Introduction

The genus Salix commonly known as willow of the family Salicaceae is a deciduous tree or shrub found primarily on moist soils in cold and temperate regions of the northern hemisphere (Anonymous 2003). There are about 400 species of willow. Many willow species are very vigorous growers and are characterized by particular physiological adaptation (Park et al. 2008). The economic importance of the various species is currently increasing and emerging in a wide array of practical application to restore damaged ecosystems including phytoremediation and bioengineering such as the production of biofuels (Perttu and Kowalik 1997; Granel et al. 2002; Vervaeke et al. 2003; Kuzovkina and Quigley 2005). Salix tetrasperma Roxb. also known as Indian willow is a valuable tree that could be used as a potential tree for phytoremediation. Besides, the plant is also used as an antipyretic, analgesic, and astringent. The dried and powdered leaves mixed with sugar is given for the treatment of several diseases, such as rheumatism, epilepsy, swellings, piles, venereal diseases, and stones in bladder (Anonymous 2003). The drug ‘Aspirin’ derived from Salix is used as non-steroidal anti-inflammatory drug (NSAIDS). Recently willows have been extensively utilized for production of biofuels because of their fast and rapid growing habit.

Rapid urbanization coupled with multifaceted and ever increasing demands of humankind has taken a toll on the natural population of many forest trees. Regeneration and restoration of these populations in the natural conditions is very low resulting in shrinking forest resource (Giri et al. 2004). In such scenarios, commercialization and exploitation of valuable forest resource needs a serious concern and steps or efforts need to be taken towards conservation and sustainable utilization of important forest trees. Conventional propagation cannot fulfill the need for rapid introduction of newly selected genotypes and in vitro propagation is the ultimate solution for this and several other woody species (Thorpe et al. 1991; Ahuja 1993; Vinocur et al. 2000). In addition, the germplasm of economically significant species may be preserved ex situ in culture collections and gene banks, circumventing the need to continuously take germplasm from natural environments. Furthermore, tissue culture of utilizable species can be used as a direct source of natural products. In vitro propagation from adventitious or axillary buds is a useful technique for mass propagation of many valuable plant species (Faisal and Anis 2002; Ahmad and Anis 2007). There are several reports on in vitro propagation of willows (Bhojwani 1980; Chalupa 1983; Dhir et al. 1984; Bergman et al. 1985; Grönroos et al. 1989; Stoehr et al. 1989; Neuner and Beiderback 1993; Agrawal and Gebhardt 1994; Amo Marco and Lledo 1996; Lyyra et al. 2006; Park et al. 2008) but, so far no reports on in vitro propagation of Salix tetrasperma is available. Therefore, the objective of the present investigations was to study the in vitro regeneration potential of S. tetrasperma from nodal explants and mass multiplication by manipulation of culture media and Plant Growth Regulators (PGRs).

Materials and methods

Establishment of aseptic culture

Young, green and actively growing shoots of S. tetrasperma were collected from a 30-year-old mature plant maintained at the University Department of Botany, Aligarh during the month of August. These were washed under running tap water for 20 min, treated with a laboratory detergent (Labolene, Qualigens, India) 5% (v/v) for 5 min followed by 3–4 washing with sterile distilled water (DW). The plant material was surface sterilized with 0.1% (w/v) HgCl2 for 4 min following repeated washes with sterile distilled water. 0.5–1 cm sized nodal segments were excised aseptically and cultured on sterile shoot induction medium.

Media and culture conditions

The nutrient medium used in all the experiments consisted of Woody Plant Medium (Lloyd and McCown 1981) with 3% (w/v) sucrose (Qualigens, India) supplemented with various concentrations of different PGRs. All the salts used were of analytical grade. The medium was solidified with 0.8% (w/v) bacteriological grade agar (Qualigens, India) and the pH of the medium was adjusted to 5.8 before autoclaving at 121°C for 15 min. All the culture vials were incubated in culture room at 25 ± 2°C under 16/8 h (light/dark) cycle with a light intensity of 50 μmol−2 s−1 supplied by cool white fluorescent lamps (2 tubes × 40 W, Philips, India) and with 60–65% relative humidity.

