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Stigma Receptivity in Eucalyptus Camaldulensis DEHNH

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Stigma Receptivity in Eucalyptus Camaldulensis DEHNH Stigma Receptivity in Eucalyptus camaldulensis DEHNH. By R. L. A. ODDIE and J. A. MCCOMB Biological Sciences, Murdoch University, Murdoch, Western Australia, 6150, Australia (Received 27th January 1998) Summary were at an earlier or later stage were cut off. The stamens of Stigma receptivity of Eucalyptus camaldulensis DEHNH. the selected flowers were removed using a thumb nail. They grown near Perth, Western Australia, was assessed by seed were then washed with deionised water to ensure all pollen production. Pollination three days after emasculation, when was removed from the stigmas. Leaves surrounding the flowers styles had just turned red and stigmas were enlarged, yellow were trimmed back to about three quarters of their original and sticky gave maximum seed set (45 to 55 seeds per capsule length. The section of branch containing the emasculated and 95% to 100% capsule set). Reasonably high levels of seed flowers was isolated in a double layer of crispy wrap bags (bags (> 25 seed per capsule and > 65% capsule set) could also be made from transparent film with very small perforations). A produced when flowers were pollinated at the time of wire coil was placed inside each bag to protect the stigmas from emasculation. The period of stigma receptivity varied between rubbing against the bag. The bags were then secured onto the cultivars. Capsules matured 14 to 16 weeks after pollination. branches with electrical ties (Figure 1d). Each bag contained Key words: stigma receptivity, pollination, Eucalyptus camaldulensis, about 15 to 25 flowers. All flowers in an isolation bag were seed set. pollinated on a particular day following emasculation ranging FDC: 181.521/.522; 164.6; 176.1 Eucalyptus camaldulensis; (941). from day 0 (the day of emasculation) to day 10. Pollen from clone 84 (originally from Mt. Fouracre, Western Introduction Australia) that had been stored for eleven months was used for the experiments. To collect the pollen, branches bearing flowers E. camaldulensis DEHNH. is grown throughout the world for on which the operculum was turning from green to yellow were land rehabilitation, paper pulp, timber and firewood (ELDRIDGE et al., 1993). In Australia it has been under utilised as a placed in jars of water in the laboratory. When the operculum commercial species and breeding programmes are only now of a flower lifted anthers were removed, placed in gelatin being established to produce genotypes with pulp quality and capsules over silica gel and stored at 4°C ± 2°C. Before use the growth form. To produce intra or interspecific hybrids, it is pollen and anthers were transferred to glass vials with rubber essential to have an understanding of the reproductive biology bungs. In the field the glass vials of pollen were kept over ice in an insulated container. When the vials were shaken the pollen of the species of interest. VISUTHITEPKUL and MONCUR (1993) stuck to the rubber bung, which was then used to apply the studied the floral biology of a natural stand of E. camaldulensis pollen to stigmas at the designated times (Figure 1c). from Petford, Queensland, but gave no information on the timing of stigma receptivity. We report here on the stigma Five styles from each treatment were harvested for histo- receptivity of two E. camaldulensis clones growing in a logical processing when the first signs of style abscission were Mediterranean climate in Western Australia. observed. About 14 weeks after pollination (March) the capsules were harvested. Each capsule was placed in a vial and Materials and Methods stored over silica gel to dry. The percent capsule set was calculated and the number of seeds in each capsule was count- Controlled pollinations were carried out using a E. camaldu- ed. lensis field trial at Kwinana, 30 Km south of Perth, Western In 1993 the flowers pollinated on day 0 in both clones 85 and Australia, in December 1993 and December 1994. The trees 87 were lost because of parrot damage or broken branches. For were made available by the ‘Tree Tech project’ a cooperative this reason pollination on day 0 was repeated the following programme between the University of Western Australia, Mur- year. Clone 84 pollen was unavailable so fresh clone 42 doch University and ALCOA of Australia Ltd. and had been (Capelis, WA) pollen was used for the crosses. There was no selected for salt tolerance and cloned in vitro (BELL et al., 1994; significant difference (P < 0.05) in the number of seeds produc- VAN DER MOEZEL and BELL, 1990). ed per capsule between 85 x 42 and 85 x 84 and between Three trees of each of clones 85 and 87 were used as female 87 x 42 and 87 x 84 