Teleost embryos have not been successfully cryopreserved. To formulate successful cryopreservation protocols, the distribution and cellular permeability to water must be understood. In this paper, the zebrafish (Brachydanio rerio) was used as a model for basic studies of the distribution to permeability to water. These embryos are a complex multi-compartmental system composed of two membrane-limited compartments, a large yolk (surrounded by the yolk syncytial layer) and differentiating blastoderm cells (each surrounded by a plasma membrane). Due to the complexity of this system, a variety of techniques, including magnetic resonance microscopy and electron spin resonance, was used to measure the water in these compartments. Cellular water was distributed unequally in each compartment. At the 6-somite stage, the percent water (VA'^) was distributed as follows: total in embryo = 74%, total in yolk = 42%, and total in blastoderm = 82%. A one-compartment model was used to analyze kinetic, osmotic shrinkage data and determine a phenomenological water permeability parameter, Lp, assuming intracellular isosmotic compartments of either 40 or 300 mosm. This analysis revealed that the membrane permeability changed (P< 0.05) during development. During the 75% epiboly to 3-somite stage, the mean membrane permeability remained constant (Lp = 0.022 ± 0.002 |xm x min'^atm"^ [mean ± S.E.M.] assuming isosmotic is 40 mosm or Lp = 0.049 ± 0.008 |im x min'^atm"^ assuming isosmotic is 300 mosm). However, at the 6-somite stage, Lp increased twofold (Lp = 0.040 ± 0.004 |j,m x min'^atm"^ assuming isosmotic is 40 mosm or Lp = 0.100 ± 0.017 |J,m x min'^atm"^ assuming isosmotic is 300 mosm). Therefore, the low permeability of the zebrafish embryo coupled with its large size (and consequent low area to volume ratio) led to a very slow osmotic response that should be considered before formulating cryopreservation protocols.

Conservation of plant genetic resources is important to prevent genetic erosion. Seed banks are the most common method of ex situ conservation; however, coffee seeds can not be stored by conventional methods. Cryopreservation is a viable alternative for long-term conservation of species that produce intermediate or recalcitrant seeds, as coffee. The aim of this work was to cryopreserve Coffea arabica L. cv Catuaí Vermelho IAC 144 zygotic embryos, and analyse the effects of dehydration prior cryopreservation and osmotic rehydration after thawing, in embryos germination and seedlings formation after cryopreservation. Prior to cryopreservation, different dehydration times (0, 15, 30, 60 and 120 min) were tested. Dehydrated embryos were cryopreserved in liquid nitrogen for 1 hour, and after thawing were rehydrated by osmotic solutions. Dehydrated and non-cryopreserved embryos were also analysed. The test with 2,3,5 triphenyl tetrazolium chloride (TTC) was used to evaluate the embryos viability. Non-dehydrated embryos did not survive after freezing. Embryos that were dehydrated until 20% of the moisture content did not germinate when osmotic rehydration was not performed. In contrast, cryopreserved embryos with the same moisture content presented 98% germination when they were rehydrated slowly in osmotic solution. According to tetrazolium tests, embryos presented maximum viability (75%) after dehydration for 60 minutes (23% moisture content). Therefore, coffee zygotic embryos (Coffea arabica L. cv. Catuaí Vermelho) can be successfully cryopreserved using physical dehydration in silica gel for 60 minutes (23% moisture content), followed by osmotic rehydration after thawing. This method allowed a germination of 98% of cryopreserved zygotic embryos.Index terms: Coffea arabica L.; ex situ conservation; embryos dehydration; long-term storage; gene bank. RESUMOA conservação dos recursos genéticos vegetais é uma medida importante para prevenção do processo de erosão genética. Bancos de sementes são as formas mais comuns de conservação ex situ, entretanto, sementes de cafeeiro não podem ser armazenadas por métodos convencionais. A criopreservação é uma alternativa viável para conservação em longo prazo de espécies, como o cafeeiro, que produzem sementes intermediárias ou recalcitrantes. O objetivo deste trabalho foi criopreservar embriões zigóticos de Coffea arabica L. cv Catuaí Vermelho avaliando o efeito da desidratação antes da criopreservação, e da reidratação osmótica após o descongelamento. Antes da criopreservação foram testados e determinados os teores de umidade em diferentes tempos de desidratação (0, 15, 30, 60 e 120 min). Após a desidratação os embriões foram criopreservados por uma hora em nitrogênio líquido, e após o descongelamento foram reidratados utilizando solução osmótica. Para avaliação da viabilidade de embriões desidratados e/ou criopreservados, utilizou-se o teste com o cloreto de 2,3,5-trifenil tetrazólio. Embriões não desidratados não sobreviveram ao congelamento. Embriões desidratados até...

