146
Capsicum and Eggplant Newsletter 22 (2003): 147-150. IN VITRO INDUCTION OF HAPLOID IN EGGPLANT (SOLANUM MELONGENA L.) Sanjeev Kumar, Major Singh, Prabhavathi K. and Amit Mathews Indian Institute of Vegetable Research, # 1, Gandhi Nagar (Naria),PB# 5002, PO. BHU, Varanasi 221 005 (India), Email : majorsingh@satyam.net.in Introduction Eggplant (Solanum melongena L.) is an important vegetable crop of Indian origin holds a coveted position in Indian sub-continent in summer and rainy season. Due to good nutritional value and its suitability to grow under a wide range of growing conditions, it has been extensively cultivated in different parts of world. Immense variability exists in this crop, which provides an opportunity to the researchers to develop plants with good quality and productivity (Daunay et al. 2000). Recently much work has been done on development of hybrids with good yield and quality. But availability / development of homozygous / inbred line is a bottleneck in efficient exploitation of heterosis as the development of inbred takes 3-4 generations of selfing. Another alternative way is to develop homozygous lines using in vitro method such as anther or pollen culture. Haploids produced through anther / pollen culture is of great importance in plant breeding programmes as well as in basic genetic studies. Haploid and doubled haploid lines obtained by efficient anther culture technique are now opened up new possibilities for rapid fixation of genotypes and genetic analysis. Moreover it can be an excellent support for molecular analysis and location of quantitative traits. Among the various methods available for haploid production, anther culture is the most practical, viable and widely used technique. Anther culture has been applied to many species and it is recognized that the production of haploid lines greatly assist in to subsequent production of F 1 hybrids (Rotino et al. (1992), Arnison et al. (1990). However, usefulness of this approach is limited in solanaceous species because some respond very poorly to anther culture (Yadav et al. 1989). The culture response is affected by numerous factors, such as genotype, donor plant culture conditions, pretreatment of flower buds or anthers, stage of development of microspore, culture medium composition and culture conditions (Arnison et al. 1990). Haploid production for practical application in breeding programme of several solanaceous crops such as tomato and potato were well documented. However, very little progress has been made in case of anther culture for haploid development in eggplant. In present study an attempt was made to standardize protocol for anther culture in eggplant to obtain haploid plants. Materials and methods The experimental material comprised of three eggplant F1 hybrids. Young anthers of Solanum melongena were cultured on MS medium (Murashige and Skoog medium, 1962) and GD medium (Gresshoff and Doys, 1972) containing various growth regulators like 2-4 D, BA, NAA (0.5, 1.0, 2.0 mg/l) for callus induction. The anther was crushed in acetocarmine to test the stage of pollen development. 147
The young isolated buds were pretreated in cold for 2-3 hrs. These buds were surface sterilized with 0.1 % HgCl 2 for 3 minutes and washed thrice with sterile water. The anthers were aseptically cultured on MS as well as GD medium. Anthers alongwith their filaments were excised under aseptic condition and placed on a sterile petri dish horizontally and sealed with parafilm. The cultures were incubated in dark for initial 20-30 days at 25 ± 2 o C and after callus initiation all the cultures were transferred to light for 16 hour photoperiod. A minimum of 200 anthers from 5 plants were cultured for each treatment. After four week of incubation the anthers were observed for callus initiation. Cultures were scored for frequency of callus induction at the end of four weeks. The frequency was calculated as the ratio between the numbers of anthers responded to callus induction or regeneration to that of total number of anthers inoculated. The developed plantlets from the anther culture were transferred to small pots. After hardening (20-30 days) they were transplanted to the green house. Results and discussion Culture of anthers to raise haploids followed by chromosome doubling is an easy method of developing homozygous lines in relatively less time than traditional methods. In eggplant callus initiation from anther has been reported earlier by Valiux and Chambonnet (1982), Yadav et al. (1989) and Karakullucku and Abak (1993). However, regeneration was poor in these cases. In present study, in vitro regeneration was carried out to produce haploid through anther culture. Selection of anther / correct stage of pollen development were found to be the most critical factor for haploid production. Fig. 1. Direct embryogenesis from anther Fig. 2. Callus initiation from anther Plantlet derived from anther culture (in pots and in field) 148
