Effect of exogenous polyamines enhances somatic embryogenesis via suspension cultures of spine gourd (Momordica dioica Roxb. ex. Willd.)

An efficient method of somatic embryogenesis using exogenous polyamines through suspension culture protocol was developed from leaf derived callus of spine gourd (Momordica dioica Roxb. ex. Willd.). Embryogenic callus was originated from cut slices of leaf explants on MS solid medium supplemented with 4.4 μM 2,4-D with addition of polyamines (putrescine, spermidine and spermine). Putrescine at a concentration of 1.0 μM showed maximum increase in fresh weights of embryogenic calli. The maximum frequency of somatic embryos (30.3%) was observed on MS medium supplemented with 3% (w/v) sucrose and 3.3 μM 2,4-D for three weeks of culture. The MS liquid medium augmented with 3.3 μM 2,4-D and 0.5 μM putrescine was effective to achieve high frequency of somatic embryo induction (59.0%). Moreover addition of PAs to the embryogenic media resulted in lowering of endogenous free PA level of 21-day-old embryogenic calli. Thus, when the media was supplemented with exogenous PAs a positive correlation was found to exist between somatic embryogenesis enhancement and decrease in endogenous free PA levels. Sustained cell division resulted in the formation of cell aggregates and then progressed to globular, heart and further to torpedo and cotyledonary stages within 5 weeks. Transfer of individual embryos on to a fresh MS basal medium with no plant growth regulators was able to achieve complete maturation. Conversion of embryos into plants was achieved on 1/2 strength MS semi solid medium containing 0.5 μM gibberellic acid (GA3) and 1.5% sucrose. Twenty two percent of somatic embryos were converted into true-to-type fertile plants. Regenerated plantlets were successfully hardened, with a survival rate of approximately 76%, and established in the field.