Wood Quality and Growth Characterization Across Intra- and Inter-Specific Aspen Hybrid Clones

WOOD QUALITY AND GROWTH CHARACTERIZATION ACROSS INTRA- AND INTER-SPECIFIC ASPEN HYBRID CLONES

James Foster Hart

B.Sc., The University of British Columbia, 2005

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF

THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

THE FACULTY OF GRADUATE STUDIES

(Forestry)

THE UNIVERSITY OF BRITISH COLUMBIA

(Vancouver)

February 2013

Abstract

Trembling aspen (Populus tremuloides Michx) is Canada’s most abundant poplar species; it is native, found nationwide, and is an ecologically and economically important hardwood species. As the demand for raw material continues to rapidly increase, there is an incentive to improve tree quality and growth rates through breeding, particularly in fast-growing species suitable for the Canadian landscape. Hybridization is considered one of the best options to accelerate tree productivity and improve wood quality. Two aspen species showing particular promise for hybridization with trembling aspen are European aspen (P. tremula) and Chinese aspen (P. davidiana) because their native climates are similar to that of western Canada.

In 2003, poplar clones were planted in Athabasca, Alberta from the following species crosses: open pollinated (OP) P. tremuloides (NN), OP P. davidiana (CC), P. tremula × P. tremula (EE), P. tremula × P. tremuloides (EN), and P. tremuloides × P. davidiana (CN). In,

November 2010, growth measurements and core samples were taken from the clones.

Productivity was quantified through analysis of stem volume. Wood quality attributes were quantified via an assessment of fibre length, fibre width, coarseness, wood density, microfibril angle, total cell wall carbohydrate and lignin content, and lignin composition.

Comparisons of the mean values for each attribute were made between crosses using generalized linear model least squares means tests.

The NN cross had lower volume growth than the CC and EE crosses. The EN and CN crosses had greater volume than the NN cross. The NN cross had shorter fibre length, but greater syringyl-guaiacyl ratio (S:G) relative to the CC cross. It also had lower density and

S:G compared to the EE cross. The EN cross had longer, wider fibres and a greater carbohydrate concentration compared to the NN cross. The CN cross had greater fibre length

and lignin concentration, but lower S:G compared to the NN cross. The results indicated that the inter-specific crosses were more desirable wood sources than the pure P. tremuloides cross. Specifically, the P. tremula x P. tremuloides cross showed the best potential to improve future generations of aspen on the Canadian landscape.

iii

Table of Contents

Abstract ...... ii

Table of Contents ...... iv

List of Tables ...... vii

List of Figures ...... viii

List of Symbols and Abbreviations ...... ix

Acknowledgements ...... x

Dedication ...... xi

Chapter 1: Introduction ...... 1

1.1 Populus tremuloides ...... 1

1.2 Aspen Breeding ...... 2

1.3 Heterosis ...... 3

1.4 Wood Density ...... 4

1.5 Microfibril Angle ...... 6

1.6 Fibre Properties ...... 9

1.7 Wood Chemistry ...... 10

1.8 Thesis Statement ...... 14

Chapter 2: Materials and Methods ...... 15

2.1 Sample Procurement ...... 15

2.2 Growth Traits and Core Sampling ...... 17

2.3 Wood Density ...... 17

2.4 Microfibril Angle ...... 18

2.5 Fibre Length, Width, and Coarseness ...... 18

2.6 Cell Wall Chemistry ...... 19

2.7 Syringyl-Guaiacyl Ratio ...... 20

2.8 Data Analysis ...... 21

Chapter 3: Results ...... 23

3.1 Models ...... 23

3.2 Least Squares Means Comparisons ...... 24

3.2.1 Volume ...... 27

3.2.2 Wood Density ...... 28

3.2.3 Microfibril Angle ...... 29

3.2.4 Fibre Length ...... 30

3.2.5 Fibre Width ...... 31

3.2.6 Coarseness ...... 32

3.2.7 Syringyl-Guaiacyl Ratio ...... 33

3.2.8 Total Carbohydrate Concentration ...... 34

3.2.9 Total Lignin Concentration ...... 35

3.2.10 Soluble/Insoluble Lignin Concentrations ...... 35

3.3 Correlations ...... 36

Chapter 4: Discussion ...... 39

4.1 Growth ...... 39

4.2 Wood Density ...... 40

4.3 Microfibril Angle ...... 42

4.4 Fibre Length, Width, and Coarseness ...... 44

4.5 Syringyl-Guaiacyl Ratio ...... 46

4.6 Lignin Concentrations ...... 48

4.7 Carbohydrate Concentration ...... 49

4.8 Correlations ...... 50

4.9 Heterosis ...... 52

Chapter 5: Conclusion ...... 53

5.1 Thesis Summary ...... 53

5.2 Research Significance ...... 55

5.3 Future Research ...... 55

References ...... 57

Appendix A: Least Squares Means ...... 71

List of Tables

Table 1: Species crosses and their associated abbreviations ...... 16

Table 2: Growth and wood quality trait sample sizes ...... 17

Table 3: PROC GLM ANOVA of traits using the class variable species ...... 24

Table 4: Least squares means comparison ...... 26

Table 5: Correlation coefficient ranges ...... 36

Table 6: Pearson’s correlation coefficient between traits...... 37

Table 7: Least squares means and standard error (in brackets) for each trait by species ...... 71

vii

List of Figures

Figure 1: Microfibril angle of the S2 layer in wood cells ...... 7

Figure 2: Partial structure of cellulose ...... 11

Figure 3: Monomer components of hemicelluloses ...... 11

Figure 4: Syringyl and guaiacyl phenylpropane units of lignin ...... 12

Figure 5: Location of tree plots ...... 15

Figure 6: Boxplot of mean volume by species cross ...... 27

Figure 7: Boxplot of mean wood density by species cross ...... 28

Figure 8: Boxplot of mean MFA by species cross ...... 29

Figure 9: Boxplot of mean fibre length by species cross ...... 30

Figure 10: Boxplot of mean fibre width by species cross ...... 31

Figure 11: Boxplot of mean coarseness by species cross ...... 32

Figure 12: Boxplot of mean S:G by species cross ...... 33

Figure 13: Boxplot of mean carbohydrate concentration by species cross ...... 34

Figure 14: Boxplot of mean lignin concentration by species cross ...... 35

viii

List of Symbols and Abbreviations

CC P. davidiana x P. davidiana CN P. davidiana x P. tremuloides CS Coarseness D Wood Density DBH Breast Height Diameter DP Degree of Polymerization EE P. tremula x P. tremula EN P. tremula x P. tremuloides FL Fibre Length FW Fibre Width GLM Generalized Linear Model HT Height INSOL Insoluble Lignin Concentration LS Least Squared MDF Medium Density Fiberboard MFA Microfibril Angle MOE Modulus of Elasticity MOR Modulus of Rupture NN P. tremuloides x P. tremuloides OP Open Pollinated OSB Oriented Strand Board RMSE Root Mean Squared Error R-squared Explained Variance S:G Syringyl-Guaiacyl Ratio SOL Soluble Lignin Concentration TC Total Carbohydrate Concentration TL Total Lignin Concentration VOL Volume

Acknowledgements

I would like to thank my supervisor, Professor Shawn Mansfield for all of his guidance and support in the duration of my studies. I enjoyed working in his lab group and the learning environment he provided. I would also like to thank my committee members, Professor

Simon Ellis and Professor Stavros Avramidis, for their helpful suggestions during my degree and constructive criticism of my thesis.

Many thanks to the lab colleagues for helping me with any questions I had regarding equipment and experiments. I would not have been able to do it without all your support. In particular, thanks to Francis for helping with sample collection. I would also like to thank my office mates Li, Grant, Francis, and Sarah for creating a good-natured atmosphere in the office on a daily basis. Also, I would like to thank Professor Tony Kozak for his help with statistical analysis.

I would also like to thank my family for their support during my post-secondary education. In particular, I’d like to thank my mother and father for their support and patience

- it made a huge difference over the years and without you I would not have been able to accomplish what I have done.

Dedication

To my parents

Chapter 1: Introduction

1.1 Populus tremuloides

Populus spp. is Canada’s most widespread and fastest growing tree genus (Zsuffa et al., 1996). It comprises over 50% of all Canadian-grown hardwoods and approximately 11% of all timber resources in Canada (Peterson & Peterson, 1995). It can be found in North America, Europe, and

Northeast Asia. Specifically, trembling aspen (Populus tremuloides Michx) is Canada’s the most abundant poplar species; it is native nationwide and is considered a commercially important hardwood species in western Canada (Einspahr & Wyckoff, 1990; Ondro, 1989; Peterson &

Peterson, 1992). Its importance is due to a rapid growth rate resulting in shorter rotation times compared to other hardwood species grown in similar environments (Cisneros et al., 2000). The primary uses for trembling aspen include oriented strand board (OSB) manufacture and bleached

Kraft hardwood pulp production (Ondro, 1989; Peterson & Peterson, 1992). Aspen pulp end-use is suited for books, newsprint, fine printing papers, as well as toiletry products (Mackes &

Lynch, 2001). In addition, but to a lesser extent, aspen is used in the manufacture of waferboard, pallets, containers, matchsticks, and chopsticks (Einspahr & Wyckoff, 1990). There is also potential for aspen to be used as a source for energy production through its conversion into biofuels such as ethanol. However, challenges remain which currently limit the effective conversion of lignocellulosic material into ethanol and need to be overcome before aspen can be utilized in this way (Mansfield, 2009). As the demand for raw material increases, there is an added incentive to improve tree quality and growth rates through breeding, particularly in fast- growing species suitable for the Canadian landscape.

1 1.2 Aspen Breeding

One putative mechanism of enhancing aspen wood quality and productivity is breeding focused on improvements in desirable wood attributes and growth rates. Early work on aspen improvement successfully demonstrated increased growth rates, and to a lesser extent improved wood fibre quality traits (Einspahr & Benson, 1970; Einspahr, 1984; Zsuffa, 1973). Naturally, there is substantial genetic diversity available in native aspen that can be exploited via selective breeding, inter-specific hybridization, and cloning; and a combination of the three would be ideal for substantial realized gains (Einspahr & Winton, 1976). In particular, hybridization is considered the best approach to improve productivity and wood quality (Einspahr, 1984; Li et al., 1993; Melchior, 1985).

