Abstract
Objective: Exacum bicolor Roxb. is an endangered medicinal herb due to overexploitation by humans and its inefficient vegetative reproduction. Here, we report an efficient and simple procedure for the regeneration of E. bicolor Roxb. using leaf as an explant.
 Methods: The optimal concentrations of the hormones needed for callus induction were determined by full factorial method using DOE (Design expert ver. 8.0). The hormones selected based on literature were kinetin, indole acetic acid, and 6-Benzylaminopurine (BAP). Multiple shoot regeneration was carried out in liquid and solid media with the optimal concentrations of the hormones obtained by DOE. Rooting was initiated using Murashige and Skoog media containing naphthalene acetic acid 0.5 mg/l, indole butyric acid (IBA) 1.0 mg/l, and gibberellic acid 3 0.5 mg/l along with 0.2% of activated charcoal.
 Results: Analysis of full factorial design run showed that BAP in combination with kinetin was effective for the growth of callus and multiple shoot regeneration was higher in liquid media (81.25%). The rate of rooting was observed to be 88.23% and the average number of roots was 0.26. Plantlets with budding apical region and well-established leaves and roots were observed in 30 days.
 Conclusion: The protocol reported here can be used for effective production of E. bicolor plants in a shorter duration compared to the conventional approach.
Highlights
Plants of genera Exacum (Gentianaceae) are well known for their ornamental [1,2] and medicinal properties [3,4,5]
The Murashige and Skoog (MS) media and the hormones naphthalene acetic acid (NAA), indole butyric acid (IBA), BAP, and kinetin were procured from HiMedia, India, and gibberellic acid 3 (GA3) was procured from LOBA Chemie, India
Rooting Individual shoots (>1.2 cm height) obtained from liquid culture were cut off and transferred into rooting media, namely, MS media containing NAA 0.5 mg/l, IBA 1.0 mg/l, and GA3 0.5 mg/l and 0.2% of activated charcoal
Summary
Plants of genera Exacum (Gentianaceae) are well known for their ornamental [1,2] and medicinal properties [3,4,5]. These plants are mostly found in the temperate and subtropical regions and include about 1738 species. There has been a rapid decline of its population due to loss of its habitat. The approach currently being used to increase their population is by seed germination. It is difficult to reproduce these plants due to the high rate of seed dormancy [7,9,10]
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