An asymmetric protoplast fusion and screening method for generating celeriac cybrids

Abstract

Celeriac F₁ hybrid seed production is currently complicated due to the instability of cytoplasmic male sterile lines. To develop alternative alloplasmic CMS lines, an asymmetric protoplast fusion and hybrid screening methodology was established. Celeriac suspension cells protoplasts were used as the acceptor and carrot, coriander and white celery mesophyll protoplasts as the donor for protoplast fusion experiments. Acceptor cytoplasmic inheritance was inhibited by iodoacetamide treatment and donor nuclear genome inheritance was prevented by UV exposure. Protoplasts were selectively stained and fused using electroporation and polyethylene glycol, and candidate hybrid shoots were obtained. One chloroplast and three mitochondrial markers that could distinguish acceptor and donors organelles were used to characterize over 600 plants obtained after fusion events, without identifying any cybrid. In order to increase the testing efficiency a high number of micro plantlets were pooled and hence the presence of the carrot specific Atp1 marker in one of the pooled samples was detected. We demonstrated that fusion took place between celeriac and a carrot indicating that the creation of viable hybrids is possible although at a very low frequency. These findings open the path for new cytoplasmic hybridization and the isolation of novel CMS lines of celeriac.

Figure 1

Celeriac and coriander protoplast fusion. Celeriac protoplasts were stained with FDA (A) and coriander protoplasts with calcein blue (B). Overlap between the photos taken with separate filters showing dual staining (C). All cells were visible in bright field (D). The arrow indicates the hybrid cell. The scale bar is 50 µm.

Figure 2

Discrimination between the plasmotypes of the different genera used during fusion experiments with the HRM marker Cox1. Distinct coloured curves are significantly different as calculated by the Gene Scanning software (Roche Diagnostics). Melting profiles differed between carrot ‘Dolanka’ (green), carrot ‘Parmex’ (red) and celeriac ‘Diamant’ (blue).

Figure 3

Type of samples used for molecular characterization. (A) Rooting plants were pooled by 5 per sample; (B) very tiny plants that were not rooting and were growing in bundles were pooled per jar, (microplant samples). The scale bar is 5 cm.

Figure 4

Alignment of the amplicon Atp1 from the electrofusion D microplant sample to the ‘Diamant’ celeriac acceptor and ‘Dolanka’ carrot donor amplicons. The fusion derived samples is identical to the donor.