INDUCTION OF TYLOSES IN EUCALYPTUS GLOBULUS 'CHIPS' - Brill
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IAWA Journal, Vol. 20 (2),1999: 193-201
INDUCTION OF TYLOSES IN EUCALYPTUS GLOBULUS 'CHIPS'
by
Mathew A. Leitch!", Rodney A. Savidge-, GeoffM. Downest-' &
Irene L. Hudson 4
SUMMARY
Cambial stern chips containing intact cambium between xylem and
phloem, or with the phloem layer removed, were cut from the main stern
axis of four-year-old Eucalyptus globulus during the winter and grown
under controlled environmental conditions for seven week s on fully
defined culture media . Light microscopy revealed that tyloses were in-
duced in sapwood vessels in the region adjacent to the cambium within
these stern chips. When incubated in autoclaved double-distilled water
(control medium) tyloses were produced in 3.7% and 4.7% of vessels
in chips with the phloem layer intact and removed, respectively. When
non-hormonal ingredients were included, tyloses developed in 69.5%
and 76.1% of vessels in chips with the phloem layer intact and removed,
respectively. Addition of 1.0 mg I-I of l-naphthalene acetic acid (NAA,
a synthetic auxin) to the medium had a slight, but significant, inhibitory
effect on tylosis formation.
Key words: Cambium, phloem, ray, sapwood, tyloses , tylosis, vessel.
INTRODUCTION
A tylosi s in hardwoods is defined as an outgrowth ofthe protoplasm of a living paren-
chyma cell through a vessel-parenchyma pit into the adjacent vessel element (Panshin
& DeZeeuw 1980). Formed usually in the aging inner sapwood, the development of
tyloses has been regarded as anormal physiological process marking the transforma-
tion from sapwood to heartwood in many hardwood species (Meyer 1967; Panshin &
DeZeeuw 1980; Wilson & White 1986). However, due to their resistance to penetra-
tion of solutions, tyloses in vessels of commercial species create difficulties during
wood preservation, chemical pulping, and match-making (Streslis & Green 1962; Du
Toit 1964; Murmanis 1975; Panshin & DeZeeuw 1980; Wilson & White 1986).
1) CRC for Hardwood Fibre and Paper Science , University of Melbourne, School of Forestry,
Creswick, Victoria, Australia, 3363.
2) Univer sity of New Brunswick , Faculty of Forestry & Environrnental Management, Fred-
ericton, NB, Canada, E3B 6C2.
3) CSIRO Forestry & For. Prod . Sandy Bay, Tasmania , Australia , 7005 .
4) University of Canterbury, Mathematics and Statistics Dept, Christchurch, New Zealand.
*) Author to whom correspondence should be addressed;
E-mail : m.leitch@landfood.unimelb .edu.au
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In most hardwood speeies, tyloses normally do not form in the outer sapwood, but
may develop in response to meehanieal injury, fungal invasion or viral infeetion (Gerry
1914; Sehmitt et al. 1997). For example, Babos (1993) found that diseased Quercus
petraea Liebl. sterns eontained three to four times more tyloses than sound sterns.
When so indueed, they are termed traumatic tyloses (Babos 1993). Whether traum at-
ie or normal, tyloses effeetively seal vessels, preventing water movement and patho-
gen spread (Salisbury & Ross 1992) .
The oeeurrenee of tyloses formation has been attributed to ehanges in water poten-
tial. When the turgor pressure inside a parenehyma eell beeomes greater than the
pressure potential of the adjaeent vessel element, through emboli sm, bloekage or
wounding of the vessel element, the lower pressure in the vessel element permits an
area of the ray eell's protoplasm that is eovered by a 'protective layer' to distend into
the vessel eavity (Streslis & Green 1962; Sehmid 1965; Murmanis 1975; Deseh 1981).
However, this protrusion of protoplasm is not merely a physieal proeess. It is neees-
sarily preeeded by the enzymatie degradation of the unlignified pit membrane of the
pit pair (Panshin & DeZeeuw 1980). Degradation of the swollen pit-pair membrane
by hydro Iytie enzymes has been reported (Foster 1967; Meyer 1967; Meyer & Cöte
1968; Murmanis 1975). As the pit membrane breaks down, the proteetive layer ap-
pears in the vessellumen initially as a small bud, followed by expansion of the grow-
ing tylosis (pan shin & DeZeeuw 1980). The strueture of the tylosis wall has been
described in detail by many authors and has been shown to be distinet from vessel-
parenehyma pit and seeondary walls in the immediate area (Isenberg 1933; Chattaway
1949; Koran & C öte 1964, 1965; Kato & Kishima 1965; Foster 1967; Meyer 1967;
Meyer & C öte 1968; Murm anis 1975).
