Resumen Introducción. La vainilla (Vanilla planifolia Andrews) es una orquídea que se cultiva comercialmente para la producción de vainillina, una sustancia saborizante que se emplea principalmente en la industria alimenticia y como cosmético. Aunque la demanda ha motivado su cultivo, hay poca disponibilidad de semilla vigorosa y libre de enfermedades lo cual ha sido una limitante. Se ha demostrado que los sistemas de inmersión temporal son eficientes. Para la micropropagación masiva de la vainilla, aún no se han establecido estudios de tiempo, frecuencia de inmersión y volumen óptimo en la proliferación de brotes en Biorreactores de Inmersión Temporal (BIT) y además se desconoce una ruta morfogénica que garantice la estabilidad genética y la optimización durante el proceso de micropropagación. Objetivo. Caracterización morfogénica de la vainilla (Vanilla planifolia Andrews) en un BIT. Materiales y métodos. Se realizaron 3 experimentos, cada uno con 3 tratamientos. Para el primer experimento, se consideraron tiempos de 5, 10 y 15 min para los tratamientos, en el segundo se evaluaron 3 frecuencias en tiempos de 6, 12 y 18 horas y en el tercero se consideraron 20, 30 y 40 ml/explante como volumen de medio de cultivo. Para los diferentes tratamientos se utilizaron 5 repeticiones, con una unidad experimental de 10 explantes y segmentos de tallo con 3 yemas axilares. El medio de cultivo consistió en las sales de Murashige Skoog (MS) suplementado con 1 mg.L-1 de BA y 1 g.L-1 de caseína. El fotoperiodo se ajustó a 16 horas luz, con una temperatura de 27°C±3 y una humedad relativa de 80% Resultados. Se obtuvieron diferencias estadísticas (p<0,05) respecto a los tiempos evaluados, frecuencia de inmersión y el volumen por explante. Se determinó como mejor tiempo de inmersión un periodo de 15 minutos, 6 horas como frecuencia de inmersión y 40 ml como volumen de medio por explante. Al comparar la respuesta de desarrollo, el sistema BIT superó al medio semisólido 1,8 veces en el número de brotes y 4 veces en el incremento de peso fresco. También con BIT se obtuvieron 36 brotes por explante mientras que en el sistema de cultivo en medio semisólido, se obtuvieron 20 brotes por explante. Conclusiones. Mediante el empleo del sistema BIT, se optimizó el mecanismo de micropropagación de V. planifolia comparado con el medio de cultivo semisólido en la ruta organogénica directa, lo cual fue demostrado con la formación de brotes preformados y brotes adventicios.

Abstract Introduction. Vanilla (Vanilla planifolia Andrews) is an orchid that is commercially grown for the production van, a flavoring substance that is used mainly in the food industry and as a cosmetic. Although the demand has motivated its cultivation, there is little availability of vigorous seed and free which has been a limited. Temporary immersion systems have been shown to be efficient. For their massive micropropagation but studies of time, immersion, frequency and optimal volume have not yet been established in the proliferation of shoots in Temporary Immersion Bioreactors (BIT), furthermore, a morphogenic pathway that guarantees genetic stability and optimization during the micropropagation process is unknown. Objective. Morphogenic characterization of vanilla (Vanilla planifolia Andrews) in a BIT. Materials and methods. Three experiments were carried out, each with 3 treatments. For the first experiment, the immersion time considered the treatments of 5, 10 and 15 min, in the second experiment 3 frequencies were evaluated at times of 6, 12 and 18 hours and in the third experiment were considered 20, 30 and 40 ml/explant as volume of culture medium. For the differents treatments used 5 repetitions, with an experimental unit of 10 explants and stem segments with 3 axillary buds. The culture medium consisted of Murashige Skoog (MS) salts supplemented with 1 mg.L-1 of BA and 1 g.L-1 of casein. The photoperiod was adjusted to 16 light hours, with a temperature of 27°C±3 and a relative humidity of 80%. Results. Statistical differences (p<0,05) were obtained with respect to the evaluated times, frequency of immersion and the volume per explant. A period of 15 minutes it was determined as the best immersion time, 6 hours as the immersion frequency and 40 ml as the volume of medium per explant. When comparing the development response, the BIT system exceeded the semisolid medium 1.8 times in the number of shoots and 4 times in the increase in fresh weight. In addition, 36 shoots were obtained per explant in BIT, while in the culture system in semi-solid medium, a multiplication rate of 20 shoots per explant. Conclusions. By using the BIT system, the micropropagation mechanism of V. planifolia was optimized compared to the semi-solid culture medium in the direct organogenic route, which was proved with the formation of preformed shoots and adventitious shoots.

