Withania somnifera (L.) Dunal, is an important herb in the ayurvedic and indigenous medical systems for over 3000 years (Sharma et al., 2010). Both leaves and roots of the plant are used as the drug and steroidal lactones occur in both parts. Roots are prescribed as medicines for hiccups, several female disorders, bronchitis, rheumatism, dropsy, stomach and lung inflammation, and skin diseases. The active pharmacological components of W. somnifera are steroidal lactones of the withanolide type. Several chemotypes  have  been  found  differing  in their withanolide content. The principal withanolide in Indian W. somnifera are withaferin A and withanolide D (Ganzera et al., 2003).

According to red list of threatened species, W. somnifera proved to be 99.75% of the endangered medicinal plant (Siddique et al., 2005; Rahman, 2001). This medicinally important plant species has been depleted from their natural habitat and is now included in the list of threatened species by The International Union for  Conservation   of   Nature   and   Natural   Resources (Kavidra et al., 2000). Usually, Withania is propagated commercially by seeds because of the lack of natural ability for vegetative propagation, but the seed viability is limited to one year making the long duration seed storage futile (Sen and Sharma, 1991; Rani and Grover, 1999; Farooqi and Sreeramu, 2004). Seed propagation, however, is not always satisfactory, since percentage of germination is low, due to the presence of certain inhibitory compounds in the fruit and high risk of catching various diseases (De Silva and Senarath, 2009). This resulted in the adulteration of plant materials, making the plant endangered (Antonisamy et al., 2000). Again, multiplication through cuttings give rise to less ramified plants and is consequently less productive than plants obtained from seeds (Supe et al., 2006). However, the conventional propagation method cannot meet the increasing demand of this plant used as raw material for the preparation of pharmaceutical products. Due to poor viability of stored seed and little information regarding seed germination of W. somnifera an alternative procedure of propagation through in vitro seed germination and seedling development is essential. In vitro propagation of W. somnifera through sequential procedure of induction of callus, shoot regeneration and rooting take more time and costly comparing to in vitro seedling regeneration using single media. The immature seeds obtained from green pods of W. somnifera can be germinated asymbiotically in vitro for rapid micro propagation (Murashige and Skoog, 1962). The method can be exploited for the rapid propagation and conservation of W. somnifera.

Therefore, the present study was carried out to optimize the concentration of gibberellic acid (GA₃) and Kinetin (Kn) in MS media for in vitro seed germination and seedling development of W. somnifera by using in vitro technique.

The seeds of Indian cultivar W. somnifera were collected from the plants grown at Horticultural Research Farm of Uttar Banga Krishi Viswavidyalaya, Pundibari, Cooch Behar, West Bengal, India. The health and density of seeds were tested by dipping them in water. Seeds that float were discarded and healthy seeds were selected for sterilization. The viability of seeds were tested by the 2,3,5-triphenyltetrazolium chloride (TTC) test (Hartman et al., 1990). All the chemicals and reagents were purchased from Hi Media (Mumbai, India) and plant growth regulators were procured from Sigma-Aldrich (Bangalore, India).

Seeds were washed thoroughly with running tap water for 5 to 10 min to remove surface dirty particles and then sterilized by immersing in 70% ethanol for 1 min with vigorous shaking followed by 20 min in 4% sodium hypochlorite containing one drop of Tween-20. The seeds were then rinsed three times with sterile distilled water in a laminar air flow cabinet to remove minor amounts of disinfection liquid. The surface-sterilized seeds were used for the treatments of in vitro germination trials (Figure 1a).

For germinating, the surface-sterilized seeds were cultured in jam bottle of standard Murashige and Skoog medium containing 3% sucrose   and    0.6%    agar    alone    and    along    with   different concentrations of GA₃ (0.5 to 5.0 mg/l) and Kn (0.5 to 5.0 mg/l) in combination for their synergistic action. For induction of culture for seed germination and seedling development, the following media were used: (a) M {MS without growth regulators (control)}; (b) MG₁K₁ {MS + GA₃  1.0 mg l^-1  + Kn 1.0 mg l^-1}; (c) MG₂K₂ {MS + GA₃  2.0 mg l^-1 + Kn  2.0 mg l^-1}; (d) MG₃K₃ {MS + GA₃  3.0 mg l^-1  + Kn 3.0 mg l^-1}; (e) MG₄K₄ {MS + GA₃  4.0 mg l^-1  + Kn 4.0 mg l^-1}; (f) MG₅K₅ {MS + GA₃  5.0 mg l^-1  + Kn 5.0 mg l^-1}.

