Fragmentation and asymmetric fusion of Cichorium protoplasts

Cytoplasmic male sterility (CMS) has already been introduced in Cichorium intybus through asymmetric protoplast fusion between chicory and sunflower and symmetric fusion between chicory and endive. Both approaches have significant drawbacks, as respectively GMOs and tetraploids with undesired endive genes are produced. Therefore, we aimed to create asymmetric Cichorium somatic hybrids, that combine a nuclear Cichorium intybus (C. ‘VL52’, C. ‘K1093’) acceptor genome and endive (C. ‘Despa’) or wild chicory (C. ‘Pi531291’, C. ‘Ames22531’) donor mitochondria. To monitor genome fragmentation effects on subsequent regeneration, we used a standard method for plantlet regeneration from Cichorium protoplasts, based on low melting point agarose (LMPA) beads. First, cytoplasmic inactivation of C. ‘VL52’ and C. ‘K1093’ by 1 – 2 mM iodoacetamide (IOA) was attempted; 1.625 mM was the lowest concentration that nearly completely inhibited sustained cell division. Following, we monitored the effects of 1-30 min UV-C irradiation (254 nm, 13.4 W) on protoplast survival of wild chicory and endive. Microcolony formation after 14 days significantly diminished after 2 – 4 min of irradiation, depending on the genotype; after 6 min of irradiation microcolony formation decreased from 55% to 14% (C. ‘Pi531291’) or from 7% to less than 2% (C. ‘Despa’). In a next phase, three different fusions (‘VL52’ + ‘Despa’, ‘VL52’ + ‘Pi531291’ and ‘K1093’ + ‘Ames22531’) were made by combining 1.625 mM treated acceptors with 1 – 15 min UV-C irradiated donors, using a standard PEG mediated chemical fusion protocol. Prior to regenerated plant characterization, a screening tool was developed based on high resolution melting (HRM) analysis of mitochondrial and chloroplast DNA fragments containing insertions, deletions, or single nucleotide polymorphisms between the fusion partners. ‘VL52’ + ‘Despa’, ‘VL52’ + ‘Pi531291’ and ‘K1093’ + ‘Ames22531’ yielded 176, 213 and 128 regenerants after asymmetric fusion, respectively. Within the first group, HRM analysis demonstrated a plasmotype with a mitochondrial mixture of both fusion partners.