Time-Dependent Proteomic Signatures Associated with Embryogenic Callus Induction in Carica papaya L.

Sex segregation increases the cost of Carica papaya production through seed-based propagation. Therefore, in vitro techniques are an attractive option for clonal propagation, especially of hermaphroditic plants. Here, we performed a temporal analysis of the proteome of C. papaya calli aiming to identify the key players involved in embryogenic callus formation. Mature zygotic embryos used as explants were treated with 20 µM 2,4-dichlorophenoxyacetic acid to induce embryogenic callus. Total proteins were extracted from explants at 0 (zygotic embryo) and after 7, 14, and 21 days of induction. A total of 1407 proteins were identified using a bottom-up proteomic approach. The clustering analysis revealed four distinct patterns of protein accumulation throughout callus induction. Proteins related to seed maturation and storage are abundant in the explant before induction, decreasing as callus formation progresses. Carbohydrate and amino acid metabolisms, aerobic respiration, and protein catabolic processes were enriched throughout days of callus induction. Protein kinases associated with auxin responses, such as SKP1-like proteins 1B, accumulated in response to callus induction. Additionally, regulatory proteins, including histone deacetylase (HD2C) and argonaute 1 (AGO1), were more abundant at 7 days, suggesting their role in the acquisition of embryogenic competence. Predicted protein-protein networks revealed the regulatory role of proteins 14-3-3 accumulated during callus induction and the association of proteins involved in oxidative phosphorylation and hormone response. Our findings emphasize the modulation of the proteome during embryogenic callus initiation and identify regulatory proteins that might be involved in the activation of this process.

Callus Type, Growth Regulators, and Phytagel on Indirect Somatic Embryogenesis of Coffee (Coffea arabica L. var. Colombia).

Coffee is a crop of global relevance. Indirect somatic embryogenesis has allowed plants of different coffee genotypes to be massively regenerated. The culture medium composition can affect the calli characteristics that are generated and their ability to form somatic embryos. This research aimed to determine the influence of the type of callus, growth regulators, and phytagel concentration on the embryogenic capacity of the Colombia variety. Leaf explants were cultured on Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.5-1.0 mg L-1), benzylaminopurine (BAP, 1.0 mg L-1), and phytagel (2.3-5.0 g L-1). The explants generated two types of calli: friable (beige, soft, watery, easy disintegration, polyhedral parenchyma cells) and compact (white, hard, low water content, difficult disintegration, elongated parenchyma cells). About 68% of the total callus generated was compact; this type of callus produced a greater number of embryos (71.3) than the friable one (29.2). The number of differentiated embryos was significantly affected by the concentration of phytagel; higher concentrations (5.0 g L-1) resulted in larger quantities (73.7). The highest number of embryos (127.47) was obtained by combining 1.0 mg L-1 2,4-D, 1.0 mg L-1 BAP, 5.0 g L-1 phytagel, and compact callus.

In Vitro Mass Propagation of Coffee Plants (Coffea arabica L. var. Colombia) through Indirect Somatic Embryogenesis.

Coffea arabica is one of the two most consumed coffee species in the world. Micropropagation through somatic embryogenesis has allowed the large-scale propagation of different coffee varieties. However, the regeneration of plants using this technique depends on the genotype. This study aimed to develop a protocol for the regeneration of C. arabica L. var. Colombia by somatic embryogenesis for its mass propagation. Foliar explants were cultured on Murashige and Skoog (MS) supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP), and phytagel for inducing somatic embryogenesis. In total, 90% of the explants formed embryogenic calli with a culture medium containing 2 mg L-1 of 2,4-D, 0.2 mg L-1 BAP, and 2.3 g L-1 phytagel. The highest number of embryos per gram of callus (118.74) was obtained in a culture medium containing 0.5 mg L-1 2,4-D, 1.1 mg L-1 BAP, and 5.0 g L-1 phytagel. In total, 51% of the globular embryos reached the cotyledonary stage when they were cultured on the growth medium. This medium contained 0.25 mg L-1 BAP, 0.25 mg L-1 indoleacetic acid (IAA), and 5.0 g L-1 of phytagel. The mixture of vermiculite:perlite (3:1) allowed 21% of embryos to become plants.

