CRISPR/Cas-mediated plant genome editing: outstanding challenges a decade after implementation.

The discovery of the CRISPR/Cas genome-editing system has revolutionized our understanding of the plant genome. CRISPR/Cas has been used for over a decade to modify plant genomes for the study of specific genes and biosynthetic pathways as well as to speed up breeding in many plant species, including both model and non-model crops. Although the CRISPR/Cas system is very efficient for genome editing, many bottlenecks and challenges slow down further improvement and applications.

Evaluation of CRISPR/Cas9 Constructs in Wheat Cell Suspension Cultures.

Despite intensive optimization efforts, developing an efficient sequence-specific CRISPR/Cas-mediated genome editing method remains a challenge, especially in polyploid cereal species such as wheat. Validating the efficacy of nuclease constructs prior to using them in planta is, thus, a major step of every editing experiment. Several construct evaluation strategies were proposed, with PEG-mediated plasmid transfection of seedling-derived protoplasts becoming the most popular.

Cell Wall Properties Determine Genotype-Specific Response to Cold in × Plants.

The cell wall plays a crucial role in plant growth and development, including in response to environmental factors, mainly through significant biochemical and biomechanical plasticity. The involvement of the cell wall in C plants' response to cold is, however, still poorly understood. × , a perennial grass, is generally considered cold tolerant and, in contrast to other thermophilic species such as maize or sorgo, can maintain a relatively high level of photosynthesis efficiency at low ambient temperatures.

Functional Validation of /guideRNA Constructs for Site-Directed Mutagenesis of Triticale .

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although /guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated /gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons.

Primary carbon metabolism-related changes in cucumber exposed to single and sequential treatments with salt stress and bacterial infection.

This study examines how salt stress interacts with bacterial infection at the metabolic level. We measured chlorophyll a fluorescence as well as profiles of phosphoenolpyruvate carboxylase (PEPC), NADP-malic enzyme (NADP-ME), NADP-isocitrate dehydrogenase (NADP-ICDH) and fumarase activities, malic and citric acids contents and the expression of NADP-ICDH and NADP-ME in the organ-dependent (root vs leaves) response of cucumber plants exposed to individual or sequential action of salt stress (50 mM or 100 mM NaCl) and Pseudomonas syringae pv lachrymans (Psl). NaCl treatment, Psl infection and the combination of these stresses caused disturbances in the activity of photosystem II which were suggested to specifically transmit the stress signals.