1. CP 3x seedlings We thank FSIV and the Australian Centre for International Agricultural Research (ACIAR) collaboration projects (FST 2003/002 and FST 2008/007) for plant material, and Hong, Xuan and Dzung for field assistance. Funding and in-kind support for N.Q.Chi was provided by a John Allwright Fellowship from ACIAR. PLOIDY EFFECTS ON REPRODUCTIVE BIOLOGY OF ACACIA MANGIUM 1. Introduction 2. Methods 3. Key results An Acacia hybridizing seed orchard was established in southern Vietnam. Ramets of clones of colchicine-induced autotetraploid A. mangium (AM-4x) [1], diploid A. mangium (AM-2x) and diploid A. auriculiformis (AA-2x) were planted in alternate rows to promote inter-ploidy pollination. Despite heavy and largely synchronous flowering and seed production of all three species/ploidy types, the orchard produced very few open-pollinated triploid (3x) seeds. We examined possible barriers to fertilisation and/or successful development of triploids. References 1 School of Plant Science, University of Tasmania, Hobart, TAS. 7001, Australia 2 CSIRO Ecosystem Sciences, Hobart, TAS. 7001, Australia 3 Research Centre for Forest Tree Improvement, Forest Science Institute of Vietnam, Hanoi, Vietnam Acknowledgements 4. Conclusions N. Q. Chi 1,3, C. Harwood 2, R. Griffin 1, J. Harbard 1, H. H. Thinh 3, and A. Koutoulis 1 Hand pollination was conducted following [2] Floral morphology and pollen tube growth was studied using light and fluorescence microscopy Pollen tube growth was studied using a clear squash technique [3] One ramet of each of 3 different clones of AM-2x, AM-4x, and AA-2x was selected for CP treatment Treatment mean comparisons were performed by Tukey-Kramer’s multiple range test at α = 0.05 Stigma cup width was greater than polyad diameter for all species/ploidy combinations [1] Blakesley D, Allen A, Pellny TK, Roberts AV (2002) Natural and induced polyploidy in Acacia dealbata Link. and Acacia mangium Willd. Annals of Botany 90, 391-398. [2] Sedgley M, Harbard J, Smith RM, Wickneswari R (1992) Development of hybridisation techniques for Acacia mangium and Acacia auriculiformis. ACIAR Proceedings Series, 63-69 [3] Martin FW (1959) Staining and observing pollen tubes in the style by means of fluorescence. Stain Technology 34, 125-128. [4] Nghiem CQ, Harwood CE, Harbard JL, Griffin AR, Ha TH, Koutoulis A. (2011). Floral phenology and morphology of colchicine-induced tetraploid Acacia mangium compared with diploid A. mangium and A. auriculiformis: implications for interploidy pollination. Australian Journal of Botany 59, 582-592. 3.1 Floral morphology [4] AM-4x flowers were intermediate between those of AM-2x and AA-2x Species/ploidy SpikeFlower Spike length (cm) Flowers per spike Flower length (mm) Style length (mm) No. of ovules per ovary A. mangium 2x10.8 a230.4 a5.1 a4.2 a 14.2 ab A. mangium 4x 8.0 b161.4 b4.8 a3.9 b13.0 b A. auriculiformis 2x 5.8 c 99.4 c4.3 b3.4 c 15.5 a Mean diameter of stigma cups and polyads. Error bars show critical difference (P<0.05) between treatments. 3.2. Pollen tube growth and ovule penetration (72 h after pollination) pt TreatmentsNormal seeds Abnormal seeds Type IType IIType III Intra-ploidy crosses AM-2x X AM-2x1520 5 7 AA-2x X AA-2x 6211921 AA-2x X AM-2x16251742 Inter-ploidy crosses AM-2x X AM-4x 8 67 1918 AA-2x X AM-4x24180286 5 Species/ploidy (taxa) Types of cross-combination (types) Ovaries with at least one ovule penetrated (%) Mean no. of ovules penetrated per ovary Mean no. of pollen tubes per style AM-2xAM-2x X AM-2x 29.7 abc 1.36 bc 6.7 abc AM-2x X AA-2x 17.6 bc 1.64 bc 6.0 abc AM-2x X AM-4x 14.3 bc 1.33 bc 4.2 c AM-4xAM-4x X AM-4x 20.6 bc1.00 c 4.6 bc AM-4x X AM-2x 35.9 abc 1.29 bc 5.3 bc AM-4x X AA-2x 35.6 abc 1.33 bc 6.0 abc AA-2xAA-2x X AA-2x 46.0 ab 1.85 bc 7.8 ab AA-2x X AM-2x 59.9 a 2.77 a 8.9 a AA-2x X AM-4x 34.7 abc 1.92 b 7.0 abc Significance of differences among taxa-by-types******* Note: ** = 0.001≤ P ≤ 0.01 and *** = P ≤ 0.001. Different letters denote significant (P < 0.05) differences among nine cross-types Pollen tubes grew well in the style Ovules penetrated in the ovary Ovule development 5 weeks post-pollination Differences in floral morphology were not sufficient to prevent interploidy pollination No pre-zygotic reproductive barriers were detected for intra- and inter ploidy mating of A. mangium and A. auriculiformis However, yields of pods and normal seeds from inter-ploidy crosses were extremely poor, compared to those from intra- ploidy crosses. Post-fertilization genic imbalance may limit the production of viable triploid progeny. pt pollen tube AA-2x AM-4x AM-2x 3.3. Normal and abnormal seeds harvested from different cross-combinations Note: Different letters denote significant (P < 0.05) differences within species/ploidy combinations AA-2x pollen tube AM-2x x AM-4x AA-2 x AM-2 x AM-4 x 60  m Mature seed category ovule penetrated 30 µm Normal seed 5 mm Abnormal seed Type I Type II Type III Normal seed 5 mm Abnormal seed Type I Type II Type III AM-2x AM-4x Style polyad stigma AM-2 x x AM-4 x 50  m pollen tubes polyad 45  m