University of Bath PHD The use of tissue culture techniques to study pathogenicity of and select for resistance to three major diseases (E. Carotovora subspp., Pythium violae, Erysiphe heraclei) of carrot (Daucus carota L) Daüstü, Nazan Award date: 1996 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 09. Oct. 2021 THE USE OF TISSUE CULTURE TECHNIQUES TO STUDY PATHOGENICITY OF AND SELECT FOR RESISTANCE TO THREE MAJOR DISEASES (E. carotovora subspp., Pythium violae, Erysiphe heraclei) OF CARROT (Daucus carota L.) Submitted by Nazan Dagiistii, M.Sc. for the degree o f Doctor o f Philosophy o f the University o f Bath 1996 COPYRIGHT Attention is drawn to the fact that copyright of this thesis rests with its author. This copy of the thesis has been supplied on the condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from this thesis and no information derived from it may be published without prior written consent of the author. This thesis may be made available for consultation within the University Library and may be photocopied or lent to other libraries for the purpose of consultation. Signed ^ UMI Number: U601711 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U601711 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 THS0! ACKNOWLEDGEMENTS I wish to express my sincere gratitude to Dr. Richard M. Cooper for his invaluable guidance, continuous encouragement and most of all, his patience, throughout the course of this project. I would also like to thank Prof. G. G. Henshaw for his input into this thesis at its critical times. May I also express my gratitude for Dr. Nigel Taylor for his guidance and help at various stages of this work. My thanks also go to Dr. Dave Blakesley for his interest. I would also like to thank members of the School of Biology and Biochemistry, especially, Dr. Dave Dymock, Terence Percival and Mike Skinner. May I also express my special thanks to Chris Davey for his technical assistance, Ursula Potter for her help in the Scanning Electron Microscopy work and Paul Christie, Dr. Roger Mepsted, and P. K. Nandi for their advice in Statistics. I would particularly like to thank Dr. Recep Akme§e for his encouragement and preparation of this thesis. I am also indebted to my “Dear Teacher”, Professor Halis Ruhi Ekingen, for his ever-lasting encouragement and support which, more than anything else, enabled me to undertake this Ph.D. program and see its successful conclusion. I am also thankful to Uludag Universitesi (Bursa, Tiirkiye) for their financial support. Finally, I am grateful to my parents, sisters and brother for all their patience, endurance and encouragement which provided the strength to face the various challenges over the last few years. ABSTRACT Highly embryogenic callus and suspension cultures of carrot ( [Daucus carota L.) were developed in order to generate, via somaclonal variation, disease resistance to three major pathogens of carrot in Europe for which there are no commercially used resistant cultivars i.e. Erwinia carotovora spp. (soft rot), Pythium violae (cavity spot) and Erysiphe heraclei (powdery mildew). Twenty seven percent of the regenerant seedlings screened from callus cultures expressed resistance to E. heraclei. Regenerant Somaclone-4 was significantly more resistant to powdery mildew than the original seed line NRI-92. However, this frequency of resistance among the regenerants was much higher than expected from somaclonal variation. It was concluded that variation may have come from the original seed used, because a high percentage of plants (ca. 40%) in the original seed line was free from powdery mildew. Nevertheless, it is clear that effective resistance already exists within the species. Scanning electron microscopy revealed no differences in terms of E. heraclei infection structures on leaf surfaces of resistant Somaclone-4 and susceptible NRI-92; resistance must be expressed after penetration. Six percent of roots of regenerants from embryogenic suspension cultures was resistant to P. violae and also the proportion of inoculated sites infected with P. violae was less than the original seed line cv. Morot Duke of which all roots developed cavity spot. Three percent of