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ABOVEGROUND BIOMASS OF SEVEN HYBRID POPLARS

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6/01 cJClRCJDateOuopss-p. 1 5

THE EFFECT OF MECHANICAL PERTURBATION ON
THE CONDUCTIVITY, MECHANICAL STRENGTH, AND ABOVEGROUND
BIOMASS OF SEVEN HYBRID POPLARS
By

Kristine A. Kern

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Botany and Plant Pathology

2003

ABSTRACT
THE EFFECT OF MECHANICAL PERTURBATION ON THE
CONDUCTIVITY, MECHANICAL STRENGTH, AND ABOVEGROUND BIOMASS
OF SEVEN HYBRID POPLARS
By
Kristine A. Kern
To mimic the wind-induced back and forth motion of plants under greenhouse

conditions, mechanical perturbation, MP, (20 flexures per day) was applied to stems of
sapling poplar clones for 70-90 days. This study quantifies the effect of MP on the wood
properties of hydraulic conductivity and mechanical strength, and total aboveground
biomass for seven hybrids of a cross between Populus trichocarpa (black cottonwood)
and P. deltoides (eastern cottonwood). Mechanical perturbation of stems was expected to
decrease hydraulic conductivity (kh) and aboveground biomass, and increase mechanical
strength. Mechanical perturbation significantly decreased specific conductivity (ks or kh
per conductive xylem cross sectional area) and aboveground biomass. Flexural rigidity
(El), wood density, and xylem transverse area at point of flexure increased while
modulus of elasticity (MOE) and modulus of rupture (MOR) decreased in most of the
mechanically perturbed stems. Surprisingly, kh max was positively correlated with El
(P<0.0001, R2=0.507) with some hybrids having both great mechanical strength and
water conduction (e. g. Hybrid 19-61) while others were deficient in both properties.

Similarly, both RS and MOE were positively correlated with percent vessel lumen area

(P<0.0001 and P=0.002, respectively).

ACKNOWLEDGEMENTS

I would like to thank Dr. F. Ewers, my advisor, for his critical role in the
development and completion of this thesis. I would especially like to thank him for his
invaluable advice, encouragement, and humor. Special thanks to Dr. F. Telewski for the
opportunity to work with hybrid poplars and Drs. P. Murphy and R. Snider for their help
and encouragement during the writing process. Thanks to D. Freville and the Plant
Science Greenhouse staff for pesticide application and assistance with greenhouse
maintenance; Dr. B. Marks, for assistance with the Universal Instron Machine; Dr. B.
Cregg, for instruction in using the leaf area meter and determining wood specific gravity.

Many thanks to E. Steere for helping conduct the mechanical perturbation of 210
clones in the greenhouse during Spring 2002 and assisting with conductivity
measurements in the lab. I would also like to thank C. Woodrum for demonstrating the
use of the Sperry apparatus, Instron, and NIH Image as well as helping me enroll in
classes that first week and reminding me to dance once in awhile; W. Paddock for
willingness to discuss statistical analysis and helping me manipulate Excel; J. Kilgore
and MB. Collins for friendship, endless support, and abundant advice during this entire

process.

TABLE OF CONTENTS

List of Tables ...................................................... , .................................. v
List of Figures ....................................................................................... vi
Introduction .......................................................................................... 1
Materials and Methods ............................................................................ 12
Culture of the Plants ...................................................................... 12
Hydraulic Conductivity ................................................................... 13
Mechanical Properties .................................................................... 15
Aboveground Biomass ................................................................... 17
Stem Cross-Sections ..................................................................... 17
Data Analysis .............................................................................. 18
Results ............................................................................................... 20
General Morphology ..................................................................... 20
Hydraulic Conductivity .................................................................. 20
Mechanical Properties .................................................................... 21
Aboveground Biomass ................................................................... 22
Stem Cross-Sections ..................................................................... 22
Correlations ................................................................................ 23
Discussion .......................................................................................... 35
Summary of major findings ............................................................. 35
The mechanical perturbation (MP) treatment ......................................... 35
Positive correlations between mechanical and hydraulic properties ............... 42
Literature Cited .................................................................................... 47

iv

LIST OF TABLES

Table 1. General Morphology .................................. . .................................. 25
Table 2. Hydraulic Conductivity ................................................................ 26
Table 3. Mechanical Properties .................................................................. 27
Table 4. Stem Cross-Sections .................................................................... 28

LIST OF FIGURES

Figure 1. Maximum hydraulic conductivity (kl1 max) versus flexural rigidity (EI) ........ 29
Figure 2. Specific conductivity (ks) versus modulus of elasticity (MOE) ................. 29
Figure 3. Specific conductivity (ks) versus modulus of rupture (MOR) ................... 30
Figure 4. Maximum hydraulic conductivity (kh max) versus aboveground biomass ...... 30
Figure 5. Flexural rigidity (EI) versus aboveground biomass .............................. 31
Figure 6. Leaf area versus aboveground biomass ............................................. 31
Figure 7. Modulus of rupture (MOR) versus modulus of elasticity (MOE) ............... 32
Figure 8. Modulus of rupture (MOR) versus specific gravity of wood .................... 32
Figure 9. Specific conductivity (ks) versus vessel lumen area ............................... 33
Figure 10. Modulus of elasticity (MOE) versus vessel lumen area ........................ 33
Figure 11. Specific conductivity (ks) versus vessel frequency .......... . ................... 34

Figure 12. Modulus of elasticity (MOE) versus vessel frequency .......................... 34

vi

INTRODUCTION

In recent years poplar plantations have emerged as a new form of tree production,
occupying approximately 15,000 ha in the region between southern Oregon and British
Columbia (Heilman et a1. 1995). Washington State University Hybrid Poplar Research
Program reports there are currently 40,470 ha of hybrid poplar plantations in the Pacific
Northwest (Johnson and Ekuan 2002). Populus trichocarpa (black cottonwood) was
successfiilly hybridized with P. deltoides (eastern cottonwood) in 1971 at Washington
State University (Johnson 2000). Some of the first hybrids outgrew their parents by 20-
30% due to heterosis (Stettler et a1. 1996). Heterosis is the increased vigor or superior
quality of particular traits arising from crossbreeding genetically different plants or
animals (Allard 1960). Heterosis drives hybrid poplar breeding.

Hybrid poplars are a source of a wide range of wood products as well as
environmental protection and improvement (historically, windbreaks and erosion
control). Presently, the majority of plantations in the Pacific Northwest are used as fiber
for the pulp and paper industry (Zsuffa et a1. 1996). Interest in using hybrid poplar for
other wood products has increased over the years. Wood from hybrid poplar is now
utilized in products such as lumber, veneer and plywood, composite panels (e.g.
particleboard, fiberboard, waferboard), structural composite lumber (e. g. parallel strand
lumber, laminated strand lumber), pallets, furniture components, fruit baskets, containers,
and chopsticks (Balatinecz and Kretschmann 2001). In some cases hybrid poplars are
even used as biomass for energy, carbon sequestration, and phytoremediation of

environmental problems (Kuhn and Nuss 2000).

