?f.aw TISSUE CULTVRE GENETIC MANIPULATION AN^ 40bWTIC HYBRIDISATION OF PLANT CELLS edited hy P.S. Rao, M.R. Hehle and M.S. Chadt,~ Proceedir~gs of National Symposium held at Bhab~.~ Ator~~ic Research Centre Bombay, India 1980. IN VlTRO CULTURE OF AMCHIS OVARIES AND OVULES. D.C.SASTRI. M.S.IULIN1 and J.?.MOSS Groundnut Improv-nt Progrvn ICRISAT. ICRISAT Patanchru P.O. 502 $4. A.P.. INDIA. INTRODUCTION The cultlvatcd groundnut hywaea L.) Is suscsptlbl. to a number of dlseases and pests. There cause a canslderablc loss In ~~!tl.tlsn In vltro. There twln objecttrts arc aimed at ohtalnlm) f mll.blc mrthod Tor cmsslng desirable bit ~ncanrat lble stmcler of i. -. In thls report we presant out attamt, to cullure 011r1es and avules a: dlfferent lntenals mftcr polllnatlm, uld also tc pollinate mules, In rltro. vlald (1) and this Justlflas the urgent attmt~on of groundnut b~orop~st. but not a11 tha rmslstance genes hwa been found In A.hypgaee. Fort,,. nately, In the wlld collecttonr of the genus a number of species are knam to be raslstant to ma dlseases (2). In the genus Ar.chil there are swan sections; hypogaea has bean crossad only wlth spec1.s from Its an sectla, Arachls. though rme other Intcrscctlsnal hybrldr have baen produced (3). Ssme Inte.3.. 7 lonal crosses for- pads, but :c. I IZ-d ovules Gossyplm arborem zvc-' r stage x G.hlrsutVn t.hlrsutlm C hlrsutm Zn r h Ze~hyranthes sp NtUre seeds are mt posslbla due tc s,rly degmeratlon of cnd0sp.m. as In A.hypogaca x A.dlwol (41, or of embryo or both. naberr of t h Sactlan Rhlzamatosae ara particularly valwble for dlscrse and rmslst*nce. but cannot be crossed wlth A.hypogaea. A proj-ct was stortad In the G:.,.. 2.it Improv-t Prq-nm at ICRISAT to Investigate Intersect Ion,' incmpatlbllltlar and to d*vel0: tachnlques to wercmr them. Enco~-. :e bi the reports of r~ccc.sf*~ culturm of wular at various stagr n' ot,elopmcnt In a nwlber of tu' (Tabla 1; to0 also 6); we started cult,:re of Arachls nmtlcol* OVJ~~' In vltro. V. have also attmpted to nlrml.ta p.9 mlmgatlan wd pd ~ntrlbutlon of the Internatlunal Crops Research Instltut. for tk *'- 1 Tropics. ICRISAT J,ur...' i r t.cle Arprwal k. 5 C - 3.
betwn 15W avd 1/00 hrs end pol'i -an 0600 anu tha follalng day. Tha flwrs or 5 ~ - ~ 3 - ovaries +. i Ilsc wlth chlorlne water, and ovules and L., >r the ter 3' m of t dweloplng peg wre dlrsected under awc- condltlons, I rmsferr to culture tub. at dally Intervmll fra!'.la dry of polllnatlar untl 10 days after POlltnmtlon (Table 2). Ovules were elm polllneted In -'lesults,.,hm~d ~llght e?-~at~or 2r - - -'ant rf'--,cqtci- 711 ations -.I.~a. n &as.~li d51ed, 2eqs *+,-. 7,ere no: 35 th-v eared '.; And :, oa ran the tlp batore panetrat "3 'he 301 1 vltro. A mrl- of lof of the mlla geminated a n -viler are polll- 2. kt* or w dwalowmnt after PI I ~ n ~ t ~ m - nated i - "if-o. "any 6 ' -k.~e=,rw long but ier -- "ere obsewed Dayr after P q Impth No. of C~~IS No. or OVL~.