Transgenic aspens (Populus tremula) with the xyloglucanase gene from Penicillium canescens keep faster growth under semi-natural conditions

Background Recombinant carbohydrases genes are used to produce transgenic woody plants with improved phenotypic traits. However, cultivation of such plants in open �eld is challenged by a number of problems. Therefore, additional research is needed to alleviate them. Results Results of successful cultivation of the transgenic aspens (Populus tremula) carrying the recombinant xyloglucanase gene (sp-Xeg) from Penicillium canescens in semi-natural conditions are reported in this paper for the �rst time. Change of carbohydrate composition of wood was observed in transgenic aspens carrying the sp-Xeg gene. The transformed transgenic line PtXVXeg1b demonstrated accelerated growth and increased content of cellulose in wood of trees growing in both greenhouse and outside in comparison with the control untransformed line Pt. The accelerated growth was observed also in the transgenic line PtXIVXeg1c. Thicker cell-wall and longer xylem �ber were also observed in both these transgenic lines. Undescribed earlier considerable reduction in the wood decomposition rate of the transgenic aspen stems was also revealed for the transformed transgenic lines. The decomposition rate was approximately twice as lower for the transgenic line PtXVXeg3b in comparison with the control untransformedline Pt. Conclusion Our results showed that the plants carrying the recombinant sp-Xeg gene do not demonstrate a decrease in growth parameters in semi-natural conditions. However, in some transgenic lines, a change in the carbohydrate composition of the wood, an increase in the cell wall thickness and a decrease in the rate of decomposition of wood are observed.


Background
Forests play a huge role in the economy as a source of timber and many types of raw materials.The logging is growing every year worldwide [1].Hardwood trees are of particular interest due to a lower content of resins and lignins, making them a convenient source of raw materials for various industries [2][3][4].Aspen (Populus tremula) is currently one of the most promising hardwood species, fast-growing, with a wide distribution range.Aspen wood is increasingly used in the pulp, paper, and viscose industries, and for container, oriented strand board, and bioethanol production [5].However, with a current high rate of logging of hardwood species, especially aspen, a shortage of raw materials is expected.Therefore, it is necessary not only to increase the e ciency and rationality of the use of wood, but also to establish forest plantations with increased productivity based on the new and improved tree forms and varieties.
Their creation by traditional breeding is a long and not very e cient process [6], while the genetic engineering approach is considered as one of the promising methods for obtaining trees with desired properties [7].
Increase in plant cell size is possible by enzymatic softening the cell wall followed by its expansion due to increased intracellular pressure [8].Xyloglucan is involved in formation of a strong cellular framework, a plant cell wall hemicellulose polysaccharide that cross-links nearby cellulose micro brils [9].
Accordingly, softening of the cell wall occurs through separation of micro brils by enzymes that cleave xyloglucans and weaken the bond between micro brils [10].Xyloglucanase is one of these enzymes belonging to the carbohydrase group, which hydrolyses xyloglucans, weakening the cross-linking of cellulose micro brils [8].
Currently, the overexpression of carbohydrases splitting xyloglucan is considered as a promising way of modifying the tree phenotype, increasing the productivity of woody plants and changing the composition and properties of wood.Shani et al. [11] produced transgenic aspen trees (Populus tremula) with an endoglucanase gene cel1 from Arabidopsis thaliana, which had signi cant phenotypic changes, such as increased plant height, leaf size, stem diameter and a higher percentage of cellulose and hemicellulose in comparison with the untransformed control under greenhouse conditions.However, the results of eld trials were not reported.Park et al. [12] showed that in white poplar (Populus alba) transformed with the xyloglucanase gene AaXEG2 from Aspergillus aculeatus, increased length of stems and discoloration of leaves were observed in comparison with the control in the climatic chambers.There was also an increase in cellulose content and a reduction in hemicellulose in transgenic trees [12].However, the growth rate of the transgenic trees in the eld was lower than that of wild-type control trees [13].
The analysis of the world experience has shown that there are successful results in using of recombinant carbohydrases and xyloglucanases to increase the growth rate and improve the quality of aspen wood (genus Populus).However, not all issues in this area are completely solved, especially for trees grown in eld conditions.Therefore, the aim of this work is to analyze aspen with altered properties of wood carrying the recombinant xyloglucanase sp-Xeg gene from Penicillium canescens under semi-natural conditions as a preliminary stage before the eld trials.
This article reports successful tests of transgenic aspen trees carrying a recombinant xyloglucanase gene sp-Xeg and growing under semi-natural conditions.Effects of xyloglucanase gene incorporation on growth parameters, chemical wood composition and rate of wood decomposition are also presented and discussed.

