Cannabis sativa L. (Cannabaceae) is an annual flowering plant well known worldwide since ancient times. After the discovery and elucidation of cannabinoids and of the endocannabinoid system, C. sativa has attracted the interest of the scientific community more and more in the last fifteen years, as new evidences of its potential role in medicine has arisen; nevertheless, the presence of the psychoactive compound Δ⁹-tetrahydrocannabinol (THC) still represents an important issue for clinical uses. For this reason, many authors have taken into account the biological effects of the non-psychotropic constituents of C. sativa, in particular cannabidiol (CBD). Some clinical trials and animal models have shown that CBD could be considered in the treatment of central and peripheral inflammation, gastrointestinal upset, epilepsy and neurodegenerative diseases.

THC containing C. sativa is approved in many countries in the world for medical purposes and some herbal preparations are enlisted in monographs of official Pharmacopoeias. On the contrary, non-psychotropic C. sativa has still a confuse regulatory status, very variable between different countries and herbal preparations are not specified.

With the aim of defining the best way to extract CBD and other secondary metabolites from non-psychotropic C. sativa, starting from the female inflorescences of C. sativa L. var. carmagnola, we tested different extraction methods by using ethanol (30-60-96% V/V) and olive oil as solvents, by varying the extraction time and the heat-decarboxylation of the herbal material. We applied two methods obtained from the Italian Pharmacopoeia FUI XII (maceration and percolation) and an automatic extraction method by means of Naviglio Estrattore^®.

Each extract was compared for the content in cannabinoids by means of TLC/HPTLC and HPCL-DAD, total phenolic compounds and total flavonoids by means of colorimetric methods and volatile constituents using a GC-MS analysis.

Each extract was evaluated for its antiradical capacity, assessed through the 2,2-diphenyl-1-picrylhydrazyl (DPPH) test.

The extract with the best phytochemical profile and antiradical capacity was then compared to CBD for its in vitro anti-inflammatory activity by dosing the production of TNF-α in LPS-stimulated microglial cells (BV2), through non-competitive sandwich ELISA.

The 96% V/V ethanolic extract (drug:extract ratio 1:10) obtained by maceration of the decarboxylated herbal material gave the best yield of active compounds: cannabidiol 466 mg/l, total flavonoids 90 mg/l (of which vitexin 12 mg/l), sesquiterpenes (being beta-caryophyllene the most representative) and very few amount of monoterpenes (<0.1 mg/l).

The better phytochemical profile correlated well with the antiradical capacity, with the IC₅₀ of the ethanolic extract being 18.7 mg/ml.

Differently from cannabidiol alone (1 µg/ml), the selected extract, used at the same concentration, was able to significantly reduce by 28.9 % the LPS-induced production of TNF-α in BV2 microglial cells.

Arnebia euchroma is a high value herbaceous perennial plant distributed in the alpine region of Himalayas. It belongs to family Boraginaceae. Its root contains naphthoquinone pigments that are used as colourant and have numerous pharmaceuticals properties such as anti-microbial, anti-cancer, antipyretic and anti‐inflammatory. There is huge demand of these natural pigments and are collected from the wild. Overexploitation of natural habitat has led to reduction in its population and therefore listed as critically endangered plant species. In this regard, plant cell and tissue culture technology could be useful as an alternate system to produce such pigments. In this study, adventitious root cultures were induced from leaf explant of Arnebia euchroma on Murashige and Skoog (MS) medium augmented with different auxin. Among different auxins tested, IBA resulted in highest root numbers (12.0±4.99) and length (1.10±0.23 cm). Growth kinetics of adventitious roots showed significant accumulation of fresh weight (85.62±6.30 g/L) and dry weight (12.00±0.00) after four weeks of cultivation under dark condition. Out of different strength of medium used, full-strength leads to highest fresh weight (52.50± 3.37 g/L) and dry weight (8.25 ± 0.75 g/L) production. The phytochemical analysis after four weeks of cultivation showed 4122.31 µg.g⁻¹ DW naphthoquinone content in these adventitious roots as compared to 3-5 years old parent plant rhizome (5563.34 µg.g⁻¹ DW). Hence, these adventitious roots can be used as an alternate source to meet the growing demand for such bioactives.

