In vitro Culture of Ovules and Embryos from some Incompatible Interspecific Crosses in the Genus

Arachis L.

Nalini Mallikarjuna and D.C. Sastril

Abstract

In vrtro culture of ovules and embyros IS commonly used to produce hybrrds from rncompalrble crosses. In several mlerspecrlrc crossar in Arachlr, ovule and or embryo cullure rs necessary because the ovules do nor develop lully in vrvo

Ovule srzes from drfferml crosses and dliierenr hormone lrealmenrs range lrom less then 0 3 mm lo 5 0 mm, rarely 7 0 mm Ovules smaller than 3 0 mm had to be cultured mlrre as the proembryos rhey conlamed were roo small lor drsseclron and cullure Ovules larger rhan 3 Omm were drssecred and lherr embryos cullured

All the ovules were culturedon MS medra with or wrlhoul agar and with diilerenl concenlrrtrons of various hormones

Dependrng on the hormones used and therr concenlratrons, drilerenr responses were observed Surface callus formatron, greenmg, swellrng. and browntng were common rn cullured ovules Embryos emerged from some of the ovules end planllers have been oblarned Cultured embryos showed srmrler responses, and some cultured embryos germinated, and developed rnlo planllsls

Culture in vrlro d'ovules el d'embryons rssus de cerrains crorsements rnterspbcrlrques rncompalrbles dans le genre Arach~s L La culture d'ovules el d'embryons rn vrtro eslune mblhode courante de poducfron d'hybrrdes 1) partrr de crorsemenls rncompalrbles La culture d'ovules el ou d'embryons s'avdre nkessarre dens plusreurs crorsemenls rnterspbcrfrques dans le genre Arachis dtant donne le ddveloppement rncompkt des ovules m vrtro

La tarlle des ovules provenant de drftdrents crorsements el ayanl sub1 drlfbrents trartemenls hormonaux varre de morns de 0,3 mm 1) 5 mm. rerement7.0 mm Les ovules de tarlle rnlbrreure b 3 mm dolvenl Btre cultrvbs enllers, les proembryons qu'tls conlrennent blant trop p ~ l r l s pour la drssectron el la culture Les o v u l e ~ de drmensron superrsure b 3,O mm son! drssbqubs el leurs embryons cultrvbs.

Tous les ovules sonl cullrvds sur des milreux MS gdlosds oti non el b drffbrsntes concentrations de ~ I V B I S B S hOrmOMS

On observe des rdponses d~ifdrentes selon k s hormones utrlrsbes et leurs concentrations Formetron de cals en surlace, verdrssement, gonlkment el brunrssement son! couranls chez lea ovules cullrvbs Des embryons se Son1 dbveloppds dans certarns de ces ovules el de leunes plants ont pu Bbe obrmus. On constate 10s mOme~ r b a ~ r l O n ~ chez les embryons cullrvds cerlarns ernbryons culhvds on1 germb el se sonl dbveloppes en plants

Introduction the most commonly used, and hybr~ds have been produced In about 50 lnlerspeclt~c crosses by

01 the several methods ava~lable to overcome bar- embryo culture (Raghavan 1977, Sastrl 1984) end riers to hybridization, in vitro culture of ovules and in more than ten lnterspeclflc crosses by ovule embryos, which would otherwise abort. has been culture (Sastrl 1984, Saslfl el el. 1980. 1982.1983)

1. R-rch Auocirtr and Cylog.cwtici8t. Groundnut Imptwml Progrrm. ICRISAT, Pl(lnch.ru P . 0 . A.P 502 324, India

Our earlier investigalions on Interspecific incom- patibility in the genus Arachrs indicated the need to culture ovules or embryos for hybrid production (Nalini and Sastri 1985). In Ihe crosses between Arachis hypogaea x Arachrs sp PI 276233, and other tetraploid accessions of the section Rhrzo- matosae, pegs were rarely lormed. If pegs were formed, they degenerated before they entered the s0il;and the seeds aborted. The ovules and the proembryos in these aerial pegs were very small. Our preliminary attempts lo culture such immature ovules even from selfed pegs were not successful (Sastri et al. 1980). We have induced pegs and pods from some incompatible pollinations by hor- mone treatments (Nalini el al 1983; Sastri and Moss 1982; Sastri et al 1982, 1983) in sufflclent numbers and maintained growth to a stage at which it was posslble to dissecl and culture ovules and embryos. The objective of the present Investl- gation was to study the hormonal requirements In Ihe medlum for survival, sustalnlng growth, and promoting subsequent development of Immature ovules or proembryos to produce hybrid plants from these crosses.

Methods All pegs and pods induced from lncompatlble crosses (Nalinl and Sastri 1985) were carefully removed from the soil about 30 to 40 days after the last hormone treatment. The soil was washed from the pods in running water, surface sterilized with Clorox for ten minutes, and washed thoroughly twice with sterile water. Washing in sterile water and subsequent operations were performed under aseptic conditions.

