Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat

Since the first report of wheat transformation by Agrobacterium tumefaciens in 1997, various factors that influence T-DNA delivery and regeneration in tissue culture have been further investigated and modified. This paper reviews the current methodology literature describing Agrobacterium transformation of wheat and provides a complete protocol that we have developed and used to produce over one hundred transgenic lines in both spring and winter wheat varieties

Genome editing: the promise and the politics

Efficient and sustainable agriculture depends on a high degree of predictability. Both in the short-term, for growers and agronomists to make informed management decisions for the immediate upcoming seasons, but also in the long term to establish future agricultural policy and trade agreements, to define crop and livestock breeding goals, and to stimulate innovation in new products with transparent regulatory frameworks for pesticides and biotechnology; all of which require decade-long timeframes or longer. However, there are many factors with implications for world agriculture that are becoming increasingly unpredictable and which pose significant challenges for sustainable future food production.  I will highlight two major areas of uncertainty, one which is environmental and beyond the control of humankind in the medium-term and the other, involving regulatory policy that is absolutely with in our short-term grasp. I will argue that providing certainty and transparency in the latter will make a significant contribution to global food security by ameliorating the effects of the former.

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 degrees 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

Functional genomic and transformation resources for commercially important red macroalgae (Rhodophyta)

Red macroalgae underpin many commercially important food, pharmaceutical and other important industries. To date, research into these species has generally focused on improving seaweed cultivation, developing new methods to extract useful compounds, or identify novel applications. Due to their economic importance, there is a requirement to develop a more complete understanding of the genome and metabolic pathways in these key seaweed species. This review describes progress in genomics, transcriptomics, protoplast isolation, and transformation approaches. It also explores the potential of genome editing using the CRISPR/Cas system to further our understanding of gene function related to different metabolic pathways and resolving unexplored aspects of macroalgal physiology traits linked to crop improvement. The application of functional genomics is essential to gain a complete understanding of both physiological and metabolomic processes, that will ultimately enhance the commercial resilience of macroalgae related industries that are subject to numerous pressures, including climate change. Although the use of genetic manipulation to alter growth characteristics or composition in seaweed will not readily apply to the macroalgae industry in the short term, it is likely to be critical for sustaining future commercial growth. The functional characterisation of macroalgal genes through the CRISPR/ Cas approach promises to open new avenues for translational research on utilising macroalgal resources for the sustainable development of these aquaculture systems

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