MATERIALS AND METHODS

Plant material and explant preparation: Young finger shaped rhizomes of C. haritha were collected during the months July-October from the Zingiberaceae germplasm of Medicinal Garden of the Tropical Botanic Garden and Research Institute, Palode. About 3.0 cm apical buds from the rhizomes were excised and washed thoroughly in running tap water and soaked in 2% (v/v) commercial bleach and 0.2% (v/v) Teepol (Reckitt Benckiser Ltd., Kolkata, India) initially for 20 min. They were treated with 15% (v/v) bleach solution for 15 min and subsequently with 0.1% (w/v) HgCl₂ for 6 min. Then the buds were rinsed in sterile distilled water thrice and trimmed into 3.0-4.0 mm size sections with terminal bud which served as the primary explant. After 30-35 days of culture, the explants showing 1.0-2.0 cm shoots were subcultured in the same media. Initially, this was done in two ways ie., half of them were longitudinally split into two equal halves (LS explants) and the rest subcultured as whole explants. Considering the suitability, all further experiments were carried out using split explants.

Media and culture conditions: Basal MS medium (Murashige and Skoog, 1962), supplemented with different combinations and concentrations of PGRs (Table 1) and sucrose at a concentration of 30.0 g L^-1 was used. Standard procedures were followed for media preparation and culture maintenance (Vincent et al., 1992). Appropriate changes were made to this basic composition in accordance with following different experiments.

Effect of plant growth regulators: As no protocol for tissue culture of C. haritha has yet been published, a broad range of experiments were designed to determine the most effective PGR condition for multiplication. The LS explants were inoculated onto basal media containing various combinations of N⁶-benzyladenine (BA), indole-3-acetic acid (IAA), α-naphthalene acetic acid (NAA) and kinetin (KN) to study their effect (Table 1). Initially, individual cytokinins (BA 4.4-13.3 μM and KN 2.3-4.6 μM) were also tested. Based on the above treatments the PGR regime of 4.4 μM BA and 2.9 μM IAA was selected for all further experiments.

Effect of nutrient media: Four mineral salt media, viz. MS, White (1963), Nitsch (1969) and Mitra et al. (1976) formulations were evaluated.

Effect of pH: Basal media with five different pH levels (5.3, 5.5, 5.7, 5.9, 6.1) were also studied to find out the suitable pH of the medium for maximum production.

Table 1:	Effect of growth regulators on shoot multiplication from rhizome tip explants of C. haritha
*Basal medium: MS + 30.0 g L-1 sucrose + 7.0 g L-1 agar and pH 5.7. **All data after 6 weeks of culture. Means in a column with the same letter do not differ significantly (p<0.05, LSD test)

Effect of agar concentration: Multiplication medium fortified with various concentrations of agar (0, 4.0, 7.0 and 10.0 g L^-1) were studied to demonstrate the most effective level of the gelling agent.

Deflasking and greenhouse transfer: Healthy shoot clumps were washed thoroughly and then immersed in a commercial fungicide (Indofil M-45) for 5 min before planting in earthen pots containing a potting mixture of river sand and broken charcoal (3:1). The plantlets were maintained in a greenhouse under semi-shade (75%) and high humid (RH 75-85%) conditions for hardening. After 60 days of hardening individual plantlets were isolated carefully and re-potted in small poly bags for further establishment.

Statistical analysis: Each treatment contained 10 replicates and was repeated twice. All experiments were conducted in a completely randomised manner. Microsoft Excel 98 PC programme was used for statistical analysis. Data were analysed by ANOVA (p< 0.05) and means were compared using Least Significant Difference (LSD) test.

RESULTS

Effect of growth regulators: Two sets of experiments were conducted to find out the suitable inocula. In the first set, whole explants were utilised for the entire culture period. In the second, the explants were split longitudinally (LS) after the initiation of cultures (Fig. 1A). LS inocula always showed more multiplication response than whole explants. This procedure resulted in approximately 45% more shoots regardless of the media (data not shown). Therefore, all further experiments were conducted with LS explants.

