Establishment Of High Frequency Regeneration System In Vitro And Transformation From Cotyledons With Petiole In Radish

Eleven radish (Raphanus sativus L.) genotypes used as material, several main factors affecting in vitro regeneration from cotyledons of radish were studied in this research. Z3 genotype was selected as the genetic transformation material, The genetic transformation system of radish was studied with Agrobacterium-mediated pWR-BcA9-orf138 plasmid carried cytoplasmic male sterility gene orf138. these factors that influenced genetic transformation, including pre-culture time, co-culture time, infection time, bacterial concentration, antibacterial agent type and delay screening and so on, were optimized. the results as followed:1. Radish in vitro regeneration system from cotyledons was established, which main procedure is that the best explant was the whole cotyledon-petiole, the most suitable seeding age was 4 days, and the optimum medium is MS + 6-BA 6 mg·L^-1 +NAA 0.05 mg·L^-1 . the regeneration ratio could reach 86.95%, and the regeneration coefficient reach 1.80.2. The regeneration ratio of different radish genotype was quite different. In the same concentration of hormone combinations, the highest regeneration ratio was 64.33% of B2, and the lowest is 1.73% of B5 among the 11 genotypes.3. The suitable medium for radish adventitious rooting was 1/2 MS added 0.3 mg·L^-1 NAA, the rooting rate reached 100%, and the strong roots was 75%. These roots were stout, main and fibrous roots were obvious, the seedlings were survival easily after transplanted.4. In the genetic transformation process of Z3, 500 mg·L^-1 Cef was selected as antibacterial agent, which could control the pollution of Agrobacterium effectively. As the transferring plasmid carrying the Hyg resistant loci, the 5 mg·L^-1 Hyg was selected to screen the resistant shoots.5. The radish genetic transformation system was established: First, the explants were cultured for 2 days' as pre-culture, Then infected 5-7 min by the EHA105 Agrobacterium of OD600=0.3-0.6, and continuely cultured for 5 days' as co-culture, then the explants were transferred to MS +6-BA 6 mg·L^-1 + NAA 0.05 mg·L^-1 + 500 mg·L^-1 Cef medium for 7 days' delay screening. Then the explants were transferred to MS +6-BA 6 mg·L^-1 + NAA 0.05 mg·L^-1 + 500 mg·L^-1 Cef +5 mg·L^-1 Hyg medium for 10 days resistance screening, lastly, successive transfer culture was took every 10 days.6. When EHA105 Agrobacterium with pWR-BcA9-orf138 plasmid was transferred to the radish genotype Z3, 126 resistant shoots were got, and 8 resistant shoots were amplified the target gene about 850bp. The preliminary validation shown that the target gene has been transferred into radish regeneration shoots.