Study On Efficient Regeneration System And Transfer By Agrobacterium Mediation Into Cucumber

Cucumber plays an important role in vegetable supply, but its quality and agricultural yield tends to be reduced constantly by various plant diseases. Traditional method of breeding has many disadvantages, such as long period, low efficiency, and cucumber exists serious hamper of sexual hybridization. So it is difficult to obtain the superior cultivar with high resistance.This study used cotyledons of S05, S06 as explants, established a highly efficient regeneration system of cucumber, then introduced luc gene and ATT1 gene into cucumber through Agrobacterium mediation. The ATT1 gene encodes a cytochrome P450 oxidase required for biosynthesis. It enhances the cutin so it will increase the risistance to diseases. The factors influencing regeneration rate and transformation rate were discussed.The results were summarized as follows:1 Add AgNO3 and ABA to the inducing medium, the explants can regenerate adventitious shoots. By this means, improve the regeneration rate and No.of shoots per explant. To S05, the best inducing medium is MS+BA2.0mg L-1+ABA1.0mg L-1'+AgNO32.0mg L-1; To S06, MS+BA1.5mg L-1+ABA0.5mg L-1+AgNO32.0mg L-1 is the best. The regeneration rate are 81.6% and 91.2% respectively.2 Cefotaxime promotes shoot organogenesis, but carbenicillin prevents inversely. So we use 400 mg L-1 Cefotaxime to eliminate Agrobacterium.3 The optimum procedure in cucumber transformation as follows S05:The cotyledons were precultured for 2 days, infected 15 min, co-cultivated for 3 days in inducing medium. S06: The cotyledons were precultured for 1 day, infected 20 min, co-cultivated for 3 days in inducing medium.4 Detection of transforming regenerated cucumber We used PCR to detect the transforming plantlets. 20 plants of S05 and 14 plants of S06 were positive with lucgene; 3 plants of S05 and 5 plants of S06 were positive with ATT1 gene. The results of PCR demonstrated the integration of target gene into cucumber genome primarily.