Submitted by Leticia Schettini (leticia@ufrrj.br) on 2016-09-20T12:51:17Z No. of bitstreams: 1 2009 - Maria Beatriz de Oliveira Monteiro.pdf: 6872732 bytes, checksum: c5bedd487727835e97ae3e6b493349b3 (MD5) / Made available in DSpace on 2016-09-20T12:51:17Z (GMT). No. of bitstreams: 1 2009 - Maria Beatriz de Oliveira Monteiro.pdf: 6872732 bytes, checksum: c5bedd487727835e97ae3e6b493349b3 (MD5) Previous issue date: 2009-05-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior-CAPES / In spite of the technological progresses, the understanding of the lignin structural formation is still matter of scientific investigations. This research aimed to utilize Eucalyptus urophylla S.T. Blake callus for lignin production. This callus were obtained from stems segments explants grown in culture medium added with a combination of the cytokine TDZ and the auxins acid indole-3-acetic (IAA), acid ?-naphthaleneacetic (NAA) and acid 2,4-dichlorophenoxyacetic (2,4-D). This growth regulators were utilized either alone or mixtured. For cell suspension production it was utilized callus obtained in medium culture added with 20?M IAA + 3?M TDZ, after 30 days of growth in vitro. Once the cells suspension was obtained, the lignin production was induced by four elicitors: jasmonic acid (JA), NAA, sucrose and control (without elicitor). It was used a completely randomized design with four replications. Each plot consisted of an Erlenmeyer with 125 ml of cells suspension culture. The Wiesner test confirmed the lignin presence in all treatments. In 3 of the 4 replicatons it was performed another evaluation to the production of DHPs (polymers by oxidative dehydrogenation) utilizing suspension filtrate added with H2O2, H2O2 + peroxidase and peroxidase. These new treatments were analyzed through polilignols production utilizing infrared (IR) and nuclear magnetic resonance of the hydrogen (NMR H). The suspension filtrate analysis of the 4th replication through ultraviolet (UV), IR, NMR 13C and NMR H evidenced the production of extra cellular lignin. Of this, the largest content was obtained in presence of sucrose as elicitor, followed by ANA and AJ. The cells in suspension increased the cellular wall lignin content in all treatments in relation to the control and the largest values were with the medium containing sucrose. DHPs were also analyzed utilizing as mattress the MS medium added of the same elicitors tested in the cellular suspension phase. For this, it was utilized as precursors the following alcohols: coniferyl or sinapyl, H2O2 and peroxidase; amd the analyses were done in RMN H and RMN 13C. The results showed DHPs synthesis of both coniferyl alcohol (DHP1c, DHP2c and DHP4c) and sinapyl alcohol (DHP2s). Nevertheless, It was not synthesized DHP in the treatment containing sucrose when the precursor was the coniferyl alcohol. On the other hand, when the sinapyl alcohol was the precursor, DHPs were only synthesized in the presence of ANA as elicitor. It was concluded that sucrose is an appropriate elicitor for the lignin production in cells suspension both at cellular and extra cellular level. However, this result was not observed in relation to DHP production. NAA auxin had a better functionality in the DHP2c and DHP2s formation. These results may be considered a progress in the lignification studies with the use of E. urophylla cell suspension. Once all these questions were answered and solved, it will be possible to develop E.urophylla suitable plants with better quality forest products and able to cause smaller environmental impact in the industrial processes. / Apesar dos avan?os tecnol?gicos, a compreens?o da forma??o estrutural da lignina ainda ? alvo de in?meras investiga??es cient?ficas. Esta pesquisa foi realizada com calos de Eucalyptus urophylla S.T. Blake para a produ??o de lignina. Os calos foram obtidos a partir de explantes de segmentos caulinares desenvolvidos em meios de cultura acrescidos de uma combina??o da citocinina TDZ e das auxinas ?cidos: 3-indolac?tico (AIA), ?