High-efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated <Emphasis Type="Italic">in planta</Emphasis> transformation in black gram (<Emphasis Type="Italic">Vigna mungo</Emphasis> (L.) Hepper) |
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| Authors: | Gnanajothi Kapildev Arunachalam Chinnathambi Ganeshan Sivanandhan Manoharan Rajesh Venkatachalam Vasudevan Subramanian Mayavan Muthukrishnan Arun Murugaraj Jeyaraj Sulaiman Ali Alharbi Natesan Selvaraj Andy Ganapathi |
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| Institution: | 1.Plant Molecular Biology Laboratory, Department of Biotechnology and Genetic Engineering,School of Biotechnology, Bharathidasan University,Tiruchirappalli,India;2.Department of Botany and Microbiology, College of Science,King Saud University,Riyadh,Kingdom of Saudi Arabia;3.Genetics and Genomics Laboratory,Chungnam National University,Daejeon,South Korea;4.International Institute of Tropical Agriculture (IITA),Nairobi,Kenya;5.Molecular Genetics and Epigenomics Lab, CIBER, Department of Agriculture and Natural Research,Delaware State University,Dover,USA;6.Department of Horticultural Science,Kyungpook National University,Daegu,Republic of Korea;7.Centre for Nanoscience and Nanotechnology,University of Madras,Chennai,India;8.Periyar E.V.R College,Tiruchirappalli,India |
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| Abstract: | In vitro culture and genetic transformation of black gram are difficult due to its recalcitrant nature. Establishment of gene transfer procedure is a prerequisite to develop transgenic plants of black gram in a shorter period. Therefore, genetic transformation was performed to optimize the factors influencing transformation efficiency through Agrobacterium tumefaciens-mediated in planta transformation using EHA 105 strain harbouring reporter gene, bar, and selectable marker, gfp-gus, in sprouted half-seed explants of black gram. Several parameters, such as co-cultivation, acetosyringone concentration, exposure time to sonication, and vacuum infiltration influencing in planta transformation, have been evaluated in this study. The half-seed explants when sonicated for 3 min and vacuum infiltered for 2 min at 100 mm of Hg in the presence of A. tumefaciens (pCAMBIA1304– bar) suspensions and incubated for 3 days co-cultivation in MS medium with 100 µM acetosyringone showed maximum transformation efficiency (46 %). The putative transformants were selected by inoculating co-cultivated seeds in BASTA® (4 mg l?1) containing MS medium followed by BASTA® foliar spray on 15-day-old black gram plants (35 mg l?1) in green house, and the transgene integration was confirmed by biochemical assay (GUS), Polymerase chain reaction, Dot-blot, and Southern hybridisation analyses. |
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