Optimized protoplast isolation and establishment of transient gene expression system for the Antarctic flowering plant Colobanthus quitensis (Kunth) Bartl.
Colobanthus quitensis is one of two terrestrial plants that grow in the maritime Antarctic. Despite its important ecological niche in extreme environments, the molecular mechanisms of its adaptation and tolerance have not been elucidated due to difficulties with genetic or transgenic approaches. For this reason, in many other plant species mesophyll protoplasts as a versatile cell-based system have been developed and used to analyze the biological functions of genes of interest. Here we report an optimized method of protoplast isolation from C. quitensis leaves. The main parameters evaluated to reach the highest protoplast yield were the use of a cell wall-degrading enzyme, an osmotic stabilizer, and different pH conditions. Moreover, transient expression and subcellular localization of proteins were validated by an immunoblot assay and a confocal microscopy, respectively, using C. quitensis protoplasts. Therefore, these results suggest that protoplasts can provide a useful cell-based system to facilitate the molecular, biochemical, and cellular characterizations of C. quitensis genes.
C. quitensis protoplasts can provide a physiologically relevant cell system to facilitate the molecular, biochemical, and cellular characterization of C. quitensis genes.
KeywordsCell-based assay Low temperature Molecular adaptation PEG–CaCl2-mediated transfection Subcellular localization
This research was supported by a grant from the Korea Polar Research Institute (PE18290).
JL and HL developed concept and supplied plant materials. JL, HSL, and HL designed the research. HL wrote the manuscript and OKC performed all experiments.
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
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