Strategies for Enhanced Production of Commercially Important Secondary Metabolites from in Vitro Established Cultures of Linum Usitatissimum L

Strategies for Enhanced Production of Commercially Important Secondary Metabolites from In Vitro Established Cultures of Linum usitatissimum L.

ADNAN ZAHIR

Department of Biotechnology

Faculty of Biological Sciences

Quaid-I-Azam University

Islamabad Pakistan

2019 Strategies for Enhanced Production of Commercially Important Secondary Metabolites from In Vitro Established Cultures of Linum usitatissimum L. Thesis submitted to The Department of Biotechnology Quaid-i-Azam University, Islamabad In the partial fulfillment of the requirements for the degree of Doctor of Philosophy In BIOTECHNOLOGY

BY ADNAN ZAHIR

Supervised by

Dr. BILAL HAIDER ABBASI Department of Biotechnology Faculty of Biological Sciences Quaid-i-Azam University, Islamabad, Pakistan 2019

DECLARATION

The whole of the experimental work included in this thesis was carried out by me in the Plant cell culture Laboratory, Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan. The findings and conclusions are of my own investigation with discussion of my supervisor Assoc. Prof. Dr. Bilal Haider Abbasi. No part of this work has been presented for any other degree.

ADNAN ZAHIR

God does not charge a soul with more than it can bear It shall be required for whatever good and whatever evil it has done. Our Lord! Take us not to task if we forget, or Lapse into error our Lord! Charge us not with the burden You laid upon those before us. Our Lord! do not burden us beyond what we have the strength to bear and pardon us and forgive our Sins, and have mercy on us You alone are our protector and help us against people who deny the Truth. (Ameen)

Dedicated

to My Parents

At this day, this moment, What I am is because of my Parents. If I want to thank them, I could not, as they deserve to be thanked, But my whole life is at their service.

Contents LIST OF FIGURES ...... iv LIST OF TABLES ...... vii ACKNOWLEDGEMENTS ...... viii LIST OF ABBREVIATIONS ...... x SUMMARY ...... xii CHAPTER 1 ...... 1 1. GENERAL INTRODUCTION ...... 2 1.1. Medicinal uses ...... 2 1.2. Phytochemistry ...... 4 1.3. In vitro Propagation of Linum usitatissimum ...... 10 1.3.1. Regeneration ...... 10 1.3.2. Anther Cultures ...... 18 1.4. Bioactive Metabolites Production Using in vitro Established Cultures of Linum usitatissimum ...... 33 1.5. Aim and Objectives ...... 38 CHAPTER 2 ...... 39 2. IN VITRO CULTURES OF LINUM USITATISSIMUM L.: SYNERGISTIC EFFECTS OF MINERAL NUTRIENTS AND PHOTOPERIOD REGIMES ON GROWTH AND BIOSYNTHESIS OF LIGNANS AND NEOLIGNANS ...... 40 2.1. ABSTRACT ...... 40 2.2. INTRODUCTION...... 41 2. 3. MATERIALS AND METHODS ...... 43 2.3.1. Inoculum Preparation ...... 43 2.3.2. Determination of Fresh and Dry Weight ...... 44 2.3.3. Determination of Callus Growth Rate ...... 44 2.3.4. Phytochemical Analysis ...... 45 2.3.5. Free Radical Scavenging Assay ...... 45 2.3.6. HPLC Analysis ...... 45 2.3.7. Statistical Analysis ...... 46 2.4. RESULTS AND DISCUSSION ...... 46 2.4.1. Trends in Growth Kinetics and Morphological Changes ...... 46 2.4.2. Trends in Biomass, Phenolics and Flavonoids Accumulation ...... 49 2.4.3. Trends in Free Radical Scavenging Assay ...... 53 2.4.4. Trends in Lignans and Neolignans Accumulation ...... 55

2.5. Conclusions ...... 56 CHAPTER 3 ...... 59 3. CHEMOGENIC SILVER NANOPARTICLES ENHANCE LIGNANS AND NEOLIGNANS IN CELL SUSPENSION CULTURES OF LINUM USITATISSIMUM L...... 60 3.1. ABSTRACT ...... 60 3.2. INTRODUCTION...... 61 3.3. MATERIALS AND METHODS ...... 62 3.3.1. Inoculum Preparation ...... 62 3.3.2. Characteristics of Ag-NPs ...... 62 3.3.3. Optimization of Ag-NPS Concentration ...... 62 3.3.4. Ag-NPs Treatments ...... 63 3.3.5. Biomass Determination ...... 63 3.3.6. Phytochemical Analysis ...... 64 3.3.7. Free Radical Scavenging Assay ...... 64 3.3.8. HPLC Analysis ...... 64 3.3.9. Statistical Analysis ...... 65 3.4. RESULTS AND DISCUSSION ...... 65 3.4.1. Effects of Repeated Elicitation with Ag-NPs on Biomass Accumulation ...... 65 3.4.2. Effects of Repeated Elicitation with Ag-NPs on Phenolics and Flavonoids Content ...... 67 3.4.3. Effects of Repeated Elicitation with Ag-NPs on Production of Lignans and Neolignans ...... 73 3.5. Conclusions ...... 73 CHAPTER 4 ...... 77 4. BIOGENIC ZINC OXIDE NANOPARTICLES ENHANCE PRODUCTION OF LIGNANS AND NEOLIGNANS IN CELL SUSPENSION CULTURES OF LINUM USITATISSIMUM L...... 78 4.1. ABSTRACT ...... 78 4.2. INTRODUCTION...... 79 4.3.1. Inoculum preparation ...... 80 4.3.2 Characteristics of ZnO-NPs ...... 80 4.3.3. Optimization of ZnO-NPs Concentration ...... 80 4.3.4. ZnO-NPs Treatments ...... 80 4.3.5. Biomass Determination ...... 81 4.3.6. Phytochemical Analysis ...... 81 4.3.7. Free Radical Scavenging Assay ...... 82

4.3.8. HPLC Analysis ...... 82 4.3.9. Statistical Analysis ...... 83 4.4. Results and Discussion ...... 83 4.4.1. Effects of Elicitation with ZnO-NPs on Biomass Accumulation ...... 83 4.4.3. Effects of Repeated Elicitation with ZnO-NPs on Free Radical Scavenging Capacity ...... 90 4.4.4. Effects of Repeated Elicitation with Ag-NPs on Production of Lignans and Neolignans ...... 91 4.5. Conclusions ...... 91 CHAPTER 5 ...... 94 5. CONCLUSIONS AND FUTURE PROSPECTS ...... 95 5.1. CONCLUSIONS ...... 95 5.2. FUTURE PROSPECTS ...... 97 PUBLICATIONS FROM THESIS ...... 98 CHAPTER 6 ...... 102 6. REFERNCES ...... 103

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LIST OF FIGURES

Figure 1.1. Morphological features of Linum usitatissimum. (retrieved from google images) ...... 3 Figure 1.2. Scientific classification of Linum usitatissimum...... 3 Figure 2.1. Chemical structures of lignans and neolignans produced by callus cultures of Linum usitatissimum. a. Secoisolariciresinol diglucoside (SDG), b. lariciresinol diglucoside (LDG), c. dehydrodiconiferyl alcohol glucoside (DCG), d. guaiacylglycerol-b-coniferyl alcohol ether glucoside (GGCG) ...... 41 Figure 2.2. A; Calli cultured on Murashige and Skoog, Gamborg B5 and Schenk and Hildebrandt media, respectively, in 16/8 h light and dark, B; Calli cultured on Murashige and Skoog, Gamborg B5, and Schenk and Hildebrandt media, respectively, in continuous dark, C; Calli cultured on Murashige and Skoog, Gamborg B5 and Schenk and Hildebrandt media, respectively, in continuous light...... 47 Figure 2.3. Fresh and dry weight of of Linum usitatissimum calli cultured on Murashige and Skoog, Gamborg B5, and Schenk and Hildebrandt media, respectively, in 16/8 h light and dark, continuous dark, and continuous light photoperiod regimes. Values are mean of triplicates ±SD...... 51 Figure 2.4. Total phenolic content of Linum usitatissimum calli cultured on Murashige and Skoog, Gamborg B5, and Schenk and Hildebrandt media, respectively, in 16/8 h light and dark, continuous dark, and continuous light photoperiod regimes. Values are mean of triplicates ±SD...... 52 Figure 2.5. Total phenolic production of Linum usitatissimum calli cultured on Murashige and Skoog, Gamborg B5, and Schenk and Hildebrandt media, respectively, in 16/8 h light and dark, continuous dark, and continuous light photoperiod regimes. Values are mean of triplicates ±SD...... 52 Figure 2.6. Total flavonoid content of Linum usitatissimum calli cultured on Murashige and Skoog, Gamborg B5, and Schenk and Hildebrandt media, respectively, in 16/8 h light and dark, continuous dark, and continuous light photoperiod regimes. Values are mean of triplicates ±SD...... 54 Figure 2.7. Total flavonoid production of Linum usitatissimum calli cultured on Murashige and Skoog, Gamborg B5, and Schenk and Hildebrandt media, respectively, in 16/8 h light and dark, continuous dark, and continuous light photoperiod regimes. Values are mean of triplicates ±SD...... 54

Figure 2.8. Free radical scavenging assay of Linum usitatissimum calli cultured on Murashige and Skoog, Gamborg B5, and Schenk and Hildebrandt media, respectively, in 16/8 h light and dark, continuous dark, and continuous light photoperiod regimes. Values are mean of triplicates ±SD...... 55 Figure 3.1. A; in vitro established callus culture of Linum usitatissimum. B; Inoculum for cell suspension cultures of Linum usitatissimum, derived from in vitro established callus culture. C; Cell suspension cultures of Linum usitatissimum fed on day 10, day 10 and 15, and day 10 and 20, respectively...... 63 Figure 3.2. Temporal effects of repeated elicitation with chemogenic Ag-NPs on fresh biomass of cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD...... 66 Figure 3.3. Temporal effects of repeated elicitation with chemogenic Ag-NPs on dry biomass of cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD...... 67 Figure 3.5. Temporal effects of repeated elicitation with chemogenic Ag-NPs on total phenolic production in cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD...... 69 Figure 3.6. Temporal effects of repeated elicitation with chemogenic Ag-NPs on total flavonoid content in cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD...... 70 Figure 3.7. Temporal effects of repeated elicitation with chemogenic Ag-NPs on total flavonoid production in cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD...... 71 Figure 3.8. Temporal effects of repeated elicitation with chemogenic Ag-NPs on free radical scavenging assay of cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD...... 72 Figure 3.9. Temporal effects of repeated elicitation with chemogenic Ag-NPs on A; secoisolaricerisinol diglucoside content (SDG), B; SDG production, C; laricerisinol diglucoside (LDG) content and D; LDG production in cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD...... 75 Figure 3.10. Temporal effects of repeated elicitation with chemogenic Ag-NPs on A; dehydrodiconiferyl alcohol glucoside (DCG) accumulation, B; DCG production, C; guaiacylglycerol-b-coniferyl alcohol ether glucoside (GGCG) accumulation, and D;

GGCG production in cell suspension cultures of Linum usitatissimum. values are mean of triplicates ± SD...... 76 Figure 4.1. A; in vitro established callus culture of Linum usitatissimum. B; Inoculum for cell suspension cultures of Linum usitatissimum, derived from in vitro established callus culture. C; Cell suspension cultures of Linum usitatissimum fed at day 0, day 0 and 15, and day 0 and 25, respectively...... 81 Figure 4.2. Temporal effects of repeated elicitation with biogenic ZnO-NPs on fresh biomass of cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD...... 84 Figure 4.3. Temporal effects of repeated elicitation with biogenic ZnO-NPs dry biomass of cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD...... 85 Figure 4.4. Temporal effects of repeated elicitation with biogenic ZnO-NPs on total phenolic content in cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD...... 86 Figure 4.5. Temporal effects of repeated elicitation with biogenic ZnO-NPs on total phenolic production in cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD...... 87 Figure 4.6. Temporal effects of repeated elicitation with biogenic ZnO-NPs on total flavonoid content in cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD...... 88 Figure 4.7. Temporal effects of repeated elicitation with biogenic ZnO-NPs on total flavonoid production in cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD...... 89 Figure 4.8. Temporal effects of repeated elicitation with biogenic ZnO-NPs on free radical scavenging assay of cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD...... 90

LIST OF TABLES

Table 1.1. Pharmacological activities of Linum usitatissimum...... 6 Table 1.2. Biochemical profile of Linum usitatissimum...... 7 Table 1.3. Classification of lignans and neolignans into major sub-groups with examples...... 8 Table 1.4. Summary of some published reports on organogenesis of Linum usitatissimum...... 13 Table 1.5. Summary of some published reports om anthers derived cultures of Linum usitatissimum ...... 20 Table 1.6. Summary of some published reports on somatic embryogenesis of Linum usitatissimum...... 28 Table 1.7. Strategies applied for secondary metabolites production in in vitro established cultures of Linum usitatissimum...... 35 Table 2.1. Major differences among Murashige and Skoog, Gamborg B5, and Schenk and Hildebrandt media...... 44 Table 2.2. Growth index of in vitro established cultures of Linum usitatissimum on Murashige and Skoog, Gamborg B5, and Schenk and Hildebrandt media, respectively, in 16/8 h light and dark, continuous dark, and continuous light photoperiod regimes...... 48 Table 2.3. HPLC quantified data of lignans and neolignans. SDG; secoisolariciresinol diglucoside, LDG; lariciresinol diglucoside, DCG; dehydrodiconiferyl alcohol glucoside, GGCG; guaiacylglycerol-b-coniferyl alcohol ether glucoside...... 58 Table 4.1. Temporal effects of repeated elicitation with biogenic ZnO-NPs on content of DCG; dehydrodiconiferyl alcohol glucoside, GGCG; guaiacylglycerol-b-coniferyl alcohol ether glucoside, SDG; secoisolariciresinol diglucoside and LDG; lariciresinol diglucoside...... 92 Table 4.2. Temporal effects of repeated elicitation with biogenic ZnO-NPs on production of DCG; dehydrodiconiferyl alcohol glucoside, GGCG; guaiacylglycerol- b-coniferyl alcohol ether glucoside, SDG; secoisolariciresinol diglucoside and LDG; lariciresinol diglucoside...... 93

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ACKNOWLEDGEMENTS All the praises for the almighty ALLAH the most omnipotent, the most merciful, who bestowed us with the ability and potential to seek knowledge of his creatures, and his Prophet Muhammad (S.A.W.W), who is forever a source of guidance and knowledge for humanity as a whole. I also pay my gratitude to the Almighty for enabling me to complete this research work within due course of time.

The successful completion of this journey is due to the combined efforts of many people. Ph.D. is a long and complicated journey. During this journey, I was helped by many and I would like to show my gratitude to all of them. This work would not have been finished without their help. Primarily, I would greatly appreciate my supervisor Dr. Bilal Haider Abbasi, Associate Professor, Department of Biotechnology, Quaid- i-Azam University, for his dynamic supervision. It is his confidence, imbibing attitude, splendid discussions and endless endeavors through which I have gained significant experience. My special thanks are due to Assoc. Prof. Dr. Muhammad Naeem, Chairman Department of Biotechnology, Quaid-i-Azam University Islamabad, for his immense concern throughout my Ph.D. period. I feel pleasure in acknowledging the nice company of my lab fellows Tariq Khan, Munazza Nazir, Mehreen Zaka, Asad Ullah, Ayesha Siddiqa, Muhammad Younas, Wali Muhammad. I will always remember the nice company of my friends Waqar Ahmad, Jalal Shah, Aman Ullah, Muzammil Shah, Muhammad Nadeem, Muhammad Azhar and Syed Salman Hashmi.

I wish to express my deep gratitude and respect to all my teachers especially to Dr. Muhammad Zia whose knowledge and guidance enabled me to attain this target.

I am also thankful to Professor Dr. Muhammad Shahab, Dean of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan for their support and help in submission of my work. Furthermore, I am indebted to Dr. Christophe Hano, Lecturer, Université d’Orléans, Chartres, France for HPLC analyses of Linum cell culture samples. Data extracted from these analyses enhances the quality of research work presented in this thesis.

I must acknowledge the role of Higher Education Commission of Pakistan which provided financial and administrative support through indigenous Ph.D. fellowship program.

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I feel pleasure to extend my deepest regards and gratitude to my family for their consistent support. My sisters, brothers, wife always prayed for my success. I must say that all the happiness in my life is because of my beloved father Zahir Rehman and my ever caring and sweetest mother. Their constant prayers and appreciations always kept me satisfied.

ADNAN ZAHIR

LIST OF ABBREVIATIONS

µg Micro-gram

Ag-NPs Silver nanoparticles

ANOVA Analysis of variance

B5 Gamborg B5 cm Centimetre

DPPH 1, 1-diphenyl-2-picrylhydrazyl

DW Dry weight

FRSA Free Radical scavenging activity

FTIR Fourier transform infrared

FW Fresh weight

HPLC High performance liquid chromatography mg/g DW milligram/gram-Dry Weight mg/L milligram/liter ml Milliliter

MS Murashige and Skoog

NAA α-Naphthalene acetic acid

PAL Phenylalanine ammonia lyase

POD Peroxidase

PPO Polyphenol oxidase

SEM Scanning electron microscope

SH Shenck and Hildebrandt

TEM Transmission electron microscope

TFC Total flavonoid content

TFP Total falvonoid production

TPC Total phenolic content

TPP Total phenolic production

XRD X-ray diffraction

ZnO-NPs Zinc oxide nanoparticles

SUMMARY Linum usitatissimum is an important cultivated, commercial and medicinal plant in the family Linacaea. This plant is also known as flax and their seeds are famous for their nutritional, medicinal and industrial purposes. Exploitation of Linum usitatissimum is context dependent. Commercially, it is used for manufacturing of linen fiber hence the name flaxseed is given to such a variety of Linum usitatissimum, on the other hand seeds of a variety used for many nutritional and industrial applications are called linseed. Linum usitatissimum is generally cultivated in areas having moderate to cold climate. The plant has a slender stem, lanceolate leaves and pale blue flowers with five petals. Canada, Russia, United States, China and India are among the top growers of Linum usitatissimum. Worldwide production of flax is 2.5-3 million tonnes per year. This plant is rich in pharmaceutical compounds along with some important nutraceuticals. Novel compounds found in flax are polyphenols, comprising of two major classes—lignans and neolignans. Lignans like secoisolariciresinol diglucoside (SDG) and lariciresinol diglucoside (LDG) are potential anticancer compounds whereas, neolignans such as dehydrodiconiferyl alcohol glucoside (DCG) and guaiacylglycerol-β-coniferyl alcohol ether glucoside (GGCG) are used in antimicrobial and anti-inflammatory drugs. However, the yield of these medicinal compounds in natural habitat is low and their extraction from wild is an expensive process. For this reason, researchers are busy devising cost effective, reproducible and practical strategies to enhance producton of lignans and neolignans in in vitro established infrastructure.

In the first experiment, we established callus cultures of Linum usitatissimum following protocol of Anjum et al. (2016). These cultures were derived from stem explants obtained from in vitro seed derived aseptic plantlets. The objective of this study was to evaluate the effects of qualitative and quantitative differences in culture medium along with varying photoperiod treatments, on growth kinetics and secondary metabolites production in callus cultures of Linum usitatissimum. To this end, ~1 g callus was inoculated on three different culture media, namely Murashige and Skoog, Gamborg B5 and Schenk and Hildebrandt, and each culture on specific growth medium was kept under the influence of different photoperiod treatments viz. 16/8 h light and dark, continuous light and continuous dark, respectively. We observed differential effects of nutrient and photoperiods variation on growth kinetics and secondary metabolites

xii accumulation in callus cultures of Linum usitatissimum. Significant growth rate was observed on Gamborg B5 medium as compared to Murashige and Skoog medium, while Schenk and Hildebrandt medium showed a slow but steady growth response. Similarly, we observed that Gamborg B5 medium enhanced callus biomass (fresh weight 413 g/l and dry weight 20.7 g/l), phenolics production (667.60 mg/l), and lignan content (secoisolariciresinol diglucoside 6.33 and lariciresinol diglucoside 5.22 mg/g dry weight respectively) at 16/8 h light and dark-week 4, while that of neolignans (dehydrodiconiferyl alcohol glucoside 44.42 and guaiacylglycerol-β-coniferyl alcohol ether glucoside 9.26 mg/g dry weight, respectively) at continuous dark-week 4. Conversely, maximum flavonoid production occurred at both Murashige and Skoog, Schenk and Hildebrandt media in the presence of continuous light. Generally, continuous dark had no significant role in any growth associated parameter.

