More 'friendly' selectable markers: the positive selection method

In some instances there are disadvantages in using antibiotic or herbicide resistant genes in a selection system, such as toxicity or allergenicity of the gene product and interference with antibiotic treatment. [72, 100] Other problems are linked to the capacity for cross-fertilization of some domestic crop species with wild varieties. Oat, for instance, is cross-fertile with wild oat species and transference of phosphinothricin resistance from transgenic oat to weedy wild oats has been reported. [30] The concerns are that phosphinothricin-resistant wild oat would eliminate control of wild oats using phosphinothricin and compromise the usefulness of transgenic crops resistant to this herbicide such as wheat. [34] Therefore, the use and release of selectable genes into the environment has been the cause of concern among environmental authorities. While many of such concerns may prove unfounded [101] they may nevertheless lead to governmental restrictions on the use of selectable genes in transgenic plants, and it is therefore desirable to develop new selection methods.

In contrast to the traditional selection where the transgenic cells acquire the ability to survive on selective media while the non-transgenic cells are killed (negative selection), the positive selection method, first developed by Joersbo and Okkels, [102] favors regeneration and growth of the transgenic cells while the non-transgenic cells are starved but not killed. The positive selection method exploits the fact that cytokinin must be added to plant explants in order to obtain optimal shoot regeneration rates. By adding cytokinin as an inactive glucuronide derivate, cells which have acquired the GUS gene by transformation are able to convert the cytokinin glucuronide to active cytokinin while untransformed cells are arrested in development. In this system, GUS serves the dual purpose of being both a selectable and screenable marker gene. Another interesting system of positive selection uses the xylose isomerase gene from Thermoanaerobacterium thermosulforogenas as a selectable gene, which expression allows effective selection of transgenic plan cells using D-xylose as the selection agent. [103] The transformation frequencies obtained by positive selection appear to be higher than using the negative selection method. This could be related to the fact that during negative selection the majority of the cells in the explants die. Such dying cells may release toxic substances which in turn may impair regeneration of the transformed cells. In addition, dying cells may form a barrier between the medium and the transgenic cells preventing uptake of essential nutrients.