Sains Malaysiana 47(6)(2018): 1093–1099

http://dx.doi.org/10.17576/jsm-2018-4706-03

Highly Efficient Proliferation and Regeneration of Protocorm-like Bodies (PLBS) of the Threatened Orchid, Phalaenopsis bellina

(Proliferasi dan Penjanaan Semula yang Cekap ke atas Jasad seperti Protokorm (PLBS) Orkid

Terancam, Phalaenopsis bellina)

YEE-CHERN CHEW¹, MUHAMMAD HUSNI ABD HALIM¹, WAN MUHAMAD ASRUL NIZAM WAN ABDULLAH², JANNA ONG ABDULLAH² & KOK-SONG LAI^2*

¹Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia

²Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia

Diserahkan: 6 November 2017/Diterima: 1 Februari 2018

ABSTRACT

Phalaenopsis bellina is an important indigenous fragrant orchid threatened with extinction. In this study, we evaluated the effect of medium strength, sucrose, nitrogen (NH4NO3) and potato extract on proliferation of P. bellina protocorm-like bodies (PLBs) to improve micropropagation in this species. Optimal treatment for PLBs proliferation rate with an average fresh weight (FW) of 0.97±0.16 g was obtained through culturing on half strength (½) MS medium containing 20 g/L sucrose, 15 mM NH4NO3 and 20% w/v potato extract supplemented with 0.8 μM 2,4 dichlorophenoxyacetic acid (2,4-D). The optimal treatment produced large, healthy and greenish PLBs with reduction in the occurrence of culture browning. In contrast, treatments with high potato extract (>20% w/v) or NH4NO3 (>30 mM) concentrations tend to have inhibitory effect and resulted in low PLBs proliferation rate, with an average FW of 0.77±0.15 g and 0.69±0.15 g, respectively. Plant regeneration of PLBs was achieved on plant growth regulator (PGR)-free ½ MS medium. In total, 60 healthy PLBs from the optimal treatment were successfully regenerated, acclimatized with 100% survival percentage and grew well in a mixture of soil, sand and vermicompost (8:4:2 (w/w/w)). With the optimal treatment, PLBs proliferation rate was enhanced by 27.63%. Our findings offer an improved micropropagation protocol of the endangered P. bellina for conservation and commercial production.

Keywords: Indigenous orchid; Phalaenopsis bellina; proliferation; protocorm-like bodies; regeneration

ABSTRAK

Phalaenopsis bellina merupakan orkid harum asli yang diancam kepupusan. Menerusi kajian ini, kami menilai kesan sukrosa, nitrogen (NH4NO3) dan ekstrak kentang terhadap proliferasi jasad seperti protocorm (PLBs) P. bellina untuk meningkatkan perambatan spesies ini. Rawatan optimum untuk kadar proliferasi PLBs dengan purata berat segar 0.97±0.16 g diperoleh dengan pengkulturan pada separuh kekuatan (½) media MS yang mengandungi 20 g/L sukrosa, 15 mM NH₄NO₃ dan 20% w/v ekstrak kentang ditambah dengan 0.8 μM 2,4 asid diklorofenoksiasetik (2,4-D). Rawatan optimum menghasilkan PLBs yang besar, sihat dan hijau dengan pengurangan keperangan kultur. Sebaliknya, rawatan menggunakan kepekatan ekstrak kentang (>20% w/v) atau NH4NO3 (>30 mM) yang tinggi cenderung memberikan kesan rencatan dan menyebabkan kadar proliferasi PLBs yang rendah dengan purata berat segar masing-masing 0.77±0.15 g dan 0.69±0.15 g. Regenerasi tumbuhan daripada PLBs dihasilkan dengan ½ media MS tanpa zat pengatur tumbuhan (ZPT). Secara keseluruhan, 60 PLBs yang sihat berjaya dijana semula dan PLBs dapat menyesuaikan diri dengan 100% kelangsungan hidup dan tumbuh dengan baik di dalam campuran tanah, pasir dan vermikompos (8:4:2 (w/w/w)). Kadar proliferasi PLBs meningkat 27.63% dengan rawatan optimum. Penemuan kami mengusul satu penambahbaikan protokol pembiakan P. bellina yang terancam untuk tujuan pemeliharaan dan penghasilan komersial.

