MSc.

The present investigation on “Induction of somaclones in vetiver “[Chrysopogon zizanioides(L.)Roberty]” was undertaken at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during 2010-2012 with an aim to induce variation in vetiver through callus mediated culture and evaluation of the same using morphological, molecular and biochemical markers.

The explants collected from AMPRS, Odakkali was used for the study. Callus formation was induced in full MS media with growth regulator combinations of 0.5mgl-1 2,4-D, 0.5mgl-1 2,4-D+0.5mgl-1 KIN, 1.0mgl-1 2,4-D, 1mgl-1 2,4-D+1.0mgl-1 KIN, 1.0mgl-1 2,4-D+1.0 mgl-1 KIN +1.0 mgl-1 BA, 1.5mgl-1 2,4-D and 1.5mgl-1 2,4-D+1.5mgl-1 KIN. The regeneration and rooting were done in full MS media with various combinations of BA and IAA respectively. Hardening of the rooted plantlets were done in paper cups filled with sand under poly house. The hardened plantlets were established into pots containing potting mixture.
Standardisation of DNA extraction was done with the CTAB method. Optimum PCR conditions for RAPD were standardised with various quantities of DNA, dNTPs, MgCl2, primers and Taq polymerase. Initially 40 RAPD primers were screened against genomic DNA of the mother plant for their ability to amplify DNA fragments. Of these, 10 RAPD primers were selected for further detailed RAPD profiling. All selected primers produced robust amplification patterns. The PCR products obtained were separated on agarose gel stained with ethidium bromide. A total of 92 RAPDs were generated of which average percentage of polymorphism was 7.4. The scored data based on RAPD banding was used to construct a dendrogram using NTSYS pc (ver. 2.02i). The RAPD assay confirmed the existence of considerable variation at the DNA level in the somaclones obtained..

MSAP analysis was done with 250 ng of template DNA for each reaction with four primers. The results showed that cytosine methylation varied from 10.1 per cent to 17.0 per cent. It also revealed that there was considerable variation between the somaclones

The oleoresin extraction was done using solvent extraction method by reflexion and condensation. Highly volatile acetone and less volatile hexane were used for extraction. The oleoresin obtained was light yellow in colour. The content varied from 0.3 (SC 7) to 2.67 (SC 4) percentage. GC Analysis was carried out using DB-5 column. The chromatographic profiles varied for all the somaclones and the mother plant. More number of components was obtained for SC 6 and the least number for SC 2. SC 4 has shown more variation from the mother plant.

In all the analysis, there is variation reported between the mother palnt and somaclones and also in between the somaclones. The oil extraction can be done further at 18 months of age of somaclones and the GC analysis result obtaining can be correlate with the molecular analysis and morphological analysis.