Características fenotípicas de semillas de soya mexicana y su correlación con la inducción de brotes in vitro y con la susceptibilidad de infección por Agrobacterium

Background and Aims: Soybean regeneration and transformation are considered highly genotype-specific; however, little is known about desirable seed traits that could be useful indicators of their regeneration and transformation capacities. In the present study, eight commercially available Mexican soybean varieties, as well as the Jack genotype as a control, were phenotypically characterized to determine the seed traits that are important factors for their in vitro performance and susceptibility to Agrobacterium tumefaciens infection.Methods: Grain dimensions, seed weight, moisture, oil, protein, ash, carbohydrate content and macro and micro elements (Mg, K, Ca, P, Na, Mn, Fe, Cu and Zn) were measured and correlated with the corresponding direct shoot organogenesis capability and Agrobacterium infection susceptibility using the cotyledonary node as explant.Key results: Notably, the ash content was the only important factor that inversely correlated with the capacity for shoot organogenesis, whereas carbohydrate and phosphorus content were positively correlated with susceptibility to Agrobacterium infection. The best response in terms of multiple shoot formation and Agrobacterium transient transformation was observed with the soybean varieties Huasteca-100, Nainari and Suaqui-86, which have lower ash content and a higher carbohydrate and phosphorus content.Conclusions: In most reported studies, seed phenotypic traits have been overlooked as factors that influence their regeneration and transformation potential. In the present study, we present evidence of associations between some specific seed traits and regeneration and transient transformation of soybean.Antecedentes y Objetivos: La regeneración y transformación de soya son consideradas altamente genotipo-específicas; sin embargo, muy poco se sabe acerca de rasgos deseables de la semilla que pudieran ser indicadores útiles de sus capacidades de regeneración y transformación. En el presente estudio, ocho variedades de soya mexicanas comercialmente disponibles, así como el genotipo Jack como control, se caracterizaron fenotípicamente con el fin de determinar qué características de la semilla son factores importantes para su desempeño in vitro y su susceptibilidad de infección por Agrobacterium tumefaciens.Métodos: Se midieron las dimensiones del grano, el peso de semilla, contenido de humedad, aceite, proteína, cenizas, carbohidratos, así como el contenido de macro y microelementos (Mg, K, Ca, P, Na, Mn, Fe, Cu y Zn) y se correlacionaron con la correspondiente capacidad de organogénesis directa y susceptibilidad de infección por Agrobacterium, usando el nodo cotiledonario como explante.Resultados clave: Notablemente, el contenido de cenizas fue el único factor importante que se correlacionó inversamente con la capacidad de organogénesis, mientras que los contenidos de carbohidratos y fósforo se correlacionaron positivamente con la susceptibilidad de infección por Agrobacterium. La mejor respuesta en términos de formación de brotes múltiples y transformación transitoria con Agrobacterium se observó con las variedades de soya Huasteca-100, Nainari y Suaqui-86, las cuales tienen un menor contenido de cenizas y un mayor contenido de carbohidratos y fósforo.Conclusiones: En la gran mayoría de estudios reportados, se han pasado por alto los rasgos fenotípicos de las semillas como posibles factores que influyen en su potencial de regeneración y transformación. En el presente estudio, presentamos evidencia de asociaciones entre algunos rasgos específicos de las semillas con la regeneración y la transformación transitoria de soya

TETX: a novel nuclear selection marker for Chlamydomonas reinhardtii transformation

Abstract Background Transformation of microalgae to obtain recombinant proteins, lipids or metabolites of economic value is of growing interest due to low costs associated with culture growth and scaling up. At present there are only three stable nuclear selection markers for the transformation of Chlamydomonas reinhardtii, which is the most commonly transformed microalgae, specifically: the aminoglycoside phosphotransferaseses aph7and aphVIII and the phleomycin resistance ble gene. As several microalgae are resistant to some of the antibiotics associated with the mentioned resistance genes, we have developed another alternative, tetX, a NADP-requiring Oxidoreductase that hydroxylates tetracycline substrates. We provide evidence that tetX can be used to obtain nuclear transformants of Chlamydomonas reinhardtii. Results We obtained nuclear transformants harbouring the tetX gene under the control of beta 2 tubulin or HSP70ARBCS2 promoters at an efficiency of transformation of 3.28 and 6.18 colony forming units/μg DNA respectively. This is the first report of a eukaryotic cell transformed using tetracycline as a selectable marker. Conclusions We developed a protocol for the nuclear transformation of Chlamydomonas reinhardtii using tetX as a selectable marker that confers stable resistance to tetracycline up to 100\ua0μg/mL. We believe tetX can be used to transform Chlamydomonas reinhardtii chloroplasts, related microalgae and other aerobic organisms sensitive to any tetracycline antibiotic.Abstract Background Transformation of microalgae to obtain recombinant proteins, lipids or metabolites of economic value is of growing interest due to low costs associated with culture growth and scaling up. At present there are only three stable nuclear selection markers for the transformation of Chlamydomonas reinhardtii, which is the most commonly transformed microalgae, specifically: the aminoglycoside phosphotransferaseses aph7and aphVIII and the phleomycin resistance ble gene. As several microalgae are resistant to some of the antibiotics associated with the mentioned resistance genes, we have developed another alternative, tetX, a NADP-requiring Oxidoreductase that hydroxylates tetracycline substrates. We provide evidence that tetX can be used to obtain nuclear transformants of Chlamydomonas reinhardtii. Results We obtained nuclear transformants harbouring the tetX gene under the control of beta 2 tubulin or HSP70ARBCS2 promoters at an efficiency of transformation of 3.28 and 6.18 colony forming units/μg DNA respectively. This is the first report of a eukaryotic cell transformed using tetracycline as a selectable marker. Conclusions We developed a protocol for the nuclear transformation of Chlamydomonas reinhardtii using tetX as a selectable marker that confers stable resistance to tetracycline up to 100\ua0μg/mL. We believe tetX can be used to transform Chlamydomonas reinhardtii chloroplasts, related microalgae and other aerobic organisms sensitive to any tetracycline antibiotic.CONACYT Beca N° 20965