Expression of xyloglucanase
The expression of recombinant gene sp-Xeg in the plants growing in semi-natural conditions was con rmed by RT-PCR.The ampli cation product of the expected size (762 bp) was found in all transgenic lines with the inserted xyloglucanase gene taken for the analysis, which con rms the presence of transcripts of the recombinant sp-Xeg gene (Fig. 1).
Western blotting con rmed the presence of a recombinant XegA protein of the appropriate size (25 kDa) in the obtained 25 independent transgenic lines of aspen carrying the xyloglucanase gene sp-Xeg [14,15].Fig. 2 shows the results of Western blotting for 10 of the 25 lines, in which the recombinant protein was detected stably in all replicates of the assay.

Growth rate
Earlier, we noted that in a greenhouse environment, some transgenic lines showed an increase in the growth rate [15].Transgenic and control plants of aspen were transferred from the greenhouse conditions to an open air and grown under semi-natural conditions up to the age of 18 months.For the analysis, six lines were chosen that demonstrated the presence of a recombinant protein.Four of them had an increased plants height in greenhouse conditions after two months in comparison with the Pt control line by 24.6% for the PtXIVXeg1a line, 15.7% for PtXIVXeg1b, 26.6% for PtXVXeg1b and 15.3% for PtXVXeg3b.
The other two lines (PtXIVXeg1c and PtXVXeg5a) were not different from the control [15].It should also be mentioned that although a tendency in increased tree height was observed for most of the transgenic trees in semi-natural conditions, statistically signi cant increase in tree height, as well as in stem diameter and volume, was observed only for the PtXVXeg1b line (Table 1, Fig. 3).In the greenhouse, after two months of vegetation, this line was taller than non-transgenic Pt control by 26.6%, in the open air after 6 months of vegetation by 25.4%, and after 18 months by 14.6% (Fig. 4).

<Table 1>
Integration of the xyloglucanase gene had little effect on the leaf area of transgenic plants.Only two aspen lines showed a signi cant decrease in area -PtXIVXeg1b by 28% and PtXVXeg3b by 17% compared to the control plants (Table 2).

<Table 2>
However, almost all transgenic lines changed the leaf shape -they signi cantly reduced the circularity.
This was due to an increase in the length and decrease in the width of the leaf blade of transgenic plants.
As a result, all the transgenic lines signi cantly changed the length-to-width ratio of the leaf by 14-23% compared to the control plants (Fig. 5).For all transgenic plants, there was a tendency to increase the number of leaves, but not statistically signi cant (Table 1).

Cellulose content in wood
The content of cellulose was measured in wood of all six studied transgenic lines.A signi cant increase in cellulose content was observed only in the PtXIVXeg1c and PtXVXeg1b lines (Table 1).The content of cellulose in other lines was on average at the control level.The reduction of cellulose content in transgenic lines was not detected.Thus, in terms of cellulose content, the PtXIVXeg1c line can be also considered as a prospective line in addition to the PtXVXeg1b line.

Carbohydrate composition of xylem
Xyloglucanase promotes cleavage of xyloglucan, thereby affecting the carbohydrate composition.A decrease in the content of pentosans (the main component of hemicellulose) was observed in all greenhouse plants [15].In plants under semi-natural conditions, there was also a decrease in the content of pentosans in all transgenic lines in the youngest parts of the stem in comparison with control (Table 3).Less difference from control was observed in the older (18-month-old) parts of the stem, and four out of six lines demonstrated an increase in the content of pentosans.However, all changes were statistically insigni cant.

<Table 3>
The ratio of hemicellulose components was also affected in transgenic lines (Fig. 6).In the youngest parts of the plant, a signi cant decrease in the xylose content was observed in most of the lines, accompanied by an increase in the content of glucose and sometimes arabinose (Fig. 6a).The content of galactose and mannose varied insigni cantly.In the most mature wood, the proportions of xylose and glucose did not differ signi cantly from control, but the content of arabinose and mannose was slightly, but signi cantly increased (Fig. 6b).The relative content of rhamnose and fucose in aspen wood was relatively low: 1.5-2% of rhamnose, independently of the wood age, and 0.2-0.5% and 0.1-0.2% of fucose in the younger and older wood, respectively.

