Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana
TL;DR: The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.
Abstract: Summary The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA
Citations
More filters
TL;DR: A Gateway-compatible Agrobacterium sp.
Abstract: The current challenge, now that two plant genomes have been sequenced, is to assign a function to the increasing number of predicted genes. In Arabidopsis, approximately 55% of genes can be assigned a putative function, however, less than 8% of these have been assigned a function by direct experimental evidence. To identify these functions, many genes will have to undergo comprehensive analyses, which will include the production of chimeric transgenes for constitutive or inducible ectopic expression, for antisense or dominant negative expression, for subcellular localization studies, for promoter analysis, and for gene complementation studies. The production of such transgenes is often hampered by laborious conventional cloning technology that relies on restriction digestion and ligation. With the aim of providing tools for high throughput gene analysis, we have produced a Gateway-compatible Agrobacterium sp. binary vector system that facilitates fast and reliable DNA cloning. This collection of vectors is freely available, for noncommercial purposes, and can be used for the ectopic expression of genes either constitutively or inducibly. The vectors can be used for the expression of protein fusions to the Aequorea victoria green fluorescent protein and to the β-glucuronidase protein so that the subcellular localization of a protein can be identified. They can also be used to generate promoter-reporter constructs and to facilitate efficient cloning of genomic DNA fragments for complementation experiments. All vectors were derived from pCambia T-DNA cloning vectors, with the exception of a chemically inducible vector, for Agrobacterium sp.-mediated transformation of a wide range of plant species.
2,490 citations
Cites background from "Floral dip: a simplified method for..."
...Promoter-reporter constructs are frequently used to provide supporting evidence of the functional role of genes by identifying the likely spatial and temporal domains of the expression of a gene (Batni et al., 1996; Curtis et al., 1997)....
[...]
TL;DR: By cloning and characterizing an Arabidopsis defence-related gene (SID2) defined by mutation, it is shown that SA is synthesized from chorismate by means of ICS, and that SA made by this pathway is required for LAR and SAR responses.
Abstract: Salicylic acid (SA) mediates plant defences against pathogens, accumulating in both infected and distal leaves in response to pathogen attack. Pathogenesis-related gene expression and the synthesis of defensive compounds associated with both local and systemic acquired resistance (LAR and SAR) in plants require SA. In Arabidopsis, exogenous application of SA suffices to establish SAR, resulting in enhanced resistance to a variety of pathogens. However, despite its importance in plant defence against pathogens, SA biosynthesis is not well defined. Previous work has suggested that plants synthesize SA from phenylalanine; however, SA could still be produced when this pathway was inhibited, and the specific activity of radiolabelled SA in feeding experiments was often lower than expected. Some bacteria such as Pseudomonas aeruginosa synthesize SA using isochorismate synthase (ICS) and pyruvate lyase. Here we show, by cloning and characterizing an Arabidopsis defence-related gene (SID2) defined by mutation, that SA is synthesized from chorismate by means of ICS, and that SA made by this pathway is required for LAR and SAR responses.
2,089 citations
TL;DR: The identification of a new locus, FLS2, is described, which is ubiquitously expressed and encodes a putative receptor kinase and shares structural and functional homologies with known plant resistance genes and with components involved in the innate immune system of mammals and insects.
2,056 citations
TL;DR: The identification of JASMONATE-INSENSITIVE 3 (JAI3) and a family of related proteins named JAZ (jasmonate ZIM-domain), in Arabidopsis thaliana and the existence of a regulatory feed-back loop involving MYC2 and JAZ proteins, which provides a mechanistic explanation for the pulsed response to jasmonate and the subsequent desensitization of the cell.
Abstract: Jasmonates are essential phytohormones for plant development and survival. However, the molecular details of their signalling pathway remain largely unknown. The identification more than a decade ago of COI1 as an F-box protein suggested the existence of a repressor of jasmonate responses that is targeted by the SCF(COI1) complex for proteasome degradation in response to jasmonate. Here we report the identification of JASMONATE-INSENSITIVE 3 (JAI3) and a family of related proteins named JAZ (jasmonate ZIM-domain), in Arabidopsis thaliana. Our results demonstrate that JAI3 and other JAZs are direct targets of the SCF(COI1) E3 ubiquitin ligase and jasmonate treatment induces their proteasome degradation. Moreover, JAI3 negatively regulates the key transcriptional activator of jasmonate responses, MYC2. The JAZ family therefore represents the molecular link between the two previously known steps in the jasmonate pathway. Furthermore, we demonstrate the existence of a regulatory feed-back loop involving MYC2 and JAZ proteins, which provides a mechanistic explanation for the pulsed response to jasmonate and the subsequent desensitization of the cell.
1,991 citations
References
More filters
TL;DR: It was found that the gene 5 promoter is active in a tissue-specific fashion whereas this is not the case for the NOS promoter, providing the first documented instance of a gene derived from a procaryotic host the expression of which is apparently regulated by plant growth factors.
Abstract: A “plant gene vector cassette” to be used in combination with various Escherichia coli gene-cloning vectors was constructed. This cassette contains a replication and mobilization unit which allows it to be maintained and to be transferred back and forth between E. coli and Agrobacterium tumefaciens hosts provided these hosts contain plasmid RK2 replication and mobilization helper functions. The cassette also harbors a transferable DNA unit with plant selectable marker genes and cloning sites which can be combined with different bacterial replicons, thus facilitating the reisolation of transferred DNA from transformed plants in E. coli. The vector cassette contains two different promoters derived from the T-DNA-encoded genes 5 and nopaline synthase (NOS). By comparing the levels of expression of the marker enzymes linked to each of these promoter sequences, it was found that the gene 5 promoter is active in a tissue-specific fashion whereas this is not the case for the NOS promoter. This observation provides the first documented instance of a gene derived from a procaryotic host the expression of which is apparently regulated by plant growth factors.