when pollinated at peak receptivity. Five parents in experiments to determine the timing of stigma styles of the day 0 crosses were harvested 6, 24, 48, 72 and 96 receptivity. Clone 85 originated from Broken Hill, New South hours following pollination to examine pollen germination. For Wales and clone 87 from Erudina, South Australia. Both of the comparison, 5 styles were also harvested from flowers pollinat- clones flowered in December so flowers developed and fruit ed 3 days after emasculation 0, 3, 6, 24 and 48 hours after they matured under similar weather conditions. During the period had been pollinated. of flowering in 1993 the mean daily temperature maximum was 28.9°C and the minimum 14.8°C. The figures for 1994 Histological processing were a mean maximum of 29.3°C and a minimum of 15.8°C. Styles were fixed in Carnoy’s fixative (6:3:1 absolute alcohol: Flowers for the trials were emasculated over a 3 day period. chloroform: glacial acetic acid) and stored at 4°C ± 2°C for at For the experiment that examined the time interval between least 24 hours. The tissue was then hydrated through an emasculation and pollination, flowers of both clones 85 and 87 ethanol series (70% ethanol, 30% ethanol, 2 changes of were emasculated and pollinated on the same days. distilled water for at least 10 minutes), softened in 0.8 N Flowers were emasculated when the operculum had turned NaOH at 60°C for 30 to 60 minutes and then stained in 0.1% from green to yellow and was beginning to lift from the water soluble aniline blue in 0.1 N K3PO4. Aniline blue solution hypanthium (Figure 1a, b). Flowers on the selected branch that was prepared by dissolving the stain then placing it in the dark 142 Silvae Genetica 47, 2–3 (1998) Figure 1. – Controlled pollination of E. camaldulensis. a. Buds at operculum shed, the stage of emasculation, b. Emasculated buds, c. Pollination using pollen on a rubber bung, d. Pollinated buds isolated in crispy wrap bags. Bars represent 1 cm. for 24 hours before filtering through number 1 Whatman filter Statistical analysis paper. Styles were stained for at least 10 minutes then the tis- The number of seeds per capsule was log transformed and sue was squashed in 80% glycerol and viewed under a Zeiss analyses of variance and Tukey’s B tests were performed to photomicroscope III utilising violet excitation with an exciter determine statistical differences between crosses and pollina- filter, 390 nm to 420 nm and a barrier filter, 450 nm. tion times using Statistical Package for Social Sciences X The styles of Eucalyptus species are covered by a thick (Anon., 1988). cuticular layer (BOLAND and SEDGLEY, 1986) that obscures the observations of pollen tubes. To enable clear observations a Results longitudinal slit was made through the styles. The style was Appearance of E. camaldulensis flowers from emasculation to placed with the slit uppermost on the slide before the tissue style abscission was squashed to ensure the cuticular layer was beneath the The nature and timing of the visual changes that took place pollen tubes when microscopic observations were made. from emasculation to style abscission were almost identical in 143 the two clones. When the flowers were emasculated the style, seed production was very low, but some seeds were set up to top of the ovary and stigma were green and dry, the receptacle day seven (Table 1). was yellow / orange and anther dehiscence had commenced. By the following day nectar was present in the receptacle. Two to The timing of stigma receptivity in E. camaldulensis - pollen four days (most usually three days) after emasculation, the tube growth style and the top of the ovary had turned red and the stigma In both E. camaldulensis clones pollen tubes grew the entire had become enlarged, sticky and yellow. The first signs of style length of the style (4 mm) when flowers were pollinated on day abscission were observed on day 10 to 11 (after emasculation) zero, one, two or three (Table 2). In most of these styles the in clone 85 and day 9 to 10 in clone 87. number of pollen tubes present was so prolific they could not be quantified. The timing of stigma receptivity in E. camaldulensis - capsule and seed set Table 2. – Pollen tube growth in the styles of clones 85 and 87 when Capsules matured in March 14 to 16 weeks after pollination. pollinated from 0 to 7 days after emasculation. Five styles were examined at each harvest which was made when styles showed the first The timing of stigma receptivity as assessed by capsule set and signs of style abscission. the number of seeds per capsule varied between clones 85 and 87 (Table 1). In clone 85 the capsule set was high (above 65%) and there were more than 25 seeds per capsule when flowers were pollinated from zero to five days after emasculation.
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