Production of selectable marker-free transgenic tobacco plants using a non-selection approach: chimerism or escape, transgene inheritance, and efficiency.
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.
Li
Baochun
B
Kentucky Tobacco Research and Development Center, University of Kentucky, Lexington, KY 40546, USA. bli2@uky.edu
Xie
Claire
C
Qiu
Hui
H
D016960:Agrobacterium tumefaciens / Q000235:genetics; D003470:Culture Media; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D040582:Inheritance Patterns; D018520:Plant Shoots / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics*; D014026:Nicotiana / Q000235:genetics*; D014170:Transformation, Genetic; D019076:Transgenes
D003470:Culture Media