Articles
GENETIC TRANSFORMATION OF APPLE WITHOUT USE OF A SELECTABLE MARKER
Article number
738_36
Pages
319 – 322
Language
English
Abstract
Selectable marker genes are widely used for the efficient transformation of crop plants.
In most cases, selection is based on antibiotic or herbicide resistance.
These marker genes are preferred because they tend to be most efficient (e.g. in apple up to 80% transformation). Due mainly to consumer and grower concerns, considerable effort is being put into developing a suite of strategies (site-specific recombinetion, homologous recombination, transposition and co-transformation) to eliminate the marker gene from the nuclear or chloroplast genome after selection.
Current efforts concentrate on systems where the marker genes are eliminated efficiently soon after transformation.
However, these methods are laborious and of doubtful reliability.
For the commercialization of transgenic plants, use of a completely marker-free technology would be greatly preferable, since there would be no involvement of antibiotic resistance genes or other marker genes with negative connotations.
With this goal in mind, we have now developed a technique for apple transformation without any selectable marker.
Transformation of the apple rootstock M.26 with the constructs pwiAtt35Sgus-intron and pin2MpNPR1 without the kanamycin resistance gene has been achieved. 1500 regenerants were harvested from leaf-piece transformation plates for each transformation.
Between 250 and 300 regenerants were chosen randomly and tested by PCR for the presence of the transgenes (attacin E, gus-intron, NPR1, or the pin2 promoter). Depending on the experiment, 22 to 25% of these regenerants showed integration of the transgene.
Southern analysis provided additional confirmation of transformation.
Some of these transgenic lines have been propagated and will be used to determine the uniformity of tissue transformed in the plantlets.
A second genotype of apple, Galaxy, was also transformed with these two constructs.
The preliminary results show 12 to 15% of the Galaxy regenerants have integrated transgenes.
Although the technique we have used is relatively simple for anyone familiar with plant transformation techniques, the results we have obtained are unprecedented for fruit crops as far as we know.
In most cases, selection is based on antibiotic or herbicide resistance.
These marker genes are preferred because they tend to be most efficient (e.g. in apple up to 80% transformation). Due mainly to consumer and grower concerns, considerable effort is being put into developing a suite of strategies (site-specific recombinetion, homologous recombination, transposition and co-transformation) to eliminate the marker gene from the nuclear or chloroplast genome after selection.
Current efforts concentrate on systems where the marker genes are eliminated efficiently soon after transformation.
However, these methods are laborious and of doubtful reliability.
For the commercialization of transgenic plants, use of a completely marker-free technology would be greatly preferable, since there would be no involvement of antibiotic resistance genes or other marker genes with negative connotations.
With this goal in mind, we have now developed a technique for apple transformation without any selectable marker.
Transformation of the apple rootstock M.26 with the constructs pwiAtt35Sgus-intron and pin2MpNPR1 without the kanamycin resistance gene has been achieved. 1500 regenerants were harvested from leaf-piece transformation plates for each transformation.
Between 250 and 300 regenerants were chosen randomly and tested by PCR for the presence of the transgenes (attacin E, gus-intron, NPR1, or the pin2 promoter). Depending on the experiment, 22 to 25% of these regenerants showed integration of the transgene.
Southern analysis provided additional confirmation of transformation.
Some of these transgenic lines have been propagated and will be used to determine the uniformity of tissue transformed in the plantlets.
A second genotype of apple, Galaxy, was also transformed with these two constructs.
The preliminary results show 12 to 15% of the Galaxy regenerants have integrated transgenes.
Although the technique we have used is relatively simple for anyone familiar with plant transformation techniques, the results we have obtained are unprecedented for fruit crops as far as we know.
Authors
M. Malnoy, E.E. Borejsza-Wysocka, P. Abbott, S. Lewis, J.L. Norelli, M. Flaishman, D. Gidoni, H.S. Aldwinckle
Keywords
Malus × domestica, transgenics, markerless
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