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Keywords:
n Colebrookean , Micropropagation, Shoot induction, LCMS, DNA barcoding, Genetic fidelity
In the present study, an attempt was made to optimize the micropropagation protocol for Colebrookea oppositifolia Sm. via direct organogenesis using axillary buds. We also investigated the effect of different concentrations of gibberellic acid (GA3) on seed germination. A very low seed germination percentage was observed in this plant. Murashige and Skoog (MS) medium with 1 mg/L thidiazuron (TDZ) recorded the highest shoot induction percentage of 91.66±1.66a. Further, shoot multiplication was seen to be highest with 7.85±0.057a number of shoots per explant on MS + 2 mg/L 6-benzylaminopurine (BAP) + 0.1 mg/L thidiazuron (TDZ) medium. For rooting, MS medium supplemented with various auxins was used, and the best rooting response with 12.97±0.093a average numbers of roots per shoot and mean root length of 1.205±0.015a cm was seen in half MS medium with 1 mg/L indole-3-butyric acid (IBA). The in vitro raised plantlets were transferred to pots containing the mixture of soil:sand:vermiculite in the ratio of 2:1:1 for hardening. Established plants were shifted to green house and then under field conditions with 90% survival rate. Liquid chromatography mass spectroscopy (LCMS) analysis of tissue culture raised plants marked the presence of therapeutically important plant metabolite, acetoside. DNA barcoding was done to authenticate the explants and tissue culture raised plantlets and sequences were submitted to the GenBank (MZ343371 and MZ343373, respectively). Further, the genetic stability of micropropagated plants were also determined using random amplified polymorphic DNA (RAPD) primers.
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RC is thankful to CSIR (Council of Scientific and Industrial Research) for Senior Research Fellowship. SGG acknowledges CSIR-IIIM (Indian Institute of Integrative Medicine) for support in carrying out the experiments and compilation of the manuscript.