Sub-lethal effect of diesel fuel on the development of in vitro produced callus culture of Tagetes patula L. (Marigold-Nemo mix)

Plant tissue culture has been applied to study different abiotic stress agents that were included in the culture medium for treatment of callus. Here, the requirements for high efficiency (about 95%) of callus initiation in leaf explants of Tagetes patula (marigold) were first established. Green calli were formed on Murashige and Skoog medium supplemented with 1.8 µM NAA and 66.6 µM BA. There was no prior study on the effect of diesel on T. patula callus, probably because diesel cannot be applied to plant tissue culture medium or used safely in the culture environment. These technical challenges were overcame here using a new protocol for exposure of T. patula calli to diesel fuel under non-aseptic conditions whereas earlier the diesel-treated calli were cultured under aseptic conditions. Immediately after up to 9 min of diesel exposure, the T. patula calli did not showed any sign of necrosis. Non-necrotic, proliferating T. patula calli were subsequently obtained on a callus induction medium supplemented with 1.8 µM NAA and 66.6 µM BA. In a histological study, the internal organisation of the diesel-treated T. patula calli were different from the control calli. In the transverse sections at 500 µm taken from the surface of the control calli, there were more cells with lignified cell walls and xylem vessels than the diesel-treated calli. Well-formed shoot apical meristem domes with leaf primordia were found in the transverse sections at 1500 µm from the surface of the control callus but were absent in the diesel-treated calli. This result suggests that the innovative protocol of diesel exposure was sub-lethal, and after further investigation might enable the extension for the application of in vitro elicitation technique to produce callus culture or shoots with the desired amount of secondary metabolite.