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Research Article
 

An Efficient Regeneration from Nodal Explants of Withania somnifera Dunal



I. Sivanesan and K. Murugesan
 
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ABSTRACT

An efficient protocol for in vitro propagation of Withania somnifera is described by axillary shoot multiplication. Nodal explants of W. somnifera differentiated shoot buds on MS, SH and B5 media containing different concentrations and combinations of auxin and cytokinin. Among the media tested, MS medium was found to be the most favorable for induction of multiple shoots than B5 and SH media. Maximum number of multiple shoots was obtained on MS medium containing BAP+IAA each at 1.5 mg L-1. The microshoots were separated from the multiple shoots, transferred to MS medium supplemented with 0.3 mg L-1 GA3 favoured maximum elongation of shoots. Regenerated shoots produced highest number of roots when transferred to half strength MS medium supplemented with 0.8% (w/v) agar, 3% (w/v) sucrose and 2.0 mg L-1 IBA. The regenerated plantlets were hardened in the greenhouse and successfully transferred in soil with 87% survival rate.

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  How to cite this article:

I. Sivanesan and K. Murugesan, 2008. An Efficient Regeneration from Nodal Explants of Withania somnifera Dunal. Asian Journal of Plant Sciences, 7: 551-556.

DOI: 10.3923/ajps.2008.551.556

URL: https://scialert.net/abstract/?doi=ajps.2008.551.556
 

INTRODUCTION

Withania somnifera Dunal. is an erect, evergreen, perennial shrub. W. somnifera grows in open and undisturbed lands in Australia, Eastern Asia and Africa. It is also found throughout the drier parts of India in waste places and on bunds, in Deccan, Mysore, Coimbatore and E. Carnatic (Gamble, 1918). This plant is reputed to have adaptogenic, tonic, analgesic, antipyretic, anti-inflammatory and abortifacient properties. Its roots were prescribed for hiccup, female disorders, cough, rheumatism and dropsy (Kiritikar and Basu, 1975). The leaves contain withanolides like withaferin A that exhibit anti-bacterial and anti-tumor properties (Devi and Sharada, 1992; Devi, 1996). W. somnifera an important medicinal shrub is being exploited on a large scale on a commercial basis for its medicinal value. Because of over exploitation, this plant is becoming a member in the endangered plant species (Antonisamy and Manickam, 1999). Micropropagation is an useful tool for conservation and multiplication of several medicinal plants (Sivanesan, 2007b; Sivanesan and Jeong, 2007a, b). There have been few reports to date on micropropagation in this plant species using various explants (Kulkarni et al., 1999; Rani and Grover, 1999; Kulkarni et al., 2000; Manickam et al., 2000; Sivanesan and Murugesan, 2005; Sivanesan, 2007a). However, only few reports are available axillary shoot multiplication through nodal explants.

Furthermore, they used in vitro grown shoots or seedlings as explants source not field grown mature plants. The majority of plants used for medicines are harvested from the wild. Hence, there is an urgent need for conservation and cultivation of this plant species for future use. Multiplication of elite clone has potential impact on pharmaceutical industries, thus, we investigate an efficient in vitro propagation method using mature field grown plants. In this study, it is reported that an in vitro method for rapid regeneration using nodal explants of W. somnifera.

MATERIALS AND METHODS

The plants were collected from in and around Shevaroy hills, Salem District, Tamil Nadu, India during the month of Jan 1999. The plants were grown in a net house at the Madras University, Botany Field Research Laboratory, Chennai, India and used for experimental studies. Explants were excised from the field grown plants and were washed thoroughly in running tap water for 30 min then in Teepol for 10 min and washed several times with distilled water. Explants immersed in 70% (v/v) ethanol for 1 min followed by mercuric chloride solution 0.2% (w/v) for 5 min and washed 4 times with sterile distilled water. The explants were blotted in sterile filter paper to remove excess water and then remove the cut ends. The nodal segments were cultured on MS (Murashige and Skoog, 1962), SH (Schenk and Hildebrandt, 1972) and B5 (Gamborg et al., 1968) media supplemented with different concentrations and combinations of plant growth regulators (auxins and cytokinins). The pH of the medium was adjusted to 5.7 using 0.1 N NaOH or 0.1 N HCl prior to autoclave at 121°C for 15 min. Cultures were incubated at 25±2°C under 16 h photoperiod with 45 μmol m-2 sec-1 irradiance provided by cool white fluorescent light (PHILIPS 40 W tubes). The microshoots were transfer into shoot elongation medium containing different concentrations of GA3. Individual shoots, which were grown about 3-4 cm long transferred to half strength MS medium containing IAA, IBA or NAA for rooting. The MS medium was supplemented with 2.0 mg L-1 IBA and glucose, maltose, fructose, lactose or sucrose at 3% (w/v), to study the effect of carbon source on in vitro rooting. The rooted plantlets were transferred into plastic cups containing soil: sand: vermiculite (1:1:1). The plantlets were maintained under the same controlled environmental conditions for 3 weeks and watered once in 2 days with half strength MS basal salts, subsequently they were transferred to polythene cover and kept in greenhouse after four weeks the plantlets transfer to the field. Data were statistically analyzed by analysis of variance (ANOVA) followed by Duncan multiple range test at 5% probability level. Data analysis was performed using SAS computer package (SAS Institute Inc., NC, USA).

