JXB Advance Access originally published online on April 8, 2004
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Journal of Experimental Botany, Vol. 55, No. 401, pp. 1307-1313, June 1, 2004
© 2004 Oxford University Press
RESEARCH PAPER |
Strategies for precise quantification of transgene expression levels over several generations in rice
Received 29 October 2003; Accepted 16 February 2004
John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK
* Present address: Agronomy Department, University of Florida, Gainesville, FL 32611-0300, USA. To whom correspondence should be addressed. Fax: +44 (0)1603 450023. E-mail: philippe.vain{at}bbsrc.ac.uk
Variation in transgene expression levels can result from uncontrolled differences in experimental protocols. Studies conducted over generations could, by their design, generate additional unwanted variation. To study sources of spurious variation, transgene expression levels were quantified over five homozygous generations in two independent transgenic rice lines created by particle bombardment. Both lines contained the same gus expression unit and had been shown to exhibit stable inheritance of transgene structure and expression. All plants were cultured and sampled using previously developed standardized protocols. Plants representative of each generation (T2, T3, T4, T5, T6) were grown either all together or across several different growth periods. GUS activity in plants from different generations was quantified either in the same assay or over multiple independent assays. Strategies in which plants were grown and phenotyped independently, significantly increased (up to 3-fold) extraneous variation in transgene expression level quantification, thus reducing the precision of molecular genetic studies and generating artefactual results in transgenic studies conducted over generations. Identification of sources of unwanted variation and quantification of their effect allowed the development of new strategies designed to control spurious variation. Growth and phenotyping of all plants from all generations together, using standard operating procedures (SOP), led to a reduction in extraneous variation associated with transgene expression level quantification. Adoption of such strategies is key to improving the reproducibility of transgenic studies conducted over generations.
Key words: Generational study, matrix attachment regions (MARs), Oryza sativa L., reproducibility, spurious variation, transgenic plants.