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The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has become the most important tool for targeted genome editing in many plant and animal species over the past decade. The CRISPR/Cas9 technology has also sparked a flood of applications and technical advancements in genome editing in the key cereal crops, including rice, wheat, maize, and barley. Here, we review advanced uses of CRISPR/Cas9 and derived systems in genome editing of cereal crops to enhance a variety of agronomically important features. We also highlight new technological advances for delivering preassembled Cas9-gRNA ribonucleoprotein (RNP)-editing systems, multiplex editing, gain-of-function strategies, the use of artificial intelligence (AI)-based tools, and combining CRISPR with novel speed breeding (SB) and vernalization strategies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Cereal crops, including triticeae species (barley, wheat, rye), as well as edible cereals (wheat, corn, rice, oat, rye, sorghum), are significant suppliers for human consumption, livestock feed, and breweries. Over the past half-century, modern varieties of cereal crops with increased yields have contributed to global food security. However, presently cultivated elite crop varieties were developed mainly for optimal environmental conditions. Thus, it has become evident that taking into account the ongoing climate changes, currently a priority should be given to developing new stress-tolerant cereal cultivars. It is necessary to enhance the accuracy of methods and time required to generate new cereal cultivars with the desired features to adapt to climate change and keep up with the world population expansion. The CRISPR/Cas9 system has been developed as a powerful and versatile genome editing tool to achieve desirable traits, such as developing high-yielding, stress-tolerant, and disease-resistant transgene-free lines in major cereals. Despite recent advances, the CRISPR/Cas9 application in cereals faces several challenges, including a significant amount of time required to develop transgene-free lines, laboriousness, and a limited number of genotypes that may be used for the transformation and in vitro regeneration. Additionally, developing elite lines through genome editing has been restricted in many countries, especially Europe and New Zealand, due to a lack of flexibility in GMO regulations. This review provides a comprehensive update to researchers interested in improving cereals using gene-editing technologies, such as CRISPR/Cas9. We will review some critical and recent studies on crop improvements and their contributing factors to superior cereals through gene-editing technologies., Competing Interests: Declaration of Competing Interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Flowering plants with indeterminate inflorescences often produce more floral structures than they require. We found that floral primordia initiations in barley ( Hordeum vulgare L.) are molecularly decoupled from their maturation into grains. While initiation is dominated by flowering-time genes, floral growth is specified by light signaling, chloroplast, and vascular developmental programs orchestrated by barley CCT MOTIF FAMILY 4 ( HvCMF4 ), which is expressed in the inflorescence vasculature. Consequently, mutations in HvCMF4 increase primordia death and pollination failure, mainly through reducing rachis greening and limiting plastidial energy supply to developing heterotrophic floral tissues. We propose that HvCMF4 is a sensory factor for light that acts in connection with the vascular-localized circadian clock to coordinate floral initiation and survival. Notably, stacking beneficial alleles for both primordia number and survival provides positive implications on grain production. Our findings provide insights into the molecular underpinnings of grain number determination in cereal crops.

Food security is one of the main topics of today's agriculture, primarily due to increasingly challenging environmental conditions. As most of humankind has a daily intake of cereal grains, current breeding programs focus on these crop plants. Customized endonucleases have been included in the breeders' toolbox after successfully demonstrating their use. Due to technological restrictions, the main focus of the new technology was on above-ground plant organs. In contrast, the essential below ground components were given only limited attention. In the present review, the knowledge of the root system architecture in cereals and the role of phytohormones during their establishment is summarized, and the underlying molecular mechanisms are outlined. The review summarizes how the use of CRISPR-based genome editing methodology can improve the root system architecture to enhance crop production genetically. Finally, future research directions involving this knowledge and technical advances are suggested., (© 2022 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

Cereal grains contribute substantially to the human diet. The maternal plant provides the carbohydrate and nitrogen sources deposited in the endosperm, but the basis for their spatial allocation during the grain filling process is obscure. Here, vacuolar processing enzymes have been shown to both mediate programmed cell death (PCD) in the maternal tissues of a barley grain and influence the delivery of assimilate to the endosperm. The proposed centrality of PCD has implications for cereal crop improvement.

Triticeae cereals are among the most important crop plants grown worldwide and being used for animal feed, food and beverages. Although breeding efforts evolved over the last ten thousand years our today's crop plants, biotechnological methods would help to speed up the process and incorporate traits impossible by conventional breeding. The main research topics were related to cover the future demand on our agricultural practices to supply sufficient food for a growing world population. Target traits are resistances against viral and fungal diseases, improvement of water and nitrogen use efficiency, to tackle plant architecture, both below and aboveground and to develop varieties that could grow on dry or salty locations. Other applications are considering accumulation of useful compounds or decreasing allergenicity. This review will summarize methods to generate the material including a section how genome engineering using gRNA/Cas (CRISPR/Cas) technology could further improve the methodology and will give an overview about recent and future applications., Competing Interests: Declaration of Competing Interest The author declare that he has no competing interests., (Copyright © 2019 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK). Published by Elsevier Inc. All rights reserved.)

