Novel protocol for isolation and transient transformation of Rhodes grass (Chloris gayana) protoplasts

Abstract Rhodes grass ( Chloris gayana ) is a warm-season C4 grass currently grown as a forage in some tropical regions, with anticipated future application in areas affected by climate change. However, there are few resources available for this grass, with few genomic resources and only one established transformation system, thus limiting the application of biotechnology methods for its improvement. Protoplast transformation can be used as a time- and resource-efficient way to examine gene pathways and functions of transcriptional elements via proteomics and transcriptomics, and to validate gene constructs. The aim of this work was therefore to establish the first Rhodes grass leaf mesophyll protoplast isolation and transient transformation protocol. A range of protoplast isolation factors were examined, including enzyme quantity and vacuum infiltration time. Up to 4.13 × 10 6 protoplasts were isolated per gram of fresh weight with an average 94.8% viability from an overnight digestion protocol with 4% (w/v) cellulase R10, 0.8% (w/v) Macerozyme R10, and no vacuum infiltration. Transformation conditions were optimized via Taguchi’s orthogonal array, which compared combinations of three levels each of green fluorescent protein (GFP) plasmid DNA quantity, polyethylene glycol (PEG) concentration, and transformation time. A transient transformation efficiency of up to 27.88% was observed by GFP expression. DNA quantity was identified to be the only factor from those tested effecting transformation efficiency ( p < 0.001) in a linear way. Work here represents the first report for Rhodes grass protoplast isolation and transformation, which could facilitate genome editing and transcriptome and proteome studies.