Biotechnology1682-296x1682-2978Asian Network for Scientific Information10.3923/biotech.2019.77.88In vitro Immature Embryo Culture and Propagation of Paphiopedilum villosum Var. Boxallii (Rchb. f.) Pfitzer]]>DebChitta Ranjan JakhaHutoka Yetokhu 22019182Background and Objective:Paphiopedilum Pfitzer (Orchidaceae) is among the most popular orchid genera that have been successfully commercialized as potted plants and cut flowers due to the peculiarity of its flowers. Paphiopedilum villosum var. boxallii is a rare and vulnerable horticultural important species. The aim of this study was to develop the in vitro propagation protocol from immature seeds. Materials and Methods: Seeds of various developmental age (150-300 days after pollination, DAP) of P. villosum var. boxallii were cultured on different nutrient media supplemented with different organic carbon source, coconut water, activated charcoal, NAA and BA at different combination. Results: Seeds from ~240 DAP old pods registered 60% germination on MS medium containing sucrose (3%, w/v), NAA and BA (2 and 6 μM, respectively in combination) within 59 days under diffused light (20 μmol m2 sec1). Incorporation of coconut water did not support seed germination but supported early differentiation to protocorm-like bodies (PLBs). Germinated seeds converted to PLBs and differentiated to plantlets. The PLBs converted to rooted plantlets full strength MS medium fortified with sucrose (3%), NAA+BA (2+6 μM in combination) where as many as 7 shoot developed/explants per sub-culture cycle. The rooted plantlets were hardened by culturing on 1/10th MS medium containing sucrose (1%) and hardening matrix (sand, decaying organic matter, brick pieces, charcoal and dried cow dung at 1:1:1:1:1 ratio). The hardened plants were transferred to potting mix along with the hardening matrix where ~65% transplants survived in the green house condition. Conclusion: This study opens up an effective alternate route of propagation of this vulnerable species Paphiopedilum villosum var. boxallii (Rchb. f.) Pfitzer.]]>Liu, Z.J., S.C. Chen, L.J. Chen and S.P. Lei,2009pp: 13-349pp: 13-349Kumar, P. and H. Rankou,2015Paphiopedilum villosum.]]>2015Huang, L.C., C.J. Lin, C. Kuo, B.L. Huang and T. Murashige,2001Paphiopedilum cloning in vitro.]]>91111121Liao, Y.J., Y.C. Tsai, Y.W. Sun, R.S. Lin and F.S. Wu,2011In vitro shoot induction and plant regeneration from flower buds in Paphiopedilum orchids.]]>47702709Arditti, J.,20082nd Edn.,Chugh, S., S. Guha and I.U. Rao,2009122507520Murashige, T. and F. Skoog,196215473497Gamborg, O.L., R.A. Miller and K. Ojima,196850151158Mitra, G.C., R.N. Prasad and A. Roychoudhury,197614350351Knudson, L.,194615214217Schenk, R.U. and A.C. Hildebrandt,197250199204Darlington, C.D. and L.F. La Cour,19766th Rev. Edn.,Pages: 201Pages: 201Temjensangba and C.R. Deb,2005Arachnis labrosa (Lindl. ex Paxt.) Reichb: A rare and threatened orchid.]]>8819661969Ding, C.C., H. Yu and F.Y. Liu,2004Paphiopedilum armeniacum.]]>26673677Deb, C.R. and Temjensangba,2006In vitro propagation of threatened terrestrial orchid, Malaxis Khasiana Soland ex. Swartz through immature seed culture.]]>44762766Deb, C.R. and Temjensangba,2007Cleisostoma racemiferum (Lindl.) Garay: An endangered orchid.]]>3499105Deb, C.R. and Sungkumlong,2008In vitro regeneration and mass multiplication of Taenia latifolia (Lindl.) using immature seeds a threatened terrestrial orchid.]]>3516Lee, Y.I., E.C. Yeung, N. Lee and M.C. Chung,2006Paphiopedilum delenatii, with emphasis on the ultrastructure of the suspensor.]]>9813111319Zeng, S., K. Wu, J.A.T. da Silva, J. Zhang, Z. Chen, N. Xia and J. Duan,2012Paphiopedilum wardii Sumerh., an endangered terrestrial orchid.]]>138198209Nhut, D.T., P.T.T. Trang, N.H. Vu, D.T.T. Thuy, D. Van Khiem, N. Van Binh and K.T.T. Van,2005Paphiopedilum delenatii propagation.]]>5158163Long, B., A.X. Niemiera, Z.Y. Cheng, C.L. Long,2010In vitro propagation of four threatened Paphiopedilum species (Orchidaceae).]]>101151162Zhang, J.J., N. Yan and H. Hu,2013Paphiopedilum species in relation to asymbiotic germination.]]>353340Nagashima, T.,1982Goeringii Rchb. F. and Paphiopedilum insigne var. sanderae Rchb.]]>5194105Sauleda, R.P.,197645305308Zeng, S.J., W. Huang, K. Wu, J. Zhang, J.A.T. da Silva and J. Duan,2016In vitro propagation of Paphiopedilum orchids.]]>36521534Pierik, R.L.M., P.A. Sprenkels, B. van der Harst and Q.G. Van der Meys,1988Paphiopedilum ciliolare Pfitz. in vitro.]]>34139153Temjensangba and C.R. Deb,2006Cleisostoma racemiferum (Lindl.) Garay.]]>5223228Hossain, M.M., R. Kant, P.T. Van, B. Winarto, S.J. Zeng and J.A.T. da Silva,20133269139Sungkumlong and C.R. Deb,2008Coelogyne suaveolens (Lindl.) Hook.]]>46243248Curtis, J.T.,1943Cypripedium L.]]>30199206Thomas, T.D.,200826618631Da Silva, J.A.T.,20137152Li, Z.Y. and L. Xu,2009In vitro propagation of white-flower mutant of Rhynchostylis gigantea (Lindl.) Ridl. through immature seed-derived protocorm-like bodies.]]>1093097Chen, Z.L., X.L. Ye, C.Y. Liang and J. Duan,2004in vitro of Paphiopedilum armeniacum and P. micranthum.]]>31540542Deb, C.R. and T. Imchen,2010in vitro hardening technique of tissue culture raised plants.]]>97983