Construction of chloroplast transformation vector and its functional evaluation in Momordica charantia L.

Abstract

Chloroplast transformation vectors require an expression cassette flanked by homologous plastid sequences to drive plastome recombination. The 16-23 plastome region was selected and using this region, a new species-specific plastid transformation vector CuIA was developed with pKSII as a backbone by inserting the 16- and -23 sequences from L. An independent expression cassette with gene encoding aminoglycoside 3'-adenylyltransferase with controlling elements is added into the- intergenic region that confers resistance to spectinomycin. An efficient plastid transformation in bitter melon ( 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 gene was verified by PCR analysis in transplastomes. Transplastomic technology developed may be a novel approach for high level expression of pharmaceutical traits.

Improved plastid transformation efficiency inScoparia dulcisL.

“…Stable integration of chloroplast transformation was achieved in tobacco, lettuce, Cedrelaodorata, Capsicum and Marchantia polymorpha L. using the insertion sites of trnA and trnI fragments from IR region (Bock and Maliga 1995;Chiyoda et al 2007;K Mühlbauer et al 2002;Lelivelt et al 2005;López-Ochoa et al 2015;Staub and Maliga 1992). In our lab, the same trnA/trnI of IR region was targeted to recover transplastomic lines of Momordica charantia by a Cucumis sativus based heterologous vector (CuIA) (Narra et al 2018b) and obtained 2 transplastomic lines from 15 bombarded plates using petiole explants. Two different plastid vectors, KNTc, and pFaadAII targeting trnR/trnN and rpl32/trnL of IR and SSC regions of plastid genomes were employed to transform chloroplasts of S.dulcis (Muralikrishna et al 2016;Narra et al 2018a).…”

Technical Advances in Chloroplast Biotechnology

“…Chilling tolerance as well as growth was observed to be increased in tropical forage Stylosanthes guianensis expressing chloroplast protein 12 [20]. Recently, plastid transformation has been reported in a valuable vegetable Momordica charantia [21], marine microalga Nannochloropsis oceanica [22], and Cyanidioschyzon merolae [23], a red alga having ability to survive in high sulfur acidic hot spring environments. This may open new horizons in understanding stress adaptability and role of transplastomic technology in developing stress-tolerant plants.…”

Plastid Transformation: How Does it Work? Can it Be Applied to Crops? What Can it Offer?

“…To date, the plastids of over 20 flowering plants have been transformed [15] (Table 1). In addition to the crops mentioned above, recent successes in plastid transformation have been reported in the plant species bitter melon [16], and the medicinal plant sweet wormwood (Artemisia annua) [17] and licorice weed (Scoparia dulcis) [18,19] (Table 1). Based on these successful cases, plastid transformation should be applicable to many plant families, whether they are monocots or dicots.…”