Germination behaviour of seeds of Withania somnifera (L.) Dunal: a high value medicinal plant

In order to evolve a quick method for smooth and optimum germination for Withania somnifera- a medicinally efficacious multipurpose plant, present investigation was carried to study the effect of physico-chemical treatments, storage, temperature, photoperiod and growth regulators (GA₃, IAA, IBA, 2–4 D and BA) on germinability. The most effective treatment is GA₃ at 150 μg/ml concentration at 25 °C. The optimal temperature for germination is 25 °C and continuous light favored germination showing that photoperiod has a significant role. The seedlings derived from seeds performed well when grown in a glasshouse. The data have implications for conservation and cultivation of the species studied.     

Biotechnological interventions in Withania somnifera (L.) Dunal

Withania somnifera is one of the most valued plants and is extensively used in Indian, Unani, and African systems of traditional medicine. It possess a wide array of therapeutic properties including anti-arthritic, anti-aging, anti-cancer, anti-inflammatory, immunoregulatory, chemoprotective, cardioprotective, and recovery from neurodegenerative disorders. With the growing realization of benefits and associated challenges in the improvement of W. somnifera, studies on exploration of genetic and chemotypic variations, identification and characterization of important genes, and understanding the secondary metabolites production and their modulation has gained significant momentum. In recent years, several in vitro and in vivo preclinical studies have facilitated the validation of therapeutic potential of the phytochemicals derived from W. somnifera and have provided necessary impetus for gaining deeper insight into the mechanistic aspects involved in the mode of action of these important pharmaceutically active constituents. The present review highlights some of the current developments and future prospects of biotechnological intervention in this important medicinal plant.     

Regeneration of plants from alginate-encapsulated shoot tips of Withania somnifera (L.) Dunal, a medicinally important plant species

A protocol was developed for plant regeneration from encapsulated shoot tips collected from in vitro proliferated shoots of Withania somnifera. The best gel composition was achieved using 3% sodium alginate and 75 mM CaCl2.2H2O. The maximum percentage response (87%) for conversion of encapsulated shoot tips into plantlets was achieved on MS medium supplemented with 0.5 mg/l IBA after 5 weeks of culture. The conversion of encapsulated shoot tips into plantlets also occurred when calcium alginate beads having entrapped propagules were directly sown in autoclaved soilrite moistened with 14-MS salts.     

Exploring plant tissue culture in Withania somnifera (L.) Dunal: in vitro propagation and secondary metabolite production

Withania somnifera (L.) Dunal (family: Solanaceae), commonly known as “Indian Ginseng”, is a medicinally and industrially important plant of the Indian subcontinent and other warmer parts of the world. The plant has multi-use medicinal potential and has been listed among 36 important cultivated medicinal plants of India that are in high demand for trade due to its pharmaceutical uses. The medicinal importance of this plant is mainly due to the presence of different types of steroidal lactones- withanolides in the roots and leaves. Owing to low seed viability and poor germination, the conventional propagation of W. somnifera falls short to cater its commercial demands particularly for secondary metabolite production. Therefore, there is a great need to develop different biotechnological approaches through tissue and organ culture for seasonal independent production of plants in large scale which will provide sufficient raw materials of uniform quality for pharmaceutical purposes. During past years, a number of in vitro plant regeneration protocols via organogenesis and somatic embryogenesis and in vitro conservation through synthetic seed based encapsulation technology have been developed for W. somnifera. Several attempts have also been made to standardize the protocol of secondary metabolite production via tissue/organ cultures, cell suspension cultures, and Agrobacterium rhizogenes-mediated transformed hairy root cultures. Employment of plant tissue culture based techniques would provide means for rapid propagation and conservation of this plant species and also provide scope for enhanced production of different bioactive secondary metabolites. The present review provides a comprehensive report on research activities conducted in the area of tissue culture and secondary metabolite production in W. somnifera during the past years. It also discusses the unexplored areas which might be taken into consideration for future research so that the medicinal properties and the secondary metabolites produced by this plant can be exploited further for the benefit of human health in a sustainable way.     

