Investigations Into the Potential of Plant Tissue Culture for the Development of Diesel-Resistant Petunia Grandiflora Juss

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.

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).

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.

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

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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 induction and proliferation in Petunia grandiflora and Tagetes patula…………………………………………………………………………27 2.2.5 Morphology and selection of calli formed in Petunia grandiflora and Tagetes patula………………………………………………………………...28 2.2.6 Maintenance of callus culture of Petunia grandiflora and Tagetes patula…………………………………………………………………………28 2.3 Exposure of callus to diesel fuel………………………………………………….28 2.3.1 Exposure of Petunia grandiflora and Tagetes patula callus to diesel toxicity………………………………………………………………………..28 2.3.2 Culture of diesel-treated Petunia grandiflora and Tagetes patula calli under aseptic conditions………………………………………………………29 2.3.3 Selection of Petunia grandiflora and Tagetes patula calli that survived after diesel exposure………………………………………………………….29 2.3.4 Exposure of Petunia grandiflora calli to sterile distilled water, diesel, and Plant Preservative Mixture (PPM)……………………………………………30 2.4 Plant regeneration from callus……………………………………………………31 2.4.1 Plant regeneration attempts in Tagetes patula…………………………..31

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2.4.2 Plant regeneration in Petunia grandiflora from non-diesel-exposed calli and those that survived diesel treatment………………………………………31 2.4.3 Morphology of different types of shoots formed in Petunia grandiflora.32 2.4.4 Shoot multiplication and root formation in Petunia grandiflora……….32 2.4.5 Petunia grandiflora plantlet lines and growth conditions………………32 2.4.6 Growth analyses……………………………………………………..….33 2.5 Hardening off and acclimatisation of Petunia grandiflora plantlets………………33 2.5.1 Under conditions of a controlled growth chamber………………………33 2.5.2 Under glasshouse conditions……………………………………………34 2.6 Glasshouse study design………………………………………………………….34 2.6.1 Experimental Petunia grandiflora plant lines tested……………………34 2.6.2 Site description…………………………………………………………35 2.6.3 Potting mix and diesel-contaminated potting mix………………………35 2.6.4 Experimental design……………………………………………………36 2.7 Analyses of plants grown under the glasshouse conditions………………………36 2.7.1 Number of leaves and leaf chlorosis rating……………………………..36 2.7.2 Shoot Analyses…………………………………………………………36 2.7.3 Biomass production…………………………………………………….39 2.8 Microbial plate counts……………………………………………………………39 2.9 Analysis of diesel in potting mix…………………………………………………40 2.9.1 Ultrasonic extraction (Method 3550C by USEPA (2007))…………….40 2.9.2 Total petroleum hydrocarbon (TPH) Analysis…………………………40 2.10 Statistical Analyses……………………………………………………………...40 Chapter 3 Phytotoxicity testing of diesel-contaminated water using Petunia grandiflora Juss. mix F1 and Marigold-Nemo mix (Tagetes patula L.)………………………………..42 3.1 Introduction………………………………………………………………………42 3.2 Materials and Methods……………………………………………………………43 3.2.1 Seeds……………………………………………………………………43 3.2.2 Treatment of seeds using water contaminated with diesel……………..43 3.2.3 Experimental design and statistical analysis……………………………44 3.3 Results……………………………………………………………………………45 3.3.1 Toxic effect of diesel-contaminated water on seed germination……….45 3.3.2 Toxic effect of diesel-contaminated water on seedling growth………...49 3.3.3 Elongation inhibition rate……………………………………………….52

3.4 Discussion………………………………………………………………………...56 3.5 Conclusion………………………………………………………………………..58 Chapter 4 Induction of callus in leaf explants of Petunia grandiflora Juss. mix F1 for experimental exposure to diesel and establishment of plant lines from calli that survived diesel treatment……………………………………………………………………………..59 4.1 Introduction………………………………………………………………………59 4.2 Materials and methods……………………………………………………………61 4.2.1 Plant materials and culture conditions…………………………………………..60 4.2.1.1 Seed…………………………………………………………………..60 4.2.1.2 Seed germination under aseptic conditions…………………………..60 4.2.1.3 Callus induction and proliferation…………………………………….60 4.2.1.4 Morphology of the calli formed……………………………………….60 4.2.1.5 Maintenance of callus culture…………………………………………61 4.2.1.6 Exposure of callus to diesel fuel………………………………………61 4.2.1.7 Culture of diesel-treated callus under aseptic conditions……………...61 4.2.1.8 Selection and multiplication of calli that survived after diesel treatment……………………………………………………………………...61 4.2.1.9 Plant regeneration from non-diesel-exposed calli and those that survived diesel treatment……………………………………………………………….62 4.2.1.10 Morphology of different types of shoots formed……………………62 4.2.1.11 Shoot multiplication and root formation…………………………….62 4.3 Statistical analysis………………………………………………………………...65 4.4 Results……………………………………………………………………………65 4.4.1 In vitro seed germination and seedling growth…………………………65 4.4.2 Efficiency of callus induction…………………………………………..66 4.4.3 Morphology of the calli formed…………………………………………67 4.4.4 Appearance of callus on subculture medium…………………………..73 4.4.5 Manipulations of diesel-treated callus under aseptic conditions……….75 4.4.6 Diesel-treated callus under aseptic conditions…………………………..78 4.4.7 Effect of IAA, NAA, Zeatin and BA concentrations on shoot regeneration from different groups of calli…………………………………………………79 4.4.8 Morphology of the different types of shoots formed……………………80

4.4.9 Shoot multiplication and root formation……………………………….95 4.5 Discussion………………………………………………………………………..97 4.6 Conclusion………………………………………………………………………103 Chapter 5 Callus induction in leaf explants of Marigold-Nemo mix (Tagetes patula L.) and exposure to diesel……………………………………………………………………..104 5.1 Introduction……………………………………………………………………..104 5.2 Materials and methods…………………………………………………………..105 5.2.1 Plant materials and culture conditions…………………………………………105 5.2.1.1 Seeds………………………………………………………………..105 5.2.1.2 Seed germination under aseptic conditions………………………….105 5.2.1.3 Callus induction and proliferation…………………………………...105 5.2.1.4 Morphology of the calli formed……………………………………...105 5.2.1.5 Maintenance of callus culture……………………………………….105 5.2.1.6 Exposure of callus to diesel fuel…………………………………….105 5.2.1.7 Culture of diesel-treated callus under aseptic conditions……………106 5.2.1.8 Selection of calli that survived after diesel exposure……………….106 5.2.1.9 Plant regeneration trials……………………………………………..106 5.3 Statistical analysis……………………………………………………………….108 5.4 Results…………………………………………………………………………..109 5.4.1 In vitro seed germination and seedling growth……………………….109 5.4.2 Efficiency of callus induction…………………………………………110 5.4.3 Appearance of calli formed…………………………………………....110 5.4.3 Appearance of callus after subculture…………………………………119 5.4.4 Manipulations of diesel-treated callus under aseptic conditions………120

5.4.5 Effect of IAA, NAA, zeatin, GA3 and BA concentrations on shoot regeneration from different groups of calli………………………………….124 5.5 Discussion……………………………………………………………………….134 5.6 Conclusion………………………………………………………………………140 Chapter 6 A histological comparison of Petunia grandiflora Juss. mix F1 and Marigold- Nemo mix (Tagetes patula L.) calli after exposure to diesel……………………………..141 6.1 Introduction……………………………………………………………………..141 6.2 Materials and Methods…………………………………………………………..141 6.2.1 Callus source…………………………………………………………..141 6.2.2 Callus fixation in FAA (formaldehyde–acetic acid–ethanol)………….141

6.2.3 Dehydration in a series of tertiary butanol alcohol (TBA)……………142 6.2.4 Callus infiltration in paraffin and tertiary butanol alcohol ……………142 6.2.5 Callus embedding……………………………………………………..143 6.2.6 Callus sectioning………………………………………………………143 6.2.7 Callus staining and coverslip………………………………………….143 6.2.8 Microscopic observations……………………………………………..144 6.3 Results…………………………………………………………………………..144 6.3.1 Transverse section of Petunia grandiflora (petunia) callus 500 µm from the top……………………………………………………………………….144 6.3.2 Transverse section of Petunia grandiflora (petunia) callus 1500 µm from the top……………………………………………………………………….148 6.3.3 Transverse section of Tagetes patula (marigold) callus 500 µm from the top…………………………………………………………………………...151 6.3.4 Transverse section of Tagetes patula (marigold) callus 1500 µm from the top…………………………………………………………………………..154 6.4 Discussion………………………………………………………………………157 6.5 Conclusion………………………………………………………………………163 Chapter 7 Growth performance of Petunia grandiflora Juss. mix F1 plant lines developed from calli after diesel exposure: Evaluation under in vitro and glasshouse conditions…………………………………………………………………………………..165 7.1 Introduction……………………………………………………………………..165 7.2 Materials and methods…………………………………………………………..166 7.2.1 Plant material and growth conditions………………………………….166 7.2.1.1 Morphological parameters…………………………………..166 7.2.2 Hardening off and acclimatisation of Petunia grandiflora plantlets……………………………………………………………………..167 7.2.3 Glasshouse study design………………………………………………167 7.2.3.1 Tested plant materials……………………………………….167 7.2.3.2 Site description……………………………………………...167 7.2.3.3 Potting mix and diesel-contaminated potting mix…………..167 7.2.4. Leaf growth…………………………………………………………...167 7.2.5 Shoot growth…………………………………………………………..168 7.2.6 Biomass production…………………………………………………...168 7.2.7 Microbial plate counts…………………………………………………168

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7.2.8 Analysis of the diesel-contaminated potting mix used………………..168 7.2.8.1 Ultrasonic extraction (Method 3550C by USEPA (2007))….168 7.2.8.2 Total petroleum hydrocarbon (TPH) contents……………….168 7.3 Statistical analysis……………………………………………………………….169 7.4 Results…………………………………………………………………………..169 7.4.1 Morphological characteristics…………………………………………169 7.4.2. Growth of experimental and control Petunia grandiflora lines under glasshouse conditions……………………………………………………….182 Line L1……………………………………………………………...182 Line L2……………………………………………………………...183 Line L3……………………………………………………………...183 Line L4……………………………………………………………...184 7.4.3. Total microbial plates count…………………………………………206 7.4.4. Total petroleum hydrocarbon analyses………………………………208 7.5 Discussion……………………………………………………………………….210 7.6 Conclusion………………………………………………………………………220 Chapter 8 Overview of results and recommendations…………………………………..221 8.1 Significance of the main research strategy and findings………………………..221 8.2 Insight from the histological study of calli following diesel exposure………….222 8.3 Plant regeneration was a key to establishment of potentially novel plantlet lines from diesel-treated petunia calli…………………………………………………….223 8.4 Growth performance of petunia plant lines under in vitro and glasshouse conditions…...... 223 8.5 Application of the findings in this thesis………………………………………..224 8.6 Limitation of the study………………………………………………………….225 8.7 Recommendations for future research…………………………………………..227 References……………………………………………………………………………...... 230 Appendices………………………………………………………………………………….279 Appendix A: plant tissues culture media……………………………………………279 A1. Murashige and Skoog Stock (MS) medium (1962)…………………….279 Appendix B: Histology analysis…………………………………………………….281 B1. Staining procedures…………………………………………………….281 Appendix C: Potting mix analysis…………………………………………………..282

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List of figures

Chapter 1

Figure 1.1 Chemical structure of United State Environmental Protection Agency 16 priority polycyclic aromatic hydrocarbons (Bruzzoniti et al., 2010)…………………………………..4

Figure 1.2 Schematic representation of pollutant fates during phytoremediation (Pilon-Smits, 2005)………………………………………………………………………………………….12

Chapter 2

Figure 2.1 Complete randomised block design. (a) Regenerated Petunia grandiflora plantlet line originated from diesel-exposed callus, (b) regenerated Petunia grandiflora plantlets from callus without any exposure to diesel, (c) Petunia grandiflora plantlets from the germinated seed in vitro, and (d) complete randomised block design of (a), (b) and (c)………………….38

Chapter 3

Figure 3.1 Gas chromatogram scan of the diesel used……………………………………….45

Figure 3.2 Effect of diesel-contaminated water on (a) root and (b) shoot length of Tagetes patula seedlings after 10 days from sowing seeds. Means + SEM are significantly different as denoted by different capital letters……………………………………………………………50

Figure 3.3 Effect of diesel-contaminated water on (a) root and (b) shoot length of Petunia grandiflora seedlings after 15 days from sowing seeds. Means + SEM are significantly different as denoted by different capital letters……………………………………………….51

Figure 3.4 Effect of diesel-contaminated water on seedling development of Tagetes patula after 10 days of sowing seeds. Seedlings in (a) 0% and (b) 4% diesel………………………..53

Figure 3.5 Effect of diesel-contaminated water on seedling development of Petunia grandiflora after 15 days from sowing seeds. Seedlings in (a) 0% and (b) 4% diesel………..54

Figure 3.6 Elongation inhibition rate of (a) Tagetes patula and (b) Petunia grandiflora seedlings grown in the presence of different concentrations of diesel………………………..55

Chapter 4

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Figure 4.1 The arrangement of the three groups of Petunia grandiflora calli on the three regions of a Petri dish containing MS medium supplemented with 9.1 µM zeatin for shoot regeneration at the start of culture……………………………………………………………62

Figure 4.2 In vitro seed germination and seedling development of Petunia grandiflora on full- strength MS medium free of plant growth regulator. Cultures were kept in a growth room at 21 ± 1 ℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W)………………………………………………………………………………………….66

Figure 4.3 Callus induction experiment using leaf explants from 4-week-old Petunia grandiflora seedlings. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). The appearance of the calli after 4 weeks of culture on medium supplemented with 5.7 µM PIC and 4.4 µM BA……………………………………………………………………………………………69

Figure 4.4 Callus induction experiment using leaf explants from 4-week-old Petunia grandiflora seedlings. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). Appearance of the calli after 4 weeks of culture on medium supplemented with 4.5 µM 2,4-D and 2.2 µM BA……………………………………………………………………………………………71

Figure 4.5 Callus induction experiment using leaf explants from 4-week-old Petunia grandiflora seedlings. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). Appearance of the calli after 4 weeks of culture on medium supplemented with 1.8 µM NAA and 6.6 µM BA……………………………………………………………………………………………73

Figure 4.6 Appearance of Petunia grandiflora callus after subculture for 8 weeks on MS medium supplemented with 1.8 µM NAA and 6.6 µM BA. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). Front view of the callus culture (a), reverse side view of the callus culture (b); and a closer view of the callus under a stereomicroscope (40×) magnification (c)…………………………………………………………………………………………….74

Figure 4.7 Flow chart of the critical steps of a combination of manipulations under aseptic conditions (in a laminar flow cabinet) and brief non-sterile fume-cupboard conditions for

xv treatment of Petunia grandiflora callus with undiluted diesel fuel before the return of the diesel-treated callus to aseptic culture conditions……………………………………………76

Figure 4.8 Occurrence of microbial contamination in Petunia grandiflora calli after three different treatments (A-C). (A) The calli were incubated for 15 minutes in Plant Preservative Mixture (PPM, 0.2%, v/v) at the end of treatment with deionised water, or (B) undiluted diesel, for 9 minutes. (C) The control was incubation of the diesel-exposed Petunia grandiflora calli with deionised water instead of PPM. Upon subculture for 14 days following the treatments of A-C, any signs of microbial contamination were recorded………………………………….77

Figure 4.9 Subculture of Petunia grandiflora calli under aseptic conditions after 9-minutes diesel treatment outside aseptic laminar airflow environment. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). (a) Appearance of the calli without any prior diesel treatment at the beginning, and (b) after 14 days, of subculture. (c) Appearance of the diesel-treated calli at the beginning and (d) after 14 days of subculture. (e) Subculture of a piece of nonnecrotic callus isolated at the beginning and (f) after 14 days……………………………………………………………78

Figure 4.10 Survival and proliferation of Petunia grandiflora calli immediately after exposure to 9 min. diesel and at different subculture stages. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W)………………………………………………………………………………………….79

Figure 4.11 Plant regeneration in Petunia grandiflora via a callus phase after 12 weeks of culture on MS medium supplemented with 2.3 µM zeatin. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). A set of calli that survived diesel treatment exhibited emergence of shoot buds (white arrow), old callus with no sign of shoot formed (black arrow), and a piece of newly formed callus with shoots formed (red arrow)………………………………………………………...82

Figure 4.12 Plant regeneration in Petunia grandiflora via a callus phase after 12 weeks of culture on MS medium supplemented with 11.4 µM IAA and 17.8 µM BA. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). A set of calli that survived diesel treatment with no sign of shoot formed (black arrow), old callus with no sign of shoot formed (white arrow) and a piece of newly formed callus with multiple shoots formed (red arrow)………………………………………83

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Figure 4.13 Plant regeneration in Petunia grandiflora via a callus phase after 12 weeks of culture on MS medium supplemented with 1.1 µM IAA and 8.9 µM BA. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). A piece of old callus with no sign of shoot formed (red arrow), a set of calli that survived diesel treatment with no sign of shoot formed (black arrow), and two pieces of new control calli with emergence of shoots (white arrow)………………………………..85

Figure 4.14 Regeneration and multiplication of line (L1) from Petunia grandiflora calli survived from diesel treatment of the subculture medium (a–c) and on micropropagation medium (d–e). Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). (a) Appearance of the diesel-treated calli after 14 days of culture, (b) a subculture of diesel-treated callus; (c) calli-derived shoots after 8 weeks; (d) multiple shoots at the beginning of subculture, and (e) after 5 weeks…………………………………………………………………………………………88

Figure 4.15 Regeneration and multiplication of line (L2) from Petunia grandiflora calli that survived diesel treatment. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). (a) Shoots formed after 12 weeks of culture (black arrow), (b) the subculture of the shoots at the beginning (c) shoot after 3 weeks; (d) subculture shoot after 4 weeks…………………………………………….89

Figure 4.16 Regeneration and multiplication of line (L3) from Petunia grandiflora calli that survived diesel treatment. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). (a) Shoots formed after 12 weeks of culture (black arrow), (b) the subculture of the shoots at the beginning (c) shoot after 3 weeks, (d) micropropagated shoot…………………………………………………….90

Figure 4.17 Regeneration and multiplication of line (L4) from Petunia grandiflora calli that survived diesel treatment. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). (a) Shoot formed after 12 weeks of culture (black arrow), (b) the subculture of the shoots at the beginning (c) and shoot after 3 weeks; (d) subculture shoots……………………………………………………91

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12 weeks of culture (black arrow), (b) the subculture of the shoot at the beginning of 3 weeks, (c) shoots after three subsequent rounds of micropropagation and (d) shoots after 4 weeks of subculture……………………………………………………………………………………92

Figure 4.19 Regeneration and multiplication of line (L6) from Petunia grandiflora calli that survived diesel treatment. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). (a) Shoots formed after 12 weeks of culture (black arrow), (b) the subculture of the shoot at the beginning of 3 weeks; (c) shoots after three subsequent rounds of micropropagation and shoots after 4 weeks of subculture…………………………………………………………………………………….93

Figure 4.20 Regeneration and multiplication of control regenerants (C-R) from Petunia grandiflora calli without exposure to diesel treatment. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). (a) Shoots formed after 12 weeks of culture (black arrow), (b) the subculture of the shoots at the beginning (c) shoots after 3 weeks, (d) micropropagated plantlets at four weeks…………………………………………………………………………………………96

Chapter 5

Figure 5.1 In vitro seed germination and seedling development of Tagetes patula on full- strength MS medium without any added plant growth regulators. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro- Lux lamps 36W)…………………………………………………………………………….109

Figure 5.2 Callus induction experiment using leaf explants from 4-week-old Tagetes patula seedlings. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). Appearance of the calli after 4 weeks of culture on medium supplemented with 2.7 µM NAA and 6.6 µM BA…………………………………………………………………………………………..112

Figure 5.3 Callus induction experiment using leaf explants from 4-week-old Tagetes patula seedlings. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). (a) Appearance of the calli after 4 weeks of culture on medium supplemented with 4.5 µM 2, 4-D and 4.4 µM BA, and (b)

xviii appearance of the calli after 4 weeks of culture on medium supplemented with 13.5 µM 2, 4-D and 2.2 µM BA……………………………………………………………………………...114

Figure 5.4 Callus induction experiment using leaf explants from 4-week-old Tagetes patula seedlings. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). Appearance of the calli after 4 weeks of culture on medium supplemented with 5.7 µM PIC and 4.4 µM BA……………………..116

Figure 5.5 Callus induction experiment using leaf explants from 4-week-old Tagetes patula seedlings. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). Appearance of the calli after 4 weeks of culture on medium supplemented with 1.8 µM NAA and 66.6 µM BA…………………..118

Figure 5.6 (a) Appearance of callus in Tagetes patula explants after 6 weeks on callus subculture medium containing 1.8 µM NAA and 66.6 µM BA, and (b) appearance of callus at 40× magnification under a stereo microscope. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W)…….119

Figure 5.7 Flow chart of the critical steps of a combination of manipulations under aseptic conditions (in a laminar flow cabinet) and brief non-sterile fume-cupboard conditions for treatment of Tagetes patula callus with undiluted diesel fuel before the return of the diesel- treated callus to aseptic culture conditions………………………………………………….121

Figure 5.8 Subculture of Tagetes patula calli under aseptic conditions after 9 minutes diesel treatment outside aseptic laminar airflow environment. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). (a) Appearance of the calli without any prior diesel treatment at the beginning, and (b) after 14 days, of subculture. (c) Appearance of the diesel-treated calli at the beginning and (d) after 14 days, of subculture. (e) Subculture of a piece of non-necrotic callus (f) isolated from 14 days, (g) which grew bigger and was subcultured as two pieces for another round of 14 days…………………………………………………………………………………………122

Figure 5.9 Survival and proliferation of Tagetes patula calli immediately after exposure to 9 minutes diesel and at different subculture stages. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W)………………………………………………………………………………………...123

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Figure 5.10 Experiments to induce plant regeneration in Tagetes patula callus after 10 weeks of culture on MS medium supplemented with (a) 1.8 µM NAA, 6.6 µM BA and 5 µM GA3 and

(b) 1.8 µM NAA, 6.6 µM BA and 10 µM GA3. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). (a) A piece of new callus with root formation (black arrow), (b) a set of old calli with no sign of shoot formed (red arrow)……………………………………………………………………125

Figure 5.11 Experiments to induce plant regeneration in Tagetes patula callus after 10 weeks of culture on MS medium supplemented with (a) 2.2 µM NAA, 1.8 µM BA and 50 µM GA3;

(b) 2.2 µM NAA, 3.6 µM BA and 50 µM GA3 and (c) 2.2 µM NAA, 5.4 µM BA and 50 µM

GA3. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). The red arrow in (a), (b) and (c) points at a piece of old callus with no sign of shoot formation, while the black arrow in (a), (b) and (c) points at a set of new calli with no sign of shoot formation………………………………….127

Figure 5.12 Experiments to induce plant regeneration in Tagetes patula callus after 10 weeks of culture on MS medium supplemented with (a) 2.2 µM NAA, 1.8 µM BA and 100 µM GA3;

(b) 2.2 µM NAA, 3.6 µM BA and 100 µM GA3 and (c) 2.2 µM NAA, 5.4 µM BA and 100 µM

Figure 5.13 Experiments to induce plant regeneration in Tagetes patula callus after 10 weeks of culture on MS medium supplemented with (a) 11.4 µM IAA, 3.6 µM BA, and 50 µM GA3 and (b) 11.4 µM IAA, 6.6 µM BA and 10 µM GA3. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). A set of calli that survived diesel treatment in (a) and (b) with no emergence of shoot buds…………………………………………………………………………………………130

Figure 5.14 Experiments to induce plant regeneration in Tagetes patula callus after 10 weeks of culture on MS medium supplemented with (a) 11.4 µM IAA, 26.6 µM BA and 86 µM GA3 and (b) 2 µM IAA, 66.6 µM BA and 86 µM GA3. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). A set of calli that survived diesel treatment in (a) and (b) with no emergence of shoot buds………………………………………………………...... 131

Figure 5.15 Experiments to induce plant regeneration in Tagetes patula callus after 10 weeks of culture on MS medium supplemented with (a) 2.3 µM zeatin; and (b) 4.6 µM zeatin and (c) 9.1 µM zeatin. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). A set of calli that survived diesel treatment in (a), (b) and (c) with no emergence of shoot buds………………………………133

Chapter 6

Figure 6.1 Overall view (40×) of the transverse sections of Petunia grandiflora calli at 500 µm from the top of the callus (a) without diesel treatment and (b) diesel-treated callus. The red arrow points to the xylem vessels surrounded by the region of active cell division and the black arrow points to the lignin wall. Abbreviation: MZ, meristem zone; PCs, parenchyma cells; V, vacuole; N, nucleus. The red-circled sections of the micrograph in (a) and (b) are shown at 100× magnification in Figure 6.2 a and b. Scale bar (a) and (b) = 10 µm……………………146

Figure 6.2 Transverse sections viewed under 100× magnification of Petunia grandiflora calli at 500 µm from the top of callus (a) without diesel treatment and (b) diesel-treated. The red arrow points to the xylem vessels and the black arrowhead points to the lignin wall. Abbreviation: MZ, meristem zone; PCs, parenchyma cells; V, vacuole; TEs, tracheary elements; N, nucleus. Scale bar (a) and (b) = 10 µm…………………………………………147

Figure 6.3 Overall view (40×) of the transverse sections of Petunia grandiflora calli at 1500 µm from the top of the callus (a) without diesel treatment and (b) diesel-treated. The red arrow points to rows of meristematic cells with many large nuclei, indicative of mitotic activities, and the black arrow points to tracheary elements (a) and (b). Abbreviation: MZ, meristem zone; PCs, parenchyma cells; V, vacuole; N, nucleus; LCW, lignin cell wall. The red-circled sections of the micrograph in (a) and (b) are shown at 100× magnification in Figure 6.4 (a) and (b). Scale bar (a) and (b) = 10 µm……………………………………………….149

Figure 6.4 Transverse sections viewed under 100× magnification of Petunia grandiflora calli at 1500 µm from the top of callus (a) without diesel treatment and (b) diesel-treated. The nuclei are very large, indicative of mitotic activities (a). The black arrow points to tracheary elements (a) and (b). Abbreviation: MZ, meristem zone; PCs, parenchyma cells; V, vacuole; N, nucleus. Scale bar (a) and (b) = 10 µm………………………………………………………………..150

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Figure 6.5 Overall view (40×) of the transverse sections of Tagetes patula calli at 500 µm from the top of the callus without (a) diesel treatment and (b) diesel-treated. The black arrow points to meristematic cells in group. Abbreviation: MZ, meristem zone; PCs, parenchyma cells; V, vacuole; N, nucleus; LCW, lignin cell wall. The red-circled sections of the micrograph in (a) and (b) are shown at 100× magnification in Figure 6.6 (a) and (b). Scale bar (a) and (b) = 10 µm…………………………………………………………………………………………...152

Figure 6.6 Transverse sections viewed under 100× magnification of Tagetes patula calli at 500 µm from the top of callus (a) without diesel treatment and (b) diesel-treated. The black arrow points to meristematic cells in a group. Abbreviation: MZ, meristem zone; PCs, parenchyma cells; V, vacuole; N, nucleus; LCW, lignin cell wall. Scale bar (a) and (b) = 10 µm……………………………………………………………………………………..153

Figure 6.7 Overall view (40×) of the transverse sections of Tagetes patula calli at 1500 µm from the top of the callus (a) without diesel treatment and (b) diesel-treated. Abbreviation: MZ, meristem zone; PCs, parenchyma cells; V, vacuole; MD, meristematic dome; LP, leaf primordia; N, nucleus; MCs, meristem cells. The red-circled sections of the micrograph in (a) and (b) are shown at 100× magnification in Figure 6.8 (a) and (b). Scale bar (a) and (b) = 10 µm…………………………………………………………………………………155

Figure 6.8 Transverse sections viewed under 100× magnification of Tagetes patula calli at 1500 µm from the top of callus (a) without diesel treatment and (b) diesel-treated. Abbreviation: MZ, meristem zone; PCs, parenchyma cells; V, vacuole; N, nucleus; LCW, lignin cell wall; MCs, meristem cells. Scale bar (a) and (b) = 10 µm………………………..156

Chapter 7

Figure 7.1 The average number of leaves in different Petunia grandiflora plant lines after 4 weeks of in vitro culture. There were two leaves at day 0. Values represent means ± SEM of four replicates containing eight jars in each of the treatment (L1 to L6) and controls. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)…………………………………………………………………………………………170

Figure 7.2 The average shoot height in different Petunia grandiflora plant lines after 4 weeks of in vitro culture. The height of the shoot cuttings was 1 cm at day 0. Values represent means ± SEM of four replicates containing eight jars in each of the treatment (L1 to L6) and controls.

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Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)…………………………………………………………………………………………170

Figure 7.3 Root length of Petunia grandiflora plantlets of different lines after 4 weeks of in vitro culture. Values represent means ± SEM of four replicates containing eight jars in each of the treatment (L1 to L6) and controls. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)………………………………………………171

Figure 7.4 Fresh mass of Petunia grandiflora plantlets of different lines after 4 weeks of in vitro culture. Values represent means ± SEM of four replicates containing eight jars in each of the treatment (L1 to L6) and controls. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)………………………………………………171

Figure 7.5 Dry mass of Petunia grandiflora plantlets of different lines after 4 weeks of in vitro culture. Values represent means ± SEM of four replicates containing eight jars in each of the treatment (L1 to L6) and controls. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)………………………………………………172

Figure 7.6 Appearance of Petunia grandiflora plantlets from seeds germinated in vitro (C-G) on plant-growth-regulator-free half-strength MS medium. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). The photo was taken after 4 weeks from culturing 1-cm shoot cuttings……………..173

Figure 7.7 Appearance of Petunia grandiflora plantlets after 4 weeks of culture on plant- growth-regulator-free half-strength MS medium. The plantlets were regenerated from callus that was not treated with diesel before (C-R plantlets). Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). The photo was taken after 4 weeks from culturing 1-cm shoot cutting…………………….174

Figure 7.8 Appearance of Petunia grandiflora plantlets after 4 weeks of culture on plant- growth-regulator-free half-strength MS medium. The plantlets of line L1 were regenerated from diesel-treated callus culture. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). The photo was taken after 4 weeks from culturing 1-cm shoot cuttings………………………………..175

Figure 7.9 Appearance of Petunia grandiflora plantlets after 4 weeks of culture on plant- growth-regulator-free half-strength MS medium. The plantlets of line L2 were regenerated

xxiii from diesel-treated callus culture. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). The black arrow on the left side is used to show the shoot with dead leaves, while the arrow on the right side is used to show the tiny chlorotic-looking leaves. The photo was taken after 4 weeks from culturing 1-cm shoot cuttings……………………………………………………………….176

Figure 7.10 Appearance of Petunia grandiflora plantlets after 4 weeks of culture on plant- growth-regulator-free half-strength MS medium. The plantlets of line L3 were regenerated from diesel-treated callus culture. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). The photo was taken after 4 weeks from culturing 1-cm shoot cuttings………………………………...177

Figure 7.11 Appearance of Petunia grandiflora plantlets after 4 weeks of culture on plant- growth-regulator-free half-strength MS medium. The plantlets of line L4 were regenerated from diesel-treated callus culture. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). The photo was taken after 4 weeks from culturing 1-cm shoot cuttings………………………………..178

Figure 7.12 Appearance of Petunia grandiflora plantlets after 4 weeks of culture on plant- growth-regulator-free half-strength MS medium. The plantlets of line L5 were regenerated from diesel-treated callus culture. The black arrow is used to show the white friable callus formed at the base of a shoot. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). The photo was taken after 4 weeks from culturing 1-cm shoot cuttings…………………………………………..179

Figure 7.13 Appearance of Petunia grandiflora plantlets after 4 weeks of culture on plant- growth-regulator-free half-strength MS medium. The plantlets of line L6 were regenerated from diesel-treated callus culture. The black arrow points to plantlet with hyperhydrated leaves. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). The photo was taken after 4 weeks from culturing 1-cm shoot cuttings………………………………………………………………………………..180

Figure 7.14 Establishment of Petunia grandiflora plantlet lines, for example, line L1 under in vitro culture (a), plantlet in a growth room after agar had been washed (b), and an established plantlet in the potting mixture under glasshouse conditions…………………………………181

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Figure 7.15 The stem diameter of Petunia grandiflora shoot cuttings of line L1, controls C-G and C-R after 5 weeks of growth under glasshouse conditions. The average stem diameter at the beginning of the experiment was 3.6 mm. Values represent means ± SEM of three replicates in each of the treatment L1, controls C-G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)………………………………...185

Figure 7.16 The number of leaves formed in Petunia grandiflora shoot cuttings of L1, C-G and C-R after 5 weeks of growth under glasshouse conditions. The average number of leaves at the beginning of the experiment was 2. Values represent means ± SEM of three replicates in each of the treatment L1, controls C-G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)…………………………………185

Figure 7.17 Visual chlorosis rating scores of Petunia grandiflora shoot cuttings of L1, C-G and C-R after 5 weeks of growth under glasshouse conditions. Values represent means ± SEM of three replicates in each of the treatment L1, controls C-G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)…………………186

Figure 7.18 The shoot height of Petunia grandiflora shoot cuttings of L1, C-G and C-R after 5 weeks of growth under glasshouse conditions. The average shoot height at the beginning of the experiment was 7 cm. Values represent means ± SEM of three replicates in each of the treatment L1, controls C-G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)………………………………………………186

Figure 7.19 (a) The fresh and (b) dry mass of Petunia grandiflora shoot cuttings of L1, controls C-G and C-R after 5 weeks of growth under glasshouse conditions. Values represent means ± SEM of three replicates in each of the treatment L1, controls C-G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)…………………………………………………………………………………………187

Figure 7.20 Morphological characteristics of Petunia grandiflora shoot cuttings of L1 (a–d) and controls C-R (e–h) and C-G (i–l). (a–d) From top to bottom of line L1: (a) a shoot cutting of L1 before sowing, (b) shoot cuttings of L1 grown in 0%, (c) 2% and (d) 7% diesel- contaminated potting mixture for 5 weeks under glasshouse conditions. (e–h) From top to bottom of control C-R: (e) a shoot cutting before sowing, (f) shoot cuttings of C-R grown under 0%, (g) 2% and (h) 7% diesel-contaminated potting mixture. (i–l) From top to bottom of control

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C-G: (i) a shoot cutting of C-G before sowing, (j) shoot cuttings of C-G grown under 0%, (k) 2% and (l) 7% diesel-contaminated potting mixture……………………………………..…188

Figure 7.21 Range of daily variations in temperatures (°C) and the light intensity (lux) in the glasshouse during the experiment (5 weeks) with Petunia grandiflora shoot cuttings of L1 and controls (C-G and C-R)……………………………………………………………………...189

Figure 7.22 The average stem diameter of Petunia grandiflora shoot cuttings of L2, C-G and C-R after 5 weeks of growth under glasshouse conditions. At the beginning of the experiment the average stem diameter of the shoot cuttings was 3.4 cm. Values represent means ± SEM of three replicates in each of the treatment L2, controls C-G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)…………………190

Figure 7.23 The number of leaves formed in Petunia grandiflora of L2, C-G and C-R after 5 weeks growth under glasshouse conditions. The average number of leaves at the beginning of experiment was 2. Values represent means ± SEM of three replicates in each of the treatment L2, controls C-G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)……………………………………………………………..190

Figure 7.24 Visual chlorosis rating scores of Petunia grandiflora shoot cuttings L2, C-G and C-R after 5 weeks of growth under glasshouse conditions. Values represent means ± SEM of three replicates in each of the treatment L2, controls C-G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)…………………191

Figure 7.25 The average shoot height of Petunia grandiflora L2, C-G and C-R after 5 weeks of growth. At the beginning of the experiment the average shoot height was 7 cm. Values represent means ± SEM of three replicates in each of the treatment L2, controls C-G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)…………………………………………………………………………………………191

Figure 7.26 The (a) fresh and (b) dry mass of Petunia grandiflora plantlet shoot cuttings of L2, C-G and C-R after 5 weeks of growth under glasshouse conditions. Values represent means ± SEM of three replicates in each of the treatment L2, controls C-G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)…………………………………………………………………………………………192

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Figure 7.27 Morphological characteristics of Petunia grandiflora shoot cuttings of L2 (a–d) and controls C-R (e–h) and C-G (i–l). (a–d) From top to bottom of line L2: (a) a shoot cutting of L2 before sowing, (b) shoot cuttings of L2 grown in 0%, (c) 2% and (d) 7% diesel contaminated potting mixture for 5 weeks under glasshouse conditions. (e–h) From top to bottom of control C-R: (e) a shoot cutting before sowing, (f) shoot cuttings of C-R grown under 0%, (g) 2% and (h) 7% diesel-contaminated potting mixture. (i–l) From top to bottom of control C-G: (i) a shoot cutting of C-G before sowing, (j) shoot cuttings of C-G grown under 0%, (l) 2% (k) and 7% diesel-contaminated potting mixture………………………………………..193

