Research On Technology Of Cotton Meristem Transformation

In recent years, cotton traits were improved by transgenic technology, a variety of finecotton varieties was obtained. Agrobacterium-mediated﹑particle bombardment andpollen-tube pathway are used, the research of mechanism was more clearly on genetictransformation of cotton by Agrobacterium-mediated.Conventionally, Agrobacterium-mediated transformation used hypocotyls as explants,the regenerated plants were obtain by tissue culture techniques, but the transformationprotocol was severely restricted by genotype-dependent reponse, a prolonged culture period,low embryogenesis rate. The shoot-tip meristem of cotton has the ability to different to plants,cotton varieties were unrestricted and culture period was short. In this paper, the genetictransformation system of cotton apical meristems was improveted.Objective: The shoot-tip meristem of Xinluzao33was wounded by the high-speed vortex ofcarborundum, the reporter gene gusA was imported by Agrobacterium-mediated, thetransformation efficiency was assessed by analysis. The different transformation conditionswere had effects on the genetic transformation.Methods: Embryos were peel stripping out from cotton seed, and cotyledon was removed.The shoot tip meristem was exposed and not damaged.2mL MS liquid medium、carborundum and embryos were in centrifuge tube, cotton embryos were infected byAgrobacterium after3000rpm high-speed vortex. The transformation conditions of differentsingle factors were seted up, for example, three kinds of bacterial concentration、the twostrains、 different amounts of carborundum、 vortex time and infection affects. Thetransformation was observated by Histochemical GUS assay and the paraffin sections ofcotton embryos. The remaining cotton embryos develop into seedlings. Leaves of the seedingwere screened by kanamycin、PCR and Histochemical GUS assay after transplanting.Results: More cells were stained with infested Agrobacterium LBA4404after cotton embryoswere infested by Agrobacteriums LBA4404and GV3101. More cells had higher expression ofgusA gene after infested by Agrobacterium LBA4404suspension OD₆₀₀=0.8. The resultsshowed that more cells were stained by the first method after using different transformation.The more the amount of carborundum, the more serious cotton embryos injuryed, and themore carborundum was inserted. More cells of cotton embryo are stained by0.2g ofcarborundum. The survival rate of seedlings was decreased with the increase of the amount ofcarborundum,12seedlings were obtained,0.2g of carborundum was more appropriate. Thelonger time vortex, the more serious cotton embryos injuryed, and the more carborundum wasinserted. The amount of seedling was lower under vortex for90s, but the transformationfrequency was improved, two GUS positive seedlings were acquired. The apical meristem was dyed after Paraffin sections of stained cotton embryos, and layers of cells were stained.78seedlings were survival after transplanting and the survival rate was72.2%. The leaves ofthe seedlings were selected with a high concentration （200g/L） Kanamycin, the effect was notobvious.3seedlings had494bp by PCR analysis, but the leaves were not stained, it showedthat they were false positives.Conclusion: The effect transformation of Agrobacterium tumefaciens LBA4404was better;the effect transformation of bacterial suspension OD₆₀₀=0.8was better; method1infectionwas better. The more the amount of carborundum and the longer time vortex, the more seriouscotton embryos injuryed, and the more carborundum was inserted. seedlings were moredifficult to develop, but it was more benefit to transformation. The transformation plants werenot obtained, but the efficiency of the method conversion was low.