Plant Regeneration And Agrobacterium Mediated Transformation Of Bt Gene And Bar Gene On Upland Cotton

As one of the most important economic crops, cotton hold the balance for development of national economy. And germplasm play an important role in breeding new cultivars with high yield, superior quality and poly-resitance. Traditional breeding is always used in new eminent cultivers breeding and germplasm innovation, With the development of biotechnology, it is a effective way to germplasm innovation and breeding in biotechnology. The enrichment of germplasm will have a great contribution to cotton genetics and breeding. By way of Agrobacterium tumefaciens-mediated embryogenic callus transformation, each of two Bt genes (CryIIA, Cry9C) and Bar gene were transformed separately into three upland cotton (EM23, EK3, SM3). Now we have obtained the regenerated plantlet carrying resistance against insect and herbicide. The results is as fellows:1. Several key parameters have been adjusted and the regeneration precedure is optimized. The medium with the combination of IBA and KT is good for callus formation. About 120 days Somatic embryogenesis were obtained with the combination of IBA(0.25mg/L) and KT (0.10mg/L) for the first time. The frequency of cotyledonary embryo germination and plantlet will be enhanced with the addition of 400mg/L activated charcoal to the medium. The grow rate of embryogenic callus is most in the medium containing 7.6g/L KNO₃. We obtained the most of cotyledonary embryo in the the medium containing 5.7g/L KNO₃. Rootstock of 4-6 days is optimum for grafting of regeneration plantlet. Excising primary root and inoculating in the medium of SH, 10-15 days later the root grow very upgrowth. Plantlets with air acclimation for 7 day and water acclimation for 1 day were transfered in soil.2. The chromosome number of embryogenic callus, somatic embryo and root tips were calculated. Embryogenic callus and immaturity embryo is the optimal material for chromosome observation. In order to obtain cells with ideal division, embryogenic callus and immaturity embryo were pretreated with saturated solution of D-dichlorobenzene for 3.5 h, then fixed in ethanol-acetic acid(3:1) fixative more than 24 h, and macerated in 5N hydrochloric acid for 20-30 minutes in 20℃-22℃. It was very difficult of obtaining ideal division cells for cotyledonary embryo and root tips.3. Several key parameters of transformation have been optimized. The optimal...