Plant regeneration from in vitro cultures of cotyledon explants and anthers of Sinapis alba and its implications on breeding of crucifers

Abstract

Protocols for plant regeneration from cotyledon explant and anther cultures of Sinapis alba have been developed for creating doubled-haploids and somaclonal variation. Among the several cultivars tested in this study, only ‘Arda’ responded well to in vitro plant regeneration both from anther-as well as cotyledoncultures. Multiple shoot formation in cotyledon explants, which always followed a brief callusing phase, was found to be the best on MS medium with ZEA (1.0mg/l) and NAA (0.1mg/l). Regeneration frequency declined sharply in the absence of auxin or presence of other cytokinins and/or auxin. The frequency of shoot regeneration also declined with reduction in the photoperiod to 16h. On MS + BAP (1.0mg/l) + NAA (1.0mg/l) medium, cotyledonary explants showed profuse callusing, which could regenerate shoots on high ZEA + low NAA/IAA medium. However, it declined with progressing time in culture. Anthers, excised from fresh as well as cold pretreated buds, cultured on 10% sucrose containing MS media with different hormonal constitution, developed calli and/or embryos. Initial culture temperature was important with embryogenesis occurring only in anthers cultured at 30°C for 3 weeks. A high temperature (35°C) treatment was lethal for both callus as well as embryo formation. While BAP + NAA and ZEA + NAA/IAA supported embryogenesis, further plant regeneration from anther-or embryo-callus could be achieved in ZEA + NAA/IAA media. Some of the regenerants flowered already in vitro and had small and sterile flowers. Cytological examination of some of the root differentiating calli indicated the presence of haploid as well as diploid cells. Shoots were rooted during prolonged incubation on the same medium or on transfer to MS (reduced)/ B5 + ZEA + NAA media.