Agrobacterium tumefaciens-mediated transformation of Vigna mungo (L.) Hepper

Transformed Vigna mungo (blackgram) calli were obtained by cocultivating segments of primary leaves with Agrobacterium tumefaciens vir helper strains harbouring the binary vector pGA472 having kanamycin resistance gene as plant transformation marker. Transformed calli were selected on Murashige and Skoog medium supplemented with 50 mg/l kanamycin and 500 mg/l carbenicillin. Transformed calli were found to be resistant to kanamycin up to 900 mg/l concentration. Expression of kanamycin resistance gene in transformed calli was demonstrated by neomycin phosphotransferase assay. Stable integration of transferred DNA into V. mungo genome was confirmed by Southern blot analysis.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - 2iP 6-(,-dimethylallylamino)purine - Kn kinetin - nptII neomycin phosphotransferaseII - MS Murashige and Skoog (1962) medium     

Agrobacterium tumefaciens-mediated genetic transformation of rye (Secale cereale L.)

A system for the genetic transformation of rye by co-cultivation with Agrobacterium tumefaciens is described. A total of 45 independent transgenic plants were regenerated with a transformation efficiency of 1 to % of the inoculated explants. The co-cultivation of Agrobacterium-strain AGL0, harboring plasmid pJFnptII and rye im-mature embryos in liquid medium allowed a high throughput and facilitated washing of the cultures to avoid Agrobacterium overgrowth. Transgenic plants were phenotypically normal and fully fertile, which might be aconsequence of the short time in tissue culture. The selection with paromomycin exclusively during the regen-eration allowed the efficient recovery of transgenic events without interfering with somatic embryogenesis. Southern blot analysis confirmed the independent nature of the analyzed plants and indicated single copy inserts in more than 50% of them. Segregation analysis confirmed single locus integration and stable transgene expression in most of the lines, while one line with multiple locus integration was also observed. The analysis of T-DNA:: plant DNA boundary sequences revealed examples of exclusion of vector sequences, deletion of a few bases of the T-DNA or insertion of up to 29 bases of the vector backbone. This stresses the importance of detailed analysis of the inserted transgenes in order to identify events with the desired integration profile.     

Molecular cloning, bacterial expression and promoter analysis of squalene synthase from Withania somnifera (L.) Dunal

Withania somnifera (ashwagandha) is a rich repository of large number of pharmacologically active secondary metabolites known as withanolides. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, but there is sparse information about the genes responsible for biosynthesis of these compounds. In this study, we have cloned and characterized a gene encoding squalene synthase (EC 2.5.1.21) from a withaferin A rich variety of W. somnifera, a key enzyme in the biosynthesis of isoprenoids. Squalene synthase catalyses dimerization of two farnesyl diphosphate (FPP) molecules into squalene, a key precursor for sterols and triterpenes. A full-length cDNA consisting of 1765 bp was isolated and contained a 1236 bp open reading frame (ORF) encoding a polypeptide of 411 amino acids. Recombinant C-terminus truncated squalene synthase (WsSQS) was expressed in BL21 cells (Escherichia coli) with optimum expression induced with 1mM IPTG at 37°C after 1h. Quantitative RT-PCR analysis showed that squalene synthase (WsSQS) expressed in all tested tissues including roots, stem and leaves with the highest level of expression in leaves. The promoter region of WsSQS isolated by genome walking presented several cis-acting elements in the promoter region. Biosynthesis of withanolides was up-regulated by different signalling components including methyl-jasmonate, salicylic acid and 2, 4-D, which was consistent with the predicted results of WsSQS promoter region. This work is the first report of cloning and expression of squalene synthase from W. somnifera and will be useful to understand the regulatory role of squalene synthase in the biosynthesis of withanolides.     

