Natural and artificial sources of genetic variation used in crop breeding: A baseline comparator for genome editing.

Traditional breeding has successfully selected beneficial traits for food, feed, and fibre crops over the last several thousand years. The last century has seen significant technological advancements particularly in marker assisted selection and the generation of induced genetic variation, including over the last few decades, through mutation breeding, genetic modification, and genome editing. While regulatory frameworks for traditional varietal development and for genetic modification with transgenes are broadly established, those for genome editing are lacking or are still evolving in many regions. In particular, the lack of "foreign" recombinant DNA in genome edited plants and that the resulting SNPs or INDELs are indistinguishable from those seen in traditional breeding has challenged development of new legislation. Where products of genome editing and other novel breeding technologies possess no transgenes and could have been generated via traditional methods, we argue that it is logical and proportionate to apply equivalent legislative oversight that already exists for traditional breeding and novel foods. This review analyses the types and the scale of spontaneous and induced genetic variation that can be selected during traditional plant breeding activities. It provides a base line from which to judge whether genetic changes brought about by techniques of genome editing or other reverse genetic methods are indeed comparable to those routinely found using traditional methods of plant breeding.

Genetic Transformation of Protoplasts Isolated from Leaves of Lolium temulentum and Lolium perenne.

Transient expression of inserted recombinant DNA in plant protoplasts is a widely used tool for functional genomics research. Recently it has been utilized to screen potential sgRNA guides for CRISPR-mediated genome editing. However, little research has been conducted into the use of transient expression of protoplasts in Lolium perenne (a globally important pasture, hay, and amenity grass), and no studies have been conducted into Lolium temulentum (a weed in cereal crops but a potentially useful model species for Lolium research). In this chapter, we describe a methodology of protoplast extraction and transformation from 14-day-old leaf mesophyll cells from L. perenne and L. temulentum. We believe this is the first report of a procedure for obtaining high density, viable protoplasts from L. temulentum. The method of polyethylene glycol (PEG)-mediated transformation is also described to achieve genetic transformation of protoplasts.

Future-proofing regulation for rapidly changing biotechnologies.

Our understanding of DNA structure and how it interacts with the environment to give form and function at the organism level is growing at an unprecedented pace which shows no sign of slowing. These developments have already led to many new products and will continue to underpin as yet unpredicted future developments in biotechnology. However, this potential will not be realised unless the mechanisms for risk assessment, regulatory approval, product claims and labelling etc. are fit for purpose, have the confidence of all stakeholders and are sufficiently agile to support this rapidly changing field. The sectors that are making particular advances in biotechnological processes include agriculture, pharmaceuticals, food, chemical and human diagnostics and therapeutics. In many of these areas the research, investment and innovation pipeline is operating well as evidenced by the many marketed products. However, developments in plant breeding methods have posed particular challenges for regulators which in turn is stifling R&D and innovation, particularly in the EU. In rapidly moving areas of research and development, it is imperative that regulatory frameworks are future-proofed by design.

Increased SBPase activity improves photosynthesis and grain yield in wheat grown in greenhouse conditions.

To meet the growing demand for food, substantial improvements in yields are needed. This is particularly the case for wheat, where global yield has stagnated in recent years. Increasing photosynthesis has been identified as a primary target to achieve yield improvements. To increase leaf photosynthesis in wheat, the level of the Calvin-Benson cycle enzyme sedoheptulose-1,7-biphosphatase (SBPase) has been increased through transformation and expression of a Brachypodium distachyon SBPase gene construct. Transgenic lines with increased SBPase protein levels and activity were grown under greenhouse conditions and showed enhanced leaf photosynthesis and increased total biomass and dry seed yield. This showed the potential of improving yield potential by increasing leaf photosynthesis in a crop species such as wheat. The results are discussed with regard to future strategies for further improvement of photosynthesis in wheat.This article is part of the themed issue 'Enhancing photosynthesis in crop plants: targets for improvement'.

Defining genetic and chemical diversity in wheat grain by 1H-NMR spectroscopy of polar metabolites.

