Accelerated in vitro propagation of elite oil palm genotypes (Elaeis guineensis Jacq.) by substituting cytokinin with putrescine

Clonal multiplication of oil palm enables the formation of commercial plantations with higher yield, which is possible through somatic embryogenesis. However, due to different modifications made in the existing protocols to avoid the appearance of the “mantled flower”, the success of this technique has been limited. Thus, this study aimed to apply a cloning protocol, using somatic embryogenesis, to 32 elite oil palm genotypes from a commercial plantation on which cytokinin was substituted with putrescine. All tested genotypes responded positively to callus induction. Moreover, the percentages of responses were differentiated: 65.62% of these genotypes produced embryogenic lines, and 40.62% out of that percentage presented lines with moderate or high multiplication capacity, which is the main factor that enabled the obtaining of clones to form a commercial clonal plantation. The somatic embryogenesis was efficient, making this protocol applicable at a commercial scale, since it allows the obtaining of clones up to 360 days. However, future evaluations on these clones are needed to investigate the appearance of the “mantled flower”.Keywords: Cloning, polyamines, tissue culture, commercial plantatio

Obtainment and maintenance of embryogenic lineages in vitro from zygotic embryos in families of Acrocomia aculeate

O estabelecimento de protocolo eficiente para a obtenção e manutenção de massas pró- embriogênicas capazes de produzir embriões somáticos é requerido para a propagação in vitro de A. aculeata em larga escala. Diante do efeito significativo do genótipo frente à indução a embriogênese somática in vitro, tornou-se indispensável a avaliação de progênies de diferentes matrizes de A. aculeata de polinização aberta. Neste intuito, os objetivos do trabalho foram avaliar progênies de matrizes de A. aculeata de polinização aberta quanto à indução de embriogênese somática in vitro e aprimorar o processo de embriogênese somática da espécie a partir de embriões zigóticos. Embriões zigóticos maduros oriundos de 19 famílias de A. aculeata foram inoculados em meio Y3 contendo 9 μM de Picloram e/ou Dicamba, ambos combinados com 0,9 μM de 2iP. As famílias apresentaram respostas significativamente diferentes para os caracteres embriões intumescidos (EI), porcentagem de embriões calejados (ECAL), porcentagem de calos embriogênicos (CEM) e porcentagem de oxidação (OXID), na fase de indução de embriogênese somática. Por meio da análise de diversidade genética foi possível separar as 19 famílias em 5 grupos no meio suplementado com Picloram, enquanto que, em meio suplementado com Dicamba, foram formados 4 grupos de dissimilaridade. O meio suplementado com Picloram foi mais eficiente em relação aos caracteres EI, ECAL, CEM, enquanto que o meio suplementado com Dicamba foi superior para os caracteres números de linhagens embriogênicas (LEs) e OXID. Das 19 famílias testadas, 16 apresentaram a capacidade de formar LEs, as quais se encontram em meio de multiplicação com 18 μM de Picloram por mais de 24 meses sem prejuízo da sua competência embriogênica. As LEs apresentaram aparência granular, coloração amarela e aspecto translúcido. As massas pró-embriogênicas (MPEs) formadas foram maturadas com maior eficiência em meio com 5,5 μM de ANA combinado com 2,5 μM de 2iP suplementado com carvão ativado, sendo caracterizadas pelo alongamento e mudança de coloração de amarelo translúcido para branco opaco. Apesar das famílias se comportarem distintamente em cada meio de germinação, a família 9 apresentou o maior número de embriões somáticos germinados (NEG) em meio contendo GA3. O processo de germinação dos embriões zigóticos e somáticos são similares, ocorrendo primeiramente o alongamento do pecíolo cotiledonar e o crescimento do haustório. A análise histológica permitiu comprovar a formação dos embriões somáticos com um sistema vascular fechado. Verificou-se também a presença de uma camada mucilaginosa peculiar na protoderme formada, característica que poderá servir de marcador do processo de embriogênese para a A. aculeata. O presente trabalho é pioneiro na obtenção e manutenção de linhagens embriogênicas e na análise da resposta in vitro de diferentes famílias de A. aculeata.The establishment of an efficient protocol for obtainment and maintenance of pro- embryogenic masses able to produce somatic embryos is required for in vitro large scale propagation of A. aculeata. The significant effect of genotype on inducing somatic embryogenesis in vitro is essential to evaluate progenies of different arrays of open- pollinated A. aculeata. To this end, the aims of this work were to evaluate progeny arrays of open-pollinated A. aculeata by induction of somatic embryogenesis in vitro and optimize the process of somatic embryogenesis of this species from zygotic embryos. Mature zygotic embryos from 19 families of A. aculeata were inoculated in Y3 containing 9 μM of Picloram and/or Dicamba, both combined with 0.9 μM of 2iP. Families showed significantly different responses to the characters swolen embryos (SE), percentage of callused embryos (CALE), percentage of embryogenic callus (EMCA), and percentage of oxidation (OXID), in the induction phase of somatic embryogenesis. Through analysis of genetic diversity, the 19 families were separated in 5 groups in medium supplemented with Picloram, whereas in medium supplemented with Dicamba dissimilarity formed 4 groups. The medium supplemented with Picloram was more efficient on the characters SE, CALE, and EMCA, while medium supplemented with Dicamba was superior to the characters number of embryogenic lineages (ELINs) and OXID. Of the 19 families tested, 16 showed the ability to form ELINs, which are in multiplication medium with 18 μM of Picloram for more than 24 months without prejudice to their embryogenic competence. The ELINs showed granular appearance, yellow color and translucent appearance. The produced pro- embryogenic masses (PEMs) were more efficiently matured in medium with 5.5 μM of ANA combined with 2.5 μM of 2iP supplemented with activated charcoal, being characterized by elongation and changes in color from translucent yellow to opaque white. Although families behave distinctly in each germination medium, the family 9 had the highest number of germinated somatic embryos (GSE) in medium containing GA3. The process of germination of zygotic and somatic embryos is similar, occurring primarily elongation of the cotyledon petiole and the growth of haustoria. Histological analysis allowed us to verify the formation of somatic embryos with a closed vascular system and the presence of a peculiar mucilaginous layer in the protoderm formed, a characteristic that may serve as a marker for the process of embryogenesis of A. aculeata. This study is a pioneer in obtainment and maintenance of embryogenic lineages and in vitro response analysis of different families of A. aculeata.Conselho Nacional de Desenvolvimento Científico e Tecnológic