Fig. 1:

Morphogenic response of rhizome tip explants of C. haritha after 6 wk of culture (A) In vitro proliferation of rhizome tip explant after 4 week (B) Induction of multiple shoots on MS + 30.0 g L-1 sucrose + 7.0 g L-1 agar + 1.0 mg L-1 BA + 0.5 mg L-1 IAA and pH 5.7 (C) Shoot multiplication on Mitra basal medium containing 30.0 g L-1 sucrose + 7.0 g L-1 agar + 1.0 mg L-1 BA + 0.5 mg L-1 IAA and pH 5.7 (D) Response of explants on White’s basal medium (E) Shoot regeneration on Nitsch basal medium (F) Hardened microplants in clay pots after 10 week of transfer

About 40% of the explants in PGR free MS basal medium produced single slender shoots with 4-5 roots. To increase the multiplication rate, the explants were inoculated on to MS medium supplemented with different PGRs, either alone or in combination. Single treatments of BA or KN showed only the growth of scale leaves and occasional development of 1-2 shoots after 35-40 days of culture (data not shown). Subsequently, another group of experiments employing various combinations of auxins and cytokinins was designed to find out the optimum PGR requirement.

The explants on different combinations and concentrations of PGRs showed a large variability in culture response. The shoot multiplication was found to occur by development of axillary buds from the axil of the scale leaves. About 92% of the explants initiated more than 2 shoots in all the PGRs tested (Table 1). The presence of BA along with IAA in the medium markedly increased the number of shoots produced per explant. The highest shoot induction was found in 4.4 μM BA and 2.9 μM IAA, which produced nearly 11 shoots in LS explants, compared with 4 shoots obtained on medium substituted with another auxin (Fig. 1B). However, this treatment resulted in reduced rhizogenic activity and shoot elongation response of the neo-formed shoots (Table 1). Higher concentrations of BA was found to be inhibitory to shoot multiplication irrespective of the IAA concentrations used. Rhizome tip explants cultured on medium containing 8.9 μM BA and 2.3 μM IAA produced an average of 5.5 shoots.

Fig. 2:	Influence of nutrient formulations on in vitro multiplication rhizome tip explants of C. haritha on a basal medium of 30.0 g L-1 sucrose + 7.0 g L-1 agar + 1.0 mg L-1 BA + 0.5 mg L-1 IAA and pH adjusted to 5.7. Vertical bars are mean±SE

Fig. 3:	Effect of pH on shoot multiplication of C. haritha explants on a basal medium of MS + 30.0 g L-1 sucrose + 7.0 g L-1 agar + 1.0 mg L-1 BA + 0.5 m g L-1 IAA. Vertical bars are mean±SE

Concentrations of IAA above 2.3 μM also showed a reduction in the rate of multiplication. When NAA was used instead of IAA, shoot bud induction and its growth were decreased, whereas the rhizogenic response was maximum compared to all other treatments. However, with this PGR combination, the shoots appeared to be healthier compared to other PGR treatments. Despite the promotive effect of cytokinins, shoot regeneration frequency was not improved significantly in BA and KN combinations. The rate of multiplication in treatments with BA and KN was intermediate between treatments with BA+IAA and BA+NAA.

Root initiation was observed after 25-30 days of incubation and the simultaneous formation of shoots and roots were observed in all treatments regardless of the concentration of the PGRs used.

Fig. 4:	Effect of gelling agent on morphogenic response of C. haritha after 6 wk of culture on MS + 30.0 g L-1 sucrose + 1.0 mg L-1 BA + 0.5 mg L-1 IAA and pH 5.7. Vertical bars are mean±SE

As compared with IAA, the response to NAA treatment of explants was better (Table 1). The best rooting response with an average of 28 roots was observed when the explants were treated with BA (4.4 μM) and NAA (5.4 μM).

