CRISPR/Cas9-mediated mutation of Eil1 transcription factor genes affects exogenous ethylene tolerance and early flower senescence in Campanula portenschlagiana.

Improving tolerance to ethylene-induced early senescence of flowers and fruits is of major economic importance for the ornamental and food industry. Genetic modifications of genes in the ethylene-signalling pathway have frequently resulted in increased tolerance but often with unwanted side effects. Here, we used CRISPR/Cas9 to knockout the function of two CpEil1 genes expressed in flowers of the diploid ornamental plant Campanula portenschlagiana.

CRISPR/Cas9 and Transgene Verification of Gene Involvement in Unfolded Protein Response and Recombinant Protein Production in Barley Grain.

The use of plants as heterologous hosts to produce recombinant proteins has some intriguing advantages. There is, however, the potential of overloading the endoplasmic reticulum (ER) capacity when producing recombinant proteins in the seeds. This leads to an ER-stress condition and accumulating of unfolded proteins.

Anthocyanin synthesis in orange carrot cv. Danvers is activated by transgene expression of the transcription factors DcMYB113_NB and DcEGL1_NB from black carrot cv. Nightbird.

Black carrots are potent sources of anthocyanin for the natural food color industry as their anthocyanins contain very high percentages of acylated anthocyanins which are much more stable than non-acylated anthocyanins. Anthocyanins are synthesized by a specific branch of the phenylpropanoid pathway activated by a triad of R2R3-MYB, bHLH and WD40 transcription factors (TFs). Recent studies in black carrots have elucidated major anthocyanin related structural genes and also regulatory TFs.

Horizontal Stacking of _a Cisgenes in Barley Is a Potent Strategy for Increasing Mature Grain Phytase Activity.

Mature grain phytase activity (MGPA) in the Triticea tribe cereals has evolved through gene duplications and neo-functionalization of the purple acid phosphatase phytase gene () in a common ancestor. Increased gene copy number of the gene expressed during seed development has augmented the MGPA in cereals like rye and wheat. PAPhy_a phytase is highly stable and a potent enzyme in feed.

Modulation of Barley (Hordeum vulgare L.) Grain Protein Sink-Source Relations Towards Human Epidermal Growth Factor Instead of B-hordein Storage Protein.

Seeds have evolutionarily developed to store protein without immediately degrading it and constitute ideal tissues for recombinant protein storage. Unfortunately, the production of recombinant protein in seeds is compromised by low yield as compared to other heterologous expression systems. In order to improve the yield of the human epidermal growth factor (EGF) in barley, protein sink-source relations in the developing grain were modulated towards EGF instead of the barley storage protein.

Heterologous microProtein expression identifies LITTLE NINJA, a dominant regulator of jasmonic acid signaling.

MicroProteins are small, often single-domain proteins that are sequence-related to larger, often multidomain proteins. Here, we used a combination of comparative genomics and heterologous synthetic misexpression to isolate functional cereal microProtein regulators. Our approach identified LITTLE NINJA (LNJ), a microProtein that acts as a modulator of jasmonic acid (JA) signaling.

Cyanidin based anthocyanin biosynthesis in orange carrot is restored by expression of AmRosea1 and AmDelila, MYB and bHLH transcription factors.

The simultaneous expression of AmRosea1 and AmDelila transcription factors from snapdragon can activate the anthocyanin pathway in orange carrots, leading to the synthesis and accumulation of anthocyanins in the taproots. Anthocyanins are phenolic compounds produced in various parts of plants. They are used as natural food dyes and are reported as beneficial antioxidants for humans.

Induced Genetic Variation in Crop Plants by Random or Targeted Mutagenesis: Convergence and Differences.

New Breeding Techniques (NBTs) include several new technologies for introduction of new variation into crop plants for plant breeding, in particular the methods that aim to make targeted mutagenesis at specific sites in the plant genome (NBT mutagenesis). However, following that the French highest legislative body for administrative justice, the Conseil d'État, has sought advice from The Court of Justice of the European Union (CJEU) in interpreting the scope of the genetically modified organisms (GMO) Directive, CJEU in a decision from 2018, stated that organisms modified by these new techniques are not exempted from the current EU GMO legislation. The decision was based in a context of conventional plant breeding using mutagenesis of crop plants by physical or chemical treatments.

Identification of transcription factor genes involved in anthocyanin biosynthesis in carrot (Daucus carota L.) using RNA-Seq.

Background: Anthocyanins are water-soluble colored flavonoids present in multiple organs of various plant species including flowers, fruits, leaves, stems and roots. DNA-binding R2R3-MYB transcription factors, basic helix-loop-helix (bHLH) transcription factors, and WD40 repeat proteins are known to form MYB-bHLH-WD repeat (MBW) complexes, which activates the transcription of structural genes in the anthocyanin pathway. Although black cultivars of carrots (Daucus carota L.

Evaluation of the mature grain phytase candidate HvPAPhy_a gene in barley (Hordeum vulgare L.) using CRISPR/Cas9 and TALENs.

