Advanced search
1 file | 965.72 KB

Production of transgenic Valencia orange suspension cells to be used as donors for chromosome transfer

Author
Organization
Abstract
The method for plant transformation and antibiotic selection has been well established for several citrus varieties. Transgenic citrus suspension cells, however, have never been produced and maintained, thus, a procedure for transformation, antibiotic selection, and maintenance of transgenic suspension cells is still required. Transgenic citrus suspension cells containing the npt II gene forresistance to an aminoglycoside antibiotic would be very valuable for chromosome transfer experiments. To determine the lethal antibiotic concentration where growth inhibition of suspension cells would occur, the antibiotics kanamycin sulfate, geneticin, and paromomycin sulfate were used. Paromomycin sulfate at 50 µg/ml was found to be the best antibiotic forselection. Geneticin and kanamycin were not appropriate forselection of transgenic Valencia sweet orange suspension cells. For protoplast transformation, the electroporation parameters and incubation procedures were established. Pulses of 400, 500, and 700 V/cm were evaluated. The best electroporation settings were pulse of 500 V/cm and capacitance of 1000 µF. To secure high survival rates of the protoplasts, it was essential to add BH3 medium immediately after electroporation. Transgenic suspension cells of Valencia sweet orange containing the npt II and the ß-glucuronidase (Gus) gene were produced and maintained in 14 day subculture cycles in medium containing 50 µg/ml of paromomycin sulfate. This concentration was also very efficient for selection of transgenic Valencia embryos, since non-transgenic embryos were bleached.
Keywords
chromosome transfer, Electroporation, paromomycin

Downloads

  • (...).pdf
    • full text
    • |
    • UGent only
    • |
    • PDF
    • |
    • 965.72 KB

Citation

Please use this url to cite or link to this publication:

Chicago
Louzada, ES, HS del Rio, Ivan Ingelbrecht, and D Xia. 2001. “Production of Transgenic Valencia Orange Suspension Cells to Be Used as Donors for Chromosome Transfer.” Subtropical Plant Science 53: 9–13.
APA
Louzada, E., del Rio, H., Ingelbrecht, I., & Xia, D. (2001). Production of transgenic Valencia orange suspension cells to be used as donors for chromosome transfer. SUBTROPICAL PLANT SCIENCE, 53, 9–13.
Vancouver
1.
Louzada E, del Rio H, Ingelbrecht I, Xia D. Production of transgenic Valencia orange suspension cells to be used as donors for chromosome transfer. SUBTROPICAL PLANT SCIENCE. Subtropical Plant Science Society; 2001;53:9–13.
MLA
Louzada, ES, HS del Rio, Ivan Ingelbrecht, et al. “Production of Transgenic Valencia Orange Suspension Cells to Be Used as Donors for Chromosome Transfer.” SUBTROPICAL PLANT SCIENCE 53 (2001): 9–13. Print.
@article{2014430,
  abstract     = {The method for plant transformation and antibiotic selection has been well established for several citrus varieties. Transgenic citrus suspension cells, however, have never been produced and maintained, thus, a procedure for transformation, antibiotic selection, and maintenance of transgenic suspension cells is still required. Transgenic citrus suspension cells containing the npt II gene forresistance to an aminoglycoside antibiotic would be very valuable for chromosome transfer experiments. To determine the lethal antibiotic concentration where growth inhibition of suspension cells would occur, the antibiotics kanamycin sulfate, geneticin, and paromomycin sulfate were used. Paromomycin sulfate at 50 {\textmu}g/ml was found to be the best antibiotic forselection. Geneticin and kanamycin were not appropriate forselection of transgenic Valencia sweet orange suspension cells. For protoplast transformation, the electroporation parameters and incubation procedures were established. Pulses of 400, 500, and 700 V/cm were evaluated. The best electroporation settings were pulse of 500 V/cm and capacitance of 1000 {\textmu}F. To secure high survival rates of the protoplasts, it was essential to add BH3 medium immediately after electroporation. Transgenic suspension cells of Valencia sweet orange containing the npt II and the {\ss}-glucuronidase (Gus) gene were produced and maintained in 14 day subculture cycles in medium containing 50 {\textmu}g/ml of paromomycin sulfate. This concentration was also very efficient for selection of transgenic Valencia embryos, since non-transgenic embryos were bleached.},
  author       = {Louzada, ES and del Rio, HS and Ingelbrecht, Ivan and Xia, D},
  journal      = {SUBTROPICAL PLANT SCIENCE},
  keyword      = {chromosome transfer,Electroporation,paromomycin},
  language     = {eng},
  pages        = {9--13},
  publisher    = {Subtropical Plant Science Society},
  title        = {Production of transgenic Valencia orange suspension cells to be used as donors for chromosome transfer},
  url          = {http://subplantsci.org/SPSJ/v53\%202001/SPSJ\%2053\%2009-13\%20Louzada\%20del\%20Rio\%20Ingelbrect\%20\&\%20Xia.pdf},
  volume       = {53},
  year         = {2001},
}