Combined sensory, volatilome and transcriptome analyses identify a limonene terpene synthase as a major contributor to the characteristic aroma of a Coffea arabica L. specialty coffee.

BACKGROUND: The fruity aromatic bouquet of coffee has attracted recent interest to differentiate high value market produce as specialty coffee. Although the volatile compounds present in green and roasted coffee beans have been extensively described, no study has yet linked varietal molecular differences to the greater abundance of specific substances and support the aroma specificity of specialty coffees. RESULTS: This study compared four Arabica genotypes including one, Geisha Especial, suggested to generate specialty coffee. Formal sensory evaluations of coffee beverages stressed the importance of coffee genotype in aroma perception and that Geisha Especial-made coffee stood out by having fine fruity, and floral, aromas and a more balanced acidity. Comparative SPME-GC-MS analyses of green and roasted bean volatile compounds indicated that those of Geisha Especial differed by having greater amounts of limonene and 3-methylbutanoic acid in agreement with the coffee cup aroma perception. A search for gene ontology differences of ripening beans transcriptomes of the four varieties revealed that they differed by metabolic processes linked to terpene biosynthesis due to the greater gene expression of prenyl-pyrophosphate biosynthetic genes and terpene synthases. Only one terpene synthase (CaTPS10-like) had an expression pattern that paralleled limonene loss during the final stage of berry ripening and limonene content in the studied four varieties beans. Its functional expression in tobacco leaves confirmed its functioning as a limonene synthase. CONCLUSIONS: Taken together, these data indicate that coffee variety genotypic specificities may influence ripe berry chemotype and final coffee aroma unicity. For the specialty coffee variety Geisha Especial, greater expression of terpene biosynthetic genes including CaTPS10-like, a limonene synthase, resulted in the greater abundance of limonene in green beans, roasted beans and a unique citrus note of the coffee drink.

Haitian coffee agroforestry systems harbor complex arabica variety mixtures and under-recognized genetic diversity.

Though facing significant challenges, coffee (Coffea arabica) grown in Haitian agroforestry systems are important contributors to rural livelihoods and provide several ecosystem services. However, little is known about their genetic diversity and the variety mixtures used. In light of this, there is a need to characterize Haitian coffee diversity to help inform revitalization of this sector. We sampled 28 diverse farms in historically important coffee growing regions of northern and southern Haiti. We performed KASP-genotyping of SNP markers and HiPlex multiplex amplicon sequencing for haplotype calling on our samples, as well as several Ethiopian and commercial accessions from international collections. This allowed us to assign Haitian samples to varietal groups. Our analyses revealed considerable genetic diversity in Haitian farms, higher in fact than many farmers realized. Notably, genetic structure analyses revealed the presence of clusters related to Typica, Bourbon, and Catimor groups, another group that was not represented in our reference accession panel, and several admixed individuals. Across the study areas, we found both mixed-variety farms and monovarietal farms with the historical and traditional Typica variety. This study is, to our knowledge, the first to genetically characterize Haitian C. arabica variety mixtures, and report the limited cultivation of C. canephora (Robusta coffee) in the study area. Our results show that some coffee farms are repositories of historical, widely-abandoned varieties while others are generators of new diversity through genetic mixing.

Transcriptomic Analysis of Alternative Splicing Events during Different Fruit Ripening Stages of Coffea arabica L.

To date, genomic and transcriptomic data on Coffea arabica L. in public databases are very limited, and there has been no comprehensive integrated investigation conducted on alternative splicing (AS). Previously, we have constructed and sequenced eighteen RNA-seq libraries of C. arabica at different ripening stages of fruit development. From this dataset, a total of 3824, 2445, 2564, 2990, and 3162 DSGs were identified in a comparison of different fruit ripening stages. The largest proportion of DSGs, approximately 65%, were of the skipped exon (SE) type. Biologically, 9 and 29 differentially expressed DSGs in the spliceosome pathway and carbon metabolism pathway, respectively, were identified. These DSGs exhibited significant variations, primarily in S1 vs. S2 and S5 vs. S6, and they involve many aspects of organ development, hormone transduction, and the synthesis of flavor components. Through the examination of research findings regarding the biological functions and biochemical pathways associated with DSGs and DEGs, it was observed that six DSGs significantly enriched in ABC transporters, namely, LOC113712394, LOC113726618, LOC113739972, LOC113725240, LOC113730214, and LOC113707447, were continually down-regulated at the fruit ripening stage. In contrast, a total of four genes, which were LOC113732777, LOC113727880, LOC113690566, and LOC113711936, including those enriched in the cysteine and methionine metabolism, were continually up-regulated. Collectively, our findings may contribute to the exploration of alternative splicing mechanisms for focused investigations of potential genes associated with the ripening of fruits in C. arabica.

