Advances in Genetics Volume 52

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Volume 52

Advances in Genetics

Advances in Genetics, Volume 52

Serial Editors

Jeffery C. Hall Waltham, Massachusetts

Jay C. Dunlap Hanover, New Hampshire

Theodore Friedmann La Jolla, California

Volume 52

Advances in Genetics Edited by

Jeffrey C. Hall

Jay C. Dunlap

Department of Biology Brandeis University Waltham, Massachusetts

Department of Biochemistry Darmouth Medical School Hanover, New Hampshire

Theodore Friedmann Center for Molecular Genetics University of California at San Diego School of Medicine La Jolla, California

Elsevier Academic Press 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 84 Theobald’s Road, London WC1X 8RR, UK

This book is printed on acid-free paper. Copyright ß 2004, Elsevier Inc. All Rights Reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (www.copyright.com), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-2004 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0065-2660/2004 $35.00 Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (þ44) 1865 843830, fax: (þ44) 1865 853333, E-mail: [email protected]. You may also complete your request on-line via the Elsevier homepage (http://elsevier.com), by selecting ‘‘Customer Support’’ and then ‘‘Obtaining Permissions.’’ For all information on all Academic Press publications visit our Web site at www.academicpress.com ISBN: 0-12-017652-1 PRINTED IN THE UNITED STATES OF AMERICA 04 05 06 07 08 9 8 7 6 5 4 3 2 1

Contents Contributors

ix

1 Therapeutic Applications of RNA Interference: Recent Advances in siRNA Design 1 Lisa Scherer and John J. Rossi I. II. III. IV.

Introduction 1 siRNA Design 2 Design of shRNAs Summary 18 References 18

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2 The Splicing of the IGF-I Gene to Yield Different Muscle 23 Growth Factors Geoffrey Goldspink and Shi Yu Yang I. Introduction 24 II. Different Ways in Which the IGF-I Gene Can Be Spliced 25 III. Cloning of IGF-I Splice Variants in Skeletal Muscle 27 IV. Transgenic Studies Involving IGF-I 29 V. Splicing of the IGF-I Gene in Neuronal Tissue 30 VI. Splicing of IGF-I Gene in Other Tissues 31 VII. Mechanical Signals in Splicing of the IGF-I Gene 31 VIII. Hormonal Influence on Splicing of the IGF-I Gene 32 IX. Peptide Products Resulting from Splicing 34 X. Receptor-Mediated Cellular Effects of IGF-I 36 XI. The Role of Binding Proteins in the Regulation of Systemic IGF-I 37 XII. Gene Transfer of MGF and IGF-IEa 38 XIII. Modus Operandi of the Different IGF-I Splice Variants 38

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vi

Contents

XIV. Conclusions References

42 43

3 Breeding Hevea Rubber: Formal and Molecular Genetics 51 P. M. Priyadarshan and A. Cle´ment-Demange I. II. III. IV. V. VI. VII. VIII. IX. X.

Introduction 52 Historical, Botanical, and Agricultural Aspects Breeding Objectives 57 Genetic Resources 59 Yield Variation 62 Reproductive Biology 66 Conventional Breeding Methodologies 71 Breeding Against Stresses 81 Biotechnologies 91 Conclusions 102 References 104

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4 Gene Transfer for Therapeutic Vascular Growth in Myocardial and Peripheral Ischemia 117 Tuomas T. Rissanen, Juha Rutanen, and Seppo Yla¨-Herttuala I. II. III. IV.

Introduction 118 Mechanisms of Vascular Growth 119 Vascular Growth Factors 121 Gene Transfer in Skeletal Muscle and Myocardium 132 V. Therapeutic Vascular Growth 136 VI. Safety Aspects 142 VII. Concluding Remarks 145 References 146

5 Metabolic Highways of Neurospora crassa Revisited 165 Alan Radford I. II. III. IV. V.

Introduction 166 Amino Acid Biosynthesis 168 Purine and Pyrimidine Biosynthesis 187 Vitamin and Cofactor Biosynthesis 192 Mainstream Carbon Metabolism 200

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Contents

VI. New Gene Designations VII. Conclusions 205 References 206

Index

209

205

This Page Intentionally Left Blank

Contributors Numbers in parenthesis indicate the pages on which the authors’ contributions begin.

A. Cle´ment-Demange (51), Centre de Coope´ration Internationale en Recherche´ Agronomique pour le De´veloppement (CIRAD), Tree Crop Departement, TA80/01, Avenue Agropolis, 34398 Montpellier, Cedex 5, France Geoffrey Goldspink (23), Department of Surgery, Royal Free and University College Medical School, University of London, London NW3 2PF, England P. M. Priyadarshan (51), Rubber Research Institute of India, Regional Station, Agartala - 799006, India Alan Radford (165), School of Biology, University of Leeds, Leeds LS2 9JT, England Tuomas T. Rissanen (117), Department of Biotechnology and Molecular Medicine, A. I. Virtanen Institute, Kuopio University, Kuopio, Finland John J. Rossi (1), Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010 Juha Rutanen (117), Department of Medicine, Kuopio University, Kuopio, Finland Lisa Scherer (1), Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010 Shi Yu Yang (23), Department of Surgery, Royal Free and University College Medical School, University of London, London NW3 2PF, England Seppo Yla¨-Herttuala (117), Department of Biotechnology and Molecular Medicine, A. I. Virtanen Institute, Department of Medicine, Kuopio University, Kuopio, Finland; and Gene Therapy Unit, Kuopio University Hospital, Kuopio, Finland

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Therapeutic Applications of RNA Interference: Recent Advances in siRNA Design Lisa Scherer and John J. Rossi Division of Molecular Biology Beckman Research Institute of the City of Hope Duarte, California 91010

I. Introduction II. siRNA Design A. Basic Considerations B. siRNA Modifications C. Antisense Versus siRNAi-Mediated RNAi III. Design of shRNAs A. shRNA Expression Vectors and Hybrid shRNAs B. miRNA/siRNA Hybrids IV. Summary References

I. INTRODUCTION RNA interference, or RNAi, is a gene-silencing mechanism originally described in plants (where it was known as post-transcriptional gene silencing, or PTGS), C. elegans and Drosophila (reviewed in Bernstein et al., 2001; Carmell et al., 2002). In the current model, the RNAi pathway is activated by a doublestranded RNA (dsRNA) ‘‘trigger’’ that is then processed into short, 21–23 nucleotide dsRNAs, referred to as small interfering RNAs (siRNAs) by the cellular enzyme Dicer. The siRNAs become incorporated into the RNAinduced silencing complex (RISC), where the siRNA antisense strand serves as a guide targeting the homologous mRNA for endonucleolytic cleavage within Advances in Genetics, Vol. 52 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2660/04 $35.00

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the siRNA/target duplex, approximately 10 bases from the 50 end of the siRNA guide. In mammalian cells, dsRNA longer than 30 nucleotides triggers the nonspecific interferon pathway rather than RNAi. However, Tuschl and colleagues demonstrated (Caplen et al., 2002; Elbashir et al., 2001a; Harborth et al., 2001) that shorter siRNAs exogenously introduced into mammalian cells bypass the Dicer step and directly activate homologous mRNA degradation without initiating the interferon response. Subsequently, a number of labs demonstrated the feasibility of expressing siRNAs and the related short hairpin RNAs (shRNAs) in vivo against human viral and cellular targets. Advances in RNAi are rapidly expanding, and considerable progress has already been made toward therapeutic applications (Couzin, 2003; Kitabwalla and Ruprecht, 2002; Martinez et al., 2002b; Scherr et al., 2003; Wilson et al., 2003; Zamore, 2001). This review will focus on providing information for the design of constructs that mediate effective silencing. Emphasis will be placed on recent research in mammalian systems to provide information most easily translated to the therapeutic setting. We will begin with the description of the basic siRNA expression cassette and review initial studies on the effects of chemical backbone modifications that are being tested for applications involving exogenous delivery. We then discuss the important features of shRNAs and the use of a variety of promoters, including those that express shRNAs as part of larger transcripts, which we refer to as hybrid RNAi molecules. Throughout, we consider the effects of RNAi/target mismatches, which have implications for specificity and escape of rapidly evolving targets. Our focus will be primarily on recent publications, and the reader will be directed to other publications as needed.

II. siRNA DESIGN A. Basic considerations Synthetic siRNAs are the method of choice for exogenous, short-term applications. Currently, unmodified siRNAs mediate RNAi effects that typically peak at 2–3 days post-transfection. The most common design for synthetic siRNAs mimics the endogenous siRNAs produced by Dicer cleavage of trigger dsRNA (Elbashir et al., 2001a), where the sense and antisense strands are 21–23 nucleotides long. The annealed portion of the duplex is completely complementary, except for two nucleotide overhangs at both 30 ends. For synthetic siRNAs, the 30 dinucleotide overhangs can be derived from the target sequence, as in their natural counterparts. More typically, they consist of uridines (though at least four can be tolerated (Miyagishi and Taira, 2002) or deoxythymidines, for cost savings, though other nucleotide combinations have been used succesfully. 50 phosphates are present on natural siRNAs and are required for in vivo function; however, it is not always

1. Therapeutic Applications of RNA Interference

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necessary to kinase synthetic siRNAs before transfection, as most mammalian cells have an endogenous kinase activity (Chiu and Rana, 2002 and Table 1.1). To ensure cleavage of the target, the antisense sequence should be completely complementary to the target (with the exception of the two, 30 penultimate nucleotides that form the overhang, as noted), though some mismatches are tolerated (see Table 1.2). As long as the guide strand maintains full complementarity to the target, mismatches between the sense and antisense strands of the siRNA duplex are tolerated to some extent (Chiu and Rana, 2002; Yu et al., 2002), whether they are simple mismatches creating a bubble or inserted nucleotides creating a small bulge. Mismatches between the siRNA antisense strand and the target tend to reduce activity to varying degrees, depending on their number and location (Table 1.2). While the rules governing the relationship between siRNA/target mismatch and RNAi activity have not been fully worked out, some generalizations can be made that probably apply to both/siRNAs and simple shRNAs (refer to Table 1.1). Mutations near the endonucleolytic cleavage site frequently, but not always, reduce the RNAi effect. Also, mutations in the first half of the antisense strand (50 end) are very detrimental (Amarzguioui et al., 2003; Holen et al., 2002; Randall and Rice, 2001). Since endonucleolytic cleavage is ‘‘measured’’ from the 50 end of the antisense siRNA strand, it is possible that mutations in the 50 end of the guide strand destabilize the antisense/mRNA target duplex in the activated RISC complex, inhibiting cleavage. The importance of complete complementarity in this part of the guide/target duplex may also explain an example of differential RNAi cross reactivity in a slightly different context. Recently, we designed siRNAS that target the fusion joint of the aberrant mRNA that codes for the EWS/Fli-1 (Ewing’s sarcoma) oncogenic protein (Dohjima, 2003). The two overlapping siRNAs have a total of 20 nucleotides complementarity against the fusion site. The distribution of homology 50 and 30 to the target fusion point is 14 and 6 nucleotides for the first and 6 and 14 for the second siRNA. By chance, however, the second siRNA has a total of 17 nucleotides of homology between the 50 end of the siRNA antisense strand and the endogenous Fli-1 transcript. This latter siRNA shows low, but measurable, cross reactivity to the endogenous Fli-1 transcript. Taken together, these results imply that, when designing RNAi constructs to target a specific isoform, it may be advisable to select a target site in the target isoform, such that mismatches between its corresponding siRNA and the nontargeted isoform fall in the 50 end of the duplex. If this not possible, as when the target is not accessible to cleavage (reviewed in Scherer and Rossi, 2003), it is important to test for cross reactivity. While siRNAs constructed from 21–23-mers are sufficient for most purposes, oligomers of up to 29 nucleotides can be used without initiating the interferon effects, which may be advisable in some applications, for example, to ensure target specificity. SiRNAs with as few as 19 nucleotides of complementarity

Table 1.1. Effects of siRNA Chemical Modifications on RNAi Effect on RNAid

siRNA characteristics Oligo S/ASa

Annealed structureb

21/21

Standard dTdT

21/21

Standard UU

21/21

Standard dTdT

Positionc

Inhibition level

OH

AS 50 terminus

Unaffected

ND

Methoxy 20 , 30 dideoxy 3-amino-propyl phosphoester 50 amino

AS 50 terminus AS 30 terminus AS 30 terminus

Abolished Unaffected Unaffected

ND ND ND

S 50 terminus AS 50 terminus S 50 þ AS 50 ends S 30 terminus AS 30 end SþAS 30 ends S/AS 30 ends S/AS 30 ends

Unaffected Abolished Abolished Unaffected Unaffected Unaffected Unaffected Unaffected

All nucleotides

Abolished

Modification

Puromycin

21/21 21/21

Standard UU Standard M

Biotin Photo-cleavable biotin 20 -O-methyl

Persistencee

Assay system Target

Cells

Refs.

Luciferase dual reporter Luciferase dual reporter

HeLa

Chiu and Rana, 2002

HeLa S100 extracts

Schwarz et al., 2002

ND ND ND Slight decrease Unaffected Slight decrease Unaffected ND

GFP/RFP dual reporter

HeLa cells

Chiu and Rana, 2002

NA

Luciferase dual reporter

HeLa S100 extracts D. mel embryo ext

Martinez et al., 2002a Elbashir et al., 2001b

21/21

Standard M

20 -O-methyl Phosphoro-thiorate Allyl Allyl

21/21 —/21 21/21 —/21 21/21

a

Standard M AS alone Standard M AS alone Standard M

Deoxyribose Deoxyribose FITC FITC 20 -deoxynucleotide substitution

S 1/2/18–21 þ AS 1/2/18–21 S 1/2/20/21 þ AS 1/2/20/21 S 20/21 þ AS 20/21 S 1/21 þ AS 1/21 AS 30 end AS 30 end AS 30 end AS 30 end Complete, S or AS up to four in 30 ends

Slight decrease (Toxic)

Increase NA

Unaffected Slight decrease Unaffected Abolished Unaffected Abolished Abolishes

ND ND

Tolerated

ND

ND ND ND ND ND

Human tissue factor

HaCaT cells

Amarzguioui et al., 2003; Holen et al., 2003

Luciferase dual reporter

D. mel embryo ext

Elbashir et al., 2001b; Holen et al., 2002

Number of nucleotides in sense (S) and antisense (AS) strands. Standard 2 nt overhangs both 30 ends: UU—uracil overhang: dTd—deoxythymidine overhang; M—completely homologous to target sequence; extra nucleotides for bubbles and bulges number relative to the 50 ends of their respective stands. c Slashes, multiple mutations within a single molecule; numbering from 50 end of indicated strand. d Change in RNAi effect relative to positive control due to mutations measured as down-regulation of target mRNA or protein. e NA, not applicable. ND, not determined. b

Table 1.2. Effect of siRNA/Target Mismatches on RNAi Activity in Mammalian Cells siRNA description Oligo (nt) S/AS

Annealed structurea

21/21 23/21 21/23 23/23

Standard dT/dT S, U10/G11 bulge AS-C11/A12 bulge S, U10/G11; AS-C11/A12 bubble Standard M

21/21

siRNA AS:target mismatches shRNA stem lengthb

Type

Positionc

Assay system Mismatch effect on RNAd

NA

None None NA NA

NA NA At bulge At bulge

NA

*

17, 18 Unaffected 9, 10, 13, 16, 19 15–20% down 4, 7 80% down 10/13, 9/10 30% down 7/10 70% down 4/10 Abolished

C:C or G:G

Positive control Unaffected Abolished Abolished

Target gene

Cells

Refs.

GFP/RFP dual reporter

HeLa cells

Chiu and Rana, 2002

**

HaCaT cells

Amarzguioui et al., 2003; Holen et al., 2002

Human tissue factor

21/21

Standard M

NA

21/21

Standard UU/dT/dT

NA

23/23

Standard dT/dT

21/21

Standard (shRNA) (shRNA) (shRNA)

NA NA NA 19 19 20

Bulges and 4/9/10 mismatches 9/12/18 9/12/18 G:A A:G * C:C/A:A U:C U:G Varied

3/7/17 3/7/17 10/11 11 18 15–20

Abolished

lin-41 (let-1siRNA) GL2 luciferase GL3 luciferase

HeLa extracts Hutvagner and Zamore, 2002 Abolished HeLa, Elbashir Abolished COS 7,293, et al., 2001a NIH/3T3 Abolished HCV Con1 NB5B Huh-7 Randall and Abolished HCV C/LB NS5B Rice, 2001 Abolished GFP Murine P19 Yu et al., 2002 ** Abolished CDH MCF Brummelkamp Abolished et al., 2002 Slight reduction **Fli-1 Dohjima, 2003

a Standard 2 nt overhangs both 30 ends: UU—uracil overhang: dTdT—deoxythymidine overhang; M—completely homologous to target sequence; extra nucleotides for bubbles and bulges number relative to the 50 ends of their respective strands. S—sense; AS—antisense. b All shRNA listed have completely complementary stems. c Commas—single mutations: different members of related siRNAs; slashes—multiple mutations within a single molecule; numbering from 50 end of antisense (AS) strand. d Change in RNAi effect relative to positive control due to mutations. * Created by inversions of standard base pairs in siRNA stem. ** Endogenous target; others transfected. NA—not applicable.

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to the target mediate effective mRNA destruction, although one study making direct comparisons of siRNAs against the same target showed that reducing complementarity from 21–20 or 19 nucleotides also reduces the RNAi effect (Schwarz et al., 2002), though this may be a target-dependent effect, as discussed in the section on design of si- and shRNAs.

B. siRNA modifications Experiments in Drosophila embryo lysates indicated a need for either free 50 -OH or 50 -phosphate on synthetic siRNA strands (Elbashir et al., 2001b; Nykanenri et al., 2001; Table 1.1). Similar results were observed in HeLa extracts (Schwarz et al., 2002) or intact cells (Chiu and Rana, 2002). Asymmetric 50 -amino modification of one or the other siRNA strand showed that 50 amino modification of the antisense strand abolishes RNAi while the same modification of only the sense strand does not inhibit the RNAi effect. Efficient 50 end radiolabeling of transfected siRNA was observed only after phosphatase pre-treatment (Chiu and Rana, 2002). Taken together, this suggests a strong requirement in vivo for a 50 phosphate on the antisense strand. This is consistent with the hypothesis that modifications of this nucleotide interfere with the ability of the antisense strand to serve as a guide for endonucleolytic cleavage in the activated RISC complex. On the other hand, modifications blocking the 30 end have little effect on duplex siRNA, and on either strand in most cases (Amarzguioui et al., 2003; Table 1.1). Interestingly, 30 FITC or deoxyribose ends on the antisense strand do block suppression effects when not a part of a siRNA duplex, in systems where antisense mediates RNAi (22; see later discussion). Up to six 20 -O-methyls per siRNA strand distributed between the 50 and 30 termini, or two 20 -O-allyl modifications at the 30 termini as shown (Table 1.1), do not adversely affect RNAi (Amarzguioui et al., 2003). Increasing the number of modifications beyond this point, or allyl modification of the 50 termini, reduce RNAi (Amarzguioui et al., 2003; Holen et al., 2003). The 20 -O-methlyl modifications shown in Table 1.1 significantly increased persistence of the RNAi effect relative to unmodified siRNAs, maintaining 60% target suppression at 5 days when the wild-type effect is gone; siRNAs with fewer modifications showed less persistence. More than two phosphorothiorate modifications are cytotoxic while not promoting significant increases in persistence (Amarzguioui et al., 2003).

C. Antisense versus siRNAi-mediated RNAi Several groups have reported that the antisense siRNA strand alone can also be an effective silencer in mammalian cells (Holen et al., 2003; Martinez et al., 2002a; Schwarz et al., 2002). In HeLa cell extracts, the antisense strand of


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siRNAs is incorporated into RISC and supports RNAi (Martinez et al., 2002a; Schwarz et al., 2002). Transfection of either 100 nM synthetic siRNA or 200 nM antisense RNA into HaCaT cells down regulated targeted endogenous human tissue factor (hTF) (Holen et al., 2003). However, the antisense was fiveto six-fold less potent than the corresponding duplex, as estimated by dose dependence (IC50 values of 5 and 30 nM, respectively). Antisense and siRNA forms show similar cleavage products, target position effects, and tolerance for mismatches and chemical backbone modifications. The exception is the effect of 30 -modifications, as already mentioned. One interpretation of this intriguing result is that exogenous antisense and siRNA duplexes enter the RNAi pathway at different stages. Since the antisense effect also has a swifter onset (if less amplitude), the antisense entry point may be downstream of the duplex entry point. From a practical perspective, similarities between the profiles of antisense and siRNA effects suggest that 21–29 oligomers that successfully down regulate target mRNAs will mediate even stronger RNAi effects as the corresponding duplex. Therefore, antisense RNAs have potential as part of a rapid and costeffective screening method for good RNAi target sites, a major bottleneck in designing effective siRNAs (reviewed in Scherer and Rossi, 2003).

III. DESIGN OF shRNAS The siRNA design mimics the natural Dicer product; the shRNA design was based in part on the structure of endogenous microRNA (Brummelkamp et al., 2002; Paddison et al., 2002). Briefly, microRNAs (miRNAs) are highly conserved RNA species that perform a number of regulatory functions in plants, invertebrates, and vertebrates (Brantl, 2002; Cerutti, 2003; Cottrell and Doering, 2003; Grishok and Mello, 2002; Grosshans and Slack, 2002; Hannon, 2002; McManus and Sharp, 2002; Voinnet, 2002). In the current model, miRNAs are transcribed in the nucleus as a transient primary transcript (primiRNA) that may be polycistronic; the pri-mRNAs undergo processing to a 70 nucleotide precursors (pre-miRNAs) in the nucleus. Processing to the mature 21–24 nt form is Dicer dependent and consequently is thought to occur in the cytoplasm; however, nuclear processing with rapid export cannot be ruled out. A number of miRNAs have now been identified in Drosophila, C. elegans, and mammals (Ambros et al., 2003; Cerutti, 2003; Dostie et al., 2003; Kidner and Martienssen, 2003; Lagos-Quintana et al., 2002, 2003; Lim et al., 2003; Reinhart et al., 2002). A comparison of the current understanding of the processing of miRNAs, based on the let-7 and lin-4 paradigms, and siRNAs, is shown in Table 1.3. Table 1.4 presents a summary of a number of reported shRNAs and their characteristics. Like siRNAs, shRNAs can be synthesized exogenously or

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Table 1.3. Comparison of Endogenous siRNA and miRNA Biogenesis siRNA Source processing intermediates Initial form

Exogenous (for example, viral) or endogenous cellular genes Long dsRNA, single target; processed by RISC in cytoplasm

Intermediate(s)

None known

Final active form

Perfectly homologous, doublestranded 21-mer; processed in cytoplasm None known

Nuclear processing complex components Cytoplasmic processing Dicer dependent? Target homology Effect on target RNA-dependent RNA polymerase dependent amplification? Cellular roles

Yes

microRNA Endogenous cellular genes Primary micro RNA (primiRNA); single stranded with internal imperfect hairpin; may be polycistronic; processed rapidly in nucleus Precursor micro RNA, (premiRNA); 65–80 nt imperfect hairpin; processed in nucleus Mature microRNA (miRNA): 1/2 side of hairpin; processed in cytoplasm Presumed to exist: not yet identified Yes

Perfect Cleavage Yes, in some organisms (not in mammals)

Imperfect Translation suppression No?

Regulation of development; defense against viral pathogens; chromatin maintainance

Regulation of development; cellular maintainance, for example, chromatin and telomere structure

transcribed in vivo. Hannon and coworkers (Paddison et al., 2002) were among the first to investigate shRNA-mediated silencing in both Drosophila and mammalian cell lines. Initially, they designed a group of constructs containing different structural features of the let-7 miRNA and tested them using a luciferase dual reporter system. Surpisingly, those shRNAs modeled most closely on the let-7 paradigm were the least effective inducers of silencing; simple hairpins with fully complementary stems were the best. In particular, the presence of mismatches with respect to the target mRNA virtually eliminated silencing. This result was interesting, since it is more characteristic of the siRNA pathway, where complete complementarity to the target is generally required to cleave the target, rather than the miRNA effect, where incomplete homology to the target mediates translational suppression (Table 1.3).


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Some other key structural parameters emerged from this work (Paddison et al., 2002). As for siRNAs, the length of the stem helix was limited to 29 bp to avoid nonspecific interferon effects in mammalian cells. Varying loop sizes from 4–23 nucleotides proved neutral in Drosophila cells; both 4- and 8-nt loops were effective in mammalian cell lines. The authors observed a loss of RNAi potency as the siRNA helix length was reduced from 29–22 nucleotides. However, since the stems were shortened relative only to the 30 end of the corresponding target sequence and not vice versa, the correlation may be secondary to a site effect. RNAi potency is known to be extremely sensitive to target site sequence; overlapping siRNAs shifted by only a few nucleotides can vary widely in potency (Holen et al., 2003). In any case, a number of very effective shRNAs with 21–23 bp stems have been documented. It should also be noted that silencing was measured by a decrease in reporter protein expression, not mRNA level; therefore, these experiments could not distinguish whether silencing was due to shRNA-mediated translation suppression or mRNA degradation. Subsequently, a number of studies have shown that shRNAs with complete target homology mediate target cleavage (Table 1.4) or an siRNAlike effect. Critical structural features continue to be defined. Hairpins in the form of 50 -sense target sequence/loop/antisense target sequence are more effective than the 50 antisense target sequence/loop/sense target sequence—30 (Czauderna et al., 2003), although the reason is not clearly understood. Despite an early report to the contrary (Brummelkamp et al., 2002), examination of the data in Table 1.4 indicates that loop size and sequence are not generally critical for silencing (though it may influence cellular localization, discussed later). A very systematic comparison of the relative effectiveness of various shRNA structures, monitored by real-time PCR, addressed this issue (Czauderna et al., 2003). Target mRNA depletion was observed in dose/response comparisons using relatively low amounts (0.06–5 nM range) of synthetic shRNAs, even when the nonphysiological polyethylene glycol linkage was substituted for loop nucleotides. Although some variations in RNAi suppression were observed, they are likely to be irrelevant at higher, more standard concentrations of 20–25 nM. The authors also addressed the issue of whether shRNAs exert their function when they adapt a bimolecular bubble form rather than a hairpin. A synthetic form of this molecule was constructed by annealing synthetic RNAs of the form 50 -target A sense/loop/target B antisense -30 and 50 - target B sense/loop/target A antisense -30 , both loops consisting of 12 adenines. This design forced efficient in vitro formation of the bimolecular complex, since the individual RNAs cannot form hairpins. Amazingly, this dual target ‘‘bubble’’ RNA efficiently knocked down both Akt-1 and Akt-2 target mRNAs and proteins. The effect was not due to cross reactivity, since standard hairpins based on the same sequences directed against the individual targets

Table 1.4. shRNA Design and Expression shRNA design and expression Promoter S/Ta

Stem length

Loop length

Loop sequence

Target gene(s) N/Cb

HI HI HI HI HI hU6þ1

T T T T T T

19 19 19 19 19 29

5 7 9 9 9 8

UUCGA CAGAGCU UUCAAGAGA UUCAAGAGA UUCAAGAGA GAAGCGUU

ND ND ND ND ND ND

hU6þ1 hU6þ1 hU6þ27 5S 7SL U6þ27 mU6þ1 mU6þ1 mU6þ1

S T T T T T T T T

27 19 19 19 19 19 21 21 21

8 4 4 4 4 4 6 6 6

GAAGCGUU UUCG UUCG UUCG UUCG UUCG CTCGAG CTCGAG CTCGAG

ND N (N) (C) (C) (N) ND ND ND

mU6þ1

T

19

3

ACA

ND

hU6þ1

S

19

8

UUCAGAGA

ND

Target reduction assay Ex/ End

mRNA

Protein

CDH1 CDH1 CDH1 p53 CD20 Luciferase dual reporter Murine p53 Lamin A/C Lamin A/C Lamin A/C Lamin A/C HIV-RT GFP dnmt-1 Lamin A/C, cdk-2

End End End End End Ex

ND ND ND Yes ND ND

Poor medium Yes Yes Yes Yes

End End End End End End Ex End End

ND ND ND ND ND ND ND ND ND

Yes Yes Yes No No Yes Yes Yes Yes

Nueronal B-tubulin CCR5

End

ND

End

IA*

ID

Cell type(s) MCF

Refs. Brummelkamp et al., 2002

HEK 293T, HeLa, Paddison COS-1, NIH 3T3 et al., 2002 MEF cells HeLa Paul et al., 2002, 2003

Sui et al., 2002

Yes

HeLa HeLa HeLa, U-2 OS, H1299, C-33A Murine P19 cells

Yes

Human PBLs

Scherer and Rossi, 2003

Yu et al., 2002

htRNAVal mU6þ1 hU6þ1 mU6þ1

T T T T

29 29 29 29

5 5 5 10

hU6þ1

T

29

10

(syn) (syn) (syn) (syn) (syn) (syn) (syn) (syn) (syn) (syn) U6þ27 U6þ2

T T T T T T T T T T T T

20–21 20–21 20–21 20–21 20–21 20–21 20–21 20–21 Bubble** 20–21 20–21 20–21

12 12 4 12 4 — 12 12 12 12 12

a

GAAAA GAAAA GAAAA CUUCCUGUCA (mir-23) CUUCCUGUCA (mir-23) A12 UAAAGCUUGCC A12 A4 A12 GAGA PEG A12 A12 A12 N12 N12

C N N C

k-ras; mutant k-ras End

C ND ND ND ND ND ND ND ND ND ND C C

p100 p100 p100 Akt-1 Akt-1 Akt-1 Akt-1 Akt-2 Akt-1 and Akt-2 PTEN p100 p100

End End End End End End End End End End End End

Yes ND ND ND

Yes No No Yes

ND

Yes

ND Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

Yes IA Yes Yes Yes Yes Yes Yes Yes

SW480 human colon cells

Kawasaki and Taisa, 2003

HeLa cells

Czauderna et al., 2003

IA IA

T, transient transfection or S, stable clone expression. N, nuclear or C, cytoplasmic localization determined; () indicate expected localization based on cassette type (syn), synthetic shRNA. ND—not determined. IA—indirect assay. * inhibition of HIV replication. ** See text. p100 and p100 ¼ PI 3 kinase subunits. b

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showed appropriate specificity. This indicates that bimolecular bubbles formed from identical components could support RNAi in vivo; however, given the efficiency of hairpin formation, it is questionable whether they would form to any great extent. From a practical point of view, dual target siRNA bubbles could be used to knockdown two unrelated messages simultaneously using a single duplex molecule, possibly avoiding potential complications arising from two individual hairpins competing for the RNAi processing apparatus.

