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.

Isolation, purification and PEG-mediated transient expression of mesophyll protoplasts in Camellia oleifera

“…The effects of PEG incubation time (10,15,20, and 25 min) on the transformation efficiency were analyzed (Fig. 6C).…”

“…In previous studies, protocols for protoplast isolation have been very successful in herbaceous plants such as wheat [ 6 ], maize [ 16 ], rice [ 17 ], carrot [ 10 ], Arabidopsis [ 18 ], and perennial ryegrass [ 19 ]. Nevertheless, in woody plants the development of protoplast isolation technology has only been reported in citrus [ 20 , 21 ], apricot [ 22 ], peach [ 23 ], tea plants [ 24 , 25 ], Populus [ 26 ] and Robinia pseudoacacia L. [ 27 ].…”

“…Plant protoplasts were the living material of plant cells by removing the cell wall and including the protoplasm and plasma membrane [6]. Due to the absence of cell walls, plant protoplasts have been widely used for genetic transformation, cell fusion, and somatic mutation [7][8][9][10]. In addition, plant protoplasts have totipotency and can regenerate into new similar individuals under appropriate conditions [11].…”

Protoplast Technology and Somatic Hybridisation in the Family Apiaceae

“…Recently, Gieniec et al developed a protocol for the real-time detection of somatic hybrids with stable non-toxic fluorescent protein (FP) tagging of mitochondria during electrofusion using carrot protoplasts [79]. Thus, either cyan (eCFP), green (sGFP), yellow (eYFP) or the mCherry variant of red FP (RFP), with a fused mitochondrial targeting sequence, were introduced to carrot cell lines by Agrobacterium-mediated transformation and were subsequently used as a source of protoplasts for electrofusion, after selection to confirm stable labelling.…”

“…They found that the protoplast response and hybrid cell formation depended on DC voltage and pulse time, and varied among protoplast sources. Heterokaryons (GFP + RFP or YFP + RFP) were identified through their dual-color fluorescence [79]. A similar approach based on dual-color fluorescence was developed many years ago for somatic hybridization in protein legumes [94] using two dyes and non GMO material, but this study using FP stable tagging of mitochondria was the first of its kind in carrot.…”