Figure 7.28 Range of daily variations in temperatures (°C) and light intensity (lux) in the glasshouse during the experiment (5 weeks) with the Petunia grandiflora plantlets L2, controls (C-G and C-R)………………………………………………………………………………194

Figure 7.29 Average stem diameters of Petunia grandiflora shoot cuttings of L3, C-G and C- R after 5 weeks of growth under glasshouse conditions. The average stem diameters were 3.3, 3.3 and 3.4 mm, respectively. Values represent means ± SEM of three replicates in each of the treatment L3, controls C-G and C-R Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)………………………………………………195

Figure 7.30 The number of Petunia grandiflora leaves formed in L3, C-G and C-R after 5 weeks of growth under glasshouse conditions. The average number of leaves at the beginning of the experiment was 2. Values represent means ± SEM of three replicates in each of the treatment L3, controls C-G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)……………………………………………..195

Figure 7.31 Visual chlorosis rating scores of Petunia grandiflora shoot cuttings of L3, C-G and C-R determined after 5 weeks of growth under glasshouse conditions. Values represent means ± SEM of three replicates in each of the treatment L3, controls C-G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)…………………………………………………………………………………………196

Figure 7.32 Shoot height of Petunia grandiflora shoot cuttings of L3, control C-G and C-R after 5 weeks of growth under glasshouse conditions. The average shoot height at the beginning of the experiment was 7 cm. Values represent means ± SEM of three replicates in each of the treatment L3, controls C-G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)……………………………………………..196

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Figure 7.33 The (a) fresh and (b) dry mass of Petunia grandiflora shoot cuttings of L3, C-G and C-R after 5 weeks of growth under glasshouse conditions. Values represent means ± SEM of three replicates in each of the treatment L3, controls C-G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)………………...197

Figure 7.34 Morphological characteristics of Petunia grandiflora shoot cuttings of L3 (a–d) and controls C-R (e–h) and C-G (i–l). (a–d) From top to bottom of line L3: (a) a shoot cutting of L3 before sowing, (b) shoot cuttings of L3 grown in 0%, (c) 2% and (d) 7% diesel- contaminated potting mixture for 5 weeks under glasshouse conditions. (e–h) From top to bottom of control C-R: (e) a shoot cutting before sowing, (f) shoot cuttings of C-R grown under 0%, (g) 2% and (h) 7% diesel-contaminated potting mixture. (i–l) From top to bottom of control C-G: (i) a shoot cutting of C-G before sowing, (j) shoot cuttings of C-G grown under 0%, (k) 2% and (l) 7% diesel-contaminated potting mixture………………………………………..198

Figure 7.35 Range of daily variations in temperatures (°C) and light intensity (lux) in the glasshouse during the experiment (5 weeks) with the Petunia grandiflora shoot cuttings of L3 and controls (C-G and C-R)…………………………………………………………………199

Figure 7.36 The stem diameters of Petunia grandiflora shoot cuttings of L4, C-G, and C-R after 5 weeks of growth under glasshouse conditions. The average stem diameters at the beginning of the experiment were 3.1, 3.3 and 3.2 mm, respectively. Values represent means ± SEM of three replicates in each of the treatment L4, controls C-G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)………………………………………………………………………………………..200

Figure 7.37 The number of Petunia grandiflora leaves formed in L4, C-G and C-R after 5 weeks of growth under glasshouse conditions. On average there were two leaves at the beginning of the experiment. Values represent means ± SEM of three replicates in each of the treatment L4, controls C-G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)……………………………………………..200

Figure 7.38 Visual chlorosis rating scores of Petunia grandiflora shoot cuttings of L4, C-G and C-R after 5 weeks of growth under glasshouse conditions. Values represent means ± SEM of three replicates in each of the treatment L4, controls C-G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)………………..201

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Figure 7.39 Root length of Petunia grandiflora shoot cuttings of L4, C-G and C-R after 5 weeks of growth under glasshouse conditions. Values represent means ± SEM of three replicates in each of the treatment L4, controls C-G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)……………………….201

Figure 7.40 Number of roots number of Petunia grandiflora shoot cuttings of L4, C-G and C- R after 5 weeks of growth under glasshouse conditions. Values represent means ± SEM of three replicates in each of the treatment L4, controls C-G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)………………………..202

Figure 7. 41 Shoot height of Petunia grandiflora shoot cuttings of L4 C-G and C-R after 5 weeks of growth under glasshouse conditions. The average shoot height at the start of the experiment was 7 cm. Values represent means ± SEM of three replicates in each of the treatment L4, controls C-G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)………………………………………………202

Figure 7.42 The (a) fresh and (b) dry mass of Petunia grandiflora shoot cuttings of L4, C-G and C-R after 5 weeks of growth under glasshouse conditions. Values represent means ± SEM of three replicates in each of the treatment L4, controls C-G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05)…………………203

Figure 7.43 Morphological characteristics of Petunia grandiflora shoot cuttings of L4 (a–d) and controls C-R (e–h) and C-G (i–l). (a–d) From top to bottom of line L4: (a) a shoot cutting of L4 before sowing, (b) shoot cuttings of L4 grown in 0%, (c) 2% and (d) 7% diesel- contaminated potting mixture for 5 weeks under glasshouse conditions. (e–h) From top to bottom of control C-R: (e) a shoot cutting before sowing, (f) shoot cuttings of C-R grown under 0%, (g) 2% and (h) 7% diesel-contaminated potting mixture. (i–l) From top to bottom of control C-G: (i) a shoot cutting of C-G before sowing, (j) shoot cuttings of C-G grown under 0%, (k) 2% and (l) 7% diesel-contaminated potting mixture………………………………………..204

Figure 7.44 Range of daily variations in temperatures (°C) and light intensity (lux) in the glasshouse during the experiment (5 weeks) with the Petunia grandiflora shoot cuttings of L4 and controls (C-G and C-R)…………………………………………………………………205

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LIST OF TABLES

Chapter 1

Table 1.1 Examples of the morphological markers used to detect somaclonal variation induced in vitro in ornamental plants………………………………………………………………….19

Chapter 3

Table 3.1 Effect of different concentrations of diesel on the changes in the percentages Tagetes patula seeds germinated during 10 days from sowing seeds…………………………………47

Table 3.2 Effect of different concentrations of diesel on the changes in the percentages of Petunia grandiflora seeds germinated during 15 days from sowing seeds………………………………………………………………………………………….48

Chapter 4

Table 4.1 Plant cell and tissue culture media used in this study……………………………..64

Table 4.2 Effect of the various combinations of picloram (PIC) with 6-benzyladenine (BA) on callus induction in Petunia grandiflora leaf explant after 4 weeks of culture………………..68

Table 4.3 Effect of the various combinations of 2, 4-dichlorophenoxyacetic acid (2,4-D) with 6-benzyladenine (BA) on callus induction in Petunia grandiflora leaf explants after 4 weeks of culture……………………………………………………………………………………..70

Table 4.4 Effect of the various combinations of 1-naphthalene acetic acid (NAA) with 6- benzyladenine (BA) on callus induction in Petunia grandiflora leaf explants after 4 weeks of culture………………………………………………………………………………………...72

Table 4.5 Callus-based shoot regeneration responses of Petunia grandiflora to various concentrations of zeatin after 12 weeks of culture……………………………………………81

Table 4.6 Callus-based shoot regeneration responses of Petunia grandiflora to various combinations of indole-3-acetic acid (IAA) with 6-benzyladenine (BA) after 12 weeks of culture………………………………………………………………………………………...81

Table 4.7 Callus-based shoot regeneration responses of Petunia grandiflora to various combinations of 1-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) with 6- benzyladenine (BA) after 12 weeks of culture……………………………………………….84

Table 4.8 Regenerated shoots formed from Petunia grandiflora calli that survived diesel treatment. Calli were cultured on the plant growth regulators (PGRs) listed…………………94

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Chapter 5

Table 5.1 Plant cell and tissue culture media used in this study……………………………107

Table 5.2 Effect of the various combinations of 1-naphthalene acetic acid (NAA) with 6- benzyladenine (BA) on callus induction in Tagetes patula leaf explants after 4 weeks of culture……………………………………………………………………………………….111

Table 5.3 Effect of the various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) with 6-benzyladenine (BA) on callus induction in Tagetes patula leaf explants after 4 weeks of culture……………………………………………………………………………………….113

Table 5.4 Effect of the various combinations of picloram (PIC) with 6-benzyladenine (BA) on callus induction in Tagetes patula leaf explant after 4 weeks of culture……………………………………………………………………………………….115

Table 5.5 Effect of the various combinations of 1-naphthalene acetic acid (NAA) with 6- benzyladenine (BA) on callus induction in Tagetes patula leaf explants after 4 weeks of culture……………………………………………………………………………………….117

Table 5.6 Experiments to induce shoot regeneration in Tagetes patula callus using various combinations of 1-naphthalene acetic acid (NAA), gibberellic acid (GA3) and 6-benzyladenine (BA) after 10 weeks of culture………………………………………………………………125

Table 5.7 Experiments to induce shoot regeneration in Tagetes patula callus using various combinations of 1-naphthalene acetic acid (NAA), gibberellic acid (GA3) and 6-benzyladenine (BA) after 10 weeks of culture………………………………………………………………126

Table 5.8 Experiments to induce shoot regeneration in Tagetes patula callus using various combinations of 1-naphthalene acetic acid (NAA), gibberellic acid (GA3) and 6-benzyladenine (BA) after 10 weeks of culture………………………………………………………………128 Table 5.9 Experiments to induce shoot regeneration in Tagetes patula callus using various combinations of indole-3-acetic acid (IAA), gibberellic acid (GA3) and 6-benzyladenine (BA) after 10 weeks of culture……………………………………………………………………130

Table 5.10 Experiments to induce shoot regeneration in Tagetes patula callus using various combinations of indole-3-acetic acid (IAA), gibberellic acid (GA3) and 6-benzyladenine (BA) after 10 weeks of culture…………………………………………………………………….131

Table 5.11 Experiments to induce shoot regeneration in Tagetes patula callus using various concentrations of zeatin after 10 weeks of culture…………………………………………..132

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Chapter 7

Table 7.1 Number of culturable microbes in non-diesel- and diesel-contaminated potting mix with or without experimental plant materials……………………………………………….207

Table 7.2 Total petroleum hydrocarbons (% TPHs) in diesel-contaminated potting mix with or without experimental plant materials………………………………………………………..209 Appendices

Table A1. The amount of major, minor and organic salts required for 500 mL stock solutions…………………………………………………………………………………….279

Table C1. General analysis…………………………………………………………282

Table C2. General analysis………………………………………………………....283

xxxii

Abbreviations % Percentage µM Micromoles per litre 2, 4-D 2,4-dichlorophenoxyacetic acid 8-oxo-dG 8-oxo-7, 8-hydro-2’-deoxyguanosine A549 cells Adenocarcinomic human alveolar basal epithelial cells AFR Auxin response factors AFR7 Auxin response factors 7 AFR9 Auxin response factors 9 Al Aluminium ANOVA Analysis of variance AQP Aquaporin ARR21-C-ox Transgenic line ARRs Arabidopsis response regulators AtFALDH Glutathione-dependent formaldehyde dehydrogenase gene BA 6-benzyladenine BaP Benzo[a]pyrene BER Base excision repair BF Bioaccumulation factor BRs Brassinosteroids ℃ Degree Celsius

C2Cl4 Tetrachloroethylene CAXcd Cadmium cations/H+ exchanger Cd Cadmium CDK Cyclin-dependent kinases CDKA Cyclin-dependent kinases A CDKB Cyclin-dependent kinases B CE Cotyledon explants CFUs Number of colonies forming units C-G Petunia line from seed germinated in vitro CHO Chinese hamster ovary cm Centimeter

xxxiii

C-R Petunia line regenerated from callus without prior exposure to diesel CRBD Completely randomised block design CYC Cyclin CYCA Cyclin A CYCB Cyclin B dH20 sterilised water DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DNA Deoxyribonucleic acid DPa Dimerization of partner protein-a E2Fa E2 promoter binding factor-a EI Elongation inhibition EPA Environmental Protection Agency ESR1 Enhancer of shoot regeneration 1 EUI1 Elongated upper internodes1 FAA Formaldehyde-acetic acid-ethanol FAME Fatty acid methyl esters FID Flame ionisation detector FPG Formamidopyrimidine-DNA glycosylase

Fv/Fm Variable fluorescence by maximum fluorescence g Gram GA Gibberellins GA20ox GA 20-oxidase GA2ox GA 2-oxidase

GA3 Gibberellic acid GA3ox GA 3ꞵ-hydroxylase GC Gas chromatographic h hour

H2O2 Hydrogen peroxide HA Humic acid HCHO Formaldehyde HCl Hydrochloric acid HDMI High-definition Multimedia Interface

xxxiv

HE Hypocotyl explants Hg Mercury HR Hypersensitive response IAA Indole-3-acetic acid IBA Indole-3-butryric acid IEA International Energy Agency KOH Potassium hydroxide KRP Kip-related protein L Litre L1 Petunia plant line 1 L2 Petunia plant line 2 L3 Petunia plant line 3 L4 Petunia plant line 4 Laccase E.C.1.10.3.2 LBD Lateral organ boundaries LBD16 Lateral organ boundaries LBD17 Lateral organ boundaries LBD18 Lateral organ boundaries LBD29 Lateral organ boundaries

Lc Root length value of the control LE Leaf explants LRP Lateral root primordia LSD Least significant difference

Lt Root length value of the treatment MC Meristematic cell min minute mL Millilitre mm Millimeter MS Murashige and Skoog (1962) MVI Morphological visual inspection

Na2SO4 Anhydrous sodium sulfate NAA 1-naphthalene acetic acid NaCl Sodium chloride NADPH Nicotinamide adenine dinucleotide phosphate

xxxv

NaOCl Sodium hypochlorite NAPH Naphthalene NBRX Navy Blue RX NE Northeast

NH3 Ammonia NW North-west OECD Organisation for Economic Co-operation and Development OGG1 Enzymes 8-oxoguanine DNA glycosylase/lyase OsAR4 4-coumarate: CoA ligases isoform 2 OsAR5 Phenylalanine ammonia-lyase OsAR6 Putative cinnamyl-alcohol dehydrogenase OsAR7 P-coumarate 3-hydroxylase PAHs Polynuclear aromatic hydrocarbons PAL Phenylalanine ammonia-lyase PAs Polyamines PCBs Polychlorinated biphenyls PCD Programmed cell death PGRs Plant growth regulators pH Scale of acidic or alkaline PHC Petroleum hydrocarbons PHEN Phenanthrene PIC Picloram POD Peroxidase PPM Plant Preservative Mixture PR1 Systemic acquired resistance PRZ1 Proporz 1 RE Root explants ROS Reactive oxygen species SAM Shoot apical meristem SAM Shoot apical meristem SB Strand breaks SE South-east SW South-west T S Transverse sections

xxxvi

T2 Transgenic TA100 Salmonella typhimurium tester strains cells TA98 Salmonella typhimurium tester strains cells TBA Tertiary butanol alcohol TEs Tracheary elements TF Translocation factor TPHs Total petroleum hydrocarbons UK United Kingdom US United States v/v Volume per volume VP16 or α-TIF Trans inducing factor w/v Weight per volume w/w weight per weight X-ray X-radiation Zn Zinc

xxxvii

Publication and conference abstracts arising from this thesis

• Wante, S. P., & Leung, D. W. M. (2018). Phytotoxicity testing of diesel-contaminated

water using Petunia grandiflora Juss. mix F1 and Marigold-Nemo mix (Tagetes patula

L.). Environmental Monitoring and Assessment, 190(7), 408. (Journal article, from

Chapter 3).

• Wante, S. P., & Leung, D. W. M. (2017). Callus induction in Marigold-Nemo mix

(Tagetes patula L) required for diesel toxicity evaluation. Paper presented at the 15th

Asia-Pacific Biotechnology Congress, Melbourne, Australia. (Poster presentation, from

Chapter 5).

• Wante, S. P., & Leung, D. W. M. (2017). Isolation of diesel-tolerant callus lines of

Marigold-Nemo mix (Tagetes patula L). Paper presented at the Plant Science Central

Palmerston North, New Zealand. (Talk presentation, from Chapter 5).

xxxviii

Chapter 1 Introduction and literature review 1.1 Petroleum hydrocarbons

Petroleum (often called crude oil) is a word derived from the Latin petra and oleum, meaning rock oil (Speight, 2007). It is an incredibly complex mixture of hydrocarbon compounds that occur naturally in sedimentary rocks in the form of gases, liquids, semisolids, or solids that contain nitrogen-, oxygen-, and sulphur-containing compounds as well as traces of metals

(Bestougeff, 1967; Colombo, 1967; Hobson and Pohl, 1973). Critical observations revealed dramatic variations in colour, odour, and viscosity of the rich fluid that reflect the diversity of its origin (Speight, 2015). Further investigations have also shown variation in the type of molecules present in petroleum, including compounds of nitrogen, oxygen, sulphur, heavy metals as well as other trace elements (Speight, 2012).

Physical and chemical properties have been used in categorising petroleum and its products into various classes. Boiling point and density (gravity) are used to describe crude oil as light or heavy oil, while odour is used to differentiate between low and high sulphur oils (Speight,

2014; Speight, 2015). On the other hand, chemical properties such as molecular composition are used to categorise the petroleum oils into two classes of compounds: aliphatic and aromatic compounds (Speight, 2015). The aliphatics are alkanes, alkenes, and alkynes while the aromatics are compounds that contain one or more aromatic benzene rings (Cermak et al.,

2010).

1.1.1 Petroleum hydrocarbons contamination

Pollution as a result of petroleum hydrocarbon spillage is a global environmental challenge with devastating effects on humans and ecological health (Döberl et al., 2013). A broad range of petroleum hydrocarbon pollution has been associated with anthropogenic activities, from the exploration and refining points to the transportation and down to the utilisation processes of the product. Osibanjo et al. (1983) explained how petroleum hydrocarbon pollution could occur in different remote areas where petroleum products are used. Molina-Barahona et al.

(2005) reported that a significant portion of the land and water resources on earth has been adversely polluted with diesel, a mixture of petroleum hydrocarbon fuel. Most of the toxic waste substances in the environment are associated with petroleum products, resulting from cracks in storage facilities, conduit seeps, and tanker collisions (Margesin et al., 2007). This may explain the reported increases in petroleum hydrocarbon contamination in the environment. A statistical report from the International Energy Agency (IEA) confirmed that global diesel consumption increased significantly by 23% during 2000–2008, and the projected demand between 2012 and 2025 is 5 million barrels/day (IEA, 2008, 2013). This projected increase in diesel usage is of great concern, as this might give rise to further pollution considering that diesel is the most widely used petroleum hydrocarbon (Ramadass et al., 2017).

1.1.2 Toxicity of petroleum hydrocarbons

At every stage of petroleum production and consumption, humans, animals, and plants are exposed to the toxic components of petroleum hydrocarbons, and many countries face serious problems of soil contamination and ecological risk from petroleum hydrocarbon at different concentrations (Hall et al., 2003; Penã-Castro et al., 2006). With the increasing demand for petroleum products around the world, this has become a global concern and a challenge

(Ramadass et al., 2017). It has been reported that the toxicity levels of petroleum hydrocarbons in biological systems increases as their molecular weight decreases (Darville and Wilhm, 1984;

Payne et al., 1995). In the case of diesel oil, a middle distillates product with more aromatics proved to be a more toxic agent in biological systems than other petroleum products (Wang and Bartha, 1990). This was further explained by the amount of polycyclic aromatic hydrocarbons, or polynuclear aromatic hydrocarbons (PAHs) discovered in diesel spills, which are quite persistent in the ecosystem (Adam and Duncan, 1999). The existence, persistence, and disposition of PAHs in the ecosystem is of toxicological concern (Heitkamp et al., 1988).

Several hundred different groups of PAHs exist, but about 16 are compounds currently listed as priority contaminants by the United States Environmental Protection Agency (USEPA)

(USEPA, 2008) (Figure 1.1).

One of the main characteristics of these toxic compounds is that they are highly hydrophobic

(Gan et al., 2009), so it will be easier for PAHs to be adsorbed onto the organic material of solid particles, creating persistent micropollutants in the ecosystem (Gan et al., 2009). Different ecosystem types act as sinks for PAHs. However, a preliminary record of PAHs in the United

Kingdom (UK) environment showed that soil was the primary sink for PAHs (Wild and Jones,

1995). The presence of these compounds has been associated with mutagenic and carcinogenic effect in humans (Mix, 1986; Mortelmans et al., 1986), and therefore, PAHs could pose potential threats to human and ecological health (Gan et al., 2009). To have a good understanding of the toxic components of hydrocarbon products, toxicity assessment of biological indicators is necessary. Hentati et al. (2013) described the significance of ecotoxicity analysis that comprises various techniques using soil-inhabiting organisms and plants as testing devices. Earthworms in soil have been extensively used as an indicator of toxic potential in infiltrated soil (Römbke et al., 2005). Avoidance reaction from both earthworms and collembolans can serve as a signal for early screening (Hentati et al., 2013).

Figure 1.1 Chemical structure of United State Environmental Protection Agency 16 priority polycyclic aromatic hydrocarbons (Bruzzoniti et al., 2010).

The toxicity assessment study of plants includes the study of root elongation, seed germination and plant growth (ISO11269-1 (1993)) recommended by the Organisation for Economic Co- operation and Development (OECD, 2000). Most of the reports show a relative sensitivity of

4 plants to contaminants (Banks and Schultz, 2005). There were also reports on the use of

Microtox to evaluate total petroleum hydrocarbon (TPH) toxicity (Chaîneau et al., 2003). It seems that the relative sensitivity shown by biological indicators to toxicity is a useful indicator to determine the extent of contamination.

1.2 Diesel

1.2.1 Effect of diesel on plants

Pollution problems related to diesel fuel may have been caused by different combinations of elements or compounds that may be present in the diesel fuel contaminant. It has been reported that diesel fuels are universal pollutants with a combination of low molecular weight alkanes and volatile alkanes (Adam and Duncan, 1999). It was also confirmed that diesel contains higher concentrations of persistent polycyclic aromatic hydrocarbons (PAHs), paraffins and total aromatic hydrocarbons than other distillate oils (Chandran and Das, 2011; Wang et al.,

1990). Studies revealed that spilled diesel oil in the ecosystem has the ability to bind to aquatic sediment and soil, thereby leading to land and groundwater contamination (Chandran and Das,

2011). The presence of PAHs in ecosystems may therefore elevate the level of trace metals and other toxic chemicals and can be found as complex mixtures in the soil (Allan et al., 2007;

Thavamani et al., 2012). The consequence is that co-contamination can change the fate and behaviour of PAHs in the soil (Couling et al., 2010; Lee et al., 2003). Even though natural trace elements take part in vital biological functions as necessary micro-elements, they can also act as environmental pollutants (Vamerali et al., 2014). Micro-concentrations of trace metals support plant growth and development, but the high bioavailability of excess metal ions in soil could affect plant development (Robinson et al., 2009). PAHs can undergo biotransformation and could alter root morphology, resulting in alterations that could have an impact on the influx of water and mineral elements needed for healthy plant development (Reynoso-Cuevas et al.,

2008). Metabolic pathway redirection and cellular structural changes are some of the changes

5 that result from elevated concentrations of PAHs and trace elements (Krämer and Chardonnens,

2001).

However, mechanisms of metabolic pathways redirection and cellular structural changes are not well understood (Krämer and Chardonnens, 2001). Doran (2009) reported that plant- assisted remediation is limited by the knowledge of the different enzymes involved and their metabolic pathways. This was also shown by the inadequate understanding of the molecular processes of plants in response to stress agents in vitro (Rai et al., 2011). The report by Zeng et al., (2014) further confirmed that morphological and physiological changes due to contaminant toxicity in plants are not well understood at the molecular level. Some plant root cultures have been shown to be useful in the study of xenobiotic decontamination, through identifying metabolic pathways without the possible complications in interpretation of findings resulting from microbial associations (Coniglio et al., 2008; Nepovím et al., 2004; Patil et al.,

2009; Shanks and Morgan, 1999). The in vitro synergistic strategy of using two different ornamental plants together was successful in the treatment of simulated textile dyes through their activated enzymes machinery (Watharkar and Jadhav, 2014). It will be of interest, therefore, to equally understand the key signalling pathways that may be involved in the decontamination of diesel fuel.

1.3 Remediation technologies of polycyclic aromatic hydrocarbons (PAHs)

1.3.1 Remediation

Decontamination or removal of PAH contaminants in the environmental media has been demonstrated by various types of remediation technologies. This includes new, traditional and conventional methods of remediation using physical, chemical and biological principles as well as new emerging ones such as electrokinetic remediation, enzyme biocatalyst treatment and vermiremediation (Falciglia et al., 2016; Gan et al., 2009; Jia et al., 2016; Kuppusamy et al.,

2017).

1.3.1.1 Non-bioremediation technologies

Previous studies reported the use of thermal treatment, vapour extraction, chemical oxidation and reduction, soil washing/solvent extraction and the electrokinetic technologies as treatment and exclusion methods for PAH-contaminated soils (Kuppusamy et al., 2017; Streche et al.,

2018). However, physicochemical and engineering procedures that involve excavation of contaminated soils and dumping of the wastes to a landfill may lead to the transfer of contaminants and pose a significant risk to public health (Vidali, 2001). Furthermore, it is challenging and progressively more expensive to find new landfill locations for the dumping of hazardous materials (Vidali, 2001).

The method of using high-temperature incineration and various types of chemical decomposition (base-dechlorination, ultraviolet oxidation, etc.) may have been a better approach to remediate PAHs (Vidali, 2001). But these technologies have their own disadvantages: the use of an incinerator system and installation of the incinerator off-gas control devices required more energy to run and was costly (Islam et al., 2012; Montanarella,

2003), as well as creating enormous quantities of toxic by-products (Wani et al., 2007).

Solvent extraction/soil washing has, however, been considered as a practical alternative to other methods of cleaning up PAHs, using a natural surfactant such as humic acid (HA) that can promote microbial activity (Gong et al., 2010). But the low reduction in the percentage of the PAHs removed after the clean-up suggested instead the use of plants that can produce fatty acid methyl esters (FAMEs) for remediation (Conte et al., 2005; Gong et al., 2010). This is because FAMEs have been reported to be more effective in the removal of PAHs than other well-established washing agents (e.g., cyclodextrin and triton X100) (Gong et al., 2010).

Different types of oxidants (Fenton’s reagents, potassium permanganate, hydrogen peroxide, sodium iron, activated persulphate and peroxy-acid) (Cheng et al., 2016; Kuppusamy et al.,

2016) have been injected into the PAH-contaminated soils for in-situ degradation studies and

7 they achieved 50–95% mineralisation of the PAHs with no reduction in the PAH percentage of the non-pretreatment soils (Liu et al., 1993; Pradhan et al., 1997; Usman et al., 2012; Yap et al., 2011), so it was concluded that low availability of the PAHs in the soil is the limiting factor for achieving efficient degradation, especially in the PAH aged soil (Lemaire et al.,

2013). Even though better remediation results were observed in the new emerging remediation technologies such as the electrokinetic method, by using a small quantity of the contaminated

PAHs soil with a higher electric intensity (Streche et al., 2018), it may be a challenge to apply to large-scale contaminated PAH soils (Streche et al., 2018). Bioremediation has been recognised worldwide for treatment of PAH-contaminated soils and is ultimately considered to be safe, eco-friendly and to cost less than other remediation technologies without the transfer of contaminants to another medium (Kuppusamy et al., 2016; Mohan et al., 2006).

1.3.1.2 Bioremediation

Biological remediation is an alternative approach that is offering the opportunity to degrade or at least reduce the environmental pollutants to less toxic materials using natural biological processes (Vidali, 2001) that include the use of microbes, plants, and animals (Adki et al.,

2014). It is commonly considered a non-toxic, cost-efficient method (Pandey et al., 2009) and is frequently carried out on site. As such, it has attracted a lot of attention from all over the world. The concept behind the use of microbes, plants, and animals for environmental clean- up can be based on certain biological principles and activities. However, the use of microbes and plants for the clean-up of PAH-contaminated sites has been experimentally tested with varying degrees of success (Zayed and Terry, 2003).

Microbial remediation is a type of degradation that involves the use of microorganisms with their associated enzymes to dissipate most organic contaminants under controlled situations to a harmless state (Israr et al., 2011). This technology depends upon many factors, including the concentrations of organic contaminants, mobility of the contaminants, access by the microbes

8 to other nutrients, presence of activated enzymes (e.g. laccase (E.C.1.10.3.2) (Dodor et al.,

2004) manganese peroxidase (Bogan and Lamar, 1996)), and the ability to stimulate the numbers of microflora or microbial groups native to the contaminated sites that are capable of performing the required activities (Agarwal, 1998; Cerniglia, 1984, 1993). In this method, microorganisms use the pollutants as a source of nutrients or energy (Agarwal, 1998; Hess et al., 1997; Tang et al., 2007). These abilities to change and utilise the contaminant as a source of energy can increase their numbers in the contaminated site and so improve their effectiveness.

Microorganisms have been described as having developed various mechanisms of reacting to different types of environmental contaminants through transport across the cell membrane, biosorption to cell walls, entrapment in extracellular capsules, precipitation, complexation and oxidation-reduction reaction (Avery and Tobin, 1993; Brady and Duncan, 1994; Huang et al.,

1990; Krauter et al., 1996; Macaskie and Dean, 1989; Rai et al., 1981; Veglio et al., 1997).

Among the many reported challenges of microbial remediation is the inability to create uniform spatial distribution on a polluted medium such as soil (Mohan et al., 2006). Also, it may take a long time to reduce the level of contaminants, which may not always be acceptable (Vidali,

2001). Moreover, the application of microbes can only be operational if the ecological conditions are optimal for their development and activity (Israr et al., 2011).

Bioremediation treatment includes land farming, biostimulation, bioaugmentation, and bioreactors. There are various limitations associated with these different bioremediation treatment options (Bosma et al., 1996; Tang et al., 2005). For instance, in the biostimulation treatment, additives are used to stimulate the functioning of microorganisms to speed up the remediation process (Mohan et al., 2006). This may affect other organisms present in the habitat (Vidali, 2001). Israr et al. (2011) described possible concerns with the use of microbes,

9 particularly if genetically modified microorganisms are released into a contaminated site during bioaugmentation treatment for remediation purposes.

Plant-assisted bioremediation (phytoremediation), however, is the in-situ clean-up of PAH- contaminated sites on a large scale by encouraging large numbers of microorganisms around the plant root zones (Petruzzelli et al., 2016). It is also defined as an environmental clean-up by plants and their related microbes (Raskin et al., 1994; Salt et al., 1995; Salt et al., 1998). In the different studies of phytoremediation of PAHs, it was reported that plants could accumulate/sequester/chemically transform and localise toxic contaminants in different environmental media (Gan et al., 2009; Roy et al., 2005; Susarla et al., 2002). It was also found that plants release enzymes that can act as a surfactant to increase the bioavailability of the contaminants and enhance the nutrient status of the soil (Gan et al., 2009; Roy et al., 2005).

Organic and inorganic pollutants in solid, liquid and gaseous substrates can be phytoremediated

(Horne, 2000). Different types of inorganic pollutants have been phytoremediated, including macronutrients (Horne, 2000), trace elements (Lytle et al., 1998), nonessential elements

(Blaylock and Huang, 2000; Horne, 2000) and radioactive isotopes (Dushenkov, 2003;

Dushenkov and Kapulnik, 2000; Negri and Hinchman, 2000). In the case of organic contaminants such as chlorinated solvents (Alkorta and Garbisu, 2001), polychlorinated biphenyls (PCBs) (Ferro et al., 1994) and PAHs (Peng et al., 2009), progressive achievements have been recorded using phytoremediation.

Flathman and Lanza (1998) and Pilon-Smits (2005) categorised the phytoremediation mechanisms into five different subcategories: phytodegradation, phytoextraction, phytovolatilisation, phytostabilisation and phytostimulation (Figure 1.2). Phytostabilisation is the use of plants to neutralise contaminants in soil (Berti and Cunningham, 2000) while phytoextraction is the ability of plants to remove and gather contaminants in their tissues

(Blaylock and Huang, 2000). However, in a process called phytostimulation, plants can also

10 increase the rate of the biodegradation processes of organic pollutants by using microorganisms around their root zones (McCutcheon and Schnoor, 2003).

Plants have shown the ability to allow some contaminants to escape in volatile form through their tissues and such process is called phytovolatilisation (Terry et al., 1995). McCutcheon and Schnoor (2003) described the process of phytodegradation as organic contaminants are degraded directly by plants using their enzymatic activities. These technologies are not independent in operations but rather can also occur simultaneously (Hansen et al., 1998).

Figure 1.2 Schematic representation of pollutant fates during phytoremediation (Pilon-Smits, 2005). 1.3.1.3 Phytoremediation studies using ornamental plants

1.3.1.3.1 Background on Petunia grandiflora and Tagetes patula

Petunia has a long history, since around 1944 in the United States, as the only bedding plant that survived a severe summer drought (Griesbach, 2006). It became national news at that time because of the drought tolerance shown (Haughton, 1978). This tolerance increased their popularity and currently it tops the world list of most popular bedding plant, with an annual

11 wholesale value exceeding US$130 million in the USA alone because of its range of colour and morphology (Schneider et al., 2003).

Today Petunia is a genus name under the Solanaceae family that is native to South America, with many classes resulting from hybridisation and propagation (Kessler, 1999). It forms flowers with a unique colour on compact plants and more branched plants with smaller to larger leaves within the family. Petunia has a base chromosome number of x = 7 rather than the typical x = 12 found in most other Solanaceae species (Griesbach, 2006; Särkinen et al., 2013).

One of these classes is the annual herb grandiflora, which was found in early 1881 as a result of a mutation in the multiflora (Ewart, 1984; Weddle, 1976). The multiflora type is also called the hybrida type, which is derived from the crosses between a white-flowered Petunia axillaris and species of the Petunia integrifolia (Griesbach, 2006). However, Petunia has long been established as a model plant for scientific research (Bombarely et al., 2016).

Tagetes patula is often called French marigold, an annual plant from the largest vascular family

Asteraceae (Compositae) (Singh et al., 2015). The plant Tagetes patula (marigold) has a chromosome number of 2n = 48 (Jalil et al., 1974) and is considered ornamental because of its floricultural use (Singh et al., 2015).

1.3.1.3.2 Phytoremediation studies

Ornamental plants have been used in the study of phytoremediation of different types of contaminants. Watharkar et al. (2013a), and Watharkar and Jadhav (2014) revealed the phytoremediation potential of Petunia grandiflora (petunia) on a dye mixture and textile effluent. Also, Watharkar et al. (2013b) reported the ability of petunia with their associated bacteria to phytotransform Navy Blue RX (NBRX) dye to a less toxic metabolite of the dye.

The most identified strategies of the phytoextraction of heavy metals by the tolerant plants are by accumulating the contaminant. An in vitro study of genetically engineered CAXcd- expressing petunia plants revealed the potential of the plant to tolerate high levels of cadmium

(Cd) and accumulate levels that were 2.5-fold higher than wild-type or untransformed plants

(Wu et al., 2011). In another study reported by Lee et al. (2015), cloned glutathione-dependent formaldehyde dehydrogenase gene (AtFALDH) (Martinez et al., 1996) was used to create transgenic petunia plants. AtFALDH-transgenic T2 plants removed 25.9% more exogenous formaldehyde (HCHO) gas than the non-transgenic plants.

The documented scientific research findings related to the phytoremediation potential of textile dyes showed that petunia was of interest (Watharkar and Jadhav, 2014). The phytoremediation potential of the plant in relation to organic contaminants, particularly diesel, has not been reported. Planting the ornamental plant may also add aesthetic and commercial values to the diesel-degraded soil (Watharkar and Jadhav, 2014).

Marigold has been used in a phytoremediation study of PAHs and heavy metals (Sun et al.,

2011) and a phytostabilisation study of iron-ore tailing (Chaturvedi et al., 2014). Also, it has been reported to phytodegrade different types of textile dyes (Patil and Jadhav, 2013; Patil et al., 2009). In a phytoremediation study marigold and Mirabilis jalapa were used to promote biodegradation of benzo [a] pyrene contaminated soil by 79.5–99.8% and 71.1–99.9%, respectively (Sun and Zhou, 2016).

The remediation potential of marigold in diesel-contaminated soil is not known. In another study, a wild ornamental plant (Iris lacteal), which has an extensive fibrous root system and rapid biomass production, showed tolerance to 4g/kg of petroleum hydrocarbons (PHC) fractions in the contaminated soil and a relatively high degradation of about 20.7% (Cheng et al., 2017).

1.4 Plant tissue culture techniques

Plant cell and tissue culture technology is based on the possibility of raising and regenerating plants and inducing genetic variation in cultured cells or tissues under aseptic conditions

(Mohamed et al., 2000). Somaclonal variation in plant cell culture is a novel source of genetic

13 variation that can be exploited for plant germplasm improvement (Ashrafzadeh and Leung,

2015; Krishna et al., 2016). The technology uses all types of plant cells, tissues, and organs under control sterile conditions (Smith and Drew, 1990).

