Background: With the accumulating omics data, an efficient and time-saving transient assay to express target genes is desired. Mesophyll protoplasts, maintaining most stress-physiological responses and cellular activities as intact plants, offer an alternative transient assay to study target genes' effects on heat and oxidative stress responses.
Results: In this study, a perennial ryegrass (Lolium perenne L.) mesophyll protoplast-based assay was established to effectively over- or down-regulate target genes. The relative expression levels of the target genes could be quantified using RT-qPCR, and the effects of heat and H2O2-induced oxidative stress on protoplasts' viability could be quantitatively measured. The practicality of the assay was demonstrated by identifying the potential thermos-sensor genes LpTT3.1/LpTT3.2 in ryegrass that over-expressing these genes significantly altered protoplasts' viability rates after heat stress.
Conclusion: This protoplast-based rapid stress regulatory gene identification assay was briefed as 'PRIDA' that will complement the stable genetic transformation studies to rapidly identify candidate stress-regulatory genes in perennial ryegrass and other grass species.
Keywords: Heat stress; Oxidative stress; Protoplast; Ryegrass; Transient gene expression.
© 2024. The Author(s).