Phenotyping Key Fruit Quality Traits in Olive Using RGB Images and Back Propagation Neural Networks.

To predict oil and phenol concentrations in olive fruit, the combination of back propagation neural networks (BPNNs) and contact-less plant phenotyping techniques was employed to retrieve RGB image-based digital proxies of oil and phenol concentrations. Fruits of cultivars (×3) differing in ripening time were sampled (~10-day interval, ×2 years), pictured and analyzed for phenol and oil concentrations. Prior to this, fruit samples were pictured and images were segmented to extract the red (R), green (G), and blue (B) mean pixel values that were rearranged in 35 RGB-based colorimetric indexes. Three BPNNs were designed using as input variables (a) the original 35 RGB indexes, (b) the scores of principal components after a principal component analysis (PCA) pre-processing of those indexes, and (c) a reduced number (28) of the RGB indexes achieved after a sparse PCA. The results show that the predictions reached the highest mean R2 values ranging from 0.87 to 0.95 (oil) and from 0.81 to 0.90 (phenols) across the BPNNs. In addition to the R2, other performance metrics were calculated (root mean squared error and mean absolute error) and combined into a general performance indicator (GPI). The resulting rank of the GPI suggests that a BPNN with a specific topology might be designed for cultivars grouped according to their ripening period. The present study documented that an RGB-based image phenotyping can effectively predict key quality traits in olive fruit supporting the developing olive sector within a digital agriculture domain.

A Lycopene ε-Cyclase TILLING Allele Enhances Lycopene and Carotenoid Content in Fruit and Improves Drought Stress Tolerance in Tomato Plants.

In the scenario of climate change, the availability of genetic resources for tomato cultivation that combine improved nutritional properties and more tolerance to water deficiency is highly desirable. Within this context, the molecular screenings of the Red Setter cultivar-based TILLING platform led to the isolation of a novel lycopene ε-cyclase gene (SlLCY-E) variant (G/3378/T) that produces modifications in the carotenoid content of tomato leaves and fruits. In leaf tissue, the novel G/3378/T SlLCY-E allele enhances ß,ß-xanthophyll content at the expense of lutein, which decreases, while in ripe tomato fruit the TILLING mutation induces a significant increase in lycopene and total carotenoid content. Under drought stress conditions, the G/3378/T SlLCY-E plants produce more abscisic acid (ABA) and still conserve their leaf carotenoid profile (reduction of lutein and increase in ß,ß-xanthophyll content). Furthermore, under said conditions, the mutant plants grow much better and are more tolerant to drought stress, as revealed by digital-based image analysis and in vivo monitoring of the OECT (Organic Electrochemical Transistor) sensor. Altogether, our data indicate that the novel TILLING SlLCY-E allelic variant is a valuable genetic resource that can be used for developing new tomato varieties, improved in drought stress tolerance and enriched in fruit lycopene and carotenoid content.

Low-Cost Hyperspectral Imaging to Detect Drought Stress in High-Throughput Phenotyping.

Recent developments in low-cost imaging hyperspectral cameras have opened up new possibilities for high-throughput phenotyping (HTP), allowing for high-resolution spectral data to be obtained in the visible and near-infrared spectral range. This study presents, for the first time, the integration of a low-cost hyperspectral camera Senop HSC-2 into an HTP platform to evaluate the drought stress resistance and physiological response of four tomato genotypes (770P, 990P, Red Setter and Torremaggiore) during two cycles of well-watered and deficit irrigation. Over 120 gigabytes of hyperspectral data were collected, and an innovative segmentation method able to reduce the hyperspectral dataset by 85.5% was developed and applied. A hyperspectral index (H-index) based on the red-edge slope was selected, and its ability to discriminate stress conditions was compared with three optical indices (OIs) obtained by the HTP platform. The analysis of variance (ANOVA) applied to the OIs and H-index revealed the better capacity of the H-index to describe the dynamic of drought stress trend compared to OIs, especially in the first stress and recovery phases. Selected OIs were instead capable of describing structural changes during plant growth. Finally, the OIs and H-index results have revealed a higher susceptibility to drought stress in 770P and 990P than Red Setter and Torremaggiore genotypes.

Image-Based Assessment of Drought Response in Grapevines.

