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.

[Acute trichinosis. Epidemics of 166 cases in Delicias City, Chih. Diagnosis with tissue compression and staining]. / Triquinosis aguda. Epidemia de 166 casos en Ciudad Delicias, Chih. Diagnóstico por compresión tisular y tinción.

The purposes of this report are: a) To show outstanding clinicopathological characteristics from the largest outbreak of trichinosis in México. b) To describe a modification to the traditional trichinoscopy through the use of compressed muscle stained with ordinary dyes an immediate observation; we have called the procedure tissular compression and dyeing technique (TCDT). The outbreak occurred in Delicias, Chihuahua, and affected 166 inhabitants who ate a not well cooked pork sausage. All patients showed malaise, 85.5 percent myalgias, and 72.7 percent facial edema; leucocytosis was seen in 75 percent of the patients, 60 percent with eosinophilia. Electromyography was performed in 21 patients in the acute phase of the illness and it was found abnormal in 85.7 percent. Only one patient died (0.6%). From 59 patients who were considered very ill, muscle biopsies were taken, and the conventional histopathological study showed larvae in 88.1 percent. TCDT demonstrated worms in 90.3 percent out of 47 biopsies; larvae were seen free and into muscle fibers with or without capsule cells. In conclusion TCDT is a simple, fast and easy reproducible procedure.