Stimulation of oleandrin production by combined Agrobacterium tumefaciens mediated transformation and fungal elicitation in Nerium oleander cell cultures

Suspension cultures derived from Agrobacterium tumefaciens-transformed calli were established in Nerium oleander L. The presence of the bacterial T-DNA in the transformed calli was detected by the polymerase chain reaction as well as plant hormone autotrophy. The ability of the cultures to accumulate oleandrin was confirmed using high performance liquid chromatography. The effect of fungal elicitors prepared from Aspergillus niger and Rhizopus stolonifer, on oleandrin production was studied during the suspension cultures of N. oleander. A rapid induction and highest oleandrin production were obtained with 2 ml dry cell powder solution of A. niger-prepared elicitor (with the concentration of 10 g l−1). The oleandrin yield reached a maximum of 3.164 mg l−1 in 25-days upon employing A. niger elicitors. It was 8.8-fold higher than that of control cultures which reached a maximum of 0.35 mg l−1. When R. stolonifer elicitor was used for the same culture period, the maximum oleandrin concentration was 0.34 mg l−1. All the transformed cultures were grown in hormone-autotrophic MS medium supplemented with 30 g l−1 sucrose at 25 °C under diffuse fluorescent light providing 37.5 mmol−2 s−1 light intensity in 12 h photoperiods.