Sinee Reiehenbaeh (1845) little work has been done on the experimental induetion
of tylosis formation (see Zimmermann 1979, 1983). Meyer (1967) studied the devel-
opment oftraumatie tyloses in Quercus alba L. by chiseling out blocks from a stand-
ing tree and growing them at 21 "C in 200 ml bottles lined with moist paper towels.
Murmanis (1975) cut sampies from Quercus rubra L. and investigated the number of
hours required for tylosis formation . In spring it took 6 h, and during active growth in
summer only 2.5 h were required . During dormancy 1.5 months or longer were nec-
essary for tyloses to appear despite temperatures conducive to metabolism.
This paper reports on an in vitro technique to study tylosis formation in stern chips
incubated in a defined liquid medium after the chips were collected from nearly dor-
mant Eucalyptus globulus Labill.
MATERIALS AND METHODS
Plant material
Material for cultures was collected during mid-winter months. Two four-year-old
southern blue gum trees (Eucalyptus globulus) between 9 and 12 metres in height and
having no visible deformities were felled in Australian Paper Plantations (Morwell,
Victoria, Australia). Following removal of all lateral shoots , three-year-old stern sec-
tions (two segments, eaeh 45 em in length) ofthe main- stem axis were transferred to
the laboratory for in vitro investigations.
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Segment preparation
The outer bark of the three-year-old stern section was pared to the level of a few
layers of crushed phloem, as previously described (Savidge 1993). The stern surface
was washed with 3% Decon 90 (vIv) for 10 min followed by I hin 3% Decon 90
(vIv). After rinsing in distilled water, the segment was immersed in 3% sodium hypo-
chlorite (2 x 15 min), rinsing with distilled water between each treatment. The stern
segment was again rinsed with distilled water, then immersed in 3% hydrogen per-
oxide (30% w/v) for 30 min. Following rinsing in double-distilled water, the seg-
ments were flame sterilised using ethanol as previou sly described (Savidge 1993).
Chip removal and culturing
An all-metal flame-sterilised knife was used to remove chips (2-3 cm axial x 2 cm
tangential x 0.5 cm radial) from the three-year-old stern segment in a laminar flow
cabinet. These stern 'chips', consisting of intact cambium between mature phloem
and xylern, were transferred to a 3% sodium hypochlorite solution for 2 min, fol-
lowed by drying on sterile filter paper. Sterilised chips were explanted to liquid media
in culture flasks . The phloem layer was aseptically removed and discarded from 50%
of the chips prior to explanting. Fifteen chips per treatment per tree harvested were
investigated.
Culture medium and conditions
The composition of the base culture medium was published by Savidge (1993) .
The exclusion of Difco-Bacto agar and I-naphthalene acetic acid (NAA) from that
medium yielded the 'nutriment' medium used in this study. NAA at 1.0 mg I-I was
added to the nutriment medium to give the ' NAA + nutriment' medium . The control
was double-distilled water with no inorganic or organic additives. The media were
adjusted to pH 5.8 with O.IN KOH prior to autoclaving (121°C, 140 kPa, 20 min).
Sterilised media were poured into 70 ml pre-sterilised culture flasks to a depth of
2-3 cm (25-35 ml of media per flask) and allowed to cool before capping. Chips
were cultured under 20 hours of light (standard cool white fluorescent , 36 Watts)
daily at a constant 24°C on an orbital shaker. After 49 days chips were fixed in etha-
nol-acetic acid-distilled water (70 I 5/25 vIv) and then sectioned for light micro-
scopy.
Microscopy and assessments
Transverse and radial sections , cut by hand with a razor blade in the central region
of each chip, were mounted in glycerol on glass slides (3 slides chipJ , 10 sections
slideI). Unstained sections were examined with brightfield and between crossed po-
larising filters using an Olympus BH-2 photomicroscope. In the region adjacent to
the cambial zone where the vessels had secondary walls, developing tyloses were
photographed and the percentage of vessels containing tyloses was recorded.
