Sains Malaysiana 48(9)(2019): 1867–1877

http://dx.doi.org/10.17576/jsm-2019-4809-07 

Development of an Efficient Particle Bombardment Transformation System for the Endemic Orchid, Phalaenopsis bellina

(Pembentukan Sistem Transformasi Pembedilan Zarah yang Cekap bagi Orkid Endemik, Phalaenopsis bellina)

YEE-CHERN CHEW², WAN MUHAMAD ASRUL NIZAM WAN ABDULLAH¹, DE-XIAN ANDREW KOK¹, JANNA ONG-ABDULLAH¹, MAZIAH MAHMOOD² & KOK-SONG LAI³*

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

²Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor Darul Ehsan, Malaysia

³Health Sciences Division, Abu Dhabi Women’s College, Higher Colleges of Technology, 41012 Abu Dhabi, United Arab Emirates

Diserahkan: 26 September 2018/Diterima: 25 Jun 2019

ABSTRACT

Phalaenopsis bellina is an important indigenous orchid with high commercial value. In this study, we established an efficient particle bombardment transformation system for P. bellina using the protocorm-like-bodies (PLBs) as target tissues. Leaf derived PLBs were proliferated on ½ strength Murashige and Skoog (MS) medium supplemented with 0.8 μM 2,4 dichlorophenoxyacetic acid (2, 4-D). Both physical and biological parameters affecting the transformation system were optimised using the green-fluorescent protein (GFP) and β-glucuronidase (GUS) as reporter systems. Optimal bombardment conditions with 6 cm target tissues distance, 1100 psi acceleration pressure, 1.0 μm gold particle size, 27 mmHg chamber vacuum pressure, single bombardment time, spermidine as DNA precipitation agent, 72 h post bombardment incubation time, 2 μg plasmid DNA in 0.15:0.12 pmol ratio (pSMCHS:p35SGFP) were successfully determined. Surviving PLBs transformants were successfully recovered from the hygromycin selection medium and verified using genomic PCR analysis. The established system is not only useful for a simple and reliable transient gene analysis but as well as generating stable transformants for selective traits improvement in orchids.

Keywords: β-glucuronidase (GUS); green fluorescent protein (GFP); particle bombardment; Phalaenopsis bellina; protocorm-like-bodies (PLBs)

ABSTRAK

Phalaenopsis bellina ialah salah suatu orkid yang mempunyai nilai komersial yang tinggi. Dalam kajian ini, kami telah membentuk sistem transformasi pembedilan zarah yang berkesan untuk P. bellina menggunakan jasad seperti protokorm (PLB) sebagai tisu sasaran. Daun daripada PLBs bercambah pada ½ kekuatan medium Murashige dan Skoog (MS) yang mengandungi 0.8 μM 2,4 asid d diklorofenok siasetik (2, 4-D). Parameter biologi dan fizikal yang mempengaruhi sistem transformasi telah dioptimumkan dengan menggunakan protein fluoresen hijau (GFP) dan β- glukuronidase (GUS) sebagai sistem pelapor. Keadaan pembedilan optimum dengan jarak tisu 6 cm, tekanan pecutan 1100 psi, saiz zarah 1.0 μm, tekanan vakum 27 mmHg, pembedilan tunggal, spermidine sebagai agen pemendapan DNA, 72 jam tempoh inkubasi selepas pembedilan, 2 μg DNA plasmid dalam 0.15:0.12 pmol nisbah (pSMCHS:p35SGFP) telah ditentukan. Transforman PLB telah berjaya dipulihkan selepas inkubasi di dalam medium pemilihan higromisin dan disahkan menggunakan analisis PCR genom. Sistem yang ditubuhkan ini tidak hanya berguna untuk analisis gen sementara yang boleh dipercayai dan mudah tetapi juga mampu menjana transformasi stabil bagi peningkatan ciri-ciri terpilih dalam orkid.

Kata kunci: β- glukuronidase (GUS); jasad seperti protokorm (PLB); Phalaenopsis bellina; pembedilan zarah; protein fluoresen hijau (GFP)

RUJUKAN

Atari, M.F.B.M. & Folta, K.M. 2019. Transformation improvement with the Standardized Pressure Agrobacterium Infiltration Device (SPAID). BMC Research Notes 12(1): 144.

Bernama. 2017. Ahmad Shabeery: Orchid Export Increases to RM13.2m Last Year. http://www.thesundaily.my/ news/2205891. Accessed on 9 June 2017.

