Agrobacterium tumefaciens-mediated transformation of common bean (Phaseolus vulgaris) var. Brunca

Common bean is a crop recalcitrant to in vitro regeneration and therefore it lacks an efficient transformation protocol that can be reproduced using A. tumefaciens. The main goal of this study was to establish a protocol for A. tumefaciens mediated transformation of Phaseolus vulgaris var. Brunca by marker genes (gusA and nptII) together with the gene for trehalose-6-phosphate synthase from Saccharomyces cerevisiae (TPS1) used in other species to increase tolerance to abiotic stress. The β-glucuronidase activity was detected in 45 % of the LBA4404 ElectroMAX® pCAMBIA1301 infected explants. Transformed explants regenerated new shoots after four to five months period in a kanamycin rich media. Surviving plants were evaluated by PCR and presented an 0.5 % efficiency of transformation. The established protocol for genetic transformation of common bean has two additional advantages with respect to previous reports: (1) it allows for obtaining transformed regenerants and (2) the genetic transformation was stable for the selective gene.

El frijol común en un cultivo recalcitrante a la regeneración in vitro y se carece de un protocolo eficiente y reproducible de transformación genética usando A. tumefaciens. Desarrollamos un protocolo de transformación genética mediada por A. tumefaciens de frijol común variedad Brunca utilizando genes marcadores (gusA y nptII) junto con el gen de la trehalosa-6-fosfato sintasa de levadura (TPS1) utilizado para incrementar tolerancia a estrés abiótico. La actividad de la β-glucoronidasa fue detectada en 45 % de los explantes infectados con la cepa LBA4404 de A. tumefaciens transformada con pCAMBIA1301. Después de 4 o 5 meses se regeneraron tallos en un medio adicionado con kanamicina. Los explantes supervivientes se evaluaron mediante PCR y presentaron una eficiencia de transformación de 0.5 %. El protocolo de transformación genética de frijol común establecido tiene dos ventajas adicionales con respecto a los reportes previos: (1) permite la obtención de regenerares transformados y (2) la transformación genética fue estable para el gen selectivo.

Simplified methodology for large scale isolation of homozygous transgenic lines of lettuce

Background: Lettuce is a globally important leafy vegetable and a model plant for biotechnology due to its adaptability to tissue culture and stable genetic transformation. Lettuce is also crucial for functional genomics research in the Asteraceae which includes species of great agronomical importance. The development of transgenic events implies the production of a large number of shoots that must be differentiated between transgenic and non-transgenic through the activity of the selective agent, being kanamycin the most popular. Results: In this work we adjusted the selection conditions of transgenic seedlings to avoid any escapes, finding that threshold concentration of kanamycin was 75 mg/L. To monitor the selection system, we studied the morphological response of transgenic and non-transgenic seedlings in presence of kanamycin to look for a visual morphological marker. Several traits like shoot length, primary root length, number of leaves, fresh weight, and appearance of the aerial part and development of lateral roots were affected in non-transgenic seedlings after 30 d of culture in selective media. However, only lateral root development showed an early, qualitative and reliable association with nptII presence, as corroborated by PCR detection. Applied in successive transgenic progenies, this method of selection combined with morphological follow-up allowed selecting the homozygous presence of nptII gene in 100% of the analyzed plants from T2 to T5. Conclusions: This protocol allows a simplified scaling-up of the production of multiple homozygous transgenic progeny lines in the early generations avoiding expensive and time-consuming molecular assays.

Optimization of coffee (Coffea arabica) transformation parameters using uidA and hpt genes: effect of osmotic pre-treatment, helium pressure and target distance / Optimización de los parámetros de transformación del café (Coffea arabica) utilizando los genes uidA y hpt: efecto del pretratamiento osmótico, presión de helio y distancia objetivo

The aim of this work was to optimize the biolistic delivery parameters that affect the DNA delivery and stable expression of marker genes into coffee tissues (Coffea arabica. L. cvs. Caturra and Catuaí). The effect of osmotic preculture length, osmotic concentration of medium, Helium pressure and target distance on transient expression of the uidA gene in coffee leaves and somatic embryos were tested. The highest transient uidA expression was obtained when Caturra (18.3±2.8) and Catuaí (6.8±2.0) leaves and Catuaí embryos (80.0±7.4) were cultured for 5h on Yasuda medium complemented with 0.5M Mannitol +0.5M Sorbitol. The combination of 1100psi and a target distance of 9cm resulted in the highest number of blue spots per Caturra leaf segment (23.6±3.9), whereas for the Catuaí variety the combination of 1100psi and a target distance of six (10.2±1.9) and nine (8.2±1.9) cm gave the highest number of blue spots per leaf segment. The optimized protocol was tested with pCAMBIA 1 301 (uidA gene and the hpt gene), pCAMBIA 1 305.2 (uidA version GUSPlus ™ and the hpt gene) and pCAMBIA 1 301-BAR (uidA gene and the bar gene). The highest number of blue spots was obtained when Caturra (54.6±5.7) and Catuaí (28.9±4.3) leaves were bombarded with pCAMBIA 1 305.2. Selection of bombarded coffee tissues with 100mg/l hygromicyn caused the oxidation of tissues. Rev. Biol. Trop. 57 (Suppl. 1): 151-160. Epub 2009 November 30.

La presente investigación tuvo como objetivo optimizar los parámetros que afectan la incorporación y expresión de genes marcadores mediante biobalística en segmentos de hoja y embriones somáticos de café (Coffea arabica. L. cvs. Caturra y Catuaí). La mayor expresión transitoria del gen uidA en segmentos de hoja de Caturra (18.3±2.8) y Catuaí (6.8±2.0) y embriones somáticos de Catuaí (80.0±7.4) se obtuvo al cultivar los explantes por cinco horas previo al bombardeo en el medio Yasuda complementado con 0.5M mannitol+0.5M sorbitol. Asimismo, se obtuvo una mayor expresión transitoria del gen uidA al bombardear los segmentos de hoja de Caturra y Catuaí y embriones somáticos de Catuaí con una presión de helio de 1 100psi y una distancia de bombardeo de 6 o 9 cm.