2,064 citations
01 Jan 1993
TL;DR: In this article, a nouvelle methode de transformation in situ par Agrobacterium is presented, based on l'infiltration sous vide de plantes d'Arabidopsis par une souche d'Agrobacteria contenant un vecteur binaire.
Abstract: Nous presentons une nouvelle methode de transformation in situ par Agrobacterium. Cette approche est basee sur l'infiltration sous vide de plantes d'Arabidopsis par une souche d'Agrobacterium contenant un vecteur binaire. Les plantes caracterisees apres leur developpement presentaient des secteurs vegetatifs transformes parmi des secteurs non transformes. En moyenne jusqu'a 5 transformants par paire inoculee ont ete selectionnes dans la descendance des plantes infiltrees. Leur analyse genetique et moleculaire suggere que la transformation a lieu tardivement, lors du dveloppement floral, car tous les transformants sont hemizygotes et possedent des sites differents d'insertion de l'ADN-T [...]
1,974 citations
TL;DR: The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is finding wide use as a genetic marker that can be directly visualized in the living cells of many heterologous organisms as discussed by the authors.
Abstract: The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is finding wide use as a genetic marker that can be directly visualized in the living cells of many heterologous organisms. We have sought to express GFP in the model plant Arabidopsis thaliana, but have found that proper expression of GFP is curtailed due to aberrant mRNA processing. An 84-nt cryptic intron is efficiently recognized and excised from transcripts of the GFP coding sequence. The cryptic intron contains sequences similar to those required for recognition of normal plant introns. We have modified the codon usage of the gfp gene to mutate the intron and to restore proper expression in Arabidopsis. GFP is mainly localized within the nucleoplasm and cytoplasm of transformed Arabidopsis cells and can give rise to high levels of fluorescence, but it proved difficult to efficiently regenerate transgenic plants from such highly fluorescent cells. However, when GFP is targeted to the endoplasmic reticulum, transformed cells regenerate routinely to give highly fluorescent plants. These modified forms of the gfp gene are useful for directly monitoring gene expression and protein localization and dynamics at high resolution, and as a simply scored genetic marker in living plants.
1,412 citations
"Floral dip: a simplified method for..." refers methods in this paper
...gfp5-ER ( Haseloff et al., 1997 ) was used to probe blots of electrophoretically separated genomic DNA from kanamycin-resistant plants or kanamycin-sensitive controls....
[...]
...A DNA fragment from the nptII gene at the border of the T-DNA region of pBINmgfp5-ER (Haseloff et al., 1997) was used to probe blots of electrophoretically separated genomic DNA from kanamycin-resistant plants or kanamycin-sensitive controls....
[...]
TL;DR: The construction of new helper Ti plasmids for Agrobacterium-mediated plant transformation using T-DNA regions deleted using site-directed mutagenesis to yield replicons carrying thevir genes that will complement binary vectorsin trans.
Abstract: We describe the construction of new helper Ti plasmids forAgrobacterium-mediated plant transformation. These plasmids are derived from three differentAgrobacterium tumefaciens Ti plasmids, the octopine plasmid pTiB6, the nopaline plasmid pTiC58, and the L,L-succinamopine plasmid pTiBo542. The T-DNA regions of these plasmids were deleted using site-directed mutagenesis to yield replicons carrying thevir genes that will complement binary vectorsin trans. Data are included that demonstrate strain utility. The advantages ofAgrobacterium strains harbouring these ‘disamed’ Ti plasmids for plant transformation viaAgrobacterium are discussed.
1,360 citations
TL;DR: A transformation procedure for Arabidopsis root explants based on kanamycin selection was established and an Agrobacterium tumor-inducing Ti plasmid carrying a chimeric neomycin phosphotransferase II gene (neo) was introduced, resulting in transformed seed-producing plants obtained with an efficiency between 20% and 80% within 3 months after gene transfer.
Abstract: Culture conditions were developed that induce Arabidopsis thaliana (L.) Heynh. root cuttings to regenerate shoots rapidly and at 100% efficiency. The shoots produce viable seeds in vitro or after rooting in soil. A transformation procedure for Arabidopsis root explants based on kanamycin selection was established. By using this regeneration procedure and an Agrobacterium tumor-inducing Ti plasmid carrying a chimeric neomycin phosphotransferase II gene (neo), transformed seed-producing plants were obtained with an efficiency between 20% and 80% within 3 months after gene transfer. F(1) seedlings of these transformants showed Mendelian segregation of the kanamycin-resistance trait. The transformation method could be applied to three different Arabidopsis ecotypes. In addition to the neo gene, a chimeric bar gene conferring resistance to the herbicide Basta was introduced into Arabidopsis. The expression of the bar gene was shown by enzymatic assay.
1,316 citations
"Floral dip: a simplified method for..." refers background in this paper
...…of earlier in planta transformation methods (Chang et al., 1994; Feldmann and Marks, 1987; Feldmann, 1992; Katavic et al., 1994), offered a substitute for widely utilized Arabidopsis transformation methods that involved root tissue culture and plant regeneration (e.g. Valvekens et al., 1988)....
[...]