Diversity of Bacillus thuringiensis strains isolated from coffee plantations infested with the coffee berry borer Hypothenemus hampei

The coffee berry borer Hypothenemus hampei Ferrari (Coleoptera: Scolytidae) was first reported infecting Costa Rican coffee plantations in the year 2000. Due to the impact that this plague has in the economy of the country, we were interested in seeking new alternatives for the biological control of H. hampei, based on the entomopathogenic bacteria Bacillus thuringiensis. Atotal of 202 B. thuringiensis isolates obtained from Costa Rican coffee plantations infested with H. hampei were analyzed through crystal morphology of the crystal inclusions and SDS-PAGE of d-endotoxins, while 105 strains were further evaluated by PCR for the presence cry, cyt and vip genes. Most of the Bt strains showed diverse crystal morphologies: pleomorphic (35%), oval (37%), bipyramidal (3%), bipyramidal and oval (12%), bipyramidal, oval and pleomorphic (10%) and bipyramidal, oval and cubic (3%). The SDS-PAGE analyses of the crystal preparations showed five strains with d-endotoxin from 20 to 40 kDa, six from 40 to 50 kDa, seven from 50 to 60 kDa, 19 from 60 to 70 kDa, 29 from 70 to 100 kDa and 39 from 100-145 kDa. PCR analyses demonstrated that the collection showed diverse cry genes profiles having several genes per strain: 78 strains contained the vip3 gene, 82 the cry2 gene, 45 the cry1 and 29 strains harbored cry3-cry7 genes. Atotal of 13 strains did not amplified with any of the cry primers used: cry1, cry2, cry3-7, cry5, cry11, cry12 and cry14. Forty-three different genetic profiles were found, mainly due to the combination of cry1A genes with other cry and vip genes. The genetic characterization of the collection provides opportunities for the selection of strains to be tested in bioassays against H. hampei and other insect pests of agricultural importance.En el año 2000 se reportó por primera vez la principal plaga del cafeto, conocida como broca (Hypothenemus hampei Ferrari) (Coleoptera: Scolitidae) en plantaciones de este cultivo en Costa Rica. Debido al impacto que esta plaga tiene en la economía del país, surgió la necesidad de encontrar una alternativa para el control biológico de esta plaga, basadas en la bacteria entomopatógena Bacillus thuringiensis. El objetivo de este trabajo fue aislar y caracterizar cepas de Bacillus thuringiensis a partir de plantaciones de café infestadas con H. hampei. Se aislaton 202 cepas que se analizaron mediante la morfología del cristal, SDS-PAGE de las d-endotoxinas, mientras que 105 cepas se evaluaron mediante PCR para determinar la presencia de genes cry, cyt y vip. La mayoría de las cepas presentaron diversas morfologías del cristal: pleomórficos (35%), ovalados (37%), bipiramidales (3%), bipiramidales y ovalados (12%), bipiramidales, ovalados y pleomórficos (10%) y bipiramidales, ovalados y cúbicos (3%). El análisis electorforético de las proteínas mostró que 5 cepas contenían d-endotoxinas con pesos moleculares entre los 20 y 40 kDa, 6 entre los 40 y 50 kDa, 7 entre los 50 y 60 kDa, 19 cepas entre los 60 y 70 kDa, 29 entre los 70 y 100 kDa y 39 cepas entre los 100 y 145 kDa. Los análisis mediante PCR mostró que la colección presenta una gran diversidad de genes cry, observándose varios genes por cepa: 78 cepas presentaron el gen vip3, 82 el gen cry2, 45 el gen cry1 y 29 cepas los genes cry3 y cry7. Un total de 13 cepas no amplificaron con los iniciadores cry1, cry2, cry3-7, cry5, cry11cry12 y cry14. Se encontraron 43 perfiles genéticos diferentes, detectándose principalmente la combinación de genes cry1A con otros genes cry o vip. La caracterización genética de esta colección provee información importante para la selección de cepas de Bacillus thuringiensis que se evaluarán mediante bioensayos contra Hypothenemus hampei u otras plagas de importancia económicaFundación para la Cooperación Costa Rica-Estados Unidos de América//CR-USA/Estados UnidosPrograma de Cooperación del Gobierno de MéxicoPrograma Iberoamericano de Ciencia y Tecnología para el Desarrollo//CYTED/EspañaUniversidad de Costa Rica//UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM

Direct somatic embryogenesis in Coffea arabica L. cvs. Caturra and Catuaí: effect of tricontanol, light condition, and medium consistency

