Journal of the Royal Society of Western Australia, 89: 101–108, 2006 Low success of controlled pollination in Eucalytpus marginata (jarrah) M A Wheeler1,2, M M Fairbanks1,3 and J A McComb1 1School of Biological Sciences and Biotechnology, Murdoch University, South Street, Murdoch, W. A. 6150, Australia 2Present address: PO Box 65, Rosebank, NSW 2480 3 Present address: Department of Agriculture and Food, Western Australia Corresponding author: M A. Wheeler, Department of Environment and Conservation, Western Australia [email protected] Manuscript received September 2005; accepted May 2006 Abstract The success of controlled pollination techniques was investigated for clones and wild trees of Eucalyptus marginata. The number of pollen tubes in the style was used as a measure of pollination success. The stigma was receptive from 3–11 days after anthesis, but it appeared that stigmatic receptivity may be highest from 4–7 days after anthesis. Flowers were isolated using aluminium foil squares, and fresh pollen was found to be more effective for pollination success than processed pollen. Results were inconclusive comparing self- to cross-pollination success, and no difference was found comparing pollination success in clones and wild trees. However clones generally tended to flower earlier than the wild trees, whatever their origins, and very few mature fruits resulted from any controlled pollinations in jarrah clones. Clones also produced a lower proportion of mature capsules from the flowers produced (Wheeler 2004) Keywords: pollination, Eucalyptus marginata, stigma, style Introduction evidence that this occurs in most of the species studied (Elridge et al. 1993; Moncur & Boland 1989; Potts and Control-pollination techniques have been developed Cauvin 1988; Pryor 1951, 1976). in several species of Eucalyptus as a tool to assist in selective breeding. Controlled pollination has been used Detection of stigma receptivity is essential for to improve seed yield (Harbard et al. 1999; Moncur 1995), successful controlled pollination and different indicators control the level of outcrossing in seed orchards and of stigma receptivity have been described. The style in improve breeding through knowledge of fertility and Eucalyptus regnans lengthens when approaching the compatibility, achieve interspecific hybridisation, and receptive period, and the surface of the stigma expands study self-incompatibility levels in Eucalyptus species. (Griffin & Hand 1979). Receptivity in this species was Detailed knowledge of the pollination biology of determined (by the lengthening of the stigma and Eucalyptus marginata (jarrah) is essential to design an increased exudate on the stigma) to be between 10 and effective seed orchard, or to conduct controlled 14 days after anthesis. Stigma receptivity in three species pollination for genetic improvement of trees. This study in Section Bisectaria was marked by stigma secretions, was undertaken to assist in the development of control- coinciding with the greatest number of pollen grains pollination techniques in E. marginata, which could be adhering to the stigma surface (Ellis & Sedgley 1992). In used for the breeding of improved lines. E. woodwardii (Eucalyptus subgen. Symphomyrtus), no style elongation was observed during the receptive The floral structure of jarrah is similar to other period, but stigma secretions were maximised when it Eucalyptus subgen. Eucalyptus species, such as was most receptive, seven days after anthesis (Sedgley & Eucalyptus stellulata (House 1997, Fig. 1a), and E. Smith 1989). regnans (Griffin 1980; Griffin & Hand 1979; Griffin et al. 1987). Flowers per umbel range between one and eleven, The method most often used for controlled pollination and trees do not usually flower prolifically every year, in eucalypts involves emasculation and isolation of but have one good flowering season every two or three flower buds, and frequently, pollen storage (Moncur, years. Flower buds are always seen in the autumn but 1995). Low fertilisation rates have occurred in several can drop off later in the year. species, and sometimes two controlled pollinations produce more seed. Griffin et al. (1987) reported many Anthesis occurs in most eucalypt species 3–28 days more fertilised ovules in E. regnans after successive prior to the stigma of the same flower being receptive pollinations two days apart. Pollen collection is usually (Griffin & Hand, 1979; Oddie & McComb 1998; Williams from buds at operculum lift stage, and the pollen is 1999). The style elongates following anthesis (operculum sieved and placed in a vial, and stored at or below 0oC. lift). While individual flowers are protandrous this does Griffin et al. (1982) reported storing pollen from E. not prevent pollen from one