Agrobacterium mediated transformation and secondary metabolite analysis of Ajuga bracteosa Wall. ex. Benth

Abstract

Ajuga bracteosa is a valuable yet endangered medicinal plant containing many important compounds, including withanolides, phytoecdysteroids, neo-clerodane and iridoid glycosides. These compounds possess a broad spectrum of biological, pharmacological and medicinal properties, but their yield is very low in wild A. bracteosa and chemical synthesis is not viable. The increased demand of natural products for medicinal purposes coupled with the low product yields and supply concerns of plant harvest has renewed interest in large-scale in vitro plant culture technology. A.bracteosa plant was subjected to in vitro culturing with the aim to study the effect of plant growth regulators on somatic embryogenesis for mass production and uniform plantlets for further genetic transformation. Leaf explants cultured on B5 medium containing 1mg/L 1-naphthaleneaceatic acid (NAA) + 0.5 mg/L 6-Benzyl amine purines (BAP) proved to be the most effective combination for somatic embryo induction. Agrobacterium tumefaciens strain GV3101 harboring pPCV002-A and pPCV002-CaMVC was used to obtain transgenic A. bracteosa plants. Transformation efficiency of 38% for rolA gene and 31% for rolC was obtained. Molecular analysis such as PCR and Southern blotting confirmed the integration of the rolA and rolC genes in the putative transgenic lines. The transcripts of these two genes were monitored by semi-quantitative RT-PCR. Furthermore, a significant difference in phytochemistry and the pharmacological activities (antioxidant, antibacterial, antiproliferative and antidiabetic) was observed not only between the transgenic and control/untransformed plants but also among the transgenic lines themselves. The rolC transgenic lines in general showed better results compared to rolA. Quantitative analysis of Electro-spray ionization mass spectrometer (ESI-TOF) revealed relatively higher contents of 20 hydroxyecdysone (20-HE) in transgenic plant lines of rolA and rolC compared to control/untransformed plant. Effect of low temperature stress on 20-HE biosynthesis revealed relatively high 20-HE content at 15°C compared to control (25°C). Principal component analysis (PCA) of the metabolic profiles obtained with non-targeted (ESI-MS) approach successfully distinguished the control A. bracteosa from the transgenic rolA and rolC population. Defense related secondary metabolites (tryptophan, mannitol, Cinnamic acid, and Shikimic acid) were identified in transgenic A. bracteosa lines. Taken together, our study suggests that low temperature stress and transformation with rol genes can be used for increased, constant and permanent production of valuable secondary metabolites from A. bracteosa plant.