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At least 19 recordsLinked to original sources

PubMed2006

A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation.

BACKGROUND: The floral dip method of transformation by immersion of inflorescences in a suspension of Agrobacterium is the method of choice for Arabidopsis transformation. The presence of a marker, usually antibiotic- or herbicide-resistance, allows identification of transformed seedlings from untransformed seedlings. Seedling selection is a lengthy process which does not always lead to easily identifiable transformants. Selection for kanamycin-, phosphinothricin- and hygromycin B-resistance commonly takes 7-10 d and high seedling density and fungal contamination may result in failure to recover transformants. RESULTS: A method for identifying transformed seedlings in as little as 3.25 d has been developed. Arabidopsis T1 seeds obtained after floral dip transformation are plated on 1% agar containing MS medium and kanamycin, phosphinothricin or hygromycin B, as appropriate. After a 2-d stratification period, seeds are subjected to a regime of 4-6 h light, 48 h dark and 24 h light (3.25 d). Kanamycin-resistant and phosphinothricin-resistant seedlings are easily distinguished from non-resistant seedlings by green expanded cotyledons whereas non-resistant seedlings have pale unexpanded cotyledons. Seedlings grown on hygromycin B differ from those grown on kanamycin and phosphinothricin as both resistant and non-resistant seedlings are green. However, hygromycin B-resistant seedlings are easily identified as they have long hypocotyls (0.8-1.0 cm) whereas non-resistant seedlings have short hypocotyls (0.2-0.4 cm). CONCLUSION: The method presented here is an improvement on current selection methods as it allows quicker identification of transformed seedlings: transformed seedlings are easily discernable from non-transformants in as little as 3.25 d in comparison to the 7-10 d required for selection using current protocols.

Journal Article↗

PubMed2000

Female reproductive tissues are the primary target of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method.

The floral-dip method for Agrobacterium-mediated transformation of Arabidopsis allows efficient plant transformation without need for tissue culture. To facilitate use with other plant species, we investigated the mechanisms that underlie this method. In manual outcrossing experiments, application of Agrobacterium tumefaciens to pollen donor plants did not produce any transformed progeny, whereas application of Agrobacterium to pollen recipient plants yielded transformants at a rate of 0.48%. Agrobacterium strains with T-DNA carrying gusA (encoding beta-glucuronidase [GUS]) under the control of 35S, LAT52, or ACT11 promoters revealed delivery of GUS activity to developing ovules, whereas no GUS staining of pollen or pollen tubes was observed. Transformants derived from the same seed pod contained independent T-DNA integration events. In Arabidopsis flowers, the gynoecium develops as an open, vase-like structure that fuses to form closed locules roughly 3 d prior to anthesis. In correlation with this fact, we found that the timing of Agrobacterium infection was critical. Transformants were obtained and GUS staining of ovules and embryo sacs was observed only if the Agrobacterium were applied 5 d or more prior to anthesis. A 6-fold higher rate of transformation was obtained with a CRABS-CLAW mutant that maintains an open gynoecium. Our results suggest that ovules are the site of productive transformation in the floral-dip method, and further suggest that Agrobacterium must be delivered to the interior of the developing gynoecium prior to locule closure if efficient transformation is to be achieved.

Arabidopsis↗

PubMed2005

Production of transgenic crops by the floral-dip method.

The application of floral dipping toward the production of transformed plants has been rather limited. However, this procedure has enabled the successful production of transformed Medicago truncatula plants (a model plant for legume genetics) at efficiencies higher than those obtained by tissue culture methods. Indeed, this simple system, without requiring any knowledge of plant tissue culture, has been a breakthrough in the production of the first transgenic radish plants. This root crop is of major importance in the Far East, and the development of such a gene transfer system in radish has enabled agronomically important germplasms to be produced. Although the radish is closely related to Arabidopsis thaliana, it appears the two plants have different mechanisms of T-DNA transfer using floral dip. This chapter describes the simple system that has been adopted in the routine production of transgenic radish.

Agrobacterium tumefaciens↗

PubMed1998

Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.

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 gene tagging, positional cloning, or attempts at targeted gene replacement.

Arabidopsis↗

PubMed2006

Arabidopsis thaliana floral dip transformation method.

