Comparation Of Ability Of Somatic Embryogenesis Of Cotton Genotypes And Production Of Ipt Gene Transgenic Plants

Cotton tissure culture and plant regeneration was the precondition of protoplast fusion, genetical transformation and creation of hybrids; Genetic improving of cotton using the genetic engineering was one of the hot regions of cotton breeding, which can overcome sexual-crossing barriers and share all genes existing in the plant, animal, and animals. Especially, the genetic engineering can impove the given characters and significantly shorten the breeding periods. However, many hinder still exisist in cotton biotechnology. The main reason is that cultivars of transgenic cotton, which requires transformation of appropriate tissue followed by regeneration, remains extremely difficult in somatic embryogenesis and regeneration. Recalcitrant cotton cultivars, long tissue culture duration, the unpredictability of tissue culture, and a high degree of genotype dependence are more troublesome in regeneration of cotton. In this study, we have optimized some factors that can significantly affect the efficiency of regeneration in cotton, and compared the regeneability of different genotypes of Seaisland cotton.(Gossypium barbadense L. ) The regeneration of transgenic plants with ipt gene is developed through transformation mediated by Agrobacterium tumefaciens, and some variations of transgenic plants are examined and analyzed. The main results obtained are listed below:1. Two kinds of hormone combinations IBA + KT and 2, 4-D+KT were all efficient for callus induction of cotton (Gossypium hirsutum L.), and the optional combinations were IBA 1.0 mg/L + KT 0.5 mg/L and 0.1 mg/L 2,4-D+0.15 mg/L KT.2. Somatic embryogenesis from calli derived from hypocotyls segments are observed only on inducing original calli medium containing NH₄NO₃. Calli do not form somatic embryos in the original calli inducing medium without NH₄NO₃. Doubled KNO₃ (3.8 g/L) but free of NH₄NO₃ can significantly improve the efficiency of somatic embryogenesis and regeneration. The percentage of embryogenic calli from all cultures reached more than 80% in Coker 201 through doubling KNO₃ and omitting NH₄NO₃. Medium supplemented with MgSO₄·7H₂O is necessary for callus induction and proliferation. And the medium supplemented with 50mg/L FeSO₄·7H₂O could promote the growth of calli, which can obtained 90-100 % callus induction rate. 28 mg/L,56 mg/L MgSO₄·7H₂O in the subculture medium can shorten the priod of somatic embryogenesis.3. To optimize the somatic embryogenesis of upland cotton, totally 22 cultivars including 10 cultivars from Yellow River valley, 10 cultivars from Yangtze River valley and two highly responsible genotypes Coker 201 and YZ-1, were used for studying the factors affecting the callus induction and differentiation. Most of the genotypes showed high regenerability under the IBA/KT hormone regime.Under the optimized culture procedure, most of the genotypes collected from the two valleys showed the similar regeneable abilities, except a relative longer time taken during the somatic embryogenesis of cultivars of the Yangtze River valley.In this study, somatic embryogenesis and plant regeneration had happened in 8 genotypes, namely, Ek3, Ek5, Em20, Em23, Ym9, Ym12, Yz73, and Ym1221 for the first time.4. Comparing to Upland cotton, the callus induction and proliferation of G. babardence was difficult, which would take 10-15 more days and obtained relatively low rate in callus induction, mainly because of the the death of the brownish calli. At the beginning of callus induction, all the calli of five different genotypes grew slowly and the texture was hardish. After several rounds of subculture, these hardish calli grew rapidly and became loosen, so this process would cost much time and lead to the delay of somatic embryogenesis of G. babardence, which would take about 8 months before somatic embrogenesis.5. 2, 4-D+IBA+KT hormone combination was very efficient for callus induction and proliferation of G. babardence species, which can obtain high efficient callus induction and shorten the period of proliferation. The optimal combinations was 0.2 mg/L 2, 4-D + 0.4 mg/L KT +1.0 mg/L IBA on callus induction medium and 0.05 mg/L 2,4-D + 0.2 mg/L KT + 0.5 mg/L IBA on callus proliferation and differentiation medium. Suspension culture of calli was helpful for calli proliferation and somatical embryogenesis, which can averagely obtain 5% higher differentiation rate than calli subcultured on solidified medium6. Various aspects of transformation processes were examined in efforts to improve the efficiency of production of transgenic cotton. Embryogenic calli which were precultured for 12 days proved to be a valuable explant to cotton transformation. The effects of concentration of A. tumefaciens inoculated, time of co-cultivation, media for co-cultivation was evaluated. OD (A₆₀₀) value 0.4 proved to be significantly better than other concentrations. Concentration of acetosyringone at 50 mg 1^-1 during co-cultivation significantly increased transformation efficiency. Under the same transgeneic procedure, four genotypes obtained different transformation efficiency.7. The isopentenyl transferases gene was introduced into cotton genome via Agrobacterium tumefaciens. Transgenic plants were obtained from four cultivars, namely Coker 201,YZ1,Ym4 and Em23. The putative transgenic plants were tested by PCR and Southern blotting. The results showed that the transgenes has inserted into cotton genome. Transgenic plants with ipt gene showed many interested characters, for example, dark green leaves and flourishing branches and buds. 8. To optimize the procedure of somatic embryos germination, rooting and transplant, many factors were studied using somatic embryos of YZ-1 as target. The results showed that low concentration of inorganic salt, namely 1/2 MS inorganic salts, in the embryo germination media could increase the ratio of normal regenerated plant and help build stronger root systems. Phytagel was proved a better agent for solidifying the medium than Agar. Activated carbon added in rooting medium was proved to be helpful for root regeneration of somatic embryos. Roots of some regenerated plantlets with 3-5 true leaves would be brown due to a long culturing period in solid medium. These plantlets could rebuild strong root system after the brown roots were removed and inoculated on fresh medium with low concentration of inorganic salt. Plantlets could healthily grow after stepwise opening the lids of culture bottles, which help the regenerated plantlets accommodate to open circumstances.