Introduction Of Antimicrobial Peptide Gene Gnk2-1into Watermelon Via Pollen-tube Pathway

Watermelon (Citrullus Lanatus.) belongs to Cucurbitaceae and it is one of the mostimportant horticultural crops in China. At present, most of the main watermelon cultivars inChina are under threat of a variety of watermelon diseases, primarily due to the lack of astrong disease resistance, especially fusarium wilt, which has a seriously impact on the yieldand the quality of watermelon. Antimicrobial peptide protein Gnk2-1, which is extracted fromGinkgo biloba, shows a strong resistance to some melon fungi such as Fusarium oxysporum.In this research, the antimicrobial peptide gene Gnk2-1was introduced into watermelonaccession04-1-2and H45by pollen-tube pathway, the influence on fruits and seedsdevelopment of treated plants was studied, and the molecular detection and disease resistanceassay of T1generation were analyzed, in order to study the feasibility of transferringexogenous gene into watermelon by pollen-tube pathway. The main results were as follows:1. The antimicrobial peptide gene Gnk2-1was introduced into ovaries of watermelonaccession04-1-2and H45by pollen-tube pathway with different concentration (100ng/μL,200ng/μL,300ng/μL,400ng/μL) at different time (24h,27h,30h,33h) afterself-pollination. The fruit setting rate of04-1-2T0generation increased with the increment oftreatment time after self-pollination, the average was33.8%, which was56.2%lower thancontrol group; by contrast, the average fruit setting rate of H45T0generation was6.2%,which was73.8%lower than control group. The average number of seeds of04-1-2and H45T0generation both rose by the increasing of treatment time, though the figures all showed asignificant decrease under all treatments compared with control group.2. The germination rate of04-1-2T1seeds treated27h after self-pollination and theseedling emergence rate of04-1-2T1seeds treated24h after self-pollination decreasedsignificantly; compared with control group, there was no distinct difference illustrated in therates of germination and seedling emergence of04-1-2T1seeds between different concentration treatments. Under all treatment time the rates of germination and seedlingemergence of H45T1seeds showed a remarkable decrease compared with control group; theseedling emergence rate of H45T1seeds was significantly lower than control group underdifferent concentrations, while the germination rate only showed the same trend at200ng/μL.3. The seedlings of04-1-2and H45T1generation were screened by kanamycin, and thekanamycin resistant rates were7.9%and1.3%respectively.32positive plants were obtainedfrom04-1-2T1kanamycin resistant plants by identification of PCR and the totaltransformation rate was2.5%; the optimum transformation time was24-27h afterself-pollination and the optimum transformation concentration was100-200ng/μL. The wasno positive plants detected from H45T1kanamycin resistant plants.4. The positive plants of04-1-2T1were inoculated with fusarium by root-immersedmethod, it’s observed that the transgenic plants showed a stronger resistance to fusarium wiltcompared with non transgenic plants after10days.