Chlorophyll fluorescence assay for kanamycin resistance screening in transgenic plants

Abstract

The applicability of a chlorophyll fluorescence assay for kanamycin (Km) resistance screening in transgenic tobacco (Nicotiana tabacum) and Arabidopsis thaliana plants was investigated. In wild-type leaves incubated in the presence of 200 mg/l Km, a decrease in maximum variable fluorescence ((Fv)m) and a significant increase in constant fluorescence (Fo) were observed. Using (Fv)m/Fo as a screening parameter, we were able to distinguish Km-treated samples from untreated samples within 4 days. This parameter was applied to Km resistance screening using tobacco plants transformed with the nptII gene via Agrobacterium. Among 74 shoots selected on medium containing 200 mg/l Km, 37 plants were scored as Km sensitive by the chlorophyll fluorescence assay. These 74 scorings proved to be accurate, as reconfirmed by (1) polymerase chain reaction amplification of the transgene, (2) enzymatic assay of neomycin phosphotransferase and (3) leaf disc assay. Using the chlorophyll fluorescence assay, we could also screen 3-week old Arabidopsis plants carrying the nptII gene. These results clearly demonstrate the reliability and efficiency of this nondestructive assay for Km resistance screening of transgenic plants.