Evaluation and application of an efficient plant DNA extraction protocol for laboratory and field testing / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Nucleic acids in plant tissue lysates can be captured quickly by a cellulose filter paper and prepared for amplification after a quick purification. In this study, a published filter paper strip method was modified by sticking the filter paper on a polyvinyl chloride resin (PVC) sheet. This modified method is named EZ-D, for EASY DNA extraction. Compared with the original cetyl trimethylammonium bromide (CTAB) method, DNA extracted by EZ-D is more efficient in polymerase chain reaction (PCR) amplification due to the more stable performance of the EZ-D stick. The EZ-D method is also faster, easier, and cheaper. PCR analyses showed that DNA extracted from several types of plant tissues by EZ-D was appropriate for specific identification of biological samples. A regular PCR reaction can detect the EZ-D-extracted DNA template at concentration as low as 0.1 ng/μL. Evaluation of the EZ-D showed that DNA extracts could be successfully amplified by PCR reaction for DNA fragments up to 3000 bp in length and up to 80% in GC content. EZ-D was successfully used for DNA extraction from a variety of plant species and plant tissues. Moreover, when EZ-D was combined with the loop-mediated isothermal amplification (LAMP) method, DNA identification of biological samples could be achieved without the need for specialized equipment. As an optimized DNA purification method, EZ-D shows great advantages in application and can be used widely in laboratories where equipment is limited and rapid results are required.

Comparison of Regulation on GM Crops Cultivation Management in USA and China / 中国生物工程杂志

The proposal to promote the commercialization of new insect-resistant GM cotton,GM corn and other major products in 13th Five Year Plan on National Science and Technology Innovation ,has put forward a test to the government's ability to manage GM crops cultivation.Only a few species of GM crops,not including food crops,have been commercialized in China,we lack experience in GM crops cultivation management.Taking USA,where the farmers cultivate huge areas of GM crops,as a comparison object,the examination and approval procedures before cultivation,GM crop growers' obligation of the management,the methods to reduce mingling of GM crops and non-GM crops are compared.And concludes that the differences in legal status of GM crops,the intellectual property regime and labeling regulation,the ability of relative stakeholders to implement segregation measures and bear loss lead to different regulation on GM crops cultivation management in USA and China.The government combine the domestic current situation of the agricultural environment in our country,and focus on substantial risk in the safety management of GM crops in cultivation management is advised.The competent department of agriculture,researchers and seed traders should work together to help guide growers to avoid the economic disputes due to mingling and reduce the impact on the ecological environment.

Diseño in silico y evaluación funcional de genes semisintéticos que confieran tolerancia a fosfinotricina / In silico design and functional assessment of semisynthetic genes that confer tolerance to phosphinothricin

La tolerancia a herbicidas es una de las características más usadas en los cultivos GM, con resultados positivos para los agricultores y el ambiente. El punto de partida, es el desarrollo de casetes de expresión que expresen la característica de interés, inicialmente construidos mediante técnicas de biología molecular convencionales. Actualmente, con herramientas de bioinformática y biología sintética, es posible diseñar y probar el constructo in silico, para luego contratar su síntesis. Esta aproximación, permite optimizar la expresión mediante la modificación del uso codónico. En este trabajo se diseñaron y evaluaron en Nicotiana benthamiana versiones semisintéticas de genes que confieren tolerancia al herbicida fosfinotricina. Se realizó un análisis de libertad de operación, con el fin de asegurar que los constructos diseñados no violen derechos de propiedad intelectual en Colombia. Se obtuvieron dos casetes de expresión con libertad de operación, que expresan versiones del gen bar.

