Stratagies for the Production of Chemically Consistent Plantlets of Silybum Marianum L

STRATAGIES FOR THE PRODUCTION OF CHEMICALLY CONSISTENT PLANTLETS OF SILYBUM MARIANUM L.

MUBARAK ALI KHAN

DEPARTMENT OF BIOTECHNOLOGY QUAID-I-AZAM UNIVERSITY ISLAMABAD, PAKISTAN 2015

Strategies for the Production of Chemically Consistent

Plantlets of Silybum marianum 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

MUBARAK ALI KHAN Supervised by

Dr. BILAL HAIDER ABBASI

Department of Biotechnology

Quaid-i-Azam University,

Islamabad, Pakistan

2015

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 and in Mass spectrometry laboratory, The University of Sheffield United Kingdom. The findings and conclusions are of my own investigation with discussion of my supervisor Dr. Bilal Haider Abbasi. No part of this work has been presented for any other degree.

MUBARAK ALI KHAN

ACKNOWLEDGEMENTS

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. Primary, I would greatly appreciate my supervisor Dr. Bilal Haider Abbasi, Assistant Professor, Department of Biotechnology, Quaid-i-Azam University, Islamabad 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 Prof. Zabta Khan Shinwari, Chairman Department of Biotechnology, Quaid-i-Azam University Islamabad, for his immense concern throughout my Ph.D period. I wish to express my deep gratitude and respect to all my teachers whose knowledge and guidance enabled me to attain this target. I feel pleasure in acknowledging the nice company of my lab fellows Mohammad Ali, Mohammad Adil, Nisar Ahmad and Ahmad Zada. I will always remember the nice company of my friends Abid Jan, Naseer Ali Shah, Akhtar Nadhaman, Mutiullah, Nazifullah, Zia Mashwani, Khalid Khattak, Zahidullah, Naveed Alam, Asad Zia, Ikramullah, Tariq Khan, Amaan and Ishtiaq Hussain.

I must acknowledge my debt to Prof. Mike Burrell, Department of Animal and Plant Sciences, The University of Sheffield, United Kingdom for providing me an opportunity to carry out part of my research project in his mass spectrometry laboratory. His constructive comments, guidance and great co-operation played a fundamental role for carrying out this epic work. I am also thankful to technical staff members Bob Keen and Heather Walker for their support and assistance during my stay in The University of Sheffield. This project was supported by Higher Education Commission of Pakistan and I acknowledge this institution for providing me Indigenous and Overseas “IRSIP” scholarship for my research in Pakistan and United Kingdom.

Deepest regards and gratitude to my brothers Dr. Sartaj Wali Khan and Dr. Abdul Ali Khan for their support. My all the four sisters prayer for my success, I am also thankful to them. My infinite thank goes to my wife Huma Ali for her moral support and tolerance in this long and hard journey. Lots of love to my kids Saad Ali Khan and Mohammad Ali Khan for making me smile whenever I met them. I must say that all the happiness in my life is because of my beloved father Prof. Muhammad Iqbal and my ever caring and sweetest mother. Their constant prayers and appreciations always kept me satisfied.

Mubarak Ali Khan

I Dedicate This Thesis To My Parents, My Wife & My Kids!

TABLE OF CONTENTS

ACKNOWLEDGEMENTS.……………………………………………………….. i DEDICATION……………………………………………………………………… ii LIST OF FIGURES………………………………………………………………… vii LIST OF TABLES ……………………………………………………………….. x LIST OF ABBREVIATIONS .……………………………………………………. xi SUMMARY………………………………………………………………………….. xii

1. GENERAL INTRODUCTION………………………………………………….... 1 1.1 SILYBUM MARIANUM L: AN IMPORTANT MEDICINAL PLANT ………… 1 1.1.1 Silymarin the medicinally active ingredient ………………………………… 2 1.1.2 Conventional propagation methods …………………………………………. 5 1.1.3 Chemical inconsistencies in Silybum products ……………………………… 6 1.1.4 Plant in vitro techniques for mass propagation ……………………………… 7 1.1.4.1 In vitro seed germination ………………………………………………… 7 1.1.4.2 Explant type and choice …………………………………………………. 8 1.1.4.3 In vitro Regeneration ……………………………………………...... 9 1.1.4.3.1 Shoot organogenesis …………………………………………………. 9 1.1.4.3.2 Somatic embryogenesis ……………………………………………… 10 1.1.4.3.3 Role of Auxins and Cytokinins in S. marianum regeneration……….. 10 1.1.4.3.4 Thidiazuron a multifaceted plant growth regulator ………………….. 11 1.1.4.3.5 Dark-light cycling and in vitro culture …………………………...... 11 1.1.4.3.6 Root organogenesis ………………………………………………... 12 1.1.4.3.7 Acclimatization and ex vitro transfer ……………...... 12 1.1.5 In vitro cultures and biochemical charaterization ………………………...... 15 1.1.5.1 DPPH free radical scavenging activity ………………………………… 15 1.1.5.2 In vitro cultures and anti-oxidative enzyme activities …………………. 16 1.1.5.3 PAL and silymarin biosynthesis………………………………………... 17 1.1.5.4 In vitro cultures and quality control …………………………………….. 19 1.1.5.5 Analysis of metabolic changes during in vitro morphogenesis ………… 20 1.1.6 Objectives …………………………………………………………………..... 22

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2. EFFECTS OF LIGHT REGIMES ON IN-VITRO SEED GERMINATION AND 23 SILYMARIN CONTENT IN SILYBUM MARIANUM L…………………………..

2.1 ABSTRACT……………………………………………………………………...... 23 2.2 INTRODUCTION………………………………………………...... 24 2.3 MATERIALS AND METHODS………………………………………………...... ……………………... 25 2.3.1 Seed germination………………………………………………………………. 25 2.3.2 Analytical methods…………………………………………………………... 26 2.3.2.1 Antioxidant enzyme activities……………………………………………… 26 2.3.2.2 Assay of PAL activity………...... 27 2.3.2.3 DPPH- Free radical-scavenging activity (FRSA)………………………….. 27 2.3.2.4 Silymarin extraction and HPLC analysis…………………………………… 28 2.3.2.5 Statistical analysis……………………………………………………………….. 28 2.4 RESULTS AND DISCUSSION………………………………………………………… 29 2.4.1 Seed germination frequency and growth parameters ………………………….. 29 2.4.2 Antioxidant enzymes activity…………………………………………………… 36 2.4.3 Silymarin content, phenylalanine ammonia lyase activity and radical scavenging 38 activity…………………………………………………………………………………….

3. THIDIAZURON-INDUCED REGENERATION AND SILYMARIN CONTENT 41 IN SILYBUM MARIANUM L……………………………………………

3.1 ABSTRACT……………………………………………………………………...... 41 3.2 INTRODUCTION………………………………………………………………….. 42 3.3 MATERIALS AND METHODS…………………………………………………… 43 3.3.1 Micro propagation………………………………………………………………. 43 3.3.1.1 Seed germination……………………………………………………………. 43 3.3.1.2 Plant growth regulators for regeneration……………………………………. 43 3.3.1.3 Silymarin assesment by HPLC ………………………………...... 44 3.3.1 Statistical analysis………………………………………………………………. 44 3.4 RESULTS AND DISCUSSION……………………...... 45 3.4.1 Callus induction frequency …………………………………………………….. 45 3.4.2 Shoot organogenesis …………………………………………………………… 47 3.4.3 Rooting and acclimatization …………………………………………………… 52

3.4.4 Silymarin content ………………………………………………………………. 53

4. ESTABLISHMENT OF AN EFFICIENT ROTOCOL FOR SOMATICE MBRYOGENESIS IN SILYBUM MARIANUM L…………………. 56

4.1 ABSTRACT……………………………………………………………………..... 56 4.2 INTRODUCTION………………………………………………………………… 57 4.3 MATERIAL AND METHODS…………………………………………………… 58 4.3.1 Plant material………………………………………………………………….. 58 4.3.1.2 Establishment of embryogenic callus……………………………………… 58 4.3.1.3 Development of somatic embryos…………………………………………. 58 4.3.1.4 Germination of somatic embryos into plantlets……………………………. 59 4.3.1.5 Histological study…………………………………………...... 59 4.3.2 Statistical Analysis……………………………………………………………. 60 4.4 RESULTS AND DISCUSSION………………………………………………….. 61 4.4.1 Induction of embryogenic callus……………………………………………… 61 4.4.2 Somatic embryo development……………………………………………………. 62 4.4.3 Embryo germination and conversion into plantlet……………………………. 65

5. TEMPORAL VARIATIONS IN METABOLITE PROFILES AT DIFFERENT GROWTH PHASES DURING SOMATIC EMBRYOGENESIS OF SILYBUM 68 MARIANUM L………………………………………………………………………..

5.1 ABSTRACT…………………………………………………………………….... 68 5.2 INTRODUCTION……………………………………………………………….. 69 5.3 MATERIAL AND METHODS………………………………………………….. 69 5.3.1 Plant samples…………………………………………………………………. 69 5.3.2 Biochemical characterization...... 70 5.3.3 Metabolite profiling………………………………………………………….. 70 5.3.3.1 Extraction of Metabolites………………………………………………….. 70 5.3.3.2 Electrospray ionisation time of flight mass spectrometry (ESI-TOF MS)… 70 5.3.3.3 Data processing……………………………………………………………. 71 5.3.3.4 Putative metabolite identification …………………………………………. 72 5.3.4 Statistical Analysis…………………………………………………………… 72 5.4 RESULTS AND DISCUSSION…………………………………………………. 72

5.4.1Phenylalanine ammonia lyase activity, antioxidant potential 72 and silymarin content………………………………………………………….

5.4.2 Metabolite profiling…………………………………………………………… 74 5.4.2.1 Mass spectra…………………………………………………………………. 74 5.4.2.2 Principal Component Analysis (PCA)………………………………………. 77 5.4.2.3 Evaluation of Putatively known metabolites ……………………………….. 82 5.4.2.3.1 Bin masses………………………………………………………………… 82 5.4.2.3.2 Distribution of key metabolites during different growth phases………….. 87 5. CONCLUSIONS AND FUTURE PROSPECTS………………………………..... 96 6. REFERENCES……………………………………………………………………… 99

LIST OF FIGURES

Fig 1.1 Taxonomic classification of Silybum marianum L. 3

Fig 1.2 Biologically active components from Silybum marianum L. 4

Proposed biosynthetic pathways showing inter-relation between PAL and Fig 1.3 17 cinnamate leading to the production of silymarin (Hu et al., 2009).

Schematic representation of various in vitro strategies for production of S. Fig 1.4 22 marianum L. plantlets.

Effects of different light treatments on % seed germination. Data were collected Fig 2.1 after 4 weeks of culture. Values are the mean ± standard error from three replicates. 33 Columns with similar alphabets are not significantly different at P<0.05.

Effects of different photoperiods on shoot and root induction response. Data were Fig 2.2 collected after 4 weeks of culture. Values are the mean ± standard error from three 35 replicates. Columns with similar alphabets are not significantly different at P<0.05.

Activities of antioxidative enzymes (SOD, APX, CAT and POD in U/mg protein) in plantlets grown under different light regimes in in vitro conditions and wild-grown Fig 2.3 37 plantlets of Silybum marianum. Values are the mean ± standard error from three replicates. Columns with similar alphabets are not significantly different at P<0.05.

Silymarin content (mg/g DW) in in vitro-grown and wild-grown plantlets of Fig 2.4 Silybum marianum. Values are the mean ± standard error from three replicates. 39 Columns with similar alphabets are not significantly different at P<0.05.

Phenylalanine ammonia lyase (PAL) activity (U/mg protein) and % antioxidant activity in in vitro-grown and wild-grown plantlets of Silybum marianum. Values Fig 2.5 40 are the mean ± standard error from three replicates. Columns with similar alphabets are not significantly different at P<0.05.

Data on callus formation parameters in Silybum marianum by the application of Fig 3.1 various concentrations and combinations of TDZ, Kn and NAA. Values are mean of 46 three replicates and observations were recorded after 4 weeks of culture.

Data on shoot induction frequency in Silybum marianum by the application of Fig 3.2 various concentrations and combinations of TDZ, Kn and NAA. Values are mean of 48 three replicates and observations were recorded after 4 weeks of culture.

Data on number of shoots in Silybum marianum by the application of various Fig 3.3 concentrations and combinations of TDZ, Kn and NAA. Values are mean of three 49 replicates and observations were recorded after 4 weeks of culture.

Data on shoot length (cm) in Silybum marianum by the application of various Fig 3.4 concentrations and combinations of TDZ, Kn and NAA. Values are mean of three 51 replicates and observations were recorded after 4 weeks of culture.

Data on silymarin content (mg/g) in regenerated plant tissues and wild grown Fig 3.5 55 plantlets of Silybum marianum. Values are mean of three replicates.

Effects of various growth media on somatic embryo maturation (%). Data were collected after 2 weeks of culture. EIM=Embryo induction medium. Values are the Fig 4.1 64 mean ± standard error from three replicates. Column bars sharing the same English letter/s are similar otherwise differ significantly at P<0.05.

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Effects of various concentrations of GA3 in MS 1/2 media on germination frequency of somatic embryos. Data were collected after 4 weeks of culture. Values Fig 4.2 66 are the mean ± standard error from three replicates. Column bars sharing the same English letter/s are similar otherwise differ significantly at P<0.05.

Somatic embryogenesis in Silybum marianum (a) Callus formation at cut ends of petiole explants after one week of culture (bar=2mm). (b) Embryogenic callus with pre-embryoid masses (PEM) after two weeks of culture period (bar=2mm). (c) Embryogenic callus with somatic emryos (arrows) (bar=2mm). (d) Embryonic cells showing isodiametric cells (blue star) non-embryonic cells (black star) with large Fig 4.3 vacuole, small starch granules and abundant intercellular spaces (bar=250µm). (e) 67 Globular somatic embryo after four weeks in embryo induction medium (bar=150µm) (f) Cotyledenary embryo, arrows show vascular bundles (bar=150µm). (g) Shoot emergence from embryo after one week in germination medium (bar=2mm). (h) Embryo converted plantlet after four weeks in germination medium, arrow shows root formation (bar=2mm).

Raw metabolic profile mass spectra for aqueous fractions of NEC, PEM, SE and Fig 5.1 GSE lines in positive ionization mode. Symbols with same shape represent the 75 common peaks in the samples.

Raw metabolic profile mass spectra for organic fractions of NEC, PEM, SE and Fig 5.2 GSE lines in positive ionization mode. Symbols with same shape represent the 76 common peaks in the samples.

PCA Score Scatter plots of aqueous fractions of NEC, PEM, SE and GSE, Fig 5.3 78 evaluated by SIMCA-P+ (12.0).

PCA Score Scatter plots of organic fractions of NEC, PEM, SE and GSE, evaluated Fig 5.4 79 by SIMCA-P+ (12.0).

PCA Loading Scatter plots of aqueous fractions of NEC, PEM, SE and GSE, Fig 5.5 80 evaluated by SIMCA-P+ (12.0).

PCA Loading Scatter plots of organic fractions of NEC, PEM, SE and GSE, Fig 5.6 81 evaluated by SIMCA-P+ (12.0).

Percentage of total ion counts of the bins (putatively identified metabolites) in NEC, PEM, SE and GSE. Bin masses were selected from the loading plots sorted Fig 5.7.A 83 by PCA. (Aqueous fractions). Data represents the values of the mean ± standard error from three replicates.

Average intensities of total ions of the bins (putatively identified metabolites) in NEC, PEM, SE and GSE. Bin masses were selected from the loading plots sorted Fig 5.7.B 84 by PCA. (Aqueous fractions). Data represents the values of the mean ± standard error from three replicates.

Percentage of total ion counts of the bins (putatively identified metabolites) in NEC, PEM, SE and GSE. Bin masses were selected from the loading plots sorted Fig 5.8.A 85 by PCA. (Organic fractions). Data represents the values of the mean ± standard error from three replicates.

Average intensities of total ions of the bins (putatively identified metabolites) in NEC, PEM, SE and GSE. Bin masses were selected from the loading plots sorted Fig 5.8.B 86 by PCA. (Organic fractions). Data represents the values of the mean ± standard error from three replicates.

Fig 5.9 Comparison of the key metabolites in NEC, PEM, SE and GSE lines. Putatively 89 known metabolites were obtained from in-house bin program on the basis of their

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distribution with total ions count (%) in the biological samples. Data represents the values of the mean ± standard error from three replicates.

LIST OF TABLES

Use of explant types and PGRs in S. marianum and other Asteraceae species, and Tab 1.1 13 their regeneration response

Effects of several plant growth regulators on seed germination frequency, mean Tab 2.1 shoot length and mean root length in culture flasks under 16-h light and 8-h dark for 30 4weeks. Data show mean of three replicates ± SE.

Reference table showing the application of different photoperiods in conjunction Tab 2.2 with combination of PGRs from T1 to T9 for in-vitro seed germination of Silybum 31 marianum.

Tab 2.3 Comparative analysis of silymarin content 39

Data on root formation parameters by the application of different concentrations of Tab 3.1 indole acetic acid (IAA) and α-napthalene acetic acid (NAA). Values are mean of 53 three replicates and observations were recorded after 4 weeks of culture.

Effects of various concentrations and combinations of 2,4-D and BA in SH media on embryogenic callus formation from leaf, petiole and root explants. Data on Tab 4.1 embryogenic callus formation were recorded after 2 weeks of culture when four 62 week old calli were sub-cultured on same media. Values are the mean ± standard error from three replicates.

Phenylalanine ammonia lyase activity (Ug-1 FW), antioxidant potential (% FRSA) and silymarin content (mg.g-1DW) in in-vitro-grown plant samples collected from different growth phases during somatic embryogenesis in Silybum marianum. The Tab 5.1 different growth phases comprised of NEC= Non- embryogenic callus, PEM= Pre- 73 embryoid massesproduced on embryogenic callus, SE= somatic embryos at mature stage, GSE= Somatic embryo germinating into plantlet. Values are the mean ± standard error from three replicates.

Correlation between (% FRSA) antioxidant potential, (PAL) Phenylalanine ammonia lyase activity (Ug-1 FW) and silymarin content (mg.g-1DW) in in-vitro- Tab 5.2 73 grown plant samples collected from different growth phases during SE in Silybum marianum. Marked correlations are significant at p < 0.05.

Putative identification of the metabolites by the personalv7qstar in-house program Tab 5.3 91 for the selected bins sorted from the aqueous extracts of biological samples

Putative identification of the metabolites by the personalv7qstar in-house program Tab 5.4 94 for the selected bins sorted from the organic extracts of biological samples

ABBREVIATIONS

2, 4 D 2, 4 dichlorophenoxyacetic acid APX Ascorbate peroxidase BA 6-Benzyladenine CAT Catalase DPPH 1, 1-diphenyl-2-picrylhydrazyl DW Dry weight EIM Embryo induction medium ESI Electro spray ionization FRSA Free radical scavenging activity FW Fresh weight GA3 Gibberellic acid GSE Embryos germinating into intact plantlets HPLC High Performance Liquid Chromatography IAA Indole acetic acid IBA Indole butyric acid Kn Kinetin Mg/g-DW milligram/gram-Dry Weight Mg l-1 milligram/litre MS Murashige and Skoog MS0 MS medium without plant growth regulators NAA α-Naphthalene acetic acid NEC Non-embryogenic callus PAL Phenyl ammonia lyase PCA Principal Component Analysis PEMs Pre-embryoid masses PGRs Plant growth regulators POD Peroxidase ROS Reactive oxygen species SE Somatic embryo SH Schenk and Hilde SOD Superoxide dismutase TDZ Thidiazuron TOF MS Time of flight mass spectrometry WHO World Health Organization

SUMMARY

Silybum marianum L. commonly known as Milk thistle is a medicinally important plant used for the treatment of hepatic disorders worldwide. Its active constituent is silymarin which is considered as a potent antioxidant and has shown multi- dimensional medicinal properties. However, collection of Silybum plant material from different regions of World has shown variation in composition of silymarin. This variation leads to inconsistencies in Silybum products and thus creates confusion in end users. To overcome such issues, feasible and reliable production system needs to be established. In vitro propagation practices are helpful to produce true-to-type and genetically stable clones in short span of time by passing conventional methods and seasonal variation. In the present research work, attempts have been made to develop different strategies for rapid multiplication and production of chemically consistent plantlets of S. marianum which can be successfully used in large scale commercial production.

In the first experiment, an efficient protocol for in vitro seed germination of S. marainum has been optimized. Viable seeds were employed on Murashige and Skoog (MS) medium containing various concentrations and combinations of different plant growth regulators (PGRs). Combination of 6-benzyladenine, BA (0.5 mg/l) and gibberellic acid, GA3 (1.5 mg/l) with thidiazuron, TDZ (1.0 mg/l) resulted into optimum seed germination frequency on MS medium and was selected for subsequent experiments with various photoperiods as described here, T1=24-h dark for 4 weeks, T2=24-h light for 4 weeks, T3=16-h light and 8-h dark for 4 weeks (control), T4=1 week dark + 3 weeks light (16/8-h), T5=2 weeks dark + 2 weeks light (16/8-h), T6=3 weeks dark + 1 week light (16/8-h), T7=1 week light (16/8-h) + 3 weeks dark, T8=2 weeks light (16/8-h) + 2 weeks dark and T9=3 weeks light (16/8-h) + 1 week dark. The germination frequency was enhanced, with the highest value (90%) observed when seeds were incubated on to MS medium containing BA (0.5 mg/l) + GA3 (1.5 mg/l) + TDZ (1.0 mg/l) kept in complete darkness for two weeks followed by subsequent transference to light condition for two weeks (T5). Optimum shoot and root length (5 cm and 3.3 cm, respectively) were recorded at this treatment. Our results revealed that exposure to continuous light (T2) produced lesser frequency (%) of seed germination compared to continuous dark conditions (T1) or 16 h light/8 h dark conditions (T3). Moreover, activities of antioxidant enzymes like Superoxide

xi dismutase (SOD), Ascorbate peroxidase (APX), Catalase (CAT) and Peroxidase (POD) were determined in this study. These enzymes were detected at highest level for T5 than other treatments, showing the involvement of these enzymes during in vitro seed germination. Silymarin was detected and quantified by HPLC and 5.48 mg/g silymarin was detected in intact plantlets derived from T5 treatment. Moreover, a linear correlation was also observed among free radical scavenging activity (FRSA), Phenylalanine ammonia lyase (PAL) activity and silymarin content.

