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.

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.

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.

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.

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.

(E)-ß-farnesene synthase genes affect aphid (Myzus persicae) infestation in tobacco (Nicotiana tabacum).

Aphids are major agricultural pests which cause significant yield losses of the crop plants each year. (E)-ß-farnesene (EßF) is the alarm pheromone involved in the chemical communication between aphids and particularly in the avoidance of predation. In the present study, two EßF synthase genes were isolated from sweet wormwood and designated as AaßFS1 and AaßFS2, respectively. Overexpression of AaßFS1 or AaßFS2 in tobacco plants resulted in the emission of EßF ranging from 1.55 to 4.65 ng/day/g fresh tissues. Tritrophic interactions involving the peach aphids (Myzus persicae), predatory lacewings (Chrysopa septempunctata) demonstrated that the transgenic tobacco expressing AaßFS1 and AaßFS2 could repel peach aphids, but not as strongly as expected. However, AaßFS1 and AaßFS2 lines exhibited strong and statistically significant attraction to lacewings. Further experiments combining aphids and lacewing larvae in an octagon arrangement showed transgenic tobacco plants could repel aphids and attract lacewing larvae, thus minimizing aphid infestation. Therefore, we demonstrated a potentially valuable strategy of using EßF synthase genes from sweet wormwood for aphid control in tobacco or other economic important crops in an environmentally benign way.

GM wheat development in China: current status and challenges to commercialization.

Genetic modification facilitates research into fundamental questions of plant functional genomics and provides a route for developing novel commercial varieties. In 2008, significant financial resources were supplied by the Chinese government for research and development (R&D) into genetic modification of the major crop species. This project was aimed at providing an opportunity for crop improvement while accentuating the development of a safe, precise, and effective wheat genetic transformation system suitable for commercialization. The focus here is on one of the key crops included in this project, wheat, to provide an insight into the main transformation methods currently in use, the target traits of major importance, and the successful applications of wheat genetic improvement in China. Furthermore, the biosafety and regulatory issues of major concern and the strategies to produce 'clean' transgenic wheat plants will also be discussed. This commentary is intended to be a helpful insight into the production and commercialization of transgenic wheat in China and to put these activities into a global context.

Metabolic engineering of plant-derived (E)-ß-farnesene synthase genes for a novel type of aphid-resistant genetically modified crop plants.

Aphids are major agricultural pests that cause significant yield losses of crop plants each year. Excessive dependence on insecticides for long-term aphid control is undesirable because of the development of insecticide resistance, the potential negative effects on non-target organisms and environmental pollution. Transgenic crops engineered for resistance to aphids via a non-toxic mode of action could be an efficient alternative strategy. (E)-ß-Farnesene (EßF) synthases catalyze the formation of EßF, which for many pest aphids is the main component of the alarm pheromone involved in the chemical communication within these species. EßF can also be synthesized by certain plants but is then normally contaminated with inhibitory compounds. Engineering of crop plants capable of synthesizing and emitting EßF could cause repulsion of aphids and also the attraction of natural enemies that use EßF as a foraging cue, thus minimizing aphid infestation. In this review, the effects of aphids on host plants, plants' defenses against aphid herbivory and the recruitment of natural enemies for aphid control in an agricultural setting are briefly introduced. Furthermore, the plant-derived EßF synthase genes cloned to date along with their potential roles in generating novel aphid resistance via genetically modified approaches are discussed.

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.

Temporal and spatial control of transgene expression using a heat-inducible promoter in transgenic wheat.

Constitutive promoters are widely used to functionally characterise plant genes in transgenic plants, but their lack of specificity and poor control over protein expression can be a major disadvantage. On the other hand, promoters that provide precise regulation of temporal or spatial transgene expression facilitate such studies by targeting over-expression or knockdown of target genes to specific tissues and/or at particular developmental stages. Here, we used the uidA (beta-glucuronidase, GUS) reporter gene to demonstrate that the barley Hvhsp17 gene promoter can be induced by heat treatment of 38-40 °C for 1-2 h in transgenic wheat. The GUS enzyme was expressed only in those tissues directly exposed to heat and not in neighbouring leaf tissues. The induction of HSP::GUS was demonstrated in all organs and tissues tested, but expression in older tissues was lower. Generally, proximal root sections showed less GUS activity than in root tips. This heat-inducible promoter provides the ability to investigate the function of candidate genes by overexpression or by down-regulation of target gene expression (for example by RNAi) in selected tissues or developmental stages of a transgenic plant, limited only by the ability to apply a heat shock to the selected tissues. It also allows the investigation of genes that would be lethal or reduce fertility if expressed constitutively.

Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat.

(1,3;1,4)-beta-d-Glucan (beta-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative beta-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total beta-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable beta-glucan was also reduced by about 50% in the transgenic lines, and the M(r) distribution of the fraction was decreased from an average of 79 to 85 x 10(4) g/mol in the controls and 36 to 57 x 10(4) g/mol in the transgenics. Immunolocalization of beta-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a beta-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.

Increasing the amylose content of durum wheat through silencing of the SBEIIa genes.

