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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. As regards Italian cultivated cardoon (Cynara cardunculus var. altilis DC) germplasm, there are few studies on its genetic characterization and identification and there is a lack of information about the genetic variability existing within and among accessions. For the wild cardoon (Cynara cardunculus var. sylvestris Lam.), no specialized crop is present and it represents mainly a weed in Italian environments. The great variability existing in Cynara spp. has not been described, the nomenclature of Italian germplasm is not always very clear since there are many cases of homonyms. In addition to this risk of genetic erosion, in the last years, Italian globe artichoke sector is facing a crisis due principally to the appearance on the market of foreign products and to the high labor cost required for crop cultivation and harvesting. In order to overcome this crisis several possible uses of Cynara spp. were considered such as i) seeds for oil, ii) roots for inulin, iii) biomass for energy, iv) fiber for pulp and paper and as potential reinforcement in polymer composites v) green forage for ruminant feeding, vi) proteins as natural rennet for traditional cheese making, and vii) entire plant for metalaccumulation. These new possible applications of the crop are linked principally to the European Union research support on new agricultural by-products (industrial raw materials) and led to an increasing interest also in aboveground globe artichoke biomass. Considering these preliminary remarks, a strategy for valorizing Italian germplasm using biomass and biocompound production has been carried out during the three years of PhD program. In particular, nine Italian spring landraces (Ascolano, Campagnano, Castellammare, Jesino, Montelupone A, Montelupone B, Bianco di Pertosa, Pisa and Tondo Rosso di Paestum), ten ‘Romanesco’ clones (S2, S3, S5, S11, S17, S18, S22,S23, Grato 1 and Campagnano), seven cultivated cardoon accessions (AFB, AFFG, AFGR, AFGI, AFM, AFM2, AFN) and AFS wild cardoon belonging to the joint germplasm collection of Tuscia University and ENEA have been considered in our PhD study. Open field trials were conducted at the Experimental Field Station of ARSIAL (Latium Regional Agency for the Development and the Innovation of Agriculture) in Cerveteri, Rome and in Tarquinia, Viterbo while greenhouse experiments were carried out at the Experimental Station of Tuscia University in Viterbo (Italy). The first objective of PhD work consisted in (i) characterizing agro-morphologically Italian germplasm using UPOV descriptors, (ii) assessing the genetic variability existing within and among landraces/clones and (iii) identifying and preserving genetic resources for the development of future plant breeding programs. As a result of this characterization, three genotypes have been selected and registered under the names of Michelangelo, Donatello and Raffaello. In order to analyze Italian Cynara spp. germplasm also from a biomass point of view, different traits explaining plant vigor and dry matter production have been considered. The aerial biomass yield resulted very high underlining the possibility of using this crop as raw industrial material. In particular, some genotypes, such as Ascolano, Campagnano, Pisa, Bianco di Pertosa for globe artichoke and AFFG and AFM for cardoon showed the highest biomass yield. A focal point of PhD program was to set up biocompound extraction methods and analysis techniques to optimize polyphenol recovery from biomass of Cynara spp at a laboratory scale. In particular, conventional (i.e. Soxhlet and maceration) and modern extraction techniques (such as Microwave-Assisted Extraction MAE, and Accelerated Solvent Extraction ASE) have been compared and, using a full two-level factorial experimental design, ASE was found as the best extraction technique which allows to reduce extraction time and solvent consumption, increase nutraceutical yield and improvement of extract quality. Moreover, the kinetics of biomass and biocompound production has been evaluated and the optimal physiological stage to collect plant material grown in open field has been identified. Biochemical