Shoot induction and multiplication

Nodal segments were placed on WPM augmented with various cytokinins (BA, Kn and 2 iP) at different concentrations (0.0, 1.0, 2.5, 5.0, 7.5, 10.0 μM) either singly or in combination with auxins (NAA, IAA, IBA) as listed in Tables 1, 2 and 3. Subculturing onto the same medium was performed every 3 weeks. The frequency of explants producing shoots, number of shoots per explants and shoot length were scored after 6 and 12 weeks of culture. Each treatment consisted of 20 replicates and all the experiments were repeated thrice.

Table 1 Effect of different concentrations of cytokinins on multiple shoot regeneration from nodal segments of Salix tetrasperma in WPM, after 6 and 12 weeks of incubation
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Table 2 Combined effect of auxins with the optimal concentration of cytokinins on shoot regeneration from nodal explants of Salix tetrasperma after 12 weeks of culture
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Table 3 Multiplication and elongation of BA (5.0 μM) treated shoot clusters on WPM using lower concentrations of BA and auxins after 12 weeks of culture
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In vitro rooting

Healthy and well-elongated shoots (4–5 cm) were excised from culture and transferred to rooting media composed of WPM or 1/2 WPM supplemented with different concentrations (0.0, 0.1, 0.5, 1.0 μM) of either of the auxins (NAA, IAA, IBA) as illustrated in Table 4. Data on percentage of rooting, mean number of roots and root length per shoot were recorded after 4 weeks of transfer to rooting media. Each treatment consisted of 20 replicates and all the experiments were repeated thrice.

Table 4 In vitro rooting of regenerated shoots of Salix tetrasperma using different treatments after 4 weeks of transfer
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Acclimatization

Plantlets with well developed shoots and roots were removed from the culture medium, washed gently under running tap water and transferred to plastic cups containing sterile Soilrite, garden soil, garden soil-vermiculite (1:1) mixture or vermiculite under diffused light (16:8 h photoperiod) conditions. Potted plantlets were covered with transparent polythene bags to ensure high humidity, maintained in a growth room and watered with 1/2 WPM solution every alternate day for 2 weeks. Thereafter, the bags were removed in order to harden the plants to field conditions. After 4 weeks, the acclimatized plants were transferred to pots containing normal garden soil and maintained in a greenhouse.

Histological analysis

For histological examination, nodal explants bearing regenerated shoot buds were excised at the time of second subculture and fixed in 5:5:90 (v/v/v) formalin:acetic acid:ethanol (FAA) for 24 h followed by storing in 70% (v/v) ethanol. Tissues were hydrated with a graded ethanol-xylol series followed by paraffin embedding using the method described by Johansen (1940). Longitudinal sections, 10 μm thick, were cut using a Spencer 820 rotatory microtome (American Optical Corporation, Buffalo, NY, USA) and the resulting paraffin ribbons were passed through a series of deparaffinising solutions, followed by staining with safranin and fast green (Johansen 1940). Sections were examined under a light microscope (Olympus CH20i; India Pvt., Ltd., New Delhi).

Statistical analysis

The experiments were based on a completely Randomized Block Design (RBD) with 20 replicates per treatment. The data on various parameters were subjected to one-way Analysis of Variance (ANOVA) using SPSS version 16 (SPSS Inc., Chicago, USA). The significance of differences among means was carried out using Duncan’s multiple range test at P = 0.05. The results were expressed as the means ± SE of three repeated experiments.