For quantitative data, a factorial ANOVA was used to compare responses at the
99% confidence level (Zar 1984). Standard errors of the mean were calculated for
results.
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RESULTS
At the time of chip preparation there were no tyloses in vessel elements in the region
adjacent to the cambium. The production of tyloses was pronounced in chips cultured
on nutriment medium compared to chips cultured on water only (Fig. 1). In the water
% Tylosis
80 -
~
:::i::::
m
T
T
~
60 -
..
Q
40
20 - :
: : : ::: ::: : :
:: : : :: :
:
o f:Jl
............ WJ :
I B. on I B. off 2 B. on 2 B.off 3 B. on 3 B. off
Treatment
Fig. 1. Production of tyloses in response to incubation of Eucalyptus globulus chips to three
treatments; 1: double distilled water ; 2: nutriments medium ; 3: nutriments + 1.0 mg 1-1 NAA
medium . - B. on = phloem intact ; B. off = phloem layer removed. - Bars are standard error s
of means. Where the standard error bar is not shown it is too small to print.
Fig. 2-7. Transverse hand sections of Eucalyptus globulus. - 2: Hydrolyti c sac structure (ar-
row) developed in the ray prior to swelling of the pit membrane (ingredients, B. off). -
3: Swelling of the ray to vessel pit membrane (nutriments + 1.0 mg I-I NAA, B. off). - 4: A
tylosis protective layer developing into the vessellumen (ingredients, B. off). - 5: The hydro-
Iytic sac structure becoming the tylosis structure in the vessel (ingredients, B. off). - 6: Tylosis
increa sing in size as the hydrolytic sac in the ray decrea ses in size (nutriments + 1.0 mg 1-1
NAA, B. on) . - 7: Vessellumen filled with tylose s at several stages of development. Note the
darker ray (arrow) adjacent to the vessel where tyloses formation is occurring (nutriments +
1.0 mg 1-1 NAA, B. on). - r = ray; v = vessel; f = fibre; hs = hydrolytic sac; pl = protective
layer ; pm =pit membrane ; sp =large spherical tylosis; tb =tylosis bud. - Scale bars =5 um in
Fig. 3 & 5; 10 um in Fig. 2,4 & 6; 20 um in Fig. 7.
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Fig. 8. Radial hand seetion showing tyloses protruding through ray to vessel pits (nutriments +
1.0 mg 1-1 NAA, B. off). - f =fibre; r =ray; rvp =ray to vessel pit; tb =tyloses bud; v = vessel;
vp = vessel pit. - Scale bar = 20 um.
only treatment, tylosis formation in the sapwood region adjacent to the cambium
occurred in 3.7% of vessels where the phloem layer remained intact and increased to
4 .7% of the vessels in chips lacking phloem (Fig. 1). When incubated in the nutriment
medium, tyloses occurred in 69.5% of the vessels where the phloem layer remained
intact and in 76.1 % of vessels in chips without phloem (Fig . 1). Chips with intact
phloem cultured on the NAA + nutriment medium produced tyloses in 64.4% of the
vessels. Removal of the phloem layer slightly increased this value to 66.5 % of ves-
sels in the sapwood region adjacent to the cambium (Fig. 1).
InitiaIly, dark-coloured (presumably tannin) substances accumulated and formed
a sac-Iike structure in the ray ceII in the region of a vessel-parenchyma pit (Fig. 2).
On the assumption that the sac-like structure contains hydrolytic enzymes encIosed
within the protective layer of the sac, we refer to this structure as the hydrolytic sac .
SweIIing of the vessel-parenchyma pit membrane (Fig. 3) foIIowed development of
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the hydrolytic sac. The tylosis protective layer subsequently pushed through the ves-
sel-parenchyma pit into the vessel (Fig. 4), and as the tylosis bud increased in volume
the hydrolytic sac reduced in size (Fig. 5) ultimately becoming very small (Fig. 6).
Eventually the lumen of the vessel element was fully occupied by the tyloses (Fig. 7).
In radial sections, tyloses protruded through the ray/ves sel vessel-parenchyma pits at
several locations (Fig. 8). Chips exposed to the control, nutriment and nutriment +
NAA media did not produce any significant cambial responses. Small amounts of
peripheral callus were produced when chips were incubated on the nutriment and
nutriment + NAA media (data not shown) .
