Standardization of Regeneration, Agrobacterium-Mediated Transformation, and Introduction of Nucleocapsid Gene of Watermelon Bud Necrosis Virus in Watermelon
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Seven types of explants (proximal cotyledon, distal cotyledon, distal cotyledon leaves, proximal cotyledon leaves, distal hypocotyls, proximal hypocotyls, and basal petioles) of watermelon (Citrullus lanatus) cv. Sugar Baby were tested for their regeneration capacity on MS medium supplemented with different concentrations and combinations of hormones (BAP 0–3 mg/l alone and IAA 0–0.5 mg/l and BAP 0–3 mg/l together). The results showed that the proximal petiole cultured in MS + BAP (3 mg/l) showed the highest percentage of callus induction (28%) and shoot production (24%). The proximal cotyledons cultured in MS + BAP (2 mg/l) + IAA (0.1 mg/l) showed the highest regeneration frequency (76%). Agrobacterium tumefaciens strain EHA 105 carrying a binary vector pBI121 containing the GUS gene (ß-glucuronidase) and kanamycin-resistance gene, nptII, was used to transform petiole explants of watermelon cv. Sugar Baby. Various conditions such as the agrobacterial concentration of OD600 0.6, infection time of 20 min, co-cultivation duration of 2 days and selection at 100 mg/l kanamycin were found as important parameters for the successful Agrobacterium-mediated transformation of watermelon. Further, a transgene construct using the nucleocapsid protein (NP) gene from watermelon bud necrosis virus (genus Tospovirus family Peribunyaviridae) was developed in pBI121 and used to transform watermelon. The successful transformation of petiole explant of watermelon with the GUS gene as well as the NP gene was confirmed by molecular assays, which showed a transformation efficiency of 14.2% and 0.375% with the GUS and NP gene, respectively.
KeywordsCitrullus lanatus Sugar Baby Watermelon bud necrosis virus Regeneration GUS assay Genetic transformation
The financial support from the Department of Biotechnology, Govt. of India (BT/PR7866/AGR/02/379/2006) and Indian Agricultural Research Institute, New Delhi is thankfully acknowledged.
Compliance with Ethical Standards
Conflict of interest
The authors declare that there is no conflict of interest regarding the publication of this manuscript.
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