Thesis Title

Physiological and Metabolic Responses of Two Rose Varieties to Plant Growth Regulators

This thesis is presented for the degree of Doctor of Philosophy of Murdoch University

By

Mohammed Muhi Ibrahim

School of Veterinary and Life Sciences Murdoch University Perth, Western Australia 2018

i DECLARATION I declare that this thesis is my own account of my research and contains as its main content work, which has not previously been submitted for a degree at any tertiary education institution.

Signature: Mohammed Ibrahim Date: December 19, 2018

ii Acknowledgements “In the name of Allah, the most Beneficent and the Merciful” بسم هللا الرحمن الرحيم Allah Almighty, the creator of everything, the most beneficent, merciful, gracious and compassionate whose bounteous blessing gave me the potential thoughts, and guidance to complete my PhD research, write my thesis and to provide with the opportunity to make this humble effort and enable me to pursue and perceive higher ideas of life.

First of all, I would like to thank my supervisor Professor Yonglin Ren Head of Postharvest Biosecurity and Food Safety Laboratory Murdoch, Western Australia. He was very kind and generous in his auspicious help, scholarly guidance, great encouragement and affectionate support. He has been a colossal source of inspiration and solace for me during my PhD studies. Secondly, it gives me great pleasure in acknowledging my associate supervisor Dr Manjree Agarwal Postharvest, Biosecurity and Food Safety Laboratory Murdoch, Western Australia. For her help during my research and her patience for many questions, valuable training, providing corrections and critical suggestions on experimental work and during my thesis writing stage. Thirdly, I take great pleasure to express my sincere and intense sense of gratitude to my associate supervisor Professor Giles Hardy Associate Dean Research/Professor in Forest Pathology in Murdoch University. For imparting all his experience, knowledge and skills in me, for his caring behaviour, moral support and his continuous efforts to accommodate a supportive environment throughout my research, and during writing stage despite his multifarious responsibilities. A sense of obligation compels me to express my cordial thanks to Professor Muslim Abdulhussein head of Horticulture Department & Landscape, Faculty of Agriculture, Kufa University, Najaf, Iraq, associate supervisor for helping me to prepare the required compounds and for critical suggestions and valuable feedback during my research.

Many thanks are passed to Mr. Bo Du Postharvest, Biosecurity and Food Safety Laboratory Murdoch University for his help with technical support with laboratory instruments and his patience and time. I also gratefully pass my thanks to Mr. James Newman Postharvest, Biosecurity and Food Safety Laboratory Murdoch University for his help in providing plant materials, time, patience, and moral assistance during my research. I sincerely acknowledge

iii and thank financial support from the Higher Committee for Education Development in Iraq (HCED) for providing me with the scholarship to undertake my doctoral study.

Special thanks to my Uncle Younes Ibrahim for his encouragement, moral support, and many thanks to my cousin Marai Younes for his love, affection, support, help, and encouragement. It would have been a very tough job for me to complete this thesis without continuous motivation and inspiration.

I am thankful to all my friends that I have known in Australia for giving the support and help in different ways, especially Charles Obiero for assistance with statistical analysis. I am also thankful to my relatives in Australia Mr. Faisal Al-Ramaid and his family especially his son Husam for their support. I take this opportunity to express my thanks to all my family members especially my affection mother, my wife Bushra, my daughter Elaf and my son Ameen “Their love, incredible patience and endless support during the entire period of my PhD study and throughout my life”, friends and my relatives in Iraq for their encouragement, moral support and good wishes.

iv Abstract

Rose (Rosa hybrida) is one of the most popular cut flowers with a worldwide production of more than 300 million stems per year. The perfume industry, which relies on a range of scented rose varieties, is also an important industrial application for roses. Among the numerous types of roses are some recent varieties including Hybrid Tea and Floribunda roses. The major problem in Australia is accelerated pre- and post-harvest flower drop and senescence, caused by deficiencies in endogenous plant growth regulators (PGRs). PGRs play important roles in the growth and development of flowers, especially in aromatic plants, stimulating the emission of volatile organic compounds (VOCs). The PGRs benzyladenine (BA) and naphthalene acetic acid (NAA) which belong to cytokinin and auxin group of PGRs respectively, are used by the floriculture industry as important growth regulators for promoting rose growth and development. However, it is still unknown that how these regulators and their application dosages influence rose plants. Therefore, this thesis aimed to evaluate and determine the efficacy of various concentrations of BA and NAA on arrange of morphological and physiological characteristics of roses to increase flowers longevity in two rose varieties (Floribunda and Hybrid Tea). In addition, this study was conducted to understand how different concentrations of BA and NAA effect the metabolic changes in different rose tissues together with a comparison of VOCs changes.

This work in this thesis developed and optimized the headspace solid-phase microextraction (HS-SPME) with three-phase fibre 50/30µm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) coupled with gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS) method for exploring and analysing VOCs emitted from the intact and excised flowers, leaves, and stems as well as from the rhizosphere and whole plants of the two rose varieties.

This study has optimized different environmental factors involved in the performance of the two rose varieties was conducted, and three physiological characteristics photosynthesis rate, respiration rate, and chlorophyll content were assessed. Three different concentrations (0, 100 and 200 mg/L) of BA and NAA were applied to the two rose varieties, and different morphological and physiological characteristics were evaluated. For physiological effects (photosynthesis rate and chlorophyll index) studies, application of 200 mg/L of both BA and NAA were shown to increase plant height, numbers of branches and flowers, stem and flower diameters, length of flower stems and flower longevity, compared to the control. However, for

v the respiration rate, the control plants had significantly superior performance to the plants treated with 100 and 200 mg/L BA and NAA, for both rose varieties.

The VOC profiles of the two rose varieties were characterized by the optimized HS-SPME-GC method. The effects of different concentrations (0, 100 and 200 mg/L) of BA and NAA on the VOCs emitted from different rose tissues for the two rose varieties were determined. The highest amounts extracted, and evaluated from the sum of peak areas were achieved after the application of 200 mg/L BA and NAA in both varieties. Of the emitted VOCs, 20 were significantly different in treated compare to non-treated Floribunda and Hybrid Tea roses from different rose tissues. Moreover, five compounds 4-heptyn-2-ol, cis-muurola-4(14)5-diene, γ - candinene, y-muurolene and prenyl acetate increased significantly after applications of 200 mg/L of BA and NAA. These five compounds have great potential to develop commercially important new rose growth regulators. The actual dosages of BA applied to the leaves was determined using filter paper as 11.16 mg/cm2 and 7.17 mg/cm2 for Hybrid Tea and Floribunda respectively. In conclusion, the application to rose plants of different concentrations of BA and NAA can promote a number of changes to both morphological and physiological parameters, and in turn have a significant effect on metabolite changes in different rose tissues. Application BA and NAA method could be applied to other floriculture plants to increase the flowers production in rose or other ornamental plants.

vi Table of Contents Contents Thesis Title ...... i Acknowledgements ...... iii Abstract ...... v Table of Contents ...... vii Publications and Presentations ...... xi Publications ...... xi Poster Presentations ...... xi List of Abbreviations ...... xii Chapter 1 ...... 1 General Introduction and Literature Review ...... 1 1.1. General Introduction ...... 2 1.2. Literature Review ...... 7 1.2.1. Roses ...... 7 1.2.1.1. History of Roses ...... 7 1.2.1.2. Importance of Rose ...... 8 1.2.1.3. Breeding Rose Varieties ...... 11 1.2.2. Volatile Organic Compounds ...... 15 1.2.2.1. Detection of VOCs ...... 17 1.2.2.1.1. VOCs Sampling and Extraction ...... 17 1.2.2.1.2. GC-MS Data Acquisition and Processing ...... 20 1.2.2.2. Biosynthesis of Volatile Compounds ...... 25 1.2.2.3. Biosynthesis of Rose Volatile Compounds ...... 26 1.2.2.4. Human Perception ...... 32 1.2.2.5. Conditions that Affect the Cultivation and Selection of Fragrant Varieties 32 1.2.3. Plant Growth Regulators ...... 33 1.2.3.1. Cytokinins (Benzyladenine) ...... 35 1.2.3.1.1 Effect of BA on Morphological and Physiological Characteristics...... 38 1.2.3.1.1. Effect of BA on Emission of VOCs...... 38 1.2.3.2. Auxins ...... 39 1.2.3.2.2. Effect of NAA on Morphological and Physiological Characteristics ... 44 1.2.3.2.3. Effect of NAA on Emission of VOCs ...... 44 1.2.4. Research Gaps...... 46

vii 1.2.5. Aims of Study ...... 46 Chapter 2 ...... 49 Optimization of Environmental Factors to Measure Physiological Parameters of Two Rose Varieties ...... 49 Abstract ...... 50 2.1. Introduction ...... 50 2.2. Materials and Methods ...... 52 2.2.1. Plant Material and Maintenance ...... 52 2.2.2. Photosynthesis Measurements ...... 52 2.2.3. Respiration Measurements ...... 53 2.2.4. Chlorophyll Measurements ...... 53 2.2.5. Statistical Analyses ...... 53 2.3. Results and Discussion ...... 53 2.3.1. Optimization of Light Intensity for Maximum Photosynthesis ...... 53 2.3.2. Analysis of Photosynthesis with Five Sampling Times ...... 54 2.3.3. Effect of Weather Conditions on Photosynthesis Rate ...... 55 2.3.4. Analysis of Respiration with Two Sampling Times ...... 56 2.3.5. Effect of Different Sampling Times on Chlorophyll Content ...... 57 2.4. Conclusion ...... 58 Chapter 3 ...... 61 Morphological and Physiological Responses of Two Modern Roses to Plant Growth Regulators ...... 61 Abstract ...... 62 3.1. Introduction ...... 62 3.2. Materials and Methods ...... 63 3.2.1. Plant Materials and Foliar Application of Growth Regulators ...... 63 3.2.2. Morphological Measurements ...... 64 3.2.3. Physiological Measurements ...... 64 3.2.4. Statistical Analyses...... 65 3.3. Results and Discussion ...... 65 3.3.1. Morphological Parameters ...... 65 3.3.2. Physiological Parameters ...... 68 3.4. Conclusion ...... 70 Chapter 4 ...... 73 Optimized Method to Analyze Rose Plant Volatile Organic Compounds by HS-SPME- GC-FID/MSD ...... 73

viii Abstract ...... 74 4.1. Introduction ...... 74 4.2. Materials and Methods ...... 76 4.2.1. Reagents ...... 76 4.2.2. Apparatus and Equipment ...... 76 4.2.3. Plant Material and Maintenance ...... 77 4.2.4. Gas Chromatogram Condition Optimization ...... 78 4.2.5. Optimization of Headspace Solid-Phase Microextraction ...... 78 4.2.6. Optimisation of Sample Chambers ...... 78 4.2.7. Optimisation of Sealing and Fibre Absorption and Desorption Time ...... 79 4.2.8. Optimization of Collection of VOCs from Different Tissues of Rose Plants 79 4.2.9. Statistical Analyses...... 79 4.3. Results and discussion ...... 80 4.3.1. Selection of Chamber Type and Determination of Extraction Time ...... 80 4.3.2. Evaluation of GC Injector (Fibre Desorption) Desorption Time ...... 80 4.3.3. Analysis of Different Extraction Time for Rose Flower ...... 82 4.3.4. Analysis of Different Extraction Time for Rose Leaves ...... 82 4.3.5. Analysis of Different Extraction Times for Rose Stems ...... 86 4.3.6. Analysis of Volatiles Organic Compounds in Rose Rhizosphere with Different Extraction Times ...... 87 4.3.7. Analysis of Different Extraction Time for Whole Rose Plants ...... 89 4.4. Conclusion ...... 91 Chapter 5 ...... 94 Study on Foliar Application of Plant Growth Regulators on Volatile Organic Compounds Composition in the Flowers of Two Rose Varieties ...... 94 Abstract ...... 95 5.1. Introduction ...... 95 5.2. Materials and Methods ...... 97 5.2.1. Standards and Reagents ...... 97 5.2.2. Apparatus and Equipment ...... 97 5.2.3. Plant Material and Maintenance ...... 98 5.2.4. Sample Extraction Using HS-SPME ...... 98 5.2.5. Optimization of Different Sample Times after PGRs Application ...... 99 5.2.6. Assessment of Peaks and Identification of Volatile Compounds ...... 99 5.2.7. Statistical Analyses...... 99

ix 5.3. Results and Discussion ...... 99 5.3.1. Comparison of Optimal Sampling Times after PGRs Application ...... 99 5.3.2. Analysis VOCs after BA and NAA Application ...... 100 5.3.3. Influence of BA and NAA on VOCs Emitted from Flowers of Two Rose Varieties ...... 102 5.3.4. Identification of VOCs Emitted from Flowers of Two Rose Varieties ..... 103 5.4. Conclusion ...... 107 Chapter 6 ...... 110 Influence of Benzyladenine on Metabolic Changes in Different Rose Tissues ...... 110 Abstract ...... 111 6.1. Introduction ...... 111 6.2. Materials and Methods ...... 113 6.2.1. Standards and Reagents ...... 113 6.2.2. Apparatus and Equipment ...... 113 6.2.3. Plant Material and Maintenance ...... 114 6.2.4. Determination the Quantity of BA Sprayed on the Plant by Filter Paper115 6.2.5. Sample Preparation and Extraction Using HS-SPME ...... 115 6.2.6. Optimization of Sample Collection Times after BA Application ...... 116 6.2.7. Identification of Peaks and Compounds ...... 116 6.2.8. Statistical Analyses...... 116 6.3. Results and Discussion ...... 117 6.3.1. Comparison of Optimal Sampling Times after BA Application ...... 117 6.3.2. The Quantity of BA Applied to Rose Plants as Determined using Filter Paper 117 6.3.3. Influence of BA on Rose Tissues VOCs ...... 118 6.3.4. Identification of VOCs Emitted from Different Rose Tissues ...... 119 6.4. Conclusions ...... 125 Chapter 7 ...... 126 General Discussion, Future Research and Conclusions ...... 126 7.1. General Discussion ...... 127 7.2. Future Research ...... 130 7.3. Conclusions ...... 131 References ...... 133

x Publications and Presentations

Publications

Ibrahim, M., Agarwal, M., Hardy, G.S.J. and Ren, Y.L. 2017. Optimized method to analyse rose plant volatile organic compounds by HS-SPME-GC-FID/MSD. Journal of Biosciences and Medicines, 5(3):13-31.

Ibrahim, M., Agarwal, M., Hardy, G., Abdulhussein, M. and Ren, Y.L. 2017. Optimization of Environmental Factors to Measure Physiological Parameters of Two Rose Varieties. Open Journal of Applied Sciences, 7(10): 585-595.

Ibrahim, M., Du, X., Agarwal, M., Hardy, G., Abdulhussein, M. and Ren, Y.L. 2018. Influence of Benzyladenine on Metabolic Changes in Different Rose Tissues. Plants, 7(4), p.95.

Ibrahim, M., Agarwal, M., Yang, J.O., Abdulhussein, M., Du, X., Hardy, G. and Ren, Y.L. 2019. Plant Growth Regulators Improve the Production of Volatile Organic Compounds in Two Rose Varieties. Plants, 8(2), p.35.

Poster Presentations

Ibrahim, M., Agarwal, M., Hardy, G.S.J. and Ren, Y.L. 2017. Effect of plant growth regulators on rose metabolism-volatile organic compounds released from rose tissues. ISER - 218th International Conference on Agricultural and Biological Science (ICABS) in Macau, China on 26th-27th August 2017. Ibrahim, M., Agarwal, M., Hardy, G.S.J. and Ren, Y.L. 2015. Can Volatile Compounds (VOCs) from Different Parts of Rose Plants be Collected and Analysed? The School of Veterinary and Life Sciences Poster Day 2015 on the 11th of November. Ibrahim, M., Agarwal, M., Hardy, G.S.J. and Ren, Y.L. 2017. Effect of plant growth regulators on rose metabolism-volatile organic compounds released from rose tissues. Murdoch Agricultural Research Symposium on Friday 1st September 2017.

xi List of Abbreviations PGRs Plant growth regulators VOCs Volatile organic compounds BA Benzyladenine NAA Naphthalen acetic acid Mg/L milligram per litter ppm Part per million HS Headspace SPME Solid-phase microextraction GC Gas chromatography FID Flam ionization detection MS Mass spectrometry DVB Divinylbenzene CAR Carboxen PDMS polydimethylsiloxane Min Minute H Hour Cm Centimetre DCM dichloromethane DHS dynamic headspace SHS static headspace CAW carbowax FPD flame photometric detector ML millilitre µl microliter EI electronic impact EV electronic energy IE ionization energy TIC total ion chromatogram RT retention time RI rendition index

xii NIST national institute of standards and technology MVA mevalonic acid IPP isopentenyl diphosphate MEP methyl-erythritol-phosphate DMAPP dimethylallyl diphosphate PAL phenylalanine OMT o-methyltransferases IAA indole acetic acid IBA indole butyric acid PAA phenyl acetic acid 2,4-D 2,4-dichlorophenoxy acetic

xiii Chapter 1 ______General Introduction and Literature Review

1 1.1. General Introduction

The floriculture industry can be divided into the categories of cut flowers (aromatic plants), cut foliage, garden plants, potted plant nursery stock (trees), annuals and perennials, bulbs, and tubers (Van Uffelen and de Groot 2005). The worldwide annual consumption of commercially grown flowers is estimated to be valued at USD $40-60 billion (Ghule and Menon 2013). In 2012, global exports of flowers had expanded by more than 10% annually and were estimated to have reached USD 25 billion (Ghule and Menon 2013). In 2013, the global exports of cut foliage, cut flowers, living plants and flower bulbs had an estimated worth of USD $20.6 billion compared with USD $21.1 billion in 2011, while in 2001 this was only USD $8.5 billion (van Rijswick 2015).

In 2010-2011, Australia exported fresh cut flowers and flower buds valued at AUD $20.2 million to many countries, including Japan, United States, Netherlands, Germany, and Canada which was a decline compared with the 2007-2008 export value of about (AUD 37.2 million) (Anon 2012). Further, in 2016-2017, Australia’s exported fresh cut flowers and flower buds worth valued at AUD 9.7 million, which was a significant reduction, compared with the previous years (Horticulture Innovation Australia Limited 2017). Although export of this amount of cut flowers is of great value, there are not enough flowers for domestic consumption, so Australia imports more flowers than it exports. Australian imports of fresh cut flowers and buds increased from 52,103 flowers in 2007-08 to 204,001 flowers during 2015-16 (Figure 1.1), and imported flowers typically come from Kenya, the Netherlands, Colombia, Ecuador and Singapore (The Australian Horticulture Statistics Handbook 2016) with an import value of AUD $66.8 million (Horticulture Innovation Australia Limited 2017). In contrast, the export of fresh cut flowers and buds declined from 37,223 flowers in 2007-08 to 6,720 flowers in 2015-16 (Figure 1.2) (Anon 2012 and Department of Foreign Affairs and Trade STARS Database, based on ABS 2017).

Recently, the total global land area for cut flower and pot plant production has been estimated at 650,000 ha (Hubner 2016). In Australia during 2013-2014, the land area of cultivated cut flowers in the open or under protection was 4470 ha (Hubner, 2016). The most popular Australian cut flowers include banksia, kangaroo paw, thryptomene, boronia, waratah, Geraldton waxflower and rose. In the Australian cut flower and foliage market, fresh and dried Australian traditional and native flowers are economically important exported floricultural commodities (Anon, 2002). The flowers are mostly exported to Western Europe, Japan, and

2 the US (Horticulture Innovation Australia Limited 2017, NSW Department of Primary Industries, 2002) equating to an estimated 20% of world production and with a value of AUD $500 million (Aldous and Hegde 2013).

In Australia, cut flowers are mainly produced in the southern states including Wimmera and Melbourne in the state of Victoria, the Central Coast of New South Wales and Perth in Western Australia, but flowers are still grown as far north as the Northern Rivers regions of New South Wales and South East of Queensland (Figure 1.3).

194,885 200,000 173,972 180,000 154,001 160,000 140,000 119,809 120,000 83,255 100,000 71,145 80,000 52,103 53,285 54,864 60,000 40,000

20,000 Number imported flowersof Number 0 2007/08 2008/09 2009/10 2010/11 2011/12 2012/13 2013/14 2014/15 2015/16

Years of production

Figure 1.1. Total Australian imports of fresh cut flower and flower buds during 2007 to 2011. (Anon 2012 and the Australian Horticulture Statistics Handbook 2016)

The rose (Rosa hybrida) belongs to the family Roseasea, and is one of the most widely cultivated garden flowers. Cut roses are commonly known as the ‘queen of the flowers’. They rank the highest in popularity and are in great demand throughout the world (Raymond 1999; Gudin 2000). Because of the rose’s beauty and economic value, it is grown in many parts of the world (Kovacheva et al. 2010). Hybrid Tea and Floribunda are two modern rose varieties (Bendahmane et al. 2013).

3 37,223 40,000 32,350 35,000

30,000 23,573 25,000 20,161 17,268 20,000 15,591 12,301 15,000 9,704 7,853

10,000 Number exported flowersof Number 5,000

0 2007/08 2008/09 2009/10 2010/11 2011/12 2012/13 2013/14 2014/15 2015/16 Years of production Figure 1.2. Total Australia exports of fresh cut flowers and flower buds during 2007 to 2016. (Anon 2012 and the Australian Horticulture Statistics Handbook 2016).

Figure 1.3. Major Cut Flower Production Areas in Australia (The Australian Horticulture Statistics Handbook 2016)

The rose has become one of the most important commercial cut flowers in Australia in recent times (AGDHAOGTR 2009). The Department of Foreign Affairs and Trade’s STARS Database (2016) reported that the annual production of roses globally is estimated at

4 approximately 6,720,221 million stems. The shelf life of various species of plants is determined by the senescence of flowers petals, sepals, and stamens and leaves. The longevity of these plant parts is mainly regulated by different PGRs such as auxins, cytokinins and ethylene (Ascough et al. 2006; Scariot et al. 2014). With advancements in technology, growers main objectives in the cut flower industry are the quality of flowers and increased flower production. Various chemicals are now being trialled to control the growth and flowering of roses with a view to creating plants that are compact as well as stretching out or retarding the rates of plant growth (Hashemabadi 2010).

In recent years, scientists have given due attention to application of growth regulators as the most important factor in improving growth rates, yields and quality of floriculture plants (Hedayat 2001). PGRs play vital roles in the coordination of the many growth or behavioural processes, which regulate the morphological and physiological characteristics and biological processes in rose plants. In terms of the morphological and physiological characteristics, PGRs improve the growth and development of plants. In addition, application of PGRs can help achieve increased flower production and improvement in flower quality (Bari and Jones 2009). Various researchers have reported that the application of growth regulators and chemicals help to increase the yields of good quality flowers and vegetative growth in floriculture plants especially rose plants (Anjum et al. 2011). Application of many PGRs, such as naphthalene acetic acid (NAA), benzyladenine (BA) and gibberellic acid (GA3), or their compounds, is becoming a popular method to ensure efficient production. In recent years, remarkable accomplishments, such as manipulating plant growth, crop yield and physiological processes have been actualized with the use of PGRs (Peleg and Blumwald 2011).

BA and NAA play a cardinal role within plants, and these PGRs have an important effect on morphological and physiological characteristics (Zhao 2008). Foliar application of BA and NAA is helpful in improving growth parameters of fruits and cut flowers (Sajid et al. 2009). BA can reduce ethylene sensitivity of cut flowers (Yuan et al. 2012). Cytokinins, including BA, influence the processes of cell division (Francis and Sorrell 2001), biosynthesis of chloroplast pigments (Bondok et al. 1995), nutrient uptake, especially potassium (Guo et al. 1994), and increasing photosynthetic efficiency (Oosterhuis and Zhao 1998). The beneficial use of BA in ornamental plants has been reported numerous times but the useful concentration of application varies with ornamental plants (Werbrouck et al. 1996a). The positive effect of

5 BA has been indicated in many plants, including Dendrobium orchid (Sakai et al. 1998), Lilium (Ohkawa 1979), and Achimenes longiflora (Vlahos 1984).

Little research has been done into the effect of BA application on morphological and physiological parameters within rose plants. Mondal and Sarkar (2017) indicated that application of BA at 200ppm increased all morphological characteristics and chlorophyll content for Hybrid Tea Rose cv. Bugatti. Similarly, Son et al. (1994) showed that using 200ppm of BA increased photosynthesis and the chlorophyll content of leaves but reduced the respiration of leaves of the rose cultivar 'Sonia'. Moreover, Ansari and Zangeneh (2008) reported that rose (Rosa hybrida) treated with 30 mg/L of BA improved vase life, flower diameter, and bud opening and decreased the occurrence of bent neck in flower stems as well increasing the concentration of the soluble carbohydrate content of leaves and petals. Similarly, the effect of NAA on some morphological and physiological factors that increase plant production have made it worthy enough for study by researchers. It is quite clear that endogenous and exogenous application of NAA play an important role in modifying and regulating many of these processes in plants. A study by Iqbal et al. (2009) indicated that foliar application of NAA alone significantly increased morphological, physiological and biological characteristics in plants. Furthermore, NAA is a growth regulator and a common ingredient in many commercial pre-post-harvest horticultural products. It is also used as a rooting agent for propagation of plants from stem and leaf cuttings (Khandaker et al. 2017b). For rose plants, Khan et al. (2007) indicated that an application of NAA at 75 mg/L improved plant height, spread, number of primary shoots, secondary shoots and survival percentage of the Damask rose. Akhtar et al. (2015) and Akhtar et al. (2016) indicated that NAA applied on Rosa centifolia increased most morphological characteristics. Furthermore, Saffari et al. (2004) showed that Rosa damascena treated with 25 mg/L of NAA effected plant height, flower yield and oil content.

Although PGRs have been used in agriculture for decades, little is known about the effects of these compounds on secondary metabolite production (Povh and Ono 2006). Recently, VOCs have received special attention by the scientific community, as they have a wide range of biological and pharmacological uses (Edris 2007). To date, there is no information about how PGRs affect the VOCs produced by roses. Results by Affonso et al. (2007) indicated that treating Lantana camara plants with concentrations (0.44 and 4.4 µmol/L) of BA increased the production of volatile compounds detected by solid phase microextraction (SPME). There are only a few studies about the extraction of VOCs from different rose tissues by headspace solid

6 phase microextraction (HS-SPME). HS-SPME analysis can be used to determine and monitor the composition of living natural materials or natural products and to provide information on the olfactory profiles released from plants or other living natural materials (De Navarre and Schlossman 2009). The SPME technique has become increasingly common because it is a simple, fast, and sensitive sample preparation method that reduces solvent usage, while integrating sampling, and sample preparation steps prior to instrumental analysis (Zhu et al. 2013; Zhang and Pawliszyn 1993).

This chapter sets out to review the literature critically concerning the horticultural industry, especially in terms of application of PGRs, the growth and development of two rose plants, and the morphological, physiological, biochemical and metabolism effect of PGR application. A steadily increasing demand for high-quality flowers and essential oils is evident worldwide. In light of this global trend, we set out to review the literature on the morphological and physiological parameters of roses, as well as the biology, metabolism and volatile compounds emitted from rose plant. The scope of this review covers the period from 1946 until October 2018. A Dialog computer-based evaluation search was conducted in the CAB Abstracts, 1984- 2018; Biosis Previews, 1980-2018; Life Sciences Collection, 1987-2017; Agricola, 1981-2018; Agris International, 1946-20118; European Directory of Agrochemical Products; and Google and internet search accessed until 15th October 2018. In this chapter, we also outline the study aims.