In the second experiment, we established cell suspension cultures of Linum usitatissimum by modifying method of Anjum et al. Simply, ~3g callus was inoculated into 100 ml liquid Murashige and Skoog medium and kept on a shaking incubator for 15 days in controlled conditions. Ten milliliters media containing viable cells was used as inoculum for establishment of cell suspension cultures. This experiment was aimed to enhance growth associated parameters and secondary metabolites accumulation with the help of repeated introduction of chemogenic silver nanoparticles at different stages, into cell suspension cultures of Linum usitatissimum. Repeated addition of silver nanoparticles enhanced biomass and polyphenols accumulation in cell suspension cultures. Adding silver nanoparticles on day 10 resulted in comparatively, highest production of lignans (secoisolariciresinol diglucoside, 252.75 mg/l; lariciresinol diglucoside, 70.70 mg/l), neolignans (dehydrodiconiferyl alcohol glucoside, 248.20 mg/l; guaiacylglycerol-β-coniferyl alcohol ether glucoside, 34.76 mg/l), total phenolic content (23.45 mg GAE/g DW), total flavonoid content (11.85 mg QUE/g DW) and biomass (dry weight: 14.5 g/l), respectively. Optimum production of both lignans and neolignans occurred on day 20 of culture; a 10-fold increase in secoisolariciresinol diglucoside, 2.8 fold increase in lariciresinol diglucoside, 5 fold increase in dehydrodiconiferyl alcohol glucoside and 1.75-fold increase in guaiacylglycerol-β-coniferyl alcohol ether glucoside was observed in production levels compared to control treatments, respectively.

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In the third experiment, cell suspension cultures of Linum usitatissimum were exposed to repeated addition of biogenic zinc oxide nanoparticles in order to evaluate its effect on biomass accumulation and secondary metabolites production. Zinc oxide nanoparticles were added at three different stages and their results were compared to control treatments. Repeated elicitation of cell suspension cultures on day 0 and 15 resulted in highest fresh weight (412.16 g/l) and lignans production (secoisolariciresinol diglucoside 284.12 mg/l: lariciresinol diglucoside 86.97 mg/l). Contrarily, repeated elicitation on day 0 and 25 resulted in highest dry biomass (13.53 g/l), total phenolic production (537.44 mg/l), total flavonoid production (123.83 mg/l) and neolignans production (dehydrodiconiferyl alcohol glucoside 493.28 mg/l: guaiacylglycerol-β-coniferyl alcohol ether glucoside 307.69 mg/l). Enhancement of plant growth, free radical scavenging capacity and secondary metabolites accumulation was several fold greater than control treatments.

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CHAPTER 1

1. GENERAL INTRODUCTION Linum usitatissimum commonly known as flax is a valuable medicinal and commercial plant in the family Linaceae. The plant derives its name from two different languages, Linum for Celtic word “lin” meaning thread and usitatissimum is the Latin word for “most useful” (Kolodziejczyk and Fedec, 1995). It is called the most useful thread because the commercial fiber “linen” is produced from this plant. In the agricultural and industrial sectors Linum usitatissimum is also known as flaxseed or linseed. The two terms have their particular meanings. Europeans use flax for the seeds grown for fiber (linen) production and linseed is referred to seeds grown for other industrial and nutritional purposes (Muir and Westcott, 2003). Both the types of flax belong to the same species but are different varieties and the same crop usually do not produce the same products (BeMiller, 1973). Prominent morphological features of Linum usitatissimum are a slender stem, lanceolate leaves and pale blue flowers with five petals. The seeds enclosed in a dry capsule are similar to apple pip in shape (Fig. 1.1). Flax is being cultivated worldwide but bulk production is limited to few countries. Data compiled in 2014 revealed Canada to be the leading producer and exporter of flax. Of the 2.65 million tonnes flax produced worldwide in that particular year, Canada produced almost 9 lacks tonnes which was 33 % of the total production. Other major producers include Kazakhstan, China and Russia, each country produces nearly half of the total production of Canada. India and United States also have some contribution towards worldwide production (Flax “linseed” production, 2014).

1.1. Medicinal uses Historically there have been two major uses of flax, human consumption and fabric manufacturing. Humans utilized flax as cereal and source for oil extraction used for frying food, as lamp oil and an important constituent of paints. However, the best documented earliest use is the fabric made from the fibers of flax stalks (Muir and Westcott, 2003). Alongside its nutritional and industrial uses the plant is also famous for carrying some novel medicinal potentials. Traditional medicinal uses include treatment of cough, cold, constipation and urinary tract infections. The bioactive molecules which contribute to the medicinal aspects of Linum usitatissimum will be comprehensively discussed in a separate section but here we shall mention some of the notable present-day medicinal uses of this plant species. The most notable biological

2 General Introduction CHAPTER 1 activity of Linum usitatissimum is the anticancer effects in breast, prostate and colon cancers owing to the presence of lignans.

Figure 1.1. Morphological features of Linum usitatissimum. (retrieved from google images)

Figure 1.2. Scientific classification of Linum usitatissimum.

3 General Introduction CHAPTER 1

Though seeds of Linum usitatissimum are considered as a rich source of phytochemicals, literature review reveals roots to be biologically more effective. Abarzua et al. (2007) studied the effects of various explants derived phytoestrogen extracts from Linum usitatissimum on human trophoblast cell line (jeg3) proliferation. They found root extract to be significantly inhibiting cell proliferation as compared to leaf and stem. In a similar study conducted by Szewczyk et al. (2014) on MCF-7 and BT-20 cancer cell lines, root extract proved highly effective. Root extract significantly inhibited cell proliferation of MCF-7 cells without showing any sign of cytotoxicity but was highly toxic (60-100 % in a dose dependent manner) to BT-20 cells. In another study performed by the same author, root extract obtained at various ages of maturity (3, 6 and 9 weeks) of Linum usitatissimum also inhibited MCF-7 cell proliferation and showed cytotoxicity in an age and dose dependent manner respectively (Szewczyk et al., 2014). The vitality and proliferation of the above mentioned cancer cell lines were strongly suppressed by higher concentrations of ethanolic flax root extracts (Theil et al., 2013). Cardio protective action is another important medicinal feature exhibited by Linum usitatissimum. Zanwar et al. (2011) subjected male Wistar rats to isoprenalin induced cardiotoxicity. Results showed that rats pre-inoculated with flax lignin concentrate extracted from flax seeds reversed the toxic marker-biochemical changes caused by isoprenalin as compared to rats injected only with isoprenalin. Angiotensin is a peptide hormone that acts to increase blood pressure thus is a major drug target. Marambe et al. (2011) successfully demonstrated in a simulated gastrointestinal digestion system that flaxseed produced inhibitory peptides against enzyme responsible for conversion of angiotensin I. Derbali et al. (2015) also showed that pre-co treatment of linum oil with isoproterenol prevented all the parameters associated with myocardial infarction by inhibiting angiotensin converting enzyme. Many among other reported prominent health-enhancing effects (Table 1.1) include anti-inflammatory, analgesic (Kaithwas et al., 2011), Reno protective (Ghule et al. 2011) and antidiabetic activities (Ghule et al. 2012). 1.2. Phytochemistry A plant’s novelty is always attributed to its phytochemical profile. Being a medicinal plant, Linum usitatissimum is blessed with a tremendous range of bioactive compounds. The plant is enriched with several classes of bioactive metabolites including proteins/amino acids, phenolics, flavonoids, lignans and neolignans each having distinct functional roles. Several authors (Wang et al. 2017; Zuk et al. 2015; Shim et al.

4 General Introduction CHAPTER 1

2014) have extensively reviewed and analytically studied biochemical composition of Linum usitatissimum, but here we concisely discuss the major bioactive compounds occurring in this species. The chemical compounds which impart both nutritional and medicinal values to Linum usitatissimum are the polyunsaturated fatty acids alpha- linolenic acid (ALA) which is omega (ω)-3 fatty acid and linolenic acid (LA) which is omega (ω)-6 fatty acid, both compounds cannot be synthesized de novo by mammals (Cunnane et al. 1995). Traditional sources of ω-3 fatty acids are marine foods particularly fishes, which are rather expensive. Flaxseed is being considered an alternative to marine foods as it is a rich source of ALA. ω-3 fatty acids not only serve as a nutritional agent but it has multiple health effects too. Commonly known health benefits of ω-3 fatty acids if consumed routinely are protection against cancer (Heinze and Actis, 2012), cardiovascular (Miller et al. 2014) and inflammatory diseases (Li et al. 2014). Similarly, LA intake also depends on various food sources and linoleate (the salt form of LA) deficient diet leads to mild skin scaling, hair loss and poor wound healing in experimental rat models (Ruthing and Meckling-Gill, 1999). Furthermore, flaxseed oil contains special proteins known as cyclolinopeptides that possess strong immunosuppressive and antimalarial activities and also have the ability to protect liver from cholate (bile salt) (Gui et al. 2012).

The key metabolites that make Linum usitatissimum distinctive are lignans. Lignans are chemically a large group of polyphenols and functionally phytoestrogenic. They are classified into eight subgroups based upon the incorporation of oxygen into the carbon skeleton and cyclization pattern (Table 1.3) (Suzuki and Umezawa, 2007). A number of lignans naturally occur in flax such as secoisolariciresinol, matairesinol, lariciresinol, pinoresinol. However, the principal lignin precursor is secoisolariciresinol diglucoside (SDG) based on its presence in comparatively higher quantities. These dietary lignans when consumed are converted into mammalian lignans “enterolactones” and “enterdiols” by the action of gut microbes that exhibit the same biological effects (Heinonen et al. 2001). Biosynthesis of lignans involves the participation of coniferyl alcohol as a precursor produced through the cinnamte/monolignol pathway. A reaction sequence in the following order has been proposed; Coniferyl alcohol (+)-Pinoresinol (+)-Laricerisinol (-)- secoisolaricerisinol (-) -Matairesinol (Xia et al., 2000)

5 General Introduction CHAPTER 1

All the lignans exist as enantiomers (mirror images/optical isomers) and both enantiomers are present in unequal amounts that is why lignans are biologically active, therefore for the metabolism of a specific enantiomer special class of proteins known as dirigent proteins catalyze biosynthesis of lignans (Davin and Lewis, 2000).

Table 1.1. Pharmacological activities of Linum usitatissimum.

Pharmacological Tissue used Mode of application Reference activities Thrombosis Flax oil In vivo (human) Allman-Farinelli et al. 1999 anti-inflammatory Flax oil In vivo (rat) Bhathena et al. 2002 Lowering of blood Milled flax In vivo (human) Jenkins et al. 1999 cholesterol Flax seeds In vivo (human) Chan et al. 1991 Hyperglycemia Flax oil/ In vivo (human) Basch et al. 2007 fiber Indigestion and Flax fibre In vivo (human) Blumenthal 2000 constipation Defatted In vivo (mice) Xu et al. 2012 flax Hypertension Flax seeds In vivo (human) Billinsky et al. 2013

Type-2 diabetes mellitus Flax seeds In vivo (rat) Hano et al. 2013

Diarrhea Flax seeds In vivo (mice) Palla et al. 2015

Root extract in vitro (BT20, MCF7) Szewczyk et al. 2014 Breast cancer Flax seeds In vivo (human) Thompson et al. 2005 Flax seeds In vivo (mice) Flax seeds In vivo (human) Demark-Wahnefried et Flax seeds In vivo (rat) al. 2004 Prostate cancer Flax seeds in vitro (LNCaP, PC-3 and Lin et al. 2002 DU-145) Colon cancer Flax seeds In vivo (rat) Serraino and Thompson, 1992 Atherosclerosis Flax lignan In vivo (rabbit) Prasad 2008 Cardiovascular diseases Flax seeds In vivo (human) Bassett et al. 2009 Antiapoptosis Flax seeds In vivo (mice) Razi et al. 2011 Neuroprotective Flax oil In vivo (rat) Moneim, 2012

6 General Introduction CHAPTER 1

Table 1.2. Biochemical profile of Linum usitatissimum.

Phenolics Amino acids Peptides Miscellaneous Caffeic acid Alanine CLA Hydrolysable tannins

Caffeic acid glucoside Arginine CL B β-Carotene

Coumaric acid Aspartic acid CL ℽ-Tocopherol

Coniferyl aldehyde Cystine CLD, CLK, CLO Vitexin

Coumaric acid glucoside Glutamic acid CLE Plastochromanol-8

Ferulic acid Glycine CLF, CLL Lutein

Ferulic acid glucoside Hisitidne CLG, CLM Proanthocyanidin

3,4-dihydroxybenzoic acid Isoleucine CLH Saturated fatty acids

SDG Leucine CLI Polyunsaturated fatty acids

Vanillin Lysine CLJ, CLP

Syringic aldehyde Methionine CLQ

Phenylalanine CLS

Proline

Serine

Threonine

Tryptophan

Tyrosine

Valine

7 General Introduction CHAPTER 1

Table 1.3. Classification of lignans and neolignans into major sub-groups with examples.

Lignan sub-groups Examples Neolignan sub-groups Examples

Justicidin B Miliumollinone Taiwanin Linderanoside Arylnaphthalene Chinensis Benzofurans Meliasendanins Dehydrodeoxy-phodophyllotoxin Radulignan Retrojusticidin B Chushizisins Podophyllotoxin Caffeicinic acid ovafolinin 3-o-dimethyleusiderin Aryltetralin Cleistantoxin Benzodioxanes Cusiderin A Sinolignan 5-o-dimethylbilagrewin Arisantetralone D Streblusol A Biphenyl Neglignan D Secoisolariciresinol Coccilignan dehydrodiconiferyl alcohol Dibenzylbutane Saururin B guaiacylglycerol-β Kadsurindutin E -coniferyl alcohol ether Ananonins Alkyl aryl ethers Hispidacine Schisanwilsonins Myrifralignan Dibenzocyclooctadiene Steganacin Callislignan B Schinlignan Miliusfragranol B Neglschisandrins Ocophyllolos

8 General Introduction CHAPTER 1

Lariciresinol Nectamazins Ribesins Cymosalignan Furan Saurufurins Didymochlaenon Olivil Biseugenol Pinoresinol Prebalanophonin Epipinoresinol Saccharnans Furofuran Lactuberins A Others neolignans Conchigeranal A Schamin Acortatarinwin E Matairesinol Penthorins Yatein Neotaiwanensol A Dibenzylbutyrolactone Hinokinin Taiwandimerol Cellobioside Rufesenolide Cubebin Truxinate Dibenzylbutyrolactol Liriodenol Myrisfrageals A

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1.3. In vitro Propagation of Linum usitatissimum Various established modern plant tissue culture methods have been successfully adopted for the in vitro multiplication and production of quality controlled cultivars of Linum usitatissimum. Being a commercial plant, the primary focus of the in vitro tissue culture studies has been the production of cultivars with desired morphological and genotypic traits. The in vitro methods utilized for this purpose include organogenesis, anther cultures, somatic embryogenesis and adventitious root culture, among others. Initially in vitro cultures were established to meet the agronomical demands of Linum usitatissimum, however, recent trends lay stress on the production of biologically active metabolites through callus and cell suspension culture technologies. 1.3.1. Regeneration in vitro culturing of Linum usitatissimum has been carried out for more than 40 years. A number of reports are available for whole plant regeneration of flax using various explants. The first successful attempt to regenerate shoots from stem explants was made by Murray et al. (1977). The authors optimized conditions for optimum shoot regeneration from haploid and diploid flax. It was observed that high number of shoots was obtained from stem explants having length of 15 mm at 30 °C. It was interesting to know that shoots regenerated directly retained the same chromosome number as the donor haploid (n=15) and diploid (2n=30) plants but the shoots regenerated via callus were diploid (2n=30) and tetraploid (4n=60) respectively. Generally, the change in ploidy level is linked to culture incubation period. Subculturing results in somaclonal variation as an adaptive mechanism to avoid culture stress which is considered a positive aspect in some breeding programs as cultivars with novel features are produced. The authors extrapolated multiplication frequency of 3000 to 4000 plantlets from the donor plant. Subsequent years also saw an influx of research articles with similar scope and results, details of which are summarized in Table 1.4. Many authors have proposed several biochemical changes during in vitro morphogenesis of Linum usitatissimum including enzymatic, proteomic and stress related phenomenon. Auxins are capable to initiate roots formation but for this to happen their catabolism by several chemical entities is a prerequisite. Tissue peroxidases influence rooting by altering auxins catabolism. It was demonstrated that secreted peroxidases had strong affinity for oxidation of IAA during root formation from hypocotyl explants (MeDougall et al. 1993). Alteration in peroxidase levels is

10 General Introduction CHAPTER 1 thought to be a “front line” response from explant to address the presence of auxins in the medium. Nutrient choice and energy sources are central to a successful in vitro regeneration scheme, therefore carbohydrates are the most exploited culture medium constituent. Different carbohydrate sources including monosaccharides and polysaccharides were tested to affect shoot and root regeneration potential of Linum usitatissimum. Monosaccharides had least affect while the disaccharides maltose and sucrose were found to promote shooting and rooting respectively (Millam et al. 1992). Similarly, David et al. (1994) identified various polysaccharide levels, maximum for glucose and a constant for galacturonic acid as limiting factors for rhizogenesis from protoplast- derived calli over the course of morphogenesis. In addition, environmental conditions also affect organogenesis of Linum usitatissimum. Light exposure positively affects both shooting and rooting as compared to dark treatment (Siegien et al. 2013). The presence of heavy metals in soil regulates various biological and physiological mechanisms in vivo, some researchers tried to mimic this situation in vitro by applying cadmium (Cd) stress as selection pressure for morphogenic response. They were able to achieve complete regeneration of plantlets but based on the results and observations they concluded that sensitivity to Cd was not affected by the presence or absence of Cd ion and the ability to tolerate Cd stress might be predetermined by genotypes (Chakravarty and Sarivastava, 1997). The articles published so far suggests Linum usitatissimum as a totipotent species for carrying out in vitro propagation studies. The optimum culture conditions include in vitro germinated seeds as explant source, use of MS medium as nutrient source, pH range 5.6-5.8, PGRs generally auxins, 16h light and 8h dark photoperiod treatment, temperature 25 ±2C and 20-30 days incubation period. For obtaining hypocotyl explants seeds are generally kept in dark on MS medium supplemented with 10 g/l sucrose at 22±2C for maximum 7-10 days. Mostly all the types of Linum usitatissimum explants tested for regeneration until now are responsive, however root segments and cotyledonal explants have been described as non-morphogenic. The lack of regeneration capacity of these two tissues has been associated with blocking of morphogenic response which in turn has been ascribed to a) genetic/absence of totipotency, b) epigenetic/cell’s inability to respond to hormonal or other signaling system driving them towards morphogenesis, c) cells are totipotent but lack presence of required elements in optimum concentrations and conditions or

11 General Introduction CHAPTER 1 presence of inhibitors. Explants of somatic origin not only give high frequency of callus induction but shoot regeneration incidence too. In contrast, explants of reproductive origin such as anthers have low callus induction frequency and shoot regeneration efficiency.

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Table 1.4. Summary of some published reports on organogenesis of Linum usitatissimum.

