Plant Material Essay -- Plants, Seeds

Plant material The seeds of A. precatorius were collected from the medicinal plant garden of Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar, M.P., India. Seeds were sterilized and germinated by following the protocol described in our previous publication .[15] Initiation of A. precatorius cell cultures Different explants from aseptically germinated seeds viz. leaves, epicotyle and petiole were tested for culture initiation by variation in plant growth regulators (PGR) and Agrobacterium mediated transformation. Non-transformed callus cultures were initiated by placing explants on solidified MS medium supplemented separately with the hormones: 1 mg/l naphthalene acetic acid (NAA); 1 mg/l Kinetin (Kn); 0.5 – 2.0 mg/l 2, 4- dichlorophenoxy acetic acid (2, 4-D) and there combinations (Data not shown). For transformation experiments, leaves were excised from 30 d old in vitro germinated plantlets of A. precatorius. A. tumefaciens strains (MTCC 431, MTCC 609, MTCC 2250 and MTCC 2251) were used to establish transformed callus cultures. These strains were procured from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India. A minimum of 30 explants were used for each experiment. All explants cultured on sterilized petriplates comprising MS medium solidified with 1.0 % agar and supplemented with 30 g/l sucrose. The pH was adjusted to 5.7 Â ± 0.2. The medium was autoclaved under 15 psig pressure at 121Â ºC for 20 min. The explants were co-cultivated with Agrobacterium strains for infection to induce transformed callus. For this purpose, Agrobacterial colonies were cultured for 48 h on solid nutrient agar medium at 28 Â ± 2Â °C. Ten loopful bacteria were then... ... in a maximum synergistic promotion of glycyrrhizin accumulation i.e. 4.9-fold higher compared to transformed control culture. The present study indicates the potential of these biotechnology-based methodologies for large-scale production of glycyrrhizin. Furthermore, in order to develop a process for commercial production of glycyrrhizin by plant cell cultures some additional yield enhancement strategies may be worked out like, optimization of medium composition, environmental condition and addition of precursors. Acknowledgments The authors are thankful to Dr. Ashish Baldi, Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Hauz Khas, New Delhi, India for his valuable and timely assistance. The author VSK wishes to acknowledge All India Council for Technical Education, New Delhi for providing junior research scholarship.