A General Protocol for Gene Expression Analysis in Chemically Mutant Coffee Plants in Response to Rust (Hemileia vastatrix Berk & Broome) Infection.

Coffea spp. is the source of one of the most widely consumed beverages in the world. However, the cultivation of this crop is threatened by Hemileia vastatrix Berk & Broome, a fungal disease, which reduces the productivity and can cause significant economic losses. In this protocol, coffee leaf segment derived from a chemical mutagenesis process are inoculated with uredospores of the pathogen. Subsequently, the gene expression changes are analyzed over the time (0, 5, 24, 48, and 120 h) using quantitative real-time polymerase chain reaction (RT-qPCR). The procedures and example data are presented for expression analysis in the CaWRKY1 gene. This procedure can be applied for quantitative analysis of other genes of interest to coffee breeders and scientists for elucidating the molecular mechanisms involved in the interaction between the plant and pathogen, potentially leading to the development of more efficient approaches for managing this disease.

Transcriptomic Data Analysis Using the Galaxy Platform: Coffee (Coffea arabica L.) Flowers as Example.

Coffee, an important agricultural product for tropical producing countries, is facing challenges due to climate change, including periods of drought, irregular rain distribution, and high temperatures. These changes result in plant water stress, leading to significant losses in coffee productivity and quality. Understanding the processes that affect coffee flowering is crucial for improving productivity and quality. In this chapter, we describe a protocol for transcriptome analysis using available Internet software, mainly in the Galaxy Platform, using RNA-Seq data from flowers collected from different parts of the coffee tree. The methods presented in this chapter provide a comprehensive protocol for transcriptome analysis of differentially expressed genes from flowers of coffee plant. This knowledge can be utilized in coffee genetic improvement programs, particularly in the selection of cultivars that are tolerant to water deficit.

Agroinfiltration for Enhanced Transgene Expression in Coffee Leaves (Coffea arabica L.).

The Coffea spp. plant is a significant crop in Latin America, Africa, and Asia, and recent advances in genomics and transcriptomics have opened possibilities for studying candidate genes and introducing new desirable traits through genetic engineering. While stable transformation of coffee plants has been reported using various techniques, it is a time-consuming and laborious process. To overcome this, transient transformation methods have been developed, which avoid the limitations of stable transformation. This chapter describes an ex vitro protocol for transient expression using A. tumefaciens-mediated infiltration of coffee leaves, which could be used to produce coffee plants expressing desirable traits against biotic and abiotic stresses, genes controlling biochemical and physiological traits, as well as for gene editing through CRISPR/Cas9.

Coffee Cell Suspensions as a Platform for Transient Gene Expression Analysis.

Coffea arabica L. is a crucial crop globally, but its genetic homogeneity leads to its susceptibility to diseases and pests like the coffee berry borer (CBB). Chemical and cultural control methods are difficult due to the majority of the CBB life cycle taking place inside coffee beans. One potential solution is the use of the gene cyt1Aa from Bacillus thuringiensis as a biological insecticide. To validate candidate genes against CBB, a simple, rapid, and efficient transient expression system is necessary. This study uses cell suspensions as a platform for expressing the cyt1Aa gene in the coffee genome (C. arabica L. var. Catuaí) to control CBB. The Agrobacterium tumefaciens strain GV3101::pMP90 containing the bar and cyt1Aa genes are used to genetically transform embryogenic cell suspensions. PCR amplification of the cyt1Aa gene is observed 2, 5, and 7 weeks after infection. This chapter describes a protocol that can be used for the development of resistant varieties against biotic and abiotic stresses and CRISPR/Cas9-mediated genome editing.

The genome and population genomics of allopolyploid Coffea arabica reveal the diversification history of modern coffee cultivars.