1.2. Literature Review 1.2.1. Roses 1.2.1.1. History of Roses

Roses belong to the family Rosaceae, the third-largest plant family including many ornamental landscape plants, such as berries and fruits, apples, raspberries, cherries, and pyracantha. However, the rose is considered the most important plant in this family (Hummer and Janick 2009). The first wild roses found in nature predate humans by thousands of millennia. Roses (Rosa) have been cultivated as ornamental plants for more than 2,000 years, since the time of the Chinese dynasties (141-87 BC) where they were common in the gardens of the imperial palace (Wang 2005).

The reason for the spread of roses throughout the Northern Hemisphere is unknown but there are North American fossils from 35 million years ago as well as fossil evidence in Asia. Much

7 later, roses were discovered in the tombs of Hawara, Egypt, by the English archaeologist William Flinders Peteri (Shepherd 1954). The modern rose industry started in 1896 in Holland where the first greenhouse was built. At the beginning of the 19th century, modern rose varieties began to be cultivated in France and England. A significant time in the history of the rose was in the early 1950s when they became increasingly popular. The varieties of that time were short-lived and production was centred near local markets. The rose industry rapidly grew, expanding into Europe and near the turn of the 20th century, rose hybrids had spread across North America (Guoliang 2003; Joyaux 2003). The quality of the roses quickly improved due to extensive testing of new varieties. After that, vase life became longer and productivity higher due to improved breeding techniques (Roberts 2003). Today, rose cultivation is extremely popular with very few home or public gardens without at least one.

It is thought that the first roses introduced into Australia were by John and Elizabeth Macarthur of Camden Park in the early 19th century. In 1845, the first record of rose hybridisation occurred in Australia (DHAOGTRA, 2005). Throughout these years of breeding, selections have focused on characteristics such as long vase life, high productivity, and novel colours and consequently only a few varieties are highly fragrant (Roberts 2003).

1.2.1.2. Importance of Rose

The rose has been called the 'Queen of Flowers' as well as the 'King of Flowers’. No other flower surpasses its beauty, colour and fragrance, which is why it is universally considered a favourite flower. Global trade has ensured that the number of ornamental and horticultural plants continue to increase worldwide, and the rose hybrids the most economically important genus (Rosa hybrida) (Gitonga et al. 2014). Rosa hybrida is recognized as economically valuable because it consists of many varieties, which is an important source of raw material for use in agro-based industries such as cosmetics and perfumery. Additionally, roses play a vital role in the manufacturing of various products of medicinal and nutritional importance (Winther et al. 2015). However, another important aspect of rose production is the floricultural trade for cut flowers (Butt 2003). In Australia, rose varieties belong to R. hybrida are considered important, especially Hybrid Tea and Floribunda, both of these are considered modern rose varieties (Scalliet et al. 2008). Australian imports of fresh cut roses increased from 66,127 flowers in 2012-13 to 116,586 flowers during 2016-17 (Figure 1.4). Whilst, exports flowers (Figure 1.5) (Department of Foreign Affairs and Trade STARS Database, based on ABS 2017).

8 There are many uses for roses and including cut flowers for industrial purposes such as perfumes, essential oils, pharmaceutical uses, floristry, rose water and other secondary uses. The major commercial use of roses is the perfume industry (Leghari et al. 2016). Essential rose oil is extracted from rose petals and is considered one of the world's most valuable scented oils. Commercially, rose oil is used in perfumes and room refreshers. Another rose by-product, rose water, has uses in medicines for such benefits as soothing the mind. Rose petals are antiseptic in nature and are used as an ingredient in eyewash. Also, they are rich in vitamins such as vitamin A, C, D, E and B3 (Kumar 2017). For skin products, rose water maintains skin pH and controls extra oil so is used in facemasks for fair skin. It is also used to create healthy, shiny hair. As a food additive, rose water is added to jams, jellies and soups. The rose is a sign of love and peace and is used as gifts in parties, wedding ceremonies, and many religious events (Leghari et al. 2016).

The import and export of cut roses have occupied the top spot in the total import and export of fresh flowers and contributed to boosting the economy of Australia. The reason for the increasing import and export market of cut roses is because of the increase of Australian population as well as more frequent demand for cut roses. Another important use of roses is rose volatiles, which contribute to boosting the value of roses. As mentioned above, like many other flowers, roses are able to produce various pleasant fragrance compounds, and highly scented flowers are used for reproduction of plants (Sakai et al. 2007). Moreover, by comparing the volatile profiles of entomophilous and anemophilous plants, it was found that entomophilous plants released more floral volatile compounds and released them more quickly than anemophilous plants, indicating fast emission of a complex blend of floral volatile compounds required to facilitate biotic pollination (Farré-Armengol et al. 2015).

A scent-baited experiment using water-bowl traps explained that some volatile compounds, such as phenylacetaldehyde, were strong attractants to pollinators but also attracted their unwelcome enemies (Theis 2006). Away from the general biological applications of volatiles, rose volatiles also have significant influence in industry. For instance, both geraniol and 2- phenylethanol give a sweet floral rose-like scent (Burdock 2016); therefore, they are in large demand in the flavour and fragrance markets.

9 116,586 112,458 120,000 98,546 86,972 100,000 66,127 80,000

60,000

40,000

20,000

0 Number of flowers imported imported flowersof Number 2012/13 2013/14 2014/15 2015/16 2016/17

Years of production

Figure 1.4. Total Australian imports of fresh rose cut flower during 2012 to 2017. (Department of Foreign Affairs and Trade STARS Database, based on ABS 2017)

1200 1,106 1,012

1000 831 765 800 546 600

400

Number exported flowersof Number 200

0 2012/13 2013/14 2014/15 2015/16 2016/17

Years of production

Figure 1.5. Total Australian exports of fresh rose cut flower during 2012 to 2017. (Department of Foreign Affairs and Trade STARS Database, based on ABS 2017)

Until 2006, the annual consumption of both compounds and their ester derivatives reached more than 10,000 tonnes (Schwab et al. 2008). For the production of these scented compounds, roses are still widely used in the perfumery industry. However, most of the commercial fragrance compounds are now chemically synthesized via environmentally damaging processes because of the high cost and lack of natural materials for flavour extraction, such as

10 the use of heavy metal catalysts (Guentert 2007). Bio production, including extraction from natural materials and transformation of natural precursors using microorganism methods or isolated enzymes, is a more desirable process from a sustainable point of view (Schwab et al. 2008). Thus, in recent years, some volatile compounds such as monoterpenoids and phenylpropanoids have become the most important target compounds for scientific research and have particular interest for the industry. Furthermore, roses are valuable natural resources for the flavour and fragrance industries. Moreover, there is now a significant research to find economically efficient methods to produce large quantities of the desired compounds.

1.2.1.3. Breeding Rose Varieties

The genus Rosa includes up to 150 species and thousands of varieties. Most of the modern roses are complex hybrids derived from only 10-20 genotypes (Figure 1.6) of the former species, and it was thought that the selection of these 10-20 species was based on easy availability, attractive characteristics or favourable seed (Korban 2007). Thus, only a limited number of progenitors are available for desirable seedlings with specific characteristics, leading to a reduction in genetic variation. Often the wild species are diploids, and nearly all cultivated roses are tetraploids (Korban 2007). Many rose species have resulted by crossing with each other (Figure 1.7) and most of these hybrids are considered modern roses (Bendahmane et al. 2013).

Modern roses are the most common roses of the 20th century and have resulted from crosses of European and Chinese roses as well as Mediterranean types, and various other species of roses during the 1700's and 1800's (Figure 1.6). These modern roses include several classes of roses, such as Hybrid Teas, Polyantha, and Floribunda roses. Hybrid Tea roses are the class of modern roses most often associated with the florist markets. Modern roses are characterized by their large flowers and are usually borne bear one per stem. Hybrid Teas are medium to tall with long stems and include the varieties ‘Peace’ and ‘Dainty Bess’ (Estabrooks 2004). Polyantha roses are a class of modern roses characterized by small-sized flowers borne in large clusters. The word ‘polyantha’ is derived from the Greek word for “many-flowered”. Moreover, Polyantha roses are normally compact in habit with short to medium length stems. Polyantha rose varieties include ‘Mothersday’, ‘Margo Koster’ and ‘China Doll’. Floribunda roses have resulted from crosses between Polyantha and Hybrid Tea roses.

The word ‘floribunda’ literally means ‘flowering in abundance’. Their large clusters of small to medium-sized blossoms characterize floribunda roses. In addition, they are compact in habit

11 and have medium length stems. Floribunda roses include the varieties ‘Betty Prior’, ‘Gruss an Aachen’, and ‘Livin’ Easy’ (Estabrooks 2004). Varieties with new characteristics such as colour, fragrance, shape and size were created by spontaneous mutations (De Vries and Dubois 1995).

Figure 1.6. Schematic representation of major steps of classification of genus Rosa (Nagar et al. 2007).

Many characteristics such as plant morphology, plant physiology, recurrent flowering and scent have been introduced to new varieties by rose genetics, studies, and molecular biology techniques. Currently, breeding roses in greenhouses is popular, due to the fact that the royalties for greenhouse varieties are 3 to 5 times as many as that of garden roses or other types of cultivation. Greenhouse roses can be classified according to the flower size as small-flower, medium-large flower, and large flower varieties. In general, rose varieties show similarity in

12 bud shape, stem length, thorniness, flower shape, flower size, and number of petals within each flower (De Vries and Dubois 1995).

Figure 1.7. Schematic representation of major steps of hybrid rose and modern rose genealogy (Bendahmane et al. 2013)

Recently, breeding efforts have concentrated on varieties that allow a reduction in the use of fungicide and pesticide applications as well as water optimization parameters significant for the environment (De Vries and Dubois 1995). On the other hand, characteristics such as fragrance are important and have a high economic value so are also being studied to be introduced into rose varieties (Pichersky and Dudareva 2007). Perhaps the inclusion of fragrance in new cut rose varieties in the future will be performed by presenting fragrant genes into rose plants using biotechnological tools. The target would be to introduce a fragrance without compromising other important characteristics such as long vase life. There are many studies that have mentioned rose genes, and how these genes effect different characteristics, such as physiological, morphological and biological studies.

13 Recent studies have indicated that roses are an important source of novel genes involved in the biosynthesis of important volatile compounds (Guterman et al. 2001). Another study by Gitonga et al. (2014) showed that by using particular genes of rose plants, particularly from hybrid rose (Rosa hybrida), they could estimate genotype by environment interactions (G × E) as well as the implications of (G × E) for rose breeding. A rose database exists and includes about 877 genes, with some accumulating only in the petals and stamens and a few in the other parts, and are involved in the biosynthesis of volatile compounds characteristic of the rose’s floral scent (Gutierrez 2009). As an example, there are two novel fragrance related genes (OOMT1 and OOMT2) described by Guterman et al. (2001); and these genes are involved in the production of 3,5-dimethoxytoluene, a volatile organic compound that is a main contributor to the scent of Rosa hybrida varieties. Furthermore, these genes are centralized in the petals and are more abundant in the adaxial epidermal cells. The regulation of these genes is critical in the evolution of scent production (Scalliet et al. 2006). Moreover, another gene, identified as RhAAT1, is expressed in developing rose petals and its expression coincides with peak fragrance emission in petal tissue (Shalit et al. 2003). The discovery of these rose fragrance genes has provided the basis for the possibility of using morphology, and biotechnology to produce very fragrant cut rose varieties. However, more physiological studies are needed to introduce these genes without compromising other important characteristics.

Even though rose fragrance contributes a significant commercial value to the cut rose and other industrial rose uses, sufficient studies have not yet been performed to understand the physiological and biochemical relationships between fragrance and pre or post-harvest performance for roses and how to introduce effective methods, such as PGRs, to improve these relationships. Thus, no rapid methods have been established for researchers to understand the effect of growth regulators on the emission of volatile compounds in commercial varieties without affecting plant performance. Moreover, the time required for the release of new varieties of rose, from the time of pollination, is about six to ten years and the rate of success in producing a new variety of rose eligible of being named is only one in every 30,000 to 40,000 seedlings (Dole and Wilkins 1999). Therefore, new volatile compounds studies are important to improve the quality of rose varieties, both to achieve commercial value for industrial purposes such as for flavours and perfume or for better cut flower attributes.

14 1.2.2. Volatile Organic Compounds

Most plants have the ability to store and release a wide range of volatile compounds through their specialized organs and tissues and these have a significant impact on the earth’s atmospheric composition (Das et al. 2013). Volatile Organic Compounds (VOCs) emitted by plants are generally a complex mixture of low-molecular-weight organic compounds with high lipophilic properties, relatively high vapour pressure and low boiling points at ambient temperature (Stashenko and Martínez 2008). While the term “volatile” in relation to these compounds is usually not completely defined, it is typically the vapour pressures of compounds that are considered volatile and this can vary over several orders of volume (Wei et al. 2011). The study of the different volatile mixes of compounds released by plants has been defined as the “volatilome” (Paredes et al. 2013) and specifically, the study of the plant volatilome is termed “volatilomics”. These include the qualitative and quantitative analysis of VOCs, headspace analysis of volatile compounds emitted by whole plant systems, organs, or enzymes, as well as advanced on-line analysis of methods for simultaneous measurements of VOC emissions.

VOCs of plant origin spread through the troposphere where they are very reactive, with lifetimes of minutes to hours (Vogel et al. 2008). They are responsible for the normal functioning of many important terrestrial life processes such as the regulation of the oxidative capacity of the troposphere in terms of the concentration of carbon monoxide, ozone, and the overall aerosol balance. There are many concepts and processes associated with VOCs; on an organismal level the essential functions of airborne VOCs are: 1) semi chemicals that evoke behavioural or physiological responses in other organisms, such as repelling herbivore attackers or attracting beneficial insects for pollination and as well as seed dispersion purposes (Huang et al. 2009); 2) toxic volatiles used by plants as a defence against predators and these toxins usually make the plants smell or taste bad, resulting in herbivores generally not wanting to eat them; 3) “eavesdrop” volatiles are released from attacked neighbours to activate defences before being attacked in a process called plantto-plant signalling (Shirey 2014); and 4) active volatile components are used in the manufacturing of chemicals for flavours, fragrances, preservatives, insecticides, and in drug discovery (Nongonierma et al. 2006; Raguso and Pellmyr 1998).

Analytical chemists have provided the methodological tools and information that may lead to better understanding of the properties, functions, ecology and applications of plant VOCs.

15 These methodological tools should highlight three important points: 1) the evaluation of the most suitable extraction method; 2) the development of methodical and systematic data analysis and processing; and 3) the translation of results that would lead to understanding a specific biological problem.

Depending on the biological problem or purpose, headspace (HS) analysis of volatiles can be a challenge. Here are some examples of biological problems in which HS may be applied: 1) analysis of disrupted plant cells and/or organs (Schütz et al. 1996); 2) volatile profiles from separated and undisrupted plant organs or tissues (Vuorinen et al. 2005); 3) studies in whole plants or plant organs at various development stages (Azam et al. 2013); 4) experimentation performed in the wild, garden, laboratory or greenhouse conditions (Kallenbach et al. 2014); 5) volatile compound studies when plants are under any abiotic or biotic stress (Birkett et al. 2003); 6) VOC extraction at different periods of time or specific times of the day or night (Webster et al. 2010; Harren and Cristescu 2013); 7) volatile profiles can be used for establishment of the differences between plants of the same family but different species or between wild and genetically modified plants (Tattini et al. 2014); and 8) gene and pathway correlation for release of VOCs (Rodríguez et al. 2014).

Furthermore, besides the biological problems, there are several challenges in the analysis of VOCs. Firstly, they are very complex in composition and have a large dynamic range resulting in the obscuring of compounds at low concentrations by the more abundant ones. Secondly, there is a need for sensitive methods that may provide unbiased information on volatiles changes due to biotic and abiotic stress such as herbivore and pathogen attack and environmental factors such as light intensity, pressure, and humidity. Thirdly, in plant biotechnology where plants are genetically modified or naturally cultivated, there is a need for a strategy that permits the identification of volatile secondary metabolites or products where the transformation is important in the understanding of a variety’s function(s) and their impact in the biosynthetic pathway mechanisms. Fourthly, it is important to understand the physiological and others factors which effect the emission of volatiles from different plant varieties. Finally, there is a need to develop improved data analysis, as existing approaches to analyse volatile data still do not allow a fast and unbiased comparative analysis of the volatile composition for a wide range of plant species and varieties that are often the target of investigations; this is especially true for the non-targeted approaches. Additional to VOC recognition, improvements need to occur in the detection and identification of the thousands of

16 molecular fragments that constitute a typical GCMS profile, thus compounds need to be systematically ordered to be able to make comparisons between the volatile profiles of varieties.

1.2.2.1. Detection of VOCs

Several techniques are available for volatile compound detection and quantification. Advanced high-throughput analysis and statistical analysis tools are continuously being modified and developed to enable researchers to obtain high-quality data and to improve data normalization methods. A few important techniques used to extract, detect and analyse VOCs are briefly summarized in the following two sections. The principal methods that have been reported in the literature for the HS extraction and SPME fibre technique and analysis of plant VOCs in different areas of research will be described.

1.2.2.1.1. VOCs Sampling and Extraction

Since the objective of the study was the identification of VOCs in rose plants in terms of an authentic profile of volatile blends emitted by the plants, the selection of the most suitable strategy of plant volatile sampling and extraction becomes the first and most important step. The second objective of this study was to understand the effect of the application of PGRs on VOCs emitted from rose pants, and this will be discussed in more detail later (Sections 1.2.3.1.2 and 1.2.3.2.1). In general, a complete plant or sample of a plant tissue needs to be enclosed in a system with or without air circulation. These are termed “dynamic” or “static” headspace (HS) collection, respectively (Elmore et al. 1997). In the dynamic headspace (DHS) method or trap, ultra-purified inert gas (carrier gas) is pushed into a system containing the plant material or tissue sample and pulled out into a sorbent tube containing an adsorbent matrix (Zhang and Li 2010). Simply, the materials of the system containing the plant or plants, should not bleed, absorb, react or in any way affect VOCs. Usually materials that do not show bleeding include glass vials and containers which can be connected to metal and/or Teflon devices and tubing (Tholl et al. 2006), although these materials may not be completely inert. Usually, the narrow glass or metal sorbent tubes that can be made in the lab or distributed by the industry are packed with the adsorbent most suitable for the compounds of interest. There are some examples of sorbent matrices for the collection of a wide range of VOCs such as silica gel, activated carbon, porous polymers like, 2, 6-diphenylene-oxide, ethenylbenzene, styrene-divenylbenzene and vinylpyrolidone among many others. After different periods of time, which can range from minutes, hours or days, VOCs can be extracted from the sorbent matrix with HS or with pure

17 solvents or mixtures of low point organic solvents such as acetone, n-octane, dichloromethane (DCM), and n-hexane or by the means of thermal desorption into the gas chromatography (GC) instrument’s injection port.

In a static headspace (SHS) system, air is not circulated and volatiles from the HS plant material are absorbed in or adsorbed onto the sorbent matrices. An example of a static system is the use of a solid phase microextraction (SPME) fibres, which is a simple and fast method for compound extraction (Pawliszyn 2001). SPME fibres are usually coated with one or combinations of up to three inert matrices such as polydimethylsiloxane (PDMS), divinylbenzene (DVB), carboxen (CAR), and carbowax (CAW). Table 1.1 lists the most common commercially available polymer coatings. With the help of a holder containing a needle (Figure 1.8), fibres are exposed into the plant HS until an equilibrium occurs between the VOCs and the fibre matrix. The time for this to occur varies from minutes to a few hours. The fibre is retracted into the holder’s needle, and VOCs are then thermally desorbed in the GC injection port for separation and then analysis.

Solid-phase microextraction (SPME) is a solvent-free adsorption and desorption technique where desorption occurs in the GC injector. As mentioned above, it consists of fused-silica fibres coated with different polymers to isolate and concentrate chemicals based on equilibrium. The SPME technique is relatively quick, easy to use, and practical; the extraction, concentration, and introduction all occur in a single step (Picó et al. 2007).

Table 1.1. Summary of commercially available SPME fibres (Vas and Vekey 2004)

18

Figure 1.8. Solid phase micro extraction (SPME) fibre: (a) SPME fibre holder and (b) cross- section of SPME fibre assembly (Abdulra’uf et al. 2012)

The primary limitation is the reduced adsorption capacity of the fibre because of the small volume of polymer coating on the fibre. For instance, heavier materials can be preferentially adsorbed onto fibres but extract preservation is not possible. Combining SPME with other techniques, such as SPME GC-FID/MS, has been successful in profiling living plant, fungal, and soil samples (Jassbi et al. 2010; Stoppacher et al. 2010; Tait et al. 2014). Samples can be collected to analyse over a relatively long time, and at different locations from the analytical instrumentation. However, these techniques are limited to measuring stable compounds that can be easily collected and stored.

To detect large and heavy volatile compounds, extra procedures need to be implemented before the analysis such as using a resin trap or rotary evaporator (Winberry and Jungclaus 1999; Comandini et al. 2012). One type of analytical method involves a direct connection between the analytical instrument and the setup used to generate the gas samples to be measured (Comandini et al. 2012).

Since, this SPME method can be employed in the field, fast analysis of the samples provides the advantage of detecting VOCs emitted from plant organs or from short-lived plant species such as radicals and larger compounds that readily oxidize, condense, or are adsorbed by different surfaces. However, it cannot be used when dealing with large volumes or high- pressured samples.

19 The identification of VOCs by headspace or thermal desorption gas chromatography (GC) uses different columns in combination with appropriate detection methods: mass spectrometry (MS), a flame ionization detector (FID), a flame photometric detector (FPD), an infrared analyser (IR), or photoionization detector (PID) (Dettmer-Wilde and Engewald 2016; Hübschmann 2015). Every type of GC column is selective for a specific compound or chemical group, thus no individual detection method is capable of total VOC estimation. Analysis by GC-MS requires pre-concentration of VOCs in adsorption traps incorporating hydrocarbon or other adsorbents packed in stainless steel or glass tubes. The air sample is transferred through the adsorbent tube, the compounds are trapped inside the tube, and once the compounds are collected, the trap is thermally desorbed at high temperature. Then individual compounds are identified by using a database (library) of mass spectra or by comparing retention times, retention indices and spectra with known standard compounds. Presently, GC-MS is the dominant method used to characterize volatile profiles from soils, housing materials, plant samples and other microorganisms (Serrano and Gallego 2006; Leff and Fierer 2008; Betancourt et al. 2013). Most of the compounds found in the volatile libraries available today are comprised of volatile chemicals identified from both animals and plants. It may be possible that there are many unknown VOCs emitted by microorganisms yet to be identified.

There are many advantages associated with the HS-SPME fibre technique. Headspace analysis with SPME is the main choice for studies related to volatile compounds from small plants, plant organs or disrupted plant tissues. Some of the advantages of using HS systems with SPME include the following: analysis is fast, easy to use and eliminates the use of solvents for the extraction of volatiles, avoiding contamination; by careful selection of the physical and chemical fibre, properties for polarity and thickness, and compounds of different polarity and boiling points (volatiles and semi-volatiles) can be easy and fast to extract; extraction efficiency is increased in small HS volumes (1 to 50 mL); according to manufacturer’s recommendations (Supelco, Bellefonte, PA, USA), and after thermal conditioning, fibres can be reused up to 100 times.

1.2.2.1.2. GC-MS Data Acquisition and Processing

The preferred instrument used in the separation of VOCs is gas chromatography (GC) in which extracted samples can either be injected into the GC inlet port as a liquid solution or by thermal desorption of the adsorbent matrix. A small part of the solution (0.5 up to 5μL) is usually injected into the heated injector in a split or splitless mode from samples that had been extracted

20 by solvents. In thermal desorption, volatile compounds already extracted by SPME fibres or sorbent tubes are heated in the inlet port at temperatures ranging from about 200-350˚C. Volatile compounds are commonly separated on fused silica capillary columns (Tholl et al. 2006) with different stationary phases and different polarities, as in the case of non-polar dimethylpolysiloxanes, and more polar polyethylene glycol polymers.

GC separation of volatile components is performed by gradually increasing the temperature of the oven in which a column is held. Furthermore, individual components are separated and sent into the GC detector by partitioning properties of the volatiles between the inert carrier gas, such as helium or nitrogen and the stationary phase. Detectors can be classified based on their nature and their desirable characteristics in non-selective, selective and specific categories. Non-selective detectors respond to all compounds present in the carrier gas stream except the carrier gas itself. Selective detectors respond with normal physical or chemical characteristics, while specific detectors respond to a single precise component only.

GC detectors can be concentrated and are dependent on mass flow. Some of the desirable characteristics of a detector are the reproducible responses to changes in eluent composition in a carrier gas stream, the low noise signal, high sensitivity and large linear dynamic range. There are more than 15 different types of detectors for GC instruments, however, the most common detectors used in plant volatile analysis are flame ionization detectors (FID), which are very stable, mass sensitive and suitable in the detection of volatile compounds in a wide dynamic range. FID does not respond to inorganic and permanent gases such as CO, CO2, CS2, NH3, and N2; while a photoionization detector (PID), which has more accurate calibration, can be sensitive in the detection of terpenes or molecules having reactive double bonds. The most popular detector in VOC analysis is mass spectrometry (MS). As mentioned earlier, MS relies on the ionization of individual volatiles, commonly by electron impact (EI), and the resulting positively charged molecules and molecule fragments are selected according to their mass-to- charge (m/z) ratio by entering a quadrupole ion trap or a quadrupole mass filter. EI involves shooting energetic electrons in a gaseous neutral electron and some of the energy of the electron is transferred to the neutral when an active electron carrying several tens of electron volts (eV) of kinetic energy hits a neutral electron. If the electron, in terms of energy transfer, collides quite effectively with the neutral electron, the energy transferred can exceed the ionization energy (IE) of the neutral electron. After that, from the mass spectrometric point of view, the most desirable process can occur, for example, ionization by ejection of one electron generating

21 a molecular ion a positive radical ion M + e- M+. + 2e. The total ion chromatogram (TIC) and mass spectra are obtained as outputs, supplying information on compound abundance and retention time (RT), rendition index (RI) and typical ion fragmentation patterns of the molecules as an intensity vs m/z plot. For VOC identification, the mass spectral databases, Wiley and the National Institute of Standards and Technology (NIST), provide possible molecule names for the compounds based on matching the proximity of the fragmentation pattern of the molecule analysed with the fragmentation pattern of a molecule from the database. Figure 1.9 illustrates an example of a TIC and mass spectrum of the volatile compound humulene, extracted from the HS of Arabidopsis thaliana flowers by using an SPME fibre as a source of extraction.

Figure 1.9. (A) Total Ion Chromatogram for Arabidopsis thaliana floral headspace; only 24 minutes are shown. (B) Mass spectrum resulting when a molecule is fragmented by ionizing energy; the base peak 93 m/z and fragment 121 m/z are shown as coloured lines. (C) The database reveals the mass spectrum of humulene, which is compared by the software with the mass spectrum of the molecule in the sample (Caceres 2015).

Databases suggest the possible molecules found in samples, but information needs to be further processed. In order to remove possible technical errors and generate robust and reliable data in the form of normalized peak areas that reflect volatile amounts, different data pre-processing

22 steps should be applied. These pure data can be used as the input for data analysis and in statistical processes. The amount of raw data obtained for each sample varies according to the method applied. For example, GC-MS data recorded could compromise intensity information for approximately 400-800 ions over a separation time of 20-90 min with 20-30 scans per second, so yielding a maximum of 1.3 × 108 data points (Steinfath et al. 2008). Standard processing of these raw data commonly includes many steps, comprising baseline correction, smoothing and spectrum extraction called deconvolution. The analyses are performed on a rather limited number of metabolites in targeted approaches where molecules are known based on previous experiments or knowledge. Samples are quite often compared with other findings by using a reference standard, which has a predetermined mass spectrum, and retention time followed by the export of signal intensities and/or integrated peak areas for further evaluation.

