2. Materials and methods
2
.
1
. Plant material Chrysanthemum Dendranthema x grandiflorum
Tzvelev cv. Cassa plants were grown in a green- house at Wageningen Agricultural University in
14 cm diameter plastic pots containing a commer- cial potting soil. The average temperature was
18°C and a 16 h photoperiod was maintained until the plants had formed 15 – 17 leaves longer
than 0.5 cm 3 – 4 weeks. Thereafter, an 8 h photoperiod was maintained until harvest. When
necessary, lengthening of the natural photoperiod was achieved by high-pressure sodium lamps
Philips SONT. Conversely, shortening of the natural photoperiod was by use of black screens.
2
.
2
. Har6est Flowering stems were harvested in the green-
house at commercial maturity by cutting the stalks at soil level. Length of the flower stems at
harvest varied between 0.7 and 1.0 m in various experiments. Flowers were brought to the labora-
tory as soon as possible. Lower leaves were re- moved up to 35 – 40 cm from the cut stem base.
Thereafter, cut stem ends were trimmed by 1 cm in air to get clean stem ends without soil particles,
and the stalks were placed for 3 h in a bucket with a mixture of ice and water 3:1 by volume in
darkness at 4°C to regain full turgidity van Meeteren, 1992. Because cutting height influences
water balance of cut chrysanthemum flowers Marousky, 1973; van Meeteren and van Gelder,
1999, the lower 30 of the stems were cut off in air after the hydration treatment. Previous experi-
ments suggested that this effect of cutting height is related to total plant length at harvest. There-
fore, a constant percentage of the stem length was cut off instead of cutting at a constant length.
Thereafter, the fresh weight of the flowers was determined as the initial weight, and flowers were
placed in the various vase solutions.
2
.
3
. Vase period conditions and 6ase solutions During the vase period, each flower was indi-
vidually placed in a 300 ml Erlenmeyer flask filled with 300 ml of the desired solution. The cut
flowers were kept in a room at 20 9 1°C, 60 9 5 RH and a light intensity of 14 mmol·m
− 2
·s
− 1
Philips, TLD 50W84HF with a light period of 12 h.
All vase solutions were made with deionized water 0.5 – 1.0 mS·cm
− 1
, with the exception of ‘tap water’ which was regular drinking water in
our laboratory. The composition of tap water according to the water company is given in Table
1. Data about some of the vase solutions used are given in Table 2. The pH of all solutions used
varied between 8.7 and 8.9. Osmolality of the solutions was measured by micro-osmometry
Roebling, Berlin.
The main components of ‘Wageningen tap wa- ter’ are HCO
3 −
and Ca
2 +
. By dissolving 110 mg
Table 1 Composition of ‘Wageningen tap water’ according to the
water-company
a
Compound Concentration
mg·l
− 1
mM Chloride Cl
−
7 0.197
B 0.02
Nitrite NO
2 −
B 4.35×10
− 4
B 1
Nitrate NO
3 −
B 0.0016
0.0625 Sulphate SO
4 2−
6 1.492
Hydrogencarbonate 91
HCO
3 −
2 0.045
Carbon dioxide CO
2
Carbonate CO
3 2−
B 0.0167
B 1
8.42×10
− 4
0.08 Phosphate PO
4 3−
B 0.05
Ammonium NH
4 +
B 2.78×10
− 3
3.58×10
− 4
0.02 Iron Fe
B 1.82×10
− 4
B 0.01
Manganese Mn 0.228
7.3 Oxygen O
2
27 0.675
Calcium Ca
2+
Magnesium Mg
2+
0.091 2.2
6 0.261
Sodium Na
+
Potassium K
+
0.013 0.5
0.6 Copper dissolving
mg Cu·l
− 1
ability 7.9
PH Electrical conductivity
0.017 S·m
− 1
a
Water source: Wageningse Berg, Wageningen. Analysed by Waterlaboratorium Oost, Doetinchem, The Netherlands.
Table 2 Composition, electrical conductivity and osmolality of vase solutions used
a
Concentration Solution number
Electrical conductivity mS·m
− 1
Compounds Osmolality mOsmol·kg
− 1
mg·l
− 1
mM 110
1.3 1.
13 NaHCO
3
2 CaCO
3
15 0.2
2. 125
NaHCO
3
1.5 32
5 99
0.7 CaCl
2
· 2H
2
O 3.
NaHCO
3
125 1.5
32 5
99 0.7
CaCl
2
· 2H
2
O 1.2
0.005 CuSO
4
· 5H
2
O 4.
NaHCO
3
125 1.5
34 5
100 1.3
KCl 1.2
0.005 CuSO
4
· 5H
2
O 125
1.5 5.
32 NaHCO
3
6 135.5
1.3 KNO
3
CuSO
4
· 5H
2
O 1.2
0.005
a
Solutions were made with deionized water.