Herbicide tolerance is one of the features most used in GM crops, which has shown positive results for farmers and the environment. The starting point is the development of expression cassettes that express the characteristic of interest, they are initially constructed by standard molecular biology techniques. Currently, by bioinformatics and synthetic biology tools, it is possible to design and test the construct in silico, and then hire their synthesis. This approach allows optimizing expression by modifying the codon usage. In this work there were designed and evaluated semi-synthetic versions of genes in Nicotiana benthamiana, these genes confer tolerance to the herbicide phosphinothricin. It was made an analysis of freedom to operate in order to ensure that the designed constructs not violate intellectual property in Colombia. There were obtained two expression cassettes with freedom to operate, which express versions of the bar gene.

La ingeniería gnnética de plantas en Colombia: un camino en construcción / Genetic Engineering of Plants in Colombia: A Road Under Construction

Se reportan en Colombia trabajos en el desarrollo de líneas genéticamente modificadas (GM) de arroz, yuca, algodón, papa, caña de azúcar, café, maíz, soya, estevia y crisantemo, por parte de cinco centros de investigación (CIAT, CENICAÑA, CENICAFE, CIB, CORPOICA) y tres universidades (Universidad Nacional de Colombia, Universidad Javeriana, Universidad de Antioquia). Para llegar a la liberación comercial de variedades GM, se requiere de verdaderas políticas públicas en ciencia y tecnología, que den financiación suficiente y oportuna, así como resolver problemas con la maraña de patentes que limitan o impiden el desarrollo de innovaciones biotecnológicas, además de con el paquete regulatorio, que incrementa significativamente los costos de desarrollo.

Here in Colombia have been reported efforts on the development of genetically modified (GM) crops, like: rice, cassava, cotton, potato, sugar cane, coffee, corn, soy, stevia and chrysanthemum, by five research center (CIAT, CENICAÑA, CENICAFE, CIB, CORPOICA) and three universities (Universidad Nacional de Colombia, Universidad Javeriana, Universidad de Antioquia). To get to the commercial release of GM varieties, real public policies on science and technology are needed, giving sufficient and timely funding, as well as to solve problems with the tangle of patents that limit or prevent the development of biotechnological innovations, and it is also required suffice funding to go through the regulatory issues, which significantly increase the cost of those developments.

STUDY OF GENE FLOW FROM GM COTTON (Gossypium hirsutum) VARIETIES IN "EL ESPINAL" (TOLIMA, COLOMBIA) / Estudio del flujo de genes desde variedades GM de algodón (Gossypium hirsutum) en El Espinal (Tolima, Colombia)

In 2009, 4088 hectares of genetically modified (GM) cotton were planted in Tolima (Colombia), however there is some uncertainty about containment measures needed to prevent the flow of pollen and seed from regulated GM fields into adjacent fields. In this study, the gene flow from GM cotton varieties to conventional or feral cotton plants via seed and pollen was evaluated. ImmunostripTM, PCR and ELISA assays were used to detect gene flow. Fifty six refuges, 27 fields with conventional cotton and four feral individuals of the enterprise "Remolinos Inc." located in El Espinal (Tolima) were analyzed in the first half of 2010. The results indicated seed mediated gene flow in 45 refuges (80.4 %) and 26 fields with conventional cotton (96 %), besides pollen mediated gene flow in one field with conventional cotton and nine refuges. All fields cultivated with conventional cotton showed gene flow from GM cotton. Two refuges and two feral individuals did not reveal gene flow from GM cotton.

En el 2009 se plantaron 4088 hectáreas de algodón genéticamente modificado en el departamento de Tolima (Colombia), sin embargo, hay ciertas incertidumbres acerca de las medidas de contención necesarias para impedir el movimiento de polen y semillas desde los campos GM regulados, hacia los campos adyacentes de cultivos convencionales. En este estudio se evaluó el flujo de genes mediado por polen y semillas desde variedades GM hacia variedades convencionales o individuos ferales, en el cultivo del algodón. Para detectar el flujo de genes se utilizaron ImmunostripTM, PCR y ELISA. Cincuenta y seis refugios, 27 campos con algodón convencional y cuatro individuos ferales de la empresa "Remolinos S.A." localizada en El Espinal (Tolima) fueron analizados en el primer semestre de 2010. Los resultados indicaron presencia de plantas GM en 45 refugios (80,4 %) y 26 campos de algodón convencional (96 %), además de un flujo génico mediado por polen en un campo de algodón convencional y nueve refugios. En todos los campos cultivados con algodón convencional se evidenció flujo de genes desde algodón GM. Solo en dos refugios y en dos individuos ferales no se evidenció flujo de genes desde algodón GM.