Explant Explant Explant Genotype PGRs Aim Remarks Reference source age combination (days) (mg/l) Cotyledonary In vitro 4 Ariane 2,4 D Morphogenic Glucose and David et al. protoplasts germinated Viking response and cell galacturonic 1994 seeds wall’s sugar acid are central composition to morphogenesis which is strongly dependent on genotype Hypocotyl In vitro 10 Garima 2 IBA, 0.5 Kin for Proline and total Kiran is best Chakravarty germinated Gaurav callusing protein content for producing and seeds Kiran 0.1 BA for shooting estimation in in vitro Cd tolerant Srivastava, cultures under Cd plants 1997 stress Garima is best for general purpose regeneration

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Hypocotyl In vitro 7 Szafir 0.005 2,4 D, 1 BA Cyanogenic potential Light Siegien et germinated for shooting of in vitro cultures positively al. 2013 seeds 0.005 BA, 1 NAA kept in light and dark affects for rooting treatments organogenesis and their cyanogenic potential Stem Potted ------Atalante 0.1 BAP Genetic analysis of Best period Bonell and plants Tape callogenesis and after culture Lassaga, Parana regeneration for 2002 INTA callogenesis Paisano evaluation is INTA 20 days, 10 Buenis days for early Aires 106 regeneration and 30 days for shoots production capacity

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Cotyledon Potted 10 ------1 NAA, 0.5 2,4 D, in vitro cultures Suitable Rosu et al. (fragments and plants 0.5 Kin for establishment protocol for 2010 nodes) callusing Isolation of serine reliable callus 1 NAA, 0.5 TDZ, protease inhibitors biomass 200 Caseine increase and Hydrosylate for higher serine callus biomass protease increase inhibitors accumulation Hypocotyl In vitro 6 Antares 2.7x105 NAA Enzymes involved Root MeDougall germinated during root formation is et al. 1993 seeds development governed by characteristic changes in peroxidase enzymes that alter catabolism of auxins

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Stem In vitro 7 Stormont 10 um Zeatin, I um Effect of various Disaccharides Millam et germinated Silver IAA polysaccharides on promote both al. 1992 seeds organogenesis shooting and rooting capacity Hypocotyl In vitro 6 Natsaja 5um 2,4 D Interactive effects of Use of sucrose Millam and germinated auxin and and 1 day Davidson, seeds carbohydrates exposure to 1992 2,4-D results in higher shoots number 3 days exposure to 2,4-D on maltose results in higher roots number Rapid callogenesis occur at low exposure times

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on sucrose treatment Hypocotyls In vitro 4-5 Belinka 0-0.1 NAA, 0-2 Genotypic and PGRs Adventitious Burbulis et germinated Dangiai BAP, 0-2 TDZ, 0-2 effect on buds al. 2012 seeds Sartai 2iP organogenesis regeneration Snaigiai incidence Vaizgantas varies with genotype and medium composition Vaizganta gives the best results Hypocotyls In vitro 4-5 Barbara 2 TDZ, 0.1 NAA Genotypic and PGRs Shoot Burbulis et germinated Szaphir for Barbara and effect on adventitious formation is al. 2009 seeds Mikael Mikael shoots formation dependent on 2 BAP, 0.1 NAA genotype for Szaphir Szaphir gives comparatively better results

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1.3.2. Anther Cultures Anther cultures are the most exploited in vitro propagation method for flax as they are capable of producing pure homozygous lines having genetic stability in a short period, normally in one generation compared to conventional inbreeding techniques that take six generations to achieve complete homozygosity. The success ratio of anther derived plant and ploidy level of anther derived callus cells vary significantly than for the somatically derived callus but the driving force to make anther cultures a superior and priority procedure is the exhibition of higher level of main agricultural traits in comparison to the available cultivars. For instance, Russian scientists while deploying anther cultures as a tool for production of new flax genotypes obtained regenerants that surpassed initial genotypes and the standard cultivar in the second to third generation. The main improved agronomical traits exhibited by the anther derived regenerants include high seed yield, dry mass and length of stems, fiber content, disease resistance, grain weight and some other aspects. Like somatic explant derived calli anther derived calli also exhibit variation in ploidy level however the regenerated plants are predominantly diploid and in rare cases haploidy has been observed. To deal with these problems researchers are trying to identify responsive genotypes by genotype screening such as a group of researchers has successfully used F1 hybrids as donor plants obtained from the cross between responsive and recalcitrant genotypes (Burbulis et al. 2006) or optimize physiological and environmental conditions for selected genotypes. As observed for somatic tissues derived cultures the importance of carbohydrates in organogenesis has also been evaluated for anther cultures. Pre-culture of anthers on high sucrose medium (15%) for 2-7 days before transfer to low sucrose medium (6%) increases morphogenic potential compared to culturing anthers directly on low sucrose medium (Chen et al. 2004). However, it has also been reported that high sucrose content is influential in initial anthers/microspores division and survival but inhibits embryo maturation and plant regeneration (Matsubayashi and Kuranuki 1975; Dunwell and Thurling, 1985). High sucrose content retards the growth of anther derived shoots and medium without sucrose completely inhibits the growth, but the most elongated shoots are obtained at low sucrose content (Chen et al. 2003). Sucrose not only serves as an energy source but also as an osmotic regulator in the medium (Chen and Dibnenki, 2004). Furthermore, sugars other than sucrose such as glucose, maltose and lactose also exhibited similar

18 General Introduction CHAPTER 1 pro-morphogenesis effects (Table 1.5). Likewise, pre-culture treatments with different photoperiod regimes, hot and cold conditions also improve anthers performance (Table 1.5). The regeneration capacity of anthers is more inclined towards direct route as the microspores present in the anthers are unable to switch the in vivo phenomenon “alternation of generation”, thus microspores behave similarly to zygote however most of the studies have described indirect plant regeneration in flax anther cultures. Another major issue presented by anther cultures is the regeneration of plantlets from the somatic cells of the anther wall which serves no value in breeding programs therefore, microspore cultures have been tried as an alternative to overcome this issue. A generalized scheme for establishment of flax anther cultures has not been yet devised but the unifying components include incubation of anthers at low temperature in dark and pre-culture treatment of anthers, mid to late stage uni-nucleate microspores as an explant, use of 0.4 to 0.6% agar as solidifying agent and higher sugar concentrations (3-12%) in contrast to 0.8% agar and 3% sugar respectively in callus cultures. The morphogenic response of Linum usitatissimum is highly affected by genotype, medium composition, culture incubation period and environmental conditions, therefore all these parameters have been extensively studied to develop optimized protocols for feasible in vitro propagation schemes, see description in Table 1.5.

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Table 1.5. Summary of some published reports om anthers derived cultures of Linum usitatissimum

Genotype Explant Medium Pre-culture/culture Conclusion Reference treatment Atalante Anthers MS media with varied 27/24 (day/night) Genotype and medium Burbulis et al. Barbara concentrations of under 16/8h composition dependent 2012 Lirina sucrose photoperiod regime response Mikael Norman Dnepr-2 Atalante Ovaries MS Dark for callogenesis 1. Highest adventitious Burbulis et al. Avangard and 27/24C day/night shoot frequency was 2011 Barbara under 16/8h observed for Mikael Linola photoperiod for callus 2. Highest number of shoots Lirina subculturing per explant was observed in Mikael second subculture Norman Symphonia Szaphir Norman Anthers MS media with varied Callus induction at Substitution of sucrose with Burbulis and Linola concentration of 25C in the dark and combination of sucrose and Blinstrubiene, Lirina sucrose and maltose maintenance at 2011

20 General Introduction CHAPTER 1

Zaltan-1 27/24C (day/night) maltose (in equal ratio) Barbara under 16/8h increases organogenesis Mikael photoperiod Szaphir Atalante Dnepr-2 Indus-2 Alba Anthers MS with different Anthers maintained at 1. Dark treatment promotes Rutkowska- Nike concentrations of high temperature callogenesis and plant Krause et al. Orshansky 2 sucrose and glucose (20C) or low regeneration 2003 temperature (4C) in 2. 2.5 % of both sucrose and dark for 14 days glucose supports callogenesis 2% sucrose is effective for shooting whereas 1% sucrose and reduced minerals enhances rooting efficiency Lirina Anthers MS media containing Donor plants grown at 1. Genotype dependent Burbulis et al. Barbara 6%, 9% and 12% two different sets of response 2005 Mikael sucrose temperature

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Szaphir ; lower 18/14C and 2. preconditioning of donor Atalante higher 22/18C plants results in higher Callus induction at callogenesis 25C in the dark and 3. higher level of sucrose in regeneration at induction medium is critical 27/24C (day/night) to some genotypes under 16/8h photoperiod regime 98–200 (AC Emerson Anthers Callus induction Inoculated explants 1. Culturing of anthers on Chen and x McGregor F1) medium: A22C were kept at 35C for 1 15% sucrose medium for 2- Dibnenki, A proprietary flax line (modified MS) with day and then at 25C 7 days before transfer to a 2004 96-3 varied concentrations for 27 more days in 6% sucrose medium

96-45 of sucrose and PEG dark increases callogenesis Regeneration medium: Genotype 98-200 calli percentage and regeneration modified N6 were pre-cultured at efficiency in contrast to high sucrose medium somatic cell-derived plants before transfer to low 2. Concentration sucrose medium combinations of sucrose and PEG that sums up to 15% results in similar

22 General Introduction CHAPTER 1

morpohogenic efficiency as 15% sucrose without PEG 3. No embryogenesis occurs at 15% PEG without sucrose, thus sucrose works both as energy source and as an osmotic regulator in the medium Szegedi 30 Anthers Callus induction Cold pretreatment Genotype and medium Obert et al. Flanders media; MS, Mo, N6, (8C for 7 days) composition dependent 2004 Carolin N&N with varied response with positive Viking concentrations of effects of cold pretreatment Super sucrose and maltose Belinka MS for shoot Red Wing elongation and rooting PR FGL 77 San Elias 192/22 F1 hybrids of AC Anthers MS media with various Anthers incubated at 1. flax microspores may Chen et al. McDuflf/AC Emerson concentrations of 35C for one day and synthesize thiamine 1998 and F1 hybrids of AC thiamine then cultured at 25C hydrochloride in vitro that’s Emerson/McGregor hydrochloride, sucrose for 27 more days why this vitamin presence in

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and maltose, the medium is not essential respectively for callus induction and shoot regeneration 2. higher concentration of sucrose and maltose (> 10%) results in smaller calli however moderate concentration (6-9% maltose and up to 6% sucrose promotes organogenesis F1 hybrids (AC Anthers Callus induction: A22 Maintenance of Anthers culture at 35C for Chen et al. McDuff/AC Emerson modified with 10 mg/l anthers at 35C for 1-4 one day increases the overall 1998 F1 hybrids thiamine days prior to culture at regeneration efficiency most (M3696/NorLin) hydrochloride 25C in darkness for probably by switching the in Shooting medium: total of 28 days vivo microspore’s modified N6 gametophytic pathway into Rooting medium: MS in vitro sporophytic pathway modified with 0.1% activated charcoal

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F1 hybrid of AC Anthers Same protocol as 27/24 (day/night) Moderate quantities of Chen et al. McDuff/96-150 mentioned above but under 16/8 h sucrose result in significant 2003 with addition of an photoperiod regime shoot growth whereas zero extra step of shoot or extremely high quantities elongation MS media result in no growth with 0, 5, 10 and 30 g/l levels of sucrose Three F1 hybrids: 99– Anthers A22C media Anthers incubated at Lactose only media Chen et al., 179, 99–181 and 99– containing 9% sucrose, 35C for one day and increased overall callus 2002 182 maltose and lactose then cultured at 25C induction however shoot respectively in the dark for 27 regeneration was noticed to more days be genotype dependent F1 plants of five Microspores NLN with added 24 h cold treatment at High seed yield and 4 days Steiss et al. different crosses glutamine and 4C of buds shorter vegetation period 1998

1. Linola 2 x DH57/1 NH4NO3 observed n some double 2. Linola 2 x haploid lines compared to Avantgard 3. Linola 1 conventional lines x CB47 4. Atlante x L.u. elegans

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5. McGregor x L.u. elegans Hella Anthers Callus induction Donor pants grown in 1. Genotype and medium Nichterlein et Atalante medium: A22 4 different sets of composition dependent al. 1991 Midas Shooting medium: temperature response AR2 modified N6 ; 18/16C, 12/10C, 2. Overall 14/8C (day/night) Rooting medium: MS 16/10C, 14/8C improved organogenesis (day/night respectively) F1 hybrid Microspores Callus induction Microspores 1. Incubation temperature of Nichterlein and (Atalante/Szegedi) medium: modified incubated at 30C and 30C showed better response Friedt, 1993 F2 (Pedigree NLN 82 35C in the dark comparatively for F1 2/Kiszombon 41) Shooting medium: Calli were maintained hybrids modified N6 at 27/24C (day/night) 2. Success ratio can be Rooting medium: MS under 16/8h enhanced by transfer of non- photoperiod rooted shoots into vermiculite hydroculture 3. Haploidy can be reduced to some extent by colchicine treatment of cuttings

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1.3.3. Somatic Embryogenesis Somatic embryogenesis from flax is a disputed topic, because some authors have claimed successful production of somatic embryos (Pretova and Williams, 1986; Tejavathi et al. 2000) but many others reported formation of embryos like structures (ELS) (Dedicova et al. 2000; Petrovska et al. 2010) and it was unconvincing to pronounce them true embryos solely based on histological observations. Salaj et al. (2005) critically assessed the findings of those authors who claimed formation of flax somatic embryos. Citing contemporary research work they ruled out the justification of the embryogenic character of the ELS and opinionated, although globular ELS are formed, it is not possible to confirm their embryogenic nature as further development into next stages is stopped. For a regenerating ELS to be called a true embryo it is necessary to develop via stages similar to those of a zygote with clear root and shoot polarity (bipolar embryos). However, the published reports lack convincing pictorial evidences or if available, is misinterpreted. They concluded that common morphological markers and histological tools such as SEM analysis and microscopy techniques were insufficient and unreliable therefore, focus must be directed to molecular markers and functional genomics.

Despite the fact that there are no complications involved in inducing somatic embryos/ELS from flax explants, the number of corresponding available research articles are limited. The first report came in 1986 and the last one in 2010 with few articles published during this time span. Both direct and indirect somatic embryogenesis have been carried out successfully mainly from hypocotyl segments (Cunha and Ferriera, 1999, 2001). The primary purpose of somatic embryogenesis is the clonal propagation of genetically uniform plant material and provision of source tissue for genetic studies (Park et al. 1998) and anther cultures have already been proven as a top- notch method to achieve these goals. This statement can be further supported by the fact that a couple of authors had tried to regenerate whole flax plants through somatic embryogenesis and they reported low shooting efficiency (Ling and Binding, 1992; Dedicova et al. 2000), these may be the reasons researchers are lacking interest to exploit somatic embryogenesis as a biotechnological tool for flax. Furthermore, the scope of these studies has been limited to optimization and histological studies of flax somatic embryos. The important features of flax somatic embryogenesis have been summarized in Table 1.6

27 General Introduction CHAPTER 1

Table 1.6. Summary of some published reports on somatic embryogenesis of Linum usitatissimum.

Explant Medium Histological Result Conclusion Reference Examination/analysis Immature MS supplemented with SEM BAP alone induced Glutamine, yeast extract and Pretova flax or without BAP, yeast callogenesis from BAP together stimulates and embryos extract and glutamine cotyledon and direct flax somatic Williams, hypocotyl tissues and embryogenesis 1986 also preserved green color of embryos Glutamine promotes culture growth SEM analysis revealed root pole formation Protoplasts MS Not mentioned Globular structures Callogenesis was induced but Ling and Induction phase;2,4-D were formed no embryogenic calli could Binding, Developmental phase; be determined 1992 NAA Germination phase; cytokinin

28 General Introduction CHAPTER 1

Hypocotyl MS SEM Embryonic structures Bipolar flax embryos can be Cunha and at the torpedo, heart induced at very low Ferriera, and true cotyledonary concentrations of 2,4-D (0.4 1996 stages were observed mg/l) and zeatin (1.6 mg/l) Hypocotyl MS media with varying Not mentioned Sucrose and maltose High sucrose concentration Cunha and concentration of induced (4%) inhibited induction and Ferriera, monosaccharides and embryogenesis at development of somatic 1999 disaccharides, total lower concentrations (1 embryos. nitrogen content (nitrate and 2%) compared to Nitrate was important to calli and ammonium glucose and fructose growth and development and formulations) and (4%). high ammonium content calcium and zeatin in Comparatively low enhanced somatic different proportions total nitrogen content embryogenesis. (30mM) with nitrate and ammonium ions in 1:1 gave better results. Calcium/zeatin interaction is inversely related.

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Hypocotyl MS GC Total lipids were Lipids and hydrocarbons are Cunha and GC-MS reduced in calli as central to growth of Ferriera, compared to primary embryogenic calli and their 2001 explant and somatic development into somatic embryos. embryos. In contrast, higher n- alkanes were found in hypocotyl segments and somatic embryos as compared to embryogenic calli. Hypocotyl MS Microscopically, Maximum number of 2,4-D Pre-treatment and Dedicova SEM bipolar globular ELS subsequent transfer to et al. 2000 were formed after pre- cytokinins containing media treatment of hypocotyl could result in ELS formation segments with 2,4-D (5 but with abnormal root and mg/l) for 24 h and shoot poles, had fused or subsequent transfer to poorly defined cotyledon and Mo medium containing normal leaf primordial failed 2 mg/l zeatin. to develop.

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Medium containing NAA (further exposure to auxin) did not induce embryogenesis. Hypocotyl MS media Microscopically Media supplemented Although SE were formed Tejavathi supplemented with with NAA induced successfully, reduced shoot et al. 2000 PGRs, ABA, glycine, somatic apex growth was observed lysine, glutamine, casein embryogenesis. their survival rate on transfer hydrolysate and high SE were characterized to soil was only 2-3% sucrose concentrations by the typical stages of furthermore, ABA (3-10%) embryo development. (considered to prevent Rapid rooting from the secondary embryogenesis) radical portion was and amino acids added to observed. medium had not any promoting effects, in addition higher sucrose concentrations (above 5%) induced necrosis Hypocotyl MS Microscopically Globular ELS ELS formed although had Salaj et al. originated from callus morphological resemblances 2005 failed to develop both to globular somatic embryos root and shoot poles but lacked real polarity and

31 General Introduction CHAPTER 1

however in some auxin their further development treated segments only was stopped into next stages, root pole was formed therefore it was unconvincing to call them SE based on the basis of morphological characterization Immature Non-conditioned and Microscopically Only non-conditioned Wide spectrum of chitinases Petrovska zygotic conditioned MS with iZE suspension were found in the conditioned et al. 2010 embryos liquid from cultures developed media but embryogenesis (iZE) embryogenic Pinus ELS resembling typical could not be induced, the nigra suspension culture growth stages of SE but reason behind this failure failed to develop could not be addressed. further. It was concluded that the Chitinolytic activity presence of was manifested during protein(s) with chitinolytic embryogenesis as activity in flax liquid culture observed in discriminated between the embryogenic Pinus non-embryogenic cultures nigra suspension and culture cultures with embryogenic potential

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1.4. Bioactive Metabolites Production Using in vitro Established Cultures of Linum usitatissimum In vitro plant cell, tissue and organ culture technology has provided all the means to transform plants into living factories. Adapting various controlled mechanisms plants are subjected to different conditions, varying in nature and intensity levels. These mechanisms when enforced, bring about metabolic shuffling, consequently plants switch primary metabolism to secondary metabolism to withstand the repercussions, and the production levels of the secreted metabolites are generally higher than those produced in natural environment. Such an approach is rarely utilized for obtaining secondary metabolites from Linum usitatissimum compared to other important medicinal plant species. Both callus and cell suspension cultures of Linum usitatissimum are capable of producing sufficient quantities of lignans, neolignans and many other bioactive metabolites under controlled conditions. PGRs not only promote morphogenesis but they also hold a potential to regulate plant metabolic profile. Anjum and Abbasi (2016) obtained significantly different levels of TPC and TFC from Linum usitatissimum callus and whole plant extract. TPC and TFC were highest for stem derived calli and this enhanced accumulation was attributed to the difference in ploidy level and enhancing effects of TDZ. Similarly, Anjum et al. (2017) used flax leaf and stem explants for PGRs induced accumulation of several lignans and neolignans. They concluded that leaf derived calli comparatively accumulated greater biomass (fresh and dry weight) but stem derived calli exhibited maximum accumulation of TPC and TFC endorsed by HPLC analysis that revealed highest levels of secondary metabolic content in stem derived calli. The presence of auxin (NAA) alone or in combination was essential for bioactive metabolites buildup. As discussed earlier that flax roots are pharmacologically more active, both adventitious and hairy root cultures have been shown to accumulate higher secondary metabolites than callus and non-transformed cultures respectively (Abbasi et al. 2017; Gabr et al. 2016). The role of auxin (NAA and 2,4-D) in these studies was prominent too.

