Optimization of micropropagation for commercial medicinal plant of Stevia (Stevia rebaudiana) through in vitro culture of shoot tip explant

Stevia (Stevia rebaudiana) is the sweetest plant in the world that is considered as suitable alternative to sugar for treating problems of obesity and diabetes, but its seeds are hard to germinate and most of them are often sterile due to incompatibility phenomenon during their formation. Also, it is difficult to produce homogenous population by seed propagation. For this reason, tissue culture is suitable approach for fast multiplication of this plant. The aim of this research was to find a rapid and efficient protocol for micropropagation of commercial medicinal stevia and mass propagation of its clones in large scale. This research was conducted in two separate experiments. In the first experiment, the shoot tip explants were cultured on MS media supplemented with 0.5, 1, 1.5 and 2 mg/l concenteratians for each of plant growth regulators include IAA, NAA, IBA, BAP, Kin and GA3 as individual treatments with control treatment (MS medium without plant growth regulators). The experiment was carried out in a completely randomized design with four replications. The second experiment was designed based on the results of the first experiment. So, in the second experiment, the shoot tip explants were cultured on MS media supplemented with BAP (0, 1, 2, 3 and 4 mg/l) and Kin (0, 0.5, 1 and 1.5 mg/l). The tratment was arranged as factorial experiment in a completely randomized design with three replications. In rooting experiment, shootlets were cultured on MS and 1/2 MS medium supplemented with different concentrations of NAA (0, .5, 1 and 1.5 mg/l). This expriment was carried out in the same way as the second expriment. In all experiments, the traits were measured after 30 days of culture and incubation in growth chamber. Finally, the rooted plantlets were initially hardened to culture room conditions and then transferred to greenhouse. The effect of plant growth regulators was significant for all measurd traits (P<0.01); MS medium containing 1.5 mg/l BAP was the most efficient treatment for the highest shoot number and internode number (with average of 5.75 shoot and 19.5 internode per plant, respectively) and the longest shoot length related to 2 mg/l Kin and 1.5 & 2 mg/l GA3 and The best treatments for callusing were 1.5 mg/l IBA, 1.5 & 2 mg/l BAP. In the second expriment, the interaction effect of BAP×Kin was significant for all traits. The highest shoot multiplication rate and internode number were obtained from MS medium supplemented with 1 mg/l BAP + 1.5 mg/l Kin (with average of 17.67 shoot and 38.67 internode per plant, respectively). This treatment was incubated in growth chamber for another one month and the shoot number was significantly increased from 17.67 to 55 shoot per plant. Most efficient condition for root production (with average of 55 root per plant) was observed on 1/2 MS medium containing 1.5 mg/l NAA. About 92% of in vitro developed plantlets were successfully established to greenhouse conditions. general, the results, like previous studies, indicated a positive and good response of the stevia plant to tissue culture. MS medium supplemented with 1 mg/l BAP + 1.5 mg/l Kin was recommended for shoot proliferation of Stevia shoot tip explant and also, incubation of cultured explants in growth chamber for two months was remarkably increased the shoot number. The 1/2 MS medium containing 1.5 mg/l NAA was resultful in root proliferation.