Abstract: A simplified and efficient transformation method was developed to produce a transgenic Lycopersicum esculentum plant. Primarily the regeneration system was standardized with 10-12 days old cotyledon explants inoculated on Regeneration Transfer (RT) medium (RT = MS basal medium supplemented with 8.88µM of BAP and 1.14µM of IAA) for callus, shoot induction and MS basal hormone-free medium for root induction. Transformation work was carried out with Agrobacterium tumefaciens strain LBA4404 harbouring binary plasmid PGPTV/hevein (Plasmid Gus Plant Transformation Vector) with kanamycin resistance marker gene -neomycin phosphotransferase (npt-II). Transformation efficiency of cotyledon explants showed higher percentage on 24hrs pre-culture on petri-dish containing RT medium, infection with bacterial suspension having optical density 0.4 at OD600nm on with 1:1 dilution for 10min and 48hrs co-cultivation period on petri-dish containing RT medium in the dark. Putative transformed explants were cultured on Selection Transfer (ST) medium (ST medium = RT medium containing antibiotics Augmentin - 250mg/l + Cefatoxin - 200mg/l + Kanamycin - 050mg/l). The Polymerase Chain Reaction (PCR) molecular confirmation of amplified npt-II gene from genomic DNA of the transformed tomato plant was an indication of the integration of the hevein gene in the tomato plant. The antifungal activity was confirmed by treating the transformed plant with Fusarium oxysporum and had resistance against the fungal disease.
Keywords: Regeneration, Pre-culture, Co-cultivation, hevein gene, Agrobacterium.
Title: Agrobacterium-Mediated Transformation of hevein gene to Lycopersicum esculentum Mill. Cultivar Arka Abha
Author: Arulananthu Gnanadurai, Srither Bhat, Rajesh Govindan, Ramesh Narayanaperumal
International Journal of Life Sciences Research
ISSN 2348-313X (Print), ISSN 2348-3148 (online)
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