RESULTS

Effect of cytokinins on shoot induction: BAP was supplemented into various media at concentration ranging from 0.1-5.0 mg L-1. Each nodal segment cultured on MS medium without plant growth regulators was differentiated into a single shoot within a period of 2 weeks (Fig. 1a). When BAP was supplemented in MS medium at concentration ranging from 0.1-3.0 mg L-1 promote multiple shoot formation (Fig. 1b). However, at 4.0 and 5.0 mg L-1 there was no regeneration from the nodal explants. A maximum of 17 shoots were induced from the nodal explants at 1.0 mg L-1 BAP and the percentage of shoot induction was 84. In SH medium the maximum shoot regeneration giving 9 shoots at 2.0 mg L-1 BAP (Table 1). Where as in the case of B5 medium 7 shoots produced at the same concentration and percentage of shoot regeneration was 70% which was comparatively less than those produced by the MS medium. MS medium containing 1.0 mg L-1 kin induced highest number of shoots 7. At higher concentrations (4.0 and 5.0 mg L-1) there was no regeneration. Nodal explants produced an average of 5 shoots when the SH medium was supplemented with 1.0 mg L-1 kin. However, the maximum number of shoots when compared to others was 8 in B5 medium with 1.5 mg L-1 kin and the percentage of shoot induction was 68% (Table 1).

Effect of combination of BAP and auxins on shoot induction: BAP and IAA each at 1.5 mg L-1 optimum for shoot multiplication. The number of shoots obtained was 22 and the percentage of shoot induction was 89. 0.1-0.5 mg L-1 (Fig. 2a, b), poor shoot induction was observed which gradually increased from 1.0-2.0 mg L-1 and there was a decrease at 3.0-5.0 mg L-1 BAP. A mean of 12 shoots were induced from the nodal explants at a concentrations of 1.5 mg L-1 each of BAP and NAA and the percentage of shoot induction was 78 (Table 2). At lower concentrations only a few shoots were produced.

Shoot elongation: GA3 was supplemented into MS medium at concentrations ranging from 0.01-2.0 mg L-1 to test its effect on shoot length. Maximum shoot length of 12 cm was obtained at 0.3 mg L-1 GA3. About 9.42 cm of shoot length was obtained at 0.5 mg L-1 and there was poor effect at higher concentrations (1.0, 1.5 and 2.0 mg L-1), the shoot length measured 4.8, 5.0 and 4.0 cm, respectively (Table 3, Fig. 3).

Table 1: Effect of cytokinins on multiple shoot formation from nodal explants of W. somnifera
Means having same letter in a column were not significantly different by Duncan`s comparison test p<0.05 level

Fig. 1: Effect of cytokinins on multiple shoot induction from nodal explants of W. somnifera (2 weeks old), (a) axillary shoot initiated from nodal explants cultured on MS basal medium with out plant growth regulators, (b) multiple shoot initiated from nodal explants cultured on MS basal medium supplemented with 1.0 mg L-1 BAP

Fig. 2: Multiple shoot induction from nodal explants cultured on MS medium fortified with BAP and IAA each at 1.5 mg L-1 (Different stages of multiplication), (a) 2 weeks old and (b) 5 weeks old

Table 2: Effect of different concentration and combination of BAP and auxins on multiple shoot regeneration from nodal explants of W. somnifera
Means having same letter in a column were not significantly different by Duncan`s comparison test p<0.05 level

Rooting and acclimatization: Regenerated shoots were subjected to root induction using MS half strength medium supplemented with IAA, IBA and NAA. Sixteen roots were produced when I BA was supplemented into the medium at 2.0 mg L-1 and 100% root induction was observed at this concentration (Table 4). The corresponding root length was 10.5 cm. The carbon source, each at 3% concentration (Fructose, glucose, lactose, maltose and sucrose) was tested for root induction. Among the carbon source sucrose gave 100% root induction (Table 5, Fig. 4). The rooted plantlets successfully transferred to the field and the percentage of survival was 87% (Fig. 5).

