Functional analysis and expression profiling of HcrVf1 and HcrVf2 for development of scab resistant cisgenic and intragenic apples

Apple scab resistance genes, HcrVf1 and HcrVf2, were isolated including their native promoter, coding and terminator sequences. Two fragment lengths (short and long) of the native gene promoters and the strong apple rubisco gene promoter (PMdRbc) were used for both HcrVf genes to test their effect on expression and phenotype. The scab susceptible cultivar ‘Gala’ was used for plant transformations and after selection of transformants, they were micrografted onto apple seedling rootstocks for scab disease tests. Apple transformants were also tested for HcrVf expression by quantitative RT-PCR (qRT-PCR). For HcrVf1 the long native promoter gave significantly higher expression that the short one; in case of HcrVf2 the difference between the two was not significant. The apple rubisco gene promoter proved to give the highest expression of both HcrVf1 and HcrVf2. The top four expanding leaves were used initially for inoculation with monoconidial isolate EU-B05 which belongs to race 1 of V. inaequalis. Later six other V. inaequalis isolates were used to study the resistance spectra of the individual HcrVf genes. The scab disease assays showed that HcrVf1 did not give resistance against any of the isolates tested regardless of the expression level. The HcrVf2 gene appeared to be the only functional gene for resistance against Vf avirulent isolates of V. inaequalis. HcrVf2 did not provide any resistance to Vf virulent strains, even not in case of overexpression. In conclusion, transformants carrying the apple-derived HcrVf2 gene in a cisgenic as well as in an intragenic configuration were able to reach scab resistance levels comparable to the Vf resistant control cultivar obtained by classical breeding, cv. ‘Santana’

GATE : a simulation toolkit for PET and SPECT

Monte Carlo simulation is an essential tool in emission tomography that can assist in the design of new medical imaging devices, the optimization of acquisition protocols, and the development or assessment of image reconstruction algorithms and correction techniques. GATE, the Geant4 Application for Tomographic Emission, encapsulates the Geant4 libraries to achieve a modular, versatile, scripted simulation toolkit adapted to the field of nuclear medicine. In particular, GATE allows the description of time-dependent phenomena such as source or detector movement, and source decay kinetics. This feature makes it possible to simulate time curves under realistic acquisition conditions and to test dynamic reconstruction algorithms. A public release of GATE licensed under the GNU Lesser General Public License can be downloaded at the address http://www-lphe.epfl.ch/GATE/

Performance and long-term stability of the barley hordothionin gene in multiple transgenic apple lines

Introduction of sustainable scab resistance in elite apple cultivars is of high importance for apple cultivation when aiming at reducing the use of chemical crop protectants. Genetic modification (GM) allows the rapid introduction of resistance genes directly into high quality apple cultivars. Resistance genes can be derived from apple itself but genetic modification also opens up the possibility to use other, non-host resistance genes. A prerequisite for application is the long-term performance and stability of the gene annex trait in the field. For this study, we produced and selected a series of transgenic apple lines of two cultivars, i.e. ‘Elstar’ and ‘Gala’ in which the barley hordothionin gene (hth) was introduced. After multiplication, the GM hth-lines, non-GM susceptible and resistant controls and GM non-hth controls were planted in a random block design in a field trial in 40 replicates. Scab resistance was monitored after artificial inoculation (first year) and after natural infection (subsequent years). After the trial period, the level of expression of the hth gene was checked by quantitative RT-PCR. Four of the six GM hth apple lines proved to be significantly less susceptible to apple scab and this trait was found to be stable for the entire 4-year period. Hth expression at the mRNA level was also stable

Review of Factors Affecting Organogenesis,Somatic Embryogenesis and Agrobacterium tumefaciens-Mediated Transformation of Strawberry

Standardization of an efficient regeneration system for each strawberry genotype is generally an indispensible pre-requisite for the successful development of transgenic plants. In this paper, we review some key factors affecting the regeneration of strawberry plants via adventitious organogenesis or somatic embryogenesis, such as type of explant, growth regulators or dark/light treatmentsPeer reviewe

