DNA methylation and differentiation in plants.

DNA methylation in plants appears to be a phenomenon more complex than in animals both for site specifity and the possible occurrence of two types of DNA methyltransferases. As in animals, DNA methylation appears to play a role in plant gene expression and differentiation (though recently acquired evidence indicated a less immediate relationship); other suggested roles for DNA methylation include genomic imprinting, paramutation and, to some extent, the gene silencing phenomenon observed in transgenic plants. However, while results obtained in mouse strictly link the presence of an active DNA methyltransferase with normal embryogenesis, in plants only circumstantial evidence points to the involvement of this enzyme in carrot somatic embryogenesis. What appears puzzling is the observation that hypomethylating mutants of Arabidopsis thaliana show normal development and morphology. The involvement of DNA methylation in plastid development is also debated. Of some interest appears the occurrence in plant cells of inhibitors of DNA methyltransferase activity although their biological significance and chemical nature remain to be elucidated

Agrobacterium-mediated transformation of the wild orchid Cattleya maxima Lindl

Protocorms are unique anatomical structures; they are akin to rhizoids and are formed by young orchid seedlings under physiological conditions. Explanted orchid tissues produce similar structures called protocorm-like bodies (PLBs) when exposed to appropriate in vitro growing conditions. Both the propagative nature of PLBs and the easiness by which they can be generated, make these structures an attractive alternative to seed-mediated production for growing large numbers of plants. To increase somatic embryogenesis and optimize the procedure, PLBs of Cattleya maxima were transformed using the Agrobacterium tumefaciens method. The T-DNA carried a Hygromycin-resistance gene, a visible marker (GFP5-GUSA) and a rice gene encoding the Somatic Embryogenesis Receptor Kinase, deemed to be important for somatic embryogenesis. Treated PLBs generated somatic embryos developing Hygromycin-resistant plantlets. The insertion of T-DNA was confirmed by PCR, and GFP expression was observed using a fluorescent stereomicroscope. Transformed Cattleya maxima PLBs were more efficient in forming somatic embryos (60-80%) than untransformed controls (45-57%), and this contrast was maximized in hormone-free, Murashige and Skoog (MS) medium (80% of the transformed plants compared to 57% of the untransformed ones). This finding supports the notion that SERK plays an important role in Orchid embryogenesis

Isolation, phylogenetic analysis, and expression of a Somatic Embryogenesis Receptor like Kinase (SERK) gene in Cattleya maxima Lindl

Orchidaceae is a central family of vascular plants in Ecuador because of its huge diversity and endemism. A prominent commercial native species well known for its richness of color is Cattleya maxima Lindl. Somatic embryogenesis is recognized as an important process for mass propagation of many ornamental crops including orchids. Somatic Embryogenesis Receptor like Kinases (SERK) genes, in particular, are highly expressed during the early phases of somatic embryogenesis and therefore, the study of their involvement in this process is of paramount importance to improve the commercial propagation of orchids. For this reason, we decided to isolate and characterize a SERK orthologue from Cattleya maxima L. The deduced amino acid sequence of a partial CmSERK cDNA shows the presence of all SERK typical domains, suggesting that it may be functionally active in C. maxima. Its role in somatic embryogenesis is further supported by its high expression level during embryogenesis and in protocorm-like bodies. Moreover, the basal expression of CmSERK in roots, leaves, and ovaries points to a broader developmental role of this gene. Keywords: CmSERK, Somatic embryogenesis, Orchids, Leucine Rich Repeat, Molecular characterizatio

Oxidative DNA damage bypass in Arabidopsis thaliana requires DNA polymerase λ and proliferating cell nuclear antigen 2

The oxidized base 7,8-oxoguanine (8-oxo-G) is the most common DNA lesion generated by reactive oxygen species. This lesion is highly mutagenic due to the frequent misincorporation of A opposite 8-oxo-G during DNA replication. In mammalian cells, the DNA polymerase (pol) family X enzyme DNA pol l catalyzes the correct incorporation of C opposite 8-oxo-G, together with the auxiliary factor proliferating cell nuclear antigen (PCNA). Here, we show that Arabidopsis thaliana DNA pol l, the only member of the X family in plants, is as efficient in performing error-free translesion synthesis past 8-oxo-G as its mammalian homolog. Arabidopsis, in contrast with animal cells, possesses two genes for PCNA. Using in vitro and in vivo approaches, we observed that PCNA2, but not PCNA1, physically interacts with DNA pol l, enhancing its fidelity and efficiency in translesion synthesis. The levels of DNA pol l in transgenic plantlets characterized by overexpression or silencing of Arabidopsis POLL correlate with the ability of cell extracts to perform error-free translesion synthesis. The important role of DNA pol l is corroborated by the observation that the promoter of POLL is activated by UV and that both overexpressing and silenced plants show altered growth phenotypes

Cryopreservation of orchid seeds through rapid and step freezing methods [version 1; referees: 1 approved, 2 approved with reservations]

