Tri-parental protoplast fusion of Brassica species to produce somatic hybrids with high genetic and phenotypic variability

Plating efficiencies and the plant regeneration frequencies of bi-parental fusion of Brassica campestris pekinensis with Ogura cytoplasmic male sterility (CMS), B. oleracea L. var. italica with Ogura CMS, and Brassica juncea var. crispifolia were compared for illustrating the advantages of tri-parental somatic hybridization. The results showed that in the triparental fusion combination, 73 plants regenerated from 712 calli and the plant regeneration frequency was 10.3%, three or four times that obtained with the 2 fusion combinations. These hybrids were classified into 12 types based on morphology, and most showed intermediate characteristics between 2 or more of the parental species. The somatic hybiridity were confirmed by flow cytometry, genomic in situ hybridization (GISH) and PCR analysis, indicating that these regenerated plants were all true hybrids. Most of the progenies with normal pollen showed varied number of seed set after backcrossing with B. juncea. The high variability in the hybrids obtained illustrated that somatic hybridization may be useful in broadening existing Brassica gene pools and obtaining material for breeding.