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A Technique to Screen American Beech for Resistance to the Beech Scale Insect (Cryptococcus fagisuga Lind.)
Published on: May 27, 2014
American Chestnut [Castanea dentata (Marsh.) Borkh]
Charles A Maynard1, Linda D Polin, Sharon L LaPierre
1SUNY College of Environmental Science and Forestry, State University of New York, Syracuse, NY, USA.
Methods in Molecular Biology (Clifton, N.J.)
|October 13, 2006
Summary
Successful American chestnut transformation relies on specific somatic embryo tissues, the bar gene for selection with phosphinothricin (PPT), and the mgfp5-ER gene for non-destructive scoring, achieving ~20% efficiency.
Area of Science:
- Plant Biotechnology
- Forest Genetics
- Molecular Biology
Background:
- American chestnut restoration faces challenges in genetic transformation.
- Efficient regeneration and selection systems are crucial for developing disease-resistant or otherwise improved American chestnut varieties.
Purpose of the Study:
- To establish a reliable protocol for the genetic transformation of American chestnut using somatic embryos.
- To identify optimal explant tissues, selectable markers, and regeneration conditions for high transformation efficiency.
Main Methods:
- Utilized rapidly dividing somatic embryos (proembryogenic masses) as the primary explant tissue.
- Employed the bar gene as a selectable marker with phosphinothricin (PPT) at 1.0–10 mg/L.
- Used the mgfp5-ER gene as a nondestructive scorable marker.
- Optimized gentle handling of somatic embryos during inoculation and co-cultivation.
Main Results:
- Achieved an average transformation efficiency of approximately 20 stable and embryogenic transformation events per gram of somatic embryo tissue.
- Demonstrated that gentle treatment of somatic embryos significantly increases transformation efficiency.
- Confirmed the efficacy of the bar and mgfp5-ER genes for selection and scoring, respectively.
- Somatic embryos were successfully induced to form shoots for subsequent micropropagation and acclimatization.
Conclusions:
- The developed protocol provides a robust method for the genetic transformation of American chestnut.
- The combination of specific somatic embryo tissues, the bar/PPT system, and the mgfp5-ER marker offers a reliable approach for genetic improvement.
- Gentle handling is a critical factor for maximizing transformation success in American chestnut.
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