Libriform ber analysis
The diameter and length of the libriform ber measured in wood samples of six studied lines are presented in Table 4.A slight increase in the ber diameter was observed in transgenic plants.The ber length was signi cantly higher than in the control in two lines PtXVXeg1b and PtXVXeg3b.(Table 4).

Microscopy of xylem
Electron microscopy of xylem of 18-month-old aspen showed an increase of the cell wall thickness in prospective lines PtXVXeg1b (1.80 microns on average) and PtXIVXeg1c (1.47 microns on average) in the xylem cells of the rst year of vegetation in comparison with non-transgenic control (1.16 microns) (Fig. 7).

Decomposition rate
Transgenic lines PtXIVXeg1b and PtXVXeg3b and control Pt were used in the decomposition experiment.These two transgenic lines were the most morphologically and biochemically similar to Pt. Analysis of the carbon dioxide emission during the decomposition of plant material showed that the stems of transgenic plants had a decomposition rate lower than in the control, especially for PtXVXeg3b, where it was about two times slower than in the control line Pt (Fig. 8).However, during root decomposition no signi cant difference in the carbon dioxide emission was detected between transgenic and control plants.
A signi cant decrease of nitrogen was observed in stems of transgenic plants in comparison with the control line, while no change for nitrogen was found in the roots.No change for carbon was found in both roots and stems of the transgenic lines in comparison with the control line (Table 5).

