Adaptation to ex vitro conditions of Stevia rebaudiana (Bertoni) Hemsl. regenerants
Abstract
L.P. Khlebova, A. Orazov, A.M. Titova, A.V. Pirogova
Stevia rebaudiana (Bertoni) Hemsl. belongs to Asteraceae family and is of great importance for pharmaceutical and food industries. Stevioside obtained from the leaves of this plant is regarded as a valuable natural sweetener. Low seed fertility is one of the most important problems in stevia production. It multiplies almost exclusively in a vegetative way. Plant tissue culture is an efficient method for mass propagation of S. rebaudiana. We studied the effect of various concentrations of auxins on rooting stevia shoot cuttings under in vitro conditions. We found that adding 0.6-1.0 mg l-1 IBA or 0.2 mg l-1 IAA to the В5 medium is effective forrooting the shoot fragments of this species. The regenerants were adapted to ex vitro conditions for 3 weeks on a hydroponic setup filled with a solution of mineral salts according to the quarter-strength Murashige and Skoog (MS) basal medium modified by the content of KH2PO4 and NH4NO3. Using a triple concentration of KH2PO4 (510 mg l-1) during the first week of adaptation and a fullconcentration of NH4NO3 (1650 mg l-1) over the next 2 weeks ensures 100% acclimatization of stevia regenerants to ex vitro conditions. The replacement of agar in the nutrient medium with a perlite-vermiculite mixture in the ratio of 1 : 3 stimulated the transition of regenerants to the photomyxotrophic type of nutrition. The use of a porous substrate provided a decrease in humidity inside the culture vessels, which led to forming both leaves with well-functioning stomata and a branched root system with root hairs. The stevia regenerants propagated in vitro on a porous substrate did not require special conditions for the ex vitro acclimatization. The yield of surviving plants in the greenhouse was 100%.
Key words: Stevia rebaudiana Bertoni; Natural sweetener; In vitro propagation; Auxin; Rooting; Acclimatization; Porous substrate; Photomyxotrophic micropropagation
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