Resposta do sistema antioxidativo à indução do estresse gradativo e choque osmótico pelo NaCl em cana-de-açúcar

A cana-de-açúcar (Saccharum officinarum) é uma gramínea considerada moderadamente sensível à salinidade. Embora tradicionalmente cultivada nas zonas de Mata úmida e Litoral da região Nordeste, o cultivo da cana-de-açúcar vem se expandindo também para regiões semi-áridas. A má qualidade da água de irrigação, altas taxas de evaporação e baixa precipitação pluviométrica, aliada a outros fatores contribuem para o processo de salinização dos solos do semi–árido. Para o desenvolvimento e seleção de genótipos tolerantes, é necessário conhecer os mecanismos fisiológicos com os quais as plantas enfrentam o estresse salino. Técnicas de cultivo in vitro para estudos da fisiologia e bioquímica de plantas, em condições de estresse, são importantes ferramentas por permitirem o controle e homogeneidade das condições de cultivo. No presente trabalho, dois genótipos de cana-de-açúcar (RB931011 e RB872552) desenvolvidos pelo Programa de Melhoramento Genético da Cana-de-Açúcar da RIDESA, para a região Nordeste, foram submetidos ao estresse salino in vitro mediante o acréscimo de NaCl ao meio de cultura sob uma ação gradativa e súbita para avaliar o comportamento fisiológico e bioquímico dessas plantas. As variedades de cana-de-açúcar foram submetidas a duas concentrações de NaCl (56mM, 112mM e o controle) de forma gradativa e repentina. Aos 35 dias de experimento, atividades enzimáticas da Catalase (CAT), Peroxidase (POX), Ascorbato peroxidase (APX), Polifenoloxidase (PPO), teores de sódio (Na+), potássio (K+), concentração de proteínas solúveis e prolina livre foram determinadas. Foi possível observar diferenças nas respostas das variedades em função da condição de indução do estresse salino, gradativo ou por choque, mais do que em função das concentrações de NaCl no meio de cultura. A resposta ao estresse é, portanto, condicionada não só pela concentração dos sais, mas pela forma que a planta é exposta ao meio salino.Sugarcane (Saccharum officinarum) is a grass considered moderately sensitive to salinity. Although traditionally cultivated in the soil of the Atlantic Coast and the Northeast, the cultivation of sugarcane is expanding to semi-arid regions. The poor quality of irrigation water, high rates of evaporation and low rainfall, combined with other factors contribute to the process of soil salinization in semi-arid. For the development and selection of tolerant genotypes, it is necessary to understand the physiological mechanisms with which plants face the salt stress. Techniques for in vitro plant tissue for studies of physiology and biochemistry of plants under stress are important tools to allow control and uniformity of culture conditions. In this study, two genotypes of sugarcane (RB931011 and RB872552) developed within the Program of Genetic Improvement of Sugarcane of rides, for the Northeast region (RIDESA) were evaluated for sudden and gradual action of salt stress-induced by adding NaCl to the culture medium during in vitro development of plants. The varieties of sugarcane were subjected to three concentrations of NaCl (0 mM, 50 mM and 100 mM) in a gradual and sudden. At 35 days of experiment, enzyme activities (CAT, POX, APX, PPO), levels of sodium (Na+), potassium (K+), soluble protein content and free proline were recorded. It was possible to observe differences in responses of varieties depending on the condition of induced salt stress, shock or gradual, rather than depending on the concentration of NaCl in the culture medium. The stress response is therefore not only conditioned by salt concentration, but by way of exposure to salt.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPE

ANTIOXIDANT ENZYMES ACTIVITY IN EMBRYOGENIC AND NON-EMBRYOGENIC TISSUES IN SUGARCANE

The objective of this work was to induce direct somatic embryogenesis from segments of immature leaves of the RB872552 variety of sugarcane and to correlate this morphogenic event with oxidative stress. Two previously described protocols were utilized for the induction of somatic embryogenesis in sugarcane with different supplementations of the culture medium and different incubation conditions. For the conversion of embryos into plants was used MS medium without phytoregulators. Histological analyses and activity of antioxidant enzymes were also conducted for the embryogenic and non-embryogenic tissues. The formation of somatic embryos was obtained in 81 % of the explants with the combination of regulators 2,4-D (2,4-dichlorophenoxyacetic acid)and BAP (6-benzylaminopurine) when incubated under 16 h photoperiod. With regards to the antioxidant enzymes, there was increased activity of peroxidase and an increase in the soluble protein content in embryogenic tissues, whereas lower activities of polyphenol oxidase and catalase appeared in these tissues compared to nonembryogenic tissues. It could be inferred that oxidative stress plays an important role in the induction of somatic embryogenesis in sugarcane

Response to in vitro salt stress in sugarcane is conditioned by concentration and condition of exposure to NaCl

Salinity is one of the major environmental stress factors that affect crop productivity, as well as interfering with plant growth and development, resulting in reduced production quality. Given this, we highlight the importance of research in response to plants subjected to salt stress in order to assess the physiological and biochemical behavior of genotypes, with the objective of selecting the more tolerant. One way to ensure the uniformity of the response of the plants is through in vitro cultivation, which allows control of the cultivation conditions. Therefore, the objective of this work was to evaluate the response of two commercial varieties of sugar cane exposed to saline stress in different conditions (gradual and abrupt). Two varieties of sugarcane (RB931011 and RB872552) were subjected to in vitro salt stress by NaCl (56 mM and 112 mM), either gradually or suddenly. The responses of the enzymatic antioxidant system (catalase, peroxidases and ascorbate peroxidase) and free proline, as well as the Na+ and K+ contents, were assessed 30 days after the beginning of the treatments. Differences were observed in the responses of the varieties as a function of the induction mode of salt stress, graded or by shock, rather than as a function of the NaCl concentrations in the culture medium. The stress response is therefore conditioned not only by salt concentration, but also by the form of exposure to salt

Response to in vitro salt stress in sugarcane is conditioned by concentration and condition of exposure to NaCl

Salinity is one of the major environmental stress factors that affect crop productivity, as well as interfering with plant growth and development, resulting in reduced production quality. Given this, we highlight the importance of research in response to plants subjected to salt stress in order to assess the physiological and biochemical behavior of genotypes, with the objective of selecting the more tolerant. One way to ensure the uniformity of the response of the plants is through in vitro cultivation, which allows control of the cultivation conditions. Therefore, the objective of this work was to evaluate the response of two commercial varieties of sugar cane exposed to saline stress in different conditions (gradual and abrupt). Two varieties of sugarcane (RB931011 and RB872552) were subjected to in vitro salt stress by NaCl (56 mM and 112 mM), either gradually or suddenly. The responses of the enzymatic antioxidant system (catalase, peroxidases and ascorbate peroxidase) and free proline, as well as the Na+ and K+ contents, were assessed 30 days after the beginning of the treatments. Differences were observed in the responses of the varieties as a function of the induction mode of salt stress, graded or by shock, rather than as a function of the NaCl concentrations in the culture medium. The stress response is therefore conditioned not only by salt concentration, but also by the form of exposure to salt

EFFECT OF EXOGENOUS PROLINE IN TWO SUGARCANE GENOTYPES GROWN IN VITRO UNDER SALT STRESS

<p class="TextosemFormatao1">Sugarcane production is influenced by various abiotic stresses including salt stress. Salinity can induce oxidative stress, which in turn damages biomolecules and cellular structures. However, such damage can be avoided or minimized by the enzymatic and non-enzymatic defense systems of plants. The aim of the study was to determine the effect of exogenous proline on Na⁺ and K⁺ content and on the activity of catalase, ascorbate peroxidase and peroxidase in two sugarcane genotypes (RB931011 and RB872552) grown <em>in vitro</em> under salt stress. The plants were grown for 20 days with or without 100 mM NaCl and exposed to 20 mM proline for varying period of time. The data were subjected to ANOVA factorial (two genotypes and five treatments), and the mean values were compared by Tukey test at a 5% probability. Under salinity stress, both genotypes exhibited membrane integrity reduction, reduced total soluble protein content and unaltered or increased endogenous proline content. Exogenous proline reduced Na⁺ accumulation in a manner proportional to the exposition period at the amino acid. In both genotypes, antioxidant enzymes activity increased with the addition of NaCl. In conclusion, RB931011 genotype showed higher proline accumulation and increased in activity of the antioxidant enzymes, indicating better salt stress tolerance than in RB872552 genotype.</p