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United States Department of Agriculture

Agricultural Research Service

Research Project: USING FUNCTIONAL AND APPLIED GENOMICS TO IMPROVE STRESS AND DISEASE RESISTANCE IN FRUIT TREES

Location: Appalachian Fruit Research Laboratory: Innovative Fruit Production, Improvement and Protection

Title: Genetic transformation of apple (Malus x domestica) without use of a selectable marker gene

Authors
item Malnoy, Mickael - CORNELL UNIVERSITY
item Boresjza-Wysocka, Ewa - CORNELL UNIVERSITY
item NORELLI, JOHN (JAY)
item Flaishman, Moshe - ARO, VOLCANI CENTER
item Gidoni, David - ARO, VOLCANI CENTER
item Aldwinckle, Herb - CORNELL UNIVERSITY

Submitted to: Tree Genetics and Genomes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 11, 2009
Publication Date: January 21, 2010
Citation: Malnoy, M., Boresjza-Wysocka, E., Norelli, J.L., Flaishman, M., Gidoni, D., Aldwinckle, H.S. 2010. Genetic transformation of apple (Malus x domestica) without use of a selectable marker gene. Tree Genetics and Genomes. 6:423-433.

Interpretive Summary: Antibiotic and herbicide resistance genes are widely used as selectable markers to facilitate the efficient transformation of crop plants. Due to the negative public connotations associated with the use of selectable markers, a completely marker-free transformation technology would be desirable for the commercialization of genetically transformed plants. With this goal in mind, a technique was developed to genetically transform apple without the use of selectable marker genes. The technique takes advantage of the apple's capacity for high efficiency transformation and allows for the generation of marker-free transgenic plants without the need for repeated transformation or sexual crossing. When two different marker-gene free vectors containing different genes-of-interest were used to transform 'M.26' apple rootstock, 22.0% to 25.4% of the plants recovered showed integration of the gene-of-interest. When the 'Galaxy' fruiting variety of apple was transformed with the same vectors, 13% of the recovered plants contained the gene-of-interest. To our knowledge, this is the first procedure that omitted selectable markers and efficiently accomplished gene transformation in tree fruit crops.

Technical Abstract: Selectable marker genes are widely used for the efficient transformation of crop plants. In most cases, antibiotic or herbicide resistance marker genes are preferred, because they tend to be most efficient. Due mainly to consumer and grower concerns, considerable effort is being put into developing strategies (site-specific recombination, homologous recombination, transposition and co-transformation) to eliminate the marker gene from the nuclear or chloroplast genome after selection. For the commercialization of genetically transformed plants, the use of a completely marker-free technology would be desirable, since there would be no involvement of antibiotic resistance genes or other marker genes with negative connotations. With this goal in mind, a technique for apple transformation was developed without the use of any selectable marker. Transformation of the apple genotype 'M.26' with the constructs pwiAtt35Sgusintron and pinMpNPR1 was achieved. In different experiments, 22.0- 25.4% of regenerants showed integration of the gene of interest. Southern analysis in some transformed lines also confirmed the integration of one copy of the gene. Some of these transformed lines have been propagated and used to determine the uniformity of transformed tissues in the plantlets. The majority of lines are solidly transformed, although some lines are chimeric, as occurs with the conventional transformation procedure using a selectable marker. A second genotype of apple, 'Galaxy', was also transformed with the same constructs and with a transformation efficency of 13%.

Last Modified: 7/28/2014
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