RESUMO
J. curcas has been studied in different countries and some interesting agronomic, pharmacological and industrial properties have been reported. More recently, it has been considered an important alternative source for biofuel production. The objective of this study was to establish a long-term method for the maintenance of calli and cell suspension cultures of the local species J. curcas and J. gossypifolia, in order to allow future studies for novel compounds with pharmaceutical or industrial applications. For this, friable calli were successfully induced from hypocotyl segments of.. curcas and J. gossypifolia that were cultured in semisolid MS media supplemented with 1.5 mg/L, and 0.5 mg/L of 2,4-D, respectively. Cell suspension cultures of J. curcas were established using 1 g of 35 and 60-day calli, in 50 mL of liquid MS media supplied with 1.5 mg/L of 2,4-D; sucrose and maltose were additionally evaluated as carbon sources. After 35 days, cell suspension cultures initiated with 35-day calli, showed greater cell growth with a maximum biomass of 194.9 g/L fresh weight, 6.59 g/L dry weight and 17.3% packed volume. The exponential phase ended at day 35 for cultures initiated with 35-day calli, and at day 21 for cultures initiated with 60-day calli. Higher biomass production was obtained with sucrose. Cell cultures were established with 35-day calli in MS media with the same 2,4-D concentration used for calli induction and 30g/L sucrose. This medium was considered optimum for the maintenance and growth of cell suspensions for both species, with sub-cultures every 20 days. The biotechnological potential for the production of bioactive compounds in these species for pharmacological, agricultural and industrial applications is being evaluated.
Assuntos
Técnicas de Cultura de Células/métodos , Jatropha/crescimento & desenvolvimento , Ácido 2,4-Diclorofenoxiacético/administração & dosagem , Biomassa , Jatropha/efeitos dos fármacos , Maltose/administração & dosagem , Sacarose/administração & dosagem , Suspensões , Fatores de TempoRESUMO
Abstract The influence of silver nitrate (AgNO3), benzyladenine (BAP), and indole-3-acetic acid (IAA) on low frequency somatic embryogenesis (LFSE) induction in Caturra and Catuaí arabica coffee was evaluated. For the Caturra cultivar, the production of somatic embryos was significantly increased by adding AgNO3 to the semisolid culture medium. The highest average number of somatic embryos for this cultivar was obtained using 6.6 μM BAP, 2.85 μM IAA, and 40 μM AgNO3. In contrast, for the Catuaí cultivar, the highest average number of somatic embryos was obtained using semisolid medium supplemented with 8.8 μM BAP, and 2.85 μM IAA. Using these protocols, somatic embryos were directly induced using leaf sections of in vitro plants of both coffee cultivars within 8 weeks. The somatic embryos developed into rooted plants with a 100% survival rate upon transfer to the greenhouse.
Assuntos
Reguladores de Crescimento de Plantas , Sementes/química , Nitrato de Prata/administração & dosagem , Coffea , Técnicas de Cultura de TecidosRESUMO
Intensive exploitation of mahogany wood (Swietenia macrophylla, Meliaceae) has resulted in the loss of natural populations. Somatic embryogenesis offers an alternative to clonal propagation and conservation of mahogany. This study describes biochemical (carbohydrates, total phenols, total flavonoids, protein, and plant growth regulators content) and histological characteristics of the somatic embryogenesis process in mahogany. Calli were obtained by culturing cotyledons of seeds from immature fruits for six weeks on semi-solid MS medium supplemented with 1.0 mgL-1 of kinetin and 4.0 mgL-1 of 2, 4-D. Primary callus was cultured on half strength semi-solid MS medium supplemented with 1.0 mgl-1 6-BA (6-benzylaminopurine) and embryogenic structures were obtained. Embryo development from globular-shaped somatic embryos to the cotyledonary stage was confirmed by histology and scanning electron microscopy. Shoot initiation was observed after somatic embryos were transferred to germination and maturation medium. Endogenous concentrations of carbohydrates, total phenols, total flavonoids, protein, and plant growth regulators were determined in embryogenic (EC) and non-embryogenic (NEC) calli of mahogany. Embryogenic cultures contained significantly higher concentrations of IAA (indoleacetic acid), ABA (abscisic acid), and GAs (Gibberellins 1+3+20), whereas non-embryogenic calli contained more total phenols, flavonoids and resistant starch. Fructose and glucose were not present at detectable levels in EC or NEC, whereas soluble starch and sucrose were only detectable in EC. Concentrations of total proteins, Z/ZR (Zeatin/zeatin riboside) and iP/iPA (N6-(Δ2-isopentenyl) adenine and N6-(Δ2-isopentenyl) adenosine) were similar in EC and NEC.
