Sequência de DNA, construção gênica, sequência de aminoácidos e método de aumento de valor nutricional de planta

Universidade Federal do Rio Grande do SulCiências BiológicasDepositad

Regeneração de soja através de culturas embriogênicas em suspensão

In an attempt to establish an alternative plant regeneration system for soybean [Glycine max (L.) Merrill] cultivars used in Brazilian breeding programs, ten genotypes were tested for their embryogenic potential. Cotyledons were removed as explants from immature seeds harvested from field-grown plants. After 45 days on induction medium, the number of responding cotyledons and the number of somatic embryos per immature cotyledon were evaluated. The percentage of explants that produced somatic embryos varied from 1 to 70% among cultivars. The average number of somatic embryos produced per cotyledon pair ranged from 0.01 to 10.3 with a mean of 3.4. Suspension cultures were initiated with three Agrobacterium tumefaciens susceptible cultivars. Suspensions were successfully developed from Bragg and IAS5 cultivars. The packed cell volume, in one-month growth, increased 8.1 fold for Bragg and 3.5 fold for IAS5 and the fresh weight increased 6.6 and 2.8 fold, respectively. The cultivars differed for the analysed parameters. All tissue from each cultivar was transferred to the maturation medium and subsequently to the germination medium. The germination frequency was 45.7 and 54.9% for Bragg and IAS5, respectively. Plants were gradually exposed to ambient humidity over one week and then planted in soil. All plants yielded seeds in the greenhouse.Com o objetivo de estabelecer um sistema alternativo de regeneração de plantas para cultivares de soja [Glycine max (L.) Merrill] utilizadas em programas de melhoramento no Brasil, dez genótipos foram testados quanto ao seu potencial embriogênico. Os cotilédones utilizados como explantes foram excisados de sementes imaturas de plantas provindas do campo. Após 45 dias em meio de indução, o número de cotilédones embriogênicos e o número de embriões somáticos por cotilédone imaturo foram avaliados. A porcentagem de explantes que produziram embriões somáticos variou de 1 a 70% entre cultivares. O número médio de embriões somáticos produzidos por par de cotilédones variou de 0,01 a 10,3, com uma média de 3,4. Culturas em suspensão foram iniciadas a partir de três cultivares suscetíveis a Agrobacterium tumefaciens. Suspensões foram estabelecidas com sucesso para os cultivares Bragg e IAS5. Em um mês, o volume celular aumentou 8,1 vezes para Bragg e 3,5 vezes para IAS5 e o peso fresco aumentou 6,6 e 2,8 vezes, respectivamente. Todo o tecido de cada cultivar foi transferido para meio de maturação e, subseqüentemente, para meio de regeneração. A freqüência de germinação foi de 45,7 e 54,9% para Bragg e IAS5, respectivamente. Durante uma semana, as plantas foram expostas gradualmente à umidade do ambiente, sendo então plantadas em solo em casa de vegetação. Todas as plantas produziram sementes

Reference gene selection for quantitative reverse transcription-polymerase chais reaction normalization during in vitro adventitious rooting in Eucaliptus globulus Labill

Background: Eucalyptus globulus and its hybrids are very important for the cellulose and paper industry mainly due to their low lignin content and frost resistance. However, rooting of cuttings of this species is recalcitrant and exogenous auxin application is often necessary for good root development. To date one of the most accurate methods available for gene expression analysis is quantitative reverse transcription-polymerase chain reaction (qPCR); however, reliable use of this technique requires reference genes for normalization. There is no single reference gene that can be regarded as universal for all experiments and biological materials. Thus, the identification of reliable reference genes must be done for every species and experimental approach. The present study aimed at identifying suitable control genes for normalization of gene expression associated with adventitious rooting in E. globulus microcuttings. Results: By the use of two distinct algorithms, geNorm and NormFinder, we have assessed gene expression stability of eleven candidate reference genes in E. globulus: 18S, ACT2, EF2, EUC12, H2B, IDH, SAND, TIP41, TUA, UBI and 33380. The candidate reference genes were evaluated in microccuttings rooted in vitro, in presence or absence of auxin, along six time-points spanning the process of adventitious rooting. Overall, the stability profiles of these genes determined with each one of the algorithms were very similar. Slight differences were observed in the most stable pair of genes indicated by each program: IDH and SAND for geNorm, and H2B and TUA for NormFinder. Both programs indentified UBI and 18S as the most variable genes. To validate these results and select the most suitable reference genes, the expression profile of the ARGONAUTE1 gene was evaluated in relation to the most stable candidate genes indicated by each algorithm. Conclusion: Our study showed that expression stability varied between putative reference genes tested in E. globulus. Based on the AGO1 relative expression profile obtained using the genes suggested by the algorithms, H2B and TUA were considered as the most suitable reference genes for expression studies in E. globulus adventitious rooting. UBI and 18S were unsuitable for use as controls in qPCR related to this process. These findings will enable more accurate and reliable normalization of qPCR results for gene expression studies in this economically important woody plant, particularly related to rooting and clonal propagation

Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill

<p>Abstract</p> <p>Background</p> <p><it>Eucalyptus globulus </it>and its hybrids are very important for the cellulose and paper industry mainly due to their low lignin content and frost resistance. However, rooting of cuttings of this species is recalcitrant and exogenous auxin application is often necessary for good root development. To date one of the most accurate methods available for gene expression analysis is quantitative reverse transcription-polymerase chain reaction (qPCR); however, reliable use of this technique requires reference genes for normalization. There is no single reference gene that can be regarded as universal for all experiments and biological materials. Thus, the identification of reliable reference genes must be done for every species and experimental approach. The present study aimed at identifying suitable control genes for normalization of gene expression associated with adventitious rooting in <it>E. globulus </it>microcuttings.</p> <p>Results</p> <p>By the use of two distinct algorithms, <it>geNorm </it>and <it>NormFinder</it>, we have assessed gene expression stability of eleven candidate reference genes in <it>E. globulus</it>: <it>18S, ACT2, EF2, EUC12, H2B, IDH, SAND, TIP41, TUA, UBI </it>and <it>33380</it>. The candidate reference genes were evaluated in microccuttings rooted <it>in vitro</it>, in presence or absence of auxin, along six time-points spanning the process of adventitious rooting. Overall, the stability profiles of these genes determined with each one of the algorithms were very similar. Slight differences were observed in the most stable pair of genes indicated by each program: <it>IDH </it>and <it>SAND </it>for <it>geNorm</it>, and <it>H2B </it>and <it>TUA </it>for <it>NormFinder</it>. Both programs indentified <it>UBI </it>and <it>18S </it>as the most variable genes. To validate these results and select the most suitable reference genes, the expression profile of the <it>ARGONAUTE1 </it>gene was evaluated in relation to the most stable candidate genes indicated by each algorithm.</p> <p>Conclusion</p> <p>Our study showed that expression stability varied between putative reference genes tested in <it>E. globulus</it>. Based on the <it>AGO1 </it>relative expression profile obtained using the genes suggested by the algorithms, <it>H2B </it>and <it>TUA </it>were considered as the most suitable reference genes for expression studies in <it>E. globulus </it>adventitious rooting. <it>UBI </it>and <it>18S </it>were unsuitable for use as controls in qPCR related to this process. These findings will enable more accurate and reliable normalization of qPCR results for gene expression studies in this economically important woody plant, particularly related to rooting and clonal propagation.</p

Somatic embryogenesis and plant regeneration derived from mature embryos of oat

Calo embriogênico tem sido o tecido-alvo mais utilizado para transformação genética de cereais. O objetivo deste trabalho foi investigar o estabelecimento de calos embriogênicos e a regeneração de plantas in vitro a partir de embriões maduros de genótipos de aveia (Avena sativa L.). Embriões maduros foram retirados das sementes e colocados em meio MS (Murashige & Skoog), contendo 30,0 g L-1 de sacarose e 2,0 mg L-1 de ácido 2,4-diclorofenoxiacético (2,4-D). Após o período de indução de calos, agregados embriogênicos foram isolados e subcultivados a cada 21 dias para meio fresco. Os calos embriogênicos foram então transferidos para meio de indução de parte aérea, e, na seqüência, as partes aéreas foram transferidas para meio de indução de raízes. Houve diferenças entre genótipos quanto à capacidade de embriogênese somática e regeneração de plantas in vitro a partir de embrião maduro. Este explante permitiu a indução de calos embriogênicos, que se multiplicaram, e que regeneraram in vitro um grande número de plantas de genótipos como UFRGS 7 e UFRGS 19, o que o faz passível de ser utilizado na transformação genética da aveia.Embryogenic callus has been the most used target tissue for cereal genetic transformation. Therefore, the objective of this study was to investigate the establishment of embryogenic calli and the in vitro plant regeneration from mature embryos of oat genotypes (Avena sativa L.). Mature embryos were taken out of the seeds and placed on a culture medium MS (Murashige & Skoog), containing 30,0 mg L-1 of sucrose and 2,0 mg L-1 of 2,4-dichlorophenoxyacetic acid (2,4-D). From the induction period, embryogenic aggregates were isolated and subcultivated each 21 days into a fresh medium. After this period, embryogenic calli were transferred to a medium for shoot regeneration. Subsequently, the shoot was transferred to a medium for root induction. There was variability among genotypes for somatic embryogenesis and in vitro plant regeneration from mature embryos. This explant allowed the induction of calli with ability to multiply and regenerate high number of plants from genotypes as UFRGS 7 and UFRGS 19, what makes it capable for oat genetic transformation