In the present study, we utilized TALEN- and CRISPR/Cas9-induced mutations to analyze the promoter of the barley phytase gene HvPAPhy_a. The purpose of the study was dual, validation of the PAPhy_a enzyme as the main contributor of the mature grain phytase activity (MGPA), as well as validating the importance of a specific promoter region of the PAPhy_a gene which contains three overlapping cis-acting regulatory elements (GCN4, Skn1 and the RY-element) known to be involved in gene expression during grain filling. The results confirm that the barley PAPhy_a enzyme is the main contributor to the MGPA as grains of knock-out lines show very low MGPA.

Barley HvPAPhy_a as transgene provides high and stable phytase activities in mature barley straw and in grains.

The phytase purple acid phosphatase (HvPAPhy_a) expressed during barley seed development was evaluated as transgene for overexpression in barley. The phytase was expressed constitutively driven by the cauliflower mosaic virus 35S-promoter, and the phytase activity was measured in the mature grains, the green leaves and in the dry mature vegetative plant parts left after harvest of the grains. The T -generation of HvPAPhy_a transformed barley showed phytase activity increases up to 19-fold (29 000 phytase units (FTU) per kg in mature grains).

Barley plants over-expressing the NAC transcription factor gene HvNAC005 show stunting and delay in development combined with early senescence.

The plant-specific NAC transcription factors have attracted particular attention because of their involvement in stress responses, senescence, and nutrient remobilization. The HvNAC005 gene of barley encodes a protein belonging to subgroup NAC-a6 of the NAC family. This study shows that HvNAC005 is associated with developmental senescence.

High mature grain phytase activity in the Triticeae has evolved by duplication followed by neofunctionalization of the purple acid phosphatase phytase (PAPhy) gene.

The phytase activity in food and feedstuffs is an important nutritional parameter. Members of the Triticeae tribe accumulate purple acid phosphatase phytases (PAPhy) during grain filling. This accumulation elevates mature grain phytase activities (MGPA) up to levels between ~650 FTU/kg for barley and 6000 FTU/kg for rye.

The barley HvNAC6 transcription factor affects ABA accumulation and promotes basal resistance against powdery mildew.

Barley HvNAC6 is a member of the plant-specific NAC (NAM, ATAF1,2, CUC2) transcription factor family and we have shown previously that it acts as a positive regulator of basal resistance in barley against the biotrophic pathogen Blumeria graminis f. sp. hordei (Bgh).

TAL effector nucleases induce mutations at a pre-selected location in the genome of primary barley transformants.

Transcription activator-like effector nucleases (TALENs) enable targeted mutagenesis in a variety of organisms. The primary advantage of TALENs over other sequence-specific nucleases, namely zinc finger nucleases and meganucleases, lies in their ease of assembly, reliability of function, and their broad targeting range. Here we report the assembly of several TALENs for a specific genomic locus in barley.

Intragenesis and cisgenesis as alternatives to transgenic crop development.

One of the major concerns of the general public about transgenic crops relates to the mixing of genetic materials between species that cannot hybridize by natural means. To meet this concern, the two transformation concepts cisgenesis and intragenesis were developed as alternatives to transgenesis. Both concepts imply that plants must only be transformed with genetic material derived from the species itself or from closely related species capable of sexual hybridization.

Transformation of barley (Hordeum vulgare L.) by Agrobacterium tumefaciens infection of in vitro cultured ovules.

Agrobacterium-mediated transformation of in vitro cultured barley ovules is an attractive alternative to well-established barley transformation methods of immature embryos. The ovule culture system can be used for transformation with and without selection and has successfully been used to transform cultivars other than Golden Promise indicating minor genotype dependency. The method allows for the rapid and direct generation of high-quality transgenic plants where the transgenes are stably expressed and show Mendelian inheritance in subsequent generations.

Cisgenic barley with improved phytase activity.

The cisgenesis concept implies that plants are transformed only with their own genetic materials or genetic materials from closely related species capable of sexual hybridization. Furthermore, foreign sequences such as selection genes and vector-backbone sequences should be absent. We used a barley phytase gene (HvPAPhy_a) expressed during grain filling to evaluate the cisgenesis concept in barley.

Transformation of different barley (Hordeum vulgare L.) cultivars by Agrobacterium tumefaciens infection of in vitro cultured ovules.

Most cultivars of higher plants display poor regeneration capacity of explants due to yet unknown genotypic determined mechanisms. This implies that technologies such as transformation often are restricted to model cultivars with good tissue characteristics. In the present paper, we add further evidence to our previous hypothesis that regeneration from young barley embryos derived from in vitro-cultured ovules is genotype independent.

Transformation of barley (Hordeum vulgare L.) by Agrobacterium tumefaciens infection of in vitro cultured ovules.

We report on a novel transformation procedure for barley by Agrobacterium infection of in vitro cultured ovules. Ovules of the cultivar Golden Promise were isolated a few hours after pollination and infected with the Agrobacterium tumefaciens strain AGL0 carrying the binary vector pVec8-GFP. The vector harboured a hygromycin resistance gene and the green fluorescence protein (GFP) gene.