Paper Title
Effect Of Agrobacterium Mediated Transformation On Rosmarinic Acid Production In Basil

Abstract
Rosmarinic acid (RA) is a natural phenolic compound produced by variety species of Lamiaceae and Boraginaceae families. In the recent few years, RA has attracted attention due to its antiviral, antibacterial, anti-inflammatory and anti-allergic activities. In the present study, the efficiency of using hairy root culture technique as a promising alternative method for the production of RA from basil was tested. Seeds of basil were germinated on MS culture medium and the obtained seedlings were used as a source for explants for Agrobacterium transformation and hairy root production. A. rhizogenes strains A4 and R1000 were used for transformation. Leaves and stem of 1-1.5 month old seedlings were used as explants for transformation. Explants were cut and immersed in bacterial suspension for 1hr at RT then cultured on MS solid medium free of growth regulators. Cultures incubated at RT for 3 days in the dark then, explants were transferred on MS medium supplemented with 500 mg/l carbencillin, 100 mg/l kanamycin. Cultures incubated at 25°C in complete darkness. Bacterial free explants that produced hairy roots were subcultured on fresh MS plates containing 400 mg/l carbencillin. Hairy roots were excised from explants when they were 2-3cm long and transferred to new MS plates then they were transferred into liquid MS medium. Hairy root cultures were maintained on a rotary shaker (80-100 rpm) in complete darkness. Results showed that hairy roots were produced when leaf explants were infected with A4 or R1000 strain while, stem explants were unable to produce hairy roots when both Agrobacterium strains used. Transformation frequencies reached 87 % in case of A4 strain and 77 % in R 1000 strain. Hairy roots resulted from R1000 infections were yellowish in color, thick, branched and forming callus at the root base. Hairy roots produced after infection with A4 strain were white, had thin and long shape. Rosmarinic acid levels were determined using HPLC. Results showed that hairy root samples produced higher levels of rosmarinic acid compared to control. Hairy roots accumulated 7.3 folds higher rosmarinic acid levels than control. The results indicated that hairy root culture can be used successfully for enhancement RA production in basil.