Field study of the fire-blight-resistant cisgenic apple line C44.4.146.

Cisgenesis, the genetic modification of a plant with genes from a sexually compatible plant, was used to confer fire blight resistance to the cultivar 'Gala Galaxy' by amendment of the resistance gene FB_MR5, resulting in the line C44.4.146. To verify whether cisgenesis changed other tree-, flower- or fruit-related traits, a 5-year field trial was conducted with trees of C44.4.146 and multiple control genotypes, including members of the 'Gala' sports group. None of the 44 investigated tree-, flower- or fruit-related traits significantly differed between C44.4.146 and at least one of the control genotypes in all observation years. However, fruits of C44.4.146 and its wild-type 'Gala Galaxy' from tissue culture were paler in color than fruits of 'Gala Galaxy' that had not undergone tissue culture. There was no significant and consistently detected difference in the fruit flesh and peel metabolome of C44.4.146 compared with the control genotypes. Finally, the disease resistance of C44.4.146 was confirmed also when the fire blight pathogen was inoculated through the flowers. We conclude that the use of cisgenesis to confer fire blight resistance to 'Gala Galaxy' in C44.4.146 did not have unintended effects, and that the in vitro establishment of 'Gala Galaxy' had a greater effect on C44.4.146 properties than its generation applying cisgenesis.

No adverse dietary effect of a cisgenic fire blight resistant apple line on the non-target arthropods Drosophila melanogaster and Folsomia candida.

Genetic modification of apple cultivars through cisgenesis can introduce traits, such as disease resistance from wild relatives, quickly and without crossing. This approach was used to generate the cisgenic apple line C44.4.146, a 'Gala Galaxy' carrying the fire blight resistance gene FB_MR5. In contrast to traditionally bred apple cultivars, genetically modified (GM) plants need to undergo a regulatory risk assessment considering unintended effects before approval for commercial release. To determine potential unintended effects of C44.4.146, we assessed major leaf components and effects on the fitness of the decomposers Drosophila melanogaster (fruit fly) and Folsomia candida (collembolan), which were fed a diet amended with powdered apple leaf material. Leaf material of 'Gala Galaxy', several natural 'Gala' mutants, and the unrelated apple cultivar 'Ladina' were used for comparison. The genetic modification did not alter major leaf components and did not adversely affect survival, growth, or fecundity of the two decomposers. Consistent with previous studies with other GM crops, the differences between conventionally bred cultivars were greater than between the GM line and its non-GM wild type. These data provide a baseline for future risk assessments.

Generation of advanced fire blight-resistant apple (Malus × domestica) selections of the fifth generation within 7 years of applying the early flowering approach.

MAIN CONCLUSION: The approach presented here can be applied to reduce the time needed to introduce traits from wild apples into null segregant advanced selections by one-fourth. Interesting traits like resistances to pathogens are often found within the wild apple gene pool. However, the long juvenile phase of apple seedlings hampers the rapid introduction of these traits into new cultivars. The rapid crop cycle breeding approach used in this paper is based on the overexpression of the birch (Betula pendula) MADS4 transcription factor in apple. Using the early flowering line T1190 and 'Evereste' as source of the fire blight resistance (Fb_E locus), we successfully established 18 advanced selections of the fifth generation in the greenhouse within 7 years. Fifteen individuals showed the habitus expected of a regular apple seedling, while three showed very short internodes. The null segregants possessing a regular habitus maintained the high level of fire blight resistance typical for 'Evereste'. Using SSR markers, we estimated the percentage of genetic drag from 'Evereste' still associated with Fb_E on linkage group 12 (LG12). Eight out of the 18 selections had only 4% of 'Evereste' genome left. Since genotypes carrying the apple scab resistance gene Rvi6 and the fire blight resistance QTL Fb_F7 were used as parents in the course of the experiments, these resistances were also identified in some of the null segregants. One seedling is particularly interesting as, beside Fb_E, it also carries Fb_F7 heterozygously and Rvi6 homozygously. If null segregants obtained using this method will be considered as not genetically modified in Europe, as is already the case in the USA, this genotype could be a very promising parent for breeding new fire blight and scab-resistant apple cultivars in European apple breeding programs.