Study On The System For Agrobacterium-mediated Genetic Transformation Of Cabbage

In this study, the system for in-planta Agrobacterium-mediated transformation with vacuum infiltration in cabbage was investigated with three cabbage varieties (Brassica Oleracea Var. capitata), mediated by two Agrobacterium strains-LBA4404(carrying npt II gene and gus gene) and GV3101 (carrying Bari-1 gene and hpt gene), which includes methods of building the suitable plant shape > transformation influencing factors, transformant screening. In addition, high frequency of shoot regeneration in cabbage was studied. The conclusions are as followed:l.Jinxin and Huangmiao varieties are easy to build the optimum plant shape for vacuum infiltration on August 5th and 10th in autumn under natural conditions. The optimum plant shape of two varieties:No. of leaves of Jinxin variety are over 30,the plant height is over 23.03cm, and diameter of root is 1.758cm ;and No.of leaves of Huangmiao variety are over 37,the plant height is over 18.40cm,and diameter of root is 1.739cm.2. The study on the influencing factor of transformation shows that, (1) the transformation frequency of Jinxin is distinctly higher than Huangmiao;(2) LBA4404 is more efficient than GV3101 for transformation;(3) the culture medium including AS and the pH being 6 have obvious effect on the transformation, but the higher or lower of sue. concentration has no evident effect on the transformation;(4) the best infiltration tune for Huangmiao is 8min+intermission 2min+30S and for Jixin is 6min+intermission 2min+30S,and increasing the times of intermission has no evident effect on the transformation;(5) in bud stage, the buds being treated by the vacuum infiltration have higher transformation than roots, leaves and stems.3. On screening Jixin and Huangmiao seeds through medium with Kan. Optimum concentration is 30mg/Lfor Jinxin and for Huangmiao is 40mg/L, and optimum time of screening is 20 days. The identified standard of the transformant is that the plant keeps normal in the medium including Kan, has green leaves or lateral roots or both.4.On screening Jixin and Huangmiao seeds through growth point selection with Hyg. Optimum concentration is 800mg/L for Jinxin and Huangmiao, and optimum time of screening is 10 days after inoculated. The Hyg resistant seedlings: leaf buds appear, become green and grow normal.5.150 Kan resistant seedlings and 55 Hyg resistant seedlings were achieved. 7 Hyg resistant seedlings were at random selected for PCR and all got the target strip. It proved that the foreign gene had been integrated into the plant genome.6.Using Niuxin variety, the type of explants, hormone compositions, AgNO3, age of seedlings and preculture affecting shoot regeneration were also studied. The results showed the highest frequency of shoot regeneration is hypocotyls explants from 6-d-old in vitro grown seedling was 64.28% and No. of per explant with shoots was 2.28,using the MS media containing 4.0mg/L6-BA.It was also found AgN03(8mg/L) was advantageous to the shoot formation; via a preculture procedure of 2 days, the frequency of shoot regeneration increased 28.57%.