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Somatic embryogenesis, as a promising biotechnological tool for many conifer trees, has never been applied for the Abies nebrodensis species. Although all the encouraging results previously obtained by the EU LIFE (European LIFE program) funded projects in over ten years, the critically endangered Sicilian fir remains alarmingly close to extinction. In this study, we reported the first protocol of somatic embryogenesis obtained from mature zygotic embryos of the Abies nebrodensis. Seeds from Abies adult trees with specific identification numbers (IN) were collected and full seeds were identified by X-ray. Different experiments were carried out for callus initiation, from both zygotic immature and mature embryos, testing different culture media. The immature embryos did not give embryogenic tissue (ET). Embryogenic callus (EC) was successfully induced from mature embryos with variable frequencies (0–40%). Schenk and Hilderbrandt (SH) was the most suitable initiation medium where the obtained callus initiation rate reached up to 40% for IN7 (first experiment). 6-benzylaminopurine (BAP) showed to be essential to induce EC (second experiment). IN8 presented the highest callus initiation rate (40%) among all tested donor trees, whereas IN13 recorded the lowest rate with 4% (third experiment). ET maturation from each singular embryo of IN7, IN8, IN10 and IN21 was successfully achieved in SH medium containing 37,83 µM abscisic acid (ABA), 8% of polyethylene glycol (PEG-4000) and 4% maltose. The encapsulation technology was assessed on the obtained ET and its proliferation was observed after encapsulation.

This chapter deals with microspore embryogenesis in Citrus. Microspore embryogenesis allows to induce immature gametes (microspores) and to deviate them, in this case, the male one, from the normal gametophytic developmental route in the direction of the sporophytic one, yielding homozygous organisms (embryos and plants).

Seven Mediterranean Ficus carica genotypes, i.e. cv. Palazzo, Severoni precoce, Bianca, Pilusedda, Dottato bianco, Bifara and Zidi, were screened for the presence of seven fig-infecting viruses associated with fig mosaic disease (FMD) in order to explore their distribution in different plant organs (leaf, bud and syconium) that will be utilized as a plant source material in different sanitation techniques. RT-PCR assays conducted on reverse-transcribed TNA extracted from leaves, apical buds and syconia (1.5-2cm) of each genotype for the presence of Fig leaf mottle-associated virus 1 (FLMaV-1), Fig mild mottling-associated virus (FMMaV), Fig mosaic virus (FMV), Fig latent virus 1 (FLV-1), Fig Badnavirus1 (FBV-1) and Fig fleck-associated virus (FFkaV) showed that the infection rates in tested cultivars were 72.2% for FBV, followed by FFkaV (27.4%), FLMaV-1 (18,7%) and FMV (11.1%); whereas FMMaV and FLV-1 were absent. Virus distribution within the different explants showed that FBV1 was the most prevalent in all explants with infection rates ranging between 50% and 83%, whereas FFkaV was mostly concentrated in syconia (39%) and to a lesser extent in buds (32%) and leaves (10%). The highest infection with FLMaV-1 was found in syconia (50%), where, as buds and leaves, showed a mild level of infection (25%). FMV was present in 25% and 8% of tested buds and leaves, respectively, whereas it was absent in syconia

In this study, the encapsulation technology based on the calcium alginate coating was applied to some Ficus carica L. genotypes. Uninodal microcuttings (3-4 mm long), excised from in vitro proliferating shoots of three Italian cultivars (‘Bifera’, ‘Palazzo’ and ‘Catalanisca’), were employed. The influence of three different plant growth regulators (PGRs): 6-benzylaminopurine (BAP), meta-topolin (MT) and zeatine (ZEA), added to the artificial endosperm, were evaluated. Particularly, the viability, regrowth and conversion parameters of the synthetic seeds were registered after 60 days from the sowing on a hormone-free medium. Results showed that the cultivars ‘Catalanisca’ and ‘Palazzo’ showed the highest viability average rate (90%), followed by ‘Bifera’ (80%). Encapsulated microcuttings regrowth percentage was highest for ‘Palazzo’ (86.7%), followed by ‘Catalanisca’ (81.7%) and ‘Bifera’ (66.7%), while the conversion rate was comparable for the three cultivars (43.4, 40.0 and 48.3%, respectively). A strong interaction between cultivars and PGRs was observed. Moreover, regarding the effect of the PGRs, BAP and MT provided the best results about regrowth and conversion, even if the cultivar ‘Catalanisca’ presented 100% of regrowth with ZEA. To our knowledge, this is the first time, that encapsulation technology has been applied to Italian cultivars of Ficus carica.

