Embriogênese somática indireta em coffea: aspectos genéticos, epigenéticos e do ambiente in vitro

Abstract

The genus Coffea comprises about 103 species with different levels of ploidy and evolutionary histories. The species of the genus have diversified mainly through changes in the DNA sequence (mutations), and also by events of allopolyploidy and other genomic changes involving structural chromosomal changes. These changes in the structure of the genome resulted in different phenotypes for some morphological, physiological and reproductive characteristics. Indirect somatic embryogenesis (ISE) is a morphogenetic expression pathway that has been used to investigate plant development and related “omic” events (genome, epigenome, transcriptome, metabolome), and also for mass propagation and germplasm conservation. Considering that genetic, epigenetic and/or physiological factors of explant donor plants and that the in vitro environment interfere in the establishment of ISE, the present study aimed to: (a) establish ISE in Coffea arabica (2C = 2.62 pg and 2n = 4x = 44 chromosomes), Coffea canephora (2C = 1.41 pg and 2n = 2x = 22 chromosomes) “Hybrid of Timor” (HT) allotriploid (1C = 2.10 pg and 2n = 3x = 33) and synthetic hexaploid (2C = 4.20 pg and 2n = 6x = 66); (b) investigate and compare the influence of evolutionary history (origin and time of formation), karyotype characteristics (number of chromosomes, ploidy level and 2C nuclear value) and global levels of methylated cytosine (5-mC%) in ISE ; and (c) assess the effect of the in vitro environment on the establishment of ISE. In the first study, the liquid and semisolid system influenced the establishment of ISE in HT allotriploid. The semisolid system resulted in a higher mean number of responsive explants (mean value of ~100%) and cell mass (mean value of 1.21 g), in a shorter period of time. Somatic embryos (SE) were regenerated when the callus of the liquid and semisolid systems were transferred to the SE regeneration medium in a liquid system. Therefore, the establishment of ISE in a liquid system was more effective, since it showed an embryogenic response after 45 days and a higher mean number of SE (38.85 per callus). In addition, karyotype variations were not observed among the regenerated plantlets. In the second study, ISE was established for allotriploid and hexaploid HT, and the evolutionary origin, karyotypic divergences, the global level of 5-mC% and the concentrations of activated charcoal influenced the in vitro response. The hexaploid HT showed a lower mean number of responsive explants and a higher level of 5-mC%. During the SE regeneration stage, the hexaploid HT again showed a higher level of 5-mC%, however both hybrids showed an mean value of 1.3 normal SE per callus at 330 days. The concentrations of 8 and 16 g L-1 of activated charcoal increased the levels of 5-mC% and the regeneration of abnormal SE in the hexaploid HT. The 5-mC% variations in hexaploid HT callus correspond to an adaptive response to in vitro conditions and, consequently, correspond to the somaclonal epigenetic variation. In the third study, C. arabica lines differed in relation to the in vitro response. 'Oeiras' exhibited the highest mean number of responsive leaf explants, followed by 'Caturra' and 'Catuaí Vermelho'. The global levels of 5-mC% increased gradually over time in the friable callus of 'Catuaí Vermelho' (20.73% at 60 days and 30.79% at 90 days) and 'Caturra' (38.70% at 60 days and 53.40% at 90 days) and remained constant for 'Oeiras' (34.34% at 60 days and 33.51% at 90 days). 'Catuaí Vermelho' exhibited the highest mean number of SE in all evaluated moments and no regeneration of SE was observed for 'Oeiras'. The embryogenic friable callus of 'Catuaí Vermelho' and 'Caturra' exhibited distinct values of 5-mC%, with mean values of 54.09% for 'Caturra' and 43.35% for 'Catuaí Vermelho'. Based on the results, the global increase of 5-mC% is necessary for the regeneration and maturation of SE in C. arabica lines. This global increase of 5-mC% is associated with a remodeling of chromatin from euchromatic to heterochromatic state. As observed in this study, DNA methylation is a dynamic and variable mechanism that interferes with the establishment of ISE. In the fourth study, the species C. arabica and C. canephora differed in relation to in vitro responses. The addition of 2,4-D to the induction medium increased the number of responsive explants in C. arabica and C. canephora and changed the patterns of global methylation in the different stages of ISE. In addition, the addition of 2,4-D promoted the regeneration of abnormal SE. The abnormal ES exhibited higher levels of global methylation and nuclei with DNA damage. In addition, the present study showed that variations in global methylation levels are an adaptive response to environmental conditions in vitro.