Establishment of pomegranate hairy root cultures and elucidating the role of transcription factor for hydrolysable tannins biosynthesis

Pomegranate (Punica granatum L.) renowned globally due to its nutritional and medicinal properties such as antioxidant, antimicrobial, cardiovascular disease, diabetes, obesity, prostate cancer and anti-inflammatory. The major healthy metabolites in pomegranate belong to hydrolysable tannins that can be detected mainly in the fruit peels and roots. Hairy root (HR) cultures have become an alternative approach for last one decade towards the biosynthesis of important secondary metabolites from diverse medicinal plants. The present study reports HR establishment in Pomegranate with two Agrobacterium rhizogenes strains- LBA1334 and Paraqual with 90 and 86% transformation efficiencies respectively. Optimization of liquid media and growth kinetics analysis showed maximum biomass in HR derived from LBA1334 strain in full strength woody plant media with 3% sucrose after 8 weeks. The metabolite profiling through HPLC revealed higher production of punicalagin A and B in the HR of LBA1334 as compared to that with Paraqual cultivated in woody plant medium. Furthermore, to understand the metabolic flux of gallic acid and shikimic acid towards the hydrolysable tannins and anthocyanins, few biosynthetic pathway related transcription factors (selected from RNA seq data) over-expressed in transgenic HR culture of P. granatum. Moreover, these transcription factors also heterologous expressed in other plant systems such as Nicotiana tabacum and Populus species to investigate their exclusive role in the shikimic acid pathway. In conclusion, the positive outcomes of the present study opened up a new horizon for pharmaceutical industries pertaining to the utilization of pomegranate HR clones as natural alternative resource of such bioactive tannins.