Efficient use of the PMI/mannose selection system in Agrobacterium-mediated transformation of tobacco (Nicotiana tabacum)

Abstract

A safe and environment friendly selectable marker system using phosphomannose isomerase (pmi) gene was employed for Agrobacterium tumefaciens mediated transformation of tobacco (Nicotinia tabacum L.). Sensitivity of tobacco leaf explants to mannose as selective agent was determined before genetic transformation. Twenty mg/L mannose was chosen for selection to suppress the emergence of untransformed shoots from the explants. Leaf explants from 4-week old seedlings of tobacco cv. Gewone Groene were transformed using Agrobacterium harbouring plasmid pCAMBIA3300-PMI carrying the pmi gene. Regenerating transformed shoots were selected on Murashige & Skoog basal medium supplemented with 1.0 mg/L 6-benzylaminopurine, 0.1 mg/L indole-3-butyric acid, and 20 g/L mannose as carbon source. The mannose-resistant shoots were rooted on a plant growth regulator-free Murashige & Skoog basal medium and the potted transgenic plants were acclimatized successfully in the greenhouse, with a survival rate of 100%. Selection efficiency and transformation efficiency based on PCR analysis of individual putative transformants were achieved as high as 94.56% and 21.75%, respectively, with a lower rate of untransformed plant escapes. Southern blot analysis of selected putative transformed plants confirmed the transgene integration into the tobacco genome. The following reverse transcription PCR analysis showed transcriptional activity of the pmi transgene in all T0 transgenic plants analysed with differences in the level of pmi transcripts, and chlorophenol red assay confirmed the activity of PMI in transgenic plants. The PMI/mannose-based transformation system presented here is efficient and reproducible, and can be used for the mass production of transgenic tobacco plants to produce novel products with industrial and medicinal values.