The fleshy fruit of tomato (Solanum lycopersicum) is a commodity used worldwide as a fresh or processed product. Like many crops, tomato plants and harvested fruits are susceptible to the onset of climate change. Temperature plays a key role in tomato fruit production and ripening, including softening, development of fruit colour, flavour and aroma. The combination of climate change and the drive to reduce carbon emission and energy consumption is likely to affect tomato post-harvest storage conditions. In this study, we investigated the effect of an elevated storage temperature on tomato shelf life and fungal susceptibility. A collection of 41 genotypes with low and high field performance at elevated temperature, including different growth, fruit and market types, was used to assess post-harvest performances. A temperature increase from 18–20 °C to 26 °C reduced average shelf life of fruit by 4 days ± 1 day and increased fungal susceptibility by 11% ± 5% across all genotypes. We identified tomato varieties that exhibit both favourable post-harvest fruit quality and high field performance at elevated temperature. This work contributes to efforts to enhance crop resilience by selecting for thermotolerance combined with traits suitable to maintain and improve fruit quality, shelf life and pathogen susceptibility under changing climate conditions.

A wide variety of fresh market and processing tomatoes (Solanum lycopersicum) is grown and consumed worldwide. Post-harvest losses are a major contributing factor to losses in crop productivity and can account for up to 50% of the harvest. To select and breed elite tomato varieties, it is important to characterize fruit quality and evaluate the post-harvest properties of tomato fruits. This includes the analysis of shelf life (the period during which a fruit remains suitable for consumption without qualitative deterioration), color, and pathogen susceptibility. Tomato shelf life depends upon the rate of fruit softening which accompanies fruit ripening and exacerbates damage during transport and handling. Furthermore, the susceptibility of tomatoes to fruit pathogens is also often linked to fruit ripening, especially for necrotrophic fungi such as Botrytis cinerea, also known as gray mold. The methods described here are critical for determining fruit quality and fungal susceptibility during storage. © 2020 The Authors. Basic Protocol 1: Fruit color as a determinant of fruit quality Basic Protocol 2: Shelf life test of tomato fruits Basic Protocol 3: Botrytis cinerea pathogen test of tomato fruits Support Protocol: Preparation of Botrytis spore inoculum.

Background Flavonoids are produced in all flowering plants in a wide range of tissues including in berry fruits. These compounds are of considerable interest for their biological activities, health benefits and potential pharmacological applications. However, transcriptomic and genomic resources for wild and cultivated berry fruit species are often limited, despite their value in underpinning the in-depth study of metabolic pathways, fruit ripening as well as in the identification of genotypes rich in bioactive compounds. Results To access the genetic diversity of wild and cultivated berry fruit species that accumulate high levels of phenolic compounds in their fleshy berry(-like) fruits, we selected 13 species from Europe, South America and Asia representing eight genera, seven families and seven orders within three clades of the kingdom Plantae . RNA from either ripe fruits (ten species) or three ripening stages (two species) as well as leaf RNA (one species) were used to construct, assemble and analyse de novo transcriptomes. The transcriptome sequences are deposited in the BacHBerryGEN database ( http://jicbio.nbi.ac.uk/berries ) and were used, as a proof of concept, via its BLAST portal ( http://jicbio.nbi.ac.uk/berries/blast.html ) to identify candidate genes involved in the biosynthesis of phenylpropanoid compounds. Genes encoding regulatory proteins of the anthocyanin biosynthetic pathway (MYB and basic helix-loop-helix (bHLH) transcription factors and WD40 repeat proteins) were isolated using the transcriptomic resources of wild blackberry ( Rubus genevieri ) and cultivated red raspberry ( Rubus idaeus cv. Prestige) and were shown to activate anthocyanin synthesis in Nicotiana benthamiana . Expression patterns of candidate flavonoid gene transcripts were also studied across three fruit developmental stages via the BacHBerryEXP gene expression browser ( http://www.bachberryexp.com ) in R. genevieri and R. idaeus cv. Prestige. Conclusions We report a transcriptome resource that includes data for a wide range of berry(-like) fruit species that has been developed for gene identification and functional analysis to assist in berry fruit improvement. These resources will enable investigations of metabolic processes in berries beyond the phenylpropanoid biosynthetic pathway analysed in this study. The RNA-seq data will be useful for studies of berry fruit development and to select wild plant species useful for plant breeding purposes.

