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, 22 (4), 575-581

Stable Plastid Transformation in Scoparia dulcis L

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Stable Plastid Transformation in Scoparia dulcis L

Narra Muralikrishna et al. Physiol Mol Biol Plants.

Abstract

In the present investigation we report stable plastid transformation in Scoparia dulcis L., a versatile medicinal herb via particle gun method. The vector KNTc, harbouring aadA as a selectable marker and egfp as a reporter gene which were under the control of synthetic promoter pNG1014a, targets inverted repeats, trnR/trnN of the plastid genome. By use of this heterologous vector, recovery of transplastomic lines with suitable selection protocol have been successfully established with overall efficiency of two transgenic lines for 25 bombarded leaf explants. PCR and Southern blot analysis demonstrated stable integration of foreign gene into the target sequences. The results represent a significant advancement of the plastid transformation technology in medicinal plants, which relevantly implements a change over in enhancing and regulating of certain metabolic pathways.

Keywords: Particle bombardment; Plastid transformation; Scoparia dulcis L.; aadA; egfp.

Figures

Fig. 1
Fig. 1
Physical map of plastid transformation vector
Fig. 2
Fig. 2
Generation of transplastomic Scoparia dulcis L. a Multiple shoot induction from leaf explants on regeneration medium without spectinomycin (Wild type). b Complete suppression of regeneration on selection medium containing 75 mg/L spectinomycin (Wild type). c Initial stage of green shoots after 3-weeks of bombardment. d Sorting of green spectinomycin resistant shoots in second round of selection. e Elongation of shoots into complete plantlet with roots. f Reconfirmation of transgenic nature. g Plant transferred into the soil and maintained in the green house
Fig. 3
Fig. 3
PCR analysis of transgene integration. a Analysis of the aadA gene in transformed S. dulcis plants using primers P2 + P3 showing 1.2 kb size amplicon. b Confirmation of gfp using primers P4 + P5 generating 0.9 kb size amplicon. c Analysis of the expression cassette using primers P1 (anneals to 3′ end of INSR) & P3 (anneals to 3′ end of aadA) showing 2.4 kb size amplicon. Lane 1 DNA marker (1 kb, Fermentas). Lane 2 Wild type plant. Lane 3& 4 transgenic plants in a and c. Lane 37 transgenic plants in b
Fig. 4
Fig. 4
Fluorescence analysis of gfp expression in transplastomic lines
Fig. 5
Fig. 5
Southern blot and protein analysis. a DNA blot confirmation generating 2.4 kb size hybridization signal. Lane 1 Wild type, Lanes 2& 3 Transgenic plants. b Protein analysis corresponding to 26 and 29 kDa of the expected sizes to gfp and aadA respectively. Lane 1 Marker, Lane 2 Wild type, Lane 3& 4 Transgenic plants
Fig. 6
Fig. 6
Physical map of the vector showing primers and probe

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