Bhattacharyya, J., Chakraborty, A., Roy, S., Pradhan, S., Mitra, J., Chakraborty, M., Manna, A., Sikdar, N., Chakraborty, S. & Sen, S.K. 2015. Genetic transformation of cultivated jute (Corchorus capsularis L.) by particle bombardment using apical meristem tissue and development of stable transgenic plant. Plant Cell, Tissue and Organ Culture 121(2): 311-324.

Chew, Y.C., Abd-Halim, M.H., Wan-Abdullah, W.M.A.N., Ong- Abdullah, J. & Lai, K.S. 2018. Highly efficient proliferation and regeneration of protocorm-like bodies (PLBs) of the threatened endemic orchid: Phalaenopsis bellina. Sains Malaysiana 47(6): 1093-1099.

Chin, C.F. 2016. The proteome of orchids. In Agricultural Proteomics Volume 1: Crops, Horticulture, Farm Animals, Food, Insect and Microorganisms, edited by Salekdeh, G.H. Switzerland: Springer International Publishing. pp. 127-135.

da Silva, T.J.A. & Tanaka, M. 2011. Optimization of particle bombardment conditions for hybrid Cymbidium: Part II. Transgenic Plant Journal 5(1): 78-82.

Dhir, S., Knowles, K. & Pagan, C.L. 2010. Optimization and transformation of Arundo donax L. using particle bombardment. African Journal of Biotechnology 9(39): 6460-6469.

Ghorbanzade, Z. & Ahmadabadi, M. 2015. Stable transformation of the Saintpaulia ionantha by particle bombardment. Iran Journal of Biotechnology 13(1): 11-16.

Gnasekaran, P., Antony, J.J.J., Uddain, J. & Sreeramanan, S. 2014. Agrobacterium-mediated transformation of the recalcitrant Vanda Kasem’s Delight orchid with higher efficiency. Scientific World Journal 2014(1): 583934.

Hafiza, M.R., Roowi, S., Subramaniam, S., Kamaruzaman, R. & Zuraida, A.R. 2010. Factors affecting delivery and transient expression of gusA gene in Malaysian indica rice MR 219 callus via biolistic gun system. Journal of Biotechnology 9(51): 8810-8818.

Hsiao, Y.Y., Pan, Z.J., Hsu, C.C., Yang, Y.P., Hsu, Y.C., Chuang, Y.C., Shih, H.H., Chen, W.H., Tsai, W.C. & Chen, H.H. 2011. Research on orchid biology and biotechnology. Plant and Cell Physiology 52(9): 1467-1486.

Hsing, H.X., Lin, Y.J., Tong, C.G., Li, M.J., Chen, Y.J. & Ko, S.S. 2016. Efficient and heritable transformation of Phalaenopsis orchids. Botanical Studies 57(1): 30. doi: https://doi. org/10.1186/s40529-016-0146-6.

Jin, T., Wang, J., Zhu, X., Xu, Y., Zhou, X. & Yang, L. 2015. A new transient expression system for large-scale production of recombinant proteins in plants based on air-brushing an Agrobacterium suspension. Biotechnology Reports 6(1): 36-40.

Lai, K.S. 2016. Analysis of EXO70C2 expression revealed its specific association with late stages of pollen development. Plant Cell, Tissue and Organ Culture 124(1): 209-215.

Lai, K.S. & Yusoff, K. 2013. Production of tENDO1 in stably transformed tobacco cell cultures for mismatch detection. Plant Cell, Tissue and Organ Culture 114(2): 287-294.

Lai, K.S., Yusoff, K. & Mahmood, M. 2013. Functional ectodomain of the hemagglutinin-neuraminidase protein is expressed in transgenic tobacco cells as a candidate vaccine against Newcastle disease virus. Plant Cell, Tissue and Organ Culture 112(1): 117-121.

Lai, K.S., Yusoff, K. & Mahmood, M. 2012. Heterologous expression of hemagglutinin-neuraminidase protein from newcastle disease virus strain AF2240 in Centella asiatica. Acta Biologica Cracoviensia. Series Botanica 54(1): 142-147.

Lai, K.S., Abdullah, P., Yusoff, K. & Mahmood, M. 2011. An efficient protocol for particle bombardment-mediated transformation of Centella asiatica callus. Acta Physiologiae Plantarum 33(6): 2547-2552.