In non-targeted approaches, researchers are usually interested in comparing volatile compounds between plants of different species or varieties in the same family, between healthy and diseased plants, or between wild types of plants and genetically modified versions. In all these cases, replication of the samples is very important to recognize components’ signatures present within the data and then building an unbiased reference per experiment (Styczynski et al. 2007).

Since there are an enormous variety of volatile compounds per sample, in a non-targeted experiment, metabolite levels are not commonly quantified as absolute but are relative to a standard or to each other. Figure 1.10 shows a perfect GC-MS-based on a metabolomics approach to data analysis. Firstly, volatile compounds are extracted from the sample or sample matrix according to the biological problem being solved. Secondly, in the situation of volatilomic studies, volatile compounds are usually separated by using gas chromatography techniques. Thirdly, a fingerprint of the volatile compounds (fragmentation) is obtained by the impact of ionization energy. Fourthly, VOCs have to be identified from the sample from the thousands of molecular fragments that constitute an ideal GC-MS profile (databases and/or analytical standards). Finally, in order to detect and evaluate differences between the samples, comparative analysis is commonly used, using multivariate exploratory techniques dedicated for metabolomics such as principal component analysis, hierarchical clusters analysis, and organizing maps amongst other methods.

23

Figure1.10. Conventional GC-MS-based metabolomics approach; (A) VOC extraction based on the biological problem; (B) gas chromatography separation of VOCs; (C) molecular fragments and mass spectrum interpretation; (D) suggested VOCs identification based on a library match and analytical standards; (E) group clustering based on plant varieties (Caceres 2015).

In any case, in gas chromatography for volatile compound separation, volatile metabolites still co-elute prior to being subjected to detection by MS. As a result, overlapping of unique fragmentation patterns can arise which is the main drawback in volatilomic analysis. Moreover, for the co-elution problem, a high variability in metabolite quantity within huge numbers of biological samples further complicates metabolite identification, thus, limiting the full analysis of a metabolite subset that includes only those volatile compounds that can be reliably identified from the experiment through all the samples (Tikunov et al. 2005).

24 1.2.2.2. Biosynthesis of Volatile Compounds

Fragrance (volatile) of flowers results from the biosynthesis and emission of the low molecular weight of volatile compounds. These compounds have a high enough vapour pressure to be released into the air under room temperature conditions. Most of the VOCs are products of three main biosynthetic pathways. There are products more than “pathways” for example, terpenoids, fatty acid derivatives and phenylpropanoids (Knudsen et al. 1993).

First, terpenes are produced by the terpenoid pathways, which use Acetyl CoA to produce isopentenyl diphosphate (IPP), one of the precursors of all terpenes. IPP is synthesized in the cytosol by the mevalonic acid (MVA) pathway and the plastid is derived from pyruvate and glyceraldehyde-3-phosphate via the methyl-erythritol-phosphate (MEP) pathway Dimethylallyl diphosphate (DMAPP) is the other essential precursor for terpenes, which is synthesized from the MEP pathway in the plastids (Lichtenthaler et al. 1997). Terpenes are the main class of secondary metabolites existing in plants. These compounds are mostly insoluble in water and can be classified according to the number of carbons existing in their structure as monoterpenes, (10 carbons), sesquiterpenes (15 carbons) and diterpenes (20 carbons) (Taiz and Zeiger 2002; Dudareva et al. 2006). Secondly, volatile compounds derived from fatty acids constitute the second largest group of floral volatiles. The main precursors of these volatile compounds are membrane lipids such as linoleic acid or linolenic acid and are the result of the lipogenase pathways, the synthesis of fatty acid derivate volatiles occur (Feussner and Wasternack 2002). Lipogenases (LOX) are the major enzymes included in the synthesis of these volatile compounds. Examples of volatiles synthesized via this pathway include cis -3- hexenol, trans-2-hexenal, and methyl jasmonate (Dudareva et al. 2006). Finally, the volatiles produced in the phenylpropanoid pathway are volatile compounds, which are mainly involved in plant reproduction and defence against their enemies (War et al. 2012). The precursor for phenylpropanoids is phenylalanine (Phe) and the main enzyme that stimulates the biosynthetic reaction is l-phenylalanine ammonia-lyase (PAL). In addition, there is a side branch of the general phenylpropanoid pathway, and some other important volatile compounds known as benzenoids, which originate from trans-cinnamic acid are produced (Boatright et al. 2004). Some of the volatile compounds originating from this pathway are eugenol, phenyl ethanol and methyl benzoate. Besides phenylalanine, other amino acids such as alanine, leucine, valine, isoleucine, and methionine are precursors of more volatile compounds for instance aldehydes, alcohols and esters (Dudareva et al. 2006).

25 1.2.2.3. Biosynthesis of Rose Volatile Compounds

Rose volatiles are a trait particular for each rose species, and humans associate the rose fragrance with the fragrance of “old” European garden roses. The volatiles of roses can be classified into five main fragrance groups: fruity, musk, myrrh, old rose, and tea (Bent 2007). Volatiles in each of these groups are characterized by the composition of different volatile compounds produced in different amounts. The fragrant note of rose plants comes from the release of a wide range of volatile compounds such as alcohols, aldehydes, alkenes, sesquiterpenes, monoterpenes, esters, and ketones (Flament et al. 1993; Kim et al. 2000). Some of these volatile compounds are produced in certain rose species, whereas in others they are not present at all, meaning each rose species has a very specific fragrance note. What makes volatile compounds in one rose species and not in another is the expression of specific genes in the rose that code for enzymes and the presence of the required substrates. The enzyme that stimulates the conversion of one or more compounds may be present in a rose species that emits this particular compound but when the enzyme is absent, the rose cannot emit this specific compound. The enzymes that play a key role in the biosynthesis process of rose volatiles are mainly released from the petals, but there are some enzymes present in other rose tissues that emit other types of volatile compounds (Guterman et al. 2002; Lavid et al. 2002).

Many researchers have studied in detail the biosynthetic pathways from some of the main volatile components of rose fragrance to expand the knowledge of the major genes and enzymes that contribute to emitting the scent of this special flower (Lavid et al. 2002; Scalliet et al. 2002; Watanabe et al. 2002; Wu et al. 2004; Scalliet et al. 2006). For instance, one of the major volatile compounds in the scent of the European rose, Rosa damascene, is 2- phenylethanol, and two hypothetical biochemical pathways have been suggested for the production of this specific compound. One of them is L-phenylalanine, mainly by its conversion into phenylpyruvic acid which is later transformed into either phenylacetaldehyde or phenyllactic acid and after that converges into 2-phenylethanol (Watanabe et al. 2002). Another recent study indicated that the converts of 2-phenylethanol from shikimic acid occurred via chorismic acid, phenylacetaldehyde, and L-phenylalanine (Yang et al. 2009). Another important component of rose scent is 3,5 dimethoxytoluene (orcinol dimethyl ether), present in nearly all types of the modern Hybrid Tea roses, and gives the characteristic tea note fragrance for this rose group (Scalliet et al. 2008). The biosynthesis processes of this compound occur by the action of O-methyltransferases (OMT) which acts on non-methylated precursors such as orcinol, eugenol and orcinol monomethyl ether guaiacol (Lavid et al. 2002; Scalliet et

26 al. 2002; Wu et al. 2003). The other main volatile acetate ester component responsible for the fragrant note of Rosa hybrida is geranyl acetate produced from the monoterpene geraniol by the action of RhAAT1 enzyme (Shalit et al. 2003). The volatile 1,3,5-trimethoxybenzene is considered a major volatile compound of Rosa chinensis scent, and is synthesized in three methylation steps from phloroglucinol. The first step is catalysed by phloroglucinol o- methyltransferase (POMT) enzyme and the second and third steps are catalysed by OMT (Wu et al. 2004). Briefly, in roses, enzymes that are potentially involved in the biosynthesis of rose volatile compounds have been identified (Chen et al. 2011; Farhi et al. 2010; Guterman et al. 2002; Hirata et al. 2012; Hirata et al. 2016; Huang et al. 2009; Lavid et al. 2002; Magnard et al. 2015; Sakai et al. 2007; Scalliet et al. 2002; Scalliet et al. 2006; Shalit et al. 2003; Yan et al. 2012; Wu et al. 2004; Wu et al. 2003), and these are summarized in Figure 1.11.

Although three main biosynthetic pathways occur, the diversity of volatile compounds released from plant flowers is very wide. As an example, more than 400 floral volatile compounds have been identified from roses (Flament et al. 1993), and these volatile compounds result from three groups of roses: species rose, old garden rose and modern roses (Table 1.2). The enormous variety of volatile compounds come from enzymatic modifications such as acetylation, hydroxylations, and methylations, which occur in synchronism with the main biosynthetic pathways. This study will concentrate on the VOCs emitted from these three main biosynthetic pathways in different rose varieties.

The production of rose volatiles is a complex trait. Factors such as the capability of aroma detection by humans, the physiology of volatile emission, pre and post-harvest transport conditions and the physiology of roses during senescence are some of the reasons that may explain why fragrances are not as predominant as they should be in cut roses.

27

Figure 1.11. Summary of known biosynthesis pathways of volatile compounds in cytosol and plastid in rose. Terpenes precursors are normally synthesized via the cytosolic acid (MVA) pathway and the plastidial metherythritol phosphate (MEP) pathway. Abbreviations: ACC: acetyl-CoA carboxylase; AAAT: Aromatic amino acid aminotransferase; AAT: alcohol acetyltransferase; CCD: carotenoid cleavage dioxygenase; DMAPP: dimethylallyl diphosphate; DMT: 3,5-dimethoxytoluene; EGS: eugenol synthase; FPP: farnesyl diphosphate; FPS: E,E-FPP synthase; GDS: germacrene D synthase; GGPP: geranylgeranyl diphosphate; GGPS: GGPP synthase; GPP: geranyl diphosphate; GPS: GPP synthase; IDI: isopentenyl diphosphate isomerase; IGS: isoeugenol synthase; IPP: isopentenyl diphosphate; LIS: linalool synthase; NUDX: NUDX hydrolase; OOMT: orcinol O-methyltransferase; OMT: O- methyltransferase; PAAS: phenylacetaldehyde synthase; PAR: phenylacetaldehyde reductase; POMT: phloroglucinol O-methyltransferase; PPDC: phenylpyruvate decarboxylase; TMB; 1,3,5-trimethoxybenzene. *, the location and the function of these proteins are unproven. Double black arrow indicates multiple enzymatic reactions; single purple arrow indicates transportation (Sun 2017).

28 Table 1.2. Several major volatile compounds present in individual roses that belong to one of the three groups: species rose, old garden rose and modern roses. Volatile data originate from Antonelli et al. (1997); Joichi et al. (2005) and Magnard et al. (2015).

Relative abundance of volatile compound* 1 5 Name 2 3 4 (Most abundant) (Least abundant) Species roses DMT 2-Phenylethanol Dihydro-β-ionol Rosa gigantean R. multiflora Old Garden roses R. × alba cv. ‘Felicité 2-Phenylethanol Benzyl alcohol Geraniol Nerol Geranyl acetate Parmentier’ R. × alba cv. ‘Maidens 2-Phenylethanol Benzyl alcohol Geraniol Nerol Geranyl acetate Blush’ R. × alba cv. ‘Mme Plantier’ 2-Phenylethanol Benzyl alcohol Geraniol Nerol 2-Phenylethyl benzoate R. × alba cv. ‘Queen of 2-Phenylethanol Benzyl alcohol Geraniol Nerol Benzyl benzoate Denmark’ Bourbon R. cv. ‘Louise 2-Phenylethanol Benzyl alcohol Geraniol Nerol Isoeugenol Odier’ Bourbon R. cv. ‘Mme Isaac Benzyl 2-Phenylethanol Geraniol Nerol Geranyl acetate Pereire’ alcohol Bourbon R. cv. ‘Variegata di Benzyl 2-Phenylethanol Geraniol Nerol Benzyl benzoate Bologna’ alcohol R. × centifolia 2-Phenylethanol Benzyl alcohol Nerol Geraniol Geranyl acetate R. × centifolia cv. ‘Blanche 2-Phenylethanol Geraniol Geranyl acetate Nerol Isoeugenol Moreau’ R. × centifolia cv. ‘Old Pink 2-Phenylethanol Benzyl alcohol Nerol Geraniol E,E-farnesol Moss’ R. × centifolia cv. ‘Soupert et 2-Phenylethanol Geraniol Benzyl alcohol Nerol Geranyl acetate Notting’ R. chinensis cv. Dihydro-β- Z-3-Hexenyl acetate Z-3-Hexenol 2-Phenylethanol TMB ‘Semperflorens’ ionol Z-3-Hexenyl R. chinensis var.spontanea TMB Z-3-Hexenol Hexyl acetate Dihydro-β-ionol acetate Dihydro-β- R. chinensis cv. ‘Single Pink’ Z-3-Hexenyl acetate Z-3-Hexenol TMB ionol & Linalool Geraniol R. chinensis cv. ‘Viridiflora’ Z-3-Hexenyl acetate Z-3-Hexenol TMB R. chinensis cv. ‘Mutabilis’ Geraniol Geranial Germacrene D E-2-Hexenal Z-3-Hexenyl acetate &

29 Linalool R. chinensis cv. ‘Miss Lowe’ Z-3-Hexenyl acetate Z-3-Hexenol 2-Phenylethanol TMB Dihydro-β-ionol Z-3-Hexenyl acetate R. chinensis cv. ‘Hermosa’ 2-Phenylethanol TMB & Z-3-Hexenol R. chinensis cv. ‘Old Blush’ Geraniol Dihydro-β-ionol TMB E-2-Hexenal Germacrene D Rosa. x damascena 2-Phenylethanol Citronellol Geraniol Linalool Nerol R. x damascena cv. Benzyl 2-Phenylethanol Geraniol Nerol Geranyl acetate ‘Kazanlik’ alcohol R. x damascena cv. ‘Marie Geranyl 2-Phenylethanol Geraniol Nerol Benzyl alcohol Louise’ acetate R. x damascena cv. ‘Pompon Benzyl 2-Phenylethanol Geraniol Nerol Isoeugenol des Princes’ alcohol R. x eglanteria cv. ‘Manning’s Benzyl alcohol Geraniol 2-Phenylethanol Nerol Benzyl benzoate Blush’ R. x gallica cv. ‘Belle Isis’ 2-Phenylethanol Geraniol Benzyl alcohol Nerol Geranyl acetate R. x gallica cv. ‘Charles de 2-Phenylethanol Geraniol Benzyl alcohol Nerol Geranyl acetate Mills’ R. x gallica cv. ‘Jenny Benzyl 2-Phenylethanol Geraniol Geranyl acetate Nerol Duval’ alcohol R. x gallica cv. ‘Rose du Maitre 2-Phenylethanol Benzyl alcohol Geraniol Nerol Benzyl benzoate d’Ecole’ R. x gallica cv. ‘Tour de Benzyl 2-Phenylethanol Geraniol Nerol E,E-farnesol Malakoff’ alcohol Nonanal + Geraniol & Noisette R. cv. ‘Aimée 2-Phenylethanol Benzyl alcohol Zrose Benzyl Nerol Vibert’ oxide benzoate Noisette R. cv. ‘Alister Stella 2-Phenylethanol Phenylacetaldehyde Geraniol Geranial Geranic acid Grey’ Old Garden roses Perpetual R. cv. ‘Ferdinand 2-Phenylethanol Geraniol Nerol Z-3-Hexenol Benzyl benzoate Pichard’ Portland R. cv. ‘Compte de Geranyl 2-Phenylethanol Geraniol Nerol Benzyl alcohol Chambord’ acetate R. x rugosa cv. ‘Roseraie de Benzyl Geraniol Nerol 2-Phenylethanol Benzyl benzoate l’Hay’ alcohol β- Dihydro-β- Tea R. cv. ‘Lady Hillingdon’ DMT Hexenyl acetate Geraniol Caryophyllene ionol

30 Modern roses R. x hybrida cv. ‘Diorama’ DMT Geraniol Germacrene D β-Ionone Z-3-Hexenyl acetate R. x hybrida cv. ‘Grand Z-3-Hexenyl β- E,E-α-Farnesene & DMT β-Ionone Mogul’ acetate Caryophyllene β-Ionol R. x hybrida cv. ‘Papa Geraniol 2-Phenylethanol Geranial Farnesol Nerol Meilland’ R. x hybrida cv. ‘Rouge DMT E-2-Hexenal Benzyl alcohol Hexanal Hexanol Meilland’ R. x hybrida cv. ‘Hacienda’ 2-Phenylethanol Geraniol Citronellol Geranial Farnesol R. x hybrida cv. ‘Pariser Citronellol & Geraniol Geranial Nerol Farnesol Charme’ Eugenol Z-3-Hexenyl R. x hybrida cv. ‘Baccara’ Benzyl alcohol Germacrene D Z-3-Hexenol DMT acetate Linalool & Dihydro- R. x hybrida cv. ‘Blue Moon’ Geraniol Citronellol Nerol Citral β-ionol 3,4-Dihydro-β- R. x hybrida cv. ‘Anna’ Germacrene D DMT Dihydro-β-ionol E-2-Hexenal ionone R. x hybrida cv. ‘Black Benzyl alcohol Benzaldehyde DMT Hexanal Z-3-Hexenol Baccara’ Note: * 1-5: indicates relative abundance of the indicated compound within the volatile blend, 1 indicates the most abundant and 5 indicates the least abundant. Abbreviation: TMB, 1,3,5-trimethoxybenzene; DMT, 3,5-dimethoxytoluene.

31 1.2.2.4. Human Perception

Fragrances are subjective traits. Not all human individuals are capable of smelling and detecting the same concentration and volatiles emitted from plant flowers. One person may consider the rose a nice fragrant flower, whilst another might not even consider that the same flower as fragrant at all.

There are many factors effecting the response of humans to fragrances such as mood, the instinctive physiological sensitivity to the specific compound, and familiarity with a similar fragrance stimulus (Stefanowicz 2013). The physiology of human smell does not participate in the optimum detection of flower volatiles. The olfactory receptors in humans are located in a very high position in the nasal cavity, which may affect the proper detection of volatiles during exposure to them, since only a small percentage of volatile gases in the nose reach the sensory organs (Lawless and Heymann 2010). In fact, some organic molecules stimulate the olfactory sense of humans more than others, and some of these are recognized at parts per billion concentrations, whilst others are detected at parts per thousand. In other words, rose volatiles vary in their ability to be perceived with some requiring only small amounts to be detected while others are only detected when large amounts are present. The threshold of detection may also differ between individuals, and increased periods of constant stimulation may decrease the perceived intensity over time (Lawless and Heymann 2010). Furthermore, one person can be sensitive to a special scent and be completely insensitive to another. Thus, at the time of asking human individuals to classify a rose as fragrant or not and this will depend on many different factors such as the ability to sense the main components of the floral trait and to the personal experience to the fragrance.

1.2.2.5. Conditions that Affect the Cultivation and Selection of Fragrant Varieties

Fragrant roses are considered luxury products since they are distributed for sale at very high prices at low volumes (Mouchette 2001; Bent 2007). Even though the fragrances are very desirable to consumers, the majority of cut rose varieties lack this important feature. From the 3,900 rose varieties described as far back as 1956, only approximately 20% of them are described as highly fragrant (Bent 2007). There are many other factors that affect fragrant varieties, among these are PGRs. In recent years, scientists have given due attention to the idea of regulating plant growth for the purpose of improving the growth, yield and quality (Hedayat 2001). Furthermore, a study by Sangwan et al. (2001) indicated that one of the factors that affect the emission of volatile compounds from aromatic plants are PGRs.

32 1.2.3. Plant Growth Regulators

Plant growth regulators are organic compounds, not nutrients that influence all physiological processes that occur in the plants. PGRs include two groups, naturally occurring such as phytohormones, and synthetic compounds. The use of both naturally occurring and synthetic PGRs has generally increased in agriculture and horticulture since the 1940s. They are used to control the developmental processes that occur in plants from germination, vegetative growth, reproductive development and maturity, through to post-harvest preservation (Basra 2000). PGRs are compounds that act at very low concentrations and have various regulatory roles during growth and development, and are also used in the defence of plants against threats. There are five well-established PGRs involved in the growth of plants: auxins, cytokinins, gibberellins, ethylene and abscisic acid (Rademacher 2000; Sadava et al. 2009). In contrast, Davies (2010) suggested that there are six groups of PGRs (Table 1.3). PGRs are able to modify plant growth and development, acting in a regulatory capacity rather than a nutritional role (George et al. 2008). They are mostly only applied at low rates and show no phytotoxic effects (Rademacher 2015).

The response of plants to PGRs may vary due to differences in species, varieties, environmental conditions, physiological and nutritional status, endogenous hormonal balance and stage of development. Most plants have the ability to store excessive amounts of exogenously supplied growth regulators in the form of reversible conjugates, which emit active hormones when the plant needs them during growth periods. Amanullah et al. (2010) noticed that PGRs enhance the source-sink relationship and translocation of photo-assimilates to the sink, thereby helping effective flower formation, fruit and seed development and enhancing the productivity of crops. The use of PGRs is becoming popular to ensure efficient production of auxins, cytokinins, gibberellins, or their synthetic compounds inside the plants. It has been indicated that the application of PGRs enhances plant growth and crop yields (Hernández 1996). PGRs modify growth and development in different ways under natural and stressful environmental conditions.

The role of PGRs on various physiological processes in plants is well known, and they enable a change in the phenotype of plants within one season to achieve desirable results. They are now being widely used to increase the production of many crop types (Barrett 2001). Whilst nutrients are required for natural growth and development of a crop, attempts to improve the quality of plant production has looked at combining both organic and inorganic chemicals

33 (Falticeanu 2004). The most important factor for success is that application of PGRs at a stage in growth where the physiological processes are at their peak. Furthermore, PGRs have the ability to influence several morphological, physiological and biological processes in plants. Some characteristics such as height and number of branches are important morphological parameters contributing to increase yields (Darasteanu et al. 2005). Plant growth regulates as the other substances have many factors that effecting and limiting on their work in plants. There are several factors that limit or effect plant hormones, such as light, soil fertility, temperature, parasitic plants and chlorophyll (Gustafson 1946).

Table 1.3. Hormones classification and function (Davies 2010)

PGRs Name Function in Plants Brassinosteroids (BRs) Control of division; Growth by elongation; differentiation of the vascular system; inhibiting root growth, and BRs are needed for fertility. Cytokinin Control of cell division; bud development; development of the leaf blade; senescence retardation and promote shoot initiation.

Abscisic acid Control of stomata apparatus function; growth inhibition; seed dormancy; inhibits shoot growth and induces storage protein synthesis in seed.

Auxin Induction of elongation growth and stem growth; stimulates cell division; differentiation of phloem elements; apical dominance; tropisms and initiation of root formation.

Ethylene Senescence induction; initiation of defensive responses; decrease elongation and Leaf and fruit abscission.

Gibberellins Stem elongation; initiation of seed germination; cell division and elongation and Enzyme production during germination.

This study will concentrate on just two groups of PGRs; cytokinins, including benzyladenine (BA) and auxins including naphthalene acetic acid (NAA). In the next two sections, BA, NAA, and their effects will be discussed as well as their roles in improving and developing different plants processes such as morphological, physiological characteristics and biological processes.

34 1.2.3.1. Cytokinins (Benzyladenine)

Cytokinins have long been recognized as essential plant hormones that affect almost all aspects of plant physiology and development (Mok 1994). Cytokinins are known to stimulate or inhibit a large number of physiological processes, and are closely associated with functions of other PGRs, for instance auxins, a group of endogenous plant growth substances. The main site of cytokinin biosynthesis in tall plants is in the roots from where they are then transported to the other aerial portions of the plant through the xylem. These hormones have strong effects on plant physiological processes and are intimately involved in the regulation of cell division, chloroplast development, apical dominance, anthocyanin production and maintenance of the source-sink relationship (Bultynck and Lambers 2004). Cytokinins have been used for decades for a number of purposes such as in research laboratories, tissue culture, pomology, turf management, woody plant production and the cut flower industry. However, they have mainly been used in commercial floriculture, containerised crops and bedding plants. In addition, Cytokinins promote branching in some plant species, but the concentrations and effects differ widely among species (Carey Jr 2008). Cytokinins are considered the most important senescence-retarding hormones and their exogenous application has been observed to prevent the degradation of chlorophyll and photosynthetic proteins in plants as well as reversing leaf and fruit abscission. Benzyladenine and kinetin are synthetic cytokinins well known to significantly improve plant growth and plant development, even under environmental stress. Also, Cytokinins stimulate leaf expansion and the development of reproductive organs, and delay senescence (Bultynck and Lambers 2004). This group of PGRs are active in promoting cell division and the regulation of many physiological processes during growth and adaptation to environmental conditions. Moreover, cytokinins are involved in root architecture development including root nodulation in leguminous plants. One of the earliest signs of exogenous cytokine applications to roots is the enhanced formation of pseudo-nodule structures on legumes and even non-leguminous plants (Barrett 2001). Furthermore, exogenous application of cytokinins on leguminous plants affected root-induced responses similar to node factors (Falticeanu, 2004).

There are more than 200 known natural and synthetic cytokinins. Isopentenyl adenine, zeatin and dihydrozation are examples of natural cytokinins abundant and physiologically active in plants (Figure 1.12). Benzyladenine and kinetin are examples of synthetic cytokinins (Figure 1.13) with high biological activity (Newman 2014). For ornamental plants, one registered cytokinin product for use is a benzyladenine (BA) based PGR. It is labelled only for

35 Hosta, Echinacea and Christmas cactus; however, its expanded use label allows it to be trialled on any ornamental plant (Carey 2008). It can be applied as a foliar spray or as a substrate drench at concentrations from 1-3000 mg/L

Figure 1.12: Structures of natural cytokinins(Newman 2014).

Figure 1.13: Structures of synthetic cytokinins (Newman 2014).

The useful application concentration varies greatly among ornamental plants and is mostly unknown. There is evidence that cytokinins mixed with anti-GAs may have synergistic effects

36 on branching (Werbrouck et al. 1996b). There is also evidence that BA mixed with anti- ethylene products might have synergistic effects on preventing senescence in plants. However, mixes of these substances with other PGRs are not common. Another BA based product called ProShear was used in the past to promote lateral branching in woody plants (Crovetti and Shafer 1988).

BA is utilized in many different ways within plants and it is applied to plants, especially ornament plants (Floriculture plants) for a number of functions and these include growth, development, morphological and physiological processes. BA plays a key role in many plant processes such as cell division, morphogenesis, lateral bud growth / apical dominance, flower promotion or inhibition, leaf expansion, delayed senescence, stomatal opening, chlorophyll biosynthesis, flower sex ratios, vernalization, parthenocarpy, nutrient signalling, photosynthate partitioning, seed development and germination, cellular differentiation, maturation, stress resistance, and mycorrhizal interactions (Carey 2008). Endogenous BA levels have been observed to fluctuate during floral induction (Bernier et al. 1993), and while BA does not excite flowering, it does regulate events such as cell division that occur in plants when flowering has occurred. Furthermore, BA has been used to enhance flower bud formation in Easter cactus after application in the initiation floral phase of plant growth (Boyle 1992), and for modification of plant architecture in 'Crimson Giant' Easter cactus. When managed properly, growers can easily multiply the number of flower buds with the application of BA. Mixes of BA + Gibberellin (GA), or only BA, have been used to thin flowers on apple trees to avoid uneven fruit yield. In this situation, it was applied on flowering trees in heavy years to cause flowers to drop off (Western Plant Growth Regulator Society 2000).

The results of applications of BA to other ornamental plants to determine its influence on flowering are inconsistent because of the wide range of physiological attributes of each plant. BA has been shown to delay flowering on many plants, but in others, it increases bud set and promotes flowering. Furthermore, BA has been used in the vegetable industry to change flower sex proportion of dioecious and monoecious plants to increase the number of female flowers in plants ready to produce fruits (Khryanin 2002). It has been used to prevent bud blast in tulips (De Munk and Gijzenberg 1977), and delay flower senescence in lilies (Ranwala and Miller 1998). There is also some evidence indicating that cytokinins such as BA can help increase the size of the flowers of some plants, for example, petunia flowers (Nishijima et al. 2006).