Effect of basal media: Experiments to ascertain the effect of selected nutrient formulations revealed visible variability on in vitro responses in C. haritha. The use of relatively low salt formulations especially White’s medium showed least response with regard to shoot growth and rooting. The shoots were also weak with narrow leaves in these formulations compared to shoots obtained on MS medium. About 35 and 40% of the explants initiated more than 2 shoots in Mitra and White’s basal media respectively while 80% developed 5 shoots or above on MS medium with the same PGR regime. The best shoot multiplication response of an average of 10.8 shoots and moderate rooting (11.3 roots) were recorded on MS basal medium supplemented with BA (4.4 μM), IAA (2.9 μM) and sucrose (30.0 g L^-1), proving its suitability as basal nutrient formulation for the in vitro multiplication of C. haritha system (Fig. 2). Though the shoot growth and rooting were better in Mitra medium than other formulations, shoot production was significantly reduced (Fig. 1C). The explants also showed reduced multiplication in both Nitsch (Fig. 1D) and White’s (Fig. 1E) formulations.

Effect of pH: Of the different levels of pH tested, 5.5 was found best for the induction of multiple shoots, with a mean number of 11.1 shoots. An alteration from this optimum pH showed an unfavourable response and reduced the rate of multiplication (Fig. 3). The explants incubated at various pH levels did not show significant difference in shoot elongation while slight incremental effect of rooting response was recorded when the pH was enhanced from 5.5 to 6.1. At lower pH level (5.3) the shoots were weak compared to the regenerants from 5.5 and above.

Effect of agar: In agar free media, sizeable difference in shoot multiplication, shoot elongation and rooting were detected in C. haritha (Fig. 4), besides, showing healthy shoot and root growth. The medium containing a low concentration of agar (4.0 g L^-1) supported shoot elongation, but exhibited a low frequency of axillary shoot formation. With an increase in the level of agar to 7.0 g L^-1 in the medium, the number of adventitious shoots and roots also increased to 10.8 and 11.3 respectively, while shoot growth was reduced. With further increased level of agar (10.0 g L^-1), the explants showed reduced growth and development of shoots from rhizome explants. Furthermore, rhizogenic response from the neo formed shoots was also enhanced.

Greenhouse transfer: About 90% of the shoots survived in non-sterile potting mixture. But most of the shoots below 2.0 cm perished mostly due to necrosis or decay. The plantlets isolated from the clumps after 60 day of hardening showed a survival rate of 98% after 4 weeks of repotting. After 35-45 days of transfer, new leaves were also developed from the in vitro shoots (Fig. 1F).

DISCUSSION

Morphogenic responses vary with different combinations of PGRs. In C. haritha, the use of whole explants after shoot initiation always showed less response than LS explants, regardless of the culture media used. This is in agreement with the results reported in Allium wallichii Kunth. (Warwrosch et al., 2001), Narcissus sp. (Squires and Langton, 1990) or Yucca glauca (Bentz et al., 1988). In the present study, the combination of two PGRs was found to be more effective than when used singly. Similar results were observed in Kaempferia galanga (Vincent et al., 1992) and in K. rotunda (Anand et al., 1997). A combination of BA (4.4 μM) and IAA (2.9 μM) was found to be most effective in C. haritha for shoot multiplication. The effect of BA along with IAA on multiple shoot induction was also reported in Alpinia calcarata (Agretious et al., 1996) and A. galanga (Anand and Hariharan, 1997) whereas in Curcuma amada (Prakash et al, 2004) and in C. longa (Salvi et al., 2002) a combination of BA+NAA produced best results, while in K. galanga (Vincent et al., 1992) a combination of BA and KN gives increased shoot number. In the present investigation, higher concentrations of BA reduced the frequency of shoot induction. This was in agreement with the studies in K. rotunda (Anand et al., 1997). Copious root formation, obtained along with the shoots were observed in many zingibers like, turmeric and ginger (Balachandran et al., 1990) Paracautleya bhatii (Rai and Thoyajaksha, 2001) and Alpinia galanga (Barthakur et al., 1999). The present study also confirms the same results. According to Agretious et al. (1996) it may be due to the root inducing factors which are ‘intrinsic’ in the rhizomes of rhizomatous plants.

