Field study to investigate consequences of the genetic modification resulting in the fire blight resistant cisgenic apple line C44.4.146
Embargoed until 2024-12-07
Author
Date
2021Type
- Doctoral Thesis
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Abstract
Apple is the third main produced fruit crop globally, with a production of around 87 million tons in 2019. Apple production relies on a few, mainly disease-susceptible, cultivars, which require the recurrent use of plant protection products during the growing season. One of the most devastating diseases in apple is fire blight, a bacterial disease caused by Erwinia amylovora. The management of fire blight produced estimated annual costs of more than US$5 million in Switzerland between 1993 and 2013. The most effective and sustainable strategy to reduce yield losses caused by E. amylovora is the use of fire blight resistant cultivars. To achieve widespread success, resistant varieties must at least reach the quality level of cultivars currently available on the market. For this goal, breeders must face the challenges of apple's reproductive biology. The long juvenility phase and self-incompatibility of apple make the breeding and selection of new cultivars a long process. This is especially true for the introduction of resistance from wild apple material, where the most effective resistance genes have been identified so far. Cisgenesis exploits genetic modification to transfer genes, including their own regulatory sequences, between sexually compatible plants. Thereby, it allows introducing a disease resistance gene from a wild apple in an established apple cultivar. While conventionally bred varieties always represent a novel genotype that needs to outcompete established cultivars with a superior combination of traits, cisgenic apple lines should resemble its untransformed cultivar except for the added trait. Therefore, cisgenesis could be the ideal tool to rapidly deploy disease resistances in apple production. However, the high regulatory requirements pose a challenge in the use of genetically modified crops in Europe. Although 30 years of research on genetically modified plants have not revealed any particular risks associated with the technology compared to conventionally bred plants, these regulatory requirements have not changed substantially. Nevertheless, each genetically modified apple line has the transferred gene integrated in a different location in the genome, which has the potential for a unique expression pattern and has to be evaluated case-by-case. Therefore, the main goal of this work was to investigate the potential unintended effects of a cisgenic modification on apple in a multi-year field trial. The investigated cisgenic line C44.4.146 was derived from the fire blight susceptible cultivar ‘Gala Galaxy’ and was amended with the fire blight resistance gene FB_MR5 which originated from the wild apple Malus × robusta 5. C44.4.146 carries a single copy of FB_MR5 on chromosome 16 and was found to be resistant to fire blight when young shoots were inoculated with E. amylovora. This thesis describes the most extensive study aiming at the discovery of potential unintended effects of a cisgenic modification in apple. In addition, growing potted trees in the field allowed producing flowers that were inoculated with the pathogen in a quarantine greenhouse to assess if flowers of C44.4.146 were also resistant to fire blight. In Chapter 1 the importance of apple, the process and challenges of apple breeding, the current status of disease resistance breeding and applications of genetic modifications in apple are summarized. Chapter 2 describes the establishment of the field trial and phenotypic assessments of the trees over five years. The design of the field trial gave a unique opportunity to compare trees and fruits of C44.4.146 to its untransformed wild type ‘Gala Galaxy’ as well as to ‘Gala’ and selected ‘Gala’ sports. Sports are spontaneous mutants of a cultivar. They derive from shoots in which one or more mutations in the meristem generated a new or modified phenotype. This phenotype is then maintained by grafting. In order to evaluate the extent of cisgenic modifications in C44.4.146, sports are ideal comparators, because they share the same genetic origin and maintain most of the characteristics of the original cultivar. In total 44 traits were investigated on tree- and fruitlevel and two metabolome studies were carried out in two years. None of the assessed traits and no metabolite feature showed a significantly different expression pattern in C44.4.146 compared to its untransformed wild type and the ‘Gala’-related genotypes in all years of investigation. In addition, the results showed that the in vitro cultivation of ‘Gala Galaxy’ introduced more evident changes than the genetic modification that led to C44.4.146. It was shown that the in vitro cultivation of ‘Gala Galaxy’ resulted in a disruption of a ‘Gala Galaxy’ fruit property, i.e., a significantly reduced area of red over color of the fruits. This reduction in red over color was inherited by C44.4.146. Transient deviations of tree-related traits within the same genotype were also observed, which derived from different propagations before plantation. Finally, greenhouse experiments with field-grown trees confirmed that FB_MR5 conferred fire blight resistance when the trees were inoculated through the flowers. Chapter 3 investigates the molecular changes in C44.4.146 using a multi-omics approach, for generating RNA transcript, protein, and metabolite profiles of leaf material collected in the field. RNA transcript profiling was repeated after three years. The differences found in these profiles between C44.4.146 and its untransformed in vitro grown 'Gala Galaxy' were always within the range of differences found among 'Gala' and its sports. While no protein or metabolite feature were found to be differentially abundant, five and four genes showed a differential expression in C44.4.146 compared to the ‘Gala’-related genotypes. Two out of these genes were identified in both years. As their gene function was mostly unknown, future experiments should investigate the function of these genes to see if the expression of these genes was modulated in a basal defense reaction caused by FB_MR5 prior pathogen recognition or if it is a consequence of the transformation process. Although the results of Chapters 2 and 3 found no clear risk associated to C44.4.146, two sensitive bioassays are described in Chapter 4 and were performed to assess whether cisgenic leaf material from the field affects the fitness of two non-target soil arthropods. Arthropods are suitable for this risk assessment as they are an essential part of the soil ecosystem and sensitive to environmental contaminants. The study showed that leaf material of C44.4.146 did not affect the fitness of Folsomia candida or Drosophila melanogaster larvae. Additionally, the studies revealed that differences of the conventionally bred cultivar were greater than differences between the cisgenic line and its untransformed wild type or ‘Gala’ sports. Finally, Chapter 5 connects the extensive results from five years of phenotypical evaluations including metabolite profiles from fruits (Chapter 2), transcript, protein, and metabolite profiles from leaves (Chapter 3), as well as biosafety studies on leaf material (Chapter 4) to conclude that the cisgenic apple line C44.4.146 is not associated with higher alterations or risks than its in vitro cultured wild type or ‘Gala’ sports. It was shown that in vitro culture and the mutations leading to ‘Gala’ sports can induce more evident differences than the cisgenic modification. Unlike genetically modified plants, tissue cultured plants and sports have not raised biosafety concerns, even though they have the potential to induce molecular and phenotypic alterations. This illustrates the unequal treatment of crops developed through genetic modification and supports that risk assessments should be adapted case-by-case to the plant and not be based on the technology used to generate it. The knowledge gained from the different approaches will be valuable in deciding how to setup future evaluations of cisgenic crops. Next to apple, other perennial, vegetatively propagated, and outcrossing fruit crops will benefit from this knowledge too. All in all, this extensive study on the unintended effects of a cisgenic modification in apple support the use of cisgenesis as complementary breeding tool for the development of improved cultivars. Show more
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https://doi.org/10.3929/ethz-b-000518977Publication status
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Contributors
Examiner: Studer, Bruno
Examiner: Patocchi, Andrea
Examiner: Broggini, Giovanni
Examiner: Krens, Frans A.
Publisher
ETH ZurichOrganisational unit
03969 - Studer, Bruno / Studer, Bruno
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