The M. lunu-ankenda plants grown and maintained in a glass house was used as explant. Glass-house conditions were maintained at temperatures 29˚C ± 3˚C with a 12L:12D photoperiod and the relative humidity fluctuated between 50% - 70%. Lateral shoots segments were used to initiate shoot cultures. Explants were cleaned in a detergent (Teepol) solution for 20 min and rinsed in distilled water. Surface sterilization was preceded by immersed in 20% (v/v) Clorox^® containing several drops of Tween-20 for 20 min and repeated for another 20 min in 5% (v/v) concentration. Subsequently, rinsed with sterilized distilled water few times. The death tissue were excised and shoot explants were inoculated onto MS medium supplemented with 3% sucrose and 1.0 mg/L BAP. The medium was adjusted to pH 5.8 prior solidified with 0.3% (w/v) gelrite and sterilized by autoclaving at 121˚C and 104 kPa for 15 min. All cultures were incubated in culture room under white light provided with white fluorescent light at intensity of 3000 lux at a photoperiodic 16 h. The room temperature was maintained at 25˚C ± 2˚C. The established in vitro plantlets were sub-cultured every three month interval Figure 1(h). These plantlets was used as explant for callus induction.

Under aseptic condition, stem of the in vitro plantlets were excised in 1 - 2 cm length and inoculated onto callus initiation medium. The medium consisted of MS [9] basal medium, 3% (w/v) sucrose, 3 g/L phytogel and 2,4-D at concentrations of 0, 0.5, 1.0, 3.0 or 5.0 mg/L or NAA at concentrations of 0, 1.0, 3.0, 5.0 or 10 mg/L, or combination of both phytohormones (Table 1). All cultures were maintained under light (1200 lux) and monitored weekly. The percentage of explants produced callus and day of callus formed were scored as amount of callus produced at every seven days intervals. Callus induction success was expressed as Percentage of explants induce

callus and weight of green callus after second sub-culture (two months).Any further changes in the coloursof the callus were also recorded.

The yellow-green and dark-green calli were used for induction of shoot and plant regeneration. A small pieces of callus (app.3 gram) were aseptically transferred onto regeneration medium consisted of MS [9] basal medium, 3% (w/v) sucrose, 0.3% (w/v) phytogel and phytohormones; BAP at concentrations of 0, 0.5, 1.0, 3.0 or 5.0 mg/L singly or in combination with NAA at concentrations of 0.5 or 1.0 mg/L, or with 2,4-D at concentrations of 0.5 or 1.0 mg/L, respectively (Table 2). All cultures were maintained under a 16 h photoperiod with lighting provided by cool-white fluorescent lamps (100 - 2000 lux). The percentage of calli regenerated to plant and number of plantlets or shoots per clump of green callus were recorded after 45 days of culture. The completely regene-

rated plantlets were separated to single plantlet and transferred to MS basal medium containing 0.5 mg/L IBA for root induction. After shoots, the rooted and healthy plantlets were individually transplanted to plastic pots containing hardening medium consisted of top soil:compost mixture (2:1). The plantlets were kept under controlled environment in a net house with 75% shading for 60 days. The survival rate of plantlets was recorded during the period of acclimatization.

The data (20 replicates per treatment) were subjected to one way analysis of variance (ANOVA) to assess treatment differences and interaction using SPSS version 11.0 software. Significance of differences between means was tested by Duncan’s Multiple Range Test (p < 0.5).

The effect of different phytohormone on callus induction from stem of M. lunu-ankenda were successfully

established (Table 1). Yellow to green, friable, non-embryogenic callus were produced from the wounded side of the stems within a month of culture on 2,4-D alone and in combination with NAA (Figures 1(a)-(c)). The best response of callus induction (75%) was observed in 1.0 mg/L of 2,4-D + 10 mg/L of NAA with dark-green colour of calli with the highest percentage (30%) in 1.0 mg/L 2,4-D. This was followed with 1.0 mg/L of 2,4-D + 5.0 mg/L of NAA (dark-green callus) and 3.0 mg/L of 2,4-D + 1.0 mg/L of NAA (yellow-greenish), which 65 and 55%, respectively. These results are in conformity with some of the earlier studies on different Rutaceae, which showed good callus induction response under the influence 2,4-D in combination with other phytohormone [10] [11] . Interestingly, 2,4-D combined with NAA at high concentrations (>3 mg/l) was enhanced the size or biomass the green colour of the calli. Green colour normally due to the formation of photosynthetic apparatus, the chlorophyll. A similar phenomena was previously reported in few plant species [11] .

The greenish callus were successfully regenerated to plantlets (Figures 1(d)-(g)). The highest percentage of plant regeneration (80%) and the highest number of shoot per calli clump (31 shoots per 3 gram callus) were obtained in 1.0 mg/L BAP (Table 2). In BAP (1.0 mg/L) + NAA or 2,4-D with increasing concentrations (0.5 - 1.0 mg/L), shown a decreases in the plant regeneration as well of number of shoot produced. A similar response was reported [10] on shoot regeneration of citrus callus a member of Rutaceae family. The in vitro rooted plantlets transplanted into plastic pots in 75% shading net house were exhibited 98% survival rate after 60 days of transplanting (Figures 1(h)-(i)).