%N VUE DE LOBTENTION DU %0$503"5%&-6/*7&34*5²%&506-064& $ÏLIVRÏ PAR Institut National Polytechnique de Toulouse (INP Toulouse) $ISCIPLINE OU SPÏCIALITÏ Sciences des Agroressources 0RÏSENTÏE ET SOUTENUE PAR Anna CIANCOLINI LE vendredi 6 juillet 2012 4ITRE CHARACTERIZATION AND SELECTION OF GLOBE ARTICHOKE AND CARDOON GERMPLASM FOR BIOMASS, FOOD AND BIOCOMPOUND PRODUCTION %COLE DOCTORALE Sciences de la Matière (SDM) 5NITÏ DE RECHERCHE ENEA et Universita degli Studi della Tuscia (Italie), -$"UMR 1010 INRA/INP-ENSIACET (France) $IRECTEURS DE 4HÒSE Pr Mario Augusto PAGNOTTA et Dr Gérard VILAREM 2APPORTEURS Dr Antonio ELIA (Professeur, Università degli Studi di Foggia, Italy) Dr Olivier DANGLES (Professeur, Université d'Avignon, France) MEMBRES DU JURY : Dr Mario Augusto PAGNOTTA (Professeur, Università degli Studi della Tuscia, Italy) Dr Gérard VILAREM (Directeur adjoint du LCA, Institut National Polytechnique de Toulouse) Dr Giovanni MAUROMICALE (Professeur, Università degli Studi di Catania, Italy) Dr Paola CRINO’ (ENEA Casaccia, Italy) Dr Marion ALIGNAN (Institut National Polytechnique de Toulouse, France) UNIVERSITÀ DEGLI STUDI DELLA TUSCIA PHD in HORTICULTURE– XXIV cycle Thesis CHARACTERIZATION AND SELECTION OF GLOBE ARTICHOKE AND CARDOON GERMPLASM FOR BIOMASS, FOOD AND BIOCOMPOUND PRODUCTION PhD student Anna Ciancolini Research units: Unità Tecnica Sviluppo Sostenibile ed Innovazione del Sistema Agro-Industriale (ENEA) Dipartimento di Scienze e Tecnologie per l'Agricoltura, le Foreste, la Natura e l'Energia (UNITUS) Laboratoire de Chimie Agro-industrielle (UMR 1010 INRA/INP-ENSIACET) Directors of the PhD thesis: Prof. Mario Augusto PAGNOTTA and Dr. Gérard VILAREM Tutors of the PhD thesis: Dr. Paola CRINO‘ and Dr. Marion ALIGNAN Coordinator of the PhD course: Prof. Alberto GRAIFENBERG Academic Year 2011/2012 A mio marito, gioia infinita dei miei giorni. Ai miei genitori, punto fermo del mio essere. Alla vita che porto dentro con tutto l’amore che posso. TABLE OF CONTENTS INTRODUCTION 2 1.1 CYNARA SPP. ORIGIN AND DOMESTICATION 4 1.2 TAXONOMY AND NOMENCLATURE 5 1.3 BOTANICAL DESCRIPTION 6 1.4 BIOLOGY AND ECOLOGY 9 1.5 GEOGRAPHIC DISTRIBUTION AND PRODUCTION 10 1.6 GENETIC RESOURCES 13 1.7 PROPAGATION 15 1.7.1 AGAMIC 15 1.7.2 GAMIC 17 1.8 AGRONOMIC ASPECTS 18 1.8.1 PLANTING 18 1.8.2 FERTILIZATION 18 1.8.3 IRRIGATION 19 1.8.4 WEED CONTROL 19 1.8.5 PARASITES AND PATHOGENS 20 1.8.6 AGRICULTURAL MANAGEMENT OF THE PLANTS 21 1.8.6.1 STALK REMOVAL 21 1.8.6.2 OFFSHOOT REMOVAL 21 1.8.6.3 BLEACHING 22 1.9 EDIBLE PRODUCT HARVESTING AND COMMERCIALIZATION 22 1.10 USES 23 1.10.1 TRADITIONAL USES 23 1.10.2 INDUSTRIAL USES 24 1.10.3 PHARMACEUTICAL USES AND MAJOR PHENOLIC COMPOUNDS 27 1.11 FOCUS ON PHENOLIC EXTRACTION TECHNIQUES 34 EXPERIMENTAL PART 38 CHAPTER 1. MORPHOLOGICAL ANALYSIS AND GERMPLASM SELECTION FOR EDIBLE USE GLOBE ARTICHOKE 42 1.1 INTRODUCTION 42 1.2 ITALIAN SPRING LANDRACES 44 1.2.1 MATERIALS AND METHODS 44 1.2.1.1 GENETIC RESOURCES AND OPEN FIELD EXPERIMENTS 44 1.2.1.2 MORPHOLOGICAL CHARACTERIZATION USING UPOV DESCRIPTORS 47 1.2.1.3 STATISTICAL ANALYSES 