Journal of Experimental Botany, Vol. 47, No. 294, pp. 135-143, January 1996
Journal of
Experimental
Botany
A comparison of methods for studying pressure and
solute potentials in xylem and also in phloem laticifers
of Hevea brasiliensis
John A. Milburn1 and Muditha S. Ranasinghe
Department of Botany, University of New England, Armidaie, NSW 2351, Australia
Received 20 March 1995; Accepted 25 September 1995
Abstract
In this study, several ways have been examined to
measure latex pressure, including some novel
methods, with the aim of finding better methods than
are presently available. A novel method, similar to the
pressure probe as used for single cells, has been
applied to laticifers, exerting a back-pressure to prevent exudation. The type of capillary bubble manometer developed by Bourdeau and Schopmeyer for
resin pressures (1958) and then used for latex pressures by Buttery and Boatman (1964, 1966) has been
improved and the construction and standardization of
bubble manometer gauges for routine field use has
been greatly simplified.
It is concluded that the back-pressure method in its
present state of development provides neither sufficient rapidity nor precision on account of the slow
response time. This method does not justify the two
operators needed. The requirement for relatively large
quantities of escaping latex limits its applications and,
in certain instances, pressurization might be incomplete. Nevertheless, it has provided some additional
confidence in the accuracy of the other methods
because readings can often be repeated many times
on a single puncture, which is impossible with a bubble
manometer. Also the gauge used is linear and therefore more accurate at high pressures than a bubble
manometer.
The newly designed Buttery and Boatman gauge
requires fewer parts and utilizes standard plastic pippetor tips. These greatly speed and simplify construction of the gauges, which are needed in large numbers
as they are essentially disposable. These gauges have
proved to be remarkably reliable, even with minimal
1
latex volumes, and have always indicated the highest
latex pressures of all methods used at a particular
site, suggesting that leakage rates are minimal at the
junction between gauge and laticifers. The plastic tip
holders facilitate the manual insertion of these delicate gauges into the tree bark.
Xylem sap is contaminated by phloem sap if collected from petioles using a pressure bomb. However,
this problem can be overcome in stems by bark ringing.
Alternatively, the vacuum extraction method can be
used which does not seem to suffer from this contamination problem.
Several ways have been tested to maximize the reliability of latex serum samples for analysis in a vapour
osmometer. Pressure and solute components of water
potential can be integrated to give total water potentials. Centrifugation provides the best method to
remove rubber particles in Hevea latex: valid osmotic
potentials of serum can be made providing appropriate
precautions are observed.
Key words: Latex pressure potentials, laticifer solute potentials, pressure pump, manometer, xylem solute potentials.
Introduction
Laticifers have been mainly studied in Hevea brasiliensis
(Willd. ex. Adr. de Juss.) Muell. Arg. (rubber tree) on
account of its great commercial importance. In addition
to providing 'commercially useful indices', latex pressures
and volumes, in the field, this system allows interesting
physiological measurements on other laticiferous plant
species of which there are great numbers. Thus if both
pressure and solute potentials of the latex can be measured
it should be possible to compare overall laticifer water
To whom correspondence should be addressed. Fax: +61 67 73 3283. E-mail: jmilbum©metz.une.edu.au
6 Oxford University Press 1996
136
Milburn and Ranasinghe
potentials with similar water potentials determined on the
xylem sap system.
It is critical to ensure that pressure and osmotic measurements made on the latex system are both reliable and
accurate and existing field methods have been compared
with new techniques. By far the best published results
for latex pressures are those of Buttery and Boatman
from bubble-gauges. However, their gauges are labourintensive to construct and are needed in large numbers
because they are essentially disposable, as they become
contaminated by coagulated latex. A new version of their
bubble-gauge was developed as a baseline for other
comparisons and, eventually, the whole instrument was
redesigned.
A problem affecting bubble-gauges is that, as pressures increase, their accuracy declines rapidly: standard
Bourdon pressure gauges remain accurate at higher pressures. Hence a pump system was designed and constructed
which could be used to stop exudation of latex, so that
a 'null point' pressure could be read (analogous with
a pressure probe used for single cells), repeatedly if
necessary.
