Development Of Large-scale And Efficient Transformation Method For Cucumis Melo (L.cv. Hetao) Using Agrobacterium-mediated Plant Genetic Transformation Via Plant Apical Meristem

It is an important modern means of genetics and breeding to transform foreign genes into plant cells using genetic engineering technology. Many systems have been established for plant transformation and gained a lot of valuable transgenic plants through 20 years research. At present transgenic technology includes physical, chemical and biological method. Although these methods have different advantages, there are still some deficiencies, which promote emergence of new transgenic technology. Using plant meristem for gene transformation will avoid callus culture, so somaclonal variation can be reduced and the foreign gene can be inherited stably.The object of the experiment is Cucumis melo L. cv. Hetao meristems of axillary buds. Using SPSS software, designed 16 groups of orthogonal experiments and investigated the parameters of transformation, including the bacterial concentration. the time of transformation and co-cultivation. The procedure is as follows: cut down the Cucumis melo L. cv. Hetao axillary buds and inoculated by engineering suspension of A. tumefaciens after being pricked with a needle. Then the stem which sprouts from axillary buds were cut off and induced to takes root in the medium to obtain regeneration plants. The results showed that the optimum concentration of A. tumefaciens is OD₆₀₀ = 0.7; the adequate time of transformation and co-cultivation is 10 min and 2 days respectively. The optimum concentration of gentamicin for selecting transformed plant on the leaves and rooting was 20 mg/L and 40 mg/L, respectively. Using this favourable transformation system, the positive rate of transgentic plants is 76% and the living rate is 76.3%.Based on this transformation system, two plant expression vectors (pAMS-CTR1,pAMC-CTR1) were transfered into the apical growing points of Cucumis melo L. cv. Hetao seedling through Agrobacterium-mediated plant genetic transformation. Each kind of 50 transgenic plants have been tested through PCR. The results are as follows: there are 35 plants containing pAMS-CTR1 and 34 plants containing pAMC-CTR1. The transformation efficiency is 70% and 68%, respectively.