Common bean (Phaseolus vulgaris L.) is the most important grain legume for human consumption providing up to 15% of total daily calories and 36% of total daily protein in parts of Africa and the Americas. As a legume, it also has a role in sustainable agriculture owing to its ability to fix atmospheric nitrogen. Wild common bean is organized in two geographically isolated and genetically differentiated wild gene pools (Mesoamerican and Andean) that diverged from a common ancestral wild population more than 100,000 years ago. From these wild gene pools, common bean was independently domesticated in Mexico and in South America nearly 8,000 years ago, and these domestication events were followed by local adaptations resulting in landraces with distinct characteristics (Schmutz et al. 2014). Domestication led to morphological changes in seed and leaf sizes, in the growth habit and photoperiod responses, variation in seed coat color and pattern that distinguish culturally adapted classes of beans. This unique example of parallel domestication is the subject of the PARDOM project that, starting from the Phaseolus replicated experiment, aims at understanding common bean genome evolution and adaptation. In the framework of the PARDOM project, we are developing TILLING-by-sequencing and genome editing technological platforms for the functional validation of candidate domestication genes in common bean. For the development of the TILLING-by-seq platform, DNA from seeds of a P. vulgaris TILLING population developed in the Mesoamerican genotype BAT93 (Porch et al. 2009; Cominelli et al. 2018) was extracted. A three-dimensional pooling system of 54 pools, each of 96 samples on average, at resolution of a population of 1728 individuals was used for NGS targeted sequencing based on custom capture probes. For the genotyping, a total of 719 genes of interest were chosen, based on the presence of one or more signals of domestication, differential expression between the Andean genotype and Mesoamerican genotype, known involvement in the phenomenon of shattering, seed development and in the cytokinin hormonal pathway. Among these genes, 27 had a complete CDS sequence coverage, whereas for the others the first 1-3 exons were covered, for a total of approximately 491Mb. The validation of candidate genes for domestication is currently in progress also via forward genetics, following the identification of target regions in coding sequences for genome editing based on CRISPR/Cas9 technology. Fifteen target candidate domestication genes have been selected, based on the presence of one or more signals of domestication. Current editing approach is directed toward MYB26, encoding a transcription factor involved in pod shattering phenotype. Given the challenges posed by common bean transformation (biolistic transgenesis), the genome editing approach is being simultaneously carried out also on soybean ( Glycine max) homologous genes.

Common bean (Phaseolus vulgaris L.) is the most important grain legume for human consumption providing up to 15% of total daily calories and 36% of total daily protein in parts of Africa and the Americas. As a legume, it also has a role in sustainable agriculture owing to its ability to fix atmospheric nitrogen. Wild common bean is organized in two geographically isolated and genetically differentiated wild gene pools (Mesoamerican and Andean) that diverged from a common ancestral wild population more than 100,000 years ago. From these wild gene pools, common bean was independently domesticated in Mexico and in South America nearly 8,000 years ago, and these domestication events were followed by local adaptations resulting in landraces with distinct characteristics (Schmutz et al. 2014). Domestication led to morphological changes in seed and leaf sizes, in the growth habit and photoperiod responses, variation in seed coat color and pattern that distinguish culturally adapted classes of beans. This unique example of parallel domestication is the subject of the PARDOM project that, starting from the Phaseolus replicated experiment, aims at understanding common bean genome evolution and adaptation. In the framework of the PARDOM project, we are developing TILLING-by-sequencing and genome editing technological platforms for the functional validation of candidate domestication genes in common bean. For the development of the TILLING-by-seq platform, DNA from seeds of a P. vulgaris TILLING population developed in the Mesoamerican genotype BAT93 (Porch et al. 2009; Cominelli et al. 2018) was extracted. A three-dimensional pooling system of 54 pools, each of 96 samples on average, at resolution of a population of 1728 individuals was used for NGS targeted sequencing based on custom capture probes. For the genotyping, a total of 719 genes of interest were chosen, based on the presence of one or more signals of domestication, differential expression between the Andean genotype and Mesoamerican genotype, known involvement in the phenomenon of shattering, seed development and in the cytokinin hormonal pathway. Among these genes, 27 had a complete CDS sequence coverage, whereas for the others the first 1-3 exons were covered, for a total of approximately 491Mb. The validation of candidate genes for domestication is currently in progress also via forward genetics, following the identification of target regions in coding sequences for genome editing based on CRISPR/Cas9 technology. Fifteen target candidate domestication genes have been selected, based on the presence of one or more signals of domestication. Current editing approach is directed toward MYB26, encoding a transcription factor involved in pod shattering phenotype. Given the challenges posed by common bean transformation (biolistic transgenesis), the genome editing approach is being simultaneously carried out also on soybean (Glycine max) homologous genes.