Discussion
Studies of the effect of the introduced recombinant construct in transgenic woody plants are usually based on a small sample of the analyzed lines selected from the entire panel of generated transgenic lines.Therefore, it is typical that only six out of 25 transgenic lines were selected for further analysis in open ground conditions in our study.For instance, in a similar study of the recombinant GS1 gene expression in poplar hybrids (Populus tremula × P. alba), only eight transgenic lines were analyzed in eld trials [17] selected from 22 initially generated transgenic lines that were grown rst under greenhouse conditions [16].Moreover, a detailed study of the recombinant gene was based only on one line [18].In another similar study of the PIP1 aquaporin gene the initial panel of PIP1-de cient transgenic Populus tremula x alba poplar hybrids included 22 lines, but only six lines were selected for initial tests in the greenhouse [19], and then ve of them were selected for the detailed study [20].In the initial study of the isoprene synthase gene ISPS 29 transgenic Grey poplar (Populus•× canescens) lines were generated [21], but only one line was used in the further semi-natural studies [22].Therefore, following the common practice in the analysis of transgenic woody plants, six from 25 transgenic greenhouse lines generated in our previous study [15] were selected for this study under semi-natural conditions.
The creation of highly productive woody plants with altered wood properties by increasing the cleavage of xyloglucan was considered in some studies as the most promising direction [12,23].However, successful results obtained in climatic chambers and greenhouse were not always con rmed in the eld [13].We tested our transgenic aspen lines with recombinant sp-Xeg gene also under semi-natural conditions: they had a closed root system and were grown in an open air during the entire vegetation period.The increase of height is considered as the main indicator of rapid growth in many similar studies related to selection for the faster growing plants [11,12,18,24].Measurements of their heights demonstrated that not all of the previously identi ed promising lines [15] have maintained a higher plant height.Convergence of phenotypic traits between transgenic and control lines was observed.PtXVXeg1b was the only line that maintained a high degree of productivity throughout the studies compared to the control.The increase in growth rate of the PtXVXeg1b line was relatively high in comparison with data published by other researchers [12].Transgenic white poplars with xyloglucanase from A. aculeatus also tended to increase growth rates, but they were grown in climatic chambers [12].When these plants were grown under greenhouse conditions, no difference in the growth rate was found [25].Further eld trials of these transgenic poplar plants showed that two transgenic white poplar lines trg300-1 and trg300-2 with overexpression of recombinant xyloglucanase from the fungus A. aculeatus showed a decrease in the growth rate and total biomass compared with non-transgenic control, while previously demonstrated an increase in productivity in climatic chambers.It was found that the deterioration in growth rates was due to a change in the transpiration process.In transgenic plants stomata dysfunction was observed [13].
The xyloglucanase XegA, which we used, is structurally identical to xyloglucanase from A. aculeatus (AaXEG2) at 70.46% based on the data from the NCBI database.Therefore, similar effects of the recombinant xyloglucanase XegA and xyloglucanase AaXEG2 on plants were observed.The transgenic plants of the line PtXVXeg1b obtained by us maintained the tendency to increase the growth rate in all experimental conditions.It was observed that incorporation of hormonal control or transcription factor genes can affect the size of plant organs [26].A similar effect was observed in aspens with xyloglucanase gene in our study.We observed the change in a leaf shape in all transgenic lines -the ratio of length to width increased, mainly due to a decrease in the leaf width (Table 3).The nal size of plant organs, such as leaves, is controlled by two processes -cell division and cell expansion [27].The size change can occur not in both, but only in the one dimension.For instance, the overexpression of the cytochrome P450 gene in Arabidopsis increased the length of the leaves without any change of their width [28].Reducing leaf size is possibly associated with a decrease in the number and / or size of cells [29].We found that a signi cant increase in the length of the wood ber was observed in transgenic lines without any change in diameter (Table 4).Since xyloglucan is involved in changing the cell shape in growth and differentiation zones [30], the expression of the xyloglucanase gene could also alter the wall extensibility in the leaf cells, which changed the shape of the cells and subsequently the leaf shape.Moreover, the observed in our study tendency of increasing the number of leaves in transgenic plants may have a compensatory effect on the growth characteristics of these plants [15].
The main structural motif of xyloglucan in genus Populus is a repeating block of the XXXG type consisting of three glucose residues containing the xylose substituent (X) and one unsubstituted glucose residue (G) [31].In the two-year-old wood of the control aspens, the content was quite similar to this indicator, both in absolute values and in the ratio of xylose, 3.6.However, in young areas the ratio was sharply reduced to 1.8, because xylose decreased and cellulose increased simultaneously at the same time.The incorporation of the xyloglucanase gene has altered the composition of the polysaccharide matrix, which primarily resulted in an increase in glucose and a decrease in xylose that caused defects in the formation of xylan.It was most signi cant in young plants, but was also observed in more mature wood, although to a lesser extent (most signi cantly in the PtXIVXeg1a and PtXIVXeg1b lines).This suggests that the incorporation of the xyloglucanase gene promoted the formation of hemicellulose, which is characteristic of the earlier stage of plant development.In the more mature wood, the effect of the xyloglucanase gene was minimal -the content of the main components did not differ much from the control, although the share of the minor components -mannose and arabinose -increased slightly.The third component, galactose, practically did not change.However, we noted signi cant changes in the relative content of other hemicellulose polysaccharides, which may be due to an indirect effect on the biosynthesis of the secondary cell wall.Our data partially agree with the results of Baba et al. [32], according to which 10% decrease in polysaccharide matrix was noted in the poplar with xyloglucanase gene, as well as a slight decrease in xylose due to galactose and arabinose.Unfortunately, in this work the age of plants was not speci ed.A signi cant decrease in xylose was observed in all Populus deltoides lines with suppression of the glycosyltransferase gene by interfering RNA [33], where it occurred due to galactose and mannose, but the glucose did not change.The reverse effect was in Populus euramericana with interfering RNA on endoglucanase gene, where xylose increased substantially, and glucose was reduced, while other hemicellulose sugars -mannose, galactose, arabinose, rhamnose and fucose remained unchanged [34].We found that the incorporation of the xyloglucanase gene in our transgenic aspens led to a decrease in the content of hemicellulose in young wood.In addition, the gene xyloglucanase has changed the composition of xyloglucan, making it more characteristic for an early age.We also showed that this effect is weakened with the age of the plant, and it is practically absent in the second year wood.It is possible that compensatory mechanisms for the formation of hemicellulose are ampli ed with age, and, in addition, it could be promoted by our plants being grown under seminatural conditions.
It was demonstrated earlier that the cellulose formation can indirectly affect the binding of micro brils to each other by xyloglucan laments, and, therefore, the cleavage of which can probably have a stimulating effect on the cellulose biosynthesis [35].Our plants had an increase in the cellulose content by 7.4% and 11.3% in the lines PtXVXeg1b and PtXIVXeg1c, respectively.Our cellulose content data are in consensus with the data on electron microscopy of xylem of transgenic plants, as well as with the data on libriform measurement.The length of the bers has increased in some transgenic lines.This is due to the speci c interaction of xyloglucanases with the cell walls resulted to the separation of the cellulose micro brils that led to the cell size increase due to the increase of intracellular pressure [8].An increase in the cellulose content may be associated with an increase in the thickness of the cell walls, especially if the gelatinous layer increases [36].We noted that the thickness of the cell wall of xylem in transgenic plants exceeded the control values and averaged 1.63 microns, whereas in control plants it was 1.16 microns.This is comparable to the increase in cellulose content in these plants.
Changes in the wood composition can affect also biogeochemical processes in ecosystems.In our earlier studies, we measured the decomposition rate of aspen wood in lines with the xyloglucanase gene using the method of mass loss [37].Signi cant differences in the decomposition rate between transgenic and control plants were found at the early stages in the transgenic roots, but not in stems.However, this method is not accurate enough, since it does not take into account the coe cient of microbial conversion, which includes the increase in the biomass of microorganisms during decomposition [38].
For a more accurate determination of the decomposition rate in plants, it is recommended to measure the intensity of the CO 2 emission [39].In our study we used this method in stems and roots, which are rarely used both in decomposition experiments.According to Zhang et al. [40], the overwhelming number of decomposition experiments were conducted with leaves or needles, and branches and roots are only occasionally used.Meanwhile, leaves and roots constitute a signi cant proportion of annual litter, and the built-in gene has an effect on the composition of the wood.Although our main experiment was conducted for two years, we used 6-month-old samples to study decomposition because they better represent the structure of litter in the natural conditions.According to Freschet et al. [41], 41% of the annual forest litter consists of leaves, 11% of the branches with up to 5 mm in diameter and 48% of the roots with up to 2 mm in diameter.
Measurement of the intensity of the CO 2 emission showed that for wood of stems of transgenic plants, a signi cant decrease in the rate of decomposition is characteristic.Such a change may be due to a decrease in the nitrogen content of these plants by 15% on average.A number of studies reported that the nitrogen content was directly related to the decomposition rate of plant residues [40,42,43].The higher nitrogen content increased the rate of decomposition, and the lower content reduced it.Perhaps, for the same reason, differences in the emission of carbon dioxide during the decomposition of the roots of our transgenic plants were not detected.It is likely that an increase in the thickness of the cell wall can also affect the rate of wood decomposition [44].
Our analysis of six transgenic lines under semi-natural conditions showed that the effect of the recombinant gene is observed in all transgenic lines.Thus, based on the results of the aspen tests in semi-natural conditions, it can be concluded that the recombinant sp-Xeg gene has a complex effect on the plant organism.Recombinant gene in uenced not only the growth parameters of transgenic plants, but also the content of cellulose, plant ber, and decomposition rate of wood.Among all transgenic aspen lines with the sp-Xeg gene, PtXVXeg1b was identi ed as the most promising line, which under all test conditions maintained an increased growth rate, had a higher cellulose content and a thicker cell wall of xylem bers.According to the published studies for woody plants, it is typical that researchers focus on detail study of recombinant gene effects in semi-natural and eld trials only in a one or a few transgenic lines [18, 20,22].We con rmed fast-growing only in one line, but it has a great practical value.Moreover, it supports further study of other remaining lines that may also contain lines fast-growing under semi-natural cultivation conditions.
Modi cation of plant properties through genetic transformation should always be assessed for its environmental impact.In particular, how transgenic aspen can affect forest ecosystems and soil.A computer simulation of virtual forest plantations consisted of the PtXVXeg1b clone and non-transgenic plants was carried out earlier using the EFIMOD simulation model that can predict carbon and nitrogen ows in forest ecosystems with strong feedback mechanism between soil and stand [45].This model experiment showed that effect of growing transgenic aspens on carbon and nitrogen ows was not different from effect of non-transgenic plants in controls.