Populus trees establish easily and have rapid juvenile growth compared to other
tree species, advantages important to their successful use as windbreaks. Hybrids derived
from species adapted to riparian areas act as vegetation filters for use in stream
stabilization and in riparian buffers to protect and improve water quality, taking up excess
nutrients and chemicals moving from adjacent farmlands (Johnson 2000). For scientists,
Populus has rapidly become a model organism for the study of tree biology through the
coordinated collection and distribution of clones supported by
industry/government/academic partnerships, and funding from multi-agency grants
(Bradshaw et al. 2000). In fact, one of the standard materials that is most widely used by
researchers is a three-generation pedigree developed at the University of Washington
which includes one parent each of P. trichocarpa and P. deltoides, two of their F.
offspring, and an F 2 family (N = 379) derived from them (Stettler and Bradshaw 1996).
Additionally, Populus has become a model organism for the study of tree biology
becauSe our current understanding of secondary tissue development in woody
angiosperms is based on the detailed analysis of vascular development within the stem of
P. deltoides (Larson and Isebrands 1974, Larson 1979, Richards and Larson 1982,
Meicenheimer and Larson 1983).

Most Populus plantations are established on former agricultural lands, which are
often unsheltered and windy (Harrington and DeBell 1996). At harvest (6-8 yrs old),
commercial clones of hybrid poplars grown under intensive culture in plantations average
21 m in height and 25 cm in diameter (Johnson 2000). Understanding the effect of wind
stress on the wood properties of hybrid poplar is important for continuing to select

hybrids that improve the production of woody biomass for energy and fiber.

In nature, trees are constantly subjected to winds that apply external loads or
mechanical tension to stems and branches (Mattheck 1991). Temporary bending of tree
stems in response to wind stress or MP is considered an elastic strain and subsequently
induces the formation of flexure wood (Telewski 1989, 1995). In this case, reaction
wood is not formed because the displacement of the stem from its original vertical
position is not permanent. Instead, the stem oscillates back and forth, alternating between
compressional and tensional strains, until the motion diminishes completely and the stem
returns to the vertical position.

The most obvious effect of wind on trees in the field is decreased height growth
and increased radial growth. Jacobs (1954) found that free-swaying Pinus radiata
(Monterey pine) grew more in diameter over the lower part of the trunk than trees held by
guy wires to prevent wind sway and had increased eccentric trunk development along the
line of the main winds. In the greenhouse, using wind generated by oscillating electric
fans, Larson (1965) found that Larix Iaricina (tamarack) held upright with guy wires
grew much taller and thinner than their unrestricted neighbors.

Telewski and Jaffe (1986a) compared Abiesfraseri (Fraser fir) growing on
windward and leeward sites located along the Roan High Bluff ridge crest of Roan
Mountain, NC. They found significant changes in growth of trees on the windward sites:
longitudinal growth of seedling intemodes was inhibited, radial growth was reduced
along both major axes in the young leaders exposed to prevailing winds, and the cross-
sections of both saplings and mature trees were substantially elliptical. In another field
study, Cordero (1999) compared growth of C ecropia schreberiana saplings in two

different wind regimes (mean wind speed at windy sites was 32 times greater than at

protected sites) in an elfin cloud forest. He found that wind exposure caused significant
reductions in height, basal diameter, leaf area, and total biomass.

In the Cordero (1999) study, decreased basal diameter of wind-exposed stems was
the opposite of expected. Two observations may explain this discrepancy. Cordero
(1999) reported a significant increase in root to shoot ratio or greater investment in
anchorage in response to wind treatment. Decreased aboveground biomass may have
been great enough to reduce radial growth of wind-exposed stems. Additionally, the
relative size of the pith cavity in wind-protected stems and wind-exposed stems may have
affected the relative diameter of stems in the two treatments. C. schreberiana stems at
the windy sites probably had a greater proportion of parenchymatous tissues (less fibers
and vascular tissues) and a relatively greater pith, whereas the larger stems of the wind-
protected plants probably had greater proportions of vascular and supportive tissues, and
less pith (Cordero 1999).

I Damage from a Windstorm at a three-year-old Populus research trial revealed that
clonal differences in susceptibility to wind toppling or leaning were associated with both
above- and below-ground characteristics (Harrington and DeBell 1996). For example,
Hybrid 47-174, the clone most resistant to wind toppling (wind tolerant), had the lowest
aboveground woody biomass per unit of cross-sectional root area and highest rates of
lower stem taper. In comparison, Hybrid 11-11, the clone most susceptible to wind
damage (wind intolerant), had the highest aboveground woody biomass per mean root
area and lowest rates of lower stem taper.

Various plant responses to wind occur simultaneously, making it difficult to

isolate their individual effects on plant growth (Biddington 1986, Telewski 1995). The

wind-induced back and forth motion of plants has been simulated in the laboratory by
mechanical perturbation (MP) including rubbing, vibrating, shaking, or flexing of stems
(Neel and Harris 1971, Jaffe 1976, Telewski and Jaffe 1986a, Niklas 1998, Cipollini
1999, Pruyn et al. 2000). Shaking of Liquidambar styraciflua (sweet gum) trunks
resulted in decreased intemode length, fewer nodes, and a 20% decrease in height growth
(Neel and Harris 1971). Jaffe (1976) found that rubbing the first true intemode of
Phaseolus vulgaris (beans) retarded stem elongation (due to decreased intemode length)
and flower production, but increased intemode diameter. F lexure of Pinus taeda
(loblolly pine) inhibited stem and needle elongation, bracing of branch nodes, and
increased radial growth in the direction of MP (Telewski and Jaffe 1986b). The vibration
of Capsella bursa-pastoris (shepard’s purse) resulted in more massive root systems and
less massive vegetative shoots, reduced dry weight of reproductive structures at maturity,
delayed formation of first mature flower and fruit, and accelerated onset of plant
senescence (N iklas 1998). Cipollini (1999) found that flexing Brassica napus
(Cruciferae) reduced plant height and root mass; increased stem diameter while
decreasing pith diameter; delayed both cotyledon senescence and date of anthesis;
reduced flower number, leaf area, and aboveground biomass. Pruyn et al. (2000) found
that flexing Populus hybrids increased radial growth especially in the direction of MP
and decreased the height to diameter growth ratio.

The morphological and physiological responses of plants to MP are collectively
termed thigrnomorphogenesis (J affe 1976). Several authors have reviewed the effects of
thigrnomorphogenesis on plants: Biddington (1986), Jaffe and Forbes (1993), Telewski

(1995), and Jaffe et al. (2002). The total effect of flexure stress on tree growth and

development (thigmomorphogenesis) results in a more compact growth form including
greater stem taper, shorter branches, and smaller leaves (Telewski 1995).

Mechanical perturbation of stems in the laboratory produces growth
characteristics that mimic the growth of plants exposed to wind in the field. In 1980,

J affe published a photograph showing that hand-rubbed beans grown in the greenhouse
and untreated beans exposed to wind were stunted to a similar extent when compared to
untreated controls grown in the greenhouse. Telewski and Jaffe (1986a) reported that
flexure of greenhouse-grown A. fiaseri reduced extension growth, increased stem
diameter, and produced an elliptical stem cross-section, characteristics that were all
present in field grown A. fraseri exposed to high velocity winds.

Clearly wind stress affects the development of wood (secondary xylem) in trees
(Jacobs 1954, Larson 1965, Telewski and Jaffe 1986a, Pruyn et al. 2000). Since wood
functions as mechanical support in trees, the mechanical properties of wood are also
subject to change. Responses of both field-grown and greenhouse-grown A. fraseri to
MP included increased flexural rigidity (E1) or a stronger stem, and decreased modulus of
elasticity (MOE) or more flexible stem tissue (Telewski and Jaffe 1986a). Pruyn et al.
(2000) reported similar results for two hybrid poplars: EI increased 31% and MOE
decreased 14% in MP clones compared to controls. F lexural rigidity is a measure of the
rigidity of the whole stem while MOE is a measure of the elasticity per unit stem cross-
sectional area. Mechanical perturbation of P. taeda decreased flexibility and increased
elasticity and plasticity of the stem (Telewski and Jaffe 1986b). Using a different
approach, Holbrook and Putz (1989) examined the biomechanics of four-year-old L.

styraciflua after the seedlings were prevented from wind sway for two years in the field.

