Cultivation of Medicinal Plants

Medicinal plants have been given great significance in recent years due to its demand in industry for human and animal welfare and alluring market prices. India is called as the “Botanical Garden” of the world due to variegated climatic ecosystem which is suitable for cultivation for medicinal plants. India being one of the world’s 12 mega biodiversity countries needs to conserve its resources where they are being exploited and should be grown commercially to avoid their susceptibility to extinction because of indiscriminate use.

Among the various medicinal plants, Withania somnifera (L.) Dunal (Winter cherry, Ashwagandha or Asgandh) of family Solanaceae is an important medicinal plant that finds extensive use as a potential herb in the traditional system of medicine as a “rasayana” and “medhya rasayana’. The similarities between roots of Ashwagandha and ginseng roots have led to it being called as Indian ginseng.

W. somnifera is a genetically simple species (2n = 48; n = 24; largely self-pollinated) most suited to develop cultivars for commercial production of novel sterols and alkaloids (Singh and Kumar 1998). It grows in dry and sub-tropical regions. The major Ashwagandha cultivating states are Madhya Pradesh, Rajasthan, Punjab, Uttar Pradesh, Haryana, Gujarat and Maharashtra among which Madhya Pradesh alone is having more than 4000 ha area for cultivation. Due to presence of alkaloids in roots, leaves and seeds, these are used in preparation of Ayurvedic and Unani medicines, to combat a wide range of diseases from tuberculosis to arthritis. Important part of ashwagandha is its roots, followed by leaves and berries due to presence of “Withanolides”. The major biochemical constituents of W. somnifera are steroidal alkaloids and lactones, a class of constituents together known as withanolides (steroidal lactones with ergostane skeleton).

Ongoing trials and research on animal support the role of ashwagandha’s root and leaf extracts in different disorders and diseases and possess properties like anticancer, antioxidant etc. and act as source of a restorative drug.

Molecular markers remain unaffected by physiological condition and environmental factors that is the reason for their wide application in genetic diversity assessment among W. somnifera (L.) Dunal genotypes and to identify duplicated accessions within the germplasm collections. Due to same reason, molecular markers are reliable for informative polymorphisms since genetic composition is unique for each species. Most important development has occurred in the field of molecular genetics with the emergence of molecular marker since for breeders it is effective tool for investigating novel sources of variations and genetic factors controlling quantitatively inherited traits. These markers are used for the detection and exploitation of DNA polymorphism (Semagn et al. 2010). For differentiating plants at inter- and/or intra-specific level genetic polymorphism plays significant role, not only in medicinal plants but also in cereals, cash, plantation and horticulture crops.

The most important role of conservation is to preserve the process of genetic diversity and development in the viable population of ecology and commercially viable varieties / genotypes to avoid possible extinction (Rout et al. 2010). Different types of marker systems have been used for biodiversity analysis. These include RFLP, SSR, RAPD and the AFLP. RAPD and ISSR markers are two molecular approaches that have been used to detect variation among plants. Systematic evaluation and quantification of the variability from the present study will serve as one step towards providing accurate genetic information for further breeding programmes for Withania improvement. The assessment of variation would provide us a correct picture of the extent of variation, further helping us to improve the genotypes for biotic and abiotic stresses. The main objective of this study was to characterize the Withania genotypes using morphological and molecular markers in order to evaluate the genetic diversity and relationships among genotypes lines.

The present field investigation were carried out during late kharif of 2013 and 2014 Instructional Farm, Rajasthan College of Agriculture, MPUAT, Udaipur (24035’N, 70042’E), Rajasthan (India).

Plants of 25 genotypes lines which include native and foreign plants collected from different parts of India, were maintained and considered for the present study (Table 1). Newly emerged leaf samples of the cultivars were used for DNA extraction.

Seven morphometric characters were evaluated from 25 genotypes lines of plant specimens. Standardization of data on morphological characters was done using the YBAR option of the Stand program from the NTSYS-pc 2.1 software (Rohlf 2004). Duplicate measurements for each line were averaged and were used to design a data matrix of pairwise similarities between genotypes lines. The simple matching coefficient (SMC) was used to measure the similarity, as it was the coefficient with the best results following a cophenetic test. Principal component analysis (PCA) was also used for non-hierarchical relationships among the genotypes. Eigenvalues and eigenvectors were calculated by the Eigen program using a correlation matrix as input (calculated using standardized morphological data), and a 2-D and 3-D plot was used to generate the two-dimensional PCA plot from NTSYS-pc 2.1 (Rohlf 2004).

Total genomic DNA was isolated from 25 genotypes lines using a cetyltrimethylammonium bromide (CTAB) extraction protocol (Doyle and Doyle 1990) and was then quantified spectrophotometrically on Nanospectrophotometer, Implen (Germany).

Twenty decamer primers (Operon Technologies Inc.) were screened in the ashwagandha genotypes, of which 15 primers generated polymorphic and reproducible banding patterns and were selected for final analysis. PCR amplification was carried out in a 20 µL reaction volume containing 200 µM of dNTP mix, 1.5 mM MgCl2, 1U of Taq polymerase, 1X of reaction buffer, 0.5 µM of primer and double distilled water and 20 ng genomic DNA.