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The following method enables the rapid production of transgenic potato plants and microtubers for gene validation and expression, or promoter studies. The method is highly efficient, with reproducible transformation efficiencies of at least 50% to 60% with potato cv. Desiree, and can produce transgenic microtubers within 6 months of initiation of the experiment. Microtubers are produced in the absence of hormones, giving an in vitro gene testing system broadly analogous to the natural state. © 2018 by John Wiley & Sons, Inc.

Oilseed rape (Brassica napus) is a commercially important member of the Brassicacea family. It is grown for its edible and industrial oils as well as for animal feed. Genetic transformation technology has been used to study gene function and produce oilseed rape with improved agronomic characteristics. This protocol describes a method for the Agrobacterium tumefaciens-mediated transformation of oilseed rape cotyledonary petioles. The method is reproducible and has been used to transform both spring and winter cultivars. Modifications have been made to the rooting stage, which have reduced the vitrification of shoots. This has not only increased the number of phenotypically normal shoots but has also resulted in an increase in transformation efficiency, concomitant with a dramatic reduction in the number of escapes regenerated. Transformation frequencies typically range from 5% to 10%, with an average of 12% using doubled haploid model varieties, but a maximum efficiency of 20% has been achieved. © 2017 by John Wiley & Sons, Inc.

Summary Lysophosphatidate acyltransferase (LPAAT) catalyses the second step of the Kennedy pathway for triacylglycerol (TAG) synthesis. In this study we expressed Trapaeolum majus LPAAT in Brassica napus (B. napus) cv 12075 to evaluate the effects on lipid synthesis and estimate the flux control coefficient for LPAAT.We estimated the flux control coefficient of LPAAT in a whole plant context by deriving a relationship between it and overall lipid accumulation, given that this process is a exponential.Increasing LPAAT activity resulted in greater TAG accumulation in seeds of between 25% and 29%; altered fatty acid distributions in seed lipids (particularly those of the Kennedy pathway); and a redistribution of label from 14C‐glycerol between phosphoglycerides.Greater LPAAT activity in seeds led to an increase in TAG content despite its low intrinsic flux control coefficient on account of the exponential nature of lipid accumulation that amplifies the effect of the small flux increment achieved by increasing its activity. We have also developed a novel application of metabolic control analysis likely to have broad application as it determines the in planta flux control that a single component has upon accumulation of storage products.

Background Wheat grains contain gluten proteins, which harbour immunogenic epitopes that trigger Coeliac disease in 1–2% of the human population. Wheat varieties or accessions containing only safe gluten have not been identified and conventional breeding alone struggles to achieve such a goal, as the epitopes occur in gluten proteins encoded by five multigene families, these genes are partly located in tandem arrays, and bread wheat is allohexaploid. Gluten immunogenicity can be reduced by modification or deletion of epitopes. Mutagenesis technologies, including CRISPR/Cas9, provide a route to obtain bread wheat containing gluten proteins with fewer immunogenic epitopes. Results In this study, we analysed the genetic diversity of over 600 α- and γ-gliadin gene sequences to design six sgRNA sequences on relatively conserved domains that we identified near coeliac disease epitopes. They were combined in four CRISPR/Cas9 constructs to target the α- or γ-gliadins, or both simultaneously, in the hexaploid bread wheat cultivar Fielder. We compared the results with those obtained with random mutagenesis in cultivar Paragon by γ-irradiation. For this, Acid-PAGE was used to identify T1 grains with altered gliadin protein profiles compared to the wild-type endosperm. We first optimised the interpretation of Acid-PAGE gels using Chinese Spring deletion lines. We then analysed the changes generated in 360 Paragon γ-irradiated lines and in 117 Fielder CRISPR/Cas9 lines. Similar gliadin profile alterations, with missing protein bands, could be observed in grains produced by both methods. Conclusions The results demonstrate the feasibility and efficacy of using CRISPR/Cas9 to simultaneously edit multiple genes in the large α- and γ-gliadin gene families in polyploid bread wheat. Additional methods, generating genomics and proteomics data, will be necessary to determine the exact nature of the mutations generated with both methods. Electronic supplementary material The online version of this article (10.1186/s12870-019-1889-5) contains supplementary material, which is available to authorized users.

The comparative phenotypic analysis of mutants is often hampered by their diverse and poorly characterised genetic backgrounds. To overcome this problem, a suite of recombinant spring barley lines was developed for four starch biosynthesis genes in a common elite background. Rapid breeding progress was made by combining foreground and background selection with the screening of bulked families. A toolkit of perfect co-dominant PCR assays was developed for the four target genes, based on the causative single nucleotide polymorphisms underlying their starch phenotypes. These were used for foreground selection during backcrossing and selfing, and may be applied to bulks of up to ten plants. Screening bulks meant that large numbers of individuals with known family structure were rapidly assessed and that breeding effort was accurately targeted. These markers were also used for quality control during field multiplication and should be readily transferable to any crosses involving these four mutations. Background selection amongst BC1 progeny known to be heterozygous for the target starch alleles identified individuals which were relatively enriched for the recurrent parent across the rest of the genome. These were further advanced and true-breeding recombinants were selected which carry the target starch mutations in a largely recurrent parent background. The resulting set of BC2F5 pre-breeding lines should enable meaningful analysis of the starch phenotypes and facilitate their transfer into commercial breeding programmes.