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Real-Time Polymerase Chain Reaction Monitoring of Recombinant DNA Entry into Soil from Decomposing Roundup Ready Leaf Biomass

David J. Levy-Booth^†, Rachel G. Campbell^‡, Robert H. Gulden^‡, Miranda M. Hart^†, Jeff R. Powell^§, John N. Klironomos^§, K. Peter Pauls^‡, Clarence J. Swanton^‡, Jack T. Trevors*^† and Kari E. Dunfield*

Departments of Environmental Biology, Plant Agriculture, Integrative Biology, and Land Resource Science, University of Guelph, Guelph, ON, N1G 2W1, Canada

J. Agric. Food Chem., 2008, 56 (15), pp 6339–6347

DOI: 10.1021/jf800767g

Publication Date (Web): June 21, 2008

†

Department of Environmental Biology.

‡

Department of Plant Agriculture.

Department of Integrative Biology.

* To whom correspondence should be addressed. (J.T.T.) Tel: 519-824-4120 ext. 53367. Fax: 519-837-0442. E-mail: jtrevors@uoguelph.ca. (K.E.D.) Tel: 519-824-4120 ext. 58088. Fax: 519-824-5730. E-mail: dunfield@uoguelph.ca.

Department of Land Resource Science.

Abstract

Glyphosate-tolerant, Roundup Ready (RR) soybeans account for about 57% of all genetically modified (GM) crops grown worldwide. The entry of recombinant DNA into soil from GM crops has been identified as an environmental concern due to the possibility of their horizontal transfer to soil microorganisms. RR soybeans contain recombinant gene sequences that can be differentiated from wild-type plant and microbial genes in soil by using a sequence-specific molecular beacon and real-time polymerase chain reaction (PCR). A molecular beacon-based real-time PCR system to quantify a wild-type soybean lectin (le1) gene was designed to compare amounts of endogenous soybean genes to recombinant DNA in soil. Microcosm studies were carried out to develop methodologies for the detection of recombinant DNA from RR soybeans in soil. RR soybean leaf litterbags were imbedded in the soil under controlled environmental conditions (60% water holding capacity, 10/15 °C, and 8/16 h day/night) for 30 days. The soybean biomass decomposition was described using a single-phase exponential equation, and the DNA concentration in planta and in soil was quantified using real-time PCR using sequence-specific molecular beacons for the recombinant cp4 epsps and endogenous soybean lectin (le1) genes. The biomass of RR soybean leaves was 8.6% less than nontransgenic (NT) soybean leaves after 30 days. The pooled half-disappearance time for cp4 epsps and le1 in RR and of le1 in NT soybean leaves was 1.4 days. All genes from leaves were detected in soil after 30 days. This study provides a methodology for monitoring the entry of RR and NT soybean DNA into soil from decomposing plant residues.

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