MATERIALS AND METHODS

The nodal explant was harvested from a one-year old plant of R. cordifolia (Fig. 1a) growing in the polyhouse of the Department of Botany, Shivaji University, Kolhapur. Healthy nodal stem segments were washed in running tap water for 30 min. The nodal explants were thoroughly rinsed with commercial detergent (labolene) for 4-5 times and then again washed in running tap water. Under aseptic conditions these explants were surface sterilized with 0.1% (w/v) aqueous mercuric chloride solution for 7 min followed by several repeated washings of sterilized double distilled water. Further, explants were cut into appropriate sizes (~1 cm) with a single node and placed on sterile nutrient medium.

In the study of Murashige and Skoog (1962), medium; containing different concentrations and combinations of growth hormones was used for the multiple shoot induction studies. The medium was supplemented with 3% sucrose and pH of the medium was adjusted to 5.8±0.1 prior to the addition of agar (0.8%; w/v). The agarified medium was dispensed in culture tubes (15 mL tube), plugged with non-absorbent cotton and autoclaved at 1.06 kg^-1 cm² pressure for 20 min.

For the root induction, half strength of MS basal medium supplemented with different concentrations of auxins was used. All the cultures were maintained at 25±2°C with 16 h light 8 h dark photoperiods (Philips TL 34, 25 μmol m^-2 sec^-1) and the relative humidity of the culture room was maintained >80%.

Rooted shoots were transferred directly to small plastic pots using different potting mixtures containing variable proportions of sterile sand, soil and compost mixtures. The hardened plantlets were transferred subsequently to the field.

Statistical analysis of data: Analysis of variance and comparison of means were carried out on all data using Statistical Analysis System (SAS). The effects of different hormones on the induction of shoots, as well as on induction of in vitro rooting, were compared by one-way ANOVA. Differences between means were assessed for significance at p<0.05 and p<0.001 using Tuke-Kramers multiple comparison test.

All the experiments were repeated at least twice. For each of the treatments stated, twenty replicates in case of multilple shoot induction and twelve replicates in case of in vitro rooting experiments were maintained. The data was collected after 4 weeks interval.

Fig. 1:	(a) Rubia cordifolia (b, c) shoot multiplication on MS basal medium supplemented with TDZ (1 m g-1) (d) in vitro rooting of micropropagated shoots on half strength MS basal medium supplemented with NAA (2 mg L-1) with callusing at base (e) hardened plant after 30 days

RESULTS AND DISCUSSION

Multiple shoots were initiated from the nodal segments (Fig. 1b) on all media combinations tried in present investigation except MS basal medium (Table 1, Fig. 1c). Among the different cytokinins tested, TDZ was found to be the most effective in shoot induction (85%) and subsequent shoot proliferation with an average of 12.67 shoots per axillary bud.

Table 1:	Effect of different concentrations of growth hormones on shoot multiplication in cultured nodal explant of Rubia cordifolia
Value represents Mean±Standard error of twenty replicates per treatment and all the experiments were repeated at least twice. Data were statistically analysed using Tukey-Kramer multiple comparison test of ANOVA where a indicates a significant difference at a level of p<0.05 and b indicates difference at a level of p<0.001

Effectiveness of TDZ in shoot multiplication has been previously reported in R. cordifolia with 0.5 mg L^-1 concentration (Shrotri and Mukundan, 2004). In the present investigation, 1 mg L^-1 TDZ was found most effective in shoot multiplication in R. cordifolia. The average number of shoots is less as compared with reports of Shrotri and Mukundan (2004) where they reported 15.6 shoots per node. BA (2 mg L^-1) and zeatin (2 mg L^-1) singly were also found effective in shoot multiplication in R. cordifolia (7.25 shoots per node and 6.08 shoots per node respectively). However, the number of multiple shoots in BA concentrations was found less as compared with those in TDZ. Use of BA singly in the medium neither accelerated the percent response, nor could increase the number of shoots per explants. These reports are in contradiction with the recent studies performed by Radha et al. (2011) in R. cordifolia, where BA alone gave better results at certain optimum concentrations.

The overall effect of BA in shoot multiplication studies in R. cordifolia was found better than that of kinetin and zeatin. The effect of zeatin (2 mg L^-1) showed good percentage of response, although it is very poor in increasing the number of multiple shoots (Table 1). However, hormone TDZ showed a promising effect with respect to the multiplication of axillary buds.

In the present study it was found that TDZ had an imperative role in shoot induction and multiplication as well, than in somatic embryogenesis in R. cordifolia. The noteworthy effect of TDZ on shoot multiplication was reported previously by several workers in number of plant species like Arachis hypogea (Murthy et al., 1995), Artemisia judaica (Liu et al., 2003), Hevea brasiliensis (Seneviratne and Flagmann, 1996), Kigelia pinnata (Thomas and Puthur, 2004), Morus alba (Thomas, 2003), Primula sp. (Schween and Schwenkel, 2002), Saintpaulia ionantha (Mithila et al., 2003) and Selenicereus megalanthus (Pelah et al., 2002) which are well in coordination with the present results.

