MICROPROPAGATION In vitro seed germination, proliferation, and ISSR marker-based clonal fidelity analysis of Shorea tumbuggaia Roxb.: an endangered and high trade medicinal tree of Eastern Ghats Shastri Prasad Shukla 1 & Abhishek Sharma 1 Received: 9 October 2016 /Accepted: 21 March 2017 / Editor: Harold Trick # The Society for In Vitro Biology 2017 Abstract An in vitro plant tissue culture approach provides a feasible alternative for extensive mass manufacturing and con- servation of elite and critically endangered medicinal plant spe- cies. The presented work describes a proficient in vitro seed germination and proliferation procedure for the conservation of an endangered high trade medicinal tree, Shorea tumbuggaia Roxb. Given the precarious position of this species, effective conservation is highly indispensable for its continued existence, primarily for ecological reasons and also for forestry purposes as this tree species is a valuable source of timber and oleoresin. In the present study, over 90% in vitro seed germination was achieved. Multiple shoot formation was established from seed- lings with a maximum of 4.60 ± 0.30 multiple shoots per ex- plant using MS (Murashige and Skoog) medium supplemented with a combination of 6.66 μM 6-benzylaminopurine and 0.454 μM thidiazuron. Rooting of multiple shoot cultures of 85% was achieved on half strength MS medium supplemented with 2.24 μM indole 3-butyric acid with 6.48 ± 0.58 roots per plantlet. Rooted plantlets showed more than 70% acclimatiza- tion in potting conditions and were established successfully in a greenhouse, with morphological similarities to mother plants. Clonal fidelity assessment of micropropagated plants with ten inter-simple sequence repeat primers reproduced a total of 24 monomorphic bands. The presented work represents the first report of in vitro proliferation, conservation, and clonal fidelity assessment of tissue culture generated S. tumbuggaia plantlets. Keywords Shoreatumbuggaia . Invitro proliferation . Invitro conservation . Clonal fidelity . ISSR Introduction Shorea tumbuggaia Roxb. (family Dipterocarpaceae) is an endangered tree species commonly known as Bgreen dammer,^ which is endemic to Southern Eastern Ghats of India (Rani and Pullaiah 2002). This is an important tree spe- cies that is native to Southeast Asia, from northern India to Malaysia, Indonesia, and Philippines. S. tumbuggaia is listed in the IUCN Red List of Endangered Species (B1ab+2ab, Haridasan et al. 2015) and under habitat conservation with Talakona, Medicinal Plants Conservation Areas (MPCA), Tamil Nadu, India. The phylogeny, genetics, and geographical habitat of this species denote it as an important true relict. It is a semi-evergreen woody tree and occurs in clusters on moun- tain slopes and peaks, dry savannah, and mixed deciduous forest, with an average age of 15 yr (Haridasan et al. 2015). Being classified of an endangered tree species, S. tumbuggaia requires effectual conservation that is highly indispensable for its continued existence, primarily for ecological reasons and subsequently for forestry purposes as a valuable source of timber and oleoresin. The wood of S. tumbuggaia has an ample market for timber and for an oleoresin-based drug (Haridasan et al. 2015). Oleoresin is extracted from the stem bark of this species and has vital medicinal properties like alexiteric and used to cure hyperhidrosis. It is also used in indigenous medicine as an external stimulant and a substitute for Abietis, Resina, and Pix Burgundica of European pharmacopeias (Ragini 2004). Extracts from S. tumbuggaia also have antinociceptive, anti- inflammatory, and wound-healing properties and provide pro- tection from oxidative stress in cancer (Jyothi et al. 2008; * Shastri Prasad Shukla sps.cimap@gmail.com 1 Plant Tissue Culture Laboratory, Plant Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants (CIMAP), Council of Scientific and Industrial Research, Lucknow, Uttar Pradesh 226 015, India In Vitro Cell.Dev.Biol.—Plant DOI 10.1007/s11627-017-9818-5