AVOCADO SUNBLOTCH VIROID (ASBVd) IN CELL AND TISSUE CULTURES OF AVOCADO ( Persea americana Mill.) By ISIDRO ELIAS SUAREZ PADRON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2003 Copyright 2003 by Isidro E. Suarez Padron To my wife Sahara, for her love and comprehensive patience during these difficult years of sacrifice, and to my sons, Sebastian and Elias David for my absence in the most precious moments. A mi esposa Sahara por su amor y comprensiva paciencia durante estos dificiles anos de sacrificio, y a mis hijos Sebastian y Elias David por mi ausencia en los momentos mas hermosos de sus vidas. AKNOWLEDGMENTS I wish to express my deepest gratitude to Dr. Richard Litz, my major professor, for his unlimited support, advice, help, patience and time, and particularly for his direction in the completion of this dissertation. His encouragement, guidance and permanent stimulation for better work will always be remembered. I want to extend my gratitude for their help, guidance, and stimulating discussion to the members of my graduate committee, Drs. Raymond J. Schnell, Dennis Gray, Paul Lyrene, and Nigel Harrison. Sincere thanks go to Mrs. Pamela Moon for her help with statistical analyses, graphic designs and lab support. The invaluable cooperation of Drs. David N. Kuhn, Martha Heath and Alan Meerow, and Cecile Olano, Wilber Quintanilla, Cheryl Krol, Mike Winterstein, Jason Clayton, Emilio Power, Tyrone McArthy, Wilhelmina Wasik and Nannette Langiven at the Plant Sciences lab of the ARS-USDA in Miami, FL, will always be appreciated. Special appreciation goes to Dr. Simon Raharjo, Darda Efendi and his lovely wife, Neneng, and Sadanand Dekhney for making this long time more enjoyable and unforgettable. The help and friendship of other staff and faculty members at TREC will be remembered. The development of this doctoral program was granted by the support of my home institution, La Universidad de Cordoba, and I am honored to acknowledge a scholarship provided by the Instituto Colombiano de la Ciencia y la Tecnologia IV (COLCIENCIAS) and the Fulbright Commission, and administered by the Latin American Program for American Universities (LASPAU). Last but not least, a scholarship provided by the Dade County Agri-Council and a research assistantship provided by the California Avocado Society were critical for the completion of this program and therefore are deeply appreciated. IV TABLE OF CONTENTS ACKNOWLEDGMENTS iv LIST OF TABLES viii LIST OF FIGURES « ABSTRACT xi CHAPTERS INTRODUCTION 1 LITERATURE REVIEW 5 Avocado 5 Somatic Embryogenesis 13 Micrografting 19 Avocado Cell and Tissue Culture 22 Viroids 32 MICROGRAFTING OF ASBVd INFECTED PLANTS 52 Introduction 52 Materials and Methods 53 Results 61 Discussion 67 REGENERATION OF AVOCADO PLANTS FROM THE NUCELLUS AS A STRATEGY FOR ELIMINATING ASBVd 73 Introduction 23 Materials and Methods 24 Results 84 Discussion 102 SUMMARY AND CONCLUSION 116 Summary H6 Conclusion 118 vi APPENDIX 120 LIST OF REFERENCES . 134 BIOGRAPHICAL SKETCH 155 vii LIST OF TABLES Table2- page 3- 2-1. Avocado production and area planted (in parenthesis) by region during 3- 1996-2002 12 4- 2-2. Largest country avocado producers during 1996 - 2002 13 3. Viroid Classification 34 1 Percentages of success of micrografting of avocado 62 2. ASBVd in micrografted plants and scion-donor plants 65 86 1 . Number and type of embryogenic cultures 4-2. Average number and standard errors (in parenthesis) of proembryonic masses (PEMs), globular somatic embryos (GSE), hyperhydric and opaque somatic embryos (HSE) and total number of somatic embryos (SE) proliferated on MSP medium in 2002 and 2003 87 4-3. Settled cell volumes (SCVs) of embryogenic cultures (in ml) in liquid medium 91 4-4. ANOVA of the total somatic embryos that developed on SED medium 93 4-5. t-test of hyperhydric somatic embryos that developed on SED medium 94 4-6. Sequence variants of ASBVd isolated from ‘Vero Beach’ SE2 99 4-7. Distribution of the number of variants isolated from in vitro tissues with respect to nucleotide exchanges in the ASBVd molecule 104 A-l. ANOVAS of number and type of embryogenic cultures that developed on MSP medium 121 A-2. ANOVAs of settled cell volumes (SCVs) in liquid medium 122 viii 2- LIST OF FIGURES 3- Figure page 1. ASBVd nucleotide sequence and secondary structure showing the areas of the hammerhead formation in the plus (+) strand 38 1. Micrografting showing localization of the scion on the rootstock (left), initial stages of elongation (center) and growth and development (right) 62 3-2. Electropherograms of RT-PCR positively indexed leaves (left) of a and b: micrograft No. 103 and its respective scion-donor seedling; c and d: 3- micrograft No. 133, and its respective scion-donor seedling and e: flower tissue of positive control ‘Vero Beach’ SE2, and negatively indexed 