Class III peroxidases (EC 1.11.1.7) have an affinity for a wide range of substrates and perform numerous functions, including the formation of lignin precursors - monolignol radicals using hydrogen peroxide. The activity and tissue localization of guaiacol (GPOX) and benzidine peroxidases (BPOX), which differ in the optimum pH (7.0 and 5.0, respectively) were determined in the first internode (relative to the hypocotyl) of zinnia plants of different age. Its length increased during 40 days from seed germination, and did not change then. The Klason lignin content increased linearly up to 60 days. Histochemical analysis revealed that in juvenile plants (20 days) lignin was found mainly in protoxylem, and in adult plants (60 days) – in sclerenchyma, protoxylem and metaxylem.

Hydrogen peroxide is a marker of lignification; together with phenolic compounds, it is used by class III peroxidases to form monolignol radicals. H₂O₂ content increased in internode tissues for 40 days, and then did not change up to 60 days. According to Ros Barcelo (2004), H₂O₂ is localized mainly in non-lignified cells of stem parenchyma and protoxylem in juvenile plants, and in mechanical tissues, metaxylem, and phloem in adult.

Histochemistry of enzymes revealed that BPOX was localized in endoderm, phloem, and protoxylem, while GPOX – in the metaxylem and sclerenchyma. A moderate increase of GPOX activity during internode growth was shown, and it correlated with lignin content. In contrast, BPOX activity was high at the initial growth stage, and declining to 60 days.

So the active lignification in mechanical tissue and xylem occurs during the period from 20 to 40 days. BPOX is likely involved in the processes at the early stages of growth, while GPOX is responsible for sclerenchyma and metaxylem lignification at the later stages.

Because of their plant-growth-promoting (PGP) properties, microbes play crucial roles in plant development, making them potential candidates for substituting chemical fertilizers, especially when the soil conditions are not the most appropriate to obtain maximum crop yields. This is the case of strain Pseudomonas brassicacearum CDVBN10, a bacterial endophyte isolated from rapeseed (Brassica napus) roots. Previous in vitro, in planta and in silico experiments have demonstrated that strain CDVBN10 have interesting PGP potential. This endophyte synthetizes cellulose, produces siderophores, solubilizes P, promotes plant height and root length in rapeseed seedlings, and carries genes implicated in several PGP pathways. Based on these data, we conducted greenhouse experiments using this strain as biofertilizer under normal and salinity stress conditions, finding significant improvements in those inoculated plants, compared to the negative control. To prove its biofertilizer potential under field conditions, we carried out a field trial in which plants inoculated with CDVBN10 showed a 216%, 174.3% and 197.8% increase in number of pods, seed weight and aerial part weight, respectively. Along-read (SMRT; PacBio) 16S rRNA gene amplicon sequencing was performed in root samples from this field trials to study possible shifts on the rapeseed root microbiome due to the inoculation with CDVBN10. Interestingly, despite the great plant improvements, there were no significant differences in root bacterial communities of inoculated plants; thus, other potential beneficial members of the plant microbiome had not been displaced. According to the results, the strain P. brassicacearum CDVBN10 is suggested as a promising bacterial biofertilizer with great performance on B. napus crops.

