Effects of glutathione supplementation and carbon source duringsomatic embryogenesis of Acca sellowiana (O. Berg) Burret(Myrtaceae) / Efeitos da suplementação de glutationa e fontes de carbono durante a embriogênesesomática de Acca sellowiana (O. Berg) Burret (Myrtaceae)

The present study intended to investigate the effects of different glutathione (GSH) levels (0, 0.1, 0.5 and 1 mM) on the somatic embryogenesis (SE) induction of Acca sellowiana. Besides, we evaluated the effect of different carbon sources (sucrose and maltose) on the somatic embryos conversion. GSH-supplemented treatments resulted in improved SE induction rates (~70%) as compared to the control GSH-free (~35%) after 50 days of culture. The total number of somatic embryos obtained did not differ between treatments, but significant differences were observed for the embryonic stages after 80 days of culture. After 80 days of culture, 0.5 and 1 mM GSH-supplemented treatments showed the largest amount of torpedo-staged somatic embryos. In contrast, treatments supplemented with 0 and 0.1 mM GSH showed equal amounts of somatic embryos at all embryonic stages. These results indicate that GSH accelerates the SE induction process and increases the synchrony of the somatic embryo formation of A. sellowiana. The use of maltose for the somatic embryos conversion, as compared to sucrose, did not influence the conversion rate of normal chlorophyllous somatic embryos, but increased the formation of normal achlorophyllous somatic plantlets. This finding can be attributed to the rapid hydrolysis of sucrose, contributing to an enhanced chlorophyll synthesis.

O presente estudo teve como objetivo investigar o efeito de diferentes níveis de glutationa (GSH) (0, 0,1, 0,5 e 1 mM) na indução da embriogênese somática (ES) de Acca sellowiana. Além disso, avaliamos o efeito de diferentes fontes de carbono (sacarose e maltose) na conversão de embriões somáticos em plântulas. Os tratamentos suplementados com GSH resultaram em melhores taxas de indução de ES (~70%) em comparação com o controle isento de GSH (~35%) após 50 dias de cultivo. Após 80 dias as taxas de indução foram iguais. O número total de embriões somáticos obtidos não diferiu entre os tratamentos, mas diferenças expressivas foram observadas nos estágios embrionários. No dia 80 em cultura, os tratamentos suplementados com 0,5 e 1 mM de GSH mostraram a maior porção de embriões somáticos no estádio torpedo. Diferentemente, tratamentos suplementados com 0 e 0,1 mM de GSH mostraram quantidades iguais de embriões somáticos em todos os estágios embrionários. Estes resultados indicam que o GSH acelera o processo de indução do ES e aumenta a sincronia na formação de embriões somáticos de A. sellowiana. O uso de maltose no meio de cultura de conversão de embriões somáticos, em comparação com a sacarose, não influenciou a taxa de conversão de embriões somáticos clorofílados normais, mas aumentou a formação de plântulas aclorofiladas normais. Esse resultado pode ser atribuído à rápida hidrólise da sacarose, apresentando translocação de plantas mais eficiente e aumento da osmolaridade do meio de cultura, contribuindo para uma síntese melhorada de clorofila.

Multiplicación in vitro y embriogénesis somática de Perezia pinnatifida (Asteraceae) planta medicinal andina

Se presentan los procedimientos para la propagación in vitro de Perezia pinnatifida (Humb. & Bonpl.) Wedd., conocida como "valeriana". Se utilizaron las metodologías de multiplicación de brotes y embriogénesis somática indirecta. El medio de cultivo basal para todas las etapas fue Murashige y Skoog a mitad de sales, suplementado con sacarosa 2.0%, phytagel 0.3% y pH 5.67; y fue usado con o sin fitohormonas en los diferentes tratamientos. Los suplementos hormonales fueron: para la multiplicación de brotes BAP 1.0 mg.L-1 + ANA 0.01 mg.L-1 ó BAP 1.0 mg.L-1; para la inducción de callos embriogénicos ANA ó 2,4-D (1.0 mg.L-1 y 2.0 mg.L-1); y para la germinación de embriones BAP (0.5 y 1.0 mg.L-1) o BAP 0.5 mg.L-1 + ANA 0.05 mg.L-1. El mayor número de brotes se obtuvo en el medio suplementado con BAP 1.0 mg.L-1. En la embriogénesis somática, ANA a 1.0 mg.L-1 indujo mayor área de callos embriogénicos, y BAP a 0.5 mg.L-1 permitió mayor germinación de los embriones somáticos