37 1.2.3.1.1 Effect of BA on Morphological and Physiological Characteristics

Applying BA may modify morphological and physiological characteristics of plants and induce the best adaptations to the environment through improving the growth and yield by increasing fruit set, fruit number, size and other parameters. Furthermore, productivity in horticultural systems, especially ornamentals, is often dependent on manipulation of morphological and physiological activities of the crops by chemicals or PGRs (Yeshitela et al. 2004). As mentioned earlier, BA is considered one of the most active cytokinins in plant processes and recently, BA has been identified as a natural cytokinin in a number of plants (Davies 2010). Furthermore, BA can be considered an endogenous-like compound with little risk to the environment (Davies 2010). Nevertheless, morphological and physiological responses to BA application may be connected with increased levels of endogenous cytokinin concentrations (Van Staden and Crouch 1996). Morphological and physiological characteristics can be promoted or inhibited with the application of BA in plants. There have been numerous studies on BA and its morphological and physiological effects in plants; these have been reviewed by Askari-Khorasgani and Mortazaeinezhad (2016) and Akhtar et al. (2016) respectively. In contrast, there have only been a few studies on the effect of BA application on morphological and physiological parameters on rose plants (Table 1.4.).

1.2.3.1.1. Effect of BA on Emission of VOCs

Several gaseous components are present in the atmosphere, mostly nitrogen, oxygen, carbon dioxide and different types of volatile compounds produced by surrounding organisms, including plants (Buddendorf-Joosten and Woltering 1994; Shiojiri et al. 2006). Recently, it has been shown that most plants have evolved the capacity to release and detect VOCs in their environment (Yamagiwa et al. 2011). A study by Sangwan et al. (2001) showed that many factors affect the emission of aromatic compounds in plants and among these are PGRs.

Essential oils are one type of aromatic compound and their production and quality are important in the aromatic industry. Among the factors influencing the quality of aromatic compounds and essential oil production are PGRs, including BA (Prins et al. 2010). Exogenous application of PGRs on plants can affect essential oil production and chemical composition (Shukla and Farooqi 1990), thus influencing not only the quantity, but also the quality of essential oils. Exogenous application of BAs in different concentrations can result in different responses from treated plants. A study by Farooqi and Sharma (1988) confirmed that a decrease in plant size

38 associated with a higher leaf production was the cause of increased essential oil yield in Japanese mint plants treated with BA and NAA.

Another study by Eid et al. (2010) indicated that tuberose plants treated with 100ppm BA increased carbohydrate content and percentage of essential oils from different plant organs. In addition, a study on gladiolus plants after application of 100ppm BA showed increased phenol compound content (Koley 2015). Similarly, a study by Sudriá et al. (2001) showed that using 0.1 mg/m3 BA in in vitro culture control plantlets of Lavandula dentata increased the content of essential oils by 150%. Whilst, Swathi (2012) reported that application of 10ppm BA on coriander plants increased leaf essential oil content. A study by Asangi and Vasundhara (2013) demonstrated that patchouli plants treated with 300ppm BA showed a high oil content compared with a control. Using another technique, a study by Zielińska et al. (2011) using the headspace solid-phase microextraction–gas chromatography–mass spectrometry method on volatile compounds showed that in the presence of BA in the culture medium significantly influenced the composition of volatiles. In roses, there much work has been done on the subject of volatile organic compounds (Table 1.5).

However, there have been from my knowledge no work on the effects of BA on VOCs emitted from rose plants. All previous work only related to the morphological and physiological parameters effected by PGRs in roses as well as the effect of PGRs (BA) on essential oil production.

1.2.3.2. Auxins

Based on studies conducted in the 1960 and 1970s, auxins were shownto exist in many living organisms such as brown (Macrocystis and Laminaria), red (Botryocladia), and green (Enteromorpha, Chlorella, and Cladophora) algae and in cyanobacteria (Oscillatoria) (Tarakhovskaya et al. 2007). Auxins are a class of PGRs involved in many aspects of plant growth and development at the molecular and whole-plant level. There are natural auxins, such as indole-3-acetic acid (IAA), phenylacetic acid (PAA) and indole-3-butyric acid (IBA) (Figure 1.14A) (Finet and Jaillais 2012) and synthetic auxins such as Naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 3,6- dichloro-2-methoxybenzoic acid (dicamba), 4-amino-3,5,6-trichloropicolinic acid (tordon or picloram) (Figure 1.14B).

39 Table 1.4. Summary of research on morphological and physiological effects of BA application on rose plants

Rose varieties Notes Reference Concentrations: 0, 10 and 20 mg/L Morphological effects: 20 mg/L improved flowers diameters and longest shelf life (Askari-Khorasgani and Rosa ‘Yellow Finesse’ Physiological effects: improved leaf chlorophyll content and produced petal Mortazaeinezhad 2016) carotenoid content Concentrations: 10, 20 and 30 mg/L Morphological effects: 30 mg/L improved Vase life, flower diameter, bud opening (Ansari and Zangeneh Rose (Rosa hybrida) and decreased bent neck 2008) Physiological effects: increased concentration of leaf and petal soluble carbohydrate content Concentrations: 0, 100, 200ppm Morphological effects: 200ppm increased plant height, secondary shoots, leaf area, Hybrid Tea stalk diameter, flowering duration and vase life, while 100ppm showed maximum (Mondal and Sarkar 2017) Rose cv. Bugatti spread, flower diameter at cup shape and number of flowers per plant (yield). Physiological effects: 200ppm increased chlorophyll content Concentrations: 0 and 200ppm Rose 'Sonia' Physiological effects: 200ppm increased photosynthesis and chlorophyll content of (Son et al. 1994) leaf, but reduced respiration of leaf Concentrations: 0.5 mg/L Rosa centifolia (Akhtar et al. 2016) Morphological effects: increased number of shoots, number of roots and root length

40 Table 1. 5. Summary of various studies on volatiles compounds emitted from rose plants

Rose species Extraction or detection methods References Rosa hybrida L. cv. Honesty Headspace with GC-MS (Helsper et al. 1998) (Sparinska and Rostoks Hybrid Rugosa Headspace solid-phase microextraction (SPME) with GC-MS 2015) Rosa damascena Solvent extraction with GC-MS (Rusanov et al. 2011b) Rosa hybrida Solvent extraction with GC-MS (Shalit et al. 2003) R. rugose, R. canina, R. vosagiaca,R. caryophyllaceae, R. coriifolia, R. subcanina, R. Hydrodistillation extraction (essential oils) with FID/ GC-MS (Nowak 2005) eglanteria, R. villosa, R. tomentosa Headspace solid-phase microextraction (HS-SPME) and Rosa hybrida ‘Anna’ (Bergougnoux et al. 2007) Solvent extraction with FID/ GC-MS Rosa hybrida cv Pariser Charme Headspace with GC-MS (Zvi et al. 2012) Rosa damascena Head-space or solid/liquid extraction with GC-MS (Caissard et al. 2005) Headspace solid-phase microextraction (HS-SPME) with GC- Rosa chinensis ‘Pallida’, R damascene, R. centifolia (Zhou et al. 2015) MS Rosa damascena Mill. Headspace with TCT-GC/MS (Yang et al. 2014) Headspace solid-phase microextraction (HS-SPME) with GC- Rosa hybrida 'Orange Flash' and 'Blue Moon' (Lee et al. 2008) MS Rosa Hybrida Hydrodistillation with GC-MS (Bazaid 2009) Rosa damascena Mill. HS-SPME with GC-MS (Baydar et al. 2016) (Rosa damascena Hydrodistillation extraction solvent (essential oils) with FID/ (Dobreva 2013) Mill. and Rosa gallica L GC-MS

41 Damask Rose and (Karami and Jandoust Headspace auto-sampler with FID/ GC-MS Persian Musk Rose 2016) Rosa damascena Mill. HS-SPME with GC-MS (Koksal et al. 2015) Rosa hybrida L. cv. Bacara Hydrodistillation extraction (essential oils) with GC-MS (Salmi et al. 2018) Rosa x hybrida cv. ‘Yves Piaget’ Solvent extraction with GC-MS (Chen et al. 2015) Rosa brunonii Lindl Hydrodistillation extraction (essential oils) with FID/ GC-MS (Kaul et al. 1999) Rosa hybrida HS-SPME with GC-MS (Héthelyi et al. 2010) (Jandoust and Karami Persian Musk Rose (Rosa moschate Hermm.) Headspace with FID/ GC-MS 2015) Rosa damascena Mill. Hydrodistillation extraction (essential oils) with FID/ GC-MS (Verma et al. 2011) Hydrodistillation and Dry Distillation with HRGC-FID and Rosa canina L. (Hosni et al. 2010) GC-MS Rosa damascena Wild Rose Steam distillation (essential oils) with GC-MS (Khan et al. 2017) Rosa roxburghii and Rosa sterilis Solvent extraction with GC-MS; UFLC/Q-TOF-MS (Liu et al. 2016) Rosa damascena Solvent extraction with GC-MS (Rusanov et al. 2011a)

42 These auxins, in low concentrations, play key roles in the growth and development plants. They regulate processes such as cell enlargement, cell division, vascular tissue differentiation, root initiation, flowering, fruit set, and growth. Furthermore, it has been demonstrated that auxins in the green alga Bryopsis plumosa stimulate rhizoid formation and activate growth in some cultured microalgae and cyanobacteria. On the other hand, high concentrations of auxin have inhibitory effects (Davies 2013; Tarakhovskaya et al. 2007). NAA, a synthetic auxin, is the most widely used plant growth regulator with a range of applications, including for the rooting of stem cuttings in horticultural crops, bud bole shedding, and the inhibition of flower drop and sprouting, button shedding and the development of suckers. NAA induces similar physiological responses as natural auxins in bioassays (Imin et al. 2005). Moreover, a study by Siddik et al. (2015) showed that NAA enhanced endogenous levels of PGRs, including gibberellins, because auxin is necessary for the biosynthesis of gibberellins, which effects growth, physiological attributes and finally yield.

Figure 1.14. Structures of (A) naturally occurring the auxins indole-acetic acid (IAA), indole- butyric acid (IBA), 4-chloroindole-3-acetic acid (4-Cl-IAA), and phenyl-acetic acid (PAA), and (B) synthetic auxins, 1-Naphthalene-acetic acid (NAA), 2,4-dichlorophenoxy-acetic acid (2,4-D), 2,4,5-trichlorophenoxy-acetic acid, 3,6-dichloro-2-methoxybenzoic acid (dicamba), and 4-amino-3,5,6-trichloropiconiölinic acid (picloram) (Sauer et al. 2013).

43 1.2.3.2.2. Effect of NAA on Morphological and Physiological Characteristics

NAA is considered one of the most important exogenous synthetic auxins, and this makes it worthy enough to study its effect on some morphological and physiological factors that may increase plant production (Scacchi et al. 2009). A study by Iqbal et al. (2009) indicated that foliar application of NAA significantly increased morphological, physiological and biological characteristics in plants. In roses, as for other ornamental plants, the application of NAA at different concentrations effects many morphological, physiological and biological processes. Some research has focussed on the application of NAA on roses (Table 1.6).

1.2.3.2.3. Effect of NAA on Emission of VOCs

Essential oils, also known as volatile or ethereal oils, are obtained from different parts of aromatic plant such as flowers, leaves, roots, barks, wood, seeds, buds, twigs and these contain scented, volatile hydrophobic and highly concentrated compounds (Brenes and Roura 2010; Negi 2012). Around the world, the pursuit of aromatic plants containing essential oils has increased exponentially and it is expected that in the future, the demand for essential oils could significantly increase. PGRs effect the essential oil storage structures and biosynthesis of essential oil in plants, which can change the yield of essential oils, control plant growth and their primary and secondary metabolite production. PGRs including NAA play a significant role in enhancing essential oils production (Bano et al. 2016). In most aromatic plants, such as rose-scented geranium, lemongrass and peppermint, the quality and content of their essential oils can be enhanced with the application of PGRs (Sangwan et al. 2001).

As mentioned earlier, NAA is an important synthetic auxin, and it is important to study the performance of NAA on the growth, yield, and biochemical attributes of many plant varieties, especially those used for production (Yamamoto and Yamamoto 1998). A study by Stefanache et al. (2010) indicated that application of NAA on Arnica montana increased and improved the quality of volatile oils. Moreover, concentrations of 70ppm of NAA on mustard plants increased the oil content and quality (Begum et al. 2018). Another study by Swathi (2012) showed that application of 20ppm NAA on coriander plants increased leaf essential oil content. A study by Passinho-Soares et al. (2013) indicated that 2.68 µmol and 5.37 µmol NAA in vitro media increased production of VOCs (monoterpenes and sesquiterpenes) which was analysed with HS-SPME coupled with GC-MS.

44 Table 1. 6. Summary of research conducted on the morphological and physiological effects of NAA application to rose plants

Rose varieties Notes Reference Concentrations: 0, 25, 50, 75, 100 mg/L Damask rose Morphological effects: 75 mg/L improved plant height, plant spread, number (Khan et al. 2007) of primary shoots, secondary shoots and survival percentage Concentrations: 8ppm Morphological effects: increased the percentage of flower opening , vase life, Rose cv. ‘Grand Prix’ flower diameter and flowers fresh weight percentage (Zaky 2013) Physiological effects: increased water uptake and reducing the depletion of sugars content and increasing anthocyanin content in flowers Concentrations: 25 mg/L (Saffari et al. 2004) Rosa damascena Mill. Morphological effects: effected on plant height and flower yield

Physiological effects: effected on oil content Concentrations: 450, 700 and 950ppm (Akhtar et al. 2015) Rosa centifolia Morphological effects: increased shoot length, shoot dry weight, number of

roots, root length and root dry weight Concentrations: 1.5 and 3.0 mg/L Rosa centifolia Morphological effects: increased number of shoots, number of roots and root (Akhtar et al. 2016) length

45 1.2.4. Research Gaps

Many studies have been done on the effect of different types of PGRs on the morphological and physiological characteristics of horticulture plants. However, little is known about the effect of PGRs on physiological and metabolite processes, especially in rose plants of BA and NAA. To my knowledge, no research has been conducted on:

 How BAs and NAAs affect the morphological and physiological changes of two rose varieties.  How morphological and physiological changes relate to changes in metabolism following BA or NAA application.  If VOCs released from rose tissues could be indicators of metabolites.  If different application rates of BA and NAA affect morph-physiological changes and VOCs.

1.2.5. Aims of Study

Hybrid Tea and Floribunda are important and popularized rose varieties in Australia. However, there is a paucity of knowledge about the effects of PGRs on these two newly developed modern roses in regional and international scientific literature. Therefore, the aims of this research were to:

1- Investigate different PGRs for their efficacy to increase physiological and morphological characteristics of two rose varieties, 2- Explore the most effective concentration from Aim 1, that extend flower longevity by reducing flower drop, 3- Determine the chemical changes in terms of secondary metabolites and VOCs produced by different rose tissues when treated with PGRs, and to understand the physiological changes in the life of the plants and flowers, and 4- Investigate whether the benefits of these compounds could contribute to the development of post-production technology to extend the flowers longevity flower crop.

46

47

48 Chapter 2 ______

Optimization of Environmental Factors to Measure Physiological Parameters of Two Rose Varieties

49 Abstract

Rose (Rosa hybrida L.) is one of the most important specialty cut flowers produced. Characterization of physiological variability in photosynthetic efficiencies, respiration rate and chlorophyll content is one of the greatest challenges in assessing rose net primary production of flowering, quality of cut flowers and other rose uses. Two modern rose varieties Floribunda and Hybrid Tea were used to optimize different methods for analysing physiological characteristics (photosynthesis rate, respiration rate and chlorophyll content). Many parameters were optimized, five different lights intensity were used (600, 900, 1200, 1500 and 2000 μmol/m2/s) of which 1200 μmol/m2/s gave the highest photosynthesis rate. Five different measuring times were used (8 am, 10 am, 12 pm, 2 pm and 4 pm) and 12 pm was shown to be the optimum time for measuring photosynthesis rate. Among two different weather conditions (sunny day and cloudy day) sunny day was selected. For respiration rate, two different measuring times (1 and 2 hours) after darkness were studied and 1 hour was chosen. Among three different times (10 am, 12 pm and 2 pm) for measuring chlorophyll content, 12 pm was selected. Using these optimized variables will allow researchers to collect robust and reproducible results to be obtained from different studies, and in turn lead to improved yields of horticultural plants.

2.1. Introduction

Roses plants (Rosa hybrid L.) Hybrid Tea Rose cv. Mr. Lincoln and Floribunda Rose cv. Iceberg are two of the most important ornamental flowering shrubby and odorous plants (Kandil et al. 2007). These two rose varieties (Hybrid Tea and Floribunda) are considered as modern roses and are very popular in Australia. They are commonly cultivated in gardens, for cut flowers and industry use (Scalliet et al. 2008; Joichi et al. 2005). Hybrid Tea rose is originated from a cross between European and Chinese roses, while Floribunda is the result of a cross between Hybrid Tea and Polyantha (Guterman et al. 2002; Myers 2013). Temperate zones of the northern hemisphere and Asia are where the majority of Rosa species are found (Boertjes and van Harten 1978). As a commercial crop, roses are distributed worldwide. There are several natural environmental factors, which affect the physiological parameters and production of rose flowers, but few systematic studies have been done on this aspect for rose production. A recent study by Ibrahim et al. (Chapter 4) on Hybrid Tea and Floribunda roses to optimize methods to analyse volatile organic compounds was conducted. There are many

50 physiological parameters, which affect rose plant production such as rates of photosynthesis, transpiration, and respiration, together with stomatal conductance and stem water potential.

Photosynthesis is an important process in all plants, and especially for rose growers wanting to improve the quality of cut flowers and whole rose plants, and for leaf area (Kim et al. 2004). Leaf distribution pattern, and the amount of light all affect the photosynthesis rate of rose plants (Baille et al. 2006; Matloobi et al. 2009). Some physiological and morphological changes occur under different light intensities leading to different growth responses (Walters 2005). A study by Kool and Lenssen (1997) showed that high quality of plant cut flowers could be achieved by increased light intensity and photosynthesis rates. Moreover, a photosynthesis rate at 1500 μmol/m2/s light intensity increased the production of plant cut flowers (Kim and Lieth 2003). Few studies have examined the effect of different light intensities on photosynthesis rate of rose plants, and there is no research on the optimization of light intensity, sampling time and weather condition on photosynthesis. Therefore, the optimization of these different photosynthesis parameters is important in order to optimize the health of rose plants and the production of flowers.

In addition to photosynthesis, respiration is another important process, which effects the growth, development and production of rose plants (Chapter 4). Leaf dark respiration is among the most fundamental of plant physiological processes and plays a major role in the carbon cycle at a leaf and on a global scale (Reich et al. 1998). Respiration rate is negatively correlated with tissue longevity in pre- and post-harvest plant physiology (Sudheer and Indira 2007). Furthermore, low respiration rate has been related to increased flower longevity in floriculture plants, for example, respiration rates have been related to the longevity of potted flowering chrysanthemum (Monteiro et al. 1991). Respiration rate and chlorophyll content in rose leaves (Rosa hybrida) increased when the temperature and photosynthesis rate were high (Urban et al. 2001). Likewise, Jiao et al. (1991) showed that high temperature (35˚C) increased respiration rate and photosynthesis rates, which were measured under ambient CO2 levels on rose plants (Rosa hybrida). There is a relationship between the photosynthesis rate and chlorophyll content, as plants tend to respond to ambient light by adjusting their chlorophyll content and composition (Walters 2005). Studying chlorophyll content in rose leaves showed rapid and highly dynamic acclimation of leaves to the changing light, which in turn influenced photosynthesis and other physiological processes (Calatayud et al. 2007). A study on marigold plants demonstrated that the chlorophyll content was high at mid-day with the application of

51 growth regulators giving high chlorophyll content as well as photosynthetic pigments (Sardoo 2016). Frak et al. (2001) reported that when trees leaves were exposed to low light they had lower chlorophyll content than those exposed to high light.

The objectives of this study were to optimize: 1) light intensity, sampling time and weather conditions in rose plants for maximum photosynthesis; and 2) different measurement times for optimal respiration rate and chlorophyll content.

2.2. Materials and Methods

2.2.1. Plant Material and Maintenance

This research was conducted at Murdoch University, Western Australia in an evaporatively cooled glasshouse under a natural photoperiod and temperatures maintained between 18±2˚C to 25±2˚C during night and day, respectively, whilst the humidity was maintained at 60±2% and 75±2% day and night, respectively. Two rose varieties (Hybrid Tea cv. Mr Lincoln) and (Floribunda cv. Iceberg) at flowering stage were used. These were purchased from Dawson’s Garden World nursery (Perth, Western Australia). These were immediately transferred to free- draining plastic pots 24 × 24 cm (diameter × height) in a 2:2:1 potting mix (2 parts composted pine bark, 2 parts course river sand and 1-part coco peat) purchased from W. A. Richgro Perth, Western Australia. There were 36 two-year-old rose plants in total. The plants were watered manually daily to container capacity. The rose pots were arranged in a factorial complete randomized design with two rose varieties, three physiological characteristics (photosynthesis, respiration and chlorophyll contents) and three replicate plants for each treatment.

2.2.2. Photosynthesis Measurements

A photosynthesis system (ADC; BioScientific, LCpro+, UK, serial number 32125) with a red/blue LED light source (LCpro+) mounted onto a 6-cm2 clamp-on leaf chamber was used to determine photosynthesis rate under various light intensities. Three fully expanded leaves for each plant (developed between September and November 2016) were used. The instrument programme was configured to detect the sum of three readings, which was then averaged. Five levels of light intensity (600, 900, 1200, 1500 and 2000 μmol/m2/s) were used. In total, 108 leaves were investigated for both rose varieties. Also, five different times were tested (8 am, 10 am, 12 pm, 2 pm and 4 pm) to determine the optimum time for measuring photosynthesis during the day. In addition, measurements were taken on three sunny days and three cloudy days.

52 2.2.3. Respiration Measurements

The respiration rate measurements were determined with an ADC (BioScientific, UK, serial number 32125) using a 6 cm2 area of a leaf that had been dark adapted for 1 and 2 hours. Measurements after 1 and 2 hours of dark acclimation were recorded following a low intensity pulse. Three leaves per plants were measured and each measurement was replicated three times.

2.2.4. Chlorophyll Measurements

Chlorophyll content was determined with a CCM-200 plus, Chlorophyll Content Meter (serial number NH 03051, Opti-Sciences, USA). Three different times (10 am, 12 pm and 2 pm) were studied for measuring chlorophyll content from two rose varieties. Three fully expanded, healthy leaves were used for each rose and time combination, and replicated three times.

2.2.5. Statistical Analyses

All physiological parameters were analysed using Statistical Analysis Software (SAS®) University edition, and the results were presented by analysis of variance (ANOVA). Least Significant Difference (LSD) was used and the level of statistical significance was P ≤ 0.05.

2.3. Results and Discussion 2.3.1. Optimization of Light Intensity for Maximum Photosynthesis

The total photosynthesis rate increased from 600 to 1200 μmol/m2/s, but there after decreased at 1500 and 2000 μmol/m2/s for both rose varieties. Consequently, 1200 μmol/m2/s was selected as the optimum light intensity for photosynthesis measurements (Figure 2.1). In contrast, the study by Kim and Lieth (2003) on rose plants (Rosa hybrida L.) showed maximum photosynthesis rate at 1500 μmol/m2/s and this led to an increase in cut flower production. Many studies have shown how environmental factors affect physiological processes in plants, such as temperature, light intensity, CO2 concentration and photosynthesis (Moriana et al. 2002).

An optimum light intensity leads to increased rose cut flower production and this is generally ascribed to an increased source of carbohydrates (Kim et al. 2004). In another study, the maximum photosynthesis rate was reached at a light intensity of 750 μmol/m2/s in rose plants (Rosa rugosa) (Ueda et al. 2000). In contrast, Samartzidis et al. (2005) indicated that using a light intensity of 180.7 μmol/m2/s significantly increased the photosynthesis rate and

53 production of cut flowers for rose plants. This is may be because different rose species response differently to the low light intensity. 2.3.2. Analysis of Photosynthesis with Five Sampling Times

There were significant (P<0.05) differences between the five sampling times, and the sampling time of 12 pm was selected because the highest photosynthesis rate was recorded at this time (Figure 2.2). The photosynthesis rate increased from 8 am to 12 pm and then it decreased from 2 pm and 4 pm onwards (Figure 2.2). Photosynthesis decreased as light intensity decreases, and increased significantly at high light intensities for rose plants (Rosa hybrida “Haban”) (Matloobi et al. 2009). Similar observations were made by Sardoo (2016) on marigold plants and rose plants (Lieth and Pasian 1990), where increases in showed that light intensity increased photosynthesis, and for rose plants. A decrease in light level below the optimum level would lead to reduced photosynthesis and in turn yield reduction in roses (Zieslin and Mor 1990).

25

a /s /s

2 ab

20 b b μmol/m

15 c a

b 10 b b

Net Photosynthesis Photosynthesis Net bc

5 Floribunda Hybrid Tea

0 600 900 1200 1500 2000

Photon flux density μmol/ m2/s

Figure 2.1. Evolution of photosynthesis rate with exposure to different light intensity of Floribunda and Hybrid Tea roses (Error bars were LSD at P ≤ 0.05).

54 25

a /s 2 20 b b

bc μmol/m c 15

a

10 b b bc

c Net Photosynthesis Photosynthesis Net

5 Floribunda Hybrid Tea

0 8:00 AM 10:00 AM 12:00 PM 2:00 PM 4:00 PM

Time of measurment

Figure 2.2. The effect of different sampling times on photosynthesis rate during different periods of the day for Floribunda and Hybrid Tea roses (error bars were LSD at P ≤ 0.05).

2.3.3. Effect of Weather Conditions on Photosynthesis Rate

There were significant (P ≤ 0.05) differences in photosynthesis between sunny and cloudy days (Figure 2.3), and with sunny days were chosen for subsequent measurements. This is because sunny days have high levels of light intensity leading to increased photosynthesis rates. Similar observations have been made in vines exposed to sunny days compared with the cloudy days (Chaumont et al. 1994), for Maple trees (Schindler and Lichtenthaler 1996), and for two Populus clones (Michael et al. 1990). To date, no research has been conducted on rose plants and optimum photosynthesis rates between sunny and cloudy days.

55 14 Sunny day Cloudy day 12

10

/s 8 2

6 μmol/m

Net Net photosynthesis 4

2

0 Hybrid Tea Floribunda

Figure 2.3. Response of photosynthesis rate to changing weather conditions for Floribunda and Hybrid Tea roses (error bars were LSD at P ≤ 0.05).

2.3.4. Analysis of Respiration with Two Sampling Times

The respiration rate did not differ significantly between the two sampling times at 1 and 2 hours after sunset (Figure 2.4). Therefore, 1 h after sunset was selected for subsequent studies on respiration. A study on rose plants (Rosa hybrida) by Lieth and Kim (2001) indicated that gas exchange (respiration rate) increased with fully expanded, young and sunlit leaves. Whilst Monteiro et al. (2001) indicated that rose plants (Rosa hybrida) exposed to long photoperiods will be photosynthetically more active and in turn would have increased respiration rates. In contrast, Murray et al. (2013) indicated that increasing light intensity inhibited respiration in algae. Furthermore, respiration in tobacco leaves was inhibited by light, while exposure to darkness after a period of light resulted in an increase of CO2 exchange and increased respiration rate (Atkin et al. 1998). Whilst Poorter et al. (1992) demonstrated that, the dark respiration rate increased with increasing leaf area, as demonstrated by the increase in CO2 levels released from plant leaves. Similarly, dark respiration rate increased with fully expanded leaves of white poplar plants during respiration measurements (Loreto et al. 2007). To date, no work has been done on optimizing the sampling times for measuring dark respiration rate in rose plants.