Photoperiod fluctuations influence plant physiology and induce stress related responses which often results in production of secondary metabolites. Bruyant et al. (1996) and Anjum et al. (2017) observed that continuous darkness was positively correlated with cellular growth and metabolites production, whereas continuous light had inhibitory effects, furthermore metabolites production was elicited by the latter group when

33 General Introduction CHAPTER 1 cultures were exposed to Ultraviolet-C radiations before maintaining in darkness. Contrasting results had been reported by Siegien et al. (2013) who achieved highest organogenesis frequency and cyanogenic potential of flax by employing continuous light, however the role of the cyanoglucosides was limited to morphogenetic actions as these compounds released free HCN that improved regeneration efficiency.

The quest for enhanced production of flax bioactive metabolites is continued and the available literature is not sufficient enough to deduce an undivided opinion about what really affects flax secondary metabolism. The efforts being made to this effect involve application of methods for increasing the production levels (employing established strategies) and carrying on molecular studies (specifically metabolic engineering) to decipher the actual mechanisms involved in biosynthesis of these metabolites. Some of these efforts are summarized in Table 1.7.

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Table 1.7. Strategies applied for secondary metabolites production in in vitro established cultures of Linum usitatissimum.

Explant Culture type Medium/Optimum Production strategy Metabolites accumulation status References PGRs concentration Hypocotyl Cell culture MS/8.88 uM BA, 2.68 Elicitation with fungal Decrease in secreted levels of lignans Hano et al. uM NAA extracts (SECO and DCG) was observed 2006 Hypocotyl Callus culture MS/growth stage Agrobacterium Various amounts of α-carotene, β- Fujisawa et dependent application mediated metabolic carotene and phytoene (estimated total al. 2008 of PGRs engineering of carotenoid amounts carotenoid were 65.4–156.3 μg/g FW) in different biosynthetic pathway transgenic lines of flax were found with crtB gene whereas none of these compounds were detected in untransformed controls Hypocotyl Callus culture MS/ growth stage RNAi-mediated Down regulation of LuPR1 gene brought Renouard et dependent application LuPR1 (pinoresinol about 30-50 fold decrease in SDG yield al. 2014 of PGRs lariciresinol reductase) but synthesis of new compounds DCG gene silencing and DDCG in seeds was observed for the first time Stem Hairy root B5/1.0 2,4-D + 0.5 or Agrobacterium Significantly higher levels of SECO, Gabr et al. culture 1.0 GA3 rhizogenes mediated MAT, SDG and various phenolic acids 2016 transformation were found in hairy roots, furthermore

35 General Introduction CHAPTER 1

SDG did not accumulate in untransformed calli Leaf and Callus culture MS/1.0 NAA PGRs induced Maximum values for TPC (6.5 and 6.87 Anjum et al. stem metabolic regulation mg/g DW) and TFC (3.2 and 3.22 mg/g 2016 DW) occurred at lower concentrations of PGRs particularly NAA Stem Cell culture MS/1.0 NAA Interactive effects of Highest levels of SDG, LDG and DCG Anjum et al. photoperiod regimes (4.6, 13.6 and 44.1 mg/g DW 2017 and UV-C radiation respectively) occurred in cultures maintained in continuous dark, however UV-C radiation + 16/8 h photoperiod exhibited maximum accumulation of SDG, LDG and GGCG (7.1, 21.6 and 9.2 mg/g DW respectively) Stem Callus culture MS/2.0 TDZ TDZ induced Significantly higher levels of TPC (5.1 Anjum and enhanced- mg/g DW) and TFC (2.72 mg/g DW ) Abbasi, accumulation of were recorded in TDZ induced stem 2016 bioactive compounds derived calli than whole plant extract Root Adventitious MS/0.5 NAA Using root culture as Root cultures exhibited maximum Abbasi et al. root culture an effective source for accumulation of TPC, SDG, LDG, DGC 2017 and GGCG (9.91, 5.50, 11.9, 21.6 and

36 General Introduction CHAPTER 1

bioactive compounds 4.90 mg/g DW respectively) compared accumulation to calli

37 General Introduction CHAPTER 1

1.5. Aim and Objectives The current study was aimed to establish in vitro callus and cell suspension cultures of Linum usitatissimum, respectively, under controlled conditions for enhanced production of commercially important secondary metabolites—lignans and neolignans—. Likewise, attempts were also made to assess biochemical and physiological response of Linum usitatissimum to varying growing conditions such as manipulating photoperiod treatments, nutrient medium composition and exposure of cultures to novel and exogenous elements. Aim of the current study encompassed following major objectives;

1. Establishment of in vitro callus cultures of Linum usitatissimum. 2. To evaluate the effects of mineral nutrients variation and different photoperiod treatments on growth kinetics and secondary metabolites production in callus cultures of Linum usitatissimum. 3. Establishment of in vitro callus cultures of Linum usitatissimum. 4. To evaluate the effects of chemogenic silver nanoparticles on growth and secondary metabolites production in cell suspension cultures of Linum usitatissimum. 5. To evaluate the effects of biogenic zinc oxide nanoparticles on growth and secondary metabolites production in cell suspension cultures of Linum usitatissimum.

38 General Introduction

CHAPTER 2

2. IN VITRO CULTURES OF LINUM USITATISSIMUM L.: SYNERGISTIC EFFECTS OF MINERAL NUTRIENTS AND PHOTOPERIOD REGIMES ON GROWTH AND BIOSYNTHESIS OF LIGNANS AND NEOLIGNANS

2.1. ABSTRACT The multipurpose plant species Linum usitatissimum famous for producing linen fibre and containing valuable pharmacologically active polyphenols, lignans and neolignans, has rarely been tested for its in vitro biosynthesis potential of lignans and neolignans. The current study aims at the synergistic effects of mineral nutrients variation and different photoperiod treatments on growth kinetics and biomass accumulation in in vitro cultures of Linum usitatissimum. Both nutrient quality and quantity affected growth patterns, as cultures established on Gamborg B5 medium had comparatively long exponential phase compared to Murashige and Skoog medium, while growth was slow but steady until last phases of the culture on Schenk and Hildebrandt medium. Similarly, we observed that boron deficiency and nitrogen limitation in culture medium (Gamborg B5 medium) enhanced callus biomass ( fresh weight 413 g/l and dry weight 20.7 g/l), phenolics production (667.60 mg/l), and lignan content (secoisolariciresinol diglucoside 6.33 and lariciresinol diglucoside 5.22 mg/g dry weight respectively) at 16/8 h light and dark-week 4, while that of neolignans ( dehydrodiconiferyl alcohol glucoside 44.42 and guaiacylglycerol-β-coniferyl alcohol ether glucoside 9.26 mg/g dry weight, respectively) at continuous dark-week 4. Conversely, maximum flavonoid production occurred at both Murashige and Skoog, Schenk and Hildebrandt media (both media types contain comparatively higher boron and nitrogen content) in the presence of continuous light. Generally, continuous dark had no significant role in any growth associated parameter. This study opens new dimension for optimizing growing conditions and evaluating underlying mechanisms in biosynthesis of lignans and neolignans in in vitro cultures of Linum usitatissimum.

40 In vitro cultures of Linum usitatissimum L.: Synergistic effects of mineral nutrients and photoperiod regimes on growth and biosynthesis of lignans and neolignans

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2.2. INTRODUCTION Linum usitatissimum commonly known as flax is an important multipurpose plant which has both medicinal and commercial applications. Historically, it has been used to manufacture “linen” fibre, but it also holds several medicinal potentials owing to the presence of some novel classes of polyphenols—the lignans and neolignans (Muir and Westcott, 2003). Lignans are polyphenols that exhibit anticancer activity mainly against breast cancer, prostate cancer, and colon cancer. Lignans include compounds like secoisolariciresinol diglucoside (SDG) and lariciresinol diglucoside (LDG) etc. On the other hand, neolignans are principal agents used in anti-inflammatory and antifungal drugs. Major neolignans are dehydrodiconiferyl alcohol glucoside (DCG) and guaiacylglycerol-β- coniferyl alcohol ether glucoside (GGCG) (Anjum et al. 2017). The difference in pharmacological activities of lignans and neolignans may be attributed to their different structural chemistry (Fig. 2.1).

Figure 2.1. Chemical structures of lignans and neolignans produced by callus cultures of Linum usitatissimum. a. Secoisolariciresinol diglucoside (SDG), b. lariciresinol diglucoside (LDG), c. dehydrodiconiferyl alcohol glucoside (DCG), d. guaiacylglycerol-b-coniferyl alcohol ether glucoside (GGCG)

41 In vitro cultures of Linum usitatissimum L.: Synergistic effects of mineral nutrients and photoperiod regimes on growth and biosynthesis of lignans and neolignans

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In principle, lignans are formed by bonding of phenyl propane dimers through the carbon atoms (C8/C8՜) of their side chains whereas neolignans contain phenyl propane dimers connected through linkage other than C8/C8՜ (Wallis, 1998). The aforementioned medicinal importance of flax has resulted in increasing in vitro exploitation for enhanced, economical and chemically consistent production of lignans and neolignans.

Various abiotic factors including water, air, light, temperature and nutrient availability interactively affect plant physiology, growth and development processes. Naturally growing plants are confronted by challenging growing conditions e.g. some plants may have access to water resources but others may face water deficiency (Zahir et al. 2014). Similarly, poor nutrients consumption due to unfavorable circumstances such as salinity and drought stress, competition for nutrients from neighbor invasive plant species, presence of insoluble/untransformable compounds, and infertile soil lacking many of the macro and micronutrients (nutrient deficiency) are detrimental to normal plant growth and development (Marschner et al. 1987; Bedford et al. 1999; López- Buico et al. 2003). Nutrients-compromised conditions not only affect primary plant growth and development processes like plant height, expansion, vegetative parts maturation, seeds and fruit size (Le Corff, 1993; Kolodziejek, 2017) but also secondary metabolic profile associated with different plant defense mechanisms (Gershenzon, 1984; Chishaki and Horiguchi, 1997). Some major nutrients required for normal plant physiology, growth and development processes comprise a set of chemical elements such as: nitrogen forms a major component of proteins and chlorophyll (deficiency may lead to stunted growth); phosphorus contributes to photosynthesis, cellular growth and division as it forms a greater part of nucleic acids (deficiency may lead to slow growth, poor rooting and low fruit yield); calcium plays a major role in cell wall synthesis and is a co-factor of many enzymes (deficiency results in necrotic tissue formation) and magnesium acts as an integral part of chlorophyll and is also critical for functioning of various enzymes (Uchida, 2000; Maathuis, 2009). Likewise, minor but vital nutrients include boron, which is involved in lignin and phenolic acids metabolism (deficiency results in failed development of flowers and fruits), and manganese and zinc are required in many enzyme dependent activities (deficiency may lead to chlorosis) (Uchida, 2000; Hansch and Mendel, 2009). It has been documented that macronutrients determines the plant growth and development status, whereas micronutrients define the 42 In vitro cultures of Linum usitatissimum L.: Synergistic effects of mineral nutrients and photoperiod regimes on growth and biosynthesis of lignans and neolignans

CHAPTER 2 secondary metabolism status (Poschenrieder et al. 2008). Similarly, along with nutrients availability changing light conditions also has stimulatory effects on biomass accumulation (Ahmad et al. 2016; Tariq et al. 2014). Illumination of in vitro plant cultures has been reported to induce biosynthesis of secondary metabolites such as phenolics, alkaloids, flavonoids and anthocyanins primarily as a result of light induced development of chloroplast; whereby synthesis of precursors involved in various biosynthetic pathways occur, and improved callus morphogenesis that is associated with biomass accumulation. In contrast, absence of light (dark) during plant growth and development generally results in inactivation of certain genes and enzymes thus lowering biomass accumulation potential of the plant (Zhao et al. 2001).

These facts and findings strengthened the suggestion that almost each and every plant species requires different growing conditions. Therefore, researchers have formulated various types of plant culture media. These culture media have some qualitative and quantitative variations in key nutritional contents depending on plant’s structural and functional diversity i.e. monocots and dicots (Morel and Wetmore, 1951; Schenk and Hildebrandt, 1972; Vasil and Thorpe, 2013). Mostly used culture media for in vitro culturing of important commercial and medicinal plants include Murashige and Skoog (MS) medium, Gamborg B5 (B5) medium and Schenk and Hildebrandt (SH) medium (Abbasi et al. 2017; Murashige and Skoog, 1962; Gamborg et al. 1968; Schenk and Hildebrandt, 1972). The differences among these three media are described in Table 2.1. The aim of current study was to evaluate the synergism produced by variation in mineral nutrients levels and photoperiod regimes in in vitro cultures of Linum usitatissmum; in terms of culture growth and biomass accumulation with a focus on biosynthesis of lignans and neolignans.

2. 3. MATERIALS AND METHODS 2.3.1. Inoculum Preparation Callus formation was induced following the protocol of Anjum et al. (2017). Simply, stem explants were excised from 25 days old in vitro seed derived plantlets and placed on MS medium containing 1 mg/l NAA, 30 g/l sucrose and 8 g/l agar as a solidifying agent. The resultant callus was kept growing for 35 days and then subcultured on the same MS (callus inducing) medium for 21 days. For the establishment of callus cultures on MS, B5 and SH media, ~1 g callus was inoculated on each media type in a 100 ml

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Erlenmeyer flask. All the three media types contained same 3% sucrose, 0.8% agar and 1 mg/l NAA, except for concentrations of salts mixture, which were 4.4 g/l (MS), 3.21 g/l (B5), and 3.20 g/l (SH) respectively. PH was adjusted to 5.6 prior autoclaving. Callus cultures were maintained at 25±2ºC in three different photoperiod regimes viz. 16/8 h light and dark, continuous dark and continuous light, for 7 weeks.

Table 2.1. Major differences among Murashige and Skoog, Gamborg B5, and Schenk and Hildebrandt media.

Nutrients Concentration mg/L Murashige and Skoog Gamborg B5 Schenk and Hildebrandt

Potassium nitrate 1900 2500 2500 Ammonium sulphate 1650 134 - Ammonium phosphate - - 300 Calcium chloride 332.16 113.24 151.02 Magnesium sulphate 180.69 112.09 195.34 Manganese sulphate 16.9 10 10 Zinc sulphate 8.6 2 1 Sodium phosphate - 130.42 - Potassium phosphate 170 - - Boric acid 6.2 3 5

2.3.2. Determination of Fresh and Dry Weight Calli from all three cultures were harvested on weekly basis. Fresh calli were pressed gently between layers of filter paper to remove excess water and then weighed. For dry weight determination, harvested calli were oven dried at 60ºC until constant weight.

2.3.3. Determination of Callus Growth Rate Following equation was used to determine weekly growth rate. 푊푎 1 퐺푅 = [( ) ∧ − 1] × 100 푊푏 푛

Where Wa is present weight (fresh or dry), Wb is previous weight (fresh or dry) and n is particular number of weeks.

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2.3.4. Phytochemical Analysis For determination of both total phenolic and flavonoid content (TPC and TFC), we added 500 μl methanol (100 % pure) to 100 mg powdered dry calli, followed by vortexing and then sonication for 30 minutes. The mixture was vortexed again to ensure proper shaking and mixing. Centrifugation of the samples was performed at 13,000 rpm for 5 minutes. The supernatant was isolated and used for further analysis. Aluminium chloride colourimetric method (Haq et al. 2012) was used for TFC determination. Quercetin derived standard curve was used as a reference for TFC determination. Extracts were subjected to UV spectroscopy for determination of TFC at 415 nm, using UV–Vis spectrophotometer (Halo DR-20, UV–Vis spectrophotometer, Dynamica Ltd., Victoria, Australia). Total flavonoid production (TFP) was calculated by using the following formula and expressed in mg /l.

TFP (mg/l) =DW (g/l) ×TFC (mg/g)

Gallic acid derived standard curve was used as a reference for TPC determination. For TPC determination, Folin-Ciocalteu (FC) method was used (Singleton et al. 1999). Extracts were subjected to UV spectroscopy for determination of TPC at 630 nm. Total phenolic production (TPP) was calculated by using the following formula and expressed in mg/l.

TPP (mg/l) =DW (g/l) ×TPC (mg/g)

TPC and TFC values were expressed in milligram equivalents per gram dry weight of calli.

2.3.5. Free Radical Scavenging Assay The method of Lee et al. (1998) was adopted to determine the free radical scavenging potential of samples spectrophotometrically at 517 nm using 1,1-diphenyl-2- picrylhydrazyl (DPPH). Following equation was used to calculate the DPPH free radical scavenging capacity of samples:

% scavenging= [(Absorbance of control - Absorbance of the sample)/Absorbance of control] ×100

2.3.6. HPLC Analysis HPLC technique was utilized to quantify the presence of some important lignans and neolignans in powdered calli samples. Only samples harvested at week 3, 4, 5, and 6 45 In vitro cultures of Linum usitatissimum L.: Synergistic effects of mineral nutrients and photoperiod regimes on growth and biosynthesis of lignans and neolignans

CHAPTER 2 respectively were analyzed through HPLC. Varian liquid chromatographic system (composed of Varian Prostar 410 auto sampler, Metachem Degasit, and Varian Prostar 335 Photodiode Array Detector) was used for the quantification important secondary metabolites including SDG, LDG, DCG and GGCG as described by Fliniaux et al. 2014. Protocol devised by Corbin et al. (2015) was followed by ultrasonic extraction of samples. Simply, centrifugation of the samples was performed at 3000 rpm for 15 minutes followed by filtration using nylon syringe membrane (0.45 μm). Purospher (Merck) reverse phase (RP-18) column was utilized for separation, and detection was performed at 280 nm wavelength using UV–Vis spectrophotometer. The compounds were identified by comparing their retention times and UV spectra to those of reliable reference standards. The mobile phase consisted of two solvents, solvent A and solvent B which were 0.2% acetic acid aqua solution and methanol respectively. For mobile phase variation, a nonlinear gradient was applied with a flow rate of 0.8 ml/min as follows: from 0 to 40 min of A–B: 90:10 (v/v) to 30:70 (v/v), from 41 to 50 min of A– B: 30:70 (v/v) to 0:100 (v/v), and A–B: 0:100 (v/v) from 51 to 60 min (Corbin et al. 2015).

2.3.7. Statistical Analysis All experiments were carried out at once. Each treatment consisted of three replicates and repeated twice. Mean values of various treatments were subjected to analysis of variance by using Origin Pro software (8.5). Also, Duncan’s multiple range test (Windows version 7.5.1, SPSS Inc., Chicago) was used to determine the significance at P < 0.05 (Duncan 1955). MS Excel (2013) was used for generation of all figures and tables.

