Genetic improvement of crops by utilization of characters from

wild species, which often have desirable traits not present in the

cultivated forms, has been the subject of serious attention. The

interspecific transfer of genes has not always been simple and

straightforward because of reproductive barriers and genetic incompatibility.

This is true in the genus Arachis where a number of

species have been identified as sources of resistance to fungal and

viral pathogens as well as to insect pests (1 1. A few species have

been crossed with A. hypogaea and their derivatives produced (1 -

41, but there are 'l; number of species which are not crossable with

oundnut (5). This limits the scope for crop improvement, and the

sS. tuation necessitates the adoption of existing methods, or develop- ment of new ones for the production of hybrids following these desirkle

wide crosses

Moss, J P

Nalini, M

Sastri, D C

ICRISAT Open Access Repository

THE USE OF -- IN VITRO TECHNIQUES IN GROUNDNUT IMPROVEMENT D.C. Sastri, J.P. Moss and M. Nalini Internatioaal Crops Research Institute for the Semi-Arid Tropics ( ICRISAT) Patancheru P.O. 502 324 A. P., INDIA INTRODUCTION Genetic improvement of crops by utilization of characters from wild species, which often have desirable traits not present in the cultivated forms, has been the subject of serious attention. The interspecific transfer of genes has not always been simple and straightforward because of reproductive barriers and genetic incom- patibility. This is true in the genus Arachis where a number of species have been identified as sources of resistance to fungal and viral pathogens as well as to insect pests ( 1  1. A few species have been crossed with A. hypogaea and their derivatives produced ( 1  - 41,  but there are 'l; number of species which are not crossable with oundnut ( 5 ) .  This limits the scope for crop improvement, and the .tuation necessitates the adoption of existing methods, or develop- sS ment of new ones for the production of hybrids following these desir- k l e  wide crosses. The genus is predominantly diploid (2n = 20) with its species grouped into seven sections ( 5 ) .  The cultivated species is a tetra- ploid in the section ARACHIS and cannot easily be crossed with other tetraploid species except A. monticola, the only other tetraploid in section ARACHIS. The other tetraploid species belong to the section RHIZOMATOSAE, and the attractive feature of these rhizomatous species is that they possess multiple resistance (6). Current know- ledge of the genus is insufficient to decide on any specific method for breaking incompatibility, In earlier attempts to cross four wild species including - A. plabrata, the one named tetraploid species 366 D. C. SASTRI ET AL. of the section RHIZOMATOSAE, with three cultivars of A. hypogaea, 25 pods were obtained which were 1/2 to 3/4 of the normai size with eborted seeds about 1/16'' long (7), although no details of number of pollinations were given. Gregory (8) also reported pod production in these crosses, but no embryos or seeds were formed. Our results show the frequency of peg formation in such crosses was up to a maxi- mu& of 21.3%, none of which bore pods with well formed seeds (3). Johansen and Smith (9) observed 20 ovules of A. hypogaea, 2-7 days after pollinations with A. glabrata, and foun? that there were no pollen tubes in the ovarTes, and fertilization had failed in all. Recently Murty et al. (10) have recorded similar observations and reported that the time taken for fertilization varied from 6 - 48 hours, and the maximum frequency of fertilization observed was 56%. It should be emphasized here that Arachis is one of the few geocarpic taxa. After fertilization, a stalk (the gynophore) de*, ops between the ovary and the node on which the sessile flowers a& borne. This stalk elongates downwards until it has pushed the o into the soil. Seed development is almost absent during the aeri '61 I growth of teh gynophore and is resumed only after the peg is buried in the soil. Therefore, it was decided that priority should be given to meth- ods to produce plants from the fertilized ovules that were formed after wide crosses, but did not develop in vivo. -- EMBRYO CULTURE Culture of embryos in incompatible crosses in Arachis was sug- gested by Gregory in 1946 but was not taken up. Dissecting and cul- turing proembryos from about 1.5 mm long ovules is technically labor- ious and not always successful. Gynophore development and pod initi- ation