Experience showed that the pump system was slow
and labour-intensive, so it was decided to streamline the
design and construction of bubble-gauges. An invaluable
set of short-cuts has been developed which greatly reduce
costs, time and failure rates and precision is apparently
increased. All positive-pressure gauges are susceptible to
leakage at the junction with the numerous vascular cells
concerned (Fig. I A). It is argued that the highest pressure
measured indicates minimal leakage and hence is the most
Sieve Tubes
BARK
/
Latex Vessels
Medullary rays
Wood
Cor1<
Cork Cambium
Stom
(a)
:ambium
ARALDITE
GRAPH PAPER
SCALE
SEAL
MICRO CAP
Fig. 1. (A) Block diagram of bark and wood of Hevea at the tapping
stage. The hard bark is r. 3 mm thick; soft bark containing the tapped
laticifers c. 2 mm thick and the sieve tube layer c. 0.3 mm thick. (B)
Bubble manometer (Buttery and Boatman type).
accurate result. Only in exceptional situations could pressures be artificially elevated above this maximum (e.g.
from a ramming effect on trapped liquid), something
which has not been experienced.
Osmotic potentials of clarified laticifer sap, i.e. serum,
has been measured a number of times since Pakianathan
(1967). Rubber particles, which coagulate easily, render
accurate measurements very fickle, blocking tubes and
contaminating thermocouple probes, such as those within
the vapour pressure osmometer. Both whole latex and
serums have been tested using high speed centrifugation
to remove nearly all the rubber particles. In addition,
refractometer readings have been tested to see if they can
be used to provide rapid estimates of osmotic potentials
as used previously on phloem sieve-tube saps (Milburn,
1974; Milburn and Zimmermann, 1977).
Materials and methods
Plant materials
All plants and samples used were field-grown Hevea brasiliensis
(Willd. ex. Adr. de Juss.) Muell. Arg. trees (Clone PB86 and
RRIC100) in Dartonfield, Agalawatta, Sri Lanka unless
otherwise stated.
Estimating laticifer pressure potentials (latex i//p)
Bubble manometer: Buttery and Boatman type
Construction: Injection needles (23 gauge, length 1.5"; Neolus,
USA) were cut from their plastic connectors using a threecornered file and a pair of pliers. Care was taken to make all
the needle tips the same length. Some epoxy cement (Araldite
Rapid, 5 min) was placed near the cut end of the needle. It was
'swirled' then pushed inside a glass capillary (100 /il microcap—
Drummond, USA). After the epoxy cement had hardened, the
open end of the glass microcap was sealed by heating it in a
small gas flame. Graph-paper scales were fixed along microcaps
to complete construction (Fig. IB).
Use in latex </ip measurements: In measuring latex ifip, a small
hole was bored into the bark towards, but not into, the xylem,
using a punch made from an injection needle with the tip
removed and sharpened to make a 'micro' cork-borer. The
manometer 'needle' was inserted immediately the borer was
withdrawn. Normally latex flowed in rapidly from many severed
laticifers. Once this latex column had stabilized within the
capillary (usually after c. 4-10 min), the pressure was estimated
from the length of the entrapped air column using a calibration
graph; this was cross-checked with a pressure bomb.
Bubble manometer: pippetor design
Construction: Using the same basic principles outlined above,
the new manometers were constructed using 50 ftl microcaps
with micropipette tips (Eppendorf, USA). A microcap was
pushed tightly into the pipette tip, the gap between pipette tip
wall and microcap was then filled with fresh epoxy cement and
stood vertically in a 60 "C oven to set. When sealed, the open
end of the microcap was heat-sealed in a small flame. A graphpaper scale was attached to the microcap (Fig. 2).
Methods to study vascular potentials in Hevea brasiliensis
137
ARALDITE
GRAPH PAPER
SCALE
DIAL TO READ
PRESSURE
~SEAL
\
EPPENDORFTIP
M1CROCAP
Fig. 2. Bubble manometer, Pippetor design.
MANOMETER
AIR
CX)LUMN
TUBING END
CONNECTS TO
THE TREE BARK
SCALE
Fig. 4. Portable high-pressure pump manometer. The tubing end ( x )
was connected to tree bark (laticiferous phloem) through the connectors
illustrated in Figs 5 and 6.
BLUE DYE
Fig. 3. Calibration of plastic tip manometer using the pressure bomb.
The length of air column (i) was measured while increasing the chamber
pressure (P).
Calibration check: The plastic tip of the manometer was fitted
into a plastic vial containing dye ( 1 % (w/v) aqueous Aniline
Blue). The gap between the manometer tip and the dyecontaining vial was sealed with epoxy cement. A pinhole
connection was made in the vial wall which was set up (Fig. 3)
in a pressure bomb (Soil Moisture Equipment Ltd., USA). As
the pressure increased within the chamber (P) the length of the
air column (i) inside the manometer was read on a graph paper
scale. Using P and l/i a calibration graph was constructed.