Coffea arabica, an allotetraploid hybrid of Coffea eugenioides and Coffea canephora, is the source of approximately 60% of coffee products worldwide, and its cultivated accessions have undergone several population bottlenecks. We present chromosome-level assemblies of a di-haploid C. arabica accession and modern representatives of its diploid progenitors, C. eugenioides and C. canephora. The three species exhibit largely conserved genome structures between diploid parents and descendant subgenomes, with no obvious global subgenome dominance. We find evidence for a founding polyploidy event 350,000-610,000 years ago, followed by several pre-domestication bottlenecks, resulting in narrow genetic variation. A split between wild accessions and cultivar progenitors occurred ~30.5 thousand years ago, followed by a period of migration between the two populations. Analysis of modern varieties, including lines historically introgressed with C. canephora, highlights their breeding histories and loci that may contribute to pathogen resistance, laying the groundwork for future genomics-based breeding of C. arabica.

Crop-to-wild gene flow in wild coffee species: the case of Coffea canephora in the Democratic Republic of the Congo.

BACKGROUND AND AIMS: Plant breeders are increasingly turning to crop wild relatives (CWRs) to ensure food security in a rapidly changing environment. However, CWR populations are confronted with various human-induced threats, including hybridization with their nearby cultivated crops. This might be a particular problem for wild coffee species, which often occur near coffee cultivation areas. Here, we briefly review the evidence for wild Coffea arabica (cultivated as Arabica coffee) and Coffea canephora (cultivated as Robusta coffee) and then focused on C. canephora in the Yangambi region in the Democratic Republic of the Congo. There, we examined the geographical distribution of cultivated C. canephora and the incidence of hybridization between cultivated and wild individuals within the rainforest. METHODS: We collected 71 C. canephora individuals from home gardens and 12 C. canephora individuals from the tropical rainforest in the Yangambi region and genotyped them using genotyping-by-sequencing (GBS). We compared the fingerprints with existing GBS data from 388 C. canephora individuals from natural tropical rainforests and the INERA Coffee Collection, a Robusta coffee field gene bank and the most probable source of cultivated genotypes in the area. We then established robust diagnostic fingerprints that genetically differentiate cultivated from wild coffee, identified cultivated-wild hybrids and mapped their geographical position in the rainforest. KEY RESULTS: We identified cultivated genotypes and cultivated-wild hybrids in zones with clear anthropogenic activity, and where cultivated C. canephora in home gardens may serve as a source for crop-to-wild gene flow. We found relatively few hybrids and backcrosses in the rainforests. CONCLUSIONS: The cultivation of C. canephora in close proximity to its wild gene pool has led to cultivated genotypes and cultivated-wild hybrids appearing within the natural habitats of C. canephora. Yet, given the high genetic similarity between the cultivated and wild gene pool, together with the relatively low incidence of hybridization, our results indicate that the overall impact in terms of risk of introgression remains limited so far.

The terroir of Brazilian Coffea canephora: Characterization of the chemical composition.

FTIR spectroscopy and multivariate analysis were used in the chemical study of the terroirs of Coffea canephora. Conilon coffees from Espírito Santo and Amazon robusta from Matas of Rondônia, were separated by PCA, with lipids and caffeine being the markers responsible for the separation. Coffees from Bahia, Minas Gerais, and São Paulo did not exhibit separation, indicating that the botanical variety had a greater effect on the terroir than geographic origin. Thus, the genetic factor was investigated considering the conilon and robusta botanical varieties. This last group was composed of hybrid robusta and apoatã. The DD-SIMCA favored the identification of the genetic predominance of the samples. PLS-DA had a high classification performance regarding the conilon, hybrid robusta, and apoatã genetic nature. Lipids, caffeine, chlorogenic acids, quinic acid, trigonelline, proteins, amino acids, and carbohydrates were identified as chemical markers that discriminated the genetic groups.

The evolutionary history of three Baracoffea species from western Madagascar revealed by chloroplast and nuclear genomes.

