Photoperiod-1 regulates the wheat inflorescence transcriptome to influence spikelet architecture and flowering time.

Photoperiod insensitivity has been selected by breeders to help adapt crops to diverse environments and farming practices. In wheat, insensitive alleles of Photoperiod-1 (Ppd-1) relieve the requirement of long daylengths to flower by promoting expression of floral promoting genes early in the season; however, these alleles also limit yield by reducing the number and fertility of grain-producing florets through processes that are poorly understood. Here, we performed transcriptome analysis of the developing inflorescence using near-isogenic lines that contain either photoperiod-insensitive or null alleles of Ppd-1, during stages when spikelet number is determined and floret development initiates. We report that Ppd-1 influences the stage-specific expression of genes with roles in auxin signaling, meristem identity, and protein turnover, and analysis of differentially expressed transcripts identified bZIP and ALOG transcription factors, namely PDB1 and ALOG1, which regulate flowering time and spikelet architecture. These findings enhance our understanding of genes that regulate inflorescence development and introduce new targets for improving yield potential.

Increasing amyloplast size in wheat endosperm through mutation of PARC6 affects starch granule morphology.

The determination of starch granule morphology in plants is poorly understood. The amyloplasts of wheat endosperm contain large discoid A-type granules and small spherical B-type granules. To study the influence of amyloplast structure on these distinct morphological types, we isolated a mutant in durum wheat (Triticum turgidum) defective in the plastid division protein PARC6, which had giant plastids in both leaves and endosperm. Endosperm amyloplasts of the mutant contained more A- and B-type granules than those of the wild-type. The mutant had increased A- and B-type granule size in mature grains, and its A-type granules had a highly aberrant, lobed surface. This morphological defect was already evident at early stages of grain development and occurred without alterations in polymer structure and composition. Plant growth and grain size, number and starch content were not affected in the mutants despite the large plastid size. Interestingly, mutation of the PARC6 paralog, ARC6, did not increase plastid or starch granule size. We suggest TtPARC6 can complement disrupted TtARC6 function by interacting with PDV2, the outer plastid envelope protein that typically interacts with ARC6 to promote plastid division. We therefore reveal an important role of amyloplast structure in starch granule morphogenesis in wheat.

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.

Population genomic analysis of Aegilops tauschii identifies targets for bread wheat improvement.

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. tauschii geographically restricted to present-day Georgia contributed to the wheat D subgenome in the independent hybridizations that gave rise to modern bread wheat. Through k-mer-based association mapping, we identified discrete genomic regions with candidate genes for disease and pest resistance and demonstrated their functional transfer into wheat by transgenesis and wide crossing, including the generation of a library of hexaploids incorporating diverse Ae. tauschii genomes. Exploiting the genomic diversity of the Ae. tauschii ancestral diploid genome permits rapid trait discovery and functional genetic validation in a hexaploid background amenable to breeding.

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.

Cas9-mediated mutagenesis of potato starch-branching enzymes generates a range of tuber starch phenotypes.

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 protoplasts. Outcomes included lines with mutations in all or only some of the homoeoalleles of SBE genes and lines in which homoeoalleles carried several different mutations. DNA delivery into protoplasts resulted in mutants with no detectable Cas9 gene, suggesting the absence of foreign DNA. Selected mutants with starch granule abnormalities had reductions in tuber SBE1 and/or SBE2 protein that were broadly in line with expectations from genotype analysis. Strong reduction in both SBE isoforms created an extreme starch phenotype, as reported previously for low-SBE potato tubers. HPLC-SEC and 1 H NMR revealed a decrease in short amylopectin chains, an increase in long chains and a large reduction in branching frequency relative to wild-type starch. Mutants with strong reductions in SBE2 protein alone had near-normal amylopectin chain-length distributions and only small reductions in branching frequency. However, starch granule initiation was enormously increased: cells contained many granules of <4 µm and granules with multiple hila. Thus, large reductions in both SBEs reduce amylopectin branching during granule growth, whereas reduction in SBE2 alone primarily affects numbers of starch granule initiations. Our results demonstrate that Cas9-mediated mutagenesis of SBE genes has the potential to generate new, potentially valuable starch properties without integration of foreign DNA into the genome.

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.

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.

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.

Barley transformation using biolistic techniques.

Microprojectile bombardment or biolistic techniques have been widely used for cereal transformation. These methods rely on the acceleration of gold particles, coated with plasmid DNA, into plant cells as a method of directly introducing the DNA. The first report of the generation of fertile, transgenic barley plants used biolistic techniques. However, more recently Agrobacterium-mediated transformation has been adopted as the method of choice for most cereals including barley. Biolistic procedures are still important for some barley transformation applications and also provide transient test systems for the rapid checking of constructs. This chapter describes methods for the transformation of barley using biolistic procedures and also highlights the use of the technology in transient assays.

Barley transformation using Agrobacterium-mediated techniques.

Methods for the transformation of barley using Agrobacterium-mediated techniques have been available for the past 10 years. Agrobacterium offers a number of advantages over biolistic-mediated techniques in terms of efficiency and the quality of the transformed plants produced. This chapter describes a simple system for the transformation of barley based on the infection of immature embryos with Agrobacterium tumefaciens followed by the selection of transgenic tissue on media containing the antibiotic hygromycin. The method can lead to the production of large numbers of fertile, independent transgenic lines. It is therefore ideal for studies of gene function in a cereal crop system.