Studies On Transformation Of Torenia Fournieri L. Using The PMI Gene As The Selectable Marker

It is well-known that transgene technology provides a powerful tool for developing traits that are otherwise difficult to achieve through conventional breeding. And we all know that the current protocol of transformation can deliver the gene to only a minor fraction of the treated cells, while the majority of the cells remain untransformed and need to be eliminated by selection procedures usually using the antibiotic or herbicide resistant genes as selectable markers. But our inadequate knowledge of just how antibiotic or herbicide resistant genes may affect the environment and human health, has raised widespread public concern and limited to extend the using of transgenic crops. The best way to solve this problem is using the safe marker genes in plant transformation. The object of this report is the use of the PMI gene as a safe mark in Agrobacteriyum-mediated transformation of Torenia, an important annual ornamental occurs in tropical and subtropical.Firstly, the Torenia regeneration system from leaf was optimized based on some previous results. The results conformed that MS + 6-BA 1.0 mg-L~-1 +2,4-D 0.1 mg L~-1, 1/2MS + 6-BA 1.0 mg-L~-1 + NAA 0.1 mg L~-1 and 1/2MS are the best media for callus induction, direct shoot induction from leaf explants and root induction of plantlet respectively, the induction frequencies were 90.91%, 90% and 84.21%. The regeneration system from root explants was also established, on 1/2MS contains 0.5-1.Smg L~-1 TDZ, highly efficient callus induction (100%) and shoot regeneration (100%) were obtained.To transform Torenia with PMI as selectable marker, the binary plasmid pHQMK was constructed by inserting the 1.2kb PMI coding region cloned from wild E.coli genome into pHQSN2 which has a CaMV35S promoter for PMI. The plasmid was introduced into A. tumefaciens EHA101 by freeze-thaw method.Based on the above regeneration system, further experiments were conducted to transform Torenia useing the PMI as safe marker. At first, the selection pressure for mannose were determined, then the explants of leaves segment from vitro-germinated plants of Torenia were transformed by EHAlOl containing the plasmid pHQMK. 18 mannose resistant plantlets were obtained, PCR southern blot and CPR assay showed that all the plants are transformed. The transformation efficiency rate ranged from 1% to 4%. These results proved that PMI can be used as an efficient and safe selectable maker for the transformation of flower plants.