Agrobacterium-mediated transformation of the wild orchid Cattieya maxima Lindi / Transformación mediada por Agrobacterium de la orquídea silvestre Cattleya maxima Lindl / Transformação da orquídea silvestre Cattleya maxima Lindl mediada por Agrobacterium

Abstract Protocorms are unique anatomical structures; they are akin to rhizoids and are formed by young orchid seedlings under physiological conditions. Explanted orchid tissues produce similar structures called protocorm-like bodies (PLBs) when exposed to appropriate in vitro growing conditions. Both the propagative nature of PLBs and the easiness by which they can be generated, make these structures an attractive alternative to seed-mediated production for growing large numbers of plants. To increase somatic embryogenesis and optimize the procedure, PLBs of Cattleya maxima were transformed using the Agrobacterium tumefaciens method. The T-DNA carried a Hygromycin-resistance gene, a visible marker (GFP5-GUSA) and a rice gene encoding the Somatic Embryogenesis Receptor Kinase, deemed to be important for somatic embryogenesis. Treated PLBs generated somatic embryos developing Hygromycin-resistant plantlets. The insertion of T-DNA was confirmed by PCR, and GFP expression was observed using a fluorescent stereomicroscope. Transformed Cattleya maxima PLBs were more efficient in forming somatic embryos (60 - 80%) than untransformed controls (45 - 57%), and this contrast was maximized in hormone-free, Murashige and Skoog (MS) medium (80% of the transformed plants compared to 57% of the untransformed ones). This finding supports the notion that SERK plays an important role in Orchid embryogenesis.

Resumen Los protocormos son estructuras anatómicas únicas: son similares a los rizoides y se forman por vástagos jóvenes de orquídeas bajo condiciones fisiológicas. Los tejidos explantados de orquídeas producen estructuras llamadas Cuerpos Similares a Protocormos (PLBs) cuando están expuestos a condiciones apropiadas de crecimiento in vitro. Tanto la naturaleza propagativa de los PLBs como la facilidad con que se generan, hacen de estas estructuras una alternativa atractiva, frente a la mediada por semillas, para la producción de gran número de plantas en crecimiento. Para aumentar la embriogénesis somática y optimizar el procedimiento, se transformaron PLBs de Cattleya maxima usando el método de Agrobacterium tumefaciens. El T-DNA portaba un gen de resistencia a la Higromicina, un marcador visible (GFP5-GUSA) y un gen de arroz que codificaba para el receptor tipo quinasa de embriogénesis somática (SERK), considerado importante en la embriogénesis somática. Los PLBs tratados generaron embriones somáticos y desarrollaron plántulas resistentes a la Higromicina. La inserción del T-DNA se confirmó por PCR, y la expresión de GFP se observó usando un estereomicroscopio fluorescente. Los PLBs transformados de Cattleya maxima fueron más eficientes en desarrollar embriones somáticos (60-80%) que los controles no transformados (45-57%) y este contraste se maximizó en medio Murashige y Skoog (MS) libre de hormonas (80% de las plantas transformadas en comparación con 57% de las no transformadas). Estos hallazgos apoyan la noción de que SERK juega un papel importante en la embriogénesis de orquídeas.

Resumo Os protocormos são estruturas anatômicas únicas: são similares aos rizoides e se formam por hastes jovens de orquídeas sob condições fisiológicas. Os tecidos explantados de orquídeas produzem estruturas chamadas Corpos Similares a Protocormos (PLBs) quando estão expostos a condições apropriadas de crescimento in vitro. Tanto a natureza propagativa dos PLBs como a facilidade com que se generam, fazem com que estas estruturas sejam uma alternativa atrativa, comparativamente a mediada por sementes, para a produção de grandes números de plantas em crescimento. Para aumentar a embriogênesis somática e otimizar o procedimento, se transformaram PLBs de Cattleya maxima utilizando o método de Agrobacterium tumefaciens. O T-DNA carregava um gen de resistencia a Higromicina, um marcador visível (GFP5-GUSA) e um gen de arroz que codificava para o receptor tipo quinasa de embriogênesis somática (SERK), considerado importante na embriogênesis somática. Os PLBs tratados geraram embriões somáticos e desenvolveram plântulas resistentes a Higromicina. A inserção do T-DNA se confirmou por PCR, e a expressão de GFP se observou utilizando um estereomicroscópio de fluorescência. Os PLBs transformados de Cattleya maxima foram mais eficientes em desenvolver embriões somáticos (60-80%) que os controles não transformados (45-57%) e este contraste se potencializou em meio Murashige y Skoog (MS) livre de hormônios (80% das plantas transformadas em comparação com 57% das não-transformadas). Estes resultados apoiam a noção de que SERK desempenha um papel importante na embriogênesis de orquídeas.

