Real-time detection of somatic hybrid cells during electrofusion of carrot protoplasts with stably labelled mitochondria

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.

Figure 1

Fluorescence of carrot callus cells obtained after transformation with CFP, GFP, YFP and RFP plasmid vectors. Spread cells (upper row) and single cells at higher magnification (bottom row) with fluorescing mitochondria labelled with FPs and observed using dedicated excitation and emission filter sets. From the left: ‘Amsterdamska’-CFP, DH-GFP, DH-YFP, DH-RFP. Scale bar: 100 μm (upper row), 50 μm (bottom row).

Figure 2

Protoplasts observed in bright field mode (upper row) and in reflected light (bottom row) after excitation with a mercury lamp using dedicated excitation and emission filter sets. From the left: DH-GFP, ‘Koral’-YFP, DH-mCherry. Scale bar of 50 μm is the same for the upper and bottom images.

Figure 3

Schematic effect of DC voltage and electric pulse combinations on ‘Koral’ protoplast integrity and hybrid formation. The number of circles represents the relative frequency of hybrid or single protoplasts.

Figure 4

Identification of the heterofusants after DH-RFP and ‘Amsterdamska’ (A)-GFP protoplast fusion. Hybrid protoplasts were observed in transmitted light and in reflected light using excitation/emission filters matching FP used for labelling. Hybrid in transmitted light (a), hybrid emitting green fluorescence due to GFP present in A-GFP component protoplast (b), hybrid emitting red fluorescence due to RFP present in DH-RFP component protoplast (c), merged images using Image J2x v.2.1.4.7 computer software (https://imagej.net/ImageJ) showing dual-colour fluorescence emitted by both components in the heterofusant (d). Scale bar: 25 μm.

Figure 5

Mean efficiency (%) of heterofusant formation depending on DC voltage and pulse time during electrofusion. Means followed by the same letters do not differ significantly according to the Tukey’s test at P = 0.05; n = 16. Whiskers represent standard errors.

Figure 6

A 20-day-old hybrid cells aggregate after DH-GFP and DH-RFP protoplast fusion. The aggregate was observed in transmitted light and in reflected light using excitation/emission filters matching FP used for labelling. Cell aggregate in transmitted light (a), cells emitting green fluorescence (b), cells emitting red fluorescence (c), merged images using Image J2x v.2.1.4.7 computer software (https://imagej.net/ImageJ) showing dual-colour fluorescence emitted by hybrid cells of the aggregate (d). Scale bar: 100 μm.