A. shRNA expression vectors and hybrid shRNAs Synthetic si- and shRNAs can only mediate short-term RNA interference, as they are gradually degraded over time or diluted by cellular division after transfection. Stable gene silencing requires endogenous expression from a promoter. Early experiments (Brummelkamp et al., 2002; Czauderna et al., 2003; Lee et al., 2002; McManus et al., 2002; Miyagishi and Taira, 2002; Paddison et al., 2002; Paul et al., 2002; Sui et al., 2002; Zamore, 2001), utilized U6 and H1 pol3 promoters. These polymerase III promoters have a number of advantages: They are relatively compact, support high levels of transcription, initiate transcription at a defined start point from external (upstream) promoter elements, and terminate transcription at runs of four or more thymidines in the DNA template, leaving 1–4 uridines at the 30 terminus of the transcribed RNA. (As an aside, it should be noted that 5– 7 thymidines may be required in some constructs for efficient termination (Paul et al., 2003). The U6 promoter comes in several versions. The complete U6 promoter is often referred to as U6 þ 27, since the first 27 nucleotides of any transcript are derived from the 50 end of the U6 snRNA. The U6 þ 1 transcripts contain only the initial þ1 G. A recently described U6 þ 2 version includes the first two naturally transcribed U6 nucleotides (Czauderna et al., 2003). The U6 þ 27 leader directs nuclear localization of the transcript; U6 þ 1 transcripts are primarily nuclear but with substantial ‘‘leakage’’ into the cytoplasm. The HI promoter is similar to U6 þ 1, though it is smaller and can initiate transcription at any nucleotide, while U6 can only initiate at a G. The U6 and H1 promoters continue to be used extensively for endogenous siRNA (requiring dual promoter constructs (Lee et al., 2002) and shRNA stable expression (Hannon, 2002; McManus and Sharp, 2002; Qin et al., 2003; Tuschl, 2002), since they add little extra sequence to the transcripts. For that reason, the U6 þ 1 and H1 promoters have also been used for expressing the majority of shRNAs, especially in the light of reports that extraneous sequences inhibit shRNA function; for example, a CMV-driven hairpin with extra appended 50 vector and 30 polyA sequences is inactive (Paddison et al., 2002). Somewhat surprisingly, the U6 þ 27 promoter has been used for expression of effective shRNAs (Table 1.4). U6 þ 27 typically results in nuclear localization of ribozyme and aptamer transcripts (Paul et al., 2002,


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2003); the same is true for primary transcripts of U6 þ 27 lamin A/C shRNA (Paul et al., 2003 and Table 1.4). For reasons that are not understood, this construct reduces levels of the target RNA significantly despite the localization of both the target mRNA and RISC complexes in the cytoplasm. In contrast, another recent paper reports a U6 þ 27-Pl 3 kinase p110 shRNA transcript that is localized in the cytoplasm and supports silencing, indicating that it is possible to override the nuclear retention signal in U6 þ 27 transcripts (Table 1.4 and Czauderna et al., 2003). The U6 þ 2 Pl 3 kinase p110 shRNA was more potent, however. These results may relate to different efficiencies of processing to the final 21–24-mer effector molecules, which were not assayed in these studies. The other subgroup of pol III transcripts utilize internal promoters and start transcription upstream of conserved promoter elements; these include the 5S ribosomal, the signal recognition particle 7SL RNA, adenoviral VA, and all the tRNAs. All are partially or completely localized to the cytoplasm, which has advantages in other contexts, such as expressing ribozymes or protein-binding aptamers. Cloning shRNAs into these vectors results in hybrid transcripts containing both the entire structural/promoter RNA and shRNAs. In the case of tRNAs, the shRNA is transcribed downstream of the entire tRNA sequence and is usually engineered to terminate immediately after the 30 end of the hairpin base. Cloning sites in the 7SL, VA, and 5S are imbedded in the middle of the larger transcripts in regions where an additional sequence will not disrupt the promoter functional elements; therefore, the shRNA is transcribed with a considerable amount of both 50 and 30 vector sequence. Nonetheless, a tRNAVal-shRNA transcript is processed to a mature siRNA size despite the fact that the entire tRNA is transcribed as part of the initial transcript; this construct also reduces endogenous target k-ras mRNA and protein levels in SW480 cells (Kawasaki and Taira, 2003 and Table 1.4). Likewise, we have observed that a tRNALys3-shRNA directed against a site in the HIV tat/rev transcript can inhibit HIV replication, although not as effectively as a U6 þ 1 driven counterpart (Scherer, L. personal communication). A direct comparison of other pol3 shRNAs showed that 7SL- and 5S-lamin A/C shRNA derivatives do not mediate RNAi, unlike the U6 þ 1 and U6 þ 27 versions (Paul et al., 2002, 2003 and Table 1.4. The reason for these differences is unclear, though the shRNAs may not be correctly processed to mature form (Engelke, D. personal communication). One interpretation is that addition of 50 sequences upstream of an shRNA (as with tRNAs and U6 þ 27) may not be as detrimental as surrounding it with foreign, potentially highly structured sequence (5S and 7SL), which could prevent processing of the active siRNA form. The problem could lie specifically with additional 30 sequences, as the additions to the 50 side of the shRNA, as in the tRNA-expression systems, are less problematic. We have some data that long

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30 tails inhibit shRNAS (Castonotto, D., personal communication). However, we have found that anti-HIV shRNAs embedded in VA promoters can be processed and inhibit HIV replication (Lee, N., personal communication), so that the mere presence of extraneous sequences does not automatically interfere with shRNA function and suggests that some specific contexts are more permissive than others. It is also interesting to note that the localization of a hybrid transcript to the nuclear compartment does not automatically prevent shRNA inhibition, as demonstrated with U6 þ 27 lamin A/C shRNA (Paul et al., 2002). One caveat is that localization may not be as strict in transient transfections, where RNAs are relatively highly expressed compared with stable cell lines, and expressed RNAs may ‘‘leak’’ in to other cellular compartments. A localization requirement may be more stringent under conditions of long-term stable expression. The resolution of these issues will require further experimentation.

B. miRNA/siRNA hybrids There are several recent reports on the use of miRNA/siRNA hybrid constructs (Zeng and Cullen, 2003; Zeng et al., 2002). The miR-30 71-nucleotide precursor (pre-miR30) expressed from the CMV-IE promoter alone, or as part of an intron or 30 UTR in an irrelevant mRNA, can be processed to the mature miR-30 form. In addition, it can suppress translation of a reporter construct containing 4 tandem miR-30 target sites in the 30 UTR, where the target sites are based on the let-7 paradigm and contain a central mismatch to the miR-30 effector strand. An analogous construct with mature miR-30 is unable to suppress the target. This indicates that the entire pre-miR-30 is required for processing miR30 to its final active form. This may explain why the CMV promoter works in this context, but not with a simple shRNA, as mentioned earlier (Paddison et al., 2002). These authors also substituted unrelated base-paired sequences in the pre-miR-30 that remain in mature miR-30; the substituted sequences maintained the original miR-30 precursor structure but were fully complementary to a heterologous target in a reporter construct. The resulting hybrid miRNA was correctly processed and directed cleavage of the target, or an siRNA effect, rather than translation suppression, the standard miRNA effect. The pre-miR30 sequence and general structure, therefore, contains the necessary information for effective processing and can direct either cleavage or translation suppression depending on the degree of complementarity with the target (Zeng et al., 2002). This is consistent with the observation that while the human endogenous let-7 miRNA has no perfectly complementary endogenous target, it will cleave one supplied ex vivo (Hutvagner and Zamore, 2002). Further investigation of structural features involved in miR-30 processing revealed that shortening the terminal loop from 15–4 nucleotides, creating a large bulge in the middle of the stem, or disrupting the base pairing at the base of the


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Figure 1.1. Effect of guide siRNA/target structure on RNAi.

predicted precursor stem, all adversely effected processing of pre-miR-30 (Zeng and Cullen, 2003). Not all miRNAs may tolerate stem substitution for target redirection, since, in some cases, forming the structure that directs correct processing may depend more on flanking sequences in the endogenous miRNA precursor. This could explain why shRNAs designed to mimic the let-7 miRNA structural features most closely were consistently less effective than simple hairpins in both Drosophila S2 and mammalian cells (Paddison et al., 2002). One question raised by these results is if a miRNA can be engineered to mediate an siRNA-like effect, is the converse also possible—can an siRNA be engineered to mediate an miRNA-like effect, translational repression? A recent report shows that an altered siRNA can function as an miRNA (Doench et al., 2003). In this case, a synthetic CXCR4 siRNA directed cleavage of a Renilla luciferase reporter mRNA containing a single, perfectly matched target CXCR4 target sequence in the 30 UTR. An otherwise identical reporter containing four tandem repeats of a mismatched target in the 30 UTR was translationally repressed, not cleaved. These results demonstrate that the mechanism is dictated by the degree of siRNA/target pairing—complete versus mismatched—as has been suggested previously. However, other parameters may also be relevant. An important issue is the relative position of the target sequence within the mRNA. SiRNAs with similar mismatches in the coding region may not mediate translation repression or any RNAi effect. A second consideration is the number of targets: RNAi-mediated translational suppression is not as robust as RNAimediated cleavage (Doench et al., 2003), and translational repression in the paradigm systems involves multiple 30 UTR targets. A third parameter is

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the exact sequence of the mismatch, though initial studies indicated this may not be particularly important (Doench et al., 2003). Finally, the structure of the mismatched region may also be a factor: A simple bulge caused by insertion in the siRNA relative to the target may not be recognized by RISC, unlike a bulge or bubble (Fig. 1.1).

IV. SUMMARY There remain a number of questions to be answered, which have implications not only for RNA applications, but also for the pathways involved in RNAi biogenesis. How are hybrid RNAi molecules processed and localized? Does delivery route make a difference, particularly with those constructs such as the short hairpin RNAs that share features with miRNAs, which undergo nuclear processing steps? Are processing and export coupled? For instance, in one case, a U6 þ 27-shRNA is retained in the nucleus (Paul et al., 2002, 2003) and in another it is exported to the cytoplasm (Czauderna et al., 2003). One obvious difference is that the nuclear version has a 4-nt loop, the cytoplasmic a 12-nt (Table 1.4). While loop size and sequence does not seem to have an impact on the RNAi effect, there may be an effect on localization, which may be related to the observation that versions of miR-30 with smaller loops are processed less efficiently. Does this suggest that these molecules use part of the miRNA pathway? Is this a reflection of a separation of function among different structural features of pre-miRNAs? On the other hand, tRNA-shRNAs with small loops are efficiently exported to the cytoplasm; are they converted to active form by the same pathway involved in processing pre-miRNAs to a mature form? Understanding the basis for processing efficiency could be very important in a therapeutic setting where stable expression levels are typically much lower than in the transient transfections used for screening. Understanding the nature of the variables governing when siRNA/target mismatches lead to loss of RNAi or translational suppression has implications for cross reactivity and the evolution of escape mutants. Further research will require taking these issues into account in siRNA design and applications.

References Amarzguioui, M., Holen, T., Babaie, E., and Prydz, H. (2003). Tolerance for mutations and chemical modifications in a siRNA. Nucleic Acids Res. 31, 589–595. Ambros, V., Bartel, B., Bartel, D. P., Burge, C. B., Carrington, J. C., Chen, X., Dreyfuss, G., Eddy, S. R., Griffith-Jones, S., Marshall, M., Matzke, M., Ruvkun, G., and Tuschi, T. (2003). A uniform system for microRNA annotation. Rna 9, 277–279. Bernstein, E., Denli, A. M., and Hannon, G. J. (2001). The rest is silence. Rna 7, 1509–1521. Brantl, S. (2002). Antisense-RNA regulation and RNA interference. Biochim. Biophys. Acta 1575, 15–25.


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Brummelkamp, T. R., Bernards, R., and Agami, R. (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550–553. Caplen, N. J., Taylor, J. P., Statham, V. S., Tanaka, F., Fire, A., and Morgan, R. A. (2002). Rescue of polyglutamine-mediated cytotoxicity by double-stranded RNA-mediated RNA interference. Hum. Mol. Genet. 11, 175–184. Carmell, M. A., Xuan, Z., Zhang, M. Q., and Hannon, G. J. (2002). The Argonaute family: Tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis. Genes Dev. 16, 2733–2742. Cerutti, H. (2003). RNA interference: Traveling in the cell and gaining functions?. Trends Genet. 19, 39–46. Chiu, Y. L., and Rana, T. M. (2002). RNAi in human cells: Basic structural and functional features of small interfering RNA. Mol. Cell 10, 549–561. Cottrell, T. R., and Doering, T. L. (2003). Silence of the strands: RNA interference in eukaryotic pathogens. Trends Microbiol. 11, 37–43. Couzin, J. (2003). RNA interference. Mini RNA molecules shield mouse liver from hepatitis. Science 299, 995. Czauderna, F., Fechtner, M., Aygun, H., Arnold, W., Klippel, A., Giese, K., and Kaufmann, J. (2003). Functional studies of the Pl(3)-kinase signalling pathway employing synthetic and expressed siRNA. Nucleic Acids Res. 31, 670–682. Doench, J. G., Petersen, C. P., and Sharp, P. A. (2003). siRNAs can function as miRNAs. Genes Dev. 17, 438–442. Dohjima, T., Lee, N. S., Li, H., Ohno, T., and Rossi, J. J. (2003). Small interfering RNAs expressed from a Pol III promoter suppress the EWS/Fli-1 transcript in an Ewing sarcoma cell line. Molecular Therapy 7, 811–816. Dostie, J., Mourelatos, Z., Yang, M., Sharma, A., and Dreyfuss, G. (2003). Numerous microRNPs in neuronal cells containing novel microRNAs. Rna 9, 180–186. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001a). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498. Elbashir, S. M., Martinez, J., Patkaniowska, A., Lendeckel, W., and Tuschl, T. (2001b). Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20, 6877–6888. Grishok, A., and Mello, C. C. (2002). RNAi (nematodes: Caenorhabditis elegans). Adv. Genet. 46, 339–360. Grosshans, H., and Slack, F. J. (2002). Micro-RNAs: small is plentiful. J. Cell Biol. 156, 17–21. Hannon, G. J. (2002). RNA interference. Nature 418, 244–261. Harborth, J., Elbashir, S. M., Bechert, K., Tuschl, T., and Weber, K. (2001). Identification of essential genes in cultured mammalian cells using small interfering RNAs. J. Cell Sci. 114, 4557–4565. Holen, T., Amarzguioui, M., Babaie, E., and Prydz, H. (2003). Similar behaviour of single-strand and double-strand siRNAs suggests they act through a common RNAi pathway. Nucleic Acids Res. 31, 2401–2407. Holen, T., Amarzguioui, M., Wiiger, M. T., Babaie, E., and Prydz, H. (2002). Positional effects of short interfering RNAs targeting the human coagulation trigger tissue factor. Nucleic Acids Res. 30, 1757–1766. Hutvagner, G., and Zamore, P. D. (2002). A microRNA in a multiple-turnover RNAi enzyme complex. Science 297, 2056–2060. Kawasaki, H., and Taira, K. (2003). Short hairpin type of dsRNAs that are controlled by tRNA(Val) promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells. Nucleic Acids Res. 31, 700–707.

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Kidner, C. A., and Martienssen, R. A. (2003). Macro effects of microRNAs in plants. Trends Genet. 19, 13–16. Kitabwalla, M., and Ruprecht, R. M. (2002). RNA interference—A new weapon against HIV and beyond. N. Engl. J. Med. 347, 1364–1367. Lagos-Quintana, M., Rauhut, R., Meyer, J., Borkhardt, A., and Tuschl, T. (2003). New microRNAs from mouse and human. Rna 9, 175–179. Lagos-Quintana, M., Rauhut, R., Yalcin, A., Meyer, J., Lendeckel, W., and Tuschl, T. (2002). Identification of tissue-specific microRNAs from mouse. Curr. Biol. 12, 735–739. Lee, N. S., Dohjima, T., Bauer, G., Li, H., Li, M. J. et al. (2002). Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat. Biotechnol. 20, 500–505. Lim, L. P., Glasner, M. E., Yekta, S., Burge, C. B., and Bartel, D. P. (2003). Vertebrate microRNA genes. Science 299, 1540. Martinez, J., Patkaniowska, A., Urlaub, H., Luhrmann, R., and Tuschl, T. (2002). Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110, 563–574. Martinez, M. A., Clotet, B., and Este, J. A. (2002). RNA interference of HIV replication. Trends Immunol. 23, 559–561. McManus, M. T., Haines, B. B., Dillon, C. P., Whitehurst, C. E., van Parijs, L., Chen, J., and Sharp, P. A. (2002). Small interfering RNA-mediated gene silencing in T. lymphocytes. J. Immunol. 169, 5754–5760. McManus, M. T., and Sharp, P. A. (2002). Gene silencing in mammals by small interfering RNAs. Nat. Rev. Genet. 3, 737–747. Miyagishi, M., and Taira, K. (2002). U6 promoter-driven siRNAs with four uridine 30 overhangs efficiently suppress targeted gene expression in mammalian cells. Nat. Biotechnol. 20, 497–500. Nykaneri, A., Haley, B., and Zamore, P. D. (2001). ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309–321. Paddison, P. J., Caudy, A. A., Bernstein, E., Hannon, G. J., and Conklin, D. S. (2002). Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16, 948–958. Paul, C. P., Good, P. D., Li, S. X., Kleihauer, A., Rossi, J. J., and Engelke, D. R. (2003). Localized expression of small RNA inhibitors in human cells. Mol. Ther. 7, 237–247. Paul, C. P., Good, P. D., Winer, I., and Engelke, D. R. (2002). Effective expression of small interfering RNA in human cells. Nat. Biotechnol. 20, 505–508. Qin, X. F., An, D. S., Chen, I. S., and Baltimore, D. (2003). Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc. Natl. Acad. Sci. USA 100, 183–188. Randall, G., and Rice, C. M. (2001). Hepatitis C virus cell culture replication systems: Their potential use for the development of antiviral therapies. Curr. Opin. Infect. Dis. 14, 743–747. Reinhart, B. J., Weinstein, E. G., Rhoades, M. W., Bartel, B., and Bartel, D. P. (2002). MicroRNAs in plants. Genes Dev. 16, 1616–1626. Scherer, L. J., and Rossi, J. J. (2003). Recent applications of RNAi in mammalian systems. In ‘‘Peptide nucleic acids, morpholinos, and related antisense biolmolecules’’ (C. Jensen and M. During, eds.), Landes Bioscience (press). Scherr, M., Morgan, M. A., and Eder, M. (2003). Gene silencing mediated by small interfering RNAs in mammalian cells. Curr. Med. Chem. 10, 245–256. Schwarz, D. S., Hutvagner, G., Haley, B., and Zamore, P. D. (2002). Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways. Mol. Cell 10, 537–548. Sui, G., Soohoo, C., Affar el, B., Gay, F., Shi, Y., and Forrester, W. C. (2002). A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 5515–5520. Tuschl, T. (2002). Expanding small RNA interference. Nat. Biotechnol. 20, 446–448.


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Voinnet, O. (2002). RNA silencing: Small RNAs as ubiquitous regulators of gene expression. Curr. Opin. Plant Biol. 5, 444–451. Wilson, J. A., Jayasena, S., Khvorova, A., Sabatinos, S., Rodrigue-Gervais, I. G. et al. (2003). RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. Proc. Natl. Acad. Sci. USA 100, 2783–2788. Yu, J. Y., DeRuiter, S. L., and Turner, D. L. (2002). RNA interference by expression of shortinterfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 6047–6062. Zamore, P. D. (2001). RNA interference: Listening to the sound of silence. Nat. Struct. Biol. 8, 746–760. Zeng, Y., and Cullen, B. R. (2003). Sequence requirements for micro RNA processing and function in human cells. Rna 9, 112–123. Zeng, Y., Wagner, E. J., and Cullen, B. R. (2002). Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol. Cell 9, 1327–1333.

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The Splicing of the IGF-I Gene to Yield Different Muscle Growth Factors Geoffrey Goldspink and Shi Yu Yang Department of Surgery Royal Free and University College Medical School University of London London NW3 2PF, England

I. Introduction II. Different Ways in Which the IGF-I Gene Can Be Spliced III. Cloning of IGF-I Splice Variants in Skeletal Muscle A. Mechano Growth Factor B. Systemic IGF-Is Are Also Produced by Active Muscle IV. Transgenic Studies Involving IGF-I V. Splicing of the IGF-I Gene in Neuronal Tissue VI. Splicing of IGF-I Gene in Other Tissues VII. Mechanical Signals in Splicing of the IGF-I Gene VIII. Hormonal Influence on Splicing of the IGF-I Gene IX. Peptide Products Resulting From Splicing X. Receptor-Mediated Cellular Effects of IGF-I XI. The Role of Binding Proteins in the Regulation of Systemic IGF-I XII. Gene Transfer of MGF and IGF-IEa XIII. Modus Operandi of the Different IGF-I Splice Variants A. Muscle Mass Regulation by IGF-I B. Satellite Cells, Muscle Hypertrophy, and Repair C. Other Actions of IGF-I Splice Variants XIV. Conclusions Acknowledgments References Advances in Genetics, Vol. 52 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2660/04 $35.00

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I. INTRODUCTION The human genome has now been sequenced, and somewhat to our surprise, there are only about 40,000 individual genes, although we know there are about 200,000 different proteins. However, in the vertebrates, splicing of a ‘‘gene,’’ or transcriptional unit, in different ways has been one way of increasing the possibilities for changing the phenotype. Another way has been via gene duplication, followed by further specialization of each individual gene (allele) during evolution. The latter takes up more space and increases the number of genes that have to be replicated each time a cell divides. Therefore, gene splicing provides an economic versatile way of increasing the quantitative as well as qualitative way of producing different, albeit related, gene products. The insulin-like growth factor (IGF) genes appear to have evolved from a single insulin-like gene that is expressed in the invertebrates. An insulin-like receptor gene is present in the C. elegans (Kenyon et al., 1993), and this has attracted considerable interest, as it determines the life span of the worm by suppressing apoptosis. This involves a system in which a transcription factor, named forkhead, is retained in the cytoplasm when the receptor is occupied by IGF. When IGF is not present, forkhead enters the nucleus and induces apoptosis (Ghaffari et al., 2003). Further studies have elucidated the signaling pathway, including age-1 and daf-1 members of the forkhead family, apparently doubling the life span of the worm by preventing sexual maturation (Dorman et al., 1995; Lin et al., 1997). The IGF-I gene and its receptor gene have proven to be very interesting, as experiments have shown that this system is involved in maintaining terminally differentiated cells and hence determining its life span in the nematode (Murphy et al., 2003). A similar signaling system seemingly exists in yeast, and therefore it is present even in a single-cell organism (Gems, 2001). Therefore, it appears to be a very ancient system that controls cellular growth (Puig et al., 2003) and tissue maintenance (Kops et al., 2002), and hence longevity of the organism (Tissenbaum and Guarente, 2003). Extrapolation to mammals has shown that IGF-I also extends the life span of mice and this appears to be associated with resistance to oxidative stress (Holzenberger et al., 2003). Therefore, it is interesting to see how this system may have evolved. In the chordate amphioxus, which is closely related to the common ancestor of the vertebrates, there is just a single insulin-like gene (Chan et al., 1990; Chu et al., 1994). During the evolution of the vertebrates, the gene has apparently been duplicated to give rise to the insulin gene, IGF-I gene, and IGF-II gene. The system is even more versatile because the insulin-like growth genes can be spliced to produce different RNA transcripts and different polypeptides with different biological activity. IGF-I, which was originally called somatomedin, was regarded as a general growth factor that is produced by the liver under the influence of growth hormone (GH). It is now realized that IGF-I is expressed by most tissues but

2. The Splicing of the IGF-I Gene to Yield Different Muscle Growth Factors

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exists as different forms of splice variants, each of which has a somewhat different action. IGF-I is known to be up regulated by skeletal muscle when subjected to exercise. Increase in the expression of the mRNA of IGF-I as a result of exercise has been known for sometime (Czerwinski et al., 1994; DeVol et al., 1990). Exercise was also shown to increase the circulating levels of IGF-I (Brahm et al., 1997), which is interesting, as it suggests that the circulating levels are not solely the result of the effect of growth hormone on the liver. As well as skeletal muscle, IGF-I is expressed by smooth and cardiac muscle. Its role in smooth muscle has not been studied to any extent, and in the heart, the experiments have usually involved administration of growth hormone. Significant improvements in cardiac function have been reported following growth hormone administration in animal models (Buerke et al., 1995) and in clinical trials involving end-stage heart failure (Fazio et al., 1996). Growth hormone induces the expression of IGF-I, which is known to be antiapoptotic (Haunstetter and Izumo, 1998), and it is assumed that this explains the beneficial response to this treatment. Using a general IGF-I antibody (Matthews et al., 1999) showed that following ischemia in the ovine heart there was a marked expression of IGF-I in the region near to the infarct. However, this study did not differentiate between the different splice variants of IGF-I. Likewise, the suggested treatment of cardiac cachexia (Rosenthal and Musaro, 2002) does not differentiate between liver systems IGF-IEa and MGF (mechano growth factor), which we have shown to be the type of IGF-I that is produced immediately after an infarct. Together with the University of Illinois, Division of Cardiology, we have studied the expression of IGF-IEa and MGF in the heart following ischemia and pressure overload and found that it is the MGF splice variant that is markedly up regulated following myocardial damage. Although this review is concerned mainly with skeletal muscle, it appears that the MGF splice variant, which was cloned from mechanically challenged skeletal muscle, may be regarded as a local tissue repair factor as well as a growth factor. Therefore, the expression and role of the different IGF-I splice variants in other tissues will be discussed. Muscle is of particular interest, as the splicing is initiated by mechanical as well as hormonal signals, producing gene products with different actions.

II. DIFFERENT WAYS IN WHICH THE IGF-I GENE CAN BE SPLICED The human gene for IGF-I resides on the long arm of chromosome 12 (Brissenden et al., 1984; Hoppener et al., 1985; Tricoli et al., 1984). It consists of at least six exons spanning a region of more than 95 KB of chromosome DNA (De PagterHolthuizen et al., 1986; Gilmour, 1994; Rotwein et al., 1986). The structure of the human IGF-I gene is show in Fig. 2.1. Exons 1 and 2 are alternative leader exons

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Figure 2.1. Structure of the mammalian IGF-gene. The larger boxes represent the deduced exons (1–6) of the IGF-I gene (not drawn to scale). Two small boxes marked by P1 and P2 are two promoters (regulatory sequences), following which are exon 1 and exon 2, respectively. Exons 1 and 2 are alternative leader exons, which are differently spliced to the common exon 3. Exons 3 and 4 code for the mature IGF-I (B, C, A, and D domains), as well as for the first 16 amino acids of the E domain. Exons 5 and 6 each contain the sequences encoding the alternative parts of the E domain together with the 30 untranslated regions, which is followed by the polyadenylation signals.

