| Cucumber(Cumcumis sativus L.) is an important vegetable in the worldwide, occupies an extremely important role in agricultural production and the national economy. But cucumber often suffers form the influence of such factors as continuous rainy weather or low temperature and weak light haze weather, short-day etc, leads to poor pollination fertilization and abortive fruit especially in the protected cultivation,it has such a great deal of influence on cucumber cultivation of high yield, high quality and efficiency that causes huge economic.In recent years, the rapid development of genetic engineering provides us with a new, fast breeding platform. Due to the factors such as the narrow genetic basis of cucumber,, the long conventional breeding period and low efficiency, it is difficult to get breakthrough of using conventional breeding,therefore, it is an effective way of using plant gene engineering method for cucumber genetic improvement. High efficiency, stable system of regeneration in vitro and genetic transformation is the base of cucumber transgenic breeding. In this experiment, the explant types and different combinations of hormones and hormone concentrations were studied by using "Jinyou No.1" cotyledon and cotyledon node as explants, a highly efficient and stable regeneration was established. The effects of different concentration of Kan, the AS concentration, infection time, pre-culture and co-culture time on cucumber genetic transformation were studied as well, established and improved a highly efficient cucumber transgenic system. On this basis, the protein gene CsEXP10 was transferred into cucumber medied by Agrobacterium tumefaciens to obtain transgenic plants. It provided theoretical basis for the study of cucumber germplasm innovation and variety improvement.The main results were as follow:1. Regeneration system of cotyledonary and cotyledonary node: Cucumber seeds were disinfected on the super-clean worktable with 70% alcohol for 1 min, first disinfected with 0.1% mercuric chloride for 15~20 min, then washed with sterile water rinse 5~6 times, finally,the seeds were inoculated on MS medium; for 5-6d, when the two cotyledons had not fully opened, the procedure involved slicing the embryonic axis(2 mm) while still attached to the cotyledons, removing the growing point, slicing the embryonic axis into two halves while still attached to the cotyledons and removing 1/2~2/3 of the cotyledon end and the cotyledon edge so that the explants contained one cotyledon with a small portion(2 mm) of split embryonic axis attached to it(including the axillary meristem). These explants were placed on shoot regeneration media(MS+2.0 mg/L 6-BA+2.0 mg/L of AgNO3), the highest bud differentiation frequency is 96.2%, regeneration number could be up to 4.38, rooting rate could reach 100%, after 40-50 d, regenerated plants could be obtained.2. Established and optimized genetic transformation. Mainly included: Pre-culture time,infection time,and co-culture time, the AS concentration and root induction. After pre-cultivation for 2d, the cotyledon node explants were then immersed in the Agrobacterium tumefaciens suspension containing 100 μmol/L AS for 15 min under constant shaking, following which, the explants were removed, blotted dry with sterile filter paper to remove excess bacteria, and then the explants were removed to selected medium: MS+2.0 mg/L 6-BA+2.0 mg/L AgNO3+300 mg/L Cef+100 mg/L Kan.3. Molecular detection of transgenic plants. we had got 9 transgenic plants through PCR analysis; they would be further tested with GUS which found that nine plants showed blue, the initial preliminary positive rate was 1.81%. |
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