Somatic embryogenesis in Carica papaya as affected by auxins and explants, and morphoanatomical-related aspects

ABSTRACT The aim of this study was to evaluate somatic embryogenesis in juvenile explants of the THB papaya cultivar. Apical shoots and cotyledonary leaves were inoculated in an induction medium composed of different concentrations of 2,4-D (6, 9, 12, 15 and 18 µM) or 4-CPA (19, 22, 25, 28 and 31 µM). The embryogenic calluses were transferred to a maturation medium for 30 days. Histological analysis were done during the induction and scanning electron microscopy after maturing. For both types of auxin, embryogenesis was achieved at higher frequencies with cotyledonary leaves incubated in induction medium than with apical shoots; except for callogenesis. The early-stage embryos (e.g., globular or heart-shape) predominated. Among the auxins, best results were observed in cotyledonary leaves induced with 4-CPA (25 µM). Histological analyses of the cotyledonary leaf-derived calluses confirmed that the somatic embryos (SEs) formed from parenchyma cells, predominantly differentiated via indirect and multicellular origin and infrequently via synchronized embryogenesis. The secondary embryogenesis was observed during induction and maturation phases in papaya THB cultivar. The combination of ABA (0.5 µM) and AC (15 g L-1) in maturation medium resulted in the highest somatic embryogenesis induction frequency (70 SEs callus-1) and the lowest percentage of early germination (4%).

Desenvolvimento e multiplicação in vitro de embriões obtidos por embriogênese somática direta de Coffea arabica L. CV. Acaiá Cerrado / Development and multiplication in vitro of embryo obtained by direct somatic embryogenesis of Coffea arabica L. CV. Acaiá Cerrado

Um importante método de multiplicação in vitro de plantas de Coffea é a embriogênese somática, que consiste no desenvolvimento de embrióides a partir de células haplóides ou somáticas possibilitando a micropropagação acelerada de clones superiores e a manutenção de híbridos interespecíficos. Entretanto, há poucos relatos da obtenção de embriogênese direta na espécie Coffea arabica. Objetivou-se com este trabalho estabelecer protocolo para desenvolvimento in vitro de embriões somáticos e produção de mudas de C. arabica cultivar Acaiá Cerrado. Avaliaram-se a influência de sacarose (0; 30; 60; 90 e 120 g L-1) x BAP (0; 2; 4 e 8 mg L-1) em embriões somáticos. No desenvolvimento das plântulas, testaram-se ANA (0; 0,25; 0,5 e 1mg L-1) x GA3 (0; 2,5; 5,0; 10 e 20 mg L-1) em brotações, com tamanho médio de 1 a 1,5 cm, oriundas de embriões somáticos in vitro. Durante a etapa de indução de embriões, o experimento foi conduzido em sala de crescimento em condições de escuro e, na etapa de desenvolvimento dos embriões e plântulas, os explantes foram submetidos à irradiância em torno de 32 µM m-2 s-1 e fotoperíodo de 16 horas, à temperatura de 25 ± 1 ºC. Na aclimatização, avaliou-se o efeito da presença e ausência de raízes em plântulas de cafeeiro oriundas de embriogênese somática direta. A adição de 90 g L-1 de sacarose e 2 mg L-1 de BAP ao meio de cultura proporciona melhor crescimento in vitro de embriões de cafeeiro. Utilizando-se 0,5 mg L-1 de ANA e 14,2 mg L-1 de GA3 obtém-se maior comprimento da parte aérea de brotações de C. arabica L. cv. Acaiá Cerrado. Conclui-se que é possível a obtenção de plântulas micropropagadas de C. arabica L. cultivar Acaiá Cerrado pela embriogênese somática direta. O enraizamento de brotações de cafeeiro cultivar Acaiá Cerrado ocorre simultaneamente ao processo de aclimatização.