Conclusions
The representative panel of transgenic aspen lines with the constitutive expression of recombinant xyloglucanase gene sp-Xeg from Penicillium canescens was analyzed.It was proved that the expression of sp-Xeg recombinant xyloglucanase leads to a change of thickness of wood ber and the plant growth.Electron microscopy showed an increase in cell wall thickness in transgenic lines.Libriform analysis also showed an increase in the length and width of the vascular ber in transgenic plants.An increase in wood ber parameters is likely to affect growth.For the rst time, it was shown that transgenic aspen plants with the gene of recombinant xyloglucanase of fungal origin under test conditions close to the eld (semi-natural conditions) do not demonstrate growth reduction.In transgenic plants, sp-Xeg recombinant xyloglucanase alters the composition of carbohydrate-containing substances in wood.The change in the content of cellulose and hemicellulose is con rmed by the data obtained in the analysis of the xylem monomeric sugars composition.For the rst time, an analysis of carbon dioxide emissions during the decomposition of plant material was carried out.It showed that the stems of transgenic plants had a decomposition rate lower than the control ones.This is a preliminary study based on a relatively small panel of the obtained transgenic lines.Although this study is in agreement with many similar studies of transgenic woody plants based also on a small number of transgenic lines selected from a larger panel of generated transgenes (e.g., [17,19,24]), we believe that more studies are needed.However, the presented here study has generated important practical data regarding performance of transgenic lines in open ground conditions (semi-natural and eld cultivation conditions) and effects of recombinant xyloglucanase sp-Xeg affecting not only productivity, but also other parameters.This study supports the trend in the action of recombinant xyloglucanase sp-Xeg observed earlier in greenhouse conditions [15].We plan to continue monitoring eld trials with six selected transgenic lines, as well as to analyze the remaining lines under semi-natural cultivation conditions.

