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

Authors

  • Y. A. KHIDR Plant Biotechnology Dept., Genetic Eng. and Biotech. Res. Inst., Sadat City, Menofia University
  • AMAL MAHMOUD Plant Biotechnology Dept., Genetic Eng. and Biotech. Res. Inst., Sadat City, Menofia University
  • E. EL-ABSAWY Department of Bioinformatics, Eng. and Biotech. Res. Inst., Sadat City, Menofia University, Egypt
  • A. A. HEMEIDA Department of Bioinformatics, Eng. and Biotech. Res. Inst., Sadat City, Menofia University, Egypt
  • M. E. HASAN Department of Bioinformatics, Eng. and Biotech. Res. Inst., Sadat City, Menofia University, Egypt

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.

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2016-01-12

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