This work inform on in vitro propagation of the "valeriana" Perezia pinnatifida (Humb. & Bonpl.) Wedd. Shoot multiplication and indirect somatic embryogenesis methodologies were performed. The basal culture medium for all stages was Murashige and Skoog, middle of salts supplemented with 2.0% sucrose, 0.3% phytagel and pH 5.67; the treatments were prepared with or without phytohormones. The hormonal supplements for the shoot multiplication were: BAP 1.0 mg.L-1 + ANA 0.01 mg.L-1, and BAP 1.0 mg.L-1; for embryogenic callus induction were: ANA or 2,4-D (1.0 mg.L-1 and 2.0 mg.L-1); and for the embryo germination were: BAP (0.5 and 1.0 mg.L-1) or BAP 0.5 mg.L-1 + ANA 0.05 mg.L-1. BAP 1.0 mg.L-1 produced the higher number buds. For somatic embryogenesis, ANA 1.0 mg.L-1 induced a greater area of embryogenic callus, and BAP 0.5 mg.L-1 allowed major germination of the somatic embryos

EFFECT OF GENOTYPE ON THE in vitro REGENERATION OF Stevia rebaudiana VIA SOMATIC EMBRYOGENESIS / Efecto del genotipo sobre la regeneración in vitro de Stevia rebaudiana a través de embriogénesis somática

Stevia rebaudiana (Asteraceae) is a plant of economic importance because of its medicinal properties and the presence of sweetener compounds on its leaves. These compounds can be a substitute for sucrose in a wide variety of products used by persons with diabetes and obesity problems. To standardize an efficient and effective propagation method for the different Stevia genotypes grown in Colombia, this study evaluated the effect of different combinations of the plant growth regulators 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 6-(gamma, gamma-dimethylallylamino) purine (2iP) and Zeatin on the induction and development of somatic embryos. Adenine and coconut water were also evaluated as supplements in the basal culture medium Murashige and Skoog Basal Salt Mixture (MS) with glutamine. The combination of 2,4-D (18.09 µM) and 2iP (7.38 µM) produced the highest number of somatic embryos per explant, which had well-defined characteristics. The genotype showed a significant effect on the embryogenic response. In the "SRQ-93" genotype, the formation and development of somatic embryos was achieved, whereas the genotypes "Bertoni" and "Morita II" only yielded embryogenic and non-embryogenic calli, respectively. The conversion to seedlings was achieved on the regeneration medium containing gibberellic acid (GA3) (0.29 µM) and activated charcoal.

Stevia rebaudiana (Asteraceae), es una planta de gran importancia económica debido a sus propiedades medicinales y a la presencia de compuestos endulzantes en sus hojas, los cuales pueden sustituir la sacarosa en gran variedad de productos utilizados por personas con problemas de diabetes y obesidad. Con el propósito de estandarizar un método de propagación eficiente y efectivo para diferentes genotipos de Stevia cultivados en Colombia, en la presente investigación se evaluó el efecto sobre la inducción y desarrollo de embriones somáticos de diferentes combinaciones de los reguladores de crecimiento vegetal 2,4-D, IAA, IBA, 2iP y Zeatina, además de los suplementos adenina y agua de coco en el medio de cultivo basal Murashige y Skoog (1962), adicionado con glutamina. Con la combinación 2,4-D (18.09 µM) y 2iP (7.38 µM) se obtuvo el mayor número embriones somáticos por explante con características bien definidas. El genotipo tuvo un efecto significativo en la repuesta embriogénica, en el genotipo "SRQ-93" se logró la formación y el desarrollo de embriones somáticos, mientras que en los genotipos "Bertoni" y "Morita II", solo se obtuvo callo embriogénico y no embriogénico respectivamente. La conversión a plántulas se alcanzó en el medio de regeneración conteniendo GA3 (0.29 µM) y carbón activado.

