Cultivation of Medicinal Plants

Introduction

Medicinal plants have gained significant attention in recent years due to their demand in the industry for human and animal welfare and alluring market prices. India is often referred to as the "Botanical Garden" of the world due to its varied climatic ecosystems, which are suitable for the cultivation of medicinal plants. As one of the world's 12 mega biodiversity countries, India needs to conserve its resources where they are being exploited and should grow them commercially to prevent their susceptibility to extinction due to indiscriminate use (Rout et al., 2010).

Cultivation of Medicinal Plants

Among various medicinal plants, Withania somnifera (L.) Dunal, also known as Winter Cherry or Ashwagandha, of the family Solanaceae, is an important medicinal plant extensively used in traditional medicine systems as a "rasayana" and "medhya rasayana." The similarities between the roots of Ashwagandha and ginseng roots have led to it being called Indian ginseng (Singh & Kumar, 1998). W. somnifera is a genetically simple species (2n = 48; n = 24; largely self-pollinated) that is well-suited for developing cultivars for commercial production of novel sterols and alkaloids. It thrives in dry and subtropical regions. The major Ashwagandha cultivating states in India include Madhya Pradesh, Rajasthan, Punjab, Uttar Pradesh, Haryana, Gujarat, and Maharashtra, with Madhya Pradesh alone having more than 4,000 hectares dedicated to its cultivation. Due to the presence of alkaloids in its roots, leaves, and seeds, Ashwagandha is used in preparing Ayurvedic and Unani medicines to combat a wide range of diseases, from tuberculosis to arthritis.

An important part of Ashwagandha is its roots, followed by leaves and berries, due to the presence of "Withanolides," which are steroidal alkaloids and lactones (steroidal lactones with an ergostane skeleton). Ongoing trials and research support the role of Ashwagandha's root and leaf extracts in different disorders and diseases, demonstrating properties such as anticancer and antioxidant effects, and serving as a source of restorative drugs.

Molecular Markers in W. somnifera

Molecular markers remain unaffected by physiological conditions and environmental factors, making them widely applicable in genetic diversity assessment among W. somnifera (L.) Dunal genotypes and in identifying duplicated accessions within germplasm collections (Semagn et al., 2010). Due to this reason, molecular markers are reliable for informative polymorphisms, as the genetic composition is unique for each species. Significant developments have occurred in molecular genetics with the emergence of molecular markers, which serve as effective tools for breeders in investigating novel sources of variations and genetic factors controlling quantitatively inherited traits. These markers are used for the detection and exploitation of DNA polymorphism, playing a crucial role in differentiating plants at inter- and/or intra-specific levels, not only in medicinal plants but also in cereals, cash crops, plantation crops, and horticulture.

Conservation and Biodiversity Analysis

The most important role of conservation is to preserve the process of genetic diversity and development in viable populations of ecology and commercially viable varieties/genotypes to avoid possible extinction (Rout et al., 2010). Various marker systems have been used for biodiversity analysis, including RFLP, SSR, RAPD, and AFLP. RAPD and ISSR markers are two molecular approaches that have been employed to detect variation among plants. Systematic evaluation and quantification of variability from present studies serve as a step towards providing accurate genetic information for further breeding programs for Withania improvement. Assessing variation offers a correct picture of the extent of variation, further aiding in improving genotypes for biotic and abiotic stresses. The main objective of this study was to characterize Withania genotypes using morphological and molecular markers to evaluate the genetic diversity and relationships among genotype lines.

Methodology

The present field investigations were carried out during the late kharif of 2013 and 2014 at the Instructional Farm, Rajasthan College of Agriculture, MPUAT, Udaipur (24°35'N, 70°42'E), Rajasthan, India. Plants of 25 genotype 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 genotype 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 used to design a data matrix of pairwise similarities between genotype lines. The simple matching coefficient (SMC) was used to measure the similarity, as it yielded 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 genotype lines using a cetyltrimethylammonium bromide (CTAB) extraction protocol (Doyle & Doyle, 1990) and was then quantified spectrophotometrically on a Nanospectrophotometer, Implen (Germany).

Results and Discussion

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, double distilled water, and 20 ng genomic DNA.

The results of this study provide a deeper understanding of the genetic makeup and diversity of Withania somnifera, which is crucial for its conservation and sustainable utilization. The insights gained can guide future breeding programs aimed at enhancing the medicinal properties and resilience of this valuable plant.

References

Doyle, J. J., & Doyle, J. L. (1990). Isolation of plant DNA from fresh tissue. Focus, 12(13), 13-15.

Rohlf, F. J. (2004). NTSYS-pc: Numerical Taxonomy System (Version 2.1). Exeter Software.

Rout, G. R., Samantaray, S., & Das, P. (2010). Biotechnology of the medicinal plants: Recent advances and potential. In Medicinal Plants (pp. 1-25). Springer.

Semagn, K., Bjørnstad, Å., & Xu, Y. (2010). The genetic dissection of quantitative traits in crops. Electronic Journal of Biotechnology, 13(5), 16-17.

Singh, S., & Kumar, S. (1998). Withania somnifera: The Indian ginseng Ashwagandha. Central Institute of Medicinal and Aromatic Plants, 1-12.