DataSheet_1_Microparticle-mediated CRISPR DNA delivery for genome editing in poplar.pdf

The use of CRISPR/Cas9 is currently the method of choice for precise genome engineering in plants, including in the biomass crop poplar. The most commonly used method for delivering CRISPR/Cas9 and its components in poplar is via Agrobacterium-mediated transformation, that besides the desired gene-editing event also results in stable T-DNA integration. Here we explore the delivery of the gene-editing reagents via DNA-coated microparticle bombardment into the model tree Populus tremula x P. alba to evaluate its potential for developing transgene-free, gene-edited trees, as well as its potential for integrating donor DNA at specific target sites. Using an optimized transformation method, which favors the regeneration of plants that transiently express the genes on the delivered donor DNA, we regenerated gene-edited plants that are free of the Cas9 and the antibiotic resistance-encoding transgenes. In addition, we report the frequent integration of donor DNA fragments at the Cas9-induced double-strand break, opening opportunities toward targeted gene insertions.</p

Additional file 1: of Structural variability and niche differentiation in the rhizosphere and endosphere bacterial microbiome of field-grown poplar trees

Community estimators. Values represent averages (±standard deviation) of 15 biological replicates (rhizosphere soil and root samples) and 11 replicates (stem and leaf samples) after normalization to 2000 sequences. Normal distributions of the data were checked with the Shapiro–Wilk test and homoscedasticity of variances was analyzed using either Bartlett’s or the Fligner-Killeens test. Significant differences in the variance of parameters were evaluated with ANOVA, and post hoc comparisons were conducted by the Tukey’s honest significant differences tests. Plant compartment effects show the overall ANOVA results: F(DFn, DFd) and P value. Significant differences at the 95% confidence interval (P < 0.05) between the plant compartments are indicated in lowercase letters (P).(XLSX 11 kb

Integration, Disintegration and the Protection of Competition: Of Myths, Stories and Images

OTU distribution across the plant compartments. Venn diagram showing the overlap in operational taxonomic unit (OTU) composition between the different plant compartments.(TIFF 471 kb

MOESM3 of Stacking of a low-lignin trait with an increased guaiacyl and 5-hydroxyguaiacyl unit trait leads to additive and synergistic effects on saccharification efficiency in Arabidopsis thaliana

Additional file 3. Relative increase in saccharification efficiency compared to wild type. The relative increase in saccharification efficiency of the parental lines and the double mutants compared to wild type was calculated based on the average of the cellulose-to-glucose conversion at the plateau. The variation was taken into account to determine whether there is an additive or synergistic effect

MOESM5 of Stacking of a low-lignin trait with an increased guaiacyl and 5-hydroxyguaiacyl unit trait leads to additive and synergistic effects on saccharification efficiency in Arabidopsis thaliana

Additional file 5. Values and significances of the released glucose/CWR over time. Released glucose/CWR of the tra2 comt-1, c4h-3 comt-4 and 4cl1-1 comt-4 double mutants, wild type and the corresponding parental lines (n = 10) (± SD), without, with acid and with alkaline pretreatment at the different timepoints. Significance groups are indicated with the same letter in superscript and different letters represent significant differences at the 0.01 significance level (ANOVA, pairwise comparison with Holm correction)

MOESM6 of Stacking of a low-lignin trait with an increased guaiacyl and 5-hydroxyguaiacyl unit trait leads to additive and synergistic effects on saccharification efficiency in Arabidopsis thaliana

Additional file 6. Relative increase in saccharification efficiency compared to wild type. The relative increase in saccharification efficiency of the parental lines and the double mutants compared to wild type was calculated based on the average of the released glucose/CWR at the plateau. The variation was taken into account to determine whether there is an additive or synergistic effect

MOESM4 of Stacking of a low-lignin trait with an increased guaiacyl and 5-hydroxyguaiacyl unit trait leads to additive and synergistic effects on saccharification efficiency in Arabidopsis thaliana

Additional file 4. Graphs of the glucose release/CWR over time. Glucose release/CWR of the tra2 comt-1, c4h-3 comt-4, and 4cl1-1 comt-4 double mutants, wild type, and the corresponding parental lines, without, with acid and with alkaline pretreatment at the different timepoints. A) Glucose release/CWR for tra2 comt-1 and its respective control lines. B) Glucose release/CWR for c4h-3 comt-4 and its respective control lines. C) Glucose release/CWR for 4cl1-1 comt-4 and its respective control lines. Measurements were done 6, 24, 48, 72, and eventually 96 h after the saccharification enzymes were added to the samples. The error bars represent standard deviations (n = 10). The exact values and significances are presented in Additional file 5. The relative increases in saccharification efficiency compared to wild type are presented in Additional file 6

MOESM7 of Stacking of a low-lignin trait with an increased guaiacyl and 5-hydroxyguaiacyl unit trait leads to additive and synergistic effects on saccharification efficiency in Arabidopsis thaliana

Additional file 7. Inflorescence stem pieces after saccharification. Inflorescence stem pieces of A) tra2 comt-1, B) c4h-3 comt-4, and C) 4cl1-1 comt-4 and their respective control lines after the plateau of saccharification was reached, without, with acid and with alkaline pretreatment. Bar 1 mm

MOESM1 of Stacking of a low-lignin trait with an increased guaiacyl and 5-hydroxyguaiacyl unit trait leads to additive and synergistic effects on saccharification efficiency in Arabidopsis thaliana

Additional file 1. Graphs of the cellulose-to-glucose conversions over time. Cellulose-to-glucose conversions of the tra2 comt-1, c4h-3 comt-4, and 4cl1-1 comt-4 double mutants, wild type, and the corresponding parental lines, without, with acid and with alkaline pretreatment at the different timepoints. A) Cellulose-to-glucose conversion for tra2 comt-1 and its respective control lines. B) Cellulose-to-glucose conversion for c4h-3 comt-4 and its respective control lines. C) Cellulose-to-glucose conversion for 4cl1-1 comt-4 and its respective control lines. The conversions were calculated based on the saccharification efficiency and cellulose content (both on CWR basis) and are expressed as % cellulose converted to glucose. Measurements were done 6, 24, 48, and 72 and eventually 96 h after the saccharification enzymes were added to the samples. The error bars represent standard deviations (n = 10). The exact values and significances are presented in Additional file 2. The relative increases in saccharification efficiency compared to wild type are presented in Additional file 3

MOESM2 of Improving total saccharification yield of Arabidopsis plants by vessel-specific complementation of caffeoyl shikimate esterase (cse) mutants

Additional file 2. Semi-quantitative analysis of the irx phenotype in stem cross sections from the wild type, cse-2 mutant, and the cse-2 proVND::CSE lines after Mäule staining. A) Scoring of the irx phenotype in the cse-2 proVND7::CSE lines; B) Scoring of the irx phenotype in the cse-2 proVND6::CSE lines. The values plotted on the graph are the averages of measurements made by two independent monitoring researchers in a double blind experiment. These values were calculated by dividing the total number of collapsed vessels found in each genotype by the total number of xylem vessels found in all vascular bundles scored per genotype. A minimum of 11 and an average of 17.5 vascular bundles were scored, by inspecting sections from three individual plants per genotype. Error bars indicate the standard deviation. One-way ANOVA and Duncan’s Multiple Range Test were performed to reveal significant (P < 0.05) differences between the various lines, which are indicated by different letters