Among grain legumes, common bean (Phaseolus vulgaris L.) is the most important for human consumption and is a recognized component of healthy diets of the Mediterranean basin. Bean seeds are a rich source of energy (mainly proteins and complex charbohydrates), valuable compounds (folates, vitamins, polyphenols), essential minerals (iron and zinc) and their consumption can contribute to reduce risk of diseases such as obesity, diabetes, cardiovascular problems and colon, prostate and breast cancer. However, despite all these positive characteristics, bean seeds contain important amounts of bioactive compounds (such as the lectins, digestive enzyme inhibitors, phytate, raffinosaccharides, phenolic compounds) considered as antinutritional, that may cause adverse negative effect to those who consume them as staple food and/or improperly processed/cooked.

Cultivated cardoon (Cynara cardunculus var. altilis L.) is a promising candidate species for the development of plant cell cultures suitable for large-scale biomass production and recovery of nutraceuticals. We set up a protocol for Agrobacterium tumefaciens-mediated transformation, which can be used for the improvement of cardoon cell cultures in a frame of biorefinery. As high lignin content determines lower saccharification yields for the biomass, we opted for a biotechnological approach, with the purpose of reducing lignin content; we generated transgenic lines overexpressing the Arabidopsis thaliana MYB4 transcription factor, a known repressor of lignin/flavonoid biosynthesis. Here, we report a comprehensive characterization, including metabolic and transcriptomic analyses of AtMYB4 overexpression cardoon lines, in comparison to wild type, underlining favorable traits for their use in biorefinery. Among these, the improved accessibility of the lignocellulosic biomass to degrading enzymes due to depletion of lignin content, the unexpected increased growth rates, and the valuable nutraceutical profiles, in particular for hydroxycinnamic/caffeoylquinic and fatty acids profiles.

Cardoon (Cynara cardunculus L. var. altilis) is a traditional crop with high yields of seed oil with a good fatty acid profile and a vigorous lignocellulosic biomass characterized by valuable phenolic compounds (e.g. chlorogenic acid and other polyphenols) (1). To overcome the hurdles of seasonal availability and high biochemical variability of the biomass, standardized cardoon cell cultures represent an alternative to field cultivation (2). We tested a biotechnological approach via Agrobacterium-mediated transformation, to obtain stable transgenic cardoon cell lines overexpressing the Arabidopsis MYB4 gene, encoding a transcription factor known to be a master regulator of the phenylpropanoid pathway (3)

