MSc

Investigations on “Agrobacterium rhizogenes mediated genetic transformation in Koduveli (Plumbago spp. L.)” were carried out at the Department of Plant Biotechnology, College of Agriculture, Vellayani during 2003-2006. Two plant species viz., Plumbago zeylanica and Plumbago rosea having great medicinal value by virtue of plumbagin, a naphthoquinone, present in them were selected for the study. Standardisation of rapid in vitro propagation of these medicinal plants was attempted in the present study.

Enhanced release of axillary buds from nodal explants, with the highest shoot proliferation of 5.5 was obtained in MS medium supplemented with BA 1.0 mg l-1 + IAA 0.05 mg l-1 for P. zeylanica . In P. rosea the highest shoot proliferation of 5.0 was recorded in MS medium supplemented with BA 1.50 mg l-1 + IAA 0.05 mg l-1. Among the different basal media tested full strength MS medium was found to be the best for shoot proliferation.

In indirect organogenesis, the highest callus index (300) was recorded in MS medium with BA 2.00 mgl-1 and 2,4-D 0.50 mg l-1 for P. zeylanica. In P. rosea also maximum callus index (300) was obtained in the same medium.

In P. zeylanica BA 1.75 mg l-1, IAA 0.05 mgl-1 and adenine sulphate 20 mg l-1 in MS medium was identified as the best medium for shoot regeneration from leaf derived callus. Whereas in P. rosea BA 2.00 mg l-1, IAA 0.05 mg l-1 and adenine sulphate 20 mg l-1 in MS medium obtained the highest rate of shoot regeneration from callus. Coconut water (100 ml l-1) was found to increase the rate of shoot regeneration in P. rosea.

Rooting of in vitro raised shoots was achieved by sub culturing them on MS medium containing IAA 0.50 mg l-1. In vitro root culture was carried out successfully in semi solid MS medium containing 1.50 mg l-1 NAA.

Genetic transformation was carried out by co-culture method. Agrobacterium rhizogenes strains A4, MTCC 532 and MTCC 2364 were utilized for transformation. YEP medium was used for culturing the bacteria.

The strain A4 was the best for transforming Plumbago spp. There was no significant difference between the two species P. zeylanica and P. rosea in their response to transformation.

Nodal explants recorded the highest transformation percentage followed by, leaf segments. Calli and root explants did not respond to transformation. Bacterial density of one (O.D600) during transformation resulted in high transformation percentage. Co-culturing for two days resulted in high transformation percentage, whereas co culturing for more than two days resulted in bacterial over growth on the tissues and low transformation per cent.

After two days of co-culture, the bacteria were killed by transferring the tissues to MS medium containing cefotaxime or ampicillin 500 mg l-1. The transformed tissues induced hairy roots in a period of seven to ten days. The hairy roots produced by transformation were negatively geotropic and possessed numerous root hairs. Transformation was confirmed by opine analysis, which was carried out by high voltage paper electrophoresis. Opines were detected only in transformed samples and not in normal roots. The hairy roots induced in P. zeylanica and P. rosea is a potential source for production of plumbagin in vitro.