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Eucalyptus Occidentalis Endl Developing systems to identify and deploy saline and waterlogging tolerant lines of Eucalyptus occidentalis Endl. By Rina Laksmi Hendrati (M.For.) This thesis is presented for the degree of Doctor of Philosophy Of The University of Western Australia School of Plant Biology Faculty of Natural and Agricultural Sciences 2009 DECLARATION FOR THESES CONTAINING PUBLISHED WORK AND/OR WORK PREPARED FOR PUBLICATION (The candidate must include a statement to this declaration that makes the student’s contribution clear to the examiners. In the case of co-authored publications included in the thesis, each author must give their signed permission for the work to be included. Where this is not possible the statement detailing the student’s contribution to the work must be signed by the coordinating supervisor.) This thesis contains work prepared for publication and all will be co-authored. Details of the works and where they appear in the thesis are outlined below. Chapter 3: Selected genotypes from 25 Eucalyptus occidentalis provenances were taller under salt-waterlogging conditions and in field trials and selection had little impact on diversity Contribution of this work is 60% by the candidate and 25% for Lucy Mulcahy and 15% for other authors Chapter 4: Selected genotypes within families of Eucalyptus occidentalis were taller under salt and/or waterlogged conditions in controlled tanks and field trials Contribution of this work is 80% by the candidate and 20% for other authors Chapter 5: Deployment for superior Eucalyptus occidentalis genotypes Contribution of this work is 80% by the candidate and 20% for other authors Chapter 6: Effect of genetic relatedness on gain in salt tolerance in crosses of Eucalyptus occidentalis Contribution of this work is 70% by the candidate and 20% for M. Byrne and 10% for other authors Candidate, Rina Laksmi Hendrati Signature: Co-ordinating supervisors, A/Prof. Julie Plummer Hans Lambers for Prof. Julie Plummer who is on sabbatical leave ii Statement of Original Contribution The results presented in this thesis are original contributions to the provision of breeding materials for high salt-tolerant tree lines for plantation under saline conditions. This thesis is my own composition and the result of my own research. Important contributions especially from my supervisors in developing the research topic, forming the hypotheses and writing up the thesis are acknowledged. All sources referred to in writing this thesis, including preliminary experiment and references are acknowledged. Rina Laksmi Hendrati September 2009 iii Thesis Structure This thesis consists of a General Introduction (Chapter 1), Literature Review (Chapter 2), four research chapters (Chapter 3, 4, 5 and 6) and a General Discussion (Chapter 7). Preliminary results in Chapter 4 have been presented as a poster in Conference Proceedings (below). All research chapters are presented in manuscript form for submission to scientific journals. Conference Proceedings Hendrati R., Barbour E.L. and Plummer J. A. (2006). Salt-and waterlogging tolerance of nine Eucalyptus occidentalis provenances under controlled conditions and in field trials: preliminary results. Proceedings of the 13th Australasian Plant Breeding Conference. Breeding for Success: Diversity in Action, Christchurch, New Zealand, 18- 21 April 2006 iv Abstract Eucalyptus occidentalis, a timber species from south Western Australia, is highly salt and waterlogging tolerant. Screening identified genotypes tolerant of high salt concentrations and waterlogging. Tolerance at provenance, family and individual level, and how phenotypic performance under salt and waterlogging was inherited was explored to provide a breeding population. Salt and/or waterlogged screening was carried out under controlled conditions up to extreme salt levels to determine tolerance between genotypes. This tank method was shown to produce repeatable results. Seedlings of 30 families from 9 provenances were used for screening. At low salt concentration (up to 300 mM NaCl), differentiation occurred for some traits but in general there was only a slight reduction in growth under salt, and waterlogging alone was not detrimental. At high salt concentration (550 mM) differentiation occurred among genotypes for all traits. Equivalent genotypes were also planted in field trials at three sites, two with medium (583 - 847 mm) and one with low rainfall (372 - 469 mm), in southern Western Australia. Survival was low (<53%) after 9 months due to an exceptional dry season followed by 3 months waterlogging in Kirkwood (38 - 1360 mSm-1), but was high >89% after 33 months in saline fields in Sandalwindy (96 - 976 mSm-1) and Roberts (88 - 1424 mSm-1). Some families were similarly in high rank for height