Ovules larger than 3.0 mm were generally dls- sected to extract the proembryos for examination and culture.

Ovules and embryos were cultured on Mura- shige and Skoog's (MS) med~um (1962) with or withoul agar, with different concentralions of sucrose, and with hormones [klnetin (Kn). Senzyl- amino purine (BAP), lndole acetic acld (IAA), Naphthalene acetic acid (NAA)] at different con cent ratio^ is.

When liquid media were used ovules were sup- ported on filter paper bridges in 15 mm x 50 mm screw-topped glass vials such that the placenta was in direct conlact with the filter paper. When agar media were used, the placenta or the placen- tal areas of cultured ovules were kept in direct contact with the surface of the medium in (25 mm x

150 mm) rlmless glass tubes All the cultures were Incubated at 25°C t 3" with 10h pholoper~od (fluo- rescent and incandescent illumlnatlon a1 about 4000 lux)

Results and Discussion Ovule Cultures

Mosl ovules were between 1 0 and 3 0 mm, with some up to 5 0 mm, but rarely reachlng 7.0 mm Slnce all the pods from hormone-aided pollinations on a plant were harvested on the same day their ages ranged from 50 to 70 days after pollination. There appeared to be no correlallon between sues of ovules and age after pollination.

The response of cullured ovules depended on their size at the tlme of culture, and Ihe medium employed. The responses could be broadly categ- orlzed Into the following

a, Increase in slze of ovules with no change- in color,

b Increase In slze and greening of ovules,

c Increase In size and brownlng of ovules,

d growth and emergence of embryos,

e callus production from surface of ovules.

I. greening of ovules, and

g. brown~ng (necrosis) and shrinkage of ovules.

The percentage of cultured ovules showlng desirable responses on a few comb~nations of media are presented In Tables 1 and 2. Among the ovules from crosses of four cultlvars with Arachrs sp PI 276233 cultured on differenl media, cultlvars MK 374 and TMV 2 were found to give better results on both llquid and sem~solid media than those of other cultlvars (Table 1. Plate 4b,c,d).On semisolid I medla the responses were slower than on liquid' medla Ovules surv~ved longer In both agar and liquid media containing BAP-NAA than when Kn- IAA comblnations were used. But Kn-IAA appeared to be better than BAP-NAA at stimulating embryo growth, development and emergence from the ovules (Tables 1 and 2).

Martin (1970) observed a beneficial effect on ovule growth with kinetin and gibberellin; kinetin at 0.5-1.0 mg/l had the best effect. In our studies, even lower concentrations of kinetin, (0.1 or 0.2 mgll), were better than BAP.

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Figure 1. Poorly-differentiated embrjos from A. hypogaea cv MK 374 x Arachis sp PI 276233

a. globular proembryo dissected from an ovule (1.5 mm) cultured on liquid medium (MS Suc. 5% + Kn 0.1 pprn + IAA 0.1 ppm) for 20 days. ( x 122)

b. swollen globular proembryo dissected from an ovule (2.5 mm) cultured on liquid medium (MS + Suc. 5% + BAP 0.5 ppm) for 74 days. (x 106)

c. heart-shaped, but elongated proembryo dissected from an ovule (3.0 mm) cultured on liquid medium (MS + Suc. 5% + Kn 0.25 pprn + IAA 1.0 ppm) for 46 days. (x 96)

d. overgrown late heart-shaped proembryo from an ovule (3.5 mm) cultured on liquid medium (MS + Suc. 5% + Kn 0.25 pprn + IAA 1.0 ppm) for 77 days. (x 54)

Figure 2 a. callusing ovule (0.5 mm) cultured on liquld medium (MS + Suc. 5% * Kn 0.1 ppm) for 41 days from A. hypogaee cv TMV 2 x Arachis sp Coll 9640. ( x 11.7)

b. differentiating callus from a globular embryo dissected from a 3.0 mm ovule cultured on semisolid medium (MS + Suc. 3% + NAA 0.75 ppm + BAP 0.5 ppm) for 79 days from the cross A. hypogaea cv MK 374 x A. glabrate. ( x 11.7)

c, late heart-shaped embryo dissected from an ovule (3.5 mm) culturedon liquid medium (MS + Suc. 5% + Kn 0.1 ppm) for 19 days from the cross A. hypogaea MK 374 *Arechrs sp PI 276233. (x 1 1.4)

d. cotyledonary embryo with one well-formed cotyledon, dissected from an ovule (3.0 mm) cultured on liquid medium (MS + Suc. 5% + Kn 0.1 ppm) for 85days from thecross A. hypogaea cv Chico x Arechis sp PI 276233. ( x 13.3)

e, late cotyledonary embryo (note poorly-developed embryo axis) dissected from an ovule (2.0 mm) cultured on liquid medium (MS + Suc. 5% + Kn 0.1 ppm + IAA 0.2 ppm) for 47 days from the cross Arechis hypogaea cv Chico x Arachis sp Coll 8649. (x 12.2)

1. embryo similar to that shown in e, from the cross A, hypogaea cv MK 374 x Arachls sp PI 276233, 20 days after culture. (x 14.4)

Table 3. Percentage embryos responding from different Arechla hypogwr cuttivan crossed with Arachia rg PI 276233 on MS medium.

