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Cassava is a staple food used in many countries around the world, despite deficiencies in micronutrients such as provitamin A carotenoids. Unfortunately, improvement of the cassava nutritional content by use of conventional breeding is slow and difficult. Therefore, there is an urgent need to develop and standardize protocols using biotechnological tools to improve cassava. The Alliance of Biodiversity International and the International Center for Tropical Agriculture (CIAT) have worked on cassava genetic transformation over the last 30 years. Here, we describe, step by step, the procedures used for genetic transformation of cassava variety TMS60444, to improve carotenoids and other traits. This protocol includes stock setup, reagents, media preparation, materials, and equipment, for the genetic transformation of embryogenic tissues. The main expected output in publishing this protocol is to provide the basis for a reproducible and reliable method to genetically modify and/or gene edit Latin American and Asian cassava varieties.

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KEY MESSAGE: The overexpression of RXam2, a cassava NLR (nucleotide-binding leucine-rich repeat) gene, by stable transformation and gene expression induction mediated by dTALEs, reduce cassava bacterial blight symptoms. Cassava (Manihot esculenta) is a tropical root crop affected by different pathogens including Xanthomonas phaseoli pv. manihotis (Xpm), the causal agent of cassava bacterial blight (CBB). Previous studies have reported resistance to CBB as a quantitative and polygenic character. This study sought to validate the functional role of a NLR (nucleotide-binding leucine-rich repeat) associated with a QTL to Xpm strain CIO151 called RXam2. Transgenic cassava plants overexpressing RXam2 were generated and analyzed. Plants overexpressing RXam2 showed a reduction in bacterial growth to Xpm strains CIO151, 232 and 226. In addition, designer TALEs (dTALEs) were developed to specifically bind to the RXam2 promoter region. The Xpm strain transformed with dTALEs allowed the induction of the RXam2 gene expression after inoculation in cassava plants and was associated with a diminution in CBB symptoms. These findings suggest that RXam2 contributes to the understanding of the molecular basis of quantitative disease resistance.

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Epigenetic mechanisms are able to regulate plant development by generating non-Mendelian allelic interactions. An example of these are the responses to environmenal stimuli that result in phenotypic variability and transgression amongst important crop traits. The need to predict phenotypes from genotypes to understand the molecular basis of the genotype-by-environment interaction is a research priority. Today, with the recent discoveries in the field of epigenetics, this challenge goes beyond analyzing how DNA sequences change. Here we review examples of epigenetic regulation of genes involved in carotenoid synthesis and degradation, cases in which histone- and/or DNA-methylation, and RNA silencing at the posttranscriptional level affect carotenoids in plants.

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Las raíces almacenadoras de yuca representan una fuente importante de almidón. La ruta metabólica del almidón ha sido reconstruida recientemente en yuca gracias a la liberación de la secuencia completa de su genoma. En este estudio se evaluó la expresión de los genes que codifican para las enzimas pululanasa, isoamilasa, α-amilasa, enzima desproporcionante, ADP-glucosa pirofoforilasa, almidón sintasa unida al gránulo, enzima ramificante del almidón y sintasa soluble del almidón, en las raíces almacenadoras de plantas de cinco y 11 meses de edad, en un grupo de cinco variedades de yuca. Se evidenciaron diferencias importantes en la expresión de estos genes entre las variedades evaluadas y entre los dos tiempos. Las variedades CM523-7 y SM1219-2 presentaron uno de los niveles más altos de expresión para los genes ADP-glucosa pirofoforilasa y almidón sintasa unida al gránulo mientras que el gen para α-amilasa fue el más bajo en estas dos variedades. Aunque la variedad TMS60444 presentó niveles de expresión similares en genes implicados en la síntesis de almidón, fue la que presentó el mayor nivel de expresión de la α-amilasa. Estos datos se pueden correlacionar con el relativo bajo contenido de materia seca en esta variedad. Los datos de expresión génica presentados en este trabajo permitirán complementar información sobre actividad enzimática con miras a identificar los elementos más importantes en la acumulación diferencial de almidón entre variedades de yuca.

Cassava storage roots represent an important starch source. Recently, the starch metabolic pathway in cassava has been reconstructed thanks to the full release of its genome. In this study gene expression was evaluated for genes coding pullulanase, isoamylase, α-amylase, deproportionating enzyme, ADP-glucose pyrophosphorylase, granule bound starch synthase, starch branching enzyme and soluble starch synthase, in cassava storage roots five and 11 months old, in five cassava varieties. Important gene expression differences were detected both at the variety and time level. CM523-7 and SM1219-2 showed one of the highest expression levels for AGPase and GBSS genes, while α-amylase showed the lowest level in these two varieties. TMS60444 variety showed similar expression levels in starch biosynthesis-related genes, but conversely also showed the highest α-amylase expression. This correlates with the relative low dry-matter content in TMS60444. Gene expression data reported here will allow complementing actual information on enzymatic activity, in order to identify the most relevant factors in differential starch accumulation between cassava varieties.