(Gilmour, 1994; Tobin et al., 1990) with distinct transcription start sites, which are differentially spliced to the common exon 3 and produce class 1 and 2 IGF-I mRNA transcripts, respectively (Welle et al., 1993). Exons 3 and 4 code for the mature IGF-I (B, C, A, and D domains) as well as the first 16 amino acids of the E domain. Exons 5 and 6 each contain the sequences encoding the alternative parts of the E domain together with the 30 untranslated regions, which is followed by alternative polyadenylation signals. Alternative splicing or inclusion of exons leads to a family of IGF-I mRNAs that all encode the same mature IGF-I but differ in sequences 50 and 30 to the IGF-I coding sequence (Daughaday and Rotwein, 1989; Hepler and Lund, 1990). Exons 1 and 2 are each termed 50 leader exons and result in two different sets of transcripts. The 30 alternative splicing of the primary IGF-I transcript may result in as many as six different IGF-I mRNAs, that is, class 1 IGF-IEa (exons 1, 3, 4, and 6) (Gilmour, 1994; Sussenbach et al., 1992), class 1 IGF-IEb (exons 1, 3, 4, and 5) (Gilmour, 1994; Rotwein et al., 1986) class 1 Ec (exons 1, 3, 4, and part of 5 and 6) (Chew et al., 1995), class 2 IGF-IEa, class 2 IGF-IEb, and class 2 IGF-IEc mRNAs. The 30 alternative splicing of the IGF-I gene generates three different proIGF-I proteins, that is, IGF-IEa, IGF-IEb and IGF-IEc. Of these proIGF-I proteins, IGF-IEb is the longest and has 147 amino acid residues. IGF-IEa is the shortest and has 105 amino acid residues; IGF-Ec has 110 amino residues. Most of the isoforms that arise from the IGF-I gene are glycosylated, apparently with the exception of the rat IGF-IEb and the human IGF-IEc. Therefore, the number of amino acids is not necessarily a good indicator of molecular size. All three proteins contain the mature IGF-I peptide and share a common 16 amino acid sequence at the N-terminal portion of the E domain. The E domain of IGF-IEb shares homology with part of the E domain of IGF-IEc. Both of these contain a potential heparin-binding domain and a consensus nuclear localization signal.


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III. CLONING OF IGF-I SPLICE VARIANTS IN SKELETAL MUSCLE It was apparent that there must be local as well as systemic regulation of muscle growth, as when a muscle is exercised it is that muscle that undergoes an increase in mass, not all the muscles of the body. Our group used differential display to attempt to clone the growth factor or growth factors involved. For this purpose, we needed an animal model in which we could make muscle grow rapidly. Previous work had shown that if the tibialis anterior in the mature rabbit was electrically stimulated while held in the stretched position by plaster cast, immobilization increased in mass by 35% within 7 days (Goldspink et al., 1992). It has previously been shown that muscles adapt to a new functional length by adding sarcomeres in series at the ends of the existing myofibrils. However, if the muscle is also subjected to electrical stimulation, it increases in girth as well as length. It was found that the RNA in the stretch/stimulated muscles increased considerably, but most of this is ribosomal RNA. Using differential display with a variety of oligonucleotide primers and RT-PCR, it was possible to detect a RNA transcript that was only expressed in the stretch/ stimulated muscle and not in the resting control muscle. This was cloned and sequenced, and it became evident that it was derived from the IGF-I gene by alternative splicing (Yang et al., 1996). Another splice variant was also expressed in muscle including human muscles, even at rest, and this was similar to the liver or systemic type of IGF-I called IGF-IEa (see Fig. 2.2). As will be seen, the terminology of the IGF-Is is a problem when attempting to apply it to nonhepatic tissues. Therefore, we named this newly discovered splice-variant mechano growth factor (MGF), as it is expressed in response to mechanical stimulation. Also, it has a different E-domain and carboxy peptide sequence to the liver type of IGF-I, and thus distinguishing it from the systemic type of IGF-I (IGF-IEa). There is a further problem with the hepatic IGF-I terminology, as MGF would be classified as IGF-IEb in the rat but IGF-IEc in the human. Also, in the human muscle, we cloned the equivalent of IGF-IEb, which is not the same as the rat IGF-IEb. Therefore, it is apparent that the muscle IGF-I isoforms, although they are related to the liver isoforms, have to be characterized separately (Hameed et al., 2003b,c).

A. Mechano growth factor Besides MGF being expressed in response to mechanical activity, it was noted that its exon 5, which encodes the E domain, has an insert that introduces a reading frame shift (see Fig. 2.2). In the rat, this is a 52-base insert, and in the human the insert is 49-base insert, neither of which are multiples of 3. As a consequence, the 30 RNA sequence codes for a different carboxy peptide sequence than that of the liver type of IGF-I. This has functional importance,

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Figure 2.2. This shows the way in which the IGF gene is spliced in muscle as a result of exercise and/or muscle damage (Regulatory Sequence 1) and hormones (Regulatory Sequence 2). In human muscle, a 49 base insert changes the reading frame in MGF, resulting in a different carboxy peptide. Muscle also expresses an IGF-IEb form, but its function is not known. The IGF-receptor domain is included in all these splice variants and is encoded by exons 3 and 4, and all presumably have an anabolic effect, but there is evidence that the MGF carboxy peptide has a specific function of activating the muscle satellite cells.

as the carboxy peptide is involved in the recognition of the binding proteins that stabilize these growth factors (see Section IX). Also in the case of MGF, the carboxy peptide (encoded in exons 5 and 6) acts as a separate and different growth factor to the mature IGF-I (IGF-I receptor binding domain). The Edomain peptide alone has been shown to induce the divisions of mononucleated myoblasts and thus activate the muscle satellite (stem) cells that are required for muscle hypertrophy and repair (Yang and Goldspink, 2002). As MGF is apparently not glycosylated and there is evidence that it has a short half life unless it is bound to its specific binding protein(s), which may be intracellular. MGF has the IGF-I receptor-binding domain encoded to exons 3 and 4, as have the other splice variants. Therefore, it presumably has the same anabolic activity as the other IGF-Is. However, as the E domain has a different action that is not blocked by the antibody to the IGF-I receptor (Yang and Goldspink, 2002), it is concluded that there is a second receptor that is involved in muscle satellite (stem) cell activation.

B. Systemic IGF-Is are also produced by active muscle As previously mentioned, IGF-IEa is also expressed in skeletal muscle as well as several other nonhepatic tissues. It has a similar sequence to the main liver IGF-I isoform. Therefore, it is assumed that it has a systemic action. However,


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muscle expresses at least two of the major binding proteins for the systemic form of IGF-I, and their expression tends to be up regulated, as is IGF-IEa by exercise. As the IGF-IEa produced by muscle will bind to these binding proteins in the extracellular matrix as well as in the serum, it is expected to have more effect on the muscles that produce it than on other muscles. Its action can then be regarded as paracrine as well as endocrine. It has also been shown that during extensive exercise, much of the circulating IGF-I is derived from muscle and not liver (Brahm et al., 1997). Although the type of IGF-I was not determined, the increased expression of IGF-I by exercised muscle is important, as it helps to explain some of the general beneficial effects of exercise, as this will influence the whole body. As well as having a different carboxy peptide sequence, IGF-IEa expression kinetics are different than those of MGF. This was first shown by Greg Adams’ group in the United States (Haddad and Adams, 2002), who found that MGF is expressed earlier than IGF-IEa in response to exercise. Hill and Goldspink (2003a) also showed that following muscle damage, MGF is produced as a pulse lasting a day or so and that IGF-IEa increases as MGF declines and IGF-IEa stays elevated for much longer. Another type of systemic IGF-I has been detected and cloned from human muscle IGFIEb (Hameed et al., 2003a), but the specific function of this isoform is not known.

IV. TRANSGENIC STUDIES INVOLVING IGF-I Transgenic mice that overexpress the IGF-I gene have been produced in several laboratories, the first being Brinsters’ and Palmiters’ group (Mathews et al., 1988) at the University of Pennsylvania, which produced the first transgenic mouse. The group produced the first mice that over expressed the growth hormone (GH) gene, and like the transgenic pigs that were produced later, they had several abnormalities. Their IGF-I transgenic mice, however, produced 30% more muscle and more bone and did not exhibit the same pathology as the GH transgenics even though the same metallothenin promoter was used. Somewhat later, IGF-I transgenes were introduced that were under the control of muscle regulatory elements, such as the chicken -actin promoter as used by Robert Schwartz’ group at Baylor College. Although the serum levels of IGF-I were not significantly elevated in these mice, there was considerable increase in muscle mass (Coleman et al., 1995). More recently, this group has shown that muscle regeneration and motoneuron regeneration is more rapid in the IGF-I transgenic mice (Rabinovsky et al., 2003). The data from these experiments is somewhat confusing as the transgene in these experiments it is not always clear, that is, whether the full-length cDNA or the entire IGF-I gene has been introduced.

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This is important as the expression of the transgene may be systemic or muscle specific, depending on whether it is under the control of a muscle-specific regulatory sequence, and also if the introduced IGF-I gene includes a binding protein domain sequence. If these binding proteins are located in the muscle, the IGF-I will then be retained within the tissue and not enter the circulation. In some studies, the IGF-I produced by the muscle has been presented as though the particular type of IGF-I is muscle specific and referred to as mIGF-I (Musraro et al., 1999, 2001). However, it appears that the systemic type of IGF-I was being expressed as it was under the control of a muscle specific promoter. Knockout mice have also been produced, but in this case the whole gene has been disabled. Therefore, none of the IGF-I peptide can be transcribed (Baker et al., 1993; Powell-Braxton et al., 1993). These mice, however, do not survive very long after birth, as their muscles appear to be dystrophic. A more sophisticated approach was made by Yakar et al. (1999) using the Cre Lox method to selectively knock out the IGF-I gene in liver cells. Surprisingly, these mice grew reasonably normally and had similar IGF-I serum levels. This is interesting as it emphasizes the role of nonhepatic IGF-I in local-tissue muscle growth regulation.

V. SPLICING OF THE IGF-I GENE IN NEURONAL TISSUE The splicing of the IGF-I gene in neuronal tissue is somewhat similar to muscle tissue. MGF is expressed in brain and spinal cord as well as IGF-IEa. However, in the CNS these are only expressed as the class 1 splice variants (Fig. 2.3) unlike muscle, which expresses both class 1 and 2 IGF-IS (Aperghis and Goldspink, unpublished obervations). This has been shown to have neuroprotective properties. This generates an N-terminal tripeptide, Gly–Pro–Glu, and

Figure 2.3. Possible splicing or inclusion of exons lead to a family of IGF-I mRNA that all encode the same mature IGF-I domain (exon 2 or 3) but differ in sequences 50 or 30 to exons 2 and 3. This leads to six different types of IGF-I mRNA: class 1 IGF-IEa, class 1 IGFIEb, class 1 IGF-IEc, class 2 IGF-IEa, class 2 IGF-IEb and class 2 IGF-IEc.


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des-N-(1–3)-IGF-I peptide. Interestingly, this truncated IGF-I, des-N-(1–3)IGF-I has a 100-fold reduction in affinity for IGFBP1 (Carlsson-Skwirut et al., 1989) and 2–3 times lower for IGFBP3 (Forbes et al., 1988), whereas it has strongly increased biological activity, varying between 4 and more than 10 times the potency of intact IGF-I (Ballard et al., 1987, 1988; Carlsson-Skwirut et al., 1989; Ogasawara et al., 1989; Szabo et al., 1988). It is also interesting to note that the tripeptide Gly–Pro–Glu appears to have intrinsic neurotransmitter activity (Sara et al., 1989).

VI. SPLICING OF IGF-I GENE IN OTHER TISSUES The complementary DNA of liver IGF-I (IGF-IEa) was reported in 1983 by Jansen et al. (1983) and the mRNA of another splice variant expressed in human liver IGF-IEb was reported by Rotwein in 1986. Although both have the 70 amino acid mature peptide, the latter has exon 5, while the terminal exon in IGF-IEa is exon 6. Later, a third splice variant was detected by Chew et al. (1995) in a hepatic cell line (HepH2) after treatment with growth hormone. However, the IGF-IEc in the human muscle cells has an almost identical sequence to the IGF-IEb in the rat. Therefore, it may be concluded that although the liver is the main source of the circulating IGF-IEa, under certain conditions nonhepatic cells are also able to splice the IGF-I gene to generate these other two splice variants. When muscle is subjected to intensive exercise, this becomes one of the main source of circulating IGF-I (Brahm et al., 1997; Hameed et al., 2003a).

VII. MECHANICAL SIGNALS IN SPLICING OF THE IGF-I GENE A number of studies have reported the IGF-I response to exercise and overload in human as well as animal skeletal muscle. In a study of young military recruits, Hellsten et al. (1996) reported an increase in IGF-I immunoreactivity of muscle samples after 7 days of strenuous exercise. More recently, in a study investigating IGF-I mRNA levels, Bamman et al. (2001) reported a 62% increase in IGF-I mRNA concentration in muscle 48 h after an acute bout of eccentric but not concentric contractions. Therefore, there is little doubt that the amounts of IGF-I expressed is influenced by exercise. However, it was not known what type of IGF-I was expressed and the nature of the mechano-transduction involved. Vanderburgh’s group measured IGF-I production by cultural muscle cells that were subjected to stretch (Perrone et al., 1995; Vandenburgh et al., 1991) and found that IGF-I production was considerably increased, as was the cell mass. It was also found that the media from these cells had a greater affect on cell mass

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than adding recombinant IGF-I. It is now realized that the type of IGF-I produced by these cells was probably MGF. The locally expressed splice variant of IGF-I, including MGF, also explains the results of the earlier experiments by Goldberg (1967), that muscles that have been overloaded surgically will still undergo hypertrophy even in hypophysectomized rats. Recently, our group has grown C2C12 muscle cells in a 3-D collagen matrix that is subjected to a given amount of mechanical strain applied at different rates and different frequencies. This was achieved by attaching the collagen matrix to a linear motor controlled by a computer. Both IGF-IEa and MGF were very markedly up regulated by a single ramp stretch. However, IGF-IEa expression was expressed less with a cyclical stretch and decreased as more cycles of stretch were applied. This was not the case for MGF (Cheema et al., 2004). Therefore, even this type of mechanical signals that induce IGF-IEa and MGF differ. One interesting question concerns the mechano-transduction mechanism via which muscle cells switch gene expression in response to mechanical signals. Gene expression is influenced by mechanical signals in several cell types, and this is believed to involve conformational changes in the cytoskeleton (Ingber, 1997). The cytoskeleton in muscle involves the dystrophin complex. It is interesting that in muscular dystrophy in which it is known that there is a defect in this cytoskeletal assembly, the muscles cannot respond to mechanical strain by producing MGF (Goldspink et al., 1996). The dystrophin complex is believed to stiffen the membrane, and when it is defective this means that not only is the membrane more susceptible to mechanical damage, but the muscle fibers are unable to undergo local repair because of the lack of MGF and probably the inability to activate the satellite cells. Strong support of this paradigm was recently provided by Frank Luyten’s group in Belgium. They found that when mesenchymal stem cells were introduced into dystrophic muscles of the mdx mouse this restores the sarcolemmal expression of dystrophin and reinstated the expression of MGF (De Bari et al., 2003).

VIII. HORMONAL INFLUENCE ON SPLICING OF THE IGF-I GENE The original somatomedin hypothesis originated in the 1950s in early efforts to understand how somatic growth was regulated by a factor secreted by the pituitary that became known as pituitary-derived growth hormone. This did not act directly on its target tissues to promote growth, because intermediary substances were involved (Daughaday and Reeder, 1966). The term somatomedin was later adopted to reflect the growth-promoting actions of these substances (Daughaday et al., 1972), which were characterized by IGF-I and called insulinlike growth factors (Klapper et al., 1983; Rinderknecht and Humble, 1978). Green et al. (1985) proposed the ‘‘dual effector hypothesis,’’ which suggested


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that GH had direct effects on peripheral tissues not mediated by IGF-I and that GH stimulated local IGF-I production. It is now realized that in addition to stimulating hepatic IGF-I production, GH up regulates the formation of a ternary IGF binding complex, including IGFBP-3 and the acid labile subunit (ALS), which stabilizes IGF-I in the serum. However, as previously mentioned, the results of recent tissue-specific gene-deletion experiments using the CreloxP model of gene deletion have questioned the role of liver GH and liver IGF-I (Sjorgren et al., 1999; Yakar et al., 1999). This homologous recombination system was used to create a hepatic-liver-specific deletion of the IGF-I gene but allowed normal expression of this gene in other nonhepatic tissues, such as heart, muscle, fat, spleen, and kidney. This liver-specific gene deletion of IGF-I reduced circulating IGF-I levels at 6 weeks of age compared with normal control mice. However, measurements of body size and individual organ weights at 6 weeks showed no difference between the knockout animals and their wild-type litter mates. Hence, postnatal growth and development was considered normal without the contribution of liver-derived IGF-I (Sjorgren et al., 1999), thus emphasizing the role of the local IGF-I system in the process. Unquestionably, the GH/IGF-I axis plays a role in postnatal growth and development, and these hormones reach their peak during adolescence. With increasing age, however, there is a marked decline in the circulating levels of GH and a somewhat smaller decline in circulating IGF-I (Rudman et al., 1981). In young adults who were growth hormone deficient, the administration of recombinant GH (rhGH) was shown to have positive effects on muscle mass and function (Cuneo et al., 1991). Another study where GH deficient adults were treated with GH for an extended period of time concluded that there was not only increased muscle strength but also decreased body fat (Beshyah et al., 1995). This led to the belief that older individuals with decreased levels of circulating GH and IGF-I would also benefit from rhGH therapy. The administration of GH to patients in end-stage heart failure has proven to be beneficial, and the evidence suggests that this increases the mass and contractility of the myocardium. Animal experiments in which recombinant IGF-I have been administered (Buerke et al., 1995) indicate that this reduces the effects of temporary ischaemia. The roles of circulating GH and IGF-I, particularly with regard to muscle adaptation in later life, are still unclear. In support of an argument that systemic growth factors may be of relatively minor importance in muscle hypertrophy is one study that showed that the overloaded muscles in hypophysectomized rats were still able to hypertrophy despite significantly reduced systemic IGF-I levels (Adams and Haddad, 1996). These findings, coupled together with the simple observation that it is only challenged muscles that hypertrophy and not all the muscles of the body, highlights the importance of a ‘‘local’’ system of muscle adaptation. Together with Michael Kjaer’s group in

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Copenhagen, our group has recently studied the relationship between GH administration and exercise and the expression of main muscle insulin-like growth factor-I (i.e., MGF and IGF-IEa) were studied. The subjects (age 74 1 year) were assigned to either resistance training with placebo, resistance training combined with GH administration, or GH administration alone. Real-time quantitative RT-PCR was used to determine mRNA levels in biopsies from the vastus lateralis muscle at baseline after 5 and 12 weeks of training. GH administration did not change MGF at 5 weeks but significantly increased IGFIEa (237%). In contrast, after 5 weeks of training combined with GH treatment, MGF increased significantly (456%) and IGF-IEa by 167%. No further significant changes were noted at 12 weeks. These data suggest that when mechanical loading in the form of resistance training is combined with GH, MGF mRNA levels are enhanced. This is believed to be due to an overall up regulation of transcription of the IGF-I gene prior to splicing, which results in more primary transcript of IGF-I. This more can be spliced to MGF with resistance exercise than would normally be the case in elderly muscle. There is some question as to whether there would be same effect if GH is administered to young healthy subjects (Rennie, 2002) who are not GH deficient. This needs to be addressed, as there is considerable use of rGH by professional athletes as well as others who can easily obtain this over the Internet. Also, the type of exercise is clearly important as this would be applicable to events in which increased muscle mass is an advantage.

IX. PEPTIDE PRODUCTS RESULTING FROM SPLICING Although the different splice variants of the IGF-I gene can be detected and quantified at the mRNA level, it is the peptides that are responsible for the biological activity. These are initially produced in the pre-pro, then proform (Fig. 2.4), and in some cases glycosylated. Bona fide medical trials have included the use of IGF-I peptides for the treatment of diabetic neuropathy, for pituitary dwarfism, and motoneuron disease (ALS). Recombinant IGF-I, which is commercially available, was used, but in the latter case, an altered IGF-I was produced by a biotechnology company for ALS treatment. Regrettably, misuse of IGF-I for enhancing athletic performance is occurring, and it is assumed that this involves the commercially available mature IGF-I, which is advertised over the Internet. This peptide is injected directly into individual muscles. Growth hormone is also misused, as previously mentioned, particularly in this way, by body builders, although it is often injected intravenously. For these reasons, and because of its role in cardioprotection, the IGF-IEa and MGF peptides are of considerable interest.


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Figure 2.4. The 30 alternative splicing of the IGF-I gene in muscle generates three different proIGF-I proteins, that is, proIGF-IEa, proIGF-IEb, and proIGF-IEc.

The tertiary structure of IGF-I was predicted sometime ago using computer graphics (Blundell et al., 1978). This was based on the three-dimensional crystalline structure of insulin as determined by x-ray diffraction. IGF-I is somewhat longer than insulin, although its receptor domain is very similar. This acts as a landmark for interactive molecular graphics for visualizing the conformation of IGF-I and IGF-II peptides. IGF-I peptides exist as single chain polypeptides consisting of about 70 amino acid residues. Not only are these derived by alternative splicing of the IGF-I gene, but like insulin, the initial peptide undergoes post-translational alteration. The A and B regions of IGF-I are similar to those of proinsulin; however, IGF-I also contains a D-region extension and an E domain and is therefore longer than insulin. Similar to proinsulin, IGF-I contains an amino-terminal B region and an A region that are separated by a short connecting C region. The A and B region of IGF-I are similar to those of proinsulin, giving a sequence homology to proinsulin of 43%. Unlike proinsulin, however, IGF-I also contains a D-region extension peptide at the carboxy terminus. Especially important is the conservation of the cysteine and glycine residues between IGF-I and proinsulin, which allows the prediction that the tertiary structure of the IGF-I will be rather similar to that of insulin. The hydrophobic core of insulin: A2 Iie, A16 Leu, B12 Val, B15 Leu, and B24 Phe (insulin notation) is conserved as well. The most obvious differences between IGF-I and insulin are in the C domain. The extracarboxy terminal regions endow the IGF-I splice variant with special properties that determine their modus operandi. In addition to a hydrophobic core similar to that of proinsulin, IGF-I has three S–S bridges that determine the three-dimensional conformation of this polypeptide. The presence of the disulphide links make it difficult to synthesize IGF-I chemically and to ensure native biological function

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and stability as these require the presence of all the three S–S bridges (Narhi et al., 1993).

X. RECEPTOR-MEDIATED CELLULAR EFFECTS OF IGF-I The biological activity of any hormone depends on the ability of the target cells to respond to the signal in extracellular milieu. This is a function of the cell receptors as well as postreceptor mechanisms. The IGF-I bind with high affinity to two cell surface receptors, IGF type I and II receptors. Although there is considerable cross reactivity between IGF-I and the insulin and IGF-II receptors, the type I receptor preferentially binds IGF-I and is commonly called the IGF-I receptor. Similarly, the type II receptor preferentially binds IGF-II and is commonly referred to as IGF-II receptor. IGF-I receptor is a tyrosine specific protein kinase with an extracellular ligand binding site, and this seems to mediate most IGF-I actions in skeletal muscle. For example, the IGF-I receptor appears to mediate amino acid and hexose uptake in rat soleus muscle (Yu and Czech, 1984) and in BC3H1 muscle cells (De Vroed et al., 1984) and DNA synthesis in chick muscle satellite cells (Duclos et al., 1991). In summary, IGF-I receptor mediated several actions of IGF-I, such as stimulation of amino acid uptake, proliferation, and differentiation, and inhibition of protein degradation (Ewton et al., 1987). It is very interesting to note that among known muscle growth factors, IGF-I is unique in its ability to stimulate both proliferation and terminal differentiation to form myotubes and then muscle fibers. Recent evidence suggests, however, that this duplicity of action is due to different IGF-I splice variants within muscle tissue. A recent study showed that different IGF-I splice isoforms have different roles in myoblast proliferation and differentiation (Yang and Goldspink, 2002). It also showed that different actions of different IGF-I isoform are mediated via a different receptor. Indeed, there is emerging evidence that the splice variant MGF induces the proliferation of the muscle satellite (stem) cells which are in the main residual mononucleated myoblasts (Hill and Goldspink, 2003a,b). As muscle is a postmitotic tissue, the extra nuclei required for postnatal growth and repair are provided by those satellite cells that fuse with the muscle fibers. As mentioned, IGF-I preferentially binds to the IGF-I receptor; it also has a considerable cross reactivity with IGF-II and insulin receptors. Interestingly, however, IGF-I binds with different regions of these receptors (Cascieri and Bayne, 1994). Analysis of the interaction of different IGF-I analogues with the IGF-I, IGF-II, and the insulin receptors showed that residues at positions 24 and 31, and the C domain of IGF-I, are important for the IGF-I binding to the IGF-I receptor and that this binding is not affected by the presence of the D domain. For the insulin receptor, the residue at 15 and 16 are within the helical


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region of the B domain, and the presence of the D and/or C domains of the IGF-I impairs the binding of the peptide to the insulin receptor. This may explain the antagonistic relationship between the IGFs and insulin and why rIGF-I was not deemed suitable as a replacement for insulin in non-insulin-resistant types of diabetes.

XI. THE ROLE OF BINDING PROTEINS IN THE REGULATION OF SYSTEMIC IGF-I In recent years, it has become increasingly apparent that the specific IGF-I binding proteins (IGFBP) determine to some extent the autocrine and paracrine actions of the IGF system. Their action can be regarded as an example of a prereceptor regulation. Among the seven IGFBPs described so far (Damon et al., 1997; Florini et al., 1996), four of them (IGFBP-2, -4, -5, and -6) are produced by different myoblast cell lines, whereas only IGFBP-4, IGFBP-5, and IGFBP-6 are expressed by adult skeletal muscle (Florini et al., 1996; Putzer et al., 1998). IGFBPs have been associated with systemic delivery, but if they are expressed in skeletal muscle they would have the effect of retaining IGF-I within the muscle tissue. When in the unbound state, the IGF-I peptides have a very short half life, and in the serum they are thought of as carrier proteins. However, when expressed within the muscle, they modulate the endocrine as well as the local influences of IGF-I (Mohan and Baylink, 1996). The precise actions of IGFBPs at the tissue level are unknown, but it is apparent that they stabilize and augment local IGF-I bioavailability (Clemmons, 1998; Jones and Clemmons, 1995; Mohan and Baylink, 1996). However, the previous work did not distinguish between the different types of IGF-I. At the present time, the author’s laboratory is using a monoclonal MGF antibody to isolate the specific binding protein(s) for MGF. What is known is that MGF does not bind to any of the previously described IGF-IBPs. This is not surprising, as if the carboxy sequence recognizes the specific binding proteins (Clemmons et al., 1992), and this is different in MGF. Both mRNA and protein levels of IGF-I and IGFBPs have been shown to be up regulated during regeneration after ischemic injury (Jennische and Hall, 2000). In situ hybridization showed IGFBP-5 restricted to regenerating muscle cells, whereas connective tissue cells expressed IGFBP-4. Exercise has shown to regulate several of the binding proteins in the serum and in the muscle (Hopkins et al., 1994). For example, Awede et al. (1999) reported that overloaded muscles in mice increased the expression of IGF binding protein 4 mRNA, whereas that of IGFBP 5 mRNA was decreased. In contrast, unloading of mouse muscle was reduced in IGFBP5 mRNA but did not affect IGFBP4 level. Thus, IGFBP4 and IGFBP5 were assumed to mediate the effects of IGF-I

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via regulation of the free IGF-I concentration in muscle and possibly via competition with IGF receptors for IGF-I.