An important method of in vitro plant's multiplication of Coffea is somatic embryogenesis, which consists in developing embryoid from haploid or diploid somatic cells, without the fusion of gametes allowing the accelerated micropropagation and maintenance of superior clones interspecific hybrids. However there are few reports of direct embryogenesis in Coffea arabica. The objective of this work to establish protocol for in vitro development of somatic embryos and seedlings of C. arabica Acaiá Cerrado. The influence of sucrose (0, 30, 60, 90 and 120 g L-1) x BAP (0, 2, 4 and 8 mg L-1) on somatic embryos from embryogenic were evaluated. In seedling development, NAA (0, 0.25, 0.5 and 1 mg L-1) x GA3 (0, 2.5, 5.0, 10 and 20 mg L-1) in shoots were tested, with average size of 1 to 1.5 cm, derived from somatic embryos in vitro. During the development stage of embryos and embryonic seedling induction, the experiment was conducted in a growth chamber under conditions of darkness and the stage of development of embryos and seedlings, the explants were subjected to irradiation around 32 mM m- 2 s-1 and a photoperiod of 16 hours at a temperature of 25 ± 1 º C. In acclimatization, the effect of the presence and absence of roots in coffee seedlings originating from direct somatic embryogenesis were evaluated. The addition of 90 g L-1 sucrose and 2 mg L-1 BAP to the culture medium provides a better in vitro growth of embryos from coffee. Using 0.5 mg L-1 NAA and 14.2 mg L-1 GA3 obtains greater shoot length of shoots of C. arabica L. cv. Acaiá Cerrado. It was concluded that it is possible to obtain plantlets C. arabica L. Acaiá Cerrado by direct somatic embryogenesis. The rooting of shoots of coffee Acaiá Cerrado occurs simultaneously with the process of acclimatization.

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.

The effects of cadmium chloride on secondary metabolite production in Vitis vinifera cv. cell suspension cultures

BACKGROUND: Plant secondary metabolites are possess several biological activities such as anti-mutagenic, anti-carcinogenic, anti-aging, etc. Cell suspension culture is one of the most effective systems to produce secondary metabolites. It is possible to increase the phenolic compounds and tocopherols by using cell suspensions. Studies on tocopherols production by cell suspension cultures are seldom and generally focused on seed oil plants. Although fresh grape, grape seed, pomace and grape seed oil had tocopherols, with our best knowledge, there is no research on tocopherol accumulation in the grape cell suspension cultures. In this study, it was aimed to determine the effects of cadmium chloride treatments on secondary metabolite production in cell suspension cultures of grapevine. Cell suspensions initiated from callus belonging to petiole tissue was used as a plant material. Cadmium chloride was applied to cell suspension cultures in different concentration (1.0 mM and 1.5 mM) to enhance secondary metabolite (total phenolics, total flavanols, total flavonols, trans-resveratrol, and α-, ß-, γ- δ-tocopherols) production. Cells were harvested at two days intervals until the 6th day of cultures. Amounts of total phenolics, total flavanols and total flavonols; trans-resveratrol and tocopherols (α-, ß-, γ- and δ-tocopherols) and dry cell weights were determined in the harvested cells. RESULTS: Phenolic contents were significantly affected by the sampling time and cadmium concentrations. The highest values of total phenolic (168.82 mg/100 g), total flavanol (15.94 mg/100 g), total flavonol (14.73 mg/100 g) and trans-resveratrol (490.76 µg/100 g) were found in cells treated with 1.0 mM CdCl2 and harvested at day 2. Contents of tocopherols in the cells cultured in the presence of 1.0 mM CdCl2 gradually increased during the culture period and the highest values of α, ß and γ tocopherols (145.61, 25.52 and 18.56 µg/100 g) were detected in the cell cultures collected at day 6. CONCLUSIONS: As a conclusion, secondary metabolite contents were increased by cadmium chloride application and sampling time, while dry cell weights was reduced by cadmium chloride treatments.

