Abstract
This paper reports the first successful transformation of Gracilaria gracilis (Stackhouse) M. Steentoft, L.M. Irvine and W.F. Farnham with lacZ-containing plasmids by microparticle bombardment. Transient expression of the lacZ reporter gene was compared under the control of three different viral promoters including the Simian virus 40 (SV40) promoter, the Cytomegalovirus (CMV) promoter and the Cauliflower mosaic virus 35S (CaMV 35S) promoter. In thalli transformed with vectors containing either the SV40 or CMV promoter, lacZ presence was confirmed by histological staining 2 and 3 days postbombardment. In thalli transformed with the vector containing the CaMV 35S promoter, lacZ presence was confirmed by histological staining 1, 2, 3 and 5 days post-bombardment. Sectioning and histological staining of bombarded thalli showed that recombinant bombarded DNA penetrated cells to below the epidermal layer of the thallus. PCR analysis verified the presence of the lacZ gene in plasmid-bombarded thalli from the first day post-bombardment onwards. β-Galactosidase activity varied depending on the type of promoter used. These results form an important foundation for the development of a successful transformation protocol for G. gracilis . Key words: Gracilaria , lacZ, microparticle bombardment, viral promoter.
Highlights
Transient expression of the lacZ reporter gene was compared under the control of three different viral promoters including the Simian virus 40 (SV40) promoter, the Cytomegalovirus (CMV) promoter and the Cauliflower mosaic virus 35S (CaMV 35S) promoter
To test for successful transformation of lacZ into G. gracilis tissue, macroalgal thalli were bombarded with the recombinant vectors pSV-β-Gal, pCMV-β-Gal and pCaMV-β-Gal
LacZ expression was visible on days 1, 2, 3 and 5 post-bombardment in the samples bombarded with pCaMV-β-Gal (Figure 1A)
Summary
In thalli transformed with vectors containing either the SV40 or CMV promoter, lacZ presence was confirmed by histological staining 2 and 3 days postbombardment. In thalli transformed with the vector containing the CaMV 35S promoter, lacZ presence was confirmed by histological staining 1, 2, 3 and 5 days post-bombardment. To test for successful transformation of lacZ into G. gracilis tissue, macroalgal thalli were bombarded with the recombinant vectors pSV-β-Gal, pCMV-β-Gal and pCaMV-β-Gal. Thalli were sampled at 5 time points postbombardment (days 1, 2, 3, 5 and 7) and stained for the presence of lacZ using an in situ histochemical stain.
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