Study On Transformation Of Cowpea Trypsin Inhibitor Gene Into Cauliflower (Brassica Oleracea Var.botrytis)

There are some researches about transferring B. t gene into cauliflower, bui few were reported about transferring CpTI gene into cauliflower. In this paper, cotyledons and hypocotyls were used as explants, by means of plant genetic engineering, foreign insect-resistance gene can be transferred into plant cell. Cowpea trypsin inhibitor (CpTI) gene was transferred into cauliflower by agrobacterium-mediated transformation method, and the transgenic cauliflower plants were obtained. The main results are as below:1. High efficiency regeneration system of cauliflowerThe high efficiency regeneration system is the base of plant genetic transformation. The explants were cotyledons and hypocotyls and MS was used as basic midium. By supplying different concentration of 6-BA, NAA, we got the optimum media which induced the adventitious bud differentiation of two cauliflower explants types. The optimum media is MS+BA 2. Omg/L+NAAO. 2mg/L. Between the two explants types, the hypocotyls expressed the higher adventitious bud differentiation capacity.2. The best Kan concentration used to select the transformant plantsExplants were restrained from differentiating when Kan was added to the medium, moreover, some callus turned white gradually and died. When Kan concentration was higher than 15mg/L, all of the explants and callus became dead. So the best selective concentration of Kan is 15mg/L3. Transformation system of cauliflowerThe procedures described were derived from numerous regeneration and transformation designed to test factors that might affect shoot regeneration. Tested parameters include length of preculture(2d, 3d), length of inoculation with agrobacteriu/n(30-60sec, 5-10min), length of co-cultivat ion(Id, 2d, 3d), Kan cocentration(0,5,10,15,30,50mg/L). Only those parameters producing the best result are described as below:The cauliflower explants were precultured on regeneration medium. After 3d preculture, the explants were inoculated with agrohncterium. Inoculated explants were co-cultivated 2d when tiny strain was seen onthe wound of the explants. The explains were then transferred to regeneration medium with 500mg/L carbanici I I in, culterecl 7-10d to kill the agrobacterium. To select the transferred cells, explants weremaintained on the selective media for about 4-6 monthes, transferred to fresh medium every 2 weeks. Finally we got transformant plants.4. Screen of characterization of transgenic cauliflower plantsThe kanamycin-resistant plants were obtained by the procedure asabove. The putative transformants were assayed by PCR, PCR-Southern blotand Southern blot analysis. All analysis showed that most of the transgenic plants have the predicted band. The results indicated that CpTIgene was transferred into cauliflower successfully. The result of insect -resistance showed that the transgenic plants are more resistant thannon-transgenic plants.