In the second experiment; an efficient, rapid and reproducible plant regeneration protocol was optimized for consistent production of silymarin by exploitation of leaf explants derived from in vitro germinated 28 days old plantlets (Experiment 1). Incubation of leaf explants on to MS medium incorporated with TDZ at various levels, either alone or in combination with other PGRs like kinetin (Kn) or α- naphthalene acetic acid (NAA), induced callus. TDZ alone at lower concentrations (2.2µM) produced optimum callus (73%). Calli developed were soft, friable and greenish white in color with a diameter of 1.5 cm. Upon transfer into shoot organogenesis media; the fast, friable and competent calli produced shoots. Highest shoot regeneration frequency (86%) with a maximum number of shoots (26 shoots) was recorded for MS medium supplemented with 11µM TDZ. However, a decrease in shoot organogenesis parameters was observed with increase in concentration of TDZ beyond the optimal level. Addition of NAA in medium containing TDZ increased mean shoot length with highest length (4.1 cm) observed at combination of 6.6µM TDZ and 4.4µM NAA. Moreover, highest rooting frequency (87%) was observed at 0.4 µM IAA, with mean number of roots (3.2 roots) and mean root length (3.4 cm). Finally the rooted shoots were transferred to sterilized vermiculite and kept under growth room conditions for two weeks prior to transfer to pots. The survival rate of rooted shoots was 73% through the hardening-off process. After optimization of protocol for regeneration, different in vitro culture extracts were subjected to HPLC for analysis of silymarin content. 8.47 mg/g silymarin was detected in regenerated plantlets. Our results demonstrated an ascending variation of silymarin from callus culture to plantlets development, thus depicting the influence of growth pattern on silymarin production during in vitro regeneration.

In the third experiment, an efficient regeneration system through somatic embryogenesis was optimized for production of chemically consistent plantlets of S.

xii marianum. The callus formation frequency was enhanced when the petiole explants were cultured on SH medium supplemented with 2.5 mg/l 2,4-D in combination with 1.5 mg/l BA. Sub culturing of these calli on media with similar hormonal composition produced embryogenic calli and highest embryogenic potential (72%) was observed for calli previously derived from petiole explants. The embryogenic calli with PEMs were transferred on to an embryo induction medium containing varying concentrations of 2,4-D alone or in combination with BA. Optimum number of somatic embryos (33 somatic embryo per callus) were recorded at (0.5 mg/l) 2,4-D. These embryos were observed on the outermost cell layer of the callus and were globular in shape. However, the presence of BA in combination with 2,4-D increased the number of somatic embryos per callus and highest number of somatic embryos (46 somatic embryo per callus) was observed at 1.5 mg/l 2,4-D in combination with 2.0 mg/l BA. Moreover, embryos were germinated on half strength MS medium supplemented with various concentrations of GA3. Highest germination rate (70%) was observed on 1/2 MS medium containing 1.5 mg/l GA3. Subsequently, the cotyledonary embryos developed the shoot and the root apex, and formed a complete plantlet. In the fourth experiment, a feasible protocol has been optimized to investigate the metabolic events happening at different growth phases during in-vitro somatic embryogenesis of S. marianum. For metabolite profiling, plant material was collected from different growth phases of non-embryonic callus (NEC), pre-embryoid masses (PEM), globular somatic embryos (SE) or cotyledonary embryos germinating into intact plantlets (GSE) during somatic embryogenesis. Extraction of metabolites was carried out by solvent-solvent extraction method in methanol:chloroform:water into aqueous and organic fractions, respectively. Electro spray ionization, time of flight mass spectrophotometer (ESI-TOF MS) was performed for evaluation of metabolite profiles at different growth phases during embryogenesis. The PCA analysis of both aqueous and organic fractions produced the PCA score plots. The samples were distinguished on the basis of differences in developmental stages during embryogenesis. The principal components PC1 and PC2 accounted for 72% variation in aqueous fractions and 54% variation in organic fractions. In addition, the loading scatter plots enabled the detection of several bin masses responsible for separating samples from different growth phases. Comparison of the selected bins among the biological samples was carried out by calculating the percentage of their total ion counts and average intensity counts. Based on the values of % total ions count and

xiii average intensity of selected bins in all biological samples, putatively known metabolites were obtained from in-house bin program. Among amino acids, arginine, asparagine and serine were abundantly detected in GSE, and were detected at negligible level in NEC. Furthermore, serine, cysteine and proline were detected at increased signals in PEM; however, glutamine and tryptophan were abundantly found in SE. Moreover; glucose, fructose, fructose-6-phosphate and sorbitol were accumulated abundantly in NEC during the process of embryogenesis. However, sucrose was measured with maximum ion counts in SE followed by GSE while lowest value was detected in NEC. Significant secondary metabolites like cinnamic acid, kaempferol, quercetin, myricetin, linolenic acid, and 5-enolpyruvyl-shikimate-3- phosphate were found at higher levels in SE when compared to other embryogenic lines. Furthermore, our results revealed a strong corroboration between these important secondary metabolites with higher levels of PAL, FRSA and silymarin content in SE which might indicate an up-regulation of plant secondary metabolite production during this growth phase.

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Chapter 1

1. GENERAL INTRODUCTION

1.1 SILYBUM MARIANUM L: AN IMPORTANT MEDICINAL PLANT

Silybum marianum is a widely recognized medicinal herb belongs to family Asteraceae and is originated in the Mediterranean basins; but now naturalized throughout the world. In Pakistan it grows wild in Punjab, Khyber Pakhtunkhwa and Kashmir (Abenavoli et al., 2010; Sedghi et al., 2010; Shah et al., 2011). Its common name, Milk Thistle is derived from its white-veined leaves which ooze a milky sap upon cut (Burgess, 2003).

Although Germany, Spain and Romania produce Silybum for commercial purposes, however, Poland is the major producer in Europe, where the cultivation area covers over 2000ha (Andrzejewska et al., 2011; Zheljazkov et al., 2006). In the last decade, the cultivation areas of Milk Thistle extended beyond Europe, into North and South America, Australia, China and other parts of the world where effective cultivation programs have been initiated for domestication and large scale production of this important plant species (Andrzejewska et al., 2011; Kavallieratos et al., 2007; Shokrpour et al., 2008).

In old history this plant is known to be used for better treatment against many ailments, e.g. Theophrastus in 4th century B.C. was the first to describe it with the name Pternix and later on in 1st century A.D it was named as “Sillybom” in Materia Medica by Diocurides who prescribed its tea for treatment of snake bites (Corchete, 2008; Kren and Walterova, 2005). Looking into its high medicinal, economic and commercial importance, Silybum products have become a multibillion-dollar industry in the developed countries (Hurrell and Puentes, 2013). Approximately 8 to 33% of patients with hepatic disorders consume this plant frequently as herbal remedy worldwide (Polyak et al., 2010). In 2007, Milk thistle extract was the 8th most important herbal supplement sold in USA with annual sales of about US$ 8 billion. However, among the consumers the demand for its extract varies from 18 to 20 tons/year (Jacob et al., 2011). Retail sales of Silybum products as seed tinctures exported from Bulgaria and UK in 2013 were US$ 9 thousands per ton (Kortbech- Olesen, 2014). Medicinal importance of S. marianum can be revealed from the list of Strategies for the production of chemically consistent plantlets of Silybum marianum. L

Chapter 1

diseases against which it is used as a better remedy. Although seeds are considered to be the most important part of this plant, however whole plants including leaves, roots and flowers were also used as herbal supplements in past (Ahmad et al., 2013b; Grieve, 1971).

It contains anti-inflammatory (Sharifi et al., 2013), hepatoprotective (Flora et al., 1998; Krecman et al., 1998), antioxidant and immuno-modulatory (Das and Mukherjee, 2012), antibacterial (Bhattacharya, 2011), anticancer (Deep and Agarwal, 2010), antitumor (Wing et al., 2010), anti-diabetic (Huseini et al., 2006) and antiviral activities (Wagoner et al., 2010). It is also used to treat hypotension (Duke, 2010), asthma (Breschi et al., 2002), hypocholesterolemic (Shaker et al., 2010), hyperprolactinemic effect (Capasso et al., 2009), obsessive-compulsive disorder (Sayyah et al., 2010), hypoglycemia (Zhan et al., 2011) and is nephroprotective (Shahbazi et al., 2012).

1.1.1 Silymarin the medicinally active ingredient

Silymarin is the main bioactive ingredient of milk thistle. It is an isomeric mixture of different flavonolignans, the important one are silybin also called silibinin, silidianin, and silichrystin (Figure 1.2). However, some other flavonolignans identified in S. marianum include dehydrosilybin, deoxysilycistin, deoxysilydianin, silandrin, silybinome, silyhermin, and neosilyhermin (Wichtl, 2004). Importance of silymarin as an important phytochemical is clearly evidenced by a substantial increase in the number of publications on this topic (Gazak et al., 2007). It has a multitude of biological activities (Polyak et al., 2007). Silymarin promotes the release of superoxide dismutase (SOD) which clears the liver from free radicals caused by alcohol metabolism; it also stimulates RNA polymerase, an enzyme in the nucleus of liver cells responsible for protein synthesis and regeneration of hepatic cells (Tawaha et al., 2007). Additionally, it inhibits the action of leukotrienes, which can also damage the liver (El-Samaligy et al., 2006; Sunitha et al., 2001).

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Scientific classification

Kingdom: Plantae

Angiosperms

Eudicots

Core eudicots

Order: Asterales

Family: Asteraceae

Genus: Silybum

Species: marianum

Common name: Milk thistle Binomial name: Silybum marianum

Figure 1.1: Taxonomic classification of Silybum marianum (Adopted from Abenavoli et al., 2010; Burgess, 2003; Sedghi et al., 2010; Shah et al., 2011).

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Figure 1.2: Biologically active components from Silybum marianum L. (Adopted from Pliskova et al., 2005).

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1.1.2 Conventional propagation methods

Conventionally S. marianum is propagated through seeds which are sown directly into soils at a depth of 1-1.5 cm. Germination of the seeds usually occurs in late autumn and early spring. However, efficiency of germination is highly dependent on the physiological health of seeds, soil fertility, pH, moisture and temperature (Andrzejewska et al., 2011). Although the optimum temperature is 15 ºC, however, the seed germination can occur at 2-15 ºC as well (Ghavami and Ramin, 2008).

Furthermore, the non-desiccated seeds harvested at maturity have higher germination rates than seeds harvested after drying (Andrzejewska et al., 2011). Nevertheless, seeds can fail to germinate even in favorable conditions due to any physical or physiological dormancy. Silybum shows confined growth to specific environmental and soil conditions but grows well in rocky soils and can be cultivated successfully on light sandy as well heavy clay soils with adequate nutrient supply at a wide range of soil pH but grows well at a pH of 5.5-7.6 (Karkanis et al., 2011). The growth pattern of S. marianum can be divided into four distinct stages like; germination, plantlet development, flowering and seed maturity.

However, the vegetative phase can be further divided into rosette stage and stem elongation (Andrzejewska et al., 2011; Haban et al., 2009; Karkanis et al., 2011). Although various cultivation strategies have been adopted to increase the healthy biomass of this important plant, but the conserved germplasm in its wild habitat is susceptible to various biotic and abiotic stresses. This plant is also under continuous serious threat from a wide range of pests and insects and is prone to poor crop yield due to some diseases caused by these pathogens and insects (Karkanis et al., 2011). The major pathogens reported in Silybum are Septoria silybi, Puccinia punctiformis and Microbotryum silybum (Berner et al., 2002; Kavallieratos et al., 2007) and the insects include Larinus latus, Cleonus piger, Terellia fuscicornis and Nezara viridula (Abdel-Moniem, 2002; Andrzejewska et al., 2006; Kavallieratos et al., 2007; Rezwani, 2008).

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1.1.3 Chemical inconsistencies in Silybum products

Significance of plant based medicines has been increasing worldwide, since many of active ingredients in medicinal plants cannot yet be prepared synthetically (Siddiqui and Hakim, 1991). It is very common with medicinal plants that there exists an extreme variability in the phytochemical contents (Wu et al., 2009), therefore (most of the time) one cannot attribute the therapeutic properties of medicinal plants to a single compound (Roscetti et al., 2004). This high variability in the phytochemical constituents of a specific plant can be explained with different reasons like;

(1) Lack of uniform cultivation technologies for optimized growth

(2) Non existing procedures for production, harvest, manufacturing, marketing, and distribution of plant material

(3) The influence of physiological, genetic, geographic and environmental variables on the biochemical profiles and production of secondary metabolites in plants (Abbasi et al., 2007; Briskin and Gawienowski, 2001; Murch et al., 2000; Murch et al., 2006; Seidler-Lozykowska and Dabrowska, 2003).

Similarly, reasons like biological contaminants and environmental pollutants, adulteration with misidentified species, quantitative and qualitative variations of bioactive compounds as well as the concern of unsustainable harvest are the major bottlenecks for production of effective Silybum products from wild grown plants (Haban et al., 2009; Lee and Liu, 2003). These problems have produced inconsistencies in the results of various clinical trials and difficulties in identifying a specific medicinal molecule with defined pharmaceutical functions in S. marianum products (Ram et al., 2005). Production of chemically consistent S. marainum crops is essential for consistent production of phytochemicals which can be achieved via in-vitro clonal propagation (Parveen and Shahzad, 2010). In-vitro plant technology is capable of producing large number of aseptic, genetically uniform, and chemically consistent plants in a short period of time and limited space (Victorio et al., 2012).

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1.1.4 Plant In vitro techniques for mass propagation

In vitro clonal propagation provides a suitable platform for consistent production of many medicinally and commercially important plants (Honda et al., 2001). In vitro plant technology is advantageous over conventional methods for conserving rare and endangered plants and for the rapid production of genetically stable and true-to-parent type progenies in short period of time (Fay, 1992). In vitro techniques are being adopted and recognized worldwide for commercial propagation of plants that are difficult to be cloned by conventional methods (Dodds, 1991; George and Sherrington, 1984). These techniques include seed germination, regeneration, micropropagation, somatic embryogenesis, meristem culture and callus culture (Fay, 1992). Moreover, in vitro cultures and regeneration of plant cells and tissues may offer a promising source for the production of metabolites that are difficult to obtain by conventional propagation methods (Cimino et al., 2006).

1.1.4.1 In vitro seed germination

Milk thistle plants, including seeds, are commonly infected by many pathogens (Berner et al., 2002; Kavallieratos et al., 2007). Seed is an important explant in establishing Silybum in-vitro cultures (Manaf et al., 2009). It is very common that contamination in in-vitro cultures frequently originates with the introduction into culture of explants contaminated with endophytic micro-organisms. These include pathogens of the plant and common environmental micro-organisms. Heterotrophic plant tissue media are capable of supporting the growth of many common environmental micro-organisms which can outburst as contamination in culture flasks (Abbasi et al., 2010). Contamination is the main limiting factor which can delay and

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inhibit the in-vitro culture growth; therefore, the best strategy is to control in-vitro culture contamination by establishing aseptic culture (Cassells et al., 1999). Different procedures have been employed for disinfection of Milk thistle seeds including surface sterilization with ethanol and sodium hypochlorite (Cimino et al., 2006; Hetz et al., 1995). However, treatment of explants with 70% ethanol and 0.2% mercuric chloride significantly reduced the level of contamination in in-vitro cultures of S. marianum (Manaf et al., 2009).

1.1.4.2 Explant type and choice

Selection and choice of appurtenant explant has an indispensable role in determining the efficiency of in-vitro propagation and varies with plant species (Thomas, 2003). Different explants derived from in-vivo or in-vitro grown Silybum plants are reported for establishment of various regeneration protocols in S. marianum (Asghari and Salimian, 2008; Bekheet et al., 2013; Gikloo et al., 2012; Hetz et al., 1995; Iqbal and Srivastava, 2000).

In early experiments mesophyll, protoplasts, seed, shoot apex, nodal sections, hypocotyls, cotyledons, stem, and leaf sections, have been used as explants for in- vitro regeneration via direct regeneration or through callus formation in S. marianum (Table 1.1). However, the most efficient explants reported for successful regeneration of S. marianum L. are seed derived shoot tips and leaf sections. Notwithstanding, explants derived from juvenile plant tissues have higher organogenic competence than mature plant tissues (Koroch et al., 2003). Furthermore, the type, size and physiological age of explant play an important role in determining the morphogenetic potential during in-vitro regeneration of complete plantlets either through callus cultures or through direct organogenesis (Debergh and Maene, 1981; Debergh and Read, 1991). In-vitro seed germination can be a continuous source of various explants at different growth stages and more importantly can provide fresh, healthy and sterile explants in aseptic condition due to maintenance of the donor plants in hygienic and controlled conditions (Koroch et al., 2003; Lai et al., 2005). Therefore, it is concluded that the most appropriate explant types for regeneration of S. marianum are shoot tip and leaf explants.

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1.1.4.3 In vitro Regeneration

In vitro regeneration of S. marianum can occur both by organogenesis and by somatic embryogenesis (Manaf et al., 2009; Radice and Caso, 1997). Solid MS (Murashige and Skoog, 1962) has been the first choice in most micropropagation studies on this species. However, the use of other media such as B5 and KM have also been reported (Cimino et al., 2006; Hetz et al., 1995).

1.1.4.3.1 Shoot organogenesis

Shoot organogenesis means the regeneration of shoots directly from differentiated tissues or indirectly from undifferentiated cells under in vitro conditions (Vanisree et al., 2004).

In vitro shoot morphogenesis is governed by many biochemical processes (Kumar et al., 2005). Type of explants, plant growth regulators, basal media and orientation of explants in the culture medium are the important factors in regulating the process of differentiation (Jones et al., 2007; Kumar et al., 2005). Additionally media supplements, alteration and modification also play a key role in the morphogenesis (Jones et al., 2007).

Previously, Liu and Cai (1990) determined callus mediated shoot organogenesis from hypocotyl explants of S. marianum with different auxin/cytokinin combinations. Application of BA (0.5 mg/l) in combination with NAA (0.5 mg/l) on to MS medium induced optimum callus formation. These calli upon sub-culturing into MS medium containing BA (2.0mg/l) and NAA (0.8 mg/l) resulted in 75% shoot formation. Nevertheless, BA alone at (3.0 mg/l) resulted in the highest frequency of direct shoot regeneration from cotyledonary node explants in S. marianum (Manaf et al., 2009). In another study, pre-treatment of the calli with lower levels of TDZ increased the shoot formation frequency up to 22 % when the calli were sub cultured on the medium containing BA (Hetz et al., 1995). Iqbal and Srivastava (2000) found best shoot regeneration steps for S. marianum on MS medium supplemented with NAA and Zeatin (Zn). Addition of BA significantly enhanced direct shoot regeneration from nodal explants. However, Radice and Caso (1997) found MS0 medium optimum for shoot regeneration. Moreover, Gikloo et al. (2012) induced direct shoot regeneration Strategies for the production of chemically consistent plantlets of Silybum marianum. L

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from mature leaf and petiole explants of in-vitro seed derived plantlets with various levels of BAP, NAA and Zn whereas combination of NAA (0.2 mg/l), BAP (0.3 mg/l) and Zn (0.3 mg/l) was more effective for direct shoot organogenesis from leaf explant.

1.1.4.3.2 Somatic embryogenesis

The ability of a plant somatic cell to produce embryo is referred to as somatic embryos and the process is known as somatic embryogenesis (Kumar and Thomas, 2012). The in-vitro system for embryogenesis produces uniform plants rapidly and easily which provides an ideal experimental means for investigation of plant differentiation as well as the large scale production of plants with consistent phytochemical profiles (Moon et al., 2013). An important application of somatic embryos is that it can be utilized for the preparation of artificial seeds or synthetic seeds which are analogous to the natural seeds (Kumar and Thomas, 2012). In the literature cited only one report is available on somatic embryogenesis of S. marianum. Somatic embryos were induced in cotyledon explants on MS medium containing (5.7 µM) IAA and (22 µM) BA (Radice and Caso, 1997).

1.1.4.3.3 Role of Auxins and Cytokinins in S. marianum regeneration

In various regeneration studies with a range of different explants tested, the medium augmented with BAP alone or in combination with other PGRs induced high rates of shoot organogenesis in S. marianum (Bekheet et al., 2013; Manaf et al., 2009). Nonetheless, Kinetin with IAA has also showed significant role in inducing shoot organogenesis in this species (Bekheet et al., 2013).