Kata kunci: Jasad seperti protocorm; orkid asli; penjanaan semula; Phalaenopsis bellina; proliferasi

RUJUKAN

Budi, W. & Teixeira Da Silva, J.A. 2015. Use of coconut water and fertilizer for in vitro proliferation and plantlet production of Dendrobium 'Gradita 31'. In Vitro Cell. Dev. Biol. Plant 51: 303-314.

Chadburn, H. 2013. Phalaenopsis violacea. The IUCN red list of threatened species 2013.e.T44393331A44529144. http:// dx.doi.org/10.2305/IUCN.UK. 20131.RL TS.T44393331A44 529144.en.

Chandran, S., Toh, C.L., Zuliana, R., Yip, Y.K., Nair, H. & Boyce, A.N. 2006. Effects of sugars and aminooxyacetic acid on the longevity of pollinated Dendrobium (Heang Beauty) flowers. J. Appl. Hort. 8(2): 117-120.

Chen, Y. & Piluek, C. 1995. Effects of thidiazuron and N6- benzylaminopurine on shoot regeneration of Phalaenopsis. Plant Growth Regul. 16: 99-101.

Christenson, E.A. & Whitten, M. 1995. Phalaenopsis bellina (Rchb. f.) Christenson, a separate from P. violecea Witte (Orchidaceae: Aeridinae). Brittonia 47: 57-60.

Cohen, D. 1995. The culture medium. Acta Hort. 393: 15-24.

Dani, F., Spirindon, K., Atthanasion, S.E., Georgia, M. & Helen- Isis, A.C. 2010. Effect of different strength of medium on organogenesis, phenolic accumulation and antioxidant activity of spearmint (Mentha spicata I.). The Open Hort. J. 3: 31-35.

Hossain, M.M., Kant, R., Van, P.T., Winarto, B., Zeng, S.J. & Teixeira Da Silva, J.A. 2013. The application of biotechnology to orchids. Crit. Rev. Plant Sci. 32: 69-139.

Huang, J.Z., Lin, C.P., Cheng, T.C., Bill, C.H.C., Cheng, S.Y., Chen, Y.W., Lee, C.Y., Chin, S.W. & Chen, F.C. 2015. A de novo floral transcriptome reveals clues into Phalaenopsis orchid flower development. PloS ONE 10(5): e0123474.

Islam, M.O., Akter, M. & Prodhan, A.K.M.A. 2011. Effect of potato extract on in vitro seed germination and seedling growth of local Vanda roxburgii orchid. J. Bangladesh Agril. Univ. 9(2): 211-215.

Islam, M.O., Mitsui, S. & Ichihashi, S. 2000. Effect of complex organic additives on seed germination and carotenoid content in Cattleya seedlings. Lindleyana 15(2): 81-88.

Jing, C., Xin, L., Kevin, V., Sebastian, P., Wen, C.T., Ke, W.L., Li, J.C., Ying, H., Qing, X., Chao, B., Zhijun, Z., Fengming, S., Weiqing, L., Yu, Y.H., Zhao, J.P., Chia, C.H., Ya, P.Y., Yi, C.H., Yu, C.C., Anne, D., Jean-Francois, D., Xun, X., Jun, Y.W., Jun, W., Xin, J.X., Xue, M.Z., Rong, D., Guo, Q.Z., Meina, W., Yong, Y.S., Gao, C.X., Guo, H.L., Li, Q.L., Lai, Q.H., Yi, B.L., Hong, H.C., Yves, V.P. & Zhong, J.L. 2015. The genome sequence of the orchid Phalaenopsis equestris. Nat. Genet. 47(1): 65-72.

Johnson, T.R., Kane, M.E. & Perez, H.E. 2011. Examining the interaction of light, nutrients and carbohydrates on seed germination and early seedling development of Bletia purpurea (Orchidaceae). Plant Growth Regul. 63: 89-99.

