Agrobacterium-mediated Sea-island Cotton (Gossypium Barbadense Linn.) Genetic Transformation Technology Research

Sea Island cotton is a fiber excellent quality cotton cultivation, with its yield accountingfor3-8%of the total world cotton production, cotton production and the textile industryoccupies an important position. Most of the cotton transgenic reported from the Upland,ButSea Island cotton genetic transformation of less reported at home and abroad, On Sea Islandcotton regeneration system and genetic transformation conditions of exploration, is expectedto establish an efficient island cotton transformation system. Agrobacterium-mediated genetransformation method is the most widely used, the mechanism is most clearly, however, theSea island cotton Agrobacterium-mediated genetic transformation of many obstacles, mainlyin Sea Island cotton by somatic embryogenesis access to plant regeneration difficulty andlarge genotype-dependent, long cycle, low conversion efficiency. Therefore explore anon-through callus dedifferentiation, direct access to the regenerated plant tissue culturetechnology, is currently part of Sea Island cotton conversion technologies to be solved. ByAgrobacterium-mediated the shoot tips transformation and use the Agrobacterium infectionflower stigma transformed exogenous gene, its simple operation, saving time and effort, nogenotype-dependent, it is a simple, fast and efficient genetic transformation method. Currently,about Sea Island cotton in the domestic and foreign reports of research in this area less. In thisstudy, self-fertile island cotton in Xinjiang, the study of clustered buds induction andAgrobacterium-mediated the shoot tips transformation of factors; Explore theAgrobacterium infection flower stigmas，Exogenous genes into plant，In this way to establishan efficient regeneration and Sea Island cotton genetic transformation system for the use ofgenetic engineering technology improvements island cotton varieties provide technicalsupport.To obtain the experimental results will be summarized as follows:1:4four Xinjiang’s islands self-sterile cotton as material, using the the flower stigmamethod Agrobacterium infection, after flowering2h,6h,12h and24h exogenous gene genetictransformation, the results show that: Four varieties and four hours of processing3,000flowers, harvest378bell, average boll rate12.60%, the934seedlings into the fieldkanamycin through initial screening,89resistant plants were obtained, the average resistancerate of9.87%; PCR detection obtained17transgenic plants, the positive rate was1.72%.2:3Island Cotton clustered buds as explants, the ratio of different hormones, the resultsshow that:6-BA concentration of0.5-1.0mg/L of MSB （30g/L glucose+2.5g/L Phytagel,）multiple shoots induction medium most conducive to Sea Island cotton shoot induction,induction of clustered buds37-41%, the number of induced clustered buds2-3, well quality, is conducive to the formation of complete plantlets.3: shoot tip of Sea Island cotton receptor material, the establishment ofAgrobacterium-mediated transformation system of sea-island cotton: grown for4days novaccine shoot tip explants, Agrobacterium bacterial concentration OD₆₀₀of0.5disseminated10minutes, incubated for48-72hours, moved to kanamycin concentration of150mg/Lselection medium （MSB+1.0mg/L6-BA+150mg/LKm+400mg/LCef）Filter,25days after thetransfer of to rooting medium （1/2MSB+20g/L glucose+3.0g/LPhytagel+3.0mg/LIB+0.3mg/LNAA+1.0g/L activated carbon） culture, resistant seedlings; resistantseedlings grow to3-5cm when domesticated, grafting or directly transplanted to pots, resistantplants.4: Total to obtain transgenic plants29,detected by PCR, of which21positive strains,from the positive strains were randomly take six by RT-PCR, which five positive strains,preliminary proof of anti-weed control agent gene EPSPS imported the island cotton genome.