Establishment Of Regeneration And Genetic Transformation System In Cucumber (cucumis Sativus L.)

Cucumber (Cumcumis sativus L.) is one of the important vegetable crops widely cultivated around the world. It plays a very significant role to the annual supply of vegetables. But cucumber often suffers from a number of diseases and pests, and has caused huge economic losses in actual cultivation and production. Because the traditional methods of insect prevention and control have many disadvantageous factors, selection and breeding resistant variety is the best way to improve the resistance to disease and pect of cucumber. However, conventional breeding is difficult to obtain breakthrough for the lack of resistant materials. In this study, the effects of genotype and phytohormone on regeneration in vitro were studied by using different cucumber materials cotyledon and cotyledon node as explants, a highly efficient regeneration was established. The effects of AS, infection time, PH value on cucumber genetic transformation was studied as well. Then, the GUS gene and MiMPK1 gene were transferred into cucumber, which medied by Agrobacterium tumefaciens. It provided theoretical basis for the study of cucumber genetic transformation.The main results were as follow:1. Genotype was one of the influence factors on regeneration. The regeneration capacity of different genotypes explants was very significantly. In the study of cucumber cotyledon regeneration, Jilinhangua had the highest differentiation ability among the six materials (Zhongnong No.8, Jilinhangua, Sichuanbaigua, Gy14, 65G, 9110Gt). Its regeneration frequency and shoots per explants were 96.7% and 5.2 in the medium MS+1.5mg/L6-BA+0.5mg/LABA+2mg/LAgNO₃. In the study of cucumber cotyledon node regeneration, Sichuanbaigua had the highest differentiation ability among the eight materials (Jilinhangua, Sichuanbaigua, Gy14, 65G, 0513, 228, 185, K2148), its regeneration frequency and shoots per explant were 100 % and 6.4 in the medium (MS+0.5mg/L6-BA+2mg/LAgNO₃).2. Plant hormone is necessary for explants regeneration. 6-BA is one of the most common hormones in cucumber differentiation in vitro . In the study of cucumber cotyledon regeneration, 6-BA was the dominant factor of adventitious buds regeneration, but the addition of ABA significantly increased the number of adventitious buds. The best inducing medium of shoots for Gy14 and 9110Gt was MS+1.0mg/L6-BA+1.0mg/LABA+2mg/LAgNO₃, the best inducing medium for Jinlinhangua and Sichuanbaigua was MS+1.5mg/L6-BA+0.5mg/LABA+2mg/LAgNO₃, the best inducing medium for 65G was MS+1.5mg/L6-BA+1.0mg/LABA+2mg/LAgNO₃, and the best inducing medium for Zhongnong No.8 was MS+2.0mg/L6-BA+0.5mg/LABA+2mg/LAgNO₃. In the study of cucumber cotyledon node regeneration, The best inducing medium of shoots for 228,185 and 65G was MS+2.0 mg/L 6-BA+2 mg/L AgNO₃, The best inducing medium for Sichuanbaigua, Jilinhangua, K2148 and 0513 was MS+0.5mg/L6-BA+2mg/LAgNO₃, and in the medium MS+1.0 mg/L 6-BA+2 mg/L AgNO₃, Gy14 had the best regeneration frequency.3. The tolerance and sensitivity of cucumber cotyledon to Km and Hyg was studied. The selection pressure concentration of Km and Hyg were 75 mg/L and 10 mg/L.4. The results of transgenic system improvement were that the number of resistant shoots significantly increased, while AS added to the pre-culture medium, bacteria liquid and co-culture medium, the best concentration was 250 ul/L, 500 ul/L and 250 ul/L. The regeneration frequency and resistant shoots per explants were highest when the infection time was 15 min. The lower PH value selective medium promoted explants differentiation while it was 5.4.5. The GUS gene was transferred into cucumber medied by Agrobacterium tumefaciens. There were 14 resistant plantlets showed blue via gus histochemical identification. This indicated that GUS gene had been integrated into the cucumber genome, and expressed in tissues, the preliminary positive rate was 7.4 %.6. The MiMPK1 gene was transferred into cucumber medied by Agrobacterium tumefaciens. PCR analysis showed that a total of 28 resistant plantlets amplified were specific bands about 500 bp, the the preliminary positive rate was 5.3 %.