AN INVESTIGATION INTO IN VITRO CULTURE AND PHYTOCHEMICAL ASPECTS OF SOME MEMBERS OF THE ORDER CYCADALES.

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1 AN NVESTGATON NTO N VTRO CULTURE AND PHYTOCHEMCAL ASPECTS OF SOME MEMBERS OF THE ORDER CYCADALES. by ROY OSBORNE B.SC. (HONS) (RHODES) M.SC. (NATAL). Submitted in fulfilment f the requirements fr the degree f DOCTOR OF PHLOSOPHY N THE Department f Btany, Faculty f Science University f Natal, Pietermaritzburg, Suth Africa JUNE 988

2 CONTENTS PREFACE ACKNOWLEDGEMENTS DEDCATON ABSTRACT LST OF FGURES LST OF PLATES Page: i i i iv v vi i ix LST OF TABLES LST OF APPENDCES x xii CHAPTER CYCADS N PERSPECTVE CHAPTER N VTRO CULTURE OF CYCADS 37 CHAPTER 3 CYCAD PHYTOCHEMSTRY 7 REFERENCES 76 APPENDCES 06

3 PREFACE Planning and initial cllectin f plant material fr this prject cmmenced in January 984. The majr prtin f the experimental wrk was carried ut in the Departments f Bichemistry and Btany, University f Durban-Westville, v~r the perid December 985 t December 986. Additinal experimental wrk was dne in the Departments f Bilgy and Chemistry, University f Natal, Durban, frm January 986 t April 988. The prject was supervised by Prfessr J. van Staden f the Department f Btany, University f Natal, Pietermaritzburg. Except where the wrk f thers is acknwledged in the text, these studies are the result f my wn investigatins and have nt been submitted in any frm t anther university fr degree purpses. SGNED: Ry Osbrne, B.Sc. (Hns), M.Sc. June 988.

4 ; i ACKNOWLEDGEMENTS wuld like t recrd my sincere appreciatin t Prfessr J. van Staden, his clleagues and pst-graduate students at the Department f Btany, University f Natal, Pietermaritzburg, fr guidance, encuragement and many helpful suggestins during the curse f this wrk. Fr the use f labratry facilities, am indebted t Prfessr A.O. Hawtrey and staff and Prfessr T. Steinke and staff f the Departments f Bichemistry and Btany f the University f Durban-Westville, and t Prfessr W. Meester and staff and Prfessr J.W. Bayles and staff f the Departments f Bilgy and Chemistry f the University f Natal, Durban. Amngst the many thers wh have assisted in varius ways, wish t pay tribute t the fllwing: Prfessr Mari Ariatti and Mrs. Barbara Huckett, Department f Bichemistry, University f Durban-Westville; Or. Peter Brain, Natal nstitute f mmunlgy; Prfessr Pat Berjak and Dr. Nrman Pammenter, Department f Bilgy, University f Natal, Durban: Or. Jack Cannn. Schl f Chemistry, University f Western Australia; Prfessr Pal de Luca and clleagues, Ort Btanic, Naples; the Directrs and staff f the Btanical Research nstitute, Pretria and Durban units and the Sugar Milling Research nstitute, Durban, and Or. R.A. Dyer f the frmer nstitute; Prfessr Nat Grbbelaar and clleagues, Department f Btany, University f Pretria; Mr. Jhn G. Hendricks, Statesville, Nrth Carlina, U.S.A.; Mr. Danie Nel, Natal Chairman and Seedbank Officer f the Cycad Sciety f Suthern Africa, and members f the Sciety and ther cycad cllectrs; Or. Knut Nrstg and his assciates at the Fairchild Trpical Garden, Miami, Flrida; Prfessr Msa Mtara, Department f Zlgy, University f Durban-Westville; Prfessrs Karl Pegel and David Taylr. Department.f Chemistry, University f Natal, Durban; Or. Clin Rgers, University f Durban-Westville; Prfessrs Antni and Maria Luiza Salatin, nstitut de Biciencias, University f Sa Paul, Brazil; Prfessr Dennis Stevensn, New Yrk Btanical Garden; Dr. Piet Vrster, Department f Btany, University f Stellenbsch; Or. David Webb, BC Research, Vancuver, Canada; and Mr. Ken Wyman and Mrs. Ann Lambert, Durban Btanic Gardens.

5 iii am grateful t Mrs. Lrraine van Hf fr technical assistance with the phtgraphic wrk and t Mrs. Dreen Denby, Mrs. Denise Allen, Mrs. Eileen Farr and Mrs. Jenny Hardman fr typing the varius drafts f this thesis. Financial supprt fr the experimental wrk was received frm the University f Natal, Durban and Pietermaritzburg. The Universities f Natal, Minnesta and Naples and the French Department Alpes-Maritimes kindly cntributed twards the csts f my attendance at the Sixth nternatinal Cngress f Plant, Tissue and Cell Culture, Minneaplis, Minnesta, 986, and the First nternatinal Cnference n Cycad Bilgy, Beaulieu-sur-Mer, France, 987. Finally, recrd my appreciatin t my wife and family fr prfreading the text and fr their patience and understanding thrughut this prject.

6 iv DEDCATON The wrk which is reprted in this thesis is dedicated with respect t the memry f my father, the late HARDE WARWCK OSBORNE Es tat in nachtlich he;l ger Stund ' der Herr sich durch ein Wunder kund. Den durren Stab in Priesters Hand hat er geschmuckt mit frischem GrUn (Tannhauser, Act 3, Wagner, Dresden, 845).

7 v ABSTRACT The present-day cycads represent the diverse, mdified remnants f a much larger grup f gymnsperms which flurished in the Meszic Era. The apprximately 48 surviving species f the Cycadales are sparsely distributed thrugh trpical and sub-trpical flras in a variety f habitats. Abut ne-half f the extant taxa are cnsidered endangered, vulnerable r rare and, because f their scarcity and decrative appeal, have attracted much public interest. Their slw grwth rate, the paucity f viable seeds and limited ptential fr vegetative reprductin severely limit bth the natural regeneratin and the cntrlled prpagatin f cycads. Over the past 40 years, varius attempts have been made t establish in v~ systems fr cycad cu l ture but nne has been successful in establishing a functinal prtcl fr the artificial prpagatin f these plants. The authr has made renewed attempts t establish in v~ cultures frm a range f haplid and diplid tissues frm Suth African Eneephal~~ and Stang~ species. Callus prliferatin was readily btained frm mst explant surces f mst species using a variety f media. Additin f the grwth factrs,4-dichlrphenxyacetic acid and kinetin in the 0-7 t 0-6 M range was beneficial but nt essential. Culture vessels which allwed relatively free gaseus interchange were advantageus and dark cnditins were marginally better than cnstant light. Explants frm cycad taxa which are mesic in habit gave a mre rapid respnse than similar explants frm xeric plants. Attempts t induce any frm f differentiatin ther than, r after, cal Jus frmatin were unsuccessful in all Eneephal~~ cultures, but tw frms f mrphgenesis were btained frm Stang~-derived material. Megagametphytic tissue ccasinally develped spherical utgrwths analgus t crallid rt primrdia. Mre significantly, primary rt cultures after callus frmatin, subculture and transfer t a light envirnment, regularly gave rise t meristematic znes and subsequent leaf emergence. This is the first recrded case f ~n v~ mrphgenesis f a Suth African cycad.

8 vi The rder Cycadales shws several distinctive phytchemical features, principally the presence f the unique methylazxymethanl glycside txins and a-amin-s-methylaminprpinic acid tgether with sme unusual phenlic cmpunds, flavanids, cartenids and cyclitls. Stang~ differs frm ther cycads in at least tw phytchemical aspects; the absence f biflavnids in the leaves and the absence f rhamnse and methyl rhamnse in the hydrlysed mucilages. These diffe.rences may indicate brader physilgical differences which wuld in turn explain the bserved differences in mrphgenetic cmpetence f tissues frm Sta.ng~ and Enc.e.phaaJr;t.6. Analyses f varius tissues frm these and ther cycad taxa were perfrmed with respect t misture, prtein, enzyme, txin and hydrcarbn cntent. Significant differences, bth between rgans and between taxa, were nted. The results f perxidase analyses were particularly imprtant in that high levels f this enzyme crrelate with the rapidity f callus frmatin in v~. Furthermre, a sharp increase in perxidase activity signals the nset f callgenesis in Sta.ng~ megagametphyte cultures. An imprtant incidental aspect f the phytchemical analyses is that f ptential value f these data t the taxnmist. n particular, the leaf wax hydrcarbn prfiles appear t be species-specific and are ideally suited t prcessing by numerical taxnmy cmputer prgrammes. t is anticipated that extensin f this wrk will make a signifi~ant cntributin t the reslutin f existing prblems in cycad taxnmy and, additinally, prvide a means t cnstruct phylgenetic sequences in the rder.

9 vi i Figure Figure LST OF FGURES Classificatin f gymnsperms as prpsed in PANT'S (957) mdificatin f ARNOLD'S (948) system. :.JOHNSON'S (959) classificatin f the Cycadales and STEVENSON'S (98, 985) prpsed mdificatins. Page: 4 6 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 0 Figure Figure Fi gure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 The wrld distributin f cycads 8 Deta i s f leaf structure and male cnes 4 f E~cephal~~ w~. Develpment f the male gametphyte in 9 cycads. Develpment f the female archegnium 9 in cycads. Lngitudinal sectin thrugh the vule 0 shrtly befre fertilizatin. Embrylgy f cycads. 0 Megaspraphyll frm female cne shwing attachment f tw vules. Germinatin f cycads. A generalized diagrammatic life-cycle 3 f a "typical" cycad. Scale drawings f karytypes f sme 7 E~ce.phal~~ speci es. Structure and nmenclature f cycad phyttxins. Sme simple phenlic cmpunds islated frm cycads. 4 Sme biflavnids islated frm cycads. 5 Sme cartenids islated frm cycads. 6 Mnsaccharides btained n hydrlysis f cycad 7 mucil ages. A prpsed structure fr the plysaccharide in 9 E~cephal~~ cne exudate. t

10 vii; List f Figures (cntd) Page: Figure 9 Cyclitls islated frm cycads. 9 Figure 0 (a) and (b). Perxidase zymgrams 40 frm leaf extracts f varius cycads. Figure (a) t (f). Variatins in fresh weight, 44 dry weight, misture cntent, ttal sluble prtein and perxidase activity f Stang~a megagametphytes in culture. Fi gure Figure 3 Duble diffusin serlgical tests: with 50 cycad seed prteins. mmun-electrphresis f sme cycad seed 5 prteins. Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 TLC analysis f cycad seed extracts t 53 detect phyttxins. Leaf wax n-alkane prfiles fr seedling 58 and mature Encephal~~ plants. Leaf wax n-alkane prfiles fr sme 6 Enceph~~ species and their hybrids. Leaf wax n-alkane prfiles fr E. natale~~ 63 plants frm different lcalities. Leaf wax n-alkane prfiles f Encephal~~ 68 species A dendrgram cnstructed n the basis f 73 unweighted pair-grup analysis f taxnmic distances calculated frm the alkane leaf wax cmpsitin f 4 Encep~~ taxa.

11 ix LST OF PLATES Page: Plate Encephalant~ wdl Sander, Durban Btanic Gardens, April 985. (a) One f fur large plants frm the riginal stck cllected in habitat, (b) a yunger specimen prpagated by sucker frm the plant in (a). Plate (a) Mature female and male cnes f Stang eju.a. eju pw., (b) Tw mature female cnes f Encephalant~ 6~x. 3 7 Plate 3 Plate 4 Plate 5 Plate 6 Plate 7 " Plate 8 Plate 9 Plate 0 Pl ate Callus grwth n megagametphyte frm Stang~a ~p~. (a) and (b) Phtmicrgraphs f chrmsmes frm callus tissue frm Stang~a ~p~ megagametphytes (a) Megagametphytic tissue f Stang~a ~p~ shwing the develpment f spherical structures; (b) transverse sectin thrugh ne such structure. (a) Micrspres f E. wdll; (b) pllen tubes arising frm E. wdll micrspres.in vltlt. n v..i;ta culture f EncephalaJLt.6 embrys; (a) juvenile embry f E. paucide~; (b) mature embry f E. natale~~: (c) i ntermed i ate stage embry f E. nataien.6~ (d) vigrus callus grwth n n embry f E. VmM~. Extensive callus grwth n a primary rt explant frm E. natale~~. Phtmicrgraph f chrmsmes in callus arising frm a lateral rt explant f E. wdll. (a), (b) and (c). Stages in the regeneratin f an apical meristem and a leaf arising frm a primary rt explant frm Stang~a ~p~. Callus initiatin n lngitudinally-cut surfaces f a leaflet f E. wdll

12 x LST OF TABLES Table Table 4( Table 3 ~ Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 Table 0 Table Table Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 The cnservatin status f Suth African Encepha.~~ and Stang~a species. Karytypes f varius cycad taxa as reprted in the literature. Selected media used by varius wrkers in cycad tissue culture experiments. Fur media used in tissue culture experiments. Macr-elements in varius culture media. Micr-elements in varius culture media. The effect f autclaving and shrt strage perids n the ph value f ph-adjusted Schenk-Hildebrandt media. The effect f cntainer n callus frmatin frm megagametphyte and primary rt explants f Encephal~~ natale~i6. n v~ culture f megagametphyte explants frm Stang~ ~OpU4.. Subculture f megagametphyte-derived callus frm Stang~a ~OpU4. n v~ culture f megagametphyte explants frm varius Encephal~~ species. n v~ culture f cycad micrgametphytes. n v~ culture f cycad embrys. Analysis f respnses in subculture f embry-derived callus frm Encephal~~ n v~ culture f primary rt explants frm E. natale~i6 seedlings. n v~ culture f primary rt explants frm ther Encephal~~ species. n v~ culture f primary rt explants f cycads ther than Encephal~~. Analysis f respnses by primary rt explants t varius culture cnditins. species. Page

13 xi List f Tables (cntd) Page: Table 9 Subculture f primary, rt-det~ed 06 callus frm varius cycad species. ~ Table 0 ~ v~ culture f cycad eafl ets. 0 Table ~ v~ culture f ther diplid tissues 3 frm E~ceph~~. Table Misture cntent in cycad material. 3 Table 3 Ttal sluble perxidase activity and 36 ttal sluble prtein in cycad material. Table 4 Variatin in fresh weight, dry weight, 43 ttal sluble prtein and perxidase activity f Sta~g~a megagametphytes in culture. Table 5 A cmparisn f the leaf wax alkane 57 distributin in seedling and mature E~cephaZ~~ plants. Table 6 A cmparisn f the leaf wax alkane 60 distributin in sme E~ceph~~ sp~cies and their hybrids. Table 7 A cmparisn f the leaf wax alkane 6 distributin f E~cephal~~ MtaleM~ frm different lcalities. Table 8 A cmparisn f the leaf wax alkane distributin f different species f 65 E~cephalaJLt~.

14 xii LST OF APPENDCES Appendix Appendix Appendix 3 -* Appendix 4 : Appendix 5 : On the evlutin f cycads (OSBORNE 986c) Cycad Research in the 80's (OSBORNE, 986b) The Wrld list f cycads (OSBORNE, HENDRCKS and STEVENSON, (in preparatin) n vi~ regeneratin f Stang~a ~pu (OSBORNE and VAN STADEN, 987). Suth African cycad research - prgress and prspects (OSBORNE, GROBBELAAR and VORSTER, in preparatin) Page:

15 CHAPTER ONE CYCADS N PERSPECTVE NDEX TO CHAPTER ONE Page. ntrductin. Taxnmy f the Cycadales 4.3 Distributin 7.4 Cnservatin status 9.5 Status f Encephal~~ wdil.6 General life cycle 6.7 Chrmsme numbers and karylgy Apparent sex reversals Crallid rts Cnventinal methds f prpagatin Cnclusin

16 . ntrductin The present-day grup f plants knwn as cycads* cmprise the diverse, mdified, remnants f a much larger grup f gymnsperms which flurished in the Meszic Era, reaching their zenith in the Jurassic Perid, sme 60 millin years ag. Althugh many palaebtanic recrds exist (WELAND, 906; WORSDELL, 906; ARNOLD, 953; HARRS, 96; DELEVORYAS, 975; MAMAY, 976), the exact rigln f the cycads remains uncertain. n essence, it is believed that the Palaezic pteridsperms gave rise t tw brad sectins within the grup: the cycadalean~, ancestrs f the extant cycads, and the cycadeidaleans (Bennettitales), which later became extinct. Clearly the cycads, with mtile sperm cells like their ancestral ferns, but bearing true embry-cntaining seeds like thse f mdern flwering plants, are a vital part in the btanical evlutinary jig-saw puzzle. Hw clsely related the present cycads are t their frerunners is anther pint f cntentin. The ppular view is that the extant cycads are relatively unchanged survivrs frm pre-histry ('living fssils', 'celacanths f the plant wrld'. etc.). Perhaps a better-infrmed pinin is that these plants cnstitute a vigrus and successful grup w~ich is still evlving and capable f respnding even' nw t changing envirnmental cnditins (ECKENWALDER, 980). Sme further detail f the speculatin n the evlutin f cycads is given in a ppular article by the authr (OSBORNE, 986c. which is included in this thesis as Appendix ). Althugh fr a majr,grup the number f taxa is relatively small (Sectin.l, there is cnsiderable diversity within these diecius cne-bearing plants. Amngst the grwth frms are the tall, usually unbranched, arbrescent species such as the Australian Le.p.<.dza.nu.a hpe-<- * Ftnte The cmmn name 'cycad' refers t all members f the rder Cycadales and is derived frm C!!ClL6 L.,. tl,le first genus t be described. Tl;le Oxfrd English Dictinary (978) g~es. the ngn as frm kykll6, as errr f k.ckll6, the accusative plural f ki.ox....mcll S a Greek name fr the dum palm f Egypt. Despite this etymlgy, there is n direct relatinship between the palms and the cycads.

17 3 Regel (stems up t 8 m) and the Suth African Enc.e.phalaJL.tM.tJtaMve.ltMU.6 Stapf and Burtt Davy (up t 3 m). These arbrescent frms, with a terminal crwn f frnd-like leaves, certainly d have a superficial resemblance t palms. Persistent leaf bases remain when the leaves are shed and lend structural supprt t the trunk, which is strengthened internally by a cmplex series f girdling leaf traces. Because f these systems, the amunt f secndary develpment is small in cmparisn with that f ther gymnsperms and wdy plants in general. Althugh cncentric and definable grwth znes are present in sme taxa, these d nt cnstitute seasnal increments and the typical annual rings assciated with wdy plants are absent. A substantial text has been published n the detailed stem anatmy f extant cycads (GREGUSS, 968). Apart frm the arbrescent cycads, there are many taxa in which the stem is partially r cmpletely subterranean. n sme f these frms the fliage is nevertheless cnsiderable with leaves reaching up t 3 m in ength,. as in sme specimens f En.c.e.pha.aJL.tM vill-6u.6 Lem. By cntrast the apprpriately-named Caribbean species, Za.m.ia punula subsp. pygmae.a (Sims) Eckenwalder, has a subterranean caudex nly 5 cm lng and has crrespndingly small leaves. A particularly unusual cycad is Zam~a P-6e.uciOPaJtMilic.a Yates in Seemann, which lives as an epiphyte in frest canpies in Csta Rica and Panama. Apart frm the abve vegetative features, the cycads have unique reprductive rgans and prcesses (Sectin.6). Add t these an apgetrpic rt system with symbitic Cyanbacteria (Sectin.9). and the resence f a seri es f exc us i ve phyttx i ns (Secti n 3.) and it is clear that this plant grup is far frm rdinary. The past decade has seen a resurgence f interest in the cycads in btanical and hrticultural disciplines. The frmatin f the Cycad Sciety (U.S.A.), the Palm and Cycad Scieties f Australia and New Zealand. and in particular the Cycad Sciety f Suthern Africa, has met with enthusiastic respnse and has brught tgether interactive grups f academic~, nurserymen. cnservatinists and laymen with cmmn interests. A recent bibligraphy f the living cycads (READ and SOLT, 986) extends t 67 pages. A ppular article, illustrating smething f the brad range in current cycad research prjects, has been published by the authr (OSBORNE, 986b, and is included in this thesis as Appendix ). Further testimny t the hi gh eve f interest in these plants is the success f the fi rst

18 4 nternatinal Cngress n Cycad Bilgy in France in 987. All this activity has nt been withut its prblems, and many cycads must nw be cnsidered endangered species (Sectin.4).. Taxnmy f the Cycadales A number f prpsals have been put frward which attempt t express the relatinships between and within the extant and extinct cycads and ther gymnsperms. The scheme f PANT (957) was drawn up n the basis f all available btanical and palaebtanical evidence and remains a useful phylgenetic system. The relative psiti~ns f the seven rders f living gymnsperms, in fur divisins, is clearly evident (Figure ). n this scheme, the Cycadales ;s the nly rder within the class Cycadps;da f the divisin Cycadphyta. GYMNOSPERMS CLASSFCATON Divisin l. CYCADOPHYTA Divisin. CHLAMYDOSPERMOPHYTA C ass l. Pteridspermpsida Class l. Gnetpsida Order l. Lyginpteridaes Order. Gnetales Order. Medullsales Order. Welwitschiaes Order 3. Glsspteridales -Order 4. Peltaspermales Order 5. Crystspermales Divisin 3. CONFEROPHYTA Order 6. Caytniales C ass l. Cniferpsida Class. Cycadpsida Order. Order. Crdaitales Cniferales Order 3. Ginkgales Order 7. Cycada es Class 3. Order 8. Pentxypsida Pentxylaes Class 4. Bennettitpsida (Cycadeidepsida) Order 9. Bennettitales (Cycadeideaes) Class. Ephedrpsida Order 4. Ephedraes Class 3. Order 5. Czekanwskipsida Class 4. Taxpsida Order 6. Taxales Czekanwskiaes FGURE : Classificatin f gymnsperms as prpsed in PANT's (957) mdificatin f ARNOLD's (948) system. The seven rders bxed are thse with present-day representatives.

19 5 The taxnmy wa/tilt the Cycada es is f mre di rect re evance t th is prject. Here t, the number f taxa at family, tribe, genus and species level has been variusly accmmdated and revised and early prpsals have been substantially mdified. n a cmprehensive mngraph n the subject, SCHUSTER (93) assigned all representatives t ne family, the Cycadaceae. Schuster's wrk has met with very substantial criticism. JOHNSON (959) has this t say "... Schuster s pr~fusely intrduces new and prfund cnfusins in taxnmic cncepts f every rank and, in nmenclature s blatantly cntravenes the rules f pririty, and s unreliably cites bth synnymy and specimens that the wrk is quite egreglus Virtually identical plants, even the same specimens, are referred t entirely different species. his arrangement and circumscriptin... bear almst n relatin t the real affinities.... lithe result is a jumble which almst defies disentanglement. One may sympathise with Schuster t sme extent; any attempt at drawing up a cycad classificatin based largely n herbarium specimens and with reference t live plants cultivated in quite atypical cnditins, is unlikely t succeed. Using parameters such as leaflet structure and the mrphlgy f female cnes, JOHNSON (959) drew up an authritative reclassificatin which has frmed the basis fr all subsequent revisins (Figure ). Jhnsn created three families t accmmdate the nine knwn genera and added a tenth genus, Lepidzamia Regel, species f which had been gruped previusly with MaC40zamia Miq. Tw families, Stangeriaceae and Cycadaceae, cntain nly single genera, while the family Zamiaceae is split int three tribes t encmpass all ther genera. STEVENSON (98) has separated ut Bwertia Hk. ex Hk. f. int a furth family, the Bweniaceae. Mre recently, STEVENSON (985) has revised the placement f Oitt Lindley, intrduced tw subrders t accmmdate the fur families, and has included an eieventh genus, Chigua, tw species f which have recently been fund in Clmbia (STEVENSON, p~. cmm.). n addi ti n t the new genus, C/t-i.gua. several ther speci es are in the prcess f descriptin and mre are undubtedly yet t be fund. Only the genera Bwert-i.a Hk. ex Hk. f., Lepidzamia Regel, MiC40CYC~ (Miq.) A. DC. and Staltg~a T. Mre are cnsidered btanically cmplete at this time. Oilt Lindley, ltcepltalajt.t0.6 Lehm, and MaC40zamia Miq. are largely

20 6 defined but sme changes are anticipated. The genera CyQ~ L. and Z~ L. present a number f taxnmic prblems and majr revisin is required. A survery f the current btanically-valid species (OSBORNE, and HENDRCKS, 985, 986) reveals a glbal ttal f 48 species in which EnQep~~ and Z~ cllectively cntribute abut 60% f the ttal. The current "Wrld List f Cycads" (OSBORNE, HENDRCKS and STEVENSON, in preparatin) is included in this thesis as Appendix 3. JOHNSON (959) rder CYCADALES : PRESENT CLASSFCATONS Cycadales families Stangeriaceae- Cycadaceae Zamiaceae tribes Encephalarteae i Dieae Zamieae genera StangelUa. CyC4Lll Lep.wzam.i.a V.iOOlt M-tCJt.cyC4Lll STEVENSON (98 ) rder famil ies Cycadales MaCJt.zam.i.a EltcepiU.laAt-6 Stangeriaceae Bweniaceae Cycadaceae Zamiaceae CeJt.atz.:un.ia Z.:unia. 80wenia ( tribes Encephalarteae Dieae Zamieae genera staltgelua. BwelLia CYCa-6 Lep.wzam.i.a V'<'OOt M.iCJt.OC!lC4Lll MaCJt.zam.i.a CeJt.atzam.i.a Encepha ilt.:t), Zam.i.a STEVENSON (985 and pell6. cnvn. ) rder Cycadales subrders Stangerinae Cycadinae i families Stangeriaceae Bweniaceae Cycadaceae i Zamiaceae tribes Encephalarteae Zamieae genera StaHgeJt..W 80welLia CYCa-6 Lep.wzam.i.a () M.{C.lt.cYC4Lll ( ) ( ) () (7) MaCJt.zam.i.a (4) C ejt.a.t z.:un.ia. (0) i, FGURE :.. EllceplU.laAtO-6 (46) lam.<:.a. (43) V.tOOlt (0) Cluqua (?) () JOHNSON'S (959) classificatin f the Cycadales and STEVENSON'S (98, 985 and pell6. cnvn.) prpsed mdificatins. Numbers in parenthesis indicate the number f valid species in crrespnding genera (OSBORNE, HENDRCKS and STEVENSON, in preparatin).

21 7 n parallel with many ther plant grups, it appears that the taxnmy f the cycads has reached the situatin where it is necessary t adpt a much brader perspective. Bichemical, immun-serlgical, karylgical, palynlgical and ultra-structural evidence shuld be cllectively and carefully assessed in future revisins. An verall cnsistency in taxnmic philsphy thrughut the rder is perhaps the majr lng-term bjective in this respect. The Suth African species f Eneep~~ Lehm. are fairly well defined (DYER, 965; GDDY, 984). A review f E. eugene-m~~ Verdrn currently prpses the establishment f ne new subspecies, E. eugenem~~ subsp. ~detb~ge~~ and tw new -species, E. dlm~~ and E. dy~n~ (LAVRANOS and GOODE, in preparatin)". The status f E. "reinwaldii/cerinus", E. "Msinga" and E. lebmbne~~ "var. Piet Retiefii" requires clarificatin, which may result in further new species being cnstituted. The taxnmy f the central, east, and west African species f Eneep~~ is less well defined and bth plitical and gegraphic cnstraints cntinue t inhibit research in these areas (MALASSE, F., p~. emm.).3 Distributin. There is ample palaebtanical evidence t supprt the view that cycadlike plants were well represented in Meszic times. Well-preserved cycadphyte material has been lcated in strata frm the late Triassic t early Cretaceus perids frm surces in Siberia, Manchuria, Oregn, Alaska, several islands in the Arctic Ocean, Greenland, Sweden, England, central Eurpe, ndia, Australia and the Antarctic cntinent (ARNOLD, ~53). Significantly, cycads were abundant at the time when the suthern part f Africa, Suth America, ndia, Antarctica and Australia were all jined in the massive supercntinent f Gndwanaland (DELEVORYAS, 975). n present times distributin f the extant cycads is limited t the trpical and mild temperate regins in bth hemispheres (Figure 3). The highest densities, in terms f prliferatin f species, are fund in Mexic (34 species in 3 genera), Queensland, Australia ( species in 4 genera) and Suth Africa (9 species in genera) (OSBORNE, HENDRCKS and STEVENSON, (in preparatin). Few f the present genera are internatinally widespread. Lepidzamia and Btuenia are endemic in Australia, while Cye~ extends frm the African cast thrugh the ndian cean islands t suth-east Ma~zamia,

22 8 Asia, Australia and the islands in the western Pacific. E~cephai~~ and Sta~g~a are cnfined t the African cntinent. lamia is fund frm Flrida, thrugh the Caribbean and central America t a large part f Suth America. C~atzamia and O~~ have a mre restricted distributin in central America, principally in Mexic, while M~~cyc~ is cnfined t Cuba. Mentin has already been made f the new species, C~gua, fund in Clmbia. FGURE 3: The wrld distributin f cycads. Areas marked indicate sites with the highest frequency f ccurrence fr the better-knwn species. After GDDY (984). Within this brad gegraphical distributin there is a wide variety f lcal habitats, frm the trpical rain-frests f nrthern Queensland, Australia, and sme central American lcatins, t the fairly arid savannah f sme parts f Mexic and the karrid scrub in the Eastern Cape f Suth Africa. n Suth Africa, E~cephai.~M gheij.ltciu:l.. Lem. thrives in high-altitude mntane grassland in the Drakensberg muntains. As previusly stated, Zamia p.~eudpajt~ilica Yates in Seemann ives as an epiphyte in the central American rain-frests. t is nt surprising that many species shw prnunced xeric mdificatins, typified by criaceus leaves ften wi~h spines r prickles, thick waxy cuticles and deeply recessed stmata. Cycads are usually sparsely distributed in their particular assciatins but in sme lcalities they may be at

23 9 east c-dmi nant. The "Mdj adji Pal m" frest f Ertc.e.phaiMt.6.tJt.aMve.rtO.6U.6 Stapf and Burtt Davy near Duiwesk00f in the nrthern Transvaal (DYER, 965), certain Mexican ppulatins f V-i..OOrt.6p-i..mLi.0.6um Dyer (CHAMBERLAN, 99) and stands f Ma~zam-i..a ~e.d!e.-i.. (Gaud.) Gardn. in suth-western Australia (BURBDGE and WHELAN, 98), are examples where cycads apprach dminance in the lcal vegetatin..4 Cnservatin status. Apprximately ne-half f the ttal number f cycad species have been classified as "endangered", "vulnerable" r "rare" by the Threatened Plant Unit (TPU) f the nternatinal Unin fr the Cnservatin f Nature and Natural Resurces (UCN) (GLBERT, 984). Prir t the appearance f man, it was the effect f lng term climatic changes and shrt term envirnmental catastrphes which mdified the wrlds cycad ppulatins: n the last 00 years it has been the activities f man which have placed pressure n the existing stands. Factrs cntributing t this pressure are mans cntinuing dmestic and agricultural demands n finite land resurces, the eradicatin prgramme fr sme txic species in Australia, activities f the tribal "medicine-men ll in sme cuntries and the widespread remval f specimen plants frm habitat fr hrticultural purpses. The cnservatin prblems are exacerbated by the slw grwth rate f cycads and the paucity f viable seeds. n the New Wrld, the Mexican species V-i..n c.~art-i.. De Luca and Sabat and V. C.~3utO-i.. De Luca, Sabat and Vasquez-Trres are cnsidered endangered and the Cuban M-i..~c.yc.a. c.aic.ma (Miq.) A.DC. is at risk (GLBERT, 984); The wrst threat seems t be that f large-scale cmmercial cllectin f habitat specimens fr exprt, particularly t the U.S.A. TANG (983) Qutes infrmatin f the shipment in 98 f plants f the newly-described C~atzam-i..a rto~tgi-i.. D. Stevensn frm Mexic t the U.S.A., resulting in the cmplete decimatin f ne f the tw knwn ppulatins f this species. n additin, TANG (985a) describes an peratin in which Zam-i..a ~~ac.e.a L.f. in Aitn was trucked frm Mexic t ne Califrnian nursery at the rate f plants per mnth in 98. Similarly, between 979 and 98, ver specimens f Zam-i..a de.b~ L.f. in Aitn were exprted frm the Dminican Republic t the U.S.A. (TANG, 985a). Japan has als "emerged as a majr imprter f Mexican cycads; in 98 ne shipment alne cnsisted f plants (TANG, 985a).

24 0 n the Orient, the Chinese species eyc.m panz'<:'huae.m-l6 L.Zhu and S. Y. Yang is endangered, C. nu.c.ltutzu Dyer has nt been seen in habitat since 905 and the natural ppulatin f C. ltabtatte.m-l6 C.J. Chen and C.Y. Chen was almst entirely destryed by a vilent typhn in 973 (HENDRCKS, J.G., (.Je.M. c.mm.) Of the Suth African cycads, the Threatened Plant Unit list (GLBERT, 984) describes three as endangered, seventeen as vulnerable, five as rare and the remaining three as 'insufficiently knwn' (Table ). A Natal cycad, Enc.e.pltalant~ wdil Sander, is extinct in nature; its unique status is cnsidered separately in this text (Sectin.5). Much f the trade in cycads results frm a cmbinatin f cllectr bsessin and business avarice. A number f cllectrs (ften perating thrugh dealers) and sme unscrupulus nurserymen find it mre expedient t remve mature specimens frm habitat, rather than wait the time required t btain plants thrugh prpagatin frm seed, ffsets r divisin (Sectin.0'). This plundering cntinues despite legislatin t the cntrary. At the internatinal level, the law is administered by the Cnventin n nternatinal Trade in Endangered species f Wild Fauna and Flra (CTES). mplemented in 975, CTES lists MlctOC.YC.M c.aic.ma, Stangwa wp~ and all species f Enc.e.phalant~ n "Appendix " which means that cmmercial trade in wild-cllected specimens is prhibited. All ther cycads fall under "Appendix " which requires that a permit must be btained frm the lcal management authrity t cver the exprt f any plants (CTES SECRETARAT, 98). Hwever, administratin f CTES is nt withut difficulties; f the 5 cuntries which pssess native cycads, 6 were nt signatries t the Cnventin (CTES SECRETARAT, 98). Furthermre, many f the signatries hav~ failed t prduce the required annual reprts and many f the reprts received appear t be incmplete r incrrect. Such defects can seriusly reduce the efficacy f the mnitring and cntrl prgrammes (GLBERT, 984). Apart frm the internatinal legislatin, many cuntries have attempted t restrict cycad explitatin by means f dmestic laws. Fairly stringent legislatin exists in Zimbabwe, Australia and Suth Africa but effective prsecutins are infrequent. n Suth Africa, mst f the relevant legislatin is prmulgated in varius Prvincial Cnservatin Ordinances and is administered by bdies such as the Natal Parks Bard

25 TABlE : The cnservatin status f Suth African Enc.e.phaf.Q.lL.C-6 and S-CWlgrua. species Classificatin Species Lcal ity Extinct in nature Endangered (3 species) Vulnerable (7 species) E. w~ Sander E. c.up~ R.A. Dyer E..tlOP.tnLLO R.A. Dyer E. a.u6.ltn-6 Lehm. E. ~e.~ R.A. Dyer E. ~~ (Thunb.) Lehm. E. c.y~~ (Jacq.j Lehm. E. e.uge.lte.-m~'u>~..u. Verdrn E. glteui.nc.hil. Lem. E. teelul.n-u. R.A. Dyer E. tjvl..-i..d.w., (Jacq. ) Lehm. E. ~ Verdrn E. aev~~ Stapf and Burtt Davy E. a.natu4 Stapf and Burtt Davy E. ebmbem4~ Verdrn E. Oltg~~ (Jacq.) Lehm. E. ngya.lll6 Verdrn E. pauc.idel'lta.tll6 Stapf and Burtt Davy E. p~nc.ep4 R.A. Dyer E. tu.p.tl04ll6 (Hle) R.A. Dyer E. wnbe.uz.telt-6~ R. A. Dyer Natal Transvaal Transvaal E. Cape E. Cape E. Cape E. Cape Transvaal Transkei. Natal Swaziland, S.E. Transvaal E. Cape E. Transvaal E. Transvaal, Swaziland Transvaal N. Natal, S.E. Transvaal, Swaziland, Mzambiqua E. Cape N. Natal. Zululand, S.E. Transvaal, Swaziland E. Transvaal, Swaziland E. Cape, Transkei E. Cape Swaziland, Mzambique Rare (5 species) nsufficiently knwn (3 species) E. af.tell-6.tun-u. Lehm. E. t,judvuu -gu.tue m.t Lehm. E. Cape. Transkei E. Cape, Transkei E. ehma.mtil Lehm. E. Cape E. lulta.lell-6~ R.A. Oyer and Verdrn Natal E. ~a.l-6ven04ll6 Stapf and Burtt Davy N. Transvaal E. ~~~ Bertl. f. E. vitto-6ll6 Lem. S. ~OPLL6 (Kunze) Baillard Zululand, Natal, MzambiQue E. Cape, Natal, S.E. Transvaal Transkei, Zululand, Swaziland, Mzambique E. Cape. Natal, Transkei, Zululand lglbert (984), see als HALL. DE WNTER. DE WNTER and VAN OOSTERHOUT (980). Bth these classificatins suffer frm the limitatins f being based largely n numerical representatins in herbaria. 0SBORNE, HENDRCKS and STEVENSON (in preparatin). 3The status f this species in currently under review by bth LAVRANOS and GOODE (in preparatin) and ROBBERTSE. VORSTER and VAN OER WESTHUZEN (in preparatin)

26 and varius reginal Nature Cnservatin Departments (OSBORNE, 985c). The effective patrlling f large areas is difficult and remval f plants frm habitat cntinues. Furthermre, theft f specimen plants frm btanic gardens, and even frm private cllectins seems t have ccurred with increasing frequency ver the past decade..5 The status f Enc.ephalaJLtM wclu.. Sander. Because f its extreme rarity and its majestic appearance, Enc.ephalaJLt~ wclu.. Sander is much sught after by private cllectrs and fr use in btanical gardens (Plate, Figure 4). Only ne specimen was ever discvered, the male multi-stemmed clump fund in 895 in the Ngye Frest, Zululand, by Medley Wd, funder f the Natal Herbarium and Curatr f the Natal (later Durban) Btanic Gardens (DYER, 965). Several schls f thught exist as t the rigin f this unique plant. HUTCHNSON and RATTRAY (933) state that the species is clsely allied t. aitelitultu: Lehm. (frm which. a.talem.{. was separated by DYER andverdoorn in 95) and t E. Uldebltatte:i.t)..i A. Braun and Buche frm East Africa. HENDERSON (945) had n dubt that the plant was a distinct species. DYER and VERDOORN (966) speculate that the slitary representative f the speci es is ei ther a re i c frm a arger ppu ati n whi ch became extinct r that it arse as a mutant frm E. na.talem.{.. The latter viewpint seems t be cnsistent with that f Medley Wd when he prpsed the name var. b.{.p..ttma (PRAN, 94). Each f these views is based n the classical cncept f species. The less rigid mdern view seems t suggest a series f taxa, in varius stages f speciatin, frm E. alten~te..<.n..{...{. in the Eastern Cape and Transkei, thrugh E. na.talem.{. in Natal. E. iebmbem.{. in Nrthern Natal. E. manike~~ Gilliland and related types in Mzambique, t varius east and central African taxa t the nrth. f each f these is accepted as a diffuse unit with a number f intermediate frms f indeterminate rank, then E. wci.u. may well be accmmdated in the series. A systematic bichemical and karylgical survey wuld be particularly useful in this regard and may als allw speculatin as t which taxa represent primitive r advance cnditins. An the trunk and sucker material frm the riginal E. wclu.. clump was remved ver the perid (OSBORNE, 986a). Fur plants were re-established in the Durban Btanic Gardens and remain as very

27 3 PLATE Ene~phatant~ w~ Sander Durban Btanical Gardens Apri 985. (a) One f fur large plants frm the riginal stck cllected in habitat. (b) A yunger specimen, estimated at 5 years f age, prpagated by sucker frm the plant in (a) abve. a. b.

28 . FGURE 4: Details f leaf structure and male cnes f Eneep~ w~ (frm "Cycads f Africa" by D. Gde, presently in print, and reprduced in reduced scale by kind permissin f the authr). a: Lwer, mid- and terminal leaf detail. b: Transverse sectin f rachis shwing attachment f median leaflets. c: Cluster f male cnes at the crwn. d: Details' f male cne scales. 4

29 5 impressive specimens at present. One was planted in the grunds f the Unin Buildings at Pretria but was subsequently lst in a transplanting peratin (DYER, R.A. p~. cmm.). Anther segment was planted at the Durban hme f Medley Wd's assciate, Maurice Evans and has since fund its way int a private cllectin in Klf, Natal (THORPE, E., p~. cmm.). The final prtin t be accunted fr was shipped t Kew Gardens and remains n display in their trpical cllectin (OSBORNE, 986a). The presence f a large plant. thught t be E. wdii (DYER, R.A., p~. cmm.), in the Old Frt Gardens in Durban. and anther large plant thught t be the same species, in a private cllectin in a garden in the Durban area, remains unaccunted fr. Despite numerus excursins int the Ngye and Mtunzini areas, n ther specimen f E. wdii was ever lcated althugh it is nt impssible that ne r mre plants may exist in sme remte psitin (GARLAND,., p~. cmm.). Peridic rumurs f a secnd plant are smetimes heard but are regarded with sceptism; all the 'wdii's' s far investigated have turned ut t be rather brad-leaved varieties f E. nataie~~. Regrettably then the species must nw be accepted as extinct in nature. Frtunately, E. wdii prduces ffsets readily. Careful vegetative prpagatin has resulted in a fair number f plants being established in ther majr btanical gardens and private cllectins thrughut the wrld (OSBORNE, 986a). Since all these plants represent ne male clne, it is nt pssible t prpagate the species frm seed. T avid the ttal dependence n cntinued prpagatin frm the riginal clne, tw avenues may be explred. The first apprach is being adpted by Cynthia Giddy at her cmmercial nursery at Umlaas Rad in Natal, and has been described by SPERS (98). Pllen frm cnes f the existing E. wdii stck is used t fertilize E. nataie~~, the species which seems t be mst clsely related. Females frm this hybrid generatin (F l, 50% E. wdii) are back-crssed with mre E. wdii t give anther generatin a little clser t pure E. wdii (F, 75% E. wdii). This prcess is repeated until an almst pure strain is established, e.g. the Fs generatin wuld have 97% E. wdii character. A disadvantage lies in the length f time frm ne generatin t anther. Assuming years fr each batch t reach maturity, the 5-generatin prces~ will take 60 years.

30 6 The secnd pssibility frms in large part the mtivatin fr this prject. Either haplid cells in the frm f E. wdii pllen, r diplid tissue frm apprpriate meristematic regins f E. wdii plants, wuld be used in establishing cultures (Sectin.). Smatic fusin f the haplid cells may be artificially induced and the resulting tissue, after mrphgenesis, may give rise spntaneusly t a prprtin f female prgeny. Alternatively, successful culture f diplid tissue with mrphgenesis wuld prvide a substantial number f plantlets which may be used in experimentatin, under varius chemical treatments r envirnmental cntrl regimes with the pssibility f sex-reversal (Sectin.8), t yield the female phentype. The prductin f a female E. wdii plant thrugh these means wuld be f enrmus benefit t grwers and cnservatinists alike and wuld als have majr academic valu~~.6 General life-cycle. The sexual reprductin prcess in cycads is well-dcumented; the fllwing general ntes are drawn frm the texts f CHAMBERLAN (99), DYER (965), PANT (973), FOSTER and GFFORD (974), and GDDY (984); this sectin als frms the basis fr a ppular paper by the authr (OSBORNE, 986d). t is stressed that the life-cycle and ntgeny described belw refer t an entirely hypthetical 'typical cycad'; cnsiderable minr variatins ccur between different taxa - details f which are beynd the scpe f this text. n nature cycad plants ~enerally prduce male r female ~ cnes (strbili) seasnally, and it is nly at the time f cning that the gender f the individuals can be psitively established. Each cne cmprises a large number f mdified leaves (sprphylls) arranged spirally abut a central axis. Each plant can prduce several male r female cnes at a particular cning 'seasn'. The male cne is cmpact at the time f emergence and elngatin f the central axis allws separatin f the micrsprphylls t reveal numerus pllen-sacs (micrsprangia) (Plate a, Figure 4). Within each micrsprangium at an early stage f develpment meisis results in the prductin f a micrspre nucleus, the first cell f the male haplid gametphyte. Divisin f this nucleus gives a prthallial cell and an

31 7 PLATE a Stangeria eripus, Durban Btanic Garden, April 985. Mature female cne (centre) with a male cne frm an adjacent plant (right). The male cne is at the pllen-shedding stage. Part f a leaf is seen n the left. PLATE b EncephaZarts ferx, Durban Btanic Garden, April 985. Tw large female cnes at a pllen-receptive stage.

32 8 antheridial initial which in turn divides t give a generative cell and a tube cell. At this three-celled stage the partially-develped male gametphyte, enclsed within the riginal spre wall, is released frm the micrsprangium as a pllen grain (Figure 5). Earlier wrkers believe the pllen t be wind transprted (CHAMBERLAN, 99, 935) but there is nw.substantial evidence f species-specific insect vectrs in pllen transfer (NORSTOG and STEVENSON, 980; NORSTOG, STEVENSON and NKLAS, 986; TANG 987a). The pllen dehiscence event is usually accmpanied by a prnunced temperature increase in the male cne (JACOT-GULLARMOO, 958); this thermgenesis fllwing a circadian rhythm (TANG, 987b) and assciated with high rates f starch and lipid cnsumptin (TANG, STERNBERG and PRCE, 987). t is thught that the heat prductin results in the vlatilizatin durs which are recgnized by the spec; fi c insect p i natrs (TWl, 987b-; TWl,. STERNBERG and PRCE, 987). ' The larger, female cnes (Plate b) are made up f megasprphylls each f which bears tw vules n its lwer surface. Within the nucellar tissue f the vule, meisis results in a haplid cell which frms a linear tetrad f megaspres, nly ne f which develps int the female gametphyte. Numerus divisins take place t give a cellular mass, certain cells at the micrpylar end (i.e. ppsite the pint f attachment t the megasprphyllj becme differentiated int archegnial initials. These' divide t give a small primary neck cell, which in turn divides t give tw r mre cells, and a large central cell (Figure 6). Shrtly befre the fertilisatin event, the nucleus f the central cell divides int a ventral canal cell nucleus and a large egg cell, but n wall is laid dwn between these nuclei. At this time elngatin f the female cne axis, particularly at the uppermst zne, allws entry f the pllen grains ver a 3 t 4 day perid. The pllen grains becme trapped in a mucilaginus liquid secreted thrugh the micrpyle f the vule and, as this fluid dehydrates, the micrspres are drawn int the pllen chamber, the desiccated gel effectively sealing ff the micrpylar rifice. After entry int the vule, the micrspre extends its tube cell int an archegnial chamber arising thrugh degeneratin f the neck cells (Figures 5 and 7). At this time the micrspre generative cell divides t prduce a sterile cell and a terminal spermatgenus cell (bdy cell) frm which tw large spermatzids with numerus cilia differentiate. Fusin f the spermatzid with the female egg cell cnstitutes the

33 9 Tube cell Prthallial Exine fgure 5: Develpment cf the male gametphyte in cycads (much enlarged), Left: The pllen-grain in transverse sectin shwing the three-celled-stage at the time f dehiscence. Right: Extensin f the pllen tube after pllinatin; the spermatgenus cell is sti l l t divide t frm tw mtile spermatzids. (Redrawn frm SWAMY, 948) We'".: ". '; " ':.~ Neck cells Central cell ~~~~ Archegnial jacket ~, ~. fl~re 6: Develpment f the female archegnium in cycads (much enlarged) Left: An archegnial initial differentiated in the female gametphyte. Centre: Divisin int a primary neck cell and a central cell. Right: The archegnium shrtly befre fertilisatin; the central cell nucleus is still t divide t frm the ventral canal cell nucleus and the egg cell nucleus. (Redrawn frm CHAMBERLAN, 935).

34 0.. i Micrpylar pening Male gametphytes One f tw archegnia after divisin f the central cell nucleus -. ~. "!")mt----- Fema e gametphyte ut nner stny layer 7"'" _ Outer fleshy layer flgllle 7: Lngitudinal sectin thrugh the vule shrtly befre. fertilisatin (Actual size). Suspensr Clerhiza Suspensr Apical Meristem Hard seed cat Embry Ctyledn fgure 8: Embrylgy f cycads (actual size). Left: Right : Early stage f embry develpment, at abut the time f shedding f the seed, shwing the characteristic ciled suspensr. Later stage f embry develpment, shrtly befre germin atin.

35 fertilisatin event. The spermatzids f cycads (abut 0,3 mm in diameter) are the largest sperm cells in either the plant r the animal kingdm (NORSTOG, 980b). The whle prcess, frm ingress f the pllen t fertilisatin, takes several mnths. The zygte resulting frm the fertilizatin prcess underges numerus successive divisins fllwing which cell walls are laid dwn. Certain f these cells differentiate int the embry, with a cnspicuus merismatic regin frm which the functinal rgans arise, and a psterir regin f elngating cells which mark the rigin f a massive ciled suspensr unique t the cycads. This is clearly visible in lngitudinal sectin f the seed and is the nly true, albeit destructive, methd f assessing seed viability (Figure 8). At this stage the cne disintegrates and the megasprphylls bearing the seed are shed. Sme f the female cycad cnes are the largest knwn in either living r extinct plants. ndividual Encephai~~ cnes may attain a mass f 30 t 40 kg and each bears several hundred seed. Cycad seeds vary widely in clur, shape, size and rnamentatin. Such variatin seems t be species specific and seed mrphlgy may well prvide useful additinal evidence fr future taxn9mic reviews in cycads (OSBORNE. 988). After the seed has been shed, the embry cntinues t develp fr abut six mnths t give a. meristematic apex surrunded laterally by tw welldevelped ctyledns which fuse at their base int a zne f tissue knwn as the clerhiza (Figure 8). t is this rgan which ruptures the seed cat at the micrpylar end and prtrudes t give the first external evidence f germinatin. The ctyledns remain permanently within the seed functining as haustrial rgans until the gametphyte reserve is depleted. The emergent clerhiza ;s penetrated by the rt tip and later by the plumule frm which the first f the true leaves f the new plant arise (Figures 9 and 0). The sequence f events described abve is in general terms similar t that fr all higher plants: a dminant sprphytic generatin bears specialised sexual rgans in which meisis reduces the chrmsme number frm the diplid (n) t the haplid (n) cnditin; the haplid gametphyte is much reduced and increasingly dependent n the hst sprphytic tissue; and fertilisatin is accmplished by plasmgamy and karygamy. t restre a diplid zygte, the first cell f the new sprphyte. A generalized life-cycle diagram fr cycads is given in Figure.

