Inger Bæksted Holme

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. The cleavage activity of individual TALENs was first tested in vivo using a yeast-based, single-strand annealing assay. The most efficient TALEN was then selected for barley transformation. Analysis of the resulting transformants showed that TALEN-induced double strand breaks led to the introduction of short deletions at the target site. Additional analysis revealed that each barley transformant contained a range of different mutations, indicating that mutations occurred independently in different cells.

Callus induction and plant regeneration from different explant types of Miscanthus x ogiformis Honda ‘Giganteus’

Different explants of Miscanthus x ogiformis Honda ‘Giganteus’ were tested in order to develop an efficient tissue culture system. Shoot apices, leaf and root sections from in vitro-propagated plants, and leaf and immature inflorescence sections from 6-month-old greenhouse-grown plants were used. The explants were cultured on Murashige and Skoog medium supplemented with 4.5, 13.6, 22.6 or 31.7 μM 2,4-dichlorophenoxyacetic acid. Three types of callus were formed but only one was embryogenic and regenerated plants. Callus induction and formation of embryogenic callus depended on the type and developmental stage of the explants. Shoot apices formed the highest percentage of embryogenic callus. There was a difference in the formation of embryogenic callus between leaf explants from in vitro-propagated shoots and greenhouse-grown plants. The best results were obtained from newly formed leaves of in vitro-propagated shoots and older leaves of greenhouse-grown plants. Immature inflorescences smaller than 2.5 cm produced a higher percentage of embryogenic callus than larger more mature inflorescences. Embryogenic callus derived from immature inflorescences had the highest regeneration capacity. Differences in 2,4-dichlorophenoxyacetic acid concentrations had no significant effect on callus induction, embryogenic callus formation and plant regeneration.

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. The marker gene elimination method was used to obtain marker-free plant lines. Here, the gene of interest and the selection gene are flanked by their own T-DNA borders to allow unlinked integration of the two genes. We analysed the transformants for co-transformation efficiency, increased phytase activities in the grain, integration of the kanamycin resistance gene of the vector-backbone and segregation between the HvPAPhy_a insert and the hygromycin resistance gene. The frequencies of the four parameters imply that it should be possible to select 11 potentially cisgenic T(1) -lines out of the 72 T(0) -lines obtained, indicating that the generation of cisgenic barley is possible at reasonable frequencies with present methods. We selected two potential cisgenic lines with a single extra copy of the HvPAPhy_a insert for further analysis. Seeds from plants homozygous for the insert showed 2.6- and 2.8-fold increases in phytase activities and the activity levels were stable over the three generations analysed. In one of the selected lines, the flanking sequences from both the left and right T-DNA borders were analysed. These sequences confirmed the absence of truncated vector-backbone sequences linked to the borders. The described line should therefore be classified as cisgenic.

Vanillin formation from ferulic acid in Vanilla planifolia is catalysed by a single enzyme

Vanillin is a popular and valuable flavour compound. It is the key constituent of the natural vanilla flavour obtained from cured vanilla pods. Here we show that a single hydratase/lyase type enzyme designated vanillin synthase (VpVAN) catalyses direct conversion of ferulic acid and its glucoside into vanillin and its glucoside, respectively. The enzyme shows high sequence similarity to cysteine proteinases and is specific to the substitution pattern at the aromatic ring and does not metabolize caffeic acid and p-coumaric acid as demonstrated by coupled transcription/translation assays. VpVAN localizes to the inner part of the vanilla pod and high transcript levels are found in single cells located a few cell layers from the inner epidermis. Transient expression of VpVAN in tobacco and stable expression in barley in combination with the action of endogenous alcohol dehydrogenases and UDP-glucosyltransferases result in vanillyl alcohol glucoside formation from endogenous ferulic acid. A gene encoding an enzyme showing 71% sequence identity to VpVAN was identified in another vanillin-producing plant species Glechoma hederacea and was also shown to be a vanillin synthase as demonstrated by transient expression in tobacco.

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. GFP-expressing embryos were isolated from the ovules, regenerated to plants and investigated by Southern blot analysis. Transformation frequencies amounted to 3.1% with hygromycin selection and 0.8% without selection. Mendelian inheritance and stable expression of the GFP gene was confirmed in 18 independent lines over two generations. We conclude that the described technique allows for the rapid and direct generation of high quality transgenic plants.

