Tetracycline-Based Binary Ti Vectors pLSU with Efficient Cloning by the Gateway Technology for <i>Agrobacterium tumefaciens</i>-Mediated Transformation of Higher Plants — Oak Academic Publishing
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Tetracycline-Based Binary Ti Vectors pLSU with Efficient Cloning by the Gateway Technology for <i>Agrobacterium tumefaciens</i>-Mediated Transformation of Higher Plants
Department of Plant Pathology and Crop Physiology, Louisiana State University and LSU AgCenter, Baton Rouge, USA;
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Department of Plant Pathology and Crop Physiology, Louisiana State University and LSU AgCenter, Baton Rouge, USA;
,
Department of Plant Pathology and Crop Physiology, Louisiana State University and LSU AgCenter, Baton Rouge, USA;
,
Department of Plant Pathology and Crop Physiology, Louisiana State University and LSU AgCenter, Baton Rouge, USA;
1 Department of Plant Pathology and Crop Physiology, Louisiana State University and LSU AgCenter, Baton Rouge, USA;
2 Department of Plant Pathology and Crop Physiology, Louisiana State University and LSU AgCenter, Baton Rouge, USA;
3 Department of Plant Pathology and Crop Physiology, Louisiana State University and LSU AgCenter, Baton Rouge, USA;
4 Department of Plant Pathology and Crop Physiology, Louisiana State University and LSU AgCenter, Baton Rouge, USA;
We constructed small high-yielding binary Ti vectors with a bacterial tetracycline resistance gene to facilitate efficient cloning afforded by the Gateway Technology (Invitrogen) for Agrobacterium tumefaciens -mediated transformation of higher plants. The Gateway Technology vectors are kanamycin-based, thus tetracycline-based destination and expression vectors are easily selected for the antibiotic resistance in the Escherichia coli media. We reduced the size of the tetracycline resistance gene Tet C from pBR322 to 1468 bp containing 1191 bp of the coding region, 93 bp of 5’-upstream, and 184 bp 3’-downstream region. The final size of binary Ti vector skeleton pLSU11 is 5034 bp. pLSU12 and 13 have the kanamycin resistance NPTII gene as a plant-selectable marker. pLSU13 and 15 contain the hygromycin resistance HPH gene as a selection marker. pLSU13 and 15 also have the β -glucuronidase ( GUS ) reporter gene in addition to the plant selection marker. We also constructed a mobilizable version of tetracycline-based binary Ti vector pLSU16 in which the mob function of ColE1 replicon was maintained for mobilization of the binary vector from E. coli to A. tumefaciens by tri-parental mating. The final size of binary Ti vector skeleton pLSU16 is 5580 bp. New tetracycline- based binary Ti vectors pLSU12 were found as effective as kanamycin-based vector pLSU2 in promoting a 10-fold increase in fresh weight yield of kanamycin-resistant calli after A. tumefaciens -mediated transformation of tobacco leaf discs. Using the Gateway Technology we introduced the plant-expressible GUS gene to the T-DNA of binary Ti vector pLSU12. Expression of the β -glucuronidase enzyme activity was demonstrated by histochemical staining of the GUS activity in transformed tobacco leaf discs.
Keywords<i>Agrobacterium tumefaciens</i>Binary Ti VectorsGateway TechnologypLSUTobacco Leaf Disk TransformationTetracycline Resistance
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