La explotación intensiva de la madera de caoba (Swietenia macrophylla) ha provocado la pérdida de poblaciones naturales. La embriogénesis somática ofrece una alternativa a la propagación clonal y la conservación de esta especie. Este estudio describe las características bioquímicas (contenido de carbohidratos, fenoles totales, flavonoides totales, proteínas y reguladores del crecimiento) e histológicas del proceso de embriogénesis somática en caoba. Los callos se obtuvieron cultivando cotiledones de semillas de frutos inmaduros durante seis semanas en medio MS semisólido suplementado con 1.0 mgL-1 de kinetina y 4.0 mgL-1 de 2, 4-D. Luego se cultivó el callo primario en medio MS semisólido y un suplemento de 1.0 mgl-1 BA y se obtuvieron estructuras embriogénicas. El desarrollo de embriones somáticos de forma globular a la etapa cotiledonar se confirmó por histología y microscopía electrónica de barrido. La iniciación del brote se observó después de que los embriones somáticos se transfirieron a un medio de germinación y maduración. Se determinaron las concentraciones endógenas de carbohidratos, fenoles totales, flavonoides totales, proteínas y reguladores del crecimiento en callos embriogénicos (EC) y no embriogénicos (NEC) de caoba. Los cultivos embriogénicos contenían concentraciones significativamente más altas de IAA, ABA y GA, mientras que los callos no embriogénicos contenían más fenoles totales, flavonoides y almidón resistente. La fructosa y la glucosa no estaban presentes en niveles detectables en EC o NEC, mientras que el almidón soluble y la sacarosa solo se detectaron en el EC. Las concentraciones de proteínas totales, Z / ZR e iP / iPA fueron similares en EC y NEC.
RESUMO
J. curcas has been studied in different countries and some interesting agronomic, pharmacological and industrial properties have been reported. More recently, it has been considered an important alternative source for biofuel production. The objective of this study was to establish a long-term method for the maintenance of calli and cell suspension cultures of the local species J. curcas and J. gossypifolia, in order to allow future studies for novel compounds with pharmaceutical or industrial applications. For this, friable calli were successfully induced from hypocotyl segments of J. curcas and J. gossypifolia that were cultured in semisolid MS media supplemented with 1.5mg/L, and 0.5mg/L of 2,4-D, respectively. Cell suspension cultures of J. curcas were established using 1g of 35 and 60-day calli, in 50mL of liquid MS media supplied with 1.5mg/L of 2,4-D; sucrose and maltose were additionally evaluated as carbon sources. After 35 days, cell suspension cultures initiated with 35-day calli, showed greater cell growth with a maximum biomass of 194.9g/L fresh weight, 6.59g/L dry weight and 17.3% packed volume. The exponential phase ended at day 35 for cultures initiated with 35-day calli, and at day 21 for cultures initiated with 60-day calli. Higher biomass production was obtained with sucrose. Cell cultures were established with 35-day calli in MS media with the same 2,4-D concentration used for calli induction and 30g/L sucrose. This medium was considered optimum for the maintenance and growth of cell suspensions for both species, with sub-cultures every 20 days. The biotechnological potential for the production of bioactive compounds in these species for pharmacological, agricultural and industrial applications is being evaluated.
J. curcas es un importante recurso alternativo de biocombustible. Por otro lado, propiedades de interés agronómico, farmacológico e industrial han sido reportadas para esta especie. El objetivo de este estudio fue el establecimiento y mantenimiento a largo plazo de callos y cultivos celulares en suspensión de J. curcas y J. gossypifolia, con el objetivo de permitir futuros estudios para nuevos compuestos con aplicaciones farmaceúticas e industriales. Los callos friables fueron exitosamente inducidos a partir de segmentos de hipocótilos J. curcas and J. gossypifolia cultivados en medio MS semisólido suplementado con 1.5mg/L y 0.5mg/L of 2,4-D, respectivamente. Los cultivos celulares en suspensión de J. curcas fueron establecidos utilizando 1g de callos de 35 y 60 días de edad en 50mL de medio MS líquido adicionado con 1.5mg/L de 2,4-D. Después de 35 días, los cultivos en suspensión celular iniciados con callos de 35 días, mostraron mayor crecimiento celular con una biomasa máxima de 194.9g/L de peso fresco y 6.59g/L de peso seco y 17.3% de volumen empacado. La fase exponencial finalizó al día 35 en los cultivos iniciados con callos de 35 días, y al día 21 en los cultivos iniciados con callos de 60 días. Dos fuentes de carbono fueron evaluadas: sacarosa y maltosa. La producción de mayor biomasa fue obtenida con sacarosa. Los cultivos celulares se establecieron con callos de 35 días cultivados en medio MS con la misma concentración de 2,4-D utilizada para la inducción de callos y 30g/L de sacarosa. Este medio fue considerado el óptimo para el mantenimiento y crecimiento de suspensiones celulares en ambas especies con subcultivos cada 20 días. El potencial biotecnológico para la producción de compuestos bioactivos en estas especies, para aplicaciones farmacológicas, agrícolas e industriales está siendo evaluado.
