Embriogênese somática e potencial uso de marcadores moleculares embriogênicos em Byrsonima intermedia A. Juss. (murici-pequeno)

Abstract

Byrsonima intermedia A. Juss., a Brazilian vegetation species, is commonly known as ‘murici-pequeno’, due to its small size compared to similar species. Evidence from previous studies shows the plant has great medicinal potential; though the plant has great difficulty in propagating naturally, due to its low germination, slow growth and tegumental dormancy. In this context, tissue culture techniques are presented as an alternative to sexual propagation, the use of somatic embryogenesis being of particular interest. Therefore, this study aimed to establish a protocol for, and analyze aspects of, the gene expression associated with the process of inducting somatic embryogenesis in murici-pequeno. For the induction of calluses from seedling leaves in vitro, different subcultures were tested (60 days) and concentrations of different cytokinins (BAP, TDZ, KIN and ZEA) combined with concentrations of NAA. Different concentrations of NAA were also analyzed in the induction of pro-embryogenic calluses. For the induction of embryogenic calli and somatic embryos, the pro-embryogenic calli were subcultured on MS area without adding growth regulators. The somatic embryos were inoculated in a maturation medium containing different concentrations of GA3 and ABA. The formation of secondary embryos, the control of hyperhydricity, the effect of different sucrose concentrations and culture media, different agar seals and concentrations were also analyzed. Molecular analyses were performed on different explants (pro-embryogenic and embryogenic calluses and somatic embryos). The results show that for the induction of somatic embryos, the use of kinetin with NAA presented the formation of somatic embryos in the second (4.76 µM CIN + 0.54 µM NAA) and third (5.17 µM CIN + 10.54 µM NAA) subcultures. The use of high concentrations of NAA (537.06 µM) favored the formation of somatic embryos. The use of GA3 in the concentration of 28.87 µM favored the formation of seedlings. The use of caps without PVC and Biosama® caps presented better results for test tubes and vials, respectively. The WPM medium, supplemented with 3% of sucrose and solidified with 0.8% of agar, reduced the hyperhydricity of somatic embryos. Even by observing the amplified fragments of genes AGL1, LEC1, SERK and BBM, it was not possible to perform the RT-qPCR because of difficulties in carrying out gene sequencing; this due to murici-pequeno being a native plant