Somatic Embryogenesis in Citrus (Citrus spp.), var. Valencia.

Somatic embryogenesis has been obtained in many citrus cultivars; however, the efficiency of the system is genotype dependent and culture synchronization is important to reach more efficient systems. In this chapter we present a detailed protocol of somatic embryogenesis induction from nucellar tissue and the use of an alternative method of callus sieving for culture synchronization and embryo production. This is a simple method which can also be evaluated for other species aiming at better culture efficiency and somatic embryo production.

Perspectives of Somatic Embryogenesis: Concluding Remarks.

One of the main objectives to achieve in plant tissue culture is the multiplication of the available plant material, taking full advantage of the regenerative capacities of plant cells. Somatic embryogenesis leverages cell totipotency to produce new explants from a cell, thus obtaining many propagules for scientific research, industrial, or exploitation purposes. Somatic embryogenesis (ES) characterizes by being one of the most efficient techniques in plant micropropagation. However, developing an efficient plant ES protocol requires several key factors to consider, as demonstrated throughout the chapters of this book. These chapters highlight the major drivers of the success of ES in different plant species: plant growth regulators, concentration of auxins and cytokines, water deficit, photoperiod, and type of culture medium; techniques such as the use of bioreactors and Thin Cell Layer (TCL); and the influence of stress on the formation of somatic embryos. Research has been conducted to address each phase of somatic embryogenesis, either individually or for all phases. The chapters of this book cover in detail the techniques used and provide guidance that will allow readers to successfully develop all the somatic embryogenesis phases in different cultures, from cell dedifferentiation to differentiation.

Optimization of the Transformation Protocol for Increased Efficiency of Genetic Transformation in Hevea brasiliensis.

The recurring growth of bacterium in newly developed resistant cells and a minimal level of bacterial infection rate are the main limiting factors of Agrobacterium-mediated transformation experiments in Hevea brasiliensis. The current study aimed to optimize crucial factors of the transformation protocol in order to obtain an efficient transformation experimental model for Hevea using cotyledonary somatic embryos as explants. Transformation conditions such as antibiotic concentration, preculture duration, Agrobacterium concentration, sonication and cocultivation conditions were analyzed using the binary vector pCAMBIA2301. Transient transformation was confirmed by GUS histochemical staining. The best transformation efficiency was observed when the explants were not cultured on a preculture medium that contained acetosyringone at a level of 100 µM. The best results were obtained using a bacterial density of 0.45 at OD 600 nm, 50 s of sonication of explants in a bacterial liquid culture and a total incubation time of 18 min in the same bacterial suspension. Transmission electron microscopical analysis confirmed the impacts of sonication on bacterial infection efficiency. Cocultivation conditions of 22 °C and 84 h of darkness were optimal for the transfer of T-DNA. Agrobacterium was eliminated with 500 mg/L of timentin, and the selection of transformants was performed using 100 mg/L of kanamycin in the selection medium. The presence of transgene was confirmed in the resistant embryos by polymerase chain reaction (PCR). The improved method of genetic transformation established in the present study will be useful for the introduction of foreign genes of interest into the Hevea genome for the breeding of this economically important plant species in the future.

Indirect somatic embryogenesis of Piper hispidinervum L. and evaluation of the regenerated plants by flow cytometry.