Results and discussion

Effect of cytokinins

In the present study, nodal explants cultured on WPM basal without any growth regulator failed to produce shoots, even after 6 weeks of culture necessitating the requirement of cytokinins for multiple shoot induction. Generally, a cytokinin is required for in vitro axillary shoot induction and proliferation. However, its effective type and optimal concentration varies with the system (Park et al. 2008). Cytokinins were reported to play a key role in DNA synthesis and cell division, which might be the reason for induction of multiple shoots. A varied response was observed with different concentrations of different cytokinins (BA, Kn and 2 iP) as summarized in Table 1. WPM supplemented with different concentrations of BA (1.0–10.0 μM) stimulated axillary shoot sprouting after 1 week of culture, and 5.0 μM BA was found to be the optimum for maximum (4.40 ± 0.50 shoots) shoot induction after 6 weeks of incubation (Fig. 1a). Further increase in BA concentration beyond 5.0 μM, did not show any improvement in shoots induction, rather a decreasing trend in all the parameters evaluated was evident. The reduction in the regeneration potential appeared to be due to the detrimental effect of high concentration on the cells predetermined to form vegetative buds. However, the shoots regenerated on BA supplemented WPM were stunted and no elongation was observed even after 12 weeks of culture. Upon exposure at higher concentrations of BA (5.0 μM and above) beyond 6 weeks and up to a maximum exposure duration of 12 weeks, the regenerated shoots tend to show hyperhydricity. Hyperhydricity of shoots exposed to higher concentrations of BA has been reported earlier by Amo Marco and Lledo (1996) on Salix tarraconensis and by Read et al. (1989) for some clones of Salix viminalis. When Kn was used as the sole source of cytokinin, a slight basal callusing was observed with a low regeneration frequency (Table 1). A maximum of 3.00 ± 0.44 shoots with the highest percentage (78%) of responding explants was produced at 5.0 μM Kn after 6 weeks of incubation. These results clearly showed that Kn was inferior to BA in this species which is in accordance with earlier reports on many woody plant species (Anis et al. 2010; Ahmad et al. 2008). However, in this case, the shoots exhibited a better elongation as compared to those induced using BA; 2 iP was also used for shoot induction and comparatively few shoots were formed with a low regeneration frequency, the highest being 2.40 ± 0.74 shoots with 70% response at 5.0 μM concentration after 6 weeks of culture, but the elongation of regenerated shoots was maximum compared to BA or Kn (Table 1), indicating a tendency of the regenerated shoots towards apical dominance. Among the three cytokinins tried, BA was the most effective followed by Kn and 2 iP which is in accordance with earlier report on hybrid willow (S. matsudana × alba NZ-1002) by Bhojwani (1980). The effectiveness of BA on multiple shoot bud differentiation has also been demonstrated in a number of cases using a variety of explants (Anis et al. 2009; Husain and Anis 2009; Siddique and Anis 2009).

Fig. 1
figure 1

a Multiple shoots induction on WPM + BA (5.0 μM) after 6 weeks of culture. Inset—an excised nodal segment b Shoot multiplication on WPM + BA (1.0 μM) after 12 weeks of culture. c LS of shoot buds showing leaf primordial and vascular bundles (LP leaf primordia, VB vascular bundles, SA shoot apex; Bar = 200 μm) d Mass multiplication WPM + BA (1.0 μM) + NAA (0.5 μM) after 12 weeks of culture. e Rooting of in vitro raised shoots on 1/2 WPM + IBA (0.5 μM)

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The differentiation of shoot buds was confirmed by histological examination of cultured explants. Most shoot buds revealed no visible connection with the original vascular tissue, although they appeared to have originated from the meristematic zone beneath the epidermis. It is possible that the initial shoots developed from pre-existing meristems, as seen in histological slides (Fig. 1c).

Multiplication and proliferation of shoots

In vitro raised shoots of S. tetrasperma multiplied and elongated onto the same medium when either Kn or 2 iP was used as supplement to WPM. The optimum proliferation response was recorded at 5.0 μM for both the PGRs yielding 7.80 ± 0.96 and 4.40 ± 0.60 shoots, respectively, after 12 weeks of culture (Table 1). BA at lower concentrations (0.5, 1.0 and 2.0 μM) were effective for multiplication and proliferation. However, at higher concentrations (2.5 or above), the shoots failed to elongate even though the number of shoots continued to increase thereby forming shoot clusters. For this, a different strategy was adopted and the induced shoots were transferred to WPM containing lower concentrations of BA (0.5, 1.0 and 2.0 μM) after 6 weeks exposure. At the end of 12 weeks, healthy and well-elongated shoots were formed, the maximum being at 1.0 μM BA. The decrease in the concentration of BA from 5.0 μM in explants establishment stage to 1.0 μM BA in multiplication stage improved shoot proliferation and nodal segments which were earlier exposed to 5.0 μM BA for 6 weeks yielded 14.40 ± 1.07 shoots with a mean shoot length of 4.76 ± 0.35 cm after 12 weeks on transfer to a multiplication medium containing 1.0 μM BA (Table 3). This increase in shoot proliferation on lowering the concentration of BA may be due to the residual cytokinin present in the shoots at multiplication stage (Shukla et al. 2009). Such a stimulating effect of lower concentrations of BA on shoot elongation and proliferation was well documented in earlier reports on some Salix species by Bergman et al. (1985) and Read et al. (1989). Similarly Lyyra et al. (2006) also achieved shoot elongation in Salix nigra upon transfer of shoot buds to one tenth of the concentrations of PGRs used in the callus initiation medium. These results clearly suggested that a continuous high dosage of either Kn or 2 iP was required for shoot multiplication in the species investigated while for BA, lowering the concentration proved beneficial for further growth and proliferation.

