Medicinal Plant Biotechnology

From Basic Research to Industrial Applications

Volume II

Edited by

Oliver Kayser and WimJ. Quax

B I C E N T E N N I A L .

WILEY-VCH Verlag GmbH & Co. KGaA

Contents

Preface XVII

Foreword XIX

List of Contributors XXIII

Color Plates XXIX

Volume I

Part 1 Linking Plants, Genes, and Biotechnology 1

1 The Engineering of Medicinal Plants: Prospects and Limitationsof Medicinal Plant Biotechnology 3Matthys K. Julsing, Wim J. Quax, and Oliver Kayser

1.1 Introduction 31.2 Genetic Transformation and Production of Transgenic Plants 51.3 Pathway Engineering and Combinatorial Biosynthesis 51.4 Bioprocessing 61.5 Plant Propagation 71.6 Bioanalytics and Metabolomics 71.7 Future Prospects 8

References 8

2 Metabolomics 9Young Hae Choi, Hye Kyong Kim, and Robert Verpoorte

2.1 Introduction 92.2 Analytical Methods 112.2.1 Chromatography 112.2.1.1 Gas Chromatography (GC) 112.2.1.2 High-Performance Liquid Chromatography (HPLC) 132.2.1.3 Capillary Electrophoresis (CE) 142.2.1.4 Thin-Layer Chromatography (TLC) 14

Medicinal Plant Biotechnology. From Basic Research to Industrial ApplicationsEdited by Oliver Kayser and Wim ]. QuaxCopyright © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN 978-3-527-31443-0

VI Contents

2.2.2 '2.2.2.12.2.2.22.2.32.2.42.32.42.52.6

3

3.13.2

3.33.3.13.3.23.3.33.43.4.13.53.63.7

4

4.14.24.34.44.54.64.74.7.1

4.7.24.7.2.14.7.2.24.7.2.34.7.2.4

Spectroscopy 14Mass Spectrometry (MS) 14Nuclear Magnetic Resonance (NMR) Spectrometry 16Identification of Metabolites 18Sample Treatment 18Data Handling 21The General Set-Up of a Metabolomics Project 25Applications 26Conclusions 26References 27

H PLC-N M R Techniques for Plant Extract Analysis 29

Dan Stcerk, Maja Lambert, and Jerzy W. JaroszewskiIntroduction 29Hyphenation of Separation Techniques and SpectroscopicMethods 30Direct HPLC-NMR Methods 32Continuous-Flow HPLC-NMR 32Stopped-Flow HPLC-NMR 33Loop-Collection HPLC-NMR 36Indirect HPLC-NMR Methods 36HPLC-SPE-NMR 37Cryogenically Cooled NMR Probes 41Miniaturization 41Conclusions 43References 43

Plant-Associated Microorganisms (Endophytes) as a New Sourceof Bioactive Natural Products 49Gary StrobelAbstract 49Introduction 49Why Are There Needs for New Medicines? 50Natural Products in Medicine 51Endophytic Microbes 52Rationale for Plant Selection 53Endophytes and Biodiversity 55Endophytes and Natural Products 56Isolation, Preservation and Storage of Endophytic Cultures for ProductIsolation 57Some Examples of Bioactive Natural Products from Endophytes 57Endophytic Fungal Products as Antibiotics 57Endophytic Bacterial Products as Antibiotics 61Endophytic Streptomycetes as Antibiotic Producers 62Volatile Antibiotics from Endophytes 63

Contents VII

4.7.2.5 Antiviral Compounds from Endophytes 644.7.2.6 Endophytic Fungal Products as Anticancer Agents 65Al.21 Endophytic Fungal Products as Antioxidants 674.7.2.8 Endophytic Fungal Products as Immunosuppressive Compounds4.8 Surprising Results from Molecular Biology Studies

