Toward robust and high-throughput detection of seed defects in X-ray images via deep learning.

BACKGROUND: The detection of internal defects in seeds via non-destructive imaging techniques is a topic of high interest to optimize the quality of seed lots. In this context, X-ray imaging is especially suited. Recent studies have shown the feasibility of defect detection via deep learning models in 3D tomography images. We demonstrate the possibility of performing such deep learning-based analysis on 2D X-ray radiography for a faster yet robust method via the X-Robustifier pipeline proposed in this article. RESULTS: 2D X-ray images of both defective and defect-free seeds were acquired. A deep learning model based on state-of-the-art object detection neural networks is proposed. Specific data augmentation techniques are introduced to compensate for the low ratio of defects and increase the robustness to variation of the physical parameters of the X-ray imaging systems. The seed defects were accurately detected (F1-score >90%), surpassing human performance in computation time and error rates. The robustness of these models against the principal distortions commonly found in actual agro-industrial conditions is demonstrated, in particular, the robustness to physical noise, dimensionality reduction and the presence of seed coating. CONCLUSION: This work provides a full pipeline to automatically detect common defects in seeds via 2D X-ray imaging. The method is illustrated on sugar beet and faba bean and could be efficiently extended to other species via the proposed generic X-ray data processing approach (X-Robustifier). Beyond a simple proof of feasibility, this constitutes important results toward the effective use in the routine of deep learning-based automatic detection of seed defects.

High throughput phenotyping dataset related to seed and seedling traits of sugar beet genotypes.

Several seed and seedling traits are measured to evaluate germination and emergence potential in relation with environmental conditions. More generally, these traits are also measured in the field of ecology as simple traits that can be correlated to other adaptative traits more difficult to measure on adult plants, as for example traits of the rooting system. Methods were developed for deep high throughput phenotyping of hundreds of genotypes from dry seed to the end of heterotrophic growth. The present dataset comes from a project on genotyping and phenotyping of populations of genotypes, with different geographic and genetic origins so as to increase genotypic diversity of sugar beet in terms of germination and early growth traits, evaluated at low temperatures. Data were collected in relation to the creation of the first sugar beet crop ontology. This dataset corresponds to the first automated phenotyping of a population of 198 genotypes and 4 commercial control varieties and is hosted on INRAE public depository under the reference number doi.org/10.15,454/AKNF4Q. The equipment and methods presented here are available on a phenotyping platform opened to collaborative research and adaptable for specific services for characterizing thousands of genotypes on different crops or other species. The phenotyping values can also be linked to genomic information to study the genetic determinism of the trait values.

Combined Effects of Temperature, Irradiance, and pH on Teleaulax amphioxeia (Cryptophyceae) Physiology and Feeding Ratio For Its Predator Mesodinium rubrum (Ciliophora)1.

The cryptophyte Teleaulax amphioxeia is a source of plastids for the ciliate Mesodinium rubrum and both organisms are members of the trophic chain of several species of Dinophysis. It is important to better understand the ecology of organisms at the first trophic levels before assessing the impact of principal factors of global change on Dinophysis spp. Therefore, combined effects of temperature, irradiance, and pH on growth rate, photosynthetic activity, and pigment content of a temperate strain of T. amphioxeia were studied using a full factorial design (central composite design 23 *) in 17 individually controlled bioreactors. The derived model predicted an optimal growth rate of T. amphioxeia at a light intensity of 400 µmol photons · m-2 · s-1 , more acidic pH (7.6) than the current average and a temperature of 17.6°C. An interaction between temperature and irradiance on growth was also found, while pH did not have any significant effect. Subsequently, to investigate potential impacts of prey quality and quantity on the physiology of the predator, M. rubrum was fed two separate prey: predator ratios with cultures of T. amphioxeia previously acclimated at two different light intensities (100 and 400 µmol photons · m-2 s-1 ). M. rubrum growth appeared to be significantly dependent on prey quantity while effect of prey quality was not observed. This multi-parametric study indicated a high potential for a significant increase of T. amphioxeia in future climate conditions but to what extent this would lead to increased occurrences of Mesodinium spp. and Dinophysis spp. should be further investigated.

An artificial miRNA as a new tool to silence and explore gene functions in apple.