36 . fgllre 9' Megasprphyll frm female cne shwing attachment f tw vules. (Actual size). FGURE 0: Germi nat i n f cycads (reduced x t) Left: Right: Rt tip emerging thrugh cl erhiza. First-leaf stage with welldevelped tap rt and evidence f crallid rt develpment, abut 3 mnths after germinatin. (redrawn frm GDDY, 984).

37 3...J CL LFE CYCLE ADULT PLANT- f 9 ADULT PlANT '" embry Megastrbilus suspensr, MegaSprPhtls Zygte Ovu les, M icrstrbil us M icrsprphylls micrsprangia...j a.. «MEOSS egg nucleus ventral canal nucleus 4 Megaspres \ central cell megagametphyte micrspres neck cell / '"---archegnial"""""" generative cell initials tube cell sperm cells /prthallial cell ~ Spermatgenus cell ~ FGURE : A generalized diagrammatic life,-cycle f a "typical" cycad..7 Chrmsme numbers and karylgy. Examinatin f the chrmsmes frm diplid cycad material is cnveniently carried ut using actively-grwing rt tips and yung leafets, while expanding megagametphytes and pllen mitses prvide samples fr haplid

38 4 chrmsme wrk. Because f the large size f cycad chrmsmes it is usually advisable t pre-treat the material chemically prir t fixing, hydrlysis and staining. A number f reagents including ~-brmnaphthalene, clchicine, 8-hydrxyquinline and. especially isprpyl-n-phenyl carbamate are emplyed fr this purpse. Acet-rcein, acet-carmine and the Feulgen system are used t.stain the chrmsmes althu.gh mre sphisticated techniques have applicatins in detailed mrphlgical analyses. Within the gymnsperms generally, chrmsmes frm different taxa are remarkably unifrm (KHOSHOO, 96); this is true t in mst cycad genera. Generally cnsistent diplid (n) numbers have been reprted fr StaltgVLia (6), CeJtatzanU.a (6), Etlc.e.phai.aJLt0.6, MaCJtzanU.a, Le.p'<'dzanU.a, V'<'OOt, Bwe.tt.<.a (all 8), Cyc.~ () and M'<'CJtc.yc.~ (6) (SAX and SEAL, 934; KHOSHOO, 96; ABRAHAM and MATHEW, 96; MARCHANT, 968; STOREY, 968; SEGAWA, KSH and TATUNO, 97; MORETT, 98; VOVDES, 983). Of these authrs, the mst extensive wrk was that f MARCHANT (968) wh surveyed the cycad cllectin f the Ryal Btanic Gardens at Kew and included in his wrk a detailed cytlgical study f 6 species f Etc.e.phai.aJLt~. The exceptin in this pattern f cnsistency is seen in the genus ZanU.a where variatin has been reprted bth between and within species. NORSTOG (980a) examined 4 species f ZanU.a and fund diplid number f 6 and 8 t ccur inmst taxa-. Z. ac.umi.nata Oersted frm Panama and Z. ~c.ata Willd. frm Venezuela had 4 chrmsmes. n three speci es, Z. -6fU.tmVLi Warcz. frm Central Ameri ca, Z. R.ddi.9e.-6U Mi q. frm Mexic and Z. dt.<.gua Seemann frm Clmbia, mre than ne chrmsme number was apparent. n particular, the l. c.hj.gua material revealed a range frm n = t 6 (NORSTOG, 980a, 98). An analgus situatin seems t be present in Z. paucijuga Wieland frm Mexic where MORETT and SABATO (984) reprt chrmsme cunts n = 3, 5, 6, 7 and 8. The discussin f chrms6me mrphlgy and cycad karytypes which fllws requires sme terminlgy t be intrduced. Chrmsmes typically have tw arms attached at a central pint f cnstrictin, the centrmere, such chrmsmes being called 'biarmed '. Where the arms are equal in length s that the centrmere is median, the chrmsme is described as 'metacentric ' (M). When the arms differ in length, the shape is called submetacentric' (S). When there is nly ne arm, i.e. the centrmere is terminal, the structure is described as 'telcentric' (T). The kartype is the cmbinatin f these different types f chrmsme t

39 5 make up the ttal cmplement and is usually expressed as xm + ys + zt where x + y + z = n, the diplid number. Occasinally the wrd 'acrcentric' (A) is used t describe a chrmsme with a subterminal centrmere. The accmpanying table (Table ) lists mst f ~he knwn cycad karytypes, while Figure illustrates the patterns btained in 0 species f E~cephal~~ with a cnsistent karytype (8M + los, n = 8) (MARCHANT, 968). A great deal f speculatin has been made as t which karytypes represent primitive r advanced cnditins, with prcesses r centrmeric fissin and fusin prcesses being invked t explain increases and decreases in chrmsme numbers respectively (HENDRCKS, 98). The fissin prcesses lead t a greater prprtin f telcentric chrmsmes and a karytype which is described as asymmetric. By cntrast, fusin reduces the number f telcentrics and leads t a mre symmetric pattern. NORSTOG (980a) pints ut that cycads have a lng histry f gegraphic islatin and warns against ver-zealus attempts at seeking crrelatins between karytype and mrphlgical characters. This is particularly true in intergeneric cmparisns as nearly all cycad genera shw cmbinatins f primitive and advanced mrphlgical characters and diversity in degree f eclgical specialisatin. The unique situatin in lamia deserves mre attentin. n l. chigua, which is a trpical arbrescent lng-lived species where the habitat is unlikely t create strng selectin pressures, ne may anticipate a primitive status (NORSTOG, 980a, 98). The high-numbered asymmetric karytypes may thus be cnsidered a starting pint in lamia evlutin and the prgressin twards reductin and symmetry in karytype leading ultimately t the Caribbean types, exemplified by l. pumiia. Against this argument is the reprt n l. paucijuga (MORETT and SABATO, 984) which has a range f kartypes similar t l. chigua, but is cnsidered an advanced species in view f its subterranean stems and reduced vegetative and reprductive structures. The pssibility exists that karytype changes, in terms f fissin and fusin, might be bidire~tinal (NORSTOG, 980a), thus making any crrelatin between karytype and phentype even mre difficult t access. Althugh there is substantial evidence f mnecius character in the Cycadeidales, an extinct rder f Cycadphyta, the present-day cycads

40 6 TABLE. Ka~ytypes f vari us cycad taxa as reprted in the i terature. M - metacentric, S - submetacentric, A - acrcentric and T - telcentric Genus/species Diplid Number (n = ) M Kartype S A T Authrs 80welUa Cyc.a { SAX and 8EAL, 934 MARCHANT, 968 SAX and 8EAL, 934 MARCHANT, 968 SAX and 8EAL, 934 MARCHANT, 968 V.wn lamia l. lltgu4.t40ua Z. ~cjtl!ll.i. l. plltjt..ic.emi, Z. pwnda. Z. pygmaea { MARCHANT, 968 VOVDES, 983 SAX and SEAL, 934 MARCHANT, 968 { MOREn, 98 MARCHANT, 968 SAX and SEAL, 934 SAX and 8EAL, 934 MARCHANT, 968 { VOVDES, 983 SAX and SEAL, 934 MARCHANT, 968 NORSTOG, 980a VOVDES, 983 Z. Unden.U l. P4e.udp~ca Z. tuejtckhwn.u: NORSTOG, 980a l. ~ac.ea. Z. td.cu.gu.i.(. l. buqua Z. 4k.Utnelt..i NORSTOG, 980a Z. td.cu.gu.i.(. var. angll4.t40ua VOVDES, 983 Z. /lllllt..ic.ata Z. ac.wninata NORSTOG, 980a Z. 6wt.6wtac.ea. X z..e.d.cu.g u.i..i.? NORSTOG, 980a Z. cjt.i.qua Z. pau.c..4uga NORSTOG, 98 MOREn and SABATO, 984

41 7 """lllllii.. j ""/ / " " " /!! f!. : b 9..!i./ / ;J. / " / " / c h : i. n ' /.. d " / ". " / " " " " e n ' j FGllE : Scale drawings f karytypes f sme Encep(talCVLtO-6 spp. x 900. a. E. cyc..d.ilt~, b. E. wnbe.fuz..tem.i-6, c. E. lna.ukem.i-6, d. E. auemtun-u, e. E. (.U-i. M f. E. ehmanlm. g. E. viil-6l6 h. E. (u:.e.debll.a.ndt-u i. E a.ev4urv., j. E ftll.jl.uill6. After MARCHANT (968)..

42 8 are unifrmly diecius (CHAMBERLAN, 935). The questin as t whether ne can predict the gender f a particular cycad plant frm karylgical studies has nt been cmpletely reslved. ABRAHAM and MATHEW (96), in a cytlgical study f Cyca~ pectinata Griff. (n = ). fund that ne f the submetacentric chrmsme pairs shwed a satellite attachment t bth chrmsmes in female nuclei but that nly ne f the pair was similarly satellited in material frm male plants. The authrs thus cnclude that the chrmsme pair which is hetermrphic in the male must be assciated with sex expressin. Accrding tshetty and SUBRAMANYAN (96), the karytypes f Cyc~ beddmei Dyer, C. ~cin~ Land C. ~eviuta Thunb. are all similar except fr a pair f hetermrphic chrmsmes frm male specimens f the latter tw f the three species. MARCHANT (968) reprts a similar hetermrphic pair frm Stang~a ~p~ (Kunze) Baillard (n = 6) but was unable t crrelate the cytlgical infrmatin with sexual phentype. The Japanese wrkers, SEGAWA, KSH and TATUNO (97) ha~e reprted distinctly-recgnisable sex chrmsmes in Cyc~ ~eviuta. A general indicatin is thus that the male plants are hetergametic with ne XY pair f chrmsmes while the female are hmgametic with an XX chrmsme pair, where X represents the satellited chrmsme. MARCHANT's (968) wrk n ncephaiau~ des indicate sme apparently nnidentical chrmsme hmlgues (Figure ) but the authr stresses that slight changes d ccur during slide preparatin and that final differences d nt necessarily mean hetermrphism. Furthermre, the presence f a hetermrphic pair des nt necessarily have sexual significance as it may arise frm a variety f surces such as satellite suppressin. Clearly, a great deal f detailed cytlgical wrk is necessary befre a final cnclusin is reached as t whether recgnizable sex-chrmsmes are a cmmn feature thrughut the Cycadales. T cmplete the review n cytlgical wrk, tw ther aspects shuld be mentined. One is the phenmenn f smatic reductin, a prcess in which cells divide withut chrmsme replicatin and the chrmsme number thus becmes prgressively reduced. This ccurs spradically in a number f unrelated vascular plants but seems t be a cmmn feature in the apgetrpic cycad rt system. STOREY (968) fund recurrent smatic reductin in 7 species frm 6 genera, the reductin ften. prceeding t a stage where nly ne r tw chrmsmes remain in each

43 9 cell. These cells becme charged with slime and the resulting envirnment appears cnducive t infectin by Cyanbacteria. This sequence f stages terminates in establishment f the symbitic crallid rt system fund in all cycads (Sectin.9). A final aspect in this sectin refers t the use f mre sphisticated cytlgical techniques. MORETT (98) used quinacrine flurescenc~ analysis t examine chrmsmes frm 3 Australian cycads in 3 genera. Enhanced flurescent banding was fund at the 'centrmeric regin in all species. f Mac~zamia but nt in Lep~dzamia r Bweltia. Differences in the banding patterns were bserved in different species f Ma~zamia. t is clear that this and ther techniques such as Giemsa banding (SHARMA and SHARMA, 975) can extend the range f this wrk. DNA-sequencing investigati ns have been carri ed ut n p en frm tw spec i es f EttCephalaJL.tM (BRANDT and VON HOLT, 975, 986) and this als may prvide data t extend the current cytlgical investigatins..8 Apparent sex reversals. Higher plants are divided int three grups insfar as sexuality is cncerned. V~e~~ plants bear male and female reprductive parts n separate i ndi vi dua s; mne~u6 plants have bth rgans n, the same individual but n separate parts f the plant. These tw grups are cmparatively small in terms f numbers f species. Mst plants fall int the third grup, the h~ap~dite6, where each flwer has bth male and female rgans. Cycads are unifrmly diec;us in this classificatin (CHAMBERLAN, 935). The classical cncept f sexuality in diecius plants invkes the ccurrence f sex-determining chrmsmes, which mayr may nt be mrphlgically recgnizable. n these terms the gender f each plant is laid dwn at the time f fertilisatin and is immutable. Hwever, the fact that hermaphrdites d ccur amngst prevailingly diecius plants, and that sex changes may be induced r may happen spntaneusly in individual plants, requires 'a re-evaluatin f the fundamental principles f plant sexuality. n a recent review, FREEMAN, HARPER and CHARNOV (980) list 5 plant families cmprising mre than 50 species where individuals have functined as males at ne time and females at anther, r have prduced hermaphrditic prgeny. ncluded in this

44 30 list are several species with mrphlgically recgnizable sex chrmsmes, generally with the male karytype shwing a difference in ne chrmsme pair. A number f authrs have addressed this apparent cnflict between gentypic and phentypic sexuality (HESLOP-HARRSON, 97; FREEMAN, HARPER and CHAR NOV, 980; POLCANSKY, 98; DURAND and DURAND, 984; LLOYD and BAWA, 984). A revisin f the classical cncept seems t be suggested in which the chrmsme mechanism is ~ex-~ectl~g rather than ~ex-e~tli~g, the nrmal rgangenetic prgramme being influenced by a number f factrs. These factrs can be separated int endgenus and exgenus hrmne effects n ne hand and envirnmental effects n the ther, althugh it is reasnable t pstulate that envirnmental influences will perate thrugh hrmnal actin. Amngst the hrmnes which influence the gender f plants are the cytkinins, gibbereins, auxins, ethylene, and abscisic acid (CHALAKHYAN, 979; FREEMAN, HARPER and CHARNOV, 980; DURAND and DURAND, 984) and sterid hrmnes (GEUNS, 978, 98). Hwever, it is undesirable t categrise sex hrmnes in terms f specific respnses as the same hrmne may have ppsite effects accrding t the plant species under investigatin (DURAND and DURAND, 984). With respect t the envirnmental factrs, it appears that there is a strng tendency fr stress situatins (e.g. lw sil fertility, dry sil, extreme temperatures, lw light intensity) t be assciated with a change frm female t male (FREEMAN, HARPER and CHARNOV, 980). Undubtedly the mechanism is hrmnal but the adaptive significance may be ratinalised in terms f physilgical cst. t is less cstly fr the plant t functin as a pllen prducer than as a seed prducer, thus a change frm female t male in terms f stress has a distinct survival value (FREEMAN, HARPER and CHARNOV, 980; LLOYD and BAWA, 984). n an analysis f ppulatins f the Australian cycad Mactzamia ~ediei (Gaud.) C.A. Gardn. and Zamia pumiia L. in the Dminican Republic, ORNDUFF (985) fund there was an increase in male cning frequency under adverse cnditins and, when cmpetitin was lwer, a greater prprtin f plants bearing female cnes. n a survey f Zamia pumiia ppulatins in Puert Ric, NEWELL (985) reprted that female plants had a larger number f leaflets per leaf than did the males. Bth these bservatins are. cnsistent with the thery that the physilgical cst t the plant is higher when it behaves as a female. This wuld be especially true in

45 3 cycads when ne cmpares the mass f large seed-prducing female cnes t that f the smaller pllen-bearing male cnes. Hwever, these bservatins d nt infer that any sex change takes place in individuals within the ppulatins. The literature n specific sex changes in individual cycads is limited but it is clear that apparently spntaneus gender reversals d ccur and usually appear t be assciated with sme traumatic incident. SCHUSTER (93) reprts ne case where a plant f Cyc~ ~evtuta Thunb. which was lngitudinally bisected, the tw pieces ging t different places. Apparently the tw plants which develped frm these sectins were f ppsite gender. CHAMBERLAN (935) mentins a change frm female t male with respect t a specimen f Cyc~ ~evtuta in Australia and als tells f the bud frm a female Cyc~ ~cin~ L. frm a Chicag cnservatry which eventually cned as a male. MENNNGER (967) dcuments tw sex change incidents: a specimen f Cyc~ ~cin~ L. which changed frm female t male after being mechanically damaged, and a male f the same species which prduced a female cne after severe frst expsure. KEMP (985) reprts a change frm male t female in a specimen f Cyc~ ~evtuta after being transplanted. An apparently authritative reprt f a sex change in Encephata.-u:. umbetuz~en.~ R.A. Dyer is given by VAN WYK and CLAASSEN (98). They fllwed the histry f several specimens, planted in a Pretria garden, ne f which prduced a male cne in 970 but a female cne in 979. This particular plant was situated in a mre expsed psitin than ther similar specimens, and it is pssible that a freak spell f sub-zer temperatures in August 97 may have initiated the change. Several ther incidents f sex reversals in cycads have been reprted t the authr persnally. A female lamia plant is said t have prduced a branch bearing a male cne (KOELEMAN,.A., p~. cmm.). A specimen f Encephata.-u:. v~~u. Lem., subjected t particularly dry cnditins, suppsedly altered frm female t male (SWANEPOEL, R., p~. cmm.). A plant f Eltcephata.-u:~ ta..u,.6jtn. Lehm., transplanted in 970 t a farm near East Lndn, bre tw successive crps f female cnes with viable seed, but fllwing a severe drught at the end f 983, the plant prduced tw successive crps f male cnes (BURSERY, B., p~~. cmm.).

46 3 n attempting t induce sex reversal thrugh transplantatin, TANG (986) mved 97 lamia pumiia L. plants f knwn gender frm ne site t anther. Of the 6 specimens which prduced cnes within a three year perid, nne shwed any evidence f sex change. Thus, if trauma can induce an alteratibn in phentypic sex expressin in cycads, the cnditins under which it ccurs must be fairly restrictive. ndirectly related t this tpic is an bservatin that the percentage f male and female cycad plants arising frm a particular batch f seed may be influenced by the temperature at which the seed is stred during its maturatin perid (HENDRCKS, J.H., p~. cmm.). All the abve phenmena can be ratinalised in terms f actin f hrmnal agents r their inhibitrs. An bservatin that gibberellic acid induces maleness in lamia seedlings (NORSTOG, K., p~. cmm.) adds weight t this and it might be anticipated that cytkinins wuld have a feminising effect. The cytkinin!gibberrellin rati appears t be critical in the sex 'expressin f several diecius plants (CHALAKHYAN, 979). n evaluating the relative gentypic directin and phentypic expressin f plant sex, further cmplexity is intrduced when multiple gene lci are pstulated. n extensive wrk with M~e~ali~ annua L. (Euphrbiaceae). DURAND and DURAND (984) suggest the existence f three sex genes which wuld give 64 pssible allelic cmbinatins (4 3 ) and 7 pssible gentypes (3 3 ) ranging frm 'supermale ' t 'superfemae '. The fact that nly ne site n ne chrmsme pair is generally mrphlgically recgnizable when sex chrmsmes have been bserved micrscpically des nt preclude the pssibility f multiple sex genes being present. f this is the case,. then ne may speculate that it is the 'supermales ' and 'superfemales' which will be cnstant in sex expressin thrughut their lives, while the intermediates may be sufficiently influenced by envirnmental factrs s as t shw sex reversals. Experimental wrk n the influence f envirnmental factrs and exgenus plant grwth regulatrs n the sexual expressin f cycads is inhibited by their slw grwth rate. Encephai~~ ~e4x Bertl. f. requires years t reach sexual maturity (DYER, 965) althugh sme species f lamia can cne within 3 years frm the seedling stage (NORSTOG, K., p~. cmm.). Assessment f gender by chrmsmal mrphlgy has nt been

47 33 cnvincingly demnstrated and, as yet. there are n bichemical r serlgical tests which are sexual l y diagnstic in cycads. Thus it is unlikely that the precise mechanisms which cntrl sexual expressin will be knwn in the immediate future. The nly hpe f gender cntrl will be as the result f field trials under varius chemical and envirnmental regimes using fairly large numbers f adult r near-adult plants..9 Crallid rts. A cmmn feature in cycads is the frmatin at an early seedling stage f dichtmusly-branching apgetrpic rts. These are initiated terminally n nrmal lateral rts at varius distances away frm the main tap rt and are fund at varius depths frm the sil surface', ften being prminently visible as crallid masses at the sil surface. These unusual rt frms have s far been recrded in 49 cycad species, in all genera except MiC40CYCa4 (GROBBELAAR, 985). Mentin has already been made f the prcess f smatic reductin in which the chrmsmes in the cells f these structures may be reduced t as few as ne r tw in number (STOREY, 968; Sectin.7). The crallid rts are ften, but nt always, invaded by varius Cyanbacteria which then exist in a symbitic relatin with their hst plant. The symbints are cnfined t an aerenchymatus zne at the junctin f the inner and uter crtex (GRLL CAOLA, 980). They exist as lng filamentus strands in which single larger hetercyst cells are regularly placed in chains f smaller vegetative cells (LNDBLAD, HALLBOM and BERGMAN, 985). n early surveys, the clnising rganisms were thught t be species f Anabaena r N tc (GRLL CAOLA, 980). Hwever, in an extensive survey f symbints frm suthern African cycads, in which 4 cultures were islated frm 3 cycad species, all but ne were identified as species f N tc, the mst cmmnly-ccurring being N. cmmune Vaucher (GROBBELAAR, SCOTT, HATTNGH and MARSHALL, 987). in ndules frm Encephal~ l~eb~an~, the same authrs identified the islate as a species f Calt~~. Significantly t, there was mre than ne symbint in a number f the assciatins.

48 34 n cmmn with the situatin in many similar plant-cyanbacterial assciatins, the cycad symbints pssess the reductase enzyme system which allws fr fixatin f atmspheric nitrgen. n an adult cycad plant it is believed that a well-established symbisis can adequately furnish the plants entire nitrgen requirement (GROBBELAAR, N., p~. cmm.). n field studies with the suth-western Australian cycad, Ma~zamia niedle (Gaud.) C.A. Gardn., it has been estimated that the symbisis leads t the fixatin f between,4 and 8,4 kg nitrgen per hectare per year, depending n the time elapsed in burning cycles (GROVE, O'CONNELL and MALAJClUK, 980). Certainly this can be a significant cntributin t the verall ecsystem particularly in the situatin where the cycad vegetatin is the dminant understry in Eucaiypt~ frests n pr sil..0 Cnventinal methds t brpagatin. Cycad plants are usually grwn frm seed, bth in nature and by man. Hwever, seed prductin and viability varies enrmusly; even in dense stands the prductin f fertile seed may be limited (NEWELL, 983). The main reasns fr this appear t be either nn-synchrny f the male and female cnes r ineffective pllinatin. Althugh early wrkers believed that cycads were generally wind pllinated, the issue has been pen t questin and there is nw increasing evidence that many taxa may be assciated with very specific insect vectrs (Sectin.6). The prblem f effective pllinatin is exacerbated when the cycad plulatin is thinly scattered ver large areas. n nn-habitat situatins, as in plantings in btanic r private gardens, there is little chance f spntaneus pllinatin and techniques have been develped fr artificially-induced pllinatin (GDDY, 984; TANG, 985b). n current bilgical terms, cycad seeds fall int the grup knwn as 'wet' r 'recalcitrant'. This type f seed is shed with high misture cntent, is susceptible t desiccatin, des nt shw any true drmancy and has a quantitatively limited strage life (ROBERTS, 973). Fr successful germinatin it is essential that grwers are aware f the nging maturatin prcesses typical f recalcitrant seed and that strage and swing times are crrectly planned. n an investigatin n. the germinatin f Encephaia4t~ nataie~~ Dyer and Verdrn, it was

49 35 fund that strage in a mist envirnment at 0 C, fllwed by transfer t 30 C when the embry was mature, lead t imprved germinatin rates (FORSYTH and VAN STADEN, 983). t is prbable that ptimal strage and incubatin times, temperatures and humidities will vary widely with different species. Germinatjn may smetimes be enhanced by cutting the seed cat r by chemical pre-treatments (BURCH, 98b; DEHGAN, 983). Many cycads prduce adventitius ffsets r basal suckers which may be used in vegetative prpagatin (GDDY, 984). Divisin f aerial stems frm a branched crwn is als pssible, as is the separatin f multiheaded clumps in thse taxa with undergrund stems (DEHGAN. 983; GDDY, 984). Prpagatin f sme species using leaf-bases with segments f stem attached has been reprted (GDDY, 984) and further research int this pssibility is necessary. While vegetative prpagatin f cycads is restricted by availability f material. many f the rarer species can be usefully increased in this manner. As an example, ne may cite the fact that the entire glbal ppulatin f Encephala4t~ wdii, thught t be sme 500 in number (OSBORNE, 986a) has been prpagated frm suckers frm the slitary specimen f this plant discvered in 895. The effrts f the Cycad Sciety (U.S.), the Cycad Sciety f Suthern Africa and the Palm and Cycad Scieties f Australia and New Zealand in public awareness prgrammes and their establishment f varius cycad seedbanks and pllen strage facilities are t be cmmended. By encuraging prpagatin frm garden-grwn material. the pressure n habitat stands frm ver-zealus cllectrs will at least be partially relieved.. Cnclusin. Sectins. t.0 f this text represent a necessarily cndensed intrductin t the bilgy f the rder Cycadales and sme f the relevant literature. n essence, these trpical and sub-trpic plants f early evlutinary rigin shw cnsiderable diversity in vegetative and reprductive mrphlgy and pssess several features unique in the plant kingdm. Because f the antiquity f the rder and in view f

50 36 their relative scarcity "and decrative appeal, cycads have attracted much public interest. n view f the generally slw grwth rates, the paucity f viable seeds and the limited ptential fr vegetative reprductin f cycads, it is evident that a vital cntributin culd be "made t cycad bilgy if these plants culd be successfully prpagated by the ~ v~ methds bradly defined as "tissue culture" techniques. Thus a detailed review f the literature specific t this tpic is presented in Chapter Tw (Sectin.3) f this text. This review als frms the basis f a separate reprt by WEBB and OSBORNE (in print). The first part f the authr's experimental wrk (Sectin.4) has thus been directed at explring techniques fr the ~ v~ prpagatin f the Suth African Cycadales.

51 37 CHAPTER TWO N VTRO CULTURE OF CYCADS NDEX TO CHAPTER TWO Page. ntrductin 38. Tissue culture f gymnsperms 39 (ther than cycads).3 Tissue culture f cycads 44.4 Experimental : materials and methds.4. Surces f plant material Nursery prcedure Disinfectin prcedures Chice and preparatin f media Chice f culture cntainers Chice f culture cnditins Cytlgical examinatin 73.5 Experiments with haplid material.5. Megagametphyte cultures Micrgametphyte cultures 80.6 Experiments with diplid material.6. Embry cultures Primary rt explant cultures Leaflet explant cultures Other explant cultures.7 Suspensin cultures 4.8 Cnclusin 4

52 38. ntrductin The term "ti~sue culture embdies a brad cncept which presently includes the culture in vj~ f single cells (r even i.ell cmpnents), grups f cells, part~ f rgans and whle rgans. This science is nt new; experiments with the culture f plant material in vit~ cmmenced early this Century. Ntable cntributins t the advancement f the science have been made by a large number f researchers including Haberlandt, Rbbins, White, Nbecurt, Gautheret, L, Ball, Wetmre, Mrel, Skg, Miller, Krikrian, Murashige, Steward, Street, Vasil, Thrpe and their c-wrkers. A vast literature has been prduced n techniques, implicatins and applicatins "and general verviews f the subject are given in the texts f WHTE (943, 963), STREET (973), MURASHGE (974), THOMAS and DAVEY (975), BUTCHER and NGRAM (976), RENERT and BAJAJ (977), CONGER (98), THORPE (98) and KRKORAN (98). Much f the early wrk n plant tissue culture was dne n herbaceus angisperms and a few bulbus mnctyledns. Many ecnmicallyimprtant crps fall utside this grup and nly fairly recently has there been success with in v~ culture f cereal crps, palms, cnifers and ther wdy plants. A brief review f the wrk in gymnsperms and especially cycads fllws (Sectins. and.3). The science f tissue culture has evlved int a recgnised and imprtant majr discipline with much future tential. Quite apart frm the usefulness f in v~ techniques as a pwerful tl in mrphgenetic and physilgical research, there are vital applied benefits. Amngst these can be listedapplicatins such as the prpagatin f plants which d nt readily give viable seed r are particularly slw t germinate, the rapid clnal multiplicatin t give unifrm crps f hrticultural and agricultural imprtance, the pssibility f genetic imprvement thrugh selectin prcesses in cmpetitive cell culture systems, the eliminatin f virus and ther diseases frm infested plant stck, the availability f guaranteed pathgen-free material fr internatinal trade, the ptential f crypreservatin f valuable germ-plasm and the prductin f natural plant prducts itt v~. Refinement f the current methdlgy will be clsely assciated with any prcedures invlving recmbinant DNA r plant 'genetic engineering'. Numerus prblems remain t be slved and pprtunities abund fr thse with imaginatin and perseverance t cntribute further t the theretical and applied aspects f this exciting field.

53 39. Tissue culture f gymnsperms (ther than cycads). Early wrk in gymnsperm tissue culture was carried ut primarily t investigate varius develpmental stages and t gain insight int gymnsperm tissue culture was carried ut primarily t investigate varius develpmental stages and t gain insight int gymnsperm mrphlgy. As the methdlgies develped, it became clear that the techniques had a significant applicatin t prpagatin systems. Because f their ecnmical imprtance, mst f the recent wrk has been dne n cniferus timber trees. Within this grup f sftwds, the mst valuable timber is fund in the fami 'ly Pinaceae and especially the genera P..tYltL6. P.<..c.e.a. Ab..te,-6, P-6e.udt-6uga, Ce.~tL6 and L~x. The histry and advances in this field have been reviewed by DURZAN and CAMPBELL (974), BROWN and SOMMER (975), DAVD (98),,JOHN (983) and BONGA and DURZAN (987). Mst attentin has been fcussed n the use f se~d-derived material, ntably embrys, ctyledns and hypctyls, which can give rise t adventitius 'budding r smatic embrygenesis with r withut an intervening callus stage. ' The respnses btained vary with the species, the explant material, its physilgical cnditin, the cmpsitin f the basa 'j culture medium, additin f grwth factrs, temperature, light intensity and phtperid. A review by NORSTOG (98) prvides inslght t the wrk n embry-derived material. LA RUE (936) successfully cultured a large number f excised gymnsperm embrys and shwed that plantlets culd be raised in the absence f the megagametphyte. Other wrk sn revealed that the develpment f embrys -tt v-u:jr.. di d nt always f w the pattern..tt v..tv and ften ca us prliferatin rather than plarised differentiatin ccurred. One explanatin fr this was that in culture situatins the embrynic explants ' had unifrm accessibility t nutrients (NORSTOG, 98). Callus frmatin n immature embrys f P-tlttL6 and G..t!tflgO was reprted as early as 50 years ag (RADFORTH. 936; RADFORTH and PEGORARO. 955; RADFORTH. TRP and BONGA. 958). There was evidence that the rientatin f the embry and the phtperid culd have an influence n the grwth pattern (BERLYN and MKSCHE, 965). When embrys were dissected and the varius segments cultured separately, there was evidence f respnse gradients. Hypctyl sectins frm the seed f P.tltL6 tambejt.u.alta Dugl. gave a rhizgenic r,espnse which increased with distance frm the ctyledns (GREENWOOD and BERLYN, 965).

54 40 The discvery f the natural auxins and cytkinins and the synthesis f their analgues prvided new impetus t this wrk. Hypctyl segments frm embrys f P..ttlu.6 gejt.ajtcuatta Wall gave ri se t a subcuturabe callus n a medium supplemented with 4,5 x la-m,4- dichlrphenxyacetic acid; sme vascular differentiatin was present within the callus mass (KONAR, 974, 975). Excised embrys and ctyledns frm P-6e.ud.,uuga me.ttz..tuu (Mirb.) Franc frmed callus which became mrphgenetic when treated with a slutin cntaining 0,5 -,0 mm 6-benzyadenine; subsequent transfer t a medium withut cytkinins stimulated grwth f the adventitus buds s frmed (CHENG, 975). The buds arse frm epidermal r subepidermal layers and primrdia became visible after 0 days in culture (CHEAH and CHENG, 978). The cytkinin _ -benzyladenine was effective in initiating adventitius buds n hypctyl segments frm P..tce.a giauca (Mench) Vss (CAMPBELL and DURZAN, 975, 976), P..tce.a ab..tu (L) Karst. (CHALUPA, 975) and B..tta 4..ie.tttai..t-6 (L) Endl. (THOMAS. DUHOUX and VAZART~ 977) but subsequent develpment was inhibited until subculture t media withut grwth factrs (CAMPBELL and DURZAN, 975; CHALUPA, 977). mmature embrys frm P..tttu.6 ~acuata D.Dn generated callus n a medium supplemented with auxin and cytkinin and mrphgenesis again ccurred when the calli were transferred t a hrmne-. free medium (RELLY and BROWN, 976). Embrys frm P-6e.ud.t ~uga me.ltz..tuu gave shts alng cty ednary surfaces, the number 'f shts increasing with increasing cncentratins f 6-benzyadenine (WNTON and VERHAGEN, 977). As an alternative rute, the ctyledns culd be induced t frm callus n a medium with auxin and cytkinin, this callus subsequently giving rise t shts (WNTON and VERHAGEN, 977). Embrys and hypcty segments frm P..tttu.6 watucluattcl A. B. Jacksn frmed callus n a medium cntaining 5.3 x 0-7M -naphthaleneacetic acid; subculture nt a medium supplemented with 4.4 x la-m 6-benzyladenine resulted in sht lnitiatin (KONAR and SNGH. 980). Excised ctyledns frm P..tttu.6 tae.da L. frmed buds alng the surfaces after 6-0 weeks in culture and these extended t 5-0 mm while still cnnected t the exp ant (MOTT, 98). Call us induced n mature embrys f P.i.ce.a -6aclte.t-6..t-6 (Bng. ) Carr. and P-tttu.6 cttt~a Dug. ex Lud. gave shts n transfer t hrmne-free medium (JOHN, 983). Because f its natinal timber industry, a great deal f gymnsperm tissue culture wrk has been carr i ed ut in Sweden. Embrys f P..tllu.6 cttt~a gave ri se t adventitius buds n media cntaining 0~7 t 0-4 M 6-benzyladenine; at the higher cytkinin levels, buds were als frmed n ctyledns and hypctys

55 4 (VON ARNOLD and ERKSSON, 98). Fr the initiatin phase it was fund that a diluted (X /4) basal medium was ptimal but f the cmpnents in the medium, nly the cncentratin f glycine was critical (VON ARNOLD and ERKSSON, 98). t was necessary t transfer the explants after abut ne mnth t a medium withut cytkinins t allw further develpment f the buds (VON ARNOLD and ERKSSON~ 98). Similar results were fund with P~cia ab~~ (L) Karst. (VON ARNOLD, 98) and iater wrk shwed that increasing the cncentratin f agar in the bud-develpment medium frm 0,5 t,0% decreased vitrificatin prblems but als reduced sht grwth and rting ptential (VON ARNOLD and ERKSSON, 984). A refinement in the technique fr bud initiatin frm P-ic.e.a ab.te. was the use f t 3 hojy' lpul~ ' prp.-t~at",ent f the exci sed embrys in,5 t 6.5 x 0-~M 6-benzyladenine at ph 5.5 rir t setting up the cultures (BORNMAN, 983; VON ARNOLD and ERKSSON, 985). A preincubatin perid fr ~even days i n di luted hrmne-trep. medium accelerated bud frmatin and frequency when embrys frm P~n~ c.~bae.a Mrlet were innculated n medium cntaining, x 0- s M 6-benzyladenine (WEBB and SANTAGO, 983). The use f a medium cntaining 0- s M 6-benzyladenine was used t initiate sht frmatin n embrys~ ctyledns and hypctyl f P-in~ c.nt~a; subsequent bud develpment and elngatin was enhanced by eliminatin f hrmnes, reductin f the cncentratin f mineral salts, vitamins and sucrse and by the inclusin f charcal (PATEL and THORPE, 984). Changes in the cncentratins f nuclear prteins, enzymes and reducing substances have been studied in a cytchemical investigatin f bud frmatin frm embrys f P-in~ c.uit~ D. Dn (PATEL and BERLYN, 983). Despite the cnsistent pattern f cytkinin-stimulated bud initiatin in gymnsperms, mrphgenesis is nt always achieved. Aplicatin f ver 00 different auxin and cytkinin treatments t callus frm Pbt~ tjtb~ L. failed t induce any mrphgenic respnse (MNOCHA, 980). Similarly, excised embrys frm the unusual Namibian plant, We.iw~c.h.ia ~ab~ Hk. f.; gave auxin-induced subculturable callus but all attempts at mrphgenesis were unsuccessful (BUTTON, BORNMAN and CARTER, 97). Quite apart frm the prcess f bud initiatin n gymnsperm explants. it was nticed that under sme circumstances the material gives rise. t embry-iike utgrwths variusly described as embryids, aaventive r ' adventitius embrys, pseudembrys r smatic embrys). Such structures were nticed n the ctyledns f 8-ita ~e.nt~ (L). Endl. and they later

56 4 develped int plantlets (KONAR and OBEROr, 965).. Embrys f P~~l~ pat~~ Mill similarly generated smatic embrys n the ctylednary surfaces and these t culd be excised t frm viable plantlets (SOMMER, BROWN and KORMANK, 975). mmature embrys frm P~cea ab~~ gave rise t embrygenic callus when cultured fr tw mnths in the dark n a medium supplemented with 0- s M.4-dichlrphenxyacetic acid and 0-6 t 0-~ 6-benzy adeni ne r ther cytki ni n (HAKMAN and VON ARNOLD, 985). This callus prduced numerus small smatic embrys which devel~ ' int nrmal plantlets n subculture under a 6-hur phtperid n a medi urn supp emented n y wi th 6-benzyiadenine (HAKMAN AND VON ARNOLD. 985). Bth fresh and stred seed f Pi~~.tambe.iliana was used t prvle embrys which develed a muci laqinus embrygenlc callus when cultured n a medium cntaining,4 x 0- a M.4-dichlrhenxyacetic acid; n transfer t a medium withut the auxin but cntaining 4,4 x 0-7 M 6-benzyiadenine, develpment analgus t that f nrmal zygtic embrys ccurred (GUPTA and DURZAN. 986). The use f nn-seed-derived material fr gymnsperm tissue culture has als received attentin. Early wrk shwed that callus culd be frmed frm cambial explants f P~~~ P~ltQ..6.teJt Ait. (GAUTHERET, 934), while callus derived frm Sequ~a ~empejtv~e~ Endl. culd be subcultured with eventual mrphgenesis (BALL, 950). Juvenile and mature needles. brachyblasts (needle fascicles). apical meristems and varius cambial regins have been brught int culture with varying degrees f success (JOHN, 983). Yung needles frm P~cea ab~~ prduced adventitius buds n a medium supplemented with 5 x 0-6 M 6-benzyladenine and 5 x 0- s M -naphthaleneacetic acid. Outgrwth f the buds was prmted n transfer t diluted medium (x /3) withut hrmnes (JANSSON and BORNMAN, 980). The frequency f this bud initiatin was inversely crrelated with needle size (JANSSON and BORNMAN, 98). Bud yield culd be increased by initial incubatin at reduced temperatures (VON ARNOLD and ERKSSON, 979a). Use f 6-benzyladenine initi~ted callus n needle expiants frm p~~~ ~adia.ta seedlings: transfer t hrmne-free medium allwed frmatin f meri~ stems which gave shts after excisin and further transfer (RELLY and BROWN, 976). With the same species, as many as 80 viable shts culd be btained frm needles f a single seedling (ATKEN, HORGAN and THORPE, 98). Mrphgenic callus 'was initiated frm vegetative buds f PiH~ c~.tua and P~cea ~dclte~~ (WEBB AND STREET, 977).

57 43 Vegetative buds f Picea abie6 were induced t frm buds and shts in respnse t 6-benzyladenine and subsequent transfer t hrmne-free medium (VON ARNOLD and ERKSSON, 979b). Of the methdlgies used in the wrk describea abve, nly the smatic embrygenesis rute can give rise directly t an entire plant. The techniques which give rise t bud initiatin generally require excisin. utgrwth and a rt-inductin prcess. Rting may be achieved in v~ r Wl th the use f a prcedure analgus t that f rti ng cnifer cuttings ' in mist-prpagatin units (JOHN, 983. The auxin indle-3-utyric acid is favured by mst wrkers fr prmting rt inductin (JOHN. 983). The technique f suspensin culture has nt yet fund wide applicatin in gymnsperms, but sme pinters exists fr future wrk. With Picea abie6, repeated subculture f cell aggregates in suspensin lead eventually t the frmatin f small embryids (CHALUPA and DURZAN, 973). Sht ti ps f P6euduuga mettz..i.e6,u, prvi ded materi a fr suspensin cultures which ultimately gave biplar embryids(durzan, 979). With seedling ctylednary material f the same species, individual prtplasts were islated and culd be multiplied t a callus stage n a fabric supprt saturated with glutamine-enriched medium (KRBY and CHENG, 979). Prtplasts have als been islated a.nd cu tured frm the cty edns f P.{nU6 piltcl6tvt. and they subsequently regenerated cell walls and cmmenced aggregatin int callus structures (DAVD and DAVD, 979). With respect t the culture f haplid, rather than diplid, plant material, the gymnsperms prvide greater pprtunitie-s than the angi' sperms. This is largely because f the well-defined micr- and megagametphytic stages in their life cycle and the accessibility f this material in dissectin. The first successful pllen culture wrk was carried ut by TULECKE (957) wh was able t initiate haplid callus frm mature m;crspres f varius gymnsperms. Micrsprphylls frm PinU6 gave a mixed haplid and diplid cellular callus in which sme glbular empryids were bserved (BONGA, 974). Micrspres frm P..i.nU6 Le6..ttw6a Ait. subjected t cld treatment and centrifugati n gave increased callus prliferatin (B~NGA and McNNS, 975). There has been little indicatin f any type f regeneratin frm this ~ material, but it may be that a precise stage in develpment ;s critical

58 44 t any mrphgenic respnse (NORSTOG, 98). The female gametphyte frm G~~g bilba gives a variety f utgrwths and subcultureable tissue ~n vdft (ROHR, 977). Megagametphytes frm varius species f P~HLL6 give callus cultures but, apart frm the presence f tracheidal elements, little differentiatin has been recrded (NORSTOG, 98). An exceptin t this generalisatin is the reprt by KONAR and SNGH (979) that megagametphytes frm Ep/te.dJta -&Uata Biss gave auxininitiated callus which culd give shts and rts after subculture n a medium supplemented with kinetin. Additin f 6-benzyladenine stimulated mre prfuse sht frmatin and allwed plantlet develpment. Publicatins n the tissue culture f gymnsperms cntinue t prliferate. A mst useful series f reviews is presented in the wrk f BONGA and DURlAN (987), the third vlume f which deals with ih V~~O case histries f material frm cniferus trees..3 Tissue culture f cycads Prir t the establishment f aseptic plant tissue culture techniques, certain bservatins had been recrded n the regeneratin f viable structures frm bth haplid and diplid cycad material. DUCHARTE (888) bserved that megagametphytes frm infertile seed f CyQa thu~~ Gaud., maintained fr prlnged perids in germinatin beds, prduced numerus adventitius rts. COULTER and CHRYSLER (904) reprted that whle lamia plants culd regenerate frm slices f the undergrund rt, an bservatin which has recently frmed the basis fr a prpagatin technique (BURCH, 98a). t is als cmmn knwledge amngst cycad enthusiasts that when whle cycad leaves are remved frm the parent plant and placed in a warm prus medium, as in a 'cmpst heap' situatin, callgenesis and ccasinal rhizgenesis may be fund at the cut leaf base surface. Finally, present methds f vegetative prpagatin invlve regeneratin, as in the rting f lateral branches and basal suckers (Sectin.0).

59 45 Much f the early tissue culture wrk n cycaas was directed at bserving regeneratin f haplid material i~ vita, and prvided imprtant infrmatin as t ntgenic develpment in these plants. LA RUE (948) cultured megagametphytes f lamia ~~da~a A. DC. (= l. pumiia L.) n simple media and fund that pustularl grwth ccurred which, in a small number f cases, led t the frmatin f rts r shts r bth. LA RUE (954) shwed that the same tissue material, grwn aseptically n mist sand, culd ccasinally regenerate small detachable balls f meristematic tissue which he called pseudbulbils. These culd be sub-cultured, eventually with the frmatin f prly-vascularised rts and leaves. The supply f minerals r carbhydrates did nt appear t be beneficial. n the same paper LA RUE (954) reprted the lw frequency prductin f presumably haplid rts and buds frm megagametphytes f Cyca ~ev uta Thunb. after tw years in culture. Furthermre, immature embrys frm bth species matured and gave rise t seedlings when fertilised vules were cultured n mist sand. NORSTOG (965) investigated regeneratin frm bth haplid and diplid tissue f lamia integ~ ia Ait. (= Z. pumiea L.) using megagametphytes and embrys respectively n a variety f media which were essentially supplemented mdificatins f WHTEls (943) basal medium (Table 3). Cultures were kept at 5 C in darkness except fr brief times f examinatin. Callgenesis was bserved in a significant number f tw-mnth-ld cul tures f "megagametphytic explants n media cntaining auxin, kinetin and rganic nitrgen surces. Optimum results were btained using medium 8 (Table 3) which cntained glutamine, asparagine, alanine and adenine in additin t 4,5 x lo-sm,4-dichlrphenaxyacetic acid and kinetin. n this case, 68% f the half-gametphyte explants frmed callus within tw mnths. After anther three mnths there was evidence f rt and subsequently leaf initiatin. The auxin,4-0 appeared t be mre successful than indle 3-acetic acid when used with kinetin in terms f mrphgenetic respnse. Hwever, elngatin f these primrdia was inhibited until the explants were transferred t a medium withut grwth factrs. NORSTOG (965) extended La Rues earlier wrk by ascertaining chrmsme numbers f the resulting tissue. Regenerated rt

60 46 TABLE 3 : SELECTED MED A USED BY VAR OUS WORKERS N CYCAD TSSUE CULTURE EXPERMENTS Basal Mdified White's Media Cns t i tuents Medium Mdified Murashige-5kg Media mg per 000 ml Whi te 8" "63,, "59,,3 K"" ( 9~3),, MgSO,.7H O NaS0, Ca(NO)).~H CaCl z 33 33, KCl KNO) , NaH PO... HO 3,5 3,5 800 KH PO NH.. NO) H]B0 3,5,5 0,5 6, 6,? MnS... 4H O 6,7 6,7 3,3,3 ZnSO... 7H O,7,7 0,5 8,6, 8,6 K 0,75 0,75 0,83 O,R] NaMO... H0 0,05 0,5 0,5 CuSO,.5H O 0,05 0,05 0,05 CC.6H 0 0,05 0,05 0,05, Fe citrate. 5H O 0 NaEOTA.H0 37,3 37,3 FeSO... 7H0 7,8 7,8 Fe (SO,)],5,5 (NH.. ) malate 00 Asparagine 00 (mal ic acid adj. t phs with NH.. OH) Glycine 3 00 Alanine Glutamine 00 ~OO ~OO ~OO Tyrsine Adenine SO , ~! ~ Kinetin 5 Auxin (, ~-O), 0(,4-0) (,4-0) 0 (NAA) Ca pantthenate 0,5 Thiamine HC 0, 0, 0,5 0,4 Ascrbic acid 0.5,- Nictinic acid 0,5 0,5,5 Ribflavin 0,5 Pyr i dxi ne HCl 0, 0, 0,5 nsi tt Sucrse Bact-agar , ~... ~~=--= NORSTOG (965);,3 NORSTOG & RHAMSTNE (967);.. KOELEMAN & SMALL (98).

