Genomic Methylated Cytosine Level during the Dedifferentiation and Cellular Competence in Coffea arabica Lines: Insights about the Different In Vitro Responses

Coffea arabica genotypes present distinct responses in vitro, and somaclonal variation occurrence has been reported. Global cytosine methylation is one of the epigenetic mechanisms that influences the Coffea in vitro responses. We aimed to establish the indirect somatic embryogenesis in C. arabica ‘Catuaí Vermelho’, ‘Caturra’ and ‘Oeiras’, associate the distinct responses to the methylated cytosine genomic level, and check the ploidy stability. Leaf explants were cultured in callus induction and proliferation medium. The resulted calli were transferred to the regeneration medium, and the mature cotyledonary somatic embryos were transferred to the seedling medium. ‘Oeiras’ exhibited the highest number of responsive leaf explants, followed by ‘Caturra’ and ‘Catuaí Vermelho’. Global methylated cytosine level increased over time in the ‘Catuaí Vermelho’ and ‘Caturra’ friable calli, remaining constant in ‘Oeiras’. ‘Oeiras’ did not regenerate somatic embryos, while ‘Catuaí Vermelho’ exhibited the highest number. Somatic embryo regeneration was associated with the increase of the methylated cytosine level. However, the ‘Catuaí Vermelho’ embryogenic calli showed a lower methylated cytosine level than ‘Caturra’. Recovered plantlets exhibited the same 2C value and chromosome number to the explant donors. Therefore, cytosine hypermethylation occurred during C. arabica indirect somatic embryogenesis, influencing cell competence and somatic embryos regeneration.

Micropropagation of Hibiscus moscheutos L. ‘Luna White’: effect of growth regulators and explants on nuclear DNA content and ploidy stability of regenerants

Hibiscus moscheutos L., also known as hardy hibiscus, is valued for its medicinal and ornamental attributes. It is usually propagated via seeds or cuttings. The purpose of this investigation was to develop a dependable micropropagation for H. moscheutos ‘Luna White’. To that end, the effect of four explant types (leaf, root, node, shoot tip) and two growth regulators 6-benzylaminopurine (BA) and meta-Topolin (mT) (6-(3-hydroxybenzylamino) purine) on in vitro growth of H. moscheutos was investigated. Genetic stability of the in vitro grown plants was assessed using flow cytometry, and chromosome count was investigated. No shoots were obtained from leaf or root explants. An efficient protocol for micropropagation of H. moscheutos using two explant types, 2-node and shoot tip explants, and two cytokinins (BA and mT) capable of producing true-to-type regenerants was established. Both BA and mT can be used at 2 μM or 4 μM using either 2-node or shoot tip explants. No significant difference was found between the nuclear DNA contents of seed-derived and in vitro grown plants (P < 0.05). The mean 2C DNA and monoploid 1Cx-values of seed-derived plants were 3.25 ± 0.08 pg and 1.62 ± 0.04 pg, respectively, compared with 3.26 ± 0.06 pg and 1.63 ± 0.02 pg, respectively, for in vitro grown plants. The chromosome number of both seed-derived plants and regenerants was determined to be 2n = 2x = 38. The mature regenerants obtained were fertile and phenotypically similar to seed-derived plants.

The Role of Anaphase-Promoting Complex/Cyclosome (APC/C) in Plant Reproduction

Most eukaryotic species propagate through sexual reproduction that requires male and female gametes. In flowering plants, it starts through a single round of DNA replication (S phase) and two consecutive chromosome segregation (meiosis I and II). Subsequently, haploid mitotic divisions occur, which results in a male gametophyte (pollen grain) and a female gametophyte (embryo sac) formation. In order to obtain viable gametophytes, accurate chromosome segregation is crucial to ensure ploidy stability. A precise gametogenesis progression is tightly regulated in plants and is controlled by multiple mechanisms to guarantee a correct evolution through meiotic cell division and sexual differentiation. In the past years, research in the field has shown an important role of the conserved E3-ubiquitin ligase complex, Anaphase-Promoting Complex/Cyclosome (APC/C), in this process. The APC/C is a multi-subunit complex that targets proteins for degradation via proteasome 26S. The functional characterization of APC/C subunits in Arabidopsis, which is one of the main E3 ubiquitin ligase that controls cell cycle, has revealed that all subunits investigated so far are essential for gametophytic development and/or embryogenesis.

MutS-Homolog2silencing generates tetraploid meiocytes in tomato (Solanum lycopersicum)

SUMMARYMSH2 is the core protein of MutS-homolog family involved in recognition and repair of the errors in the DNA. While other members of MutS-homolog family reportedly regulate mitochondrial stability, meiosis, and fertility, MSH2 is believed to participate mainly in mismatch repair. The search for polymorphism inMSH2sequence in tomato accessions revealed both synonymous and nonsynonymous SNPs; however, SIFT algorithm predicted that none of the SNPs influenced MSH2 protein function. The silencing ofMSH2gene expression by RNAi led to phenotypic abnormalities in highly-silenced lines, particularly in the stamens with highly reduced pollen formation.MSH2silencing exacerbated formation of UV-B induced thymine dimers and blocked light-induced repair of the dimers. TheMSH2silencing also affected the progression of male meiosis to a varying degree with either halt of meiosis at zygotene stage or formation of diploid tetrads. The immunostaining of male meiocytes with centromere localized CENPC (Centromere protein C) protein antibody showed the presence of 48 univalent along with 24 bivalent chromosomes suggesting abnormal tetraploid meiosis. The mitotic cells of root tips of silenced lines showed diploid nuclei but lacked intervening cell plates leading to cells with syncytial nuclei. Thus we speculate that tetraploid pollen mother cells may have arisen due to the fusion of syncytial nuclei before the onset of meiosis. It is likely that in addition to Mismatch repair (MMR), MSH2 may have an additional role in regulating ploidy stability.