Influence of toxic diesel fuel on calli and after plant regeneration.

The toxic effects of diesel fuel on whole plants have been reported before, but little is known about the toxic effect of diesel fuel on callus cultures. This knowledge is a pre-requisite for exploring the possibility of using a sub-lethal diesel concentration as an agent for in vitro cell line selection to obtain novel somaclonal variants resistant to diesel toxicity. These novel variants could be useful for the phytoremediation of diesel-contaminated soil. Here, a callus induction medium [Murashige and Skoog medium supplemented with 1.8 µM of naphthlene-1-acetic acid (NAA) and 6.6 µM of 6-benzyladenine (BA)] was found to induce 85% of leaf explants to form light green calli. Since it was not possible to include diesel in aseptic culture, the calli were exposed to diesel under non-aseptic conditions. It was found that the calli did not exhibit any sign of necrosis immediately after up to 9 min of diesel exposure. The diesel-treated calli were subsequently subcultured successfully on the callus induction medium using the proliferating, non-necrotic cells. Transverse sections of the control and diesel-treated calli after 2 weeks of culture revealed that the control calli exhibited more small meristematic cells while diesel-treated calli exhibited larger, empty-looking parenchyma cells. Moreover, it was possible to induce, though at a low frequency (< 15%), shoot formation in the control calli and those derived from the diesel treatment on the Murashige and Skoog medium supplemented with 1.1 µM of indole-3-acetic acid (IAA) and 13.3 µM of BA. Under glasshouse conditions, the shoots regenerated from the calli derived from the diesel treatment exhibited higher biomass than those from the control calli and seedlings when grown in a potting mix spiked with 0%, 2% and 7% diesel. Taken together, these results suggest that up to 9 min of diesel exposure of calli was sub-lethal.