Periodic Reporting for period 1 - MalusEdit (Malus genome editing via CRISPR/Cas9 to develop sustainable pest free apples)
Periodo di rendicontazione: 2019-10-16 al 2021-10-15
Apple cultivation is often threatened by various diseases, such as powdery mildew and fire blight. A common practice to preserve plant health and thus apple yield and quality is to use pesticides with a negative impact on human health and the environment. Ancient apple varieties contain all the information on how to resist these pests, but these characteristics are often not found in the commercial varieties. What makes the difference is often only a single letter in the DNA sequence but introducing only this small modification by common breeding practices is impossible. In this EU-funded project, gene editing was used to introduce changes at four targeted gene positions in the plant DNA which are known to confer sensitivity to powdery mildew and fire blight. This was achieved by a DNA-based Agrobacterium-mediated integration of the CRISPR/Cas9 gene-editing machinery in the plant genome. The editing machinery was then removed from the plant genome via a heat-induced recombination system to facilitate the acceptance of the resulting varieties by the customer within a less restrictive regulation of gene-edited and cisgenic varieties within the EU. The resulting plants showed enhanced resistance to powdery mildew and fire blight and can potentially replace sensitive and unsustainable varieties in a few years.
Another important outcome of the MalusEdit project was the development of a procedure to regenerate a whole plant from a single cell. This will allow the use of gene editing applications that do not make any use of DNA intermediates. The regeneration procedure can now be used to speed up apple breeding projects and thus deliver in short time apples that are improved for multiple important agronomical traits towards real sustainable agriculture.
Another important outcome of the MalusEdit project was the development of a procedure to regenerate a whole plant from a single cell. This will allow the use of gene editing applications that do not make any use of DNA intermediates. The regeneration procedure can now be used to speed up apple breeding projects and thus deliver in short time apples that are improved for multiple important agronomical traits towards real sustainable agriculture.
Plants edited in three out of four genes for pathogen sensitivity (Dipm4, Hipm1, and Mlo19) were obtained, the CRISPR/Cas9 gene-editing machinery was successfully eliminated from the plant genome, and edited lines were tested for disease resistance. Superior lines will be further evaluated for resistance and agronomic traits, and potentially registered as new lines with improved resistance to pathogens. Gathered data will be published in a peer-review journal. A protocol to regenerate apple plants from protoplasts was developed and it is now planned to be used to regenerate gene-edited apple lines from single cells edited with DNA-free transfection technologies. The protocol will be published in a peer-review journal.
The stacking of mutations in the MalusEdit project allowed us to obtain plants that not only are resistant to different pests but are also expected to show a stronger and more durable resistance. Gene editing applications to stack mutations at several target DNA sites are still limited and thus our knowledge of the impact of combined targeted modifications on plant physiology is still missing. For the first time, this project provided apple plants that are simultaneously mutated in several S-genes allowing us to study the overall impact of such mutations on plant resistance and agronomic performances.
The single-cell regeneration protocol which was developed during MalusEdit will permit unlocking the full potential of gene editing allowing DNA-free applications of this technology in apple. Within a less restrictive EU regulation on DNA-free gene-edited plants, this regeneration procedure will speed up apple breeding programs aimed at developing sustainable disease-resistant and quality-enhanced apples with a huge positive impact on human health and the environment.
The single-cell regeneration protocol which was developed during MalusEdit will permit unlocking the full potential of gene editing allowing DNA-free applications of this technology in apple. Within a less restrictive EU regulation on DNA-free gene-edited plants, this regeneration procedure will speed up apple breeding programs aimed at developing sustainable disease-resistant and quality-enhanced apples with a huge positive impact on human health and the environment.