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. In this review we discuss the challenges that can occur during tissue culture, transformation, regeneration, and mutant detection. We also review the opportunities provided by new CRISPR platforms and specific applications related to gene regulation, abiotic and biotic stress response improvement, and de novo domestication of plants.

AbstractNew genomic techniques (NGTs) are challenging from an ethical perspective as well as from the legal and regulatory point of view. Acceptance of solutions by society is a factor that can determine the success of a technology. Increasingly, there is a growing gap between the solutions offered by biotechnology and the public, which with certain ambiguity evaluates biotechnological innovations. Here we investigate the level of knowledge of Polish society on NGTs and the perception of this techniques. Our survey was carried out on a representative group of Polish citizens from August 2022 till January 2023, and was based on a 3-part self-developed questionnaire. Questions concerned: (i) demographic characteristics, (ii) the knowledge of the NGTs including the legal state of the NGT after a ruling European Court of Justice (Case C-528/16) and (iii) respondent’s attitudes towards the NGTs. As result, only 15% of respondents (n = 194) gave the correct definition of NGTs, 43% the respondents never encountered the term NGTs, and 35% could not define it. The majority of respondents recognised the potential benefits of using the technology in agriculture as well as in healthcare, but they were not convinced about the potential personal use of NGTs if they were allowed in Poland.

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. However, the usefulness of this approach is affected by associated construct copy number bias and chromatin relaxation, both influencing the outcome. Therefore, to achieve a reliable evaluation of CRISPR/Cas9 constructs, we proposed a system based on an Agrobacterium-mediated transformation of established wheat cell suspension cultures. This system was used for the evaluation of a CRISPR/Cas9 construct designed to target the ABA 8′-hydroxylase 1 gene. The efficiency of editing was verified by cost-effective means of Sanger sequencing and bioinformatic analysis. We discuss advantages and potential future developments of this method in contrast to other in vitro approaches.

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 C4 plants’ response to cold is, however, still poorly understood. Miscanthus × giganteus, 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. This unusual response to chilling among C4 plants makes Miscanthus an interesting study object in cold acclimation mechanism research. Using the results obtained from employing a diverse range of techniques, including analysis of plasmodesmata ultrastructure by means of transmission electron microscopy (TEM), infrared spectroscopy (FTIR), and biomechanical tests coupled with photosynthetic parameters measurements, we present evidence for the implication of the cell wall in genotype-specific responses to cold in this species. The observed reduction in the assimilation rate and disturbance of chlorophyll fluorescence parameters in the susceptible M3 genotype under cold conditions were associated with changes in the ultrastructure of the plasmodesmata, i.e., a constriction of the cytoplasmic sleeve in the central region of the microchannel at the mesophyll–bundle sheath interface. Moreover, this cold susceptible genotype was characterized by enhanced tensile stiffness, strength of leaf wall material, and a less altered biochemical profile of the cell wall, revealed by FTIR spectroscopy, compared to cold tolerant genotypes. These changes indicate that a decline in photosynthetic activity may result from a decrease in leaf CO2 conductance due to the formation of more compact and thicker cell walls and that an enhanced tolerance to cold requires biochemical wall remodelling. Thus, the well-established trade-off between photosynthetic capacity and leaf biomechanics found across multiple species in ecological research may also be a relevant factor in Miscanthus’ tolerance to cold. In this paper, we demonstrate that M. giganteus genotypes showing a high degree of genetic similarity may respond differently to cold stress if exposed at earlier growing seasons to various temperature regimes, which has implications for the cell wall modifications patterns.

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/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 cas9/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. For exemplification, we addressed the triticale ABA 8′-HYDROXYLASE 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in triticale can be increased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in triticale genes.

The protective effects of Trichoderma asperellum IZR D-11 as a biocontrol agent against the powdery mildew Erysiphe alphitoides infecting leaves of Quercus robur were evaluated for the first time. A strain of Trichoderma had earlier been isolated in Belarus, and was identified in this study as T. asperellum by sequencing of three genomic markers: internal transcribed spacer, translation elongation factor 1 alpha and RNA polymerase II subunit 2, with over 99.2% identity to corresponding genomic sequences in GenBank. When applied once in the first year just after onset of powdery mildew disease, T asperellum IZR D-11 reduced disease progression and continued to reduce powdery mildew levels during the following three years. Photosynthetic activity as represented by chlorophyll fluorescence measured in oak seedlings was increased in treated plants, and greater assimilate production was also found. The use of this antagonistic fungus increased the total water content in oak leaves suggesting that T. asperellum IZR D-11 can serve as a preventive measure to reduce energy losses in the process of water transpiration. GC-MS analysis detected 49 volatile compounds in the headspace of pure cultures of T. asperellum . Sesquiterpenes represented mainly by daucene, dauca-4(11),8-diene and isodaucene were the largest group of compounds emitted. We speculate that these volatiles from T. asperellum IZR D-11 may be involved in induced resistance in the plant, but further research is needed. The above results suggest that T. asperellum strain IZR D-11 has potential as a biocontrol agent of oak powdery mildew in forest nurseries.

