Callus Induction from Diverse Explants and Genotypes Enables Robust Transformation of Perennial Ryegrass (Lolium perenne L.)

Genetic transformation of perennial ryegrass (Lolium perenne L.) is critical for fundamental and translational research in this important grass species. It often relies on Agrobacterium-mediated transformation of callus tissue. However, callus induction is restricted to a few genotypes that respond well to tissue culture. Here, we report callus induction from different perennial ryegrass genotypes and explants, such as shoot tips, seeds, and anthers, which were transformed with several plasmids for functional genomics. β-glucuronidase (GUS) histochemical staining showed the LmdsRNAbp promoter sequence was active in stigmas, spikelets, anthers, and leaves. We also transformed calli with plasmids allowing gene silencing and gene knock-out using RNA interference and CRISPR/Cas9, respectively, for which genotypic and phenotypic investigations are ongoing. Using 19 different constructs, 262 transgenic events were regenerated. Moreover, the protocol regenerated a doubled haploid transgenic event from anther-derived calli. This work provides a proof-of-concept method for expanding the range of genotypes amenable to transformation, thus, serving research and breeding initiatives to improve this important grass crop for forage and recreation

Neues Wissen dank alten Apfelsorten – die Schweizerische Apfel-Kernsammlung

Erhaltungssammlungen pflanzengenetischer Ressourcen stellen ein wertvolles Reservoir an genetischer Vielfalt dar. Beim Obst ist die Anzahl der inventarisierten alten Sorten aber meistens zu gross, um alle Sorten ohne einen übermässigen Aufwand erhalten und ausführlich beschreiben zu können. Kernsammlungen stellen die kompakteste Form der Vielfalt einer Kulturart dar. Die Auswahl von Sorten für eine Kernsammlung soll einerseits die genetische und phänotypische Vielfalt wiedergeben, anderseits auch Sorten von öffentlichem Interesse berücksichtigen. Neben der Erhaltung dient eine Kernsammlung besonders der Beschreibung der genetischen Vielfalt und somit der Identifikation von sortenspezifischen Eigenschaften, die von der Züchtung effizient und gezielt genutzt werden können. Insbesondere in der Apfelzüchtung, wo die genetische Vielfalt in den wenigen, kommerziell erfolgreichen Sorten gering ist, ist die Verbreiterung der genetischen Basis ein wichtiges Element, um den Kulturapfel an neue Herausforderungen wie klimatische Veränderungen oder das Auftreten von neuen Krankheiten anzupassen. Um die Apfelzüchtung in der Schweiz weiter voranzubringen, entschied das BLW nach Absprache mit Agroscope und der ETH Zürich eine Schweizerische Apfel-Kernsammlung zu etablieren. Diese besteht aus 330 alten Schweizer Apfelsorten, die zusammen mit heute genutzten Sorten als Referenz an drei klimatisch unterschiedlichen Standorten gepflanzt wurde. Die Beschreibung möglichst vieler Eigenschaften mit Hilfe modernster Technologie soll ermöglichen, interessante Merkmale bezüglich Fruchtqualität sowie Resistenz und Toleranz gegenüber Krankheiten und Klimaveränderungen zu. Einbezug dieser Eigenschaften in der Apfelzüchtung wird die Entwicklung von vielfältigen, robusten und lokal angepassten Sorgen weiter voranbringen

Detection of the fire blight biocontrol agent Bacillus subtilis BD170 (Biopro®) in a Swiss apple orchard

Fire blight, caused by Erwinia amylovora, is a major disease threat to apple, pear and other pome fruit worldwide. The disease is widespread in Europe and has recently become established in Switzerland. Antibiotics are the most effective controls used in North America but these are not permitted for agricultural use in most European countries. A newly registered biological control product Biopro®, based on the antagonist Bacillus subtilis strain BD170, is being used as an alternative strategy for fire blight management. A specific molecular marker was developed for monitoring the spread of this agent on blossoms after Biopro® spray application in a Swiss apple orchard throughout the bloom period for 2years. Direct spraying resulted in efficient primary colonisation of pistils in flowers that were open at the time of treatment. Subsequent bacterial dissemination (secondary colonisation) of flowers that were closed or at bud stage at the time of treatment was observed but was found to be dependent on the timing of treatments relative to bloom stage in the orchard. Foraging honeybees were shown to be disseminators of Biopro®. We also report detection of the biocontrol agent in honey collected from hives where bees were exposed by placing Biopro® at the entrance or in the hatching nest and from hives that were simply placed in sprayed orchards.ISSN:0929-1873ISSN:1573-846

Gnomoniopsis castanea is the main agent of chestnut nut rot in Switzerland

Nuts of sweet chestnut have been an important food source for the alpine population in Switzerland since the Middle Ages and are still valued today for the preparation of traditional food commodities. Nut quality is reduced by insect damage and by various pathogenic fungi. In the last few years, producers and consumers perceived an increase of brown nut rot; while the nut rot agent Gnomoniopsis castanea was reported locally in southern Switzerland, its presence has not been investigated over large areas until now. This study assessed the incidence of brown nut rot and identified the causal agent present in Switzerland. Fully ripened nuts were collected from the main sweet chestnut growing areas of Switzerland. A filamentous fungus morphologically identified as G. castanea was isolated from 10 to 91% of the sampled nuts, despite only 3 to 21% of the sampled nuts showing brown rot symptoms. This fungus was isolated from symptomatic chestnuts as well as from apparently healthy chestnuts. Our results suggest a possible endophytic lifestyle in ripened nuts as well as in branches, leaves and unripe nuts as previously found. Species identity of 45 isolates was confirmed by EF-1alpha, beta-tubulin and ITS sequencing. Concatenation of β-tubulin and calmodulin sequences showed that several haplotypes were present at each sampling locality. No other nut rot pathogens could be isolated in this study, suggesting that G. castanea is the main causal agent of nut rot in Switzerland. The presence of this species is reported for the first time in a site in northern Switzerland. Further studies are needed to assess the influence of meteorological conditions and chestnut varieties on the incidence of G. castanea in order to provide prevention strategies for chestnut growers.ISSN:0031-9465ISSN:1593-209

Microsatellite markers for population studies of the ascomycete Phyllosticta ampelicida, the pathogen causing grape black rot

Grape black rot, caused by the homothallic ascomycete Phyllosticta ampelicida, is a disease originating from North America and is widespread in Europe. To investigate population structure and epidemics of this pathogen, we developed 11 microsatellite markers. A multiplex PCR assay was used to amplify genomic DNA from environmental samples including mummified berries and foliar lesions, and from fungal cultures. Environmental samples were collected from five countries (Switzerland, France, Germany, Luxembourg and the USA), and consisted of 64 different genotypes. Five additional genotypes were identified from pure cultures isolated in Switzerland and Germany. The allele rarefaction approach indicated that French vineyards in the region of Bordeaux displayed the greatest mean allelic richness, probably related to the fact that France is the country where the disease was first reported outside North America, in 1885. Our results also suggest the absence of links between the species/cultivar of Vitis hosts and the infecting P. ampelicida genotypes. This is the first report of development of microsatellite markers and their deployment for population studies of P. ampelicida.ISSN:0031-9465ISSN:1593-209

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.ISSN:0960-7412ISSN:1365-313

High-resolution genetic map of the Rvi15 (Vr2) apple scab resistance locus

ISSN:1380-3743ISSN:1572-978

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

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.ISSN:0032-0935ISSN:1432-204

Development of a Taqman real-time PCR method to quantify nptII in apple lines obtained with ‘established’ or ‘new breeding’ techniques of genetic modification

ISSN:1438-2377ISSN:1438-238