on Pestalotiopsis microspora 68

4.9 Concluding Statements 69Acknowledgments 70References 70

68

5 DNA Profiling of Plants 73Hilde Nybom and Kurt Weising

5.1 Introduction 735.2 Methodology of Plant DNA Profiling 745.2.1 DNA Sequencing 755.2.2 Multilocus DNA Profiling 765.2.2.1 Hybridization-Based RFLP Fingerprinting 775.2.2.2 PCR with Arbitrary Primers 775.2.2.3 PCR with Microsatellite-Complementary Primers 775.2.2.4 AFLP Analysis 785.2.3 Locus-Specific Microsatellite DNA Markers 785.2.4 PCR-Based RFLP Analysis of Organellar and Nuclear Genomes 795.2.5 Other DNA Marker Methods 805.2.6 The Next Generation: SNPs and DNA Microarrays 805.3 Applications 815.3.1 Genotype Identification 815.3.1.1 Plant Species 815.3.1.2 Plant Cultivars and Accessions 835.3.1.3 Jn-Vitro-Propagated Plant Material 835.3.2 Genetic Diversity 845.3.2.1 Variation and Relatedness Among Cultivars and Accessions 845.3.2.2 Amount and Distribution of Variability in Wild-Growing Plants 855.3.2.3 Plant Systematics 865.3.3 Gene Tagging 875.4 Conclusions 88

References 89

6.16.26.36.3.1

Bioprospecting: The Search for Bioactive Lead Structuresfrom Nature 97Michael WinkAbstract 97Introduction 97The Function of Secondary Metabolites 102Modes of Action 103Biomembranes 106

VIII Contents

6.3.2 Proteins and Protein Conformation 1076.3.3 DNAandRNA 1126.4 The Utilization of Secondary Metabolites in Medicine 1146.5 Conclusions 115

Key references 115

7 Biotechnological Approaches for the Production of some PromisingPlant-Based Chemotherapeutics 117Ashish Baldi, V.S. Bisaria, and A.K. SrivastavaAbstract 117

7.1 Introduction 1187.2 Production by Plant Cell Cultures 1247.3 Biotechnological Approaches for Production 1257.3.1 Dedifferentiated Cultures 1257.3.2 Differentiated Cultures 1297.3.3 Optimization of Culture Media Composition

and Culture Conditions 1317.3.4 Immobilization 1327.3.5 Feeding of Precursors 1327.3.6 Elicitors 1337.3.7 In-Situ Product Removal Strategies 1357.3.8 Biotransformation Studies 1367.3.9 Bioreactor and Scale-Up Studies 136.3.10 Biosynthetic Pathway Mapping and Metabolic Engineering 138.3.10.1 Biosynthesis of Podophyllotoxin 138

7.3.10.2 Biosynthesis of Paclitaxel 1417.3.10.3 Biosynthesis of Camptothecin 1437.4 Future Prospects 146

References 147

Part 2 Genetic Modifications, Transgenic Plants and Potential of MedicinalPlants in Cenetechnology and Biotechnology 157

8 In-Vitro Culturing Techniques of Medicinal Plants 159Wolfgang Kreis

8.1 Introduction 1598.2 Basic Methods and Techniques 1618.2.1 Seed Germination In Vitro 1618.2.2 Embryo Culture 1628.2.3 Callus Culture 1628.2.4 Organogenesis 1648.2.5 Haploid Technology 1658.2.6 Somatic Embryogenesis 1668.3 Protoplast Technology 167

Contents IX

8.3.1 Protoplasts 1678.3.2 Somatic Hybridization 1688.4 Special Techniques 1698.4.1 Gene Transfer 3698.4.2 Germplasm Storage 1698.5 Permanent In-Vitro Cultures 1708.5.1 Cell Suspension Culture 1708.5.2 Root Culture 1718.5.3 Shoot Culture 1738.5.4 Bioreactors 1748.6 Methods and Techniques Related to Secondary Metabolism 2778.6.1 Inducing Variability 1778.6.2 Selection 1788.6.3 Biotransformation 1788.6.4 Elicitation 1798.6.5 Immobilization and Permeation 1808.7 Conclusions 380

References 181

9 Biotechnological Methods for Selection of High-Yielding Cell Linesand Production of Secondary Metabolites in Medicinal Plants 187Donald P. Briskin

9.1 Introduction 1879.2 Medicinal Plant Tissue Cultures and the In-Vitro Production

of Phytomedicinal Secondary Metabolites 1889.3 Factors Leading to Elevated Production of Secondary Metabolites

in Medicinal Plant Cell Cultures 189

9.3.1 Cell Culture Media Components 1899.3.2 Elicitors and Jasmonates 1919.3.3 Exogenous Substances 1929.3.4 Immobilization 1939.3.5 Physical Stress 1939.4 Enhancement of Secondary Metabolite Production

through Agrobacterium rhizogenes Transformation 1949.5 Metabolite Engineering of Medicinal Plants in Culture 1959.6 Large-Scale Production of Medicinal Secondary Metabolites

in Bioreactor Systems 1979.7 Summary and Perspective 198

References 199

10 Impact of Whole-Genome and Expressed Sequence Tag Databaseson the Study of Plant Secondary Metabolism 203Jillian M. Hagel, Jonathan E. Page, and Peter J. Facchini

