Using Random Amplified Polymorphic DNA to Assess Genetic Diversity and Structure of Natural Calophyllum brasiliense

The objective of this study was to assess the genetic variability in two natural populations of Calophyllum brasiliense located along two different rivers in the state of Minas Gerais, Brazil, using RAPD molecular markers. Eighty-two polymorphic fragments were amplified using 27 primers. The values obtained for Shannon index (I) were 0.513 and 0.530 for the populations located on the margins of the Rio Grande and Rio das Mortes, respectively, demonstrating the high genetic diversity in the studied populations. Nei’s genetic diversity (He) was 0.341 for the Rio Grande population and 0.357 for the Rio das Mortes population. These results were not significantly different between populations and suggest a large proportion of heterozygote individuals within both populations. AMOVA showed that 70.42% of the genetic variability is found within populations and 29.58% is found among populations (ФST=0.2958). The analysis of kinship coefficients detected the existence of family structures in both populations. Average kinship coefficients between neighboring individuals were 0.053 (P<0.001) in Rio das Mortes and 0.040 (P<0.001) in Rio Grande. This could be due to restricted pollen and seed dispersal and the history of anthropogenic disturbance in the area. These factors are likely to contribute to the relatedness observed among these genotypes

USO DE BIORREATORES DE IMERSÃO CONTINUA, TEMPORÁRIA E MEIO DE CULTURA SEMISSÓLIDO NA PRODUÇÃO DE CLONES DE Eucalyptus camaldulensis

The plant micro-propagation in bioreactor systems is regarded as one way to reduce cost by automation and production scheduling. This research was carried out in order to obtain an efficient procedure for clone production of Eucalyptus camaldulensis on different types of bioreactor including continuous and temporary immersion bioreactor. To do so, the apical meristems (1 mm) and the apical meristems with adjacent tissue (2,5 mm) were used as initial explants. These tissues were cultured, for 60 days, in semisolid culture medium supplemented with 1 mg L-1 indole acetic acid (IAA) and 0.32 mg L-1 benzylaminopurine (BA). After 60 days, the meristems with adjacent tissue were transferred to a continuous immersion bioreactor and maintained in dark or light conditions. In order to verify the effect of the explant source on bioreactor multiplication, the explants subcultured from meristems multiplied in semisolid culture medium and the meristems multiplied in continuous immersion bioreactor were tested and maintained in dark conditions. After establishing this parameters, the multiplication experiments were carried out in continuous and temporary immersion and the multiplied explants were then rooted in MS medium supplemented with 0, 2, 4, 8 and 20 mg L-1 indole butyric acid (IBA) and kept in the dark or under controlled lighting conditions. After that, the rooting the plants were acclimatized in mist chamber. The meristem with adjacent tissue favored a greater number of buds/explants. The continuous immersion bioreactor in the dark provided higher shoots number and multiplication rate. The rooting was better on culture medium without auxin and kept in the dark for 15 days or the culture medium supplemented with auxin and maintained under light with 100% plantlet rooting. The Eucalyptus camaldulensis acclimatization was efficient, with high survival rate (76%). It was possible to establish the procedure for bioreactor micro-propagation of Eucalyptus camaldulensis large-scale clones.A micropropagação em sistemas de biorreatores é considerada como uma forma de reduzir os custos de produção por meio do escalonamento de automatização do processo. O objetivo desse trabalho foi desenvolverum protocolo eficiente de produção de mudas de Eucalyptus camaldulensis em diferentes tipos de sistema, incluindo biorreator de imersão continua e temporária. Para isso, meristemas apicais (1 mm) e meristemas apicais com tecido adjacente (2,5 mm) foram usados como explantes iniciais. Esses tecidos foram cultivados, por 60 dias, em meio de cultura suplementado com 1 mg L-1 de ácido indolacético (AIA) e 0.32 mg L-1 de benzilaminopurina (BAP). Após 60 dias, os meristemas com tecidos adjacentes foram transferidos para biorreatores de imersão contínua ou temporária e mantidos no escuro ou sob condições controladas de luminosidade. Para verificar o efeito da fonte de explante na multiplicação em biorreator foram testados explantes subcultivados de meristemas multiplicados em meio de cultura semissólido e meristemas multiplicados em biorreator de imersão contínua e mantidos no escuro. Despois de estabelecer esses parâmetros, os experimentos de multiplicação foram realizados em biorreatores de imersão contínua e temporária. Os explantes multiplicados foram enraizados em meio de cultura MS suplementado com 0, 2, 4, 8 e 20 mg L-1 de ácido indolbutírico (AIB) e mantidos no escuro ou sob condições controladas de luminosidade. Depois do enraizamento as plantas foram aclimatizadas em câmara de nebulização. Os meristemas com tecidos adjacentes favoreceram um maior número de gemas/explantes. O biorreator de imersão contínua e mantido no escuro promoveu maior número de brotações e maior taxa de multiplicação e o melhor enraizamento ocorreram no meio de cultura isento de auxina, mantido no escuro por 15 dias ou o meio de cultura suplementado com auxina, mantido na luz apresentando 100% de enraizamento. A aclimatização do Eucalyptus camaldulensis foi eficiente com taxa de sobrevivência de 76%. Portanto, foi possível desenvolver um método eficiente de micropropagação em biorreator para a produção de mudas Eucalyptus camaldulensis em larga escala