61 47 and leaf material shwed the expected haplid number f 8 chrmsmes but nevertheless the structures resembled clsely thse f similar stages frm diplid material, at least in the early develpment. Use f a medium riginally devised fr the culture f excised barley embrys prmted the develpment f smatic embrys frm a few expants f bth gametphytic and embrynic material f the same lamia species. These embryids were mstly dictylednus and seemed t be analgus t La Rue's pseudbubis. Fllwing this wrk, NORSTOG and RHAMSTNE (967) excised megagametphytes and juvenile embrys frm lamia ~nteg~~a Ait. (=l. pumiia L.) and Cyc~ ~~~~ L. "and explred their respnse t a wide variety f culture media. Bth slid and liquid media were used, the latter being held in reciprcating shakers r rtating flasks. T quantify the results, the mass f tissue was recrded at varius stages as the experiment prgressed. On simple media with n auxin r cytkinin supplements, lamia prembrys grew in essentially a nrmal fashin althugh in sme instances the usual dictylednus develpment gave way t plyctyledny. On ther media cntaining lw cncentratins f,4-0 and kinetin the pr-embrys frmed sub-culturable callus. Much mre rapid grwth ccurred n transfer t medium '59' (Table 3) which is a MURASHGE and SKOOG (96) medium mdified by LNSMAER and SKOOG (965) cntaining amin acids, adenine and 4,5 ~ 0- s M,4-0 and kinetin. On further transfer t media lacking auxin and cytkinin, 'pseudbulbils' appeared which later develped int adventive embrys. This latter type f grwth was prmted n media withut rganic nitrgen supplements and eventually gave rise t 'frnds' abut 7 cm lng. The resulting pantlets did nt develp further and were nt transferred t sil. Diplid cultures frm Cyc~ pr-embrys were unsuccessful. n the experiments with haplid material, using media similar t thse previusly described but relatively high in auxin (4,5 x 0- s M,4-0), megagametphytlc explants frm bth lamia and Cyc~ culd be maintained, sub-cultured, and gave every indicatin f " lng-term sustained grwth. Abundant undifferentiated tissue appeared

62 48 within days n bth slid and liquid media althugh the frmer was mre successful. Light and darkness had n bvius affect. As with the diplid experiments, subsequent transfer f the Zamia-derived callus t media withut auxin and kinetin resulted in the frmatin f smatic embrys. These structures frmed haplid plantlets in minimal media but the plants were nt established ;n sil. NORSTOG and RHAMSTNE (967) mentin specifically that a decisive factr in inductin f embrygenesis is the islatin f cells frm rganised tissue. Prliferat0n f small clumps f undifferentiated cellular material led t,matic embryny whereas the use f larger explants favured direct rgangenesis. Anther bservatin by these authrs is the imprtance f nitrgen frm amin acids rather than frm inrganic surces. t is als evident that the develpment frm embry t plantlet required a depaupe rate medium; the authrs cnclude that the situatin ~~ v~ is analgus t the usual pattern ~~ v~v, where the embry, initially dependent n its cmplex rganic fd reserve, becme auttrphic and grws n a largely inrganic substrate. tal ian wrkers ~E LUCA, MORETT and SABATO, 979) used haplid explants frm the cycads CeJr..cU:zami.a meuc.ala Brng., Cyc.CUJ flevtuta Thunb. and the Suth Afri can species Etlc.epitataJt.t.6 wnbetuz.tet.6~ R. A. Dyer, and explred respnses t a wide variety f media under different temperature and light cnditins. Verificatin cunts f the resulting tissue cnfirmed the plidy status. CeJr..cU:z~ megagametphytes n medium '8' (Table 3) cntaining 4,5 x 0- s M..4-0 and kinetin swelled and became green in a shrt time. Thereafter the results were smewhat variable;

63 49 With CyQ~ haplid n medium '8', callusing was fllwed by 'pseudbulbil' develpment in relative prfusin. Hwever, these spherical ndules were vascularised with tracheids and later shwn t be analgus t crallid rt ndules, a nrmal feature f cycad ntgeny (DE LUCA and SABATO, 980). EtQe.pltalaJLtO.6 material n the same medium frmed callus withut 'pseudbulbil' develpment. Bth CyQ~ and EitQe.pltalaJLt0.6 megagametphytes prduced callus n medium '59' with 4,5 x 0-~M,4-0, but there was n indicatin f subsequent mrphgenesis. DE LUCA, MORETT AND SABATO (979) cnclude that the ptential fr regeneratin appears t be favured by ambient (5 C) rather than elevated (35 C) temperatures, by diffuse light (50~Em-s-) rather than darkness, and by media generally with lwer cncentratins f auxins, cytkinins and amin acids. There was n reprt f attempted subculturing t. different media and it ;s nt clear whether plantlets were grwn beynd the stage illustrated by the authrs. DE LUCAAND SABATO (980) cultivated megagametphyte tissue frm CYQ~ ~e.vluta Thunb. n WHTE's (943) basal medium supplemented with,4-0 and kinetin at varius levels under cntinuus lw-intensity (50~Em-s-) light. After three mnths n the medium with 4,5 x lo-sm levels f each grwth factr, a large number f explants had develped craliid rts frm spherical ndules identical t thse btained in earlier wrk (DE LUCA, MORETT and SABATO, 979). N micr-rganisms were assciated with these rt ndules which thus appear t be an inherent feature f cycad rt systems and nt necessarily a respnse t a particular symbitic assciatin. The regeneratin f crallid rts i~ v~ extends an earlier bservatin that apgetrpic rts are frmed when seeds f Ma~zamia ~e.dle.i (Gaud.) Gardn. are grwn in sterile cnditins (LAMONT and RYAN, 977). Similar results t thse frm CyQ~ ~e.vluta were btained in an experiment with explants frm Ma~zamia Qmmuni.6 L. Jhnsn (DE LUCA, SABATO, BALDUZZ and NAZZARO, 980 ). Megagametphyte halves, cultured n WHTE's (943) medium supplemented with 4,5 x 0- s M,4-0 and kinetin gave callus frmatin after tw mnths! Abut half these calli subsequently gave rise t crallid rt primrdia, again free frm any micr-rganisms. The authrs speculate that the cycad crallid rts represent remnants f primitive pneumatphres which acquired the symbint in an

64 50 eviutinary advance. Further investigatin int regeneratin frm z. p~a megagametphytes was carried ut by RVERA ROSA (unpublished results). Using Murashige Skg medium with varius levels f -naphthaleneacetic acid and 6-benzyladenine, ndular callus was btained at different frequencies. With 4.5 x lo-7m levels f each grwth factr, half the explants gave ndular callus which in turn gave rise t biplar dictylednus embryids. These failed t develp further. With 0- s M NAA and 0-6M BA, a friable callus was btained n 50% f the explants and ccasinal rts were frmed. Sme sht mrphgenesis was seen n transfer t hrmne-free medium but autnmus plants culd nt be raised. When entire Z. p~a seeds were grwn ~n v~, the germinating rt prduced a friable callus. f this callus was excised, regeneratin ccurred frm the cut end f the embry and frm the megagametphyte, the latter develping utgrwths which became rts. ' Rts als ccasinally arse frm ~he area in these circumstances, and nrmal leaf develpment was bserved in 6% f the explants. nypctyl-r.tyled~nary Small cylindrical explants f megagametphyte frm the particularly rare Cuban cycad, ~~cyca6 calcma (Miq.) A.DC. were cultivated by PENA and GRLLO (98) n Murashige-Skg medium mdified by the inclusin f NAA and ccnut milk, but lacking ptassium idide. With 0% ccnut milk a yellw, friable callus develped and rts were prduced when 5 x 0-6M NAA was present. ncreasing the auxin level inhibited rhizgenesis and altered the texture f the callus. With 5% ccnut milk and 5 x 0- sm NAA, rapid callgenesis ccurred with rt frmatin fllwing transfer t medium with reduced NAA levels. At very lw r zer NAA cncentratins, the frmatin f pink 'pseudbulbils' was reprted. Callus grwth culd be maintained thrugh five successive passages but grwth rates steadily decreased.

65 5 Yet anther prject n megagametphytic tissue was that f LALBERTE, BERTRAND and VETH (983) wh used explants frm infertile seeds f Encephai~~ v ti~~ Lem. n a mdified WHTE's (943) medium cntaining thiamine Hel (0, mg - ), insitl (50 mg - ), glutamine and asparagine (00 mg - each) and adenine sulphate (40 mg - ) under medium intensity light (50~Em-s-) fr ~ l6-hur phtperid. Surface ndulatin fllwed callgenesis and was mst abundant when the - medium was supplemented with 4.5 x 0-6 M,4-0 and,3 x lo-tim kinetin. Micrscpic studies shwed that these meristematic areas ccurred internally as well as superficially. Althugh sme tracheid frmatin was fund in the callus, there was n significant mrphgenesis. N attempt was made t subculture callus r t prmte rganised develpment. Cmparatively little in v~ wrk has been attempted with cycad micrgametphytes. LA RUE (954) carried ut experiments with Zamia pumiia micrsprangia and cited earlier reprts f cycad pllen germinatin n a variety f media. Since mature pllen grains were killed during disinfestatin prcesses, unpened micrsprangia cntaining micrspres at the uninucleate stage were used. These were cultured n WHTE's (943) medium with a wide variety f additives. The supplements had little effect; nrmal pllen develpment ccurred in all cases, with pllen tube frmatin but withut apparent spermatgenesis. While pllen tubes were viable fr a prlnged perid, grwth was limited and the cultures eventually senesced. n v~ spermatgenesis was achieved when micrsprangia f Encep~~ aitenteinii Lehm. were cultivated n a medium which TULECKE (957) had p~eviusly used fr pllen studies n G~nkg biiba L. (DE LUCA and SABATO, 979). Micrspres were in a trinucleate cnditin at the time f inculatin. After abut ne week the micrsprangia burst pen and a mass f pllen tubes appeared. Thereafter spermatgenesis prceeded nrmally, giving ~ise t the characteristic large mtile spermatzids after 6 mnths. There was n difference in bservatins when the medium was supplemented with,7 x 0-6 M,4-0. n a similar experiment, using the same media, spermatgenesis f C~atzamia mexicana Brng. and Cyc~ ~eviuta Thunb. was investigated

66 5 (DE LUCA, LA VALVA and SABATO, 980). With the latter species, micrgametphytes remained at the immature sperm mther stage after mnths in culture. n the case f the C~atzamia, callgenesis was recrded after mnths in tw f the cultures which had been prvided with the,4-0 supplement. This callus had a superficial periderm and a parenchymatus cre but it was nt pssible t ascertain frm which tissue the grwth had riginated. Chrmsmal assessment was nt carried ut t cnfirm the presumably haplid nature f the callus. The amunt f published research wrk n cycad tissue culture, ther than that f gametphytic rigin, is limited. Pssibly the ntriusly slw ~~ v~v grwth rate has influenced wrkers against the chice f these plants fr shrt-term prjects. Frtunately mre attentin is nw being paid t the ptential benefits f tissue culture as a prpagative tl; this is especially true fr cycads where the cntinued survival f s many taxa is in jepardy (Sectin.4i. BROWN and TEAS (966) btained callus grwth frm mature embrys f lamia ~~da~a A.DC. (=l. pumiia L.) and frm the leaf rachi s f Cyea/.) Jtev'tut.a Thunb. usi ng Knp s salts and 3% sucrse in slutin. Rhizgenesis fllwed n the embry-derived callus with sme evidence f leaf prductin later. MUSTOE (967) reprted the prductin f callus frm cambial explants f the same tw species. A fairly extensive prject n the ptential f cycad tissue culture fr prpagatin was carried ut by HENSON (980) at the micrprpagatin unit f the Ryal Btanic Gardens at Kew. Using relatively large explants, up t 3 cm in length, frm yung leaves and ther parts f the plant, including vules, mega- and micr-sprphylls, crallid rts and seedling hypctyls, and using mainly Murashige Skg media with a range f cncentratins f auxins, cytkinins and gibberellic acid, Hensn grew callus frm 35 cycad species. Callus frmed readily with explants frm C~atzamia, Sta~g~a and lamia withut exgenus grwth factrs, but either NAA r AA was needed fr ca us ; niti at; n in eyea/.), V-i~, EtteephataJtt0.6, Lep~dzamia and

67 53 Ma~zamia. Whilst the abslute cncentratins were nt critical, gd results were generally btained with 5,3 x 0-6 M NAA and 4,5 ~. 0-7 M BA. Callus mrphlgy and clur were variable and independent f explant rigin r grwth regulatr applicatin. Friable callus prliferated n sub-culture but did nt differentiate. A slwer-grwing, mre cmpact callus, which develped in abut half the species tested, gave rise t spherical structures ana9gus t LA RUE's (954) pseudbulbils. These were mst readily btained in cultures kept in the dark fr several mnths and did nt necessarily require any exgenus grwth substances. The structures cmprised an range-brwn epidermis and a parenchymatus cre with internal tracheidal develpment. Rhizgenesis was bserved frm the spherical structures btained frm yung lamia leaves in a few instances, but plantlets were nt successfully regenerated. Anther majr prject with a prpagative bias was that undertaken by Suth African wrkers (KOELEMAN and SMALL, 98). Stem and rt explants frm E. c.upiclu.6 R.A. Dyer, E. eugette-majtal6u Verdrn, E.. ittpittll6 R.A. Dyer, E. R.anatLL6 Stapf and Burtt Davy, E. R.ebmbelt6.t.6 Verdrn, E. R.efunanttU Lehm., E. tatar.elt6.t.6 R. A. Dyer and Verdrn, E. pauc.. cietttatll6 Stapf and Burtt Davy and E. t4alt6ven LL6 Stapf and Burtt Davy were used. The aim was t develp a prtcel generally applicable fr clnal prpagatin f the rarer cycad species. Callgenesis was achieved in each f the nine species tested, requiring abut tw t fur mnths fr stem explants and an additinal tw mnths fr rt tissue. Optimum results were btained using medium 'Kl' (Table 3) which is a MURASHfGE SKOOG medium cntaining amin acids amides and supplemented with 5,4 x 0-5 M NAA and 7,0 x 0-5 M kinetin. Explants frm mesic species generally grew faster than thse frm xeric habitats. Of the species tested, E. R.ehmbell-6.t.6 was the mst vi grus wh i e. R.attatM gave the prest respnse. KOELEMAN (pe~. cmm) subsequently reprted that n rgangenesis ccurred in this series f experiments. Extensive use f itt v~ culture techniques has been made by WEBB and clleagues f Queen's University. Kingstn. Canada (WEBB. 98, 98a, 98b, 983, 984; WEBB and DE JESUS, 98; WEBB, NEVAREZ and DE JESUS, 984). Wrk was initially cncerned with the assessment f light n cycad apgetrpic rt ndule develpment. Embrys frm several taxa were cultured in darkness n a medium based n WHTE's

68 54 macrnutrients with MU~ASH[GE-SKOOG micrnutrients, and the resulting tissues then expsed t different light regimes. A general cnclusin was that light stimulated ndule develpment but inhibited bth primary and secndary rt initiatin and elngatin. f leaf initiatin did ccur, this fllwed much later. Partial r cmplete ctylednary excisin r remval f the gametphyte suppressed grwth (WEBB, 98a) as did the reductin r dilutin f nitrate in the medium (WEBB. p~. cmm.) Light-induced rt ndules had the same anatmy as algal free ndules frm sil-grwn plants (WEBB. 983) but there has nt yet been any attempt at establishing the cycad-cyanbacterium symbisis ~~ v~. Using mature embrys f)'m Zam{a pumita, WEBB, RVERA STARSZAK and MATOS (983) reprt n callus frmatin and differentiatin n MURASHGE SKOOG medium with different NAA and BA supplements. The auxin was necessary fr callus initiatin while the cytkinin appeared t affect the frequency f callgenesis and the mrphlgy f the prduct. Gd results were btained with 4,5 x 0-6 M NAA and BA. Friable callus, with pr regenerative ptential, was prduced when the NAA/BA rati was high. Mre cmpact callus develpmen~ when the BA level was increased and this yielded rts, shts and ccasinal embry-like structures resembling the smatic, embrys reprted by NORSTOG and RHAMSTNE (967). Althugh there was cnsiderable leaf elngatin ~~ v~, auttrphic plants culd nt be established. Cntinuing the studies n Z. pumila, MONNER and NORSTOG (984) cultivated immature embrys and their separate embry. suspensr and egg membrane cmpnents n varius media mdified frm a barley embry culture frmulatin, essentially t cmpare develpment ~~ vul and ~~ v~. Maximum grwth arse at the embry base and after tw mnths prtruberances identifiable as ctyledns appeared, but these had a mre callus-like appearance than the cmparative stage ~~ v~v. The suspensr and the egg membrane were nt required fr embry develpment ~~ v~ except in particularly immature embry systems. Further wrk was carried ut by the same authrs (MONNER and NORSTQG, 986) in rder t cmpare the ~~ v~ grwth f Z~a embrys excised at different stages in their develpment. Embrys excised shrtly after fertilizatin gave an initial extensin f the suspensrs fllwed by enlargement f the embry t a club-shaped structure with callus-like cellular appearance.

69 55 Embrys excised tw mnths later were still undifferentiated but rapidly develped tw ctyledns in culture. When these embrys were lngitudinally bisected, each regenerated a biplar structure. Mature, differentiated embrys did nt have this ptential fr regeneratin. The authrs prpse three stages f ~~ vui develpment : an early unprgrammed phase is fllwed by a time when inductive stimuli cause prgrammed rgangenesis; the final phase is a perid in which cell lineages are n lnger labile..4 Experimental : materials and methds.4. Surces f plant material Seeds, seedlings and vegetative material used in the tissue culture wrk described in this Chapter and fr the bi~chemical studies described in Chapter 3, were btained frm a large number f surces. n each case, care was taken t ensure that the material was crrectly identified; where any dubt existed as t the validity f the name, the material was rejected. Seeds and yung seedlings f E~cephal~~ and Sta~g~a species were btained frm members f the Cycad Sciety f Suthern Africa either directly r via the Sciety s central seedbank. Seeds f the Australasian cycads, Bwe.tua, CJC~, Le.p.<..dza.mi.a and MaCJtzarrU.a, were supplied by the Palm and Cycad Sciety f Australia. Seeds frm the New Wrld genera, C~atzarrU.a, V~~ and Za.mi.a, were sent frm the seedbank f the Cycad Sciety (U.S.) and frm Fairchild Trpical Garden, Flrida, U.S.A. Vegetative material (leaves, rts, etc.) and samples frm reprductive rgans (cne-scales, cne-axes, pllen, etc.) were made available by the Durban Btanic Gardens, the Durban unit f the Btanic Research nstitute and by cycad enthusiasts with large plant cllectins f knwn prvenance, including Mr G.C. Cx, Mr. R. Heerman, Mr. R.E. Levitt and Mr. H.E. Whlberg, all f the greater Durban area.

70 56.4. Nursery prcedure Seeds were germinated in seed trays cntaining a friable, humus-rich ptting sil in which the seeds were placed hrizntally and buried t apprximately ne-third f their depth. After the hypctyl emerged frm the micrpyle, the seeds were transferred individually t black plastic nursery bags, 00 mm x 00 mm x 55 mm, cntaining humus-rich garden lam supplemented with a cmmercial 3:: (5) granular fertiliser at the rate f 50 g per 50 kg. Watering was carried ut s as t maintain mist cnditins. Pest cntrl WaS accmplished by the ccassinal use f a cmmercial systemic insecticide. Seeds and seedlings were kept in a shade huse cvered with "50%"shade clth and at ambient temperature cnditins..4.3 Disinfectin prcedures The disinfectin prcedures adpted in this prject generally emplyed ethanl and hypchlrite slutins as surface sterilants; these prcedures being mdified slightly dependent n the nature f the material. Fresh seeds were freed frm their fleshy sarctestae and washed thrughly in water. The seeds were then immersed fr 4 minutes in 70% (v/v) ethanl cntaining a few drps f "Teepl/ wetting agent, washed with sterile distilled water and placed fr 30 minutes in a 0% (v/ ) v slutin f "Jik" (3~5% NaOel when packed, Reckitt Husehld Prducts), als cntaining a few drps f wetting agent. The disinfected seeds were then rinsed three times in sterile distilled water, allwing 5 minutes saking time between each change. The sclertestae were then cracked with sterile pliers and the intact megagametphytes subjected t a further hypchlrite treatment and washing prcess as described abve. S~eds treated in this manner were then aseptically dissected under a laminar-flw hd t give embrys r sectins f megagametphytic tissue. Primary rt tissue was btained frm seedlings grwn as described in Sectin.4.. The seedlings were carefully remved frm their

71 57 cntainers, freed frm the bulk f the sil medium and washed in running tap water. After remval f the leaves and lateral rtlets, the primary rts were scrubbed with a sft bristle brush t remve adherent sil particles tgether with the epidermal layer. The cleaned rts were then immersed in 70% ethanl and 0% "Jik" with water rinses as described abve. Aseptic dissectin then prvided cubes r cylinders f rt tissue fr transfer t culture. Cycad leaves were fund t give the mst difficulties with regard t disinfectin. The cmbined effect f a waxy cuticle, cvering f leaf hairs and deeply-recessed stmata necessitated a fairly rigrus treatment. Hwever, the yung, actively-grwing leaflets, which ffered the best ptential fr grwth ~n v~, were prne t damage during the disinfectin prcess. Thus the fine balance between incmplete disinfectin and necrtic damage f the leaf tissue was nt easily btained. The prcedure adpted was as fllws. The intact leaflets were washed with tap water cnt~ining % "Teepl", swabbed briefly with tluene and chlrfrm in successin, immersed fr 4 minutes in 70% ethanl, rinsed in sterile distilled water, immersed in 0% "Jik" fr 30 minutes, rinsed in sterile distilled water, immersed fr 30 minutes in 0,5% mercuric chlride slutin, rinsed in sterile distilled water, immersed again in 70% alchl fr 4 minutes and finally rinsed three times with sterile distilled water. Despite this rigrus treatment, fungal infectins were frequent and between 50 and 00% f leaflet explant cultures shwed cntaminatin within 3 weeks. seem t have been addressed by ther wrkers. This prblem des nt Fr instance, HENSON (980), strngly favured the use f cycad leaflets as an explant surce but did nt describe his disinfectin prcedure. t;s speculated that the deeply-recessed stmata in cycad leaflets (BAJNATH, NADOO and RAMCHARUN, 980) act as reservirs fr micrbial infectin but the pssibility f natural fungal endphytes cannt be eliminated. Other cycad tissues used in this prject, e.g. leaf petiles, cne. ' scales and cne axes, were disinfected by the sequent.ial treatment with ethanl and hypchlrite slutins as previusly described. These surces f tissues, as with the seed and rt material, resulted in spradic cntaminatin by bacteria and fungi, but the infectin f~equency (generally less than 5%) was cnsidered acceptable.

72 Chice and preparatin f media Mentin has been made in Sectin.3 f several specific media used fr the i~ v~ culture f cycad material; these being essentially mdificatins f WHTEls (943) medium and that f MURASHGf and SKOOG (96)~ as detailed in Table 3. Table 4 gives details f fur ther widely-used media: the riginal MURASHGE and SKOOG (96) (MS) frmulatin and the subsequent LNSMAER and SKOOG (965) mdificatin; the SCHENK and HLDEBRANDT (97) (SH) medium; the medium used fr cell suspensin cultures develped by GAMBORG, MLLER and OJMA (968) (B5) and that used fr pllen grain wrk by NTSCH and NTSCH (969) (NN-69). These media reflect nly a few f the wide number f frmulatins develped and mdified ver the last 50 years. Since the nutritinal requirements f plants i~ v~ cannt be uncnditinally extraplated t culture requirements i~ v~~, almst all wrk invlves an empir cal prcess f media selectin and ptimizatin. Thus the usual prcedure invlves the use f ne r mre f the mre ppularli media as a starting pint with prgressive mdificatin f individual cmpnents n the basis f experimental results, until near-ptimal grwth is btained. Often the macr-nutrients f ne frmulatin are cmbined with the micrnutrients f anther and even the rganic supplements f a third. Anther mdificatin, ften ap~lied t mineral-rich media like the MS frmulatin, is the use f half - r quarter-strength mixtures.. Finally, it must be nted that the medium ften requires mdificatin fr different stages f grwth - i.e. initiatin, prliferatin and mrphgenesis. n rder t prvide a basis fr initial medium selectin, it is useful t review briefly the rles f particular cnstituents. Macr-nutrients The levels f each f the macrnutrient elements (N, P, K, Ca and Mg) and the lincidental ll elements (S, Cl and Na) fr the varius frmulatins discussed, are shwn in Table 5. A detailed discussin f the functin f each element is beynd the scpe f this text; such infrmatin is readily btained frm reviews n plant nutritin tmurashge. 973; CLARKSON and HANSON, 980). Besides their specific majr structural and functinal purpses, the elements may cntribute t buffer capacity and smtic ptential, act in in-balance rles and catins may serve as Lewis acids.

73 59 TABLE 4 FOUR MEDA USED N TSSUE CULTURE EXPERMENTS Cnstituents mg per 000 ml "HS,, "SH,,3 "B5"~ "NN-69" 5 HgSO~.7H CaC.H zo KN KHzPO~ 70 NH,H PO, 300 NaHPO~.HzO (NH,)zSO, 34 NH,N H 3 B0 3 6, 5,0 3,0 HnSO~.4H0,3 0 * ~ ZnSO~.7H0 8,6,0,0 K 0,83,0 0,75 NaHO~.H0 0,5 0, 0,5 CuSO,.5H zo 0,05 0, 0,05 CCl z.6h zo 0,05 0, 0,05 NazEDTA.H zo 37,3 0 37,3 FeSO,.7H zo 7,8 5 7,8 Glycine,0 7 Fl ic acid Bit in Thiamine.HC 0, 7 5,0 0,0 lictinic acid 0,5 7 5,0,0 Pyri dx i ne. HC 0,5 7.0,5,0 ns it Sucrse Sact-agar SO , ,5 0,05 37,3 7,8,0 0,5 0,05 0,5 5,0 0, ~ Ntes: () () (3) (4) (5) (6) (7) Auxin and cytkinin are added accrding t specific requirements. MURASHGE and SKOOG (96). SCHENK and HLDEBRANDT (97). GAHBORG, HLLER and OJHA (968). NTSCH and NTSCH (969). * These media ist this as the mnhydrate, HnSO~.HzO. The LHSAER and SKOOG (965) mdificatin f the HS frmula mits nictinic acid, pyridxine and glycine, and increases the thiumine level t 0,4 mg.e.-

74 TABLE 5 MACRO-ELEMENTS PRESENT N VAROUS CULTURE MEDA : ELatENT SUPPLED White's basal Frmulatin (refs. given n text, see als Tables ); all figs. as mg per 000 ml (Figures n parenthesis represent the crrespnding mlar cncentratins) "8" "63" "59" "K" "HS" "SH" "B5"....=-==--=:~ -- :a r:~ N (ex NHt) (,5 x 0-3 ) (, x 0- ) (, x 0- ) (, x 0- ) (,6 x 0-3 ) (,0 x 0-3 ) "NN-69" 6 (9,0 x 0-3 )..J c ; J - : f r, ~ z w ' ' w / /:, -J / /, c J,. ~ z f, w, - Q i u z -,, ~ /; r - N (ex NO;) N (ttal) , x 0-3 ) 0, x 0-3 ) C7,lxl0-3 ) 0,9 x 0- ) 0,9 x 0- ) 0,9 x 0- ) (,5 x lq-) (,5 x 0-~) 45' , x 0-3 ) 0, x 0-3 ) (8,6 x 0-3 ) (6,0 x 0- ) (6,0 x 0- ) (6,0 x 0- ) (,8 x 0- ) (,6 x 0- ) p (,Ox 0 -~ ) (,0 x O-~) (5,8 x 0-3 ) (,xl0-3 ) (, x 0-3 ) (, x 0-3 ) (,6 x 0-3 ) (, x 0-3 ) K (,7 x 0-3 ) (,7 x 0-3 ) (,4 x 0- ) (,0 x 0- ) (,0 x 0- ) (,0 x 0- ) (,5 x 0- ) (,5 x 0- ) Ca (, x 0-3 ) (, x 0-3 ) (, x 0-3 ) 0,0 x 0-3 ) 0,0 x 0-3 ) 0,0 x 0-3 ) (,4 x 0-3 ) (,0 x 0-3 ) Mg ,0 x 0-3 ) (3,0 x 0-3 ) 0,0 x 0-3 ) (,5 x 0-3 ) (,5 x 0-3 ) (,5 x 0-3 ) (,6 x 0-3 ) (,0 x 0-3 ) S (4,5 x 0-3 ) (4,5 x 0-3 ) (4,4 x 0-3 ) (,7x 0-3 ) (,7 x 0-3 ) (,7x 0-3 ) (,7 x 0-3 ) (, x 0-3 ) C (8,7 x 0-~) (8,7 x 0-~) (9,4 x 0-3 ) (6,0 x 0-3 ) (6,0 xl 0-3 ) (6,0 x 0-3 ) (,7 x 0-3 ) (,0 x 0-3 ) Na (,9 x 0-3 ) (,9 x 0-3 ) (8,6 x 0-3 ) (, x 0-3 ) NOTE ; The nitrgen figures d nt include cntributins frm the amin acids present in "8", "63", "59" and "K". = 58 (,8x 0- ) 384 (,7 x 0- ) 6 (5,0 x 0-~) 387 (9,9 x 0-3 ) 45! (, x 0-3 ) 8 (7,6 x 0-~) 3 : i (,0 x 0-3 ) : 80 (, x 0-3 ), -,, ~ 0'\

75 6 Nitrgen is supplied in three frms: inrganic ammnium ins, as frm ammnium nitrate, dihydrgen phsphate, sulphate and malate; nitrate ins, as frm calcium, ptassium and ammnium nitrate; and rganic nitrgen cmpunds such as amin-acids, ther amines and ami des and mre cmplex nitrgenus substances. A drift in ph during culture may ften be ascribed t the absrptin f NH+ r NO- with the cnsequent 4 3 release f prtns r anins int the medium. Variatin n the nature and quantity f nitrgen used is clearly seen in Table 5. WHTE's (943) medium has the lw ttal N f 3, x 0-3 M while the MS medium is 3,9 x la- M. Nitrate is cnsistently supplied at levels greater than that prvided by the ammnium in, which is smetimes cnsidered inhibitry (MURASHGE, 973). The lw nitrgen in WHTE's medium is ffset in the "8" and "63" mdificatins with amine supplements. Phsphrus is emplyed in all frmulatins as the dihydrgen phsphate anin, HPO:~ supplied as its ptassium, ammnium r sdium salt, and has usefulness in buffering capacity apart frm being a surce f the element. Medium "63" has the highest level f phsphrus, at 5,8 x la- 3 M but the use f the sdium salt prvides the high catinic level f 8,6 x la- 3 M which may be disadvantageus. Ptassium is supplied as the nitrate, the dihydrgen phsphate r the chlride, with levels frm,7 x la- 3 M in WHTE's frmulatin t,5 x la- M in bth "SH" and "65". Use f the chlride anin at 9,4 x lq-3m in "63" may create adverse effects. Calcium is present frm nitrate r chlride surces and varies frm,0 x la- 3 M t 3,0 x la- 3 M. Magnesium is cnsistently supplied frm Epsm salts and the quantities used yield the catin at between 7,6 x 0-4 M and 3.0 x la- 3 M. A cmparisn f the varius media shws that the MS frmulatin is mst "nutrient-rich" with WHTE's medium at the ther end f the scale. An interesting quantificatin f this is btained in cmparisn f the ttal. macrnutrient levels; MS prvides 9,3 x la- M, SH has 6,3 x la-m and WHTE's,7 x la- 3 M. A further cmparisn f general interest is that f the N:P:K ratis; MS has 50::7, SH prvides ::0 and WHTE's shws 3::7. Whilst this may be useful reference infrmatin, it is nt apprpriate t select anyne medium as being superir t anther n this basis; such decisinscan be made nly n the basis f experimental bservatins.

76 6 Micr-elements The levels f micrnutrients supplied in the varius media described are shwn in Table 6. Less variatin is seen in bth the element leveis supplied and the range f surce cmpunds used. Micr-elements are required by plants fr specific structural purpses (e.g. metallprteins r structural chelates), participatin in redx reactins via electrn transfer, and by the catalytic rle f the transitin metals. They may als serve as Lewis acids. The irn level is variable and a range frm, x 0-sM t,0 x 0-*M is seen. Older literature refers t the use f ferric citrate and ferric sulphate as irn surces but the mre recent frmulatins cnsistently use ferrus sulphate with a stichimetric equivalent f EOTA. t shuld be nted that sme f this EOTA will inevitably 'migrate' in the prepared medium t thse trace metals with higher cmplex-frmatin cnstants (C, Cu and Zn) but, since these are present at much lwer cncentratins, the net lss is small. Use f the cmplexing reagent inhibits undesirable precipitatin reactins in the medium. Carbhydrates n all frmulatins quted, carbn is supplied as sucrse at 5,8 x la- M t 8,8 x la- M (0-30 g - ). The sucrse serves the threefld purpse f being a surce f elemental carbn, prviding an energyrich substrate fr the hetertrphic metablism ~l vi)bt, and has the majr influence n smtic ptential f the medium. The mnsaccharides glucse, arabinse, mannse, ribse and xylse, and the hexl carbhydrate, srbitl, have smetimes been used in media but withut any apparent general advantage. The substance my-insitl (), ne f the fur naturally-ccurring ismers f hexahydrxycyclhexane, has been used widely in media. Thught t be invlved with the synthesis f phsphlipids, ~el wall pectins and cytplasmic membranes, and a cnstituent f the vitamin B cmplex, it is ften incrprated int media at levels frm 5,6 x 0-~M t 5,6 x la- 3 M ( mg - ).

77 TABLE 6 "CRO-ELE"ENTS PRESENT N VAROUS CULTURE "EDA Frmulatin (refs. given in text, see als Tables & ); all figs. as mg per 000 ml (Figures in parenthesis.represent the crrespnding mlar cncentratins) ELE"ET SUPPLED White's basal "8" "63" "59" "K" "MS" "SH" "85" "NN-69", Fe 0,70 (, x 0-5 ) Mn,65 (3,0 x 0-5 ) Zn 0,6 (9,4 x 0-6 ) 0,70,67 5,58 5,58. 5,58 3,0 5,58 (, )( 0-5 ) 0,0 x 0-5 ) (,D x 0-~) (,D x O-~) (,OXO-~) (5,4 x 0-5 ) (l,xlo-~),65 0,74 5,50 5,50 5,50,9,9 0,0 x 0-5 ) (,3 x 0-5 ) (,0 x 0-~) (,0 x 0-~) (,0 x 0-~) (5,8 x 0-5 ) (5,8 x O-~) - 0,6 0,,95,95,95 0,3 0,45 (9,4 )( 0-6 ) (,7 x 0-6 ) 0,0 )( 0-5 ) 0,0 x 0-5 ) 0,0 x 0-5 ) (3,5 x 0-6 ) (l,o x 0-6 ) 5,58 (,D x 0-~). 6,6 (, x 0-~),7 0,5 x 0-5 ) i B 0,7 (,4 x 0-5 ) 0,57 (4,5 x 0-6 ) ~- M - 0,7 0,09,0,0,0 0,89 0,53 (,4 x 0-5 ) (8,0 x 0-6 ) (,Oxl0-~) (,D x 0-~) (,D x O-~) (8, x 0-5 ) (4,8 x 0-5 ) 0,57-0,63 0,63 0,63 0,76 0,57 (4,5 x 0-6 ) (5,0 x 0-6 ) (5,0 x 0-6 ) (5,0 x 0-6 ) (6,0 x 0-6 ) (4,5 x 0-6 ) - 0,00 0,0 0,0 0,0 0,040 0,0 (,0 x 0-7 ) (,D x 0-6 ) (,D x 0-6 ) (,D x 0-6 ) (4, x 0-7 ) (,0 x 0-6 ),77 (,6 x O-~) - 0,0 (,0 x 0-6 ) i Cu - C - - 0,006 0,006 0,006 0,006 0,05 0,006 (,D x 0-7 ) (,D x 0-7 ) (,0 x 0-7 ) (,D x 0-7 ) (f,o x 0-7 ) (,0 x 0-7 ) 0,006 0,006 0,006 0,006 0,006-0,05 (,D x 0-7 ) (,0 X 0-7 ) (,0 )( 0-7 ) (,D x 0-7 ) (4, x 0-7 ) (,D x 0-7 ) 0,006 (,0 x 0-7 ) - i m w

78 64 OH OH H () MYO-NOSTOL OH H"'( y'"' ex CH,CH,OH ~C/+ Hz () THAMNE Cl- CH3 ClOHH, (3) NCOTNAMDE (N') ~COOH (4) NCOTNC ACD H 3C N HOV,",OH CHzOH (5) PYRDOXDE (CH z).. COOH (6) BOTN CH3 0H HOCHz--C--C--COHN-tHzCHzCOOH CH 3H (i) PANTOTHENC ACD ceoh NHz (8) PARA-AMNOBENZOC ACD ( 9 i FO L C AC 0 (lor CHOLNE () RBOFLAVNE

79 65.CH,CH,CONH, CONH,./ M CH CONH,," Me ~ CHJ ~. ~ ~ M.,. CH,CH,CONH, M... 't!--n'" ~N~ H~/jO~ J, :::.:... Me CH J Me ~O~H, / M. CH,CH,CONH, i H CO-CH,CH, /.-'' <"::0.' -<~Hr.."M. HOH,K/ NH., - HN=C-NHCH.C~ CH. ---C---COOH., NH z H (3) L-ASCORBC ACD NH z f H z NCOCH z, ---C---COOH H () CYANOCOBALAMN (4) L-ARG NE ( 5) L-ASPARAGNE ~H HOOC---C---~ H CH CONH, H (6) L-GLUT~NE HzNC H COOH (7) GLYCNE NHz H3 C---C---CJOH H (8) L-ALMWlE -0- HO ~ CHz---i---COOH (9) L - TYROS NE NHz, NH: HSCHz---C---COOH A (0) L-CYSTENE H (Xl N~ CHz--!--COOH, H () L-TRYPTOPHANE H eyco'h ( ) L -PROL NE (3) ADENNE co"h (4) NDOLE-3-ACETC ACD AA (5) l-naphthaleneacetc ACD NAA

80 66 OCHCOOH C C (6),4-CHLOROPHENOXYACETC ACD,4-0 H N ~CH')'COOH (7) NOLE-3-BUTYRC "AC0 BA OCHCOOH ~C C J (B),4,S-TRCHLOROPHENOXYACETC ACD,4,S-T {9) 4-CHLOROPHENOXYACETC ACD CPA (30) 4-AMNO-3,S,6-TRCHLOROPCOLNC ACD PCLORAM COOH Cf~OCHJ U Cf (3) 3,6-CHLORO-0-ANSC ACD (OCAMBA) HN-CHz-D J-vN~ 0 l,j-nh (3) KNETN, KN HN-CH,-O ~'~ lnj-nh (33) 6-SfNZYLAOENNE, SA CH,~'N~-<N: lnj-nh (34) SO-PENENYLAOENNE. ip, PA

81 67 Vitamins n their wrk with tbacc callus culture, LNSMAER and SKOOG (965) demnstrated that many water-sluble vitamins are nt essential; indeed sme f these cmpunds are grwth inhibitrs. One f the mre imprtant plant vitamins is thiamine (). (vitamin B ) which is invlved with the c-enzyme ccarbxylase, essential in the KREBS cycle, and als with the transketlase enzyme in the pentse shunt pathway in carbhydrate metablism. Thiamine is generally supplied as the hydrchlride, at levels frm 3 x 0-7 M t 3 x 0- s M. The usefuiness f ther B-grup vitamins remains equivcal and synergistic interactins further cmplicate matters. Despite this, mst frmulatins include nictinamide (3) (niacin) r nictinic acid (4) which are imprtant c-enzymes in bilgical dehydrgenase reactins. and pyridxine (5 (ne f the vitamins B ), supplied as the hydrchlride, and which is 6 an essential c-enzyme in amin acid metablism. These three cmpnents may be incrprated at levels frm 0-7 M t 0- s M. Less cmmnly used are bitin (6) (vitamin H), pantthenic acid (7), as its calcium salt, and p-am;nbenzic acid (8). The latter is a part f the structure f flic acid (9), itself ccasinally used in culture media. Chline (0) is anther cnstituent f the vitamin B cmplex and ccasinally used in its chlride frm. Ribflavin () (vitamin B ) is rarely used in view f its pssibly inhibitry effects (LNSMAER and SKOOG, 965). Finally, amng the B-grup vitamins, the unusual cbalt-cntaining natural prduct, cyancbalamin () (vitamin B ), has infrequently been added t media at very lw cncentratins. Amng the ther vitamins it is nly vitamin C that has an applicatin in ~n v~ culture. A cmmn l y-bserved event in tissue cultures ;s the prgressive darkening f explants and callus, due t secretin and xidatin f phenlic substances. The xidatin prducts are ften txic t the tissue and severe inhibitin r necrsis usually results. The phenlic metablites als appear t inactive auxins with bvius cnsequences. Ascrbic acid (3) (vitamin C), especially in cmbinatin with citric acid, may ften alleviate this prblem (MURASHGE, 974). Varius ther natural and synthetic anti-xidants have been used fr the same purpse. Activated charcal has als been utilised, but this material is nt specific in its adsrptin prperties and may remve essential grwth factrs frm the medium in additin

82 68 t the txic metablites. There is n panacea t the phenlic prblem which cntinues t plague many wrkers, especially thse dealing with wdy explant material. Supplementary rganic cmpunds Many f the earlier media cntained rganic supplements in the frm f materials such as ccnut milk, yeast extract, casein hydrlysate, tmat juice, range juice, banana puree and ther mre extic mixtures. Useful as they may have been, these supplements result in undefined media with cnsequent difficulties in exact reprducibility. Largely as the result f islatin and identificatin f the active cnstituents in such mixtures, current media are mre fully defined; the previusly-used extracts, juices and suspensins have been replaced by specific amin acid mixtures and ther rganic cmpunds. Amin acids and ami des have been shwn t be beneficial in many cases. Perhaps the mst frequently-used amin acids and amides are arginine (4). asparagine (5), glutamine (6) and glycine (7) (MURASHGE, 973). The first tw f these are reprted t be stimulatry in gymnsperms like P-ic.ea ab-i~ (STENHART, STANDFER and SKOOG, 96). Other amin acids smetimes used are alanine (8), tyrsine (9), cysteine (0), tryptphane () and prline (). Only the L-ismers f the pticallyactive cmpunds are effective. The D-frm is usually withut effect and smetimes negates the value f the L-ismer when racemic mixtures are used (MURASHGE, 974). Amin acid supplementatin must be dne with cautin as these cmpunds can be quantitatively and qualitatively inhibitry t sme tissues. The purine, adenine (3), is very cmmnly added t media, usual l y in the frm f its sulphate. Adenine, vital in nucletide bisynthesis, appears t be mre necessary in mrphgenesis stages than in callus initiatin r prl"iferatin (MURASHGE, 974). Despite the vluminus literature n vitamins and ther rganic supplements, their use remains disputed. LETHAM (967) claimed that if the apprpriate cytkinin was used, the nly rganic cmpunds necessary wuld be sucrse, insitl and an auxin, with thiamine as a 'desirable extra'.

83 69 Grwth regulating substances The selectin f the grwth regulating auxins and cytkinins is ne f the mst critical aspects f any plant tissue culture prject. Different phythrmnes bring abut different respnses, cncentratins must be within specific limits and synergistic and antagnistic interactins add t the cmplexity. Where large explants are used, r where an active meristem is included in the explant, there may be sufficient endgenus phythrmnes fr sustained grwth. When smaller explants are used, r when specific mrphgenetic respnses are sught, then an exgenus supply becmes essential. The classical experimentatin f SKOOG and MLLER (957) demnstrated clearly that an auxincytkinin interactin critically cntrls mrphgenesis. n general, relatively high cncentratins f cytkinin favur sht initiatin and repress rting while high auxin levels stimulate rting and inhibit shting. n develpment f the MS medium, MURASHGE and SKOOG (96) used auxin and cytkinin at,0 and 0, mg - fr callus cultures and increased these t 4,0 and,5 fr sht differentiatin. Rting was induced by transfer t a medium withut phythrmnes. Manipulatin f the auxin-cytkinin rati t achieve the desired grwth respnse is nw standard technique in.tit vj.tjt. wrk. t is als nted that well~established callus cultures smetimes underg a spntaneus change manifested by a lack f dependence n further exgenus hrmne supply, such cultures being described as habituated. Amng the auxins used in tissue cultures are the naturally-ccurring cmpund indle-3-acetic acid (4) (laa) and a large number f related synthetic substances such as -napthaleneacetic acid (5) (NAA),,4-dichlrphenxyacetic acid (6) (,4-0), indle-3-butyric acid (7) (BA),,4,5,-trichlrphenxyacetic acid (8) (,4.5-T). 4-chlrphenxyacetic acid (9) (CPA), 4-amin 3,5,6-trichlrpiclinic acid (30) ('Piclram') and 3,6-dichlr--anisic acid (3) ('Oicamba'). Of these the first three are mst cmmnly used,,4-0 and NAA being preferred t AA n accunt f better stability. The cytkinins are all derivatives f adenine (aminpurine) and include 6-furfurylaminpurine (3) (kinetin, KN), 6-benzylaminpuri ne (33) (BA), 6-~-~-dimethylaminpurine (34) (ispententyladenine, ip) and the naturally-ccurring 6-(4-hydrxy- 3-methyl-t4an--butenylamin)purine (35) (zeatin, Z). Other plant

84 70 regulating substances are ethylene, abscisic acid and the gibberellins which are less cmmnly used fr ~~ v~ wrk. Of the latter grup, the mst imprtant cmpund used ;s gibberellic acid (GA 3 ) On the basis f the infrmatin summarized in the preceeding paragraphs, it was felt that the media f SCHENK and HLDEBRANT (97) (SH) and medium 59 f NORSTOG and RHAMSTNE (967) prvided a useful starting pint. nitial experimentatin shwed these media t be generally satisfactry fr bth haplid and diplid cycad material and hence an elabrate multi-factrial experiment in ptimising the level f each cmpnent was nt cnsidered necessary. Preparatin f media All media in this prject were prepared freshly in ne-litre batches. The ph was adjusted t 5,8 by additin f 0, M KOH. Bacterilgical grade agar (BiLab) was used at 7,5 g - as the slidifying agent. Media were dispensed int the apprpriate cntainers and sterilized by autclav;ng at 03,4 k Pa ( C) fr 0 minutes. The ph f the medium ;s imprtant insfar as slubility and agar-gel ;~g prperties are cncerned, quite apart frm its effect n tissue grwth and develpment. At a ph f 4,0 the agar fails t gel while at a ph f 7,0 incipient precipitatin f the least sluble salts (calcium sulphate, Ksp,45 x 0-5, and magnesium ammnium phsphate, Ksp,5 x 0-~ bth at 5 C) cmmences. The effect f autclaving and strage n phadjusted SH media ;s shwn in Table 7. TABLE 7 : The effect f autclaving and shrt strage perids n the ph value f ph-adjusted SH media. nitial ph value - ph value immediatedly.after 4,0 4,6 5,0 ~, 6,0 5,8 7,0 6,7 ph value ne week autclaving after autclaving, 4,6 5,0 5,8 6,6

85 7 t is evident that at ph values less than 5, the ph drift is upwards while at ph values greater than 5, the drift is dwnwards. Further strage des nt greatly affect ph values. Frm the abve results it appears reasnable t cntinue the practice f adjusting ph t Chice f culture cntainers A wide variety f differently-shaped glass and plastic cntainers is available fr use in plant tissue culture and insufficient attentin is ften paid t the selectin f cntainer and clsure. Minr influences n in v~ respnses culd arise frm the chemical nature f the cntainer, usually brsi Licate glass r plycarbnate plastic, and frm the vlume and surface area f the medium in relatin t headspace vlumes. The majr effect appears t be assciated with the relative rates f gaseus exchange between the internal headspace and the external atmsphere. n cntainers with restricted gas exchange it may be anticipated that 00% relative humidity is maintained, that xygen depletin may ccur and that carbn dixide ethylene and ther vlatile substances may accumulate. These circumstances may lead t grwth inhibitin; in general an hermetically sealed cntainer is mre detrimentao t culture grwth than a cntainer allwing gaseus exchange (DE PROFT, MAENE and EBERGH, 985). n rder t test the effect f the cntainer n the grwth f haplid and diplid cycad tissue in vita, a series f 69 explants f megagametphyti c ti ssue and 48 cubes f primary rt ti ssue frm lc.e.phaxa.u0.6 natale.~~ were grwn in SH medium supplemented with 4,5 x 0-6 M,4-0 and kinetin in three different types f cntainers. The respnse, in terms f callgenes;s within 50 days, ;s shwn in Table 8. nspectin f these results shws that the frequency f callus frmatin is clearly related t the type f cntainer. The best respnse is shwn by cultures in Erlenmeyer flasks with cttn-wl clsures, hence this type f cntainer was used wherever pssible in subsequent experimentatin.

86 7 TABLE 8 : The effect f cntainer n callus frmatin frm megagametphyte and primary rt explants f EncephaZal ts natazensis. (A) Megagametphyte explants Cui ture vessel Clsure Number f explants % shwing callus within 50 days Universal bttles, 8mls. Rubber seal and screw cap 3 0% Tissue culture tubes, "Magenta" ribbed 40mls. plastic clsures; 3 6% "Parafi m" seal Erlenmeyer flasks, Cttn wl bung, 5mls. fi cap and masking 3 70% tape seal (B) Primary rt explants, i Culture vessel Clsure Number f ~ shwing explilnts cililus wi thin 50 dlys Tissue culture tubes, "Magenta" ribbed 40ml s. plastic clsures; 4 58% "Parafi m" seal Erlenmeyer flasks, Cttn wl bung, 5mls. fi cap and masking 4 7 % tape seal.4.6 Chice f culture cnditins Cultures n media cntaining sucrse shuld bypass the need fr light; many species are rutinely cultured in the dark. Hwever, light des playa rle in inducing mrphgenesis. MURASHGE (974) reviewed light requirements in terms f phtperidic, intensity and spectral qality parameters. He makes a general recmmendatin f cntinuus 00 ~E m- s- flurescent light fr establishment and maintenance stages --- fllwed by an increase in intensity t 300 t 000 ~E m- s- fr mrphgenesis. t is,nted in this r.espect that the daylight quantum flux measurement at slar nn is typically abut 000 ~E m- s- (Lambda nstrument Crpratin, Brchure B-777).

87 73 Tissue culture experiments are usually kept at cnstant cntrlled temperatures in the 0-8 C range but, where cntrlled temperature facilities are nt available, ambient temperatures ften suffice. A diurnal temperature cycle may be used, especially in assciatin with cntrlled phtperids in grwth cabinets. An incidental advantage f a cntrlled fluctuating temperature cycle is the effect in prmting a mre rapid gaseus exchange. n this prject, unless therwise stated, cultures were initiated in the dark at 5! C and then transferred aft.r -3 weeks t a cntrlled envirnment cabinet. The latter was held at 5! C under either cntinuus light r under a 6/8 hur phtperidic cycle. The light was prvided by a cmbined bank f flurescent tubes and incandescent bulbs at mean intensities frm 30 t 00 ue m- s-.4.7 Cytlgical examinatin Since explants frm well-defined haplid and diplid cycad tissues were used in this prject, it was cnsidered adviseable t mnitr the chrmsmal status f cultures. Small segments f tissue were remved frm the bulk f the material and fixed vernight in Carny s fluid (ethanl: chlrfrm: acetic acid :: 6:3:). After rinsing in distilled water, the samples were sftened in M hydrchlric acid fr.5 hurs at rm temperature and rinsed well with water. Small sub-samples were squashed in % acet-rcein in glacial acetic acid fr 30 minutes n a glass slide and the preparatin carefully flattened under a cver slip. After visual micrscpic examinatin, representative grups f metaphase chrmsmes were cunted and phtgraphed using a Zeiss cmpund phtmicrscpe with Kdak Pan 45 technical film which was subsequently prcessed with 063 develper (:3) fr 5 minutes at 0 C..5 Experiments with haplid material.5. Megagametphyte cultures Segments frm megagametphytes frm Sta~g~a ~p~ and 4 species f Enc.e.y.>halMt-6 were subjected t a variety f pre-treatments and cultured

88 74 n slid media with different grwth factr supplements under different envirnmentall cnditins. The results btained are summarized in Tables 9, 0 and. Wi th, Sta.ngeJLia vu.pu..6 (Tab e 9), vi grus grwth f a sft, wh i te friable callus was readily btained in 5-30 days (Plate 3). N significant difference was fund in the perfrmance f lngitudinally r transversely bisected megagametphytes r 5mm cubes f the internal tissue. The selectin f the cntainer had a majr effect n callgenesis, the explants in Universal bttles giving a cnsistently prer respnse than thse in tissue culture tubes r Erlenmeyer flasks (Sectin,.4.5 and Table 8). The presence, absence r intensity f light did nt affect callus frmatin, but the riginal explants in light cnditins became green and presumably phtsynthetic. Cytlgical examinatin cnfirmed a cnsistent n = 8 haplid chrmsme number (Plates 4a & b) in the callus material. Culture f the explants n a medium cntaining 0,0% clchicine smewhat inhibited the rate' f grwth and resulted in the ' appearance f cells with diplid, tetraplid and ctaplid chrmsme cmplements. The material culd nt tlerate the higher cncentratin f 0,05% clchicine and became necrtic. The megagametphyte-derived callus frm StangvU.a vu.pu..6 culd be maintained in subculture ver several passages at 3-4 week intervals. Results f several experimenta in this regard are summarized in Table 0. n general, callus grwth cntinued and was unaffected by the strength r nature f the medium emplyed and the presence, absence r nature f grwth factr supplements. Hwever, in six explants the presence f a number f small, cmpact "spherical structures" was seen (Plate 5a). These structures attained abut 5mm in diameter. A transverse sectin revealed a central p~renchymatus zne within a cambial layer surrunded by peripheral crk cells (Plate 5b). There was n evidence f any vascular develpment. These structures appear t be analgus t the crallid rt ndu~es reprted t have arisen in several ther cycad megagametphyte culture experiments (DE LUCA & SABATO, 980: DE LUCA~ SABATO, BALDUZZ & NAZZARO, 980) and als in cycad seedling and embry cultures, particularly at lw t medium light intensities (WEBB, 98, 98b, 983, 984: WEBB & DE JESUS, 98: WEBB, NEVAREZ & DE JESUS, 984), (Sectin.3). N further differentiatin was bserved, either n the

89 TABLE 9 : n vitr culture f megagametphyte explants frm Stange ria eripus Explant Number f explants Cu ture medium Cntainer Li gh t reg i me Observatins 5nrn cubes 3 SH, 4,5xl0-6 M,4-0 and KN 50ml Erlenmeyers Dark Vigrus callus grwth in 5-30 days Transverse '-megas. 6 SH, 4,5xl0-6 M,4-0 and KN Ti ssue cu tu re Dark Vigrus callus grwth in 5- tubes 30 days Trans verse, -megas. 6 SH, 4,5xl0-6 M,4-0 and KN T ssue -cu ture 00llE m- s -, Greening f explant, vigrus tubes cnstant callus grwth Transverse,-megas. SH, 4,5xl0-6 M,4-0 and KN 5ml Erlenmeyers Dark Vigrus callus in 5 days = n = 8 Transverse,-megas. as abve + 0,0% clchicine 5ml Erlenmeyers Dark Less vigrus callus, n = 8, 6, 3, 64. Transverse '-megas. as abve + 0,05% clchicine 5ml Erlenmeyers Dark All cultures necrtic Lng i tud i na '-megas. SH, 4,5+0-6 M,4-0 and KN 5ml Erlenmeyers Dark Vigrus callus grwth in 5-30 days i Lng i tud i na '-megas. 5 59, n grwth factrs Universal bttles Dark Limited callus grwth in 30 days Lngitudinal '-megas. 5 59,,4xl0-6,4-D; Universal bttles Dark Mderate callus grwth in 30 :,4xO- s KN days : Lngitudinal '-megas. 5 59,,4xl0-6 AA; Universal bttles Dark Mderate callus grwth in 30,4xl0-5 ip days! Lngitudinal,-megas. 5 59, n grwth factrs Universal bttles 30lEm- s-, Greening f explant, mderate cnstant callus grwth lngitudinal,-megas. 5 59,,4xl0-6,4-0; Universal bttles 30 Em- s-, ' Greening f explant, mderate,4xl0- s KN cnstant callus grwth Ntes: SH, medium f SCHENK and HLDEBRANDT (97); 59, medium f NORSTOG and RHAMSTNE (967) ;,4-0,,4-dichlrphenxyacetic acid; KN, kinet in; M, indle-3-acetic acid; ip, ispentenyladenine; n = chrmsme number bserved in cytlgical examinatin; megas., megagametphytes ~ <.T

90 76 PLATE 3 Prl ific crumbly white callus gr.wth n an explant f megagametphytic tissue frm Stange ria eripus seed after 4 days n SH medium supplemented with 4,5 x. 0~6M.,4-D and kinetin and grwn in the dark at ambient temperatures (-5 C). The explant was taken frm the micrpylar end f the megagametphyte as evidenced by the dark area f the micrpylar rifice. Sca le x.

