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Previous issue date: 2009-05-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The main objective of this research was to work out models for the study of the
lignification. Eucalyptus grandis seedlings were produced in vitro, and the stem
segments explants were used to obtain friable callus. For such, the auxins tested were
TDZ, 2,4-D, NAA and IAA. The treatments with 50,0 ?M 2,4-D; 3,0 ?M TDZ and in
the absence of growth regulator didn't present callus formation. After 210 days, it was
chosen a callus formed in the culture medium added with 2,5 ?M of TDZ. The selected
callus was maintained in the same treatment, however, in liquid medium and under
agitation for 60 days at 25 ?C in the darkness, to induce cellular wall and extracellular
lignin formation. After this period, the cells suspensions were treated in the following
order: MS medium supplemented with sucrose, 2,4-D + kinetin, coumaric acid and
without extra supplement. The cells were maintained in these treatments for 30 days. It
was used a completely randomized design with four replications. Each plot consisted of
an Erlenmeyer with 125 ml of cells suspension culture. For cellular wall lignin
extraction and determination, it was used the method of lignin thyoglicolate. The
concentration of cellular wall and extracellular lignins were evaluated by UR. For data
normality verification it was used the Lilliefors test. The results were statistically
analyzed by variance analysis and by Tukey test at 5% level of significance. The highest
lignin concentration in the cellular wall (371.4200 ppm) was verified in the treatment
EG1pc 0,0584 ?M of sucrose. Extracellular lignins were also analyzed with Wiesner
reagent, H NMR and 13C NMR. The highest concentration (134.3167 ppm) of
extracellular lignins was verified in the treatment EG3e of 100?M coumaric acid).
However, with the results obtained in H NMR and ??CNMR analysis, it was not possible
to characterize the polymerization of pure extracellular lignin. A very useful tool for
lignification study is the dehydrogenized polymerization (DHP) in vitro. DHPs were
produce in cells suspension filtered medium (substrate) at different treatments. For each
treatment, it was added H2O2, peroxidase or H2O2 + peroxidase. The DHPs production
was done in MS culture medium (without the prior cultivation of cells) as a substrate. It
was added to MS medium three different solutions (coniferyl or sinapyl alcohol,
peroxidase and the H2O2) by dripping. The products of DHP and the filtered were
analyzed by IR and H NMR. The DHPs produced in medium MS without the previous
growth of the cells, were analyzed by H NMR and ??CNMR. The IR spectra didn t
present any sign in the zone of 1500 cm-1. H NMR also showed no formation of DHPs.
Nevertheless, in the, cells suspensions both in H NMR and in ??CNMR analysis
presented signs of DHPs, except the DHP1c (MS + 0,0584 ?M sucrose). H NMR
presented: 3.86; 6.92; and 4.71 ppm and the ??CNMR: 134,26; 88,21; 65,17; 55,90; and
20,75 ppm. / O objetivo principal desse trabalho foi a elabora??o de modelos para o estudo da
lignifica??o. Mudas de Eucalyptus grandis, obtidas a partir da germina??o de sementes
in vitro, foram desenvolvidas visando ? obten??o de explantes de segmentos caulinares.
Para tal, foram testadas as auxinas TDZ, 2,4-D, ANA e AIA. N?o foi constatada a
forma??o de calos nos tratamentos contendo 50,0 ?M de 2,4-D; 3,0 ?M de TDZ e na
aus?ncia de regulador de crescimento. Ap?s 210 dias, foi selecionado um calo formado
no tratamento contendo 2,5 ?M de TDZ. Este calo foi cultivado no mesmo tratamento,
por?m, em meio l?quido e sob agita??o por 60 dias a 25 ?C no escuro, para indu??o da
forma??o de lignina da parede celular e ligninas extracelulares. Ap?s este per?odo, as
c?lulas em suspens?o foram submetidas aos seguintes tratamentos: meio MS
suplementado com sacarose, 2,4-D + cinetina, ?cido p-cum?rico e sem suplemento
extra. As c?lulas permaneceram nestes tratamentos por 30 dias. O delineamento
experimental utilizado foi o inteiramente casualizado, com 4 repeti??es, sendo a parcela
experimental constitu?da por um Erlenmeyer contendo 125 ml de cultura de c?lulas em
suspens?o. Para extra??o e determina??o da lignina da parede celular, foi utilizada a
t?cnica baseada na lignina tioglicolato. A concentra??o de lignina da parede das c?lulas
e das ligninas extracelulares foi avaliada em UV. Para verificar a normalidade dos dados
foi usado o teste Lilliefors. Os dados foram submetidos ? an?lise de vari?ncia e as
m?dias comparadas pelo teste de Tukey a 5% de probabilidade. A maior concentra??o
de lignina na parede celular (371.4200 ppm) foi verificada no tratamento EG1pc 0,0584
?M de sacarose. Ligninas extracelulares tamb?m foram analisadas com o reagente
Wiesner, RMN H e RMN13C. A maior concentra??o (134.3167 ppm) de ligninas
extracelulares foi constatada no tratamento EG3e (100?M ?cido p-cum?rico). Por?m, os
resultados de RMN H e RMN13C, n?o permitiram caracterizar a polimeriza??o de
lignina extracelular pura. No estudo da lignifica??o utilizou-se a t?cnica de
polimeriza??o desidrogenativa (DHP) in vitro. DHPs foram elaborados em meios
obtidos a partir de filtrados das culturas de c?lulas em suspens?o em diferentes
tratamentos. Para cada tratamento, foi adicionado H2O2, peroxidase ou H2O2 +
peroxidase. A produ??o de DHPs foi efetuada em meio MS (sem o pr?vio cultivo de
c?lulas) como substrato no qual tr?s solu??es (?lcool conifer?lico ou sinap?lico,
peroxidase e o H2O2) foram adicionadas por gotejamento. Os produtos das DHPs,
juntamente com os filtrados, foram analisados por IV e RMN H. Nos tratamentos
realizados em meio MS sem o pr?vio cultivo de c?lulas em suspens?o, os DHPs
produzidos foram analisados por RMN H e RMN13C. Os espectros de IV n?o
apresentaram sinal na regi?o de 1500 cm-1, bem como as an?lises em RMN H
mostraram que n?o houve forma??o de DHPs. Por?m, nas suspens?es celulares tanto em
RMN H quanto RMN13C apresentaram sinais de DHP, exceto no tratamento com
DHP1c (MS + 0,0584 ?M sacarose). As an?lises em RMN H apresentaram: 3.86; 6.92 e
4.71 ppm e de RMN13C: 134,26; 88,21; 65,17; 55,90 e 20,75 ppm.
| Identifer | oai:union.ndltd.org:IBICT/oai:localhost:tede/489 |
| Date | 28 May 2009 |
| Creators | Pereira, Regina Paula Willemen |
| Contributors | Abreu, Heber dos Santos |
| Publisher | Universidade Federal Rural do Rio de Janeiro, Curso de P?s-Gradua??o em Ci?ncias Ambientais e Florestais, UFRRJ, Brasil, Ci?ncias Agr?rias |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFRRJ, instname:Universidade Federal Rural do Rio de Janeiro, instacron:UFRRJ |
| Rights | info:eu-repo/semantics/openAccess |
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