10.1 Introduction 20310.2 Whole-Genome Sequences 204

Contents

10.310.410.510.610.7

Expressed Sequence Tags 207Terpenoids 209Phenylpropanoids 215Alkaloids 219Conclusion 225Acknowledgments 225References 225

11 Biotechnology of Solanaceae Alkaloids:A Model or an Industrial Perspective? 237Birgit Drager

11.1 Culture Systems 23911.1.1 The Early Days of Solanaceae Culture 23911.1.2 Hairy Roots 24011.1.3 Further Culture Systems 24211.2 Alkaloid Production 24411.2.1 Choice of the Best Culture System 24411.2.2 Optimization Strategies 24411.2.2.1 Large-Scale Culture, Light, and Aeration 24511.2.2.2 Feeding of Alkaloid Precursors 24611.2.2.3 Nutrient Medium Variation 24611.2.2.4 Elicitation 24711.3 Alkaloid Biosynthesis 25111.3.1 Tropane Alkaloid Metabolites, Enzymes and Products11.3.2 Putrescine N-methyltransferase (PMT) 25211.3.3 Tropinone Reductases 25311.3.4 Hyoscyamine-6P-Hydroxylase 25411.4 Solanaceae Model Systems for Transformation

and Overexpression 25511.5 Conclusion and Future Aspects 25711.5.1 More Genes and Enzymes 25711.5.2 Compartmentation, Transport, and Excretion 258

Acknowledgment 259References 259

251

Volume II

12 Plant Cell Cultures: Production of Biologically ImportantSecondary Metabolites from Medicinal Plants of Taiwan 267Mulabagal Vanisree and Hsin-Sheng TsayAbstract 267

12.1 Introduction 26712.2 Cell Culture Protocols for Biologically Important

Secondary Metabolites 269

12.2.112.2.2

12.2.3

12.2.4

12.2.5

12.2.6

12.2.7

12.2.8

12.2.9

12.2.1012.3

13

13.113.2

13.313.3.113.3.213.413.4.113.4.213.4.313.4.413.513.6

14

14.114.214.2.1

Contents XI

Production of Imperatorin from Angelica dahurica var.formosana 270Production and Analysis of Corydaline and Tetrahydropalmatinefrom Corydalis yanhusuo 272Identification of Alkyl Ferulates in Micropropagated Plantsof Dendrobium tosaense MAKIN0 273Production of Diosgenin from Dioscorea doryophoraby Cell Suspension Cultures 274Establishment of Gentiana davidii var.formosana (Hayata)T. N. Ho Cell Suspension Cultures 275Quantitative Analysis of Anthraquinones Emodinand Physcion formed in In-Vitro-Propagated Shootsand Plants of Polygonum multijlorum 276Production of Cryptotanshinone from Callus Culturesof Salvia miltiorrhiza Bunge 278Harpagoside Production in Micropropagated Plantsof Scrophularia yoshimurae Yamazaki 279Production of Anthocyanins in Callus Culturesof Solanum melongena 280Taxol Synthesis in Cell Suspension Cultures of Taxus mairei 281Conclusions and Future Perspectives 281References 282

Exploring and Accessing Plant Natural Product Biosynthesisin Engineered Microbial Hosts 287Erin K. Marasco and Claudia Schmidt-DannertIntroduction 287Heterologous Expression of Plant Natural Product Genesand Pathways 288Alkaloids 290Monoterpenoid Indole Alkaloids 290Benzoisoquinoline Alkaloids 293Isoprenoids 296Sesquiterpenes and Diterpenes 297Taxol 299Artemisinin 301Carotenoids 302Flavonoids 304Conclusions 308References 309

Production of Therapeutic Antibodies in Plants 319Richard M. Twyman, Stefan Schillberg, and Rainer FischerIntroduction 319The Evolution of Recombinant Antibody Technology 321The Importance of Recombinant Antibodies 321

