Somatic Embryogenesis: Identified Factors that Lead to Embryogenic Repression. A Case of Species of the Same Genus

Somatic embryogenesis is a powerful biotechnological tool for the mass production of economically important cultivars. Due to the cellular totipotency of plants, somatic cells under appropriate conditions are able to develop a complete functional embryo. During the induction of somatic embryogenesis, there are different factors involved in the success or failure of the somatic embryogenesis response. Among these factors, the origin of the explant, the culture medium and the in vitro environmental conditions have been the most studied. However, the secretion of molecules into the media has not been fully addressed. We found that the somatic embryogenesis of Coffea canephora, a highly direct embryogenic species, is disrupted by the metabolites secreted from C. arabica, a poorly direct embryogenic species. These metabolites also affect DNA methylation. Our results show that the abundance of two major phenolic compounds, caffeine and chlorogenic acid, are responsible for inhibiting somatic embryogenesis in C. canephora.     

Biotechnological applications for the improvement of coffee (Coffea arabica L.)

Summary The important advances in coffee biotechnological techniques which have been made particularly during the last 10yr could benefit the coffee breeder in practice and open new perspectives for the development of new varieties. The molecular phylogeny of Coffea species has been established using DNA sequence data. The molecular markers have revealed an extremely reduced genetic diversity in Coffea arabica L. in comparison to C. canephora. However, wild accessions collected in the Ethiopian highlands appeared to constitute a valuable gene reservoir. A complete genetic linkage map of C. canephora was reported and additional ones are being constructed, particularly on C. arabica. The integration of Molecular Assisted Selection in coffee breeding promises to drastically increase the efficiency of breeding programs. Economically important genes of the caffeine biosynthetic pathway or genes encoding for seed storage proteins have been isolated. The high performance already achieved in the in vitro propagation process by somatic embryogenesis offers the possibility to mass propagate superior hybrids in different countries of both C. arabica (selected F₁ hybrids) and C. canephora (rootstock variety). Pilot productions by somatic embryogenesis currently permit preparation for commercial application. Somaclonal variation was observed. The percentage of the off-types can vary between 3 and 10% depending on the genotype. Seed cryopreservation enables a routine use for long-term conservation of coffee genetic resources. Transgenic plants have been obtained for the C. arabica and C. canephora cultivated species through Agrobacterium-mediated transformation which constitutes the technique now currently used to transfer directly genes in coffee plants.     

Histology of somatic embryogenesis in <Emphasis Type="Italic">Coffea arabica</Emphasis> L.

The objective of this study was to characterize the histodifferentiation of somatic embryogenesis obtained from leaf explants of C. arabica. Therefore, we histologically analyzed the respective stages of the process: leaf segments at 0, 4, 7, 15 and 30 days of cultivation, Type 1 primary calli (primary calli with embryogenic competence) and 2 (primary calli with no embryogenic competence), embryogenic calli, globular, torpedo and cotyledonary embryos, and mature zygotic embryos. Callus formation occurred after seven days of culture, with successive divisions of procambium cell. In this cultivation phase, it was found that Type 1 primary calli are basically formed by parenchymal cells with reduced intercellular spacing, whereas Type 2 primary calli are predominantly composed of parenchymal cells with ample intercellular spaces and embryogenic calli composed entirely of meristematic cells. After 330 days, it was evident from the differentiation of somatic embryogenesis that there was formation of globular somatic embryos, consisting of a characteristic protoderm surrounding the fundamental meristem. With the maturation of these propagules after 360 days, torpedo-stage somatic embryos arose, in which tissue polarization and early differentiation of procambial strands were verified. After 390 days, cotyledonary somatic embryos were obtained, where the onset of vessel elements differentiation was verified, a characteristic also observed in mature zygotic embryos. We concluded that somatic embryogenesis obtained from C. arabica leaves initiates from procambium cell divisions that, in the course of cultivation, produce mature somatic embryos suitable for regenerating whole plants.     

