Immature Pollen as a Target for Gene Targeting
Immature pollen grains were used for gene targeting (GT) to evaluate the potential of higher plant male gametophyte as a target for gene targeting experiments. The artificial B18/4 target locus inserted to tobacco genome has been used to assess gene targeting in tobacco mid-bi-cellular pollen. In this system, a neomycin-phosphotransferase (nptII) gene, exclusively expressed in seeds, was converted into a constitutive nptII gene by insertion of the CaMV 35S promotor between the HMW seeds-specific promotor and a functional restored nptII gene at the target locus. The tobacco mid-bi-cellular pollen isolated from the B18/4 target locus plants was transformed by the biolistic approach with the repair construct. GT experiments led to the recovery of seven kanamycin resistant plants. Southern analysis confirmed that in one transgenic line an ectopic GT event occurred arising by modification of the repair construct by the target locus and subsequent integration elsewhere in the tobacco genome.
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