Genetic transformation of Paulownia elongata S. Y. Hu., mediated by Agrobacterium tumefaciens and biolistic system

Objective: The most appropriate conditions for genetic transformation through direct (bioballistic) and indirect (Agrobacterium tumefaciens) transformation systems in Paulownia elongata were established. Design/methodology/approach: Starting from in vitro propagation through both direct and indirect organogenesis, internodal stem segments with 0.5 to 1 cm length were determined as the best explant. The optimum dose for selection media was determined to be 15 mg L-1 of kanamycin. It was possible to obtain transgenic plants under both transformation systems. In the case of Agrobacterium tumefaciens, two hours of incubation, 48 h of co-cultivation, and optical density of 0.9 were used; while for bioballistics, the best conditions were 120 PSI of shot pressure, shot height at level 6 (16 cm), and vacuum pressure of 22 Hg mm, with particle inflow gun system (PIG). Results: Both systems produced complete transformants, chimeras, as well as those confirmed by histochemical X-GLUC and PCR analysis, producing a total of 14 positive plants by A. tumefaciens transformation from 26 trials and ten positive plants by the bioballistic system from 30 trials; a construction with chitinase and glucanase, NPT II selection gene and the GUS reporter gene were used. Findings/conclusions: So far, this has been the first report including integration of chitinase and glucanase genes

Enhanced Transgene Expression in Sugarcane by Co-Expression of Virus-Encoded RNA Silencing Suppressors

Post-transcriptional gene silencing is commonly observed in polyploid species and often poses a major limitation to plant improvement via biotechnology. Five plant viral suppressors of RNA silencing were evaluated for their ability to counteract gene silencing and enhance the expression of the Enhanced Yellow Fluorescent Protein (EYFP) or the β-glucuronidase (GUS) reporter gene in sugarcane, a major sugar and biomass producing polyploid. Functionality of these suppressors was first verified in Nicotiana benthamiana and onion epidermal cells, and later tested by transient expression in sugarcane young leaf segments and protoplasts. In young leaf segments co-expressing a suppressor, EYFP reached its maximum expression at 48-96 h post-DNA introduction and maintained its peak expression for a longer time compared with that in the absence of a suppressor. Among the five suppressors, Tomato bushy stunt virus-encoded P19 and Barley stripe mosaic virus-encoded γb were the most efficient. Co-expression with P19 and γb enhanced EYFP expression 4.6-fold and 3.6-fold in young leaf segments, and GUS activity 2.3-fold and 2.4-fold in protoplasts compared with those in the absence of a suppressor, respectively. In transgenic sugarcane, co-expression of GUS and P19 suppressor showed the highest accumulation of GUS levels with an average of 2.7-fold more than when GUS was expressed alone, with no detrimental phenotypic effects. The two established transient expression assays, based on young leaf segments and protoplasts, and confirmed by stable transgene expression, offer a rapid versatile system to verify the efficiency of RNA silencing suppressors that proved to be valuable in enhancing and stabilizing transgene expression in sugarcane

Regeneración de maíces blancos subtropicales vía embriogénesis somática

El maíz (Zea mays L.) se destina principalmente a la alimentación animal (maíces amarillos) y en menor proporción a la humana (blancos). Los maíces blancos subtropicales son importantes para los humanos. Sin embargo existen problemas de regeneración, vía embriogénesis somática, que limitan su transformación genética. Por tanto, el objetivo de esta investigación fue evaluar la embriogénesis somática en nueve líneas de maíces blancos subtropicales. En la inducción de embriogénesis somática se evaluaron 1, 2 y 5 mg L-1 de 2,4-D y Dicamba. Las líneas 78, 395 y 444 produjeron 70.1 a 87.2% de callos embriogénicos, similar a los testigos (67.2 a 74.7%). Las líneas 442 y 332 presentaron una tasa media de inducción de callos de 48.4 a 60.6%, mientras que las 330, 202, 204 y 331 mostraron los menores porcentajes (3.9 a 26.2%). Las líneas 330, 331 y el testigo 216×72 presentaron los mayores porcentajes con Dicamba, mientras que la línea 442 tuvo mayor porcentaje con 2,4-D. La inducción de las líneas varió entre dosis. La línea 395 regeneró 1.11 plantas por callo, similar a los tres testigos (0.74-0.95 plantas), mientras que las 442, 78 y 332 regeneraron 0.67, 0.37 y 0.33. Se obtuvieron plántulas enraizadas (92-99%) y aclimatación (95-100%) en las líneas 395, 442, 78 y 332. El número de plantas fértiles regeneradas fue 228, 187, 108 y 79 en las líneas 395, 442, 78 y 332. Se identificaron cuatro líneas embriogénicas de maíz blanco subtropical (395, 442, 78 y 332) con capacidad de regeneración de plantas fértiles en 17 semanas y con potencial para ser sometidas a transformación genética