However, the development of plant cell and tissue technology has passed many milestones, just like other fields of science (Bhojwani and Razdan, 1986; Gamborg et al., 1976). Gottlieb

Haberlandt established the theoretical concept of tissue culture in his address to the German

Academy of Science in 1902 on his experiments on the culture of single cells (Haberlandt,

1902). At that time the aim was to provide information about the interrelationships and complementary effects to which cells within the entire multicellular organism are exposed

(Krikorian and Berquam, 1969).

Until then no significant contributions were made in the area as conceived by Haberlandt, but in the mid-1930s, a major push led to the development of plant tissue culture techniques

(Steward et al., 1958). The investigation identified some of the critical problems in cultivating isolated cells in nutrient solutions through a new technique (Bhojwani and Razdan, 1986).

Today plant tissue culture has tremendous practical potential in the understanding of the morphology, physiology, biochemical and molecular changes in plants and their commercial applications.

1.4.1 Explant and in vitro cultures

The responses of a plant cell, tissue or organ to the in vitro culture conditions may largely depend on the suitability of the culture composition, which is mostly determined by the presence of the artificial hormonal component in the medium. The genetic and epigenetic make-up of cultured explant tissue (leaf, shoot tip or other source of explant) may be reprogrammed to fit in the hormonal environment and ultimately determine its adaptability to in vitro cultures (Neelakandan and Wang, 2012). The age and types of explant used determine the fate of the cultures in vitro, even when the requirements and culture conditions are

14 optimised (Granja et al., 2018); for instance, various explants, of the leaf (LE), cotyledon (CE), hypocotyl (HE) and root (RE) have been used to initiate callogenesis (Gourguillon et al., 2016;

Gourguillon et al., 2018).

1.4.2 Callus culture and plant regeneration

Callus cultures are undifferentiated masses of cells induced from different explant types. Shoot bud or plantlet formation can be initiated when they are provided with the optimal conditions in the presence of plant growth regulators (PGRs) under aseptic conditions (Hare and Cress,

1997). The in vitro regenerants may be used to obtain useful information in the studies of the morphology, and the biochemical and molecular events taking place during the early development stages of the plantlets (De Klerk, 1996). The technique is also attracting interest in studies of physiology, genetics, toxicity and resistance of plants under controlled conditions

(Benderradji et al., 2007; Leung, 2017).

1.4.3 Somaclonal variation

1.4.3.1 Cell line selection

It has been reported that at the cellular level, isolation of variants is more precise than the whole plant level, and takes place without chimeras (Huynh et al., 2013). It has also served as a valuable tool in clarifying the mechanisms of salt tolerance at the cellular level (Huynh et al.,

2013). Progeny lines with improved metal tolerance and extraction characteristics have also been regenerated following in vitro cell-line selection (Guadagnini et al., 1999; Herzig et al.,

1997). In vitro selection of abiotic stress-tolerant cell lines and regenerated plants have been reported in species of Setaria italic (Rout et al., 1998), Tagetes minuta (Mohamed et al., 2000),

Echinochloa colona (Samantaray et al., 2001), Foeniculum vulgare (Khorami and Safarnejad,

2011) and soybean genotype (Huynh et al., 2013). These were obtained by subjecting plants to different stress agents in a controlled environment and initiating tolerance in plants with the desired developmental phenotypes (Purohit et al., 1998; Vázquez and Linacero, 2010);

15 however, there is no prior study on somaclonal variation of diesel-tolerant plants. Conceivably, this is most probably related to the technical challenges associated with the use of diesel fuel as a selection pressure in the aseptic culture medium during plant cell culture. This practical attempt has therefore involved the development of a novel method using in vitro cell line selection to regenerate variant lines in plants that will be resistant to diesel pollution. Tissue culture plants of petunia have been reported to degrade and disperse disulphonated triphenylmethane textile dye (Watharkar et al., 2013b). The combination of tissue culture plants of petunia and Gaillardia grandiflora have also been reported as degrading a mixture of textiles dyes (Watharkar and Jadhav, 2014). These plants have exhibited practical potential in phytoremediation and will likely be suitable phytoremediators because they are not food plants

(Watharkar and Jadhav, 2014), and therefore, the use of petunia and marigold to generate somaclonal variant diesel-tolerant plants would further open the possibilities of their phytoremediation potential.

1.4.3.2 Histological analyses of callus culture

For in vitro cell-line selection studies using various stress agents, some have reported the use of histological characterisation of the putative lines. For instance, in a study of in vitro selection of copper-tolerant Nicotiana tabacum, the stem cross section of Cu-tolerant plant lines of N. tabacum had normal xylem tissue formation whereas the control had few xylem elements in the central cylinder (Gori et al., 1998). However, in most of the studies of in vitro cell-line selection, morphological observations of the selected lines were reported without considering the importance of histological characterisation analysis. In contrast to the previously reported studies, histological analysis has been employed in the present study of callus culture of petunia and marigold exposed to undiluted diesel fuel.

1.5 Morphological analyses of somaclonal variants generated in vitro

It seems reasonable to evaluate the performance of plantlets regenerated in vitro for their

quality with respect to growth and development under in vitro as well as ex vitro conditions

(Palombi and Damiano, 2002). For example, somaclonal variants from sugarcane have been

evaluated under stringent field testing conditions, to gain a better understanding of the effect

of the genetic changes in putatively beneficial somaclones (Gilbert et al., 2005).

1.5.1 In vitro evaluation

As far as the cell and tissue culture technique is concerned, its operation is conducted under strict aseptic conditions; therefore, results obtained on the morphological parameters of plantlets raised in vitro would not be complicated by the potential interference/interactions with microorganisms in field trails (Ashrafzadeh and Leung, 2017). In the case of in vitro cell-line selection, it has been common practice to evaluate the variant lines obtained in vitro for the selected stress agent to uncover the improved tolerant lines with desirable characteristics that might be of advantage over the control (Clemens, 2001; Zhu et al., 2007). Morphological and physiological traits are the most common phenotypic parameters used to observe variations among the cell lines. For example, there have been studies of in vitro selection of strawberry

(Fragaria ananassa) plants tolerant to salt stress (Dziadczyk et al., 2003), development of salt- tolerant lines in Chrysanthemum morifolium (Hossain et al., 2007) and copper-tolerant tobacco plant lines (Rout and Sahoo, 2007). Morphological methods have been used in these studies.

Such methods were not possible in this study because of the technical challenges associated

with the use of diesel fuel under in vitro conditions. The approach in the present study was that

the in vitro growth performance of the putative diesel-resistant plant lines of petunia obtained

(first reported in this study) were first compared with the parent plants (the control) on a

medium without diesel fuel. Then a parallel study was carried out that evaluated the different

lines of petunia growing under glasshouse conditions in potting mix contaminated with diesel

17 fuel. It was anticipated that this might have the potential to unveil variants with respect to the relative sensitivity to diesel toxicity and also possible enhanced tolerance to diesel that might be of use for future phytoremediation trials in the field.

Most of the in vitro assessments of putative resistant plant lines to biotic and abiotic stress have reported the detection of variations using morphological parameters and to a lesser extent at the DNA level (Evans et al., 1984). This has always been the basis of the argument for further possible methods of investigation for variation (Cloutier and Landry, 1994). However, many somaclones have been easily identified based on certain growth parameters such as differences in plant stature, leaf morphology and pigmentation abnormality (Israeli et al., 1991).

Somaclonal variations in the different ornamental plants have been identified using the morphology methods listed in Table 1.1: the significance of morphological methods as a quick check assessment tool for detecting somaclonal variation cannot be overemphasised, as morphological characters are often influenced by environmental factors (Mandal et al., 2001).

Table 1.1 Examples of the morphological markers used to detect somaclonal variation induced in vitro in ornamental plants Species Common name Source of variation Detection References method

Chrysanthemum morifolium Ramat Chrysanths Gamma-ray Morphology (Lamseejan et al., 2000)

Aponogeton ulvaceus Baker Madagascar water lettuce Callus culture Morphology (Kam et al., 2016)

Dianthus L. hybrid Carnation × Sweet X-ray Morphology (Cassells et al., 1993) William

Dieffenbachia spp. Mother-in-law’s tongue Number of subcultures Morphology (Shen et al., 2007)

Tulipa gesneriana L. Tulips Duration in culture Morphology (Podwyszynska, 2005)

Heliconia bihai Lobster claw Number of Morphology (Rodrigues, 2008) micropropagation

Limonium perezii Hubbard Statice Chimeric effect Morphology (Kunitake et al., 1995)

Ledebouria graminifloria (Baker) __ BA, NAA Morphology (Shushu et al., 2009) Jessop

Note: (BA) 6-benzyladenine; (NAA) 1-naphthalene acetic acid

1.5.2 Evaluation under glasshouse conditions

One common challenge facing the successful establishment of plantlets raised in vitro under ex vitro conditions is their low survival rate when transferred from in vitro cultures (Xiao et al., 2011). For instance, in vitro plantlets are often grown under a control level of light intensity

(1,200-3,000 lux) and temperature (25 ± 2°C), on a medium with sufficient sugar and nutrient contents, and at a high level of humidity (Chandra et al., 2010; Hazarika, 2003). The in vitro conditions favour the formation of certain physiological and anatomical features in plantlets that are well adapted to the culture conditions. (Hazarika, 2003; Marin et al., 1988). These may have led to the development of plantlets that would not survive outside the culture environment if taken or grown directly under glasshouse or field conditions (Hazarika, 2003).

To increase the survival chances of in vitro plantlets with better growth performance under glasshouse conditions, it is therefore necessary to gradually expose plantlets to similar but artificial ex vitro conditions within the in vitro control growth chamber before the final transfer to the real ex vitro environment through the process of acclimatisation (Mathur et al., 2008).

However, the acclimatisation steps may be different between species of plantlets generated in vitro, but the principal steps remain the gradual lowering of air humidity inside the culture jars and increasing light intensity and temperature in the culture jars (Bolar et al., 1998; Kadleček,

1997; Preece and Sutter, 1991). Considerable efforts have been made to optimise the acclimatisation steps of plantlets raised in vitro for better growth performance ex vitro. For example, Chrysanthemum morifolium (Lamseejan et al., 2000), Dieffenbachia spp. (Shen et al., 2007) and Dianthus L. hybrid (Cassells et al., 1993) have been successfully established ex vitro. In this study, the potentially diesel-resistant variants of petunia were also successfully established in the glasshouse (first reported in this study).

Recently, the use of ornamental plants for phytoremediation of environmental contaminants including diesel is of great interest because of its significance and the realistic potentials of the

20 plants as phytoremediators (Liu et al., 2008; Liu et al., 2006). This has prompted the variously reported research findings on the assessment of plants for phytoremediation of petroleum contaminations (Cheng et al., 2017; Ikeura et al., 2016; Peng et al., 2009; Zhang et al., 2010).

Many of the reported research findings on the evaluation of plants under greenhouse conditions were obtained in pots experiments containing simulated petroleum-contaminated soil (Cheng et al., 2017; Liu et al., 2012). In many studies, the starting plant materials were seeds (Ikeura et al., 2016; Liu et al., 2014; Xiao et al., 2015; Zhang et al., 2010), and only in a few reports were cuttings used from the mother plants (Cheng et al., 2017).

The reported research findings of the toxicity bioassay tests of diesel contamination on different seed types have better informed our understanding of variation in the sensitivity responses among the different germinated seeds (Adam and Duncan, 2002; Bamgbose and Anderson,

2015; Wante and Leung, 2018). In addition, other factors may also be involved, such as soil type, history of the plant materials used and climatic conditions (Kaur et al., 2017).

Alternatively, uniformity of plant materials can be possible with cuttings from different plantlets/lines of the same age for comparison of growth performance under glasshouse conditions (Peterson and Baldwin, 2004; Xiaoling et al., 2011).

Soil-less media such as potting mix are suitable for use in horticulture for growing seedlings and propagation of ornamental plants (Chavez et al., 2008), because they have capacity for adequate water retention and aeration (Erstad and Gislerød, 1994). Physical and chemical properties of the medium can be managed for plant cultivation (Chavez et al., 2008).

These advantages of the soil-less medium are the important factors that guided the choice of potting mix as the best medium for the screening of petunia cuttings for growth performance in the presence of diesel contamination under glasshouse conditions. Potting mix is also considered to be a pristine medium compared to soil. This is because it may have less

21 interference from the microbial density in the medium than from inhabitant microbes in natural soil (Koohakan et al., 2004).

1.6 Aims, objectives and structure of the thesis

Considering the toxic effects of PAHs found in diesel, which have the potential to cause mutagenic effects and cancer in humans and some mammals, there is a need for research into the potential of diesel-resistant ornamental plants. The main aim in this research was to develop plants cell lines using in vitro cell-line selection to assist remediation technology

(phytoremediation and phytomanagement) of diesel-contaminated land. This may be low-cost and eco-friendly technology for the biodegradation of diesel contaminants and providing ecosystem services supplied by the ornamental plants (their roots to help soil functionality and their flowers to provide for pollination). In addition to beautifying the environment, these two ornamental plants have demonstrated strong ability in the phytoremediation of various contaminations (Liu et al., 2017). In this research, putative novel diesel-resistant petunia plants were generated using the non-genetic engineering method of in vitro cell-line selection under aseptic conditions. Furthermore, in the remainding time available within the PhD time limit, the morphological, histological changes and growth performance of these putative plant lines derived in vitro were evaluated in the lab and under glasshouse conditions. The thesis was structured and formatted toward accomplishing the following specific objectives:

Chapter 1

The general overview of current knowledge, the literature review and major aspects related to the scope of the study were provided.

Chapter 2

In this chapter, the materials and methods used repeatedly in the different parts of the study were described. In addition, the common statistical software and methods used for analyses of

22 data were also described. The methods specific to particular experimental parts (Chapters 3, 4,

5 and 7) were also described.

Chapter 3

There was no prior study on the effect of diesel on petunia and marigold. Here, the relative sensitivities of petunia and marigold seeds and seedlings to diesel were investigated.

Chapters 4 and 5

The requirements for high efficiency of callus initiation, subculture and plant regeneration in

Petunia grandiflora (Chapter 4) and Tagetes patula callus cultures (Chapter 5) were investigated. An innovative procedure to expose callus to diesel and then subculture the diesel- treated Petunia grandiflora callus was first established in Chapter 4. It was then validated with another plant Tagetes patula and described in Chapter 5.

Chapter 6

To find out the effect, if any, diesel exposure might have on the internal organisation of Petunia grandiflora and Tagetes patula calli, a histological study of the calli was carried out.

Chapter 7

Six experimental lines of plants (L1–L6) were regenerated from Petunia grandiflora calli exposed to undiluted diesel for 9 min. Two control lines of plants, one from germinated seed

(C-G) and one from a 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 diesel tolerance mechanism in plants. It might also be of use for phytoremediation of diesel contamination.

Chapter 8

The final chapter highlights an overall discussion, the conclusion, and main findings of the thesis to better inform our understanding of the in vitro selection of diesel-resistant Petunia grandiflora plants. Suggestions for future research directions and the limitations of the research from thesis results are also provided.

Chapter 2 General materials and methods 2.1 Plant materials 2.1.1 Seeds

Seeds of Marigold-Nemo mix (Tagetes patula L.) and Petunia grandiflora Juss. mix F1 were purchased from Kings Seeds, Katikati, New Zealand. Within 18 months of seed storage in a closed container in the dark at 22 ℃, the seeds were used for this study. There did not appear to be any change in seed germinability kept in this way.

2.1.2 Analysis of diesel fuel

A typical gas chromatographic (GC) scan of the diesel used in this study with abundant peaks of low molecular weight components (C12–C20, Figure 3.1) was obtained using a GC flame ionisation detector (FID) by Hill Laboratories, Hamilton, New Zealand.

2.1.3 Preparation of diesel-contaminated water solution

The diesel-contaminated water was prepared with deionised water at 0, 0.5, 1.0, 1.5, 2.0, 2.5,

3.0 and 4.0% (v/v). The 0% (v/v) is the deionised water without diesel and is the control treatment. The 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 4.0% (v/v) are the simulated diesel-contaminated, deionised water treatments.

2.1.4 Treatment of seed using water contaminated with diesel Petunia grandiflora (petunia) or Tagetes patula (marigold) seeds were sown in 10 mL of a test solution (0–4% diesel, prepared in deionised water, v/v) in a plastic Petri dish (90 mm diameter). There were four replicates (3 Petri dishes each with 18 seeds) for a test solution. All the replicates were arranged in a completely random way on the bench in a growth room at 25

℃. Germination rates were scored daily up to 10 and 15 days from sowing marigold and petunia, respectively.

2.1.5 Measurement of root length, shoot height and plant size

Root length and shoot height of marigold and petunia seedlings were measured using a ruler after 10 and 15 days from sowing seeds, respectively. The petunia seeds, whether germinated or not after 15 days in the different treatments, were rinsed several times in deionised water before they were returned to Petri dishes with deionised water only for a further 15 days to score for any sign in the recovery of seedling growth.

2.1.6 Elongation inhibition rates

The elongation inhibition (EI) rates (in %) of the roots of marigold and petunia incubated in diesel-contaminated water compared to the control were determined according to Visioli et al.

(2014). The differences between the mean root length value of the control Lc and the different diesel treatments (Lt0.5 to Lt4) were calculated and expressed as EI % using the formula:

(퐿푐 − 퐿푡) 퐸퐼 % = × 100 퐿푐

2.2 In vitro medium composition and culture conditions 2.2.1 Medium composition and culture conditions for seeds of Petunia grandiflora and

Tagetes patula germinated in vitro

The basal MS medium (Murashige and Skoog, 1962) was the nutrient medium used in the in vitro germination of petunia and marigold seeds. The medium prepared was supplemented with

3% sucrose (w/v) and adjusted to a pH of 5.7–5.8 using 1 N KOH or 1 N HCl. Then 0.8% (w/v) agar (Oxoid Ltd., UK) was added before the medium was autoclaved at 121 ℃, 103,421 Pa for

15 minutes.

2.2.2 Seed germination under aseptic conditions

Seeds of marigold and petunia were germinated under in vitro conditions. Before surface sterilisation, the seeds were firstly washed with 0.8% (w/v) soapy water (Pyroneg L88z,

ThermoFisher Scientific Ltd., Christchurch, New Zealand) for 3 min., and then rinsed four times with sterile distilled water. For surface sterilisation, the washed seeds were (a) soaked

26 for 5 min. in 70% ethanol, (b) washed four times with sterile distilled water, (c) immersed in

1% (w/v) sodium hypochlorite (NaOCl) for 15 min., and (d) finally rinsed thoroughly with sterile distilled water. Five surface-disinfected seeds were placed, under aseptic conditions, on the surface of sterile basal MS (Murashige and Skoog, 1962) medium described in section

2.2.1.

2.2.3 Medium composition and culture conditions for callus induction and sub-culturing in Petunia grandiflora and Tagetes patula

Full strength MS basal (Murashige and Skoog, 1962) medium was the nutrient medium used for the callus initiation and sub-culturing, and it was supplemented with 3% sucrose (w/v), plant growth regulators and 0.8% (w/v) agar before the medium was autoclaved at 121 ℃,

103,421 Pa for 15 minutes.

2.2.4 Callus induction and proliferation in Petunia grandiflora and Tagetes patula

Four-week-old leaves of seedlings of petunia and marigold germinated in vitro were aseptically excised for callogenesis. The adaxial side of the leaves of petunia or marigold was placed on the MS medium supplemented with various combinations of auxin types, 1-naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and picloram (PIC), in combination with different concentration of the cytokinin 6-benzyladenine (BA). There were 15 replicate explants in each treatment of petunia and marigold. The cultures were kept in a growth room at 21 ± 1 ℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps

36W) for four weeks. For the determination of callus induction frequency, the percentage of explants forming a callus was calculated. All calli obtained were separated from the explants and carefully divided into two parts for sub-culturing in the same type of medium that initiated calli of petunia or marigold. Subsequent sub-culturing was achieved after every two weeks to a fresh medium to propagate the callus and to avoid browning. The calli were divided into two

27 groups: the first group was maintained as stock while the second group was used for further experiments.

2.2.5 Morphology and selection of calli formed in Petunia grandiflora and Tagetes patula

Different types of calli that formed on the callus induction medium of petunia and marigold in response to various types and different concentrations of plant growth regulators were visually assessed using the following parameters: colour, size, and texture. Additionally, photographs of the calli were taken at the beginning and at the end of four weeks of culture. A medium that was associated with the highest percentage of leaf explants forming non-friable, green compact calli was selected as the medium for subculture of petunia and marigold.

2.2.6 Maintenance of callus culture of Petunia grandiflora and Tagetes patula

Pieces of non-friable, green compact callus were maintained for one month on the optimised callus subculture medium selected for petunia and marigold. Calli were physically examined and observed under a stereomicroscope for any change in colour and texture. Subsequent sub- culturing was achieved after every two weeks to fresh optimised subculture medium to propagate the callus and to avoid browning.

2.3 Exposure of callus to diesel fuel

2.3.1 Exposure of Petunia grandiflora and Tagetes patula callus to diesel toxicity

About 50 pieces of petunia callus or marigold callus were removed from the callus subculture medium under aseptic conditions in a laminar flow cabinet and placed in a sterile polycarbonate jar (250 mL). The jar with the calli was taken to a fully running fume cupboard (EcoairTM ,

Thermoplastic Engineering Ltd., Wellington, New Zealand) with the fan turned on. Then the lid of the jar was opened slightly and long enough so that 100 mL of diesel could be poured into the jar. The jar was swirled continuously by hand to keep the callus pieces wholly submerged in the diesel for varying times of 5, 7 and 9 min. Then with, the lid of the jar opened slightly, the diesel was decanted and replaced with 100 mL of sterile distilled water. The jar

was closed with the lid for a brief rinsing of the callus pieces and this was repeated four more

times. At the end of the last round of rinsing, 100 mL of 0.25% (v/v) of Plant Preservative

MixtureTM , (PPM, Plant Cell Technology, Washington, USA) was poured into the jar which

was swirled (with the lid on) to immerse the callus pieces for 15 min. The jar was brought back

to the laminar flow cabinet to resume plant tissue culture under aseptic conditions. Without

rinsing, the callus pieces were removed from the PPM solution and placed on the surface of

the callus subculture medium (5 pieces each in a pre-sterilised plastic Petri dish).

2.3.2 Culture of diesel-treated Petunia grandiflora and Tagetes patula calli under aseptic

conditions

After two weeks of diesel exposure and culture on the callus medium, the diesel-treated callus

pieces of petunia and marigold were assessed visually and compared with those not exposed to

diesel. The non-necrotic regions of the diesel-treated calli were then excised, and one piece

was placed in a Petri dish (9 cm diameter) with fresh callus subculture medium. After 14 days,

the calli were assessed visually for necrosis and increase in size. The calli were then subdivided

into two smaller pieces, and each was placed on a Petri dish with fresh callus medium for

another 14 days before visual assessments. Signs of any contamination in the cultures were

monitored throughout the 6-week period of experiment.

2.3.3 Selection of Petunia grandiflora and Tagetes patula calli that survived after diesel

exposure

For the purpose of this investigation, each non-necrotic region from diesel-treated calli of petunia and marigold that had been cultured on MS subculture medium was selected and subcultured. Then, signs of any contamination in the cultures were monitored throughout three subcultures stages (each of two weeks duration). At the start of the third subculture, calli of petunia or marigold that proliferated to twice their initial size were subdivided into two pieces and each placed on MS subculture medium for petunia or marigold. After six weeks of culture,

29 a callus that was able to proliferate on the subculture medium was considered as a culture of interest that was successfully derived from the initial callus pieces exposed to harmful diesel.

2.3.4 Exposure of Petunia grandiflora calli to sterile distilled water, diesel, and Plant

Preservative Mixture (PPM)

Three sterile polycarbonate jars (250 mL) were labelled 1, 2 and 3 and filled with about 50

pieces of calli, each under aseptic conditions in a laminar flow cabinet. The three jars with the

calli were taken to a fully running fume-cupboard (EcoairTM , Thermoplastic Engineering Ltd.,

Wellington, New Zealand) with the fan turned on. Then the lids of the jars were opened slightly

and for long enough so that 100 mL of diesel could be poured into each of jar-1 and jar-2; in

jar-3, 100 mL of sterile distilled water was poured. The jars were swirled continuously by hand

to keep the callus pieces wholly submerged in the diesel and sterile distilled water for 9 min.

Then, with the lid of the jars opened slightly, the diesel was decanted off and replaced with

100 mL sterile distilled water. The jars were closed with the lid and swirled briefly for rinsing

the callus pieces. This rinsing step was repeated four more times. At the end of the last round

of rinsing, 100 mL of 0.25% (v/v) of Plant Preservative MixtureTM, (PPM, Plant Cell

Technology, Washington, USA) was poured only into jar-1 and jar-3 which were swirled (with

the lid on) to immerse the callus pieces for 15 min. In jar-2, 100 mL of sterile distilled water

was poured and was swirled (with the lid on) to immerse the callus pieces for 15 min. The jars

were brought back to the laminar flow cabinet to resume plant tissue culture under aseptic

conditions. Without rinsing the callus pieces from the jars, calli were removed from the PPM

solution or sterile distilled water and, placed separately on the surface of the callus subculture

medium (5 pieces in a Petri dish). Contaminations were monitored during two weeks of culture.

2.4 Plant regeneration from callus

2.4.1 Plant regeneration attempts in Tagetes patula

Calli were cultured on basal MS medium supplemented with 1.8, 3.6, 5.4 and 6.6 µM BA in combination with different concentrations of GA3 (5,10, 50 to 100 µM) or NAA (1.8 to

2.2 µM). For this investigation, two different types of calli were used: old (more than three months old after the second subculture from induction) and new callus (one month old from the second subculture). There were 18 old and 18 new pieces of calli in each treatment, and there were four replicates. They were separated with a straight line drawn at the back of each pre-sterilised plastic Petri dish and cultured for ten weeks. Another set of MS basal medium with different plant growth regulators (PGRs) at various concentrations was used for the culture of diesel-treated calli only for ten weeks.

2.4.2 Plant regeneration in Petunia grandiflora from non-diesel-exposed calli and those that survived diesel treatment

For this investigation, all calli originated from the optimised callus culture induction medium/callus subculture MS medium. Three different types of calli were used: putative diesel-resistant calli, non-diesel-exposed (control) calli including old (more than 3 months old after the second subculture from callus induction) and new callus (one-month-old from the second subculture) were cultured on basal MS medium supplemented with 2.3, 4.6 and 9.1 µM zeatin alone (Table 4.4), or a combination of different concentrations (1.1, 11.4 and 17.1 µM) of IAA or 0.5 µM NAA with different concentrations (4.4, 8.9,13.3 and 17.8 µM) of BA

(Tables 4.6 and 4.7). There were at least three pieces of calli from each of the three groups: calli that survived diesel exposure, and old and new control calli that were placed in a Petri dish. In each treatment, there were eight Petri dishes and each Petri dish had the three groups of calli placed on the three regions of the dish separated with straight lines as shown in Chapter

4 (Figure. 4.1).

2.4.3 Morphology of different types of shoots formed in Petunia grandiflora

Shoots formed in the calli that survived diesel-treatment, and new and old control petunia calli

were visually assessed, and their photographs were taken after 12 weeks of culture on MS

medium with different plant growth regulators.

2.4.4 Shoot multiplication and root formation in Petunia grandiflora

The calli that regenerated shoots were removed from the agar, and shoot-forming locations were carefully noted. Multiple shoots formed at the same point on the callus were considered the same group of plant lines. Shoots arising from a different position or point location on a piece of callus were regarded as different plant lines. Then, shoots were isolated and immediately transferred to PGR-free half-strength MS (Murashige and Skoog, 1962) medium supplemented with 3% sucrose (w/v) and gelled with 0.8% (w/v) agar for shoot proliferation and rooting for 3–5 weeks.

Shoots that formed roots and 4 to 5 leaves were micropropagated using the same medium composition. Shoots with no sign of root formation after three weeks were transferred to half- strength MS medium supplemented with 2.5 µM IBA for four weeks. Finally, rooted and non- rooted shoots were micropropagated and maintained on PGR-free, half-strength MS medium for plant line establishment purposes.

2.4.5 Petunia grandiflora plantlet lines and growth conditions

The petunia plantlets used for the evaluation of in vitro growth performance study included:

(a) a line of plantlets from the seed germinated in vitro (C–G) and (b) lines of plants regenerated

from callus cultures that were with (L1–L6) or without exposure (C–R) to 9 min. of undiluted

diesel. The plantlets from the germinated seed and from callus cultures without exposure to

diesel were the control treatments, while the plantlets regenerated from callus cultures exposed

to diesel were the treatment lines (L1 to L6). For each of the plant line of (a), (b) and (c), shoot

tip segments of 1 cm with two leaves were excised from the plantlets. Five shoot tips were

32 cultured for four weeks in a jar containing 50 mL half-strength MS medium without addition of PGRs. There were four replicates containing eight jars in each of the treatments (L1 to L6) and controls.

2.4.6 Growth analyses

At the end of the four-week experiment, plantlets were carefully removed from the culture jars, and any agar attached to the roots were washed using running tap water. Signs of any root were visually examined, counted and measured using a ruler and the averages (means) were expressed in centimetre (cm). Further, shoot height was measured from the shoot base to the apical tip using a ruler, and the means expressed in centimetre (cm). The leaves were counted, and the mean of the leaf numbers determined. The fresh and dried biomass of the plantlets was determined at the end of the four weeks. Fresh plantlets of the treatments, L1 to L6, and the controls were weighed, and then plantlets were dried in an oven (Contherm Scientific Limited,

Hutt City, New Zealand) at 80℃ for 48 h and weighed using a balance. The mean mass value was expressed as fresh and dried biomass in grams (g).

2.5 Hardening off and acclimatisation of Petunia grandiflora plantlets

2.5.1 Under conditions of a controlled growth chamber

Petunia plantlets of (a) germinated, (b) regenerated petunia plant lines (L1–L4) from diesel- exposed callus cultures, and (c) non-diesel-exposed cultures were micropropagated in sterile polycarbonate jars containing 50 mL of half-strength MS medium (Murashige and Skoog,

1962) without any added plant growth regulators. All plantlets were maintained in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36 W). Five jars of each category of plantlets (a, b and c) with well-established roots were randomly selected. The lids of the culture jars with the selected plantlets were opened for about 48 hours in the growth room at 21 ± 1℃ with continuous lighting. Then, the jars were transferred to a washing room, and the agar attached to roots was carefully washed off using

Milli-Q ultrapure water (ThermoFisher Scientific, France). Washed plantlets were returned to their jars, and about 50–60 mL of the Milli-Q ultrapure water was added to cover the roots of the seedlings.

After this, the jars were returned to the growth room for another week, after which, about 10–

15 plantlets were selected from each of the three categories of experimental materials. All the selected plantlets were taken to the glasshouse.

2.5.2 Under glasshouse conditions

The potting mix with a slow-release fertiliser was the medium used for growing the plantlets.

The composition of the potting mix was sent to the Hill’s Laboratory, New Zealand for analysis. The selected pre-acclimatised petunia plantlets with no sign of wilting (see Chapter

7, Figure 7.14b) were transferred to a plastic pot (100 mm in diameter × 77 mm in height, containing the potting mixture with a slow-release fertiliser) under glasshouse conditions. One plantlet was transferred to each pot. The potting mix was watered and kept in a misting area of the glasshouse for five weeks. The plantlets that survived were then transferred to a shaded area (with 50 % normal lighting) in the glasshouse and watered for 1 minute at about 8 pm every day. Well established petunia plantlets were further propagated to increase the numbers of the useable plantlets for experiments.

2.6 Glasshouse study design

2.6.1 Experimental Petunia grandiflora plant lines tested

The eight-week-old micropropagated petunia plantlets of a uniform size that survived the hardening step under the glasshouse conditions included: (a) a line of plantlets from the seed germinated in vitro, and (b) lines (L1 to L4) of plants regenerated from callus cultures that were exposed to undiluted diesel for 9 min. and from callus culture without exposure to diesel. The plantlets were propagated at the same time, then only those of similar size were kept under

50% shaded area in the glasshouse for 10–12 weeks. After this, the shoot tips (7 cm long) of

34 the propagated cuttings were used in the diesel evaluation experiments under the glasshouse conditions.

2.6.2 Site description

A glasshouse room with six tables of the same size was used in setting up the experiment design

(Figure 2.1). There were four legs to each table with its flat top surface (2430 mm long and 920 mm wide). There was an upper roof deck the same length and breadth as the tables. The distance between the flat top surface of the table and the roof deck was 810 mm, and the distance of the flat top surface to the ground floor was 770 mm.

The transparent polyethene film sheets were used to cover the upper top of the roof deck and the four sides on top of the tables. The six tables were divided into two groups forming a set of three tables in a row. The first set of the three tables were in a south-west (SW) direction and north-west (NW) when coming through the entrance door, while the second set of tables was located south-east (SE) and northeast (NE) of the entrance door. An automated temperature and light-intensity data logger (Hobo data logger and devices, version 3.7.7) was attached to the table at the middle and the start of each experiment. They were taken off to read the data at the end of each experiment. In each of the six tables, there were 40 water drippers connected through plastic pipes that were attached to edges of the tables. The drippers were designed to water the potting mix inside the pots at the rate of 1 litre/hour, but for this experiment, the drippers were reset to water for 1 min on odd days at about 8 pm. In each table, there were 40 randomly arranged 100 mm squat pots of the same size (diameter 100 mm × height 77 mm).

2.6.3 Potting mix and diesel-contaminated potting mix

Potting mix with a slow-release fertiliser and the diesel fuel were supplied by the glasshouse unit and the School of Biological Sciences of the University of Canterbury. At the initial stage of the experiment, both the pristine potting mix sample and the diesel fuel was analysed by Hill

Laboratories, Hamilton, New Zealand. Potting mix was mechanically mixed with the diesel

35 fuel at the concentrations of 0%, 2% and 7% (w/w). The 0% is the clean potting mix without the diesel fuel and was the control treatment. The 2% and 7% of diesel added to the potting mix was the simulated diesel-contaminated potting mix treatments. There were 40 pots, and

250g of the clean potting mix or the diesel-contaminated potting mix were weighed out and placed in a pot at the start of each experiment.

2.6.4 Experimental design

Three experimental blocks (A, B and C) were designed for the experiment. There were 8-rows and 5 columns in each block with a total of 40 pots. The pots in blocks A, B, and C (see Figure

2.1) were arranged in a complete randomised block design (CRBD).

2.7 Analyses of plants grown under the glasshouse conditions

2.7.1 Number of leaves and leaf chlorosis rating

After five weeks of growth, at least three replicates in each of the treatments (clean potting mix and potting mix spiked with 2% and 7% diesel were randomly selected, and the leaves were counted and examined for chlorosis using a visual chlorosis score: 1 (green), 2 (slightly chlorotic), 3 (slightly green), 4 (chlorotic) and 5 (severely chlorotic) (Başar, 2003). The purpose of this was to determine the mean leaf numbers and to classify different chlorosis ratings in relation to diesel concentrations.

2.7.2 Shoot Analyses

At the beginning and the end of the experiment (5 weeks later), the diameter of the plantlet lines used was determined. The average height of shoot cutting used was 7 cm long, with about

2 cm of the base of the cutting planted into the potting mix. The diameters of the cuttings were measured just about 2 cm above the surface of the potting mix using an electronic digital vernier caliper. The average mean of the diameter was expressed in millimetre (mm). At the end of the experiment, plantlets were carefully removed from the potting mix using a mini digging fork and any adhered potting mix on the plantlets was removed. Signs of any roots were visually

36 examined. The shoot heights of at least three replicates in each treatment were measured from the shoot base to the apical tip using a ruler and the means were expressed in centimetre (cm).

2.7.3 Biomass production

At the end of the experiment (5 weeks), the fresh mass of the plantlets without any adhered potting mix was measured. The plantlets were then dried in an oven (Contherm Scientific

Limited, Hutt City, New Zealand) at 80℃ for 48 h before their mass was measured. The mean mass values were expressed as fresh and dried biomass in grams (g).

2.8 Microbial plate counts

The potting mix for each of the three randomly selected replicate pots in the treatment controls was collected at the end of the experiment (5 weeks). For plantlets that formed roots or those without root formation, potting mix was removed directly from the roots or around part of the buried plantlets while for pots without plantlets, potting mix was taken randomly around a different part of the pot using a spatula that was cleaned with 70% ethanol after every sampling.

The root regions or part of plantlets buried without root formation in the potting mix were regarded to be “regions with high rhizosphere activities” (Euliss et al., 2008). All potting mix samples were placed in a sterilised glass container, labelled, capped, and placed on ice for transport to the lab from the glasshouse. A sample of the potting mix was stored at 4 ℃. Finally, the potting mix samples collected were analysed for the total number of culturable microbial populations (plate counts). To determine the total microbial numbers, the microbes were extracted from the potting mix using a shaking method. One gram of the potting mix was placed in a sterilised glass container with 9 mL of sterilised dH2O. The containers with the potting mix were shaken for 5 min. and allowed to settle for 1–2 min. before plating. If the samples settled for more than 5 min. before plating, the potting mix was resuspended by vortexing and allowed to settle again (Euliss et al., 2008). Serial dilutions of 10−3, 10−4, and 10−5 of the supernatants were spread on the plates (yeast extract-peptone-glucose agar plates). The composition of the agar medium was as follow: 0.5% yeast extract, 1.0% peptone, 1.0% glucose, 1.5% agar (Euliss et al., 2008).