characterization has been performed using the methods set up and collecting plant material in the optimal physiological stage identified in order to distinguish which genotypes were more suitable for bio-compound production purpose. As a result of morphological and biochemical characterization, genotypes were well distinguished from each other and were identified those which are the most suitable for food use, for biomass production and/or for dual-production (food and non-food). The last focal point of PhD program was the development of an alternative technique for biomass and biocompound production in greenhouse grown conditions. The adaptation of globe artichoke and cardoon genotypes to floating system has been evaluated and the biochemical and molecular responses of the plant to the salinity stress have been also investigated. Results obtained in the three PhD years, highlighted the possibility of using successfully some Cynara spp. genotypes for biomass and bio-compound production, in particular in open field condition. Also the real prospect of using some globe artichoke genotypes for food and non-food dual-production (biomass for biocompound extraction and heads for human food) has been underlined. Il carciofo e il cardo, appartenenti alla famiglia delle Asteraceae (Compositae), sono delle piante erbacee, perenni, originarie del bacino del Mediterraneo e tradizionalmente coltivate a scopo alimentare rispettivamente per i capolini e le foglie carnose. L’Italia rappresenta una delle più ricche riserve di germoplasma autoctono di carciofo (Cynara cardunculus var. scolymus L.), il quale è propagato vegetativamente e ben adattato alle differenti condizioni pedoclimatiche del nostro Paese. Sulla base dell’epoca di maturazione commerciale dei capolini, i genotipi di carciofo vengono classificati in tipologie precoci e autunnali. Un altro criterio di classificazione, basato sulle caratteristiche morfologiche dei capolini, suddivide i suddetti genotipi in quattro gruppi varietali: ‘Violetto di Sicilia’, dello ‘Spinoso Sardo’, del ‘Catanese’ e del ‘Romanesco’. La tipologia ‘Romanesco’, caratterizzata da capolini inermi di forma rotonda o ellittica e di colore verde-viola, è molto diffusa negli ambienti di coltivazione cinaricola dell’Italia centrale. Negli ultimi anni, la messa a punto della tecnica di propagazione in vitro per il carciofo, ha portato ad una rapida diffusione di cloni della tipologia ‘Romanesco’. In particolare, il clone C3 propagato in vitro ha conosciuto una grande diffusione nelle aree di coltivazione cinaricola, sostituendo molte varietà afferenti alla tipologia ‘Romanesco’ tradizionalmente coltivate nel Lazio e determinando rischi di erosione genetica per queste risorse genetiche. Inoltre, l’introduzione sul mercato di nuovi ibridi F1 come Madrigal, Concerto, Opal, Tema, and Romolo, meglio rispondenti alle richieste dei consumatori, rappresenta un ulteriore fattore di rischio per la perdita del germoplasma autoctono. Per quanto riguarda il cardo coltivato (Cynara cardunculus var. altilis DC), esistono pochi studi sulla caratterizzazione genetica e sull’identificazione del germoplasma autoctono e mancano dati riguardanti la variabilità genetica esistente tra ed entro accessioni diverse. Per il cardo selvatico (Cynara cardunculus var. sylvestris Lam.), non sono presenti coltivazioni specializzate ed esso rappresenta un’infestante negli ambienti di coltivazione cinaricola italiani. L’ampia variabilità esistente nel germoplasma di Cynara spp. non è stata descritta; la nomenclatura delle risorse genetiche non è spesso chiara e spesso si trovano casi di omonimia. Oltre a questo rischio di erosione genetica, negli ultimi anni assistiamo anche ad una forte crisi del settore cinaricolo dovuta alla comparsa sul mercato di prodotti stranieri e all’alto costo di produzione e di raccolta previsti dalla coltura. Negli ultimi decenni, per far fronte a questa crisi, sono stati considerati diversi possibili usi per la specie quali, ad esempio, i) la produzione di olio dai semi; ii) l’estrazione di inulina dalle radici; iii) la produzione di energia dalla biomassa; iv) l’estrazione della fibra per l’industria cartaria; v) la produzione di foraggio da destinare all’alimentazione