The pH of the medium was adjusted to 5.8 before the addition of 0.8% (w/v) agar. All cultures were incubated under controlled condition at 25 ± 2°C temperature, 60 ± 10% relative humidity and 16 h photoperiod with a photosynthetic photon flux density (PPFD) of 20 μmol m^-2s^-1 provided by cool white fluorescent lamps (2 × 40 W, Phillips, India).  

The cultures were observed daily and the data on daily seed germination was collected until the completion of the germination (maximum up to 30 days). The seeds with 0.5 mm or more radical growth were counted as germinated seeds (Figure 1b). The final germination percentage (Gp) was calculated from the total seeds that germinated on the day of completion. The other germination parameters, such as germination speed (GS), germination rate (Rs) (Rajabi and Poustini, 2005), and germination value (GV) (Djavanshir and Pourbeik, 1976) were calculated. Different growth parameters such as seedling vigour index (SVI) (Abdual Baki and Anderson, 1973) and growth value (GV) (Meredith, 1978) were calculated. Root length and shoot length of the seedlings were recorded (Figure 1e) and root to shoot ratio was calculated. Fresh weight (FW) of seedlings was recorded and dried in hot air oven at 60°C until constant weight and then dry weight (DW) of seedlings were recorded. Moisture content of seedlings was calculated using formula:

(Fresh weight – Dry weight / Fresh weight) × 100

Plantlets with well developed shoots and roots (Figure 1c and d) were transferred to plastic cup containing autoclaved perlite (Figure 1. f) and maintained for four weeks in culture room. The plantlets were then transferred to poly cups containing garden soil and were maintained in a shade net house (Figure 1g).

The experiments were designed in Completely Randomized Design (CRD). In each treatment, 50 seeds were inoculated at 5 seeds per jam bottle and each treatment was replicated four times. The statistical analysis was done by employing the O.P Stat software packages (O.P. Sheoran, 1968) and the mean were compared using Duncan’s multiple range test (DMRT) at the 0.05% probability level.

Seed germination and seedling growth are known to be regulated by exogenous hormones. Growth regulators used in pre-sowing seed treatment with growth regulator play an important role in regulating germination and vigour (Raghav and Kasera, 2012). Gibberellins are a family of 136 tetracyclic diterpenes, a small subset of which are active as plant hormones and known to stimulate seed germination in a wide range of plant species, the predominant active GA depends on the species (Thomas et al., 2005)

Seed germination

It is evident from the data presented in Table 1  that  days required for germination were decreased by increasing concentration of GA₃ with higher Kn rate. Among the different combinations of GA₃ and Kn, minimum days required for germination (8.3 days) was noted in treatment MS medium supplemented with 5.0 mg l^-1 GA₃ and 5.0 mg l^-1 Kn which was at par with the days required in treatment MG₄K₄. Maximum days (14.23) for germination were recorded on MS medium without growth regulators. The results corroborate the findings of the experiments conducted by Mello (2009). The significantly highest germination percentage (92.67%) was recorded in MS medium supplemented with 3.0 mg l^-1 GA₃ along with 3.0 mg l^-1 Kn followed by MG₄K₄ (85.67%). The least germination percentage (68.53%) was observed in MS medium containing no growth regulators. An improvement in seed germination with application of GA₃ was evidenced, but its concentration beyond optimum dose causes reduction in germination percentage (Dhoran and Gudadhe, 2012). The present investigation showed that seed germination percentage increases with increasing rate of GA₃ along with increasing concentration of Kn, but not beyond 3 mg l^-1 each of GA₃ and Kn.  The  finding  is  in line with that of Kaur et al. (1998) which reported the enhanced germination and seedling growth of chick pea when seeds were treated with Kn in combination with GA₃. The maximum germination speed (6.15) was observed in MS medium supplemented with 5.0 mg l^-1 GA₃ + 5.0 mg l^-1 Kn, while the least germination speed (3.37) was recorded under control (M). This may be due to the fact that germination speed is greatly enhanced by higher rate of GA₃ application (Dhoran and Gudadhe, 2012). The highest germination rate (1.83) was recorded in MS medium supplemented with 3.0 mg l^-1 GA₃+ 3.0 mg l^-1 Kn, while the minimum (1.12) was recorded in MS medium which is devoid of growth regulators. The result is in agreement with the findings of Mello (2009) who reported that GA₃ increases the rate of seed germination. This might be due to effectiveness of GA₃, which at higher concentrations overcome dormancy, causing rapid germination of seed. Higher concentration of GA₃ and Kn proved more effective from their respective lower concentration.  Application of GA₃ and Kn at various combinations significantly influenced the germination value over the control (M).  The  maximum  germination  value  of  56.07 was recorded in MS medium supplemented with GA₃3.0 mg l^-1 + Kn 3.0 mg l^-1. The control treatment (M) showed minimum germination value (23.09). The observation supported the report that higher concentrations of GA₃ improve germination value (Naeem et al., 2004). ). MS medium when supplemented with 3.0 mg l-1 GA3 + 3.0 mg l-1 Kn produced maximum seedling vigour index (875.73) followed by the treatment MG4K4; while the minimum seedling vigour (509.86) was noticed in control treatment. The result is in agreement with the finding of Mello et al. (2009) which reported that the GA3 treated seedlings of Penstemon digitalis cv Husker Red showed highest vigour index during light period.