BACKGROUND: Piper hispidinervum is a species native from the Amazon region with great economic potential, given its scientifically proven insecticidal properties. In this study, an efficient protocol of plant regeneration via indirect somatic embryogenesis has been established for the first time. In a first experiment, for the induction of calluses, foliar explants of non-discriminated accesses of P. hispidinervum were inoculated in MS medium supplemented with α-naphtalenacetic acid (NAA) and 6-benzylaminopurine (BAP), in different combinations. For a second experiment, foliar explants from five different accesses of P. hispidinervum (PH17, PH21, PH28, PH37, and PH39) were analyzed regarding the formation of calluses when cultivated in MS medium with 5 mg L-1 NAA + 2.5 mg L-1 BAP. To obtain somatic embryos-like structures, calluses were cultivated in MS medium with 10 mg L-1 NAA + 2.5 mg L-1 of BAP. The somatic embryos-like structures obtained were inoculated in MS medium devoid of growth regulators and the plantlets were subjected to acclimatization. Calluses and somatic embryos-like structures were subjected to anatomical analysis and genetic stability of regenerated plants was analyzed by flow cytometry. RESULTS: The treatments 2.5 mg L-1 BAP and 5 mg L-1 NAA + 2.5 mg L-1 BAP, after 60 days of cultivation, provided each 32% of primary callus, not being verified the formation of calluses in medium devoid of BAP. It was found that accesses differed among them with respect to the formation of primary calluses, with emphasis on accesses PH28, PH37, and PH39, with mean percentage of 95.3%. Regarding the percentage of embryogenic calluses and formation of somatic embryos-like structures, there were no statistical differences between accesses, with mean values of 90.6% and 77.3%, respectively. The somatic embryos-like structures of P. hispidinervum have conspicuous morphoanatomical similarities with the zygotic embryo, and flow cytometry analysis showed no significant variation in nuclear DNA size among plants regenerated in vitro and plants coming from seed germination, which indicates ploidy level stability. CONCLUSION: This protocol is the first cited in the literature that demonstrates an efficient micropropagation process by somatic embryogenesis of P. hispidinervum. It can be used either to enable large-scale vegetative production or to subsidize germplasm conservation or genetic engineering of P. hispidinervum.

Somatic embryogenesis from flower tepals of Hippeastrum aiming regeneration of virus-free plants.

Hippeastrum hybridum is an important bulbous flower plant in world floriculture, which are propagated conventionally by the technique known as double or twin scales to obtain plants with clonal origin. However, this technique promotes the propagation of systemic diseases, particularly mosaic-inducing viruses. The aim of this paper was to evaluate the somatic embryogenesis (SE) from tepals as an alternative to provide a technique for SE induction and to obtaining virus-free plantlets of Hippeastrum from infected plants. The concentrations of 2,4-Dichlorofenoxiacetic Acid (2,4-D) and thidiazuron (TDZ) was evaluated in SE induction pathway. The monitoring of viruses during the assays with tepals was performed by Reverse Transcription-Polymerase Chain Reaction. SE induction was obtained, for the first time, in tepal segments from flower buds of Hippeastrum. The 2,4-D was the main factor for embryogenic callus induction, and TDZ increased the SE induction rate. However, conversion of somatic embryos into plantlets were only developed in free-2,4-D media, replaced by 1.0 mg L-1 6-Benziladenine. Out of five virus species monitored during the experiment, Cucumber mosaic virus was detected in tepals and Hippeastrum mosaic virus in leaves of donor plants. The SE-derived plantlets that germinated in vitro were acclimatized and tested negative for all viruses assayed.

Optimized somatic embryogenesis and plant regeneration in elite Argentinian sugarcane (Saccharum spp.) cultivars.

BACKGROUND: Biotechnological breeding of elite sugarcane cultivars is currently limited because of the difficulty of regenerating plants by tissue culture. Here, we report that commercially elite sugarcane genotypes, which are adapted to Argentinian agro-ecological conditions, are capable of being regenerated via indirect somatic embryogenesis. Leaf rolls of five elite genotypes were cultured following two callus induction protocols using different concentrations of 2,4-D as the growth regulator. Embryogenic calluses were regenerated under light conditions. Regenerated plants were subsequently acclimatized in the greenhouse under two acclimatization procedures before being transplanted to the field. RESULTS: Four of the five genotypes were able to form somatic embryos following the two induction protocols. The variables related to embryogenic callus production were influenced by the interaction between genotype and culture conditions. For plant regeneration, the embryogenic calluses were further cultured on an IBA-supplemented medium, where we observed a high genotype dependence. Calluses from the four cultivars regenerated a good number of plants. With the procedures described here, we obtained more than 90% of well-acclimatized plants both in the greenhouse and in the field. CONCLUSIONS: This protocol provides a simple way to regenerate sugarcane plants through indirect somatic embryogenesis. Also, the results confirm that tissue culture ability is highly genotype-dependent in sugarcane. Our findings suggest that these elite cultivars could be good candidates for biotechnological breeding.