Latex i/ip measurements: Before using the manometer, the tree
bark was scraped lightly with a blunt blade, a hole was drilled
in the tree bark using a 1 mm diameter bit; the tapenng
manometer tip was then quickly pressed tightly into the hole.
Once the latex column had stabilized within the microcap, the
air column length (i) was recorded and the pressure (P) was
determined from the calibration curve.
Portable high-pressure pump manometer
The pump system (utilizing a high pressure pump as used to
inflate SCUBA compressed-air harpoon guns, Mares, Italy) is
described in Fig. 4. Air pressure could be applied to slow, stop,
or even reverse exudation from wounded laticifers.
This instrument consists of three main parts.
1. A cylinder with a piston which can generate pressure by
pushing a plunger.
2. A gauge to read the pressure inside the system.
3. A long transparent plastic tube connecting the pressure
system and the laticifers. The latex position could be
observed through the transparent tubing. An attachment
was needed for the tree junction.
The minimum balancing pressure, at which latex flow through
the tubing was stopped, provided the turgor pressure inside the
laticifer </!„ (latex tpp). A perfect seal without gas leaks between
the laticifers and the pump system is essential for correct
readings. Various attachments were tested to anchor the tube
within the tree with a perfect leak-free connection while
puncturing as many laticifers as possible.
Brass wood screw connectors: A hollow, tapered and threaded,
brass wood-screw with a pointed tip and a side port for latex
entry and a riffled base, was attached to the pressure pump
with metal clamps. At a height of 1.5 m above-ground a hole
was drilled (3 mm drill-bit) into a tree trunk almost into the
wood. As soon as the drill-bit was withdrawn, the brass tip of
the connector was quickly inserted. Latex was expected to flow
via the port into the tubing. By applying a back-pressure with
the hand pump, latex flow could be slowed down until, as the
pressure was increased, it eventually stopped. At this point the
gauge pressure was noted. At first trials failed, because the side
port hole was too small and was blocked readily by bark
fragments. In later tests a larger hole was made: a little
ammonium carbonate powder in the tubing serving as an
anticoagulant.
Injection needle connectors: An injection needle (gauge 23,
length 1.5", Neolus, USA) was pushed from the base of it into
PVC tubing with the same internal diameter as the injection
needle base. Another connection tube was fitted tightly to hold
and seal the clamped needle. The basal end of this system was
attached to the pressure pump as indicated in Fig. 5. When the
needle tip was retracted c. 2 mm, latex began to exude via the
PVC tubing. Latex flow could be stopped using the pump to
apply positive pressures. Then pressure opposing flow equals
the laticifer pressure potential (latex i/>p). Possible gas leaks in
the unit itself were detected by testing the equipment under a
container of water before latex </rp measurements were made.
For each new measurement, a fresh (reusable) 'tip unit' replaced
138
Milburn and Ranasinghe
convenient location, for comparative measurements of
turgor.
Latiafer solute potential (latex tpj
INJECTION
NEEDLE
END CONNECTS TO
THE PUMP TUBING
Fig. 5. Connector used between laticifers and pressure pump at early
stages of work (injection needle connector).
the previous attachment. This pressure pump with the needle
attachment was used to study diurnal latex turgor pressure
variations in wintering and non-wintering trees.
Eppendorf pipette tip connectors: A transparent tube (o.d. 3 mm)
was pushed tightly into an Eppendorf pipette tip. A gap
between the tubing and the pipette tip was sealed with epoxy
cement. An injection needle was cut at the tip, using a three
cornered file, and then polished with fine emery paper. It was
coated with epoxy cement and pushed into the free end of the
transparent tubing. Both ends of this tube were held vertically
while the glue was setting. This tube, connected to the pump,
was inserted into an injection syringe-barrel and the gap
between them was filled with epoxy cement. The needle base
(the Luer female tip) and the syringe barrel was conveniently
detachable, but rapid and leak-free attachment between pump
and latex system occurred (Fig. 6). The tip inserted into the
tree was cut obliqely to minimize blockage. A 3 mm hole was
drilled almost as deep as the wood in the tree trunk. The
Eppendorf pipette tip end was quickly inserted into the hole.
By applying pressure from the pump the latex flow was stopped
and the latex turgor pressure recorded. This plastic tip
attachment proved to be so satisfactory that it was used in all
subsequent experiments.
The tree bark was wounded using a needle. About 0.5 cm3
escaping latex was collected into an Eppendorf vial kept in ice.