Additional file 15: Table S9. Differential expression of candidate transcripts homologous to enzymes involved in the phenylpropanoid pathway of R. genevieri and R. idaeus cv. Prestige during three fruit ripening stages: Examples of candidate enzymes of the general phenylpropanoid pathway, flavonoid regulatory enzymes, transporters, decorating and modifying enzymes.

BACterial Hosts for production of Bioactive phenolics from bERRY fruits (BacHBerry) was a 3-year project funded by the Seventh Framework Programme (FP7) of the European Union that ran between November 2013 and October 2016. The overall aim of the project was to establish a sustainable and economically-feasible strategy for the production of novel high-value phenolic compounds isolated from berry fruits using bacterial platforms. The project aimed at covering all stages of the discovery and pre-commercialization process, including berry collection, screening and characterization of their bioactive components, identification and functional characterization of the corresponding biosynthetic pathways, and construction of Gram-positive bacterial cell factories producing phenolic compounds. Further activities included optimization of polyphenol extraction methods from bacterial cultures, scale-up of production by fermentation up to pilot scale, as well as societal and economic analyses of the processes. This review article summarizes some of the key findings obtained throughout the duration of the project.

Systemic, posttranscriptional silencing of transgenes in Nicotiana benthamiana was initiated in localized regions of the plant by introduction of transgene-homologous DNA fragments, including those without a promoter. Following this initiation step, a sequence-specific signal of gene silencing spread from cells that had received the ectopic DNA via a relay mechanism that employs plasmodesmatal and phloem channels. The nature of this mechanism, the mode of its initiation, and the ability of the signal to move long distances indicate the existence of a sequence-specific signaling mechanism in plants that may have roles in developmental control as well as in protection against transposons and viruses.

Brachypodium distachyon is an attractive genomics and biological model system for grass research. Recently, the complete annotated genome sequence of the diploid line Bd21 has been released. Genetic transformation technologies are critical for the discovery and validation of gene function in Brachypodium. Here, we describe an efficient procedure enabling the Agrobacterium-mediated transformation of a range of diploid and polyploid genotypes of Brachypodium. The procedure relies on the transformation of compact embryogenic calli derived from immature embryos using either chemical selection alone or a combination of chemical and visual screening of transformed tissues and plants. Transformation efficiencies of around 20% can routinely be achieved using this protocol. In the context of the BrachyTAG programme (BrachyTAG. org), this procedure made possible the mass production of Bd21T-DNA mutant plant lines.

During the past decade, Brachypodium distachyon has emerged as an attractive experimental system and genomics model for grass research. Numerous molecular tools and genomics resources have already been developed. Functional genomics resources, including mutant collections, expression/tiling microarray, mapping populations, and genome re-sequencing for natural accessions, are rapidly being developed and made available to the community. In this article, the focus is on the current status of systematic T-DNA mutagenesis in Brachypodium. Large collections of T-DNA-tagged lines are being generated by a community of laboratories in the context of the International Brachypodium Tagging Consortium. To date, > 13 000 lines produced by the BrachyTAG programme and USDA-ARS Western Regional Research Center are available by online request. The utility of these mutant collections is illustrated with some examples from the BrachyTAG collection at the John Innes Centre-such as those in the eukaryotic initiation factor 4A (eIF4A) and brassinosteroid insensitive-1 (BRI1) genes. A series of other mutants exhibiting growth phenotypes is also presented. These examples highlight the value of Brachypodium as a model for grass functional genomics.