Programs attempting to utilize aspen hybridization strategies have been successful in the

United States, Germany, and China (Einspahr, 1984; Kailong et al., 1999; Li et al., 1993;

Melchior, 1985). And, testing in Finland also resulted in faster juvenile growth in hybrid aspen trees compared to the parents (Yu et al., 2001). More specifically, productivity in the United

States was substantially improved where rotation ages were shown to decrease from 40 years to as low as 20 years (Li et al., 1993). There is also potential for improved wood quality in combination with superior growth using hybrid aspen breeding strategies (Aziz et al., 1996). It was suggested that similar breeding programs are possible in Canada, if well designed programs are developed (Farmer, 1991; Li et al., 1993).

Two promising species for hybridization with trembling aspen are European aspen (P. tremula L.) and Chinese aspen (P. davidiana.) because their native climates are similar to that of

Western Canada. In the United States, P. tremuloides and P. tremula crosses have already demonstrated improved growth, form, and wood quality; while, P. tremuloides and P. davidiana

2 crosses had superior growth characteristics on relatively poor and dry sites (Li et al., 1993). It is anticipated that similar results can be achieved in Canada, if the same inter-specific crosses of parent trees from northern European countries such as Finland and from north-eastern China are employed (Li, 1995).

1.3 Heterosis

A theory for the improved growth observed in inter-specific hybrid crosses of aspen is heterosis.

Heterosis (i.e. hybrid vigor) is the superiority of hybrid offspring over either the average value between the two parents, called the mid-parent value, or the better parent’s value for a given trait

(Hayes & Gowen, 1952). Numerous studies have shown both significant and stable heterosis in

F1 hybrids of Populus spp. However, the genetic cause is not yet fully understood (Heimburger,

1936; Li et al., 1993; Stettler et al., 1988; Wu et al.,1992; Zsuffa, 1973). There are two major hypotheses commonly used to explain the genetic basis of heterosis: dominance and overdominance effects (Falconer, 1989). The dominance effect hypothesis suggests heterosis is caused by the suppression of one parent’s recessive, deleterious alleles by the other parent’s dominant, superior alleles in their heterozygous offspring (Bruce, 1910; Keeble & Pellew, 1910).

The overdominance hypothesis suggests a heterozygous combination of the two parent’s alleles at a single locus in the offspring is superior to either homozygous allele combination at that locus

(East, 1936; Shull, 1908). A review of research into the mechanisms involved with the phenomenon of heterosis proposes there are, in addition to dominance and overdominance effects, epigenetic machinery regulating multigenic traits causing increased efficiencies and greater growth (Baranwal et al., 2012). Specifically, increased energy input, by photosynthetic

3 efficiency, and less energy expenditure for basic metabolism are both proposed to improve growth in hybrid plants.

Hybrid aspens demonstrating heterosis were analyzed using a quantitative approach to determine the genetic cause (Li & Wu, 1996). The results suggest heterosis between P. tremuloides and P. tremula may be caused by overdominance. In contrast, improved growth in hybrids compared to their parents could be accounted for by a later bud set in hybrid aspen (Li et al., 1998). Similar findings (Yu et al., 2001) also concluded that the perceived hybrid vigor was a function of the observed extended growth period. As such, the authors suggested the improved vigor observed was not necessarily true heterosis commonly assumed to occur in aspen.

Nonetheless, there was a desirable increased growth in the hybrids.

1.4 Wood Density

Aspen’s wood density across North America is comparable to values common to European studies. Trembling aspen basic density has been reported to fall between 320-402 kg/m3 for mature trees (Yanchuk & Micko, 1990; Yanchuk et al., 1983; Yanchuk et al., 1984). In accordance with the values observed in Canada, the average basic density of P. tremuloides in the United States was documented to be 350 kg/m3 (Green et al., 1999), however, higher basic density values have also been reported (391 kg/m3 and 380 kg/m3) (Horn, 1978; Wengert et al.,

1985). Studies of Finnish P. tremula trees reported average basic densities of 379 and 376 kg/m3

(Junkkonen & Heräjärvi, 2006; Kärki, 2001).

Wood density primarily impacts the performance of wood in structural products (Downes et al., 2003). Lower density wood generally results in OSB panels with superior and more isotropic mechanical properties compared to higher density wood because it can be pressed to a

4 higher compactness ratio; that is, the inter wood furnish contact surface area increases improving internal bond strength and ultimately board performance (Hsu, 1997; Semple et al., 2007). A study of various hardwood species providing a wide range of wood densities concluded that increased wood density results in decreased modulus of elasticity (MOE), modulus of rupture

(MOR), and tensile strength in medium-density fiberboard (MDF) at the same panel density

(Woodson, 1976). Aspen’s inherent lower density makes it a preferred raw material for OSB and low-to-medium density fiberboard manufacturing; it can be pressed to a high compression ratio resulting in boards with high bending strength and low porosity (Geimer, 1976; Mackes &

Lynch, 2001). In Canada, five hybrid poplar clones were tested for their mechanical performance in OSB production and compared to native P. tremuloides produced OSB as a benchmark

(Semple et al., 2007). The fast growing and low density hybrid poplar was highly suited for use in OSB manufacturing. It is very likely that a fast growing hybrid aspen would be a viable raw material as well.

Increased specific gravity of wood employed in pulping operations generally results in lower paper strength and stiffness (Via et al., 2004). However, this correlation is likely attributed to the increased fibre coarseness associated with higher wood density. Specific gravity has also been inversely correlated with sheet density (Semen et al., 2001). The relationship is attributed to the ease of compressing fibres; lower density improves the fibres compressibility within a sheet.

While density appears to be related to end product properties, it is not a good predictor of pulp yield (Mansfield & Weineisen, 2007). In contrast, increased density does have advantages, as wood of higher density permits a higher packing density in the digester, and as such results in an increased pulp yield per chemical cook.

5 Density relationships with other wood properties are variable and may be species dependent. A recent review summarized several studies and found either a slight negative correlation or no correlation between density and tracheid length (Via et al., 2004). In Douglas- fir, density was negatively correlated with fibre length and coarseness (Ukrainetz et al., 2008).

Wood density also had a general inverse relationship with fibre length and width, and a slight positive correlation with coarseness, however, all three comparisons were not significant

(Gerendiain et al., 2008). In contrast, density and fibre length were positively correlated in

Eucalyptus globulus (Wimmer et al., 2002). A high correlation between density and coarseness was also found in Loblolly pine (Veal et al., 1987), however, no significant correlation was found in Radiata pine (Kibblewhite & Uprichard, 1996).

1.5 Microfibril Angle

Microfibril angle (MFA), as shown in Figure 1 as phi, is the angle between the longitudinal axis of the fibre cell and the cellulose microfibrils in the secondary cell wall (Barnett & Bonham,

2004). MFA has been shown to range from 18 to 22o in P. tremuloides fewer than three years-old

(Horvath et al., 2012; Maloney & Mansfield, 2010). In contrast, the MFA of poplar clones have been shown to decrease with age, from 28o to as low as 7.8o by year 11 (Fang et al., 2006).

Similarly, in two 31-year-old natural aspen trees, mean MFAs were estimated at 12.5 and 14.7o

(Francis et al., 2006). MFA variation in wood has been related to the performance of solid wood and, to a lesser extent, pulp and paper properties.

Figure 1: Microfibril angle of the S2 layer in wood cells (Ye, 2007)

MFA impacts solid wood in structural applications. Specifically, in Eucalyptus, MFA alone was shown to account for 86% of the observed variation in MOE, and MFA together with density accounted for 96% of the MOE variation (Evans & Ilic, 2001). These results are supported by another study with Eucalyptus that showed that MFA accounted for 87% MOE variation, and the combination of MFA and density accounted for 92% variation in MOE (Yang

& Evans, 2003). Likewise, the changes in wood stiffness within a tree can be primarily attributed to the change in MFA (Cave & Walker, 1994). A larger MFA in solid wood generally corresponds to lower stiffness making it suitable only for low-grade use; thus, its value is reduced (Barnett & Bonham, 2004). In addition, increased MFA causes greater longitudinal shrinkage, and uneven MFA within boards causes increased warping during drying, both of which further reduce structural suitability and overall market value.

MFA has less of an impact on wood quality used in pulp and paper manufacturing.

Several studies, reviewed by (Donaldson, 2008), implicate MFA with the strength properties of

Kraft pulp; however, it was proposed that any possible paper strength relationships with MFA were likely obscured by changes in other fibre properties (Wimmer et al., 2002). In contrast,

7 additional studies found MFA had limited influence on most paper properties (French et al.,

2000; Wimmer et al., 2002). MFA was, however, a partial factor impacting the stretch property of handsheets derived from aspen, as well as other hardwood species (Downes et al., 2003;

Gurnagul et al., 1990; Horn, 1978; Watson & Dadswell, 1964).

There have been many studies investigating the relationship of MFA with other wood properties. It is generally accepted that MFA and fibre length are negatively correlated (Barnett

& Bonham, 2004; Cisneros et al., 2000; Donaldson, 2008; Porth et al., 2013; Ukrainetz et al.,

2008; Wimmer et al., 2002). However, no correlation between MFA and fibre length or width was observed in Picea abies (Bergander et al., 2002). In addition, MFA and tracheid coarseness were not correlated in Pinus taeda L. (Courcheneet al., 2006) but was positively correlated in

Pseudotsuga menziesii (Ukrainetz et al., 2008).

MFA tends to have a variable relationship with wood density; namely, correlated in some cases, while no correlation was found in others (Davison et al., 2006; Donaldson, 2008;

Ukrainetz et al., 2008). Pinus radiata showed a strong correlation between density and MFA

(Schimleck & Evans, 2002). In Eucalyptus nitens, MFA was poorly correlated to density but, that might have been due to the narrow range in MFA observed (Schimleck et al., 2003). A positive relationship was found between density and MFA in Eucalyptus globulus (Wimmer et al., 2002) but no significant correlation was found between density and MFA in P. trichocarpa

(Porth et al., 2013). MFA in 11-12 year old poplar clones was shown to be negatively correlated with density and other wood properties namely: cellulose content, fibre length, and fibre width

(Sheng-zuo et al., 2004). In contrast, a strong negative correlation was observed between MFA and density in Eucalyptus nitens however, the relationship was only valid over a few growth rings, not across larger distances (Evans et al., 2000).

8 1.6 Fibre Properties

Aspen wood fibres are commonly described as short, with thin to medium cell wall thickness and high length-to-width ratio (Dhak et al., 1997; Mackes & Lynch, 2001; Mansfield & Weineisen,

2007). Trembling aspen fibre length and width generally range between 0.4-1.25 mm and 12-25

µm, respectively (Einspahr & Winton, 1976; Groover et al., 2010; Gurnagul et al., 1990; Horn,

1978; Mansfield & Weineisen, 2007; Yanchuk & Micko, 1990; Yanchuk et al., 1984). Trembling aspen coarseness averaged 0.0859 mg/m in the United States and ranged from 0.10 to 0.18 mg/m in Canada (Horn, 1978; Karaim et al., 1990; Mansfield & Weineisen, 2007). These properties make trembling aspen an ideal resource for producing high-density paper sheets with good optical properties and smooth surfaces (Dhak et al., 1997; Mansfield & Weineisen, 2007).