The sample was then centrifuged for 30 min at 12 000 rpm.
After opening the lid of the vial, a hole was made in the vial
wall, using a needle. By stirring the needle tip, serum could be
mixed without disturbing the uppermost 'cream rubber' layer.
This layer was then pushed down gently allowing the removal
of a single drop of clear serum. The osmotic concentration of
this droplet was measured using a vapour pressure osmometer
(Model 5500, Wescor, USA). The aim of centrifugation of latex
is mainly to protect the osmometer thermocouple head from
contamination by rubber particles from bursting latex bubbles.
Latex I/I, and latex serum zlRI: Latex samples were collected
into Eppendorf vials from holes drilled into Ficus bark. These
samples were taken to the laboratory in ice, centrifuged for 30
min, and the serum was used for latex I/I, determinations using
the same procedures as for Hevea latex.
A drop of centrifuged latex serum was used to measure ARI
on a bench refractometer (Atago Optical Co. Ltd., Japan) at
25 °C. All results are normally expressed, as previously, as the
RI difference from water {ARI x 10"*), which provides a very
convenient scale.
Xylem pressure potential (xylem \j/pj
For each measurement, a leaf was detached from the tree,
sealed in a humidified polythene bag, then earned rapidly to
the laboratory. The minimum pressure needed to make xylem
sap exude using a pressure bomb was measured (Scholander
et al, 1965; Waring and Cleary, 1967).
To prevent interference from latex exuding while pressure
was being applied, the petiole was ring-barked about 2 cm
below the distal end. The surface of the petiolar ending was
recut before each reading of xylem i/rp to minimize terminal
blockages.
Comparison of methods in latex \pp determination
Xylem solute potential (xylem \j/J
1. The simple manometer (Buttery-Boatman), and the plastic
tip manometer were both used to measure latex </>p simultaneously in Hevea brasiliensis.
2. Two operators used two pressure pumps with different
attachments, injection needle and Eppendorf tip, to measure
latex i/ip simultaneously. Manometers were also used to
measure latex i/rp, wherever possible to provide further
comparative checks.
3. Plastic tip manometers and the pressure pump, with the
Eppendorf tip attachment system, were used to make several
latex </>p measurements on a Ficus elastica (Roxb.) tree
growing at Con's Harbour, NSW, selected for its size and
Two methods were used to collect xylem sap.
1. After measuring xylem tensions using a pressure bomb, an
extra pressure was applied. Xylem sap exuding from the
petiolar stump was collected into a micro-pipette.
2. The vacuum xylem sap extraction method (Bollard, 1953,
1957) and since used extensively (Pate, 1975, 1980) was also
used. The apparatus used is shown in Fig. 7. A small branch
was harvested from the tree and quickly carried to the
laboratory sealed in a polythene bag. Bark was then removed
from the cut end of the branch to minimize contamination of xylem sap with cellular or phloem sap. Next, while
ENDFIXWrm
NEEDLE BASE
INJECTION SYR1NCER
BARREL
CONNECTING
TUBE TO THE PUMP
Fig. 6. (A) Connector used between laticifers and pressure pump after improvements (Eppendorf-tip connector). (B) Modified pressure pump
tubing end to hold the connector described in Fig. 6A.
Methods to study vascular potentials in Hevea brasiliensis
SHOOT PROGRESSIVELY
TRIMMED BACK WITH
SECATEURS
LEAFY SHOOT
FROM TREE
VIAL
HOLDING'
THE
BRANCH
BARK IS REMOVED
FROM THE BRANCH
RUBBER
BUNG
VIAL
COLLECTING
XYLEM SAP
139
Results
Laticifer pressure potential (latex tj/^
Results for the various manometer systems, in comparison
with other techniques, are exemplified by data collected
over 2 weeks in March 1992, summarized in Table 1.
Portable high-pressure pump manometer
iEPARATING
FUNNEL
PACKING TO Hi
THE COLLECTING
VIAL 4 SUPPORT
THEVACCUM
END CONNECTED TO
VACUUM PUMP
Fig. 7. Apparatus used to collect xylem sap for ifi, determination. While
applying near vacuum, the tree branch was trimmed in small pieces
sequentially from the distal end (Bollard, 1957).
applying vacuum, portions of shoot tips were cut progressively from the top of the branch using pruning shears. Xylem
sap exuding from the enclosed stump was thus collected.
Osmotic concentrations of xylem sap were measured by
vapour pressure osmometry (Model 5500, Wescor, Logan,
USA).
