Molecular Breeding

, Volume 16, Issue 3, pp 247–260

Transformation of Plants with Multiple Cassettes Generates Simple Transgene Integration Patterns and High Expression Levels

  • Pawan K. Agrawal
  • Ajay Kohli
  • Richard M. Twyman
  • Paul Christou

DOI: 10.1007/s11032-005-0239-5

Cite this article as:
Agrawal, P.K., Kohli, A., Twyman, R.M. et al. Mol Breeding (2005) 16: 247. doi:10.1007/s11032-005-0239-5


We transformed rice (Oryza sativa L.) simultaneously with five minimal cassettes, each containing a promoter, coding region and polyadenylation site but no vector backbone. We found that multi-transgene cotransformation was achieved with high efficiency using multiple cassettes, with all transgenic plants we generated containing at least two transgenes and 16% containing all five. About 75% of the plants had simple transgene integration patterns with a predominance of single-copy insertions. The expression levels for all transgenes, and the overall coexpression frequencies, were much higher than previously reported in whole plasmid transformants. Four of five lines analyzed for transgene expression stability in subsequent generations showed stable and high expression levels over generations. A simple model is proposed, which accounts for differences in the molecular make-up and the expression profile of transgenic plants generated using whole plasmid or minimal cassettes. We conclude that gene transfer using minimal cassettes is an efficient and rapid method for the production of transgenic plants containing and stably expressing several different transgenes. Our results facilitate effective manipulation of multi-gene pathways in plants in a single transformation step.


Plant transformationCassette transformationMultigene transgenicsPathway engineeringGene expressionTransgenic loci

Copyright information

© Springer 2005

Authors and Affiliations

  • Pawan K. Agrawal
    • 1
  • Ajay Kohli
    • 2
  • Richard M. Twyman
    • 3
  • Paul Christou
    • 4
  1. 1.Central Rice Research InstituteCuttackIndia
  2. 2.Institute for Research on Environment and Sustainability, School of BiologyUniversity of Newcastle-upon-TyneNewcastle-upon-TyneUK
  3. 3.Department of BiologyUniversity of YorkHeslingtonUK
  4. 4.Department de Produccio Vegetal i Ciencia ForestalICREA, Universitat de LleidaLLEIDASpain