How to transform Arabidopsis（拟南芥）

Arabidopsis can be stably transformed using Agrobacterium tumefaciens -mediated transfer of T-DNA. A breakthrough in Arabidopsis research was the invention of the vacuum-infiltration procedure, a simple and reliable method of obtaining transformants at high efficiency while avoiding the use of tissue culture.

Vectors and Agrobacterium hosts

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. 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 coil , and then transferred to the Agrobacterium host by electroporation or direct transformation. Previously, plasmids were mobilized by triparental mating, but this method has largely fallen out of use.

T-DNA vectors

Numerous T-DNA vectors 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 (2000a)

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 marker next to the left border to ensure that resistant plants have 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 lac Z α-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 (no 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. 2000b) 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 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.

We use, for example, derivatives of pCGN1578 (McBride and Sumerfelt 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.