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La yuca (Manihot esculenta Crantz) es un cultivo de alta importancia en países tropicales. La transformación genética de yuca ha sido posible desde hace 15 años mediante la producción que callo embriogénico friable (CEF) a partir de embriones somáticos. En el presente trabajo se evalúan la inducción de embriones somáticos usando tres diferentes auxinas sintéticas y la producción de CEF a partir de éstos en los cultivares de yuca SG107-35 y BRA685. Estos cultivares son resistentes a la bacteriosis vascular de yuca cuyo agente causal es Xanthomonas axonopodis pv. manihotis, una de las principales limitantes del cultivo. Los resultados obtenidos muestran que en ambos cultivares la hormona Picloram a una concentración de 12 mg/l fue más eficiente que 2,4-D y Dicamba para producir embriones somáticos. Adicionalmente se consiguió la producción de CEF y la regeneración de plantas mediante embriogénesis somática en el cultivar BRA685. Los resultados del presente trabajo son importantes para evaluar la transformabilidad de distintos cultivares de yuca. Actualmente este número es bastante reducido principalmente porque la producción de CEF es fuertemente influenciada por el genotipo. Por tal razón solo se transforma de manera rutinaria y eficiente en el cultivar 60444. La posibilidad de transformación de distintos cultivares de yuca permitirá explotar la enorme variabilidad del cultivo, invitándonos a aumentar los esfuerzos para mejorar y universalizar los protocolos de transformación de yuca.

The cassava (Manihot esculenta Crantz) crop has a very important role as a food, feed and a raw material in developing countries; therefore it is a priority to develop technologies oriented to the solution of problems and agronomic improvement of the crop. The genetic transformation of cassava was developed 15 years ago by producing friable embryogenic callus (FEC) from somatic embryos as target tissue for transformation. In the present work we evaluated the induction of somatic embryos by using three different synthetic auxins and the production of FEC from both SG107-35 and BRA685 cassava cultivars; both are resistant to cassava bacterial blight caused by Xanthomonas axonopodis pv. manihotis, the most important bacterial disease affecting the crop. Our results showed that in both cultivars gave rise to somatic embryos in media containing Picloram at a concentration of 12 mg/l being more efficient than using 2,4-D or Dicamba. Additionally the cultivar BRA685 produced regenerative FEC giving rise to plants through somatic embryogenesis. However compared to the model cultivar 60444, FEC production was greatly lower. This work shows new efforts to increase the number of transformable cultivars of cassava and take advantage of the enormous genetic variability of the crop.

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Los combustibles obtenidos a partir de materia vegetal, como el etanol y el biodiesel, están tomando importancia en la dinámica energética mundial, gracias principalmente a que son más económicos y menos contaminantes del medio ambiente que los combustibles fósiles. El mercado de biocombustibles ha producido un incremento en las áreas de cultivo tanto de plantas regularmente usadas como materia prima para su obtención como de aquellas con potencial de ser nuevas fuentes de producción, al igual que ha impulsado la investigación básica orientada hacia el incremento en calidad y producción de diferentes cultivos. Las plantas que almacenan cantidades importantes de almidón, azúcares simples o aceites son el blanco principal para la producción de biocombustibles, aunque nuevas tecnologías están permitiendo la utilización de celulosa como materia prima. El cultivo de yuca (Manihot esculenta) está ampliamente distribuido en toda la zona tropical y es la base alimenticia de cerca del 10% de la población mundial. El alto contenido de almidón en las raíces almacenadoras de la yuca hace de este cultivo una opción para la obtención de etanol. El uso de técnicas de mejoramiento no convencional de variedades de yuca permitirá la generación de plantas más aptas para la industria de biocombustibles. En este artículo de reflexión se revisa el estado actual de los biocombustibles a nivel mundial y nacional, y se comentan los beneficios y retos a afrontar en cuanto a las implicaciones respecto al medio ambiente y la alimentación humana. Finalmente se discute el potencial de la yuca como fuente eficiente de materia prima para la obtención de biocombustibles en Colombia.

Fuels such as ethanol and biodiesel, obtained from plants and their constituents, have recently received the world’s attention as a true alternative to the global energy supply, mainly because they are cheaper and less contaminant of the environment than the currently used, non-renewable fossil fuels. Due to the pushing biofuel market, the world is currently experiencing an increase of agricultural land devoted to grow crops used to obtain them, like maize and sugar cane, as well as crops that have the potential to become new sources of biofuels. Similarly, this emerging market is boosting the basic research oriented towards obtaining better quality and yield in these crops. Plants that store high quantities of starch, simple sugars or oils, are the target of the biofuel industry, although the newest technologies use also cellulose as raw material to produce fuels. Cassava (Manihot esculenta) is widely grown in the tropics and constitutes a staple food for approximately 10% of the world population. The high starch content of its storage roots, together with the use of conventional and non-conventional breeding turn this crop into an option to obtain better adapted varieties for ethanol production. This reflexion article reviews the current status of biofuels worldwide and at the national level, and discusses the benefits and challenges faced in terms of effect on the environment and the human food chain. Finally, it discusses the potential of cassava as a source of raw material for obtaining biofuels in Colombia .