Effect of over-expression of Linum usitatissimum PINORESINOL LARICIRESINOL REDUCTASE (LuPLR) gene in transgenic Phyllanthus amarus

Shoot tip explants of Phyllanthus amarus were cocultivated with Agrobacterium tumefaciens strain LBA 4404 carrying plasmid pCAMBIA 2301 harbouring genes coding for betaglucuronidase (gus), kanamycin (kan), and neomycin phosphotransferase II (nptII) along with a gene coding for Linum usitatissimum PINORESINOL LARICIRESINOL REDUCTASE (Lu-PLR). Transformed shoot tip explants were maintained in a Murashige and Skoog (MS) medium containing TDZ 1.54 mg l(-1), kan 50 mg l(-1) and cephotaxime 62.5 mg l(-1). The optimum medium for regeneration of multiple shoots was MS supplemented with TDZ 1.54 mg l(-1), kan 50 mg l(-1). Efficient and effective rooting of plantlets was achieved by culturing the in vitro regenerated shoots on liquid 1/2 MS medium containing 0.7 mg l(-1) indole 3-butyric acid (IBA) and 5 mg kan. Rooted plants were acclimatized in the mixtures of vermiculite and soil. The transformation of kan-resistant plantlets regenerated from shoot-tip explants was confirmed by GUS and polymerase chain reaction (PCR) analysis. Southern blot and reverse transcribed PCR (RT-PCR) analysis confirmed successful integration and expression of Lu-PLR gene. Quantitative analysis of phyllanthin performed on transgenic and wild plants using high-performance liquid chromatography (HPLC) revealed that transgenic lines contained higher phyllanthin content (0.3-0.81% w/w) than wild plants (0.09% w/w). The highest yield of phyllanthin was detected in transgenic lines was up to 1.16, 1.22 and 1.23 folds higher than that of wild plant. This report highlights the transgenic approach to enhance the contents of phyllanthin and hypophyllanthin.