Development of in vitro mass multiplication procedure for Stevia rebaudiana Bertoni and determination of genetic fidelity of regenerated tissue culture plants
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Date
2017
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MPUAT, Udaipur
Abstract
Stevia rebaudiana Bertoni is a perennial herb of Astereace family and its Leaves of Stevia produce diterpene glycosides (stevioside and rebaudiosides). Five superior mother plants (S36, S53, S81, S34, S37) on the basis of morphological characters were selected. Among the selected plants ‘S36’ mother plant exhibited highest leaf length (7.84 cm), leaf width (2.68 cm) and stevioside (3.4%) and rebaudioside (1.0%) content. Leaf length and leaf width was positively correlated with stevioside content so we had selected the mother plant ‘S36’ for micropropagation studies. The highest shoot induction was observed 68.3 % at 2.0 mg/l TDZ from in vivo nodal explants. Hundred per cent shoot induction was observed at 2.5 mg/l TDZ from in vitro nodal explants of selected mother plant. We observed that in vitro nodal explants were superior in shoots induction compared to in vivo nodal explants. In cold treatment of 48 hr showed highest shoot induction (25.8 shoots/explant) from in vitro leaf explant. Clumps of shoot buds were transferred to shoot multiplication media for 3-weeks. Maximum numbers of shoots were obtained from 1.0 mg/l BAP in combination with 0.5 mg/l Kin i.e. 7.3 shoots per explants. Highest mean shoot length (1.63 cm) with maximum multiplication was recorded at 1.0 mg/l BAP in combination 0.5 Kin mg/l. Highest rooting response (91.66 %) was noticed on hormone free media with quality roots, in respect to root numbers and root length. We also observed that rooting media without growth regulator induce the in vitro shoot length, significantly thereby reducing one more step for shoot elongation. Highest survival of regenerated plants (98.66 %) was recorded in cocopeat with combination of vermiculite. Randomly selected five mature hardened plants regenerated from S36 mother plant were evaluated for stevioside and rebaudioside-A content by HPLC. Results revealed that stevioside content (3.2 to 4.1) was comparatively similar to mother plant. However, the rebaudioside-A content (1.0 to 1.4) was higher in tissue culture raised plants comparative to S36 mother plant. Monomorphic banding pattern was observed for all the amplified band classes across all the micropropagated plants, along with mother plant with all the tested RAPD and ISSR markers. RAPD and ISSR markers confirmed the genetic stability of tissue cultured plants. We developed a simple, efficient and reproducible micropropagtion method for mass production of true-to-type plants which may feasible for commercial exploitation.
Description
Development of in vitro mass multiplication procedure for Stevia rebaudiana Bertoni and determination of genetic fidelity of regenerated tissue culture plants
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Citation
Singh and Sharan, 2017