SCOPE: The application of high-throughput 1H nuclear magnetic resonance (1H-NMR) of unpurified extracts to determine genetic diversity and the contents of polar components in grain of wheat. METHODS AND RESULTS: Milled whole wheat grain was extracted with 80:20 D2 O:CD3 OD containing 0.05% d4 -trimethylsilylpropionate. 1H-NMR spectra were acquired under automation at 300°K using an Avance Spectrometer operating at 600.0528 MHz. Regions for individual metabolites were identified by comparison to a library of known standards run under identical conditions. The individual 1H-NMR peaks or levels of known metabolites were then compared by Principal Component Analysis using SIMCA-P software. CONCLUSIONS: High-throughput 1H-NMR is an excellent tool to compare the extent of genetic diversity within and between wheat species, and to quantify specific components (including glycine betaine, choline, and asparagine) in individual genotypes. It can also be used to monitor changes in composition related to environmental factors and to support comparisons of the substantial equivalence of transgenic lines.

Visualisation of plastid degradation in sperm cells of wheat pollen.

Like most angiosperms, wheat (Triticum aestivum) shows maternal inheritance of plastids. It is thought that this takes place by cytoplasmic stripping at fertilisation rather than the absence of plastids in sperm cells. To determine the fate of plastids during sperm cell development, plastid-targeted green fluorescent protein was used to visualise these organelles in nuclear transgenic wheat lines. Fewer than thirty small 1-2-µm plastids were visible in early uninucleate pollen cells. These dramatically increased to several hundred larger (4 µm) plastids during pollen maturation and went through distinct morphological changes. Only small plastids were visible in generative cells (n = 25) and young sperm cells (n = 9). In mature sperm cells, these green fluorescent protein (GFP)-tagged plastids were absent. This is consistent with maternal inheritance of plastids resulting from their degradation in mature sperm cells in wheat.

Potential impact of genome editing in world agriculture.

Changeable biotic and abiotic stress factors that affect crop growth and productivity, alongside a drive to reduce the unintended consequences of plant protection products, will demand highly adaptive farm management practices as well as access to continually improved seed varieties. The former is limited mainly by cost and, in theory, could be implemented in relatively short time frames. The latter is fundamentally a longer-term activity where genome editing can play a major role. The first targets for genome editing will inevitably be loss-of-function alleles, because these are straightforward to generate. In addition, they are likely to focus on traits under simple genetic control and where the results of modification are already well understood from null alleles in existing gene pools or other knockout or silencing approaches such as induced mutations or RNA interference. In the longer term, genome editing will underpin more fundamental changes in agricultural performance and food quality, and ultimately will merge with the tools and philosophies of synthetic biology to underpin and enable new cellular systems, processes and organisms completely. The genetic changes required for simple allele edits or knockout phenotypes are synonymous with those found naturally in conventional breeding material and should be regulated as such. The more radical possibilities in the longer term will need societal engagement along with appropriate safety and ethical oversight.

Generation of Marker- and/or Backbone-Free Transgenic Wheat Plants via Agrobacterium-Mediated Transformation.

Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide resistance genes to the weedy relatives are perceived as major biosafety concerns in genetically modified (GM) crops. In this study, five novel vectors which used gusA and bar as a reporter gene and a selection marker gene, respectively, were constructed based on the pCLEAN dual binary vector system. Among these vectors, 1G7B and 5G7B carried two T-DNAs located on two respective plasmids with 5G7B possessing an additional virGwt gene. 5LBTG154 and 5TGTB154 carried two T-DNAs in the target plasmid with either one or double right borders, and 5BTG154 carried the selectable marker gene on the backbone outside of the T-DNA left border in the target plasmid. In addition, 5BTG154, 5LBTG154, and 5TGTB154 used pAL154 as a helper plasmid which contains Komari fragment to facilitate transformation. These five dual binary vector combinations were transformed into Agrobacterium strain AGL1 and used to transform durum wheat cv Stewart 63. Evaluation of the co-transformation efficiencies, the frequencies of marker-free transgenic plants, and integration of backbone sequences in the obtained transgenic lines indicated that two vectors (5G7B and 5TGTB154) were more efficient in generating marker-free transgenic wheat plants with no or minimal integration of backbone sequences in the wheat genome. The vector series developed in this study for generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium-mediated transformation will be useful to facilitate the creation of "clean" GM wheat containing only the foreign genes of agronomic importance.

