Transformation Study Of Antibacterial Peptides Gene In Watermelon

Watermelon is one of the major vegetable crops widely cultivated in many areas of the world. Its cultivating area covers the second place among all the vegetable crops and its agricultural yield covers the third place.Fusarium wilt of watermelon ( Fusarium oxysporm f. sp. niveum (E. f. Smith) Synder et Hansen) , one of the soil-borne pathogens, is now well established throughout the watermelon-growing regions of the world. While the availability of highly disease resistant materials is qui te rare. It is a limiting factor in production.Antibacterial peptides, synthesized by induction in organism, show strong bactericidal action. Glucanase and chitinase genes are reported quite frequently with high anti-fungal action in recent years. In this paper, the transformation of glucanase and chitinase genes into watermelon by the method of Agrobacterium tumefaciens mediation were conducted, in order to enhance the wilt resistance in watermelon significantly. The main results were as following:1. High efficient plant regeneration system was established by using cotyledonary explants. Cotyledonary explants were taken from 5-day-old watermelon seedlings. Ninety five percent of shoot regeneration rate was obtained on medium consisted of MSO basal medium supplemented with 2 mg/L benzylaasenine and 0.2mg/L indole-3-aceticacide. Cultures were maintained in a tissue culture chamber of 26癈 under a 16h light/8h dark photoperiod. The regenerated shoots were elongated on the same basal medium(MSO) supplemented with 0.2 mg/L kinetin. The elongated shoots were rooted on the basal medium(MSO) supplemented with 0. 1 mg/L naphthalene acetic acid. Seventy nine percent of root generation rate was obtained.2. Kanamycine was used as the selective antibiotics. We used thekanamycine concentration from 0 to 125 mg/L in watermelon shoots regeneration. Shoots regeneration rate decreased with the increase of kanamycine concentration. When kanamycine concentration reached 125 mg/1, shoots regeneration were quite difficult.3. GUS assay was used to screen the putative transgenic plants. The leaf of transgenic plants turned into blue after GUS stain, which demonstrated that GUS gene was expressed in putative transgenic plants.4. We used PCR analysis to screen the NPTII, glucanase and chitinase genes of putative transgenic plants. The NPTII, Glucanase and chitinase genes were all present in all transgenic plants with the fragments of 240bp, 201bp and 178bp respectively. The none-transgenic plants had no such fragments. PCR analysis demonstrated that glucanase and chitinase genes had integrated into the watermelon.