Effect of combined treatments of cytokinins along with an auxin was evaluated for shoot multiplication rate by taking the optimized concentration of different cytokinins in combination with different concentrations (0.1, 0.5, 1.0 μM) of auxins (NAA, IAA, IBA). For shoot proliferation, the reduced concentration of BA (1.0 μM) along with NAA (0.5 μM) was more effective over others (Tables 2, 3). Shoot clusters regenerated on WPM containing 5.0 μM BA proliferated and produced 18.40 ± 0.92 well-elongated shoots upon transfer to a medium containing BA (1.0 μM) and NAA (0.5 μM) (Table 3; Fig. 1b). Such stimulating effects of combinations of these two hormones are well supported by earlier studies on Balanites aegyptiaca (Anis et al. 2010) and Vitex negundo (Ahmad et al. 2008; Ahmad and Anis 2010). Combinations of auxins along with Kn (5.0 μM) or 2 iP (5.0 μM) also yielded favorable results and multiplication and elongation of shoots were enhanced with an optimum response at 0.5 μM of NAA supplementation (Table 2). However, combination of 5.0 μM BA with auxins (NAA, IAA and IBA) at different levels (0.1, 0.5, 1.0 μM) did not significantly improve the regeneration and number of shoots and shoot length were not significantly affected (Table 2). Effectiveness of auxins for stimulating axillary shoot proliferation synergistically with cytokinins (BA, Kn, and 2 iP) follows the order NAA > IAA > IBA (Tables 2, 3).

Rooting

Rooting of in vitro raised shoots were carried out by transferring the healthy, well-elongated shoots to rooting media composed of either full-strength or half-strength WPM with or without different concentrations of auxins (NAA, IAA, IBA). Half-strength growth-regulator-free WPM was found superior to full-strength WPM for root development as this was able to induce 2.0 roots per shoot as against 1.0 in case of full-strength (Table 4). Rooting of willow species in hormone-free WPM was earlier reported by Gebhardt (1992) and Park et al. (2008). The production of adventitious roots on auxin free basal media may be due to the endogenous level of salicylic acid which plays an important role in plant growth and development (Raskin 1992) including in vitro rooting (Khalafalla and Hattori 2000). Of the various treatments evaluated for in vitro rooting, the best rooting response could be observed in half-strength WPM containing 0.5 μM IBA where about eight roots were formed with a length of about 5 cm (Table 4; Fig. 1e). Similar results were reported in Tylophora indica by Faisal et al. (2007).

Hardening and acclimatization

The rooted plantlets were successfully hardened off inside the culture room in selected planting substrate for 4 weeks and eventually established in garden soil in the green house. Among the tested potting substrates, Soilrite™ (Keltech Energies Ltd, Bangalore) was found to be the most suitable for the successful establishment (Fig. 2) of S. tetrasperma plantlets where maximum (85%) survival rate was recorded (Table 5). The plantlets were subsequently transferred to pots containing garden soil followed by shifting to green house and finally to field conditions under full sun.

Fig. 2
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An acclimatized plant in Soilrite (4 weeks old)

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Table 5 Effect of different potting substrates on the survival of plantlets of Salix tetrasperma after 4 weeks of transfer
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Conclusion

The present study demonstrates the successful use of nodal explants from an elite clone of S. tetrasperma for multiple shoot induction and proliferation. Nodal segments carry a high potential for rapid shoot regeneration on a medium containing 5.0 μM BA followed by transfer to a medium containing lower concentrations of PGRs viz., 1.0 μM BA and 0.5 μM NAA after 6 weeks, thereby reducing the input cost. Such a system would be useful for mass propagation and multiplication of this valuable plant species. Since the regeneration of plantlets was achieved without an intervening callus phase, somaclonal variation if any may be avoided and thus ensures clonal multiplication. Commercial exploitation of the developed protocol is possible, as the nodal segments from in vitro raised shoots may be employed as propagules for further multiplication, obviating the dependence on field material.