Use of selected techniques to estimate water potentials of latex
and xylem sap
Utilizing the technique described above, phloem and xylem
water potentials were estimated in the following situations.
Water potentials in non-wintering trees: The following parameters
were studied in four field-grown untapped non-wintering Hevea
brasiliensis trees (clone PB86) hourly. Latex i/»p was measured
on the tree trunk, 1.5 m above ground, using the pressure pump
with the needle attachment. Centrifuged latex serum from
appropriate experiments were utilized for latex ifip determination.
Xylem </ip was determined using the pressure bomb and I/I, was
measured in the osmometer, using both the pressure bomb
collection and the vacuum-extracted sap.
Brass wood screw connectors: All pressures measured were
less than 0.2 MPa. Complete cessation of latex flow could
not be achieved reliably by application of pressure from
the pump on account of persistent leaks in the system.
This system was eventually abandoned.
Injection needle connectors: Results (Fig. 8) indicate latex
</ip values ranging from 0.35 to 0.85 MPa in the two trees.
Early morning latex tfip values were higher, then towards
the end of the day the magnitude of latex </rp declined
seeming to indicate true changes in laticifer turgor pressures. Tree 1 has a higher value at the beginning of the
experiment and by midday latex </ip in both trees changed
to a similar degrees.
Eppendorf pipette tip connectors: Eppendorf connectors
were compared with the other methods used previously.
Results are presented in Table 1 and discussed below in
comparison with other techniques.
Comparison of methods: latex \pp determination
Measurements made with two pressure pumps using
different attachments and also bubble-gauges, about the
same time on the same tree, are listed in Table 1.
The plastic tip manometer gave higher values than the
(conventional) Buttery and Boatman construction of
bubble manometer. The newly-designed manometer readings accord with both pressure-pump readings with
Eppendorf-tip connectors. These two devices usually gave
higher pressure values than the two other devices (conventional manometer and needle-tip connector with the pressure pump). Higher pressure values probably indicate
Water potentials in wintering trees: Trie following measurements
were made on trees while they were 'wintering' (i.e. leafless).
Latex i/ip and ip, were estimated by the same methods used on
non-wintering trees. Xylem ifip was determined on bare twigs
using a pressure chamber. Only the vacuum xylem sap extraction
method (Fig. 7) was used to collect xylem sap. Xylem sap if>,
was determined using the vapour pressure osmometer.
Contamination of xylem sap with phloem sap: A further
experiment was performed to check the results obtained under
(a) above. Xylem sap was collected from Hevea leaves and
branches using the pressure bomb. Prior to sealing into the
pressure bomb a bark ring (about 0.5 cm) was removed from
the cut end of the branch. Xylem sap was collected in branches
of Hevea using the vacuum sap extraction method. Collected
sap was analysed estimating ARJ, Osmotic potentials and
sucrose concentrations (Ashwell, 1957).
11
12
13
TIME (HOUR)
Fig. 8. Latex </rp values obtained for two Hevea trees simultaneously
using needle connector and pressure pump.
140
Milburn and Ranasinghe
Table 1. Latex <pp values obtained using different devices
Values given are the average of several readings, number of readings is given in brackets:figuresfollowing indicate the range of the readings, lowest
then highest
Date
17.03.92
21.03.92
21.03.92
22 03.92
22 03.92
22.03.92
22.03.92
24.03.92
24.03.92
24.03.92
24 03.92
26.03.92
26.03.92
29.03 92
29.03.92
Manometer reading (bar = MPa x 10 ')
Pump reading (bar = MPa x 10 ')
Buttery/Boatman
Needle tip
Plastic tip
5 8(2. 5.6-5.9)
64(2, 6.1-67)
7.6(2, 7.6-7 6)
7.1(2, 6.8-7.3)
6.3(3, 4.6-7.3)
7 50(1)
6 00(1)
6 4(1)
8.9(2, 7.5-10.2)
8.9(2, 8.5-9 3)
5.6(2, 5.6-5.6)
6.0(2, 6.0-6.0)
9.5(3,9 3-9.5)
10 1 3, 9.8-10.6)
8.1(3, 7.5-8.5)
5 1(3, 4.5-5.6)
minimized gas and fluid leaks in the system. Readings
from needle tip connectors with the pressure pump were
not consistent with the other readings. The values
obtained are always somewhat lower than might be
expected. This indicates gas and fluid leakage may have
occurred from the needle connections from a relatively
small number of wounded laticifers, hence underestimating actual values.
