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, 22 (1), 61-8

Biolistic Transformation of Scoparia Dulcis L

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Biolistic Transformation of Scoparia Dulcis L

Kota Srinivas et al. Physiol Mol Biol Plants.

Abstract

Here, we report for the first time, the optimized conditions for microprojectile bombardment-mediated genetic transformation in Vassourinha (Scoparia dulcis L.), a Plantaginaceae medicinal plant species. Transformation was achieved by bombardment of axenic leaf segments with Binary vector pBI121 harbouring β-glucuronidase gene (GUS) as a reporter and neomycin phosphotransferase II gene (npt II) as a selectable marker. The influence of physical parameters viz., acceleration pressure, flight distance, gap width & macroprojectile travel distance of particle gun on frequency of transient GUS and stable (survival of putative transformants) expressions have been investigated. Biolistic delivery of the pBI121 yielded the best (80.0 %) transient expression of GUS gene bombarded at a flight distance of 6 cm and rupture disc pressure/acceleration pressure of 650 psi. Highest stable expression of 52.0 % was noticed in putative transformants on RMBI-K medium. Integration of GUS and npt II genes in the nuclear genome was confirmed through primer specific PCR. DNA blot analysis showed more than one transgene copy in the transformed plantlet genomes. The present study may be used for metabolic engineering and production of biopharmaceuticals by transplastomic technology in this valuable medicinal plant.

Keywords: Axenic leaf explants; Biolistic transformation; GUS assay; Scoparia dulcis L.; Stable expression.

Figures

Fig. 1
Fig. 1
Production of transgenic Scoparia dulcis using axenic leaf explants via microprojectle -mediated genetic transformation. a Profuse shoot organogenesis from 95 % of axenic leaf explants after 4 weeks of culture on RMBI medium. b Complete inhibition of shoot organogenesis in all control explants within a week days of culture, on selective shoot regeneration medium (RMBI-K). c c1 Transient gus expression shown by bombarded explant at 650 psi with flight distance of 6 cm. c2 Transient gus expression shown by bombarded explant at 1100 psi with flight distance of 9 cm. c3 No transient gus expression shown by control (non-bombarded explants). d Putative transgenic Shoots developed from bombarded explants after three rounds of selection on selective shoot regeneration medium (RMBI-K). e Shoot cluster showing gus expression after three rounds of selection on selective shoot regeneration medium (RMBI-K). f Elongation of putative transgenic shoots after last round of selection on selective shoot regeneration medium (RMBI-K). g gus stained elongated putative transgenic shoots after last round of selection on selective shoot regeneration medium (RMBI-K). h gus -positive transgenic plantlet ready for rooting on RMRI-K medium. i Phenotypically normal trangenic plant hardening on soil in the greenhouse
Fig. 2
Fig. 2
PCR analyses of transgenic plant lines of Scoparia dulcis produced via microprojectle -mediated genetic transformation. a PCR detection of uidA gene specific band (1812 bp) in transgenic line 1–4; No such band detected in wild (Wt) type; M, Molecular marker. b PCR detection of npt II (1355 bp specific band in transgenic line 1–4; No such band detected in wild (Wt) type; M, Molecular marker)
Fig. 3
Fig. 3
Southern blot detection of transgene integration in transgenic lines of Scoparia dulcis produced via microprojectle -mediated genetic transformation Wt- Wild type; 1–4, Different transgenic plant lines

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