Induction of Hairy Roots on Somatic Embryos of Rhizoclones from Typha domingensis Seedlings.

A protocol for the induction of hairy roots on somatic embryos of rhizoclones from Typha domingensis seedlings grown in hydroponic rhizotron systems was established for the first time. Rhizogenesis was induced through the agrotransformation of somatic embryos in oblong and scutellar states of development using the K599, LBA9402, and A4 strains of Agrobacterium rhizogenes. The transfection to the embryos was performed by cocultivation of rhizoclones on a Murashige and Skoog mineral medium at 50% strength (MS0.5), in the dark, at 28 ± 2 °C for 72 h. In contrast to nontransformed embryos that did not exhibit any root tissue, transformed embryos presented hairy roots that varied in number, length, and density depending on the bacterial strain, and K599 was the most effective strain. After analysis via optical microscopy, the transformed embryos were collected and transferred to fresh culture media supplemented with 400 mg mL-1 cefotaxime and 10 mg L-1 ascorbic acid. The efficiency of transformation and survival of the oblong and scutellar embryos were similar among the three bacterial strains. The results show that agrotransformation of somatic embryos of rhizoclones from T. domingensis is a novel and viable strategy for the generation of genetic transformants of Typha that have potential applications in bioremediation technologies.

Influence of Silver Nitrate on Somatic Embryogenesis Induction in Arabica Coffee (Coffea arabica L)

Abstract The influence of silver nitrate (AgNO3), benzyladenine (BAP), and indole-3-acetic acid (IAA) on low frequency somatic embryogenesis (LFSE) induction in Caturra and Catuaí arabica coffee was evaluated. For the Caturra cultivar, the production of somatic embryos was significantly increased by adding AgNO3 to the semisolid culture medium. The highest average number of somatic embryos for this cultivar was obtained using 6.6 μM BAP, 2.85 μM IAA, and 40 μM AgNO3. In contrast, for the Catuaí cultivar, the highest average number of somatic embryos was obtained using semisolid medium supplemented with 8.8 μM BAP, and 2.85 μM IAA. Using these protocols, somatic embryos were directly induced using leaf sections of in vitro plants of both coffee cultivars within 8 weeks. The somatic embryos developed into rooted plants with a 100% survival rate upon transfer to the greenhouse.

2- Isopentenyladenine in the induction of direct somatic embryogenesis capacity of Coffea arabica L / 2- Isopenteniladenina na capacidade de indução da embriogênese somática direta de Coffea arabica L

This study evaluated the effect of different concentrations of 2-isopentenyladenine (2-iP) on the direct somatic embryogenesis capacity of the Mundo Novo cultivar of Coffea arabica. Leaf explants were cultivated with half the MS salt concentration and the addition of sucrose (20gL-1) and 2-iP (0; 2.5; 5; 7.5 and 10µM). The 2-iP doses of 7.5 and 10µM produced the greatest responses with respect to the percentage of explants with embryogenic structures and the size of the embryogenic structures. However, the greatest production of somatic embryos occurred on the explants treated with 10µM of 2-iP, followed by 7.5µM, whereas their production was absent or reduced with 0 and 5µM, respectively.(AU)