-to t~ 3v.' -=,t growth r-' the war hen pol! Inatton In mbryo(aqpmd cultur 'rey rsre wlturmd and wll ->tad In vltro on htrasnlgc ard Ska>p'r madlum In vltro pol llneted 134 uppl-ted vlth caleln hydr9l~rrte 600 mg/l). within 2-3 w~+k, the Day of polllnetlan 22 wlar grc* to 5 tlmr their original size, In I2 hrs Ilght altematlnp I - 3 days less th.n 1-2 13 Ith 12 hr) jark. Later they kc- bran end necrorlc, attar poll lnqtlm 1.0 a 4-6 days 1-2 a 3-7 10 The mler dld not rerpnd *rl l to othor media (~ablm 3). There after pot IIM~I~ was mly l rnarglnal Incrwre in th. size 7-10 dbp 3-4.5 of t b mules which bwr b m Q 8-16 IZ eftar poll l~tlon rlthln threa mks and curd daveloplng lrre p.ctlve of th. photocondlbrr thn 10 dap 7-10 ca 2 b after pol I mat ran (In ~ 1 1 ) tlcnr. h a than ZW -lay of A.montlsole nn culturbd at varlarl 7-10 a I 12 (mt In wlfk 1 mom t h ltwer, but no trdllngs awld k rrld. Elghtvfwr of there wla* 5-7 a L 16 33 Cln wll) 1 nr. tmrferred to culture more thm 7 days after ~ollln.tlon, l st.9. 5-7 a I 11 (not In -11) L nt at& ~ r t l n (It) reparted he sorld recover "ran ~wdllno1 frm For tk tnltlbl mxmrlnmmta th &la of hreshlga md SLrOOp (26) Wlra (27). ~t.o md nfchayluk 08). brtln (12) md flv mnd Zmsky (29 *lth or *Itkt mdlcfutlma nra usd. 74 owlu. A rml[.bla dlu, techfgu* *d fr.4tmrrt stlll evelop.d kfora mutlnelt culmr. wuln frm 1norp.tIble c*s*@.
Table 3. Responses of A.montiwla ovults to some rnedls Wrl:c wlth Ovules HS MS wlth KF Hart In - IAAO nlgll) Kn I AA CH Fran NC Sl lght Hochange,but Undertrlal HT N C NC In ovules poll lnrted swcl l lng; ovules when tra pegs up fiawarl runatn green trrnsferred frm to 1L a long for 6 wcks KM show rmi I Ing then become fm NC after Brown In NC N C NT NC 8 unpol l lnrtrd 9 weeks b ~ e k s f lmrr Pol I lnated Sl lght Inltla) Slight rmlllny Swlllng 6 Very slow Sane ovules -- In vltro Swtlllng swlllngthen thennochange ocorlonally ce1'.15 form callus In 8 wecl. s no chanse, callus In for- ~tlon brow In j weeks 4 weeks I I AbbravIrt Ions: CH Casein hydrolysate HS Rurrshlge anc Skoog'~ wdlum IAA Indole acet lc rcld NC No change Kn K.o C Mlchyluk's dlun NT Not trled KN Klnet in ZZ ZIvalmsky's~lun
varlos Zlv and Zm3ky could culture prps md Induce C.ud fonwtlm In rachia hyposaaa cultured m t varlws stager of devmlopnont (19).. h samm ~dltions of culturm, A.nontlcola pegs dld not respond. i i ~ urashlg- and Skoog's mdlun with klnrtln (4mg/l1 1 1 2, uiar (s. cldl.~,, I i l hq.d prollflc callus Inltiatlon wtthln a ru(; aftmr culture. Th. ~varl-s whlch r-sponded hat rr- tram fl-ra 2 and 1 d.ys aft-r polll- atlon, particularly vhon they bar- cold tlrated m t boc for 4 or lore wrs. In three raks th- callurn rrr profusr and a frr org-tlc Iraas auld br obsarvad in tho form of bolblls. Hand md mlcrotol. mactlons ravamlad that rylra hd b6.n fomd and tbt thr- wn rwlons filch wore dlffarentlatlng Into rhmts. rhrn such ullus.nm Xylopmasls war a r m frrqvrnt trmsfrrrd to Uhlta's adlu rlth IM (4 q/l).,a of rhos* protukrmnces alongetmd to form pal* gmul stalk IIke irructurms which rmlnod organlrod but dld not dlff-rmtlatr furrhcr. uhan tha wgs wsrm cultured on Uhlte's nodlun rlth IM (*q/l). me culturmd p.9 showd m lnltlal callus at a rmqlon Just blow th ~vulas and lacar tha tlp rhorod gootropism, tha latter bolw th normal.aspens- of tho p.9 In vlvo. A few of th. formulatl~s trlod w far hm supportad smth or callus fomtlbn whn usd to culturn owles, so our efforts are ro Iiroct-d at tasting th* mfflusy ot vmrlous w t s In t* d l a md athr factors.