Ficus carica L. is considered one of the oldest fruit trees in the Mediterranean basin and is widely grown and harvested for the consumption of its fruits dry and fresh. This species is affected by different virus diseases, especially by Fig mosaic disease (FMD), for which Fig leaf mottle-associated virus 1 (FLMaV-1), Fig leaf mottle-associated virus 2 (FLMaV-2), Fig mild mottling-associated virus (FMMaV), Fig mosaic virus (FMV), Fig latent virus 1 (FLV-1), Fig badnavirus 1 (FBV-1) and Fig fleck-associated virus (FFkaV) are associated. FMD is the most widespread disorder of this species, which represents a threat and a constraint for healthy fig production and germplasm exchange. Thus, the objective of the present doctoral research was the establishment of an efficient and rapid in vitro F. carica propagation, sanitation and conservation of free-FMD plant material for future large-scale commercialization. Initially, FMD-related viruses distribution was screened within the different fig plant organs (buds, leaves, syconia and seeds) of 14 Mediterranean genotypes (Palazzo, Severoni precoce, Bianca, Pilusedda, Dottato bianco, Bifera, Zidi, Baiyadi, Biancu, Brogiotto nero, Catalanisca, Houmairi, Triboiti and Turca 'Serilop') which were utilized afterward as in vitro plant source material. RT-PCR assays revealed that all the aforementioned viruses were present without any exception in seeds, whereas only 4 viruses (FBV, FFkaV, FLMaV-1 and FMV) were detected in buds, leaves and syconia with highly variable infection rates. Moreover, encapsulation technology proved to be a powerful multiplication technique to sustain standard fig tissue culture protocol for three cultivars (Catalanisca, Palazzo and Bifera) and it gave high, almost similar, viability, regrowth and conversion rates. Microcutting rooting in one-step was achieved and conversion rate was comparable for the three cultivars. Furthermore, in order to eliminate FMD associated viruses, with the exception of FBV-1 which resisted to all the sanitation attempts, Caulogenesis and Meristem Tip Culture Protected by the Synthetic Seeds technique (MTC-SS) gave the best sanitation rates. Finally, F. carica (cv. Houmairi) artificial seeds conservation, for final delivery, was achieved. A high viability and moderate regrowth rates were registered with a lesser conversion rate strictly related to the plant growth regulators (PGRs) used.Keywords: Fig, mosaic, RT-PCR, virus distribution, cytokinins, encapsulation, micropropagation, synthetic seed., Resumen La higuera (Ficus carica L.) es considerada como uno de de los árboles frutales más antiguos de la cuenca mediterránea y es ampliamente cultivado y cosechado para el consumo de sus frutos tanto secos como en fresco. Esta especie se ve afectada por diversas enfermedades virales, especialmente por la denominada "Fig mosaic disease" (FMD) asociada actualemnte a los virus: Fig leaf mottle-associated virus 1 (FLMaV-1), Fig leaf mottle-associated virus 2 (FLMaV-2), Fig mild mottling-associated virus (FMMaV), Fig mosaic virus (FMV), Fig latent virus 1 (FLV-1), Fig badnavirus 1 (FBV-1) y Fig fleck-associated virus (FFkaV). Esta enfermedad representa una amenaza y un obstáculo para la producción de higos y el intercambio de germoplasma. El principal objetivo del presente trabajo fue establecer un método de propagación de higuera in vitro para el saneamiento y la conservación de material vegetal libre de FMD para su posterior comercialización. Inicialmente, se estudió la distribución de los virus implicados en la enfermedad en diversos órganos de 14 genotipos de F. carica (Palazzo, Severoni precoce, Bianca, Pilusedda, Dottato bianco, Bifera, Zidi, Baiyadi, Biancu, Brogiotto nero, Catalanisca, Houmairi, Triboiti y Turca 'Serilop'), los cuales fueron utilizados posteriormente como fuente material vegetal in vitro. Los resultados obtenidos mediante RT-PCR revelaron que todos los virus mencionados estaban presentes sin excepción en las semillas, mientras que sólo cuatro de ellos (FBV, FFkaV, FLMaV-1 y FMV) fueron en brotes, hojas y siconios con tasas de infección variables. Además, la tecnología de encapsulación demostró ser una técnica de multiplicación eficaz para poder aplicar el protocolo estándar de cultivo de tejidos de higo para tres cultivares (Catalanisca, Palazzo y Bifera) dando altas tasas de viabilidad, rebrote y conversión. Se logró el enraizamiento de microcortes en un solo paso y el índice de conversión fue comparable para los tres cultivares. La callogénesis