Volume 91, Issue 5, May (2004), pp. 673-681 © The Author 2004
doi:10.1079/BJN20041100

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Relative stability of transgene DNA fragments from GM rapeseed in mixed ruminal cultures

Ranjana Sharma¹, Trevor W. Alexander^1,2, S. Jacob John³, Robert J. Forster¹ and Tim A. McAllister^1
1Agriculture and Agri-Food Canada Research Center, P.O. Box 3000, Lethbridge, Alberta , Canada, T1J 4B1
²Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada, T6G 2P5
³Aquaculture Centre for Excellence, Lethbridge Community College, Lethbridge, Alberta, Canada, T1K 1L6

 (Received 24 April 2003–Revised 10 October 2003–Accepted 15 January 2004)

The use of transgenic crops as feeds for ruminant animals has prompted study of the possible uptake of transgene fragments by ruminal micro-organisms and/or intestinal absorption of fragments surviving passage through the rumen. The persistence in buffered ruminal contents of seven different recombinant DNA fragments from GM rapeseed expressing the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) transgene was tracked using PCR. Parental and transgenic (i.e. glyphosphate-tolerant; Roundup Ready^®, Monsanto Company, St Louis, MO, USA) rapeseed were incubated for 0, 2, 4, 8, 12, 24 and 48h as whole seeds, cracked seeds, rapeseed meal, and as pelleted, barley-based diets containing 65g rapeseed meal/kg. The seven transgene fragments ranged from 179 to 527bp and spanned the entire 1363bp EPSPS transgene. A 180bp ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit fragment and a 466bp 16S rDNA fragment were used as controls for endogenous rapeseed DNA and bacterial DNA respectively. The limit of detection of the PCR assay, established using negative controls spiked with known quantities of DNA, was 12·5pg. Production of gas and NH₃ was monitored throughout the incubation and confirmed active in vitro fermentation. Bacterial DNA was detected in all sample types at all time points. Persistence patterns of endogenous (Rubisco) and recombinant (EPSPS) rapeseed DNA were inversely related to substrate digestibility (amplifiable for 48, 8 and 4h in whole or cracked seeds, meal and diets respectively), but did not differ between parental and GM rapeseed, nor among fragments. Detection of fragments was representative of persistence of the whole transgene. No EPSPS fragments were amplifiable in microbial DNA, suggesting that transformation had not occurred during the 48h incubation. Uptake of transgenic DNA fragments by ruminal bacteria is probably precluded or time-limited by rapid degradation of plant DNA upon plant cell lysis.

Keywords:
Genetically modified, EPSPS gene fragments, Roundup Ready^®rapeseed, Rubisco, Foreign DNA stability

Abbreviations:
CS, cracked seed, D, pelleted diet, EPSPS, 5-enolpyruvylshikimate-3-phosphate synthase, M, meal, P, parental rapeseed, R, Roundup Ready^® rapeseed, Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase, WS, whole seed