Proteomic Analysis of Developing Somatic Embryos of Coffea arabica

Differential protein profiles of three stages of somatic embryogenesis, including globular, torpedo, and cotyledonary somatic embryos, of Coffea arabica cv. Catuaí Vermelho were analyzed in an attempt to better understand somatic embryogenesis in coffee plants. Somatic embryos at these different stages of development were collected from in vitro-grown cultures, and then macerated in liquid nitrogen. Proteins were extracted with phenol and further quantified using the Bradford method. The bidimensional electrophoresis analysis revealed a wide range of proteins ranging between 10 and 160?kDa and of pH values ranging from 3 to 10. Several differentially expressed proteins were identified by mass spectrometry, and some were found to be specific to these different stages of somatic embryogenesis in coffee. The enolase and 11S storage globulin proteins, for example, could be used as molecular markers for somatic embryo development stages and for embryogenic and non-embryogenic genotype differentiation, respectively.     

Comparison between a coffee single copy chromosomal region and Arabidopsis duplicated counterparts evidenced high level synteny between the coffee genome and the ancestral Arabidopsis genome

The Arabidopsis thaliana genome sequence provides a catalogue of reference genes that can be used for comparative analysis of other species thereby facilitating map-based cloning in economically important crops. We made use of a coffee bacterial artificial chromosome (BAC) contig linked to the S_H3 leaf rust resistance gene to assess microsynteny between coffee (Coffea arabica L.) and Arabidopsis. Microsynteny was revealed and the matching counterparts to C. arabica contigs were seen to be scattered throughout four different syntenic segments of Arabidopsis on chromosomes (Ath) I, III, IV and V. Coffee BAC filter hybridizations were performed using coffee putative conserved orthologous sequences to Arabidopsis predicted genes located on the different Arabidopsis syntenic regions. The coffee BAC contig related to the S_H3 region was successfully consolidated and later on validated by fingerprinting. Furthermore, the anchoring markers appeared in same order on the coffee BAC contigs and in all Arabidopsis segments with the exception of a single inversion on AtIII and AtIV Arabidopsis segments. However, the S_H3 coffee region appears to be closer to the ancestral genome segment (before the divergence of Arabidopsis and coffee) than any of the duplicated counterparts in the present-day Arabidopsis genome. The genome duplication events at the origin of its Arabidopsis counterparts occurred most probably after the separation (i.e. 94 million years ago) of Euasterid (Coffee) and Eurosid (Arabidopsis).     

In Silico and Quantitative Analyses of the Putative FLC-like Homologue in Coffee (Coffea arabica L.)

The sequential pattern of coffee flowering is a major constraint that directly affects productivity, increases harvest costs, and generates a final product of lower quality for mixing dry fruits with ripe and unripe ones. The objective of this work was to identify and analyze one of the main genes involved in flowering regulation, FLOWERING LOCUS C (FLC) in coffee (Coffea arabica L.). The identification of this gene was conducted in silico using a coffee EST database (CAFEST) and bioinformatics tools. Quantitative PCR results suggest that the identified CaFLC-like homologue is directly involved in flowering regulation in coffee. This expands our knowledge on evolutionary conservation of flowering pathways in dicot species. The functional studies of CaFLC-like with mutants of a more tractable species will lead to a better understanding of the molecular regulation as well as the specific functions of each gene flowering during floral induction in coffee.     

Lipid transfer proteins in coffee: isolation of Coffea orthologs,Coffea arabica homeologs,expression during coffee fruit development and promoter analysis in transgenic tobacco plants

The aim of the present study was to perform a genomic analysis of non-specific lipid-transfer proteins (nsLTPs) in coffee. Several nsLTPs-encoding cDNA and gene sequences were cloned from Coffea arabica and Coffea canephora species. In this work, their analyses revealed that coffee nsLTPs belong to Type II LTP characterized under their mature forms by a molecular weight of around 7.3 kDa, a basic isoelectric points of 8.5 and the presence of typical CXC pattern, with X being an hydrophobic residue facing towards the hydrophobic cavity. Even if several single nucleotide polymorphisms were identified in these nsLTP-coding sequences, 3D predictions showed that they do not have a significant impact on protein functions. Northern blot and RT-qPCR experiments revealed specific expression of Type II nsLTPs-encoding genes in coffee fruits, mainly during the early development of endosperm of both C. arabica and C. canephora. As part of our search for tissue-specific promoters in coffee, an nsLTP promoter region of around 1.2 kb was isolated. It contained several DNA repeats including boxes identified as essential for grain specific expression in other plants. The whole fragment, and a series of 5′ deletions, were fused to the reporter gene β-glucuronidase (uidA) and analyzed in transgenic Nicotiana tabacum plants. Histochemical and fluorimetric GUS assays showed that the shorter (345 bp) and medium (827 bp) fragments of nsLTP promoter function as grain-specific promoters in transgenic tobacco plants.     

