فصلنامه زیست شناسی گیاهی ایران
سال چهاردهم شماره 1 (پیاپی 51، بهار 1401)

One of the environmental problems is soil pollution with heavy metals from urban and industrial wastewater, and the treatment of these pollutants by plants is a cost-effective and environmentally friendly method. In this study, an experiment was conducted to evaluate the phytoremediation rate of Aloe vera in terms of metal accumulation index (zinc, chromium, lead, copper, manganese, nickel, magnesium, cadmium, mercury) and its morphological characteristics under two types of urban and industrial wastewater treatment. The design was completely randomized with three replications. The results showed that urban and industrial wastewater treatments caused significant changes (P≤0.05) in all examined parameters including leaf fresh weight, leaf dry weight, relative leaf water content, leaf length, leaf gel weight, the ratio of gel to leaf, root fresh weight, fresh plant weight, total biomass, root length, plant height, number of ramets, ramets height, ramets weight and root tolerance index in Aloe vera. The highest index of metal accumulation was observed in plants treated by urban wastewater and all the mentioned parameters showed a more significant decrease by treatment of urban wastewater than industrial wastewater. In the industrial wastewater treatment, all parameters decreased, but the average number of ramets increased compared to the control. In general, based on the parameters studied in this study, it can be said that the Aloe vera plant had better performance in soils containing industrial wastewater than in soils containing urban wastewater. IntroductionOne of the environmental problems is soil pollution with heavy metals from urban and industrial wastewater. Human activities such as the disposal of household waste and industrial activities mainly import heavy metals into the soil (European Commission, 2013; USEPA, 2021). Heavy metal pollution of soil can harm human life and the ecosystem through direct intake or entering the food chain (soil-plant-human or soil-plant-animal-human) (European Commission, 2013). Phytoremediation is a biological and cost-effective method to remove or reduce environmental pollutants. Some plants can remove, stabilize or transfer contaminants of soil or groundwater (Gajic and Pavlovic, 2018). Therefore, choosing a suitable plant for phytoremediation can be a crucial step in eliminating pollution based on region and type of pollution and expansion of urban wastewater.The province of Guilan, located in the north of Iran, has a humid subtropical climate and is a fertile province in terms of agriculture. The increased urban population in this province can cause pollution transfer into the river or land fields. This problem threatens the agricultural products of Guilan and causes a critical challenge. It seems that the cultivation of plants with high phytoremediation ability and the creation of a green belt in the landfill site of urban and industrial wastewaters is the most feasible and low-cost method to decrease the risk of soil and water pollution. In this study, we examined the ability of Aloe vera for phytoremediation of urban and industrial wastewater (UWW and IWW) and the possible alterations of, its morphological and physiological characteristics under wastewater pollution. Materials and Methods For this study, Aloe vera plants gathered from the local nursery were planted in the pots containing 11 kilos of soil suitable for A. vera cultivation. After a month of transferring the plants to the pots and adapting them to the new conditions, they were divided into three groups. Each group was irrigated for 14 months (every twice a week) with 300 mL of one type of solutions consideredas treatments for this experiment, including urban water (drinking water), UWW collected from the landfill site of Saravan, and IWW collected from the paper factory. Moreover, pots without plants were irrigated like pots with plants to determine the amounts of heavy metals (zinc, chromium, lead, copper, manganese, nickel and magnesium) after wastewater addition to the soil and compare it with maximum standard levels, pots with plants.Soil samples were homogenized and dried in an oven at 30°C for 24h and the concentration of heavy metals was determined. The relative water content (RWC) of leaves was measured before harvesting plants (Barrs and Weatherley, 1962). After harvesting the whole plants, leaf fresh weight, leaf length, leaf gel weight, ratio gel to leaf, root fresh weight, plant fresh weight, root length, plant height, number of ramets, ramets height, ramets weight, and tolerance index were measured. Leaf dry weight and total biomass were determined after drying plant tissues (oven 50 ºC for 72 hours). All dried plant tissues were turned into powder. Then the concentration of metal elements in plant samples was determined using ICP-OES based on the method of Salt et al. (1998) and Al-Oud Saud (2003).Metal Accumulation Index (MAI) was calculated using the following formula to determine the amount of metals accumulated in plants. (Liu et al., 2007):  Results and discussionComparison of means showed that urban and industrial wastewater treatments caused significant changes in all examined parameters, including the fresh weight of leaf, root, whole plant, and ramets, dry weight of leaf, length of leaf and roots, the height of plants and ramets, relative leaf water content, leaf gel weight, ratio gel to leaf, total biomass, number of ramets and root TI. The highest index of MAI was observed in plants treated by UWW, and the reduction of all mentioned parameters was more under treatment with UWW than IWW. Under the IWW treatment, all parameters decreased, but the average number of ramets increased compared to the control.Considering that MAI shows the overall performance of plants for co-accumulation of metal elements (Liu et al., 2007), A. vera was successful in accumulating Zn, Cr, Pb, Cu, Mn, Ni, and Mg. Previous studies have reported that plant species with a high MAI value should be used as barriers between contaminated and vulnerable areas, such as parks and residential areas (Nadgorska–Socha et al., 2017). It has been reported that high levels of metals in the soil inhibit various metabolic functions of plants and cause a delay in growth. The toxicity of heavy metals in different plants varies according to factors such as the type of plant, the bioavailability of metals, and the amount of metal displacement in the plant organs (Wang et al., 2006; Usman et al., 2005).The results indicate that an increase in the concentration of heavy metals probably decreases the amount of plant production, the size of the cells, and the dry weight of the organs. (Sharma and Dubey, 2005; Yadollahi et al., 2016). ConclusionAccording to MAI, morphological and physiological characteristics of the A.vera under UWW and IWW treatments, A.vera can be a good refining plant for cultivating and creating a green belt in contaminated soils. Due to the remarkable purifying ability of A.vera in removing heavy metals from contaminated soils, it is recommended to cultivate A.vera in areas polluted with industrial and urban wastewater to prevent the spread of pollution to the surrounding lands.  AcknowledgementThis article is extracted from the results of Sareh Ebrahimi Nokande’s Ph.D. thesis from the Faculty of Sciences, University of Mohaghegh Ardabili, Ardabil, Iran. The authors would like to thank University of Guilan for the technical supports.

