Nuevas perspectivas sobre la histopatología de la infección de Pinus Radiata por Fusarium Circinatum

223 p.

Targeted gene modification in Fragaria vesca mediated by CRISPR/Cas9 system

Genome editing is becoming an important biotechnological tool for gene function analysis and crop improvement, being the CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated protein 9) system the most widely used. The natural CRISPR/Cas9 system has been reduced to two components: a single-guide RNA (sgRNA) for target recognition via RNA-DNA base pairing, which is commonly expressed using a promoter for small-RNAs (U6 promoter), and the Cas9 endonuclease for DNA cleavage (1). To validate the CRISPR/Cas9 system in strawberry plants, we designed two sgRNAs directed against the floral homeotic gene APETALA3 (sgRNA-AP3#1 and sgRNA-AP3#2). This gene was selected because ap3 mutations induce clear developmental phenotypes in which petals and stamens are missing or partially converted to sepals and carpels respectively (2). In this work, we used two different U6 promoters to drive the sgRNA-AP3s expression: AtU6-26 from Arabidopsis (4), and a U6 promoter from Fragaria vesca (FvU6) (this work). We also tested two different coding sequences of Cas9: a human- (hSpCas9) (3) and a plant-codon optimized (pSpCas9) (this work). Transient expression experiments using both CRISPR/Cas9 systems (AtU6-26:sgRNA-AP3#1_35S:hSpCas9_AtU6-26:sgRNA-AP3#2 and FvU6:sgRNA-AP3#1_35S:pSpCas9_FvU6:sgRNA-AP3#2) were performed infiltrating Agrobacterium tumefaciens into F. vesca fruits. PCR amplification and sequencing analyses across the target sites showed a deletion of 188-189 bp corresponding to the region comprised between the two cutting sites of Cas9, confirming that the CRISPR/Cas9 system is functional in F. vesca. Remarkably, the two systems showed different mutagenic efficiency that could be related to differences in expression of the U6 promoters as well as differences in the Cas9 transcripts stability and translation. Stable transformants for both F. vesca (2n) and Fragaria X anannassa (8n) are currently being established to test whether is possible to obtain heritable homozygous mutants derived from CRISPR/Cas9 strategies in strawberry. Thus, our work offers a promising tool for genome editing and gene functional analysis in strawberry. This tool might represent a more efficient alternative to the sometimes inefficient RNAi silencing methods commonly used in this species.

Estudo da associação dos genes GGR, GGPPS2 e GGPPS6 em Arabidopsis thaliana L. (Brassicaceae) a compostos alelopáticos

Dissertação (mestrado)—Universidade de Brasília, Departamento de Botânica, Programa de Pós-Graduação em Botânica, 2016.

Influência de diferentes meios de cultura na transformação genética de milho tropical.

2016

Metodología para la detección de Agrobacterium vitis a partir de material vegetal de Vitis vinífera

