Transformation of Kalanchoe blossfeldiana mediated by Agrobacterium tumefaciens and transgene silencing

Abstract

A method for Agrobacterium-mediated transformation of Kalanchoe blossfeldiana Poelln. (Crassulaceae) and quantitative analysis of the expression pattern of β-glucuronidase (GUS) gene are reported. Leaf segments were infected with the Agrobacterium strain LBA4404 having a binary vector plasmid pBI121 or its derivatives and cultured on Murashige and Skoog medium supplemented with 0.5 mg/l benzyladenine, 2 mg/l indoleacetic acid, 300 mg/l carbenicillin and 20 mg/l kanamycin (selection medium) for regeneration. Primary transformants (R 0) with three kinds of chimeric GUS genes were obtained in different experiments, and Southern blot analysis confirmed the presence of the GUS gene in their genome. The integrated GUS gene(s) seemed to be silenced in about half of the transformants. There was no clear correlation between copy number and expression level of the GUS gene. Observed levels of GUS activity within transformants were divided into three classes: (1) high activity, (2) low activity and (3) near-zero activity. Selfed progeny (R 1) of a near-zero activity and a low activity transformant segregated to high, low and near-zero activity. Southern blot analysis unexpectedly revealed no clear difference in the band pattern of the GUS gene; some progeny showed high activity while some progeny showed near-zero activity derived from the near-zero-activity transformant.