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The transient gene expression of protoplasts in plant is a considerable tool for gene functional research that has been widely used in gene analysis and functional characterization. Therefore, the objectives of this study were to develop a protocol for the isolation and purification of sugarcane protoplasts (Saccharum spp. hybrids), conduct transient PEG-mediated protoplast transfection with D27, and localize the D27 protein in sugarcane protoplasts. Total yield and viability of protoplasts were optimized for enzyme combination, mannitol concentration, and duration and temperature of enzymatic hydrolysis. High production of intact protoplasts (10.94 × 106 protoplasts g−1 FW) and a survival rate of > 80.0% was achieved through enzymatic hydrolysis at constant temperature of 28 °C, 60–70 rpm min−1 for 8 h in a solution containing 2.0% cellulase R-10, 0.5% macerozyme R-10, 0.6% pectolyase Y-23, 20 mM 2-(N-morpholine) ethanesulfonic acid (MES), 20 mM KCl, and 400 mM mannitol (pH 5.7). Using GFP as the reporter gene, the protoplasts were transformed most efficiently with 25% PEG 4000 for 25 min and the ScD27 protein was localized in the chloroplasts. The localization of ScD27 protein in sugarcane protoplast demonstrated that the newly developed protocol was functionally effective. This optimized sugarcane protoplast isolation, purification, and transient expression protocol lays a foundation for future molecular biology research in sugarcane.

The trehalose-6-phosphate synthase (TPS) gene family plays important roles in conferring plant stress resistance, but a comprehensive analysis of the gene family is lacking for sugarcane (Saccharum spp. hybrids). The objective of this study is to document functional classification, evolutionary characterization, and expression profiling of sugarcane TPS gene (ScTPS) family. Nine putative ScTPS genes were identified and assigned to two distinct classes based on gene structure and phylogeny. Phylogenetic analysis showed that 31 TPS genes from Arabidopsis, rice and sugarcane could be divided into five distinct clades, suggesting that there were at least five orthologous groups in monocot and dicot plants. Evolution analysis of TPS genes revealed that TPS family members appeared to have undergone strong negative selection. The strength of the selective pressure differed in most clades, especially Class I TPS genes, experienced significantly stronger negative selection pressure than Class II TPS genes. There were also cis-regulatory elements related to phytohormones and abiotic stresses. Additionally, ScTPS genes were found to exhibit divergent expression in response to simulated drought, salinity, and ABA stresses. Since ScTPS genes function in sugarcane adaptation to environmental stimuli, it might be used as a molecular marker in screening sugarcane germplasm for increased stress resistance.