4- leaves (right) of f and g: micrograft No. 10 and its scion-donor seedling; h and i: micrograft No. 1 1 1 and its scion-donor seedling; and j: leaf tissue of negative control ‘Simmonds’ 64 3-3. Gel agarose electrophoresis of PCR amplified fragments of micrografts 12, 15 and 52 (Lines 1, 3 and 5) and their respective scion-donor plants (lines 2, 4, 6). Line 7 = positive control ‘Vero Beach’ SE2 floral tissue. M = 100 bp ladder 66 4. ASBVd variant J02020 secondary structure molecule (Daros and Flores, 2000) isolated from micrograft No. 133 67 1. Induction and proliferation of embryogenic cultures on solid medium. Embryogenic culture induction from nucellus (left) and PEM proliferation (right) 85 4-2. Mean and standard errors from the ANOVA of a = somatic embryo development; b = globular somatic embryos; c = hyperhydric and opaque somatic embryos <0.5 cm diam.; d = hyperhydric and opaque somatic embryos >0.5 cm dia., and e = PEMs of cultivars ‘Irwin’, ‘Vero Beach’ 1, ‘Vero Beach’ SE2 and ‘Yama’ 381 87 4-3. Suspension cultures of ‘Vero Beach’ SE2 90 4-4. Means and standard errors of settled cell volumes (SCVs) of suspension cultures 92 4-5. Average of somatic embryos that developed on SED medium in each dish 93 IX 4-6. Average number of hyperhydric somatic embryos per Petri dish that developed on SED medium from approx. 100 mg embryogenic culture inoculum 94 4-7. Shoot development (left) and plantlet recovery from somatic embryos of ‘Vero Beach’ SE2 95 4-8. Electropherograms of RT-PCR amplified fragments of: a: PEMs of ‘Hass’ 11.4.3, b: somatic embryo of ‘Vero Beach’ SE2, c: ‘Vero Beach’ SE2 PEMs one year after induction, d: PEMs of ‘Yama’ 381 one year after induction, e: ‘Vero Beach’ SE2 nucellar plant number 1 and f: floral tissue of ‘Vero Beach’ SE2 97 = 4-9. Agarose gel electrophoresis of RT-PCR amplified products. 1 and 10 PEMs of ‘Hass’ 11.4.3; 2 to 4 = PEMs of ‘Irwin’, ‘Yama’ 381 and ‘Vero = Beach’ 1 respectively; 5 = Somatic embryo of ‘Vero Beach’ SE2; 6 to 8 Nucellar plants number 1, 2 and 3 respectively; 9 = Floral tissue of ‘Vero Beach’ SE2; M= 100 bp ladder 98 4-10. Nucleotide variation, frequency of changes and number of variants with these changes in a: embryogenic cultures, b: floral tissue, c: nucellar plant 1, d: nucellar plant 2 and e: nucellar plant 3 of ‘Vero Beach; SE2 105 A-l. ASBVd variants isolated from micrograft No 133 123 A-2. ASBVd variants isolated from PEMs, somatic embryos, floral tissue and nucellar plants 124 x Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy AVOCADO SUNBLOTCH VIROID (ASBVd) IN CELL AND TISSUE CULTURES OF AVOCADO (Persea americana Mill.) By Isidro Elias Suarez Padron December, 2003 Chairman: Richard E. Litz Major Department: Horticultural Sciences Avocado sunblotch is an important disease of avocado (Persea americana Mill.). The symptoms of the disease include marked decrease in the health of the plant, streaks on stems, bleaching and variegation of leaves and sunken yellow or reddish areas on the fruit surface. The disease is caused by the avocado sunblotch viroid (ASBVd), a single- stranded RNA molecule with a rod-like circular secondary structure. ASBVd-affected avocado trees have >25% reduction in fruit yield and >50% undergraded fruits. ASBVd can be transmitted via seeds, vegetative tissue and pollen, and can withstand high temperatures when thermotherapy treatments have been applied. Nucellus culture and micrografting have been used for the recovery of healthy plants of several citrus species infected with citrus exocortis viroid (CEVd). Likewise, healthy potato ( Solanum tuberosum ) and grapevine ( Vitis vinifera ) plants have been recovered from potato spindle xi tuber viroid (PSTVd)-infected and grapevine viroid-infected materials, respectively. This study has examined the use of nucellar culture and micrografting in order to recover healthy avocado plants from ASBVd-infected material. Embryogenic cultures were induced from the nucellus of ASBVd-infected cultivars ‘Irwin’, ‘Vero Beach’ 1, ‘Vero Beach’ SE2 and ‘Yama’ 381. The cultures proliferated and nucellar plants were recovered from white-opaque somatic embryos of cultivar ‘Vero Beach’ SE2. Embryogenic nucellar cultures and regenerated plants were indexed for the presence of ASBVd using RT-PCR. ASBVd was persistent in embryogenic cultures and nucellar plants. Shoot tips from ASBVd-infected seedlings were micrografted onto ASBVd-free seedlings and plants were recovered under in vitro conditions. RT-PCR indexing established that ASBVd was persistent in scions that developed from shoot tips <0.5 mm. Pospiviroidae viroids such as PSTVd and CEVd move in the phloem and therefore cannot infect the nucellus and the shoot apical meristem, both of which lack vascular tissue. These viroids can therefore be eliminated either by micrografting or nucellar culture.