USE OF IN VITRO TISSUE CULTURE IN PROPAGATION AND GENETIC IMPROVEMENT OF FRUIT TREES

La coltura in vitro, applicata alla propagazione e miglioramento genetico della biodiversità vegetale, può rappresentare uno strumento efficace per affrontare i problemi attuali come i cambiamenti climatici, le nuove esigenze dei consumatori e indirettamente lo sviluppo delle aree rurali. Inoltre, può assumere un ruolo strategico nel miglioramento genetico e nella propagazione delle cultivar al fine di ottenere genotipi resistenti, con frutti migliori dal punto di vista organolettico e piante capaci di adattarsi ai cambiamenti climatici. Il miglioramento genetico attraverso i metodi convenzionale è limitato da molti fattori infatti, gli alberi da frutto sono caratterizzati da un lungo periodo di giovanilità, elevata eterozigosi e molto spesso incompatibilità sessuale. La tecniche in vitro della coltura di antere e/o microspore isolate, per ottenere piante aploidi o doppi aploidi, riescono a superare alcuni limiti del miglioramento genetico tradizionale e fornire in un solo passaggio linee completamente omozigoti interessanti per studi di genomica, mutazioni, mappatura e trasformazione genetica. La tecnologia in vitro oltre ad essere applicata al miglioramento genetico, risulta un metodo complementare ai metodi convenzionali di propagazione agamica, in quanto, il materiale vegetale risulta protetto da eventuali attacchi di patogeni, il processo è indipendente dalle condizioni stagionali, permette uniformità genetica ed elevati tassi di moltiplicazione in uno spazio ridotto. La coltura in vitro è resa possibile dal fenomeno della totipotenza; cioè la capacità che hanno le cellule, organi o tessuti di de-differenziarsi e acquisire capacità meristematiche dando origine ad organi, tessuti o a un individuo intero, anche diverso da quello di partenza. Questa ricerca ha avuto come obiettivo l’induzione dell’embrio enesi ametica in vari genotipi di agrumi, tramite coltura in vitro di antere. Inoltre, si è studiata la messa a punto di un efficiente protocollo di conservazione e propagazione, attraverso la tecnologia del seme sintetico, rispettivamente in Fico, Luppolo e Cappero. Gli esperimenti sono stati condotti nel 2017, 2018 e 2019 presso l’Universit de li Studi di Palermo (UNIPA) e l’Instituto Valenciano de Investi aciones A rarias (IVIA). Per quanto riguarda la coltura di antere di agrumi è stata ottenuta embriogenesi somatica nella varietà Moro (2x), portando alla rigenerazione di embrioni che dopo le analisi della ploidia e molecolari sono risultati tetraploidi eterozigoti; nei genotipi Marisol (4x), Clemenules (4x), Moro Los Valles (4x), Sanguinelli (4x) e Sanguinelli (2), è stata osservata divisione simmetrica del nucleo, strutture multinucleate e la formazione di embrioni che in se uito all’analisi citofluorimetrica sono risultati per la maggior parte diploidi. L’analisi molecolare, effettuata con i marcatori SSR invece, ha rilevato risultati singolari, infatti, nei genotipi Marisol, Clemenules e Sanguinelli (2x e 4x), anche se sono state osservate microspore multinucleate e la maggior parte dei rigenerati erano diploidi, dall’analisi sono risultati tutti eterozi oti, mentre in Moro Los Valles, imarcatori hanno rilevato che dei diploidi rigenerati tre erano omozigoti, due omozigoti mutati (variabilità gametoclonale) e due eterozigoti mutati. La tecnologia del seme sintetico è stata applicata alla varietà Houmairi di Ficus carica L., in particolare, è stato valutato l’effetto di due Regolatori di crescita (PGR): 6-benzilaminopurine (BAP) e meta-Topolina (mT), aggiunti all’endosperma artificiale, e della conservazione a 4° C per 30 iorni delle microtalee incapsulate. La più alta percentuale di semi vitali e che hanno portato ad una maggiore ripresa vegetativa si è avuta nella tesi che non ha previsto conservazione a freddo prima della semina e quando l’endosperma artificiale veniva addizionato con il BAP. In maniera diversa, la percentuale maggiore di conversione si è avuta, sia per i semi conservati che non quando l’endosperma veniva addizionato con mT. La propagazione in vitro, è