Optimized protoplast isolation and establishment of transient gene expression system for the Antarctic flowering plant Colobanthus quitensis (Kunth) Bartl.

Abstract

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.