Three replicates were made for each dilution. The plates were incubated at 26 ℃ for 36–48 h before the number of colony forming units (CFUs) was counted.

2.9 Analysis of diesel in potting mix 2.9.1 Ultrasonic extraction (Method 3550C by USEPA (2007))

About 1 g of three replicates of the potting mix was sampled randomly from a vegetated and unvegetated pot, and the potting mix was contaminated with 0%, 2%, and 7% diesel (w/w) at the beginning and end of the experiment (5 weeks). One gram of anhydrous sodium sulphate

(Na2SO4) (1:1 (w/w)) was added to 1 g of the potting mix to chemically dry the potting mix

(USEPA, 2007). Ten mL of tetrachloroethylene (C2Cl4) (analytical grade, Acros, Germany)

[1:10 M (sample)/V (solvent)] was added to the potting mix samples. Ultrasonic extraction

(Elma Elmasonic, Model S30, Germany) was performed for 30 minutes each time and repeated three times on each replicate (USEPA, 2007). Then the mixture was filtered using disposable

10 mL plastic syringes (ThermoFisher Scientific, Germany) and disposable 3.1 µM glass microfiber (ThermoFisher Scientific, United States). The extract obtained was decanted into a glass vial and stored at 4 ℃ until gas chromatograph (GC) scan flame ionisation detector (FID) analysis.

2.9.2 Total petroleum hydrocarbon (TPH) Analysis The extract was analysed in a GC equipped with a flame ionisation detector (GC-FID,

Shimadzu QP-2010) and an autosampler using a capillary column (Restek, RTi-5Sil-ms,

30 min × 0.32 mm i.d. × 0.25 micron) with helium as the carrier gas. Column pressure was controlled to keep the linear velocity constant. The column temperature was held at 50°C for 1 minute and then ramped at 15°C per minute to 320°C and held at that temp for 10 minutes.

Total petroleum hydrocarbons (TPHs) were analysed using the hydrocarbon standard. The hydrocarbon standard was made at the Department of Chemistry, University of Canterbury,

New Zealand. One gram was measured for each of nonane, decane, dodecane, tetradecane, and hexadecane into a mix, then diluted 10 µL of the mix to 1 mL of pentane. The TPHs in the

40 diesel were determined and expressed as a percentage of residual TPHs in diesel by the following equation:

푓푖푛푎푙 푇푃퐻 푃푒푟푐푒푛푡푎푔푒 푟푒푠푖푑푢푎푙 = × 100 푖푛푖푡푖푎푙 푇푃퐻

2.10 Statistical Analyses

All the experiments were conducted using a completely randomised block design with at least three replications in each treatment and were repeated twice. Where data transformation was required, they were transformed before statistical analysis. Genstat for Windows 19th Edition and IBM SPSS (version 24) software were used to analyse the data using the analysis of variance (ANOVA) for a randomised block design. The mean difference between the independent replicate at the 5% level of significance was calculated and tested using the post hoc test of Duncan's multiple range. The Fisher’s unprotected least significant difference (LSD) test was also performed and presented as a measure of variability. All graphs were plotted using the GraphPad Prism software Version 7.0.

Chapter 3 Phytotoxicity testing of diesel-contaminated water using Petunia grandiflora Juss. mix F1 and Marigold-Nemo mix (Tagetes patula L.) 3.1 Introduction

With the increasing demand for petroleum products around the world, contamination of terrestrial ecosystems has become a widespread anthropogenic pollution concern (Gkorezis et al., 2016). There is also a global concern for possible leakage from storage tanks of petroleum hydrocarbons such as diesel into the aquatic environment (Ramadass et al., 2017). The toxicity of petroleum hydrocarbons increases as their molecular weights decrease (Darville and Wilhm,

1984). Diesel oil was found to be more toxic than other products of petroleum (Wang and

Bartha, 1990). This was explained by the increased levels of polycyclic aromatic hydrocarbons or polynuclear aromatic hydrocarbons (PAHs) present in diesel spills (Adam and Duncan,

1999). The PAHs are quite persistent in the ecosystems (Adam and Duncan, 1999). There is also a need to assess the toxicity of diesel contamination in the environment (Ramadass et al.,

2017).

Phytoremediation is a green technology that could help mitigate the undesirable effects of various environmental pollutants (Pittarello et al., 2017). Conceivably, the use of ornamental plants would be preferable to food plants for phytoremediation purposes as this would minimise the chance of pollutants entering the food chain. Moreover, another potential benefit is that planting ornamental plants useful for phytoremediation would add a more pleasant aesthetic value to the polluted landscape (Cheng et al., 2017).

In recent studies, Tagetes patula (marigold) and Petunia grandiflora (petunia) are two ornamental plants that have been shown to exhibit phytoremediation potential in the contamination of water by textile dye (Patil and Jadhav, 2013; Patil et al., 2009; Watharkar and

Jadhav, 2014). Marigold was also investigated in relation to phytoremediation of

42 benzo[a]pyrene and heavy metals (Sun et al., 2011), and the phytostabilisation of iron-ore tailings (Chaturvedi et al., 2014). Although there is no prior work on the response of these two plants to diesel-contaminated water, it seems reasonable to hypothesise that these two plants could exhibit cross-tolerance (Perez and Brown, 2014) to multiple pollutants, not restricted to those studied previously but also including diesel.

It is possible, therefore, that these two plants might also be useful for phytoremediation of diesel contamination. An important prerequisite towards a deeper evaluation of this possibility is a rapid and simple assessment of the phytotoxicity of diesel contamination of water on these two ornamental plants. The objective of this chapter is to investigate the effects of different concentrations of diesel on seed germination, early seedling root elongation and shoot growth.

In addition, it was of interest to compare the relative tolerance of the two plants to diesel contamination during germination and early seedling growth.

3.2 Materials and Methods

3.2.1 Seeds

Seeds of Marigold-Nemo mix (Tagetes patula L.) and Petunia hybrida-mix F1 (Petunia grandiflora Juss.) were purchased from Kings Seeds, Katikati, New Zealand and stored as described in section 2.1.1.

3.2.2 Treatment of seeds using water contaminated with diesel

Tagetes patula or Petunia grandiflora seeds were sown in 10 mL of a test solution (0–4% diesel, prepared in deionised water, v/v) in a plastic Petri dish (90 mm diameter) as described in section 2.1.3 The diesel used in this study was analysed using a GC flame ionisation detector

(FID) as described in section 2.1.2 There were four replicates for a test solution, and all the replicates were arranged in a completely random way, as described in detail in section 2.1.4.

The number of seeds germinated was scored and root length and shoot height of marigold, and

43 petunia seedlings were also measured using a ruler after 10 and 15 days, as described in sections

2.1.4 and 2.1.5. The elongation inhibition rates (in %) of marigold and petunia were determined according to Visioli et al. (2014), as described in section 2.1.6.

3.2.3 Experimental design and statistical analysis

All the experiments were conducted using a complete randomised block design with four replications in each treatment and were repeated twice. Genstat for Windows 19th Edition software was used to analyse the data using the analysis of variance for a randomised block design, which included two orthogonal contrasts. These were (1) the comparison of the average of the diesel treatments with the "no diesel" (control) treatment, and (2) the linear contrast between the non-zero rates of diesel (which is equivalent to looking at the slope of the regression line for non-zero rates). The least significant difference (LSD (5%)) is presented as a measure of variability, as suggested by Saville (2015). All graphs were plotted using the

GraphPad Prism software Version 7.

3.3 Results

3.3.1 Toxic effect of diesel-contaminated water on seed germination

The germination data during the first two days from sowing marigold and petunia seeds were not included in Tables 3.1 and 3.2, respectively, to avoid issues with ANOVA analysis. There was no germination after one day of sowing marigold seeds in deionised water (control) and water contaminated with different concentrations of diesel (0.5–4%). A majority (about 72%) of the marigold seeds germinated, evident from the emergence of the radicle after two days from sowing in the control. Only 8.3% of the seeds germinated in 0.5% and 1% diesel while there was still no germination in the presence of 1.5% or higher concentrations of diesel.

Figure 3.1 Gas chromatogram scan of the diesel used.

After three days from sowing, virtually all the marigold seeds in the control had completed germination, but a much lower percentages of seeds began to germinate in water contaminated with diesel (Table 3.1). Overall, in the presence of all the levels of diesel-contaminated water studied, rates of seed germination were slowed down throughout the experiment. In addition, by the end of the experiment (10 days after sowing seeds), the severity of inhibition on seed germination compared to the control was related to the level of diesel present in water in a dose-dependent manner. Therefore, the least inhibition (about 10%) was observed in 0.5% diesel while the greatest inhibition (about 40%) was observed in 4% diesel.

Pairwise comparison between the percentages of marigold seed germination in the control and water contaminated with the different concentrations (0.5–4%) of diesel showed significant differences (Table 3.1). For example, it was highly significant (p < 0.001) from day 5 up to day

10 when the seed germination percentages between the control and 1% or higher concentrations of diesel were compared. There were also significant differences in the percentages of marigold seed germination among the different levels of diesel treatment.

The seeds of petunia did not germinate in any of the treatments in the first 2 days from sowing.

Then a majority (about 86%) of the seeds sown in deionised water without addition of diesel

(control) germinated the following day (Table 3.2). By day 6, all the seeds in the control Petri dishes had germinated. The toxic effect of diesel-contaminated water on seed germination was evident after three days from sowing, as seed germination in 0.5% to 4% diesel appeared to be lower than the control in a dose-dependent manner. Most notably among all the treatments, there was still no seed germination on day 3 in the presence of 4% diesel. At the end of the incubation (15 days from sowing) with 0% to 2% diesel, the percentage of seed germination was not significantly different (about 88% to 100%). The most severe toxic effect on seed germination (75%) was found in the 4% diesel treatment (p < 0.001, compared with control) after 15 days from sowing.

Table 3.1 Effect of different concentrations of diesel on the changes in the percentages Tagetes patula seeds germinated during 10 days from sowing seeds. Diesel concentrations in deionised water Days from sowing Contrasts of the mean germination (%) between day 10 and days 3 and 6 (%, v/v) Mean germination %

3 4 5 6 7 8 9 10 10-3 10-6

0 95.8(a) 98.6(a) 98.6(a) 100(a) 100(a) 100(a) 100(a) 100(a) 4.2(b) 0.0(c)

0.5 30.6(b) 44.4(b) 66.7(b) 77.8(b) 80.6(b) 84.7(b) 87.5(ab) 90.3(a) 59.7(a) 12.5(b)

1 26.4(bc) 41.7(bc) 52.8(bc) 61.1(bc) 68.1(bc) 69.4(c) 73.6(bc) 73.6(b) 47.2(a) 12.5(b)

1.5 11.1(c) 19.4(e) 25(e) 37.5(d) 45.8(d) 51.4(d) 58.3(cd) 63.9(bc) 52.8(a) 26.4(a)

2 22.2(bc) 38.9(bd) 50(cd) 61.1(bc) 63.9(bc) 69.4(c) 72.2(bc) 72.2(bc) 50.0(a) 11.1(bc)

2.5 15.3(bc) 23.6(de) 37.5(de) 50(cd) 54.2(cd) 56.9(cd) 63.9(cd) 63.9(bc) 48.6(a) 13.9(b)

3 12.5(c) 25(cde) 36.1(de) 52.8(cd) 56.9(cd) 62.5(cd) 63.9(cd) 65.3(bc) 52.8(a) 12.5(b)

4 12.5(c) 19.4(e) 30.6(e) 38.9(d) 45.8(d) 48.6(d) 52.8(d) 58.3(c) 43.1(a) 19.4(ab)

LSD (5 %) 15.9 17.7 14.4 14.5 14.6 13.1 13.9 13.5 17.4 10.9

Significance of contrasts

Control (0) vs diesel concentrations *** *** *** *** *** *** *** *** *** ***

Linear rate in non-zero rates of diesel con. * ** *** *** *** *** *** *** ns ns

Germination percentages at day 1 and 2 have been omitted from ANOVA because there was no germination in most treatments with various diesel concentrations including the control. Means values within the same column with different letters are statistically different according to the p values of significant comparisons.

*= p < 0.05, ** = p < 0.01, *** = p < 0.001 and ns = no significant difference detected

Table 3.2 Effect of different concentrations of diesel on the changes in the percentages of Petunia grandiflora seeds germinated during 15 days from sowing seeds.

Contrasts of the mean Diesel germination (%) concentrations in Days from sowing between day 15 with deionised water Mean germination % 3 and 6 (%, v/v)

3 4 5 6 7 8 9 10 11 12 13 14 15 15-3 15-6 0 86.(a) 91.7(a) 95.8(a) 100(a) 100(a) 100(a) 100(a) 100(a) 100(a) 100(a) 100(a) 100(a) 100(a) 13.9(a) 0.0(c) 0.5 63.9(b) 72.3(b) 87.5(a) 91.7(ab) 94.4(ab) 98.6(ab) 98.6(a) 98.6(a) 98.6(a) 100(a) 100(a) 100(a) 100(a) 36.1(d) 8.3(bc) 1 50(c) 59.7(b) 70.8(b) 76.4(bc) 83.3(bc) 86.1(ac) 88.9(ab) 93.1(a) 94.4(a) 94.4(a) 97.2(a) 97.2(a) 98.6(a) 48.6(cd) 22.2(ab) 1.5 36(d) 44.4(c) 58.3(c) 69.4(cd) 75(cd) 81.9(bcd) 83.3(ac) 84.7(ab) 87.5(ab) 87.5(ab) 88.9(ab) 88.9(ab) 88.9(ab) 52.8(bd) 19.4(ab) 2 18.1(e) 40.3(cd) 56.9(c) 69.4(cd) 73.6(cd) 77.8(ce) 84.7(a) 84.7(ab) 87.5(ab) 87.5(ab) 87.5(ab) 87.5(ac) 87.5(ac) 69.4(ab) 18.1(ab) 2.5 18.1(e) 38.9(cd) 54.2(c) 66.7(cd) 68.1(ce) 72.2(ce) 72.2(bcd) 75(bc) 77.8(bc) 79.2(bc) 79.2(bc) 80.6(bc) 83.3(bc) 65.3(abc) 16.7(ab) 3 9.7(ef) 27.8(de) 41.7(d) 56.9(d) 62.5(de) 68.1(de) 69.4(cd) 69.4(bc) 73.6(c) 76.2(bc) 80.6(bc) 80.6(bc) 81.9(bc) 72.2(ab) 25(a) 4 0(f) 20.8(e) 40.3(d) 54.2(d) 56.7(e) 63.9(e) 63.9(d) 66.7(c) 68.1(c) 69.4(c) 72.2(c) 75(c) 75(c) 75(a) 20.8(ab) LSD (5 %) 11.2 12.6 12.9 18.4 16.8 16.3 17.5 16.2 13.5 14.5 13.7 12.7 12.7 17.7 16.4 Significance of contrasts Control (0) vs diesel *** *** *** *** *** ** ** ** ** ** * * * *** ** concentrations Linear rate in non- *** *** *** *** *** *** *** *** *** *** *** *** *** *** ns zero rates of diesel concentration Germination percentages at days 1 and 2 have been omitted from ANOVA because there was no germination in most of the treatments with various diesel concentrations including the control. Mean values within the same column with different letters are statistically different according to the p values of significant contrasts.

*= p < 0.05, ** = p < 0.01, *** = p < 0.001 and ns = no significant difference detected

3.3.2 Toxic effect of diesel-contaminated water on seedling growth

A decrease compared to the control by about 40% and 60% in root length of marigold seedlings germinated in 0.5% and 1% diesel respectively was found (Figure 3.2a). There was a small further reduction in root length in response to 1.5% to 2.5% diesel. The root length in 3% or

4% diesel was about 15% of the control. Generally, the toxic effects of the diesel treatments on shoot length seemed to be less severe than on root length (Figure 3.2 a & b). Incubation in as little as 0.5% diesel led to a substantial inhibition of root and shoot growth of petunia; these lengths were not measurable (radicle was just visible) in the seeds germinated in water contaminated with more than 1% diesel (Figure 3.3 a & b). The marigold and petunia seedlings germinated in water (control) typically had a pair of green, expanded cotyledons, a reddish hypocotyl and a long primary root (Figures 3.4a & 3.5a). Some petunia seedlings had also started to form lateral roots after ten days from sowing seeds (Figure 3.4a & 3.5a). In comparison, the marigold seedlings incubated in 4% diesel appeared stunted in cotyledon development and root-shoot growth (Figure 3.4b). The severity of 4% diesel toxicity on petunia seedlings was more pronounced as there was hardly any cotyledon or root-shoot development

(Figure 3.5b) but some sign of necrosis (presence of some black spots at the emerged radicle).

Therefore, to ascertain if this severe inhibition of seedling development was temporary or due to more permanent damage, the petunia seeds in the different test solutions were rinsed with deionised water. Then after 15 days of further incubation in deionised water only, there was no change in severe inhibition of seedling development; that is, the results obtained were the same as shown in Figure 3.3 a & b: there was no sign of recovery.

5 0 A

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( B

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t g

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2 0

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0 0 0 .5 1 .0 1 .5 2 .0 2 .5 3 .0 4 .0

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m 1 5

m (

B C t B D h C D g C D

i 1 0 D E

t E

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0 0 0 .5 1 .0 1 .5 2 .0 2 .5 3 .0 4 .0

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(b)

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3.3.3 Elongation inhibition rate

The seedling growth of petunia seems to be more sensitive to the toxicity of diesel than marigold seedlings (Figures 3.2 and 3.3). Based on a calculation of the elongation inhibition rate of roots in diesel-contaminated water, the differential sensitivity of marigold and petunia seedlings to diesel was evident (Figure 3.6). In response to 0.5% diesel, marigold and petunia seedlings exhibited about 40% and 80% inhibition in root elongation, respectively, compared with the control (Figure 3.6). Another major difference in the root elongation inhibition rates of marigold and petunia seedlings was that the former exhibited a gradual increase in root elongation inhibition in response to 1–4% diesel (Figure 3.6a), while the latter exhibited 100% inhibition in root elongation in response to as little as 1% diesel (Figure 3.6b).

Figure 3.4 Effect of diesel-contaminated water on seedling development of Tagetes patula after 10 days of sowing seeds. Seedlings in (a) 0% and (b) 4% diesel.

Figure 3.5 Effect of diesel-contaminated water on seedling development of Petunia grandiflora after 15 days from sowing seeds. Seedlings in (a) 0% and (b) 4% diesel.

1 0 0

a r

8 0

i t

i 6 0

n i

4 0

i t

a 2 0

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E 0 0 .0 0 .5 1 1 .5 2 2 .5 3 4 % o f d ie s e l in w a te r v /v

(a) Marigold

% 1 5 0

n o

i 1 0 0

n 5 0

o l

0 E 0 .0 0 .5 1 1 .5 2 2 .5 3 4 % o f d ie s e l in w a te r v /v

(b) Petunia

Figure 3.6 Elongation inhibition rate of (a) Tagetes patula and (b) Petunia grandiflora seedlings grown in the presence of different concentrations of diesel.

3.4 Discussion

In an earlier study by Ogbo (2009), seed germination of Arachis hypogaea, Vigna unguiculate,

Sorghum biocolor and Zea mays in response to 2% diesel was only half of that in the control

(germination without diesel contamination) (Ogbo, 2009). However, the radicle length of V. unguiculate was not affected by up to 5% diesel, while that of A. hypogaea was significantly reduced in comparison with the control (by about 50%). The phytotoxicity evaluation study here has also revealed that Tagetes patula (marigold) and Petunia grandiflora (petunia) seeds exhibited both similar and differential responses to the toxicity of diesel-contaminated water.

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. This was also found in other studies (for example, diesel contamination retarded lettuce seed germination (Fatokun et al., 2015). However, the possible mechanism(s) for this adverse effect of diesel contamination is currently not known, as this has never been investigated. In terms of percentage of seeds germinated at the end of the experiment, petunia seeds seemed to be less sensitive than marigold seeds to the toxic effect of diesel-contaminated water. It has been shown before that there is a variation in the sensitivity of germination of different seeds to diesel contamination. For example, lettuce seed germination was found to be more sensitive than sweet potato to diesel-contaminated soil

(Fatokun et al., 2015). In another soil contamination study, 0.85% diesel greatly inhibited lettuce and wheatgrass seed germination but had no effect on the germination of radish and alfalfa (Bamgbose and Anderson, 2015).

There is a paucity of information about the differential sensitivity of plants to diesel at the postgerminative seedling growth stage. The present study has revealed that in relation to root growth in particular, petunia seedlings seemed to be more sensitive or less tolerant to the toxic effect of diesel than marigold seedlings.

In fact, the lack of petunia seedling development (cotyledon expansion and root-shoot growth) was largely inhibited in the presence of the diesel solutions tested. Moreover, there was no sign of recovery even following several rinses in deionised water and incubation in water for an additional 15 days, suggesting that more permanent damage occurred during prior incubation in diesel-contaminated water, particularly 1.5% to 4% diesel.

There is considerable interest in phytoremediation of environmental pollutants (Pulford and

Watson, 2003). An important prerequisite of phytoremediation is to screen for plants more tolerant to a pollutant of interest. Conceivably, phytotoxicity evaluation of a plant at the germination stage in the laboratory should be more attractive (more rapid, time saving and lower cost) than evaluation of the effect of a pollutant on plant growth and development in the field (Freemark et al., 1990). However, as a commonly accepted practice, germination data are operationally based on scoring the visible emergence of the radicle. As shown in this and other studies, although a plant may be tolerant to a pollutant as far as germination is concerned, it might exhibit poor seedling growth in the presence of the pollutant. Phytotoxicity evaluation at the postgerminative seedling growth stage in the lab might have more implications for selecting tolerant plants for phytoremediation and is still more attractive than the more time- consuming and expensive field screening of more tolerant plants for phytoremediation. In this study, based on comparing phytotoxicity at both the germination and postgerminative seedling growth stages, marigold would seem to show more potential for phytoremediation study of diesel contamination in addition to its previously studied potential in phytoremediation of heavy metals (Sun et al., 2011). Although petunia was of interest in the phytoremediation of water polluted by textile dye, its seedling growth was shown to be significantly affected by diesel contamination. Work is underway in our lab to investigate if the utility of petunia could be extended for phytoremediation of diesel by discovering new variants of petunia with improved tolerance to diesel contamination.

3.5 Conclusion

The main finding that marigold and petunia exhibited differential sensitivity to diesel contamination during germination and early seedling growth has not been reported before. The inhibition of seedling development by incubation of petunia seeds in diesel-contaminated water compared to the control remained unchanged, even after rinsing and further incubation in deionised water. This suggests that the action of diesel contamination might be more than just delaying seedling development in petunia.

Chapter 4 Induction of callus in leaf explants of Petunia grandiflora Juss. mix F1 for experimental exposure to diesel and establishment of plant lines from calli that survived diesel treatment

4.1 Introduction

Petunia is one of the most important and common ornamental bedding plants cultivated around the world (Regalado et al., 2017). In many studies, petunia is considered as a model experimental plant system in plant biology. For example, in the study of self-incompatibility and floral development, flavonoid biosynthesis, male sterility transposons, retroelement activity, and identification and selection of plant genotypes (Conner et al., 2009; Gerats and

Vandenbussche, 2005). Different techniques of cell and plant tissue culture were used in many of these studies. Novel plants were generated using in vitro plant cell line selection under the pressures of different environmental pollutants (Ashrafzadeh and Leung, 2015; Rai et al.,

2011). However, there is no similar study using diesel as the selection pressure for the in vitro plant cell line selection. Specific examples of ornamental plants used for the development of drought-tolerant and NaCl-tolerant plant lines are Tagetes minuta (Mohamed et al., 2000) and

Chrysanthemum morifolium (Hossain et al., 2007). Recent studies have shown that Petunia grandiflora is of interest in phytoremediation of textiles dyes (Watharkar and Jadhav, 2014) and its seedling growth was shown to be significantly affected by diesel-contaminated water

(Chapter 3 of this thesis; Wante and Leung, 2018). It seems worthwhile to investigate the in vitro potential of Petunia grandiflora for phytoremediation of diesel fuel. The aim was to extend the application of in vitro plant cell line selection to generate diesel-resistant plants.

Toward this, the experimental work was structured and carried out as follows: 1) establishment of optimised callus culture conditions using four-week-old leaf explants from seedlings of

Petunia grandiflora grown in vitro, 2) optimised callus culture conditions following exposure

59 to 9 min. of diesel treatment, and 3) plantlet regeneration from diesel-treated callus on MS medium supplemented with various concentrations of auxin types (1-naphthalene acetic acid

(NAA) and indole-3-acetic acid (IAA)) in combination with different concentrations of cytokinin types (6-benzyladenine (BA) and zeatin). The objective of the work described in chapter was to establish plant lines of Petunia grandiflora from calli that survived 9 min. of diesel treatment.

4.2 Materials and methods

4.2.1 Plant materials and culture conditions

4.2.1.1 Seed

Seeds of ornamental cultivars of Petunia hybrids-mix F1 (Petunia grandiflora Juss) were purchased from Kings Seeds Company, Katikati, New Zealand. Seeds were kept in a closed container, as described in section 2.1.1.

4.2.1.2 Seed germination under aseptic conditions

Seeds of Petunia grandiflora (petunia) were germinated under in vitro conditions. They were washed with 0.8% (w/v) soapy water (Pyroneg L88z, ThermoFisher Scientific Ltd.,

Christchurch, New Zealand) and then surfaced sterilised with 1% (w/v) sodium hypochlorite

(NaOCl) for 15 minutes, as described in detail in sections 2.2.1 and 2.3.2.

4.2.1.3 Callus induction and proliferation

Four-week-old leaves of seedlings germinated in vitro were aseptically excised for callogenesis. The adaxial side of the leaves was placed on the callus induction medium (Table

4.1), described in detail in sections 2.2.3 and 2.2.4.

4.2.1.4 Morphology of the calli formed

The different types of calli that formed on the callus induction medium (Table 4.1) in response to various types and different concentrations of plant growth regulators were visually assessed,

as described in section 2.2.5. A medium that formed the selected callus was considered for the

medium for callus subculture.

4.2.1.5 Maintenance of callus culture

Selected pieces of callus were maintained on the optimised callus subculture medium (Table

4.1) supplemented with 1.8 µM NAA and 6.6 µM BA as described in section 2.2.6. The second

group of calli was used for further experiments.

4.2.1.6 Exposure of callus to diesel fuel

In a preliminary trial, 30 pieces of petunia calli were each totally submerged in 50 mL diesel fuel for 3, 5, 7 and 9 min. Diesel exposure time of 9 min. was then chosen after morphological visual inspection morphological visual inspection (MVI) of calli before and after 2 weeks of culture. All the critical steps involved in the exposure of callus to diesel fuel are described in detail in Chapter 2 (2.4.1 and 2.4.4) and summarised in Figure 4.5. There were about 15 pieces of callus for each of the three replications, and the calli were exposed to undiluted diesel for 9 min. It was then rinsed with sterile distilled water and finally with 0.2% (v/v) of Plant

Preservative Mixture (PPM) as described in section 2.4.1. The callus pieces were placed on the surface of the selected MS medium supplemented with 1.8 µM NAA and 6.6 µM BA (callus subculture medium) in Petri dishes) as described in section 2.3.1.

4.2.1.7 Culture of diesel-treated callus under aseptic conditions

After 2 weeks of diesel exposure and then culture on the callus subculture medium, the diesel- treated marigold callus pieces were assessed using the MVI approach throughout the experiment (6 weeks) as described in section 2.3.2.

4.2.1.8 Selection and multiplication of calli that survived after diesel treatment

A non-necrotic region from a diesel-treated callus that had been cultured on MS subculture medium (Table 4.0) was selected, as described in section 2.3.3. A callus at 6 weeks of culture was considered as one that putatively survived diesel exposure.

4.2.1.9 Plant regeneration from non-diesel-exposed calli and those that survived diesel

treatment

For this investigation, all calli were originated from the optimised callus culture induction

medium/callus subculture MS medium, as described in detail in Chapter 2 (2.4.2).

4.2.1.10 Morphology of different types of shoots formed

Shoot formed from the calli that survived diesel-treatment and the controls were visually

assessed as described in section 2.4.3.

4.2.1.11 Shoot multiplication and root formation

The calli that regenerated shoots were carefully removed from the agar and transferred for shoot proliferation and rooting as describe in Chapter 2 (2.4.4).

Table 4.1 Plant cell and tissue culture media used in this study.

Serial/Number Composition Plant growth regulators Purpose/ use

1 Full-strength MS basal (Murashige and Skoog, 1962) medium No added PGRs In vitro germination of seed

2 Full-strength MS basal (Murashige and Skoog, 1962) medium NAA, 2, 4-D & PIC to Callus induction BA (Tables 4.2, 4.3 & 4.4)

3 Full-strength MS basal (Murashige and Skoog, 1962) medium 1.8 µM NAA & 6.6 Optimised callus µM BA induction/callus subculture medium

4 Full-strength MS basal (Murashige and Skoog, 1962) medium 1.8 µM NAA & 6.6 Shoot regeneration µM BA, and (Tables 4.5,4.6 & 4.7)

5 Half-strength MS basal (Murashige and Skoog, 1962) medium Shoot proliferation, micropropagation of shoot No added PGRs tip and root formation of regenerants

6 Half-strength MS basal (Murashige and Skoog, 1962) medium 2.5 µM IBA Root formation of some specific plantlet regenerants

Note: All the media were supplemented with 3% sucrose(w/v) and gelled with 0.8% (w/v) agar.

4.3 Statistical analysis

All the experiments were carried out in a complete randomised block design (CRBD) and repeated at least twice. Where data transformation was required, they were transformed before statistical analysis. Data were statistically analysed using the IBM SPSS (version 24) software by calculating the mean difference of the independent replicate at the 5% level of significance one-way analysis of variance (ANOVA) and the post hoc test of Duncan's multiple range and

Fisher’s unprotected least significant difference (LSD) tests. All the graphs were plotted using the GraphPad Prism software Version 7.0.

4.4 Results

4.4.1 In vitro seed germination and seedling growth

In the preliminary trials, about 94% of the surface sterilised seeds of Petunia grandiflora germinated on the germination medium. The Petunia grandiflora seeds were germinated on

PGR-free MS medium (Figure 4.2a), and checked 72 hours after sowing, and when radicle emergence from the germinated seeds was observed, and when 1-week cotyledons were observed (Figure 4.2b). The seedlings developed further at 2 weeks (Figure 4.2c), and after 4 weeks, several leaves had formed on each seedling (Figure 4.2d).

a b c d

(a) 0 day (b) 1 week (c) 2 weeks (d) 4 weeks

Figure 4.2 In vitro seed germination and seedling development of Petunia grandiflora on full-strength MS medium free of plant growth regulator. Cultures were kept in a growth room at 21 ± 1 ℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W).

4.4.2 Efficiency of callus induction

In the preliminary experiments, several different combinations of auxin types including

indoleacetic acid (IAA), NAA, PIC and 2,4-D with different concentrations of BA were tested

on the young in vitro leaf explants for callus induction. The combinations of IAA with BA

concentrations seemed not to be promising in inducing callus from the leaf explants (results

not shown). Therefore, NAA, PIC and 2,4-D with different concentrations of BA were used to

optimise the higher percentage of leaves forming the callus. Among the combinations of PIC

and BA used in one of the three experiments, the highest percentage (about 22.5%) of petunia

leaf explants formed masses of calli after 4 weeks of culture on medium supplemented with a

combination of 5.7 µM PIC and 4.4 µM BA (Table 4.2).

Among the combinations of 2,4-D and BA used, the highest percentage (about 37.5%) of the

leaf explants formed calli after culture for 4 weeks on the medium supplemented with a

combination of 4.5 µM 2,4-D and 2.2 µM BA (Table 4.3).

In the final experiment, using NAA, a different auxin type, and BA to improve the efficiency of callus formation, it was found that 85% of the leaf explants formed calli when cultured on the medium supplemented with the combination of 1.8 µM NAA and 6.6 µM BA (Table 4.4).

4.4.3 Morphology of the calli formed

Calli with different colours, sizes, and textures were formed in response to different plant growth regulators. Friable calli were formed on the medium supplemented with PIC and BA and appeared largely yellowish in the initial experiment (Table 4.1, Figure 4.2). The calli formed on the medium supplemented with a combination of BA and 2,4-D was light green/whitish and friable (Table 4.3, Figure 4.4). Both compact and friable calli were formed on the medium supplemented with NAA and BA and appeared largely light green/green in the final experiment (Table 4.4, Figure 4.5). The medium supplemented with 1.8 µM NAA and 6.6

µM BA which led to the highest percentage (85%) of leaves forming the callus was selected as the callus subculture medium.

Table 4.2 Effect of the various combinations of picloram (PIC) with 6-benzyladenine (BA) on callus induction in Petunia grandiflora leaf explant after 4 weeks of culture.

Plant growth regulators % of explants formed callus Colour Texture Estimated amount of callus formation

PIC (µM) BA (µM) Leaf

0 0 0 (± 0)c - - 0

5.7 4.4 22.5 (± 0.6)a Creamy yellowish/light green Friable †

5.7 44.4 5 (± 0.5)bc Light green Friable †

17.2 4.4 10 (± 0)b Creamy yellowish Friable †

17.2 22.2 5 (± 0.3)bc Creamy yellowish Friable †

17.2 44.4 0 (± 0)c - - -

28.5 4.4 0 (± 0)c - - -

28.5 44.4 0 (± 0)c - - -

28.5 66.6 0 (± 0)c - - -

Data were collected from the three replications after 4 weeks of culture and presented as mean ± SEM. Means within a column having the same letter are not significantly different by Duncan's multiple range test at p < 0.05.

† Slight amount of callus on an explant Light green Creamy yellowish

†† Moderate callus formation ††† High callus formation

Table 4.3 Effect of the various combinations of 2, 4-dichlorophenoxyacetic acid (2,4-D) with 6-benzyladenine (BA) on callus induction in Petunia grandiflora leaf explants after 4 weeks of culture.

Plant growth regulators % of explants formed callus Colour Texture Estimated amount of callus formation 2, 4-D (µM) BA (µM) Leaf

0 0 0 (± 0)c - - 0

4.5 2.2 37.5 (± 0.3)a Light green/whitish Friable †

4.5 4.4 5 (± 0.3)c Light green/whitish Friable/compact †

4.5 8.8 1.5 (± 0.29)b Whitish Friable †

9 2.2 2.5 (± 0.3)c Light green/whitish Friable †

13.5 2.2 0 (± 0)c - - 0

13.5 4.4 0 (± 0)c - - 0

13.5 8.8 5 (± 0.3)c Light green/whitish Friable †

Data were collected from the three replications after 4 weeks of culture and presented as means ± SEM. Means within a column having the same letter are not significantly different by Duncan's multiple range test at p < 0.05.

† Slight amount of callus on an explant Light green Whitish

†† Moderate callus formation

††† High callus formation

Table 4.4 Effect of the various combinations of 1-naphthalene acetic acid (NAA) with 6-benzyladenine (BA) on callus induction in Petunia grandiflora leaf explants after 4 weeks of culture.

Plant growth regulators % of explants formed callus Colour Texture Estimated amount of callus formation NAA (µM) BA (µM) Leaf

0 0 0 (± 0)d - - -

1.8 2.2 47.5 (± 0.5)c Light green/whitish and green Friable/compact ††

1.8 4.4 70 (± 0.4)b Green/whitish Friable/compact †††

1.8 6.6 85 (± 0.3)a Light green/whitish and green Friable/compact †††

2.7 2.2 60 (± 0.4)bc Light green/whitish Friable/compact ††

2.7 4.4 57.5 (± 0.3)bc Light green/whitish and green Friable/compact †††

2.7 6.6 65 (± 0.9)b Green/whitish Friable/compact †††

† Slight amount of callus on an explant Light green Green Whitish

†† Moderate callus formation

††† High callus formation

4.4.4 Appearance of callus on subculture medium

Pieces of callus were subcultured every two weeks on MS medium supplemented with 1.8 µM

NAA and 6.6 µM BA for 6 weeks without any visible changes in the green colour and compact texture of the calli in both the front view and the reverse side of the plastic Petri dishes (Figure

4.5 a and b). A closer view of the callus under a stereomicroscope under 40× magnification showed a lump of nodulated green callus (Figure 4.6c).

a b

4.4.5 Manipulations of diesel-treated callus under aseptic conditions

The flow chart in Figure 4.7 highlights an innovative step used to expose petunia calli to undiluted diesel fuel outside the laminar flow cabinet, that is, in potentially non-aseptic environment. After this, the subcultured calli under aseptic conditions that survived the diesel treatment were able to proliferate (Figure 4.9f).

In the flow chart (Figure 4.7), for the calli that were prior-exposed to diesel (for 9 min.), there was no contamination when PPM was used suggesting that the protocol was useful and successful in treating calli under otherwise potentially non-aseptic conditions (Figure 4.8).

More importantly, petunia calli did not exhibit extensive necrosis during the undiluted fuel treatment for up to 9 min. (Figure 4.9d), suggesting that the treatment was sub-lethal.

Immediately after treatment, the treated petunia calli were cultured for 14 days (Figure 4.9d).