Wood fibres’ physical properties are primarily important in paper production, as sheets made from hardwoods are substantially affected by fibre morphology (Horn, 1978). Some wood fibre properties of particular importance are fibre length, fibre width, and coarseness. Fibre length and the length to width ratio both affect various paper properties, including the tear-tensile strength relationship, as well as paper stiffness (Amidon, 1981; Horn, 1978). Increased fibre length generally improves coherence between fibres and improves the physical characteristics of the ensuing paper. Coarseness also impacts product strength; lower coarseness is associated with higher strength (Via et al., 2004). However, it has been shown that coarseness is secondary to fibre length in predicting burst strength. Decreased coarseness may improve bleachability and result in increased pulp brightness (Mansfield & Weineisen, 2007). Furthermore, fibres with lower coarseness are thought to collapse more easily, which improves paper smoothness (Yu et al., 2001).

9 Weighted fibre length may be moderately correlated with coarseness (Kibblewhite &

Uprichard, 1996). Fibre length and coarseness were positively correlated in Pseudotsuga menziesii (Ukrainetz et al., 2008) as well as Picea abies (Gerendiain et al., 2008); furthermore, fibre length and coarseness were positively correlated with fibre width.

1.7 Wood Chemistry

Wood cell-walls are primarily comprised of cellulose, hemicellulose, and lignin (Baucher et al.,

2003). In hardwoods, the proportion by dry weight is approximately 40 to 45%, 15 to 25%, and

18 to 35% of cellulose, hemicellulose, and lignin, respectively (Rowell, 2005). P. tremuloides cell wall composition has been reported to be 78% holocellulose (cellulose and hemicellulose combined) and 21% lignin (Pettersen, 1984). A study of several aspen clones found similar concentrations with lignin ranging from 19.2-22.8%, and total carbohydrate concentration ranging 71.5-75.5% (Mansfield & Weineisen, 2007). In contrast, lower total carbohydrate and lignin concentrations for several aspen clones ranged from 66.3- 71.0% and 18.6-20.3%, respectively (Stewart et al., 2006). The aforementioned average value of holocellulose was a general average across the United States, while the latter concentrations were specific to clones from sites in northern British Columbia, Canada which might explain the discrepancy between the clones and the general average values reported.

Cellulose (Figure 2) is a long-chain, linear carbohydrate composed solely of glucose monomers and it forms fibrils that are the main constituent of the cell wall (Baucher et al., 2003;

Parham & Gray, 1984; Rowell, 2005).

Figure 2: Partial structure of cellulose (Balat et al., 2009)

Hemicellulose (Figure 3) is made up of mixed linkage polysaccharide heteropolymers that contribute to the structural integrity of the cell wall as a matrix (Rowell, 2005). Hemicellulose is comprised of a combination of sugars; namely, xylose, arabinose, glucose, mannose, and galactose (Parham & Gray, 1984).

Figure 3: Monomer components of hemicelluloses (Pettersen, 1984)

Cellulose has an average degree of polymerization (DP) around 10,000, while other carbohydrates such as hemicelluloses have lower DP around 100 to 200. Higher cellulose DP may increase the propensity for the cellulose chains to crystallize and reduces the amount that is accessible to moisture sorption, pulping, and chemical modification (Rowell, 2005). Specifically, a higher DP has been suggested to negatively affect carbohydrate’s ability to be hydrolyzed

(Mansfield et al., 1999).

11 Lignin is a heterogenous hydrophobic phenolic polymer which encrusts the cellulose and hemicellulose matrix, and strengthens the cell wall (Baucher et al., 2003). Hardwood lignin polymers are primarily comprised of syringyl and guaiacyl phenylpropanoid units (Figure 4).

Their relative concentrations are commonly expressed as the syringyl-guaiacyl ratio (S:G), and have been shown to vary with species (Boerjan et al., 2003).

Figure 4: Syringyl and guaiacyl phenylpropane units of lignin (Balat et al., 2009)

The cell wall composition affects the end use of wood in several ways. Lignin content has a large role in the structural stability of wood products. Transgenic aspen with reduced lignin content exhibits marked reduction in both compression strength parallel to the grain and in modulus of elasticity (Horvath et al., 2010). For paper products, an increase in cellulose content increases the raw material’s pulp yield. Lignin must be removed from the cellulosic component of wood during the pulping process and must be balanced with cellulose degradation (Baucher et al., 2003); therefore, higher lignin content will have adverse effects on cellulose yield.

Along with concentration, the composition of lignin is an important factor for wood pulping. The high-syringyl concentration common in Populus spp. facilitates a reduced chemical load required during fibre production (Stewart et al., 2006). This inherent trait in poplars can be

12 realized through increased acid hydrolysis of hemicelluloses (Davison et al., 2006). It was also speculated that a higher S:G accounted for improved pulping ease of aspen clones (Mansfield &

Weineisen, 2007). Populus spp. with higher S:G resulted in lower kappa number (residual lignin) following chemical pulping and improved pulp bleachability (Huntley et al., 2003). Similar results were observed by (Bose et al., 2009). The improved processing may be a trade-off because there appears to be a direct negative relationship between S:G and yield (Mansfield &

Weineisen, 2007). It is possible, however, that the difference in yield might be attributed to more residual lignin being removed from the pulp in wood with higher syringyl concentration

(Huntley et al., 2003).

The S:G relationship with cellulose and lignin concentrations in wood is not completely clear. In some cases there is either a weak or no correlation, while others have shown a relationship. Lignin concentration and S:G were independently increased and decreased, respectively, in transgenic trembling aspen (Li et al., 2003). A change in S:G had no correlation to lignin concentration in six transgenic poplars (Lapierre et al., 1999). Furthermore, no correlation was found between total carbohydrate concentration and S:G in E. nitens, and only a weak correlation in E. globulus (Wallis et al., 1996). In contrast, the ratio of lignin to carbohydrates and S:G were positively correlated in E. globulus (del Río et al., 2005). This relationship could be due to a positive correlation between S:G and carbohydrate concentration and/or a negative relationship between S:G and lignin concentration. In P. trichocarpa, S:G was negatively and positively correlated to lignin and holocellulose concentrations, respectively

(Porth et al., 2013). Also, lignin and S:G exhibited a strong negative correlation in poplar (Bose et al., 2009). The authors suggested additional research is required to clarify apparent site dependence of the relationship.

13 1.8 Thesis Statement

The aim of this thesis is to 1) characterize the wood quality and tree growth of P. tremuloides, P. tremula, and P. davidiana clones grown in Alberta, Canada, and 2) investigate the effects of P. tremula and P. davidiana hybridization with P. tremuloides on wood quality and tree growth.

Productivity will be quantified through analysis of volume calculated from tree height and breast height diameter. Wood quality attributes will be quantified through the analysis of the core samples for fibre length, fibre width, coarseness, wood density, microfibril angle, total cell wall carbohydrate and lignin content, and lignin composition.

The characterized tree and wood properties may offer insight into whether or not interspecific crossing of these aspen species can be achieved in western Canada and, more importantly, if this breeding strategy can be used to improve potential future generations of aspen on the Canadian landscape.

14 Chapter 2: Materials and Methods

2.1 Sample Procurement

In 2003, poplar clones were planted in two adjacent plots, consisting of the main growth trial plot and a secondary plot for wood evaluation, which were located within one kilometer of each other near the Alberta-Pacific pulp mill in Athabasca, Alberta (Figure 5). The trees represent clones from one of the following seven species crosses: two different open pollinated (OP) P. tremuloides families, two different OP P. davidiana families, a P. tremula × P. tremula family, a

P. tremula × P. tremuloides family, and a P. tremuloides × P. davidiana family. All four OP crosses are assumed to be an intra-specific cross due to the mother parent’s proximity to its own species.

Figure 5: Location of tree plots (Alberta-Pacific, 2012)

15 The OP P. tremuloides parents were all from Rock Island Lake, Alberta, while the OP P. davidiana and parents were from Al-Pac’s breeding program. The inter-specific cross of P. tremuloides × P. davidiana was made from a controlled cross between P. tremuloides (mother) and P. davidiana (father) from Al-Pac’s breeding program. All P. tremula material was of

Finnish origin, and the inter-specific cross between P. tremula × P. tremuloides consisted of the female P. tremula with the male P. tremuloides parent of Canadian origin that has previously been used for breeding in Finland.

Table 1: Species crosses and their associated abbreviations Genetics classification Species Abbreviation

Intra-specific (OP) P. tremuloides x P. tremuloides NN Intra-specific (FS) P. tremula x P. tremula EE Intra-specific (OP) P. davidiana x P. davidiana CC Inter-specific (FS) P. tremula x P. tremuloides EN Inter-specific (FS) P. tremuloides x P. davidiana CN

In the growth trial plot trees were planted in rows consisting of groups of five trees by species cross. The placement of each five-tree grouping was randomly selected. The growth tree plot was surrounded by two rows of buffer trees around the entire perimeter of the plot. The wood quality plot grouped each species type separately into seven sections with no tree buffer along the perimeter. Both plots used 1m × 1m tree spacing.

In November 2010, growth measurements were taken from the trial plot, while the excess plot was used to collect 5 mm increment wood cores. Twenty to thirty trees from each species cross were measured for height and diameter at breast height (DBH). At the same time, twenty to thirty trees from each species were cored (5 mm increment core) facing North to South, with

16 the exception of the P. tremula x P tremula cross, which only had twelve trees available for coring.

Table 2: Growth and wood quality trait sample sizes Species MFA, S:G, D FL, FW, CS TL, TC, SOL, INSOL VOL NN 50 45 50 40 CC 60 57 57 51 EE 12 12 12 29 EN 30 29 30 30 MFA = Microfibril Angle, S:G = Syringyl-Guaiacyl Ratio, D = Wood Density, FL = Fibre Length, FW = Fibre Width, CS = Coarseness, TL = Total Lignin Concentration, TC = Total Carbohydrate Concentration, SOL = Soluble Lignin Concentration, INSOL = Insoluble Lignin Concentration, VOL = Volume.

2.2 Growth Traits and Core Sampling

Tree height was measured using an extendable meter stick. Trunk diameter was measured at breast height (1.3 m) using electronic calipers. Stem volume was calculated using equation [1] for Aspen according to British Columbia Forest Inventory Division (1976).

logV = -4.538904 + 1.834410 logD + 1.208970 logH [1]

where, V is the volume (m3), D is the diameter at breast height (cm), and H is the tree height (m).

