Exploring bridge-grafting as technique to restore growth in girdled Ocotea bullata and Curtisia dentata in the Southern Cape forest area by ANNA SUSANNA VAN WYK submitted in accordance with the requirements for the degree of MASTER OF SCIENCE in the subject NATURE CONSERVATION at the UNIVERSITY OF SOUTH AFRICA SUPERVISOR: MRS. E.M. VAN STADEN CO-SUPERVISORS: PROF. W.A.J. NEL DR. G PRINSLOO MAY 2017 ii ACKNOWLEDGEMENTS I would first and foremost like to express my sincerest appreciation to my supervisors, Mrs. E.M. van Staden and Prof. W.A.J. Nel, for their professional guidance and unequivocal support, and the trust shown in me during the entire study period. I would furthermore like to thank Dr. Gerhard Prinsloo for introducing me to metabolomics as a method for analyzing plant metabolites and for the training and assistance provided regarding the analysis of the bark samples. A special thank you to UNISA as an institution, for the opportunities I was presented with during this study period, with special reference to Professor Nel, Mrs. Van Staden and Dr. Prinsloo who made these opportunities possible. I would like to extend my gratitude to the SANParks staff involved in making this study possible – Dr. Wessel Vermeulen and his team of scientists at Scientific Services in Knysna, Jessica Hayes, regional ecologist, Sandra Taljaard, Park Manager of the Wilderness National Park, and Leslie-Ann Meyer, Park Manager of the Tsitsikamma National Park. Your inputs are highly valued and appreciated. My appreciation goes further to include all SANParks staff members who assisted me during the entire study period. The National Research Fund assisted in the funding of this project. Without this assistance, it would not have been possible to complete this study as it was an extremely expensive study. I thank you with the highest sincerity. My children Bianca, Jean, Renier and Madeleine and their respective families, and my brother, Dr. Dirk de Klerk, thank you for the support during all my years of study. iii ABSTRACT In South Africa, there is a growing concern regarding the sustainability of bark harvesting due to the reduced availability of medicinal trees in natural areas and the slow growing and slow-reproducing nature of South Africa’s indigenous trees, of which some have specific habitat requirements and a limited distribution. With an estimated 80% of all Africans in South Africa still relying on plant material for their basic healthcare needs, approximately 200 000 traditional healers and an estimated 63 000 commercial harvesters, medicinal plants are being exploited to extinction. The aim of the study was to determine whether bridge-grafting could be used to restore growth in girdled Ocotea bullata and Curtisia dentata trees as these two medicinal tree species are listed on the South African Red List as ‘endangered’ and ‘near threatened’ due to overexploitation. These trees were also historically used in furniture production and general carpentry, which increased these trees’ vulnerability to extinction. Bridge-grafting is a technique widely described in horticultural literature but has not been used to restore growth in medicinal tree species with extensive harvesting damage. Metabolomics as analytical method is a relatively new science, but it is very useful, accurate and repeatable in obtaining knowledge on the metabolites present in a plant, and for determining the concentrations of metabolites. NMR metabolomics is, however, not sensitive enough to quantify metabolites with very low concentrations such as plant hormones. The results achieved showed that O. bullata and C. dentata responded differently to girdling and bridge-grafting regarding location of callus initiation, callus development, rate of callus development, basal shoot initiation and development and their sucrose source-sink relationships. NMR metabolomics showed that seasonality was an important factor affecting metabolite responses in both species. NMR metabolomics also showed that after one year, there were no differences in responses above and below the girdle wounds, nor were there differences between the grafted trees and the normal control trees. iii Although the method of bridge-grafting was successful in restoring the growth of O. bullata and C. dentata with this study, much more research needs to be conducted to prevent endangered girdled medicinal trees from becoming extinct, to maintain tree species diversity, genetic diversity and biodiversity. Key terms: Commercial bark harvesting, Medicinal trees, Girdling, Bridge-grafting, Callus tissue, Bark recovery, Sucrose concentrations iv ABBREVIATIONS AND ACRONYMS AIDS : Acquired Immuno-Deficiency Syndrome CBD : Convention on Biological Diversity CITES : Convention on the Trade of Endangered Species C.S.I.R. : Council for Scientific and Industrial Research DBH : Diameter at Breast Height IAA : Indole-3-Acetic Acid IAM : Indole-3-Acetamide IBA : Indole-3-Butyric Acid IAOx : Indole-3-Acetaldoxime IPA : Indole-3-Pyruviv Acid GC-MS : Gas chromatography – Mass spectroscopy HPLC-PDA : High Performance Liquid Chromatography – Photo Diode Array Detector IUCN : International Union for the Conservation of Nature LC-MS : Liquid chromatography – Mass spectroscopy NEMBA : National Environmental Management: Biodiversity Act NEMBA ToPS : National Environmental Management: Biodiversity Act: Trade of Protected species NEMPA : National Environmental Management: Protected Areas Act NGO : Non-Governmental Organization NMR : Nuclear Magnetic Resonance (spectroscopy) v OPLS : Orthogonal Partial Least squares or Orthogonal Projections of Latent structures OSC : Orthogonal Signal Correction PAA : Phenol Acetic Acid PCA : Principle Component Analysis ppm : parts per million SANBI : South African National Biodiversity Institute SANParks : South African National Parks WCMC : World Conservation Monitoring Centre UNEP : United Nations Environmental Program. UNESCO : United Nations Educational, Scientific and Cultural Organization WCED : World Commission on Environment and Development WHO : World Health Organization WWF : World Wide Fund for Nature vi GLOSSARY Abscission: A leaf or fruit in which the cells at the base of the organ dies and tears, permitting the organ to fall cleanly way from the stem with a minimum of damage (Mauzeth, 1995: G - 1). Alleles: The alternative forms of a gene (Hartl, 2000:2). Anomalous secondary growth: Any form of secondary growth that does not conform to that typically occurring in gymnosperms or dicots (Mauzeth, 1995: G - 2). Anticlinal cell division: Cell division perpendicular to the surface of the meristem (Hopkins and Hüner, 2009:490). Apical dominance: Inhibiting effect of a terminal bud upon the development of lateral buds (Tainton, 1999:412). Apoplastic movement of water: Water movement that takes place through the non- living cell-walls and intercellular spaces of the plant (Moore, 2013:90). Auxin: A plant hormone produced in the apical meristemic regions of plants and in actively growing coleoptile apices, germinating seeds, root tips and the apical buds of growing stems (Hopkins and Hüner, 2009:306). Biodiversity: The complete range of species, biological communities, and their ecosystem interactions and genetic variation within a species (Primack, 2012:315). vii Biparental inbreeding: Mating between genetically related individuals or biparental inbreeding occurring because of spatial genetic structuring within populations. Plant populations are likely to develop spatial structure because gene dispersal through pollen and seed is often localized around the female parent (Nason and Ellstrand, 1995:307). Bulbs: A short subterranean, vertical stem that has fleshy, scale-like leaves such as an onion (Mauzeth, 1995: G - 3). Callus tissue: A term used to describe undifferentiated tissue in tissue cultures and wound healing (Hopkins and Hüner, 2009:491). Carbohydrate metabolism: Denotes the various biochemical processes responsible for the formation, breakdown and interconversion of carbohydrates in living organisms (Chapman and Reiss, 1999:307). Carrying capacity: Potential of an area to support life through the production of plant material for consumption over an extended number of years without deterioration of the overall ecosystem (Tainton, 1999:413). Chloroplast: Plastids in the leaves of plants in which chlorophyll occurs and where the process of photosynthesis takes place (Mauzeth, 1995: G - 4). Coleoptile: It is the pointed protective sheath covering the emerging shoot in a germinating monocot seed viii such as maize or grass (Hopkins and Hüner, 2009:492). Cultural harvesting methods: The harvesting methods applied by traditional healers for many years. Includes limitations regarding harvesting methods, harvesting sizes, times collected, tools collected with, the rituals performed before collection, to name a few (Kambizi and Afolayan, 2006:28). Cytokinin: A plant hormone produced in the roots of plants and is primary responsible for regulation cell division and delayed senescence (Hopkins and Hüner, 2009:493). Cytoplasm: Protoplasm consists of nucleus, vacuoles and cytoplasm, or fluid (Mauzeth, 1995: G - 5). Dedifferentiation: A process whereby structures that were specialized for a specific function lose their specialization and become simplified or generalized. (www.dictionary.com/browse/dedifferentiation, viewed 30 September 2016). Embolisms: An obstruction caused by the formation of gas bubbles in the xylem (Hopkins and Hüner, 2009:493). Endangered Species: One of the categories which