Standardization of Regeneration, Agrobacterium -Mediated Transformation, and Introduction of Nucleocapsid Gene of Waterm
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RESEARCH ARTICLE
Standardization of Regeneration, Agrobacterium-Mediated Transformation, and Introduction of Nucleocapsid Gene of Watermelon Bud Necrosis Virus in Watermelon Rakesh Kumar1,4,3 • A. Swapana Geetanjali1,5 • M. Krishnareddy2 P. K. Jaiwal3 • Bikash Mandal1
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Received: 1 June 2019 / Revised: 1 June 2019 / Accepted: 25 July 2019 The National Academy of Sciences, India 2019
Abstract 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
Significant Statement In this study, the nucleocapsid protein gene of watermelon bud necrosis virus (WBNV) was cloned and used to develop a transgene construct in a binary vector. The transgene construct was used to transform watermelon cv. Sugar Baby using the optimized regeneration and transformation protocol. This study will help to develop resistant watermelon plants against WBNV. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s40011-019-01130-9) contains supplementary material, which is available to authorized users. & Bikash Mandal [email protected] 1
Division of Plant Pathology, Advanced Centre for Plant Virology, Indian Agricultural Research Institute, New Delhi, India
2
Division of Plant Pathology, Indian Institute of Horticultural Research, Hessaraghatta, Bangalore, India
3
Maharishi Dayanand University, Rohtak, Haryana, India
4
Present Address: JK Agri Genetic Ltd, Hyderabad, India
5
Present Address: SRM University, Chennai, India
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. Keyw
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