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

Received: 26 September 2018/Accepted: 25 June 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)

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*Corresponding author; email: lkoksong@hct.ac.ae