Efficient isolation of protoplasts from freesia callus and its application in transient expression assays
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Freesia hybrida is one of the most famous cut flower variety domesticated from wild species in Freesia genus, distinguished for its colorful flower colors and sweet scents. It has potential to be a model plant to unravel the molecular basis of plant specialized metabolites biosynthesis in family Iridaceae, which are crucial to genetically modify ornamental traits such as flower color and flower scents. However, the lengthy life cycle propagated from tissue culture and extremely low transformation rate severely decelerate functional characterization of genes through transgenic method in this iridaceous plant. Using calluses induced from young flower buds from one popular cultivar of Freesia hybrida, ‘Red River®’, an efficient protoplast isolation method was established and optimized, which was further employed in the gene transient expression system. Results demonstrated that the newly explored transient system was versatile and could be applied in numerous aspects, such as protein immunoblotting, enzyme activity, protein subcellular localization, protein–protein interaction, protein–DNA interaction and regulatory gene transregulation property assays. Moreover, calluses induced from another cultivar of Freesia hybrida, ‘Ambiance’ could also be used to isolate protoplasts for gene transient transfection analysis, indicating the wide applicability of this system. Results in this study provided new methods for the elucidation of gene function in Freesia in vivo, which might also be inspirational in other species with long life cycles or hardly to be transformed, such as plants in family Iridaceae.
Freesia protoplast isolation and transfection methods were established and illustrated the utilization in protein immunoblotting, enzyme activity, subcellular localization, protein-protein or protein-DNA interaction, and investigation of transactivation capacities of regulators.
KeywordsFreesia hybrida Protoplast isolation Transient transfection Flavonoid Gene function
We thank Professor Shucai Wang and Dr. Yuzhu Dong for their expertise and helps in the isolation and transfection of plant protoplasts.
XS, YL, XG and LW conceived the study. XS and YL established and optimized the protoplast system and drafted the manuscript. LZ performed the FDA staining, Western blot and quantitative real-time PCR assays. LT performed the enzyme assay and subcellular localization fluorescence measurements. CW and LQ prepared materials, including freesia calluses and vectors. XG supervised the study and critically revised the manuscript. All authors have participated in this research and approved the final manuscript.
This work was supported by the National Natural Science Foundation of China (31570295, 31300271), the China Postdoctoral Science Foundation funded project (2018M641761), the Department of Science and Technology of Jilin Province (20190201299JC, 20190303095SF), the Programme for Introducing Talents to Universities (B07017) and the Fundamental Research Fund for the Central Universities. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Compliance with ethical standards
Conflict of interest
The authors have no conflicts of interest to declare.
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