Arabidopsis floral dip transformation is notable for a number of reasons. First, it is strikingly simple to perform. Agrobacterium is applied to flowering Arabidopsis plants that subsequently set seed, and transgenic plants are then selected among the progeny seedlings. Because no plant tissue culture is required, somaclonal variation is avoided, and the procedure can be performed easily by nonspecialists. Success rates are high: it is common that 1% of the progeny seedlings are transgenic. The biology behind the method is interesting: Arabidopsis and some related Brassicaceae are apparently unique in allowing exogenously applied Agrobacterium to colonize the interior of developing ovaries, where female gametophyte cell lineages are transformed. The availability of the method has had a transformative effect on the overall practice of plant molecular biology, as the generation and analysis of large numbers of transgenic plants is now routine in hundreds of laboratories. The method has been exploited in a genomics context to make stable gene knockout plant lines for most Arabidopsis genes.

Agrobacterium tumefaciens↗

PubMed2001

Transgenic radish (Raphanus sativus L. longipinnatus Bailey) by floral-dip method--plant development and surfactant are important in optimizing transformation efficiency.

Transgenic radish (Raphanus sativus L. longipinnatus Bailey) plants were produced from the progeny of plants which were dipped into a suspension of Agrobacterium carrying both the beta-glucuronidase (gusA) gene and a gene for resistance to the herbicide Basta (bar) between T-DNA border sequences. The importance of development of the floral-dipped plant and presence of surfactant in the inoculation medium were evaluated in terms of transgenic plant production. Plants dipped at the primary bolt stage of growth, into a suspension of Agrobacterium containing 0.05% (v/v) Silwet L-77 resulted in optimum transformation efficiency, with 1.4% from 1110 seeds. The presence of Pluronic F-68 or Tween 20 in the inoculation medium was beneficial towards transgenic plant output compared to treatments without surfactant. Putative transformed T1 plants were efficiently selected by spraying with 0.03% (v/v) Basta and all herbicide-resistant plants tested positive for GUS activity when analysed both histochemically and fluorometrically. Southern analysis revealed that both the gusA and bar genes integrated into the genome of transformed plants and segregated as dominant Mendelian traits. These results demonstrate that radish can be genetically modified for the improvement of this important vegetable crop.

Aminobutyrates↗

PubMed2005

Floral dip: agrobacterium-mediated germ line transformation.

Many researchers use the flowering plant Arabidopsis thaliana to study gene function and basic plant biology. This easy-to-grow, small plant is ideal for genetic studies as it has a relatively simple genome compared to crop plants and its genetic material has been recently sequenced. Another very useful feature of Arabidopsis is that it is extremely simple to transform genetically. The ability to insert genes of interest stably into a given plant is essential to understand and verify gene function. Transformation is also a means of introducing specific traits that are difficult or impossible to introduce by conventional breeding techniques. This chapter provides detailed explanations on the floral dip protocol, a simple method to transform Arabidopsis by inoculating immature flowers with Agrobacterium tumefaciens.

Arabidopsis↗

PubMed2004

Genomic stability in Arabidopsis thaliana transgenic plants obtained by floral dip.

The occurrence of DNA modification is an undesired phenomenon accompanying plant cell transformation. The event has been correlated with the stress imposed by the presently utilised transformation procedures, all depending on plant differentiation from in vitro cell culture, but other causes have not been excluded. In this work, transgenic Arabidopsis thaliana plants have been produced by an approach that does not require cell dedifferentiation, being based on in planta Agrobacterium-mediated gene transfer by flower infiltration, which is followed by recovery and selection of transgenic progeny. Genomic DNA changes in transgenic and control plants have been investigated by AFLP and RAMP analysis. Results show no statistically relevant genomic modifications in transgenic plants, as compared with control untreated plants. Variations were observed in callus-derived A. thaliana plants, thus supporting the conclusion that somaclonal variation is essentially correlated with the stress imposed by the in vitro cell culture, rather than with the integration of a foreign gene.

Animals↗

PubMed2000

Floral spray transformation can efficiently generate Arabidopsis transgenic plants.

