Agrobacterium.mediated transformation and stable expression of the green fluorescent protein in Brassica rapa.
2003
Wahlroos T. | Susi P. | Tylkina L. | Malyshenko S. | Zvereva S. | Korpela T.
An efficient Agrobacterium-mediated method for transformation, regeneration and screening of Brassica rapa subsp. oleifera (synonym to B. campestris) was developed. For transformation of B. rapa subsp. oleifera, 5-d-old cotyledons were co-cultivated for 2 d with Agrobacteria (strain AGL1) harbouring a binary vector carrying a gene for green fluorescent protein (GFP). For regeneration, cultivation of explants in Murashige-Skoog-based media supplemented with 2 mg/l alpha-naphthaleneacetic acid, 4 mg/l 6-benzylaminopurine, 3 mg/l abscisic acid and 11.9 mg l-6 silver nitrate was found to be most optimal. Regeneration and screening steps were performed in the presence of low level of antibiotic selection and visually screened based on GFP fluorescence. GFP did not prove to be very useful in regeneration steps but reduced the time and number of plants to be handled in screening process. Using the method, up to 9 per cent of fluorescing transformants (T0 generation) were obtained. Expression of GFP in T1 generation was further confirmed by Western blotting and fluorescence/confocal microscopy.
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