Characterization of putative germline-specific promoters from Arabidopsis thaliana and their application in DNA modification strategies

PhD thesis A big challenge in Plant Biotechnology – both for the scientific community and the industry – is to develop strategies that allow the removal of selectable marker genes and the introduction of targeted modifications in genomic sequences (gene targeting). In this manuscript such strategies, based on the activation of a recombinase in the germline of Arabidopsis thaliana, are described. The removal of the selectable marker in the germline cells has the advantage that additional handling can be avoided, limiting the extra work to the bare minimum. Furthermore, a gene targeting strategy based on the introduction of DNA modifications in germline cells such as meiocytes has the benefit that the system is activated when the plant cells favor homologous recombination over non-homologous recombination which could increase gene targeting efficiencies. First, a set of Arabidopsis thaliana promoters were tested for their ability to activate a site-specific recombination system (e.g. CRE/lox) in the germline. Promoter fragments of the SOLO DANCERS (SDS), APETALA1 (AP1) and SUPERMAN (SUP) genes showed the highest efficiency of the promoters that were tested in the frame of this work. The promoter fragment of the SDS gene conferred functionality in one germline; 80% of the progeny contained one recombined allele. The promoter fragment of the AP1 gene was functional in the common germline; 84% of the progeny plants contained a recombined allele (32% one recombined allele, 52% two recombined alleles). Five promoter fragments of the SUP gene were evaluated. Three of them (SUP, SA and SCAN) resulted in functionality in both germlines. The SUP, SA and SCAN promoter fragments resulted in progeny plants of which 90%, 82% and 80% contained a recombined allele, respectively. The amount in which both alleles were excised varied: 59%, 35% and 24% respectively. Subsequently, the AP1 and SDS promoters were used to develop an efficient gene marker removal system. By introducing a germline-specific auto-excision (GSA) construct containing a cre recombinase gene under control of a germline-specific promoter, transgenic plants become programmed to lose their selectable marker after selection of primary transformants. Two modules of this genetic program were developed. In the first module, a promoter functional in both germlines was used (AP1 promoter). In the second module, a promoter functional in one germline was used (SDS promoter). Germline excision was observed in 10 out of 12 single-locus lines (83%) by using the AP1 promoter and in 12 out 12 single-locus lines (100%) by using the SDS promoter. Within the independent lines varying efficiencies from relatively low to high were observed. Furthermore, the SDS promoter from A. thaliana was also successfully used in N. tabacum. Finally, the SDS and AP1 promoters were also used in the gene targeting strategy in which a donor molecule was placed between two tandemly repeated lox sites and became activated upon expression of the cre gene which was placed under control of the SDS or AP1 promoter. Recombination events were obtained. However, at this point it is not shown that the target locus was altered. Therefore we can not exclude the possibility that ectopic GT events were detected.