Optimization Of Cotyledonary Node Regeneration System And Bar Genetic Transformation By Agrobacterium In Cotton

Cotton is an important cash crop. Cultivating cotton varieties by genetic engineering is a new field. It is a kind of effective method to improve the insect resistance, herbicide resistance, resistance to adversity, disease resistance, production and fiber quality of cotton through transferring exogenous genes into cotton to cultivate new varieties by the transgenic technology. Cotton tissue culture is a core technology of cotton genetic engineering. Currently, there are still many problems in cotton tissue culture and genetic transformation system. This study was designed to research the physiological mechanism in the process of shoot regeneration in vitro, and to perfect the regeneration system of cotton cotyledons nodes, meanwhile, the agrobacterium-mediated transformation method of cotton cotyledons nodes was used to research the receptor materials, and mature cotton genetic transformation system was established to improve the frequency of regeneration and genetic transformation of cotton. The main experimental results are presented as follows:1. In the process of optimizing regeneration system of cotton cotyledons nodes,’Baimian1’’Baimian2’ and’Coker312’were used as the test materials, and two different ways of disinfection were compared, and it was found that explants were soak in20%peroxide+0.1%twain for the12h was a better way of disinfection, not only because the rate of pollution was lower but also the toxin of Mercuric chloride was avoided;The rate of induce between three genotypes was different,the induction rate of’Baimian1’ was higher than’Baimian2’and’Coker312’.2. The research of physiological mechanism shows that:physiological change was closely related with adventitious shoot differentiation.The contents of soluble protein, soluble sugar and endogenous hormones all appeared regular changes in the process of adventitious shoot differentiation from cotton cotyledons nodes. On the whole, the content of soluble protein was changed with multiple shoot clumps differentiation in’Baimian1’,’Baimian2’and’Coker312’, decreasing in the4th days after the explants were inserted into the culture medium and then increased in rest time; the content of soluble sugar changed similarly in’Baimian1’and’Coker312’, i.e. the content dropped in0-4th day, increased during4th-8th day, decreased again in8th-12th day; but the content of soluble sugar in’Baimian2’did not change obviously in the first8days and it increased after8days. During the process of adventitious shoot differentiation, endogenous hormones content changed regularity with time of culture, content of IAA, GA3, ZR and ABA reduced greatly in0-4th day after inoculation, and then minor rise or reduce, IPA reach a maximum in the8th and12th day, respectively; CTK remained at a high level in the whole growth period; GA3/IAA, ABA/IAA and CTK/IAA were in the trend of increasing on the whole; the rate of CTK/IAA was the highest one, and GA3/IAA was the lowest one. The change of physiological indexes had very good guidance function for optimizing the induction rate,and improving the rate by increase the content of ABA, CTK, GA3or reduce the IAA.3. Combined with the results of physiological mechanism, the rate of induce, the average number of adventitious bud, elongation condition of adventitious bud and so on were used to choose the best inductive medium suitable for’Baimian1’which was’MSB+1.0mg/L6-BA and0.5mg/L IBA’, and ’MSB+2.5mg/L6-BA+2.5mg/L KT’was suitable for’Baimian2’, and’MSB medium supplemented with2.0mg/L6-BA’was appropriate for’Coker312’. For rooting culture,’MSB+0.5mg/L NAA’was suitable.4. Cotyledonary nodes from’Baimian1’and’Baimian2’were used as explants in the genetic transformation. The bar gene was transfered to cotton by Agrobacterium-mediated technology. The results showed that:the rate of Gus instantaneous expression and transformation was highest when concentration of Agrobacterium (OD600) was between0.5-0.8, infected for20min and co-cultured for3days in the dark. Through the herbicide screening and PCR assays proved that the technology herbicide-resistant gene had been successfully transferred into cotton.