8. J.*.Stmart and C. I.dsu, J.tieredl rr L ( 779). Arachls ovules do rerwnd better on a Alum upp1rvlt.d 4th caraln hydro1-3te. Careln fdmlysate rams esmtlal for m Inltlal graith ar has 'eon shcn for ovules In Abelmorchus (11). Goacyplw (Zn a 417) \:Jt Nlcotlna rustlca and U.tabacum (19). Haaver. on a mdfum composed of HS nucmlamnts, Haller'r mlcmlants, CvlIu'r vltvnlnr with klnetln md glbberellln, Rartln (12) has abla to rear ram reds and seedllngr fmm A.hypogaea wules. Harcvar, the brt grath of set fed A.mntlcol8 owlar occurred when the tlp of t h young gynophors was cultured, wltbut exclstlng th ovul*~, on RS wlth caraln hydrolyrata (500 d l ). d a Klnatln rsgularly In&-d profus* callus fonrtlm within thrm when ycung gynophoras 2 and 3 d~ys attar polllnatlm wra cultured on lls with klnetln (%/I). ct.d krt tkrs did not form shoots or roots. A feu dlffarentlatlng zmas carld br detc Only 3mt like structurar mra raan. ha. cultures MY need to br trmrfarnd to othr mdla to contlnua dlfferentlatlm. To our Lmo*ldga, ullus lnltlatlwr from warlar md rubssquant rogmmratlon of plants In Iegws tus kerr wuerar- ful In only au I nstnu. Wlth Trlfol lu pratmn (31). 11. V.P.S.Bajaj. Proc. Vat1 Inrr. Scl. 8 I J E B, 175 (1364) 12. J.P.Hartln. OICa~insux u, 155 (I?:') 13. P.C.Jo hl. "Plant 5bryology: A r y rum Councll of 5' i c and Ind~~strlai Research N -151, pp 199 (19621 4. A.A.H.Eld. E.De.Lsnghs and L.Vaterkeyn, La Ccllule 69, 359 (I:i~l. 5. C.A.8easley and I.P.Tlng. hr.j.bot. 60. 130 (1973) 6. R.N.Chopra and P.S.Sabharwa1, 'Plant tltsue wrd organ wlture - A' svwoslum" (P.rahsshrarl d M f. 9angawaq.ed) pp.257. lo?srnatlonal Soclety of Plant *orpblogl~t$.delhl(i%j). 7. H.Kuwada and T.Mbuchl. Japan J.Bred. 26, 298 (1376). 8. S.m.Read and G.B.Co,lliir, J.Yeredlty 2, 311 (1978). 9. K.l(.nta and P.Mahe$hwarl. Phyranorphologr z, 210 (1%3), 10. N.#aheshuu.rl. Science x, 3hZ (1958).!I. H.Hahashrar1, and M.La1. Phytmrphology x. 107 (1961) 2. T.Uakizuka and T.Nakajlm., Japan J.lr.cd. 2. 182 (1974). :3 T.'dakizuka and T.Nakajims. Japan J.Brtcd. 3, 161 (19751,.4. F.L.Harner, her. Potato, Jour. 1, 282 (1554). 5. T.Nalojlma, Y.Doymu and H.Eatsumoto, Japan J.Breed, 373 (1%)). 26. T.Hurashlga and F.Skccq, Phvslologla P1.2, 473!1%1). 27. P.R.Vhlta, A nond book of Plant Tlssue Culture. J.C.ttall?raSS. Mcastsr (1943). 28. K.N.Km and M.R.Rlchayluk. Planta s, I05 (1975). 29. m.zlv and E.Zamsky. Ann.lkt. 579 (1975). 30. P.C.Joah1 and B.tI.Johr1. PhyfomrphOlogy z, 1% (1972). 31. K.Horvath Buch and R.B.Snlth. Pl.Sclencr Letters & 231 Cl979). REFEREMCES 1. II.U.Glbbon8. Dovalopmnt u ~ Trmshr d of Techmloqy for th. ralnted agrlailtun md th SAT farrr: ICRlSAT (1979). 2. J? s 8 hoe. tnt. lagw Con?. bu (1978) (In pmrr). 3. ~.?.Gmr). L W.C.6~~~kradlty 185 (1979). 5. E.L.Johmsm L B.V.Snlth, k*r.j.bt. bj, 250 (19%). I. ~.kg$.n, (nbryopmslr In vaswlar plmtr. kd..lc Prmr. (1976). 6.M.S.hndIr. 80t,Cu.&7(1972). 7. C.A.Bmalq, Blosclula 906 (1971).