Transformation Of CsTril And Optimization Of Regeneration System In Cucumber (Cucumis Sativus L.)

Although cucumber tissue culture and genetic transformation have been developed for several decades,there are still many problems in technology,such as low ratio of regeneration in vitro and low efficiency of genetic transformation.Therefore,in cucumber,optimizing in vitro regeneration system and improving the efficiency of genetic transformation are urgently needed for the application of genetic engineering in germplasm innovation and breeding.In this study,the cucumber cultivar ‘Xintai Mici’ was selected as the experimental material to be transferred the cucumber Tril gene through constructing vector,getting explant,co-culturing with agrobacterium,screening transformant and plant regeneration.For improving cucumber transformation,the concentration of 6-Benzylaminopurine(6-BA)in the differentiated medium and kanamycin(Kan)in the rooting medium were optimized.The germination rate,rooting rate and transformation rate were further improved to lay the foundation for the genetic transformation system of cucumber.The main results are as follows :By constructing the plant expression vector containing pBI121-9930TrilPro-TrilCDS-3xflag and the Agrobacterium guidance method,the cucumber ‘Xintai Mici’ varieties were transformed and 11 seedlings were successfully obtained.After PCR identification,qRT-PCR detection,and P1 single plant offspring identification,one plant was identified as a transgenic seedling,and the transformation rate was 0.33 %.In view of the low conversion rate of the transgenic seedlings,the result showed that the rate of shoot regeneration was more than 76% in thedifferentiation stage of 6-BA concentration of 0.5 mg/L,which effect was the best.When the optimal concentration of Kan at the rooting medium is60 mg/L,it could screen effectively as the lowest concentration.The effects of three different Kan-screening methods on the rate of positive individuals were studied in the transformation course based on the optimal concentrations of 6-BA and Kan.The result showed that the transformation period was the shortest(45 d),shoot rate(62.6%),rooting rate(22.5%)and the positive seedling rate was highest(1.33%)with the selecting medium on rooting stage only,adding 60 mg /L Kan.