In Vitro Conversion of Coffea spp. Somatic Embryos in SETIS™ Bioreactor System.

Somatic embryogenesis (SE) is an excellent example of mass plant propagation. Due to its genetic variability and low somaclonal variation, coffee SE has become a model for in vitro propagation of woody species, as well as for large-scale production of vigorous plants that are advantageous to modern agriculture. The success of the large-scale propagation of an embryogenic system is dependent on the development, optimization, and transfer of complementary system technologies. In this study, two successful SE systems were combined with a SETIS™ bioreactor immersion system to develop an efficient and cost-effective approach for the in vitro development of somatic embryos of Coffea spp. This study used an efficient protocol for obtaining somatic embryos, utilizing direct and indirect SE for both C. canephora and C. arabica. Embryos in the cotyledonary stage were deposited in a bioreactor to complete their stage of development from embryo to plant with minimal manipulation. Following ten weeks of cultivation in the bioreactor, complete and vigorous plants were obtained. Different parameters such as fresh weight, length, number of leaves, and root length, as well as stomatal index and relative water content, were recorded. In addition, the survival rate and ex vitro development of plantlets during acclimatization was assessed. The best substrate combination was garden soil (GS), peat moss (PM), and agrolite (A) in a 1:1:0.5 ratio, in which the bioreactor-regenerated plants showed an acclimatization rate greater than 90%. This is the first report on the use of SETIS™ bioreactors for the in vitro development of somatic embryos in Coffea spp., providing a technology that could be utilized for the commercial in vitro propagation of coffee plants. A link between research and innovation is necessary to establish means of communication that facilitate technology transfer. This protocol can serve as a basis for the generation and scaling of different species of agroeconomic importance. However, other bottlenecks in the production chains and the field must be addressed.

Characterization of the PIN Auxin Efflux Carrier Gene Family and Its Expression during Zygotic Embryogenesis in Persea americana.

Auxins are responsible for a large part of the plant development process. To exert their action, they must move throughout the plant and from cell to cell, which is why plants have developed complex transport systems for indole-3-acetic acid (IAA). These transporters involve proteins that transport IAA into cells, transporters that move IAA to or from different organelles, mainly the endoplasmic reticulum, and transporters that move IAA out of the cell. This research determined that Persea americana has 12 PIN transporters in its genome. The twelve transporters are expressed during different stages of development in P. americana zygotic embryos. Using different bioinformatics tools, we determined the type of transporter of each of the P. americana PIN proteins and their structure and possible location in the cell. We also predict the potential phosphorylation sites for each of the twelve-PIN proteins. The data show the presence of highly conserved sites for phosphorylation and those sites involved in the interaction with the IAA.

Auxin-Cytokinin Cross Talk in Somatic Embryogenesis of Coffea canephora.

Cytokinins (CK) are plant growth regulators involved in multiple physiological processes in plants. One less studied aspect is CK homeostasis (HM). The primary genes related to HM are involved in biosynthesis (IPT), degradation (CKX), and signaling (ARR). This paper demonstrates the effect of auxin (Aux) and CK and their cross talk in a Coffea canephora embryogenic system. The transcriptome and RT-qPCR suggest that Aux in pre-treatment represses biosynthesis, degradation, and signal CK genes. However, in the induction, there is an increase of genes implicated in the CK perception/signal, indicating perhaps, as in other species, Aux is repressing CK, and CK are inducing per se genes involved in its HM. This is reflected in the endogenous concentration of CK; pharmacology experiments helped study the effect of each plant growth regulator in our SE system. We conclude that the Aux-CK balance is crucial to directing somatic embryogenesis in C. canephora.

Differences in the Abundance of Auxin Homeostasis Proteins Suggest Their Central Roles for In Vitro Tissue Differentiation in Coffea arabica.