ABSTRACT. Torch ginger (Etlingera elatior (Jack) RM Smith) can be propagated in vitro, but there is currently little information about the stages of acclimatization and adaptation for torch ginger in a greenhouse. The objective was to study the growth responses, survival and photosynthetic response during the acclimatization of plants maintained on three different substrates (Plantmax forestry ® , sand and 1:1 Plantmax forestry ® and sand) under three shading conditions (50% red and blue shading nets and control without shading nets). The highest leaf and shoot numbers per plant and survival rate were observed in treatments with the Plantmax forestry ® type substrate in the absence of shading. In vitro culture plants behaved similarly or better than rhizome propagated control plants with regard to the net photosynthesis rate, carboxylation efficiency, stomatal conductance and transpiration rate. In general, for both conditions, in vitro and control plants had similar efficiency of biochemical functions and of photosystem II. These results show that plants derived from in vitro culture exhibit satisfactory physiological yield and greenhouse acclimation capacity.Keywords: torch ginger, growth curve, gas exchange, photoautotrophy.Crescimento e respostas fotossintéticas durante aclimatização ex vitro de Bastão-do-imperador (Etlingera elatior Jack RM Smith) RESUMO. O bastão-do-imperador (Etlingera elatior Jack Smith RM) pode ser propagado in vitro, mas não há informações sobre as fases de aclimatização e adaptação no campo. O objetivo foi estudar as respostas de crescimento e sobrevivência durante a aclimatização ex vitro da espécie, quando mantida em diferentes substratos: Plantmax florestal ® ; areia; Plantmax florestal ® e areia 1:1; e malhas de sombreamento a 50% na cor vermelha; azul e controle sem a presença de malha, assim como estudar respostas fotossintéticas durante a aclimatização em casa de vegetação. A maior taxa de sobrevivência das plantas, número de folhas e número de brotos por planta, foram observados no tratamento com substrato tipo Plantmax florestal ® puro, na ausência de sombreamento. Quanto à taxa de fotossíntese líquida, eficiência de carboxilação, condutância estomática e taxa de transpiração, plantas de cultivo in vitro apresentaram comportamento semelhante ou superior quando comparadas com plantas controle obtidas a partir de rizomas. Em geral, a eficiência do funcionamento bioquímico e do fotossistema II foram semelhantes em plantas de cultivo in vitro e controle. Conclui-se que tais resultados mostram que as plantas derivadas do cultivo in vitro apresentam rendimento fisiológico satisfatório, conferindo capacidade de aclimatação no campo.Palavras-chave: Bastão-do-imperador, curva de crescimento, trocas gasosas, fotoautotrofia.

As a consequence of the difficulty in conventional coffee seed storage, biotechnological alternatives such as cryopreservation have been investigated. The objective of this study was to develop a protocol for the cryopreservation of Coffea arabica L. (cv. 'Catuaí Vermelho' -IAC 144) zygotic embryos by vitrification. For the cryopreservation study, the embryos were immersed in Plant Vitrification Solution 2 at different times (0, 10, 25, 50, 100, and 250 min) and two temperatures (0 and 25 °C). Subsequently, the best thawing time was determined in a water bath (1, 3, 5 minutes or directly in Recovery Solution). An anatomical study was conducted on non-stored and stored embryos, with or without the use of Plant Vitrification Solution 2. The immersion in cryoprotectant solution for 100 min at 0 °C allows embryo cryopreservation. Embryos can be directly thawed in Recovery Solution after storage in liquid nitrogen. It was observed that Plant Vitrification Solution 2 reduced internal water content in the cells, allowing subsequent embryo growth resumption.

