INTRODUCTION

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Thevetia peruviana (Pers.) K. Schum. is a small ornamental shrub belonging to the family Apocyanaceae. It is commonly called as Be still tree, Digoxin, Lucky nut, Nerium Oleander, Yellow oleander and milk bush. It is an evergreen, dicotyledonous shrub, with origin from the forest of tropical America and now widely spread out in various tropical and subtropical regions of the world (Pearn, 1989). It grows to a height of about 10 to 15 feet and continues its blooming throughout the year. Flowers are bright yellow and funnel shaped with 5 petals spirally twisted. The fruits are green in color and changes from green to brownish black when it achieves maturity. Each fruit contains a nut which is longitudinally and transversely divided. Propagation is primarily by means of seeds, can also propagate from cuttings in spring early summer with hardwoods cuttings (Prajapati et al., 2003).

Besides being a very popular ornamental tree, the medical and toxicological properties of this plant have also been recognized. It has been used, for example, in the treatment of hemorrhoids, ulcers, leprosy, snake bites, in the induction of abortion and also reported to have anti-cancerous properties. The whole plant exudes milky juice which is very poisonous, quite unsafe for internal use. It has also been regarded as a potential source of biologically active compounds, namely insecticides, rodenticides, fungicides and bactericides (Patil et al., 2007). Its seeds, leaves, fruits and roots are being used in traditional medicine as an acrid purgative, as emetic and for intermittent fever treatment (Gata-Gonclaves et al., 2003). In recent years the plant has been reported to have strong anti-HIV activity also (Tewtrakul et al., 2002). Such diverse medicinal applications of the plant clearly indicate towards presence of diverse secondary metabolites in various parts. Many secondary metabolites like cardiac glycosides thevetin A and B, thevetoxin, peruvoside, iridoid glycosides, cerberin, perusitin, peruvoside, quercetin, flavonoids, triterpenes, monoterpenes, thevefolin, ruvoside, digitonin, peruvianoside kokiphin, theviridoside, theveside, nerifolin and oleandrin have been reported in different extracts of the plant (Abe et al., 1996). Oleandrin is reported to be a potent inhibitor of human tumor cell growth (Afaq et al., 2004). So there is a sharp rise in the commercial demand of Thevetia peruviana in the pharmaceutical industry, during recent years. Naturally occurring plants are unable to meet this demand because of many problems like non-availability of large scale true-to type planting material, natural propagation being vulnerable to environmental changes and the non applicability of genetic improvement techniques. These problems can be easily dealt with plant tissue culture techniques.

Plant cell cultures were introduced as an important tool for studying and producing plant secondary metabolites in the mid 1960s. Improved cell and tissue culture technologies would help in producing the active compounds in vitro with better productivities without cutting down the natural resources. Some highly effective secondary metabolites that are used in pharmaceuticals, food industry and cosmetics have been produced through plant cell cultures, callus cultures, shoot cultures and root cultures (Ramachadra-Rao and Ravishankar, 2002; Zhang et al., 2002; Mathur et al., 2007; Taha et al., 2008). The direct manipulation of plant cell and tissue culture systems has many advantages over the conventional isolation of secondary metabolites (Kim et al., 1995). Such manipulations have been reported to enhance the secondary metabolites production in many species (Zhang et al., 2002; Mathur et al., 2007).

Till now, there is no report regarding the use of callus cultures of Thevetia peruviana for secondary metabolites isolation. To the best of our knowledge, there are only a few reports about the tissue culture work on Thevetia peruviana. The available ones report the callus induction and plant regeneration using leaf discs as the explant (Kumar, 1992; Zibbu and Batra, 2010). No studies are available regarding the use of stem segments for callus or shoot regeneration. In the present study we report a highly efficient protocol for callus induction from stem segments of Thevetia peruviana and its subsequent maintenance for a period of consecutive nine months for further use in regeneration studies and biochemical analysis.

