In vitro freezing in microtitre plates applied to tobacco plants transformed with the inaZ gene of Pseudomonas syringae.

Abstract

High throughput assays have been developed to measure the ice nucleation activity of transgenic tobacco, Nicotiana tabacum L. cv. Petit Havana SR1 plants expressing the ice nucleation gene, inaZ, from Pseudomonas syringae at a young seedling stage, as well as in leaf tissue. Both assays are carried out in 96-well microtitre plates. The first assay involves direct seeding in vitro, one seed per microtitre plate well containing Murashige-Skoog agar. When seedlings reach the two-leaf stage, they are exposed to freezing temperatures by floating the plates on a circulating alcohol bath set at temperatures colder than -9 degrees C. The second assay involves placing small leaf discs individually in microtitre plate wells containing sterile distilled water. The assays complement each other, give highly reproducible results, are technically simple and enable the detection of freezing events in large numbers of plants. The utility and limitations of these assays are discussed.