Abstract
Cre-lox mediated site-specific integration in tobacco or Arabidopsis used polyethylene glycol or Agrobacterium, respectively, to deliver the integrating DNA. The polyethylene glycol method is inconvenient since it requires the use of protoplasts. The Agrobacterium method is inefficient as the single-stranded T-DNA is not a substrate for Cre-lox recombination. In this study, we tested the biolistic method for the site-specific insertion of DNA into the rice genome. Two target callus lines, each harboring a single genomic lox target, were generated by Agrobacterium-mediated transformation. The target callus lines were subjected to a second round of transformation by particle bombardment with a construct designed to excise the plasmid backbone from the integrating DNA, followed by the recombination of the integrating DNA into the genomic lox target. Site-specific integration was obtained from both target callus lines. Three integrant plants were regenerated from one target line and were found to have a precise copy of the integrating DNA at the target site, although only one plant has the integrating DNA as the sole copy in the genome. Site-specific integration through the biolistic delivery of DNA can be considered for other plants that are transformable via particle bombardment.
References
Albert H., Dale E.C., Lee E. and Ow D.W. 1995. Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant J. 7: 649–659.
Day C.D., Lee E., Kobayashi J., Holappa L. D., Albert H. and Ow D.W. 2000. Transgene integration into the same chromosomal location can produce alleles that express at a predictable level, or alleles that are differentially silenced. Genes Dev. 14: 2869–2880.
Hajdukiewiecz P., Svab Z. and Maliga P. 1994. The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol. Biol. 25: 989–994.
Hohn B., Levy A.A. and Puchta H. 2001. Elimination of selection markers from transgenic plants. Curr. Opin. Biotech. 12: 139–143.
Iyer L.M., Kumpatla S.P., Chandrasekharan M.B. and Hall T.C. 2000. Transgene silencing in monocots. Plant Mol. Biol. 43: 323–346.
Lyznik L.A., Rao K.V. and Hodges T.K. 1996. FLP-mediated recombination of FRT sites in the maize genome. Nucl. Acid Res. 24: 3784–3789.
Maqbool S.B. and Chritou P. 1999. Multiple traits of agronomic importance in transgenic indica rice plants: analysis of transgene integration patterns, expression levels and stability. Mol. Breed. 5: 471–480.
Matzke M.A., Mette M.F. and Matzke A.J.M. 2000. Transgene silencing by the host genome defense: implications for the evolution of epigenetic control mechanisms in plants and vertebrates. Plant Mol. Biol. 43: 401–415.
Meyer P. 2000. Transcriptional transgene silencing and chromatin component. Plant Mol. Biol. 43: 221–234.
Muskens M.W.M., Vissers A.P.A., Mol J.N.M. and Kooter J.M. 2000. Role of inverted DNA repeats in transcriptional and posttranscriptional gene silencing. Plant Mol. Biol. 43: 243–260.
Ow D.W. and Medberry S.L. 1995. Genome manipulation through site-specific recombination. Critical Rev. Plant Sci. 14: 239–261.
Puchta H. and Hohn B. 1996. From centiMorgans to base pairs: homologous recombination in plants. Trends Plant Sci. 1: 340–348.
Vergunst A.C., Jansen L.E.T. and Hooykaas P.J.J. 1998. Site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana mediated by Cre recombinase. Nucl. Acids Res. 26: 2729–2734.
Vergunst, A.C. and Hooykaas, P.J.J. 1998. Cre/lox-mediated sitespecific integration of Agrobacterium T-DNA in Arabidopsis thaliana by transient expression of cre. Plant Mol. Biol. 38: 393–406.
Vergunst, A.C. and Hooykaas, P.J.J. 1999. Recombination in the plant genome and its application in biotechnology. Crit. Rev. Plant Sci. 18: 1–31.
Zuo J., Niu Q.-W., Moller S.G. and Chua N.-H. 2001. Chemicalregulated, site-specific DNA excision in transgenic plants. Nat. Biotechnol. 19, 157–161
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Srivastava, V., Ow, D.W. Biolistic mediated site-specific integration in rice. Molecular Breeding 8, 345–349 (2002). https://doi.org/10.1023/A:1015229015022
Issue Date:
DOI: https://doi.org/10.1023/A:1015229015022