GENERATION OF MARKER-FREE TRANSGENIC PLANTS
Transgenic technologies have enormous potential to improve crops of interest in a relatively precise way. However, the methods to introduce foreign DNA in a plant cell, either by Agrobacterium, microinjection, particle gun, or protoplast transformation, are relatively inefficient. For identifying those cells that have integrated the DNA into their genome, a selectable marker gene is co-introduced with the gene of interest. Approximately fifty different selection systems have been developed over the past several years. Despite the large number of systems, marker genes that confer resistance to the antibiotics kanamycin (nptII) and hygromycin (hpt) or the herbicide phosphinothricin (bar) have been used in most plant research and crop development techniques. Selection markers are not required in mature plants, especially when they are grown in fields. The European Union suggests avoiding the use of selectable markers in genetically engineered crops, and the ultimate goal is to introduce as few foreign sequences, in addition to the gene of interest, as possible. Moreover, the generation of marker-free transgenic plants responds not only to public concerns over the safety of genetically engineered (GE) crops, but supports multiple transformation cycles for transgene pyramiding.
Transformation without selection
De Vetten et al.1 reported transformation of potato without the use of selectable markers. The best results were obtained with the potato variety Karnico, using the Agrobacterium tumefaciens strain AGL0, which exhibits extremely high transformation efficiency because it contains a DNA region originating from a super virulent A. tumefaciens strain. In this experiment, approximately 5000 regenerated shoots were isolated and analyzed by PCR. Transgenic lines were obtained with an average frequency of 4.5%. However, vector backbone sequences, which are as undesirable and unacceptable as selectable markers, were transferred along with the gene of interest in 60 out of the 99 transgenic lines. Moreover, only 10 vector-free lines contained a single T-DNA insertion, which is another important criterion for commercialization of GM crops.
Marker elimination strategies
The simplest marker elimination strategy is the co-transformation of genes of interest with selectable marker genes followed by the segregation of the separate genes through sexual crosses. Co-transformation has been accomplished in a number of ways, including co-inoculation of plant cells with two Agrobacterium strains, each containing a simple binary vector, dual binary vector systems, and modified two-border Agrobacterium transformation vectors.2
2. Ipt selection
The isopentenyl transferase (ipt) gene that leads to cytokinine overproduction and results in transgenic shoots with abnormal shoot morphology can also be used as a selectable marker. In this case, appearance of normal-looking plants emerging from abnormal tissues indicates excision of the ipt gene, resulting in marker-free plants. Ipt selection was combined with a plant-derived T-DNA-like P-DNA fragment and used to generate marker- and backbone-free potato lines in a dual binary vector system with negative selection provided by codA against nptII marker gene integration. CodA is a conditionally lethal dominant gene encoding an enzyme that converts the non-toxic 5-fluorocytosine to cytotoxic 5-fluorouracil. Using this highly efficient way of selection hundreds of marker- and backbone-free Ranger Russet potato plants displaying reduced expression of a tuber-specific polyphenol oxidase gene were produced by Rommens et al.3
Efficient transformation systems using ‘shooter’ mutant Agrobacterium strains are also reported.4 These strains possess defective auxin-synthesis genes, but carry an intact ipt gene on the T-DNA of their own Ti plasmid that results in proliferation of transgenic cells and differentiation of adventitious shoots. Using a ‘shooter’ strain, regeneration on growth regulator-free media only occurs after successful infection of the plant tissues by agrobacteria. Furthermore, in a ‘shooter’ mutant / binary vector experiment, more than 60% of the transgenic lines proved to be ipt-free.4 Thus this system is a potentially useful alternative for marker-free gene transfer.
3. Recombination methods
This strategy is based on the use of site-specific recombinases, under the control of inducible promoters, to excise the marker genes. Successful use of the Cre/lox, FLP/FRT, or R/Rs systems has been reported in different plant species in which Cre, FLP, and R are the recombinases, and lox, FRT, and Rs are the recombination sites, respectively.5
Recently, a binary vector designated PROGMO was constructed to assess the potential of the Zygosaccharomyces rouxii R/Rs recombination system for generating marker- and vector backbone-free transgenic plants with high transgene expression and low copy number insertion.6 The PROGMO vector utilizes a constitutively expressed plant-adapted R recombinase and a codA-nptII bi-functional, positive/negative selectable marker gene. It carries only the right border (RB) of T-DNA and consequently the whole plasmid will be inserted as one long T-DNA into the plant genome. The Rs recognition sites are located at certain positions such that recombinase enzyme activity will recombine and delete both the bi-functional marker genes as well as the backbone of the binary vector, leaving only the gene of interest flanked by a copy of Rs and RB (Fig. 1).
The efficiency of PROGMO transformation was tested by introduction of the β-glucuronidase (GUS) reporter gene into potato. It was shown that after 21 days of positive selection and using 300 mgl-1 5-fluorocytosine for negative selection (Fig. 2), 29% of regenerated shoots carried only the GUS gene flanked by a copy of Rs and RB.
The PROGMO vector approach is simple and might be widely applicable for the production of marker- and backbone-free transgenic plants of many crop species.
References and further reading
1. de Vetten N et al. (2003) A transformation method for obtaining marker-free plants of a cross-pollinating and vegetatively propagated crop. Nat. Biotechnol. 21, 439-442
2. Miki B & McHugh S (2004) Selectable marker genes in transgenic plants: applications, alternatives and biosafety. J. Biotechnol. 107, 193-232
3. Rommens CM et al. (2004) Crop improvement through modification of the plant’s own genome. Plant Physiol. 135, 421-431
4. Bukovinszki A et al. (2006) Engineering resistance to PVY in different potato cultivars in a marker-free transformation system using a ‘shooter mutant’ A. tumefaciens. Plant Cell Rep. DOI 10.1007/s00299-006-0257-8
5. Hare PD & Chua N-H (2002) Excision of selectable marker genes from transgenic plants. Nat. Biotechnol. 20, 113-122
6. Kondrák et al. (2006) Generation of marker- and backbone-free transgenic potatoes by site-specific recombination and a bi-functional marker gene in a non-regular one-border Agrobacterium transformation vector. Transgenic Res. 15, 729-737
Ingrid M. van der Meer