Food legumes are crucial for all agriculture-related societal challenges including climate change, agrobiodiversity conservation, sustainable agriculture, food security and human health. Indeed, the transition to novel plant-based diets largely based on food legumes could present major opportunities for climate change mitigation strategies while generating significant co-benefits in terms of human health. Common bean (Phaseolus vulgaris L.) is the world's most important food legume and is considered a valuable source of proteins and of many macro and micronutrients. Several studies have suggested that consumption of beans is associated with a number of beneficial effects on human health including the reduction of cardiovascular diseases and diabetes mellitus, the prevention of different types of cancer and the control of some metabolic functions. Despite all their positive characteristics, beans also contain many antinutritional compounds, such as phytic acid, lectins, enzyme inhibitors, oligosaccharides, that could affect their nutritional value. Analysis of the content of these compounds may bring out traits of interest in order to promote nutrition and preserve health, and furthermore, allow to use them in breeding programs to eliminate adverse components or to modify their levels in new common bean varieties. In this context, in order to identify promising parental lines, the content of certain antinutritional compounds (including phytic acid and oligosaccharides) was evaluated in flours obtained from a hyper-core collection of 50 domesticated genotypes (single seed descent, SSD) of common bean from America and Europe including both Andean and Mesoamerican gene pools (BEAN_ADAPT and INCREASE projects). Moreover, since cooking time of the common bean represents an important issue for consumer preference, with consequences for nutrition, health, and environment, this trait has also been assessed. The seeds of the genotype "INCBN_00201" (from Greece, Andean genepool) was found to accumulate significantly low levels of phytic acid (0.81 g/100 g), while seeds of the genotype "INCBN_00091" (from Costa Rica, Mesoamerican genepool) accumulate lower level of oligosaccharides (3.89 g/100 g) if compared to the average content (respectively 1.36 and 5.13 g/100 g). As expected from the different shape and size of seeds, variability was also found for cooking time. Further analyses are ongoing in order to investigate the level of other nutritional and antinutritional compounds of seeds, however these first results revealed levels of variability high enough to be exploited in future breeding programs.

A comprehensive quality evaluation was performed on dried sweet bell pepper samples obtained from two years of production, two genotypes and two drying technologies, using one conventional and two organic cropping systems. A total number of 35 quality indexes, comprising non-volatile and volatile flavor compounds and health related compounds were evaluated for their possible role in the flavor of dried bell pepper: the most significant variations in the data analysis due to the year, drying technique, genotype and cropping system resulted in 25, 25, 16 and 10 significant interactions, respectively. The PCA analysis confirmed the main role of drying technique and sampling year. The malic acid for non-volatile compounds, linalool and 2-methoxy-3-isobutylpyrazine for volatiles represent the most relevant chemical indexes significantly correlating with many sensory descriptors scored by panellists, and are, therefore, proposed as quality markers to be carefully monitored for a correct quality evaluation of dried bell pepper.

Cardoon (Cynara cardunculus L. var. altilis) is a traditional Italian vegetable crop, with high yields of seed oil with a good fatty acid profile (high oleic acid) and a vigorous lignocellulosic biomass characterized by valuable compounds that can be recovered both from the apical part (e.g. chlorogenic acid and other polyphenols) and from roots (inulin). To overcome the hurdles of seasonal availability and high biochemical variability of the biomass, standardized cardoon cell cultures represent an alternative to field cultivation. In this frame, we tested a biotechnological approach via Agrobacterium-mediated transformation to obtain stable transgenic cardoon cell lines with the aim of increasing the accessibility of the cellulose fraction and of boosting the production of high-value nutraceuticals. Our strategy revolved around the overexpression of the Arabidopsis MYB4 gene, encoding a transcriptional factor known to be a master regulator of the phenylpropanoid pathway. We provide a detailed characterization of the transgenic lines based on molecular, metabolic and transcriptomic comparisons with wild-type controls. The research activities here described are part of the larger project BOBCAt (funded by Cariplo) devoted to optimize and scale-up the use of cardoon cell cultures in economically and environmentally sustainable conditions, in line with the principles of Circular Economy.

Phytic acid (PA), the main form of phosphorus storage present in seeds, is an antinutritional factor for its ability to chelate cations important for human nutrition. Plant breeders have spent many efforts to isolate and develop low phytic acid (lpa) mutants in different important crops. We isolated different common bean (Phaseolus vulgaris L.) lpa mutants with reduction of PA content at different extent. The consumption of common bean seeds harboring the lpa1 mutation, affecting the PvMRP1 transporter and causing a reduction of 90% in PA content, improved iron status of volunteers in human trials, but caused adverse gastrointestinal effects, presumably due to the increased stability of lectin phytohemagglutinin L (PHA-L) in these seeds, compared to the wild type (wt) ones. A hard-to-cook (HTC) defect observed in the lpa1 seeds intensified the problem. We confirmed and quantified the HTC phenotype of the lpa1 common bean seeds in three different genetic backgrounds, giving a genetic demonstration of the so-called "phytase-phytate- pectin" theory and found differences depending on the background. In one of them, we correlated the HTC defect to the redistribution of calcium, whose concentration in all parts of the seed and, particularly in the cell walls, was larger in the lpa1 compared to the wt. Furthermore, the lpa1 mutation, combined with the presence of different PHA alleles, affected the stability of the PHA-L lectin, due to an excess of free cations.