They found that whole-tree flexibility (a parameter similar to El) and MOE tended to
decrease, although not significantly, in free swaying trees compared to constrained trees.

Many of the responses observed in wind-stressed or mechanically perturbed trees
are examples of developmental acclimation, resulting in altered biomass allocation that
enables trees to withstand bending or swaying stresses by reducing drag and increasing
mechanical strength (Telewski and Jaffe 1986a). However, the function of wood is not
solely mechanical support. A second important function of wood is the long distance
transport of water from roots to leaves (water conduction).

In gymnosperrns a single cell type, the tracheid, is responsible for both water
conduction and mechanical support. Spicer and Gartner (2002) bent seedlings of
Pseudotsuga menziesii (Douglas-fir) such that the angle of the stem was 25° or 50° from
vertical during their entire fourth growing season to assess the impact of compression
wood (CW) formation on whole-plant hydraulic properties. Compression wood is the
type of reaction wood formed on the lower sides of inclined stems and branches of
gymnosperms (Esau 1977). Spicer and Gartner (2002) found that %CW increased and
specific conductivity (ks, conductivity normalized for sapwood area) decreased in
seedlings induced to form CW. Compression wood shares some of the characteristics of
flexure wood: lower MOE, greater density, and shorter, narrower tracheids (Telewski
and Jaffe 1986a, Timell 1986, Telewski 1989). Compression wood of P. menziesii is
expected to have tracheids that are shorter and narrower, one explanation for the decrease
in ks. Telewski (1989) concluded that wood formed under the influence of flexural stress
has a morphology and function more closely related to compression wood than normal

wood despite several differences (i.e. flexure wood tracheids differ from CW tracheids in

that they are not rounded with a thicker 82 cell wall layer and intercellular spaces). For
this reason, the formation of flexure wood in trees may also decrease ks. Despite this
tradeoff, Spicer and Gartner (2002) found that the water potential and stomatal
conductance of inclined seedlings remained similar to controls indicating that even a
severe reduction in stem ks caused by compression wood had little impact on leaf-level
processes.

The question of a trade-off between the functions of mechanical support and
water conduction may also be applied to woody angiosperms. With increasing
specialization, trees with conducting elements that were more efficient in conduction than
in support evolved (Esau 1977). As a result, two different cell types in woody
angiosperms are specialized to function in mechanical support (fibers) and water
conduction (vessel elements). For example the xylem of poplar is a complex
combination of cell types including vessel elements (28-34%), fibers (53-60%), and axial
(0. 1 -0.3%) and ray parenchyma (1 1-14%) (Balatinecz and Kretschmann 2001).

Preliminary work on Quercus ilex (holly oak) showed that inclined seedlings had
the same ks as vertical controls, suggesting an effective decoupling of transport and
support functions in angiosperms (Spicer and Gartner 2002). In the case of these inclined
seedlings, tension wood, the type of reaction wood formed on the upper sides of inclined
stems and branches of arboreal dicotyledons, is expected to form and not flexure wood.
The anatomical characteristics of flexure wood in angiosperms are not as extensively
documented as in gymnosperrns, however, MP of L. styraciflua showed a decrease in
fiber length and that vessel members decreased in length and diameter (Neel and Harris

1971). In addition, Pruyn (1997) showed that vessel diameter and frequency decreased in

the flexure wood of two hybrid poplars. Given that vessel diameter and frequency
decreased, conductivity is likely to decrease in woody angiosperm stems subjected to
MP.

Despite this information, the relationship between the mechanical strength of
wood and conductivity has received little study. In one study, Wagner et al. (1998)
reported an inverse relationship between the efficiency of water transport and the
mechanical strength of wood when comparing closely related chaparral shrubs growing
in the same habitat. Greater vessel lumen area and two-fold greater ks corresponded to
lower stem density, MOE, and modulus of rupture (MOR). However, in another study
the lack of correlation among xylem specific conductivity and vessel diameter with MOR
and xylem implosion resistance was reported for 22 species of chaparral shrubs,
suggesting the mechanical properties of chaparral xylem have little impact on water
transport efficiency (Davis et al. 2002).

Similarly, Woodrum et al. (2003) assessed hydraulic conductivity and mechanical
properties of lateral, woody branches of five species of Acer (Aceraceae) and found no
trade-off between ks max and MOE or MOR. However, fiber lumen diameter was
inversely correlated to MOE and MOR suggesting that differences in mechanical
properties may be due to fiber lumen differences that do not influence the efficient
transport of water.

The relationship between conductivity and mechanical support, the two primary
functions of wood, must be considered in order to optimally select hybrid poplar clones
that continue to improve the production of woody biomass for energy and fiber. Poplar

hybrids continue to be developed via selection in breeding programs and by genetic

engineering. Studying the effect of MP on conductive capacity, mechanical strength, and
aboveground biomass of greenhouse grown hybrid poplars is one step towards
understanding the dynamic effect of wind stress on hybrid poplars grown in plantation.

The present study quantifies the effect of MP on hydraulic conductivity and
mechanical strength, two of the primary functions of wood, and aboveground biomass for
seven hybrids of a cross between P. trichocarpa and P. deltoides. This study expands
upon an investigation conducted by Pruyn et al. (2000) in which the mechanical
responses of two poplar hybrids (Hybrid 11-11, characterized as wind intolerant; 47-174,
characterized as wind tolerant) subjected to MP were reported. Mechanical perturbation
increased EI and decreased MOE, in effect reducing susceptibility of both clones to wind
damage. In the present study, one of the hybrids, Hybrid 19-61, is a known tri/aneuploid
(Bradshaw and Stettler 1993) while the other six hybrids are diploid. Attractive features
of triploids have been their greater volume growth and longer, wider fibers (Stettler et al.
1996). Differences in conductivity, mechanical properties, and aboveground biomass
among hybrids are reported. Thin cross-sections of stems are analyzed to determine
whether there is an anatomical explanation for differences in conductivity and
mechanical properties due to MP. Given current knowledge, the following predictions
are made for greenhouse grown clones subjected to MP or control treatments:

1. Mechanical perturbation will alter biomass allocation of all clones, resulting in
decreased height growth of the leader stems, increased radial growth of stems at point of
flexure, and reduced leaf area.

2. Mechanical perturbation will decrease conductivity, increase mechanical

strength, and decrease aboveground biomass of all clones.

3. Different hybrids will vary significantly in conductivity, mechanical
properties, and aboveground biomass allocation.

4. Conductivity will be inversely proportional to mechanical strength of controls
and clones subjected to MP.

5. Aboveground biomass will be proportional to conductivity and inversely
proportional to mechanical strength of controls and clones subjected to MP.

6. Percent vessel lumen area will be positively correlated with conductivity.

ll

MATERIALS AND METHODS

Culture of the Plants

Seven F 1 hybrids of Populus trichocarpa x P. deltoides (T x D) were used in this
study: 11-11, 11-5, 46-158, 49-177, 47-174, 55-260, and 19-61. These hybrids were
originally developed as part of a joint hybrid poplar breeding program between the
University of Washington and Washington State University. Hybrids 11-11 and 47-174
have been planted extensively in commercial plantations in western Oregon and
Washington (DeBelI et al. 2002). Stem cuttings of the trees came from several sources:
the breeding program’s field station in Puyallup, Washington, the USDA Forest Service
in Olympia, Washington, and the Fort James Corporation. These hybrids have been
cultivated at the Plant Science Greenhouses at Michigan State University since 1994,
with photoperiod ranging from 12 to 14 hr and diurnal temperature maintained at
approximately 25°C.