Wheat germ agglutinin (WGA) is an active participant in ABA-controlled plant reactions to biotic and abiotic stresses; part of this wheat lectin is excreted in the area of the root apical meristem and becomes an exogenous agent for the plant. We have previously shown that 24-epibrassinolide (EBR) increased the WGA mRNA level independently of ABA because it did not change ABA content. The 0.4 μM EBR root pretreatment of wheat seedlings under the influence of 1 mM cadmium acetate in the presence of fluridone was found to inhibit the stress-induced accumulation of ABA, while the WGA content in plants was higher than in stressed EBR-untreated wheat roots. This indicates the possible existence of ABA-independent ability of EBR to regulate the quantitative level of WGA in wheat roots under cadmium stress. The protective effect of EBR is also proved by the prevention of cadmium growth-inhibiting effect on mitotic index. Moreover, EBR-pretreated wheat plants under cadmium stress form in the root cell walls Casparian bands and suberin lamellae, the critical places in the apoplastic barriers of endo- and exodermis. Thus we have demonstrated the involvement of wheat lectin in the realization of EBR-induced protective effect on plants under cadmium stress. An important contribution to the EBR-induced strengthening the barrier properties of the cell walls of the studied tissues have the ability of EBR to induce ABA-independent accumulation of WGA, which further promotes the increasing lignin and suberin biopolymers deposition in the cell walls of wheat roots.

This work was supported by the Russian Foundation for Basic Research (grant no. 20-04-00904 a), partly within the framework of the state assignment (theme no. AAAA-A16-116020350029-1).

Nitric oxide (NO) is an important signaling molecule capable of increasing the plant resistance to environmental stress of different nature. However, the mechanisms underlying the protective effect of NO on the plants still remains poorly understood. In the present study we have investigated the effect of 200 μmol SNP (Sodium Nitroprusside) on the growth and ascorbate acid (AsA) and glutathione metabolism in wheat plants (Triticum aestivum L.) under the influence of 12 % PEG. The results showed that application of SNP to wheat plants increased AsA and reduced glutathione (GSH) contents and enhancement of glutathione reductase activities. Meanwhile, growth stabilization and a decrease in the damaging effect of drought on the state of membrane structures were noted in plants, as judged by the level of MDA in them. Our results suggested that SNP can regulate the ascorbate and glutathione metabolism and has important roles in alleviating oxidative damage and enhancing drought tolerance in wheat.

This work is supported by Grant RFBR (20-04-00904 а) and partly within the framework of the state assignment (state registration number АААА-А16-116020350029-1).

Raffinose family oligosaccharides (RFOs) are major soluble carbohydrates in soybean seeds that cannot be digested by human and other monogastric animals. Hence, a major goal is to reduce RFO levels to improve the nutritional quality of soybean. In this study, we utilized a dual gRNAs CRISPR/Cas9 system to induce knockouts in two soybean galactinol synthase genes, GmGOLS1A and its homeolog GmGOLS1B. Genotyping of T0 plants showed that the construct design was efficient in inducing various deletions in the target sites or sequences spanning the two target sites of both GmGOLS1A and GmGOLS1B genes. A subset of induced alleles was successfully transferred to progeny and, at the T2 generation, we identified null segregants of single and double mutant genotypes without off-target induced mutations. The seed carbohydrate analysis of double mutant lines showed a reduction in the total RFO content of soybean seed from 64.7 mg/g dry weight to 41.95 mg/g dry weight, a 35.2% decrease. On average the stachyose content, the most predominant RFO in soybean seeds, decreased by 35.4% in double mutant soybean, while the raffinose content increased by 41.7%. A slight decrease in verbascose content was also observed in mutant lines. Aside from changes in soluble carbohydrate content, some mutant lines also exhibited increased protein and fat contents. Otherwise, no difference in seed germination, plant development and morphology was observed in the mutants. Our findings indicate that GmGOLS1A and GmGOLS1B contribute to soybean oligosaccharides profile through RFO biosynthesis pathways, and are promising targets for future investigation, as well as crop improvement efforts. Our results also demonstrate the potential in using elite soybean cultivar for transformation and targeted genome editing.