2.4. RESULTS AND DISCUSSION 2.4.1. Trends in Growth Kinetics and Morphological Changes While analyzing growth rate index, we observed that maximum growth activities after inoculation occurred at MS, B5 and SH, respectively, for the first two weeks. Both MS and B5 provided suitable conditions for rapid adaptation of cells to the medium. Cultures maintained in continuous dark exhibited retarded growth and those in continuous light exhibited rapid growth as compared to moderate growth activities observed in cultures maintained in 16/8 h light and dark. However, growth was slowed down at the end of the 3rd week on MS medium. Maximum growth activities were

46 In vitro cultures of Linum usitatissimum L.: Synergistic effects of mineral nutrients and photoperiod regimes on growth and biosynthesis of lignans and neolignans

CHAPTER 2 extended to 4th week on B5 medium, while SH medium exhibited slow but steady growth pattern up to 6th week irrespective of photoperiod regimes applied (Table 2.2). Some minor variations were observed while recording these parameters.

Figure 2.2. A; Calli cultured on Murashige and Skoog, Gamborg B5 and Schenk and

Hildebrandt media, respectively, in 16/8 h light and dark, B; Calli cultured on Murashige and Skoog, Gamborg B5, and Schenk and Hildebrandt media, respectively, in continuous dark, C; Calli cultured on Murashige and Skoog, Gamborg B5 and Schenk and Hildebrandt media, respectively, in continuous light.

The growing conditions had effects on morphology and texture of calli too. Generally, calli grown on SH medium had compact texture compared to friable calli obtained on both MS and B5 media. Overall, calli kept in the continuous dark had yellowish color compared to the bright green color of those kept in continuous light (Fig. 2.2B and 2.2C). However, it was noticed in 16/8 h light and dark that B5 had a more greenish look as compared to calli derived on MS and SH media (Fig. 2.2A). Excluding some inconsistent changes, we could break weekly intervals into specific growth phases i.e.

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Table 2.2. Growth index of in vitro established cultures of Linum usitatissimum on Murashige and Skoog, Gamborg B5, and Schenk and Hildebrandt media, respectively, in 16/8 h light and dark, continuous dark, and continuous light photoperiod regimes.

Photoperiod Media type Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 regime FW DW FW DW FW DW FW DW FW DW FW DW FW DW 56.3 54.8 43.5 24.1 - - - Murashige and Skoog 34.60 21.34 -3.29 1.15 0.70 -0.67 -63.21 4 6 7 9 3.19 1.79 275.21 52.2 52.7 29.2 24.8 10.5 - - - - - 16/8 h Gamborg B5 67.33 52.08 3.27 -32.67 7 3 1 3 7 3.31 6.74 3.25 10.37 537.56 Schenk and 53.2 64.3 15.6 16.7 11.2 - - 11.35 5.90 4.67 3.72 2.94 3.05 1.95 Hildebrandt 8 5 0 7 6 517.14 459.53 73.6 53.5 13.6 - - - Murashige and Skoog 97.66 67.58 9.57 2.57 1.69 1.26 -5.07 -93.41 5 7 3 1.67 1.95 519.74 70.3 64.8 24.8 23.5 - - - - Continuous dark Gamborg B5 20.33 5.04 8.43 2.34 -6.47 79.25 8 8 6 8 1.23 2.65 2.34 106.80 Schenk and 30.5 31.5 32.2 26.7 12.7 - - - 17.52 31.20 1.01 1.29 6.85 -1.22 Hildebrandt 2 5 0 2 5 4.22 219.75 529.71 122.4 107.7 40.9 38.8 13.8 16.7 13.2 - - - - - Murashige and Skoog 7.75 -0.86 1 9 3 3 1 9 7 0.75 1.09 1.78 226.43 301.76 149.7 142.9 38.2 22.0 13.6 13.8 15.2 10.3 - - Continuous light Gamborg B5 1.53 0.83 -1.04 -26.77 8 3 2 0 3 5 2 7 3.24 135.04 Schenk and 79.4 60.1 12.3 10.0 - - - - 75.25 83.81 9.77 6.75 3.42 2.53 Hildebrandt 0 8 4 3 2.52 5.43 225.78 286.52

48 In vitro cultures of Linum usitatissimum L.: Synergistic effects of mineral nutrients and photoperiod regimes on growth and biosynthesis of lignans and neolignans

CHAPTER 2 week 1 was regarded lag phase, week 2 early log phase, week 3 and 4 exponential log phase, week 5 and week 6 stationary phase, and week 7 was declining or death phase. Growing conditions affect plant physiology, morphology and development in several ways. Current study provided an insight into how fluctuations in nutrient availability can regulate plant’s growth morphological and metabolic response. We observed optimum growth associated parameters on B5 medium compared to MS and SH media. Our observations are in agreement with Condori et al. (2010), who also compared effects of modified MS and B5 media on hairy root cultures of peanut. Similar to our findings, they concluded that roots cultured in B5 medium had exhibited a longer exponential growth phase as compared to modified MS medium. This extended growth phase was considered to be a result of the early depletion of ammonium but retention (availability) of nitrate till the end of exponential phase in the B5 medium compared to contrasting observations made in MS medium. Likewise, they reported marked color changes in B5 medium that were absent in MS medium.

2.4.2. Trends in Biomass, Phenolics and Flavonoids Accumulation Increase in callus biomass of Linum usitatissimum followed an individually similar pattern on each media type, but differential effects of various photoperiod regimes applied were observed too. Generally, initially, biomass (fresh weight) doubled each week till the end of 4th week, with some minor variations observed in dark photoperiod. Although, comparatively higher biomass values were recorded in calli grown in continuous light at the end of week 5, however almost similar values were recorded for calli grown in 16/8 h light and dark at the end of week 4 (Fig. 2.3). Time and energy consumptions are critical during in vitro culturing, therefore 16/8 h light and dark-week 4 data were considered as optimum. We obtained highest FW (413 g/l) and DW (20.7 g/l) (Fig. 2.3), and TPC (32.22 mg GAE /g DW) (Fig. 2.4) on B5 medium followed by some considerable results on SH and MS medium. Calli cultivated in dark showed minimum growth response. Furthermore, marked differences among biomass values in exponential phase were not observed in dark photoperiod as for the other two over the course of time. Biomass accumulation is directly associated with primary metabolism, particularly carbon and nitrogen metabolism. Carbon metabolism caters to the energy needs as a result of carbohydrates synthesis and thus contributes to cellular growth and structural components of the cell (Sturm and Tang, 1999). We supplied same concentrations of sucrose (3%) as a carbon source to all the callus cultures, therefore 49 In vitro cultures of Linum usitatissimum L.: Synergistic effects of mineral nutrients and photoperiod regimes on growth and biosynthesis of lignans and neolignans

CHAPTER 2 the role of carbon will not be discussed here. On the other hand, products of nitrogen metabolism are mainly amino acids and proteins which consequently are utilized in regular housekeeping cellular functions (Causin, 1996). Both cellular growth and division culminate in enhanced fresh weight and dry weight over the course of time. In our study similar enhancing effects were observed when we monitored calli growth on weekly intervals. Onyango et al. (2012) argued that ammonium to nitrate ratio matters greatly in cellular growth, and lower ammonium to nitrate ratio favours cellular growth. Among the different media tested in our experiment highest total nitrogen content was found in MS, SH and B5, respectively, but MS was the only media having almost similar ammonium to nitrate ratio, whereas B5 and SH contained least ammonium to nitrate ratio, hence maximum growth occurred at B5 and SH accordingly. Our results are also in agreement with those of Rahayu et al. (2005) who reported that nitrate application compared to ammonium coupled with positive phytohormonal regulation results in rapid tomato leaf expansion. Likewise, low phosphate levels also result in enhanced biomass accumulation (Panda et al. 1992). Phosphate levels were found comparatively lower in B5 and SH media and highest in MS medium.

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Figure 2.3. Fresh and dry weight of Linum usitatissimum calli cultured on Murashige and Skoog, Gamborg B5, and Schenk and Hildebrandt media, respectively, in 16/8 h light and dark, continuous dark, and continuous light photoperiod regimes. Values are mean of triplicates ±SD.

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Figure 2.4. Total phenolic content of Linum usitatissimum calli cultured on Murashige and Skoog, Gamborg B5, and Schenk and Hildebrandt media, respectively, in 16/8 h light and dark, continuous dark, and continuous light photoperiod regimes. Values are mean of triplicates ±SD.

Figure 2.5. Total phenolic production of Linum usitatissimum calli cultured on Murashige and Skoog, Gamborg B5, and Schenk and Hildebrandt media, respectively, in 16/8 h light and dark, continuous dark, and continuous light photoperiod regimes. Values are mean of triplicates ±SD.

Contrasting results were obtained for flavonoid production. Enhanced flavonoid accumulation occurred on MS and SH media respectively as compared to B5 medium. Maximum TFC (7.4 mg QUE /g DW) was observed at MS medium16/8 h light and

52 In vitro cultures of Linum usitatissimum L.: Synergistic effects of mineral nutrients and photoperiod regimes on growth and biosynthesis of lignans and neolignans

CHAPTER 2 dark-week 3 (Fig. 2.6). Interestingly, enhanced accumulation was observed in those weeks which produced the least amount of phenolics. Generally, MS medium at week 4 and SH medium at week 6 resulted in higher flavonoid production (124 and 117 mg QUE/l, respectively) (Fig. 2.7), with continuous light having pronounced effects. Dark photoperiod resulted in comparatively lower productions of both phenolics and flavonoids (Fig. 2.5 and fig. 2.7). Both phenolics and flavonoids represent a major portion of a medicinal plant’s secondary metabolic profile. Linum usitatissimum is generally recognized for containing some novel phenolics. In the current study, enhanced phenolic accumulation on B5 medium can be the result of comparatively lower boron concentration in the medium. Several authors have reported that lower boron content positively regulates shikimic acid pathway, the primary phenol metabolic pathway. Heidarabadi et al. (2011) concluded that boron deficiency had positively affected activities of certain enzymes such as PAL, PPO and PO that are involved in the biosynthesis of phenols in roots of Linum usitatissimum. Similar results were also obtained by Camacho-Cristóbal et al. (2002), who exposed tobacco plants to short-term boron starvation. In the current study, phenol production was not greatly affected by photoperiod regimes but, interestingly, continuous light stimulated flavonoid accumulation, probably as a result of the stressed condition induced by continuous light. Under such circumstances, flavonols play a key role as photoprotectants and free radical scavengers (Koes et al. 2005). It has been proposed that light intensity and quality highly influence flavonoid biosynthesis, and higher solar radiation apparently increases flavonoid content in plants (Zorrati et al. 2014). Our results are also supported by Uleberg et al. (2012). These researchers enhanced levels of anthocyanins in bilberry fruits by extending day length to 24 h from 12 h day length treatment. Enhancing effects of light on flavonoid biosynthesis has also been reported in many other plant species including Chinese bayberry (Niu et al. 2010), cranberry (Zhou and Singh, 2004), and tomato (Løvdal et al. 2010).

2.4.3. Trends in Free Radical Scavenging Assay All the samples tested for DPPH-FRSA exhibited significant potential to scavenge reactive radicals. The scavenging capacity enhanced in a time-dependent manner i.e. a gradual increase with the passage of each week was observed (Fig. 2.8). Maximum scavenging capacity occurred at week 3 and onwards, irrespective of both media type and photoperiod regimes applied. Maximum values recorded were in the range of 90- 53 In vitro cultures of Linum usitatissimum L.: Synergistic effects of mineral nutrients and photoperiod regimes on growth and biosynthesis of lignans and neolignans

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95% whereas minimum values were in the range of 60-75%. Maximum scavenging activities occurred generally at week 4 and 5, while lowest scavenging activities occurred at week 1 and 7.

Figure 2.6. Total flavonoid content of Linum usitatissimum calli cultured on Murashige and Skoog, Gamborg B5, and Schenk and Hildebrandt media, respectively, in 16/8 h light and dark, continuous dark, and continuous light photoperiod regimes. Values are mean of triplicates ±SD.

Figure 2.7. Total flavonoid production of Linum usitatissimum calli cultured on Murashige and Skoog, Gamborg B5, and Schenk and Hildebrandt media, respectively,

54 In vitro cultures of Linum usitatissimum L.: Synergistic effects of mineral nutrients and photoperiod regimes on growth and biosynthesis of lignans and neolignans

CHAPTER 2 in 16/8 h light and dark, continuous dark, and continuous light photoperiod regimes. Values are mean of triplicates ±SD.

2.4.4. Trends in Lignans and Neolignans Accumulation Results obtained could not reveal a specific pattern of lignans and neolignans accumulation, rather random values were recorded throughout the culture period, however, it was observed that samples harvested from the B5 medium at week 4 contained higher amounts of both lignans and neolignans (Table 2.3). Maximum accumulation of lignans (SDG; 6.33 and LDG; 5.22 mg/g DW, respectively) occurred at 16/8 h light and dark-week 4 while that of neolignans (DCG; 44.42 and GGCG; 9.26 mg/g, DW respectively) occurred at dark-week 4.

Influence of nutrient elements on biomass accumulation and secondary metabolites biosynthesis during in vitro culturing systems have been realized long ago. According to a strong hypothesis; the carbon-nutrient balance (CNB), nitrogen limitation results

55 In vitro cultures of Linum usitatissimum L.: Synergistic effects of mineral nutrients and photoperiod regimes on growth and biosynthesis of lignans and neolignans

CHAPTER 2 in enhanced polyphenols accumulation in plant tissues by stimulating the assimilation of excessive carbon to produce carbon-based secondary compounds and their precursors (Kováčik & Bačkor 2007, Ibrahim et al. 2011, Li et al. 2005). B5 medium that proved optimum for enhanced production of lignans and neolignans in the current study was initially devised for suspension cultures of soybean root cells. B5 medium contains considerably lower nitrogen concentrations compared to MS and SH, and thus fits the CNB hypothesis. Several authors have successfully utilized B5 medium for the in vitro production of important secondary metabolites, such as Rosmarinic acid from Anchusa officinalis (De-Eknamkul and Ellis, 1985), Cryptosin from Cryptolepis buchanani Roem (Venkateswara et al. 1987), Robustaquinones from Cinchona robusta (Schripsema et al. 1999), and taxol from Taxus spp. (Wu et al. 2001). Coincidentally, B5 medium has been also used effectively for related Linum species. Earlier, Baldi et al. (2008) and more recently, Renouard et al. (2017) have reported enhanced biomass accumulation and lignan production in hairy root cultures of Linum album and Linum flavum, respectively. The same lower ammonium to nitrate ratio principle (mentioned earlier in case of biomass accumulation) also applies to the enhanced accumulation of secondary metabolites. Praveen and Murthy (2013) obtained higher shoot biomass and bacoside A content when they kept nitrate concentration higher to that of ammonium. In many similar studies, increasing nitrate and reducing ammonium had positive effects on production of gymnemic acid and withanolide A (Praveen et al. 2011; Praveen and Murthy, 2011).

2.5. Conclusions Enhanced production of secondary metabolites from in vitro established cultures, though is a superior process to the existing traditional production systems, however, it is a complex, meticulous and expensive process. The more complex is a process the better the results are. In this study, a comprehensive strategy was devised to propagate in vitro cultures of Linum usitatissimum. We observed key changes in growth kinetics and biomass accumulation. Major findings included: the regulatory role of nitrogen content, particularly lower ammonium/nitrate ratio contributing to enhanced calli growth and accumulation of commercially important polyphenols; lignans and neolignans, the effect of light quality and intensity (photoperiod regimes) on flavonoid production, and the most noticeable, boron deficiency triggered upregulation of

56 In vitro cultures of Linum usitatissimum L.: Synergistic effects of mineral nutrients and photoperiod regimes on growth and biosynthesis of lignans and neolignans

CHAPTER 2 polyphenol metabolism. Current findings obviously provide the basis for future scale- up productions of the valuable lignans and neolignans.

57 In vitro cultures of Linum usitatissimum L.: Synergistic effects of mineral nutrients and photoperiod regimes on growth and biosynthesis of lignans and neolignans

CHAPTER 2

Table 2.3. HPLC quantified data of lignans and neolignans. SDG; secoisolariciresinol diglucoside, LDG; lariciresinol diglucoside, DCG; dehydrodiconiferyl alcohol glucoside, GGCG; guaiacylglycerol-b-coniferyl alcohol ether glucoside.

week 3 week 4 week 5 week 6

DCG GGC SDG LDG DCG GGC SDG LDG DCG GGC SDG LDG DCG GGC SDG LDG G G G G

Photoperiod regime Media type M 2.7±0. 2.15± 0.48±0 0.36± 1.5±0. 0.97± 1.12±0 0.17± 2.17±0 1.46± 0.93±0 1.4±0. 0.21±0 0.16± 0.28±0 0.27± S 038cd 0.030b .006d 0.006c 026d 0.017d .019cd 0.003c .030cd 0.020c .013d 049b .003d 0.002d .004d 0.040c B 0.22±0 0.21± 0.12±0 0.33± 16.13± 1.83± 6.33±0 5.22± 10.79± 1.27± 5.34±0 1.29± 0.37±0 0.3±0. 0.19±0 0.34± 5 .003 0.002d .001d 0.005c 0.285b 0.032c .111b 0.022a 0.152 b 0.017c .075bc 0.184b .006d 005d .003d 0.007c

S 6.5±0. 0.41± 3.06±0 1.4±0. 1.37±0 0.66± 7.6±0. 1.93± 2.17±0 0.28± 0.06±0 0.3±0. 0.17±0 0.22± 0.1±0. 0.26± H 091c 0.005d .043c 024b .024d 0.011d 134b 0.034b .030cd 0.003d .001d 010c .003d 0.003d 001d 0.002c

16/8 h M 1.68±0 1.04± 0.15±0 0.38± 15.42± 3.85± 6.38±0 1.75± 2.75±0 3.27± 23.3±0 4.59± 0.19±0 0.33± 1.62±0 0.24± S .011d 0.014c .002d 0.013c 0.272b 0.068b .112b 0.030b .038cd 0.046b .329a 0.162a .010d 0.005d .028cd 0.008c

B 0.17± 0.12±0 0.3±0. 44.42± 9.26± 1.21±0 0.57± 5.28±0 1.82± 8.85±0 1.56± 0.62±0 0.19± 0.69±0 0.41± 0.1±0d 5 0.002d .001d 010c 0.785a 0.163a .021cd 0.010c .074c 0.025c .125b 0.055b .212d 0.003d .012d 0.007c S 4.31±0 0.31± 0.09±0 0.25± 2.62±0 3.12± 3.95±0 0.26± 15.74± 4.47± 0.72±0 1.74± 12.01± 4.82± 1.75±0 2.89± H .030c 0.004d .001d 0.008c .046cd 0.055b .069c 0.004c 0.222b 0.063b .101d 0.061b 0.003b 0.085b .030cd 0.051b

Continuous Dark M 2.29±0 0.74± 0.12±0 0.38± 4.32±0 0.58± 2.07±0 2.27± 1.48±0 1.1±0. 2.19±0 1.03± 5.35±0 1.1±0. 4.82±0 0.33± S .03cd 0.010d .001d 0.013c .076c 0.010d .036c 0.040b .020d 016c .030c 0.036b .094c 019c .085c 0.005c

B 1.37±0 0.52± 0.13±0 0.3±0. 2.83±0 1.93± 2.23±0 2.37± 0.81±0 0.41± 1.17±0 0.65± 2.57±0 0.41± 0.58±0 0.62± 5 .019d 0.007d .001d 010c .050cd 0.034c .039c 0.041b .011d 0.005d .016cd 0.022c .045cd 0.007d .010 0.010c S 3.34±0 0.46± 1.51±0 1.29± 2.46±0 1.68± 1.95±0 2.07± 0.27±0 0.16± 0.18±0 0.3±0. 0.16±0 0.16± 0.15±0 0.41± H .047cd 0.006d .021cd 0.045b .043cd 0.029c .034cd 0.036b .003d 0.002d .002d 010c .002d 0.002d .002 0.007c

Continuous Light

58 In vitro cultures of Linum usitatissimum L.: Synergistic effects of mineral nutrients and photoperiod regimes on growth and biosynthesis of lignans and neolignans

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3. CHEMOGENIC SILVER NANOPARTICLES ENHANCE LIGNANS AND NEOLIGNANS IN CELL SUSPENSION CULTURES OF LINUM USITATISSIMUM L.