and development from these incompatible crosses have been indu- ced by hormone treatments (11, 12). Embryos in such cases grow to late globular or heart stages. Embryos at later stages were also % observed in very rare instances. The younger embryos, when cultu . ed, occasionally formed callus on MS with IAA or NAA (1 or 2 ppm) 4 with Kinetin or BAP (0.5 or 1 ppm), but then became brown and necl ' tic. The embryos later than heart stages formed healthy callus ano' shoots. The rare well-developed embryos grew normally when cultured on White's as well as on MS medium. Culture conditions favoring the growth of embryos at globular stages are being investigated. IN OVULO EMBRYO CULTURE -- In ovulo culture of embryos has been useful in a number of -- plants where the dissection and culture of proembryos is difficult (13 ) .  Martin (14) cultured ovules from aerial gynophores of - A. hypo- TECHNIQUES IN GROUNDNUT IMPROVEMENT 367 gaea and obtained plants, using MS with Heller's microelements, vita- mins, amino acids and casein hydrolysate as in Dulieu's medium, and 0.5 - 1.0 mg per litre of kinetin. Sastri et al. (15) used a number of media but were unable to culture _A. hypogaea ovules, even on Martin's (14) medium, and attempts at ovule cultures were discontinued. However, in vitro -- pollination of ovules had been achieved (15). Culture of whole ovaries, or gynophore tips, was, therefore, attempted. OVARY AND GYNOPHORE TIP CULTURE Ziv and Zamski (16)  were the first to successfully culture the rminal segments of aerial gynophores and to obtain pods. Using Q ir medium with various concentrations of kinetin and NAA and dark incubation, we failed to induce pod formation in peg tip cultures of monticola and A. hpyogaea. However, MS supplemented with 2 mg of b and 0.5 rng O£-BAP per litre has induced pod formation in dark incubated peg- tip cultires of A. hypogaea and A. pusilla (a diploid wild species incompatible with-all other species of ~rachis). Some pegs from incompatible pollinations were cultured on this medium but without any success. Attempts with ovary cultures have resulted in callus formation and details have been reported earlier (15). An- other observation worthy of mention is that on this medium pegs form callus which differentiates into plants. Rangaswamy et al. (17) reported root formation from callus in pericarp cultures. THE FUTURE The importance of groundnut as a crop in developing countries, and as a source of edible oil and protein for developing and develop- ed countries, necessitates an objective realization of the problems reducing productivity and demands diversification of approaches to IK: p improvement, particularly when conventional methods of breeding e been unable to alleviate certain problems. The recent interest in, and application of, tissue culture methods in the improvement of me crops, notably potato, rapeseed and sugarcane, has stimulated roundnut scientists to turn their attentions to a wider range of B approaches. Some of the initial problems associated with utiliza- tion of wild species have been overcome. Despite the ploidy differ- ences between some wild species and the cultivated groundnut, Spielman et al. (18) produced fertile interspecific hybrids in large numbers. More recent reports of plant regeneration in tissue and organ cultures (19, 20) have now created hopes of regeneration from single cells and application of these techniques to some of the refractory problems in groundnut breeding. The importance of haploids in plant breeding is now too well 368 D. C. SASTRI ET AL. recognized to elaborate. The success of Mroginski and Fernandez (21, 22) in inducing plantlets in anther cultures from the cultiva- ted and a few diploid species of Arachis has stimulated our efforts to produce haploids at ICRISAT. Plantlets have been obtained from anther cultures of - A. pusilla and - A. sp. P. I. No. 276233. . Production of plants form meristem cultures (19, 23) and their survival after freezing for 28 days (24) should be utilized for gen. eration of disease-free populations, long term storage and inter- national transfer of germplasm. Whilst our present knowledge is being utilized to realize these objectives, efforts to explore othe: possibilities will be initiated. Foremost amongst these must be spontaneous and induced variability in cultures and selection of cell lines with desirable traits. In a very recent report Bajaj et al. (25) have obtained genetically variable plants from the anther derived calli of - A. hypogaea and a diploid wild species, - A. villod The recent developments in methodologies should, therefore, soon lead to realization of hybrids in large numbers from incompatible crosses as was achieved in compatible species (2, 18). In the meantime optimization of conditions for culturing a range of tissues should stimulate efforts to produce agriculturally desirabll plants by other methods. REFERENCES 1. J. P. Moss, Wild species in the improvement of groundnuts, in: "Advances in Legume Science, I' J. Summerfield and A.H. -Bunting, eds. , Royal Botanic Gardens, England (1980). 