الفهرس | Only 14 pages are availabe for public view |
Abstract Commercial cucumber cultivars were explored for embryogenesis and plant regeneration induced in somatic tissues on plant growth regulators. An efficient in vitro regeneration protocol for Cucumis sativus L. via somatic embryogenesis has been developed. Embryogenic callus cultures were established from mature seeds, cotyledons and shoot tips on Murashige and Skoog (MS) medium containing 87.64 μM sucrose, 0.8% agar, 2,4- dichlorophenoxyacetic acid 2,4-D and kinetin. Maximum callus induction (94%, 92%) was observed in cotyledons and mature seeds on MS medium supplemented with 1mg/l 2,4-D, respectively. Somatic embryos were observed on mature seed explants after 17 weeks on MS medium supplemented with 5mg/l 2,4-D (33%) and 0.5mg/l α-naphthaleneacetic acid (NAA) (27%). while somatic embryos were observed on cotyledon explants after 18 weeks on MS medium supplemented with 2mg/l 2,4-D (10%) and 0.5mg/l NAA (25%). The highest percentage of somatic embryogenesis (40%) was obtained with 2 mg/l 2,4-D and 0.5 mg/l NAA (30%) on shoot tip explants after 13 weeks. Regeneration of adventitious buds from callus of cotyledon and mature seeds were achieved after 8 weeks on MS medium supplemented with 0.5mg/l and 1mg/l 6-benzyladenine (BA), respectively. Generally, auxin was found critical for induction of callus and formation of somatic embryos, while the cytokinin was essential for callus differentiation and plant regeneration. Cucumber explants (Cucumis sativus L. cv faris) were transformed by LBA4404 strain of Agrobacterium tumefaciens harboring the binary vector pPZPnptCat-WMV. The T-DNA region contained Neomycin Phosphotransferase II (NPT II) as a selectable marker gene and sequence of Watermelon Mosaic Virus (WMV-II) fused with GFP gene. Agrobacterium-mediated transformation was optimized using GFP as a reporter gene. Optimized parameters were Agrobacterium concentration, pre-culture period, co-cultivation period and immersion time. Obtained results were based on the percentage of GFP expression. Agrobacterium concentration at OD600nm 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. Following co-cultivation, leaf, cotyledon, callus and shoot-tip explants were cultured on selective medium containing 200 mg/l kanamycin + 300 mg/l cefotaxime. Kanamycin resistant shoots were induced from these explants after four weeks. Putative transgenic plantlets were obtained from leaf, cotyledon and shoot-tip explants after 8 weeks and after 12 weeks from callus. Molecular evidences by PCR demonstrated the effectiveness of the transformation procedure. These results show that the Agrobacteriummediated gene transfer system and regeneration via organogenesis is an effective method for producing transgenic cucumber plantlets containing virus sequences of WMV-II to confer resistance against this virus. |