Global food security depends on cereal crops with durable disease resistance. Most cereals are colonized by rust fungi, which are pathogens of major significance for global agriculture 1 . Cereal rusts display a high degree of host specificity and one rust species or forma specialis generally colonizes only one cereal host 2 . Exploiting the non-host status and transferring non-host resistance genes between cereal crop species has been proposed as a strategy for durable rust resistance breeding. The molecular determinants that define the host status to rusts, however, are largely unknown. Here, we show that orthologous genes at the Rphq2 locus for quantitative leaf rust resistance from cultivated barley 3 and Rph22 from wild bulbous barley 4 affect the host status to leaf rusts. Both genes encode lectin receptor-like kinases. We transformed Rphq2 and Rph22 into an experimental barley line that has been bred for susceptibility to non-adapted leaf rusts, which allowed us to quantify resistance responses against various leaf rust species. Rphq2 conferred a much stronger resistance to the leaf rust of wild bulbous barley than to the leaf rust adapted to cultivated barley, while for Rph22 the reverse was observed. We hypothesize that adapted leaf rust species mitigate perception by cognate host receptors by lowering ligand recognition. Our results provide an example of orthologous genes that connect the quantitative host with non-host resistance to cereal rusts. Such genes provide a basis to exploit non-host resistance in molecular breeding.

Tailoring defense responses to different attackers is important for plant performance. Plants can use secondary metabolites with dual functions in resistance and defense signaling to mount herbivore-specific responses. To date, the specificity and evolution of this mechanism are unclear. Here, we studied the functional architecture, specificity, and genetic basis of defense regulation by benzoxazinoids in cereals. We document that DIMBOA-Glc induces callose as an aphid resistance factor in wheat. O -methylation of DIMBOA-Glc to HDMBOA-Glc increases plant resistance to caterpillars but reduces callose inducibility and resistance to aphids. DIMBOA-Glc induces callose in wheat and maize, but not in Arabidopsis , while the glucosinolate 4MO-I3M does the opposite. We identify a wheat O -methyltransferase (TaBX10) that is induced by caterpillar feeding and converts DIMBOA-Glc to HDMBOA-Glc in vitro. While the core pathway of benzoxazinoid biosynthesis is conserved between wheat and maize, the wheat genome does not contain close homologs of the maize DIMBOA-Glc O -methyltransferase genes, and TaBx10 is only distantly related. Thus, the functional architecture of herbivore-specific defense regulation is similar in maize and wheat, but the regulating biosynthetic genes likely evolved separately. This study shows how two different cereal species independently achieved herbivore-specific defense activation by regulating secondary metabolite production.

The angiosperm embryo and endosperm are limited in space because they grow inside maternal seed tissues. The elimination of cell layers of the maternal seed coat by programmed cell death (PCD) could provide space and nutrition to the filial organs. Using the barley (Hordeum vulgare L.) seed as a model, we elucidated the role of vacuolar processing enzyme 4 (VPE4) in cereals by using an RNAi approach and targeting the enzymatic properties of the recombinant protein. A comparative characterization of transgenic versus wild-type plants included transcriptional and metabolic profiling, flow cytometry, histology and nuclear magnetic imaging of grains. The recombinant VPE4 protein exhibited legumain and caspase-1 properties in vitro. Pericarp disintegration was delayed in the transgenic grains. Although the VPE4 gene and enzymatic activity was decreased in the early developing pericarp, storage capacity and the size of the endosperm and embryo were reduced in the mature VPE4-repressed grains. The persistence of the pericarp in the VPE4-affected grains constrains endosperm and embryo growth and leads to transcriptional reprogramming, perturbations in signalling and adjustments in metabolism. We conclude that VPE4 expression executes PCD in the pericarp, which is required for later endosperm filling, and argue for a role of PCD in maternal control of seed size in cereals., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Background: While the genetic transformation of the major cereal crops has become relatively routine, to date only a few reports were published on transgenic triticale, and robust data on T-DNA integration and segregation have not been available in this species., Results: Here, we present a comprehensive analysis of stable transgenic winter triticale cv. Bogo carrying the selectable marker gene HYGROMYCIN PHOSPHOTRANSFERASE (HPT) and a synthetic green fluorescent protein gene (gfp). Progeny of four independent transgenic plants were comprehensively investigated with regard to the number of integrated T-DNA copies, the number of plant genomic integration loci, the integrity and functionality of individual T-DNA copies, as well as the segregation of transgenes in T1 and T2 generations, which also enabled us to identify homozygous transgenic lines. The truncation of some integrated T-DNAs at their left end along with the occurrence of independent segregation of multiple T-DNAs unintendedly resulted in a single-copy segregant that is selectable marker-free and homozygous for the gfp gene. The heritable expression of gfp driven by the maize UBI-1 promoter was demonstrated by confocal laser scanning microscopy., Conclusions: The used transformation method is a valuable tool for the genetic engineering of triticale. Here we show that comprehensive molecular analyses are required for the correct interpretation of phenotypic data collected from the transgenic plants.

The control of transgene expression is vital both for the elucidation of gene function and for the engineering of transgenic crops. Given the dominance of the Triticeae cereals in the agricultural economy of the temperate world, the development of well-performing transgene expression systems of known functionality is of primary importance. Transgenes can be expressed either transiently or stably. Transient expression systems based on direct or virus-mediated gene transfer are particularly useful in situations where the need is to rapidly screen large numbers of genes. However, an unequivocal understanding of gene function generally requires that a transgene functions throughout the plant's life and is transmitted through the sexual cycle, since this alone allows its effect to be decoupled from the plant's response to the generally stressful gene transfer event. Temporal, spatial and quantitative control of a transgene's expression depends on its regulatory environment, which includes both its promoter and certain associated untranslated region sequences. While many transgenic approaches aim to manipulate plant phenotype via ectopic gene expression, a transgene sequence can be also configured to down-regulate the expression of its endogenous counterpart, a strategy which exploits the natural gene silencing machinery of plants. In this review, current technical opportunities for controlling transgene expression in the Triticeae species are described. Apart from protocols for transient and stable gene transfer, the choice of promoters and other untranslated regulatory elements, we also consider signal peptides, as they too govern the abundance and particularly the sub-cellular localization of transgene products., (Copyright © 2010 Elsevier GmbH. All rights reserved.)