In this study, floral spray and floral dip were used to replace the vacuum step in the Agrobacterium-mediated transformation of a superoxide dismutase (SOD) gene into Arabidopsis. The transgene was constructed by using a CaMV 35S promoter to drive a rice cytosolic CuZnSOD coding sequence in Arabidopsis. The transgene construct was developed in binary vectors and mobilized into Agrobacterium. When Arabidopsis plants started to initiate flower buds, the primary inflorescence shoots were removed and then transformed by floral spray or floral dip. More than 300 transgenic plants were generated to assess the feasibility of floral spray used in the in planta transformation. The result indicates that the floral spray method of Agrobacterium can achieve rates of in planta transformation comparable to the vacuum-infiltration and floral dip methods. The floral spray method opens up the possibility of in planta transformation of plant species which are too large for dipping or vacuum infiltration.

Arabidopsis↗

PubMed2002

Expression of an antisense GIGANTEA (GI) gene fragment in transgenic radish causes delayed bolting and flowering.

A late-flowering transgenic radish has been produced by the expression of an antisense GIGANTEA (GI) gene fragment using a floral-dip method. Twenty-five plants were dipped into a suspension of Agrobacterium carrying a 2.5 kb antisense GI gene fragment from Arabidopsis, along with the gusA and bar reporter genes, all under the control of a CaMV 35S promoter. From a total of 1462 seeds harvested from these floral-dipped plants, 16 Basta-resistant T1 plants were found to have GUS activity (transformation efficiency of 1.1%). Southern analysis confirmed the integration of one or two copies of the gusA gene in these herbicide-resistant plants. Expression of the GI gene in T1 plants was much reduced compared to both wildtype plants and plants transformed with pCAMBIA3301 (positive control). In the progenies of eleven T1 plants analysed (T2 generation), all lines showed a significant delay in both bolting and flowering times compared to wildtype and positive control plants, and that, the level of GI transcript was inversely proportional to the time of bolting and flowering. At a maximum, bolting and flowering times were delayed by 17 and 18 days respectively, compared to wildtype plants (in positive control plants, the delay was 23 and 26 days, respectively). Ten of the 11 lines exhibited a significant reduction in plant height compared to wildtype and positive control plants. This study provides evidence that down-regulation of the GI gene by co-suppression could delay bolting in a cold-sensitive long-day (LD) plant. Production of late-flowering germplasms of radish may allow this important crop to be cultivated over an extended period and also provide further food to the famine countries of S/E Asia.

Agrobacterium tumefaciens↗

PubMed2003

Seed-expressed fluorescent proteins as versatile tools for easy (co)transformation and high-throughput functional genomics in Arabidopsis.

We demonstrate that fluorescent proteins can be used as visual selection markers for the transformation of Arabidopsis thaliana by the floral dip method. Seed-specific expression of green fluorescent protein (GFP) variants, as well as DsRed, permits the identification of mature transformed seeds in a large background of untransformed seeds by fluorescence microscopy. In planta visualization of transformed seeds in siliques shows that susceptibility to floral dip transformation is limited to a small, defined window in flower development. In the competent stage, the random transformation of up to 25% of the seeds within a single silique may occur. The use of fluorescent proteins with different spectral characteristics allows a rapid identification and genetic analysis of seeds that have received multiple genes-of-interest in co-transformation experiments. The data reveal that co-transformation does not occur at random, since the co-transformed genes are integrated at a single genetic locus in approximately 70% of the cases. This genetic linkage of the co-transformed genes greatly simplifies metabolic pathway engineering by reverse genetics in Arabidopsis. Additional advantages of using visual selection instead of antibiotic resistance include a rapid identification of the effect of the T-DNA insertion or the transgene on seed development and/or germination. This technology, of tagging and identifying transformed seeds by fluorescence provides a novel high-throughput screening system with many potential applications in plant biotechnology.

Journal Article↗

PubMed2026

Optimization of functional genetics tools for a model tetraploid Capsella bursa-pastoris, with focus on homoeolog-aware gene editing.

