Regeneration of plants from somatic embryos of Verticillium dahliae-resistant wild olive genotypes

Regeneration capacity, via somatic embryogenesis, of several wild olive genotypes differing in their response to Verticillium wilt (resistant genotypes Stop Vert, Out Vert, Ac4 and Ac 18 and the susceptible Ac 15) has been evaluated. To induce somatic embryogenesis, methodologies previously used in cultivated (high ratio cytokinin/auxin) or wild olive (low ratio cytokinin/auxin) were used. Obtained results revealed the importance of genotype, explant type, mineral formulation and hormonal balance in the induction process, ca. use of apical buds obtained from micropropagated shoots following the methodology of Mazri et al. (2013) in cultivated olive (4 days in liquid medium MS ½, 30 µM TDZ – 0.54 µM ANA, 8 weeks in basal medium MS ½, followed by subculturing in ECO basal medium supplemented with 0.5 µM 2iP, 0.44 µM BA and 0.25 µM IBA) was adequate to obtain somatic embryos in 2 genotypes, Stop Vert and Ac18, but no embryogenic response was observed in the other three. An analysis of genetic stability on Stop Vert, using SSR and RAPDs markers, was carried out in embryogenic callus, plants regenerated form this callus and micropropagated shoots in comparison with the mother plant. Polymorphism was only observed in the banding pattern generated by RAPDs in one of the 10 callus samples evaluated, resulting in a variation rate of 0.07%. This is the first time in which plants have been regenerated via somatic embryogenesis in wild olive.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Research project: Junta de Andalucía P11-AGR799

Somatic embryogenesis in explants of adult wild olive trees

In this investigation, somatic embryogenesis in explants of adult wild olive trees, using the protocol developed by Mazri et al. (2013, Sci. Hort. 159: 88-95), was induced. Four genotypes differring in the level of resistance to the fungal pathogen Verticillium dahliae were used: Ac18, StopVert and OutVert (symptomless resistant genotypes, Jiménez-Fernández et al. 2015, Plant Pathology, in press) and Ac15 (susceptible genotype, Jiménez-Díaz, IAS-CSIC, Córdoba, personal communication). Three types of explants from actively growing cultures were used: first pair of leaves, petioles and shoot apex. Firstly, all explants were cultured on a liquid induction medium with MS mineral elements at 0.5X and 30 μM TDZ-0.5 μM NAA, for 4 days at 80rpm. Afterwards, explants were transferred to basal MS with 0.5X macroelements, for 8 weeks. Finally, calli were cultured on expression ECO medium supplemented with 0.25 μM IBA, 0.5 μM 2ip and 0.44 μM BA, for several subcultures. Cultures were incubated under darkness at 25 degrees. Embryogenic calli were observed on shoot apex (StopVert) or leaf primordia (Ac18) explants.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Bicriteria food packaging process optimization in double-layered upright and diagonal multihead weighers

[EN] Double-layered multihead weighing machines contain twice the number of hoppers as present in a simple machine with the same number of heads, which enables additional objective optimization possibilities considering the increased number of combinations among hoppers. This research study deals with bicriteria optimization for double-layered upright and diagonal machines using brute force as the optimization criteria. One of the optimization objectives is related to the target weight; the target weight must be at least and as close as possible to the weight to pack. Furthermore, this study also aims to minimize the time for which a certain portion of a product remains in the hopper while waiting to be selected for package formation. This time is known as priority and is measured based on the number of iterations or the number of packages produced by the machine while the hopper waits to be discharged. For these purposes, Different strategies were tested for both machines, which simultaneously optimize the target weight and the priority of the hoppers, showing the reduction of the extraction of the process in addition to reducing the costs of excess product and its reprocessing.We express our gratitude for the support from Universidad Simon Bolivar, Colombia, and Universitat Politecnica de Valencia.Garcia-Jimenez, R.; García-Díaz, JC.; Pulido-Rojano, AD. (2023). Bicriteria food packaging process optimization in double-layered upright and diagonal multihead weighers. Journal of Computational and Applied Mathematics. 428:1-10. https://doi.org/10.1016/j.cam.2023.11516811042

Packaging Process Optimization in MultiheadWeighers with Double-Layered Upright and Diagonal Systems

[EN] In multihead weighers, packaging processes seek to find the best combination of passage hoppers whose product content provides a total package weight as close as possible to its (nominal) label weight. The weighing hoppers arranged in these machines dispense the product quantity that each package contains through computer algorithms designed and executed for this purpose. For its part, in the packaging process for double-layered multihead weighers, all hoppers are arranged in two levels. The first layer comprises a group of weighing hoppers, and the second comprises a set of booster hoppers placed uprightly or diagonally to each weighing hopper based on design of the machine. In both processes, the initial machine configuration is the same; however, the hopper selection algorithm works differently. This paper proposes a new packaging process optimization algorithm for double-layer upright and diagonal machines, wherein the hopper subset combined has previously been defined, and the packaging weight is expressed as actual values. As part of its validation, product filling strategies were implemented for weighing hoppers to assess the algorithm in different scenarios. Results from the process performance metrics prove that the new algorithm improves processes by reducing variability. In addition, results reveal that some machine configurations were also able to improve their operation.We express our gratitude for the support from Universidad Simon Bolivar, and Universitat Politecnica de Valencia.Garcia-Jimenez, R.; García-Díaz, JC.; Pulido-Rojano, ADJ. (2021). Packaging Process Optimization in MultiheadWeighers with Double-Layered Upright and Diagonal Systems. Mathematics. 9(9):1-20. https://doi.org/10.3390/math9091039S1209

