Fast seed histology protocols: Benzene derivatives-free vs xylene-dependent

Introduction. Seeds are complex structures that allow the biological and crop propagation of plants. Seed histology can be used for teaching, researching, and for pathological diagnostic. Histology protocols are commonly divided into 5 different stages: fixation, processing, cutting, staining, and mounting. Xylene is a dangerous reagent used during the processing, staining, and mounting of histological specimens that can contaminate the environment and is toxic for users. Objective. To compare two new protocols for seed histology accelerated with microwave, tested on seeds of economic importance species. Materials and methods. The experiments were done between January and May of 2022 at the Centro de Investigaciones en Granos y Semillas (CIGRAS) of the Universidad de Costa Rica. The compared protocols were: a benzene derivatives-free (BDF) and a xylene-dependent (XD). Seeds of Carica papaya L. (Caricaceae) var. Pococí, Coffea arabica L. (Rubiaceae) var. Obata, Glycine max L. (Fabaceae) var. CIGRAS-06, Phaseolus vulgaris L. (Fabaceae) var. Tayni, Oryza sativa L. (Poaceae) var. Lazarroz FL, and Zea mays L. (Poaceae) var. EJN-2 were used. Three technical replicates of five seeds of each species were tested with the XD and BDF protocols, on different days each replicate. Results. The photomicrographs obtained with both protocols showed that the samples maintained the morphology integrity of embryo, endosperm or cotyledons, and other seed structures. BDF and XD protocols produced seed histology slides and microphotographs. PAS-Coomassie Blue staining made a good differentiation of carbohydrates and proteins. Fastness of both protocols is a benefit compared with other protocols for plant histology that can take several days or even weeks. Conclusions. The BDF and XD protocols were suitable for seed histology analysis of bean, coffee, maize, papaya, and soybean, slides were obtained in less than 5 hours. BDF protocol is the first for plant tissue processing that does not use benzene derivatives and that uses paraffin as embedding medium.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Centro para Investigaciones en Granos y Semillas (CIGRAS

Caracterización genética, genómica e histológica de una mutación sexual en Carica papaya (caricaceae)

La papaya es una planta frutal de gran importancia económica como cultivo agrícola. Se considera un modelo para el estudio de la determinación sexual debido a que puede tener hembras, hermafroditas y machos. Las flores de papaya pueden sufrir de carpelodia. Se encontró una mutación en una planta hermafrodita que produce unan mezcla de flores perfectas, femeninas e intermedias, cuyo fenotipo se heredó a lo largo de cuatro generaciones por medio de cruces y retrocruces. El objetivo de la investigación fue caracterizar aspectos genéticos, genómicos e histológicos de la mutación que revierte el sexo, de hermafrodita a femenino, en Carica papaya. Se encontró que macroscópica y microscópicamente las flores femeninas provenientes de plantas hermafroditas mutadas son iguales a las flores femeninas provenientes de plantas femeninas silvestres. El fenotipo mutado no segrega de forma mendeliana. Es posible que el fenotipo mutado sea causado por un cambio epigenético transgeneracional. Por medio del análisis de la secuenciación por tGBS se encontró un QTL en el cromosoma dos de la papaya que está correlacionado con el fenotipo mutado, este se ubica entre los 35.85 cM y los 42.81 cM. Además, se encontraron nueve marcadores moleculares correlacionados de forma significativa con el fenotipo mutado que permitieron realizar la diferenciación molecular de plantas mutantes y no mutantes. Se encontró que es posible el cambio fenotípico de una planta hermafrodita en una planta femenina, por lo que, es posible que el ancestro original de la papaya haya sido un organismo monoico. Es posible que los genes AGL8-related, el transportador GABA 1 y el NAD dependiente de epimerasa/dehidratasa se encuentren relacionados con la generación del fenotipo mutante ya que se encuentran dentro de la región del QTL encontrado. El estudio del transcriptoma y metiloma de esta mutación puede ayudar a identificar o comprobar de forma específica el o los genes que causan este fenotipo y, si estos están relacionados con la carpelodia, pueden ayudar al mejoramiento genético de líneas con carpelodia disminuida o nula.Fondo de Apoyo a Proyectos Finales de Graduación de Posgrado brindado por la Vicerrectoría de Investigación de la Universidad de Costa Rica.Sistema de Estudios de Posgrado de la Universidad de Costa Rica, por el Fondo de Becas CeNAT – CONARE.Universidad de Costa Rica/[734-B6-269]/UCR/Costa RicaUniversidad de Costa Rica/[736-B6-501]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Sistema de Estudios de Posgrado::Ciencias Agroalimentarias::Maestría Académica en Ciencias Agrícolas y Recursos Naturales con énfasis en Biotecnologí