In this study, the effects of chitosan nanoparticles at two levels of 0.2 and 0.4 g/L under salinity stress conditions (at two levels of 75 and 150 mM) on biochemical and physiological indicators of Okra (Abelmoschus esculentus L.) were studied. The experiment was conducted as a two-factor factorial based on a completely randomized design in four replications. The results of variance analysis of the data showed that the effect of chitosan nanoparticles on fresh weight, dry weight, length, malondialdehyde, soluble sugar, starch, total phenol content, and DPPH inhibition percentage in aerial and root parts, chlorophylls, carotenoid, flavonoid, and anthocyanin content in the aerial part was significant at p<1%.  Comparison of average data showed that at 150 mM salinity in the aerial and root parts, fresh weight 1.46 and 1.31, dry weight 1.5 and 1.4, and length 1.27 and 1.52 times showed the most significant decrease compared to the control. Also, chlorophyll a 1.8, b 1.2, and carotenoid 1.6 times in the aerial part, starch 1.8 and 3.6, IC50 inhibition percentage 1.61 and 4.4 times in the aerial and root parts showed the most significant decrease in salinity of 150 mM compared to the control. Treatment with nanoparticles of 0.2 and 0.4 g/L increased chlorophyll a (1.38 and 1.39), b (1.19 and 1.18), carotenoid (1.3 and 4.1) in leaves, and flavonoid (1.7 and 1.5) and anthocyanin (1.3 and 1.5) times in aerial part and increase of soluble sugar (1.6 and 1.8; 1.6 and 1.5), total phenol (1.11 and 1.16; 1.19 and 1.3) times, respectively in shoots and roots and reduction of malondialdehyde (1.6, 1.8, 1.7, 2.8), and starch concentration (1.38, 1.43, 1.9 and 2.4) and IC50 inhibition percentage (1.13, 1.32, 1.14 and 1.75) times were in aerial and root parts. Chitosan nanoparticles at two levels of 0.2 and 0.4 g per liter improved the negative effects of salinity, and in general, chitosan nanoparticles at a concentration of 0.4 g/L showed better performance in improving both levels of salinity.IntroductionOne of the main problems in arid and semi-arid areas is soil salinity, which reduces the growth and productivity of crops (Sheikhalipour et al., 2021). Chitosan is a defense stimulant, but its use is extremely limited due to the lack of solubility in water environments. Nanoparticles have many applications in agriculture and industry due to their properties of high permeability, solubility, biodegradability, non-toxicity, and affordability. Chitosan nanoparticles are unique due to their high ratio of surface-to-volume, size-dependent quality, and optical properties (Sathiyabama and Manikandan, 2016). In maize (Zea mays) (Oliveira et al., 2016) and thistle (Silybum marianum) (Mosavikia et al., 2020), the use of chitosan nanoparticles in increasing salinity tolerance has also been studied. The Okra plant belongs to the Malvaceae family and will experience a yield decrease of up to 50% with an increase in soil salinity (Salehi Salmi and Daneshvar, 2016). Due to the increasing population and the daily food needs of the people, the use of plant growth regulators and nanotechnology for cultivation in maximum areas will be efficient strategies for improving plant resistance to adverse environmental conditions, especially the salinity of soils during the plant growth period.In the present study, an attempt has been made to investigate the biochemical and physiological indicators of the Okra plant under salt stress and the effect of chitosan nanoparticles on mitigating the negative effects of salinity stress. Materials and Methods In this research, green Okra seeds of the Clemson Spineless variety with registration number: GB-34265 were prepared by Pakan Seed Company of Isfahan. The treatment was done with 0.2 and 0.4 grams per liter of chitosan nanoparticles as foliar spraying. Salt stress was applied at the levels of 75 and 150 mM along with Hoagland's solution. Different growth and biochemical parameters were measured after taking the samples of the control and treatment groups. The parameters were the length of the aerial part and the root,  the dry weight (after 72 hours of drying in the oven),  chlorophyll and carotenoid content  (Lichtenthaler, 1987),  the amount of total flavonoid (Chang et al., 2002),  total phenol (Marinova et al., 2005), anthocyanin (Fulcki and Francis, 1968), the percentage of radical collection (2, 2-diphenyl 1-picrylhydrazyl DPPH) (Burits and Bucar, 2000), the amount of soluble sugars (by the phenol sulfuric acid method),  starch (Magne et al., 2006), and malondialdehyde (Heath and Packer, 1968). To minimize the experimental errors, sampling was done for each treatment in 4 replicates and three plants in each replicate. The experiment was conducted as a two-factor factorial based on a completely randomized design in four replications. SAS 9.4 software was used for statistical data analysis. Results and discussionComparison of means showed that urban and industrial wastewater treatments caused The results of variance analysis of the data showed that the effect of Chitosan nanoparticles on wet and dry weight, length, the content of Malondialdehyde, Soluble sugar, Starch, total Phenol, and percentage of DPPH inhibition