Título:	Estabelecimento e Cultivo de Células em Suspensão e Uso de Biorreatores como Estratégias de Propagação de Bambus do Gênero Guadua
Autor(es):	Queiroz, Fernanda Furlan
Orientador(es):	Scherwinski-Pereira, Jonny Everson
Assunto:	Poaceae Bambusoideae Guadua morfogênese sistemas de cultivo análises bioquímicas
Data de publicação:	4-Nov-2021
Data de defesa:	28-Jul-2020
Referência:	QUEIROZ, Fernanda Furlan. Estabelecimento e Cultivo de Células em Suspensão e Uso de Biorreatores como Estratégias de Propagação de Bambus do Gênero Guadua. 2020. 208. f., il. Tese (Doutorado em Botânica)—Universidade de Brasília, Brasília, 2020.
Abstract:	This work aimed to improve in vitro clonal multiplication techniques of Guadua magna and Guadua aff. chaparensis, developing studies related to callogenesis, somatic embryogenesis and cells suspension culture, in addition to evaluating the application of bioreactors in stages of the process. Anatomical and histochemical investigations, in addition to molecular analyzes, were also carried out to better characterize the different steps involved in the processes studied. Therefore, initially, induction was performed the callogenesis in explants that consisted of nodal segments, internodal segments and leaf sheaths of plants established in vitro of G. magna. The explants were submitted to MS medium culture supplemented with dicamba, 2,4-D or picloram in concentrations of 0; 6.77; 13.54; 20.31 and 27.08 μM. At 60 days of cultivation, it was found that all treatments showed yellowish-white calli and mucilaginous appearance. The histological analysis revealed meristematic zones with cells in intense division and formation of adventicius roots. The highest average percentages of calli formation were obtained in the concentrations 20.31 μM (88.0 %), 13.54 μM (85.3 %) and 27.08 μM (74.7%). The highest increment of fresh mass of calli was verified in 13.54 μM of picloram (402.5 mg). The calli formed were inoculated into Erlenmeyer flasks (125 mL) containing 20 mL of liquid MS medium, supplemented with 4.44 μM dicamba, 2.4-D or picloram, which were evaluated four different calli origins to induction calli step to establishment: 13.54 μM dicamba (T1), 13.54 μM 2.4-D (T2), 13.54 μM picloram or 20.31 μM picloram (T4). After six subcultures of 30 days each, T3 and T4 showed better rates of establishment (100 % and 83.3 %), increase in fresh mass and dry mass of the cell suspension cultures. From cytochemical analysis, cultures in T3 and T4 showed a greater cluster of reddish cells, which qualifies them as positive in terms of embryogenic viability, giving these treatments the best culture conditions for this purpose. In a second chapter, nodal segments of the species of G. aff. chaparensis were inoculated into MS medium with 2,4-D and picloram, in concentrations of 11.1 μM or 22.2 μM to induce somatic embryogenesis. After 120 days of cultivation, four morphologically distinct calli were observed: (1) gelatinous in 11.1 μM of 2,4-D, (2) compact in 22,2 μM of 2,4-D, (3) friable in 11.1 μM picloram and (4) nodular compacts in 22.2 μM picloram. In friable calli, the formation of pro-embryos was observed, a decisive characteristic for the progression of somatic embryos, and also the presence of starch grains. The friable calli were subjected to liquid culture under agitation to obtain cell suspension cultures in the treatments T1- control, T2- 4.44 μM of 2.4-D, T3- 4.44 μM of picloram or T4- in combination with auxins. All treatments provided cells with embryogenic viability verified with cytochemical analysis, except T1- control. After the multiplication of cultures, calli recovery was performed, using aliquots of 20 μL of the cultures resuspended in MS medium in the treatments with 2,4-D and picloram, in the concentrations of 0; 6.77; 13.54; 20.31 and 27.08 μM. Such calli showed a friable aspect and, from the anatomical analysis, somatic pro-embryos were observed. Finally, in a third chapter, it was realized the multiplication of vegetative propagules of G. magna in different in vitro culture systems: T1 CLM- Conventional Liquid Medium, T2 LMA- Medium under Agitation, T3 PERIB- Permanent Immersion Bioreactors and T4 TIB- Temporary Immersion Bioreactor. After 90 days of cultivation, PERIB and TIB showed higher means of the vegetatives propagules survival, number of shoots, size of shoots and mass fresh, as well as chlorophyll content a, b and carotenoids. Through the analysis of electrolyte leakage, it was possible to verify that in all treatments the damage to the membrane was relatively low in the culture systems (≤ 33.5), except T2-LMA (60.4). In the analysis of the biochemical components (AST / Starch), the G. magna seedlings grown in T3- PERIB and T4-TIB, consumed their carbohydrate reserves (AST) in greater quantity. The anatomical analysis revealed that the propagated seedlings contained the basic cellular structure necessary for the development of new shoots and roots and also evidenced the presence of meristematic regions, cells of leaf and root pimordial, and stem apical meristem. It was also possible to visualize the connection between the vascular system of the stem and the leaves, where one or more vascular bundles are oriented towards the leaves. As for acclimatization, the optimal survival rates were obtained from the G. magna plants from T3-PERIB and T4- TIB, with the occurrence of spontaneous adventitious rooting. For genetic fidelity analysis, using ISSR's molecular markers, the individuals analyzed showed 100 % similarity in all treatments, and the somaclones differed on average less than 0.2 %. It is concluded that the new information generated in this study can be useful to contribute to the understanding of the biological, physiological and biochemical aspects during the in vitro cultivation process of G. magna and G. aff. chaparensis, as well as the improvement of in vitro clonal multiplication in cultivation systems, including other bamboo species.
Unidade Acadêmica:	Instituto de Ciências Biológicas (IB) Departamento de Botânica (IB BOT)
Informações adicionais:	Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Botânica, Programa de Pós-Graduação em Botânica, 2020.
Programa de pós-graduação:	Programa de Pós-Graduação em Botânica
Licença:	A concessão da licença deste item refere-se ao termo de autorização impresso assinado pelo autor com as seguintes condições: Na qualidade de titular dos direitos de autor da publicação, autorizo a Universidade de Brasília e o IBICT a disponibilizar por meio dos sites www.bce.unb.br, www.ibict.br, http://hercules.vtls.com/cgi-bin/ndltd/chameleon?lng=pt&skin=ndltd sem ressarcimento dos direitos autorais, de acordo com a Lei nº 9610/98, o texto integral da obra disponibilizada, conforme permissões assinaladas, para fins de leitura, impressão e/ou download, a título de divulgação da produção científica brasileira, a partir desta data.
Agência financiadora:	Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e Fundação de Apoio à Pesquisa do Distrito Federal (FAP/DF).
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