Patrones de cantidad y distribución de cristales de oxalato de calcio en el tallo de Hylocereus costaricensis (Cactaceae) en respuesta a condiciones lumínicas en Liberia, Guanacaste, Costa Rica, en el 2018

La histocitotecnología vegetal permite realizar la descripción microscópica tisular y citológica de los componentes de una célula, estructura, órgano, u organismo vegetal, ya sea para fines anatómicos, funcionales, o moleculares Las técnicas funcionales de la histoquímica marcan un compuesto de forma más o menos específica. La técnica de Von Kóssa marca sales de calcio, por relaciones argentafines, que permitiría marcar, por ejemplo, el carbonato de calcio de los cistolitos, o el oxalato de calcio (CaOx) de los idioblastos. La pitahaya comprende un grupo de cactus originario de México, Centroamérica y Suramérica, que produce frutos en forma de bayas de color amarillo o púrpura de interés económico. Hylocereus es el género de pitahaya que se comercializa predominantemente en Costa Rica. Este género presenta hábitos epífitos en árboles vivos, rocas y muros de forma natural, y en plantaciones agrícolas para la producción de sus frutos. El objetivo de esta investigación es describir la cantidad y distribución de cristales de CaOx en el tallo de Hylocereus costaricensis como marcador de estrés abiótico de acuerdo con el grado de exposición solar durante febrero del 2018 a abril del 2018 en la finca El Salto de Liberia, Guanacaste, Costa Rica. Se encontró que la técnica de Von Kóssa modificada permitió el marcaje específico de cristales de CaOx y que, junto con el programa ImageJ, permitió la cuantificación por microfotografía de los cristales de CaOx. Además, se observó que el tejido en condiciones de sombra es el que tuvo una mayor cantidad de cristales de CaOx. Dentro de las zonas tisulares, la zona de la cumbre, que es la cresta superior de una costilla del cladodio de la pitahaya, fue la que tuvo la mayor cantidad de cristales de CaOx seguido de la corteza y de la médula. Esto coincidió con una mayor concentración de clorofilas y carotenoides en la zona de la cumbre, seguido de la corteza y de la médula. Esto sugiere que el CaOx puede tener la función de almacenamiento de carbono, además de otras funciones, como la protección contra herbivoría. Los cristales en el tejido se encontraron en una distribución agrupada, y presentaron diferentes morfologías: aciculares o rafidios, prismáticos rectangulares, romboides, cuadrados y tetragonales, que estaban formados por CaOx monohidrato, y drusas compactas, drusas compuestas por cristales prismáticos tetragonales, drusas tipo laberinto y drusas amorfas que estaban compuestas por CaOx dihidrato. Esto sugiere que la morfología de los cristales de CaOx se determina de forma multifactorial, por regulación de la concentración de los precursores del CaOx, por proteínas relacionadas a los cristales de CaOx y por la morfología de la cámara de los cristales de los idioblastos.UCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de Tecnologías en Salu

