Highly Efficient Protoplast Isolation and Transient Expression System for Functional Characterization of Flowering Related Genes in Cymbidium Orchids
by
Rui Ren 1
, Jie Gao 1, Chuqiao Lu 1, Yonglu Wei 1, Jianpeng Jin 1, Sek-Man Wong 2,3,4, Genfa Zhu 1,* and Fengxi Yang 1,*
Rui Ren 1, Jie Gao 1, Chuqiao Lu 1, Yonglu Wei 1, Jianpeng Jin 1, Sek-Man Wong 2,3,4, Genfa Zhu 1,* and Fengxi Yang 1,*
1
Guangdong Key Laboratory of Ornamental Plant Germplasm Innovation and Utilization, Environmental Horticulture Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
2
Department of Biological Sciences, National University of Singapore (NUS), 14 Science Drive 4, Singapore 117543, Singapore
3
National University of Singapore Suzhou Research Institute (NUSRI), Suzhou Industrial Park, Suzhou 215000, China
4
Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore 117604, Singapore
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(7), 2264; https://doi.org/10.3390/ijms21072264 (registering DOI)
Received: 28 February 2020 / Revised: 23 March 2020 / Accepted: 24 March 2020 / Published: 25 March 2020
(This article belongs to the Special Issue Cell-Specificity in Plants)
Protoplast systems have been proven powerful tools in modern plant biology. However, successful preparation of abundant viable protoplasts remains a challenge for Cymbidium orchids. Herein, we established an efficient protoplast isolation protocol from orchid petals through optimization of enzymatic conditions. It requires optimal D-mannitol concentration (0.5 M), enzyme concentration (1.2 % (w/v) cellulose and 0.6 % (w/v) macerozyme) and digestion time (6 h). With this protocol, the highest yield (3.50 × 107/g fresh weight of orchid tissue) and viability (94.21%) of protoplasts were obtained from flower petals of Cymbidium. In addition, we achieved high transfection efficiency (80%) through the optimization of factors affecting polyethylene glycol (PEG)-mediated protoplast transfection including incubation time, final PEG4000 concentration and plasmid DNA amount. This highly efficient protoplast-based transient expression system (PTES) was further used for protein subcellular localization, bimolecular fluorescence complementation (BiFC) assay and gene regulation studies of flowering related genes in Cymbidium orchids. Taken together, our protoplast isolation and transfection protocol is highly efficient, stable and time-saving. It can be used for gene function and molecular analyses in orchids and other economically important monocot crops.
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Keywords:
Cymbidium orchids; protoplast isolation; protoplast-based transient expression system (PTES); subcellular location; bimolecular fluorescence complementation (BiFC) assay; gene regulation
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Ren, R.; Gao, J.; Lu, C.; Wei, Y.; Jin, J.; Wong, S.-M.; Zhu, G.; Yang, F. Highly Efficient Protoplast Isolation and Transient Expression System for Functional Characterization of Flowering Related Genes in Cymbidium Orchids. Int. J. Mol. Sci. 2020, 21, 2264.
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