The influence of triacontanol (TRIA) concentration and its interaction with indole-3- acetic acid (IAA) on direct somatic embryogenesis (DSE) of Coffea arabica L. cvs Caturra and Catuaí was determined. Additionally, light conditions and culture medium consistency (semisolid vs. liquid) were evaluated. A higher average of somatic embryos per explant from Caturra (3.9±0.5) and Catuaí (3.6±0.5) leaves was obtained when 4.55 μM TRIA was added to the half-strength Murashige and Skoog (1962) medium supplemented with 1.1 μM benzilaminopurine (BAP) and 2.85 μM IAA. In relation to medium consistency, the highest number of embryoids in Caturra (3.2±0.2) and Catuaí (6.0±0.4) explants was obtained using Yasuda et al. (1985) semisolid medium. Regarding photoperiod, the number of embryoids per explant obtained for Catuaí cultured under a 16 h light photoperiod was 7.6±1.0 and in the dark 6.2±0.6. In Caturra, 4.2±0.4 embryoids were produced in the dark and 3.8±0.5 with 16 h light. No somatic embryos were observed in Caturra and Catuaí explants after 12 weeks of culture in liquid Yasuda et al. (1985) medium, and under any of the light conditions.Se investigó la influencia de la concentración de triacontanol (TRIA) y su interacción con el ácido indolacético (AIA) en la inducción de la embriogénesis somá- tica directa en Coffea arabica L. cvs. Caturra y Catuaí. Adicionalmente, se evaluó el efecto de la condición de la luz y de la consistencia del medio de cultivo (semisólido vs. líquido). Se determinó que la mayor cantidad de embriones somáticos por explante fue de 3,9±0,5 en Caturra y 3,6±0,5 en Catuaí, en el medio de cultivo Murashige y Skoog (1962) al 50%, complementado con BAP (1,1 µM), IAA (2,85 µM) y TRIA (4,55 µM). Para la consistencia del medio, la mayor cantidad de embriones somáticos en Caturra (3,2±0,2) y Catuaí (6,0±0,4) se obtuvo con el medio semisó- lido de Yasuda et al. (1985). En Catuaí, la mayor cantidad de embrioides se obtuvo con 16 h luz (7,6±1,0) y oscuridad (6,2±0,6) y para Caturra, con oscuridad (4,2±0,4) y 16 h luz (3,8±0,5). No se observó la formación de embriones somáticos en los explantes de Caturra y Catuaí después de 12 semanas de cultivo, en el medio líquido descrito por Yasuda et al. (1985) ni bajo ninguna de las condiciones de luz

Comparison of three in vitro protocols for direct somatic embryogenesis and plant regeneration of coffea arabica L. CVS. Caturra and Catuaí

Comparación de tres protocolos para la embriogénesis somática directa y la regeneración de plantas de Coffea arabica L. cvs. Caturra y Catuaí in vitro. La presente investigación tuvo como objetivo establecer una metodología para la inducción de la embriogénesis somática directa en las variedades de café Caturra y Catuaí. Se observó un efecto del genotipo en la inducción de la embriogénesis somática directa; con la variedad Caturra se obtuvo un mayor número de embriones somáticos (2,71±0,45). Los explantes de Caturra provenientes de plantas de 3 meses mostraron una mejor respuesta a la inducción de la embriogénesis somática directa que los explantes provenientes de plantas de 12 meses. De los explantes provenientes de plantas de 12 meses de Catuaí rojo no se obtuvo embriones somáticos en ninguno de los medios de cultivo evaluados. No hubo diferencias significativas en el número de embriones somáticos producidos a partir de la primera y segunda hoja de vitroplantas y los explantes provenientes de la parte distal, media y basal de las hojas de plantas de café. El medio de cultivo Yasuda resultó el más indicado para inducir la embriogénesis somática y el desarrollo de embriones somáticos en plántulas en Caturra. Mientras que para Catuaí rojo, fue el de Hatanaka.A coffee (Coffea arabica L. cvs. Caturra and Catuaí) plant regeneration protocol via direct somatic embryogenesis was established. Vitroplant explants from first and second pair of leaves and explants from the distal, middle, and basal sections of 3 and 12 month-old plants were cultivated on the protocols described by Yasuda, Hatanaka and CATIE. Caturra somatic embryos were cultured on the same embryo induction media or DEV medium. The maximum number of somatic embryos (2.71±0.45) was obtained from Caturra vitroplants. Explants from 3 month-old plants showed better response than 12 month-old Caturra explants. No somatic embryos were obtained in the in vitro protocols evaluated with 12 month-old Catuaí rojo plants. No differences were observed on the number of embryos produced from the first and second of vitroplant leaves and sections of the distal, middle, and basal coffee plant leaves. The Yasuda protocol was the most efficient to induce direct somatic embryogenesis and embryo-toplant conversion for the Caturra variety, whereas Hatanaka’s was the most suitable for the Catuaí variety.UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Biologí