En nuestro país no está desarrollada una técnica sensible y precisa que permita confirmar la presencia de la bacteria Agrobacterium vitis; por lo tanto es muy importante contar con herramientas analíticas que permitan identificar la bacteria en vides y/o suelos para controlar la diseminación y propagación de la misma, permitiendo así a las empresas, mejorar la competitividad de sus productos en el mercado y garantizar la seguridad y calidad de la materia prima principal de la industria vitivinícola. El objetivo de este estudio es poner a punto una metodología de detección de Agrobacterium vitis sensible y de bajo costo basada en la técnica de PCR (Reacción en cadena de la polimerasa). El desarrollo de esta metodología pretende ser una herramienta importante para la industria vitivinícola al permitir detectar la presencia o ausencia de la bacteria en programas de monitoreo, tomar decisiones a tiempo y así proteger la calidad y sanidad de las vides. En el presente trabajo se empleó la metodología propuesta por Eastwell y colaboradores en 1995. La misma consistió en el aislamiento de la bacteria en medio selectivo RS, en la extracción/purificación de ADN bacteriano, amplificación por PCR, separación del producto amplificado por electroforesis y revelado por tinción, pudiéndose observar que el 100% del material con sintomatología que se utilizó presentó desarrollo de colonias características de Agrobacterium vitis. Al hacer la amplificación de los ADN y posterior revelado se observó, en primera instancia, que la amplificación no fue óptima, pero cuando se realizaron las distintas adaptaciones de la técnica se vieron diferentes resultados encontrándose que podría tratarse de: A. vitis virulento, no virulento o A. tumefaciens. Para ello fue necesario adecuar la técnica y estudiar algunos aspectos tales como las temperaturas de incubación de los medios de cultivos, evaluación de la concentración de ADN para determinar si existía una concentración mínima del mismo para su posterior amplificación. También se trabajó con diferentes concentraciones del gel de agarosa y se modificaron los volúmenes de siembra y los tiempos de corrida del gel en la cuba de electroforesis. Fue posible adaptar la metodología para la identificación de cepas de Agrobacterium vitis. Los mejores resultados se evidenciaron trabajando con una temperatura de incubación de las colonias de 28 °C, con una concentración del gel de agarosa del 2 % tal como lo indicaba la técnica original, volúmenes de siembra de electroforesis de 10 μl de PCR producto y un tiempo de corrida del gel en la cuba de electroforesis de 70 min a 90 Voltios. Además observamos que concentraciones de ADN superiores a 49 ng/μl registraron bandas visibles siendo las de 116 ng/μl o superiores las concentraciones más adecuadas y optimas para la obtención de bandas bien definidas y nítidas.

Marker gene elimination from transgenic barley, using co-transformation with adjacent 'twin T-DNAs' on a standard Agrobacterium transformation vector

We have tested a methodology for the elimination of the selectable marker gene after Agrobacterium-mediated transformation of barley. This involves segregation of the selectable marker gene away from the gene of interest following co-transformation using a plasmid carrying two T-DNAs, which were located adjacent to each other with no intervening region. A standard binary transformation vector was modified by insertion of a small section composed of an additional left and right T-DNA border, so that the selectable marker gene and the site for insertion of the gene of interest (GOI) were each flanked by a left and right border. Using this vector three different GOIs were transformed into barley. Analysis of transgene inheritance was facilitated by a novel and rapid assay utilizing PCR amplification from macerated leaf tissue. Co-insertion was observed in two thirds of transformants, and among these approximately one quarter had transgene inserts which segregated in the next generation to yield selectable marker-free transgenic plants. Insertion of non-T-DNA plasmid sequences was observed in only one of fourteen SMF lines tested. This technique thus provides a workable system for generating transgenic barley free from selectable marker genes, thereby obviating public concerns regarding proliferation of these genes.

Agrobacterium-mediated transformation of Australian rice cultivars Jarrah and Amaroo using modified promoters and selectable markers

We report the first successful Agrobacterium-mediated transformation of Australian elite rice cultivars, Jarrah and Amaroo, using binary vectors with our improved promoters and selectable markers. Calli derived from mature embryos were used as target tissues. The binary vectors contained hph (encoding hygromycin resistance) or bar (encoding herbicide resistance) as the selectable marker gene and uidA (gus) or sgfpS65T as the reporter gene driven by different promoters. Use of Agrobacterium strain AGL1 carrying derivatives of an improved binary vector pWBVec8, wherein the CaMV35S driven hph gene is interrupted by the castor bean catalase 1 intron, produced a 4-fold higher number of independent transgenic lines compared to that produced with the use of strain EHA101 carrying the binary vector pIG121-Hm wherein the CaMV35S driven hph is intronless. The Ubiquitin promoter produced 30-fold higher β-glucuronidase (GUS) activity (derivatives of binary vector pWBVec8) in transgenic plants than the CaMV35S promoter (pIG121-Hm). The two modified SCSV promoters produced GUS activity comparable to that produced by the Ubiquitin promoter. Progeny analysis (R1) for hygromycin resistance and GUS activity with selected lines showed both Mendelian and non-Mendelian segregation. Lines showing very high levels of GUS activity in T0 showed a reduced level of GUS activity in their T1 progeny, while lines with moderate levels of GUS activity showed increased levels in T1 progeny. Stable heritable green fluorescent protein (GFP) expression was also observed in few transgenic plants produced with the binary vector pTO134 which had the CaMV35S promoter-driven selectable marker gene bar and a modified CaMV35S promoter-driven reporter gene sgfpS65T.

