The presence of 1-10 μM aminoethoxyvinylglycine (AVG) or 5-30 μM AgNO3 markedly enhanced shoot regeneration from cotyledon and hypocotyl cultures of eight recalcitrant Brassica campestris and B. juncea genotypes tested. Expiants of B. campestris ssp. chinensis and ssp. parachinensis grown with a high AVG concentration (20 μM), regenerated poorly. All cytokinins tested were equally effective in promoting shoot formation, except that kinetin was inhibitory to shoot regeneration from hypocotyls of B. campestris ssp. pekinensis (cv. Wong Bok). Both AgNO3 and AVG had no effect on percent rooting and number of roots per rooted cutting of Wong Bok, White Sun and Leaf Heading, but AgNO3 was inhibitory to rooting of India Mustard. However, root elongation of all cuttings was markedly inhibited by AVG at concentrations of 5 and 10 μM.

An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90-100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg/L AgNO3 and 0.5-2 mg/L BA in combination with 0.01-0.05 mg/L 2,4-D or 0.1-1 mg/L NAA. Of all the Agrobacterium strains tested, A. tumefaciens A208-SE, carrying the disarmed Ti plasmid and a binary vector pROA93, was the most effective for B. juncea transformation. pROA93 carries the coding sequences of the NPTII and the GUS genes, both driven by a common CaMV 35S promoter in two divergent directions. Inoculated explants grown on the selection medium in the presence of 0.5 mg/L BA and 0.1 mg/L NAA gave rise to transgenic shoots at the highest frequency (9%). All Ro transgenic plants were phenotypically normal, but variation in expression patterns of the GUS gene occurred among the transgenic plants in an organ- and tissue-specific manner. Both the NPTII and the GUS genes were transmitted to the R1 seed progeny and showed co-segregation.

In this paper we describe procedures for transforming micro colonies derived from mesophyll protoplasts of Petunia hybrida with Agrobacterium tumefaciens. The method is efficient, up to 70% of the colonies were transformed, and we used a similar method to transform cells from a suspension culture of haploid Nicotiana plumbaginifolia.