Induction of secondary metabolite production by UV-C radiation in Vitis vinifera L. Öküzgözü callus cultures

BACKGROUND: The aim of the present work was to examine the role of UV-C irradiation on the production of secondary metabolites (total phenolic, total flavanols, total flavonols, catechin, ferulic acid and trans-resveratrol in phenolic compounds and α-, ß-, γ- δ-tocopherols) in callus cultures. Studies on the effects of UV-C treatment on callus culture are seldom and generally focused on UV-B. However UV-C radiation play an important role in accumule secondary metabolites. RESULTS: In this study, callus cultures from Öküzgözü grape cultivar were initiated from leaf petiole explants. Calli formed after 6 weeks on the medium supplemented with 0.5 mg L-1 benzylaminopurine (BA), 0.5 mg L-1 indole acetic acid (IAA) on B5 media. Callus tissues were exposed to UV-C irradiation at 10, 20 and 30 cm distances from the UV source for 5 and 10 minutes and samples were collected at hours 0, 24 and 48. CONCLUSIONS: The greatest total phenolic content (155.14 mg 100 g-1) was detected in calli exposed to UV-C for 5 min from 30 cm distance and sampled after 24 h. 24 h and 48 h incubation times, 30 cm and 5 min were the most appropriate combination of UV-C application in total flavanol content. Maximum total flavonol content (7.12 mg 100 g-1) was obtained on 0 h, 5 min and 20 cm combination. The highest (+)- catechin accumulation (8.89 mg g-1) was found in calli with 10 min UV-C application from 30 cm distance and sampled after 48 h. Ferulic acid content increased 6 fold in Öküzgözü callus cultures (31.37 µg g-1) compared to the control group. The greatest trans-resveratrol content (8.43 µg g-1) was detected in calli exposed to UV-C for 5 min from 30 cm distance and sampled after 24 h. The highest α-tocopherol concentration was found in calli exposed to UV-C for 10 min from 30 cm distance and sampled after 24 h. As a conclusion, it was showed that UV-C radiation had remarkable promoting effects on the accumulation of secondary metabolites in the calli of Öküzgözü grape cultivar.

Effective B-lactam antibiotics for Agrobacterium tumefaciens suppression in indica rice calli / Antibióticos beta-lactámicos efectivos para eliminar el Agrobacterium tumefaciens en callos de arroz índica

It was evaluated the antibacterial activity of seven β-lactam antibiotics against A. tumefaciens strain EHA105, living in indica rice calli. For detection of persistent Agrobacterium in callus tissues, homogenates of infected calli were spread over LB agar plates to count colony forming units per gram of calli. Agrobacterium growth was completely suppressed during plant regeneration at 250 mg/l of all of the antibiotics tested. Also it was appraised the effect of β-lactams on callus growth and plant regeneration. A similar tendency of increased calli fresh weight with 100 mg/l and 250 mg/l of all antibiotics was proven. But the use of 500 mg/l caused decreasing of callus growth. About 80% of calli formed shoots in a month when we used 100 mg/l or 250 mg/l of β-lactam during the regeneration stage. Two morphogenic responses were distinguished during regeneration stage: somatic embryogenesis and adventitious shoots organogenesis.

Se evaluó la actividad antibacteriana de siete antibióticos β-lactámicos sobre la cepa EHA105 de Agrobacterium tumefaciens inoculada en callos de arroz. Para detectar el Agrobacterium persistente en los callos, se cultivó en medio LB sólido un extracto de callos infectados, y se contó el número de unidades formadoras de colonias por gramo de callo. La bacteria se eliminó totalmente en la fase de regeneración utilizando 250 mg/l de cualquiera de los antibióticos probados. Se evaluó además el efecto de los β-lactámicos sobre el crecimiento de los callos y la regeneración de plantas. El incremento del peso de los callos fue similar cuando se utilizó 100 y 250 mg/l de antibiótico, pero disminuyó significativamente cuando la concentración se elevó a 500 mg/l. La regeneración de plantas ocurrió a partir del 80% de los callos cultivados con 100 y 250 mg/l de antibiótico. En la regeneración se distinguieron dos respuestas morfogénicas: embriogénesis somática y organogénesis adventicia.

Assessment of somatic embryogenesis potency in Indian soybean [Glycine max (L.) Merr.] cultivars.

