Embryonic abnormalities and genotoxicity induced by 2,4-dichlorophenoxyacetic acid during indirect somatic embryogenesis in Coffea

Abstract

Indirect somatic embryogenesis (ISE) is a morphogenetic pathway in which somatic cells form callus and, later, somatic embryos (SE). 2,4-dichlorophenoxyacetic acid (2,4-D) is a synthetic auxin that promotes the proliferation and dedifferentiation of somatic cells, inducing the ISE. However, 2,4-D can cause genetic, epigenetic, physiological and morphological disorders, preventing the regeneration and/or resulting abnormal somatic embryos (ASE). We aimed to evaluate the toxic 2,4-D effect during the Coffea arabica and C. canephora ISE, assessing the SE morphology, global 5-methylcytosine levels (5-mC%) and DNA damage. Leaf explants were inoculated in media with different 2,4-D concentrations. After 90 days, the friable calli were transferred to the regeneration medium, and the number of normal and abnormal SE was monthly counted. The increase of the 2,4-D concentration increased the number of responsive explants in both Coffea. At 9.06, 18.08 and 36.24 μM 2,4-D, C. arabica presented the highest values of responsive explants, differing from C. canephora. Normal and abnormal SE regeneration increased in relation to the time and 2,4-D concentration. Global 5-mC% varied at different stages of the ISE in both Coffea. Furthermore, the 2,4-D concentration positively correlated with global 5-mC%, and with the mean number of ASE. All ASE of C. arabica and C. canephora exhibited DNA damage and showed higher global 5-mC%. The allotetraploid C. arabica exhibited greater tolerance to the toxic effect of 2,4-D than the diploid C. canephora. We conclude that synthetic 2,4-D auxin promotes genotoxic and phytotoxic disorders and promotes epigenetic changes during Coffea ISE.

Production of Black Cumin via Somatic Embryogenesis, Chemical Profile of Active Compounds in Callus Cultures and Somatic Embryos at Different Auxin Supplementations

“…In addition, the mechanism of natural accrued auxin on SE is related to the type of exogenous auxin, which added to the medium [27]. The effect of synthetic auxins on SE could be observed as NAA in Picea abies and P. omorika [28], 2,4-D (2,4-dichlorophenoxyacetic acid) in Coffea [29], and IBA in Digitalis lanata [30].…”

Scanning Electron Microscopy Analysis of Tea’s Embryo Axis Explant Cultured on Murashige and Skoog Medium Containing 2,4-Dichlorophenoxyacetic acid

“…These explants then cultured on MS medium containing 2,4-D 2 mg.L -1 for inducing somatic embryogenesis. The selection of 2,4-D as PGR for inducing somatic embryogenesis was based on consideration that 2,4-D is a powerful embryogenesis inducer and has succeeded in triggering explant response to embryogenesis (Caeiro et al 2022;Gustian et al 2022) although it was also reported can result in abnormalities related to epigenetic and genetic changes (Fraga et al 2016;de Morais Oliveira et al 2023). All of explants showed the mountain-like structure exactly at centre of former incision of SAM after 7-DAC (Figure 1B).…”