In December 2001 all of the trees were cut back to force new shoot growth. In
January 2002 leader stems of uniform height and stem width were selected on all trees.
Each leader stem was then staked. The trees were grown for 4 wk before treatment
began.

Thirty trees of each hybrid were arranged in a complete randomized block design
on five benches in the greenhouse. Fifteen trees of each hybrid were mechanically
perturbed (20 flexures back and forth along the same plane) once daily from February 18
until May 8, 2002. The remaining 15 were controls (without mechanical perturbation).

A location just above the fourth node from the base of the stem was designated as the

12

point of flexure. The point of flexure is the place on the stem that is grasped with the

thumb and forefinger of one hand while the other hand is used to flex the stem by

grasping it approximately 10 cm above the flexure point. Flexing did not exceed 45°.
Fertilizer (20-20-20 N-P-K, 7.5 g L") was applied to each individual tree three

times between December 2001 and April 2002. During the experiment, Mavrik® (0.6 ml

L'l H20) was applied once and Sanmite® (0.3 ml L'l H20) twice to control spider mites.
To lower soil pH, 1.0 L of a sulfuric acid solution (0.1 ml H2804 L" H20) was applied to
each pot in February and March 2002.

Before and after the experiment, the height of the leader stem and diameter of the
stem at the point of flexure were recorded. Diameter was measured with a digital caliper
sensitive to 0.1 mm, in the direction of flexure (parallel to flexure) as well as

perpendicular to flexure.

Hydraulic Conductivity

Sampling was completed over a 3 wk period, April 18 to May 8, 2002. A single
stem segment was taken from each healthy tree (166 of 210 trees sampled). Using plastic
water-filled bags to wrap around the stem, the stem was cut 15 cm below the point of
flexure while locally submerged, to prevent the introduction of air embolism and re-cut
20 cm above the point of flexure while under water. The submerged stern segments were
transported to the laboratory in a bucket of water. The stem portion distal to the cut
segment was prepared for measurement of leaf area and oven dry weight.

To ensure a fresh-cut surface, the stem segments were re-cut under water in the

laboratory such that the point of flexure was 5 cm above the base of the segment and 10

cm below the top. Hydraulic conductivity per unit pressure gradient (kh, kg m MPa'1 5")
was measured as described by Sperry et al. (1988). To discourage microbial growth,
degassed 10 mMol citric acid was run through a 0.2 pm Gelman filter and allowed to
flow via gravity from a known height through tubing connected to the distal end of the
stem segment. Volumetric flow rate was determined by recording the time required for
0.1 g of citric acid to flow through the stem segment and collect in a beaker on an
analytic balance (model BP 1215, Sartorius AG Gottingen Germany).

Emboli were removed from the stem segments via perfusion with citric acid at
175 kPa for 15 min. Hydraulic conductivity measurements and perfusion were alternated
until the rate of flow remained constant (less than a 10% difference observed) between
consecutive trials for a given stem segment. Conductive vessels were stained using 0.5%
Crystal Violet immediately following measurements of conductivity. Stem segments
were stored in plastic bags and refrigerated until mechanical properties were measured 2
wks later.

Hydraulic conductivity (initial and maximum) were calculated using the equation:

kh = F/(dP/dx) (Tyree and Ewers 1991)
where F is the water flux (kg s") and dP/dx is the pressure difference causing the flow
(MPa m'l). Specific conductivity was calculated as described by Tyree and Ewers (1991)
using the equation:

kS = kh max / AS
where As is the conductive xylem cross-section area (m2). The degree of embolism in the
stem segment was calculated using the initial conductivity as a percentage of the

maximum afier emboli were removed via the equation:

% embolism = {(kh max — kh initial) / kh max} * 100%
A subsample of eight trees of each hybrid, four control and four treatment, were
randomly selected to measure the leaf area distal to each segment using a LI-COR area
meter (model LI-3100, Ll-COR Inc., Lincoln, NE). The leaves of all trees were oven
dried at 45°C to a constant weight. Leaf area of the remaining trees was estimated by
linear regression using equations derived from plots of leaf area and oven dry leaf weight.
The total leaf area distal to the segment was calculated for each segment and used to
calculate leaf specific conductivity using the equation:

LSC = kh / A; (Tyree and Ewers 1991)
where Al is the total leaf area (m2). In cases where leaves were present on the 15 cm
segment, 50% of this leaf area was included in the LSC calculation (Ewers et al. 1989).
The Huber value, defined as:

HV = A5 / A. (Tyree and Ewers 1991) .
was also calculated so any difference in LSC could be attributed to differences inherent to

the wood.

Mechanical Properties

The 15 cm stem segments used to measure hydraulic conductivity were also used
to measure mechanical properties: flexural rigidity (El, N mmz), second moment of cross-
sectional area (I, mm4), modulus of elasticity (MOE, N mm'z), modulus of rupture (MOR,
N mm'z), and the specific gravity of wood at green volume (sp gr). Segments were kept
at approximately 10°C until testing with an Instron Universal Machine (model 4202,

Instron Corporation, Canton MA) at the Department of Agricultural Engineering at

15

Michigan State University. A four-point bending test with a compression load cell of 500
N was conducted as described by Pruyn et al. (2000). The span length (L), the distance
between the two supported ends, was 135 mm. The distance (a) between one supported
end and the nearest load (F) was equal to 45 mm. The load was applied at two points
along the span length at a crosshead speed of 20 mm/min. Stress versus strain data were
collected every 0.1 m using a computer with Cy4200 software networked to the Instron.
Stem segments were deflected until the load reached a maximum value, known as
the critical strain, reaching the limit of the elastic range (Spatz and Bruechert 2000).
Stem segments did not rupture under stress due to the juvenility of the wood. However,
after testing the stem segments did not return to their original shape either, indicating the
elastic limit had been exceeded. The modulus of rupture (MOR) was estimated using the
load value at the asymptote of the curve and the equation:
MOR = (F max*a*Rmajor)/I (modified from Ugural 1991)
where Fmax is the load at stem failure, Rmajo, is the major radius of the stem segment
minus the pith, and I is the second moment of cross sectional area for a hollow ellipse:
I = («*Rmajo,.Rmi,.,,3)/4 (Wainwright et al. 1982)
Maximum and minimum stem diameters were measured with a digital caliper at three
points along the stem segment (at the center and 2 cm from each end) to determine mean
maximum and minimum radii, Rmajo, and Rminor, as stems were elliptical in cross-section.
To calculate the second moment of cross sectional area (I), pith radius was measured at
the center of the stem segment, raised to the 4‘h power, and subtracted from Rmajor*R'ninor3.
Flexural rigidity (E1) was calculated using the slope (F/V) of the linear portion

(elastic part) of the curve and the equation:

16

E1 = (F/V)(a2/12)(3L-4a) (modified from Gere and Timoshenko 1997)

F lexural rigidity was divided by second moment of cross sectional area to calculate the
modulus of elasticity (MOE) (Pruyn et al. 2000).

