A rapid method for qualitative assay of both neomycin phosphotransferase II and β-glucuronidase activities in transgenic plants

Abstract

A rapid and efficient method for assaying both NPT II and GUS activities was developed. In this method, which is modified from that of McDonnell et al. (1987), and Jefferson (1987), no sample processing procedures such as grinding and centrifugation are necessary. Cut plant tissues (leaves) or intact calli or cells expressing the genes of interest are placed in wells of a microtiter plate containing reaction mixture, and after incubation the reaction mixture is directly used for both NPT II and GUS assays. For the NPT II assay, aliquots of the reaction mixture are blotted onto Whatman P81 paper through a manifold, and the product of the reaction is detected by autoradiography. For GUS activity, aliquots or the rest of the reaction mixture are observed for fluorescent emission under a hand-held UV light or read in a fluorimeter after adding stop buffer to the reaction mixture. This method is the simplest, cheapest, and quickest assays for NPT II and GUS reported to date, and is extremely efficient and suitable for assaying small amounts of samples (as little as 0.3 mg tissue), such as in transient expression assays, or for the quick screening of large numbers of samples, such as in studies of gene inheritance in transgenic plants. In our laboratory, it has been used successfully in assaying NPT II activities for transient and stable gene expression in transformed protoplasts, calli, and leaf tissues of various transgenic plants. It has also been used for detecting both NPT II and GUS activities in transgenic rice plants, in which more than 400 samples could be assayed per day per person.