Establishment Of Regeneration And Agrobacterium Mediated Genetic Transformation System Of Cauliflower And Its Application

On the basis of regeneration system establishment with caulflower hypocotyls, Agrobacterium mediated genetic transformation system of cauliflower had been established. Using the transformation system , overexpression vectors harboring BpERF1 gene of cabbage and CaNAC1 gene of pepper were introduced into caulfower plants Xuebai and Songhua respectively, and transgenic plantlets were acquired and the effect of BpERF1 overexpression on disease resistance was evaluated, the main results were as follow:1.Establishment of cauliflower regeneration systemSome factors that might affect the transformation of cauliflower were studied,such as genotype seeding age,hormone concentration,AgNO3 et al. The main results were as follow:Regeneration frequency varied dramatieally with genotype. Shoot regeneration rate of Xuebai was 95% and shoot regeneration rate of Taibao was 40% in the suitable condition;Seeding age had significant effect on shoot induce and the seedlings of 6 day age gave the best result. The combination of MS+6-BA 3.0mg/L+IAA0.2mg/L were found to be the most efficient for shoot formation, which result in 95.7% of shoots regeneration for Xuebai with 3.9 buds in average. In addition,it was found that adding AgNO3 in 4.0 mg/L to the medium could enhance the frequeney of regeneration and depress browning.2. Establishment of cauliflower agrobacterium mediated transformation systemThe minimum lethal homomycin concentration of cauliflower shoot regeneration was 5mg/L, and the minimum lethal carbenicillin concentration for growth of agrobaetium in the initial select medium was 500mg/L carbenicillin and that for the continuous medium was 200 mg/L. On the other hand. In the case of vectors harbouring NPT II selecting gene,10mg/L of kanamycin was determined as the minimum lethal homomycin concentration for selection of resistant transgenici plants.Some factors that affect the transformation rate of cauliflower were also studied,such as pre-culture time,EHA105 growth stage and concentration,infecting time,co-culture time et al. We had preliminarily determined an effective system for genetic transformation. The protocol was: numerical OD600 0.4-0.6, 3-5 minutes for infection,3days for co-culture,selection after delay for 14days.3. PCR validation and disease resistance detection of the transgenic plantsBy the agrobacterium mediated genetic transformation system we established, 10 regenerated homomycin resistant plants of the overexpression vectors of BpERF1 were acquired. The result of PCR detection with the specific primers of BpBPERF1 showed 6 of the 10 resistance plants were transgenic plants. The result of disease resistance analysis showed that the overexpression of BpERF1 enhanced the resistance of transgenic plants to plasmid by delay the appearance of the symptom of disease after inoculation of the pathogen.We also transformed the overexpression vector of CaNAC1 to cauliflower, and 6 resistance regenerating plants were acquired, among which, 4 regenerated homomycin resistant plants were PCR positive plants .