Specific gravity (sp gr) of wood at green volume was calculated using the
equation given by Panshin and de Zeeuw (1980): sp gr = (oven dry weight of
wood)/(weight of displaced volume of water using green wood). A subsample from each
segment was taken by cutting at the point of flexure and 3.5 cm above the point of
flexure. Stem diameter, xylem diameter, and pith diameter were measured with a digital
caliper before the cortex was removed from each subsample. Each subsample was
submerged in a 10 ml graduated cylinder of distilled water and the displacement of water

was recorded. The subsamples were oven dried at 45°C to a constant weight.

Aboveground Biomass
Stems and leaves from the apex of each tree to 5 cm below the point of flexure

were oven dried at 45°C to a constant weight.

Stem Cross-Sections

Sixteen stems of Hybrids 46-158 and 19-61 (eight control and eight MP) were
used for analysis of anatomical differences due to flexure treatment. Hybrids 46-148 and
19-61 were chosen because their mean ks and MOE showed the greatest response to
flexure treatment. Additionally, Hybrid 46-148 is known to be diploid while Hybrid 19-

61 is triploid.

Transverse sections were taken approximately 3.5 cm above the point of flexure
on each stem segment. Thin sections (approximately 40 pm thick) were made using a
sliding microtome. Sections were subjected to a dehydration series of ethanol and xylene
(modified from Johansen 1940) and then mounted on slides using Permount.

A light microscope interfaced with a CCD video camera and multi-scan analog
monitor was used to analyze the sections. NIH Image 1.5 sofiware was used to measure
vessel lumen area per unit area of wood. Two areas were chosen on both sides of the pith
along the same axis in all stems. In mechanically perturbed stems this axis was in the
direction of flexure. The total area per stem scanned was 0.240 mmz. Vessel lumen
areas of each stem were used to calculate percent vessel lumen area, average and
maximum vessel lumen diameter, hydraulic diameter, and vessel frequency. Hydraulic
diameter was calculated using the equation:

HD = Eds/Ed“ (Sperry et al. 1994)

where d is the diameter of the vessel lumen.

Data Analysis

All data were analyzed using SAS Version 8.1. Analysis of variance for seven
hybrids and two treatments (control and MP) was conducted for each variable using two-
way ANOVAs to show the main effect of each factor. Tukey-Kramer was used for
multiple comparisons among means of hybrids to identify which hybrids were
significantly different from each other. The specific effect of treatment (control and MP)
within each hybrid was conducted by slicing to identify which hybrids had a significant

difference between control and MP. Some variables were transformed before analysis of

18

variance because the data were not normally distributed according to at least 3 out of 4
tests for normality (Shapiro-Wilkinson, Kolmogorov-Smimov, Cramer-von Mises, and
Anderson-Darling). Variables transformed using square root were leaf area, kh max, ks,
LSC, aboveground biomass, sp gr, EI, and MOE. HV and kh initial were transformed using
logm. Regression analysis was used to determine correlations between variables.

Exce12000 was used to graph means and plot trendlines.

l9

RESULTS

General Morphology

Multiple comparisons among means showed only Hybrids 11-5 and 47-174
differed significantly (P=0.01) with respect to height growth (Table 1). Hybrid 47-174
had greater height grth (125.5 :t 7.0 cm) than 11-5 (90.6 :I: 7.0 cm). None of the
hybrids differed significantly in the change of transverse area of stem at point of flexure
or leaf area distal to stem segments. Hybrid 46-158 had the greatest Huber value.

Mechanical perturbation had a significant effect on several morphological
characteristics (Table 1). It decreased the mean height growth of all hybrids (P<0.0001)
and increased the change in transverse area of stem at point of flexure (P<0.0001). With
the exception of Hybrid 49-177, leaf area distal to stem segments generally decreased
(P=0.04) when clones were mechanically perturbed. Huber value (P<0.0001) increased

in all hybrids with clones subjected to MP.

Hydraulic Conductivity

Multiple comparisons among means showed some hybrids inherently differed in
conductive capacity on a whole stem basis, k2. initial and kh max, or on a tissue area basis, ks
(Table 2). Hybrid 19-61, a known triploid, had significantly higher kh initial, kh max, and k5
than several of the diploid hybrids. Hybrids 47-174 and 19—61 had lower percent
embolism than Hybrids 11-11, 11-5, and 46-158 (P<0.0001). Hybrid 47-174 had the

lowest LSC while LSC in Hybrids 46-158, 49-177, and 19-61 were significantly greater.

20

Mechanical perturbation of clones significantly decreased kS (P<0.0001) when
compared to controls (Table 2). Mechanical perturbation did not have a significant effect
on k}, initial, kh max, ks, percent embolism, or LSC. However, kn max tended to decrease in

mechanically perturbed clones with the exception of Hybrid 49-177.

Mechanical Properties

Mechanical properties on a whole stem basis (EI) and a tissue area basis (MOE,
MOR) are shown in Table 3. Multiple comparisons among means showed that some
hybrids differed significantly. Hybrid 19-61 had the highest EI, while E1 of 49-177 was
significantly lower (P=0.04). Hybrid 19-61 had the greatest MOE and MOR. Compared
to Hybrid 19-61, MOE of Hybrids 11-11, 46—158, and 49-177 were significantly lower as
was MOR of Hybrids 11-5, 46-158, 49-177, and 55-260. Hybrids with low mean specific
gravity included 11-5 and 49-177 while Hybrids 47-174 and 19-61 had significantly
greater wood specific gravity. Second moment of cross sectional area (I) did not
significantly differ among hybrids.

Flexural rigidity (P=0.01) increased in clones subjected to MP, except in Hybrid
47-174. MOE (P<0.0001) and MOR (P=0.0006) decreased with MP with one exception:
MOR of Hybrid 49-177 was not affected by flexure (80.14 3: 4.32 N mm'2 for control and
80.34 :t 2.82 N mm'2 for MP). Wood specific gravity at green volume significantly
increased (P<0.0001) in clones subjected to MP compared to controls. Mechanical

perturbation significantly increased second moment of cross sectional area (P<0.0001).

21

Aboveground Biomass
Statistically, all seven hybrids were similar in aboveground biomass accumulation
(Table 1). However, clones of all hybrids showed decreased aboveground biomass

accumulation when subjected to MP (P=0.04).

Stem Cross-Sections

Analysis of vessels from stem cross-sections of Hybrids 46-158 and 19-61
(hybrids that showed the most pronounced response to MP in terms of k5 and MOE) are
shown in Table 4. When comparing hybrid means, Hybrid 46-158 had lower vessel
lumen area than 19-61 (P=0.0003). Hybrid 46-158 had higher VDalvg than Hybrid 19-61
(P<0.0001). There was no significant difference between hybrids for VDmax, HD, and
vessel frequency.

Mechanical perturbation of clones decreased vessel lumen area, VDavg, VDmax,
HD, and vessel frequency compared to controls for both hybrids; however, there was a
significant Hybrid x MP interaction for each variable except vessel frequency.
Mechanical perturbation decreased lumen area by 38% for Hybrid 46-158 and only 22%
for Hybrid 19-61. Mechanical perturbation decreased VDavg by 49% for Hybrid 46-158
and only 19% for Hybrid 19-61, but the average vessel diameters were similar for
mechanically perturbed clones of both hybrids. Mechanical perturbation decreased
VDmax by 31% for Hybrid 46-158 and only 9% for Hybrid 19-61. Maximum vessel
diameters of mechanically perturbed clones of Hybrid 19-61 were 23% wider than
Hybrid 46-158. Mechanical perturbation decreased HD by 29% for Hybrid 46-158 and

only 6% for Hybrid 19-61. Hydraulic diameter of mechanically perturbed clones of

22

Hybrid 19-61 was 22% greater than Hybrid 46-158. Mechanical perturbation of clones

significantly decreased (P=0.05) vessel frequency of both hybrids.

