IMPROVED CONDITIONS FOR AGROBACTERIUM-MEDIATED TRANSFORMATION OF ALMOND

An improved system for Agrobacterium-mediated almond transformation was developed.
A 100 fold increase in efficiency (12.3%) in comparison to the available transformation method (0.1%) was observed in spite of the lower regeneration ability of the almond explants (20-30%, instead of 70-90% in the existing method). Key modifications included use of 150 µM acetosyringone (AS) during the induction period (21 days), and a different selection scheme.
Bacteria were directly applied onto wounds.
After three days of co-cultivation, bacteria were removed by washing with a 200 µg ml^-1 cefotaxime solution and explants were then transferred to fresh induction medium with 300 µg ml^-1 cefotaxime, 150 µM AS and 15 µg ml^-1 kanamycin.
Kanamycin was maintained at 15 µg ml^-1 during sub-cultures on shoot elongation medium and gradually raised after shoot transfer to micropropagation medium (one sub-culture at 15 µg ml^-1, one sub-culture at 30 µg ml^-1, and the following at 50 µg ml^-1). Surviving shoots were propagated on 50 µg ml^-1 kanamycin for 2.5 years.
Transformed shoots tested positive by PCR, GUS assay and Southern blotting.
The improved strategy is being used with cultivar material and is available for gene function analyses in almond.

International Symposium on Biotechnology of Temperate Fruit Crops and Tropical Species

M. Costa, C. Miguel, M.M. Oliveira

acetosyringone, kanamycin, Prunus dulcis

https://doi.org/10.17660/ActaHortic.2007.738.74