Articles
DEVELOPMENT OF AGROBACTERIUM-MEDIATED TRANSFORMATION SYSTEM FOR SNAPDRAGON (ANTIRRHINUM MAJUS)
Article number
508_35
Pages
241 – 242
Language
Abstract
Agrobacterium-mediated gene transfer to snapdragon could be achieved within a few months with a reasonable high frequency of transformation.
Segments of hypocotyls from axenic seedlings of snapdragon, Antirrhinum majus cv.
Madame Butterfly Orchid (Sakata seed Co.), were inoculated with Agrobacterium tumefaciens harbouring binary vector plasmid pIG121 (Ohta et al., 1990) which carried NPTII and intron-GUS genes.
After co-cultivation, the explants were transferred to shoot-regeneration medium followed by root-inducing medium.
Both media contains two antibiotics, cefotaxime for elimination of Agrobacterium and kanamycin for selection of transformed shoots.
Multiple shoots were directly induced from explants 4–5 weeks after co-cultivation, and rooted 2–3 weeks after transfer to the root-inducing medium.
Shoots with kanamicyn resistance were sampled for assay of histochemical GUS. Adventitious shoots from hypocotyls inoculated with A. tumefaciens exhibited GUS expression.
This GUS expression was the result of transformation event of GUS gene into the plant genome, because the GUS gene construct used in this experiment possessed intron sequences.
No GUS expression was observed in non-transformed adventitious shoots.
All of the regenerated shoots inducing roots on the selection medium were analysed for the presence of NPTII and GUS genes by PCR Transformation frequency with respect to the number of regenerated shoots was 2–3 %.
Segments of hypocotyls from axenic seedlings of snapdragon, Antirrhinum majus cv.
Madame Butterfly Orchid (Sakata seed Co.), were inoculated with Agrobacterium tumefaciens harbouring binary vector plasmid pIG121 (Ohta et al., 1990) which carried NPTII and intron-GUS genes.
After co-cultivation, the explants were transferred to shoot-regeneration medium followed by root-inducing medium.
Both media contains two antibiotics, cefotaxime for elimination of Agrobacterium and kanamycin for selection of transformed shoots.
Multiple shoots were directly induced from explants 4–5 weeks after co-cultivation, and rooted 2–3 weeks after transfer to the root-inducing medium.
Shoots with kanamicyn resistance were sampled for assay of histochemical GUS. Adventitious shoots from hypocotyls inoculated with A. tumefaciens exhibited GUS expression.
This GUS expression was the result of transformation event of GUS gene into the plant genome, because the GUS gene construct used in this experiment possessed intron sequences.
No GUS expression was observed in non-transformed adventitious shoots.
All of the regenerated shoots inducing roots on the selection medium were analysed for the presence of NPTII and GUS genes by PCR Transformation frequency with respect to the number of regenerated shoots was 2–3 %.
Authors
M.L. Cui, T. Handa, K. Takayanagi, H. Kamada
Keywords
Agrobacterium tumefaciens, intron-GUS, NPTII
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