The Establishment Of Transformation Systems Of Cotton (Gossypium Hirsutum L.) Via Particle Bombardment

In this paper, Biolistics transformation systems were established with apical meristems of "CCRI 19", "CCRI 24","CCRI 27" and the embryogenic callus cells of "CCRI 24". Thought these systems the bivalent gene of Bt+CpT I were introduced into cotton, and the transgenic plants resistant to Kanamycin were obtained. The main results were as follow:1 , The establishment of Biolistics tranformation systems for cotton on apical meristemsThe kernels of cotton were incubated into the MSB medium. When the seedings were 40~60 hours age, they were dissected and remained the state of meristem and 2 leaf primordia. These meristems were placed straightly into the plates with some MSB medium and bombarded by gold powders with the Helium pressure of 1300 psi, the bombardment distance of 9cm, and the vacuum pressure of about 27~28 inches Hg. Subsequently the bombarded meristems were cultured into MSB medium with activated charcoal 1.0g/L for 4~7 days and then were transferred to the selective medium MSB+KM 45, 60, 80, 85-100mg/L. Finally the regenerated plants resistant to kanamycin were obtained with the transformation rate amounting to 2.94% . 2, The etablishment of Biolistics tranformation systems for cotton on embryogenic callus cellsThe hypocotyls of 7-day-old seedlings were incubated into MSB+0.01mg/L IAA+0.01 mg/L KT+0.01 mg/L 2,4-D to induce the callus issues, then the callus issues were transferred to another medium for inducing embryogenic callus issues. By the osmotic treatment (mannitol, the concentration of 0.3 mg/L, pre-treatment 2-4 hours, post-treatment 16 hours), the embryogenic callus which were fresh and the shape of grain were placed into the districts of diameter of 3cm in the plates. Thebombardment parameters: the Helium pressure of 1300 psi, the bombardment distance of 9cm, and the vacuum pressure of about 27~28 inches Hg. By transition culture, the embryogenic callus were transferred to selective medium to select transformation and the plants resistant to kanamycin were obtained. 3 , Confirmation of transgenic plantsThe PCR assay of kanamycin-resistant transformants showed that the target gene had been integrated into cotton genome accompanying with Bt+CpT Igene .