Inducción "in vitro" de raíces de "Carica papaya" mediante "Agrobacterium rhizogenes" y ácido 3-indolbutírico

In vitro rooting of seedlings is one of the most important steps in the process o f micropropagatio n and in C. Papaya has been one of the major limiting, having survival percentages below 50%. The present research was conducted in the laboratory of Biotechnology of the University of Colima in 2009. In order to induce an efficient root system, evaluated papaya shoots var. "Maradol" of approximately 3 cm length, inoculated with A. rhizogenes at concentrations of 1x107, 1x108 and 1x109 cells ml-1 and established on MS medium with acetosyringone (1 00 mM) and phloroglucinol (25 mg / L). For the experiment with auxin shoots were subcultured in MS liquid medium containing 0, 1, 2, 3, 4 and 5 µM IBA; incubated in the dark for 126 h, followed by subculture in substrate BM2 + MS mineral salts. None of the inoculated shoots with bacterial concentrations formed roots; in contrast, 100% of the treated shoots with IBA to a concentration of 3 µM formed roots. With the above demonstrates that seedlings of C. papaya may not be susceptible to strain A4 A. rhizogenes, while the use of IBA proved to be the most viable and effective option for in vitro root induction in shoots of papayaEl enraizamiento de plántulas in vitro es una de las etapas más importantes en el proceso de micropropagación y en C. papaya ha sido una de las principales limitantes, obteniéndose porcentajes de sobrevivencia inferiores al 50%. El presente trabajo de investigación se realizó en el laboratorio de Biotecnología de la Universidad de Colima en 2009. Con el propósito de inducir un sistema radicular eficiente, se evaluaron brotes de papaya var. "Maradol" de aproximadamente 3 cm de longitud que fueron inoculados con A. rhizogenes a concentraciones de 1x107, 1x108 y 1x109 células mL-1 y establecidos en medio MS con acetosiringona (100 mM) y floroglucinol (25 mg/L). Para el experimento con la auxina, los brotes se subcultivaron en medio líquido MS con 0, 1, 2, 3, 4 y 5 µM de AIB; se incubaron en oscuridad durante 126 h, seguido del subcultivo en sustrato BM2+sales minerales del MS. Ninguno de los brotes inoculados con las concentraciones bacterianas formó raíces; en contraste, 100% de los brotes tratados con AIB a una concentración de 3 µM formaron raíces. Con lo anterior se demuestra que las plántulas de C. papaya posiblemente no son susceptibles a la cepa A4 de A. rhizogenes, mientras que el empleo de AIB resultó ser la opción más viable y efectiva para la inducción in vitro de raíces en brotes de papay

Biological control of late leaf rust disease [Pucciniastrum americanum (Farl.) Arthur] in raspberry (Rubus idaeus L.) using two biological products: Bacillus subtilis (Fungizard®) and Larrea tridentata botanic extract (CleanCrop®) under screenhouse conditions.