3.1. ABSTRACT Cell suspension cultures of Linum usitatissimum are a great source of the novel and multipurpose medicinal compounds⸺ lignans and neolignans. Conventional culturing practices usually result in low yield of plant secondary metabolites; therefore, we conceived a successful mechanism to elicit production of lignans and neolignans in cell suspension cultures, simply, by addition of chemogenic Ag-NPs into the culture medium. A three stage feeding strategy (day 10, 10 and 15, and 10 and 20, respectively, after inoculation) spanning the log growth phase (day 10-20), was implemented to elicit cell suspension cultures of Linum usitatissimum. Though enhancing effects of Ag-NPs were observed at each stage, feeding Ag-NPs at day 10 resulted in comparatively, highest production of lignans (secoisolariciresinol diglucoside, 252.75 mg/l; lariciresinol diglucoside, 70.70 mg/l), neolignans (dehydrodiconiferyl alcohol glucoside, 248.20 mg/l; guaiacylglycerol-β-coniferyl alcohol ether glucoside, 34.76 mg/l), total phenolic content (23.45 mg GAE/g DW), total flavonoid content (11.85 mg QUE/g DW) and biomass (dry weight: 14.5 g/l), respectively. Furthermore, a linear trend in accumulation of lignans and neolignans was observed throughout log phase as compared to control, wherein growth non-associated trend in biosynthesis of these metabolites was observed. Optimum production of both lignans and neolignans occurred on day 20 of culture; a 10-fold increase in secoisolariciresinol diglucoside, 2.8 fold increase in lariciresinol diglucoside, 5 fold increase in dehydrodiconiferyl alcohol glucoside and 1.75-fold increase in guaiacylglycerol-β-coniferyl alcohol ether glucoside was observed in production levels compared to control treatments, respectively.

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3.2. INTRODUCTION Linum usitatissimum, also known as flax, is a novel plant in the family Linaceae. Historically, it has been used for commercial production of linen fiber but, currently, it is gaining importance as a source of novel phytomedicine (Muir and Westcott 2003). Linum usitatissimum contains a mixture of pharmacologically active polyphenols called lignans and neolignans. These polyphenols differ structurally due to the difference in bonding patterns of their respective phenyl propane units (Wallis 1998). Polyphenols that exhibit anticancer activity mainly against breast cancer, prostate cancer and colon cancer are lignans. Some notable lignans include compounds like secoisolariciresinol diglucoside (SDG) and lariciresinol diglucoside (LDG), etc. Many polyphenols used in anti- inflammatory and antifungal medications are neolignans. Important neolignans include guaiacylglycerol-β-coniferyl alcohol ether glucoside (GGCG) and dehydrodiconiferyl alcohol glucoside (DCG) (Zahir et al. 2018). Presence of these novel polyphenols has resulted in overgrowing in vitro exploitation of Linum usitatissimum in the recent flourishing era of phytomedicine.

Production of biologically active plant secondary metabolites using plant cell tissue and organ culture system has long been in practice. This in vitro technology is the best alternative to the cumbersome conventional harvesting and extraction method from wild and cultivated plants. Though the process is superior in terms of product’s controlled quality and chemical consistency, yet production level is a major limitation. Production level of such a system is comparatively lower than conventional production techniques (Murthy et al. 2014). Since emergence of in vitro techniques, researchers have been busy devising strategies for enhanced production levels, mainly, using plants as in vitro green factories. To this effect, among several reported successful methodologies (Namdeo 2007), elicitation is considered the simplest and the most effective strategy to increase products yield in vitro (Karwasara et al. 2010). Several compounds of both biotic and abiotic origins can act to elicit enhanced accumulation of key metabolites upon introduction to in vitro plant culture systems (Farag et al. 2017), especially during the log growth phase. Though, various abiotic elicitors have successfully been reported to elicit cell cultures, metallic nanoparticles (NPs) have hardly been tested for its elicitation potential. Limited number of publication is available that cite the use of NPs as elicitors; however, the available literature described their potential enhancing effects. Furthermore, if not the nanoparticles per se, the source

61 Chemogenic silver nanoparticles enhance lignans and neolignans in cell suspension cultures of Linum usitatissimum L.

CHAPTER 3 metallic compounds have been greatly utilized to this end (Mahendran et al. 2018; Osman et al. 2018), which could lay the basis for using NPs as elicitors. To the best of our knowledge, this is the first report on successful elicitation of flax cell suspension cultures with chemogenic Ag-NPs. The scope of current study encompassed the temporal additive effects of Ag-NPs on biomass accumulation and production of lignans and neolignans in cell suspension cultures of Linum usitatissimum in controlled environment.

3.3. MATERIALS AND METHODS 3.3.1. Inoculum Preparation Callus cultures were established on solidified MS medium. in vitro seeds derived aseptic plantlets (4 weeks old) were used as source for stem explants. Explants of ~1 cm size were inoculated in a 100 ml Erlenmeyer flasks, on medium containing 1 mg/l NAA, for 30 days (Fig. 3.1A). To prepare inoculum for cell suspension culture, ~3 g callus was inoculated into 100 ml liquid MS medium in 250 ml Erlenmeyer flasks, containing 30 g/l sucrose, 4.43 g/l salt mixture and 1 mg/l NAA. pH of the culture media was set at 5.6 prior to autoclaving. These flasks were placed in a shaking incubator (120 rpm) for 15 days in 16/8h light and dark photoperiod at 25±2 °C (Fig. 3.1B).

3.3.2. Characteristics of Ag-NPs Ag-NPs were prepared and reported by our group earlier (Zaka et al. 2016). Polyols process, an ecofriendly approach was utilized for synthesis of Ag-NPs.

Polyethyleneimine (C2H5N)n and silver nitrate (AgNO3; 1 mM) were added in a 1:2 proportion. This mixture was heated at 150 °C for 15 min. Formation of Ag-NPs was noted by color change and confirmed by UV-visible spectroscopy. XRD was employed for calculating particles size and texture, while, TEM revealed the shape. Resultant NPs obtained were spherical in shape, having crystalline appearance, and average size was up to 18 nm. The stability of Ag-NPs was confirmed using UV-visible spectroscopy (absorbance value of 0.68 and peak value of 451 nm were used as reference parameters), before its application.

3.3.3. Optimization of Ag-NPS Concentration Previously, day 30 of cell suspension culture of Linum usitatissimum has been reported to show maximum growth and phytochemicals accumulation (Nadeem et al. 2018), therefore, current experiment spanned for 30 days. Initially, we optimized Ag-NPs

62 Chemogenic silver nanoparticles enhance lignans and neolignans in cell suspension cultures of Linum usitatissimum L.

CHAPTER 3 concentration for elicitation of cell suspension cultures of Linum usitatissimum. Different concentrations of chemogenic Ag-NPs i.e. 1, 5, 10, 20, 30, 40, 50, 75, 100, 200 and 500 µg/l, respectively, were added to cell cultures on day zero. Optimization and subsequent experiments were carried out in 100 ml Erlenmeyer flasks containing 30 ml liquid MS medium and 10 ml inoculum.

Figure 3.1. A; in vitro established callus culture of Linum usitatissimum. B; Inoculum for cell suspension cultures of Linum usitatissimum, derived from in vitro established callus culture. C; Cell suspension cultures of Linum usitatissimum fed on day 10, day 10 and 15, and day 10 and 20, respectively.

3.3.4. Ag-NPs Treatments Among the various concentrations tested for optimization, 30 µg/l Ag-NPs showed optimum biomass accumulation (FW and DW) on day 30, therefore, it was selected for further experimentation. For repeated elicitation of cell suspension cultures, different periodic intervals were specified for addition of Ag-NPs to cell suspension cultures. Cell cultures were fed with Ag-NPs on three occasions, initially on onset of log phase i.e. day 10, then on day 10 and 15 (mid log phase), and finally day 10 and 20 (end of log phase), (Fig. 3.1C). Cells were harvested at 5 days interval after each feeding step until day 30 of the cell suspension culture. Cell cultures without addition of NPs were used as control treatment.

3.3.5. Biomass Determination Cells harvested at different intervals were gently pressed between layers of filter papers to drain excessive water. Fresh weight was determined instantly; using weighing balance, whereas, cells were oven dried at 50 °C until for 24 hours and then dry weight was determined.

63 Chemogenic silver nanoparticles enhance lignans and neolignans in cell suspension cultures of Linum usitatissimum L.

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3.3.6. Phytochemical Analysis For determination of both total phenolic and flavonoid content (TPC and TFC), we added 500 μl methanol (100 % pure) to 100 mg powdered dry calli, followed by vortexing and then sonication for 30 minutes. The mixture was vortexed again to ensure proper shaking and mixing. Centrifugation of the samples was performed at 13,000 rpm for 5 minutes. The supernatant was isolated and used for further analysis. Aluminium chloride colourimetric method (Haq et al. 2012) was used for TFC determination. Quercetin derived standard curve was used as a reference for TFC determination. Extracts were subjected to UV spectroscopy for determination of TFC at 415 nm, using UV–Vis spectrophotometer (Halo DR-20, UV–Vis spectrophotometer, Dynamica Ltd., Victoria, Australia). Total flavonoid production (TFP) was calculated by using the following formula and expressed in mg /l.

TFP (mg/l) =DW (g/l) ×TFC (mg/g)

TPP (mg/l) =DW (g/l) ×TPC (mg/g)

TPC and TFC values were expressed in milligram equivalents per gram dry weight of calli.

3.3.7. Free Radical Scavenging Assay The method of Lee et al. (1998) was adopted to determine the free radical scavenging potential of samples spectrophotometrically at 517 nm using 1,1-diphenyl-2- picrylhydrazyl (DPPH). Following equation was used to calculate the DPPH free radical scavenging capacity of samples:

% scavenging= [(Absorbance of control - Absorbance of the sample)/Absorbance of control] ×100

3.3.8. HPLC Analysis HPLC technique was utilized to quantify the presence of some important lignans and neolignans in powdered calli samples. Only samples harvested on day 10, 15, 20, 25

64 Chemogenic silver nanoparticles enhance lignans and neolignans in cell suspension cultures of Linum usitatissimum L.

CHAPTER 3 and 30, respectively. were analyzed through HPLC. Varian liquid chromatographic system (composed of Varian Prostar 410 auto sampler, Metachem Degasit, and Varian Prostar 335 Photodiode Array Detector) was used for the quantification important secondary metabolites including SDG, LDG, DCG and GGCG as described by Fliniaux et al. 2014. Protocol devised by Corbin et al. (2015) was followed by ultrasonic extraction of samples. Simply, centrifugation of the samples was performed at 3000 rpm for 15 minutes followed by filtration using nylon syringe membrane (0.45 μm). Purospher (Merck) reverse phase (RP-18) column was utilized for separation, and detection was performed at 280 nm wavelength using UV–Vis spectrophotometer. The compounds were identified by comparing their retention times and UV spectra to those of reliable reference standards. The mobile phase consisted of two solvents, solvent A and solvent B which were 0.2% acetic acid aqua solution and methanol respectively. For mobile phase variation, a nonlinear gradient was applied with a flow rate of 0.8 ml/min as follows: from 0 to 40 min of A–B: 90:10 (v/v) to 30:70 (v/v), from 41 to 50 min of A–B: 30:70 (v/v) to 0:100 (v/v), and A–B: 0:100 (v/v) from 51 to 60 min (Corbin et al. 2015).

3.3.9. Statistical Analysis All experiments were carried out at once. Each treatment consisted of three replicates and repeated twice. Mean values of various treatments were subjected to analysis of variance by using Origin Pro software (8.5). Also, Duncan’s multiple range test (Windows version 7.5.1, SPSS Inc., Chicago) was used to determine the significance at P < 0.05 (Duncan 1955). Origin Pro software (8.5) was used for generation of all figures.

3.4. RESULTS AND DISCUSSION 3.4.1. Effects of Repeated Elicitation with Ag-NPs on Biomass Accumulation Presence of NPs in a plant growing medium causes a couple of systemic changes, sometimes characterized by a measurable or detectable component (Nair 2016). In current study, primary effects of NPs feeding during log phase of cell suspension cultures of Linum usitatissimum were the changes observed in biomass accumulation. We obtained several fold enhancements in maximum fresh and dry weight quantities, respectively, compared to control treatment (Fig. 3.2 and 3.3). Highest fresh weight (290 g/l, observed on day 25 of culturing) was obtained in cultures repeatedly elicited

65 Chemogenic silver nanoparticles enhance lignans and neolignans in cell suspension cultures of Linum usitatissimum L.

CHAPTER 3 on day 10 and 15 (Fig. 3.2), and dry weight (14.5 g/l, observed on day 20 of culturing) in cultures elicited on day 10 only (Fig. 3.3). Interestingly, feeding NPs at early log phase enhanced dry weight spontaneously, however maximum fresh weight accumulation was observed at later stages.

300 a a Control Day 10 250 Day 10+15 Day 10+20 b 200

b b b c 150

100 d d d d

Fresh weight (g/l) weight Fresh d d d

50 e e e e e

0 10 15 20 25 30 Days

Figure 3.2. Temporal effects of repeated elicitation with chemogenic Ag-NPs on fresh biomass of cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD.

The effects of Ag-NPs or source compound on plant growth are inconclusive. Few reports discussed the growth promoting effects of Ag-NPs (Das et al. 2018; Gupta et al. 2018; Sharma et al. 2012), while many publications have reported negative impacts (Li et al. 2018; Tripathi et al. 2017; Yin et al. 2011). It has also been observed that effects of Ag-NPs on plant growth are dose dependent i.e. lower concentrations promote plant growth and higher concentrations have inhibitory effects (Al-Huqail et al. 2018; Lee et al. 2012; Thuesombat et al. 2014). In current study, we have utilized lower concentrations of Ag-NPs. Navarro et al. (2008) has argued that interaction of plants with NPs may be positive or negative depending on plant type and characteristics

66 Chemogenic silver nanoparticles enhance lignans and neolignans in cell suspension cultures of Linum usitatissimum L.

CHAPTER 3 of NPs. Furthermore, this area has received the least attention so far, and requires detailed studies that would provide insight into such interactions.

16 a a a Control 14 Day 10 Day 10+15 12 Day 10+20 b

bc bc 10 bc

c c c 8 cd cd cd cd cd 6

Dry weight (g/l) Dryweight

4 d

e e e e 2

10 15 20 25 30 Days

Figure 3.3. Temporal effects of repeated elicitation with chemogenic Ag-NPs on dry biomass of cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD.

3.4.2. Effects of Repeated Elicitation with Ag-NPs on Phenolics and Flavonoids Content Accumulation of phenolic compounds is considered a front line element in antioxidative defense system against ROS. These compounds are commonly known as non-enzymatic antioxidants. Their production varies in different environments and can be upregulated adding foreign substances to the culture medium (Zahir et al. 2014). In current study, adding Ag-NPs on day 10 enhanced TPC and TPP (23.45 mg/g DW and 340 mg/l, respectively, observed on day 20 of culturing), (Fig. 3.4 and 3.5). This increase in production was almost 6.5 folds compared to control. Repeated elicitation at later stages (day 10 and 15, and day 10 and 20) had least additive effects than onset of log phase, however compared to control; repeated elicitation had marked differential

67 Chemogenic silver nanoparticles enhance lignans and neolignans in cell suspension cultures of Linum usitatissimum L.

CHAPTER 3 accumulation. Our findings are in agreement with studies of Chung et al. (2018) and Fazal et al. (2016), who demonstrated elicitation effects of Ag-NPs on phenolics accumulation in calli cultures of Prunella vulgaris L. and hairy root cultures of Cucumis anguria, respectively.

27 Control a a 24 Day 10 Day 10+15 ab 21 Day 10+20 b b

) b b 18 c c

/g DW

GAE

d d d

mg ( 12 de

TPC 9 e e

e 6 f f f f

3 10 15 20 25 30

Days

Figure 3.4. Temporal effects of repeated elicitation with chemogenic Ag-NPs on total phenolic content in cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD.

Contrasting results were obtained for TFC and TFP (Fig. 3.6 and 3.7). Elicitation generally tended to enhance flavonoids production in the first phase, but a major decline was observed in the later phase. Highest TFC and TFP (23.45 mg/g DW and 340 mg/l, respectively, observed on day 20 Of culturing) occurred when cultures were elicited on day 10 (Fig. 4A and B). Zhao et al. (2010a) suggested, adding elicitors at early phase of cell suspension cultures of Saussurea medusa promotes flavonoids biosynthesis, whereas decreases at later phase. Similar trends in phytochemicals accumulation (on addition of elicitor after 10 days of inoculation) have also been reported by Ketchum et al. (1999) and Moreno et al. (1996) in different cell suspension cultures, respectively. Furthermore, a positive correlation existed between FRSA (Fig. 3.8) and phenolics 68 Chemogenic silver nanoparticles enhance lignans and neolignans in cell suspension cultures of Linum usitatissimum L.

CHAPTER 3 content. A significant free radical scavenging activity is attributed to presence of a wide range of phytochemicals (Ahmad et al. 2013).

400

Control a a 350 Day 10 Day 10+15 300 Day 10+20 b

bc 250

c 200 c c 150 cd TPP (mg GAE/l) d d d d 100 e e e 50 ef f f f f 0 10 15 20 25 30

Days

Figure 3.5. Temporal effects of repeated elicitation with chemogenic Ag-NPs on total phenolic production in cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD.

69 Chemogenic silver nanoparticles enhance lignans and neolignans in cell suspension cultures of Linum usitatissimum L.

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14 Control a a 12 Day 10 Day 10+15 Day 10+20 10 b b b b b b b 8

c c c 6

d d TFC (mg QUE/g DW) 4 d

d d d d d 2

0 10 15 20 25 30 Days

Figure 3.6. Temporal effects of repeated elicitation with chemogenic Ag-NPs on total flavonoid content in cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD.

70 Chemogenic silver nanoparticles enhance lignans and neolignans in cell suspension cultures of Linum usitatissimum L.

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180 a a

Control 160 Day 10 140 Day 10+15 Day 10+20 120 b

100 bc

c c 80 c c c cd

TFP (mg QUE/l) 60

d d 40 e e e 20 f f f f f 0 10 15 20 25 30 Days

Figure 3.7. Temporal effects of repeated elicitation with chemogenic Ag-NPs on total flavonoid production in cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD.

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100 Control a a a a a Day 10 ab ab ab ab ab ab ab Day 10+15 80 Day 10+20 b b bc c c c c 60 c

% FRSA 40

0 10 15 20 25 30

Days

Figure 3.8. Temporal effects of repeated elicitation with chemogenic Ag-NPs on free radical scavenging assay of cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD.

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3.4.3. Effects of Repeated Elicitation with Ag-NPs on Production of Lignans and Neolignans Production of bioactive secondary metabolites is generally associated with plant defense mechanisms. Secondary metabolites consist of a range of structurally complex molecules, such as, alkaloids and polyphenols, depending on plant’s biochemistry. Their biosynthesis is stimulated when plants are exposed to unfavorable environment (Mulabagal and Tsay 2004; maVerma and Shukla 2015). Exposure of cell suspension cultures of Linum usitatissimum to Ag-NPs also triggered some significant changes in its polyphenols profile. HPLC analysis of some key lignans and neolignans exhibited a positive correlation with TPC. Optimum production of lignans (Fig. 3.9) and neolignans (Fig. 3.10) occurred on day 20 when cell suspension cultures were treated with Ag-NPs on day 10. Highly produced lignans and neolignans included SDG (252.75 mg/l) and DCG (248.20 mg/l), (Fig 3.9B and 3.10B), respectively. Secondary metabolites accumulation reaches to maximum, near end of log phase or during stationary phase when major nutrients are depleted in the medium (Heble and Staba 1980; Phillips and Henshaw 1977; Zhao et al. 2010b). Similar observations were also made in our study in control treatments, however a linear relationship of polyphenols accumulation was observed throughout log phase in Ag-NPs treated cultures, possibly as a result of activation of cell’s signal transduction system, stimulating early and enhanced biosynthesis of polyphenols (Ramirez-Estrada et al. 2016).