XII. GENE TRANSFER OF MGF AND IGF-IEA The finding that muscle fibers can be transfected using a single intramuscular injection using a plasmid vector containing a foreign cDNA provides a basis for a method of treating medical conditions that are associated with marked muscle loss. IGF-I has been introduced in this way by Goldspink and Yang (2001), including the splice variant MGF. The latter resulted in a 25% increase in the mean muscle fiber size in injected muscle within 3 weeks. Similar experiments by other groups have also been carried out using a viral construct containing the liver type of IGF-I. This also resulted in a 25% increase in muscle mass, but this took over 4 months to develop (Barton-Davis et al., 1998; Musaro et al., 2001). The effectiveness of MGF is apparently because it initiates the activation of muscle satellite (stem) cells, as well as up regulating protein synthesis and its action is more specific than that of the systemic IGF-I. Besides treating certain medical conditions, gene transfer of this type may well be open to abuse. If it involves an adenoviral vector, then its detection would be relatively simple using PCR, as this virus infects most cell types. Plasmid vectors will be somewhat more difficult to detect, but new approaches that involve determining the different actions of the gene produces from those of introduced genes are under way.

XIII. MODUS OPERANDI OF THE DIFFERENT IGF-I SPLICE VARIANTS The anabolic effects of IGF-I have been clearly demonstrated by numerous in vitro studies, where it has been shown that IGF-I acts to increase the diameter of myotubes to suppress protein degradation and to increase amino acid uptake and stimulate protein synthesis (Bodine et al., 2001; Florini, 1987; Florini et al., 1996; Rommel et al., 2001; Semsarian et al., 1999; Vandenburgh et al., 1991). Its expression during muscle hypertrophy has been shown by using several animal models, including stretch-induced hypertrophy of the muscle, in which Schlechter et al. (1986) and Czerwinski et al. (1994) reported an increased expression of muscle IGF-I mRNA. DeVol et al. (1990) demonstrated that there was a threefold increase in IGF-I mRNA levels in the soleus and plantaris muscles in 11- to 12-week-old female rats following tenotomy-induced hypertrophy. This particular study employed hypophysectomized rats, which further suggests that the observed increase in IGF-I mRNA expression was GH


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independent. Later studies utilizing a similar model of functional overload in both normal and hypophysectomized rats found that both mRNA and protein levels of IGF-I were increased in muscle, prior to the attainment of significant hypertrophy, and remained elevated for up to 28 days during the hypertrophy process (Adams and Haddad, 1996). In another study with utilized treadmill training of GH-suppressed rats, levels of IGF-I mRNA and protein increased by 55% and 250%, respectively (Zanconato et al., 1994).

A. Muscle mass regulation by IGF-I There is evidence that experimental manipulation of the levels of IGF-I in muscle can induce hypertrophy both in vitro (Vandenburgh et al., 1991) and in vivo (Adams, 2002; Adams and McCue, 1998; Coleman et al., 1995). For example, overexpression (Coleman et al., 1995) or direct infusion (Adams and McCue, 1998) of IGF-I results in hypertrophy, whereas inhibition of intracellular signaling components associated with IGF-IR activation can prevent this response (Bodine et al., 2001). In another study, Criswell et al., (1998) concluded that overexpression of IGF-I in the muscles of transgenic mice was not shown to prevent unloading-induced atrophy stimulated by hind limb suspension. The signaling pathways by which IGF-I promotes skeletal muscle hypertrophy remains unclear, with roles suggested for both the calcineurin/ NFAT (nuclear factor of activated T cells) pathway (Musaro et al., 1999; Semsarian et al., 1999) and the P13-kinase/Akt pathway (Rommel et al., 1999). More recently, studies investigating the hypertrophic response both in vitro and in vivo have reported that it is the Akt/mTOR pathway and not the calcineurin pathway that is involved in promoting hypertrophy by activating downstream targets such as p70 S6 kinase (Bodine et al., 2001; Rommel et al., 2001). In addition, it was also reported that IGF-I might in fact act via Akt to inhibit the calcineurin/NFAT signaling pathway during this process (Rommel et al., 2001). Most studies conducted to date have looked at the expression of total IGF-I in response to muscle overload. As mentioned earlier, IGF-I exists as different isoforms, which are derived from the IGF-I gene by a process of alternative splicing. Yang et al. (1996) demonstrated that following acute stretch by hind-limb immobilization in the extended position, rabbit EDL muscles expressed both IGF-IEa and MGF mRNAs. However, very little MGF mRNA, if any could be detected in the control muscles not subjected to stretch. This led to the conclusion that MGF was activated by mechanical signals indicating micro cellular damage (Hill and Goldspink, 2003a,b). The notion that MGF is mechano sensitive has been supported by several further studies. For example, when stretch was combined with electrical stimulation of the rabbit tibialis anterior muscle, MGF mRNA was up regulated more so than by stretch alone (McKoy et al., 1999). Interestingly, Owino et al. (2001) showed that older

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muscles in the rat were less able to respond to overload, and this was also confirmed in the human by Hameed et al. (2003c). In older animals, overexpression of the different IGF-I isoforms has been associated with the prevention of some of the age-related effects on skeletal muscle, namely, the decline in muscle mass (Barton-Davis et al., 1998; Musaro et al., 2001). For example, Barton-Davis and coworkers injected a recombinant adeno-associated virus (AAV) directing overexpression of IGF-I (Ea isoform) into the extensor digitorum longus (EDL) muscles of young and old mice. The construct, containing a myosin light chain MLC/3 promoter, resulted in an overexpression of IGF-I in the muscles but did not increase circulating IGF-I levels. Four months postinjection, the injected muscles of the younger animals were on average 15% larger and 14% stronger than the noninjected muscles. In the older animals, age-related changes in muscle were therefore prevented, and in the injected muscles that were 27% stronger than the noninjected older animals. However, intramuscular injection of a plasmid gene construct containing MGF cDNA (under the control of muscle regulatory elements) into the tibialis anterior muscle of mice was shown to have a 25% increase in muscle mass after just 3 weeks (Goldspink and Yang, 2001), which has been shown to be reflected in a marked increase in muscle strength.

B. Satellite cells, muscle hypertrophy, and repair Satellite cells in skeletal muscle were first described by Mauro (1961), and it is now realized that these cells provide the extra nuclei for postnatal growth (Moss and Leblond, 1970; Schultz, 1996) and that they are also involved in repair and regeneration following local injury of muscle fibers (Grounds, 1998). In normal adult undamaged tissue, the satellite cells are quiescent and usually detected just beneath the basal lamina. They express M-cadherin (M-cad) (Bornemann and Schmalbruch, 1994; Irintchev et al., 1994) and when activated commence to coexpress myogenic factors, including c-met, MyoD and myf5, and later myogenin (Beauchamp et al., 2000; Cornelison and Wold, 1997; Qu-Petersen et al., 2002). The origins of satellite cells are still somewhat uncertain as they were thought to be residual myoblasts (reviewed by Seale and Rudnicki, 2000), but there is accumulating evidence that some may also originate from pluripotent stem cells derived from progenitor cells of the vasculature (Qu-Petersen et al., 2002). Pluripotent stem cells from bone marrow cells (Ferrari et al., 1998), as well as epidermal cells (Pye and Watt, 2001), have also been shown to fuse and adopt the muscle phenotype when introduced into dystrophic muscle. However, very few muscle stem cells are derived from sources other than residual myoblasts so the feeling is that these cells make an insignificant contribution, if any as their percentage is so low.


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It has been established that even in normal muscle, local injury does occur from time to time (Wernig et al., 1990), but in certain diseases such as the muscular dystrophies, the muscle fibers are markedly more susceptible to damage, in particular to the membrane (Cohn and Campbell, 2000). The contractile system of muscle fibers also sustains damage during eccentric contractions, that is to say, when the muscle is activated while being stretched. It is interesting to note that the forces generated by activation of muscle contraction combined with stretch exceed even those of maximal isometric contraction, thus causing damage (Lieber and Friden, 1999). During regeneration of skeletal muscle in young rats following ischemia- or myotoxin-induced damage, elevated expression of IGF-I has been reported (Edwall et al., 1989; Jennische and Hansson, 1987; Jennische et al., 1987), which was diminished by the 15th day of recovery (Marsh et al., 1997). However, there was no distinction between the types of IGF-I splice variant involved. Results of experiments (Hill and Goldspink, 2003a,b) in which damage was induced by stretch and stimulation or bupivacaine, demonstrated that MGF was produced as a pulse of about 1 day after damage was inflicted. On the other hand, the latter increased expression of IGF-IEa was inversely related to the decline in MGF. Following the initial surge in MGF, it seems that the IGF-I gene is then spliced toward IGF-IE. It seems that in both myotoxin- and mechanical activity–induced damage models, the temporal expression pattern for each IGF-I splice variant in both models showed similar differential gene splicing sequences, with MGF peaking before IGF-IEa. This temporal difference in expression of the two muscle IGF-I RNA transcripts has also been described in the rat following commencement of resistance exercise (Adams, 2002). As M-cad expression, a marker of satellite cell activation whether measured as mRNA or protein, peaked well before IGF-IEa, it is therefore unlikely that the systemic type of IGF-IEa is responsible for initial activation of satellite cells. However, it is not possible to say from these data whether this was due to an increase in the number of satellite cells, as it is known that quiescent satellite cells do stain to some extent for M-cad protein (Rosenblatt et al., 1999). Nevertheless, it represents a marked increase of M-cad whether it is in existing satellite cells or an increase in the number of these cells or both. Our in vivo experiments, however, showed that MGF caused proliferation of the monnucleated myoblasts and that these stayed in the monucleated state (Yang and Goldspink, 2002). This indicates that MGF replenished the muscle satellite (stem) cell pool. In contrast Miranda Grounds group (Shavlakadze et al., 2004) found that IGF-IEa overexpression in transgenic MLC/MiGF mice (Barton-Davis et al., 1998, 1999) was not associated with an increased number of satellite cells. This makes physiological sense as IGF-IEa is present in reasonably high concentrations even in resting muscle and in the main sources of mature IGF-I whereas MGF is produced in response to exercise and/or damage.

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C. Other actions of IGF-I splice variants MGF and IGF-IEa splice variants apparently yield the same mature peptide, which is derived from the highly conserved exons 3 and 4 of the IGF-I gene. These exons present in all the known IGF splice variants are known to code for the IGF-I receptor ligand domain. A mechanism of extracellular endoproteolysis of the IGF-I prohormone results in the same mature peptide (Gilmour, 1994), even though the splice variants of IGF-I may have different 30 sequences, including the E domain. It has been suggested that IGF-I precursors could be pluripotent, in a form analogous to that of prohormone propiomelanocortin and proglucagon (Siegfried et al., 1992). The observation that a synthetic peptide derived from the rat Eb domain induces proliferation in epithelial cells is noteworthy (Siegfried et al., 1992). However, the use of recombinant IGF-I in many experiments indicate that not only do the IGF-I splice variants have a general anabolic effect but they inhibit apoptosis.

XIV. CONCLUSIONS The results of these studies provide additional insight into the complexity and implication of the IGF-I system in conditions of damage and subsequent regeneration. IGF-IEa and MGF are produced by active muscle in rodents and have been shown to be positive regulators of muscle hypertrophy (Goldspink, 1999; McKoy et al., 1999; Owino et al., 2001). We have now shown that MGF is expressed in human muscle after a single bout of exercise, although the response was impaired in the case of the elderly subjects (Hameed et al., 2003b). However, elderly men are given growth hormone treatment MGF expression, and muscle cross-sectional area was increased, presumably because growth hormone results in more of the primary transcript being available to be spliced toward MGF (Hameed et al., 2004). Animal experiments have shown that the MGF isoform is acutely induced, whereas IGF-IEa has a delayed effect that is sustained during the later phase of tissue regeneration and adaptation. As the expression of the autocrine splice variant (MGF) precedes satellite cell activation, it is likely that this form of IGF-I is associated with satellite cell activation, not the systemic IGF-IEa type. This is in accordance with the finding that dystrophic muscles do not respond by up regulating MGF expression and are more susceptible to damage because they are unable to undergo local muscle repair. Future experiments investigating the signaling involved in the splicing of the IGF-I gene are of importance, as they may result in a therapeutic application for ameliorating muscle loss in a range of medical conditions.


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Acknowledgments This work was supported by grants from the Wellcome Trust, the International Olympic Games WADA Committee, and an EC (PENAM) grant for studying the effects of exercise, including muscle damage.

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Jennische, E., and Hansson, H. A. (1987). Regenerating skeletal muscle cells express insulin-like growth factor-1. Acta Physiol. Scand. 130, 327–332. Jennische, E., Skottner, A., and Hansson, H. A. (1987). Satellite cells express the trophic factor IGF-I in regenerating skeletal muscle. Acta Physiol. Scand. 129, 9–15. Jones, J. I., and Clemmons, D. R. (1995). Insulin-like growth factors and their binding proteins: Biological actions. Endocr. Rev. 16, 3–34. Kenyon, C., Chang, J., Gensch, E., Rudner, A., and Tabtiang, R. (1993). A C. elegans mutant that lives twice as long as wild type. Nature 366, 461–464. Klapper, D. G., Svoboda, M. E., and Van Wyk, J. J. (1983). Sequence analysis of somatomedin-C: Confirmation of identity with insulin-like growth factor I. Endocrinology 112, 2215–2217. Kops, G. J., Dansen, T. B., Polderman, P. E., Saarloos, I., Wirtz, K. W., Coffer, P. J., Huang, Y. Y., Box, J. L., Medema, R. H., and Burgering, B. M. (2002). Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 419, 316–321. Lieber, R. L., and Friden, J. (1999). Mechanisms of muscle injury after eccentric contraction. J. Sci. Med. Sport. 2, 253–265. Lin, K., Dorman, J. B., Rodan, A., and Kenyon, C. (1997). Daf-16: An HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 278, 1319–1322. Marsh, D. R., Criswell, D. S., Carson, J. A., and Booth, F. W. (1997). Myogenic regulatory factors during regeneration of skeletal muscle in young, adult, and old rats. J. Appl. Physiol. 83, 1270–1275. Mathews, L. S., Hammer, R. E., Behringer, R. R., D’Ercole, A. J., Bell, G. I., Brinster, R. L., and Palmiter, R. D. (1988). Growth enhancement of transgenic mice expressing human insulin-like growth factor I. Endocrinology 123, 2827–2833. Matthews, K. G., Devlin, G. P., Conaglen, J. V., Stuart, S. P., Mervyn Aitken, W., and Bass, J. J. (1999). Changes in IGFs in cardiac tissue following myocardial infarction. J. Endocrinol. 163, 433–445. Mauro, A. (1961). Satellite cells of skeletal muscle fibres. J. Biophys. Biochem. Cytol. 9, 493–495. McKoy, G., Ashley, W., Mander, J., Yang, S. Y., Williams, N., Russell, B., and Goldspink, G. (1999). Expression of insulin growth factor-I splice variants and structural genes in rabbit skeletal muscle induced by stretch and stimulation. J. Physiol. 516, 583–592. Mohan, S., and Baylink, D. J. (1996). Insulin-like growth factor system components and the coupling of bone formation to resorption. Horm. Res. 45, (Suppl), 59–62. Moss, F. P., and Leblond, C. P. (1970). Satellite cells as the source of nuclei in muscles of growing rats. Anat. Rec. 170, 421–435. Murphy, C. T., McCarroll, S. A., Bergmann, C. I., Fraser, A., Kamath, R. S., Ahringer, J., Li, H., and Kenyon, C. (2003). Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature 424, 277–283. Musaro, A., McCullagh, K. J., Naya, F. J., Olseon, E. N., and Rosenthal, N. (1999). IGF-I induces skeletal myocyte hypertrophy through calcineurin in association with GATA-2 and NF-ATel. Nature 400, 581–585. Musaro, A., McCullagh, K., Paul, A., Houghton, L., Dobrowolney, G., Molinaro, M., Barton, E. R., Sweeney, H. L., and Rosenthal, N. (2001). Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle. Nat. Genet. 27, 195–200. Narhi, L. O., Hua, Q-X., Arakawa, T., Fox, G. M., Tsai, L., Rosenfeld, R., Holst, P., Miller, J. A., and Weiss, M. A. (1993). Role of native disulfide bonds in the structure and activity of insulin-like growth factor 1: Genetic models of protein-folding intermediates. Biochemistry 32, 5214–5221. Ogasawara, M., Karey, K. P., Marquardt, H., and Sirbasku, D. A. (1989). Identification and purification of truncated insulin-like growth factor I from porcine uterus. Evidence for high biological potency. Biochemistry 28, 2710–2721.

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Breeding Hevea Rubber: Formal and Molecular Genetics P. M. Priyadarshan Rubber Research Institute of India, Regional Station Agartala - 799006, India

A. Cle´ment-Demange Centre de Coope´ration Internationale en Recherche´ Agronomique pour le De´veloppement (CIRAD) Tree Crop Departement, TA80/01, Avenue Agropolis, 34398 Montpellier, Cedex 5, France

I. II. III. IV.

Introduction Historical, Botanical, and Agricultural Aspects Breeding Objectives Genetic Resources A. Collections from Amazonia B. Induced Polyploidy, Mutagenesis, and Dwarf Genotypes V. Yield Variation A. Root Heterogeneity and Stock–Scion Interactions B. Laticiferous System, Physiological Attributes, and Yield Thresholds VI. Reproductive Biology A. Seasonal and Nonsynchronous Flowering B. Low Fruit Set C. Apomixy VII. Conventional Breeding Methodologies A. Primary Clones, Natural Pollination, and Seed Gardens B. Derivation of Recombinants and Clonal Selection C. Genetic Analysis and Variability Management

Advances in Genetics, Vol. 52 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2660/04 $35.00

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D. Early Selection, Estimation of Genetic Value, and Development of Clones VIII. Breeding Against Stresses A. Nontraditional Environments B. Abiotic Stress Factors C. Phenology Under Differential Geo-Climates D. G E Interactions and Specific Adaptation E. Resistance to Leaf Diseases IX. Biotechnologies A. In Vitro Culture B. Molecular Genetics X. Conclusions Acknowledgments References

I. INTRODUCTION Hevea (Para rubber) is a tall, deciduous, perennial ligneous tree, with rhythmic growth and orthotropic ramification, belonging to the Euphorbiaceae family. It can reach 30 m high and a girth of over 3 m in its region of origin, the Amazon basin. With a history spanning more than 450 years, it attained prominence only during later half of the nineteenth century as a consequence of the discovery of vulcanization by Goodyear in 1839, which gave to natural rubber the required level of quality and durability for wide industrial utilization. Introduced from the Amazon basin to southeast Asia, it staked almost 40% of the export revenue of Brazil, nearly equaling coffee in importance until 1940 (Dean, 1987). Rubber statistics during 2002 kept Thailand (2.3 million tons) at the helm of rubber producers, with Indonesia, India, Malaysia, China, Vietnam, Coˆte d’Ivoire, Liberia, Sri-Lanka, Brazil, Philippines, Cameroon, Nigeria, Cambodia, Guatemala, Myanmar, Ghana, Democratic Republic of the Congo, Gabon, and Papua New Guinea in the descending order of production. The southeast Asian countries continue to enjoy dominance in rubber production and trade by maneuvering more than 90% of the 7.97 million tons of rubber produced worldwide in 2003 (Sekhar, 2004). Thailand, Indonesia, India, and Malaysia together contributed 74% of the rubber produced. The center of origin, Latin America, shares only 2%, mainly due to the occurrence of South American leaf blight (SALB), caused by Microcyclus ulei P. Henn. Von. Arx (Chee, 1976). Confronted with the competition of synthetic elastomers, 75% of the natural rubber produced is consumed by the automotive industry (mainly in tires). More than 50% of natural rubber is consumed in Asia.

3. Breeding Hevea Rubber

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From the beginning of its domestication, rubber cropping was developed in the industrial sector, consisting of large estates with monospecific plantations. But the small-holding sector progressively increased its share and now owns more than 80% of the world’s natural rubber production. Smallholders’ plantations sometimes look similar to estates, but most are rubber-based agroforests (in Indonesia) or multicropping systems, especially in the young age (Gouyon, 1996; Viswanathan and Rajasekharan, 2001). The aim of rubber breeding is to provide small-holders as well as agroindustrial estates with an easily reproducible and genetically superior planting material in the form of a varietal type, currently represented by grafted clones, for latex production and, more recently, wood production. Although breeding is a long-term activity, introducing superior clones in plantation and replantation projects proved to be a convenient and cheap way to improve or maintain the profitability of rubber cropping. Now, biotechnologies hold new promise for deriving improved planting material and increasing the efficiency of rubber breeding. This review is an attempt to take stock of the progress made in breeding Hevea rubber worldwide.

II. HISTORICAL, BOTANICAL, AND AGRICULTURAL ASPECTS Rubber is synthesized in over 7500 plant species (Compagnon, 1986), confined to 300 genera of seven families, namely, Euphorbiaceae, Apocynaceae, Asclepiadaceae, Asteraceae, Moraceae, Papaveraceae, and Sapotaceae (Backhaus, 1985; Cornish et al., 1993; Heywood, 1978; Lewinsohn, 1991). At least two fungal species are also known to make natural rubber (Stewart et al., 1955). Hevea brasiliensis (Willd. Ex. A. de. Juss. Mu¨ ll-Arg.) is the almost exclusive contributor toward natural rubber produced worldwide (Greek, 1991). Rubber is a hydrocarbon polymer constructed of isoprene units (C5H8), and natural rubber is a secondary metabolite (cis 1,4polyisoprene) chiefly originating in the secondary phloem of the tree. The possible roles of latex in plants, though unclear so far, have been attributed to a protection from predation, a source of stored carbon and moisture, and a counteractant to ozone injury (Hunter, 1994). However, further detailed research will only give insight into the phenomenon of functions of latex, which is essentially an extensive subject. The genus Hevea is composed of 10 species: H. brasiliensis, H. guianensis, H. benthamiana, H. pauciflora, H. spruceana, H. microphylla, H. rigidifolia, H. nitida, H. camporum, and H. camargoana (Schultes, 1990). There are no biological barriers between them, and some species are proven to be intercrossable by hand pollination (Cle´ ment-Demange et al., 2001). Consequently, the Hevea species can be considered a species complex. H. paludosa has been identified in Brazil as an 11th species (Gonc˛ alves et al., 1990; Priyadarshan and Gonc˛ alves, 2003). An elaborate

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Table 3.1. Occurrence and Features of Hevea Species Species H. benthamiana Muell.-Arg. H. brasiliensis (Willd. ex. A. L. Juss.) Muell.-Arg. H. camargoana Pires

H. camporum Ducke

H. guianensis Aublet

H. microphylla Ule

H. nitida Mart. ex Muell.-Arg.

H. pauciflora (Spr. ex Benth.) Muell.-Arg.

Occurrence

Notable featuresa

North and west of the Amazon rain forest basin, upper Orinoco basin (Brazil) South of the Amazon river (Brazil, Bolivia, Ecuador, Peru)

Complete seasonal defoliation. Medium-size tree. Habitat: swamp forests Complete defoliation. Medium to large tree size. Habitat: well-drained soils Possibility of natural hybridization with H. brasiliensis. 2–25 m tree height. Habitat: seasonally flooded swamps Retains old leaves until new leaves appear. Maximum 2 m tall. Habitat: dry savannahs Retains old leaves until new leaves and inflorescences appear. Grows at higher altitudes (1100 m msl). Medium-size tree. Habitat: well drained soils Complete defoliation. Small trees that live in flooded areas (igapo´ s). Habitat: sandy soils

Restricted to the Marajo island of the Amazon river delta (Brazil)

South of the Amazon between the Marmelos and Manicore´ rivers’ tributaries of the Madeira river Throughout the geographic range of the genus (Brazil, Venezuela, Bolivia, French Guyana, Peru, Colombia, Surinam, Ecuador)

Upper reaches of the Negro river in Venezuela. It is not found in other regions of the geographic range of the genus Between the rivers Uaupes and Icana, tributaries of the upper Negro river (Brazil, Peru, Colombia) North and west of the Amazon river (Brazil, Guyana, Peru). Distribution discontinuous due to habitat preferences

H. rigidifolia (Spr. ex Benth.) Muell.-Arg.

Among the Negro river and its effluents. Uaupes and Ic˛ ana rivers (Brazil, Colombia, and Venezuela)

H. spruceana (Benth.) Muell.-Arg.

Banks of the Amazon, Negro river and lower Madeira (Brazil)

H. paludosa Uleb

Marshy areas of Iquitos, Peru

Inflorescences appear when leaves are mature. Small to medium-size trees (2 m) Retains old leaves until new leaves and inflorescences appear. No wintering. Small to big trees. Habitat: well-drained soils, rocky hillsides Retains old leaves even after inflorescences appear. Small tree from savannas. Sometimes tall, with small crown on the top. Habitat: well drained soils Retains old leaves until new leaves and inflorescences appear. Flowers reddish purple. Medium-size tree. Habitat: muddy soils of islands Small leaflets, narrow and thin in the fertile branches. Habitat: marshy areas


55

description of the taxonomical and botanical aspects of Hevea is beyond the scope of this article. Schultes (1977a, 1987, 1990) and Wycherley (1992) refer the readers to excellent narrations on the subject. However, an account of the salient features of different species of Hevea is given in Table 3.1. Hevea species in its natural habitat occur in Bolivia, Brazil, Colombia, French Guyana, Guyana, Peru, Surinam, and Venezuela. These countries need a special mention since they are around the center of origin. All species except H. microphylla occur in Brazil. Four species have been found in Colombia, and three occur in Venezuela. Two occur in Bolivia and the Guyanas. H. guianensis is the most widely adapted species. Temperate-type rubber thrives even up to 2500–3000 m in the Andes mountains (Senyuan, 1990). These Hevea species probably evolved in Amazonian forests over 100,000 years (Cle´ ment-Demange et al., 2001). With the possible exception of one triploid clone of H. guianensis (2n ¼ 3 x ¼ 54), and the possible existence of one genotype of H. pauciflora, with 18 chromosomes (Baldwin, 1947), all species display 2n ¼ 36 chromosomes (x ¼ 9), and H. brasiliensis behaves as an amphidiploid (Ong, 1975, Priyadarshan and Gonc˛ alves, 2003; Raemer, 1935; Wycherley, 1976). Hevea rubber is depicted in ancient religious documents from Mexico dating back to 600 A.D. (Serier, 1993). Christopher Columbus gave the first description of rubber in the fifteenth century, and the astronomer Charles Marie de la Condamine was the first to send samples of the elastic substance called ‘‘caoutchouc’’ from Peru to France in 1736, with full details about the habit and habitat of the trees and procedures for processing (Dijkman, 1951). As a botanist, Fuse´ e Aublet described the genus Hevea in 1775. History recapitulates the names of five distinguished men: Clement Markham (of the British India Office), Joseph Hooker (director of Kew Botanic Gardens), Henry Wickham (naturalist), Henry Ridley (director of Singapore Botanic Gardens), and R. M. Cross (Kew gardner), with Kew Botanic Gardens playing the nucleus of rubber procurements and distribution. According to the directions of Markham, Wickham collected 70,000 seeds from the Rio Tapajoz region of the upper Amazon (the Boim district) and transported it to Kew Botanic Gardens in June 1876 (Baulkwill, 1989; Schultes, 1977b; Wycherley, 1968). Of the 2700 seeds germinated, 1911 were sent to the botanical gardens in Ceylon in 1876, and 90% of them survived. In September 1877, 100 Hevea plants specified as ‘‘cross material’’ were sent to Ceylon. Earlier, After Wicherley (1992), Schultes (1977a), Goncalves et al. (1990), Pires (1973), and Brazil (1971). Modified from Priyadarshan and Goncalves (2003). a The deciduous characteristics mentioned here have a bearing on the incidence of fungal diseases, especially through secondary leaf fall (Oidium). since retention of older leaves may make the tree ‘‘escape’’ Oidium. Dwarf types are desirable for possible wind tolerance. All species are diploid (2n ¼ 36) (Majumder, 1964) and are intercrossable (Clement-Demange et al., 2001). b Pires (1973) considered 11 species, including H. paludosa; Brazil (1971) considers 11 species.

