Department of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow, AL. 29 Listopada 54, 31-425, Krakow, Poland.
Sci Rep. 2020 Nov 2;10(1):18811. doi: 10.1038/s41598-020-75983-w.
Somatic hybridisation in the carrot, as in other plant species, enables the development of novel plants with unique characteristics. This process can be induced by the application of electric current to isolated protoplasts, but such electrofusion requires an effective hybrid cell identification method. This paper describes the non-toxic fluorescent protein (FP) tagging of protoplasts which allows discrimination of fusion components and identification of hybrids in real-time during electrofusion. One of four FPs: cyan (eCFP), green (sGFP), yellow (eYFP) or the mCherry variant of red FP (RFP), with a fused mitochondrial targeting sequence, was introduced to carrot cell lines of three varieties using Agrobacterium-mediated transformation. After selection, a set of carrot callus lines with either GFP, YFP or RFP-labelled mitochondria that showed stable fluorescence served as protoplast sources. Various combinations of direct current (DC) parameters on protoplast integrity and their ability to form hybrid cells were assessed during electrofusion. The protoplast response and hybrid cell formation depended on DC voltage and pulse time, and varied among protoplast sources. Heterofusants (GFP + RFP or YFP + RFP) were identified by detection of a dual-colour fluorescence. This approach enabled, for the first time, a comprehensive assessment of the carrot protoplast response to the applied electric field conditions as well as identification of the DC parameters suitable for hybrid formation, and an estimation of the electrofusion success rate by performing real-time observations of protoplast fluorescence.
体细胞杂交在胡萝卜中，与其他植物物种一样，可以培育出具有独特特性的新型植物。该过程可以通过对分离的原生质体施加电流来诱导，但这种电融合需要有效的杂交细胞识别方法。本文描述了原生质体的无毒荧光蛋白（FP）标记，允许在电融合过程中实时区分融合成分和识别杂种。将四个 FP 中的一个（青色（eCFP）、绿色（sGFP）、黄色（eYFP）或红色 FP（RFP）的 mCherry 变体）与融合的线粒体靶向序列一起引入到三种品种的胡萝卜细胞系中通过农杆菌介导的转化。经过选择，一组具有 GFP、YFP 或 RFP 标记的线粒体的胡萝卜愈伤组织系被用作原生质体来源，这些系表现出稳定的荧光。在电融合过程中，评估了各种直流（DC）参数对原生质体完整性及其形成杂交细胞的能力的影响。原生质体的反应和杂种细胞的形成取决于 DC 电压和脉冲时间，并且在原生质体来源之间有所不同。异核杂种（GFP+RFP 或 YFP+RFP）通过检测双荧光来识别。这种方法首次能够全面评估胡萝卜原生质体对施加电场条件的反应，以及确定适合杂交形成的 DC 参数，并通过实时观察原生质体荧光来估计电融合成功率。