Two 5 mm diameter increment cores were taken bark to bark at breast height from the wood quality plot trees. One core was used for density, microfibril angle, and fibre measurements, while the second core was retained for chemical analysis.

2.3 Wood Density

The average wood density was measured of the southern section of one core from pith to bark, along the radial face. The cores were precision sawn to 1.68 mm thick sections using a twin blade pneumatic circular saw. Cut sections were extracted in a Soxhlet apparatus with hot

17 acetone for 24 hours and allowed to acclimate to 7% moisture. The average density was determined for the entire length of the section, using an X-ray densitometer scanned at a 0.254 nm resolution (Quintek Measurement Systems Inc., Knoxville, USA).

2.4 Microfibril Angle

MFA was measured on the radial face of the growth ring closest to the bark by X-ray diffraction.

The 002 diffraction spectra were screened for T value distribution using a Bruker D8 discover X- ray diffraction unit equipped with an arc array detector (GADDS). Measurements were performed using wide-angle diffraction in the transmission mode with CuKα1 radiation (λ=1.54

Å). The X-ray source was fit with a 0.5mm collimator and the scattered photon were collected by the GADDS detector. Both the X-ray source and detector were set to theta=0o. The average of the T-values from each of the two 002 diffraction arc peaks was used to calculate the MFA using the regression equation [2], established for Populus species (R2=0.9686):

MFA = -3.8364 – 0.9583T [2]

MFA is the microfibril angle in degrees and T is the average of the 002 diffraction arc peak T- values. The regression was developed for the diffraction unit by the Mansfield group and validated using microscopy.

2.5 Fibre Length, Width, and Coarseness

Samples, representing wood from pith to bark, were macerated in Franklin’s solution (1:1 glacial acetic acid and 30% hydrogen peroxide) at 70 oC for 48 hours. Following the reaction, the

18 solution was decanted, and the fibres washed with 1 L deionized water to remove residual

Franklin’s solution from the wood fibres. The fibres were then suspended in deionized water and gently stirred in a Waring blender until fully separated. The suspension was strained through filter paper under vacuum and dried in an oven at 50 oC for 24 hours. Approximately 0.350 mg of dried fibre was weighed on an analytical balance and re-suspended in deionized water. The solution was again gently stirred in a Waring blender until until the fibres separated and their attributes determined on a Fibre Quality Analyzer (OpTest Equipment Inc., Hawkesbury Ontario,

Canada). Length weighted fibre length and fibre width were measured in millimetres, while fibre coarseness was measured in milligrams per meter.

2.6 Cell Wall Chemistry

Wood from pith to bark was ground in a Wiley mill until it passed through a 40 mesh screen and

Soxhlet extracted with hot acetone for 24 hours to remove extractives. The extracted wood was oven dried at 105 oC for 24 hours, and ~200 mg of dried material used for cell wall characterization. To the wood flour, 3 mL of 72% (w/w) H2SO4 was pipetted into a test tube and mixed for 30 seconds every 10 minutes. After 2 hours, the contents of the test tube were transferred to a serum bottle. Residue in the test tube was transferred into the serum bottle with

112 mL nanopure water. The serum bottles were then sealed and autoclaved at 121 oC for 60 minutes. The samples were allowed to cool and stored overnight in a 4 oC fridge. The contents of the bottles were then vacuum filtered through a pre-weighed medium coarseness crucible (Pyrex,

USA) and 15 mL filtrate was collected for further analysis. The retentate was rinsed with 60 mL of deionized water to remove any residual sugars and acid. The crucibles containing retentate were oven dried at 105 oC for 24 hours and then placed in a desiccator over phosphorus

19 pentoxide to cool before being weighed to obtain the insoluble lignin content of the wood. A sample of filtrate was diluted 50:50 with 4% H2SO4 (w/w) and analyzed for acid-soluble lignin using a Cary 50 Bio UV/ Visible Spectrophotometer (Varian Inc., Australia) at 205 nm to obtain an absorbance reading between 0.2 – 0.7. The soluble and insoluble lignin values were combined to determine the total lignin concentration measured as a proportion of the initial extractive free wood.

Next, 0.9 mg of the solubilized filtrate and 0.1 mg of fucose (5mg/mL) internal standard was pipetted and weighed into a microfuge tube. The solution was filtered through a 0.45 µm nylon filter. The total carbohydrate content (arabinose, galactose, glucose, mannose, and xylose) was determined using an anion exchange high-performance liquid chromatograph (Dx-600;

Dionex, Sunnyvale, CA, USA) equipped with an ion exchange PA1 (Dionex) column, a pulsed amperometric detector with a gold electrode, and a SpectraAS3500 auto injector (Spectra-

Physics, USA). The concentrations of arabinose, galactose, glucose, mannose, and xylose were calculated in proportion to the initial extractive free wood and combined as total carbohydrate concentration.

2.7 Syringyl-Guaiacyl Ratio

Ten mg of extract-free wood was used to determine the lignin monomer composition. One mL of a reaction mixture (8.75 mL dioxane, 250 µL BF3, and 1 mL ethaniol) was added to a 6 mL reaction vial and purged with N2 gas before the lid was tightly sealed. Vials were placed in a heating block at 100 oC for 4 hours with periodic agitation every hour. The vials were transferred to a -20 oC fridge for 5 minutes to halt the reaction. Then, 200 µL of internal standard (5 mg tetracosane /1 mL methylene chloride) and 300 µL 0.4 M NaHCO3 were added to the vial to

20 bring the pH between 3 and 4. Next, 2 mL of nanopure water and 1 mL methylene chloride were added to the vial, which was then recapped, vortexed, and allowed to separate into upper

(aqueous) and lower (non-aqueous and containing lignin-breakdown products) phases to allow extraction of the reaction products from the aqueous mixture. One mL of the lower phase was drawn by pipette, filtered by passing through a Pasteur pipette packed with small quantity of tissue paper and a centimeter of granular anhydrous Na2SO4, and finally transferred directly into a 2 mL polypropylene safe-lock microfuge tube. The sample was evaporated to dryness in a

Speedvac set to 45 oC and then resuspended in 700 µL of methylene chloride and kept at -20 oC overnight. 20 uL of resuspended sample was derivatized by combining it with 20 µL of pyridine and 100 µL of N,O-bis(trimethylsilyl) acetamide in a glass insert within an amber-glass GC vial.

The vial was sealed and inverted to mix. It was incubated for at least 2 hours at 25 oC and then 1

µL of solution was analyzed by a gas chromatograph (HP 5890 Series II, Agilent Tech., Ontario,

Canada) on an HP 6890 series II column equipped with an auto injector and flame ionization detector (FID) (Agilent Tech., Ontario, Canada) as per Robinson & Mansfield (2009).

2.8 Data Analysis

The phenotypic traits assessed in this thesis compared species crosses for statistical differences among their mean values. In order to simplify the comparisons, the observations from clones with the same intra-specific cross, but different parents were grouped together, reducing the number of comparisons. A generalized linear model (GLM) was utilized using PROC GLM in

SAS (SAS Institute V 9.2). The following linear model [3] was used for analysis of each phenotypic trait:

21 Yij = u + Tj + Eij [3]

where, Yij is the individual phenotypic observation of the ith clone of the jth species cross, u is the overall mean, Tj is the class variable for species cross, and Eij is the residual error of the observation.

Each trait was tested for the assumptions of analysis of variance, significance of the regression, normal distribution of the sample population’s residuals, and homogeneity of interclass residual variance. The ANOVA procedure was used to check the significance of the regression. The univariate procedure was used to test the residual’s distribution for normality and finally Bartlett’s test was used to test for the residual’s homogenous variation between species crosses. If a trait failed to meet these assumptions, the data were transformed using the dependent variable and/or removing outliers. The pre-planned comparisons between species cross types were performed using the least squares means procedure. Finally, the SAS CORR procedure was used to determine the correlations between wood quality phenotypic traits for all species crosses.

22 Chapter 3: Results

3.1 Models

Table 3 summarizes the results for the PROC GLM class variable models, Kolmogorov-Smirnov tests for normality, and Bartlett’s tests for heteroscedasicity. A PROC GLM model ANOVA F- test for significance was performed for each regression (Ho: regression is not significant, Ha: regression is significant, alpha = 0.05). Each model’s p-value was smaller than alpha; therefore, all comparisons were considered significant. Next, each PROC GLM model’s residuals were tested for normality using the Kolmogorov-Smirnov test. The p-value for each model was again greater than alpha, thus, each model’s residuals were normally distributed. Finally, each PROC

GLM model’s species groups were tested for homoscedasticity using the Bartlett’s test. The p- value for all but one model was greater than alpha and had homogenous variance. The exception was the volume model, where the Bartlett test p-value was 0.005; since this value is extremely close to alpha, the model can still be used for the least squares (LS) means comparisons, but the results should be interpreted with caution.

23 Table 3: PROC GLM ANOVA of traits using the class variable species (Ho: regression is not significant, Ha: regression is significant, Alpha = 0.05), Kolmogorov-Smirnov test for normality (Ho: data is normally distributed, Ha: data is not normally distributed, Alpha = 0.01), and Bartlett’s test for homogeneity (Ho: variances are homogeneous, Ha: variances are not homogeneous, Alpha = 0.01). Proc GLM Kolmogorov- Bartlett Smirnov Trait R-squared RMSE F-value D - statistic Chi-Square (P-value) (P-value) (P-value)

MFA 0.12 1.07 5.18 0.07897 3.2338 (0.0006) (0.0582) (0.5195) D 0.10 29.25 4.86 0.056371 8.8251 (0.001) ( >0.1500) (0.0656) FL 0.41 0.05 22.06 0.055896 2.1548 (<.0001) ( >0.1500) (0.7073) FW 0.13 1.60 4.66 0.058263 8.3866 (0.0015) (>0.1500) (0.0784) CS 0.11 0.23 3.63 0.054734 3.1562 (0.0078) ( >0.1500) (0.532) S:G 0.52 0.25 47.71 0.058548 6.7663 (<.0001) (0.1321) (0.1488) VOL 0.47 0.000746 34.28 0.051991 14.84 (<.0001) (>0.1500) (0.005) TC 0.10 4.53 4.42 0.055846 9.9356 (0.002) (>0.1500) (0.0415) TL 0.13 1.14 6.27 0.041626 3.9646 (0.0001) ( >0.1500) (0.4108) SOL 0.36 0.51 21.37 0.0556349 12.9529 (<.0001) (>0.1500) (0.0115) INSOL 0.32 1.1459 18.04 0.047491 3.6516 (<.0001) (>0.1500) (0.4552) MFA = Microfibril Angle, D = Wood Density, FL = Fibre Length, FW = Fibre Width, CS = Coarseness, S:G = Syringyl-Guaiacyl Ratio, VOL = Volume, TC = Total Carbohydrate Concentration, TL = Total Lignin Concentration, SOL = Soluble Lignin Concentration, INSOL = Insoluble Lignin Concentration, R-squared = Explained Variance, RMSE = Root Mean Squared Error.

