Generation Of The Transgenic Antiviral Watermelon And Melon

Watermelon and melon are important cucurbit crops in China, the cultivation areas and the consumptions amount top the list in the world. But the plant virus diseases seriously affect the qualities watermelon and melon, also reduce the production, and thus result in much less yield. By means of Agrobacterium mediated genetic transformation method, the virus derived gene is imported transferred into the genome of watermelon and melon, and explore the relationship between virus and cucurbitaceae crops host, create new antiviral cucurbitaceae germplasm. Thus our research project is of important huge theoretical and practical significance and practical application value. The main results are as follows:1.Optimization of regeneration system for the watermelon tissue culture regeneration system. Watermelon plantlets easily become yellowish and gradually die with subsequent subculture. The result showed that when doubling the concentration of Fe-EDTA, the shoots in vitro grew stronger after several subculture. The roots of watermelon plantlets are fragile during transplanting. The re sults showed that 2-3 cm watermelon plantlets were cut as scion and then grafted onto bottle gourd rootstock with two cotyledons completely opened. It can not only produce high survival rate and stronger watermelon plantlets, but also yield mature fruits with one month in advance. It paves a way for transgenic technology in watermelon.2.We established an effective transformation protocol mediated by Agrobacterium for watermelon. Half cotyledons proximal to stem from 5-day-old seedlings were used as explants, when the cotyledons changed from yellow to green. Watermelon cotyledons that changed from yellow to green were pre-cultured for five days and then co-cultured for three days. After five day pre-culture and three day co-cultivation, the explants were transferred onto the selection medium for adventitious shoots induction. After that, the putative transgenic shoots were sub-cultured for three times, and then transferred to rooting medium or grafted onto rootstocks to obtain transgenic plants. The exogenous gene in watermelon genome was verified by PCR and Southern hybridization. CMV virus attack experiment preliminary indicated the disease resistance of watermelon.3. Artificial micro RNA has the capacity of binding and degrading RNA of virus genome, thus make the plants resistant to the virus. This study used artificial micro RN A construct which can identify the 2a/2b gene of CMV. The ELISA detection index was less than 3 after being infected by CMV virus, which suggested that transgenic watermelon was conferred resistance to CMV. In this study, our transgenic watermelon lines obtained by artificial micro RNA technology were resistant to the CMV. It provides a new way for genetic improvement of cucurbit crops.4. We established an effective transformation protocol mediated by Agrobacterium for melon. Half of cotyledon proximal to stem(without stem apex) from 5-day-old seedlings were used as explants. After five day pre-culture and three day co-cultivation in medium with MS+BA1.2mg/L, the explants were transferred onto the selection medium for adventitious shoots induction. The exogenous gene in melon genome was verified by PCR, Southern hybridization and GUS detection.5. An expression vector containing hairpin structure sequences was transferred into plant and induce silencing of the virus genes. They can also improve disease resistance by a similar mechanism. A plant expression vector with three inverted repeats of coat protein genes from three viruses(ZYMV, WMV and CMV) were constructed. Agrobacterium mediated transformation was used for transformation melon varieties. They provide a new method to study the transgenic cucurbits.This study provided a new strategy for the transgenic technology of cucurbit crops. Transgenic watermelon and melon lines with res istance to virus were obtained by the improved method.