Kang Hyunhee, Naing Aung Htay, Park Soon Ki, Chung Mi Young, Kim Chang Kil
Department of Horticulture, Kyungpook National University, Daegu, 41566, Korea.
School of Applied Biosciences, Kyungpook National University, Daegu, 41566, Korea.
Protoplasma. 2023 Jan;260(1):271-280. doi: 10.1007/s00709-022-01776-9. Epub 2022 May 27.
The protocol optimized for Petunia hybrida cv. Mirage Rose produced high protoplast yields in 3 out of other 11 cultivars (Damask White, Dreams White, and Opera Supreme White). Factors optimized in the protoplast transfection process showed that the best transfection efficiency (80%) was obtained using 2.5 × 10 protoplast density, 40% polyethylene glycol (PEG) concentration, 10 µg plasmid DNA, and 15 min of transfection time. Assessing the usability of the protocol for other cultivars (Damask White, Dreams White, and Opera Supreme White), a reasonable protoplast transfection efficiency (⁓50%) was observed in the cultivars Dreams White and Opera Supreme White, with lower efficiency (⁓50%) observed in the cv. Damask White. The transient expression of enhanced green fluorescent protein (eGFP) in the nucleus of the transfected protoplasts of all cultivars was confirmed using PCR. This system could be valuable for genome editing of unwanted genes in petunias using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology. Furthermore, it could contribute to other studies on protein subcellular localization, protein-protein interactions, and functional gene expression in the petunias.
针对矮牵牛品种“幻影玫瑰”优化的方案在其他11个品种（大马士革白、梦幻白和至尊歌剧白）中的3个品种中产生了较高的原生质体产量。原生质体转染过程中优化的因素表明，使用2.5×10的原生质体密度、40%的聚乙二醇（PEG）浓度、10μg质粒DNA和15分钟的转染时间可获得最佳转染效率（80%）。评估该方案对其他品种（大马士革白、梦幻白和至尊歌剧白）的适用性时，在梦幻白和至尊歌剧白品种中观察到了合理的原生质体转染效率（约50%），而在大马士革白品种中观察到的效率较低（约30%）。使用PCR证实了所有品种转染原生质体细胞核中增强型绿色荧光蛋白（eGFP）的瞬时表达。该系统对于利用成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白9（Cas9）技术对矮牵牛中不需要的基因进行基因组编辑可能具有重要价值。此外，它还可以为矮牵牛中蛋白质亚细胞定位、蛋白质-蛋白质相互作用和功能基因表达的其他研究做出贡献。