stata applicata anche, alla varietà Cascade di Humulus lupulus L. con l’obiettivo di realizzare un protocollo efficiente di micropropagazione. Partendo dal prelievo in campo del materiale vegetale, si è proceduto alla sterilizzazione, stabilizzazione della coltura asettica nonché alla moltiplicazione, radicazione e acclimatazione alle condizioni ex vitro. Per la moltiplicazione sono stati testati 3 PGR: BAP, mT e Thidiazuron (TDZ) in diverse combinazioni, mentre per la radicazioni 2 PGR auxinici: Acido indol-3-acetico (IAA) e Acido indol-3-butirrico (IBA). L’interazione TDZ/BAP ha mostrato il più alto tasso di germogliamento anche se con un maggiore sviluppo di callo tale da considerare il mezzo con 2mg Lˉ¹ di TDZ il migliore nonostante dia un germogliamento inferiore così come per la presenza di callo. Per quanto riguarda la maggiore percentuale di radicazione si è avuta nel mezzo contenente 2 mg Lˉ¹ di IBA anche se differiva per pochi punti percentuali dal mezzo che conteneva 1 mg Lˉ¹ di IAA. Infine, è stata valutata l’influenza del rivestimento di alginato di calcio e di tre diversi regolatori di crescita (PGR): 6-benzilaminopurina (BAP), meta-topolina (mT) e zeatina (ZEA), sulla vitalità, la ricrescita e la conversione dei propaguli. di due genotipi siciliani di Capparis spinosa (L.). Le microtalee di cappero sono state sezionate e collocate in diversi endospermi artificiali a base di sali e vitamine Murashige e Skoog, arricchiti di mT o ZEA o BAP. I semi sintetici ottenuti sono stati seminati su un mezzo MS addizionato con 0,4 mg / Lˉ¹ di acido acetico naftalenico e 0,7 mg / Lˉ¹ di acido gibellerico. Dopo 60 giorni, sono stati rilevati i seguenti parametri: vitalità, ricrescita, numero e lunghezza dei germogli e delle radici, conversione. I risultati confermano che l'incapsulamento non ha influito negativamente sulla vitalità, che ha mostrato la percentuale più alta con BAP in Tracino e con ZEA in Scauri. Risultati simili sono stati ottenuti nella ricrescita, con differenze statisticamente significative tra i tre PGR testati: Tracino ha mostrato la migliore ricrescita in capsule arricchite con BAP, Scauri con ZEA. Inoltre, la conversione dei semi sintetici è stata fortemente influenzata dalla PGR ed era più elevata nell'endosperma artificiale addizionato con BAP in Tracino e ZEA in Scauri.In vitro culture, applied to the propagation and genetic improvement of plant biodiversity, can be an effective tool to face current problems such as climate change, and new consumer needs. Moreover, it can take on a strategic role in genetic improvement and propagation of cultivars in order to obtain genotypes resistant to biotic and abiotic stresses, with fruits improved from an organoleptic point of view and plants able to adapt to climate change. Genetic improvement through conventional methods is limited by many factors. Fruit trees are characterized by a long period of juvenitlity, high heterozygosity and, very often, by sexual incompatibility. The in vitro techniques of the cultivation of isolated anthers and/or microspores, to obtain haploid or double haploid plants, are able to overcome some limits of traditional genetic improvement and provide in a single step completely homozygous lines of interest for genomic studies, mutations, mapping and genetic transformation. In addition, in vitro technology is a complementary method or can even support conventional methods of agamic propagation, since the plant material is protected from possible attacks by pathogens, their production is independent of seasonal conditions, this tecquique allows genetic uniformity and high multiplication rates, in a small space. In vitro culture is made possible by the phenomenon of totipotency, that is, the ability of cells, organs or tissues to de-differentiate and acquire meristematic skills, giving rise to organs, tissues or to an entire individual, even different from the original one. This research had as its objective the induction of gametic embryogenesis in various citrus genotypes through in vitro culture of anthers, and the development of an efficient protocol for Fig, Hop