Evaluation of the effect of plant growth regulators and explant type on Stevia rebaudiana micropropagation

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, callusing and regeneration of commercial medicinal stevia and mass propagation of its clones in large scale. In micropropagation experiment, the highest shoot multiplication rate and internode number were obtained from shoot tip explants in 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. The longest shoot length related to 2 mg/l Kin, 1.5 & 2 mg/l GA3. The highest diameter and weight of callus were obtained from leaf explant in MS medium supplemented with 0.5 mg/L BAP + 2 mg/l of 2,4-D (with average of 2.87 cm and 2.94 gr, respectively). In regeneration experiment, Maximum shoot induction was happened in MS medium supplemented with 3 mg/l BAP (with an average of shoot number 7.75 per explant) and 3 mg/l BAP + 1.5 mg/l Kin + 0.5 mg/l NAA (with an average of shoot number 8.67 per explant) from leaf explants and was not observed regeneration from cotyledon and inter-node explants. The best treatment for rooting of regenerated shoots was MS medium fortified with 1 mg/l IBA (with an average of root number 58.33). Also, about 92% of in vitro developed plantlets were successfully established to greenhouse conditions.