Micropropagação, embriogênese somática, conservação in vitro e duplicação cromossômica em Cattleya tigrina A. Richa

• Ano de defesa: 2019
• Autor(a) principal: Sá, Thays Saynara Alves Menezes de
• Orientador(a): Arrigoni-Blank, Maria de Fátima
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Programa de Pós-Graduação: Pós-Graduação em Agricultura e Biodiversidade
• Palavras-chave em Português: Agricultura; Orchidaceae; Micropropagação; Plantas; Crescimento (plantas); Citometria de fluxo; Multiplicação in vitro; Análise estomática; Poliploidia
• Palavras-chave em Inglês: In vitro multiplication; Stomatal analysis; Polyploidy; Flow cytometry
• Área do conhecimento CNPq: CIENCIAS AGRARIAS
• Link de acesso: http://ri.ufs.br/jspui/handle/riufs/11644

Cattleya tigrina A. Rich., of the family Orchidaceae, is endemic in Brazil, with its distribution in the Northeast, in the Southeast and in the Southern regions. Due to its exuberance, this species is threatened with extinction, due to the devastation of the forests and by great extractive activities that are carried out by collectors and merchants. The objective of this study was to develop technologies for micropropagation, somatic embryogenesis, in vitro conservation, and chromosome duplication for the C. tigrina orchid. For the in vitro establishment, ripe fruits were collected. The capsules were opened and the seeds were isolated and inoculated in an MS culture medium, with half of the macronutrients supplemented with 30 g L-1 sucrose, 7 g L-1 agar, and 1 g L-1 activated carbon. For the in vitro multiplication, the experimental design was completely randomized in a 4x2 factorial scheme, with four volumes of the medium (10, 15, 20 and 25 mL) and with two different consistencies of the culture medium (steady and semisolid liquid). For the acclimatization, the substrates containing pine bark, charcoal, vermiculite, and coconut powder, were supplemented with 1 g.L-1 of limestone and were tested. In the somatic embryogenesis process, the young leaf explants were inserted into the MS culture medium that was supplemented with different concentrations of 2,4-D. Somatic embryos at different stages of the development were used for the histological studies. In the in vitro conservation experiments, when under slow growth, with different concentrations of the MS salts and the osmotic regulators, were tested (sucrose; sucrose: mannitol; sucrose: sorbitol) at two temperatures (18°C and 25°C). For the induction of polyploidy, the explants were treated with colchicine at concentrations of 0, 2.5, 7.5, and 12.5 mM for 24 and 48 hours, together with oryzalin at concentrations of 0, 10, 30, and 50 μM for 3 and 6 days. The confirmation of ploidy was performed by flow cytometry and stomatal analysis. The results obtained allowed for the researchers to infer that for the in vitro multiplication of the plants, the liquid culture media was stationary and that the semi-solids could be used at 10 mL for the liquid medium and at 25 mL for the semisolid medium. Acclimatization was performed by only using the pinus bark. The MS medium that was supplemented with 2,4-D (0,3 mg.L-1) induced the direct somatic embryogenesis. The histological analyses indicated that the somatic embryos originated from the cells of the epidermal layers of the leaf. The species can be conserved under a slow growth regime for a period of 730 days, by using a culture medium with 25% of the MS salts and at temperatures of 18°C or 25°C. For the osmotic regulator experiment, 20 g.L-1 of sucrose was stored at 25ºC. At the induction of polyploidy, all of the colchicine solutions and the immersion times were effective. These procedures obtained a greater number of polyploid individuals that were confirmed by flow cytometry and stomatal analysis. Oryzalin did not induce chromosome duplications at the concentrations tested.