AN ABSTRACT OF THE THESIS OF Joanne C. Ruttum for the degree of Master of Science in Botany and Plant Pathology presented on June 27, 1991. Title: Development of in vitro Lily Scale Bulblets as Related to Virus Elimination -Redacted for Privacy_ Abstract approved: Thomas C. Allen, Jr. Lily hybrids vary in their ability toproduce virus-free (VF) bulblets when grown fromvirus-infected scalesintissue culture. Asiatic hybrids typically produce a higher percentage of in vitro VFscale bulblets than do Lilium longiflorum cultivars. Three hypotheses concerning the cause of this variation are tested onfive lily hybrids: an Asiatic hybrid, two L. longiflorum cultivars, an Oriental hybrid and L. candidum. The first hypothesis states that VFscale bulblets originate from wound tissue that is naturallylow in virus concentration and blocks the passage of virusparticles from one cell to the next. The second hypothesis says that scale-to-bulblet vascular connections, which serve asvirus pathways, occur inhybrids showing high percentages of virus-infected scale bulblets, while connections are absent in those hybridswithlow numbersof virus-infected bulblets. The third hypothesis concerns the virus concentration in the scale at the site of bulblet origin: bulblets of hybrids producing large numbers of VF bulblets originate from scale tissues low in virus concentration; bulblets of low percentage VF bulblet hybrids originate from scale tissues high in virus concentration. The first two hypotheses are not supported by the results of thisstudy. First, lily bulblets do not originate from wound tissue. Second,scale-to-bulblet vascular connections consistently occur in 'Enchantment,' an Asiatic hybrid, and occasionally occur in L. candidum. Vascular connections are not detected in the low VF bulblet producers, L. longiflorum cultivars 'Ace' and 'Nellie White,' nor are they seen in the Oriental hybrid 'Stargazer.' Speculative support exists for the third hypothesis concerningunevenvirus concentration in the scale. Distinct virus particles are observed with the electron microscope in the double virus-infected L.longiflorum cultivars and not in the other singly-infected lilies. The doubly-infectedliliesproduce a continuouslayer of divided cells in the adaxial subepidermis of the scale where bulbletsoriginate, whereasthesingly-infected lilies produce cell division masses in the same area but only beneath forming bulblets. This studysuggests that virus particles in L. longiflorum cultivars are more uniformly distributed than particles in the other lilies examined. This occurs not only at the site of bulblet origin but also throughout the scale mesophyll. Whether this is due to concurrent viral infection or to hybrid variation is unknown. Development of in vitro Lily Scale Buiblets as Related to Virus Elimination by Joanne C. Ruttum A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Completed June 27, 1991 Commencement June 1992 APPROVED: Redacted for Privacy Professor of Botany and Plant Pathol6R9 charge of major Redacted for Privacy Head of Department of Botany and Plant Pathology Redacted for Privacy Dean of Gradue School 1 Date thesis is presented June 27, 1991 Typed by Joanne C. Ruttum for Joanne C. Ruttum ACKNOWLEDGEMENTS I wish to thank the following people: Dr. Thomas C. Allen,Jr. of Oregon State University for accepting and supporting me as a graduate student, without whom I may never have discovered the world of lilies and plant viruses; Drs. Fred R. Rickson and Machteld C. Mok of Oregon State University for serving on my committee and for their support and guidance; Richard Samson,Alfred H.Soeldner,and Blaine Baker of Oregon State University for their lasting patience and assistance; Oregon Bulb Farms of Aurora, Oregon and the Easter Lily Research Foundation of Brookings, Oregon for supplying the bulbs used in this study; Dr. Edwin R. Florance of Lewis and Clark College, Portland, Oregon and Dr. Wolf H. Fahrenbach and Michael Webb of the Oregon Regional Primate Research Center of Beaverton, Oregon for the use of their labs and for their technical assistance; and Kurt Ruttum, my husband, for moral and financial support. LIST OF ABBREVIATIONS BYMV = bean yellow mosaic virus CMV = cucumber mosaic virus DAE = days after explanting ELISA = enzyme-linked immunosorbent assay ISEM = immunosorbent electron microscope ISMV = iris severe mosaic virus LSV = lily symptomless virus TBV = tulip breaking virus TBV-L = tulip breaking virus longiflorum strain TEM = transmission electron microscope TMV = tobacco mosaic virus VF = virus free VI = virus inclusion VP = virus particle TABLE OF CONTENTS INTRODUCTION 1 REVIEW OF LITERATURE 5 Tissue Culture and Virus Elimination 5 Plant Regeneration through Tissue Culture 