Phenotypic traits of Mexican soybean seeds and their correlation with in vitro shoot induction and susceptibility to Agrobacterium infection

Abstract: Background and Aims: Soybean regeneration and transformation are considered highly genotype-specific; however, little is known about desirable seed traits that could be useful indicators of their regeneration and transformation capacities. In the present study, eight commercially available Mexican soybean varieties, as well as the Jack genotype as a control, were phenotypically characterized to determine the seed traits that are important factors for their in vitro performance and susceptibility to Agrobacterium tumefaciens infection. Methods: Grain dimensions, seed weight, moisture, oil, protein, ash, carbohydrate content and macro and micro elements (Mg, K, Ca, P, Na, Mn, Fe, Cu and Zn) were measured and correlated with the corresponding direct shoot organogenesis capability and Agrobacterium infection susceptibility using the cotyledonary node as explant. Key results: Notably, the ash content was the only important factor that inversely correlated with the capacity for shoot organogenesis, whereas carbohydrate and phosphorus content were positively correlated with susceptibility to Agrobacterium infection. The best response in terms of multiple shoot formation and Agrobacterium transient transformation was observed with the soybean varieties Huasteca-100, Nainari and Suaqui-86, which have lower ash content and a higher carbohydrate and phosphorus content. Conclusions: In most reported studies, seed phenotypic traits have been overlooked as factors that influence their regeneration and transformation potential. In the present study, we present evidence of associations between some specific seed traits and regeneration and transient transformation of soybean.Resumen: Antecedentes y Objetivos: La regeneración y transformación de soya son consideradas altamente genotipo-específicas; sin embargo, muy poco se sabe acerca de rasgos deseables de la semilla que pudieran ser indicadores útiles de sus capacidades de regeneración y transformación. En el presente estudio, ocho variedades de soya mexicanas comercialmente disponibles, así como el genotipo Jack como control, se caracterizaron fenotípicamente con el fin de determinar qué características de la semilla son factores importantes para su desempeño in vitro y su susceptibilidad de infección por Agrobacterium tumefaciens. Métodos: Se midieron las dimensiones del grano, el peso de semilla, contenido de humedad, aceite, proteína, cenizas, carbohidratos, así como el contenido de macro y microelementos (Mg, K, Ca, P, Na, Mn, Fe, Cu y Zn) y se correlacionaron con la correspondiente capacidad de organogénesis directa y susceptibilidad de infección por Agrobacterium, usando el nodo cotiledonario como explante. Resultados clave: Notablemente, el contenido de cenizas fue el único factor importante que se correlacionó inversamente con la capacidad de organogénesis, mientras que los contenidos de carbohidratos y fósforo se correlacionaron positivamente con la susceptibilidad de infección por Agrobacterium. La mejor respuesta en términos de formación de brotes múltiples y transformación transitoria con Agrobacterium se observó con las variedades de soya Huasteca-100, Nainari y Suaqui-86, las cuales tienen un menor contenido de cenizas y un mayor contenido de carbohidratos y fósforo. Conclusiones: En la gran mayoría de estudios reportados, se han pasado por alto los rasgos fenotípicos de las semillas como posibles factores que influyen en su potencial de regeneración y transformación. En el presente estudio, presentamos evidencia de asociaciones entre algunos rasgos específicos de las semillas con la regeneración y la transformación transitoria de soya

Transient co-expression with three O-glycosylation enzymes allows production of GalNAc-O-glycosylated Granulocyte-Colony Stimulating Factor in N. benthamiana