BACKGROUND: High amylose starch has attracted particular interest because of its correlation with the amount of Resistant Starch (RS) in food. RS plays a role similar to fibre with beneficial effects for human health, providing protection from several diseases such as colon cancer, diabetes, obesity, osteoporosis and cardiovascular diseases. Amylose content can be modified by a targeted manipulation of the starch biosynthetic pathway. In particular, the inactivation of the enzymes involved in amylopectin synthesis can lead to the increase of amylose content. In this work, genes encoding starch branching enzymes of class II (SBEIIa) were silenced using the RNA interference (RNAi) technique in two cultivars of durum wheat, using two different methods of transformation (biolistic and Agrobacterium). Expression of RNAi transcripts was targeted to the seed endosperm using a tissue-specific promoter. RESULTS: Amylose content was markedly increased in the durum wheat transgenic lines exhibiting SBEIIa gene silencing. Moreover the starch granules in these lines were deformed, possessing an irregular and deflated shape and being smaller than those present in the untransformed controls. Two novel granule bound proteins, identified by SDS-PAGE in SBEIIa RNAi lines, were investigated by mass spectrometry and shown to have strong homologies to the waxy proteins. RVA analysis showed new pasting properties associated with high amylose lines in comparison with untransformed controls. Finally, pleiotropic effects on other starch genes were found by semi-quantitative and Real-Time reverse transcription-polymerase chain reaction (RT-PCR). CONCLUSION: We have found that the silencing of SBEIIa genes in durum wheat causes obvious alterations in granule morphology and starch composition, leading to high amylose wheat. Results obtained with two different methods of transformation and in two durum wheat cultivars were comparable.

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.

Transgenic wheat, barley and oats: production and characterization.

Ever since the first developments in plant transformation technology using model plant species in the early 1980s, there has been a body of plant science research devoted to adapting these techniques to the transformation of crop plants. For some crop species progress was relatively rapid, but in other crop groups such as the small grain cereals, which were not readily amenable to culture in vitro and were not natural hosts to Agrobacterium, it has taken nearly two decades to develop reliable and robust transformation methods.In the following chapters of this book, transformation procedures for small grain cereals are presented, together with methods for gene and protein expression and the characterization of transgenic plants. In this introductory chapter we try to put these later chapters into context, giving an overview of the development of transformation technology for small grain cereals, discussing some of the pros and cons of the techniques and what limitations still exist.

Selection of transformed plants.

The low frequency and randomness of transgene integration into host cells, combined with the significant challenges of recovering whole plants from those rare events, makes the use of selectable marker genes routine in plant transformation experiments. For research applications that are unlikely to be grown in the field, strong herbicide- or antibiotic resistance is commonly used. Here we use genes conferring resistance to glufosinate herbicides as an example of a selectable marker in wheat transformation by either Agrobacterium or biolistics.

Biolistics transformation of wheat.

We present a complete, step-by-step guide to the production of transformed wheat plants using a particle bombardment device to deliver plasmid DNA into immature embryos and the regeneration of transgenic plants via somatic embryogenesis. Currently, this is the most commonly used method for transforming wheat and it offers some advantages. However, it will be interesting to see whether this position is challenged as facile methods are developed for delivering DNA by Agrobacterium tumefaciens or by the production of transformants via a germ-line process (see other chapters in this book).

Agrobacterium-mediated transformation of bread and durum wheat using freshly isolated immature embryos.

Agrobacterium-mediated transformation of wheat is becoming a viable alternative to the more established biolistic protocols. It offers advantages in terms of simple, low-copy-number integrations and can be applied with similar efficiencies to specific durum wheat and spring and winter bread wheat types varieties.

Promoter sequences for defining transgene expression.

The design of reverse genetic experiments that utilize transgenic approaches often requires transgenes to be expressed in a predefined pattern and there is limited information regarding the gene expression profile for specific promoters. It is important that expression patterns are predetermined in the specific genotype targeted for transformation because the same promoter-transgene construct can produce different expression patterns in different host species. This chapter compares constitutive, targeted, or inducible promoters that have been characterized in specific cereal species.

Stable transformation of plants.

This chapter provides an overview of the main steps in the process to produce stably transformed plants. Most transformation methods use tissue culture to recover adult plants from regenerable explants and can be divided into three stages: (1) choice and preparation of explant tissue, (2) deoxyribonucleic acid (DNA) delivery, (3) callus induction/regeneration and selection. Each of these stages is introduced from a general perspective and a detailed protocol for our exemplar species, wheat, is given. We focus here on DNA delivery by particle bombardment as Agrobacterium-mediated transformation methods for wheat are reported elsewhere.

Transient transformation of plants.

Transient expression in plants is a valuable tool for many aspects of functional genomics and promoter testing. It can be used both to over-express and to silence candidate genes. It is also scaleable and provides a viable alternative to microbial fermentation and animal cell culture for the production of recombinant proteins. It does not depend on chromosomal integration of heterologous DNA so is a relatively facile procedure and can lead to high levels of transgene expression. Recombinant DNA can be introduced into plant cells via physical methods, via Agrobacterium or via viral vectors.