Proposed theoretical and experimental work
Theoretical work
Over the years there have been a number of attempts to mathematically model plant
cell walls (usually fibres or tracheids) from cell wall constituents e.g. \citet{mark1967cell,Astley_1998,Yamamoto_2002} and \citet{Kojima_2004}. However very few efforts have used these
techniques to investigate the formation of growth stresses \citep{Archer_1987,Yamamoto_1998,Guitard_1999}).
Currently the most advanced model for how growth stresses develop within the
cell wall was presented by \citet{Alm_ras_2005} using the unified hypothesis
\cite{okuyama1986,Okuyama_1994,yamamoto1991,Yamamoto_1998,Yamamoto1992} utilising both the lignin swelling and
cellulose contraction hypotheses. For details see Section \ref{section:GS}.
Proposed model of the cell:
Modelling of a generic single cell with variable cell wall parameters to
investigate the required geometry and constituents to create maximum
longitudinal and tangential extension and contraction via the lignin swelling
hypothesis. The single cell model should have the capacity to put limits on the
magnitude of stress generation by lignin swelling under different constituent and geometric makeups.
Because the proposed experiments (see Section 1.2) induce tension wood in species
both with and without G-layers an experimental upper limit of the stress
generation by lignin swelling can be measured and
compared to the theoretical data derived from the model.
It is expected that the base model and parameters will be similar to those
utilised to describe lignin swelling by \citet{Alm_ras_2005} and \citet{Yamamoto_1998}. Cell wall layer radii, thickness, MFA, moduli of the CMF bundles and matrix will all be included. Additional variables will be included
as necessary. It is intended to add the standard deviation of the MFA within the
cell wall layers, as in \citet{Harrington_1998}, pore size (or conversely fibril
aggregate size) \citep{Fahl_n_2005,Chang_2014,BIORESBioRes_07_1_0521_Salmen_OSSR_Struc_Organis_Wood_Polymers,Kim_2011} and cell wall
constituents \citep{Baba_2009,Donaldson_2001} and layer properties/geometries
\cite{Bergander_2002,grozdits1984differentiation,Alm_ras_2005,Yamamoto_1998,Chang_2014,Yamamoto1995} to form a model, conceptually similar to the qualitative
architecture presented by \citet{Mellerowicz_2011}, \citet{Salm_n_2009} and others. Boundary conditions will be initially derived from those
presented by \citet{Alm_ras_2005} and further modified for increased realism
and/or usability of later models.
One of the major differences between the model presented here and those in previous
literature is the inclusion of intertwining cellulose fibril aggregates. Recently \citet{Chang_2014} measured the pore size and shape within tension wood and opposite
wood of poplar during cell wall maturation. With this recent advancement,
reasonable assumptions around how regularly fibrils interact with each other can be made. It is thought that these pores
occur between joining fibrils connecting the macrofibrils into the larger
structure that is the forming cell wall. If the deposition of lignin into the
pores forcing the fibrils apart is the mechanism by which growth stresses
develop the quantity and geometry of pores are important parameters to
investigate as they will largely affect the ability of the mechanism to cause
stress.
The model is limited to a single unconstrained cell, which differs significantly
from a cell within a tree, due to the constraining effects of the surrounding
wood. Boundary conditions will be used to minimize this problem because modeling
a significantly large volume of wood is not feasible. The minimum resolution which
will be considered is the fibril aggregate, these are thought to be the smallest
structural components responsible for growth stresses under the lignin swelling
hypothesis.
Experimental work
Currently neither lignin swelling or cellulose contraction (described in Section
\ref{section:GS}) have any direct experimental evidence. The tension which cellulose is under
on the stem periphery has been directly measured using x-ray diffraction showing
a strain reduction of 0.2% in cellulose when the stress is released \citep{Clair_2006}.
Experimental evidence of the G-layer providing contraction within tension wood
has been presented by \citet{Goswami_2008}. Longitudinal extension and tangential
contraction were observed when the G-layer was enzymatically removed from
poplar tension wood samples. The S2 layer was reported to have a high MFA (36
degrees) as has been reported previously for other G-layer producing species
\citep{M_ller_2006}. \citet{Goswami_2008} suggested lateral swelling of the G-layer caused the
contraction.
The primary goal of the set of experiments which will be presented within this
chapter is to attempt to identify which cell wall constituents contribute to
stress generation. In order to evaluate stress generation mechanisms a number of
experimental techniques have been identified.
Basic cell wall anatomy and geometry needs to be investigated for the NZDFI
species involved in this project. Where possible literature values will be used
to approximate values for model parametrization.
The following properties are required:
The cell wall anatomy of different wood types (tension, normal and opposite)
needs to be investigated for the various NZDFI species (principally E.
bosistoana ). The anatomy study will consist of investigating which species
produce a G-layer (microscopy with staining \citep{Qiu_2008}), cell wall architecture
(atomic force and electron microscopy), and the MFA and MFA standard deviation in all three wood types (X-ray diffraction).
Within
tension wood the G-layer (in G-layer producing species) will be
removed in order to determine the secondary cell wall properties of tension wood
(enzymatic removal).