The plastic tip manometer and Eppendorf-tip connections with the pressure pump were selected for more
detailed studies in latex </>p on account of their ease of
use and freedom from leakage, as discussed below, along
with their similarity in performances.
Eppendorf tip
5.6(3, 5.2-5.9)
5.0(2, 5 0-5.0)
5.5(2,4 5-6.2)
4 8(3,4.8-5.0)
4.3(2,4.0-4.5)
4.8(4 4.7-5.0)
3.9(2, 3.8-4 0)
6.4(2, 6.3-6 5)
8.6(3, 7.9-9.0)
7.4(2, 7.3-7.4)
7 0(3, 6.8-7.1)
8.6(3, 8 5-8.7)
5.7(2, 5.5-5 9)
5.9(2, 5.2-6 6)
7.9(2, 7.8-7.9)
8.9(2, 8.9-8.9)
8.0(3, 7.9-8.2)
5 4(2, 3 8-6 9)
-1.2
10
12
14
HOUR OF DAY
16
Latex I/IS and J R I : The virtually linear relationship
between latex >p, and ARI is presented in Fig. 9.
Variation in potentials of latex and xylem sap
Non-wintering trees: Figure 10A represents the anticipated
behaviour pattern of trees in relation to both latex and
10
12
HOUR OF DAY
14
16
Fig. 10. Typical diurnal behaviour in potentials of latex and xylem sap
in non-wintering (leafy) Hevea trees (A) represents changes in latex </ip
and xylem </ip and (B) represents changes in latex i/r, and xylem i/r,.
Xylem i/r, values, obtained from vacuum collection, are close to zero
and are free from contamination. High xylem ^, values in pressure
bomb collections are due to probable contaminations
20
Fig. 9. Relationship between refractive difference from water (x 10 4)
and osmolarity or I/I, of Ficus elaslica latex (1 osmolen-2.4 MPa
at 25 C).
xylem </ip. An increase in xylem tension towards midday
was followed by a decrease towards the end of the day.
Latex i/ip changes followed the xylem sap tensions.
Maximal values of 1.4 MPa occurred in the early morning,
decreasing to 0.35 MPa towards midday, then increasing
again towards the end of the day. Figure 10B represents
Methods to study vascular potentials in Hevea brasiliensis
results for latex and xylem i/i,. Latex tp, remained practically constant at around -1.0 MPa throughout the day.
Leaf xylem </>, measured from sap collected using the
pressure bomb, remained almost constant at -0.3 MPa.
These values seemed too low (i.e. high in solutes) for the
pure xylem sap, which is normally almost pure water
(Salisbury and Ross, 1969). These results apparently
indicate possible contamination. Osmotic values measured
on sap samples, collected by vacuum sap extraction, were
close to zero, which was not unexpected.
141
fairly constant around -1.1 MPa throughout the day.
Xylem </>, values also remained constant and stayed close
to zero (virtually pure water).
Contamination of xylem sap with phloem sap: The results
presented in Table 2 show significant concentrations of
solutes (indicated by ARI, osmotic potentials and sucrose
concentrations) within xylem sap extracted from leaf
petioles using the pressure bomb. The other two methods,
extracting sap by pressure bomb from ringed branches,
and using vacuum, indicated that it was virtually pure
water. These results indicate potential contamination
from pressurized bark.
Wintering trees: Figure 11A indicates latex and xylem i/>p
throughout the day. Under wintering conditions, the latex
</rp value in trees remained relatively constant around
0.8 MPa. Xylem i/ip also remained constant at around
-0.35 MPa. Figure 1 IB shows </is values for latex and Discussion
xylem. Latex tfi, behaved as in non-wintering trees, being
The aim of this research has been to simplify measurements of pressure while maximizing accuracy at minimal
cost, in terms of both time and expenditure. After extensive work on laticifers, and also other systems such as
sieve tubes, it is considered that the manometer design
originally devised by Buttery and Boatman (1966) is the
best conventional reference field instrument against which
any possible improvements should be tested.
The Buttery and Boatman design has, however, definite
disadvantages. Firstly, and most seriously, it becomes
-5
inaccurate at high pressures, on account of the fact that
10
12
14
HOUR OF DAY
a doubling of the pressure reduces the gas volume by
half. If continued, the gas volume quickly reaches a
fraction of a millimetre at high pressures, which can not
be estimated very accurately, even aided by optical magnification. Secondly, there is another potential problem,
because some of the pressurized entrained gas might
dissolve in the column of entrained sap. Thirdly, all such
pressure manometers suffer from a possible escape of sap,
especially at the junction with the laticifers themselves.
