Efficient In Vitro Regeneration, Analysis of Molecular Fidelity and Agrobacterium tumifaciens - Mediated Genetic Transformation of Grewia asiatica L.
- Corresponding Author:
- Surrinder K Lattoo
Plant Biotechnology Division, CSIR - Indian
Institute of Integrative Medicine, Canal Road
Jammu Tawi-180 001, Jammu and Kashmir, India
E-mail: [email protected]
Received March 17, 2016; Accepted May 14, 2016; Published May 21, 2016
Citation: Wani TA, Rana S, Bhat WW, Pandith SA, Dhar N, et al. (2016) Efficient In Vitro Regeneration, Analysis of Molecular Fidelity and Agrobacterium tumifaciens - Mediated Genetic Transformation of Grewia asiatica L. J Plant Biochem Physiol 4:167. doi:10.4172/2329-9029.1000167
Copyright: © 2016 Wani TA, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Grewia asiatica is a dietotheraphtically important fruit bearing shrub, indigenous to India. It is a rich resource of triterpinoids and flavonoids and possesses many putative health benefits. Two of the drawbacks which include short shelf life of its fruits and larger seed volume impedes its full exploitation. Seed abortion for developing seedless cultivars through biotechnological interventions is a viable option. One of the prerequisites for such strategy is to develop an efficient plant regeneration and transformation protocols in G. asiatica. Against this backdrop multiple shoot induction was achieved from nodal explants with axillary buds, on culturing in Woody Plant medium (WM) fortified with 3% (w/v) sucrose, 2 × 10-5M Kinetin (Kn) and 1 × 10-5M indole-3-butyric acid (IBA) giving rise to an average of 4.25 ± 0.71 microshoots per explant. More than 90% of the explants formed micro-shoots with mean shoot length of 10.5 ± 1.96 cm leading to whole plant regeneration. Healthy regenerated shoots showed prolific rooting of more than 95% on WM supplemented with 4.8 × 10-6M indole-3-butyric acid (IBA). Following simple hardening procedures, rooted plantlets, were transferred to soil-sand (1:1; v/v) with about 92% success. Genetic fidelity was assessed using random amplified polymorphic DNA (RAPD). Additionally, Agrobacterium-mediated genetic transformation protocol was developed using A. tumefaciens strain GV2260 harboring binary vector p35SGUSINT containing hygromycin phosphotransferase gene (hpt). Transformation was verified by GUS assay and detection of the hygromycin phosphotransferase (hpt) by polymerase chain reaction. In vitro regeneration and ensuing molecular fidelity of regenerated plants and transformation studies are hitherto unreported for G. asiatica.