in aerial and root parts, Chlorophylls, Carotenoid, Flavonoid, and Anthocyanin in the aerial part was significant at the probability level of 1%. The comparison of the average data showed that in the salinity of 150 mM in the aerial and root parts, fresh weight was 1.46 and 1.31, dry weight was 1.5 and 1.4, and length was 1.27 and 1.52 times, respectively. Also, Chlorophyll a (1.8), b (1.2), and Carotenoid 1.6 times in the aerial part, Starch 1.8 and 3.6 times, and IC50 inhibition percentage 1.61 and 4.4 times in the aerial part and roots in salinity 150 mM showed the largest decrease compared to the control. Treatment with nanoparticles 0.2 and 0.4 g/liter increased Chlorophyll a (1.38 and 1.39), b (1.19 and 18), and Carotenoid 1.3 and 1.4 times in leaves, and Flavonoid 1.7 and 1.5 and Anthocyanin 3 times 1.1 and 5.1 times in the aerial part, and it also increases the dissolved Sugar 1.6, 1.8, 1.6 and 1.5, total Phenol 1.11 and 1.16, 1.19 and 1.3 times in the aerial parts and roots, respectively. And reducing the content of Malondialdehyde 1.6, 1.8, 1.7, 2.8, Starch 1.43, 1.38, 1.4, 2.9 and IC50 inhibition percentage (1.32, 1.13, 1.14, 1.75) was equal in the aerial and root parts. ConclusionFoliar spraying or soil application of particles with a diameter of less than 100 nanometers has created a promising perspective in the agricultural sector and environmental stress management. Foliar spraying of chitosan nanoparticles on the Okra plant can play a vital role in inhibiting oxidative stress caused by salt. In the present study, chitosan nanoparticles increased the content of chlorophyll a, b, carotenoids, anthocyanins, phenol, and flavonoid and decreased the amount of malondialdehyde, which maintained membrane function and increased tolerance against salt stress. Chitosan nanoparticles at two levels of 0.2 and 0.4 g per liter improved the adverse effects of salinity. Overall, chitosan nanoparticles at a concentration of 0.4 g per liter showed a better performance in improving plant tolerance under both levels of salinity. Chitosan nanoparticles can be introduced as a suitable candidate for mitigating the adverse effects of salinity stress on plants, which can be attributed to the presence of its amine and hydroxyl groups, which are easily accessible in chemical reactions, leading to the improvement of the salt stress and an increase in the productivity of crops.

In order to investigate the stimulating effect of TiO2 nanoparticles on the growth rate and the increase of some metabolites in the microalgae Spirulina platensis, the anatase and rutile forms of the TiO2 nanoparticle were compared to the bulk form, in the presence or absence of citrate, on the microalga. The highest chlorophyll, carotenoid, phycocyanin (PC), allophycocyanin (APC), and phycoerythrin (PE) were observed on 3rd day. The highest PC and APC/PE were obtained by rutile and anatase, respectively. Rutile NPs could stimulate the increase of dry biomass and maximum specific growth rate (µm) better than anatase and even the bulk form. Carbohydrate production was stimulated by bulk and rutile. Low concentrations of nanoparticles often had a better effect in increasing PC, APC, and PE pigments. The addition of citrate without TiO2 stimulated the production of astheaxanthin, lipid, protein, and ROS. The simultaneous treatment by citrate-TiO2 was able to reduce the negative effect of nanoparticles on dry weight, but totally, its effect was dependent on the form of TiO2 and stress time. Antioxidant activity was stimulated by the bulk form and caused a decrease in Malondialdehyde (MDA), but an increase in MDA was observed by nanoparticles. The highest ROS was observed in the treatment with rutile (with citrate) on the 3rd day. In general, the stimulating effect of TiO2 on the improvement of indices or its negative effects on S.platensis microalga depended on the form and concentration of TiO2, the presence or absence of citrate, and the duration of treatments.IntroductionThe titanium element, in some cases, improves plant metabolic processes (Gohari et al., 2022; Carvajal and Alcaraz 1998). TiO2 nanoparticles in different forms have many applications in industry, and it is possible to find their way into aquatic ecosystems (Yang et al., 2015). The two forms of anatase and rutile have different surfaces, structural, and toxicity properties (Parrino et al., 2021). Some studies have shown that anatase has more cytotoxicity and photocatalytic activity than the rutile form (Clément et al., 2013). Of course, some sources mention TiO2 with low toxicity (Parrino et al., 2021). However, the effects of TiO2 nanoparticles depend on the type of organism under investigation, nanoparticle concentration, physicochemical and morphological characteristics such as its size and crystal structure. S. platensis, a spiral filamentous blue-green microalgae, has attracted the attention of researchers due to its numerous economic applications, including as a fertilizer and enhancer in agriculture, and has valuable minerals, vitamins, and nutritional compounds (Godlewska et al., 2019; Deng and Chow, 2010). Citric acid is required for various biological processes (Mudunkotuwa and Grassian, 2010). The citrate-nanometal complex can facilitate the entry of NPs along with citrate into the cell (Rupasinghe, 2011); hence the citrate form of nanometals has been recommended for the growth of plants (Chandrika et al., 2021). Some studies on plants have also shown that citrate reduces the adverse effects of nanoparticles (Tirani et al., 2018). Citrate can be adsorbed on the oxides of nanoparticles through Van der Waals bonds and prevent particles from aggregation (Park and Shumaker-Parry 2014; Rupasinghe 2011; Mudunkotuwa and Grassian, 2010).The aim of researchThe goal of this research was a comparative evaluation of the possible stimulating or adverse effects of bulk forms and nanoparticles of TiO2 (anatase and rutile), with or without citrate (in concentrations of 12.5, 25, 50, 100 and 200 mg. L-1) on the growth, biomass and intracellular compounds of microalgae S. platensis in 5 days. Materials and Methods Zarrouk nutrient medium was used for the growth of microalgae. Anatase nanoparticles 10-25 nm (Purity 99.9%, Specific surface area, SSA 200-240 m2 g-1), rutile nanoparticles 30 nm, (Purity 99.9%, SSA 35-60 m2 g-1) and bulk sample (diameter <500 nm) were used. Thirty-two (32) treatments were designed as follows: sample S, control culture without any treatment; Samples B1 to B5 treatments containing concentrations of 12.5, 25, 50, 100 and 200 mg L-1 of bulk TiO2 respectively; samples A1 to A5 containing concentrations of 12.5, 25, 50, 100 and 200 mg L-1 NPs anatase respectively; Samples R1 to R5 containing concentrations of 12.5, 25, 50, 100 and 200 mg L-1 of NPs rutile, respectively; C, samples containing citrate. In samples containing both citrate and TiO2, citrate to titanium dioxide was used in a molar ratio of 3.5 to 1 (Mudunkotuwa and Grassian, 2010). In sample C, which only had citrate, the amount of citrate was considered equal to it in 200 mg/liter TiO2. Then all the samples were placed in controlled conditions of 100 µmol m-2s-1,16/8 h of light/darkness and a temperature of 28 ± 2 °C for five days. Sampling and measurement of indicators were done on days zero (day of inoculation), 3, and 5 experiments. Various parameters were evaluated, including dry weight, maximum specific growth rate (µm), pigments chlorophyll a, total carotenoid, phycobilins, astaxanthin, total soluble sugar, total soluble protein, lipid, lipid peroxidation, antioxidant activity (DPPH radical inhibition), and ROS levels. Results and discussionRutile nanoparticles in the presence of citrate or without it caused an increase in dry biomass and µm index, while anatase nanoparticles caused a decrease. This can be attributed to the difference in particle size and the difference in specific surface area (SSA) of rutile particles (about 30 nm) and Anatase (about 10-25 nm) (Parrino et al., 2021). The high content of ROS was not observed in most of the anatase treatments, but the level of ROS was high on the 5 d in control C, while the simultaneous presence of titanium and citrate caused a significant reduction of it. Surface adsorption of citrate on TiO2 nanoparticles prevents the aggregation of TiO2 and increases cell access to nanometal (Mudunkotuwa and Grassia 2010). However, exopolymers attached to the outer surface of the cells can increase the contribution of citrate-nanometal complex surface adsorption on the cell surface and reduce its entry into the cells (Zhou et al., 2016). On the 3 d, antioxidant system activity increased in anatase treatments, which caused at least a partial reduction of ROS in anatase samples without citrate. The amount of antioxidant activity was often higher in bulk nanoparticles. Sendra et al. (2017), showed that the rate of exopolymers in the treatment with TiO2 nanoparticles was much higher than when they were treated with TiO2 bulk form. The concentration of 100 mg L-1 caused the highest amount of chlorophyll a, carotenoid, and astaxanthin, and the lowest concentration (12.5 mg L-1) caused the highest levels of APC, PC, PE and total phycobilin pigments. Anatase stimulated the production of the highest amount of chlorophyll, APC, and PE, especially on the 3 d, and rutile specifically induced the production of carotenoid, astaxanthin, and PC. The carbohydrate amount was increased by bulk and nano-rutile form. Lipid content was increased by the treatment of citrate, TiO2 (with or without citrate), and bulk form with citrate. ConclusionThe effects of the treatments were observed as stimulating and positive or negative effects depending on the form and concentration of TiO2, the presence or absence of citrate, and the duration of treatment with nanoparticles. Biomass (dry weight) increased by the treatment with rutile nanoparticles and, to some extent, by treatment with a bulk form of rutile. An increase in chlorophyll, carotenoid, astaxanthin, phycobilins, carbohydrate, lipid, and protein was also observed. It is possible that Spirulina with the improved indices containing a certain level of nanoparticles can be used as a fertilizer in agriculture. Citrate did not always show the same effects, and its effects depend on the type of accompanying nanoparticle, TiO2 concentration, and duration of stress.

Cellulose is the most abundant linear homopolysaccharide in nature, which has always raised great attention from investigators and industrial investors for use in wood and paper pulp industries, biofuel, and nanocellulose products due to its semi-crystalline nature and unique physicochemical properties. However, the cell wall recalcitrance and high cellulose crystallinity of wood biomass are the most challenging issues during cellulose extraction and its saccharification in bioethanol and nanocellulose production industries. Here, we have investigated the effect of CRISPR/Cas9-mediated mutagenesis on the Pro435 and Try436 residues at the binding site of PalCESA4-specific P-CR domain from white poplar. As the result, we generated a PalCESA4P435del_W436del homozygous T0 mutant plant with normal growth that showed a significant decrease in cell wall area (21.89%), cell wall thickness (7.5%), cellulose content (~ 44%), and cellulose crystallinity (19.5%) compared to the WT plant. Our findings reveal a promising approach to achieving genetically edited woods with suitable physicochemical properties of cellulose microfibrils in different plant species for industrial applications.IntroductionCellulose is the most abundant linear homopolymer in wood biomass of woody plants. Due to its semi-crystalline nature, it uses in various industrial applications, including wood and paper, textile, pharmaceutical and medicinal, agriculture, biofuel, and bio-nanomaterials (Littlewood et al., 2014; Trache et al., 2020). Despite its wide applications, high cellulose crystalization is the most important challenge in the production processes of bioethanol and nanocellulose from wood biomass (Vermerris and Abril, 2015). As the previous reports associated with the cellulose biosynthesis pathway in Arabidopsis and poplar, the CrI and DP of cellulose, the number of microfibrils in the cell wall architecture is highly influenced by the arrangement of cellulose synthase A (CESA) subunits during the formation of the cellulose synthase complex (CSC) (Luan et al., 2011; Scanlon and Timmermans, 2013; Speicher et al., 2018). In our previous study, we identified the strong binding site containing four amino acids Pro435, Trp436, Pro437, and Gly438 in the P-CR domain of poplar CESA4 subunit, which is involved in the CESA4 and CESA8 heterodimerization. The CRISPR/Cas9-mediated mutagenesis in Trp436 and Pro437 residues could alter plant growth and photosynthesis rates, a cell wall thickness of xylem and phloem cells, lignocellulose content, CrI, and DP of cellulose fibers in poplar (Nayeri et al., 2022). Materials and Methods The CESA4-specific sgRNA (5′-CAGGATGGTACCCCCATGGCCTGG-3′) was designed using CRISPRdirect, CHOPCHOP, and CRISPOR-Tefor tools. The genome sequence data of P. trichocarpa v 4.1 was used to determine the sgRNA on-target and off-target sites. The molecular cloning of the recombinant CRISPR/Cas9 expression vector (Figure 1) was performed according to the method described by Nayeri et al., 2022. The pulvini tTCL explants from the petioles of 4th nodes close to apical buds of P. alba L. were transformed using competent bacterial cells of Agrobacterium tumefaciens strain LBA4404 as described by Nayeri et al., 2022. The T0 plants were screened and validated using PCR analysis of bar and pcoCas9 genes. The bacterial contaminations of to plants were examined using PCR analysis of the Vir G gene. The screening of T0 mutants and identification of mutation types in the PalCESA4-sgRNA target site gene were performed using heteroduplex PCR genotyping methods (Guo et al., 2018), amplification of PCR fragments using mutation site-specific primers (MSBSP-PCR) (Bhattacharya and Van Meir, 2019) and sanger sequencing analysis. The sequence data of primer sets were presented in Table 1. The wood anatomy of the T0 mutant was investigated using light microscopy (LM) and field emission scanning electron microscopy (FE-SEM) analysis. The contents of lignocellulosic components were determined according to the TAPPI international standard guidelines. The cellulose crystallinity index (CrI) and its degree of polymerization (DPN, DPW, PDI) were determined using XRD analysis patterns and gel permeation chromatography (GPC), respectively. The statistical analysis was performed using one-way ANOVA analysis of variance and mean comparison based on LSD posthoc's minimum significant difference using SPSS IBM v. 22.0 (SPSS Inc, Chicago, USA). The maximum significance level was set at 5% (p-value ≤0.05). Results and discussionAs shown in the PCR analysis results, 14 out of 15 regenerated T0 plantlets had DNA bands of PCR products belonging to bar and pcoCas9 genes. Furthermore, 11 out of 14 T0 plants showed a lack of DNA bands from the PCR2 products (196 bp), which indicates homozygous or biallelic mutations in T0 mutants. In the rest of the T0 plants, no mutation or heterozygous mutations were observed due to the sharp DNA bands from the PCR2 products. The heteroduplex genotyping results showed the double DNA band of CESA-PCR3 fragments (160 bp) in 5% agarose gel, indicating heterozygous mutation in the T0 mutant poplar. However, the DNA hybridization in potent biallelic/homozygous T0 mutants indicates homozygous mutation in all 11 T0 mutants. Among the T0 mutants, we identified a homozygous T0 mutant plant (L7) with double deletion mutations in P435 and W436 residues (Figure 3E). As the wood anatomy analysis results, the Vd value of the homozygous palCESA4P435del_W436del (L7) T0 plant showed a significant decrease of 74.85% compared with the WT. The cell wall area (21.89%), cell wall thickness (7.5%), cellulose content (approximately 44%), and CrI of cellulose (19.5%) significantly decreased. According to the previous reports, the knockdown or knockout of PtriCESA4, PtriCESA7a/b, and PtriCESA8a/b genes cause a remarkable decrease in the thickness of the cell wall and cellulose content (~90%), resulting in the production of unhealthy and abnormal mutants (Abbas et al., 2020; Xu et al., 2021). Our previous findings reveal that substitution and deletion of Trp436 and Pro437 residues in the binding site of CESA4-specific P-CR domain led to a 13-25% decrease in the cellulose CrI from wood biomass of T0 poplar mutant (Nayeri et al., 2022).   ConclusionThese findings can be used in the production of gene-edited plants using the CRISPR/Cas9 system with desirable traits such as low cellulose crystalization, leading to saving in costs and time in the pure cellulose extraction process and preparation of cellulose-based biomaterials. Therefore, this isa promising technology for the mass production of cellulose-based products, which increases export volume and develop new and environmental-friendly technologies in the country. AcknowledgementThis article was extracted from the final report of the research project under contract number 346/27/D, which was applied to the research grant of the University of Tabriz, Tabriz, Iran. Our special thanks to Iranian Research Institute for Information Science and Technology (IranDoc) for technical and financial suports of our genetic engineering lab, the center of graduate education, faculty of agriculture, university of Tabriz, Tabriz-51666, Iran IR.

In order to investigate the effect of humic acid and mycorrhiza on cut flower of Gerbera cv. Dune, an experiment was performed as a completely randomized design with two factors and three replications in hydroponic conditions. First factor was included: humic acid in 4 concentrations of 0 (control), 500, 1000 and 2000 mg/l as drench and the second factor: two levels of mycorrhizal fungi (without mycorrhiza and with mycorrhiza) were applied by root inoculation. Indicators such as leaf number and leaf area, root volume, fresh and dry weight of flowers, vase life, chlorophyll a, b and total chlorophyll, total soluble sugar and some postharvest traits such as petal ion leakage and petal malondialdehyde were measured. The results showed that humic acid and mycorrhizal fungus improved the morphological and biochemical properties of gerbera. Concomitant use of humic acid at a concentration of 1000 mg/l and mycorrhizal fungus on the difference between leaf length and root volume, fresh and dry weight of flowers and vase life almost doubled compared to the control. Application of humic acid and mycorrhizal fungus significantly increased total chlorophyll content and soluble sugar content. Application of humic acid and mycorrhizal fungus reduced ion leakage and malondialdehyde. IntroductionGerbera (Gerbera jamesonii) is a rosette, herbaceous, perennial plant belonging to the Asteraceae family. The leaves have long petioles up to 15 cm long. The flower diameter in wild gerbera is 7-10 cm and in hybrid plants is 15-25 cm. Gerbera is a day-neutral plant and flowers better in more light intensity. This plant flowers from late spring to late autumn and even early winter (Rashidi, 2009). Humic substances are obtained from the decomposition of soil organic matter, peat, lignin, etc., and their actions are similar to auxin and cytokinin. These substances improve the absorption of some elements by being placed in the cell membrane, which can to maintain the stability of the membrane (Balazadeh and Hassanpour Asil, 2014). Humic acid increases the length and weight of roots and the number of lateral roots (Ambreen and Khetran, 2014). Due to its cytokinin-like properties, humic acid delays the decomposition of chlorophyll and protein in leaves and senescence in flowers. These compounds play an essential role in the metabolism of carbohydrates and their transfer to the growing buds, thereby increasing the dry matter in flowers and their longevity (Hosseini Darvishani and Chamani, 2013). In many plants, the root system of the plant is symbiotically connected with different species of fungi, and they form a complex association called mycorrhizae (Alizadeh, 2011). Mycorrhizal fungi in the hydroponic system lead to the expansion and development of the roots, which increase the yield of the product. Three main groups of ornamental plants that are suitable for symbiosis with mycorrhizal fungi have been identified, including pot plants, biennial and perennial plants, and ornamental plants grown under hydroponic conditions (Crisan et al., 2017).Considering the positive effects of mycorrhizal fungi and humic acid on the quantitative and qualitative traits of plants, this study investigated the effects of different concentrations of humic acid were investigated on some growth, flowering, and post-harvest characteristics of gerbera flowers in symbiosis with mycorrhizal fungi in hydroponic conditions. Materials and MethodsTo perform this experiment, tissue-cultured plantlets of gerbera (Gerbera jamesonii ‘Dune’) were used. These plants were grown in pots of size 20 (volume 7 liters, height and diameter of pots 19 and 24 cm, respectively) under soilless conditions (a mixture of 65% peat moss, 30% perlite, and 5% cocopeat). The daily temperature of the greenhouse was 20-25/13-16ºC (day/night), and the light intensity was 400-500 µmol.m-2s-1. This research was carried out in a factorial trial based on a completely randomized design with three replications. Each replication contained three pots with one plant in each pot. The experimental factors included: four concentrations of humic acid (0, 500, 1000, and 2000 mg/l) as a drench, two-week intervals for three months, and the second factor: two levels of mycorrhizal fungi (with or without mycorrhizal inoculation). The culture medium was inoculated with a mixture of three types of mycorrhizal fungi, including Rhizophagus fasciculatus, Diversispora versiformis, and Funneliformis mosseae. Inoculation was done at the time of planting plants in pots and in the root zone. Sixty grams of inoculum (including soil, fungal spores, hyphae, and mycorrhizal roots) were added to half of the pots (plants inoculated with mycorrhizal fungi). The same amount of autoclaved inoculum was added to the rest of the pots (plants without mycorrhizal inoculation). At the end of the experiment, parameters were measured, such as leaf number and leaf area, root volume, fresh and dry weight of flowers, vase life, chlorophyll a, b, and total chlorophyll, total soluble sugar, and some postharvest traits such as petal ion leakage and petal malondialdehyde. Statistical analysis of data was done with SAS Ver. 9.2, and the comparison of means was done with Tukey's multiple range test. Results and DiscussionThe results showed that humic acid and mycorrhizal fungus improved the morphological and biochemical properties of gerbera. A comparison of means showed that the length of leaves and leaf area increased with the increase of humic acid in plants inoculated with a mycorrhizal fungus. The highest root volume was observed in the concentrations of 1000 and 2000 mg/l of humic acid in inoculated plants. Increasing the concentration of humic acid led to a significant increase in the flower fresh and dry weight of inoculated plants so that the highest flower fresh and dry weight was observed at a concentration of 2000 mg/l of humic acid in inoculated plants. The results showed that by increasing the concentration of humic acid up to 1000 mg/l, the vase life of gerbera flowers increases, but at higher concentrations, a decreasing trend was observed. The use of mycorrhizal fungi also increased the vase life of the flowers. The application of humic acid (up to 1000 mg/l) and inoculation with mycorrhizal fungi led to an increase in the amount of total chlorophyll. In non-inoculated plants, the concentrations of 1000 and 2000 mg/l of humic acid caused a significant increase in total soluble sugar compared to the control. Also, the application of humic acid and mycorrhizal inoculation reduced ion leakage and malondialdehyde of flowers during the vase life period. Due to the formation of complexes with mineral ions, humic substances cause the absorption of more elements and, as a result, increase the amount of photosynthesis in the plant, thus increasing the growth of the aerial parts of the plant (Abbas and Hammad, 2017). It should be noted that the hormone-like property of humic acid in plants accelerates the growth of roots leading to the growth of aerial parts and the improvement of the flowering characteristics of the plant (Youssef et al., 2004). ConclusionFrom the results obtained, it can be concluded that the use of treatments with humic acid and mycorrhizal fungi improves some parameters of the plants, including the morphological, physiological, biochemical, and post-harvest characteristics. According to the results, among different concentrations of humic acid, the concentration of 1000 mg/l of humic acid with mycorrhizal fungi inoculation had the highest effect on improving the measured traits.

This study was conducted to investigate the effect of salinity caused by sodium chloride on the content of ions and understand the level of resistance in four varieties of Canola (Brassica napus L.) namely Talaye, Sarigol, Zarafam, and Opera. The plants were grown in hydroponic solution (1/4 strength Hoagland solution) and greenhouse conditions. When the plants reached the 5-leaf stage, they were treated with concentrations of 0 (control), 50, 75, and 100 mM sodium chloride for 14 days and then harvested. In order to measure the content of nutritional elements of the plant organs, extracts of the desired organs were prepared.
The results of the experiments showed that with increasing salinity, the potassium content of shoots and roots decreased in all 4 cultivars in response to salinity stress, while the content of chlorine and sodium increased. The highest increase of chlorine and sodium elements in response to salinity stress was observed in Talaye and Sarigol cultivars, about 1000 times more than the control plants. The increase in the sodium content of the shoot was higher than that of the root and it increased about 800 times in the roots of Talaye and Sarigol cultivars, it increased about 800 times. Proline and glycine betaine contents of leaves increased in response to salt stress. The amount of soluble sugar in shoot and root also increased in some cultivars, so in the treatment of 100 mM, its amount in the leaves of Talaye variety increased by 43% and in Sarigol by 80%. According to the obtained results, it can be concluded that among the cultivars studied, Opera and Zarafam cultivars have better performance under salt stress than the other two cultivars, and the performance of Sarigol is weaker than other cultivars.