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. PEPC and NADP-ME were induced in cucumber plants under stress, confirming that in C3 plants they function in defence responses. The profiles of malate and citrate contents, PEPC as well as NADP-ICDH and NADP-ME activities and gene expression in response to a combination of salt and pathogen stresses differed from those provoked by individual stress with respect to the direction, intensity and timing. The results indicated that the most pronounced defence response related to the readjustment of the carbon metabolism was observed in the leaves of plants exposed to combined stress. Intense activity changes of NADPH-generating enzymes, NADP-ICDH and NADP-ME, characterized the tailored response to combined stress and could be important for the integration of defence mechanisms between organs.

Bioeconomy, biotechnology and genetically-modified organisms in particular have been the subject of discussion for a long time. Biotechnology is applied in a variety of economic areas which include biopharmaceuticals, biobased products and agriculture. During the last 20 years, innovative biotechnological techniques for plant genome improvement have been developed. Many factors worldwide have led to the status quo : different legislations around the world, the lack of public acceptance in the EU and high expectations for new strategies for sustainability and food security. Therefore, a clear regulatory status for new techniques is crucial for research and development, as well as for their practical implementation. This should be based on solid science which plays a critical role in developing the bioeconomy.

ENWEndNote BIBJabRef, Mendeley RISPapers, Reference Manager, RefWorks, Zotero AMA Żurawska-Zajfert M, Grelewska-Nowotko K, Żmijewska E, et al. In-house harmonization of quantitative PCR method validation to determine GM maize events. BioTechnologia. 2016;97(4):305-313. doi:10.5114/bta.2016.64548. APA Żurawska-Zajfert, M., Grelewska-Nowotko, K., Żmijewska, E., Linkiewicz, A., Nowosielski, J., & Zimny, J. et al. (2016). In-house harmonization of quantitative PCR method validation to determine GM maize events. BioTechnologia, 97(4), 305-313. https://doi.org/10.5114/bta.2016.64548 Chicago Żurawska-Zajfert, Magdalena, Katarzyna Grelewska-Nowotko, Ewelina Żmijewska, Anna Linkiewicz, Jarosław Nowosielski, Janusz Zimny, and Sławomir Sowa. 2016. "In-house harmonization of quantitative PCR method validation to determine GM maize events". BioTechnologia 97 (4): 305-313. doi:10.5114/bta.2016.64548. Harvard Żurawska-Zajfert, M., Grelewska-Nowotko, K., Żmijewska, E., Linkiewicz, A., Nowosielski, J., Zimny, J., and Sowa, S. (2016). In-house harmonization of quantitative PCR method validation to determine GM maize events. BioTechnologia, 97(4), pp.305-313. https://doi.org/10.5114/bta.2016.64548 MLA Żurawska-Zajfert, Magdalena et al. "In-house harmonization of quantitative PCR method validation to determine GM maize events." BioTechnologia, vol. 97, no. 4, 2016, pp. 305-313. doi:10.5114/bta.2016.64548. Vancouver Żurawska-Zajfert M, Grelewska-Nowotko K, Żmijewska E, Linkiewicz A, Nowosielski J, Zimny J et al. In-house harmonization of quantitative PCR method validation to determine GM maize events. BioTechnologia. 2016;97(4):305-313. doi:10.5114/bta.2016.64548.

AbstractBiological insecticides are an effective method used in plant protection. One of the most widely used active substances in biological insecticides is Cry1Ab protein, which is toxic for lepidopteran insects. This protein is produced during bacterial sporulation byBacillus thuringiensis.Other sources of Cry1Ab protein are genetically modified plants (GM) with expression ofcry1Abgene. Cry1Ab protein in both bioinsecticides and GM plants is present in the form of protoxin, which requires activation by enzymatic treatment in the gut of susceptible insects. So far, Cry1Ab mode of action is not fully understood, but there are 3 main concepts describing it. Two of them assume that a toxic protein after binding to receptors in the insect gut penetrates into the cells, causing pore formation in the gut, which leads to the death of the sensitive insect. In the third model Cry1Ab toxic action is a result of toxin-induced chemical processes initiating a cell death pathway. This work describes the structure and mode of action of Cry1Ab protein, present in biological insecticides and genetically modified plants.

