Wendy A Harwood

John Innes Centre · Department of Crop Genetics

Potato is an important part of the traditional Norwegian diet, and the crop faces several challenges with respect to pests and diseases, as well as the increasingly challenging changes in climate. Genome editing may provide tools to improve the resilience of Norwegian potato cultivars to new climate challenges. We have altered the skin colour of tw...

The model plant Nicotiana benthamiana is an increasingly attractive organism for the production of high‐value, biologically active molecules. However, N. benthamiana accumulates high levels of pyridine alkaloids, in particular nicotine, which complicates the downstream purification processes. Here, we report a new assembly of the N. benthamiana gen...

The growth and development of plants are dependent upon the transcription factors. In plants, the basic leucine zipper (bZIP) family of transcription factor is known for its largest size and diversity. Using newly available wheat genome data, we compared our identification of 181 Triticum aestivum bZIP (TabZIP) genes to those reported in earlier st...

To safeguard bread wheat against pests and diseases, breeders have introduced over 200 resistance genes into its genome, thus nearly doubling the number of designated resistance genes in the wheat gene pool¹. Isolating these genes facilitates their fast-tracking in breeding programs and incorporation into polygene stacks for more durable resistance...

Medicago truncatula is the model plant species for studying symbioses with nitrogen-fixing rhizobia and arbuscular mycorrhizae, where edited mutants are invaluable for elucidating the contributions of known genes in these processes. Streptococcus pyogenes Cas9 (SpCas9)-based genome editing is a facile means of achieving loss of function, including...

CRISPR/Cas has been established for targeted mutagenesis in many plant species since 2013, including Brassica napus and Brassica oleracea. Since that time, improvements have been made in terms of efficiency and choice of CRISPR systems. This protocol encompasses improved Cas9 efficiency and an alternative Cas12a system, allowing more challenging an...

Previous studies of gene function rely on the existing natural genetic variation or on induction of mutations by physical or chemical mutagenesis. The availability of alleles in nature, and random mutagenesis induced by physical or chemical means, limits the depth of research. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repea...

The most abundant phenolic compound in Solanaceous plants is chlorogenic acid (CGA), which possesses protective properties such as antimicrobial and antioxidant activities. These properties are particularly relevant when plants are under adverse conditions, such as pathogen attack, excess light, or extreme temperatures that cause oxidative stress....

The model plant Nicotiana benthamiana is an increasingly attractive organism for the production of high-value, biologically active molecules. However, N. benthamiana accumulates high levels of pyridine alkaloids, in particular nicotine, which complicates the downstream purification processes. Here, we report the assembly of an improved N. benthamia...

Plant breeding for centuries has relied on the availability of genetic variation to introduce new desirable traits into crops. Biotechnology has already accelerated the ability to induce and utilize new genetic variation, through approaches such as mutation breeding and using technologies such as marker assisted breeding to rapidly identify the req...

Many plants associate with arbuscular mycorrhizal fungi for nutrient acquisition, while legumes also associate with nitrogen-fixing rhizobial bacteria. Both associations rely on symbiosis signaling and here we show that cereals can perceive lipochitooligosaccharides (LCOs) for activation of symbiosis signaling, surprisingly including Nod factors pr...

Granule-bound starch synthase I (HvGBSSI) is encoded by the barley waxy (Wx-1) gene and is the sole enzyme in the synthesis of amylose. Here, a Wx-1mutant was identified from an ethyl methane sulfonate (EMS)-mutagenizedbarley population. There were two single-base mutations G1086A andA2424G in Wx-1 in the mutant (M2-1105). The G1086A mutation is lo...

Granule-bound starch synthase I (HvGBSSI) is encoded by the barley waxy ( Wx-1 ) gene and is the sole enzyme in the synthesis of amylose. Here, a Wx-1 mutant was identified from an ethyl methane sulfonate (EMS)-mutagenized barley population. There were two single-base mutations G1086A and A2424G in Wx-1 in the mutant (M2-1105). The G1086A mutation...

Monoterpene indole alkaloids (MIAs) are a diverse class of plant natural products that include a number of medicinally important compounds. We set out to reconstitute the pathway for strictosidine, a key intermediate of all MIAs, from central metabolism in Nicotiana benthamiana. A disadvantage of this host is that its rich background metabolism res...

