RESUMEN

KEY MESSAGE: Transgenic A. hypochondriacus and A. hybridus roots were generated. Further, a distinct plant regeneration program via somatic embryos produced from hairy roots was established. Work was implemented to develop an optimized protocol for root genetic transformation of the three grain amaranth species and A. hybridus, their presumed ancestor. Transformation efficiency was species-specific, being higher in A. hypochondriacus and followed by A. hybridus. Amaranthus cruentus and A. caudatus remained recalcitrant. A reliable and efficient Agrobacteruim rhizogenes-mediated transformation of these species was established using cotyledon explants infected with the previously untested BVG strain. Optimal OD600 bacterial cell densities were 0.4 and 0.8 for A. hypochondriacus and A. hybridus, respectively. Hairy roots of both amaranth species were validated by the amplification of appropriate marker genes and, when pertinent, by monitoring green fluorescent protein emission or ß-glucuronidase activity. Embryogenic calli were generated from A. hypochondriacus rhizoclones. Subsequent somatic embryo maturation and germination required the activation of cytokinin signaling, osmotic stress, red light, and calcium incorporation. A crucial step to ensure the differentiation of germinating somatic embryos into plantlets was their individualization and subcultivation in 5/5 media containing 5% sucrose, 5 g/L gelrite, and 0.2 mg/L 2-isopentenyladenine (2iP) previously acidified to pH 4.0 with phosphoric acid, followed by their transfer to 5/5 + 2iP media supplemented with 100 mg/L CaCl2. These steps were strictly red light dependent. This process represents a viable protocol for plant regeneration via somatic embryo germination from grain amaranth transgenic hairy roots. Its capacity to overcome the recalcitrance to genetic transformation characteristic of grain amaranth has the potential to significantly advance the knowledge of several unresolved biological aspects of grain amaranths.

Asunto(s)

Agrobacterium/genética , Amaranthus/genética , Raíces de Plantas/química , Raíces de Plantas/crecimiento & desarrollo , Técnicas de Embriogénesis Somática de Plantas/métodos , Transformación Genética , Amaranthus/fisiología , Cotiledón/genética , Medios de Cultivo/química , Regulación de la Expresión Génica de las Plantas , Marcadores Genéticos , Germinación , Glucuronidasa/genética , Proteínas Fluorescentes Verdes/genética , Raíces de Plantas/citología , Raíces de Plantas/microbiología , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa

RESUMEN

BACKGROUND: Animal skin provides an ideal medium for the propagation of microorganisms and it is used like raw material in the tannery and footware industry. The aim of this study was to evaluate and identify the microbial load in oropharyngeal mucosa of tannery employees. METHODS: The health risk was estimated based on the identification of microorganisms found in the oropharyngeal mucosa samples. The study was conducted in a tanners group and a control group. Samples were taken from oropharyngeal mucosa and inoculated on plates with selective medium. In the samples, bacteria were identified by 16S ribosomal DNA analysis and the yeasts through a presumptive method. In addition, the sensitivity of these microorganisms to antibiotics/antifungals was evaluated. RESULTS: The identified bacteria belonged to the families Enterobacteriaceae, Pseudomonadaceae, Neisseriaceae, Alcaligenaceae, Moraxellaceae, and Xanthomonadaceae, of which some species are considered as pathogenic or opportunistic microorganisms; these bacteria were not present in the control group. Forty-two percent of bacteria identified in the tanners group are correlated with respiratory diseases. Yeasts were also identified, including the following species: Candida glabrata, Candida tropicalis, Candida albicans, and Candida krusei. Regarding the sensitivity test of bacteria identified in the tanners group, 90% showed sensitivity to piperacillin/tazobactam, 87% showed sensitivity to ticarcillin/clavulanic acid, 74% showed sensitivity to ampicillin/sulbactam, and 58% showed sensitivity to amoxicillin/clavulanic acid. CONCLUSION: Several of the bacteria and yeast identified in the oropharyngeal mucosa of tanners have been correlated with infections in humans and have already been reported as airborne microorganisms in this working environment, representing a health risk for workers.