Embriogénesis somática en el cultivar de plátano 'FHIA - 25' (AAB) a partir de ápices meristemáticos / Somatic embryogenesis in plantain cultivar 'FHIA - 25' (AAB) from meristem tips

El cultivar de plátano 'FHIA - 25' (AAB), posee excelente rendimiento y alta resistencia a "Sigatoka negra", pero con la limitante del bajo contenido de azúcar en su fruto, lo cual hace que sea necesario disponer de un método de regeneración de plantas a nivel celular como la embriogénesis somática, que se complemente a técnicas biotecnológicas de transformación genética para mejorar la calidad del fruto. El presente trabajo se realizó con el objetivo de establecer una metodología de regeneración vía embriogénesis somática a partir del explante inicial ápices de brotes axilares establecidos directamente en medio de cultivo líquido. Se obtuvieron suspensiones celulares embriogénicas homogéneas a partir del explante antes mencionado. Se lograron las mayores tasas de multiplicación a la densidad celular de 3,0%. La incubación de los embriones somáticos durante 30 días en el medio de cultivo de maduración permitió incrementar la germinación de los mismos. Durante la fase de aclimatización las plantas provenientes de los embriones somáticos, así como las plantas regeneradas por organogénesis, mostraron un alto porcentaje de supervivencia (98 y 97 %, respectivamente), sin la presencia de variación somaclonal.

Plantain cultivar 'FHIA - 25' (AAB) shows high yielding qualities and high resistance to Black Sigatoka disease, but its sugar content in the fruit is low, so a regeneration method at cell level is necessary, such as somatic embryogenesis supported by biotechnological tools to improve fruit quality. This work was performed with the aim of establishing a plant regeneration method via somatic embryogenesis using initial explants of shoot apices from axillary buds in liquid culture medium. Homogenous embryogenic cell suspensions were obtained from mentioned explants. The highest cellular multiplication rates were achieved at 3,0% density. The incubation of somatic embryos during 30 days in the maturation culture medium permitted to increase germination. During the acclimatization stage, plants regenerated from somatic embryos, as well as plants from organogenesis, showed a high survival percentage (98 and 97 respectively), without somaclonal variation.

Comparing the Effect of Two Promoters on Cassava Somatic Embryo at Transient GUS Assay Level.

35S promoter from the Cauliflower Mosaic Virus (pCaMV) is a constitutive promoter commonly used in plant genetic transformation while Cassava Mosaic Virus (pCsVMV) is another promoter which is underutilized. The combination of the two promoters was used to form (pOYE153). The method adopted includes the insertion of a β–glucuronidase reporter gene (UidA) into a promoter cassette comprising the CsVMV promoter. The second construct (pCAMBIA2310) had (pCaMV) used for the selectable marker and gene of interest. This construct was mobilized into Agrobacterium tumefaciens strain LBA4404 and then tested for expression of the UidA gene in transient assays in cassava somatic embryos. After co-cultivation of these Agrobacterium with the plant tissues, histochemical β–glucuronidase (GUS) assays were performed to determine the level of UidA gene expression in transient assays. The results showed that the pCsVMV was able to drive high gene expression of β–glucuronidase reporter gene (UidA) in the transient assays in cassava somatic embryo. Expression of the gene also increases with the increase in the day of cocultivation and likewise expression of the gene was higher for the sample in the light than the dark.

Improvement Germination and Conversion of Somatic Embryos and Production of Normal Plantlets in Ferula Assa-foetida L. (A Rare Medicinal Plant).

Secondary somatic embryogenesis leads to the formation of abnormal somatic embryos and produces abnormal seedlings. Normal plants are difficult to obtain from these embryos, due to the asynchronous maturation of the embryogenic tissues and low germination and conversion rates. The effects of some media additives and different strengths of MS medium on germination and plantlet formation of in vitro derived somatic embryos of Ferula Assa-foetida were studied. The highest number of normal embryos was observed in MS medium containing 30g/l sucrose with 0.5% or 1% AC and in MS medium supplemented with PEG and 0.5% or 1% AC. The treatments of MS medium with 30g/l sucrose and 0.5% AC × MS medium containing sorbitol and MS medium containing PEG and 1% AC × ½ MS had maximum number of normal germinated embryos without secondary somatic embryogenesis (SSE). In some of the treatments the embryos were converted better than the others, such as; the interaction effect of MS medium with 30g/l sucrose and 0.5% AC× MS, MS medium with 30g/l sucrose × MS medium with glutamine. Using different strength of MS medium and presence of some media additives is effective on germination and conversion of somatic embryos into normal plantlet. Presence of Activated Charcoal in the culture medium can reduce secondary somatic embryogenesis.