Majority of the Indian soybean cultivars are recalcitrant to tissue culture regeneration. The present communication reports the development of somatic embryogenesis in a liquid culture medium from immature cotyledons of G. max. Following induction with 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthalene acetic acid (NAA), the number of somatic embryos and percentage of explants that responded were higher with 45.24 µM 2,4-D. The proliferation of somatic embryos for three successive cycles was achieved in 22.62 µM 2,4-D. Histodifferentiation of somatic embryos under NAA (10.74 µM) indicated that better embryo development and maturation was achieved without any growth regulator. The amino acids such as L-glutamine favoured the somatic embryo induction and histodifferentiation at 20 and 30 mM respectively, where as L-asparagine at 10 mM concentration enhanced the somatic embryo proliferation. In addition, somatic embryos that were desiccated (air-drying method) for 5 days showed better germination (40.88%). The Indian soybean cultivars also showed strict genotypic influence and cv. Pusa 16 was emerged as a best responding cultivar for somatic embryo induction with 74.42% of response.

Somatic embryogenesis and plant regeneration capacity in Argentinean maize (Zea mays L. ) inbred lines

Somatic embryogenesis, which is still the method of choice for tissue culture, regeneration and transformation of maize, is largely considered highly genotype-dependent. The Hi II, a highly embryogenic genotype, has been extensively used in transformation protocols. However, this is not an inbred line; instead, it has a proportion of the undesirable A-188 background, and the progeny segregates for phenotypic characteristics and shows poor agronomic performance. In an effort to identify genotypes that combine a high somatic embryogenic response and good agronomic performance, we evaluated 48 advanced inbred lines developed at INTA. Callus development and somatic embryogenesis capacity were measured in 200 immature embryos per line. Embryogenic capacity [EC (mature somatic embryos/callus evaluated) x 100], Regeneration Capacity (RC) and Fertile Plant Recovery in greenhouse (FPR, fertile plants/regenerated plants) were recorded. A total of 17 lines reached an EC > 50 percent, and 14 out of those 17 lines regenerated seedlings. The FPR ranged between 50 and 100 percent. Also, we selected three promising lines with high agronomic performance, as alternatives to Hi II, in order to be included in a maize transformation scheme via somatic embryogenesis. In addition, we report the usefulness of Single Sequences Repeat (SSRs) in the determination of genetic diversity among 14 divergent lines for somatic embryogenesis response. The seven lines displaying good in vitro behaviour can be crossed to obtain hybrids combining desirable alleles for somatic embryogenesis response and different genetic backgrounds.

Study on plant regeneration from somatic embryos of vulnerable medicinal plant Glehnia littoralis / 中国中药杂志

<p><b>OBJECTIVE</b>To study the cause of the seeds dormancy of Glehnia littoralis in vitro and to establish plant regeneration methods via somatic embryos.</p><p><b>METHOD</b>The effects of endosperm and exogenous hormone on the seed dormancy breaking of G. littoralis and the effect of hormone concentration on embryonic callus induction and plant regeneration via somatic embryos were observed,</p><p><b>RESULTS</b>The germination rate of the seeds with 1/3 endosperm was the highest which achieved 31%. TDZ, 6-BA and GA3 treatment could not break seed dormancy but easily lead to abnormal seedlings. Embryogenic callus induction rates was up to 57% on MS supplemented with 1.0 mg x L(-1) 2,4-D. After 20 days culture, embryogenic calli were transferred to MS medium and cotyledonary embryos were formed in 40 days. The regenerated plants were obtained in 20 days.</p><p><b>CONCLUSION</b>An effective system of plant regeneration of G. littoralis was established in this study.</p>

Production and characterization of a somatic hybrid of Chinese cabbage and cabbage / 生物工程学报

