A large variety of binary vectors for plant transformation have been described. Among them the pPZP series of vectors [6
] and the derived pCAMBIA vectors [7
] are especially popular. A prominent feature of these vectors is their stability in bacteria, the high copy number, and the relatively small size. One disadvantage was the use of the CaMV promoter for the plant selectable marker and the use of spectinomycin and chloramphenicol as selectable markers for bacteria which led us to construct the vector pPZP3425 [8
]. In this vector the CaMV35S promoter for the plant selectable marker (kanamycin) was replaced by the weaker nos
promoter and a kanamycin resistance gene for selection in bacteria was included in the vector backbone.
Although pPZP3425 proved useful for our purposes in producing promoter:: GUS
lines (by replacing the CaMV promoter in the GUS
cassette with a promoter of interest) or overexpression lines (by replacing GUS
in the GUS
cassette with a gene of interest) [8
], selection of homozygous transgenic lines with a strong expression of the gene of interest was still a lengthy procedure. Since we were interested to produce a large number of transgenic Arabidopsis overexpression lines with a strong expression level for putative antimicrobial peptides we set out to construct a pPZP vector that would have three important features: First, it should allow us to use selection or screening on soil to avoid growing Arabidopsis under sterile conditions. Second, we wanted to easily select lines with a strong expression level. Third, the vector should be used for the expression in syncytia using specific promoters that would not be active in seedlings or leaves.
The first precondition excluded the use of antibiotic resistance such as kanamycin or hygromycin or the use of metabolite resistance genes such as the E. coli-
derived phosphomannose isomerase which allows growth on mannose [25
] or the Streptomyces rubiginosus
xylose isomerase (xylA
) gene which allows growth on xylose [26
] as a plant selectable marker for which sterile conditions would have to be used. Herbicide resistance would avoid the need for sterile growth but all T1 seedlings have to be grown for selection. In contrast, if using a fluorescent marker that is expressed in seeds, only the transgenic seeds obtained after transformation would have to be grown further and no additional treatment would be needed. Such an approach is especially important if a large number of transgenic lines have to be produced. An example is the work of [27
] who conducted a high-throughput screen in Arabidopsis for castor genes that would lead to changes in hydroxy fatty acid composition in seeds. We decided to try the DsRed gene as a screenable marker [13
]. This leaves the possibility to use in addition GFP or other most frequently used fluorescent markers as reporter genes. Furthermore, DsRed has a rather weak fluorescence which might seem to be a disadvantage. However, this weak fluorescence makes it easier to identify seeds with a different level of fluorescence.
Expression of the gene of interest varies largely in independent transgenic lines [24
] due mainly to position effects. One way of reducing this variation is the inclusion of matrix attachment regions in the T-DNA. However, this only worked with gene silencing mutants, which limits the use of these vectors [28
]. We have demonstrated here that the use of the vector pMAA-Red allows an easy and efficient selection of transgenic lines with a strong expression of the gene of interest in wild-type Arabidopsis plants. Of course, mutants could also be used, if needed. Pre-selection of lines with a strong DsRed expression in seeds according to their fluorescence reduces the number of lines that have to be tested at the transcript or protein level.
Our third precondition was that the vector should allow the use of syncytium-specific promoters instead of the CaMV promoter which is active in most tissues of Arabidopsis plants. We have recently shown that several genes are expressed in syncytia which are normally expressed in pollen [23
], such as MIOX4
, or in seeds, such as Pdf2.1
whose promoter is used here to drive the expression of DsRed in seeds. Using the MIOX4
promoter would allow a specific expression in syncytia which could be useful for genes with a negative effect on plant growth. However, screening such transgenic lines would require the analysis of expression of the gene of interest in syncytia. Such a screening is very time consuming because syncytia have to be cut out from infected roots. In this case a pre-screening that would reduce the number of lines would lead to a significant reduction of time and effort. Thus, the pMAA-Red vector that we have constructed is especially useful for the syncytium-specific expression of transgenes or for similar cases where the expression of the transgene would be restricted to tissues that could not be easily screened. Promoter::reporter constructs and overexpression lines can be produced from this vector as from pPZP3425 [8
]. The CaMV promoter can be replaced by a promoter of interest using NcoI and one of the unique sites in the polylinker. The GUS sequence can be replaced with a sequence for overexpression of a gene of interest by using NcoI and BamHI. If the sequence to be cloned contains NcoI or BamHI site, it is usually possible to use a restriction enzyme that produces compatible cohesive ends with NcoI and BamHI. For instance, BspHI, PciI, and FatI produce cohesive ends that are compatible with NcoI.
After transformation of Arabidopsis plants it takes about 3–4
weeks until T1 seeds can be harvested and inspected for fluorescent seeds. A big advantage of transformation with the vector pMAA-Red is that only these selected seeds have to be grown on soil to produce the T2 generation. After another 4
weeks, the first siliques of these plants can be screened for a 3:1 segregation of fluorescent seeds which can then be used to produce homozygous T3 seeds for further analysis. Again, the first siliques of these plants can be used to select homozygous lines and only those will be grown for maturity, which will take a total of 6 to 8
weeks. Depending on the growth conditions, the whole procedure from transformation to harvesting homozygous seeds could be completed within four month.