Artificial miRNA (amiRNA) is a powerful technology to silence genes of interest. It has a high efficiency and specificity that can be used to explore gene function through targeted gene regulation or to create new traits. To develop this gene regulation tool in apple, we designed two amiRNA constructs based on an apple endogenous miRNA backbone previously characterized (Md-miR156h), and we checked their efficiency on an easily scorable marker gene: the phytoene desaturase gene (MdPDS in apple). Two pairs of miRNA:miRNA* regions were designed (named h and w). The monocistronic Md-miR156h with these MdPDS targets was placed under the control of the CaMV 35S promoter to generate the two plasmids: pAmiRNA156h-PDSh and pAmiRNA156h-PDSw. Two Agrobacterium-mediated transformation experiments were performed on the cultivar 'Gala'. A total of 11 independent transgenic clones were obtained in the first experiment and 5 in the second. Most transgenic lines had a typical albino and dwarf phenotype. However, six clones had a wild type green phenotype. Molecular analyses indicated clear relationships between the degree of albino phenotype, the level of MdPDS gene expression and the amount of mature amiRNAs. This study demonstrated for the first time in apple the functionality of an artificial miRNA based on an endogenous miRNA backbone. It provides important opportunities for apple genetic functional studies as well as apple genetic improvement projects.

Efficient Targeted Mutagenesis in Apple and First Time Edition of Pear Using the CRISPR-Cas9 System.

Targeted genome engineering has emerged as an alternative to classical plant breeding and transgenic methods to improve crop plants. Among other methods (zinc finger nucleases or TAL effector nucleases) the CRISPR-Cas system proved to be the most effective, convenient and least expensive method. In this study, we optimized the conditions of application of this system on apple and explored its feasibility on pear. As a proof of concept, we chose to knock-out the Phytoene Desaturase (PDS) and Terminal Flower 1 (TFL1) genes. To improve the edition efficiency, two different single guide RNAs (gRNAs) were associated to the Cas9 nuclease for each target gene. These gRNAs were placed under the control of the U3 and U6 apple promoters. Characteristic albino phenotype was obtained for 85% of the apple transgenic lines targeted in MdPDS gene. Early flowering was observed in 93% of the apple transgenic lines targeted in MdTFL1.1 gene and 9% of the pear transgenic lines targeted in PcTFL1.1. Sequencing of the target zones in apple and pear CRISPR-PDS and CRISPR-TFL1.1 transgenic lines showed that the two gRNAs induced mutations but at variable frequencies. In most cases, Cas9 nuclease cut the DNA in the twenty targeted base pairs near the protospacer adjacent motif and insertions were more frequent than deletions or substitutions. The most frequent edition profile of PDS as well as TFL1.1 genes was chimeric biallelic. Analysis of a sample of potential off-target sequences of the CRISPR-TFL1.1 construct indicated the absence of edition in cases of three mismatches. In addition, transient transformation with the CRISPR-PDS construct produced two T-DNA free edited apple lines. Our overall results indicate that, despite the frequent occurrence of chimerism, the CRISPR-Cas 9 system is a powerful and precise method to induce targeted mutagenesis in the first generation of apple and pear transgenic lines.

High-affinity nitrate/nitrite transporter genes (Nrt2) in Tisochrysis lutea: identification and expression analyses reveal some interesting specificities of Haptophyta microalgae.

Microalgae have a diversity of industrial applications such as feed, food ingredients, depuration processes and energy. However, microalgal production costs could be substantially improved by controlling nutrient intake. Accordingly, a better understanding of microalgal nitrogen metabolism is essential. Using in silico analysis from transcriptomic data concerning the microalgae Tisochrysis lutea, four genes encoding putative high-affinity nitrate/nitrite transporters (TlNrt2) were identified. Unlike most of the land plants and microalgae, cloning of genomic sequences and their alignment with complementary DNA (cDNA) sequences did not reveal the presence of introns in all TlNrt2 genes. The deduced TlNRT2 protein sequences showed similarities to NRT2 proteins of other phyla such as land plants and green algae. However, some interesting specificities only known among Haptophyta were also revealed, especially an additional sequence of 100 amino acids forming an atypical extracellular loop located between transmembrane domains 9 and 10 and the function of which remains to be elucidated. Analyses of individual TlNrt2 gene expression with different nitrogen sources and concentrations were performed. TlNrt2.1 and TlNrt2.3 were strongly induced by low NO3 (-) concentration and repressed by NH4 (+) substrate and were classified as inducible genes. TlNrt2.2 was characterized by a constitutive pattern whatever the substrate. Finally, TlNrt2.4 displayed an atypical response that was not reported earlier in literature. Interestingly, expression of TlNrt2.4 was rather related to internal nitrogen quota level than external nitrogen concentration. This first study on nitrogen metabolism of T. lutea opens avenues for future investigations on the function of these genes and their implication for industrial applications.

Medicago truncatula stress associated protein 1 gene (MtSAP1) overexpression confers tolerance to abiotic stress and impacts proline accumulation in transgenic tobacco.