E. carotovora inoculated roots of regenerants was free from soft rot, in contrast to 18% of roots of cv. Morot Duke which remained symptomless after inoculation. These findings suggest that taproots showing resistance to E. carotovora may have arisen by one or a combination of: variation induced by tissue culture; variation resulting from segregation in the seed line used; “escapes” resulting from an imperfect inoculation procedure. Reinoculation of cloned putative resistant individuals is required to confirm these possibilities. Co-culture of carrot cells with both E. carotovora wild type and mutants and with P. violae was attempted with the aims of i) investigating determinants of pathogenicity and ii) regenerating putative, novel resistant plants from surviving embryogenic cells. Mycelial fragments of P. violae killed carrot cells very rapidly (< 24 hrs) such that this did not provide a suitable selection procedure. The pathogenicity determinants of P. violae are unknown, but cellulase, suberinase and pectin lyase were the major cell wall degrading enzymes, which may aid in penetration of the suberinized endodermis which surrounds carrot taproots. Carrot cells were killed rapidly (ca. 70% of the cell units and 80% of single cells in 24 hrs) with wild type E. carotovora (SCRI 193). Results in vitro and in vivo implied that the major pathogenicity factor of E. carotovora involved in maceration and killing of carrot cells was the extracellular cell wall degrading enzyme, pectate lyase (PGL). Thus pectate lyase killed carrot cells, enzyme deficient mutants (RJP 116 and RJP 243) had reduced pathogenicity and killed plant cells more slowly and to lower levels than wild type SCRI 193. However, in some cases cell killing in co-culture was initiated just before appearance of PGL, suggesting the involvement of another factor. The possible value of enzyme deficient mutants in screening carrot cells for disease resistance in vitro is discussed. Kanamycin at 25 pg/ml was used successfully for removing bacteria from co-culture whilst retaining viability of embryogenic cell units; however the antibiotic markedly reduced their regeneration capacity. No mature somatic embryo was regenerated from co-culture. However, it should be possible with a more suitable antibiotic to select and regenerate plants from this developed co-culture technique. CONTENTS ACKNOWLEDGEMENTS i ABSTRACT ii CONTENTS iv ABBREVIATIONS xiv CHAPTER 1 GENERAL INTRODUCTION 1 1. CARROT 1 1.1. ORIGIN, DISTRIBUTION, DESCRIPTION AND TAXONOMY 1 1.2. ECONOMIC IMPORTANCE OF CARROT 2 2. SOMATIC EMBRYOGENESIS IN CARROT 3 2.1. FACTORS INFLUENCING SOMATIC EMBRYOGENESIS 7 3. SOME MAJOR CARROT DISEASES CAUSED BY FUNGI AND BACTERIA 11 3.1. BACTERIAL SOFT ROT DISEASE CAUSED BY Erwinia carotovora 11 3.1.1. Pathogenesis by Soft Rot Erwinias 15 3.1.1.1. Cell Wall Degrading Enzymes 15 3.2. CAVITY SPOT DISEASE CAUSED BY Pythium viola 24 3.2.1. Pathogenesis by Pythium spp. 28 iv 3.3. POWDERY MILDEW DISEASE CAUSED BY Erysiphe heraclei 33 4. SOMACLONAL VARIATION AND DISEASE RESISTANCE 34 4.1. In Vitro SELECTION FOR DISEASE RESISTANCE 40 4.1.1. Selection Using Obligate Parasite 41 4.1.2. Selection Using Facultative Parasites 43 4.1.3. Selection in vitro with Toxins and Culture Filtrates 45 5. AIMS 49 CHAPTER 2 MATERIALS AND METHODS 50 1. CARROT TISSUE CULTURE 50 1.1. PLANT MATERIAL 50 1.2. PREPARATION AND SURFACE STERILIZATION OF EXPLANTS 51 1.3. CULTURE MEDIA 51 1.4. ESTABLISHMENT OF CALLUS AND SUSPENSION CULTURES 52 1.4.1. Initiation, Subculture and Maintenance of Callus Cultures 52 1.4.2. Initiation, Subculture and Maintenance of Suspension Cultures 52 1.4.3. Subculture Regime for Stock Cell Suspension Cultures 53 1.4.4. Preparation of Carrot Cell Suspension Culture for Co-Culture 56 1.4.5. Assessment of Cell Viability of Carrot Suspension Culture 56 1.4.6. Determination of Fresh Weight of Cell Fractions from Suspension Cultures 59 1.5. ACCLIMATIZATION OF PLANTLETS REGENERATED FROM CALLUS AND SUSPENSION CULTURES 59 1.6. PRODUCTION OF SEEDS FROM REGENERATED PLANTS 60 2. BACTERIAL ISOLATES 60 2.1. CULTURE MEDIA AND PREPARATION OF BACTERIAL INOCULUM 60 2.2. MAINTENANCE AND STORAGE OF BACTERIAL ISOLATES 61 2.3.