Articles
AGROBACTERIUM TUMEFACIENS VERSUS BIOLISTIC-MEDIATED TRANSFORMATION OF THE CHRYSANTEMUM CVS. POLARIS AND GOLDEN POLARIS WITH NUCLEOCAPSID PROTEIN GENES OF THREE TOSPOVIRUS SPECIES
Article number
482_31
Pages
209 – 218
Language
Abstract
Agrobacterium tumefaciens and Biolistic transformation procedures were developed for Polaris and Golden Polaris, two important commercial chrysanthemum Dendranthema grandiflora Tzvelev (syn. Chrysanthemum morifolium Ramat.) cultivars.
The disarmed A. tumefaciens strains LBA4404, C58sZ707 and EHA 105 containing the binary plasmids pBIN19 or pGA482GG were used for transformation.
Both plasmids contained within the T-DNA borders of the nucleocapsid (N) protein genes of either tomato spotted wilt (TSWV), impatiens necrotic spot (INSV), or groundnut ringspot (GRSV) Tospoviruses, and the marker gene neomycin phosphotransferase (NPT II); pGA482GG contained also the
-glucuronidase (GUS) gene.
Transgenic plants were recovered using leaf and stem explants, and using a step-wise kanamycin selection procedure to optimize recovery of transformed plants.
PCR and Southern blot analyses confirmed the integration of the N genes into the chrysanthemum genome.
Most of the putative transgenic plants tested were PCR positive (97%) indicating that the kanamycin selection procedure was effective and helped reduce the number of escapes.
One hundred and fifty eight transgenic Polaris and sixty six transgenic Golden Polaris plants were obtained with different N gene constructs.
The cultivar Iridon was also transformed with the three N gene constructs, and two hundred and seventy three independent transgenic lines were recovered.
These results demonstrate the efficiency of the procedures used to transfer genetic material into the genome of chrysanthemum cultivars recalcitrant to regeneration.
The disarmed A. tumefaciens strains LBA4404, C58sZ707 and EHA 105 containing the binary plasmids pBIN19 or pGA482GG were used for transformation.
Both plasmids contained within the T-DNA borders of the nucleocapsid (N) protein genes of either tomato spotted wilt (TSWV), impatiens necrotic spot (INSV), or groundnut ringspot (GRSV) Tospoviruses, and the marker gene neomycin phosphotransferase (NPT II); pGA482GG contained also the
-glucuronidase (GUS) gene.Transgenic plants were recovered using leaf and stem explants, and using a step-wise kanamycin selection procedure to optimize recovery of transformed plants.
PCR and Southern blot analyses confirmed the integration of the N genes into the chrysanthemum genome.
Most of the putative transgenic plants tested were PCR positive (97%) indicating that the kanamycin selection procedure was effective and helped reduce the number of escapes.
One hundred and fifty eight transgenic Polaris and sixty six transgenic Golden Polaris plants were obtained with different N gene constructs.
The cultivar Iridon was also transformed with the three N gene constructs, and two hundred and seventy three independent transgenic lines were recovered.
These results demonstrate the efficiency of the procedures used to transfer genetic material into the genome of chrysanthemum cultivars recalcitrant to regeneration.
Authors
L.M. Yepes, V. Mittak, S.-Z. Pang, D. Gonsalves, J.L. Slightom
Keywords
pathogen-derived resistance
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