3.5. Conclusions Cell suspension cultures are versatile in vitro plant culturing practices, enabling researchers to manipulate growing conditions for scaling up biomass accumulation and bioactive metabolites production. Current study adapted elicitation mechanism as a tool, for early and enhanced biomass and secondary metabolites production in cell suspension cultures of Linum usitatissimum. Generally, plant secondary metabolites are accumulated during late growth phases, but presence of elicitors can stimulate biosynthesis at early stages as a counter defense mechanism. This is a convenient and an economical approach towards secondary metabolites production. We successfully applied chemogenic Ag-NPs as an elicitor, and obtained significantly higher amounts of lignans and neolignans in a short course of time. Our findings suggest extending in vitro application of metallic NPs to enhance novel phytochemical accumulation across a wide range of important medicinal plants. This strategy seems a modern, feasible,

73 Chemogenic silver nanoparticles enhance lignans and neolignans in cell suspension cultures of Linum usitatissimum L.

CHAPTER 3 economical and pragmatic approach to address ever-growing needs of plant derived natural compounds.

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A B 20 300 Control Control 18 a a Day 10 Day 10 a a Day 10+15 250 16 Day 10+15 Day 10+20 Day 10+20 14 b 200 b b b b 12

10 150 b

8 c c b b b 100 b 6

SDG SDG content (mg/g) cd SDG SDG production (mg/l) c 4 d 50 c c d d d d d c 2 d d c d d d d d d d d d 0 0 C D 6 80

a a a a 70 5 60 4 ab 50 b b b

3 b 40

c c b 30 b b b 2 b

LDG content (mg/g) d d d d d

LDG production (mg/l) 20 d d c de 1 d d d d d d d de de e e e 10 e e e e 0 0 10 15 20 25 30 10 15 20 25 30 Days Days

Figure 3.9. Temporal effects of repeated elicitation with chemogenic Ag-NPs on A; secoisolaricerisinol diglucoside content (SDG), B; SDG production, C; laricerisinol diglucoside (LDG) content and D; LDG production in cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD. 75 Chemogenic silver nanoparticles enhance lignans and neolignans in cell suspension cultures of Linum usitatissimum L.

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A B 300 18 a a Control Control Day 10 16 Day 10 a a Day 10+15 250 Day 10+15 Day 10+20 14 Day 10+20 b 200 12 b b b bc 10 bc bc 150 8 c c c c c c c c c 6 100 d d d d cd

DCG content DCG (mg/g) d DCG productionDCG (mg/l) d d d d d d d 4 d 50 2 de e e e e 0 0 C 5.0 40 D

4.5 a a 35 4.0 a 30 b 3.5 a b 25 3.0 b b b 2.5 b b 20

2.0 bc bc bc bc bc bc bc 15 c 1.5 c c c c c c

GCGG content (mg/g) 10

GCGG production (mg/l) 1.0 cd d d d d d d d d 5 d d d d 0.5 d e 0.0 0 10 15 20 25 30 10 15 20 25 30 Days Days

Figure 3.10. Temporal effects of repeated elicitation with chemogenic Ag-NPs on A; dehydrodiconiferyl alcohol glucoside (DCG) accumulation, B; DCG production, C; guaiacylglycerol-b-coniferyl alcohol ether glucoside (GGCG) accumulation, and D; GGCG production in cell suspension cultures of Linum usitatissimum. values are mean of triplicates ± SD.

76 Chemogenic silver nanoparticles enhance lignans and neolignans in cell suspension cultures of Linum usitatissimum L.

CHAPTER 4

4. BIOGENIC ZINC OXIDE NANOPARTICLES ENHANCE PRODUCTION OF LIGNANS AND NEOLIGNANS IN CELL SUSPENSION CULTURES OF LINUM USITATISSIMUM L.

4.1. ABSTRACT Zinc oxide nanoparticles have emerged as a novel elicitor for enhanced biosynthesis of secondary metabolites in in vitro established plant cell cultures in recent times. For this reason, current study was aimed to explore elicitation abilities of zinc oxide nanoparticles for enhanced accumulation of polyphenols—lignans and neolignans— in cell suspension cultures of Linum usitatissimum, which is a famous commercial and medicinal plant in the family Linaceae. We optimized concentration of zinc oxide nanoparticles before carrying out a full-fledged experiment. Subsequently, an optimum dose of 100 µg/l was introduced into the culture medium on day 0, day 0 and 15, and finally day 0 and 25. We observed that repeated elicitation positively affected various growth parameters and stimulated physiological responses in Linum usitatissimum cell suspension cultures as compared to one-time elicitation (day 0 only). Repeated elicitation of cell suspension cultures on day 0 and 15 resulted in highest fresh weight (412.16 g/l) and lignans production (secoisolariciresinol diglucoside 284.12 mg/l: lariciresinol diglucoside 86.97 mg/l). Contrarily, repeated elicitation on day 0 and 25 resulted in highest dry biomass (13.53 g/l), total phenolic production (537.44 mg/l), total flavonoid production (123.83 mg/l) and neolignans production (dehydrodiconiferyl alcohol glucoside 493.28 mg/l: guaiacylglycerol-β-coniferyl alcohol ether glucoside 307.69 mg/l). Enhancement of plant growth and secondary metabolites accumulation was several fold greater than the control treatments. Furthermore, a linear relationship existed between total phenolic and flavonoid contents which in turn was correlated with higher antioxidant activities.

78 Biogenic zinc oxide nanoparticles enhance production of lignans and neolignans in cell suspension cultures of Linum usitatissimum L. CHAPTER 4

4.2. INTRODUCTION Linum usitatissimum, a novel plant belonging to the family Linaceae, has a rich history regarding industrial applications —linen fiber production and linseed oil extraction— however, currently it is increasingly exploited as a great source of some health promoting phenolic metabolites known as lignans and neolignans (Muir and Westcott 2003). These metabolites are naturally occurring polyphenols, having potential therapeutic applications, mainly against different types of cancer, some microbial infections and inflammation as well. Major lignans are secoisolariciresinol diglucoside (SDG) and lariciresinol diglucoside (LDG), whereas dehydrodiconiferyl alcohol glucoside (DCG) and guaiacylglycerol-β-coniferyl alcohol ether glucoside (GGCG) are well-known neolignans (Zahir et al. 2018a). Both lignans and neolignans share similar structural conformation, but they can be easily characterized by difference in (a minor but significant) bonding pattern. Generally, lignans consist of phenyl propane dimers, that are bonded through the carbon atoms (C8/ C8՜) of their side chains whereas in neolignans, these dimers are connected through bond other than C8/C8՜ (Wallis 1998). A variety of established techniques and methods exist for commercial production of phytomedicines. Among these, biotechnology based plant cell cultures are superior and modern methods, employed for extraction and isolation of phytomedicinal products. However, certain modifications and adaptations to such a system on the whole or some operational conditions, are essential for enhanced accumulation of plant derived secondary metabolites because production potential of such systems is intrinsically low, therefore they need stimulation (Murthy et al. 2014). For this purpose, elements of diverse nature are used as stimulatory agents, which elicit enhanced biosynthesis of secondary metabolites in in vitro plant cell cultures. These agents include elements from both biotic and abiotic sources (Namdeo 2007). Nanoparticles (NPs) are emerging abiotic elicitors used for obtaining improved yields of plant derived medicinal compounds, such as recently, we have reported successful elicitation of Linum usitatissimum cell suspension cultures with chemogenic silver NPs (Zahir et al. 2018b). Generally, NPs have distinct physicochemical properties that enable them to interact with plant metabolic systems in a conducive way, rarely observed while using source/candidate material in raw form (Fazal et al. 2016; Zaka et al. 2016), thus current study was also aimed along the same lines to enhance accumulation of lignans and neolignans in cell suspension cultures of Linum usitatissimum using biogenic ZnO-NPs as an abiotic elicitor. 79 Biogenic zinc oxide nanoparticles enhance production of lignans and neolignans in cell suspension cultures of Linum usitatissimum L. CHAPTER 4

4.3. Materials and methods 4.3.1. Inoculum preparation Callus cultures were established on MS medium as described previously (Zahir et al. 2018a). in vitro seeds derived aseptic plantlets (4 weeks old) were used as source for stem explants. Explants of ~1 cm size were inoculated in a 100 ml Erlenmeyer flasks, on medium containing 1 mg/l NAA, for 30 days (Fig. 4.1A). Inoculum for cell suspension culture was prepared by modified method of (Anjum et al. 2017). simply, ~3 g callus was inoculated into 100 ml liquid MS medium in 250 ml Erlenmeyer flasks (Fig. 4.1B), containing 30 g/l sucrose, 4.43 g/l salt mixture and 1 mg/l NAA. pH of the culture media was set at 5.6 prior to autoclaving. These flasks were placed in a shaking incubator (120 rpm) for 15 days in 16/8h light and dark photoperiod at 25±2 °C.

4.3.2 Characteristics of ZnO-NPs ZnO-NPs were prepared and reported by our group earlier (Abbasi et al. 2017). Biogenic process, an ecofriendly approach was adapted for green synthesis of ZnO- NPs. Root extract of Linum usitatissimum (10g/100 ml) and zinc nitrate hexahydrate

(0.1 mM) were added in a 1:1 proportion. This mixture was heated at 60 °C for 3 h under continuous stirring. Formation of ZnO-NPs was noted by precipitation of yellow particulates and confirmed by UV-visible spectroscopy. XRD was employed for calculating particles size and texture, while, TEM revealed the shape. Resultant NPs obtained were hexagonal in shape, having crystalline appearance, and average size was up to 35 nm. The stability of ZnO-NPs was confirmed using UV-visible spectroscopy (absorbance value of 2.5 and peak value between 230-800 nm were used as reference parameters), before its application.

4.3.3. Optimization of ZnO-NPs Concentration Current experiment spanned for a total of 40 days. Initially, we optimized ZnO-NPs concentration for elicitation of cell suspension cultures of Linum usitatissimum. Different concentrations of biogenic ZnO-NPs i.e. 1, 5, 10, 20, 30, 40, 50, 75, 100, 200 and 500 µg/l, respectively, were added to cell cultures at day zero. Optimization and subsequent experiments were carried out in 100 ml Erlenmeyer flasks containing 30 ml liquid MS medium and 10 ml inoculum.

4.3.4. ZnO-NPs Treatments Among the various concentrations tested for optimization, 100 µg/l ZnO-NPs showed optimum biomass accumulation (FW and DW) on day 25, therefore, it was selected for 80 Biogenic zinc oxide nanoparticles enhance production of lignans and neolignans in cell suspension cultures of Linum usitatissimum L. CHAPTER 4 further experimentation. For repeated elicitation of cell suspension cultures, different periodic intervals were specified for addition of ZnO-NPs to cell suspension cultures. Cell cultures were fed with ZnO-NPs on three occasions, initially on day 0, then on day 0 and 15, and finally day 0 and 25 (Fig. 4.1C). Cells were harvested at 5 days interval after each feeding step until day 40 of the cell suspension culture. Cell cultures without addition of NPs were used as control treatment.

Figure 4.1. A; in vitro established callus culture of Linum usitatissimum. B; Inoculum for cell suspension cultures of Linum usitatissimum, derived from in vitro established callus culture. C; Cell suspension cultures of Linum usitatissimum fed at day 0, day 0 and 15, and day 0 and 25, respectively.

4.3.5. Biomass Determination Cells harvested at different intervals were lightly pressed between layers of filter papers to remove excessive water. Fresh weight was determined directly; using weighing balance, whereas, cells were oven dried at 50 °C until for 24 hours and then dry weight was recorded.

4.3.6. Phytochemical Analysis For determination of both total phenolic and flavonoid content (TPC and TFC), we added 500 μl methanol (100 % pure) to 100 mg powdered dry calli, followed by vortexing and then sonication for 30 minutes. The mixture was vortexed again to ensure proper shaking and mixing. Centrifugation of the samples was performed at 13,000 rpm for 5 minutes. The supernatant was isolated and used for further analysis. Aluminium chloride colourimetric method (Haq et al. 2012) was used for TFC determination. Quercetin derived standard curve was used as a reference for TFC determination. Extracts were subjected to UV spectroscopy for determination of TFC at 415 nm, using UV–Vis spectrophotometer (Halo DR-20, UV–Vis spectrophotometer, Dynamica Ltd., Victoria, Australia). Total flavonoid production (TFP) was calculated by using the following formula and expressed in mg /l.

81 Biogenic zinc oxide nanoparticles enhance production of lignans and neolignans in cell suspension cultures of Linum usitatissimum L. CHAPTER 4

TFP (mg/l) =DW (g/l) ×TFC (mg/g)

TPP (mg/l) =DW (g/l) ×TPC (mg/g)

TPC and TFC values were expressed in milligram equivalents per gram dry weight of calli.

4.3.7. Free Radical Scavenging Assay The method of Lee et al. (1998) was adopted to determine the free radical scavenging potential of samples spectrophotometrically at 517 nm using 1,1-diphenyl-2- picrylhydrazyl (DPPH). Following equation was used to calculate the DPPH free radical scavenging capacity of samples:

% scavenging= [(Absorbance of control - Absorbance of the sample)/Absorbance of control] ×100

4.3.8. HPLC Analysis HPLC technique was utilized to quantify the presence of some important lignans and neolignans in powdered calli samples. Only samples harvested on day 15, 20, 25, 30, 35 and 40, respectively, were analyzed through HPLC. Varian liquid chromatographic system (composed of Varian Prostar 410 auto sampler, Metachem Degasit, and Varian Prostar 335 Photodiode Array Detector) was used for the quantification important secondary metabolites including SDG, LDG, DCG and GGCG as described by Fliniaux et al. 2014. Protocol devised by Corbin et al. (2015) was followed by ultrasonic extraction of samples. Simply, centrifugation of the samples was performed at 3000 rpm for 15 minutes followed by filtration using nylon syringe membrane (0.45 μm). Purospher (Merck) reverse phase (RP-18) column was utilized for separation, and detection was performed at 280 nm wavelength using UV–Vis spectrophotometer. The compounds were identified by comparing their retention times and UV spectra to those of reliable reference standards. The mobile phase consisted of two solvents, solvent A and solvent B which were 0.2% acetic acid aqua solution and methanol respectively.

82 Biogenic zinc oxide nanoparticles enhance production of lignans and neolignans in cell suspension cultures of Linum usitatissimum L. CHAPTER 4

For mobile phase variation, a nonlinear gradient was applied with a flow rate of 0.8 ml/min as follows: from 0 to 40 min of A–B: 90:10 (v/v) to 30:70 (v/v), from 41 to 50 min of A–B: 30:70 (v/v) to 0:100 (v/v), and A–B: 0:100 (v/v) from 51 to 60 min (Corbin et al. 2015).

4.3.9. Statistical Analysis All experiments were carried out at once. Each treatment consisted of three replicates and repeated twice. Mean values of various treatments were subjected to analysis of variance by using Origin Pro software (8.5). Also, Duncan’s multiple range test (Windows version 7.5.1, SPSS Inc., Chicago) was used to determine the significance at P < 0.05 (Duncan 1955). Origin Pro software (8.5) was used for generation of all figures and tables.

4.4. Results and Discussion 4.4.1. Effects of Elicitation with ZnO-NPs on Biomass Accumulation Physiological responses are the first key changes which occur when plants are exposed to elements of diverse nature. NPs trigger plant physiological response in both positive and negative ways (Gupta et al. 2018; Tripathi et al. 2017), primarily depending on concentration of NPs and plant species (Navarro et al. 2008; Thuesombat et al. 2014). Generally, application of NPs in lower concentrations has been found promoting plant growth and secondary metabolites production (Karimi et al., 2018). We observed positive impacts of ZnO-NPs on biomass accumulation in cell suspension cultures of Linum usitatissimum, as we obtained maximum fresh weight, 412.16 g/l — on day 35 after initial culturing when cell suspension cultures were repeatedly elicited on day 0 (Fig. 4.2) and 15— and dry weight, 13.53 g/l —on day 30 after initial culturing when cell suspension cultures were repeatedly elicited on day 0 and 25— (Fig. 4.3), respectively. Our observations are in accordance with the raising perspective on using ZnO-NPs as a potential nanofertilizer, which is gaining wide acceptance because of the increasing reports on their positive impacts on plant growth (Khanm et al. 2018; Panwar 2012). These reports described growth promoting effects of ZnO-NPs as a result of effective physiological changes including regulation of plant pigments directly associated with biomass buildup such as chlorophyll and carotenoids (Latef et al. 2017; Tarafdar et al. 2014); increase in nutrients uptake efficiency (Chanu and Upadhyaya 2019), upregulation of antioxidant metabolism (Venkatachalam et al. 2017) and most

83 Biogenic zinc oxide nanoparticles enhance production of lignans and neolignans in cell suspension cultures of Linum usitatissimum L. CHAPTER 4 important, involvement of zinc as an essential micronutrient in metabolism of different plant biomolecules (Bonnet et al. 2000).

450 a 400 Control Day 0 ab ab 350 Day 0+15 Day 0+25 b b 300 bc bc

250

c 200

150 d d

Fresh g/l weight d d d d d de de 100 e e e e e e e

50 f f f f f f f f

0 5 10 15 20 25 30 35 40

Days Figure 4.2. Temporal effects of repeated elicitation with biogenic ZnO-NPs on fresh biomass of cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD.

84 Biogenic zinc oxide nanoparticles enhance production of lignans and neolignans in cell suspension cultures of Linum usitatissimum L. CHAPTER 4

14 Control a Day 0 Day 0+15 ab ab ab 12 ab Day 0+25 ab

10 b b b b

8 bc bc c c 6 c c c c

Dry g/l weight c c c 4 cd cd d d d d e 2 e e e e

0 5 10 15 20 25 30 35 40 Days Figure 4.3. Temporal effects of repeated elicitation with biogenic ZnO-NPs dry biomass of cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD.

4.4.2 Effects of Repeated Elicitation with ZnO-NPs on Phenolics and Flavonoids Content

Accumulation of phenolics and flavonoids in response to novel substances as a primary defense line is the principal indicator of active plant secondary metabolism Zahir et al. (2014). Linum usitatissimum contains a wide range of phenolics and some flavonoids. In current study, we observed similar trends for both phenolics and flavonoids accumulation, we noted that one-time elicitation had not impacted phenolic and flavonoid contents but repeated addition of ZnO-NPs into culture medium, especially at a later stage of culture —log phase— significantly enhanced both total phenolic and flavonoid content/production, respectively, as compared to control treatments. Optimum TPC (39.71 mg GAE/g) and TPP (537.44 mg/l) occurred on day 30 after initial culturing when cell suspension cultures were repeatedly elicited on day 0 and 25 (Fig 4.4 and 4.5). This increase in phenolics production was 15.8 fold as compared to

85 Biogenic zinc oxide nanoparticles enhance production of lignans and neolignans in cell suspension cultures of Linum usitatissimum L. CHAPTER 4 control. Optimum TFC and TFP also occurred during the same period. Highest TFC and TFP were recorded to be 9.15 mg QUE/g and 123.83 mg/l (Fig 4.6 and 4.7), respectively. This increase in flavonoids production was 8.8 fold as compared to control. This increase in flavonoids production was 8.8 fold as compared to control. These results are in agreement with our previous finding (Zahir et al. 2018b), wherein similar trends in total phenolic and falvonoid contents were observed.

42 Control a Day 0 39 Day 0+15 b 36 Day 0+25 33 bc

) 30 c c c c c c 27

/g DW c c c c cd cd cd c c 24 cd

GAE 21 d

( d d d 18 d 15

TPC

12 e e e e 9 e e 6 f f 3 5 10 15 20 25 30 35 40

Days Figure 4.4. Temporal effects of repeated elicitation with biogenic ZnO-NPs on total phenolic content in cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD.

