Abstract
An efficient system for shoot regeneration and Agrobacterium-mediated gene transfer into Brassica napus was developed through the modification of the culture conditions. Different concentrations of benzyladenine (1.5, 3.0 and 4.5 mg dm−3) and thidiazuron (0.0, 0.15 and 0.30 mg dm−3) were evaluated for shoot regeneration of 7, 14 and 21-d-old hypocotyl explants. Maximum shoot regeneration frequency was obtained in 21-d-old explants using 4.5 mg dm−3 benzyladenine and 0.3 mg dm−3 thidiazuron. Under above culture condition, the highest percentage of shoot regeneration frequency was 200 %. Agrobacterium-infected explants grown on the selection medium gave rise to transgenic shoots at a frequency of 11.8 %. Transformed shoots rooted when cultured on a medium supplemented with 2 mg dm−3 of indolebutyric acid and 10 mg dm−3 kanamycin. The rooted plantlets were successfully established in the soil and developed fertile flowers and viable seeds. Evidences for transformation were confirmed by GUS assay and PCR analysis.
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Abbreviations
- BA:
-
benzyladenine
- B5 medium:
-
Gamborg medium
- CaMV35S:
-
cauliflower mosaic virus 35S promoter
- CIM:
-
callus induction medium
- CTAB:
-
cetyl trimethyl ammonium bromide
- 2,4-D:
-
2,4-dichlorophenoxyacetic acid
- GUS:
-
β-glucuronidase
- IBA:
-
indole-3-butyric acid
- MS:
-
Murashige and Skoog
- NPTII:
-
neomycin phosphotransferase II
- RIM:
-
root induction medium
- SIM:
-
shoot induction medium
- SMM:
-
shoot maturation medium
- TDZ:
-
thidiazuron
- X-gluc:
-
5-bromo-4-chloro-3-indolyl-β-D-glucuronide
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Jonoubi, P., Mousavi, A., Majd, A. et al. Efficient regeneration of Brassica napus L. hypocotyls and genetic transformation by Agrobacterium tumefaciens . Biol Plant 49, 175–180 (2005). https://doi.org/10.1007/s10535-005-5180-2
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DOI: https://doi.org/10.1007/s10535-005-5180-2