Abstract Background Expression of economically relevant proteins in alternative expression platforms, especially plant expression platforms, has gained significant interest in recent years. A special interest in working with plants as bioreactors for the production of pharmaceutical proteins is related to low production costs, product safety and quality. Among the different properties that plants can also offer for the production of recombinant proteins, protein glycosylation is crucial since it may have an impact on pharmaceutical functionality and/or stability. Results The pharmaceutical glycoprotein human Granulocyte-Colony Stimulating Factor was transiently expressed in Nicotiana benthamiana plants and subjected to mammalian-specific mucin-type O-glycosylation by co-expressing the pharmaceutical protein together with the glycosylation machinery responsible for such post-translational modification. Conclusions The pharmaceutical glycoprotein human Granulocyte-Colony Stimulating Factor can be expressed in N. benthamiana plants via agroinfiltration with its native mammalian-specific mucin-type O-glycosylation

Application of metabolic engineering to enhance the content of alkaloids in medicinal plants

Plants are a rich source of bioactive compounds, many of which have been exploited for cosmetic, nutritional, and medicinal purposes. Through the characterization of metabolic pathways, as well as the mechanisms responsible for the accumulation of secondary metabolites, researchers have been able to increase the production of bioactive compounds in different plant species for research and commercial applications. The intent of the current review is to describe the metabolic engineering methods that have been used to transform in vitro or field-grown medicinal plants over the last decade and to identify the most effective approaches to increase the production of alkaloids. The articles summarized were categorized into six groups: endogenous enzyme overexpression, foreign enzyme overexpression, transcription factor overexpression, gene silencing, genome editing, and co-overexpression. We conclude that, because of the complex and multi-step nature of biosynthetic pathways, the approach that has been most commonly used to increase the biosynthesis of alkaloids, and the most effective in terms of fold increase, is the co-overexpression of two or more rate-limiting enzymes followed by the manipulation of regulatory genes.ISSN:2214-030

Antimicrobial Activity of Rhoeo discolor Phenolic Rich Extracts Determined by Flow Cytometry

Traditional medicine has led to the discovery of important active substances used in several health-related areas. Phytochemicals in Rhoeo discolor extracts have proven to have important antimicrobial activity. In the present study, our group determined the antimicrobial effects of extracts of Rhoeo discolor, a plant commonly used in Mexico for both medicinal and ornamental purposes. We evaluated the in vitro activity of phenolic rich extracts against specifically chosen microorganisms of human health importance by measuring their susceptibility via agar-disc diffusion assay and flow cytometry: Gram-positive Listeria innocua and Streptococcus mutans, Gram-negative Escherichia coli and Pseudomonas aeruginosa, and lastly a fungal pathogen Candida albicans. Ten different extracts were tested in eight different doses on all the microorganisms. Analytical data revealed a high content of phenolic compounds. Both agar-disc diffusion assay and flow cytometry results demonstrated that Pseudomonas aeruginosa was the least affected by extract exposure. However, low doses of these extracts (predominantly polar), in a range from 1 to 4 μg/mL, did produce a statistically significant bacteriostatic and bactericidal effect on the rest of the microorganisms. These results suggest the addition of certain natural extracts from Rhoeo discolor could act as antibacterial and antimycotic drugs or additives for foods and cosmetics

Novel Bacillus thuringiensis Binary Insecticidal Crystal Proteins Active on Western Corn Rootworm, Diabrotica virgifera virgifera LeConte

A new family of insecticidal crystal proteins was discovered by screening sporulated Bacillus thuringiensis cultures for oral activity against western corn rootworm (WCR) larvae. B. thuringiensis isolates PS80JJ1, PS149B1, and PS167H2 have WCR insecticidal activity attributable to parasporal inclusion bodies containing proteins with molecular masses of ca. 14 and 44 kDa. The genes encoding these polypeptides reside in apparent operons, and the 14-kDa protein open reading frame (ORF) precedes the 44-kDa protein ORF. Mutagenesis of either gene in the apparent operons dramatically reduced insecticidal activity of the corresponding recombinant B. thuringiensis strain. Bioassays performed with separately expressed, biochemically purified 14- and 44-kDa polypeptides also demonstrated that both proteins are required for WCR mortality. Sequence comparisons with other known B. thuringiensis insecticidal proteins failed to reveal homology with previously described Cry, Cyt, or Vip proteins. However, there is evidence that the 44-kDa polypeptide and the 41.9- and 51.4-kDa binary dipteran insecticidal proteins from Bacillus sphaericus are evolutionarily related. The 14- and 44-kDa polypeptides from isolates PS80JJ1, PS149B1, and PS167H2 have been designated Cry34Aa1, Cry34Ab1, and Cry34Ac1, respectively, and the 44-kDa polypeptides from these isolates have been designated Cry35Aa1, Cry35Ab1, and Cry35Ac1, respectively