Przed dopuszczeniem na rynek UE produkty zmodyfikowane genetycznie (GM) muszą zostać ocenione pod względem bezpieczeństwa dla zdrowia człowieka i zwierząt a w przypadku uprawy również pod względem bezpieczeństwa dla środowiska. Niezbędnym elementem autoryzacji GMO w UE jako żywność i pasza jest również opracowanie wiarygodnych metod wykrywania i oznaczania ilościowego modyfikacji genetycznych. Metody te umożliwiają identyfikację każdego autoryzowanego GMO i są stosowane przez państwa członkowskie dla potrzeb kontroli rynku. Zgodnie z zaleceniami Komisji Europejskiej do analiz jakościowych i ilościowych GMO wykorzystuje się metody molekularne oparte na technikach PCR i Real Time PCR. Walidacja tych metod, spełniająca określone wymagania, pozwala na uzyskiwanie wiarygodnych, powtarzalnych i porównywalnych wyników pozwalających kontrolować GMO na rynku europejskim.

The ability to edit DNA sequences in plant genomes via site-directed mutagenesis is of great importance for basic plant biology and agriculture biotechnology. Targeted genome modifications become possible using site-specific nucleases- new synthetic biology tools. Recent advances in genetic engineering allow to modify naturally occurring nucleases. Designed domains derived from zinc finger transcription factors or transcription activator-like effectors fused to endonuclease domain of a class II restriction enzyme are responsible for identifying and introducing desired changes in the selected locus. Precise editing of DNA sequence occurs by introducing double strand break and repair by subsequent mechanism involving either non-homologous end joining or homologous recombination. Site-specific nucleases comprise meganucleases, zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regulatory interspaced short palindromic repeats/CRISPR-associated system. The application of these tools can help overcome legal problems related to use of genetically modified organisms. We present and discuss commonly used systems for site-specific genome engineering and highlight the ability to create non-transgenic plants for modern plant breeding.

Abstract. The objective of the study was to evaluate the impact of genetically modified (GM) ingredients (soya bean meal and maize grain) used in poultry diets on birds' performance, as well as the accumulation of the transgenic DNA in eggs, breast muscle and internal organs. In the present experiment four generations of Japanese quails, which were subject to three different diets, two containing genetically modified organisms (GMO, soya and maize) and one GMO-free control, were analysed. Birds' performance traits were monitored along the trial. A screening molecular method – polymerase chain reaction amplification (PCR) – was used to detect CaMV 35S promoter and nos terminator in the collected samples. Results showed no presence of modified DNA in the analysed products. According to the obtained results, it was concluded that there was no negative effect of the use of genetically modified soya bean meal or maize grain found with regard to final product safety for consumers or to birds' productivity.

Maize can be a valuable source of pollen when plants attractive for bees are not available. Honeybees can forage from conventional maize as well as from genetically modified (GM) maize. The Court of Justice of the European Union (EU) ruled that pollen in honey must be treated as a food ingredient and therefore falls within the scope of Regulation 1829/2003/EC on GM food and feed and requires authorization. GM pollen unauthorized in the EU cannot be present in honey at any level, and honey must be labelled if it contains more than 0.9% of pollen from authorized GM plants in relation to total pollen content. However, currently available analytical methods allow only for estimation of GM pollen quantity in honey. Therefore, Directive 2001/110/EC related to honey needs to be amended so that pollen can be regarded as a natural constituent of honey. Because the EU is a big honey importer, validated and harmonized detection methods are necessary for the control of GM pollen in honey.

Kukurydza (Zea mays L.) MON810 Bt z białkiem Cry1Ab stanowi najbardziej efektywną ochronę przeciwko omacnicy prosowiance (Ostrinia nubilalis Hbn.). Jest ona uprawiana w Polsce od 2007 roku. Odmiany typu MON810 produkują 92 kDa N-terminalny fragment białka Cry1Ab z Bacillus thuringiensis (Bt) ssp. kurstaki szczep HD1, warunkujący odporność na owady z rzędu Motyle. Jednym z istotnych parametrów określających potencjał do rozwoju odporności populacji szkodników kukurydzy oraz wpływu upraw GM na owady niedocelowe jest poziom ekspresji białka w częściach roślin z którymi wchodzą w kontakt. W odmianach typu MON810 ekspresja białka Cry1Ab powinna mieć charakter konstytutywny ze względu na obecność promotora CaMV35S. Dane wskazują jednak, że zawartość białka w organach roślinnych jest różna. Ilościowy test immunoabsorpcji enzymatycznej (ELISA) został użyty do oznaczenia poziomu białka Cry1Ab w odmianach DKC60-16Bt i DKC3421YG uprawianych w warunkach zamkniętego użycia i w polu. Przedmiotem pracy było określenie poziomu białka Cry1Ab w dwu odmianach kukurydzy z cechą MON810 w Polsce i przeanalizowanie jego akumulacji w wybranych organizmach niedocelowych.