Study On Transfer Of CsGRX4 Gene By Grobacterium Mediation Into Cucumber

Transgenic technology is an effective way for the development of new cucumber cultivars for disease resistance, while its application has also been restricted owing to the low frequency of regeneration and genetic transformation on cucumber. So it has become a critical and urgent work to build a highly efficient regeneration and genetic transformation system. This study uses C1 and Y4 as materials, and established a highly efficient regeneration system of cucumber. Then the effect of some factors in cucumber genetic transformation which transferred the CsGRX4 gene into cucumber, mediated by Agrobacterium tumefaciens was analyzed. This study built the foundation of high frequency genetic transformation.The main results were as follow:Firstly the best disinfection system was established. The best disinfection way of Y4 was 3% NaClO for 15 min, and C1 was 0.1% HgCl2 for 3 min.Secondly the best regeneration system was established. Stage b3 of seed growth, in which the seed coat was completely off and the cotyledons hadn’t expanded yet showed the maximum number of inducing buds. The best way of cutting the explant was to cut hypocotyl longitudinally, remove the terminal bud,keep one cotyledon with 1mm petiole and cut 2/3 of the cotyledon.And the best way to plant the explants was to insert at a little diagonal angle with underside down. The best buds induction medium for Y4 was 3.0 mg/L 6-BA+1.5 mg/L ABA+1.5 mg/L AgNO3+MS. While the best buds induction medium for C1 was 2.0 mg/L 6-BA+1.0mg/L ABA+1.5 mg/L AgNO3+MS. Under the optimal condition, the rate of budding explants and number of induced buds of per explant was 90.00% and 2.71 for Y4, while 87.22% and 2.53 for C1.Thirdly the tolerance and sensitivity of cucumber cotyledon to Basta and Cef was studied. The selected concentration of Basta and Cef was 3.0 mg/L and 500 mg/L.Moreover, the effect of some influencing factors on the frequency of cucumber genetic transformation were analyzed and discussed. Stage b2 of seed development, in which the seeds coat wasn’t entirely off and the uncover part of cotyledons was green was the best stage of seed development to induce resistant buds.The best way of cutting the explant was to cut hypocotyl longitudinally, remove the terminal bud, keep one cotyledon with 1mm petiole and cut 2/3 of cotyledon.And the best way to plant the explants was to insert at a little diagonal angle with underside down. The best length of explant preculture was 3 days for C1 and 2 days for Y4.Then the best infection system was to infect the explants using Agrobacterium tumefacious of which OD value was 0.4 with 0.1 g/L AS for 10 minutes, and cocultured for 2 days on co-culture medium.In the end, the resistant plants were transplanted. Then the survival plants were analyzed with Basta resistance screening and PCR test. The number of survival plants for Y4 was 12, while for C1 was 15.Under the treatment of 1.0 g/L Basta, all the C1 plants died. According to the PCR results, no Y4 plants were positive.