animale; vi) la produzione di formaggi come caglio vegetale mediante impiego di estratti fiorali; vii) l’accumulo di metalli pesanti nella pianta. Lo studio di questi nuovi possibili utilizzi è stato reso possibile grazie ai finanziamenti della Comunità Europea che ha incentivato e supportato la ricerca nel settore delle agrorisorse e ha portato anche ad un forte interesse nell’uso della biomassa di carciofo. Nel corso dei tre anni di dottorato è stata portata avanti una strategia di ricerca volta alla valorizzazione del germoplasma italiano di carciofo e cardo basata sull’uso della biomassa per l’estrazione di biomolecole. In particolare, sono stati considerati nove genotipi autoctoni primaverili (Ascolano, Campagnano, Castellammare, Jesino, Montelupone A, Montelupone B, Bianco di Pertosa, Pisa and Tondo Rosso di Paestum), dieci cloni della tipologia ‘Romanesco’ (S2, S3, S5, S11, S17, S18, S22, S23, Grato 1 and Campagnano), sette genotipi di cardo coltivato (AFB, AFFG, AFGR, AFGI, AFM, AFM2, AFN) e un genotipo di cardo selvatico (AFS), tutti appartenenti alla collezione di germoplasma dell’Università della Tuscia e dell’ENEA. Le prove sperimentali di campo sono state svolte presso l’Azienda sperimentale ARSIAL (Agenzia Regionale per lo Sviluppo e l’Innovazione in Agricoltura) di Cerveteri (Roma) e di Tarquinia (Viterbo), mentre le prove di serra sono state condotte nella Azienda Sperimentale dell’Università della Tuscia, a Viterbo (Italia). Il primo obiettivo del dottorato ha riguardato i) la caratterizzazione agro-morfologica del germoplasma italiano per mezzo dei descrittori UPOV, ii) la valutazione della variabilità genetica esistente tra ed entro i genotipi, e iii) l’identificazione e la conservazione delle risorse genetiche in collezione per lo sviluppo futuro di programmi di miglioramento genetico. La caratterizzazione del germoplasma ha portato alla selezione di tre genotipi validi per la produzione edule che sono stati validati e proposti per l’iscrizione al Registro Nazionale delle Varietà con i nomi di Michelangelo, Donatello e Raffaello. Al fine di caratterizzare il germoplasma anche da un punto di vista della biomassa, sono stati anche considerati diversi caratteri morfologici atti a descrivere il vigore della pianta e a quantificare la produzione di sostanza secca. La produzione di biomassa è risultata davvero molto interessante nel nostro lavoro e si è sottolineato il grande potenziale di utilizzo della coltura nel settore industriale. Alcuni genotipi come Ascolano, Campagnano, Pisa e Bianco di Pertosa, per il carciofo, e AFFG e AFM, per il cardo, sono risultati davvero interessanti dal punto di vista della produzione di biomassa. Un altro importante obiettivo del dottorato è stato quello di mettere a punto un metodo per l’estrazione di polifenoli al fine di ottimizzare le rese. Sono state messe a confronto tecniche convenzionali (ad esempio metodo Soxhlet e macerazione) ed altre più innovative (ad esempio Microwave Assisted Extraction - MAE e Accelerated Solvent Extraction - ASE). La tecnica ASE ha fornito i migliori risultati, consentendo anche di ridurre i tempi d’estrazione e il consumo di solvente, fornendo al tempo stesso alte rese e elevata qualità degli estratti. E’ stato anche condotto lo studio della cinetica di accumulo della biomassa e delle biomolecole in campo al fine di individuare il momento migliore per la raccolta del materiale in campo ed ottimizzare le rese. Una volta messo a punto il metodo d’estrazione e individuata l’epoca ottimale per il prelievo delle biomasse in campo, si è andati ad effettuate le estrazioni al fine di caratterizzare biochimicamente i genotipi in prova e di selezionere quelli più rispondenti a tale attitudine. La caratterizzazione morfologica e biochimica hanno permesso di ben identificare i genotipi e di selezionare quelli più adatti per la produzione di capolini (‘food production’), di biomassa o per la duplice attitudine (food and non-food). Nel corso del dottorato si è anche valutato un sistema alternativo per la produzione di biomassa e di biomolecole in serra. E’ stata valutata l’adattabilità del carciofo e del cardo al sistema di coltivazione fuori suolo (floating system), soprattutto per evidenziare risposta biochimica e molecolare delle piante sottoposte allo stress salino in termini di aumento nella produzione di sostanze fenoliche. I risultati ottenuti nel corso del dottorato hanno messo in luce la reale possibilità di usare alcuni genotipi di carciofo e di cardo per la produzione edule, altri per quella di biomassa e di biomolecole, soprattutto in condizioni di pieno campo. Si è inoltre delineata una reale prospettiva per l’impiego di alcuni genotipi di carciofo in una duplice destinazione commerciale (biomassa per l’estrazione di biomolecole e capolini per la destinazione alimentare). Dottorato di ricerca in Ortoflorofrutticoltura