ABSTRACT This study aimed to evaluate the suitability of native Trichoderma strains for the biological control of Late Leaf Rust Disease (LLRD) in an organic Rubus idaeus plantation under screenhouse conditions. Four treatments were evaluated: T1) Trichoderma sp. strain Clombta; T2) Trichoderma sp. strain Chlorolota; T3) Co-application of both Trichoderma strains (Clombta + Chlorolota) and T4) Control, which consisted in the application of two biological products: Bacillus subtilis (Fungizard®) and Larrea tridentata botanic extract (CleanCrop®)]. Disease severity, disease severity index (DSI) and Area Under the Disease Progress Curve (AUDPC) were evaluated. The Co-application of Trichoderma sp. strain Chlorolota and Trichoderma sp. Clombta (from 43,44 to 35,73%) reduce the LLRD severity at the same level than the Control (from 44,61 to 34,33%). For DSI, Trichoderma sp. train Chlorolota (64,13) and the Co-application (61,11) showed similar values than those from the Control (59,84). Co-application of both Trichoderma strains showed the lowest AUDPC (71,2), at the same level that the Control (68,7). However, Trichoderma sp. Chlorolota (72,1) achieved the same AUDPC that the Co-application. The use of Trichoderma sp. Chlorolota or its Co-application with Trichoderma sp. strain Clombta was able to reduce the LLRD in R. idaeus.RESUMEN El objetivo del presente estudio fue evaluar la idoneidad de cepas nativas de Trichoderma para el control biológico de la roya tardía de la hoja (RTH), en una plantación orgánica de Rubus idaeus, bajo condiciones de malla sombra. Se evaluaron cuatro tratamientos: T1) Trichoderma sp. cepa Clombta; T2) Trichoderma sp. cepa Chlorolota; T3) Co-aplicación de ambas cepas de Trichoderma (Clombta + Chlorolota) y T4) Control, el cual consistió en la aplicación de dos productos biológicos: Bacillus subtilis (Fungizard®) y extracto botánico de Larrea tridentata (CleanCrop®)]. Se evaluó la severidad de la enfermedad, el índice de severidad de la enfermedad (IS) y el área bajo la curva de progreso de la enfermedad (ABCPE). La co-aplicación de Trichoderma sp. cepas Chlorolota y Clombta (de 43,44 a 35,73%) redujo la severidad de RTH al mismo nivel que el Control (de 44,61 a 34,33%). Para el IS, Trichoderma sp. cepa Chlorolota (64,13) y la Co-aplicación (61,11) mostraron valores similares al Control (59,84). La co-aplicación de ambas cepas de Trichoderma obtuvo el menor ABCPE (71,2), al mismo nivel que el Control (68,7). Sin embargo, Trichoderma sp. Chlorolota (72,1) logró el mismo ABCPE que la Co-aplicación. El uso de Trichoderma sp. Chlorolota o su Co-aplicación con la cepa Clombta fueron capaces de reducir la RTH en R. idaeus

Optimización de un protocolo para aislamiento de DNA de hojas de "Saccharum officinarum".

The DNA extraction free polysaccharides, polyphenols, plant protein and RNA is the initial step to several studies in molecular biology. There are various methods of extracting specific DNA of sugar cane (Saccharum officinarum), however, are time consuming and expensive equipment and reagents used. The aim of this study was to optimize a fast, efficient and inexpensive protocol for extracting DNA from leaves of S. officinarum. The variables evaluated were yield, purity, integrity and functionality of DNA purified from leaves of sugarcane 6 months old. Techniques spectrophotometry, agarose gel electrophoresis and molecular markers to evaluate the above variables were used. The performance variables (mg/g) and purity (A260:280 and A260:230) the extracted DNA showed highly significant differences (p˃ 0.05) protocol I. The best results were obtained with the protocol interaction I +N2, DNA yield 1.17 mg/g and 1.95 and 1.91 purities (A260:280 and A260:230). Functionality DNA by PCR amplification with the RAPD markers and TRAP bands sharp profiles generated good quality and easy to interpret. The use of the protocol I+N2 for extracting DNA from S. officinarum to obtain high yields of DNA with optimal quality and purity for applications of molecular markers is recommended.La extracción de DNA libre de polisacáridos, polifenoles, proteínas y RNA de plantas constituye el paso inicial para diversos estudios en Biología molecular. Existen diversos métodos de extracción de DNA específicos para caña de azúcar (Saccharum officinarum), sin embargo, consumen mucho tiempo y utilizan reactivos y equipos costosos. El objetivo del presente estudio fue optimizar un protocolo rápido, eficiente y de bajo costo para extraer DNA de hojas de S. officinarum. Las variables evaluadas fueron rendimiento, pureza, integridad y funcionalidad del DNA purificado de hojas de caña de azúcar de 6 meses de edad. Se utilizaron técnicas de espectrofotometría, electroforesis en geles de agarosa y marcadores moleculares para evaluar las variables anteriores. Las variables rendimiento (mg/g) y pureza (A260:280 y A260:230) del DNA extraído mostraron diferencias altamente significativas (p˃ 0.05) con el protocolo I. Los mejores resultados se obtuvieron con la interacción del protocolo I +N2, con un rendimiento de DNA de 1.17 mg/g y purezas de 1.95 y 1.91 (A260:280 y A260:230). La funcionalidad del DNA por amplificación por PCR con los marcadores RAPD y TRAP generaron perfiles de bandas nítidas de buena calidad y de fácil interpretación. Se recomienda el uso del protocolo I +N2 para la extracción de DNA de S. officinarum para obtener elevados rendimientos de DNA con calidad y pureza óptima para aplicaciones de marcadores moleculare