Recent advances in development of marker-free transgenic plants: Regulation and biosafety concern.

During the efficient genetic transformation of plants with the gene of interest, some selectable marker genes are also used in order to identify the transgenic plant cells or tissues. Usually, antibiotic- or herbicide-selective agents and their corresponding resistance genes are used to introduce economically valuable genes into crop plants. From the biosafety authority and consumer viewpoints, the presence of selectable marker genes in released transgenic crops may be transferred to weeds or pathogenic microorganisms in the gastrointestinal tract or soil, making them resistant to treatment with herbicides or antibiotics, respectively. Sexual crossing also raises the problem of transgene expression because redundancy of transgenes in the genome may trigger homology-dependent gene silencing. The future potential of transgenic technologies for crop improvement depends greatly on our abilities to engineer stable expression of multiple transgenic traits in a predictable fashion and to prevent the transfer of undesirable transgenic material to non-transgenic crops and related species. Therefore, it is now essential to develop an efficient marker-free transgenic system. These considerations underline the development of various approaches designed to facilitate timely elimination of transgenes when their function is no longer needed. Due to the limiting number of available selectable marker genes, in future the stacking of transgenes will be increasingly desirable. The production of marker-free transgenic plants is now a critical requisite for their commercial deployment and also for engineering multiple and complex trait. Here we describe the current technologies to eliminate the selectablemarker genes (SMG) in order to develop marker-free transgenic plants and also discuss the regulation and biosafety concern of genetically modified (GM) crops.

Cultivos transgnnicos:: entre los riesgos biolóicos y los beneficios ambientalles y económicos / Gm Crops:: Between Biological Risk and Environmental and Economics Benefits

Los cultivos transgénicos, biotecnológicos o genéticamente modificados (GM) son el resultado de la aplicación de la tecnología del ADN recombinante en agricultura. Este tipo de organismos se constituyen con la transferencia de genes foráneos (transgenes) de cualquier origen biológico (animal, vegetal, microbiano, viral) al genoma de especies cultivadas de plantas. Los cultivos GM se utilizan en el mundo desde 1996 y en diciembre de 2010 se llegó a mil millones de hectáreas, sembradas en todo el periodo. En solo el pasado año 2010 se sembraron 148 millones de hectáreas, cultivadas por 15,4 millones de agricultores en 29 países. Los cultivos GM que se usan en agricultura global son principalmente soya, algodón, maíz y colza, que expresan transgenes derivados de bacteria y que confieren resistencia a insectos lepidópteros (RIL) o, tolerancia a algunos herbicidas (TH) como glifosato y glufosinato de amonio. Las primeras variedades transgénicas contenían solo un transgen de interés, o evento simple, mientras que las variedades actuales expresan varios transgenes, o eventos apilados, que en algunos casos confieren resistencia a diferentes especies de insectos lepidópteros y coleópteros, así como tolerancia a dos tipos diferentes de herbicidas. Para el año 2009 se sembraron en Colombia, 18.874 hectáreas de cultivos GM de algodón, 16.793 hectáreas de cultivos GM de maíz, y cerca de cuatro hectáreas de cultivos GM de clavel y rosas. Maíz y algodón se sembraron en los departamentos de Sucre, Cesar, Córdoba, Huila y Tolima. Solo maíz en los departamentos de Antioquia, Valle del Cauca, Meta, Cundinamarca y Santander, y clavel y rosas en el departamento de Cundinamarca. Las variedades transgénicas de maíz y algodón, expresan características RIL y TH, como eventos simples o como eventos apilados. En el caso de clavel y rosa, se trata de genotipos que expresan color azul. Desde la academia se ha tratado de organizar el debate sobre la adopción de los cultivos GM, alrededor del análisis ponderado de los riegos biológicos y beneficios ambientales y económicos. Los riesgos biológicos se definen por los posibles efectos negativos sobre consumidor humano o ambiente en que se liberan. Los beneficios ambientales tienen que ver con los efectos de la reducción en el uso de agroquímicos (insecticidas y herbicidas), y beneficios económicos con la reducción en las pérdidas debidas al ataque de insectos y a la competencia de malezas, así como a la reducción de costos de producción.

