Background: Since its discovery, somatic hybridization has been used to overcome the sexual barriers between cultivated and wild species. A combination of two somatic cells might provide a novel set of features, often of agronomical importance. Here, we report a successful approach for production and selection of interspecific somatic hybrid plants between cultivated and wild carrot using dual-labelling of protoplasts and an early selection of fused cells via micromanipulator. Both subspecies used in this study are characterised by a very high regenerative ability in protoplast cultures. Thus, a precise and effective method of hybrid selection is essential to assure the development and regeneration of much less numerous heterokaryons in the post-fusion cell mixture.
Results: Electrofusion parameters, such as alternating current and direct current, were optimised for an efficient alignment of protoplasts and reversible membrane breakdown followed by a cell fusion. Four hundred twenty-nine cells emitting green-red fluorescence, identified as hybrids, were obtained. Co-culture with donor-derived protoplasts in the alginate feeder layer system stimulated re-synthesis of the cell wall and promoted cell divisions of fusants. Somatic embryogenesis occurred in hybrid-derived microcalli cultures, followed by plant regeneration. Regenerated hybrids produced yellowish storage roots and leaves of an intermediate shape between cultivated and wild subspecies. The intron length polymorphism analysis revealed that 123 of 124 regenerated plants were hybrids.
Conclusions: The developed protocol for protoplast fusion and an early selection of hybrids may serve as an alternative to combining genomes and transferring nuclear or cytoplasmatic traits from wild Daucus species to cultivated carrot.
Keywords: Daucus carota L.; Dual-labelling; Electrofusion; ILP markers; Micromanipulation; Protoplasts.
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