Articles
DIRECT REGENERATION AND SELECTION OF POPULUS TREMULA L. TRANSGENIC SHOOTS FROM AGROBACTERIUM TUMEFACIENS-TRANSFORMED STEM EXPLANTS.
Article number
447_71
Pages
347 – 348
Language
Abstract
An Agrobacterium-mediated transformation procedure involving direct regeneration of shoot-buds from stem explants of in vitro-grown Populus tremula plants is described.
Disarmed Agrobacterium tumefaciens, strain EHA105 was used for transformation, harboring the binary plasmid pKIWI105 (carrying the uidA and nptII genes, coding for
-glucuronidase and neomycin phosphotransferase, respectively). The overall procedure included: (1) inoculation of stem explants, (2) regeneration of adventitious shoots from the cut surface of the explants, (3) kanamycin selection of transformants, (4) rooting and plantlet establishment, (5) molecular analysis of transformed plants.
Transient GUS expression was observed in the cut surface of the stem explants, and an inoculation period of up to 72 hours was found to be most effective.
Adventitious shoots regenerated after 2-3 weeks of culture on a Woody Plant Medium supplemented with TDZ and carbenicillin.
Transformants were selected in three different ways: (1) explants were cultured with 50 mg/l kanamycin in the regeneration medium immediately; (2) explants were cultured with 50 mg/l kanamycin in the regeneration medium 10–14 days after infection; (3) plantlets regenerated from stem segments on non-selective medium were selected with 100 mg/l kanamycin in half-strength MS rooting medium.
Selection procedures (1) and (2) yielded green resistant shoots and/or white non-resistant shoots.
Kanamycin-resistant shoots were transferred to half-strength MS medium for rooting.
With selection procedure (3), non-resistant plantlets formed pale-green or white leaves and failed to root, whereas resistant plantlets formed dark-green leaves and well-developed roots.
Selection of adventitious shoots immediately was found to be optimal with regard to both the time required and the transformation efficiency, the latter being much higher than with the other procedures.
Leaf samples from resistant shoots or plantlets were tested for GUS expression, and subjected to PCR analysis of uidA and nptII genes.
A Southern blot of PCR-amplified fragments confirmed their authenticity.
A Southern blot of total plant DNA confirmed the integration of uidA and nptII genes into plant cells.
Disarmed Agrobacterium tumefaciens, strain EHA105 was used for transformation, harboring the binary plasmid pKIWI105 (carrying the uidA and nptII genes, coding for
-glucuronidase and neomycin phosphotransferase, respectively). The overall procedure included: (1) inoculation of stem explants, (2) regeneration of adventitious shoots from the cut surface of the explants, (3) kanamycin selection of transformants, (4) rooting and plantlet establishment, (5) molecular analysis of transformed plants.Transient GUS expression was observed in the cut surface of the stem explants, and an inoculation period of up to 72 hours was found to be most effective.
Adventitious shoots regenerated after 2-3 weeks of culture on a Woody Plant Medium supplemented with TDZ and carbenicillin.
Transformants were selected in three different ways: (1) explants were cultured with 50 mg/l kanamycin in the regeneration medium immediately; (2) explants were cultured with 50 mg/l kanamycin in the regeneration medium 10–14 days after infection; (3) plantlets regenerated from stem segments on non-selective medium were selected with 100 mg/l kanamycin in half-strength MS rooting medium.
Selection procedures (1) and (2) yielded green resistant shoots and/or white non-resistant shoots.
Kanamycin-resistant shoots were transferred to half-strength MS medium for rooting.
With selection procedure (3), non-resistant plantlets formed pale-green or white leaves and failed to root, whereas resistant plantlets formed dark-green leaves and well-developed roots.
Selection of adventitious shoots immediately was found to be optimal with regard to both the time required and the transformation efficiency, the latter being much higher than with the other procedures.
Leaf samples from resistant shoots or plantlets were tested for GUS expression, and subjected to PCR analysis of uidA and nptII genes.
A Southern blot of PCR-amplified fragments confirmed their authenticity.
A Southern blot of total plant DNA confirmed the integration of uidA and nptII genes into plant cells.
Authors
C.S. Jensen, T. Tzfira, A. Vainstein, A. Altman
Keywords
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