Identification and characterization of SSR markers of Guadua chacoensis (Rojas) Londoño & P.M. Peterson and transferability to other bamboo species.

The aim of this study was to develop simple sequence repeat (SSR) markers for genetic studies on G. chacoensis, as well as to evaluate their transferability to other bamboo species. Genomic DNA was isolated from G. chacoensis and its partial sequencing was used to find SSR loci. The obtained sequencing data were de novo assembled using the software CLC Genomics Workbench® 8.0v. The SSR loci primers were identified and designed with the software SSRLocator. The selected markers were validated using 56 plants sampled in seven populations from southern Brazil. The markers with potential polymorphism were selected and fluorescently labeled for characterization by capillary electrophoresis. In total, 92 SSR loci were found in G. chacoensis contigs. Suitable primers were designed for 70 SSR loci, and the remaining 22 SSR loci did not have sequences for primer development. Out of 35 selected SSR markers, after PCR optimization, 10 with high polymorphism potential were characterized. These loci can be used in genetic analyses of G. chacoensis and all of them were successfully transferred to other bamboo species. Non-polymorphic loci require further tests with additional plants, from different populations, to identify possibilities of their use.

Storage and in vitro germination of orchids (Orchidaceae) seeds from Atlantic Forest - Brazil.

The orchid seed banks of Atlantic Forest may be considered a key strategy for the conservation of species threatened with extinction by indiscriminate collection or habitat destruction. The aim of this study was to evaluate the seed viability, to choose the best culture medium for the asymbiotic germination and evaluate germination, after storage for different periods and temperatures for the Brazilian native orchids: Gomesa praetexta (Rchb.f.) M.W.Chase & N.H.Williams, Gomesa forbesii (Hook.) M.W.Chase & N.H.Williams, Gomesa recurva R.Br. and Grandiphyllum divaricatum (Lindl.) Docha Neto. Knudson C (KC), Murashige & Skoog (MS), half-strength MS (1/2 MS macro- and micro-nutrients) and Woody Plant Medium (WPM) culture media were tested for germination. The WPM culture medium was the best for asymbiotic germination of all species evaluated, with high germination percentages and improved seedling development. Seeds of G. divaricatum, G. praetexta, G. recurva and G. forbesii indicated orthodox behavior, with high viability rates after 12 months of storage, being recommended the storage temperature of -80°C for the first three species and -20°C for G. forbesii. The protocol developed in the present study was efficient for seed bank storage, in vitro germination and seedling production of G. divaricatum and G. praetexta, contributing to conservation strategies of these species.

5-Azacytidine combined with 2,4-D improves somatic embryogenesis of Acca sellowiana (O. Berg) Burret by means of changes in global DNA methylation levels.

UNLABELLED: DNA methylation is an epigenetic regulatory mechanism of gene expression which can be associated with developmental phases and in vitro morphogenetic competence in plants. The present work evaluated the effects of 5-azacytidine (AzaC) and 2,4-dichlorophenoxyacetic acid (2,4-D) on Acca sellowiana somatic embryogenesis (SE) and global DNA methylation levels by high-performance liquid chromatography mass spectrometry (HPLC/MS/MS). 2,4-D-free treatments revealed no somatic embryo formation in both accessions tested. Treatments supplemented with 2,4-D pulse plus AzaC in the culture medium resulted in increased embryo formation. In AzaC-free treatment, HPLC/MS/MS analysis showed a gradual increase in methylation levels in cultures of both accessions tested during SE induction. Treatment with AzaC and 2,4-D-free resulted in a marked decrease in methylation for both accessions, ranging from 37.6 to 20.8 %. In treatment with 2,4-D and AzaC combined, the 85 accession showed increasing global methylation levels. Otherwise, the 101X458 accession, in the same treatment, showed a decrease between 10 and 20 days, followed by an increase after 30 days (39.5, 36.2 and 41.6 %). These results indicate that 2,4-D pulse combined with AzaC improves SE induction. However, the conversion phase showed that although positively influencing SE induction, AzaC had a dysregulatory effect on the stage of autotrophic plant formation, resulting in significantly lower conversion rates. The results suggest that DNA methylation dramatically influences SE in Acca sellowiana, and global DNA methylation dynamics are related to morphogenetic response. KEY MESSAGE: 5-Azacytidine combined with 2,4-D increases the number of Acca sellowiana somatic embryos. Global DNA methylation is directly affected by these compounds.