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Abstract 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.

Regeneration of cellulose by direct solvent dissolution essentially involves the pretreatment of cellulose with a solvent for swelling the cellulose chains and weakening the cellulose molecules bonds. Aqueous solutions based on sodium hydroxide (NaOH) are low cost, non-volatile, nontoxic, environmentally friendly cellulosic solvent with fast dissolution power at low temperatures. The mechanism of cellulose dissolution in this solvent is not yet fully understood because there are parameters such as temperature, crystallization and degree of cellulose polymerization that affect the solubility of cellulose in the NaOH / urea system. Therefore, this study investigated the effect of kappa factor changes in chlorine dioxide bleaching on the dissolution of OCC recycled pulp on chemical compounds (lignin and hemicellulose) and the degree of polymerization. The results showed that with further reduction of lignin, the dissolution of pulp decreases and this reduction in dissolution is more severe in lignin levels below 3% (reduction from 44% to about 37% dissolution). It seems that during the bleaching process and part of lignin and hemicellulose removal, a part of cellulose with low degree of polymerization is also removed and the average polymerization of the remaining cellulose increases which is more resistant to the phenomenon of pulp dissolution. With increasing kappa factor (to 0.2 and 0.3), there was no significant change in dissolution and hemicellulose. In general, it seems that lignin and the degree of polymerization are not only the effective factors in OCC pulp dissolving and hemicellulose content also can be effective. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

One of the common applications of cellulosic fibers as dissolving pulp production requires high purity. Different cellulosic fiber sources are used to produce dissolving pulp. Due to deforestation and lack of agricultural land and problems with cultivation, the use of wood and non-wood sources of such fibers is declining. Another source of cellulose is recycled pulp. Chlorine dioxide acts as a completely selective bleaching chemical. Increasing kappa factors is very effective in removing lignin levels. In this research, dissolving pulp from recycled fibers is obtained using soda cooking and bleaching with chlorine dioxide. Then, the effect of changes in kappa factor of the second stage of chlorination on chemical composition (holocellulose and lignin), structural properties (degree of polymerization, alpha-cellulose and degree of brightness) and accessibility (water retention value) are investigated. The results showed that the degradation of holocellulose levels was not severe and did not cause cellulose damage and decreased alphacellulose levels as well as the degree of polymerization. The removal of lignin as a hydrophobic component and the presence of hemicellulose as a hydrophilic component increase accessibility (water retention) and decrease brightness degree. [ABSTRACT FROM AUTHOR]

To evaluate the percentage and degree of polymerization and yield of inulin, and also root fresh yield of 13 native accessions and two imported cultivars of chicory (Cichorium intybus L.), an experiment was conducted using randomized complete block design with three replications in Khoshkedaran Research Station of Mazandaran province, Iran, in three consecutive years (2016-2018). The roots were harvested at the end of the rosette stage in the second year of cultivation, then weighed and relevant calculations were performed. Samples were extracted by using aqueous extraction method, and their total sugar content and reducing sugar and the percentage and degree of polymerization of inulin were calculated. Means comparison showed that accessions "Rasht" and "Chamestan" and imported eultivar "Selenite" had the highest percentage of inulin (15.8, 13.9, and 14.3%, respectively) among the chicory genotypes. Findings showed that eultivar "Selenite" and accession "Tonekabon" were more superior to other genotypes in terms of fresh root yield and inulin and ranked first (35.458 t ha"؛ fresh root and 2818.99 kg ha"؛ inulin yield) and second (12.6921 ha"؛ fresh root and 802.5 kg ha"؛ inulin yield), respectively. In addition, "Selenite" and "Tonekabon" had good stability in terms of percentage and degree of inulin polymerization in two years of research and were found to be more suitable for the western region of Mazandaran province than other genotypes. [ABSTRACT FROM AUTHOR]

In this research, the influence of paper drying condition, as one of the most important variable in paper recycling, on the characteristics of fibers was studied. In this respect, bagasse soda pulp was collected from Pars Paper mill in Khozestan province and handsheets were made. The control hand sheet samples were dried at room temperature and the rest of the handsheets were dried using drum dryer set at 60° C for 3 hours, 100° C for 50 minutes and 120° C for 25 minutes to reach the same moisture content as the sheets dried at room temperature. Then, these handsheets were slushed in water and the pulp properties were determined. The results showed that with increasing the drying temperature, cellulose content did not change while hemicelluloses were reduced. Although the assessment of changing the lignin content with micro kappa method showed an increasing trend, but based on previous literature, it was concluded that the result are not justified. Also, the results indicated that changing the degree of polymerization and crystallinity did not show a consistent trend, showing initial reduction following by increased values. These results were attributed to interaction effect of drying temperature and time.

This research was performed to study the effect of Murray red gum tree age (4, 6, 8 years) on the chemical components, viscosity and the cellulose degree of polymerization. The Eucalyptus trees were cut at the ages of 4, 6, and 8 from hand planted forests. The contents of cellulose, hemicellulose, lignin, extractives and ash for each age were then determined. The results show that with increasing age the contents of cellulose, hemicellulose and lignin increased but the extractives and ash contents decreased. After measuring the viscosity of cellulose solution, the degree of polymerization(DP) was also determined by using the standard equation. The viscosity numbers for 4, 6 and 8 year old trees were 290, 503 and 566 mL/g, respectively, and the DP were 272, 568 and 652, respectively. Finally after analyzing and comparing the results, the age of 8 was found to have best properties for viscose industry. But in order to reduce the forestry and production costs, 6 year old tree instead of 8 could be cut because of the close results in DP and cellulose content.

Background and Aim: Sufficient polymerization is a critical factor to obtain optimum physical properties and clinical efficacy of resin restorations. The aim of this study was to evaluate wear rates of composite resins polymerized by two different systems Light Emitting Diodes (LED) to and Halogen lamps. Materials and Methods: In this laboratory study, 20 specimens of A3 Tetric Ceram composite were placed in brass molds of 2*10*10 mm dimensions and cured for 40 seconds with 1 mm distance from surface. 10 specimens were cured with LED and the other 10 were cured with Halogen unit. A device with the ability to apply force was developed in order to test the wear of composites. After storage in distilled water for 10 days, the specimens were placed in the wear testing machine. A chrome cobalt stylus with 1.12 mm diameter was applied against the specimens surfaces with a load of 2 kg. The weight of each samples before and after 5000, 10000, 20000, 40000, 80000 and 120000 cycles was measured using an electronic balance with precision of 10-4 grams. Data were analyzed using t test and paired t test. P0.05). Conclusion: Based on the results of this study, LED and halogen light curing units resulted in a similar wear rate in composite resin restorations.