Embryo development in Carica papaya Linn

Proyecto interdisciplinarioPapaya (Carica papaya Linn.) is a tropical plant whose draft genome has been sequenced. Papaya produces large fruits rich in vitamins A and C and is an important cash crop in developing countries. Nonetheless, little is known about how the female gametophyte develops, how it is fertilized and how it develops into a mature seed containing an embryo and an endosperm. The Papaya female gametophyte displays a Polygonum-type architecture consisting of two synergid cells, an egg cell, a central cell, and three antipodal cells. Reports are available of the presumed existence of varieties in which cross fertilization is bypassed and autonomous development of embryos occurs (e.g., apomixis). In this study, we analyzed the development of female gametophytes in a commercial Hawaiian parental line and in the presumed apomictic Costa Rican line L1. Samples were collected before and after anthesis to compare the overall structure, size and transcriptional patterns of several genes that may be involved in egg and endosperm cell fate and proliferation. These genes were the putative papaya homologs of ARGONAUTE9 (AGO9), MEDEA (MEA), RETINOBLASTOMA RELATED-1 (RBR1), and SLOW WALKER-1 (SWA1). Our results suggest that its feasible to identify the contour of structural features of Polygonum-type development, and that in bagged female flowers of line L1 we might have observed autonomous development of embryo-like structures. Possible downregulation of papaya homologs for AGO9, MEA, RBR1 and SWA1 was observed in embryo sacs from line L1 before and after anthesis, which may suggest a tentative link between suspected apomixis and transcriptional downregulation of genes for RNA-directed DNA methylation, histone remodelers, and rRNA processing. Most notably, the large size of the papaya embryo sac suggests that it could be a cytological alternative to Arabidopsis thaliana for study. Significant variation in embryo sac size was observed between the varieties under study, suggesting wide differences in the genetic regulation of anatomical features.Universidad de Costa Rica/[736-B5-A13]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Estación Experimental Agrícola Fabio Baudrit Moreno (EEAFBM)UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de BiologíaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Jardín Botánico Lankester (JBL

Reproductive biology of Phragmipedium longifolium and floral anatomy associated with pollinators attraction in the genus

Phragmipedium is a genus with 21 species restricted to the tropics and subtropics from Central and South America. As it is a genus capable of producing seeds from both self- and cross-pollinated flowers, it is an ideal group to investigate the components of its reproductive success. In this research some of those components are described in P. longifolium as well as anatomical floral structures possibly related to the attraction of pollinators. To study the reproductive system in P. longifolium, manual self- and cross pollinations were carried out, and seed viability was measured by tetrazolium staining. No significant differences in fruit production between self- and cross-pollinated flowers were found. Higher seed viability was found in the self-pollinated flowers, which indicates that this species is frequently self-pollinated. Sections of the petals of P. humboldtii, P. longifolium, and P. warzewiczianum were stained with toluidine blue in order to identify and characterize anatomical structures possibly involved in the attraction of pollinators. Three kinds of glandular trichomes were observed on the petals of all three species, which are possibly involved in the secretion of fragrances to attract pollinators.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Jardín Botánico Lankester (JBL

<i>NTH2</i> 1271_1272delTA Gene Disruption Results in Salt Tolerance in <i>Saccharomyces cerevisiae</i>

Trehalose is a common energy reservoir, and its accumulation results in osmotic protection. This sugar can accumulate through its synthesis or slow degradation of the reservoir by trehalase enzymes. Saccharomyces cerevisiae contains two neutral trehalases, NTH1 and NTH2, responsible for 75% and 25% of the enzymatic metabolism. We were interested in the loss-of-function of both enzymes with CRISPR/Cas9. The later NTH2 was of great importance since it is responsible for minor metabolic degradation of this sugar. It was believed that losing its functionality results in limited osmotic protection. We constructed an osmotolerant superior yeast capable of growing in 0.85 M NaCl after independent nth21271_1272delTA mutation by CRISPR/Cas9 technology, compared with nth1 893_894insT and wild type. We suggest that this yeast model could give clues to breeding commercial yeast resulting in non-GMO salinity-tolerant strains

NTH2 1271_1272delTA Gene Disruption Results in Salt Tolerance in Saccharomyces cerevisiae

