Introduction

Plants have been an importants source of medicine for thousands of year. The World Health Organization estimates that up to 80% of people still rely on herbal remedies for their health care. The demand for medicinal plants is therefore very high leading to their over- exploitation from the wild. There are various medicinal plants which are known to have several medicinal properties since ancient times.Among these medicinal plants, Withania somnifera (Solanaceae) is such a wonderful herb having great potential for the treatment of several maladies. Withania somnifera , also known as ashwagandha, Indian ginseng and winter cherry, has been an important herb in the Ayurvedic and indigenous medical systems for over 3000 years. The roots and leaves of the plant are categorized as rasayans, which are reputed to promote health and longevity by augmenting defence against disease, arresting the ageing process, revitalizing the body indebilitated conditions (Agrawal and Paridhavi,2007).

Historically the plant has been used as an antioxidant, adaptogen, livertonic, anti-inflammatory agent, astringent and more recently to treat ulcers, bacterial infection  venom toxins and senile dementia.Clinical trials and animal research support the use of Withania somnifera for anxiety, cognitive and neurological disorders, inflammation, hyperlipidemia and Parkinson’s disease.Recently it has been also used to inhibit the development of tolerence and dependence of chronic use of various psychotropic drugs (Sharma et al., 1985).

Though India has a rich biodiversity, the burgeoning demand of Withania somnifera due to its enormous medical properties, is putting heavy strain on the existing resources, hence it is increasingly being threatened in its natural habitat. Plant invitro regeneration is a biotechnological tool that offers a potential solution to the problem of medicinal plants decimation in India and other countries. Hence, the micropropagation of Withania somnifera was attempted in our tissue culture laboratory.

Materials and methods

Withania somnifera were collected from the vicinity of Bhopal after proper identification and explants were taken from the leaves, hypocotyls, and axillary nodal segments.Explants were washed thoroughly with running tap water for 10 min and then washed with distilled water an   then surface sterilized with 0.1% (w/v) mercuric chloride solution for five minutes followed by three times washing in sterilized distilled water to remove traces of mercuric chloride. For initiation of multiplication, M.S basal medium supplemented with different concentrations of cytokinin, BAP (0.1-1.0mg/l). Kn (0.1-1.0mg/l) in combination with Auxin, IAA (0.4mg/l) was used (Ray and Jha,2001).

The p^H of the medium was adjusted to 5.8 before addition of 0.8(w/v) agar. All cultures were maintained under continuous16h dark/light period for four weeks. For further elongation and multiplication of regenerated shoots, the primary shoots formed invitro were separated aseptically and cultured in M.S supplemented with BAP (0.5-1.0mg/l) and IAA (0.1-1.0mg/l). Subsequent subcultures were placed on MS medium.For root induction, excised shoots with three or more leaves were transferred to MS basal medium fortified with IAA (0.2-0.6mg/l) and IBA (0.2-0.6mg/l). Plantlets with well developed roots were removed from culture medium and transferred to half strength of MS basal medium after gentle washing with sterilized double distilled water and carefully transferred to earthern pots containing sand and soil in 1:1 ratio. Plantlets were maintained in controlled condition and after two weeks transformed outside under the full sun (Sen and Sharma,1991).

Results and Discussion:

The invitro propagation of wonder herb Withania somnifera was found to be successful as significant regeneration of shoots occured from Withania somnifera axillary nodal explants cultured on M.S.media containing various carbon sources also supplemented with varying concentrations of BAP, IBA and other important plant hormones. A satisfactory shoot proliferation from axillary nodal explants was obtained on 3 %( w/v) of sucrose. The number of roots and root length was found to be satisfactory. IBA was found effective for root induction. All micro propagated plants were free from external defects.

Figure 1: Showing explant regeneration; Withania somnifera (a) shoot induction (b) root induction.   Click here to View figure

In the present study, attempt to micro propagate the plant of Withania somnifera, using commercially available well known, Murashige and Skoog (MS) culture medium supplemented with Cacl₂ and varying concentrations of BAP along, with IBA was done. The micro propagation proceeded successfully in the conditions of keeping temperature 25 ± 2º C, relative humidity 55-60% and 16 hours light and 8 hours dark period alternatively. It was shown that appropriate concentration of IBA, BAP and Auxin gave significant growth. It took 23 days for axial shoot formation and 52 days for root regeneration. Thus it can be concluded that the choice of culture medium, concentration of growth regulators and their ratios, culture conditions and the parts of plant taken as explant are the factors, which regulate of micro propagation of medicinal plants. Micro propagated plants can be used to supplement the natural stock of plants in wild populations as well as provide a ready supply to the herbal medicinal trade.

References

1. Agrawal S.S. and Paridhavi M. (2007), Herbal drug technology, Hyderabad, Universities press (India) Pvt. Ltd; 1 – 27.
2. Ray S and Jha S (2001) production of withaferin A in shoots cultures of Withania somnifera. Planta Med. 67: 432-6.
3. Sen J., Sharma A.K. (1991), Micropropagation of Withania somnifera from Germinating Seeds & Shoots Tips, Plant Cell Tiss. Org. Cut; 26: 71 – 73.
4. Sharma S.P. (1985), Varanasi, Chowkhamba Orientalia, India, Charaka Samhita vol. I- VI.