There is also a danger, in the case of latex measurements
especially, that rubber coagulation may block the system
10
12
14
HOUR OF DAY
producing a reading which underestimates the true value.
Another possibility, perhaps only a slim one, is that the
Fig. II. Typical diurnal behaviour in potentials of latex and xylem sap
pressures determined exceed the true pressures being
in wintering (leafless) Hevea trees. (A) represents changes in latex and
xylem i/rp; (B) represents changes in latex and xylem </*,.
generated if, during the insertion of the needle, plant
Table 2. ARI, osmolanty and sucrose level in xylem sap collected from different organs using two techniques
Sample
Method
1
2
3
1
2
3
1
2
3
Pressure
Pressure
Pressure
Pressure
Pressure
Pressure
Bollard
Bollard
Bollard
bomb
bomb
bomb
bomb
bomb
bomb
Organ
Expressed sap
(ARIx 10" 4 )
Sucrose
(%)
Sucrose
(mM)
Osmolanty
(mOsm)
Leaf
Leaf
Leaf
Branch
Branch
Branch
Branch
Branch
Branch
25
65
20
5
5
4
3
10
3
3.03
5.39
1.21
0.14
0 18
0.09
0.06
0.14
0.07
88.4
157.4
35.4
4.2
5.4
2.7
1.8
4.2
2.0
_
—
50
—
»0
«0
*0
142
Milburn and Ranasinghe
material is driven plunger-like into the syringe needle.
Finally, on account of contamination by rubber particles,
it is virtually impossible to use the same gauge twice;
hence they must be disposable and so simple and cheap
construction is essential.
The attraction of setting up a pressure pump system is
that it should overcome some of these problems. Firstly,
only a single instrument would be required. Secondly, it
should give an accurate pressure measurement, even at
high pressures, because it could utilize a linear pressure
gauge. Thirdly, it should allow repeated measurements
on the same system, allowing the continuity of the latex
flow to be checked throughout by applying extra pressure,
so forcing latex back into the tree and then allowing a
new balance point to be determined as often as required.
To a certain extent our new device has allowed us to
check on the validity of the bubble manometers in exactly
this way. The results indicate that systematic errors, such
as those caused by gas dissolution in the bubble manometer, do not seem to generate a serious source of error.
However, the very nature of latex, which tends to form
a deposit on the wall of transparent tubes, has rendered
a reversal of the meniscus difficult to observe. In this
sense the system is unlike a pressure bomb when used to
estimate xylem sap tensions, because water is a liquid
which does not leave a deposit, being truly reversible so
that readings can be taken repeatedly. Such a 'null point'
system requires time for raising and lowering pressures:
in the case of latex these adjustments are slow (indeed
sluggish) on account of the considerable latex viscosity,
when compared with virtually water (xylem sap via pressure bomb). On the other hand a great attraction of both
manometers and also pressure pumps (as used here) is
that they can be used easily under field conditions on
intact trees, rather than on destructively sampled twigs,
as in the case of the pressure bomb.
However, the most serious problem with the pressure
pump in practice, is its slowness in achieving a balance
point and as presently developed, it certainly works best
in the hands of two operators. Hence it is both slow and
time-consuming in operation. Further, the connection
tube needs to be cleaned thoroughly to minimize latex
coagulation between readings; it has been found desirable
to have an array of connection tips when performing a
series of measurements. In the best cases, the connection
with the tree has remained in position over several hours,
allowing several measurements to be repeated. But in
reality the latex pressures do not seem to vary much over
short periods of time, so such measurements are not
particularly useful. On the other hand if laticifers to
which the pressure pump is already connected are punctured elsewhere on the tree, even though some distance
away, this can be detected rapidly and easily, providing
latex deposits have not obscured the transparent tube.
Latex then retreats backwards rapidly into the tree,
moving towards the puncture.
In any attempt to measure latex pressures, there is a
critical relationship between the size of the potential
reservoir, the flow rates through the system and the
amount of sap required to operate the pressure-measuring
system. The 'connection volume' between the gauge and
the laticiferous system is critical; it must also exert only
a minimal resistance to flow. Hence it is desirable to
make a connection with as many severed laticifer vessels
as possible. However, as the size of the bore-hole is
increased, it becomes increasingly likely that there will be
serious leakage and escape of latex. Also it is essential
that there is minimal tissue brei near the connector tip,
which could cause blockages. For this reason we advocate
a sharp drill to make a clean hole which is stopped just
outside the cambium. Into this hole a gradually-tapered
conical tip is sealed by friction-fit, providing an efficient
seal. Attempts to use syringe-needle tips alone, without a
drill, suffered probably because insufficient numbers of
laticifers were punctured to compensate for the leakage
and also there were relatively serious blockages within
the unprotected needle caused by brei.