Globe artichoke gives an important contribution to the Mediterranean agricultural economy. However, in the Mediterranean countries, where this crop was first grown and its cultivation developed and increased over the centuries, production has recently remained stagnant since the end of last century. The principal problems related to the development of globe artichoke as a modern cultivation consist in the irregularity both of production and commercialization calendar as well as in the traditionally adopted vegetative propagation system, which requires expensive agronomical practices and does not guarantee well-being propagation material. No intensive breeding programs have been carried out. The only real novelty in this sense is represented by the development of few seed propagated hybrids. Male sterility was used to allow production of many experimental F1 hybrids eliminating the long and expensive operation of floral emasculation. Seed-planted cultivars have the following major advantages: 1) labor saving and a cheap operation of the mechanical seed sowing; 2) conversion of globe artichoke into annually grown crops and introduction into crop rotation; 3) respect to vegetatively propagated plants, more efficient use of both moisture and fertilizers due to the long vertical tap roots penetrating into the soil deeper than the adventitious roots produced by planted suckers; 4) protection from pathogens and pests by annual cultivation coupled with crop rotation; 5) more vigorous and healthier growth of the plants with a low input of pesticides, fungicides and fertilizers, so making globe artichoke as a valid rotation choice in an organic cultivation system. For the development of globe artichoke as a modern crop, it is important to produce new stable seed propagated hybrids, but also to set up the techniques necessary to rationalize the cultivation system. In the process of F1 hybrid production, it needs to consider some aspects such as (i) the correct management of the cultural cycle, (ii) the adaptability of the new F1 hybrids to the different cultivation environments as well as to the different production and commercialization calendars, (iii) the economical production of F1 hybrid seed because the adequate pollination of male-sterile plants still remains difficult to achieve. IV During the three years of PhD program, a strategy of development of F1 hybrids was carried out in order to reach some objectives. The first one was to develop stable male and female parental lines, selecting the germplasm already available and trying to improve their stability. Some male sterile clones were selected and individuated as good female parents; some male fertile genotypes were selected as stable. Moreover, in order to deep the knowledge on the morphological and functional differences between male sterile and male fertile globe artichoke flowers, a study of the floral biology was carried out. The floral male and female structures were staged and studied in order to highlight the differences. The male sterile flowers showed either a normal development of the male reproductive structures or a normal meiosis in the cytological analyses of pollen formation. The block of pollen viability was post-meiotic and the low viability of the male sterile pollen was connected with a less developed external exine structure. Female organs of the male sterile flowers were more elongated than those of the male fertile flowers at the same stages. Different cross-combinations were developed either in Italy (2006 and 2007) or in USA 82008) in order to compare the different hybrids and to individuate the most