Trehalose is a common energy reservoir, and its accumulation results in osmotic protection. This sugar can accumulate through its synthesis or slow degradation of the reservoir by trehalase enzymes. Saccharomyces cerevisiae contains two neutral trehalases, NTH1 and NTH2, responsible for 75% and 25% of the enzymatic metabolism. We were interested in the loss-of-function of both enzymes with CRISPR/Cas9. The later NTH2 was of great importance since it is responsible for minor metabolic degradation of this sugar. It was believed that losing its functionality results in limited osmotic protection. We constructed an osmotolerant superior yeast capable of growing in 0.85 M NaCl after independent nth21271_1272delTA mutation by CRISPR/Cas9 technology, compared with nth1&nbsp;893_894insT and wild type. We suggest that this yeast model could give clues to breeding commercial yeast resulting in non-GMO salinity-tolerant strains

Reproductive biology of Phragmipedium longifolium and floral anatomy associated with pollinator attraction in the genus

Phragmipedium is a genus with 21 species restricted to the tropics and subtropics from Central and South America. As it is a genus capable of producing seeds from both self- and cross-pollinated flowers, it is an ideal group to investigate the components of its reproductive success. In this research some of those components are described in P. longifolium as well as anatomical floral structures possibly related to the attraction of pollinators. To study the reproductive system in P. longifolium, manual self- and cross-pollinations were carried out, and seed viability was measured by tetrazolium staining. No significant differences in fruit production between self- and cross-pollinated flowers were found. Higher seed viability was found in the self-pollinated flowers, which indicates that this species is frequently self-pollinated. Sections of the petals of P. humboldtii, P. longifolium, and P. warzewiczianum were stained with toluidine blue in order to identify and characterize anatomical structures possibly involved in the attraction of pollinators. Three kinds of glandular trichomes were observed on the petals of all three species, which are possibly involved in the secretion of fragrances to attract pollinators.Universidad de Costa Rica/[814-B5-200]/UCR/Costa RicaBosque de Paz Biological Reserve/[]//Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Jardín Botánico Lankester (JBL)UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Biologí

Phenotypic and genotypic characterization of virulence factors and susceptibility to antibiotics in Salmonella Infantis strains isolated from Chicken meat: First findings in Chile

Salmonella Infantis is a zoonotic pathogen that causes gastroenteritis in humans and animals, with poultry being its main reservoir. In Chile, there are no data to characterize S. Infantis strains in poultry production. In this study, 87 S. Infantis strains were isolated from chicken meat for sale in supermarkets in Santiago, Chile, and characterized according to their virulence genes, biofilm formation abilities, antibiotic susceptibility, and resistance genes. Through polymerase chain reaction or PCR, the strains were analyzed to detect the presence of 11 virulence genes, 12 antibiotic resistance genes, and integrase genes. Moreover, disc di usion susceptibility to 18 antimicrobials and the ability to form biofilm in vitro were evaluated. Results demonstrated six di erent virulence gene profiles. Ninety-four percent of the strains were multi-resistant to antibiotics with weak biofilm formation abilities, 63.2% of the strains were broad spectrum - lactam resistant, and the bla CTX-M-65 gene was amplified in 13 strains. Only 3.4% of the strains were fluoroquinolone resistant, and the qnrB gene was amplified in two strains. Colistin resistance was exhibited in 28.7% of the strains, but mrc genes were not amplified in any strain under study. The isolated S. Infantis strains are pathogenic and antibiotic multi-resistant, and thus, this Salmonella serotype should be under surveillance in the poultry food production chain with the aim of protecting public health.Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) CONICYT FONDEF ID18I1000

A Coffee Berry Borer (Coleoptera: Curculionidae: Scolytinae) Bibliography

Native to Africa, the coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae: Scolytinae), has gradually invaded most coffee-growing areas worldwide. Adult females colonize the coffee berry and oviposit within galleries in the coffee seeds. Larvae and adults consume the seeds, resulting in drastic reductions in yields and quality, negatively affecting the income of approximately 20 million coffee-growing families (~100 million people) in ~80 countries, with losses surpassing more than $500 million annually (Vega et al. 2015). It has become evident that the coffee berry borer scientific community could greatly benefit from having access to a bibliography of the literature related to the insect. Such an information source would allow scientists to find out what research areas have been explored throughout the many coffee berry borer-infested countries after more than 100 years of research on the topic. It could also help to direct lead future research efforts into novel areas, and away from topics and ideas that have been thoroughly investigated in the past