Plant regeneration via indirect somatic embryogenesis and optimisation of genetic transformation in Coffea arabica L. cvs. Caturra and Catua\ued

A protocol for Coffea arabica L. cvs. Caturra and Catua\ued plant regeneration via indirect somatic embryogenesis (ISE) was established. Furthermore, a biolistic mediated genetic transformation protocol was optimized for Catua\ued callus aggregates. Maximum callus induction was obtained when Caturra (87%) and Catua\ued (67%) leaves were cultured on Murashige and Skoog medium with 18.56 \u3bcM kinetin and 4.52 \u3bcM2,4-dichlorophenoxyacetic acid (2,4-D). Catua\ued suspension cultures were established from embryogenic callus using liquid proliferation CP and Sli media and diffused light and darkness. The higher suspension cultures fresh weight was obtained using Erlenmeyer (1425.4 \ub1 354.9 mg) than Recipient for Automated Temporary Immersion System (RITA\uae) (518.6 \ub1 55.1 mg), whereas the dry weight of suspension cultures was not significantly affected by the culture system used. Higher number of embryos per vessel (307.6 \ub1 49.0) and their fresh weight (9.6 \ub1 1.5 mg) were obtained with semisolid R medium than S3 medium. The highest somatic embryo development (25.0 \ub1 2.7) and fresh weight (780.0 \ub1 85.4 mg) were obtained with 1 min of immersion every 8 hrs. Higher fresh weight of regenerated plantlets was obtained with liquid Yasuda medium in RITA\uae (124.6 \ub1 16.3 mg) than semisolid media (36.3 \ub1 11.3 mg). For genetic transformation, the effect of helium pressure (900 and 1550 psi), and target distance (9 and 12 cm) and plasmid (pCAMBIA 1301, pCAMBIA 1305.2 and pCAMBIA 1301-BAR) on transient uidA expression Catua\ued suspension cultures were evaluated. The highest number of blue spots was obtained using 900 psi and 9 cm (125.8 \ub1 17.3). Stable uidA expression was observed on Catua\ued callus aggregates transformed with pCAMBIA 2301 and cultured on 100 mg l-1 of kanamycin

Adapting Agriculture to Climate Change: A Synopsis of Coordinated National Crop Wild Relative Seed Collecting Programs across Five Continents

The Adapting Agriculture to Climate Change Project set out to improve the diversity, quantity, and accessibility of germplasm collections of crop wild relatives (CWR). Between 2013 and 2018, partners in 25 countries, heirs to the globetrotting legacy of Nikolai Vavilov, undertook seed collecting expeditions targeting CWR of 28 crops of global significance for agriculture. Here, we describe the implementation of the 25 national collecting programs and present the key results. A total of 4587 unique seed samples from at least 355 CWR taxa were collected, conserved ex situ, safety duplicated in national and international genebanks, and made available through the Multilateral System (MLS) of the International Treaty on Plant Genetic Resources for Food and Agriculture (Plant Treaty). Collections of CWR were made for all 28 targeted crops. Potato and eggplant were the most collected genepools, although the greatest number of primary genepool collections were made for rice. Overall, alfalfa, Bambara groundnut, grass pea and wheat were the genepools for which targets were best achieved. Several of the newly collected samples have already been used in pre-breeding programs to adapt crops to future challenges.info:eu-repo/semantics/publishedVersio

Relación de los cultivos modificados genéticamente con el ambiente y la salud de la población costarricense