However, auxin in synergistic combination with a cytokinin has a remarkable influence on the regeneration process in Silybum. Iqbal and Srivastava (2000) reported that BA in combination with NAA is important for callus induction and shoot regeneration of S. marianum. Such synergism of PGRS is also reported by Asghari and Salimian (2008) for soft friable callus formation. Recently, Gikloo et al. (2012) reported that BA and Zn in combination with NAA stimulated direct shoot regeneration in Silybum. It means that BA is one of the important growth regulators for callogenesis and organogenesis of S. marianum. Strategies for the production of chemically consistent plantlets of Silybum marianum. L

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1.1.4.3.4 Thidiazuron a multifaceted plant growth regulator

Thidiazuron (TDZ) is a phenyl urea (N-phenyl-N-1,2,3-thidiazol-5-yl urea), initially it was described as a cotton defoliator but later on it is proven as an efficient plant growth regulator (Guo et al., 2013). TDZ is capable of dual function of fulfilling both the cytokinin and auxin requirement of various regenerative responses, but is structurally different from conventional PGRs (Arshad et al., 2012). It has been extensively used as a highly effective bio-regulator for plant morphogenesis in many micropropagation studies during last decade (Guo et al., 2013). At lower concentrations TDZ is reported to be much more effective (10-1000 times) in organogenesis via callogenesis in many plant species than the commonly employed conventional cytokinins (Wannakrairoj and Tefera, 2012). Furthermore, TDZ application has shown a profound effect on enhancing the levels of secondary metabolites in the in vitro cultures of many medicinal plants (Liu et al., 2007).

1.1.4.3.5 Effects of Dark-light cycling on in vitro culture

Light is an important factor influencing plant growth and development and has a significant role in regulation of metabolic pathways for the biosynthesis of valuable secondary metabolites (Shohael et al., 2006a). Acting as a stress inducer, it is widely believed that light has a stimulating effect on biosynthesis of plant flavonoids and anthocyanins (Zhong et al., 1991).

Application of dark-light cycling on in vitro cultures revamp the morphogenic potential in many elite medicinal plants, and act as external stress stimuli for determining in vitro plant development as well biosynthesis of important secondary metabolites (Shohael et al., 2006a). Since light is necessary for the formation of chlorophyll content which carries the precursors responsible for secondary metabolites biosynthesis, therefore, periodic dark-light cycling may alter these biosynthetic pathways (Zhong et al., 1991). These metabolic modifications as a consequence of stress conditions lead to the production of higher concentrations of secondary metabolites than mother plant (Shohael et al., 2006b).

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1.1.4.3.6 Root organogenesis

In vitro induction and development of roots in the regenerated shoot is a very critical step in any plant regeneration study and is normally achieved in an auxin containing medium (Zhong et al., 1991). Considerable work has been done to enhance rooting efficiency in S. marianum. Nevertheless, root induction is not restricted to a specific medium. The most potent auxins reported for development of root system in Silybum are IBA and NAA (Iqbal and Srivastava, 2000). However, addition of activated charcoal into rooting medium containing IBA uplifts root formation frequency (Gikloo et al., 2012). Radice and Caso (1997) reported root development from somatic embryos in the MS medium containing IBA. Moreover, MS medium devoid of any plant growth regulator (MS0) is also reported as an optimum medium for root development in S. marianum (Abbasi et al., 2010; Hetz et al., 1995).

1.1.4.3.7 Acclimatization and ex vitro transfer

Acclimatization and transfer of micropropagated plants in the field are critical steps determining the success of in vitro plant technology for commercial exploitation (Liu and Cai, 1990). Different strategies can be accomplished in the greenhouse as well as in the open field for acclimatization to achieve highest survival rates. The failure in these procedures is a deteriorating factor to the time and cost expenditures (Savangikar, 2004).

Initial optimization of the ex-vitro growth conditions prior transfer of the freshly regenerated plants is a prerequisite step in the acclimatization process (Savangikar, 2004). This can be achieved by transference of the well rooted plants to a mist chamber for a week to alleviate the low humidity, and then transfer into pots covered by polythene bags. The bags are later removed for acclimatization of the plants and no misting is then further required (Lai et al., 2005). Finally the plants are acclimatized in compost (peat: sand: soil) before transfer to field. However, the prevailing ex vitro conditions like humidity and temperature of the transplanting season greatly influence the initial survival of potted plantlets (Savangikar, 2004). Abbasi et al. (2010) reported 74% acclimatization and growth in the greenhouse of micropropagated S. marianum.

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Table 1.1: Use of explant types and PGRs in S. marianum and other Asteraceae species, and their regeneration response

Species Explant Response Medium + PGRs References S. marianum Hypocotyl Adventitious shooting MS + NAA, BA Liu and Cai (1990) S. marianum Mesophyll and protoplasts Adventitious shooting KM + BA, TDZ Hetz et al. (1995) S. marianum Young cotyledon Somatic embryogenesis MS + IAA, BA, IBA Radice and Caso (1997) P. hybridus Leaf and petiole Direct shooting MS + BA, NAA, Kn, Wildi et al. (1998) S. marianum Leaf and shoot tips In vitro micropropagation MSTDZ + NAA, BA, Zn Iqbal and Srivastava (2000) S. marianum Cotyledons Callogenesis B5 + BA, 2,4-D, NAA Cimino et al. (2006) A. scoparia Shoot tips, leaf and petiole Direct shooting MS + 2,4-D, NAA Aslam et al. (2006) S. marianum Seedlings Callogenesis MS + 2,4-D, Kn Asghari and Saliman (2008) S. marianum Hypocotyl In vitro micropropagation B5 + BA, 2,4-D Manaf et al. (2009) G. jamesonii Shoot tips and axillary buds Adventitious shooting MS + BA, 2,4-D, IBA Altaf et al. (2009)

S. marianum Leaf In vitro regeneration MS + BA, NAA, GA3 Abbasi et al. (2010) S. marianum Cotyledons Direct shooting, MS + BA Bekheet (2011) S. marianum Leaf In vitro micropropagation MS + BA, NAA, IBA Gikloo et al. (2012) S. marianum Shoot tips Direct shooting MS + BA, NAA Rady et al. (2013)

S. marianum Leaf, petiole and stem Direct shooting MS + Kn, IAA, GA3 Bekheet et al. (2013)

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1.1.5 In vitro cultures and biochemical characterization

Secondary metabolites are the compounds which serve within the plant as defense mechanism against pathogen, biotic and abiotic stresses (Tardif et al., 2008). These active metabolites are of great medicinal interest for their potential impact on human health. They exhibit antimicrobial or medicinal properties because of their unique biological functions (Luximon Ramma et al., 2003). Nonetheless, many secondary metabolites like phenolic acids, flavonoids‐ and anthocyanins are strong antioxidant in their nature (Khanavi et al., 2009). In vitro culture is the most attractive technology for the production of important antioxidant compounds than wild grown plants (Ahmad et al., 2013a). It provides many benefits like simple extraction and purification, independence of climatic factors and of seasonal variation, control over targeted metabolite production, shorter production cycle, fulfilling market demands and understanding the biosynthetic pathways regulating the production of metabolites (Grassmann, 2005). The hepatoprotective nature of S. marianum is due to the production of its important secondary metabolites, which are phenylpropanoids and are collectively called as silymarin. Silymarin is a mixture of different flavonoid isomers and have a strong antioxidant potential (Koksal et al., 2009). Moreover, the liver protecting effect of silymarin is most likely due to its free radical scavenging activity (Wallace, 2008). There are different methods available for the determination of antioxidant potentials in plant derived materials. Among these methods, the free radical scavenging activity by using colored or synthetic stable radicals of 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay is frequently used (Aruoma, 2003; Prieto et al.,1999).

1.1.5.1 DPPH free radical scavenging activity

Among the different tests used for the determination of antioxidant potentials of plant/s and its derived materials, DPPH free radical scavenging activity is the most simplest, inexpensive and robust method (Ali and Abbasi, 2013). This method determines the overall antioxidant potential of a sample whether it contains a solid or liquid component.

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Moreover, this assay is widely used for the quantification of significant antioxidants in in vitro regenerated plant tissues (Li et al., 2010). Different factors affect the production of important secondary metabolites during in vitro cultures (Duangporn and Siripong, 2009). Application of plant growth regulators like auxin and cytokinin alone or in combinations in in vitro cultures modulate the metabolic pathways and thus alter the production of secondary metabolites. Kinetin is reported for the enhanced production of anthocyanins in Haplopappus gracilus but inhibits anthocyanins in populous cell cultures (Seitz and Hinderer, 1988). Since these secondary metabolites may also include some antioxidants as a single compound or mixture which can easily be determined by DPPH free radical scavenging assay. Higher DPPH activity in reported for regenerated plantlets of Dendrobium tosaense than its wild grown plants (Lo et al., 2004). Numerous reports are available on free radical scavenging activity of wild-grown Silybum (Ligeret et al., 2008; Tawaha et al., 2007; Wojdyło et al., 2007). However, Abbasi et al. (2010) observed higher DPPH activity in callus cultures of S. marianum than wild grown plants.

1.1.5.2 In vitro cultures and anti-oxidative enzyme activities

Under normal growth conditions plants produce reactive oxygen species (ROS) important - - - ones are: superoxide (O2 ), peroxide (H2O2), hydroxyl (OH ) and singlet oxygen (O ) as metabolic by-products but in lower amount. However, increased amount of these ROS are produced when plant is under stress conditions which leads to oxidative damage of the plant cell (Cai Hong et al., 2005). Naturally, plants through its antioxidant system via enzymatic and non‐ enzymatic routes scavenge the ROS by coupling oxidation and reduction to minimize the oxidative damage caused by the free radicals (Molazem and Azimi, 2011). The antioxidant enzyme system in plants comprised of important enzymes like, ascorbate peroxidase (APX), peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) as they are normally involved in the regulation of processes for plant growth and developmental by giving protection to plant system from various stresses (Mitrovic and Bogdanovic, 2009).

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Peroxidases are used by the plants to remove H2O2 produced in chloroplast (Wood et al., 2003). However, they are reported to regulate in vitro morphogenesis in different plant species (Gupta and Datta, 2003; Meratan et al., 2009). POD has a significant role in auxin catabolism and participate in differentiation and in vitro regeneration (Mitrovic and Bogdanovic, 2009). Catalase converts hydrogen peroxide in to water and oxygen and regulate plant growth and development. During in vitro cultures CAT plays a crucial role in cell differentiation and is mostly involved in shoot organogenesis in different plant species (Meratan et al., 2009). Moreover, SOD exists in three different types depending on the metal type in its catalytic site, which are Cu/Zn-SOD, Mn-SOD and Fe-SOD (Halliwell et al., 1995). All of these SODs participate in overall defense system against ROS and regulate the normal plant growth and development (Gupta and Datta, 2003). Many researchers have reported the involvement of antioxidant enzyme system in plant organogenesis, differentiation, growth and regeneration (Gupta and Datta, 2003; Meratan et al., 2009). However, these enzymes are not tissue specific but fluctuate in different quantities during different growth stages (Abbasi et al., 2011).

1.1.5.3 PAL and silymarin biosynthesis

Silymarin, the most potent antioxidant so far is obtained from plant sources through phenylpropanoid metabolism of plants via shikimmate pathway (Koksal et al., 2009; Limem et al., 2008). Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is a key plant enzyme in the biosynthesis of silymarin and other phenolic compounds. It is involved in the enzymatic catalysis of phenylalanine to cinammic acid. The intermediate by product of the reaction is Trans-cinammic acid, which act as the principal modulator of PAL turnover (Figure 1.2). A variety of stimuli can induce PAL, the important stimuli comprised of radiation, light, temperature, plant hormones, wounding, and disease. This induction is often characterized by the concurrent in vivo development of a PAL inactivation system (Koksal et al., 2009; Limem et al., 2008). Evaluation of PAL activity in in vitro cultures can act as a best biochemical marker to monitor various regenerative responses during Silybum regeneration and to check the concurrent production of Strategies for the production of chemically consistent plantlets of Silybum marianum. L

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silymarin synchronized by this strategic enzyme. Nonetheless, PAL activity has been found in many plants, and is encoded by multigene family (Azzi et al., 2004).

Shikimate pathway

Phenyl ammonia lyase (PAL)

Gallic acid Phenyl alanine

Benzoic acid Ellagic acid Cinnamic acid

P -coumaric acid P-hydroxy benzoic acid

P-coumaroyl CoA

Quercitin Chalcone

Iso-flavones Flavanone Dihydro quercitin

Flavones Dihydro kaempferol Myricetin

Silymarin Kaempferol

Figure 1.3: Proposed biosynthetic pathways showing inter-relation between PAL and cinnamate leading to the production of silymarin (Hu et al., 2009).

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1.1.5.4 In vitro cultures and quality control

Plants produce a broad spectrum of medicinally important phytochemicals as secondary metabolites in order to cope with the stress conditions (Tardif et al., 2008). These compounds are of great medicinal importance to human health. Generally these compounds are accumulated and stored in enormous quantity in different parts of the plant like leaves, stem, flowers, root and seeds with varied chemical and physical properties (Luximon Ramma et al., 2003). These natural products are detected and isolated from different‐ parts on the basis of their unique properties like chemical polarity or physical boiling point. However, most of the isolated plant crude extract is a mixture of multiple compounds which pose a challenge in extraction and isolation procedures to target a single compound of interest in bulk of mixtures (Naczk and Shahidi, 2004). As isolation of plant based products is laborious and requires a highly skilled expertise, It usually becomes very difficult to isolate the compound of interest from mixtures if the nature of the compound is unknown or the required reference standard is missing or not available (Rimando et al., 2001). Nevertheless, these issues have been addressed by the exploitation of different chromatographic and separation techniques for isolation of maximum active compounds from plant parts by using appropriate solvent for extraction. Different solvents are frequently used for extraction of phytochemicals, these include ethanol, methanol, water, chloroform, petroleum ether and ethyl acetate (Naczk and Shahidi, 2004; Rimando et al., 2001).

Numerous chromatographic techniques are available, based on the common principle that separation of specific compound from the mixture is possible through the use of its differential distribution in mobile and stationary phases (Ullman, 2006). These techniques are widely demanding in pharmaceutical preparations for isolation of medicinally active natural products (Ikan, 1991). Since some of the important medicinally active compounds are present in very less amount in plants, isolating all existing compounds becomes troublesome in a mixture present in crude extract. This variation affects the quality and efficacy of many commercial therapeutic agents. The comprehensive protocols have Strategies for the production of chemically consistent plantlets of Silybum marianum. L

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devolved by WHO which ensure the safety and efficacy of the raw materials by guiding the farmers for correct package of practices including cultivation and post-harvest handling practices. Furthermore, micropropagation based quality control in medicinal species provide guarantee for the protection and efficiency of crude drugs before final pharmaceutical products formation (Liu et al., 2008).

High performance liquid chromatography (HPLC) is one of the most versatile chromatographic techniques which lead to the rapid and uniform separation of compound of interest with high accuracy. HPLC is a famous analytical tool for chemical analysis to assure quantification and characterization of bioactive materials in medicinal plants with a qualitative degree of reliability (Ahmad et al., 2013a). In addition to better separation of the components of a mixture, HPLC is the much improved and automated method as it uses a detector (Ikan, 1991).

1.1.5.5 Analysis of metabolic changes during in vitro morphogenesis

Metabolic profiling is the analysis to identify and quantify maximum possible metabolites in a biological sample and is a rapidly expanding field of interest in the biological sciences (Dunn et al., 2005; Fiehn, 2002). Metabolite finger printing can describe the metabolic events happening at a specific point in time as a consequence of cellular interaction to different environmental stimuli and thus can provide a snapshot to understand how an organism adapts to its environment (Bundy et al., 2009).

Organisms contain thousands of metabolites, with individual eukaryotic cells estimated to contain between 4,000 and 20,000 metabolites, whereas estimates of all metabolites in the plant and fungal kingdoms are up into the hundreds of thousands (Gummer et al., 2009). Therefore, to analyze as many of these as possible requires an instrument with good mass resolution and which can process samples quickly. Time of flight (TOF) mass spectrometers have the advantage that measurements are rapid and a scan of a full mass spectrum can be done in a few microseconds. TOF instruments also provide a better mass resolution than traditional quadrupole instruments (Dunn et al., 2005), and this higher

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resolution allows the detection of metabolites of different monoisotopic masses which have the same nominal mass (Marshall and Hendrickson, 2008). However, the choice of any technique to be used for metabolite profiling depends on the nature of the sample, metabolite type under analysis and the available equipment. Every approach has its own pros and cons, sufficient literature is available on the choice of approach (Dunn et al., 2005; Zhang et al., 2012) as well as direct comparisons between techniques (Barding et al., 2012) but eventually, a complete picture of metabolome can only be obtained by using a combination of techniques, although this is not always possible.

Electrospray ionisation (ESI) mass spectrometer is a rapid, highly sensitive and soft ionization technique which mainly produces the protonated molecular species for a wide range of compounds, thereby making data interpretation easier (Smedsgaard and Nielsen, 2005). During in vitro regeneration plant cell differentiates and dedifferentiates to progress its development from one growth stage to another. Furthermore, each growth phase is influenced by expression of some rare metabolites so evaluation of such unique metabolites during in vitro regeneration can give some significant insights to understand the metabolic pattern of plant growth. As a series of practical events are executed in-vitro for induction, multiplication, maturation and germination of somatic embryos (Helmersson et al., 2004). Using metabolic profiling, regulation of developmental events can be further elucidated at the metabolic level (Businge et al., 2012).

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1.1.6 Objectives

The aim of the present work was to optimize strategies for in vitro cultures to enhance growth parameters and silymarin content in S. marianum. Moreover, an attempt was made to evaluate the interplay of plant antioxidant elements in relation to silymarin production as a consequence of in vitro stress conditions. Within this aim the following objectives have been undertaken.

 Development of a feasible protocol for in vitro seed germination.

 Exploitation of TDZ for establishment of efficient regeneration protocol from leaf explants of S. marianum L.

 Development of a feasible protocol for somatic embryogenesis using leaf and petiole explants for production of chemically consistent plantlets.

 Evaluating the biochemical markers and key metabolites responsible for metabolic events at different growth phases during in-vitro somatic embryogenesis of S. marianum L.

 HPLC based fingerprinting of silymarin for quality control.

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Silybum marianum L.

Viable seeds

Surface sterilization

Culture on MS

medium + Hormones

In vitro seed germination

Acclimatization In vitro explants

Culture on solid

medium +Hormones

Rooted Embryogenesis plantlets Callus culture Somatic embryos

Roots Shoot organogenesis Embryos converting to plantlets

Figure 1.4: Schematic representation of various in vitro strategies for production of S. marianum L. plantlets. Strategies for the production of chemically consistent plantlets of Silybum marianum. L

Chapter 2

2. EFFECTS OF LIGHT REGIMES ON IN-VITRO SEED GERMINATION AND SILYMARIN CONTENT IN SILYBUM MARIANUM L.

2.1 Abstract

Silybum marianum is an economically important crop worldwide. It is renowned for production of biologically important silymarin. Average sale of silymarin is about US$ 8 billion/annum and its demand varies from 18-20 tons per year. Despite of its demand, there is lack of research efforts on cultivation and improvement of this plant. We hereby established feasible seed germination protocol for production of healthier and chemically consistent plantlets. Combination of Benzyladenine (BA, 0.5 mg/l) +

Gibberellic acid (GA3, 1.5 mg/l) + Thidiazuron (TDZ, 1.0 mg/l) produced optimum germination frequency in seeds kept in 2 weeks dark and subsequently transferred to 2 weeks light (16 hrs photoperiod) conditions. Correlation among mean shoot length, mean root length, set of antioxidative enzyme activities was also observed in current report. Silymarin was determined by High performance liquid chromatography (HPLC). Considerable amount of silymarin (5.48 mgg-1 DW) was detected in our study, which was comparative to other reports available. Antioxidant activity (1, 1- diphenyl-2-picrylhydrazyl; FRSA) and phenylalanine ammonia lyase (PAL) activity was also determined. Silymarin content had shown direct relationship with these activities. It showed that silymarin was a major antioxidant in current report. This study provides basis for expedited production of S. marianum plantlets with feasible content of silymarin.

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2.2 Introduction

Silybum marianum L. is an important medicinal plant belongs to family Asteraceae (Abbasi et al., 2010). Its major bioactive component is silymarin; which is an isomeric mixture of flavonolignans (Pliskova et al., 2005; Vaid and Katiyar, 2010). Silymarin is the most proven phytochemical with known mechanism of action against viral hepatitis in various clinical studies (Huseini et al., 2006; Thabrew and Hughes, 1996). Furthermore, silymarin has antioxidant, anti-inflammatory, antifibrotic, immuno-modulatory and antitumor effects (Agarwal et al., 2006; Flora et al., 1998; Katiyar et al., 1997; Kuki et al., 2012).

It is very common for this species to have optimum germination at 2–15 °C in autumn and spring seasons in soil; with low to moderate nutritional requirements. It is also adaptable to poor quality soils. However, the poor nutritional status of the soil is the limiting factor for the production of healthy Silybum plantlets with enhanced and consistent chemical profiles (Karkanis et al., 2011). In-vitro growth conditions can overcome these issues, which lead to extensive chemical and genetic variability in many wild and cultivated medicinal plant species. This variability occurs as a consequence of abiotic and biotic contamination, and is the major bottleneck in the production of high-quality plant material for preparation of effective phytomedicine (Murch et al., 2000). Furthermore, advantages of seed explants are also available in literature (Malik and Saxena, 1992; Victor et al., 1999).