The non-necrotic regions from the diesel-treated calli were then excised and placed individually on fresh subculture medium used (Figure 4.9e). At the end of 14 days of the first subculture, about 20% of the 40% of the calli of diesel-treated petunia survived (Figure 4.10). At the end of 14 days of the second subculture, the number of subculturable calli pieces increased to 37%

(Figure 4.10).

Callus

9 min–sterile deionised- 9 min–diesel-treated water-treated callus pieces In a laminar flow callus pieces

cabinet under aseptic conditions In a non-sterile

fume cupboard water

with with

Rinsed5x with

sterile deionised deionised sterile

× In a non-sterile fume cupboard water Submerge callus pieces in 0.2%

Rinsed5 Submerge callus piece sterile deionised deionised sterile (v/v) PPM for 15 Submerge callus in sterile deionised min pieces in 0.2% (v/v) water for 15 min PPM for 15 min In a laminar flow In a laminar flow cabinet under aseptic cabinet under aseptic conditions

Place diesel-treated callus Place sterile deionised- pieces on the surface of sterile Place diesel-treated water-treated callus pieces callus subculture medium callus pieces on the on the surface of sterile surface of sterile callus callus subculture medium In a laminar flow subculture medium cabinet under aseptic conditions

2 weeks incubation of treated calli cultures

Figure 4.7 Flow chart of the critical steps of a combination of manipulations under aseptic conditions (in a laminar flow cabinet) and brief non-sterile fume-cupboard conditions for treatment of Petunia grandiflora callus with undiluted diesel fuel before the return of the diesel-treated callus to aseptic culture conditions.

2 5

) 2 0

(

o 1 5

i M

m 1 0

n o

c 5

A B C T re a tm e n ts

4.4.6 Diesel-treated callus under aseptic conditions a b

c d

e f

a f te r d ie s e l e x p o s u r e

e n d o f 1 s t s u b c u ltu re

e n d o f 2 n d s u b c u ltu re

5 0

a a

4 0

)

(

l 3 0

o b

n a

e 2 0 M

1 0

N um ber o f su bcu ltu re after initial d iesel treatm ent

4.4.7 Effect of IAA, NAA, Zeatin and BA concentrations on shoot regeneration from different groups of calli

In an experiment using different concentrations of zeatin, the highest percentage (about 25%) of shoot formation response was exhibited by new (1-month-old from second subculture after induction) control petunia calli. There were about 1.8 shoot buds per callus after 12 weeks of culture on the medium supplemented with 2.3 µM zeatin (Table 4.5). Fewer shoots (about 4.1) were formed in the diesel-treated calli cultured on the same medium (Table 4.5).

Within the two different combinations of IAA and BA used, the best shoot formation response of the three groups (about 25%) was exhibited in the new calli: there was an average of 4.8 shoot buds per callus after culture for 12 weeks on the medium supplemented with the combination of 11.4 µM IAA and 17.8 µM BA (Table 4.6).

In the final experiment, use of IAA at much lower concentrations and lower BA concentrations

(compared to one of the higher BA concentrations in the initial experiment, see Tables 4.6 and

4.6), was investigated with the aim of improving the efficiency of shoot formation. It was found that 33.3% of the new petunia calli formed shoots, with about 1.8 shoot buds per callus when cultured on the medium supplemented with the combination of 1.1 µM IAA and 8.9 µM BA

(Table 4.7).

4.4.8 Morphology of the different types of shoots formed

Shoots of different shape, size and orientation were formed in response to different plant growth regulators. The new petunia calli produced the highest percentage of shoots on the MS medium supplemented with 2.3 µM zeatin (Figure 4.11 red arrow). A majority of the shoots were formed at the proliferated edges of the callus, and some had begun to differentiate into leafy shoots (Table 4.5, Figure 4.11). Multiple shoots were also formed in the new calli on the

MS medium supplemented with a combination of 11.4 µM IAA and 17.8 µM BA (Table 4.6,

Figure 4.12, red arrow). Some shoots were embedded in agar (Figure 4.12). Leafy shoots were formed at the edges of new calli on the MS medium supplemented with 1.1 µM IAA and

8.9 µM BA (Table 4.7, Figure 4.13, white arrow).

Table 4.5 Callus-based shoot regeneration responses of Petunia grandiflora to various concentrations of zeatin after 12 weeks of culture.

Plant growth regulator % of callus formed shoot Number of shoot buds formed/callus

Zeatin (µM) Old New Diesel-treated Old New Diesel-treated

2.3 0 (± 0)a 25 (± 2.1)a 4.1 (± 2.5)a 0 (± 0)a 1.8 (± 0.5)a 0.3 (± 0.3)a

4.6 0 (± 0)a 4.1 (± 2.5)b 0 (± 0)a 0 (± 0)a 0.3 (± 0.3)b 0 (± 0)a

9.1 0 (± 0)a 4.1 (± 2.5)b 4.1 (± 2.5)a 0 (± 0)a 0.5 (± 0.3)b 0.3 (± 0.3)a

Table 4.6 Callus-based shoot regeneration responses of Petunia grandiflora to various combinations of indole-3-acetic acid (IAA) with 6- benzyladenine (BA) after 12 weeks of culture.

Plant growth regulators % of callus formed shoot Number of shoot buds formed/callus

IAA (µM) BA (µM) Old New Diesel-treated Old New Diesel-treated

17.1 4.4 0 (± 0)a 12.5 (± 2.5)b 0 (± 0)a 0 (± 0)a 1.3 (± 0.6)b 0 (± 0)a

11.4 17.8 0 (± 0)a 25 ± (2.7)a 0 (± 0)a 0 (± 0)a 4.8 (± 1.0)a 0 (± 0)a

Data were collected from the four replications after 12 weeks of culture and presented as means ± SEM. Means within a column having the same letter are not significantly different by Duncan's multiple range test at p < 0.05. Percentage values were arcsine transformed prior to analysis. n = 72 in Table 4.5 and 96 in Table 4.6 per treatment.

Plant growth regulators % of callus formed shoot Number of shoot buds formed/callus

IAA (µM) NAA (µM) BA (µM) Old New Diesel-treated Old New Diesel-treated

- 0.5 4.4 0 (± 0)a 0 (± 0)b 0 (± 0)a 0 (± 0)a 0 (± 0)a 0 (± 0)a

1.1 - 13.3 8.3 (± 2.9)a 12.5 (± 4.3)ab 4.1 (± 2.5)a 0.8 (± 0.5)a 1 (± 0.7)a 0.3 (± 0.3)a

1.1 - 8.9 0 (± 0)a 33.3 (± 2.8)a 0 (± 0)a 0 (± 0)a 1.8 (± 0.5)a 0 (± 0)a

11.4 - 4.4 4.1 (± 2.5)a 12.5 (± 4.3)ab 4.1 (± 2.5)a 0.5 (± 0.5)a 1.5 (±1.0)a 0.5 (± 0.5)a

4.4.9 Shoot multiplication and root formation

The non-necrotic part of the diesel-treated calli forming leafy shoots grew slowly on callus subculture medium for 8 weeks with undifferentiated dark-brown, shoot-like structures. A plantlet was established on micropropagation medium after 5 weeks (Figure 4.14 (a-e)) and this plantlet and the clonal plantlets derived from it are herein referred to as line L1 regenerants from diesel-treated calli (Table 4.8).

On medium supplemented with 11.4 µM IAA and 4.4 µM BA, diesel-treated calli formed two shoots at different positions. Shoot 1 was formed at the top of the callus (Figure 4.15a; black arrow) with clusters of leaves, while shoot 2 was embedded in the agar (Figure 4.16a; black arrow). Shoot 1 was transferred to a half-strength MS medium containing no PGRs (Figure

4.15b) for 3 weeks with no sign of root formation (Figure 4.15c). This shoot grew strongly and produced numerous roots after transfer to a fresh PGR-free medium for a further 4 weeks

(Figure 4.15d). This plantlet and clonal plantlets derived from it are herein called line L2 regenerants from diesel-treated calli (Table 4.8).

Similarly, shoot 2 was also well established on the micropropagation medium after three subsequent rounds of micropropagation, each of 4 weeks duration (Figure 4.15 (a-d)). The plantlets from this source are herein referred to as line L3 regenerants from diesel-treated calli

(Table 4.7)

A shoot formed from the diesel-treated callus on medium supplemented with 1.1 µM IAA and

13.3 µM BA had three long visible leaves (Figure 4.17a; black arrow). The shoot was transferred to PGR-free, half-strength MS medium (Figure 4.17b) and it then grew slowly on micropropagation medium for 3 weeks with no sign of root formation (Figure 4.17c).

After further subculture of the shoot for three consecutive periods of 4 weeks each from the initial subculture on micropropagation medium, a plantlet was established with multiple roots.

The plantlet from this source is referred to as line L4 regenerants of diesel-treated calli (Table

4.8).

On the MS medium containing 9.1 µM zeatin, shoot formation was induced in a diesel-treated callus (Figure 4.18a; black arrow). The shoot was transferred to PGR-free half-strength MS medium (Figure 4.18b) and allowed to grow for 3 weeks. Then shoots were subcultured for three successive rounds, each of 4 weeks’ duration to produce many pale-yellow leaves with

86 no sign of root formation (Figure 4.18c). In a preliminary experiment, no root formation was observed on the medium with 0.5 µM IBA and 0.5 µM NAA for 10 days.

The shoots grew well in the first week on medium supplemented with 2.5 µM IBA but after the fourth week large friable white calli formed around the stem base without any sign of root formation (Figure 4.18d). The plantlets from this source were maintained on micropropagation medium and referred to herein as line L5 regenerants from diesel-treated calli (Table 4.8).

Shoots formed from a diesel-treated callus on the medium supplemented with 2.3 µM zeatin

(Figure 4.19a; black arrow) were transferred to PGR-free half-strength MS medium (Figure

4.19b) and allowed to grow for 3 weeks. The shoots produced no roots on the micropropagation medium for three consecutive rounds of subculture, each of 4 weeks’ duration (Figure 4.19c).

In a preliminary experiment, no root formation was observed on the medium with 0.5 µM IBA and 0.5 µM NAA for 10 days. The shoots continued to grow on medium supplemented with

2.5 µM IBA and formed thin white roots apparently within the friable white callus after 4 weeks

(Figure 4.19d). The plantlet from this source was also maintained on micropropagation medium and referred to herein as line L6 regenerants from diesel-treated calli (Table 4.8).

a b

c d

Figure 4.14 Regeneration and multiplication of line (L1) from Petunia grandiflora calli survived from diesel treatment of the subculture medium (a–c) and on micropropagation medium (d–e). Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro- Lux lamps 36W). (a) Appearance of the diesel-treated calli after 14 days of culture, (b) a subculture of diesel-treated callus; (c) calli-derived shoots after 8 weeks; (d) multiple shoots at the beginning of subculture, and (e) after 5 weeks.

a b a

c d

a b

c d

a b

c d

a b

c d

Figure 4.18 Regeneration and multiplication of line (L5) from Petunia grandiflora calli that survived diesel treatment. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). (a) Shoots formed after 12 weeks of culture (black arrow), (b) the subculture of the shoot at the beginning of 3 weeks, (c) shoots after three subsequent rounds of micropropagation and (d) shoots after 4 weeks of subculture.

Table 4.8 Regenerated shoots formed from Petunia grandiflora calli that survived diesel treatment. Calli were cultured on the plant growth regulators (PGRs) listed.

Plant growth regulators (µM) Regenerated shoot from diesel-treated callus

IAA NAA Zeatin BA Number of shoots formed Lines

- 1.8 - 6.6 1 L1

11.1 - - 4.4 2 L2 and L3

1.1 - - 13.3 1 L4

- - 9.1 - 1 L5

- - 2.3 - 1 L6

The medium supplemented with 1.1 µM IAA and 8.9 µM BA produced shoots with clusters of leaves from non-diesel treated calli (Figure 4.20a; black arrow). Shoots were easily established with root formation on the micropropagation medium after three consecutive rounds of micropropagation each of 4 weeks. The plantlet (Figure 4.20 b and c) became well established after subculture on fresh medium for 4 weeks, with multiple roots formed (Figure 4.20 (d)).

The plantlets are herein called control regenerants from non-diesel treated calli.

a b

c d

4.5 Discussion

It was reported by Sharma and Mitra (1976) that explants, such as in vitro shoot apical meristem and leaf of Petunia hybrida, formed callus on a medium supplemented with 2.2 µM

6-benzyladenine (BA) and 5.7 µM indole-3-acetic acid (IAA). This contrasted with the findings of the present study that showed a higher BA concentration (6.6 µM) in combination with a lower concentration of auxin (1.8 µM 1-naphthalene acetic acid (NAA)) led to the highest percentage of leaf explants (about 85%) forming callus. Although NAA is a different auxin type and a different Petunia species was used in the present study, the use of IAA in the preliminary experiments suggested that the optimisation of IAA concentrations on the callus induction medium seemed to be less effective than the other auxin types tested in this study

(data not shown). In 1990, Nissen and Sutter reported that IAA concentration in the culture medium was unstable and was easily degraded, even in the presence of cool-white fluorescent lighting in the culture room. However, in this study, even lower concentrations of 2,4- dichlorophenoxyacetic acid (2,4-D) or picloram (PIC) with combinations of BA did not induce a higher percentage of callus in leaf explants. For example, 2.2 µM BA with a combination of

9 µM 2,4-D, induced only about 2% of leaf explants to form callus. The variability in the percentage of leaf explants forming callus in this study demonstrated that explant type alone was only one factor. Other factors, such as the medium (largely influenced by the added plant growth regulators), was also a determinant for establishing efficient callus formation

(Ashrafzadeh and Leung, 2017). In this present study, calli of different types were formed in relation to the ratio of exogenous PGRs in callus inducing medium, the combination of PIC and BA formed calli that appeared largely friable and yellowish. Callus mass is heterogeneous and consists of a mixture of different cell types (Silvertand et al., 1996). This was demonstrated by the formation of a light green/whitish friable callus mass from a combination of BA and 2,

4-D. Also, in this study, light green/whitish and green compact calli were induced when the

97 leaf explants were cultured on the callus inducing medium with the highest concentrations of

6.6 µM BA. The calli were formed along the leaf margin of the adaxial side of leaf explants that was in contact with the medium. The calli that formed after four weeks of culture in this medium appeared to be the largest, compared to those on the other callus inducing media, trialled. For this reason, this medium was considered to be the optimal callus inducing medium for use in this study. It was also used as a subculture medium to maintain the texture and colour of the primary calli during growth and following exposure to diesel.

Recent studies, however, suggest that auxin is a recognised inducer of Arabidopsis lateral organ boundaries domain (LBD)/asymmetric leaves2-like (ASL) transcription factors. They are involved in callus induction (Fan et al., 2012; Ikeuchi et al., 2013). The LBD, also known as

ASL, are proteins that belong to a family of plant-specific transcription factors, identified by an N-terminal-conserved LOB/AS2 domain (Husbands et al., 2007; Majer and Hochholdinger,

2011). For example, during the formation of callus in Arabidopsis thaliana (Arabidopsis) root explants, the LDB16, LDB17, LDB18 and LDB29 were induced on the callus inducing medium

(Fan et al., 2012). Ikeuchi et al. (2013) suggested that, during this process of callus induction, the gradual suppression of the mitotic cell cycle might have eventually led to the termination of cell differentiation and the reacquisition of cell proliferation competence. The LBDs have been demonstrated to act downstream of the auxin response factor7 (ARF7) during callus induction in Arabidopsis. Cell proliferation competence was activated through the stimulation of E2 promoter binding factor-a (E2Fa) that plays a central role in cell cycle re-entry (Inzé and

De Veylder, 2006). In the leaf explants of Nicotiana tabacum, E2Fa and dimerisation of partner protein-a (DPa) were overexpressed leading to callus formation (Kosugi and Ohashi,

2003). This was supported by the finding that callus induction required activation of both E2Fa and DPa (Ikeuchi et al., 2013). In this study, and other related findings of callus induction

(Chakraborty et al., 2013; Mori et al., 2005), the challenge remained to study how the different

auxin and cytokinin had influenced the formation of friable and non-friable, creamy yellowish

to light green, and green calli. Certain types of genes were identified to be associated with the

formation of white friable callus, green compact callus, embryonic callus, and embryonic and

root callus in the presence or absence of auxin and cytokinin (Anzola et al., 2010; Banno et al.,

2001; Chanvivattana et al., 2004; Stone et al., 2001). These are discussed in Chapter 5 of this

thesis.

There is much interest in the use of plants to assist remediation (phytoremediation) of environmental pollutants, including diesel fuel (Gerhardt et al., 2017). There are studies to generate novel plants using in vitro plant cell line selection under the pressure of different environmental pollutants (Ashrafzadeh and Leung, 2015; Rai et al., 2011). However, there is no prior study of exposing in vitro plant cell cultures to diesel. Recent studies have shown that

Petunia grandiflora (petunia) is of interest for phytoremediation of textile dyes (Watharkar and

Jadhav, 2014). Petunia plants will also be of interest for phytoremediation of diesel- contaminated land because the use of ornamental plants would be preferable to food plants for phytoremediation of toxic pollutants. This approach would minimise the chance of pollutants entering into the food chain (Wante and Leung, 2018) but the germination of petunia seeds in diesel-contaminated water was found to be affected to a significant extent by the contaminants

(Wante and Leung, 2018). It appears, therefore, that the development of novel diesel-resistant petunia plants is necessary. The mutagenic properties of diesel has been studied extensively in both animals and human cell lines (Danielsen et al., 2008; Hemmingsen et al., 2011; Mitchell et al., 1981) and these were associated with the high-level content of polycyclic aromatic hydrocarbons (PAHs) found in diesel fuel (Wang et al., 1990). For example, in the in vitro study of L5178Y mouse lymphoma cells mutagenicity was observed following exposure of the cells to diesel toxicity, suggesting that diesel contained substances act as mutagens and DNA- damaging agents (Mitchell et al., 1981). In another similar study, short-time exposure of

Salmonella typhimurium bacterial, L5178Y mouse lymphoma cells and Chinese hamster ovary

(CHO) cells to diesel seemed to result in gene mutation, DNA damage, and chromosomal aberrations (Lewtas, 1983). There was a limited study on the mutagenicity effect of diesel to plants, but Aina et al. (2006) reported that benzo[a]pyrene (BaP) and naphthalene (NAPH) has induced mutation and random DNA changes in the root and shoot of Trifolium repens.

Diesel fuel is highly volatile and flammable and must be handled in a fully ventilated environment such as an operating fume cupboard with a fan turned on. A new protocol was developed for this study, which enabled the exposure of petunia calli to toxic diesel within a safe testing environment. A closer look at the petunia callus under a stereomicroscope revealed its friable to nodulated appearance. It is reasonable to assume that there would be residual diesel in the calli even after several rounds of rinsing of the diesel-exposed calli. After two weeks of a subculture of the diesel-exposed calli, there were signs and smell of diesel evident on the agar medium. There was no contamination when calli were incubated with 100 mL of 0.2% (v/v)

Plant Preservative Mixture (PPM) for 15 minutes compared to the calli without the PPM treatment. PPM is a branded combination of isothiazolinone biocide, methylisothiazolinone and methylchloroisothiazolinone (Plant Cell Technology, Inc., USA) (Miyazaki et al., 2010), a broad-spectrum biocide, that has been reported to be effective in preventing contamination of plant tissue culture (Orlikowska et al., 2012; Rihan et al., 2012). This suggested that the use of

PPM in the protocol was useful and successful to treat calli under otherwise potentially non- aseptic conditions. There was also a significant increase in the proportion of non-necrotic sectors in the calli that survived diesel exposure at the end of the second subculture (about

37%). For shoot regeneration from callus culture with or without exposure to diesel, culture media containing different auxin types, singly or in combination with a cytokinin, were tested.

In a similar study, culture of petunia calli on a medium supplemented with 4.6 µM zeatin resulted in the highest percentage of shoot formation in (Meyer et al., 2009). In contrast to this

100 study, calli cultured on a medium supplemented with 4.6 µM zeatin resulted in the lowest percentage of shoot regeneration (about 4%). In the present study, shoot regeneration was stimulated in about 25% and 4% of new calli (control) and diesel-treated calli, respectively, cultured on a medium supplemented with 2.3 µM zeatin. When 1.1 µM IAA in combination with 8.9 µM BA was added to the medium, the number of calli forming shoots increased to about 33% in the new calli (control), but there was no shoot formation in either the old calli

(control) or diesel-treated calli. The combinations of 1.1 µM IAA with 13.3 µM BA, and 11.4

µM IAA with a 4.4 µM BA were the only media formulations that induced shoot regeneration in both the controls (new and old calli) and the diesel-treated calli. It may be that the mechanism that was involved in the regeneration of shoots in this study was the acquisition of competence of the calli and shoot commitment to respond to shoot induction signal in the cultures (Cary et al., 2001; Che et al., 2007). During the acquisition of competence in Arabidopsis root explants,

Che et al. (2007) observed that callus formed turned green, and this was presumed to be due to the up-regulation of genes involved in the development of the photosynthetic apparatus (Che et al., 2006). Most of these genes that were responsive to cytokinin induction. For example, the

Arabidopsis response regulator 5 (ARR5) marker gene was expressed in Arabidopsis root explants cultured on callus induction medium (Che et al., 2007). The ARR is an A-type response regulator that was upregulated through shoot development (Brandstatter and Kieber, 1998;

D'Agostino et al., 2000; Hwang and Sheen, 2001; Rashotte et al., 2003; Sakai et al., 2000;

Sakai et al., 2001; To et al., 2004). In this study, it is reasonable to speculate that the use of green callus induced on the optimised callus induction medium with the high cytokinin concentration of 6.6 µM BA may have contributed to the acquisition of competence during shoot regeneration development. Although information about the mechanism of action of PGRs in shoot regeneration is limited, in the wild-type of Arabidopsis thaliana root explants, overexpression of enhancer of shoot regeneration 1 (ESR1) was detected after the acquisition

101 of competence and before the induction of shoots (Banno et al., 2001). Another study suggests that overexpression of ESR1 had no effect on callus induction nor root formation but that it increased the efficiency of shoot regeneration in the presence of high cytokinin concentrations

(Zuo et al., 2000). In relation to the syngenetic relationship between the overexpression of

ESR1 and the requirement for an optimal cytokinin level, one can argue that the exogenous concentration of BA in different media formulations in this present study had contributed to shoot regeneration in both the controls (new and old calli) and the diesel-treated calli. Despite these arguments, in this study, the use of cytokinin alone, for example, 2.3 µM, 4.6 µM and 9.1

µM zeatin did not induce shoot regeneration in the old calli, and therefore, in this case, one can only presume that the calli had lost regeneration ability, possibly because of the age of the calli or the acquisition of competence had not been yet acquired. The older calli (control) that turned dark green may acquire the competence to form shoots, but this has not been determined yet.

Although there are reports of shoot regeneration in petunias, most are protocols for shoot regeneration from protoplast culture. It is, therefore, important to note that this is the first report on shoot regeneration in diesel-treated petunia calli. An important step forward that has been achieved here is that shoots were regenerated in calli on all the different media tested.

The ability to produce shoots is an important mechanism that contributes to the developmental

plasticity of plants (Barton, 1998; Steeves and Sussex, 1989). However, on the basis of the

results in this study, the number of shoot buds formation rate per callus on all the different

media tested were not considered to be efficient. This is because in this study, the highest

number of shoot buds formed was about five shoots per callus (only in new calli) on the

medium with a high cytokinin concentration of 17.8 µM BA but this is still low compared with

about 27 shoots per stem explants of Phragimate commums (Reed) on a medium with 53 µM

BA (Guo et al., 2004). Although in the experiment using Reed (Guo et al., 2004), the method

of regeneration was a direct type (without a callus phase), which may have also contributed to

102 the high number of shoot buds formed. In another regeneration study, the callus cultures of Zea mays on a medium with a low concentration of cytokinin of 2 µM BA induced the highest number of shoot buds of about nine shoots per callus, and it was considered to be an efficient regeneration protocol (Pathi et al., 2013). Because of this, it is reasonable to assume that the average number of shoot buds formed using an efficient in vitro regeneration protocol may depend on the type of regeneration methods (that is, whether direct or indirect).

4.6 Conclusion

Petunia calli of different morphologies can be induced in young leaf explants of in vitro germinated seedlings. As well as this, a medium for callus induction in leaf explants has been optimised: MS medium supplemented with 1.8 µM NAA and 6.6 µM BA. This medium was highly efficient and consistent in all the repeated experiments. It was also selected as a callus subculture medium. A new protocol was successfully established for exposing petunia calli to the toxicity of diesel fuel under non-aseptic conditions, and the diesel-treated calli were successfully re-cultured under aseptic conditions.

More importantly, the diesel-treated calli exhibited non-necrotic regions during re-culture under aseptic conditions and proliferated during subculture. The shoot regeneration protocol was probably not optimal in the petunia calli because only few shoots were regenerated that were further established as the plantlet lines on PGRs-free medium in this study. At present, there is a paucity of knowledge about the physiology and biochemistry of diesel resistance/tolerance in plants (Silva et al., 2017). These plantlet lines could be novel tools/system in the studies aiming to obtain a better understanding of the physiological and biochemical basis of diesel resistance in petunia. To achieve this, a comparative study (Chapter

7) was carried out to evaluate the performance of the plantlet lines regenerated from diesel- treated calli, from in vitro-germinated seeds, and from calli without exposure to diesel.

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Chapter 5

Callus induction in leaf explants of Marigold-Nemo mix (Tagetes

patula L.) and exposure to diesel

5.1 Introduction

There are many different species in the genus Tagetes, and their practical uses have been well established (Soule, 1993; Vasudevan et al., 1997). For example, Tagetes erecta has been widely used in the studies of secondary metabolites like thiophenes with nematocidal properties

(Bohlmann and Zdero, 1978; Ketel, 1986) and carotenoid production (Benítez-García et al.,

2014). These studies were achieved using in vitro cultures of T. erecta following optimisation of different protocols through callus culture, organogenesis or embryogenesis (Belarmino et al., 1992; Bespalhok and Hattori, 1998; Gupta and ur Rahman, 2015; Kothari, 2004; Kothari and Chandra, 1984, 1986). Tagetes patula is an important ornamental plant because of its floricultural use around the world (Singh et al., 2016). In several studies, it was found to tolerate adverse environmental conditions and to be a good phytoremediator of textile dyes, and iron tailing (Belarmino and Mii, 2000; Chaturvedi et al., 2014; Patil and Jadhav, 2013); however, a drought-tolerant clone of Tagetes minuta has been selected and characterised in vitro

(Mohamed et al., 2000). To my knowledge, until now there has been only one report on in vitro culture of Tagetes patula studying optimisation and haploid plant regeneration from anther culture (Qi et al., 2011).

In this study, four-week-old Tagetes patula seedlings grown under in vitro conditions were used. The objectives were: 1) to establish an optimised medium for efficient callus induction in Tagetes patula leaf explants, 2) to investigate the effect of exposure of Tagetes patula callus culture to diesel, and 3) to investigate the plant regeneration requirements in Tagetes patula.

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5.2 Materials and methods

5.2.1 Plant materials and culture conditions

5.2.1.1 Seeds

Seeds of ornamental cultivar of Marigold-Nemo mix (Tagetes patula L.) were purchased from

Kings Seeds Company, Katikati, New Zealand. Seeds were kept in a closed container as

described in detail in section 2.1.1.

5.2.1.2 Seed germination under aseptic conditions

Seeds of Tagetes patula (marigold) were germinated under in vitro conditions. They were

washed with soapy water and then surfaced sterilised with 1% (w/v) sodium hypochlorite

(NaOCl) for 15 minutes, as described in detail in sections 2.2.1and 2.2.2.

5.2.1.3 Callus induction and proliferation

Leaves were aseptically excised from 4-week-old marigold seedlings grown under in vitro

conditions for callogenesis. The adaxial side of the leaves was placed on the MS medium as

described in sections 2.2.3 and 2.2.4.

5.2.1.4 Morphology of the calli formed

Different types of calli formed on the callus induction medium (Table 5.1) in response to

various types and different concentrations of plant growth regulators and were visually

assessed, as described in section 2.2.5.

5.2.1.5 Maintenance of callus culture

Selected pieces of callus were maintained on the optimised callus subculture medium (Table

5.1) supplemented with 1.8 µM NAA and 66.6 µM BA, as described in section 2.2.6.

5.2.1.6 Exposure of callus to diesel fuel

In a preliminary trial, 30 pieces of marigold calli were totally submerged in 50 mL of diesel fuel for 3, 5, 7 and 9 min. Finally, the diesel exposure time of 9 min. was chosen after morphological visual inspection (MVI) of calli before and after 2 weeks of culture. All the

105 critical steps involved in the exposure of callus to diesel fuel were described in detail in Chapter

2 (2.3.1) and are summarised in Figure 5.7. There were about 15 pieces of calli each in the three replications, and the calli were exposed to undiluted diesel for 9 min. They were then rinsed with sterile distilled water and finally with 0.2% (v/v) of Plant Preservative Mixture

(PPM), as described in section 2.3.1. The callus pieces were placed on the surface of the selected MS medium supplemented with 1.8 µM NAA and 66.6 µM BA (callus subculture medium) in Petri plates, as described in section 2.3.1.

5.2.1.7 Culture of diesel-treated callus under aseptic conditions

After 2 weeks of diesel exposure followed by culture on the callus subculture medium, the diesel-treated marigold callus pieces were assessed using the MVI approach throughout the experiment (6 weeks), as described in section 2.3.2.

5.2.1.8 Selection of calli that survived after diesel exposure

A non-necrotic region from a diesel-treated callus that had been cultured on MS subculture medium (Table 5.1) was selected, as described in section 2.3.3. A callus that proliferated after

6 weeks of culture was considered as a callus derived from the callus previously exposed to diesel.

5.2.1.9 Plant regeneration trials

Calli were cultured on basal MS medium supplemented with 1.8, 3.6, 5.4 to 6.6 µM BA in

combination with different concentrations of GA3 (5, 10, 50 to 100 µM) or NAA (1.8 to

2.2 µM) (Table 5.6–5.11), as described in section 2.4.1.

106

Table 5.1 Plant cell and tissue culture media used in this study

Serial/Number Composition Plant growth Purpose/ use regulators

1 Full-strength MS basal (Murashige and Skoog, 1962) medium No added PGRs In vitro germination of seed

2 Full-strength MS basal (Murashige and Skoog, 1962) medium. NAA, 2, 4-D & PIC Callus induction to BA (Tables 5.2, 5.3, 5.4 & 5.5)

3 Full-strength MS basal (Murashige and Skoog, 1962) medium 1.8 µM NAA & 66.6 Optimised callus µM BA induction/callus subculture medium 4 Full-strength MS basal (Murashige and Skoog, 1962) medium (Tables 5.6–5.11) Shoot regeneration

Note: All the media were supplemented with 3% sucrose (w/v) and gelled with 0.8% (w/v) agar.

107

5.3 Statistical analysis

The mean difference between the replicates at the 5% level of significance was calculated using one-way analysis of variance (ANOVA) and was tested using the post hoc test of Duncan's multiple range and Fisher’s unprotected least significant difference (LSD) test. All graphs were plotted using the GraphPad Prism software Version 7.0.

108

5.4 Results

5.4.1 In vitro seed germination and seedling growth

In the preliminary trials, about 87% of the surface sterilised seeds of marigold germinated on the basal MS basal medium without any added PGR. The marigold seeds were germinated on

PGR-free MS medium (Figure 5.1a): after 48 hours from sowing, radicle emergence from the germinated seeds was observed, and cotyledons were observed after 1 week (Figure 5.1b).

The seedlings developed further (Figure 5.1c), and after 4 weeks, several leaves were formed in each seedling (Figure 5.1d).

a b c d

(a) 0 day (b) 1 week (c) 3 weeks (d) 4 weeks

Figure 5.1 In vitro seed germination and seedling development of Tagetes patula on full-strength MS medium without any added plant growth regulators. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W).

109

5.4.2 Efficiency of callus induction

Among the combinations of NAA and BA used in one of the three initial experiments, the highest percentage (about 40%) of the marigold leaf explants formed masses of calli after 4 weeks of culture on medium supplemented with a combination of 1.8 µM NAA and 4.4 µM

BA or 2.7 µM NAA and 6.6 µM BA (Table 5.2).

Among the combinations of 2,4-D and BA used, the highest percentage (about 43%) of the leaf explants formed calli after culture for 4 weeks on the medium supplemented with a combination of 4.5 µM 2,4-D and 4.4 µM BA or 13.5 µM 2,4-D and 2.2 µM BA (Table 5.3).

In contrast, more explants (63% and 53%) of leaf explants formed calli after culture on medium supplemented with a combination of 5.7 µM PIC and 4.4 µM BA, or 5.7 µM PIC and 66.6 µM

BA, respectively.

In the final experiment, mainly by using much higher BA concentrations than in the initial experiment (compare Tables 5.2 and 5.5), to improve the efficiency of callus formation, it was found that 95% of the leaf explants formed calli when cultured on the medium supplemented with the combination of 1.8 µM NAA and 66.6 µM BA (Table 5.5).

5.4.3 Appearance of calli formed

Calli with a different colour, size and texture were formed in response to different plant growth regulators. Both compact and friable calli were formed on the medium supplemented with

NAA and BA, which appeared largely light green in the first initial experiment (Table 5.2,

Figure 5.2). The calli formed on the medium supplemented with a combination of BA and

2,4-D or PIC were creamy yellow and friable (Tables 5.3 and 5.4, Figure 5.3 (a and b) and

Figure 5.4). The combination of the lowest concentration of NAA (1.8 µM) and the highest BA concentration (66.6 µM) used in the final experiment resulted in calli that appeared green and compact (Table 5.5 and Figure 5.5).

110

Table 5.2 Effect of the various combinations of 1-naphthalene acetic acid (NAA) with 6-benzyladenine (BA) on callus induction in Tagetes patula leaf explants after 4 weeks of culture. Plant growth regulators (%) of explants Colour Texture Estimated amount of callus formed callus formation NAA (µM) BA(µM) Leaf

0 0 0 (± 0)c - - -

1.8 2.2 25 (± 0.3)b Light green Friable †

1.8 4.4 40 (± 0.4)a Green and light green with whitish Compact and friable † part

1.8 6.6 35 (± 0.3)ab Light green and whitish part Friable ††

2.7 2.2 37.5 (± 0.5)ab Light green Friable ††

2.7 4.4 25 (± 0.6)b Green Compact ††

2.7 6.6 42.5 (± 0.5)a Green and light green with whitish Compact and friable ††† part

† A slight amount of callus on an explant Light green Green Whitish

†† Moderate callus formation

††† High callus formation

111

Figure 5.2 Callus induction experiment using leaf explants from 4-week-old Tagetes patula seedlings.

Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light,

Sylvania Gro-Lux lamps 36W). Appearance of the calli after 4 weeks of culture on medium supplemented with 2.7 µM NAA and 6.6 µM BA.

112

Table 5.3 Effect of the various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) with 6-benzyladenine (BA) on callus induction in Tagetes patula leaf explants after 4 weeks of culture. Plant growth regulators % of explants formed callus Colour Texture Estimated amount of callus formation

2,4-D (µM) BA (µM) Leaf

0 0 0 (± 0)d - - -

4.5 2.2 20 (± 0.6)c Creamy yellowish Friable ††

4.5 4.4 43.3 (± 0.3)a Creamy yellowish Friable ††

4.5 8.8 26.6 (± 0.7)bc Creamy yellowish Friable ††

9 2.2 33.3 (± 0.3)ab Creamy yellowish Friable †

13.5 2.2 43.3 (± 0.3)a Creamy yellowish Friable ††

13.5 4.4 16.6 (± 0.3)c Creamy yellowish and whitish spot Friable ††

13.5 8.8 33.3 (± 0.3)ab Creamy yellowish Friable ††

† A slight amount of callus on an explant Creamy yellowish Whitish

†† Moderate callus formation

††† High callus formation

113

114

Table 5.4 Effect of the various combinations of picloram (PIC) with 6-benzyladenine (BA) on callus induction in Tagetes patula leaf explant after 4 weeks of culture. Plant growth regulators % of explants formed callus Colour Texture Estimated amount of callus formation

PIC (µM) BA(µM) Leaf

0 0 0 (± 0)f - - - 5.7 4.4 63 (± 0.3)a Light green Friable †† 5.7 22.2 23 (± 0.3)de Light green and brownish Friable † 5.7 44.4 26 (± 0.3)de Light green and brownish Friable † 5.7 66.6 53 (± 0.7)ab Extremely light yellow Friable †† 17.2 4.4 36 (± 0.3)cd Light green brownish spot Friable †† 17.2 22.2 33 (± 0.7)ce Extremely light yellow Friable †† 17.2 44.4 40(± 0.6)c Extremely light yellow Friable †† 28.5 44.4 20 (± 0)e Brownish Friable †† 28.5 4.4 43 (± 0.3)bc Extremely light yellow Friable †† 28.5 66.6 26 (± 0.3)de Extremely light green Friable ††

† A slight amount of callus on an explant Light green Extremely light green Brownish Extremely light yellow

†† Moderate callus formation

††† High callus formation

115

116

Table 5.5 Effect of the various combinations of 1-naphthalene acetic acid (NAA) with 6-benzyladenine (BA) on callus induction in Tagetes patula leaf explants after 4 weeks of culture. Plant growth regulators % of explants formed Colour Texture Estimate amount of callus callus formation NAA (µM) BA (µM) Leaf

0 0 0 (± 0)c Brownish/light green 0 0 5.7 22.2 60 (± 0.9)b Creamy yellowish/whiting spot Compact and †† friable 5.7 44.4 65 (± 0.6)b Light green with whiting sections Compact and ††† friable 5.7 66.6 70 (± 0.7)b Creamy yellowish/light green /whiting Friable ††† spot 1.8 44.4 67 (± 0.5)b Green/light brown spot around the calli Compact ††† 1.8 66.6 95 (± 0.3)a Green Compact †††

† A slight amount of callus on an explant Light green Green Creamy yellowish Brownish Whitish

†† Moderate callus formation

††† Massive callus formation

117

118

5.4.3 Appearance of callus after subculture

Pieces of callus were subcultured every two weeks on the subculture medium (Table 5.1) for 6

weeks without any visible changes in the green colour and compact texture of the calli (Fig.