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in June 1877, 22 seedlings, not specified either as Wickham or Cross, were sent from Kew to Singapore, which were distributed in Malaya and formed the prime source of 1000 tappable trees found by Ridley in 1888. An admixture of Cross and Wickham materials might have occurred, as the 22 seedlings were unspecified (Baulkwill, 1989). One such parent tree planted during 1877 was available in Malaysia even after 100 years (Schultes, 1987). Seedlings from the Wickham collection of Ceylon were also distributed worldwide. As a matter of fact, rubber trees covering millions of hectares in southeast Asia are derived from a very few plants of Wickham’s original stock from the banks of the Tapajoz (Imle, 1978). This contention is, however, debatable (Thomas, 2001). The first introduction of rubber to India was in 1879 from Ceylon (now Sri Lanka), when 28 Hevea plants were planted in the Nilambur Valley of the Kerala state in southern India. (Haridasan and Nair, 1980). Between 1880 and 1882, plants on an experimental scale were raised in different parts of southern India and the Andaman Islands. The research of P. J. S. Cramer (in Bogor, Indonesia) on the propagation and breeding of Hevea between 1910 and 1918 is noteworthy. He made a trip to the Amazon and succeeded in getting seeds of allied species like H. spruceana and H. guianensis. Cramer also conducted experiments on variations observed in 33 seedlings imported from Malaysia in 1883, from which the first clones of the East Indies were derived (Dijkman, 1951). Along with van Helten, a horticulturist, he could standardize vegetative propagation by 1915. The first commercial planting with bud-grafted plants was undertaken in 1918 on Sumatra’s east coast. The clones Ct3, Ct9, and Ct38 were the first identified by Cramer (Dijkman, 1951; Tan et al., 1996). Commercial ventures gradually spread to China, Thailand, and Vietnam, and rubber became an integral part of the economy of southeast Asia toward latter half of the twentieth century. Synthetically speaking, breeding was initiated with very strict mass selection among the trees at the beginning of twentieth century. With the introduction of bud grafting, ‘‘generative’’ and ‘‘vegetative’’ selection methodologies were simultaneously used that resulted in seedlings and grafted clones (Dijkman, 1951). Around 1950, the advantages of grafted clones proved to be overwhelming for yield potential compared with genetically improved seedlings, and the focus shifted to the derivation of clones for latex productivity. Progress in yield improvement in Hevea resulted in a gradual increment, from 650 kg/ha in unselected seedlings during 1920s to 1600 kg/ha in the best clones during the 1950s. The yielding potential was further enhanced to 2500 kg/ha in PB, RRIM, RRII, RRIC, IRCA, BPM, and RRIV clones during 1990s. During these 70 years of rigorous breeding and selection, notable clones like RRIM 501, RRIM 600, RRIM 712, PB 217, PB 235, PB 260, RRII 105, RRIC 100, IRCA 18, IRCA 230, IRCA 331, and BPM 24 were derived (Cle´ mentDemange et al., 2001; Priyadarshan, 2003a; Simmonds, 1989; Tan, 1987). Some


57

of the primary clones, like PB 56, Tjir 1, Pil B84, Pil D65, Gl 1, PB 6/9, and PB 86, selected during the aforesaid period, became parents of improved clones. It must also be acknowledged that primary clones like GT 1 and PR 107 are still widely used, although their identification traces back to the 1920s. In Latin America, every breeding effort is focussed on the derivation of clones having acceptable yield, along with durable resistance to SALB (Dean, 1987). It must be emphasized that this disease represents a permanent threat for the whole rubber industry (Davies, 1997). In the more humid areas of Asia, susceptibility to Corynespora leaf fall disease has become important for the rubber industry and breeders. Rubber is currently planted in the form of grafted trees at a density of about 450 trees per hectare. It experiences an immature phase that may vary from 5–9 years, depending on climate, soil conditions, and management. When the trunk girth of the trees reaches 50 cm, tapping is initiated that may last between 15 and 30 years. The tapping, a periodically renewed cut incised in the bark of the trunk, generates latex (cell cytoplasm containing rubber particles) throughout the year (Jacob et al., 1995). The tapping intensity is a result of the combination of tapping frequency (tapping every 2, 3, 4, or 5 days) and of chemical stimulation intensity by the application of Ethephon, an ethylene releaser (Abraham et al., 1968). In Asia, rubber wood has become an increasingly important economic product and represents a new challenge for breeders, which was first addressed by RRIM (Othman et al., 1995). Many of the clones issued from the RRIM 2000 series claim to be latex-timber clones. The trunk and the branches are used for varied transformation (furniture, plywood, particle wood, and fuel wood). Latex and wood are two complementary ways for atmospheric carbon sequestration (d’Auzac, 1998).

III. BREEDING OBJECTIVES Latex yield has long been the exclusive objective, with a close relationship between land productivity and labor productivity. The main components of productivity are: (a) growth of the trunk that determines the duration of immature phase, (b) evolution of yield per tree along time, and (c) stability of the stand (number of tapped trees per unit area) related with resistance to stress factors like tapping panel dryness (TPD), wind damage, varied diseases, low temperature, higher altitude, and moisture deficit. Latex yield and growth are hardly separable. Now, breeding and selection are quite exclusively applied to the clonal aerial part of the tree. The choice of seedling families to be used as rootstock is very limited. Among different potential advantages, the possibility of cloning a whole plant in vitro would allow breeding to be applied to the root system for

58


resistance to root diseases, for better adaptation to specific soils, and for better anchorage. This raises the theoretical idea of developing a rubber tree with three different genetic components, namely, roots, trunk, and canopy, each selected for its own requirements, according to the concept of a ‘‘compound tree’’ (Simmonds, 1985, 1989). Such a combination of high-yielding trunks with canopies resistant to SALB had been experimented by way of crown budding, but it proved to be quite impossible to develop this technique at a commercial level. Characteristics related to rubber technological quality has not deserved much attention so far, mainly because the most important quality factors, cleanliness of the harvest and conservation process, are not linked with genetics. Although some traits like viscosity or susceptibility to oxidation have a genetic basis, they widely vary according to different factors, such as climate, seasonal variations, age of the trees, and tapping systems. Multilocation trials and localized experimentation is set in order to test and optimize the adaptation and yielding potential of clones in specific environments. Characterization of the architecture of the trees in connection with wind risk is also important. Phenology is assessed in connection with susceptibility to different leaf diseases. Adaptation of clones to new environments, especially to suboptimal or marginal areas, has become important (Priyadarshan, 2003). Traditionally, rubber breeding is performed in close relationship with agro-industrial estates, which provide land and facilities for large-scale and longterm experimentation so as to make available a diversified list of 10–15 clones that can be recommended. Diversification of clones allow those large plantations to mitigate risks. Among those clones, the more stable ones are identified for recommendation to small-holders. But as far as small-holders represent a predominant share of all rubber production, with specific constraints, a selection focused on these constraints and on small-holders’ cropping techniques is required (e.g., fast-growing trees able to compete efficiently with weeds, canopy adapted to multicropping, clones adapted to uneven and intensive tapping systems, climatic variations, etc.). More recently, selection for timber has become a very important objective. An estimation from RRIM shows that a hectare of rubber plantation can yield around 190 m3 of rubber wood, and 2.7 million m3 of Hevea wood would be available from Malaysia (Arshad et al., 1995). Approximately 741 million m3 of wood must be available from 8,927,000 ha worldwide. The demand is expected to increase fast, and the RRIM has been making earnest efforts to derive latex timber clones (Othman et al., 1995). Lately, there is some interest generated among the scientists to evolve rubber as a factory producing useful chemicals, especially lifesaving drugs (Yeang et al., 2002). In the future, new requirements linked with environmental concerns such as reforestation or


59

carbon sequestration might appear, but implication for breeding is not clear for now.

IV. GENETIC RESOURCES Hevea brasiliensis is supposed to have become a stabilized amphidiploid (4x ¼ 36) during the course of evolution. This contention is amply supported by the observance of tetravalents during meiosis (Ong, 1975). Nevertheless, for practical purposes, Hevea is considered a diploid genus (2n ¼ 36). In situ hybridization studies revealed two distinct 18S–25S rDNA loci and one 5S rDNA locus, suggesting a possible allotetraploid origin with the loss of 5S rDNA during the course of evolution (Leitch et al., 1998). Hence, as long as a potential ancestor with 2n ¼ 18 is not known, the rubber tree would be considered an amphidiploid (Priyadarshan and Goncalves, 2003). Segregation analysis of isozyme or RFLP markers show a majority of nonduplicated loci (Seguin et al., 1996b). Consequently, the two ancestral genomes of Hevea would have strongly diverged. Locus duplications are infrequent in the Hevea genome, and they could have occurred due to chromosomal modifications posterior to the polyploidization event (Seguin et al., 2003). Only a comprehensive molecular analysis with this objective will reveal the details of origin.

A. Collections from Amazonia Allied species of Hevea make up a gene pool for breeding purposes, especially for the identification and introduction of genes of resistance to leaf diseases (Priyadarshan and Gonc˛ alves, 2003). Within Hevea brasiliensis, the basic species for natural rubber production, a very clear distinction needs to be made between the ‘‘Wickham’’ population and the series of wild accessions from the Amazonian forest, usually called Amazonian population. The Wickham population has been the basis for rubber domestication and has evolved through a breeding history of one century, with a current high level of adaptation to modern rubber cropping, except in SALB affected areas (many areas of Latin America). Conversely, the Amazonian populations, still under evaluation, have not been much modified by human selection. They display an average latex yield of around 12% of the level of currently developed Wickham clones and a fairly high resistance to Microcyclus or Corynespora cassiicola Berk et. Curt. Wei. (Cle´ ment-Demange et al., 2001). Different expeditions for the collection and transfer of allied species and Amazonian accessions have been organized since 1890. Between 1951 and 1952, 1614 seedlings of five Hevea species (H. brasiliensis, H. guianensis, H. benthamiana, H. spruceana, and H. pauciflora) were introduced to Malaysia

60


(Tan, 1987). Brookson (1956) has given an account of these introductions. In Sri Lanka, 11 clones of H. brasiliensis and H. benthamiana, and 105 hybrid materials were imported between 1957 and 1959 through triangular collaboration of the U.S. Department of Agriculture, Instituto Agronomico do Norte (IAN, Brazil), and Liberia. Many of these clones were later given to Malaysia (Tan, 1987). Introductions to the germplasm collection of CNRA in Coˆ te d’Ivoire, with CIRAD cooperation, were made of 40 accessions from the French–Brazilian collection of 1974 from Acre and Rondonia, 19 accessions from a Firestone collection in the Madre de Dios basin in Peru (MDF accessions), 24 accessions by the Brazilian Research Centre, Embrapa, in Manaus (CNSAM accessions), and 10 accessions from allied Hevea species. Part of the collections made by R. E. Schultes has also been rescued from two conservation sites in Columbia, thanks to a France–Columbia agreement, with 302 accessions from the Calima site and 41 accessions from the Palmira site, which were transferred to Coˆ te d’Ivoire in 1987 after a quarantine period on Martinique. Between 1945 and 1982, collections from Brazil (mostly Rondonia) have been undertaken at least 10 times (Gonc˛ alves et al., 1990). These materials can be used for the selection of clones under stressful conditions, like low temperature, moisture stress, wind, and disease incidence, and they may be subjected to continuous selection pressure under natural conditions or artificial situations. In 1981, due to an initiative taken by the International Rubber Research and Development Board (IRRDB), 63, 768 seeds, 1413 meters of bud wood from 194 high yielding trees and 1160 seedlings were collected from Brazilian Amazonia (Simmonds, 1989; Tan, 1987). This collection was carried out over three states—Acre, Rondonia, and Mato Grosso—in 16 different districts and 60 different locations overall. Of this, 37.5% of the seeds were sent to Malaysia and 12.5% to Coˆ te d’Ivoire. Half of the collections were maintained in Brazil. The accessions from bud wood collection were brought to Malaysia and Coˆ te d’Ivoire after quarantine against SALB. After the establishment of two germplasm centers of the IRRDB in Malaysia and Coˆ te d’Ivoire, other IRRDB-member countries were supplied with material according to their request. Crosses between Wickham and Amazonian accessions are relevant due to possible introgression of more variation. A specific program has been carried out by Coˆ te d’Ivoire and France (CIRAD and CNRA, with European Union funding) for the characterization and utilization of Amazonian accessions from 1985–1997. The analysis of genetic diversity of H. brasiliensis germplasm, by the use of genetic markers applied to the nuclear and cytoplasmic genomes (isozymes, RFLP, and microsatellites), showed a general structure made of six groups; four groups within the IRRDB 1981 collection, one group made up of the Schultes–Palmira collection, and the other made of the Wickham population


61

(Seguin et al., 1996, 2003). At field level, it was found that the average latex yield in Wickham–Amazonian crosses was still rather low, ranging between 30% and 50% of the level of GT1, probably due to the important gap between the two populations. Conversely, a wide variability was found within these crosses for growth, with probable heterotic effects enabling the selection of very vigorous Wickham–Amazonian clones. A clear difference in branching habits could be observed between accessions from Acre and Rondonia on the one hand, which more often have tall trunks with poor branching located at high height, and those from Mato Grosso on the other hand, which more often display trees with abundant branching at low height (Cle´ mentDemange et al., 1998). In 1995, an expedition was launched by RRIM to collect rubber seeds from Brazil. From this collection, about 50,231 seedlings were planted in Malaysia, including allied species (Malaysian Rubber Board, 1999; Rubber Research Institute of Malaysia, 1997).

B. Induced polyploidy, mutagenesis, and dwarf genotypes As part of the process for widening the genetic base, induced mutations and polyploidy have been tried on a limited scale (Markose et al., 1977; Mendes and Mendes, 1963; Zheng et al., 1981). There has been some indication of enhanced yield and useful secondary attributes in the induced polyploids (Saraswathyamma et al., 1984). Polyploidy breeding has not increased the enthusiasm of scientists due to chimeras (Zheng et al., 1980). An artificial triploid has been produced by crossing a diploid and a tetraploid (Saraswathyamma et al., 1988). Naturally occurring triploids have also been reported elsewhere (Nazeer and Saraswathyamma, 1987), but it must be acknowledged that all planting materials being currently utilized throughout the rubber-growing countries are common diploid clones. Alongside polyploidy and mutations, somaclonal variations could also play a role in raising genotypes with a wide range of variation. The existence of some putative genetically dwarf or semidwarf genotypes was mentioned (Ong et al., 1983; Simmonds, 1989), but not well documented. It was also attempted to associate some molecular genetic markers with the dwarfing trait (Venkatachalam et al., 2001). As a matter of fact, genetic resources so far have been viewed as populations of clones, more often conserved in bud wood gardens as living collections in order to widen the genetic base for breeding. Biotechnology is now producing many types of molecular genetic markers, issued either from nonexpressed or expressed DNA, as well as cloned genes of agronomic interest. Consequently, genetic resources increasingly include the molecular level represented as the ‘‘molecular resources’’ of Hevea germplasm. With the new possibilities of gene transfer (see

62


Section IX.B.4), genes belonging to other genera might also get involved in rubber breeding in the future.

V. YIELD VARIATION A. Root heterogeneity and stock–scion interactions A major part of rubber breeding efficiency can be attributed to the grafting technique, which enables the multiplication of elite genotypes at the level of the budded part (aerial part of the tree) and which determines the use of clones as the almost exclusive varietal type in rubber cropping. Unfortunately, cloning the whole tree (aerial part and roots) for the development of single-component clonal trees by the cutting technique (self-rooted marcots and mist propagated cuttings) generates a high ratio of uprooting due to lack of taproot and inadequate anchorage. Bud-grafted population has a high level of homogeneity and should exhibit intraclonal variation in yield to a minimum, barring factors like: (a) soil heterogeneity, (b) difference in juvenility of buds, and (c) variable seedling rootstocks. However, our own experience with RRII 105 monoclonal populations indicated a difference of 10–310 ml in total volume of latex/tap and a range of 28.1–43.9% in drc during peak yielding period (October–January). An estimate showed that 20% of the trees yielded more than 150 ml/tree/tap, and an equal percentage showed higher drc (more than 38%). In another experiment with RRII 105, total volume of latex and dry rubber yield was 5.0–325.0 ml and 1.8–144.0 g, respectively (Chandrashekar et al., 1997). Such variation was reported from countries like Malaysia (Hardon, 1969). The differences exhibited are significant and refutable for a homogeneous population. The soil heterogeneity is indeed a key attribute maneuvering the overall yield of a stand and can be geared at will through the observance of appropriate agronomic practices. Soil can be tested for deficiency and a fertilizer dosage can be followed in cognizance with the soil test data. The second factor will be the difference of juvenility in budded plants. This factor has actually not been assessed so far. However, this should not make a significant difference, since budded plants of a given population would normally arise from the same budding generation. The effect of rootstock has been most intriguing. Studies conducted in the past have proven that there are reliable and marked effects of rootstock on the yield of scion when budding was made onto illegitimate seedlings, where variation in terms of yield and girth were significant (Buttery, 1961). Further, it has been demonstrated that monoclonal or selfed seedlings from monoclonal blocks of bud-grafted plants showed marked differences in yield (Ng et al., 1982). From these results, it was suggested that vigorous hybrid seedlings issued from polycross seed gardens would provide better rootstocks


63

(Simmonds, 1985). Also, monoclonal seedlings of PB 5/51 and RRIM 623 were found to be significantly superior to other stocks (Ng, 1983). In an unpublished experiment (CNRA–CIRAD, Coˆ te d’Ivoire), monoclonal seedlings of GT 1 used as rootstock were found significantly better than three other monoclonal seedling rootstocks (issued from clones RRIM 600, PB 5/51, and LCB 1320). This confirms the good performance of GT 1 seedling rootstock already published earlier (Combe and Gener, 1977). This performance of GT 1 seedling rootstock is assumed to be linked with the male-sterile behavior of GT 1, which displays exclusive unselfed seedling progenies, with no inbreeding effect and better growth. Another experiment confirms that the most important part of yield variation is due to differences between the budded clones rather than between the seedling rootstocks, therefore mitigating the importance of the choice of seedling rootstock, which is heavily dependent on the availability of monoclonal seedlings in the planting areas (Cle´ ment-Demange, A. personal communication, from Hevego results). It was also shown that the rootstock can affect the thickness of scion bark and the number of latex vessel rings. Bobilioff (1923) made a notable observation that during the formation of new bast, when the scion cambium made 12 latex vessels, stock could make 9 latex vessels, therefore making the superiority of the scion cambium more prominent. The rationale enunciated indicates that, though the soil heterogeneity and juvenility of buds may not necessarily influence the yielding pattern of a bud-grafted population, the variable rootstock should exert effects over yield that are largely uncontrollable. Although poorly addressed by breeding in the lack of an efficient cloning technique, the root system directly affects soil–plant relationships, water and mineral uptake, and water stress resistance (Ahmad, 2001), as well as resistance to wind uprooting. Moreover, efficient breeding for the growth of budded clones and the increasing use of fast-growing clones may have generated an imbalance between stock and scion, therefore emphasizing uprooting hazard (Cle´ ment-Demange et al., 1995b). Consequently, cloning the root system is a major challenge for rubber tree breeding, as it would greatly facilitate growth and yield improvement as well as adaptation to various environments.

B. Laticiferous system, physiological attributes, and yield thresholds Hevea has articulated laticifers located in every tissue of the tree, notably in the soft bark (liber) of the trunk from which it can be extracted by tapping. These laticifers develop into ramose tubelike structures only through the addition of new primordia to existing ones and not through the growth of individual cells (Scott, 1886). These primordia are seen in longitudinal rows, and, during initial stages of germination, the end walls of these primordia break down and cell rows are seen converting into vessels. Laticifers are found to differentiate acropetally

64


in newly formed plant parts, such as the stem, leaves, flowers, and fruits. Vessels lying nearby are found to produce protuberances fusing together (anastomosis), therefore forming a paracirculatory ramified structure of a laticiferous system (syncytium). The different laticifer rings successively generated by the cambium are not interconnected. There are no plasmodesmata between the latex vessels and their surroundings (de Fay¨ and Jacob, 1989). The phloem and xylem vessels are linked by horizontal medullary rays that allow exchange of resources like water, carbohydrates, nutrients, and hormones by the way of intercell diffusion or active transportation (He´ bant and de Fay¨ , 1980). There use to be connections between laticiferous systems of stock and scion, evidenced by the transport of latex (Bonner and Galston, 1947). Yet there is marked difference in the rate of development of latex vessel formation in scion and stock, due to genotypic differences. Mahlberg (1993) gives an excellent historical perspective of the laticifers. A cross section of the bast would have cork, rings of stone cells, rows of latex vessels interspersed with medullary rays, cambium, and wood. The internal structure of the bast is apparently dependent on hereditary factors, since periodicity exists in the formation of vessels (Bobilioff, 1923). The latex is a cytoplasm that contains rubber particles, lysosomal microvacuoles known as lutoids, and double-membrane organelles rich in carotenoids assimilated to plastids, the Frey-Wyssling particles, the role of which has not yet been fully elucidated (Paardekooper, 1989). Water, which specifically plays a key role in latex flow after tapping, is another important constituent. Neither the mitochondria nor the nuclei are present in the latex collected upon tapping; they remain adherent to the plasmalemma, which enables regeneration of the latex. However, various types of RNA are found in the latex, enabling the study of genes expressed within the laticifers. Rubber particles are surrounded by a phospholipoglycoprotein monomembrane, with a negative charge ensuring the colloidal stability of latex, as well as protection against oxidative degradation. Two major components namely, biochemical and nonbiochemical, mainly determine the latex yield. While biochemical components include reactions involving the bursting of lutoid particles and their subsequent interactions with rubber particles and latex vessel plugging, the nonbiochemical components are variables linked with carbohydrate assimilation (photosynthesis) and water status (turgor pressure, vapor pressure, and transpiration). Coagulation plays a key role as it stops latex flow. Electron microscopic studies of Southorn (1968) showed that the latex vessels form internal plugs of latex coagulum. Lutoid particles, a prominent component of latex, contain hydrolytic enzymes. A protein/lipid membrane encloses them, and their rupture, caused by the high osmotic gradient near the cut, leads to the plugging of laticifers (Southorn and Edwin, 1968), thereby indicating that plugging is a major event that controls latex flow and yield. Plugging index, a ratio between


65

initial and final flow of latex, has been considered a clonal characteristic (Milford et al., 1969) and is an attribute that is responsive toward yield stimulants used for prolonging latex flow by subsiding the activity of lutoids. The plugging of latex vessels leads to gradual decrease in latex flow. Spillage of the internal contents of lutoids will be sufficient to reduce the elastic contraction of laticifers through the plugging mechanism. Turgor pressure in laticifers is vital for the flow of latex. Turgor pressure as high as 10–14 atmospheres is observed before sunrise, and studies on diurnal variations in latex yield gave a correlated response between latex yield and variations in atmospheric vapor pressure. On the other hand, an increased transpiration rate is found to decrease phloem turgor pressure by increasing xylem tension. In short, the aforesaid components (biochemical and nonbiochemical) are not mutually exclusive and exhaustive. Both components have to work in cognizance with each other, and the effect in total would amply influence the net yield of latex. There are two factors limiting latex production in Hevea brasiliensis namely, latex flow and in situ latex regeneration (Serres et al., 1994). More yield is retrievable when latex flow is easier and longer, and the latex regeneration must be substantially active to compensate the loss of cellular material through tapping. In a kinetic study of latex regeneration conducted in PB 235, PB 217, and GT1 (Serres et al., 1994), it was estimated that 72 h are needed to regain the cellular material in full. Studies on partition coefficient spelled that, for the preparation of one mole of isoprene, five moles of ATP would be necessary (Moraes, 1977). Combining the highest primary production and highest partition coefficient, Templeton (1969) estimated the potential yield of 4–6 tons of rubber/ha/year, which is very high if compared with actual figures. Even in the current best clones, the average yield recorded is little more than 2500 kg/ha/year, and considering the estimates of Templeton, it is evident that we are yet to have the best-suited genetic combination for gaining yield according to calculated threshold levels. A recent comprehensive review (d’Auzac, 1998) takes stock of the accumulated knowledge related to the laticiferous system, latex composition, and regulation of the production of latex. Related to this knowledge, upstream and downstream aspects are exposed. They are: (a) latex diagnosis methodology, (b) the metabolic typology of clones issued from this methodology (c) underand overexploitation of the plots due to tapping intensity, (d) possible induction of tapping panel dryness (TPD disease), (e) impact of the level and loading of sucrose into the laticifer, and (f) competition between vegetative growth and latex production. From these results, many consequences for selections based on physiological characteristics can be drawn.

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VI. REPRODUCTIVE BIOLOGY As a perennial crop, rubber breeding is space and time consuming. For genitors to be recombined together by hand or natural pollination a first-time constraint is related to the onset of flowering (4–5 years) even though some early flowering techniques can be applied. A second constraint is related to the long period required for an accurate assessment of the behavior of experimental clones at the level of one tree, as well as at the level of the tapped stand in one plot, especially for estimating traits such as susceptibility to tapping panel dryness or to wind damage. As far as space is concerned, one tree planted at normal density needs around 20 m2 (500 trees/ha). Consequently, the breeding and selection schemes have to be optimized by considering these constraints.

A. Seasonal and nonsynchronous flowering Similar to other tropical trees, Hevea normally takes 4–5 years to attain reproductive stage—a phase called ripeness to flower (Kramer and Kozlowski, 1979). Though the capacity to flower is retained thereafter, the periodicities as well as the quantitative importance of flowering vary from clone to clone, as in other tropical trees (Owens, 1991). Precocious flowering is also rarely observed in rubber seedlings (Sasikumar, 2000). Hevea shows seasonal flowering in response to alteration of the seasons. In the Northern Hemisphere, March and April is the main flowering season, and a short spell of secondary flowering prevails from August to September in many areas. It seems reasonable to presume that geographic location has a bearing on the trees to flower during the secondary season. While it flowers and sets seeds during both the seasons in Malaysia, the southern parts of India experience flowering in March and April only. In northeast India, Tripura state experiences flowering and seed set during both seasons, but the viability of seeds is largely less during the secondary season. This prompts hand pollination experiments to be centered to March and April when a substantial number of clones undergo flowering for a short span of 10–15 days. In Malaysia, too, hand pollination experiments are restricted to the main flowering season. The shift in flowering coincides with the latitudinal changes. Flower emergence occurs toward mid-February. However, toward mid-March, the emerging flowers remain apparently dormant until the onset of congenial environmental conditions. The appearance of female flowers takes 10–12 more days than male flowers (dichogamy), and due to incomplete protrandry, some of the male flowers emerge after the appearance of female flowers (Webster and Paardekooper, 1989). In Manaus and Sao Paulo (Brazil), which are located south of the equator, Hevea flowers only during August and September (Priyadarshan et al., 2001).


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The inflorescence is a branched panicle consisting of monoecious flowers that are borne in the axils of basal leaves that newly emerge after wintering. Flowers are greenish yellow, with a bell-shaped calyx having five triangular lobes but no petal. The male flowers are with 10 anthers arranged over a staminal column in rows of five each. The female flowers consists of a threecelled ovary with three short sessile stigmas. For each pistillate flower, about 70 staminate flowers are found. Hermaphrodite flowers are reported in PR107, AVROS 1328, GT 1 and Tjir 16 (Cuco and Bandel, 1994). However, such a phenomenon could not be confirmed in PR 107 grown in northeastern states of India, perhaps due to altered environmental conditions. GT 1 is a male sterile clone, and the impediment to normal process of gametogenesis has been characterized at a histological level (Leconte, 1983). Some other clones having GT 1 as female parent, such as IRCA 41 and IRCA 319, also proven to be male sterile, indicating that cytoplasmic genes might be responsible for male sterility (Nouy and Leconte, 1985, unpublished observations). BPM 24 is also reported to be male sterile in Thailand. Male sterility can be visually observed by the fact that stamens remain small and flat and produce no pollen. Hevea appears to be obligately insect pollinated (Rao, 1961) and predominantly cross pollinated (Simmonds, 1989). The strongly scented flowers of mature inflorescence attract insects (pollinators) that are mostly midges and ants of the families Heleidae and Ceratoponoidae. A consequence of nonsynchronous flowering on one site of breeding is that it is impossible to apply hand pollination for crossing early flowering clones with late-flowering clones, which is a restriction to genetic recombination between available genitors. Pollen storage, although possible (Hamzah et al. 1999), cannot be easily mastered for practical use.

B. Low fruit set Low fruit set in rubber, and its variation among clones, is a major limitation to genetic recombination in rubber breeding. It affects the number of full-sib families that can be evaluated, the size and the balance in sizes of these families, the cost of hand-pollination campaigns, and the quality of matting designs that can be established for genetic analysis. A great number of female flowers are shed soon after the period of fertilization. This predominant flower and fruit drop occurs 5–15 days following pollination (Leconte, 1983). By contrast, most of the young fruits able to initiate their growth will produce viable seeds. At the time of the maturation of fruits, a secondary shedding may occur due to Phytophthora attacks. Low fruit set is not due to natural pollination deficiency (Warmke, 1952). Pollen fertility, varying in a range from 50–98%, also does not seem to be a limitation. The development of flowers to fruits is estimated to be very low, around 5% (Husin, 1990). In fact, fruit-set success rate, assessed by controlled pollination, varies

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widely depending on the pollinated clones, from no success at all to a maximum of 5–10% for ‘‘fertile’’ clones such as PB 5/51 or PB 260. The success rate varies from year to year, with a coefficient of variation of 45% (Cle´ ment-Demange et al., 1995a).