MATERIALS AND METHODS
Plant material and explants preparation: The plant material used in the present investigation was collected from 6-7 year-old shrub of Thevetia peruviana, growing near the science block of the Chaudhary Devi Lal University, Sirsa, Haryana, India, during the months of January to July, 2009. Three explants viz., nodal segments, inter-nodal segments and shoot apices (each of 3-4 cm in size) were excised from healthy and juvenile shoots and were washed under running tap water for 10 min to remove the traces of dust and dirt. These were then swabbed with an alcohol (50% v/v) soaked muslin cloth followed by washing in tween-20 (2% v/v) for 10 min. After it, these were subjected to surface sterilization by treating with 0.1% (w/v) mercuric chloride solution for 8 min. Subsequently, explants were thoroughly rinsed 5 times with sterilized distilled water. The explants were trimmed to a final size of 0.5-2 cm and were placed aseptically in 150 mL Erlenmeyer flasks/Magenta boxes containing 35-40 mL medium.

Callus induction
Effect of various concentrations of 2,4-D and kinetin, individually: All the three types of explants were cultured vertically on MS (Murashige and Skoog, 1962) medium supplemented with five different levels of 2,4-D (2.26, 4.52, 6.79, 9.05, 11.31 and 13.57 μM) and kinetin (2.32,4.65, 6.97, 9.29 and 11.61 μM), individually, to select out the best explant and most favoring growth regulator regime with regard to callus induction and proliferation.

The medium was fortified with 3% (w/v) sucrose and pH was adjusted to 5.8 before being solidified with 0.8% (w/v) agar. The culture vessels containing the media were autoclaved at 15 lb and 121°C for 20 min. Cultures were maintained at 25±2°C, at a photoperiod of 16 h (80 μmol m^-2 sec^-1) for 30 days. On 30th day, observations were made for % callus induction, fresh weight of callus and callus morphology. Though dry weight is more reliable criteria to assess the proliferation, we needed to grow more amount of callus tissue for subsequent biochemical investigation (unpublished data). So fresh weight recordings were done by placing the weighing balance in Laminar air flow chamber, under strict aseptic conditions and the cultures were then used for further sub-culturing experiments. Observations were made daily for noting the number of days required for callus initiation.

Effect of various combinations of 2,4-D and kinetin: The best responding explant, as observed in the above study, was subjected to fifteen different combinations of 2,4-D and kinetin (five levels of 2,4-D viz., 2.26, 4.52, 6.79, 9.05, 11.31 and 13.57 μM, with three levels of kinetin viz., 0.46, 0.93 and 1.39 μM, in 5x3 factorial combinations) and observations were made as reported eariler. Medium sterilization and culture conditions were as described earlier.

Effect of sucrose concentration: Retaining the best explant and two most favoring growth regulator regimes with regard to % callus induction and fresh weight of callus, two more levels of sucrose (2 and 2.5%) were tested and observations were made as reported earlier. Medium sterilization and culture conditions were as described earlier.

Callus maintenance: Calli, initiated from nodal segments were selected for long term growth because of maximum proliferation as observed after one month of culture. Out of twenty five media (having different concentration and combinations of growth regulators) used for callus induction study, two were selected for callus maintenance study because of high fresh weight of callus observed on them after one month of culture.

Calli were routinely sub cultured for consecutive eight months, each sub culture after a regular interval of 30 days, on the two selected media formulation. Calli pieces weighing 20-25 mg (fresh weight), obtained by division of larger callus, were inoculated on fresh medium and allowed to proliferate for 30 days. On 30th day, fresh weight increase (fwi) was calculated as the difference between the final and the initial fresh weight. Fresh weight recording was done under aseptic conditions.

Experimental design and statistical analysis: All the experiments were conducted using a Complete Random Design (CRD). There were 10 replicates per treatment and each treatment was repeated in three sets. Data were subjected to analysis of variance (ANOVA) using the SPSS version 12. The significant differences among the treatments were compared using Duncan’s Multiple Range Tests (DMRT). Treatment values were represented as the Mean±SE.

RESULTS
Callus induction
Effect of various concentrations of 2,4-D on the three explants: Inter-nodal segments and shoot apices did not respond on any of the five concentrations of 2,4-D. The two way ANOVA for the differences among various concentrations of 2,4-D and among experimentally replicated sets with regard to % callus induction, number of days for callus induction and fresh weight of callus indicated that all the three responses from nodal segments were significantly affected by concentration of 2,4-D (p<0.01).

