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.

Protoplast Preparation for Algal Single-Cell Omics Sequencing

“…Protoplast preparation and fusion is a standard genetic engineering procedure developed in the early 1960s for plants and algae, which is now integrated with state-of-art genome editing CRISPR [ 11 , 12 , 53 , 54 , 55 ]. In the early stage, this method is based on enzymatic lysis of cell walls and quickly applied to all major algal phyla, including Blue-green algae (cyanobacteria) and green, red, and brown algae [ 10 ].…”

“…Algal protoplasts are cells from which the cell wall is removed, which can then be processed like animal cells for SCS ( Figure 1 ). Fortunately, the methods for generating protoplasts for all major algal phyla have been readily available, as protoplast production and fusion are the starting materials for traditional genetic engineering and state-of-art genome editing with CRISPR ( Clustered Regularly Interspaced Short Palindromic Repeats ) [ 9 , 10 , 11 , 12 ].…”

Protoplasts: From Isolation to CRISPR/Cas Genome Editing Application

“…This strategy became a solution for crop breeders in cases where sexual incompatibility was a barrier or as a means to incorporate traits from wild species into related crops without the need for sexual reproduction (Louzada et al, 1993). Variations on this method have been used to introduce a variety of desired traits into crops, including stress resistance (Hennig et al, 2015), pathogen resistance (Kumari et al, 2020), seedlessness (Wu et al, 2005), male sterility (Bruznican et al, 2021), and increased photosynthetic efficiency (Takahata and Takeda, 1990). This can be performed to create a symmetric cell fusion, in which the complete nuclear genomes of the two species are combined (Narasimhulu et al, 1992;Laiq et al, 1994;Ling and Iwamasa, 1994;Desprez et al, 1995;Kirti et al, 1995); an asymmetric cell fusion, in which DNA fragments or partial chromosomes from one species are introduced into the other (Zhou and Xia 2005;Sigeno et al, 2009); or a cybrid, in which chloroplast or mitochondrial genomes from one species are introduced into cells of another (Kochevenko et al, 2000;Guo et al, 2004).…”

Impact of Genomic and Transcriptomic Resources on Apiaceae Crop Breeding Strategies

“…Recently, to develop alternative CMS lines, an asymmetric protoplast fusion was accomplished in celeriac: carrot, coriander and celery mesophyll protoplasts were used as a donor for protoplast fusion experiments while celeriac suspension cell protoplasts were used as acceptors. The constitution of viable alloplasmic hybrids, although at a very low frequency, was demonstrated [ 58 ]. It is clear that for the success of this methodology, an efficient protocol for protoplast regeneration is a pivotal starting point.…”