and Caper through synthetic seed technology and micropropagation, respectively. The experiments were conducted in 2017, 2018 and 2019 at the University of Palermo (UNIPA) and the Valencian Institute of Agricultural Research (IVIA). As far as the cultivation of citrus anthers is concerned, somatic embryogenesis was obtained in the Moro variety (2x), leading to the regeneration of embryos which after ploidy and molecular analyzes were heterozygous tetraploids; and Marisol (4x), Clemenules (4x), Moro Los Valles (4x), Sanguinelli (4x) and Sanguinelli (2) genotypes, symmetric division of the nucleus, multinucleated structures and formation of embryos that following the analysis was observed cytofluorimetric results for the most part were diploid, molecular analysis, carried out with SSR markers instead, found singular results, in fact, in the Marisol, Clemenules and Sanguinelli genotypes (2x and 4x), even if multinucleated microspores and the most regenerated were diploid, from the analysis they were all heterozygous, while in Moro Los Valles, the markers found that three regenerated diploids were homozygous, two mutated homozygotes (gametoclonal variability) and two mutated heterozygotes.In this research, synthetic seed technology has been applied to the Houmairi variety of Ficus carica L., in particular, the effect of two “Plant rowth Re ulators” (P R): 6-benzylaminopurine (BAP) and meta-Topolina (mT), added to the artificial endosperm, and of the preservation at 4 ° C for 30 days of the encapsulated microcuttings. The highest percentage of viable seeds that led to a greater vegetative recovery occurred in the thesis that it did not foresee cold storage before sowing and when the artificial endosperm was added with BAP. In a different way, the highest percentage of conversion occurred, both for the stored seeds and not when the endosperm was added with mT. In vitro propagation was applied to the Cascade, variety of Humulus lupulus L. with the aim of achieving an efficient micropropagation protocol. Starting from the collection in the field of the plant material, sterilization, stabilization of the aseptic culture as well as the multiplication, rooting and acclimatization to ex vitro conditions were carried out. For the multiplication 3 PGR were tested: BAP, mT and Thidiazuron (TDZ) in different combinations, while for the rooting 2 PGR auxinic: indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). The TDZ / BAP interaction showed the highest germination rate even though with a greater callus development such as to consider the medium with 2 mg lˉ¹ of TDZ the best despite giving a lower budding as well as for the presence of callus. As regards the greater percentage of rooting, it occurred in the medium containing 2mg lˉ¹ of IBA without significant difference from the medium that contained 1 mg L of IAA. Finally, the influence of the calcium alginate coating the cuttings along with the three different growth regulators (PGR) were evaluated: 6-benzylaminopurine (BAP), meta-topolina (mT) and zeatin (ZEA), on vitality, regrowth and the conversion of the propagules of two Sicilian genotypes of Capparis spinosa (L.) (Tracino and Scauri, from Pantelleria Island). Caper microcutting have been sectioned and placed in different artificial endosperms based on Murashige and Skoog, enriched with mT or ZEA or BAP. The synthetic seeds obtained were seeded on an MS medium added with 0.4 mg / L of naphthalene acetic acid and 0.7 mg / L of gibelleric acid. After 60 days, the following parameters were detected: vitality, regrowth, number and length of shoots and roots, conversion. The results confirm that encapsulation did not negatively affect vitality, which showed the highest percentage with BAP in Tracino and with ZEA in Scauri. Similar results were obtained in regrowth, with statistically significant differences between the three PGRs tested: Tracino showed the best regrowth in capsules enriched with BAP, Scauri with ZEA. Furthermore, synseed conversion was strongly influenced by PGR and was higher in artificial endosperm added with BAP in Tracino and ZEA in Scauri genotype