Abstract The objective was the development of an efficient system for the micropropagation of the caper (Capparis spinosa L.), a woody shrub of great interest due to its products, its remarkable resistance to drought and its tolerance to high temperatures. In vitro cultures were first established through disinfection and seed germination. A scarification of these with concentrated H2SO4 (98% v/v) was necessary to break dormancy. Only 15% of the seeds germinated. Nodal segments were obtained from the germinated seedlings, which were cultivated in basal medium of Murashige and Skoog (MS) semisolid added with benzyladenine (BA), 2-isopentenyladenine (2iP) and metatopoline (MT), this in order to induce multiple sprouting. The best response was obtained with 2 mg L-1 of these cytokinins, with an average number of well-differentiated sprouts per nodal segment of 3.6 with MT, 2.3 with BA and 1.5 with 2iP, this after 54 d of incubation. The combination of cytokinins with an auxin, naphthaleneacetic acid, was also tested. This combination improved the BA response, reaching an average of 3.2 sprouts per nodal segment. With the other cytokinins, it did not show a positive effect, maintaining very similar values with and without auxin. In addition to well-differentiated sprouts, masses or clusters of numerous small sprouts were generated, unsuitable for transfer to the rooting medium. These masses were transferred to RITA type temporary immersion bioreactors, where an average of 89 well-differentiated sprouts were generated per original explant, this in a medium with 1 mg L-1 of BA with 2 min immersions every 6 h. The sprouts took root with an efficiency of 80% in the basal environment, generating well-differentiated plants suitable for transfer to the ground. The survival already in the external environment of the plants generated in vitro was 85%, showing an apparently normal development already in ex vitro conditions.

Resumen El objetivo fue el desarrollo de un sistema eficiente para la micropropagación del alcaparro (Capparis spinosa L.), arbusto leñoso de gran interés por los productos derivados del mismo, su notable resistencia a la sequía y su tolerancia a las altas temperaturas. Primero se establecieron cultivos in vitro se a través de la desinfección y germinación de semillas. Fue necesaria una escarificación de estas con H2SO4 concentrado (98% v/v) para romper la dormancia. Solo 15% de las semillas germinaron. Se obtuvieron segmentos nodales a partir de las plántulas germinadas, mismos que se cultivaron en medio basal de Murashige y Skoog (MS) semisólido adicionado con benciladenina (BA), 2-isopenteniladenina (2iP) y metatopolina (MT), esto con el fin de inducir la brotación múltiple. La mejor respuesta se obtuvo con 2 mg L-1 de estas citocininas, con un número promedio de brotes bien diferenciados por segmento nodal de 3.6 con MT, 2.3 con BA y 1.5 con 2iP, esto a los 54 d de incubación. Se probó también la combinación de las citocininas con una auxina, el ácido naftalenacético. Esta combinación mejoró la respuesta de la BA, alcanzando un promedio de 3.2 brotes por segmento nodal. Con las otras citocininas no mostró un efecto positivo, manteniéndose valores muy similares con y sin la auxina. Además de brotes bien diferenciados, se generaron masas o racimos de numerosos brotes pequeños, no aptos para su transferencia a medio de enraizamiento. Estas masas se transfirieron a biorreactores de inmersión temporal tipo RITA, en donde se generó un promedio de 89 brotes bien diferenciados por explante original, esto en un medio con 1 mg L-1 de BA con inmersiones de 2 min cada 6 h. Los brotes enraizaron con una eficiencia de 80% en el medio basal, generando plantas bien diferenciadas y aptas para su transferencia a suelo. La supervivencia ya en el medio externo de las plantas generadas in vitro fue de 85%, mostrando un desarrollo en apariencia normal ya en condiciones ex vitro.

ABSTRACT. In vitro culture systems based on liquid culture media are considered to be more effective than semisolid culture medium systems. Liquid culture media systems provide better nutrient availability for plant tissues, easier culture handling, and the potential for scaling up and automation. However, in vitro liquid culture requires more careful handling due to the potential for contamination and the possibility of negative effects, such as hyperhydricity or vitrification, that hinder the growth and development of the plant material. Temporary immersion bioreactors have emerged as a workable alternative for capturing the benefits of liquid media, though semisolid systems are still traditional. Many studies have shown that silicon (Si) is a beneficial plant nutrient. Silicon might have a positive effect in both semisolid and liquid in vitro systems. The objective of this study was to evaluate the effect of silicon on the micropropagation and acclimatization of banana plants cultivated in vitro by comparing liquid temporary immersion bioreactor technology and semisolid traditional culture systems. Different silicon concentrations (0 and 1 mL L-1) and culture systems (liquid temporary immersion bioreactor and semisolid traditional culture) were evaluated over a 36-day period. The growth characteristics plant size, fresh and dry weight, and number and length of leaves and roots were evaluated. After the 36-day in vitro growth period, plants were transferred to a greenhouse for acclimatization and were evaluated after 30 days for the same growth characteristics used in the in vitro studies. The temporary immersion bioreactor system resulted in greater growth of banana plants compared to the traditional semisolid system. Temporary immersion bioreactors also showed a positive interaction with Si and resulted in higher values for all growth characteristics in the acclimatization phase.