56 2.4 1 h 2 h

2 /s /s 2 1.6

μmol/m 1.2 Dark Dark Respiration

0.8

0.4

0 Hybrid Tea Floribunda

Figure 2.4. Effect of sampling times on dark respiration rate of leaves of Floribunda and Hybrid Tea roses (error bars were LSD at P ≤ 0.05).

2.3.5. Effect of Different Sampling Times on Chlorophyll Content

Three sampling times (10 am, 12 pm and 2 pm) were used to measure the chlorophyll content, and 12 pm was determined as the best time since it gave a high level of chlorophyll content (Figure 2.5). The chlorophyll content increased significantly from 10 am to 12 pm and then decreased by 2 pm. Many factors affect chlorophyll content in leaves, these include light, weather conditions and leaf area. In rose plants (Rosa hybrida cv. Habari), total chlorophyll content decreased with low light exposure, shade or cloudy day and with small leaf areas, and in turn increased with high light levels and with increasing leaf area (Matloobi et al. 2009).

Furthermore, Esk and Esk (2013) indicated that chlorophyll content of Satureja khuzestanica plants increased with light intensity at 1500 μmol/m2/s during daylight and with fully intact leaves. Whilst Urban et al. (2001) reported that at a light intensity of 700 μmol/m2/s on rose (Rosa hybrida cv. First red and Twing) plants increased chlorophyll content. Moreover, Ueda et al. (2000) showed that increasing light intensity and sun light increased the chlorophyll content for rose (Rosa rugosa) plants. Recently, Sardoo (2016) and Salehi et al. (2014) showed that chlorophyll content increased with increasing the light on marigold and for Ficus banjamina L. plants, respectively.

57 60

a 50 ab b

40

30 a b ab

20 Total Chlorophyll concentration indexChlorophyll Total 10 Floribunda Hybrid Tea

0 10:00 AM 12:00 PM 2:00 PM

Time of mesurment

Figure 2.5. Analysis of different sampling times on chlorophyll content of Floribunda and Hybrid Tea roses (error bars were LSD at P ≤ 0.05).

2.4. Conclusion

This study has shown a light intensity of 1200 μmol/m2/s gives the most efficient photosynthesis rates in Hybrid Tea and Floribunda roses. Whilst, 12 pm was the optimal sampling time for determining high levels of photosynthesis in both rose varieties, and sunny days were much better than cloudy days for obtaining high rates of photosynthesis. For respiration rate, 1 hour after darkness was determined to be suitable for measuring dark respiration. Finally, the optimum chlorophyll content in both rose varieties was obtained at 12 pm. It is recommended that future studies use these optimized parameters in order to obtain robust and reproducible results. These optimized parameters are used in the following chapters.

58

59

60 Chapter 3 ______

Morphological and Physiological Responses of Two Modern Roses to Plant Growth Regulators

61 Abstract

Rose is a very popular ornamental plant especially in floriculture where it is mainly used for cut flowers and garden plants. The present study was conducted to investigate the influence of foliar application of benzyladenine (BA) and naphthalene acetic acid (NAA) on growth, flowering, pre-harvest and physiological parameters of two-years-old roses (Hybrid Tea and Floribunda). BA and NAA were applied at three concentrations (0, 100 and 200 mg/L). Results revealed that 200 mg/L for both BA and NAA increased plant height, number of branches and flowers, stem and flower diameter, length of flower stem and flower longevity and for physiological effects (photosynthesis rate) and chlorophyll index with Hybrid Tea 30.67 and 27.57 index for BA and NAA, respectively. Whilst, Floribunda was 30.03 and 20.71 for BA and NAA, respectively as compared with the control. However, for respiration rate, the results demonstrated that the control plants had higher respiration rates compared with plants treated with 100 and 200 mg/L (BA and NAA) for both rose varieties. There were no significant differences between 100 and 200 mg/L BA and NAA applications, in terms of chlorophyll index and plant height for both roses. Whilst the results revealed that, there were no differences between the treatments of 100 and 200 mg/L for both PGRs in terms of stem diameter, number of flowers and flower diameter for both rose varieties. All these findings could be applied successfully to other ornamental plants especially floricultures to enhance all morph- physiological characteristics and then increase the production.

3.1. Introduction

Rose (Rosa hybrida) belongs to family Roseasea and is one of the most cultivated garden plants for cut flowers. Among different flowers, roses enjoy worldwide popularity and their flowers are in great demand worldwide (Raymond 1999, Gudin 2003). They are grown in many parts of the world owing to their excellent beauty and economic values (Kovacheva et al. 2010). Hybrid Tea and Floribunda belong to the modern roses (Bendahmane et al. 2013, Chapter 2). Today with the advancement of technology, the main objectives in flower production are perfection in the quality of flowers and an increase in flower production. Various chemicals are now being tried to control growth and flowering of rose varieties with a view to either stretch out or retard the rate of plant growth (Hashemabadi 2010). In recent years, scientists have attempted to regulate plant growth with the application of PGRs to improve the growth, yield and quality of flowering plants (Hedayat 2001). Despite many studies on the influence of PGRs on roses, there are few studies on the mode of application of PGRs to modern roses.

62 PGRs play vital roles in the coordination of many growth or behavioural processes in rose, and they regulate the amount, type and direction of plant growth. In addition, PGRs can increase flower production and improve of flower quality in floriculture (Bari and Jones 2009). Various studies have reported that the application of growth regulators and chemicals help to increase the yield of good quality flowers and vegetative growth in floricultural plants especially rose plants (Anjum et al. 2011; Hashemabadi 2010). Many PGRs, such as the auxin NAA, benzyladenine BA and gibberellic acid GA3 are being used to ensure efficient production. Foliar application of PGRs (BA and NAA) improve the quality of growth parameters of fruits and cut flowers (Sajid et al. 2009). PGRs have been successfully used to manipulate plant growth, crop yields and physiological processes (Peleg and Blumwald 2011). BA and NAA when applied to plants will influence morphological and physiological characteristics within plants (Zhao 2008).

BA can reduce ethylene sensitivity of cut flowers (Yuan et al. 2012), and cytokinins including (BA) and influence cell division (Francis and Sorrel 2001), the biosynthetic processes of chloroplast pigments (Bondok et al. 1995), nutrient uptake especially potassium (Guo et al. 1994), and increase photosynthetic efficiency (Oosterhuis and Zhao1998). The beneficial uses of BA in ornamental plants have been reported numerous times, but the effective concentration of its application varies among ornamental plants (Werbrouck et al. 1996). The positive effects of BA have been demonstrated on many plant species including Dendrobium orchid (Sakai et al. 1998), Lilium (Ohkawa 1979) and Achimenes longiflora (Vlahos 1984).

Many studies have shown NAA to have positive effects on the growth and development of the ornamental plants (Umrao et al. 2007, Rana et al. 2005). Hybrid Tea and Floribunda are important and popular varieties in Australia, and an understanding of how PGRs on these two modern rose varieties is lacking in the scientific literatures. Therefore, the purpose of this study was to investigate the comparative effects of BA and NAA on the morphological and physiological parameters in these two rose varieties.

3.2. Materials and Methods 3.2.1. Plant Materials and Foliar Application of Growth Regulators

The study was conducted at Murdoch University, Western Australia in an evaporatively cooled glasshouse under natural photoperiods, and the temperature was maintained between 18±2˚C to 25±2˚C during night and day, respectively, and the humidity was between 60±2% and

63 75±2% day and night, respectively. Two-year-old rose plants of Hybrid Tea and Floribunda were purchased from Dawson’s Garden World nursery (Perth, Western Australia). The plants were approximately 1m high and maintained in 5 L pots. In total, 36 rose plants were used and the plants were watered manually daily to container capacity. The rose plants were arranged in a factorial complete randomized design with the two rose varieties, two PGRs, three concentrations and three replicate plants for each treatment. Benzyladenine (BA) at 98% purity, (product number B3408) and Naphthalene acetic acid (NAA) at 95% purity (product number N0640) were purchased in crystalline form from Sigma-Aldrich (Castle Hill, NSW, Australia). For application of growth regulators, solutions of BA and NAA (0, 100 and 200 mg/L) were prepared. Foliar applications of BA and NAA were applied one month prior to flower blooming by spraying rose plants uniformly to the point of run-off (approximately 100 mL/plant) using a mist sprayer (450 mL) (Bunnings Warehouse, Perth, Australia). Control rose plants were treated with double distilled water. Each rose plant was sprayed individually with the specific solution and the treatments were applied late in the afternoon. The concentrations of each PGR were selected based on a previous experiment (Chapter 6) conducted to establish optimum concentration for various rose varieties.

3.2.2. Morphological Measurements

The morphological parameters measured included plant height (cm), number of branches/plant, stem diameter (mm), leaf area (cm2), length of flower stem (cm), number of flowers/plant, flower diameter (mm), and flower longevity (days). A tape measure was used to measure plant height and length of flower stems, whilst a digital Vernier calliper was used to measure stem and flower diameters. Leaf area was measured using a Portable Laser Leaf Area Meter - CI- 202 (CID Bio-Science, Inc. USA). For each plant, three leaves were used from different locations (top, middle and bottom) and the average was taken.

3.2.3. Physiological Measurements

Chlorophyll content was determined with a CCM-200 plus, Chlorophyll Content Meter (serial number NH 03051, Opti-Sciences, USA). The chlorophyll content was measured from different locations (top, middle and bottom) of the rose plants and the average was taken. For each location, three healthy fully expanded leaves were randomly selected for each rose plant (a total of nine leaves). To determine the photosynthesis rate, a photosynthesis system ADC (BioScientific, LCpro+, UK) (serial number 32125) with a red/blue LED light source (LCpro+) mounted onto a 6-cm2 clamp-on leaf chamber was used under various light intensities. Three

64 fully expanded leaves from each plant were used and the data averaged. A light intensity of 1200 μmol/m2/s was used, and the samples were measured at 12 pm on sunny days for both chlorophyll and photosynthesis measurements. Whilst, respiration rates were determined the ADC, as described above using a 6 cm2 area of leaf. Measurements were recorded after 1 h of dark acclimation following a low intensity. Each respiration measurement was replicated three times for each plant (Chapter 2).

3.2.4. Statistical Analyses

Analyses of variances (ANOVA) were made with the general liner model procedure of Statistical Analysis Software (SAS®) University edition version. The Least Significant Difference (LSD) was used to determine the treatment effects on each variable derived from these analyses to establish significant effects at P ≤ 0.05.

3.3. Results and Discussion 3.3.1. Morphological Parameters

Plant heights, number of branches, stem diameters, leaf areas and length of stems were significantly affected by foliar applications of BA and NAA at 200 mg/L for both rose varieties (Table 1). Hashemabadi (2010) indicated that different PGRs applied at different concentrations to Rosa hybrida plants significantly affect morphological characteristics such as bud length, vase life, yield, flower parameters, pre-harvest stage and the quality and production of flowers. Furthermore, Malik et al. (2017) showed that different PGRs applied to Dahlia plants effected all morphological parameters measured include number of flowers, number of leaves, diameter of flowers, diameter of stem and plants height. In the current study, BA applied at 200 mg/L increased plant heights for Hybrid Tea and Floribunda, compared with the control. There were no significant differences between the 100 and 200 mg/L BA and NAA applications, but there were significant differences between the control (0) and 200 mg/L BA and NAA applications. These findings might be because NAA promote vegetative growth by inducing active cell division in the apical meristem. Similarly, BA promotes vegetative growth by inducing cell elongation and division in plant tissues. These findings agree with the reports of Sharma et al. (2004) and Awasthi et al. (2012) for gladiolus. Aier et al. (2015) reported that gladiolus plants treated with 250ppm BA were significantly taller than the control plants. In the case of numbers of branches/plant, the results indicated that there were significant differences between the control and 200 mg/L BA and NAA applications (Table 1). In addition, 200 mg/L of BA and NAA enhanced stem diameters, and for both rose varieties after BA and

65 NAA applications compared with the control (Table 1). These findings correspond to those of Salehi (2014a) on three ornamental plants (Ficus benjamina, Schefflera arboricola and Dizigotheeca elegantissima) after application of 200ppm BA. The results indicated that the number of shoots per plant and stem diameters in these plants increased with this concentration compared to the control plants. Whilst, Kumar et al. (2008) showed that gladiolus plants treated with 100ppm NAA significantly increased growth parameters such as number of stems and stem diameters compared to the control.

In the current study, there were significant differences in leaf area after the 100 and 200 mg/L BA and NAA applications for both rose varieties compared with the control (Table 1). For both rose varieties the leaf areas after the application of 200 mg/L of both BA and NAA were significantly greater than those with the 100 mg/L and control treatments. In another study, the application of NAA at 100ppm on gladiolus plants showed the treated plants had higher leaf areas (Sudhakar and Kumar 2012). Similarly, the length of flower stems were significantly longer after 100 and 200 mg/L applications of NAA and BA than that of the control treatments for both varieties, although for Floribunda there were differences between the 100 and 200 mg/L treatments of BA (Table 1). Aier et al. (2015) also reported similar results in gladiolus plants after they were treated with 250ppm BA with increased leaf area, number of flowers, flower diameter and length of flower stems. Matin et al. (2015) showed Narcissus plants treated with 200ppm BA, significantly increased their vegetative growth and improved flower quality.

In the current study, for both flower varieties BA and NAA increased the numbers of flowers produced, flower diameter and flower longevity compare with the control (Table 1). Also, for both rose varieties, the application of 200 mg/L doubled or more than doubled flower production (Table 1). This may be because BA and NAA enhance cell division and cell enlargement. Pan and Xu (2011) reported the application of BA increased the number of Jatropha curcas flowers compared with the control.

66 Table 3.1. Effect of the plant growth regulators benzyladenine and naphthalene acetic acid on chlorophyll index and morphological growth parameters of two modern rose varieties (Hybrid Tea and Floribunda)

V PGRs Treatments Chlorophyll Plant No. of Stem Leaf area Length of No. of Flower Flower (mg/L) index height branches diameter (cm2) flower flowers diameter longevity (cm) per plant (mm) stems (cm) per plant (mm) (days) BA Control 20.67 b 49.10 b 2.00 c 12.10 b 63.29 b 22.20 c 1.33 b 88.66 b 5.50 b 100 30.97 a 60.46 a 3.33 b 15.19 a 70.30 b 37.33 b 2.33 ab 118.33 a 6.16 b

200 30.67 a 54.47 ab 4.00 a 16.03 a 80.60 a 48.95 a 3.33 a 116.33 a 8.50 a NAA Control 18.58 b 46.25 b 1.66 b 11.65 b 63.83 c 20.83 b 1.55 b 90.66 b 6.16 b

Hybrid Tea 100 23.10 ab 49.93 ab 2.33 ab 13.85 a 70.95 b 29.83 b 2.66 a 113.67 a 8.23 a 200 27.57 a 61.70 a 3.33 a 14.09 a 80.66 a 55.27 a 3.33 a 133.00 a 9.16 a BA Control 19.50 b 41.10 b 7.00 b 10.35 b 57.47 b 23.98 b 12.00 b 75.88 b 5.55 c 100 25.05 ab 51.50 a 9.00 ab 11.17 b 71.13 a 37.73 a 13.00 b 78.35 b 7.00 b

200 30.03 a 57.23 a 11.66 a 14.47 a 73.86 a 42.23 a 23.66 a 89.39 a 8.60 a NAA Control 17.25 b 39.20 c 7.33 b 10.79 b 50.80 c 21.83 c 9.33 c 74.56 b 5.73 b

Floribunda 100 19.70 ab 44.16 b 7.33 b 12.13 ab 59.69 b 37.33 b 16.33 b 75.97 b 7.33 a 200 20.71 a 51.50 a 10.33 a 13.11 a 72.37 a 48.43 a 24.33 a 83.96 a 8.00 a Within a column, means sharing similar letters are not statistically ( P< 0.05) significant, V represent (varieties) and PGRs represent plant growth regulators

67 Furthermore, Joyce et al. (2004) demonstrated that Grevillea plants treated with 10ppm BA increased the longevity of flowers compared with control plants, while Pradhan (2015) reported that application of NAA (100 and 125ppm) to gladiolus plants increased flower numbers, flower size and flower longevity. Whilst a study by Sevik and Guney (2013) found the application of NAA to Melissa officinalis plants increased morphological and flower characteristics.

3.3.2. Physiological Parameters

The chlorophyll content increased significantly with application of BA and NAA 200 mg/L for both rose varieties (Table 3.1). This increase in chlorophyll content might assist in the growth and development of rose plants. The results agree with those of Mondal and Sarkar (2017) who used BA on Hybrid Tea plants and also found chlorophyll content was increased. Furthermore, a study on Spathiphyllum wallisii plants by Salehi (2014b) reported that BA at 200ppm increased chlorophyll contents. While Reddy et al. (2014) showed that 20ppm of NAA to Greengram plants significantly increased chlorophyll content in their leaves. In contrast, Khandaker et al. (2017b) indicated that Orchid plants treated with 100ppm NAA, showed chlorophyll content did not change significantly between the control and treated plants.

Photosynthesis increased with increasing concentrations of BA and NAA from 0 to 200 mg/L for both rose varieties (Figure 3.1). The highest photosynthesis rate was recorded with 200 mg/L for BA and NAA, and there were significant differences between the control and 200 mg/L BA and NAA and between 100 and 200 mg/L with BA for Hybrid Tea, and for NAA in Floribunda. In contrast, there were no significant differences between 100 and 200 mg/L BA with Floribunda, and NAA with Hybrid Tea. This may be because BA and NAA increase leaf area, which in turn increase the chlorophyll content, which is an important factor for enhancing photosynthesis. These results are similar to those of Salehi (2014c) who found the application of BA at 150 mg/L to Dizigotheeca elegantissima plants increased photosynthesis. A study by Salehi (2014a) on three ornamental plants found that 200ppm of BA significantly increased photosynthesis compared with control plants. Furthermore, Hejnák (2010) showed 100ppm BA applied to beet plants increased photosynthesis compared with the control. Whilst, Khandaker et al. (2017a) showed when 40ppm of NAA applied to Syzygium samarangense plants significantly increased photosynthesis. BA and NAA play important roles in plant growth and development, and these responses vary with the concentration and application method (Latimer 2001).

68 0(Control) 100 mg/L 200 mg/L

20 BA 16 a a 12 b b

bc bc /s

2 8

4 μmol/m

0 20 NAA a 16 a ab 12 b

c Net Photosynthesis Photosynthesis Net

8 c

4

0 Hybrid Tea Floribunda

Figure 3.1. Effect of BA and NAA concentrations on photosynthesis rate (μmol/m2/s) for two rose varieties (Hybrid Tea and Floribunda). Error bars were LSD at p ≤ 0.05 (n=3).

Applications of BA and NAA at 100 and 200 mg/L reduced the respiration rates of both rose varieties compared with the control. The applications of 200 mg/L BA and NAA for both rose varieties were significantly different to the non-treated control plants (Figure 3.2).

There were no significant differences between 100 and 200 mg/L BA and NAA for both rose varieties with regards to the results (Figure 3.2). PGRs have direct and indirect effects on respiration rates in plants and in turn play important roles in physiological and metabolic processes (Bi et al. 2011). Tsantili et al. (2002) showed the application of BA to plants pre and post-harvest lead to decreased respiration rates because BA effected ethylene production and metabolic processes. A study by Franco and Han (1997) showed that Easter lily plants treated with BA 500 mg/L significantly reduced respiration rates by one-third to one-half compared with untreated plants

69 Hybrid Tea Floribunda 6 5 a 4 BA b a

/s 3 bc 2

/m 2 b 1 μmol bc 0

6 a 5

4 b a Dark Respiration Respiration Dark NAA 3 bc b

2

1 bc 0 0(CONTROL) 100 MG/L 200 MG/L

Figure 3.2. Effect of BA and NAA concentrations on respiration rate for two rose varieties (Hybrid Tea and Floribunda). Error bars were LSD at p ≤ 0.05 (n=3).

Furthermore, MacLean and Dedolph (1962) indicated that Chrysanthemum morifolium and Dianthus caryophyllus plants treated with 20ppm BA had significantly reduced respiration rates in the post-harvest stage compared with the non-treated controls. There are no studies on the effect of NAA on respiration rates in rose plants.

3.4. Conclusion

The application of either BA or NAA at 200 mg/L resulted in significantly higher values for both growth promotion and physiological parameters. The foliar application of BA and NAA improved flower growth, number of flowers and flower longevity, all economically important traits for commercially produced rose flowers. The use of BA and NAA also helped to improve other morphological parameters including the number of branches, stem length, leaf area and length of flower stems. In addition, these PGRs helped to increase the chlorophyll content and photosynthesis rates, while at the same time decreasing respiration rates, all of which assist in

70 enhancing the value of the treated rose plants. Thus, the use of BA and NAA in commercial production systems could potentially help growers to improve the flower traits of their roses, ultimately leading to increased prices in the domestic and international markets.

71

72 Chapter 4 ______

Optimized Method to Analyze Rose Plant Volatile Organic Compounds by HS-SPME-GC-FID/MSD

73 Abstract

A method involving headspace solid-phase microextraction (HS-SPME) fibre combined with gas chromatography (GC) coupled with flame ionization detection (FID) and gas chromatography with mass spectrometry (GC-MS) was developed and optimized to investigate VOCs from different tissues (flowers, leaves, stems, rhizosphere and whole plants) of Floribunda and Hybrid Tea roses (intact and cut). A three-phase fibre 50/30μm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) was used. Two types of chambers (Tedlar bag and glass jar) were evaluated for collection of VOCs and the glass jar was selected. Absorbed compounds on the fibre were completely desorbed in the GC injector port at three desorption times (5, 10 and 15 min), and 5 min at 250˚C was used. The maximum extraction efficiency for flower tissues (equilibrium absorption) was achieved 2 h after fibre exposure in the headspace for intact and cut Floribunda and Hybrid Tea flowers. Under the optimized HS-SPME and GC-FID/MS conditions, 1h extraction time was chosen for intact and cut Floribunda and Hybrid Tea leaves and stems. A 5 cm depth was selected for roots and soil (rhizosphere) for both rose varieties, and optimum 6 h and 12 h extraction times of VOCs from rhizosphere were selected for Floribunda and Hybrid Tea, respectively. One hour was chosen for VOCs released from whole rose plants for both varieties. The different tissues of rose plants gave a wide range of the VOCs; also the chromatograms of the two varieties were quite different and the specific VOC pattern of depended on the variety. This study demonstrated the feasibility of this HS-SPME-GC-FID/MSD method for identifying VOCs from two rose varieties, and the potential use of this method for physiological studies on rose plants or on other floriculture plants.

4.1. Introduction

Roses (Rosa hybrida L.) are an important ornamental plant of the family Rosacea. The genus Rosa includes about 100-200 species and more than 18,000 varieties (Gudin 2000; Weiss 1997). The family Rosacea is recognized for its high economic value and use in agro-based industries, especially in cosmetics, perfumes, food and pharmaceutical industries (Guterman et al. 2002; Kovacheva et al. 2010). Roses are the most important cut flower crop in the world, with Australia exporting about 6,720,221 stems with an approximate value of $3,743,000 in 2016. In contrast, Australia imports about 204,001,778 stems at a cost of about $63,838,000 in 2016 (Department of Foreign Affairs and Trade 2016). The main industrial application of roses comes from the perfume industry, which relies on different scented rose varieties (Özel et al.

74 2006; Rusanov et al. 2011a; Baydar and Baydar 2005; Rusanov et al. 2011b; Pellati et al. 2013). Among these varieties are the Hybrid Tea rose and Floribunda rose, both are modern roses. The Hybrid Tea rose is a cross between European and Chinese roses (Scalliet et al. 2008; Joichi et al. 2005). Floribunda is the result of crossing hybrid tea with Polyantha (Myers 2013).

Roses emit a wide range of volatiles organic compounds (VOCs) from flowers, leaves, stem and roots (Kigathi et al. 2009). These can be extracted by various techniques, such as hydro- distillation, microwave extraction, and solvents, all of these methods require grinding and adding solvents. Thus, some chemicals, which are released from the plants, will not be detected. In contrast, recently the solid phase microextraction (SPME) method was developed to capture VOCs in order to profile and quantify these compounds. Most studies have focused on volatile emissions from flowers or fruits, with little known about emissions from other plant tissues, such as leaves, stems, roots and even the whole plant including rhizosphere soil. There are many methods to obtain rose volatiles, such as from rose oil (essential oil) and these methods have been extensively reviewed (Hosni et al. 2010; Naquvi et al. 2014; Rusanov et al. 2011b; Dobreva 2013). Unfortunately, the aroma of extracted oils rarely represents the delicate natural aroma of floriculture plants, because of thermal artefacts produced during the steam distillation process (which typically occurs at 60-70˚C) (Deng et al. 2006). Likewise, other extraction methods using ground samples and solvents also frequently fail to capture the natural aromas (Sparinska and Rostoks 2015; Koksal et al. 2015; Kiralan 2015). Studies by Shunying et al. (2005) and Chang and Kim (2008) indicated that essential oils extracted from Chrysanthemum did not fully represent the delicate natural aroma. To identify VOCs produced by roses or other plants, it is necessary to develop sensitive, non-destructive, rapid and systematic methods from different plant tissues that are also cost effective. There are no studies about the use of headspace solid phase microextraction HS-SPME for fresh intact or excised tissues obtained from roses. The HS-SPME method is a new, simple, fast, and highly sensitive and solvent-free sample preparation technique for the extraction of volatile compounds (Prosen and Zupančič- Kralj 1999; Bicchi et al. 2000; Wardencki et al. 2004). The SPME method is widely used for the analysis of volatile compounds, and it is successfully employed to monitor the extraction of aromatic components for Jasminum (Pragadheesh et al. 2011).

The HS-SPME method also provides interesting results when gas chromatography (GC) is combined with a Flame Ionization Detector (FID) and mass spectrometric detection (MS). The SPME method gives simultaneously tens or hundreds of possible volatile compounds, but it

75 must be optimized for the volatiles being targeted (Dorea et al. 2008; Jeleń et al. 2012). There are many factors that affect the optimization of extraction conditions. These include the correct fibre and an appropriate chamber for capturing the VOCs, the temperature used during extraction and the extraction time from the headspace (Nongonierma et al. 2006). In the past, optimization experiments have only analysed a variable at a time, this approach does not allow any investigation into the possible interactions between variables, and hence it is not possible to explore fully the opportunities for optimization (Ferreira et al. 2007). To date, there has been no systematic work on optimizing extraction conditions for individual tissues of rose plants or the whole plant. Therefore, developing robust and practical methods for the extraction of VOCs from rose plants has important commercial implications for the aroma industry. The objective of the present work is the systematic optimization of HP-SPME methods, and the analysis of VOCs from different rose tissues, and intact plants by gas chromatography coupled with a Flame Ionization Detector and Mass Spectrometry (GC–FID/MS).

4.2. Materials and Methods 4.2.1. Reagents

Ethanol was purchased from Merk (high-performance liquid chromatography [HPLC] grade). An n-hexane 95% was purchased from Sigma-Aldrich Australia, catalogue number 270504- 2L, and the n-Alkane standard (C7-C30) was purchased from Sigma-Aldrich Australia, catalogue number 49451-U.