Este estudo avaliou o efeito de diferentes concentrações de 2-isopentalinadenina (2-iP) na capacidade de embriogênese somática direta da cultivar Mundo Novo de Coffea arabica. Explantes foliares foram cultivados em meio com metade da concentração de sais de MS e adição de sacarose (20gL-1) e de 2-iP (0; 2,5; 5; 7,5 e 10µM). Os tratamentos com 7,5 e 10µM de 2-iP induziram respostas mais elevadas de porcentagem de explantes com estruturas embriogênicas e de tamanho de estrutura embriogênica. Porém, os explantes tratados 10µM de 2-iP tiveram maior produção de embriões somáticos, seguido de 7,5µM, enquanto nos tratamentos controle e com 5µM, as respostas foram respectivamente de ausência e de baixa formação destes.(AU)

2- Isopentenyladenine in the induction of direct somatic embryogenesis capacity of Coffea arabica L / 2- Isopenteniladenina na capacidade de indução da embriogênese somática direta de Coffea arabica L

ABSTRACT: This study evaluated the effect of different concentrations of 2-isopentenyladenine (2-iP) on the direct somatic embryogenesis capacity of the Mundo Novo cultivar of Coffea arabica. Leaf explants were cultivated with half the MS salt concentration and the addition of sucrose (20gL-1) and 2-iP (0; 2.5; 5; 7.5 and 10µM). The 2-iP doses of 7.5 and 10µM produced the greatest responses with respect to the percentage of explants with embryogenic structures and the size of the embryogenic structures. However, the greatest production of somatic embryos occurred on the explants treated with 10µM of 2-iP, followed by 7.5µM, whereas their production was absent or reduced with 0 and 5µM, respectively.

RESUMO: Este estudo avaliou o efeito de diferentes concentrações de 2-isopentalinadenina (2-iP) na capacidade de embriogênese somática direta da cultivar Mundo Novo de Coffea arabica. Explantes foliares foram cultivados em meio com metade da concentração de sais de MS e adição de sacarose (20gL-1) e de 2-iP (0; 2,5; 5; 7,5 e 10µM). Os tratamentos com 7,5 e 10µM de 2-iP induziram respostas mais elevadas de porcentagem de explantes com estruturas embriogênicas e de tamanho de estrutura embriogênica. Porém, os explantes tratados 10µM de 2-iP tiveram maior produção de embriões somáticos, seguido de 7,5µM, enquanto nos tratamentos controle e com 5µM, as respostas foram respectivamente de ausência e de baixa formação destes.

Morpho and Cytological Differentiation of Calli of Eucalyptus grandis x Eucalyptus urophylla During Somatic Embryogenesis

ABSTRACT The aim of this study was to induce and analyze embryogenic calli from two types of explants (leaves and meristems) of the hybrid Eucalyptus grandis x Eucalyptus urophylla. Leaves and meristems of plants kept in a nursery were disinfected and inoculated in Petri dishes containing MS culture medium supplemented with different concentrations of the growth regulator dicamba (1.13, 4.52, and 9.04 µM) and without it. At 60 days of culturing, the calli were analyzed by scanning electron microscopy and at 90 days were evaluated by light microscopy in regard to the embryogenic characteristics of the cells. Different type of calli were induced in leaf explants, designated as Type I with light yellow coloring, Type II with dark yellow coloring, and Type III of brown coloring; however, only Type I had embryogenic characteristics. In the meristematic explants, only one type of callus was induced, and it had embryogenic characteristics. At 90 days of culturing, the formation of somatic embryos in the different embryogenic stages was observed and the formation of procambium, protoderm, and ground meristem tissues. At 150 days of culturing, the concentration of 1.13 µM of dicamba was prominent in the formation of somatic embryos in the different embryogenic stages.

Efecto de Dicamba y de ácido 2,4 diclorofenoxiacético sobre la embriogénesis somática en caña de azúcar / Effect of Dicamba and 2,4 dichlorophenoxiacetic acid on sugarcane somatic embryogenesis

