An improved Nicotiana benthamiana bioproduction chassis provides novel insights into nicotine biosynthesis.

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 genome as well as the generation of low-nicotine lines by CRISPR/Cas9-based inactivation of berberine bridge enzyme-like proteins (BBLs). Triple as well as quintuple mutants accumulated three to four times less nicotine than the respective control lines. The availability of lines without functional BBLs allowed us to probe their catalytic role in nicotine biosynthesis, which has remained obscure. Notably, chiral analysis revealed that the enantiomeric purity of nicotine was fully lost in the quintuple mutants. In addition, precursor feeding experiments showed that these mutants cannot facilitate the specific loss of C6 hydrogen that characterizes natural nicotine biosynthesis. Our work delivers an improved N. benthamiana chassis for bioproduction and uncovers the crucial role of BBLs in the stereoselectivity of nicotine biosynthesis.

Reconstitution of monoterpene indole alkaloid biosynthesis in genome engineered Nicotiana benthamiana.

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 results in the derivatization of some heterologously produced molecules. Here we use transcriptomic analysis to identify glycosyltransferases that are upregulated in response to biosynthetic intermediates and produce plant lines with targeted mutations in the genes encoding them. Expression of the early MIA pathway in these lines produces a more favorable product profile. Strictosidine biosynthesis was successfully reconstituted, with the best yields obtained by the co-expression of 14 enzymes, of which a major latex protein-like enzyme (MLPL) from Nepeta (catmint) is critical for improving flux through the iridoid pathway. The removal of endogenous glycosyltransferases does not impact the yields of strictosidine, highlighting that the metabolic flux of the pathway enzymes to a stable biosynthetic intermediate minimizes the need to engineer the endogenous metabolism of the host. The production of strictosidine in planta expands the range of MIA products amenable to biological synthesis.

An Efficient Agrobacterium-Mediated Transformation Protocol for Hexaploid and Tetraploid Wheat.

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 efficiencies in tissue culture and the lack of transformable genotypes. We developed, in the hexaploid spring wheat cultivar "Fielder," a robust, reproducible Agrobacterium tumefaciens-mediated transformation system with transformation efficiencies of up to 33%. The system requires immature embryos as starting material and includes a centrifugation pretreatment before the inoculation with Agrobacterium. This high-throughput, highly efficient, and repeatable transformation system has been used effectively to introduce genes of interest for overexpression, RNA interference, and CRISPR-Cas-based genome editing. With slight modifications reported here, the standard protocol can be applied to the hexaploid wheat "Cadenza" and the tetraploid durum wheat "Kronos" with efficiencies of up to 4% and 10%, respectively. The system has also been employed to assess the developmental gene fusion GRF-GIF with outstanding results. In our hands, this technology combined with our transformation system improved transformation efficiency to 77.5% in Fielder. This combination should help alleviate the genotype dependence of wheat transformation, allowing new genome-editing tools to be used directly in more elite wheat varieties. © 2021 The Authors. Basic Protocol 1: Growing of donor plants Basic Protocol 2: Transformation of Agrobacterium with vector by electroporation Basic Protocol 3: Starting material collection, sterilization, and embryo inoculation Basic Protocol 4: Selection, regeneration, rooting, and acclimatization of transformants.

CRISPR-Cas9 Based Genome Editing in Wheat.

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 enabling targeted site-specific double stranded breaks in DNA which, when incorrectly repaired, result in gene knockout. The two most widely cultivated wheat types are the tetraploid durum wheat (Triticum turgidum ssp. durum L.) and the hexaploid bread wheat (Triticum aestivum L.). Both species have large genomes, as a consequence of ancient hybridization events between ancestral progenitors. The highly conserved gene sequence and structure of homoeologs among subgenomes in wheat often permits their simultaneous targeting using CRISPR-Cas9 with single or paired single guide RNA (sgRNA). Since its first successful deployment in wheat, CRISPR-Cas9 technology has been applied to a wide array of gene targets of agronomical and scientific importance. The following protocols describe an experimentally derived strategy for implementing CRISRP-Cas9 genome editing, including sgRNA design, Golden Gate construct assembly, and screening analysis for genome edits. © 2021 The Authors. Basic Protocol 1: Selection of sgRNA target sequence for CRISPR-Cas9 Basic Protocol 2: Construct assembly using Golden Gate (MoClo) assembly Basic Protocol 3: Screening for CRISPR-Cas9 genome edits Alternate Protocol: BigDye Terminator reactions for screening of CRISPR-Cas9 genome edits.