In vitro flowering – A system for tracking floral organ development in Dendrocalamus hamiltonii Nees et Arn. ex Munro.

Dendrocalamus hamiltonii plants are slender and tall (15-25 m) thereby, rendering tagging, sampling and tracking the development of flowers difficult. Therefore, a reproducible system of in vitro flowering was established for tracking the stages of flower development. MS medium supplemented with 2.22 µM 6-benzylaminopurine, 1.23 µM indole-3-butyric acid and 2% sucrose was optimized as the flower induction medium (FIM) wherein 28 and 42 days were required for the development of gynoecium and androecium, respectively. Six distinct stages of in vitro flower development were identified, and the flowers were comparable with that of in planta sporadic flowers. Pollen viability of the in vitro flowers was higher than those of in planta ones. The in vitro system developed in the present study facilitates easy tracking of different stages of flower development under controlled environmental conditions. It can also be used for medium- or long-term storage of pollens and manipulation of in vitro fertilization.

Somatic embryogenesis and plant regeneration in Wedelia calendulacea Less. an endangered medicinal plant

In this work, plant regeneration via somatic embryogenesis was achieved from leaf and internode derived callus of Wedelia calendulacea, an endangered medicinal plant. Primary callus was induced by culturing leaf disc and internode explant on Murashige and Skoog medium supplemented with 2.0 mg L-1 of 2,4-D under light condition. Transfer of embryogenic callus on a reduced concentration of 2,4-D facilitated somatic embryo development while calluses remained unorganized at the same 2,4-D level. A histological analysis confirmed somatic embryo by revealing the presence of a closed vascular system in the developing embryos and lack of a vascularconnection with surrounding callus tissues. Somatic embryos germinated into plantlets upon transfer on MS medium containing 1.0 mg L-1 BAP plus 0.5 mg L-1 GA3. Plantlets were acclimatized successfully and survived under soil condition. This is the first on somatic embryogenesis of W.calendulacea. This result could facilitate genetic transformation of this important medicinal plant.

Propagação in vitro de genótipos de alface via embriogênese somática / In vitro propagation of lettuce genotypes via somatic embryogenesis

A propagação in vitro via embriogênese somática é uma alternativa eficiente para a propagação em larga escala do material vegetal. No entanto, não há relatos do desenvolvimento de protocolos completos de embriogênese somática, com produção de plantas de alface. O presente trabalho teve como objetivo estabelecer a propagação in vitro de genótipos de alface, Paris White e Red Salad Bowl, avaliando a indução dos calos embriogênicos, regeneração dos embriões somáticos e posterior conversão em plantas. Para a indução de embriogênese somática, duas fontes de explante (folhas cotiledonares inteiras e seccionadas) foram cultivadas em meio MS+10,75µM de ANA+0,89µM de BA. A proliferação dos calos embriogênicos foi realizada em meio MS+24µM de AIA+0,15µM de BA. Para a maturação dos embriões somáticos e conversão em plantas, utilizou-se meio MS sem regulador de crescimento (semi-sólido sem carvão ativado e com carvão ativado e meio líquido). A fonte do explante seccionada foi estatisticamente superior apenas para o genótipo Paris White. Para a regeneração dos embriões somáticos, observou-se que, no genótipo Paris White, o meio líquido foi superior estatisticamente, quando comparado aos meios semi-sólidos e, para o genótipo Red Salad Bowl, foi o meio semi-sólido com acréscimo de carvão ativado. Tendo em vista o potencial da aplicabilidade comercial da embriogênese somática para a produção em larga escala de plântulas de alface do genótipo Paris White, os protocolos de indução, proliferação, maturação dos embriões somáticos e conversão em plantas, foram adequados, em especial, em sistema líquido.

