Construction of chloroplast transformation vector and its functional evaluation in Momordica charantia L.
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Chloroplast transformation vectors require an expression cassette flanked by homologous plastid sequences to drive plastome recombination. The rrn16-rrn23 plastome region was selected and using this region, a new species-specific plastid transformation vector CuIA was developed with pKS+II as a backbone by inserting the rrn16-trnI and trnA-rrn23 sequences from Cucumis sativus L. An independent expression cassette with aadA gene encoding aminoglycoside 3′-adenylyltransferase with psbA controlling elements is added into the trnI-trnA intergenic region that confers resistance to spectinomycin. An efficient plastid transformation in bitter melon (Momordica charantia L.) was achieved by bombardment of petiole segments. The frequency of transplastomic plants yielded using standardized biolistic parameters with CuIA vector was two per 15 bombarded plates, each containing 20 petiole explants. Integration of aadA gene was verified by PCR analysis in transplastomes. Transplastomic technology developed may be a novel approach for high level expression of pharmaceutical traits.
KeywordsChloroplast transformation vector Expression cassette Flanking regions Plastome sequence
MK would like to acknowledge the financial assistance provided by the Department of Science and Technology -INSPIRE (DST/INSPIRE Fellowship/2011/426), New Delhi. AS is grateful to UGC for BSR-Faculty Fellowship. The authors are also thankful to UGC, New Delhi for financial support under SAP-DRS phase-II to the Department of Biotechnology, Kakatiya University.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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