PRODUCTION OF TRANSGENIC CUCUMBER PLANTLETS CONTAINING SEQUENCES FROM WATERMELON MOSAIC VIRUS-II FUSED WITH GFP GENE

Abstract

Cucumber (Cucumis sativus L. cv. Faris) explants were transformed by LBA4404 strain of Agrobacterium tumefaciens harboring the binary vector pPZPnptCat-WMV. The T-DNA region contains Neomycin Phosphotransferase II (NPT II) as a selectable marker gene and sequences of Watermelon Mosaic Virus-II (WMV-II) fused with Green Fluorcent Protein (GFP) gene under control of 35S promoter. Agrobacterium-mediated transformation was optimized using GFP as a reporter gene. The optimized parameters were Agrobacterium concentration, preculture period, co-cultivation period and immersion time. Results were recorded based on the percentage of green fluorescent protein (GFP) expression. Agrobacterium concentration at (OD 600 nm 0.8), four days of pre-culture, three days of co-cultivation and sixty minutes of immersion time gave the highest percentage of GFP areas (78%, 46%, 82% and 52%, respectively). Cotyledon was the best explants to give the highest percentage of GFP areas in all tested parameters. Following co-cultivation, leaf, cotyledon, callus and shoot-tip explants were cultured on selective and regeneration Murashige and Skoog (MS) medium containing 1mg/L 6-Benzylaminopurine (BA), 200 mg/L kanamycin and 300 mg/L cefotaxime. Kanamycin resistant shoots were induced from these explants after four weeks. Putative transgenic plantlets were produced from leaf, cotyledon and shoot-tip explants at 8 weeks and at 12 weeks from callus. Integration of the transgenes in the cucumber genome was confirmed by PCR analysis. This study showed that the Agrobacterium-mediated gene transfer system and regeneration via organogenesis is an effective method for producing transgenic cucumber plantlets. Cloning construct of Catgfp-WMV-2 into binary vector pPZPnpt was done successfully. Agrobacterium concentration at 0.8, four days of pre-culture, three days of co-cultivation and sixty minutes of immersion time gave the highest number of GFP positive percentage (78%, 46%, 82% and 52%, respectively). Cotyledon was the best explants to give the highest number of GFP positive percentage in all tested parameters. Putative transgenic plantlets were obtained from (leaf, cotyledon and shoot-tip explants after 8 weeks) and after 12 weeks from callus.