Novel versions of Cas12a, together with optimised guide architecture led to barley target genes being mutagenised in around 90% of transgenic lines. This system also functioned in Brassica oleracea.

Nitrogen (N) is an important element for plant growth and development. Although several studies have examined plants’ response to N deficiency, studies on plants’ response to excess N, which is common in fertilizer-based agrosystems, are limited. Therefore, the aim of this study was to examine the response of barley to excess N conditions, specific...

The granule-bound starch synthase I (GBSSI) encoded by the waxy gene is responsible for amylose synthesis in the endosperm of wheat grains. In the present study, a novel Wx-B1 null mutant line, M3-415, was identified from an ethyl methanesulfonate-mutagenized population of Chinese tetraploid wheat landrace Jianyangailanmai (LM47). The gene sequence...

To safeguard bread wheat against pests and diseases, breeders have introduced over 200 resistance genes into its genome, thus nearly doubling the number of designated resistance genes in the wheat gene pool. Isolating these genes facilitates their fast-tracking in breeding programs and incorporation into polygene stacks for more durable resistance....

We report the first successful, high efficiency use of Lb Cas12a in barley and describe the development and application of two novel Cas12a variants. In total we compared five coding sequence (CDS) variants including the two novel ones and two guide architectures over 5 different target genes using twenty different guides. We found large difference...

We report highly efficient genome editing in Brassica species. We compare the efficiency of targeted mutagenesis using four Streptococcus pyogenes Cas9 ( Sp Cas9) systems in Brassica oleracea and Brassica napus over 3 target genes and five guides to identify two which show a striking improvement to our first published system (Lawrenson et al., 2015...

The wild relatives and progenitors of wheat have been widely used as sources of disease resistance (R) genes. Molecular identification and characterization of these R genes facilitates their manipulation and tracking in breeding programmes. Here, we develop a reference-quality genome assembly of the wild diploid wheat relative Aegilops sharonensis...

Aegilops tauschii, the diploid wild progenitor of the D subgenome of bread wheat, is a reservoir of genetic diversity for improving bread wheat performance and environmental resilience. Here we sequenced 242 Ae. tauschii accessions and compared them to the wheat D subgenome to characterize genomic diversity. We found that a rare lineage of Ae. taus...

In this study, a range of barley allelic mutants lost ADPG binding structure of starch synthase IIa (SSIIa) were created through targeted mutagenesis of SSIIa by RNA-guided Cas9. The transcriptomic and qRT-PCR results showed the increased mRNA expression of HvGBSSI and the decreased HvSSIIa and HvSBEI levels in ssIIa mutant grains, which were consi...

The wild relatives and progenitors of wheat have been widely used as sources of disease resistance ( R ) genes. Molecular identification and characterization of these R genes facilitates their manipulation and tracking in breeding programmes. We developed a reference-quality genome assembly of the wild diploid wheat relative Aegilops sharonensis an...

The APETALA2/Ethylene-Responsive factor (AP2/ERF) gene family is a large plant-specific transcription factor family, which plays important roles in regulating plant growth and development. A role in starch synthesis is among the multiple functions of this family of transcription factors. Barley (Hordeum vulgare L.) is one of the most important cere...

The APETALA2/Ethylene-Responsive factor (AP2/ERF) gene family is a large plant-specific transcription factor family, which plays important roles in regulating plant growth and development. A role in starch synthesis is among the multiple functions of this family of transcription factors. Barley (Hordeum vulgare L.) is one of the most important cere...

Advances in the use of RNA-guided Cas9-based genome editing in plants have been rapid over the last few years. A desirable application of genome editing is gene targeting (GT), as it allows a wide range of precise modifications; however, this remains inefficient especially in key crop species. Here, we describe successful, heritable gene targeting...

Discoveries in model plants grown under optimal conditions can provide important directions for crop improvement. However, it is important to verify whether results can be translated to crop plants grown in the field. In this study, we sought to study the role of MYB28 in the regulation of aliphatic glucosinolate (A-GSL) biosynthesis and associated...

Wheat, though a key crop plant with considerable influence on world food security, has nonetheless trailed behind other major cereals in the advancement of gene transformation technology for its improvement. New breeding technologies such as genome editing allow precise DNA manipulation, but their potential is limited by low regeneration efficienci...