91 77 PLATE 4a & b Phtmicrgraphs f chrmsmes frm callus tissue cultured frm Stange ria eripus megagametphytic tissue. Squash preparatin with acet-rcein stain. The haplid (n = 8) chrmsme number is evident in bth early (upper picture) and late (lwer picture) metaphase stages. Scale x 400.

92 TABLE 0 : Subculture f megagametphyte-derived callus frm Stanger i a eripus Number f explants , Culture medium Cntainer Li gh t reg i me Observat ins SH, n grwth factrs 5ml Erlenmeyers 50JEm-s-, hr pht- Cntinued callus grwth; perid "spher i ca structures" n ne explant in 60 days!-strength SH, n grwth factrs 5ml Erlenmeyers 50lJEm-s -, hr pht- Cntinued callus grwth perid SH, 5-hur pre-treatment f 5ml Erlenmeyers 50JEm - s -, hr pht- Cntinued callus grwth; explant with 4,5xl0-~H BA J perid "spher i ca structures" n ne explant in 60 days SH, 5-hur pre-treatment f 5m Erlenmeye rs 50lJEm-s-, hr pht- Cntinued callus grwth; explant with 3xl0-~H GA3 perid "spherical structures" n tw explants in 60 days SH, 4,5xl0-6 M BA 5ml Erlenmeyers 50lJEm-s-, hr pht- Cntinued callus grwth; per id "spherical structures" n tw explants in 60 days SH, 4,5xl0- s M BA 5ml Erlenmeyers 50lJEm- s -, hr pht- Cntinued callus grwth perid SH, 3xl0-6 M GA3 5ml Erlenmeyers 50lJEm-s-, hr pht- Cntinued callus grwth per ld SH, 3xl0- s M GAl 5ml Erlenmeyers 50lJEm-s-, hr pht- Cntinued callus grwth, per id, 59, 0, % activated charcal 50ml Erlenmeyers 50lJEm- s-, hr pht- Cntinued callus grwth perid 59, N grwth factrs 50ml Erlenmeyers 30lJEm-s-, cnstant Cntinued callus grwth 59, lxl0-5 M ip 50ml Erlenmeyers Dark Cntinued callus grwth Ntes: SH, medium f SCHENK and HLDEBRANDT (97); 59, medium f NORSTOG and RHAMSTNE (96]) ; BA, 6-benzyladenine; GA 3, gibberellic acid; ip, ispentenyladenine... CO

93 79 PLATE Sa Megagametphytic tissue f Stange ria eripus shwing the develpment f spherical structures analgus t crallid rt ndules. Scale x 3.,PLATE 5 b Transverse sectin thrugh the utermst tissues frm the abve structure. A cambial zne (CA) separates the uter crk layer (CO) frm the internal parenchyma (p). Tluidine blue stain. Scale x 00.

94 80 spherical structures r the remaining tissues. Results with cultures frm E~cephai~~ megagametphytes are recrded in Table. N significant difference was nticed with respect t the tw different basal media emplyed, but the grwth factr supplements did appear t influence callus prductin. Cmbinatins f 0-6 and 0-7 M,4-0 and kinetin, and NAA and 6-benzyladenine, generally resulted in vigrus callus grwth in days. ndleacetic acid and ispentenyladenine supplements did nrmally nt prmte callgenesis. n the varius pre-treatment tests, the slvent dimethylsulphxide appears t be a useful vehicle t facilitate hrmne absrptin by the explant (BLAKELY, BLAKELY and GALLOWAY, 986); the slvent itself having n deleterius affect. n these pre-treatment experiments the tw auxins,4-0 and NAA prmted callgenesis, while the auxin AA and the three cytkinins tested did nt result in callus grwth. Tests t evaluate the perfrmance with different levels f sucrse shwed that the levels f - 5% were satisfactry, but that an increase t 0% inhibited callus frmatin. As fund with the Sta~g~a explants, the use f universal bttles lead t unsatisfactry results. n the evaluatin f light influences, it appears that the presence, absence r intensity f light des nt affect cal jus prductin, but des cause the riginal explant t becme phtsynthetic. The additin f clchicine seems t inhibit the rate f callus grwth, but unlike the situatin in Sta~g~a, did nt result in any multiplicities f chrmsme number. There was n evidence f any type f differentiatin r mrphgenesis in the E~cephai~~ megagametphyte cultures..5. Micrgametphyte cultures An initial attempt at establishing ~~ v~ cultures f cycad micrspres was carried ut using pllen cllected, at the time f dehiscence, frm the male cnes f E~cephai~~ C~et. After disinfectin by sequential treatment with alchl, hypchlrite and sterile distilled water, the. pllen, in suspensin in sterile distilled water, was innculated nt slid SH media supplemented with varius cmbinatins f,4-0, NAA; kinetin and 6-benzyladenine. Cultures were incubated at 5 C in bth light and dark cnditins. N evidence f any develpment was seen

95 Species TABLE : n vi tp culture f megagametphyte explants frm,varius Encephalapts spp. Explant N. f explants Cu tu re med i um Cntainer Light regime Observat ins E. natalensis!-megas. 3 SH, ~,5x0-6H,~-0 & KN Universal bttles Dark N respnse in 50 days E. natalensis i -megas. 3 SH, ~,5x0-6H,~-0 & KN Tissue culture Dark 6% f explants with tubes callus in 50 days E. nat alensi s i -megas. 3 SH, ~,5x0-6H,~-0 & KN 5ml Erlenmeyers Dark 70% f explants with, callus in 50 days E. natalensis Transverse!- 6 59, ~-hr pretreatment Tissue culture OO)JEm - S - Greening within ~O megas in 5% OHSO tubes cnstant days E. na t alensis Transverse!- 6 59, 4-hr pretre~tment Tissue cui t ure 00)JEm-s - Greening within 40 megas i n 5% OHSO with 3x0-~H tubes cnstant days AA E. natalensis Transverse!- 6 59, 4-hr pret reatment Tissue culture 00)JEm- s- Greening, w it~ callus megas in 5% OMSO with x0-~m tubes cnstant n all explants with i n, days E. natalensis Transverse!- 6 59, 4-hr pretrea~ment Tissue culture 00)JEm- s- Greening, with callus megas. in 5% OMSO with 3x0-~M tubes cnstant n all explants within NAA 40 days E. natalensis Transverse!- 6 59, 4-hr pretreatment Tissue culture 00)JEm- s- Greening within 40 megas. in 5% OMSO with x0-~ KN tubes cnstant days E. natalensi s Transverse! , 4-hr pretreatment Tissue culture 00)JEm- s- Greening within 40 megas. in 5% OMSO with x0-~ BA tubes cnstant days E. natalensis Transverse!- 6 59, ~-hr pretreatment Tissue culture 00)JEm- s- Greening within 40 megas. in 5% OMSO with x0-~ tubes cnstant days ip, E. nat alensis Transverse!- 6 59, 4,5x0-6 M,~-0 & KN Tissue culture 00)JEm- s - Greening, with callus megas. tubes cnstant n all explants within 40 days c

96 TABLE - cntinued Species Explant N. f explants Cu ltu re med i um Cntainer Light regime Observatins E. natazensis Trans verse!- 6 megas. 59, ~,5xl0-6H,~-0 & KN Tissue culture sucrse level raised t 5% tubes 00JEm- s- cnstant Greening, with callus n all explants within ~O days E. natazensis Transverse!- 6 megas. 59, ~,5xl0- H,~-0 & KN Tissue culture sucrse level raised t tubes 0% 00JEm- s- cnstant Greening within ~O days E. natazensis Transverse!- 6 megas. 59, ~,5xl0-6H AA & ip Ti ssue cu ture tubes 00JEm-s- cnstant, Greening within ~O days E. natazensis Transverse!- megas. 6 natazensis Transverse E.!- megas. 6 59, ~,5xl0-6H AA and ip Tissue cui ture sucrse level raised t 5% tubes 59, ~,5xl0-6H AA & ip Tissue culture sucrse level raised t tubes 0% 00JEm- s- cnstant 00JEm-s- cnstant Greening, callus n explants within ~O days Greening within ~O days E. natazensis Transverse ;- 6 megas. 59, ~,5xl0-6H,~-0 and Tissue culture ~,5xl0-'H KN tubes 00JEm- s- cnstant Greening, callus n 3 explants within ~O days E. natazensis Transverse!- 6 megas. 59, ~,5xl0-'M,40 and Tissue culture ~,5xl0-6H KN tubes 00JEm- s- cnstant Greening, callus n all explants within 40 days E. natazensis Transverse!- 6 megas. 59, ~,5xl0-tH NAA & BA Tissue culture tubes 00JEm-s- cnstant Greening, callus n all explants within 40 days E. natazensis Transverse ;- 6 megas. 59, 4,5xl0-6 M NAA and Tissue culture ~,5xl0-'H BA tubes OOJEm-s - cnstant Greening, callus n all explants within 40 days E. natazensis Transverse!- 6 megas. 59, 4,5xl0-'M NAA and Tissue culture 4,5xl0- E H BA tubes 00JEm- s- cnstant Greening, callus n 3 explants within 40 days E. nataze7sis Transverse!- 6 megas. 59, 4,5xl0-6 M AA and Tissue culture ~,5xl0-'M ip tubes 00JEm- s- cnstant Greening within ~O days E. nata Zen sis Transverse!- 6 megas. 59, 4,5xl0-'M AA and Tissue culture 4,5xl0-6 H ip tubes 00JEm- s- cnstant Greening within 40 days c N

97 Species TABLE - cntinued Explant N. f explants Culture medium Cntainer Light regime Observat ins E. villsus Lngitudinal , varius supplements Universal Light and dark All explants becme megas. bttles cnditins necrtic E. Zebmb-! -megas. 90 SH, varius pretreat- Universal Dark Spradic callus frmatin ensib ments and varius btt les in sme cultures in 50 supplements days E. ptel'gnus Transverse , 4,5xl0-6 M,4-0 Tissue culture 00\lEm- S- Vigrus callus in 40 megas. and KN tubes cnstant days n = 9 E. ptel'gnus Transverse - 0 as abve + 0,0% Ti ssue culture 00\lEm- s- Mderate callus n 40 megas. clchi cine tubes cnstant days n '" 9, E. ptel'gnus Transverse - 0 as abve + 0,0% Tissue culture 00\lEm- s- Slight callus in 40 days megas. clchicine tubes cnstant n = 9 Ntes: SH, medium f SCHENK and HLDEBRANDT (97); 59, medium f NORSTOG and RHAMSTNE (967);,4-0,,4-dichlrphenxyacetic acid; KN, kinetin; DMSO, dimethylsulphxide; AA, indle-3-acetic acid; NAA, l-naphthaleneacetic acid; BA, 6-benzyladenine; ip, ispentenyladenine., (» w

98 84 ver a 3-mnth perid; intact, ungerminated, micrspres. micrscpic examinatin shwed the presence f Althugh TULECKE (957) managed t induce prliferatin and callus develpment in pllen cultures frm G~nkg bilba L., mst wrkers have had difficulties using pllen grains fr ~n v~ cultures. t may be that pllen material is susceptible t necrsis during the disinfestatin treatment (LA RUE, 954). Mst current wrk n haplid male tissue culture frm angispermus material has favured the use f whle anthers fr the inductin f andrgenesis (RENERT and BAJAJ, 977). By analgy, in cycads, results. have been reprted frm micrsprangia rather than micrspres. (DE LUCA and SABATO, 979; DE LUCA, LA VALVA and SABATO, 980). Althugh in their wrk the fcus f attentin has been n develpment f pllen tubes, nuclear divisins and spermatge~is~ callus initiatin was bserved in at least tw cases frm C~atzamia mexiqana when the medium was supplemented with,4-0. Thus attempts were made t establish micrspre cultures using unpened micrsprangia rather than the pllen cllected after dehiscence. n the case f EnQephalant~ ~~x and E. wdii, the unpened micrsprangia were cllected frm male cnes at a perid apprximately ne week prir t dehiscence. The micrsprangia were disinfected by treatment with alchl and hypchlrite slutins and innculated nt varius media as indicated in Table. Micrscpic examinatin f the material shwed that, at the time f cllectin and innculatin, mst f the spres were in a mnnucleate cnditin (Plate 6a); a small number were in the mre advanced tw-celled stage with the small prthallial cell separated frm the larger antheridial cell. All cultures respnded by shwing germinatin t give a mass f interwven pllen tubes within -3 weeks, the culture having an almst callus-like appearance n superficial inspectin. Micrscpic examinatin shwed the pllen tubes t be variusly extended in length and width, but generally unbranched, with granular cytplasmic cntents (Plate 6b). A large number f the micrspres had nt germinated but had develped t the three-celled stage. Subculture f the abve pllen-tube masses allwed cntinued extensin. N evidence f spermatgenesis r any ther frm f mrphgenesis was bserved.

99 Species Number f cultures TABLE : n vi t )' cu ture f cycad micrgametphytes Culture medium Cntainer light regime Observat ins E. f er x 4 SH, n _grwth factrs Universal bttles Dark E. f er x 4 SH, 4,5xl0-6 H,4-0 and KN Universal bttles Dark E. f er x 4 SH, 4,5xl0- s H NAA Universal bttles Dark E. f er:r 4 i-strength SH Universal bttles Dark Germinatin f micrspres and develpment f mass f pllen tubes in -3 weeks E. L.'dii 9 SH, 4,5xl0-6 H,4-0 and KN Unive r sal bttles Dark subcul tures 9 59,0,% activated Universal bttles 00lJEm- s- Cntinued grwth f pllen fr_m E. w)dii charcal cnstant tubes Ntes : SH, medium f SCHENK and HLDEBRANDT (97); 59, medium f NORSTOG and RHAMSTNE (967);,4-0,,4-dichlrphenxyacetic acid; NAA, l-naphthaleneacetic acid; KN, kine tin. (X) U"

100 PLATE 6a Micrspres f E. wdii at the time f culture -initiatin. The mnnucleate cnditin and peripheral cytplasm are clearly evident. Scale x 350., ".. '. ;. -- " - r,. -, ~ " e.... e ' 0 e ~,. '. ~,~ -, J> t 0 ~ PLATE 6b P en tubes f E. wdii 4,5x 0-6 M Scale x 33. arising frm germinating micrspres after 35 days n SH medium with,4-0 and kinetin. Acet-rcein stain.

101 87.6 Experiments with diplid material.6. Embry cultures As is the case in ther recalcitrant r "wet" seeds, the embry in cycad seeds shws mre-r-less uninterrupted develpment frm the time f fertilisatin until germinatin (Sectin.6). Thus seed cllected at the time f shedding frm the parent cnes cntains immature embrys while seed dissected just prir t germinatin will reveal fully-devel~ embrys. Embrys at different stages were aseptically remved frm surface-sterilised seeds f three species f Encephaiant~ and Stang~a ~pu, subjected in sme cases t hrmne pre-treatments, and cultured n a variety f media in different cntainers under different cnditins. The results are summarised in Table 3. n general, immature embrys cntinued essentially nrmal develpment fr a perid f abut 6 weeks (Plate 7a) but thereafter became necrtic. On the ther hand, mature embrys gave a variety f respnses, dependent n the pre-treatment, medium, cntainer and envirnmental cnditins. Withut pre-treatment and with n grwth factrs, the embrys again cntinued essentially nrmal grwth (Plate 7b), althugh in sme cases the ctyledns became highly ciled (Plate 7c). Prlific crumbly ff-white callus arse frm mature embrys f E. nataie~~ and E. vm~u after - 3 weeks n bth the SH and the "59 medi a when lw auxin and cytkinin supplements were present (Plate 7d). n cmmn with the situatin in many ther gymnsperms (Sectin.), the callus develped alng the ctylednary surfaces. Cytlgical examinatin f 5 such cultures frm E. vm~u cnfirmed its cnsistent diplid (n = 8) status. The cnditins which prduced the highest frequency f callgenesis in these embry cultures were similar in many respects t thse which gave the best results with the megagametphyte cultures (Sectin.5.). There was n significant difference in perfrmance f the explants n the tw different basal media. Cytkinin pre-treatment ffered n particular advantage but an auxin/cytkinin supplement was essential fr callus develpment. Cncentratins in the range 0-6 t 0-5 M were sati~factry in this respect but an increase in the 6-benzyladenine level abve 0-5 M resulted in necrsis. Sugar cncentratins in the - 5%

102 TABLE 3 : vitl'o cu tu re f cycad embrys, Species Embry cnditin N. f explants Cu tu re med i um Cntainer Li gh t reg i me Observatins E. natazensis intermediate and 4 SH, 4-hr pre-treatment mature in,5xl0-~m BA Universal btt les Dark Callus alng "suspensr in 3 explants after mnths E. natazensis intermediate and 4 SH, n pre-treatment mature Universal btt les Dark Highly ciled ctyledns and callus at suspensr in explants after mnths E. natazensis intermediate and 4 SH, 4-hr pre-treatment mature i n, 5x 0 -~ M BA Universal bttles 50)JEm-s - / hr phtpe r i d All cultures necrtic E. natazensis intermediate and 4 SH, n pre-treatment mature Universal btt les 50)JEm- s- / hr phtperid Cntinuus nrmal grwth with greening f ctyledns in explant,, E. natazensis mature 4 SH, 4,5xl0-6 M.,4-0 and KN E. natazensis mature 4 SH, 4,5xl0-6 M,4-0 and KN 50ml Er enmeyers Dark 50ml Erlenmeyers 00)JEm- s- cnstant Vigrus callus n ctyledns in 3 explants after 0 days Vigrus callus n ctyledns in explants after 0 days! E. natazensis mature 4 59, 4,5xl0-6 M,4-0 and KN, E. natazensis mature 4 59, 4,5xl0-6 M,4-0 and KN 50ml Erlenmeyers 50ml Erlenmeyers Oark 00JEm-s- cnstant Vigrus callus n ctyledns in all explants after 0 days All cultures necrtic E. natazensis mature 8 SH, 4,5xl0-6 M,4-0 and BA 5ml Er enmeye rs 00)JEm- s- 6/8 hr ph tpe r i d Callus n ctyledns f 5 explants after 4 weeks E. natazensis mature 8 SH, 4,5xl0-6 M,4-0; 4,5xl0- s M BA 5ml Erlenmeyers 00JEm- s- 6/8 hr phtpe r i d Cntinued grwth f all explants c CO

103 TABLE 3 : cntinued - ' Species Embry cnditin N. f explants Cu ture medium Cntainer Li gh t reg i me Observatins E. natazensis mature 8 SH, 4,5xlO- 6 M,4-0; 5ml Erlenmeyers OOJEm-s- All cultures necrtic,xl0-~m BA 6/8 hr phtperid E. natazensis mature 8 SH, 4,5xl0-6 M,4-0; 5ml Erlenmeyers 00JEm-s - All cultures necrtic 4,5xl0-~M BA 6/8 hr phtperid E. pauciden- immature 6 SH,!-hr pretreatment 5ml Erlenmeyers 50JEm- s- Cntinued develpment in all tatus in,5xl0-~m BA / hr explants fr 6 weeks; therephtperid after necrtic E. vilzsus mature 5 59,,4xl0-6 M,4-D; 5ml Erlenmeyers Dark Vigrus callus n ctyle-,4xl0- s M BA; % sucrse dns in 4 explants after 3 weeks E. vilzsus mature 5 59,,4xl0- E M,4-0; 5ml Erlenmeyers 00JEm-s- Vigrus callus n ctyle-,4xl0- s M BA; % sucrse cnstant dns in 3 explants after, 3 weeks E. vi ZZsus mature 5 59,,4xl0-6 M,4-0; 5ml Erlenmeyers Dark Vigrus callus n ctyle-,4xl0- s M BA; 5% sucrse dns in all explants after 3 weeks E. vi ZZsus mature 5 59,,4xl0-6 M,4-0; 5ml Erlenmeyers 0 0JEm- s- Vigrus callus n ctyle-,4xl0- s M BA; 5% sucrse cnstant dns in 4 explants after 3 weeks E. vi ZZsus mature 5 59,,4xl0- f M,4-0; 5ml Erlenmeyers Dark Slight callus grwth in 4,4xl0- s M BA; 0 % e'xplants after 3 weeks sucrse E. vi ZZsus mature 5 59,,4xl0-6 M,4-0; 5ml Erlenmeyers 00JEm- s- S ight callus grwth in,4xl0- s M BA; 0 % cnstant explant after 3 weeks sucrse s. el"ipus immature 6 SH, 4,5xl0-6 M,4-0 Universal bttles Dark Cntinued nrmal develpment and KN in all explants fr 6 weeks; thereafter necrtic Ntes : SH, medium f SCHENK and HLDEBRANDT (97); 59, medium f NORSTOG and RHAMSTNE (967) ;,4-0,,4-dichlrphenxyacetic acid; KN, kinetin; BA, 6-benzyladenine ex> U)

104 90 PLATE 7a Develpment f a juvenile embry f EncephaZarts paucidentatus n S H medium, with n grwth factrs, after six weeks in culture. The pr-embry (lwer left), the massive ciled suspensr and tw egg membranes are well defined. Acet-rcein stain. Scale X 6. PLATE 7b Develpment f a mature embry f EncephaZarts natazensis n SH medium, with n grwth factrs, after tw mnths in culture. Behaviur in vitr is analagus t that in viv.

105 9 PLATE 7c Develpment f an intermediate-stage embry f EncephaZarts natazensis n SH medium, with n grwth factrs, after tw mnths in culture. Prnunced curvature f the ctyledns is apparent. Scale X 8. PLATE 7d Vigrus callus grwth ccurring n the distal (ctylednary) end f a mature embry f EncephaZarts vizzsus after 40 days n medium '59' supplemented with,4 x 0-6 M,4-D and,4 x 0-sM BA and grwn in the dark at C. Scale X.

106 9 range were satisfactry, but 0% was inhibitry. The use f Erlenmeyer flasks gave better results than use f Universal bttles. The frequency f callgenesis was higher in the dark than in cnstant light r under a diurnal phtperid. Hwever, explants expsed t light cnditins became green and presumably phtsynthetic with pssible lng term advantages. N evidence was seen f any differentiatin in any f the cultures. T investigate the respnse t sub-culture, explants frm the mre vigrus callus frm E. natale~~ and E. v~~~ were transferred t fresh medium with varius grwth factr supplements, as shwn in Table 4. When,4-0 and kinetin in cncentratins frm 0-7 t 0-6 were used, callus prliferatin cntinued. The absence f grwth factrs inhibited this cntinued develpment but several f the E. v~~~ subcultures n media withut hrmnes gave rise t green areas f tissue with pssible meristematic activity. These later became necrtic. Cncentratins f x 0- s M cytkinin. gibberellic acid and vitamin 0 3 all resulted in necrsis f the cultures. TABLE 4 : Analys i s f respnses btained in subculture f embry-derived callus frm E. vizzsus and E. natazensis n medium '59' with different grwth factr supplements. Species Grwth factr supplements Number f Observatins cu ltu res E. vizzsus n grwth factrs 0 Sme greening, pssibly meristematic 4,4xl0- s M,4-0 and KN 5 Cntinued callus prliferatin 4,5xl0-'M,4-0; 4,5xl0- s M KN 5 Cntinued callus prliferatin 4,sx 0-6M,5-0, 4,5xlO-'M KN 5 Cntinued callus prl iferatin xlo- s M KN N respnse xl0- s M BA 3 N respnse xl0-~m ip N respnse x 0 -sm GAl N respnse xl0- s M Vitamin N respnse E. natazensis n grwth factrs 8 N respnse

107 93.6. Primary rt explant cultures n the first few mnths after germinatin f cycad seeds, a well-defined primary tap-rt is established (Figure 0). Depending n the species and the grwing cnditins, this attains smewhat carrt-like dimensins within ne t three years. n crss sectin, rts at this stage shw a brad, well-defined central vascular zne, usually with a tetrach arrangement f xylem elements, t the utside f which is an abundant parenchymatus crtex. n view f the abundance f this tissue, the ease with which it can be surface-sterilized, and the relative absence f endgenus micr-rganisms, it was lgical t explre this material as an explant surce. This extends the previus wrk f KOELEMAN and SMALL (98). Cubes f primary rt tissue (5mm-0mm sides) were aseptically dissected frm rts f cycad species, each cube cntaining sme vascular tissue. These explants were cultured n a variety f media with different hrmne supplements, smetimes after specific pre-treatments, in different culture vessels and expsed t different light regimes. Due t the scarcity f certain rt samples, several f the treatments culd be carried ut nly in small replicate numbers; interpretatin f the results must take this limitatin int accunt. The parameters tested and the respnses btained are presented in Table 5 (E. nataie~~l, Table 6 (ther Encephai~~ spp.), and Table 7 (ther cycad genera). Table 8 gives an verview f the rt explant experimentatin. A vigrus fast-grwing callus is quite readily btained in fairly high frequency (>50%) frm mst rt explants under mst cnditins within 0-30 days (Plate 8). There was little evidence f fungal r bacterial cntaminatin. The callus was generally initiated in the rt cambial zne but spread rapidly ver the entire cut surface f the explant. t was usually crisp, ff-white in clur and cmprised large vaculated cells with prnunced nuclei. The size f tbe explant in the cubes seemed t be imprtant. Mst cubes were in the range 5mm-0mm per side and these gave the generally successful callus grwth described abve. Hwever, smaller cubes, such as 3mm per side, did nt give any respnse, even after several mnths. These bservatins are cnsistent wit~ the cmment by MURASHGE (974) that the explant size is ften critical in callus initiatin. n evaluating the respnse t the psitin in the rt frm which the explant is

108 - TABLE S : n vitr culture f primary rt explants frm Ecephalarts natal ensis seedl ings, Origin f explant N. f explants Pre-treatment cnditins Culture medium Cntainer Light regime Observat ins, i Tp /3rd Tp /3rd 6 - SH, 4,5x0-6 M,4-0 and KN 5ml Erlenmeyers Dark 8 - SH, 4,5x0-6 M,4-0 and KN Ti s sue cu tu re Dark. tubes Vigrus callus n 5 explants in S days Vigrus callus n explants in days Mid /3rd 6 - SH, 4,5x0-6 M,4-0 and KN 5ml Erlenmeyers Dark Vigrus callus 'in all explants n days Mid /3rd 7 - SH, 4,5x0-6 M,4-0 and KN Tissue culture Dark tubes Vigrus callus in 3 explants in days, Bttm l3rd 6 - SH, 4,5x0-6 M,4-0 and KN 5ml Erlenmeyers Dark Mderate callus n 5 explants in 30 days, Bttm l3rd Tp 3rds Tp 3rds Tp 3rds Tp 3rds Tp 3rds Tp /3rds Tp 3rds 6 - SH, 4,5x0-6 M,4-0 and KN Tissue culture Dark tubes 7 - SH, 4,5x0-6 M, 4-0 and KN Universal bttles Dark 7 - SH, 4,5x0-6 M,4-0 and KN Universal bttles 5 Oil Em- s- /hr phtperid 4 - SH, 4.5x0- E M,4-0 and KN 5ml Erlenmeyers Dark 3 - SH, 4.5x0-6 M,4-0 and KN Tissue cu ture Dark tubes - SH, 4.5x0-6 M,4-0 and KN Tissue culture OOlEm - S- tubes cnstant ~ 3-59, 4,5x0-6 M.~-0 and KN Tissue cu ture Dark tubes 6-59, ~,5x0-6M,~-0 and KN Ti ssue cu tu re 30lEm- s- tubes cnstant._ Mderate callus in 6 explants in 30 days Mderate callus in 4 explants in 40 days Mderate callus n explant 40 days Vigrus callus in all explants in 3 days Vigrus callus in explant in 30 days Vigrus callus n explant in 30 days N respnse in 50 days Vigrus calls in all explants in 5 days.0 +=-

109 , TABLE 5 cntinued... Origin f explant N. f explants Tp /3rds - Tp /3rds 6 - Tp /3rds 6 - Tp /3rds 6 - Tp /3rds 6 - Tp /3rds 6 - Tp /3rds 6 - Tp /3rds 6 - Tp /3rds 6 - Tp /3rds 6 + 6,. Tp /3rds Pre-treatment cnditins Tp /3rds 6 xl0-'h.4-0 in 5% OHSO. 4 hrs. Tp /3rds 6 xl0-'h NAA in 5% OHSO. 4 hrs. ~ Culture medium Cntainer xl0-6 H.4-0 and KN Tissue culture tubes xl0-6 H.4-0 and Tissue culture 4.5xl0-'H KN tubes xl0-'H.4-0 and Tissue cu ture 4.5 x 0-6M KN tubes xl0-6 H NAA and Tissue culture BA tubes xlO- 6 H N~A and Tissue culture 4.5xlO-'H BA tubes xlO-'H NAA and Ti ssue cu tu re 4.5 x 0-6 BA tubes xlO- 6 M AA and Ti s sue cu tu re ip tubes xl0-6 H AA and Tissue cu tu re 4.5xlO-'M i P tubes xlO-'H AA and Tissue culture 4.5xl0-6 H ip tubes xlO- 6 M.4-0 Tissue cu ture and KN sucrse t 5% tubes and 0% xlO- 6 H AA and Tissue cu lure ip sucrse t 5% and tubes 0% 59. n grwth factrs Tissue cu ture tubes 59. n grwth factrs Tissue cu lture tubes Li gh t reg i me 00)JEm- s-} cnstant 30)JEm- s -} cnstant 30)JEm- s -} cnstant 30)JEm- s-} cnstant 30)JEm- s -} cnstant 30)JEm- s- cnstant 30)JEm- s- cnstant 30)JEm- S- cnstant 30JEm- s- cnstant 30)JEm-s - cnstant 30)JEm- s- cnstant 30)JEm- s -} cnstant 30)JEm- s- cnstant Observatins Vigrus callus in explant in 30 days N respnse in 50 days N respnse in 50 days Vigrus callus in all explants in 5 days N respnse in 50 days : N respnse in 50 days N respnse in 50 days N respnse in 50 days N respnse in 50 days N respnse in 50 days N respnse in 50 days N respnse in 50 days N respnse in 50 days ~ 0

110 TABLE 5 cntinued.. Origin f explant N. f explants Pre-treatment cnditins Tp /3rds 6 xl0-~h laa in 5% DHSO, 4 hrs. Tp /3rds 6 xl0-~h KN in 5% DMSO, 4 hrs. Tp /3rds 6 xl0-~m BA in 5% DMSO, 4 hrs. Tp /3rds 6 xl0-~h ip in 5% DMSO, 4 hrs. Tp /3rds 3 xl0-~h BA, 4 hrs. Tp /3rds xl0-~m BA, 4 hrs. Tp 3rds 3 xl0-~m NAA, 4 hrs. Tp /3rds xl0-~m NAA, 4 hrs. Tp /3rds 4 4xl0 - ~M NAA, 4 hrs. Tp /3rds 4 lxl0- l M NAA, 4 hrs. Tp /3rds 0 4xl0-~M NAA, 4 hrs. Tp /3rds xl0-~m NAA and BA, 4 hrs. Cu ture med i um Cntainer 59, n grwth factrs Tissue culture tubes 59, n grwth factrs Tissue culture tubes 59, n grwth factrs Tissue culture tubes 59, n grwth factrs Ti ssue cu tu re tubes SH, n grwth factrs SH, n grwth factrs SH, n grwth factrs SH, n grwth factrs SH, n grwth factrs SH, n grwth factrs MS, 4,5xl0-6 M BA SH, n grwth factrs 5ml Erlenmeyers 5ml Erlenmeyers 5ml Erlenmeyers 5ml Erl enmeyers Universal bttles Universal btt les Universal btt les 5ml Erlemeyers light regime 3D\lEm- S-l cnstant 30\lEm - s - cnstant 30\lEm - S - cnstant 30\lEm- s- cnstant Dark 30" Em- s- cnstant Dark 30"Em- s- cnstant Dark Dark Dark Dark Observat ins N respnse n 50 days N respnse in 50 days N respnse in 50 days N respnse in 50 days Callus n explant in 6 days Callus in explant in 30 days Callus in all explants in 5 days Callus in explant in 9 days Callus in 3 explants in days Callus in explants in; 8 days Callus in all explants in 5 days Callus n explant in days \.0 en

111 TABLE 5 cntinued.. Origin f explant N. f explants Pre-treatment cnditi,ns Culture medium Cntainer Li ght reg i me Observatins Tp 3rds 3 xl0-'m NAA and BA, SH, n grwth factrs 4 hrs. Tp /3rds - SH liquid medium; explants n filter paper bridge 5ml Erlenmeyers 30"Em- s- Callus in all explants cnstant n 5 days Universal Dark Callus in 6 cultures bttles in days Ntes : SH, medium f SCHENK and HLDEBRANDT (97) 59, medium f NORSTOG and RHAMSTNE (967) MS, medium f MURASHGE and SKOOG (96),4-0,,4-dichlrphenxyacetic acid, NAA, l-naphthaleneacetic acid laa, indle-3-acetic acid KN, kinetin BA, 6-benzyladenine, P, ispentenyladenine DMSO, dimethylsulphxide u:> -...J

112 TABLE 6 : n vitp culture f primary rt explants f ther Ell cep llq~apts species Species N. f explants Cui ture medium Cntainer light regime Observat ins!e. a~t e nst ei n i i 4 SH, 4,5xlO- 6 M,4-0 and KN 5ml Erlenmeyers Dark Callus in explants in 5 days E. caffe p 8 SH, 4,5xlO- 6 M,4-0 and KN 5ml Ed enmeyers Dark Callus in explants in days ; E. caffep 3 SH, 4,5xlO- 6 M,4-0 and KN Tissue culture tubes Dark Callus in 3 explants in 0 days E. caffep SH, 4,5xlO- 6 M,4-0 and KN Tissue culture tubes 00~Em-s-, cnstant C~llus in e'xplants in 5 days E. caffe p, : E. caffep 3 59, 4,5xlO- 6 M,4-0 and KN Tissue culture tubes Dark 59, 4,5xlO- 6 M,4-0 and KN Tissue culture tubes 00~Em- s-, cnstant Callus in 3 explants in 6 days Callus in explant in 5 days, E. cycadif ~iu8 4 SH, 4,5xlO- 6 H,4-0 and KN 5ml Erlenmeyers Dark Callus in explant in 50 days : E. cycadif ~ iu s 3 SH, 4,5xlO- 6 H,4-0 and KN Tissue culture tubes Dark Callus in 3 explants in 6 days : E. cycadi f lius SH, 4,5xlO- 6 H,4-0 a ~d KN Tissue culture tubes 00~Em- s-, cnstant Callus in explants in 8 days ' E. cycadiflius 3 59, 4,5xlO- 6 H,4-0 and KN Tissue culture tubes Dark N respnse in 50 days E. cy cadif~ius 59, 4,5xlO- 6 H,4-0 and KN Tissue culture tubes 00~Em- s-, cnstant N respnse in 50 days E. f e px 4 SH, 4,5xlO- 6 H,4-0 and KN 5ml Erlenmeyers Dark Callus in explant in days E. f e'x 3 SH, 4,5xlO- 6 H ~4-O and KN Tissue culture tubes Dark Callus in 3 explants in 6 days E. f e px SH, 4,5xlO- 6 H,4-0 and KN Tissue culture tubes OO~Em- s-, cnstant Callus in explants in 5 days E. f epx 3 59, 4,5xlO- 6 H,4-0 and KN Tissue culture tubes Dark Callus in 3 explants n 6 days E. f e px 59, 4,5x0-6 H,4-0 and KN Tissue culture tubes OO~Em- s-, cnstant Callus in explants in '5 days cnt i nued.... \.0 c:

113 TABLE 6 cntinued. Species N. f explants Cu ture medium Cntainer light regime Observatins E. f'idel'ici- 4 SH, 4,5xl0-6 M,4-0 and KN guilielmi 5ml Erlenmeyers Dark Callus in 3 explants in 37 days E. fl'idel'ici- 3 SH, 4,5xl0-6 M,4-0 and KN {Tuilielmi Tissue culture tubes Dark Callus in explant in 4 days E. fl'idel'ici- SH, 4,5xl0-6 M,4-0 and KN guilielmi Tissue culture tubes 00~Em- s-, cnstant N respnse in 50 days E. fl'idel'ici- 3 59, 4,5xl0-6 H,4-0 and KN guilie lmi Tissue culture tubes Dark N respnse in 50 days E. fl'idel'ici- 59, 4,5xl0-6 H,4-0 and KN guilielmi : Tissue culture tubes 00~Em-s-, cnstant N respnse in 50 days E. ghellinckii 4 SH, 4,5xl0-6 H,4-0 and KN 5ml Erlenmeyers Dark N respnse in 50 days E. hol'l'idlls 4 SH, 4,5xl0-6 H,4-0 and KN E. hol'l'idus 3 SH, 4,5xl0-6 H,4-0 and KN E. hol'l'idub SH, 4,5xtO~6M,4-0 and KN E. hol'l'idub 3 59, 4,5xtO- 6 H,4-0 and KN E. hol'l'idub 59, 4,5xl0-6 H,4-0 and KN E. lanatub 4 SH, 4,5xl0-6 M,4-0 and KN E. lanatus 3 SH, 4,5xtO- 6 H,4-0 and KN E. lallatub SH, 4,5xtO- 6 M,4-0 and KN E. lana tub 3 59, 4,5x0-6 H,4-0 and KN E. lanatus 59, 4,5xl0-6 H,4-0 and KN 5ml Erlenmeyers Dark Tissue culture tubes Dark Tissue culture tubes 00~Em- s-, cnstant Tissue culture tubes Dark Tissue culture tubes too~em- s -, cnstant 5ml Erlenmeyers Dark Tissue culture tubes Dark Tissue culture tubes 00~Em- s-, cnstant Tissue culture tubes Dark Tissue culture tubes too~em- s-, cnstant Callus in 3 explants in 3 days Callus in 3 explants in 4 days Callus in explants in 0 days N respnse in 50 days N respnse in 50 days Callus in 3 explants in 48 days Callus in t explant in 4 days N respnse in 50 days N respnse in 50 days Callus in explants in 0 days,, E. lebmbensis 5 SH, 4,5xl0-6 H,4-0 and KN 5ml Erlenmeyers Dark Call us in 5 explants in days E. lehmarinii 4 SH, 4,5xl0-6 H,4-0 and KN 5ml Erlenmeyers Oark Callus in explant in 4 days Cnt inued..... '-0 '-0

114 TABLE 6 cntinued... Species N. f explants Cu ture med i urn Cntainer Light regime Observat ins E. lngiflius 4 SH, 4,5xl0-6 M,4-0 and KN 5ml Erlenmeyers Oark Callus n 4 explants in 8 days E. l ngiflius MS, 4,5xl0-6 M BA; explants Universal bttles Dark Callus in all explants in 5 pre-treated with 4xl0-~M days NM fr 4 hurs E. lngiflius 3 SH, 4,5xl0-6 M,4-0 and KN Tissue culture tubes Dark Callus in 3 explants in 8 days E. lngi fuus SH, 4,5xl0-6 M,4-0 and KN Tissue culture tubes 00vEm-s-, cnstant Callus in explants" in 4 days E. lngiflius 3 59, 4,5xl0-6 M,4-0 and KN Tissue culture tubes Dark Callus in explants n 4 days E. lngi f Uus 59, 4,5xl0-6 M,4-0 and KN Tissue culture tubes 00vEm-s-, cnstant Callus in explants in 5 days E. ngqftlus 5 SH, 4,5xl0-6 M,4-0 and KN 5ml Erlenmeyers Dark N respnse in 50 days E. tl"ansvensue 4 SH, 4,5xl0-6 M,4-0 and KN 5ml Erlenmeyers Dark Callus in 4 explants in days E. tl"ispinsus 4 SH, 4,5xl0-6 M,4-0 and KN 5ml Erlenmeyers Dark Callus in 4 explants in 3 days E. vizlsus 4 SH, 4,5xl0- M,4-0 and KN 5ml Erlenmeyers Dark Callus in 4 explants in 38 days E. vizlsus 3 SH, 4,5xl0-6 M,4-0 and KN Tissue culture tubes Dark Callus in 3 explants in 3 days E. vizlsus SH, 4,5xl0-' M,4-0 and KN Tissue culture tubes 00vEm- s-, cnstant Callus in explants in 0 days E. vizlsus 3 59, 4,5x0-6 M,4-0 and KN Tissue culture tubes Dark Callus in 3 explants in 8 days E. vilzsus 59, 4,5xl0-6 M,4-0 and KN Tissue culture tubes 00vEm- s-, cnstant Callus in explants in 5 days E. wdii 5 SH, 4,5xl0-6 M,4-0 and KN Universal bttles Dark N respnse in 50 days (lateral rt frm mature plant) 8 SH, 4,5xl0-6 M,4-0 and KN 5ml Erlenmeyers Dark Callus in 7 explants in 40 days NOTES : SH, medium f SCHENK and HLDEBRANDT (97) 59, medium f NORSTOG and RHAMSTNE (96]) MS, medium f HURASHGE and SKOOG (96),4-0,,4-dichlrphenxyacetic acid NM, l-naphthaleneacetic acid KN, kinetin BA, 6-benzyladenine

115 Species TABLE 7 : n vitr cu ture f pr lmary rt exp lants f cycads ther than Encephalarts N. f explants Culture medium Cntainer light regime Observat ns : Stangel>ia eripus 4 SH, 4,5xlO- 6 M,4-0 and KN Universal bttles Dark Callus in all explants in. 4 days Stange ria eripu8 3 SH, 4,5xl0-6 M,4-0 and KN Tissue culture tubes Dark Callus n 6 explants in ~ 5 days Stangel>ia eripu8 4 SH, 4,5xl0-6 M,4-0 and KN 5ml Erlenmeyers Dark Callus in explants in 5 days Stangel'ia eripu8 3 SH, 4,5xl0-6 M,4-0 and KN Tissue culture tubes 00vEm- s-, Callus in all explants in cnstant 5 days Stangeria eripus 3 59, 4,5xl0-6 M,4-0 and KN Tissue culture tubes Dark N respnse in 50 days Stangeria eripu8 59, 4,5xl0-6 M,4-0 and KN Tissue cuiture tubes loojem-s-, Callus in explants in cnstant 30 days Bwenia 8err~lata SH, 4,5xl0-6 M,4-0 and KN Tissue culture tubes Dark Callus in explant in days Bwenia spectabilis 0 SH, 4,5xl0-6 M,4-0 and KN Tissue culture tubes Dark Callus in 6 explants in 9 days,zamia f... rfuracea 8 SH, 4,5xlO- 6 M,4-0 and KN Tissue culture tubes Dark Callus in explant in days, Zamia pumila 5 SH, 4,5xl0-6 M,4-0 and KN Tissue culture tubes Dark Callus in explant in 5 days Ntes : SH, medium f SCHENK and HLDEBRANDT (97) 59, medium f NORSTOG and RHAMSTNE (967),4-0,,4-dichlrphenxyacetic acid KN, kinetin _._- - i a

116 0 TABLE 8 : Analysis f respnses by primary rt explants t vari us parameters A. Origin f.explant in the rt Psitin N. f explants % shwi ng callus Average time t shw tested' frmat in in 50 days callgenesis (days) Tp ne-third Middle ne-third 3 83 Bttm ne-third B. Chice f basal medium Basal medium N. f explants % shwing callus Average time t shw tested frmat i n in 50 days callgenesis (days) Schenk & Hildebrandt (97) Nrstg & Rhamstine's 59 (96]) C. Chice f cntainer Cntainer N. f explants % shwing callus Average time t shw tested frmat in in 50 days callgenesis (days) 5ml Erlenmeyers Tissue culture tubes D. Light regime Light cnditin N. f explants % shwing callus Average time t shw tested frmat in in 50 days callgenesis (days) Cnstant dark Cnstant light ( 00\JEm- s E. Respnse by sp~,c ies Sp.ecies N. f explants % shwing callus Average time t shw tested frmat in in 50 days callgenesis (days) E. auensteinii E. aaffer E. cycadifuus E. ferx E. frid~iai-guitielmi E. ghellinakii 4 a - E. hrridus E. lanatus E. lebmbensis 5 00 E. lehmannii E. lngiflius E. natalensis E. ngojanus 5 a - E. transvensus 4 00 E. trispinsus E. viusus s. eripus

117 03 PLATE 8 Extensive callus grwth ccurring n the upper surface f ca. 5 mm cube frm primary rt tissue f ne-year ld EncephaZarts natazensis after 37 days in culture n SH medium supplemented with 4,5 x 0-6 M,4-D and kinetin and grwn i~ the dark at C. Scale x 6. dissected, there was n significant difference between central and peripheral explants. Transverse cylinders f tissue gave a respnse similar t that f cubes f tissue. There was, hwever, a difference with respect t explant psitin relative t the lng axis f the rt. A higher frequency f callgenesis in a shrter time perid was bserved in explants frm tp and particularly the middle ne-third f the rt, as cmpared t a lwer frequency and lnger time requirement in explants frm the bttm ne-third (Tabl& 8a). t was nted in dissectin that the upper rt material was firmer in texture, and cntained a mre well-defined cambium, than the lwer rt tissue. Furthermre the pssibility exists f a hrmne gradient alng the rt axis. t is shwn later that the perxidase activity in cycad rts is cnsistently higher in the upper rt tissue than in the lwer tissue (Sectin 3.6)~ this may well cmprise part f a gr~-respnse cntrl mechanism. Analgus examples are readily fund in the literature; such as a decline in regenerative respnse dwn the length f tbacc stems, differences in grwth respnses f leaf explants f different ages and a plarisatin in regenerative ability f Lit um bulb scales (MURASHGE, 974).