Abstract Objective : To evaluate the in vitro multiplication of Morus alba L., Criolla variety, in temporary immersion systems (SETIS™). Materials and Methods : Two trials were conducted for the evaluation of three immersion times (1,0; 3,0; 5,0 min.) and three immersion frequencies every four, six and eight hours. Afterwards, another experiment was conducted for the analysis of the morphological response of the sprouts, at 28, 45 and 60 days of cultivation. A complete randomized design was used, with three repetitions in all the experiments. Results : The results related to the immersion time showed significant differences in the evaluated variables for p < 0,05, except in the number of sprouts. In addition, it was found that the highest number of axillary buds was obtained in the immersion time of three minutes, with a value of 4,67. Meanwhile, in the length of the sprouts, the maximum value was achieved with three minutes of immersion (3,0 cm) with significant differences (p < 0,05) with regards to the other treatments. With the use of temporary immersion systems, with three minutes of immersion every eight hours, during 45 days of cultivation, the highest number of axillary buds and 1,90 sprouts were obtained; the sprout length reached 9,82 cm. Conclusions : In the sprouts no morphophysiological changes were observed, ascribed to the cultivation conditions. It was proven that the in vitro micropropagation of mulberry, Criolla variety, in temporary immersion systems SETIS™, is possible.

Resumen Objetivo : Evaluar la multiplicación in vitro de Morus alba L., variedad Criolla, en sistemas de inmersión temporal (SETIS™). Materiales y Métodos : Se realizaron dos experimentos para la evaluación de tres tiempos de inmersión (1,0; 3,0; 5,0 min.) y tres frecuencias de inmersión cada cuatro, seis y ocho horas. Posteriormente, se desarrolló otro experimento para el análisis de la respuesta morfológica de los brotes, a los 28, 45 y 60 días de cultivo. Se empleó un diseño completamente al azar, con tres repeticiones en todos los experimentos. Resultados : Los resultados relacionados con el tiempo de inmersión mostraron diferencias significativas en las variables evaluadas para p < 0,05, excepto en el número de brotes. Además, se encontró que el mayor número de yemas axilares se obtuvo en el tiempo de inmersión de tres minutos, con un valor de 4,67. Mientras, en la longitud de los brotes, el máximo valor se logró con tres minutos de inmersión (3,0 cm) con diferencias significativas (p < 0,05) respecto al resto de los tratamientos. Con el empleo de los sistemas de inmersión temporal, con tres minutos de inmersión cada ocho horas, durante 45 días de cultivo, se obtuvo el mayor número de yemas axilares y 1,90 brotes; la longitud de los brotes alcanzó 9,82 cm. Conclusiones : En los brotes no se observaron cambios morfofisiológicos, atribuidos a las condiciones de cultivo. Se demostró que es posible la micropropagación in vitro de morera, variedad Criolla, en sistemas de inmersión temporal SETIS™.

Resumo A técnica de propagação in vitro de explantes através de biorreatores de imersão temporária é uma ferramenta que possibilita, a partir do cultivo em meio líquido, aumentar a eficiência da produção de mudas. Diversas pesquisas com a cultura do morangueiro mostraram a maior eficiência do sistema quando comparado ao processo convencional da micropropagação, em meio sólido. Nesse sentido, o objetivo foi estabelecer um protocolo de multiplicação e enraizamento de morangueiro ‘Pircinque’, em biorreatores de imersão temporária. Realizaram-se dois estudos distintos e independentes, caracterizados pelas etapas de multiplicação e enraizamento de explantes de morangueiro recém-introduzidos e registrados no Brasil. Foram estudados dois meios de cultura (MS e KNOP) e, como tratamento-testemunha, foi avaliado o crescimento dos explantes em meio de cultivo sólido, com a adição de 5 g L-1 de ágar. Tempos distintos de imersão do meio de cultura em contato com os explantes foram estudados: cinco ou oito vezes ao dia, durante 15 minutos. O estudo compreendeu os fatores meio de cultura e tempo de imersão, assim como o tratamento testemunha (sólido). Verificou-se que o uso de sistema de biorreatores de imersão temporária é uma técnica eficiente para a multiplicação e enraizamento de explantes de morangueiro cv. Pircinque, quando comparada ao método convencional de micropropagação com uso de meio de cultura sólido, possibilitando otimizar a produção de mudas em biofrábricas. O meio de cultivo MS líquido, em contato cinco vezes ao dia com explantes de morangueiro ‘Pircinque’, favorece o crescimento da parte aérea e do sistema radicular.

Abstract The in vitro propagation technique via temporary immersion bioreactors is a tool that, through the culture in a liquid medium, allows an increase in the efficiency of seedling production. Several researches with the strawberry crop have shown greater efficiency of the system compared to the conventional process of micropropagation in solid medium. In this sense, the objective herein was to establish a protocol of multiplication and rooting of the ‘Pircinque’ strawberry, in temporary immersion bioreactors. Two distinct and independent studies were carried out, characterized by the multiplication and rooting stages of strawberry explants, newly introduced and registered in Brazil. Two culture media (MS and KNOP) were studied and, as a control treatment, the growth of the explants in solid culture medium was evaluated with the addition of 5 g L-1 of agar. Different immersion times of the culture medium were explored: five or eight times a day, for 15 minutes. The study was composed of the culture medium and immersion time factors, as well as the control (solid) treatment. It was verified that the use of temporary immersion bioreactors system is an efficient technique for the multiplication and rooting of explants of strawberry cv. Pircinque, when compared to the conventional method of micropropagation with the use of solid culture medium, making it possible to optimize the production of seedlings in biofactories. The MS liquid medium, in contact with explants of ‘Pircinque’ strawberry five times a day, increased the growth of the aerial part and the root system.

