MATERIALS AND METHODS

Single node cutting, taken from one year old lower and lateral branches of a 20 year old vegetatively propagated plant (mother bush) of D. hamiltonii growing at the Institute nursery at Kosi, District Almora (79°38’10"E and 29°28’15"N; 1150 m altitude) were collected during May 2004 and used for developing in vitro cultures. The cutting (about 3.0 cm in length) was soaked in distilled water containing Labolene (Liquid detergent; 0.2% v/v; Qualigens, India) for about 2-3 min, washed with distilled and sterilized water 3-4 times, disinfected with 0.2% mercuric chloride (w/v, BDH, India; 5 min) and again rinsed with distilled and sterilized water (x4). Subsequently both the ends were trimmed and segments cultured on Murashige and Skoog’s (1962) medium (20 mL) with out any Plant Growth Substances (PGSs) by placing them vertically in test tubes (15x1.5 cm). The pH of the medium was adjusted to 5.8 and gelled with 0.2% phytagel (w/v, Sigma) before autoclaving (1.05 kg cm^-2, 121°C, 20 min). All the cultures were maintained at 25±1°C in a culture room with 14/10 h day/night cycle. All laboratory experiments and hardening of in vitro raised plants were carried out at the Institute and its nursery located at Kosi-Katarmal, District Almora (1200 m altitude).

After 3-4 weeks of incubation the axillary buds sprouted from the nodal explants; these buds (2.0-2.5 cm long) were excised from the mother stumps and cultured on MS medium supplemented with different concentrations of BAP (2.0-12.0 μM) and 1.0 μM NAA. The buds produced small clusters of 3-5 multiple shoots (2.5-3.0 cm) with a rhizomatous portion, hereafter called propagules. The shoots were individually cut about 1.5 cm above the base and discarded, while the propagules, now referred to as shoot cut was separated and further cultured for multiplication. For rooting these propagules (with shoot cut) were separated again and placed in 1/2 MS media containing IBA at different concentrations (10.0-200.00 μM) for 7 days followed by placing in medium without IBA for 2 weeks. The plantlets were removed from the flasks, thoroughly washed with water, transferred to plastic cups (6x6x7 cm) containing 250 g of autoclaved soil and placed under greenhouse conditions (25°C air temperature, relative humidity 65%; 18/6 h day/night cycle) for 15 days. Following another 15 days, they were shifted to polythene bags (16x10 cm) containing equal proportion of soil and farm yard manure (1:1) and transferred to a polyhouse/nethouse for hardening. Following one month of hardening, the plants were planted in the field and their growth performance was monitored.

SD was calculated following the method of Snedecor and Cochran (1967).

RESULTS AND DISCUSSION

The sprouted axillary buds on the nodal explants (Fig. 1A) were excised and transferred on MS medium containing different concentrations of BAP (2.0-12.0 μM) and 1.0 μM NAA. Subsequently within the next 3-4 weeks, differentiation was observed and multiple shoots were formed. Out of the various concentrations of BAP used, the medium supplemented with 8.0 μM BAP and 1.0 μM NAA resulted in profuse shoot formation with increased rhizomatous portion. Shoot portions were removed and the propagules were separated and multiplied on media containing different concentrations of BAP (2.0-12.0 μM) and 1.0 μM NAA (Fig. 1B, Table 1). The medium containing 8.0 μM BAP and 1.0 μM NAA further enhanced multiplication, for example, starting with 1 explant, more than 20 propagules could be obtained; the average shoot length and leaf size were also highest when compared to other treatments of BAP (Table 1). This is the first report from these species where adopting this method higher number of propagules and multiple shoots were obtained by the end of the 2nd subculture (Table 1, Fig. 1C). Data on multiplication of propagules and shoots (Table 2) indicates that by following in vitro shoot cut method there was a 5-fold increase when compared to control (without shoot cut); moreover, length of shoots was also found to nearly double following shoot cut.

Fig. 1:	Various stages during the development of in vitro propagation protocol of D. hamiltonii. (A) establishment of explant in MS medium without PGSs; bar = 1 cm, (B) a representative photo depicting culture of propagules following in vitro shoot cut method on MS medium supplemented with 8.0 μM BAP and 1.0 μM NAA; bar = 1.5 cm, (C) profuse shoot multiplication after in vitro shoot cut on medium containing BAP and NAA as in Fig. B, during 2nd subculture; bar = 1.5 cm, (D) and (E) profuse rooting in propagules after shoot cut and following culture on MS medium containing 100 μM IBA and subsequently placing in IBA-free medium; bar = 1.5 cm and (F) transfer of in vitro raised plants to soil and hardening in a polyhouse; bar = 8 cm

Table 1:	Effect of BAP and in vitro shoot cut on multiplication of propagules and shoots of D. hamiltonii
A propagule consisted of 3 shoots, each treatment consisted of 5 propagules cultured per flask and conducted in triplicate, data were recorded after 3 weeks, all results were obtained using propagules during 2nd subculture, aall concentrations of BAP in the medium contained 1.0 μM NAA, bMean±SD

Table 2:	Effect of shoot cut on multiplication of propagules and shoots of D. hamiltonii
A propagule consisted of 3 shoots, aeach set represents treatment given to various batches using 3 propagules per flask and conducted in triplicate, data were recorded after 3 weeks during 2nd subculture, bMean±SD

Compared to an earlier study (Bag, 2001) wherein a combination of 5.0 μM BAP and 2.0 μM NAA supplemented to MS medium was used for multiplication, this study resulted in generation of 20% higher number of multiple shoots (Table 1, Fig. 1C). Studies using bunches, each containing 3-5 shoots, for shoot proliferation in bamboo have been reported earlier, e.g., Thamnocalamus spathiflorus (Bag et al., 2000) and D. hamiltonii (Bag, 2001; Sood et al., 2002).

