Efficient shoot regeneration of Brassica campestris using cotyledon explants cultured in vitro

A system has been developed for efficient regeneration of shoots from Brassica campestris in vitro. Using 4-day old cotyledons with petioles as expiants and a combination of BA and NAA in the regeneration media, up to 70% of expiants produced shoots after 2 weeks in culture. The optimal conditions for regeneration were found to include a BA concentration of 2mgL(-1) and NAA concentration of 1mgL(-1). Light intensity had a profound effect on regeneration potential. The use of silver ions as an inhibitor of ethylene action reduced regeneration rates in this system. Rooting occured simultaneously with shoot formation on these media and the resultant shoots could be rooted readily on minimal medium. The genotype dependency was investigated and indicated that this method would be widely applicable to B. campestris cultivars. Regeneration of one cultivar, a high erucic acid type (R-500), was inefficient in the system described here. Histological studies indicated the development of multiple shoot primordia from the petiolar cut ends of the expiants after the initiation of meristematic activity in the cells about 100μm from the cut site within 2 days of culture initiation. The system described is compatible with previously reported Agrobacterium - mediated transformation protocols involving cotyledonary petioles.