The wild species of the Coffea genus present a very wide morphological, genetic, and biochemical diversity. Wild species are recognized more resistant to diseases, pests, and environmental variations than the two species currently cultivated worldwide: C. arabica (Arabica) and C. canephora (Robusta). Consequently, wild species are now considered as a crucial resource for adapting cultivated coffee trees to climate change. Within the Coffea genus, 79 wild species are native to the Indian Ocean islands of Comoros, Mayotte, Mauritius, Réunion and Madagascar, out of a total of 141 taxa worldwide. Among them, a group of 9 species called "Baracoffea" are particularly atypical in their morphology and adaptation to the sandy soils of the dry deciduous forests of western Madagascar. Here, we have attempted to shed light on the evolutionary history of three Baracoffea species: C. ambongensis, C. boinensis and C. bissetiae by analyzing their chloroplast and nuclear genomes. We assembled the complete chloroplast genomes de novo and extracted 28,800 SNP (Single Nucleotide Polymorphism) markers from the nuclear genomes. These data were used for phylogenetic analysis of Baracoffea with Coffea species from Madagascar and Africa. Our new data support the monophyletic origin of Baracoffea within the Coffea of Madagascar, but also reveal a divergence with a sister clade of four species: C. augagneurii, C. ratsimamangae, C. pervilleana and C. Mcphersonii (also called C. vohemarensis), belonging to the Subterminal botanical series and living in dry or humid forests of northern Madagascar. Based on a bioclimatic analysis, our work suggests that Baracoffea may have diverged from a group of Malagasy Coffea from northern Madagascar and adapted to the specific dry climate and low rainfall of western Madagascar. The genomic data generated in the course of this work will contribute to the understanding of the adaptation mechanisms of these particularly singular species.

Evaluation of suitable reference genes for expression profile analyses of target genes in the coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae).

The coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae), is a major destructive insect pest of coffee, which impacts the coffee crops negatively. As a draft genome has been completed for this insect, most molecular studies on gene transcriptional levels under different experimental conditions will be conducted using real-time reverse-transcription quantitative polymerase chain reactions (RT-qPCR). However, the lack of suitable internal reference genes will affect the accuracy of RT-qPCR results. In this study, the expression stability of nine candidate reference genes was evaluated under different developmental stages, temperature stress, and Beauveria bassiana infection. Data analyses were completed by four commonly used programs, BestKeeper, NormFinder, geNorm, and RefFinder. The result showed that RPL3 and EF1α combination were recommended as the most stable reference genes for developmental stages. EF1α and RPS3a combination were the top two stable reference genes for B. bassiana infection. RPS3a and RPL3 combination performed as the optimal reference genes both in temperature stress and all samples. Our results should provide a good foundation for the expression profile analyses of target genes in the future, especially for molecular studies on insect genetic development, temperature adaptability, and immune mechanism to entomogenous fungi in H. hampei.

Genetic diversity and structure of the coffee leaf rust fungus Hemileia vastatrix across different coffee management systems in Ethiopia.

Although coffee leaf rust (CLR), caused by Hemileia vastatrix, poses an increasing threat to coffee production in Ethiopia, little is known regarding its genetic diversity and structure and how these are affected by coffee management. Here, we used genetic fingerprinting based on sequence-related amplified polymorphism (SRAP) markers to genotype H. vastatrix samples from different coffee shrubs, across 40 sites, covering four coffee production systems (forest coffee, semi plantation coffee, home garden coffee, and plantation coffee) and different altitudes in Ethiopia. In total, 96 H. vastatrix samples were successfully genotyped with three primer combinations, producing a total of 79 scorable bands. We found 35.44% of amplified bands to be polymorphic, and the polymorphic information content (PIC) was 0.45, suggesting high genetic diversity among our CLR isolates. We also found significant isolation-by-distance across the samples investigated and detected significant differences in fungal genetic composition among plantation coffee and home garden coffee and a marginally significant difference among plantation coffee and forest coffee. Furthermore, we found a significant effect of altitude on CLR genetic composition in the forest coffee and plantation systems. Our results suggest that both spore dispersal and different selection pressures in the different coffee management systems are likely responsible for the observed high genetic diversity and genetic structure of CLR isolates in Ethiopia. When selecting Ethiopian coffee genotypes for crop improvement, it is important that these genotypes carry some resistance against CLR. Because our study shows large variation in genetic composition across relatively short geographical distances, a broad selection of rust isolates must be used for coffee resistance screening.

High-Density Chitosan Induces a Biochemical and Molecular Response in Coffea arabica during Infection with Hemileia vastatrix.