Abstract: Background and Aims: Moringa oleifera is a source of phytochemicals with potential applications in medicine. The medicinal use of its leaves is recognized worldwide, to which the presence of functional bioactive compounds is attributed. This work aimed to standardize a protocol for in vitro germination of M. oleifera in temporary immersion bioreactors, using different culture media. Methods: The seeds were sown with and without seed coat, in liquid basal media composed of water (control) and salts of Murashige and Skoog (MS) in their original concentration or diluted to 50% and 25%, with 0, 15 and 30 g l-1 sucrose. Germination percentage, indicators of vigor and growth in seedlings were evaluated. Key results: In seeds with seed coat, 23.33±5.77% germination was obtained in a single treatment, without being able to evaluate the proposed parameters. These results were complemented with the tetrazolium viability test of the ungerminated seeds, registering a 34.16±18.73% viability. The removal of the seed coat and liquid culture medium favored germination. The highest percentage of germination, germination capacity and coefficient of uniformity, as well as greater leaf development, ratio of aerial and root biomass, increase of dry weight of plant material per unit of time, and the accumulation of dry matter, were obtained with MS as a basal medium. Conclusions: For the standardization of germination in vitro of Moringa oleifera it is recommended to remove the seed coat and to use MS as a basal medium, since they accelerate the process and make it possible to obtain seedlings that can be used for experiments of indirect and / or direct regeneration for a massive escalation of germplasm.

Resumen: Antecedentes y Objetivos: Moringa oleifera es fuente de fitoquímicos con aplicación potencial en medicamentos. Su uso medicinal, sobre todo de las hojas, es reconocido mundialmente; se les atribuye la presencia de compuestos bioactivos funcionales. El objetivo de este trabajo fue estandarizar la germinación in vitro de M. oleifera en biorreactores de inmersión temporal utilizando distintos medios de cultivo. Métodos: Las semillas se sembraron con y sin testa, en medios basales líquidos compuestos de agua (control) y sales de Murashige y Skoog (MS) en su concentración original o diluido al 50 y 25%, con 0, 15 y 30 g l-1 de sacarosa. Se evaluaron los porcentajes de germinación, indicadores de vigor y crecimiento en plántulas. Resultados clave: En las semillas con testa se obtuvo un 23.33±5.77% de germinación en un solo tratamiento sin lograr evaluar los parámetros propuestos. Se complementaron estos resultados con la prueba de viabilidad de tetrazolio de las semillas no germinadas, registrando un 34.16±18.73% de viabilidad. La remoción de la testa y medio de cultivo líquido favorecieron la germinación. El mayor porcentaje de germinación, capacidad germinativa y coeficiente de uniformidad, así como mayor desarrollo foliar, relación de biomasa aérea y radical, incremento de peso seco de material vegetal por unidad de tiempo, al igual que la acumulación de masa seca, se obtuvieron con MS como medio basal. Conclusiones: Para la estandarización de la germinación in vitro de Moringa oleifera se recomienda la remoción de la testa y la utilización de MS como medio basal, ya que aceleran el proceso y posibilitan obtener plántulas que podrán ser utilizadas para experimentos de regeneración indirecta y/o directa para un escalado masivo de germoplasma.

SUMMARY In the ornamental industry, gladiolus (Gladiolus spp) is one of the main cut flowers worldwide. The propagation is through corms, with low multiplication rates, which makes it necessary to use in vitro techniques to obtain vegetative material with identical, uniform and disease-free physiological characteristics. However, the research about in vitro multiplication of gladiolus is scarce. The temporary immersion system (RITA) was used successfully to produce gladiolus microcorms in vitro. To date, the temporary immersion bioreactor system (BIT) has not been used for production of in vitro gladiolus microcorms, which represents only 25 % of the cost of a RITA system. The objective was to evaluate three in vitro culture systems: semi-solid medium, liquid medium in partial immersion and liquid medium in temporary immersion bioreactors (BIT), for the multiplication of gladiolus microcorms of the variety “Ámsterdam”. In the BIT system there was an increase of 91 % in microcorms multiplication compared to the semisolid system and 100 % in relation to the partial immersion system. The number of shoots was also higher (41.3) in the BIT system than in the semi-solid culture system and in the liquid culture system with partial immersion (5.8 and 6.5 shoots per explant). These results allowed the use of temporary immersion bioreactors to be established as the best system for the massive multiplication of gladiolus microcorms.

RESUMEN En la industria ornamental, el gladiolo (Gladiolus spp) se encuentra dentro de las principales flores de corte a nivel mundial. La propagación es a través de cormos, con tasas de multiplicación bajas, lo que hace necesario el uso de técnicas de cultivo in vitro para obtener material vegetativo con características fisiológicas idénticas, uniformes y libres de enfermedades. Sin embargo, la investigación en multiplicación in vitro de gladiolo es escasa. El sistema de inmersión temporal (RITA) se usó con éxito para producir microcormos de gladiolo in vitro. A la fecha, el sistema de biorreactores de inmersión temporal (BIT) no se ha usado para producción de microcormos de gladiolo in vitro, lo cual implica sólo 25 % del costo de un sistema RITA. El objetivo fue evaluar tres sistemas de cultivo in vitro: medio semisólido, medio líquido en inmersión parcial y medio líquido en biorreactores de inmersión temporal (BIT), para la multiplicación de microcormos de gladiolo variedad “Ámsterdam”. En el sistema BIT hubo un incremento de 91 % en la multiplicación de microcormos en comparación con el sistema semisólido y de 100 % en relación con el sistema de inmersión parcial. También el número de brotes fue mayor (41.3) en el sistema BIT que en el sistema de cultivo en medio semisólido y en el sistema de cultivo en medio líquido con inmersión parcial (5.8 y 6.5 brotes por explante). Estos resultados permitieron establecer el uso de biorreactores de inmersión temporal como mejor sistema para la multiplicación masiva de microcormos de gladiolo.

