Articles
AGROBACTERIUM-MEDIATED TRANSFORMATION OF THE APPLE CULTIVARS ‘GALA’, ‘GOLDEN DELICIOUS’ AND ‘ELSTAR’, AND THE STRAWBERRY CULTIVARS ‘GARIGUETTE’, ‘POLKA’ AND ‘ELSANTA’
Article number
484_93
Pages
547 – 556
Language
Abstract
In vitro leaf segments of the apple cultivars ‘Gala’, ‘Golden Delicious’ and ‘Elstar’, and leaf segments from greenhouse-grown material of the strawberry cultivars ‘Gariguette’, ‘Polka’, ‘Elsanta’ and CPRO-DLO breeding line no. 88312, were co-cultivated with a supervirulent Agrobacterium strain, containing the nptII and gus genes.
Putative transgenic callus clumps were excised from the apple leaf explants and GUS-positive shoots were induced on calli which proliferated on medium containing kanamycin.
Southern blot analysis of GUS-positive clones of the three cultivars confirmed the integration in the apple genome of both genes with one or two copies per genome being present.
The transformation frequencies per leaf explant, based on the number of GUS-positive shoots, were 0.7–8% for ‘Gala’, 0.2–6% for ‘Golden Delicious’ and 0.4–0.8% for ‘Elstar’. Adventitious shoots appearing directly on the strawberry leaf segments (‘Gariguette’, no. 88312) or via callus (‘Polka’) were transferred to a medium containg kanamycin and subcultured for several periods of 4–6 w.
The percentage of green, kanamycin-resistant shoots per leaf explant was up to 2% for ‘Gariguette’ and line no. 88312, and up to 5% for ‘Polka’. A number of shoots were tested for both GUS-expression and rooting capacity on a medium containing kanamycin. ‘Elsanta’ proved to be recalcitrant for regeneration and transformation.
However, one kanamycin-resistant, GUS-positive plant has been obtained.
Southern blot analysis of this plant and of kanamycin resistant plants of ‘Gariguette’ and line no. 88312 confirmed the integration into the strawberry genome of 1–3 copies of both genes.
Future experiments are aimed at introducing antifungal genes and/or genes influencing the metabolic pathways.
Putative transgenic callus clumps were excised from the apple leaf explants and GUS-positive shoots were induced on calli which proliferated on medium containing kanamycin.
Southern blot analysis of GUS-positive clones of the three cultivars confirmed the integration in the apple genome of both genes with one or two copies per genome being present.
The transformation frequencies per leaf explant, based on the number of GUS-positive shoots, were 0.7–8% for ‘Gala’, 0.2–6% for ‘Golden Delicious’ and 0.4–0.8% for ‘Elstar’. Adventitious shoots appearing directly on the strawberry leaf segments (‘Gariguette’, no. 88312) or via callus (‘Polka’) were transferred to a medium containg kanamycin and subcultured for several periods of 4–6 w.
The percentage of green, kanamycin-resistant shoots per leaf explant was up to 2% for ‘Gariguette’ and line no. 88312, and up to 5% for ‘Polka’. A number of shoots were tested for both GUS-expression and rooting capacity on a medium containing kanamycin. ‘Elsanta’ proved to be recalcitrant for regeneration and transformation.
However, one kanamycin-resistant, GUS-positive plant has been obtained.
Southern blot analysis of this plant and of kanamycin resistant plants of ‘Gariguette’ and line no. 88312 confirmed the integration into the strawberry genome of 1–3 copies of both genes.
Future experiments are aimed at introducing antifungal genes and/or genes influencing the metabolic pathways.
Authors
K. Puite, J. Schaart
Keywords
Malus pumila, Fragaria x ananassa
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