4.2.2. Apparatus and Equipment

An Agilent Technologies gas chromatograph 7829A (serial number CN14272038) fitted with a HP-5MS column non-polar (30 m x 0.25 mm, film thickness 0.25 μm, RESTEK, catalogue number 13423), with a flame ionization detector (FID) was used throughout the study. Three replicate solid phase microextraction (SPME) fibres 50/30µm divinylbenzene/carboxen /polydimethyl siloxane (DVB/ CAR/PDMS; Sigma-Aldrich Australia, catalogue number 57347-U), attached to a manual SPME holder (Supelco Inc.) were used. The fibres were conditioned as recommended by the manufacturer before analyses were conducted. In order to collect samples from the rhizosphere profile of rose plants, hollow sample probes 20 cm long were made from stainless steel, and one-millimetre diameter holes were cross drilled at 5 mm increments from the tapered end up the probe to 3.5 cm from the top. A 1/8 cm compression fitting with a septum was installed into the top of the probe to facilitate insertion of the SPME fibre. A single tube was inserted into each pot to capture the VOCs. For whole plants, a glass

76 chamber (30 × 35 × 60 cm) with a 5 mm port drilled into one side, into which a septum was placed to accommodate the SPME fibre and was designed for the extraction of VOCs (Table 4.1). For excised rose tissues, a 500 mL Pyrex (Silverlock Packaging; JG2701 FL) glass jar with a 5 mm port drilled into one side, into which a septum was placed (Table 4.1). As a comparison to the glass jar, a Tedlar bag about 0.5 L was purchased from SKC, Inc (catalogue number 236-001, 0.5 L from SKC). Aluminium foil 150 m × 44 cm (Vital Packaging Company) was used to cover the glass chambers and jars top, from which VOCs were extracted and the foil was also used to extract VOCs from intact stems (Table 4.1).

Table 4.1. Different rose tissues and type of chambers used to capture and detect the VOCs from two rose varieties in this study.

Rose tissue Type Chamber type Measurements Sample Amount

Flowers Intact Glass jar 500 mL One flower Cut Glass jar 500 mL

Leaves Intact Glass jar 500 mL Three leaflets Cut Glass jar 500 mL

Stems Intact Aluminium foil 30 × 30 cm Length 13 cm Cut Glass jar 500 mL

Root Intact Stainless steel sample probe 3.5 cm drilled 5 cm depth

Whole Intact Glass chamber 30 × 35 × 60 Whole plant plant cm

4.2.3. Plant Material and Maintenance

This study was conducted on two-year-old rose plants (Hybrid Tea cv. Mr Lincoln) and (Floribunda cv. Iceberg), purchased from Dawson’s Garden World nursery (Perth, Western Australia). These were immediately transferred to free-draining plastic pots 24 × 24 cm in a 1:1, v:v (potting mix:soil) and placed into an evaporatively cooled glasshouse at Murdoch University. The temperature ranged between 18±2˚C to 25±2˚C during night and day, respectively with humidity 60±2% to 75±2% during night and day, respectively. The rose plants were watered manually daily to container capacity with about 400 mL water. The rose pots were arranged in a factorial based complete randomize design with two rose types and five rose tissues and three replicates for each.

77 4.2.4. Gas Chromatogram Condition Optimization

The optimization step of the SPME conditions was necessary to identity the compounds using the flame ionization detector (FID). The oven column temperature ranged from 50-250˚C, programmed at 5˚C/min, with a final hold time of 5 min. Helium (He) was used as the carrier gas at 1.1 mL/min constant flow, and FID temperatures of 290˚C, injection port temperature 250˚C, and the GC-FID instrument was operated under the splitless mode.

4.2.5. Optimization of Headspace Solid-Phase Microextraction

For optimization of the HS-SPME, the variables chosen were extraction time (min), desorption time (min), and the four types of chambers (glass chamber, glass jar, stainless steel sample probe, and aluminium foil) for extraction, while the SPME fibre (DVB/CAR/PDMS), the fibre extraction temperature of 24±1˚C, the amount of sample and the headspace volume were kept constant. Different extraction times, different desorption times and different extraction chambers were used for the different rose tissues. In order to optimize the adsorption time and extraction times, all factors influencing the equilibrium between the analyses and the fibre were taken into consideration. The fibres were cleaned between each extraction by placing them into the GC injection port for 15 min at 250˚C to ensure absence of carry over peaks and contaminants in blanks to have good repeatability between the injections. The three replicate fibres were calibrated using standard n-alkene C7-C30 after dilution in the ratio of 1/10 mL in n-hexane, and then desorbed for one hour at room temperature, and this procedure was repeated twice with three replicates before statistical analysis. The results are presented as mean values.

4.2.6. Optimisation of Sample Chambers

To determine the most efficient extraction method of the VOCs emitted by intact and excised tissues of the rose plants, a comparison was made between the Tedlar bag and a Pyrex glass jar (0.5 L). The results showed that there were no differences between the Tedlar bag and glass jar, so the jar was chosen because it was easy to handle, and readily captured the VOCs emitted. Individually, rose flowers, leaves or cut stems were placed into the Pyrex glass jar and the opening covered with aluminium foil and incubated at 25˚C for 1, 2 and 3 h, respectively. Aluminium foil was used for intact stems. Whilst for the analysis of VOCs from the whole plant including the soil, a glass chamber (30 × 35 × 60 cm) was used. For the capture of VOCs from the roots and soil, a single stainless steel probe was inserted into the soil.

78 4.2.7. Optimisation of Sealing and Fibre Absorption and Desorption Time

To determine the best absorption time; the fibres were exposed to the headspace of the different chambers with each of the plant tissues for either 1, 2 and 3 h. After exposure, the fibre was retrieved and injected into the heated GC injection port (250˚C) for desorption. For each chamber type, plant tissue type and time combination, there were three replicate samples repeated twice. To optimize the GC injector desorption time, three different desorption times (5, 10 and 15 min) were used at 250˚C.

4.2.8. Optimization of Collection of VOCs from Different Tissues of Rose Plants

The rose tissues studied were intact or excised flowers, stems, or leaves and whole plants including the roots in soil. Each was replicated three times. Briefly, rose plants were transferred to the laboratory and either an excised or intact single fully open flower, leaves (three fully expanded and undamaged leaves), or stem (13 cm) was inserted into the 500 mL glass jar at room temperature (25±2˚C). The jar was covered with aluminium foil and then the SPME fibre was immediately inserted into the headspace for 1, 2 or 3 h. For the roots, the rhizosphere was analysed by inserting the sample probes at two different depths (5 and 10 cm) into which the SPME fibre was inserted overnight and then analysed.

Lastly, the whole rose plants without the plastic pot were sampled by placing into the 30 × 35 × 60 cm glass chamber, the open top was covered with aluminium foil and the fibre inserted into the headspace for 1, 2 or 3 h to record the volatiles emitted from the entire plant. Blank runs of the fibre and of the two glass chambers and Tedlar bags were run before starting each set of daily sampling. The fibres were removed from the chambers/bags and immediately placed into the injection port of the GC for 5 min at 250˚C. After analysis, the fibres were thermally desorbed in the GC injector to prevent contamination.

4.2.9. Statistical Analyses

Data were analysed using Statistical Analysis Software (SAS®) University edition, and the results were presented by analysis of variance (ANOVA). The areas of ten main peaks were used in the analyses. For significant differences, means were compared using Least Significant Difference (LSD) and significant differences are reported at 5% level of significance.

79 4.3. Results and discussion 4.3.1. Selection of Chamber Type and Determination of Extraction Time

The extraction efficiency of the two different chambers (0.5 L Tedlar bag and 0.5 L glass jar) was evaluated by comparing the main peak areas of the ten compounds from Floribunda and Hybrid Tea rose flowers under the same extraction time, SPME fibre, desorption time, and GC conditions. There were no significant differences between the glass jar and Tedlar bag, so the jar was chosen because it was easier to use than the Tedlar bag, also the Tedlar bag is not as easy to clean as the jar, and the jar was considered to optimum chamber in which to capture the VOCs emitted (Figure 4.1). Therefore, the jar was selected as appropriate jar to optimize the other parameters. These observations agree with a study by Pragadheesh et al. (2011) who used an SPME fibre to extract a number of high-quality VOCs from Jasminum.

25

6 20 Bag Jar 10

15

10

5 GC GC readings (Area)*

0 Floribunda Hybrid

Figure 4.1. Peaks of volatiles organic compounds produced by intact Floribunda and Hybrid Tea rose flowers extracted by two types of chambers (Tedlar bag and jar). Error bars are LSD at p≤ 0.05 (n = 3).

4.3.2. Evaluation of GC Injector (Fibre Desorption) Desorption Time

The various desorption times (5, 10 and 15 min) of the three-phase fibre did not differ significantly. Therefore, 5 min was selected as desorption time (Figure 4.2). Desorption time is an important process and it depends on speed of desorption of the VOCs from the fibre through the injection port. Theoretically, desorption time should be long enough to completely release all the absorbed volatiles from the fibre. For this reason, 250˚C was chosen as the optimum desorption time for the whole experiment. In fact, there are two factors that should

80 be considered: (1) in the SPME technique, injection port temperature is important because every fibre has a temperature stability range based on the fibre coating (e.g. for the three-phase fibre, 270˚C is recommended as the limit of temperature tolerance), and (2) exposure of the fibre to very high temperatures can shorten the fibre’s life, a result in the loss of its polymer coating (Qiu et al. 2014).

30 Hybrid

25

20

15

10

6

10

* 5

0 Floribunda 1.5

1 GC readingsGC (Area)

0.5

0 4 6 8 10 12 14 16 Desorption time (min)

Figure 4.2. Effects of different desorption times on desorption of VOCs from rose flowers. Bars represent LSD at p ≤ 0.05 (n = 3).

Moreover, high temperatures used for the release of the VOCs from the fibre in the injection port can reduce GC sensitivity; this could be due to denaturation, destruction, or decomposition of the chemicals at higher temperatures (Magan and Evans 2000). Therefore, there is a need to optimize the temperatures used to ensure maximum release of VOCs from the fibre without compromising the composition of VOCs released.

81 4.3.3. Analysis of Different Extraction Time for Rose Flower

The three-phase fibre was exposed in the head-space of the jar for three extraction times (1, 2 and 3 h), with cut and intact Floribunda and Hybrid Tea flowers under the optimized HS-SPME GC conditions. The total area of VOCs increased between 1 h to 2 h, but there was no significant difference between 2 and 3 h. Hence, the 2 h extraction time was chosen as the optimum extraction time (Figure 4.3). The differences in GC readings between cut and intact flowers were significant for Floribunda, and the VOCs emitted from the intact flowers were superior to those from the cut flowers. In contrast, there were no significant differences between the cut and intact flowers for Hybrid Tea. Therefore, intact flowers for both rose varieties were chosen for further work (Figure 4.4).

The optimal extraction time from the fibre for both intact and excised flowers was 2 h, which is longer than the time used by Pragadheesh et al. (2011) who reported 15 min as the optimum extraction time to extract the volatiles compounds produced by Jasminum sambac flowers. This difference is most likely due to the different plant species being tested. Also maybe the size of the flowers of Jasmine comparison to rose flowers since, the extraction time depends on the chemical nature of the compounds present, the distribution constant, the fibre polymeric phase, and to the size of the molecular mass (e.g., polyunsaturated fatty acids and other compounds are expected to require longer extraction times depending on their lower partitioning and diffusion coefficient). The HS-SPME method has been used for extracting volatile compounds from flowers at room temperature, this method is favourable for flower VOCs analysis, because it minimizes high temperature artifacts (Chen et al. 2013)

In the present study, the results indicated that there were differences between intact and cut flower for the Floribunda variety, and intact flowers were superior to excised flowers as determined by total peak area. In contrast, there were no differences between excised and intact flowers for the Hybrid Tea variety. Apparently, more volatiles are emitted from the Hybrid Tea variety than Floribunda variety (Figure 4.12), and this is because different species have different types and amounts of compounds. Furthermore, Chinese roses and European roses including the hybrid tea have higher scent than the other rose types (Guterman et al. 2002; Joichi et al. 2005; Flament et al. 1993; Nakamura 1987).

4.3.4. Analysis of Different Extraction Time for Rose Leaves

The optimized HS-SPME and GC conditions were applied to collect the VOCs from the cut and the intact leaves for both rose varieties. There were no differences between different

82 extraction times (1, 2 and 3 h), for both varieties, so 1 h was selected (Figure 4.5). Each variety emitted different numbers and amounts of volatiles from the leaves, with larger numbers produced from the intact leaves than the cut leaves for both varieties (Figure 4.6). In general, all plants have the ability to emit VOCs and the content and composition of these VOCs will depend on the plant species and plant organ.

8 1 hour 2 hours 3 hours 7 A 6 5 4 3

2

6 1 0 80

60

40 B GC readings (Area) *10 readings (Area) GC

20

0 Cut Intact

Figure 4.3. (a) Effects of extraction time with Floribunda rose (cut and intact) on the extraction efficacy of the total compounds identified from rose flowers at 1, 2 and 3 h for the glass jar; and (b) as for (a) for Hybrid Tea rose. Error bars were LSD at p ≤ 0.05 (n = 3).

83 6

Intact Cut

5

6 4

3

2

1 GC readings (Area) *10 (Area) readings GC

0 Floribunda Hybrid

Figure 4.4. Gas chromatograph readings for volatiles organic compounds emitted from cut and intact rose flowers from Floribunda and Hybrid tea rose varieties. Error bars were LSD at p ≤ 0.05 (n = 3).

The VOCs emitted from plant leaves are considered the main challenges in plant VOCs research because they are difficult to sample accurately due to the low concentrations of VOCs present and the inherent reactivity of some VOC compounds that makes them hard to detect directly (Materić et al. 2015). Using the optimized HS-SPME method developed in this study, we were able to accurately detect volatiles from excised and intact rose leaves, and there were no significant differences between the different extraction times, and a 1 h extraction time was chosen for both varieties. In contrast, Zini et al. (2002) using an SPME fibre with a glass chamber used an extraction time of 1 min performed every 30 min to identify VOCs from intact Eucalyptus leaves.

Moreover, Materić et al. (2015) showed that VOCs from foliage could be analysed using different sampling methods and available analytical techniques used for this purpose. In the present study, we found significant differences between the cut and the intact leaves for the two varieties, with more VOCs obtained from attached rose leaves for both varieties. This may be because for the intact leaves, respiration and photosynthesis are be higher than the cut leaves, and consequently more plant secondary metabolites are be produced. To our knowledge, this is the first comprehensive study on the VOCs of cut and intact rose leaves

84 1 hour 2 hours 3 hours 9 (a) 8 7 6 5 4

3

6 2 1

0

9

8 (b) GC readingsGC (Area)*10 7 6 5 4 3 2 1 0 Cut Intact

Figure 4.5. (a) Volatile organic compounds collected from cut and intact leaves following extraction times of 1, 2 or 3 h for (a) Floribunda and (b) Hybrid Tea leaves. Error bars were LSD at p ≤ 0.05 (n = 3).

85 9

8 Intact Cut

7

6 6

10 5

4

3

2 GC GC readings (Area)*

1

0 Floribunda Hybrid

Figure 4.6. Effect of gas chromatograph readings on emission of volatile organic compounds from cut and intact rose leaves from Floribunda and Hybrid Tea were different. Error bars were LSD at p ≤ 0.05 (n = 3).

4.3.5. Analysis of Different Extraction Times for Rose Stems

There were no significant differences in the emission of VOCS from the intact and the excised rose stems for the two rose varieties. There were ten compounds produced and there were no differences between the different extraction times (1, 2 and 3 h) (Figure 4.7). For the Floribunda variety there was no significant difference between the cut and the intact stems, whilst there were significant differences in the Hybrid Tea variety. Overall, the results indicated that the cut stems gave superior results compared with the intact stems (Figure 4.8); therefore, the cut stems were selected for further studies. Using the optimized HP-SPME fibre method developed in this study, we found that the GC chromatogram for different extraction times were not different between the intact or the excised rose stems for both rose varieties and a 1 h extraction was selected. A recent study on Thymus stems indicated that VOCs extracted using hydro distillation and solvent microwave extraction techniques were higher than those from root volatiles (Azar et al. 2011).

86 1 hour 2 hours 3 hours (a) 0.7

0.6

0.5

0.4

0.3

0.2

6 0.1

0.70

(b) 0.6

0.5 GC readingsGC (Area)*10 0.4

0.3

0.2

0.1

0 Cut Intact

Figure 4.7. Volatile organic compounds collected from cut and intact stems following extraction times of 1, 2 or 3 h for (a) Floribunda and (b), Hybrid Tea leaves. Error bars were LSD at p ≤ 0.05 (n = 3).

Likewise, a study by Ahmad et al. (2014) found VOCs emitted from Polygnum minus stems, were more effectively detected on an SPME fibre after the stems were extracted with hydro distillation and the VOCs analysed. Most of the volatiles that were detected from the intact and the cut Floribunda stems indicated that there were no differences between the cut and the intact Floribunda stems. In contrast, there were significant differences between the intact and the cut Hybrid Tea stems, with more VOCs obtained from the intact stems than the cut stems.

4.3.6. Analysis of Volatiles Organic Compounds in Rose Rhizosphere with Different Extraction Times

The amount of the volatile compounds did not differ significantly between those collected at 5 cm than 10 cm depth (Figure 4.9). Therefore, the 5 cm depth was selected for subsequent

87 studies. The VOCs emitted from the Floribunda rhizosphere did not differ significantly between the different extraction times (6, 12 and 16 h). Therefore, 6 h was chosen for subsequent studies for Floribunda (Figure 4.10). In contrast, in Hybrid Tea there were significant differences in the amounts of VOCs produced at the different extraction times from the rhizosphere (Figure 4.10). Therefore, 12 h was selected as the best extraction time to absorb the VOCs emitted from rhizosphere of Hybrid Tea.

0.6 Intact Cut

6 0.5 10 0.4

0.3

0.2

GC readings (Area) * (Area) readings GC 0.1

0 Floribunda Hybrid

Figure 4.8. Gas chromatograph readings for volatile organic compounds emitted from cut and intact rose stems from Floribunda and Hybrid Tea rose varieties. Error bars were LSD at p ≤ 0.05 (n = 3).

0.8

5cm 10cm

6 10 0.6

0.4

0.2 GC readings (Area) * (Area) readings GC

0 Floribunda Hybrid

Figure 4.9. Effects of soil depth (5 and 10 cm) on desorption of volatile organic compounds of Floribunda and Hybrid Tea rose varieties. Bars represent LSD at p ≤ 0.05 (n = 3).

88 The results clearly show that the HS-SPME combined with GC-FID can be reliably used to analyse VOCs from the rhizosphere of rose plants, and that depth of extraction did not affect the total peak area. This may be because the VOCs have small molecular weight and are readily move into the extraction tube inserted into the soil.

Hybrid

16h 12h 6h Floribunda

0 0.2 0.4 0.6 0.8 1 GC readings (Area) *106

Figure 4.10. Effects of extraction time with Floribunda and Hybrid Tea rose varieties on the extraction efficacy of the total volatile organic compounds identified from the rhizosphere at 6, 12 and 16 h. Error bars were LSD at p ≤ 0.05 (n = 3).

Also, extraction time depends on the distribution constant, chemical nature of desired compounds, and fibre polymeric phase. Most of the volatiles detected from whole rose plant indicated there were no significant differences between different extraction times for both rose varieties; consequently 1 h was considered an optimal extraction time for whole plants.

4.3.7. Analysis of Different Extraction Time for Whole Rose Plants The three-phase fibre was exposed to a whole rose plant in a glass chamber for three extraction times (1, 2 and 3 h). There were no differences for the total area of VOCs for Floribunda and Hybrid Tea rose varieties (Figure 4.11) with the extraction time. Therefore, a 1 h extraction time was used in the subsequent studies.

89 4

6 1 hour 2 hours 3 hours 10 3

2

1 GC GC readings (Area)*

0 Floribunda Hybrid

Figure 4.11. Volatile organic compounds collected from whole rose plants following extraction times of 1, 2 or 3 h for Floribunda and Hybrid Tea rose varieties. Error bars were LSD at p ≤ 0.05 (n = 3).

HS-SPME can reduce the time used for extraction significantly because of the faster diffusion rate of the molecules in the gaseous phase. Furthermore, the fibre is suspended in the HS above the sample, so there is no contamination of the fibre by the sample. From our study, the fibres can be reused more than 200 times before they deteriorate, therefore reducing the cost of the materials. Finally, HS-SPME GC does not require skilled technicians as it can be readily and reliably used after a short period of training.

To our knowledge, this is the first study on the detection of VOCs from different organs of roses, and it is the first time the HP-SPME technique has been used to identify VOCs from attached and excised flowers, leaves, stems, roots and soil (rhizosphere), and the whole plant for roses. The HS-SPME technique coupled with GC is a robust and cost effective system for the identification of rose volatiles. Further studies are in progress to detect and identify the VOCs that are released from different rose organs by GC-MS. Therefore; the optimized methods developed in this study can be used either as a detection tool or as a new qualitative methodology for physiological studies of roses and other horticultural and agricultural plants.

90

ormalization N

Retention time (min)

Figure 4.12. Gas chromatography (GC) chromatograms of (a) Hybrid Tea rose and (b) Floribunda rose

4.4. Conclusion

This study showed that the use of a glass jar with a 5 min desorption time gave good results for the extraction of VOCs from roses. A 2 h extraction time was found to be optimum for the extraction of the VOCs from flowers of both rose varieties. Whilst, a 1 h extraction time was found to be optimum for leaves, stems and whole rose plants for both rose varieties. In contrast, there was a difference between the two rose varieties for the optimum extraction time of VOCs from the rhizosphere. The optimum extraction times from rhizosphere of Floribunda and Hybrid Tea were 6 h and 12 h, respectively. These findings provide a sound baseline for further studies different horticulture plants as they are easy and fast to get the results.

91

92

93 Chapter 5 ______

Study on Foliar Application of Plant Growth Regulators on Volatile Organic Compounds Composition in the Flowers of Two Rose Varieties

94 Abstract

The study focused on the influence of the plant growth regulators (PGRs) benzyladenine (BA) and naphthalene acetic acid (NAA) on volatile compounds composition from flowers of two modern rose varieties. Forty-five plants each of Hybrid Tea and Floribunda rose were tested. BA and NAA were applied at 0, 100 and 200 mg/L to both rose varieties. Gas chromatography (GC) coupled with flame ionization detection (FID) and mass spectrometry (GC-MS) was used to analyse and identify the volatile organic compounds (VOCs) from the flowers. A three-phase fibre 50/30µm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) was used to capture VOCs at 2, 4 and 8 weeks, and 4 weeks was selected as it had the highest peak area. In total, 81 and 76 VOCs were detected after treatment in both rose varieties with BA and NAA, respectively. In addition, 20 compounds, which had significant differences between different treatments, were identified from Floribunda and Hybrid Tea. The majority of VOCs were extracted after the application of 200 mg (BA and NAA)/L of formulation, and the results showed that four important compounds, cis-muurola-4(14)5-diene, γ -candinene, y-muurolene and prenyl acetate increased significantly compared with the controls. These compounds are commercially important aroma chemicals. This study used the rapid and solvent-free SPME method to show BA and NAA treatments and could result in significant volatiles composition in the flowers of two rose varieties, enhancing the aromatic values of the flowers. This method has the potential to be applied to other valuable aromatic floricultural plant species.

5.1. Introduction

Most floral scents are considered pleasant; they have played key roles in the early development of flowers (Caruso 2000). The genus Rosa is comprised of between 100-200 species and more than 18,000-24,000 commercial cultivars most of which are hybrids, and collectively these are based on only eight wild species (Horn 1992; Thomas 2000 and Kim et al. 2004). Hybrid Tea and Floribunda roses are considered modern roses and are popular in Australia (Chapter 2). The main commercial and industrial uses of roses are based on the perfume and floriculture industries, the former of which relies on scented rose varieties (Özel et al. 2006). There are many studies on the emission of rose volatiles, but few discuss modern rose varieties (Pellati et al. 2013). Many factors affect the emission of aromatic compounds and among these are plant growth regulators PGRs (Sangwan et al. 2001).

95 Plant growth regulators (PGRs) are produced naturally by plants or synthetically by chemists, and they play a key role in life cycles of plants (Davies 2010). PGRs include cytokinins and auxins, which are small molecular compounds that regulate at low concentrations plant development processes (Fu et al. 2011). BA and NAA have both been applied pre- and post- harvest to many ornamental plant species. Cytokinins such as BA are used to improve the quality and vase life of cut flowers. Moreover, BA promotes cell elongation and division, decreases flower drop and increases flower production (Krug et al. 2006). BA has also been used to improve the growth and development of aromatic plants. Treating Lantana camara plants with BA at 0.44 and 4.4µmol/L increased volatile organic compounds production, which was detected by solid phase micro-extraction (SPME) (Affonso et al. 2007). Similarly, Zielińska et al. (2011) indicated that the production of VOCs increased with application of different PGRs such as 0.57 µmol indole-3-acetic acid (IAA), 0.45 µmol thidiazuron, 9.3 µmol kinetin and 4.4 µmol BA when applied to Agastache rugosa plants grown in vitro. Another study on Stevia rebaudiana plants reported that BA and IAA added to tissue culture media significantly influenced the in vitro production and distribution of secondary metabolites to produce strong antioxidant activity (Radic et al. 2016). Auxins such as NAA coordinate many growth and behavioral processes in plant life cycles. Furthermore, NAA induces cell division and elongation, and plays a key role in shoot development and increased flower production (Fu et al. 2011). A study on Rosa damascene indicated that the application of NAA at 25 mg/L to rose plants, increased flower longevity, plant height, flower yields and flower oil content (Saffari et al. 2004). A study by Bota and Constantin (2015) showed that the addition of 0.1 mg/L NAA and 1 mg/L BA to a cell suspension culture of Digitalis lanata increased the production of flavonoids, which are related to secondary metabolite production. Another study by Coste et al. (2011) indicated that the addition of BA (0.2 mg/L), Kinetin (0.1 mg/L) and NAA (0.05 mg/L) together to mineral salts (MS) medium enhanced the production of secondary metabolites in shoot cultures of Hypericum hirsutum and H. maculatum plants. Little information is known about the influence of these PGRs on secondary metabolite production (Povh and Ono 2006). To date, to our knowledge there has been no research reported on the influence of NAA on volatiles composition in rose plants.

The detection of VOCs is widely used by scientists, because of their wide range of beneficial pharmacological and biological activities (Edris 2007). Currently, more than 400 VOCs have been identified from rose flowers (Lavid et al. 2002). The headspace solid phase micro- extraction (HS-SPME) is a new method for the extraction of VOCs from plants; it is now

96 commonly used (Zhu et al. 2013). HS-SPME is a pre-concentration technology, which integrates extraction, concentration and sampling, and is a simple, fast and solvent-free preparation method for the extraction of VOCs (Zhang and Pawliszyn 1993). It also prevents the production of artifacts compared with conventional solvent extraction procedures (Bicchi et al. 2000). This method coupled with GC- FID/MS analysis, and using the SPME fiber divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) has been successfully applied for the analysis of VOCs emitted from rose plants (Chapter 4 and Chapter 6). To our knowledge, there are no studies on the influence of BA and NAA on volatiles composition from rose flowers using the SPME technique. Consequently, the aim of this study was to study metabolite accumulation in the flowers of two rose varieties following the application of BA and NAA, using the HS-SPME technique with GC-FID/MS.

5.2. Materials and Methods 5.2.1. Standards and Reagents

The n-alkane standard (C7-C30) reference materials at 1000 μg/mL in hexane were purchased from Sigma-Aldrich (catalog number 49451-U; Castle Hill, NSW, Australia). Naphthalene acetic acid (NAA) (95% purity, product number N0640) was purchased in crystalline form from Sigma-Aldrich (Castle Hill, NSW, Australia). Mohammed et al. (Chapter 6) described all other standards and regents. Briefly, these included high-performance liquid chromatography grade ethanol, n-hexane (95%), benzyladenine (BA) (98%), sodium hydroxide as a pellet (97%), rooting hormone (Clonex) in gel form, Vapor guard (anti-transpirant concentrate) liquid, deionized water (Milli-Q Ultrapure water system) and double distilled water.