pGreen : a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation

Binary Ti vectors are the plasmid vectors of choice in Agrobacterium-mediated plant transformation protocols. The pGreen series of binary Ti vectors are configured for ease-of-use and to meet the demands of a wide range of transformation procedures for many plant species. This plasmid system allows any arrangement of selectable marker and reporter gene at the right and left T-DNA borders without compromising the choice of restriction sites for cloning, since the pGreen cloning sites are based on the well-known pBluescript general vector plasmids. Its size and copy number in Escherichia coli offers increased efficiencies in routine in vitro recombination procedures. pGreen can replicate in Agrobacterium only if another plasmid, pSoup, is co-resident in the same strain. pSoup provides replication functions in trans for pGreen. The removal of RepA and Mob functions has enabled the size of pGreen to be kept to a minimum. Versions of pGreen have been used to transform several plant species with the same efficiencies as other binary Ti vectors. Information on the pGreen plasmid system is supplemented by an Internet site (http://www.pgreen.ac.uk) through which comprehensive information, protocols, order forms and lists of different pGreen marker gene permutations can be found.

A guide to Agrobacterium binary Ti vectors

Agrobacterium-based plasmid vectors allow the transformation of a wide range of plant species by capitalizing on a natural bacterial system to introduce DNA into the nuclear genome of plants. It is often a complex task to consider fully all the possible plasmid vectors and Agrobacterium strains available, and it can thus be difficult to take full advantage of these research tools. This practical guide is a survey of the many binary Ti plasmid vectors and Agrobacterium strains available, and aims to help researchers to make an informed decision about the system that is best suited to their needs...

The Introduction of Transgenes to Control Blackheart in Pineapple: Biolistics Vs Agrobacterium Transformation, in 'Contributing to a Sustainable Future'

Techniques for the introduction of transgenes to control blackheart by particle bombardment and Agrobacterium co-transformation have been developed for pineapple cv. Smooth Cayenne. Polyphenol oxidase (PPO) is the enzyme responsible for blackheart development in pineapple fruit following chilling injury. Sense, anti-sense and hairpin constructs were used as a means to suppress PPO expression in plants. Average transformation efficiency for biolistics was approximately 1% and for Agrobacterium was approximately 1.5%. These results were considered acceptable given the high regeneration potential of between 80-90% from callus cultures. Southern blot analysis revealed stable integration of transgenes with lower copy number found in plants transformed with Agrobacterium compared to those transformed by biolistics. Over 5000 plants from 55 transgenic lines are now undergoing field evaluation in Australia

Genetic transformation of Brassica nigra by agrobacterium based vector and direct plasmid uptake

Genetic transformation systems have been established for Brassica nigra (cv. IC 257) by using an Agrobacterium binary vector as well as by direct DNA uptake of a plasmid vector. Both the type of vectors carried nptII gene and gus gene. For Agrobacterium mediated transformation, hypocotyl tissue explants were used, and up to 33% of the explants produced calli on selection medium. All of these expressed B-glucuronidase gene on histochemical staining. Protoplasts isolated from hypocotyl tissues of seedlings could be transformed with a plasmid vector by FEG mediated uptake of vector DNA. A number of fertile kanamycin resistant plants were obtained using both the methods, and their transformed nature was confirmed by Southern blot analysis and histochemical staining for GUS. Backcrossed and selfed progenies of these transformed plants showed the presence of npt and gus genes.