Highly Efficient Protoplast Isolation and Transient Expression System for Functional Characterization of Flowering Related Genes in Cymbidium Orchids.
Int J Mol Sci
; 21(7)2020 Mar 25.
Article
en En
| MEDLINE
| ID: mdl-32218171
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.
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Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Protoplastos
/
Orchidaceae
Idioma:
En
Revista:
Int J Mol Sci
Año:
2020
Tipo del documento:
Article
País de afiliación:
China