Many plants can modify their leaf profile rapidly in response to environmental stress. Image-based data are increasingly used to retrieve reliable information on plant water status in a non-contact manner that has the potential to be scaled to high-throughput and repeated through time. This paper examined the variation of leaf angle as measured by both 3D images and goniometer in progressively drought stressed grapevine. Grapevines, grown in pots, were subjected to a 21-day period of drought stress receiving 100% (CTRL), 60% (IRR 60%) and 30% (IRR 30%) of maximum soil available water capacity. Leaf angle was (i) measured manually (goniometer) and (ii) computed by a 3D reconstruction method (multi-view stereo and structure from motion). Stomatal conductance, leaf water potential, fluorescence (F v /F m ), leaf area and 2D RGB data were simultaneously collected during drought imposition. Throughout the experiment, values of leaf water potential ranged from -0.4 (CTRL) to -1.1 MPa (IRR 30%) and it linearly influenced the leaf angle when measured manually (R 2 = 0.86) and with 3D image (R 2 = 0.73). Drought was negatively related to stomatal conductance and leaf area growth particularly in IRR 30% while photosynthetic parameters (i.e., F v /F m ) were not impaired by water restriction. A model for leaf area estimation based on the number of pixels of 2D RGB images developed at a different phenotyping robotized platform in a closely related experiment was successfully employed (R 2 = 0.78). At the end of the experiment, top view 2D RGB images showed a ∼50% reduction of greener fraction (GGF) in CTRL and IRR 60% vines compared to initial values, while GGF in IRR 30% increased by approximately 20%.

Identification and Characterization of a Thermotolerant TILLING Allele of Heat Shock Binding Protein 1 in Tomato.

The identification of heat stress (HS)-resilient germplasm is important to ensure food security under less favorable environmental conditions. For that, germplasm with an altered activity of factors regulating the HS response is an important genetic tool for crop improvement. Heat shock binding protein (HSBP) is one of the main negative regulators of HS response, acting as a repressor of the activity of HS transcription factors. We identified a TILLING allele of Solanum lycopersicum (tomato) HSBP1. We examined the effects of the mutation on the functionality of the protein in tomato protoplasts, and compared the thermotolerance capacity of lines carrying the wild-type and mutant alleles of HSBP1. The methionine-to-isoleucine mutation in the central heptad repeats of HSBP1 leads to a partial loss of protein function, thereby reducing the inhibitory effect on Hsf activity. Mutant seedlings show enhanced basal thermotolerance, while mature plants exhibit increased resilience in repeated HS treatments, as shown by several physiological parameters. Importantly, plants that are homozygous for the wild-type or mutant HSBP1 alleles showed no significant differences under non-stressed conditions. Altogether, these results indicate that the identified mutant HSBP1 allele can be used as a genetic tool in breeding, aiming to improve the thermotolerance of tomato varieties.

Corrigendum: Can High Throughput Phenotyping Help Food Security in the Mediterranean Area?

[This corrects the article DOI: 10.3389/fpls.2019.00015.].

Can High Throughput Phenotyping Help Food Security in the Mediterranean Area?

According to the IPCC 2014 report the Mediterranean region will be affected by strong climatic changes, both in terms of average temperature and of precipitations regime. This area hosts some half a billion people and the impact on food production will be severe. To implement a climate smart agriculture paradigm and a sustainable increase of agricultural productivity different approaches can be deployed. Agriculture alone consumes 70% of the entire water available on the planet, thus the observed reduction of useful rainfall and growing costs for irrigation water may severely constrain food security. In our work we focused on two typical Mediterranean crops: durum wheat, a rainfed crop, and tomato, an irrigated one. In wheat we explored the possibility of identifying genotypes resilient to water stress for future breeding aims, while in tomato we explored the possibility of using biostimulants to increase the plant capacity of using water. In order to achieve these targets, we used high throughput phenotyping (HTP). Two traits were considered: digital biovolume, a measure based on imaging techniques in the RGB domain, and Water Use Efficiency index as calculated semi-automatically on the basis of evaporation measurements resulting in a high throughput, non-destructive, non-invasive approach, as opposed to destructive and time consuming traditional methods. Our results clearly indicate that HTP is able to discriminate genotypes and biostimulant treatments that allow plants to use soil water more efficiently. In addition, these methods based on RGB quality images can easily be scaled to field phenotyping structure USVs or UAVs.

