Optimization of protoplast isolation, transformation and its application in sugarcane (Saccharum spontaneum L)
Qiongli Wang,
Guangrun Yu,
Zhiyong Chen,
Jinlei Han,
Yufang Hu,
Kai Wang
Affiliations
Qiongli Wang
Key Laboratory of Genetics, Breeding and Multiple Utilization of Crops, Ministry of Education/Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Center for Genomics and Biotechnology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China; Corresponding author.
Guangrun Yu
Key Laboratory of Genetics, Breeding and Multiple Utilization of Crops, Ministry of Education/Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Center for Genomics and Biotechnology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
Zhiyong Chen
Key Laboratory of Genetics, Breeding and Multiple Utilization of Crops, Ministry of Education/Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Center for Genomics and Biotechnology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
Jinlei Han
Key Laboratory of Genetics, Breeding and Multiple Utilization of Crops, Ministry of Education/Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Center for Genomics and Biotechnology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
Yufang Hu
Key Laboratory of Genetics, Breeding and Multiple Utilization of Crops, Ministry of Education/Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Center for Genomics and Biotechnology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
Kai Wang
Key Laboratory of Genetics, Breeding and Multiple Utilization of Crops, Ministry of Education/Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Center for Genomics and Biotechnology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China; National Engineering Research Center of Sugarcane, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China; Correspondence to: Kai Wang, National Engineering Research Center of Sugarcane, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China.
Sugarcane is a prominent source of sugar and ethanol production. Genetic analysis for trait improvement of sugarcane is greatly hindered by its complex genome, long breeding cycle, and recalcitrance to genetic transformation. The protoplast-based transient transformation system is a versatile and convenient tool for in vivo functional gene analysis; however, quick and effective transformation systems are still lacking for sugarcane. Here, we developed an efficient protoplast-based transformation system by optimizing conditions of protoplasts isolation and PEG-mediated transformation in S. spontaneum. The yield of viable protoplasts was approximately 1.26 × 107 per gram of leaf material, and the transformation efficiency of 80.19% could be achieved under the optimized condition. Furthermore, using this approach, the nuclear localization of an ABI5-like bZIPs transcription factor was validated, and the promoter activity of several putative DNase I hypersensitive sites (DHSs) was assessed. The results indicated this system can be conveniently applied to protein subcellular localization and promoter activity assays. A highly efficient S. spontaneum mesophyll cell protoplast isolation and transient transformation method was developed, and it shall be suitable for in vivo functional gene analysis in sugarcane.