-naftalenoac?tico (ANA) e diclorofenoxiac?tico (2,4-D), nas formas isoladas e conjugadas. Para a produ??o de c?lulas em suspens?o foram utilizados os calos formados no tratamento contendo 20?M de AIA + 3?M de TDZ, ap?s 30 dias de cultivo in vitro. Obtidas as c?lulas em suspens?o, a produ??o de lignina foi induzida empregando-se quatro elicitores: ?cido jasm?nico (AJ), ANA, sacarose e testemunha (sem o emprego de elicitores). O delineamento experimental utilizado foi o inteiramente casualizado, 4 repeti??es e um Erlenmeyer contendo 125 ml de cultura de c?lulas em suspens?o. O teste de Wiesner confirmou a presen?a de lignina, em todos os tratamentos testados. Em 3 das 4 repeti??es foram realizados subtratamentos para a produ??o de DHPs (pol?meros por desidrogena??o oxidativa) a partir do filtrado da suspens?o com H2O2, H2O2 + peroxidase e peroxidase. A an?lise desses subtratamentos foi realizada pela detec??o da produ??o de polilign?is atrav?s de raios infravermelho (IV) e resson?ncia magn?tica nuclear do hidrog?nio (RMN H). O filtrado da suspens?o da repeti??o 4 foi analisado por ultravioleta (UV), IV, RMN 13C e RMN H, sendo constatada a presen?a de lignina extracelular, com o maior teor sendo observado na presen?a do elicitor sacarose, seguido do ANA e AJ. As c?lulas em suspens?o apresentaram aumento no teor de lignina na parede celular em todos os tratamentos em rela??o ? testemunha e os maiores valores foram com o meio contendo sacarose. Foram analisadas tamb?m as DHPs tendo como colch?o o meio de cultura MS acrescido dos mesmos elicitores testados na fase de suspens?o celular. Para isto foram utilizados como precursores os ?lcoois conifer?lico ou sinap?lico, H2O2 e peroxidase, com a lignina analisada em RMN H e RMN 13C. Os resultados mostraram que houve s?ntese de DHPs do ?lcool conifer?lico (DHP1c, DHP2c e DHP4c) e do ?lcool sinap?lico (DHP2s). Porem quando se utilizou como precursor o ?lcool conifer?lico n?o foram sintetizadas DHPs no tratamento contendo sacarose como elicitor. E, quando foi utilizado como precursor o ?lcool sinap?lico, somente foram formadas DHPs na presen?a do elicitor ANA. Concluiu-se, ent?o, que a sacarose apresentou-se como um elicitor adequado para a produ??o de lignina nas c?lulas em suspens?o, tanto em n?vel celular quanto extracelular. Entretanto, isto n?o foi observado em rela??o ? produ??o de DHP. A auxina ANA teve funcionalidade maior na forma??o de DHP2c e DHP2s. Estes resultados devem ser considerados como um avan?o nos estudos de lignifica??o com a utiliza??o de c?lulas em suspens?o de E. urophylla. Quando esses questionamentos forem identificados e solucionados ser? poss?vel o desenvolvimento de novos indiv?duos desta esp?cie que conduzam ? produ??o de produtos florestais de melhor qualidade, com menor impacto ambiental nos processos industriais.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-09-12T17:27:40Z No. of bitstreams: 1 2009 - Maria Beatriz de Oliveira Monteiro.pdf: 6859044 bytes, checksum: 68c2b98ad22b807fdd1e5779b401a4aa (MD5) / Made available in DSpace on 2017-09-12T17:27:40Z (GMT). No. of bitstreams: 1 2009 - Maria Beatriz de Oliveira Monteiro.pdf: 6859044 bytes, checksum: 68c2b98ad22b807fdd1e5779b401a4aa (MD5) Previous issue date: 2009-05-29 / CAPES / In spite of the technological progresses, the understanding of the lignin structural formation is still matter of scientific investigations. This research aimed to utilize Eucalyptus urophylla S.T. Blake callus for lignin production. This callus were obtained from stems segments explants grown in culture medium added with a combination of the cytokine TDZ and the auxins acid indole-3-acetic (IAA), acid ?-naphthaleneacetic (NAA) and acid 2,4-dichlorophenoxyacetic (2,4-D). This growth regulators were utilized either alone or mixtured. For cell suspension production it was utilized callus obtained in medium culture added with 20?M IAA + 3?M TDZ, after 30 days of growth in vitro. Once the cells suspension was obtained, the lignin production was induced by four elicitors: jasmonic acid (JA), NAA, sucrose and control (without elicitor). It was used a completely randomized design with four replications. Each plot consisted of an Erlenmeyer with 125 ml of cells suspension culture. The Wiesner test confirmed the lignin presence in all treatments. In 3 of the 4 replicatons it was performed another evaluation to the production of DHPs (polymers by oxidative dehydrogenation) utilizing suspension filtrate added with H2O2, H2O2 + peroxidase and peroxidase. These new treatments were analyzed through polilignols production utilizing infrared (IR) and nuclear magnetic resonance of the hydrogen (NMR H). The suspension filtrate analysis of the 4th replication through ultraviolet (UV), IR, NMR 13C and NMR H evidenced the production of extra cellular lignin. Of this, the largest content was obtained in presence of sucrose as elicitor, followed by ANA and AJ. The cells in suspension increased the cellular wall lignin content in all treatments in relation to the control and the largest values were with the medium