El cultivo in vitro de la caña de azúcar ha sido establecido en muchas variedades comerciales con el propósito de producir material libre de enfermedades microbianas, conservar germoplasma, detectar resistencia a enfermedades y plagas, etc. En este sentido, el objetivo de este trabajo fue analizar la efectividad de las auxinas sintéticas ácido 2,4-diclorofenoxiacético (2,4D) y ácido 3,6-dicloro-2-metoxibenzoico (Dicamba), en la inducción del proceso de embriogénesis somática y la regeneración de vitroplántulas de distintas variedades de caña de azúcar (C26670, RB855546, V99245, V756, V781, V0050, CC8592, CC8475). Para esto se cultivaron discos de hojas en fase de macollamiento, de 1 cm de diámetro y 2 mm de grosor, en medio Murashige-Skoog, 1962 (MS) suplementado con 50 ml.l-1 agua de coco, 30 g.l-1 sacarosa y dos tratamientos diferentes: 3 mg.l-1 2,4-D ó 6.63 mg.l-1 Dicamba, ambos en completa oscuridad a 25ºC, durante 1 mes. Los callos obtenidos se colocaron en medio de regeneración, conteniendo ½ sales MS, 200 ml.l-1 agua de coco y 60 g.L-1 sacarosa, incubándose bajo luz continua, 25ºC, por 2 meses. El mayor porcentaje de callo embriogénico se obtuvo en medios suplementados con Dicamba un promedio de 70,83 % de callo embriogénico por variedad ; mientras que en los medios con 2,4D se obtuvo 62,08 % de callo embriogénico por variedad. Se obtuvo un promedio de 89,00 % de plantas regeneradas a partir de los callos obtenidos en medios con Dicamba y 66,12 % de plantas a partir de callos obtenidos en medios con 2,4D. Con el uso de Dicamba se estableció un sistema eficiente de embriogénesis somática para estas variedades de caña de azúcar.

In order to conserve sugarcane germplasm, produce microbial disease-free material, detect resistance to diseases and pests, etc., in vitro propagation of sugarcane has been established in many commercial varieties. In this sense, the aim of this work was to analyze the efficiency of 2,4-dichlorophenoxyacetic acid (2,4D) and 3,6-dichloro -2- methoxybenzoic acid (Dicamba) to induce somatic embryogenesis and regeneration of plantlets from sugarcane varieties C26670, RB855546, V99245, V756, V781, V0050, CC8592, CC8475. For induction of embryogenic callus, leaf discs in tillering stage of 1 cm diameter and 2 mm thick, were inoculated on Murashige-Skoog, 1962 medium (MS), supplemented with 50 ml.l-1 coconut water, 30 g.l-1sucrose and two different treatments: 3 mg.l-1 2,4D or 6.63 mg.l-1 Dicamba, both of them in total darkness at 25 °C, during 1 month. For plant regeneration, embryogenic calli were transferred to ½ MS salts supplemented with coconut water 200 ml.l-1 and sucrose 60 g.l-1 and incubated under continuous light, 25 °C, for 2 months. The highest percent of embryogenic callus induction was obtained in media supplemented with Dicamba, an average of 70.83 % of embryogenic callus by variety, while in media with 2,4-D, 62.08 % of embryogenic callus was obtained by variety. An average of 89,00 % of plantlets was obtained from calli induced on media with Dicamba and an average of 66.12% of plantlets was obtained from calli induced on media supplemented with 2,4D. Using Dicamba it was possible to establish an efficient somatic embryogenesis protocol for these sugarcane varieties.

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.

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.

Somatic embryogenesis and pant regeneration from callus cultures of Cleome rosea Vahl

This paper describes a protocol for the efficient vegetative propagation of Cleome rosea by somatic embryogenesis. Leaf and stem explants from nursery-grown seedlings of C. rosea were cultivated on Murashige and Skoog (MS) medium supplemented with indole-3-acetic acid (IAA), a -naphthaleneacetic acid (NAA), 4-amino-3,5,6-trichloropicolinic acid (picloram) or 2,4-dichlorophenoxyacetic acid (2,4-D). Nodular calli were produced from both explant types in the presence of 4.5 and 9.0 µM 2,4-D. Embryo development and maturation were achieved when calli from stem explants were transferred to media containing a ten-fold reduction of 2,4-D concentration initially used (0.45 and 0.90 µM). Leaf-derived calli did not form embryos with the same treatments. The highest frequency of embryogenic callus formation (85 percent) and number of embryo per callus (13.45 ± 2.8) were achieved during the first subculture on medium supplemented with 0.90 µM 2,4-D. Embryo conversion into plantlets was achieved following transfer to growth regulator-free MS medium solidified with 2 g.L-1 phytagel. An acclimatization rate of 53 percent was found three months after transfer to ex vitro conditions and the recovered plants presented a normal phenotypic aspect.