Stress associated proteins (SAP) have been already reported to play a role in tolerance acquisition of some abiotic stresses. In the present study, the role of MtSAP1 (Medicago truncatula) in tolerance to temperature, osmotic and salt stresses has been studied in tobacco transgenic seedlings. Compared to wild type, MtSAP1 overexpressors were less affected in their growth and development under all tested stress conditions. These results confirm that MtSAP1 is involved in the response processes to various abiotic constraints. In parallel, we have performed studies on an eventual link between MtSAP1 overexpression and proline, a major player in stress response. In an interesting way, the results for the transgenic lines did not show any increase of proline content under osmotic and salt stress, contrary to the WT which usually accumulated proline in response to stress. These data strongly suggest that MtSAP1 is not involved in signaling pathway responsible for the proline accumulation in stress conditions. This could be due to the fact that the overexpression of MtSAP1 provides sufficient tolerance to seedlings to cope with stress without requiring the free proline action. Beyond that, the processes by which the MtSAP1 overexpression lead to the suppression of proline accumulation will be discussed in relation with data from our previous study involving nitric oxide.

The carnitine biosynthetic pathway in Arabidopsis thaliana shares similar features with the pathway of mammals and fungi.

Carnitine is an essential quaternary ammonium amino acid that occurs in the microbial, plant and animal kingdoms. The role and synthesis of this compound are very well documented in bacteria, fungi and mammals. On the contrary, although the presence of carnitine in plant tissue has been reported four decades ago and information about its biological implication are available, nothing is known about its synthesis in plants. We designed experiments to determine if the carnitine biosynthetic pathway in Arabidopsis thaliana is similar to the pathway in mammals and in the fungi Neurospora crassa and Candida albicans. We first checked for the presence of trimetyllysine (TML) and γ-butyrobetaine (γ-BB), two precursors of carnitine in fungi and in mammals, using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Both compounds were shown to be present in plant extracts at concentrations in the picomole range per mg of dry weight. We next synthesized deuterium-labeled TML and transferred A. thaliana seedlings on growth medium supplemented with 1 mM of the deuterated precursor. LC-ESI-MS/MS analysis of plant extracts clearly highlighted the synthesis of deuterium labeled γ-BB and labeled carnitine in deuterated-TML fed plants. The similarities between plant, fungal and mammalian pathways provide very useful information to search homologies between genomes. As a matter of fact the analysis of A. thaliana protein database provides homology for several enzymes responsible for carnitine synthesis in fungi and mammals. The study of mutants affected in the corresponding genes would be very useful to elucidate the plant carnitine biosynthetic pathway and to investigate further the role of carnitine in plant physiology.

Overexpression of a Medicago truncatula stress-associated protein gene (MtSAP1) leads to nitric oxide accumulation and confers osmotic and salt stress tolerance in transgenic tobacco.

The impact of Medicago truncatula stress-associated protein gene (MtSAP1) overexpression has been investigated in Nicotiana tabacum transgenic seedlings. Under optimal conditions, transgenic lines overexpressing MtSAP1 revealed better plant development and higher chlorophyll content as compared to wild type seedlings. Interestingly, transgenic lines showed a stronger accumulation of nitric oxide (NO), a signaling molecule involved in growth and development processes. This NO production seemed to be partially nitrate reductase dependent. Due to the fact that NO has been also reported to play a role in tolerance acquisition of plants to abiotic stresses, the responses of MtSAP1 overexpressors to osmotic and salt stress have been studied. Compared to the wild type, transgenic lines were less affected in their growth and development. Moreover, NO content in MtSAP1 overexpressors was always higher than that detected in wild seedlings under stress conditions. It seems that this better tolerance induced by MtSAP1 overexpression could be associated with this higher NO production that would enable seedlings to reach a high protection level to prepare them to cope with abiotic stresses.

The effect of carnitine on Arabidopsis development and recovery in salt stress conditions.

Carnitine exists in all living organisms where it plays diverse roles. In animals and yeast, it is implicated in lipid metabolism and is also associated with oxidative stress tolerance. In bacteria, it is a major player in osmotic stress tolerance. We investigate the carnitine function in plants and our present work shows that carnitine enhances the development and recovery of Arabidopsis thaliana seedlings subjected to salt stress. Biological data show that exogenous carnitine supplies improve the germination and survival rates of seedlings grown on salt-enriched medium, in a manner comparable to proline. Both compounds are shown to improve seedling survival under oxidative constraint meaning that they may act on salt stress through antioxidant properties. A transcriptome analysis of seedlings treated with exogenous carnitine reveals that it modulates the expression of genes involved in water stress and abscisic acid responses. Analyses of the abscisic acid mutants, aba1-1 and abi1-1, indicate that carnitine and proline may act through a modulation of the ABA pathway.