flower pollinating and regnans satisfactorily at room temperature for 36 days, fertilising another flower on the same tree, and there is after grinding the anthers to release the pollen in a glass tissue grinder with distilled water. The homogenate was © Royal Society of Western Australia 2006 then filtered through a nylon cloth (37µm pore size) then 101 Journal of the Royal Society of Western Australia, 89(3), September 2006 a millipore filter (3µm) and the pollen on the filter dried with the stigma placed on a microscopic slide in 1% over silica gel. Turner et al. (1994) kept branches with aniline blue, and gently squashed under a cover slip. buds in water for three to five days in the laboratory so This tissue was observed under a fluorescence that the buds could open and pollen easily collected for microscope for pollen tubes (Fig. 1d). processing. Time of stigmatic receptivity was tested in In E. marginata pollination is affected by a range of emasculated and intact flowers by pollinating0–11days insect and bird vectors (Yates et al. 2005). Purple after anthesis in 1999 and2–7days after anthesis in crowned lorikeets have been observed feeding on E. 2000, 2001 (Table 1). Pollination success variation within marginata flowers (House 1997). A range of insect species the flowering season was observed by comparing have been seen to visit E. marginata flowers, such as pollination success at the early, middle and late periods flies, bees, wasps, beetles and moths (Yates et al. 2005), in of the flowering season (20 flowers for two clones for common with other Monocalypts such as E. regnans each of the three times using processed pollen). A trial (Griffin & Ohmart 1986). Wind pollination is regarded as was also conducted to determine whether multiple being unlikely as a major pollinating vector in pollinations of the same flower were advantageous. In Eucalyptus. Pollen is thought to be dispersed up to jarrah, the flowers were pollinated on day four followed approximately 100 metres (Eldridge et al. 1993), and in E. by a repeat pollination on day five after anthesis (20 kochii sometimes up to 500 metres (Byrne, pers. comm.), flowers for two clones using fresh pollen). and, given the evidence from certain natural hybrids, apparently much further in some species (Potts et al. Tests of pollination techniques 2003). Preliminary work was conducted in 1999 to test While it could be expected that similar pollination stigmatic receptivity and emasculation and isolation rates could be seen in E. marginata as other eucalypts, as techniques. In 2000 and 2001 a number of trials were well as capsule production, it is not necessarily the case. conducted to test various aspects of the pollination This study, while following similar techniques to process using two clones, and two wild trees. successful methods used in other species, is surprising in Two pollination techniques were compared. These are: the low levels of pollination success achieved. the ‘processed’ technique after Moncur (1995), and the ‘fresh’ technique. In each case pollen from several genotypes was used for cross-pollination. The ‘processed’ Materials and Methods technique involved brushing the flowers of several genotypes over a 200mm sieve and collecting pollen in a Trees used small bottle with a rubber stopper and storing it at 4°C or at room temperature. Pollen was applied to the stigma Wild trees (endemic E. marginata trees) in the Harry using the rubber stopper or a toothpick. Secondly the Waring Mammal Reserve (32° 9' 47"S, 115° 49' 22"E), at ‘fresh’ technique involved wiping a freshly picked flower Wattleup, Western Australia (WA) and clones (developed with the operculum removed across a receptive style. using tissue culture techniques for resistance to Each style was wiped with several buds from several Phythophthora cinnamomi, McComb et al. 1996) growing genotypes. Branches with buds were sometimes picked at Murdoch University, WA (32° 2' 54"S, 115° 50' 46"E) and kept in water for up to five days in the laboratory, and Marrinup Orchard, (32° 42' 5"S, 116° 3' 5"E) near and the flowers used when they were at operculum lift Dwellingup, W.A. were used for control pollination stage. Only pollen with viability over 50% was used, experiments. Wild trees were chosen for their similar size determined by using the method described in Wheeler to the clonal trees. and McComb (2006). This technique involves Stigmatic Receptivity and Pollination Success germinating fresh pollen in vitro using the wettable cellophane technique (Alexander and Ganeshan 1989) Stigmatic receptivity in Eucalyptus marginata was with a germination medium of 300 ppm hydrated first investigated using clones growing at Murdoch calcium nitrate, 200 ppm hydrated magnesium sulphate, University