The wheat Sr22, Sr33, Sr35 and Sr45 genes confer resistance against stem rust in barley.

In the last 20 years, stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt), has re-emerged as a major threat to wheat and barley production in Africa and Europe. In contrast to wheat with 60 designated stem rust (Sr) resistance genes, barley's genetic variation for stem rust resistance is very narrow with only ten resistance genes genetically identified. Of these, only one complex locus consisting of three genes is effective against TTKSK, a widely virulent Pgt race of the Ug99 tribe which emerged in Uganda in 1999 and has since spread to much of East Africa and parts of the Middle East. The objective of this study was to assess the functionality, in barley, of cloned wheat Sr genes effective against race TTKSK. Sr22, Sr33, Sr35 and Sr45 were transformed into barley cv. Golden Promise using Agrobacterium-mediated transformation. All four genes were found to confer effective stem rust resistance. The barley transgenics remained susceptible to the barley leaf rust pathogen Puccinia hordei, indicating that the resistance conferred by these wheat Sr genes was specific for Pgt. Furthermore, these transgenic plants did not display significant adverse agronomic effects in the absence of disease. Cloned Sr genes from wheat are therefore a potential source of resistance against wheat stem rust in barley.

Correction to: An efficient and reproducible Agrobacterium- mediated transformation method for hexaploid wheat (Triticum aestivum L.).

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

An efficient and reproducible Agrobacterium-mediated transformation method for hexaploid wheat (Triticum aestivum L.).

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 transformation system for the spring wheat cv 'Fielder' that yields transformation efficiencies of up to 25%. We report on some of the important factors that influence transformation efficiencies. In particular, these include donor plant health, stage of the donor material, pre-treatment by centrifugation, vector type and selection cassette. Transgene copy number data for independent plants regenerated from the same original immature embryo suggests that multiple transgenic events arise from single immature embryos, therefore, actual efficiencies might be even higher than those reported. CONCLUSION: We reported here a high-throughput, highly efficient and repeatable transformation system for wheat and this system has been used successfully to introduce genes of interest, for RNAi, over-expression and for CRISPR-Cas9 based genome editing.

Impacts of the EU GMO regulatory framework for plant genome editing.

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, the uptake of these new technologies has been slowed down due to the uncertainty associated with the regulation of genome edited crops. For many European consumers, their view of new breeding technologies is influenced by many factors. Those who have never faced a major food crisis may not sufficiently appreciate the challenges posed by a projected rise of 2 billion in the human population by 2050. In addition, consumers with a regular and plentiful supply of food may not have to consider how their food is produced, or appreciate the challenges EU farmers are already facing to meet future demand. Misleading online articles, questioning the safety and ethics of these "new" biotech foods, can also lead consumers to be reluctant to accept them. Consequently, Europe's mixed view on biotech crops may also be hindering their adoption in countries who have even more to gain from the technology. In this review, we discuss the current data on global and EU GM crop adoption and the potential impact a new wave of crop development may have for agriculture. We reflect on how the EU has viewed GM crops, and we consider the future of both genetic modification (GM) and genome editing (GE) in the EU. We explore lessons learnt from the adoption of GM crops and examine the potential impact the recent decision not to exempt genome edited crops from the EU GMO Directive, will have on EU farmers, scientists, consumers, trading countries, and the rest of the world.

Characterizing standard genetic parts and establishing common principles for engineering legume and cereal roots.

Plant synthetic biology and cereal engineering depend 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 of codon optimization, intron-mediated enhancement and varying terminator sequences. Most of these alternative approaches for regulating transgene expression have only been tested in small-scale experiments, typically testing a single gene of interest. It is therefore difficult to interpret the relative importance of these approaches and to design engineering strategies that are likely to succeed in different plant species, particularly if engineering multigenic traits where the expression of each transgene needs to be precisely regulated. Here, we present data on the characterization of 46 promoters and 10 terminators in Medicago truncatula, Lotus japonicus, Nicotiana benthamiana and Hordeum vulgare, as well as the effects of codon optimization and intron-mediated enhancement on the expression of two transgenes in H. vulgare. We have identified a core set of promoters and terminators of relevance to researchers engineering novel traits in plant roots. In addition, we have shown that combining codon optimization and intron-mediated enhancement increases transgene expression and protein levels in barley. Based on our study, we recommend a core set of promoters and terminators for broad use and also propose a general set of principles and guidelines for those engineering cereal species.

An Introduction to Barley: The Crop and the Model.

