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Rye (Secale cereale L.)
1University of Florida-IFAS, Agronomy Department, PMCB, Genetics Institute, Gainesvilles, FL, USA.
Methods in Molecular Biology (Clifton, N.J.)
|September 22, 2006
Summary
Developing transgenic rye (Secale cereale L.) is challenging. This study optimized Agrobacterium cocultivation in liquid medium, successfully regenerating over 40 fertile, phenotypically normal transgenic plants.
Area of Science:
- Plant Science
- Agricultural Biotechnology
- Molecular Biology
Background:
- Rye (Secale cereale L.) exhibits significant recalcitrance in tissue culture and genetic transformation.
- Embryogenic rye callus rapidly loses regenerative capacity under stress, including high Agrobacterium density.
Purpose of the Study:
- To develop an efficient method for Agrobacterium-mediated genetic transformation of rye.
- To regenerate fertile, phenotypically normal transgenic rye plants.
Main Methods:
- Cocultivation of Agrobacterium and immature rye embryos in a liquid medium.
- Optimization of washing steps to prevent Agrobacterium overgrowth.
- Selection of transgenic events using paromomycin and confirmation via Southern blot, Western blot, and T-DNA boundary sequence analysis.
Main Results:
- Regeneration of over 40 independent transgenic rye plants from 100 inoculated immature embryos.
- Stable integration of the nptII selectable marker gene confirmed by molecular analyses.
- Generated transgenic plants were phenotypically normal and fully fertile.
Conclusions:
- Liquid medium cocultivation is an effective strategy for high-throughput Agrobacterium-mediated rye transformation.
- The optimized protocol overcomes recalcitrance issues, enabling the production of viable transgenic rye.
- Short tissue culture duration may contribute to the normal phenotype and fertility of the regenerated transgenic plants.
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