Capsella bursa-pastoris is a recent allotetraploid and a promising model for studying early consequences of polyploidy. One of the intriguing questions in polyploid research is how new functions arise from initially identical or nearly identical homoeologous genes. Functional genetics tools, including genetic editing, can help to understand this process, but they have not been developed for C. bursa-pastoris yet. We present here the results of our study aimed at filling this gap. In particular, we compared the efficiency of floral dip transformation in six accessions of C. bursa-pastoris representing distant populations. The Asian clade accession PGL0025 had the highest efficiency of transformation (~ 1.1%). Comparison of Agrobacterium tumefaciens strains EHA105 and GV3101 (pMP90) showed that the latter is more effective. Also, we created a genome-wide gRNA database for all pairs of homoeologs of the PGL0001 accession of C. bursa-pastoris and integrated it into publicly available genome browser: https://t2e.online/igv_capsella_bursa-pastoris/ . We assessed the possibility of differential editing for two pairs of homoeologous genes with high sequence similarity (> 90%) both in vitro and in silico. Despite the test results that indicated off-target activity, we have succeeded in obtaining lines of plants with homozygous frameshift mutations in each of the homoeologs separately in vivo. We expect that these findings and resources will promote the use of C. bursa-pastoris as a model in functional genetics experiments, in particular, the studies of the fate of duplicated gene after polyploidization event.

Capsella↗

PubMed2007

Plant native tryptophan synthase beta 1 gene is a non-antibiotic selection marker for plant transformation.

Gene transformation is an integral tool for plant genetic engineering. All antibiotic resistant genes currently employed are of bacterial origin and their presence in the field is undesirable. Therefore, we developed a novel and efficient plant native non-antibiotic selection system for the selection of transgenic plants in the model system Arabidopsis. This new system is based on the enhanced expression of Arabidopsis tryptophan synthase beta 1 (AtTSB1) and the use of 5-methyl-tryptophan (5MT, a tryptophan [Trp] analog) and/or CdCl2 as selection agent(s). We successfully integrated an expression cassette containing an AtT-SB1 cDNA driven by a cauliflower mosaic virus 35S promoter into Arabidopsis by floral dip transformation. Transgenic plants were efficiently selected on MS medium supplemented with 75 microM 5MT or 300 microM CdCl2 devoid of antibiotics. TSB1 selection was as efficient as the conventional hygromycin selection system. Northern blot analysis of transgenic plants selected by 5MT and CdCl2 revealed increased TSB1 mRNA transcript whereas uneven transcript levels of hygromycin phosphotransferase II (hpt) (control) was observed. Gas chromatography-mass spectrometry revealed 10-15 fold greater free Trp content in AtT-SB1 transgenic plants than in wild-type plants grown with or without 5MT or CdCl2. Taken together, the TSB1 system provides a novel selection system distinct from conventional antibiotic selection systems.

Arabidopsis↗

PubMed2003

The noble radish: past, present and future.

Recent developments in plant biotechnology have revealed that radish can be genetically modified by a technique called "floral-dipping". This system has been used successfully to delay both bolting and flowering in radish by the co-suppression of the photoperiodic gene, GIGANTEA. Future research could use this system to improve the pharmaceutical value of the crop for global usage.

Arabidopsis Proteins↗

PubMed2001

Germ-line transformation of Arabidopsis lasiocarpa.

In planta transformation methods have opened up the possibility of transforming plant species for which no regeneration protocols currently exist. In this study, the suitability of the germ-line transformation method developed for Arabidopsis thaliana was examined for four taxa in the Brassicaceae that have not been previously transformed: Arabidopsis griffithiana, Arabidopsis lasiocarpa, Arabidopsis petraea and Capsella bursa-pastoris. Numerous transformants were obtained for A. lasiocarpa. Transformation of A. lasiocarpa was confirmed at the phenotypic and molecular levels for stably transformed lines and for backcrossed lines segregating the T-DNA insert. Parameters affecting transformation efficiency of A. lasiocarpa were also explored. As with A. thaliana, sucrose and surfactant in the inoculation medium are required for high levels of transformation, although the suitable concentrations of these are different for A. lasiocarpa. Other components present in earlier versions of the inoculation medium had little effect on transformation efficiency. Vacuum infiltration (rather than simple floral dipping) led to higher rates of transformation and did not seriously affect seed production in A. lasiocarpa. Identification of species susceptible to germ-line transformation will aid in determining the factors important for applying this technology to more recalcitrant species.