La ingeniería genética y los alimentos derivados de los cultivos genéticamente modificados (CGMs) han sido objeto de debate a escala mundial, como ocurre históricamente con el surgimiento de tecnologías novedosas. Se cuestiona si los CGMs son seguros al ambiente y si los productos derivados de ellos son inocuos para los consumidores. Sobre la primera de esas inquietudes, la práctica se ha orientado a estudiar caso por caso, según las propias características del CGM, para minimizar su impacto en el ambiente. Estudios científicos en diversas latitudes han demostrado que no ha habido efectos dañinos en este particular. En cuanto a los alimentos derivados de los CGMs se han desarrollado sistemas de evaluación rigurosa para permitir su consumo y comercialización, y hay protocolos aprobados por la FAO y la OMS para garantizar su inocuidad. Hasta el momento, en ningún caso se han detectado contraindicaciones para la salud humana en los productos disponibles hoy en el mercado. Por su parte, Costa Rica estableció desde los años 90 un marco regulatorio en bioseguridad que se encarga de garantizar la seguridad para la salud y el ambiente en el manejo de los CGMs. Asimismo, se ha hecho una gran inversión estatal y privada en esta área, lo que le permitió a Costa Rica posicionarse en la región como uno de los primeros países con fortalezas en materia de bioseguridad y de investigación en ingeniería genética en centros de investigación nacionales. Cualquier intento de restringir o prohibir dicha actividad pondría en riesgo esa inversión, afectaría la generación de nuevo conocimiento para la toma de decisiones y el liderazgo alcanzado en el campo y nos privaría de los beneficios de esta prometedora tecnología.Relationship of genetically modified crops with the environment and health of the Costa Rican human population. Genetic engineering and the food derived from genetically modified crops (GMCs) have been the center of debate worldwide, as has occurred historically with the advent of new technologies. Questions are derived from the potential impact of GMCs to the environment and the safety of the products to the consumers. In relation to the first inquiry, practice has been oriented to a case-by-case-study, according to the own characteristics of the GMC, in order to minimize its impact in the environment. Scientific studies in diverse latitudes of the world have demonstrated that GMCs in the market showed no adverse effects related to this issue. In relation to food derived from the GMCs, rigorous evaluation protocols have been developed and approved by FAO and WHO to guarantee the innocuousness of these products. Up to the moment, no contraindications for human health have been pointed out for the products that are available today in the market. In the particular case of Costa Rica, the country has established since the 90s a regulatory biosafety framework for the management of the GMCs, safeguarding the biodiversity of the country and the health of consumers. At the same time the country has made significant public and private investments in the field that allowed the country to obtain a leading position in biosafety in the region and genetic engineering research at national research centers. Any attempt to restrict or prohibit these activities in the country, will put in risk the previously described investment, will affect the generation of new knowledge for decision making and the leadership in the field, preventing the benefits derived from this promising technology. Rev. Biol. Trop. 52(3): 727-732. Epub 2004 Dic 15

Relationship of genetically modified crops with the environment and health of the Costa Rican human population

La ingeniería genética y los alimentos derivados de los cultivos genéticamente modificados (CGMs) han sido objeto de debate a escala mundial, como ocurre históricamente con el surgimiento de tecnologías novedosas. Se cuestiona si los CGMs son seguros al ambiente y si los productos derivados de ellos son inocuos para los consumidores. Sobre la primera de esas inquietudes, la práctica se ha orientado a estudiar caso por caso, según las propias características del CGM, para minimizar su impacto en el ambiente. Estudios científicos en diversas latitudes han demostrado que no ha habido efectos dañinos en este particular. En cuanto a los alimentos derivados de los CGMs se han desarrollado sistemas de evaluación rigurosa para permitir su consumo y comercialización, y hay protocolos aprobados por la FAO y la OMS para garantizar su inocuidad. Hasta el momento, en ningún caso se han detectado contraindicaciones para la salud humana en los productos disponibles hoy en el mercado. Por su parte, Costa Rica estableció desde los años 90 un marco regulatorio en bioseguridad que se encarga de garantizar la seguridad para la salud y el ambiente en el manejo de los CGMs. Asimismo, se ha hecho una gran inversión estatal y privada en esta área, lo que le permitió a Costa Rica posicionarse en la región como uno de los primeros países con fortalezas en materia de bioseguridad y de investigación en ingeniería genética en centros de investigación nacionales. Cualquier intento de restringir o prohibir dicha actividad pondría en riesgo esa inversión, afectaría la generación de nuevo conocimiento para la toma de decisiones y el liderazgo alcanzado en el campo y nos privaría de los beneficios de esta prometedora tecnología.Genetic engineering and the food derived from genetically modified crops (GMCs) have been the center of debate worldwide, as has occurred historically with the advent of new technologies. Questions are derived from the potential impact of GMCs to the environment and the safety of the products to the consumers. In relation to the first inquiry, practice has been oriented to a case-by-case-study, according to the own characteristics of the GMC, in order to minimize its impact in the environment. Scientific studies in diverse latitudes of the world have demonstrated that GMCs in the market showed no adverse effects related to this issue. In relation to food derived from the GMCs, rigorous evaluation protocols have been developed and approved by FAO and WHO to guarantee the innocuousness of these products. Up to the moment, no contraindications for human health have been pointed out for the products that are available today in the market. In the particular case of Costa Rica, the country has established since the 90s a regulatory biosafety framework for the management of the GMCs, safeguarding the biodiversity of the country and the health of consumers. At the same time the country has made significant public and private investments in the field that allowed the country to obtain a leading position in biosafety in the region and genetic engineering research at national research centers. Any attempt to restrict or prohibit these activities in the country, will put in risk the previously described investment, will affect the generation of new knowledge for decision making and the leadership in the field, preventing the benefits derived from this promising technology

