EFFECT OF SUCROSE AND MEDIA STRENGTH ON IN VITRO MULTIPLICATION IN SWERTIA CHIRATA BUCH.-HAM EX WALL: AN ENDANGERED MEDICINAL HERB Original Article MANU PANT * , PRABHA BISHT ** , MANJU P. GUSAIN *** * Department of Life Sciences, Graphic Era (Deemed to be University), 566/6, Bell Road, Clement Town, Dehradun 248002, Uttarakhand, India, ** Tissue Culture Lab, G and TP Division, Forest Research Institute, Dehradun, Uttarakhand, 248006, India, ** Received: 26 Jul 2018 Revised and Accepted: 26 Sep 2018 Department of Zoology and Biotechnology, HNB Garhwal University, Srinagar Garhwal, Uttarakhand, India Email: himaniab@gmail.com ABSTRACT Objective: The present study was performed to investigate the role of varying concentrations of carbohydrate source and strengths of nutrient medium in growth and development of in vitro shoots of Swertia chirata-an endangered medicinal plant. Methods: MS medium supplemented with 6-Benzylaminopurine (4.44 µM), Indole-3 acetic acid (2.85 µM) and Adenine sulphate (271.45 µM) was used to test the efficiency of of sucrose at concentrations of 1-5% and of media strength varying from full to one-fourth. The data were analysed using analysis of variance (ANOVA) of Completely Randomized Design (CRD) in GenStat 5 Edition 3.2 for PC/Windows NT (Copyright 1995, Lawes Agricultural Trust (Rothamsted Experimental Station)) Results: Observations on axillary shoot multiplication indicated that sucrose at a concentration of 3% and MS medium in its full strength proved to be most optimal for in vitro culture multiplication. On this medium combination mean number of 11.80 shoots (after 4 w) and 18.50 shoots (after 8 w) could be obtained On sucrose free medium the shoots exhibited necrosis while at lower concentrations of 1-2% sucrose, the shoots developed were thin and unsuitable for further growth in vitro. At higher levels of sucrose in the medium, the shoots became thick and stunted. Similarly, reduction in medium strength resulted in a decline in shoot number and shoot length to an average of 6.50 shoots (1.33 cm mean length) on half strength medium and 5.60 shoots (0.88 cm mean length) on one-fourth strength; as observed after 4 w. Conclusion: The experimental findings suggest that any decline from the standard had a significant effect on the number, size and overall health of shoots developed in vitro. The conditions so standardized augment the production of healthy shoots that shall aid in subsequent rooting and survival after transplantation of tissue-culture raised plantlets. Keywords: Swertia chirata, In vitro propagation, Medicinal plant, Shoot length, Shoot multiplication © 2018 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open-access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) DOI: http://dx.doi.org/10.22159/ijpps.2018v10i11.28135 INTRODUCTION Swertia chirata is a native of temperate Himalaya and widely acknowledged as a medicinal herb to cure chronic fever, anaemia, blood, liver, lung and skin disorders [1-4]. The bitter active ingredients of the plant viz. amarogentin, xanthones, iridoid glycosides, mangiferin and C-glucoflavones [5-7] account for its wide range of medicinal and curative powers. The plant, therefore, enjoys a good national and international market. However, Swertia chirata has now been categorized as critically endangered [8-11] owing to unscientific collection practices from the wild; aided by poor seed germination rate in nature (only 2-4%) and low seed viability [12- 14]. Consequently, development of alternative propagation strategies for this important medicinal plant becomes the call of the hour. Some studies have already been performed to develop large-scale propagation protocol for S. chirata via tissue culture technology. However, there is a lacuna in studies to standardize different physical parameters involved in the propagation of plant in vitro. The present work was undertaken to investigate the effect of different carbohydrate concentrations and medium strength on in vitro shoot multiplication of S. chirata. The findings shall ensure optimal utilization of resources in establishing a standardized system for maximal production of healthy plantlets of Swertia chirata. MATERIALS AND METHODS Chemicals and reagents Standard analytical grade chemicals of Merck, India, and Hi-media laboratories, India were used in the present study. The culture media comprised of inorganic salts, organic compounds, amino acids, plant growth regulators, carbohydrate source and gelling agents. All chemicals used for stock preparation were of analytical grade. All major and minor salts components of MS medium used were of Hi-media laboratories, India. Plant growth regulators, adjuvants, sucrose (carbohydrate source), agar (gelling agent) used were also of Hi-media laboratories, India. Auxins, cytokinins used were dissolved in dilute 1.0 N NaOH or 1.0 N HCl. General methodology In vitro cultures established via axillary bud proliferation and multiplying on MS medium containing BAP (4.44 µM)+IAA (2.85 µM)+Ads (271.45 µM) (Pant et al. 2010) were used to assess the effect of varying concentrations of sucrose and media strength on shoot multiplication in Swertia chirata. For the purpose, shoots were separated from established cultures and transferred to MS medium fortified with PGRs and gelled with 0.7% agar. Sucrose was used as the carbohydrate source at concentrations of 1-5% in the multiplication medium while MS medium strength was tried at three levels: (1/4 X, 1/2 X, 1X) to standardize conditions for best results of in vitro multiplication. In all experiments, the pH of the medium was adjusted to 5.8 prior to autoclaving the medium at 121 °C and 1.0 x 10 5 Data collection and analysis Pa for 15 min. Observations on average shoot number and average shoot length were recorded after an interval of 4 w and 8 w. All experiments were repeated thrice. Each treatment consisted of minimum ten replicates. The data were analysed using analysis of variance (ANOVA) of Completely Randomized Design (CRD) in GenStat 5 Edition 3.2 for PC/Windows NT (Copyright 1995, Lawes Agricultural Trust (Rothamsted Experimental Station)). The significance level International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 10, Issue 11, 2018