Enhanced production of withaferin-A in shoot cultures of Withania somnifera (L) Dunal

Withania somnifera (L) Dunal, commonly known as ashwagandha or Indian ginseng, is the source of large number of pharmacologically active withanolides. Withaferin-A (WS-3), a major withanolide of W. somnifera, has been proven to be an effective anti-cancer molecule. In this study, a liquid culture system for shoot proliferation, biomass accumulation and withaferin-A production of an elite accession (AGB002) of W. somnifera was investigated. The nodal explants cultured on Murashige and Skoog (MS) semi-solid medium supplemented with various concentrations of 6-benzyl adenine (BA) and Kinetin (Kn) elicited varied responses. The highest number of regenerated shoots per ex-plant (35?±?3.25) and the maximum average shoot length (5.0?±?0.25 cm) were recorded on MS medium supplemented with BA (5.0 μM). The shoots were further proliferated in half and full strength MS liquid medium supplemented with the same concentration BA. It was interesting to note that shoots cultured on MS half strength liquid medium fortified with 4 gL-1 FW (fresh weight) shoot inoculum mass derived from 5 week old nodal explants of W. somnifera showed highest accumulation of biomass and withaferin A content in 5 weeks. Withaferin A was produced in relatively high amounts (1.30 % and 1.10 % DW) in shoots cultured in half and full strength MS liquid media respectively as compared to natural field grown plants (0.85 % DW). A considerable amount of the withaferin A was also excreted in the culture medium. Successful proliferation of shoots in liquid medium and the synthesis of withaferin A in vitro opens new avenues for bioreactor scale-up and the large-scale production of the compound.     

Improved production of withanolides in shoot suspension culture of Withania somnifera (L.) Dunal by seaweed extracts

The influence of Gracilaria edulis and Sargassum wightii extracts was investigated for the production of biomass and withanolides in the multiple shoot suspension culture of Withania somnifera. Supplementation of 40 % G. edulis extract in MS liquid medium for 24 h exposure time in the culture recorded the highest biomass accumulation [62.4 g fresh weight and 17.82 g dry weight (DW)] and withanolides production (withanolide A 0.76 mg/g DW; withanolide B 1.66 mg/g DW; withaferin A 2.80 mg/g DW and withanone 2.42 mg/g DW) after 5 weeks of culture, which were 1.45–1.58-fold higher than control culture. This naturally available G. edulis extract-treated multiple shoot suspension culture protocol offers a potential alternative for the optimum production of biomass and withanolides utilizing shake-flasks.     

Class-based stratification matrix for physical leaf traits in phenetic relations of Withania somnifera (L.) Dunal accessions

Withania somnifera (L.) Dunal is a promising herb with many pharmaceutical and therapeutic uses ranging from immunomodulation to anticarcinogenicity. It is commonly known as Indian ginseng, as it is comparable to Panax ginseng, which is a widely studied and utilized herb. There are limited studies on the genetic diversity of W. somnifera from the northeastern region of the Indian Subcontinent. This paper describes the characterization of wild accessions collected from Tamil Nadu State. A total of 15 accessions collected from wild populations were studied for their physical leaf traits such as leaf fresh weight (g), dry weight (mg), leaf dry matter content (mg g^?1), specific leaf area (mm² mg^?1), leaf size (mm²), total carbon and nitrogen, and total withaferin-A content in leaves. An attempt was made to correlate physical leaf traits with withaferin-A content. The molecular traits, which were treated in a presence–absence matrix, failed to group the hyper-withaferin-A accessions. The quantified physical leaf traits were converted into a presence–absence matrix using a novel method of class-based stratification. The phenetic relations inferred from the Fitch–Margoliash algorithm applied to physical leaf trait data resulted in grouping of accessions with high withaferin-A content. These traits were used in the selection of promising accessions which can be further used for breeding programmes.     

Factors influencing the Agrobacterium tumefaciens-mediated transformation of carrot (Daucus carota L.)

