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

/pdf/floral-dip-a-simplified-method-for-agrobacterium-mediated-4anwrgidds.pdf

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

Two genes, rd29A and rd29B, which are closely located on the Arabidopsis genome, are differentially induced under conditions of dehydration, low temperature, high salt, or treatment with exogenous abscisic acid (ABA). It appears that rd29A has at least two cis-acting elements, one involved in the ABA-associated response to dehydration and the other induced by changes in osmotic potential, and that rd29B contains at least one cis-acting element that is involved in ABA-responsive, slow induction. We analyzed the rd29A promoter in both transgenic Arabidopsis and tobacco and identified a novel cis-acting, dehydration-responsive element (DRE) containing 9 bp, TACCGACAT, that is involved in the first rapid response of rd29A to conditions of dehydration or high salt. DRE is also involved in the induction by low temperature but does not function in the ABA-responsive, slow expression of rd29A. Nuclear proteins that specifically bind to DRE were detected in Arabidopsis plants under either high-salt or normal conditions. Different cis-acting elements seem to function in the two-step induction of rd29A and in the slow induction of rd29B under conditions of dehydration, high salt, or low temperature.

/pdf/a-novel-cis-acting-element-in-an-arabidopsis-gene-is-s54v2xhkdn.pdf

A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress.

The coi1 mutation defines an Arabidopsis gene required for response to jasmonates, which regulate defense against insects and pathogens, wound healing, and pollen fertility. The wild-type allele, COI1, was mapped to a 90-kilobase genomic fragment and located by complementation of coi1-1 mutants. The predicted amino acid sequence of the COI1 protein contains 16 leucine-rich repeats and an F-box motif. It has similarity to the F-box proteins Arabidopsis TIR1, human Skp2, and yeast Grr1, which appear to function by targeting repressor proteins for removal by ubiquitination.

https://science.sciencemag.org/content/sci/280/5366/1091.full.pdf

COI1: An Arabidopsis Gene Required for Jasmonate-Regulated Defense and Fertility

http://www.biologie.uni-freiburg.de/data/bio3/laux/publications_files/Mayer1998.pdf

Role of WUSCHEL in Regulating Stem Cell Fate in the Arabidopsis Shoot Meristem

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.

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

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.

https://www.pnas.org/content/pnas/85/15/5536.full.pdf

Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection.

Isolation of genes expressed in specific tissues of Arabidopsis thaliana by differential screening of a genomic library.

The crucifer Arabidopsis thaliana has become widely used as a model system for plant molecular biology [1]. In this chapter we describe a simple and highly reproducible procedure for regeneration and transformation of Arabidopsis thaliana (L.) Heynh. [1, 2]. For this method the explant source is the root system of the plant.

Arabidopsis regeneration and transformation (Root Explant System)

The small, rapidly cycling crucifer Arabidopsis thaliana has been generally accepted as an experimental model to study plant molecular biology and developmental genetics (National Science Foundation 1990). One of the important contributions to turn this weed into a model has been the elaboration of an efficient transformation system. We developed a simple and highly reproducible tissue-culture procedure for transformation of Arabidopsis using Agrobacterium tumefaciens as the gene delivery system and the roots of axenically grown plants as the explant source (Valvekens et al. 1988). Arabidopsis root explants have a high potential for rapid shoot regeneration. Critical for roots to form shoots over their entire length, is a short preculture of maximum 7 days on 2,4-dichlorophenoxyacetic acid (2,4-D)-containing medium prior to culture on medium with high concentration of the cytokinin 6(y,ydimethylallylamino)purine (2-ip). We incorporated this efficient regeneration method with Agrobacterium infection and developed a selection procedure for the regeneration of transgenic Ti shoots, transformed with the neomycin phosphotransferase II (nptll) gene conferring resistance to the antibiotic, kanamycin. With this method, initially developed for the Arabidopsis ecotype C24, transgenic T2 seeds are routinely obtained within 3 months of tissue culture at high frequency.

Dirk Valvekens

Papers

Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection.

Isolation of genes expressed in specific tissues of Arabidopsis thaliana by differential screening of a genomic library.

Arabidopsis regeneration and transformation (Root Explant System)

Transformation of Arabidopsis thaliana Using Root Explants