Summary: A Temporary Immersion System (TIS) enables plant micropropagation in unsterilized sealed environments. However, it is a complicated and expensive technology. In this paper, an automation alternative based on open hardware and software platforms is presented, with the objectives of reducing costs and increasing performance as compared with commercial systems, through software reconfiguration of routines for nutrient application, constant monitoring and remote control of the system. For hardware development, the Bottom Up methodology was used, and Kanban methodology was used for the software. The cost of the proposed system is on average 10% that of commercial electronic devices used in the industry to solve process automation problems. Growth assessment of the explants is necessary to corroborate the competitive advantages of the prototype relative to the traditional systems of temporary immersion.

Resumen: Un Sistema de Inmersión Temporal (SIT) permite la micropropagación de plantas en ambientes esterilizados y herméticos; sin embargo, es una tecnología complicada y costosa. En este trabajo, se presenta una alternativa de automatización con base en plataformas abiertas de hardware y software, con los objetivos de reducir costos y aumentar las prestaciones frente a sistemas comerciales, mediante la reconfiguración por software de rutinas para la aplicación de nutrientes, monitoreo constante y control a distancia del sistema. Para el desarrollo del hardware se utilizó la metodología Bottom Up, y para el software, la metodología Kanban. El sistema propuesto tiene un costo promedio de un 10% en comparación con dispositivos electrónicos comerciales utilizados en la industria para resolver problemas de automatización de procesos. Sin embargo, es necesario una evaluación de crecimiento de los explantes para corroborar las ventajas competitivas del prototipo equiparadas a los sistemas tradicionales de inmersión temporal.

Stevia rebaudiana Bertoni, Asteraceae, is a natural sweetener called “Stevia”. Its properties came from the diterpenoid glycosides presented in the leaves: a stevioside and rebaudisioside. The percentage of seed germination of S. rebaudiana is very low and the plants produced are heterogeneous, so it is not suitable for mass propagation in the field.The tissue culture in temporary immersion systems, is an effective tool for micropropagation, since it increases the multiplication coefficient and produces the improvement in the quality of in vitro regenerated plants. This research determined the efficieness of propagation process using temporary immersion devices for scaling up the mass production of Stevia rebaudiana, with the use of liquid media in automated temporary immersion system (RITA®) and twin flasks system (BIT), compared with semisolid culture. Most of the treatments involving temporary immersion systems (BIT y RITA®), produced green plants and vigorous plants, with low levels of hyperhydricity. The highest average of regenerated leaves and shoots was obtained in the RITA® system, with 10 minutes of immersion every 8 hours. It was determined also that 10 minutes on inmersion each 12 hour with the BIT produced vigorous plants, with an increased length, fresh and dry weight mass. The use of in vitro culture in temporary immersion bioreactors (RITA® and BIT), offers many advantages in the process of scaling the production of this kind of commercial plant, related to connentional propagation system and encouraging the process of acclimatization

Stevia rebaudiana Bertoni, familia Asteraceae, es conocida como “yerba dulce” por poseer un edulcorante natural. Sus propiedades provienen de la presencia de glicósidos diterpenos denominados esteviósidos y rebaudiósidos en las hojas. El porcentaje de germinación de las semillas de S. rebaudiana es muy bajo y las plantas producidas son heterogéneas, por lo que no es conveniente para la propagación masiva en campo. El cultivo en sistemas de inmersión temporal, es una herramienta eficaz para la micropropagación, ya que incrementa el coeficiente de multiplicación y produce el mejoramiento en la calidad del material regenerado in vitro. En esta investigación se determinó el proceso de propagación de inmersión temporal más eficiente para el escalamiento de la producción masiva de Stevia rebaudiana, con el empleo de medios líquidos en sistemas de inmersión temporal automatizado (RITA®) y vasos gemelos (BIT), en comparación con el cultivo en medio semisólido. La mayoría de los tratamientos en sistemas de inmersión temporal BIT y RITA® produjeron plantas verdes y vigorosas, con bajos niveles de hiperhidricidad. El mayor número promedio de regeneración de hojas y brotes se obtuvo en RITA® con 10 minutos de inmersión cada ocho horas. Se determinó que en el tratamiento que consistió en 10 minutos de inmersión cada 12 horas en BIT, produjo plantas muy vigorosas, con el mayor incremento en longitud, masa fresca y masa seca promedio. El empleo de cultivo in vitro en biorreactores de inmersión temporal (RITA® y BIT), ofrece muchas ventajas en el proceso de escalamiento de la producción de esta especie de interés comercial, respecto a los sistemas de cultivo convencional, favoreciendo el proceso de aclimatación

