An asymmetric protoplast fusion and screening method for generating celeriac cybrids

Silvia Bruznican,Johan Van Huylenbroeck,Ellen De Keyser,Danny Geelen,Hervé De Clercq,Tom Eeckhaut

doi:10.1038/s41598-021-83970-y

Silvia Bruznican, Johan Van Huylenbroeck + Show 4 more

Open Access

https://doi.org/10.1038/s41598-021-83970-y

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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.

Highlights

Celeriac F1 hybrid seed production is currently complicated due to the instability of cytoplasmic male sterile lines
Iodoacetamide (IOA) inactivated B. napus protoplasts fused with X-ray treated B. tournefortii resulted in alloplasmic B.napus cytoplasmic male sterility (CMS) that was caused by mitochondrial gene rearrangements[5]
The establishment of a protocol for asymmetric protoplast fusion of celeriac with related genera is an important step toward the creation of hybrids and cybrids

Summary

Introduction

Celeriac F1 hybrid seed production is currently complicated due to the instability of cytoplasmic male sterile lines. CMS was introgressed in celery from a wild relative as Dawson reported in 1993 to the ‘Grower’ magazine, but is unstable None of these lines are currently used for mass scale seed production. Et al.[8] generated novel CMS types in tomato by fusing Lycopersicon esculentum IOA treated protoplasts with Solanum acaule or Solanum tuberosum γ or X-ray irradiated protoplasts They obtained fertile plants only when the donor was Solanum lycopersicoides, in spite of the presence of donor mitochondria fragments in the acceptor. Et al.[14] reported the regeneration of 3 hybrids after a symmetric fusion between celery mesophyll and Daucus carota (carrot) root protoplasts. Et al.[13] performed asymmetric fusion between IOA treated celery and UV treated carrot protoplasts and regenerated 11 petaloid celery CMS plants

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