5 Virus Elimination with Meristems 7 Virus Elimination in Lilies by 8 Adventitious Bud Formation Virus Elimination through Heat Treatment 11 and Chemotherapy Virus Assay Techniques and Lily Viruses 13 External Symptomatology 13 Serology 14 Electron Microscope Assay 15 Lily Viruses 17 Symptoms 17 Identification 17 Detection with ELISA 18 Cytological Changes Due to Virus Infection 21 Movement of Virus Particles within Plants 21 Virus Particle Movement within Lily 23 Tissues Virus Effects on Plant Cells 23 Organelle Degeneration 23 Viral Synthesis 24 Unknown Effects 25 Virus Inclusions 26 Virus Effects on Lilies 27 Organelle Abnormalities 27 Virus Inclusions 27 Cytology of Lily Scale Bulblet Formation 28 Scale Bulblet Formation by Scaling 28 In vitro Scale Bulblet Formation 30 TABLE OF CONTENTS (CONTINUED) MATERIALS AND METHODS 32 Lily Selection 32 Tissue Culture 32 Preparation for Light Microscope Examination 33 Virus Assay 34 ELISA 34 Electron Microscope Assay 34 RESULTS 36 Tissue Culture 36 General Plantlet Formation 36 General Variation among Cultures 37 Specific Variation among Lily Varieties 37 Anatomical Examination 39 Virus Assay 42 ELISA on Mother Plant Tissue 42 Electron Microscope Assay of in vitro 43 Tissues Ultrastructural Abnormalities 44 DISCUSSION 46 Hypothesis 1: Wound Tissue 46 Hypothesis 2: Vascular Connections 47 Hypothesis 3: Virus Concentration 50 BIBLIOGRAPHY 91 LIST OF FIGURES Figure Page 1. Diagram of tissue culture initiation 56 procedure. 2. Diagram of in vitro scale explants shows 57 the three areas samples were sectioned for TEM examination. 3. Diagram of in vitro bulblet development 58 and location on explant. 4. Comparison of root development. 59 5. Comparison of scale vascular bundles. 60 6. Collapse and cell wall thickening of cells 61 at edge of original transverse cut of scale explant. 7. Cell division in epidermal and subepidermal 62 scale layers initiate bulblet development. 8. 'Enchantment' bulblets develop above scale 63 vascular bundles. Cell division between bulblet and vascular bundle in mesophyll is characteristic of this lily. 9. Easter lily bulblet positioned above scale 64 vascular bundle with little cell division in mesophyll between bulblet and vascular bundle. 10. Easter lily section with cell division 65 across entire top (adaxial) surface of scale explant. Bulblet base shows cells with secondary thickening of walls. 11. 'Enchantment' section with subepidermal 66 cell division only at origin of bulblet. 12. Vascular connections between bulblet 67 procambium and scale vascular bundle occur in 'Enchantment.' LIST OF FIGURES (CONTINUED) Figure Page 13. 'Ace' scale section without vascular 68 connection between bulblet procambium and scale vascular bundle. 14. Comparison of scale bulblet root origin. 69 15. LSV and TBV particles present in Easter 70 lily cells. 16. TBV pinwheel inclusions present in Easter 74 lily cells. 17. Aggregated mitochondria found in Madonna 77 bulblet cell. 18. Chloroplasts -- swollen, aggregated, and 78 with or without starch grains found in 'Stargazer.' 19. Chloroplast with vesiculated outer membrane 79 found in Easter lily bulblet cell. 20. Cell wall deformations present in Easter 80 lily cells. 21. Vesiculated cytoplasm with many aggregated 81 ribosomes as seen in 'Ace' mesophyll cell. 22. Advanced degenerate cell, covered with dark 82 osmiophilic material, present in 'Nellie White' section. 23. Osmiophilic globules present in 'Enchantment' 83 cells. 24. Osmiophilic globules present within 84 plasmodesmata in 'Enchantment' and 'Stargazer' sections. LIST OF TABLES Table Page 1. Absorbance for cucumber mosaic virus, 87 tulip breaking virus and lily symptomless virus on lily leaf tissue 2. Number of cell sections per lily type 88 containing virus particles (VP) by DAE and tissue area 3. Number of cell sections per lily type 89 containing virus inclusions (VI) by DAE and tissue area 4. Total number of lily cell sections 90 containing virus particles and virus inclusions by DAE and tissue area DEVELOPMENT OF IN VITRO LILY SCALE BULBLETS AS RELATED TO VIRUS ELIMINATION INTRODUCTION Tissue culture methods for producing virus-free (VF)1 lilies have been known and applied for over twenty years.In vitro procedures and virus assay techniques have been modified and perfected during this period and are now available to commercial bulb growers worldwide. One of the first research teams to develop a reliable in vitro method of producing VF lilies was Allen and Fernald (1972). They diced lily bulb scales and incubated the small pieces on nutrient medium in culture tubes. Some of the bulblets, produced on the surface of the virus-infected scale pieces, were free of virus. The virus elimination procedure developed by Allen