Assessment of production and qualitative characteristics of different populations of Salvia sclarea L. found in Sicily (Italy)

Salvia sclarea L. is an important industrial crop, valued for its herbal-aromatic properties and high quality essential oils, that is used in food, pharmaceuticals and cosmetics. In this study, carried out from 2009 to 2010, the morphological and production characteristics and essential oil content and composition of three Sicilian populations were studied. In particular, the composition of essential oils extracted from primary and secondary inflorescences using steam distillation was assessed. Morphological, production and qualitative data from the three populations were subjected to analysis of variance and cluster analysis. Regarding the quality of the oils, only the most prevalent compounds were taken into consideration in this study. The three populations were linalyl acetate/ linalool chemotypes. Highly significant variations were found for the effective local population and inflorescence type in the composition of the essential oil principal components. In particular, the primary inflorescences were found to be accumulation sites favoured by monoterpenes, and secondary inflorescences were favoured by sesquiterpenes and sclareol. Populations “S. Stefano Quisquina” and “Alcara Li Fusi” performed best on a morphological and production level, whereas populations “Prizzi”and “Alcara Li Fusi” performed best in terms of quality. Population “S. Stefano Quisquina” produced high levels of sclareol. Biotype selection from within the populations should be based on both morphological, production and quality analyses

Effect of irrigation with treated wastewater on bermudagrass (Cynodon dactylon (L.) Pers.) production and soil characteristics and estimation of plant nutritional input

In recent years, climate change has greatly affected rainfall and air temperature levels leading to a reduction in water resources in Southern Europe. This fact has emphasized the need to focus on the use of non-conventional water resources for agricultural irrigation. The reuse of treated wastewater (TWW) can represent a sustainable solution, reducing the consumption of freshwater (FW) and the need for mineral fertilisers. The main aim of this study was to assess, in a three-year period, the effects of TWW irrigation compared to FW on the biomass production of bermudagrass [Cynodon dactylon (L.) Pers.] plants and soil characteristics and to estimate the nutritional input provided by TWW irrigation. TWW was obtained by a constructed wetland system (CWs) which was used to treat urban wastewater. The system had a total surface area of 100 m2. An experimental field of bermudagrass was set up close to the system in a Sicilian location (Italy), using a split-plot design for a two-factor experiment with three replications. Results highlighted a high organic pollutant removal [five days biochemical oxygen demand (BOD5): 61%, chemical oxygen demand (COD): 65%] and a good efficiency in nutrients [total nitrogen (TN): 50%, total phosphorus (TP): 42%] of the CWs. Plants irrigated with TWW showed higher dry aboveground dry-weight (1259.3 kg ha-1) than those irrigated with FW (942.2 kg ha-1), on average. TWW irrigation approximately allowed a saving of 50.0 kg TN ha-1 year-1, 24.0 kg TP ha-1 year-1 and 29.0 kg K ha-1 year-1 on average with respect to commonly used N-P-K fertilisation programme for bermudagrass in the Mediterranean region. Soil salinity increased significantly (p � 0.01) over the years and was detected to be higher in TWW-irrigated plots (+6.34%) in comparison with FW-irrigated plots. Our findings demonstrate that medium-term TWW irrigation increases the biomass production of bermudagrass turf and contributes to save significant amounts of nutrients, providing a series of agronomic and environmental benefits

Effect of Foliar Treatments with Calcium and Nitrogen on Oregano Yield

Oregano, Origanum vulgare L., is a perennial herbaceous plant belonging to the Lamiaceae family. Oregano shows variations in biomass yield and essential oil (EO) content due to the influence of abiotic and biotic factors. The aim of this study was to assess the effect of different foliar applications based on calcium (Ca) and nitrogen (N) on morphological and productive parameters in oregano. Tests were carried out in Sicily (Italy) in 2020–2021. In each year, eight foliar applications were applied. Only flowers and leaves were used for the extraction of the EO. For all parameters in the study, except for plant height and inflorescence length, the highest values were found in treated plants with respect to the control. In plants treated with calcium and nitrogen, an increase of between 0.6 and 1.6 t ha−1 was observed for fresh yield, and an increase of between 0.5 and 0.9 t ha−1 was observed for dry yield. The increase in biomass yield led to an increase in EO yield of between 4 and 12 kg ha−1. The results highlight that foliar treatments with Ca in combination with N enable growth in crop production in environments that show poor water availability

Treatment of combined dairy and domestic wastewater with constructed wetland system in Sicily (Italy). Pollutant removal efficiency and effect of vegetation