3.2 Least Squares Means Comparisons

Appendix A summarizes the mean values and standard errors for each trait for the species crosses. Least squares means comparisons were performed between pre-determined pairings of the five species crosses to ensure the overall protection level of the test. The three intraspecific

24 crosses were compared to one another, and the two interspecific crosses were compared to their corresponding intraspecific crosses for a total of seven species comparisons. The LS-means comparison t-tests and corresponding p-values are found in Table 4.

25 Table 4: Least squares means comparison t-values and p-values (in brackets). Ho: LS-means are equivalent, Ha: LS-means are not equivalent, Alpha = 0.0036. Values in bold are statistically significant. Comparison D MFA FL FW CS S:G VOL TC TL SOL INSOL

NN vs CC -1.49711 1.225158 -8.27532 -2.54536 -2.04838 9.152155 -8.34487 -1.91891 -0.65065 - - (0.1362) (0.2224) (<.0001) (0.0122) (0.0427) (<.0001) (<.0001) (0.0567) (0.5162) - -

NN vs EE -3.9884 -2.47147 -2.90398 1.539314 -0.08546 -3.30187 -4.73953 -0.3364 1.378634 - - (<.0001) (0.0145) (0.0044) (0.1263) (0.9320) (0.0012) (<.0001) (0.7370) (0.1699) - -

CC vs EE -3.1478 -3.28206 0.759125 2.737636 0.839913 -8.89836 2.706956 -1.74186 1.806361 - - (0.0019) (0.0013) (0.4492) (0.0071) (0.4026) (<.0001) (0.0075) (0.0834) (0.0727) - -

EN vs NN 2.16285 -2.00997 8.229494 3.316554 2.327621 -1.10323 10.76648 3.659188 -1.03221 4.92152 -3.24524 (0.0319) (0.0462) (<.0001) (0.0012) (0.0216) (0.2714) (<.0001) (0.0003) (0.3035) (<.0001) (<.0001)

EN vs EE -2.2912 -3.71115 1.322527 3.222536 1.104507 -3.85339 5.449184 0.686445 0.599252 - - (0.0231) (0.0003) (0.1884) (0.0016) (0.2715) (0.0002) (<.0001) (0.4934) (0.5498) - -

CN vs NN 0.05608 1.299655 4.552577 1.402141 -1.03579 -9.37302 3.023491 0.605803 3.782373 -1.6793 4.46007 (0.9553) (0.1956) (<.0001) (0.1634) (0.3023) (<.0001) (0.0029) (0.5455) (0.0002) (0.0951) (<.0001)

CN vs CC -1.20968 2.547126 -2.32553 -0.71326 -2.97552 -1.92474 -4.21241 0.346783 3.357428 - - (0.2280) (0.0118) (0.0217) (0.4770) (0.0035) (0.0559) (<.0001) (0.7292) (0.0010) - -

MFA = Microfibril Angle, D = Wood Density, FL = Fibre Length, FW = Fibre Width, CS = Coarseness, S:G = Syringyl-Guaiacyl Ratio, VOL = Volume, TC = Total Carbohydrate Concentration, TL = Total Lignin Concentration, SOL = Soluble Lignin Concentration, INSOL = Insoluble Lignin Concentration.

26 3.2.1 Volume

The mean tree volume and variation for each species cross is shown in Figure 6, and were shown to range from 1.14x10-3-3.11x10-3 m3. The EN cross had the largest average volume, while the

NN cross had the lowest. The LS-means tests (Table 4) demonstrated that volume was the trait with the greatest number of differences.

0.004

0.002

Volume (m^3)

0.000 CC CN NN EN EE Species

Figure 6: Boxplot of mean volume by species cross

The point located within the box represents the average and the horizontal line is the median. The box represents the 25th and 75th percentiles. The whiskers represent the 5th and 95th percentile. The ‘x’ symbols mark the 1st and 99th percentile. Finally, the ‘-’ symbols mark the range of the sample set.

The NN cross showed significantly less growth compared to the CC and EE cross, showing 55.0% and 43.8% less volume, respectively. The tree volume of CC and EE crosses were not significantly different. The EN cross had 172.8% and 53.1% larger volume than the NN and EE crosses, respectively. The CN cross was 51.8% larger than the NN cross, but the CN cross was significantly smaller than the CC cross (31.6% less growth).

27 3.2.2 Wood Density

The mean wood density and variation for each species cross is shown in Figure 7. The species crosses average densities ranged from 399-436 kg/m3. The EE cross had the highest average wood density, while the NN cross had the lowest average wood density.

550

500

450

400

Density (kgm^-3)

350

300 CC CN NN EN EE Species

Figure 7: Boxplot of mean wood density by species cross

The LS-means tests (Table 4) revealed the EE cross was significantly different from NN and CC crosses, and that the EE cross had 9.54% and 7.15% greater density than the NN and CC crosses, respectively. All other species cross comparisons evaluated were similar.

28 3.2.3 Microfibril Angle

The mean MFA and variation for each species cross is shown in Figure 8. The species crosses

MFA averages ranged from 22.2-23.7o. The EE cross had the largest average MFA, while the EN cross had the smallest average MFA.

MFA (degrees)

CC CN NN EN EE Species

Figure 8: Boxplot of mean MFA by species cross

The LS-means test (Table 4) shows statistically significant differences between the EE cross and both the CC and EN crosses, with the EE cross being significantly greater than the CC and EN crosses by 5.3% and 6.8%, respectively. All other species cross comparisons evaluated were similar.

29 3.2.4 Fibre Length

The mean fibre length and variation for each species cross is shown in Figure 9. The average fibre length of the species crosses ranged from 0.428-0.537 mm. The EN cross had the longest average fibre length, while the NN cross had the shortest average fibre length.

0.7

0.6

0.5

0.4

Fibre Length (mm)

0.3

0.2 CC CN NN EN EE Species

Figure 9: Boxplot of mean fibre length by species cross

The LS-means tests (Table 4) showed that the NN cross was significantly different from several other species crosses. The NN cross was 20.3%, 19.4% and 13.7% shorter than the EN, CC, CN crosses, respectively, while it was similar to the EE cross. The remaining species cross comparisons were not significantly different from one another.

30 3.2.5 Fibre Width

The mean fibre width and variation for each species cross is shown in Figure 10. The fibre width averages ranged from 19.4-22.2 µm. The EN cross had the largest average fibre width, while the

EE cross had the thinnest average fibre width.

Fibre Width (um)

CC CN NN EN EE Species

Figure 10: Boxplot of mean fibre width by species cross

The LS-means tests (Table 4) revealed the EN cross had significantly wider fibres compared to the NN and EE crosses, 7.2% and 14.4% respectively. There were no other significant differences between the species crosses compared.

31 3.2.6 Coarseness

The mean fibre coarsenesses and variation for each species cross is shown in Figure 11. The fibre coarseness averages ranged from 0.063-0.075 mg/m, and the EN cross had the largest average fibre coarseness values, while the CN cross had the smallest values.

0.12

0.10

0.08

0.06

Coarseness (mg/m) 0.04

0.02 CC CN NN EN EE Species

Figure 11: Boxplot of mean coarseness by species cross

The LS-means test (Table 4) indicated that the CN cross was significantly different from the CC cross; the CN cross’s average coarseness was had a12.5% lower than the CC cross. However, the p-value was 0.0035 and very close to alpha, and therefore the result should be interpreted with caution.

32 3.2.7 Syringyl-Guaiacyl Ratio

The mean S:G and variation for each species cross is shown in Figure 12. The species crosses average S:G ranged from 1.74-2.56. The EE cross had the highest molar ratio of syringyl monomers in the lignin polymer, while the CN cross had the lowest ratio.

3.0

2.5

2.0

S:G (ratio)S:G

1.5

CC CN NN EN EE Species

Figure 12: Boxplot of mean S:G by species cross

The LS-means tests (Table 4) showed several statistically distinct crosses. The S:G for the NN cross was 23.8% and 31.6% greater than those of the CC and CN crosses, respectively. The EE cross was 11.8% and 15.3% greater than those of the NN and EN crosses, respectively. The greatest comparative difference in S:G was between the EE and CC crosses; the EE cross was

38.3% greater than the CC cross. The comparisons between the NN and EN crosses, as well as between the CN and CC crosses were not significantly different.

33 3.2.8 Total Carbohydrate Concentration

The mean carbohydrate concentration and variation for each species cross is shown in Figure 13.

The species crosses total cell wall carbohydrate concentrations averages ranged from 63.8-

67.7%. The EN cross had the highest average carbohydrate concentration and the NN cross the lowest average carbohydrate concentration.

Carbohydrate (%) 55

CC CN NN EN EE Species

Figure 13: Boxplot of mean carbohydrate concentration by species cross

The LS-means test (Table 4) revealed the EN cross was significantly different from the NN cross. The EN cross was 3.9% greater than the NN cross. All other species cross comparisons evaluated were similar.

34 3.2.9 Total Lignin Concentration

The mean total cell wall lignin concentration and variation for each species cross is shown in

Figure 14. The species crosses averaged lignin concentrations ranged from 21.4-22.9%. The CN cross had the highest average lignin concentration and the EE cross had the lowest average lignin concentration.

Lignin (%) 22

CC CN NN EN EE Species

Figure 14: Boxplot of mean lignin concentration by species cross

The LS-means tests (Table 4) revealed the CN cross was significantly higher than the NN and

CC crosses, and that the CN cross had 1.06% and 0.92% greater average lignin concentrations than the NN and CC crosses, respectively. All other species cross comparisons were similar.

3.2.10 Soluble/Insoluble Lignin Concentrations

The soluble and insoluble lignin concentrations were compared between the inter-specific crosses and the NN cross. The EN cross’s soluble and insoluble lignin concentrations were

0.64% greater and 1.08% less (total wood mass) than the NN cross’s, respectively. In contrast,

35 the CN cross’s insoluble lignin concentration was 1.33% greater than the NN cross’s concentration and its soluble lignin concentration was not significantly different.