Assuntos
Técnicas de Cultura de Células/métodos , Jatropha/crescimento & desenvolvimento , Biomassa , Jatropha/efeitos dos fármacos , Maltose/administração & dosagem , Suspensões , Sacarose/administração & dosagem , Fatores de Tempo , /administração & dosagemRESUMO
A method for regeneration of the commercially important common bean (Phaseolus vulgaris ) using N6-benzylaminopurine(BAP) and adenine sulphate (AS) was established. Embryogenic axes of the Costa Rican common bean cultivars Bribrí, Brunca, Guaymí, Huetar and Telire were cultured on Murashige and Skoog medium supplemented with 100 mgl-1 myo-inositol, 1 mgl-1 thiamine, 30 gl-1 sucrose, BAP (0, 5 and 10 mgl-1), AS (0, 20 and 40 mgl-1) and 8 gl-1 agar. Regardless of the concentration of BAP and AS in the induction medium, the number of shoots and leaves differed significantly among the common bean cultivars evaluated. The higher average of shoots was obtained for Brunca > Telire > Bribrí > Guaymí > Huetar. Moreover, independently of the cultivar, the induction medium supplemented with 5 mgl-1 BAP and 20 or 40 mgl-1 AS resulted in the higher average of shoots formation. Culture of Bribrí, Brunca, Guaymí, Huetar and Telire embryogenic axes on induction medium supplemented with different BAP and AS resulted in a differential response. Successful acclimatization of common bean in vitro plants were achieved in the greenhouse, and plants appeared morphologically normal. The regeneration system developed in this investigation for this important crop could be a useful tool for the genetic modification through mutagenesis or genetic transformation.
Assuntos
Phaseolus/anatomia & histologia , Phaseolus , Phaseolus/metabolismo , Adenina/administração & dosagem , Adenina/uso terapêutico , Estruturas Vegetais , Sulfatos/metabolismoRESUMO
Rice (Oryza sativa cv. 5272) embryogenic calli were obtained from mature zygotic embryos culture on Murashige & Skoog (1962) medium supplemented with 2.5 mg/l 2,4- dichlorophenoxyacetic acid. Histological analysis of somatic embryogenesis revealed that after two weeks of culture of explants on the callus induction medium, somatic embryo development began with a cluster of proembryogenic cells in the peripheral region of the calli. The outer cell layer of embryogenic calli consisted of small and isodiametric cells with a dense cytoplasm and a prominent nucleus and nucleolus; whereas the inner cell layer is composed of large cells with small nucleus and large vacuole. These embryogenic cells underwent a series of organized divisions and formed the proembryo with a well-defined protodermis. Rev. Biol. Trop. 57 (Suppl. 1): 141-150. Epub 2009 November 30.
Los estudios anatómicos e histológicos de los callos embriogénicos de arroz (Oryza sativa) mostraron que estos se originan del epitelio escutelar de los embriones cigóticos maduros. Al crecer en un medio Murashige y Skoog (1962) suplementado con 2.5 mg/l 2,4-D, presentan grupos de células embriogénicas en las zonas periféricas, las cuales a su vez darán lugar a la formación de proembriones y de embriones somáticos. Las células de los callos embriogénicos se caracterizan por tener núcleo y nucleolo conspícuos, forma isodiámetrica, citoplasma denso y están acompañadas de células adyacentes con abundantes gránulos de almidón. Los embriones somáticos completan su desarrollo para dar formación a plántulas completas, al estar en un medio de regeneración. Los estudios histológicos permitieron observar la expresión transitoria del gen uidA en grupos de células de las capas más externas de los callos embriogénicos sometidos al método de transformación genética por biobalística.
Assuntos
Oryza/embriologia , Desenvolvimento Embrionário , Técnicas de Embriogênese Somática de Plantas , Costa Rica , Histologia , MorfogêneseRESUMO
An efficient and reproducible method for regeneration of commercial and pure lines of tropical butternut squash (Cucurbita moschata) plants via somatic embryogenesis was developed. The influence of genotype, explant source, N6-benzylaminopurine (BAP), 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) concentration on somatic embryogenesis induction was investigated. Friable embryogenic calli was produced from zigotic embryos (53-56%) and cotyledons from seedlings (70%) of C. moschata cv. Sello de Oro cultured on callus induction medium (CIM) supplemented with 0.5 mg/l or 3.5 mg/l 2,4-D. No embryogenic calli was obtained from leaf segments of C. moschata cv. Sello de Oro cultured on CIM supplemented with different concentrations of BAP and 2,4-D and cotyledons from seedlings of C. moschata cv. PVG 04 cultured on CIM with BAP and 2,4,5-T. Embryogenic calli induction was achieved in 75% C. moschata pure lines evaluated and calli percentage frequency range from 5% to 34%. Successful acclimatization of squash in vitro plants was achieved in the greenhouse and in the field. Regenerated plants appeared morphologically normal and set flowers and fruits with seeds that could germinate normally. Rev. Biol. Trop. 57 (Suppl. 1): 119-127. Epub 2009 November 30.
