Establishment of callus and in vitro enhancement of glucosinolates in 'malunggay' (Moringa oleifera Lam.)

The in vitro protocol for callus culture of Moringa oleifera Lam was successfully established utilizing cotyledon from green and dried mature seeds.High percentage sterile culture was obtained in green mature seeds pre-treated with fungicide prior to disinfection with 5% Calcium hypochlorite (CaOCl)solution for 30 mins (15:15 mins) following double sterilization procedure. High percentage sterile culture was also obtained in dried mature seeds by following the above procedure, except that decoated seeds were disinfected with 2% CaOCl. Final disinfection was completed with 1% streptomycin in both green and dried mature seeds. The green mature seeds were utilized for callus and short induction while dried mature seeds were used to determine the effect of pH and ascorbic acid on growth and development of malunggay seedlings in vitro. Highest percent callusing was observed in culture inoculated in MS+ 2.5 um 2,4-D + 0.5 um TDZ, which was comparable with MS basal medium containing BA(0.5-.5 um) and 2,4-D (1.0-5.0 um) singly or in combination. Loose crystallins calli were observed in media containing 2,4-D and B singly or in combination, while compact, nodular and loose calli were obtained when the culture media was further enriched with TDZ. Highest callus weight was significantly achieved in MS + 1.0 um 2,4-D + 0.5 um BA + 5.0 um TDZ. Slight greening of calli was observed 4 weeks after the first transfer onto the shoot induction media, however, not significantly different among treatments. Microscopic observation of hand-sectioned protrusions on green calli revealed heterogenous and asynchronous cell growth showing actively dividing cells. Xylem differentiation was also evident,indicating early initiation of bud initials. Results of preliminary studies on the effects of increasing pH levels (4-10) showed no significant effect on the number of root,shoot and trifoliate leaves and root length, except for pH 4 at which longest shoot was significantly observed. Highest glucose level was significantly observed in roots of seedlings of pH 10, whereas, highest glucose level was significantly obtained in 3rd trifoliate leaves at pH 4. Length of shoots and roots and the number of roots and trifoliate leaves were significantly reduced with increasing levels of ascorbic acid (2.5-15 mM),while shoot number was not affected. High glucose level was significantly obtained in roots and 3rd trifoliate leaves with 10.0 and 15.0 mM ascorbic acid, respectively. Moreover, glucose content increased with increasing level of ascorbic acid added directly in the cotyledon extracts.