Abstract:

La regeneración de plantas a partir de embriones inmaduros en trigo fue descrita en numerosas publicaciones. Está demostrado que bajo condiciones específicas de cultivo in vitro la regeneración de plantas ocurre sólo a través de embriogénesis somática inducida sobre las capas endémicas superficiales de los escutelos. Asimismo es ampliamente aceptado el origen unicelular de los embriones somáticos. Estas características unidas a la alta reproductibilidad y alta frecuencia de regeneración de plantas, han hecho de la embriogénesis somática sobre escutelos de trigo el procedimiento preferido de los biotecnólogos para la transformación para la transformación genética a través de la biolística o Agrobacterium. De allí la importancia práctica de investigar los factores que influyen en el proceso de embriogénesis in vitro de trigo.

Author affiliation: Bossio, Adrián Ezequiel. Universidad Nacional de Luján. Departamento de Ciencias Básicas.

Abstract:

Somatic embryogenesis in scutella of wheat (Triticum aestivum L.) is a well documented phenomenon and it has been shown through transcriptome analysis that genes involved in antioxidant responses, particularly in glutathione (GSH) biosynthesis, participate in the process. Thus, we investigated the influence of post-transcriptional silencing (PTGS) of the glutathione biosynthesis genes GSH1 and GSH2 on somatic embryogenesis in wheat. We found that PTGS of either of the target genes drastically inhibits callus regeneration and overall efficiency of transformation, in a similar manner as the GSH biosynthetic inhibitor buthionine sulfoximine. Supplementing the medium with glutathione did not overcome the observed low efficiency of wheat transformation. Furthermore, of the small number of obtained transformants, none exhibited altered GSH1 and GSH2 levels of transcription. Thus, it is concluded that GSH is essential for somatic embryogenesis and, as a consequence, it is difficult to regenerate wheat plants with silenced GSH1 and GSH2 genes. © 2012 Springer Science+Business Media B.V.

Author affiliation: Bossio, Adrian Ezequiel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina

Author affiliation: Díaz Paleo, Antonio Horacio. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina. Universidad Nacional de Luján. Departamento de Ciencias Básicas; Argentina

Author affiliation: del Vas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina

Author affiliation: Baroli, Irene Mabel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina

Author affiliation: Acevedo, Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria; Argentina

Author affiliation: Rios, Raul Daniel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina. Universidad Nacional de Luján. Departamento de Ciencias Básicas; Argentina

Abstract:

Somatic embryogenesis in scutella of wheat (Triticum aestivum L.) is a well documented phenomenon and it has been shown through transcriptome analysis that genes involved in antioxidant responses, particularly in glutathione (GSH) biosynthesis, participate in the process. Thus, we investigated the influence of post-transcriptional silencing (PTGS) of the glutathione biosynthesis genes GSH1 and GSH2 on somatic embryogenesis in wheat. We found that PTGS of either of the target genes drastically inhibits callus regeneration and overall efficiency of transformation, in a similar manner as the GSH biosynthetic inhibitor buthionine sulfoximine. Supplementing the medium with glutathione did not overcome the observed low efficiency of wheat transformation. Furthermore, of the small number of obtained transformants, none exhibited altered GSH1 and GSH2 levels of transcription. Thus, it is concluded that GSH is essential for somatic embryogenesis and, as a consequence, it is difficult to regenerate wheat plants with silenced GSH1 and GSH2 genes.

Instituto de Genética

Author affiliation: Bossio, Adrian Ezequiel. INTA. Instituto de Genética "Ewald A. Favret"; Argentina

Author affiliation: Diaz Paleo, Antonio Horacio. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Universidad Nacional de Luján. Departamento de Ciencias Básicas; Argentina

Author affiliation: Del Vas, Mariana. INTA. Instituto de Biotecnología; Argentina.

Author affiliation: Baroli, Irene. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Acevedo, Alberto. INTA. Centro de Investigación de Recursos Naturales; Argentina

Author affiliation: Ríos, Raúl Daniel. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Universidad Nacional de Luján. Departamento de Ciencias Básicas; Argentina

Abstract:

Plant stress studies are increasingly being based on gene expression. The analysis of gene expression requires sensitive, precise, and replicable measurements for specific mRNA sequences. Real-time RT-PCR is nowadays the most sensitive method for the detection of low abundance mRNA. A stable reference gene is mandatory to obtain reliable quantitative real-time PCR (qPCR) analysis results. Real-time RT-PCR is referred with one or several internal control genes, which should not fluctuate during treatments. In this study, we have chosen eight genes as candidates of possible reference genes for maize (Zea mays L) during water deficit stress at flowering time: a-tubulin, 3`phosphate glyceraldehyde dehydrogenase (GAPDH), 18S ribosomal subunit, protein 13S ribosomal, actin, zein, invertase, and starch synthase IIB. The eight reference genes candidates were tested on maize plants around flowering time, under three different conditions: before water deficit (BWD), under water deficit (WD) and after water deficit (AWD). Results from the three experimental conditions indicated that protein 13S ribosomal gene was the most stable among all the reference genes tested. This result suggests that protein 13S ribosomal gene can be used as internal control (housekeeping) for qPCR analysis in maize plants under water deficit stress during flowering time.