In these experiments, the generation of maximal pressures have been exploited as the guide for efficacy. It is
easy to measure a pressure less than the true value,
because of leakage. On the other hand it is virtually
impossible to produce overestimates of these pressures.
For the above reasons, the highest pressures measured at
a site have been accepted as the best estimate of true
values, rather than a mean from several readings, on the
grounds that leaks would produce invalid underestimates
for some readings, which should not be allowed to detract
from the most accurate readings.
The new design of disposable manometer combines the
advantages from the Buttery and Boatman design with
what we have learned from our pressure-pump trials. It
is very easy to construct and handle, having only three
components (pipettor tip, glass micropipette and graphpaper scale). Apart from having to seal the glass tubes at
one end, all components are standardized and available
directly from the manufacturer. The readings are almost
non-invasive; virtually no sap escapes. The gauges do not
require prolonged or constant monitoring, one can set up
several in rapid series and read the values when convenient. Because the amount of latex required is small the
effects of viscosity are not serious and a stable reading is
usually obtained within seconds or, at worst, after a few
minutes. The rapid equilibration also probably indicates
minimal readjustment of water from living cells surrounding the disturbed laticifers.
The pressures measured by the above techniques
seemed to be valid and accurate. In non-wintering trees,
early morning latex ipp had the highest value of 1.4 MPa.
At that time the xylem tension was —0.3 MPa. Towards
Methods to study vascular potentials in Hevea brasiliensis
midday, on account of transpiration, xylem </>p decreased
down to — l.OMPa. Because of this transpirational pull,
water should be withdrawn from laticifers; they actually
became less turgid, giving a low latex ifrp value around
0.4 MPa. By the end of the day, transpiration was reduced
and both xylem and laticiferous tissues returned overnight
to their most hydrated status. These observations accord
with the Buttery and Boatman (1966) observations on
Hevea in Malaysia, which explain the loss of turgor in
laticifers in daytime as due to high transpirational rates.
Latex and xylem </i, did not fluctuate, in contrast with
large differences in tpp values. Latex serum osmotic potentials remained relatively constant around — 1.0 MPa with
only slight variations from that value. The values obtained
for xylem </>, using two sap extraction methods indicates
contamination of pressure bomb samples from Hevea
petioles, probably by phloem sap expressed from sieve
tubes. Xylem sap collected by this method must be
considered unreliable for xylem tf>, determinations, and so
it is better replaced by the Bollard (1957) vacuum xylem
sap extraction method unless combined with bark ringing.
Xylem I/I, did not exhibit any significant diurnal variation,
nor is it reasonable to suppose that xylem </i, should
suddenly change while flowing from branches and twigs
into petioles and leaves. The difference between these
tissues is that stems can be bark-ringed to eliminate sieve
tube effects, but petioles can not be ringed in this way.
Latex i/ip and xylem if>v in wintering trees remained
relatively constant throughout the day. Since the tree was
devoid of leaves, little transpiration was expected. The
laticifers remained fully turgid throughout the day
because water was not extracted via a transpirational
pull. The slight decrease of latex i/ip towards the end of
the day may be caused by some slight transpiration from
the leafless aerial parts of the plant, in accordance with
Buttery and Boatman's (1966) observations. Latex I/I,
remained constant around —1.1 MPa throughout the
day. Xylem </r, remained constant with a value close
to zero.
The comparison of different xylem sap extraction
methods for xylem </«, determinations, confirmed that the
contamination of extracted xylem was from sieve tube
sap. AR1, sucrose concentration and osmolarity of leaf
xylem sap, collected using a pressure bomb, were greater
in comparison with sap collected from bark-ringed stems
by pressure bomb or vacuum sap-extraction techniques.
These results suggest equally accurate and satisfactory
results from both of these latter methods.
143
Acknowledgements
The authors wish to thank the Australian International
Development Bureau for funding this work and D Jenkins and
staff in the Physics Workshop, UNE, for construction of the
pump assembly. We thank the Director, Dr LMK Tillekeratne,
and also MissTushara Peris and Mr NLD Daynaratne of the
Rubber Research Institute of Sri Lanka who provided general
assistance.
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