In order to broaden Chinese cabbage gene pool, we conducted interspecific somatic hybridization between Chinese cabbage (Brassica campestris, 2n=20, AA) and Cabbage (B. oleracea, 2n=18, CC). Protoplasts were isolated from 10-day-old cotyledons and hypocotyls of young seedlings, and fused by 40% polyethylene glycol (PEG). Fused cells were cultured in modified K8p liquid medium supplemented with some plant growth regulators. Fusion products were characterized by their morphological, cytological and molecular biological traits. The results showed that, a total of 35 regenerated green plants were obtained from 320 calli, the plant regeneration frequency was 10.94%, and eleven of which were survived in greenhouse. All regenerants were true hybrids as confirmed by randomly amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) analysis. Ploidy levels of hybrid plants were determined by chromosome counting and flow cytometry. The sum of the chromosome number (2n = 38) from the two fusion patents were found in 36.4% of regeneratns; another 36.4% had chromosomes range to 58-60; 27.2% had more chromosomes ranges to 70-76. All regenerated plants produced normal flowers. We investigated the pollen fertility and seed set after self-pollination and backcrossing with the parental species. For hybrids with chromosomes more than 38 it was possible to obtain some seeds when they after self-pollination. Within the group of hybrids with 38 chromosomes, seed set were very variable, only 0.11 seeds per pod by self-pollination, 0.23-0.76 by open-pollination, 0.02-0.04 by backcrossing with Chinese cabbage. Progeny lines obtained by self-pollination had larger leaves and leaf shapes intermediate of the parental species. Pollen fertility was gradually recovered in the first and second progenies. The backcrossing progeny lines, as a whole, exhibited morphologies were similar to Chinese cabbage. Morphological variations were observed among the somatic hybrids and their progenies.

Agrobacterium-mediated genetic transformation of secondary somatic embryos in alfalfa / 生物工程学报

We describe a genetic transformation method of secondary somatic embryogenesis in alfalfa through cotyledon-stage somatic embryos of alfalfa infected by Agrobacterium strain GV3101. The Agrobacterium strain GV3101 contained binary vector pCAMBIA2301 that had gus gene as reporter and npt II gene as selectable marker. The infected primary embryos were induced through series of medium under 75 mg/L kanamycin selection. We obtained the transgenic alfalfa plants. Then, GUS expression in different tissue of transgenic alfalfa was tested by GUS histochemical analysis. Further, the stable integration and transformation efficiency were tested by polymerase chain reaction and Southern blotting hybridization. The result showed that GUS expression was different in different organs of transgenic alfalfa; the copy number of integrated npt II gene was from 1 to 4; the transformation efficiency via primary somatic embryogenesis was 65.82%.

Regeneración vía embriogénesis somática de una variedad colombiana élite de Theobroma cacao L / Regeneration through somatic embryogenesis of an elite colombian Theobroma cacao L variety

El objetivo de esta investigación fue evaluar dos protocolos de propagación vía embriogénesis somática a partir de explantes florales en dos clones élite BIOB e ICS95 de Theobroma cacao L. Se obtuvo un 50 y 32% de callo embriogénico en ICS95 y BIOB respectivamente con el protocolo de Fontanel et al. (2002), modificado después de un periodo de cultivo de tres meses. Los embriones pasaron por fases que se correspondieron con medios de cultivo diferenciales: Inducción, Formación, Maduración y Mantenimiento. Para la embriogénesis somática secundaria se obtuvo un 23% de embriones a partir de embriones somáticos primarios en un medio, conteniendo 1mg/L de 2,4,5 T (2,4,5 Triclorofenoxiacético). Se logró, además, desarrollar enraizamiento adventicio aplicando pulsos de IBA (Ácido Indol Butírico) a 0.5mg/L y 0.5g/L durante un minuto. Las plantas enraizadas se llevaron a una mezcla de tierra: arena (1:1) para su adaptación ex vitro, obteniéndose un 66% de plantas aclimatadas. Los estudios histológicos mostraron diferentes características típicas del desarrollo embriogénico. Este es el primer reporte en el que se logra de manera exitosa la conversión hasta plántula (68%) y la adaptación ex vitro de una variedad colombiana de cacao vía embriogénesis somática primaria y secundaria.

In this research we evaluate two protocols of propagation via somatic embryogenesis from floral explants using two elite clones BIOB and ICS95 of Theobroma cacao L. We obtained 50 and 32% of embryogenic callus on ICS95 and BIOB respectively with Fontanel et al., (2002) protocol modified after three months of culture. The embryos went through four phases; Induction, Formation, Maduration and Mantenimiento which corresponded each one with different media culture. For secondary somatic embryogenesis we obtained 23% of embryos from primary somatic embryos in a medium with 1mg/L of 2,4,5 T (2,4,5 Triclorofenoxiacetic). Also we obtained plants that developed new roots applying pulses with IBA (Indol Butiric Acid) 0.5mg/L and 0.5g/L for a minute. The developed plants were moved to a mix of potting soil and sand (1:1) for their ex vitro adaptation, getting 66% of acclimatized plants. The histological analysis showed the typical characteristics of the embryogenic development. This is the first report where it is achieved the successful conversion to plantlets (68%) and ex vitro adaptation of a colombian cocoa variety via primary and secondary embryogenesis.