GM foods: is there a way forward?

There are many quality targets in cereals that could generate step-change improvements in nutritional or food-processing characteristics. For instance, levels of acrylamide, soluble and insoluble fibre, antioxidants, allergens and intolerance factors in food are, to a large extent, determined by the genetics of the raw materials used. However, improvements to these traits pose significant challenges to plant breeders. For some traits, this is because the underlying genetic and biochemical basis of the traits is not fully understood but for others, there is simply a lack of natural genetic variation in commercially useful germplasm. One strategy to overcome the latter hindrance is to use wide crosses with more exotic germplasm; however, this can bring other difficulties such as yield loss and linkage drag of deleterious alleles. As DNA sequencing becomes cheaper and faster, it drives the research fields of reverse genetics and functional genomics which in turn will enable the incorporation of desirable traits into crop varieties via molecular breeding and biotechnology. I will discuss the evolution of these techniques from conventional genetic modification to more recent developments in targeted gene editing and the potential of biotechnology to complement conventional breeding methods. I will also discuss the role of risk assessment and regulation in the commercialisation of GM crops.

The first crop plant genetically engineered to release an insect pheromone for defence.

Insect pheromones offer potential for managing pests of crop plants. Volatility and instability are problems for deployment in agriculture but could be solved by expressing genes for the biosynthesis of pheromones in the crop plants. This has now been achieved by genetically engineering a hexaploid variety of wheat to release (E)-ß-farnesene (Eßf), the alarm pheromone for many pest aphids, using a synthetic gene based on a sequence from peppermint with a plastid targeting amino acid sequence, with or without a gene for biosynthesis of the precursor farnesyl diphosphate. Pure Eßf was produced in stably transformed wheat lines with no other detectable phenotype but requiring targeting of the gene produced to the plastid. In laboratory behavioural assays, three species of cereal aphids were repelled and foraging was increased for a parasitic natural enemy. Although these studies show considerable potential for aphid control, field trials employing the single and double constructs showed no reduction in aphids or increase in parasitism. Insect numbers were low and climatic conditions erratic suggesting the need for further trials or a closer imitation, in the plant, of alarm pheromone release.

Molecular characterization of two isoforms of a farnesyl pyrophosphate synthase gene in wheat and their roles in sesquiterpene synthesis and inducible defence against aphid infestation.

Aphids are important pests of wheat (Triticum aestivum) that affect crop production globally. Herbivore-induced emission of sesquiterpenes can repel pests, and farnesyl pyrophosphate synthase (FPS) is a key enzyme involved in sesquiterpene biosynthesis. However, fps orthologues in wheat and their functional roles in sesquiterpene synthesis and defence against aphid infestation are unknown. Here, two fps isoforms, Tafps1 and Tafps2, were identified in wheat. Quantitative real-time polymerase chain reaction (qRT-PCR) and in vitro catalytic activity analyses were conducted to investigate expression patterns and activity. Heterologous expression of these isoforms in Arabidopsis thaliana, virus-induced gene silencing (VIGS) in wheat and aphid behavioural assays were performed to understand the functional roles of these two isoforms. We demonstrated that Tafps1 and Tafps2 played different roles in induced responses to aphid infestation and in sesquiterpene synthesis. Heterologous expression in A. thaliana resulted in repulsion of the peach aphid (Myzus persicae). Wheat plants with these two isoforms transiently silenced were significantly attractive to grain aphid (Sitobion avenae). Our results provide new insights into induced defence against aphid herbivory in wheat, in particular, the different roles of the two Tafps isoforms in both sesquiterpene biosynthesis and defence against aphid infestation.

Future of breeding by genome editing is in the hands of regulators.