Genome editing of polyploid crops : prospects, achievements and bottlenecks

Plant breeding aims to develop improved crop varieties. Many crops have a polyploid and often highly heterozygous genome, which may make breeding of polyploid crops a real challenge. The efficiency of traditional breeding based on crossing and selection has been improved by using marker-assisted selection (MAS), and MAS is also being applied in polyploid crops, which helps e.g. for introgression breeding. However, methods such as random mutation breeding are difficult to apply in polyploid crops because there are multiple homoeologous copies (alleles) of each gene. Genome editing technology has revolutionized mutagenesis as it enables precisely selecting targets. The genome editing tool CRISPR/Cas is especially valuable for targeted mutagenesis in polyploids, as all alleles and/or copies of a gene can be targeted at once. Even multiple genes, each with multiple alleles, may be targeted simultaneously. In addition to targeted mutagenesis, targeted replacement of undesirable alleles by desired ones may become a promising application of genome editing for the improvement of polyploid crops, in the near future. Several examples of the application of genome editing for targeted mutagenesis are described here for a range of polyploid crops, and achievements and bottlenecks are highlighted

Towards the production of genetically modified strawberries which are acceptable to consumers

-This manuscript discusses different aspects that are relevant to genetically modified strawberry plants with improved characteristics and ‘acceptable’ to consumers and growers of strawberry. It starts with a consumer acceptance survey, held in Norway, Denmark and the UK, studying public perception of genetic modification in general and specifically of genetically modified strawberries with altered properties. This study revealed that genetically modified plants are better accepted by consumers if only genes from the species itself are used for the genetic modification. Subsequently, the results of a functional analysis of the strawberry polygalacturonase inhibiting protein gene (FaPGIP) are described. This indicates that this gene is a possible candidate to induce resistance to Botrytis cinerea when upregulated in strawberry fruits. For this analysis, the FaPGIP gene was overexpressed in transgenic strawberry plants using the cauliflower mosaic virus 35S (CaMV35S) promoter. This showed that FaPGIP overexpression led to resistance to Botrytis in transgenic leaves. For the generation of intragenic (i.e. genetically modification using native genetic elements only) strawberry plants, a transformation vector was constructed in which FaPGIP was combined with a strawberry fruit-specific promoter and terminator that were isolated from a strawberry expansin gene (FaExp2). This vector also included elements that allow the elimination of (foreign) selectable marker genes after genetically modified plant lines have been established. Using this vector, genetically modified strawberry plants were produced that contained only genes from the species itself, and therefore these plants were called intragenic, rather than transgenic. Unfortunately, further evaluations of the intragenic strawberry plants could not demonstrate any enhanced level of resistance to Botrytis in fruits

Optimisation of droplet digital PCR for determining copy number variation of α-gliadin genes in mutant and gene-edited polyploid bread wheat

Targeted and random mutagenesis of gene families require accurate quantification. Droplet digital PCR (ddPCR) enables high-throughput screening of copy number variation (CNV). We tested the accuracy of ddPCR for CNV analysis in the large α-gliadin gene family, using degenerate primers. First, duplex ddPCR assays measured α-gliadins in diploid (15–17 copies) and tetraploid (70–76 copies) wheat accessions and a corresponding number in resulting Synthetic Hexaploid Wheat, demonstrating linear amplification up to 86–95 genes. Second, we amplified 61 α-gliadin genes in Chinese Spring. Most α-gliadins of the homoeologous chromosomes 6A and 6D were correctly amplified, as corroborated using deletion and nullisomic-tetrasomic lines, but one group of genes from 6B were not amplified with these primers. Third, in Paragon we amplified 61 α-gliadin genes while selected γ-irradiated mutant lines revealed reductions of 25–50%. Finally, using two duplex ddPCR assays, we showed that CRISPR/Cas9-targeting of α-gliadins in Fielder produced indels (1–50 bp) in up to 10 α-gliadin genes plus large deletions (>300 bp) in 20 of 87 amplified α-gliadin genes. ddPCR is suitable for high-throughput screening of CNV and gene-editing-induced mutations in large gene families, in polyploids. In wheat, ddPCR enables screening of gliadins in breeding programs towards hypoimmunogenic gluten for coeliac patients.</p

Expression of anthocyanin biosynthesis-related genes during flower development in Lilium spp.