In Vivo Phenotyping for the Early Detection of Drought Stress in Tomato.

Drought stress imposes a major constraint over a crop yield and can be expected to grow in importance if the climate change predicted comes about. Improved methods are needed to facilitate crop management via the prompt detection of the onset of stress. Here, we report the use of an in vivo OECT (organic electrochemical transistor) sensor, termed as bioristor, in the context of the drought response of the tomato plant. The device was integrated within the plant's stem, thereby allowing for the continuous monitoring of the plant's physiological status throughout its life cycle. Bioristor was able to detect changes of ion concentration in the sap upon drought, in particular, those dissolved and transported through the transpiration stream, thus efficiently detecting the occurrence of drought stress immediately after the priming of the defence responses. The bioristor's acquired data were coupled with those obtained in a high-throughput phenotyping platform revealing the extreme complementarity of these methods to investigate the mechanisms triggered by the plant during the drought stress event.

Ectopic Expression of PII Induces Stomatal Closure in Lotus japonicus.

The PII protein in plants has been associated to many different tissue specialized roles concerning the Nitrogen assimilation pathways. We report here the further characterization of L. japonicus transgenic lines overexpressing the PII protein encoded by the LjGLB1 gene that is strongly expressed in the guard cells of Lotus plants. Consistently with a putative role played by PII in that specific cellular context we have observed an alteration of the patterns of stomatal movement in the overexpressing plants. An increased stomatal closure is measured in epidermal peels from detached leaves of normally watered overexpressing plants when compared to wild type plants and this effect was by-passed by Abscisic Acid application. The biochemical characterization of the transgenic lines indicates an increased rate of the Nitric Oxide biosynthetic route, associated to an induced Nitrate Reductase activity. The phenotypic characterization is completed by measures of the photosynthetic potential in plants grown under greenhouse conditions, which reveal a higher stress index of the PII overexpressing plants.

Selenium-enriched durum wheat improves the nutritional profile of pasta without altering its organoleptic properties.

Two field experiments were conducted over three growing seasons (2006-07, 2008-09 and 2009-10) to evaluate Se-enriched pasta through foliar fertilization at various rates and timing of application on 4 durum wheat varieties. Our findings confirm the effectiveness of foliar Se fertilization to increase Se concentrations in durum wheat grain, even at high Se rates (120gSeha(-1)). Se fortification was significant across different genotypes, with greater Se accumulation in landraces ('Timilia') and obsolete varieties ('Cappelli'), with respect to modern varieties. The Se content in the grain was increased by up to 35-fold that of the untreated control. The Se concentration decreased during milling (11%), while processing and cooking of pasta did not show significant decreases. This biofortification stategy had no effects on grain quality parameters, except for reduced gluten index in the high-gluten variety PR22D89, as well as for the sensorial properties of the spaghetti.

Induced mutations in tomato SlExp1 alter cell wall metabolism and delay fruit softening.

Fruit ripening and softening are key traits for many fleshy fruit. Since cell walls play a key role in the softening process, expansins have been investigated to control fruit over ripening and deterioration. In tomato, expression of Expansin 1 gene, SlExp1, during fruit ripening was associated with fruit softening. To engineer tomato plants with long shelf life, we screened for mutant plants impaired in SlExp1 function. Characterization of two induced mutations, Slexp1-6_W211S, and Slexp1-7_Q213Stop, showed that SlExp1 loss of function leads to enhanced fruit firmness and delayed fruit ripening. Analysis of cell wall polysaccharide composition of Slexp1-7_Q213Stop mutant pointed out significant differences for uronic acid, neutral sugar and total sugar contents. Hemicelluloses chemistry analysis by endo-ß-1,4-d-glucanase hydrolysis and MALDI-TOF spectrometry revealed that xyloglucan structures were affected in the fruit pericarp of Slexp1-7_Q213Stop mutant. Altogether, these results demonstrated that SlExp1 loss of function mutants yield firmer and late ripening fruits through modification of hemicellulose structure. These SlExp1 mutants represent good tools for breeding long shelf life tomato lines with contrasted fruit texture as well as for the understanding of the cell wall polysaccharide assembly dynamics in fleshy fruits.

Growth responses of crop and weed species to heavy metals in pot and field experiments.

