SOMATIC HYBRIDIZATION OF CYTOPLASMIC MALE STERILE BRASSICA OLERACEA AND ATRAZINE RESISTANT B. CAMPESTRIS (PROTOPLAST)

Techniques for plant regeneration in Brassica oleracea L. var. italica cv. Green Comet broccoli were developed. Using media developed by Pelletier et al. (Mol. Gen. Genet, 191: 244-252), high frequency regeneration from leaf protoplasts was obtained when plants regenerated from hypocotyl explants (GCR) were used as a protoplast source. Over 70% of protoplast-derived cells divided and up to 77% of the calli produced shoots. Some of the 178 plants grown to maturity had variant phenotypes. Cotyledon protoplasts from selfed progeny of GCR showed higher totipotency than those from seed-grown plants.; Brassica lines carrying the ogu cytoplasmic male sterility (cms) derive from Raphanus show temperature sensitive chlorosis due to nuclear-plastid incompatibility. Protoplast fusion was used to replace Raphanus chloroplasts in this cytoplasm with atrazine resistant Brassica chloroplasts. An assay using nitro-blue tetrazolium was developed to verify the presence of atrazine resistant chloroplasts. After 30 min light, protoplasts from atrazine sensitize B. oleracea remained green in photosynthesis medium with 50 uM atrazine and 0.01% nitro blue tetrazolium; protoplasts from atrazine resistant B. campestris stained blue black.; Protoplasts from cms Green Comet and atrazine resistant B. campestris were fused using polyethylene glycol. The facts that B. campestris does not regenerate on the media used and that atrazine inhibits the growth of light-grown Green Comet calli were used to select for hybrid tissue. One plant, regenerated on medium containing 50 uM atrazine, contained nuclear genomes from both parents, as determined from rDNA hybridization, chromosome counts and morphology. This plant contained atrazine resistant chloroplasts and was male fertile. Mitochondrial DNA showed homology with both parents; novel fragments were also present. On medium lacking atrazine a plant regenerated that contained the Green Comet nucleus and atrazine sensitive chloroplasts. Mitochondrial DNA showed homology with cms mitochondria but also contained novel fragments. The original goal, cms plants with Brassica chloroplasts, was not accomplished; however the regenerated plants may be of value in studying mitochondrial genetics.