Glyphosate selection of gene amplification in suspension cultures of 3 plant species

Stepwise selection was carried out with increasing glyphosate concentration s to produce suspension cultures of Medicago sativa L. (alfalfa), Glycine m ax L. (Merr.) (soybean) and Nicotiana tabacum L. (tobacco) (two lines) that were at least 100-fold more resistant than the original culture as measure d by the I-50. The selection process required from 8 to I I transfers to fr esh medium over a total period from 161 to 312 days. The alfalfa and soybea n lines contained 62- and 21-fold higher activity levels of the glyphosate target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), respect ively. The tobacco lines had EPSPS enzyme activity levels more than 800-tim es higher than the original cultures. The EPSPS gene copy number and mRNA w ere increased in all of the lines as measured by southern and northern hybr idization, respectively. Thus, as has been found before with most glyphosat e-resistant suspension cultures, the resistance is caused by high EPSPS enz yme activity due to EPSPS gene amplification. Alfalfa and soybean EPSPS gen e amplification and the very high EPSPS enzyme activity increases found in the tobacco cultures have not been reported before. These studies show that EPSPS gene amplification can occur in many plant species to confer glyphos ate tolerance.