Seedling growth

The data presented in Table 2 shows that MS medium when supplemented with 3.0 mg l^-1 GA₃  + 3.0 mg l^-1 Kn produced maximum seedling vigour index (875.73) followed by the treatment MG₄K₄; while the minimum seedling vigour (509.86) was noticed in control treatment. The result is in agreement with the finding of  Mello  et  al.  (2009) which reported that the GA₃ treated seedlings of Penstemon digitalis cv Husker Red showed highest vigour index during light period. Maximum shoot length (5.72 cm) was recorded to be significantly higher than the rest of the treatment combinations when the germinated seeds were transferred to the MS media supplemented with 5.0 mg l^-1 GA₃ + 5.0 mg l^-1 Kn which is at par with the treatment MG₄K₄. The minimum shoot length (4.21 cm) was observed in MS containing no growth regulators. Similar result was observed by Tolera et al. (2009) in Saccharum officinarum where application of GA3 and Kn showed significant higher value for shoot length as compared to control.

This might be due to the fact that increase in both the concentrations of GA₃ and Kn triggers the cell elongation and faster multiplication of the cells that results in rapid growth and development of the seedlings as compared to the treatments with lower hormonal concentrations. The maximum root length (4.98 cm) was observed in MS medium supplemented with 5.0 mg l^-1 GA₃ + 5.0 mg l^-1 Kn which is at par with the treatment MG₄K₄, while the control treatment (M) produced minimum root length (3.23 cm). This  finding  is  in close affirmation with the findings of Naeem et al. (2004). This may be due to the fact that GA₃ is better for inducing root growth and has the tendency of increasing root length with the increase of GA₃ concentration in the MS medium (Ribeirio et al., 2009). Significantly, the least shoot/root ratio (1.15) was recorded in MS medium supplemented with 5.0 mg l^-1 GA₃+ 5.0 mg l^-1 Kn (MG₅K₅) whereas the maximum shoot/root ratio was recorded in the treatment MG₁K_1.

The maximum shoot weight (4.48 g) was produced in treatment MG₅K₅, that is, MS supplemented with  5.0 mg l^-1  GA₃ + 5.0 mg l^-1 Kn followed by the treatment MG₄K₄ (4.16 g). Least shoot weight (1.7 g) was observed in control. The result is in conformity with the findings of Ribeirio et al. (2009) in Lentil; Kaul and Farooq (1994) in Morning Glory; and Chaudhary and Khan (2000) in Okra where they concluded that the hormone Kn show inhibition in shoot length and number of internodes. It may also be associated with a significant expansion in diameter of shoot and an increase in area of leaves as well as their number resulted in overall gain in weight of seedlings (Naeem et al., 2004). The maximum weight of  the root (1.83 g) was recorded in the treatment MG₅K₅ followed by the treatment MG₄K₄. While the minimum root weight (0.87 g) was recorded in control (M). This might be due to the addition of biomass per plant with increasing concentration of GA₃ with respect to the root number. Increase in GA₃ concentration might also result in an exponential increase in the number of roots without any phytotoxic effect of GA₃ on root formation even at higher concentration of used GA₃ (Ribeirio et al., 2009). Application of GA₃ and Kn at various combinations significantly influenced the shoot/root weight ratio. The least shoot/root weight ratio of 1.98 was recorded in control treatment (M) where the more shoot/root weight ratio was found in treatment MG₃K₃.