In vitro propagation via somatic embryogenesis is an efficient alternative to large-scale propagation of plant material. Nevertheless, there are no reports of development of complete somatic embryogenesis protocols, with plant production of lettuce. This study aimed to establish lettuce in vitro propagation of Paris White and Red Salad Bowl genotypes, evaluating embryogenic callus induction, somatic embryos regeneration and subsequent conversion into plants. For somatic embryogenesis induction, two sources of explants (whole and sectioned cotyledons) were grown on MS medium containing 10.75µM NAA and 0.89µM BA. Callus proliferation occurred in MS medium supplemented with 24µM AIA and 0.15µM BA. For somatic embryos maturation and conversion into plants, we used MS medium devoid of growth regulator under three conditions: semi-solid medium with and without activated charcoal, and liquid medium. The sectioned explant source was statistically superior only for genotype Paris White. For somatic embryos regeneration, we observed that, in genotype Paris White, the liquid medium was statistically higher than semi-solid media, and for genotype Red Salad Bowl, activated charcoal-added semi-solid medium was the better. Considering the potential commercial applicability of somatic embryogenesis for seedlings mass production of lettuce Paris White genotype, somatic embryos induction, proliferation, maturation and conversion into plants protocols were adequate, especially in liquid system.

In vitro culture of petiole longitudinal thin cell layer explants of vietnamese ginseng (PANAX VIETNAMENSIS HA ET GRUSHV.) and preliminary analysis of saponin content.

The present work describes a procedure that allows for the easy and rapid induction of somatic embryos, calli, shoots and adventitious roots of Vietnamese ginseng (Panax vietnamensis Ha et Grushv.) from longitudinal thin cell layers (lTCLs). In order to investigate the morphogenesis of this medicinal plant, the effect of separately–supplemented plant growth regulators and combinatorial effect of co–supplemented auxins and cytokinins in dark or under 16-hour photoperiod was examined. After eight weeks of culture, the lTCL explants excised from petiole of three-month-old in vitro plants and cultured on a semi solid basal Murashige and Skoog (MS) media supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1 mg/l thidiazuron (TDZ) in dark, 2.0 mg/l α-napththaleneacetic acid (NAA) in dark and 1.0 mg/l 2,4-D under light gave the highest rate of callogenesis (100%), embryogenesis (53.3%) and adventitious root formation (100% with a mean of 16.7 roots), and shoot formation (26.7%), respectively. The metabolite of petiole lTCL-derived calli qualitative and quantitative analyses were performed by using high-performance liquid chromatography and thin layer chromatography. The simple procedure, together with similar saponin profiles between the resulted in vitro tissues and plants grown in nature, suggest its potential use in generating Vietnamese ginseng in large amount for medicinal purpose.

Efecto de la densidad de inóculo en la multiplicación y diferenciación de suspensiones celulares embriogénicas en el cultivar híbrido de banano FHIA-18 (Musa spp AAAB) / The effect of inoculum density on embryogenic cell suspension proliferation and differentiation in banana hybrid cultivar FHIA-18 (Musa spp AAAB)

La obtención de un sistema de regeneración eficiente por medio de la embriogénesis somática en las Musaceas, es hoy una gran herramienta ante los enormes problemas que presenta este género con el ataque de enfermedades como la sigatoka negra. El objetivo del trabajo es determinar las densidades celulares adecuadas para las etapas de multiplicación de suspensiones celulares embriogénicas y formación de los embriones somáticos en medios de cultivo líquidos. Como material vegetal se usaron brotes inmaduros de la inflorescencia masculina de Musa AAAB, cv. FHIA-18. Los resultados demostraron que es posible el establecimiento de suspensiones celulares homogéneas a partir de embriones somáticos en etapa globular, y obtener los mayores volúmenes de biomasa celular al multiplicar dichas suspensionescon una densidad del 3% del volumen de células sedimentadas. A partir del decimoquinto día en el medio de cultivo de formación de embriones comenzaron a formarse estructuras compuestas por proembriones y embriones somáticos en etapa globular; entre las densidades estudiadas los mejores resultados se obtuvieron con 100 mgMF en la cual se formaron 1 871 ES.l-1 con un peso de 248 mgMF.l-1.