86 Biogenic zinc oxide nanoparticles enhance production of lignans and neolignans in cell suspension cultures of Linum usitatissimum L. CHAPTER 4

600 Control a Day 0 500 Day 0+15 Day 0+25 ab 400 ab

b b b 300

c c 200 c c

TPP (mg GAE/l) c c c c c d 100 cd cd cd cd cd cd d d d d d d d d e e 0 5 10 15 20 25 30 35 40 Days

Figure 4.5. Temporal effects of repeated elicitation with biogenic ZnO-NPs on total phenolic production in cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD.

87 Biogenic zinc oxide nanoparticles enhance production of lignans and neolignans in cell suspension cultures of Linum usitatissimum L. CHAPTER 4

10 a Control Day 0 b 8 Day 0+15 b Day 0+25

) c 6 c c c c c cd

/g DW cd cd cd cd cd

QUE d d 4 d d d d d d

( d d d de de de

TFC e 2 e

0 5 10 15 20 25 30 35 40 Days

Figure 4.6. Temporal effects of repeated elicitation with biogenic ZnO-NPs on total flavonoid content in cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD.

88 Biogenic zinc oxide nanoparticles enhance production of lignans and neolignans in cell suspension cultures of Linum usitatissimum L. CHAPTER 4

140

a Control 120 Day 0 Day 0+15 100 Day 0+25 b b

60 c c c c c c c

TFP (mg QUE/l) 40

d d d d d de de de de de 20 e e e e e e e e e e e e 0 5 10 15 20 25 30 35 40 Days

Figure 4.7. Temporal effects of repeated elicitation with biogenic ZnO-NPs on total flavonoid production in cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD.

89 Biogenic zinc oxide nanoparticles enhance production of lignans and neolignans in cell suspension cultures of Linum usitatissimum L. CHAPTER 4

4.4.3. Effects of Repeated Elicitation with ZnO-NPs on Free Radical Scavenging Capacity Increased polyphenol accumulation is indicated by higher antioxidant activities (Ahmad et al. 2013). A positive correlation between FRSA and phenolic content was recorded in current study. Maximum FRS activity observed was up to 90 % in repeatedly elicited cultures (Fig. 4.8). Generally, repeated elicitation resulted in enhanced FRS activity at last stages of culture as compared to control. Our results are supported by the findings of García-Lopez et al. (2018) and Zafar et al. (2016) who successfully enhanced phenolics and flavonoids, and also observed maximum antioxidant activities as a result of applying ZnO-NPs during germination of Capsicum annum and stem explants response of Brassica nigra, respectively. Significant changes in all the growth parameters as compared to control were reported in these studies.

Control 100 Day 0 ab ab a ab Day 0+15 ab ab a a ab ab a ab ab ab a a Day 0+25 80 ab ab ab ab ab ab ab ab ab ab ab ab b 60 b b

FRSA % FRSA 40

0 5 10 15 20 25 30 35 40

Days

Figure 4.8. Temporal effects of repeated elicitation with biogenic ZnO-NPs on free radical scavenging assay of cell suspension cultures of Linum usitatissimum. Values are mean of triplicates ± SD.

90 Biogenic zinc oxide nanoparticles enhance production of lignans and neolignans in cell suspension cultures of Linum usitatissimum L. CHAPTER 4

4.4.4. Effects of Repeated Elicitation with Ag-NPs on Production of Lignans and Neolignans Elicitation of cell suspension cultures of Linum usitatissimum with ZnO-NPs also enhanced accumulation of the secondary metabolic compounds lignans and neolignans. This accumulation correlated with the total phenolic content, as both lignans and neolignans are also polyphenols by nature. We obtained highest lignans content/production (SDG 25.01 mg/g and 284.12 mg/l: LDG 7.65 mg/g and 86.97 mg/l) after 25 days of initial culturing when cell suspension cultures were repeatedly treated with ZnO-NPs on day 0 and 15; whereas highest neolignans content/production (DCG 36.44 mg/g and 493.28 mg/l: GGCG 22.73 mg/g and 307.69 mg/l) occurred after 30 days of initial culturing when cell suspension cultures were repeatedly treated with ZnO-NPs on day 0 and 25. The mechanism underlying enhanced production of secondary metabolites in response to ZnO-NPs is still unclear but a number of reports are available on their role as a potential abiotic elicitor during in vitro cultures such as Sharafi et al. (2013), Karimi et al. (2018) and Javed et al. (2017). Bhardwaj et al. (2018) made an attempt to decipher the transcriptional regulation of ZnO-NPs induced enhanced accumulation of bacoside A content in cell suspension cultures of Bacopa monnieri. They suggested that reduced transcriptional levels of HMG-CoA reductase gene in response to ZnO-NPs might be a result of diverging biosynthetic pathway from mevalonate to isoprenoid pathway. Furthermore, Mosavat et al. (2019) have recently reported elicitation of thymol in callus cultures of Thymus kotschyanus and carvacrol in callus cultures of Thymus daenesis under ZnO-NPs stress.

4.5. Conclusions Biotechnological approach towards enhanced production of commercially important secondary metabolites from a wide range of medicinal plants is so far the best methodology as it offers flexibility in terms of manipulating growing/operational conditions and reproducibility. Application of ZnO-NPs in cell suspension cultures of Linum usitatissimum proved a simple and effective procedure to enhance biomass and most important, accumulation of polyphenols—lignans and neolignans— in a short course of time, possibly as a result of ZnO-NPs induced metabolic shuffling. Results obtained during this study support the positive role of ZnO-NPs as an elicitor during in vitro established cultures of a variety of medicinal plants.

91 Biogenic zinc oxide nanoparticles enhance production of lignans and neolignans in cell suspension cultures of Linum usitatissimum L. CHAPTER 4

Table 4.1. Temporal effects of repeated elicitation with biogenic ZnO-NPs on content of DCG; dehydrodiconiferyl alcohol glucoside, GGCG; guaiacylglycerol-b-coniferyl alcohol ether glucoside, SDG; secoisolariciresinol diglucoside and LDG; lariciresinol diglucoside.

Harvesting Time (Days) Elicitation Stages Control Day 0 Day 0 and 15 Day 0 and 25 Neolignans content Lignans content Neolignans content Lignans content Neolignans content Lignans content Neolignans content Lignans content

DCG GGCG SDG LDG DCG GGCG SDG LDG DCG GGCG SDG LDG DCG GGCG SDG LDG

2.02± 6.49± 2.15±0.02 0.95±0.01 0.276±0.00 0.78±0.01 0.43±0.10 0.27±0.10 0.953±0.01 0.78±0.02 0.43±0.00 15 1.59±0.04e 0.04 de 0.95±0.03e 0.78±0.02e 0.43±0.0 d 0.27±0.00d 0.13d bc e 3d e d d e e d

7.74± 3.32±0.05 4.27± 8.63±0.13 0.249±0.00 0.80±0.01 0.73±0.02 12.60±0.39 1.79±0.05 17.47±0.54 0.74±0.00 0.24±0.00 0.80±0.00 0.73±0.02 20 3.87±0.12b 8.63±0.01c 0.15d b 0.13 d d 5d e d b c b d d e d

10.36 11.85 3.68±0.05 2.05±0.04 1.40±0.02 0.512±0.01 0.65±0.01 1.53±0.03 15.75±0.98 3.25±0.10 25.01±1.17 7.65±0.23 0.51±0.001 0.65±0.00 25 ± ± 1.40±0.01c 1.53±0.04c b bc e 5d e c b b a a d e 0.09c 0.23 c 6.77± 2.35±0.04 6.51± 7.04±0.10 0.630±0.02 0.54±0.07 0.17±0.03 0.79±0.01 2.48±0.07d 3.41±0.10 36.44±0.21 22.73±0.16 1.86±0.12 1.74±0.01 30 1.25±0.04c 7.78±0.44c 0.13d be 0.18 d d 0d e d d e b a a e bc

4.86± 2.22±0.08 3.76± 1.85±0.02 0.81±0.01 0.240±0.00 0.10±0.02 0.13±0.00 1.18±0.01 0.88±0.02 1.18±0.01 35 0.57±0.02e 0.42±0.01e 1.51±0.15c 0.78±0.03d 1.28±0.01c 0.09d be 0.07 de bc e 7d e d c d e

2.96± 4.65± 2.37±0.01 1.97±0.08 0.56±0.10 0.067±0.00 0.02±0.00 0.13±0.00 0.23±0.01 0.52±0.02 0.06±0.00 0.17±0.00 40 0.01 de 9.20±0.28c 0.30±0.10e 0.07±0.00e 0.09±0.00d 0.02d be bc e 2d e d d d e d

92 Biogenic zinc oxide nanoparticles enhance production of lignans and neolignans in cell suspension cultures of Linum usitatissimum L. CHAPTER 4

Table 4.2. Temporal effects of repeated elicitation with biogenic ZnO-NPs on production of DCG; dehydrodiconiferyl alcohol glucoside, GGCG; guaiacylglycerol-b-coniferyl alcohol ether glucoside, SDG; secoisolariciresinol diglucoside and LDG; lariciresinol diglucoside.

Elicitation Stages

Harvesting Time (Days) Control Day 0 Day 0 and 15 Day 0 and 25 Neolignans Neolignans production Lignans production Lignans production Neolignans production Lignans production Neolignans production Lignans production production

DCG GGCG SDG LDG DCG GGCG SDG LDG DCG GGCG SDG LDG DCG GGCG SDG LDG

2.23±0. 1.41±0.0 2.23±0.0 4.00±0.2 24.36±0. 5.99±0.09 8.09±0.0 4.86± 1.41±0.0 4.00±0. 4.86±0.1 4.00±0.12 4.86±0.04 1.41±0.01 2.23±0.0 15 7.60±0.15de 04e 4e 2e 5e 48d c 8c 0.07e 1d 06e 5e e e d 2e

19.64±1. 8.18±0.5 3.59±0.0 50.51±1. 21.65±0.3 27.893±0.55 25.24±0. 38.75± 1.11±0.0 3.36±0. 3.29±0. 137.83± 191.02±4. 38.75±0.0 1.11±0.09 3.29±0.1 20 57bc 8c 3e 01c 2b 8d 78b 0.58d 1d 25e 06b 6.7b 12b 3d d 2e

36.98±1. 86.97±2. 3.35±0.0 76.57±1. 27.384±0. 88.060±1.76 15.27±0. 7.15± 2.61±0.0 3.35±0. 7.84±0. 179±3.4 284.12±6. 7.15±0.07 2.61±0.02 7.84±0.2 25 14b 69a 1e 53c 71b 1c 67bc 0.10e 2d 05e 15c 4b 80a e d 3c

5.49±0. 9.61±0.3 41.54±1. 25.28± 42.05±0. 14.64±0.2 40.439±1.08 7.79±0.1 61.40±2. 4.77±0. 1.51±0. 94.74±1. 30.26±1.9 493.28±2. 307.69±2. 23.57±0. 30 17d 4c 28ab 1.51d 84d 9c 9d 4c 92c 09e 02e 9c 3d 96a 46a 19b

1.99±0. 9.56±0.2 13.55±0. 14.09±0. 6.45±0.22 10.916±0.81 5.38±0.0 6.74±0.3 0.88±0. 1.11±0. 6.16±0.2 12.77±0. 4.57±0.14 17.31±0.7 9.03±0.45 14.74±0. 35 06d 6c 13de 28d c 8de 5c 0e 03e 01e 9e 08c e 7d c 18bc

0.38±0. 0.77±0. 4.59±0.5 0.60±0.0 17.20±0. 8.80±0.17 10.983±0.0 7.30±0.2 3.23±0.0 0.13±0. 80.41±3. 2.06±0.1 2.62±0.04 0.70±0.07 0.83±0.04 1.57±0.0 40 01d 01e 4c 6e 34d c 44de 9c 4e 02e 49c 0d e e d 3e

93 Biogenic zinc oxide nanoparticles enhance production of lignans and neolignans in cell suspension cultures of Linum usitatissimum L.

CHAPTER 5

5. CONCLUSIONS AND FUTURE PROSPECTS

5.1. CONCLUSIONS The present study was conducted in correlation to the available literature on in vitro propagation and secondary metabolites production in callus/cell cultures of Linum usitatissimum. After thorough review and analysis, we opinionate that rapid multiplication and readily availability of germplasm is a prerequisite to a successful plant breeding and in vitro culturing program. in vitro culturing techniques play an important role to help scientists, farmers and researchers to achieve novel cultivars and extract medicinal compounds in a cost effective process, short time and above all in vast quantities under controlled conditions. The in vitro propagation studies conducted on Linum usitatissimum has provided promising results for obtaining plants, not only with desirable traits but also for rapid multiplication and production of novel secondary metabolites. There are no real constraints either genotypic, phenotypic or biochemical that could undermine in vitro culturing of Linum usitatissimum, however pursuit for the best is always on and researchers are working on optimization of various factors that influence a regeneration and production scheme. The most prominent factor that could be taken into consideration during in vitro studies is the genotypic response, as it has been observed mostly with organogenesis and anther cultures establishment, that physical parameters, growth conditions and productivity varied according to the specific genotype being employed. Somatic embryogenesis on the other hand is still an unreliable method because the existing techniques are deemed inefficient and suggestions came for the exploitation of more sophisticated and advanced molecular techniques to examine the underlying mechanisms involved in embryogenesis, thus it may help to resolve the disagreement that exists among researchers.

Secondary metabolites production in in vitro cultures of Linum usitatissimum is attracting the scientific fraternity because these bioactive compounds are being considered as best alternative to the expensive, structurally complex and insufficiently available compounds. Linum usitatissimum cell/callus culture provides a feasible production system for the extraction of novel anticancer compounds which is evident from the successful demonstration of enhanced accumulation of several lignans and neolignans in vitro by many scientists, which are unable to achieve in vivo. Furthermore, metabolic engineering and molecular manipulations have enabled

researchers to express certain metabolites that are not present actually in Linum usitatissimum such as certain types of carotenoids. These reports encourage scientists to keep continuing the efforts for obtaining novel flax compounds through development of a system which is economical, reproducible and sustainable. Following the same approach, we also devised our current research work in similar fashion and made a successful attempt to enhance biomass accumulation and secondary metabolites production in in vitro established cultures of Linum usitatissimum. Our main findings are described below;

 In the first experiment, we evaluated the synergistic and differential effects of nutrient medium composition and photoperiod treatments on growth and secondary metabolites accumulation in callus cultures of Linum usitatissimum. Among the various media tested, Gamborg B5 medium produced optimum biomass accumulation (fresh biomass 413 g/l and dry biomass 20.7 g/l) and secondary metabolites content (secoisolariciresinol diglucoside 6.33 lariciresinol diglucoside 5.22, dehydrodiconiferyl alcohol glucoside 44.42 and guaiacylglycerol-β-coniferyl alcohol ether glucoside 9.26 mg/g dry weight, respectively). It was observed during this study that several nutrient medium qualities such as ammonium to nitrate ratio, total nitrogen content, phosphate levels and most important boron content impact growth response and polyphenols accumulation in callus cultures of Linum usitatissimum. Photoperiod treatments in many combinations also regulated biochemical profile of Linum usitatissimum, dark treatment slowed down the growth while presence of light was influential in biomass accumulation.  In the second experiment, Ag-NPs were supplemented into the culture medium to enhance biomass and polyphenols accumulation in cell suspension cultures of Linum usitatissimum. Inoculation stages were determinant in enhancing effects, especially feeding of Ag-NPs during log phase of cell suspension cultures provided best results. Feeding Ag-NPs on day 10 resulted in highest growth associated parameters (dry biomass; 14.5 g/l, secoisolariciresinol diglucoside, 252.75; lariciresinol diglucoside, 70.70; dehydrodiconiferyl alcohol glucoside, 248.20 and guaiacylglycerol-β-coniferyl alcohol ether glucoside, 34.76 mg/l, respectively)

 In the third experiment, we applied biogenic ZnO-NPs as an elicitor to evaluate its enhancing effects on growth and secondary metabolism in cell suspension cultures of Linum usitatissimum. We observed that repeated inoculation of ZnO- NPs was more effective as compared to one-time inoculation. Repeated inoculation on two occasions viz. day 0 and 15, and day 0 and 25 resulted in optimum growth and secondary metabolic activities. Repeated elicitation of cell suspension cultures on day 0 and 15 resulted in highest fresh biomass (412.16 g/l) and lignans production (secoisolariciresinol diglucoside 284.12 mg/l: lariciresinol diglucoside 86.97 mg/l). Whereas, repeated elicitation on day 0 and 25 resulted in highest dry biomass (13.53 g/l), total phenolic production (537.44 mg/l), total flavonoid production (123.83 mg/l) and neolignans production (dehydrodiconiferyl alcohol glucoside 493.28 mg/l: guaiacylglycerol-β- coniferyl alcohol ether glucoside 307.69 mg/l).

5.2. FUTURE PROSPECTS  Current research design was based according to laboratory setup. Results obtained are promising and should be reproduced on a larger scale like bioreactor based studies. Operation performed on a large scale would result in production of secondary metabolites in bulk amount.  The nutrient medium influenced enhancement of biochemical compounds can also be replicated in cell suspension cultures of Linum usitatissimum, wherein effects of individual macro and micro nutrients can be evaluated and compared for various physiological and biochemical responses.  Elicitation of cell suspension cultures resulted in enhanced biosynthesis of polyphenols, this strategy provides an easy approach towards metabolic engineering of Linum usitatissimum. Each elicitor stimulates a particular step in biosynthetic pathways of secondary metabolism, targeting expression of some important genes and enzymes in response to elicitors would help identify limiting steps and factors in secondary metabolism of Linum usitatissimum.  Major finding of the current study includes synergism in plant physiological and metabolic response, as we observed for mineral nutrients variation and different photoperiod treatments. Similar strategies can be chalked out by applying combinations of various biotic and abiotic elicitors simultaneously in order to evaluate synergistic effects on various growth parameters.

PUBLICATIONS FROM THESIS

Zahir A, Ahmad W, Nadeem M, Giglioli-Guivarc'h N, Hano C & Abbasi BH (2018a) In vitro cultures of Linum usitatissimum: Synergistic effects of mineral nutrients and photoperiod regimes on growth and biosynthesis of lignans and neolignans. Journal of Photochemistry and Photobiology B: Biology Zahir A, Nadeem M, Ahmad W, Giglioli-Guivarc’h N, Hano C & Abbasi BH (2018b) Chemogenic silver nanoparticles enhance lignans and neolignans in cell suspension cultures of Linum usitatissimum L. Plant Cell, Tissue and Organ Culture (PCTOC):1-8 Abbasi BH, Zahir A, Ahmad W, Nadeem M, Giglioli-Guivarc’h N, Hano C. Biogenic zinc oxide nanoparticles-enhanced biosynthesis of lignans and neolignans in cell suspension cultures of Linum usitatissimum L. Artificial cells, nanomedicine, and biotechnology. 2019 Dec 4;47(1):1367-73.

Publications as a co-author during Ph.D.

Nadeem, M., Abbasi, B.H., Garros, L., Drouet, S., Zahir, A., Ahmad, W., Giglioli- Guivarc’h, N. and Hano, C., (2018). Yeast-extract improved biosynthesis of lignans and neolignans in cell suspension cultures of Linum usitatissimum L. Plant Cell, Tissue and Organ Culture (PCTOC), 135(2), pp.347-355.

Nadeem, M., Ahmad, W., Zahir, A., Hano, C. and Abbasi, B.H., Salicylic acid‐ enhanced biosynthesis of pharmacologically important lignans and neolignans in cell suspension culture of Linum ussitatsimum L. Engineering in Life Sciences. https://doi.org/10.1002/elsc.201800095

Ahmad, W., Zahir, A., Nadeem, M., Garros, L., Drouet, S., Renouard, S., Doussot, J., Giglioli-Guivarc’h, N., Hano, C. and Abbasi, B.H., (2018). Enhanced production of lignans and neolignans in chitosan-treated flax (Linum usitatissimum L.) cell cultures. Process Biochemistry.

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