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Investigation into the Arabidopsis transformant selection time and escapes frequency reduction

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Author(s): Ali DEHESTANI | Gholamreza AHMADIAN | Ali Hatef SALMANIAN | Nadali Babaeian JELODAR | Mehdi SEYEDI | Kamal KAZEMITABAR

Journal: Biharean Biologist
ISSN 1843-5637

Volume: 3;
Issue: 1;
Start page: 51;
Date: 2009;
Original page

Keywords: In planta transformation | Secondary leaves | seedling morphology | selection efficiency | Arabidopsis | Antibiotic concentration

ABSTRACT
Selection of putative transformants after Arabidopsis in planta transformation and the main factors involved in are comprehensively discussed. Different antibiotic concentrations, basal medium composition, incubation condition of selection plates and various selection criteria were investigated. Results showed that although kanamycin concentrations from 35 to 75 mgl-1 could discriminate transformants from non-transformed seedlings, optimum selection was carried out on plates with 75 mgl-1 kanamycin. This concentration of kanamycin along with three days dark treatment before light incubation of selection plates resulted in etiolated seedlings which were distinguished quickly by their small dark green secondary leaves. Secondary leaves development was demonstrated to be the most reliable criteria for rapid and accurate selection of transformants with a selection efficiency of 97.5%. In contrast, variations in root length and seedling colour complicated the selection process resulting in more wrong selections. Addition of sucrose to selection medium deferred the selection procedure by repressing antibiotic toxicity; while sucrose omission accelerated discrimination of transformants form bleached non-transformed seedlings. We strongly recommend use of 75 mgl-1 kanamycin supplemented medium, 3 day dark treatment before light incubation of plates, and selection of transformants using secondary leaves development criteria for rapid and accurate selection of transformed seedling. These modifications would eliminate false positive transformants while reducing selection period to 6 days.

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