Maria Filomena Figueiredo Nazaré Gomes Strategies for the improvement of Arbutus unedo L. (strawberry tree): in vitro propagation, mycorrhization and diversity analysis 2011 Departamento de Ciências da Vida Universidade de Coimbra Maria Filomena Figueiredo Nazaré Gomes Orientador: Prof. Doutor Jorge Manuel Pataca Leal Canhoto Strategies for the improvement of Arbutus unedo L. (strawberry tree): in vitro propagation, mycorrhization and diversity analysis 2011 Dissertação apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra para obtenção do grau de Doutor em Biologia, especialidade de Fisiologia. ACKNOWLEDGMENTS My first thanks must go to my supervisor, Prof. Dr. Jorge Canhoto who helped me, for his encouragement, scientific support and careful review of this work. To Dr. Rita Costa, Dr. Helena Machado and Dr. Maria Margarida Ribeiro, a very special thanks for their scientific support, encouragement and careful review of Chapters 4 and 5. To Dr. António Portugal, Dr. Maria Teresa Gonçalves and Dr. Nuno Mesquita I am very grateful for their scientific support. To Prof. Justina Franco, Dr. Conceição Costa and to the team work of Laboratory of Plant Science and Chemistry from ESAC, I am very grateful for the fruit analyses performed. To Dr. Ludovina Lopes, Dr. Isabel Evaristo and Dr. Elisa Figueiredo, I express my gratitude for their support, scientific advises and encouragement. To Prof. Dr. Fernando Páscoa, I would like to thank for his support, encouragement and friendship always stated. I would also like to express my gratitude to my colleagues José Maia, Fernando Casau, David Rodrigues, Célia Ferreira, Carmo Magalhães, and Cristina Cameira for their support, encouragement and friendship. My acknowledgements to: Escola Superior Agrária de Coimbra, Departamento de Ciências da Vida, Universidade de Coimbra and INRB, I.P./INIA - Polo de Oeiras the availability of their facilities for experimental work; to Portuguese Foundation for Science and Technology (FCT) for PhD fellowship (SFRH/BD/37170/2007 to 2009) and to PROTEC program for PhD fellowship (SFRH /BD / 50263 / 2009 to 2011). I would like to thank to students Mafalda Simões, Esteban San Martin, Ana Ponce Díaz, Jorge Agrela, Vera Santos, Gema Requena, Iker Sorzabalbere and Filipe Moreira for their collaboration. To Beatriz and Eugénio! TABLE OF CONTENTS INDEX OF ABBREVIATIONS........................................................................................... III GENERAL SUMMARY ......................................................................................................... 1 RESUMO GERAL ................................................................................................................... 2 1 INTRODUCTION ............................................................................................................. 5 1.1 ARBUTUS UNEDO L., A BRIEF DESCRIPTION ............................................................................... 5 1.2 ARBUTUS UNEDO , ECONOMIC AND BIOLOGICAL RELEVANCE ........................................... 8 1.3 MICROPROPAGATION OF WOODY PLANTS .............................................................................. 11 1.3.1 Factors affecting in vitro micropropagation...................................................................................... 13 1.4 MYCORRHIZAL SYNTHESIS ............................................................................................................ 17 1.4.1 Different types of mycorrhiza ........................................................................................................... 18 1.4.2 Function and potential applications of mycorrhiza ........................................................................... 21 1.5 GENETIC DIVERSITY STUDIES USING DNA MARKERS .......................................................... 24 1.6 OBJECTIVES ......................................................................................................................................... 29 1.7 REFERENCES ....................................................................................................................................... 31 2 MICROPROPAGATION OF STRAWBERRY TREE ( ARBUTUS UNEDO L.) FROM ADULT PLANT ........................................................................................................ 46 2.1 ABSTRACT ............................................................................................................................................ 46 2.2 INTRODUCTION .................................................................................................................................. 48 2.3 MATERIAL AND METHODS ............................................................................................................. 50 2.3.1 Plant material .................................................................................................................................... 50 2.3.2 Surface-sterilization procedures and culture establishment .............................................................. 50 2.3.3 Shoot multiplication .......................................................................................................................... 51 2.3.4 Rooting ............................................................................................................................................. 52 2.3.5 Acclimatization ................................................................................................................................. 54 2.3.6 Anatomical studies ............................................................................................................................ 55 2.3.7 Statistical analysis ............................................................................................................................. 55 2.4 RESULTS ................................................................................................................................................ 56 2.4.1 Culture establishment ....................................................................................................................... 56 2.4.2 Shoot multiplication .......................................................................................................................... 58 2.4.3 Rooting ............................................................................................................................................. 60 2.4.4 Acclimatization ................................................................................................................................. 66 2.4.5 Anatomical studies ............................................................................................................................ 68 2.5 DISCUSSION .......................................................................................................................................... 73 2.5.1 Culture establishment ....................................................................................................................... 73 2.5.2 Shoot multiplication .......................................................................................................................... 74 2.5.3 Rooting ............................................................................................................................................. 76 2.5.4 Acclimatization ................................................................................................................................. 79 2.6 REFERENCES ....................................................................................................................................... 81 3 EFFECT OF PLANT GROWTH REGULATORS AND GENOTYPE ON THE MICROPROPAGATION OF ADULT TREES OF ARBUTUS UNEDO L. (STRAWBERRY TREE) ...................................................................................................... 91 3.1 ABSTRACT ............................................................................................................................................ 91 3.2 INTRODUCTION .................................................................................................................................. 93 3.3 MATERIAL AND METHODS ............................................................................................................. 95 3.3.1 Establishment and propagation ......................................................................................................... 95 3.3.2 Effect of plant growth regulators ...................................................................................................... 95 3.3.3 Effect of the genotype ....................................................................................................................... 96 3.3.4 Rooting and acclimatization ............................................................................................................. 96 3.3.5 Experimental design and statistics .................................................................................................... 97 3.4 RESULTS ................................................................................................................................................ 98 3.4.1 Effect of PGRs .................................................................................................................................. 98 i Table of Contents 3.4.2 Effect of the genotype ....................................................................................................................