Transgenic aspens
Transgenic aspen lines with the introduced recombinant sp-Xeg xyloglucanase gene from the fungus Penicillium canescens under the transcriptional control of the 35S promoter and nopaline synthase terminator were analyzed.The sp-Xeg gene encodes a chimeric xyloglucanase XegA with a white poplar cellulase signal peptide [14].The in vitro-derived trees were adapted to in vivo conditions in the climatic chambers for a month and were grown in greenhouses for a further month.In total, 25 transgenic lines, two control lines -non-transgenic wild-type line (Pt) and a transgenic line with the inserted gene βglucuronidase (PtIGus5a) were studied, respectively.Each line (genotype) was represented by 50 plants (ramets).The plants were grown in individual plastic containers with a volume of 1 liter (with peat to perlite ratio of 3:1) and after two months of growth in the greenhouse moved to semi-natural conditions in an open air with additional watering and feeding.Semi-natural conditions are the cultivation of potted plants outside of the greenhouse [46].Such growing conditions are close to the eld and allow us to estimate the resistance of plants to various biotic and abiotic factors.After four months of vegetation under semi-natural conditions, a random sample of six transgenic lines and a non-transgenic control line (Pt) were selected for the further analysis.Selected lines were transplanted into plastic containers with a volume of 2 liters.Nontransgenic control aspens (natural variant) were randomized with aspens of transgenic lines and grown together alongside each other under the same conditions.Under these conditions the plants were grown up to the age of one and a half years with wintering in natural conditions.
After two months of growth in the greenhouse and before transfer to semi-natural conditions, the height and number of leaves were measured.After another 4 months of vegetation, growth was measured, samples were taken for molecular analyses and carbohydrate composition measurement, and some of the plants were used to analyze the decomposition rate of the stem wood.The second part wintered and continued to grow for another year, and then, at the age of 18 months, the growth, content of cellulose and pentosans were measured, and samples were taken for carbohydrate composition, microscopy and libriform, and the annual results of the decomposition experiment were evaluated.
The presence of the recombinant gene and protein expression were con rmed in all 25 transgenic lines.
Following common practice used in studies of transgenic woody plants we selected six transgenic lines from 25 previously transformed greenhouse plants for our analysis under semi-natural conditions [15].The sampling was random (except excluding abnormally developing dwarf line PtXVXeg1c).This subsample included PtXIVXeg1a, PtXIVXeg1b, PtXIVXeg1c, PtXVXeg1b, PtXVXeg3b, and PtXVXeg5a.Four of them demonstrated increased growth in comparison with the control line (Pt), but only for one line (PtXVXeg1b) it was statistically signi cant (Table 1).In addition to growth parameters, analyses of speci c content of cellulose, pentosans, carbohydrate composition of xylem, measurement of libriform were also performed on all these six transgenic lines and the control line.Electron microscopy was carried out on the plants of the PtXIVXeg1c, PtXVXeg1b, and Pt lines.The decomposition rate was measured in the plants of the PtXIVXeg1b, PtXVXeg3b and Pt lines.To carry out the decomposition analysis, we selected these two lines that fell into the same group with the control plants according to Duncan's ANOVA-1 rank tests based on measuring biometric indicators obtained for greenhouse plants, and they differed only by the content of pentosans [15].

