Production of synthetic genotypes of Brassica juncea via somatic and sexual hybridization

The major objective of this study was to produce synthetic genotypes of Brassica juncea from its parental species B. rapa and B. nigra via somatic and sexual hybridization. As prerequisites for somatic hybridization experiments, methods were developed to improve the culture of mesophyll and hypocotyl protoplasts of B. nigra and B. rapa , to obtain reliable plant regeneration from mesophyll protoplast cultures of B. nigra , and to fuse protoplasts of B. nigra and B. rapa . A modified Kao's medium (1977), was found suitable for the culture of mesophyll protoplasts of B. nigra and B. rapa . At a density of approximately $110\sp5$ protoplasts/ml within a culture plate insert surrounded by culture medium, mesophyll protoplast cultures of B. nigra accessions R890, R1819, R3392 and U1218 and B. rapa cvs. R500 and Wong Bok formed colonies. Genotypic differences in cell division and colony formation were observed. Hypocotyl protoplasts of B. nigra and B. rapa were successfully isolated from 6 day-old seedlings cultured in a modified Kao's medium (1977). With B. nigra accession R890 and B. rapa cv. R500, cell division and colony formation were optimal when hypocotyl protoplasts were cultured at a density of 0.5 to $1.010\sp5$ protoplasts/ml within a culture plate insert surrounded by a nurse culture of 4 to 6 day-old mesophyll protoplasts of B. nigra . Plant regeneration was obtained from mesophyll protoplast-derived calli of B. nigra accession R890 originally cultured in inserts; a shoot regeneration frequency of 8.1% was obtained on a medium containing the salts and vitamins of medium K3 (Nagy and Maliga 1976) with 3 g/l sucrose, 18.2 g/l mannitol, 2 mg/l ZR, 0.1 mg/l NAA, 10 g/l agarose, pH 5.6. For somatic hybridizatian studies, methods were developed to select out parental protoplasts using iodoacetic acid and to efficiently fuse protoplasts on the bottom of a petri dish using PEG. Twenty-nine plants were recovered from fusion experiments between mesophyll protoplasts of B. nigra accession R890 and hypocotyl protoplasts of B. rapa cv. Tobin. The somatic hybrid plants resembled natural B. juncea , had $2n=36$ chromosomes and had pollen viabilities ranging from 30 to 45%. Twenty-one plants, derived from one callus colony, possessed the mitochondrial and chloroplast genomes of B. rapa , as found in natural B. juncea . Eight plants, derived from another callus, had a novel cytoplasmic combination consisting of the mitochondrial genome of B. rapa and the chloroplast genome of B. nigra . Synthetic genotypes of B. juncea were also produced from reciprocal sexual crosses between B. rapa and B. nigra . Seventy-eight interspecific hybrid plants from the cross B. rapa x B. nigra and six hybrid plants from the reciprocal cross were identified by their morphology, pollen viability and chromosome number. The colchicine-induced allotetraploids resembled natural B. juncea in morphology, had 18 bivalents at metaphase I, and had between 35 and 70% pollen viability