14

J

XII Contents

14.2.2 Structure of Naturally Produced Antibodies 32114.2.3 Antibody Derivatives 32214.2.4 Humanized Antibodies 32314.3 Production of Recombinant Antibodies in Plants 32314.3.1 Traditional Expression Systems 32314.3.2 The Advantages of Plants 32414.3.3 Limitations that Need to be Addressed 32514.4 Current Objectives in the Field 32614.4.1 Economic Considerations 32614.4.2 Choice of Plant Species and Platform for Antibody Production 32714.4.3 Product Yields: Strategies to Enhance Transgene Expression

and Protein Accumulation 33014.4.4 Protein Folding, Assembly, and Glycosylation 33214.4.5 Biosafety Concerns 33314.5 Plant-Derived Antibodies in Clinical Trials 33614.6 Regulatory Considerations 33714.6.1 Downstream Processing 33714.6.2 Regulation of Plant-Based Production 33814.7 Summary 338

References 339

15 Glycosylation of Recombinant Proteins in Plants 345Gilbert Gorr and Friedrich Altmann

15.1 Introduction 34515.2 A Word on Names 34615.3 General Aspects of Glycosylation 34615.3.1 Asn-Linked Oligosaccharides (N-Glycans) 34615.3.2 Mucin-Type and Other O-Linked Glycans (O-Glycan) 35215.3.3 Hydroxyproline-Linked Oligosaccharides (Hyp-O-Glycans) 35215.4 Why Antibodies? 35415.5 "Humanization" of N-Glycans in Plants 35615.5.1 Authentic or Human-Like Glycosylation as the Benchmark 35615.5.2 Towards a Humanized N-Glycosylation in Plants 35915.5.3 Improving Antibodies by Glycoengineering 36115.6 Trust is Good, Control is Better: A Structural Analysis 36215.7 Conclusions and Outlook 365

References 365

16 Frontiers in Transgenic Medicinal Plants Research 375Ya'akov Tadmor and Efraim Lewinsohn

16.1 Introduction 37516.1.1 Biosynthesis: Active Principles in Medicinal Plants 37816.1.2 Identification of the Biosynthetic Pathways 37916.2 Sources of Key Genes that Affect the Biosynthesis

of Plant Natural Products 380

Contents XI I I

16.316.416.516.5.16.5.16.5.16.5.16.5.

11.1

1.21.31.4

16.5,216.5.16.5.16.5.16.6

2.12.23

Genomic Approaches Applied to Medicinal Plants Research 383Generation of Transgenic Medicinal Plants 385Metabolically Engineered Medicinal Plants 387Alkaloids 387Amino Alkaloids 387Terpenoid Indole Alkaloids 387Tropane Alkaloids: Hyoscyamine and Scopolamine 388Isoquinoline Alkaloids: Morphine, Codeine, and Thebaine 388Metabolic Engineering of the Terpenoid Pathway 388Monoterpenes 388Sesquiterpenes 389Cannabinoids 390Conclusions 390References 391

Part 3 Plants as Drug Factories 397

17 Intellectual Property Protection of Plant Biotechnology 399Konrad Sechley and Clare Salisbury

17.1 Introduction 39917.1.1 Utility Patents 40017.1.2 Trade-Related Aspects of Intellectual Property Rights (TRIPS) 40117.1.3 Plant Variety Protection 40117.1.4 Genetic Resources 40317.2 Protection of Plant Biotechnology in Europe 40417.2.1 Patent Protection: European Patent Convention (EPC) 40417.2.2 Biotechnology Directive 40717.2.3 Ethics of Patenting Plant Biotechnology 40817.3 North America 40917.3.1 US Plant Patent 40917.3.2 Utility Patents 41017.3.2.1 USA 41017.3.2.2 Canada 41117.4 Conclusion 413

References 414

18 Breeding of Medicinal Plants 417Friedrich Pank

18.1 Introduction 41718.2 Demands on MAP Cultivars 41818.3 Specifics of MAP Breeding 41918.4 Genetic Resources 42018.5 Breeding Methods 42018.5.1 Utilization of Available Natural Diversity 420

XIV Contents

18.5.2 Generation of New Variability 42118.5.2.1 Combination Breeding by Crossing 42218.5.2.2 Hybrid Breeding 42318.5.2.3 Synthetic Cultivars 42518.5.2.4 Induced Mutation 42618.5.2.5 Somaclonal Variation 42618.5.2.6 Somatic Hybridization (Protoplast Fusion) 42618.5.2.7 Molecular Gene Transfer 42718.5.3 Selection 42818.5.3.1 Positive and Negative Mass Selection 42818.5.3.2 Recurrent Selection 42918.5.3.3 Individual Selection with Progeny Testing 42918.5.3.4 Breeding in Apomicts 43018.5.3.5 Clonal Breeding 43218.6 Improvement of Selection Response by Specific Techniques 43318.6.1 Accelerating the Succession of Generations 43318.6.2 Early Selection 43718.6.3 Doubled Haploids 43818.6.4 Determination of Characteristics 43818.6.4.1 Markers 43818.6.4.2 Rapid Analytical Methods 43918.6.4.3 Spectrophotometric Color Measurement 44118.6.4.4 Breeding for Resistance to Pests and Diseases 44218.6.4.5 Bioassay 44318.7 Outlook 444