Greenhouse and field studies were performed to examine the growth responses and possible phytoremediation capacity towards heavy metals of several Brassicaceae (Brassica alba, Brassica carinata, Brassica napus and Brassica nigra) and Poaceae (durum wheat and barley). Soils used featured total concentrations of Cr, Cu, Pb and Zn largely exceeding the maximum levels permitted by the Italian laws. Different organic amendments were tested such as a compost and the plant growth-promoting rhizobacterium Bacillus licheniformis. In the greenhouse experiment, plant length, leaf area index and shoots dry matter were evaluated periodically for the Brassicaceae examined. Whereas plant length, grains production, weight of 1,000 seeds, ear fertility and tiller density were determined under field conditions at the end of the crop cycle for wheat and barley. In general, the species tested appeared to be tolerant to high heavy metal concentrations in soil, and slightly significant differences were found for all parameters considered. A marked growth increase was shown to occur for Brassicaceae cultivated on compost- and bacillus-amended contaminated soils, with respect to non-amended contaminated soils. With some exception, higher growth parameters were measured for wheat and barley plants cropped from contaminated soils in comparison to non-contaminated soils. Further, bacillus amendment enhanced the length of wheat and barley plants in both non-contaminated and contaminated soils, while different effects were observed for the other parameters evaluated.

Engineered drought tolerance in tomato plants is reflected in chlorophyll fluorescence emission.

Drought stress is one of the most important factors that limit crop productivity worldwide. In order to obtain tomato plants with enhanced drought tolerance, we inserted the transcription factor gene ATHB-7 into the tomato genome. This gene was demonstrated earlier to be up-regulated during drought stress in Arabidopsis thaliana thus acting as a negative regulator of growth. We compared the performance of wild type and transgenic tomato line DTL-20, carrying ATHB-7 gene, under well-irrigated and water limited conditions. We found that transgenic plants had reduced stomatal density and stomatal pore size and exhibited an enhanced resistance to soil water deficit. We used the transgenic plants to investigate the potential of chlorophyll fluorescence to report drought tolerance in a simulated high-throughput screening procedure. Wild type and transgenic tomato plants were exposed to drought stress lasting 18 days. The stress was then terminated by rehydration after which recovery was studied for another 2 days. Plant growth, leaf water potential, and chlorophyll fluorescence were measured during the entire experimental period. We found that water potential in wild type and drought tolerant transgenic plants diverged around day 11 of induced drought stress. The chlorophyll fluorescence parameters: the non-photochemical quenching, effective quantum efficiency of PSII, and the maximum quantum yield of PSII photochemistry yielded a good contrast between wild type and transgenic plants from day 7, day 12, and day 14 of induced stress, respectively. We propose that chlorophyll fluorescence emission reports well on the level of water stress and, thus, can be used to identify elevated drought tolerance in high-throughput screens for selection of resistant genotypes.

A new mutant genetic resource for tomato crop improvement by TILLING technology.

BACKGROUND: In the last decade, the availability of gene sequences of many plant species, including tomato, has encouraged the development of strategies that do not rely on genetic transformation techniques (GMOs) for imparting desired traits in crops. One of these new emerging technology is TILLING (Targeting Induced Local Lesions In Genomes), a reverse genetics tool, which is proving to be very valuable in creating new traits in different crop species. RESULTS: To apply TILLING to tomato, a new mutant collection was generated in the genetic background of the processing tomato cultivar Red Setter by treating seeds with two different ethylemethane sulfonate doses (0.7% and 1%). An associated phenotype database, LycoTILL, was developed and a TILLING platform was also established. The interactive and evolving database is available online to the community for phenotypic alteration inquiries. To validate the Red Setter TILLING platform, induced point mutations were searched in 7 tomato genes with the mismatch-specific ENDO1 nuclease. In total 9.5 kb of tomato genome were screened and 66 nucleotide substitutions were identified. The overall mutation density was estimated and it resulted to be 1/322 kb and 1/574 kb for the 1% EMS and 0.7% EMS treatment respectively. CONCLUSIONS: The mutation density estimated in our collection and its comparison with other TILLING populations demonstrate that the Red Setter genetic resource is suitable for use in high-throughput mutation discovery. The Red Setter TILLING platform is open to the research community and is publicly available via web for requesting mutation screening services.