Ecuador has a great variety of climatic regions that potentiate biodiversity. The family Orchidaceae constitutes one of the most important of the country, having identified about 4032 species with a high degree of endemism, therefore the development and research of alternative methods of storage and conservation of species is a strategy of primary interest for researchers and for society in general. In cryopreservation, temperatures reach below -190°C in order to paralyze the chemical reactions and keep the plant material viable for long periods. The present research focuses on the development of protocols for cryopreservation of seeds, aimed at the preservation of biodiversity, focusing on the family Orchidaceae, for the subsequent generation of a seed bank. The assays were performed on seeds of Epidendrum quitensium, Sobralia rosea, and Epidendrum anderssonii. Two freezing rates were tested: rapid freezing at -196°C; and step freezing at -22°C, -60°C to 196°C, further analyzed four combinations from Dimethylsulfoxide DMSO, glycerol and sucrose (DMSO 1M; DMSO 1M + glycerol 1M; DMSO 1M + sucrose 1M; DMSO 1M + glycerol 0,5M + sucrose 0,5M). The best results were obtained both in rapid and stepped freezing without the use of cryo-protective substances, by introducing the seeds directly into liquid nitrogen. Species of the genus Epidendrum presented a more efficient response in comparison to Sobralia. The viability of the seeds was evaluated by the tetrazolium test

Agrobacterium-mediated transformation of the wild orchid Cattleya maxima Lindl

Protocorms are unique anatomical structures; they are akin to rhizoids and are formed by young orchid seedlings under physiological conditions. Explanted orchid tissues produce similar structures called protocorm-like bodies (PLBs) when exposed to appropriate in vitro growing conditions. Both the propagative nature of PLBs and the easiness by which they can be generated, make these structures an attractive alternative to seed-mediated production for growing large numbers of plants. To increase somatic embryogenesis and optimize the procedure, PLBs of Cattleya maxima were transformed using the Agrobacterium tumefaciens method. The T-DNA carried a Hygromycin-resistance gene, a visible marker (GFP5-GUSA) and a rice gene encoding the Somatic Embryogenesis Receptor Kinase, deemed to be important for somatic embryogenesis. Treated PLBs generated somatic embryos developing Hygromycin-resistant plantlets. The insertion of T-DNA was confirmed by PCR, and GFP expression was observed using a fluorescent stereomicroscope. Transformed Cattleya maxima PLBs were more efficient in forming somatic embryos (60-80%) than untransformed controls (45-57%), and this contrast was maximized in hormone-free, Murashige and Skoog (MS) medium (80% of the transformed plants compared to 57% of the untransformed ones). This finding supports the notion that SERK plays an important role in Orchid embryogenesis

Effect of exogenous amino acids on the intracellular content of proline and other amino acids in Daucus carota cells.

The effect of tryptophan on the biosynthesis of proline has been investigated. Cells of Daucus carota grown in B5 medium supplemented with 5x10 - 4 M tryptophan acquired the ability to grow in the presence of inhibitory concentrations of azetidine-2-carboxylic acid, an analog of proline. When trp was added to carrot cell cultures at sub-growth inhibiting concentrations, overproduction of intracellular free proline was observed. An increase was also observed for lys, his, ala, leu and phe. Likewise, the addition of asparagine, glutamic acid and phenylalanine to the medium stimulated the intracellular increase of free proline and other amino acids

Release of nucleotide-cleaving phosphatase from carrot cell grown in suspension culture.

Higher plants contain unspecific phosphatases which can hydrolyse a wide variety of phosphate esters including sugar phosphates, nucleotides and phosphoenolpyruvate (Shaw 1966, Matile 1975). These enzymes can be released by plant tissue culture into the culture medium (Straus and Campbell 1963). Also terminal pyrophosphate bonds are hydrolysed by these enzymes (Shaw 1966, Hirasawa et al. 1979). While studying polydeoxyribonucleotide synthesis in cultured carrot cells it was found that deoxyribonucleoside 5'-triphosphates were hydrolysed and used as DNA precursors by intact cells at a rate approaching that of deoxyribonucleotides. This behaviour is different from that of bacteria and mammalian cells which are not able to rapidly utilize exogenous deoxyribonucleotides as DNA precursors. In this report we show that phosphohydrolases are excreted into the medium by carrot cell suspension cultures, and thus they are responsible for a rapid transformation of nucleotides into permeable compounds, which can be taken up by the cell and incorporated into DNA

Agrobacterium-mediated transformation of strawberry cultivar MarmoladaROnebor*.

We have established an efficient protocol for the Agrobacterium-mediated transformation of strawberry cultivar Marmoladam onebor*. Explants consisting of leaves with stipules were co-cultivated for two days with bacteria and then transferred to selective medium. Green shoots, that regenerated on selective medium, were rooted in medium containing kanamycin. Results of molecular analyses by PCR, slot and Southern blots, performed on rooted shoots, confirmed the integration of nptII gene into the genome of this cultivar