In-vitro seed germination is often considered as the most sensitive stage in the life cycle of economically important plant species, and it normally shortens the period required for establishment of vegetative clones by simplifying the methods for obtaining regenerants by bypassing callogenesis and retaining the integrity of the seedling (Nikolic et al., 2006). Under normal growth conditions plants produce - - reactive oxygen species (ROS); like superoxide (O2 ), peroxide (H2O2), hydroxyl (OH - ) and singlet oxygen (O2 ) as metabolic byproducts (Cai et al., 2004). However, stress conditions enhance production of these ROS; which leads the plant cell to switch on its antioxidant system via enzymatic and non enzymatic routes to scavenge the

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reactive oxygen species by coupling redox to minimize the oxidative damage (Molazem and Azimi, 2011).

Antioxidative enzymes like superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD) are of significant importance in plant growth and development. These antioxidative enzymes regulate various processes including germination, growth of seedling and the process of senescence through acting against various stresses (Mitrovic and Bogdanovic, 2009). Silymarin released SOD has shown a significant role in destroying free radicals caused by alcohol in the liver (Ahmad et al., 2013b). Light is an important factor influencing plant growth and development and has a significant role in regulation and synthesis of valuable phytochemicals (Abbasi et al., 2007; Shohael et al., 2006b). Acting as a stress inducer, it has a stimulating effect on biosynthesis of important secondary metabolites (Zhong et al., 1991). The main objective of present study was to evaluate effects of different light regimes on in vitro seed germination of S. marianum and determination of silymarin content in germinated plantlets by HPLC. Furthermore, role of antioxidative enzymes and other biochemical parameters were also determined.

2.3 Materials and Methods

2.3.1 Seed Germination

The seeds of S. marianum were collected from wild plants grown in Main Campus of Quaid-i-Azam University, Islamabad, Pakistan in 2011. These seeds were surface sterilized by immersion in 0.5% HgCl2 solution for ~1 min, 70% ethyl alcohol for ~3 min and washed three times with sterile distilled water. Sterilized seeds were then placed on to (Murashige and Skoog, 1962) medium containing 30 g l-1 sucrose, and solidified with 8 g l-1 agar. Plant growth regulators [Benzyladenine (BA), Gibberellic acid (GA3), α-Naphthalene acetic acid (NAA) and Thidiazuron (TDZ)] were added to the medium either alone or in combinations, the pH was adjusted to 5.8. All media were autoclaved at 121°C for 20 min.

In preliminary studies BA, GA3, NAA and TDZ were added to MS medium either alone at concentrations of (0.5, 1.0 and 1.5 mg/l) or in combination of; TDZ (0.5, 1.0, and 1.5 mg/l) + BA (0.5 mg/l) / GA3 (1.5 mg/l), BA (0.5 mg/l) + GA3 (1.5 mg/l) as Strategies for the production of chemically consistent plantlets of Silybum marianum. L

Chapter 2

shown in Table 1. BA (0.5 mg/l) + GA3 (1.5 mg/l) + TDZ (1.0 mg/l) showed optimum response and selected for subsequent experiments with various photoperiods as described in Table 2.

For dark treatments, the culture flasks were wrapped in aluminium foils. For light treatments, the flasks were exposed to white tubular fluorescent lights with a light intensity ranges from ~40-50 µmol m-2 s-1. All cultures were maintained in growth room at temperature of 25±2°C.

2.3.2 Analytical methods

Plant tissues were collected from T1 to T7 and growth room potted wild plantlets after one month (Table 2.2). For fresh weight (FW) determination, the plant samples were gently pressed on filter paper to remove excess water and weighed. Subsequently, the plant materials were dried in an oven at 60 °C for 24 h and dry weight (DW) was recorded.

2.3.2.1 Antioxidant enzyme activities

For antioxidative enzyme activities, the extract was prepared from fresh plant tissue by modifications in the method of Abbasi et al. (2011); briefly homogenate of in-vitro germinated plantlets was extracted with ice-cold 0.5 M Tris-HCl (pH 6.8) buffer. The extracts were centrifuged at 10000 rpm for 20 min at 4°C and resulting supernatant was kept in freezer and used for enzyme assays. UV-visible spectrophotometer (Halo DR-20, UV-VIS spectrophotometer, Dynamica Ltd, Victoria, Australia) was used to determine absorption of extracts. Superoxide dismutase (SOD; EC 1.15.1.1) was determined by the method of Giannopolitis and Ries (1977), Catalase (CAT; EC 1.11.1.6) was determined by the method of Arrigoni et al. (1992), Peroxidase (POD; EC 1.11.1.7) was determined according to Abeles and Biles (1991) and Ascorbate peroxidase (APx; EC 1.11.1.11) was determined by the method of Miyake et al. (2006).

2.3.2.2 Assay of PAL activity

PAL activity (EC 4.3.1.5) was determined by the method of Wakabayashi et al. (2012), briefly the freeze dried plant material (80-100mg FW) was homogenized in a

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mortar pestle with ice-cold 100 mM potassium borate buffer (pH 8.8) containing 2 mM mercaptoethanol, and then centrifuged at 12,000 rpm for 10 min at 4 ◦C. After centrifugation, the supernatant was used for the assay of PAL activity. For the assay of PAL activity, the reaction mixture (2 mL) contained 0.5 mL of 4 mM-1 phenylalanine, 0.5 mL of extract, and 1 mL of 100 mM potassium borate buffer (pH 8.8). The mixture was incubated at 30°C; the reaction was terminated by addition of 0.2 ml of 6 M HCl and the absorbance was recorded at 290 nm after 30 min. The amount of product formed was calculated from the increase in absorbance. One unit of the PAL activity (U) is defined as the amount of absorbance variation of 0.01 (Asample-Acontrol).

2.3.2.3 DPPH- Free radical-scavenging activity (FRSA)

Antioxidant activity was determined by some modifications in the method of Abbasi et al. (2010). Briefly, 10.0 mg of dried plant tissue was dissolved in 4 ml of methanol and then added to methanolic solution of DPPH° (1, 1-diphenyl-2-picrylhydrazyl; 1 mM, 0.5 ml). The mixture was vortexed and absorbance of the resulting solution was read spectrophotometrically at 517 nm. A methanolic solution of DPPH that had decayed and hence no longer exhibited purple color (2 mg of butylated hydroxyanisole (BHA) dissolved in 4 ml of methanol with 0.5 ml of DPPH solution added) was used for background correction instead of pure methanol. Finally, the radical scavenging activity was calculated as %age of DPPH discoloration using the equation:

% scavenging DPPH free radical = 100 × (1-AE/AD)

Where AE is absorbance of the solution when an extract was added at a particular concentration and AD is the absorbance of the DPPH solution with nothing added.

2.3.2.4 Silymarin extraction and HPLC analysis

Extraction and quantification of silymarin from dried plant samples was determined according to the method of Hasanloo et al. (2005). Briefly, 200 mg of dry plant sample was ground, and ultrasonicated in methanol and 0.1% phosphoric acid (70:30, v/v; 1ml for each) for 30 min. The High Performance Liquid Chromatography

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(HPLC) system (Shimadzu Lc8A, Japan) was set with binary pump, a variable wavelength () detector, solvent vacuum degasser, and an auto sampler with a 10 l injection loop. The column was C18 (ODS) with 150 × 4.6 mm, 5m particle size.

Ultra pure water containing 0.1% phosphoric acid (A) and acetonitrile (B) were used as chromatographic eluents. The gradient elution for silymarin was programmed as follows: 0-30 min, 10-20% B; 30-110 min, 20-80% B. The flow rate was 1.0 ml/min and the injection volume was 10 µl. Reference standard of silymarin was purchased from Sigma (CA, USA). Identification was achieved by comparison of the sample retention time (Rt) with that of the standard. Quantification of silymarin was expressed in mg g-1 of dry weight, was accomplished using a known concentration of standard and peak areas.

2.3.2.5 Statistical analysis

For statistical analysis, five culture flasks were used and data were collected from duplicates. ANOVA (Analyses of variance) and DMRT (Duncan multiple range test) were used for comparisons of means of different treatments. SPSS (Windows version 7.5.1, SPSS Inc., Chicago) was used to determine the significance at P<0.05.

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Chapter 2

2.4 Results and Discussion

2.4.1 Seed germination frequency and growth parameters

Several PGRs were exploited in preliminary experiments (Table 2.1). However, a combination of BA (0.5mg/l) + GA3 (1.5 mg/l) + TDZ (1.0 mg/l) produced optimum response in controls (16 h light/8 h dark) and performed extensive experiments with different photoperiods in subsequent research (Table 2).

Optimum % seed germination was observed for T5 and lowest was recorded for T9 (Figure 2.1). This shows that incubation of seeds in complete darkness for 2 week and subsequent transference to 16 h light/8 h dark significantly enhanced seed germination frequency in S. marianum. In preliminary experiments, it was found that combination of TDZ, GA3 and BA produced optimum seed germination frequency (Table 2.1). Nikolic et al. (2006) concluded from their study on Lotus corniculatus that all cytokinins stimulated % seed germination under 16 h light. Furthermore, % germination was found to be improved by presence of TDZ in culture medium in Carica papaya under 16 h light (Bhattacharya and Khuspe, 2001). Contrarily, MS medium incorporated with BA and TDZ and light and dark treatment had no effect on seed germination in Grammatophylum speciosum (Khampa et al., 2007). In our study, MS basal medium produced lowest % seed germination frequency. Although, hormone free MS basal medium was found most effective for seed germination of Dendrobium densiflorum (Pradhan and Pant, 2009). Exposure to continuous light produced lesser % seed germination than continuous dark conditions or 16 h light/8 h dark conditions (Figure 2.1). Contrarily, seeds of some plant species failed to germinate in the absence of light (Dissanayake et al., 2010). Our data indicated that pre-incubation of seeds to darkness and subsequent transfer to light conditions produced better results than pre-incubation in light and subsequent transfer to darkness (Figure 2.1). Dissanayake et al. (2010) concluded from their research that light and GA3 strongly interact to overcome dormancy in ``Guayule``. Some species are affected by brief exposure to light, other require intermittent illumination or require exposure for long periods to induce germination (Penfield et al., 2005).

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Table 2.1: Effects of several plant growth regulators on seed germination frequency, mean shoot length and mean root length in culture flasks under 16-h light and 8-h dark for 4weeks. Data show mean of three replicates ± SE.

S # % Seed Mean length (cm) MS + PGRS (mg/l) germination Shoot Root 1 MS (0) 14 ± 2.7 0.9 ± 0.6 0.7 ± 0.3 2 BA (0.5) 43 ± 4.2 1.3 ± 0.3 0.8 ± 0.4 3 BA (1.0) 38 ± 4.0 1.1 ± 0.5 0.6 ± 0.4 4 BA (1.5) 31 ± 2.4 0.8 ± 0.4 0.3 ± 0.4

5 GA3 (0.5) 22 ± 3.6 1.1 ± 0.5 0.4 ± 0.5

6 GA3 (1.0) 28 ± 6.1 2.3 ± 0.6 0.9 ± 0.4

7 GA3 (1.5) 33 ± 3.4 3.0 ± 0.4 1.6 ± 0.3 8 NAA (0.5) 18 ± 3.7 2.4 ± 0.6 1.8 ± 0.6 9 NAA (1.0) 14 ± 1.2 2.1 ± 0.3 1.4 ± 0.6 10 NAA (1.5) 09 ± 1.5 1.9 ± 0.4 1.1 ± 0.5 11 TDZ (0.5) 22 ± 3.6 1.1 ± 0.5 0.3 ± 0.3 12 TDZ (1.0) 28 ± 1.4 1.6 ± 0.3 0.9 ± 0.4 13 TDZ (1.5) 18 ± 1.4 1.3 ± 0.3 0.4 ± 0.5 14 TDZ (0.5)+BAA (0.5) 32 ± 3.5 2.0 ± 0.4 1.4 ± 0.6 15 TDZ (1.0) + BAA (0.5) 40 ± 6.8 2.3 ± 0.6 1.6 ± 0.4 16 TDZ (1.5)+BAA (0.5) 33 ± 3.4 1.9 ± 0.4 1.2 ± 0.4

17 TDZ (1.0)+GA3 (1.5) 27 ± 7.5 1.1 ± 0.5 0.2 ± 0.4

18 TDZ (1.5)+GA3 (1.5) 33 ± 3.4 1.9 ± 0.6 0.8 ± 0.4

19 TDZ (1.5)+GA3 (1.5) 30 ± 5.5 1.6 ± 0.3 0.4 ± 0.5

20 BA (0.5)+GA3 (1.5) 48 ± 5.2 2.8 ± 0.5 1.6 ± 0.3

21 BA (1.0)+GA3 (1.5) 41 ± 5.2 2.5 ± 0.2 1.3 ± 0.3

22 BA (1.5)+GA3 (1.5) 36 ± 3.7 2.1 ± 0.3 1.1 ± 0.5 23 BA (0.5)+GA3+(1.5)+TDZ(0.5) 58 ± 5.1 2.9 ± 0.6 1.7 ± 0.6 24 BA (0.5)+GA3(1.5) +TDZ(1.0) 64 ± 4.9 3.2 ± 0.4 1.9 ± 0.6

25 BA (0.5)+GA3(1.5)+TDZ(1.5) 53 ± 5.0 2.7 ± 0.6 1.5 ± 0.3

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Table 2.2: Reference table showing the application of different photoperiods in conjunction with combination of PGRs from T1 to T9 for in- vitro seed germination of Silybum marianum.

Treatment MS + PGRS (mg/l) Light regimes Incubation period

T1 BA (0.5) + GA3 (1.5) + TDZ (1.0) Darkness 24-h dark (4weeks)

T2 BA (0.5) + GA3 (1.5) + TDZ (1.0) Light 24-h light (4weeks)

T3 BA (0.5) + GA 3 (1.5) + TDZ (1.0) Photoperiod 16-h light and 8-h dark (4weeks)

T4 BA (0.5) + GA3 (1.5) + TDZ (1.0) Photoperiod 1 week dark + 3 weeks light (16/8-h)

T5 BA (0.5) + GA3 (1.5) + TDZ (1.0) Photoperiod 2 weeks dark + 2 weeks light (16/8-h)

T6 BA (0.5) + GA3 (1.5) + TDZ (1.0) Photoperiod 3 weeks dark + 1 week light (16/8-h)

T7 BA (0.5) + GA 3 (1.5) + TDZ (1.0) Photoperiod 1 week light (16/8-h) + 3 weeks dark

T8 BA (0.5) + GA3 (1.5) + TDZ (1.0) Photoperiod 2 weeks light (16/8-h) + 2 weeks dark

T9 BA (0.5) + GA3 (1.5) + TDZ (1.0) Photoperiod 3 weeks light (16/8-h) + 1 week dark

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Nonetheless, in vitro conditions are reported to enhance seed germination frequency in different cultivars of papaya (Bhattacharya and Khuspe, 2001). Preconditioning treatments are not necessarily enhancing seed germination in some economically important plant species (Jorge et al., 2006). Furthermore, light spectral quality is also reported to affect seed germination frequency in Calanthe (Baque et al., 2011).

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100

90 a ab 80 b b 70 bc 60

40 d d 30 de

% seed germination % seed 20 e ef 10

T1 T2 T3 T4 T5 T6 T7 T8 T9 MS0 Treatments

Figure 2.1: Effects of different light treatments on % seed germination. Data were collected after 4 weeks of culture. Values are the mean ± standard error from three replicates. Columns with similar alphabets are not significantly different at P<0.05.

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Data of mean shoot length and mean root length is shown in (Figure 2.2). Optimum shoot and root length (5 cm and 3.3 cm, respectively) was recorded for T5. Similar findings were made by Li et al. (2010) for black pepper. Lowest shoot and root length was recorded for T9. This data showed that shoot and root length were correlated. MS basal medium produced better data than T9. Application of GA3 produced higher response in seed germination than Kinetin and Indole acetic acid (IAA) in black pepper (Li et al., 2010). Plants collected from T5 treatment were healthier than other treatments. Addition of peptone is reported to enhance shoot and root length in seedlings of Dactylorhiza spp. (Vejsadova, 2006). Pre-incubation with light (T9) produced retarded plants. However, normal plants were produced on MS basal medium. This data shows that pre-incubation in dark enhanced growth parameters.

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6.5 Mean shoot length 6.0 a Mean root length 5.5 5.0 4.5 4.0 bc bc bc 3.5

3.0 c c c 2.5 c c cd 2.0 cd bc

Mean length (cm) length Mean 1.5 d d d d d d 1.0 de de 0.5 0.0

T1 T2 T3 T4 T5 T6 T7 T8 T9 MS0 Treatments

Figure 2.2: Effects of different photoperiods on shoot and root induction response. Data were collected after 4 weeks of culture. Values are the mean ± standard error from three replicates. Columns with similar alphabets are not significantly different at P<0.05.

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2.4.2 Antioxidant enzymes activity

The sequence of the metabolic pattern that occurs during germination involves the activation of specific enzymes at the appropriate times and regulation of their activity. Antioxidative enzyme system (SOD, CAT, APX and POD) occurs in plants to check and balance this stress (Vatankhah et al., 2010). We determined activities of SOD, CAT, APX and POD to evaluate their role against light-induced stress (Figure 2.3). Highest activity of SOD was recorded for T5. However, comparative levels of SOD were - recorded for T2 and T7. SOD is involved in the initial catalytic conversion of O2 into

H2O2 and O2. The higher SOD activity in the T5 plantlets might be attributed to plant antioxidant system for elimination of ROS to attain highest germination frequency with longest shoots and roots. The subsequent transference of culture flasks from dark treatment to light treatment in conjunction with PGRs in T5 might have induced stress and consequently caused production of ROS. Comparatively, higher levels of POD and CAT were recorded for T5 and T7 than other treatments (Figure 2.3). Ducic et al. (2003) detected enhanced CAT and SOD activity during early stages of germination of Chenopodium seeds. However, they also found higher levels of POD in later stages of germination. Kang and Saltveit (2002) observed negative effect of chilling and heat shock on antioxidative enzyme activities of germinated seeds of Rice. Tang and Newton (2005) found that POD and CAT activities were enhanced by addition of TDZ in to growth medium. APX was significantly higher in T5 than other treatment conditions tested. APX is a member of ascorbate- glutathione cycle and has a vital role in the detoxification of plant tissues from poisonous H2O2 (Shohael et al., 2006a). Jowkar et al. (2011) demonstrated higher APX and POD activity in osmo-priming treated seeds of Silybum marianum ecotypes. Correlation in data of antioxidative enzymes and seed germination frequency and growth parameters showed involvement of these enzymes.

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80 SOD 70 POD CAT a ab 60 APX ab

50 b b b b bc 40 bc bc bc c c

30 c cd cd cd 20 cd cd d dcd d cd 10 d d d d d dd d 0

Antioxidative enzyme activity (U/mg protien) (U/mg activity enzyme Antioxidative

T1 T2 T3 T4 T5 T6 T7 MS0 Treatments

Figure 2.3: Activities of antioxidative enzymes (SOD, APX, CAT and POD in U/mg protein) in plantlets grown under different light regimes in in vitro conditions and wild- grown plantlets of Silybum marianum. Values are the mean ± standard error from three replicates. Columns with similar alphabets are not significantly different at P<0.05.

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2.4.3 Silymarin content, phenylalanine ammonia lyase activity and radical scavenging activity

Silymarin was detected and quantified by High performance liquid chromatography (HPLC) in different samples of S. marianum (Figure 2.4). 5.48 mg/g silymarin was detected in T5 plantlets. Our data is comparative to other reports available (Table 2.3). However, considerably higher content of silymarin is reported for fruits of S. marianum by Hasanloo et al. (2005). Hasanloo et al. (2008) obtained higher silymarin content in cells of S. marianum kept in dark. Picloram, jasmonic acid and L-phenylalanine are reported to elicit accumulation of silymarin in submerged cultures of S. marianum (Hasanloo et al., 2008; Rahimi et al., 2011). Biosynthesis of silymarin involves activity of phenylalanine ammonia lyase (PAL). PAL is a strategic enzyme in biosynthesis of phenylpropanoid metabolism (Koksal et al., 2009). Highest PAL activity was recorded for T5. Correlation in PAL activity and silymarin was observed in our results (Figure 2.5). Light and exposure to ultrasound waves are reported to enhance activity of PAL in hairy root cultures of some medicinal plant species (Abbasi et al., 2007). Naringenin is also reported to enhance PAL activity in S. marianum hairy root cultures (Rahimi et al., 2011). Free radical scavenging activity (FRSA) was determined by DPPH method (Figure 2.5). This activity was higher in plants collected from T5 treatment. FRSA of silymarin is reported in literature (Abbasi et al., 2010; Koksal et al., 2009), and our data endorsed these reports. Correlation was observed among FRSA, PAL and silymarin content in our report. Similar findings were made by Abbasi et al. (2007) for Echinacea hairy roots.

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6 a

5 ab

3 bc

2 c

Silymarin content (mg/g) content Silymarin c c 1 cd cd cd

T0 T1 T2 T3 T4 T5 T6 T7 MS0 Treatments

Figure 2.4: Silymarin content (mg/g DW) in in vitro-grown and wild-grown plantlets of Silybum marianum. (T0= Wild grown plants). Values are the mean ± standard error from three replicates. Columns with similar alphabets are not significantly different at P<0.05.

Table 2.3: Comparative analysis of silymarin content

Reference Plant tissues Silymarin (mg/g DW) This study Intact plantlets 5.48 Rahimi et al. (2011) Hairy roots 7.5 Rahnama et al. (2008) Hairy roots 0.11 Hasanloo et al. (2008) Cell culture 0.413 Hasanloo et al. (2005) Fruits 27.1

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90 DPPH free radical scavanging activity 80 PAL activity a 70 b ab 60 b bc 50 bc bc bc

40 c c 30 cd cd cd d cd d PAL activity (U/g FW) activity(U/g PAL 20

Free radical scavanging activit (RSA%) radicalscavanging Free

T1 T2 T3 T4 T5 T6 T7 MS0 Treatments

Figure 2.5: Phenylalanine ammonia lyase (PAL) activity (U/mg protein) and % antioxidant activity in in vitro-grown and wild-grown plantlets of Silybum marianum. Values are the mean ± standard error from three replicates. Columns with similar alphabets are not significantly different at P<0.05.