Arabidopsis↗

PubMed2003

Transgene-induced RNA interference: a strategy for overcoming gene redundancy in polyploids to generate loss-of-function mutations.

Gene redundancy in polyploid species complicates genetic analyses by making the generation of recessive, loss-of-function alleles impractical. We show that this problem can be circumvented using RNA interference (RNAi) to achieve dominant loss of function of targeted genes. Arabidopsis suecica is an allotetraploid (amphidiploid) hybrid of A. thaliana and A. arenosa. We demonstrate that A. suecica can be genetically transformed using the floral dip method for Agrobacterium-mediated transformation. Transgenes segregate as in a diploid, indicating that chromosome pairing occurs exclusively (or almost so) among homologs and not among homeologs. Expressing a double-stranded (ds) RNA corresponding to the A. thaliana gene, decrease in DNA methylation 1 (DDM1) caused the elimination of DDM1 mRNAs and the loss of methylation at both A. thaliana- and A. arenosa-derived centromere repeats. These results indicate that a single RNAi-inducing transgene can dominantly repress multiple orthologs.

Arabidopsis↗

PubMed2004

Salt cress. A halophyte and cryophyte Arabidopsis relative model system and its applicability to molecular genetic analyses of growth and development of extremophiles.

Salt cress (Thellungiella halophila) is a small winter annual crucifer with a short life cycle. It has a small genome (about 2 x Arabidopsis) with high sequence identity (average 92%) with Arabidopsis, and can be genetically transformed by the simple floral dip procedure. It is capable of copious seed production. Salt cress is an extremophile native to harsh environments and can reproduce after exposure to extreme salinity (500 mm NaCl) or cold to -15 degrees C. It is a typical halophyte that accumulates NaCl at controlled rates and also dramatic levels of Pro (>150 mm) during exposure to high salinity. Stomata of salt cress are distributed on the leaf surface at higher density, but are less open than the stomata of Arabidopsis and respond to salt stress by closing more tightly. Leaves of salt cress are more succulent-like, have a second layer of palisade mesophyll cells, and are frequently shed during extreme salt stress. Roots of salt cress develop both an extra endodermis and cortex cell layer compared to Arabidopsis. Salt cress, although salt and cold tolerant, is not exceptionally tolerant of soil desiccation. We have isolated several ethyl methanesulfonate mutants of salt cress that have reduced salinity tolerance, which provide evidence that salt tolerance in this halophyte can be significantly affected by individual genetic loci. Analysis of salt cress expressed sequence tags provides evidence for the presence of paralogs, missing in the Arabidopsis genome, and for genes with abiotic stress-relevant functions. Hybridizations of salt cress RNA targets to an Arabidopsis whole-genome oligonucleotide array indicate that commonly stress-associated transcripts are expressed at a noticeably higher level in unstressed salt cress plants and are induced rapidly under stress. Efficient transformation of salt cress allows for simple gene exchange between Arabidopsis and salt cress. In addition, the generation of T-DNA-tagged mutant collections of salt cress, already in progress, will open the door to a new era of forward and reverse genetic studies of extremophile plant biology.

Abscisic Acid↗

PubMed2004

Characterization of the Arabidopsis lysine-rich arabinogalactan-protein AtAGP17 mutant (rat1) that results in a decreased efficiency of agrobacterium transformation.

Arabinogalactan-proteins (AGPs) are a family of complex proteoglycans widely distributed in plants. The Arabidopsis rat1 mutant, previously characterized as resistant to Agrobacterium tumefaciens root transformation, is due to a mutation in the gene for the Lys-rich AGP, AtAGP17. We show that the phenotype of rat1 correlates with down-regulation of AGP17 in the root as a result of a T-DNA insertion into the promoter of AGP17. Complementation of rat1 plants by a floral dip method with either the wild-type AGP17 gene or cDNA can restore the plant to a wild-type phenotype in several independent transformants. Based on changes in PR1 gene expression and a decrease in free salicylic acid levels upon Agrobacterium infection, we suggest mechanisms by which AGP17 allows Agrobacterium rapidly to reduce the systemic acquired resistance response during the infection process.

Arabidopsis↗