5.2.2. Apparatus and Equipment

A semi-quantitative analysis of VOCs from flowers of Hybrid Tea and Floribunda rose varieties was performed to obtain the relative amount of each volatile component. For this procedure, a gas chromatograph with a flame ionization detection (GC-FID) system, 7829A GC (serial number CN14272038; Mulgrave, Victoria, Australia), equipped with an HP-5MS column non-polar (30 m × 0.25 mm, film thickness 0.25 μm, catalogue number 13423) was used. The column temperature program was between 50-250˚C with gradual increases of 5˚C/min; helium was used as carrier gas with a flow of 1.1 mL/min. The total run time was 45 min; and the GC-FID mode was splitless, and the temperatures of the injector was 250˚C and 290˚C, respectively.

97 The flower samples were analysed and identified using a GC-MS Agilent 7820A gas chromatograph coupled to a 5977E mass spectrometer. The analytical conditions used were as follows; splitless injection at 250˚C, and an HP-5MS capillary column (30 m × 0.25 mm fused- silica, with a film thickness of 0.25 μm, Agilent Technologies Inc). The column temperature program was 50-250˚C with gradual increases of 5˚C/min; helium was used as carrier gas with a flow of 0.7 mL/min. The total GC-MS run time was 45 min and the constituents were identified by matching their spectra with those recorded in a mass spectra library (Wiley, Hoboken, New Jersey, USA). Relative amounts were calculated in relation to the total area of the chromatogram. Mass spectra were compared with the retention indices (RI) or GC retention time data of synthetic standards and compounds published in the literature. The same conditions of the GC-MS analysis were employed to ensure reproducibility by using the RESTEK website to translate the conditions (the pure chromatography EZGC method translator) of the GC-FID to GC-MS. A fibre 50/30µm divinylbenzene/ carboxen/polydimethyl siloxane (DVB/CAR/PDMS) coated fused-silica fibre was used as it is the most suitable fibre for adsorbing VOCs emitted from the headspace of different rose tissues (Chapter 4 and Chapter 6).

5.2.3. Plant Material and Maintenance

The experiment was conducted in March 2018 (summer). Ninety plants of two rose plants (45 plants each of Hybrid Tea cv. Mr Lincoln and Floribunda cv. Iceberg) were grown in an evaporatively cooled greenhouse at Murdoch University (Perth, Western Australia, Australia). 100 and 200 mg of BA and NAA were mixed with 4 to 6 drops of liquid sodium hydroxide (NaOH) 1 N and then added to 1 L of double distilled water. The growing conditions, age of plants, application of BA and NAA, and plant maintenance were described previously (Chapter 6). The rose plants were prepared in a factorial based completely randomized design with two rose varieties, two hormones, three concentrations of each hormone and three replicate plants for each treatment.

5.2.4. Sample Extraction Using HS-SPME

The rose plants were transferred to the laboratory for sampling. The samples were analyzed in biological triplicates. Three undamaged and fully open flowers for each rose variety were individually sampled within a chamber for the extraction of VOCs as previously described (Chapter 4). Briefly, the flowers were placed in a 500 mL glass jar with a septum, sealed with aluminum foil to trap the volatiles inside the glass jar. The SPME fiber was inserted through a

98 5 mm port within the septum to absorb the VOCs. The HS-SPME protocol included three steps; firstly, the SPME fiber was activated for half an hour in the GC-FID, secondly, the extraction time for the samples was 2 hours with the HS-SPME, and finally the SPME fiber was injected into the GC port for 45 min. at 250˚C to collect the VOCs from the lowers of the two rose varieties.

5.2.5. Optimization of Different Sample Times after PGRs Application

To determine the most efficient collection time for VOCs after the application of BA and NAA, samples were collected at 2, 4 and 8 weeks post-PGRs treatment.

5.2.6. Assessment of Peaks and Identification of Volatile Compounds

The volatile compounds were identified using quantitative analysis software (MS quantitative analysis) for GC identification. Each treatment was compared with the other treatments according to the total peak area. The volatile compounds identified from samples were also compared with the compounds identified from the internal standards. Moreover, the identified volatile compounds were also compared with the literature using the retention index (RI) relative to a mixture of n-alkanes (C7-C30) in n-hexane by using DVB-CAR-PDMS fibre. Comparison of the MS-fragmentation pattern of the target analyses with those of pure components was also performed. The VOC peaks produced by the GC-MS were identified using AMDIS (Chapter 6).

5.2.7. Statistical Analyses

The Analyses of variances (ANOVA) were made using the general liner model procedure of the statistical analysis software (SAS®) University edition version. Means of 10 main peak areas were = analysed at each of the sampling times. Least significant differences (LSD) and the MetaboAnalyst 4.0 (http://www.metaboanalyst.ca/faces/home.xhtml) were used to compare means and significant differences reported at the level of statistical P ≤ 0.05 (Chapter 6).

5.3. Results and Discussion 5.3.1. Comparison of Optimal Sampling Times after PGRs Application

The 2, 4 and 8 weeks sampling times were significantly (P<0.05) different from each other for both the BA and NAA treatments. A sampling time of four weeks was shown to the optimum based on both high peak and total peak area (Figure 5. 1). The peak areas of VOCs increased from 2 to 4 weeks and decreased at 8 weeks, so 4 weeks was selected as the optimum sampling

99 time for both BA and NAA. This sampling time corresponds with the results of (Chapter 6 and Baghele et al. 2016) on rose plants. In contrast, a study by Salehi et al. (2013) indicated that samples collected from Aloe barbadesis plants at 8 weeks after the application of PGRs was the optimum time for sampling.

3

2 weeks 4 weeks 8 weeks

6 10 2

1 GC GC readings (Area)*

0 Floribunda Hybrid

Figure 5. 1. Comparison of the main peak area using different sampling times for the analysis of volatile compounds collected from flowers of two rose varieties. Bars represent LSD at (p ≤ 0.05) (n=3).

5.3.2. Analysis VOCs after BA and NAA Application

A total of 81 components were identified from both rose varieties after the application of BA. Eight VOCs produced by Hybrid Tea flowers were significantly (P≤0.05) different from the controls (double distilled water) as determined by one-way ANOVA & post-hoc Tests (Figure 5. 2a). In contrast, the Floribunda flowers produced 5 VOCs that were significantly (P≤0.05) different from the controls (Figure 5. 3a). The accurate collection of VOCs is very important for the analysis of flower volatiles. Various methods have been used to analyse volatile compounds, and include HS-SPME (Zhu and Chai 2005). Unfortunately, the VOCs obtained from extracted oils rarely provide the natural aroma of flowers because of thermal artefacts produced during the steam distillation process that typically occurs at 60-70˚C (Deng et al. 2006). In contrast, the HS-SPME fibre method is favourable for the analysis of volatiles emitted from flowers because high-temperature artefacts are minimized during the process (Chen et al.

100 2013). Overall, the different rose varieties showed differences in the quantity and quality of volatile compounds produced, with only a few differences in the emission of VOCs (Table 5. 1).

A total of 76 compounds were identified from the two rose varieties after NAA application. Five compounds showed significant (P≤0.05) differences for Hybrid Tea (Figure 5. 2b), while two compounds were significant (P≤0.05) for Floribunda (Figure 5. 3b), compared with the control. The two PGRs induced different types, amounts, and quantities of VOCs emitted from the flowers. The HS-SPME technique has been used to analyse the emission of volatile organic compounds from fresh flowers of two rose varieties (Chapter 4) and chrysanthemum (Sun et al. 2015). The results correspond to a recent study by Ibrahim et al. (Chapter6), which showed that as the concentration of BA applied to Hybrid Tea and Floribunda increased from 0-200 mg/L, the amount of metabolites produced increased significantly.

Figure 5. 2. Volatile organic compounds (VOCs) collected from Hybrid Tea flowers after application of BA (A) and NAA (B), the red dots represent the VOCs, which are significantly (P<0.05) different to the total collected volatile compounds.

101

Figure 5. 3. Volatile organic compounds (VOCs) collected from Floribunda flowers after the application of BA (A) and NAA (B), red dots represent the VOCs, which have significant (P<0.05) different from total collected VOCs.

5.3.3. Influence of BA and NAA on VOCs Emitted from Flowers of Two Rose Varieties

For both BA and NAA, applications of 200 mg/L gave the highest peaks compared with 0 or 100 mg/L for both rose varieties (Figure 5. 4). Similarly, Ibrahim et al. (2018; Chapter 6) showed that BA at 200 mg/L increased the peak areas and the emission of volatile compounds from different rose tissues in two rose varieties (Hybrid Tea and Floribunda). Farooqi et al. (1993) indicated that the amount of essential oil extracted from Rosa damascena plants after the application of kinetin increased compared with that of the control plants. Moreover, another study on Plectranthus ornatus plants grown in tissue culture indicated that BA application significantly increased VOCs and these helped callus induction (Passinho-Soares et al. 2017). Prior to the current study, no work had been done on how NAA effects the production of VOCs in vivo in roses.

102 20 BA Control 100 mg/L 200 mg/L

15

10

5 5

0 20 NAA

15

GC readingsGC (Area) *10 10

5

0 Floribunda Hybrid

Figure 5. 4. Effects of BA and NAA application on the extraction efficacy of the total volatile organic compounds VOCs identified from flowers of two rose varieties as determined by GC- MS. Error bars are LSD at (p ≤ 0.05) (n=3).

5.3.4. Identification of VOCs Emitted from Flowers of Two Rose Varieties

Each PGR has different numbers of identifying compounds for both rose varieties (Table 5. 1). The amount of some of these VOCs increased with increasing levels of BA, for instance, cis- muurola-4(14), 5-diene, which is an important compound in the perfume industry, increased from 7.61 to 33.45 in total peak area as BA levels increased from 0-200 mg/L in Hybrid Tea flowers (Table 5. 1). Ma et al. (2015) showed that this compound was found in different tissues of Artemisia annua plants. Another aroma chemical γ-cadinene has been used for essential oils and medicinal purposes (Khoo et al. 2016), and this compound increased from 0.13 to 4.14 with an increase in BA from 0 to 200 mg/L in Hybrid rose. Similarly, γ-muurolene increased from 9.24 to 29.91 as BA was increased from 0 to 200 mg/L in Floribunda. Folashade and Omoregie (2012) demonstrated that γ-muurolene was an important compound found in Lippia multiflora used in the essential oils industry. Moreover, a study on tissue cultured Plectranthus ornatus plants using BA and NAA showed that many compounds such as cis-muurola-4(14),5-

103 diene, γ-cadinene and γ-muurolene increased significantly and stimulated callus induction and plant growth (Passinho-Soares et al. 2017). In the current study, after NAA application at 200 mg/L, the amount of myrcene increased in Hybrid Tea rose. This compound is very important as an essential oil, flavor, and fragrance, as well as for health and medicinal purposes and corresponds to the findings by Hartsel et al. (2016) from Cannabis sativa plants. In addition, prenyl acetate produced in high quantities in Hybrid Tea after NAA application at 200 mg/L is also used for commercial and chemical purposes.

Specifically, in this study, we only selected 81 and 76 VOCs produced from BA and NAA, respectively for both rose varieties. These were analyzed by MetaboAnalyst 4.0, and from this only 20 VOCs were shown to increase significantly compared with those of the controls. None of the BA and NAA applications significantly decreased the concentrations of VOCs below the controls.

104 Table 5. 1. Identification per cent area of VOCs that differed significantly (p ≤ 0.05) between flowers of two rose varieties after being treated with different concentrations of BA and NAA.

RT Compounds RI V Mean of peaks at BA (mg/L) Mean of peaks at NAA (mg/L)

0 100 200 0 100 200

8.82 Propyl glycolate 928 F 1.33 1.23 2.64* 0.32 0.30 0.58

10.11 Prenyl acetate† 932 H 2.28 2.09 2.69 1.98 12.95 17.01*

10.55 α-Pinene 939 F 3.56 4.02 6.40* 4.61 5.60 5.43

12.77 Myrcene 979 H 12.25 16.60 13.20 14.12 17.48 30.44*

13.52 D-Limonene 1018 F 0.11 0.84 1.09* 2.52 2.03 2.38

19.19 Cuminal 1214 H 1.24 1.88 4.38* 1.52 1.84 1.04

21.29 Geranial 1249 H 37.57 51.06 70.71* ND ND ND

22.03 Methyl geranate 1299 H 2.26 3.25 5.21* 4.46 2.23 6.86

24.19 Geranyl acetate 1360 H 2.58 10.84 11.09* 9.01 12.42 15.01

25.24 Caryophyllene 1396 F 21.56 27.90 24.88 25.38 31.83 51.21*

25.42 Isocaryophyllene 1424 F 27.46 28.72 22.86 17.03 34.62 37.11*

25.44 Caryophyllene 1428 H 2.26 3.25 5.21 4.04 6.18 7.39*

25.61 α,β-Dihydro-β-ionone 1437 H 45.47 49.27 47.49 42.86 69.98 71.17*

105 25.62 Dihydro-β-ionone 1439 H 44.82 56.10 76.17* 25.22 24.75 38.47

25.88 cis-Muurola-4(14),5-diene† 1455 H 7. 61 10.83 33.45* 5.07 10.43 13.17

† * 26.59 γ-Muurolene 1494 F 9.24 12.06 29.91 26.66 23.97 27.09 30.90 trans-3-Octadecene 1695 H 1.07 1.26 2.20* 4.28 6.99 7.04

34.27 Z-5-Nonadecene 1818 H 0.87 1.03 1.16* 13.07 13.17 19.09

35.15 9-Nonadecene 1880 H 16.39 15.82 15.93 13.57 39.87 43.62*

35.23 γ-Cadinene† 1905 H 0.13 0.16 4.13* 1.27 0.79 2.06

(*) represents significant differences between different concentrations of BA and NAA (0 (control), 100 and 200 mg/L); (RT) retention time; (RI) retention index based on alkane series; (V) rose variety; (F) Floribunda; (H) Hybrid Tea; (ND) not detected; (†) compounds that increased significantly with LSD P ≤ 0.05 in both rose varieties with increasing concentrations of BA and NAA compared to the control.

106 5.4. Conclusion

The best sampling time was 4 weeks after application with the two PGRs. Eighty-one and 76 VOCs were emitted from flowers for both varieties after the application of BA and NAA, respectively. Of these, twenty VOCs were produced at significantly higher concentrations than those of controls. The application of both NAA and BA at 200 mg/L was the optimum concentration and superior to the control and 100 mg/L treatments. To our knowledge, this is the first comprehensive study on the influence of BA and NAA on aromatic volatile compounds emitted from fresh rose flowers. These findings will benefit industries where there is an interest in increasing the quantity and quality of volatile compounds from plant products. In addition, four VOCs known to be precursors of fragrant compounds were increased significantly after BA and NAA treatments, these were cis-muurola-4(14),5-diene, γ-cadinene, prenyl acetate and γ-muurolene. All of these compounds play important roles in aromatic plants. Consequently, BA and NAA at 200 mg/L could be used to increase and improve the quantity and quality of VOCs produced from roses and potentially other floricultural plants. Future research could also explore using higher concentrations than 200 mg/L for both BA and NAA. Furthermore, future research could look at applying both BA and NAA together; this has not been done before. This is likely to get more VOCs might be better quality or more abundance.

107

108

109 Chapter 6 ______

Influence of Benzyladenine on Metabolic Changes in Different Rose Tissues

110 Abstract

Two modern rose varieties, Floribunda and Hybrid Tea, were used to analyse and identify metabolic changes after foliar application with benzyladenine (BA). Volatile organic compounds (VOCs) were assessed as metabolites. Two doses of BA were applied at 11.16 and 17.87 mg/cm2 for Hybrid Tea and 7.17 and 12.26 mg/cm2 for Floribunda. Sampling time was optimized and treatment duration was 4 weeks. After treatment, the volatiles from the treated and untreated roses were extracted using headspace solid-phase microextraction (HS-SPME) technology using a three-phase fibre 50/30µm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) and analysed by gas chromatography (GC) coupled with a flame ionization detector (FID) together with mass spectrometry (GC-MS). The results showed that BA and its dose rate led to metabolic changes of treated roses in comparison with untreated controls. The number of VOCs extracted and detected from leaves, stem, rhizosphere and whole plants of the two rose varieties following dose rates of 17.87 and 12.26 mg/cm2 were 43, 65, 40 and 68 compounds for each plant tissues, respectively. Whilst the VOCs extracted and detected from leaves, stems, rhizosphere and whole plants from both rose varieties following dose rates11.16 and 7.17 mg/cm2 were 38, 61, 34 and 66 compounds from each plant tissues, respectively. The results demonstrated that some volatiles, such as 4-heptyn-2-ol, phenyl methyl ether and 3-methyl-apopinene, increased with increasing doses of BA. These compounds are aroma chemicals with a very powerful smell. This study shows that BA treatments could have a significant effect on metabolite changes in different rose tissues. This method could be applied to other floriculture plants.

6.1. Introduction

The rose is one of the most common flowers cultivated in containers, glasshouse and gardens. Roses are important to the cut flower industry and for pharmaceutical purposes (Guterman et al. 2001; Kovacheva et al. 2010). Thus, the rose is commercially very important. It is also highly sought after for interior decoration and for the perfume industry, of which the latter relies on different scented varieties (Özel et al. 2006; Rusanov et al. 2011a; Rusanov et al. 2011b; Baydar and Baydar 2005; Pellati et al. 2013). There are numerous varieties of modern roses that are considered important; among these varieties are Hybrid Tea and Floribunda (Ibrahim et al. 2017a; Chapter 2).

111 Recently, PGRs such as cytokinins (CKs) have become widely used to ensure efficient production of aromatic plants. There are many studies indicating that PGRs affect the growth and development of various aromatic plants (Sangwan et al. 2001). Ramesh et al. (2001) reported that benzyladenine (BA), a cytokinin applied as a foliar spray at different concentrations, might reduce flower drop and senescence. Although PGRs have been used in agriculture and horticulture for decades, little is known about the effects of PGRs on the production of metabolic compounds (Povh and Ono 2006).

Currently, VOCs have received special attention from the scientific community, as they have a wide range of biological and pharmacological uses and there are many publications related to the emission of volatiles (Edris 2007). Affonso et al. (2007) treated Lantana camara plants with high concentrations (0.44 and 4.4 µmol/L) of BA and showed an increase in the production of volatile compounds detected by solid phase microextraction (SPME). These include myrcene, α-phellandrene, α-copaene, trans-caryophyllene and β-gurjunene. Another study indicated that incorporating PGRs, such as 4.4 µmol BA, 9.3 µmol kinetin, 0.45 µmol thidiazuron, 0.57 µmol indole-3-acetic acid (IAA) and 0.41 µmol picloram, into in vitro media of Agastache rugosa plants increased the emission of volatile compounds, such as α-pinene, limonene, isomenthone and pulegone (Zielinska et al. 2011). To date, no work has been conducted on the influence of BA on metabolite accumulation in roses. Moreover, there are few studies on modern rose varieties and the extraction of VOCs from different rose tissues by headspace solid phase microextraction (HS-SPME) (Riffault-Valois et al. 2016). HS-SPME analysis can be used to determine and monitor the composition of living natural materials or natural products and provide information on the olfactory profiles released from these (Draelos 2011). The SPME technique has become increasingly common because it is a simple, fast and sensitive sample preparation method that reduces solvent usage while integrating sampling and sample preparation steps prior to instrumental analysis (Zhu et al. 2013; Zhang and Pawliszyn 1993). In Chapter 4, the HS-SPME technique was successfully used to extract VOCs emitted from different rose tissues with the SPME fibre divinylbenzene/carboxen/ polydimethylsiloxane (DVB/CAR/PDMS) coupled with gas chromatography (GC) mass spectrometry (MS). However, there has been no work on the effect of BA on metabolic changes by using VOCs from rose plants. Therefore, the aim of this study was to increase the metabolite accumulation in two rose varieties through the addition of BA, and to determine this using HS- SPME with a GC-flame ionization detector (FID)/MS.

112 6.2. Materials and Methods 6.2.1. Standards and Reagents

Standard n-alkane (C7-C30) reference materials, at 1000 µg/mL of each component in hexane, was purchased from Sigma-Aldrich (catalogue number 49451-U; Castle Hill, NSW, Australia). These included decane, docosane, dodecane, eicosane, heneicosane, heptacosane, heptadecane, hexacosane, hexadecane, heptane, nonacosane, nonadecane, nonane, octacosane, octadecane, octane, pentacosane, pentadecane, tetracosane, tetradecane, triacontane, tricosane, tridecane and undecane. High-performance liquid chromatography grade ethanol was purchased from Merk (Kenilworth, NJ, USA). An n-hexane (95%) was purchased from Sigma-Aldrich (catalogue number 270504-2L; Castle Hill, NSW, Australia). Benzyladenine (BA) (98% purity, product number B3408) was purchased in crystalline form from Sigma-Aldrich (Castle Hill, NSW, Australia). Sodium hydroxide (97%) in pellet form was purchased from Merck (catalogue number ME9M591064; Kenilworth, NJ, USA). Rooting hormone (Clonex) in gel form was purchased from Hydroponic Xpress (Code: 9313656614189; Perth, Western Australia, Australia). Vapor guard (anti-transpirant concentrate) liquid was purchased from Miller Chemical (Hanover, PA, USA). Deionized water (Milli-Q Ultrapure water system) was obtained from Diversified Equipment Company (Lorton, VA, USA). Double distilled water was obtained from Refresh Pure Water (Perth, Western Australia, Australia).

6.2.2. Apparatus and Equipment

Aromatic compounds of the two rose varieties (Hybrid Tea and Floribunda) were analyzed by an Agilent Technologies 7829A GC system (serial number CN14272038; Mulgrave, Victoria, Australia), fitted with a non-polar HP-5MS column (30 m × 0.25 mm, film thickness 0.25 µm, catalogue number 13423). The oven column temperature ranged from 50-250◦C, and it was programmed at 5◦C/min, with a final hold time of 5 min, using helium (He) as carrier gas at 1.1 mL/min constant flow, in a split ratio of 1:1. The GC was coupled to a flame ionization detector (FID). The GC-FID instrument was operated under the splitless mode, and the FID temperature was 290◦C. The volatile compounds were identified by GC-MS using an Agilent 7820A GC (Mulgrave, Victoria, Australia), equipped with a HP-5MS 30 m × 0.25 mm fused-silica capillary column (Santa Clara, CA, USA), with a film thickness of 0.25 µm, coupled to an Agilent model 5977E mass spectrometer detector (MSD) under the splitless mode. The injector temperature was maintained at 250◦C. The GC conditions in the GC-MS analyses were the same as for the GC analyses, and used the pure chromatography (EZGC) method translator from the Restek website to translate the conditions of the GC-FID to GC-MS. The He carrier

113 gas was supplied by BOC Gas (Sydney, Australia). The total GC-MS run time was 45 min and the temperature of the injector port was 250◦C. The VOCs were identified by matching their spectra with those recorded in a Mass Spectral library (National Institute of Standards and Technology (NIST) mass spectral search program for the NIST/EPA/NIH Mass Spectral Library version 2.2, 2014, Wiley, Hoboken, New Jersey, USA). In addition, the VOCs were confirmed by comparing the Kovats Indices (KI) or GC retention time data with those of authentic standards or from the published literature. A 50/30µm divinylbenzene/carboxen/ polydimethyl siloxane (DVB/CAR/PDMS, catalogue number 57347-U; Sigma-Aldrich Castle Hill, NSW, Australia) fibre was attached to a manual SPME holder (Supelco Inc, Sigma- Aldrich Castle Hill, NSW, Australia). Three SPME fibres of medium polarity were used for extraction and the fibres were conditioned at the manufacturer’s recommended conditioning temperature (270˚C) before analysis.

6.2.3. Plant Material and Maintenance

The experiment was carried out in March 2018 (summer). Ninety two-year-old rose plants grown in free-draining containers were used. These plants were prepared from cuttings of Hybrid Tea cv. Mr Lincoln and Floribunda cv. Iceberg, procured from Roworth Rose Nursery (Perth, Western Australia, Australia) and treated with rooting hormone (Clonex) in gel form and planted into seedling trays containing peat: perlite (1:1, v/v). The trays were then placed in a greenhouse at Murdoch University. After three months, the rooted cuttings were transferred to individual 16 cm × 16 cm (diameter × height) free-draining plastic pots. One year later, these were transferred to 24 cm × 24 cm (diameter × height) free-draining plastic pots consisting of two parts composted pinebark, two parts coarse river sand and one-part coco peat, all obtained from Richgro (Perth, Western Australia, Australia). These were then placed in an evaporatively cooled glasshouse at Murdoch University. Glasshouse temperatures were maintained between 18±2◦C and 25±2◦C during the night and day, respectively, while the humidity was maintained at 60±2% and 75±2% day and night, respectively. The plants were fertilized every three months with a complete fertilizer (Miracle-Gro Maxfeed Soluble Plant Food All Purpose, including NPK) (Scotts Australia, NSW, Australia)) and watered manually daily to container capacity with about 400 mL of water. BA powder (0 (control), 100 and 200 mg) was mixed with 4 to 6 drops of liquid sodium hydroxide (NaOH) 1 N and then added to 1 L of double distilled water. BA at 0 (control), 100 and 200 mg/L was then applied to selected rose plants using a hand mist sprayer (450 mL). The foliage of each plant received 100 mL of solution to near run-off, while the controls were sprayed with double distilled water. The containers were arranged in a

114 factorial based completely randomized design with two rose varieties, two hormones, three concentrations of each hormone and three replicate plants for each treatment.

6.2.4. Determination the Quantity of BA Sprayed on the Plants by Filter Paper

To determine the actual dosage of BA applied to the plants, filter paper (MACHEREY- NAGEL, Duren, Germany) was used. The filter paper was cut to different shapes and sizes according to leaves sourced from the top, middle and bottom of plants from both rose varieties. The filter papers were weighed before use and then placed at the top, middle and bottom of the rose plants. After application of BA, as described above, the filter papers were removed and weighed again. This method was repeated twice and the average was taken (Table 6.1). Methods to quantify spray retention by leaves and the actual dosage, following the application of PGRs is lacking, despite growing interest by researchers.

Table 6. 1. The actual dosage (mg/cm2) of two benzyladenine (BA) formulations (100 and 200 mg/L) after application to two rose varieties as determined by using filter paper

Hybrid Tea (mg/cm2) Floribunda (mg/cm2)

Canopy Leaves 100 mg/L 200 mg/L 100 mg/L 200 mg/L

Top 8.82b 14.53b 7.70a 13.38a

Middle 12.43a 18.11a 5.76a 8.99b

Bottom 12.25a 20.98a 8.06a 14.42a

Whole plant 11.16b 17.87a 7.17b 12.26a

LSD 3.73 3.25 4.58 4.11

Similar letters mean there are no significant differences between different leaf levels with least significant difference (LSD) p ≤ 0.05. Numbers were calculated according to the equation: mg/cm2 = 푾풂−푾풃 (풎품) . (Wa) means weight after application, (Wb) means weight before application, while LA means 푳푨 (풄풎ퟐ) leaf area

6.2.5. Sample Preparation and Extraction Using HS-SPME

At sampling, the rose plants were transferred to the laboratory. The samples were analysed in biological triplicates. Three each of intact fully expanded and undamaged leaves, cut stems (13 cm long), rhizosphere soils and whole plants were sampled in different chambers for the

115 extraction of VOCs as previously described (Chapter 4). Briefly, for leaf and stem samples, a 500 mL glass jar with septum, covered with aluminium foil was used. For rhizosphere sampling, a stainless-steel probe 20 cm long with 1-millimeter diameter holes throughout and a septum installed in the top of the tube was inserted into the soil. The SPME fibre was inserted through the septum. For whole plants, a glass chamber (30 × 35 × 60 cm) with a 5 mm port for the septum was used.