Jelena Brkljacic, Erich Grotewold, Randy Scholl, Todd Mockler, David F. Garvin, Philippe Vain, Thomas Brutnell, Richard Sibout, Michael Bevan, Hikmet Budak, Ana L. Caicedo, Caixia Gao, Yong Gu, Samuel P. Hazen, Ben F. Holt III, Shin-Young Hong, Mark Jordan, Antonio J. Manzaneda, Thomas Mitchell-Olds, Keiichi Mochida, Luis A.J. Mur, Chung-Mo Park, John Sedbrook, Michelle Watt, Shao Jian Zheng, and John P. Vogel*

The combination of a tractable experimental system, molecular tools, facile genetics and comprehensive genomics resources places Brachypodium distachyon in a unique position to promote and accelerate biological investigations in cereal and grass crops. Brachypodium has a proven track-record as a bridge into Triticeae cereal crops by helping to navigate and assemble their large and complex genomes. Brachypodium’s compact genome already provides a useful reference for map-based cloning and comparative genomics. In this review, I argue that Brachypodium will also fulfill a more generic role as a functional genomics model. In this context, Brachypodium will play a pivotal role in multi-system approaches to underpin tomorrow’s grass research landscape.

In a survey of the BrachyTAG mutant population of Brachypodium distachyon, we identified a line carrying a T-DNA insertion in one of the two eukaryotic initiation factor 4A (eIF4A) genes present in the nuclear genome. The eif4a homozygous mutant plants were slow-growing, and exhibited reduced final plant stature due to a decrease in both cell number and cell size, consistent with roles for eIF4A in both cell division and cell growth. Hemizygous plants displayed a semi-dwarfing phenotype, in which stem length was reduced but leaf length was normal. Linkage between the insertion site and phenotype was confirmed, and we show that the level of eIF4A protein is strongly reduced in the mutant. Transformation of the Brachypodium homozygous mutant with a genomic copy of the Arabidopsis eIF4A-1 gene partially complemented the growth phenotype, indicating that gene function is conserved between mono- and dicotyledonous species. This study identifies eIF4A as a novel dose-dependent regulator of stem elongation, and demonstrates the utility of Brachypodium as a model for grass and cereals research.

Over the past several years, Brachypodium distachyon (Brachypodium) has emerged as a tractable model system to study biological questions relevant to the grasses. To place its relevance in the larger context of plant biology, we outline here the expanding adoption of Brachypodium as a model grass and compare this to the early history of another plant model, Arabidopsis thaliana. In this context, Brachypodium has followed an accelerated path in which the development of genomic resources, most notably a whole genome sequence, occurred concurrently with the generation of other experimental tools (e.g. highly efficient transformation and large collections of natural accessions). This update provides a snapshot of available and upcoming Brachypodium resources and an overview of the community including the trajectory of Brachypodium as a model grass.

This chapter contains sections titled: Introduction Description of the pCLEAN Vector System Benefits of the pCLEAN Vector System Conclusion Acknowledgments References

A collection of 4117 fertile T-DNA lines has been generated by Agrobacterium-mediated transformation of the diploid community standard line Bd21 of Brachypodium distachyon. The regions flanking the T-DNA left and right borders of the first 741 transformed plants were isolated by adapter-ligation PCR and sequenced. A total of 1005 genomic sequences (representing 44.1% of all flanking sequences retrieved) characterized 660 independent T-DNA loci assigned to a unique location in the Brachypodium genome sequence. Seventy-six percent of the fertile plant lines contained at least one anchored T-DNA locus (1.17 loci per tagged line on average). Analysis of the regions flanking both borders of the T-DNA increased the number of T-DNA loci tagged and the number of tagged lines by approximately 50% when compared to a single border analysis. T-DNA integration (2.4 insertions per Mb on average) was proportional to chromosome size, however, varied greatly along each chromosome with often low insertion level around centromeres. The frequency of insertion within transposable elements (5.3%) was fivefold lower than expected if random insertion would have occurred. More than half of the T-DNAs inserted in genic regions. On average, one gene could be tagged for every second fertile plant line produced and more than one plant line out of three contained a T-DNA insertion directly within or 500 bp around the coding sequence. Approximately, 60% of the genes tagged corresponded to expressed genes. The T-DNA lines generated by the BrachyTAG programme are available as a community resource and have been distributed internationally since 2008 via the BrachyTAG.org web site.

