Development of a phosphomannose isomerase-based <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation system for chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) |
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Authors: | Gunvant Patil Amit Deokar P K Jain R J Thengane R Srinivasan |
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Institution: | (1) National Research Centre on Plant Biotechnology, IARI Campus, New Delhi, 110012, India;(2) Department of Botany, University of Pune, Pune, 411007, India; |
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Abstract: | To develop an alternative genetic transformation system that is not dependent on an antibiotic selection strategy, the phosphomannose
isomerase gene (pmi) system was evaluated for producing transgenic plants of chickpea (Cicer arietinum L.). A shoot morphogenesis protocol based on the thidiazuron (TDZ)-induced shoot morphogenesis system was combined with Agrobacterium-mediated transformation of the pmi gene and selection of transgenic plants on mannose. Embryo axis explants of chickpea cv. C-235 were grown on a TDZ-supplemented
medium for shoot proliferation. Embryo axis explants from which the first and second flush of shoots were removed were transformed
using Agrobacterium carrying the pmi gene, and emerging shoots were allowed to regenerate on a zeatin-supplemented medium with an initial selection pressure of
20 g l−1 mannose. Rooting was induced in the selected shoots on an indole-3-butyric acid (IBA)-supplemented medium with a selection
pressure of 15 g l−1 mannose. PCR with marker gene-specific primers and chlorophenol red (CPR) assay of the shoots indicated that shoots had been
transformed. RT-PCR and Southern analysis of selected regenerated plants further confirmed integration of the transgene into
the chickpea genome. These positive results suggest that the pmi/mannose selection system can be used to produce transgenic plants of chickpea that are free from antibiotic resistance marker
genes. |
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