Inst. de Genética "Ewald A. Favret"- IGEAF

Author affiliation: Decima Oneto, Cecilia Andrea. INTA. Instituto de Genética; Argentina

Author affiliation: Bossio, Adrian Ezequiel. INTA. Instituto de Genética; Argentina

Author affiliation: Faccio, Paula Daniela. INTA. Instituto de Genética; Argentina

Author affiliation: Beznec, Ailin. INTA. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Blumwald, Eduardo. University of California Davies. Department Plant Sciences; Estados Unidos

Author affiliation: Lewi, Dalia Marcela. INTA. Instituto de Genética; Argentina

Abstract:

Senescence can be delayed in transgenic plants overexpressing the enzyme isopentenyltransferase (IPT) due to stress-induced increased levels of endogenous cytokinins. This trait leads to sustained photo-synthetic activity and improved tolerance to abiotic stress. The aim of this study was to generate andcharacterize transgenic plants of maize (Zea mays L.) transformed with the IPT gene sequence underthe regulation of SARK promoter (protein kinase receptor-associated senescence). Three independenttransgenic events and their segregating null controls were evaluated in two watering regimes (WW:well watered; WD: water deficit) imposed for two weeks around anthesis. Our results show that the WD treatment induced IPT expression with the concomitant increase in cytokinin levels, which prolongedthe persistence of total green leaf area, and maintained normal photosynthetic rate and stomatal con-ductance. These trends were accompanied by a minor decrease in number of grains per plant, individualgrain weight and plant grain yield as compared to WW plants. Plants expressing the IPT gene under WD had PGR, anthesis and silking dates and biomass levels similar to WW plants. Our results demonstratethat expression of the IPT gene under the regulation of the SARK promoter helps improve productivity under WD conditions in C4 plants like maize.

Inst. de Genética "Ewald A. Favret"- IGEAF

Author affiliation: Decima Oneto, Cecilia Andrea. INTA. Instituto de Genética "Ewald A. Favret"; Argentina

Author affiliation: Otegui, Marí­a Elena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Agronomía; Argentina

Author affiliation: Baroli, Irene. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Beznec, Ailin. INTA. Instituto de Genética "Ewald A. Favret"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Faccio, Paula Daniela. INTA. Instituto de Genética "Ewald A. Favret"; Argentina

Author affiliation: Bossio, Adrian EzequielINTA. Instituto de Genética "Ewald A. Favret"; Argentina

Author affiliation: Blumwald, Eduardo. University of California. Department Plant Sciences; Estados Unidos

Author affiliation: Lewi, Dalia Marcela. INTA. Instituto de Genética "Ewald A. Favret"; Argentina

Abstract:

Senescence can be delayed in transgenic plants overexpressing the enzyme isopentenyltransferase (IPT) due to stress-induced increased levels of endogenous cytokinins. This trait leads to sustained photosynthetic activity and improved tolerance to abiotic stress. The aim of this study was to generate and characterize transgenic plants of maize (Zea mays L.) transformed with the IPT gene sequence under the regulation of SARK promoter (protein kinase receptor-associated senescence). Three independent transgenic events and their segregating null controls were evaluated in two watering regimes (WW: well watered; WD: water deficit) imposed for two weeks around anthesis. Our results show that the WD treatment induced IPT expression with the concomitant increase in cytokinin levels, which prolonged the persistence of total green leaf area, and maintained normal photosynthetic rate and stomatal conductance. These trends were accompanied by a minor decrease in number of grains per plant, individual grain weight and plant grain yield as compared to WW plants. Plants expressing the IPT gene under WD had PGR, anthesis and silking dates and biomass levels similar to WW plants. Our results demonstrate that expression of the IPT gene under the regulation of the SARK promoter helps improve productivity under WD conditions in C4 plants like maize.

Author affiliation: Décima Oneto, Cecilia Andrea. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina

Author affiliation: Otegui, Maria Elena. Universidad de Buenos Aires. Facultad de Agronomía; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Baroli, Irene Mabel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Beznec, Ailin Yanina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Faccio, Paula. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina

Author affiliation: Bossio, Adrian Ezequiel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina

Author affiliation: Blumwald, Eduardo. University of California at Davis; Estados Unidos

Author affiliation: Lewi, Dalia Marcela. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Genética; Argentina