Progress in Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz)

To help solve breeding problems in cassava that have long eluded conventional strategies, a routine transformation protocol is esential. The agropine-mannopine nondisarmed strain of Agrobacterium tumefaciens (CIAT 1182) was selected and used with the binary vector pGV1040. This vector contains the npt II, gus, and bar genes. Inoculations were carried out on cotyledonary leaves obtained from somatic embryos of cassava cultivar M Peru 1983. Following co-cultivation, somatic embryos were induced in a selection medium, supplemented with 16-32 mg/l of Basta. Several Basta-resistant plantlets were obtained. Positive lines were screened, using the polymerase chain reaction (PCR), together with specific primers for the gus and products analyzed on Southern blots under highly stringent conditions. Only lines showing the presence of both genes were transplanted to pots. Genomic Southern analysis of these plants are new under wa

Standardization of a Method for Analysis of Cocaine in Urine Samples using Solid-Phase Extraction (SPE) and High Performance Liquid Chromatography (HPLC).

Los altos costos que implica el consumo de cocaína pura, llevó a la producción de base de cocaína o bazuco (crack), un producto de menor valor económico, gran demanda y generalmente más usado por consumidores de bajo nivel socioeconómico. El bazuco es una sustancia blanca, o pardusca, semisólida o sólida, cuyo principio activo es la cocaína. La cocaína es un alcaloide con una larga historia de uso y abuso. Su determinación, tanto en muestras no biológicas como en fluidos biológicos continúa siendo una tarea de primer orden. En el presente trabajo se reporta la validación de un método de extracción de la cocaína en muestras de orina, usando la técnica de extracción en fase sólida (SPE). Porcentajes de recuperación entre el 99.4 y el 99.8 % fueron obtenidos para cocaína empleando SPE. La técnica cromatográfica fue estandarizada, empleando una columna C8, un detector ultravioleta (254 nm), una fase móvil de agua-acetonitrilo-ácido acético (58-40-2) a un flujo de 0.4 mL/min. El rango de linealidad estuvo entre 0.01 μg/mL y 1.0 μg/mL. Se crearon tres curvas de cuantificación: 10 – 20 ppb, 20 – 100 ppb y 100 – 1,000 ppb con coeficientes de determinación r2 de 0.9965, 0.9947 y 0.9973 respectivamente. El método fue aplicado en muestras de orina de un grupo de personas voluntarias que consumen bazuco en la ciudad de Popayán, Cauca, Colombia

Genetics of esterase and glutamate oxaloacetate transaminase isozymes in cassava (Manihot esculenta Crantz)

Genetic studies of two isozyme loci have been carried out in root tips of eight cassava F1 progenies using polyacrylamide gel electrophoresis. Two loci, esterase-1 (East-1), the most distant cationic region, and glutamate oxaloacetate transaminase-1 (Got-1), the most proximal anionic region, were evaluated among the segregating populations. Est-1 locus has five multiple alleles, including one null allele, and behaves as monomer, with a diploid inheritance pattern with only two alleles presenting each individual. These data were confirmed by esterase characterization of CIAT cassava germplasm bank accessions; a total of 11 different phenotypes representing 15 different genotypes for this locus were demonstrated. Got-1 locus comprises three allels with diploid inheritance. This work provides evidence for a diploid inheritance pattern of the two isozyme loci supporting the allotetraploid nature of cassava. Many other regions of the zimograms are still to be elucidated. This information will be valuable to interpret studies on isozyme fingerprinting of cassava genetic diversity

Towards the development of Agrobacterium tumefaciens and particle bombardment-mediated cassava transformation

Our strategy for Aqrobacterium-mediated cassava transformation involves the utilization of cotyledonary leaves from somatic embryos and infection with both wild and disarmed strains. Virulence tests for wild Aqrobacterium strains as well as regeneration protocols have been developed; the plasmid used in all the experiments was the pGV 1040 containing the uid A, the bar and the npt ll genes (provided by Plant Genetic Systems. Gent). Sensitivity levels of cassava tissues to the commercial herbicide Basta, used as selection agent, were determined. Minimum lethal doses for the herbicide were 1 mg/l for immature leaves and 18 mg/l for somatic embryos. Results with kanamycin as selection agent were inconsistent. Only somatic embryos showed endogenous uid A gene expression; endogenous activity was reduced by 70-80 using the Kosugi protocol (1990), which includes methanol. Expression of opine synthesis genes of a wild Aqrobacterium strain, isolated from cassava tumors, was obtained after transformation of cotyledonary leaves of somatic embryos with the wild strain. Transformation experiments are now under way using the wild Aqrobacterium strain carrying the pGV 1040 plasmid. A biolistic device was used to bombard early stage somatic embryos using the pGV 1040 construct harboring the same genes as in the Aqrobacterium experiments. An average of 20 GUS expressing spots per 0,5 cm2 of embryogenic tissue was obtained. GUS expression in proliferating somatic embryos has been observed after one and two months from bombardment

Transgenic plants of casssva (Manihot esculenta) with resistance to basta obtained by Agrobacterium-mediated transformation

Transgenic plants of cassava (Manihot esculenta) resistant to the herbicide Basta were obtained through Agrobacterium-mediated transformation. The plants also expressed the uidA gene and two were positive for PCR- and/or Southern-based detection of the nptII gene. Somatic-embryo-derived cotyledons were used as source of explants. A non-disarmed Agrobacterium strain (CIAT 1182) was used to transfer the genes of interest into cassava cultivar MPer183. Greenhouse tests of resistance to Basta (Hoechst) showed three plant lines with different levels of tolerance to the herbicide. Based on Southern tests of transgenesis, the transformation efficiency was 1%. The results constitute the first report of the bar gene conferring herbicide resistance to cassava plants

Future initiatives in biotechnology research for tropical agriculture: the case of cassava

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