Correlations

Contrary to the hypothesis, conductivity was not inversely proportional to
mechanical strength but, instead, positively correlated to mechanical strength. At the
whole stern level, kh max showed a significant positive correlation with E1 (P<0.0001,
Figure 1). This relationship was also evident at a per unit xylem level as kS was
positively correlated with MOE (P=0.02, Figure 2) and MOR (P<0.0001, Figure 3).
When Hybrid 19-61, the triploid hybrid that exhibited great conductive capacity and
mechanical strength, was removed from the regression analysis, the correlation between
khmax and EI (P<0.0001) remained significantly positive, as did the correlation between kS
and MOR (P=0.0004). However, when Hybrid 19-61 was removed from regression
analysis for ks and MOE, no correlation existed (P=0.4).

Aboveground biomass was positively correlated with kh max (P<0.0001, Figure 4),
El (P<0.0001, Figure 5), and leaf area (P<0.0001, Figure 6). Modulus of rupture was
positively correlated with MOE (P<0.0001, Figure 7) and wood specific gravity
(P<0.0001, data not shown). There was a significant positive correlation between MOR
and wood specific gravity of the controls (P=0.009, Figure 8) but this relationship was
not observed in the clones subjected to MP. Modulus of elasticity was not correlated
with wood specific gravity (P=0.4, R2=0.0048, data not shown).

Analysis of vessel anatomy of Hybrids 46-158 and l9-6l showed ks and vessel

lumen area were positively correlated (P<0.0001, Figure 9) as were MOE and vessel

23

lumen area (P=0.002, Figure 10). On the other hand, EI tended to decrease as vessel
lumen area increased but this was not a significant negative correlation (P=0.9,
R2=0.0012, data not shown).

Specific conductivity tended to increase when vessel frequency increased but this
was not a significant positive correlation (P=0.3, Figure 11); however, MOE and vessel
frequency were positively correlated (P=0.001, Figure 12). Average vessel lumen
diameter did not significantly correlate with ks, kh max, El, MOE, or MOR (data not

shown).

24

 

 

 

 

 

 

 

 

 

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Figure 2. Specific conductivity (ks) versus modulus of elasticity (MOE). Means of each
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horizontal bars are 1 SE of MOE.

29

 

 

 

 

 

 

 

 

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Figure 3. Specific conductivity (ks) versus modulus of rupture (MOR). Means of each
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Figure 4. Maximum hydraulic conductivity (kh max) versus aboveground biomass. Means
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30

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 5. Flexural rigidity (E1) versus aboveground biomass. Means of each hybrid are
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Figure 6. Leaf area versus aboveground biomass. Means of each hybrid are shown as
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31

 

 

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Figure 7. Modulus of rupture (MOR) versus modulus of elasticity (MOE). Means of each

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Figure 8. Modulus of rupture (MOR) versus specific gravity of wood. Means of each
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horizontal bars are 1 SE of specific gravity of wood.

32

 

 

 

 

 

 

 

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Figure 9. Specific conductivity (ks) versus vessel lumen area. I = Hybrid 46-158
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N=32. Vertical bars are 1 SE of RS and horizontal bars are 1 SE of vessel lumen area.

 

 

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Figure 10. Modulus of elasticity (MOE) versus vessel lumen area. I = Hybrid 46-158
Control; 1:: = Hybrid 19-61 Control; A = Hybrid 46-158 MP; A = Hybrid 19-61 MP;
N=32. Vertical bars are 1 SE of MOE and horizontal bars are 1 SE of vessel lumen area.

33

 

 

 

   

 

 

 

 

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Vessel Frequency (mm'z)

 

 

 

Figure 11. Specific conductivity (k5) versus vessel frequency. I = Hybrid 46-158
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N=32. Vertical bars are 1 SE of kS and horizontal bars are 1 SE of vessel frequency.

 

 

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1500 5 . ‘
110 120 130 140 150 160 170 180 190 200
Vessel Frequency (mm'z)

 

 

 

Figure 12. Modulus of elasticity (MOE) versus vessel frequency. I = Hybrid 46-158
Control; D = Hybrid 19-61 Control; A = Hybrid 46-158 MP; A = Hybrid 19-61 MP;
N=32. Vertical bars are 1 SE of MOE and horizontal bars are 1 SE of vessel frequency.

34

DISCUSSION

Summary of major findings

The allocation of biomass of these seven hybrid poplars in response to mechanical
perturbation (MP) was consistent with results from the literature as detailed in the
discussion below: height grth and leaf area decreased, and radial growth increased.
Mechanical perturbation of clones increased the mechanical strength on a whole stem
basis, another result consistent with the literature. Previously unknown, specific
conductivity (ks) of stems decreased when subjected to MP. Total aboveground biomass
also decreased with MP. Despite decreased conductivity and increased mechanical
strength when subjected to MP, overall, maximum conductance (kh max) was positively
correlated with flexural rigidity (EI) and percent vessel lumen area, suggesting some
hybrids have superior conductivity and mechanical strength. As expected hybrids varied
in conductivity and mechanical strength, but not total aboveground biomass, suggesting
there is an opportunity to select clones with superior mechanical strength and

conductivity.

The mechanical perturbation (MP) treatment

The present results support thigmomorphogenetic theory: radial growth
increased, stem elongation decreased and leaf area decreased with MP (Neel and Harris
1971, Jaffe 1976, Telewski and Jaffe 1986a, Telewski 1995). The total effect of MP on
tree growth and development resulted in a more compact growth form. Telewski (1995)

described how this ultimately reduces the speed-specific drag of the crown and maintains

35

geometric similarity between the stem diameter and height growth, producing a greater
margin of safety against mechanical failure under windy conditions.

Radial growth increased with MP, a result consistent with Hybrids 47-174 and 11-
1 1 studied by Pruyn et al. (2000). The effect of MP on stem elongation of Hybrids 47-
174 and 11-11 was significant in the present study. Pruyn et al. (2000) found that
elongation of stems subjected to MP was decreased in Hybrid 11-11 and increased in
Hybrid 47-174; however, neither was significantly different from controls. One
explanation for this discrepancy may be that significantly more growth occurred in the
70-90 day MP treatment of this study than Pruyn et al. (2000) found in a 60-day
treatment. It is also possible that the time of year the studies were conducted, despite
being grown in the greenhouse, influenced the grth rate as well. This study was
conducted March through May while the study by Pruyn et al. (2000) was conducted in
July and August, the hottest months in a greenhouse in Michigan and the end of the
growing season.

Mean leaf area per tree decreased for each hybrid poplar when subjected to MP, a
result consistent with Telewski and Jaffe (1986a), Telewski and Pruyn (1998), Cipollini
(1999), and Cordero (1999). Net photosynthesis was not measured in this study, however
Ferree and Hall (1981) reported a decrease in net photosynthesis and transpiration in
response to MP. It is possible MP decreased the net production of photosynthate in these
clones in addition to altering biomass allocation. Clearly, total aboveground biomass and
leaf area were positively correlated in this study.