Table 3: Effect of GA3 on shoot elongation
Means having same letter in a column were not significantly different by Duncan`s comparison test p<0.05 level

Table 4: Effect of auxins on root induction
Means having same letter in a column were not significantly different by Duncan`s comparison test p<0.05 level

Table 5: Effect of different carbon source on root induction in W. somnifera
Means having same letter in a column were not significantly different by Duncan`s comparison test p<0.05 level

Fig. 3: Elongation of microshoots cultured on MS medium supplemented with 0.3 mg L-1 GA3

Fig. 4: Root induction of excised regenerated shoots cultured on half strength MS medium supplemented with 2.0 mg L-1 IBA after 30 days

Fig. 5: Acclimatized plants were ready for field transfer

DISCUSSION

Withania somnifera is used in several indigenous drug preparations. Because of overexploitation, this plant had entered in the endangered plants` list. Tissue culture is an important technique wherein numerous plants (clones) could be produced from a single explant. Therefore, this plant was micro propagated using nodal explants, which would lead to a large-scale multiplication of this plant. Kulkarni et al. (2000) stated that nodal explants formed multiple shoots both from pre-existing and de novo buds on MS medium containing 0.1-0.5 mg L-1 BAP. Similar results were obtained in the present study. Manickam et al. (2000) reported that the greatest number of shoots 8 was obtained from the nodal explants when MS medium was supplemented with 1.0 mg L-1 BAP. In this present study we obtained an average of 17 shoots at the same concentration. Handique and Bora (1999) also observed the same in Houttuynia cordata and in Pistacio vera (Onay, 2000). In this study, three different media (MS, SH and B5) was used to test the efficacy of shoot induction. Among the three media MS was found to be the best medium. The need of MS salts for shoot multiplication shows the moderate salt requirement for the growth of W. somnifera. The MS medium was supplemented with different combinations and concentrations of auxins and cytokinin to promote shoot multiplication BAP and IAA together in the medium each at 1.5 mg L-1 produced maximum shoots. It was similar to the findings of Salvi et al. (2001) in Neem where, the highest number of shoots was produced in combination of BAP and IAA. Similar results were also observed by others Tivarekar and Eapen (2001) in Mung bean; Singh and Sehgal (1999) in Ocimum sanctum. Synergistic effect of auxins in combination with BAP on the enhancement of shoot multiplication was observed in Dubrisia myoporoides (Kukreja and Mathur, 1985) and Velariana wallichi (Mathur et al., 1988). Shoot buds induced on the optimum medium failed to elongate rapidly. Therefore, microshoots were transferred to MS medium supplemented with different concentrations of GA3. GA3 when tried at 0.3 mg L-1 increased the shoot length by upto 12 cm in W. somnifera. This was supported by the observations of Amutha et al. (2003) in Vigna radiata. Complete root induction was obtained in W. somnifera, cultured in half strength MS medium that contained 2 mg L-1 IBA, which was similar to the results observed by Manickam et al. (2000), Rani and Grover (1999), Sivanesan and Murugesan (2005) and Sivanesan (2007a). Root induction was tried using half strength MS medium supplemented with different carbon sources. Among them, sucrose proved to be the best carbon source. This was similar to the findings of Marin and Marin (1998) in Prunus sp. The root growth in glucose or fructose containing medium was remarkably slower than that was in sucrose amended medium (Inomata et al., 1993). A successful tissue culture method of propagation must result in re-establishment in soil of a high frequency of the tissue culture derived plants. In this study, regenerated plantlets of W. somnifera were transferred to the field with 87% of survival rate. Rani and Grover (1999) obtained 83% of survival rate in the same plant. Manickam et al. (2000) observed only 84% while Kulkarni et al. (2000) observed 100% of survival rate in W. somnifera. Micropropagation through tissue culture enables the large scale production of the plants. In this study, an efficient protocol was developed for regeneration of W. somnifera by using nodal explants.

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