Enhanced expression of the <i>GUS</i> reporter gene by stable co-expression of the TBSV-encoded <i>P19</i> suppressor in transgenic sugarcane.

<p>(a) Relative abundance of <i>P19</i> and <i>GUS</i> transcripts was determined by northern blot and quantitative RT-PCR (qRT-PCR) analyses in two representative <i>P19-GUS</i> transgenic sugarcane lines co-expressing <i>GUS</i> and <i>P19</i> (two plants per line). Lines expressing <i>GUS</i> with no suppressor were used as a control. Blots of RNA (15 µg per sample) were probed with radioactively labeled <i>P19</i> DNA, stripped and then reprobed with <i>GUS</i> DNA. Normalized qRT-PCR <i>P19</i> expression levels of the <i>P19-GUS</i> lines are reported as a percentage, relative to that of the highest expressing plant. GUS activity (pmoles of 4-methylumbelliferone/min/µg protein) of the <i>P19-GUS</i> lines is also indicated. Values represent three biological samples and three technical repeats, and are reported with the standard error. (b) Methylation status of the coding region and promoter of the <i>GUS</i> reporter gene in the <i>P19-GUS</i> transgenic sugarcane lines. Southern blot of genomic DNA (10 µg per sample) of two representative <i>P19-GUS</i> lines, one non-silenced (Line 1, plants 4 and 5) and one silenced (Line 3, plants 12 and 16), digested with methylation-sensitive <i>Hpa</i>II (H), and methylation-insensitive <i>Msp</i>I (M), restriction endonucleases, were probed with the <i>GUS</i> gene or the Ubi promoter. Shifts in DNA hybridization fragments indicate methylation.</p

Dosage effect of the TBSV-encoded P19 RNA silencing suppressor on transient expression of the <i>GUS</i> reporter gene in sugarcane protoplasts.

<p>DNA (5 µg) from pUbi:<i>GUS</i>:Tnos (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066046#pone.0066046.s003" target="_blank">Figure S3</a>) was co-transfected into a protoplast suspension (100 µL; 1×10⁵ protoplasts) with three concentrations of <i>P19</i> DNA (pUbi:<i>P19</i>:Tnos) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066046#pone.0066046.s003" target="_blank">Figure S3</a>), respectively, and GUS activity of protoplasts was measured at 24 h post-transfection. Vector with no <i>P19</i> suppressor (pUbi:Tnos; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066046#pone.0066046.s003" target="_blank">Figure S3</a>) and sterile water were used as controls. Values represent three biological samples and six technical repeats, and are reported with the standard error. Means with the same letter are not significantly different (<i>p</i>>0.05).</p

Quantitative assessment of the effect of viral RNA silencing suppressors on transient expression the <i>GUS</i> reporter gene in sugarcane protoplasts.

<p>The GUS activity of protoplasts was monitored for 24 h after co-transfection of protoplasts (1×10⁵ protoplasts; 100 µL) with 5 µg of <i>GUS</i> (pUbi:<i>GUS</i>:Tnos; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066046#pone.0066046.s003" target="_blank">Figure S3</a>) and 10 µg each of RNA silencing suppressors (under the control of the Ubi promoter and nos terminator; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066046#pone.0066046.s003" target="_blank">Figure S3</a>), <i>P1/HC-Pro</i>, <i>γb</i>, <i>P19</i>, <i>P19/R43W</i> and <i>SCBV OrfI</i>. Vector with no suppressor (pUbi:Tnos; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066046#pone.0066046.s003" target="_blank">Figure S3</a>) was used as a negative control. Values represent means with standard error from three independent experiments and six replicates per experiment. Means with the same letter are not significantly different (<i>p</i>>0.05).</p