Dairy wastewater (DWW) contains large amounts of mineral and organic compounds, which can accumulate in soil and water causing serious environmental pollution. A constructed wetland (CW) is a sustainable technology for the treatment of DWW in small-medium sized farms. This paper reports a two-year study on the performance of a pilot-scale horizontal subsurface flow system forDWWtreatment in Sicily (Italy). The CW system covered a total surface area of 100 m2 and treated approximately 6 m3 per day of wastewater produced by a small dairy farm, subsequent to biological treatment. Removal efficiency (RE) of the system was calculated. The biomass production of two emergent macrophytes was determined and the effect of plant growth on organic pollutant RE was recorded. All DWW parameters showed significant differences between inlet and outlet. For BOD5 and COD, RE values were 76.00% and 62.00%, respectively. RE for total nitrogen (50.70%) was lower than that of organic compounds. RE levels of microbiological parameters were found to be higher than 80.00%. Giant reed produced greater biomass than umbrella sedge. A seasonal variation in RE of organic pollutants was recorded due to plant growth rate Our findings highlight the efficient use of a CW system for DWW treatment in dairy-cattle farms

Growth and development of succulent mixtures for extensive green roofs in a Mediterranean climate

Green roof systems, aimed at reducing anthropic impact on the environment, are considered environmental mitigation technologies and adopted by many countries across the world to strengthen urban ecosystem services. This study evaluates two mixtures of succulent: one of Crassulaceae and the other of Aizoaceae, used in the creation of a continuous and homogenous plant groundcover in Mediterranean environments. To assess the species mixtures, the parameters plant height, growth index, cover percentage and flowering were observed. Hydrological observations were also carried out to evaluate the rainfall retained by the test system in any given month. All data were subjected to analysis of variance. Growth indicators in the study showed trends characteristic of xeric plants, which tend to slow down in dry, summer climate conditions to the point of halting plant vertical growth and ground cover development completely. The Aizocaeae mix, during the initial stage, showed prevalent horizontal growth, confirmed by greater a greater growth index (13,21) and cover percentage (45%) compared to Sedum (Growth index: 3,61; Cover: 36%). In contrast, the Sedum mix recorded greater vertical growth at the beginning (Sedum mixture: 7.53 cm; Aizoaceae mixture: 6,11 cm). During the final stages of observations, however, greater vertical growth in the Aizoaceae (7,88 cm) became apparent together with a recovery in horizontal growth in the Sedum (79%), albeit not sufficient to outperform the Aizoaceae mixture (87%). Flowering in the two mixtures occurred between late spring and late summer. The Sedum mixture guaranteed a longer flowering period (130 days) compared to the Aizoaceae (93 days), with a gradual start followed by steady flower emission. Regarding rainfall water retention, a comparison of the mixtures in late winter/early spring revealed that the Sedum performed best (44.9 L m2 vs 37.4 L m2), whilst the Aizoaceae outperformed the Sedum in Autumn (63 L m2 vs 55 L m2), in conjunction with favorable growth rates in both species mixtures. Both mixtures demonstrated satisfying results and are considered suited to a Mediterranean environment. Furthermore, based on the different growth rates of the species in the two test mixtures, this study suggests that new combinations of Sedum and Aizoaceae together might prove more resilient in Mediterranean environments

Effects of irrigation, peat-alternative substrate and plant Habitus on the morphological and production characteristics of Sicilian rosemary (Rosmarinus officinalis L.) biotypes grown in pot

Irrigation and growing substrate are considered as essential cultivation practices in order to obtain good productive and qualitative performance of potted rosemary plants. In pot growing, the chemical, physical and biological characteristics of the substrate must be stable over time in order to allow regular plant growth. However, the effects of cultivation techniques on the characteristics of potted rosemary are little known. Peat is traditionally used as the organic growing medium; however, despite numerous advantages, its use has determined a degradation of peatlands in the northern hemisphere and an increase in greenhouse gases in the atmosphere. The aim of the present study was to assess the effects of irrigation and peat-alternative substrates on the morphological, aesthetic and production characteristics of potted Sicilian rosemary biotypes with different habitus types. Two years, two different irrigation levels, three peat-alternative substrates and three types of rosemary plant habitus were tested in a split-split-split-plot design for a four-factor experiment. The results highlight that irrigation and substrate determined significant differences for all tested parameters. Rosemary plants demonstrated the best performances when irrigation was more frequent; vice versa, the greatest percent content in essential oil was obtained when irrigation events were less frequent. The chemical–physical characteristics of peat-alternative substrates changed with decreases in the peat content and increases in the compost content. The erect habitus biotype showed the best adaptation capacity to the various treatments. Our results suggest that irrigation and peat-alternative substrates significantly affect the growth of rosemary plants and should, therefore, be taken into consideration in order to improve the cultivation of this species in pots for ornamental purposes

