| Cauliflower, a variety of Brassica in Cruciferae, is popular with many people as a kind of vegetable. However, cauliflower production always suffers from diseases such as black rot, blackleg, club root and so on, affecting the yield and the quality of cauliflower seriously.Somatic hybridization can overcome the sexual incompatibility barriers to obtain the hybrid between different species, and transfer the alien resistant genes from other species. It can enrich the diversity of pathogen resistant genes of the breeding material, and realize the innovation of crop germplasm.The objective of this study was to establish an asymmetrical somatic hybridization technique between Brassica oleracea and B. nigra, and to achieve the transfer of multi-resistant genes from wild mustard to cauliflower. The genotype of B.nigra used has resistance to pathogen Phoma Lingam, Plasmodiophora and Xanthomonas Campestris pv. Campestris, which result in the black leg, club root and black rot diseases respectively. Mesophyll protoplasts of B.nigra were irradiated by different doses of UV light first, and fused with hypocotyl protoplasts of B. oleracea var botrytis in PEG-induced fusion experiments . About 170 regenerated plants were obtained, and 40 of them were investigated on morphology, RAPD or SRAP molecular marker, isozyme pattern, chromosome number and genome size. Results revealed that 30 of the 40 plants were true hybrids, and the allopolyploid frequency was about 75%. Comparing the different identification method, it was thought that the morphological observation was more simple and direct, and with enough veracity in most of the cases, but the SRAP molecular marker was more exact and dependable, especially for the exceeding asymmetrical hybrids.The culture and regeneration ability of fused protoplast with different UV treatments were investigated and the regenerated plants were analysed by several means: morphology, DNA molecular marker, isozyme analysis, chromosome counting, relative DNA content measurment. Results indicated that asymmetric somatic hybrids could be obtained when UV doses were in the range from 0.001 J·cm-2 to 0.200 J· cm-2. Fused protoplasts lost capability of continuous division and differentiation at 0.300 J·cm-2. Morphology of the regenerated plants varied a lot with either intermediate or cauliflower-like shape. DNA molecular marker analysis (SRAP) of the hybrids demonstrated that the genetic information of the recipient parent was retained more or less complete and intact, while genetic information from the donor parent was lost in the hybrids from 2.27%-93.33%. About 23% of the hybrids had a chromosome number less than thesum of donor and recipient. About 20% of the hybrids had a DNA content less than the sum of donor and recipient. These results revealed that UV radiation could result in the chromosome deletion of the donor genome in the hybrid cells, and promote the obtaining of asymmetrical hybrid plants. However, the degree of chromosome elimination seems to be independent on UV dose.In the meantime, black-rot resistant test was performed in 25 regenerated plants. Nineteen of which showed a good resistance against Xamthomonas campestris pv campestris, while 6 plants showed 3-5 grade symptoms. That gave the elementary proof of transferring of alien pathogen resistant genes from wild B.nigra to B. oleracea via UV mediated asymmetrical somatic hybridization.
|
PDF Full Text Request