RT-PCR analysis
Total plant RNA was extracted from the leaves of all 6-month-old transgenic and control lines collected in the semi-natural conditions by the Invitrogen commercial method with the TRIzol® reagent (Thermo Fisher Scienti c Inc., USA).Genomic DNA was removed from the samples using DNAse I (Fermentas, USA) according to the manufacturer's protocol.cDNA synthesis was performed using M-MuLV reverse transcriptase, RNaseH-(SibEnzyme, Russia) and oligo-d(T)18 primer (Synthol, Russia) at a temperature of 37°C for 75 minutes.For the revertase inactivation, the mixture was heated to 70°C.We took 2 µl of the cDNA mixture as a template for PCR ampli cation using primers Xeg-up (GAAATGGCTAATGCCACTACATT) and Xeg-low (GATTTAGGCAACATCGGCAG) (Evrogen, Russia) under conditions similar to the PCR analysis of total DNA [15].To control RNA contamination with residues of the genomic DNA, PCR was performed with RNA preparations of each of the clones without revertase treatment.

Western blot analysis
Total protein extracts were obtained from the leaves of 6-month-old transgenic and control plants ex vitro by the addition of an extraction buffer [0.175 M Tris / HCl (pH 8.8), 5% SDS (w/v), 15% glycerol (v/v), 0.3 M mercaptoethanol] [47].Electrophoretic separation of proteins was performed according to Laemmli [48] in a 12% polyacrylamide gel.Electrotransfer of the separated polypeptides was carried out on a BioTrace nitrocellulose membrane (Pall, USA) by semi-dry transfer on a TE70PWR transblotter (Amersham, USA).Polyclonal rabbit anti-xyloglucanase antibodies from P. canescens fungus were used as the primary antibodies.Monoclonal goat anti-rabbit antibodies conjugated with alkaline phosphatase (Sigma, USA) were used as the secondary antibodies.Immunocomplexes were detected using a stabilized Western Blue substrate (Promega, USA).It was a qualitative Western blot analysis.

Growth indicators
To study each line, 40 plants were used.The length of the stem was measured from the root neck to the apical bud.Stem diameter was measured at the base of the root neck.The volume (V, cm 3 ) was measured by the formula: V = SD 2 H, where SD -stem diameter (cm), H -plant height (cm) [11].The number of leaves was calculated from the apical bud and to the root neck.The parameters of the leaf blade were measured using the LAMINA software [49] in the second year of vegetation.Height was measured at the age of 2, 6 and 18 months and analyzed using the Statistica 7.0 software (https://www.tibco.com/products/tibco-statistica).

Analysis of the speci c cellulose content
Median internodes of 20 plants per each 18-month-old line were used to determine the content of cellulose by the Kurschner-Hanak nitrogen-alcohol method [50].The content was recalculated taking into account the weight of an absolutely dry sample.

Analysis of the speci c pentosan content
The speci c content of pentosans in wood was estimated using the modi ed Tollens method [51,52] by converting them to furfural during distillation in the presence of HCl.For this analysis, two 10 cm long cuts of stem per plant, representing the 1st and the 2nd year growing wood, respectively, were taken from 18-month-old plants.The cuts were stripped off their bark, and the air-dried sawdust samples weighing 0.1 g were prepared.The optical absorption of the distillate was measured by a two-beam spectrophotometer at a wavelength of 277 nm.The dryness coe cient of wood (С dry ) was calculated from the following formulas: dry where W -the relative humidity of the wood, m -the mass of the empty bag (g), m1 and m2 -the weights (g) of the bag with the sample before and after drying, respectively.
Then, the content of pentosans in dry matter was calculated according to the formula: , where A -the obtained from the distillation, n -the conversion factor for the percentage of furfural to pentosans (2.434 for hardwoods), m -the mass of the sawdust sample (g), С dry -coe cient of dryness.For clarity, the percentage of pentosans was converted to absolute values as mg / g dry weight.

