Development of a phosphomannose isomerase-based Agrobacterium-mediated transformation system for chickpea (Cicer arietinum L.)

Gunvant Patil, Amit Deokar, P. K. Jain, R. J. Thengane, R. Srinivasan

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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 Agrobacteriummediated 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.

Original languageEnglish
Pages (from-to)1669-1676
Number of pages8
JournalPlant Cell Reports
Volume28
Issue number11
DOIs
StatePublished - Aug 28 2009

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Keywords

  • Agrobacterium
  • Chickpea
  • Cicer arietinum
  • Genetic transformation
  • Mannose
  • Phosphomannose isomerase (PMI)

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