NONRECOMBINANT BACKGROUND IN GENE TARGETING - ILLEGITIMATE RECOMBINATION BETWEEN A HPT GENE AND A DEFECTIVE 5' DELETED NPTII GENE CAN RESTORE A KM(R) PHENOTYPE IN TOBACCO

Previously we have demonstrated gene targeting in plants after Agrobac terium-mediated transformation. In these initial experiments a transge nic tobacco line 104 containing a T-DNA insertion with a defective neo mycin phosphotransferase (nptII) gene was transformed with a repair co nstruct containing an otherwise defective nptII gene. Homologous recom bination between the chromosomally located target and the incoming com plementary defective nptII construct generated an intact nptII gene an d led to a kanamycin-resistant (Km(r)) phenotype. The gene targeting f requency was 1 x 10(-5). In order to compare direct gene transfer and Agrobacterium-mediated transformation with respect to gene targeting w e transformed the same transgenic tobacco line 104 via electroporation . A total of 1.35 x 10(8) protoplasts were transformed with the repair construct. Out of nearly 22 1000 transformed cells 477 Km(r) calli we re selected. Screening the Km(r) calli via PCR for recombination event s revealed that in none of these calli gene targeting had occurred. To establish the origin of the high number of Km(r) calli in which gene targeting had not occurred we analysed plants regenerated from 24 Km(r ) calli via PCR and sequence analysis. This revealed that in 21 out of 24 plants analysed the 5'-deleted nptII gene was fused to the hygromy cin phosphotransferase (hpt) gene that was also present on the repair construct. Sequence analysis of 7 hpt/nptII gene fusions showed that t hey all contained a continuous open reading frame. The absence of sign ificant homology at the fusion site indicated that fusion occurred via a process of illegitimate recombination. Therefore, illegitimate reco mbination between an introduced defective gene and another gene presen t on the repair construct or the chromosome has to be taken into accou nt as a standard byproduct in gene targeting experiments.