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Chapter 3

3. THIDIAZURON-INDUCED REGENERATION AND SILYMARIN CONTENT IN SILYBUM MARIANUM L.

3.1 Abstract

Silybum marianum, of family Asteracea is renowned for production of biologically important silymarin, which has shown multi-dimensional medicinal properties. It has a high protective role against jaundice and hepatitis C worldwide. We hereby established a feasible and efficient method for indirect regeneration of S. marianum for production of consistent plantlets. Calli were induced from leaf explants of seed-derived plantlets on Murashige and Skoog (MS) medium supplemented with several concentrations of different plant growth regulators (PGRs). Highest callogenic response (89%) was recorded for 4.4µM Thidiazuron (TDZ) in combination with 6.6µM Kinetin (Kn). Subsequent sub- culturing of callus after 4 weeks of culture, on medium with similar compositions of PGRs induced shoot organogenesis. Highest shoot induction frequency (86%) with maximum mean multiple shoots (26 shoots per explant) were recorded for 11µM TDZ after 4 weeks of transfer. Longest shoots (4.1 cm) were recorded for MS medium augmented with 6.6µM TDZ and 4.4µM α-naphthalene acetic acid (NAA). Furthermore rooted plantlets were developed on MS medium containing different concentrations of indole acetic acid (IAA). Silymarin was determined by High performance liquid chromatography (HPLC) and 8.47 mg/g DW silymarin was detected in the regenerated plantlets. This study contributes to a better understanding of the different mechanisms involved in morphogenesis and production of biologically active principle in Silybum marianum.

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3.2 Introduction

Silybum. marianum L. Gaertn. belongs to family Asteracea and is commonly known as Milk thistle, it is native to the Mediterranean basin and is now widespread throughout the world. It is cultivated as a medicinal plant (Khan et al., 2009) and has been used from ancient times (Kren and Walterova, 2005). It has an isomeric mixture of multiple flavonolignans called silymarin that is considered as the most potent antioxidant (Khan et al., 2013). Approximately 8 to 33% of patients with hepatic disorders consume this plant frequently as herbal remedy worldwide (Polyak et al., 2007). However, there are some issues with Silybum products, as existing high variability in the end products has resulted inconsistent results regarding its efficacy in various clinical trials (Haban et al., 2009; Lee and Liu, 2003). Therefore, control, characterization and standardization of silymarin appear to be a necessary mandate for formulation of end products with consistent efficacy (Aruoma, 2003).

Consistent production of phytochemicals can be achieved via in vitro clonal propagation (Parveen and Shahzad, 2010). The increasing world’s demand for silymarin (18-20 tons per year) is endangering the sparse populations of this species, still this economically important herb has been ignored in research programs for its improvement and cultivation (Ahmad et al., 2008). Plant in-vitro technology is capable of producing large number of aseptic, genetically similar, and chemically consistent plants in a short period of time and limited space for the production of high quality medicines (Victorio et al., 2012).

Thidiazuron (N-phenyl-N-1,2,3-thidiazol-5-yl urea) has proven to be highly effective bio regulator of plant morphogenesis. It is reported to be much more effective in organogenesis via callogenesis in many plant species than the commonly employed conventional cytokinins (Hussain et al., 2013; Wannakrairoj and Tefera, 2012). Initially TDZ was classified as cytokinin due to its cytokinin like responses. However, later on research showed that TDZ is capable of fulfilling both the cytokinin and auxin requirement of various regenerative responses in many plant species (Arshad et al., 2012). Furthermore TDZ application has a profound effect on enhancing the

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levels of secondary metabolites in the in vitro cultures of many medicinally important plant species (Liu et al., 2007).

The main objective of present study was to exploit TDZ for establishment of efficient regeneration protocol for S. marianum and to evaluate its impact on accumulation of silymarin in regenerated tissues and plantlets by HPLC.

3.3 Materials and Methods

3.3.1 Micro propagation

3.3.1.1 Seed germination

In vitro seed germination was achieved by the feasible protocol reported by Khan et al. (2013) as described in chapter 2.

3.3.1.2 Plant growth regulators for regeneration

Leaf explants (1.5 cm2) were excised from in vitro germinated 28-days old seedlings and were placed onto Murashige and Skoog (1962) basal medium (MS, 1962; Phytotechnology Labs, USA) containing 3% sucrose and 0.8% (w/v) agar (Phytotechnology Labs, USA) in 150 ml conical flask supplemented with various PGRs like Thidiazuron (TDZ), Kinetin (Kn) and α-napthalene acetic acid (NAA). Different concentrations (1.0, 2.2, 4.4, 6.6, 8.8, 11, 13.2 and 15.4µM) of TDZ or (1.0. 2.2, 4.4, 6.6 and 8.8 µM) of Kn or NAA either alone or in combinations were tested. The pH (Eutech Instruments pH 510, Singapore) of media was adjusted to 5.8 before autoclaving (121°C, 20 min, Systec VX 100, Germany). MS basal medium (MS0) without any plant growth regulator (PGR) was used as control for different phases of regeneration.

After 4 weeks, number of responding explants (% callus induction) was recorded. Callus was refreshed by sub-culturing to MS medium with similar composition of PGRs for further growth and multiplication. Data on % shoot induction response; number of shoots per explant and mean shoot length (cm) was collected after 4 weeks of callus sub-culturing. Elongated shoots were transferred to rooting medium for root induction. Rooted plantlets were removed from flasks, washed with double distilled

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water and transferred to pots for acclimatization. For fresh weight (FW) determination, the plant samples were gently pressed on filter paper to remove excess water and weighed. Subsequently, the plant materials were dried in oven at 35°C for 24 h and dry weight (DW) was recorded.

3.3.1.3 Silymarin assesment by HPLC

For silymarin content determination, different samples (calli, regenerated shoots, regenerated plantlets, and growth room potted plantlets) were subjected to High Performance Liquid Chromatography (HPLC; Shimadzu Lc8A, Japan). Extraction and quantification of “silymarin” from dried plant samples was done by the valuable protocol of Khan et al. (2013) as described in chapter 2.

3.3.2. Statistical Analysis

For statistical analysis, each treatment consisted of 10 culture flasks and data was collected from triplicates. Analysis of variance (ANOVA) and Duncan’s multiple range test (DMRT) was used for comparison among treatment means. However, for chromatographic quantification analysis, data were collected from triplicates and represented as values of mean.

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3.4 Results and discussion

3.4.1 Callus Induction frequency

TDZ is considered as one of the most potent bioregulator for indirect regeneration in many plant species (Murthy and Saxena, 1998). TDZ induced callogenesis from different explants of many medicinal plants has been reported (Mithila et al., 2003), suggesting TDZ as propitious PGR for callus induction than other cytokinins (Thomas, 2003). Incubation of leaf explants on to MS medium incorporated with different concentrations of PGRs, induced callus in 7 days of culture time (Figure 3.1). Negligible (5%) microbial contamination was observed during explants culturing. Explants taken from in vitro grown plantlets (Zheng et al., 2009) are often more responsive in micropropagation than explants derived from in vivo grown plants (Abbasi et al., 2010).

In present study, TDZ alone at lower concentrations (2.2 µM) produced optimum callus (73%). Notwithstanding, a decrease in callus formation was observed with increase in levels of TDZ. At concentration ≥ 2.2 µM, pale yellow calli were induced. These observations are in agreement with (Radhika et al., 2006). No callus induction response was observed in controls (Figure 3.1). Moreover, the callogenic response was enhanced (89%) when MS medium was augmented with TDZ (4.4µM) in combination with Kn (6.6µM) (Figure 3.1). Calli developed were soft, friable and greenish white with a diameter of 1.5 cm after four weeks of explant culturing. Similar morphological parameters in callus formation were observed by Mroginski et al. (2004). Similarly, a number of reasons can be anticipated for the development of different characteristics in callus cultures like nature, color and biochemical composition, which are usually determined by the composition of the medium; PGR type and concentration along with explants culturing practices (Danya et al., 2012) and such variations in calli have been reported in many plant species (Ishii et al., 1998).

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a ab 80 ab ab b b bc b 60 bc bc bc bc c c c c c c

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TDZ 13.2uM Plant Growth regulators

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TDZ8.8+NAA2.2uM

TDZ 4.4+NAA6.6uM TDZ 6.6+NAA4.4uM

TDZ 2.2+NAA8.8uM Figure 3.1: Data on callus formation parameters in Silybum marianum by the application of various concentrations and combinations of TDZ, Kn and NAA. Values are mean of three replicates and observations were recorded after 4 weeks of culture.

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3.4.2 Shoot Organogenesis

The fast, friable and competent calli were transferred into shoot organogenesis media. Highest shoot regeneration frequency (86%) with a maximum number of shoots (26 shoots per explants) was recorded for MS medium supplemented with 11µM TDZ. However, shoot regeneration was induced on a wide range of TDZ concentrations (Figure 3.2 and 3.3). In current report, TDZ alone induced better response than other PGRs tested. Similarly, Banerjee et al. (2012) reported TDZ alone as the best PGR for shoot induction in Hypericum perforatum L. In our data the highest concentration of TDZ in the medium profoundly enhanced the shoot regeneration frequency. However, a decrease in shoot organogenesis parameters was observed with increase in concentration of TDZ beyond the optimal level. Kumar and Reddy (2012) have reported optimum shoot induction frequency in Jatropha curcas at higher concentration of TDZ (9.08 µM). However, Parveen and Shahzad (2010) reported optimum shoot number in Cassia sophera Linn at lower concentration of TDZ (2.4µM). Furthermore, Mukhtar et al. (2012) concluded from their work that increase in level of TDZ beyond an optimum level has an inhibitory effect on shoot organogenesis. In contrast to our findings, lower values of TDZ have been advocated for higher frequency of shoot organogenesis in many plant species (Chhabra et al., 2008; Raghu et al., 2006). In our study, calli transferred to MS0 produced no shoots. This is consistent with the results of Yucesan et al. (2007) but contrary to the work of Radice and Caso (1997) who found MS0 as an optimum medium for shoot regeneration of S. marianum. The stimulating effect of TDZ on shoot organogenesis in many plant species from various explants has been documented (Ahmad and Anis, 2007; Banerjee et al., 2012; Ismail et al., 2011; Wannakrairoj and Tefera, 2012; Yucesan et al., 2007). Combinations TDZ with NAA produced lower values for shoot organogenesis in current report. Similarly, combination of TDZ with NAA failed to induce shoot organogenesis in Lycopersicon esculentum (Osman et al., 2010). Findings of Mirici (2004) supported our data.

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TDZ 11.0uM Plant Growth regulatorsTDZ 13.2uM

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TDZ8.8+NAA2.2uM

TDZ 4.4+NAA6.6uM TDZ 6.6+NAA4.4uM

TDZ 2.2+NAA8.8uM

Figure 3.2: Data on shoot induction frequency in Silybum marianum by the application of various concentrations and combinations of TDZ, Kn and NAA. Values are mean of three replicates and observations were recorded after 4 weeks of culture.

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a 25

ab 20 b b b bc bc c 15 c c c cd d d d de de 10 de de de e e e e

Number of shoots per explant per shoots of Number ef 5 f

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TDZ 13.2uM Plant GrowthTDZ 11.0uM regulators

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TDZ8.8+NAA2.2uM

TDZ 4.4+NAA6.6uM TDZ 6.6+NAA4.4uM

TDZ 2.2+NAA8.8uM

Figure 3.3: Data on number of shoots in Silybum marianum by the application of various concentrations and combinations of TDZ, Kn and NAA. Values are mean of three replicates and observations were recorded after 4 weeks of culture.

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Contrarily, TDZ in combination with other PGRs could be more effective than TDZ alone (Lincy and Sasikumar, 2010). In another report, combination of NAA, Zn and BAP produced best shoot proliferation in S. marianum (Gikloo et al., 2012). For shoot elongation, clumps of proliferated shoots were separated into individual shoot (0.2-0.4 cm) and were sub-cultured to medium with similar composition of PGRs (Figure 3.4). Significant differences were observed in elongation at different concentrations and combinations of plant growth regulators. Contrary to data on shoot/explant, addition of NAA in medium containing TDZ increased mean shoot length. Longest mean shoot length (4.1 cm) was observed on medium augmented with 6.6µM TDZ and 4.4µM NAA. Previously, Abbasi et al. (2010) observed longest shoots of S. marianum on MS medium fortified with BA and NAA. Combination of TDZ & Kn produced smaller shoots (1.2 to 2.3 cm in length). However, NAA showed inhibitory action when applied individually at all concentrations (Figure 3.4). The pivotal role of TDZ in profuse shoot organogenesis in present work can be ascribed to its stimulating effect on plant morphogenesis in the de novo synthesis of auxins and cytokinins by increasing the level of indigenous precursors for their profound production (Zhongguang et al., 2012). Furthermore, TDZ is supposed to inhibit cytokinin oxidase, the enzyme responsible for the inactivation of cytokinin, thereby increasing the levels of endogenous cytokinins (Nikolic et al., 2006). This explains the higher activity of TDZ in achieving highest shoot organogenesis in our study.

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a 4 ab ab b b bc bc 3 c c c c c c cd cd

d d 2 d de de e e ef Mean shoot length (Cm) length shoot Mean 1 ef f f 0

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TDZ8.8+NAA2.2uM

TDZ 4.4+NAA6.6uM TDZ 6.6+NAA4.4uM

TDZ 2.2+NAA8.8uM

Figure 3.4: Data on shoot length (cm) in Silybum marianum by the application of various concentrations and combinations of TDZ, Kn and NAA. Values are mean of three replicates and observations were recorded after 4 weeks of culture.

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3.4.3 Rooting and acclimatization

Elongated shoots were transferred to MS0 and MS medium containing several concentrations (0.2, 0.4, 0.8, 1.6 or 3.2 µM) of NAA or IAA (Table 3.1). IAA showed more effective results than NAA by promoting highest rooting frequency (87%) at 0.4 µM IAA with mean number of roots (3.2 roots) and mean root length (3.4 cm). However, increasing concentrations of both NAA and IBA resulted in a linear decrease in rooting; maximum inhibition of the rooted shoots was observed for NAA at 3.2 µM. In present study reduced levels of auxins considerably induced roots as generally reported for root induction and proliferation in many herbaceous plants (Victorio et al., 2012). Recently, Gikloo et al. (2012) has reported combination of activated charcoal with IBA (0.25mg l-1) as best medium for roots development in S. marianum. But previously, Abbasi et al. (2010) induced optimum rooting of S. marianum on MS0. Finally the rooted shoots were transferred to sterilized vermiculite and kept under growth room conditions for two weeks prior transfer to pots. The survival rate of rooted shoots was 73% through the hardening off process.

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Table 3.1: Data on root formation parameters by the application of different concentrations of indole acetic acid (IAA) and α-napthalene acetic acid (NAA). Values are mean of three replicates and observations were recorded after 4 weeks of culture.

Rooting (%) No. of roots/shoot Root length (cm) Conc (µM) NAA IAA NAA IAA NAA IAA 0.2 52.45ab 73ab 2.5ab 2.9ab 3.1a 2.8ab 0.4 61a 87.32a 2.9a 3.2a 2.7ab 3.4a 0.8 46b 61b 2.3ab 2.7ab 2.2ab 3.1a 1.6 33bc 53bc 1.9bc 2.1bc 1.9bc 2.7bc 3.2 13c 44.53c 1.3c 1.8c 1.4c 1.9c

3.4.4 Silymarin content

Callus, in vitro shoots, micropropagated plantlets and growth room potted plantlets were collected at various growth stages for HPLC analysis to determine the content of Silymarin (Figure 3.5). To standardize the fingerprints of tissue cultured materials through HPLC, similar peaks present in all samples were denoted as “common peaks” for silymarin production in S. marianum. The chromatographic data along with silymarin standard (Sigma) representing valuable information based on the relative peak area and retention time for assessment and quantification of silymarin in all in- vitro and in-vivo Silybum samples. 8.47 mg/g silymarin was detected in regenerated plantlets. Differentiated tissues possess higher levels of silymarin than callus (Figure 3.5).

Our results demonstrate an ascending variation of silymarin from callus culture to plantlets development thus depicting the influence of growth pattern on silymarin production in the in vitro regeneration of this medicinally important herb. Growing conditions have a direct relationship with accumulation of secondary metabolites (Naz et al., 2013) governed by stress conditions, which consequently influence the metabolic pathways responsible for the accumulation of the related natural products (Abbasi et al., 2011; Nikolova and Ivancheva, 2005). In case of silymarin, TDZ Strategies for the production of chemically consistent plantlets of Silybum marianum. L

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supplementation has shown significant effect on its accumulation in current study. Biosynthesis of silymarin involves activity of phenylalanine ammonia lyase (PAL) in phenylpropanoid pathway (Koksal et al., 2009). Furthermore, TDZ treatment is reported to enhance the levels of secondary metabolites via modifications of plant growth and development, since its biological activity is generally higher than most of the plant growth regulators when applied to in vitro cultures (Liu et al., 2007). Our results are comparable with the findings of El Sherif et al. (2013) who observed considerable silymarin content in multiple shoot cultures of S. marianum by different biotic elicitors.

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Silymarin content (mg/g) content Silymarin 2

0 Micropropagated Wild plant Growth room Callus Regenerated potted plants shoots plantlets

Figure 3.5: Data on silymarin content (mg/g) in regenerated plant tissues and wild grown plantlets of Silybum marianum. Values are mean of three replicates.

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4. Establishment of a feasible and efficient protocol for somatic embryogenesis in Silybum marianum L.

4.1 Abstract

Silybum marianum is an economically and medicinally important crop worldwide. It is renowned for production of biologically important silymarin. We hereby established a feasible and efficient protocol of indirect somatic embryogenesis for production of healthier and chemically consistent S. marianum plantlets. Highest embryogenic potential was observed for calli previously derived from petiole explants on Schenk and Hildebrandt (SH) medium containing 2.5 mg l-1 2,4- dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l-1 N6-benzyladenine (BA). Somatic embryos were induced when embryogenic calli with pre-embryoid masses (PEMs) were subcultured on same media as used for induction of embryogenic callus. Highest number of somatic embryos (46 somatic embryo per callus) was observed at 1.5 mg l- 1 2,4-D and 1.5 mg l-1 BA, however ½ strength MS medium showed optimal response -1 for maturation followed by germination of somatic embryos at 1.5 mg l GA3.

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4.2 Introduction

Silybum marianum (L.) Gaertn. (Milk thistle) belonging to Asteracea is an important medicinal herb used in treatment of liver diseases with a history of use spanning more than centuries (Abbasi et al., 2010). It is valued for its distinct compound called silymarin having several pharmacological activities such as anti inflammatory, anti hepatitis, anti viral and antioxidant (Khan et al., 2014). Average sale of silymarin is about US$ 8 billion/annum and its demand varies from 18 to 20 tons/year (Khan et al., 2013). A very common problem associated with medicinal plant preparations is the extreme variability in the phytochemical contents (Wu et al., 2009). Similarly, the efficacy of silymarin from wild grown Silybum plants has been compromised by contamination with biological and environmental pollutants, adulteration with misidentified species, quantitative and qualitative variations of bioactive compounds as well as the concern of unsustainable harvest (Haban et al., 2009; Lee and Liu, 2003). These problems have produced inconsistencies in the results of various clinical trials and difficulties in identifying a specific medicinal molecule with defined pharmaceutical functions in S. marianum products (Ram et al., 2005).

However, development of elite varieties with predictable phytochemical profiles towards plant tissue culture application might probably circumvent these issues of variability in Silybum end products (Khan et al., 2014). Clonal plant production in vitro have potential to ensures vigorous growth of pharmacologically superior plants, and thus significantly reduces contamination of plants, and facilitates biochemical characterization supported by chromatographic fingerprint analysis for quality control (Murch et al., 2000; Murch et al., 2006).

The in vitro system for embryogenesis produces uniform plants rapidly and easily which provides an ideal experimental mean for investigation of plant differentiation as well as the large scale production of plants with consistent phytochemical profiles (Moon et al., 2013). It also gives a clear insight into the factors controlling somatic embryogenesis, and an understanding towards morphology of embryogenesis in-vitro compared with zygotic embryo formation. Furthermore, somatic embryos can be utilized for the preparation of artificial seeds or synthetic seeds which are analogous

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to the natural seeds (Kumar and Thomas, 2012). Based on our literature survey, there has been no any somatic embryo study in S. marianum as yet. The main objectives of present study were to establish a feasible protocol for somatic embryogenesis using different explants, basal media containing various combinations and concentrations of PGRs.

4.3 Materials and methods

4.3.1 Plant material

Mature seeds of S. marianum were collected from wild grown plants in main campus of Quaid-i-Azam University Islamabad in 2012. The seeds were dipped into 70% ethanol (v/v) for 5 minutes followed by surface sterilization with 0.1 % (w/v) freshly prepared aqueous mercuric chloride solution (HgCl2) for 3 minutes. Then, washing step was employed three times with sterile distilled water.

