Fig. 1. Embryogenic callus (left) and somatic embryos. Fig. 2. Clump of developing somatic embryos.
Plant Biotechnology Section, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India
A tissue culture system with efficent degree of somatic embryo production would be useful in genetic manipulation studies aimed at rice improvement. Several potentially useful genes can now be introduced into cultivated rice and prospects for future rice biotechnology are promising (Toenniessen and Khush 1991). Compared to japonica rice, indica rice has been less responsive to in vitro culture (Kyozuka et al. 1988) and high frequency somatic embryogenesis with distinct somatic embryo formation has been limited (Biswas and Zapata 1992). Indica rice cultivars, Pusa Basmati 370, Pokkali, Mahsuri, Tellahamsa, HR22, IR50, and IR64 were screened for somatic embryogenesis. Callus cultures were initiated from mature seed embryos on MS medium supplemented with 2 mg/l 2.4-D+50mg/l L-tryptophan. During the subcultures, callus was yellowish white, but also exhibited some white compact callus tissues. These embryogenic clusters were selectively picked and cultured on MS medium with 1 mg/l kinetin, 1 mg/l ABA, 3% mannitol and 3% sucrose with 1 g/l casein hydrolysate. The embryogenic callus proliferated on this medium and after 2-3 passages (of 3-week duration each) produced somatic embryos. The white compact callus exhibited globular embryos which later turned green and differentiated into plantlets with a coleoptile and roots (Fig. 1). Of the cultivars tested, Pokkali and HR22 gave high frequency of somatic embryogenesis (>60%) compared to other cultivars (<40%). The rate of somatic embryo to plantlet conversion was in the range of 65-90% for these two cultivars. For each culture, several embryos (50-60) were observed (Fig. 2) which were singled out and were grown into complete plantlets on basal medium without any hormones. Plantlets were transferred to filter paper wicks in liquid basal medium for further growth for 2 weeks. At 8-10 cm height, seedlings were transplanted in polybags initially and were then grown to maturity in pots in the greenhouse. Plants were normal and identical to control seed derived plants and exhibited 74-92.8% fertility. The seed size was normal in Pokkali but slightly lower in other cultivars. Lowered fertility (55.9-79.2%) was noted in Basmati 370.
Selective subculturing of white, compact embryogenic tissues led to optimised cultures with potential for long-term culture. These embryogenic cultures maintained on MS medium with kinetin, ABA, CH, mannitol and sucrose exhibited better revival and retention capacity for regeneration (58-60%). On other media (media with 0.2 mg/l 2,4-D and media with no hormones) the frequency of somatic embryogenesis was only 12-38%. The revival of embryogenic response was observed even after 24 months of culture in Basmati 370, Pokkali and HR22 although in other cultivars a lower response was noted. Green plantlets regene- rated from these cultures grew normally upto maturity.
Fig. 1. Embryogenic callus (left) and somatic embryos.
Fig. 2. Clump of developing somatic embryos.
The potential of the embryogenic callus to produce somatic embryos and their conversion into plants provides the best and accessible target tissue for studies in mutant selection and also genetic transformation.
References
Biswas-Ghosh, G. C. and F. J. Zapata, 1992. Plant regeneration from long term cell suspension cultures of indica rice (Oryza sativa L. cv. IR43). J. Plant Physiol. 139: 523-527.
Kyozuka, J., E. Otoo and K. Shimamoto, 1988. Plant regeneration from protoplasts of indica rice: genotypic difference in culture response. Theor. Appl. Genet. 76: 887-896.
Toenniessen, G. H. and G. S. Khush, 1991. Prospects for the future. In Rice Biotechnology. G. S. Khush and G. H. Toenniessen (eds). p. 309-313. IRRI, Philippines and CAB International, U.K.