118 04 As with the cultures f megagametphytes and embrys, the selectin f basal medium had n material effect n callus frmatin by rt explants (Table l8b). Hwever, as in the afrementined cases, the chice f culture cntainer was imprtant. The use f 5ml Erlenmeyer flasks gave rise,t a higher frequency f callus frmatin, althugh requiring smewhat mre time, than the explants in tissue culture tubes (Table l8c). The pssible explanatins fr this phenmenn have already been stated, (Sectin.4.5) and cnfirm previus results (Table 8). As with the embry cultures, the rt explants gave a smewhat better respnse in dark cnditins than in the light (Table l8d). The presence f bth an auxin and a cytkinin appeared beneficial; ptimal results were fund when 4,5 x lo-6m,4-0 and kinetin were used in cmbinatin. Other cncentratins and different hrmne cmbinatins were generally unsuccessful in prmting callgenesis, with the exceptin f the 4,5 x lo-6m NAA and 6-benzyladenine cmbinatin (Table 5). A pulse li pre-treatment f the explant with relatively high auxin and cytkinin cncentratins was unsuccessful with 4-hur expsure, but sme respnse was nted when the treatment time was reduced t 4 hurs (Table 5). The use f NAA appeared t lead t the best results in the series f pre-treatments, but this ffered n particular advantage ver culture nt medium incrprating lwer auxin and cytkinin levels. n the analysis f callus-prductin respnses by the different species f cycads tested, there is evidence that the fast-grwing species f " mesic rigin (e.g. E. ~~x, E. lebmbe~~, E. lng~li~, E. natale~~, E. ~a~ven~~ and E. v~~~) respnd mre quickly and at a higher frequency, than species which are adapted t mre xeric habitats (e.g. E. eyeadi li~, E. ~~~-g~etm, E. gheteine~, E. lanat~ and E. lehmannii). This bservatin is cnsistent with the reprt by KOELEMAN and SMALL (98) wh fund rt explants f E. lebmbe~~ the mst respnsive f 9 species tested, E. lanat~ being the least successful. Cytlgical examinatin f a sample f the callus derived frm a lateral rt explant f Eneephala4t~ w~ shwed a diplid (n = 8) chrmsme cmplement in each f 8 c~lls with clear metaphase stages (Plate 9). This prvides useful evidence f the status f this species;

119 05 PLATE 9 Phtmicrgraph f chrmsmes in callus tissue arising frm a lateral rt explant f E. wdii. The diplid number (n = 8) is evident. Squash preparatin in acet-rcein, scale x 400. the bserved (n = 8) number, which is characteristic f the genus as a whle, is likely t represent the plidy cnditin f the parent material. This finding thus discunts speculatin (OSBORNE, 986a) that E. w~ may be aneuplid r plyplid var i ant f E. natale~~ r anther clselyrelated species. Subculture f primary rt-derived callus Callus derived frm primary rt explants f E. natale~~ and ther Encephala4t~ species was subcultured t varius media in different cntainers and under different light regimes as shwn in Table 9. n these transfers it was fund essential t include at least a prtin f the riginal explant material in the subcultures; where this was nt dne, the subcultured material failed t cntinue grwth. The subcultures

120 TABLE 9 : Subculture f primary rt-derived callus frm varius cycad species Species Number f subcultures Culture medium Cntainer Light regime E. natazensis 4 SH, n grwth factrs 5ml Erienmeyers 50~Em- s-, cnstant E. natazensis 0 SH, explants trea'ted wi th 5ml Erlenmeyers 50~Em- s-, cnstant xo-~h BA fr 4 hurs E. natazensi s 6 SH, 4,5xl0-6,4-0 and KN Universal bttles Dark E. natazensis 7 SH, 4,5x0-~H BA 5ml Er enmeyers 00~Em- s-\ cnstant E. natazensis 9 MS, 4,5xl0-6 M BA 5ml Erlenmeyers Dark E. natazensis 0 59, 4,5xl0-6 H,4-0 and KN Tissue culture 00~Em- s -, cnstant tubes E. nata Zell si s 0 SH, O,5ml 0-~ t 0-3 M 5ml Erlenmeyers 00~Em-s-, cnstant BA added t callus E. varius spp. 3 SH, n grwth factrs 5m Erlenmeye rs 50~Em- s-, cnstant Observat ins Cntinued callus grwth Cntinued callus grwth Necrtic in 60 days Cntinued callus grwth Cntinued callus grwth Cntinued callus grwth Necrtic in days Cntinued callus grwth E. varius spp. SH, explants treated with x0 - ~H BA fr 4 hurs 5ml Erlenmeyers 50~Em- s -, cnstant Cntinued callus grwth E. va r ius spp. 0 SH, n grwth factrs 5ml Erlenmeyers 00~Em- s-, cnstant E. varius spp. 7 59,,5xl0-6 M KN Ti ssue culture 00~Em- s-, cnstant tubes E. varius spp. 7 59, n grwth factrs Ti ssue cu ture 00~Em- ~ s-l, cnstant tubes E. varius spp. 8 SH, 4,5xl0-6 M,4-0 and KN Tissue culture 00 ~ Em-s-, cnstant tubes Bwenia spectabizis 7 SH, 4,5xl0-6 M,4-0 and KN 5ml Erlenmeyers 00~Em- s-, phtper id Stangel'ia er ipus 5 SH, 4,5xl0-6 M,4-0 and KN 5ml Erlenmeyers Dark Stangel'ia eripus 5 SH, 4,5xl0-6 M,4-0 and KN 5ml Erlenmeyers 00 ~ Em- s-, cnstant Ntes : SH, medium f SCHENK and HLDEBRANDT (97) 59, medium f NORSTOG "and RHAMSTNE MS, medium f MURASHGE and SKOOG (96 ) BA, 6-benzyladenine,4-0,,4-dichlrphenxyacetic acid KN, kinetin Cntinued callus grwth Cntinued callus grwth Cntinued callus grwth Cntinued callus grwth Cntinued callus grwth Cntinued callus grwth Apical meristem and leaf regeneratin in all cultures (96]) '\

121 07 with prtins f the riginal material generally shwed cntinued callus grwth which culd be maintained ver several passages at abut 3-mnthly transfer intervals. There was little difference in perfrmance with respect t the presence r absence f hrmne supplements r the light cnditins emplyed. Cultures in Universal bttles failed t cntinue grwth after abut 60 days. Treatment with 6-benzyladenine at varius cncentratins during the transfer prduced n change in develpment. Spt applicatins f higher levels f the cytkinin directly nt the callus resulted in necrsis after days, smewhat dependent n the cncentratin used. There was n evidence f any type f differentiatin in any f the cultures. n the case f Stang~a ~p~, differentiatin was cnsequent n subculture. Thirty primary rt tissue explants which gave rise t a fairly cmpact callus n SH medium with 4,5 x 0-6M,4-0 and kinetin were subcultured after 5 days nt similar medium in 5ml Erlenmeyer flasks. Fifteen f these subcultures were expsed t cnstant (00~Em-s-) light while the remainder were maintained in darkness. Each f the subcultures in the light envirnment develped a small green meristematic zne 'within 4 days (Plate loa) fllwed by the emergence (Plate lob) and expansin (Place 0c) f a typical circinate leaf. This pattern f mrphgenesis was cnsistent in all cultures after transfer t the light envirnment, but did nt ccur in any f the flasks held in the dark. Attempts t re-establish plantlets in axenic cnditins 'were unsuccessful due t necrsis fllwing fungal infectins. The abve i~ the first instance f ~n v~ mrphgenesis in a Suth African cycad. n view f the significance f these results, this experimentatin has been separately reprted (OSBORNE and VAN STAOEN, 987) in a paper which als cmprises Appendix 4 t this thesis..6.3 Leaflet explant cultures The ready availability f leaf material, particularly f the rarer species f cycads, cupled with the indicatins frm the wrk f HENSON (980) that cycad l~af explant material ffers gd ptential fr ~n v~ culture, made it imprtant t explre this avenue further. Explants f apprximately lcm were dissected frm unblemished, surface-sterilized, juvenile and mature leaflets f 6 species

122 08 AM (a) Appearance f apical meristem (AM), tw weeks after subculture and transfer t a light envirnment. EL (b) Emergence f a circinate leaf (EL), ne week subsequent t (a) abve. (c) Prnunced extensin f the leaf petile, tw weeks subsequent t (b) abve. PLATE 0 Light-induced mrphgenesis frm a primary rt explant f Stan~eria eripus n SH medium supplemented with mg t- each f,4-d and kinetin. Scale x.

123 09 f Enc.ephaWLtM and frm StangvUa vuopu6. These were set n different media in a variety f cntainers as shwn in Table 0. Despite a vigrus disinfectin treatment, between 50 and 00% f the leaflet explant cultures shwed fungal infectin within 3 weeks. t is thught that the deeply-recessed leaf stmata act as reservirs hlding fungal spres and these cavities escape penetratin by the disinfecting reagents (Sectin.4.3). ncreasing the cncentratin f the reagents and the use f lnger expsure times resulted in necrsis f the leaf tissue itself. This infectin prblem is nt mentined in HENSON's (980) reprt.. "A~ PLATE Callus initiatin n lngitudinally-cut surfaces f a leaflet f f EncephaZarts wdii after 37 days in culture n SH medium cntaining 5 x 0- s M NAA and KN. Scale x 0.

124 TABLE 0 : n vitp culture f cyc~d leaflets Species N. f explants Culture medium Cntainer Light regime Observatins E. natalensis 0 SH, 4,5xl0-'H,4-0 5m Erlenmeyers Dark Callus in 5 explants in 70 days (juveni e leaf) and KN Remaining 5 cultures fungus-infected E. natalensis 0 SH, 4,5xl0-'H,4-0 Universal bttles Dark N respnse in explants in 00 days (mature leaf) and KN fungal-infectin n 9 cultures E. natalensis 0 MS, 4,5xl0-'H,4-0 Petrl-dlshes Dark Callus in 4 explants n 45 days ' (seedl ing leaf) and KN Remaining 6 cultures fungus-nfected E. wdii 4 SH, 4,5xl0-'H NAA 5ml Erlenmeyers Dark Callus in explant n 30 days (juven i e leaf) Remaining 3 cultures fungus-infected E. wdii 4 SH, 5xl0- s H NAA and 5ml Erlenmeyers Dark Callus in explant in 30 days (juveni e leaf) KN Remaining 3 cultures fungus-infected E. wdii 8 SH, varius,4-0 Universal bttles Dark All cultures fungus-infected (juvenile leaf) and KN cmbinatins E. wdii 6 SH, varius NAA and 5ml Erlenmeyers Dark All cui tures fungus-infe'cted (juvenile leaf) BA cmbinatins E. wdii 5 SH, n grwth 5ml Erlenmeyers Dark All cultures fungus-infected (juvenile leaf) factrs, explants and Universal pre-treated with 0 btt les t xo-~m NAA fr ' 4 hurs E. wdii 50 59, 4,5xl0-6 M,4-0 5ml Erlenmeyers Dark and N respnse in explants in 00 days (juveni e leaf) and KN 00JEm- s-, Remaining 38 cultures fungus-infected cnstant E. cupidus 5 MS, 4,5x0-6 M BA 5ml Erlenmeyers Dark N respnse in 9 explants in 00 days (mature leaf) and Universal Remaining 6 cultures fungus-infected bttles

125 - TABLE 0 cntinued. Species N. f explants Culture medium Cntainer Light regime Observatins E. eugena- MS, 4,5xl0-6 H SA 5ml Erlenmeyers Dark N respnse in explants in 00 days maraisii Remaining culture fungus-infected (mature leaf) E. lehmannii 5 SH, varius NAA ~nd 5m Erlenmeyers Dark N respnse in 8 explants in 00 days (mature leaf) BA cmbinatins Remaining 7 cultures fungus-infected E. inpinus 5 SH, varius NAA and 5ml Erlenmeyers Dark N respnse in 3 explants n 00 days (mature leaf) BA cmbinatins Remaining cultures fungus-infected Stangeria 0 SH, 4,5xl0-6 H,4-0 Universal bttles Dark N respnse in 8 explants in 00 days eripus and KN Remaining cultures fungus-infected (mature leaf) Ntes SH, medium f SCHENK and HiLDEBRANDT (97) MS, medium f MURASHGE and SKOOG (96) 59, medium f NORSTOG and RHAMSTNE (967),4-0,,4-dichlrphenxyacetic acid NAA, l-naphthaleneacetic acid BA, 6-benzyladenine

126 n thse instances where fungal infectin was absent, nly the explants frm juvenile leaflets r frm seedling leaves had the ptential fr callgenesis; n callus frmatin ccurred n any explants frm mature, fully-expanded leaves. Cal lus frmatin was favured by the incrpratin f the auxins,4-0 r NAA at lw levels in the medium, and arse between the upper and lwer epidermis alng the cut surfaces f the explant (Plate \. The callus required a lnger perid in initiatin, and was mre cmpact in appearance and less vigrus in grwth than callus frm megagametphyte, embry r rt tissues. n the limited experimentatin n subculturing this material, callus prliferatin culd nt be maintained; all explants with callus becming necrtic in abut mnths..6.4 Other explant cultures n additin t the use f embrys. rts and leaflets as diplid tissues fr ~~ v~ culture, it was cnsidered useful t explre ther surces. Explants frm leaf petiles, leaflet petilules, leaf bases, cne axes and sprphylls f 5 species f E~cephal~~ were dissected and set n SH medium with varius supplements, in either Universal bttles r Erlenmeyer flasks and incubated in the dark at 5 C as indicated i n Table. Callgenesis ccurred in varying frequency in all explants except thse f the leaflet petilules. The absence f any respnse with the latter explants may be due t the cmparatively small explant size since leaf petiles, f similar ntgeny, did give at least sme evidence f callus frmatin. The ptimum perfrmance in terms f frequency f callus develpment and time fr this t ccur, was fund with explants frm the cne axes and micrsprphylls f E. ~~x and E. wdli. Thse explants which had given rise t callus were subcultured n SH medium supplemented with 4,5 x 0-6 M,4-0 and kinetin. n all cases the callus cntinued t develp fr a perid f - mnths but subsequently became necrtic. was bserved. N evidence f any frm f differentiatin

127 Species TABLE : n vitr culture f ther diplid tissues frm Encephalarts, Explant surce N. f explants Cu ture medium Cntainer Light regime Observatins E. caffer male cne axis 8 SH, 4,5xl0-'H,4-0 and KN 5ml Erlenmeyers Dark N respnse in 70 days E. fer-x male cne axis 6 SH, n grwth factrs Universal bttles Dark N respnse in 70 days E: fer-x male cne axis 4 SH, 5xl0-5 H NAA Universal bttl~s Dark Callus in explant in days E. fer-x micrsprphylls 4 SH, 4,5xl0-'H,4-0 and KN 5ml Erlenmeyers Dark Callus in all explants in 4 days E. f er-x micrsprphylls 4 SH, 5xl0.. s H NAA 5ml Erlenmeyers Dark Callus in all explants in 4 days E. latifrns leaf petiles 3 SH, 4,5xl0-'H,4-D and KN Universal bttles Dark Ca llus in explants in 40 days E. latifr-ns leaf petiles 3 SH, 5xl0- s H NAA Universal bttles Dark N respnse in 70 days E. natalensis leaflet petilules 30 SH, 4,5xl0"'H,4-D and KN Universal bttles Dark N respnse in 70 days : and 5ml Erl enmeyers E. wdii leaf bases 8 SH, 4,5xl0-'M,4-D and KN 5ml Erlenmeyers Dark Callus in 4 explants in 30 days E. wdii male cne axis SH, 4,5xl0- t M,4-D and KN 5ml Erlenmeyers Dark Callus in all explants in days! E. wdii micrsprphylls SH, 4,5xl0- t H,4-D and KN 5ml Erlenmeyers Dark Ca llus in all explants in days Ntes S.H, medium f SCHENK and HLDEBRANDT (9]),4-0,,4-dich)rphenxyacetic acid NAA, l-naphthaleneacetic acid KN, kinetin w

128 4.7 Suspensin cultures A brief investigatin was made int the feasibility f establish~ng viable suspensin cultures frm cycad material. VigruslY-9~W'ng haplid callus derived frm StangvUa vuopl6 megagametphytlc tissue (Sectin..5. ) was used as the starting mate~ial, and tw prcedures were used t initiate the suspensin cultures. n the first prcedure. apprximately 9 prtins f the callus were macerated in a Dunce glass hmgenizer in small quantities f the B5 liquid medium f GAMBORG, MLLER and OJMA_ (968). The crude suspensins were diluted with further prtins f the medium and maintained n an rbital shaker. After 48 hurs, the carse aggregates were remved by filtratin thrugh 00~m nyln mesh, and the resulting suspensins subcultured t fresh medium. After five days aliquts frm the cultures were plated nt slid media in Petri dishes and subsequently examined under an inverted micrscpe. n the secnd prcedure, similar quantities f the callus were suspended in liquid SH medium and incubated with cellulase and pectinase enzymes n a shaking table. After 48 hurs, the cultures were centrifuged, washed and resuspended i n fresh medium. Aliquts were withdrawn ver a 4-day perid and set n slid medium f the same cmpsitin in Petri dishes. n bth cases utlined, further subcultures t fresh liquid media were made at intervals and the prgresses mnitred by all density cunts. Bth the mechanical ~nd the enzymatic preparatins gave rise t suspensins with single cells and aggregates f up t 0 cells. These did nt prliferate during the tw week perid~ all cell density cunts n subcu l turing were simply related t slutin dilutin factrs. Plating ut f a iquts n slid media appeared t prmte cell divisin and grwth ; wi thin abut -3 weeks aggregates f 00 cells were visible. There was n evidence f any rganizatin within these cell clusters which subsequently slwly became necrtic..8 Cnclusin Attempts have been made t establ ish ~n v~ cultures, using varius hapl~id and diplid cycad surce material, n varius media with

129 5 different hrmne supplements and expsed t different envirnmental cnditins. Effective surface disinfectin f leaflets prved difficult but ther explant material was cnveniently disinfected with sequential 70% ethanl and 0,75% sdium hypchlrite treatments. Callus prductin was readily achieved frm mst explant surces f mst cycad speci es t e ted. Crisp white callus generally arse within twenty t thirty days n cut surfaces f these explants. There was n particularly significant difference in the respnses n MURASHGE-SKOOG (96), NORSTOG and RHAMSTNE (967) and SCHENK and HLDEBRANDT (97) basal media. Additin f an auxin and a cytkinin appeared beneficial but nt essential; inclusin f,4-0 and kinetin in the 0-7 t 0-6 M range gave a generally satisfactry respnse. The selectin f culture vessels was imprtant; c.ntainers which allwed a relatively free gaseus exchange lead t a higher frequency f callgenesis than cntainers where gaseus exchange was inhibited. Dark cnditins appeared t give a marginally greater success rate than cnstant light. Cycad taxa which are mesic in natural habitat gave mre rapid respnses at higher frequencies than their xeric cunterparts. Differences in respnses between juvenile and mature leaflet explants implied that physilgical age f the explant tissue is imprtant.. Similarly. differences in respnse frm explants taken at var~us psitins in primary rt tissue may imply the existence f endgenus hrmne gradients in the rt. Attempts t induce any frm f differentiatin n the callused explants met with little success. Subculture t media with different hrmne supplements, and envirnmental changes, bth failed t induce any frm f mrphgenesis in all cultures frm Encephal~~. Sme degree f success was achieved with Stang~-derived cultures. Subculture f megagametphytic callus lead ccasinally t the frmatin f spherical structures which may be crallid rt primrdia. Subculture f callused Stang~ primary rt explants t SCHENK and HLDEBRANDT (97) medium with 4,5 x 0-6 M,4-0 and kinetin, assciated with simultaneus transfer frm a dark t a cnstant light envirnment lead t the appearance f meristematic znes and subsequent leaf appearance. This is the first knwn reprt f ~n v~ mrphgenesis f a Suth African cycad. t is clear that the aim f establishing a

130 6 prtcl fr the clnal prpagatin f cycads by ~ v~ techniques, is an ambitius ne. Sme cnsiderable effrt has resulted in nly a very mdest degree f success, and a large element f serendipity makes the design f any prject f this nature smewhat "pen-ended". t is pssible that future advances in tissue culture techniques, perhaps assciated with a much wider understanding f the physilgical rle f plant grwth regulatrs and envirnmental interactins, may lead t a mre predictable cntrl ver mrphgenesis. n rder better t understand the physilgy f cycads, the sectin f wrk which fllws was directed primarily at explring at least sme aspects f cycad phytchemistry. Cmparative chemical and bichemical analyses bth f different rgans and between different taxa may prvide evidence f fundamental differences which may in turn relate t different ~ v~ respnses. Thus the questin as t why Stang~ rt explants give rise t meristema~ic znes in culture when the identical material frm ther cycads des nt, may be answered in terms f inherent bichemical differences. Apart frm the bvius benefits f establishing ~ v~ prpagatin systems, researches cmbining this wrk with phytchemical investigatins may yield clues t the mechanism f sex-expressin and hence ffer a means f sex cntrl. The creatin f a female clne f Eneephal~~ wdii may eventually be pssible. n v~ systems may als lead t a better understanding f the cycad-cyanbacterial rt symbisis and t an assessment f cnditins influencing pllen viability. Yet a further applicatin wuld: be the establishment f haplid cultures f all cycad taxa fr the purpses f detailed karylgical wrk. t is imprtant that further prjects f this kind are encuraged. The value f systems fr the prpagatin f endangered plants remains unquestinable.

131 7 CHAPTER THREE CYCAD PHYTOCHEMSTRY NDEX TO CHAPTER THREE Page 3. ntrductin 8 3. Cycad phyttxins Other phytchemical aspects 3". 4" Analysis f misture cntent Analysis fr sluble prtein'cntent Analysis f perxidase enzyme activity Bichemical changes during callgenesis frm StangetUa. etuptl Serlgical investigatins Analysis f cycad txins Analysis f leaf wax hydrcarbns Cnclusin

132 8 3. ntrductin Despite the significant evlutinary psitin f the Cycadales, little wrk has been dne t investigate the phytchemistry f the rder until fairly recently. mpetus here was prvided by the discvery f the first f a series f unique txins in cycad seeds in the 940 ' s (sectin 3.). Mre recently, sme investigatins have been made int cycad phenlics, flavnids, carbhydrates and ther cmpunds (sectin 3.3). Althugh mst f this wrk is funded n chem-taxnmic bjectives, the data cntribute t ur knwledge f cycad physilgy and further relate t develpmental mrphlgy. n the experimental sectin f this Chapter (sectins 3.4 t 3.0), the authr reprts n varius phytchemical investigatins bth f different rgans and between different taxa. ntegratin f these varius researches will undubtedly result in a better understanding f the fundamental factrs cntrlling mrphgenetic cmpetence ~ v~. 3. Cycad phyttxins Cycads have been used as a surce f edible starch in different parts f the wrld fqr many years, particularly in circumstances when nrmal fd supplies have been restricted. The 'arrwrt' starch, generally btained frm rhizmes f M~antha, can als be extracted frm Zamia rts while sag l starch, usually derived frm the stem f certain palms, is als h r ested frm Cyc~, Zamia and Ma~zamia plants (THERET, 958; WHTNG, 963). The Cape Httentt peple prduced a palatable frm f bread frm the stems f certain ~cephal~~ species (DYER, 965). a prcess which gave rise bth t the vernacular name 'brdbm ' and t the genus name (e~ = within, ~ephaii = head, ~~ = bread). Althugh the vegetative parts f certain species can prvide a surce f starch, this harvest is invariably destructive t the plant. By cntrast, the seed crp presents a fd surce which is bth readily and repeatedly cllectabl~. Hwever, it is in the starch-rich megagametphytes where cycad txins are present at their highest cncentratins. t is quite remarkable that many indigenus peple in different parts f the wrld have apparently quite independently fund ut hw t detxify

133 9 the seeds. All 'recipes' are based n steeping the crushed seeds in water which allws degradatin and extractin f the txins (WHTNG, 963; OSBORNE, 985a). mprperly detxified r untreated seed have caused severe illness and smetimes death t humans and t livestck. nteresting circumstantial evidence f cycad txins was recrded by the btanist Banks, wh accmpanied Captain Ck's expeditin. n 770 a number f the crew suffered frm a vilent fit f vmiting and tw f the ship's hgs died after eating seed f Cye~ media R. Br. (THERET, 958). Anther ntable case was th~t f General J.C. Smuts and his Cmmand wh were incapacitated fr several days during the Suth African War f when they resrted t cycad seed fr emergency fd supplies (RETZ, 969), the plant respnsible believed t be Eneephal~~ lng~iiuq (Jacq) Lehm. (OYER, 965). The high pst-war incidence f amytrphic lateral sclersis and assciated cnditins such as Parkinsnism and Alzheimer's disease amngst the Chamrr peples n the Marianas islands f Guam and Rta has been ascribed t the accumulated effect f many years' cnsumptin f Cye~ ~cin~ seeds in which cmplete remval f the txin is never certain (PALEKAR and DASTUR, 965; SPENCER, NUNN, HUGON, LUDOLPH, ROSS, ROY and ROBERTSON, 987). Evidence that it is the cycad seed which is respnsible is prvided by the cmparatively lw incidence f the afrementined diseases n the neighburing island f Saipan, where the cycad ppulatins have been cleared fr agricultural purpses, and by the declining frequency f the diseases as the Chamrr ppulatin has becme westernized and dietary habits have changed. (SPENCER, NUNN, HUGON, LUDOLPH, ROSS, ROY and ROBERTSON, 987). The uter fleshy layer and/r the megagametphyte f the Suth African spec i es Enee.pha.iaJLt0.6 eyc0.di.6-uw.:.. E. e.uge.ne.- majta..uu, E..fi.vr.x., E. h~w.:., E. lehmannu, E. lngi6uw.:. and E. viil~w.:. have either prved t be acutely txic when administered t rabbits r have been suspected r knwn t be pisnus when ingested by man (STEYN, VAN DER WALT and VEROOORN, 948). Txic and carcingenic effects have been bserved in rats fed with varius parts f the female cnes f E. umbduz-iem..t.6, E. viil~uq, E. lebmbem..t.6 and E. laevi6iiw.:. (TUST N, 974). The kernels f E. lanatw.:. have als been recgnized as being txic and carcingenic (TUSTN, 983). t is nt nly the cycad seeds which are txic. Grazing by stck n

134 0 leaves f certain Ma~zamia species in Australia and Zamia species in the Americas has resulted in the partial r ttal paralysis f the hind legs, a cnditin knwn as the 'wbbles' r 'staggers'. Whilst this rarely kills the animals directly, they are unable t find fd and water and perish as a result. The Australian Gvernment has embarked n quite extensive eradicatin prgrammes t remve. the ffending species, particularly Ma~zamia m~el F. Muell. but frtunately a specific reserve has been set aside t prevent the species becming extinct (OSBORNE, 985a). The active principles fall int tw grups, the methylazxymethanl glycsides and at least ne nn-prtein amin acid. Of these tw, the frmer have received cnsiderable attentin wh)le the latter has mre recently been reinvestigated fllwing earlier reprts. The methylazxymethanl glycsides COOPER (940) btained a crystalline substance, which he named macrzamin, frm the seeds f the Australian cycad Ma~zamia ~p~~ (Salisb.) Miq. This cmpund was shwn t cmprise a methylazxylmethanl unit linked t a primeverse sugar residue (LYTHGOE and RGGS, 949; LANGLEY, LYTHGOE and RGGS, 95), (Figure 3, structure 36). Macrzamin was islated frm Suth African species Encephai~~ tanat~ and E. ~an6ven~~ (ALTENKRK, 974). A similar txin, cycasin (Figure 3, structur~ 37) cnsists f the same aglycne unit linked t D-glucse and was fund initially in Cy~ ~evtuta Thunb. (NSHDA, KOBAYASH and NAGAHAMA, 955) and C. ~C n~ L. (RGGS, 956; KORSCH and RGGS, 964). Althugh macr~zamin and cycasin are the majr cycad txins, several clsely-related substances, the necycasins (Figure 3, structures 37a t 37g) have been islated frm Cy~ species; all share the cmmn methylazxymethanl aglycne with a ~-glycsid;c linkage t a variety f different sugar residues (NAGAHAMA, 964). Cycasin and macrzamin have nw been fund in all ten cycad genera but nt in any ther plants (DE LUCA, MORETT, SABATO and SNSCALCO GGLANO, 980; MORETT, SABATO and SNSCALCO GGLANO, 98a, 983). Macrzamin 'is generally mre abundant than cycasin, ccurring in levels frm abut 0, t 5% by fresh weight in mature seeds. The range in cncentratins is genus dependent and thus has chemtaxnmic significance. n the Suth African cycads, Encephat~~ seed have,09 t,86% macrzamin while Stang~ ~p~ cntains 4,70% (MORETT, SABATO and SNSCALCO GGLANO, 983).

135 (36) OH HACROZAMN * = (Methyl-ONN-azxy) methyl r Methylazxymethanl B - primeverside (37a) (37b) (37c) NEOCYCASN A NEOCYCASN B NEOCYCASN C (37d) NEOCYCASN 0 (37e) (37f) (37g) NEOCYCASN E NEOCYCASN F NEOCYCASN G OH CYCASN = (Methyl-ONN-azxy) methyl r Methylazxymethanl B-D-glucpyranside Methylazxymethanl B-laminaribiside Methylazxymethanl B-gentibiside Methylazxymethanl B-laminaritetraside Methylazxymethanl B-laminaritriside Methylazxymethanl B-cellbiside Cycasin 6-0-S-laminaribiside Cycasin 3-0-B-gentibiside '" Nte: ' n (36) and (37) abve, the trivial names are fllwed by a full chemically-descriptive name defined by UPAC rules, and an abridged alternative. The names S-primeversylxyazxymethane fr (3 6) and S-D-glucsylxyazxymethane fr (37) are als encuntered in the literature. NH H3C-HN-HC---f--- COOH (38) a-amno-s-methylamnoproponc ACD = S-N-methylamin-L-alanine, BMAA. H FGURE 3 Structure and nmenclature f cycad phyttxins. Whilst references t the bisynthesis f these natural prducts are lacking, there has been sme wrk n the txic actin. The glycsides are hydrlysed sn after ingestin by vertebrate animals by the actin f the enzyme ~-glycsidase in the gut; it is the resulting aglycne and its subsequent decmpsitin prducts which are txic. Evidence fr this is prvided by the fact that cycasin is nn-txic when administered rally t germ-free rats (SPATZ, SMTH, McDANEL and LAQUEUR, 967) and is similarly withut ill-effect when injected directly int the bldstream (SEELY, FREED. SLVERSTONE and RPPERE,

136 985). Oxidative degradatin f the aglycne methylazxymethanl gives methylazxyfrmaldehyde, methylazxycarbxylic acid and the methyldiaznium in as principle metablites; the carcingenicity is thught t arise frm the methylating actin f ne r mre f these units n DNA residues (KKUCH, KARASAWA, SUZUK and HOPFNGER, 98). t is f interest t nte that certain insects which feed n cycads, ntably the hairstreak butterfly Eumaeu atala ~~da Reber which eats Zamia fliage, accumulate fairly large quantities f the txins internally (ROTHSCHLD, NASH and BELL, 986). t is believed that the cmpunds remain in the glycsylated frm and are therefre harmless t the insect but a valuable defence mechanism; n ingestin by a predatr, the mlecules are hydrlysed and exert their txic influence (ROTHSCHLD, M., p~ cmm.). a-amin-e-methylaminprpinic acid The cmpund a-amin-e-methylaminprpinic acid (synnym e-n-methylamin L-alanine r BMAA, Figure 3, structure 38) was first islated frm seeds f Cyc~ ~cin~ L. (VEGA AND BELL, 967). t was later shwn t be widespread ~n free r bund frm in seeds and leaves thrughut the genus but nt in any ther cycad genera (DOSSAJ and BELL, 973). Bilgical activity f this txin is cnfined t the L-ismer which is txic t chicks, rats and mice (VEGA, BELL and NUNN, 968) and causes neurlgical disrders t macaque mnkeys (SPENCER, NUNN, HUGON, LUDOLPH, ROSS, ROY and ROBERTSON, 987). These authrs speculate that it is the amin acid derivative, rather than the azxy cmpunds, which is respnsible fr the cycad-assciated neurlgical diseases in the Pacific slands. 3.3 Other phytchemical aspects While the majr bichemical interest in cycads has been fcussed n their phyttxins (Sectin 3.), a number f ther phytchemical investigatins have been carried ut and many f these are significant in applicatin t cycad taxnmy. The lder wrk is summarized in the review f TH..ERET (958), which includes a discussin f the smewhat limited uses f cycad material as surces f fibres, gums and ils. An entry int the current phytchemical literature f the grup ;s prvided by HEGNAUER (986).

137 3 Phenlic cmpunds and flavanids n a survey f leaves f species f cycads, WALLACE (97) fund evidence f caffeic, prtcatechuic, p-cumaric, p-hydrxybenzic, ferulic and vanillic acids (Figure 4, structures 39-44) in all examples. Since these cmpunds are ubiquitus in the pteridphytes, gymnsperms and angisperms, their presence is f limited significance. Sinapic acid (Figure 4, structure 45) ccurred in V~n ~p~nut~um Dyer and C~z~ mexicana Brngniart and pssibly ther taxa while,4- dihydrxybenzic acid (Figure 4, structure 46) was fund in tw species f Encep~~ and in Bwenia ~~ (W. Bull), Chamberlain. Syringic acid (Figure 4, structure 47) was tentatively identified in C~z~ mexicana. The presence f sinapic and syringic acids in cycads indicates that the distributin f these cmpunds in plants is wider than thught previusly (WALLACE, 97). The biflavnid chemistry f cycads has attracted a certain amunt f attentin, principally because f the taxnmic implicatins. A survey f biflavnes in the leaves f 8 species has shwn that the pattern f ccurrence f amentflavne, hinkiflavne (Figure 5, structures 48-49), their methyl ethers and ther derivatives is cnsistent within mst genera (DOSSAJ, MABRY and BELL, 975). Leaf biflavnes are cnfined t the uter periclinal wall and anticlinal walls f epidermal cells and their presence is believed t cntribute t the plant's defense against micrbial and predatr attack (GADEK, QUNN and ASHFORD, 984). The biflavnid distributin in the seed testae is quite different t that in the leaves. n an analysis f 8 species, GADEK (98) fund that representatives frm Ma~z~ are characterised by the ccurrence f cupressuflavne and amentflavne derivatives while Cy~ species cntained nly the amentflavne-based cmpunds. Encep~~, Lep~dz~ and Z~ samples gave nly trace amunts f biflavnids. The ccurrence f cupressuflavne (Figure 5, structure 50) is unusual utside the gymnsperm families Cupressaceae and Araucariaceae. An unusual feature is the cmplete absence f biflavnids in Stang~ (DOSSAJ, MABRY and BELL, 975). Cartenids The highly-clured seed cats f cycads,cntain simple cartenid mixtures. The bright yellw seed cat f Cyc~ ~evluta has zeaxanthin as the majr cmpnent with smaller amunts f cryptxanthin and s-cartene (Figure 6,

138 4 HO HOb-CH-CHCOOH (39) CAFFEC AC D HO HD-COOH (40) PROTOCATECHUC ACD H0- CH-CHCOOH (4) p-cdumarc ACD H0D-COOH (4) p-hydrdxybenzoc ACD MeO HOOCOOH (43) FERULC ACD (44) VANLLC ACD OH H-O-COOH (45) SNAPC ACD (46),4-DHYDROXYBENZOC ACD HO~COOH MeO (47) SYRNGC ACD FGURE 4 Sme simple phenl ic cmpunds islated frm cycads.

139 5 HO OE (48) AMENTOFLAVONE HO (49) HNOKFLAVONE OH OH HO OH HO OH OH 0 (50) CUPRESSUFLAVONE FGURE 5 Sme biflavnids islated frm cycads.

140 OH HO (5) ZEAXANTHN HO (5) CRYPTOXANTHN (53) -CAROTENE (54) LYCOPENE (55) SEM - -CAROTENOt:lE FGURE 6 Sme cartenids isqlated frm cycads.

141 7 structures 5-53) (BOUCHEZ, ARPN, DERUAZ and GULLUY, 970). The seed cat f Zamia has lycpene (Figure 6, structure 54) as the principal pigment while extracts frm Eneep~~, V~n and Mactzamia cntained a mixture f unsubstituted mn- and dihydrxy- 8- cartenes (BAUMAN and YOKOYAMA, 976). An attractive red-brwn cluratin, characteristic f emergent C~zamia leaflets, has been ascribed t the presence f semi-s-cartenne (Figure 6, structure 55), an unusual seccartenid fund previusly nly in the fruits f the citrus relative, M~ja extiea (CARDN, GNANNESCH, SELVA and CHELL, 987). Carbhydrates All cycads have a well-develped system f mucilage ducts and excisin f the leaf rachis r cne peduncle, r injury t the caudex allw cllectin f the exudate. This mucilage cnsists f a cmplex plysaccharide which may be hydrlysed t its cmpnent sugars. The exudate frm a female cne f Eneep~~ lng~6~~ yielded fucse, rhamnse and 3-0-methyl rhamnse, arabinse, xylse, galactse, mannse, glucurnic acid with its 4-0-methyl ether (Figure 7, structures 56-6) (STEPHEN and DE BRUYN, 967). CH H~H OH (56) a-o-fucose H~H OR OH (57) a-l-rhahnose (R-H) 3-0-HETHYL RHAMNOSE (RaHe) HV:-0~H ~ OH (58) a-l-arabnose ;:-0\ H~~ 6H OH (59) a-d-xylose HO (60) a-d-galactose (6) a-d-mannose OCOOH 0 OH RO OH (6) a-d-glucuronc ACD (R=H) 4-0-HETHYL GLUCURONC ACD (R~He) OH FGURE 7 Mnsaccharides btained n hydrlysis f cycad mucilages.

142 8 n a survey f hydrlysed mucilages frm excised leaf raches f cycad species DE LUCA, MORETT, SABATO and SNSCALCO GGLANO (98) shwed the taxnmic usefulness f these data at a generic level. The African and Australasian genera cntain mainly arabinse and galactse while the American genera have higher prprtins f fucse and galactse in V~n; rhamnse and arabinse in C~zamia; rhamnse, fucse and methyl rhamnse in lamia; and gal act se and methyl rhamnse in M.{.CJtc.yc.cL6. Sta.ngeM.a. is unusual in having virtually n rhamnse r methyl rhamnse. Lep~dzamia and Enc.ephaiant~ are the nly genera which shw similar patterns. With E. lng~6~ at least, the mnsaccharide pattern is nt significantly affected by plant age, sex r envirnment (SNSCALCO GGLANO, 980). A mre cmprehensive survey f the genus Enc.ephaiant~ has shwn a mre-rless identical mnsaccharide pattern in 4 species investigated (MORETT, SABATO and SNSCALCO GGLANO, 98b). Analyses f the exudates frm cnes f three species f Enc.ephalant~ have highlighted certain structural features (STEPHENS and STEPHEN, 988). One is the ccurrence in varying amunts f 3-0-methyl-L-rhamnse which, tgether with the parent L-rhamnpyranse unit, can cmprise mre than 0% f the ttal carbhydrate cmpsitin. Anther is the high prprtin f acidic units, ntably D-glucurnic acid and its related 4-methyl ether. Sequential degredatin f the plysa~charide has lead t the pstulated structure shwn in Figure 8. Cycl itl s The ccurrence f plyhydrxycyclhexanes (cyclitls) is cmmn in gymnsperms. n the cycads, the cmpunds my-insitl, sequyitl and pinitl (Figure 9, structures 63-65) have been fund. n particular, sequyitl has been islated frm leaves f several species f Cy~, Enc.ephaiant~, Lep~dzami..a. and CeJULtzami..a. (PLOUVER, 965) and it is extracted tgether with macrzamin frm the seeds f MaCJtzamia ~edl~ (Gaud.) C.A. Gardn. (CANNON, RASTON, TOA and WHTE, 980). Enzymes Despite the wide applicatin f enzymlgical and serlgical techniques in bilgical investigatins, little attentin has been paid t their use in cycad research. MERRAM (974) studied the lamia ppulatins in Flrida. using isenzyme ban~ ' ing systems and fund the perxidases t prvide useful 'fingerprints'. An indicatin was that the Flrida l~ cmprise ne

143 9 FGURE 8 : A prpsed structure fr the plysaccharide in Enc.ephalaJLt.6 cne exudate. Numbers indicate linkage cnfiguratins where knwn; legend as belw (STEPHENS. and STEPHEN, 988). ~ a.-l-rhamnose 0 ~-D-MANNOSE " a.-l-arabnose D ~-D-GALACTOSE ~ ~-D-XYLOSE ~-D-GLUCURONC ACD OH OH OH OH OH OH ~H OH ~H OMe ~ HO HO HO OH OH OH OH (63) MYO-NOSTOL (64) SEQUOYTOL (65) -p NTOL FGURE 9 Cylitls islated frm cycads.

144 30 plymrphic species. A study f the perxidase isenzymes in the seeds f CyQ~ ~~n~ shwed a difference in the enzyme pattern at different stages during seed maturatin (PENA, GRllO and PEREX, 983). Other cmpunds Mentin f ther cmpunds islated frm cycads is scattered sparsely in the literature. The bibligraphy f READ and SOlT (986) and the review by HEGNAUER (986) are useful reference surces. The reviews by OSBORNE (986b, Appendix ) and OSBORNE, GROBBElAAR and VORSTER (in preparatin), Appendix 5) may als be cnsulted in this regard. 3.4 Analysis f misture cntent 3.4. Materials and methds An apprpriate mass f the sample material (Qa. g) was cut int smallish prtins and ven dried at 05 C t cnstant mass, which was generally achieved within hurs. The percentage lss in mass is reprted as misture althugh this is mre crrectly "misture and vatiles i' 3.4. Results and discussin Althugh the purpse f the misture determinatins was primarily t enable expressin f ther analytical results n a dry basis, the data als prvided sme inherently useful infrmatin. The results are recrded in Table Z. ROBERTS (973) classifies seeds int tw majr grups described as ~hdx, nrmally desiccated prir t dispersal and with misture cntents reduced t the 0-5% range, and wet r ~eqal~ant seeds which retain high misture levels and in which the embry cntinues t develp. n this classificatin the cycad seeds fall int the recalcitrant grup. The analyses recrded shw that cycad megagametphytes at the time f seed fall have mistures in the 4-55% range and that partial dehydratin t 3-4% ccurs during strage. Hwever, the enclsed embrys appear prtected frm desiccatin and retain relatively. high misture levels (Qa. 64%). This indeed wuld be the anticipated situatin in all recalcitrant seeds as any significant dessicatin f the cntinuusly develping embry itself wuld lead t lss f viability.

145 3 TABLE : M.lSTURE C.NTENT N CYCAD MATERAL CYCAD SPECES SAMPLE MATERAL USED NUMBER.F SAMPLES MEAN M.lSTURE C.NTENT, % E. lebmbensis mature fresh seed, sarctesta 73,5 mature fresh seed, megagametphyte 43,8 E. natalensis 3-year ld seedling, leaf 60.,5 6-year ld plant, emergent leaflet 8, 6-year ld plant, emergent leaf rachis 87,7 mature plant, mature leaflet 49,5 3-year ld seedling, pr imary rt 9,8 mature fresh seed, sarctesta 57,3 mature fresh seed, megagametphyte 54,6 mature stred seed, megagametphyte 0 4,8 mature stred seed, embry 4 64, E. villsus fresh inferti l e seed, megagametphyte 4,6 mature stred seed, megagametphyte 36,8 mature stred seed, embry 64,4 E. wdii mature plant, emergent leaflet 7,0. mature plant, emergent leaf rachis 88,4 mature plant, lateral rts 76, s. eripus mature fresh seed, megagametphyte 50,0 mature stred seed, megagametphyte 3, Cycas :t'evluta Cycas thuarsii Din edul.e Lepidzcunia pe:t' ffskyana callus culture ex megagametphyte, mature plant, mature leaflet mature plant, mature leaflet mature plant, mature leaflet 3-year ld seedling, leaflet 9, 58,4 67,6 43,9 70.,4 Zcunia fu:t'fu:t'acea 3-year ld seedling, leaflet 65,4

146 3 t is als reasnable t assume that any misture deficit in the meqagametphyte wuld be reversed by imbibitin prcesses prir t germinatin. The fact that germinatin ccurs naturally during the wet summer perids reinfrces this hypthesis. The findings utlined abve appear t be cnsistent with a reprt by FORSYTH and VAN STADEN (983) where ~tc.e.r.>ftaiajltq.6 Y.a..taie.ML6 seed were fund t have 48% misture when shed and, when expsed after strage t mist germinatin-inducing cnditins, rapidly imbibed water t establish a final misture cntent f 66%. Anther pint f interest is the change in mis~ure cn~ent f leaf material frm the time f emergence t maturity. The figures frm. Y.a..taie.ML6 shw the emergent leaflet and rachis with misture levels f 8, and 87,7% respectively while a fully-mature leaflet yields 49,5%. Fullyexpanded seedling leaves appear t be intermediate in this range. The misture cntent may thus be a useful indicatr f leaflet physilgical age and hence callgenic and mrphgenic cmpe~ence. 3.5 Analysis fr sluble prtein cntent 3.5. Materials and methds The determinatin f sluble prtein is an imprtant rutine bichemical test. A cmmnly-used technique is the spectrphtmetric methd f lowry, ROSEBROUGH, FARR and RANDALL (95) which makes use f the Flin and Cicalteau reagent. A knwn mass f fresh materlal (e.g.,00 g) was grund in a mrtar with a suitable vlume (e.g. 9,0 mls) f precled (4 C) extractin buffer (0,076 M Tris, 0,005 M citric acid, ph 8,65; POUlK, 957). The macerate was centrifuged at x g fr 30 minutes at 4 C. The clear supernatant slutin was used fr the sluble prtein determinatins, the perxidase analyses (Sectin 3.6) and fr serlgical wrk (Sectin 3.8). Fr the sluble prtein determinatin the extract was diluted x 5, with buffer slutin, prir t analysis. A series f standard slutins prviding 0-0 ~g prtein was prepared using bvine serum albumin (Merck Art. 08). _ The alkaline reagent cmprised ml % CuS0 4.5H 0 and ml % ptassium sdium

147 33 tartarate t which 00 ml % Na Z C0 3 in 0, M NaOH was added. Sample aliquts (e.g. 0, ml, equivalent t mg fresh material in the example) were made up t a vlume f,0 ml. Standard slutins were similarly treated. The alkaline reagent (5,0 ml) was added and the mixture ieft fr 0 minutes, after which 0,5 ml dilute (:) Flin reagent was added. The absrbance was read at 660 nm in cm glass cells after 30 minutes against a reagent blank. The prtein amunts were derived frm a calibratin curve and expressed as mg ttal sluble prtein per g fresh weight f sample material Results and discussin The prtein analysis figures are recrded in Table 3 tgether with the crrespnding perxidase figures (Sectin 3.6), thus als allwing expressin f perxidase/prtein ratis. The sluble prtein cntents vary widely. Emergent leaves and - year ld seedling leaflets shw generally lwer levels than fully-expanded leaves r thse frm lder seedlings and mature plants. Thus accumulatin f sluble prtein appears t be a cmbined functin f leaf and plant age. There is n particular crrelatin between prtein levels and taxnmic rank r plant habit. The highest leaf prtein amunt recrded (00 mg g-l) was that frm a mature leaf frm a mature plant f V;~ edule. High prtein cntents were als bserved in megagametphytes and cne scales, in particular the micrsprphylls frm Eneephal~~ wdji prvided 3 mg g~l. Generally lw sluble prtein cncentratins «0 mg g-l) were fund in seed sarctestae, embrys and rts. n the primary rts there is sme evidence f a prtein gradient frm tp t bttm. The material in culture des nt appear t shw any significant prtein accumulatin at callus-grwth stages. There are few references with which t cmpare these results. The 'seed kernels' f Cye~ ~eviuta are said t cntain - 4% crude prtein and 66-70% starch n a fresh weight basis, while the stem f this species has 9,5% prtein n a dry basis (THERET, 958). n an Australian cycad, Ma~zamJa ~ediej, the megagametphyte n dry basis is reprted as cntaining 3% prtein and 60,7% starch. N details have been published n the cmpsitin f Suth African cycads.

148 Analysis f perxidase enzyme activity 3.6. Materials and methds Determinatin f ttal s l uble perxidase activity Preparatin f the sample material is described in Sectin The clarified : extract was again diluted x 5 with buffer slutin prir t analysis. The assay fr ttal slub le perxidase activity fllws a methd mdified frm that f GASPAR, SERVLLE and DARMONT (974). Aliquts f 0, ml f this slutin (equivalent t mg material in the example given) were added t 8,0 m phsphate buffer (ph 6,),,0 ml ~phenyenediamine (% m/v) and,0 m hydrgen perxide (~, vlumes) and the absrbance read at 449 nm after exactly and 5 minutes n a Beckman Mdel 4 spectrphtmeter in em glass cells against a reference slutin cntaining buffer and rganic substrate nly. The difference between the tw absrbance values was used in calculatins. A calibratin curve was prepared fllwing the identical prcedure and using amunts f 0,0 t 0,50 ~g hrseradish perxidase (ref....7, BDH) as the standard enzyme. Results are expressed in terms f ~g HRP equivalent per g fresh weight f material, i.e. in p.p.m. by mass. Quantitative analysis f perxidase isenzymes by starch gel electrphresis Preparatin f the sample material is described in Sectin 3.5., but in this case the centrifugatin f the abstract was unnecessary. The technique used fllws the methd f BRAN (986). A gel was prepared by suspending 0 g f hydrlysed starch (Sigma S-450) in 00 ml gel buffer (as fr extractin buffer, abve; POULK, 957) and heated ver a flame until a viscus slutin resulted. This was de-aerated under vacuum, cast int a perspex frame (internal measurements x x 0,5 cm) and chilled t a firm cnsistency. The sample slutins were absrbed nt pieces f thick filter paper (5 x 5 mm) which were inserted int slits (ca. 5 per frame) cut acrss the gel cms frm the cathdic edge. The gel assembly was munted in a cnventinal hrizntal electrphresis tank cntaining electrde buffer (0,05 M NaOH, 0,30 M H BO; POULK, 957) and run at a starting current 3 3

149 35 f 5 ma until the buffer frnt, indicated by a brwn line in the gel, had advanced abut 60 mm in the andic directin (ca. 4 hurs). The gel assembly was chilled briefly and then sliced hrizntally by means f a length f surgical nyln under tensin. The half remaining in the frame was stained by flding with detecting reagent (5 ml water, 0,3 ml glacial acetic acid, 0,05 g benzidene, drp 00 vl. H 0 ; BRAN, 986) fr 5 minutes. Gels were then phtgraphed and/r careful sketches made t recrd the psitins f the varius andic (+ve) r cathdic (-ve) bands. The electrphretic mbility f each band was then calculated as a fractin f the distance travelled by the buffer frnt Results and discussin Ttal sluble perxidase activity The results are recrded tgether with the sluble prtein analyses in Table 3. Fr the in viv situatin, the perxidase levels are higher in leaves than in ther parts f the plant. n Encephal~~, the fast grwing speci es f mesi c ri g; n (e. g. E..tJtaMVetl.O~lL6,. nataiem~) have higher seedling leaf perx idase cncentratins than slwe~ grwing taxa with xeric mdificatins (e.g. E. ielunamt-il. E. gtle.tunciu:..<:.). t is nted that thse species with the higher leaf perxidase levels are thse which respnded mst rapidly in terms f callgenesis frm rt explants (Sectin.6.). There is als a clear indicatin that leaf perxidase activity falls as the plants get lder (e.g. E. natalem~, s. ~OPlL6) but a cmparisn f juvenile and mature leaf samp es fr'm the same specimen (E. natalem~) des nt shw thi s effect. There is n clear evidence that the enzyme cncentratins are influenced by the plant's gender (E. ttatalem-w, E..tebmbeM~ var. Piet Reti~). n the limited evaluatin f the leaf perxidase levels in the different genera, it seems that Cyc~, D~tt and Lepidz~a are higher in enzyme cncentratin than- Etlcephal~~ which is, in turn richer in perxidase that Statlg~a and Z~a. Relatively high levels f the enzyme are seen in sme mature seeds where

150 36 TABLE 3 : Ttal sluble perxidase activity and ttal sluble prtein in cycad material. Cycad species Sample material used (Number f samples if > ) Mean perxidase ljg g- fresh weight Mean prtein mg g- fresh "eight ljg perxidase per mg prtein E. aztensteinii. mature plant, mature leaf,3 0, 75 E. arenari us 3-yr ld sr.edl i ng, leaf,6 5 0,077 E. f erx 5-yr ld plant, mature leaf 34,3 75 0,96 E. ghezzinckii 3-y r a d seed ling, lea f 7,4 34 0,30 E. Zebmbensis mature male plant, mature leaf 4,3 00 0,43 fresh seed, inferti e, sarctesta 3,4 37,S 0,09 fresh seed, inferti e, megagametphyte 3,4 9 0,08 E. Zebmbensis mature male plant, mature leaf 39,0 5 0,750 VQ'. Piat Reti ef mature female plant,,mature leaf 50,0 0 0,495 E. Zehmannii -yr ld seedling, leaf, 48,8 0,50 E. ZngifZius 6-mnth ld seedling, upper half f primary rt 0,88 5,6 0,056 6-mnth ld seedl ing, lwer half f primary rt 0,48,9 0,040 3-yr ld seedling, leaf 30,3 90 0,337 cal us tissue ex culture frm pr i mary rt 8 5,8 4,96 E. natal ensi s -yr ld seedl ing, upper half f primary rt (),4 8,4,36 -yr ld seedling, lwer half f primary rt (3) -y r 0 d seed ling, lea f 3-yr ld seedl ing, leaf () 3, ,9 6,0 6,0 97 0,567 3,94 0,380 6-yr ld plant, emergent leaf () 30,S 8,,68 6-yr ld plant, petile (),8 4,4,68 E. princeps E. transvensus E. vizzsus E. wdii mature male plant, juveni e leaf mature male plant, mature leaf () mature female plant, mature leaf (3) mature ferti e seed, megagametphyte mature fertile seed, embry 3-yr ld seedl ing, leaf -yr ld seedl ing leaf -yr ld seedling, leaf mature male plant, mature leaf male cne, micrsprangia male cne, micrsprphyl s male cne, cen~ral axis 3,6 9,5,0 37,8 3,8 34,8 74 8,3 6,0 0,9 6,0 3, , , ,0 0,39 0, 0,6 0,440,7 0,300,64,9 0,4' 0,099 0,070 0,83

151 37 TABLE 3: Ttal sluble perxidase activity.. an d tta sluble prtein in cycad material. (Cntinued) Cycad species Sample material used (Number f samples if > ) Mean perxidase vg g- fresh weight Mean prtein mg g- fresh weight Vg perxidase per mg prtein S. el'ipus -yr ld seedl ing, uppe r ha f f primary rt (), 8, O,3c -yr ld seedl ing, lwe r ha f f primary rt () 0,66 7, -yr ld seedl ing, leaf () 5,8 5,5,0 0,09 mature plant, mature leaf () 0, 5 0,96 immature ferti e seed, sarctesta - 8,9 - immature fertile seed, megagametphyte (3) 0, 79 0,00 callus tissue ex culture frm megagametphyte () 60,9 4,4 4, 3 Cyaas revl"t a mature plant, mature leaf () , 4 mature seed, sarctesta 6, 38, 0,60 mature" seed, megagametphyte, 93 0,0 Cyaas thual'sii yung plant, leaf 94 0,847 Din edule mature plant, mature leaf ,30 Lepidzamia perff skyana 3-yr ld seedling, leaf 69 69,00 Zamia furf uraea yung plant, leaf () 6,6 6 0,49 the activity is lcated in the megagametphytic tissue rather than the embry (. nataie.tt6-w) r sarctesta (CYC.M Jte.v.luta. The enzyme may increase in cncentratin as a functin f maturity and fertilisatin as the immature (S. e.jtipuo) and infertile (E. ie.bmbe.tt6-w) megagametphytes have particularly lw levels. Rt perxidase levels are generally lw and there appears t be a gradient frm the upper t the lwer parts f the rts (E. nataie.tt6-w, E. ing~~ ~~u, S. e.jtipu). This may be assciated with the grwth respnses reprted previusly (Sectin.6.) where explants frm the upper ne- third f the primary rts shwed mre rapid callgenesis th~ " thse frm the lwer prtins. Particularly high perxidase cncentratins were fund in fast-grwing callus ~n v~. n the case f S. e.jtipu megagametphyte cultures, there is an apprximately SOO-fld increase while the rt explants f E. Zng~~u shw almst a 00-fld increase. This aspect f massive enzyme increase S examined in greater detail in Sectin 3.7.