Sterility of hybrids produced from interspecific hybridization in lilies (Lilium, Liliaceae) is a great limitation in the breeding program, especially for Lilium longiflorum, which only has white-flowered cultivars. Because modification of flower colour in L. longiflorum by conventional breeding is limited by pre- and post-fertilization barriers, we think genetic modification could be used as an alternative in the future. For this, we need to understand what determines white colouration in L. longiflorum and other species and identify the molecular mechanisms regulating flower colour. In this study, we determined the accumulation of anthocyanins and related compounds in flower tissues during flower developmental stages in L. longiflorum cultivar ‘Lincoln’ and in the Oriental hybrid lily cultivars ‘Rialto’, ‘Perth’ and ‘Gran Tourismo’, respectively with white, pink and red flowers. Furthermore, the presence/absence and the expression of eight structural genes (CHSa, CHSb, CHIa, CHIb, F3H, F3’H, DFR, ANS) and three transcription factor genes (MYB12, MYB15, bHLH2) in flower tissues were investigated. Two structural genes (LlLinF3’H and LlLinDFR) and one transcription factor gene (LlLinbHLH2) were not detected in ‘Lincoln’ flowers. In ‘Rialto’, an amino acid substitution in the R2 repeat of LhRiaMYB12 which was previously reported to be responsible for the white flower colour is also found in the LhPerMYB12 of the pink ‘Perth’ flowers. Moreover, LhRiaDFR is present but not expressed in ‘Rialto’ flowers. Accumulation of cyanidin was observed in the flowers of ‘Perth’ and ‘Gran Tourismo’. High amounts of dihydrokaempferol accumulated in flowers of all four lily cultivars confirming the expression and functionality of early structural genes in the pathway. The elevated expression of the structural genes is strongly correlated with the expression of LhMYB12 and LhMYB15. This information can be used in the future to generate new L. longiflorum or Oriental lily hybrid cultivars with novel flower colours

Towards the production of genetically modified strawberries which are acceptable to consumers

This manuscript discusses different aspects that are relevant to genetically modified strawberry plants with improved characteristics and ‘acceptable’ to consumers and growers of strawberry. It starts with a consumer acceptance survey, held in Norway, Denmark and the UK, studying public perception of genetic modification in general and specifically of genetically modified strawberries with altered properties. This study revealed that genetically modified plants are better accepted by consumers if only genes from the species itself are used for the genetic modification. Subsequently, the results of a functional analysis of the strawberry polygalacturonase inhibiting protein gene (FaPGIP) are described. This indicates that this gene is a possible candidate to induce resistance to Botrytis cinerea when upregulated in strawberry fruits. For this analysis, the FaPGIP gene was overexpressed in transgenic strawberry plants using the cauliflower mosaic virus 35S (CaMV35S) promoter. This showed that FaPGIP overexpression led to resistance to Botrytis in transgenic leaves. For the generation of intragenic (i.e. genetically modification using native genetic elements only) strawberry plants, a transformation vector was constructed in which FaPGIP was combined with a strawberry fruit-specific promoter and terminator that were isolated from a strawberry expansin gene (FaExp2). This vector also included elements that allow the elimination of (foreign) selectable marker genes after genetically modified plant lines have been established. Using this vector, genetically modified strawberry plants were produced that contained only genes from the species itself, and therefore these plants were called intragenic, rather than transgenic. Unfortunately, further evaluations of the intragenic strawberry plants could not demonstrate any enhanced level of resistance to Botrytis in fruits

The ROSEA1 and DELILA transcription factors control anthocyanin biosynthesis in Nicotiana benthamiana and Lilium flowers

The activity of anthocyanin biosynthesis genes is regulated at the transcriptional level, thus manipulation of transcription factors (TFs) is an ideal strategy to alter the expression of multiple target genes. In this study, we investigated the effect of introducing ROSEA1 (ROS1, a MYB-type) and DELILA (DEL, a bHLH-type) TFs from snapdragon under control of a flower specific promoter, Floral Binding Protein 1 (FBP1) from petunia into Nicotiana benthamiana flowers and Lilium tepals. The usefulness of the FBP1 promoter was demonstrated by the generation of purplish flowers in otherwise normal-growing plants of N. benthamiana, while the expression by the 35S promoter led to the development of stunted plants with anthocyanins in all parts. N. benthamiana was successfully transformed by ROS1 alone and by a combination of ROS1 + DEL. The observed accumulation of delphinidin corresponded to the expression of NbCHS, NbF3H, NbDFR and NbANS. The effect of ROS1 + DEL on Lilium flower colour was investigated using agroinfiltration. A higher cyanidin accumulation was observed in tepals of the Oriental hybrid lily cv. ‘Perth’ resulting in deeper pink colouration at the infiltrated area. Nevertheless, the introduction of ROS1 + DEL did not produce any phenotypic changes to the white-flowered L. longiflorum cv. ‘Lincoln’ and the white Oriental hybrid lily cv. ‘Rialto’ due to other deficiencies in their anthocyanin biosynthetic pathway. Co-expression of ROS1 + DEL under control of the FBP1 promoter together with active structural anthocyanin biosynthetic genes can result in modification of Lilium flower colour.</p