Studies On The Establishment Of Cotton (G.barbadense L.) Shoot Apexes Regeneration System And The Transformation Of Resistant Pest Gene (Bt; SATI) By Agrobacterium-mediated

In this experiment, XinHai16, XinHai20, K-222 and 97006 as the transformation accepters, Introduct resistant insect gene in cotton apex by Agrobacterium-mediated.Through studies on influence factors of shoot culture and transformation, establishment of regeneration system for cotton transformation.the study can offer technical support. The main results of the research are given belowing:1. Establishment of regeneration system for cotton.Soak seed in Ethyl Alcohol for 30 sec,soak seed in 10% HgCl2 solution for 30 min. Slosh the solution Continue.Remove solution and rinse seed 4X in sterilized in water.Leave seed in final water about 12-16 hours. Squeeze seeds out of the seed coat and culture onto 1/2MSB5 medium to germinate 0-3 days ,at 25-28.Removing the seed coat pior to germination reduces contamination and easy to inoculation. .Then culture the four variety of cotton apex onto MSB5 medium with diffent hormones. Among 9 different combinations of hormones, 6-BA0.5mg/L+NAA0.1mg/L and 1/2MSB5+0.1mg/ L IBA combined were the best for the generation.The result showed, when the cotton shoot apex is induced bud, the difference between genetypes don't distinct. And the way cotton shoot apex is induced bud derectly is better method.2. Construction of the transgenic system of cotton shoot apexAccording to studies on the sterilization concentration of Cef, the ascertainment of Kan screening pressure,and the effect factors of transform frequence,the transgenic system with Agrobacterium tumefaciens was constructed: the shoot apex without pre-cultured should be infected 20-30min in Agrobacterium tumefaciens with OD600=0.8,co-cultivated 48-72hours,and screened with 200mg/L Cef for sterilizing Agrobaclerium tomefaciens, 150mg/L Kan for highest selecting resistant bud . The transformation rate of EHA105 is higher than LBA4404.3. Transgenic plants were tested by PCRThe result of electrophoresis of PCR of resistant plant was that some plants get the same line as the positive control Bt (1080 bp). The transformation rate is 0.7%. Transgenic plants of Bt obtained were grafted and make it grow in order to testing its on next step. The results of PCR analysis showed that the BT gene in the T-DNA had been integrated into island cotton genome preliminarily.