The data presented in Table 3 showed that the maximum fresh weight of seedling (5.91 g) was observed in the treatment MG5K5, that is, MS + 5.0 mg l-1 GA3 + 5.0 mg l-1 Kn followed by the treatment MG4K4 (5.19 g). While the minimum seedling fresh weight (3.13 g) was recorded in control (M). Significantly, the maximum dry weight of the seedling (0.78 g) was recorded in MS medium supplemented with 5.0 mg l^-1  GA₃ +  5.0 mg l^{- 1}  Kn   followed   by  MG₄K₄ (0.67 g) and the least was observed in control (0.30 g). The minimum moisture content in seedling (86.80%) was found in MS medium supplemented with GA₃ 5.0 mg l^-1 and Kn 5.0 mg l^-1 whereas maximum (90.42%) was observed in control treatment. The result is in line with the finding of Stojicic et al. (2012) where the highest seedling dry weight of Pinus peuce was obtained by application of higher concentration of GA₃. The reason might be due to the addition of biomass per plant with increasing concentration of GA₃ with respect to the root number.

The maximum leaf number (5.57) of the seedling prior to transfer to the hardening media was observed in the treatment MG₅K₅ which is followed by the treatment MG₄K₄, while the least (4.12) was observed in control. The finding is in close concurrence with that of Kedia et al. (2012) which reported the significant increase in the number of internodes as well as number of leaves on application of combined of GA_­3, IAA and Kn. Moreover, the similar effect of GA_­3 application on leaf number was found in Annona crassiflora (Ribeirio et al., 2009). The maximum seedling height (8.79 cm) at the stage prior to transfer to hardening media was  observed  in  the  treatment MG₄K₄ containing MS medium with 4.0 mg l^-1 GA₃ + 4.0 mg l^-1 Kn, while the minimum seedling height (5.25 cm) was observed in the control treatment (M). The present observation is in harmony with the result obtained by various workers and they reported that application of growth regulators enhance plant growth (Hernandez, 1997; Ashraf et al., 1987; Ashraf et al., 1989) and stem length (Lee et al., 1999; Kabar, 1990). Application of GA₃ accelerates bud development and stem elongation, but the best results can be achieved if GA₃ is applied in combination with kn (Hernandez, 1997; Bagatharia and Chanda, 1998).

From the present investigation, it may be concluded that growth hormones gave significantly better response than control both in seed germination and seedling development in W. somnifera (L.) Dunal. MS medium when supplemented with 3.0 mg l^-1 GA₃ and 3.0 mg l^-1 Kn was found more effective for maximum germination percentage (92.67), germination rate (1.83), germination  value  (56.07)  and   seedling vigour index (875.73). Whereas minimum days required for germination (8.30), maximum germination speed (6.15), shoot length (7.72 cm), weight of shoot (4.48 g), weight of root (1.83 g), fresh weight of seedlings (5.91 g), dry weight of seedlings (0.78 g), number of leaves per plantlet (5.57) and plant height (8.79 cm) were recorded in MS medium supplemented with 5.0 mg l^-1 GA₃   and 5.0 mg l^-1 Kn. 

So, the present protocol clearly describes that for in vitro seed germination of W. somnifera (L.) Dunal, MS basal medium supplemented with 3.0 mg l^-1 GA₃ and 3.0 mg l^-1 Kn is recommended best while subculture of the germinated seedlings in MS basal medium supplemented with 5.0 mg l^-1 GA₃  and 5.0 mg l^-1 Kn is recommended best for in vitro seedling development. Since the germination percentage of Withania seeds in the natural environment is very poor, the present protocol will be helpful to produce quality seedlings in large quantities and hence, conserve the rare species from natural collection.

The  authors have not declared any conflict of interests.