containing sucrose. DHPs were also analyzed utilizing as mattress the MS medium added of the same elicitors tested in the cellular suspension phase. For this, it was utilized as precursors the following alcohols: coniferyl or sinapyl, H2O2 and peroxidase; amd the analyses were done in RMN H and RMN 13C. The results showed DHPs synthesis of both coniferyl alcohol (DHP1c, DHP2c and DHP4c) and sinapyl alcohol (DHP2s). Nevertheless, It was not synthesized DHP in the treatment containing sucrose when the precursor was the coniferyl alcohol. On the other hand, when the sinapyl alcohol was the precursor, DHPs were only synthesized in the presence of ANA as elicitor. It was concluded that sucrose is an appropriate elicitor for the lignin production in cells suspension both at cellular and extra cellular level. However, this result was not observed in relation to DHP production. NAA auxin had a better functionality in the DHP2c and DHP2s formation. These results may be considered a progress in the lignification studies with the use of E. urophylla cell suspension. Once all these questions were answered and solved, it will be possible to develop E.urophylla suitable plants with better quality forest products and able to cause smaller environmental impact in the industrial processes. / Apesar dos avan?os tecnol?gicos, a compreens?o da forma??o estrutural da lignina ainda ? alvo de in?meras investiga??es cient?ficas. Esta pesquisa foi realizada com calos de Eucalyptus urophylla S.T. Blake para a produ??o de lignina. Os calos foram obtidos a partir de explantes de segmentos caulinares desenvolvidos em meios de cultura acrescidos de uma combina??o da citocinina TDZ e das auxinas ?cidos: 3-indolac?tico (AIA), ?-naftalenoac?tico (ANA) e diclorofenoxiac?tico (2,4-D), nas formas isoladas e conjugadas. Para a produ??o de c?lulas em suspens?o foram utilizados os calos formados no tratamento contendo 20?M de AIA + 3?M de TDZ, ap?s 30 dias de cultivo in vitro. Obtidas as c?lulas em suspens?o, a produ??o de lignina foi induzida empregando-se quatro elicitores: ?cido jasm?nico (AJ), ANA, sacarose e testemunha (sem o emprego de elicitores). O delineamento experimental utilizado foi o inteiramente casualizado, 4 repeti??es e um Erlenmeyer contendo 125 ml de cultura de c?lulas em suspens?o. O teste de Wiesner confirmou a presen?a de lignina, em todos os tratamentos testados. Em 3 das 4 repeti??es foram realizados subtratamentos para a produ??o de DHPs (pol?meros por desidrogena??o oxidativa) a partir do filtrado da suspens?o com H2O2, H2O2 + peroxidase e peroxidase. A an?lise desses subtratamentos foi realizada pela detec??o da produ??o de polilign?is atrav?s de raios infravermelho (IV) e resson?ncia magn?tica nuclear do hidrog?nio (RMN H). O filtrado da suspens?o da repeti??o 4 foi analisado por ultravioleta (UV), IV, RMN 13C e RMN H, sendo constatada a presen?a de lignina extracelular, com o maior teor sendo observado na presen?a do elicitor sacarose, seguido do ANA e AJ. As c?lulas em suspens?o apresentaram aumento no teor de lignina na parede celular em todos os tratamentos em rela??o ? testemunha e os maiores valores foram com o meio contendo sacarose. Foram analisadas tamb?m as DHPs tendo como colch?o o meio de cultura MS acrescido dos mesmos elicitores testados na fase de suspens?o celular. Para isto foram utilizados como precursores os ?lcoois conifer?lico ou sinap?lico, H2O2 e peroxidase, com a lignina analisada em RMN H e RMN 13C. Os resultados mostraram que houve s?ntese de DHPs do ?lcool conifer?lico (DHP1c, DHP2c e DHP4c) e do ?lcool sinap?lico (DHP2s). Porem quando se utilizou como precursor o ?lcool conifer?lico n?o foram sintetizadas DHPs no tratamento contendo sacarose como elicitor. E, quando foi utilizado como precursor o ?lcool sinap?lico, somente foram formadas DHPs na presen?a do elicitor ANA. Concluiu-se, ent?o, que a sacarose apresentou-se como um elicitor adequado para a produ??o de lignina nas c?lulas em suspens?o, tanto em n?vel celular quanto extracelular. Entretanto, isto n?o foi observado em rela??o ? produ??o de DHP. A auxina ANA teve funcionalidade maior na forma??o de DHP2c e DHP2s. Estes resultados devem ser considerados como um avan?o nos estudos de lignifica??o com a utiliza??o de c?lulas em suspens?o de E. urophylla. Quando esses questionamentos forem identificados e solucionados ser? poss?vel o desenvolvimento de novos indiv?duos desta esp?cie que conduzam ? produ??o de produtos florestais de melhor qualidade, com menor impacto ambiental nos processos industriais.