i EFFECTS OF PLOIDY LEVEL ON THE REPRODUCTIVE BIOLOGY OF TROPICAL ACACIA SPECIES Nghiem Quynh Chi Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Plant Science UNIVERSITY OF TASMANIA July – 2012 ii Declaration of Originality I hereby declare that this submission is my own work and contains no material which has been accepted for a degree by the University or any other institution, and to the best of my knowledge and belief, it contains no material previously published or written by another person except where due acknowledgement is made in the text of the thesis, nor does the thesis contain any material that infringes copyright. (signed): ………………………………………………….. 2nd October 2012 Authority of Access This thesis may be made available for loan and limited copying in accordance with the Copyright Act 1968. Statement regarding published work contained in thesis The publishers of the papers comprising Chapters 3 and 4 hold the copyright for that content, and access to the material should be sought from the respective journals: Australian Journal of Botany and Journal of Tropical Forest Science. The remaining non published content of the thesis may be made available for loan and limited copying in accordance with the Copyright Act 1968. iii ABSTRACT Acacia mangium Willd., A. auriculiformis A. Cunn. ex Benth and their natural hybrid, A. mangium x A. auriculiformis , are important plantation species for timber production in the tropics. However, their potential to invade natural ecosystems has been a concern because of their prolific production of long-lived seeds. Deployment of triploid Acacia clones in plantations might reduce fertility and associated weed risk. Studies were conducted in a hybridizing orchard that was established in southern Vietnam in 2003 with alternate rows of diploid A. mangium (AM-2x), diploid A. auriculiformis (AA-2x) and colchicine-induced autotetraploid A. mangium (AM-4x) clones. Heavy flowering and seed production was obtained for all three species/ploidy combinations; however, the yield of viable open-pollinated triploid (3 x) seeds from open pollinated seed had been very low. The reproductive behaviour of the three species/ploidy combinations in the orchard was therefore investigated to determine whether there are barriers to the production of triploid progeny within and between these two species. Peak flowering period for both AM-2x and AM-4x (November – December) was slightly earlier than for AA-2x (December – January). The spikes of AM-4x were shorter than those of AM-2x, resulting in fewer flowers per spike, but they were longer and had more flowers than AA-2x. The proportion of male to hermaphrodite flowers was similar for all three species/ploidy combinations. AM-4x flowers had shorter styles, but the stigma and polyad diameters were greater than those of AM-2x. Differences in stigma and polyad size between cytotypes were not iv sufficient to adversely affect inter-cytotype pollination. Pollen recovered from the bodies of the main insect pollinators (honeybees) indicated that they did not discriminate in their foraging behaviour. Therefore, neither the floral phenology and morphology of species/ploidy categories nor the pollinator foraging behaviour created barriers to inter-cytotype pollination. Pollen-pistil interactions following different mating combinations within and between each of AM-4x, AM-2x, and AA-2x were investigated. Following controlled pollinations, AM-4x ovules exhibited more attraction to self- than outcross- pollen tubes, in contrast to AM-2x and AA-2x; however, this trend was not consistent for all of the genotypes examined. The reciprocal crosses of AA-2x and AM-2x were successful as pollen tubes grew well in both AM-2x and AA-2x styles and penetrated their ovules. For inter-cytotype crosses, inter-species, particularly those with AA-2x as the maternal parent, had a significantly greater number of ovules penetrated than did intra-species crosses. However, yields of pods and filled seeds following all inter-cytotype crosses were extremely poor, compared to those from the intra-cytotype crosses. Thus, there were strong barriers to production of viable 3 x progeny, despite the demonstrated absence of pre-zygotic isolation. Ovule abortion occurred more frequently in all inter-cytotype crosses than in the open-pollinated flowers at 5 and 7 weeks after pollination. Consequently, the proportion of filled seeds set per pod for inter-cytotype crosses was far lower than in pods arising from intra-cytotype crosses and open-pollination. Moreover, the weight of filled seeds from inter-cytotype crosses was significantly lighter than filled seeds from open-pollination, and they were unable to germinate. Analysis using v microsatellite markers of DNA obtained from these non-germinated seeds confirmed that most were triploid and had resulted from the target inter-cytotype crosses. It was concluded that abnormal seed development and abortion occurred throughout the 18-week period of pod development, resulting in failure of this set of inter-cytotype crosses to produce any viable triploid progeny. Possible reasons for this are discussed. Research on application of in vitro culture techniques to immature embryos may be required to recover triploid progeny from inter-cytotype crosses of these Acacia species, as has been achieved for other species. vi Acknowledgement I would like to thank my supervisors, Dr Chris Harwood, Prof Rod Griffin, Ms Jane Harbard, Assoc/Prof Anthony Koutoulis, and Assoc/Prof Rene Vaillancourt, for their support, advice and encouragement throughout the course of my study. Much appreciation to Dr Ha Huy Thinh, Mr Do Huu Son, Mr Kieu Thanh Tinh for their support and Mr La Ngoc Hong, Ms Nguyen Thi Xuan, Mr Nguyen Van Loc and Mr. Nguyen Van Dzung for technical assistance during field work in Vietnam. I also thank Mr. Tran Duc Vuong, Ms Sascha Wise and Mr. Adam Smolenski for their assistance in molecular genetics laboratory and Mrs Aina Price, Mrs Helen Bond, Mrs Leesa Borojevic, Mr Mark Cozens, Mr Ian Cummings, Mrs Tracey Jackson and Mr Alvin Lam for their assistance in microscopy and glasshouse. I thank all the staff members within School of Plant Science and within International Service, Division of the Pro-Vice Chancellor (Student & Education) of University of Tasmania who helped me in many ways during my study period. I am grateful to the Australian Centre for International Agricultural Research (ACIAR) for financial support provided by projects (FST 2003/002 and FST 2008/007) and the John Allwright Fellowship. Thanks also go to the Research Centre for Forest Tree Improvement (RCFTI) and Eastern South Vietnam Forest Scientific and Production Centre of the Forest Science Institute of Vietnam (FSIV) for plant material and assistance. Finally, special thanks to my family and friends for their love and understanding. 1 TABLE OF CONTENTS ABSTRACT ................................................................................................................ iii CHAPTER 1. INTRODUCTION ................................................................................. 8 1.1. The genus Acacia .................................................................................................. 8 1.2. The importance of Acacia mangium , A. auriculiformis , and Acacia hybrid ....... 10 1.2.1. Acacia mangium Willd. ............................................................................... 10 1.2.2. Acacia auriculiformis A. Cunn. ex Benth. .................................................. 11 1.2.3. Natural Acacia hybrid ( A. mangium x A. auriculiformis ) ........................... 12 1.2.4. History of introduction and utilization ........................................................ 13 1.3. Breeding programs for tropical Australian Acacia species in Vietnam .............. 14 1.4. Some aspects of reproductive biology of Acacia species .................................... 16 1.4.1. Phenology .................................................................................................... 16 1.4.2. Flower morphology and biology ................................................................. 17 1.4.3. Pollination ecology ...................................................................................... 20 1.4.4. Breeding system .......................................................................................... 21 1.5. The problem and potential solution for invasiveness of Acacia species. ............ 22 1.6. The potential for using polyploidy for Acacia breeding ..................................... 24 1.7. Aims of the study ................................................................................................ 25 CHAPTER 2. MATERIALS AND METHODS ....................................................... 26 2.1. Plant materials ..................................................................................................... 26 2.2. Hand pollination technique ................................................................................. 28 2.2.1. Pollen collection .......................................................................................... 28 2.2.2. Hand controlled-pollination ......................................................................... 28 2.3. Microscopy .......................................................................................................... 30 2.4. Microsatellite markers ......................................................................................... 31