Simple multiplication and effective genetic transformation (four methods) of in vitro-grown tobacco by stem thin cell layers

Tobacco is one of the principal model plants used in the study of physiological, developmental and genetic processes, often through the use of tissue culture. The success of genetic transformation of Nicotiana tabacum, as with any other plant, requires the selection of non-chimeric transformed tissues and their subsequent effective regeneration. In this paper, the use of transverse thin cell layers (tTCLs) in conjunction with high initial antibiotic selection levels supported the greatest regeneration of transgenic material (adventitious shoots, callus or somatic embryos) and the lowest number of escapes when different plasmid constructs were used, independent of the use of Agrobacterium tumefaciens, biolistics, Agrolistics or SAAT (sonication-assisted Agrobacteriumtransformation). Shoot regeneration capacity (SRC) in tobacco is influenced by gene introduction method (GIM), SRC being GIM-dependent. SAAT stimulated SRC in in vitro stem tTCLs while other GIMs (Agrobacterium, biolistics, Agrolistics) inhibited SRC. SRC was also influenced by the explant and regeneration media. Plants derived from all GIM treatments grew and flowered normally under in vitro and greenhouse conditions. The integration of the GUS transgene is also GIM-dependent, but in all cases is shown to occur in the venation. Flow cytometric and RAPD analyses demonstrate that despite the presence of high levels of endopolyploidy (up to 16C), regenerated propagules (adventitious shoots or somatic embryos) do not exhibit polyploidy and subsequently regenerated plants are genetically stable in vitro.