O objetivo deste trabalho foi averiguar a melhor densidade de explantes e o melhor tipo de sistema de cultivo visando desenvolver um protocolo de micropropagação de baixo custo para a Carobinha. Foram realizados experimentos de multiplicação in vitro com quatro tipos de frascos: R.I.T.A. (50 explantes/frasco), erlenmayer, (50 explantes/frasco), potes tipo maionese (6 explantes/frasco) e cubetas (1 explante/frasco). O co-cultivo de explantes, tanto em meio sólido quanto em meio líquido (R.I.T.A.), promoveu maiores taxas de explantes com brotação e de sobrevivência. O sistema de imersão temporária proporcionou melhores índices de desenvolvimento, brotação, sobrevivência e altura dos explantes. Concluímos que biorreatores podem ser utilizados eficientemente para a micropropagação de carobinha.

The aim of this study was to identify the best explant density and the best cultivation system with the goal of developing a micropropagation protocol of low cost for "carobinha" (Jacaranda decurrens CHAM.). Experiments of in vitro multiplication were carried out using four flask types: R.I.T.A. (50 explants/flask), Erlenmeyer (50 explants/flask), mayonnaise pots (6 explants/flask) and cuvettes (1 explant/flask). The co-cultivation of explants, in both solid and liquid medium (R.I.T.A.), led explants to show higher sprouting and survival rates. The temporary immersion system provided better rates of development, sprouting, survival and height of explants. We concluded that bioreactors may be efficiently used for the micropropagation of "carobinha".

La automatización de la producción de plantas con el empleo de Biorreactores de Inmersión Temporal (BIT) constituye una herramienta eficaz para la propagación in vitro con la cual se elevan los coeficientes de multiplicación y se aumenta la calidad del material propagado. Algunos factores que influyen en este proceso son las características de los explantes que se emplean para inocular los BIT, la manipulación de los mismos y su estado fisiológico. Estos, aparejados a los indicadores de eficiencia del sistema, son elementos a tener en cuenta en el estudio de la morfogénesis de cultivos como el plátano, que presenta índices de proliferación bajos en medio semisólido. Con el objetivo de optimizar el protocolo descrito por Roels, et al. (2005), se evaluó el efecto del manejo del explante (con división y sin división), el volumen de medio de cultivo durante tres ciclos de proliferación de brotes de plátano cv. CEMSA ¾ sobre el coeficiente de multiplicación y la calidad de los mismos. El mayor coeficiente de multiplicación se logró con el tratamiento en el que se incrementó el volumen del medio de cultivo en función de la biomasa sin seccionar el explante durante los tres ciclos de propagación. Estas modificaciones al protocolo de Roels, et al. (2005), al reducir la manipulación de los explantes durante cada ciclo de cultivo y aumentar las tasas de multiplicación, lo hacen más eficiente y simple, y de esta manera más aplicable para la propagación de plátano a gran escala.

The automation of the production of plants with the use of Temporary Immersion Bioreactors (TIB) is an effective tool for in vitro propagation, with which rates of multiplication and increases the quality of the material spread are increased. Some factors influencing this process are the characteristics of the explants used to inoculate the BIT, the manipulation of the data and the physiological state. These factors, coupled with efficiency indicators of the system, are elements to be considered in the study of morphogenesis in crops such as plantain, which has low growth rate in semisolid medium. In order to optimize the protocol described by Roels, et al. (2005), we evaluated the effect of management of the explants (with division and without division), thevolume of culture medium for three cycles of shoot proliferation of banana cv. ¾ CEMSA on the coefficient of multiplication and their quality. The highest multiplication rate was achieved with the treatment which increased the volume of culture medium in terms of biomass without severing the explants during the three cycles of propagation. Since they reduce the manipulation of the explants for each crop cycle, and they increase the multiplication rate making it more efficient and simple, these modifications to the protocol of Roels, et al. (2005), make more applicable the propagation of plantain on a large scale.

A busca por novas técnicas de cultivo in vitro vem sendo amplamente estudadas, visando otimizar e baixar o custo de produção das mudas que tenham interesse agronômico. O objetivo deste trabalho foi comparar diferentes sistemas de cultivo in vitro de Ananas comosus L. Merril. Para tanto, o crescimento in vitro de plântulas foi promovido em sistemas de biorreatores (B.I.B.® e R.I.T.A.®) com ciclo de imersão a cada 2 horas por 15 minutos e o sistema tradicional em frascos de 200 mL. Em todos os sistemas de cultivo, foram utilizadas solução nutritiva líquida MS, suplementado com 1 mg L-1 de BAP, 0,25 mg L-1 de ANA, 30 g L-1 de sacarose e 0,5 µL de Tween® 20, pH ajustado para 5,8 e autoclavagem a 120°C por 15 minutos. As culturas foram mantidas em sala de crescimento durante 30 dias, temperatura controlada de 25±2°C, sob luz branca fria (46,8 µmol.m-2.s-1), com 16 horas de fotoperíodo. O delineamento foi inteiramente casualizado, com três tratamentos, três repetições e dez plantas por estágios. Para os sistemas avaliados, o biorreator de imersão por bolhas apresentou os melhores resultados dentre as variáveis analisadas, com destaque ao número total de brotações, sendo 2,3 e 3,1 vezes superiores quando comparado com o sistema R.I.T.A.® e sistema tradicional respectivamente. Portanto, o sistema de imersão por bolhas torna-se um equipamento eficaz na produção de mudas de abacaxizeiro em larga escala.

