Agrobacterium Tumefaciens-mediated Embryogenenic Calluses Transformation And Plant Regeneration Of Gossypium Hirsutum Containing Chitinase And Glucanase Genes

Upland cotton (Gossypium hirsutum) is one of the most important world wide economical crops, playing a vital role in the economic, political, and social affair. It is widely planted in our country, about 400 million hectors per year, and made a significant contribution to the development of rural area .while cotton is highly susceptible to many biotic and abiotic stresses, with the enlargement of its planting area, the disease of epiphyte is become increasingly overwhelming, thus endangered the quantity and quality of cotton production, and caused great calamity to cotton industry of our country .for the difficulty of obtain resistance material. It is a better strategy of useing abroad antifungal genes for transformation. To the property of chitinase and (3-(l,3)-glucanase can degrade the wall of fungal cell, thus it can be used to suppress fungal disease .In this study chitinase and p-(l,3)-glucanase was transferred into Gossypium hirsutum E-kang-9 variety callus by Agrobacterium tumefaciens .The main results as follows:1. In this research the affect of IBA & KT and inorganic salts to the regeneration of hypocotyls was studied, the primary callus initiation on the medium containing the optimal combination of 1.0 mg/L IBA and 0.5 mg/L KT, When primary callus are induced and proliferated on the medium orderly supplemented with 120 mg/L MgSO4and 42 mg/L FeSO4, calli was prone to form embryogenic calli. 1000 mg/L KCl and 200 mg/l CaCl2, and the callus is quickly promoted to form embryogenic calli. During calli inducing NH4NO3 is inevitable otherwise somatic embryo will not formed ,but during the stage of somatic embryo transformed to calli, NIHUNO3 must deserted and supplied with 1900mg/lKNO3, for it can benefit to the form of calli; Asn and Gin should be added during embryos germinate to young plants.2. In this study efficient callus transformation system mediated by Agrobacterium was developed using E-R 9 embryogenic callus as explants, optimized the parameter about transformation such as pre-cultivation, co-cultivation temperature, AS and Km concentration .The result as follow: The efficiency of transformation for 5 to 10 days pre-cultivation is super to not pre-cultivation ; the best choice temperature of co-cultivation is 19℃; AS is not necessary to this experiment; The suitable concentration of Km is 100mg/l.3. In this study 60 resistant plantlets were obtained, random select 20 plantlets, PCR result verified 14 plantlets to be positive.4.Preliminary study of embryogenic regeneration on Gossypium barbadense was investigated .The results show us: The combination of 2,4-D 0.15 mg/1 and KT 0.5 mg/1 isthe best one on calli inducing of Gossypium barbadens...