MS medium has been frequently used as nutrient medium for tissue culture programmes (George, 1996). This medium was also found suitable for the micropropagation of C. haritha. Zingiberaceae members generally prefer MS medium as in the case of Curcuma sp. (Yasuda et al., 1987) and Alpinia calcarata (Agretious et al., 1996). Where as in C. amada, B₅ media was found more effective than MS (Barthakur and Bordoloi, 1992). Similarly, foliar explants of Juniperus oxycedrus produced double the number of buds by using modified SH medium (Schenk and Hildebrandt, 1972) compared to MS (Gomez and Segura, 1994). The nutrient formulation is critical in determining the success of in vitro propagation. The mineral components in the culture medium have a swaying role in the rate of multiplication in C. haritha, as suggested by Ramage and Richard (2002). The extent of shoot multiplication in C. haritha could be increased to almost three-fold when MS represented the mineral formulation. It appears that the sensitivity of explanted tissues to PGRs could be changed with the change in the mineral salt nutrition in the culture medium. The regenerative capacity of the immature embryo-derived callus of indica rice differs when N₆ minerals (Chu et al., 1975) were substituted with MS under identical 2,4-D regime (Koetje et al., 1989).

The present study also revealed that an unfit nutrient medium reacted poorly, regardless of the shoot multiplication potential of the explant or optimum PGR levels. By simply replacing it with a favourable formulation, the shoot initiating ability of the explant was enhanced. According to Preece (1995) optimisation of nutrient salts in the medium could help to reduce the concentration of PGRs. Nutrient composition, like PGR, also plays similar and crucial role in achieving optimum explant response in vitro and changing to a suitable formulation can have a favourable influence on explant survival, growth and development.

In this study, the pH level of the culture medium did not show very significant differences in culture response compared to other treatments. However, pH 5.5 was found to be most effective for achieving optimum shoot multiplication. Better rhizogenic response was observed at pH 6.1 than at lower pHs. These data were similar to those of Venus fly trap (Jang et al., 2003) that the number of shoots was highest on the medium at pH 5.5 while better rhizogenesis at pH 6.5.

The concentration of gelling agent affects the physical and chemical properties of the medium and it could modify the accessibility of soluble substances (Marga et al., 1997). In the present investigation, optimum shoot multiplication was obtained in media fortified with 7.0 g L^-1 agar. Contrary to this, in C. longa (Salvi et al., 2002) liquid medium was found more favourable than semisolid medium for shoot multiplication and in solid media, 4.0 and 6.0 g L^-1 agar produce best results. In the present study when the concentration was increased to 10.0 g L^-1, a reduction in shoot number was observed. Similarly, Igawa et al. (2002) reported reduced shoot differentiation in Limonium sinuatum at higher concentrations of agar. This may be due to the reduced uptake of PGRs, due to higher rigidity of the medium. Addition of agar reduces the shoot length in corn shoots (Mohamed-Yasseen, 2001). C. haritha also shows the same effect in liquid media, having elongated shoots and healthy roots.

The present study describes an efficient protocol for the micropropagation of C. haritha, which is endemic to Western Ghats of peninsular India. As this system produces shoots directly without the involvement of a callus phase, it is an efficient method for clonal multiplication and conservation of this endemic species.

ACKNOWLEDGMENTS

The authors acknowledge Prof. G.M. Nair, Former Director, Tropical Botanic Garden and Research Institute for the facilities, Dr. S. Seeni, HOD (Plant Biotechnology Division) for encouragement and the Western Ghats Cell, Planning and Economic Affairs Department, Government of Kerala for financial support. The second author is also thankful to Dr. P.J. Mathew, Head, Medicinal, Aromatic and Spice Plants Unit for encouragement.