152 38 Observatins n the ct'relatin between perxidase level and -ttl v-u:jt grwth are fund in the literature. Leaf segments f C-tchOJUwn -t~yb~ L. with different endgenus perxidase cncentratins shwed a crrelated variatin in grwth ptential and rgan-frming ability (VASSEUR and LEGRAND, 97). With callus tissue frm Ppui~ ~~nui-tde6 Michx. perxidase activity is clsely and psitively related t grwth rate; there is als evidence that exgenus auxin and cytkinin applicatins have different qualitative and quantitative effects n the perxidase enzyme system (WOLTER and GORDON, 975). The perxidase activity in stems f N-tctia~a tabacum L. increases basipetally (i.e. with increasing age f interndal segments) (THORPE, TRAN THANH VAN and GASPAR, 978). These wrkers als pint ut the relatin between perxidases and auxin metablism; changes in the enzyme pattern are manifestatins f the differentiatin derepressin and repressin mechanisms. Further evidence is becming available frm recent -t~ v~ studies n several plant species. Fr instance, the passage f stem tissue f Act.t~d.a cfu:.~el..6-w L. t ca us is accmpan i ed by an increase in ttal sluble perxidase (HRSCH and FORTUNE, 984). Studies f the isperxidase levels in gymnsperms nave been limited, but a recent paper by PATEL and BERLYN (983) details the cytchemical events which ccur in cultured embrynic explants f P-tn~ cultejt..i.. D. Dn. t was fund that perxidase was strngly lcalised in regins f grwth and differentiatin, especially in develping vascular strands. The nly reference t perxidase in cycads is an bservatin that there is increased enzyme activity during germinatin f seeds f Cyc~ ~c.t~~ (PENA, GRLLO and PEREZ, 983). Quantitative analysis f perxidase isenzymes n view f the usefulness f plant enzyme "fingerprints" t the taxnmist (SMTH, 976), a brief investigatin int this field was cnsidered wrthwhile. A preliminary survey using crude extracts frm a few cycad plants and well-knwn enzyme-detecting spt tests, failed t detect any significant quantities f alchl dehydrgenase, esterase, acid and alkaline phsphatase, leucine amin peptidase r malate dehydrgenase. The benzidine test hwever, readily shwed perxidase in all samples. A further test n a starch gel system shwed that andic perxidases were present in all cycad material

153 39 samples and that a cathdic respnse was very much less cmmn. Leaf extracts gave the best 'zymgrams ' and hence the electrphresis methd was emplyed t cmpare leaf perxidases frm different individuals and frm different taxa. The results are shwn in diagrammatic frm in Figures 0a and 0b. t is emphasized that many f the bands were diffuse and hence the exact psitin and electrphretic mbility were difficult t ascertain. Ntwithstanding this, it is clear that the perxidase enzyme patterns can prvide useful input fr the taxnmists. Plymrphism w~hi~ species appears t be absent. indeed several species give apparently identical patterns; this is nt surprising with clsely-related species E. ~atalen~ and E. iebmben~ but it is unexpected that E. trispinqsus gives the same pattern. The presence f cathdic bands in E. V~~U6, E. ~gya~u6 and E. c~~ is cnsistent with their phylgeny and it is remarkable that a strng cathdic band is als seen in Sta~g~a ~OpU6; the nly cmmn feature amngst these fur taxa being a subterranean habit. n the extic cycads examined, an apparently identical pattern is shared b,y the three Ma~zamia species and Lep~zamia hpei and it, ;s significant that the latter genus was previusly incrprated in the frmer. The variatin in the tw Zamia species examined is cnsistent with reprts n variatin in chrmsme number and karytype within the genus (NORSTOG, 980a, 98). t is interesting t nte that starch gel electrphresis has been used t examine plymrphism in Cyc~ ~umph i (LEGENGUTH, 984). Leaf material frm 48 plants was examined fr per~idase and esterase activity and the authr reprts pr reslutin and weak reprducibility f the perxidase pattern but it was nted that perxidase activity in the lder leaves is increased tgether with an increase in iszyme cmplexity. The esterase pattern revealed cnsiderable dissimilarity frm ne plant t anther but there was sme evidence f plymrphism in certain bands. The authr suggests that this may indicate sme genetic variability which may have evlutinary ptential. These preliminary tests shw the usefulness f the perxidase iszyme system in taxnmic wrk. The starch gel medium ffers a rapid and inexpensive analytical system 'and althugh the reslutin S ften limited, a trial run using a plyacrylamide gel system aid nt indicate that an alternative matrix culd i~prve reslutin.

154 40 r!. tw!jms. +s elf i! " _ - ' _.. _ ,, i i! :, '- i!ii!::iiiii!! -ve t 0,0 - _. J, +ve jl. j ; :!,..... ' - ' :!, : -! - - p, 0 = 0,4 0,6 0,.8 j.. 'r. _. A R C D E F G H J K L M.,. 0 Figure 0a: Diagrammatic representatin f zymgram! btained in starch gel electrphresis f leaf extracts frm varius species f EncephaZarts. A. E. ZngifZius, B. E. aztensteinii, C. E. natazencis, D. E. Zebmbensis, E. E. transvensus, F. E. vilzsus, G. E. ngyanus, J. E. friderici- H. E.caffer, guiliezmi, M. E. hrridus.. E. ferx, K. E. Zehmannii, L. E. trispinsus,

155 4 r -! i!! ;-ve i t i 0, ! - - -, _. - i, ; ;!,,! i,, ~ i ~ '+ve i ~ 4 ~', f--,, - ~ ll.l! i ~!!. ~ ;!.... Ki!a -... ~ ~ &iz:::: A B C D E F G H,. J ~..,.a... ;,0 Figure 0b: Diagrammatic representatin f zymgrams btained in starch gel electrphresis f leaf extracts frm varius cycads. A. EncephaLarts natalensis, B. Stangeria eripus, C. Maarzamia mrei, D. M. cnununis, E. M. mique Zii, F. Lepidzamia hpei, G. Bwenia spectabizis, H. Cycas nrmanbyana,. Zamia pumi La var. pumila, J. Z. furfurac~a.

156 4 3.7 Bichemical changes during callgenesis frm Sta~g~a ~ruq 3.7. Materials and methds Because f the ease and reprducibility with which callus grwth culd be btained frm megagametphyte cultures f Sta~g~a ~p~. (Sectin.S.), this explant material was selected fr a brief investigatin int the changes in fresh weight, dry weight, sluble prtein and perxidase activity, which ccur prir t and during callgenesis i~ v~. Transversely bisected megagmetphytes were aseptically transferred t preweighed culture vessels cntaining SH medium supplemented with 4,S x 0-6 M,4-0 and kinetin, 48 such cultures being prepared and reweighed t establish the initial fresh mass f each explant. All cultures were incubated in the dark at SC At intervals f 0, 7, 4,, 8, 3S, 4 and 49 days, sets f six cultures were remved and prcessed as fllws. The explants were remved, washed free frm residual medium and weighed t determine fresh weight at the time f harvest. A sample (Qa. 0,3g) frm each culture was ven dried t cnstant mass at 0SC t determine misture cntent and hence dry mass at the time f harvest. The fresh and dry mass increase frm the time f inculatin culd then be estimated. A further sample (,00 g) was grund in a mrtar with 9,0 mls f pre-cled (40C) extractin buffer (0,076 M Tris, O,OOS M citric acid, ph 8,6S, POULK, 9S7) and the resulting mixture centrifuged at SO 000 x g fr 30 minutes at 4 C. Aliquts ffm the clarified extract were taken fr ttal sluble prtein and ttal sluble perxidase determinatins as described in Sectins 3.S. and Results and discussin The data btained in this experiment are presented in Table 4 are shwn in graphical frm in Figures a - f. and t is clear that several significant physilgical changes ccur in the explant tissue prir t macrscpic evidence f callgenesis, the latter event being evident frm 4 days f the day f inculatin. There is an almst linear increase in fresh weight ver the first fur

157 43 TABLE 4 : Variatin in fresh weight, dry weight, ttal sluble prtein and perxidase activity f megagametphyte explants frm Stangeria eripus in cu tu re. PRMARY DATA Time Fresh weight Fresh weight Misture Ttal sl uble Perxidase (days) at start (g) at end (g) (g/oog prtein activity fr.esh (mg g- fresh (ljg g - fresh weight) weight) weight) 0,0 + 0,37 t,0 + 0,37 48,6.!. 3,3 83, + 7,3 8,9 +, ,96 + 0,48, + 0,47 54,.!. 5,9 4,.!. 4,0 33,8.!. 40, ,87 + 0,43,3 + 0,47 60,4.!. 5,0 63,6 + 9,4 5,3 +, - - -,95.!. 0,54,4.!. 0,68 57,4.!. 4,4 64,9 + 3,,7 + 4, ,96 + 0,33,5 + 0,40 60,4.!.,9 6,6 + 5,4 8,0 + 4, ;09 + 0,34,33 + 0,39 63,4.!.,9 65,9.!. 4,6,4 + 9,0 4,87.!. 0,5,8 + 0,9 6,3.!. 3,7 60, + 6,6 94, + 6, ,0 + 0,44,58 + 0,55 6,7.!. 3,8 58,4 +,3 77,5 + 33, DERVED DATA Time Adjusted fresh * Adjusted dry * Misture Ttal sluble Perxidase (days) weight end (g) weight at end (g/loog prtein activity (g) dry weight) (mg g-dry (ljg g - d ry weight) weight). 0 (,00 ini tial) (J,03 i ni tial) 95.! ,.!. 5,7-7,8.!. 0,08 0,99.!. 0,0.! !. 4 77,7.!. 99,9 4,8.!. 0, 0,90.!. 0,08 59.! ,9 + 6,5 - -,44.!. O,t,03.!. 0,07 37.! !. 0 55,4.!. 37,5 8, , !. 58.!. 3 9,8.!. 0,3 - -, 35,3.!. 0,4 0, , ! ,0.!. 5,5 4,45.!. 0, 0,94.!. 0,07 60.! ! ,59.!. 0,3 0,99.!. 0,05 63.! * Prprtinally adjusted t an initial explant weight f,00 g. t The range is indicated thrughut as + ne standard deviatin. -

158 44,8 Figure ta Z,6 FRESH WEGHT (9) \,4 \ \, \,8 Screw-capped bttles Cnical flasks \ ) TME (days), Figure tb DRY WEGHT (9), 0 0,8 O'~~----?-----r- ~ ~ ~ ~ TME (days) Figure a and b.: Variatin in fresh and dry weight f megagametphyte explants frm Stang~ ~pu in the dark n SH medium with 4,5 x 0-6 M,,4-0 and KN. Vertical bars represent +/- ne standard deviatin.

159 45 90 Figure c MOSTURE 30 (g/00g dry weighd TME (days) 70 Figure d 60 MOSTURE (g/00g fresh 50 wei ght ) TME (days) - Figure c and d : Variatin in misture cntent f megagametphyte explants frm Stang~ ~PU4 in the dark n SH medium with 4,5 x 0-6 M,4-0 and KN. Vertical bars represent +/- ne standard deviatin.

160 46 TOTAL SOLUBLE 50 PROTEN (mg g-l dry we ight) Figure e TME (days) 300 Figure '( PEROXDASE ACTVTY 50 (ll9 HRP 9- dry weight) TME (days) Figure e and f : Variatin in ttal sluble prtein and perxidase activity f megagametphyte explants frm Stang~ ~OPU6 in the dark n SH medium with 4,5 x 0-6 M,4-0 and KN. Vertical bars represent +/- ne standard deviatin.

161 47 weeks (Figure a) and this increase may be ascribed t an increase in mi sture cntent (Fi gure c). By cntrast, the dry we i ght shws an initial decline with a recvery later (Figure b). These effects are analagus t seed behaviur when misture is imbibed and fd reserves are utilised in the perid just befre germinatin. An interesting and entirely frtuitus bservatin is assciated with the chice f culture cntainer. Due t limitatins f glassware supply, tw types f cntainers had been used in the experiment. The learl i' cultures were cntained in 8 ml screw-capped Universa " bttles while the "l ater" explants were- held in 5 ml cnical flasks with cttn-w. bung clsures. Fi gure a shws that the fresh wei ght increase f explants in the screw-capped cntainers is cnsiderably mre advanced than thse in the cnical flasks. This effect is quite different t that previusly bserved in megagametphytes frm E~cephai~~ (Sectin.4.5) where use f Universal bttles inhibited callus grwth altgether. n the case f Sta~g~a, it appears that use f the Universal bttles may result in an initially favurable respnse, but this is nt sustained. There is abut a three-week time delay fr the expants in the cnical flasks t reach the same stage as thse in the screw-capped bttles. t is speculated that the restricted gaseus exchange in the screw-capped cntainers results in the headspace having cnstant 00% relative humidity while the cnical flasks underg slight desiccatin with a cncmitant slight but significant increase in water deficit in the medium. Other influences arising frm differences in the gaseus exchange rates, such as carbn dixide and ethylene accumulatin, and restrictin f xygen supply, may have an effect (Sectin.4.5). The ttal sluble prtein shws an initial sharp decline (Figure e), pssibly due t the utilisatin f readily-available prtein reserves. This fall is restred within tw weeks after which the sluble rtein stays mre-r-less cnstant. The perxidase activity is widelyvariable as shwn by the large standard deviatins (Figure f). Hwever, a significant increase in the enzyme level des ccur frm 8 t 4 days, the perid just befre visible evidence f callus frmatin. This cnfirms the earlier reprt (Sectin 3.6.) where bth haplid and diplid callus shw dramatic increases ver the initial perxidase levels. Again, the situatin is parallel t that f the

162 48 seed, where large increases in enzyme activity are a fundamental aspect f the germinatin prcess. 3.8 Serlgical investigatins 3.8. Materials and methds Fresh seed f E. la.talem.i..6, derived frm plants in the Kranskp area f Natal, were suplied t the Natal nstitute f mmunlgy where a quantity f the antiserum was prepared using standard techniques. These invlved preara~in f the crude seed extract and emulsificatin with Freund's cmplete adjuvant after which a rabbit was injected intramuscularly at fur sites at several intervals ver a six-week perid. At the end f this perid the antiserum was btained after - -- cltting f a bld sampie frm' a test bleed. Duble-diffusin serlgy An Ouchterlni duble-diffusin plate (0 cm x 0 cm) was prepared using 0,8% agarse in barbital buffer (0,05 M barbitne, ph 8,6). Extracts frm varius cycad seeds were prepared by grinding apprximately 0,5 g fresh megagametphytic material in abut ml barbital buffer and centrifuging the prduct at 9000 x g fr 5 minutes. The E. la.talem.i..6 antiserum was placed in the central wells and the test samples in the peripheral wells. The reactin was allwed t prceed fr at least 6 hurs at ambient temperature and the resulting plates examined ver a light bx t ascertain the nature f any precipitin reactins. mmun-electrphresis mmun-electrphresis plates (0 cm x 0 cm) were prepared using 0,8% agarse in barbital buffer (0,05 M barbitne, ph 8,6). Wells were cut int the matrix half-way alng the plate (relative t the cathde-ande axis). Test samples f varius cycad seeds were prepared as described abve, aliquts f the clarified suspensin being placed in the sample wells. The plates were electrphresed, using the barbital slutin as an electrde buffer, at a starting current f 5 ma and running fr 5 hurs.

163 49 Channels were then cut int the matrix parallel t the current directin, abut 5 mm frm the sample wells, and these trughs laded with antiserum prepared as described previusly. The nature and extent f the antiserum-antigen reactin was assessed by viewing ver a light bx after abut 6 hurs Results and discussin Duble-diffusin serlgy The results are shwn diagrammatically in Figure. Althugh nly a few taxa have been examined, it is clear that the respnses btained crrelate with the present taxnmy at the family level. The E. ~atate~~ (Kranskp) antiserum gives the expected respnse with a fresh extract frm the same batch f seed, cnfirming the validity f the methd. A similar intense single band was btained with ther samples f E. tla.tate~~ and indeed with all species f tncephatajd.6 tested. Other representatives frm the Zam;aceae (Ma~zamia, Lepidzamia, C~atzamia and Zamia) all shwed a similar band f lwer intensity. The respnse frm Sta~g~a (Stangeriaceae) was quite different; a dule faint bahding pattern being seen. N reactin was fund in any f the samples f CyCa.6 (Cycadaceae), again indicating significant difference frm the EtlcephataJd.6 prtein. Fllwing the initial experiment with extracts frm the cycad megagametphytes, a similar test was run using the same antiserum with extracts frm cycad leaflets in the peripheral wells. Thirteen species f E~cephataJd.6 and five extic cycads were tested in this manner. N reactin was seen in any f these samples. The inference drawn ;s that the leaflet prteins differ markedly frm thse in the seeds. Applicatin f the methdlgy is thus limited t cmparisns f antiserum and antigen derived frm similar rgans. mmun-electrphresis The results are shwn diagrammatically in Figure 3. While the duble-diffusin serlgical tests gave crrelatins with taxa at the fami iy level~ the immun-electrphresis patterns appear

164 50 GROUP A. E. natazensis. Kranskp area. B. E. natazensis. Msinga area. C. E. natazensis. B. R.. Durban. D. E. vinsus. E. Cape E. E. vinsus. Durban. F. E. ferx E. natazensis. (Kranskp) antiserum in central well GROUP G. E. natalensis. Kranskp area. H. E. pauidentatus.. E. lanatus J. E. trispinsus K. Stangeria eripus. Umzint area. L. Stangez'ia eripus. B. R.. Du rban. E. natazensis (Kranskp) antiserum in central well. GROUP 3 M. Marzamia hetermera N. Marzamia Zuida O. Marzamia mrei P. Marzamia pzatyrahis Q. Lepidzamia hpei R. Lepidzamia perffskyana E. natazensis (Kranskp) antiserum in central well. GROUP 4 S. Cyas aliza T. Cyas media U. Cyas revzuta V. Ceratzamia mexiana. Belize W. Zamia flcf'flcf'aea X. Zamia latifzia E. natazensis (Kranskp) antiserum in central well. FGURE : Duble-diffusin serlgical tests with cycad seed prteins shwing antiserum-antigen precipitatin reactins in samples with similar prteins.

165 5 i : + + FGURE 3: mmun-electrphresis f sme cycad seed prteins against E. natalensis antiserum. A. E. natalensis, B. E. ferx, C. E. ngyanus, D. E. pauaidentatus, E. E. trispinsus, F. E. vilzsus, G. Stangeria eripus, H. Maarzamia hetermera,. M. mrei, J. M. pzatyraahis, K. Lepidzamia hpei, L. L. perffskyana, M. Cyaas aazaiaza, N. Ceratzamia mexiaana,. Zamia jurfuraaea, P. Z. Zatiflia.

166 5 t be cnsistent at the genus level. n the genera tested, each gave a characteristic prfile althugh it was nt pssible t distinguish between Cyc~~ and C~atzamia. There was n separatin int discreet bands alng the directin f current travel as is usually bserved with higher plant prteins (SMTH, 976); thus it may be inferred that cycad prteins are smewhat less cmplex with greater hmgeneity within each genus. The results with the tw species f lamia are unusual in that the reacting prtein is largely cathdic; all ther genera shw andic migratin. Whilst, the serlgical results are useful in a cnfirmatin f grupings at the family and genus level, in the examples tested there was n evidence f individuality at the species level. This line f investigatin was nt therefre persued. 3.9 Cycad txins 3.9. Materials and methds The methd used was mdified frm that f MORETT, SABATO and SNSCALCO GGLANO (98). A,0 9 fresh weight sample f the material was grund in a mrtar with 9,0 ml f 50% aqueus ethanl and a prtin f the resulting suspensin centrifuged in an Eppendrf tube at 9000 x g fr 0 minutes. Fr the qualitative tests, 0~i aliquts f the clear extracts were applied t a 0 x 0 cm T.L.C. silica gel plate (Merck Art. 5554) which was develped in ne dimensin using n-butanlacetne-water (4:5:) as the slvent system. Samples f pure macrzamin kindly supplied by Dr J Cannn f the University f Western Australia and Dr. P. Casri f the University f Naples, were used fr reference purpses. After drying ff the residual slvent, the T.L.C. plates were sprayed with aniline-diphenylamine-acetne-phsphric acid regent ( ml: g:50 ml:8,5 ml) and heated at 80 C fr 5 minutes. Simple sugars and glycsides, including macrzamin, adpt a blue-grey clur n this treatment Results and discussin A representatin f the results frm the TLC plates is shwn in Figure 4. The methd is a cnvenient and rapid technique which

167 A B C D E F G H J K L M N 0 P Q R S T U rstds) MAC GLU SUC C) C) a.=- C) C- O t 0 C) C) C> 7~ 0 C> c~ C> C ) 0 0 <).68 C:J 0 c- 0 c <:::) D C> 0 0 C> ~. 44 <0000 CL) a (?]) 8 c) C) C!) C0 a c C' C) C) C) C) 00 0 C' (::) C:> C) c::::> C> C> C> C) C3 c::::;.. 7 FGURE 4: TLC analysis f cycad seed extracts. A. E. natazensis, Kranskp area, after 3 mnths' strage, B. E. natalensis, Msinga area, after 3 mnths' strage, C. E. natalensi s, B.R.., Durban, fresh megagametphyte, C. E. natalensis, B.R.., Durban, after 6 mnths' strage, E. E. vizlsus, Durban, fresh megagametphyte, F. E. villsus, Durban, after 6 mnths' strage, G. E. Zebmbensis, Westville, after 3 mnths' strage at OC, H. S. epipus, Umzint area, fresh megagametphyte,. S. epipus, Umzint area, after 6 mnths' strage, J. S. epipus, B.R.., Durban, fresh megagametphyte, K. S. epipus, B.R.., Durban, after 3 mnths' strage at OC, L. Macpzamia hetepmepa, after 6 mnths' strage, M. M. mpei, after 6 mnths' strage, N. M. pzatypachis, after 4 mnths' strage, O. Lepidzami a pepffskyana, after 6 mnths' strage, P. Cycas cazcicza, after 4 mnths' strage, Q. C. pevzuta, U.N. Pietermaritzburg, fresh sarctesta, R. C. pevluta, U.N. Pietermaritzburg, fresh megagametphyte, S. C. pevluta, U.N. Pietermaritzburg, after 3 mnths' strage, T. Zamia fupfupacea, after 4 mnths' strage, U. Z. latifzia, after 4 mnths' ~trage, MAC, authentic macrzamin, GLU, glucse, SUC, sucrse. Slvent system n-butanl-acetne-water, 4:5:. <.n w

168 54 detects macrzamin at levels greater than abut 0,% by fresh weight f material. The txin (R 0,5) was fund in nearly all samples F tested and did nt appear t be affected by shrt term strage at ambient r cld cnditins. On the basis f clur intensity, the stang~a seed cntains mre macrzamin than any f the Encephai~~ seed samples. This is cnsistent with the finding f MORETT, SABATO and SNSCALCO GGLANO (983) wh reprt Encephai ~~ seed as having,,09 -,86% macrzamin and Stang~a ~p~ as having 4,70%. Of the varius samples tested, nly the megagametphytes f Cgc~ ~evtuta failed t shw the presence f macrzamin, althugh the txin was present in the sarctesta f the same seeds. Either sucrse (R 0,34) r glucse (R F F 0,44) r bth sugars, culd be seen in the extracts and there appears t be sme degree f change in sugar distributin n strage. Several unidentified spts with RF values in the range 0,6-0,74 appeared n the chrmatgrams. t is pssible that these represent cycasin r varius necycasins. Cycasin, being a mnsaccharide glycside, wuld be expected t exhibit a higher RF value than macrzamin, a disaccharide glycside, but n standard material was btainable t establish this. An attempt t quantify the txin cncentratins by HPLC techniques, using a cnventinal C-8 reverse phase clumn, was unsuccessful. N respnse at all was btained with the standard macrzamin sample. This may have been because the instrument used recrded respnse by refractive index f the eluant; if the alternate ultra-vilet system had been available the respnse may have been detected. ndeed, this has been emplyed successfully in the simultaneus determinatin f cycasin and its degradatin prducts (YAG, TADERA and KOBAYASH, 980). 3.0 Analysis f leaf wax hydrcarbns NOTE: The wrk described in this sectin refers t results f a jint prject carried ut in assciatin with Prfessrs Antni and Maria Luiza Salatin f the nstitut de Biciencias, University f sa Paul, sa Paul, Brazil, ver the perid August 986 t April 988.

169 Materials and methds Fresh leaf material was cllected frm different habitat and garden grwn plants f as many Encephai~~ species as pssible, and frm Stang~a ~pu. Samples were taken frm mature unblemished leaves f adult plants where n dubt existed as t their existing taxnmic status. Vucher specimens f the leaf samples were depsited at the herbarium f the University f Durban-Westville. Only the median leaflets were used in the analysis. Sample~ were als cllected frm seedling plants and juvenile leaves fr cmparisn. The selected leaf prtins were ven-dried at 50 C fr apprximately 7 hurs. The epicuticular waxes were extracted by means f three successive immersins f 30 secnds in chlrfrm. The extracts were cmbined and the slvent evaprated. The crude residue was fractined in a clumn (30 x,5cm) f silica gel (Merck, mesh), the alkane fractin being eluted by 30ml f petrleum ether (biling range C). The purity f the alkane fractin was mnitred by TLC using silica gel 60 G plates (Merck) with flurescein indicatr and visualized under lng wave ultravilet light. The presence r absence f lefinic hydrcarbns was assessed by cmparing the abve plates with thse btained under identical,cnditins but using silica gel impregnated with O,lM silver nitrate. Analyses f the cnstituent nrmal and branched-chain alkane hmlgues were perfrmed n a CG-37 gas chrmatgraph (CG-nstruments Cientifics Ltda) which is similar in capability t the current Varian instrumentatin. The clumn f stainless steel (m x 3,mm internal diameter) was packed with % QV-0 n Gas Chrm Q. Nitrgen was used as' the carrier gas. njectr and detectr temperatures were set at 80 C and the clumn temperature ramped frm 60 t 80 C at 4 C min-. A flame inizer detectr system prvided input t a CG-300 prcessr-integratr (CG-nstruments Cientifics L~da). dentificatin f the alkane hmlgues was based n cmparisn f peak retentin times with thse f authentic samples f linear alkanes. Peaks with retentin times intermediate between tw successive linear hmlgues were ascribed t branched chain alkanes since there was n evidence f an lefins being present in the extracts. A cluster analysis and phylgenetic evaluatin f the data was perfrmed using the NTSYS numerical taxnmy analytical

170 56 system (Applied Bistatistics nc.). The SMNT rutine was used t cmpute taxnmic distances which were in turn analysed using an unweighted pair-grup methd in the SAHN prgramme. This resulted in the prductin f a matrix which culd be displayed in the frm f a phengram by use f the TREE rutine. Gdness f fit f the clustering analysis was measured by prcessing the tree matrix thrugh the COPH rutine t give a cphenetic value matrix which culd be cmpared with the riginal taxnmic distance matrix by use f the MXCOMP rutine. The manipulatins invlved in the varius aspects f these analyses are described in detail by ROHLF, KSHPAUGH and KRK (97) and SNEATH and SOKAl (973) Results and discussin The analytical data btained frm the varius samples are shwn in Tables 5, 6, 7 and 8 and Figures 5, 6, 7 and 8. n nearly all cases the cntributin f the n-alkane hmlgue far exceeds the ttal cntributin frm the crrespnding ismers. Thus, fr clarity in the graphical representatins, nly the n-ism~r figures are pltted. Similiarly, the discussin f results is cnveniently limited t a cnsideratin f the n-alkane prfiles. Seedling and mature leaves Table 5 shws a cmparisn f the alkane distributin in seedling and mature plant leaves f seven species f Enceph~~. The n-alkane prfile fr fur f these pairs, selected as representative, ;s given in Figure 5. With E. ngyanu, the rather irregular prfile fr the seedling leaves diff~rs markedly frm the "skewed-nrmal" pattern in the mature leaves. The reverse trend is seen in E. ianatu. n E. ~~~ and E. v~~u there is cnsiderable similarity between the juvenile and adult leaf patterns. There is therefre little verall cnsistency between the prfiles f seedling and mature leaves. This statement must be qualified by the cnstraint that the seedling and mature leaf samples tested, althugh frm the same species, are nt necessarily frm the same clne. Except in the case f E. ~e40~, the results btained in these tests are nt generally cnsistent with the situatin in Angisperms where yunger leaves usually bear a higher prprtin f shrt-chain hmlgues (FABOYA, OKOGUN and GODDARD, 980; BAKER and HUNT, 98;

171 57 TAULE 5 : A COMPAUSOH OF THE LEAF WAX ALKANE ljstr~uton N ~EEOLNG AND MATuHE i'''''~i'",.",l't"" PLANTS Alka... ~ hlllo lgue by c"rbn 4L(. t.lllut'r (nrlklll i soulers) Percentilge abunance f alkane hmlgues by species in seedl ing alia (ikiture pl.nt le_ves ::. /':l'v'& 5' S c7n c7i c8n cl8i c9n clsi c0n c0i cn c i cn cui cln c3i c4n c4i c5n c5i c6n c6i c7n c7 i c6n cdi c9n c9i clo" c30i c3n e3 i c3n e3i c33n c3li c34n c34i c35n c3~j c36n,36i b tr tr 0 tr tr 3 0 tr 9. 3 tr tr 3 4 tr tr tl' 4 tl' 7 tr 3 tr l 4 b 3 0 tr tr tr tr 0 tr tr tr 0 0 tr 0 tr tr ~ 3 tr j tr 6 8 tr tr tr 4 5 tr 7 tr tr 0 9 g tr tr tr tr lr 0 tr tr 8 3 l tr U tr tr 0 J l tr 0 tr tr U U tr tr tr 4 9 ZZ tr U 0 3 tr tr b tr 0 tr tr tr 0 4 tr tr tr tr tr tr 0 tr tr tr tr U tr 0 tr J U Ntes:. "n" represents the nrmal r unbranched hololgue. "i" represents the ttal SODerie cntributn. "5' represents seedlng leaves. "M" leaves frm mature plants. tne numbers being an average cmpsitin ver the nulliber f saalples inicated n parentnesls,

172 58 E. ngyanus, seedling, Ref E. ngyanus, mature, refs. 5, ~ E. Lanatus, seedling, ref. 37. Z5 E. Lanatus, mature, ref \ \ \ E. ferx, seedling, ref E. ferx, mature, refs. 4, E. villsus, seedling, ref. 34. (43) (33 ) ~ " E. villsus, mature, refs. 0,, ~ :;;000' (35) (3 ) FGURE 5 : LEAF WAX n-alkane PROFLES FOR SEEDLNG AND MATURE ENCEPHALARTOS PLANTS. The percentage cntributin f each alkane (y-axis) is pltted against the alkane carbn atm number (x-axis).

173 59 SALASOO, 983). Pure species and their hybrids Table 6 and Figure 6 shw the data relating t analyses frm tw sets f parent species and their hybrids, which ccur naturally in verlapping distributin areas. n bth cases the hydrcarbn prfile f the hybrid is intermediate in character between the patterns in the parents. This intermediacy is quantified in terms ( the fairly high crrelatin cefficients between the hybrid data and the arithmetic mean f results f the tw parents. The crrelatin is higher in the E. h~ X E. lehma~~ hybrid (0,96) than in the E. h~~ X E. l~g~li~ hybrid (0,8). n the latter crss, the prfile is much clser crrelated with the E. h~~ parent (0,90) than with the E. l~g~~ parent (0,57). t is tempting t evaluate the hybrid prfiles in terms f dminant and recessive gene cntributins frm the parents but the intermediacy in the hybrid results wuld seem t indicate a multiple-gene system is perative. There is insufficient evidence available at this stage t speculate further in this regard. Lcalities Table 7 and Figure 7 give the data frm the analyses f leaves f mature E~cep~~ natale~~ plants in different lcalities thrughut the distributin area fr the species. n the plants frm several lcalities (Ngeli Frest, Kranskp and Msinga) there is a bimdal alkane prfile. The lwer-carbn-hmlgue series is centred at abut C-0 while the higher alkanes are distributed nre-r-less nrmally abut the C-30 peak. This bimdal distributin is less bvius in the Estn, Klf grge, Jllivet and Vryheid samples while the Melmth sample is irregular. The cnclusin infered is thus that a certain amunt f variatin ccurs within at least this species. Since nly ne sample frm each lcality was tested, it is nt pssible t say whether the variatin is indeed lcality-specific. Furthermre, it is nt knwn whether similar infraspecific variatin ccurs in ther E~cephalaAt~ species. The data available d nt preclude the pssibility f plymrphism within E. ~atale~~.

174 60 r TAilLE 6 : A COMPARSON OF THE LEAf WAX ALKANE DSTRl uutlon N SOME t:"""ph.. ",'."" S~(CES AND THER HYBRDS Alk"e hllllio yue ~y carbn atou umber (rma / i SOllll!rs) c7n c 7i c80 c8i c90 c9i c00 c0i cn c i cun cui cjn c3 c40 c4i c5n c5i c6/ c6 c7n c7 i abn c~bi c% c.9i c30n c30i c3n c3 i c30 c3i c330 c33i c340 c34; c35n c35i cj6n c36i Crre i t i n cefficients Ntes : Percenla~~ abundance f a kane hm lgues ill lhe lil". i di CaLcd E'..Jl'l-iJ~" E. lolli/ijl,,," Arithmetic Ref. 47 kef. 9 mean Refs. 47,9 lr 6 lj ~ 4 tr lr tr 4 4 ti 7 4 tr tr tr 3 5 tr l tr tr 0,90 tr tr 3 tr tr 6 tr tr tr,57 E.'.!v/,, i,u.; k E. lullyi)"'l,,," Refs. 54,64 0,8 tr _ 8 J J ~ ~ tr ~ 4 tr 3 tr tr 0,86 t. }... JJlJ.uaJi i kef. 46 tr 6 ] ~ 8 4 ~ ~ tr tr tr 3 Ari thllll:l ic mean fiefs. 47, 46., "0" re~resents the n0'il r unbranched hml09ue, "i" represets the ttal i sollieri c cntribut in, The r~ference nuillber refers t the cllectin nuillber fr the ruaterial and crrespnds t herb'r' uld voucher specmen nullbers. K tr l' tr tr tr tr 0,95 }:.'.. rt.jl'/ id"u x E. Z4:iUtunnii. Ref. 5~ 0, l 3 l ~ G 3 D

175 6 E. hrridus, ref E. LngifLius, ref. 9., 0 5 0,,, E. hrridus x E. LngifLius, 5 Arithmetic mean, refs. 47 and 9. refs. 54,64 (natural hybrid) \ E. hrridus, ref E. Lehmannii, ref , E. hrridus x E. Lehmannii, 5 ref. 58 (natural hybrid) Arithmetic mean, refs. 47 and ? FGURE 6 : LEAF WAX n-alkane PROFLES FOR SOME EncephaLarts species and their hybrids. 5

176 6 TAlL 7 : A CuMPAklSON OF THE LEAF WAX ALKANE D STRBUTON Of l::ni.'t''tia!..lrw;; NM,lU',\'J;; FROM DfFERENT LOCAL ltl ES AH,ane holllo gue by e4lrbun atm nulliber (nun.. / su",rs) M.i e Ms inga Kef. Mili: ""lagata kef. 7 Estn Ref. 3 Male Me uluth Ref. 4 Mal~ Vryhe id Ref. 5 feu... le Kluuf k"f. 6 Feu... " Jull ; vet lid. U 7 Ngel; Rd. 60 F~llile Kr.nskp Ref, 6 Ar; tlllilet C Average (uf 9) C 7n e7i e8 ei el9n e9i e0 e0i en c i c0 ei e3n c3i c4 c4; c5n c5i efill e6 i cvn e7i c8n asi c9n c9; c30n c30i c3n c3 ; e3n eni cj3 c33; d4n c34 i c35n c35; e3(,n c36; Nutes : : : u tr is U tr tr 7 7 J tr tr tr tr J tr J tr tr 3 tr (j l 0 9 lr 3 4 D tr _ "n" represents the nna r unbranched hmlgue. "" r'epres~nts tile tta ; solller; c cntr; but jn The refer~nce number refers t the clectln number ir the lat~r;.l and crrespnds t herbariuul vucher spec en nuwbers. tr 4 tr tr tr tr tr tr 4 tr 4 9 ~ 8 3 lr tr tr tr tr 4 ~ 3 tr tr 3 6 tr tr tr tr 3 U tr tr lr tr

177 63 E. natalensis, Ngeli Frest, ref. 60. E. natalensis, female, 'Kranskp, 5 ref E. natalensis, male, Msinga, ref.. S 0 E. natalensis, Estn, ref S 7 9 E. natalensis, female, Klf Grge, ref E. natalensis, female, J i vet, ref , S E. natalensis, male, Melmth, ref E. natalensis, male, Vryheid, ref ,, 7 S 3 S S FGURE 7 LEAF WAX n-alkane PROFLES FOR E. natalensis PLANTS FROM DFFERENT LOCALTES. 5

178 64 jnter-specific variatin, Details f the leaf wax alkane analyses fr 4 E~cephal~~ taxa are given in Tables 8 and Figure 8. nspectin f the data shws that a wide range f variatins ccurs frm ne taxn t anther. The variatin between taxa appears t be wider than that within E. ~atale~~ (see previus paragraph) and hence there is a ptential usefulness f these data fr taxnmic evaluatin. The bimdal distributin between lwer and higher carbn hmlgues ccurs widely thrughut the genus, with the C-0 and C-30 fractins being apprximate centres fr the tw grups. n the higher hmlgue zne there is a bias twards dd-numbered C-atm hmlgues, this tendency exe!'llll ified best by E. -ts-vtx. and E. VillM L6 where the C-3 and C-33 alkanes are particularly prminent. An explanatin fr this tendency is fund in the bisynthesis f alkanes. Bisynthesis f alkanes The prductin f plant waxes is a cnsequence f a series f enzymicallycntrlled, and hence genetically-determined, bisynthetic steps. The metablic pathways fr alkane bisynthesis are fairly well knwn (KOLATTUKUDY, 976; HARWOOD and RUSSELL,984). A first stage invlves the elngatin f ubiquitus saturated fatty acid mlecules such as palmitic acid (CH (CH) COOH) and stearic acid (CH (CH) COOH) by successive C- unit additins, the prduct thus retaining an even-c number. The secnd stage is a decarbxylatin prcess which yields an dd-c alkane. This bisynthetic rute is clearly cnsistent with the typical ccurrence f high prprtins f C-9, C-3 and C-33 alkanes in epi~uticular waxes f a large number f higher plants. The prcess is cnsistent with the analytical results btained frm leaf wax samples frm E. -ts-vtx. and E. vill~ L6 as described in the previus paragraph. An alternative pathway which invlves a cndensatin f tw fatty acids fllwed by reductive decarbxylatin, the s-called "acceptrdnr" cndensatin mechanism (KOLATTUKUDY, 976), has since lst favur (HARWOOD and RUSSELL, 984). n sme higher plants, there are significant quantities f even-c alkanes.

179 65 TAillE 8 : A COHPAASON Of THE LEAf WAX ALKANE DSTRBUTON Of OlffERElll SPECES Of t""':'... lul"u~ Alkane! hllll,,'v9ye lly c.. rbn alo nyluber (nn... ' /,0lli:(5) Percentdge abyndance f., kane holllo' gues based n average CUl'US i t in per ta'(ll f. ('u (';/KJ~ ikj R~i. E. c.:uflid"':j Ref. E. t:uoi;f~mu"clitlii (waterth. r9) Ref. 5 E. e"(lclle lj'\liuii (Middleburg) Refs. 6,48 :.'. d.vll'i.ri("''':fj (sellsa LAYRANOS.nd equoe) Ref. 7 ::. J'~/V;S; /lefs. 4, ::. ['iel.: 'i,," yuilic:lflri. Refs, 43,44 ::. "h"hi,.ukii Refs. 4,4, 65,66 (7 c7i CHll c8i c9n c9i c0n cui cn c i cn c~i c3n c3i c~n c4 i c~n c5i c6 cl6i c7n c7i c8n c8i c90 c9i c300 c3ui c3n C) i cln cji c33/ c33i c34 c34 i c3~ cj5i c)6 c36i u a tr tr 3 tr tr tr tr Lr 6 tr U 9 U 3 l u tr tr J tr 4 tr tr tr 5 tr a 3 5 J tr tr a a 4 tr 6 3 tr S a tr 9 5 U U u a tr tr tr tr 4 tr 3 4 tl' ) tr ' tr 0 8 u 4 U U U U tr J J ) 5 tr tr u Lr J J tr tr

180 66 TAilLE 8 lcntinu~d)... P"rc.nLdge abulud"c. f ill ka"e hulgues bils~d n averilge ClVO> Hli VE:r L/,AOn Al~ane hmlgue E. l.:~otj,;u';l/..:i~ by cilrbn.'. ),..l J"~;./UlJ L'. lauut",., E. lut:ii'lvwj!'. t:mb,,,md ~;s t '... JU4l,," i- i::. lo/'!,{i!,vli la;;j i:, l... m~k.":'hj i.d VAr "Pi~t k~tiefii" HOU nuluber kef. 47 kef, 4 Ref, 3 Ref, 7 Refs, 8,9 kef. 46 Ref, 9 R~f, 50 - c7n 0 0 a c7i tr tr 0 0 chin 6 4 tr a 0 ~ 5 c8i tr cwn 3 ~O 4 3 & 4 cl~i 0 a 0 a c0n c0i cn c.i tr cn c i c c3i tr a a 0 a tr 0 c4n Z c4i a a a 0 0 a c5" c~ tl' tr tr c6n tr c6i tr 0 a 0 0 c7n 6 5 tr 4 c7i a tr tr 0 t,' cbn : 0 c8i 0 tr tr tr 0 cgn c9i a 0 0 a a 0 0 a c30" c30i 0 0 a a c3n c3 i a c tr 7 eni a 0 c c33i a a a a 0 a a a c34n tr a 4 tr 0 0 a c34 i 0 a a '0 a 0 a a c35n tr 0 0 a a a c35i 0 0 a a 0 0 a 0 c36n a c36i

181 67 TA&~E 8 (COll Hlued}... Percentage adund.nce f a kane h0ld 09ues bas~d n a vera9c CO~US i t i n ~er tdxn Alkalic 'OU ~u. ::. t.:. ' l.u ~ l ":.;i... flia.u..:jdi t:.,. ;J.J!lU"'t; E. pau.:'wtjlfl U t.uf t:. llj il.. ~~ ~.; t l'l.l. :';~....J'; la'; t'. "i. ll ~... wd uy CrDun t ". "",uji'i Rd. 5 c'.d" 7 Refs. 5,6 Ref. 49 R.f. 4~ R.f. U kd~. 0,\, Ref. 9 4 LOU nytocr (""" r 9) C C 7 i a 0 0 tr clbn 3 tr clsi tr lr 0 tr c9n tr 4 c9i 3 0 tr 3 tr 0 tr c0/l c0i tr c ,_ c 8 tr tr tr cn 3. a 4 l ci tr 0 0 eln Y 0 c3i 0 tr 0 0 0,4n e4i c c5i tr 0 0 e ctii tr tr c7n 5 ~ c7 i tr 0 0 cl csi 0 0 t r cy, C 6 0 c9i c c30i c c3i 0 0 a a a tr 0 tr e3n 4 3 J 7 e3i c:03n e33i c34 tr tr tr tr tr 0 e34i c35/ 0 tr c35i U U e a c3ui

182 69 E. paucidentatus, ref E. princeps, ref E. hrridus, ref E. munchii, ref E. friderici-gui~ie~mi, refs. 43, E. gheuinckii, refs. 4,4,65, E. ~anatus, ref E. ~ehmanii, ref L FGURE 8 (cntinued): LEAF WAX n-alkane PROFLES OF Encepha~arts SPECES.

183 70 E. ngyanus, refs. 5,6. 5 E. lngiflius, ref \, E. cupidus, ref E. eugene-maraisii, Waterberg frm, ref E: latifrns, ref E. transvensus, ref E. ferx, refs. 4, E. viusus, refs. 0,,. 0 (35) i w FGURE 8 (cntinued) : LEAF WAX n-alkane PROFLES OF Encephalarts SPECES.

184 7 An explanatin fr their ccurrence may lie in the lss f ne carbn unit by means f an 'a-xidatin prcess at sme undefined stage in the bisynthetic sequence (KOLATTUKUDY, 976; HARWOOD and RUSSELL, 984). This type f prcess may accunt fr the cmmn ccurrence f large prprtins f even-c alkanes in Encep~ species ther than E. ~~x and E. viii u. The bimdal distributin f alkanes which ccurs s cmmnly in Encephat~ is nt readily accunted fr in bisynthetic terms. t may be speculated that tw cmpeting bisynthetic rutes ccur, ne f which terminates at abut C-0 and the ther at C-30 alkane units. Alternatively, the pssibility f the same prcess perating n different precursrs cannt be eliminated. The alkane prductin prcesses in lwer plants are nt well knwn, but a general indicatin exists that algae, msses and ferns have smewhat greater cncentratins f lwer alkane hmlgues (C-5 t C-3), and that the rati f dd-c t even-c cmpunds is clser t unity, than in the Angisperms (STRANSKY, STREBL and HEROUT, 967). Thus, if the bimdal alkane prfile in Encephai~ is assciated with tw separate bisynthetic mechanisms, it may be that the rute leading t the lwer hmlgues is that which ccurs in lwer plants while the prductin f higher hmlgues fllws an Angisperm bisynthetic rute. Clearly there is an pprtunity fr useful research wrk t test this hypthesis but such a prject is beynd the scpe f the current thesis. Taxnmic implicatins The taxnmic usefulness f leaf wax analyses is well dcumented and has been fcussed in particular n alkane distributin patterns in Angisperms (EGLNTON, GONZALES, HAMLTON and RAPHAEL, 96; KOLATTUKUDY, 976; SMTH, 976). Develpment f mdern gas chrmatgraphy techniques has greatly facilitated data cllectin. Whilst there is usually cnsiderable crrelatin between hydrcarbn prfiles and taxnmic rank, the influend$ f factrs such as physilgical age f the material, seasnal effects, sil type and ther lcal envirnment~l influences must nt be discunted (STRANSKY, STREBL and HEROUT, 967). The variatin f alkane prfiles between juvenile and mature Encephai~ leaves, and the variatin with

185 7 ne species frm different lcalities, have already been shwn t be significant in the preceding paragraphs. _Despite the abve cnstraints, it is believed that the leaf-wax alkane distributin in E~cepnai~~ has cnsiderable taxnmic value. Figure 9 gives a dendrgram cnstructed n the basis f unweighted pair-grup analysis f taxnmic distances calculated frm the alkane distributins in 4 E~cephai~~ taxa (see Sectin 3.0.). nspectin f the dendrgram reveals many relatinships which are cnsistent with mrphlgical similarities, but there are a number f instances where the alkane-based relatinships differ markedly frm thse anticipated n mrphlgical grunds. n view f the prevalence f the C-3 and C-33 alkanes in E. ~~x and E. v~~u6 (as previusly discussed) the separatin f these frm the remaining taxa is nt surprising. These tw species are similar in sharing mesic habitats and having well-develped subterranean caudices. Hwever, they differ substantially in leaf and cne mrphlgy and are nt cnsidered t be clsely related. A grup f mesic plants f arbrescent habit, with relatively few adaptatins, cmprises E. ~aie~~, E. lebmbe~~, E. mani~e~~ and E. wdii. These ccur in juxtrapsitin in the dendrgram. On mrphlgical grunds, it may be anticipated that E. l~gi6~, E. paucident~ and E. ~a~ve~~u6 wuld be included but the alkane data indicate therwise. The presence f E. ~e~ and E. dlmiticu6 in this area wuld nt be expected n mrphlgical evidence. A secnd grup which is usually cnsidered t be related cmprises E. n~u6, E. lehma~nii and E. p~~cep~ (and E. ~pi~o~u6 n which data are nt available). These ccur in the dendrgram within the same brad area but tgether with several ther species nt cnsidered t be related. Within this zne f the dendrgram, the clse relatin f E. ~~c -g~elmi and E. gnelli~c~ is cnsistent! with sme degree f mrphlgical similarity cupled with an unusual degree f frst tlerance. The psitins f the varius taxa within the E. euge~e-m~ii cmplex

186 73 Relative taxnmic distances (SNEATH and SOKAl) Matrix crrelatin = 0,8 r- E. arena'ius E. dz,mitius E. natalensis, '- C[ r--- r E. lebmbensis E. lcbmbensis var "Piet Retiefii" E. manikensis E. wdii E. eugene-mamivi-i Mi dde burg frm E. pauidentatus E. princeps E. hrl'idus E. munhi-i E. friderii-gui lie Z,mi E. gheuinkii E. lanatus E. lehrzannii E. ngyanus E. Z,ngi f Hus - ~C E. upidus E. eugene-mal'aisii Waterberg frm E. latifl'ns E. transvensus E. fel'x E. ViUOSU8 FGURE 9 A dendrgram cnstructed n the basis f unweighted pair-grup analysis f taxnmic distances calculated frm the alkane leaf wax cmpsitin f 4 Enephalal'ts taxa.