1. Controlled pollination Maas (1919), who first experimented with controlled pollination in rubber, showed that the success rate was much higher in cross-pollination than in self-pollination. The estimation of the proportion of cross-pollination can be judged by undertaking isozyme analysis of seedlings and their female parents, as done in other tree species, where both megagametophyte (n) and embryo tissues (2n) are assayed for allozyme loci (Adams and Joly, 1990). Paiva et al. (1994) indicated cross-pollination to be 64% through isozyme studies. However, since many allozymes are produced at different stages of development, it is always reliable to use DNA analysis as a means to spell out the proportion of crosspollination. Dijkman (1951) argued that a clone like LCB 510 (PR107) is practically self-sterile and that over 3000 self-pollinations yielded only one seed, therefore indicating that there is clonal variation toward self-incompatibility. The clone PB 5/51 is heterozygous for a recessive yellow gene, and open pollination led to an estimation of 16–8% selfing (Simmonds, 1989). As a matter of fact, the self-pollination rate in open pollination is strongly assumed to be much influenced by the specific context of the female trees and of the possible neighboring cross-pollinators (monoclonal or polyclonal plots, size of the plots, etc.). By comparing eight hand-pollinated full-sib families, including two self-pollinated crosses, Leconte (1983) found no difference in the share of pollinated flowers with pollen-tubes growing from stigmas to ovules (an average of 77%). Sedgley and Attanayake (1988) confirm that there is no difference in pollen tube growth for self- or cross-pollinated flowers. These results show that low fruit set as well as poor success in selfing are not due to incompatibility between the pollen on one hand and the stigma or the style on the other hand, but are operating in the ovary at the prezygotic level (the same incompatibility alleles for the two parents) or postzygotic level (accumulation of homozygous loci in the embryo, therefore producing inbreeding effects). The rationale is that the main cause for low fruit set in rubber would be related to a major ‘‘female fertility’’ factor, with important differences between clones. Studies on the effect of environmental attributes over the fruit set during hand pollination though meager, demonstrated that fruit-set success could be negatively correlated with evaporation (Yeang et al., 1986). Maximum temperature and relative humidity of postpollinated days are also found to influence fruit set. The distribution of fruits on the floral shoots was found to conform to a negative binomial distribution that supports aggregated distribution therefore


69

indicating that some of the shoots are favored for fruit setting (Yeang and Ong, 1988). Leconte (1983) found that most of the fruits were borne by flowers issued from the buds located at the base of assimilatory leaves rather than from the buds of the scale leaves; consequently, he suggested focusing hand pollination on that type of flower.

2. Postfertilization events The probable postfertilization events are illustrated in Fig. 3.1. Micro- and mega-sporogenesis were studied to reveal developmental abnormalities that can cause low fruit set, and no such irregularities were seen in these processes (Cuco and Bandel, 1995). Gandhimathi and Yeang (1984) observed that all three ovules need to be developed for fruit setting. This result is confirmed by Sedgley and Attanayake (1988). As rubber fruits mostly have three carpels (sometimes four, and very rarely five), it must be emphasized that fruits with only one or two seeds can be very rarely observed. A further study (Hamzah et al., 2002) confirms the particularly high fertility of clone PB 5/51 (female flowers having a greater propensity for successful fruit set). This study also shows that flowers with no ovule penetrated on the one hand, as well as flowers with all three ovules penetrated on the other hand, are greatly overrepresented, which raises the notion of the ‘‘differential receptivity’’ of the flowers on one tree. Ovule abortion decreases seed production and is a crucial factor for the survival of remaining ovules. On the other hand, the abortion of ovules never precludes fruit wall formation in the early stages, indicating that the control mechanisms of these two processes are not interdependent (Sowmyalatha et al., 1997). However, due to fruit-load compensation, when the optimum number of fruits is set, the pistillate flowers arising later may not fulfill their reproductive role. Only 25% of total pistillate flowers form fruits, and of these only about 25% attain maturity. Since Hevea is an outbreeding taxon, reproductive success is expected to be low, as demonstrated in other outbreeding species (Weins et al., 1987). Selfing, or the crossing of related parents, of an outbreeding taxon could result in inbreeding depression, and since there are no conspicuous incompatibility barriers, it can very well be presumed that an inbred progeny would bring together deleterious recessive alleles largely contributing to reduced seed set. Here, the maternal parent can selectively abort genetically inferior progeny. Such preferential abortion is prevalent in many tree species (Stephenson, 1981). The abortion of fruits is seen even 80 days after pollination. The fruits attain maturity and dehisce within 145 days from the date of pollination. With such an open-pollinated system, deriving maximum permutation gene combinations will be cumbersome, thereby indicating that even the polyclonal seedlings do not represent the factual extent of heterozygosity as anticipated theoretically,

70


Figure 3.1. Probable postfertilization events behind seed maturity and germination in Hevea.

reducing the size of legitimate families from which progenies with desired attributes can be selected.

C. Apomixy The utility of apomixis, a natural phenomenon by which embryos are formed without meiosis or fertilization, needs to be explored in a cross-pollinated taxon like Hevea, since apomictically produced embryos are genetically identical to the female parent. In many ways, apomictic embryos are analogous to somatic embryos. Many latex yielding species have the capability of reproducing apomictically, and the case of guayule (Parthenium argentatum) is a fine example. While in guayule, the expression of apomixis is evident and prominent, Hevea


71

brasiliensis owes a recession. During fruit development, adventive embryos are seen to develop from the nucellus of abortive ovules (Sowmyalatha et al., 1997). Though separate genetic control mechanisms are available for sexual and apomictic embryo developments (Koltunow, 1993), the formation of adventive embryos gets stimuli from the aborting embryos. Obviously, Hevea can be a facultative apomict, where both sexual and asexual embryogenesis can be active and adventive embryony is a prevalent phenomenon in Euphorbiaceae (Bhojwani and Bhatnagar, 1992). It is noteworthy that polyploid forms of guayule are obligate apomicts and that diploids are sexually reproducing. Three pairs of genes are determined to be involved in the determination of breeding behavior. The gene a in homozygous condition leads to the formation of unreduced egg; gene b prevents fertilization; gene c stimulates an egg to develop without fertilization. Plants with a genetic makeup of aabbcc will be apomictic (Bhojwani and Bhatnagar, 1992). Since apomictic and nonapomictic biotypes are morphologically and cytologically distinguishable, the characterization of genes at the molecular level will also be possible. Adventive embryos in Hevea are seen to be degenerating, which amply indicates the presence of genes meant for apomixis, but the lack of proper activation or stimulus stands to constrain expressivity.

VII. CONVENTIONAL BREEDING METHODOLOGIES Breeding methodologies and techniques, aimed at maximizing the genetic gain, depend first on the breeding objectives. They are also strongly conditioned by the biological constraints and opportunities of the rubber tree, which determine the choice of the clones that are efficient enough to quickly integrate genetic progress and to provide the farmers with the best efficiency (formerly seedling varieties, now the current grafted clones, and, in the future, clones with clonal roots derived in vitro). Those methodologies and techniques, backed by the theory of quantitative genetics, must derive clones adapted to the available growing environments. Grafting makes possible the multiplication of the aerial part of trees identical to mother trees evolved from controlled pollination and preliminary selection. It therefore allows a high level of exploitation of the genetic variability, with the selection and replication of elite genotypes for further assessment. In the end of the selection process, grafting makes possible the organization of the mass multiplication of recommended clones in a rather short period, with the support of nurseries and budwood gardens in accordance with the needs of the planters and at reasonable cost. Conversely, this possibility induced most breeders to practice very early selection of individual genotypes,

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therefore neglecting a more ‘‘in-depth’’ analysis of the agricultural value of full-sib families issued from controlled pollination. Elements of breeding methodologies have been synthetically described in literature, with the major contributions of Dijkman (1951), Shepherd (1969), Wycherley (1969), Tan (1987), Simmonds (1989), Cle´ ment-Demange et al. (2001), and Priyadarshan (2003a). Main ideas will be recalled and discussed hereafter. The biotechnological contributions to breeding are being dealt with toward the end of this review.

A. Primary clones, natural pollination, and seed gardens Whitby (1919) was the first to report the considerable variability in the productive capacity of seedlings. Dijkman (1951) provided a detailed report on the identification of elite seedling trees and on the very intensive mass selection that led to primary clones like GT 1 and PR 107. He also gave an account of the competition between vegetative selection and generative selection between 1920 and 1950. First clones released out of seedlings were those of Cramer’s Cultuurtuin (Ct3, Ct9, Ct88), selected from 33 seedlings planted in Penang through Java in Indonesia (Dijkman, 1951). Mixed planting of these clones gave yield over 1700 kg/ha against unselected seedlings (496 kg/ha) (Tan et al., 1996). In 1924, Major Gough selected 618 seedlings from a population of about one million in the Kajang district of Malaysia, which yielded prominent primary clones like Pil A44, Pil B84, Pil B16, PB 23, PB 25, PB 86, PB 186, and Gl 1 (Tan et al., 1996). By 1930, it was understood that the primary clones had reached a plateau of yield (Tan, 1987). Hence, the emphasis shifted from primary clones to recombinants issued from controlled pollination. While recombination breeding was under way, polyclonal seed gardens were set up with improved clones in order to yield poly-cross seedlings to ensure the provision of supplementary planting materials. Thus, the best seedlings came from the Prang Besar Isolated Gardens (PBIG), Gough Gardens (GG), and Prang Besar Further Proof Trials (Tan et al., 1996). By 1970, poly-cross seedling areas extended to 7700 ha. Shepherd (1969) explained how positive the Gough Gardens were for rubber selection and how complementary the two ‘‘vegetative’’ and ‘‘generative’’ selections had been at that period. Both yield and secondary attributes were given deserving importance while selecting clones (Ho et al., 1979). Final selection was on the basis of 65% and 35% scores for yield and secondary attributes, respectively (Tan et al., 1996). The procedure involved field selection in the estates, nursery selection applied to seedlings, small-scale selection with 16 plants per genotype, and large-scale testing with 128 plants per genotype. The constitution of polyclonal seed gardens involving clones with high general combining ability (GCA) is supposed to provide panmictic conditions


73

Figure 3.2. Two-dimensional design for the production of polycross progenies (after Simmonds, 1986).

and to ensure seedlings with high genetic divergence. Selection for both vigor and high yield can be exercised in such seedlings (Simmonds, 1986). After the popularization of clones in 1950s, the potential of extending rubber to marginal areas was understood and the concept of producing polyclonal seedlings through constituting seed gardens emerged. This seems to be an appreciable option, since results on the yield of polyclonal seedlings from nontraditional areas like Tripura and Konkan (India) are encouraging (Chandrashekar et al., 2002; Sasikumar et al., 2001). There is a contention that yield and girth variation can be largely accounted for by additive genetic variance (Gilbert et al., 1973; Nga and Subramaniam, 1974; Tan, 1981). However, phenotypic selection would be effective only within the Wickham population and cannot be extended directly to new combinations, such as Wickham–Amazonian crosses. According to general genetic principles, selection based on genotypic values as reflected by GCA would be more reliable and desirable. GCA could be estimated by the evaluation of seedling progenies in order to select the best parent clones. It is here that biotechnology can contribute significantly to assess the molecular diversity of parents (see Section IX.B.1). The number of parents is an important element in determining the constitution of polyclonal seed gardens. Though gardens with more than four clones are possible, an optimum of nine clones with heteroneighbors has been suggested (Simmonds, 1986) (Fig. 3.2). The extent of selfing within seed gardens may reduce the vigor of the first generation population (SYN1), since there is no evidence of self-incompatibility. However, it can be presumed that the seeds produced are cross-pollinated, given the argument that zygotic inability reduces germination due to inbreeding (Simmonds, 1986). Such SYN1 progenies were considered as Class I planting material in Malaysia until recently. Moreover, SYN1 progenies might just be of better use in nontraditional or marginal areas. However, the provision of seeds to

74


planters by seed gardens appears hazardous nowadays in many aspects related to: (a) the agronomic performance of such synthetic varieties, especially in marginal areas where economic profitability would be more questionable; (b) the long time between the setting of the seed garden and the seed production phase; and (c) the ability of seed gardens to provide enough seeds to satisfy demand. By contrast, seed gardens can be viewed as a recombination tool for addressing the improvement of wild Amazonian populations, so recapitulating the joined ‘‘vegetative’’ and ‘‘generative’’ strategy of the first few years. With this view, a methodological study was conducted on one seed garden established in Coˆ te d’Ivoire with 50 Amazonian parents and overall 300 trees (around six grafted trees per parent). Paternity testing was done by using microsatellite markers over samples of seeds collected from identified mother trees within the seed garden (Blanc et al., 2001). The main result was that most of the paternal contribution to the progenies was due to a restricted number of male parents with substantial flowering. Consequently, this seed garden was very far from a panmictic status, and the GCA of parents could not be exploited fully. In fact, each seed garden would probably have its own characteristics and would need evaluation before its use. As biotechnology is now providing new tools able to identify the male parents of progenies, natural pollination associated with mass selection might gain new interest in the form of what was called ortet selection.

B. Derivation of recombinants and Clonal selection The basic structure of a rubber recombination breeding program is usually made of an initial phase by controlled hand pollination between selected parents for the production of full-sib families, followed by three selection stages, namely, Seedling Evaluation Trial (SET), Small-Scale Clonal Trial (SSCT), and LargeScale Clonal Trial (LSCT). SSCT and LSCT sometimes are designated as Preliminary Proof Clone Trial and Further Proof Clone Trial, respectively (in Prang Besar, Malaysia). The process is cyclical, with the best clones becoming candidates for recombination in the next cycle. From around 500 kg/ha in primary clones to more than 2500 kg/ha in the best current clones, rubber breeding has come a long way, primarily due to recombination breeding and the selection of clones such as those from the RRIM and PB series. The RRIC 100 series, released in Sri Lanka during 1970s, is yet another example. Much of the hybridization work in Malaysia (RRIM, Prang Besar), Indonesia, India, Coˆ te d’Ivoire, Brazil, Thailand, and Vietnam further strengthened the array of hybrid clones (Table 3.2). These clones are known for their adaptability to specific hydrothermal/agro-climatic situations. At least 16 primary clones played a major role and can be considered prime progenitors of many modern clones (Fig. 3.3). However, the outstanding clones are with varied genetic setup, obviously due to selection pressure applied


75

under varied conditions. Many valuable recombinants must have been lost during the course of the assortative mating of primary clones and hybrid clones, followed by the subsequent directional selection for yield under varied geo-climates (Priyadarshan, 2003a). The breeding policy has been mainly to cross ‘‘the best with the best’’ (GAM, generation-wise assortative mating), with strong emphasis on precocious yield in selection within Wickham material (Wycherley, 1976). But it could also be considered for genetic analysis and quantitative estimation procedures, especially for the assessment of clonal general combining ability for growth and latex yield improvement. Breeding for disease resistance has to take into account specific aspects related to host–pathogen interactions (see Section VIII.E).

C. Genetic analysis and variability management Economic characteristics in rubber are generally considered polygenetically controlled, which does not mean that ‘‘many’’ genes are systematically involved in one specific trait, but that most traits can be analyzed by quantitative methods. Yield in rubber analyzed according to different types of mating designs was shown to have a large additive genetic variance. Heritability and general combining abilities for yield and growth have been investigated at the RRIM and tend to be high, thus justifying GAM (Gilbert et al., 1973; Nga and Subramaniam, 1974; Simmonds, 1989; Tan, 1977, 1978, 1981). This has been true at least at the level of variability of the Wickham population so far, but it could have been altered by selection and would need to be reevaluated. It is here that the low female fertility of many parents is a limiting factor for producing every full-sib progeny, making it necessary to set new mating designs specifically suited for newly planned genetic analysis. The need for selecting highly heterozygous clones, the risk of narrowing the genetic base and of increasing the level of relatedness between clones, and the risk of developing inbreeding within clones have been underlined (Simmonds, 1989). One of the options to circumvent these requisites is to involve Amazonian germplasm in breeding programs. So far, few results about the assessment of the genetic parameters of Wickham–Amazonian crosses have been published. Those crosses appear as the best way to introgress the new germplasm into breeding populations, notably for the introduction of components of resistance to leaf diseases into productive clones. But most of the still unselected Amazonian genotypes from recent collections bear a large part of alleles unfavorable for yield (a genetic burden in heterozygous plants). Prebreeding appears necessary within the Amazonian groups before using in crossing with Wickham in an efficient way (Baudouin et al., 1997). Based on field experiments for yield, a working population of 287 accessions was extracted at the IRRDB African Germplasm Centre

Table 3.2. Profile of Prominent Clones

Clone

Parentage

RRII 105I RRII 203I RRII 208I RRIC 100M RRIM 600M RRIM 623M RRIM 712M RRIM 936M RRIM 937M RRIM 2015M PB 217M PB 235M PB 255M PB 28/59M PR 255M PR 261M IRCA 111CD IRCA 230CD RRIT 163I HAIKEN IC REYAN 8-333C BPM 24M IAN 873B

Tjir 1 Gl 1 PB 86 Mil 3/2 Mil 3/2 AVROS 255 RRIC 52 PB 83 Tjir 1 PB 86 PB 49 PB 84 RRIM 605 RRIM 71 GT 1 PR 107 PB 5/51 RRIM 703 PB 5/51 IAN 873 PB 5/51 PB 6/9 PB 5/51 PB S/78 PB 5/51 PB 32/36 Primary clone Tjir 1 PR 107 Tjir 1 PR 107 PB 5/51 RRIM 600 PB 5/51 GT 1 PB 5/51 RRIM 501 Primary clone SCATC 88-13 SCATC 217 GT 1 AVROS 1734 PB 86 FA 1717

Yield (kg/ha)

Girth increment during tapping

2210 1618 1587 1774 2199 1622 2264 2146 2483 2760 1778 2485 2283 2023 2018 1838 1446 1807 2086 1500 2187

3 4 3 3 4 4 2 3 2 4 4 3 3 1 3 3 5 5 2 3 3

1394 1920

2 4–5

Resistance to Wind damage

Panel dryness

Pink Disease

Oidium

Colletotrichum

3 3 3 5 4 2–3 5 4 5 NA 4 2 4 3 4 4 3 3 NA 4 3

5 2 3 3 4 3 4 3 3 NA 4 2 2 3 3–4 3–4 3 3 NA 3 3

5 3 NA 3 1 2–3 3 4 4 NA 2 3 2 2 3 3 NA NA NA 2 NA

3 3 3 4 3 1–2 3 3 3 4 2 2 2 2 1 1–2 NA NA 3 NA 3

3 3

3 4

3 NA

3 4

Corynespora

Phytophthora

5 NA NA 3 3 3–4 1 4 3 4 3 2 2 2 3 4 NA NA NA NA NA

5 3 NA 5 1 4 3 4 5 4 4 4 4 4 4 3 NA NA 3 NA NA

1 3 NA NA 1 1 3 2 3 3 1 3 2 2 3 3 NA NA NA NA NA

2 4

3–4 NA

4 NA

IAC 301B IAC 40 IAC 300 Fx 3864 IAN 4493 IAC 303 PB 260VN RRIC 121VN GT 1VN RRIV 4VN

RRIM 501 AVROS 1511 RRIM 608 AVROS 1279 RRIM 605 AVROS 353 PB 86 PB 38 EX 441 Tjir 1 RRIM 505 AVROS 1511 PB 5/51 PB 49 PB 28/59 IAN 873 Primary clone RRIC 110 PB 235

2320

4

4

4

NA

4

4

NA

NA

2420

4

3

3

NA

2

3

NA

2

1887

3

2

2

NA

3

2

NA

2

1755 1711 21906Y

4 3 3

3 3 3

3 2 2

NA NA NA

2 2 2

2 2 2

NA NA NA

3 2 2

169110Y 165410Y 145910Y 210310Y

4 4–5 3 2

3–4 4 5 2

3–4 4 5 4

4–5 2 2–3 2–3

4 3–4 3 2

NA NA NA NA

3–4 4 3–4 4

3 4 3 3–4

1 ¼ poor; 2 ¼ below average; 3 ¼ average; 4 ¼ good; 5 ¼ very good. NA ¼ Not available, since the disease is not prominent. Under conditions of M ¼ Malaysia; I ¼ India; C ¼ China; CD ¼ Coˆ te d’Ivoire; B ¼ Brazil. Tapping system ¼ s/2 d/2 6d/7 86%; number of tapping days per year ¼ 158 11; trees per hectare ¼ 327 34. IAN 873 exhibits good tolerance to SALB. Tapping under Vietnamese (southeast) conditions ¼ S/2 d/3 6d/7. 6 Y ¼ average over 6 years; 10 Y ¼ average over 10 years. REYAN is the new name for SCATC.

Figure 3.3. Parentage of outstanding clones.


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(Cle´ ment-Demange et al., 1998). This was done because a detailed evaluation of whole Hevea germplasm is quite impossible for preserving genetic variability. It was proposed to combine the use of field experiments and genetic molecular markers (microsatellites) to extract a clonal population of reduced size containing maximized genetic variability (Cle´ ment-Demange et al., 2001) according to the concept of ‘‘core collection’’ (Brown, 1989; Hamon et al., 1998). Managing accessions and data on different traits raises the need for the development of a Hevea germplasm database.

D. Early selection, estimation of genetic value, and development of clones Test procedures of new clones in a perennial crop such as rubber are lengthy, still extending up to 20–30 years between pollination and yield assessment, distributed over three selection stages (Tan, 1998). This justifies efforts intended to improve early selection methods in order to optimize and shorten the cycle as much as possible. One component of early selection is made of identification of traits that can be measured at a young age and are predictive enough of behavior at maturity. Another is optimization of available information and of combined management of the different selection stages in order to improve the accuracy of estimation of genetic value. The relationships between yield and parameters, including girth, height, bark thickness, latex vessel number, latex vessel and sieve tube diameters, and rubber hydrocarbon in bark and petiole were inconsistent (Gunnery, 1935). This is probably because those simple relationships were not adequate enough to explain whole-tree functioning. According to He´ non and Nicolas (1989), the thickness of the bark cannot be considered a reliable attribute to predict yield, but the number of latex vessel rings can help to differentiate poor yielders in both Amazonian and Wickham populations. But the variability of the anatomical traits of the bark appeared too narrow for undertaking selections within the Wickham population. A method was developed in China to predict yield potential through quantifying latex oozing out of leaflets or petiolules (Zhou et al., 1982). Physiological parameters such as plugging index (Ho, 1976), bursting index (Dintinger et al., 1981), photosynthetic rates (Samsuddin et al., 1987), and morphological attributes like the number of stomata (Senanayake and Samaranayake, 1970) were also used, but only plugging index and latex vessel number showed consistent and significant correlations with yield (Huang et al., 1981). On the other hand, correlations between yield of 2- to 3-year-old budded plants and mature yield could be demonstrated (Ho, 1976). Gonc˛ alves et al. (1998a,b) analyzed selection for growth of grafted clones at different ages in order to determine the younger possible age for efficient selection.

80


The first stage of selection is normally applied to seedlings (Fernando and De Silva, 1971) that are full-sib progenies borne out of hand pollination and are evaluated in a ‘‘nursery’’ (by a seedling evaluation trial). Information from this stage is used for selecting new clones to be evaluated as grafted trees in a small-scale clone trial (SSCT). Gnagne et al. (1998) investigated different procedures for assessing yield on young seedlings at the nursery stage, such as the Mendes test or the Hammaker–Morris–Mann test (Dijkman, 1951), and analyzed the relationships (linear correlations) between SET and SSCT in order to determine some efficient selection rates at the SET stage (Gnagne et al., 1998). It was found that yield measured at SET stage can be a predictor of mature yield of grafted clones evaluated at the SSCT stage (with a moderate prediction ability), but growth before tapping appeared not acceptable as a predictor of growth or yield at the SSCT stage. Tan (1998) confirmed those results and recommended the use of mainly nursery yield as a yield predictor and to apply a mild selection at the first nursery stage. The combination of two early stages of selection (seedlings in SET and grafted clones in SSCT) can be viewed only at a statistical level, based on correlations between the two stages for a set of genotypes. In this framework, Simmonds (1985) presented a theoretical approach in order to assist decision about the selection thresholds that can be applied at the first stage. But the seedlings in SET usually have the property of belonging to different full-sib families. Based on the theory of quantitative genetics, Jayasekera and Hettiarachchi (1988) underlined the importance of taking into account the ‘‘family’’ value of the seedlings under selection, and the rightness of ‘‘family selection’’ was theoretically confirmed by Simmonds (1986). Similarly, the case of nursery selection and the relationships between the two stages of early selection in rubber were examined by Gnagne et al. (1998) in order to have the best estimation of the genetic value of genotypes under selection. A combined family individual selection was proposed in the form of a linear combination of family value and individual values. At the nursery stage, with only one seedling tree per genotype, it will almost be impossible to directly assess the environmental effect, therefore limiting the predictive efficiency of this first stage. Nursery selection could even be limited to a selection of the best families only. With this view, early selection does not aim priority at shortening the cycle but at improving the selection efficiency. Ong et al. (1986) proposed a modified selection scheme based on promotion plot clone trials, combining SSCT and LSCT at the same stage in order to reduce the time between hand pollination and the release of new clones. As early selection is based on measurement of latex yield, this process introduces the risk to select clones only with greater early yield. That such a clone was characterized by active metabolism and low level of sucrose in laticifers was estimated by measuring inorganic phosphorus and sucrose ratios in the latex (Jacob et al., 1995). This type of latex diagnosis applied to early

81


selection of rubber, and a procedure was developed satisfactorily at the level of SSCT at CIRAD and CNRA (Gnagne et al., 1998). The structure and expression of laticifer-specific genes need to be studied in detail in order to detect molecular markers having correlation with yield or other traits. Since molecular genetic markers are independent of the environment, using them as predictors can contribute to improve the accuracy of genetic value assessment according to the concept of marker-assisted selection, or MAS (Lynch and Walsh, 1998). This technique needs refinement toward the operational level (Fig. 3.4). The third stage of selection is the Large-Scale Clonal Trial (LSCT), involving evaluation of individual genotypes in small sets, on rather large plots, over a long period. These trials provide the planters and the breeders with a wide range of characteristics about the behavior of the clones in one or many different locations. Rubber-producing countries have developed their own procedures for the recommendation and dissemination of new clones. Until 1995, the threeyearly ‘‘Enviromax Planting Recommendations’’ of RRIM were geared by the approach formalized by Ho et al. (1979) and periodically published in issues of the RRIM Planters’ Bulletin. Within each producing country, direct contacts are established for clonal testing and for communication about clones between breeders and planters or extension services. Even if the selection process officially ends with the LSCT, the study of clones continues long after recommendations, in relation to wind damage, tapping panel dryness, or other biotic or abiotic stresses, and also to assess their adaptation to specific tapping systems or farming systems. As a tree and a perennial crop, rubber exhibits new aspects and differences between clones over longer periods.

