Hughes, Oliver Thomas Wesley (2018)Orchid – mycorrhiza relationships, propagation of terrestrial and epiphytic orchids from seed. Doctoral thesis (PhD), Manchester Metropolitan University.
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Orchid – Mycorrhiza relationships, propagation of terrestrial and epiphytic orchids from seed
Oliver Thomas Wesley Hughes
A thesis submitted in partial fulfilment of the requirements of Manchester Metropolitan University for the degree of Doctor of Philosophy
Division of Biology and Conservation Ecology
School of Science and the Environment
In Collaboration with the North of England Zoological Society and the University of Manchester
2018
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Contents
Abstract ...... 14
Acknowledgements ...... 16
Chapter 1...... 20
General Introduction ...... 20
References ...... 29
Chapter 2...... 38
Symbiotic germination of the terrestrial orchid Microtis media in vitro is not dependent on fungal sympatry ...... 38
Abstract ...... 39
Keywords Terrestrial orchid germination · Fungal compatibility orchids · Mycorrhizal Fungi · Symbiosis · Symbiotic propagation ...... 39
Introduction...... 39
Materials and Methods ...... 40
Study species ...... 40
Experimental design ...... 42
Results ...... 43
Discussion ...... 45
Acknowledgements ...... 50
References ...... 50
Chapter 3...... 55
Assessing fungal compatibility in the germination and development of four terrestrial orchids of the subtribe Orchidinae ...... 55
Abstract ...... 56
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Keywords Orchidaceae • Mycorrhizae • Fungal compatiibility • Germination • Conservation ...... 56
Introduction...... 56
Materials and Methods ...... 59
Organismal details ...... 59
Experimental procedure ...... 61
Results ...... 64
Anacamptis morio ...... 66
Dactylorhiza incarnata ...... 67
Serapias bergonii ...... 72
Serapias parviflora ...... 74
Discussion ...... 75
Species specific responses ...... 75
Experimental variables ...... 77
Fungal specificity and compatibility ...... 79
Acknowledgements ...... 81
References ...... 82
Appendix ...... 90
Chapter 4...... 92
Symbiotic culture enhances in vitro germination and development of two European terrestrial orchids; Anacamptis laxiflora and Ophrys apifera ... 92
Abstract ...... 93
Keywords Orchidaceae • Germination • Terrestrial • Mycorrhizae • Conservation ...... 93
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Introduction...... 94
Materials and methods ...... 97
Results ...... 101
Initial germination and development ...... 101
Further development and acclimatisation ...... 105
Discussion ...... 109
Acknowledgements ...... 114
References ...... 114
Chapter 5...... 120
The effects of substrate rugosity and complexity on in vitro germination and development in the European terrestrial orchids; Anacamptis morio and Dactylorhiza purpurella ...... 120
Abstract ...... 121
Keywords Orchidaceae • Germination • Terrestrial • Mycorrhizae • Rugosity • Substrate complexity ...... 121
Introduction...... 122
Materials and methods ...... 124
Organismal details ...... 124
Experimental procedure ...... 125
Results ...... 129
Germination and development ...... 129
Statistical analysis ...... 135
Rugosity treatments ...... 135
Negative control (treatments) ...... 136
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Substrate complexity ...... 137
Discussion ...... 138
Acknowledgements ...... 144
References ...... 144
Chapter 6...... 162
Asymbiotic and symbiotic germination of five epiphytic orchids from the subtribe Pleurothallidinae - advantage for initial symbiotic germination 162
Abstract ...... 163
Keywords Orchidaceae • Mycorrhizae • Germination • Propagation • Conservation ...... 164
Introduction...... 164
Materials and Methods ...... 168
Organismal details ...... 168
Experimental procedure ...... 169
Results ...... 173
Masdevallia melanoxantha...... 174
Germination ...... 174
Development ...... 175
Masdevellia menatoi ...... 176
Germination ...... 176
Development ...... 177
Masdevallia patchicutzae ...... 179
Germination ...... 179
Development ...... 179
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Pleurothallopsis microptera ...... 179
Germination ...... 179
Development ...... 180
Restrepia contorta ...... 181
Germination and development ...... 181
Stelis sp. Peru ...... 181
Germination and development ...... 181
Discussion ...... 186
Acknowledgements ...... 191
References ...... 192
Chapter 7...... 203
A method for using microcosms for the ex situ study of tripartite symbioses between ectomycorrhizal plants, fungi and myco-heterotrophic plants ...... 203
Abstract ...... 204
Keywords Orchidaceae • Mycorrhizae • Myco-heterotrophy • Microcosm • Symbiotic culture ...... 204
Introduction...... 204
Materials and methods ...... 206
Organismal details ...... 206
Experimental procedure ...... 207
Results ...... 209
Discussion ...... 212
Acknowledgements ...... 215
6
References ...... 215
Appendix ...... 222
Chapter 8...... 225
General Discussion ...... 225
References ...... 237
7
List of Figures
Chapter 1.……………………………………………………………………………….. 21
General Introduction………………………………………………………………….. 21
Chapter 2………………………………………………………………………………... 38
Symbiotic germination of the terrestrial orchid Microtis media is not dependent on fungal sympatry……………………………………………………... 38
Figure 1. Number of Microtis media protocorms on each of the fungal isolates……………………………………………………………………………... 44 Figure 2. Stages of development in Microtis media ………………...... 45
Figure 3. Microtis media plants with Rhizoctonia solani……………………… 46
Chapter 3………………………………………………………………………………... 55
Assessing fungal compatibility in the germination and development of four terrestrial orchids of the subtribe Orchidinae……………………………… 55
Figure 1. Stages of orchid germination used to assess development………. 65
Figure 2. Germination of Anacamptis morio, Dactylorhiza incarnata, Serapias bergonii and Serapias parviflora on each treatment……………….. 68
Figure 3. Stages of development in Anacamptis morio, Dactylorhiza incarnata, Serapias bergonii……………………………………… 70
Figure 4. Development of Anacamptis morio on each treatment…………… 71
Figure 5. Development of Dactylorhiza incarnata on each treatment…….... 72
Figure 6. Development of Serapias bergonii on each treatment……..…….. 73
Figure 7. Development of Sera