The development and application of high precision genome editing tools such as programmable nucleases are set to revolutionize crop breeding and are already having a major impact on fundamental science. Clustered regularly interspaced short palindromic repeats (CRISPR), and its CRISPR‐associated protein (Cas), is a programmable RNA‐guided nuclease...

Advances in the use of RNA-guided Cas9-based genome editing in plants have been rapid over the last few years. A desirable application of genome editing is gene targeting (GT), as it allows a wide range of precise modifications, however this remains inefficient especially in key crop species. Here we describe successful, heritable gene targeting in...

Aegilops tauschii, the diploid wild progenitor of the D-subgenome of bread wheat, constitutes a reservoir of genetic diversity for improving bread wheat performance and environmental resilience. To better define and understand this diversity, we sequenced 242 Ae. tauschii accessions and compared them to the wheat D-subgenome. We characterized a rar...

In a ruling on 25 July 2018, the Court of Justice of the European Union concluded that organisms obtained by means of techniques/methods of mutagenesis constitute GMOs in the sense of Directive 2001/18, and that organisms obtained by means of techniques/methods of directed mutagenesis are not excluded from the scope of the Directive. Following the...

In the last 20 years, stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt), has reemerged as a major threat to wheat and barley production in Africa and Europe. In contrast to wheat with 82 designated stem rust (Sr) resistance genes, barley’s genetic variation for stem rust resistance is very narrow with only ten resistance genes g...

We sought to quantify the role of MYB28 in the regulation of aliphatic glucosinolate biosynthesis and associated sulphur metabolism in field-grown B. oleracea with the use of CRISPR-Cas9-mediated gene editing technology. We describe the first characterised myb28 knockout mutant in B. oleracea, and the first DEFRA-regulated and approved CRISPR field...

In wheat/alien species hybrids, pairing homoeologous (Ph1) suppresses the pairing and recombination of wheat and alien chromosomes. Ph1b has been found to be the most effective allele in inducing meiotic homoeologous pairing/recombination and is widely used in gene introgression from wild Triticeae species into wheat. However, due to the poor agron...

In barley (Hordeum vulgare L.), Agrobacterium-mediated transformation efficiency is highly dependent on genotype with very few cultivars being amenable to transformation. Golden Promise is the cultivar most widely used for barley transformation and developing embryos are the most common donor tissue. We tested whether barley mutants with abnormally...

Understanding the function of genes within staple crops will accelerate crop improvement by allowing targeted breeding approaches. Despite their importance, a lack of genomic information and resources has hindered the functional characterisation of genes in major crops. The recent release of high-quality reference sequences for these crops underpin...

Three EMS mutant wheat lines with varied mobility of Wx-A1 and Wx-B1 were identified. In the mutant lines M2-97-8 and M2-104-2, Wx-B1 showed slightly faster mobility compared to those of wild line, and Wx-A1 in the mutant line M2-101-1 has a greatly faster mobility, which co-migrates with Wx-D1. The results of gene sequencing indicated that SNP mut...

Improving nitrogen use efficiency (NUE) is very important for crops throughout the world. Rice mainly utilizes ammonium as an N source, but it also has four NRT2 genes involved in nitrate transport. The OsNRT2.3b transporter is important for maintaining cellular pH under mixed N supplies. Overexpression of this transporter driven by a ubiquitin pro...

The waxy gene (Wx-1) encodes the granule-bound starch synthase (GBSSI or waxy protein, Wx), which is the key enzyme that catalyzes amylose synthesis in wheat. In this study, tetraploid wheat (Triticum turgidum durum L.) was mutagenized by ethyl methanesulfonate, and M2 populations of 3474 plants were generated. The Wx-A1 orWx-B1 null mutants were i...

In a ruling on 25 July 2018, the Court of Justice of the European Union concluded that organisms obtained by means of techniques/methods of mutagenesis constitute GMOs in the sense of Directive 2001/18, and that organisms obtained by means of techniques/methods of directed mutagenesis are not excluded from the scope of the Directive. Following the...

In barley (Hordeum vulgare L.), Agrobacterium-mediated transformation efficiency is highly dependent on genotype with very few cultivars being amenable to transformation. Golden Promise is the cultivar most widely used for barley transformation and developing embryos are the most common donor tissue. We tested whether barley mutants with abnormally...