In responsive anthers, the wall tissue gradually turned brown and depending on the genotype after 3-8 weeks they burst open due to pressure exerted by growing callus (Figure 1). After 4 weeks interval the anthers from all the three F 1 hybrids produced callus. However the anther of F 1 MS x Pant Rituraj showed produced maximum embryogenic callus (Figure 2). The average number of anthers giving callus initiation was between 11.8 to 20 % depending on the genotype and pretreatment. Two types of response were recorded; some anthers produced embryogenic callus, while some produced embryos directly. These embryogenic calli were again multiplied on same medium and then transferred to different media having varying concentration of NAA (0.1, 0.2, 0.5, 1.0, 2.0 and 2.5 mg/l). The embryos were taken directly for germination and embryogenic callus was cultured to stimulate development of embryos. The embryogenic callus initiation was observed from the anthers on medium containing 2.0 mg/l 2,4-D. Karakullukcu and Abak (1993) also used 2,4-D for callus initiation in eggplant in combination with kinetin. A cold treatment of anthers at 5 o C for 2-3 hours gave better frequency of callus induction. The cold or mild heat treatment has been reported to be effective for initial response of anther culture. Earlier Valiux and Chambonnet (1982) used heat treatment at 35 o C for eggplant anthers. The regeneration from the callus was initially observed as green spot in the callus. Shoot initiation from the callus was observed on the MS medium containing 0.2 0.5 mg/l NAA. These shoots were multiplied on 1.0 mg/l BAP with 0.2 mg/l IAA. The explants bearing several shoot buds were transferred to GR-free MS medium for elongation. The individual elongated shoots were cultured on half strength MS medium to induce rooting. The putative haploid plants were acclimatized under conditions of high humidity and transferred to field (Figure 3,4). The regenerated plants were found to be sterile with stunted morphology as compared to normal fertile plants. The further evaluation of these plants is still under progress. The variability observed in the callus induction of anthers of three hybrids suggests that it is better to consider behaviour of each genotype on different induction medium. Considering the frequency of callus initiation and plantlet regeneration the further work on factors involving the initial response may permit to achieve a higher frequency. References ARNISON P.G., DONALDSON P., HO L.C. and KELLAR W.A., 1990. The influence of various physiological parameters on anther culture of broccoli (Brassica oleracea var. italica) Plant Cell Tissue Organ Culture 20:147-155. DAUNAY M.C., LESTER R.N. and LATERROT H., 1991. The use of wild species for the genetic improvement of brinjal eggplant (Solanum melongena) and tomato (Lycopersicon esculentum). In. Solanaceae III: Taxonomy, chemistry, evolution. (Hawkes JG, R.N. Lester, M. Nee & N. Estradar eds.) Royal Botanic garden, Kew, U.K. pp. 380-412. GRESSHOFF P.M. and DOYS C.H., 1972. Development and differentiation of haploid Lycopersicon esculentum (tomato) Planta 107: 161-170. KARAKULLUKCU S. and ABAK K., 1993. Researches on anther culture of eggplant: II. The effects of sugar and growth regulators. Doga-Turk-Tarim-ve-Ormancilik-Dergisi 17:811-820. 149
MURASHIGE T. AND SKOOG F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiologia Plantarum 15:473-497. RAINA S.K. and IYER R.D., 1973. Differentiation of haploid plants from pollen callus in anther culture of Solanum melongena L. Z. Pflanzenzucht 70:275-280. ROTINO G.L., SCHIAVI M., FALAVIGNA A., PEDRAZZINI E., PERRONE D., SALZANO M., CORREALE A. and RESTAINO F., 1992. Comparison of eggplant doubledhaploid lines with their inbred and hybrid parental genotypes. Capsicum Newsletter. Special issue: 283-288. VAULX R.D. and CHAMBONNET D., 1982 In vitro anther culture of eggplant (Solanum melongena L.) stimulation of plant production by means of treatment at 35 C combined with low concentration of growth substances. Agronomie 2: 383-388. YADAVA N.R., VERGHESE T.M. and SHARMA D.R., 1989. Morphogenetic studies in androgenic callus of Solanum melongena L. Current Science 58:637-639. 150