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Zingiber spectabile is a tropical ornamental species with difficulties to obtain efficient propagation system. Thus, this study aimed to assess the in vitro propagation of Zingiber spectabile. Seed characterization was determined by measuring length, width and thickness, the weight of 1000 seeds and imbibition curve. In vitro germination of seeds was at constant (25 °C) or alternating temperatures (20-30 ºC). For optimization of in vitro multiplication, different concentrations of activated charcoal (0.0, 0.1 and 0.3%) and sucrose (0.0, 0.1, 0.3, 0.5 and 0.7 M) were evaluated. Plantlets were inoculated in flasks with different sealing systems (PVC covers with or without filters at the center) and culture media (MS or WPM). The plants were acclimatized in Plantmax ® substrate. Seeds were of 6.06 mm length, 3.22 mm wide and 2.83 mm thick. The weight of 1,000 seeds corresponded to 46.4 g. The seed imbibition curve approaches to a tree phase pattern. Alternating temperatures induced high germination rates (68%). The addition of 0.3% activated charcoal provided higher root growth and plants with smaller number of senescent leaves. The best plant growth was obtained by the use of 0.1 M sucrose. All acclimatized plants survived (100%). The results demonstrate that Z. spectabile respond well to in vitro propagation. Keywords: beehive ginger, micropropagation, tropical plants. RESUMO Propagação in vitro de Zingiber spectabileZingiber spectabile é uma espécie ornamental tropical com dificuldades para se obter um sistema de propagação eficiente. Assim, este estudo teve como objetivo avaliar a propagação in vitro de Z. spectabile. A caracterização das sementes foi determinada pela medição do comprimento, largura e espessura, pelo peso de 1.000 sementes e pela curva de embebição. A germinação in vitro das sementes foi avaliada em temperatura constante (25 °C) ou alternada (20-30 ºC). Para a otimização da multiplicação in vitro, diferentes concentrações de carvão ativado (0,0, 0,1 e 0,3%) e sacarose (0,0, 0,1, 0,3, 0,5 e 0,7 M) foram avaliadas. As plântulas foram inoculadas em frascos com diferentes sistemas de vedação (tampas de PVC com ou sem filtros no centro) e meio de cultura (MS ou WPM). As plantas foram aclimatizadas em substrato Plantmax®. As sementes apresentaram comprimento de 6,06 mm, largura de 3,22 cm e espessura de 2,83 mm. O peso de 1000 sementes correspondeu a 46,4 g. A curva de embebição de sementes se aproxima de um padrão trifásico. Temperaturas alternadas induziram altas taxas de germinação (68%). A adição de 0,3% de carvão ativado proporcionou maior crescimento radicular e plantas com menor número de folhas senescentes. O melhor crescimento de plantas foi obtido pelo uso de sacarose 0,1 M. Todas as plantas aclimatizadas sobreviveram (100%). Os resultados demonstraram que Z. spectabile responde bem à propagação in vitro. Palavras-chave: sorvetão, micropropagação, plantas tropicais.

Vasconcellea quercifolia A. St.-Hil. (Caricaceae) is a tropical fruit species native to Brazil, with a great importance in plant breeding programs. The V. quercifolia has a resistance to the main diseases of Caricaceae, Papaya Ringspot Virus (PRSV). Considering its potential, cryopreservation becomes a tool for the conservation of this species. The objective of this paper was to study the cryopreservation of V. quercifolia zygotic embryos through dehydration in silica gel. Excised zygotic embryos were dehydrated in silica at 0, 20, 40, 80 and 100 minutes and then inoculated in MS medium. The percentage of germinated and recovered embryos, and growth analysis were evaluated, besides water content. Subsequently, they were acclimatized in a growth room with temperature controlled. For cryopreservation, the embryos were excised and dehydrated in silica for 0, 20, 40 and 60 minutes, immersed in Liquid Nitrogen (LN) for 1 hour, thawed in Recovery Solution (RS) and inoculated in MS medium. After 30 days, the percentage of germinated and recovered embryos was evaluated. The silica gel promotes a fast dehydrate of embryos. The results showed that embryo dehydration affected seedling development, and dehydration for over 20 minutes showed a reduction in all evaluated parameters. The plantlets regenerated from embryos dehydrate survive the acclimatization. It was possible to cryopreserve the V. quercifolia zygotic embryos when the dehydration time of 20 minutes by silica gel was used.

Introgression of natural genetic variations affecting trichome development and terpenes biosynthesis to obtain insect-resistant tomatoes To deal with insect pests the tomato wild relatives produce a variety of defensive compounds in their glandular trichomes type-VI. By contrast, although cultivated tomatoes (Solanum lycopersicum L.), also display type-VI trichomes, the gland in cultivated tomato is much smaller containing mainly monoterpenes and low levels of cytosolic-derived sesquiterpenes, which have no apparent effect against insect pests. In the present work, we carried out crosses between the wild species S. habrochaites and the tomato cultivar Micro-Tom (MT) in order to create introgression lines (ILs). We successfully transferred the plastid-derived sesquiterpene pathway from S. habrochaites to type-VI trichomes of the cultivated tomato (cv. Micro-Tom). The trichomes of the introgressed line named MT-Sesquiterpene synthase 2 (MT-Sst2) showed even higher concentration of α-santalene, β-bergamotene, and α-bergamotene compared to the wild species type-VI glandular trichomes. Surprisingly, the presence of high amounts of plastid-derived sesquiterpenes was not sufficient to confer resistance to specific tomato pests in MT-Sst2. The sesquiterpene profile of MT-Sst2 and LA1777 unveils sesquiterpene-derived compounds only found in the wild species, which point for additional steps necessary to obtain insect-resistant tomatoes. This work paves the way for both the understanding of the morphology and functionality of type-VI trichomes and for breeding insect resistant tomatoes.