Plant specialized metabolites (SMs) play an important role in the interaction with the environment and are part of the plant defense response. These natural products are volatile, semivolatile and non-volatile compounds produced from common building blocks deriving from primary metabolic pathways and rapidly evolved to allow a better adaptation of plants to environmental cues. Specialized metabolites include terpenes, flavonoids, alkaloids, glucosinolates, tannins, resins, etc. that can be used as phytochemicals, food additives, flavoring agents and pharmaceutical compounds. This review will be focused on Mediterranean crop plants as a source of SMs, with a special attention on the strategies that can be used to modulate their production, including abiotic stresses, interaction with beneficial soil microorganisms and novel genetic approaches.

Cardoon (Cynara cardunculus L. var. altilis) is a traditional Italian vegetable crop, with high yields of seed oil with a good fatty acid profile (high oleic acid) and a vigorous ligno-cellulosic biomass characterized by valuable compounds that can be recovered both from the apical part (e.g. chlorogenic acid and other polyphenols) and from roots (inulin). To overcome the hurdles of seasonal availability and high biochemical variability of the biomass, standardized cardoon cell cultures represent an alternative to field cultivation. In this frame, we tested a biotechnological approa ch via Agrobacterium-mediated transgenesis in order to boost the production of desired compounds and to improve their bio-accessibility to extraction. We set up a protocol that represent the first example of stable cardoon cells transformation; as a proof of concept for this method, we prove how it is possible to tinker the transcriptional regulation of the phenylpropanoid biosynthesis. This strategy revolves around the reduction of lignin content of cells via altered transcriptions of MYB genes, encoding transcriptional factors known to be master regulators of development and stress response in plants. This approach aims at increasing the accessibility of the cellulose fraction for improved recovery of hydrophilic bioactive compounds. The research activities here described are part of the larger project BOBCAt aiming to optimize and scale-up the growth of wild type and engineered plant cell cultures in economically and environmentally sustainable conditions (e.g. using industrial by-products and wastewaters as nutritive substrates), in line with the principles of Circular Economy.

Cardoon (Cynara cardunculus L. var. altilis) is a traditional Italian vegetable crop, with high yields of seed oil with a good fatty acid profile (high oleic acid) and a vigorous ligno-cellulosic biomass characterized by valuable compounds that can be recovered both from the apical part (e.g. chlorogenic acid and other polyphenols) and from roots (inulin). Despite its value as a multifunctional industrial crop, cardoon biomass suffers from seasonal availability and high variability of its quality and quantity. To overcome these hurdles and improve the production and accessibility of bioactive compounds, in this project we investigate the potential use of cardoon cell cultures as an alternative to field cultivation, in the frame of a sustainable cell-based biorefinery. For this reason, different organs and tissues (seeds, hypocotyls, cotyledons and leaves) of cardoon cv. Spagnolo have been characterized for their biochemical and transcriptional profile of fatty acid and phenylpropanoids profile. The molecular analysis of both of these pathways allowed the identification of promising genetic targets to boost the production of the monounsaturated fatty acids/MUFAs (via modulation of committed enzymes of oleic acid biosynthesis) and to increase the accessibility of the cellulose fraction for improved recovery of hydrophilic bioactive compounds. This latest strategy revolves around the reduction of lignin content of cells via the alteration of MYB genes, transcriptional factors known to be master regulators of development and stress response in plants. The research activities also aim at optimizing and scale-up the growth of wild type and engineered plant cell cultures in economically and environmentally sustainable conditions (e.g. using industrial by-products and wastewaters as nutritive substrates), in line with the principles of Circular Economy