To adapt to the challenges of climate change and the growing world population, it is vital to increase global crop production. Understanding the function of genes within staple crops will accelerate crop improvement by allowing targeted breeding approaches. Despite the importance of wheat, which provides 20 % of the calories consumed by humankind,...

To adapt to the challenges of climate change and the growing world population, it is vital to increase global crop production. Understanding the function of genes within staple crops will accelerate crop improvement by allowing targeted breeding approaches. Despite the importance of wheat, which provides 20 % of the calories consumed by humankind,...

[This corrects the article DOI: 10.1186/s13007-019-0503-z.].

In our previous work, a novel high-molecular-weight glutenin subunit (HMW-GS) with an extremely large molecular weight from Aegilops sharonensis was identified that may contribute to excellent wheat (Triticum aestivum) processing quality and increased dough strength, and we further generated HMW-GS homozygous lines by crossing. In this study, we cr...

In this study, we successfully knock-out the d-hordein component of barley storage protein using RNA-guided Cas9. Mutation frequencies of 25% and 14% at two different target sites were obtained. Homozygous mutant plants that were T-DNA free were identified in the T1 generation. Barley grains without d-hordein proteins from T2 seeds showed a signifi...

Background: Despite wheat being a worldwide staple, it is still considered the most difficult to transform out of the main cereal crops. Therefore, for the wheat research community, a freely available and effective wheat transformation system is still greatly needed. Results: We have developed and optimised a reproducible Agrobacterium-mediated...

Phosphatidylinositol transfer protein (PITP) and phosphatidylinositol 4-kinase (PI4K) are very upstream regulatory elements of the phospholipid signaling pathway in the signal transduction network. Unlike in animal systems, their role in stress signaling is poorly understood in plants. To study this area, PITP- and PI4K-overexpressing transgenic ba...

Key message An EMS-induced single-base mutation at a splice site caused abnormal RNA splicing and resulted in the gene inactivation and the lack of Wx-A1 protein in a wheat EMS mutant line. Abstract An EMS-mutagenized population was generated using common wheat cv. SM126 consisting of 10,600 M2 plants. One Wx-A1 null mutant was identified through...

Plant synthetic biology and cereal engineering depends on the controlled expression of transgenes of interest. Most engineering in plant species to date has relied heavily on the use of a few, well‐established constitutive promoters to achieve high levels of expression; however, the levels of transgene expression can also be influenced by the use o...

We investigated whether Cas9‐mediated mutagenesis of starch‐branching enzymes (SBEs) in tetraploid potatoes could generate tuber starches with a range of distinct properties. Constructs containing the Cas9 gene and sgRNAs targeting SBE1, SBE2 or both genes were introduced by Agrobacterium‐mediated transformation or by PEG‐mediated delivery into pro...

In this study, we successfully expressed the active 1Ay subunit of Triticum urartu in barley by Agrobacterium-mediated transformation with a transformation efficiency of 19.9%. The results of SDS–PAGE revealed that the expressed proteins of 1Ay subunit were present at some grains of each of 46 original T0 plants, showing identical mobility to those...

While public and political views on genetic modification (inserting “foreign” genes to elicit new traits) have resulted in limited exploitation of the technology in some parts of the world, the new era of genome editing (to edit existing genes to gain new traits/genetic variation) has the potential to change the biotech landscape. Genome editing of...

Knockout mutants are an invaluable reverse genetics tool which have not been well developed in crop species compared to models like Arabidopsis. However, the emergence of CRISPR/Cas9 has changed this situation making the generation of such mutants accessible to many crops including barley. A single T-DNA construct can be transformed into barley imm...

Barley has a number of unique features among our crop plants. It was one of the earliest plants to be domesticated and continues to play an important role in modern agriculture today. It is a versatile crop, used both for human nutrition and for animal feed, and plays an important role as an experimental model plant allowing advances in plant genet...

Barley transformation is an essential tool for a range of functional genomics studies as well as for future crop improvement. The demand for efficient crop transformation systems continues to grow, with new genome editing technologies adding to that demand. Here we describe an efficient and routine transformation protocol for the spring barley Gold...

New plant breeding technologies, such as genome editing, are enabling new crop varieties to be developed far quicker and with greater precision and scope than achievable using conventional methods. These advances could help farmers address the challenges of climate change, sustainability, and global food security. However, despite their potential,...