4.3.1.2 Establishment of embryogenic callus

The surface disinfected seeds were germinated in vitro according to the method of Khan et al. (2013) as described in chapter 2 Petiole explants (~2.0 cm) were excised from 4 weeks old in vitro germinated seedlings, and then placed onto SH (Schenk and Hildebrandt, 1972) media containing 3% sucrose (w/v) and 0.8% (w/v) agar in 150 ml conical flask supplemented with (0.5, 1.5, 2.5, 5.0 or 8.0 mgl-1) of 2,4-D or BA alone or 1.5 mgl-1 BA in combination with 2,4-D (0.5, 1.5, 2.5, 5.0 or 8.0 mgl-1). The pH of media was adjusted to 5.8 prior to autoclaving (121°C, 20 min at 1 atm. pressure), cultures were placed in 16 hrs photoperiod with light intensity of ~40 μmol m-2 sec-1 and temperature was maintained at 25±1°C. In all sets of experiments, PGR free medium was used as control treatment. After 4 weeks of callus induction, the frequency of callus induction (%) was recorded. Of the induced callus, embryogenic callus were considered from callus tissue producing pre-embryoid masses (PEMs).

4.3.1.3 Development of somatic embryos The PEMs developed on surface of embryogenic calli were aseptically cut into small sections (~2 cm) and then transferred into SH medium containing (0.5, 1.5, 2.5, 5.0 or 8.0 mgl-1) of 2,4-D or BA alone or 1.5 mgl-1 BA in combination with 2,4-D (0.5, 1.5,

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2.5, 5.0 or 8.0 mgl-1). To further investigate the influence of PGRs and type of media on the growth of globular somatic embryos, fresh medium was provided either with 1.5 mgl-1 2,4-D in combination with 1.5 mgl-1 BA or another set of basal media [SH0, MS0 or ½ MS (Murashige and Skoog, 1962)] without PGRs. The percent maturation of somatic embryos and their growth were recorded after four weeks of culture in a flask with three replications.

4.3.1.4 Germination of somatic embryos into plantlets

After 2 weeks in embryo development medium, cotyledonary embryos were then transferred to the half strength MS medium supplemented with various levels (0.0, -1 0.5, 1.5 or 2.0 mgl ) of GA3. Plantlet conversion was evaluated by counting plantlets with well developed leaves and roots after four weeks of culture in germination medium. Plantlets were removed from flasks, washed three times with double distilled water, and then transplanted to pots with a mixture of soil, sand and perlite (1:2:1, v/v/v) for acclimatization. Pots were covered with polythene bags to maintain high humidity. The bags were perforated and the covers were removed after two weeks when the plantlets showed new leaves. The survival rates were calculated after four weeks of hardening.

For fresh weight (FW) determination, the plant samples were gently pressed on filter paper to remove excess water and weighed. Subsequently, the plant materials were dried in an oven at 35°C for 24 h, and dry weight (DW) of each sample was recorded.

4.3.1.5 Histological study

Plant samples of somatic embryos at different developmental stages were collected from culture flasks, and were prepared according to the protocol earlier published by Ahmet (2000). Briefly, samples were fixed for 24 hrs in a sodium phosphate buffer at 0.2 M and pH 7.2 containing 2% para-formaldehyde (w/v), 1% glutharaldehyde (w/w) and 1% caffeine (w/v), to precipitate the oxidised phenolic compounds in situ. The fixed samples were then rinsed continuously with distilled water for twenty minutes. The specimens were then dehydrated by passing through a series of (30, 50, 70, 90 or 100%) ethanol solutions and infiltrated with resin (Spurr, 1969). These specimens

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were then transferred into been capsules in resin, and placed in an oven at 70 ºC overnight. The specimens were then cut into blocks, and sectioned appropriately at 10 µm using a rotary microtome (Slee Technik). The sections were mounted onto slides, and then allowed to dry for ten minutes before staining. The specimens were then stained with 0.05% toluidine blue O (w/v). Cover slips were mounted with Histoclad mounting medium and dried on a 40 ºC hot plate for 35 minutes. Permanent slides were observed under a microscope (Nikon AFX-DX (Labophot) equipped with a camera connecting to the computer system.

4.3.2 Statistical Analysis

For statistical analysis, each treatment consisted of 12 culture flasks and data was collected from triplicates. Analysis of variance (ANOVA) and Duncan’s multiple range test (DMRT) was used for comparison among treatment means. All experiments were repeated three times. However, for biochemical assays all the parameters were expressed as µg g-1 fresh weight of sample, data were collected from triplicates and represented as values of mean ±SE.

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4.4 Results and Discussion

4.4.1 Induction of embryogenic callus

On SH medium containing 2.5 mg l-1 2,4-D, 86.2% of petiole segments produced calli. The initial sign of callusing was observed at the cut end of explants after 1 week of culture initiation (Table 4.1). This data is in agreement with findings of Kumar et al., (2012) who observed optimum callus formation on MS medium supplemented with 2 mg l-1 2,4-D in cotyledonary explants of Clitoria ternatea. The callus formation frequency was significantly (P<0.05) enhanced when the petiole explants were cultured on SH medium fortified with 2.5 mg l-1 2,4-D in combination with 1.5 mg l-1 BA. Four week-old calli were separated from primary explants, and incubated on the same media used for callus induction. Highest embryogenic potential (72%) was observed for calli previously derived from petiole explants at 2.5 mg l-1 2,4-D combined with 1.5 mg l-1 BA (Table 4.1). 2,4-D and BA are reported as potent bioregulators for acquiring embryogenic competency in cultures when employed in synergistic combination in a wide range of plant species (Wani et al., 2010; Singh et al., 2011). In our study, morphologically two different types of calli were observed; embryogenic and non-embryogenic. Based on visual observations, embryogenic calli were creamy-yellow, compact, nodular and contained cytoplasmically rich small embryogenic cells in a cluster form, which can be assumed as pre-embryogenic masses (PEM) [Figure 4.3,B]. Such structures are commonly observed during indirect somatic embryogenesis in many plants (Zimmerman 1993; Firoozabady and Moy 2004). However the non-embryogenic calli were pale green, friable, soft and did not contain any PEM at all.

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Table 4.1: Effects of 2,4-D measured directly against BA or combination equivalent in SH media on embryogenic callus formation (%) and mean number of somatic embryos per embryogenic callus from petiole explants. Data on embryogenic callus formation were recorded after 2 weeks of culture when four week old calli were sub- cultured on same media while data on number of somatic embryos were collected after 4 weeks of culture. Values are the mean ± standard error from three replicates.

S# SH + PGRS (mg/l) Callus Induction Embryogenic callus Number of somatic (%) formation (%) embryos (mean) 1 SH (0) 0 0 11±1.9 2 2,4-D (0.5) 69.6±4.6 21.2±1.3 33±2.1 3 2,4-D (1.5) 73.4±5.4 33.6±2.1 22±1.0 4 2,4-D (2.5) 86.2±6.5 56±4.8 18±2.3 5 2,4-D (5.0) 75.1±5.9 43±3.4 13±1.2 6 2,4-D (8.0) 67.2±4.3 32.2±2.7 10±0.9 7 BA (0.5) 22.1±1.0 18±2.3 4±0.2 8 BA (1.5) 39±2.2 26±2.0 9±0.5 9 BA (2.5) 59.4±5.3 39±2.2 6±0.4 10 BA (5.0) 46±4.0 27±1.4 4±0.2 11 BA (8.0) 22.2±1.0 19.3±2.5 2±0.08 12 2,4-D (0.5)+BAA (1.5) 63±4.5 53±4.1 29±2.6 13 2,4-D (1.5)+BAA (1.5) 76.1±5.7 61.4±4.0 46±4.0 14 2,4-D (2.5)+BAA (1.5) 90±7.6 72±5.6 18±2.3 15 2,4-D (5.0)+BAA (1.5) 78.4±5.1 64.2±4.4 11±1.9 16 2,4-D (8.0)+BAA (1.5) 64±4.4 49.4±3.7 6±0.4

4.4.2 Somatic embryo development

Optimum number of somatic embryos (33 somatic embryo per callus) were recorded at (0.5 mg l-1) 2,4-D when the embryogenic calli with PEMs were transferred on to embryo induction medium (Table. 4.1). Similarly, 2, 4-D has been reported as the most effective auxin for induction of somatic embryos in most of the plant species (Prange et al., 2010; Zhang et al., 2010; Dai et al., 2011; Sivanesan et al., 2011). Globular somatic embryos appeared on the surface of the embryogenic calli after 2–3 weeks of sub culturing (Figure 4.3,C). Comparatively, the combination of BA with 2,4-D increased the number of somatic embryos per callus. In that, highest number of somatic embryos (46 somatic embryo per callus) was observed at 1.5 mg l-1 2,4-D in

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combination with 1.5 mg l-1 BA (Table 4.1). Similar effects of BA and 2, 4-D combination was reported earlier by Omar et al., (2013) in which somatic embryogenesis of strawberry were employed using petiole explants. In present study, different stages of somatic embryos were observed simultaneously on the same embryogenic tissue (Fig. 4.3: E,F) indicating that somatic embryogenesis in S. marianum was asynchronous.

In most protocols in which auxins act as efficient inducer of somatic embryogenesis, development of somatic embryos is achieved by reducing or removing auxin from the culture medium (Pinto et al., 2008). In our data, an adequate increase in embryo maturation was observed on MS0 medium followed by SH0. However, an ample change in maturation (60%) was observed when the embryos from embryo induction medium were sub-cultured on 1/2 strength MS medium (Figure 4.1). At this stage, individual embryos enlarged and mature fully into distinct bipolar structures which later established into complete plantlets. The higher activity of auxin free medium (1/2 MS) for maturation of somatic embryos can be explained by the fact that, once embryogenesis is induced, the auxin role changes, and embryo begins to synthesize its own auxins and thus require less auxin (Gerdakaneh and Zohori, 2013).

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70 a 60

40 b

30 Embryo maturation (% ) 20 c

10 c

EIM SH 0 MS 0 MS 1/2

Figure 4.1: Effects of various growth media on somatic embryo maturation. Data were collected after 2 weeks of culture. Values are the mean ± standard error from three replicates. Column bars sharing the same letter/s are similar otherwise differ significantly at P<0.05.

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4.4.3 Embryo germination and conversion into plantlets

½ strength MS was an optimum medium for embryo maturation, while it was less effective for embryo germination. Therefore, GA3 at various concentrations was added to ½ MS medium. Highest germination rate (70%) was observed at 1.5 mg l-1

GA3 (Figure 4.2). The cotyledonary embryos developed subsequently the shoot and the root apex, and formed a complete plantlet within 4 weeks (Figure 4.3:G,H). Similarly, Baskaran and Staden (2012) found ½ strength MS medium supplemented -1 with 1.4 mg l GA3 as the best medium for somatic embryo germination in Merwilla plumbea. The role of GA3 on the germination of SE has been reported in other plant species (Xiangqian et al., 2002; Cangahuala et al., 2007; Siddiqui et al., 2011). Contrarily, PGR free medium is also reported as suitable medium for embryo conversion into plantlets (Correa et al., 2009).

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80 a 70

) 60 %

b 40 b

30 b

20 Embryo conversion ( c 10

0 -1 -1 -1 -1 -1 gl gl gl gl gl

0.0 m 0.5 m 1.0 m 1.5 m 2.0 m

Gibberellic acid (GA3)

Figure 4.2: Effects of various concentrations of GA3 in ½ MS media on germination frequency of somatic embryos. Data were collected after 4 weeks of culture. Values are the mean ± standard error from three replicates. Column bars sharing the same letter/s are similar otherwise differ significantly at P<0.05.

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A B C

D E F

G H

Figure 4.3: Somatic embryogenesis in Silybum marianum (a) Callus formation at cut ends of petiole explants after one week of culture (bar=2mm). (b) Embryogenic callus with pre-embryoid masses (PEM) after two weeks of culture period (bar=2mm). (c) Embryogenic callus with somatic emryos (arrows) (bar=2mm). (d) Embryonic cells showing isodiametric cells (blue star) non-embryonic cells (black star) with large vacuole, small starch granules and abundant intercellular spaces (bar=250µm). (e) Globular somatic embryo after four weeks in embryo induction medium (bar=150µm) (f) Cotyledenary embryo, arrows show vascular bundles (bar=150µm). (g) Shoot emergence from embryo after one week in germination medium (bar=2mm). (h) Embryo converted plantlet after four weeks in germination medium, arrow shows root formation (bar=2mm).

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5. TEMPORAL VARIATIONS IN METABOLITE PROFILES AT DIFFERENT GROWTH PHASES DURING SOMATIC EMBRYOGENESIS OF SILYBUM MARIANUM L.

5.1 Abstract

Silybum marianum, commonly known as Milk thistle, is a popular herbal supplement used for the treatment of jaundice and liver cirrhosis worldwide. Here we established methods for comparative metabolite profiling of the different growth phases during embryogenesis in S. marianum. Metabolite profiles from developmental stages of non-embryogenic callus (NEC), pre- embryoid masses (PEMs), somatic embryos (SE) and embryos germinating into intact plantlets (GSE) were obtained using Electro spray ionization mass spectrometry ESI/MS. Principal component analysis (PCA) was carried out to identify key metabolites in different growth phases during somatic embryogenesis. The loading scatter plots enabled the detection of several bin masses responsible for separating samples from different growth stages. Based on the values of % total ions count and average intensity of selected bins in all biological samples, putatively known metabolites were obtained from in-house bin program. Amino acids associated with various biosynthetic pathways like arginine, asparagine and serine were abundantly detected in GSE, while they were detected at decreased intensities in NEC. However, tryptophan was measured with increased signals in SE when compared to other growth phases. Glucose, fructose and fructose-6-phosphate were accumulated the most in NEC; however they were detected with lowest intensities in GSE. Moreover, sucrose and significant secondary metabolites like cinnamic acid, kaempferol, quercetin, myricetin, linolenic acid, and 5-enolpyruvyl-shikimate-3- phosphate were found at higher presence in SE when compared to other embryogenic phases.

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5.2 Introduction

Silybum marianum is a medicinally significant herb native to Mediterranean basin, but is now naturalized throughout the world. Its bioactive compound is rich in liver protecting phenylpropanoids, known as silymarin (Abbasi et al., 2010). The in-vitro system for embryogenesis produces uniform plants rapidly and easily which provides an ideal experimental mean for investigation of plant differentiation as well as the large scale production of plants with consistent phytochemical profiles (Moon et al., 2013). It also gives a clear insight into the factors controlling somatic embryogenesis, and an understanding towards morphology of embryogenesis in-vitro compared with zygotic embryo formation. (Kumar and Thomas, 2012).

Metabolite profiling is the analysis to identify and quantify maximum possible metabolites in a biological sample and its demand is rapidly expanding in different biological fields (Dunn et al., 2005). Furthermore, it can describe the metabolic events happening at point in specific plant tissues (Bundy et al., 2009). Since a series of events are practically executed in-vitro during different steps of somatic embryogenesis (Helmersson et al., 2004). Thus using metabolite profiling, regulation of developmental events in different growth phases of embryogenesis can be further elucidated at the metabolic level (Businge et al., 2012).

The main objectives of present study was to investigate the metabolic events at different growth phases by utilizing ESI/MS during in-vitro somatic embryogenesis of S. marianum.

5.3 Materials and methods

5.3.1 Plant samples

Plant material was collected from different growth phases of non-embryonic callus (NEC), pre-embryoid masses (PEM), globular somatic embryos (SE) or cotyledonary embryos germinating into intact plantlets (GSE) during somatic embryogenesis. Samples were collected at the time of transfer to new medium. These growth phases have been described in details in chapter 4. For each growth stage, three biological replicates were collected. All samples were transferred to air tight vials, flash frozen

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in liquid nitrogen and stored at −80 °C until further processing for metabolite extraction.

5.3.2 Biochemical characterization

DPPHº free radical scavenging activity was determined by the method of Abbasi et al. (2010). Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity and silymarin content were determined by the methods of Khan et al. (2013), these methods are described in chapter 2.

5.3.3 Metabolite profiling

5.3.3.1 Extraction of Metabolites

Extraction of metabolites was carried out according to the method of Overy et al. (2005). Briefly, 1 ml of solvent mixture A (methanol:chloroform:water, 2.5:1:1 at -20 °C) was added to the Eppendorf tube containing the fine powder of each sample. Samples were vortexed for 25 seconds and kept on ice for 5 minutes and centrifuged at 14×103 rpm for 5 minutes at 4° C. The supernatant was collected and transferred into a pre-chilled storage tube and labeled as supernatant A. The remaining pellet was re-extracted with 0.5 ml of the pre-chilled solvent B (methanol:chloroform, 1:1 at -20 °C) followed by vortexing and centrifugation at 14×103 rpm for 5 minutes at 4° C to obtain the supernatant B. In the next step, supernatants A and B were combined and the top aqueous phase (methanol plus water) containing polar metabolites were decanted into a new cooled 1.5 ml Eppendorf tube. Then organic layer was separated from aqueous layer by adding 250 µl chilled distilled water into the mixture followed by centrifugation for two minutes. Both aqueous and organic phases were then stored at -80 °C until analysis.

5.3.3.2 Electrospray ionisation time of flight mass spectrometry (ESI-TOF MS)

ESI-TOF MS was performed on a LCT spectrometer (API Q-Star, Waters Corporation, Milford, USA.) based on the methods described by Davey et al. (2008) and Walker (2011). The mass spectrometer was operated at a resolution of 4,000 (FWHM) in positive mode with a capillary voltage of 4800 V, extraction cone at 3 V and sample cone at 20 V with a range finder lens voltage of 75 V chosen for detection Strategies for the production of chemically consistent plantlets of Silybum marianum. L

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of masses from 50 to 800 Da. Source temperature was 110 °C and desolvation temperature was 120 °C. Flow rates were 100 L h-1 for nebulisation and 400 L h-1 for desolvation. Spectra were collected in centroid mode at a rate of one spectrum s-1 (0.95 s scan time, 0.05 s interscan delay) with 180 summed over a 3-min period without background subtraction or smoothing. Samples were either loaded using a syringe pump (Razel, Connecticut, USA) at a flow rate of 20 µl min-1 (aqueous phase) or loaded using an automated Waters 2695 Separations Module combining a HPLC pump and an autosampler (Waters Corporation, Milford, USA) (organic phase) with an inject volume of 150 µl at a flow rate of 50 µl min-1. The Waters LCT instrument has the capability to introduce an external standard (LocksprayTM) and the compound sulphadimethoxine with a neutral exact mass of 310.0736, was used for this purpose.

5.3.3.3 Data processing

For each sample run, the summation of 180 centroid mode spectra were exported from MassLynx data systems as text file peak lists (Accurate mass to 4 decimal places vs. ion count). These were imported into Microsoft Excel (Microsoft Corp, USA) and an in-house macro program was used to compare the accurate masses of three technical replicate analyses of each sample. Noise reduction was carried out according to the procedure defined by Overy et al. (2005). For identification of real molecules within these profiles from background noise three replicate mass spectra of each individual sample were obtained. Once a peak had been selected as a true peak, the mean of the three masses over the three replicate scans was used as the accurate mass and this value along with the corresponding average intensity made up the metabolite profile. Finally the text files contained mass spectra of respective samples were analyzed by Simca-P+ (version 12.0). Principal component analysis (PCA) was carried out using Pareto scaled 0.2 Da binned data sets in Simca-P v12.0 software (Umetrics, Sweden). Significance values of the ion counts between the samples were determined using one way ANOVA.

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5.3.3.4 Putative metabolite identification

Identification of putatively known metabolites was performed through the comparison of monoisotopic masses likely to be present in extracts, including [M+H]+, [M-H]- and [M+Na]+ against the list of metabolites in the biocyc database (http://biocyc.org/).

5.3.4 Statistical Analysis

Analysis of variance (ANOVA) and Duncan’s multiple range test (DMRT) was used for comparison among treatment means. All experiments were repeated three times. However, for analytical assays, data were collected from triplicates and represented as values of mean ±SE.

5.4 Results and Discussion

5.4.1 Phenylalanine ammonia lyase activity, antioxidant potential and silymarin content

PAL and free radical scavenging activities were found to be higher in SE in the process of embryogenesis (Table 5.1). PAL activity has been found in many plants, and is encoded by multigene family (Azzi et al., 2004). However, silymarin content quantified by HPLC in plant samples derived from different growth phases during embryogenesis, revealed the highest content (4.27 ±1.04 mg.g-1 DW) in SE followed by GSE (2.41±0.65 mg.g-1 DW). Moreover, a significant correlation was observed between FRSA% with PAL (r =0.99, p value 0.02) and between FRSA% with silymarin content (r =0.89, p value 0.045) at p < 0.05 (Table 5.2) which might indicate an up regulation of plant secondary metabolite production. The enhanced antioxidant activity exhibited by SE can be ascribed to the concurrent production of silymarin during development of somatic embryos, since silymarin is reported for its enhanced antioxidant potential (Abbasi et al., 2010). Nevertheless, PAL has a profound role in the biosynthesis of silymarin and other phenolic compounds. It converts phenylalanine to cinnamic acid through formation of trans-cinnamate as an intermediary by product, which acts as the principal modulator of PAL turnover (Koksal et al., 2009; Limem et al., 2008).

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Table 5.1: Phenylalanine ammonia lyase activity (Ug-1 FW), antioxidant potential (% FRSA) and silymarin content (mg.g-1DW) in in-vitro-grown plant samples collected from different growth phases during somatic embryogenesis in Silybum marianum. The different growth phases comprised of NEC= Non- embryogenic callus, PEM= Pre-embryoid massesproduced on embryogenic callus, SE= somatic embryos at mature stage, GSE= Somatic embryo germinating into plantlet. Values are the mean ± standard error from three replicates.

