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.

The physiological and biochemical responses of pedunculate oaks (Quercus robur L.) to heat stress (HS) and mycorrhization (individually as well in combination) were estimated. One-year-old Q. robur seedlings were grown under controlled conditions in a pot experiment, inoculated with a commercial inoculum of ectomycorrhizal (ECM) fungi, and subjected to 72 h of heat stress (40 °C/30 °C day/night temperature, relative humidity 80%, photoperiod 16/8 h) in a climate chamber, and they were compared with seedlings that were grown at room temperature (RT). An in-depth analysis of certain well-known stress-related metrics such as proline, total phenolics, FRAP, ABTS, non-protein thiols, and lipid peroxidation revealed that mycorrhized oak seedlings were more resistant to heat stress (HS) than non-mycorrhized oaks. Additionally, levels of specific polyamines, total phenolics, flavonoids, and condensed tannins as well as osmotica (proline and glycine betaine) content were measured and compared between four treatments: plants inoculated with ectomycorrhizal fungi exposed to heat stress (ECM-HS) and those grown only at RT (ECM-RT) versus non-mycorrhized controls exposed to heat stress (NM-HS) and those grown only at room temperature (NM-RT). In ectomycorrhiza inoculated oak seedlings, heat stress led to not only a rise in proline, total phenols, FRAP, ABTS, non-protein thiols, and lipid peroxidation but a notable decrease in glycine betaine and flavonoids. Amounts of three main polyamines (putrescine, spermine, and spermidine) were quantified by using high-performance liquid chromatography coupled with fluorescent detection (HPLC/FLD) after derivatization with dansyl-chloride. Heat stress significantly increased putrescine levels in non-mycorrhized oak seedlings but had no effect on spermidine or spermine levels, whereas heat stress significantly increased all inspected polyamine levels in oak seedlings inoculated with ectomycorrhizal inoculum. Spermidine (SPD) and spermine (SPM) contents were significantly higher in ECM-inoculated plants during heat stress (approximately 940 and 630 nmol g⁻¹ DW, respectively), whereas these compounds were present in smaller amounts in non-mycorrhized oak seedlings (between 510 and 550 nmol g⁻¹ DW for Spd and between 350 and 450 nmol g⁻¹ DW for Spm). These findings supported the priming and biofertilizer roles of ectomycorrhizal fungi in the mitigation of heat stress in pedunculate oaks by modification of polyamines, phenolics, and osmotica content.

Clopyralid and imazamox are successfully used for weed control during the first years of regeneration of pedunculate oak forests. Hence, the question that arises is how these herbicides affect microorganisms, especially the activity of dehydrogenase enzyme, when they reach the soil. Two study sites were selected in regenerated pedunculate oak forests, and the two herbicides were applied in two doses that are used for weed control (clopyralid, 100 g a.i. ha⁻¹ and 120 g a.i. ha⁻¹; imazamox, 40 g a.i. ha⁻¹ and 48 g a.i. ha⁻¹). The effect of the herbicides was evaluated 7, 14, 21, 30, and 60 days after application. A significant reduction in dehydrogenase activity was found on days 7 and 14 at both sites. However, after 14 days there was a recovery of dehydrogenase activity for all treatments such that the values obtained on day 21 did not differ from the control values. The effect of clopyralid and imazamox on dehydrogenase activity was not dose-dependent. Dehydrogenase activity also depended on soil properties, soil sampling time and climatic conditions during the investigation years. The results show that clopyralid and imazamox can reduce soil dehydrogenase activity, but this effect is transient. This can be attributed to the ability of microorganisms to overcome the stress caused by the herbicide by developing the capability to utilize herbicides as a nutrient source and proliferating in such an environment.