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 genetics, plant physiology, plant pathology, plant biochemistry, and more recently in plant biotechnology. In this introductory chapter, the key features of barley as both crop and model plant are considered.

Agrobacterium-Mediated Transformation of Barley Immature Embryos.

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 Golden Promise, based on Agrobacterium-mediated inoculation of immature embryos. This protocol has been widely used for overexpression and RNAi applications and more recently for CRISPR/Cas9 mediated genome editing. Average transformation efficiencies of 25% can be easily achieved.

Creating Targeted Gene Knockouts in Barley Using CRISPR/Cas9.

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 immature embryos and stable transgenic lines regenerated through tissue culture which contain targeted mutations. Mutations are detected in T0 plants and go on in subsequent T1 and T2 generations to segregate from T-DNA, leaving lines which are non-transgenic and carrying a variety of mutations at the target locus. These mutations can be targeted to a particular gene of interest in order to bring about a loss of function creating a knockout mutant.

Agrobacterium-mediated transformation systems of Primula vulgaris.

BACKGROUND: Genetic transformation is a valuable tool and an important procedure in plant functional genomics contributing to gene discovery, allowing powerful insights into gene function and genetically controlled characteristics. Primulaceae species provide one of the best-known examples of heteromorphic flower development, a breeding system which has attracted considerable attention, including that of Charles Darwin. Molecular approaches, including plant transformation give the best opportunity to define and understand the role of genes involved in floral heteromorphy in the common primrose, Primula vulgaris, along with other Primula species. RESULTS: Two transformation systems have been developed in P. vulgaris. The first system, Agrobacterium-mediated vacuum infiltration of seedlings, enables the rapid testing of transgenes, transiently in planta. GUS expression was observed in the cotyledons, true leaves, and roots of Primula seedlings. The second system is based on Agrobacterium tumefaciens infection of pedicel explants with an average transformation efficiency of 4.6%. This transformation system, based on regeneration and selection of transformants within in vitro culture, demonstrates stable transgene integration and transmission to the next generation. CONCLUSION: The two transformation systems reported here will aid fundamental research into important traits in Primula. Although, stable integration of transgenes is the ultimate goal for such analyses, transient gene expression via Agrobacterium-mediated DNA transfer, offers a simple and fast method to analyse transgene functions. The second system describes, for the first time, stable Agrobacterium-mediated transformation of Primula vulgaris, which will be key to characterising the genes responsible for the control of floral heteromorphy.

Gateway®-compatible plant transformation vectors.

Studies in functional genomics and crop improvement programs often rely on the introduction and expression of transgenes in plants. There are two essential components required for in planta transgene expression, a plasmid vector on which the transgene sequence is carried and a delivery system capable of transferring the vector to the target cells. Agrobacterium-mediated plant transformation and the binary plasmid vector system is the preferred method of transgene delivery. The cloning technologies used for DNA manipulation underpin many of these studies. Increased demand for efficient high-throughput transformation systems is driving forward improvements in gene cloning techniques. This chapter gives an overview of Gateway(®)-compatible binary vectors for use in Agrobacterium-mediated transformation systems. It describes a fast, efficient, and robust cloning protocol for the production of an over-expression binary vector using Gateway(®) recombinational cloning.

Wheat (Triticum aestivum L.) transformation using mature embryos.

In most protocols for the Agrobacterium-mediated transformation of wheat, the preferred target tissues are immature embryos. However, transformation methods relying on immature embryos require the growth of plants under controlled conditions to provide a continuous supply of good-quality target tissue. The use of mature embryos as a target tissue has the advantage of only requiring good-quality seed as the starting material. Here we describe a transformation method based on the Agrobacterium-mediated transformation of callus cultures derived from mature wheat embryos of the genotype Bobwhite S56.

Analysis of T-DNA/Host-Plant DNA Junction Sequences in Single-Copy Transgenic Barley Lines.

Sequencing across the junction between an integrated transfer DNA (T-DNA) and a host plant genome provides two important pieces of information. The junctions themselves provide information regarding the proportion of T-DNA which has integrated into the host plant genome, whilst the transgene flanking sequences can be used to study the local genetic environment of the integrated transgene. In addition, this information is important in the safety assessment of GM crops and essential for GM traceability. In this study, a detailed analysis was carried out on the right-border T-DNA junction sequences of single-copy independent transgenic barley lines. T-DNA truncations at the right-border were found to be relatively common and affected 33.3% of the lines. In addition, 14.3% of lines had rearranged construct sequence after the right border break-point. An in depth analysis of the host-plant flanking sequences revealed that a significant proportion of the T-DNAs integrated into or close to known repetitive elements. However, this integration into repetitive DNA did not have a negative effect on transgene expression.