During the past 25 years, the molecular analysis of transgene insertion patterns and sites in plants has greatly contributed to our understanding of the mechanisms underlying transgene integration, expression, and stability in the nuclear genome. Molecular characterization is also an essential step in the safety assessment of genetically modified crops. This chapter describes the standard experimental procedures used to analyze transgene insertion patterns and loci in cereals and grasses transformed using Agrobacterium tumefaciens or direct transfer of DNA. Methods and protocols enabling the determination of the number and configuration of transgenic loci via a combination of inheritance studies, polymerase chain reaction, and Southern analyses are presented. The complete characterization of transgenic inserts in plants is, however, a holistic process relying on a wide variety of experimental approaches. In this chapter, these additional approaches are not detailed but references to relevant bibliographic records are provided.

Brachypodium distachyon is emerging as a new model system for bridging research into temperate cereal crops, such as wheat and barley, and for promoting research in novel biomass grasses. Here, we provide an adapter ligation PCR protocol that allows the large-scale characterization of T-DNA insertions into the genome of Brachypodium. The procedure enables the retrieval and mapping of the regions flanking the right and left borders (RB and LB) of the T-DNA inserts and consists of five steps: extraction and restriction digest of genomic DNA; ligation of an adapter to the genomic DNA; PCR amplification of the regions flanking the T-DNA insert(s) using primers specific to the adapter and the T-DNA; sequencing of the PCR products; and identification of the flanking sequence tags (FSTs) characterizing the T-DNA inserts. Analyzing the regions flanking both the LB and RB of the T-DNA inserts significantly improves FST retrieval and the frequency of mutant lines for which at least one FST can be identified. It takes approximately 16 or 10 d for a single person to analyze 96 T-DNA lines using individual or batch procedures, respectively.

Brachypodium distachyon is a novel model system for structural and functional genomics studies of temperate grasses because of its biological and genetic attributes. Recently, the genome sequence of the community standard line Bd21 has been released and the availability of an efficient transformation system is critical for the discovery and validation of the function of Brachypodium genes. Here, we provide an improved procedure for the facile and efficient Agrobacterium-mediated transformation of line Bd21. The protocol relies on the transformation of compact embryogenic calli derived from immature embryos using visual and chemical screening of transformed tissues and plants. The combination of green fluorescent protein expression and hygromycin resistance enables early identification of transformation events and drastically reduces the quantity of tissue to be handled throughout the selection process. Approximately eight independent fully developed transgenic Bd21 plants can be produced from each immature embryo, enabling the generation of thousands of T-DNA lines. The process—from wild-type seeds to transgenic T1 seeds—takes ∼8 months to complete.

Transgenic rice plants from indica, japonica, and javanica varieties that express several foreign genes were generated via particle gun bombardment of immature embr yos. Molecular and genetic characterization of large numbers of these plants (more than 500 independent transgenic plants) provided information on structure, expression, and stability of integrated DNA through multiple generations. Such evaluations were carried out in the greenhouse (five generations) and in the field (three generations). Stability of foreign DNA was found to be dependent on the nature of the promoter and the transgene, and in specific cases, on gene copy number. A general conclusion of the results presented here is that direct DNA transfer utilizing electric discharge particle bombardment for the delivery of foreign DNA into rice tissue results in the recovery of large numbers of independently derived transgenic plants in a variety- independent fashion.