The coffee industry faces coffee leaf rust caused by Hemileia vastratix, which is considered the most devastating disease of the crop, as it reduces the photosynthetic rate and limits productivity. The use of plant resistance inducers, such as chitosan, is an alternative for the control of the disease by inducing the synthesis of phytoalexins, as well as the activation of resistance genes. Previously, the effect of chitosan from different sources and physicochemical properties was studied; however, its mechanisms of action have not been fully elucidated. In this work, the ability of food-grade high-density chitosan (0.01% and 0.05%) to control the infection caused by the pathogen was evaluated. Subsequently, the effect of high-density chitosan (0.05%) on the induction of pathogenesis-related gene expression (GLUC, POX, PAL, NPR1, and CAT), the enzymatic activity of pathogenesis-related proteins (GLUC, POX, SOD, PPO, and APX), and phytoalexin production were evaluated. The results showed that 0.05% chitosan increased the activity and gene expression of ß-1,3 glucanases and induced a differentiated response in enzymes related to the antioxidant system of plants. In addition, a correlation was observed between the activities of polyphenol oxidase and the production of phytoalexin, which allowed an effective defense response in coffee plants.

New genetic markers for 100% arabica coffee demonstrate high discriminatory potential for InDel-HRM-based coffee authentication.

Food authenticity is crucial in today's society, given the heightened consumer awareness and attention to the products they consume. Reliable and efficient techniques are needed to quickly detect potential food adulterations that can negatively impact product quality and economic value. Coffee, a globally traded agricultural product, holds immense economic importance, with an estimated value of USD 83 billion. It is widely consumed and recognized as a functional food that provides minerals (K, Mg, Mn, Cr), niacin, and antioxidants. However, the preferred coffee species, Coffea arabica, known for its superior drink quality, is often adulterated with Coffea canephora (Robusta and Conilon) beans, even in 100% Arabica coffee. To distinguish between these two coffee species, a comprehensive study was conducted using a robust approach to identify differences in Single-Ortholog Copy (SOC) based on InDel regions in these gene pairs. These differences were validated using a meticulous methodology that considered variations in amplicon size: electrophoretic profile, and high-resolution melting (HRM). The innovative combination of InDels and HRM resulted in highly distinctive HRM profiles, outperforming SNP-based methods previously used. The targeted InDel approach utilized in this study facilitated precise quantification of Coffea species beans with a detection sensitivity of 0.5%. The study's findings establish the reliability and accuracy in distinguishing between the two coffee species, showcasing the valuable application of InDels for quality control and ensuring the authenticity of coffee beans. This pioneering research contributes to the advancement of authenticity verification methods for both imported and exported coffee beans, as well as in future studies that require significant genetic differences between these species, such as C. arabica and C. canephora.

Functional Characterization of ent-Copalyl Diphosphate Synthase and Kaurene Synthase Genes from Coffea arabica L.

The biochemical profile of coffee beans translates directly into quality traits, nutraceutical and health promoting properties of the coffee beverage. Ent-kaurene is the ubiquitous precursor for gibberellin biosynthesis in plants, but it also serves as an intermediate in specialized (i.e., secondary) diterpenoid metabolism that leads to a diversity of more than 1,000 different metabolites. Nutraceutical effects on human health attributed to diterpenes include antioxidant, anticarcinogenic, and anti-inflammatory properties. Cafestol (CAF) and kahweol (KAH) are two diterpenes found exclusively in the Coffea genus. Our objective was to identify and functionally characterize genes involved in the central step of ent-kaurene production. We identified 17 putative terpene synthase genes in the transcriptome of Coffea arabica. Two ent-copalyl diphosphate synthase (CaCPS) and three kaurene synthase (CaKS) were selected and manually annotated. Transcript expression profiles of CaCPS1 and CaKS3 best matched the CAF and KAH metabolite profiles in different tissues. CaCPS1 and CaKS3 proteins were heterologously expressed and functionally characterized. CaCPS1 catalyzes the cyclization of geranylgeranyl diphosphate (GGPP) to ent-copalyl diphosphate (ent-CPP), which is converted to ent-kaurene by CaKS3. Knowledge about the central steps of diterpene formation in coffee provides a foundation for future characterization of the subsequent enzymes involved in CAF and KAH biosynthesis.

The chloroplast protein HCF164 is predicted to be associated with Coffea SH9 resistance factor against Hemileia vastatrix.