Two genotypes of sweet pepper were studied regarding their profile of quality indexes related to yield, taste and healthy components. The peppers were grown with three different types of cultivation, one conventional (CONV) and two organic (ORG), in three harvest years, 2015, 2016, and 2017. The two genotypes belong to the typology "red long horn", comparing a local cultivar selected for organic production (RTV), against a commercial F1 hybrid (Alceste F1). The main quality indexes of sweet pepper were measured, considering yield, tastants such as sugars, organic acids, volatiles and phytochemicals such as ascorbic acid, carotenoids, and tocopherols. Moreover, the Folin-Ciocalteu and DPPH assays were performed to assess the total polyphenols content and the antioxidant capacity. The results indicated that the 3 years of harvest were different, with the summer of 2016 more rainy and less warm than others, and most significant differences among the samples were due to this factor. The data collection indicates that ORG fruits had significant higher agronomic yield and sugar content, while higher content of ascorbic acid, yellow carotenoids, and Folin-Ciocalteu index was found in 2016 with respect to 2015 and 2017, in accordance with the meteorological variations. Moreover, in 2016, some volatiles showed strong depletions in ORG vs CONV samples. In some cases, considerable differences between the genotypes were observed, due to the different adaptability to ORG conditions for RTV and to CONV conditions for Alceste F1. A high content of free sugars and ascorbic acid was found in RTV ORG, while high content of organic acids, carotenoids, and 2-methoxy-3-isobutyl pirazine was observed in Alceste F1 ORG.

Phytic acid (PA) represents the major storage form of seed phosphate (P). During seed maturation, it accumulates as phytate salts chelating various mineral cations, thereforereducing their bioavailability. During germination, phytase dephosphorylates PA releasingboth P and cations which in turn can be used for the nutrition of the growing seedling.Animals do not possess phytase, thus monogastric animals assimilate only 10% of thephytate ingested with feed, whilst 90% is excreted and may contribute to cause Ppollution of the environment. To overcome this double problem, nutritional andenvironmental, in the last four decades, many low phytic acid (lpa) mutants (most ofwhich affect the PA-MRP transporters) have been isolated and characterized in all majorcrops, showing that the lpa trait can increase the nutritional quality of foods and feeds andimprove P management in agriculture. Nevertheless, these mutations are frequentlyaccompanied by negative pleiotropic effects leading to agronomic defects which mayaffect either seed viability and germination or plant development or in some cases evenincrease the resistance to cooking, thus limiting the interest of breeders. Therefore,although some significant results have been reached, the isolation of lpa mutantsimproved for their nutritional quality and with a good field performance remains a goalso far not fully achieved for many crops. Here, we will summarize the main pleiotropiceffects that have been reported to date in lpa mutants affected in PA-MRP transporters infive productive agronomic species, as well as addressing some of the possible challengesto overcome these hurdles and improve the breeding efforts for lpa mutants.

Seed phytic acid reduces mineral bioavailability by chelating minerals. Consumption of common bean seeds with the low phytic acid 1 (lpa1) mutation improved iron status in human trials but caused adverse gastrointestinal effects, presumably due to increased stability of lectin phytohemagglutinin L (PHA-L) compared to the wild type (wt). A hard-to-cook (HTC) defect observed in lpa1 seeds intensified this problem. We quantified the HTC phenotype of lpa1 common beans with three genetic backgrounds. The HTC phenotype in the lpa1 black bean line correlated with the redistribution of calcium particularly in the cell walls, providing support for the "phytase-phytate-pectin" theory of the HTC mechanism. Furthermore, the excess of free cations in the lpa1 mutation in combination with different PHA alleles affected the stability of PHA-L lectin.

