of medicinal plant is not easy. It is a challenging task because very little knowledge of seed biology. Efforts have not been made to search elite specimen and their propagation.
Withania sominifera is a green shrub found through- out the drier parts in India, Baluchistan, Pakistan, Afgan- istan, Shri Lanka, Congo, South Africa, Egypt, Morocco, and Jordan. In India, it is widely grown in the prov- inces of Madhya Pradesh, Uttar Pradesh, plains of Pun- jab andnorthwestern parts of the India like Gujarat and Rajasthan Withania sominifera (L.) Dunal, commonly called Indian ginseng is a member of the family Solan- aceae, growing up to a height of 30-150 cm. It is con- sidered as important medicinal plant in the Ayurvedic and indigenous medicinal system of India. It has many medicinal properties like anti-inflammatory, anticancer, antistress, anti-ageing, immune-modular, adaptogenic and shows the free radical scavenging activity It is used for treatment of tuberculosis, rheumatism, inflammatory conditions and cardiac diseases. It is also useful as abor- tificient, amoebicide, anodyne, bactericide, contracep- tive and spasmolytic. The roots are also used as sedative for senile debility and for the prevention and inhibition of Alzheimer’s disease (Sivanesan, 2007, Rout et. al., 2011, Udaykumar et al. 2013 and Darwesh et al., 2014).
Recently, Dharajiya et. al., (2014) have analyzed the antibacterial activity of various solvents viz. aqueous, Hexane, ethyl acetate and methanol extracts of stem of Withania somnifera was evaluated against gram nega- tive bacteria Escherichia coli, Serratia marcescens, Pseudomonas aeruginosa and gram positive bacterium Bacillus cereus by agar well diffusion method.
Traditionally W. somnifera is propagated from seeds, but the mature and healthy seeds are not always availa- ble for germination. The viability period of seeds is very short and their germination is also poor. The provision of alternative sources of Withania somnifera by encour- aging its cultivation will go a long way in reducing their heavy dependence on the wild populations. Con- ventional propagation methods have proved to be inad- equate to meet this challenge. Large scale production through plant in vitro regeneration will provide a means of putting the plant onto the market at lower prices.
Many earlier studies have reported in vitro propaga- tion of Ashwagandha by using different explants, such as shoot tips (Hadeer et al., 2014; Rani et al., 2014; Baba et al., 2013; Sivanesan, 2007; Ray and Jha, 2001and Roja and Heble, 1991), axillary bud (Rani and Grover, 1999), hypocotyl (Kulkarni et al. 2000), cotyledon (Kumar et al. 2013), leaf (Joshi and Padhya, 2010 and Kulkarni et al. 1996), seed (Supe et al., 2006), cotyledonary leaf segments (Rani et al. 2003), callus of leaf (Arumugam and Gopinath 2013), shoot tip and root (Shrivastava and Dubey, 2007;) and the nodal areas, (Kumar et al., 2011).
Autade et al.
The present study was done to determine the effect of growth hormones on shoots initiation, multiplication, rooting and hardening of Ashwagandha to standard- ize the micro propagation technique in Ashwagandha, as very less literature available for plant regeneration through nodal explant.
MATERIAL AND METHODS
The plant material of Ashwagandha was collected in plastic bags from Dhanwantari Medicinal and Aro- matic plants project, Mahatma Phule Krishi Vidyapeeth, Rahuri. The plant material was propagated in College of Agricultural Biotechnology, Loni, Tal. – Rahata, Dist. – Ahmednagar, Maharashtra for further use.
Young nodal segments were selected as explants and were washed under running tap water for 15 minutes. Later immersed in 1% tween-20 for 1 minute and washed with sterile double distilled water for 2-3 times. The pri- marily surface sterilized nodal segments then rinsed in 70% ethanol for 1 minute under laminar air flow hood and washed with sterile double distilled water for 2-3 times. Finally rinsed with 0.1% mercuric chloride (HgCl2) for 7-8 minutes and washed with sterile double distilled water for 4-5 times to remove all the surfactants.
One by one explants were placed on the filter paper to soak up the extra water. The nodal explants were then cut from both ends. Finally with the help of forcep the explants were inoculated on the surface of the MS media in such a way that ¾ part of the nodal explants would be in contact with MS media. The culture bottles containing explants inoculated on MS media supple- mented with respective different conc. of BAP and was incubated at 25±3°C under white fluorescent light (2000 lux) for 16/8 hours light and dark conditions. In total 6 weeks of initiation period one subculture was done in 3rdweek and data for average shoot number per explants and average shoot length was recorded at the end.
The best initiated grown shoots were then trans- planted on MS media supplemented with different con- centrations of BAP and NAA. Again the data for average shoot number per explant and average shoot length was recorded after 6 weeks of incubation period.
For complete plant development regenerated shoots were excised and transferred to MS medium supple- mented with different concentrations of IBA and data of average number of roots and average length of roots (cm) was recorded after 6 weeks of inoculation in growth chamber. Each treatment was repeated trice and statisti- cal analysis were done by calculating the standard error (SE) for the treatments.
The rooted plants were removed from culture vessels and washed in running tap water to remove agar. The
