M. K. Kaul

Morphological and chemical variation in 25 collections of the Indian medicinal plant, Withania somnifera (L.) Dunal (Solanaceae)

Six phenotypic characters and three withanolide markers were assessed in 25 accessions of Withania somnifera collected from different states of India for studying genetic variability. The variability ranges observed at phenotypic and chemotypic levels were polymorphic. Based on D2 values and PCA (Principal Component Analysis) of phenotypic traits like plant height, no. of branches/plant, no. of seeds/berry, root length, root diameter and root yield, these 25 accessions were grouped in five clusters. The relative contribution of each character towards genetic divergence was worked out. Five accessions–AGB002 (Rajasthan), AGB003 (J&K), AGB004 (Madhya Pradesh), AGB006 (J&K) and AGB009 (Punjab) representing clusters 2 and 4 exhibited maximum intra and inter-cluster divergence. Cluster 5 representing accession AGB053 (Andhra Pradesh) was having mixed traits. Chemically most of the accessions in cluster 3 showed uniformity in presence of three marker withanolides Withaferin A, Withanone and Withanolide A in the leaves.

Regeneration of Plantlets via Callus Cultures in Celastrus paniculatus Willd-A Rare Endangered Medicinal Plant

Plantlet regeneration in Celastrus paniculatus Willd (Celastraceae), an endangered medicinal plant has been achieved from cotyledonary leaf-derived callus. The cotyledonary leaves from mature embryo callused on MS medium supplemented with NAA (5 × 10-6 M) and Kinetin (5 × 10-6 M) with a doubling time of 16.2 days. Well developed multiple shoots of 5.6 cm could be obtained via organogenesis within 28 days on MS + BA (2 × 10-5 M). Altered shoot morphology occurred with the change in the concentration of auxins and cytokinins. Rooting (85%) of isolated shoots was achieved on McCown medium (WPM) containing IBA (5 × 10-6 M). Rooted plantlets were transferred in small pots containing sand, garden soil and vermiculite mix. The procedure can serve as a reliable and reproducible protocol for propagation and ex situ conservation ofthis species.

Assessment and characterization of genetic diversity in Withania somnifera (L.) Dunal using RAPD and AFLP markers

Genetic diversity of 23 accessions of Withania somnifera collected from different geographical regions of India was estimated by employing Random Amplification of Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP) markers. Eighteen RAPD primers and six AFLP primer combinations revealed 37.82 and 43.94% polymorphism, respectively, among 163 and 286 genetic loci amplified. The AFLP assay revealed higher levels of polymorphism among the tested W. somnifera accessions compared to the RAPD. Mean genetic diversity based on Shannon index ranged from 1.33 (RAPD) to 5.13 (AFLP). Unweighted pair group method based on arithmetic average (UPGMA) analysis was performed on Jaccard’s similarity coefficient matrix. The matrix reveals, two main clusters, wild accessions formed one cluster and the cultivated accessions formed the other. The cultivated accessions are well separated from the wild ones at a low similarity value of 0.3, indicating that cultivated and wild accessions are highly distinct. Morphologically cultivated accessions were also quite distinct from the wild ones and the cluster analysis of RAPD and AFLP fingerprints clearly discriminated the five cultivated accessions of W. somnifera . A strong correlation was observed between morphology and molecular marker systems. Identification of specific markers to wild as well as cultivated accessions is yet another important finding in the present study. Such genetic diversity is useful in facilitating the development of large number of new varieties through hybridization, transfer of useful genes, thus maximizing the use of such available germplasms as genetic resource materials for breeders. The present input, first of its kind in Ashwagandha, will thus assist the marker assisted crop improvement programme. Key words: Withania somnifera, genetic diversity, RAPD, AFLP, polymorphism, Shannon index.

Regeneration via organogenesis in callus cultures of Argyrolobium roseum

A reproducible protocol has been developed for high frequency plant regeneration from immature embryos of Argyrolobium roseum Jaub & Spach, an important medicinal legume. Green nodular calli were initiated from immature embryos excised from 10-d-old pods in 70 % of cultures within 3 weeks when grown on Murashige and Skoog (MS) medium supplemented with 0.5 mg dm−3 benzylaminopurine (BAP) + 0.25 mg dm−3 indole-3-acetic acid (IAA). Subsequent transfer of 5 mm2 callus pieces to MS medium supplemented with BAP (0.5 mg dm−3) alone or in combination with IAA (0.25 mg dm−3) facilitated regeneration of multiple shoots. Organogenic calli bearing multiple shoots when transferred to MS medium supplemented with BAP (0.5 mg dm−3) + IAA (0.25 mg dm−3) supported rapid shoot elongation. Shoot propagules subcultured to Gamborgs medium (B5) with 0.5 mg dm−3 indole-3-butyric acid (IBA) rooted with 80 % frequency and developed into phenotypically normal plants. Plantlets were successfully acclimatized in a sterile mixture of sand and garden soil (1:1) under greenhouse and thereafter transferred to field beds.

Production dynamics of Withaferin A in Withania somnifera (L.) Dunal complex

Withaferin A (WA) is an important withanolide holding promise in cancer treatment and as a relatively safe radiosensitive/chemotherapeutic agent, which is present in traces in all parts of Withania somnifera except the leaves, where as it is reported to be present in only two non-Indian chemotypes (South African chemotype/Israel chemotype 1). The present studies have marked its presence in all Indian populations (wild/cultivated), as well as two identified Indian chemotypes (AGB002 and AGB025). The quantitative dynamics of WA production in Indian populations and interchemotypic hybrids developed at our institute have been studied, and the results were compared with five previously reported chemotypes from Israel, South Africa and India. An analysis on inheritance characteristics based on presence/absence of WA in hybrid plants and their respective parents is given for future studies on the chemogenetics of this complex species in greater detail. Further, the production potential of WA in vitro propagated plants of elite varieties developed at our institute is discussed, in view of maintaining chemotypic fidelity and stability from a production point of view. Also, evidence-based clues suggesting the leaves as the site of the synthesis of WA is provided.

Variability and selection on different Argyrolobium roseum accessions for morphological traits and yield

Variability studies were carried out among different accessions of Argyrolobium roseum for 12 metric traits under natural and cultivated conditions. Almost all characters showed higher values in cultivated than natural population. Leaf breadth recorded highest CV (30.59%). Highest percentage of vitexin and D-pinitol (0.208 and 0.773% dwb) was observed in RAR-7 and RAR-6, respectively, under cultivated conditions. Phenotypic coefficient was higher than genotypic coefficient of variation. The association analysis revealed that root yield had positive significant correlation with 100 seed weight (r=0.839) biomass yield (r=0.601), where biomass yield had a positive significant correlation with leaf length, single pod weight. High heritability (97.4–99.8%) coupled with moderate genetic advance ranged between (49–67%) as a percent of mean was observed for leaf breadth, pod length, total foliage biomass yield and total root yield/plant. This suggests that direct selection for these traits is suitable for the improvement of this crop.