Impiego delle cover crop nella coltivazione del mais

Measurement and sensing of cover crop growth and nitrogen credits in conservation agriculture

The industrial transformation of tomato (Lycopersicon esculentum Mill.) produces processed foods, such as dried tomatoes. In this study two varieties (SaAb and PerBruzzo), grown in three cropping systems (one conventional and two organic ones), were processed by two types of small-scale drying (oven or sun drying), over two years of production. The dried samples were analyzed for their non-volatile and volatile composition, relating the results with sensory analysis. The multivariate analysis performed on collected data allowed a detailed comparison of the effects of processing, year-to year variation and cropping systems. Results indicated that drying methods mainly influenced the composition and flavor profile, also affected by the production year. The cropping system significantly influenced some quality indices, such as the acid and sugar amounts, and the aldehydes, respectively higher and lower in organic samples. The comprehensive PCA analysis allowed discrimination of drying methods and, to a lesser extent, cropping systems. ? 2019 Elsevier Ltd

Phytic acid (PA), the main form of phosphorus storage present in seeds, is an antinutritional factor for its ability to chelate cations important for human nutrition. Plant breeders have spent many efforts to isolate and develop low phytic acid (lpa) mutants in different important crops. We isolated different common bean lpa mutants with reduction of PA content at different extent. The consumption of common bean seeds harboring the lpa1 mutation, affecting the PvMRP1 transporter and causing a reduction of 90% in PA content, improved iron status of volunteers in human trials, but caused adverse gastrointestinal effects, presumably due to the increased stability of lectin phytohemagglutinin L (PHA-L) in these seeds, compared to the wild type (wt) ones. A hard-to-cook (HTC) defect observed in the lpa1 seeds intensified the problem. We confirmed and quantified the HTC phenotype of the lpa1 common bean seeds in three different genetic backgrounds, giving a genetic demonstration of the so-called "phytase-phytate-pectin" theory and found differences depending on the background. In one of them, we correlated the HTC defect to the redistribution of calcium, whose concentration in all parts of the seed and, particularly in the cell walls, was larger in the lpa1 compared to the wt. Furthermore, the lpa1 mutation, combined with the presence of different PHA alleles, affected the stability of the PHA-L lectin, due to an excess of free cations. Moreover, we showed a decreased seed density in the lpa1 mutant compared to the wt, but only in some of the analyzed genetic backgrounds. All these data suggest that the pleiotropic effect due to the lpa1 mutation are strictly dependent on the genetic background. Other common bean lpa mutants, have been recently isolated with milder effect on PA reduction than the previously described lpa1 one. A candidate gene approach for three of these mutants did not reveal any mutation in known PA biosynthetic genes or in genes coding for PA transport. A mapping approach is underway in order to identify the affected genes. These mutants can be assayed for their cooking and nutritional properties in order to develop useful biofortified beans devoid of negative traits.

Low-phosphorus stress is a challenging factor in limiting plant development. Soybean is cultivated in soils often low in phosphorus. However, on average 65% of total P is in the form of organic phosphates, which are unavailable to plants unless hydrolyzed to release inorganic phosphate. One approach for enhancing crop P acquisition from organic P sources is boosting the activity of acid phosphatases (APases). This study seeks to understand the role of an Arabidopsis (Arabidopsis thaliana) purple APase gene (AtPAP18) in soybean. Thus, the gene was isolated and a final vector (AtPAP18/pK7GWG2D) was built. Composite soybean plants were created using Agrobacterium rhizogenes-mediated transformation. A. rhizogenes K599 carrying the AtPAP18/pK7GWG2D vector with egfp as a reporter gene was used for soybean hairy root transformation. Analysis of Egfp expression detected fluorescence signals in transgenic roots, whereas there was no detectable fluorescence in control hairy roots. The enzyme assay showed that the APase activity increased by 2-fold in transgenic hairy roots. The transformed hairy roots displayed an increase in plant soluble P and total P contents, as compared with the control plants, leading to improved biomass production. RT-PCR analysis revealed high expression levels of AtPAP18 in transformed hairy roots. It is noteworthy that these primers amplified no PAP18 transcript in control hairy roots. Taken together, the findings demonstrated that overexpression of the AtPAP18 gene offers an operative tactic to reduce the utilization of inorganic phosphorus (Pi) fertilizer through increased acquisition of soil Pi, especially improving the crop yield on soils low in available P. ? 2018, ? 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.