DISCUSSION
Chips cultured in double-distilled water displayed significantly less tyloses formation
than chips cultured in the other two media preparations. Nutritional factors (organic
and inorganic components) in the medium were essential for the production of ty-
loses in sapwood vessels within chips. Auxin at the one concentration tested had a
slight, but significant, inhibitory effect on the nutriment medium's promotion of ty-
loses formation. NAA at the tested concentration had previously been found condu-
cive to in vitro cambial growth in cultured chips of other species (Savidge 1993;
Leitch & Savidge 1995; Savidge & Leitch 1996). The results indicate that one or
more constituents of the nutriment medium promote tylosis formation in E. globulus
chips grown in vitro. The nutriment medium with or without auxin was not, however,
conducive to promote cambial activity, indicating that the tylosis formation is inde-
pendent of cambial growth.
With the addition of nutrirnent, tyloses production in the chips occurred in a man-
ner similar to that reported in the literature (Schmid 1965; Murmanis 1975; Panshin
& DeZeeuw 1980; Desch 1981). An accumulation of substances developed in the ray
cells neighbouring vessel elements prior to the formation of the hydrolytic sac near
the pit membrane. Swelling and degradation of the pit membrane, presumably by
hydrolytic enzymes has been described previously in other species (Foster 1967; Meyer
& C öte 1968; Murmanis 1975). Murmanis (1975) observed vesicular membranous
structures that preceded expansion of the ray cell protoplast and suggested they may
carry hydrolytic enzymes. Once the pit membrane has presumably been disintegrated,
the protective layer of the parenchyma begins to protrude into the lumen of the vessel
under turgor pressure created by the parenchyma protoplast (Murmanis 1975). Gerry
(1914) suggested this occurred from a reduction in internal press ure or cessation of
sap conduction in large diameter vessel elements. This protrusion into the vessel of
the protective layer appears initially as a small bud and continues to develop until a
large spherical tylosis is produced. Murmanis (1975) suggested tyloses will develop
and increase in size until either space or metabolie supplies becomes limiting. It has
been reported previously that the majority of tyloses appear to originate from adja-
cent rays (Chattaway 1949; Foster 1967). Although it was occa sionally noted that the
tyloses appeared to originate from axial parenchyma in the current study, this was not
clearly demonstrated.
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Cambial activity studies have shown the cambium of E. globulus to be active
throughout the season with aperiod of low activity during the mid-winter months
(M. Leit ch , unpublished data) . Although endogenous factors have been shown to in-
fluence in vitro cultures until depleted (Savidge 1993, 1994; Leitch & Savidge 1995),
this study suggests that endogenous levels of plant hormones and metabolic supplies
were insufficient for cambial activity or tyloses production.
Peripheral callus may occur as a means of restoring tensional stress within the chip
to allow continued growth (Savidge 1993). Unidentified endogenous factors have
been suggested to be involved in changing cellular forces within the chip to aid in
wound healing around the periphery of the chip (Kutschera 1989). It is possible that
initial cambial wounds and callus production, described previously (Savidge 1993;
Leitch & Savidge 1995), are a result of substance translocation for the purpose of
healing and regaining tensional stress to permit developmental processes to occur.
CONCLUSIONS
Endogenous reserves from Eucalyptus globulus wood collected during winter are
insufficient für tyloses or cambial development. Tyloses have been produced using an
in vitro system. The key ingredient for tyloses formation in E. globulus is the inclu-
sion of nutriments in the medium; inclusion of NAA in the nutriment medium had a
slight inhibitory effect on tyloses production.
Meyer (1967) stated the importance of tyloses to the wood preserving industry,
and more importantly realised manipulation of this feature would be desirable. How-
ever, this would require an understanding of the genetic and physiological state of the
tree (Murmanis 1975). Current research in our facility is addressing genetic control
of tylosis development leading to heartwood formation . Many researchers have stud-
ied tyloses and their development, however, no system is yet available where the
means of promoting tyloses in the wood can be altered via a defined tissue culture
medium. Having developed an in vitro system to study tyloses formation in E. globu-
lus creates the opportunity to manipulate the media to identify specific components
that are crucial to the tyloses development. It will be desirable to include TEM studies
in future experiments to identify changes in tylosis development based on media ma-
nipulations.
ACKNOWLEDGMENTS
This research was funded by the Cooperative Research Centre for Hardwood Fibre and Paper
Science .
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