The development of powerful “omics” technologies has enabled researchers to identify many genes of interest for which comprehensive functional analyses are highly desirable. However, the production ...of lines which ectopically express recombinant genes, or those in which endogenous genes are knocked down via stable transformation, remains a major bottleneck for the association between genetics and gene function in monocotyledonous crops. Methods of effective DNA transfer into regenerable cells of immature embryos from cereals by means of Agrobacterium tumefaciens have been modified in a stepwise manner. The effect of particular improvement measures has often not been significantly evident, whereas their combined implementation has resulted in meaningful advances. Here, we provide updated protocols for the Agrobacterium-mediated generation of stably transgenic barley, wheat, triticale and maize. Based upon these methods, several hundred independent transgenic lines have been delivered, with efficiencies of inoculated embryos leading to stably transgenic plants reaching 86% in barley, 10% in wheat, 4% in triticale, and 24% in maize.
CRISPR is a prokaryotic defence system that was adapted as a tool for genome editing and has become one of the most important discoveries of this century. CRISPR‐associated endonucleases cleave DNA ...at precise sites, which are marked by complementary short‐guided RNA. The recently developed versions of endonucleases are compatible with a broad range of PAM motifs, have a higher specificity and enable a specific nucleotide to be replaced.
Use of RNA-guided endonucleases for targeted genome editing is one of the most important breakthrough discoveries of the 21st century. Recent studies have described modifications of this precise base ...editing technique that open up a new dimension to plant genome editing.
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.
•CRISPR/Cas9 approach enabled editing of cereal genomes to improve agronomic traits.•Many cereal mutants produced through CRISPR/Cas9 became alternatives in breeding.•CRISPR/Cas9 may be combined with novel breeding approaches to develop new germplasm.
Rice is a premier staple food that constitutes the bulk of the daily diet of the majority of people in Asia. Agricultural productivity must be boosted to support this huge demand for rice. However, ...production is jeopardized by soil salinity. Advances in whole-genome sequencing, marker-assisted breeding strategies, and targeted mutagenesis have substantially improved the toolbox of today’s breeders. Given that salinity has a major influence on rice at both the seedling and reproductive stages, understanding and manipulating this trait will have an enormous impact on sustainable production. This article summarizes recent developments in the understanding of the mechanisms of salt tolerance and how state-of-the-art tools such as RNA guided CRISPR endonuclease technology including targeted mutagenesis or base and prime editing can help in gene discovery and functional analysis as well as in transferring favorable alleles into elite breeding material to speed the breeding of salt-tolerant rice cultivars.
The incredible success of crop breeding and agricultural innovation in the last century greatly contributed to the Green Revolution, which significantly increased yields and ensures food security, ...despite the population explosion. However, new challenges such as rapid climate change, deteriorating soil, and the accumulation of pollutants require much faster responses and more effective solutions that cannot be achieved through traditional breeding. Further prospects for increasing the efficiency of agriculture are undoubtedly associated with the inclusion in the breeding strategy of new knowledge obtained using high-throughput technologies and new tools in the future to ensure the design of new plant genomes and predict the desired phenotype. This article provides an overview of the current state of research in these areas, as well as the study of soil and plant microbiomes, and the prospective use of their potential in a new field of microbiome engineering. In terms of genomic and phenomic predictions, we also propose an integrated approach that combines high-density genotyping and high-throughput phenotyping techniques, which can improve the prediction accuracy of quantitative traits in crop species.
Genome Engineering Using TALENs Hensel, Goetz; Kumlehn, Jochen
Methods in molecular biology (Clifton, N.J.),
2019, Letnik:
1900
Journal Article
Genome engineering involves methods of genetic modification of cells at predefined genomic sites. Here, we used transcription activator-like effector nucleases (TALENs) for the site-directed ...mutagenesis in barley. Target gene-specific TALEN-encoding expression units were designed and delivered to totipotent cells of either cultivated embryogenic pollen or immature embryos. The analysis of resulting transgenic plants revealed that the described approach allows for the generation of site-specific, heritable mutations at reasonable efficiency.
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.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Prime Editing: A New Way for Genome Editing Marzec, Marek; Brąszewska-Zalewska, Agnieszka; Hensel, Goetz
Trends in cell biology,
April 2020, 2020-Apr, 2020-04-00, 20200401, Letnik:
30, Številka:
4
Journal Article
Recenzirano
Precise and efficient use of genome editing tools are hampered by the introduction of DNA double-strand breaks, donor DNA templates, or homology-directed repair. A recent study expands the genome ...editing toolbox with the introduction of prime editing, which overcomes previous challenges and introduces insertions, deletions, and all putative 12 types of base-to-base conversions in human cells.
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