3.3 Correlations

There are no set values to define the strength ranges of correlations. Therefore, ranges were established to segregate the trait correlations in an appropriate manner. Table 5 summarizes the descriptive words associated to each correlation strength range.

Table 5: Correlation coefficient ranges

Correlation Coefficient Interpretation

.00 - .19 Slight, almost negligible correlation .20 - .39 Low, quite small correlation .40 - .69 Moderate correlation .70 - .89 High correlation

These qualifiers are used to describe the relationships between the wood quality traits and growth traits. In contrast, Table 6 summarizes correlation coefficients and their level of significance between the traits studied.

36 Table 6: Pearson’s correlation coefficient between traits and p-value (in brackets). Ho: regression is not significant, Ha: regression is significant, Alpha = 0.05. Values in bold are statistically significant. D FL FW CS S:G TL TC SOL INSOL DBH MFA 0.09 -0.02 -0.27 -0.06 -0.1 -0.02 -0.02 - - - (-0.26) (0.83) (0) (0.55) (0.2) (0.77) (0.81) D 0.2 -0.25 0.19 0.11 -0.11 0.18 - - - (0.02) (0) (0.03) (0.14) (0.14) (0.02) FL 0.4 0.45 -0.11 -0.2 0.12 - - - (<.0001) (<.0001) (0.2) (0.03) (0.18) FW 0.4 (0.02) -0.19 0.09 - - - (<.0001) (0.83) (0.03) (0.34) CS 0.06 -0.15 0.05 - - - (0.48) (0.09) (0.59) S:G -0.39 0.3 0.43 -0.58 - (<.0001) (<.0001) (<.0001) (<.0001) TL -0.19 - - - (0.01) HT 0.88 (<.0001) MFA = Microfibril Angle, D = Wood Density, FL = Fibre Length, FW = Fibre Width, CS = Coarseness, S:G = Syringyl-Guaiacyl Ratio, VOL = Volume, TC = Total Carbohydrate Concentration, TL = Total Lignin Concentration, SOL = Soluble Lignin Concentration, INSOL = Insoluble Lignin Concentration, HT = Height, DBH = Breast Height Diameter.

37 MFA had a low negative correlation with fibre width, as did density. However, density had a low positive correlation with fibre length, and slight positive correlations with coarseness and total carbohydrate concentration. Fibre length had moderate positive correlations with fibre width and coarseness; it also had a low negative correlation with total lignin concentration. Fibre width had a moderate positive correlation with coarseness and a slight negative correlation with total lignin concentration. S:G had a low negative correlation with total lignin concentration and a low positive correlation with total carbohydrate concentration. S:G had a moderate positive correlation with soluble lignin concentration and a moderate negative correlation with insoluble lignin concentration. Total lignin concentration had a slight negative correlation with total carbohydrate concentration. Height had a highly positive correlation with DBH.

38 Chapter 4: Discussion

4.1 Growth

When comparing the intra-specific crosses, the NN cross’s average growth was markedly less than the growth of either the EE or CC crosses, while the EE and CC crosses displayed similar growth rates. In Li et al.’s (1993) study, the P. tremuloides average rotation age was 40 year and the expected rotation age for hybrid crosses were based on the P. tremuloides rotation. For example, if the inter-specific cross growth doubled that of the P. tremuloides, then the hybrid’s minimum expected rotation was assumed to be 20 years. Assuming the NN cross has equivalent growth to P. tremuloides from the lake states, it would then have a minimum 40-year rotation.

Using the aforementioned method of projecting rotation age shows the relatively superior growth of the CC and EE crosses over the NN cross could potentially reach merchantable volumes within 18 and 22 years, respectively.

The CN hybrid volume was intermediate, and significantly different from those volumes of the NN and CC crosses. In contrast, the EN cross’s volume was greater than the NN and EE crosses. The improved growth observed in the EN and CN crosses is in agreement with numerous studies of heterosis using hybridization of P. tremuloides with P. tremula or P. davidiana (Li et al., 1993; Melchior, 1985; Yu, Tigerstedt et al., 2001). Again, assuming P. tremuloides has a minimum 40-year rotation, the EN cross’s relatively superior growth suggests its clones could reach harvestable size within as few as 15 years. These findings are consistent with Li et al. (1993) who concluded that stand rotation could be reduced by half in the lake states

(ca. 20 years). However, the relative growth improvements observed in the EN hybrid over the

NN cross exceeds the relative improvements observed in Li et al.’s (1993) study examining P. tremuloides × P. tremula hybrids over intraspecific P. tremuloides clones. This latest finding

39 implies the effects of heterosis are more pronounced in Canada than the United States, or the original parents have a higher breeding value than those used in the US study. Although the CN hybrid was not superior to the CC cross in volume, its improved growth over the NN cross could reduce rotation times from 40 to 26 years, which is a marked improvement over native aspen. In addition, the CN cross may have inherent drought tolerance traits derived from the P. davidiana parent, something not expected for the EN cross, which could improve its relative performance on dry sites (Li et al., 1993).

The biomass of each species cross could be estimated by multiplying their average wood density by their average volume to determine the expected mass within a stem. The average biomass estimates for each cross follows similar trends to the volume but not to the density. This trend is likely because the differences in average stem volume between crosses were more pronounced than the differences in wood density. Therefore, species crosses with superior volume growth would also be expected to have greater potential for rapid production of biomass for bioenergy applications, such as ethanol production.

4.2 Wood Density

An examination of the wood density revealed that among the crosses investigated, the lowest average density belonged to the NN cross, and the species crosses were in agreement with the upper range reported for mature aspen trees originating from Alberta (Yanchuk et al., 1983). In contrast, the EE cross had the highest wood density, displaying an average wood density of 436 kg/m3, which was greater than aspen estimates reported in the literature for P. tremula

(Junkkonen & Heräjärvi, 2006; Kärki, 2001) as well as North American P. tremuloides

(Mansfield & Weineisen, 2007; Yanchuk et al., 1984). The EN cross wood density was higher

40 than P. tremuloides × P. tremula density values reported in Finland (Junkkonen & Heräjärvi,

2006).

The EE cross developed higher density wood than the NN cross, while the EN cross average wood density was between and statistically equal to the two. The EE cross’s higher density over the NN and EN crosses was in agreement with earlier literature (Bjurhager et al.,

2008; Junkkonen & Heräjärvi, 2006; Tsoumis, 1991). The EE cross’s average wood density exhibited a 37.5 kg/m3 increase over the NN cross, which reflects an approximate 10% increase in density. The EE cross was also significantly different from, and 7% higher than, the CC cross.

Yanchuck and Micko (1990) studied the variation in radial wood density in aspen selected for their extremes (high and low density values). Density was averaged in five year increments from pith to bark, and they found that density displayed large variability both among and between trees of some clones. There was also varied density profile from pith to bark among clones; some were high near the pith, decreased, and then stabilized; while others steadily increased from pith to bark. Also, wood density decreases generally occurred between years 6-

10. In comparison, wood density in both P. tremula and P. tremula × P. tremuloides from

Finland increased radially outward from the pith. The specimen densities investigated in this study were measured as averages of 7 years growth, and therefore, if they follow similar patterns of growth trends for native North American or European aspen, their current overall wood density may not be an accurate predictor of their mature average wood density; it be an overestimation, or they could further increase as they mature.

In general, all the species crosses examined showed wood density estimates well suited for pulp and paper production. The higher the average wood density the better, as elevated density results in increased packing density in the digester, and consequently higher potential

41 chemical pulp yields. However, the relative density of wood is influenced in part by wall thickness; thus, wood density can be used as an indicator of cell wall thickness (Balatinecz &

Kretschmann, 2001; Gurnagul et al., 1990). Fibres with thinner walls collapse more easily and lead to improved fibre compression (Amidon, 1981). As such, higher densities may also manifest poorer paper properties, such as lower sheet density and reduced burst and tensile strength (Via et al., 2004).

Similarly, lower density and thinner cell walls, are preferred for OSB manufacture as they facilitate greater compressibility and thus greater inter-strand contact surface area, which in turn manifests in improved MOR, MOE, and tensile strength (Woodson, 1976). The crosses all displayed densities within the preferable lower range for MDF and OSB furnish; therefore, the aspen clones investigated in the current study would exhibit suitable physical properties for OSB and MDF production (Geimer, 1976; Hsu, 1997; Semple et al., 2007; Woodson, 1976). More specifically, the North American aspen might have a slightly higher compactness or compression ratio, but the European aspen would likely have comparable mechanical characteristics.

4.3 Microfibril Angle

The average MFA for each cross was in the low twenties and in agreement with several studies evaluating juvenile trembling aspen MFA, which has been shown to range from 18 to 22 degrees

(Horvath et al., 2012; Maloney & Mansfield, 2010). MFA is generally greatest near the pith and decreases radially toward the bark (Barnett & Bonham, 2004). The MFA of the poplar clones was shown to consistently decrease with age, from 28o in the first four years of growth to 7.8o at year 11 (Fang et al., 2006). Similarly, in two 31-year-old natural aspen trees, the mean MFAs were estimated at 12.5o and 14.7o (Francis et al., 2006). Assuming the species crosses in this

42 study follow a similar trend to those reported previously for aspen, their MFA will decrease considerably by the time they reach rotation age.

The EE cross’s MFA was largest, and was significantly higher than the EN cross with a difference of 1.5o. The EE cross was also 1.2o greater than the CC cross, but was statistically equal to the NN cross. Several studies have partially explained the variation in handsheet stretch due to MFA with higher MFA correlating to increased percent strain (Downes et al., 2003;

Gurnagul et al., 1990; Horn, 1978; Watson & Dadswell, 1964). It is possible the EE cross may have slightly higher handsheet stretch compared to the CC and EN crosses. A study of various

o poplar clones with varied MFAs ranging from 12.5 to 30.7 exhibited handsheet stretch between

3.4 and 5.1% (Francis et al., 2006). However, the MFA of the aspen crosses in this study ranged from 22.2 to 23.7o and if their papersheet properties behaved similarly to the poplar from the aforementioned study the average differences in paper stretch due to MFA would likely be negligible.

Similarly, studies found changes in MFA explained a substantial amount of variation in

MOE in Eucalyptus (Evans & Ilic, 2001; Yang & Evans, 2003). The MFA in these studies ranged from approximately from 8.5 to 23o and in both cases the MOE was relatively constant until MFA decreased below 14o at which point MOE increased at a greater rate. The MFA of species crosses in this study were all in the low twenties and their variation was only 1.5o.

Therefore, it is likely there are negligible differences in wood stiffness among crosses provided the relationships between MFA and MOE for aspen crosses in this study are similar to the one observed in Eucalyptus.