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). In this study, we use a transgenic approach to constitutively silence HvNAC6 expression, using RNA interference (RNAi), to investigate the in vivo functions of HvNAC6 in basal resistance responses in barley in relation to the phytohormone ABA. The HvNAC6 RNAi plants displayed reduced HvNAC6 transcript levels and were more susceptible to Bgh than wild-type plants. Application of exogenous ABA increased basal resistance against Bgh in wild-type plants, but not in HvNAC6 RNAi plants, suggesting that ABA is a positive regulator of basal resistance which depends on HvNAC6. Silencing of HvNAC6 expression altered the light/dark rhythm of ABA levels which were, however, not influenced by Bgh inoculation. The expression of the two ABA biosynthetic genes HvNCED1 and HvNCED2 was compromised, and transcript levels of the ABA conjugating HvBG7 enzyme were elevated in the HvNAC6 RNAi lines, but this effect was not clearly associated with transgene-mediated resistance. Together, these data support a function of HvNAC6 as a regulator of ABA-mediated defence responses for maintenance of effective basal resistance against Bgh.

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. We investigated the ovule culture ability of four cultivars Femina, Salome, Corniche and Alexis, known to have poor response in other types of tissue culture, and compared that to the data for the model cultivar, Golden Promise. Subsequently, we analyzed the transformation efficiencies of the four cultivars using the protocol for Agrobacterium infection of ovules, previously developed for Golden Promise. Agrobacterium tumefaciens strain AGL0, carrying the binary vector pVec8-GFP harboring a hygromycin resistance gene and the green fluorescence protein (GFP) gene, was used for transformation. The results strongly indicate that the tissue culture response level in ovule culture is genotype independent. However, we did observe differences between cultivars with respect to frequencies of GFP-expressing embryos and frequencies of regeneration from the GFP-expressing embryos under hygromycin selection. The final frequencies of transformed plants per ovule were lower for the four cultivars than that for Golden Promise but the differences were not statistically significant. We conclude that ovule culture transformation can be used successfully to transform cultivars other than Golden Promise. Similar to that observed for Golden Promise, the ovule culture technique allows for the rapid and direct generation of high quality transgenic plants.

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. This is notably more than other cereals. For instance, rice, maize, and oat have MGPAs below 100 FTU/kg. The cloning and characterization of the PAPhy gene complement from wheat, barley, rye, einkorn, and Aegilops tauschii is reported here. The Triticeae PAPhy genes generally consist of a set of paralogues, PAPhy_a and PAPhy_b, and have been mapped to Triticeae chromosomes 5 and 3, respectively. The promoters share a conserved core but the PAPhy_a promoter have acquired a novel cis-acting regulatory element for expression during grain filling while the PAPhy_b promoter has maintained the archaic function and drives expression during germination. Brachypodium is the only sequenced Poaceae sharing the PAPhy duplication. As for the Triticeae, the duplication is reflected in a high MGPA of ~4200 FTU/kg in Brachypodium. The sequence conservation of the paralogous loci on Brachypodium chromosomes 1 and 2 does not extend beyond the PAPhy gene. The results indicate that a single-gene segmental duplication may have enabled the evolution of high MGPA by creating functional redundancy of the parent PAPhy gene. This implies that similar MGPA levels may be out of reach in breeding programs for some Poaceae, e.g. maize and rice, whereas Triticeae breeders should focus on PAPhy_a.

HvDep1 Is a Positive regulator of culm elongation and grain size in barley and impacts yield in an environment-dependent manner

Heterotrimeric G proteins are intracellular membrane-attached signal transducers involved in various cellular processes in both plants and animals. They consist of three subunits denoted as α, β and γ. The γ-subunits of the so-called AGG3 type, which comprise a transmembrane domain, are exclusively found in plants. In model species, these proteins have been shown to participate in the control of plant height, branching and seed size and could therefore impact the harvestable yield of various crop plants. Whether AGG3-type γ-subunits influence yield in temperate cereals like barley and wheat remains unknown. Using a transgenic complementation approach, we show here that the Scottish malting barley cultivar (cv.) Golden Promise carries a loss-of-function mutation in HvDep1, an AGG3-type subunit encoding gene that positively regulates culm elongation and seed size in barley. Somewhat intriguingly, agronomic field data collected over a 12-year period reveals that the HvDep1 loss-of-function mutation in cv. Golden Promise has the potential to confer either a significant increase or decrease in harvestable yield depending on the environment. Our results confirm the role of AGG3-type subunit-encoding genes in shaping plant architecture, but interestingly also indicate that the impact HvDep1 has on yield in barley is both genotypically and environmentally sensitive. This may explain why widespread exploitation of variation in AGG3-type subunit-encoding genes has not occurred in temperate cereals while in rice the DEP1 locus is widely exploited to improve harvestable yield.

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

Highlight HvNAC005 was shown to be a strong positive regulator of senescence, involved in regulation in the cross field of different hormone and signalling pathways controlling developmental senescence in barley.