En este estudio se desarrolló un método eficiente y reproducible para la regeneración de líneas puras de la planta tropical Cucurbita moschata mediante la vía de embriogénesis somática. Además se investigó acerca de la influencia del genotipo, transplante, y la concentración de N6-benzylaminopurina (BAP), 2,4-diclorofenoxyacetico ácido (2,4-D) y 2,4,5-triclorofenoxyacetico ácido (2,4,5-T) en la inducción de embriogénesis somática. Los callos embriogenéticos viables fueron producidos de embriones zigóticos (53-56%) y cotiledones de semillas (70%) de C. moschata cv. Sello de Oro cultivados en un medio de inducción de callos (CIM) suplementado con 0.5 mg/l o 3.5 mg/l 2,4-D. Los callos no embriogénicos fueron obtenidos de segmentos de hojas de C. moschata cv. Sello de Oro cultivados con CIM suplementado con diferentes concentraciones de BAP y 2,4-D y cotiledones de semillas de C. moschata cv. PVG 04 cultivado con CIM con BAP y 2,4,5-T. La inducción de callos embriogenéticos fue exitosa en un 75% de las líneas puras evaluadas de C. moschata y el porcentaje de frecuencia de los callos fue de 5% a 34%. Se logró una adecuada aclimatización de las plantas in vitro tanto en el invernadero como en el campo. Las plantas regeneradas fueron normales morfológicamente, y las flores y frutos poseen semillas que pueden germinar normalmente.
Assuntos
Regeneração , Técnicas In Vitro , Cucurbita/classificação , Desenvolvimento Embrionário , Costa Rica , Genótipo , Ácidos IndolacéticosRESUMO
Shoot tips of maize (Zea mays L.) were cultured on Murashige and Skoog medium supplemented with 2 mg/l BA +1 mg/l 2,4-D +40 mg/l, to investigate phases of ontogenetic development. The study used light microscopy as well as scanning and transmission electronic microscopy techniques. Shoot tips of maize are composed of small cells with a dense cytoplasm and a prominent nucleus. The process of organogenesis began with swelling of the shoot tip, as the first evidence of organogenic calli formation observed three weeks after culture get started. There were two morphologically different types of cells within the organogenic calli. The layer consisted of large cells with small nucleus, free-organelle cytosol, irregular plasmatic membrane, trichome-like structures, and thick cell walls. In the inner cell layer, small and isodiametric cells with a prominent nucleus, small vacuoles, endoplasmatic reticulum, Golgi, mitochondrias and chloroplasts were observed. The presence of trichomes in the more active morphogenic zones could indicate an organogenic potential. Rev. Biol. Trop. 57 (Suppl. 1): 129-139. Epub 2009 November 30.
Los ápices de vástagos de maíz (Zea mays L.) fueron cultivados con el medio Murashige y Skoog, utilizando como suplemento 2 mg/l BA +1 mg/l 2,4-D +40 mg/l, con el fin de investigar el proceso organogénico durante las diferentes fases del desarrollo ontogenético. El estudio utilizó tanto microscopía de luz, como técnicas de microscopía electrónica. Los análisis histológicos revelaron que los vástagos de maíz están compuestos de pequeñas células con citoplasma denso y núcleo prominente. El proceso de organogénesis inicia con el engrosamiento del ápice del vástago, como primera evidencia de la formación organogénica del calli observada tres semanas después del inicio del cultivo. El estudio ultraestructural muestra dos tipos de células morfológicamente diferentes en el calli organogénico. La capa externa consiste de células grandes con núcleo pequeño, citosol sin organelas, membrana plamática irregular, estructuras similares a tricomas y paredes celulares delgadas. Mientras que en la capa interna se observaron células pequeñas e isodiamétricas con un núcleo prominente, vacuolas pequeñas, retículo endoplasmático, Golgi, mitocondrias y cloroplastos. Observaciones de microscopía electrónica revelan la organización externa de la organogénesis del calli del maíz. Los resultados obtenidos en el presente estudio contribuyen con la comprensión del proceso de organogénesis en los ápices de los vástagos del maíz. Este estudio mejorará nuestro conocimiento acerca de los estados anatómicos óptimos para la integración estable de genes externos. Además, provee información de las condiciones apropiadas en el cultivo in vitro para la regeneración de plantas transgénicas. Este estudio sugiere que la presencia de tricomas en las zonas morfogénicas más activas podría indicar su potencial organogénico.