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

Genetic transformation of Eucalyptus camaldulensis by agrobalistic method

Eucalyptus stands in the setting of worldwide forestry due to its adaptability, rapid growth, production of high-quality and low cost of wood pulp fibers. The eucalyptus convetional breeding is impaired mainlly by the long life cycle making the genetic transformation systems an important tool for this purpose. However, this system requires in vitro eficient protocols for plant induction, regeneration and seletion, that allow to obtain transgenic plants from the transformed cell groups. The aim of this work was to evaluate the callus formation and to optimize the leaves and callus genetic transformation protocol by using the Agrobacterium tumefaciens system. Concerning callus formation, two different culture media were evaluated: MS medium supplemented with auxin, cytokinin (M1) and the MS medium with reduced nitrogen concentration and supplemented with auxin, cytokinin coconut water (M2). To establish the leave genetic transformation, those were exposed to agrobiolistics technique (gene gun), to tissue injury, and A. tumesfasciens EHA 105 contening the vetor pCambia 3301 (35S::GUS::NOS), for gene transference and to establish the callus transformation thoses were exposed only to A. tumefasciens. For both experiments, the influence of different infection periods was evaluated. The M2 medium provided the best values for callus sizea and fresh and dry weight. The leaves genetic transformation using the agrobiolistics technique was effective, the gus gene transient expression could be observed. No significant differences were obtained in the infection periods (4, 6 and 8 minutes). The callus genetic transformation with A. tumefaciens also promotend the gus gene transient expression on the callus co-cultiveted for 15 e 30 minutes. The transformed callus was transfered to a regeneration and selection medium and transformed plants were obtained

THE USE OF CONTINUOUS, TEMPORARY IMMERSION BIOREACTOR SYSTEM AND SEMISOLID CULTURE MEDIUM FOR THE PRODUCTION OF Eucalyptus camaldulensis CLONES

The plant micro-propagation in bioreactor systems is regarded as one way to reduce cost by automation and production scheduling. This research was carried out in order to obtain an efficient procedure for clone production of Eucalyptus camaldulensis on different types of bioreactor including continuous and temporary immersion bioreactor. To do so, the apical meristems (1 mm) and the apical meristems with adjacent tissue (2,5 mm) were used as initial explants. These tissues were cultured, for 60 days, in semisolid culture medium supplemented with 1 mg L -1 indole acetic acid (IAA) and 0.32 mg L -1 benzylaminopurine (BA). After 60 days, the meristems with adjacent tissue were transferred to a continuous immersion bioreactor and maintained in dark or light conditions. In order to verify the effect of the explant source on bioreactor multiplication, the explants subcultured from meristems multiplied in semisolid culture medium and the meristems multiplied in continuous immersion bioreactor were tested and maintained in dark conditions. After establishing this parameters, the multiplication experiments were carried out in continuous and temporary immersion and the multiplied explants were then rooted in MS medium supplemented with 0, 2, 4, 8 and 20 mg L -1 indole butyric acid (IBA) and kept in the dark or under controlled lighting conditions. After that, the rooting the plants were acclimatized in mist chamber. The meristem with adjacent tissue favored a greater number of buds/explants. The continuous immersion bioreactor in the dark provided higher shoots number and multiplication rate. The rooting was better on culture medium without auxin and kept in the dark for 15 days or the culture medium supplemented with auxin and maintained under light with 100% plantlet rooting. The Eucalyptus camaldulensis acclimatization was efficient, with high survival rate (76%). It was possible to establish the procedure for bioreactor micro-propagation of Eucalyptus camaldulensis large-scale clones

Diversidade e estrutura genética espacial de Calophyllum brasiliense Camb. (Clusiaceae) em uma floresta paludosa

