Subjecting the ray florets to different pre-treatments resulted in a marked influence on culture establishment. Data presented in the Table 1 indicated that the pre-treatment of explants with different concentrations of fungicides namely carbendazim, mancozeb and biocide, i.e. 8-HQC significantly reduced the microbial load and improved the survival percentage as compared to control (distilled water dip). The pre-treatment of ray florets with mancozeb-45 (0.1%) + carbendazim (0.1%) + 8-HQC (200 mg/L) for 3 hours was found to be the best with regards to minimum contamination (15.56%) with highest survival (89.44%) of explants when compared to the other treatments and control. Pre-treatment with mancozeb-45 (0.2%)+carbendazim (0.2%) + 8-HQC (200 mg/L) for 3 h recorded the 8.89% microbial contamination with 75.56%survival which were statistical lower than the mancozeb-45 (0.1%)+ carbendazim (0.1%)+8-HQC (200 mg/L) for 3 hours. The present findings are in conformity with the Bala et al (2010) in rose who found the carbendazim (0.2%)+diathane M-45 Indofil® (0.2%)+ 8-hydroxy quinnoline citrate (200 mg/L) for 3 h agitation gave the highest explant survival (62.47%) and bud sprouting (56.63%). Kadam et al (2010) find the best response by pre-treated thepetals and immature flower of tuberose with carbendazim 0.1%+ mancozeb 0.1% and 8-HQC (200 mg/L) for 2½ h.

1.2 Standardization of surface sterilization for explants

Perusal data presented in Table 2 revealed that surface sterilization of explants with 0.1 % HgCl₂ for 4 min resulted in lower contamination percentage (15.56) along with maximum survival (91.04%) over control (10.00%) and other treatment. Surface sterilization of explants with HgCl₂ (0.1%) for a duration of 6 minutes significantly reduced microbial contamination (7.78%) but also reduced the survival percentage (51.77%) and caused drying of explants within 10 days of inoculation which may be due to phytotoxicity caused by long exposure of explants with HgCl₂. All the treatment was statistically significantly different to each other. Verma et al (2011) also reported that surface sterilization with 0.1% HgCl₂ gave the highest survival percentage and lower the microbialcontamination in stevia which were quite close to our findings. In another report nodal explants washed with household detergent to remove the impurities and then washed thoroughly under tap water and then sterilization was done with 70% alcohol for 2 minutes followed by 1% HgCl₂ for 2~3 minutes (Ilahi et al., 2007).

The wounded ray florets culture on MS medium fortified with different concentration of BAP and NAA for the regeneration. The data presented in Table 3 showed that MS medium supplemented with BAP and NAA significantly enhanced shoot regeneration percentage from the ray florets over the control (MS medium devoid of hormones). Significantly higher shoot regeneration (93.33%) was observed on MS medium supplemented with BAP (4 mg/L) + NAA (0.1 mg/L) than those on MS + BAP (4 mg/L) + NAA (0.5 mg/L) (71.11%) followed by MS + BAP (3 mg/L) + NAA (1.0 mg/L) (66.67%) and MS + BAP (3 mg/L) + NAA (0.5 mg/L) (51.11%). The highest (5.67) number of microshoots in minimum days (30.67) was observed on MS medium supplemented with BAP (4 mg/L) + NAA (0.1 mg/L) which were statistically significant with other treatment. These observations are quite close to the recommendations of other workers in chrysanth- emum (Kumar et al., 2004; Latado et al., 2004) who regenerates microshoots from the chimeric ray florets of chrysanthemum. Mechanical wounding in leaf explants by brushing the leaf surfaces was shown to increase the shoot regeneration capacity in D. grandiflora (de Jong et al., 1993) therefore we also cultured the wounded ray florets and find the earliest regeneration from the wounded parts of the ray florets. Medium supplemented with higher concentration of BAP in combination with low concentration of NAA initiates differentiation process following adventitious bud and shoot initial formation. It is well known that cytokinins accelerate the cell division process in the first step of their application and, then, large amount of callus is formed followed by differentiation of shoot initials, organogenesis and shoot regeneration.

1.4 Effect of GA₃ on elongation
The regenerated microshoots individually transferred to elongation media consisting of basal MS medium supplemented with various concentrations of GA3 significantly affected the growth of the shoots and made them elongated, with thick, sturdy and strong stem and well developed dark green expended leaves. In this process, the micro shoots attained an optimum height so that they could be transferred for rhizogenesis. Maximum (5.55 cm) shoot length was recorded after 30 days of culturing on MS medium supplemented with 0.50 mg/L GA₃ which was considerably higher than those cultured on MS medium supplemented with 0.25 mg/L GA₃ (3.88 cm) and 1.0 mg/L GA₃ (3.48 cm) (Figure 1). All these treatments were statistically significant with each other. The MS medium devoid of hormones (control) showed minimum (2.28 cm) length of microshoots. Shruti et al (2011) study the effect of GA₃ on shoot elongation of in vitro grown stevia and found that medium containing 0.5 mg/L GA₃ showed maximum elongation (3.5). Elongated microshoots reduce the chance of mortality during the rooting and hardening processes. Gibberellins are known for inducing stem elongation in a number of crops. The elongation of the stems is not due to increased formation of nodes and internodes but results from rapid elongation of internodes, which is due to both cell division and cell elongation. Gibberellic acid is involved in several important biochemical and morphogenetic responses which include the promotion of elongation in axial organs, such as stems and flower pedicels, along with the stimulation of root growth (Srivastava, 2005).

1.5 Acclimatization of plantlets

For obtaining the high success during the plantlet acclimatization, plantlets transferred in glass jars with polypropylene lids filled with peat + soilrite (1:1) and moistened with half-strength MS medium (devoid of growth regulators, calcium, organics and sucrose) was found to be the best in terms of maximum survival percentage (95.00), plant height (22.45 cm), number of leaves per plant (31.50) and minimum days taken for transfer to greenhouse (21.00) (Table 4). After 20~25 days of acclimati- zation the plants ready to transfer in the green house. These results are in line with the earlier workers in chrysanthemum (Mandal and Datta, 2005; Nahid et al., 2007).