Investigations into the potential of plant tissue culture for the development of diesel-resistant Petunia grandiflora Juss. mix F1 and Marigold-Nemo mix (Tagetes patula L.) plants A thesis submitted in the partial fulfilment of the requirements for the Degree of Doctor of Philosophy in Biotechnology at the University of Canterbury by Solomon Peter Wante 2019 ABSTRACT 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 ii 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 iii 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. iv DEDICATION This Ph.D. thesis is dedicated to God Almighty; my beloved parents, Peter Wante and Larei Peter; my lovely wife, Shiktira Solomon; my dear children, Joy and Neriah; and my beloved sisters (Hannatu, Rhoda and Rahila) and brother (Haruna). v ACKNOWLEDGMENTS I am deeply grateful to my supervisor, Professor David Leung for his mentorship that allowed me to explore the scientific realm and develop my research skills. I really appreciate your patience, understanding, and encouragement that helped me to go further to where I am today. Special thanks also to my co-supervisor, Professor David Collings for his support in this project. I sincerely appreciate Federal University Kashere for the award of TETFund Nigeria Government Scholarship. I am grateful to the School of Biological Sciences, University of Canterbury for the Student Travel Grant award to attend and present a poster at the 15th Asia-Pacific Biotechnology Congress, Melbourne, Australia and also for the award of UC Foundation Doctoral Publication Scholarship. I am also indebted to the New Zealand Society of Plant Biology for the Travel Grant award to attend and present a talk at the Plant Science Central Conference, Palmerston North, New Zealand. I am thankful to the School of Biological Science and Chemistry Department, in particular, the technical staff: Craig Galilee, David Conder, Reijel Gardiner, Nicholas Etheridge, Nicole Lauren-Manuera and Tomas Davison. Special thanks to Mathew Walter (photography and posters) and Mathew Polson (guidance on the use of GC FID). They have been supportive through their knowledgeable skills. I am so thankful to Simon Enochson for his generosity of his time to assist in the glasshouse experiment and to my new and old lab mates for their friendship and helping me in many ways. I am grateful to Dr. Hossein Alizadeh for his generous assistance in statistical analysis. A very special thanks to my beloved wife (Shiktira) and my lovely daughters (Joy and Neriah) for their love, understanding, and sacrifices. Many thanks to my entire family including my in-laws for their prayers, long waiting, financial support, and encouragement. vi Contents Abstract……………………………………………………………………………………..ii Dedication…………………………………………………………………………………..v Acknowledgments…………………………………………………………………………..vi Contents………………………………………………………………………………….....vii List of figures………………………………………………………………………………xiv List of tables………………………………………………………………………………xxx Abbreviations……………………………………………………………………………xxxiii Publication and conference proceedings arising from this thesis………………………xxxviii Chapter 1 Introduction and literature review…………………………………………….1 1.1 Petroleum hydrocarbons………………………………………………………….1 1.1.1 Petroleum hydrocarbons contamination………………………………..2 1.1.2 Toxicity of petroleum hydrocarbons…………………………………...2 1.2 Diesel……………………………………………………………………………..5 1.2.1 Effect of diesel on plants……………………………………………….5 1.3 Remediation technologies of PAHs………………………………………………6 1.3.1 Remediation…………………………………………………………….6 1.3.1.1 Non-bioremediation technologies…………………………….7 1.3.1.2 Bioremediation………………………………………………..8 1.3.1.3 Phytoremediation studies using ornamental plants…………..11 1.3.1.3.1 Background on Petunia grandiflora and Tagetes patula…………………………………………………………… ………………………………………………………………..11 1.3.1.3.2 Phytoremediation studies…………………………..12 1.4 Plant tissue culture techniques…………………………………………………...13 1.4.1 Explant and in vitro cultures……………………………………………14 1.4.2 Callus culture and plant regeneration…………………………………..15 1.4.3 Somaclonal variation…………………………………………………...15 1.4.3.1 Cell line selection………………………………………………...15 1.4.3.2 Histological analyses of callus culture…………………………...16 1.5 Morphological analyses of somaclonal variants generated in vitro ….…..………17 1.5.1 In vitro evaluation………………………………………………………17 1.5.2 Evaluation under glasshouse conditions………………………………..20 vii 1.6 Aims, objectives and structure of the thesis ……………………………………..22 Chapter 2 General materials and methods ………………………….……………………25 2.1 Plant materials……………………………………………………………………25 2.1.1 Seeds……………………………………………………………………25 2.1.2 Analysis of diesel fuel…………………………………………………..25 2.1.3 Preparation of diesel-contaminated water solution …………………….25 2.1.4 Treatment of seed using water contaminated with diesel ………………25 2.1.5 Measurement of root length, shoot height and plant size……………….26 2.1.6 Elongation inhibition rates ……………………………………………..26 2.2 In vitro medium composition and culture conditions …………………………….26 2.2.1 Medium composition and culture conditions for seeds of Petunia grandiflora and Tagetes patula germinated in vitro…………………………………………………………………………..26 2.2.2 Seed germination under aseptic conditions ……………………………26 2.2.3 Medium composition and culture conditions for callus induction and sub- culturing in Petunia grandiflora and Tagetes patula…………………………27 2.2.4 Callus