Production of selectable marker-free transgenic tobacco plants using a non-selection approach: chimerism or escape, transgene inheritance, and efficiency

Plant Cell Rep. 2009 Mar;28(3):373-86. doi: 10.1007/s00299-008-0640-8. Epub 2008 Nov 19.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agrobacterium tumefaciens / genetics
  • Culture Media
  • Gene Transfer Techniques*
  • Genetic Vectors
  • Inheritance Patterns
  • Nicotiana / genetics*
  • Plant Shoots / genetics
  • Plants, Genetically Modified / genetics*
  • Transformation, Genetic*
  • Transgenes

Substances

  • Culture Media