43 4.4 Fibre Length, Width, and Coarseness

All the crosses displayed average fibre lengths that were consistent with the ranges reported for

P. tremuloides (Groover et al., 2010; Yanchuk & Micko, 1990). Previously, it was shown that trembling aspen fibre length followed a common trend: fibre length near the pith was short and slowly increased toward the bark (Yanchuk et al., 1984). Therefore, it is likely that the average fibre length of the aspen clones investigated will increase as they mature, and become more consistent with values reported in other studies.

Specifically, the EE and EN crosses average fibre lengths were 0.083 and 0.109 mm longer than the NN cross, respectively. However, only the EN and NN crosses were significantly different. The non-significant difference between the EE cross and both the EN and NN crosses is likely because the standard error was large for the EE cross. Fibre length was generally longer in crosses that contained P. davidiana compared to the intraspecific P. tremuloides cross. The

CN cross was slightly shorter but statistically equal to the CC cross; while both the CN and CC crosses were significantly longer than the NN cross. They were 0.103 mm and 0.068 mm longer, respectively. Clones with P. davidiana for one or both parents, may therefore, be better suited for hardwood pulp production, as this cell trait has been shown to contribute to superior mechanical properties (Amidon, 1981; Horn, 1978). During paper manufacturing, fibres are pressed together to increase contact surface area in the sheet which is necessary for inter-fibre hydrogen bonds to occur; and increased fibre length, along with decreased coarseness, was found to promote fibre flexibility and collapse which further aids in inter-fibre bonding (Paavilainen, 1993). The relatively longer fibres observed in the CC and CN crosses, and thus the potential for increased inter-fibre bonding would likely form paper with increased tensile and tear strength (Via et al.,

2004).

44 All crosses average fibre widths were also consistent with the range previously reported for aspen (Groover et al., 2010). In contrast, the average coarseness estimates were below reported values for P. tremuloides in Canada and the US (Horn, 1978; Karaim et al., 1990;

Mansfield & Weineisen, 2007). One explanation could be the fibres have thin walls; it would allow the fibres to be short and wide but still maintain thin cell walls, and thereby have low average coarseness values.

The EN cross’s average fibre width was significantly greater than the EE and NN crosses by 2.8 and 1.5 µm, respectively. The EN cross’s fibres were also relatively long which is preferable because, in general, pulp from longer and wider fibres will exhibit superior quality

(Seth, 1990a; Seth, 1990b). Thus, the EN cross’s fibre dimensions make it a preferable fibre source for pulp manufacture.

While the CN cross was of statistically lower coarseness than the CC cross, it was equivalent to the NN cross. In addition, the NN cross was also equal to the CC cross. The p value for the CN vs. CC comparison was very close to alpha and it would be too presumptuous to expect differences in their pulp properties based on differences in coarseness.

In general, lower coarseness is beneficial in the production of bleached Kraft hardwood pulp because fibres of lower coarseness generally have the propensity to collapse during sheet formation and as a consequence improves paper smoothness and pulp brightness (Mansfield &

Weineisen, 2007; Yu et al., 2001). Lower coarseness has also been shown to be associated with higher paper strength, but is secondary to fibre length in predicting burst strength (Seth, 1990b;

Via et al., 2004). Thus, any strength improvement from increased collapsibility would likely be negatively influenced by the effect of the relatively short fibres. As the clones age to maturity, their average fibre length will likely increase and their coarseness would likely also increase with

45 age as it is related to other wood properties such as density (Via et al., 2004). Thus, the magnitude of the coarseness and fibre length’s effect on fibre quality may normalize.

4.5 Syringyl-Guaiacyl Ratio

The S:G estimates for the NN and EN crosses were slightly lower than previously reported P. tremuloides estimates (Stewart et al., 2006); while they were similar to a more expansive group of British Columbian P. tremuloides from several sites (Mansfield & Weineisen, 2007). The S:G of the EE cross was comparable to P. tremuloides clones from both previous studies, but the CC and CN crosses were below the ranges in the studies. The discrepancies in S:G ratio in this study and those from the literature might be explained by regional differences between trees from

British Columbia and those from Alberta, or possibly due to inherent differences in lignin composition between P. tremuloides and P. davidiana.

The differences in lignin composition would have notable implications on the chemical pulping efficiency of the clones. Lignin rich in G units has relatively more carbon-carbon bonds

(5-5 linkages; biphenyl) than lignin rich in S units because the aromatic C5 position of G units is free to undergo coupling reactions, while it is unavailable in S units (Baucher et al., 2003). The

β-O-4 linkages are targeted by chemicals during pulping and are less resistant than carbon- carbon linkages. As a consequence, wood lignin with higher concentrations of S units is more susceptible to Kraft delignification (Lapierre et al., 1999). One proposed cause for this improved delignification is that S units have a higher propensity to form β-O-4 linkages among subunits

(Chang & Sarkanen, 1973), and lignin with a higher proportion of S units had a lower overall molecular weight which may be a factor in pulping efficiency (Stewart et al., 2006).

46 Lignin composition may also be a factor in the efficiency of wood conversion to ethanol.

Specifically, variation in lignin monomer composition in transgenic hybrid poplar was associated with a 10% increase in cellulose recovery in organosolv pre-treatment, and it was more easily hydrolyzed by enzymes with less inhibition to fermentation (Mansfield et al., 2012). Therefore, crosses in this study with higher S:G would have higher potential yield if used as a source for such biofuel production.

Species crosses with P. davidiana generally showed lower S:G. To increase the clarity in the magnitude of differences in the S:G between species crosses, the S:G ratio can be expressed in percent syringyl lignin monomers. For example, a 2:1 S:G corresponds to 66.6% syringyl lignin monomer. If there was a 6.6% decrease in syrignyl lignin monomers, the corresponding

S:G would be 1.5:1. When compared to the NN cross, the percent syringyl lignin monomers for the CN and CC crosses were 6.1 and 4.7% lower, respectively. In contrast, the EE cross had 7.01 and 2.31% greater syringyl lignin monomers composition than the CC and NN crosses, respectively. The EN cross’s percent syringyl lignin monomer was 0.7% less than the NN cross, although it was not significantly different.

The CN and CC crosses would likely demand greater chemical/energy (H-factor) to achieve a desired pulp end quality (kappa; residual lignin) compared to the other crosses; whereas, the EE cross would be most efficient (Stewart et al., 2006). The difference in syringyl concentration between the EE and NN crosses corresponds to an approximate 2.5% yield difference using a regression for P. tremuloides (Mansfield & Weineisen, 2007). Therefore, the yield from the EE cross clones might be hindered by its higher S:G. In addition, the CN and CC crosses might be less desirable as a raw material for ethanol biofuel production, whereas the EE cross would be more desirable due to the effect of S:G on recovery.

47 There was an interesting trend when comparing the interspecific crosses with their respective pure crosses. Both interspecific crosses S:G ratio were lower than their respective pure crosses; however, they were only significantly different from the pure cross with the larger S:G ratio. This finding may indicate that aspen trees are dominant towards a lower S:G.

4.6 Lignin Concentrations

The cell wall lignin concentrations for all crosses were similar to those previously reported for P. tremuloides in the literature (Mansfield & Weineisen, 2007; Pettersen, 1984;

Stewart et al., 2006). Lignin content will have adverse effects on cellulose yield as lignin removal during the pulping process is accompanied with some cellulose degradation (Baucher et al., 2003). Also, in bioconversion processes to ethanol, lignin both significantly obstructs enzymatic accessibility and binds with cellulolytic enzymes which impact sugar release

(Mansfield et al., 2012). The authors also found that transgenic poplar trees with lowered lignin content showed as much as 15% improvement in their conversion efficiency of cellulose to the corresponding monomers. Therefore, species crosses in this study with lower lignin levels would be better suited for use either in pulp or ethanol biofuel applications.

The CN cross showed lignin concentrations that were significantly greater than the NN and CC crosses, by 0.92 and 1.06%, respectively. While the differences were small, any change in lignin concentration will have an effect on processing operations of large scale pulp mills.

Therefore, the CN cross would likely be more difficult and demanding during pulp manufacture, and as a source for ethanol bioconversion, in comparison to either the CC or NN intra-specific crosses.

48 The mean soluble and insoluble lignin concentrations were compared between interspecific crosses and the NN intra-specific crosses. The CN cross’s increased total lignin content is largely a result of the observed increased insoluble lignin. The EN cross’s soluble lignin concentration was greater than and insoluble lignin lower than, the NN cross. Therefore, the EN cross’s lignin content, while equal to the NN cross, is in a more desirable form than the NN cross as it has the potential to improved pulping efficiency due to its lower insoluble lignin concentration (Stewart et al., 2006).

The fact that the CN hybrid had more lignin than either the NN or CC crosses may indicate high lignin content is a dominant trait in inter-specific crossing; although, the trend was not apparent for the EN cross with its respective pure crosses as they were all statistically equivalent to each other. With respect to their lignin concentrations, all the intra-specific crosses, as well as the EN hybrid, would be better suited for bleached Kraft hardwood pulp production or as a biofuel source than the CN hybrid.

4.7 Carbohydrate Concentration

The total carbohydrate content of each cross was less than the general average for P. tremuloides

(Pettersen, 1984) and were less than ranges reported for trembling aspen clones in British

Columbia (Mansfield & Weineisen, 2007; Stewart et al., 2006). The differing degrees of accessibility of monomers which comprise the total carbohydrate content in the cell wall will have an effect on the availability for moisture sorption and pulping (Rowell, 2005), and affects carbohydrate’s ability to be hydrolyzed (Mansfield et al., 1999). The monomeric composition will play a role in yield for pulp manufacture and ethanol biofuel applications; but, in general, greater total cell wall concentration results in higher yields.

49 Overall, the clones in this study would have lower yields in pulp manufacture or biofuel applications compared to native P. tremuloides according to the concentrations measured. In relation to one another, the EN cross carbohydrate concentration was significantly higher than the NN cross. Using the EN hybrid instead of the NN cross as a fibre source for Kraft pulp processing would improve the relative yield. The EE cross had higher carbohydrate concentrations than the NN cross, but they were statistically equal, which is likely due to the EE cross’s large standard error. In addition, the CN and CC crosses had slightly higher carbohydrate concentrations than the NN cross but were again statistically equal and would likely have similar pulp yields.

4.8 Correlations

The growth characteristics of height and DBH were the only two traits that displayed a high correlation. Unfortunately, the growth traits could not be correlated to the wood quality traits because measurements were performed on different sets of trees. The trial plot (for growth traits) was intended to be preserved as a long-term study of tree performance so the clones were not available for wood coring.