Perennials utilize complex adaptive strategies and molecular mechanisms to cope with water-deficit conditions. Hence, to gain molecular insights regarding the water-deficit stress, two-year-old coconut seedlings of the varieties Kalpa Sree and Kalpatharu were subjected to soil water-deficit regimes (25% of available of soil moisture and control). Biochemical, physiological and growth parameters underlying water-deficit stress revealed the differential enzymatic anti-oxidants, lipid peroxidation status and water use efficiency trait between the genotypes investigated. The whole plant water use efficiency at control condition was significantly low in Kalpatharu (4.06) compared to Kalpa Sree (4.74). Nevertheless, under severe stress [25% ASM] Kalpatharu exhibited highest WUE (5.68) as against dwarf variety Kalpa Sree (3.84). Furthermore, the leaf transcriptome profiles of the control and water-deficit stressed seedlings were examined utilizing paired-end RNA-Seq. In total, ~7300 differentially expressed genes have been identified between the seedlings under water-deficit stress and control. Analysis of control and stressed Kalpasree leaf transcriptome showed significant upregulation of PHLOEM PROTEIN 2-LIKE A1-like, WRKY transcription factor 40 isoform X1 and downregulation of glycerol-3-phosphate acyltransferase 3 transcripts. On the other hand, upregulation of transcripts encoding polyamine oxidase, arabinose 5-phosphate isomerase among others and downregulation of aquaporin PIP1-2 transcript was documented in Kalpatharu leaves. Besides, long non-coding RNA and genic SSRs were also identified from the transcriptome data to further enrich the genomic resources of coconut palm which would pave way for its utilization in developing climate-smart coconut crop. The implication of this study in molecular dissection of the adaptive response of coconut to the soil-water deficit is also discussed

Peacock spot caused by Spilocaea oleagina and anthracnose caused by Colletotrichum spp., are the most important foliar and fruit diseases of olive. Applications of copper-based fungicides are the main control measures for these pathogens. However, replacement of copper –based products by more eco-friendly alternatives is a priority. In this study we investigated the efficacy of 5 novel Nano-copper (Cu-NPs) formulations against the 2 major olive diseases. Two commercial copper-based formulations were included as reference treatments. The efficacy of Cu-NPs against Spilocaea oleagina was evaluated in one-year-old olive trees (cv. Chondrolia Chalkidikis) under controlled environmental conditions in plant growth chamber, while the efficacy against Colletotrichum spp. was evaluated under field conditions on the same cultivar. Results showed that the most effective Cu-NPs against Colletotrichum spp. was 110_CN_S4_X1 that provided a mean control efficacy value of 61.03%, while the 2 commercial formulations of conventional cooper products provided significantly lower control efficacy values of 35.06 and 45.45%. Similarly 3 of the Cu-NPs tested (110_CN_S4_X1, 109_CC_S4_X2, 108_CN_S1_X1) were found to be highly effective against Spilocaea oleagina with control efficacy values ranging from 60 to 67.5%. The results of this study are expected to contribute in the optimization of olive diseases control and reduce the yield losses caused, using a new generation of biocides.

This research has been co‐financed by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH – CREATE – INNOVATE (project code: T1EDK- 01492)

Many vegetable crop plants, including tomato, have high water needs. The optimal water supply is essential for successful production since most of the tomato cultivars are drought-sensitive, especially at the reproductive stage. One of the options to overcome the negative effects of water reduction on yield is the use of deficit irrigation methods. Detailed knowledge on the effects of different irrigation methods on fruit developmental processes could be a critical factor to analyse the effect on final yield. It is well known that water reduction limits fruit growth rate and final fruit size in tomato, as a consequence of impact on cell division and expansion processes. This paper reviews the roles of cellular traits in the responses of tomato fruit growth to deficit irrigation (DI) were assessed using wild type (WT) and its flacca mutant deficient in ABA. We specifically addressed how cell number, cell size and setup of pericarp cell layers were affected by water deficit during fruit development. Micromorphological analysis of pericarp cell layers showed that DI induced in flacca a stronger negative effect on cell division and expansion than in WT at an early stage of fruit development, but in ripe fruit, the effects of DI were similar in flacca and WT. The main difference between flacca and WT responses to water restriction was a stronger negative impact during early cell division phase in flacca, which is consistent with the involvement of ABA in the cell division process and water-stress-induced ABA synthesis in WT.