More ‘Friendly’ Selectable Markers: the Positive Selection Method
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 (1996) 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 effectiveselection of transgenic plan cells using D-xylose as the selection agent (Haldrup et al., 1998). 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.
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