ASPECTS OF LEAF AND EXTRACT PRODUCTION from Tasmannia lanceolata by Chris Read, B. Agr.Sc. Tas. Submitted in fulfillment of the requirements for the Degree of Doctor of Philosophy University of Tasmania, Hobart December 1995 ' s~, ... ~~ \ ·'(11 a_C\14 \t\J. \I ' This thesis contains no material which has been accepted for the award of any other degree or diploma in any University, and to the best of my knowledge, contains no copy or paraphrase of material previously written or published by any other person except where due reference is given in the text. University of Tasmania HOBART March 1996 This thesis may be made available for loan and limited copying in accordance with the Copyright Act 1968 University of Tasmania HOBART March 1996 Abstract This thesis examines several aspects of the preparation, extraction and analysis of solvent soluble compounds from leaf material of Tasmannia lanceolata and reports a preliminary survey of extracts of some members of the natural population of the species in Tasmania. A major constituent of these extracts, polygodial, was shown to be stored within specialised idioblastic structures scattered throughout the mesophyll, and characterised by distinctive size and shape, and a thickened wall. The contents of these cells were sampled directly, analysed and compared with the composition of extracts derived from ground, dry whole leaf. This result was supported by spectroscopic analysis of undisturbed oil cells in whole leaf tissue. In a two year field trial, the progressive accumulation of a number of leaf extract constituents (linalool, cubebene, caryophyllene, germacrene D, bicyclogermacrene, cadina-1,4 - diene, aristolone and polygodial) during the growth flush was followed by a slow decline during the subsequent dormant season. These results were interpreted in relation to leaf dry matter accumulation, in order to propose a harvest period within which leaf material will produce consistent composition of extract. Under four levels of irradiance in a growth cabinet experiment the plant exhibited many characteristics of a 'shade' species, in particular, a limited ability to acclimate to high light levels. Assimilation rates were highest at 150µmol m-2s-l while elevated respiration rates and a reduced quantum yield occurred at a higher light level. Maximum assimilation rates in leaves grown at 150µmol m-2s-l were obtained at around 250µmol m-2s-l. Optimum net assimilation rate was obtained from 18-25°C. The effect of level of irradiance on the proportion of extractable compounds in the leaf, chlorophyll levels, specific leaf area, leaf thickness and percentage dry matter in the leaf are reported and discussed in relation to a probable production system in which the new canopy is largely removed at the end of each growth cycle. The ontogenetic patterns determining canopy architecture were observed in the field, and used, with support from the results of a trial pruning of mature trees, to discuss the likely outcome of various harvest methods. These results are combined to suggest a production strategy for maximum yield of leaf extract of consistent composition. The strategy proposes harvesting in late summer, after new leaf has achieved full maturity and may enable full canopy recovery in the subsequent growing season. Acknowledeemen ts My supervisor, Professor Bob Menary has supported and advised me throughout the preparation of this thesis and provided me with guidance and encouragement in the years prior to its commencement. The project was part of a Rural Industry Research and Development Corporation sponsored investigation into the Tasmannia lanceolata as a new essential oil crop, supported by North Eucalypt Technologies and Essential Oils of Tasmania. I would like to thank Sandra Garland, Matthew Gregory and Irene Jacobs for technical assistance and Dr Noel Davies for his advice, and his work with the analytical equipment at the Central Science Laboratory, University of Tasmania. My best friend Sue, and our daughter Anna have shared the burden of this process and together with my parents, who instilled in me an enthusiasm for inquiry, they have my heartfelt thanks for their support and inspiration. Tony Read, my uncle, has shown me the benefits of patience and contemplation, both of which I found invaluable during the course of the project. CONTENTS Abstract Acknowledgements Contents 1 Introduction.................................................................................. 1 2 Literature Review 2.0 Introductory...................................................................... 3 2.1 General............................................................................ 3 2.1.l Distribution of the Winteraceae 2.1.2 Medicinal and culinary uses of Winteraceae 2.2 Morphological and anatomical studies of Tasmannia........................ 5 2.3 Extracts of Winteraceae......................................................... 9 2.3.1 Extraction methods 2.3.2 Polygodial - a bioactive sesquiterpene dialdehyde 2.4 Oil Cells as storage sites for secondary metabolites......................... 21 2.4.1 Oil cell development and structure 2.4.2 Oil cell content c/whole tissue extracts 2.4.3 Isolation of oil bearing organs/cells 2.4.4 Sampling the contents of secretory structures 2.4.5 Other methods of determining oil cell contents 2.5 Seasonal changes in oil composition........................................... 30 2.5.1 Commercial production of essential oils 2.5.2 Chemosystematic studies 2.5.3 Ecological and other studies 2.6 Canopy manipulation in tree species........................................... 40 2.6.1 Growth patterns in Tasmannia lanceolata 2.6.2 Classical canopy models 2.6.3 Pruning and canopy management in evergreen tree crops 2. 7 Light acclimation in understorey species ....................................... 46 2.7.1 Photosynthesis in Tasmannia and Drimys spp. 2.7.2 Ecological significance of acclimation 2.7.3 Light level and the accumulation of secondary compounds 2. 7 .4 Effects of light level on photosynthetic and morphological characters 2.8 Harvest strategies for yield and composition of secondary products....... 53 2.9 Conclusion ........................................................................ 54 3 General Materials, Methods and Preliminary Studies 3.1 Extractions ......................................................................... 55 3.2 Extract Analyses .................................................................. 56 3.3 Plant material, growing conditions............................................. 58 3.4 Microscopy....................................................................... 58 3.5 Preliminary investigations ....................................................... 59 3.6 Propagation of Tasmannia lanceolata ........................................... 66 3.7 Other ............................................................................... 71 4 Preparation, extraction and analysis of leaf extracts of Tasmannia lanceolata. 4.0 Introduction...................................................................... 73 4.1 Time and temperature of drying ................................................ 74 4.2 Comminution of leaf material................................................... 78 4.3 Pelletisation and extraction...................................................... 82 4.4 Ethanol degradation of extract components................................... 84 4.5 Effect of storage conditions on extract quality................................ 86 4.6 Quantitative analysis of small leaf samples .................................... 89 4.7 Identification of extract components ............................................ 93 4.8 Injection artefacts of polygodial.. ............................................... 98 4.9 Summary and Conclusions ...................................................... 99 5 Oil Cells in Tasmannia lanceolata. 5.0 Introduction....................................................................... 100 5.1 Oil cell contents: direct sampling and spectroscopic methods ............... 100 5.2 Oil cell ultrastructure: microscopy.............................................. 107 6 Seasonal changes in oil composition 6.0 Introduction...................................................................... 115 6.1 Changes in extract composition during annual cycle......................... 116 6.2 Discussion...................................................................... 132 7 Harvesting for extract yield and composition 7.0 Introduction....................................................................... 134 7.1 The effect of changing light levels on leaf physiology, oil production ..... 135 7.2 Canopy architecture and pruning strategies................................... 153 8 General discussion ..................................... : .................................... 167 9 Bibliography .................................................................................. 177 Appendices .......................................................................................... 192 I CHAPTER 1: INTRODUCTION Interest in 'wild' populations of plant species with a view to cultivation for human consumption has enjoyed a long history. It is of particular contemporary significance, as concerns are raised about the loss of 'non-commercial' species (flora and fauna) through the careless or deliberate activities of agriculture, forestry, mining and urban development. Support for measures to preserve 'biodiversity' hinge on two premises: the