Ken, T. & Mashiro, M. 1993. Micropropagation of Phalaenopsis and Doritaenopsis by culturing shoot tips of flower stalk buds. Plant Cell Rep. 13(1): 7-11.

Lai, K.S., Abdullah, P., Yusoff, K. & Maziah, M. 2011. An efficient protocol for particle bombardment-mediated transformation of Centella asiatica callus. Acta Physiol. Plant 33: 2547-2552.

Lai, K.S., Yusoff, K. & Maziah, M. 2014. Morphological and physiological characterization of embryogenic and non-embryogenic tissues of Centella asiatica. Plant Tissue Cult. Biotech. 24(1): 125-129.

Maziah, M. & Chew, Y.C. 2008. Agrobacterium-mediated genetic transformation of Phalaenopsis bellina using GFP and GUS reporter genes. Pertanika J. Sci. Technol. 16(2): 129-139.

Marschner, H. 1995. Mineral Nutrition of Higher Plants. 2nd ed. London: Academic Press.

Murashige, T. & Skoog, F. 1962: A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant 15: 473-497.

Nambiar, N., Tee, C.S. & Maziah, M. 2012. Effects of organic additives and different carbohydrate sources on proliferation of protocorm-like bodies in Dendrobium Alya Pink. Plant Omics J. 5(1): 10-18.

Nowak, B., Miczyński, K. & Hudy, L. 2004. Sugar uptake and utilization during adventituous bud differentiation on in vitro leaf explants of 'Wegierka Zwykla' plum (Prunus domestica). Plant Cell Tiss. Organ Cult. 76: 255-260.

Park, S.Y., Murthy, H.N. & Paek, K.Y. 2002. Rapid propagation of Phalaenopsis from floral stalk-derived leaves. In Vitro Cell. Dev. Biol. Plant 38: 168-172.

Poddubnaya-Arnold, V.A. 1967. Comparative embryology of the Orchidaceae. Phytomorphology 17: 312-320.

Rosmah, M., Mariam, A.L., Zaleha, A.A. & Rimi, R. 2010. Effects of carbon source and potato homogenate on in vitro growth and development of Sabah's endangered Orchid: Phalaenopsis gigantea. As-Pac. J. Mol. Biol. Biotechnol. 18(1): 199-202.

Shim, S.W., Hahn, E.J. & Paek, K.Y. 2003. In vitro and ex vitro growth of grapevine rootstock '5BB' as influenced by number of air exchanges and the presence or absence of sucrose in culture media. Plant Cell Tiss. Organ Cult. 75: 57-62.

Shimasaki, K. & Uemoto, S. 1990. Micropropagation of a terrestrial Cymbidium species using rhizomes developed from seeds and pseudobulbs. Plant Cell Tiss. Organ Cult. 22: 237-244.

Shohael, A.M., Chakrabarty, D., Ali, M.B., Yu, K.W., Hahn, E.J., Lee, H.L. & Paek, K.Y. 2006. Enhancement of eleutherosides production in embryogenic cultures of Eleutherococcus sessiliflorus in response to sucrose-induced osmotic stress. Process Biochem. 41: 512-518.

Shrestha, B.R., Tokuhara, K. & Mii, M. 2007. Plant regeneration from cell suspension-derived protoplasts of Phalaenopsis. Plant Cell Rep. 26(6): 719-725.

Teixeira Da Silva, J.A. 2013. Ammonium to nitrate ratio affects protocorm like bodies PLB formation in vitro of hybrid Cymbidium. J. Ornamental Plants 3(3): 155-160.

Toor, R.K., Savage, G.P. & Hee, A. 2006. Influence of different types of fertilisers on the major antioxidant components of tomatoes. J. Food Composition Anal. 19: 20-27.

Yu, Y.H., Wen, C.T., Chang, S.K., Tian, H.H., Hei, C.W., Tian, S.W., Yann, L.L., Wen, H.C. & Hong, H.C. 2006. Comparison of transcripts in Phalaenopsis bellina and Phalaenopsis equestris (Orchidaceae) flowers to deduce monoterpene biosynthesis pathway. BMC Plant Biol. 6: 14.

*Pengarang untuk surat-menyurat; email: laikoksong@upm.edu.my