So far, our marker-free technology has been tested in a limited number of plant species, including strawberry, apple and tobacco.
We have observed no differences in transformation efficiencies as compared to transformations using pBinplus (van Engelen et al., 1995), a pBin19 derivative, which we normally use in our transformation studies. In quite a number of transgenic events we have noticed transfer of binary vector backbone sequences to the plant genome (ranging from 10 to 50%). The transfer of vector backbone sequences is a common feature in Agrobacterium mediated transformation systems. The frequency we have observed is similar to that reported in literature (see for example De Buck et al., 2000).
Induction of recombinase and negative selection
For elimination of marker-DNA we have studied timing of induction of recombination activity and application of negative selection (see Figure 1).
For early induction of recombinase activity we have treated plant tissue with dexamethasone one or two months after the start of the transformation experiment. At this time-point no regenerants were visible yet. Together with the induction of recombinase activity the positive selection on kanamycin was stopped and induction of regeneration was continued under negative selective conditions (addition of 5-FC).
In the alternative late marker elimination strategy, first transgenic plants were regenerated under continuously positive selection pressure. Then in a secondary step shoot tissue (or other appropriate explant types with regeneration capacity) from these plants was treated with dexamethasone for induction of recombinase activity and subsequently secondary regenerants were induced under negative selection conditions (using 5-FC in the regeneration media).
Both early and late induction strategies resulted in the recovery of completely marker-free transgenic plants. The early induction strategy resulted in marker-free plants quite effectively, but recovery of marker-free plants was less efficient than for the late induction strategy. Depending on the exact timing of early induction (and finishing positive selection) a considerable number of non-transgenic escapes were obtained.The late induction strategy proved to be more efficient in recovery of completely marker-free transgenic plants. However, the longer period necessary for producing marker-free plants and the double regeneration step needed may render the late induction strategy less favorable.
Application of marker-free technology to Nicotiana tabacum resulted in an unexpectedly low number of marker-free plants. Transformation of pMF1 using the supervirulent Agrobacterium strain AGL0 resulted in high transformation efficiencies, but after induction of recombination (both early and late inductions) just a single marker-free transgenic plant was recovered. The possible integration of high T-DNA copy-number may have hampered marker-removal in this case. Alternatively, incomplete T-DNA transfer may have occurred, resulting in loss of one of the recombination-sites (Rs), which is located direct upstream of the left T-DNA border (LB). Loss of Rs prevents recombination. Recently, incomplete T-DNA transfer was found to be a common phenomenon in transgenic tomato (Thomas and Jones (2007), and the authors suggested that the specific mechanism leading to incomplete T-DNA integration may be a common feature to Solanum species. Improvement of application of clean vector technology to Nicotiana will be subject of future studies.
Optimization of dexamethasone treatment and negative selection on 5-FC
For the induction of recombinase activity we usually perform an overnight incubation of the plant tissue in liquid MS-medium (including sugar; no hormones or selective agents, but Agrobacterium-eliminating agents such as cefotaxime may be included) containing 10 µM dexamethasone. After overnight incubation the plant tissue is transferred to fresh regeneration medium supplemented with 1 µM dexamethasone and 5-FC.
We have tested the effect of dexamethasone concentrations ranging from 1-50 µM on the regeneration capacity of non-transformed tissue without observing a negative effect. However, overnight incubation in liquid medium caused extreme expansion and vitrification (hyperhydricity) of the Nicotiana leaf explants during subsequent culture.
For negative selection against tissue that is still containing marker sequences (cod A) we usually apply 5-FC at a concentration of 150 mg/l. Testing higher 5-FC concentrations showed that for Nicotiana concentrations up to 1000 mg/l 5-FC had no negative effect on the regeneration capacity of non-transgenic leaf explants. Strawberry seemed to be more sensitive to 5-FC than tobacco. At 250 mg/l 5-FC a normal regeneration capacity was observed, but at 500 mg/l 5-FC and higher concentrations regeneration was prevented. Remarkably, for both strawberry and tobacco low 5-FC concentrations (100-150 mg/l) had a stimulatory effect on the regeneration capacity.
Testing for absence of marker sequences
We have designed a series of primer pairs to screen putative marker-free plants for 1.) absence of Agrobacteria, 2.) absence of vector backbone, 3.) occurrence of the recombination step and 4.) complete removal of all undesired sequences. A sheet with primer sequences will be send upon request.
For the PCR-check for the occurrence of the recombination step the reverse primer is located just behind the inserted gene of interest. An additional forward gene-of-interest-specific primer has to be designed for each new gene sequence to be transferred (see figure 2). The reverse primer is positioned on the overlap of the Rs-site and the LB, which prevents this primer from annealing to the other Rs-site which is located just behind the gene of interest in non-recombined sequences.