6.2.6. Optimization of Sample Collection Times after BA Application

A preliminary study was conducted to optimize the most efficient time for VOC sample collection after the application of BA to the rose plants, and three time periods (2, 4 and 8 weeks) were tested for each of the rose tissues. The samples from different rose tissues were prepared as described above. 6.2.7. Identification of Peaks and Compounds

Peaks were identified by using quantitative analysis software for GC identification. Total peak area for each treatment was compared with peak areas of the other treatments. Identified compounds from samples were also compared with the compounds identified from the internal standards. Identification of compounds was determined by comparing mass spectra and Kovatas Indices (KI) with authentic standards and data from published papers. GC-MS compounds, were identified using AMDIS version 2.72 (working under the NIST mass spectral search program for the NIST/EPA/NIH Mass Spectral Library) and by searching the NIST 2014 MS database with retention index confirmation. Relative percentage amounts of the separated compounds were calculated from total ion chromatograms by the computerized integrator.

6.2.8. Statistical Analyses

The results were analysed statistically by SAS® software, University edition, and the results were presented by analysis of variance (ANOVA). Means of 10 main peak areas were used to analyze and detect at different sampling times. Least significant difference (LSD) and the MetaboAnalyst 4.0 online program (http://www.metaboanalyst.ca/faces/home.xhtml) were used to compare means and significant differences were reported at the 0.05 significance level.

116 6.3. Results and Discussion 6.3.1. Comparison of Optimal Sampling Times after BA Application

There were significant differences between the three sample times post BA application. Four weeks was selected as the optimal time for the absorption of volatile compounds from the leaves, rhizospheres and whole plants for both rose varieties (Figure 6. 1A C and D). In contrast, eight weeks was chosen as the optimum collection time of VOCs from stems (Figure 6. 1B). These time periods were chosen as they gave superior total peak areas and numbers of compounds compared to the other sampling times. These findings for all tissues except stems agree with Baghele et al. (2016) on Rosa hybrida cv Poison after application with BA. The collection time for stems corresponds with that found for Aloe barbadensis by Salehi et al. (2013), who found an optimum sampling time to be eight weeks after application with BA.

Hybrid

Floribunda A B

Hybrid

Floribunda C D

0 1 2 3 4 5 6 0 5 10 15

5 GC readings (Area) *10

Figure 6. 1. Comparison of main peak area of volatile compounds collected in different rose tissues; (A) leaves, (B) stems, (C) rhizosphere, (D) whole plant using different sampling times. 8 weeks, 4 weeks and 2 weeks, bars represent LSD at (p ≤ 0.05) (n=3).

6.3.2. The Quantity of BA Applied to Rose Plants as Determined using Filter Paper

The quantity of sprayed BA that lands on leaves can be affected by many factors. These include treatment efficiency, differences arising from retention variability depending on plant species,

117 wettability, stage of growth, and architecture. Therefore, further investigation is needed on the effect of coverage and dosage on foliage as well as the methods to determine the actual dosage applied to the plant (Søgaard and Lund 2007). A few studies have used filter paper to detect and determine actual dosages of herbicides or insecticides applied to plants (Massinon et al. 2014). A study by Zabkiewicz et al. (2007) reported that using filter paper showed that between 5% and 92% of an applied product can be off-target and not reach the leaves. There have been many field studies aiming at determining the best methods of herbicide and pesticide delivery based on volumes applied, actual doses, crop growth stage and species (Butler et al. 2004). To our knowledge, there have been no studies on the detection and quantification of actual dosage of PGRs after application to plant foliage. In the present study, the number of droplets landing on the rose leaves was variable for practical reasons. When comparing the results for the whole plant, Hybrid Tea received a higher (11.16 mg/cm2) dose compared with Floribunda (7.17 mg/cm2) when treated with 200 mg/L BA (Table 6.1). The different locations of filter paper indicated the probability for different amounts of BA being retained. Leaves obtained from different parts of the rose canopy had different leaf areas, with the top, middle and lower having the small, medium and large leaves, respectively. Thus, retention and adhesion of BA on the bottom leaves will be the greatest (Table 6.1). In addition, it might be because the distribution of the filter paper, the area, spray volume and total retention of BA by the plant was different between the two roses. A study by Ramwell et al. (2002) reported the use of filter paper for quantifying herbicides deposited or sprayed on plants. In another study, the actual dosage and retention of hormones depended on the amount of sprayed product, number of droplets landing on the leaf surface and leaf area (Massinon et al. 2014). It could be useful to relate how leaf angle might influence the overall retention in future studies.

6.3.3. Influence of BA on Rose Tissues VOCs

The 200 mg/L BA application gave the highest peaks compared to 0 or 100 mg/L for the different rose tissues (Figure 6. 2A, C and D). Similarly, a study by Abad Farooqi et al. (1993) demonstrated that applying kinetin to Rosa damascena increased the amount of essential oils compared to control plants. Moreover, another study by Passinho-Soares et al. (2017) indicated that BA applied to Plectranthus ornatus in plant tissue culture significantly increased VOCs and these helped callus induction. Prior to the current study, no work had been done on how BA affects VOCs production in vivo.

118 2.5 A 2 B

1.5

5

10 1 ×

0.5

1.50 C D

1 GC readingsGC (Area) 0.5

0 Hybrid Floribunda Hybrid Floribunda

Figure 6. 2. Effects of different applied concentrations of BA on the extraction efficacy of the total compounds identified from different rose tissues; (A) leaves, (B) stems, (C) whole plant, (D) rhizosphere. Control, 100 mg/L and 200 mg/L of BA; bars represent LSD at (p ≤ 0.05) (n=3).

6.3.4. Identification of VOCs Emitted from Different Rose Tissues

All plants release VOCs from leaves, stems, flowers and rhizophores. The rose plants produce a diverse range of secondary metabolites from all tissues, as determined by the sparse partial least squares discriminant analysis (sPLS-DA) of the data, as shown by the distinct clustering of samples according to BA treatments (Figure 6. 3A and B). This indicated that different VOCs were produced in response to the different BA concentrations. There was no overlap in the VOCs produced between the three treatments of BA from the different rose tissues based on sPLS-DA (Figures 6. 3-6), respectively. There was a good separation between the data used for analysis of different BA treatments and these gave variances in the score plots for all rose tissues. The major differences that were found between Floribunda and Hybris Tea were hexanal, 3-hexen-1-ol, acetate, (E)- and cedrol; these components were found in Hybrid Tea and were not found in Floribunda. In contrast, α-pinene, (D)- and caryophyllene were found in Floribunda, but not found in Hybrid Tea. Identification of the compounds produced was based on a spectrometric data comparison. A total of 43, 65, 40 and 68 VOCs were detected from leaves, stem, rhizospheres and whole plants, respectively. Only those VOCs shown to differ significantly between the three BA concentrations tested according to ANOVA with a p value

119 of 0.05 were identified to a specific chemical compound. For Hybrid Tea there were 4, 6, 1 and 5 compounds from leaves, stems, rhizospheres and whole plants, respectively; from Floribunda, there were 2, 6, 2 and 6 compounds, respectively (Table 6. 2).

Some VOCs increased as the BA levels applied increased, for example, 4-heptyn-2-ol, one of the precursors of the fragrance compound US 7,842,660 B2 was found to increase from 20.26 to 54.43 in total peak area as the BA levels increased from 0 to 200 mg/L in whole plants for both rose varieties (Table 6. 2). In the case of Floribunda root tissue, phenyl methyl ether, an aromatic chemical used to produce essential oils, increased as BA increased. For leaves, the amount of 3-methyl-apopinene increased as BA was increased in Hybrid Tea; this compound is an important essential oil and corresponds to the findings by Mahdavi et al. (2017) on the extraction of essential oil from Etlingera brevilabrum leaves. In a study by Sun et al. (2015), different chrysanthemum varieties were shown to produce different types and numbers of volatile compounds when treated with increasing concentrations of BA. A similar observation was made by Sparinska et al. (2015) where different rose varieties gave different types and numbers of VOCs. Furthermore, a study by Ahmad et al. (2014) demonstrated that the SPME technique obtained more VOCs from Polygonum minus compare with the hydrodistillation technique. This difference may be due to the heat applied during hydro- distillation, which could degrade some heat unstable compounds; consequently, the difference could be attributed to the high sensitivity of the SPME technique. For eucalyptus leaves, SPME was successfully used to collect and identify VOCs (Zini et al. 2002). Based on the results of the present study and those of Chapter 4, HS-SPME can be used to analyse volatile compounds emitted from fresh tissues.

120

Figure 6. 3a and b. Sparse partial least squares discriminant analysis (sPLS-DA) model obtained from the classification of volatile organic compounds emitted from the leaves of Floribunda and Hybrid Tea rose samples based on VOCs according to BA treatments. (A) Score plot of Floribunda and (B) score plot of Hybrid Tea leaves. L represents Leaves, F represents Floribunda, H represents Hybrid Tea. While C (red), T1 (green) and T2 (blue) represent 0, 100 and 200 mg/L BA, respectively. Three dots in each group represent n = 3 biological replicates.

121

Figure 6. 4a and b. Sparse partial least squares discriminant analysis (sPLS-DA) model obtained from the classification volatile organic compounds emitted from the stems of Floribunda and Hybrid Tea roses samples based on VOCs according to BA treatments. (A) Score plot of Floribunda and (B) for the Hybrid Tea stems. S represents Stems, F represents Floribunda, H represents Hybrid Tea. While C (red), T1 (green) and T2 (blue) represent 0, 100 and 200 mg/L BA, respectively. Three dots in each group represent n = 3 biological replicates.

Figure 6. 5a and b. Sparse partial least squares discriminant analysis (sPLS-DA) model obtained from the classification of volatile organic compounds emitted from the rhizosphere of Floribunda and Hybrid Tea rose samples according to BA treatments. (A) Score plot of Floribunda and (B) for the Hybrid Tea rhizosphere. R represents rhizosphere, F represents Floribunda, H represents Hybrid Tea. While C (red), T1 (green) and T2 (blue) represent 0, 100 and 200 mg/L BA, respectively. Three dots in each group represent n = 3 biological replicates.

122

Figure 6. 6a and b. Sparse partial least squares discriminant analysis (sPLS-DA) model obtained from the classification volatile organic compounds emitted from whole plants of Floribunda and Hybrid Tea rose samples according to BA treatments. (A) Score plot of Floribunda and (B) for the Hybrid Tea whole plants. W represents whole plants, F represents Floribunda, H represents Hybrid Tea. While C (red), T1 (green) and T2 (blue) represent 0, 100 and 200 mg/L BA, respectively. Three dots in each group represent n = 3 biological replicates.

123

Table 6. 2. Identification of volatile organic compounds (VOCs) that differed significantly (P ≤ 0.05) between different rose tissues after being treated with different concentrations of BA.

Leaves Stems Rhizosphere Whole plants Mean of Peaks Mean of Peaks Mean of Peaks Mean of Peaks RT Compounds RI C 0 100** 200** 0 100** 200** 0 100** 200** 0 100** 200** 6.56 Hexanal 769 H 1.27 2.57 5.62 23.02 32.89 44.74 * 26.53 28.79 28.46 2.51 7.07 6.63 8.15 cis-β-Hexenyl formate 802 H 1.35 4.73 2.10 0.94 5.70 * 2.32 ND ND ND 3.66 3.57 3.98 30.61 9.42 Phenyl methyl ether † 878 F 3.88 4.95 14.20 5.55 5.86 8.24 15.46 19.46 14.75 16.35 43.55 * H 5.73 7.63 13.22 ND ND ND 27.68 43.50 27.28 20.26 16.23 54.43 * 9.56 4-Heptyn-2-ol † 897 F ND ND ND 3.74 4.71 4.83 29.98 40.95 74.59 5.74 7.77 37.03 * 10.36 3-Methyl-apopinene † 922 H 6.09 3.05 12.72 * 4.37 14.39 13.57 ND ND ND 2.28 2.85 4.56 6.43 10.44 α-Pinene, (D)- 931 F 4.25 12.64 30.66 4.73 4.53 5.51 0.17 0.69 0.25 1.95 2.01 * 12.72 3-Hexen-1-ol, acetate, (E)- 983 H 2.50 3.54 5.96 2.58 3.66 2.59 5.91 16.78 6.53 15.89 17.12 24.02 * 14.05 β-cis-ocimene 1005 F 2.31 2.92 2.70 1.42 2.09 5.97 * ND ND ND 0.45 0.76 0.74 14.15 3-Carene 1024 F ND ND ND 11.40 14.69 19.94 * ND ND ND 0.34 0.43 0.56 15.96 Nonanal 1081 H 0.12 0.26 0.43 2.20 44.23 * 5.02 ND ND ND 0.57 0.56 0.59 17.02 α-Pyronene 1115 F ND ND ND 4.81 5.14 6.75 ND ND ND 0.51 2.85 0.59 18.82 2,2-Dimethylpentane 1221 H 0.46 1.19 0.51 0.37 1.60 0.20 ND ND ND 0.23 1.24 1.66 20.27 Toluene, 3,5-dimethoxy 1245 H 1.72 2.02 1.83 ND ND ND 2.55 3.60 2.53 0.36 2.30 0.67 22.66 2,3-Dimethylundecane 1284 F ND ND ND 2.33 3.77 3.18 ND ND ND 1.83 2.40 11.47 * 23.89 Copaene 1392 F 0.17 0.43 3.13 * ND ND ND 1.32 5.00 1.43 0.18 0.18 2.73 24.40 β-Bourbonene 1408 H 0.60 7.02 * 0.34 ND ND ND ND ND ND 0.33 0.65 1.31 2,4-Diisopropenyl-1-methyl-1- H 0.33 0.38 0.46 0.20 0.40 0.56 0.72 1.37 0.86 4.31 9.11 13.15 24.76 1416 vinylcyclohexane F 0.21 0.36 0.55 0.13 0.44 3.85 0.62 1.46 4.71 14.82 1.47 0.74 H 0.20 0.20 1.05 2.86 3.34 2.89 0.32 0.41 0.31 0.17 4.11 0.71 24.92 α-Cedrene 1423 F 0.10 0.37 0.50 2.33 3.77 3.14 0.35 0.36 0.42 0.40 1.60 1.54 25.07 Caryophyllene 1431 F 0.21 0.36 0.55 0.29 0.38 0.47 0.52 0.56 2.64 * 0.35 0.59 0.36 26.10 beta-Guaiene 1469 F 0.27 0.47 2.67 0.38 2.53 5.07 * ND ND ND 0.28 2.37 1.16 H 0.55 0.32 0.56 ND ND ND ND ND ND 1.37 1.54 4.25 * 26.46 γ-Muurolene 1494 F ND ND ND 0.39 0.48 0.77 ND ND ND 2.36 3.16 6.71 * H 2.39 3.08 3.63 1.99 1.58 2.14 2.05 2.27 1.94 0.69 2.72 1.63 27.14 Froggatt ether 1509 F 1.82 3.38 1.23 2.14 2.25 3.76 ND ND ND 0.51 1.91 6.84 28.29 β-Calacorene 1548 F ND ND ND 5.14 9.80 9.34 0.50 0.57 0.54 1.04 1.13 1.27 29.66 Cedrol 1595 H 1.58 3.24 1.53 0.26 0.60 0.49 0.53 1.38 4.62 0.46 0.60 0.72 H 1.15 1.29 1.39 0.45 0.48 0.49 ND ND ND ND ND ND 34.48 N-Acetylhystrine 1935 F 0.61 0.89 0.75 0.35 0.49 0.48 1.06 2.01 1.52 0.29 1.35 3.05 (*) Means: there are significant differences between different concentrations of BA (0, 100 and 200 mg/L) with LSD P ≤ 0.05; (RT) retention time, (RI) retention index based on alkane series, (C) means varieties of Roses (F) Floribunda and (H) Hybrid Tea, (ND) not detected. (†) Means: components that increased significantly in leaves, stems, rhizosphere and whole plants with increasing level of BA as compared to the control. (**) Means mg/L.

124 6.4. Conclusions

This study demonstrated that the collection of VOCs from fresh leaves, stems, rhizospheres and whole plants by HS-SPME coupled with GC-FID/MS represented a sensitive, rapid and effective method for analysing and detecting VOCs. In total, 32 compounds were identified and the concentrations of these differed significantly depending on the concentrations of BA were applied to the roses. The highest concentration of VOCs for both rose varieties occurred four weeks after BA application, with significantly lower concentrations at two and eight weeks. Increased leaf area led to increased retention of BA, and consequently, an increase in the actual dosage of BA applied to the rose plants as determined by the filter paper method. In addition, different responses were observed for the four tissues of both rose varieties, as shown by the different VOCs detected after BA application. However, there were differences in the amounts of different volatiles between the rose tissues. Of interest to the industry, BA was shown to have a significant effect on rose metabolites, and some aromatically important compounds increased following the application of 100 mg/L or 200 mg/L BA. In addition, the compounds, 4-heptyn-2-ol, phenyl methyl ether and 3-methyl-apopinene, are precursors for making fragrant compounds. Consequently, BA can be used to increase and improve the quantity and quality of VOCs produced from roses and other floricultural plants. Thus, in the future, BA can be used to increase the fragrance of the flowers.

125 Chapter 7 ______

General Discussion, Future Research and Conclusions

126 7.1. General Discussion

This study has done significant research in two specific areas: a) understood improvement of the quality and enhancement of the production of aromatic volatiles from rose plants after applications of BA and NAA and b) examined responses of different morph-physiological characteristics following the application of BA and NAA.

Specifically, the major findings were:

 More volatiles were emitted from Hybrid Tea compared with the Floribunda rose.  Application of BA and NAA at 200 mg/L significantly increased the production and quality of the volatiles produced, and some compounds increased with increasing concentrations of BA and NAA and these were cis-muurola-4(14)5-diene, γ -candinene, y-muurolene and prenyl acetate, 4-heptyn-2-ol, phenyl methyl ether and 3-methyl- apopinene in the two rose varieties.  200 mg/L BA and NAA increased all morph-physiological characteristics compared with the control, except for the respiration rate, which decreased in both rose varieties.  Applications of 200 mg/L BA and NAA significantly increased vegetative growth and improved flower quality and longevity in both rose varieties. Although, flower longevity was superior in Hybrid Tea than in Floribunda

There they are discussed in more detail below:

Plant growth regulators play vital roles in the coordination of various growth or behavioural processes in rose, and they regulate the amount, type and direction of plant growth. The application of PGRs improve the growth, yield and quality of flowering plants (Hedayat 2001). In addition, increases in flower production and improvement of flower quality in floriculture plants can be achieved by the application of PGRs (Bari and Jones 2009). Many PGRs, such as auxin (NAA), and benzyladenine (BA) are increasingly used commercially to ensure efficient production. Foliar application of PGRs (BA and NAA) improve the quality of growth parameters and production of cut flowers (Sajid et al. 2009). BA can reduce ethylene sensitivity of cut flowers (Yuan et al. 2012), increase photosynthetic efficiency (Oosterhuis and Zhao1998), and influence cell division (Francis and Sorrel 2001), biosynthetic processes of chloroplast pigments (Bondok et al. 1995), and nutrient uptake especially potassium (Guo et al. 1994). Many studies have shown that NAA has positive effects on the growth and development of the ornamental plants (Umrao et al. 2007, Rana et al. 2005). Also, NAA plays

127 a main role in plant life cycles, such as cell division and elongation, and increases flower production and shoot development (Davies 2013). PGRs have an important role for changing the production of metabolites and VOCs that are emitted from different rose tissues. For example, the application of BA to rose plants increased the production of volatile compounds as detected by solid phase microextraction (SPME) (Affonso et al. 2007). In Chapter 6 the application of BA to rose plants increased the production of metabolites form different rose tissues. Whilst in Chapter 5 it was shown that the production of volatile compounds (fragrances) increased from rose flowers of plants treated with BA and NAA than from the control plants

A number of different physiological parameters were optimized on two modern rose varieties, Floribunda and Hybrid Tea. The results demonstrated that measurements at 12 pm on sunny days was the optimum for measuring photosynthesis rate for both rose varieties. A light intensity of 1200 μmol/m2/s gave the highest photosynthesis rate compared with other light intensities. In contrast, a study by Kim and Lieth (2003) Rosa hybrida roses showed a maximum photosynthesis rate at 1500 μmol/m2/s and led to an increase in plant growth and production. The differences between the two studies using different light intensity is likely because of different environments conditions, different seasons and different rose varieties. Therefore, these findings highlight the importance of specific light intensities and large specific leaf areas for increasing photosynthesis and chlorophyll contents, and in turn increasing production of rose flowers.

After optimization of the various physiological methods had been completed, different concentrations (0, 100 and 200 mg/L) of BA and NAA were applied to both rose varieties to understand the response of morphological and physiological parameters. All the morphological characteristics tested, including plant height, number of branches, stem diameter, leaf area and length of stem, and physiological characteristics, such as photosynthesis rate, chlorophyll content and respiration rate were significantly improved following the foliar application of 200 mg/L BA and NAA. These findings are most likely due to BA and NAA promoting vegetative growth by inducing active cell division and cell elongation in the apical meristem. Our findings are consistent with the reports of Sharma et al. (2004) and Awasthi et al. (2012) on gladiolus that BA and NAA promote vegetative growth and flower production. In the present study, the two rose varieties treated with 200 mg/L BA and NAA significantly increased vegetative growth and improved flower quality and longevity. For physiological characteristics, the

128 photosynthesis rate, chlorophyll content also increased significantly with the application of 200 mg/L BA and NAA for both rose varieties. This increase in photosynthesis and chlorophyll content may have a positive impact on the growth and development of rose plants. This is because both BA and NAA play a key role in shoot development, increased flower production, cell division and elongation, as well as enhancing the production of photosynthetic pigments. These results agree with those of Mondal and Sarkar (2017) who used BA to increase the chlorophyll content in the leaves of the Hybrid Tea rose plants compared with control plants. A study on marigold plants demonstrated that the application of growth regulators increased chlorophyll content as well as photosynthetic pigments (Sardoo 2016). BA and NAA play important roles in effecting plant growth and development and are applied for specific purposes according to plant response (Latimer and Whipker 2013).

To analyse quality and production VOCs, the SPME technique using the SPME fibre coupled to the GC FID/MS was found to be a robust, rapid and reliable. These findings agree with Pragadheesh et al. (2011) who used an SPME fibre to extract a number of high-quality VOCs from Jasminum sambac. It is important to optimize the desorption time along with temperature as high temperatures used for emission of VOCs from the fibres in the injection port can reduce GC sensitivity, possibly due to denaturation, destruction, or decomposition of the chemicals at higher temperatures (Magan and Evans 2000). Therefore, different parameters were optimized for the analysis of emitted VOCs to ensure maximum release of VOCs from the fibre without compromising the composition of VOCs released. To my knowledge, this is the first study on the use of VOCs from different rose plant tissues, and it is the first time the HP-SPME technique has been used to identify VOCs from attached and excised flowers, leaves, stems, roots and soil (rhizosphere), as well as whole rose plants.

The optimized methods were successfully used to analyse the production of VOCs emitted from different rose tissues. In total, 81 and 76 compounds from the two rose varieties were detected after application of BA and NAA, respectively. The collection of VOCs is very important for flower volatile analysis. Many methods have been used to analyse VOCs, including HS-SPME (Zhu and Chai 2005). Based on the results from the present study HS- SPME can be used to analyse volatile compounds emitted from different fresh rose tissues of two rose varieties. Consequently, BA and NAA can be used to increase and improve the quantity and quality of VOCs produced from roses and other floricultural plants. Applications of 200 mg/L of both BA and NAA gave the highest peaks compared to 0 or 100 mg/L for both

129 rose varieties. Similarly, Abad Farooqi et al. (1993) indicated that the application of kinetin to Rosa damascena increased the amount of oil compared to control plants.

Twenty volatile compounds were identified which had significant differences compared with the control. Some volatile compounds increased with 200 mg/L of BA and NAA application such as cis-muurola-4(14),5-diene, γ-cadinene, γ-muurolene and myrcene. These compounds are important aroma compounds, and have commercial applications for both rose varieties. A study by Ma et al. (2015) showed that cis-muurola-4(14),5-diene was found in different tissues of Artemisia annua plants. Moreover, an in vitro study using tissue cultures of Plectranthus ornatus treated with BA and NAA showed that many compounds such as cis-muurola-4(14),5- diene, γ-cadinene and γ-muurolene significantly increased and helped plant growth and callus induction (Passinho-Soares et al. 2017). Myrcene is an important in essential oils for flavour and fragrance, and for health and medical purposes corresponding to the findings by Hartsel et al. (2016) from Cannabis sativa plants.

Prior to the current study, no work had been done on how BA effects VOC production in vivo. A total of 43, 65, 40 and 68 VOCs were detected from leaves, stem, rhizosphere and whole plants, respectively and only those that differed significantly between the three BA concentrations tested were identified. Some VOCs increased as BA levels increased, for example 4-heptyn-2-ol, phenyl methyl ether, and 3-methyl-apopinene, which are the precursors to the production of fragrant compounds (US 7,842,660 B2). Phenyl methyl ether is an aroma chemical used in essential oil industries, whilst 3-methyl-apopinene is an important essential oil, both of which increased with increasing levels of BA. These findings correspond to a study by Mahdavi (2017) on extraction of essential oil from Etlingera brevilabrum leaves. A study by Sun et al. (2015) showed that different Chrysanthemum cultivars produced different types and numbers of VOCs, as did Sparinska and Rostoks (2015) where different rose cultivars gave different types and numbers of VOCs.

7.2. Future Research

This study has found potential avenues for future research, these include: 1- Different types of formulations of BA and NAA may be developed so that these compounds can be applied through irrigation, and dipping for a range of horticultural and floricultural produce.

130 2- It will be of value to understand the biochemical pathways by which the major VOCs of interest are produced. By understanding the pathways involved it may be possible to develop new PGRs or synthetic compounds. 3- The physiological and biochemical mechanisms of how BA and NAA modulate abscission and senescence of different rose genotypes warrant detailed study. 4- It is yet to be investigated if these two growth regulators are safe to use by the industry. Their impacts on the environment and human health when applied to different horticultural and floricultural commodities warrants investigation. 5- Further work should examine applications of BA and NAA higher than 200 mg/L as these might result in higher concentrations of VOCs of interest being produced than observed in the current study.

7.3. Conclusions

This thesis was primarily undertaken to test, detect and identify the influence of BA and NAA on morph-physiological characteristics and metabolic changes that occur after application of these two hormones to two rose varieties. During this study, all methods were optimised for physiological characteristics and the emission and absorption of volatile organic compounds. These optimised parameters provide a stable basis for future research activities relating to the production and analysis of VOCs from horticultural and floricultural crops. As well as benefiting research, these optimised parameters could be used by industry. The research also showed that the SPME method coupled to GC-FID/MS would have huge benefits to industries such as those interested in aromatic (perfumes, medicine, cosmetics, culinary and decoration) products. In particular, the SPME method is superior to distillation methods where heat can interfere with the output of VOCs and their characterization. A number of valuable VOCs were shown to be produced by the two rose varieties. These specific volatile compounds are important because they are very powerful aromatic compounds, and of significant value to (cis- muurola-4(14),5-diene, γ-cadinene, γ-muurolene and myrcene) industries. In future, these compounds could potentially be made synthetically and then applied to the plants to improve the quality and production of flowers as well as higher concentrations of VOCs. The VOC’s identified in this study could be explored as future PGRs to enhance the production and quality of flower fragrances. Moreover, these PGRs may lead to increased flower production and longevity, and consequently will have commercial value for cut flower products in local and export markets. The approaches taken to optimize rose production in the current study are

131 applicable to other horticultural or floricultural plants where there is a need to increase the quality and production.

132 References

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