The research for new techniques of in vitro cultivation is being object of many studies around the world, in order to optimize and decrease production costs of seedlings with agronomical interest. The main goal of this work was to compare different systems of in vitro cultivations using Ananas comosus L. Merril. So, the in vitro growth of the plantlets was promoted in two different bioreactors: Bioreactor of Immersion by Bubbles (B.I.B.®) and the Reactor of Temporary Immersion (R.I.T.A.®) with immersion cycle every 2 hours for 15 minutes and the traditional system in flasks with 200 mL. All cultivation systems used the MS liquid nutritive solution, supplemented with BAP (1 mgL-1), ANA (0.25 mgL-1), sucrose (30 gL-1) and Tween 20® (0.5 µL). The pH was adjusted to 5.8 and sterilized at 120°C for 15 minutes. The cultures were kept into a growth room during 30 days, with controlled temperature of 25±2°C, under white cold light (46.8 µmol.m-2.s-1), with photoperiod of 16 hours. The experimental design used was randomized, with three treatments, three repetitions and ten plants each stage. Among the evaluated systems, the BIB® presented the best results for the tested variables, mainly the total number of shoots, being 2.3 e 3.1 times superior when compared with the system R.I.T.A.® and the traditional consecutively. So the system of immersion by bubbles turns into an effective equipment to produce seedlings of pineapple in large scale.

Se evaluaron dos tipos de bioreactores para la propagación in vitro de Vaccinium corymbosum: un reactor de inmersión temporal (RITA®) y un reactor de inmersión permanente. Los resultados fueron comparados con el sistema de micropropagación habitualmente empleado de multiplicación en medio de cultivo semi-sólido, usado como control en este trabajo. Se emplearon dos variedades, ‘O`Neal’ y ‘Georgia Gem’, del triple híbrido Vaccinium corymbosum, V. ashei y V. darrowii. Los explantos fueron cultivados en medio WPM (Lloyd y Mc Cown, 1981) suplementado con ácido ascórbico (0,01 mM), sulfato de adenina (0,22 mM), sacarosa (2%) y 2iP (20µM). Para minimizar la ocurrencia de hiperhidricidad en los explantos se evaluó el efecto del ancymidol (0.4, 0.9 y 2.0 µM ) como retardante del crecimiento y del phloroglucinol (0.32mM) como promotor de la biosíntesis de lignina. Las máximas tasas de multiplicación obtenidas fueron 28 y 24 brotes por yema para las variedades ‘O’Neal’ y ‘Georgia Gem’ respectivamente, las cuales fueron alcanzadas en el sistema de inmersión permanente con phloroglucinol. Observaciones al microscopio óptico de las plantas obtenidas mostraron una mayor diferenciación de xilema y mayor lignificación de los tejidos en los explantos provenientes del tratamiento con phloroglucinol. Los brotes provenientes del tratamiento de multiplicación que alcanzaron 3 cm o más de altura fueron enraizados ex vitro en una mezcla de tierra, turba y perlita (1:1:1) obteniéndose un 80% de enraizamiento. Las plantas fueron aclimatadas y mostraron un desarrollo normal en invernáculo. Abreviaciones: IBA – ácido indol 3 butírico, 2iP – 6-(g, g-dymethilamino) purine, WPM – Woody Plant Medium (Lloyd y McCown, 1981).

Two kinds of bioreactors were evaluated for in vitro propagation of Vaccinium corymbosum: a temporary immersion system (RITA®) and a permanent immersion reactor. Results were compared with the system currently used of micropropagation in semi-solid medium, used as control for this study. Two varieties, ‘O’Neal’ and ‘Georgia Gem’, of the triple hybrid Vaccinium corymbosum, V. ashei and V. darrowii were employed. Explants were cultured in WPM medium (Lloyd and Mc Cown, 1981) containing 0.01mM ascorbic acid, 0.22mM adenine sulphate, 2% sucrose and 20mM 2iP. To minimize hyperhydricity of explants, the effect of ancymidol (0.4, 0.9 and 2.0 µM) as growth retardant and phloroglucinol (0.32mM) as promoter of lignin biosynthesis was tested. The highest multiplication rates were 28 and 24 shoots per bud for the varieties ‘O’Neal’ and ‘Georgia Gem’ respectively, and were achieved in the permanent immersion system containing phloroglucinol. Light microscopy of the shoots showed a more developed and lignified xylem in the phloroglucinol-derived plants. Shoots obtained from the multiplication system that were 3 or more centimeters long were rooted ex vitro in a mixture of soil, peat and perlite (1:1:1) resulting in 80% rooting. Plants acclimatized well and exhibited normal development in the greenhouse. Abbreviations: IBA – Indol-3-butyric acid, 2iP – 6-(g, g-dymethilamino) purine, WPM – Woody Plant Medium (Lloyd and McCown, 1981).

