Fig 1.
Schematic representation of the experimental procedure used in this study.
Leaf explants of Coffea arabica (Ca) and C. canephora (Cc) were cultivated in the somatic embryogenesis (SE) induction media. In order to evaluate the factors involved in the inhibition of SE in Ca, the next steps were followed: 1. Ca and Cc explants were co-cultured together in the same medium. 2. The explants from Ca were discarded and the conditioned medium (CM) was used either fresh (CM-∆) or autoclaved (CM+∆) to culture the Cc explants. 3. The CM from seven days was fractionated with a 5 kDa cut-off membrane and the LmmCM obtained was added into the embryogenic cultures of Cc and Daucus carota (Dc). 4. The LmmCM was analyzed by GC-MS or UPLC-ESI-MS and the commercial identified phenolic compounds were added to Cc cultures. 5. The effect of LmmCM in DNA methylation was compared with that of the 5-azacytidine. Green circles: Cc leaf explants; yellow circles: Ca leaf explants; orange circles: Dc embryogenic cells.
Fig 2.
The somatic embryogenesis process in Coffea arabica and C. canephora.
Leaf explants were cultured in liquid medium using 5 μM of 6-benzyladenine. A. Leaf explants of C. arabica during the 56th day of somatic embryogenesis induction. The samples were taken every seven days. B. Leaf explants of C. canephora during the somatic embryogenesis process. The samples were taken every seven days. Bars = 5 mm.
Fig 3.
Scanning electron microscopy (SEM) examination of the explants of Coffea arabica and C. canephora under embryogenic conditions.
A. SEM of C. arabica explants during the 56-day somatic embryogenesis process. The white star indicates the cellular growth, and the white arrows show the generation of proembryogenic mass-like structures at 42 days and the development of a globular-like stage with an aberrant surface at 56 days. B. SEM of C. canephora explants during the somatic embryogenesis process. At 21 and 28 days after induction (dai), the proembryogenic mass (Pm) emerging from the mesophyll region of the explant is shown. The globular stage (G) appears after five weeks, with a well-differentiated protoderm structure. Heart stages (H) can be observed at 42 dai, torpedo stage (T) at 49 dai and cotyledonary stage (C) at 49 and 56 dai.
Fig 4.
Inhibition assays during the somatic embryogenesis of C. canephora.
A. C. canephora control after 42 dai with any treatment. Embryogenic explants of C. canephora (Cc) 21 dai were co-cultured together with 7-day-old explants of C. arabica (Ca). B. The picture was taken one week later. C. The picture was taken three weeks later.
Fig 5.
Effect of the conditioned medium of C. arabica on the formation of somatic embryos in C. canephora.
A. Temporal course of explants from C. canephora leaves under embryogenic induction without (control) or with the presence of the conditioned medium (CM) of C. arabica fresh (-∆) or autoclaved (+∆) from 7, 14 and 21 dai, as indicated in Materials and Methods. B. The embryogenic response of C. canephora evaluated after five weeks without (control) or with the presence of CMCa-∆ and CMCa+∆. Error bars represent the SE (n = 3). Bars marked with different letters indicate statistically different values between each embryogenic stage at a given time according to a Tukey test (P ≤ 0.01). The experiment was carried out independently three times.
Fig 6.
Effect of low molecular mass compounds secreted by explants of C. arabica on the embryogenic cultures of C. canephora and D. carota.
A. Morphological effects on the explants of C. canephora and suspension cells of D. carota under embryogenic conditions treated with low molecular mass of conditioned medium (LmmCM). B. Fraction of LmmCM of C. arabica of 7, 14, 21 and 28 dai was added to the embryogenic cultures, and its effect was plotted against the number of somatic embryos generated. C. The LmmCM of 7, 14, 21 and 28 days was added at the beginning of embryogenic induction in D. carota. The LmmCM of 7, 14, 21 and 28 days was added at 21 days after the embryogenic induction in the embryogenic cultures of C. canephora. The number of somatic embryos in C. canephora and D. carota at different developmental stages was counted at 56 and 14 days, respectively. Controls were cultivated in the absence of LmmCM. Error bars represent the standard error (n = 3). Different letters in bars represent the statistical significance of mean differences between each embryogenic stage at a given time according to the Tukey test (P ≤ 0.01). The experiment was carried out three times.
Fig 7.
Selected extracted ion chromatograms obtained of LmmCM from C. arabica by UPLC-ESI-ITMS.
A. Phenolic compounds detected in negative electrospray ionization mode. B. Caffeine was detected under positive electrospray ionization. Concentrations of phenolics compound are listed in Table 1.
Table 1.
Concentration of small phenolic compounds identified in two independent samples of LmmCM from C. arabica by UPLC-ESI-ITMS.
Fig 8.
Effect of caffeine, chlorogenic acid, hydoxybenzoic acid and trans-cinnamic acid in the somatic embryogenesis of C. canephora.
Explants from C. canephora leaves under embryogenic induction were supplemented with four different concentrations (1 μM, 10 μM, 100 μM and 1,000 μM) of A. Caffeine; B. Chlorogenic acid; C. Hydroxybenzoic acid, and D. Trans-cinnamic acid, at 7 dai. In contrast to the other compounds, which were diluted in the same culture medium, chlorogenic acid was diluted with dimethyl sulfoxide, which was used as a control. The number of every somatic embryo stage (globular, heart, torpedo and cotyledonary) was counted after 56 dai with and without (control) the phenolic compounds. The bars represent the mean ± SE (n = 3). Bars marked with different letters indicate statistically different values between each embryogenic stage at a given time according to the Tukey test (P ≤ 0.01). The experiments were performed three times.
Fig 9.
Global DNA methylation levels during the embryogenic induction of C. arabica and C. canephora and the effect of LmmCM on DNA methylation.
A. Percentage of DNA methylation from explants of C. arabica and C. canephora under embryogenic induction conditions. B. Percentage of DNA methylation during the SE of C. canephora in normal conditions (control), 10 μM of 5-azacytidine (an inhibitor of DNA methylation) and with LmmCM from 7 dai C. arabica. 5-azacytidine was added every 7 days from day 7 until day 49 and the fraction of LmmCM was added only one time, at day 7 after the induction. Error bars represent the SE (n = 3). Bars marked with different letters indicate statistically different values between each embryogenic stage at a given time according to the Tukey test (P ≤ 0.01). Each experiment was carried out three times.