Development Of Biolistics-Agrobacterium Tumefaciens Transformation System For Shoot Apex Of Cotton And Introduction Of GLU-CHI Gene

Cotton is one of the important cash crops in the world. However, this crop has been suffering from the disease of Verticillium dahliae Kleb for a long time. After long time practice, it seemed that it is difficult for breeders to find a new anti-fungal materials resisting to Verticillium dahliae Kleb by traditional breeding methods. So it is a primary target for the application of genetic engineering to improve agronomic characteristics.In this paper, efficient regeneration system and Biolislics-Agrobacterium tumefaciens mediated transformation procedure were established with Chuanmian 239 and Chuanmian 45 as materials . Through these systems, the bivalent expression vecter of Maize Chitinase gene and Wheat P -1,3-Glucanase gene were introduced into cotton, and transgenic plants resistant to Kan were obtained. The main results were as follows: 1.Establishment of regeneration systemsShoot apices isolated from 1.5-day-old seedings were cultured on MSB) (MSB mediumcontaining BA O.lmg/L) for 2-3 weeks. Subsequently, the apices were transferred to MSBi(MSB medium containing active charcoal Ig/L) for 2-3 weeks. The explants were thenrecultured on MSB3 (MSB medium containing IBA 0.1 mg/L and IAA 0.1 mg/L) for 2 weeks.Finally, the apices were transferred to 1/2MSB for 2-4 weeks. Regeneration frequencyamounts to above 90%.2.Confirmation of selection pressureShoot apices isolated from 1.5-day-old seedings were cultured on MSB) medium containing Kan of different concentration (0, 10, 20, 30, 40, 50, 75, lOOmg/L) to test the sensitivity to Kan. The results showed that the selection pressure is Kan 50 mg/L. 3.Establishment of biolistics-Agrobacterium tumefaciens transformation systems for cottonShoot apices isolated from 1.5-day-old seedings were bombarded by tungsten powder with the bombardment distance of 3 cm, the vacuum pressure of about 27-28 inches Hg and the Helium pressure of 1550psi. The apices were dipped into the Agrobacterium suspension of ODeool-l and placed in vacuum apparatus for 30 min, the vacuum pressure was about 25 inches Hg. The apices were co-cultured on MSBi medium supplemented with AS lOOmg/L covered with a sterile filter disc under 28 癈 for 3d in darkness. After co-culture, the explants were transferred to the delay selection media ( MSBi medium supplemented with Cef 500mg/L) for 7d. Subsequently, the explants were placed in turn onto MSBi, MSBz , MSBs containing Kan 50mg/L and Cef 500mg/L to be screened, and the transformants were obtained with transformation frequency amounting to 7.89%. 4.Confirmation of transgenic plantsThe PCR assay of Kan-resistant transformants showed that the target gene had been integrated into cotton genome accompanying with NPTII gene.