Salinity is a widespread environmental stress that restricts crop production worldwide. Soils in Iran, particularly in arid and semi-arid zones, are affected by salinity stress. Under salt stress, plants show reduced water potential, ion toxicity, and ion uptake interference. Crop plants mainly respond to salinity using various mechanisms, including avoidance or tolerance. Almost all crops are glycophytes and cannot inhibit Na+ and Cl- accumulation in shoots and its out-coming damage to leaf tissues (Munns and Tester, 2008). Tolerant plants produce different osmolytes, such as soluble sugars, proline, glycine betaine, and toxic ion compartmentation. Canola is one of the most important oilseed crops used for edible oil production due to the lower content of saturated fatty acids (Brand et al., 2001). Iran has a potential capacity of up to 5 million tons/year of canola production, while it is not more than 190 thousand tons/year (Zarafshani et al., 2017). McNeilly and Ashraf (2004) concluded that salt-tolerant rapeseeds have lower concentrations of sodium and chlorine in their aerial parts when faced with salinity. Plant species show different capacities of tolerance when exposed to high salinity. This study was conducted to investigate the effect of salinity caused by sodium chloride on the content of ions and to determine salt tolerance ability in four varieties of Canola (Brassica napus L.): Talaye, Sarigol, Zarfam, and Opera. This study aimed to determine the salt tolerance capacity of four different cultivars of rapeseed considering, the content of ions, and the effects of chlorine and sodium ions on physiological and biochemical parameters.

One-week-oldseedlings of the above mentioned four cultivars of canola were transferred to ¼ strength Hoagland solution and grown for five weeks under greenhouse conditions and different NaCl treatments (0, 50, 75, and 100 mM).
100 mg ground dry mass of shoots and roots were added to hot water. Then, each extract (10 ml) was used to determine K+, Na+, and Cl- ions content using flame photometry and chloride analyzer based on titration with silver ions methods. The soluble sugars were measured in the samples extracted by ethanol based on the Dubois et al. (1954) method. The proline contents of leaves and roots were determined using Bates et al. (1973). The extracts of leaves and roots in sulfosalicylic acid (3%) were transferred to toluene after adding acetic acid, phosphoric acid and, ninhydrin reagents to the mixture. Then, the optical density of the upper part of the solution was read at 520 nm using a spectrophotometer. All calculations were made according to a standard curve plotted by known different concentrations of proline. The glycine betaine contents were measured according to Grattan and Grieve (1983). Ground dry mass of leaves and roots were incubated in sulfuric acid and dichloroethane for 1 and 2.5 hours, respectively, and the optical density of the extracts was determined at 360 nm 

In this study, salinity stress has increased the sodium content of shoots and roots in all four cultivars. This increase is more in Talayah and Sari-Gol cultivars than the other two cultivars. Also, the sodium content of the shoots is higher than the roots. In some cultivars of the rapeseed plant, the barrier mechanisms that prevent sodium from entering the roots, less Na accumulation in the aerial organs, and maintaining high ratios of potassium to sodium, especially in young photosynthetic tissues, probably have increased tolerance to salinity (Atlasi Pak, 2016). However, in all cultivars, the potassium contents of root and shoot significantly decreased under different salinity. Other researchers also considered the increase of sodium absorption in the roots and its competition with potassium absorption under salt stress as the cause of the decrease in the amount of potassium in the roots (Mittal and Dubey, 1991). The reduction of potassium in the roots and shoots has also been reported in rapeseed under salinity. The researchers stated that the amount of potassium in the shoot is directly related to the growth rate as brassica species that have more ability to retain potassium will be more tolerant to salinity (He and Cramer, 1993). The results of the present research showed that the chlorine content of aerial parts and roots increased with the application of salinity. It was also observed that the amount of chlorine in aerial parts is about 3 times higher than the sodium content of aerial parts. It could be due to the higher inhibitory effects of Cl on plant growth rather than Na in Canola (Teakle and Tyerman 2010). According to the results obtained in this study, changes in osmolytes concentrations showed different patterns. This variation was higher in soluble sugars than the others. However, proline and glycine betaine contents increased with increasing salinity in all cultivars. Various studies showed a positive relationship between the accumulation of osmolytes, such as GB and proline, and stress tolerance in plants. Some studies showed that an increase in the concentration of these two osmolytes under stress is not an adaptive stress response. The accumulation of GB in response to salinity has been found in many crops, including corn (Sorghum bicolor) (Weimberg et al., 1984). 

According to the results, salinity treatment causes changes in the ion content of roots and shoots including a higher content of Na and Cl and a lower content of K. In addition, based on the highest amounts of chlorine in aerial organs in all four cultivars (3 times greater than Na), it can be concluded that the decrease in growth in all cultivars could be related to chlorine toxicity, and sodium has intensified the effects. Proline and glycine betaine contents increased under salt stress in four cultivars. Overall, the results showed that Opera and Zarafam cultivars have higher capacities to tolerate salinity than the others.