2. V. S. Raman, "Cytogenetics and Breeding in Arachis ,I' Today and Tomorrow Printer and Publishers, New   el hi-6). 3. A.K. Singh, D.C. Sastri and J.P. Moss, Utilization of wild Arachis species at ICRISAT, in: "Proceedings of the Inter- national Workshop on ~roundnzs , " R. W. Gibbons, ed . , Patan- cheru, India ( 1980 ) . 4. H. T. Stalker, Cytogenetic investigations in the genus Arachi in: "Proceedings of the International Workshop on Ground- -nuts," R.W. Gibbons, ed., Patancheru, India (1980). 5. M.P. Gregory and W.C. Gregory, Exotic germplasm of Arachis L. Interspecific hybrids, J. Hered. 70:185-193 ( 1 9 7 9 r  6. R. W. Gibbons, ed. , "~rocezdin~s of the International Workshop on Groundnut s , " ICRISAT, Patancheru , India ( 1980 1. 7. F.H. Hull and W.A. Carver, Peanut improvement, Florida Agric. Exp. - -  Sta. Ann. Report 1938:39-40 (1938). 8. W.C. Gregory, Peanut breeding program underway. Research and farming, -- 69th Ann. 5. - N.C. - Agric. 9. %. 1946:42-44 (1946). 9. E.L. Johansen and B.W. Smith, Arachis hypogaea x A. diogoi. Embryo and seed failure, - Amer. - J. - Bot. 43: 250-258 (1956). TECHNIQUES IN GROUNDNUT IMPROVEMENT U.R. Murthy, N.G.P. Rao, P.B. Kirti and M.  har rat hi, Cyto- . - genetics and groundnut improvement, - IARI Reg. - Sta. 9. Hyderabad (1980). D.C. Sastri and J.P. Moss, Hormonal requirement for incom- patible crosses in the genus Arachie (in preparation) (1982). Anon., "ICRISAT Annual Report 1979-1980," ICRISAT, Pantacheru, India (1981 ). V. Raghavan, "Experimental Embryogenesis in Vascular Plants, " Academic Press, London (1976 ) . J. P. Martin, culture -- in vitro d'ovules dlArachide, Oleagineaux 25 : 155-166 (1970). D.C. Sastri, M.S. Nalini and J.P. Moss, Arachis ovule and ovary culture - in -9 vitro - in: "Proceedings of Symposium on Plant Tis- sue Culture, Somatic Hybridization and Genetic Manipulation in Plant Cells," P.S. kao et al., eds., BARC,  omb bay, India (1980). M. Ziv and E. Zamski, Geotropic responses and pod development in gynophore explants of ~rachis hypbgaea cultbred - invitro Ann. Bot. 39: 579-583 (1975). -9 - -N. S. Rangaswamy , T. S. Rangan and P. S. Rao, Morphogenesis of peanut (Arachis h o aea L.) pericarp in tissue cultures, Naturwiss. 22:l-2 -7 (1965 . I.V. Spielman, A.P. Burge and J, P. Moss, Chromosome loss and meiotic behavior in interspecific hybrids in the genus Arachis L. and their implications in breeding for-disease resistance, - Z. Pf lanzenzuchtg. 83: 236-250 (1979). D.C. Sastri, M.S. Nalini and J.P. Moss, Tissue culture and prospects of crop improvement in Arachis hypogaea and other 11 oilseed crops, in: Proceedings of Symposium on Tissue Cul- ture of ~ c o n o m i z l l ~  Important Crops in Developing Coun- tries, " National University of Singapore (in press) (1981 1. L.A. Mroginski, K.K. Kartha and J.P. Shyluk, Regeneration of peanut (A. hypogaea) plantlets by -- in vitro culture of imma- ture leaves, - - -  Can. J. Bot. 59: 826-830 (1981 ). L.A. Mroginski and A. Fernandez, Cultivo -- in vitro de anteras de especies de Arachis (~eguminosae), Oleagineaux 34:243-248 (1979). L.A. Mroginski and A. Fernandez, Obtencion de plantulas por cultivo -- in vitro de anteras de especies silvestres de Arachis (~eguminosae ) , Oleagineaux 35 : 89-92 (1980 ) . K.K. Kartha, K. Pahl, N.L. Leung and L.A. Mroginski, Plant regeneration from meristems of grain legumes: soybean, cow- pea, peanut, chickpea and bean, Can. J. bot. 59:1671-1679 - - -  (1981 I. Y,P. S Bajaj, Freeze preservation of meristems of A. hypogaea and Cicer arietinum, Indian J, . Biol. 17:1455-1407 ( 1 9 7 r  - -Y.P.S Bajaj, A.K. Ram, K.S. Ram, K.S. Labana and H. Singh, Regeneration of genetically variable plants from anther deriv- 0. C.  SASTRI ET AL. ed c a l l u s  of Arachis hypogaea and - A. v i l l o s a ,  -- Plant Sci.  Lett .  23:35-39 (1981).  

The Use Of In Vitro Techniques In Groundnut Improvement

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