Fungal diversity is one of the most important indicators of overall forest biodiversity and its health. However, scarce information exists on the state of macrofungal communities of mountain forests in Serbia, making it one of the countries with the least-published mycological data in the Mediterranean and Balkan region of Europe. This paper presents the results of the first comprehensive, long-term study of macrofungal communities in some of the most important mountain forest ecosystems in Serbia (Tara, Kopaonik and Vidlič). In the course of three consecutive years, the sampling of five permanent experimental plots resulted in 245 species of macrofungi, classified into three functional groups (terricolous saprothrophs, lignicolous, and mycorrhizal fungi). Special attention was given to protected and indicator species, which point out the great value of studied forest habitats and the importance of their conservation. It was found that precipitation, habitat humidity, and temperature significantly influence the occurrence and distribution, primarily of mycorrhizal and lignicolous group of fungi. Thus, the continuation of long-term monitoring is crucial in order to more precisely determine which groups/species of macrofungi would, and to what extent they would, adapt to a rapidly changing climate.

Halomorphic soils cover a significant area in the Vojvodina region and represent ecological and economic challenges for agricultural and forestry sectors. In this study, four economically important Serbian poplar clones were compared according to their biochemical and transcriptomic responses towards mild and severe salt stress to select the most tolerant clones for afforestation of halomorphic soils. Three prospective clones of Populus deltoides (Bora-B229, Antonije-182/81 and PE19/66) and one of hybrid genetic background P. nigraxP. deltoides, e.g., P. x euramericana (Pannonia-M1) were hydroponically subjected to NaCl as a salt stress agent in a concentration range from 150 mM to 450 mM. Plant responses were measured at different time periods in the leaves. Biochemical response of poplar clones to salt stress was estimated by tracking several parameters such as different radical scavenging capacities (estimated by DPPH, FRAP and ABTS assays), accumulation of total phenolic content and flavonoids. Furthermore, accumulation of two osmolytes, glycine betaine and proline, were quantified. The genetic difference of those clones has been already shown by single nucleotide polymorphisms (SNPs) but this paper emphasized their differences regarding biochemical and transcriptomic salt stress responses. Five candidate genes, two putative poplar homologues of GRAS family TFs (PtGRAS17 and PtGRAS16), PtDREB2 of DREB family TFs and two abiotic stress-inducible genes (PtP5SC1, PtSOS1), were examined for their expression profiles. Results show that most salt stress-responsive genes were induced in clones M1 and PE19/66, thus showing they can tolerate salt environments with high concentrations and could be efficient in phytoremediation of salt environments. Clone M1 and PE19/66 has ABA-dependent mechanisms expressing the PtP5CS1 gene while clone 182/81 could regulate the expression of the same gene by ABA-independent pathway. To improve salt tolerance in poplar, two putative GRAS/SCL TFs and PtDREB2 gene seem to be promising candidates for genetic engineering of salt-tolerant poplar clones.

Daedaleopsis spp., a lignicolous fungus causes of white rot on wild cherry and other broadleaved species and makes economic losses in Serbian forestry. The paper presents results of two morphologically distinct fungi Daedaleopsis confragosa and Daedaleopsis tricolor isolated from native populations of wild cherry (Prunus avium L.) found in the sites of Protected Forests of Serbia. Morphological appearance of D. tricolor was found more abundant in comparison to D. confragosa species. Samples from Serbia were analysed using morphometric and molecular tools and compared with isolates from United Kingdom and published sequences from Sweden, Austria, Hungary, Germany, Canada, France, USA and Czech Republic to give the taxonomic insight and their genetic relatedness using fungal barcoding region ITS rDNA. Results from BLAST search confirmed morphology of the isolates to their taxonomic affiliation as D. confragosa while sequence analysis showed mutations at several polymorphic positions that indicates genetic divergence to D. tricolor. Phylogenetic analysis presents narrow genetic relations of Serbian isolates with the one from United Kingdom while distinctness from other countries investigated specimens. Future work needs variable regions for both species to be amplified in order to evaluate species boundaries or employing NGS technologies in more detailed sequence analyses.