Study On The System For In-planta Agrobacterium-mediated Transformation With Vacuum Infiltration In Pakchoi

In this study, the system for in-planta Agrobacterium-mediated transformation with vacuum infiltration in pakchoi was investigated with three pakchoi varieties (Brassica campestris L.ssp.chinensis var communis Tsten et Lee) , mediated by two Agrobacterium strains-LBA4404(carrying . npt II gene and gus gene) and GV3101(carrying CaMV CABB-BJI gene VI and hpt gene),which includes methods of building the suitable plant shape transformation influencing factors transformant screening and the transformation mechanism. In addition, floral dip was studied, either. The conclusions are as followed.l.The research of building the suitable plant shape in natural and manpower conditions indicated that, under natural conditions in Yangling, the best sowing time of building the optimum plant shape for the three varieties is September 7th in Autumn, the best sowing time of Shanghaiqing and Huangxinwu is February 17th in Spring; under manpower conditions, when seeds are treated under 0-4, Shanghaiqing for 17d, Huangxinwu for 25d and Hiyueman for 29d and when Huangxinwu seeds are treated under 7-9 for 25d, they will form the suitable plant shape. By compared, the former is better than the latter, but the latter is not a bad method, either.2. The study on the influencing factor of transformation shows that, (1) the transformation frequency of Huangxinwu is distinctly higher than the others;(2) LBA4404 is more efficient than GV3101 for transformation;) the culture medium including AS has no evident effect on the transformation and when Ca2+ concentration is increased to O.lmol/L, the transformation rate becomes zero;(4) the best infiltration time for Huangxinwu is Imin+intermission 30S+30S and for Shanghaiqing and Siyueman is 2min+intermission 30S+30S;(5) plants lOd after clipping the emerging bolts have the highest transformation frequency in the four periods;(6)the size of plant has no distinct effect on the transformation;(7) the plants infiltrated after treated for 7h under 0-4 have higher transformation rate than plants direct infiltrated.3.The experiment of the three varieties's resistance to antibiotics finds that, the identified standard of the transformant is that the plant grows in gear, has green leaves or lateral roots or both; the Hyg for Shanghaiqing is 30mg/L, for Huangxinwu and Siyueman is 50mg/L;the best screening concentration of Kan for the three varieties is 50mg/L; the best screening time is 20d after seeds sowed on the culture medium.4.The transformation mechanism is studied by two crossing modes. The result demonstrates both the ovule and the pollen are the target.5.The result of floral dip showed that when the developing floral tissues were dipped into a solution containing Agrobacterium tumefaciens for 5 minutes or 1 Ominutes,transformants were achieved.6.170 Kan resistant seedlings and 67 Hyg resistant seedlings were achieved. 15 Kan resistant seedlings were at random selected for PCR and 8 seedlings got the target strip. The result of PCR indicated that the foreign-gene had been integrated into the plant genome.