External loading forces such as wind must be transferred down the stem to the

roots and then into the ground in order to prevent mechanical failure of the tree (Stokes

36

and Mattheck 1996). Examining the effect of MP on tree root development was not
addressed in this study, however it may be useful since reduction of wind sway and MP
modifies secondary growth in lateral roots (Jacobs 1954, F ayle 1976, Stokes et al. 1995,
Stokes et al. 1997). Jacobs (1954) found that sway increased the diameter grth of
roots as far as two feet from the base of the trunk of Pinus radiata (Monterey pine).
Stokes et al. (1995) found that although changes in shoot growth were small, significant
differences were found in root growth between wind-stressed and control Picea sitchensis
(Sitka spruce) and Larix decidua (European larch). Wind-stressed trees of both species
increased almost 60% in the number of roots important for anchorage on the windward
side and in L. decidua this also occurred on the leeward side. Stokes et al. (1997) showed
that flexing of P. sitchensis caused significant increases in: course root mass, course root
to fine root ratio, total root to shoot ratio, mean cross-sectional root area along the axis of
flexing, and the incidence of coarse branches.

These studies all examined the root growth of gymnosperrns and concluded the
alteration of biomass to the roots should improve the anchorage of trees subjected to wind
movement. Studies that examine the effect of MP or wind on root growth of hybrid
poplars are lacking in the literature, however, the structure and function of Papulus root
systems is reviewed in Pregitzer and Friend (1996) and several studies of differences
among clones in relative quantity of carbon allocation to root systems are reviewed in
Hinckley et al. (1989).

The alteration of biomass due to MP resulted in clones that were shorter and had
thicker stems. These physical changes affected the mechanical properties of the stems.

Flexural rigidity increased in clones subjected to MP while modulus of elasticity (MOE)

37

and modulus of rupture (MOR) decreased. These results are consistent with Telewski
and Jaffe (1986a, 1986b), and Pruyn et al. (2000). Flexural rigidity is the product of
MOE and the second moment of cross-sectional area, which scales with stem radius to
the fourth power. In this case, the increased radial growth of clones subjected to MP had
a greater influence on the calculation of El than the decrease in MOE which resulted in
greater rigidity of the whole stem. Ecologically, the rigidity of the entire stem is
important in reducing the susceptibility of the stem to mechanical failure due to wind
stress.

Different mechanical results were reported for Liquidambar styraciflua (sweet
gum) grown under field conditions for 2 years (Holbrook and Putz 1989). Free swaying
trees were shorter, increased in diameter, and tended to decrease in MOE. However,
whole-tree flexibility (expressed as radians N!) of free swaying trees was not
significantly different from that of constrained trees.

V Differences in cell wall composition, the presence of lignin and the angle of the
microfibrils, may explain the range of MOE values of the seven hybrid poplars and the
relative strength of the wood. The fibers of tension wood typically show the replacement
of either the S; or 83 layer by an unlignified layer which in section has a translucent
swollen and gelatinous appearance (Wardrop 1964, Panshin and de Zeeuw 1980). This
unlignified layer is termed the gelatinous (G) layer and is primarily composed of
cellulose.

The presence of gelatinous fibers and lignin content were not examined in the
present study, however, Pruyn (1997) found gelatinous fibers and observed lignin in the

fiber, vessel, and ray cell walls of control and MP stems of Hybrids 47-174 and 11-11.

38

Pruyn (1997) also found that total ligninthioglycolic acid (LTGA lignin) decreased in MP
stems. When young shoots of Populus eramericana cv ‘Ghoy’ were inclined to induce
the formation of tension and opposite wood, the formation of gelatinous fibers on the
upper face of the stem was observed (Jourez and Avella-Shaw 2003). Tension wood is
the reaction wood formed on the upper sides of branches or leaning stems in
dicotyledonous angiosperms and is characterized by the presence of gelatinous fibers,
which have lower lignin and higher cellulose content compared to normal fibers
(Telewski et al. 1996). In a study of tension wood in Magnolia abovata and M. kobus
which are considered to be primitive angiosperms, the amounts of lignin decreased in the
cell walls of fiber tracheids, especially with great decrease in proportion of guaiacyl units
in lignins (Yoshizawa et al. 2000). Pruyn (1997) concluded that flexure wood shared
some characteristics of tension wood since lignin content also decreased with MP.
Mattheck and Kubler (1995) proposed that the decrease in lignin typical of tension wood
serves to increase the ability of stems to yield under bending stress. In the present study,
decreased lignin content may be another reason for the observed decrease in MOE of
stems subjected to MP.

The second explanation for difference in relative strength of cell walls is the angle
of the microfibrils. The behavior of wood cells is based to large degree on the structure
of their secondary walls, specifically the helical arrangement of the microfibrils in the S2
layer, which provides a mechanism for absorbing energy and preventing structural
failures (Dickison 2000). Microfibril angle and density accounted for approximately 95
percent of the variation in wood stiffness (longitudinal MOE) of longitudinal sections of

Eucalyptus delegatensis (Evans and Ilic 2001).

39

Telewski (1989) found that microfibril angle increased in tracheids of Abies
fiaseri (Fraser fir) that differentiated under the influence of flexure. Lindstrom et al.
(2002) found that MOE negatively correlated with microfibril angle of young P. radiata
clones. In other words, as the microfibril angle increased the elastic properties of the
stem decreased. In the present study, a change in microfibril angle in stems subjected to
MP may explain the increase in whole stern rigidity. A change in microfibril angle could
also affect the strength of the cell walls in directions not measured using the Universal
Instron Machine for 4-point bending.

Wood with a specific gravity of 0.36 or less, like the T x D hybrid crosses used in
this study is considered light (Panshin and de Zeeuw 1980). Wood specific gravity at
green volume of Populus trichocarpa was reported as 0.31 with P. deltoides at 0.37
(Forest Products Laboratory 1999). The hybrids used in this study have wood specific
gravities that fall near P. trichocarpa. Mechanical perturbation increased the wood
specific gravity of these poplar clones, a result consistent with tension wood of P.
deltoides (Lassen 1959) and free swaying L. styraciflua (Holbrook and Putz 1989).
Wood formed under the influence of MP was denser but had lower lignin content
according to the results of Pruyn (1997). It is surprising there was no correlation between
specific gravity and MOE in the present study especially since there is a linear
relationship between MOE and wood density, the amount of cell wall material in a given
volume of wood (Lindstrom et al. 2002). This discrepancy may be due to the juvenility
of the wood of the poplar clones used in this study.

Conductivity of these hybrids was previously unknown. Mechanical perturbation

decreased stem conductance (kh mm,“ and kh max) and specific conductivity (k5. conductivity

4O

per conductive xylem area). Specific conductivity decreased 10-45% depending on the
hybrid when clones were subjected to MP. This result was expected since the flexure
wood of angiosperms shares many characteristics with tension wood. Tension wood is
characterized by the presence of fewer and smaller vessels than normal wood and a
corresponding increase in the proportion of fibers (Wardrop 1964). In other words,
conductivity decreases because the frequency of vessels decreases and the vessels are
smaller when stems are subjected to MP. In fact this was observed in the MP stems of
Hybrids 46-158 and 19-61. Reduction of k5 corresponded to decreases in percent vessel
lumen area, hydraulic diameter, vessel frequency, and average and maximum vessel
lumen diameter.

Vessel frequency of Hybrids 46-158 and 19-61 was greater than the reported
values of mature P. deltoides and P. trichacarpa (26-100 mm'z, Panshin and de Zeeuw
1980), however average vessel diameter of Hybrids 46-158 and 19-61 was lower than
reported values of either parent (51-150 um, Panshin and de Zeeuw 1980). The range of
values for vessel data reported here for Hybrids 46-158 and 19-61 are within the range
found for Hybrids 47-174 and 11-11 by Pruyn (1997).