Lao, J., Ai, O., Bromley, J.R., McInerney, P., Suttangkakul, A., Smith-Moritz, A.M., Plahar, H., Chiu, T.Y., Fernández‐Niño, S.M.G., Ebert, B., Yang, F., Christiansen, K.M., Hansen, S.F., Stonebloom, S., Adams, P.D., Ronald, P.C., Hillson, N.J., Hadi, M.Z., Vega‐Sánchez, M.E., Loqué, D., Scheller, H.V. & Heazlewood, J.L. 2014. The plant glycosyltransferase clone collection for functional genomics. The Plant Journal 79(3): 517-529.

Lau, S.E., Schwarzacher, T., Othman, R.Y. & Harikrishna, J.A. 2015. dsRNA silencing of an R2R3-MYB transcription factor affects flower cell shape in a Dendrobium hybrid. BMC Plant Biology 15(1): 194. doi: 10.1186/s12870-015-0577-3.

Li, J., Kuang, P., Liu, R.D., Wang, D., Wang, Z.N. & Huang, M.R. 2013. Transfer of the gafp and npi, two disease-resistant genes, into a phalaenopsis by Agrobacterium tumefaciens. Pakistan Journal of Botany 45(5): 1761-1766.

Li, S., Cong, Y., Liu, Y., Wang, T., Shuai, Q., Chen, N., Gai, J. & Li, Y. 2017. Optimization of Agrobacterium-mediated transformation in soybean. Frontiers in Plant Science 8(1): 246.

Low, L.Y., Yang, S.K., Andrew Kok, D.X., Ong-Abdullah, J., Tan, N.P. & Lai, K.S. 2018. Transgenic plants: Gene constructs, vector and transformation method. In New Visions in Plant Science, edited by Çelik, Ö. Croatia: Intech Open. pp. 41-61.

Lu, Y., Chen, X., Wu, Y., Wang, Y., He, Y. & Wu, Y. 2013. Directly transforming PCR-amplified DNA fragments into plant cells is a versatile system that facilitates the transient expression assay. PLoS ONE 8(2): e57171.

Mahdavi, F., Mahmood, M. & Noor, N. 2014. Optimization of particle bombardment parameters for DNA delivery into the male flowers of banana. Biologia (Bratislava) 69(7): 888-894.

Maraschin, F.D.S., Memelink, J. & Offringa, R. 2009. Auxin-induced, SCF^TIR1-mediated poly-ubiquitination marks AUX/ IAA proteins for degradation. The Plant Journal 59(1): 100-109.

Noman, A., Aqeel, M., Deng, J., Khalid, N., Sanaullah, T. & Shuilin, H. 2017. Biotechnological advancements for improving floral attributes in ornamental plants. Frontier in Plant Science 8(1): 530.

Que, Q., Elumalai, S., Li, X., Zhong, H., Nalapalli, S., Schweiner, M., Fei, X., Nuccio, M., Kelliher, T., Gu, W., Chen, Z. & Chilton, M.D. 2014. Maize transformation technology development for commercial event generation. Frontier in Plant Science 5(1): 379.

Tan, B.C., Chin, C.F., Liddell, S. & Alderson, P. 2013. Proteomic analysis of callus development in Vanilla planifolia Andrews. Plant Mololecular Biology Reporter 31(6): 1220-1229.

Wani, S.H., Sanghera, G.S., Haribhushan, A., Singh, N.B. & Gosal, S.S. 2011. Bio-physical parameters affecting transient GUS expression in Indica rice variety PAU 201 via particle bombardment. Elixir Bio Technology 34(2011): 2496-2501.

Wu, H., Awan, F.S., Vilarinho, A., Zeng, Q., Kannan, B., Phipps, T. & McCuiston, J. 2015. Transgene integration complexity and expression stability following biolistic or Agrobacterium-mediated transformation of sugarcane. In Vitro Cellular & Developmental Biology-Plant 51(6): 603-611.

Zhang, S.B., Chen, W.Y., Huang, J.L., Bi, Y.F. & Yang, X.F. 2015. Orchid species richness along elevational and environmental gradients in Yunnan, China. PLoS ONE 10(11): e0142621.

Zhao, Y., Zhang, Y., Su, P., Yang, J., Huang, L. & Gao, W. 2017. Genetic transformation system for woody plant Tripterygium wilfordii and its application to product natural celastrol. Frontiers in Plant Science 8(1): 2221.

*Pengarang untuk surat-menyurat; email: lkoksong@hct.ac.ae