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.
Highlights
Among the 350,000 plant species on earth, Orchidaceae is one of the largest and the most evolved families of monocot plants (Figure S1) [1,2]
Viable protoplasts were released from petals with variable yield and viability (Figure 1B)
The isolated protoplasts ranged from 20 to 100 μm in diameters, and a large proportion of protoplasts isolated from flowers petals were rich in cytoplasm and anthocyanidin
Summary
Among the 350,000 plant species on earth, Orchidaceae (more than 25,000 species) is one of the largest and the most evolved families of monocot plants (Figure S1) [1,2]. To obtain high yielding quality orchid plants, intense efforts have been put into orchid biology research for the past few decades. Related researches include tissue culture, protoplast fusion and regeneration [4,5]. The release of orchid complete genome sequences of Phalaenopsis [6], Dendrobium [7] and Apostasia [8] has greatly facilitated gene cloning, genetic evolutionary analysis and synthetic biology. Plant protoplasts have attracted great attention, since they can be isolated from specific plant tissues/organs and maintained their cellular identity, which practically provided a unique single cell system to answer specific questions related to cell types [12,13]. Isolation of sufficient viable protoplasts remains a challenge for orchids and many other monocot crop plants [14]
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