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

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.

Efficient Regeneration of Carrot (Daucus carota L.) plants from cell suspension derived-callus

This study succeeded in establishment carrot cell suspension cultures from stem callus in liquidMS medium containing 1.0 mg L-1of each NAA and BA. Its density approach 3.4 ×105cell ml-1atthe third day of culture. These cells ,continued, when embedded in agar drop using MultipleDrop Array (MDA) technique, in division and forming cellular colonies which producingnumerous callus primordia that developed to callus cultures. When transferred to the soliddifferentiation medium (MS+ 1.0 mg L-1 NAA+ 1.5 mg L-1 BA), two hundreds and seventy-threeshoots produced. They readily rooted in agar-solidified MSO medium after three weeks andadapted in conditions of culture room. They were not transferred to field due to the unfavorableenvironmental conditions at that time. Cell suspension-derived callus tissues contained 82.62 μganthocyanin gm-1 of callus fresh weight, and 0.555 mg beta-carotene 100 gm-1of callus freshweight in the third period of extraction compared with their quantity calculated in stems calluswhich recorded 52.4 μg gm-1and 1.988 mg gm-1respectively.