C. arabica and C. canephora ISE was established based on a tissue culture procedure proposed by Sanglard et al. [16], involving the stages of callus induction and proliferation (callogenesis), followed by SE regeneration (embryogenesis). Basically, we modified the 2,4-D concentration, adding 18.08, 36.24 and 54.36 µM in the callus induction and proliferation medium. We verified the immediate 2,4-D effect in the induction and proliferation of the C. arabica and C. canephora friable callus, as well as its remaining genotoxic effect during the SE regeneration stage in the embryogenic callus from the mean number of MCSE and ASE. In addition, we measured, compared, and associated global 5-mC% with 2,4-D concentrations during the in vitro response. Our results showed that 2,4-D has a genotoxic and phytotoxic effects on SE regeneration in both species, however C. canephora is more sensitive to the action of 2,4-D, since ASE showed more DNA damage. Despite this, C. canephora requires relatively higher 2,4-D concentration than C. arabica to ISE establishment.
2,4-D is a synthetic auxin widely used in plant tissue culture to induce ISE [14 and 16]. This compound triggers the cellular transition processes from the differentiated cell to embryogenic cell. This process is complex and includes dedifferentiation and cell division, which are modelate to metabolic and developmental reprogramming, conferring competence to the cells of the explants [1]. The 2,4-D concentration and time influenced the rate and cell proliferation of friable callus of the two Coffea, as well as the global 5-mC%. 2,4-D induces ISE in C. arabica and C. canephora and genetic, epigenetic factors and endogenous auxin levels in the cell can influence establishment in vitro. According to Vondráková et al. [12], the effectiveness of the exogenous auxin in ISE is affected by the endogenous auxin level in the cell and its homeostasis. Homeostasis is controlled by several mechanisms, such as auxin biosynthesis, as well as its degradation, transport and conjugation [25].
For C. canephora, exogenous auxin increases both free indole-3-acetic acid (IAA) and IAA amide conjugates during the callus induction, due to novel synthesis [26]. IAA is synthesized by the enzymes tryptophan aminotransferase of Arabidopsis (TAA) and YUCCA (YUC) from one of the tryptophan-dependent pathways. Uc-Chuc et al. [27] identified 10 members of the CcYUC gene family in C. canephora. Quintana-Escobar et al. [28] showed differences in the concentration of auxin homeostasis proteins in C. arabica, with a more significant accumulation of ABCs, GH3.17, UGT75C1 and IBR1 proteins. Karyotypic/genetic differences influence several traits of an individual, including the biosynthesis of auxins. C. arabica is an allotetraploid with 2C = 2.62 pg and 2n = 4x = 44 chromosomes. C. canephora is diploid with 2C = 1.41 pg and 2n = 2x = 22 chromosomes. We expected that C. arabica has more family members or copies of genes that encode proteins involved in auxin metabolism and, as a result, require fewer exogenous auxins. However, further investigations should be conducted to understand auxin biosynthesis in polyploids, as well as its spatial and temporal pattern of distribution during ISE.
The use of synthetic auxins, mainly 2,4-D, are necessary for the ISE induction and SE multiplication in Coffea, consequently potentiating the regeneration of plantlets in vitro [9, 10 and 16]. 2,4-D can cause epigenetic and genetic changes in cells during ISE, even at very low concentrations [1, 29 and 30]. In the present study, 2,4-D and, consequently, the global variations of 5-mC% influenced the in vitro response (acquisition of competence) in C. arabica and C. canephora. In addition, ASE regeneration was observed for both species. Corroborating with Oliveira et al. [9 and 10], variations in 5-mC% in ISE in Coffea were associated with species and in vitro environment. The formation of ASE was related to the prolonged effect of 2,4-D, which influences the expression, development and maturation of the SE and its subsequent conversion into a plant. The long exposure or accumulation of 2,4-D in tissue interferes in establishing internal auxin gradients and inhibits cell polarization [31]. As a consequence, 2,4-D disrupts normal endogenous auxin balance and polar auxin transport, inducing embryonic abnormalities [32 and 33].
In addition to 2,4-D, embryonic abnormalities may be associated with other chemical compounds required in plant tissue culture. These compounds may also cause mutations or epigenetic changes (somaclonal variation) and, consequently, influence embryonic development, SE morphology and, consequently, prevent plant recovering [17]. The formation of ASE has already been reported in Coffea [10 and 34] and in different species such as Medicago truncatula [35], Theobroma cacao [36] and Melia azedarach [37], being recurrent and common in plant tissue culture. For example, the ASE of the autoallohexaploid “Híbrido de Timor” (Coffea) exhibited the same ploidy level and chromosome number as the explant donor plants, and the high concentrations of activated charcoal in the regeneration medium has been appointed as one of the causes of these abnormalities due to phytotoxic effect [10]. The pattern of development of a normal SE in eudicotiledonea is characterized by the differentiation of a bipolar structure consisting of stem and root apex, passing through the stages of pre-embryonic and embryonic development, globular, cordiform, torpedo and cotyledonary [38]. However, ASE from C. arabica and C. canephora passed through these developmental stages, showing higher global 5-mC% and DNA damage due to the genotoxic action of 2,4-D.
DNA damage can be induced by exogenous chemical and physical factors by the action of genotoxic agents. Maintenance of the DNA integrity is necessary for the proper development of the organism and for the faithful transmission of the genetic information from one generation to the next [39]. Plants have specific mechanisms that repair DNA damage of the nuclear and organellar genomes. Our data show that C. canephora is more sensitive to 2,4-D, as ASE exhibited more DNA damage and higher values of 5-mC%. C. arabica is more tolerant to 2,4-D, probably because it is a polyploid. As reviewed by Schifino-Wittmann [40] and Sattler et al. [41], polyploids have a greater buffering effect in relation to adaptability, as they have more genomic copies than diploids, and can accumulate more hidden variability. Therefore, Coffea species react differently to the in vitro environment, especially to 2,4-D, and that genetic characteristics, as well as the evolution of the species, influence the in vitro response. We show, for the first time, that 2,4-D promotes DNA damage during ISE and its short-term consequences in vitro, such as the formation of ASE that present morphological and/or physiological disturbances, preventing plantlet regeneration.
The addition of 2,4-D to the culture medium stimulates the induction and proliferation of friable callus in C. arabica and C. canephora and consequently increases the regeneration of SE. However, 2,4-D promotes morphological and epigenetic changes and still causes DNA damage, compromising the development of SE. Therefore, the establishment of somatic embryogenesis requires complex cellular, biochemical and molecular processes. Epigenetics plays a key role in somatic embryogenesis, as it is a genetic regulatory mechanism that influences morphogenetic processes in vitro. Maintaining the integrity of the genome of plantlets regenerated in vitro is desirable, as DNA damage can lead to loss of genetic fidelity, alter methylation patterns and cause oxidative stress, functional disturbances and cell death.