Coffea arabica is one of the most important crops worldwide. In vitro culture is an alternative for achieving Coffea regeneration, propagation, conservation, genetic improvement, and genome editing. The aim of this work was to identify proteins involved in auxin homeostasis by isobaric tandem mass tag (TMT) and the synchronous precursor selection (SPS)-based MS3 technology on the Orbitrap Fusion™ Tribrid mass spectrometer™ in three types of biological materials corresponding to C. arabica: plantlet leaves, calli, and suspension cultures. Proteins included in the ß-oxidation of indole butyric acid and in the signaling, transport, and conjugation of indole-3-acetic acid were identified, such as the indole butyric response (IBR), the auxin binding protein (ABP), the ATP-binding cassette transporters (ABC), the Gretchen-Hagen 3 proteins (GH3), and the indole-3-acetic-leucine-resistant proteins (ILR). A more significant accumulation of proteins involved in auxin homeostasis was found in the suspension cultures vs. the plantlet, followed by callus vs. plantlet and suspension culture vs. callus, suggesting important roles of these proteins in the cell differentiation process.

Genome-Wide Analysis, Modeling, and Identification of Amino Acid Binding Motifs Suggest the Involvement of GH3 Genes during Somatic Embryogenesis of Coffea canephora.

Auxin plays a central role in growth and plant development. To maintain auxin homeostasis, biological processes such as biosynthesis, transport, degradation, and reversible conjugation are essential. The Gretchen Hagen 3 (GH3) family genes codify for the enzymes that esterify indole-3-acetic acid (IAA) to various amino acids, which is a key process in the induction of somatic embryogenesis (SE). The GH3 family is one of the principal families of early response to auxin genes, exhibiting IAA-amido synthetase activity to maintain optimal levels of free auxin in the cell. In this study, we carried out a systematic identification of the GH3 gene family in the genome of Coffea canephora, determining a total of 18 CcGH3 genes. Analysis of the genetic structures and phylogenetic relationships of CcGH3 genes with GH3 genes from other plant species revealed that they could be clustered in two major categories with groups 1 and 2 of the GH3 family of Arabidopsis. We analyzed the transcriptome expression profiles of the 18 CcGH3 genes using RNA-Seq analysis-based data and qRT-PCR during the different points of somatic embryogenesis induction. Furthermore, the endogenous quantification of free and conjugated indole-3-acetic acid (IAA) suggests that the various members of the CcGH3 genes play a crucial role during the embryogenic process of C. canephora. Three-dimensional modeling of the selected CcGH3 proteins showed that they consist of two domains: an extensive N-terminal domain and a smaller C-terminal domain. All proteins analyzed in the present study shared a unique conserved structural topology. Additionally, we identified conserved regions that could function to bind nucleotides and specific amino acids for the conjugation of IAA during SE in C. canephora. These results provide a better understanding of the C. canephora GH3 gene family for further exploration and possible genetic manipulation.

Transcriptome analysis of the induction of somatic embryogenesis in Coffea canephora and the participation of ARF and Aux/IAA genes.

BACKGROUND: Somatic embryogenesis (SE) is a useful biotechnological tool to study the morpho-physiological, biochemical and molecular processes during the development of Coffea canephora. Plant growth regulators (PGR) play a key role during cell differentiation in SE. The Auxin-response-factor (ARF) and Auxin/Indole-3-acetic acid (Aux/IAA) are fundamental components involved in the signaling of the IAA. The IAA signaling pathway activates or represses the expression of genes responsive to auxins during the embryogenic transition of the somatic cells. The growing development of new generation sequencing technologies (NGS), as well as bioinformatics tools, has allowed us to broaden the landscape of SE study of various plant species and identify the genes directly involved. METHODS: Analysis of transcriptome expression profiles of the C. canephora genome and the identification of a particular set of differentially expressed genes (DEG) during SE are described in this study. RESULTS: A total of eight ARF and seven Aux/IAA differentially expressed genes were identified during the different stages of the SE induction process. The quantitative expression analysis showed that ARF18 and ARF5 genes are highly expressed after 21 days of the SE induction, while Aux/IAA7 and Aux/IAA12 genes are repressed. DISCUSSION: The results of this study allow a better understanding of the genes involved in the auxin signaling pathway as well as their expression profiles during the SE process.