Em café, o uso de bioreatores é a mais promissora maneira de aumentar o processo de micropropagação, particularmente a embriogênese somática. A disponibilidade de um eficiente processo de embriogênese somática poderia aumentar a rápida produção em massa de materiais heterozigotos, tais como clones selecionados de Coffea canephora clones e variedades híbridas F1 de Coffea arabica. Nos últimos 15 anos, bioreatores (bioreatores mecanicamente ou pneumaticamente agitados, bioreatores de imersão temporária) têm sido predominantemente usados em café para otimizar a regeneração em massa de embriões somáticos, a partir de tecidos embriogênicos. Esta revisão apresenta os principais resultados obtidos com vários modelos de bioreatores, no que diz respeito aos vários passos do processo de micropropagação: i) a multiplicação de tecidos embriogênicos, ii) a regeneração em massa de embriões somáticos e iii) a produção de embriões pré-germinados e plântulas nos bioreatores. A literatura mostra que o escalonamento do processo de micropropagação pode ser útil, desde que uma produção muito eficiente de embriões seja atingida para C. arabica e C. canephora. Além disso, foi demonstrado que embriões pré-germinados de café - i.e. com alongamento do eixo embrionário (10-12 mm), formação de ponta de radícula, expansão do cotilédone e esverdeamento - obtidos em bioreatores de imersão temporária eram fotoautotróficos e capazes de regenerar plântulas vigorosas depois de semeados em viveiro. A possibilidade do uso da tecnologia de bioreatores em escala industrial de micropropagação é também discutida, particularmente no contexto sócio-econômico do cultivo do café.

In coffee, bioreactors are the most promising way for scaling-up micropropagation processes, particularly somatic embryogenesis. The availability of an efficient somatic embryogenesis process would allow the rapid mass production of heterozygous materials such as selected Coffea canephora clones and F1 Arabica hybrid varieties. For the last fifteen years, bioreactors (mechanically or pneumatically agitated bioreactors, temporary immersion bioreactors) have mostly been used on coffee to optimize the mass regeneration of somatic embryos from embryogenic tissues. This review presents the main results, obtained with several bioreactor models, concerning the different steps of the micropropagation process : i) the multiplication of embryogenic tissues, ii) the somatic embryo mass regeneration and iii) the production of pre-germinated embryos and plantlets in bioreactors. The literature shows that scaling-up can be successful, since very efficient embryo production has been achieved for both C. arabica and C. canephora. Moreover, it was proven that the pre-germinated coffee embryos - i.e. embryonic axis elongation (10-12 mm), root tip formation, cotyledon expansion and greening - obtained in temporary immersion bioreactors were photoautotrophic and able to regenerate vigorous plantlets after sowing under nursery conditions. The feasibility to apply the bioreactor technology in an industrial micropropagation procedure is also discussed in the particular socio-economic context of coffee growing.

Mudas micropropagadas de banana têm sido ofertadas ao mercado com o intuito de suprir a demanda de uma fruticultura cada vez mais tecnificada. Os preços mais elevados deste tipo de muda têm sido um dos maiores entraves ao seu uso. Vários são os fatores que oneram o seu preço final: mão-de-obra especializada, necessidade de laboratórios bem equipados, estrutura de aclimatização apropriada, baixa taxa de multiplicação de algumas variedades, etc. O presente trabalho relata a micropropagação de bananeiras cv. Terra, utilizando biorreatores de imersão temporária, com o objetivo de aumentar a taxa de multiplicação e diminuir os custos de produção das mudas. Os resultados obtidos mostraram que o ciclo de imersão de 4 horas e a renovação do meio de cultura aos 30 dias foram essenciais para uma maior produção de biomassa e crescimento dos explantes. A composição do meio de cultura influenciou o desenvolvimento dos explantes de banana cultivados nos biorreatores. Explantes cultivados em meio MS + 3mg/L de BAP com renovação para MS básico, após 30 dias, apresentaram maior produção de biomassa e alta taxa de multiplicação. Comparando-se o biorreator de imersão temporária com o sistema tradicional em semi-sólido, observou-se que, no primeiro, as microplantas apresentaram maior comprimento, produção de biomassa de 2,86 vezes maior e 2,20 vezes mais brotos do que no sistema tradicional.

Banana seedlings has been micropropagated and sold to the producers to supply a rather competitive fruit crop market. This high quality propagule has usually a higher price in the market than field propagated seedlings. Several factors contribute to its final costs: need of specialized labour, need of well equipped laboratory and acclimatization structure, low multiplication rate of some varieties etc. The work presented here reports the development of a new way to micropropagate banana var. Terra, a known slow seedling producing variety, by using a temporary immersion bioreactor. The aim of this work was to increase the multiplication rate of this variety of banana and to reduce the costs of production of the micropropagated seedlings. The results showed that the immersion cycle of 4 hours with medium culture renewed at 30 days was essential to a higher biomass and explants growth. The composition of the medium culture positively influenced the development of the banana explants cultured in the bioreactors. Cultured explants in MS medium + 3 mg/L of BAP changed to a basic MS medium after 30 days presented the higher biomass and multiplication rate of all treatments. Traditional cultivation using semi-solid media was compared to a temporary immersion system and the results showed that the temporary immersion system presented 2.86 times more biomass production and 2,20 times more viable shoots than the traditional semi-solid system.