Note: growth stresses for a large number of samples will be collected during
the breeding work, however because of the time consuming nature of the
experimental works presented here only a small number of specimens will be
tested as needed.
In order to produce the three types of wood required two different growth
manipulation techniques are suggested:
Technique one: Young stems (less than three month old growth from coppice) will
be restrained to a loop, similar to \citet{jacobs1945l} and allowed to
grow for approximately three months, with regular adjustments of the restraints
to make sure the cambium is not damaged.
Technique two: Straight one year old stems (from coppice, and seedlings of a
mixture of E. camadulensis, E. tricarpa and E. quadrangulata will be bent and restrained
and allowed to grow for a further three months, with regular adjustment of the
restraints to avoid cambium damage. Normal wood samples can be collected from
these stems higher up where they are straight.
The following experiment is proposed in order to investigate the proportion of
the stem reorientation that is due to the G-layer. During growth tension wood
production is induced by forcing curvature into the living stem, as described
above. By introducing an enzyme treatment to the plant while it is still
transpiring it should be possible to degrade the G-layer and reverse any straightening that was caused
by the G-layer. With the G-layer removed the remaining stress can be released.
Due to the expense, time, equipment and expertise required to test all of the
properties needed to parameterise the model presented in section 1.1 a number of
properties will be taken from literature and assumed to be a good approximation
for the current work. It is likely to be the case that a number of these
properties are not from Eucalyptus species.
Breeding
Growth stresses cause a number of issues for harvesting and milling
timber, tree breeding programs can be used in order to select for
genetics which reduce these effects. There is no reason to expect breeding for
growth stress differs significantly from breeding trees for
any other trait. Over the last few decades many advances have been made in
experimental and statistical techniques which rapidly improve the time and
accuracy of tree breeding.
It is suspected that the most efficient way to minimise the issues growth
stresses cause during the production of timber is through appropriate genetic
selection. Eucalyptus species, in particular E. bosistoana are showing
promise within the NZDFI trials to produce high value naturally durable
structural timber. To make this
product profitable, growth stresses need to be reduced to minimise the effects
discussed in Section xxxx. Within the NZDFI project there are a number of
additional concerns for breeders (such as durability, form and growth rate). Using conventional breeding methods
discussed below, growth stresses will be minimised within the NZDFI genetics.
Currently two trials have been established or will soon be established, these
include:
All trials at Harewood are set out as randomised individual tree trials. Principally
this work will be concerned with E. bosistoana of which there are two
trials. One with 8 replicates of 20 families, planted in 2012 and coppiced in
2013, due to be harvested in spring/summer 2015. The other
E. bosistoana trial has 10 replicates of 20 families, was planted in
2010 and harvested for the first time in 2012, the plants were then coppiced
and harvested again in December 2014. Four families representing the highest and
lowest growth stress generating genetics were coppiced for a second time and
will be due for harvest in 2016. Preliminary results from the 2012 and 2014
harvests show heritability of growth strain generated family
rankings. The same data was collected from E. argophloia plants planted
in 2010 measured and coppiced in 2012, with final measurements completed in 2014.
Due to the success of the previous trials NZDFI is currently setting up a 10,000
plant trial with 200 families of E. bosistoana and 336 seedlings over 13 families of E.
argophloia . The trials are set up as alpha lattices and harvest is expected in
late 2016 or early 2017.
Note: the term family is used here to mean the same mother, but not necessarily
the same father. If seeds are collected at different times even from the same
tree variability exists due to the possibly of different fathers. Also some
Eucalyptus self propagate, but it is unknown what proportion of
seeds are self propagated within the NZDFI genetic material. Any effects of self
propagation are ignored.
Within the structure of the breeding program there is the ability to
statistically check within family rankings from young stems and more mature
stems, although the trees are grown under different environmental conditions.
In order to select for low growth stress producing families experimental tests
need to be undertaken on each plant. The main test to be used to determine the
extent of growth stress within the breeding population is the split test (also
known as the Pairing Test) as described in \citet{Chauhan_2010}. The test involves
taking a straight 500mm long stem section and cutting along the pith to create
a radial split. The diameter of the stem is taken before testing and the width
of the opening measured immediately after splitting. Once the opening is
measured the stem is cut across the grain to give two samples (one from each
side of the split). Density is measured by measuring the mass (using balances)
and volume using the displacement method on each of the pieces. Acoustics are
also taken using Wood-Spec to calculate the dynamic modulus, and hence the
stress can be derived \citep{Chauhan_2010}. Due to the size of the Woodville trial it
may be the case that fewer tests are carried out. Decisions on the essential
tests will be made near the time of harvesting.
It
is expected that typical statistical techniques used in breeding will be applicable. The
two objectives to be achieved for the breeding trials are to estimate genetic
parameters, (in particular heritability of growth strain) and to predict
breeding values for families. Mixed model methods are commonly used within tree
and animal breeding for these purposes \citep{White_2007}.
The breeding work undertaken in the thesis will be limited to the investigation
of growth strain in the NZDFI Woodville and Harewood E. bosistoana
trials. The assumption is made that families which rank well at young ages will rank well at a commercially viable harvest size.