An extremely useful tool for dealing with the enormous problems involved in banana growing (Musaceae) caused by the attack of diseases such as black Sigatoka can be obtained today by ensuring an efficient regeneobration system via somatic embryogenesis. The work was aimed at defining appropriate cell densities for embryogenic cell suspension growth stages and somatic embryo formation in liquid culture medium. Immature male inflorescence buds from Musa AAAB cf FHIA-18 were used as vegetal material. The results showed that it is possible to establish homogeneous cell suspensions from somatic embryos in globular stage andobtain greater cell biomass volume by multiplying the suspension with 3% sedimented cell volume (density).Embryos began to form structures in culture medium consisting of globular stage somatic proembryos and embryos from the fifteenth day onwards. The best results amongst the densities studied were obtained with 100 mgMF, in which 1,871 ES.l -1 were formed weighing 248 mgMF.l-1.

In vitro regeneration of Didymopanax morototoni

The present study aimed at establishing a complete plant regeneration protocol for Didymopanax morototoni (matchwood), a native Brazilian forest species. Four types of explants (root, shoot, node, and cotyledonary leaves) were obtained from in vitro germinated seeds. In the first step, woody plant medium (WPM) with casein hydrolysate (250 mgL-1 ) and 2,4-D (1.0 and 5.0 mgL-1) were used combined with kinetin (0.1 and 1.0 mgL-1). Twenty days after inoculation, the material was evaluated. Embryogenic calli were split, transferred to expression medium with several combinations of NAA and KIN, and moved to fresh medium after 60 days. Light did not interfere in embryo expression. Somatic embryos were formed either from individual cells or cell clusters. Plantlets were obtained in WPM medium and 10 gL-1 of sucrose with no plant regulator, or using 0.1 mgL-1 BAP and 0.5 mgL-1GA. Plantlets from somatic embryos of D. morototoni developed in 33% of the cases.

O presente estudo visou o estabelecimento de um completo protocolo de regeneração para Didymopanax morototoni (morototó, caixeta) uma espécie florestal nativa do Brasil. Quatro tipos de explantes (raiz, caule, nódulo foliar e folha cotiledonar) foram obtidos a partir de sementes germinadas. Na primeira etapa, meio WPM com caseína hidrolisada (250 mgL-1) e 2,4D (1,0 e 5,0 mgL-1) foram usados em combinação com cinetina (0,1 ou 1,0 mg L-1). Vinte dias depois de inoculado, o material foi avaliado. Calos embriogênicos foram divididos e transferidos para meio de expressão com várias combinações de ácido naftaleno-acético e cinetina, e repicados a cada 60 dias para meio novo. A luz não interferiu na expressão embriogênica. Embriões somáticos foram formados ou de células individuais ou de agregados de células. As plântulas foram obtidas no meio WPM com 10 g L-1 de sacarose e sem reguladores de crescimento ou com 0,1 mg L-1 de Benzil-adenina e 0,5 mg L-1 de giberelina. O desenvolvimento das plântulas a partir de embriões somáticos de D. morototoni foi alcançada em 33% dos casos.

Uniform design for in vitro somatic embryogenesis and plant regeneration of Gymnadenia conopsea / 中草药

Objective Using stem tip of Gymnadenia conopsea in this experiment to investigate somatic embryogenesis and plant regeneration in vitro.Methods Uniform dedign for the most suitable media for embryogenic callus induction and embryogenic cell complex,development of somatic embryo and plant regeneration were screened.Results The results showed that Chaturvedi and Mitra(CM)+6-BA 1.0 mg/L+IAA 2.0 mg/L+2,4-D 0.1 mg/L was fit for embryogenic callus and embryogenic cell complex induction.Percentage was 98%;The medium of development of somatic embryo and plant regeneration was CM+6-BA 1.00 mg/L+IAA 0.10 mg/L.Percentage was 100% and converted into plantlets with shoots and roots after 50 d culture on the same medium.Conclusion The observation of morphostructure and ultrastructure proves the process of somatic embryogenesis of G.conopsea.