Regeneration of Cytologically Stable Plants Through Dedifferentiation, Redifferentiation, and Artificial Seeds in Spathoglottis plicata Blume. (Orchidaceae) Sk Moquammel Haque, and Biswajit Ghosh * Plant Biotechnology Laboratory, Post Graduate Department of Botany, Ramakrishna Mission Vivekananda Centenary College, Rahara, Kolkata 700118, India Received 12 December 2016; Received in revised form 8 May 2017; Accepted 14 July 2017 Available online ABSTRACT Spathoglottis plicata Blume. is a horticulturally important vulnerable ground orchid with beautiful flowers blooming round the year. High- frequency protocorm-like body (PLB) formation was established via callus culture from vegetative tissues of in vitro germinated seedlings of S. plicata. Media containing MS salts and Gamborg’s B5 vitamins supplemented with 1.0 mg·L -1 2,4-dichlorophenoxyacetic acid (2,4-D), 3.0 mg·L -1 α-naphthaleneacetic acid (NAA), 1.0 mg·L -1 kinetin (KIN), and 10% (v/v) ‘Aloe vera gel’ (AvG) were effective in fragile calli induction. A maximum of (22.3 ± 0.52) PLBs were induced from about 250 mg callus within 45–55 days in the presence of 2.0 mg·L -1 NAA and 3.0 mg·L -1 6-benzylaminopurine (BAP). Briefly, 3.0% sodium alginate was found to be most suitable for the formation of an appropriate shape and good germination rates (86.7%) of artificial seeds. Out of three different temperatures (4, 15, and 24 °C), the best result was achieved at 4 °C with 66.7% germinability even after 90 days of storage. Plantlets were acclimatized with 86.6% survival rate and 76.3% of these plants produced flowers within 12–15 months of field transfer. Chromosomal studies revealed cytological stability of all regenerants containing 2n = 40 chromosomes as in the parental plants. The present protocol can be applied reliably for the purposes of large-scale commercial propagation and short-term conservation of this orchid. Keywords: Spathoglottis plicata; Aloe vera gel; artificial seeds; cytological stability; in vitro conservation; orchid micropropagation; protocorm-like body; regeneration; dedifferentiation; redifferentiation 1. Introduction Terrestrial ground orchid Spathoglottis plicata Blume. is an eco- nomically important ‘vulnerable’ orchid with beautiful flowers blooming round the year. This orchid is native to Southeastern Asia and is also found in the Philippines, New Guinea, Australia, and the Islands of the South-West Pacific Ocean (Cribb and Tang, 1982). Because of the difficulty in seed germination and propa- gation, a significant number of orchids including Spathoglottis, used for commercial purposes, are collected from wild habitats (Aewsakul et al., 2013). Because of the illegal over-collection from its natural habitat, Spathoglottis is designated as ‘vulnerable’ under the Queensland Nature Conservation Act 1992 (https:// wetlandinfo.ehp.qld.gov.au/wetlands/ecology/components/species/ ?spathoglottis-plicata). Besides the conventional forms of protection of endangered species, advances in biotechnology, especially in the field of in vitro culture techniques, can provide an excellent tool to conserve endangered plant species. To meet the demand for orchids and for the conservation of their biodiversity, the de- velopment and deployment of innovative technologies, like in vitro propagation, artificial seeds by encapsulation of high-frequency protocorm-like bodies (PLBs), are indispensable (Hossain et al., 2013). The history of tissue culture reveals that various complex organic compounds like casin hydrolysate, coconut milk, tomato juice, banana puree, orange juice, grape juice, pineapple juice, yeast * Corresponding author. Tel.: +91 9432113696 E-mail address: ghosh_b2000@yahoo.co.in Peer review under responsibility of Chinese Society for Horticultural Science (CSHS) and Institute ofVegetables and Flowers (IVF), Chinese Academy of Agricultural Sciences (CAAS) https://doi.org/10.1016/j.hpj.2017.10.002 2468-0141/© 2017 Chinese Society for Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Agri- cultural Sciences (CAAS).This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). ARTICLE IN PRESS Please cite this article in press as: Sk Moquammel Haque, and Biswajit Ghosh, Regeneration of Cytologically Stable Plants Through Dedifferentiation, Redifferentiation, and Arti- ficial Seeds in Spathoglottis plicata Blume. (Orchidaceae), Horticultural Plant Journal (2017), doi: 10.1016/j.hpj.2017.10.002 September 2017. Horticultural Plant Journal, 3 (5): 199–208. Horticultural Plant Journal Available online at www.sciencedirect.com The journal’s homepage: http://www.journals.elsevier.com/horticultural-plant-journal