Summary
An efficient and reliable method for shoot regeneration from leaf disks of Fragaria vesca L. has been developed. This protocol has been successfully employed to obtain transformed plants using Agrobacterium tumefaciens as gene vector. Murashige and Skoog basal medium supplemented with benzyladenine (4 mg/l) and indole-3-butyric acid (0.25 mg/l) induced the maximum percentage of shoot regeneration (98%) and the highest number of shoot colonies per explant (4.6) after 8 weeks of culture. Isolated shoots would elongate and proliferate when the benzyladenine concentration was lowered to 0.5 mg/l. The established protocol for shoot regeneration was employed to transform leaf disks using Agrobacterium tumefaciens carrying the plasmid pBI121. A 7.7% of the inoculated explants showed kanamycin resistance after 10 weeks of selection in a medium containing 25 mg/l of this antibiotic. The transgenic shoots obtained were rooted in the presence of 25 mg/ kanamycin and successfully acclimatized. The final percentage of transformation obtained based on beta-glucuronidase expression was 6.9%.
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Abbreviations
- BA:
-
benzyladenine
- IBA:
-
indole-3-butyric acid
- MS:
-
Murashige and Skoog basal medium
- LSD:
-
least significant difference
- NOS:
-
nopaline synthase promoter
- NPTII:
-
neomycin phosphotransferase (EC 2.7.1.95)
- CaMV35S:
-
cauliflower mosaic virus promoter
- GUS:
-
beta-glucuronidase (EC 3.2.1.31)
- LB:
-
Luria Broth base
- CTAB:
-
hexadecil trimethyl ammonium bromide
- PCR:
-
polymerase chain reaction
- X-gluc:
-
5-bromo-4-chloro-3-indolyl-glucuronide
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El Mansouri, I., Mercado, J.A., Valpuesta, V. et al. Shoot regeneration and Agrobacterium-mediated transformation of Fragaria vesca L.. Plant Cell Reports 15, 642–646 (1996). https://doi.org/10.1007/BF00232469
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DOI: https://doi.org/10.1007/BF00232469