The transgenic crops were the result of the application of recombinant DNA technology in agriculture. These crops were developed by transfer of foreign genes (transgenes) from any biological origin (animal, plant, microbial, viral) to the genome of cultivated species of plants. The crops genetically modified (GM) have been used in the world since 1996; up to December 2010 they counted to a billion hectares planted throughout the period. In just the past year 2010 148 million hectares were planted, grown by 15.4 million farmers in 29 countries. GM crops that are used in global agriculture are mainly soybean, cotton, corn and canola, which express transgenes derived from bacteria, and confer resistance to lepidopteran insects (ILR) or herbicide tolerance (HT; glyphosate and glufosinate ammonium). The first transgenic varieties containing only a single transgene, or simple event, while the current varieties express several transgenes, or stacked, confereing resistance to different species of Lepidoptera and Coleopteran insects and tolerance to two different herbicides. In 2010 were planted in Colombia, 18.874 hectares of GM cotton, 16.793 hectares of GM corn, and 4 hectares of GM carnations and GM roses. GM corn and GM cotton were planted in Sucre, Cesar, Cordoba, Huila and Tolima. GM corn was planted in Antioquia, Valle del Cauca, Meta, Cundinamarca and Santander. Carnations and roses were planted in Cundinamarca. GM maize and GM cotton expressing ILR and HT features, as simple events or stacked. In the case of GM carnation and GM roses, these genotypes that express the color blue. Academia has tried to organize the debate on the adoption of GM crops around the analysis of biological risks and environmental vs environmental and economic benefits. Biological hazards are defined by the possible negative effects on human consumers or negative effects on the environment. The environmental benefits are related to reduce use of agrochemicals (insecticides and herbicides), and the economic benefits from the reduction in losses due to insect and weed competition to reduction of production costs.

EXPRESIÓN DE LA PROTEÍNA Cry1Ac EN TEJIDOS DE LÍNEASTRANSGÉNICAS DE PAPA (Solanum tuberosum spp. ANDÍGENA)VAR. DIACOL CAPIRO. / Cry1Ac Protein Expression in Tissues of Potato (Solanumtuberosum spp. Andígena) Transgenic Lines Var. Diacol Capiro

La papa (Solanum sp.) es el cuarto producto alimenticio más importante en el mundo. En Colombia anualmente se producen alrededor de 2,8 millones de toneladas, sirviendo como sustento económico a 90.000 familias. En el país, Tecia solanivora genera el mayor impacto económico en el cultivo con pérdidas de hasta el 100% en la producción de tubérculos. El fitomejoramiento vía introducción de genes Cry, que codifican para cristales proteicos insecticidas, constituye una alternativa para reducir el ataque de insectos en cultivos de interés comercial. En este trabajo se caracterizó la inserción, transcripción y expresión del gen Cry1Ac en diferentes tejidos y en tres etapas del desarrollo para dos líneas transgénicas de Solanumtuberosum spp. andígena variedad Diacol Capiro generadas previamente por transformación con Agrobacteriumtumefaciens. La caracterización se realizó a través de técnicas de PCR, RT-PCR y ELISA. Se corroboró la inserción y transcripción del gen utilizando primers que amplificaron una banda específica de 766pb para Cry1Ac. Los niveles de expresión de la proteína llegaron a ser mayores a 45µg/g y no mostraron diferencias significativas entre las líneas analizadas, ni entre las tres etapas del desarrollo. No se evidenciaron diferencias significativas entre las líneas transgénicas con respecto al control al hacer un análisis de algunas características fenotípicas relevantes. Los resultados encontrados sugieren la realización de seguimientos y ensayos de bioseguridad sobre este material, ya que los altos niveles de expresión en todos los tejidos analizados, pueden afectar a organismos no blanco.

