MSc

A study on “Agrobacterium tumefaciens mediated genetic transformation in kudangal (Centella asiatica L. Urban.)” was conducted at the Department of Plant Biotechnology, College of Agriculture, Vellayani during 2004-2006.

Centella asiatica is an important medicinal plant of India and is used in many ayurvedic formulations. Centella asiatica contains a blend of compounds including triterpenes (asiatic acid, madecassic acid and asiaticoside) that appear to have antioxidant, tissue regenerative and memory enhancing properties. The present study was undertaken with the main objective of evolving a protocol for Agrobacterium tumefaciens mediated genetic transformation in Centella asiatica, which could further be utilized for the metabolic engineering of Centella to enhance the level of secondary metabolites.

Callus was induced from leaf and node explants of Centella on MS medium supplemented with growth regulators. MS medium supplemented with Kn 2 mg l-1 and NAA 4 mg l-1 was proved to be the best in terms of callus induction percentage (85.7) from leaf explant in 25.50 days. With node explants, the maximum callus induction (86.67%) was obtained on MS medium supplemented either with Kn 2 mg l-1 and NAA 3 mg l-1 or with Kn 1 mg l-1 and NAA 3 mg l-1 in 23.67 and 22.00 days respectively. Of the various regeneration treatments, 16.67 per cent regeneration from callus was obtained on MS medium supplemented with Kn 2mg l-1, BA 4mg l-1, NAA 0.25 mg l-1 and ADS 20 mg l-1.

Two strains of Agrobacterium tumefaciens viz., LBA4404 and EHA105 harbouring the plasmid pCAMBIA2301 were used for genetic transformation. As the plasmid harbour nptII and gus reporter genes, the sensitivity of Agrobacterium strains and Centella callus to different concentrations of kanamycin was evaluated. The lethal dose of kanamycin to Agrobacterium and Centella callus were 350 and 125 mg l-1 respectively. The effective dose of cefotaxime for the elimination of bacterial strains LBA4404 and EHA105 was 75 mg l-1 and the lethal dose of cefotaxime to Centella callus was 150 mg l-1.

Genetic transformation was achieved by co-cultivating callus and node with bacterial suspension. Conditions like infection and co-cultivation time, type of the explant, selection agent and suitable Agrobacterium strains were optimized. The Agrobacterium strain, EHA105 with pCAMBIA2301 was more efficient for transformation in Centella. The most effective infection time was 20 minutes, followed by a co-cultivation period of four days. The survival of tissues transformed by the strains LBA4404 and EHA105 on the selection media were 80.65 per cent and 66.67 per cent respectively. Maximum transformation efficiency of 50 percent was obtained when callus was co-cultivated with EHA105 (pCAMBIA2301) for four days.

The transformation efficiency was increased when acetosyringone 100 µM was added to infection and co-cultivation media. Transformation was confirmed by histochemical GUS assay of putative transformants. This study provides a protocol for genetic transformation in Centella which can be used for transferring desirable genes.