Investigating variability and behaviour of Colletotrichum gloeosporioides strains from lesions of coffee blister spot

Coffee blister spot has been associated with species from the Colletotrichum genus, but there is no information on the variability of isolates present on leaf lesions. This study evaluated a population of Colletotrichum gloeosporioides strains from blister spot lesions in Coffea arabica. Colletotrichum spp. isolates were collected from blister spot lesions on leaves of coffee trees from Catuaí and Topázio cultivars (Coffea arabica). Monosporic cultures were obtained from colonies with sporulation. A pathogenicity test was carried out by inoculation of pathogens on the leaves of young coffee plants. C. gloeosporioides strains were characterized by morphologial, cytological and physiological analyses. The molecular analysis was carried out using Inter‐Retrotransposon Amplified Polymorphism (IRAP) markers. C. gloeosporioides strains showed no pathogenicity on coffee plants and presented a wide variability in all traits evaluated. The presence of sexual strains, formation of CATs (conidial anastomosis tubes) among conidial strains and high mycelial compatibility among strains observed suggest the occurrence of sexual and asexual recombination. The role of these C. gloeosporioides strains on the lesions of coffee plant leaves is unclear.     

Brassinosteroid increases the cytokinin efficiency to induce direct somatic embryogenesis in leaf explants of <Emphasis Type="Italic">Coffea arabica</Emphasis> L. (Rubiaceae)

The combination of different plant growth regulators can result in beneficial effects in the induction of in vitro morphogenetic pathways. The present study reports the effect of 24-epibrassinolid (24-epiBR; brassinosteroid) when added alone and in association with N⁶-(2-isopentnyl) adenine (2-iP; cytokinin) in the induction of direct somatic embryogenesis in Coffea arabica. Leaf explants were cultivated in a modified Murashige and Skoog (MS) medium with 0 or 10 µM 2-iP and different concentrations (0.01, 0.10 or 1.0 µM) of 24-epiBR. Explants cultured on MS medium supplemented with 1.0 µM 24-epiBR in association with 2-iP produced 6.8 times more somatic embryos than the explants cultured with only 2-iP. Histological analyses also provided evidence that the supplementation of brassinosteroids in the culture medium could have influenced somatic embryogenesis differentiation. Somatic embryos obtained in the presence of brassinosteroid and cytokinin were better structured morpho-histologically as compared to those obtained in the medium with just cytokinin. This study opens new perspectives for the use of brassinosteroids in the somatic embryogenesis of C. arabica, so as to optimize the in vitro regeneration systems used in genetic improvement programs in C. arabica productive systems.     

Sequences and transcriptional analysis of Coffea arabica var. Caturra and Coffea liberica plant responses to coffee berry borer Hypothenemus hampei (Coleoptera: Curculionidae: Scolytinae) attack

The coffee berry borer (CBB) is the most prevalent pest of coffee plantations. Within the Coffea genus, C. arabica is susceptible to CBB and C. liberica shows a lower susceptibility. Two EST libraries were constructed from the total RNA of C. arabica and C. liberica fruits artificially infested with CBBs for 24 h. Using 6000 clones sequenced per library, a unigene database was generated, obtaining 3634 singletons and 1454 contigs. For each contig, the proportion of sequences present in both species was determined and a differential gene expression between the species was detected. C. arabica displayed a higher relative expression of proteins involved in general stress responses, whereas C. liberica showed the induction of a higher number of insect defense proteins. In order to validate the results, quantifications through real-time PCR were done. A hevein-like protein, an isoprene synthase, a salicylic acid carboxyl methyltransferase and a patatin-like protein gene were highly upregulated in C. liberica at 24 and/or 48 h after insect infestation compared to C. arabica. The identification of metabolic pathways induced by this pest insect provides tools to take advantage of the genetic resources available for the control of CBB.     