Four-year study on the bio-agronomic response of biotypes of Capparis spinosa L. on the island of Linosa (Italy)

The caper plant is widespread in Sicily (Italy) both wild in natural habitats and as specialized crops, showing considerable morphological variation. However, although contributing to a thriving market, innovation in caper cropping is low. The aim of the study was to evaluate agronomic and production behavior of some biotypes of Capparis spinosa L. subsp. rupestris, identified on the Island of Linosa (Italy) for growing purposes. Two years and seven biotypes of the species were tested in a randomized complete block design. The main morphological and production parameters were determined. Phenological stages were also observed. Analysis of variance showed high variability between the biotypes. Principal component analysis and cluster analysis highlighted a clear distinction between biotypes based on biometric and production characteristics. Production data collected in the two-year period 2007–2008 showed the greatest production levels in the third year following planting in 2005. In particular, biotype SCP1 had the highest average value (975.47 g) of flower bud consistency. Our results permitted the identification of biotypes of interest for the introduction into new caper fields. Further research is needed in order to characterize caper biotypes in terms of the chemical composition of the flower buds and fruits

Productivity of two Brassica oilseed crops in a Mediterranean environment and assessment of the qualitative characteristics of raw materials for bioenergy purposes

Rapeseed (Brassica napus var. oleifera D.C.) and Ethiopian mustard (Brassica carinata A. Braun) are promising industrial crops for cultivation in the Southern Mediterranean area due to profitable yields under semi-arid conditions. The exploitation of raw materials produced by these crops is very convenient for farmers to produce bioenergy directly on-farm and permits them to create a short agri-energy supply chain. The purpose of this study was to determine their yield performance under rainfed conditions and make an economic assessment of a combined heat and power plant (CHP) system operating on pure vegetable oil (PVO). Tests were conducted in Sicily (Italy) from 2012 to 2014. Seed and crop residue yields were detected. The analysis of seed, defatted seed meal and crop residue, and the chemical-physical aspects of PVO were carried out according to conventional protocols. A pilot CHP system was used for cogenerating electricity and heat. In general, rapeseed had the highest seed (2.27 t ha−1) and oil (1.11 t ha−1) yields. The average oil content ranged from 44.88 % (Ethiopian mustard) to 45.73 % dry matter (rapeseed). Ethiopian mustard performed better than rapeseed in terms of aboveground biomass yield (5.49 t ha−1), in both years. The two crops showed different fatty acid profiles of the oil mainly due to diverse content of erucic and oleic acids. The CHP system had an average consumption of 14.41 kg PVO h−1. These results confirm that the productivity of the species can be appreciable in the Southern Mediterranean area and indicate the use of raw materials of these crops as crucial to the development a sustainable short agri-energy supply chain

Constructed wetlands as nature-based solution for sustainable wastewater management in urban areas: a critical assessment by experimental studies and literature

In urban areas, constructed wetland systems (CWS) have been recognized as one of the most valid technologies for sustainable wastewater management. They represent green infrastructure and provide solutions to treatment of wastewaters that cities and peri-urban areas produce in large amount. In particular, CWS offer the advantages of wastewater reuse for parklands irrigation, water storage and peak-flow attenuation, control of stormwater runoff, nutrient cycling and uptake, heavy metals sequestration, removal of organic compounds. In greener cities, these systems have the potential to perform not just hydrological and wastewater treatment functions, but also urban design and ecological aspects as demonstrated by numerous applications around the world. CWS, in fact, can function as urban wildlife sites, educational or recreational facilities, landscape engineering and ecological areas. The aim of this paper was to highlight the potential functionality of CWS in urban areas by a critical assessment of two experimental studies and literature. In particular, the two studies were carried out in Sicily (Italy) in order to evaluate: i) the pollutant removal efficiency of two CWS to treat urban wastewater and first-flush stormwater; ii) the reuse of treated wastewater for irrigation of plants. The results confirm that CWS are efficient in the treatment and reuse of wastewater for irrigation purposes in modern cities. Furthermore, CWS showed very high organic pollutant removal and a good efficiency in reducing excess of nutrients and trace metals, highlighting their important role in the treatment of stormwater in urban areas