Agrobacterium tumefaciens mediated transfer of exogenous hydroxy methyl glutaryl CoA (HMG CoA) reductase gene to Centella asiatica L
Loading...
Files
Date
2008
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Department of Plant Biotechnology, College of Agriculture, Vellayani
Abstract
A study on “Agrobacterium tumefaciens mediated transfer of exogenous hydroxyl methyl glutaryl CoA (HMG CoA) reductase gene to Centella asiatica L.” was conducted at the Department of Plant Biotechnology, College of Agriculture, Vellayani during 2006-2008. Centella asiatica is an important medicinal plant, which is used in many ayurvedic formulations. It contains a blend of compounds including triterpenes (asiatic acid, madecassic acid and asiaticoside), which are responsible for its medicinal properties. Among the various secondary metabolites in Centella asiatica, asiaticoside possesses remarkable pharmaceutical value due to its anti-inflammatory, antitumour, neuroprotective, skin care and toning effects. Since the asiaticoside content in the Indian ecotypes is less, the industrial demands are met by importing this plant from African countries. By improving asiaticoside content, utilization of Indian ecotypes could be improved and thereby the cost of medical preparations could be reduced. Metabolic engineering is becoming a popular approach for the modification of medicinal plants for altering the metabolite content. Medicinal plants with quantitatively and qualitatively improved pharmacological properties have been produced by metabolic pathway engineering. The present study was undertaken with an objective to enhance the asiaticoside content by introducing exogenous hmgr gene to Centella asiatica. This gene is responsible for coding hydroxy methyl glutaryl CoA reductase enzyme which acts at the upstream of the triterpene biosynthetic pathway and produce mevalonic acid. Callus was induced from leaf and node explants of Centella and MS medium supplemented with Kn 4 mg l-1 and NAA 2 mg l-1 was found to be the best for callus induction. Callus initiation was faster (23 days) from node compared to leaf (25 days), with 100 and 92.85 per cent induction respectively. Of the various media tried for regeneration, the highest regeneration (0.052%) was obtained on MS medium supplemented with Kn 4mg l-1 and NAA 2 mg l-1. Agrobacterium tumefaciens strain EHA105 harbouring the plasmid pBE2113 containing nptII and hmgr gene was used for transformation. The sensitivity of Agrobacterium strains and Centella callus to different concentrations of kanamycin was evaluated. The lethal doses of kanamycin to Agrobacterium and Centella callus were 350 and 125 mg l-1, respectively. The effective dose of cefotaxime for the elimination of bacteria was 50 mg l-1 and the lethal dose of cefotaxime to callus was 100 mg l-1. Genetic transformation was achieved by co-cultivating callus with bacterial suspension (OD600 of 0.1) containing 100 μM acetosyringone. An infection time of 20 min and co-cultivation period of four days were given. The transformed tissues were selected on MS medium containing 200, 250 and 300 mg l-1 of kanamycin. The survival of the tissues on these media after three weeks was 18.75, 19.35 and 13.63 per cent, respectively. Transformation was confirmed by PCR analysis with npt II gene specific primer. All the three samples gave appreciable quantity of the product of size 700 bp which was comparable to positive control. In the present study regeneration of the transformed tissues was not obtained in the media standardized. Even though an attempt was made to analyse asiaticoside content in the transformed callus using thin layer chromatography (TLC), there was no detectable quantity of asiaticoside. This could be due to the undifferentiated nature of the callus. As the accumulation of asiaticoside is mainly in the leaves, a better protocol for regeneration needs to be developed so that further studies on triterpenoid analysis could be carried out.
Description
PG
Keywords
null