Abstract
An efficient high frequency plant regeneration protocol through direct organogenesis was developed for Sevia rebaudiana Bert. Nodal segments containing axillary buds were used as an explant and inoculated on Murashige and Skoog’s (MS) medium containing 3% (w/v) sucrose, 0.8% (w/v) agar supplemented with various concentrations of benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) ranging from 1.00 to 9.00 μM. Maximum multiple shoots (96%) were obtained in MS medium supplemented with 1.0 μM TDZ with an average of 60 shoots per culture, having an average shoot length of 6.0 cm. The best in vitro root induction (89%) was achieved on half strength MS medium without any growth regulator with an average of 24 roots per culture and root length of7 cm. The rooted plantlets were successfully established in soil and grown to maturity at the survival rate of 95% in the indoor grow room. High-performance liquid chromatography was used to assess the stability in chemical profile and quantification of stevioside and rebaudioside A content of in vitro propagated S. rebaudiana plants and compared with their mother plant at the peak vegetative stage. Our results show no significant differences (p in vitro propagated plants. Furthermore, fully developed in vitro propagated S. rebaudiana plants were also compared with mother plant for their gas and water vapour exchange characteristics and leaf anatomy. The results show that in vitro propagated and hardened plants of S. rebaudiana are morphologically as well as functionally comparable to each other and to their mother plant.
Highlights
Stevia rebaudiana Bert. (Asteraceae) commonly known as a natural sweetner, is a zero calorie value natural alternative source to traditional sugar obtained from sugarcane, sugarbeet [1]
Nodal segments containing axillary buds were used as an explant and inoculated on Murashige and Skoog’s (MS) medium containing 3% (w/v) sucrose, 0.8% (w/v) agar supplemented with various concentrations of benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) ranging from 1.00 to 9.00 μM
Content Since stevioside concentrations in the S. rebaudiana leaves are reported to be maximum at peak vegetative stage i.e. just before flowering [18], leaf samples from mother plant and fully developed in vitro propagated plants were collected at that stage for the comparison
Summary
Stevia rebaudiana Bert. (Asteraceae) commonly known as a natural sweetner, is a zero calorie value natural alternative source to traditional sugar (sucrose) obtained from sugarcane, sugarbeet [1]. (Asteraceae) commonly known as a natural sweetner, is a zero calorie value natural alternative source to traditional sugar (sucrose) obtained from sugarcane, sugarbeet [1]. It is native from Paraguay and Southern Brazil, but is cultivated on a commercial scale in other countries, Japan, Taiwan, Korea, Thailand and Indonesia [2]. 118 TDZ-Induced High Frequency Plant Regeneration through Direct Shoot Organogenesis in Stevia rebaudiana Bertoni: An Important Medicinal Plant and a Natural Sweetener baudiana. We report an efficient high frequency in vitro regeneration protocol using TDZ. Metabolic profiling (HPLC analysis), eco-physiological (photosynthesis, transpiration, stomatal conductance, internal CO2 concentration and water use efficiency) and leaf anatomical (stomatal length, width, frequency and index, leaf transverse section, epidermal cells, palisade cells and spongy parenchyma cells) characteristics were studied to determine the differences, if any, exists between mother plant and in vitro propagated and hardened S. rebaudiana plants
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