Partial resistance to leaf rust (Hemileia vastatrix) in coffee (Coffea arabica L.): genetic analysis and molecular characterization of putative candidate genes

One of the major production limiting diseases in coffee is the orange leaf rust caused by the fungus Hemileia vastatrix (Berkeley and Broome). Little is known about the inheritance and genetic determinism of partial resistance in coffee (C. arabica L.) to H. vastatrix. This information would be useful to breed durable resistant cultivars efficiently. In this report, a genetic analysis of partial resistance to leaf rust in Coffea arabica was performed using nine segregating progenies from a cross between the susceptible variety Caturra and the resistant introgressed line DI200. Evolution of partial resistance was evaluated under field conditions by measuring rust incidence (RI) and defoliation (DEF) in two separate regions of productive branches per tree and during four successive years (2003–2006). Genetic components of rust resistance were estimated using the Means and Variance Generation Method, under an additive-dominant model. The most important genetic effect was the additive one, while resistance heritability estimates ranged from 73 to 53% for broad and narrow sense heritabilities, respectively. Genetic estimates for the number of segregating genes showed that at least five independent genes or genetic regions are implicated in the partial resistance to rust. We further analyzed the presence of resistance (RGC) and defense (DGC) gene candidates in the resistant and susceptible parents by using a degenerated-primer PCR approach. A total of 40 different genomic coffee sequences were isolated exhibiting strong similarity to known RGC or DGC homologous. Phylogenetic analysis clustered these sequences into nine families. One family exhibited the TIR protein element, representing the first TIR class proteins identified in coffee. While genetic analysis suggest a predictable success in the processes to improve the selection of resistant lines for future varieties with durable resistance, the molecular characterization of candidate genes represent a primary approach towards the identification of mechanisms involved in partial resistance to coffee leaf rust.     

Somatic embryogenesis and plant regeneration through cell suspension in diploid (AB) banana cultivar Elakki Bale (syn Neypoovan)

An efficient protocol was developed using cell suspensions for somatic embryogenesis and plantlet regeneration in a most popular diploid AB banana (M.accuminata X M.bulbisiana hybrid) cv. Elakki Bale (syn Neypoovan) known for its taste and keeping quality in southern India. Floral primodia from position 8–16 of male inflorescence which were more responsive for embryogenesis were used as explants for the embryogenic callus production in MS media supplemented with different concentration of 2,4-D. A concentration of 18.1 μM 2, 4-D produced maximum embryogenic calli in 1 % of the explants inoculated. Embryogenic calli on repeated sub culturing on MA2 media produced good embryogenic cell suspensions (ECS). Microscopic examination of ECS showed globular, smaller with dense cytoplasm filled with starchy granules characteristic of embryogenic cells. Highest number of somatic embryos (189) was produced on modified MA3 media. A germination percentage of 31 % were observed in BAP 22.19 μM concentration. Regenerated plants with normal shoot and root were hardened in soilrite. Direct somatic embryogenesis and plant regeneration was also noticed in embryogenic calli which did not pass through the ECS stage. The protocol optimized for somatic embryogenesis through cell suspension and also direct embryogenesis leading to plantlet regeneration can be used for the micropropagation and genetic manipulation.     

Influence of triacontanol on somatic embryogenesis in <Emphasis Type="Italic">Coffea arabica</Emphasis> L. and <Emphasis Type="Italic">Coffea canephora</Emphasis> P. ex Fr.

Summary A highly reproducible method for regeneration of Coffea arabica and C. canephora plants via direct somatic embryogenesis from cultured leaf and stem segments of regenerated plants was developed. Embryogenesis was influenced by the presence of triacontanol (TRIA) in the medium. TRIA incorporated at 4.55 and 11.38 μM in half-strength MS basal medium containing 1.1 μM 6-benzyladenine (BA) and 2.28 μM indole-3-acetic acid (IAA) induced direct somatic embryogenesis in both species. A maximum of 260±31.8 and 59.2±12.8 somatic embryos per culture were induced from in vitro leaf explants of C. arabica and C. canephora, respectively. TRIA also induced embryo formation from in vitro stem segment callus tissues along with multiplication of primary embryos into secondary embryos. By using TRIA, it was possible to obtain somatic embryogenesis in C. arabica and C. canephora.     