The various concentrations of 2,4-D used in this study did not favor the callus induction to the optimum level. The nodal segments responded with the maximum frequency of only 20% on 6.79 μM 2,4-D, significantly not different to the frequency of 18.33% observed on 4.52 μM level (Table 1). Frequency of callus induction was quite low (10%) on 2.26, 9.05 and 11.21 μM concentration of 2,4-D. Callus initiation from the nodal segments was observed from the lower cut portion, after a minimum of 19.636 days and 20.591 days on 6.79 μM and 9.05 μM 2,4-D, respectively significantly earlier than that on other concentrations (Table 1). Maximum proliferation resulting in significantly higher fresh weight, of 0.4064 g, callus with greenish white color, was observed after thirty days of culture on 6.79 μM 2,4-D (Fig. 1a), while on other concentrations of 2,4-D significantly lower proliferation was observed, as interpreted by DMRT (Table 1).

Effect of various concentrations of kinetin on the three explants: The three-way ANOVA indicated that the three responses were significantly affected by the concentration of kinetin (p<0.01) as well as the nature of explant (p<0.01). At each level of kinetin, nodal segments responded with high frequency of callus induction followed by inter-nodal segments and shoot apices (Table 2).

For nodal explants, maximum frequency of callus induction (88.33%) was observed on 6.97 μM kinetin, significantly higher from that on other concentrations, as analyzed by DMRT (Table 2). Kinetin, at 6.97 μM, was also observed as the most productive level for inter-nodal and shoots apices, in terms of maximum % callus induction (Table 2). The mean number of days required for callus initiation from nodal segments was significantly low (22.60 days) on 6.97μM kin, amongst all the five concentrations.

Table 1:	Percent callus induction, fresh weight of callus, after thirty days of culture and number of days for callus initiation recorded from nodal segments on various concentrations of 2,4-D
Means within columns followed by the same letters are not significantly (p<0.05) different

Fig. 1:	Callus, initiated from nodal segments, after 30 days of culture on MS medium supplemented with (a) 6.79 μM 2,4-D, (b) 6.79 μM kin and (c) 9.05 μM 2,4-D + 0.93 μM kin

Table 2:	Effect of various concentrations of kinetin, on the three explants, with regard to frequency of callus induction, fresh weight of callus, after 30 days of culture and number of days for callus initiation
Means within columns followed by the same letters are not significantly (p<0.05) different

On higher and lower concentration than 6.97 μM kin, the number of days for callus initiation was significantly higher than that observed on it. Same situation was observed for inter-nodal segments and shoot apices. Callus was initiated from the lower cut portion, touching the medium, of each of the three explants and was light greenish in color. Callus, initiated from nodal segments, proliferated more as compared to the callus from inter-nodal and shoot apices at each level of kinetin, as indicated by fresh weight recordings (Table 2). For nodal segments, maximum proliferation resulting in 2.075 g of callus was observed on 6.97 μM kinetin (Fig. 1b) and this was significantly not different to fresh weight of 2.03 g observed on 9.29 μM kinetin (Table 2). Inter-nodal and shoot apices initiated calli proliferated to a maximum of only 0.485 and 0.414 g, respectively both on 6.97 μM kinetin, after one month of culture.

Effect of different combinations of 2,4-D and kinetin: Since in the above two studies, nodal segments responded far better than inter-nodal and shoot apices (both in terms of frequency of callus induction and proliferation), only nodal segments were subjected to different combinations of 2,4-D and Kinetin. Two-way ANOVA indicated that the response of nodal segments was significantly affected by the combination type (p<0.01).

Among fifteen combinations, maximum of 100% callus induction was observed on medium supplemented with a combination of 9.05 μM 2,4-D and 0.93 μM kinetin (Fig. 2). For each of the three levels of kinetin, frequency of callus induction was observed maximum with 9.05 μM 2,4-D (Fig. 2). Significantly lower frequency was observed on combinations having 2.26 μM 2,4-D (lowest concentration used) with each of the three concentration of kinetin. Quite similar frequencies of callus induction were observed on media having quite dissimilar ratio of 2,4-D to kinetin (Fig. 2).