The roles of the cytokinin, particularly BAP in in vitro bud break and shoot multiplication is well known. Although, BAP has been used to induce bud break in nodal explants of Bambusa wamin (Arshad et al., 2005) and Guadua angustifolia (Jimenez et al., 2006), it was not required in this study as reported earlier by Bag (2001) and Sood et al. (2002). Like in several plant species, BAP alone has been very effective for shoot multiplication in bamboos Dendrocalamus asper (Arya et al., 1999), Bambusa bambos (Kapoor and Rao, 2006), B. vulgaris (Ramanayake et al., 2006) and G. angustifolia (Jimenez et al., 2006). However, in this investigation, BAP in combination with NAA was found to be effective; similar results were reported for this species earlier (Bag, 2001) and in the temperate bamboo T. spathiflorus (Bag et al., 2000). BAP in conjunction with kinetin was also demonstrated effective in B. wamin (Arshad et al., 2005). It must be mentioned that BAP and its metabolites have been identified as naturally occurring cytokinins in plant tissues (Nandi et al., 1989a, b).

Pulse (short) treatment of the propagules with IBA for 7 days resulted in maximum rooting at 100 μM concentration and using this method more than 90% success could be achieved (Table 3, Fig. 1D). The number of roots per plant was more than 6 and the length of longest root was about 5.0 cm (results not shown). In an previous study using IBA (2.1 or 4.2 μM) or NAA (2.7 or 5.4 μM) up to 30% rooting was observed (Sood et al., 2002). Well-rooted plantlets (Fig. 1E) following proper hardening (Fig. 1F) formed profuse root system with a survival rate of over 85% (Table 3) under polyhouse/nethouse conditions. The performance of the plants transferred in the field was satisfactory and exhibited normal growth even after 2 years. A two-step rooting procedure using IBA treatment followed by transfer to auxin-free media had been reported by Bag et al. (2000).

The auxin IBA is being widely used as an effective PGS for root induction in various plant species. In several species of bamboo, medium supplemented with IBA alone was used for successful rooting; B. wamin (Arshad et al., 2005), B. vulgaris (Ramanayake et al., 2006) and Dendrocalamus hookeri (Ramanayake et al., 2008) are some examples. In this investigation, a short treatment with IBA alone was sufficient enough to induce rooting as reported earlier in this species (Bag, 2001) and in T. spathiflorus (Bag et al., 2000). Phenolic compounds, which act as auxin protectors, have also been reported to promote rooting. While, in Bambusa tulda use of coumarin alone resulted in maximum rooting response of 98% (Mishra et al., 2007), in Dendrocalaamus giganteus IBA together with coumarin was necessary (Ramanayake et al., 2008).

Table 3:	Effect of different concentrations of IBA on rooting of propagules following in vitro shoot cut
-: Not applicable. A propagule consisted of 3 shoots, each treatment consisted of 10 propagules per flask and in replicates of 10, data were recorded following 7 days treatment in IBA and then placing in medium without IBA, acalculated on per flask basis

In Bambusa nutans, addition of glucose along with IBA supported 85% rooting (Yashodha et al., 2007). These results indicate a close interaction amongst various PGSs and/or with different compounds to evoke a physiological response.

Nodal segments taken from mature bushes (Ramanayake and Yakandawala, 1997; Bag, 2001; Sood et al., 2002; Arshad et al., 2005; Ramanayake et al., 2006; Jimenez et al., 2006) were also used in this study; this approach ensures propagation of true-to-type plants. The shoot cut method used in this study has enhanced in vitro multiplication by 5 fold (when compared to control) followed by high rooting efficiency (>90%). In an earlier report only up to 30% rooting was demonstrated in this species (Sood et al., 2002). Hardening and field performance of micropropagated plants are crucial aspects that need utmost attention. Substantial numbers of micropropagated plants do not survive after transfer from controlled in vitro conditions to ex vitro environment of the greenhouse and later in the open field. Present results demonstrated more than 70% plant survival in the field even after two years of plantation and are comparable with a previous investigation (Sood et al., 2002).

This study clearly demonstrates high frequency multiplication and enhanced rooting method for in vitro propagation of D. hamiltonii. These features are necessary for increased production of true-to-type plants for large-scale multiplication of this species. Tissue culture method allows the production of a large number of plantlets identical to the mother plant, is less labour intensive and cheaper once the protocols has been standardized and hence offers distinct advantages over conventional methods of multiplication of different species of elite bamboo clones (Saxena and Dhawan, 1999; Godbole et al., 2002; Sood et al., 2002; Das and Pal, 2005; Jimenez et al., 2006; Ramanayake et al., 2006, 2008; Yashodha et al., 2007). Therefore, this study will not only help in large scale multiplication of this useful multipurpose bamboo species for restoration of degraded land but also result in economic benefits of the inhabitants of Indian Central Himalaya.

ACKNOWLEDGMENTS

Dr. L.M.S. Palni and N. Bag are thanked for valuable suggestions during this study. Director, GBPIHED, Kosi-Katarmal, Almora is thanked for providing necessary facilities. The financial help received from Institute funds and from DBT (No. BT/PR/487/Agr/16/398/2004), Govt. of India, New Delhi is duly acknowledged.