5.6 (a)). A closer view of the callus under a light microscope of (40×) magnification revealed

the arrangement of closely attached, unorganised mass of cells (Figure 5.6 (b)).

a b

119

5.4.4 Manipulations of diesel-treated callus under aseptic conditions

The flow chart in (Figure 5.7) highlights an innovative step used to expose marigold calli to undiluted diesel fuel outside the laminar flow cabinet, that is, in a potentially non-aseptic environment. After this, the re-cultured calli under aseptic conditions that survived the diesel treatment were able to proliferate (Figure 5.8e). Marigold calli did not exhibit extensive necrosis immediately after exposure to undiluted diesel fuel for up to 9 minutes (Figure 5.8c).

Immediately after treatment, the diesel-treated marigold calli were cultured for 14 days (Figure

5.8d). The non-necrotic regions from the diesel-treated calli were then excised and placed individually on the fresh medium of the same composition (callus subculture medium) (Table

5.1). At the end of 14 days of the first subculture, about 30% of the diesel-treated marigold calli survived (Figure 5.9). At the end of 14 days of the second subculture, the number of subculturable callus pieces increased to about 60% (Figure 5.9).

120

Callus

Non-diesel treated callus 9 min-diesel-treated pieces (control) In a laminar flow callus pieces cabinet under aseptic conditions In a non-sterile fume cupboard

In a laminar flow water

cabinet under Rinsed5x with aseptic conditions Submerge callus deionised sterile pieces in 0.2% (v/v) PPM for 15 min

In a laminar flow cabinet under aseptic conditions

Place diesel-treated callus Place callus pieces of non- pieces on the surface of diesel treatment (control) sterile callus subculture on the surface of sterile medium callus subculture medium In a laminar flow cabinet under aseptic conditions

2-weeks incubation of treated calli cultures

121

a b

c d

e f

122

a f te r d ie s e l e x p o s u r e

e n d o f 1 s t s u b c u ltu re

e n d o f 2 n d s u b c u ltu re

8 0

6 0

)

(

i l

l b

f 4 0 o

e M

2 0

N um ber o f su bcu ltu re after initial d iesel treatm ent

123

5.4.5 Effect of IAA, NAA, zeatin, GA3 and BA concentrations on shoot regeneration from different groups of calli

In an experiment using eight different combinations of NAA, GA3 and BA, no shoot formation was exhibited in the new (1-month-old from second subculture after induction) and old (more than 3 months old after the second subculture from callus induction) groups of calli after 10 weeks of culture (Tables 5.6, 5.7 and 5.8; Figures 5.10, 5.11 and 5.12). In another different experiment, four combinations of IAA, GA3 and BA were used; there was no sign of shoot buds formed in the diesel-treated calli after 10 weeks of culture (Tables 5.9 and 5.10; Figures

5.13 and 5.14). In the final experiment, different concentrations of zeatin were used; no shoot was formed in the diesel-treated calli after 10 weeks of culture (Table 5.11, Figure 5.15).

124

Plant growth regulators % of callus formed Number of shoot buds formed/callus shoot (µM)

NAA BA GA3 Diesel-treated Diesel-treated

1.8 6.6 5 0 0

11.4 6.6 10 0 0

a b

Figure 5.10 Experiments to induce plant regeneration in Tagetes patula callus after 10 weeks of culture

on MS medium supplemented with (a) 1.8 µM NAA, 6.6 µM BA and 5 µM GA3 and (b) 1.8 µM NAA,

6.6 µM BA and 10 µM GA3. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). (a) A piece of new callus with root formation (black arrow), (b) a set of old calli with no sign of shoot formed (red arrow).

125

Table 5.7 Experiments to induce shoot regeneration in Tagetes patula callus using various combinations of 1-naphthalene acetic acid (NAA), gibberellic acid (GA3) and 6-benzyladenine

(BA) after 10 weeks of culture.

Plant growth regulators % of callus Number of shoot buds formed/callus formed shoot (µM)

NAA BA GA3 Diesel-treated Diesel-treated

2.2 1.8 50 0 0

2.2 3.6 50 0 0

2.2 5.4 50 0 0

126 a b

Figure 5.11 Experiments to induce plant regeneration in Tagetes patula callus after 10 weeks of culture

on MS medium supplemented with (a) 2.2 µM NAA, 1.8 µM BA and 50 µM GA3; (b) 2.2 µM NAA,

3.6 µM BA and 50 µM GA3 and (c) 2.2 µM NAA, 5.4 µM BA and 50 µM GA3. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). The red arrow in (a), (b) and (c) points at a piece of old callus with no sign of shoot formation, while the black arrow in (a), (b) and (c) points at a set of new calli with no sign of shoot formation.

127

Plant growth regulators % of callus Number of shoot buds formed/callus formed shoot (µM)

NAA BA GA3 Diesel-treated Diesel-treated

2.2 1.8 100 0 0

2.2 3.6 100 0 0

2.2 5.4 100 0 0

128

a b

Figure 5.12 Experiments to induce plant regeneration in Tagetes patula callus after 10 weeks of culture on MS medium supplemented with (a) 2.2 µM NAA, 1.8 µM BA and 100 µM GA3; (b) 2.2 µM NAA,

3.6 µM BA and 100 µM GA3 and (c) 2.2 µM NAA, 5.4 µM BA and 100 µM GA3. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). The red arrow in (a), (b) and (c) points at a piece of old callus with no sign of shoot formation, while the black arrow in (a), (b) and (c) points at a set of new calli with no sign of shoot formation.

129

Table 5.9 Experiments to induce shoot regeneration in Tagetes patula callus using various combinations of indole-3-acetic acid (IAA), gibberellic acid (GA3) and 6-benzyladenine (BA) after 10 weeks of culture.

Plant growth regulators % of callus formed Number of shoot buds formed/callus shoot (µM)

IAA BA GA3 Diesel-treated Diesel-treated

11.4 3.6 50 0 0

11.4 6.6 10 0 0

a b

Figure 5.13 Experiments to induce plant regeneration in Tagetes patula callus after 10 weeks of culture on MS medium supplemented with (a) 11.4 µM IAA, 3.6 µM BA, and 50 µM GA3 and (b) 11.4 µM

IAA, 6.6 µM BA and 10 µM GA3. Cultures were kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). A set of calli that survived diesel treatment in (a) and (b) with no emergence of shoot buds.

130

Plant growth regulators % of callus formed Number of shoot buds formed/callus shoot (µM)

IAA BA GA3 Diesel-treated Diesel-treated

11.4 26.6 86 0 0

2 66.6 86 0 0

a b

Figure 5.14 Experiments to induce plant regeneration in Tagetes patula callus after 10 weeks of culture

on MS medium supplemented with (a) 11.4 µM IAA, 26.6 µM BA and 86 µM GA3 and (b) 2 µM IAA,

66.6 µM BA and 86 µM GA3. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). A set of calli that survived diesel treatment in (a) and (b) with no emergence of shoot buds.

131

Table 5.11 Experiments to induce shoot regeneration in Tagetes patula callus using various concentrations of zeatin after 10 weeks of culture.

Plant growth regulator % of callus formed shoot Number of shoot buds formed/callus

Zeatin (µM) Diesel-treated Old

2.3 0 0

4.6 0 0

9.1 0 0

132 a b

133

5.5 Discussion

It has been difficult to culture Tagetes patula (marigold) in vitro due to browning of most explants (Ketel, 1986, 1987; Ketel et al., 1985), except the anther (Qi et al., 2011), leading to poor callus formation and growth. Different callus types were induced in the young marigold anther explants placed in contact with the basal (MS) medium (Murashige and Skoog, 1962) supplemented with different concentrations of different types of plant growth regulators

(PGRs) (Qi et al., 2011). Generally, leaf explants are suitable for in vitro cell culture establishment (Chakraborty et al., 2013; Ling et al., 2013). Plant growth regulators, such as the combination of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA) used in this, and other, studies added to MS basal medium induced friable creamy yellowish callus from the young leaf explant of the genus Tagetes. For example, in this study, about 43% of the leaf explants formed friable calli on the medium supplemented with 4.5 µM of 2,4-D and 4.4 µM

BA or 13.5 µM 2,4-D and 2.2 µM BA. Similarly, the study by Benítez-García et al. (2014) found that 9 µM 2,4-D and 8.8 µM BA was a suitable PGR combination for friable callus induction in T. erecta. There was a higher percentage of friable callus induction when picloram

(PIC), in combination with BA, was used. Picloram alone, or in combination with BA, has been reported to induce yellow friable or yellow compact callus in Lilium martagon explants (Kedra and Bach, 2005). In another similar study using cotyledons and hypocotyls of T. erecta, the auxin type 1-naphthalene acetic acid (NAA) (5.4 µM) in combination with 66.6 µM BA initiated a higher percentage of callus (about 93%) (Mohamed et al., 1998).

Here in this present study, a higher percentage (about 95%) of leaf explants formed green and compact callus when cultured on the medium supplemented with 1.8 µM NAA and 66.6 µM

BA. This type of callus formed was only found when the leaf explants were cultured on the medium with a combination of NAA and BA. The optimised combinations of the hormonal balance in the media are the important steps of success in plant tissue culture (Ye et al., 2017).

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In the present study, efficient callus inducing media were successfully established and also used as a subculture medium to maintain the colour and texture of the primary callus. It is, however, important to note that it took around 6-7 months to induce more of the green, nonfriable callus. This type of callus culture was required for exposing callus to undiluted diesel (see Chapter 4).

The different types of calli formed on callus inducing media with different auxin types and cytokinin concentrations might be required for the process of switch-over from the mitotic cell cycle to the endomitotic cell cycle (Kandasamy et al., 2001). Ikeuchi et al. (2013), described the complex molecular regulatory mechanisms of auxin to cytokinin balance effect in callus inducing media. Auxin is converted through auxin response factors (AFR) specifically AFR7 and AFR9 to trigger the expression of lateral organ boundaries (LBD) family transcription factors (LBD16, LBD17, LBD18, and LBD29) (Fan et al., 2012; Lee et al., 2009; Okushima et al., 2007). These LBDs, in turn, stimulate E2 promoter binding factor-a (E2Fa) that plays a central role in cell cycle re-entry (Inzé and De Veylder, 2006). Overexpression of E2Fa and dimerisation of partner protein-a (DPa) caused overproliferation of callus in leaf explants of

Nicotiana tabacum (Kosugi and Ohashi, 2003). This was supported by the finding that callus induction required activation of both E2Fa and DPa (Ikeuchi et al., 2013). The combination of auxin type and cytokinin concentrations in this study and other similar studies resulted in the induction of calli of a different colour, size, and texture. Tajima et al. (2004) found that hypocotyl and root explants of putative ARR21-C-ox transgenic Arabidopsis thaliana

(Arabidopsis) after 35 days on MS medium induced friable calli. White friable calli were also induced from the leaf segment explants of Withania somnifera on the media with a combination of 2,4-D and kinetin (Chakraborty et al., 2013). In a different study (Banno et al. (2001), green compact/nonfriable callus was induced in Arabidopsis root explants transformed with pSK-

ESR1, a35S:ESR1 construct on callus inducing media. It seems reasonable to presume that

135 genes involved in auxin or cytokinin signalling mighty have also influenced the formation of friable and non-friable, creamy yellowish to light green and green calli in this present study and other related findings (Chakraborty et al., 2013; Mori et al., 2005). However, a study reported by Anzola et al. (2010) showed that white friable callus was induced in Arabidopsis shoot apex, which downregulated the kip-related protein (KRP) genes encoding cyclin- dependent kinases (CDK) inhibitors, and a transcriptional adaptor protein proporz1 (PRZ1) in the cell cycle through the reacquisition of cell proliferation capability. In another similar study, where whitish/light green friable calli were induced on the hypocotyl and root explants of

Arabidopsis, the calli have overexpressed the constitutively active form of the Arabidopsis response regulator21 (ARR21) gene (Tajima et al., 2004). Tajima et al. (2004) suggested that

ARR21 may have been involved in a hormone-mediated signalling pathway which cytokinin, or auxin, or both, have been involved in mediating signals that severely disrupted the ARR21-

C-ox transgenic. In the Banno et al. (2001) study, where dark green compact callus was induced with increasing cytokinin levels, the overexpression of the enhancer of shoot regeneration

(ESR1) gene was detected. Although it was not clear how cytokinin induced overexpression of

ESR1 in callus, Banno et al. (2001) suggested that ESR1 may have functioned as a transcriptional repressor in the callus derived shoots. This presumption, however, may not hold because of overexpression of a fusion protein of ESR1 and α-trans-inducing factor (α-TIF), a peptide that functions as a strong transactivator in plants with the same effect as ESR1 on shoot regeneration (Aoyama and Chua, 1997; Zuo et al., 2000).

Novel plants resistant to environmental pollutants are of interest for phytoremediation (Wang et al., 2017). There are studies to generate novel plants using in vitro plant cell line selection under the pressure of different environmental pollutants (Ashrafzadeh and Leung, 2015; Rai et al., 2011). There is a notable absence of studies on in vitro selection of diesel-resistant plants.

However, diesel has been reported to cause mutation in both animals and human cell lines

136

(Danielsen et al., 2008; Hemmingsen et al., 2011; Mitchell et al., 1981), which were associated

with the properties of polycyclic aromatic hydrocarbons (PAHs) found in diesel fuel (Wang et

al., 1990). Here, there are indications that diesel may induce genetic variation in cultured plant

cells because following in vitro exposure of L5178Y mouse lymphoma cells, Salmonella

typhimurium bacterial, and Chinese hamster ovary (CHO) cells to toxic diesel, mutagenicity

and damaged DNA were observed (Lewtas, 1983; Mitchell et al., 1981). The studies of Lewtas

(1983) and Mitchell et al. (1981) further confirmed that some substances in diesel could act as

mutagens and DNA-damaging agents.

Diesel fuel is highly volatile and flammable and must be handled in a fully ventilated environment such as an operating fume cupboard with a fan turned on. In this study, we sought a practical approach to this perceived technical barrier. The finding in this chapter is to extend the application of in vitro plant cell line selection to generate diesel-resistant plants. Recent studies have shown that marigold is of interest for phytoremediation of textile dyes (Patil et al.,

2009), but their seeds germinated in vitro were sensitive to water contaminated with 4% diesel

(Wante and Leung, 2018). An innovative step here, in this present study, is that the marigold calli were exposed to undiluted diesel fuel outside the laminar flow cabinet, that is, potentially a non-aseptic environment. The calli that had earlier been exposed to diesel for up to 9 minutes did not exhibit extensive necrosis during the undiluted fuel treatment, suggesting that the treatment was sub-lethal, a condition required to generate somaclonal variants (Ashrafzadeh and Leung, 2015). Even so, a short exposure of S. typhimurium cells to toxic diesel has been shown to cause genetic mutation (Lewtas, 1983).

Plant Preservative Mixture (PPM) is a broad-spectrum biocide that has been reported to be

effective in preventing contamination of plant tissue culture (Orlikowska et al., 2012; Rihan et

al., 2012). The results obtained suggest that it was beneficial to include PPM in the protocol

for exposing callus to diesel. This protocol enabled exposure of marigold calli to toxic diesel

137 fuel and then the diesel-treated calli were successfully cultured under aseptic conditions. More importantly, the diesel-treated calli exhibited non-necrotic regions during aseptic culture and proliferated during subculture. In this study, the shoot regeneration experiment, using the tested combination of different auxin types and cytokinin concentrations, failed to regenerate shoots from the selected callus culture (old and new) and diesel-treated calli. In T. erecta, leaf-derived calli were tested for shoot regeneration using different NAA and BA concentrations and these also failed to generate shoots, but rather they formed roots (Belarmino et al., 1992).

Furthermore, in this present study, the majority of the new calli formed roots on the different combinations of auxin types and cytokinin concentrations, while diesel-treated calli formed roots only in the medium with 2.3 µM zeatin. A study of root formation in callus cultures by

Soh et al. (1998) demonstrated the mechanism of actions of PGRs in transforming root organogenesis. For example, yellowish compact callus was formed from the hypocotyl explants of Vigna unguiculate on MS medium supplemented with 1 µM kinetin and 1.81 µM

2,4-D. Further subculture of the callus on medium with NAA and BA, the callus changed to pale green with numerous green spots under continuous lighting (Soh et al., 1998). In the present study, the green spots did not appear visible on the calli, but the majority of the calli that formed roots have turned greener after the two to three weeks of culture on the shoot regeneration media. In the study reported by Soh et al. (1998) and other similar studies (Meyers et al., 1986; Sharma and Kothari, 1993), the nodular structures appeared from the green spots, and root organogenesis occurred directly from the structures. On this basis, it is reasonable to presume that the green spots were at the position of root primordium differentiation (Soh et al.,

1998). It was reported that 2,4-D induced adventitious root primordium in calli of Bupleurum falcatum and Lactuca sativa but failed to develop into roots on the same medium (Bae et al.,

1994; Kang et al., 1996). On transfer to auxin-free medium, roots were regenerated. In the stud of Soh et al. (1998), adventitious root primordium did not form on the medium with auxin.

138

Based on these findings, it is possible that the presence of auxin alone in a callus can inhibit induction of root primordium and differentiation (Bae et al., 1994; Kang et al., 1996), while the optimal balance of auxin and cytokinin can induce root formation in callus cultures (Soh et al., 1998). In the present study, the combination of auxin types and cytokinin concentrations used during shoot regeneration of callus could be optimal for rooting.

Haploid plants regenerated from callus-derived anther culture of marigold were reported (Qi et al., 2011). The haploid plant was not desirable in this study because of a high possibility of the occurrence deleterious traits in culture, and the doubling of haploids may not always lead to the formation of a homozygous plant (Palmer et al., 2005). Although, in the genus Tagetes, for example, T. erecta, various plant regeneration protocols have been optimised mostly through direct regeneration from different explants such as leaf, cotyledonary leaf, and hypocotyl

(Gupta and ur Rahman, 2015; Mohamed et al., 1998; Vanegas et al., 2002). The best hormonal combinations used to induce shoot regeneration of T. erecta were also part of the tested auxin and cytokinin combinations in this study. No shoot regeneration in marigold callus cultures was observed. Roots were formed in most of the calli, and increased callus formation and greening were also observed in the controls and calli that were exposed to diesel. This was also observed in Arabidopsis lines carrying ARR10 transgene (Hill et al., 2013). This suggests that one challenge could be the inability of the callus culture to synthesise and/or respond to exogenous cytokinin in the media (Hill and Schaller, 2013). Although the seven-month regeneration experiment was not successful, lessons have been learned about plant regeneration challenges in callus culture of Tagetes patula.

5.6 Conclusion

The efficient marigold callus culture protocol established here might be used in in vitro plant cell line selection for the isolation of somaclonal variants of marigold plants. If successful, the present study would have been an important step leading to the generation of marigold plants

139 that could be resistant/tolerant to toxic levels of diesel. Further investigation is required to achieve successful plant regeneration in marigold callus.

140

Chapter 6 A histological comparison of Petunia grandiflora Juss. mix F1 and Marigold-Nemo mix (Tagetes patula L.) calli after exposure to diesel 6.1 Introduction Histology analysis has been used for the study of the internal organisation of plant structures

(Yeung, 1999). For example, there is a study of the changes in callus culture of Papaver somniferum during differentiation and induction of organogenesis (Šamaj et al., 1990). There are also reports on various in vitro root and callus cultures of ornamental plants (Baskaran and

Van Staden, 2017; Chen et al., 2016; Kumar et al., 2016; Yumbla-Orbes et al., 2017).

However, there is no report until now regarding the histology of diesel-treated calli of Petunia grandiflora (petunia) and Tagetes patula (marigold).

The main objective of this chapter was to study the histology of callus produced in Petunia grandiflora and Tagetes patula at two weeks after diesel treatment. It provides insights into the effect of diesel toxicity in plants at the cellular level.

6.2 Materials and Methods

6.2.1 Callus source

Calli from the leaf explants of petunia and marigold were induced on the selected callus induction media, as explained in sections 2.2.4 and 2.2.5.

6.2.2 Callus fixation in FAA (formaldehyde–acetic acid–ethanol)

At the end of two weeks of culture, the diesel-exposed and non-diesel exposed calli of petunia and marigold were used for histological examination. About 20 callus pieces were randomly selected and carefully separated from the agar. A selected piece of callus was immediately transferred into a 10 mL scintillation glass vial containing 5 mL of formaldehyde – acetic acid

– ethanol (FAA) in the proportion of 1.5 mL of 95% ethanol, 3 mL of dH2O, 0.2 mL of 37% formaldehyde solution (histological grade) and 0.3 mL of glacial acetic acid as a fixative

141 solution, and a small piece of paper labelled with a pencil (Ruzin, 1999). Then, the vial was placed under a vacuum of approximately 60 mm Hg for at least 1h, then overnight at room temperature under vacuum ready for the dehydration processes.

6.2.3 Dehydration in a series of tertiary butanol alcohol (TBA)

Fixed calli of petunia and marigold were dehydrated using the following prepared solutions of

100 mL of tertiary butyl alcohol (TBA) (Merck, Germany) series.

1. 10 mL TBA, 40 mL 95% ethanol, 50 mL dH2O (for 1 h)

2. 20 mL TBA, 50 mL 95% ethanol, 30 mL dH2O (overnight)

3. 35 mL TBA, 50 mL 95% ethanol, 15 mL dH2O (for 1 h)

4. 55 mL TBA, 45 mL 95% ethanol (for 1 h)

5. 75 mL TBA, 25 mL 95% ethanol (for 1 h)

6. 100 mL TBA (for 1 h)

7. 100 mL TBA (for 1 h)

8. 100 mL TBA (overnight)

The changes and replacement of the TBA solutions from steps 6–8 were done in a standard oven at 40 °C to prevent the TBA from freezing (Ruzin, 1999).

6.2.4 Callus infiltration in paraffin and tertiary butanol alcohol

After the TBA series, calli were placed in embedding cassettes II tube packs (ThermoFisher

Scientific, ShandonTM Ltd., Christchurch, New Zealand) and incubated with 50:50 mix of

100% TBA and liquid paraffin (HistosecR pastilles without dimethyl sulphoxide (DMSO),

Merck, Germany) at 40 ℃ for 24 hours. Further infiltration of calli was then performed using a Tissue Processor (ThermoFisher Scientific ShandonTM Ltd., “Citadel 1000” USA) with molten embedding paraffin at 60 ℃ under vacuum for 72 hours.

142

6.2.5 Callus embedding

After 72 hours in melted embedded paraffin, calli were placed head down singly into disposable base plastic moulds and fitted with an embedding ring (ThermoFisher Scientific FisherbrandTM,

Ltd., Christchurch New Zealand) at the top, then freshly molten paraffin wax (HistosecR pastilles without DMSO, Merck, Germany) was poured into each using the tissue embedding console system (Miles Scientific 4586 Tissue-Tek,). The wax mould was then allowed to harden on the cooling section of the embedding console system, forming formed fine calli- paraffin blocks. The blocks were stored at room temperature and later transferred to a refrigerator at 4 ℃ before being sectioned.

6.2.6 Callus sectioning

Calli-paraffin blocks were removed from the refrigerator and trimmed using a single-edged razor blade. The blocks were fixed onto an automatic rotary microtome (Leica RM2165,

Germany) using the embedded ringside to be held in the machine. Sections 10 µm thick were cut at about 500 µm and 1500 µm into the calli in the form of ribbons. A piece of a ribbon with four sequential sections was placed on a glass slide and fixed on a warm plate for 2–5 minutes.

Then the glass slides were stored in boxes at room temperature for 2–3 days so that the sections were adequately fixed onto the glass slides and were ready to be dipped in staining solutions

(Ruzin, 1999). The transverse sections (TS) from two different regions of petunia and marigold calli cultured for two weeks after diesel treatment and control calli without diesel treatment were analysed. The two regions were from below the surface and middle of the calli, which were estimated to be about 500 µm and 1500 µm, respectively, from the top of the calli.

6.2.7 Callus staining and coverslip

Callus sections on the glass slides were stained in safranin O at 70% ethanol and fast green in

95% ethanol, used in Johannsen’s double staining method (Johansen, 1940) to improve the contrast and better differentiate cell structures. The glass slides were kept in 100% xylene

143 inside a fume cupboard (EcoairTM , Thermoplastic Engineering Ltd., Wellington, New Zealand) with the fan turned on. One slide at a time was removed from the solution. Then 1–2 drops of coverslip sealant were placed in the middle of the slide, which was spread around gently, covered using a coverslip and allowed to dry for 4–5 days at room temperature in a fume cupboard.

6.2.8 Microscopic observations

Three replications of each treatment of the transverse sections of petunia and marigold calli mounted on glass slides were then observed at 40× and 100× magnification and photographed under a light microscope (Olympus BX50, Olympus Optical Co. Ltd., Japan). At 100× magnification, a drop of oil (oil immersion) was added on a slide when observing under the microscope. Selected images were captured using a colour camera XCAM HDMI (Aptina Sony

ToupView, China). All the imaging was analysed using ToupView software version 3.7.

6.3 Results

The histology of the TS the calli at the two regions (500 µm and 1500 µm from the top of the calli) was first viewed under a light microscope at a low magnification (40×) to reveal the overall internal callus structure and the pattern of cellular arrangement (Figures 6.1, 6.3, 6.5, and 6.7 a and b). Closer observations of the selected regions of interest at a higher magnification of 100× confirmed the details and arrangement of the different types of cells (Figures 6.2, 6.4,

6.6, and 6.8 a and b).

6.3.1 Transverse section of Petunia grandiflora (petunia) callus 500 µm from the top

The histological overview of the (TS at 40× magnification) at 500 µm from the top of petunia callus not exposed to diesel (Figure 6.1a) showed the distribution pattern of meristematic cells

(MCs) with a visible nucleus and the presence of xylem vessels with lignin in the cell walls.

Many vacuolated parenchyma cells (PCs) were loosely arranged between the meristematic cells

144

(Figure 6.1a). In the TS of the diesel-treated petunia callus (Figure 6.1b), there was more vacuolated PCs, few meristematic cells (MCs) and xylem vessels.

In the TS (observed under 100× magnification) of petunia callus without prior exposure to diesel, the safranin-stained cell walls of the xylem vessel showed clear contrast with many of the surrounding MCs having dense cytoplasm (Figure 6.2a). The nucleus of the MCs appeared to be large. Although there were not many of them, it was an indication of active cell division

(Figure 6.2a). In comparison, in the diesel-treated petunia callus, the cluster of MZ and vascular tissue were surrounded by relatively large vacuolated PCs (Figure 6.2b). The vacuolated PCs were mostly rounded in shape and composed of thin cell wall or degraded cell wall.

145 a MZ PC MZ

N V PC

MZ V

b MZ PC

MZ V

Figure 6.1 Overall view (40×) of the transverse sections of Petunia grandiflora calli at 500 µm from the top of the callus (a) without diesel treatment and (b) diesel-treated callus. The red arrow points to the xylem vessels surrounded by the region of active cell division and the black arrow points to the lignin wall. Abbreviation: MZ, meristem zone; PCs, parenchyma cells; V, vacuole; N, nucleus. The red- circled sections of the micrograph in (a) and (b) are shown at 100× magnification in Figure 6.2 a and b. Scale bar (a) and (b) = 10 µm.

146 a

V MZ

PC MZ

V N

PC MZ

b V

V V

147

6.3.2 Transverse section of Petunia grandiflora (petunia) callus 1500 µm from the top

An overview of the TS of the petunia calli without prior diesel treatment showed three categories of cellular organisation: large vacuolated parenchyma cells mostly at the peripheral zone of the callus interspersed with large, empty intercellular spaces in the central sections of meristematic cells with some cells progressively differentiated to tracheary elements (TEs)

(Figure 6.3a). In the MZs, the cells have many large visible nuclei (Figure 6.3a). In the diesel- treated TS of the petunia callus, they appeared to have more TEs than the control. The TEs were mostly distributed in the central region, which also had the notable development of the lignified cell walls and seemed to be connected and interspersed with long, empty intercellular spaces (Figure 6.3b). Here, there were more vacuolated PCs of different shapes and sizes in the diesel-treated TS of petunia callus (Figure 6.3b). Large vacuolated PCs formed at the left side of the peripheral region, while moderate to small vacuolated PCs were found mostly toward the right side of the peripheral region with limited lignified cell walls (Figure 6.3b). At higher magnification (100×), the major differences between the callus with or without prior diesel treatment were shown clearly (Figure 6.4). For example, a high number of TEs that were more developed in the diesel-treated callus (Figure 6.4b). In the untreated callus, the nuclei showed clear contrast, indicative of mitotic activities (Figure 6.4a).

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a MZ V V

N PC MZ V

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6.3.3 Transverse section of Tagetes patula (marigold) callus 500 µm from the top

A general overview of the TS of a marigold callus without prior diesel treatment revealed prominently connected xylem vessels formed at the centre, which had a high number of cells with lignified walls stained reddish/purplish (Figure 6.5a). The xylem vessels radiated along the TS in the form of a beam of rays with localised centre points of origin. The MCs with or without nuclei formed a strand along some of the connected xylem vessels (Figure 6.5a).

Moreover, on both sides of the callus TS, there were groups of MZs and vacuolated PCs with thin cell walls.

The MCs were more organised into groups in the TS of the diesel-treated callus (Figure 6.5b) which appeared to have dense cytoplasm. Intercellular spaces were randomly distributed between MZs and PCs across the TS of the callus due to the collapse of some cells (Figure

6.5b).

At a higher magnification, the callus without prior exposure to diesel showed the connections of 3–5 rows of isodiametric cells extended across the entire cellular mass, mostly with red lignified walls (Figure 6.6a). Parenchymatous cells also formed part of the cellular mass, with some cells showing prominent nuclei (Figure 6.6a). In the diesel-treated callus, instead of xylem vessels there more MCs with some visible nuclei (Figure 6.6b).

151 a V MZ

PC N MZ

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152 a V

N V

V V

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6.3.4 Transverse section of Tagetes patula (marigold) callus 1500 µm from the top

The TS (40× magnification) at 1500 µm from the top of the control callus revealed the prominent shoot apical meristem (SAM) dome (MD) with leaf primordia (LP) (Figure 6.7a).

The cells in these regions were small, roughly spherical or polyhedral in shape with dense cytoplasm. Vacuolated PCs with thin cell walls and some having a nucleus appeared to be further away from these MZs and to radiate toward the peripheral edges (Figure 6.7a); whereas in the diesel-treated callus, more, large vacuolated PCs were found randomly distributed across the TS (Figure 6.7b) than in the control callus (Figure 6.7a). More importantly, although there were many long, slender, undifferentiated MCs, they did not appear to be mitotic active at 1500

µm from the top of the diesel-treated callus (Figure 6.7b).

At higher magnification (100×), the structure of the SAM becomes clearer (pointed with a black arrow) with the tunica and corpus layer of cells clearly shown at 1500 µm from the top of the control callus (Figure 6.8a). The meristematic cells with dense cytoplasm were compactly arranged without intercellular spaces (Figure 6.8a). These were absent in the diesel- treated callus (Figure 6.8b), and instead, some cells had differentiated into xylem with the cell wall showing a reaction to lignin stain (Figure 6.8b).

154 a PC N

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6.4 Discussion

Ornamental plants are considered to be a group of important plants due to their excellent diversity of floral cultural values worldwide (Reinten et al., 2011). Petroleum hydrocarbons, including diesel, represent one of the environmental contaminants of major concern around the world, possibly because of the presence of polycyclic aromatic hydrocarbons (PAHs) (Jagtap et al., 2014). PAHs were listed by the Environmental Protection Agency (USEPA, 2008) as priority pollutants because they were identified as or suspected to be potential mutagens/carcinogens (Keith, 2015). Diesel contains high concentrations of PAHs (Chandran and Das, 2011; Wang et al., 1990). There are various reports on the biological effects of diesel associated with metabolic activation and DNA damage that lead to mutation and transformation in both animals and human cell lines (Danielsen et al., 2008; Jantzen et al., 2012; Mitchell et al., 1981; Risom et al., 2007; Tokiwa et al., 1999). Little is known about the mutagenic/carcinogenic properties of diesel with respect to plants, although an attempt has been made to study the genotoxicity of diesel, benzo[a]pyrene (BaP) and naphthene (NAPH) related to DNA damage and ploidy alterations in roots of Acorus tatarinowii (Chen et al., 2013) and to roots and shoots of Trifolium repens (Aina et al., 2006). However, ploidy alterations in the roots and shoots of those plants were not observed. It was found that diesel and PAHs induced DNA sequence changes (Aina et al., 2006; Chen et al., 2013). These results suggest that PAHs were associated with genotoxic properties, but the concentrations used in that experiments were not adequate to cause DNA damage (Aina et al., 2006; Chen et al., 2013).

In a recent study of in vitro toxicity assay of diesel-contaminated water using two ornamental plants Tagetes patula (marigold) and Petunia grandiflora (petunia), marigold seems to show more potential for phytoremediation of diesel than petunia (Wante and Leung, 2018). Petunia has been suggested to be a good phytoremediator of textile dyes (Watharkar and Jadhav, 2014).

Despite the many reported findings over many years on diesel toxicity to plants, the tolerance

157 to diesel toxicity at the cellular level has not been reported until now. Visual observation of the diesel-exposed calli of petunia and marigold immediately after 9 minutes of treatment did not reveal signs of necrosis, but after two weeks of culture, the observed histological structures of the cells presented changes in the development of diesel-treated and non-diesel-treated calli.

For example, the petunia calli without prior exposure to diesel showed more regions with MCs than the diesel-treated calli at 500 µm from the top. Šamaj et al. (1990) reported that large clusters of MCs were observed in the first two surface layers of the organogenic calli of

Papaver somniferum compared with their older calli. This was not the case in marigold calli at the 500 µm layer; the diesel-treated calli had more groups of organised MCs than the calli without prior exposure to diesel. The MCs are undifferentiated cells with the potential to proliferate by cell division and specialise into different cell types in plants, although responses from environmental signals can influence changes in the mitotic activity (Colón‐Carmona et al., 1999; Doerner et al., 1996). It has been established that cells proliferated through the mitotic cell cycles consisting of four different phases, Gap 1 phase (G1 phase), DNA synthesis phase (S phase), Gap 2 phase (G2 phase) and mitotic phase (M phase) (Komaki and Sugimoto,

2012). During cell division, cyclin-dependent kinases (CDKs) and cyclins (CYCs) have been identified to be the primary regulators and potential reporters of mitotic activity (Colón‐

Carmona et al., 1999). CDKs are a class of heterodimeric serine/threonine protein kinases

(Mironov et al., 1999). In plants, the progression of mitotic cell cycle is determined by the constant activation of CDK with a combination of different CYCs to activate the transition from the G1 to S shape and the G2 to M phase (Komaki and Sugimoto, 2012). For example, in

Arabidopsis, CDKA;1 firstly binds to CYCDs and CYCA3 to move from the G1 to S transition and shape progression, respectively While CDKA;1 creates a complex with CYCD3 to facilitate M phase progression (Boruc et al., 2010; Dewitte et al., 2007; Schnittger et al., 2002;

Van Leene et al., 2010). In the case of the diesel-treated marigold calli, the cells may have

158 shown the state of increasing participation in transcription because of the many large nuclei in the MZs (Hancock, 2004; Miyoshi and Sugimoto, 2008). Here in this study, one can only presume that diesel might have induced the activity of cell division because the proliferated cells might have been modulated in response to stress by the plant growth hormone gibberellin

(GA) (Razem et al., 2006). Although the combinatorial interaction of the CYCs and CDKs complexes were regulated by the presence of distinct substrates which could be induced through stress and activate different cell cycle (Kitsios and Doonan, 2011). However, when plant cell stops multiplying and starts to differentiate, they might have triggered the endoreduplication cycle or endocycle in which cells repeat DNA replication without involving mitosis (Komaki and Sugimoto, 2012). For example, in shoot and root apex of Arabidopsis plants, salt stress had induced a decrease in the transcript levels of CDKA/CDKB and

CYCA/CYCB which result in transient downregulation of mitotic activity, which was characterised by fewer MCs and limited growth (Munns and Rawson, 1999; Sun et al., 2004).