48 1.2.2 RESULTS 50 1.2.3 DISCUSSION 57 1.3 ITALIAN ‗ROMANESCO‘ CLONES 58 1.3.1 MATERIALS AND METHODS 58 1.3.1.1 GENETIC RESOURCES AND OPEN FIELD EXPERIMENTS 58 1.3.1.2 MORPHOLOGICAL CHARACTERIZATION USING UPOV DESCRIPTORS 59 1.3.1.3 STATISTYCAL ANALYSES 59 1.3.2 RESULTS 59 1.3.3 DISCUSSION 68 CARDOON 69 1.4 INTRODUCTION 69 1.5 ITALIAN ACCESSIONS 70 1.5.1 MATERIALS AND METHODS 70 1.5.1.1 GENETIC RESOURCES AND OPEN FIELD EXPERIMENTS 70 1.5.1.2 MORPHOLOGICAL CHARACTERIZATION 72 1.5.1.3 STATISTICAL ANALYSES 72 1.5.2 RESULTS 72 1.5.3 DISCUSSION 78 CHAPTER 2. DEVELOPMENT OF POLYPHENOL EXTRACTION TECHNIQUE FROM CYNARA SPP. BIOMASS 2.1 INTRODUCTION 84 2.2 MATERIALS AND METHODS 85 2.2.1 PLANT MATERIAL, SOLVENTS AND REAGENTS 85 2.2.2 EXTRACTION TECHNIQUES 85 2.2.2.1 SOXHLET EXTRACTION 85 2.2.2.2 MACERATION 86 2.2.2.3 MICROWAVE ASSISTED EXTRACTION 86 2.2.2.4 ACCELERATED SOLVENT EXTRACTION 86 2.2.3 DETERMINATION OF MOISTURE CONTENT 87 2.2.4 COLORIMETRIC ANALYSIS OF TOTAL PHENOLICS 87 2.2.5 HPLC ANALYSIS 87 2.2.6 STATISTICAL ANALYSES 88 2.2.7 EXPERIMENTAL DESIGN FOR MICROWAVE ASSISTED EXTRACTION AND ACCELERATED SOLVENT EXTRACTION 88 2.3 RESULTS 89 2.3.1 OPTIMIZATION OF MICROWAVE ASSISTED EXTRACTION (MAE) AND ACCELERATED SOLVENT EXTRACTION (ASE) FOR PHENOLIC COMPOUNDS 89 2.3.2 COMPARISON BETWEEN NOVEL AND TRADITIONAL EXTRACTION METHODS 98 2.3.2.1 COLORIMETRIC ANALYSIS OF TOTAL PHENOLICS 98 2.3.2.2 HPLC ANALYSIS 100 2.4 DISCUSSION 103 CHAPTER 3. BIOMASS AND NUTRACEUTICAL KINETICS EVALUATION IN GLOBE ARTICHOKE AND CARDOON PLANTS GROWN IN OPEN FIELD 3.1 INTRODUCTION 108 3.2 MATERIALS AND METHODS 109 3.2.1 FIELD EXPERIMENTS 109 3.2.2 AGRO-MORPHOLOGICAL CHARACTERIZATION 109 3.2.3 BIOCHEMICAL CHARACTERIZATION 110 3.2.4 DETERMINATION OF MOISTURE CONTENT 110 3.2.5 SOLVENTS AND REAGENTS 110 3.2.6 EXTRACTION PROCEDURE 111 3.2.7 HPLC ANALYSIS 111 3.2.8 COLORIMETRIC ANALYSIS OF TOTAL PHENOLICS 112 3.2.9 STATISTICAL ANALYSES 113 3.3 RESULTS 113 3.3.1 AGRO-MORPHOLOGICAL CHARACTERIZATION AND PLANT GROWTH EVALUATION 113 3.3.2 BIOCHEMICAL CHARACTERIZATION AND PHENOLIC COMPOUND KINETICS EVALUATION 121 3.4 DISCUSSION 130 CHAPTER 4. MORPHOLOGICAL ANALYSIS AND GERMPLASM SELECTION FOR BIOMASS AND BIOCOMPOUND PRODUCTION 4.1 INTRODUCTION 136 4.2 GLOBE ARTICHOKE: EVALUATION OF BIOMASS AND BIOCOMPOUND PRODUCTION 137 4.2.1 MATERIALS AND METHODS 137 4.2.1.1 EXPERIMENTAL FIELD AND PLANT MATERIAL 137 4.2.1.2 MORPHOLOGICAL CHARACTERIZATION 138 4.2.1.3 PHENOLIC COMPOUND EXTRACTION AND ANALYSIS 139 4.2.1.3.1 SAMPLE PREPARATION 139 4.2.1.3.2 EXTRACTION PROCEDURE AND ANALYSIS 140 4.2.1.4 STATISTICAL ANALYSES 140 4.2.2 RESULTS 140 4.2.2.1 BIOMETRIC CHARACTERISTICS 140 4.2.2.1.1 BIOMASS PRODUCTION 140 4.2.2.1.2 EDIBLE PRODUCTION 145 4.2.2.2 BIOCHEMICAL CHARACTERISTICS 147 4.2.3 DISCUSSION 150 4.3 CARDOON: ITALIAN AUTHOCTONOUS ACCESSIONS 154 4.3.1 MATERIALS AND METHODS 154 4.3.1.1 EXPERIMENTAL FIELD AND PLANT MATERIAL 154 4.3.1.2 MORPHOLOGICAL CHARACTERIZATION 154 4.3.1.3 PHENOLIC COMPOUND EXTRACTION AND ANALYSIS 155 4.3.1.3.1 SAMPLE PREPARATION 155 4.3.1.3.2 EXTRACTION PROCEDURE AND ANALYSIS 156 