Page Banner

United States Department of Agriculture

Agricultural Research Service

Related Topics

Research Project: TRANSGENE MANAGEMENT THROUGH SITE-SPECIFIC RECOMBINATION

Location: Plant Gene Expression Center Albany_CA

2008 Annual Report


1a.Objectives (from AD-416)
Objective 1: Develop and evaluate transformation vectors that use site-specific recombination systems for more efficient and precise integration of DNA into the host genome. Test the site-specific integration and repeated stacking of DNA into designated chromosomal sites using selected reporter genes and oxidative stress tolerance genes.

Objective 2: Develop and evaluate the use of site-specfic recombination to break the linkage drag associated with the introgression of transgenes from laboratory lines to field cultivars. Test the reciprocal exchange of transgenic DNA flanked by recombination sites.


1b.Approach (from AD-416)
For objective 1, vectors will be constructed by recombinant DNA methods using as much as possible publicly available genetic components. Vectors will be designed for use in direct DNA transformation. Model dicot and monocot plant systems will be transformed with the initial empty vector harboring a reporter gene. The initial transformants will be screened for single copy insertion and acceptable expression level of the reporter construct. Several of the initial transformants that meet the above criteria will be propagate as "target lines". Progeny of these target lines will be used to test the site-specific integration of additional DNA into the same locus, mediated by the transient co-introduction of the appropriate recombinase gene. Site-specific integrants will be subjected to further introduction of a second recombinase gene to remove unneeded DNA. Characterization of the structure and expression of the introduced DNA will be performed by standard molecular analysis.

For objective 2, a construct will be made by recombinant DNA methods with sets of recombination sites designed for interchromosomal recombination and harboring a first marker gene. This construct will be transformed into line #1. The transgenic locus of the line #1 will be introduced into another genetic background such that the flanking DNA of the new line #2 differs from that of line #1. The transgenic locus of line #1 will be further modified through site-specific insertion of a second marker gene to form line #3. Line #3, with first and second marker genes, will be crossed to line #2 containing the first marker gene. The efficiency of exchange by site-specific recombination between the two transgenic loci will be examined in progeny, as determined by linkage to polymorphic flanking DNA. Formerly 5335-21000-016-00D (4/06).


3.Progress Report
This project aims to develop a way to integrate new DNA repeatedly into a known location in the plant genome (objective 1), and to test whether site-specific recombination performed between homologous chromosomes can be used to speed up the transgene introgression into elite varieties (objective).¿ Both objectives are being tested in tobacco, as rice requires muchh more labor and resources not afforded by project funds.

For objective 1, three tobacco target lines have been obtained from Agrobacterium mediated transformation and shown by molecular analysis to contain a single target molecule with correct recombination sites. The integration construct for inserting the first new DNA molecule into these target sites has been obtained. New recombinase constructs have been tested in a transient expression system and were shown effective in prromoting site-specific recombination. The integration construct along with the recombinase construct has been introduced into tobacco protoplasts. Calluses that show the selectable phenotype characteristic of the integration construct have been obtained and are being regenerated into plants.

For objective 2, tobacco lines with the construct to test chromosome recombination have been obtained from Agrobacterium mediated transformation and three single copy lines were chosen afterr molecular analysis for use to generate two different derivatives by either ParA mediated or Bxb1 mediated recombination. These recombinase genes were introduced by secondary transformation into the three chosen lines bearing the test construct and are awaiting confirmation. Additionally, tobacco transgenic lines expressing ParA recombinase or Bxb1 recombinase have been obtained from Agrobacterium mediated transformation. Crosses between the ParA or the Bxb1 recombinase lines and the linnes harboring the test construct are in progress.

This research addresses National Program 302 Component 3, “Plant Biotechnology Risk Assessment” and Problem Statement 3A, “Improving and Assessing Genetic Engineering Technology”.


4.Accomplishments
1. Experiments to target DNA through site-specific integration. The specific placement of DNA into a known location is a desired goal in plant transformation. ARS scientists in the Plant Gene Expression Center in Albany, CA are testing a new site-specific integration system to deliver the DNA into pre-defined target sites in the plant genome. Using single copy target lines generated in Fiscal Year (FY) 2007, new DNA was introduced into these plant lines during FY08, resulting in putative transformed calluses with the expected selectable phenotype. If plants regenerated from these calluses show site-specific integration, then the use of this technology would help speed the breeding of transgenes to new varieties. This research addresses National Program 302 Component 3, “Plant Biotechnology Risk Assessment” and Problem Statement 3A, “Improving and Assessing Genetic Engineering Technology”.

2. Generating derivative plant lines to test interchromosomal recombination. Due to linkage drag, the introgression of transgenes from laboratory to field varieties requires numerous backcrosses. To expedite this breeding process, ARS scientists in Albany, CA are testing the possibility of using site-specific recombination to break the linkage drag. In Fiscal Year 2008, they introduced recombinase genes into a parental plant line to derive two different alleles, such that recombination between the two alleles can reveal the efficiency of interchromosomal recombination. Working out the efficiency of interchromosomal site-specific recombination would help evaluate this technology for use in plant breeding.

This research addresses National Program 302 Component 3, “Plant Biotechnology Risk Assessment” and Problem Statement 3A, “Improving and Assessing Genetic Engineering Technology”.


5.Significant Activities that Support Special Target Populations
NONE.


6.Technology Transfer

Number of the New MTAs (providing only)1
Number of Non-Peer Reviewed Presentations and Proceedings6

Review Publications
Ow, D.W. 2008. Chance Events Shape a Career. In: C. Neal Stewart, Jr., Plant Biotechnology and Genetics: Principles, Techniques and Applications. Adobe E-Book, "lifebox." Wiley and Sons, New York, pp. 238-240.

Stewart, C., Ow, D.W. 2008. The Future of Plant Biotechnology. In: C. Neal Stewart, Jr., Plant Biotechnology and Genetics: Principles, Techniques and Applications. Adobe E-Book. Wiley and Sons, New York, pp. 357-369.

Last Modified: 4/20/2014
Footer Content Back to Top of Page