Table 2:	Effect of MS basal medium supplemented with various concentrations of auxin on in vitro rooting in Rubia cordifolia
*MS basal medium (Murashige and Skoog, 1962) containing half strength major, minor, vitamins, FeEDTA, inositol and Sucrose (1.5%). Value represents Mean±Standard error of twelve replicates per treatment and all the experiments were repeated at least twice. Data were statistically analysed using Tukey-Kramer multiple comparison test of ANOVA where a indicates a significant difference at a level of p<0.05 and b indicates difference at a level of p<0.001

In addition to this, high concentrations of TDZ resulted in stunted shoots with hyperhydric condition which is in concurrence with the preceding studies accounted in Pyrus pyrifolia (Kadota and Niimi, 2003) and Musa acuminate (Farahani et al., 2008). Indeed, various studies showed that TDZ was effective and more active than BA and zeatin especially in the micropropagation of woody plants (Lu, 1993).

In the in vitro rooting studies, NAA (2.0 mg L^-1) was found highly effective (100% root induction) with an average of 8.75±0.91 roots per shoot (Table 2, Fig. 1d). However, there was presence of unwanted callus at the base of the shoot. Increase in the NAA concentration (3 mg L^-1), does not affect the rooting percentage but the number of roots per shoot declined (Table 2) considerably. It was also observed that increased auxin concentration favours callus induction at the shoot base. Phytohormone IBA (3 mg L^-1), was also found to induce 100% root induction in the in vitro grown shoots but the concentration in any way, did not support the increase in number of roots per shoot. Infact, the number was reduced to half of that (4.58±0.77) which were recorded in NAA (2 mg L^-1).

Table 3:	The survival percentage of micropropagated plantlets of Rubia cordifolia in different potting mixtures
*Data collected after 30 days of transplantation

Among three different auxins tested, IAA was found least effective. Efficacy of an auxin is very well known for induction of rooting in several plant species such as Aloe polyphylla (Abrie and Van Staden, 2001), Cunila galioides (Fracaro and Echeverrigaray, 2001), Myrtus communis (Shekafandeh, 2007) and Morus alba (Thomas, 2003). Similarly, the role of NAA in root induction of in vitro derived shoots has been well documented in Grevillea robusta (Rajsekaran, 1994), Safflower (Radhika et al., 2006), Spondius mombin (Carvalho et al., 2002) and Vitex nigundo (Kannan and Jesrai, 1998). Combination of two auxins did not show any indicative effect for the increment in number of roots per shoot. In all the combinations of auxins tested (each combination contained two auxins), half strength MS basal medium supplemented with IBA (2 mg L^-1) and NAA (1 mg L^-1) indicated 83.33% of rooting with an average of 5.0 roots per shoot. These findings are in contradiction to the reports of Rahman et al. (2004) in Elaeocarpus robustus where they posed that when auxins are used in combination, it enhances the average number of roots/plant, percent root induction and the average length of the roots. Effectiveness of two or more auxins in the in vitro rooting has been previously reported in Baliospermum montanum (Johnson and Manickam, 2003), Ophiorrhiza mungo (Jose and Satheeshkumar, 2004), Prunus hybrid (Vaez-Livari and Salehi-Soghadi, 2006) and Rosa indica (Soomro et al., 2003).

Direct transfer of tissue culture raised plants to field or wild is not possible due to high rate of mortality, as the regenerates in the culture condition has been cosseted in an environment with very high humidity, varied light and temperature conditions (Deb and Imchen, 2010). Direct transfer to sunlight also causes charring of leaves and wilting of plants (Hiren et al., 2004; Lavanya et al., 2009). The hardening of in vitro raised plantlets is essential for better survival and successful establishment (Deb and Imchen, 2010). Hardening and acclimatization are the crucial stages of tissue culture (Chabukswar and Deodhar, 2005). In this study, it was observed that the potting mixture (soil: Sand: compost; 1: 2: 1) showed high survival rate (65%) of the in vitro derived plants (Table 3) at relative humidity 80-90% and exhibited normal growth within 30 days (Fig. 1e). This may probably be due to high sand content in the mixture which favours the adequate aeration for roots. In the present study, high content of sand was found useful because the in vitro grown plants had very delicate and fragile roots which otherwise would have been damaged by hard soil. Micropropagated plants established in potting mixture were uniform and identical to donor plants with respect to growth characteristics and vegetative morphology.

In conclusion, using axillary bud culture technique more than 1000 plants were raised from single axillary bud within one year. Due to its tremendous medicinal potential, there is rapid depletion and exploitation of this plant from its natural resources. Thus the present in vitro multiplication protocol developed for R. cordifolia will significantly contribute not only towards its conservation but also for its sustainable use.

ACKNOWLEDGMENT

One of the authors, Dr. Swaroopa Ghatge is thankful to the Department of Botany, Shivaji University, Kolhapur for funding this present work with departmental research fellowship.