The seeds of A. precatorius were collected from the medicinal plant garden of Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar, M.P., India. Seeds were sterilized and germinated by following the protocol described in our previous publication .
Initiation of A. precatorius cell cultures
Different explants from aseptically germinated seeds viz. leaves, epicotyle and petiole were tested for culture initiation by variation in plant growth regulators (PGR) and Agrobacterium mediated transformation. Non-transformed callus cultures were initiated by placing explants on solidified MS medium supplemented separately with the hormones: 1 mg/l naphthalene acetic acid (NAA); 1 mg/l Kinetin (Kn); 0.5 – 2.0
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The agrobacterial suspension was centrifuged at 6000 g for 10 min. The supernatant was discarded and the pellet was resuspended in 5 ml of the fresh liquid nutrient agar medium. This concentrated culture was used for infection of plant materials. The explants were kept in sterile petriplates; explant margins were incised manually with sterile scalpel, dipped in A. tumefaciens and incubated for 10-20 min. In another experiment, 2 ml of acetosyringone solution at different concentrations (10, 25 and 50 M) were also added along with suspension of Agrobacterium strain to be tested. Nutrient agar medium without bacteria applied to the explants served as a control. Excess Agrobacteria were then removed by blotting the infected explants on sterile filter paper. The infected leaves (with or without acetosyringone) were then co-cultivated at 27 ± 2°C for 48 h on solidified MS media in 12/12h light/dark regime. The transformed cultures were then transferred to fresh MS medium containing varying concentration of acetosyringone for better transformation efficiency and 500 mg/l cefotaxime to check the overgrowth of bacteria. Axenic cultures were obtained by subsequent subculture to fresh MS medium containing the same antibiotic (subsequently with decreased concentration) every 4 d. The transformed cultures were checked for Agrobacterium contamination by culturing samples on nutrient agar medium after