As áreas de ocorrência de florestas paludosas se encontram alteradas e restritas devido aos processos de destruição e fragmentação, com consequente redução do seu tamanho populacional. Calophyllum brasiliense Camb. é uma espécie arbórea abundante em ambientes ciliares, devido à sua preferência em colonizar solos com alta saturação hídrica, sendo considerada especialista em hábitat. Além das consequências ecológicas, como mortalidade e baixo recrutamento, poderá ocorrer perda da variabilidade genética, comprometendo a viabilidade da espécie no local. Nesse contexto, foi realizado um censo da espécie em um fragmento de floresta paludosa, sendo estabelecidas quatro classes de altura para a análise genética. Os marcadores aloenzimáticos revelaram 11 locos polimórficos, com um número médio de 2,0 alelos em cada loco, não sendo observada a perda ou a fixação de alelos e polimorfismo de 100%. As Classes I e III apresentaram excesso de homozigotos, sendo esse valor não significativo para na Classe I. A heterozigosidade foi superior à esperada nas Classes II e IV. Os valores de diversidade genética encontrados na espécie estudada foram considerados altos em relação aos valores relatados em outras espécies arbóreas. Portanto, a ocorrência de mutações e a incorporação de novos alelos na população são elevadas, além de aumentarem o número de recombinações e múltiplas paternidades nas progênies. No geral, a análise da distribuição espacial dos genótipos foi aleatória nas classes analisadas e, na Classe I, os indivíduos próximos a uma distância de 10 m apresentaram estrutura de família. A alta diversidade genética da espécie e a ausência de estruturação espacial dos genótipos na maioria das classes analisadas devem ser consideradas em planos de conservação dessa área

Using Random Amplified Polymorphic DNA to Assess Genetic Diversity and Structure of Natural Calophyllum brasiliense (Clusiaceae) Populations in Riparian Forests

The objective of this study was to assess the genetic variability in two natural populations of Calophyllum brasiliense located along two different rivers in the state of Minas Gerais, Brazil, using RAPD molecular markers. Eighty-two polymorphic fragments were amplified using 27 primers. The values obtained for Shannon index ( ) were 0.513 and 0.530 for the populations located on the margins of the Rio Grande and Rio das Mortes, respectively, demonstrating the high genetic diversity in the studied populations. Nei&apos;s genetic diversity ( ) was 0.341 for the Rio Grande population and 0.357 for the Rio das Mortes population. These results were not significantly different between populations and suggest a large proportion of heterozygote individuals within both populations. AMOVA showed that 70.42% of the genetic variability is found within populations and 29.58% is found among populations (B = 0.2958). The analysis of kinship coefficients detected the existence of family structures in both populations. Average kinship coefficients between neighboring individuals were 0.053 ( &lt; 0.001) in Rio das Mortes and 0.040 ( &lt; 0.001) in Rio Grande. This could be due to restricted pollen and seed dispersal and the history of anthropogenic disturbance in the area. These factors are likely to contribute to the relatedness observed among these genotypes

Similaridade genética entre clones de seringueira (Hevea brasiliensis), por meio de marcadores RAPD Genetic similarity among rubber tree (Hevea brasiliensis) clones using RAPD markers

A seringueira [Hevea brasiliensis (Willd. ex. Adr. de Juss) Muell.-Arg.] é uma espécie nativa da região amazônica e compreende a maior fonte produtora de borracha natural do mundo. Na busca de condições mais favoráveis ao cultivo, além da busca pela auto-suficiência na produção de borracha natural, o cultivo da seringueira migrou para outras regiões do país. Objetivou-se, com o presente trabalho, estimar a similaridade genética de genótipos de seringueira, provenientes de regiões distintas do país, Lavras-MG (UFLA) e Campinas-SP (IAC), por meio de marcadores moleculares RAPD. A análise foi efetuada em 41 indivíduos, representados por 17 genótipos diferentes, com base em 19 primers, que geraram 121 fragmentos polimórficos. Os dados foram analisados utilizando o software NTSYS-pc - 2.1, por meio do coeficiente de Dice e pelo método das médias (UPGMA). A similaridade genética entre o material analisado variou de 0,56 a 1,00. Na análise do dendrograma, foram observados 18 grupos. Os clones (RRIM600, GT1, PB235, PL PIM e FX2261), utilizados em diferentes repetições, foram idênticos, quando comparados entre si, entretanto o mesmo não foi observado para os clones identificados como RRIM 701. Os resultados obtidos sugerem que o material avaliado na UFLA é o mesmo implantado no IAC, exceto o RRIM 701, mostrando uma ampla variabilidade genética, disponível para estudos e propagação da cultura.<br>The rubber tree [Hevea brasiliensis (Willd. ex. Adr. de Juss) Muell.-Arg.] is a native species from Amazon region, and represents the biggest source of natural rubber in the world.. However, the rubber tree culture has had an expansion to other brazilian regions, in search of more favorable conditions for its cultivation and self-sufficiency in natural rubber. The aim of this work was to estimate genetic similarity among rubber tree clones, from different Brazilian regions, Lavras (UFLA) and Campinas (IAC), by using RAPD molecular markers. The analysis was made using 41 individual plants, which represent 17 different clones, based on 19 primers, which raised 121 polymorphic fragments. The data were analysed with NTSYS-pc - 2.1 software, by using Dice coefficient and UPGMA method. Genetic similarity among the materials showed variation from 0,56 to 1,00. In dendogram analysis, 18 groups were observed. The clones RRIM600, GT1, PB235, PLPIM and FX2261 used in different replications, were identical, when compared among themselves. However, results were not the same for the clones identified by RRIM 701. Results suggest that the UFLA material is the same of IAC material, except for RRIM 701, showing wide genetic variability available for studies and culture propagation