Analysis of hemicelluloses monosaccharide
Monomeric sugars of hemicelluloses (arabinose, fucose, galactose, glucose, mannose, rhamnose, and xylose) were measured by standard alditol acetate method [53,54].For the analysis of the composition of monosaccharides, two 10 cm long cuts of stem per plant without bark, representing the 1st (6 months) and the 2nd (18 months) year growing wood, respectively, were taken from 18-month-old plants.Samples of 5 mg of wood sawdust were hydrolyzed with 2M tri uoroacetic acid (TFA) at 100° C for 5 hours.The mixture of neutral monosaccharides was converted to alditol acetates and identi ed by gas chromatography-mass spectrometry (GC-MS) analysis using the GCMSQP 2010 Plus chromatograph (Shimadzu Corporation, Japan) with the HP-5MS column (60 m × 0.32 mm × 0.25 μm).Myo-inositol was used as an internal standard.Helium was used as the carrier gas.The temperature of the injector was 150° C. The column temperature was increased from 60° C to 250° C at a rate of 2° C/min, and, then, held for 10 minutes [55].

Microscopy
For the microscopy analysis, samples of 18-month-old plants were taken from the lower part of the stem (wood of the second year of cultivation) in three replicates.A total cut of all tissues of the stem was made.To prepare the cross sections, the samples were embedded in the epoxy-resin mixture containing DER-332, DER-732, DDSA, and DMP-30 [56].The transverse sections were obtained using the ultra microtome Reichert Om U2 (Austria) with glass knives, stained with methylene blue, azur-II, and basic fuchsin [57] and photographed with the AxioImager M1 light microscope (CarlZeiss, Germany).The slices were scanned, and the thickness of the cell walls was estimated using the AxioVision 4.8.1 software package (CarlZeiss, Germany).

Measurement of the libriform bers
For measurement of libriform bers, samples of the 18-month-old plants were taken from the bottom of the stem without bark (wood of the second year of cultivation) in three replicates.Samples were macerated using acetic acid and sodium chlorite, and the length and diameter of their libriform bers were measured [58].

Measurement of the aspen decomposition rate
The decomposition rate was estimated by analyzing the emission of carbon dioxide during plant material decomposition [59].In the experiment, sifted through a 0.5 mm ne sieve, washed and sterilized sand was used as a substrate.The plant tissue (stems and roots) from the 6-month-old plants was ground in a porcelain mortar, and, then, dried at 65º C for three days, and 100 mg of this dried and ground tissue were placed in a glass tube with 2 g of sand and sealed with rubber stoppers.To ensure the decomposition in the test tubes, an aqueous extract of the forest plant litter was added.Distilled water was also added to the tubes in an amount of 50% of the total moisture capacity of the sand (taking also into account the water needed to restore the initial mass of plant tissue).Then, the tubes were placed in a thermostat at 22º C for 48 weeks.Samples of air were sampled from the tubes every 8 weeks, and their carbon dioxide gas was analyzed using a gas chromatograph Crystallux 4000M (Research and Production Company «Meta-chrom», Yoshkar-Ola, Russia).All samples were analyzed also for C and N content by gas chromatography using the Euro EA-CHNSO Elemental Analyser (HEKAtech GmbH, Wegberg, Germany).Growth rate of the transgenic (PtXVXeg1b) and non-transgenic (Pt control) aspen lines

Abbreviations
Aspen leaves under semi-natural conditions presenting control (Pt) and transgenic lines PtXVXeg1b (demonstrating a statistically signi cant changed the length-to-width ratio of the leaf), PtXVXeg5a (demonstrating a statistically signi cant increase in the length of the main vein), and PtXVXeg3b (demonstrating a statistically signi cant decrease in the leaf blade area)

Figure 2 Western
Figure 2

Table 4
Mean diameter and length (± SD) of wood fiber in aspen lines

Table 5
Percentage of nitrogen and carbon (± SD) in the wood of the aspen lines