Optimization of coffee (Coffea arabica) transformation parameters using uidA and hpt genes: effect of osmotic pre-treatment, helium pressure and target distance

The aim of this work was to optimize the biolistic delivery parameters that affect the DNA delivery and stable expression of marker genes into coffee tissues (Coffea arabica. L. cvs. Caturra and Catuaí). The effect of osmotic preculture length, osmotic concentration of medium, Helium pressure and target distance on transient expression of the uidA gene in coffee leaves and somatic embryos were tested. The highest transient uidA expression was obtained when Caturra (18.3±2.8) and Catuaí (6.8±2.0) leaves and Catuaí embryos (80.0±7.4) were cultured for 5h on Yasuda medium complemented with 0.5M Mannitol +0.5M Sorbitol. The combination of 1100psi and a target distance of 9cm resulted in the highest number of blue spots per Caturra leaf segment (23.6±3.9), whereas for the Catuaí variety the combination of 1100psi and a target distance of six (10.2±1.9) and nine (8.2±1.9) cm gave the highest number of blue spots per leaf segment. The optimized protocol was tested with pCAMBIA 1 301 (uidA gene and the hpt gene), pCAMBIA 1 305.2 (uidA version GUSPlus ™ and the hpt gene) and pCAMBIA 1 301-BAR (uidA gene and the bar gene). The highest number of blue spots was obtained when Caturra (54.6±5.7) and Catuaí (28.9±4.3) leaves were bombarded with pCAMBIA 1 305.2. Selection of bombarded coffee tissues with 100mg/l hygromicyn caused the oxidation of tissues. Rev. Biol. Trop. 57 (Suppl. 1): 151-160. Epub 2009 November 30.<br>La presente investigación tuvo como objetivo optimizar los parámetros que afectan la incorporación y expresión de genes marcadores mediante biobalística en segmentos de hoja y embriones somáticos de café (Coffea arabica. L. cvs. Caturra y Catuaí). La mayor expresión transitoria del gen uidA en segmentos de hoja de Caturra (18.3±2.8) y Catuaí (6.8±2.0) y embriones somáticos de Catuaí (80.0±7.4) se obtuvo al cultivar los explantes por cinco horas previo al bombardeo en el medio Yasuda complementado con 0.5M mannitol+0.5M sorbitol. Asimismo, se obtuvo una mayor expresión transitoria del gen uidA al bombardear los segmentos de hoja de Caturra y Catuaí y embriones somáticos de Catuaí con una presión de helio de 1 100psi y una distancia de bombardeo de 6 o 9 cm

Direct somatic embryogenesis in Coffea arabica L. cvs. Caturra and Catuaí: Effect of triacontanol, light condition, and medium consistency

The influence of triacontanol (TRIA) concentration and its interaction with indole-3- acetic acid (IAA) on direct somatic embryogenesis (DSE) of Coffea arabica L. cvs Caturra and Catuaí was determined. Additionally, light conditions and culture medium consistency (semisolid vs. liquid) were evaluated. A higher average of somatic embryos per explant from Caturra (3.9±0.5) and Catuaí (3.6±0.5) leaves was obtained when 4.55 µM TRIA was added to the half-strength Murashige and Skoog (1962) medium supplemented with 1.1 µM benzilaminopurine (BAP) and 2.85 µM IAA. In relation to medium consistency, the highest number of embryoids in Caturra (3.2±0.2) and Catuaí (6.0±0.4) explants was obtained using Yasuda et al. (1985) semisolid medium. Regarding photoperiod, the number of embryoids per explant obtained for Catuaí cultured under a 16 h light photoperiod was 7.6±1.0 and in the dark 6.2±0.6. In Caturra, 4.2±0.4 embryoids were produced in the dark and 3.8±0.5 with 16 h light. No somatic embryos were observed in Caturra and Catuaí explants after 12 weeks of culture in liquid Yasuda et al. (1985) medium, and under any of the light condition