Highly Efficient Protoplast Isolation and Transient Expression System for Functional Characterization of Flowering Related Genes in Cymbidium Orchids

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.

Highly Efficient Leaf Base Protoplast Isolation and Transient Expression Systems for Orchids and Other Important Monocot Crops

“…Mesophyll protoplasts were successfully isolated from Dendrobium [∼3.97 × 10 5 /g fresh weight (FW)] ( Khentry et al, 2006 ), Cymbidium (maximum 1.1 × 10 7 /g FW) ( Pindel, 2007 ), and Phalaenopsis orchids (5.9 × 10 6 /g FW) ( Li et al, 2018 ). Additionally, flower petals were used for efficient protoplast isolation from Dendrobium ( Hu et al, 1998 ) and Phalaenopsis [1.9 × 10 5 per petal (∼0.2 g/FW)] ( Lin et al, 2018b ), and Cymbidium orchids (∼3.50 × 10 7 /g FW) ( Ren et al, 2020 ). However, the protoplast isolation efficiency is relatively lower than that of Arabidopsis (∼3.0 × 10 7 /g FW) ( Wu et al, 2009 ), maize (1–5 × 10 6 /g FW) ( Cao et al, 2014 ), rice (∼1.0 × 10 7 /g FW) ( Zhang et al, 2011 ), and cassava ( Manihot esculenta Crantz ) (4.4 × 10 7 /g FW) ( Wu et al, 2017 ).…”

Efficient Multiplex Genome Editing Tools identified by Protoplast Technology inPhalaenopsis

“…Until recently, the orchid protoplast transient expression system was reported only in two Phalaenopsis species, Phalaenopsis aphrodite subsp. formosana (m1663) 31 and Phalaenopsis hybrid cultivar 'Ruili Beauty' 32 , and Cymbidium orchid 33 . However, compared to the protoplast technology established in this study, these protoplast isolation systems are suboptimal.…”

“…Thus far, orchid protoplasts have been successfully isolated from Dendrobium 27-29 , and Phalaenopsis [30][31][32] , and the orchid protoplast transient expression system has been established in Phalaenopsis aphrodite subsp. formosana (m1663) , Phalaenopsis hybrid cultivar 'Ruili Beauty' 32 , and Cymbidium orchid 33 . However, these orchid protoplast transient expression technologies have not been applied to screen efficient CRISPR-Cas toolkits for orchid research.…”

A cassava protoplast system for screening genes associated with the response to South African cassava mosaic virus

“…Transforming plant protoplasts instead of whole plants has a relatively shorter turnaround time, and is desirable because protoplasts show comparable cell-independent responses as whole plants [ 85 , 98 ]. Transient transformation of plant protoplasts for rapid gene characterisation is well-established for several plant species [ 1 , 38 , 56 , 68 , 76 , 96 ]. More recently, cassava protoplasts were used for rapid gene characterisation [ 96 ] because they are a demonstrably reliable system for correlating in planta activities.…”