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Vectors and Agrobacterium Hosts for Arabidopsis Transformation

Adapted from "How to Transform Arabidopsis," Chapter 5, in Arabidopsis by Detlef Weigel and Jane Glazebrook. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2002.

INTRODUCTION

Arabidopsis can be stably transformed using Agrobacterium tumefaciens-mediated transfer of T-DNA. A. tumefaciens is a soil-dwelling bacterium that transforms normal plant cells into tumor-forming cells by inserting a piece of bacterial DNA (the transfer, or "T," DNA) into the plant cell genome. The T-DNA, which is flanked by left- and right-border (LB and RB) sequences, resides on a tumor-inducing (Ti) plasmid. The Ti plasmid also carries many of the transfer functions for mobilizing the T-DNA. This article provides a brief discussion of the principles of T-DNA transformation, including consideration of T-DNA vectors and their hosts.

RELATED INFORMATION

For protocols describing the transformation of Arabidopsis with Agrobacterium, see In Planta Transformation of Arabidopsis and Root Transformation of Arabidopsis.

T-DNA Vectors

Numerous T-DNA vectors (see Table 1) and bacterial hosts are now available and the choice among them will depend on the application. For an excellent discussion of the history of T-DNA vectors and comparison of many different systems, see the review by Hellens et al. (2000b).

The original T-DNA vectors were clumsy, requiring recombination of the foreign DNA with the resident Ti plasmid in Agrobacterium. It was later discovered that, with the exception of the T-DNA borders, the transfer functions of the Ti plasmid did not need to be present in cis. This discovery led to the development of the so-called binary vector systems, in which the Agrobacterium host contains a disarmed Ti plasmid. The disarmed plasmid encodes the transfer functions, but it does not harbor the T-DNA segment that will be transferred to the plant cell. Instead, the T-DNA resides on a separate plasmid, which is typically manipulated in Escherichia coli and then transferred to the Agrobacterium host by electroporation (see Transformation of Agrobacterium Using Electroporation) or by direct transformation (see Transformation of Agrobacterium Using the Freeze-Thaw Method). Previously, plasmids were mobilized by triparental mating, but this method is no longer common.

Older T-DNA vectors, such as pBIN19 (Bevan 1984), have largely fallen out of favor because of their low copy number in E. coli, which makes it difficult to obtain large amounts of DNA during various cloning steps. More recently developed vectors typically contain a high-copy-number origin of replication for E. coli. Another disadvantage of earlier vectors, such as pBIN19, is that the plant resistance marker is next to the right border. Because T-DNA transfer is directional, with the right border being transferred first, it is better to have the resistance marker next to the left border to ensure that resistant plants have received a complete (or nearly complete) copy of the T-DNA.

Additional considerations when choosing a vector include the resistance marker in bacteria, resistance marker in plants, the size of the vector, the presence of a lacZ -peptide-coding sequence surrounding the cloning site (for blue-white selection in E. coli), and finally the configuration of unique restriction sites available for cloning. The most common bacterial resistance markers are kanamycin, streptomycin or spectinomycin, gentamycin, and tetracycline. Because Agrobacterium strains typically contain resistance markers on the chromosome and/or the Ti plasmid (to select against other bacteria and for the Ti plasmid, respectively), care must be taken to use compatible vector/Agrobacterium combinations. Most Agrobacterium strains (C58C1 and GV3100 are exceptions) carry rifampicin resistance on the chromosome. GV3101 (pMP90) has gentamycin resistance on the Ti plasmid; GV3101 (pMP90RK) carries gentamycin and kanamycin resistance. Vectors that require selection for tetracycline resistance should not be used with GV3101, because mutants with resistance to 5 µg/ml tetracycline arise at very high frequency. Note that some T-DNA vectors contain only the replication origin (oriV) for Agrobacterium and that the replication functions must be provided in trans by the appropriate Ti helper plasmid. These vectors need a helper such as pMP90RK (not to be confused with pMP90).

With respect to plant resistance markers, several families of T-DNA vectors, such as the pSLJ (Jones et al. 1992), pPZP (Hajdukiewicz et al. 1994), pCAMBIA (http://www.cambia.org.au), and pGreen (Hellens et al. 2000a) series, include members conferring different resistances. This can often be convenient, because it allows retransformation of a plant that is already transgenic. The most widely used plant resistance markers are probably those for the antibiotic kanamycin (see Kanamycin Selection of Transformed Arabidopsis) and the herbicide phosphinothricin or glufosinate ammonium, better known by its trade names Basta and Finale. An advantage of the latter is that it can be used for selection of transgenic plants on soil (see Glufosinate Ammonium Selection of Transformed Arabidopsis).

We use, for example, derivatives of pCGN1578 (McBride and Summerfelt 1990) and of the pPZP series (Hajdukiewicz et al. 1994). We have found, however, that the cauliflower mosaic virus (CaMV) 35S promoter driving the resistance marker in the original pPZP vectors can lead to ectopic expression of the gene carried on the T-DNA, because the CaMV 35S promoter is right next to the multiple cloning site. This becomes a problem when predictable, tissue-specific transgene expression is required. This problem can be solved by replacing the promoter/resistance marker combination in pPZP with one from a different vector.

Agrobacterium Strains

Many of the older protocols used strain LBA4404, but this strain often does not appear to be virulent enough for the vacuum-infiltration method. Better strains are C58 derivatives such as GV3101 (pMP90), GV3101 (pMP90RK) (Koncz and Schell 1986), and AGL-1 (Lazo et al. 1991).

REFERENCES

Bevan M. 1984. Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res. 12: 8711–8721.[Abstract/Free Full Text]

Hajdukiewicz P., Svab Z., Maliga P. 1994. The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol. Biol. 25: 989–994.[Medline]

Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM. 2000a. pGreen: A versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol. Biol. 42: 819–832.[Medline]

Hellens R, Mullineaux P, Klee H. 2000b. A guide to Agrobacterium binary Ti vectors. Trends Plant Sci. 5: 446–451.[Medline]

Jones J.D., Shlumukov L., Carland F., English J., Scofield S.R., Bishop G.J., Harrison K. 1992. Effective vectors for transformation, expression of heterologous genes, and assaying transposon excision in transgenic plants. Transgenic Res. 1: 285–297.[Medline]

Koncz C. and Schell J. 1986. The promoter of the T_L-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet. 204: 383–396.

Lazo G.R., Stein P.A., Ludwig R.A. 1991. A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Bio/Technology 9: 963–967.[Medline]

McBride K.E. and Summerfelt K.R. 1990. Improved binary vectors for Agrobacterium-mediated plant transformation. Plant Mol. Biol. 14: 269–276.[Medline]