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Subcellular Localization of the D27 Protein in Sugarcane (Saccharum spp. Hybrids) Using an Optimized Protoplast Isolation, Purification, and Transient Gene Expression Protocol

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Abstract

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.

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Acknowledgments

This study was supported by Fund for the National Natural Science Foundation of China (31860405); Earmarked Fund for China Agriculture Research System (CARS-170101); Provincial Innovation Team of Sugarcane Germplasm Innovation and New Variety Breeding of Yunnan Academy of Agricultural Sciences (2019HC013); Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences (No. 1630052017020-4); The Applied Basic Research Projects in Yunnan Province (2016FB071); Applied Basic Research Projects of Yunnan Academy of Agricultural Sciences (YJM201705); Overseas Top Talents Project “Sugarcane genetic improvement and extension”; Yunnan Provincial Science and Technology Cooperation Program, China (Yunnan) -Sri Lanka Sugarcane International Joint Research Center (2018IA076); The Key Research and Development Program of Yunnan Province, the Joint Research and Development Center of Sugarcane Variety Improvement in South and Southeast Asia (2019IB008).

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  1. Zhuan-di Wu, Xin Hu and Feng-gang Zan have contributed equally to this work.

Authors and Affiliations

  1. Sugarcane Research Institute, Yunnan Academy of Agricultural Sciences/Yunnan Key Laboratory of Sugarcane Genetic Improvement, Kaiyuan, 661699, People’s Republic of China

    Zhuan-di Wu, Xin Hu, Feng-gang Zan, David M. Burner, Zheng-ying Luo, Jia-yong Liu, Li-ping Zhao, Li Yao, Yong Zhao, Xin-long Liu, Hong-ming Xia, Kun Yang, Jun Zhao, Pei-fang Zhao, Wei Qin, Xue-kuan Chen & Cai-wen Wu

  2. United States Department of Agriculture, Agricultural Research Service, Sugarcane Research Unit, Houma, LA, 70360, USA

    Feng-gang Zan, Yong-bao Pan, Xue-kuan Chen & Cai-wen Wu

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  1. Zhuan-di Wu
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Contributions

CWW and FGZ, ZDW, XKC conceived and designed the experiments. ZDW, FGZ, XH, and ZYL performed the experiments. FGZ, ZDW, and XKC result analysis, and manuscript drafting. YBP, XKC, and DMB revised the manuscript. All authors have read and approved the final manuscript. XKC, JYL, LPZ, LY, YZ, XLL, HMX, KY, JZ, PFZ, and WQ participated in experimental work.

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Correspondence to Xue-kuan Chen or Cai-wen Wu.

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Wu, Zd., Hu, X., Zan, Fg. et al. Subcellular Localization of the D27 Protein in Sugarcane (Saccharum spp. Hybrids) Using an Optimized Protoplast Isolation, Purification, and Transient Gene Expression Protocol. Sugar Tech 23, 316–325 (2021). https://doi.org/10.1007/s12355-020-00879-y

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