MATERIALS AND METHODS

Experiment I

Plant material

The plants for the experiment were purchased from Vandeputte N.V., in Belgium. Rooted cuttings were transplanted in to 10-cm pots with peat-perlite 3:1 as a substrate and placed randomly in a greenhouse where they remained until the end of the experiment.

The experiment was conducted at ‘Unifarm Experimental Center’ in Wageningen, The Netherlands, starting in September 2001.

Temperature – Lighting

Average monthly temperatures of the greenhouse are shown in Table 1.

Table 1: Average Temperature (^oC) in the greenhouse during the experiment

Plants were under natural light supplemented with high-pressure sodium lamps to provide 16-h days starting at 06:00 in the morning, as Callistemon has high light requirements. Heating was also provided during the experiment.

Fertilisation – Diseases - Irrigation

One application with Fe solution was applied as Callistemon is often suffers from iron deficiency. There were no treatments for pests and diseases since Callistemon is rarely infected, and no pests or diseases were reported. Irrigation was done manually every morning. After application with growth retardants plants were not watered for one day.

Experiment description

The purpose of this study was to evaluate the effectiveness of paclobutrazol and cycocel as a root-medium drench on the control of Callistemon citrinus growth. A completely randomised design was used with 6 single pots per treatment. In total 4 applications, applied at 2 weekly intervals, were made. Each plant received 100 ml solution in each application.

Treatments

i.                     Control

ii.                   20 ppm Cycocel

iii.                  30 ppm Cycocel

iv.                 40 ppm Cycocel

v.                   5 ppm Paclobutrazol

vi.                 10 ppm Paclobutrazol

vii.                20 ppm Paclobutrazol

Bonzi (paclobutrazol) (from Zeneca Agro Company) with 4grl^-1 concentration of active ingredient and Cycocel (from R.Van Wesemael b.v.) with 750gr l^-1 were used.

Measurements

The height of the plants was measured every week. The final height measurements were taken one month after the last application and these measurements were mainly used for the evaluation and the statistical analysis. The internode length, leaf characteristics, number of shoots per plant, total leaf area, total specific leaf area and fresh and dry weight of stem, leaves, roots were also measured at the termination of the study. The effects of growth retardants on root growth and on starch levels were also evaluated. Finally a market quality rating was conducted.

Starch analysis

A starch analysis of a sample of 20 leaves was done for all treatments after the end of the experiment, in order to observe any possible changes in starch content between treated and control plants.

Leaf samples were dried, dry weights were taken and samples pulverised using a ball-mill grinder. 5ml of 80% ethanol were added to 15mg of each of the powdered plant tissue samples in centrifuge tubes. Tubes were vortexed, put in a shaking waterbath (80°C for 20 minutes), vortexed again and centrifuged for 5 minutes at a speed of 8000rpm. The supernatants were discarded, and 3ml 80% ethanol were added to each tube which was then vortexed and centrifuged for 5 minutes more at the same speed (8000rpm). This last step was repeated 3 times. Afterwards the pellets were dried in a SpeedVac concentrator for 30 minutes. 2ml alpha-amylase solution, consisting of 1 mg Rohalase/ ml H₂O, was added to each of these pellets, which were then put in a shaking waterbath at 90°C for 30 minutes. 1ml of amyloglucosidase (1 mg/ml citrate buffer, 50mM pH=4.6) was added and tubes were put in a shaking waterbath at 60°C for 15 minutes. After that, 1ml of each sample was put in an Eppendorftube and centrifuged for 15 minutes. Samples were then diluted 10 times to be finally analysed as glucose in a high performance liquid chromatography or HPLC apparatus. The glucose data multiplied by 0.9 to converted them to starch values (Gitzels, 1996).

Root analysis

After the end of the experiment the roots of each plant were separated and cleaned carefully of soil. Measurements were made of the main root length (primary root), the number of secondary roots and the average length of secondary roots.

Market quality rating

People were asked to give a market quality rating to all treated plants. It has to be mentioned that none of them knew of the plant Callistemon citrinus before the assessment, and they judged the general appearance of each plant, giving a score from 0-4, with 1: poor quality, unmarketable; 2.5: marketable; 4: excellent. The labels, indicating the treatment that each plant was receiving during the experiment, were covered during the quality rating.

Experiment II

Plant Material

Plants were purchased from a retailer company in The Netherlands (Xotus), in the middle of March 2002. Plants were 80-100 cm height with peat as substrate. They were placed outside since a greenhouse is not necessary at this time of the year.

Irrigation was done manually every morning. After application with growth retardants plants were not watered for one day.

Experimental description

The purpose of the experiment was to evaluate the effectiveness of paclobutrazol and Cycocel as a root medium drench on the frost resistance of Callistemon.

The experiment was done in two parts. In the first, plants immediately after they were placed in a cold room (5 C) to induce cold hardiness, were treated with growth retardants and starting the next day, frost resistance was evaluated for the next 5 days. 2 plants per treatment were used and leaves from those plants were the material for the experiment.

In the second part, in order to evaluate if there is any effect of growth retardants on non-acclimated plants, the same experiment was carried out without placing the plants in the cold room.

In both parts of the experiment, the same setup was used. 3 pots per treatments per temperature were used with 2 leaves per pot.

The temperatures that frost resistance was tested were 4, 0, -4, -8, -12 and -16 ⁰C. The temperature was reduced 4 degrees every 20 minutes.

Treatments

i.   Control

ii.  20 ppm Cycocel

iii. 30 ppm Cycocel

iv. 5 ppm Paclobutrazol

v. 10 ppm Paclobutrazol

One application was done one day before the beginning of frost resistance test. Each plant received 250 ml solution of growth retardants.

Bonzi (Paclobutrazol) from Zeneca Agro Company with 4 grl^-1 concentration of active ingredient and Cycocel (from R.Van Wesemael b.v.) with 750 grl^-1 were used.

Frost Resistance Test

Healthy leaves were selected and cleaned with milliQ water (low EC) to remove any surface contaminants that could interfere with the measurements (such as algae or particles of dirt). Leaves were placed in plastic pots along with 3 cm³ of milliQ water.

These were placed on a wire mesh shelf inside a freezer fitted with a proportional response temperature controller and a fan was used to blow air over the pots and increase the cooling rate of the discs. The freezer air temperature was initially 4 ⁰C and was reduced by 4 ⁰C every 20 minutes. Samples were removed every 20 minutes and allowed to warm up initially in ice and then at room temperature before an extra 50 cm³ of milliQ water was added to each pot.

About 24 hours later the conductivity of the water in the pots was measured with a conductivity meter. After that, the pots were heated to ~80 ⁰C for at least 24 hours to ensure the leaves had been killed. This treatment allowed the release of all the mobile electrolytes contained within each leaf. Once the pots had cooled to room temperature the conductivity of the water was measured again, and the conductivity measured 24 hours after the freeze treatment was then expressed as a percentage of the conductivity after the leaves had been heated and killed. Both measurements of conductivity were corrected by subtracting the conductivity of the appropriate blanks.

The above procedure was carried out on 5 consecutive days for both of the parts of the experiment, in order to see in which day growth retardants have (if they have) an effect on the frost resistance (Harbinson, 1984).

Statistical analysis

Statistical analysis was conducted using SPSS 10.1 package. An analysis of variance was performed and treatment effects were tested using an F-test at 5% probability. When significant differences were found according to the F test, the Tukey test was then used. For market quality rating Wilcoxon test was used.

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