Genetic and metabolic diversity inStevia rebaudianausing RAPD and HPTLC analysis

Abstract

Context: Stevia rebaudiana Bertoni (Asteraceae) is an important medicinal plant and is much used due to its zero calories sweetening property. Stevia leaves as well as its extracts and pure compounds are currently used in the preparation of several medicines, food products and neutraceuticals. Objective: To study the genetic and metabolic variability in S. rebaudiana among accessions of different geographical regions of India using random amplified polymorphic DNA (RAPD) markers and high-performance thin layer chromatography (HPTLC) analysis. Materials and methods:The RAPD analysis of Stevia rebaudiana (11 accessions) was carried out using 20 random operon primers. Dendrogram was constructed for cluster analysis based on the unweighted pair group method with arithmetic means (UPGMA) using Winboot. The HPTLC analysis of all samples was carried out on silica using acetone:ethyl acetate:water (5:4:1, v/v/v) for fingerprinting and quantification of stevioside and rebaudioside A at 360 nm after spraying with anisaldehyde sulphuric acid. Results: Ten out of 20 primers screened were found most informative; amplification products of the genotypes yielded a total of 87 scorable bands (67 polymorphic), whereas genetic similarity (GS) coefficient (0.01-0.08) and polymorphism (67.24-92.40%) showed huge variability. Similarly, HPTLC analysis showed large variation among different samples with respect to their presence or absence of metabolite and their concentration. Conclusion: Out of the 11 Stevia accessions, Delhi and Mohali varieties showed much relatedness with each other and were concluded to be the superior genotype in context to RAPD and HPTLC analysis. The information obtained here could be valuable for devising strategies for cultivating this medicinal plant.

Stevia Rebaudiana Bertoni, a Source of High-Potency Natural Sweetener—Biochemical and Genetic Characterization

“…The product amplified by primer 9 had the highest polymorphism (87.5%), while fragment amplified by primer 10 exhibited the lowest polymorphism (60%). A similar proportion of polymorphism (67.24-92.40%) was observed by Chester et al [28], who analyzed 11 genotypes using 10 RAPD primers. The average polymorphism value for this study (72%) was similar to the results obtained by Thiyagarajan and Venkatachalam [8]-81%-and Gupta et al [25]-67%.…”

HI-HPTLC-UV/Vis/FLD-HESI-HRMS and bioprofiling of steviol glycosides, steviol, and isosteviol in Stevia leaves and foods

“…Furthermore, enzymatic methods [13], electroanalytical techniques [14], capillary electrophoresis [15], desorption electrospray ionization MS [16], paper spray-MS [17], near infrared [18], and Raman spectroscopy [19] were exploited as analytical tools. However, mainly HPLC with different detectors (UV [20], ELSD [21], CAD [22], and MS [23][24][25][26][27]) or high-performance thin-layer chromatography (HPTLC) with different derivatization reagents [5,[28][29][30][31][32][33] were used. Among these, a fast reversed phase HPLC-MS/MS method was reported for analysis of seven steviol glycosides (not all baseline separated), isosteviol, and steviol in Stevia leaf extracts, with a total run time of 9 min per sample [34].…”

Biotechnological approaches in Stevia rebaudiana and its Therapeutic Applications

“…This finding is important to learn more about the roles of miRNAs in Stevia development and physiology but also to provide a framework for further designing RNAi based experiments for regulation of gene expression in these species. HPTLC as a sensitive, safe, simple and high throughput method developed for quantification of stevioside and rebaudioside A, was used for the quality control of S. rebaudiana as well as to check the content of Stevia glycoside during different stages of crop for commercial production [55]. There is inulin-type fructo oligosaccharides as major and significant component of Stevia leave extracts that structure and degree of polymerisation of fructo oligosaccharides present in roots and leaves of Stevia rebaudiana (Bert.)…”

“…Chester et al (2013) studied genetic diversity in 11 accessions of Stevia rebaudiana of different geographical regions in India using random amplified polymorphic DNA (RAPD technique) markers and high-performance thin layer chromatography (HPTLC) analysis that they detected 67.24-92.40 percent polymorphism. The information obtained from HPTLC analysis of all samples were carried out on silica using acetone: ethyl acetate: water (5:4:1, v/v/v) showed large variation among samples and the same result obtained with RAPD marker [42]. Assessment of genetic fidelity of in vitro propagated Stevia rebaudiana plants Based on RAPD analysis were evaluated that no polymorphism was detected with RAPD analysis using DNA from in vitro-raised plantlets [41].…”