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United States Department of Agriculture

Agricultural Research Service

Research Project: GENETIC IMPROVEMENT OF FRUIT CROPS THROUGH FUNCTIONAL GENOMICS AND BREEDING

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

Title: A high throughput transformation system allows the regeneration of marker-free plum plants (Prunus domestica L.)

Authors
item Petri, Cesar -
item Hily, Jean-Michel -
item Vann, Cheryl
item Dardick, Christopher
item Scorza, Ralph

Submitted to: Annals of Applied Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 4, 2011
Publication Date: August 18, 2011
Citation: Petri, C., Hily, J., Vann, C.D., Dardick, C.D., Scorza, R. 2011. A high throughput transformation system allows the regeneration of marker-free plum plants (Prunus domestica L.). Annals of Applied Biology. 159:302-315.

Interpretive Summary: Prune plums are an important U.S. domestic and export fruit crop but the current industry relies essentially on one variety, and this presents a danger in terms of the genetic vulnerability to changes in climate and to pests and disease to which this variety is susceptible. Traditional breeding for plum improvement has been only marginally successful in producing new high quality adapted varieties of prune plums. Genetic engineering has the potential to speed this process and to make available useful genes from other plant species that can be transferred to plum for improvement of this important crop. Generally, genetic engineering requires the use of “marker” genes whose only function is to aid in the selection of the genetically engineered plants in the laboratory. Since these genes are not necessary once the genetically engineered plants are isolated, we have developed a method of eliminating the use of these marker genes. We developed a very productive system for producing genetically engineered plum plants. Using this system, we left out the selection genes and used highly sensitive molecular testing (polymerase chain reaction) to go through hundreds of plum plants to select those that contain the inserted gene(s) of interest for improving plums. This combination of methods has allowed us to eliminate the unnecessary marker genes in genetically engineered plums which, although safe, nevertheless are not necessary and a cause of concern for some, and can delay the acceptance of genetically improved plants.

Technical Abstract: A high-throughput transformation system previously developed in our laboratory was used for the regeneration of transgenic plum plants without the use of antibiotic selection. The system was first tested with two experimental constructs, pGA482GGi and pCAMBIAgfp94(35S), that contain selective marker and reporter genes. Transformation was monitored by GUS detection, and estimated transformation efficiencies were 5.7 percent and 17.7 percent for pGA482GGi and pCAMBIAgfp94(35S), respectively. Subsequently, an intron-hairpin-RNA (ihpRNA) construct carrying the Plum Pox Virus coat protein (ppv-cp) gene without selectable or reporter marker genes was designed. Five transgenic lines were regenerated as confirmed by DNA blot analysis. We believe that this is the first report on the production of marker-free plants transformed with a potential agronomically important trait in a Prunus species.

Last Modified: 4/20/2014
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