Optimized System For Plant Regeneration And Agrobacterium-mediated Transformation Of Watermelon (Citrullus Lanatus)

Watermelon(Citrullus lanatus Thumb.) is an important cucurbit crop species and one of the most important vegetables. It is cultivated broadly in many countries and regions in the world as an economic crop of high value. But its output and quality have been severely influenced due to plant diseases. So improvement of its disease-resistance has always been an important research field.Watermelon (Citrullus lanatus cv. Zaojia, F17, Huang Meiren mature seeds were used as explants sources. The target of this study is to establish in vitro regeneration system in watermelon, and intrduct disease-resistance transcription factor Pti4 gene in tomato into watermelon by agrobacterium-mediated transformation, and carry out PCR detection in transgenic plant. The main results are as follows:1. Establishment of in vitro regeneration system in pepper watermelon1) Dark cultivation could enhance the rate of germination and seedlings in a certain degree. Rate of germination and seedlings cultured in dark for three days were 96% and 94%, higher than that without dark cultivation.2) The optimal explants screened in this test was proximal cotyledons and entire cotyledons, and their adventitious bud inductivity were similar too,88.56% and 89.67%, the number of adventitious buds per explant was 7.45 and 7.78; both are higher than other explants.3) There was no significant difference of inductivity between mediums with 6-BA alone and with 0.2 mg/L IAA for the explants of entire cotyledons and proximal cotyledons. The optimal working concentration of 6-BA was 2 mg/L-3 mg/L in cotyledon; The best performance occurred to MS medium with 2.0 mg/L 6-BA+0.2 mg/L IAA.4) The inductivity of three cultivars was absolutely different. The adventitious buds inductivity and the number of adventitious buds per explant of Zaojia was 100% and 4.73, which was better than Huang Meiren and F17.5) The percentage of elongation was almost the same both in MS medium supplemented with 0.2 mg/L KT or not, but adventitious buds after elongated in MS medium supplemented with 0.2 mg/L KT were hard to induce roots.2. Study on Agrobacterium-mediated Genetic transformation of watermelon1) The pre-cultivation for 2 days in the dark resulted in a higher percentage of explants with resistant buds (10.0%).2) The highest percentage of kanamycin-resistant shoots was obtained at 10 min inoculation (11.7%).3) 2 days co-cultivation was found optimal for zaojia watermelon and obtained the production of significantly higher rate of kanamycin-resistant buds (16.7%) as compared to other treatment of co-cultivation.4) Optimal selective medium was:MS+6-BA (2.0 mg/L)+Kan (100 mg/L)+Cef (500 mg/L).5) Introduction of exogenous target gene Pti4 into watermelon was by agrobacterium-mediated transformation, and got 67 Kan-resistance plants in watermelon. Use NPT//primer to carry out PCR detection in these plants (blank as negative control, agrobacterium bacterial liquid with trager gene Pti4 as positive control),49 Kan-resistance plants amplified the expected gene fragment, conversion rate is about 73.1%.