187 74 deserves mentin. The Wlkberg frm (E. dimiticu sensu LAVRANOS and GOODE), the Middelburg frm and the Waterberg frm are evidently quite different in terms f alkane prfiles. This may lend supprt t a current prpsal that E. eugene-m~al il shuld be separated int fur distinct taxa (LAVRANOS AND GOODE, 988). n summary, the evidence cllected s far frm apprximately ne-half f the varius Enceph~~ taxa is seen t prvide much useful evidence t the taxnmists. This authr, in cnjunctin with Prfessr A and M.L. Salatin, will cntinue t cllect and prcess leaf wax alkane data until a fully-representative "library" f alkane prfiles is available. t is anticipated that an imprtant publicatin will arise frm this wrk and, in the lnger term, similar surveys may be cnducted n ther genera within the Cycadales. 3. Cnclusin The experimental wrk in this sectin has explred a number f facets f cycad phytchemistry with an emphasis n the Suth African species. Misture analyses f the seed shw that at the time f shedding, a relatively wet embry is surrunded by relatively dry megagametphytic tissue within the sclertesta. The latter is encased in turn within a relatively wet fleshy layer. On strage, bth the uter fleshy layer and the megagametphyte lse misture, this prcess being reversed by imbibitin prir t germinatin. This behaviur is cnsistent with the character f the brad grup described as recalcitrant seeds. Misture cntents f ther tissues shw a general tendency t decrease with age. The sluble prtein analyses f varius tissues vary widely. A tendency fr accumulatin f sluble prteins with physilgical age was evident. High levels are present in sme reprductive structures but nt in embrys r sarctestae. There is an indicatin f a prtein gradient frm the tp t the bttm f primary rts. Perxidase enzymes are higher in cycad lp.aves than ther parts f the plants. Higher perxidase activities ar~ shwn by leaves f plants frm mesic habitats than frm thse with xeric adaptatins, and the

188 75 levels decline with leaf age. Tissues with higher perxidase levels give a mre rapid callgenesis respnse ~» v~ than crrespnding tissue with lwer enzyme levels. A general indicatin emerges f a crrelatin between perxidase activity and grwth ptential, cnsistent with reprts in the literature. This is undubtedly part f the cmplex sequence f interactins between varius grwth regulating substances and specific enzymes. Further evidence f this crrelatin is prvided by the sharp increase in perxidase activity f Sta»g~ megagametphytes ~ v~ which ccurs just prir t visible signs f callus frmatin. n general, it is clear that rgan cmpsitin varies widely. Whilst this variatin is nt unexpected, it is significant in terms f explaining the respnses f different rgans in culture. Mre cmprehensive analyses may lead t a mre ratinal basis n which material culd be selected fr ~» v~ experiments. Data n the distributin f endgenus plant grwth regulatrs wuld be especially useful in t~is respect. An incidental benefit arising frm the phytchemical wrk is seen in its value in taxnmic applicatins. n this respect, the authr's experimental wrk shws sme parameters t be mre useful than thers. The thin layer chrmatgraphy f cycad txin,s, the seed prtein serlgy and immunelectrphresis data reinfrce the present separatins at family and genus levels but have little value at the species level. Perxidase electrphresis prvides a measure f separatin between species but techniques suffer frm limitatins in reslutin and reprducibility. Of the varius parameters explred, the analyses f cycad leaf wax hydrcarbns prvided the mst ptentially-useful data fr taxnmic evaluatin. At least within the genus E»eephatant~, each species prvides a unique hydrcarbn prfile. t is anticipated that use f these techniques, in cnjunctin with numerical taxnmy cmputer prgrammes, culd cntribute much t reslutin f existing taxnmic prblems and culd in additin allw the cnstructin f a phylgenetic sequence fr the rder.

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218 05 WEBB, K.J. and STREET, H.G Picea. Acta Hrticulturae 78: Mrphgenesis in vitr f Pinus and WHTE, P.R A handbk f plant tissue culture. J. Cattell Press, Lancaster, Pennsylvania. WHTE, R.P The cultivatin f plant and animal cells. Secnd editin. Rnald Press, New Yrk. WHTNG, M.G Txicity f cycads. Ecnmic Btany 7: WELAND, G.R American fssil cycads. Carnegie nstitutin, Washingtn. WNTON, L.L. and VERHAGEN, S.A Shts frm Duglas fir cultures. Canadian Jurnal f Btany 55: WOLTER, K.E. and GORDON, J.C Perxidases as indicatrs f grwth and differentiatin in aspen callus cultures. Physilgia Plantarum 33: 9-3. WORSDELL, W.C The structure and rigin f the Cycadaceae. AnnaZs f Btany 0: YAG, F., TADERA, K. and KOBAYASH, A Simultaneus determinatin f cycasin, methylazxymethanl and frmaldehyde by high perfrmance liquid chrmatgraphy. Agricultural and Bilgical Chemistry 44:

219 APPENDCES 06

220 07 ON THE EVOLUTON OF CYCADS by Ry Osbrne vvha T really did happen "in the beginning" f life n eanh is a puzzle which is likely t remain unslved, at least in ur lifetimes. Careful examinatin and re-examinatin f all the e\; dence by a great many trained minds has resulted in a stry f what might have happened. Over the last century this stry has been prgressively mdified and becmes steadily mre cnvincing. appearance. But these early plants all re~ned ne trait frm their marine ancestrs, an acnvely ~;mming male sperm cell and the cnstraint that water is essential in the reprductive prcess. Later, with the advent f the seed-bearing plants, the mtile spermatzid became redundant and (except fr a few strange anachrnism.s li~e Ginkg bilba) water was n lnger essennal reprductin. " 7 FG. : Series f female sprphylls, shwing reductin frm its leafy cnditin t the reduced sprphyll f the mst cmpact ne:. Cyeas revlula. C)'cas circinalis 3. C)'cas media 4. Din edue 5. Din spinulsum '6. Macrwmia miguelii 7. Zamia flndal.(j (Chamberlain, 99). The first plants abundant n this planet were undubtedly fairly simple algae which grew in the ceans up t three billin years ag. Their phtsynthetic ability resulted in the prductin f xygen which slwly built up t %, 5% and 0% in the earth's atmsphere. This increase in xygen had tw far-reaching cnsequences: the ptential fr life utside the marine envirnment and the establishment f the prtective zne layer high up in the atmsphere which culd screen ut lethal ultra-vilet radiatin and allw rganisms n land t sun;\ e. The stage was thus set fr the first f the land plants, the liverwrts, msses amd primitive ferns, t make their Excelsa N., 986. Smewhere in the cmplex evlutinary pattern between the great grups f 'ferns' and 'seedplants' came the cycads, which d have mtile sperm cells like their primitive ancestrs but bear seeds similar t thse f the mst advanced flwering plants. Dr Knut Nrstg f the Fairchild Trpical Garden draws an analgy between the evlutinary sequence in plants and animals; in his cmparisn the cycads are the 'reptiles' f the plant wrld since it is the reptiles that span the great gap between the amphibian restricted t its watel)' habitat and the birds and mammals with 9

221 08 internal fenilisaun. Nrstg and his clleagues are presently carrying ut detailed electrn micrscpy studies n the sperm cells f msses, ferns and cycads, and believe that the fundeme~tal differencs and similarities culd shed new hght n plant.evlutin. Within the cycads ch~cters rang~ frm the primitive t the advanced. Take fr ms~ce the way in ~'hich the leaflets f Cycas uncol - very much like the circinate character f the fern frnds. n Cycas t, the very lsely-rganised n explaini~g this apparent paradx sme f the mst useful evidence cmes frm the fssil recrds. Figure shws a 60-millin year ld cycad leaf called Zamites, a name implying prbable relatinship v.;th the present Zamia genus. Since the age f rd. strata are fairly accurately. assessed, the age f any fssil specimen trapped within may be estimated. But nt all fssils are perfea specimens; ften they are prly preserved and cnsist f incmplete fragments rather than whle plants. The laner difficulty has frequently resulted in fssil leaves, stems and ther parts f FG. : a 60-millin year ld fssilized frnd named Zamites Jencnis fund in France. (Case, 98). cllectin f mdified seed-bearing leaves (see Fig. ) is clearly earlier in evlutinary time than the cmpact cne we knw in Eruephalarts. Yet the prminent midrib in the Cycas leaflet is cnsidered advanced. Wind-pllinatin is cnsidered primitive and insect r bird-pllinatin advanced; bviusly animal-pllinated plants cannt pre-date the animals themselves! But the exp:.ns cannt set'm t agree JUSt hw cycads are ~llma.ted s that des nt help much. CnslderatlOn f. features like the anatmy f the stem and the structure f leaf stmata all add mre evidence. The verall cnclusin is that cycads usually have a mixture f sme relativdyad~ced and s?me relatively-primitive charactenscs - the Stuatin is nt as simple as was first thught. 0 the same plant being given different names at first. But painstaking wrk by many dedicated palaebtanists has allwed the theretical recnstructin f the plants and frests f many millins f years ag. Figure 3 shws sme examples f these recnstructed speci~s. Well-preserved cycad fssils have been fund, ften abundantly, in Meszic rcks frm Siberia, Manchuria, Oregn, Alaska, Greenland, Sweden, England and Central Eurpe, ndia, Australia and Suth Africa. The fssil recrd frm this part f the wrld shares the same prblems f incmpleteness and fragmentatin as d mst ther areas. The seed-fern (pteridsperm) fliage, which preceded the cycads, is quite well rep-. resented by the s-called Dicridium flra, Excelsa N., 986.

222 09 examples f which have been fund frm the upper Umkmaas \'alley and 'Little Sv.;tzerland' in Natal, RuX\'jJ)e in the O.F.S. and Drdrecht in the Eastern Cape. Later and mre typical cycad fliage called Zamitl'S and Dict)'OUlmites cmes frm the gelgical 'Uitenhage' series, and gd examples are displayed at the Pn Elizabeth and Kingwilliamstwn museums and the Bernard Price nstitute fr Palaentlgical vegetatin described as the Devnian Flra was al!!l0st entirely destryed hy a majr glaciatin in the Suthern Hemisphere at the end f the Palaezic era, abut 50 millin years ag. But the cld was fllwed by a lng perid f warm and balmy years, the Meszic era. Mean temperatures were abut 0" C higher than at present and the climate was mre r less the same wrldv.;de. t was at this time that the cycads FG 3: Hypthetical recnstructins f early cycads frm fssil recrds.. Bjlll 'ia simplex (Flrin, 933);. C)'cadeidea (Delevrvas 97)' 9 la/ '. d' ( s h. 93'.".. '.. 'J" ~. n wmsma s('war wna 9a7~~' ),4. l/zamsma ggas (Williamsn, 870); 5. LeptC)'caJ gracilis (Delevryas & Hpe, Research at Wits University. The tireless efrns f wrkers like Prfessr Aiex du Tit, Dr Edna Plumstead and Dr Heidi Andersn have dne much t sn ut the cmplexities f the early cycad flra f Suthern Africa. Anther fact?!' w.hich may 'shed sme ffght n cycad evluun S the earth's climate. Fr instance, it is nw knwn that the lycpd type Excelsa N., 986. rse t abundance in the wrld's vegetatin;just as in the animal wrld the dinsaurs were then at zenith. But the Meszic cycads were nt the same as ur present cycads, an imprtant and ften verlked pint. ndeed there were tw brad types, the cycadaleans (ancestrs f ur presen~day c\'cads) and the ycadeidaleans (smemes caed the Bennettitales) which later

223 0 became extinct. These tw grups differed s much in their reprductive habits, and in ther ways, that it is thught that bth grups evlved independent(} frm a much earlier ancestr in the seed-ferns. A final factr in the cycad equatin is that f the gegraphy f the wrld f the late Palaezic and Meszic eras. This was the time when the suthern part f Africa, Suth America, ndia, the gradie~t t the cast - the great escarpment tk its present frm and the central karr became the arid basin we knw nw. Thrughut this time the cycads were changing _ the pre!>ent wrld distributin prvides an excellent example f hw a grup f plants evlved frm presumably ne cmmn ancestr and, as a result f gegraphical is~atin and cnsequent climatic and ther dfferences, FG. 4: Prpsed cntinental re-gruping fr Gndwanaland. (Trustwell, 970). Antarctica and Australia were all jined t frm the massive supercntinent f Gndwanaland (see Figure 4). At the time when the cycads had reached their zenith, great cracks appeared in the land and thusands f tns f basalts welled up and spread ver the surface. The Suth Atlantic cean gradually widened, starting by a separatin f the Falkland tip frm the Agulhas escarpment fajrly late in the Meszic era. On the eastern side, the Mascarene Plateau reached acrss Tanzania-Kenya thrugh Madagascar t ndia - a giant dinsaur trajl which later became disrupted; nly 65 millin years ag ndia separated and rafted up t cllide with Asian landmass. T the suth, Antarctica frmed a bridge between Suth America and Australia until abut 50 millin years ag. As recently as 5 millin years ag the African cntinent had assumed its prescnt psitin, but majr uplifts were still t fllw. These rajsed the interir plateaux by mre than 800m and increased underwent a whle series f very' marginal changes which cllectively gave rise t different genera: Ena'f'lwlarts and Stangm'a in Africa, Cycas in the ndian cean slands, Macrzamia, Bwenia and Lepidz.amia in Australi.a, and Zamia, Ceralz.amia and Mirrcycas in the New Wrld. Futhermre, u'ithin each f these genera, there was the pssibility f funher speciatin under the influence flcal envirnmental cnditins. n Encephalarts fr example, we can pustulate a fairly clse relatinship bet\\ eene. altrnsteinii, E. natalmsis, E. wdii (?), E. lebmbflsis, E. manikensis and E. gratl/s. E. princeps is thught by Dr Dyer t be the first (hence the natne) in a grup including E. lehmannil~ E. tn'spinsus and E. hrridus. Clearly related are E. villsus and E, wnbduz.iensis, E. ghellinckii and E. c),wdiflius: E. amwrius and E. fcrx; E. cffer and E. 7lg.ra7ls. And s n. t is t be cnsidered smething f a miracle that the cycads survived thrugh s many atld such EXldsa N. J, J 986.

224 extreme climatic and gegraphical changes. Thse that sun'ived did s by yirtue f their - adaptability - the ability t 'change thrugh genetic prcesses, the eliminatin f weaker plants in cmpetitin with the strnger, the 'sutvival f the fittest', Thus the cycads are nt really sme sn f hangers-n frm the cal ages, but, as Dr James Eckenwalder f the University f Trnt puts it,,, 'a vigrus and successful mdem grup f plants, still evlving and capable f respnding t changing envirnmental cnditins: References \QS3. C. A. Arnld (~). Origin and relatinships f the cycads. Phl'lmrph%g:..- 3 pp. 5-{i5.. D.. Axelrd and P. H. Raven (978 ). Late cretaceus and tertiary vegetatinal histry f Africa. Chapter 5 in 'Bigegraphy and eclgv f Suthern Africa'. Ed. M. J A. Werger, Pub. Dr. Junk. S. G. R. Case (98). A piarial guide t fssils. Van Nstrand-Reinhld. 4. C. J Chamcrlain (99). The Li\;ng Cycads. Reprimf'd 965 h\' Hafner Puhlishing C. 5. T. Dl'k~."as (975). M f"'0zir c\'radph\,l'~. Gndwana G ('l~' - papers pr{'s('n.('d a,,he Srd Gndwana. S)mpmium, CanC'lTa. A us,ralia Ed. K. S. W. Campbell, Pub. Australian Natinal Uni\'er~i!Y Press. 6. J E. Eckmwalder (980). Cycads - the prime f their lives. TG Bulletin, JanuaJ)' L A. Frakf's (979). Climates thrugh gf'lgic time. Elsevier Publishers. 8. T. M. Harris (96). The Fssil cycads. Paltuanllgy 4 pp K. Nrstg (980). Reptiles f the plant wrld - cycads. TG Bulletin, January 980, 0. J F. Truswell (970). An intrductin t the histrical gelgy f Suthern Africa. PurnelL. W. C. Wrsddl (906). The SlTUctUT(' and rigin f the C)'cadaceae. Annals f Blam- 0 pp Acknwledgements The authr is indebted t Prfessr Esme Hennessy (Universit), f Durban-Westville) and Dr R. J RaYner (Bernard Price nstitute fr Palaentl~ca Research, Universit), f the Witwatersrand) fr their kind assistance in the preparatin f this feature. Exrr/.G N.,

225 CYCAD RESEARCH N THE 80'S ========rl==================~ BY ROY OSBORNE Althugh "this newsletter is nt set ut t be an in-depth scientific jurnal, it is clear that many f ur readers are deeply interested in current technical prjects invlving cycads and thus it is apprpriate t highlight sme f the cycad-related research wrk which has been carried ut ver the past few years. This article is nt intended t be a cmprehensive review f the literature, indeed nly abut ne-half f the ttal number f 'cycad' publicatins is mentined. T avid making this t invlved, have nt fr instance cvered the current wrk n palaebtany such as that f Delevryas (98). have als nt included specific cmments n the many mre ppular-style articles such as thse featured in the magazines f the fur cy c~d scieties, nr the several excellent cycad articles published in Fa irchild Trpical Garden Bulletins. ~e vertheless, this article des shw smething f the great deal f scientific wrk currently in prgress. TAXONOMY Ah0 SYSTEMATCS Dr. Dennis Wm. Stevensn (98,985) f Clumbia University in New Yrk has prpsed significant classificatin changes within the Cycadales n the basis r knwn and suspected relatinships in the rder. n his current hypthesis, the rder is divided int tw sub-rders, the Stangerineae (families Stangeriaceae and Bweniaceae) and the Cycadineae (families Cycadaceae and Zamiaceae). The family Zamiaceae is split int sub-families Diideae (Din ) and Zamiideae with tribes ~amieae (Ceratzamia~ Micrcycas~ ~ami a ) and Encephalarteae "Encer: hazarts~ Kpidzamia~ lacrzamia). The rather cnfused taxnmy f the ;enus Zamia has been reviewed by fames Eckenwalder (980) wh :nslidated 35 taxa frm the West ndies int the single species :amia pwrri Z Cl ~ a prpsal l.'hich has En.c..ephaicvr.t.6 N.6, June 986 met with sme dissentin and may becme mdified. New species in the genus are Z. fairchizdiana and Z. pseudmnticza (Gmez, 98), Z. inermis (Vvides & Rees, 983a) and Z. spzendens (Schutzman, 984). Schutzman and Vvides (985) have cllabrated ~ a systematic study f the brad cncepts f Z. Zddigesii and Z. furfuracea. An impressive amunt f taxnmic research has been carried ut by the talian team headed by Prf. Pal De Luca at the University f Naples, with assciates Sergi Sabat, Ald Hretti and Mari Vazquez-Trres and thers. The effrts f abut 5 years field wrk in Mexic have resulted in SlX new Din species being described: D. cazifani (979), D. caputi (980a), D. rzedwsk7:i (l980b), D. merzae (98a), D. hzmgrenii (98lb) and D. tmasezzii in tw varietal frms (984). Tw varieties f D. eduze~ var. eduze and var. angustifzium are nw recgnized (98). The talian schl was respnsible fr changing the name frm 'Din' back t the riginal Di n (98), a mve ppsed by Andrew Vvides and Nancy Mren (983) but resubstantiated by De Luca, Sabat and Stevensn (984). The wrk n Din has very recently been summarised by f,abat and De Luca (985) in a mst infrmative paper n distributin, eclgy and mrphlgy f the genus in which three natural grups are recgnized and which includes a key t the genus. Fur new species f Mexican Cerat zamia have been described: C. hizdae (Vvides and Rees, 980), C. kues t eriana~ clsely allied t C. zal'ag zae~ re-discvered by Ald Mretti and clleagues (98), C. nrstgii (Stevensn, 98) and C. micrstrbiza (Vvides and Rees, 983b) althugh the rank f the latter species is under review. Future reclassificatin f -several taxa, presently varieties f C. me xican a ~

226 3 is irmninent. A cmprehensive vrk n the distri~ butin (vith maps) and eclgy f the New Wrld cycads has been published by Baduzzi, De Luca and Sabat (98) and includes a discussin n the rigin f these cycads n the basis f fssil recrds and cntinental drift. A pertinent bservatin fr grwers is that mst Din species nrmally favur dry cnditins, Zamia is cnsistently heat-lving while Ceratzamia is restricted t areas vhere misture is high. Whilst there has thus been substantial vrk n the Nev Wrld cycads, little has been published n the African r Australasian species. Tw nev Cycas species are added: C. panzhihuaensis (Cheng et al, 980) and C. guizhuensis (Lan & Zu, 983), bth frm China. The relatively nev science f karytyping, characterisatin f the individual chrmsmes and chrmsme pairs, has prved t have useful taxnmic significance in cycad systematics. Dr. Knut Nrstg f Fairchild Trpical Garden has been the leader in this area f research and has sh~~ that the situatin is nt as simple as ~as first thught. Dr. Nrstg (980, 98) investigated the chrmsme numbers f 4 species f Zamia and fund cnsiderable variatin. n Zamia chigua, cnsidered t be ne f the primitive members f the genus, the chrmsme number varies frm t 6. Less variatin is bserved with the chrmsme numbers f \.Jest ndian Zarrr~as and Z. pseudparasitica which are thught t be mre advanced. Andrew Vvides (983) is als active in the field f Zamia karytyping vhile Xretti and Sabat (984) make reference t chrmsme evlutin in presenting evidence fr the advanced status f Z. paucijuga. Jhn Hendricks (98) discusses aspects f chrmsme evlutin in the brader cncept vithin the rder as a whle. Ald Mretti (98) has carried ut a chrmsme study f several Australian cycads using a flurescencestaining technique ~hich shwed clear differences betveen Macrzamia n ne hand and Bwenia and Lepidzamia n the ther. This bservatin supprts the taxnmic separatin f Lepidzamia as a genus distinct frm Macrzamia with vhich it had previusly been included. Additinal evidence in this cnnectin is given later in this text. Hveve~ Hretti als fund that Macrzamia cmmunis and M. pauli-guilielmi subsp. pauzi-guiziezmi are karytypicaly similar despite their present classificatin int the Hacrzamia and Parazamia sectins f the genus respectively, Even mre detailed in the micrscpic sense is the painstaking mapping f the infrmatin n the genes themselves. Buran Kurdi-Haidar and c-wrkers (983) frm the American University f Beirut have reprted n the DNA sequencing n the genme f Cycas revzuta. Wrk in the cycad taxnmy cntinues and it i~ hped that sme f the lse ends in the Cycas and Zamia genera vi be tied up sn. Sme lng-knwn species like Encepr~Zarts 'archeri' frm Vi in Kenya are yet t be prperly written up and several 'nev species like the EncephaZarts 'msinga' frm Nrthern Natal/Kwazulu will be described in frthcming literature. POLLEN GENESS AND DSPERSAL Because f their unique evlutinary psitin, the cycads cntinue t attract interest in develpmental studies and this is especially true with respect t pllen grain ntgeny. Pal de Luca, Vincenz la Valva and Sergi Sabat (980) have investigated the prcess f sperm frmatin in Ceratzamia mexicana and Cycas revluta, vhile the French vrker, Jean-Claude Audran (98), similarly reprted details f pllen grain develpment in Ceratzamia nexicana. Osama Terasaka (98) frm Tky has reprted his results n the develpment f the nucleus inside pllen grainsf varius gymnsperms including Cycas revluta and Bwenia serrulata,while Stuchlick and Mncada (983) have vritten up the pllen mrphlgy f Cuban gymnsperms, including Zamia and Micrcycas. Michael Zavada (983) frm ndiana University has published a reprt n the pllen vall develpment f Zamia fll'idana. A current reprt by Dehgan and Dehgan (985) n the pllen mrphlgy f

227 4 cycads prvides valuable insights int taxnmic relatinships, and supprts current re-classificatin prpsals within the rder. Karl Niklas has been invlved in a jint prject with Knut Nrstg (984) in which they make detailed measurements f pllen transprt in aerdynamic terms frm the surce t the female cne surface. The implicatins f their findings in the reprductin f Cycas, Din and Zamia are discussed. Little wrk has yet been dne n the insect vectr aspect in cycad pllinatin, ne publicatin f relevance being a descriptin by Gary Breckn and V.N. Ortiz (983) n the pllinatin f Zamia pumila thrugh the agency f fungus gnats. There is a real need fr mre studies n the relative imprtance f wind and insect factrs in pllinatin f cycad species in habi tat. LEAF MORPHOLOGY, A."U.TOHY fa PHYSOLOGY Raijnath, Naid and Ramcharuy (980) frm the University f Durban-Westville in Natal have used electrn micrscpic techniques t examine in detail the leaf surfaces f several cycad species, while Keleman, Rbbertse and Eicker (98) frm the University f Pretria have prduced an extensive paper n the anatmy f leaflets f Suth African EncephaZaY'ts species, tgether with an identificatin key based n leaf anatmy. Leaf surface mrphlgy has als been investigated by Prfe~sr Bijan Dehgan and Bart Schutzman (984) f the University f Flrida. Karatela and Gill (984) frm Bendel State University in Nigeria have similarly reprted n the leaf surface character f their lcal species, EncepJ-zaZaY'ts bay'tel'i. The detailed examinatin f certain structures within cycad cells has been carried ut by Dr. David ~ebb (98a) f Queen's University in Canada, "Th reprted n the differences in plastid structures between light-gr~~ and dark-grwn seedlings f Zamia jy'idana. The structure f plastids frm leaves f MacY'zarrr:-a m:jol~ei received the attentin f Bnatti and Sabat (984). nvestigatins int the structure f the vule include the reprt n strage cells in the vule f Cycas revluta. by Rene Rhr (980), while Japanese wrkers, Hiraka, Wada and Takada (98) hav~ fund evidence.in the same species f interactin between the nucleus and cytplasm by means f an intranuclear canal system. Dennis Stevensn, whse taxnmic wrk was referred t earlier, has als been cncerned with grwth and develpment studies f cycads, demnstrating the radial grwth pattern in 8 genera (980a) and reprting n leaf bud and subsequent leaf grwth prcesses (98); n the basis f the latter findings, the recmmendatin was made t separate ut the Bweniaceae as a family apart frm the Zamiaceae, a prpsal which is supprted by evidence frm chemical cnstituents (see later). Little wrk has been published n cyc.d physil~gy, ne study f interest being the investigatins int the phtchemical prperties f Cycas circinazis and C. beddmei by Prabhakar and Ra (98, 984). SEED K>Rl'BOLOCY, DSrEiSAL AND GERMNATON Prfessr Bijan Dehgan and his cwrkers in Flrida have been very active in research int cycad seed mrphlgy and its implicatins in dispersal and evlutin. n 983 Dehgan and Yuen shwed the buyancy in sea-water f seeds f Cycas rumphii and C. th~rsi~ a prperty arising frm an internal spngy layer within the seed. Because f their fltatin it is prbable that these seeds can travel frm ne island t anther in ndian Ocean currents. Anther interesting tpic cvered by Dehgan and Jhnsn (983) has been the effect f sulphuric acid fllwed by gibberellic acid, n seed germinatin. \--Then Zamia flridana seeds,,,ere treated in this manner, a remarkable 907. germinatin was fund within 6 weeks. Seed germinatin in cycads is dependent n the interactin f time, misture cntent, humidity and temperature. C. Frsyth and 'Hannes' van Staden (983) f the University f Natal's Pietermaritzburg campus have investigated this interactin in EncephaZapts natazensis. Optimum germinatin (90%) was fund when the seeds were stred in

228 5 mist cnditins fr 3 mnths at 0 C and then incubated at 30 C. The prcess f germinatin seems t be assciated ~ith enzymes knwn as perxidases which ~ere studied by Esperenza Pena and c-wrkers (983) at the University f Havana in Cycas circinalis seeds. Seed dispersal by animals presents an imprtant avenue fr research. Allan Burbidge and Rbert \~elan (98) have studied and quantified the transprtatin f Macrzamia riedlei seed by the pssums, ncturnal marsupials f Australia. ROOT KlRPROLOGY AND CORALLOD ROOTS The frmatin and develpment f cycad rt ndules r crallid rts has been an area f much research. De Luca and Sabat (980) and Schneider (984) have studied the situatin in Cycas r evluta. Numerus publicatins frm the team lead by Dr. David Webb f Queen's University have explred the interactin bet~een light and rt ndule develpment in such plants as Bwenia serrulata (98), Zamia flridana (98b), Z. pumila (983a),!~crzamia cmmunis (983b), M. diplmer (983c), Din edul e (984), Cycas revluta, Encephalart s altensteinii and Zamia furfuracea (984 ref. 99). The different ndulat in traits in different taxa culd prvide additinal criteria fr taxnmic decisins. Webb (98c) has als reprted n the effect f the gametphyte and ctyledns n rt grwth f Zamia j0ri dana embrys, partial r cmplete excisin f these rgans reducing bth primary and secndary rt elngatin. Within the rt ndules are the Cyanbacteria (previusly blue-green algae), Nstc as reprted by Grilli Caila (980) and Anabaena as detailed by Cheng Zhu (98) with respect t Cycas r evluta ndules. Extracts frm the crallid rts f this species appear t have anti-viral prperties, at least with respect t the cntrl f tmat viruses, an interesting bservatin frm Ra and c-wrkers (984). Anther physilgical aspect ha s been the seasnal change in phenl ic substances; Obukwicz and clleagues (98) f the University f Wiscnsin believing that these cm- punds may serve as a natural defence mechanism against unde sirable micrrganisms. The Swedish wrkers, Lindblad, Hallbm and Bergman (985) are currently explring the metablic activity f Zamia crallid rts in relatin t the hetercyst cells in the filamentus strands f Cyanbacteria. Dennis Stevensn (980b) has researched the gr~th f cycads ~ith subterranean sterns, using Zaw.ta pumi la and Stange ria as typical examples. n bth these plants, the stem and rt prgressively cntract as new grwth ccurs at the apex, effectively pulling the stem undergrund. TSSUE cm..rnre The prpagatin f viable plantlets by means f tissue culture techniques may well be the key t prtectin f endangered cycad species. Dr. Niclas Hensn (980) wrking at Kew Gardens, investigated 35 species and fund that the establishment f callus grwth frm tissue explants is relatively easily btained. Furthermre, prmising indicatins f the inductin f sht and leaf grwth frm callus stages was btained in sme Zamia cultures. Arthur Keleman and Prfessr J.G.C. Small (98) managed t btain quite vigrus callus grwth frm stern and rt tissue f several Encephalarts species, but subsequent rgangenesis was disappint ~ng. The differentiatin stage frm callus t plantlet appears t be cntrlled by a critical auzin-cytkinin interactin. David Webb and clleagues wrking at the University f Puert Ric have investigated the influence f the auxin, NAA, the cytkinin, BAP, and the amin acid, L-glutamine, n the in vi tr develpment f Zamia pumila embrys. n this wrk by \~ebb, Rivera, Starszak and Hats (983), callus grwth led t either sht r rt develpment, and embry-like structures were frmed depending n the treatment prgrammes emplyed. Hwever, plant lets capable f grwing in sil were nt btained. Esperenza Pena and Emma Grill (98) f Havana University have cultured the particularly endangered species Micrcycas calct7l t a callus sta ge \-lith sme evidence f rt frmatin. n Hntreal. Svlvie l..::ll it-.t:>..-t- ('l., "-' --

229 6 Bertrand and Jachim Vieth (983) reprt n callus frmatin and sme degree f differentiatin in megagametphyte cultures frm Encephalarts vihsus. Michel Hnnier has cllabrated with Knut Nrstg (984) studying the develpment f irr.rnature embrys f Zamia in culture. Like much research, there is an element f gd luck in this field. With sufficient wrk it is almst certain that a suitable technique fr the prpagatin f cycads en masse by tissue clning will be perfected. CHEMCAL CONSTTUENTS The txic cnstituents f cycads have been further explred. Jack Cannn and clleagues (980) frm the. University f Western Australia have determined the crystal structure f the txic cmpunds macrzamin and sequyi tl frm Macl'zamia riedl ei. De Luca, Hretti, Sabat and Siniscal Giglian (980) have reprted n the ubiquity f cycasin and the latter three authrs (98, 983) have since sh~~ bth txins t be present in all cycad genera but nt in ther plants. Macrzamin is generally mre abundant than cycasin, ccurring at levels frm 0. i. in Cycas ca i~siana t abut 5i. i n BO'wen i a spect abilis" the different cncentratin being largely genus dependent. The evidence nw available supprts the previusly-mentined prpsal t separate Bweni a frm the Zamia ceae; indeed, Bvenia may be clser t Stangeria than riginally thught. Prfessr R.C. Tustin (983) f rm the Ondersteprt Veterinary Research Unit has published details f the t ~ icity and carcingenicity f the Suth African cycads, especially Encephalarts l anatus",,'hile Gerge Hffman and R. \,7. Hrgan (984) cmment n the implicatin f cycad materials as a surce f fdstuff. Pr fessr de Luca's team (98, ref., has investigated the sugar cmpsitin f the hydrlysed mucilae frm.:> dlf ferent cycads and has fund that there is als a brad range f diff~re n c e~ between Australasian, African and ~~ erican genera. n their analyses, Lepi dzamia appears t be q u~ t e d j ~ t inc t frm /vacl'zarr.-/a.. r e nfrcng the justificatin fr its separate generic status. P.A. Gadek (98) f the University f New Suth~ales explred the class f cmpunds kn"~ as biflavnids which accumulate in the brightly-clured testae f cycad seeds as ripening ccurs. Again, different genera have different chemical patterns. nterestingly the cmpund cupressuflavne, previusly knwn in ther gymnsperms like the Cupressaceae, AUTacariaceae and Pdcarpaceae, was fund in tw Macrzamias" M. macdnnellii and M. cmmunis. Gadek, Quinn and Ashfrd (984) later reprted n the biflavnids in cycad leaves where it is thught t act as a deterrent t leaf-eating insects and micrbial invasin. Other chemical substanc~s recently islated frm cycads include sme unusual fatty acids frm Cycas revluta, fund by Japanese wrkers frm Tru, Takagi and Y. ~ tabashi (98), certain prteins which ccur in the pllen tube f C'jcas armstl'ngii, demns tra ted by J.~. Pettit (98) frm the British }useum and the nvel strage glbul in named macrzim, islated frm the seeds f Macrzamia cmmunis by R.J. Blagrve, Lilley and Higgins (984) frm the CSRO in Australia. ECOLOGY The effect f spntaneus r accidental fires n cycads in habitat has been cnsidered by varius Australian scientists. T.S. Grve, A.M. O'Cn~ell and N. Halaj czuk (980) frm the CSRO in \,7embley, Western Australia, have investigated the effects f burning cycles n grm"th, nutrient cntent and rate f nitrgen-fixatin in ppulatins f Macl'zamia r ieclei and fund that fire stimulates leaf grwth and crallid rt activity. This plant makes a significant cntributin t the bisystem f eucalyptus frests where it fixes abut 35 kg f nitrgen per hectare in each 5-7 year interval between burnings. June Dlva and Jhn Sctt (98) have studied the effect f f ire n the meal ybug/cycad assciatin and fund bth respnd favurably t increased fire frequency. A fascnatin ~ paper by J. M. Beatn (9 8) discussed the fire aspect with respect t Au stralian abriginal cycad "farming", ' while Beth Gtt (9 8 ) has written n

230 7 the usage f Macrzamia mrei rts by the indigenus peple f Suth Australia. Anther traumatic incident t plants in habitat is the effect f severe cld. Rita Hummel (984) frm the University f F] rida has published a paper n the freezing tlerance f cycads. Occasinal reprts f spntaneus sex-changes in cycads cntinue t appear. Van Wyk and Claassen (98) have prduced the mst thrugh such reprt with respect t Encephalarts wnbeluziensis, and Maans Kemp (985) recrds a male t female change in Cycas revluta. n all cases, these sex reversals appear t be assciated with sme traumatic envirnmental incident. A current publicatin by Ornduff (985) deals with the tpic f sex ratis in cycads, reprting n field studies n Macrza~a riealei and Zamia pwni la. Hungarian \.Jrkers, Brhidi and Muniz (980), have discussed the separatin in gelgical time f Cuba frm the cntinental landmass, and the cnsequent evlutin f an endemic flra including Micrcycas; a similar and brader theme is fund in the paper by Balduzzi et al mentined previusly. Mre recently, Eenevskii (984) frm }scw UniversitY,has reviewed features f Cuban flra and includes a discussin f their indigenus cycads. Sandra J. Newell, nw at the ndiana University f Pennsylvania, has published papers (983, 985) arising frm her earlier field studies n Zamia pumila in habitat in Puert Ric, n which interesting aspects f leaf mrphlgy and reprductin are discussed. OONCLUSON AND ACKNOWLEDGEMEln'S There is a surprising amunt f research wrk currently in prgress and it is clear that the centres f such prjects are in the U.S.A and Eurpe. Cuntries like Suth Africa and. Australia have the real benefit f large endemic cycad ppulatins which prvide ideal pprtunities fr many prjects, the utcme f which culd add significantly t ur knwledge f t he plants. t is hped that academics and teachers will thus mtivate students in this directin mre in the future than in the past. wuld like t thank Prfessr Nat Grbbeaar, Dr. Dave Webb and particularly Jhn Hendricks, fr their helpful cmments n an early draft f this paper. Ry Osbrne University f Natal January AUDRAN, J-C. (98) Rev. Palaebt. Plynl. 33: BAJNATH, H., NAlDOO, S. and RAMCHARUN, S. (980)?rc. Electrn Mi crscpy Sc. f S. Africa. 0: BALDUZZ, A., DE LUCA, P. and SABATO, S. (98) Delpina 3/4: BEATON, J.M. (98) Archael. Oceani a 7: BLAGROVE, R.J., LLLEY, G.G. and HGGNS (984) Aust. J. Plant..Physil. : BONAT, M.P. and SABATO, S. (984) Carylgia 37: BORHD, A. and MUNZ, O. (980) Acta. Bt. Sci. Hung. 6: BPJ ~ CKON, G. and ORTZ, V.N..(983) Amer. J. Bt. 70: BURBDGE, A.H. and ~~ELAN, R.J. (98) Aust. J. Ecl. 7: CANNON, J.R., RASTON, C.L., TOA, n.r. and '.JETE, A.H. (980) Aust. J. C7 e ~ ';. 33: CHENG, S. et al (980) J. Pt. :7.. DEHGAN, B. and DEHGAN, N.B. (985) Amer. J. Bt. 7: DEHGAN, B. and JOHNSON, C.R. (983) Sci. Hrt ie. 9: DEHGAN, B. and SCHUTZMAN, B. (983) Hrt. Sci. 8: DEHGAN, B. and SCHTJTZHAN, B. (984) Hrt..C;(! ~. Q r:,l, 7

231 8 6. DEHGAN, B. and YUEN, K.K. (98) Rrt. Sci. 7: DEHGAN, B. and YUEN, K.K. (983) 80 t. Gaz 44: DELEVORYAS, T. (98) Rev. Palaebt. & PalynZ. 37: DE LUCA, P., LA VALVA, V. and SAB~TO, S. (980) Capylgia 33: DE LUCA, P., MORETT, A., SABATO, S. and GGLANO, G.S. (980) Phytchemistry 9: DE LUCA, P., MORETT, A., SABATO, S. and GGLANO, G.S. (98) Phytcr~mistry : DE LUCA, P. and SABATO, S. (979) Brittnia 3: DE LUCA, P. and SABATO, S. (980) Plant Sci. Lett. 8: DE LUCA, P., SABATO, S. and STEVENSON, D.W. (984) Taxn 33: DE LUCA, P., SABATO, S. and VAZQUEZ-TORRES, M. (980) Brittnia 3:43-46(a} and 5-9(b). 6. DE LUCA P., SABATO, S. and VAZQUEZ TORRES, M. (98) Brittnia 33: 79-85(a) and (b). 7. DE LUCA, P., SABATO, S. and VAZQUEZ-TORRES, M. (98) Brittnia 34: DE LUCA P., SABATO, S. and VAZQUEZ TORRES, M. (984) Brittnia 36: DOLVA, J.}. and SCOTT, J.K. (98) J.R. Sc. West Aust. 65: ECKE!\'fl,.lALDER, J.E. (980),. Arnld Arb. 6: ELENEVSK, A.G. (984) BuuZZ Msk. O-Va spyt Prir Otd Bil. "89:6-7. 3~ FORSYTH, C. and VAN STADEN, J. (983) S. Afi'. J. Sc i. 79: GADEK, P.A. (98) Phytchemistry : GADEK, P.A., QUNN, C.J. and ASHFORD A.E. (984) Aust. J. Bt. 3: GOMEZ, L.D. (98) Phytlgia 50: GOTT, B. (98) Archael. Oceania 7: GRLL CAOLA, M. (980) New Phytl. 85: GROVE, T.S., O'CONNELL, A.M. and MALAJ CZUK, N. (980) Aust. J. Bt. 8: HENDRCKS, J.G. (98) Phyta HENSON, N. (980) Kew Reprt - Unpublished. 4. HROAKA, T., HADA, A. and TAKADA, Y. (98) J. Ultrastruct. Res. 77: HOFF}~, G.R. and MORGAN, R.W. (984) Envirn. Mutagen. 6: HUMMEL, R.L. (984) Hrt. Sci. 9: KARATELA, Y.Y. and GLL, L.S. (984) Feddes Reprt 95: KEMP, H.J. (985) Veld & Flra: KOELE~~, A., ROBBERTSE, P.J. and ECKER, A. (98) J.S. Afr. Bt. 47: KOELE}Ul~, A. and SMALL, J.G.C. (98) S. Afr. J. Bt. : KURD-HADAR, B., SHALHOUB, V., DB-HAJJ, S. and DEED, S. (983) Chrmsma 88: LALBERTE, S., BERTRAND, C. and VETH, J. (983) Rev. Can. Bil. Exp. 4: LAN, K. and ZOU, R. (983) Acta Phyttax. Sin. : LNDBLAD, P., HALLBOM, L. and BERG~, B. (985) New Phytl. 0:9-7.

232 9 5. HONNER, M. and NORSTOG, K. (984) z. Pjanzenphysil. 3: MORETT, A. (98) Delpina 3/4: MORETT, A. and SABATO, S. (984) Plant Syst. Evl. 46: MORETT, A., SABATO, S. and GGLANO, G.S. (98) Phytchemistry 0: MORETT, A., SABATO, S. and GGLANO, G.S. (983) Phytchemistry : MORETT, A., SABATO, S. and VAZQUEZ-TORRES, Brittnia 34: NE~~LL, S.J. (983) Bull. Trrey Bt. Club 0: NEWELL, S.J. (985) Amer. J. Bt. 7: NKLAS, K.J. and NORSTOG, K. (984) Bt. Gaz~ 45: NORSTOG, K. (980) Carylgia 33: NORSTOG, K. (98) Carylgia 34: OBUKO~~CZ, M., SCHALLER, M. and KENNEDY, G. S. (98) NeUJ PhytL. 87: ORNDUFF, R. (985) Amer. J. Bt. 7: PENA, E. and GRLLO, E. (98) Rev. Jard. Bt. Nac. 3: PENA, E., GRLLO, E. and PEREZ, D. (983) Rev. Jard. Bt. Nac. 4: PETTT, J.M. (98) J. Cell Sci. 57: PRABHAKAR, C.S. and RAO, K.R. (98) Phytsyn. Res. : PRABHAKAR, C.S. and RAO, K.R. (984) Phtchem. Phtbil. 39: 04S. 70. RAO, G.P., BAGHEZ, A.K., SNGH, K.K. and CHATTERJ, K.S. (984) Experientia 40: ROHR, R. (980) Cytlgia 46: SABATO, S. and DE LUCA, P. (985) Amer. J. Bt. 7: SCHNEDER, A. (984) Bt. Gaz SCHUTZMAN, B. (984) Phytlgia 55: SCHUTZMAN, B. and VOVDES, A.P. (985) Amer. J. Bt. 7: STEVENSON, D. (l980a) Amer. J. Bt. 67: STEVENSON, D. (980b) Bt. J. Linn Sc. 8: STEVENSON, D. (98) Amer. J. Bt. 68:04-4. ~ 79. STEVENSON, D. (98) Brittnia 34: STEVENSON, D.-l. (985) Amer. J. Bt. 7 : STUCHLCK, L. and MONCADA, M. (983) Acta Bt. Hung. 9: TAKAG, T. and TABASH, Y. (98) Lipids 7: TERASAKA, O. (98) Cytlgia 47: TUSTN, R.C. (983) J.S. ~fr. Ve:. Assc. 54: VAN ~~K, A.E. and CLAASSEN, M.. (98) Veld & Flra: VOVDES, A.P. (983) Amer. J. Bt. 70: VOVDES, A.P. and MOPENO, N.P. (983) Ta..xn. 3: VOVDES, A.P. and REES, J. (980) Bitica 5: VOVDES, A.P. and REES, J. (983a) Flra Veracruz Fasc. 6:-5. ') ')

233 0 90. VOVDES, A.P. and REES, J. (983b) Madrn 30: WEBB, D.T. (98) Phytmrphlgy 3: WEBB, D.T. (98a) Ne~ Phytl. 9: \"'EBB,D. T. (98a) Phytmrphlgy 3: WEBB, D.T. (98c) Z.Pfanzenphysil. 06: WEBB, D.T. (983a) Amer. J. Bt. 70: \"TEBB, D.T. (983b) Ann. Bt. 5: ~7. WEBB, D.T. (983c) Phytmrphlgy 3: ~'EBB, D.T. (984) Amer. J. Bt. 7: WEBB, D.T., NEVAREZ, M. and DE JESUS, S. (984) Envirn. Exp. Bt. 4: ~TEBB, D.T., RVERA, M.E., STARSZAK, E. and MATOS, J. (983) Ann. Bt. 5: ZAVADA, M.S. (983) Pllen spres 5: ZHU, C. (98) Acta Bt. Sin. 4:09-4. (An early draft f this article was pubi= shed in the Cycad Newsletter (U.S.A.), Vl. V, n. 3, Nvember 985.)

234 THE WORLD LST OF CYCADS * ~ Ry Osbrn~, Jhn G Hendricks Dennis Wm. Stevensn. Taxnmy is that discipline f btany ~hich prvides a ratinal basis fr the naming f plants. t is an bjective science in that set prcedures ~rk t~ard develping a~ systematic genealgy. H~ever, prblems relating t the selectin f crite~ia f rank result in subjective judgement and pinin; this is especially true in the rder Cycadales. Mst cycad species names riginated frm studies f islated plants r habitats. ~~en descriptins f these species ~ere later cmpared n a brader reginal basis, many appeared t be synnmus r less differentiated than thers, thus names and classificatins had t be reassigned. This prcess may repeat as ften as justified, the mst recent prperly-published names and ranks being accepted as valid. Apart frm the as yet unreslved cnfusin f the early literature ver the identificatin f sme taxa, there are als prblems f judgement and f pinin. n additin, there are kn~~htt undescribed ne~ species and there are difficulties arising frm situatins ~hen btanically invalid names applied by cllectrs t taxa that differ frm r have nt been recnciled ~ith valid taxa. Anther imprtant factr is that sme cycad habitats have yet t be fully explred and the plants prperly classified. Frtunately, V..i.n, Enc.eplla.CVLt.6 and Mac.JtzCJl..i.a are largely defined althugh sme changes are anticipated. Only Bwelua, Lep..i.dzcun.i.a, M..i.U~OC.YC.M and S.tangvUa are cns idered taxnanically cmplete at this time. At the time f submissin f this thesis, this paper is currently under cnsideratin fr publicatin in the Mem~ ~ the New Y~k Btan..i.c.al Ganden.

235 Majr advances have recently been made in Zamia taxnmy by infrmatin frm chrmsme karytyping. Bichemical studies t a~e being used t define genera and give prmise f differentiatin at lwer taxnmic rank. in future years. Other mdern analytical techniques may have applicatin Taxnmy in its purest frm reflects evlutin; the best advances will be made by cmbining the knwledge frm the many relevant interacting scientific disciplines. Our list f validly-named cycad species was first published in ENCEPHALARTOS (N.3, September 985), Jurnal f the Cycad Sciety f Suthern Africa. A supplementary list fllwed in a subsequent issue f the same Jurnal (N.5, March 986). The list which nw fllws is a further update where ~a changes have been made n the basis f recent infrmatin and itemizes the currently valid taxnmic ranking at r belw the species level t the best f ur existing knwledge. t is nt a listing f all validly published species, nr des it represent ur pinin as t 'gd' species r suitable classificatin. ndeed, ur pinins and thse f many thers are at cnsiderable variance frm this list, particularly in assignments in the :genus Cy~. Wrk is prceeding tward an imprved cycad taxnmy, thugh perhaps nt f the scpe nr at the rate which might be desired. n the interim, it is recmmended that labelling f specimen plants is supplemented with any additinal apprpriate infrmatin. Ry Osbrne Jhn G. Hendricks Dennis Wm Stevensn University f Natal 0 BrkmeadeDrive New Yrk Btanical Garden King Gerge V Avenue Statesville NC 8677 Brnx NY 0458 Durban. 400 U.S.A. U.S.A. Suth Africa Nte: An asterisk against the taxn given indicates that the publicatin is currently i~ pnint.