Embryos respond~ng' (%I Concentrattons 01 addrt~ves

Cultwars Kn (mgll) IAA (mg/l) BAP (mg 11) NAA (mgll) Sucrose (%) Agar(%) Robut 33.1 MK 374 TMv 2

010 0 1 5 0 6 66 0 22 0 3 0 7 25 00 0 0 0 50 2 00 2 8 33 0 0 0 50 0 50 3 0 7 3 57 26 1 1 0 0 0 50 0 75 3 0 7 0 0 5011 0 0 0 50 2 00 3 0 7 21 42 2000 2727

1 Embryos whlch IHSp(Xld0d 1nCreBSRd In SIN and became green

Conclusions These studies on media and hormones have increased the survival, growth and development of hybrid ovules and embryos in culture. Changes In media necessary for continued survival, and better growth and development of ovules and embryos are being investigated, but there is now a method to produce hybrids in culture between specles of Ara- chis which were previously incompalible. This is an important step towards transfer of desirable char- acters for the genet~c lmprovemenl of Arachls hypogaea

References Nalini, M., and Sastri, D.C. 1985 Ut~l~zat~on of Incom- patible specles In Arach~s Sequentlal hormone appl~ca- lions. Pages 147-151 In Proceedlngs of an lnternatlonal Workshop on Cytogenel~cs of Afach~s, 31 Oct-2 Nov ICRISAT Center, lnd~a 1983, Patancheru. A P 502 324. International Crops Research lnsl~tute lor the Seml-Ar~d Trop~cs, lndla

Nalini, M., Sastri, D.C., and Moss, J P 1983 Hormone- a~ded peg, pod and embryo development In ~ncompat~ble crosses In the genus Arachls L Page 259 In Abstracts of contr~buted papers 01 the XV lnternat~onal Congress of Genet~cs, 12-21 Dec 1983, New Delhr, lnd~a Pl 1 New Delh~, lnd~a Oxford and IBH Publlsh~ng Co

Martin, J.P. 1970. Culture In vltro d'ovules d'Arachlde Ol6ag1neaux 25: 155-1 56

Raghavan, V. 1977 Appl~ed aspecls 01 embryo culture Pages 375-397 m Fundamental and applted aspects plant cell. tlssue and organ culture (Reinert. J , and Ba

Spr~nger.Verlag Y P S . eds ) Berltn. Federal Republlc ol Germany

Sastri, D.C. 1984 lncompal~b~lrty In angiosperms and slgn~l~cance In crop lmprovement Advances In Appl~ed B~otogy 10 71.1 1 1

Sastr~, D C , and Moss, J P 1982 Enects of growlh regulators on lncompat~ble Crosses In the genus Arachls L Journal 01 Experimental Botany 33 1293-1 301

Sastri, D.C., Nalini, M. and Moss, J.P. 1980 In vltro culture 01 A~achls ovarles and ovules Pages 366-373 n Proceedlngs 01 a Nallonal Sympos~um on Plant Tlssue Culture, Genetlc Man~pulatlon and Somattc Hybr~dlrat~on 01 Plant Cells. 27-29 Feb 1980. Bombay. Maharast)tra. lndla (Rao, P S . Heble, M R . and Chadha. M S , eds ) Bombay. Maharashtra, lndla Department of Atomlc Energy

Sastri. D.C., Nalini, M., and Moss, J.P. 1982 Tlssue culture and prospects for crop Improvement of Arach~s hypogaea and other 011 Seed crops Pages 57.64 In Tlssue culture of economically Important plants pro- ceed~ngs of me Internatanal Syrnpostum 28-30 4 1 1981. Nat~onat Unlvers~ty 01 S~ngapore S~ngapore (Rao A N ed ) Committee on Sc~ence and Technology In Oevetop~ng Countr~es and AsIan Network lor B~otog~al Sc~ences

Sestri, D.C.. Moss, J.P., and Nalini, M. 1983 The use01 In vltro techn~ques In groundnut Improvement Pages 365-370 m Plant cell culture In crop tmprovement (Sen S K . and G~les. K L , eds ) New York. USA Plenum Press

Murashige, T., and Skoog, F. 1962. A revised medlum lor rapid growlh and bioassays wlth tobacco tissue cul- tures. Physiologia Plantarum 15. 473-497