In planta transformation EMBO COURSE Practical Course on Genetic and Molecular Analysis of Arabidopsis Module 5 In-planta Agrobacterium mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration and floral dip Nicole Bechtold and Georges Pelletier 5 - 1 In planta transformation 1. Introduction .......................................................................................................................3 1.1 Arabidopsis transformation ..........................................................................................3 1.2 Brief summary of T-DNA transfer ............................................................................3 2. In planta transformation methods.......................................................................................4 2.1. Growth conditions of the plant material before infiltration............................................4 2.2. Agrobacterium culture and preparation........................................................................4 2.3. Vacuum infiltration......................................................................................................5 2.4. Floral dipping ..............................................................................................................5 3. Screening of transformants.................................................................................................6 3.1. In the greenhouse ........................................................................................................6 3.2. In vitro........................................................................................................................6 4. Control of Arabidopsis pests..............................................................................................7 5. Materials............................................................................................................................7 5.1. Greenhouse materials ..................................................................................................7 5.2. Laboratory materials....................................................................................................8 5.2.1 Equipment.............................................................................................................8 5.2.2 Medium.................................................................................................................8 5.2.2.1. Agrobacterium culture medium......................................................................8 5.2.2.2. Infiltration medium ........................................................................................8 5.2.2.3. Floral dipping medium ...................................................................................9 5.2.2.4. Sterilisation solution ......................................................................................9 5.2.2.5. In vitro culture medium..................................................................................9 5.2.3. Plant materials ....................................................................................................10 5.2.4. Agrobacterium strains and vectors......................................................................10 References ...........................................................................................................................10 5 - 2 In planta transformation 1. Introduction 1.1 Arabidopsis transformation Plant genetic transformation was initiated and developed in the eighties thanks to the convergence of constant progress in (i) the protocols for regeneration from tissue culture; (ii) molecular techniques leading to well expressed marker genes after transfer in plant cells; and in (iii) the diversification of DNA delivery methods. Arabidopsis thaliana can be transformed through direct DNA uptake in protoplasts ( Damm et al, 1989) or after cocultivation of leaf or root explants with Agrobacterium (Valvekens et al, 1988)um It is also possible to transform this species by directly applying Agrobacterium to the plant and recovering transformants in the progeny. The first "in-planta" method was described by Feldmann and Marks in 1987 and consisted of the imbibition of seeds with Agrobacterium. Another whole plant transformation procedure is that of Hong-Gil Nam (1990) in which young inflorescences are cut off and the wounded surfaces are inoculated with Agrobacterium. These procedures offer two main advantages. Tissue culture and the resulting somaclonal variations are avoided and only a short time is required in order to obtain entire transformed individuals. However, the mean frequency of transformants in the progeny of such inoculated plants is relatively low and very variable. The infiltration method proposed later by Bechtold et al (1993) was based on the assumption that the stage at which the T-DNA transfer takes place with these methods is late in the development of the plant, either at the end of gametogenesis or at the zygote stage. This assumption was deduced from the observation that transformants are hemizygous for T-DNA insertions. The following protocol makes use of adult plants which are infiltrated with Agrobacterium at the reproduction stage. Each treated plant gives, on average, 10 transformants which can be selected from the progeny after four to six weeks (0.4% of the seeds). A new method based on the same principle was developed by Clough and Bent in 1998. They simplified the infiltration medium and substituted a surfactant for vacuum infiltration allowing penetration of the agrobacteria in the plant tissues, (Silwet L-77). The frequency of transformants obtained by this method is nearly the same as for infiltration: about 0.5% of the progeny of the treated plants. As with infiltration this frequency varies with experiments. The floral dip protocol is described afterthe vacuum infiltration protocol (Chap 2.4). 1.2 Brief summary of T-DNA transfer In Agrobacterium, the transferred DNA (T-DNA) is a defined part of the Tumor inducing plasmid (pTi for A. tumefaciens) and does not encode any genes important for the transfer process. These genes, the vir genes, are located elsewhere on the pTi or can be located on another plasmid and act in trans (binary vectors). The only cis-acting elements required during the transfer are two imperfect direct repeat sequences of 25 bp, the left border (LB) and the right border (RB). These sequences delimit the region of DNA to be excised from the pTi and can border any DNA sequence to be introduced into plant cells (Hooykaas and Schilperoort, 1992). The first step of the T-DNA transfer (Zupan and Zambryski, 1997; Tinland, 1996) is bacterium-plant cell recognition and the attachment of the bacterium to the plant cell. The genes required are located on the bacterial chromosome. The vir genes are then induced by the 5 - 3 In planta transformation presence of phenolic compounds (acetosyringones) produced by wounded plant cells. The constitutive VirA protein will be induced and activates the VirG protein which stimulates the expression of the other normally repressed vir genes. The VirD2 and VirD1 proteins excise the single-stranded T-DNA from the Ti plasmid. Upon this excision, VirD2 stays covalently attached to the 5'-end of the released DNA (RB); this molecule is then exported to the plant cell through a channel probably composed by VirB proteins. VirE2 proteins are associated with the single-stranded T-DNA and from part of the T-complex structure. VirD2 and VirE2 probably mediate the entry of the complex into the nucleus through the nuclear-pores. The exact mechanism of the integration of the T-DNA into the plant genome remains unknown. One model proposed by B. Tinland is that the 3'-end (LB or adjacent sequence) of the single- stranded T-DNA pairs with a short (5 bp) complementary sequence in the plant DNA, thus determining the point of intergration. The 5'-end (RB) will be ligated to the plant DNA near this region and after endonucleolytic digestion of the displaced plant DNA, the upper strand is extended by repair replication to copy the T-DNA. As a consequence, the sequence before the LB is poorly conserved whereas the sequence before the RB is frequently conserved up to the nucleotide that was attached to the VirD2 protein. 2. In planta transformation methods 2.1. Growth conditions of the plant material before infiltration 1. Prepare some plastic trays ( 20x30 cm) with compost and wet them moderately. 2. Sow 50 carefully separated seeds on the surface of the compost. 3. Place the trays at 4°C for 64h for stratification (optional). 3. Place the trays in the greenhouse (sixteen-hour day photoperiod, 15°C night and 20 to 25 °C day temperature with additional artificial light (105 mE/m2/s) and subirrigate with a layer of tap water under the trays, until germination. Afterwards water moderately for 4 to 6 weeks. Plants must be as vigorous as possible. A better development is observed when they are grown during the rosette stage under relatively short days (13 h) . It is also preferable to avoid etiolation by providing sufficient lighting. The optimal stage for infiltration is when the plants have the first siliques formed and the secondary floral stems are appearing. 2.2. Agrobacterium culture and preparation 1. Prepare a preculture by inoculating 10 ml of LB medium containing the appropriate antibiotics with 100 ml of a fresh culture or a glycerol stock, or with a colony taken from a dish. It is convenient to maintain (for 1 month) a sample of the most recent