We are witnessing the timely convergence of several technologies that together will have significant impact on research, human health and in animal and plant breeding. The exponential increase in genome and expressed sequence data, the ability to compile, analyze and mine these data via sophisticated bioinformatics procedures on high-powered computers, and developments in various molecular and in-vitro cellular techniques combine to underpin novel developments in research and commercial biotechnology. Arguably the most important of these is genome editing which encompasses a suite of site directed nucleases (SDN) that can be designed to cut, or otherwise modify predetermined DNA sequences in the genome and result in targeted insertions, deletions, or other changes for genetic improvement. It is a powerful and adaptive technology for animal and plant science, with huge relevance for plant and animal breeding. But this promise will be realized only if the regulatory oversite is proportionate to the potential hazards and has broad support from consumers, researchers and commercial interests. Despite significant progress in research and development and one genome edited crop close to commercialization, in most regions of the world it still remains unclear how or whether this fledgling technology will be regulated. The various risk management authorities and biotechnology regulators have a unique opportunity to set up a logical, appropriate and workable regulatory framework for gene editing that, unlike the situation for GMOs, would have broad support from stakeholders.

Gluten quality of bread wheat is associated with activity of RabD GTPases.

In the developing endosperm of bread wheat (Triticum aestivum), seed storage proteins are produced on the rough endoplasmic reticulum (ER) and transported to protein bodies, specialized vacuoles for the storage of protein. The functionally important gluten proteins of wheat are transported by two distinct routes to the protein bodies where they are stored: vesicles that bud directly off the ER and transport through the Golgi. However, little is known about the processing of glutenin and gliadin proteins during these steps or the possible impact on their properties. In plants, the RabD GTPases mediate ER-to-Golgi vesicle transport. Available sequence information for Rab GTPases in Arabidopsis, rice, Brachypodium and bread wheat was compiled and compared to identify wheat RabD orthologs. Partial genetic sequences were assembled using the first draft of the Chinese Spring wheat genome. A suitable candidate gene from the RabD clade (TaRabD2a) was chosen for down-regulation by RNA interference (RNAi), and an RNAi construct was used to transform wheat plants. All four available RabD genes were shown by qRT-PCR to be down-regulated in the transgenic developing endosperm. The transgenic grain was found to produce flour with significantly altered processing properties when measured by farinograph and extensograph. SE-HPLC found that a smaller proportion of HMW-GS and large proportion of LMW-GS are incorporated into the glutenin macropolymer in the transgenic dough. Lower protein content but a similar protein profile on SDS-PAGE was seen in the transgenic grain.

Comparative transcriptomic analyses revealed divergences of two agriculturally important aphid species.

BACKGROUND: Grain aphid (Sitobion avenae F) and pea aphid (Acyrthosiphon pisum) are two agriculturally important pest species, which cause significant yield losses to crop plants each year by inflicting damage both through the direct effects of feeding and by vectoring debilitating plant viruses. Although a close phylogenetic relationship between grain aphid and pea aphid was proposed, the biological variations between these two aphid species are obvious. While the host ranges of grain aphid is restricted to cereal crops and in particular wheat, that of pea aphid is wider, mainly colonizing leguminous plant species. Until now, the genetic factors underlying the divergence between grain aphid and pea aphid still remain unclear due to the limited genomic data of grain aphid available in public databases. RESULTS: Based on a set of transcriptome data of grain aphid generated by using Roche 454 GS-FLX pyrosequencing, comparative analysis between this set of transcriptome data of grain aphid and mRNA sequences of pea aphid available in the public databases was performed. Compared with mRNA sequences of pea aphid, 4,857 unigenes were found to be specifically presented in the transcriptome of grain aphid under the rearing conditions described in this study. Furthermore, 3,368 orthologous pairs which could be calculated with both nonsynonymous (Ka) and synonymous (Ks) substitutions were used to infer their sequence divergences. The average differences in the coding, 5' and 3' untranslated regions of these orthologs were 10.53%, 21.29% and 18.96%, respectively. Moreover, of 340 orthologs which were identified to have evolved in response to positive selection based on the rates of Ka and Ks substitutions, 186 were predicted to be involved in secondary metabolism and xenobiotic metabolisms which might contribute to the divergence of these two aphid species. CONCLUSIONS: The comprehensive transcriptome divergent sequence analysis between grain aphid and pea aphid provides an invaluable resource for the investigation of genes involved in host plant adaptation and evolution. Moreover, the demonstration of divergent transcriptome sequences between grain aphid and pea aphid pave the way for the investigation of the molecular mechanisms underpinning the biological variations of these two agriculturally important aphid species.

Delivering sustainable crop protection systems via the seed: exploiting natural constitutive and inducible defence pathways.