Sample FRSA (%) PAL (Ug-1 FW) Silymarin content NEC 19.9±1.1 21.9±2.1 mg.g0.56±0.01-1 DW PEM 31.1±3.1 43.8±8.2 0.71±0.02 SE 65.2±4.2 72.2±10.4 4.3±1.0 GSE 44.4±3.1 56.5±6.7 2.4±0.7

Table 5.2: Correlation between (% FRSA) antioxidant potential, (PAL) Phenylalanine ammonia lyase activity (Ug-1 FW) and silymarin content (mg.g-1DW) in in-vitro-grown plant samples collected from different growth phases during SE in Silybum marianum. Marked correlations are significant at p < 0.05.

FRSA (%) PAL Silymarin FRSA (%) 1 (Ug-1 FW) content (mg.g -1DW) PAL (Ug-1FW) 0.99 1 Silymarin content 0.89 0.83 1 (mg.g -1DW)

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5.4.2 Metabolite profiling

5.4.2.1 Mass spectra

The spectrum profiles of both aqueous and organic fractions were different although some common peaks can be seen (Figure 5.1 and 5.2). Each spectrum detected several hundred masses, although the aqueous fractions of PEM and GSE lines exhibited abundance of heavier molecules than the NEC and SE lines which showed several low m/z molecules. Furthermore, the spectra of aqueous fractions were dominated by common peaks at m/z 214 and m/z 219, however many uncommon peaks were also detected with a highest peak at m/z 116 in GSE. The spectra of organic fractions were dominated by common peaks at m/z 171, m/z 178 and m/z 183. Within all organic fractions, SE was dominated by many peaks which were not common in other samples. As some compounds are very sensitive to ESI-MS, it must be noted that peak intensity is not necessarily a reflection of the relative concentrations of these metabolites (Pitt, 2009). Due to the increased number of high mass peaks, the identification of metabolites using just accurate mass without any structural identification from a Time of Flight MS (TOF-MS) becomes more difficult due to the increasing number of isomers which are possible at a particular mass (Kruve et al., 2013).

Several problems can occur when running samples through ESI-MS. Due to the presence of very high concentration of metabolites in the sample some of the metabolites may not ionize and so do not enter the MS, for instance steroids do not ionize well using ESI because they are relatively non-polar molecules, which lack a functional group capable of carrying a charge (Pitt, 2009). These un-ionized neutral species deposited on the cones or at the end of capillary tube of the MS can lead to a gradual loss of sensitivity and eventually a blockage which can be time consuming to clear.

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Figure 5.1. Raw metabolic profile mass spectra for aqueous fractions of NEC, PEM, SE and GSE lines in positive ionization mode. Symbols with same shape represent the common peaks in the samples.

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Figure 5.2. Raw metabolic profile mass spectra for organic fractions of NEC, PEM, SE and GSE lines in positive ionization mode. Symbols with same shape represent the common peaks in the samples.

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5.4.2.2 Principal Component Analysis (PCA)

The PCA analysis of both aqueous and organic fractions extracted from all samples produced the PCA score plots (Figure 5.3 and 5.4). The samples could be distinguished on the basis of differences in developmental stages during embryogenesis. The principal components PC1 and PC2 accounted for 72% variation in aqueous fractions and 54% variation in organic fractions. For the aqueous fractions PCA separated the growth phase SE and NEC with 44% variance on PC1 having positive factor scores whereas the variance on PC 2 was 28% contributing positive factor scores for GSE and the negative factor scores exhibited for PEM (Figure 5.3). However, for organic fractions the samples NEC and PEM were clearly separated by PC1 while SE and GSE were readily discriminated by PC2 (Figure 5.4). In addition the corresponding loading scatter plots enabled the detection of several bin masses responsible for separating samples from different growth stages (Figure 5.5 and 5.6). The loadings scatter plot for a PCA analysis can provide a list of the metabolite bins that change most and whether they increased or decreased in abundance. The bins which are closest to the origin in the loadings plot are the bins that changed the least. Conversely, the bins furthest away from the origin are those that changed most, suggesting that these bins contain compounds which might be good for the differential responses in different developmental stages during embryogenesis (Song et al., 2014).

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Figure

5.3: PCA Score Scatter plots of aqueous fractions of NEC, PEM, SE and GSE, evaluated by SIMCA-P+ (12.0).

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Figure 5.4: PCA Score Scatter plots of organic fractions of NEC, PEM, SE and GSE, evaluated by SIMCA-P+ (12.0).

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Figure 5.5: PCA Loading Scatter plots of aqueous fractions of NEC, PEM, SE and GSE, evaluated by SIMCA-P+ (12.0).

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Figure 5.6: PCA Loading Scatter plots of organic fractions of NEC, PEM, SE and GSE, evaluated by SIMCA-P+ (12.0).

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5.4.2.3 Evaluation of Putatively known metabolites

5.4.2.3.1 Bin masses

Metabolites that influenced the separation pattern among the biological samples in the score scatter plot were sorted in the loading scatter plots. As shown in (Figure 5.5 and 5.3), bin mass 321.2 was found in the loading scatter plot influencing the separation of the SE lines and bin mass 137 was found contributing in the separation of the GSE lines among aqueous fractions. Similarly, the organic fractions of the biological samples showed bin mass 178.2 separating the SE lines from the other plant lines (Figure 5.6 and 5.4). Bin mass 120 was found contributing in high score scatter loadings of aqueous fractions but this bin was not identified by the in-house bin program. So, such bins that were not identified by the in-house bin program were ignored and the selected bins identified by the in-house bin program were compared among the biological samples. Comparison of the selected bins among the biological samples was carried out by calculating their percentage of total ion counts and average intensity counts (Figure 5.7, A and B; 5.8, A and B). Increased signals of the bin mass 130 was found in SE followed by GSE and PEM while decreased peak signals detected in NEC. Bin mass 214.2 was also compared among the biological samples with the increased total ion counts detected in non-embryogenic callus and decreased signals were detected in somatic embryo. Average intensities calculated also depicted the same behavior. Percentage of total ion counts of the bin mass 379.2 was found to be higher among all the bins tested in the organic fractions of the NEC and PEM. Increased response of bin mass 178.2 was found in the SE and GSE lines in comparison with NEC. Bin mass 172 showed increased peak intensity/average intensity counts in the SE samples followed by the PEM, GSE and NEC lines (Figure 5.8, A and B).

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4.0 NEC 3.5 PEM SE 3.0 GSE

2.5

2.0

1.5

1.0 Total ion counts (%)

0.5

0.0

-0.5 93 97 144 147 188 242 321.2 377 485.2 Bins sorted by PCA

Figure 5.7.A: Percentage of total ion counts of the bins (putatively identified metabolites) in NEC, PEM, SE and GSE. Bin masses were selected from the loading plots sorted by PCA. (Aqueous fractions). Data represents the values of the mean ± standard error from three replicates.

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NEC 300000 PEM SE 250000 GSE

200000

150000

100000

AverageIntensity Ion counts 50000

93 97 144 147 188 242 321.2 377 485.2 Bins sorted by PCA

Figure 5.7.B: Average intensities of total ions of the bins (putatively identified metabolites) in NEC, PEM, SE and GSE. Bin masses were selected from the loading plots sorted by PCA. (Aqueous fractions). Data represents the values of the mean ± standard error from three replicates.

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80 NEC PEM 70 SE GSE 60

Total Ion counts (%) 20

172 178 178.2 184 187.2 379.2 Bins sorted by PCA

Figure 5.8.A: Percentage of total ion counts of the bins (putatively identified metabolites) in NEC, PEM, SE and GSE. Bin masses were selected from the loading plots sorted by PCA. (Organic fractions). Data represents the values of the mean ± standard error from three replicates.

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35000000 NEC PEM 30000000 SE GSE 25000000

20000000

15000000

10000000

5000000 Average Intensity Ion counts 0

-5000000 172 178 178.2 184 187.2 379.2

Bins sorted by PCA

Figure 5.8.B: Average intensities of total ions of the bins (putatively identified metabolites) in NEC, PEM, SE and GSE. Bin masses were selected from the loading plots sorted by PCA. (Organic fractions). Data represents the values of the mean ± standard error from three replicates.

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5.4.2.3.2 Distribution of key metabolites during different growth phases

A substantial number of metabolites were detected during analysis of samples which were obtained from all the bin masses sorted by PCA loading scatter plots (Table 5.3 and 5.4). Based on the values of % total ions count and average intensity of selected bins in all biological samples, putatively known metabolites were obtained from in- house bin program (Figure 5.9). Amino acids associated with various biosynthetic pathways like arginine, asparagine and serine were abundantly detected in GSE, while they were detected at negligible level in NEC. Both arginine and asparagine are key components of nitrogen metabolism which occurs by means of recurrent interconversion of both amino acids (Canovas et al., 2007). Arginine is also important as a precursor for polyamine biosynthesis, via arginine decarboxylase pathway (Minocha et al., 2004). In current study, serine, cysteine and proline were detected with increased values of total ion counts in PEM, however glutamine and tryptophan were abundantly found in SE. Glutamine is considered as the preferred endogeneous amino acid involved in plant metabolism, providing nitrogen for the biosynthesis of amino acids, nucleic acids and acts as an amino group donor in transamination reactions (Jeyaseelan and Rao, 2005).

As the measured presence of tryptophan during SE growth phase was significantly higher than other embryogenic lines. This might be due to the establishment of auxin gradient which is required for embryo differentiation and balancing bilateral symmetry in cotyledons (Kong et al., 1997), as tryptophan, acts as a key precursor for auxin (indole-3-acetic acid, IAA) biosynthesis through the tryptophan-dependent pathway so its elevated levels in SE are indicative of the essential role auxin has during normal somatic embryo development at this stage (Michalczuk et al., 1992). Previously, tryptophan, proline and serine were reported to foster the development of somatic embryos in diverse taxa like Medicago sativa (Stuart et al., 1985).

Glucose, fructose, fructose-6-phosphate (an intermediate of the pentose phosphate pathway) and sorbitol (a sugar alcohol) were accumulated the most in NEC; however, they were detected with lowest value of total ions count in GSE (Figure 5.9). This sequential decrease in carbohydrate content might be associated with utilization of

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sugars in different growth phases during somatic embryogenesis (Correia et al., 2012). Sucrose was measured with maximum ion counts in SE followed by GSE and lowest value was detected in NEC. The presence of endogenous sucrose during development of somatic embryo has previously been positively linked with the capability of cultures to develop normal mature embryos in Pinus taeda (Robinson et al., 2009). It is well established that exogenous carbohydrates are essential for the induction, proliferation and maturation phases of somatic embryogenesis during which they act as signaling molecules, osmoticum and sources of carbon and energy (Lipavska and Hana, 2004).

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NEC 5 PEM SE GSE 4

2 % Total Ion Counts

0 Urea Serine Proline Cystein Sucrose Glucose Fructose Arginine Quercitin Myricitin Isoleucine Glutamine Kaemferol Tryptophan Asparagine Fructose-6-P hikimate-3-P Linolenic acid S Cinnamic acid

Figure 5.9: Comparison of the key metabolites in NEC, PEM, SE and GSE lines. Putatively known metabolites were obtained from in-house bin program on the basis of their distribution with total ions count (%) in the biological samples. Data represents the values of the mean ± standard error from three replicates.

Strategies for the production of chemically consistent plantlets of Silybum marianum. L

Chapter 5

Significant secondary metabolites from plant phenolics and flavanoids like cinnamic acid, kaempferol, quercetin, myricetin, linolenic acid, and 5-enolpyruvyl-shikimate-3- phosphate were found at higher presence in SE when compared to other embryogenic phases (Figure 5.9). As intermediary products of phenylpropanoid metabolism, these phenolics and flavonoids may stimulate differentiation and create a situation that is more favorable for embryogenesis (Kishor, 1989). They are also reported for their role in oxidative phosphorylation and photophosphorylation, stimulation of RNA synthesis, bud development and prevention of senescence due to strong antioxidant nature (Rathod et al., 2012).

Cinnamic acid has a stimulatory role in activating plant antioxidant system against reactive oxygen species (ROS) via antioxidative enzymes like Superoxide dismutase (SOD) and Peroxidase (POD) (Szalai and Janda, 2009). Therefore, it is likely that in our study, cinnamic acid acted as a potent antioxidant for alleviating the ROS produced as a consequence of in-vitro stress condition during embryogenesis, when the pre-embryoid masses acquired the embryogenic competency in -vitro for development of somatic embryos. Moreover, cinnamic acid is a principal intermediate in shikimate pathway and is involved in the biosynthesis of important phenolic acids such as hydroxycinnamic acids, sinapic acid, and caffeic acids which all are considered as potent antioxidants (Chen and Ho, 1997). Furthermore, the enhanced accumulation of these important secondary metabolites can be corroborated to the higher levels of PAL, FRSA and silymarin content detected in SE growth phase in current study (Table 5.1). It is evident that PAL is the strategic enzyme in the shikimate pathway for the concurrent production of these important antioxidants as natural scavengers for ROS in order to continue the normal metabolic pattern for development of somatic embryos in S. marianum.

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Chapter 5

Table 5.3: Putative identification of the metabolites by the personalv7qstar in-house program for the selected bins sorted from the aqueous extracts of biological samples

Bin Masses Metabolites Name Formula Accurate masses 93

Sulfate H2O4S 97.96738

Phosphoric acid H3PO4 97.9769

Acetaldehyde C2H4O2 60.02113

Acetic acid C2H4O2 60.02113 Urea CH4N2O 60.03236

Glycolic acid C2H4O3 76.01604

Propandiol C3H8O2 76.05243 97

Creatine C4H9N3O2 131.0695

Skatole C9H9N 131.0735

Glutamic acid C5H9NO4 147.0532

Isoleucine C6H13NO2 131.0946

Pyridoxine C8H11NO3 169.0739

N-carbamoylputrescine C5H13N3O 131.1059

Glutamate-1-semialdehyde C6H12O4 148.0736 144

Pyroglutamic acid C5H7NO3 129.0426

Pipecolic acid C6H11NO2 129.079

Glycerol C3H8O3 92.04734 147

Dimethyl trisulfide C2H6S3 125.9632

Thymine C5H6N2O2 126.0429

Citramalic acid C5H8O5 148.0372

Ribonic acid C5H10O6 148.0372

Cinnamic acid C9H8O2 148.0524

Coumaraldehyde C9H8O2 148.0524

Trans-cinnamate C9H8O2 148.0524

Mevalonic acid C6H12O4 148.0736 188

N2-acetyl-l-ornithinem C7H14N2O3 174.1004

Arginine C6H14N4O2 174.1117

L-arginine C6H14N4O2 174.1117

Gramine C11H14N2 174.1157

Glyceraldehyde-3-phosphate C3H7O6P 169.998

Dihydroxybenzoic acid C7H6O4 154.0266

Gentisic acid C7H6O4 154.0266

Gallic acid C7H6O5 170.0215

Citric acid C6H8O7 192.027

Scopoletin C10H8O4 192.0423

Quinic acid C7H12O6 192.0634 Strategies for the production of chemically consistent plantlets of Silybum marianum. L

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242

L-tryptophan C11H12N2O2 204.0899

Tryptophan C11H12N2O2 204.0899

Phenyllactic acid C9H10O3 166.063

Oxaloglutarate C7H8O7 204.027

1-pheynl-5-heptene-1,3-diyne C13H10 166.0782

Dimethoxybenzoic acid C9H10O4 182.0579 Mannitol C6H14O6 182.079 Sorbitol C6H14O6 182.079 L-lathyrine C7H10N4O2 182.0804 321.2

Caffeate C9H8O4 180.0423

Caffeic acid C9H8O4 180.0634

Alpha-d-glucose C6H12O6 180.0634

Alpha-d-mannose C6H12O6 180.0634

Beta-d-glucose C6H12O6 180.0634

Fructose C6H12O6 180.0634

Galactose C6H12O6 180.0634

Glucose C6H12O6 180.0634

Fructose-6-phosphate C6H12O6 180.0634

Mannose C6H12O6 180.0634

Myo-insitol C6H12O6 180.0634

Sorbose C6H12O6 180.0634

D-myo-inositol C6H12O6 180.0634

Myo-inositol C6H12O6 196.0524

Thiarubine a C13H8O2 196.0524

Thiarubine b C13H8O2 180.0786

Coniferyl alcohol C10H12O3 196.0583

Galactonate C6H12O7 196.0583

Galactonic acid C6H12O7 196.0583

Gluconic acid C6H12O7 180.1514 377

Ribose-5-phosphate C5H11O8P 230.0191

Ribulose-5-phosphate C5H11O8P 230.0191

Ribose-1-phosphate C5H11O8P 230.0191

Xylose-5-phosphate C5H11O8P 230.0191

Xylulose-5-phosphate C5H11O8P 230.0191

Decursinol C14H14O4 246.0892

Marmesin C14H14O4 246.0892

D3-isopentyl-pp C5H12O7P2 246.0058

Dimethylallyl diphosphate C5H12O7P2 246.0058

Isopentenyl diphosphate C5H12O7P22 246.0058

Isopimpinellin C13H10O5 246.0528

Pimpinellin C13H10O5 246.0528

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Inosine C10H12N4O5 268.0808 485.2 Cocaine C17H21NO4 303.1471 Dihydroxyquercetin C15H12O7 304.0583 Taxifolin C15H12O7 304.0583 Dihydroxymyricetin C15H12O8 320.0532 4-coumaroylshikimate C16H16O7 320.0896 Quercitin-3-galactoside C21H20O12 464.0955

Strategies for the production of chemically consistent plantlets of Silybum marianum. L

Chapter 5

Table 5.4: Putative identification of the metabolites by the personalv7qstar in-house program for the selected bins sorted from the organic extracts of biological samples

Bin Masses Metabolites Name Formula Accurate masses 172

Glyceric acid C3H6O4 105.0187836

Serine C3H7NO3 105.0425949

L-serine C3H7NO3 105.0425949

L-cysteine C3H7NO2S 121.0197525

Cysteine C3H7NO2S 121.0197525

Diethanolamine C4H11NO2 105.0789795

5-(2-hydroxyethyl)-4-methylthiazole C6H9NOS 143.0404816

Phenethylamine C8H11N 121.0891495 178

Aspartic acid C4H7NO4 133.0375061

L-aspartic acid C4H7NO4 133.0375061

L-methionine C5H11NO2S 149.0510559

Methionine C5H11NO2S 149.0510559

D-aspartate C4H7NO4 133.1040039

L-aspartate C4H7NO4 133.1040039

D-cathinone C9H11NO 149.0840607 178.2

Lactose C12H22O11 342.1162

Maltose C12H22O11 342.1162

Melibiose C12H22O11 342.1162

Sucrose C12H22O11 342.1162

Trehalose C12H22O11 342.1162 184

Selenophosphate H3PO3Se 160.9559937

2-aminoadipic acid C6H11NO4 161.0688019

O-acetyl-l-homoserine C6H11NO4 161.0688019

2-keto-glutaramate C5H6NO4 145.1150055

Normetanephrine C9H13NO3 183.0895386 187.2

3-phosphoglycerate C3H7O7P 185.9929

Sulfate H2O4S 97.96738

Orthophosphate H3PO4 97.9769

Phosphoric acid H3PO4 97.9769

Methyl allyl disulfide C4H8S2 120.0067

3-(methylthio)propionic acid C4H8S2 120.0245

Erythrose and isomers C4H8O4 120.0423

Purine C5H4N4 120.0436

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379.2

Cinchonine C19H22N2O 294.1732

GA9 C19H24O4 316.1674

α-Eleostearic acid C18H30O2 278.2246

α-Linolenic acid C18H30O2 278.2246

γ-Linolenic acid C18H30O2 278.2246

Linolenic acid C18H30O2 278.2246

Punicic acid C18H30O2 278.2246

y-linolenic acid C18H30O2 278.2246

GA12-aldehyde C20H29O3 316.2039

Sterculic acid C19H34O2 294.2559

9,10-Dihydroxystearic acid C18H36O4 316.2614

α-Ribazole C14H18N2O4 278.307

Kaempferol C15H10O8 318.0376

Myricetin C15H10O8 318.0376

Ellagic acid C14H6O8 302.0063

Kaempferol C15H10O6 286.0477

Luteolin C15H10O6 286.0477

Morin C15H10O7 302.0427

Quercetin C15H10O7 302.0427

5-enolpyruvyl-shikimate-3-phosphate C10H13O10P 324.0246 5-O-(1-Carboxyvinyl)-3- C10H13O10P 324.0246 phosphoshikimate

Strategies for the production of chemically consistent plantlets of Silybum marianum. L

Chapter 6

6. CONCLUSIONS AND FUTURE PROSPECTS

CONCLUSIONS

In the present study, we have attempted to work on establishment of different strategies for production of S. marianum plantlets with consistent phytochemical profiles. Due to its high commercial and therapeutic demands, geographic variability in silymarin content and lack of uniform cultivation practices, these strategies will be useful for production of healthy biomass. The regeneration system developed here assured efficient establishment, multiplication, rhizogenesis and successful acclimatization of S. marianum plants that could be exploited to multiply elite genotypes and develop other in vitro strategies for the conservation of this valuable medicinal plant. Moreover, this is the first report on silymarin production in different regenerated tissues of S. marianum for chromatographic fingerprint analysis and quality control. Following conclusions can be deduced from our results

 In the first experiment, an efficient in vitro seed germination protocol has been developed by application of various light regimes in conjunction to different plant growth regulators. This report showed that incubation of seeds at BA

(0.5 mg/l) + GA3 (1.5 mg/l) + TDZ (1.0 mg/l) in complete darkness for 2 week and its subsequent transference to 16 h light/8 h dark for 2 week, significantly enhanced germination parameters in S. marianum. Moreover, 5.48 mg/g silymarin was detected in this study and a correlation was observed among FRSA, PAL and silymarin content.

