Regenerative potential, metabolic profile, and genetic stability of Brachypodium distachyon embryogenic calli as affected by successive subcultures.

Brachypodium distachyon, a model species for forage grasses and cereal crops, has been used in studies seeking improved biomass production and increased crop yield for biofuel production purposes. Somatic embryogenesis (SE) is the morphogenetic pathway that supports in vitro regeneration of such species. However, there are gaps in terms of studies on the metabolic profile and genetic stability along successive subcultures. The physiological variables and the metabolic profile of embryogenic callus (EC) and embryogenic structures (ES) from successive subcultures (30, 60, 90, 120, 150, 180, 210, 240, and 360-day-old subcultures) were analyzed. Canonical discriminant analysis separated EC into three groups: 60, 90, and 120 to 240 days. EC with 60 and 90 days showed the highest regenerative potential. EC grown for 90 days and submitted to SE induction in 2 mg L-1 of kinetin-supplemented medium was the highest ES producer. The metabolite profiles of non-embryogenic callus (NEC), EC, and ES submitted to principal component analysis (PCA) separated into two groups: 30 to 240- and 360-day-old calli. The most abundant metabolites for these groups were malonic acid, tryptophan, asparagine, and erythrose. PCA of ES also separated ages into groups and ranked 60- and 90-day-old calli as the best for use due to their high levels of various metabolites. The key metabolites that distinguished the ES groups were galactinol, oxaloacetate, tryptophan, and valine. In addition, significant secondary metabolites (e.g., caffeoylquinic, cinnamic, and ferulic acids) were important in the EC phase. Ferulic, cinnamic, and phenylacetic acids marked the decreases in the regenerative capacity of ES in B. distachyon. Decreased accumulations of the amino acids aspartic acid, asparagine, tryptophan, and glycine characterized NEC, suggesting that these metabolites are indispensable for the embryogenic competence in B. distachyon. The genetic stability of the regenerated plants was evaluated by flow cytometry, showing that ploidy instability in regenerated plants from B. distachyon calli is not correlated with callus age. Taken together, our data indicated that the loss of regenerative capacity in B. distachyon EC occurs after 120 days of subcultures, demonstrating that the use of EC can be extended to 90 days.

New Insights on Coffea miRNAs: Features and Evolutionary Conservation.

Small RNAs influence the gene expression at the post-transcriptional level by guiding messenger RNA (mRNA) cleavage, translational repression, and chromatin modifications. In addition to model plants, the microRNAs (miRNAs) have been identified in different crop species. In this work, we developed a specific pipeline to search for coffee miRNA homologs on expressed sequence tags (ESTs) and genome survey sequences (GSS) databases. As a result, 36 microRNAs were identified and a total of 616 and 362 potential targets for Coffea arabica and Coffea canephora, respectively. The evolutionary analyses of these molecules were performed by comparing the primary and secondary structures of precursors and mature miRNAs with their orthologs. Moreover, using a stem-loop RT-PCR assay, we evaluated the accumulation of mature miRNAs in genomes with different ploidy levels, detecting an increase in the miRNAs accumulation according to the ploidy raising. Finally, a 5' RACE (Rapid Amplification of cDNA Ends) assay was performed to verify the regulation of auxin responsive factor 8 (ARF8) by MIR167 in coffee plants. The great variety of target genes indicates the functional plasticity of these molecules and reinforces the importance of understanding the RNAi-dependent regulatory mechanisms. Our results expand the study of miRNAs and their target genes in this crop, providing new challenges to understand the biology of these species.

Organ polarity in plants is specified through the opposing activity of two distinct small regulatory RNAs.

Small RNAs and their targets form complex regulatory networks that control cellular and developmental processes in multicellular organisms. In plants, dorsoventral (adaxial/abaxial) patterning provides a unique example of a developmental process in which early patterning decisions are determined by small RNAs. A gradient of microRNA166 on the abaxial/ventral side of the incipient leaf restricts the expression of adaxial/dorsal determinants. Another class of small RNAs, the TAS3-derivated trans-acting short-interfering RNAs (ta-siRNAs), are expressed adaxially and repress the activity of abaxial factors. Loss of maize leafbladeless1 (lbl1) function, a key component of the ta-siRNA biogenesis pathway, leads to misexpression of miR166 throughout the initiating leaf, implicating ta-siRNAs in the spatiotemporal regulation of miR166. The spatial restriction of tasiRNA biogenesis components suggests that this pathway may act non-cell-autonomously in the meristem and possibly contributes to the classic meristem-borne adaxializing Sussex signal. Here, we discuss the key participants in adaxial/abaxial patterning and point out the intriguing possibility that organ polarity in plants is established by the opposing action of specific ta-siRNAs and miRNAs.

Influence of the antibiotic timentin on plant regeneration of tomato (Lycopersicon esculentum Mill.) cultivars.

Cotyledon explants of tomato (Lycopersicon esculentum Mill. cvs 'Santa Clara', 'Firme' mutant, 'IPA-5' and 'IPA-6') were excised from 8- to 10-day-old in vitro-grown seedlings. Four different shoot induction media supplemented with timentin (300 mg l-1) were screened. When cotyledon explants were cultured on MS-based medium with 1.0 mg l-1 zeatin plus 0.1 mg l-1 IAA and supplemented with timentin, higher regeneration frequencies and a greater number of elongated shoots were obtained. It was observed that timentin caused an increase in the morphogenesis of in vitro cotyledon explants of tomato cultivars. In two of three cultivars tested, rooting of shoots was positively influenced, both in the presence and absence of timentin in the rooting medium, among shoots regenerated from explants derived from timentin-supplemented medium. The results confirm those of a previous investigation on the beneficial effects of this class of antibiotics on tomato regeneration and, consequently, its reliability for use in the transformation of this species.