Influence of in vitro environment on somatic embryogenesis of Coffea arabica L. cv. Caturra rojo: the effects of carbon dioxide on embryogenic cell suspensions

The influence of environment in the culture vessel is a factor that has very little study in the process of somatic embryogenesis. The present research was carried out with the objective to determine the effects of carbon dioxide on somatic embryogenesis of Coffea arabica cv. Caturra rojo. Embryogenic cell suspensions were cultured under different carbon dioxide concentrations (2.5%, 5.0%, and 10.0%) in the gases mixture and two control treatments, one with passive exchange and the other with forced ventilation. The results demonstrated that there were a larger number of somatic embryos formed with a concentration of 2.5% CO_2. The differentiation of these somatic embryos of coffee in embryogenic cell suspensions (130 × 10³ SE l⁻¹) was also stimulated. The effects of CO₂ on somatic embryogenesis were demonstrated when the control with passive exchange was compared with forced ventilation control, because in the former, where there was an accumulation of CO₂, the production of somatic embryos was greater. CO₂ could stimulate the formation and differentiation of somatic embryos directly, which led to a modification of the pH patterns of the culture medium or indirectly when producing changes in the pH that favored the somatic embryogenesis process.     

Analysis of alien introgression in coffee tree (<Emphasis Type="Italic">Coffea</Emphasis><Emphasis Type="Italic">arabica</Emphasis> L.)

The transfer of desired traits from related wild diploid Coffea species into the cultivated allotetraploid C. arabica is essential in coffee breeding to develop pest/disease-resistant cultivars. The present work is an attempt to gain insights into alien introgression in C. arabica. An F2 population derived from a cross between T5296 and Et6 was analysed with simple sequence repeat (SSR; microsatellite) and amplified fragment length polymorphism (AFLP) molecular markers. The T5296 is a derivative of an interspecific hybrid introgressed by the diploid C. canephora species and Et6 is a wild Ethiopian accession of C. arabica. The origin of the revealed polymorphism was determined by comparisons using representative accessions from C. arabica and its two diploid parental species, C. eugenioides and C. canephora. The number and mode of inheritance of canephora-introgressed segments were investigated, as well as their sub-genome localisation and rate of recombination. The results suggested that the transfer of desirable genes into C. arabica from C. canephora is not limited by the ploidy level differences or the suppression of recombination between the different genomes.     

Developing Single Nucleotide Polymorphism (SNP) Markers for the Identification of Coffee Germplasm

Coffee is one of the most widely consumed beverages and represents a multibillion-dollar global industry. Accurate identification of coffee cultivars is essential for efficient management, exchange, and use of coffee genetic resources. To date, a universal platform that can allow data comparison across different laboratories and genotyping platforms has not been developed by the coffee research community. Using expressed sequence tags (EST) of Coffea arabica, C. canephora and C. racemosa from public databases, we developed 7538 single nucleotide polymorphism (SNP) markers and selected 180 for validation using 25 C. arabica and C. canephora accessions from Puerto Rico. Based on the validation result, we designated a panel of 55 SNP markers that are polymorphic across the two species. The average minor allele frequency and information index of this SNP panel are 0.281 and 0.690, respectively. This panel enabled the differentiation of all tested accessions of C. canephora, which accounts for 79.2 % of the total polymorphism in the samples. Only 21.8 % of the polymorphic SNPs were detected in the 12 C. arabica cultivars, which, nonetheless, were able to unambiguously differentiate the 12 Arabica cultivars into ten unique genotypes, including two synonymous groups. Several local Puerto Rican cultivars with partial Timor pedigree, including Limaní, Frontón, and TARS 18087, showed substantial genetic difference from the other common Arabica cultivars, such as Catuai, Borbón, and Mundo Nuevo. This coffee SNP panel provides robust and universally comparable DNA fingerprints, thus can serve as a genotyping tool to assist coffee germplasm management, propagation of planting material, and coffee cultivar authentication.