Production of selectable marker-free transgenic tobacco plants using a non-selection approach: chimerism or escape,transgene inheritance,and efficiency |
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Authors: | Baochun Li Claire Xie Hui Qiu |
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Affiliation: | (1) Kentucky Tobacco Research and Development Center, University of Kentucky, 105C, Lexington, KY 40546, USA |
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Abstract: | Public concern and metabolic drain were the main driving forces for the development of a selectable marker-free transformation system. We demonstrated here the production of transgenic tobacco plants using a non-selection approach by Agrobacterium tumefaciens-mediated transformation. A. tumefaciens-infected leaf explants were allowed to produce shoots on a shoot induction medium (SIM) containing no selective compounds. Up to 35.1% of the A. tumefaciens-infected leaf explants produced histochemically GUS+ shoots, 3.1% of regenerated shoots were GUS+, and 72% of the GUS+ shoots were stably transformed by producing GUS+ T1 seedlings. When polymerase chain reaction (PCR) was used to screen the regenerated shoots, 4% of the shoots were found to be PCR+ for the transgene and 65% of the PCR+ shoots were stable transformants. Also, generation of PCR+ escapes decreased linearly as the number of subculture increased from one to three on SIM containing the antibiotic that kills the Agrobacterium. Twenty-five to 75% of the transformants were able to transmit transgene activity to the T1 generation in a Mendelian 3:1 ratio, and a transformation efficiency of 2.2–2.8% was achieved for the most effective binary vector. These results indicated that majority of the GUS+ or PCR+ shoots recovered under no selection were stable transformants, and only one-third of them were chimeric or escapes. Transgenes in these transgenic plants were able to transmit the transgene into progeny in a similar fashion as those recovered under selection. |
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Keywords: | Nicotiana tobacum L. Marker-free transgenic plants Non-selection GUS histochemical assay PCR Agrobacterium tumefaciens-mediated transformation Chimerism Escapes Transgene inheritance Transformation efficiency |
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