y el culñtivo de meristemos con la técnica de la semilla sintética (MTC-SS) fueron las técnicas que proporcionaron mayores tasas de desinfección para los virus estudiados a excepción de con FBV-1, entidad viral que no fue eliminada con ninguna de las técnicas ensayadas. Por último, se logró la conservación de las semillas artificiales de higuera (cv Houmairi), registrándose una alta viabilidad y tasas de rebrote moderadas con un menor grado de conversión estrictamente relacionado con hormonas utilizadas.Palabras clave: Higuera, mosaico, RT-PCR, la distribución de los virus, hormonas, encapsulación, micropropagación, y la semilla sintética., Resum La figuera (Ficus carica L.) és considerada un dels arbres fruiters més antics de la conca mediterrània i és àmpliament conreat i collit per al seu consum fresc i sec. Les malalties virals, especialment "Fig mosaic disease" (FMD), associada amb els viruses: Fig leaf mottle-associated virus 1 (FLMaV-1), Fig leaf mottle-associated virus 2 (FLMaV-2), Fig mild mottling-associated virus (FMMaV), Fig mosaic virus (FMV), Fig latent virus 1 (FLV-1), Fig badnavirus 1 (FBV-1) i Fig fleck-associated virus (FFkaV). Esta malaltia representa una amenaça per a la producció de figues i l'intercanvi de germoplasma. El principal objectiu d'aquest treball va ser estableixerun mètode de propagació de figuera in vitro per al sanejament i la conservació de material lliure de FMD per a su posterior commercialització. Inicialment, es va estudiar la distribució dels virus associats a FMD en diversos òrgans en 14 genotips de F. carica (Palazzo, Severoni Precoce, Bianca, Pilusedda, Dottato bianco, Bifera, Zidi, Baiyadi, Biancu, Brogiotto diners, Catalanisca, Houmairi, Triboiti i Turca 'Serilop'), els quals van ser utilitzats posteriorment com a font de material vegetal in vitro. Els resultats obtinguts del anàlisis realitzats per RT-PCR van revelar que tots els virus eren presents sense excepció en les llavors, mentre que només quatre virus (FBV, FFkaV, FLMaV-1 i FMV) van ser detectats en brots, fulles i siconis amb taxes d'infecció variables. A més, la tecnologia d'encapsulació va demostrar ser una tècnica de multiplicació eficaç per poder aplicar el protocol estàndard de cultiu de teixits de figa per a tres cultivars (Catalanisca, Palazzo i Bifera) donant taxesadequades de viabilitat, rebrot i conversió. Es va aconseguir l'arrelament de microtalls en un sol pas i l'índex de conversió va ser comparable per als tres cultivars. La calogènesi i el cultiu de meristems protegits per llavors sintètiques (MTC-SS)van ser les tècniques que proporcionarem millores tases de desinfecció per als virus estudiats amb l'excepció de FBV-1 que es va resistir a tots els mètodes de sanejament. Finalment, es va aconseguir la conservació de la llavors artificials de figuera (cv. Houmairi), registrant-ne una alta viabilitat i taxes de rebrot moderades amb un menor grau de conversió estrictament relacionat amb hormones utilitzades.Paraules clau: Figuera, mosaic, RT-PCR, la distribució dels virus, hormones, encapsulació, micropropagació, i la llavor sintètica., Yahyaoui, E. (2017). Use of standard and setup of non conventional techniques for the elimination of viruses associated with Fig Mosaic Disease (FMD) in fig germplasm (Ficus carica L.) [Tesis doctoral no publicada]. Universitat Politècnica de València. doi:10.4995/Thesis/10251/79876., TESIS

The complete genome of a Tomato black ring virus isolate (TBRV-Mirs) (RNA1, 7,366 nt and RNA2, 4,640 nt) and the RNA2 sequences (4,437; 4,445; and 4,442 nts) of three Grapevine chrome mosaic virus isolates (GCMV-H6, -H15, and -H27) were determined. All RNAs contained a single open reading frame encoding polyproteins of 254kDa (p1) and 149kDa (p2) for TBRV-Mirs RNA1 and RNA2, respectively, and 146kDa for GCMV RNA2. p1 of TBRV-Mirs showed the highest identity with TBRV-MJ (94%), Beet ringspot virus (BRSV, 82%), and Grapevine Anatolian ringspot virus (GARSV, 66%), while p2 showed the highest identity with TBRV isolates MJ (89%) and ED (85%), followed by BRSV (65%), GCMV (58%), and GARSV (57%). The amino acid identity of RNA2 sequences of four GCMV isolates (three from this study and one from GenBank) ranged from 91 to 98%, the homing protein being the most variable. The RDP3 program predicted putative intra-species recombination events for GCMV-H6 and recognized GCMV as a putative inter-species recombinant between GARSV and TBRV. In both cases, the recombination events were at the movement protein level.