Regeneración de plantas, vía embriogénesis somática, a partir de material adulto de olivo silvestre

La embriogénesis somática es una herramienta poderosa para la clonación de genotipos de interés. En algunas especies como el olivo, esta técnica se ve limitada por las dificultades que presenta la regeneración a partir de material adulto. En este trabajo, se ha inducido embriogénesis somática a partir de material adulto de Olea europaea var. sylvestris siguiendo el protocolo desarrollado por Mazri et al. (Scient. Hort. 159: 88-95, 2013) para el cultivar de olivo Dahbia. Se emplearon 4 genotipos con distinto nivel de resistencia al patógeno fúngico Verticillium dahliae: AC18, StopVert and Outvert (genotipos resistentes) y AC15 (genotipo susceptible) (Jiménez-Díaz, IAS-CSIC, Córdoba, comunicación personal). Inicialmente, los explantos se cultivaron en oscuridad en medio líquido de inducción conteniendo la formulación mineral MS con los macroelementos a la mitad, y un suplemento de 30 µM TDZ-0.5 µM NAA durante 4 días, a 80 rpm; posteriormente, fueron transferidos al mismo medio sin reguladores del crecimiento. A las 8 semanas, el callo que proliferó se cultivó en medio de expresión ECO suplementado con 0.25 µM IBA, 0.5 µM 2iP y 0.44 µM BA, durante varios subcultivos. Sólo los explantos de yema apical del genotipo Stopvert formaron callo embriogénico con una frecuencia del 5%. Tras la multiplicación de este callo, una parte del mismo se transfirió a medio de maduración ECO con membranas de acetato de celulosa para la maduración de embriones. Los embriones maduros mostraron un porcentaje de germinación del 35%, lo que ha permitido la recuperación de plantas. La estabilidad genética del material obtenido se analizó mediante marcadores microsatélites. Se compararon plantas regeneradas a partir de embriones somáticos con el callo embriogénico del que procedían, plantas iniciadas a partir de yemas laterales y mantenidas in vitro mediante proliferación de axilares y la planta donante.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Heterologous expression of AtNPR1 gene in olive for increasing fungal tolerance

The NPR1 gene encodes a key component of SAR signaling mediated by salicylic acid (SA). After a pathogen infection, the accumulation of SA releases NPR1 monomers in the cytosol that are translocated to the nucleus, activating the expression of pathogenesis-related (PR) genes. Overexpression of NPR1 has conferred resistance to fungal, viral and bacterial pathogens in several plant species. The aim of this research was to generate transgenic olive plants expressing the gene AtNPR1 from Arabidopsis thaliana to obtain material resistant to fungal pathogens. Three transgenic lines expressing AtNPR1 gene under the control of the constitutive promoter CaMV35S were obtained following the protocol of Torreblanca et al. (2010), using an embryogenic line derived from a seed of cv. Picual. Level of AtNPR1 expression in transgenic calli varied greatly among the different lines, being higher in the line NPR1-780. The elicitation of embryogenic calli in liquid medium with AS did not increase endochitinase activity, a PR protein. However, jasmonic acid induced a transient increase in chitinase activity after 24 h of treatment in all the lines, being the increment higher in transgenic NPR1 than in control. After maturation and germination of transgenic somatic embryos, plants were micropropagated and acclimated to ex vitro conditions. The expression of AtNPR1 did not alter the growth of transgenic plants neither in vitro nor in the greenhouse. Experiments are in progress to determine the resistance of transgenic AtNPR1 plants to V. dalihae and R. necatrix.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Research projects: Plan Nacional AGL2014-52518-C2-1-R; AGL2017-83368-C2-1-R and Junta de Andalucía P11-AGR799

AOLI-- Adaptive Optics Lucky Imager: Diffraction Limited Imaging in the Visible on Large Ground-Based Telescopes

The highest resolution images ever taken in the visible were obtained by combining Lucky Imaging and low order adaptive optics. This paper describes a new instrument to be deployed on the WHT 4.2m and GTC 10.4 m telescopes on La Palma, with particular emphasis on the optical design and the expected system performance. A new design of low order wavefront sensor using photon counting CCD detectors and multi-plane curvature wavefront sensor will allow dramatically fainter reference stars to be used, allowing virtually full sky coverage with a natural guide star. This paper also describes a significant improvements in the efficiency of Lucky Imaging, important advances in wavefront reconstruction with curvature sensors and the results of simulations and sensitivity limits. With a 2 x 2 array of 1024 x 1024 photon counting EMCCDs, AOLI is likely to be the first of the new class of high sensitivity, near diffraction limited imaging systems giving higher resolution in the visible from the ground than hitherto been possible from space.Comment: SPIE vol 8446, 201