under saline conditions in controlled and field trials. Height had the highest narrow-sense heritability value, especially under controlled salt- waterlogging (0.85) treatment and 20% selection enabled a gain of 8-14% under controlled conditions and in the field. Leaf production under salt was not an inherited trait. Systems were developed to hasten deployment of selected material. Extended daylength (16 h) and paclobutrazol (1 mg a.i/mm stem circumference) stimulated flowering in 2 year-old plants. Clonal propagation was possible. Grafting success varied from 0-100% depending on scion/rootstock provenances. Cutting propagation was possible but these plants did not perform as well as seedlings from similar families under salt-waterlogging. Na+ and Cl- concentrations in seedling leaves could not be used to distinguish low from high salt tolerant genotypes. Microsatellite analysis of molecular diversity indicated that genetic diversity is high in selected genotypes. There was only a slight reduction in heterozygosity from species level to provenance and family levels, and two superior genotypes maintained high diversity. v Crossing was possible using one stop pollination of cut immature styles and capsule retention varied from 0-34% and germination rate from 2-96%. Genetic distance between parents was correlated with seed set and offspring fitness. Wider genetic distances increased capsule retention, seed germination and seedling survival. Under 500 mM salt-waterlogging, offspring heights were similar when parental genetic distances were similar. High heritability value for height from ANOVA-REML parental screening was confirmed using parent-offspring regression. Screened superior genotypes, which withstood very high salt concentration, provide a breeding population for further breeding and for plantations under saline regions in low-medium rainfall areas in Western Australia and other parts of the world. These trees provide an economic return in areas where no other plants may survive and an environmental service in potentially reducing waterlogging, salinity and its spread. vi Acknowledgements I would like to express my sincere gratitude to A/Prof. Julie Plummer, Dr Liz Barbour from Forest Products Commission Western Australia and Dr Margaret Byrne from Department of Environment and Conservation Western Australia for their supervision, patience, understanding and encouragement in guiding and advising me throughout my study My sincere thanks to Dr Richard Mazanec from the Department of Environment and Conservation Western Australia, Mr Kevin Murray from School of Mathematics and Statistics the University of Western Australia and Dr Budi Leksono and Dr Arif Nirsatmanto my colleagues from Biotechnology and Tree Improvement Research Institute, Yogyakarta, Indonesia for statistical advice. Special thanks to Dr Chris Jones for spending his busy time to proof read my thesis and for kindly helping me to learn lots of things. I am deeply grateful to the Seed Technology Nursery, Forest Products Commission Western Australia for financial support and for use of the valuable data from field trials and the first screening experiment carried out by Lucy Mulcahy. I would also like to thank for assistance from Ryan Abbott and Len Norris from the Seed Technology Nursery, Forest Products Commission Western Australia. I am also sincerely grateful to Bronwyn McDonald, Jane Sampson, Melisa Miller and Sean Stankowski for their assistance during my work in the Molecular Laboratory of the Department of Environment and Conservation Western Australia, and Perry Swanborough, Garry Cash and Elizabeth Halladin from Plant and Soil Laboratories, Faculty of Natural and Agricultural Science, The University of Western Australia. I also want to thank the Australian Agency for International Development (AusAID) for providing my scholarship up to completion. Many thanks to the previous AusAID Liaison Officer, Mrs Rhonda Haskell, and the Student Contact Officer for AusAID, Mrs Christine Kerin, who were always willing to help and support my study. My sincere thanks to Dr Nur Masripatin, Dr Bambang Tri Hartono and Dr Rufi’ie, the Heads of Biotechnology and Tree Improvement Research Institute who supported me in my application and in my studies at UWA. vii Many thanks to my wonderful Indonesian friends and families who were always there with helpful hands. I also need to mention friends, who share the same room to study, Nio Song Ai, Inge Tvede, Aneeta Pradhan, Wang Xing and Tiernan O'Rourke Finally, I give thanks to my mother and sisters for their support and prayers, and for the companionship, help and prayers from my lovely kids, Kiki and Zaki, hopefully this will encourage you and help you to have a better future. You will always be in my prayers. Amen. viii Table
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