To explore the connection between chloroplast and coffee resistance factors, designated as SH1 to SH9, whole genomic DNA of 42 coffee genotypes was sequenced, and entire chloroplast genomes were de novo assembled. The chloroplast phylogenetic haplotype network clustered individuals per species instead of SH factors. However, for the first time, it allowed the molecular validation of Coffea arabica as the maternal parent of the spontaneous hybrid "Híbrido de Timor". Individual reads were also aligned on the C. arabica reference genome to relate SH factors with chloroplast metabolism, and an in-silico analysis of selected nuclear-encoded chloroplast proteins (132 proteins) was performed. The nuclear-encoded thioredoxin-like membrane protein HCF164 enabled the discrimination of individuals with and without the SH9 factor, due to specific DNA variants linked to chromosome 7c (from C. canephora-derived sub-genome). The absence of both the thioredoxin domain and redox-active disulphide center in the HCF164 protein, observed in SH9 individuals, raises the possibility of potential implications on redox regulation. For the first time, the identification of specific DNA variants of chloroplast proteins allows discriminating individuals according to the SH profile. This study introduces an unexplored strategy for identifying protein/genes associated with SH factors and candidate targets of H. vastatrix effectors, thereby creating new perspectives for coffee breeding programs.

Identification of sources of male sterility in the Colombian Coffee Collection for the genetic improvement of Coffea arabica L.

In coffee (Coffea arabica L.), male sterility is a prerequisite for the exploitation of heterosis since it provides an efficient and reliable method for the production of hybrid seeds. Given its relevance, the objective of this study was to identify male-sterile genotypes within the Colombian Coffee Collection that can be used in genetic improvement. For this purpose, Ethiopian germplasm and progenies derived from hybrids between C. arabica x C. canephora were explored between 2017 and 2021. In the first stage, genotypes without visual presence of pollen were preselected in the field, followed by selection through staining and verification of male sterility and female fertility through directed crosses (directed, reciprocal and selfing). In this stage, 9,753 trees were explored, preselecting 2.4% due to visual absence of pollen. The staining of structures allowed us to confirm the lack or sporadic production of pollen in 23 individuals of Ethiopian origin. The results of the directed crosses led to the identification of 11 male-sterile and 12 partially male-sterile genotypes belonging to 15 accessions. In all cases, the individuals were characterized by the presence of anthers but with an absence or low content of pollen, which is why the male sterility is possibly of the sporogenic type. The female receptivity values were between 2.9% and 72.6%, being higher than 30% in five genotypes. These genotypes are a valuable tool for the genetic improvement of C. arabica with the potential to facilitate the use of heterosis and to allow a deeper understanding the development of male gametophytes in the species.

An analysis of codon utilization patterns in the chloroplast genomes of three species of Coffea.

BACKGROUND: The chloroplast genome of plants is known for its small size and low mutation and recombination rates, making it a valuable tool in plant phylogeny, molecular evolution, and population genetics studies. Codon usage bias, an important evolutionary feature, provides insights into species evolution, gene function, and the expression of exogenous genes. Coffee, a key crop in the global tropical agricultural economy, trade, and daily life, warrants investigation into its codon usage bias to guide future research, including the selection of efficient heterologous expression systems for coffee genetic transformation. RESULTS: Analysis of the codon utilization patterns in the chloroplast genomes of three Coffea species revealed a high degree of similarity among them. All three species exhibited similar base compositions, with high A/T content and low G/C content and a preference for A/T-ending codons. Among the 30 high-frequency codons identified, 96.67% had A/T endings. Fourteen codons were identified as ideal. Multiple mechanisms, including natural selection, were found to influence the codon usage patterns in the three coffee species, as indicated by ENc-GC3s mapping, PR2 analysis, and neutral analysis. Nicotiana tabacum and Saccharomyces cerevisiae have potential value as the heterologous expression host for three species of coffee genes. CONCLUSION: This study highlights the remarkable similarity in codon usage patterns among the three coffee genomes, primarily driven by natural selection. Understanding the gene expression characteristics of coffee and elucidating the laws governing its genetic evolution are facilitated by investigating the codon preferences in these species. The findings can enhance the efficacy of exogenous gene expression and serve as a basis for future studies on coffee evolution.

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

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.