Here in this study, it is reasonable to hypothesise that petunia diesel-treated calli at the 500 µm layer may have entered the endoreduplication cycle or endocycle which have resulted in the formation of vascular tissue differentiation and large vacuolated PCs.

However, the significant histological differences at these layers of the calli in this study were marked by the presence or absence of different cell content and the pattern of structural arrangement in the cells. For example, petunia calli without prior exposure to diesel had large empty-looking PCs loosely arranged between the MZs. In contrast, the diesel-treated calli had many large empty-looking differentiated vascular tissues. A closer look at petunia and marigold calli without prior diesel exposure revealed that lignified cell walls were much more abundant than in the diesel-treated calli. Lignin is a polymer of phenylpropanoid compounds obtained from the lignin monomers p-coumaryl alcohol (forming H-units), coniferyl alcohol (forming

G-units), and sinapyl alcohol (forming S-units) (Gavnholt and Larsen, 2002; Moura et al.,

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2010). Biosynthesis of the polymers is formed through the stepwise combination of the three monomers followed by polymerisation (Liu et al., 2015). Barros et al. (2015) reported that cell- wall lignification is formed during the differentiation of different cell types, but is also associated with cell response to specific environmental changes. Petunia and marigold calli without prior exposure to diesel might be in a state of cell differentiation rather than under stress, because, theoretically, calli exposed to diesel toxicity should be under much more stress than calli without diesel exposure. Plants with increased tolerance to metal toxicity such as aluminium (Al) have shown a reduction in cell-wall lignin production (Lima et al., 2009). For example, Camellia sinensis grown on 500 µM AlCl3 which was considered relatively toxic showed a decrease in the activities of phenylalanine ammonia-lyase (PAL) and peroxidase

(POD) bound to the cell wall as well as a decrease in lignin content (Ghanati et al., 2005). This is contrary to other studies where cadmium (Cd) toxicity increased lignin content in soybean

(Finger-Teixeira et al., 2010; Yang et al., 2007), Matricaria chamomilla (Kováčik et al., 2008) and two varieties of Vicia sativa roots (Rui et al., 2016). It was also accompanied by increased activities of antioxidant enzymes such as POD, PAL and laccases (p-diphenol:O2 oxidoreductases) (Finger-Teixeira et al., 2010; Kováčik et al., 2008; Rui et al., 2016; Yang et al., 2007). Here in this study, one can argue that petunia and marigold calli without prior diesel exposure at the 500 µm layers were likely to have an increased activity of POD, PAL and laccases because of the high number of cells with lignified cell walls. H2O2-dependent PODs and H2O2-dependent laccases have catalysed the formation of polymers of lignin precursors

(Gavnholt and Larsen, 2002; Marjamaa et al., 2009). H2O2 is used as an oxidant to produce monolignol phenoxy radicals, which combine spontaneously to form lignin polymers (Rui et al., 2016). In the phenylpropanoid pathway, PAL is considered to be the first enzyme that catalyses the oxidative deamination of phenylalanine to trans-cinnamic acid and ammonia

(NH3) (Olsen et al., 2008). In the study on the response of two varieties of Vicia sativa to Cd

160 toxicity, it has been suggested that the induction of POD activity may be a later response (Rui et al., 2016). It was presumed that PODs had played a role in the formation of H2O2 by catalysing the linkages between some types of phenolic acid and cell-wall polymers while the laccase-like oxidase may have participated in the early stage of lignin biosynthesis during the

Cd treatment (Sterjiades et al., 1992; Yang et al., 2007). However, here in this study, the hypothesised mechanism that might have been involved in the lignin biosynthesis cannot be linked with diesel toxicity because petunia and marigold calli without prior diesel exposure at the 500 µm layer produced a high number of cells with lignified cell walls than the diesel treated calli.

In another study, Al toxicity has been reported to be associated with histological evidence of increased lignin cell-wall formation and the expression of genes coding for enzymes that catalyse the various steps of phenylpropanoid pathways, which produce precursors to secondary metabolites (Huang et al., 2010; Moura et al., 2010). For instance, both Al-tolerant and -sensitive Oryza sativa plants have been observed to up-regulate the genes such as 4- coumarate: CoA ligases isoform 2 (OsAR4), phenylalanine ammonia-lyase (OsAR5), putative cinnamyl-alcohol dehydrogenase (OsAR6) and p-coumarate 3-hydroxylase (OsAR7) that code for enzymes of the lignin biosynthesis pathways (Humphreys and Chapple, 2002; Mao et al.,

2004). Moreover, the Al-induced up-regulated genes were detected in the Al-sensitive Oryza sativa plants with a higher level of accumulated transcripts of PAL earlier than in the tolerant varieties (Mao et al., 2004). Sasaki et al. (1996) explained that the level of growth inhibition is closely associated with the level of lignin decomposition in both Al-tolerant and Al-sensitive varieties. In this study, cell differentiation in petunia and marigold calli without prior diesel exposure at the 500 µm layer may have led to cell growth inhibition and generated lignin in the cell wall.

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In a similar study, using Nicotiana tabacum callus under salinity stress, the callus cells showed structural changes such as damage to the nucleus and cytoplasmic shrinkage (Bennici and Tani,

2012). In this study, diesel toxicity could be linked to the presence of large empty cellular spaces in petunia calli at 500 µm. In contrast, the diesel-treated calli of marigold appeared to have more PCs with visible nuclei. This suggests that there were regions on the calli that were not severely affected by the diesel toxicity at that layer. A similar finding was reported in N. tabacum callus under a high dose of salinity stress, where the callus cells appeared normal in their structure (Bennici and Tani, 2012). One can argue that for the callus cultured on a medium with a high dose of salinity, it is possible that salt has diffused through the mass of the large callus, or only in some regions that were in contact with the medium (Andrea and Tani, 2009).

However, in this study, calli were submerged in undiluted diesel before they were subcultured.

Therefore, hypothetically, the cells at that region of 500 µm from the top of the callus were relatively closer to the diesel fuel, being very close to the outer surface of the callus.

In this study, the cells at 1500 µm from the top of the callus showed histological differences between the diesel-treated and non-treated calli. For example, petunia calli without prior exposure to diesel had large vacuolated PCs mostly at the peripheral zone of the callus, large empty intercellular spaces and a central area of MCs with some progressively differentiated to

TEs. In the diesel-treated callus, TEs appeared at the central region and more developed with notable lignified cell walls. Groover et al. (1997) reported that TEs are produced in plant cells by the synthesis of the secondary cell wall through the clearing of the entire cellular contents.

Cell differentiation into TEs is a characteristic of developmentally programmed cell death

(PCD) in plants (Pennell and Lamb, 1997). PCD is an active process that is different from necrosis and occurs in specific cells during the development of various tissues (Groover et al.,

1997). However, TEs have been induced in the many different studies using callus culture. For example, it has been found that light increased the formation of TEs in the callus culture of

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Pinus radiata (Möller et al., 2006). In the present study, diesel fuel may have influenced the formation of TEs in petunia calli at 1500 µm. In contrast, at 1500 µm from the top of the diesel- treated marigold calli, there were large randomly arranged vacuolated cells and few cells differentiated into xylem with their cell wall showing a reaction to lignin stain.

Salinity stress caused varied structural anatomical changes in plant cell including inhibition of cell differentiation and earlier occurrence of lignification (Robinson et al., 1997). In this study, the results confirmed these observations: at 1500 µm from the top of the marigold calli without prior exposure to diesel fuel, and the callus showed formation of dome-like regions of undifferentiated meristematic cells. This type of structure is comparable with the dome-like regions found in the shoot apical meristem (SAM) of plants. For example, in a monocotyledonous Elaeis guineensis juvenile plant (3-month-old plant) without inflorescence initiation, the development of SAM is characterised by the formation of a meristematic dome with tunica and corpus layers of cells (Jouannic et al., 2011). Here in this study, a closer view of the cells confirmed a meristematic dome and leaf primordia; MCs were small, roughly spherical or polyhedral in shape with dense cytoplasm. These are all characteristics of actively dividing cells. However, the activity of the cells is controlled by the functioning of two processes: maintaining cell division and formation of lateral organs (Jouannic et al., 2011). The present result is consistent with the formation of SAM at 1500 µm from the top of the marigold callus.

6.5 Conclusion

Petunia and marigold calli at 500 µm and 1500 µm from the top of the calli differ significantly in their responses to diesel toxicity. To my understanding, there have been no reported findings until now regarding cellular responses of plants to diesel toxicity. This novel finding, however, was obtained in the present study of the histological changes that occurred in callus cultures after diesel exposure. Interestingly, evidence for the expected total cellular collapse in the

163 diesel-treated calli was not found. Instead, there was a difference in their developmental changes compared with calli not exposed to diesel. For example, at the 500 µm from the top of the petunia calli without prior exposure to diesel, the safranin-stained cell walls of the xylem vessel showed a clear contrast with many of the surrounding undifferentiated cells. In the diesel-treated callus, the cluster of meristematic and vascular tissue was surrounded by relatively large PCs. Nevertheless, the results in this study further confirmed that the innovative protocol of diesel exposure was sub-lethal, a condition required to generate somaclonal variants

(Ashrafzadeh and Leung, 2015; Rai et al., 2011). This protocol should therefore enable the extension of the application of in vitro plant cell line selection to generate plants from diesel- treated calli of Petunia grandiflora or Tagetes patula. Further research could show a new way forward to yield novel diesel-tolerant plants.

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Chapter 7 Growth performance of Petunia grandiflora Juss. mix F1 plant lines developed from calli after diesel exposure: Evaluation under in vitro and glasshouse conditions

7.1 Introduction

Petroleum hydrocarbons are among the common environmental contaminants worldwide

(Palmroth et al., 2002). Diesel fuel consists of a complex mixture of petroleum hydrocarbons with a high content of light hydrocarbons (Jagtap et al., 2014). The light hydrocarbons found in diesel have made them more toxic to plants than other petroleum products (Jagtap et al.,

2014). Polycyclic aromatic hydrocarbons (PAHs) are also present in diesel and have been recognised as posing a serious threat to public and ecosystem health (Jagtap et al., 2014). Some plants have been shown to withstand the effects of PAH contamination in the environment

(Palmroth et al., 2002). Phytoremediation is a green technology that could help to mitigate the undesirable effects of diverse environmental pollutants using plants (Pittarello et al., 2017).

Several research studies under field and glasshouse conditions have reported the use of different systematic approaches to select suitable plant species for phytoremediation (Campbell et al., 2002; Cunningham et al., 1997; Sung et al., 2002; Wiltse et al., 1998). To my understanding, there is no published research report on selecting plants regenerated in vitro from calli that have been treated with diesel. There are many studies on plant screening for phytoremediation of petroleum hydrocarbons, but there are only a few reports on the use of ornamental plants. These include the single and joint effect of heavy metals and benzo[a]pyrene on the growth of Tagetes patula (Sun et al., 2011), evaluation of remediation capability of

Mirabilis jalapa to treat petroleum-contaminated soil (Peng et al., 2009), and the assessment of the removal rate of eight PAHs using five ornamental species (Xiao et al., 2015). The use of ornamental plants would be preferable to food plants for phytoremediation purposes as this

165 would minimise the chance of pollutants entering the food chain (Wante and Leung, 2018).

Ornamental plants also have the potential to add their aesthetic value to the polluted landscape

(Liu et al., 2017). The selection of many plants has been based on their performance in the growth rooms under control conditions (Phillips et al., 2006). There is a recognised need for further field studies to verify the findings under a controlled environment (Frick et al., 1999;

Pilon-Smits, 2005), particularly for plants generated by in vitro tissue culture. The objective of this chapter was to assess the growth performance of the plants regenerated from callus culture of Petunia grandiflora that had been exposed to diesel. Evaluation of the plants was carried out with the plants grown under aseptic culture conditions without diesel contamination and also in potting mix spiked with diesel under glasshouse conditions.

7.2 Materials and methods

7.2.1 Plant material and growth conditions

The apical shoot tips of Petunia grandiflora (petunia) used for the evaluation of in vitro growth performance were from a line of plantlets from the seed germinated in vitro (C-G) and lines of plants regenerated from callus cultures that were with (L1–L6) or without exposure (C-R) to 9 minutes of undiluted diesel, see section 2.4.5.

7.2.1.1 Morphological parameters

At the end of the experiment (4 weeks), plantlets were carefully removed from the culture jars, and any agar attached to the roots was washed away using running tap water. The plantlet growth was determined, as described in section 2.4.6, and their mean height was expressed in centimetre (cm). The morphological features of the plantlet lines and their controls C-G and C-

R were examined using the morphological visual inspection (MVI) approach.

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7.2.2 Hardening off and acclimatisation of Petunia grandiflora plantlets

Six- to eight-week-old plantlet lines were handled for the hardening process, as described in section 2.5.1 and were then transferred for acclimatisation under glasshouse conditions (see section 2.5.2).

7.2.3 Glasshouse study design

7.2.3.1 Tested plant materials

Shoot cuttings of similar size were excised from the eight-week-old micropropagated petunia plantlets of the different lines kept under glasshouse conditions (described in section 2.6.1); then the cuttings were evaluated for their response to the toxicity of diesel contamination under glasshouse conditions.

7.2.3.2 Site description

A glasshouse of the University of Canterbury was used for this experiment, as described in section 2.6.2.

7.2.3.3 Potting mix and diesel-contaminated potting mix

The medium used for the evaluation of the plantlets is a potting mix containing slow-release fertiliser, and the diesel fuel was supplied by the glasshouse unit, the School of Biological

Sciences, University of Canterbury. The analyses of the potting mix, diesel fuel, mixing of the potting mix with diesel and the experimental setting are described in sections 2.6.3 and 2.6.4.

7.2.4 Leaf growth

At the end of the experiment (5 weeks) under the glasshouse conditions, leaves of the shoot cuttings were examined, as described in section 2.7.1.

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7.2.5 Shoot growth

At the beginning and after 5 weeks of the experiment, the diameter and shoot height were measured, as described in section 2.7.2. The respective means of the shoot cutting diameter in millimetre (mm) and height in centimetre (cm) were determined.

7.2.6 Biomass production

The average fresh and dried biomass (in g) of the shoot cuttings after 5 weeks of the experiment under glasshouse conditions were determined, as described in section 2.7.3.

7.2.7 Microbial plate counts

The total number of culturable microbial populations in the potting mix with or without diesel used under glasshouse conditions after 5 weeks of the experiment were determined and analysed as explained in section 2.8. This is expressed as the number of colony forming units per gram of dry potting mix (CFUg-1).

7.2.8 Analysis of the diesel-contaminated potting mix used

7.2.8.1 Ultrasonic extraction (Method 3550C by USEPA (2007))

Three replicates of the potting mix in pots were sampled randomly with or without diesel contamination at day 0 and after 5 weeks of the experiment, as described in section 2.9.1. The extracts were stored at 4 ℃ until gas chromatography (GC) using scan flame ionisation detector

(FID) analysis.

7.2.8.2 Total petroleum hydrocarbon (TPH) contents

The three replicates of extracts described in section 7.2.8.1 were analysed in a GC FID, as described in sections 2.9.1 and 2.9.2. Total petroleum hydrocarbons TPHs were analysed using the hydrocarbon standard made by the Department of Chemistry, University of Canterbury,

New Zealand. The TPHs in diesel were determined and expressed as a percentage of the residual.

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7.3 Statistical analysis

All experiments were conducted using a complete randomised block design with at least three replications in each treatment. Where data transformation was required, they were transformed before statistical analysis. GenStat for Windows 19th Edition software was used to analyse the data using two-way analysis of variance (ANOVA) for a randomised block design. The multiple comparisons using Fisher’s unprotected least significant differences (LSD (5%)) was determined and presented as a measure of variability. All graphs were plotted using the

GraphPad Prism software Version 7.0.

7.4 Results

7.4.1 Morphological characteristics

The morphological parameters such as number of leaves, shoot height and root length of petunia plantlets were measured for 6 different experimental lines and the controls grown in vitro for 4 weeks. The plantlets of C-G and line L4 had formed a greater number of leaves

(about 16 per plantlet) than the control C-R and other lines (Figure 7.1). The shoots of lines

L1, L4 and C-G, were taller than the other lines at about 4 cm each (Figure 7.2). Similarly, the roots of lines L1, L4 and C-G were longer than the other lines (Figure 7.3). The fresh and dry weight of the whole plantlets of line L4 were higher than the other lines (Figures 7.4 and 7.5).

Most of the leaves in the petunia plantlets of the controls and the experimental lines (L1–L4) were green except the top leaves had slight chlorosis (Figures 7.6–7.13). But some dead- looking leaves were found in line L6 (Figure 7.13), and plantlets in line L6 (Figure 7.13) were largely chlorotic with some plants displaying a hyperhydrated appearance. The size of the leaves in C-R was mostly smaller than in C-G and line L4 but appeared similar to the other lines (Figures 7.6–7.13). Line L5 had a type of curly leaf (Figure 7.12). Some shoots appeared to have formed a callus at the base (Figure 7.12).

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Figure 7.6 Appearance of Petunia grandiflora plantlets from seeds germinated in vitro (C-G) on plant- growth-regulator-free half-strength MS medium. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). The photo was taken after 4 weeks from culturing 1-cm shoot cuttings.

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Figure 7.7 Appearance of Petunia grandiflora plantlets after 4 weeks of culture on plant-growth- regulator-free half-strength MS medium. The plantlets were regenerated from callus that was not treated with diesel before (C-R plantlets). Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). The photo was taken after 4 weeks from culturing 1-cm shoot cuttings.

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Figure 7.8 Appearance of Petunia grandiflora plantlets after 4 weeks of culture on plant-growth- regulator-free half-strength MS medium. The plantlets of line L1 were regenerated from diesel-treated callus culture. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). The photo was taken after 4 weeks from culturing 1-cm shoot cuttings.

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Figure 7.9 Appearance of Petunia grandiflora plantlets after 4 weeks of culture on plant-growth- regulator-free half-strength MS medium. The plantlets of line L2 were regenerated from diesel-treated callus culture. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). The black arrow on the left side is used to show the shoot with dead leaves, while the arrow on the right side is used to show the tiny chlorotic-looking leaves. The photo was taken after 4 weeks from culturing 1-cm shoot cuttings.

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Figure 7.10 Appearance of Petunia grandiflora plantlets after 4 weeks of culture on plant-growth- regulator-free half-strength MS medium. The plantlets of line L3 were regenerated from diesel-treated callus culture. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). The photo was taken after 4 weeks from culturing 1-cm shoot cuttings.

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Figure 7.11 Appearance of Petunia grandiflora plantlets after 4 weeks of culture on plant-growth- regulator-free half-strength MS medium. The plantlets of line L4 were regenerated from diesel-treated callus culture. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). The photo was taken after 4 weeks from culturing 1-cm shoot cuttings.

178

Figure 7.12 Appearance of Petunia grandiflora plantlets after 4 weeks of culture on plant-growth- regulator-free half-strength MS medium. The plantlets of line L5 were regenerated from diesel-treated callus culture. The black arrow is used to show the white friable callus formed at the base of a shoot. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). The photo was taken after 4 weeks from culturing 1-cm shoot cuttings.

179

Figure 7.13 Appearance of Petunia grandiflora plantlets after 4 weeks of culture on plant-growth- regulator-free half-strength MS medium. The plantlets of line L6 were regenerated from diesel-treated callus culture. The black arrow points to plantlet with hyperhydrated leaves. Culture was kept in a growth room at 21 ± 1℃ with continuous lighting (cool white fluorescent light, Sylvania Gro-Lux lamps 36W). The photo was taken after 4 weeks from culturing 1-cm shoot cuttings.

180

181

7.4.2. Growth of experimental and control Petunia grandiflora lines under glasshouse conditions

The acclimatisation process employed on the petunia lines generated in vitro was successful to establish L1–L4, and controls (C-G and C-R) under the glasshouse conditions (Figure 7.14).

Line L1

Results from the glasshouse experiments show how petunia plantlet growth was affected by

2% and 7% diesel-contaminated potting mix. There was a significant increase in stem diameter of line L1 over those of C-R and C-G grown in the non-diesel- and diesel-contaminated potting mix after 5 weeks (Figure 7.15). The number of leaves formed decreased in L1, C-R and C-G as the percentage of diesel was increased in the contaminated potting mix, but C-G exhibited the worst performance in this regard (Figure 7.16). Also, in the presence of 2% diesel, C-R formed the greatest number of leaves (Figure 7.16). Leaf chlorosis was relatively severe in L1,

C-G, and C-R in the presence of 7% diesel than for 0% and 2% diesel (Figures 7.17 and 7.20).

There was no significant difference in the shoot height of L1, C-G and C-R grown in the potting mix spiked with 7% diesel compared to those in the potting mix without diesel. Among all the treatments, L1 exhibited the highest shoot height of about 9 cm (Figure 7.18). The fresh and dry masses of L1, C-G, and C-R declined with the increase in the percentage of diesel added to the potting mix. However, L1 exhibited the highest fresh and dry masses compared with C-

G and C-R grown in the potting mix with 2% and 7% diesel (Figure 7.19). During this experiment, the range of daily variations in temperatures was 18–32 °C, and the light intensity was 2000–4000 lux in the glasshouse (Figure 7.21).

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Line L2

There was no difference in the stem diameter of L2, C-R and C-G grown in potting mix without diesel (Figure 7.22). L2 exhibited greater stem diameter than C-R and C-G grown in the presence of 2% and 7% diesel (Figure 7.22). L2 formed the highest number of leaves compared with C-G and C-R grown in the potting mix pots with 0%–7% diesel (Figure 7.23). Leaf chlorosis was most severe in shoot cuttings grown in the potting mix with 7% diesel (Figures

7.24 and 7,27). L2 tends to be slightly chlorotic even at 0% diesel compared with C-G and C-R

(Figures 7.24 and 7.27). There was a slight difference in the shoot height of L2, C-G and C-R grown in potting mix spiked with 2% and 7% diesel compared with the control. L2 exhibited the highest shoot height of about 9 cm when grown in the absence of diesel (Figure 7.25). The fresh and dry masses of L2, C-G, and C-R were decreased with increasing percentage of diesel in the potting mix. Overall, L2 exhibited the highest fresh and dry masses of plantlet material when grown in the presence of 2% diesel (Figure 7.26). In this experiment, the range of daily variations in temperatures was 18–35 °C, and the light intensity was 1500–5800 lux in the glasshouse (Figure 7.28).

Line L3

The stem diameter of L3, C-R, and C-G decreased slightly with increasing with level of diesel in the potting mix (Figure 7.29). C-R formed the highest number of leaves compared with L3 and C-G on diesel-contaminated potting mix, while leaf formation in L3 seemed to be lower than for C-G in the presence of an increasing level of diesel (Figure 7.30). The highest chlorosis scores were found in the leaves of C-G, C-R and L3 grown in the presence of 7% diesel (Figures

7.31 and 7.34). The leaves of C-R were more chlorotic (slightly green) than for line L3 and

C-G grown in potting mix spiked with 2% diesel (Figures 7.31 and 7.34). There was a small decrease in the shoot height of L3, C-G and C-R grown in the potting mix spiked with 2% and

7% diesel compared with the 0% pot (Figure 7.32).

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L3 exhibited the highest shoot height of about 9 cm when grown in the absence of diesel (Figure

7.32). The fresh and dry masses of L3, C-G, and C-R decreased with increasing percentage of diesel in the potting mix pots, although, C-G exhibited the highest dry mass grown in potting mix (Figure 7.33). During this experiment, the range of daily variations in temperatures was

14–31 °C, and the light intensity was 1200-7500 lux in the glasshouse (Figure 7.35).

Line L4

Only the stem diameter of L4 was not affected by 2% and 7% diesel (Figure 7.36). In terms of the number of leaves formed, L4 was the least sensitive to the levels of diesel contamination trialled (Figures 7.37 and 7.43). Leaf chlorosis was higher in C-G, C-R and L4 grown in potting mix spiked with 7% diesel than with the lower level of diesel (Figures 7.38 and 7.43). Roots were only formed here in the shoot cuttings of L4 grown in potting mix with or without 2% diesel (Figures 7.39 and 7.43). The number of roots formed in L4 seemed to be affected by the presence of increasing levels of diesel (Figure 7.40). There was a slight but significant difference between the shoot heights of L4, and the controls (C-G and C-R) grown in potting mix with no added diesel and 2% diesel (Figure 7.41). The fresh and dry masses of L4 were the least affected by the increasing level of diesel added to the potting mix (Figure 7.42). During this experiment, the range of daily variations in temperatures was 20–35 °C, and the light intensity was 1800-9500 lux in the glasshouse (Figure 7.44).

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) 6

m d

m c C -G ( b c b c b c r b C -R e b

t 4

e a

a L 1

i d

t S

0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

2 5 d

s d C -G

e v

a c

2 0 c C -R

f L 1

o 1 5

b r

e b b

b 1 0

m u

N 5 a a

0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

185

6 g C -G

n b i

t b b

a C -R

s i

s 4 L 1

f h

o a c a a a

l 2 a a

i V

0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

1 0 ) c d b c d C -G b b m a a a

c 8 a C -R

(

h L 1

g 6

t 4

o h

S 2

0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

186

3 a C -G

) C -R

g (

s 2 L 1 s

a b c

h s d d d

e 1 r

F e e

0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

(a)

0 .4 e e C -G

C -R

) 0 .3 d

g (

d L 1

s s

a c b c

0 .2 c

y b r a D 0 .1

0 .0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

(b)

187

a e i

b f j

c g k

d h l Figure 7.20 Morphological characteristics of Petunia grandiflora shoot cuttings of L1 (a–d) and controls C-R (e–h) and C-G (i–l). (a–d) From top to bottom of line L1: (a) a shoot cutting of L1 before sowing, (b) shoot cuttings of L1 grown in 0%, (c) 2% and (d) 7% diesel-contaminated potting mixture for 5 weeks under glasshouse conditions. (e–h) From top to bottom of control C-R: (e) a shoot cutting before sowing, (f) shoot cuttings of C-R grown under 0%, (g) 2% and (h) 7% diesel-contaminated potting mixture. (i–l) From top to bottom of control C-G: (i) a shoot cutting of C-G before sowing, (j) shoot cuttings of C-G grown under 0%, (k) 2% and (l) 7% diesel-contaminated potting mixture.

188

189

5 ) C -G m c d c d d d b c b b c

m 4 C -R (

a r

e a L 2 t

e 3

a i

d 2

m e

t 1 S

0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

4 0

s C -G e

v e

a C -R

e 3 0 l

d d

f L 2

r 2 0 c c

e c b

m ab b u 1 0

N a

0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

190

5 e

g C -G n

i d e

t 4 d C -R

i L 2 s

f 3 c

r e

l b c b c

f h o ab ab

c 2

a a

u s

i 1 V

0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

) 1 0 e d c d b c c d C -G

m c d

c ab ab a (

8 C -R t

h L 2 g

i 6

t 4

o h

S 2

0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

191

3 a C -G

C -R )

g b ( 2 b c L 2

s c

a m d h d s d d

e 1

r e F

0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

(a)

0 .4 f C -G

e C -R

) 0 .3

g d (

L 2

s d s

a c 0 .2 b c b c

m b

r a D 0 .1

0 .0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

(b)

192

a e i

b f j

c g k

d h l

194

) C -G f m e f

m 4 d e c d c d d e C -R (

b c r a ab

e L 3

t 3

m a

i 2

m e

t 1 S

0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

Figure 7.29 Average stem diameters of Petunia grandiflora shoot cuttings of L3, C-G and C-R after 5 weeks of growth under glasshouse conditions. The average stem diameters were 3.3, 3.3 and 3.4 mm, respectively. Values represent means ± SEM of three replicates in each of the treatment L3, controls C- G and C-R Different letters indicate means that are significantly different from each other (LSD test, p < 0.05).

d 2 5 d C -G s c

e c

v 2 0 C -R

e l

L 3

f 1 5

e b b

b 1 0 m

u a a a

N 5

0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

195

g C -G

n e f i

t e f

a 4 d e C -R

i c d L 3

f o

a 3

l o

b c b c

h o

c 2 ab

a a a

u s

i 1 V

0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

1 0 b c c d ) b C -G ab ab m ab a

c 8 C -R

(

h L 3

g 6

t 4

o h

S 2

0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

196

2 .5 a C -G

2 .0 C -R

)

g (

L 3 s

s 1 .5 b

a b

h c 1 .0 c d s c d d

e d d

r F 0 .5

0 .0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

(a)

0 .2 5 f e C -G 0 .2 0 d d d C -R

) c d g

( L 3 b c

s 0 .1 5 s

a b m

y 0 .1 0

r D 0 .0 5

0 .0 0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

(b)

197

a e i

b f j

c g k

d h l Figure 7.34 Morphological characteristics of Petunia grandiflora shoot cuttings of L3 (a–d) and controls C-R (e–h) and C-G (i–l). (a–d) From top to bottom of line L3: (a) a shoot cutting of L3 before sowing, (b) shoot cuttings of L3 grown in 0%, (c) 2% and (d) 7% diesel-contaminated potting mixture for 5 weeks under glasshouse conditions. (e–h) From top to bottom of control C-R: (e) a shoot cutting before sowing, (f) shoot cuttings of C-R grown under 0%, (g) 2% and (h) 7% diesel-contaminated potting mixture. (i–l) From top to bottom of control C-G: (i) a shoot cutting of C-G before sowing, (j) shoot cuttings of C-G grown under 0%, (k) 2% and (l) 7% diesel-contaminated potting mixture.

198

199

) 5

m C -G e e

m e d e (

4 c d C -R

r c e b t b L 4

e 3 a

a i

d 2

m e

t 1 S

0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

5 0

g C -G

s e

v 4 0 C -R

e l

f L 4

f 3 0

e d d

b c d

2 0 c m

u b

N 1 0 a a

0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

Figure 7.37 The number of Petunia grandiflora leaves formed in L4, C-G and C-R after 5 weeks of growth under glasshouse conditions. On average there were two leaves at the beginning of the experiment. Values represent means ± SEM of three replicates in each of the treatment L4, controls C- G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05).

200

g C -G

n b

i b t

a 4 b C -R

i L 4

f o

a 3

l f h a o a a a a a

c 2

u s

i 1 V

0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

5 C -G c

) 4 C -R

m b

c (

L 4

h 3

e l

o o

R 1 a a a a a a a 0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

201

2 0

C -G s t c C -R

o 1 5

o r

L 4

r b

1 0

b m

u 5 N a a a a b b b 0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

Figure 7.40 Number of roots number of Petunia grandiflora shoot cuttings of L4, C-G and C-R after 5 weeks of growth under glasshouse conditions. Values represent means ± SEM of three replicates in each of the treatment L4, controls C-G and C-R. Different letters indicate means that are significantly different from each other (LSD test, p < 0.05).

1 0 d c d b c a b c C -G ) a a ab a a

m 8 C -R

(

t L 4

h 6

t 4

o h

S 2

0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

202

4 a C -G

C -R

) 3 b

g (

L 4

s a

2 c

h d s

e d e r

F 1 fg e f g fg

0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

(a)

0 .4 C -G f C -R

) 0 .3 e g e ( L 4

s s

a 0 .2 c d d

y b c b r

D a 0 .1

0 .0 0 2 7

% o f d iesel in p ottin g m ix (w /w )

(b)

203

a e i

b f j

c g k

d h l

Figure 7.43 Morphological characteristics of Petunia grandiflora shoot cuttings of L4 (a–d) and controls C-R (e–h) and C-G (i–l). (a–d) From top to bottom of line L4: (a) a shoot cutting of L4 before sowing, (b) shoot cuttings of L4 grown in 0%, (c) 2% and (d) 7% diesel-contaminated potting mixture for 5 weeks under glasshouse conditions. (e–h) From top to bottom of control C-R: (e) a shoot cutting before sowing, (f) shoot cuttings of C-R grown under 0%, (g) 2% and (h) 7% diesel-contaminated potting mixture. (i–l) From top to bottom of control C-G: (i) a shoot cutting of C-G before sowing, (j) shoot cuttings of C-G grown under 0%, (k) 2% and (l) 7% diesel-contaminated potting mixture. 204

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7.4.3. Total microbial plates count

The number of the culturable microbial populations showed a very slight difference after 5 weeks of pot trial with the four experimental petunia lines and two controls. In the potting mix with 0% and 2% diesel that were planted with L4, there was a significantly greater number of culturable microbes than in the potting mix without any plants (Table 7.1). The number of total cultural microbial populations in the 2% diesel-contaminated potting mix was slightly raised when the C-R or L3 shoot cuttings were planted compared to that without any plants (Table

7.1). In the potting mix spiked with 7% diesel, the number of microbial counts was higher in the presence of the shoot cuttings of L1 and L3 than in their absence (Table 7.1).

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Table 7.1 Number of culturable microbes in non-diesel- and diesel-contaminated potting mix with or without experimental plant materials.

% (w/w) of Total counts (105 cfu/g) Total counts (105 cfu/g) Total counts (105 cfu/g) Total counts (105 cfu/g) diesel contaminant 5 weeks 5 weeks 5 weeks 5 weeks in potting Plant Plant Plant Plant mix lines lines lines lines Planted Unplanted Planted Unplanted Planted Unplanted Planted Unplanted

0 C-G 62 a 57 a C-G 49 a 49 a C-G 66 a 53 a C-G 68 a 50 a

C-R 56 a 51 a C-R 74 a 59 a C-R 66 a 53 a C-R 51 a 51 a

L1 66 a 55 a L2 58 a 54 a L3 67 a 51 a L4 96 b 60 a 2 C-G 93 a 84 a C-G 99 b 94 b C-G 102 a 80 a C-G 85 a 75 a C-R 100 a 78 a C-R 101 b 87 b C-R 107 a 95 b C-R 70 a 67 a L1 88 a 82 a L2 69 a 65 a L3 99 a 87 ab L4 120 b 83 a 7 C-G 146 a 143 b C-G 138 ab 116 a C-G 113 a 123 a C-G 144 a 137 a C-R 130 a 123 a C-R 158 b 141 b C-R 149 ab 143 a C-R 124 a 117 a L1 173 b 129 a L2 115 a 111 a L3 154 b 139 a L4 115 a 137 a

Means of three replicates in a column and row followed by the same letters are not statistically different according to LSD test, p < 0.05. Note: C-G are control shoot cuttings from seed germinated in vitro; C-R are control shoot cuttings from plantlets regenerated from non-diesel-exposed callus cultures and lines of plantlets; L1–L4 are regenerated from diesel-exposed callus cultures.

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7.4.4. Total petroleum hydrocarbon analyses

The residual percentage of the total petroleum hydrocarbons (TPHs) in both 2% and 7% diesel- contaminated potting mix at the start of the glasshouse trials was calculated to be 100% (data not shown). These declined drastically after 5 weeks (Table 7.2). However, there was no significant difference between the planted and unplanted pots in the residual percentage of

TPHs of the potting mix initially spiked with 2% diesel (Table 7.2). In the 7% diesel- contaminated potting mix, the residual percentages of TPHs were higher in the presence of L2 and L4 (Table 7.2).

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Table 7.2 Total petroleum hydrocarbons (% TPHs) in diesel-contaminated potting mix with or without experimental plant materials

% (w/w) of % of residual TPHs after % of residual TPHs after 5 % of residual TPHs after % of residual TPHs after diesel 5 weeks weeks 5 weeks 5 weeks contaminant Plant Plant Plant Plant in potting lines Planted Unplanted lines Planted Unplanted lines Planted Unplanted lines Planted Unplanted mix

2 C-G 14.2 (±0.8)a 14.6 (±1.2)a C-G 17.3 (±1)a 21.5(±2.9)a C-G 19 (±3.4)a 20.1 (±2.1)a C-G 19.3 (±1.5)a 19.7 (±1.4)a

C-R 14.6 (±1.4)a 18.3 (±1)a C-R 16.6 (±1.4)a 18.1 (±1.4)a C-R 15.5 (±0.9)a 17.1 (±2.5)a C-R 15.3 (±1.8)a 16.5 (±0.9)a

L1 16.5 (±1)a 20.5 (±1.4)a L2 18.8 (±0.8)a 21.5 (±1.4)a L3 16 (±2.7)a 16.5 (±1.7)a L4 17.2 (±2.8)a 17 (±0.9)a

7 C-G 57.9 (±2)b 57.2 (±3.7)b C-G 50 (±4.5)bc 46.2 (±4.8)b C-G 50.9 (±4.2)b 49.8 (±3.9)b C-G 51.4 (±1.4)b 46.7(±2.8)b

C-R 50.3 (4.1)b 51.4 (±3.2)b C-R 46.8 (±1.4)b 50.1 (±6.7)b C-R 50.4 (±1.8)b 51 (±3)b C-R 49.8 (±2.1)b 51.7 (±4)bc

L1 56.6 (±3.4)b 59 (±2.9)b L2 55.4 (±2.3)c 57.2 (±4.8)b L3 49.2(±2.6)b 49.4 (±1.8)b L4 50.2 (±2.2)b 55.5 (±2.2)c

Means ± SEM of three replicates in a column and row followed by the same letters are not statistically different according to LSD test, p < 0.05.

Note: C-G are control shoot cuttings from Petunia grandiflora seed germinated in vitro; C-R are control shoot cuttings from Petunia grandiflora plantlet regenerated from non-diesel-exposed callus culture; and Petunia grandiflora plantlet lines L1-L4 are regenerated from diesel-exposed callus culture.

