Cadmium resistance in transgenic tobacco plants expressing the Nicotiana glutinosa L. metallothionein gene

Abstract

To understand the function of metallothioneins (MTs) in plants, we introduced the Nicotiana glutinosa MT gene into tobacco (N. tabacum) plants via an Agrobacterium mediated transformation. Full-length MT cDNA was fused between the cauliflower mosaic virus 35S (CaMV 35S) promoter and the nopaline synthase (nos) terminator of the pMBP1 binary vector in sense orientation. Tobacco leaf discs which were cocultivated with Agrobacterium carrying the chimeric MT gene, formed kanamycin-resistant shoots on medium containing kanamycin. The kanamycin-resistant shoots were subsequently rooted on medium containing 200 μM CdSO4. Approximately 30% of individual transgenic plants developed normally. Nontransgenic plants promptly underwent leaf chlorosis, and their growth and development were inhibited on MS medium containing 50 μM CdSO4. Genomic Southern blot analysis showed that the MT gene was stably integrated into the nuclear genome of transgenic tobacco plants. The expression level of MT transcripts was analyzed by RNA gel blot analysis. Self-pollinated seeds obtained from transgenic tobacco plants showing cadmium tolerance were germinated on a medium containing 100 μM CdSO4. PCR analysis from sensitive and stably resistant T2 seedlings for cadmium sulfate confirmed a high correlation between the phenotypic expression of the MT gene and the transgenic genotype, indicating that the MT gene is inherited in the next generation.