236 3 BOWENA ( species) Australia B. ~~uiata (W. 'Bull) Chamberlain Queensland B. ~pectab~ Hk. ex Hk. f. Queensland CERATOZAMA (0 species) Mexic, Guatamala, Belize C. ~yphyt idia V-Trres, Sabat & D. Stevensn Veracruz C. hitdae G. Laundry & M. Wilsn San Luis Ptsi & Queretar C. ~u~t~~a Regel C. l~ua Miq. Tamaulipas San Luis Ptsi & Queretar C. matudae Lundell Chiapas, N. Guatemala C. mexica~a Brngn. C. miqueua~a H.Wendl. C. ~~tg~ D. Stevensn C. JtbMta Miq. C. zajtagzae Medellin-Leal * CHGUA ( species) * C.beJttta...U. D. Stevensn * C. Jt~tJtep'<' D. Stevensn CYCAS (4 species) Puebl, Veracru~ Veracruz Chiapas S. Mexic Guatemala & Belize San Luis Ptsi Suth America Clumbia Clumbia C. atr..m6tjtngu Miq. C. baguanhee~~ L.K. Fu & S.Z. Cheng C. b~attica C.A. Gardn. C. c.a.,uz.~.<.ana F. Muell C. calc.<.cla J.R. Macnchie C. chamb~n..<..<. W.H. Brwn & Kienhlz China Western Australia Queensland, Australia N. Territry, Australia Vietnam

237 4 c. wc.in.i~ L. subsp. wc.irta.m frma wc.irta.m Asia, Africa frma gtauqa (Miq.) Schuster frma gtha.ruj.. Schuster subsp. madag~~e~~ (Miq.) Schuster Madagascar frma madag~qajlie~~ frma ~gnqa4pid~ Schuster subsp. papuana (F.Muell) Schuster subsp. 4uiminiana (Prte) Schuster New Guinea Philippines var. 4uimi..u.a.na var. QU44arUJ.. Schuster frma QU44an..U. frma ape4t04um Schuster frma m~a Schuster var. ~Q4a;tQheeyana subsp. Ve4a Schuster (F.Muell.) Schuster New Guinea var. Ve4a var. beddmei Dyer C. gu.izhue.~~ K. Lan & R. Zu C. ha.ittane.~~ C.J. Chen & C.Y. Chen ndia China Hainan sland, China C. mecu.a R. Br. var. mecu.a var. ~aqea (W.V. Fitzg. ) Schuster var. iane-piei (C.A. Gardn.) Schuster N. Territry, Australia Western Australia Western Australia C. rru.dtllizil Dyer var. rru.qhllizil var. ~~plic.ipinna Srnitinand Vietnam, China S.E. Asia

238 5 c. nath~tii Schuster c. nnmanbya~ F. Muell c. panzhihuaen6~ L. Zhu & S.Y. Yang c. pectinata W. Griff c. pnuin~a J.R. Macnchie C. nevluta Thunb. var. nevr.u;ta Sri Lanka Queensland, Australia China Widespread, S.E. Asia Western Australia Japan, Ryukyu s. var. nbuta C. numpw Miq. S.E. Asia & Pacific s. subsp. numpftli. var. ftwtljw var. ~ubi~ceua Schuster frma ~ubi~ceua frma par.~uica frma papua~a Kaneh (F. Muell.) Kaneh Palau New Guinea frma ~eemamt.u:. (A. Braun) Kaneh Fiji subsp. zeylanica Schuster C. ~zechuanen6~ C. Chen & L.K. Fu Sri T..B.;nka, China, S.E. Asia subsp. ~iamen6~ subsp. balan6a.-e (Warburg) Schuster C. ~niana Carruth. Vietnam China, Taiwan C. undutata Desf. C. LmdU Me rr i Philippines DOON (0 species) v. cali6ani De Luca & Sabat v. caputi De Luca, Sabat & V-Trres v. edute Lindley var. edute Mexic, Hnduras Oaxaca Puebla Nuev Len, San Luis Ptsi, Tamaulipas

239 6 var. angu ti6fium (Miq.) Miq. v. h mg~eni De Luca, Sabat & V-Trres v. mej~ae Standley & L.O. Williams v. m~iae De Luca, Sabat & V~Trres Nuev Len, Tamaulipas. San Luis Ptsi Oaxaca N. Hnduras Chiapas Oaxaca v. ~zedw~~ De Luca, Mretti, Sabat & V-Trres Oaxaca Vera Cruz, Oaxaca v '~ t~e.r.j.j.. De Luca, Sabat & V-Trres var. t~e.r.j.j.. var. ~n~e~e De Luca, Sabat & V-Trres S.W. Cast, Mexic N.W. Cast, Mexic ENCEPHALARTOS (4~ species) E. a.ue~tuni Lehni. E. CVten~ R.A. Dyer Africa: E.Cape, Transkei E. Cape E. b~~ Carruth. ex Miq. subsp. b~~ subsp. aiicmu L.E. Newtn E. bub~nu Mellville E. ca.6-6~ (Thun b.) Lehm. E. chima~a~e~~ R.A. Dyer & Verdrn E. cn~nnu R.A. Dyer & Verdrn E. cup..i.d.u R. A. Dyer E. cyc0.cu..6uu (Jacq.) Lehm. E. eugene.,.~u Verdrn E. ~~x Bertl f. E. Wd~~-g'~~ Lehm. Benin, Ghana, Nigeria, Sudan, Tg Nigeria Tanzania, Kenya E. Cape Mzambique, Zimbabwe Zimbabwe Transvaal E. Cape Transvaal Zululand, N. Natal, Mzambique E. Cape, Transkei

240 7 Transkei, Natal E. gjtatu. Prain E. te.e.n.aw R.A. Dyer Malawi, Mzambique Swaziland, S.E. Transvaal, E. hiide.bjtattdtii A. Braun & Buche var. Ui.de.bJtattdtii var. de.n.tatu. Melville E. t JtJti..du. (Jacq.) Lehm. E. ~ Verdrn E. in.pin.u. R.A. Dyer * E. awue.lt6"w Bamps & Liswski E. lae.vi6~ Stapf & Burtt Davy E. lanatu. Stapf & Burtt Davy E. tati6jt t6 Lehm. E. taujte.ntian.u. De Wild. E. te.bmbe.lt6"w Verdrn E. fe.hmaylw Lehm. E. ioylg4uu. (Jacq.) Lehm. E. maylike.lt6"w Gilliland E. majtuylgue.lt6"w Devred E. munc.hli R.A. Dyer & Verdrn E. Ylatate.lt6"w R.A. Dyer & Verdrn E. YlgyaYlu. Verdrn E. Africa Tanzania, Uganda E. Cape E. Transvaal Transvaal Zaire E. Transvaal, Swaziland Transvaal E. Cape Angla. Zaire N.Natal, S.E. Transvaal, Swaziland, Mzambique E. Cape E. Cape Mzambique, Zimbabwe Zaire Mzambique Natal Zululand, N. Natal S.E. Transvaal, Swaziland E. pauc.ide.~ Stapf & Burtt Davy E. pgge.i Aschersn E. pjtin.c.e.p~ R.A. Dyer E. pte.jtgoylu. R.A. Dyer & Verdrn E. ~c.l-u.zu Ma:laisse E. Transvaal, Angla, Zaire E. Cape, Transkei Mzambique Zaire, Zambia Swaziland

241 8 ~ E. l.lcia.vai (Authrs and lcality t be advised) E.!.)epte~~ Schweinfurth Zaire, Sudan, Uganda Central Af. Rep. E. teguiayle.u..6 Melville E. btamveyl!.)u).) Stapf. ;& Burtt'Davy E. ~piyl!')u)') (Hk.)R.A. Dyer E. umbeiuziem-w R.A. Dyer E. vill!.)u).) Lem. E. vien!.)-w (Authrs and lcality t be E. wclu. Sander Kenya N. Transvaal E. Cape Swaziland, Mzambique ~~. Cape, Natal, S.E. Transvaal advised) Transk~i, Zululand, Swaziland, Mzamblque (Extinct in nature) LEPVOZAMA ( species) L. hpei Regel L. p~~~yayla Regel Australia: Queensland N.S.W. & Queensland MACROZAMA (4 species) M. cmmuyl.<..6 L.A.S. Jhnsn M. diptm~ (F.Muell.) L.A.S. Jhnsn M. ~awcetti C. Mre M. het~m~ C. Mre M. tucida L.A.S. Jhnsn M. macd YLYLe.Uli (F. Muell. ex Miq.) A. DC. M. miquetii (F. Muell.) A.DC. M. m4ei F. Muell. M 0 pauu -guiue. mi W. Hl'll & F. Muell. subsp ~exu!.)a subsp. ptu4iyl~via M. ptaty4ach.<..6 F ~M. Bailey (C.Mre) L.A.S. Jhnsn L.A.S. Jhnsn M. 4iedtei (Gaud.) C.A. Gardn. N.S.W. N.S.W. N.S.W. N.S.W. Queensland & N.S.W. Central Australia Queensland & N.S.W. Queensland & N.S.W. Queensland N.S.W. Queensland & N.S.W. Queensland SW. Australia

242 9 M. ~ecu~da C. Mre M. ~p~~ (Salisb.) Miq. M. ~te~m~a L.A.S. Jhnsn N.S.W. N.S.W. N.S.W. MCROCYCAS ( species) M. calcma (Miq.) A.D.C. Cuba W. Cuba STANGERA ( species) S. ~p~ (Kunze) Baillard Suth Africa E. Cape, Natal, Transkei & Zululand. ZAMA (43 species) Nrth, Central, Suth America, and West ndies Z. acwru.~ata Oersted ex Dyer Z. amblyphyw.cu.a D. Stevensn Z. ampt40lia Hrt. ex Masters Z. a~g~ti..6-lia Jacq. Z. buv-<'a~a (Bngn.) A. DC. Z. ducgua Seemann Z cupa..u.em,u, Ducke Z. L.D. Gmez Z. ~ch~ Miq. Z. ~~~acea L. fil. in Aitn Z. hvut~ae Caldern & Standley Z. -i~~ Vvides, Rees & V-Trres Z. -i~egjt-4lia L. fi. in Aitn Z. j~~em,u, R.E. Schultes Z. law~~ana Dyer Z. lec-it'ltu Ducke Nicaragua, Panama. Puert Ric, Cuba, Jamaica Clmbia Bahamas, Cuba Blivia Clmbia, Panama Clmbia, Brazil Csta Rica, Panama Queretar, San Luis Ptsi, Veracruz Veracruz E Salvadr, Hnduras, Guatemala, Chiapas Veracruz Flrida, Bahamas, Cuba Clmbia Oaxaca Brazil

243 30 Z. Uttdew Regel ex Andre Z. Uttdtey-<- Warsz. ex A. Dietrich Z. ld~guil Miq. Z. ma~c.a-ta Linden ex Regel Z. mt.ta~a A. Br. z. m~c.la Chamberlain Z, m~c.a-ta Willd. Z. b-uiet'l6.v., Ducke Z. buqua A. Br. Z. paucj.juga Wieland Z. p-<-c.ta Dyer Z. pepp~g~a~a Martius & Eichler Z. pjttjuc.et'l6.v., Urban Z. p-6eudm~c.la L.D. Gmez Z. p-6eudpajtmwc.a Yates in Seemann Z. pumila L. Z. pwtpwtea Vvides, Rees & V-Trres Z. pygmaea Sims Z. ~ez~ Linden Z. -6lU.ttnru Warsz. ex A. Dietrich Z. -6paJtt.ea A.DC. Z. -6pfe~det'L6 Schutzman Z. -6ylvmc.a Chamberlain Z. tu~c.k.humil J. Dnnell Smith Z. ulu Damm.. Z. v~c.ha..ueuil Miq. Z. walu-6il A. Br. Ecuadr Panama Mexic Clmbia, Panama Clmbia Veracruz Venezuela Brazil Clmbia W. Mexic Guatemala, Mexic Peru Puert Ric Csta Rica Panama, Csta Rica Caribbean (widespread) Veracruz W. Cuba & sla da Pinas Clmbia Nicaragua, Panama Csta Rica, Guatemala Oaxaca Mexic Mexic Guatemala Brazil Mexic Clmbia

244 NOTES 3 HORTSCENCE (6): n Vitr Reg~neratin f Stangeria. enpus R. Osbrne and J. van Staden Department f Chemistry, University f Natal, King Gerge V Avenue, Durban and UN/CSR Research Unit fr Plant Grwth and Devel;ment, Department f Btany, University f Natal, Pietermantzburg, Suth Africa Additinal index wrds. tissue culture, cycads, primary rt explants n Suth Africa, the indigenus cycads cmprise 8 species f Encephalarts (Zamiaceae) and Stangeria eripus (Kunze) Baillard (3), the latter species being the sle representative f the Stangeriaceae. Present cycad ppulatins are under severe pressure frm cmbined effects f agricultural and urban develpment, explitatin by the tribal " medicine-men", activities f plant cllectrs, and demands frm scientific and educatinal institutins. n additin, the relatively slw grwth rate, limited ptential fr vegetative prpagatin, and the restricted supply f viable seed further exacerbate the delicate cnservatin status f these diecius plants (). Althugh cnsiderable attentin has been given t the use f haplid material, usually the female gametphyte in cycad tissue culture, there are few reprts n in vitr culture f smatic tissue. Callus has been derived frm leaflet explants f bth Encephalarts and Stangeria (4) and frm stem and rt tissue f Encephalarts (5), but n significant rgangenesis was btained in any f the reprted studies. Regeneratin f vegetative material has been achieved using primary rt tissue frm Stangeria eripus seedlings and is reprted here. Fresh seeds f Stangeria eripus were germinated and seedlings established in plastic bags cntaining a lam sil. After 8 mnths, five healthy plants were freed frm the sil and the primary rts ('" 00 mm lng and 5 mm in diameter at the widest pint) excised. These rgans then were scrubbed t remve superficial dirt, allwed t stand in running water (30 min), and dis- Received fr publicatin 7 Feb We thank the Depts. f Bichemistry and Btany f the Univ. f Durban-Westville fr the use f their facilities. Seed material was kindly supplied by the Durban unit f the Btanical Research institute. The cst f publishing this paper was defrayed in part by the payment f page charges. Under pstal regu latins, this paper therefre must be hereby marked advertisement slely t indicate this fact. infected by sequential immersin in 70% ethanl (4 min), 0% Jik (cmmercial 3.5% NaOC slutin) cntaining % Teepl (30 min) and % HgC cntaining % Teepl (30 min). After thrugh rinsing and saking in sterile distilled water ( hr), the rts again were treated with hypchlrite and rinsed three times with sterile distilled water. Segments ("'5 mm cubes), each f which included vascular and crtical tissue, then were dissected aseptically frm the upper twthirds f the rts and transferred individually t 5-ml flasks cntaining the medium f Schenk and Hildebrandt (7) supplemented with 4.5 x 0-6 M each f,4-d and kinetin. The medium was slidified with 0.6% Bact-agar. Thirty cultures were established in this manner and they were placed in a dark cupbard at 5 ± C fr 30 days. At the end f the initial culture perid, 5 cultures were transferred t fresh medium f the same cmpsitin and placed in a grwth cabinet under cnstant light (",00 ~ml's - 'm - ) and temperature (5 ± 0. ). After abut 5 days f dark culture, n cntaminatin had ccurred. Cmpact white callus had frmed n the upper surface f all explants. On subculture and transfer t the light envirnment, each explant develped a small green meristematic zne within 4 days fllwed by emergence (Fig. ) and expansin (Fig. ) f a typical circinate leaf. This pattern f mrphgenesis was cnsistent in all cultures after transfer t light, but did nt ccur in any f the flasks remaining in the dark. This is, t ur knwledge, the first reprt f in vitr mrphgenesis in a Suth African cycad. n separate experiments, we have been unable t btain a similar respnse frm any species f Encephalarts. Thus, the success with Stangeria may be assciated with an inherent regenerative capacity f the rt material; it has been reprted that Stangeria plants may be btained frm rt cuttings (6), a phenmenn nt knwn' in ther cycads. t is als f interest that the American lamia cycad lng has been recgnized fr the regenerative ptential f its undergrund tuberus rt and stem tissue (). Fig.. Light-induced regeneratin f a vegetative bud (arrw) fllwing initial callgenesis n a primary rt explant f Srangeria eripus. Fig.. Emergence and extensin f the first leaf; weeks subsequent t the stage in Fig.. Literature Cited. Culter, J.M. and M.A. Chrysler Regeneratin in lamia. Bt. Gaz. 56: Dyer, R.A The cycads f Suthern Africa. Bthalia 8: Giddy, C. Cycads f Suth Africa. nd Ed. C Struik, Capetwn. 4. Hensn, N Thefrmatin f rganized elements and callus frm cycads in culture at Kew. int. Rpt., Ryal Bt. Gardens, Kew, Surrey, England. 5. Keleman, A. and LG.C. Small. 98. A nte n callus frmatin by stem and rt tissue f sme Encephalarts species. S.A. J. Bt. : Pant, D.D Cycads and the Cycadales. nd Ed. Central Bk Dept, Allahabad, india. 7. Schenk, R.U. and A.C. Hildebrandt 97. Medium and techniques fr inductin and grwth f mnctylednus and dictylednus plant cell cultures. Can. J. Bt. 50: HORTSCENCE, VOL. (6), DECEMBER 987

245 4th draft, May 88 3 SOUTH AFRCAN CYCAD RESEARCH PROGRESS AND PROSPECTS* ~ *R. OSBORNE, N. GROBBELAAR and P. VORSTER.. Department f Chemistry, University f Natal, King Gerge V Avenue, 400 Durban; and UN/CSR Research Unit fr Plant Grwth and Develpment, University f Natal,300 Pietermaritzburg.. Department f Btany, University f Pretria, 000 Pretria. 3. Department f Btany, University f Stellenbsch, 7600 Stellenbsch Received... Accepted ABSTRACT The rder Cycadales ccupies a key psitin in the btanical hierarchy and shws many unusual and sme unique features. With 8 species f Encephalarts and the nly representative f Stang~, Suth Africa is similar t Mexic and Australia in the richness f its cycad flra. A resurgence f interest in this plant grup is demnstrated by the extent f recent and current research prjects int their taxnmy, mrphlgy, prpagatin and bichemistry. Numerus ther research pprtunities remain t be explred. UTTREKSEL Die Cycadales is n rde wat vele ngewne en enkele unieke kenmerke vertn en gevlglik n sleutelpsisie in die planthierargie beklee. Met 8 Encephalarts spesies en die enigste verte.e.nwrdiger van Stangeria inheems in Suid-Afrika, verskil Suid-Afrika min van Meksik en Australie wat die rykheid van sy brdbmflra aanbetref. Hernude belangstelling in hierdie plantgrep wrd in die mvang van die nlangse en huidige navrsing r hul taksnmie, mrflgie, vrtplanting en bichemie weerspieel. Desndanks wag talle ander navrsingsgeleenthede p ntginning. KEYWORDS: Cycadales, cycads, ~phalarts, Stangeria. At the time f submissin f this thesis, this paper is currently under cnsideratin fr publicatin in the Suth AnJUc.an ] Wtna.f. 0 n Sue.nee..

246 33 * T whm crrespndence shuld be addressed NTRODUCTON The present-day cycads cmprise the diverse, mdified remnants f a much larger grup f plants which flurished in the Meszic Era, reaching their zenith in the Jurassic Perid. Of the 3 valid species in the Cycadales,l.l.. Suth Africa has 9 species in tw genera (Encephalarts Lehm. and Stangeria T. Mre).l Suth Africa, Mexic (apprx. 8 species in 3 genera) and Australia (apprx. 3 species in 4 genera) cllectively accunt fr abut 60 percent S the wrld's cycad flra. Yet in a recent bibligraphy f cycad literature, in which 73 references were listed, nly 03 mentin Encephalarts and 9 deal with Stangeria.~ A current resurgence f lcal interest in these plants may address this imbalance. n this text, we wish t review current knwledge f the Suth African cycads and utline sme preliminary results frm certain prjects presently underway. TAXONOMY Due very largely t the excellent wrk f Drs R.A. Dyer and. Verdrn, the taxnmy f the Suth African cycads has been fairly well defined. S -9 n essence, this cuntry has 8 species f Encephalarts (Zamiaceae) and Stangeria erirus (Kunze) Baillard, the sle representative f bth the genus and its family (Stangeriaceae) (Table ). The status f several Encephalarts taxa is currently under review (Vrster, unpublished results). Of particular interest is the taxnmic psitin f several small ppulatins which are clearly related t larger grups f defined species frm which they have becme gegraphically islated. Despite its fairly well-defined taxnmy, there has as yet been nly ne attempt t establish phylgenetic relatinships within Encephalarts.~ t is speculated that the ancestral cycad stck was arb.rescent and mesic and that all adaptatins t harsher envirnments represent advances.' n this sense E. altensteinii Lehm., E. lebmbensis Verdrn, E. natalensis Dyer & Verdrn and E. transvensus Stapf & Burtt Davy are examples f primitive species. By cntrast, thse with subterranean caudices (e.g. ~ Dggy'anus Verdrn and E. villsus Lem.); thse with much reduced leaflets (e.g. E. y.cadiflius (Jacq.)Lehm. and E. ghellinckii Lem.) and the highly-armed species in arid areas (e.g. E. hrridus (Jacq.) Lehm. and E. trispinsus (Hk) Dyer) must be cnsidered relatively mre advanced. t is anticipated that much f the research presently in hand will allw cnstructin f a mre rigrus phylgeny f the genus.

247 34 CONSERVATON Of the Suth African cycads, the Threatened Plant Unit (TPU) f the nternatinal Unin fr the Cnservatin f Nature and Natural _ Resurces (UCN) lists three as endangered, seventeen as vulnerable, five as rare and three as "insufficiently knwn" (Table )." This classificatin is a revisin f that f lcal wrkers,~ and suffers frm the same limitatins f being based largely n numerical representatin frm herbarium specimens. A Natal species, Encephalarts wdii Sander, is extinct in nature, nly ne multi-trunked specimen f this plant ever being discvered and all prtins being transplanted t btanical gardens fr safekeeping. s,r~ Pressure n existing cycad ppulatins arises frm the cmbined effects f man's cntinuing dmestic and agricultural demands n finite land resurces and the widespread remval f plants frm habitat by unscrupulus dealers r cllectrs, despite legislatin intended t prevent this. There is als sme evidence that significant numbers f ~geria erip'us have been remved fr use by tribal herbalists (Cunningham, pers.~.). Thefts frm public and private gardens als ccur with dismaying frequency, prize specimens cmmanding exhrbitant prices n lcal and verseas black markets. Cnservatin prblems are exacerbated by the slw grwth rate, paucity f viable seeds and the limited ptential fr vegetative prpagatin. Legistatin t cntrl the explitatin, sale and transprtatin.f cycads exists at an internatinal level in terms f the Cnventin n nternatinal Trade in Endangered Species f Wild Fauna and Flra (CTES)~and lcally in terms f the varius natinal and reginal nature cnservatin laws. 'S' The "Mdjadji Palms" (E. transvensus) in the Letaba area are additinally and effectively prtected in terms f the authrity f the Rain Queen f the Balbedu tribejr furthermre bth this frest and the type specimen f E. natalensis near Mnteseel in Natal have been prclaimed Natinal Mnuments.l~ The activities f the Cycad Sciety f Suthern Africa in creating a public awareness f the necessity fr cnservatin, and in establishing seedbank and pllen exchange facilities, tgether with the peratins f public and private cycad nurseries, have an effect f reducing cllectr demand n habitat specimens. The principle f "cnservatin thrugh cultivatin" making plants available thrugh authrised channels, may well be at least as effective as attempts at discipline thrugh legislatin. The establishment f cycad reserves and scientifically-managed cllectins in btanical gardens ffers much hpe fr the maintainance f cycad gene-pls in the lng term. ECOLOGY Suth African cycads are fund in a variety f habitats, ranging frm rain-frests t karr scrub and frm castal wdlands t high-altitude grasslands.~r Althugh their distributin is fairly well dcumented, there has been n detailed wrk published n their eclgy. A quantitative survey f existing ppulatins wuld prvide

248 35 invaluable data fr cnservatin planning and wuld allw fr better evaluatin f their cnservatin status. Studies n the ppulatin dynamics culd quantify and integrate with aspects such as sex-ratis, seed prductin, seed dispersal, plant regeneratin and grwth rate. n additin, the" effect f spntaneus and deliberate fires n the grassland species may cnfirm speculatin that burning cycles prmte vegetative grwth and induce cning. A further interesting aspect pertaining t cycads with subterranean caudices relates t hw the psitin f the stem apex is maintained; speculatin is made that cntactile rts perate tgether with stem cmpactin and pssibly prgressive decmpsitin f - the stem base (Grbbelaar, unpublished results).. MORPHOLOGY The grss mrphlgy f the Suth African cycads has been thrughly described and frms the basis fr their present taxnmy.s A study f leaf surface characters f Encehalarts and Stangeria describes differences in leaf cell mrphlgy, stmatal arrangement, leaf hairs and cuticular wax depsit patterns.~ Sme f these characters appear t be species-specific and pssibly sex-related in Stangeria. An investigatin int the cmparative anatmy f Encephalarts leaflets describes the stmatal distributin, and the arrangment f sclerenchyma, pallisade, vascular tissues and resin canals. q ~ ~gene-maraisii and E. cupidus leaflets are unusual in having bilateral symmetry with stmata and pallisade layers bth adaxially and abaxially. The remaining 6 species can be gruped n the basis f leaf anatmy and plant habitat. The characters described are used t create a species key and allw speculatin n the phylgeny f the genus. " Preliminary SEM studies n cycad pllen mrphlgy have indicated characteristic differences at the genus level (Fig. ),9 cnfirming earlier reprts by Wdehuse,q and Van Zinderen Bakker. t Cycad pllen micrspres are batshaped, mnsulcate and bilaterally symmetrical, measuring abut 6-37 micrn by 4-3 micrn, with an exine surface which varies frm psilate t fvelate and fssulate.\-~ Since the characteristics f the sexual reprductive structures in plants are usually highly cnserved, any differences. in the pllen mrphlgy f different species may be particularly significant. A survey f Encephalarts pllen mrphlgy is presently under way at the University f Pretria. A similar survey with respect t the seed kernel (sclertesta) mrphlgy is being planned; preliminary results here t indicate that this aspect merits further investigatin.~~

249 36 REPRODUCTVE BOLOGY Because f the antiquity f the rder, much attentin has rightly been fcussed n the reprductive bilgy f the Cycadales. The abslute dieciusness f the plants, the ntgeny f the micrgametphyte with the ultimate release f large mtile spermatzids, the develpment f a substantial megagametphyte and the embrylgy f cycads, have all been well-dcumented.. n-t~ Mst f the wrk in this regard has been dne n nn-african species. Althugh early assumptins were made that cycads were wind pllinated, recent studies n the mes-american Zamia species have shwn that beetles may be bligatry pllen vectrs at least in this genus."'-lq This wrk calls t mind the early bservatins n insect pllinatrs in Suth African Encephalarts species~o~lthe mst likely pllinatrs f ur lcal cycads appear t be the elngate weevils f the subfamily Rhyncphrinaej the activities f these hst-specific beetles undubtedly resulting in a mre efficient disperal than wind distributin.'~ A thrugh investigatin int the rle f insects in pllinatin f Suth African cycads remains t be dne. n passing, it is nted that the nble idea f re-establishing nursery-raised plants in habitats frm which the particular species had previusly disappeared, may be futile unless the pllinatin vectr is simulanteusly re-intrduced. Significant temperature increases by male Encephalarts cnes, and the assciated release f vlatile durs, at the time f pllen release is uncmmn but nt unique in plants, and is clearly assciated with insect pllinatin. A male cne frm Encephalarts altensteinii reached a temperature f 7 deg. C abve ambient shrtly after remval frm the parent plant.~~l~similar heating has been nticed in male cnes f E. transvensus (5 deg. C. abve ambient) (Grbbelaar, ~npublished results) and E. wdii (7 deg. C. abve ambient) (Osbrne, ~npublished results). A detailed investigatin has since shwn that thermgenesis is widespead thrughut the Cycadales. 3 ' Temperature rises fllw a circadian rhythm, reaching maxima in the late afternn r early evening and lasting fr t 5 hurs. Thermgenesis is cincident with the time f maximum cne axis elngatin and pllen release, the site f heating being lcated in the cne sprphylls. Cnes f Stangeria are exceptinal in nt shwing any significant heating. The pllinatin and fertilisatin prcesses in Encephalarts and Stangeria are nt well knwn. t is thught that a relatively lng perid elapses between the time f pllinatin f a female cne and the event f vule fertilisatin. Little is knwn f the site f the micrspre at that time r the germinatin prcess. Grbbelaar (unijublished results) ntes that large interspecific differences are fund in the length f the micrpylar tubes in Encephalarts. A useful reprt n spermatgenesis in Encephalarts altensteinii shws the develpment f the gametes t be similar t that in ther cycads, resulting ultimately in tw ciliate spermatzids sme 50 ~ 80 micrn in diameter.'7

250 37 Chrmsme cunts frm Encephalarts and Stangeria cells indicaie cnsistency in the diplid number n=8 and n=6 respectively. 8 A cunt frm rt tissue f Encephalarts wdii gave 'n=8, shwing that the surviving clne f this species is nt an artefact f chrmsme multiplicity.l~ Whilst detailed karytype analyses f the mes-american cycads has been given attentin, largely in elucidatin f the variable chrmsme numbers within the genus Zamia: there has been little such wrk n the African cycads. A karytype survey f these taxa culd cntribute t their phylgeny and culd als prvide answers as t whether r nt these plants have recgnizable sex chrmsmes, a cntentius issue at present.~~ The wrk f Mgfrd,~'~~ wh used flurescence techniques t find the psitin f heterchrmatin in chrmsmes frm rt tip cells f E. caffer and E. lehmannii, culd be usefully extended in this directin. The presence r absence f sex-directing chrmsmes must be viewed in relatin t the reprts n spntaneus gender changes in mature cycad plants. This unusual ccurrence has been recrded in bth extic and Suth African species. The mst authritative reprt details the histry f several specimens f Encephalarts umbeluziensis in a Pretria garden, ne f which prduced a male cne in 970 but a female in 979.~r This plant was the mst expsed f a grup during a freak sub-zer temperature spell in 97. A specimen f E. villsus subjected t particularly dry cnditins is said t have changed. frm female t male (Swanepel, pers. ~.). A plant f E. latifrns, transplanted in 970 t a farm near East Lndn, bre tw successive crps f female cnes with viable seeds but, fllwing a severe drught in 983, then prduced tw successive crps f male cnes (Bursey, pers. ~.). Cnsistent with similar bservatins in extic cycads, all these sex changes appear t be assciated with sme traumatic event; the phenmenn may be ratinalised in hrmnal terms and n the basis f higher physilgical cst t the plant in prductin f large, seed-bearing female cnes. n similar cntext, a recent study f the sex ratis f Encephalarts transvensus in the Mdjaji Nature Reserve, prvides evidence that the availability f water may influence the prprtin f plants bearing female r male cnes in a given year. 4 ' Hwever,a dilemma arises in recnciling an envirnmental influence with a gentypically-cntrlled prcess, especially if sex-chrmsmes are shwn t be present in cycads; thus the mechanism -f gender manifestatin deserves research attentin. Elucidatin f this mechanism culd lead t the artificial cntrl f the gender f prpagated plants and a particularly significant applicatin wuld be the pssible re-creatin f a female clne f Encephalarts wdii.~' A'further area f cycad bilgy t be explred lcally is that f interspecific hybridisatin. Wrk accmplished n the mes-american cycads has shwn the diagnstic value f this t the taxnmists.~b n Suth Africa, a number f natural Encephalarts hybrids have been recrded.~' Their relative scarcity is ascribed t discreet g~graphical distributin patterns and the nn-synchrny f cning tmes, rather than t physilgical reprductive barriers. This viewpint is reinfrced by the cmparative ease with which artificial interspecific hybrids can be btained.

251 38 Anther tpic f interest is the recalcitrant r "wet" nature f cycad seeds. The embry in these prpagules shws cntinuus develpment frm fertilisatin thrugh t germinatin. Thus the lng-term strage time fr cycad seeds is limited and the embrys are subject t necrsis thrugh desiccatin. String Encephalarts natalensis seeds in mist cnditins until the embry is mature, fllwed by incubatin at 30 deg C, gives satisfactry germinatin frequencies,~ but little is knwn generally f the ptimal cnditins fr pst-harvest embry develpment and fr seed germinatin. Similarly, little is knwn f the best strage cnditins fr cycad pllen r its lngevity. nfrmatin f this srt wuld be invaluable in upgrading the few existing cycad seedbank and pllen-bank facilities. CORALLOD ROOT STUDES A cmmn feature in cycad plants is the frmatin at an early seedling stage f dichtmusly-branching apgetrpic rts which ften later appear as crallid masses at sil level. These unusual rt frms have been fund in 49 species, in all genera except Micrcycas which was nt tested. S The structures are usually, but nt always, invaded symbitically in the crtical regins by filamentus nitrgen-fixing Cyanbacteria.~ n a study f the symbints in African cycads, 4 cultures were islated frm 3 cycad species and all but ne were identified as Nstc species, mst ften N. cmmune Vaucher.~3 n E. hildebrandtii Braun & Buche, an East African cycad, the islate was a species f CalthriJ. Hwever, bth the rle f and the inductin prcess f crallid rts require further study; little is knwn f cycad-cyanbint specificity r the details f the nitrgen metablism in these systems. Wrk cntinues at the University f Pretria n varius aspects f the rt symbitic assciatins. n pineering TEM studies n cycad crallid rts, large crystalline inclusins were bserved in cells frm Encephalarts altensteinii and Macrzamia cmmunis.f~ These apparently prteinaceus structures d nt ccur in crallid rts frm fycas circinalis, C. revluta r Din edule. TEM studies n the cyanbint ~n situ and after islatin have fllwed.s~ Similar studies have since been carried ut at the University f Pretria n several Suth African cycad species using bth TEM and SEM techniques (Jubert et al., in press; Chang et al., in p-ress). TSSUE CULTURE The delicate cnservatin status f many f the Suth African cycads is clear mtivatin fr attempting t establish prtcls fr in vitr prpagatin systems. Callus grwth was quite readily btained n leaf~et explants f mst cycads, including Encephalarts and Stang~, n a variety f artificial media.r~ Callgenesis was similarly easily induced n stem and rt tissue segments frm nine Encephalarts species.~7 Hwever, in bth these studies, n further mrphgenesis culd be prmted. Experiments with a wide variety f bth haplid and diplid tissue explants frm varius Encephalarts

252 39 species have been carried ut n several media with different auxin and cytkinin supplements (Osbrne, ~published results). Vigrus callus prductin was again btained in many cases but n further differentiatin culd be prmted. Sme encuraging results have been btained frm primary rt tissue cultures f Stangeria where callus frmatin can prgress t light-induced emergence f meristematic znes, buds and leaves in sequence. rg Despite the necessarily empirical nature f this experimentatin, further effrts culd prve rewarding. BOCHEMSTRY All cycad seeds cntain fairly large quantities f unusual substituted azxy cmpunds which are acutely and chrnically txic. A ntable result f this was when General J. C. Smuts and many f his cmmand were incapacitated fr several days during the Suth African War f after resrting t cycad seed fr emergency fd supplies.~q The plant respnsible is believed t be Encephalarts lngiflius.~ The fleshy sarctesta and/r the ~ch-rich megagametphyte frm ~ycadiflius, ~. ~gene-maraisii, E. ferx, E. hrridus, E. lehmannii, E. lngiflius and E. villsus have either prved acutely txic t rabbits r have been suspected r knwn t be pisnus t man.' Txic and carcingenic effects have been bserved in rats fed n female cne prtins frm E. umbeluziensis, E. villsus, E. lebmbensis and ~ laeviflius." The megagametphyte f E. lanatus has als been reprted as txic and carcingenic.'~ The cmpund macrzamin (methylazxymethanl ~-primeverside)(fig ), first fund in the Australian Macrzamia, has been islated frm E. lanatus and E. transvensus.'l This cmpund, tgether with the clsely-related cycasin (methylazxymethanl ~-D-glucside)(Fig ), has nw been fund in all cycad genera in cncentratins which are genus-dependent, but neither has been fund in any ther plant grup.4~~ Macrzamin is generally mre abundant that cycasin, ccurring at,09 -,86% in Encephalarts seeds and at 4,70% in Stangeria erip~." Further extractin and quantificatin f minr related azxy cmpunds may extend the chemtaxnmic usefulness f these txins, the bisynthesis f which remains t be explred. Recent attentin has been fcussed n the cmpund ~ -amin-~-methylaminprpinic acid (synnym ~ -N-methylaminprpinic acid r BMAA). This cmpund has been islated frm seeds f y'cas~? and has been implicated in the etilgy f human amytrphic lateral sclersis and ther neurmtr disrders such as Parkinsnism and Alzheimer's diseases.'i Whether this cmpund is present in Suth African cycads remains t be seen. The biflavnid chemistry f cycads has been partially researched and appears t be useful taxnmically at least at the genus level.,q4 An unusual feature is the cmplete absence f biflavnids frm the leaves f Stangeria.'~

253 40 All cycads cntain mucilage canals and excsn f leaves r cnes allws cllectin f the exudate. Hydrlysi~ f this material and gas-chrmatgraphy f the mnsaccharides has prvided data which crrelate with genera.~~ Encephalarts mnsaccharides cmprise mainly arabinse and galactse and are similar in this respect t the sugars frm the Australian genus Lepidzamia. Stangeria is nce again exceptinal in nt having detectable amunts f rhamnse r methylrhamnse. Lcal wrkers have shwn that the hydrlysed exudate frm the cne f Encephalarts lngiflius cmprises fucse, rhamnse and 3-0-methyl rhamnse, arabinse, xylse, galactse, mannse, glucur~nic acid and its 4-0-methyl ether.~3 With this species, the mnsaccharide pattern is nt affected by plant age, sex r envirnment.7~ A mre cmprehensive survey f the genus Encephalarts has revealed a similar pattern in 4 species investigated.'~ Current wrk at the University f Cape Twn n Encephalarts cne exudates has highlighted the high prprtins f rhamnse and its methyl ether, and D-glucurnic acid and its methyl ether~~~7 These authrs have used sequential degradatin techniques which enable pstulatin f the structure and linkages in the plysaccharide. A preliminary survey f Encephalarts leaf waxes indicates that species-specific prfiles may be readily btained using gas-chrmatgraphic techniques." n Angisperms, the bisynthetic rute typically leads t hydrcarbns with dd carbn atm numbers, usually C 9 and C 3 mlecules. n sme Encephalarts species there appear t be high- prprtins f alkanes with even-numbered carbn.atms. Thus this area f wrk is imprtant bth fr its taxnmic and its bisynthetic relevance. A cmprehensive survey f cycad leaf hydrcarbns is presently being carried ut. Of the varius enzymes present in plants, the perxidase grup is readily examined by electrphretic methds. Crude leaf extracts frm cycad leaves give characteristic "fingerprints" f several, mainly andic, bands (Osbrne, ~published results). Preliminary results suggest that plymrphism within Encephalarts is absent and that the perxidase zymgrams may cntribute useful data in interspecific relatinships. Serlgical and immun-electrphresis wrk n cycad seed prteins is useful in cnfirming grupings at the family and genus levels (Osbrne, ~npublished results). Opprtunities exist in amin acid sequencing analyses; sme wrk has been accmplished using Encephalarts pllen as a surce f histne prteins.,q-vo The Suth African cycads have been neglected in studies by plant physilgists. n view f the early rigin f the grup, it is anticipated that a C-3 phtsynthetic mechanism will be fund t perate, but experimental cnfirmatin is required. Studies n mineral nutritin and deficiency symptms wuld prvide useful infrmatin t grwers. CONCLUSON The Suth African cycads have quite deservedly been admired and appreciated by btanists, hrticulturists and laymen in view f the decrative appeal and the rarity status f these plants. ncreasing

254 4 attentin is nw being paid t varius aspects f their taxnmy, mrphlgy, prpagatin and bichemistry. n mst cases this wrk is at a fairly preliminary stage and many avenues f research are yet t be explred. Exciting pprtunities and challenges, many f an interdisciplinary nature, await cycad researchers in the future. Nte Table and Figure f this paper are nt reprduced in this Appendix. The reader is referred t the crrespnding details in the main text; Table crrespnds t Table (page ) while Figure crrespnds t Figure 3 (page.).

255 (a) (b) FGURE SEM phtmicraraphs f cycad micrspres, x 3000, scale bar 0. 6 (a) Enc.e.phala.u:-6 ajte.ncv7.-lu-6 shwing smth exine surface and regular clpus (b) Stangeria eripus shwing pitted exine and irregular clpus.

256 REFERENCES. Osbrne R. and Hendricks J. (985). A wrld list f cycads. Enceph4la~tas 3, Osbrne R. and Hendricks J. (986). A wrld list f cycads - first supplement. Enceph4la }'tas; 5, Giddy C. (984). Cycads f Suth Africa. Secnd revised editin. C Struik, Cape Twn. 4. Read R.W. and Slt M.L. (986). Bibligraphy f the living cycads. Lyani4, Dyer R.A. (965). The cycads f suthern Africa. Bathaz.i a 8, Dyer R.A. (97). A further new species f cycad frm the Transvaal, Encephala'r,tas cup idus. Batr,alia 0, Dyer R.A. (97). A new species f Encephala }'tas frm Swaziland. Bathal-i4 0, Dyer R.A. and Verdrn.C. (966). Zamiaceae. n CODD L.E. and De Winter B. (Eds). Fla~a f S.Af~ica, Keleman A., Rbbertse P.J. and Eicker A. (98). Die anatmie van die pinnas van die Suid-afrikaanse spesies van EncephaZM'ts Lehm. J.S.Af~.Bat. f7, Nrstg K. (980). Chrmsme numbers in Z4mia (Cycadales). Car':"lagi4 33, Gilbert S. (984). Cycads: Status, trade, explitatin and prtectin, Wrld Wildlife Fund, U.S.A.. Hall A.V., De Winter M., De Winter B. and van Osterhut S.A.M. (980). Threatened plants f suthern Africa ,Yat ifl4l Sc ierd iric P~g~ammes Repa~t N Osbrne R. ( 986 ). ".,. " " ', ~ncepnal4~tas waall ~ncepnala~tas 5, Cites Secretariat (98). Prceedings f cnference f the parties, New Delhi. Cnventin. UCN. Switzerland. the third meeting f the Vl. Secretariat f the 5. Osbrne R. (985).. Cycads and the law. EtCepha la }'t as f, 8.

257 6. Osbrne R. (985). Btanical histrical mnuments. Bncepha.~a.f' ts 3, Baijnath H., Naid S. and Ramcharun S. (980). Leaf surface characters in selected genera f the Cycadales. P-r'c.E JCJc.09.A'-'. 0, Whitelck L. (986). Cycad pllen studies. Erlcepr:a La.'t s 8, -3. Wdehuse R.P. (935). Pllen grains: their structure, identificatin and significance in science and medicine. McGraw-Hill, New Yrk/ Lndn, pp Van Zinderen Bakker E.M. (953). Suth African pllen grains and spres. Part. Gymnspermae. A.A. Balkema, Amsterdam/Cape Twn, pp 9-. Marshall J., Grbbelaar N. and Osbrne R. (988). SEM Cmparisn f pllen f predminantly indigenus Cycadales. Pster. S.A. Assc. f Btanists Cngress, Jan 988, Univ. f Cape Twn, Cape Twn.. Osbrne R. (988). Cycad seed kernel mrphlgy. Encepha.la.f,tS 3: Chamberlain C.J. (99). The Living Cycads 965. Facsimile f the 99.editin. Hafner, New Yrk 4. Chamberlain C.J. (935). Gymnsperms: Structure and Evlutin. 966 Facsimile f the 935 editin. Univ. Chicag Press, Chicag. 5. Pant D.D. (973). Cyca.s and the Cycadales. Secnd Edtn., Central Bk Dept, Allahabad, ndia. 6. Nrstg K. (98). Experimental embrylgy f gymsperms. n: Jhri B.M. (Ed). Experimental embrylgy f vascular plants. Springer Verlag. Berlin, pp Niklas K.J. depsitinal reprductin 45, and Nrstg K. (984). Aerdynamics and pllen grain patterns n cycad megastrbili: implicatins n the f three cycad genera (Cyca..s, D i n and Zami a) Bt.Ga.z 8. Nrstg K., Stevensn D.W. and Niklas K.J. (986). The rle f beetles in the pllinatin f Zam i a u,''''u,f'acea L. fil. (Zamiaceae). Bi tf'pi ca. 8, Tang W. (987). nsect pllinatin in the cycad (Zamiaceae). {met-'.j.bt. 74, pu,m i La 30. Pearsn H.H.W. (906). Ntes Tf'a.ns.S.A/-,.,.Phi L.Sc. 6, n Suth African cycads. 3. Rattray G. (93). Ntes n the pllinatin f sme Suth African cycads. T ~>ans.hy.sc.s {fr'. 3,

258 3. Marlth R. (94). Nte n the entmphilus nature f BncephaLa-r-ts-. r -,.'ans.liy.sc.s tj-p. 4, Cmptn S.G. (987). nsect pllinatin implicatins. Bncepha.La.f,ts-, Jact-Guillarmd A. 958). Temperature variatins in male cnes f Bncepha.La.f,tS-. Na.tu-)'e 8, Jact-Guillarmd A. 959). Variatins f temperature in male cnes f Br:cepha L a-)'t s' a L t er:s-t e-i r: -i -i Lehm. ;7.Af f '.E t. 5, Tang W. 987). Heat prductin in cycad cnes. Et.Gaz. 48, De Luca P. and Sabat S. 979). t-: ",itf' spermatgenesis f Bncepha.La.-,.,ts Lehm. Ca.f-j/Lg-ia. 3, Marchant C.J. 968). Chrmsme patterns and nuclear phenmena in the cycad families Stangeriaceae and Zamiaceae. Chf'msma. 4, Osbrne R. 986). Chrmsme cunt f BncephaLa~ts wdii. BncephaLa.~ts 7, Nrstg K. 98). Karytypes f Za.m-ia. ch-igua. Cycadales). Ca. -p}' ig-ia. 34, Mretti A. and Sabat S. 984). Karytypes f Zami a pa.uc -ij-uga. Wieland. Pia.nt System. B'ril. 46, Mehra P.N. 986). Letter t the Editr. Bncepha.la.~ts 8, Mgfrd J. 978). Nuclelar heterchrmatin in Encepha.la.f,tS-. J.S tj-,.,.bt. U, Mgfrd D.J. 979). Heterchrmatin in Encepha.la.r-ts. Cytlgia. U, Van Wyk A.E. umbe l wzi ens.. i s-. and Claassen M.. 98). Sex reversal in Bncepha.la.-),ts VeLd & Fl-,.,a.. Dec 98: Meyer J. J.M. and Grbbelaar N. 988). The sex rati f Br:cepha.la.-,.,tst",a.ns",ensus. Paper. S.A. Assc. f Btanists Cngress, Jan 988, Univ. f Cape Twn, Cape Twn. 47. Osbrne R. 985). Sex change in cycads - hpe fr Wdii? Bncepha.la.",ts, Nrstg K. 987). Cycad hybridizatin studies. Fa.i",chi ld r",pica.l Ga -,.,dens- EuL L. 4, Vrster P. 986). Hybridizatin in Er:cepha.la.-,.,ts-. Excelsa, 0-06.

259 Frsyth C. and van Staden J. (983). Germinatin f cycad seeds. S.Af~.J.Sci. 79, Grbbelaar N. (985). Kraalvrmige wrtels. Encepha.~a.r' t$ C, Grbbelaar N., Hattingh W. and Marshall J. (986). The ccurrence f crallid rts n the Suth African species f the Cycadales and their abili ty t fix nitrgen symbitically. S.Ar",J.Et. 5, Grbbelaar N., Sctt W E., Hattingh W. and Marshall J. (987). The identificatin f the crallid rt endphytes f the Suth African cycads and the ability f the islates t fix dinitrgen. S.Af~.J.Et. 53, Grilli Caila M. (970). Crystalline inclusins in cycad rt ndules. G i rn.et.ta.i. M, Grilli Caila M. (975). A light and electrn micrscpic study f blue-green algae grwing in the crallid rts f Encepha.La.ds a~tenste i n i i and in culture. PhjiCigia C, Hensn N. (980). The frmatin f rganised elements and callus frm cycads in culture at Kew. Kew nternal Reprt. Kew, England. 57. Keleman A.. and Small J.G.C. (98). A nte n callus frmatin by stem and rt tissue f sme EncephaLa.~ts species. S.Af~.J.Et., Osbrne R. and van Staden J. (987). fl ", i t 'r' regeneratin f Sta.tjger, i a. er' ~p!.l$. H0'tSc i, Reitz D. (99). Cmmand - A Ber jurnal f the Ber War. 969 reprint f the 99 editin. Faber & Faber, Lndn. 60. Steyn D.G., van der Walt S.J. and Verdrn.C. (948). The seeds f sme species f Encephaia'tO$' cycads) - a reprt n their txicity. S {rr' ).4rE'd.J., Tustin R.C. (974). Txicity and carcingenicity f sme Suth African cycad EncrE'pha.ia. r't$ ) species. S. {r,,)4ed,j. A, Tustin R.C. (983). Ntes n the txicity and carcingenicity f sme Suth African cycad species with special reference t that f EncrE'pha.ia.t,t$ ia.na.t!.ls. S. {rr,.vet {$ $ c. SC, ~lte~i~k B. (974). Occurrence f macrzamin in the seeds f :incre'pr~a. L a. r,t s t r'a.ts""re'flos!.ls and E. l a.na.t w:;. L l yd i a. 37, De Luca P., Mretti A., Sabat S. and Siniscalc Giglian G. 980). The ubiquity f cycasin in cycads. Ph.ytchem i st "y 9, Mretti A., Sabat S. and Siniscalc Giglian G. 98). Distributin f macrzamin in Australasian cycads. Ph:;d chre'm i s t 'r)' 0,

260 Mretti A., Sabat S. and Siniscalc Giglian G. significance f methylazxymethanl glycsides Ph:;.:tch~'l is tf'y, 5-8. (983). Taxnmic in the cycads. 67. Vega A. and Bell E.A. (967). new amin acid frm seeds a-amin-p-methylaminprpinic acid, a f CycrZ$ c i,pc inrzz is. Phj.:tch~m istr'ji 6, 68. Spencer P.S., Nunn P.B., Hugn J., Ludlph A.C., Rss S.M., Ry D.N. and Rbertsn R.C. (987). Guam amytrphic lateral sclersis - Parkinsnism - Dementia linked t a plant excitant neurtxin. Sci~nc~ 37, (reprts) Dssaji S.F., Mabry T.J. and Bell E.A. (975). Biflavnids f the Cycadales. Hich~m.Sy$t~m.EcZ., Gadek P.A. (98). Biflavnids frm the seed testa f Cycadales. Phytch~m is tr"i, Gadek P.A., Quinn C.J. and Ashfrd A.E. (984). Lcalizatin f the biflavnid fractin in plant leaves, with special reference t AgrZth i s r'ustrz. Aus t,",rzz.ht. 3, De Luca P., Mretti A., Sabat Sand Siniscalc Giglian G. (98). A cmparative study f cycad mucilages. Phyt ch~mi st f')', Stephen A.M. and de Bruyn D.C. Et"lc~'PhrZ i rzt't s Z f/g i j 0 h us Lehm. CrZ,'hydr'rZt~ lj~s. 5, (967). (female) The gum exudate frm (family Cycadaceae). 74. Siniscalc Giglian G. (980). Analisi gascrmatgrafica dei mnsaccaridi delle mucillagini di Et"lc~'PhrZlrZ pts loflg ijl ius Lehm. (Zamiaceae). J~ip irlrz n.s. -, Mretti A., Sabat S. and Siniscalc Giglian G. (98). Mnsaccharide cmpsitin f the mucilages in Ef/c~phrZlrZ"ts Lehm. (Zamiaceae). G ir'l". Ht. trzl. 5, Stephens D.C. and Stephen A.M. (988). Exudates frm EflcephrZlrZt'ts cnes. Enc~phrZirZ'ts 3, Stephens D.C. and Stephen A.M. (988). Exudates frm Enc~phrZlrZ'tO$ cnes as chemical taxnmic markers. S.Aj'.J.Sci. (in preparatin). 78. Osbrne R., Salatin M.L. and Salatin A. (987). Cycad leaf waxes. Enc~phrZZrZt,ts, Brandt W.F. and vn Hlt c. (975). slatin and characterizatin f the histnes frm cycad pllen. F~s' L~tt~H; 5, Brandt W.F. and vn Hlt C. (986). The primary structure f histne H3 frm cycad pllen. F~s' L~tt~ r' s 9t, 78-8.

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