As is the case in the present study, Pruyn (1997) also showed that vessel
frequency, percent vessel lumen area, and average vessel diameter decreased in stems
subjected to MP. These results are generally consistent with Telewski and J affe (1986a,
1986b) as well as studies of dicotyledonous angiosperms. Neel and Harris (1971)
observed a reduction in vessel element length and diameter in flexed L. styraciflua as
compared with the vessel elements of untreated trees. Jourez et al. (2001) found vessel

frequency and porosity were significantly lower in tension wood than in opposite wood

41

of young inclined stems of Populus euramericana cv ‘Ghoy’. J ourez et al. (2001) also
observed that vessel elements in tension wood were 2.5% longer than in opposite wood, a
result inconsistent with flexed L. styraciflua (Neel and Harris 1971). Mechanical
perturbation decreased tracheid length in A. fi'aseri (Telewski and J affe 1986a) and P.
taeda (Telewski and J affe 1986b). Data on vessel element length was not collected in the
present study and it remains unknown whether the vessel elements of these hybrid
poplars increased or decreased in length due to MP. The average vessel element length
for mature P. trichocarpa is reported as 58 i 9 pm (Panshin and de Zeeuw 1980). More
than 90% of Populus vessels are shorter than 15 cm in length (Blake et al. 1996).
Investigation of ray anatomy of flexure wood is lacking in the literature, however rays of
tension wood in P. deltoides decreased in size (Kaeiser and Boyce 1965) and were more

numerous in Populus euramericana cv ‘Ghoy’ (J ourez et al. 2001).

Positive correlations between mechanical and hydraulic properties

The present study suggests there is an opportunity to select clones with superior
mechanical strength and conductivity because there was a significant positive correlation
between kh max and EI (whole tree basis) and between kS and MOE (tissue area basis).
This correlation was not expected and contradicts the results of other research (Wagner et
al. 1998, Spicer and Gartner 2002, Woodrum et al. 2003). Several explanations are
possible for the contradiction: Wagner et al. (1998) used different stem segments to test
conductivity and mechanical strength of chaparral shrubs; Spicer and Gartner (2002) used
Pseudotsuga menziesii (Douglas-fir), a gymnosperrn having one cell type to function both

in water transport and support; and Woodrum et al. (2003) used lateral branches of five

42

Acer species (likely to have tension wood present) which differ anatomically compared to
the wood of hybrid poplars.

There was a significant positive correlation between RS and percent vessel lumen
area for these hybrids but we did not collect data on fiber lumen area. Jourez et al. (2001)
found a negative correlation between the proportion of vessel lumina and the proportion
of fiber lumina including the G-layer. This is interesting because in the present study,
stems subjected to MP had decreased percent vessel lumen area but increased specific
gravity. This would mean as wood formed when subjected to MP, fewer vessels were
produced and they occupied less of the stem cross-sectional area. As a result, more fibers
must be present. The presence of more fibers supports the observed increase of flexural
rigidity of clones subjected to MP. In the future, analysis of fibers may confirm this
assumption for these hybrid poplars.

Although fibers were not examined in the present study, three fiber variables are
considered to be important in determining the physical characteristics of pulp and paper:
fiber density, fiber length, and fiber strength (Panshin and de Zeeuw 1980). Fiber length
is particularly important since it determines to a large extent the physical and mechanical
properties of paper (Koubaa et al. 1998). The average diameter of libriform fibers of
mature P. deltoides and P. trichocarpa ranges from 26 to more than 30 um (Panshin and
de Zeeuw 1980). Fiber length of mature P. trichocarpa was reported as 1.38 i 0.19 mm
(Panshin and de Zeeuw 1980). Koubaa et al. (1998) showed that fiber length varied
considerably by clone type and height in the tree for ten different Populus x
euramericana clones. Tension wood fiber length has been described as longer, and

sometimes of equivalent length or shorter (Jourez et al. 2001). Examination of the effect

43

of MP on fiber properties may be of interest especially in hybrid poplar clones harvested
specifically for pulp.

The positive correlation between kh max and E1 is strong in the case of these seven
hybrid poplars. The relationship remained significant even when Hybrid 19-61, the
triploid that exhibited great conductance and mechanical strength, was removed from
regression analysis. Triploids have a phenotype that is biased toward the parent from
which it carries two genomes (Bradshaw and Stettler 1993). Clearly, Hybrid 19-61
exhibited greater growth compared to the six diploids. In addition, triploids typically
have longer and wider fibers (Stettler et al. 1996), anatomical characteristics that offer an
explanation for the observed greater mechanical strength compared to the diploids.

The relationship between kh max and E1 is also supported by evidence that both RS
and MOE were positively correlated with percent vessel lumen area. Clearly, some of the
clones have both great mechanical strength and water conduction. When subjected to
MP, Hybrid 19-61 had the most radial growth compared to the six other hybrids
(diploids) as well as higher vessel frequency in stem cross-section compared to Hybrid
46-158. In other words, when stems of Hybrid 19-61 were subjected to MP, more wood
developed in the stem and a greater proportion of the stem was in the form of vessels.
Both of these phenomena may explain the superior conductivity and mechanical strength
of Hybrid 19-61.

Due to the heterosis exhibited by some of these clones in terms of conductivity
and mechanical strength, it may be advantageous to select poplar clones based on
mechanical strength (MOE, MOR) and conductivity (ks). Testing conductivity and

mechanical strength of older stems is needed however. The stems tested in this study

44

were juvenile and it remains unknown whether the wood properties of these seven hybrid
poplars are similar to seven-year—old stems harvested for woody biomass production.

Silviculture of hybrid poplar in plantations is intensive and rotations are short
(five to eight years for fiber alone and up to 15 years for solid wood products); therefore,
a large portion of the wood produced will be in the juvenile core (DeBell et al. 2002).
The wood formed each year gradually assumes the character of mature wood and the
progression from juvenile to mature wood differs among wood properties and species
(Panshin and de Zeeuw 1980, Zobel and Sprague 1998). For P. deltoides, the
juvenile/mature wood demarcation point for mechanical properties and specific gravity is
about 17 to 18 years, with cell length and fibril angle stabilizing about a year or two later
(Bendtsen and Senft 1986). Therefore, hybrid poplars in plantation are harvested well
before maturity.

Juvenile wood is generally weak, lower in density, and composed of cells which
are shorter in length and vessels narrower in diameter when compared to those found in
mature wood (Telewski et al. 1996, Zobel and Sprague 1998). Large microfibril angles,
short fibers with thin walls, and low percentages of latewood in the annual ring all
contribute to low strength and stiffness in juvenile wood (Bendtsen and Senft 1986).
However differences between juvenile core and mature wood of diffuse-porous
hardwoods such as Populus are often quite small, so that sometimes product quality in
operational forestry is not affected (Zobel and Sprague 1998). In fact, DeBell et al.
(2002) showed that average wood density of Hybrids 11-11 and 47-174 was similar for
the first three years (0.37 g cm'3), decreased to 0.335 g cm'3 at age four and five, and

finally increased to 0.40 g cm'3 at age nine. On the other hand, mean fiber length of the

45

two clones consistently increased each year through age nine, starting at approximately
0.55 mm and ending at approximately 0.95 mm (DeBell et al. 2002).

In addition to the concerns about differences between properties of j uvenile and
mature wood, selection of poplar clones with great mechanical strength and water
conduction should be accompanied by other considerations. For example strong shoots
with superior water conduction may have another liability not yet identified (e. g.
vulnerability to xylem embolism or attack by insects and pathogens). Field tests of older

individuals are needed to determine the economic potential of specific clones.

46

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