Background: The naked caryopsis character in barley is a domestication-associated trait defined by loss-of-function of the NUD gene. The functional NUD gene encodes an Apetala 2/Ethylene-Response Factor (AP2/ERF) controlling the formation of a cementing layer between pericarp and both lemma and palea. The downstream genes regulated by the NUD transcription factor and molecular mechanism of a cementing layer formation are still not sufficiently described. A naturally occurring 17-kb deletion in the nud locus is associated with the emergence of naked barley. Naked barley has been traditionally used for food and nowadays is considered as a dietary component for functional nutrition. Results: In the present study, we demonstrate that targeted knockout of the NUD gene using RNA-guided Cas9 endonuclease leads to the phenotype conversion from hulled to naked barley. Using in vivo pre-testing systems, highly effective guide RNAs targeting the first exon of the NUD gene were selected. Expression cassettes harboring the cas9 and guide RNA genes were used to transform barley cv. Golden Promise via Agrobacterium-mediated DNA transfer. The recessive naked grain phenotype was observed in 57% of primary transformants, which indicates a frequent occurrence of homozygous or biallelic mutations. T-DNA-free homozygous lines with independently generated mutations in the NUD gene were obtained in the T1 generation. At homozygous state, all obtained mutations including one- and two-amino acid losses with the translational reading frame being retained invariably caused the naked grain phenotype. Conclusions: The hulled and naked barley isogenic lines generated are a perfect experimental model for further studies on pleiotropic consequences of nud mutations on overall plant performance under particular consideration of yield-determining traits. Due to the high β-glucan content of its grains, naked barley is considered as being of particular dietary value. The possibility to convert hulled into naked barley cultivars by targeted mutagenesis allows breeders to extend the potential utilization of barley by the provision of functional food.

The modifcation of crop genomes employing functional components of the microbial CRISPR/Cas immune system is a rapidly developing area of applied research. Site-directed plant genome modifcation by this technology involves the construction of Cas endonuclease- and guide-RNA-encoding vectors, delivery of the plasmid DNA into plant cells, processing of the chosen genomic target site by the corresponding gene products and regeneration of plants from modifed cells. The utilization of this technology in local breeding programs is mainly limited by the typically strong genotype dependence of gene transfer andin vitroregeneration procedures, which holds particularly true in cereals. In the present study, an evaluation ofin vitroregeneration efciency of immature embryos of ten Siberian barley cultivars revealed that only one of these is on a par with the experimental standard cultivar Golden Promise. This cultivar, namely cv. Aley, was consequently chosen for further experiments on site-directed mutagenesis in leaf mesophyll protoplasts. Two genes controlling hulledvsnaked (Nud) and two-rowedvssix-rowed barley (Vrs1) were used as targets to be modifed via polyethyleneglycol-mediated cellular uptake of guide-RNA/Cas9-encoding plasmid DNA. Deep-sequencing of amplicons obtained from protoplast genomic DNA revealed that 6 to 22 percent of the target sites were mutated. The detected modifcations comprised deletions in all three target sites and of various sizes, whereas insertions were observed in only one of the target genes (Vrs1) and were confned to the size of 1 nucleotide. This study demonstrates the possibility of site-directed genome modifcation in Siberian barley. Further steps in technology advancement will require the development of protocols with reduced genotype dependence in terms of both the gene transfer to totipotent cells and the subsequent plant regeneration originating from such cells.

With the advent of the new genome editing tool of target-specifically customizable endonucleases, a huge variety of novel opportunities have become feasible. The crop improvement is one of the main applications of genome editing in plant science and plant biotechnology. The amount of publications referring to genome editing and CRISPR/Cas system based molecular tools application in crops is permanently growing. The aim of this study is the systematization and cataloging of these data. Earlier we published the first catalog of targeted crop genome modifications as of February 10, 2017. The current review is an update of the catalog; it covers research papers on crop genome modifications from February 10, 2017 to August 17, 2018, found by searching 47 crop names in the Scopus database. Over one year and a half, 377 articles mentioning CRISPR/Cas and crop names have been published, of which 131 articles describe an experimental application of this tool for editing 193 genes in 19 crops, including rice with the largest number of genes modified (109 genes). Editing 50 of 193 genes was aimed at crop improvement. The catalog presented here includes these 50 genes, specifying the cultivars, each gene and gene product function, modification type and delivery method used. The current full list of genes modified with CRISPR/Cas with the aim of crop improvement is 81 in 16 crops (for 5 years from August 2013 to August 2018). In this paper, we also summarize data on different modifications types in different crops and provide a brief review of some novel methods and approaches that have appeared in crop genome editing research over the reviewed period. Taken together, these data provide a clear view on current progress in crop genome modifications and traits improvement using CRISPR/Cas based genome editing technology.

The characterization of major resistance genes (R genes) in the potato remains an important task for molecular breeding. However, R genes are rapidly evolving and frequently occur in genomes as clusters with complex structures, and their precise mapping and identification are complicated and time consuming. Comparative analysis of root transcriptomes of Solanum phureja genotypes with contrasting resistance to Globodera rostochiensis revealed a number of differentially expressed genes. However, compiling a list of candidate R genes for further segregation analysis was hampered by their scarce annotation. Nevertheless, combination of transcriptomic analysis with data on predicted potato NBS-LRR-encoding genes considerably improved the quality of the results and provided a reasonable number of candidate genes that provide S. phureja with strong resistance to the potato golden cyst nematode. Combination of comparative analyses of tissue-specific transcriptomes in resistant and susceptible genotypes may be used as an approach for the rapid identification of candidate potato R genes for co-segregation analysis and may be used in parallel with more sophisticated studies based on genome resequencing.

The role of gene of proline dehydrogenase (PDH) in the maintenance of stress tolerance was investigated using the model transgenic plants of tobacco (Nicotiana tabacum L.) carrying an antisense suppressor of PDH gene (a fragment of Arabidopsis PDH gene under the control of cauliflower mosaic virus 35S promoter in antisense orientation) and notable for a low activity of PDH and elevated content of proline. The progeny of transgenic plants belonging to the 5th generation (T₅) with partially suppressed PDH activity was more resistant to various types of stress as compared with the control plants of tobacco, cv. Petit Havana SR-1 (SR1). The seedlings of transgenic lines cultured in Petri dishes on agar media supplemented with stress agents were resistant to high NaCl concentrations (200–300 mM) and water deficit simulated by an increased agar content in the medium (14 g/l) as compared to the control seedlings of cv. SR1. Juvenile plants of transgenic lines grown in pots filled with a mixture of vermiculite and perlite also manifested the higher resistance to water deficit and low temperatures (2°C and −2°C) than the control plants. Thus, the partial PDH suppression correlated with an increase in nonspecific resistance to different types of abiotic stress: salinity, water deficit, and low temperatures. Such transgenic lines of tobacco are promising genetic models for thorough investigation of molecular mechanisms of stress resistance in plants.

The CRISPR/Cas system is one of the most promising genome editing tools. It can provide the development of modified nontransgenic plants with the possibility of simultaneous multiple targeted mutation