NaHCO
3
and 15 mg CaCO
3
l
− 1
deionized water solution 1, Table 2, approximately the same
concentration of CO
3 2 −
ions was achieved. Cal- cium carbonate was added to increase the pH. In
solution 2 Table 2, both CO
3 2 −
and Ca
2 +
con- centrations were similar to those in tap water.
Low concentrations of Cu
2 +
can prolong vase life of cut flowers Laurie, 1936. By atomic-ab-
sorption spectroscopy Varian AA-20 plus, Palo Alto, CA, low amounts of Cu
2 +
were found in samples of tap water at our laboratory. Therefore,
CuSO
4
was included in some of the vase solutions. In an experiment investigating possible phytotoxic
effects of CuSO
4
, initial uptake of solution was increased by a dehydration pre-treatment over 1 h
to 5 of their saturated fresh weight to simulate dry storage or transport before placing the flowers
in various concentrations.
2
.
4
. Fresh weight measurements The flowers were weighed at noon during sev-
eral days of vase life. For that purpose, flowers were taken out of water for as short a time as
possible 20 – 30 s. The fresh weight of each flower was expressed relative to the initial weight
to represent the water status of the flower.
2
.
5
. Uptake and transpiration rates Average daily uptake rate was calculated from
daily measurements of the weight of the Erlen- meyer flasks without flowers. Values were cor-
rected for direct evaporation from the flasks. Average daily transpiration rate was calculated
from the combined weights of the flower and the Erlenmeyer flask. Short-term transpiration rate
was measured by placing four Erlenmeyer flasks each with a flowering stem on a Sartorius
LC3200D balance Go¨ttingen, Germany coupled to a personal computer. Weight was logged at a
60 s sample rate. For comparisons of treatments, a running average over 13 min was used.
2
.
6
. Hydraulic resistance Hydraulic resistance per unit length R
h
of stem segments was determined from measured
flow rates through the segments under a 40 kPa partial vacuum. Resistance was measured as
R
h
= D
PDx q
where DPDx is the hydraulic pressure gradient [kPa m
− 1
] and q the water mass flow [mmol·s
− 1
].
Partial vacuum DP = P
atmospheric
− P
actual
was obtained by lowering the actual pressure P
actual
at the upper end of the stem segment by a peristaltic tubing pump Watson-Marlow 505 Di,
Cheltenham, UK controlled by a personal computer. Actual pressure was measured using a
ceramic
pressure transducer
DVR 5,
Vacuubrand, Wertheim, Germany. Flow through the stem segments was calculated from weight
changes of a container filled with solution, in which the lower end of the stem segment was
placed. Composition of the solution was varied as mentioned for the individual experiments. Weight
of the container was measured on the balance coupled to the personal computer at a 1 s sample
rate averaged over 30 s. Measured flow rates were corrected
for direct
evaporation from
the container.
All sample preparation was done under the same solution as that used in the container to
prevent air-entrance into the vessels at the cut surface. Stems were cut into 20 cm long segments.
Leaves were removed from the stem segment with a razor blade, leaving 1 cm stubs of the petioles
on the stem. The upper side of the segments was inserted into silicon tubes also under solution and
attached to the measurement system. When hydraulic resistance was measured after several
days of vase life, the solution in the container during the measurement and the vase solution
during the vase life experiment were of the same composition. During preparation and during
change of solutions a small positive pressure due to gravity was applied to the tubing system
attached to the upper side of the stem segment to avoid air-entry at the basal cut surface.
2
.
7
. Bacteria In an experiment on the specific effects of
Cu
2 +
, bacterial numbers in vase solutions of NaHCO
3
plus CaCl
2
with and without CuSO
4
respectively, solutions 2 and 3; Table 2 were monitored after 2, 3, 4 and 6 days of vase life. A
third treatment involved treating the flowers in as sterile a manner as possible and placing them in
the solution without Cu
2 +
solution 2. In this treatment, the lower 15 cm of the stem was wiped
with a tissue saturated with 96 ethanol, Erlen- meyer flasks with vase solution were autoclaved
and during the vase life experiment the Erlen- meyer flasks were covered with aluminium foil.
Solution samples were diluted, plated onto Plate Count Agar Oxoid, Unipath, Hampshire, UK
and incubated at 27°C for 48 h before the number of colony forming units cfu was counted. These
numbers were expressed per millilitre of vase wa- ter.
2
.
8
. Statistical design In the vase experiments, the design was a ran-
domised complete block with 12 flowers per treat- ment. The flowers were placed in a climate room
with four blocks, and three flowers per treatment in each block. Within a block, the three flowers of
one treatment were grouped together to form one sample unit. Analysis of variance was applied to
relative fresh weight, average daily uptake and transpiration rate, followed by mean separation
according to Tukey’s HSD-test. Each treatment was
repeated at
least once
in a
separate experiment.
3. Results
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