homogeneous ones. A focal point of the project was to find an evaluation system capable of distinguishing which hybrids were more homogeneous than others. For this reason, an evaluation of all hybrids based on the morphological traits was carried out using the Protocol for Globe Artichoke European Union CPVO (2004) and some other traits chosen by Lopez Anido et al. (1998) both in Italy and in USA. In order to analyze the hybrids also from a molecular point of view, five F1 offsprings produced during the PhD program along with their parents and a commercial hybrid (Opal F1) used as a control were analyzed by ISSR markers. Molecular work was realized in the laboratory of the Dpto. Ciencia y Tecnologia Agraria of Universidad Politecnica de Cartagena (UPCT), Spain. Hybrids were all well differentiated by both the molecular and morphological analyses. The results of the molecular analysis were compared with those of the morphological one. The clustering of clones based on the morphological characters resulted consistent with that derived from the molecular analysis. The last focal point of the research was the development of an effective pollination technique for the production of F1 hybrids by comparing different pollination systems studied. Their potentialities in seed productivity of our male sterile lines were evaluated. In 2006, the field experiment were carried out at the Regional Agency for the Development and Innovation of Agriculture (ARSIAL), in Tarquinia (Viterbo, Italy) and V both manual pollination by brush and pollination by bumblebees (Bombus terresris) were compared with an open pollination control. In 2008, open field activities were carried out in the experimental fields of Big Heart Seed Company (BHSC), in Brawley (California, USA) and the effectiveness of honey bees (Apis mellifera) as pollinators was evaluated in order to verify if, changing the percentage of both male sterile and male fertile plants, there were some differences in the seed production. The results of the first year experiment should be repeated because highlighted interesting potentialities in the use of the bumblebees as valid alternative to the honey bees. Unfortunately, some environment problems reduced seed production and it was not possible to get some definitive results. The results obtained in the third year confirmed that the honey bees are less attracted by the male sterile flowers. Dottorato di ricerca in Ortoflorofrutticoltura

Grafting represents an effective tool for controlling the race 1,2 of Fusarium oxysporum f. sp. melonis (FOM) and Didymella bryoniae in melon (Cucumis melo L.). Although not considered a soilborne pathogen, D. bryoniae survives on plant remains in the soil. The lack of effective resistant commercial hybrids and the gradual reduced use of soil fumigation with methyl bromide increase the risk of damages by both these pathogens. We determined the effectiveness of eight commercial rootstocks, ‘RS 841’, ‘P 360’, ‘ES 99-13’, ‘Elsi’ (Cucurbita maxima Duchesne × Cucurbita moschata Duchesne), and ‘Belimo’, ‘Energia’, ‘Griffin’, ‘ES liscio’ (Cucumis melo genotypes), for their resistance to FOM and D. bryoniae. During 2003 and 2004 growing seasons, the inodorus F1 hybrid Incas was grafted onto each of these commercial rootstocks and then evaluated, under greenhouse conditions, in terms of productivity and fruit quality. Cucurbita rootstocks (‘RS 841’, ‘P 360’, ‘ES 99-13’, ‘Elsi’) were highly resistant both to the race 1,2 of FOM (100% survival) and to D. bryoniae (almost absent crown lesions and low leaf disease index); this reaction clearly differed from that of both the C. melo rootstocks (‘Belimo’, ‘Energia’, ‘Griffin’, ‘ES liscio’) and the control Incas. In both years, the highest yield was recorded in the graft combination Incas/‘RS 841’ with 5.6 and 8.1 kg·m−2 during 2003 and 2004, respectively. The Cucurbita rootstock ‘RS 841’ produced yields higher than C. melo rootstocks (‘Belimo’, ‘Energia’, ‘Griffin’, ‘ES liscio’) and the control Incas. Fruit dry matter, titratable acidity, total soluble solid contents, fruit firmness, and Hunter color [L* (brightness), a* (redness), and b* (yellowness) parameters] of grafted melons were similar to those of the plants grown on their own roots.