Agrobacterium-mediated Transformation Of Cucumber With Chitinase And β-1, 3-glucanase Genes

Crop production is limited by pathogenic fungi and other diseases. Agronomic practices such as application of pesticides were main methods to solve the problem, and some pesticides can lead to heavy environmental pollution. Breeding of disease resistant cultivars is the best way to fight against fungal pathogens. Generally, conventional breeding is efficient, but time-consuming and less efficient for development of disease resistant cultivars due to the reproduction barrier among different species. Transgenic approach offers a potentially fungicide-free and environment-friendly solution for fungal pathogen control.Cucumber (Cucumis sativus L.) is one of the most popular and world-wide consumed vegetables, it has been extensively investigated for genetic engineering. The pathogenesis-related (PR) genes, such as the bean chitinase (Chi) and tobacco 1,3-glucanase (Gln) genes, appear to be a preferred avenue for engineering crop protection. It will be potential to obtain disease-resistant resources by introducing PR gene into cucumber genome.This research aimed at establishing a system of regeneration and genetic transformation of cucumber and obtaining some anti-fungal transgenic cucumber materials via Agrobacterium-mediated transformation. Two PR genes - Chi together with Glu and three cucumber cultivars - Jinyan 4, Jjinyan 7 and LBC were involved in this study. The main results were as follow:1. Three-day seedling cotyledons of cucumber were optimal explants for regeneration, and an optimal regeneration was achieved in MS median supplemented with 2.0 mg/L BA and 1 mg/L ABA. BA was more effective than TDZ (Thidiazuron) in inducing cucumber adventitious shoots.2. Shoot regeneration of cucumber cotyledons was promoted by appropriate concentration of AgNO3 (2.0 mg/L) and a higher concentration proved harmful.3. The regeneration frequencies of Jinyan 4 and Jinyan 7 were up to 92.97% and 82.43% respectively on the MS medium supplemented with 2.0 mg/L BA, 1.0 mg/L ABA and 2.0 mg/L AgNO3.4. Shoot regeneration from cotyledons of non-transformed cucumber was completely inhibited by 100 mg/L kanamycin.5. Shoot regeneration of cucumber cotyledon was not significantly affected in the optimal medium supplemented with 100 - 400 mg/L Cefotaxime.6. The transformation frequency of cucumber was not significantly enhanced by feeder layer of tobacco cell suspension during co-cultivation.7. Genomic DNA was extracted from some putative transgenic plants. The results of Multiplex PCR and Southern blotting confirmed that both the Chi and Glu genes were integrated into the cucumber genome. In this study, transgenic plants from three genotypes, Jinyan 4, Jinyan 7 , were obtained.8. Primary disease bioassay on transformants showed improved resistance to cucumber downy mildew.