.P. 35,96,97 72 Traas, J.A. 64 Elleman, C.J. 105 Mulcahy, G. Tuyl, J.M. van 135 Erdelskâ, O. 168 N Nai Yan Li 134 Franchi, G.C. 25 Nakamura, S. 99 V Franssen-Verheijen, M.A.W. 118 Nakanishi, T. Neales, T.F. 100 Viegi, L. 176 Neut, A. van der 44 G Noher de Halac, I. 47,50 W Gaude, T. 83,102 Nijkamp, H.J.J. 51 Walles, B. 56 Georgieva, I.D. 167 153,165 Went, J.L. van 135,153 Gilissen, L.3.W. 121 N.js, A.P.M. Wiermann, R. 32 Willemse, M.T.M. 57 ,118,194 H O 35,96,97 O'Neill, P.B. 100,199 Williams, E.G. 73,100,115 Habintore, E. 84 116,119,166,183 Hagemann, R. 52,53 Owens, S.J. 105 Wilms, H.J. 143,160 Harrod, G. Wojciechowska, W. 93 Harte, C. 136 P 25 Wijk, A.J.P. van 160 Herich, R. 33 Pacini, E. 102 Hervé, Y. 102 Palloix, A. 131 X Hodgkin, T. 108 Pennell, R.I. 24 Hoekstra, F.A. 121 Petitprez, M. 140 Y Hoop, S.J. de 44 Peteya, D. Yamada, Y. 68 Hough, T. 73,119 Plyushch, T.A. 155 Polito, V.S. 134 Pretovâ, A. 182 Z I Zandonella, P. 83 Zuberi, M.I. 105

206