References 445

19 Camptothecin Production in Cell Cultures

of Ophiorrhiza Species 451Takashi Asano, Hiroshi Sudo, Mami Yamazaki, and Kazuki Saito

19.1 Introduction 45119.2 Tissue Cultures 45219.2.1 Establishment of Tissue Cultures 45219.2.1.1 Aseptic Plants 45319.2.1.2 Hairy Roots 45419.2.2 Camptothecin Production and Metabolite Profiles 45419.2.3 Excretion of Camptothecin into the Culture Medium 45719.2.4 Camptothecin Production in Bioreactors 45719.2.5 Regeneration of Transformed Plants from Hairy Roots 45919.3 Camptothecin Biosynthesis Studies with O. pumila Hairy Root 46019.3.1 Camptothecin Biosynthetic Genes 46019.3.1.1 Strictosidine Synthase (OpSTR) 46119.3.1.2 Tryptophan Decarboxylase (OpTDC) 46219.3.1.3 NADPHxytochrome P450 Reductase (OpCPR) 46219.3.2 In-Silico and In-Vitro Tracer Study from [l-13C]glucose 462

Contents XV

19.4 Summary and ConclusionsAcknowledgments 463References 464

462

20 Plant Biochemistry and Biotechnology of Flavor Compoundsand Essential Oils 469Dinesh A. Nagegowda and Natalia Dudareva

20.1 Introduction 46920.2 Biosynthesis of Flavors/Essential Oils 47120.2.1 Terpenoids 47120.2.1.1 Monoterpenes 47320.2.1.2 Sesquiterpenes 47420.2.2 Phenylpropanoids 47520.3 Regulation of Essential Oil Accumulation 47720.4 Practical Applications 47820.5 Metabolic Engineering 48120.6 Conclusions 483

Acknowledgments 483References 483

21 Cinkgo biloba and Production of Secondary Metabolites 493Christine Sohier and Didier Courtois

21.1 Introduction 49321.1.1 The Ginkgo biloba Tree 49421.1.2 Terpenes and Flavonoids 49421.2 Biosynthesis and Accumulation of Ginkgolides and Bilobalide 49621.2.1 Terpene Content and Localization of Biosynthesis 49621.2.2 Biosynthetic Pathway 49821.3 Biosynthesis of Flavonoids 50021.3.1 Variations in Flavonoid Content 50021.3.2 Flavonoid Metabolic Pathway 50021.4 Selection of Plants for Terpene Content and Propagation 50121.4.1 Selection 50121.4.2 Techniques of Micropropagation 50221.4.3 Somatic Embryogenesis 50220.5 Tissue cultures 50321.5.1 Terpene Biosynthesis and Accumulation 50321.5.2 Flavonoids Biosynthesis and Accumulation 50421.5.3 4'-OMethylpyridoxine (Ginkgotoxin) Biosynthesis

and Accumulation 50521.5.4 Biotransformation 50521.5.5 Cryopreservation of Plant Cell Materials 50521.6 Genetic Transformation and Organ Cultures 50621.7 Conclusions 506

References 507

XVI Contents

22

22.122.222.322.422.5

23

23.123.223.323.3.123.3.223.3.323.3.3.1

23.4

Production of Paclitaxel in Plant Cell Cultures 515Homare TabataAbstract 515Introduction 515Plant Cell Culture Technology for Paclitaxel Production 516Paclitaxel Biosynthetic Pathway and Related Genes 517Genetic Modification of Taxus Cells 522Future Technology for the Production of Useful Compounds 523Acknowledgments 524References 524

Biological Production of Artemisinin for Malaria TherapyYan Zhao and Chunzhao LiuIntroduction 529The Biosynthesis of Artemisinin 530The Biological Production of Artemisinin 531Artemisinin Production by Plants 531Artemisinin Production by Microorganisms 535Artemisinin Production in Bioreactors 536Production of Artemisinin from A. annua L. Shootsin a Nutrient Mist Bioreactor 537Outlook 539References 540

529

Subject Index 543