Establishment Of The System For Producing Transgenic Cottons Mediated By Agrobacterium

The preferred method of cotton genetic transformation is mediated by agrobacterium.The common receptors used in cotton genetic transformation are hypocotyl, cotyledon,shoot tips and embryogenic callus, etc. Agrobacterium-mediated hypocotyl transformationsystem is the most sophisticated. But the conversion cycle is too long, being at least10-12months; Agrobacterium-mediated transformation to cotton embryogenic callus can notonly shorten the conversion cycle but also reduce the workload, and finally improve theconversion efficiency. But the transgenic seedlings appears a high frequency variation; Thetransformation that using shoot tips as a receptor is belonging to the regeneration processof organogenesis, and chimera phenomenon may occurs. In this study, we have establishedan efficient regeneration system, including callus induction, embryogenic callus culture,embryoid body formation by selecting Xinluzao45hypocotyls as explants;,. We haveobtained a few SM (a gene related to fiber iniation) transgenic seedlings by abovetragengenic procedure and selecting Xinluzao33embryogenic callus as receptors;Meanwhile, we have tried the mature embryos of mature cotton seeds as receptors toproduce the transgenic cotton and obtained about20-30d transgenic plants. This is atransformation method without relying on the tissue culture.1、On the basis of the conventional regeneration system, we selected Xinluzao45under hypocotyl as explants for tissue culture, and the efficient regeneration systemestablished. We found the most suitable medium for callus proliferation as following:MS+0.1mg.L-12,4-D+0.1mg.L-1KT+1.9g.L-1KNO3+0.75g.L-1MgCl2+30g.L-1glucose+2.0g.L-1Gelrite. Callus induction rate is82%. When callus prolifed to5-7mm,we conducted embryonic callus induction. After about2-3times subculture, embryogeniccallus appeared. The color is from green, gray-green to yellow-green, and loose texturecallus easily differentiated to embryonic callus. Differentiation rates are73%. Theembryogenic callus singled out to induce embryoid, and further regeneration seedlings. Wegot16regeneration seedlings finally, and the period is about5-7months long.2、The cotton gene SM was transformed to Xinluzao33embryonic callus by Agrobacterium-mediated transformation. We obtained10transgenic seedlings afterresistance screening, differentiation culture and embryogenesis phases. The entireconversion cycle is in a length of5-7months. The transgenic ratio of SM gene is77%confirmed by PCR detection.3、Mature embryos of four most suitable varieties were tried as receptor to establishthe transgenic system directly from emdryos, not through callus stage. The results showedno difference in the rate of four species in transient expression. But conversion rate ofXinluzao45is higher than other three species, being10%-20%. Experiments in bacteriaconcentration, infection time, kanamycin concentration with and without AS indicated that,0.6OD value,15min infection,100mg/L kanamycin concentration infection AS andSilwet-77are the best combination, having the highest conversion efficiency (25%).Finally We got28transgenic plants after about one month culture. This provide a newmethod to produce the transgenic cottons.In this research we have esterblished Agrobacterium-mediated transformation methodby using embryogenic callus and mature embryos as receptors, which were verified bymolecular methods (GUS staining, PCR) and dispensing kan+. Using this method, the SMgene was successfully introduced to Xinluzao33and Upland WC. The establishedtransgenic system will provide a new strategy for producing transgenic cottons.