Variability in Chakkarakolli (Gymnema sylvestrwe R.Br.) using morphological, biochemical nad molecular markers

Abstract

Plants represent an unparalleled source for drug development. Plant based natural products play a dominant role in the pharmaceutical industry. Plant based remedies are available for a number of different health problems. In today’s scenario, diabetes is one of the most common non-communicable diseases globally and is also the fourth major cause of death in most developed countries. So the demand for natural alternatives to blood sugar control is ever increasing. Several indigenous medicinal plants have been indicated in the Ayurveda system of medicine to possess antidiabetic activity. Native to the forests of South India, Gymnema sylvestre R.Br. is known for its hypoglycaemic property from the time immemorial. Practitioners of Ayurveda first used Gymnema to treat diabetes almost 2000 years ago. Keeping in view the importance of this medicinal plant, a study was conducted to identify the variability in Gymnema accessions collected and maintained at the Centre for Plant Biotechnology and Molecular Biology. The present study was undertaken at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during the period 2003-2005. The objectives of the study were to characterise the variability in Gymnema germplasm accessions using morphological, biochemical and molecular markers. The morphological characterisation of 93 Gymnema accessions based on vegetative characters indicated the existence of a wide variation. All the 93 accessions were then characterised using biochemical markers. The total saponin content in the leaves was chosen as the marker constituent since saponins are the major bioactive components in Gymnema. The saponins were estimated based on TLC-Densitometry technique. The techniques for detection of saponins through TLC could be standardised. The saponin content in the leaves of different accessions ranged between 0.6 per cent and 5.4 per cent. Based on the morphological and biochemical characters, variants were identified in Gymnema germplasm collection. Eighteen such plants showing high variation were subjected to molecular characterisation using isozyme and RAPD markers. Isozyme analysis was carried out using three enzyme systems viz. malate-dehydrogenase, esterase and RUBISCO. Eight isozyme loci could be clearly identified of which five were polymorphic. Ten alleles were identified over the five polymorphic loci giving an average of two alleles per polymorphic locus. A mean observed heterozygosity of 19.8 per cent and a mean expected heterozygosity of 17.2 per cent were obtained over the heterozygous loci studied. The cluster analysis grouped the 18 accessions in to two major clusters of 17 and one accession. Molecular characterisation using RAPD markers was conducted to appraise the extent of diversity among the 18 accessions of Gymnema. The protocol for genomic DNA isolation from Gymnema was standardised. The protocol suggested by Rogers and Bendich (1994) with slight modification was found to be most appropriate. The protocol for RAPD assay in Gymnema was standardised. Sixty random primers were screened and 15 were selected for RAPD profiling of Gymnema accessions. The primer OPAH 12 was found to have the highest resolving power. A total of 123 amplification products were generated by 15 primers, of which 90 were polymorphic. In the dendrogram, the 18 accessions were grouped in to two major clusters of 16 and two accessions. The accessions Peringottukurushi 137 and Kuzhalmannon 89, occurring in the third sub-cluster were the most closely related with 85 per cent similarity. A combined dendrogram was also derived from the results of morphological and molecular studies. The present study revealed the existence of sufficient genetic variation in the Gymnema germplasm collection. This variability can be used to identify useful genotypes that could be used as cultivars for the extraction of standard drugs. More precise techniques like HPLC and AFLP could further be used to get a better idea about the extent of variability in Gymnema.