Please use this identifier to cite or link to this item: https://doi.org/10.3390/ijms21072264
Title: Highly efficient protoplast isolation and transient expression system for functional characterization of flowering related genes in Cymbidium orchids
Authors: Ren, R.
Gao, J.
Lu, C.
Wei, Y.
Jin, J.
Wong, S.-M. 
Zhu, G.
Yang, F.
Keywords: Bimolecular fluorescence complementation (BiFC) assay
Cymbidium orchids
Gene regulation
Protoplast isolation
Protoplast-based transient expression system (PTES)
Subcellular location
Issue Date: 2020
Publisher: MDPI AG
Citation: Ren, R., Gao, J., Lu, C., Wei, Y., Jin, J., Wong, S.-M., Zhu, G., Yang, F. (2020). Highly efficient protoplast isolation and transient expression system for functional characterization of flowering related genes in Cymbidium orchids. International Journal of Molecular Sciences 21 (7) : 2264. ScholarBank@NUS Repository. https://doi.org/10.3390/ijms21072264
Rights: Attribution 4.0 International
Abstract: 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. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
Source Title: International Journal of Molecular Sciences
URI: https://scholarbank.nus.edu.sg/handle/10635/198926
ISSN: 1661-6596
DOI: 10.3390/ijms21072264
Rights: Attribution 4.0 International
Appears in Collections:Staff Publications
Elements

Show full item record
Files in This Item:
File Description SizeFormatAccess SettingsVersion 
10_3390_ijms21072264.pdf2.56 MBAdobe PDF

OPEN

NoneView/Download

Google ScholarTM

Check

Altmetric


This item is licensed under a Creative Commons License Creative Commons