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7.5 Discussion

A major finding from the in vitro culture of the Petunia grandiflora (petunia) lines of plantlets regenerated from callus culture (C-R, and lines L1 to L6) is that there is variability among the regenerated plantlets compared with C-G when evaluated in the absence of selective pressure

(diesel). Trauma, stress or selective pressure on plant cells can cause a reset in the genomic expression and reprogramme the developmental pathways of the entire plant system (Phillips et al., 1994). Here this variation in the experimental lines and the control plantlets was not only in the number of leaves but also in variation in the growth parameters such as shoot height, root length and fresh and dry mass of the whole plantlets. The minor or major differences in the appearances of the in vitro regenerated plantlets might be related to genetic or epigenetic changes (Radhakrishnan and Ranjitha Kumari, 2008). The chances of genetic changes in petunia plantlet lines regenerated from diesel-tolerant callus might have been increased by exposure of callus to diesel, because diesel fuel has been demonstrated to exhibit strong mutagenicity in Salmonella typhimurium (tester strains TA98 and TA100) and mammalian microsome assay (Bünger et al., 2007). The mutagenic effect of diesel has been strongly associated with the high-level content of polycyclic aromatic hydrocarbons (PAHs) (Wang et al., 1990). Mutagenicity of PAHs in diesel to plants has not been extensively investigated, although Aina et al. (2006) reported that benzo[a]pyrene (BaP) and naphthalene (NAPH) types of PAHs induced random root and shoot DNA sequence changes in Trifolium repens with a preferential site for mutation. The damaged DNA was the result of reactive metabolites of

PAHs that triggered DNA to form covalent bonds of stable or depurinating adducts, apurinic sites, and the ROS-mediated oxidative damage (Aina et al., 2006). In the human alveolar epithelial A549 cell line, damage by diesel to DNA involves the mechanistic approach of DNA strand breaks (SB) and oxidative damage (Hemmingsen et al., 2011). It was observed that in the cell, lesion of the 8-oxoguanine base is produced in DNA, and the 8-oxo-7, 8-hydro-2’-

210

deoxyguanosine (8-oxo-dG) nucleotide is potentially mutagenic if not corrected before DNA replication (Kasai, 1997). Plants have the two enzymes 8-oxoguanine DNA glycosylase/lyase

(OGG1) and formamidopyrimidine-DNA glycosylase (FPG), which may be of advantage in

DNA repair activity within the base excision repair (BER) pathway (Balestrazzi et al., 2011).

However, some morphological features found in regenerated petunia plantlet lines are likely to be genetic, because genomic changes may modify phenotype as an adaptive mechanism to withstand stressful conditions (Joyce et al., 2003). For example, the shoot height of plantlet lines L1, L4 and C-G was significantly greater than the other lines. The same trend of differences was also noted on root length, where the root length of plantlets lines L1, L4, and

C-G were greater than the other lines.

Plant growth regulators such as gibberellins (GAs) and brassinosteroids (BRs) were associated with increased biomass production of plants (de Freitas Lima et al., 2017). GA biosynthesis and metabolism have been shown to be critical in regulating plant height and aerial biomass

(Fernandez et al., 2009). For example, in rice, hybrid poplar, transgenic maize and switchgrass, low GA levels induce overexpression of GA 20-oxidase (GA20ox) and GA 3ꞵ-hydroxylase

(GA3ox). Low GA levels also promote a loss of function in GA2ox and the elongated upper internodes 1 (EUI 1) gene resulting in tall plant phenotypes with increased total biomass

(Eriksson et al., 2000; Jeon et al., 2016; Luo et al., 2006). By contrast, high production of bioactive GA inhibits the expression of the important enzymes GA20ox and GA3ox leading to overexpression of GA 2-oxidase (GA2ox), and the EUI 1 gene resulting in short plant phenotypes with reduced biomass (Luo et al., 2006). For phytoremediation purposes, fast- growing plants with high biomass production are usually more preferable candidates (Glick,

2003; Hutchinson et al., 2001). This is because the functions of bioaccumulation factor (BF), which is the ratio of contaminant concentration in a plant to soil, and translocation factor (TF),

211

which is the proportion of uptake of the contaminant concentration in shoots to roots, depend on the efficiency of the biomass production of plants (Sun et al., 2009a; Sun et al., 2009b).

Here in this study, regenerated experimental petunia plantlet lines have shown some similarity in leaf number, shoot height and root length to those in the controls. However, line L4 exhibited significantly higher fresh and dry masses because of their many, large-sized leaves, which were presumed to have higher tissue nitrogen and water concentration. Low to moderate biomass production in willow clones were related to the low concentration of tissue nitrogen and water, even though the clones were previously affected by the adverse effect of drought (Tharakan et al., 2005).

Although one of the successful applications of in vitro selection is to harness desirable traits

(Duncan, 1996; Veilleux and Johnson, 1998), in this study, some variants regenerated petunia plantlets, for example, line L2 plantlets were found to exhibit reduced growth compared with

C-G. There are a number of factors associated with undesirable abnormal traits found in plants regenerated in vitro. Some pre-exist in plants and/or are influenced by tissue culture conditions

(Orzechowska et al., 2013). Culture conditions, particularly plant growth regulator imbalance, have been strongly implicated to cause some aberrations found in plants regenerated in vitro

(Bairu et al., 2011). Nehra et al. (1992) reported that BA and 2, 4-D combinations in the range of 1–20 µM produced regenerants from callus culture of strawberry with reduced plant vigour, petiole length and leaf size. In other reported similar studies, Jain et al. (1989) observed that some regenerated Brassica juncea plants showed slow and reduced growth. Also, Guadagnini

(2000) found that a number of tobacco regenerants exhibited morphological dwarfism. These abnormalities can be related to chromosome aberrations or alteration in the gene responsible for qualitative traits (Stephens et al., 1991). For example, in five recessive maize plants with a mutation in any GA-related genes involved in early GA biosynthesis may lead to the generation of bioactive GAs and eventual dwarfism (Bensen et al., 1995; Fujioka et al., 1988; Winkler

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and Helentjaris, 1995). However, the result from isozyme analysis and measurement of the total nuclear DNA content of the dwarf strawberry plant by Nehra et al. (1992) showed no difference from the normal standard runner plant. Therefore, it is reasonable to argue that these added plant growth regulators are not mutagenic but rather influence some physiological changes in the culture cells by deactivating switchings that promote GA biosynthesis leading to an abnormal growth development pathway of the petunia regenerants (Bayliss, 1980). For example, the experimental plantlets regenerated on media with only 9.1 µM zeatin and 2.3 µM zeatin, line L5 produced a curly leaf, while in line L6 a callus formed at the base of the shoots.

In a similar study, using tobacco, wrinkled and lanceolate leaves were found on the regenerants

(Guadagnini, 2000). Poor growth performances of line L5 and L6 made them difficult to establish under glasshouse conditions, and therefore, lines L1–L4 and control plantlets (C-R and C-G) were further evaluated for their relative sensitivity/tolerance to diesel-contaminated potting mix under glasshouse conditions. The main advantage of the quick bioassay testing here was to screen for putative diesel-tolerant plantlets lines from the “escape”.

Hell (2003) suggested that a fast screening method for eliminating escape plants was preferred to target for an escape-free selection system. Therefore, morphological parameters such as stem diameter, shoot height, number of leaves, leaf chlorosis, root length, and fresh and dry mass were used to identify putative diesel-tolerant line(s). In a similar study, the morphological features of the ornamental plant Mirabilis jalapa exhibited tolerance to 1% petroleum (Peng et al., 2009). Petunia plantlet lines showed relative tolerance to diesel-contaminated potting mix.

For example, in the potting mix with 2% diesel, the stem diameters of line L1 (Figure 7.18) and L4 were large (Figure 7.36).

To my best knowledge, stem diameter has not been examined in all other similar studies.

Perhaps, in the prior studies using seedlings, achieving uniformity from seed at the start of the experiment would be a big challenge. However, stem diameter is a relatively simple, non-

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destructive parameter for estimating water in the plantlets or shoot cuttings (Meng et al., 2017).

In the present study, the results suggest that water content appeared to be higher in all the plantlet lines grown in potting mix without diesel than in the potting mix contaminated with diesel. However, diesel toxicity in the potting mix may have been caused by the presence of the different combinations of elements or compounds found in diesel; PAHs have been reported to be higher in diesel and may elevate the level of some metals in the potting mix (Allan et al.,

2007; Thavamani et al., 2012). It has been reported that the a high concentration of lead (Pb) ions decreased the amount of diffusive (short-distance) water transport in young seedlings of

Lupinus luteus roots, as there was a reduction in water transfer rate across the membranes and vacuoles (intercellular endoplasm system), as well as water diffusion along the apoplast

(Rucińska-Sobkowiak et al., 2013). Transmembrane transport permits about 75% to 95% water flow through aquaporins (AQPs), also called water channel proteins (Henzler et al.,

2004). However, it was demonstrated that zinc (Zn), Pb, Cd and mercury (Hg) toxicity altered the conductivity function of AQPs in the epidermal cells of Allium cepa, which reduced the membrane water permeability (Rucińska-Sobkowiak, 2016). The presence of toxic metals has suppressed the influx of water through AQPs (Rucińska-Sobkowiak et al., 2013). One can, therefore, argue that the significant decrease in fresh and dry masses observed in the petunia plantlet lines grown on 2% and 7% diesel-spiked potting mix compared with those grown without diesel might have been triggered by reduced permeability of water through channels

(Rucińska-Sobkowiak, 2016). It may have also interfered with the functionality of the photosynthesis process, creating disturbance in the chloroplast metabolism, which could inhibit chlorophyll biosynthesis and reduce the activity of enzymes involved in CO2 fixation in plants

(De Filippis and Ziegler, 1993).

Here in this study, the number of leaves formed in plantlet lines and the controls was highly sensitive to diesel in potting mix. In the potting mix spiked with 7% diesel, the average number

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of leaves formed was reduced by more than 50% to 11 compared with the plantlets grown in the potting mix without diesel. Presumably, there may have been an increase in production of

H2O2, protein oxidation and level of lipid peroxidation that caused plasma membrane damage, as observed in Riccia fluitans plants exposed to phenanthrene (PHEN) (Burritt, 2008). For the oxidation of PAHs in the plasma membrane, nicotinamide adenine dinucleotide phosphate

(NADPH) oxidase was involved (Alkio et al., 2005). There might be disturbance in chloroplastic or mitochondrial electron transport chains (Burritt, 2008). It is, therefore, reasonable to assume that the absence of root formation in some petunia plantlet lines may have affected water to nutrient absorption. As It was also reported in the case of soil contaminated with crude oil, suggesting a delay in the production of new leaves (Osuagwu et al., 2013). In this study, it explained the correlation between the reduced number of leaves and increased diesel concentrations.

Visual symptoms such as leaf burns and necrosis on plants grown in diesel-contaminated soil have been used as indicators to assess the impact and tolerance in plants (Palmroth et al., 2002).

Also, in the study of an avirulent plant-pathogen interaction, necrotic lesions were used as an indication of a hypersensitive response (HR), and the defence mechanisms involved local cell death to restrict the spread of the pathogen (Lamb and Dixon, 1997). Among the experimental plantlet lines and the controls, C-R had a severely chlorotic rating and was therefore more sensitive than lines L1, L2, L3, L4, and the control C-G when grown in potting mix contaminated with 7% diesel. The leaves of C-R appeared to have a burnt appearance, pale yellowish leaf and yellowish edges or mid-rib of the leaf. Palmroth et al. (2002) also observed that leaf burning was associated with plants grown on diesel-contaminated soil and the nature of the leaf burns varied from dominant at the leaf edges, covering the entire leaf, and sometimes the burn started at the leaf tips. In a study of Arabidopsis thaliana (Arabidopsis) exposed to

0–0.5 µM of PHEN, the number of leaves with stress symptoms increased over time.

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Microscopic fluorescence analyses suggested that the white spots seen on the leaves were where there was accumulation of PHEN or its derivatives (Alkio et al., 2005). It was further suggested that necrotic lesions on the leaves were localised dead cells due to the defence mechanism of endogenous fluorescent molecules produced in reaction to PHEN exposure as in an hypersensitive reaction (HR) (Alkio et al., 2005). In this study and other reports, there are no similar fluorescence analyses of different abiotic stress. In HR, fluorescent phenolic compounds were deposited in the cell wall (Mellersh and Heath, 2001), while fluorescence of

PHEN was distributed within the cytoplasm (Alkio et al., 2005).

Line L3, in contrast, was found to be more chlorosis sensitive than the other experimental plantlet lines and control plantlets when grown in potting mix without diesel. Their leaves had a widespread bleached-looking effect. Chlorotic leaves have been associated with reduced leaf expansion and size (Fernández et al., 2008; Larbi et al., 2006), but this was not found in the case of line L3.

In response to 0.6% diesel-contaminated soil, the growth of Pinus densiflora, Populus tomentiglandulosa, and Thuja orientalis was significantly reduced compared with the control

(Jagtap et al., 2014). In a similar study using diesel-contaminated soil, the growth of Spartina argentinensis declined more in the presence of diesel than in clean soil (Redondo-Gómez et al., 2014), which was in agreement with the finding in this study. Diesel-contaminated potting mix negatively affected the shoot height of the experimental lines and control plantlets. For example, when grown in potting mix without diesel or with 7% diesel, the shoot heights of line

L3 were about 9 cm and 7 cm, respectively. It has been observed that PAH contamination may not necessarily bring about the total shutdown of metabolic activities in plants because the expression levels of PR 1 genes, considered as housekeeping genes, were not disrupted by

PHEN treatment in Arabidopsis. Instead, PAH contamination is specific to suppress the expression of non-enzymatic protein expansin (Alkio et al., 2005). Plant growth affected by

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the presence of diesel or some specific PAH contamination may be attributed to a reduction in cell division or cell expansion frequency or both (Alkio et al., 2005). In the histology of petunia calli exposed to diesel, more cells in the untreated petunia calli had prominent nuclei than the treated calli, indicative of a high rate of cell division. However, exposure to PHEN of plants expressing the CyclinB1;1::GUS reporter, which is a standard construct for cell division analysis (Colón‐Carmona et al., 1999), showed that the spatio-temporal arrangement of mitotic activities was not affected (Alkio et al., 2005). The reported finding of a reduction in expansin expression further confirms that growth reduction in plants may be due to an inhibition of cell enlargement (Alkio et al., 2005), because expansins are extracellular proteins attributed to a key functional role in wall stress relaxation and, thus, in cell and tissue growth (Pien et al.,

2001).

The significant difference between the tested experimental plantlet lines and the control plantlets was the ability of line L4 to form 13 roots per plant in the absence of diesel and 8 roots per plant in potting mix contaminated with 2% diesel. Currently, little is known about the diesel-tolerant mechanisms in plants. However, in an experiment using R. fluitans’ exposure to

PHEN, for the plant to recover from or tolerate PHEN exposure, they must control ROS production and swiftly up-regulate their defence against oxidative damage (Burritt, 2008). This was shown by a significant level of increase in ascorbate and glutathione, as well as increased activities of the ascorbate-glutathione cycle (Burritt, 2008). In the same experiment, Burritt

(2008) also reported that polyamine (PA) synthesis helps the recovery of R. fluitans exposed to PHEN, by increasing the activities of enzymes arginine decarboxylase (ADC) and S- adenosylmethionine decarboxylase (SAMDC). The critical step here is that increased PA synthesis reduced amino acid leakage and photosystem damage (higher Fv/Fm values) (Burritt,

2008). Presumably, petunia line L4 tolerated the diesel stress by utilising the reported mechanism of R. fluitans to PHEN and decreasing the response time between the stress and the

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production of new roots (Balliana et al., 2017). Another interesting finding of the tolerant mechanism of PAHs in plants is that the marker gene of the systemic acquired resistance, PR1, was induced in PHEN-exposed Arabidopsis plants (Alkio et al., 2005). The induction of PR1is regulated by salicylic acid (SA), which mediates defence responses against biotrophic pathogens (Thomma et al., 1998).

However, a previous investigation reported inhibition of petunia root elongation in diesel- contaminated water (Wante and Leung, 2018). A similar result was obtained here with the plantlet line L4 forming roots in the potting mix with 2% but not 7% diesel. It is reasonable to hypothesise that at 7% diesel, petunia plantlet line L4 has not been able to control ROS production to a level that permits root formation.

It is important to note that root formation and full root development are criteria for efficient phytoremediation (Hou et al., 2001). In this pot trial, planting diesel tolerant plants did not appear to influence TPH losses from the potting mix. Also, there was no significant difference between the planted and unplanted pots in the residual percentage of TPHs in the potting mix initially spiked with 2% diesel. Since only one petunia plantlet was planted per pot, the leaves formed after 5 weeks were not enough to create a dense canopy to cover the surface area of the pot, which might have otherwise limited the volatile and PAH components of diesel from escaping. It has been reported that benzo[a]pyrene was absorbed by the aerial part of Tagetes patula from the ambient air, possibly originally volatilised from the soils (Sun et al., 2011).

A 30-day pot experiment to assess the phytoremediation potential of 14 ornamental plants in weathered petroleum-contaminated soil has also been reported (Liu et al., 2012). The bioassay testing experiment was relatively short compared with the long and time-consuming traditional practice of phytoremediation screening of plants. The present study was, however, consistent with the general aim of any phytoremediation screening method to identify the most tolerant plants within a selected time frame.

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Hernández-Ortega et al. (2012), reported that 0.75% diesel-contaminated substrate reduced the total biomass of Melilotus albus by 75%. A similar study observed a lower production of total grass and legume biomass in diesel-contaminated soil than for the control (Palmroth et al.,

2002). In another study using diesel-contaminated soil, about 88% and 75% of the total shoot and root biomasses were reduced, respectively, relative to clean soil (Jagtap et al., 2014). In this study, the results were largely consistent with these findings.

Here, the plantlet line L4 exhibited the highest total fresh biomass in clean and diesel- contaminated potting mix compared to C-G and C-R, although the total biomass of line L4 decreased with increasing levels of diesel in potting mix (Figure 7.42). Reduction in plant growth in oil-contaminated soils has been linked to the toxicity effect of light petrol constituents in soil (Li et al., 1997; Peng et al., 2009; Salanitro et al., 1997). As mentioned previously, the various components of elements and compounds found in diesel can affect the functionality of plants, particularly the photosynthesis process by creating disturbance in the chloroplast metabolism that could inhibit chlorophyll biosynthesis and reduce plant biomass production (De Filippis and Ziegler, 1993). Presumably, the levels of disruption in GA biosynthesis and metabolism in the less tolerant petunia plantlet lines were much higher due to the more significant decrease in biomass production compared with the L4 line.

The lack of root formation in the experimental plantlet lines (L1–L3) and the control plantlets could be related to the very few variations found in the microbial population of the planted and unplanted potting mix, regardless of the level of diesel contamination. The presence of roots in plants is thought to play an important role in microbial abundance in many studies (Hutchinson et al., 2003). For example, in this study, higher counts of total culturable microbes than the control were only found in the pots of line L4 that formed roots. In another similar investigation, Altai wild rye (Elymus angustus Trin.) encouraged the growth of hundreds more endophytic hexane degraders than the unplanted control (Phillips et al., 2006).

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Studies show that in plants that were under stress conditions of contaminants, their roots produced a substantial amount of certain phenolic chemicals (e.g., salicylate). These are inducers of microbial hydrocarbon degradation (Gaffney et al., 1993; Kamath et al., 2004). In most phytoremediation screening studies, soil was mainly used to enumerate the culturable microbial population. However, Graber et al. (2010) reported culturable microbial counts in potting mix, which is considered a pristine medium compared to natural soil. Potting mix was used in this study because there may be less interference of inhabitant-microbial colonies

(Koohakan et al., 2004). As was mentioned earlier in this study, the plantlet lines tested did not form roots and accelerate total petroleum hydrocarbon (TPH) depletion in the used potting mix.

The roots in plantlet line L4 grown in potting mix contaminated with 2% diesel did not cause a significant reduction in the TPHs of diesel after 5 weeks. This was probably because the root density was not enough to extend fully over the potting mix. Root intensity and depth are the major contributors to increased remediation of TPHs of diesel (Hou et al., 2001).

7.6 Conclusion

The petunia line L4 outperformed the growth of the controls and other experimental plant lines both in in vitro conditions in the absence of diesel and under glasshouse conditions in potting mix spiked with 2% diesel. The morphological characteristics of all the plantlet lines and the controls were affected by 7% diesel. The ability to form roots only in line L4 when grown in the potting mix contaminated with 0% and 2% diesel correlated with increased culturable microbial counts over those in the potting mix planted with the other plantlet lines. Therefore, the plantlet line L4 seems to be more tolerant to 2% diesel exposure than other plantlet lines.

It may be worthwhile to investigate the phytoremediation potential of line L4 grown in soil contaminated with diesel in future studies.

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Chapter 8 Overview of results and recommendations

8.1 Significance of the main research strategy and findings

There is no prior study on the response of Petunia grandiflora and Tagetes patula (petunia and marigold) to diesel-contaminated water. However, in the present study, information was obtained regarding the relative differential sensitivity of petunia and marigold seed germination, early seedling root elongation and shoots to diesel contamination. Petunia seedlings seemed to be more sensitive to, or less tolerant of, the toxic effect of diesel than marigold seedlings. At present, there is a paucity of knowledge about diesel tolerance mechanism(s) in plants. The findings from this investigation were an important prerequisite towards a deeper evaluation of (a) diesel tolerance, and (b) the potential of petunia and marigold for phytoremediation of diesel contamination of waterways and land. The main aim of this study was to make contributions to the development of plants with improved growth performance in the presence of diesel contamination. These plants could then be used in helping future studies to obtain a better understanding of diesel tolerance in plants. The main technological approach in this study was to apply in vitro culture technology to select calli of petunia and marigold that could survive diesel exposure. In this study, callus culture was initiated from young in vitro leaf explants of petunia and marigold seedlings. Efficient callus induction and subculture media for both petunia and marigold have been established. A novel, protocol was developed to obtain calli that survived diesel exposure. It was found that petunia and marigold calli did not exhibit extensive necrosis during exposure to the undiluted fuel treatment for up to 9 minutes, suggesting that the treatment was sub-lethal.

This was achieved, first under non-aseptic conditions for exposure of calli to diesel, and then the calli were re-cultured under completely aseptic conditions. The protocol is a valuable practical contribution that overcame the perceived technical barrier of using diesel in in vitro

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culture technology. The histological study of the diesel-treated calli was the first study into the possible consequences of diesel toxicity on callus. The six novel plantlet lines generated from diesel-treated calli of petunia have further confirmed that a callus with plant regeneration potential could be established after exposure to diesel. A major finding in this investigation was the variations evident in the morphological parameters and growth performance of the plantlet lines grown under in vitro conditions and in potting mix with or without diesel contamination under glasshouse conditions. The shoot cuttings of one plantlet line, L4, exhibited root formation that had a great influence on the number of culturable microbial populations in the rhizosphere regions.

8.2 Insight from the histological study of calli following diesel exposure The histological study was a first attempt to obtain insight into responses of diesel toxicity at the cellular level. Changes in the structural organisation of petunia and marigold calli were observed at two levels from the top of the calli. At 500 µm from the top of the petunia callus without diesel treatment, the distribution of meristematic cells seemed to be more extensive than in the diesel-treated callus. Further down the calli (1500 µm deep), more xylem vessel formation was observed in the diesel-treated callus than the callus without diesel exposure. It is worth mentioning that diesel-treated marigold calli layers at 500 and 1500 µm mm did not show a similar extent of xylem differentiation in response to diesel toxicity as the diesel-treated petunia calli; in contrast, in diesel-treated marigold callus, there were more parenchyma cells with visible nuclei.

Another important finding was the presence of prominent dome-like regions of undifferentiated meristematic cells at 1500 µm from the top of the marigold callus without prior exposure to diesel. It was not clear why empirical manipulations of plant growth regulators in the culture media did not result in plant regeneration in marigold callus. This anatomical insight revealed that the callus subculture protocol investigated in this study did not enable the development

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and emergence of leafy shoots from the induced meristematic regions. This suggests that further development of a successful protocol for plant regeneration from marigold callus is justified.

8.3 Plant regeneration was a key to establishment of potentially novel plantlet lines from diesel-treated petunia calli Petunia shoot regeneration was achieved from diesel-treated calli and those without exposure to diesel. Successful plant regeneration together with overcoming the technical barrier of treating calli with diesel was key in establishing the six different experimental plantlet lines and controls. Petunia plant line L1 was generated during the sub-culturing stages of the non- necrotic part of the diesel-treated callus by forming leafy shoots that grew slowly on the callus subculture medium. The new petunia calli produced more shoots, and the majority of the shoots were formed at the proliferated edges of the calli. Shoots that were induced from the non- necrotic part of diesel-treated calli on the MS medium supplemented with different zeatin concentrations had poor root development. At the initial stage of the shoot development that formed line L5, severe leaf chlorosis was observed, but later on, the shoots started to form white roots within the friable white callus.

8.4 Growth performance of petunia plant lines under in vitro and glasshouse conditions Evaluation of the growth performance of the different plantlet lines in vitro without the effect of diesel and the tolerance to diesel toxicity under glasshouse conditions led to identification of L4 as a promising line for further research and development into diesel tolerance in plants.

Line L1, generated from the diesel-treated callus, also exhibited significant growth performance compared with lines L2, L3, L5, L6 and C-R.

In the plantlet lines and the control plantlets, tests confirmed the direct effect of diesel on stem diameter. For example, decreased stem diameter was observed when the percentage of diesel was increased. With regard to their stem diameters at 2% diesel, the four experimental lines

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generated showed a significant level of tolerance compared to the plantlet controls. Also, in this study, experimental plantlet lines showed less chlorotic sensitivity to the effect of diesel than the plantlet controls.

In line L4 the allocated biomass produced fresh and dry weight that was significantly higher than all the tested lines. Another significant finding in this study was the ability of line L4 shoot cuttings to form roots not only at 0% diesel but also at 2% diesel. This unique morphological characteristic was not observed in the other experimental lines and plantlet controls. The roots formed had influenced the presence of total culturable microbial populations in the potting mix, but not enough to cause the degradation of TPHs in diesel. The morphological parameters studied were relevant and were good indicators to assess the tolerance level to diesel contamination in generated petunia lines.

8.5 Application of the findings in this thesis

Environmental pollution with petroleum hydrocarbons including diesel poses serious environmental problems worldwide and constitutes a potential threat to the ecosystem and public health. The bioassay toxicity testing of diesel-contaminated water using petunia and marigold seed has, however, provided information that together with the standard guideline protocol developed for testing chemicals by the Organization for Economic Cooperation and

Development (OECD) may be used as part of a policy document. Moreover, for the purpose of research, it serves as background knowledge into a deeper investigation of the diesel tolerance mechanism in the plants.

To the knowledge of the author, the efficient in vitro callus-inducing media protocols using the leaf explants of Petunia grandiflora and Tagetes patula developed in this study have not been previously reported. These protocols may therefore be of use to induce callogenesis both from these plants and other related ornamental plants.

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The novel in vitro technology developed in this study was successful in exposing calli of petunia and marigold to diesel toxicity under non-aseptic conditions before re-culture under fully aseptic conditions. This novel technology is expected to break the technical barrier to selecting diesel-tolerant cells because the technology is transferable and can be adapted for use with calli from different plants. Also, the histological study on the diesel-treated calli yielded some insight about the effect of diesel toxicity on petunia and marigold at the tissue differentiation level. The technique could usefully be employed to study different types of cells following exposure to diesel. Although plant regeneration was not successful in marigold calli, the information provided could be beneficial to further guide the development of an experiment to regenerate marigold plants.

The plant lines regenerated from petunia calli in this study can be added to existing horticultural germplasm used for conservation, breeding studies or other related studies. Based on the morphological characteristics presented, plant line L4 exhibited tolerance potential to 2% diesel, and therefore line L4 may be useful plant material for creating phyto-management and beautification of diesel-contaminated sites. If further characterised and confirmed to be mutant, the gene responsible for diesel tolerance could be transferred to other plants using gene transfer technology to improve the remediation efficiency in these plants. Moreover, the biomass produced can be useful in generating biofuels or bioproduction of secondary metabolites for large-scale production.

8.6 Limitation of the study

The aim of the present study was to develop diesel-tolerant Petunia grandiflora and Tagetes patula plant cell lines using cell and tissue culture technology under in vitro conditions. The

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specific objectives of the thesis were described in section 1.6. However, certain fundamental limitations in the thesis were observed and are described here.

Variability of diesel composition: In this study, the diesel used was from the same source, but considering the experimental design, where diesel was needed in each of the different series of experiments including repeat experiments, it was inevitable that the diesel used was from the same batch. However, the required conditions to store small quantities (not more than 5-gallon cans) of flammable fuels such as diesel was followed (Thompson et al., 2013). For example, the diesel was stored in an airtight polyethene container in a cupboard at 16–18 °C for not more than six months. Although, gas chromatograph profiling was obtained between the start of each group of experiments and the repeat experiment, and this showed no difference in the range of the low molecular weight hydrocarbons found in the diesel, this might not have been sufficient to reflect the variable nature of the different PAHs and some heavy metals that could have been present in the diesel.

2. In vitro culture conditions: Under aseptic conditions of the cell and tissue culture environment, tolerance mechanisms that involved the study of physiology, biochemistry and molecular biology under different abiotic and biotic stresses have been investigated. However, in this study, it was a challenge to study the tolerance mechanism of the experimental petunia plantlets lines developed under in vitro culture conditions because of the technical barrier in using diesel in the Murashige and Skoog (1962) medium.

3. Diesel exposure: The potting mix spiked with diesel was thoroughly mixed using a mechanical method, but the toxicity level to plants in the potting mix may not reflect the concentration of diesel added because the bioavailability of petroleum hydrocarbons in soil or potting mix is lower than in water (Jośko and Oleszczuk, 2014). However, the method used in

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this study was consistent with the general aim of phytoremediation screening under glasshouse conditions to identify the most tolerant plants.

4. Genotype and storage of seeds: Seeds were presumed to be similar because of their uniform phenotypic appearance and the same storage conditions; nevertheless, genotypic variation may still be highly significant. In this study, it was mentioned in Chapter 2, section 2.1.1, that the petunia and marigold seeds were stored in a closed container in the dark at 22 ℃, but as seeds were commercially purchased, it was beyond the scope of the investigator to control variations that may have occurred due to genetic variation.

8.7 Recommendations for future research

The results of the in vitro growth performance and the morphological characteristics under diesel conditions have provided an insight into the diesel tolerance of plantlet line L4, suggesting further investigations. However, more time is needed beyond the limited time frame of a doctorate to study the physiology, biochemical and the molecular aspects of diesel tolerance mechanisms. These mechanisms might be associated with activated switches for the vigorous growth and root development of line L4 plants. The physiology, biochemical and molecular mechanism associated with diesel stress tolerance in line L4 compared to C-G, for example, can be characterised by gas exchange, photosynthetic pigments, chlorophyll index and protoplasmic damage (Alkio et al., 2005; Silva et al., 2017). There is, however, little known about the physiology, biochemical and molecular changes associated with diesel tolerance mechanism in plants (Silva et al., 2017). It will also be of interest to compare the activities of peroxidase (POD), catalase (CAT) and ascorbic acid oxidase (AAO) in line L4 and the controls. Reduced oxidative stress has been associated with resistance to diesel and many other forms of abiotic stress in plants (Liu et al., 2009; Wang et al., 2011). At present, it is still unclear how the expression of the pathogenesis-related protein, PR1, was induced in response

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to PAHs in plants (Alkio et al., 2005). Investigations into the detailed defence mechanism of gene activation in line L4 will also be of interest.

The superiority of the morphological variation observed in line L4 under in vitro and glasshouse conditions may be associated with genetic changes that occurred during diesel treatment of calli. Genetic changes in plants caused by mutation have been related to cytogenetic abnormalities and specific gene sequence changes, which are often found in regenerated plants and their progeny (Kaeppler et al., 2000). In contrast, epigenetic changes are determined by mechanisms of gene silencing or gene activation and such variation might be unstable or reversible (Kaeppler et al., 2000). Somaclonal variation can be assessed using phenotypic or cytogenetic analysis, protein study, DNA or isoenzyme markers (Smýkal et al.,

2007), but the use of molecular techniques has been reported to be more precise with less effort to determine the genetic stability of regenerated plants than karyological and phenotypic analyses (Cloutier and Landry, 1994; González et al., 1996; Ruiz et al., 1992).

Biochemical markers are equally important in evaluating somaclonal variation, as these are centred on the variability of proteins and isozymes (Naseer and Mahmood, 2014). They have been reported to be effective in the evaluation of somaclonal variation in regenerated apple shoots (Liu et al., 2004), the embryogenic tissue of Dioscorea bulbifera (Dixit et al., 2003) and date palm plants (Saker et al., 2000). However, recent technological advances reveal the insensitivity of secondary metabolites and isozymes as biochemical markers for analysis of variation induced by in vitro cultures (Morell et al., 1995).

Genetic changes resulting from point mutations or initiation of mobile (transposable) elements have been detected using DNA-based marker approaches such as those using randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR) and interplay simple sequence repeats (ISSR) (Aversano et al.,

2011; Bairu et al., 2011; Phillips et al., 1994). These DNA-based marker approaches with a

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high level of molecular precision should be employed for detecting variation in petunia plantlet line L4 in response to diesel contamination.

Among these DNA-based marker approaches, ISSR has been proved to be of advantage because it could yield more consistent results quickly and reveal a high level of polymorphism

(Reddy et al., 2002). Moreover, it is user-friendly and cost-effective (Reddy et al., 2002). It was also reported to be effective with a small amount of DNA without the need for prior sequence knowledge to design the primers required (Lakshmanan et al., 2007). However, a lack of proper reproducibility of RAPD, uneconomical AFLP and the necessity to know the flanking sequences to change species-specific primers for SSR polymorphism are the main restrictions of these three DNA-based marker approaches (Viehmannova et al., 2014). Since the ISSR technique is more sensitive and reliable in evaluating the genetic variability of in vitro regenerated lines (Viehmannova et al., 2014), this method should be selected in determining the somaclonal variation induced during in vitro studies of diesel exposure.

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Appendices

Appendix A: plant tissues culture media

A1. Murashige and Skoog Stock (MS) medium (1962)

Table A1. The amount of major, minor and organic salts required for 500 mL stock solutions

1. Major salts (10x) 1 Litre 2 Litre

NH4NO3 16.5 g 33 g

KNO3 19 g 38 g

CaCl2.2H2O 4.4 g 8.8 g

MgSO4.7H2O 3.7 g 7.4 g

KH2PO4 1.7 g 3.4 g dH20-bring volume to 1 L 2 L

Label and store at 4℃

2. Minor salts (100x) 1 Litre 2 Litre

KI 0.083 g 0.166 g

H3BO3 0.620 g 1.240 g

MnSO4.4H2O 2.230 g 4.460 g

ZnSO4.7H2O 0.860 g 1.720 g

CuSO4.5H2O 0.0025 g 0.005 g

CoCl2.6H2O 0.0025 g 0.005 g

Na2MoO4.2H2O 0.025 g 0.050 g dH20-bring volume to 1 L 2 L

Label and store at 4℃

3. Organic supplement (100x) 500 mL 1L

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Myo-inositol 5,000 mg 10,000 mg

Nicotinic acid 25 mg 50 mg

Pyridoxine-HCl 25 mg 50 mg

Thiamine-HCl 5 mg 10 g

Glycine 100 mg 200 mg dH20-bring volume to 500 mL 1 L

Label and store at 4℃

4. Iron stock (100)

Solution A FeSO4.7H2O 1.39 g in 200 mL dH2O

Solution B Na2EDTA.2H2O 1.865 g in 200 mL dH2O

Mix solution A and B

Adjust volume to 500 mL

Store in dark bottle at 4℃

280

Appendix B: Histology analysis

B1. Staining procedures

1. Callus sections on the glass slide were stained in 1% Safranin-O staining solution for

2–24 h.

2. The excess stain was washed out for about 2-3 times under running water.

3. The sections were dehydrated for 10s in 95% ethanol plus 0.5% picric acid.

4. Callus sections were washed for 10s to maximum 1 min in 95% ethanol + 4 drops

ammonium hydroxide per 100 mL to stop picric acid action. Excessive ethanol washing

will completely remove Safranin-O staining.

5. Sections were dipped briefly for 10 s in 100% ethanol to finish dehydration.

6. Counterstained for 10–15 s in fast green staining solution.

7. The excess fast green was washed with a clearing solution (50% clove oil diluted with

a 1:1 mixture of ethanol: xylene solution).

8. Tissue sections on the glass slides were washed in clearing solution by dipping the

sections for 5–10 s.

9. The clearing solution was removed by dipping the sections for a few moments into

xylene (not Histo-Clear) plus 2–3 drops 100% ethanol (to remove residual water).

10. Sections were rinsed twice in xylene.

11. Tissue sections on the glass slide were kept in the final xylene solution while the

coverslip was mounted on each glass slide.

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Appendix C: Potting mix analysis

Table C1. General analysis

282

Table C2. General analysis

283