To reduce the need for seasonal inputs, crop protection will have to be delivered via the seed and other planting material. Plant secondary metabolism can be harnessed for this purpose by new breeding technologies, genetic modification and companion cropping, the latter already on-farm in sub-Saharan Africa. Secondary metabolites offer the prospect of pest management as robust as that provided by current pesticides, for which many lead compounds were, or are currently deployed as, natural products. Evidence of success and promise is given for pest management in industrial and developing agriculture. Additionally, opportunities for solving wider problems of sustainable crop protection, and also production, are discussed.

Secondary cell wall composition and candidate gene expression in developing willow (Salix purpurea) stems.

The properties of the secondary cell wall (SCW) in willow largely determine the suitability of willow biomass feedstock for potential bioenergy and biofuel applications. SCW development has been little studied in willow and it is not known how willow compares with model species, particularly the closely related genus Populus. To address this and relate SCW synthesis to candidate genes in willow, a tractable bud culture-derived system was developed in Salix purpurea, and cell wall composition and RNA-Seq transcriptome were followed in stems during early development. A large increase in SCW deposition in the period 0-2 weeks after transfer to soil was characterised by a big increase in xylan content, but no change in the frequency of substitution of xylan with glucuronic acid, and increased abundance of putative transcripts for synthesis of SCW cellulose, xylan and lignin. Histochemical staining and immunolabeling revealed that increased deposition of lignin and xylan was associated with xylem, xylem fibre cells and phloem fibre cells. Transcripts orthologous to those encoding xylan synthase components IRX9 and IRX10 and xylan glucuronyl transferase GUX1 in Arabidopsis were co-expressed, and showed the same spatial pattern of expression revealed by in situ hybridisation at four developmental stages, with abundant expression in proto-xylem, xylem fibre and ray parenchyma cells and some expression in phloem fibre cells. The results show a close similarity with SCW development in Populus species, but also give novel information on the relationship between spatial and temporal variation in xylan-related transcripts and xylan composition.

Genetic transformation of wheat via particle bombardment.

Since its first invention in the late 1980s the particle gun has evolved from a basic gunpowder driven machine firing tungsten particles to one more refined which uses helium gas as the propellant to launch alternative heavy metal particles such as gold and silver. The simple principle is that DNA-coated microscopic particles (microcarriers) are accelerated at high speed by helium gas within a vacuum and travel at such a velocity as to penetrate target cells. However, the process itself involves a range of parameters which are open to variation: microparticle type and size, gun settings (rupture pressure, target distance, vacuum drawn, etc.), preparation of components (e.g., gold coating), and preparation of plant tissues. Here is presented a method optimized for transformation of wheat immature embryos using the Bio-Rad PDS-1000/He particle gun to deliver gold particles coated with a gene of interest and the selectable marker gene bar at 650 psi rupture pressure. Following bombardment, various tissue culture phases are used to encourage embryogenic callus formation and regeneration of plantlets and subsequent selection using glufosinate ammonium causes suppression of non-transformed tissues, thus assisting the detection of transformed plants. This protocol has been used successfully to generate transgenic plants for a wide range of wheat varieties, both spring and winter bread wheats (T. aestivum L.) and durum wheats (T. turgidum L.).

Genetic transformation of wheat via Agrobacterium-mediated DNA delivery.

The method described involves an initial incubation of wheat immature embryos in a liquid culture of Agrobacterium tumefaciens. The Agrobacterium strain is engineered to contain a binary vector with a gene of interest and a selectable marker gene placed between the T-DNA borders; the T-DNA is the region transferred to the plant cells, thus harnessing the bacterium's natural ability to deliver specific DNA into host cells. Following the initial inoculation with the Agrobacterium, the embryos are co-cultivated for several days after which the Agrobacterium is selectively destroyed using an antibiotic. Tissue culture of the embryos on plant media with a correct balance of hormones allows embryogenic callus formation followed by regeneration of plantlets, and in the later stages of tissue culture a selectable marker (herbicide) is included to minimize the incidence of non-transformed plants. This protocol has been used successfully to generate transformed plants of a wide range of wheat varieties, both spring and winter bread wheats (T. aestivum L.) and durum wheats (T. turgidum L.).