 In the second experiment, a feasible protocol for micropropagation of S. marianum from leaf explants was optimized. The plant growth regulator Thidiazuron (TDZ) was exploited to improve micropropagation protocol and silymarin production. The major observations of this experiment have shown that TDZ in combination with NAA was much effective in callus formation; while alone at highest level it resulted into optimum shoot organogenic parameters. TDZ supplementation has significantly effected silymarin accumulation in present study since it was the most effective PGR in Strategies for the production of chemically consistent plantlets of Silybum marianum. L

Chapter 6

micropropagation via indirect organogenesis from leaf explant in S. marainum. A considerable content of silymarin (8.3 mg/g DW) was detected in micropropagated plantlets in this report.

 In the third experiment, an efficient procedure for somatic embryogenesis in S. marianum has been optimized. Indirect somatic embryogenesis through embryogenic callus formation, followed by embryo development and germination into plantlets was observed. Among the tested explants petiole showed best response, followed by leaf explants. SH medium was superior to B5 and MS media in induction of embryogenic callus and formation of somatic embryos. However, embryos were germinated in 1/2 MS medium

supplemented with various levels of GA3. Furthermore, significant biochemical variations were detected in plant samples raised during different growth phases. The biochemical assays assured the protective role of silymarin against ROS as a strong antioxidant. This study indicated that the process of somatic embryogenesis was characterized by some biochemical and physiological changes, influenced by interplay of explants type, basal media and plant growth regulators.

 In the fourth experiment, a feasible protocol has been optimized to investigate the metabolic events happening at different growth phases during in-vitro somatic embryogenesis of S. marianum. ESI-TOF MS was performed for evaluation of metabolite profiles at different growth phases during embryogenesis. The PCA analysis differentiated the plant samples on the basis of differences in developmental stages during somatic embryogenesis. Furthermore, the loading scatter plots of PCA enabled the detection of several key metabolites responsible for separating samples from different growth stages.

Strategies for the production of chemically consistent plantlets of Silybum marianum. L

Chapter 6

FUTURE PROSPECTS

 The optimized protocols established here, have potential for scale-up on a commercial level by different industries to further enhance the production of plantlets and silymarin content for multiple applications. Moreover, these protocols can be taken up for further research on other aspects such as mutagenesis, molecular analysis and enhanced production of other secondary metabolites.

 Future research shall focus on designing strategies for achieving higher frequencies of embryo proliferation in order to allow long term culture maintenance. Moreover, the optimized in vitro cultures need to be scaled-up to bioreactor level to produce chemically consistent plantlets and antioxidant compounds.

 MALDI-MSi procedure should be standardized for the direct visualization of the metabolites distribution pattern during in vitro somatic embryogenesis in S. marianum L.

 Role of primary and secondary metabolites at different stages of growth and development during in vitro regeneration can also be investigated.

Strategies for the production of chemically consistent plantlets of Silybum marianum. L

Chapter 7

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Thesis

ORIGINALITY REPORT

17% 12% 11% 0% SIMILARITY INDEX INTERNET SOURCES PUBLICATIONS STUDENT PAPERS

PRIMARY SOURCES

Susan J. Murch. "St. John’s wort (Hypericum 1 % perforatum L.): Challenges and strategies for 2 production of chemically-consistent plants", Canadian Journal of Plant Science, 07/07/2006 Publication

ALEMARDAN, Ali; KARKANIS, Anestis and 2 % SALEHI, Reza. "Breeding Objectives and 1 Selection Criteria for Milk Thistle [Silybum marianum (L.) Gaertn.] Improvement", Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 2013. Publication

www.academicjournals.org 3 Internet Source 1%

www.ncbi.nlm.nih.gov 4 Internet Source 1%

docs9.chomikuj.pl 5 Internet Source 1%

herkules.oulu.fi 6 Internet Source <1% www.biotehnologii.usamv.ro 7 Internet Source <1%

www.scielo.br 8 Internet Source <1%

scialert.net 9 Internet Source <1%

etd.lib.nsysu.edu.tw 10 Internet Source <1%

www.cicy.mx 11 Internet Source <1%

bashanfoundation.org 12 Internet Source <1%

Bekheet, S. A.; Taha, H. S. and Gabr, Ahmed 13 % M. M.. "Protocol for in vitro morphogenesis <1 and hairy root cultures of Milk thistle (Silybum marianum L. Gaertn)", Journal of Applied Sciences Research, 2013. Publication

www.jmedar.ro 14 Internet Source <1%

ejournal.sinica.edu.tw 15 Internet Source <1%

ekheet, S. A.. "In vitro biomass production of 16 % liver-protective compounds from Globe <1 artichoke (Cynara scolymus L.) and Milk thistle (Silybum marianum) plants", Emirates Journal of Food & Agriculture (EJFA)/2079052X, Plant Cell Tiss Organ Cult (2010) 101:371–376 DOI 10.1007/s11240-010-9692-x

RESEARCH NOTE

Shoot regeneration and free-radical scavenging activity in Silybum marianum L.

Bilal Haider Abbasi • Mubarak Ali Khan • Tariq Mahmood • Mushtaq Ahmad • Muhammad Fayyaz Chaudhary • Mir Ajab Khan

Received: 18 May 2009 / Accepted: 18 January 2010 / Published online: 2 February 2010 Ó Springer Science+Business Media B.V. 2010

Abstract The morphogenic potential and free-radical Abbreviations scavenging activity of the medicinal plant, Silybum BA 6-Benzyladenine marianum L. (milk thistle) were investigated. Callus DPPH 1 1-Diphenyl-2-picrylhydrazyl development and shoot organogenesis were induced from FRSA Free-radical scavenging activity leaf explants of wild-grown plants incubated on media GA3 Gibberellic acid supplemented with different plant growth regulators Kn Kinetin (PGRs). The highest frequency of callus induction was MS0 MS medium without plant growth regulators observed on explants incubated on Murashige and Skoog MS Half-strength macro-nutrients of MS medium (MS) medium supplemented with 5.0 mg l-1 6-benzylad- NAA a-Naphthaleneacetic acid enine (BA) after 20 days of culture. Subsequent transfer of PGRs Plant growth regulators callogenic explants onto MS medium supplemented with Zn Zeatin -1 -1 2.0 mg l gibberellic acid (GA3) and 1.0 mg l a-naphthaleneacetic acid (NAA) resulted in 25.5 ± 2.0 shoots per culture flask after 30 days following culture. Moreover, when shoots were transferred to an elongation Fruits of Silybum marianum (L.) Gaertn (milk thistle, medium, the longest shoots were observed on MS medium Asteraceae), contain isomeric mixtures of flavonolignans, supplemented with 0.5 mg l-1 BA and 1.0 mg l-1 NAA, including silychristin, silydianin, silybin, and isosilybin, and these shoots were rooted on a PGR-free MS basal collectively known as silymarin (Kurkin et al. 2001; medium. Assay of antioxidant activity of in vitro and in Morazzoni and Bombardelli 1995). Silymarin is used as a vivo grown tissues revealed that significantly higher anti- hepatoprotector for oral treatment of toxic liver damage oxidant activity was observed in callus than all other and of chronic inflammatory liver diseases and liver cir- regenerated tissues and wild-grown plants. rhosis (Valenzuela et al. 1986; Flora et al. 1998). Aside from its antioxidant properties and its role in stimulating Keywords Silybum Á Callus Á Regeneration Á protein synthesis and cell regeneration (Tawaha et al. Antioxidant Á DPPH Á Gibberellic acid 2007), silymarin may also reduce incidence of certain forms of cancer (Katiyar et al. 1997), and has been among the most investigated plant extracts with known mechanisms of action (Becker and Schrall 1977; Cimino et al. 2006; Sanchez-Sampedro et al. 2007). Although cell cul- B. H. Abbasi (&) Á M. A. Khan Á M. F. Chaudhary Department of Biotechnology, Faculty of Biological Sciences, tures of S. marianum are capable of producing silymarin, Quaid-i-Azam University, Islamabad 45320, Pakistan amounts produced are lower than those produced in fruits e-mail: [email protected] (Ferreiro et al. 1991; Cacho et al. 1999; Alikardis et al. 2000; Sanchez-Sampedro et al. 2005). T. Mahmood Á M. Ahmad Á M. A. Khan Department of Plant Sciences, Quaid-i-Azam University, Currently, all commercially available silymarin is Islamabad 45320, Pakistan obtained from intact fruits of wild plants. The increasing

123

Industrial Crops and Products 46 (2013) 105–110

Contents lists available at SciVerse ScienceDirect

Industrial Crops and Products

journa l homepage: www.elsevier.com/locate/indcrop

Effects of light regimes on in vitro seed germination and silymarin content in

Silybum marianum

a a,∗ a b

Mubarak Ali Khan , Bilal Haider Abbasi , Nisar Ahmed , Huma Ali

Department of Biotechnology, Quaid-i-Azam University, Islamabad 45320, Pakistan

Department of Biotechnology, Bacha Khan University Charsadda, KP, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: Silybum marianum is an economically important crop worldwide. It is renowned for production of biolog-

Received 24 August 2012

ically important silymarin. Average sale of silymarin is about US$ 8 billion/annum and its demand varies

Received in revised form

from 18 to 20 tons/year. Despite of its demand, there is lack of research efforts on cultivation and improve-

14 December 2012

ment of this plant. We hereby established feasible seed germination protocol for production of healthier

Accepted 19 December 2012 −1

and chemically consistent plantlets. Combination of benzyladenine (BA, 0.5 mg l ) + gibberellic acid (GA3,

−1 −1

1.5 mg l ) + thidiazuron (TDZ, 1.0 mg l ) produced optimum germination frequency in seeds kept in 2

Keywords:

weeks dark and subsequently transferred to 2 weeks light (16 h photoperiod) conditions. Correlation

Silybum

HPLC among mean shoot length, mean root length, set of antioxidative enzyme activities was also observed in

Antioxidant current report. Silymarin was determined by high performance liquid chromatography (HPLC). Consid-

−1

Plant growth regulators erable amount of silymarin (5.48 mg DW) was detected in our study, which was comparative to other

Light reports available. Antioxidant activity (1,1-diphenyl-2-picrylhydrazyl; FRSA) and phenylalanine ammo-

nia lyase (PAL) activity was also determined. Silymarin content had shown direct relationship with these

activities. It showed that silymarin was a major antioxidant in current report. This study provides basis

for expedited production of S. marianum plantlets with feasible content of silymarin.

1. Introduction a consequence of abiotic and biotic contamination, and is the

major bottleneck in the production of high-quality plant mate-

Silybum marianum L. is an important medicinal plant belongs rial for preparation of effective phytomedicine (Murch et al., 2000).

to family Asteraceae (Abbasi et al., 2010). Its major bioactive Furthermore, advantages of seed explants are also available in lit-

component is silymarin; which is an isomeric mixture of ﬂavono- erature (Malik and Saxena, 1992; Victor et al., 1999).

lignans (Vaid and Katiya, 2010; Pliskova et al., 2005). Silymarin In vitro seed germination is often considered as the most sensi-

is the most proven phytochemical with known mechanism of tive stage in the life cycle of economically important plant species,

action against viral hepatitis in various clinical studies (Thabrew, and it normally shortens the period required for establishment of

1996; Huseini et al., 2006). Furthermore, silymarin has antioxidant, vegetative clones by simplifying the methods for obtaining regen-

anti-inﬂammatory, antiﬁbrotic, immuno-modulatory and antitu- erants by bypassing callogenesis and retaining the integrity of

mor effects (Agarwal et al., 2006; Kuki et al., 2012; Flora et al., 1998; the seedling (Nikolic et al., 2006). Under normal growth condi-

Katiyar, 2005). tions plants produce reactive oxygen species (ROS); like superoxide

− − −

It is very common for this species to have optimum germina- (O2 ), peroxide (H2O2), hydroxyl (OH ) and singlet oxygen (O2 )

◦

tion at 2–15 C in autumn and spring seasons in soil; with low as metabolic byproducts (Cai et al., 2004). However, stress con-

to moderate nutritional requirements. It is also adaptable to poor ditions enhance production of these ROS; which lead the plant

quality soils. However, the poor nutritional status of the soil is cell to switch on its antioxidant system via enzymatic and non-

the limiting factor for the production of healthy Silybum plantlets enzymatic routes to scavenge the reactive oxygen species by

with enhanced and consistent chemical proﬁles (Karkanis et al., coupling redox to minimize the oxidative damage (Molazem and

2011). In vitro growth conditions can overcome these issues, which Azimi, 2011).

lead to extensive chemical and genetic variability in many wild Antioxidative enzymes like superoxide dismutase (SOD), cata-

and cultivated medicinal plant species. This variability occurs as lase (CAT), ascorbate peroxidase (APX) and peroxidase (POD) are

of signiﬁcant importance in plant growth and development. These

antioxidative enzymes regulate various processes including germi-

∗ nation, growth of seedling and the process of senescence through

Corresponding author. Tel.: +92 51 90644121; fax: +92 51 90644121.

acting against various stresses (Mitrovic and Bogdanovic, 2009).

E-mail addresses: [email protected], [email protected] (B.H. Abbasi).

THIDIAZURON ENHANCED REGENERATION AND SILYMARIN CONTENT IN SILYBUM MARIANUM L.

MUBARAK ALI KHAN, BILAL HAIDER ABBASI* AND ZABTA KHAN SHINWARI

Department of Biotechnology, Quaid-i-Azam University, Islamabad 45320, Pakistan *Correspondence e-mail: [email protected]

Abstract

Silybum marianum, of family Asteraceae is renowned for production of biologically important silymarin, which has shown multi-dimensional medicinal properties. It has a high protective role against jaundice and hepatitis C worldwide. We hereby established a feasible and efficient method for indirect regeneration of S. marianum for production of consistent plantlets. Calli were induced from leaf explants of seed-derived plantlets on Murashige and Skoog (MS) medium supplemented with several concentrations of different plant growth regulators (PGRs). Highest callogenic response (89%) was recorded for 4.4µM Thidiazuron (TDZ) in combination with 6.6µM Kinetin (Kn). Subsequent sub-culturing of callus after 4 weeks of culture, on medium with similar compositions of PGRs induced shoot organogenesis. Highest shoot induction frequency (86%) with maximum mean multiple shoots (26 shoots per explant) were recorded for 11µM TDZ after 4 weeks of transfer. Longest shoots (4.1 cm) were recorded for MS medium augmented with 6.6µM TDZ and 4.4µM α- naphthalene acetic acid (NAA). Furthermore, rooted plantlets were developed on MS medium containing different concentrations of indole acetic acid (IAA). Silymarin was determined by High performance liquid chromatography (HPLC) and 8.47 mg/g DW silymarin was detected in the regenerated plantlets. This study contributes to a better understanding of the different mechanisms involved in morphogenesis and production of biologically active principle in Silybum marianum.

Introduction showed that TDZ is capable of fulfilling both the cytokinin and auxin requirement of various regenerative Silybum marianum L. Gaertn., belongs to family responses in many plant species (Arshad et al., 2012). Asteraceae and is commonly known as Milk thistle, it is Furthermore, TDZ application has a profound effect on native to the Mediterranean basin and is now widespread enhancing the levels of secondary metabolites in the In throughout the world. It is cultivated as a medicinal plant vitro cultures of many medicinally important plant species (Khan et al., 2009) and has been used from ancient times (Liu et al., 2007). (Kren & Walterova 2005). It has an isomeric mixture of The main objective of present study was to exploit multiple flavonolignans called silymarin that is TDZ for establishment of efficient regeneration protocol considered as the most potent antioxidant (Khan et al., for S. marianum and to evaluate its impact on 2013). Approximately 8 to 33% of patients with hepatic accumulation of silymarin in regenerated tissues and disorders consume this plant frequently as herbal remedy plantlets by HPLC. worldwide (Polyak et al., 2007). However, there are some issues with Silybum products, as existing high Materials and Methods variability in the end products has resulted inconsistent results regarding its efficacy in various clinical trials (Lee Viable seeds of Silybum marianum were collected & Liu, 2003; Haban et al., 2009). Therefore, control, from main campus of Quaid-i-Azam University, Pakistan. characterization and standardization of silymarin appear In vitro seed germination was achieved by the feasible to be a necessary mandate for formulation of end products protocol reported by Khan et al., (2013). Leaf explants with consistent efficacy (Aruoma, 2003). Consistent (1.5 cm2) were excised from In vitro germinated 28-days production of phytochemicals can be achieved via In vitro old seedlings and were placed onto Murashige and Skoog clonal propagation (Parveen & Shahzad, 2010). The basal medium (MS, 1962; Phytotechnology Labs, USA) increasing world’s demand for silymarin (18-20 tons per containing 3% sucrose and 0.8% (w/v) agar year) is endangering the sparse populations of this (Phytotechnology Labs, USA) in 150 ml conical flask species, still this economically important herb has been supplemented with various PGRs like Thidiazuron (TDZ), ignored in research programs for its improvement and Kinetin (Kn) and α-napthalene acetic acid (NAA). cultivation (Ahmad et al., 2008). Plant In vitro technology Different concentrations (1.0, 2.2, 4.4, 6.6, 8.8, 11, 13.2 is capable of producing large number of aseptic, genetically similar, and chemically consistent plants in a and 15.4µM) of TDZ or (1.0. 2.2, 4.4, 6.6 and 8.8 µM) of short period of time and limited space for the production Kn or NAA either alone or in combinations were tested. of high quality medicines (Victorio et al., 2012). The pH (Eutech Instruments pH 510, Singapore) of media Thidiazuron (N-phenyl-N-1,2,3-thidiazol-5-yl urea) was adjusted to 5.8 before autoclaving (121°C, 20 min, has proven to be highly effective bio-regulator of plant Systec VX 100, Germany). MS basal medium (MS0) morphogenesis. It is reported to be much more effective without any plant growth regulator (PGR) was used as in organogenesis via callogenesis in many plant species control for different phases of regeneration. than the commonly employed conventional cytokinins After 4 weeks, number of responding explants (% (Wannakrairoj & Tefera, 2012; Hussain et al., 2013). callus induction) was recorded. Callus was refreshed by Initially TDZ was classified as cytokinin due to its sub-culturing to MS medium with similar composition of cytokinin like responses. However, later on research PGRs for further growth and multiplication. Plant Cell Tiss Organ Cult DOI 10.1007/s11240-014-0587-0

ORIGINAL PAPER

Temporal variations in metabolite proﬁles at different growth phases during somatic embryogenesis of Silybum marianum L.

Mubarak Ali Khan • Bilal Haider Abbasi • Huma Ali • Mohammad Ali • Mohammad Adil • Ishtiaq Hussain

Received: 16 May 2014 / Accepted: 28 July 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Silybum marianum, commonly known as Milk obtained using Electro spray ionization mass spectrometry thistle, is a popular herbal supplement used for the treatment ESI/MS. Principal component analysis (PCA) was carried of jaundice and liver cirrhosis worldwide. Here we estab- out to identify key metabolites in different growth phases lished methods for somatic embryogenesis and comparative during somatic embryogenesis. The loading scatter plots metabolite profiling of the different growth phases during enabled the detection of several bin masses responsible for embryogenesis in S. marianum. Highest embryogenic separating samples from different growth stages. Based on potential was observed for calli previously derived from the values of % total ions count and average intensity of petiole explants on Schenk and Hildebrandt medium con- selected bins in all biological samples, putatively known taining 2.5 mg l-1 2,4-dichlorophenoxyacetic acid (2,4-D) metabolites were obtained from in-house bin program. and 1.5 mg l-1 N6-benzyladenine (BA). Somatic embryos Amino acids associated with various biosynthetic pathways (SE) were induced when embryogenic calli with pre- like arginine, asparagine and serine were abundantly embryoid masses (PEMs) were subcultured on same media detected in GSE, while they were detected at decreased as used for induction of embryogenic callus. Highest number intensities in NEC. However, tryptophan was measured with of somatic embryos (46 somatic embryo per callus) was increased signals in SE when compared to other growth observed at 1.5 mg l-1 2,4-D and 1.5 mg l-1 BA, however phases. Glucose, fructose and fructose-6-phosphate were strength MS medium showed optimal response for mat- mostly accumulated in NEC; however they were detected uration followed by germination of somatic embryos at with lowest intensities in GSE. Moreover, sucrose and sig- -1 1.5 mg l GA3. Metabolite profiles from developmental nificant secondary metabolites like cinnamic acid, kaempf- stages of non-embryogenic callus (NEC), PEMs, SE and erol, quercetin, myricetin, linolenic acid, and 5-enolpyruvyl- embryos germinating into intact plantlets (GSE) were shikimate-3-phosphate were found at higher amount in SE when compared to other embryogenic phases.

Electronic supplementary material The online version of this Keywords Silybum Á Somatic embryo Á Metabolite Á article (doi:10.1007/s11240-014-0587-0) contains supplementary Electro spray ionization Á Bin masses material, which is available to authorized users.

M. A. Khan Á B. H. Abbasi (&) Á M. Ali Á M. Adil Abbreviations Department of Biotechnology, Quaid-I-Azam University, SH Schenk and Hildebrandt Islamabad 45320, Pakistan MS Murashige and Skoog e-mail: [email protected] PGR Plant growth regulator H. Ali SE Somatic embryo Department of Biotechnology, Bacha Khan University Charsada, NEC Non-embryogenic callus Charsada, KP, Pakistan PAL Phenylalanine ammonia lyase FRSA Free radical scavenging activity I. Hussain Department of Biological Sciences, Karakoram International ESI Electro spray ionization University, Gilgit-Baltistan, Pakistan PCA Principal component analysis

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