Generation of organotypic multicellular spheres by magnetic levitation : model for the study of human hematopoietic stem cells microenvironment

Q4Q3Background and Objective: The characteristics of human hematopoietic stem cells are conditioned by the microenvironment of the bone marrow, where they interact with other cell populations, such as mesenchymal stem cells and endothelial cells; however, the study of this microenvironment is complex. The objective of this work was to develop a 3D culture system by magnetic levitation that imitates the microenvironment of human HSC. Methods and Results: Human bone marrow-mesenchymal stem cells, umbilical cord blood-hematopoietic stem cells and a non-tumoral endothelial cell line (CC2811, LonzaⓇ) were used to develop organotypic multicellular spheres by the magnetic levitation method. We obtained viable structures with an average sphericity index greater than 0.6, an average volume of 0.5 mm3 and a percentage of aggregation greater than 70%. Histological studies of the organotypic multicellular spheres used hematoxylin and eosin stains, and an evaluation of vimentin expression by means of immunohistochemistry demonstrated an organized internal structure without picnotic cells and a high expression of vimentin. The functional capacity of human hematopoietic stem cells after organotypic multicellular spheres culture was evaluated by multipotency tests, and it was demonstrated that 3D structures without exogenous Flt3L are autonomous in the maintenance of multipotency of human hematopoietic stem cells. Conclusions: We developed organotypic multicellular spheres from normal human cells that mimic the microenvironment of the human hematopoietic stem cells. These structures are the prototype for the development of complex organoids that allow the further study of the biology of normal human stem cells and their potential in regenerative medicine.https://orcid.org/0000-0002-9152-5552https://orcid.org/0000-0003-3075-9854https://orcid.org/0000-0002-0084-0339https://orcid.org/0000-0003-1881-9367N/

The AOLI low-order non-linear curvature wavefront sensor: a method for high sensitivity wavefront reconstruction

The Adaptive Optics Lucky Imager (AOLI) is a new instrument under development to demonstrate near diffraction limited imaging in the visible on large ground-based telescopes. We present the adaptive optics system being designed for the instrument comprising a large stroke deformable mirror, fixed component non-linear curvature wavefront sensor and photon-counting EMCCD detectors. We describe the optical design of the wavefront sensor where two photoncounting CCDs provide a total of four reference images. Simulations of the optical characteristics of the system are discussed, with their relevance to low and high order AO systems. The development and optimisation of high-speed wavefront reconstruction algorithms are presented. Finally we discuss the results of simulations to demonstrate the sensitivity of the system.Comment: 10 pages. To be published in Proc SPIE 8447: Adaptive Optics Systems II

Transformación de olivo con el gen AtNPR1 para inducir tolerancia a patógenos fúngicos.

El gen NPR1 codifica un componente esencial de la respuesta SAR mediada por ácido salicílico (AS). Tras la infección por el patógeno, la acumulación de AS libera los monómeros NPR1 en el citoplasma, los cuáles son translocados al núcleo activando la expresión de genes relacionados con la patogénesis (PR). La sobreexpresión del gen NPR1 de Arabidopsis thaliana ha incrementado la resistencia a hongos, bacterias y virus, en distintas especies. El objetivo de esta investigación fue sobreexpresar este gen en olivo con objeto de evaluar su efecto en la tolerancia a dos hongos de suelo, el hemibiotrofo Verticillium dahliae (Vd), una de las mayores amenazas del cultivo y el necrotrofo Rosellinia necatrix, un patógeno emergente en nuevas plantaciones. Se obtuvieron 3 líneas transgénicas, a partir de una línea embriogénica derivada de semilla del cv. Picual. Las líneas mostraron diferencias en el nivel de expresión del transgen en hoja, aunque estas diferencias no afectaron a los niveles de actividad endoquitinasa basal, similar a la de plantas control. La respuesta a Vd varió con el patotipo; así, todas las plantas murieron 50 días tras su inoculación con la cepa defoliante (D) V-138. Por otra parte, la respuesta a patotipos no defoliantes (ND) también fue variable, en función de la raza; tras la inoculación con la cepa V1242 (ND, raza 2), los síntomas aparecieron transcurridos 44-55 días, siendo la línea NPR1-780, con mayor expresión del transgen, la que mostró menor índice de severidad de la enfermedad. Esta línea también mostró un comportamiento superior al control tras la inoculación con la cepa V1558 (ND, raza 1), aunque las diferencias no fueron tan acusadas. En la respuesta a R. necatrix, las líneas transgénicas mostraron un ligero retraso en el desarrollo de la enfermedad con valores AUDPC entre 7-15% inferiores al control.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech