Aibika (a green leafy vegetable in PNG): Biodiversity and its effect on micronutrient composition Lydia Rubiang-Y alambing A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Food Science and Technology School of Chemical Engineering Faculty of Engineering March 2014 PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Rubiang-Yalambing First name: Lydia Other name/s: Abbreviation for degree as given in the University calendar: PhD School: Chemical Sciences and Engineering Faculty: Engineering Title: Aibika (a green leafy vegetable in PNG): Biodiversity and effect on micronutrient composition Abstract 350 words maximum: (PLEASE TYPE) Over twenty different varieties of aibika or Abe/moschus manihot (L.) which is a commonly consumed green leafy vegetable in Papua New Guinea were studied with two main objectives. Firstly to determine the extent of genetic diversity between the accessions currently held at the National Agricultural Research Institute (NARI) in PNG which would aid effective management of the aibika germplasm and secondly to analyse micronutrients (total folate and minerals) in all accessions and identify any relationships between the nutrient contents and genotypes. Total folate contents ranged from 34 - 132 f..Lg/1 00 g on a fresh weight basis over two years indicating a wide range and a significant difference (p<0.05) between the two years. The mineral contents (mg/lOOg fresh weight) were in the following ranges for the 3 year period; iron, 0.8 -8.7; zinc, 0.32- 2.31; calcium, 197- 635; potassium, 265- 630; sodium, 1.0- 41; magnesium, 79-264; manganese, 0.42 - 2.09 and copper, 0.13 - 1.7. A significant (p<0.05) variation in the mineral contents was observed reflecting on variations in growing conditions between the collections. There was no significant effect of genotype on micronutrient content in all accessions. UPGMA analysis was performed to determine the clusters of accessions of aibika according to concentrations of all minerals and folate in all 3 years. Generally, the clusters of accessions were different in each of the 3 years. Environmental and other factors seem to have a greater impact on the micronutrient data compared to the genotype. The 23 accessions studied were grouped into five main groups according to the techniques of random amplification of polymorphic DNA (RAPD) and directed amplification of minisatellite region DNA (DAMD). Sequencing results from two chloroplast DNA regions studied, the psbM-trnD and the trnL-trnF intergenic spacer regions did not show any variation in the accessions. The nuclear encoded ITS region was also studied which showed that sequences from all accessions were identical with the exception of LAL Am 22 1 where the sequence data from the reverse primer suggested that there were 11 differences from the rest of the accessions. Data from this sequence has been submitted to the Genbank (accession number KC48173). Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. 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( . ~ . t.0 .3. ...J .. ;)o.l ~ ... ···· ···· ·· ··· ······· ···· ··· ACKNOWLEDGEMENTS I wish to thank the following individuals and organisations, without whose support I would never have done or completed this PhD study: Assoc. Professor Jayashree Arcot, my supervisor – I am grateful and fortunate to have a supervisor like you. You have assisted me in so many ways not just academically. Thank you for your patience, understanding and for the exposure you have given me in the area of food composition. The Australian Government (Australian Development Scholarship) – for the full scholarship to study in Australia The Papua New Guinea University of Technology, Lae, PNG – for allowing me to take up the study scholarship. The PNG National Agricultural Research Institute (NARI), for the MOA to use the aibika germplasm at Laloki. Rosa Kambuou, Janet Paofa and their staff at NARI Laloki, for the assistance with sample collection, sample exportation and information when needed. The assistance of other staff at Bubia is also acknowledged. Assoc. Professor Paul Holford, Hawkesbury Campus, University of Western Sydney, Richmond. I would never have done the DNA work without the assistance of someone so helpful, and understanding. I started the DNA work with no background knowledge on molecular biology and have learnt a lot in this study. Thank you. Professor Heather Greenfield, my co-supervisor, for her professional advice and guidance. Mr. Camillo Taraborrelli, for his assistance in the importation and quarantine procedures for the aibika samples, and the technical help in the running of my experiments. Karrie, Nisha, Veronica and Yang, thank you for the friendship and assistance during my study. My husband and son for their support, understanding and sacrifices made so I could complete my studies. i ABSTRACT Aibika or Abelmoschus manihot as it is scientifically known is the most popular and commonly-consumed indigenous green leafy vegetable in Papua New Guinea (PNG). Many different varieties of aibika can be found in the country and the PNG National Agricultural Research Institute (NARI) is overseeing the collection and maintenance of these varieties in a field gene bank at Laloki, PNG. At this stage the accessions have only been morphologically characterised and there is no data on genetic differences between the accessions. Over twenty different varieties of aibika were imported into Sydney over a three year period. The two (2) main objectives for this study were: Firstly to determine the extent of genetic diversity between the accessions with the aim of assisting PNGNARI in identifying core genotypes in the current collection which would aid its effective management. Second aim was to analyse micronutrients (total folate and minerals) in all accessions and identify any relationships between the nutrient contents and genotypes. It is known that micronutrients do vary within varieties of same species so that the identification of a nutrient rich variety or