Factores que influyen en la embriogénesis somática in vitro de palmas (Arecaceae) / Factors affecting in vitro somatic embryogenesis of palms (Arecaceae)

La embriogénesis somática (ES) es una vía de desarrollo in vitro que presenta una serie de ventajas sobre otras técnicas utilizadas para la regeneración de palmas. Esta técnica tiene gran potencial para superar las limitaciones observadas al tratar de propagar clonalmente estas plantas utilizando yemas basales. A pesar de la conocida recalcitrancia que presentan las palmas al cultivo in vitro, si se utilizan los reguladores de crecimiento apropiados, el tipo y el estado de desarrollo del explante adecuados, así como genotipos con buena respuesta, es muy probable que se obtengan buenos resultados. Esto ha sido demostrado parcialmente en Phoenix dactylifera (palma dátil), Elaeis guineensis (palma aceitera), Bactris gasipaes (pejibaye) y Cocos nucifera (coco). También se ha logrado generar protocolos eficientes en otras palmas menos estudiadas, como Geonoma gamiova (una palma ornamental), Euterpe edulis (palmito dulce) y Areca catechu (palma de betel). La inducción de ES se ha conseguido principalmente con el uso de auxinas. De ellas, la que se ha utilizado con más frecuencia es el ácido 2,4-diclorofenoxiacético (2,4-D), aunque en algunos casos (como en pejibaye y palma aceitera) se ha usado picloram y dicamba, también con buenos resultados. Los explantes más utilizados han sido inflorescencias, ápices y segmentos basales de hojas, todos con un estado de desarrollo incipiente. También se ha visto que el tamaño del explante y el medio de cultivo juegan un papel importante en la respuesta. En este trabajo se presenta una recopilación de los trabajos más importantes sobre ES en esta familia de plantas y del efecto de varios factores sobre su establecimiento y desarrollo.

Somatic embryogenesis (SE) is an in vitro developmental pathway that exhibits a number of advantages over other techniques for regeneration of palms. This technique has great potential to overcome the limitations observed when trying to propagate these plants clonally using basal buds. Despite the known recalcitrance of palms for in vitro culture, good results can be obtained by using the appropriate growth regulators, explant type and developmental stage, as well as responsive genotypes. This has been partially observed in Phoenix dactylifera (date palm), Elaeis guineensis (African oil palm), Bactris gasipaes (peach palm) and Cocos nucifera (coconut). Efficient protocols have been also generated in less-studied palms, such as Geonoma gamiova (an ornamental palm), Euterpe edulis (Assai palm) and Areca catechu (areca palm). Induction of SE has been achieved mainly through the use of auxins. Of these, 2,4-dichlorophenoxyacetic acid (2,4-D) has been used most frequently, although in some cases (such as in peach palm and African oil palm) picloram and dicamba have been employed also with good results. The most commonly used explants are young inflorescences, apical buds and leaf-basal segments. Explant size and culture medium also play an important role in obtaining good results. This review presents a compilation of the most important publications on SE in this plant family and the effect of various factors on induction and development of this pathway.

Regulatory networks of somatic embryogenesis in plant / 生物工程学报

The somatic embryogenesis in plant is a very complicated and highly ordered process, which is regulated by many internal and external factors. Among them, gene expression and regulation are key factors. Genes encoding regulatory proteins, for example PLANT GROWTH ACTIVATOR, LEAFY COTYLEDON, BABY BOOM, SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE and PICKLE, interact with each other and form a complicated regulatory network. Recent progress on this regulatory network was reviewed on the basis of our study on the PLANT GROWTH ACTIVATOR 37 gene. In addition, future research perspectives on plant somatic embryogenesis were discussed.

Histology of somatic embryogenesis in rice (Oryza sativa cv. 5272) / Histología de la embriogénesis somática en arroz (Oryza sativa cv. 5272)

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.