Study On Plant Regeneration Of Glandless Upland Cotton Jisheng-1 And Transformation Of DREB1C Gene

Cotton was one of the most difficult regenerative plants. Compared to other crops, the study of cotton tissue culture was lagging behind. However, in recent years, a great deal of progress has been made in cotton tissue culture and the regenerative technique of upland cotton has been established in some laboratorys. But these studies were mostly concentrated on a few varieties, particularly some model varieties, and there are many problems not to be solved, such as low quantity of regenerative varieties, low germination rate of somatic embryos, high abnormal embryos's proportion, long-cycle of regeneration and so on. The objective of this work was to study the somatic embryogensis and plant regeneration system in glandless upland cotton Jisheng-1 which had big cultivating areas. The system of cotton regeneration has been established. Also the factors affecting Agrobacterium-medium transformation efficiency were investigated. The results as follows:1. The high effective system of regeneration of the glandless upland cotton Jisheng-1 has been established. Several key parameters have been adjusted and the regeneration procedure was optimized. Callus could be inducted by hypocotyl, cotyledon and radicel, but compare to cotyledon and radicel the callus could be inducted quickly by hypocotyl. The medium with the combination of IBA and KT was the best medium for callus formation. Under the MSB sold medium with the concentration of 1.0mg/L IBA and 0.5mg/L KT, the callus began to exhibit somatic embryogenesis 90 days later. The MSB sold medium with KNO3 substituting for NH4NO3 could not largely increase the somatic embryogenesis rate of callus. The adding of Gln (2.0g/L) and Asn (1.0g/L) to the MSB sold medium could promote the the germination and regeneration of the somatic embryo.2. Here we first reported a stable and high-efficient transformation system about glandless upland cotton Jisheng-1 that was on the basis of embryogenic callus and the Agrobacterium-medium and could to be used to the transformation of DREB1C transfering fator. Using the Jisheng-1 embryogenic callus as explant, the major parameters including the need of pre-cultivation, the consentration of Agrobacterium solution, the temperature of co-cultivation, the time of co-cultivation, as well as the concentration of Hygromycin, were compared. The results were obtained that the appropriate pre-cultivation of embryogenic callus time was 7 days, the appropriate consentration of Agrobacterium solution was 0.4OD-0.6OD, the appropriate temperature of co-cultivation was 20℃, the appropriate time of co-cultivation was 60h, the best concentration of Hygromycin for screening transformants was 25mg/L. The transformation system had more transformation efficiency than other transformation system using hypocotyls as explants. A mass of transformants could be obtained by the help of the transformation system. Under the above conditions, the inducing rate of callus with resistence to Hygromycin could reach to 70%. PCR tests to the transformants suggested the DREB1C transfering fator fragments had been transformed to the genome of the glandless upland cotton Jisheng-1.