4.3.1.4 STATISTICAL ANALYSES 156 4.3.2 RESULTS 156 4.3.2.1 BIOMETRIC CHARACTERISTICS 156 4.3.2.1.1 BIOMASS PRODUCTION 156 4.3.2.2 BIOCHEMICAL CHARACTERISTICS 161 4.3.3 DISCUSSION 164 CHAPTER 5. BIOMASS AND BIOCOMPOUND PRODUCTION IN FLOATING SYSTEM UNDER SALT STRESS: MORPHOLOGICAL, BIOCHEMICAL AND MOLECULAR RESPONSES 5.1 INTRODUCTION 170 5.2 MATERIALS AND METHODS 172 5.2.1 GREENHOUSE EXPERIMENTS 172 5.2.2 AGRO-MORPHOLOGICAL CHARACTERIZATION 177 5.2.3 PHENOLIC COMPOUND EXTRACTION AND ANALYSIS 178 5.2.3.1 SAMPLE PREPARATION 178 5.2.3.2 EXTRACTION PROCEDURE 178 5.2.3.3 SOLVENTS AND REAGENTS 178 5.2.3.4 HPLC ANALYSIS 178 5.2.4 INULIN EXTRACTION AND ANALYSIS 178 5.2.4.1 SAMPLE PREPARATION 178 5.2.4.2 EXTRACTION PROCEDURE 178 5.2.4.3 SOLVENTS AND REAGENTS 179 5.2.4.4 HPAEC-PAD ANALYSIS 179 5.2.5 RNA EXTRACTION AND MOLECULAR ANALYSIS 179 5.2.5.1 SAMPLE PREPARATION 179 5.2.5.2 RNA EXTRACTION AND MOLECULAR ANALYSIS 180 5.2.6 STATISTICAL ANALYSES 180 5.3 RESULTS 181 5.3.1 MORPHOLOGICAL CHARACTERIZATION OF GLOBE ARTICHOKE AND CARDOON PLANTS GROWN IN FLOATING SYSTEM AND EFFECT OF SALT STRESS ON PLANT BIOMETRIC TRAITS 181 5.3.2 BIOCHEMICAL CHARACTERIZATION OF GLOBE ARTICHOKE AND CARDOON PLANTS GROWN IN FLOATING SYSTEM AND EVALUATION OF SALT STRESS ON BIOCOMPOUND CONTENT 187 5.3.2.1 PHENOLIC COMPOUNDS 187 5.3.2.2 INULIN 191 5.3.3 MOLECULAR RESPONSE TO STRESS TREATMENTS 191 5.4 DISCUSSION 192 GENERAL CONCLUSION 200 REFERENCES 204 ANNEX 1 222 ACKNOWLEDGEMENTS 224 SUMMARY Globe artichoke and cardoon, belonging to the Asteraceae (Compositae) family, are herbaceous perennial plants native to the Mediterranean area, which are traditionally grown as vegetables for the heads and the fleshy petiole leaves, respectively. Italy is the richest reserve of globe artichoke (Cynara cardunculus var. scolymus L.) autochthonous germplasm, which is vegetatively propagated and well adapted to the different pedoclimatic conditions of the Country. On the basis of head harvest time, these traditionally grown genetic resources are classified as early or late distinct clonal varietal groups. Other distinct varietal groups are also identified, according to the morphological traits of the head, into four main typologies such as Violetto di Sicilia, Spinoso Sardo, Catanese and Romanesco. In Central Italian environments, the Romanesco type, characterized by a spherical or sub- spherical and non-spiny green-violet heads, is widespread. In the last years, the development of in vitro technologies allowed the propagation of Romanesco globe artichoke type and its rapid expansion. As a result, the Romanesco clone C3 has been in vitro micropropagated and widely distributed to the farmers. This clone has replaced many Romanesco landraces traditionally grown in the Latium Region and has led either to a significant erosion of local genetic resources or a loss of diversity. Moreover, introduction of new seed-propagated F1 hybrids such as Madrigal, Concerto, Opal, Tema, and Romolo, well suited to market demands, represents a further factor increasing the risk of genetic erosion for autochthonous germplasm.