The potato plant is the fourth most important crop in the world. In Colombia around 2.8 million tons are produced annually economically supporting 90000 families. In the country, the major economic impact in the crop is caused by Tecia solanivora that originates loses up to 100% in the tuber production. The genetic plant breeding related to the introduction of Cry genes which codify insecticidal crystal proteins is an alternative for reducing the insect attack in commercial crops. In this work, the insertion, transcription and expression of Cry1Ac gen was characterized in different tissues and three development stages of two transgenic lines of Solanum tuberosum variety Diacol Capiro that were previously transformed by Agrobacterium tumefaciens method. The characterization was realized by PCR, RT-PCR and ELISA techniques. The gen insertion and transcription was confirmed using primers for Cry1Ac gen that amplified a specific band of 766 bp. The protein expression levels were higher than 45 µg/g and were not significantly different between the analyzed lines nor the three development stages. Furthermore, taking into account some relevant phenotypic features, no significant differences were found between transgenic lines and controls. The results suggest that monitoring and biosecurity assays are necessary with this vegetal material because their high level expression inside all the tissues analyzed that could affect non-targeted insects.

Insufficient regulatory supervision prior to release of genetically modified crops for commercial cultivation in India.

(1) Do we need GM crops? No. We have far better alternatives such as integrated pest management (IPM), biopesticides and appropriate agro-practices including organic farming for almost all crops. IPM is a part of the country’s stated national agriculture policy. It has been shown to be effective but is not being used. (2) Was appropriate risk assessment carried out in the case of the Bt-cotton crops that have been released? No. For example, our present system of testing for allergenicity does not take into account recent work (e.g., papers in this area – a paper titled “Allergic potential of novel foods” in the Proceedings of the Nutrition Society, volume 64, issue No.4, pp 487–490 of November 2005; a paper titled “Allergenicity Assessment of Genetically Modifi ed Crops – What makes sense?” in Nature Biotechnology of January 2008, volume 26, pp 73–81; a paper titled “Transgenic expression of bean alpha-amylase inhibitor in peas results in altered structure and immunogenecity” in Journal of Agricultural Food Chemistry, 2005, volume 53, pp 9023–9030). The Annexure lists 29 tests relevant to the release of GM crops. Only a few have been done for Bt-cotton and GM crops in the pipeline, and that too inadequately (see item 5 below).

Allergenicity of Genetically Modified Crops / 소아알레르기및호흡기학회지

During the period 1996 to 2006, 51 countries have granted regulatory approvals for genetically modified (GM) crops for import for food and feed use and for release into the environment. A total of 539 approvals has been granted for 107 events for 21 crops. One of the safety concerns regarding GM crops is potential allergenicity of the inserted protein. In safety-assessment of the allergenic potential from GM crops, the allergenic properties of the gene donor and the host organisms should be evaluated. The sequence of the newly introduced protein should be compared with all known allergens. If a sequence homology match to a known allergen is identified for the introduced protein, then this protein should be assessed for immunoreactivity using sera from allergic patients. If no sequence homology to known allergens is identified, then the stability of the protein to digestion and processing is assessed. Currently, laboratory and clinical tests have been reported and the assessment process of GM crops has worked well to prevent the unintended introduction of allergens. However, these tests may still occasionally miss a new allergen from all novel foods containing GM crops. The newly introduced protein in GM crops must be carefully assessed as proteins may induce allergic reactions in humans.