A protocol for high-throughput Agrobacterium-mediated barley transformation.

Agrobacterium-mediated transformation of barley is a valuable tool for determining gene function by over-expression of a gene of interest or by RNAi-based gene silencing. The method is based on the inoculation of immature embryos with Agrobacterium and uses a hygromycin resistance gene to allow selection of transgenic plants. The protocol described leads to average transformation efficiencies of 25 % meaning that large numbers of fertile transgenic plants can be produced.

Overexpression of cytokinin dehydrogenase genes in barley (Hordeum vulgare cv. Golden Promise) fundamentally affects morphology and fertility.

Barley is one of the most important cereal crops grown worldwide. It has numerous applications, but its utility could potentially be extended by genetically manipulating its hormonal balances. To explore some of this potential we identified gene families of cytokinin dehydrogenases (CKX) and isopentenyl transferases, enzymes that respectively irreversibly degrade and synthesize cytokinin (CK) plant hormones, in the raw sequenced barley genome. We then examined their spatial and temporal expression patterns by immunostaining and qPCR. Two CKX-specific antibodies, anti-HvCKX1 and anti-HvCKX9, predominantly detect proteins in the aleurone layer of maturing grains and leaf vasculature, respectively. In addition, two selected CKX genes were used for stable, Agrobacterium tumefaciens-mediated transformation of the barley cultivar Golden Promise. The results show that constitutive overexpression of CKX causes morphological changes in barley plants and prevents their transition to flowering. In all independent transgenic lines roots proliferated more rapidly and root-to-shoot ratios were higher than in wild-type plants. Only one transgenic line, overexpressing CKX under the control of a promoter from a phosphate transporter gene, which is expressed more strongly in root tissue than in aerial parts, yielded progeny. Analysis of several T1-generation plants indicates that plants tend to compensate for effects of the transgene and restore CK homeostasis later during development. Depleted CK levels during early phases of development are restored by down-regulation of endogenous CKX genes and reinforced de novo biosynthesis of CKs.

Electrophoretic and chromatographic evaluation of transgenic barley expressing a bacterial dihydrodipicolinate synthase.

Nutritional quality of human and animal foodstuffs is determined by the content of essential amino acids. Barley is the fourth most important cereal of the world and the second most important cereal grown in the Czech Republic. Cereal grains such as barley contain insufficient levels of some essential amino acids, especially lysine. Dihydrodipicolinate synthase is the key enzyme involved in the regulatory step for lysine biosynthesis. Two constructs pBract214::sTPdapA and pBract214::mdapA containing the dapA gene from Escherichia coli coding for the bacterial dihydrodipicolinate synthase were used for transformation of barley. An Agrobacterium-mediated technique was used for transformation of immature embryos of spring barley cv. Golden Promise. Transgenic barley plants of the T0 and T1 generations were evaluated by PCR, real-time PCR, gel electrophoresis, and Western blot. Amino acid content was analyzed by HPLC after HCl hydrolysis. The lysine content in leaves of the T1 generation plant no. 5/5 was 50% higher than in wild-type plants; the lysine content in seeds of T2 generation plant no. 5/16 was 30% higher than in wild-type seeds of spring barley cv. Golden Promise.

Advances and remaining challenges in the transformation of barley and wheat.

Highly efficient and cost-effective transformation technologies are essential for studying gene function in the major cereal crops, wheat and barley. Demand for efficient transformation systems to allow over-expression, or RNAi-mediated silencing of target genes, is greatly increasing. This is due to technology advances, such as rapid genome sequencing, enhancing the rate of gene discovery and thus leading to a large number of genes requiring functional analysis through transformation pipelines. Barley can be transformed at very high efficiency but the methods are genotype-dependent. Wheat is more difficult to transform, however, recent advances are also allowing the development of high-throughput transformation systems in wheat. For many gene function studies, barley can be used as a model for wheat due to its highly efficient transformation rates and smaller, less complex genome. An ideal transformation system needs to be extremely efficient, simple to perform, inexpensive, genotype-independent, and give the required expression of the transgene. Considerable progress has been made in enhancing transformation efficiencies, controlling transgene expression and in understanding and manipulating transgene insertion. However, a number of challenges still remain, one of the key ones being the development of genotype-independent transformation systems for wheat and barley.