Pre- and post-agroinfection strategies for efficient leaf disk transformation and regeneration of transgenic strawberry plants

• DOI: 10.1007/s00299-009-0801-4
• Publication Date: 2009-12-02T01:49:09Z
• Authors: Amjad Masood Husaini
• Keywords: 0106 biological sciences; 0301 basic medicine; DNA, Plant; Phenylurea Compounds; Acetophenones; Plants, Genetically Modified; Fragaria; 01 natural sciences; Anti-Bacterial Agents; Culture Media; Plant Leaves; Tissue Culture Techniques; 03 medical and health sciences; Agrobacterium tumefaciens; Kanamycin; Thiadiazoles; Regeneration; Transgenes; Genetic Engineering; Plant Shoots

Following previously described Agrobacterium tumefaciens-mediated transformation procedures for Fragaria x ananassa Duch. 'Chandler', we undertook several experiments to establish the importance of some parameters affecting transformation. The most important factor that increased the percent recovery of transformants was the introduction of a pre-selection phase, in-between co-cultivation and selection, in which leaf disks were cultured on pre-selection regeneration medium containing validamycin A, timentin, and cefotaxime. The average percentage of leaf disks forming shoots on selection medium containing cefotaxime (250 mg l(-1)) + timentin (250 mg l(-1)) was 5.4% and about three shoots per regenerating leaf disk. Maximum transformation percentage, based on polymerase chain reaction, was 31.25%. Transgene integration and copy number were assessed by Southern hybridization confirming single copy as well as multiple copies of transgene integration in shoots as well as roots separately. This confirmed the non-chimeric nature of these transgenic plants. The system is very promising for the regeneration of genetically transformed cells and obtaining transgenic strawberry plants at high efficiency.