The fibre characteristics had the highest correlations of the wood properties. Each fibre characteristic was positively correlated with one another, which is in agreement with several previous studies (Gerendiain et al., 2008; Kibblewhite & Uprichard, 1996; Ukrainetz et al.,

2008). Generally, fibre traits were negatively correlated with lignin content, although coarseness was not significant. There was also a general trend of positive, but non-significant, correlations between fibre traits and carbohydrate concentration. Similar significant relationships were

50 observed in Douglas-fir (Ukrainetz et al., 2008); while, no significant relationships were observed in P. trichocarpa (Porth et al., 2013).

S:G correlations with cell wall composition was favorable; it was inversely correlated with lignin concentration and directly correlated with carbohydrate concentration, and these relationships are in agreement with other studies (Bose et al., 2009; del Río et al., 2005; Porth et al., 2013). However, the correlations in this study were low in strength (0.19 to 0.39 in magnitude) which could be why weak or non-significant correlations were observed in other studies (Lapierre et al., 1999; Wallis et al., 1996).

Correlations of S:G with soluble and insoluble lignin were investigated. Treating the lignin traits as their individual components had stronger correlations compared to the total lignin concentration. The direct correlation with soluble lignin and inverse correlation of insoluble lignin with S:G ratio indicates S:G ratio could be a desirable trait to breed for; the clones in this study with higher S:G ratio would likely have improved pulping efficiency, with less lignin, more easily removed lignin, and more cellulosic fibres.

There were several other statistically significant, albeit small, correlations between other wood properties. The trees were measured during juvenile wood development and some correlations could become prominent in mature wood. Higher density was correlated with increased fibre length, coarseness, and carbohydrate concentration. In contrast, P. trichocarpa had no significant correlations between density and fibre length, but there was a positive correlation between density and alpha cellulose (Porth et al., 2013). In addition, several other poplar clones’ correlations between density and fibre length and width, as well as MFA and fibre width, were in agreement with this study (Sheng-zuo et al., 2004).

51 4.9 Heterosis

The interspecific species crosses in this study were bred from different parent crosses than those of the intraspecific crosses. As such, the interspecific crosses do not reveal true heterosis when compared to their relative intraspecific crosses. However, the comparisons are indicative of heterosis that could be prevalent in offspring of interspecific crosses.

The EN cross showed signs of heterosis as the growth was superior to both the EE and

NN crosses. In contrast, the CN cross growth was not indicative of heterosis as the growth was in between the respective intraspecific crosses. While heterosis is primarily used to qualify growth improvements, the wood quality characteristics were also investigated to determine if the hybrid crosses’ phenotypes were beyond that of the intraspecific crosses. There were signs of the hybrids surpassing in the intraspecific crosses when investigating some of their wood quality traits. However, even though the interspecific crosses wood quality values were beyond both intraspecific crosses, in most cases the interspecific crosses’ trait was only significantly different from one of the two intraspecific crosses to which it was compared according to the LS means tests (Table 4). The trend of dominance in the CN cross was prevalent for lignin concentration and S:G. Furthermore, the apparent dominance for these traits were unfavorable for wood used in pulp manufacture and ethanol biofuel applications. In contrast, the EN cross showed the trend of dominance in MFA, fibre length and width, and carbohydrates; and, the trend for all of the wood quality traits were favorable. Overall, it appears any improved growth in the interspecific hybrid crosses does not negatively affect the wood quality characteristics in the clones.

52 Chapter 5: Conclusion

5.1 Thesis Summary

Aspen clones located near the Alberta-Pacific pulp mill in Athabasca, Alberta represented five species crosses: P. tremluloides × P. tremuloides (NN), P. davidiana × P. davidiana (CC), P. tremula × P. tremula (EE), P. tremula × P. tremuloides (EN), and P. tremuloides × P. davidiana

(CN). The clones were evaluated and compared by their growth and wood quality characteristics to determine the effectiveness of interspecific crossing aspen as a breeding strategy to improve future generations of aspen on the Canadian landscape.

The most notable difference between the species crosses was in volume growth and large improvements could be realized using hybrid aspen over native aspen trees. The NN cross had lower volume growth than the CC and EE crosses with 55.0 and 43.8% less volume, respectively. The EN and CN crosses had 172.8 and 51.8% greater volume than the NN cross, respectively and could potentially decrease rotations from 40 years to 15 and 26 years. When coupled with density, the species crosses with improved growth exhibited similar trends with improved biomass production. Thus, the improved volume growth is a substantial improvement in total lignocellulosic mass produced by the clones. Furthermore, improved growth in the interspecific hybrid crosses does not appear to negatively affect the wood quality characteristics in the clones.

The NN cross had shorter fibre length, but greater S:G relative to the CC cross.

Therefore, there may be a trade-off in processing efficiency with increased pulp quality if P. davidiana trees replaced P. tremuloides. The NN cross also had lower density and S:G compared to the EE cross. The EE cross would have a higher digester packing density and greater ease of delignification over the NN cross when used in Kraft pulp production.

53 The EN cross had longer, wider fibres, higher carbohydrate concentration, a more favorable distribution of soluble and insoluble lignin compared to the NN cross, and it could potentially create better quality pulp with higher yield. The EN cross had slightly lower MFA and greater fibre width but lower S:G compared to the EE cross. The processing efficiency of the

EN cross would likely be lower compared to the EE cross.

The CN cross had greater fibre length and lignin concentration in the form of insoluble lignin, but lower S:G compared to the NN cross which indicates the CN cross would have higher

Kraft pulp processing demands and be less desirable as a biofuel source due to the higher lignin concentration and decreased S:G. However, the CN cross’s longer fibres may create paper with superior strength properties compared to using the NN cross fibres. The CN cross also had greater lignin concentration compared to the CC cross and would have a lower relative Kraft pulp yield.

Overall, the EN cross is the most promising species cross evaluated. It had the largest volume growth and its wood traits maintained or surpassed the quality of the NN cross. The EE cross was also promising due to its high wood density and volume growth which would translate to higher biomass production. In addition, its relatively high S:G may improve efficiency in pulping and biofuel applications. The CN and CC crosses displayed improved growth rates and longer fibres than the NN cross, but their lower S:G and higher lignin concentrations make them less desirable.

The wood fibre characteristics were all positively correlated with one another. Also, they showed a general trend of negative correlations with total lignin concentration and positive correlations with total carbohydrate concentration. There were significant correlations between

S:G ratio, both soluble and insoluble lignin concentration, and total carbohydrate concentration;

54 clones with higher S:G ratio would likely have improved pulping efficiency, with less lignin, more easily removable lignin, and more cellulosic fibre at a given time in the Kraft pulp process.

5.2 Research Significance

There is an increasing escalating need to improve tree quality and growth rates as the demand for raw material increases globally. The research performed in this thesis demonstrates the potential of using hybridization strategies to significantly increase the growth rate of aspen in Alberta,

Canada. It also provides insight beyond growth characteristics of hybrid aspen crosses through the extensive characterization of their wood quality traits.

The results clearly indicate that the hybrid species crosses were more desirable wood sources than pure P. tremuloides cross. Specifically, P. tremula x P. tremuloides displayed superior growth rates, while either equaling or surpassing the P. tremuloides x P. tremuloides wood quality attributes. In contrast, the P. tremuloides x P. davidiana species cross, which also displayed superior growth compared to the P. tremuloides x P. tremuloides species cross, appeared to have less desirable lignin content and composition which may affect processing energy requirements and yield in bleached Kraft hardwood pulp production or potential biofuel applications. Therefore, the P. tremula x P. tremuloides cross appears to be the most desirable candidate cross that should be considered for use to improve potential future generations of aspen on the Canadian landscape.

5.3 Future Research

The results obtained from this thesis are useful, but also limited by the experimental design. First and foremost, it would be useful to sample wood cores from the trees measured for growth to

55 establish definitive relationships of wood traits and growth. Also, increasing the sample size for each species cross would be beneficial to reduce their standard error of the mean. It would be beneficial to measure the clones’ growth performance and wood quality characteristics once they have reached merchantable volume to determine changes in growth rate and wood quality over their lifespans. Furthermore, the crossing potential of P. tremula × P. davidiana should be investigated as they each exhibit a different set of advantages/disadvantages over P. tremuloides alone, and could potentially produce clones superior to both the P. tremula x P. tremuloides and

P. tremuloides x P. davidiana hybrid crosses observed in this study. It would be useful to assess the genetic and phenotypic correlations between growth and wood properties as well as the heritability of traits. Finally, and most importantly, it would be extremely useful to assess the growth and wood traits of these crosses on trees grown on multiple sites.

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70 Appendix A: Least Squares Means

Table 7: Least squares means and standard error (in brackets) for each trait by species Type D MFA FL FW CS S:G VOL TC TL SOL INSOL

CC 407.10 22.5 0.531 21.7 0.072 1.85 0.00253 64.12 22.01 - - (3.78) (0.1) (0.007) (0.2) (0.002) (0.03) (0.00011) (0.61) (0.15) - -

CN 399.10 23.1 0.496 21.4 0.063 1.74 0.00173 64.50 22.93 2.51 20.37 (5.43) (0.2) (0.012) (0.4) (0.003) (0.05) (0.00015) (0.91) (0.23) (0.07) (0.23)

EE 436.22 23.7 0.511 19.4 0.068 2.56 0.00203 66.63 21.36 - - (8.44) (0.4) (0.027) (0.8) (0.008) (0.07) (0.00014) (1.31) (0.33) - -

EN 413.33 22.2 0.537 22.2 0.075 2.22 0.00311 67.69 21.59 3.42 18.12 (5.34) (0.2) (0.011) (0.3) (0.003) (0.05) (0.00014) (0.83) (0.21) (0.10) (0.19)

NN 398.72 22.8 0.428 20.7 0.065 2.29 0.00114 63.82 21.87 2.79 19.04 (4.14) (0.2) (0.010) (0.3) (0.003) (0.04) (0.00012) (0.66) (0.17) (0.09) (0.19) D = Wood Density (kg/m3), MFA = Microfibril Angle (degrees), FL = Fibre Length (mm), FW = Fibre Width (µm), CS = Coarseness (mg/m), S:G = Syringyl/Guaiacyl (ratio), VOL = Volume (m3), TC = Total Carbohydrate Concentration (%), TC = Total Lignin Concentration (%), SOL = Soluble Lignin Concentration (%), INSOL = Insoluble Lignin Concentration (%), CC = P. davidiana x P. davidiana, CN = P. tremuloides x P. davidiana, EE = P. tremula x P. tremula, EN = P. tremula x P. tremuloides, NN = P. tremuloides x P. tremuloides.