To develop an efficient procedure for Agrobacterium tumefaciens-mediated genetic transformation of carrot (Daucus carota L.) the effects of several factors were studied. Parameters which significantly affected the transformation frequency were the variety, the explant type, and the co-cultivation period. Under optimal conditions, using the A. tumefaciens C58C1 containing either pGSTRN943 or pGSGluc1 and 3 days of co-cultivation, the frequency of transformation of petiole explants of the variety Nanco was greater than 45%. This procedure does not require acetosyringone or prolonged precultivation period. Using kanamycin (100 mg l^-1) for selection, a large number of transgenic plantlets developed from the embryogenic calli within 8–10 weeks of culture on hormone-free medium. Transformation was confirmed by histochemical detection of -glucuronidase activity in the transformed cells, by the ability of petiole segments to produce embryogenic calli in presence of kanamycin, and by Southern hybridization analyses.     

Sonication,Vacuum Infiltration and Thiol Compounds Enhance the Agrobacterium-Mediated Transformation Frequency of Withania somnifera (L.) Dunal

In the present study, we have established a stable transformation protocol via Agrobacterium tumafacines for the pharmaceutically important Withania somnifera. Six day-old nodal explants were used for 3 day co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring the vector pCAMIBA2301. Among the different injury treatments, sonication, vacuum infiltration and their combination treatments tested, a vacuum infiltration for 10 min followed by sonication for 10 sec with A. tumefaciens led to a higher transient GUS expression (84% explants expressing GUS at regenerating sites). In order to improve gene integration, thiol compounds were added to co-cultivation medium. A combined treatment of L-Cys at 100 mg/l, STS at 125 mg/l, DTT at 75 mg/l resulted in a higher GUS expression (90%) in the nodal explants. After 3 days of co-cultivation, the explants were subjected to three selection cycles with increasing concentrations of kanamycin [100 to 115 mg/l]. The integration and expression of gusA gene in T0 and T1 transgenic plants were confirmed by polymerase chain reaction (PCR), and Southern blott analysis. These transformed plants (T0 and T1) were fertile and morphologically normal. From the present investigation, we have achieved a higher transformation efficiency of (10%). Withanolides (withanolide A, withanolide B, withanone and withaferin A) contents of transformed plants (T0 and T1) were marginally higher than control plants.     

Seasonal low temperature plays an important role in increasing metabolic content of secondary metabolites in Withania somnifera (L.) Dunal and affects the time of harvesting

Withania somnifera (L.) Dunal is a subtropical crop which is widely used in traditional medicine owing to the presence of root bioactives called withanolides. In the present study, phenological variation in withanolide content in roots and leaves was determined at various days after plantation (DAP). Results showed that during the full vegetative state, the amount of marker secondary metabolites viz., withanolide A (Wd-A) and withanone (Wn), increased significantly (p????0.05) more than early vegetative state (8.16 and 3.2 times, respectively) and maturity stage (54 and 1.33 times, respectively). To qualify the role of temperature per se in enhancing secondary metabolite content, plants during full vegetative stage were exposed to temperature similar to lowest minimum temperature found during the growing period (8°C). During recovery, the metabolic content of secondary metabolites again fell to the level of plants growing at 25°C. This indicated that seasonal temperature played a key role in increasing secondary metabolites rather than the phenological stage of the plant. The physiological importance of this increase in harvesting and its balance with biomass yield has further been discussed.     

Agrobacterium tumefaciens-mediated transformation and transgenic-plant regeneration of onion (Allium cepa L.)

 An Agrobacterium tumefaciens-mediated transformation method has been developed for onions (Allium cepa L.) using immature embryos as the explant source. Transgenic plants were recovered from the open-pollinated onion cultivar Canterbury Longkeeper at a maximum transformation frequency from immature embryos of 2.7%. The method takes between 3–5 months from explant to primary regenerant entering the glasshouse. Multiple-shoot formation from primary transgenic material made possible the clonal multiplication of transformants. The binary vector used carried the nptII antibiotic resistance gene and the m-gfp5-ER reporter gene. Transgenic cultures were initially screened for their ability to fluoresce and to grow in the presence of geneticin (5–25 mg/l). The transgenic nature of individual plants was confirmed by Southern blot analysis. Received: 12 October 1998 / Revision received: 17 May 1999 Accepted: 14 June 1999