Abstract
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.
Highlights
Somatic hybridisation in the carrot, as in other plant species, enables the development of novel plants with unique characteristics
We developed a set of carrot lines whose cells had fluorescently labelled mitochondria by different fluorescent protein (FP), and used them as sources of protoplasts for somatic hybridisation
We demonstrate that real-time observation of protoplast fluorescence during electrofusion enables the assessment of the effect of various direct current (DC) parameters on protoplast stability and hybrid formation, and the optimisation of these parameters for somatic hybridisation
Summary
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. 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. The application of electric current, instead of chemical inducers, prevents the loss of protoplasts and hybrids that commonly occurs during cell washing of fusogens, so the fusion efficiency may be h igher[27]
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