VIII. BREEDING AGAINST STRESSES A. Nontraditional environments

The traditional rubber-growing tracts extend to 10 north and south of equator, and these areas offer environmental conditions ideal for rubber cropping (Pushparajah, 1983). They are: (a) 2000 to 4000-mm rainfall distributed over 100–150 days per annum (Watson, 1989); (b) a mean annual temperature of around 28 2 C, with a diurnal variation of about 7 C (Barry and Chorley, 1976), and (c) sunshine hours of about 2000 per year at the rate of 6 per day in all months (Ong et al., 1998; Pushparajah, 1977; Yew, 1982). In a study with hydrothermal index, Rao et al. (1993) rationalized Senai of Malaysia (1 360 N; 0 103 39 E) to be the most suitable area for rubber cultivation and production. The increased global demand for rubber, and the extension in cultivation of other agricultural crops, prompted countries outside the hitherto traditional

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Figure 3.4. Scheme for the derivation of clones (modified from Priyadarshan, 2003a).

zone to focus their attention on the cultivation of rubber. Such a tendency often extended rubber to suboptimal environments. Specific areas of China, Thailand, Vietnam, India, Coˆ te d’Ivoire, and the southern plateau of Brazil fall under nontraditional zones that experience one or more stress situations, namely, drought, low temperature, high altitude, diseases, and strong winds. The annual mean temperature decreases when moving away from the equator, with more prominent winter conditions from November to January. Northeastern states of India and China lying between 18 and 24 N are regions well recognized as inhospitable for the crop, exhibiting stress situations like low temperatures and typhoons (Zongdao and Yanqing, 1992). It may also be worthwhile to note that rubber areas of China and Tripura fall under the same latitude range, though climatic conditions in vivid pockets of China vary,


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since China’s tropical and subtropical regions are undulating and diversified (Priyadarshan, 2003a; Priyadarshan and Gonc˛ alves, 2003). Four main climatic zones are prominent in Brazil, namely, the Amazonian basin, Brazilian plateau, coast lands within the tropics, and southern states. Rubber inhabits all areas except the southern states. The southern Brazilian plateau (450–500 m MSL), especially Sao Paulo, is undergoing experiments for rubber cultivation. This extension to areas seasonally affected by dry and cold conditions is mostly motivated in Brazil to escape the climatic conditions congenial to SALB. These areas, apart from high altitude, offer high rainfall that often exceeds the basic requirements. A geo-climatic comparison of various environments in Tripura, China, Brazil, Coˆ te d’Ivoire, Indonesia, Vietnam, and Thailand would amply reveal a spectrum of climatic conditions over which rubber is grown (Table 3.3). In India, marginal areas delineated as nontraditional zones, spread over the states of Maharashtra, Orissa, Tripura, Assam, West Bengal, Meghalaya, and Mizoram, pose a multitude of hazards namely, moisture stress, low temperature, wind, high altitude, and disease epidemics, apart from altered soil physical properties. Nevertheless, even if breeding can be efficiently involved, adaptation of rubber cropping to nontraditional environments will not be able to overcome growth performances and latex yields compared with favorable zones. Therefore this issue is related to socioeconomic conditions (e.g., rubber price, cost of labor, availability of land). Moreover, adaptation to new areas needs more ecophysiological research with a better evaluation of existing clones in various environments rather than the creation of new clones that can be considered only through a long-term perspective.

B. Abiotic stress factors In China, two types of cold damage (chilling injury) have been identified namely, radiative and advective (Zongdao and Xuequin, 1983). In the radiative type, the night temperature falls sharply to 5 C, whereas the day temperature ranges between 15 and 20 C or above; in the advective type, the daily mean temperature remains below 8–10 C, with a daily minimum of 5 C. In both of these types, under extreme circumstances, the complete death of the plant is the ultimate outcome. An analogous atmosphere prevails in northeastern states of India also. Reports from China point out that while clones GT 1 and Haiken 1 can withstand temperatures as low as 0 C for a short span, SCATC 93–114 can endure temperatures as low as 1 C. The cold-wave conditions in Tripura state (in northeast India) can be conveniently classified as relating to the radiative type. Chinese clones like Haiken 1, SCATC 88–13, and SCATC 93–114 are being evaluated in Tripura. The initial yielding pattern shows Haiken 1 to be a high yielder among Chinese clones, as compared with RRIM 600, which is

Table 3.3. Spectrum of Weather Variables Under Different Geo-climates

Attributes

Temperature ( C) (mean) Daily temperature range ( C) Relative humidity (%) Sunshine (% h) Wind run (m/s) Rainfall (mm/year) Number of rainy days Moisture availability index Penman ETo (mm/day) Latitude Longitude Altitude (m)

Bogor (Indonesia)*

Pindorama (Sao Paulo, Brazil)**

Kourou (French Guyana)*

Odienne (Coˆ te d’Ivoire)**

Nong Khai (Thailand)**

Hainan (China)**

Agartala (Tripura, India)**

Senai (Malaysia)*

Dak Lak (Vietnam)**

27.4

22.9

26.3

25.6

26.8

22.6

25.4

26.9

21.5

9.1

11.8

7.8

12.7

10.2

7.8

9.9

7.2

7.9

79 61 2.4 1791.5 159 0.78

67 55.1 1.6 1117.6 117 0.49

81.5 49.9 1.35 2573.53 193 1.4

67 59.2 1.3 1297.9 119 0.67

74 58.1 1.2 1455.96 128 0.7

79.9 46.8 2.7 1431.29 151 0.6

76.8 50.8 1.38 1960.1 93 1.1

82.3 47.8 2.1 2282.2 182 1.2

75.7 48.8 2.5 1669.31 163 0.8

4.4 5 90 S 106 580 E 16

3.87 20 250 S 49 590 W 505

3.78 5 70 N 52 560 W 48

4.3 9 300 N 7 340 W 451

3.97 17 510 N 102 440 E 164

3.48 19 20 N 109 300 E 671

Source: www.iwmi.org * Traditional; **nontraditional, Senai (Malaysia) is considered as the area offering the optimum environment.

3.39 23 490 N 91 160 E 31

3.9 1 360 N 103 390 E 13

3.57 14 550 N 108 100 E 655


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used as a local check. Though SCATC 93–114 is proclaimed as cold endurant under Chinese conditions, it never shows considerable yield potential under the conditions of Tripura, at least during the initial stages on B0–1 panel (Priyadarshan et al., 1998a,b). China has also developed the clone Zhanshi 86, which is more cold endurant than SCATC 93–114; this clone is issued from seedlings out of a random cross between SCATC 93–114 and Wuxing I3 (Senyuan, 1990) (see Table 3.2). Further, clones like Zhanshi 306–15 (RRIM 600 Guangxi 6–68) give around 10 kg of dry rubber per tree, but these contentions are to be proven at the block level. IAN 873, an SALB-resistant high-yielding clone developed in Brazil, shows resistance to cold weather in China (Senyuan, 1990). The areas between 15–20 N of western and eastern India have been identified as nontraditional zones for rubber cultivation. For instance, the Konkan region experiences long dry periods, high temperatures, low atmospheric humidity and zero rainfall between September and May, daytime tempera tures range between 38 and 41 C during the summer months, with occasional days getting as hot as 47 C, though the region gets rainfall of 2430 mm, the distribution is uneven (Devakumar et al., 1998). The atmosphere during summer results in a high-vapor-pressure deficit. Almost an analogous situation prevails in the eastern part of India. Wind is yet another abiotic stress influencing the establishment and growth of rubber. One impact is a contribution to the drying effect of drought conditions, especially in regions of long-lasting steady winds such as during the dry season in the highlands of Vietnam. It is argued that wind speeds of 2.0–2.9 m/s retard rubber growth and latex flow and that speeds of 3.0 m/s and above severely inhibit normal growth. Winds over a Beaufort force of 10 (more than 24.5 m/s) play havoc with branch breaks, trunk snaps, and the uprooting of trees, mainly prevalent in China from June to October (Watson, 1989). Studies in China revealed that clones PR 107 and Haiken 1 can be wind enduring, and PB 5/51 is wind enduring in Tripura (Priyadarshan, 2003a). The establishment of shelter belts, consisting of fast-growing and wind-resistant species, is one remedial measure being followed in China (Zongdao and Xuequin, 1983). But this needs proof, taking into account their effects on rubber standability as well as the economic cost of their implementation and land occupation. Alternatively, adoption of the judicial pruning of branches and the induction of branches at a lower height can reduce wind damage from 25.3–13.7% (Zongdao and Xuequin, 1983). In Coˆ te d’Ivoire, rubber plantations often experience wind damage due to storms occurring at the onset of the rainy season (April–May). Though hypotheses have been made on the relationship between architecture and the wind susceptibility of the clones, no clear factual validation is available (Combe and du Plessix, 1974). By comparison with nontapped trees (Clement-Demange et al., 1995b), tapping is heavily limiting growth rate, to some extent in height but

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mainly at the level of trunk girth increment. Following these observations, a large-scale design comparing two levels of standard girth (50 and 65 cm) for the initiation of tapping was conducted in Coˆ te d’Ivoire. Although the bigger size of the trunk improves the stiffness of the tapped trees, opening after the attainment of 65 cm delays the productive period.

C. Phenology under differential geo-climates Hevea exhibits significant changes in phenology under different geo-climates. The shift in cultivation north and south of the equator induces ample phenological changes. A comparison is made in relation to Tripura (in northeastern India) and Sao Paulo (in southern Brazil). Rubber in Tripura experiences wintering from December to January, and reflushing commences by February, followed by flowering (Priyadarshan et al., 2001). The yielding pattern of clones in Tripura shows a clear delineation of low- and high-yielding regimes (Priyadarshan et al., 1998a). There is a multitude of factors that induce a lowyielding period, normally low temperature, utilization of carbohydrate reserves for refoliation, flowering, fruit development from February to April, a low moisture period from February to March, and powdery mildew (Oidium heveae Stein.) infestation. These factors together impose a low yielding period from May to September (Priyadarshan et al., 2000). A photoperiodic stimulus, operating because of a slight change in the proportion of light to dark in day length, is sufficient to trigger flowering and has been very well established in several tropical tree species (Baker et al., 1983). However, the fall in temperature from October to November stimulates yield, while the mean temperature is 28 C, making the atmosphere most ideal for latex flow and production. The minimum temperature experienced early in the morning is 15–18 C, and after 10 a.m., the temperature rises to 27–28 C. While the former is congenial for latex flow, the latter is ideal for latex regeneration through accumulation (Ong et al., 1998). The plateau region of Sao Paulo state, an escape area for SALB, has been referred to as the most important rubber-growing region of Brazil (Costa et al., 2000). While Tripura lies at 22–24 N, Sao Paulo is at 20–22 S (400–500 m MSL), making these areas nontraditional. Unlike Tripura, trees are exploited throughout the year in Sao Paulo. Reflushing, flowering, and seed fall are experienced once a year in Sao Paulo (Ortolani et al., 1998). Plants react to different environments through phenological changes. For instance, defoliation is a phenomenon to circumvent moisture and low temperature stresses by minimizing transpiration so as to ensure reproduction, seed dispersal, and the perpetuation of generations. Flowering and fruit formation utilize a large amount of carbohydrate reserves. Hence, flowering and fruit formation precede a low yielding phase in rubber, both in Tripura and Sao


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Paulo. The environmental conditions inducing defoliation, flowering, and low- and high-yielding periods are analogous. The peak yielding period in Sao Paulo is from January to May, followed by winter and defoliation, while in Tripura from January to May is the low-yielding period. The latitudinal changes north and south of equator induce phenological changes in Hevea in vice versa fashion. It is noteworthy that Sao Paulo is a disease-free zone for SALB. Similarly, Tripura experiences only Oidium heveae, but the traditional regions experience at least two other diseases (Corynespora and Phytophthora). It may be pertinent to believe that low temperatures and dry spells extend an antagonistic effect over the environment to reduce disease incidence in a particular zone. Moreover, the nonexistence of alternate hosts would be another reason for a disease-free atmosphere. Since the peak yielding periods both north and south of equator are in a vice versa fashion, it is evident that rainfall and the minimum temperature in a region are the major factors influencing yield and the phenology of rubber. It has also known that the dry period increases and mean temperature decreases when one moves away from the equator. Minimum tem peratures in the range of 15–20 C influence the yield north and south of the equator. The effect of rainfall assumes significance in increasing yield only after 2 months. The differential phenology in Hevea on both sides of the equator might be advantageous for shortening the testing cycle. For instance, Sumatra (in Indonesia) has two conspicuous flowering seasons, since the equator passes through its center (Priyadarshan et al., 2001). This needs to be exploited fully by breeders.

D. G E interactions and specific adaptation It is imperative that there are low- and high-yielding periods in nontraditional areas. This is evident from the analysis of yielding trends in Tripura (India), Vietnam, and Sao Paulo (Fig. 3.5). Under the hydrothermal situations of Tripura, in a study involving 15 clones of vivid geographical origin, all clones show an increment in yield toward the onset of the cold season—from October to November. It is implicit that cold weather (18–20 C) is very favorable for latex flow by pushing the coagulation time to a later period of the day; the onset of the cold season renders a stimulatory effect to maximize yield, and the trend continues until the temperature falls below 15 C, during January. The clones are classified under two categories: (a) one showing a slow escalation in yield from April onward, reaching the maximum during November and receding sharply during December and January, and (b) a low-yield from April to October, with the peak yield during November and December (high-yield regime), and then receding during January. In Sao Paulo (in the Southern Hemisphere), the low- and high-yield regimes are reversed. While PB 235 comes

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Figure 3.5. Contribution toward yield in GT 1 and PB 235 over months in Vietnam (highlands), India (Tripura), and Brazil (Sao Paulo).


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under the first category, all the other clones come under the second. The trend shows that the first category is appreciable, since the clones give considerable yield during regime I, which ensures better returns to the planter. The rationale is that the fall in temperature, along with reduced evaporation and low wind speeds, prevail on the microenvironment to influence yield stimulation from October to December (Priyadarshan, 2003b). PB 235 seems to own an adaptive mechanism, whereby it yields higher when the ambient temperature ranges between 22 C and 28 C. When all clones continue with a higher yield in combination with descending temperature, PB 235 recedes yield during January, when the ambient temperature drops below 18 C. Evidently, the existence of genetic homeostasis and their subsequent expression in the changed environment might be the reason for the near uniform yield in these clones. In Sao Paulo, RRIM 526 showed higher yield during low regime in comparison with RRIM 600, RRIM 614, and AVROS 1328 (Gonc˛ alves, I.A.C. personal communication). These observations clearly rationalize the selection to be in favor of consistent yield (Priyadarshan et al., 2000). The weather variables and environmental index were used as covariates while analyzing the yield data to determine the variable that contributed to heterogeneity in the GE interactions. The test of heterogeneity for the environmental index showed high significance, so indicating that the high stability values of a few clones (s2 i) over the years were due to the linear effect of the environment (Priyadarshan, 2003b). However, under Malaysian conditions, Tan (1995) accounted GE interactions with a nonlinear effect of wind damage and disease. In fact, these hazards play a prominent role in differentiating the adaptation of clones to one or another location. The grouping of clones with a high mean and low coefficient of variation is proven to be dependable in selecting better performers in a new environment (Priyadarshan et al., 2002; Tan, 1995). GE interactions were also significant for rubber production and girth increment under the conditions of Sao Paulo (Costa et al., 2000; Gonc˛ alves et al., 1998a,b; 2003). This need to identify the specific adaptation of clones to the diversity of rubber planting tracts might lead to the emphasis of ecophysiological research, which can provide functional and predictive models for characterizing those adaptations.

E. Resistance to leaf diseases South American leaf blight (SALB) has played and still plays a major role in the history and the geographic distribution of the rubber industry, as on one hand it prevents Latin America from developing rubber cropping in all the otherwise favorable climatic conditions, and on the other hand it represents a permanent major threat to the crop in Asia and Africa (Davies, 1997; Dean, 1987). Some breeding work, mainly based on back-cross technique, has been undertaken in

90


the past to incorporate resistance to these diseases in high-yielding clones. However, the efforts were in vain due to the unknown polygenic nature of the attributes, high variability of the pathogen, and multiple interactions between fungus strains and rubber clones (Rivano, 1997a,b). Simmonds (1990, 1991) argued that the pathotype-specific resistance (vertical resistance-VR) has resulted in catastrophic failures. Horizontal resistance (HR) should be more effective and durable (Rivano et al., 1989; Simmonds, 1990). Resistance sources appear to be low in a high-yielding Wickham population but rather frequent within the Amazonian germplasm. However, the wild population is yet to be improved for yield. With these views, efforts have been reoriented toward the analysis of partial resistance components (Junqueira et al., 1990). Recently, the genetic determinism of the resistance source of H. benthamiana (F 4542), widely used in many former back-cross programs, has been characterized by a genetic map (Lespinasse et al., 2000b). A Cirad–Michelin common research and breeding program is currently carried out in Brazil to reduce the incidence of SALB on rubber cropping. Clones with an early refoliation, such as AVROS 2037, RRIC 100, RRIM 600, or PB 260, can develop their new leaves before the onset of the rainy season and therefore are able to escape the incidence of Colletotrichum goleosporioides. By contrast, the widely planted clone GT 1, which has a late defoliation, has been seriously affected in many areas of Malaysia, Indonesia (Kalimantan), and central Africa. The consequence of early defoliation was successfully used for the development and implementation of early artificial defoliation by Ethephon aerial spraying over the rubber crops in Cameroon and Gabon for escaping from Colletotrichum (Se´ ne´ chal, 1986). But from a breeding point of view, many other resistance genetic components might be also exploited for the creation of resistant or tolerant clones. Corynespora leaf fall disease (CLFD) has become a major threat for rubber cropping in southeast Asia and western Africa. An escape strategy related to early defoliating clones or by way of artificial defoliation is not operative. It was demonstrated that the fungus is acting by the emission of a toxin (cassiicoline) in the leaves (Onesirosan et al., 1975). Studies conducted under controlled conditions have not put evidence of a significant interaction between clones and strains (Breton et al., 2000). These laboratory studies demonstrated GT 1 to be tolerant and PB 260 and RRIC 100 to be highly susceptible. But, by contrast, RRIC 100 displays a high level of tolerance at field level, whereas the field susceptibility of clones like PB 260, IRCA 18, and IRCA 230 is confirmed (Gohet and Cle´ ment-Demange, personal communication). Clonal susceptibility to Oidium heveae has been investigated in India (Rajalakshmy et al., 1997). This disease seems to be favored by rather cold conditions prevalent toward the onset of refoliation. In a comparative study


91

with clones of various geographic origins, SCATC 93-114, Haiken 1, and RRIM 703 were judged resistant to Qidium in the traditional areas of India (Alice et al., 2000; Rajalakshmy et al., 1997). While studying sensitivity relationships between clones, Alice et al. (2001) confirmed these results and marked SCATC 93-114, RRIM 703, Haiken 1, RRII 208, RRII 5, and PB 310 as stable sources of resistance over the years. Other diseases may deserve the attention of the breeders. As a matter of fact, many observations related to the evolution of clonal susceptibilities to diseases are collected in different places but are left unpublished and scattered. A centralization of all these observations would be very useful for all researchers and for the rubber industry.

IX. BIOTECHNOLOGIES The attainment of yield plateau and prevalent intraclonal variations in yield prompted researchers to neutralize these problems by employing modern tools of biotechnology. However, yield alone would not encourage the cultivation of Hevea, since the species is sensitive to biotic and environmental attributes and physiological disorders. A long breeding cycle and larger size of crop also make breeding time-consuming. Biotechnology applied to Hevea can be discussed under two categories namely, in vitro culture and molecular breeding. While in vitro culture deals mainly with regeneration and propagation, molecular breeding extends the provision for the identification, characterization, introduction, and expression of novel genes.

A. In Vitro culture Experimentation with in vitro culture of rubber commenced during the 1960s, with Chua (1966) attempting derivation of callus from plumule tissues of seedlings. The effects of osmotic concentration, carbohydrates, and pH of culture media were also studied. Later, the Rubber Research Institute of Malaysia took the initiative of undertaking large-scale tissue culture work by maintaining callus cultures from various explants (Paranjothy and Gandhimathi, 1976). It later expanded to somatic embryogenesis and micropropagation through stem explants. While anther culture was employed to achieve pure lines first and exploitation of heterosis thereon, micropropagation and somatic embryogeny were worked out to have homogeneous populations. Though research on in vitro culture commenced nearly 35 years ago, even after rigorous experimentations these areas are still infant due to shortcomings toward commercial applicability. This section is devoted to discussing such aspects in some detail.

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1. Anther culture The Rubber Research Institute of Ceylon (RRIC) was the first to carry out culture of anthers to raise haploid plants (Satchuthananthavale and Irugalbandara, 1972). However, first plants from Hevea pollen were made available during 1977 at the Baoting Institute of Tropical Crops, in Hainan, China (Chen et al., 1979). Since then, at least four laboratories in China took the lead in researching production of haploid plants in vitro (Carron et al., 1989). In addition, attempts were made to produce plants through gynogenesis (Guo et al., 1982; Yang and Fu, 1997). Carron et al. (1989) enumerated three phases for the production of haploids from anther culture. In the first phase, the production of callus and embryos takes nearly 50 days. Here, the media formulation is vital, since the balance between callus development and the initiation of embryos needs to be maintained. The modified MB medium (Chen, 1984) is widely used, with the addition of NAA and coconut water, which regulate the development of microspores, and a judicial concentration of sources of nitrogen, potassium, and sugar makes the production of calli and embryos. The somatic callus then degenerates, and the embryos develop from microspores. At this stage, subculture must be carried out into a differentiation medium in order to avoid the degeneration of embryos (Chen et al., 1982). The maturity of embryos is the crucial factor in the second phase. The cultures need 2–3 months for the apical bud to develop. Coconut water at this stage will be substituted with gibberellic acid (GA3) for the better development of cotyledons. In the third phase, the progressive increment of GA3, gradual withdrawal of other growth regulators, addition of 5-bromouracil, and reduction of sugar will result in the development of plants from embryos. The cytological investigations of callus, embryos, and plantlets showed mixoploidy (Qin et al., 1979). However, when the plants develop in vitro, there is a progressive tendency toward diploidy (Carron et al., 1989). Above all, the developmental stage of anther is vital for correct results. The anthers from male flowers that have yellow corolla should not be selected, for the microspores will be in the binucleate stage. Such anthers will repress callus and embryogenesis. Only uninucleate pollen are ideal for haplogenesis, which can be obtained from greenish-yellow flowers (Chen, 1984; Shije et al., 1990).

2. Somatic embryogenesis and meristem culture Anther wall was used to achieve somatic embryogenesis in the earlier stages. The first plants raised from somatic embryogeny were obtained by Wang of the Rubber Research Institute of Baodao, China, and by Paranjothy of the Rubber Research Institute of Malaysia (Carron et al., 1989). Later, the inner integument that represents mother tissue was used to produce somatic embryos by the


93

CIRAD in France (Carron and Enjalric, 1982). For inner integument culture, 45- to 75-day-old seeds are subjected to surface disinfection (Carron and Enjalric, 1982) followed by four successive phases, namely, callogenesis, differentiation, multiplication of embryoids, and development of embryoids into plantlets (Carron et al., 1984). The judicious combination of 2,4-D, IAA, and benzylaminopurine (BAP), and an increase in sucrose concentration promotes callogenesis in the darkroom culture condition. Cultures are then taken into the light with a changed macro-element composition to increase tissue proliferation (Carron et al., 1989). The differentiation medium is enriched with naphthoxyacetic acid (NOA) and BAP with a low-sucrose concentration. It takes 5–6 months in this medium for the embryos to develop. Carron et al. (1989) claim that nearly 3000 globular or lanceolate embryoids could be achieved in a period of 4 months after the initiation of embryoids. For the plantlet development phase, the addition of indolebutyric acid (IBA) is crucial. These conditions promote both root and cotyledon formation. Successful plantlet formation and acclimatization have been achieved in Haiken 1, Haiken 2, and SCATC 88/13 (Wang et al., 1980). Anther wall requires 2,4-D, NAA, and Kinetin (KN) for both callogenesis and embryo induction. BAP and Zeatin are essential, in addition to NAA and 2,4-D. GA3 is found to increase the number of embryoids. BAP and GA3, together with lower sucrose level, are shown to improve plant regeneration. Sushamakumari et al. (2000) and Carron et al. (1998) gave a detailed account of the procedure and media formulation for somatic embryogenesis in Hevea. The aforesaid procedures experience strong genotype–medium interactions (El Hadrami et al., 1991; Montoro et al., 1993). Tissue–medium interactions are also very prominent. This is evident from a culture of integument tissue of immature seeds where an entirely different additive of 234 mM sucrose, 9 mM BAP, and 2,4-D, were needed for embryogenesis. Abscisic acid (ABA) was essential for embryo development (Etienne et al., 1993; Veisseire et al., 1994a,b). The maturation phase takes 25 days. Low germination percentage and plant conversion are seen as setbacks in this procedure, since the mechanism involved in this technology is poorly understood (Cailloux et al., 1996; Linossier et al., 1997). For instance, initiation and germination of embryos are seen to progress at a higher temperature of 24–27 C (Wang and Chen, 1995; Wang et al., 1998). Polyethylene glycol (PEG) and high CaCl2 were seen to stimulate embryo production (Etienne et al., 1997a; Linossier et al., 1997). Thus, the clone–tissue–media interactions prevail in this technology, which necessitates extensive basic studies. More recently, Etienne et al. (1997b) standardized a pulsed-air temporary immersion system for enhancing embryo production by culturing embryogenic callus under immersion in an autoclavable filtration unit RITA. Somatic embryo production was three to four times greater than that for embryos on a semisolid medium, to the tune of 400 embryos/g fresh weight, which also reduced the number of abnormal embryos. Thirteen thousand embryo-derived plants have been planted for field trials near

94


Bangkok in Thailand (RRIT) and at CNRA in Coˆ te d’Ivoire (Carron et al., 1995b). Clones PR 107 and PB 260 were highly regenerative. This is a leap toward regeneration of Hevea in vitro, since higher regeneration should be ensured so as to have a rapid gene-transfer system in Hevea. Meristem culture follows three phases, namely, primary culture, multiplication, and rooting and acclimatization. The selection of explants is very crucial. Indeed, very juvenile stem pieces have exhibited a higher rate of aseptic cultures. Culture treatments are rather rigorous due to higher anticipated infection by fungi and bacteria. A mixture of gentamycin, kanamycin, chlortetracycline, chloramphenicol, rifampicin, and the fungicide benomyl is found to be ideal for disinfecting the explants. The primary culture involves soaking explants in a solution of growth regulators (IBA and BAP) for 2–3 h. Budding is initiated in MB medium (Carron et al., 1989) without growth regulators. Isolated buds are cultured in a half strengh of Lepoivre medium with IBA and BAP. These buds are subcultured to form microshoots that will in turn be cultured as explants in the multiplication phase. The soaking base of the root in an IBA–NAA mixture for 3–4 days induces roots. Rooted microcuttings can be transferred to soil in 4–5 weeks. A number of clones, such as RRII 105, PB 5/ 51, PB 235, IRCA 438, IRCA 440, IRCA 442, PR 107, and GT 1, have been multiplied through micropropagation (Carron et al., 1995a). However, the acclimatization of plants is very crucial, with a balance between relative humidity and temperature governing the establishment of plants in the soil (Leconte and Carron, 1988). The commercial value of the aforesaid procedures needs to be debated. Though gross experimentation was conducted in the past for standardizing in vitro technologies, there had been many setbacks in commercializing these procedures (Carron et al., 1992; Thulaseedharan et al., 2000). A number of aspects inherent in the explant tissue, namely, release of phenols, contamination of bacteria and fungi, recalcitrant status, reduced axillary branches, lack of sufficient juvenility, and, above all, increased sensitivity of in vitro raised plantlets toward environmental attributes are responsible for the delay in commercialization. There are, however, remedial measures for these setbacks. Since the contamination of micro-organisms is location specific, newer chemicals are to be tried to raise aseptic cultures. Instead of treating the explants with antioxidants, the incorporation of them in the media decreased browning (Seneviratne and Wijesekara, 1996). The use of support systems like cellulose plugs in liquid media reduced the synthesis of polyphenols, and embryogenesis activity could be maintained for more than 200 days (Housti et al., 1992). On the other hand, the growth regulators used to induce axillary branches and somatic embryogenesis are more or less the same throughout. A judicious combination of new growth regulators that have shown positive results in other tree species can be tried in rubber. Also, metabolism of ethylene and polyamines during callus development


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must be controlled by appropriate adjustment of growth regulators (Carron et al., 1992). More prominently, water status of embryogenic callus is a governing factor to enhance embryogenesis (Etienne et al., 1991). Further, Lardet et al. (1999) demonstrated that protein and starch accumulation commenced from the thirteenth and fifteenth week, respectively, and the development and maturity of zygotic embryos happens accordingly. Somatic embryos also display the same accumulation abilities. However, the smaller size of somatic embryos can accomplish a relatively small mass of starch and protein reserves. This is related to lower vigor and conversion rates. The vigor is directly related to acclimatization success. Hence, the increasing the size of somatic embryos through nutrient supplies must get deserving priority. Techniques like air layering to achieve rooted cuttings and progression of this conventional multiplication for 3–4 generations can provide juvenile plants. They can be used as source plants for explants with increased juvenility in vitro. Increased turgidity and increased absorption of N, P, and K are prerequisites for embryo induction. Juvenility is yet another crucial factor, wherein the successful micropropagation of mature stem apices micrografted onto 3-week-old seedlings grown in vitro increased the success of micropropagation (Perrin et al., 1994). Such basic studies must be conducted in meristem culture also, in order to implement these technologies in a commercial way. If commercialized in the strict sense, these technologies can assist breeding programs and enhance productivity significantly.

