Alterações epigenéticas na morfogênese in vitro: estudo comparativo de padrões de metilação de DNA durante a organogênese e a embriogênese somática em Solanum melongena L. (berinjela)

• Autor(a) principal: Alencar, Amanda Santos de
• Data de Publicação: 2019
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Biblioteca Digital de Teses e Dissertações da UERJ
• Texto Completo: http://www.bdtd.uerj.br/handle/1/7916
• topic: Callogenesis; Simultaneous induction; BAP; MSAP; LFY; Calogênese; Indução simultânea; BAP; MSAP; LFY; Berinjela; Genética vegetal; Berinjela; CNPQ::CIENCIAS BIOLOGICAS::BOTANICA

Although several protocols for in vitro organogenesis induction and in vitro somatic embryogenesis have been established for eggplant, the molecular mechanisms that control these processes are poorly understood. In this work, the MSAP (Methylation Sensitive Amplified Polymorphism) technique was applied to organogenic and embryogenic calli at different developmental stages in order to identify possible epigenetic differences between the two regeneration pathways. The in vitro culture system used displays simultaneous development of both callus types in different regions of cotyledon explants in response to the same growth regulator. Eggplant seeds cv. Florida Market were washed with sodium hypochlorite and germinated in MS½ medium (half salt concentration). After 21 days, cotyledon segments were excised and inoculated in MS + BAP 5 mg/L solidified medium. DNA was extracted from the original explant and from calli formed after 7, 10 and 14 days of culture. For MSAP analysis, the DNA was digested with EcoR I, Msp I and Hpa II, followed by adapters ligation, preamplification and selective amplification with 19 primer combinations. Electrophoresis was performed on 6% denaturing polyacrylamide gels and stained with silver. Among the 19 primers tested, 15 had an analyzable band pattern, with a total of 715 bands. Considering the entire primer set, total methylation corresponded to 43.9% of epigenetic events, followed by demethylation (26.96%), external methylation (20.63%) and internal methylation (8.9%). The two types of calli showed different of complete methylation dynamics along the culture period. Changes in band patterns along the culture period (0, 7, 10 and 14 days) and polymorphisms between organogenic and embryogenic calli were observed in 11 primers. A total of 34 potential epigenetic markers associated with each regeneration pathway were visualized. The combination of EcoR I + CAG x Msp I - Hpa II + GTCA primers showed 188 amplified fragments, of which 22 were polymorphic, being considered the best performance among the tested primers. The reconstruction of the genomic sequence comprehended of the 5'-CCGG-3' cut site and the selective Msp I-Hpa II + GTCA primer forms the 5'-CGGTCA-3' motif. Database search results indicated that this motif corresponds to the part of the recognition sequence of the LFY (LEAFY) DNA binding domain in Arabidopsis thaliana, which is involved with the differentiation and identity of floral meristems, acting as a factor of transcription. Thus, it can be concluded that the MSAP technique can be used for the detection of specific methylation patterns in eggplant embryogenic and organogenic calli. MSAP can also be applied in a targeted manner by constructing selective primers with motifs of interest, allowing the inference of the activity of genes present in the primer fragment.