Fig. 2:	Frequency of callus induction from nodal segments on various combinations of 2,4-D and kinetin, as observed after 30 days of culture (Bars on the columns represent the standard error)

Fig. 3:	Fresh weight of callus (g), initiated from nodal segments, on various combinations of 2,4-D and kinetin, as observed after 30 days of culture (Bars on the columns represent the standard error)

Fig. 4:	Number of days for callus initiation from nodal segments on different combinations of 2,4-D and kinetin (Bars on the columns represent the standard error)

A similar trend was also observed for callus fresh weight increase, observed after one month of culture (Fig. 3). Significantly higher fresh weight callus mass of 2.556 g, of light green color, was observed after one month of culture on combination of 9.05 μM 2,4-D with 0.93 μM kinetin (Fig. 1c). Callus was observed to be initiated earliest (9.283 days) on combination of 9.05 μM 2,4-D with 0.93 μM kinetin, while in general the number of days required for callus initiation was significantly lower on 9.05 μM 2,4-D, with each of the three levels of kinetin, compared to other combinations (Fig. 4).

Effect of sucrose concentration: Retaining the best explant (nodal segments) and two media formulation (one with 6.97 μM kinetin alone and other with 9.05 μM 2,4-D in combination with 0.93 μM kinetin), with regard to maximum frequency of callus initiation and subsequent proliferation as reported in the previous sections, two lower levels of sucrose (2.5 and 2%) were used for studying the effect on the above said responses (Table 3). The aim was to know whether at lower level we could achieve considerably similar/higher %callus induction with same or reasonably higher proliferation.

When the responses on 2.5 and 2% sucrose were compared to that on control (3%) for both the medium formulations, the %callus induction did not differ significantly on 2.5% sucrose while on 2% level, it was significantly lower. On the other hand, fresh weight of callus (after four weeks of culture) was found to be significantly different on all the three levels of sucrose, highest being for 3%. On 2% sucrose, callus proliferation was lowest and the callus obtained was white in color with friable appearance. Day for callus initiation was not affected significantly by the change in sucrose concentration (Table 3).

Callus maintenance: Calli, obtained from nodal segments were maintained for consecutively nine months, on the two medium formulations (one with 6.97 μM kinetin alone and other with 9.05 μM 2,4-D in combination with 0.93 μM kinetin), each with 3% sucrose (as it was observed to be most optimal concentration for callus proliferation), to define the best conditions for long term maintenance and proliferation of callus cultures of T. peruviana (Table 4).

Table 3:	Percent callus induction, fresh weight of callus, after thirty days of culture and number of days for callus initiation on different levels of sucrose
Means within columns followed by the same letters are not significantly (p<0.05) different

Table 4:	Fresh weight (g) of callus as observed on 30th day of each sub-culturing, on different media formulations
Means within columns followed by the same letters are not significantly (p<0.05) different

Fig. 5:	Callus proliferation on MS medium supplemented with 6.97 μM kinetin after (a) Fifth sub-culture and (b) Eight sub-culture

Fig. 6:	Callus proliferation on MS medium supplemented with 9.05 μM 2,4-D+0.93 μM kinetin after (a) Fifth sub-culture and (b) Eight sub-culture

It was observed that calli, maintained on MS medium supplemented with 0.93 μM kinetin along with 9.05 μM 2,4-D, proliferated with an average monthly fresh weight of 2.361 g, while on other medium (MS supplemented with 6.97 μM kinetin alone) the average monthly fresh weight of callus was observed to be significantly low, being 1.19 g (Table 4). Also, on 6.97 μM kinetin, the fresh weight of callus decreased gradually after each sub-culture, while on other medium (0.93 μM kin+9.05 μM 2,4-D) a nearly consistent value of fresh weight of callus was observed after each subculture (Table 4). Callus morphology was also found to be influenced by nature of growth regulators. Calli, on kinetin supplemented media, became whiter in color with each sub-culture (Fig. 5a, b). On the other hand, calli, sub-cultured on medium having both 2,4-D and kinetin were light green in color till the last subculture (Fig. 6a, b).