B. Molecular genetics An analysis of the association between DNA sequence variation and heritable attributes can help to exploit variations in plants at the molecular level. As conventional identification and utilization of recombinants with desirable traits is time-consuming and laborious in rubber due to long generation time and the larger size of the crop, new tools could be developed in order to manage germplasm variability and to assist breeders in their recombination strategies. Development of DNA markers allows to implement gene mapping routinely. The molecular marker systems can be broadly classified into three, namely, first generation (Restriction Fragment-Length Polymorphisms [RFLPs], RAPDs, and modifications); second generation, mainly based on targeted PCR techniques with simple sequence repeats (SSRs) or microsatellites, amplified fragment length polymorphism (AFLPs) and their modifications; and third-generation markers such as expressed sequence tags (ESTs) and single nucleotide polymorphism (SNPs) (Gupta et al., 2001). All marker systems, except SNPs, have been applied in Hevea so far. This section deals with aspects of molecular breeding like molecular diversity, gene linkage maps, QTLs (Quantitative Trait Loci), laticifer-specific gene expression, and direct gene transfer.

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1. Molecular diversity Agro-morphological traits, mainly from leaf morphology, have been considered as genetic markers and proposed for describing and identifying clones (Dijkman, 1951). Such traits were submitted to the Union pour la Protection des Obtensions Ve´ ge´ tales (UPOV) for examination as rubber identification markers, but their main drawback is their high sensitivity to environmental effects. Analysis of isozymes that are proteic genetic markers were developed at CIRAD, with 13 polymorphic isozymic systems to formulate a diagnostic kit for clonal identification associated with a clonal identification database. This kit proved to be able to differentiate a large set of cultivated clones (Leconte et al., 1994). However, the analyses need to be carried out near the field sites due to the fragility of isozymes to varied temperatures, or else the samples need to be freezedried and transported to the laboratory. Besse et al. (1993b) demonstrated that fingerprinting through RFLP minisatellite probes could be more powerful, and identification of 73 Wickham clones could be done with 13 probes associated with restriction enzyme Eco RI. RFLPs and DAFs were also used for identification of progeny with two common parents such as PR 255 and PR 261; RRIM 901 and RRIM 905; RRIM 937 and RRIM 938 (Low et al., 1996). Further, Besse et al. (1994), using 92 Amazonian and 73 Wickham clones, did an assessment of RFLP profiles. Interestingly, Amazonian accessions could be categorized into genetic groups by their geographic origin, namely, Acre, Rondonia, and Mato-Grosso. The very high level of polymorphism of microsatellites (SSRs) proved to be appropriate for clonal identification. Polymorphisms in microsatellites were detected in H. pauciflora, H. guianensis, H. camargoana, H. benthamiana and H. brasiliensis (Low et al., 1996). These polymorphisms must have played a role in delineating species during the course of evolution. A microsatelliteenriched library was constructed in H. brasiliensis involving four types of simple sequence repeats such as (GACA)n (10%), (GATA)n (9%), (GA)n (34%), and (GC)n (9%) (Atan et al., 1996). In cooperation with the French National Centre for Sequencing, CIRAD developed different microsatellite-enriched libraries to identify a large collection of microsatellite markers (Seguin et al., 2003). Two possible applications are: clonal identification with the advantage of leaf samples sent through normal mail from one site to a distant laboratory and identification of parentage of seeds collected from an open-pollinated seed garden (Blanc et al., 2001). The nuclear genome contains 48% of most slowly annealing DNA (putative single copy) and 32% middle repetitive sequences with remaining highly repetitive or palindromic DNA (Low et al., 1996). The whole nuclear genome size was first estimated as 6 108 base pairs. In yet another study


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through flux cytometry, Seguin et al. (2003) estimated 2 109 base pairs for H. brasiliensis, H. benthamiana, H. guianensis, H. pauciflora, and H. spruceana. Isozymic diversity studies (Chevallier, 1988; Seguin et al., 1996a,b) showed the existence of three genetic groups. The isozymic studies showed that alleles included in the Wickham population are also included in the Amazonian populations, whereas many new alleles could be found in the Amazonian populations only, thus underlining the genetic enrichment provided by the collections. RFLPs, as ‘‘true’’ molecular genetic markers, were used with homologous probes from a CIRAD Hevea bank that showed genetic enrichment brought by Amazonian collections to Hevea germplasm, following genetic structure based on geographical collection sites (Besse et al., 1993b; Seguin et al., 1996d). A clone from Rondonia, RO/C/8/9, showed eight specific restriction fragments and a unique malate dehydrogenase (MDH) allele, indicating its interspecific origin. As expected, polymorphism among allied species was also very prevalent. The cytoplasmic genome has not been subjected to genetic recombination through meiosis, and its evolution is slower. Mean molecular size of chloroplast DNA (ct DNA) is estimated to be 152 kb (Fong et al., 1994). Mitochondrial DNA (mt DNA) was also analyzed with heterologous probes from broad bean by CIRAD and CNRA (Luo et al., 1995). A high mitochondrial polymorphism was found in Amazonian accessions. The diversity of mt DNA of the Wickham population is almost nil, as only GT 1, a male-sterile clone, exhibited a different type from that of 49 other Wickham clones analyzed. mt DNA appears to be a valuable tool for studies on classification and phylogeny in plants, resulting more from DNA rearrangements rather than nucleotide substitutions (Palmer and Herbon, 1988). Sequencing of a highly polymorphic mt DNA fragment on 23 genotypes showed real potential for phylogenetic analysis in Hevea (Luo and Boutry; 1995). In chloroplast DNA analysis, much less polymorphism was found, therefore indicating the high level of conservation of this genome. As a synthesis of these diversity studies, the Hevea genetic structure clearly appears as geographically structured (Besse et al., 1994) in relation to the hydrographic network of the Amazonian forest (Luo et al., 1995; Seguin et al., 1996b). Good relationships are found between the results from different genetic markers. Even if the contribution of isozymes is important by itself, molecular markers provided important clarifications for the distinction of different groups. There would be no barrier to the migration of Hevea genes within the Amazonian basin. However, the width of the area and the limited dispersion of Hevea seeds allowed the preservation of the current structure, which is assumed to be issued from the fragmentation of the Amazonian forest during the pleistocene

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period, according to the refuge theory presented by Haffer in 1982 (Seguin et al., 2003). The mt DNA of Wickham clones has lesser variation, since their female progenitors are restricted by a very small set of primary clones. Cytoplasmic donors for most of the improved clones are either PB 56 or Tjir 1 (Fig 3.6). Obviously, this is the reason for the mt-DNA profile showing only two clusters (Priyadarshan and Gonc˛ alves, 2003). A possible explanation for greater polymorphism in the mt DNA of wild accessions is that they must have been evolved through interspecific hybridization. mt-DNA polymorphism in wild accessions needs to be exploited fully. A molecular survey of available Amazonian accessions and isolation of competent molecular variants in their progeny are the possible exercises that can give meaningful results.

2. Gene-linkage maps and QTLs Due to its heterozygous nature, the construction of the molecular gene linkage map in Hevea requires specific methodology. Unlike annual crops, a cross between two heterozygous parents in Hevea can yield information up to four alleles, which are segregated further. A comprehensive genetic linkage map of Hevea brasiliensis has been formulated recently with the help of RFLPs, AFLPs, microsatellites, and isozyme markers (Lespinasse et al., 2000a). This was accomplished through a double pseudo-test cross according to the methodology of Grattapaglia and Sederoff (1994), and a map was constituted separately for each parent. Further, homologous markers segregating in both parents were ascertained and a consensus map was prepared. The parents used were PB 260 (PB5/ 51 PB 49) and RO 38 (F4542 AVROS 363). F4542 is a clone of Hevea benthamiana. The F1 synthetic map of 717 markers was distributed in 18 linkage groups. This was comprised of 301 RFLP, 388 AFLP, 18 microsatellite, and 10 isozyme markers. Identification of loci was based on the mobility of electrophoretic bands, necessitating verification of consistency of the location of alleles in both parental maps. The genetic length of 18 chromosomes was fairly homogeneous, with an average map length per chromosome of 120 cm. Many AFLP markers were seen in clusters, which were attributed to reduced recombination frequency regions. Though the RFLP markers were well distributed all over the 18 linkage groups, these were insufficient to saturate the map. AFLPs and few microsatellites together enriched saturating the map. However, these exercises are the initial steps for making a total genetic linkage map of Hevea in the future. The isozymes were found to inherit following a 1:1 ratio (Chevallier, 1988). On the other hand, a partially nonrandom arrangement of duplicate loci was observed by Lespinasse et al. (2000a) in their RFLP profiles, with certain chromosome pairs indicating they have homology descending from a common ancestor. The origin of such duplications is still unknown, and Hevea brasiliensis continues to behave as a diploid.


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QTLs for resistance to SALB (Microcyclus ulei) were mapped using 195 F1 progeny derived from a cross between PB 260 (susceptible) and RO 38 (resistant) clones (Lespinasse et al., 2000b), which was done in continuation of a genetic analysis done earlier (Seguin et al., 1996a). Eight QTLs were identified for resistance in a RO38 map through a Kruskel–Wallis marker-bymarker test and interval mapping method (Lander and Botstein, 1989; Oojen van et al., 1992). The F1 consensus map confirmed results obtained in parental maps. It was further rationalized that the resistance (alleles) of RO 38 inherited from a wild grandparent (H. benthamiana) and no favorable alleles came from AVROS 363, the Wickham parent. Eight different QTLs for five strains of fungi were available in RO 38, with specificity of resistance to different strains. More durable resistance would be available in other allied species and wild accessions of Hevea (Priyadarshan and Gonc˛ alves, 2003). However, the selection of clones having durable resistance with polygenic determinism is also important while undertaking such studies (Rivano, 1997a,b). Darmono and Chee (1985), while studying lesion size on leaf discs, identified SIAL 263, an illegitimate progeny of RRIM 501, as resistant to SALB.

3. Laticifer-specific gene expression The inquisitiveness to synthesize artificial rubber, of late, has increased the knowledge on rubber biosynthesis and the genes involved. Genes responsible for the key enzyme for polymerization of polyisoprenes—the rubber transferase—is one of the most abundantly expressed genes in the latex. Genes expressed in the latex can be broadly categorized into three based on their function: (a) defense genes, (b) genes for rubber synthesis, and (c) genes for allergenic proteins (Han et al., 2000). Hevein, a chitin-binding protein, is one of the defense proteins that plays a crucial role in the protection of wound sites from fungal attack. A cDNA clone (HEV 1) encoding hevein was isolated by using a polymerase chain reaction (PCR) (Broekaert et al., 1990). HEV 1 is of 1018 base pairs and includes an open reading frame of 204 amino-acids with a signal sequence of 17 amino acid residues, followed by 187 amino acid polypeptides. This polypeptide is found to contain striking features, such as an amino terminal region (43 amino acids) with homology to other chitin-binding proteins and an amino acid termini of wound-inducible proteins in potatoes and poplars. It was also seen that their genes are well expressed in leaves, stems, and latex (Broekaert et al., 1990). Nearly 12.6% of the proteins available in the latex are defense-related (Han et al., 2000). Mainly three-rubber synthesis related genes are expressed in the latex, namely, rubber elongation factor (REF) (Dennis and Light, 1989; Goyvaerts et al., 1991), HMG CoA reductase (Chye et al., 1992), and small rubber particle protein (SRPP) (Oh et al., 1999). They constitute the 200-odd distinct

Figure 3.6. Transmission of cytoplasm of PB 56 and Tjir 1 in the derivation of modern clones (see also Fig. 3.3).


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polypeptides (Posch et al., 1997). The most abundantly expressed gene is REF (6.1%) and then SRPP (3.7%) (Han et al., 2000). These expressed sequences (expressed sequence tags—ESTs) were compared with public databases of identified genes. About 16% of the database matched ESTs encoding rubber biosynthesis–related proteins. Analysis of ESTs revealed that rubber biosynthesisrelated genes are maximumally expressed followed by defense-related genes and protein-related genes (Han et al., 2000). Unlike photosynthetic genes, transcripts involved in rubber biosynthesis are 20–100 times greater in laticifers than in leaves (Kush et al., 1990). On the other hand, transcripts for chloroplastic and cytoplasmic forms of glutamine synthetase are restricted to leaves and laticifers, respectively (Kush et al., 1990), indicating thereby that the cytoplasmic form of glutamine synthetase plays a decisive role in amino acid metabolism of laticifers. Studies of laticifer-specific gene expression have important implications on selection and breeding. It would be worthwhile to use transcript levels as molecular markers for early selection (Kush et al., 1990). The transcript levels of hydrolytic enzymes, namely, polygalacturonase and cellulase, would be taken as indicators for better laticifer development. It is felt that extensive studies of the expression of genes are mandatory to unravel the intricacy of latex production. Detection and evaluation of more molecular markers must also help to breed Hevea at the molecular level.

4. Direct gene transfer The stable introduction of foreign genes into plant cells through direct gene transfer systems has opened up incredible avenues in the improvement of crops, especially perennial species, and rubber is no exception. While the in vitro plant regeneration system in rubber is getting standardized in few laboratories worldwide, efforts have been made to transform Hevea cells through Agrobacterium tumefaciens in order to complement plant breeding efforts to increase genetic variation (Arokiaraj et al., 1990, 1994). The anther-derived calli were transformed, with A. tumefaciens harboring gus- and nptII encoding, -glucuronidase and neomycin phosphotransferase, respectively. Fluorometric assay and enzymelinked immunosorbent assay (ELISA) were performed to prove the expression of gus and nptII genes, respectively, in calli and embryoids (Arokiaraj et al., 1996). The expression of foreign proteins in Hevea latex was demonstrated in 1998 (Arokiaraj et al., 1998). This transformation appeared stable even after three vegetative generations spanned to 31⁄2 years with no chimeras indicating that a single transformed plant is sufficient to have a population achieved through budding. But this exercise would not be adequate enough to take care of the stock–scion interaction and ensuing yield variation in a clonal population. Transformation of Hevea cells with genes for apomixis might be an alternative to circumvent stock–scion interaction. Lately, genes for human serum proteins

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have been expressed in rubber latex through genetic transformation (Arokiaraj et al., 2002; Yeang et al., 2002).

X. CONCLUSIONS Rubber breeding has been successful in achieving substantial yield improvements. However, research needs to be reconstructed through a multifaceted approach, and concerted efforts must take rubber to new hard areas: 1. Since the introduction of Wickham seedlings to Asia in 1876, and due to the occurrence of SALB and its containment to Latin America, rubber breeding in southeast Asia was based only on Wickham material, with a focus on yield improvement. The other confined to the Americas devoted to create Microcyclus-tolerant and productive clones. As a perennial crop, rubber breeding has been influenced deeply and positively by the grafting technique, which permitted the development of the current grafted clones evolved from handpollinated recombinations. In spite of the implementation of early selection techniques, rubber breeding is impeded by the time length of the selection process and the limited creation of full-sib families gained through low success rates of hand pollination. In order to broaden the genetic base, varied attempts were made to introduce new germplasm to Asia, including species allied to H. brasiliensis, among which the international IRRDB collection was the most significant. But, due to the low yield level of this germplasm and the length of the breeding process, benefits will be distributed only over a long time period. Apart from the creation of new clones for development, this research requires specific germplasm prebreeding programs in order to produce new parents to be included in recombination prior to selection. The spectrum of useful genetic variation needs to be enlarged, especially by utilizing variable cytoplasmic donors, since most oriental clones received cytoplasm either from PB 56 (through PB 5/51) or from Tjir 1. 2. After the fast progress in the past, and as germplasm breeding will not provide a quick answer, how can latex yield still be improved in the future? The research for complementarities within the Wickham population could provide new guidelines for the crossing programs and may be based on the splitting of the Wickham population into two different and complementary genetic populations. This could also help to limit the development of many related clones and inbreeding depression. But as far as rubber cropping in relation to overall economy is concerned (new locations, new objectives, new economic constraints), rubber breeding needs to address derivation of larger scope of clones adapted to varied biotic or abiotic stresses, and to varied specifications, including rubber quality. Consequently, it would be


3.

4.

5.

6.

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required not only to select elite clones but also to describe the behavior of a larger range of clones at small-scale experimentation level and in different environments (the development of clonal databases). Such a description will also help to suggest better arguments for the diversification of recommended clones. More emphasis on ecophysiology research could provide the necessary results for achieving some or all of those goals. Research devoted to SALB resistance involving recurrent backcrossing of Amazonian-resistant clones (mainly the H. benthamiana F 4542 or derived clones) with Wickham high-yielding clones, to evolve different resistance sources (clone or polyclonal seedling population) needs to be augmented. This strategy could also be applied to specific programs aimed at selecting clones resistant to Corynespora, Oidium, or other diseases. As rubber wood, after latex, has become an economically important product issued from rubber cropping, rubber breeding now has to integrate new traits, especially traits based on architecture and biomass production, in order to produce better-optimized ‘‘latex timber’’ clones. Biotechnologies hold new promise for rubber breeding. Although in vitro culture still meets different obstacles, this technique would pave the way for new planting material in the form of single-component clonal trees, or twocomponent trees with a clonal root system. Moreover, somatic embryogenesis is the gateway for the implementation of targeted genetic transfers to some current high-yielding clones, therefore accelerating genetic progress on agricultural traits or widening the scope of rubber cropping products (such as the possible production of proteins in the latex). The introduction of genes for apomixis to rubber for the production of somatic seeds deserves special attention. Molecular genetic markers already provide new efficient tools and methods for the analysis of genetic diversity, the identification of clones or the parents of clones, the elucidation of genetic determinism of selected traits, and for the tracing of genetic components along the selection process. Even if a real ‘‘marker-assisted selection’’ applied to rubber is still to be developed and validated, therefore contributing to early selection, it is very probable that molecular markers, especially microsatellites, will be substantially used at different levels and will improve the efficiency of rubber breeding. However, this does not mean that the quality of field testing, associated with the methodology of quantitative genetics and modern statistics, must be overlooked. It must be acknowledged that rubber breeding does not receive much funding compared with other crops such as oil palm, probably because rubber cropping appears to be less profitable due to frequent price fluctuations. Also, the current method of clone development (vegetative multiplication) cannot generate a profitable market. There is still no official registration of rubber clones, and very few producing countries belong to the varieties registration

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organization UPOV (currently China and Brazil). It must be noticed that rubber germplasm does not lie within the scope of activities of the International Plant Genetic Resources Institute. Consequently, international cooperation, as well as joint cooperation between public research institutions and the products transformation private sector, must be promoted in order to amplify the efficiency and efforts of rubber breeders. Even if the needs of small-holders are addressed with priority, industrial estates have provided a significant contribution in land facilities and logistics for large-scale and long-term testing of clones, and would continue to do so. With many projects in this direction (IRRDB, 2002), IRRDB can play a key role in this field of cooperation in rubber breeding.

Acknowledgments The authors are thankful to a number of scientists worldwide for providing data and for interaction during the preparation of the manuscript, namely, Dr. P. de S. Goncalves (Instituto Agronomico Campinas, Sao Paulo, Brazil); Dr. Pascal Montoro, Dr. M. Seguin, and Dr. Pujade-Renaud (CIRAD, France); Dr. T. T. T. Hoa (Rubber Research Institute of Vietnam); Dr. Keith Chapman (FAO, Bangkok); and Dr. H. Huasun (Chinese Academy of Tropical Agricultural Sciences), for their kind assistance in the writing of this chapter.

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Gene Transfer for Therapeutic Vascular Growth in Myocardial and Peripheral Ischemia Tuomas T. Rissanen,* Juha Rutanen,* and Seppo Yla¨-Herttuala*,{,{ * Department of Biotechnology and Molecular Medicine, A. I. Virtanen Institute, Kuopio University, Kuopio, Finland { Department of Medicine, Kuopio University, Kuopio, Finland { Gene Therapy Unit, Kuopio University Hospital, Kuopio, Finland

I. Introduction II. Mechanisms of Vascular Growth A. Vasculogenesis B. Angiogenesis C. Arteriogenesis III. Vascular Growth Factors A. Vascular Endothelial Growth Factors (VEGFs) B. Fibroblast Growth Factors (FGFs) C. Other Angiogenic Growth Factors IV. Gene Transfer in Skeletal Muscle and Myocardium A. Gene Transfer Vectors B. Gene Delivery Methods V. Therapeutic Vascular Growth A. Angiogenic Gene Therapy in Skeletal Muscle B. Angiogenic Gene Therapy in Myocardium C. Endothelial Precursor Cell Therapy VI. Safety Aspects A. Edema Related to Vascular Growth B. Pathological Angiogenesis VII. Concluding Remarks References

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ABSTRACT Therapeutic vascular growth in the treatment of peripheral and myocardial ischemia has not yet fulfilled its expectations in clinical trials. Randomized, double-blinded placebo-controlled trials have predominantly shown the safety and feasibility but not the clear-cut clinically relevant efficacy of angiogenic gene or recombinant growth factor therapy. It is likely that growth factor levels achieved with single injections of recombinant protein or naked plasmid DNA are too low to induce any relevant angiogenic effects. Also, the route of administration of gene transfer vectors has not been optimal in many cases leading to low gene-transfer efficacy. Animal experiments using intramuscular or intramyocardial injections of adenovirus encoding vascular endothelial growth factor (VEGF, VEGF-A), the mature form of VEGF-D, and fibroblast growth factors (FGF-1, -2, and -4) have shown high angiogenic efficacy. Adenoviral overexpression of VEGF receptor-2 ligands, VEGF-A and the mature form of VEGF-D, enlarge the preexisting capillaries in skeletal muscle and myocardium via nitric oxide(NO)–mediated mechanisms and via proliferation of both endothelial cells and pericytes, resulting in markedly increased tissue perfusion. VEGF also enhances collateral growth, which is probably secondary to increased peripheral capillary blood flow and shear stress. As a side effect of VEGF overexpression and rapid microvessel enlargement, vascular permeability increases and may result in substantial tissue edema and pericardial effusion in the heart. Because of the transient adenoviral gene expression, the majority of angiogenic effects and side effects return to baseline by 2 weeks after the gene transfer. In contrast, VEGF overexpression lasting over 4 weeks has been shown to induce the growth of a persistent vascular network in preclinical models. To improve efficacy, the choice of the vascular growth factor, gene transfer vector, and route of administration should be optimized in future clinical trials. This review is focused on these issues. ß 2004, Elsevier Inc.

I. INTRODUCTION Insufficient blood flow to the heart or lower limbs due to coronary artery disease or peripheral arterial disease causes severe inability and more deaths than any other disease in the developed countries. Conventional revascularization strategies, angioplasty and bypass surgery, are effective in improving both symptoms and prognosis of patients with ischemic disease. However, not all patients, such as the elderly with comorbidity or those with extensive and severe vascular occlusions, are eligible for these approaches. Furthermore, in many cases, the outcome of these therapies is not completely satisfactory even after a technically

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successful procedure, often due to reduced regenerative capacity of the aged patient population. Growth of blood vessels is required for normal embryonic development, growth, and tissue repair. In the recent years, regenerative medicine has emerged and introduced approaches to take advantage of nature’s own tools to restore compromised blood circulation in the heart and skeletal muscle. This novel treatment to promote tissue perfusion by means of gene transfer or utilization of bone marrow–derived stem cells is called therapeutic angiogenesis. In this review, we describe mechanisms of vascular growth, properties of different vascular growth factors, gene transfer techniques, preclinical and clinical experience, and safety issues of this novel therapy.

II. MECHANISMS OF VASCULAR GROWTH Vascular growth can classically be defined as the formation of blood vessels from vascular stem cells during embryonic development (vasculogenesis), the sprouting of new capillaries from preexisting ones (angiogenesis), and collateral artery growth (arteriogenesis) to circumvent the occluded main artery. In this chapter, these mechanisms are discussed.

A. Vasculogenesis Blood vasculature is formed in the beginning of the third embryonic week by the process called vasculogenesis (Risau and Flamme, 1995). The mesoderm-derived stem cells, hemangioblasts, give rise to the vascular and hematopoietic cell lines (Risau and Flamme, 1995). From these cells, angioblasts arise, which further develop into components of the vessel wall, endothelial cells (ECs), and mural cells, such as pericytes and smooth-muscle cells (SMCs). ECs and SMCs proliferate and differentiate further to form vascular plexus, which spreads by angiogenic sprouting and remodeling (Carmeliet and Collen, 1999). Finally, the functional vascular network is established by organization of the arterial, venular, and capillary circulation (Risau and Flamme, 1995). Recently, vascular stem cells such as endothelial progenitor cells (EPCs) have been found to contribute to vascular growth also in adults, at least to some degree (Asahara et al., 1997).

B. Angiogenesis Angiogenesis is traditionally defined as the sprouting of new capillaries from preexisting ones (Risau, 1997). However, recently a different type of capillary growth, enlargement, was found to occur after VEGF overexpression (Pettersson

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et al., 2000; Rissanen et al., 2003b; Rutanen et al., 2004). Angiogenesis is a crucial element of various physiological and pathological events, such as wound healing, skeletal growth, hair growth, follicular growth, development of the corpus luteum, and tumor growth (Carmeliet, 2003; Folkman, 1971). Angiogenesis is needed to provide enough oxygen and nutrients to tissues with an increasing demand. The formation and maturation of a capillary network is a complex process involving various steps. Certain growth factors have been shown to be crucial. The most important stimulus for angiogenesis is hypoxia (Carmeliet, 2003; Risau, 1997), usually mediated by vascular endothelial growth factor (VEGF, VEGF-A)–expression induced by hypoxia-inducible factor-1 (HIF-1) (Rissanen et al., 2002; Pugh and Ratcliffe, 2003; Semenza, 2000). During angiogenesis, ECs proliferate and migrate toward the stimulus (Pugh and Ratcliffe, 2003; Semenza, 2000; Vajanto et al., 2002). Pericytes also actively participate in the formation of new capillary tubes (Gerhardt and Betsholtz, 2003; Morikawa et al., 2002). Endothelium-derived platelet-derived growth factor-B (PDGF-B) is an important factor to control pericyte recruitment (Lindahl et al., 1997). Extracellular matrix (ECM) and the basement membrane are first degraded and later reassembled to provide structural support to the growing vessel (Jain, 2003; Kalluri, 2003). In this process, matrix metalloproteinases (MMPs) play a crucial role (Bergers and Benjamin, 2003; Jain, 2003; Kalluri, 2003). In the maturation process, the stabilization of ECs, pericytes, basement membrane, and ECM are needed to form new persistent capillary tubes. For example, vessels with insufficient coverage of mural cells are fragile, leaky, and prone to regression (Benjamin et al., 1998; Hellstrom et al., 2001b). This is often the case in tumors where the vasculature may be disorganized, dilated, immature, leaky, and lacks pericytes (Bergers and Benjamin, 2003; Jain, 2003). In tumors and inflammatory processes, infiltrating macrophages are an important source of angiogenic growth factors (Barbera-Guillem et al., 2002; Rehman et al., 2003).

C. Arteriogenesis After the occlusion of the main artery, the natural response is the enlargement of the preexisting arterial anastomoses to form collateral arteries to bypass the occlusion (Schaper and Ito, 1996; Schaper and Scholz, 2003). This process is called arteriogenesis (Schaper and Scholz, 2003). Arteriogenesis involves remodeling of the intima, media, and adventitia, driven by increased circumferential wall stress against the medial layer and fluid shear stress against the endothelium (Schaper and Scholz, 2003). Ischemia is not a direct trigger for arteriogenesis, since collaterals grow upstream to ischemic tissue (Ito et al., 1997a; Schaper and Ito, 1996).

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The exact mechanism of vascular adaptation to increased blood flow is unclear. Endothelium seems to play a crucial role in the process through NO production and signaling via integrins (Jin et al., 2003; Muller et al., 1997; Nadaud et al., 1996). In an animal model of myocardial ischemia, NO has been shown to be a crucial mediator of arteriogenesis (Matsunaga et al., 2000). Also, local inflammation of the vessel wall caused by the rapid increase in fluid shear stress is thought to play an important role in the initiation phase of arteriogenesis (Arras et al., 1998; Ito et al., 1997b). Cytokines and adhesion molecules have been reported to entice monocytes and macrophages to collaterals where they secrete growth factors such as fibroblast growth factors (FGFs) (Arras et al., 1998). Also, the genetic depletion of tumor necrosis factor- (TNF-) or its receptor p55 in mice has been reported to impair collateral growth after femoral artery occlusion (Hoefer et al., 2002). In ischemic myocardium or skeletal muscle, arteriogenesis is more important than angiogenesis in supplying blood to tissue because collaterals provide bulk flow to the ischemic areas, unlike capillaries, which only provide blood for the immediate cellular milieu. Unfortunately, endogenous arteriogenesis stops prematurely when the conductance of

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