O trabalho descreve um protocolo para a propagação in vitro de Cleome rosea por embriogênese somática. Explantes foliares e caulinares, obtidos de plantas germinadas sob condições in vivo, foram cultivados em meio de Murashige and Skoog (MS) suplementado com ácido 3-indolacético (AIA), ácido naftalenoacético (ANA), ácido 4-amino-3,5,6-tricloropicolínico (picloram) ou ácido 2,4-diclorofenoxiacético (2,4-D). Calos de aspecto nodular foram produzidos a partir de ambos os tipos de explante na presença de 4,5 e 9,0 μM de 2,4-D. O desenvolvimento e a maturação de embriões somáticos foram alcançados quando calos obtidos de explantes caulinares foram transferidos para meio de cultura suplementado com uma concentração de 2,4-D dez vezes menor do que aquelas utilizadas na indução do processo de calogênese (0,45 e 0,90 μM). Calos derivados de explantes foliares não produziram embriões ao serem submetidos a estes mesmos tratamentos. Os maiores valores de freqüência de calos embriogênicos (85 por cento) e número médio de embriões por calo (13,45±2,8) foram alcançados durante a primeira subcultura em meio suplementado com 0,90 μM de 2,4-D. O processo de conversão dos embriões somáticos em plantas foi observado após transferência dos embriões para meio MS sem suplementação hormonal solidificado com 2 g.L-1 de fitagel. Três meses após a transferência para condições ex vitro a taxa de aclimatização alcançada foi de 53 por cento e as plantas apresentavam um aspecto fenotípico normal.

Preservation of endangered Tunisian grapevine cultivars using embryogenic cultures

The preservation of embryogenic lines derived from several endangered local grapevine cultivars was studied. Embryogenic calluses were obtained from immature anthers of eight cultivars, sampled on both fruity-cuttings and field grown vines. Anthers at the 'separated flower' stage, derived from fruity-cuttings, resulted in an increased induction of somatic embryogenesis, compared to those derived from the field. Pro-embryogenic calluses were induced on Chée and Pool (1987) basal medium, supplemented with 9 micron M of 2,4-dichlorophenoxyacetic acid (2,4-D) and 11.35 micron M of thidiazuron (TDZ) under dark conditions. Different anther zones (filament, abaxial, adaxial, lateral zones and entire anthers) were involved in somatic embryogenesis induction. The percentages of granular and yellowish pro-embryogenic calluses ranged between 15.6 percent and 34.8 percent in 'Kahli Kerkennah' and 'Muscat Raf-raf' cultivars, respectively. Although, morphological diversifications of pro-embryogenic calluses (several necrosis and spontaneous maturation) were observed on the induction mediumafter 5 subcultures. The reduction of 2,4-D and TDZ levels to 4.52 micron M and 2.89 micron M respectively, induced granular and yellowish embryogenic material. Thus, Chée and Pool (1987) (CP) enriched with 4.52 micron M of 2,4-D and 2.89 micron M of TDZ revealed to be the most appropriate for long-term maintenance. In fact, all the cultivars presented high and regular embryo maturation rates after 12, 24, 36 and 48 months of cultivation on this medium, under light conditions. After 4 years, they still exhibit high germination and regeneration abilities. Germination of somatic embryos was achieved on Murashige and Skoog (1962) basal-medium, with rates ranging from 69 percent to 96 percent. Only 5 percent of somatic embryos were concerned by morphological variations. The regenerated plantlets presented a normal phenotype under controlled greenhouse conditions, compared to mother plants.