Genome editing of apple – establishing a workflow for the generation of homohistont mutant lines

Ongoing crop breeding is critical to react to various issues such as climatic changes, altered consumer preferences, the occurrence of new pathogens, and the rapidly growing world population.
Therefore, the use and further development of new breeding technologies, such as CRISPR/Cas-based genome editing (GE), that enable efficient and targeted breeding, are of great interest.
Furthermore, GE also offers a great benefit for breeding research, e.g., for the functional characterization of candidate genes by targeted mutagenesis.
This study aimed to establish a workflow for apple, to obtain stable, homohistont mutant lines.
For this purpose, the GUS reporter gene from a transgenic, nptII marker-free apple line (M2003) was used as a target for mutagenesis by CRISPR/Cas9. A T-DNA construct mediating the expression of the CRISPR/Cas9-system was transformed into M2003. The experimental design allowed the phenotypic detection of mutations resulting in loss-of-function of the GUS reporter activity by histological staining.
Furthermore, GE events were characterized by molecular methods such as PCR, amplicon-deep sequencing, CAPS marker analysis, and Sanger sequencing.
A chimeric composition of regenerants and shoots that exhibited multiple mutation events was observed frequently and revealed the need for de-chimerization.
Using chimeric, genome-edited leaf-like material obtained directly after T-DNA transformation for a second regeneration step, the generation of a stable, homohistont mutant line derived by CRISPR/Cas-mediated GE was demonstrated.

XXXI International Horticultural Congress (IHC2022): International Symposium on Breeding and Effective Use of Biotechnology and Molecular Tools in Horticultural Crops

S. Schröpfer, H. Flachowsky

CRISPR, Cas9, chimerism, mutation, GUS, de-chimerization, regeneration, Malus × domestica

https://doi.org/10.17660/ActaHortic.2023.1362.4