Anthropogenic use of petroleum hydrocarbons has contributed to the toxic cocktails of pollutants that could threaten the sustainability of biodiversity on the Earth. For example, there are many studies showing the toxic effects of diesel on humans and plants. Considering that plants can provide many beneficial services to the ecosystem, it would be a worthwhile contribution if diesel-resistant plants could be identified from germplasm screening or be developed with the aid of plant biotechnology such as plant tissue culture. In particular, if diesel-resistant non-food plants such as ornamental plants could be developed, the resistant plants might be deployed to add economic value to the land contaminated with diesel. With the long-term goal of producing plants that can be used in phytoremediation of diesel-contaminated land, the present project was initiated to investigate the possibility of using plant tissue culture techniques to generate diesel-resistant Petunia grandiflora (petunia) and Tagetes patula (marigold).

There was no prior study on the effect of diesel on petunia and marigold, and therefore, the present work began investigating the relative sensitivity of petunia and marigold seeds and seedlings to water contaminated with 0–4% diesel in Petri dishes under controlled laboratory conditions. Generally, in the presence of 0.5% to 4% diesel, there was a delay in the speed of seed germination of both marigold and petunia. It was also found that marigold and petunia exhibited differential sensitivities to diesel contamination during germination and early seedling growth. This key finding has not been reported before.

Plant tissue culture has been applied to develop novel plants resistant to different abiotic stress agents that were included in the culture medium or treatment of the cultures before plant regeneration. However, there was no prior report of diesel-resistant plants obtained by using plant tissue culture. There are some technical challenges in using diesel as a stress agent to select for variant plant cells resistant to diesel toxicity. For example, diesel cannot be applied to plant tissue culture medium or the culture environment.

In this study, the requirements were established for high efficiency of callus initiation, subculture and plant regeneration in petunia and marigold callus cultures. A novel protocol to expose petunia calli to diesel under non-aseptic operating conditions and then subculture of the diesel-treated petunia calli under aseptic conditions was first demonstrated. This was also validated with the callus culture of marigold.

In a histological study of the petunia and marigold after diesel exposure at 500 μm and 1500 μm from the top of the calli, it was found that the internal organisation of the petunia and marigold calli were different from the respective calli not exposed to diesel. More regions with meristematic cells were observed in the petunia calli without prior exposure to diesel at 500 μm from the top than in the diesel-treated calli. This was not the case in marigold calli at 500 μm; the diesel-treated calli had a higher density of lignified cell walls and xylem vessels than the calli without prior exposure to diesel. Deep into the marigold calli, at 1500 μm from the top of the control callus, the prominent shoot apical meristem dome with leaf primordia was revealed, but this type of feature did not appear to be found in the diesel-treated calli. In the diesel-treated petunia calli, they appeared to have more tracheary elements and lignified cell walls than the control at 1500 μm from the top.

Six experimental lines of plantlets (L1–L6) were regenerated from petunia calli exposed to undiluted diesel for 9 min. Two control lines of plants, one from germinated seed (C-G) and one line of plantlets regenerated from the calli that were not treated with diesel (C-R) were also used for comparison to determine the relative growth performance of the six experimental lines in the absence of diesel (evaluation under in vitro conditions), and in the diesel-spiked potting mix under glasshouse conditions. One line (L4) exhibited plant vigour of interest for future studies into the diesel tolerance mechanism in plants. The potential of producing diesel-resistant plants is promising for their application to phytoremediation of diesel-contaminated landscapes.