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Vegetative Propagation and Genetic Finger Printing for Eucalyptus grandis and Eucalyptus amplifolia

Permanent Link: http://ufdc.ufl.edu/UFE0024073/00001

Material Information

Title: Vegetative Propagation and Genetic Finger Printing for Eucalyptus grandis and Eucalyptus amplifolia
Physical Description: 1 online resource (97 p.)
Language: english
Creator: Yang, Zhi
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: cutting, eucalyptus, finterprinting, micropropagation, multiplication, rooting
Forest Resources and Conservation -- Dissertations, Academic -- UF
Genre: Forest Resources and Conservation thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Eucalyptus grandis and Eucalyptus amplifolia clones may be grown as short rotation woody crops in Florida. Several propagation methods were evaluated for 4 well-known and 6 new E. grandis as well as 9 new E. amplifolia clones. The propagation methods included traditional cutting (macrocutting), mini-cutting, and tissue culture. Healthy branches were cut into 40 mm long macrocuttings with two-half leaves. NAA commercial powder was added to stimulate rooting. Juvenile sprouts from felled trees were rooting better than the branches from the crown of the trees. Rooting response from cuttings made in fall was higher than that in spring. After successful propagation by cuttings, some healthy plants served as stock plants for mini-cutting. Three-month-old new shoots on the stock plants were collected for mini-cutting, and the method for mini-cutting was just like the traditional cutting. Shoots from different parts of the stock plants influenced the rooting percentage; as the basal part from the lateral branches was optimal material for mini-cutting. Although the rooting percentage for mini-cuttings was not as high as for traditional cuttings, the mini-cutting could provide consistent supply of plant material. Macrocuttings and mini-cuttings worked as stock plants for tissue culture, and nodes from 6-week-old branches worked well as explants. Driver Kuniyuki Walnut (DKW), Murashige and Skoog (MS), and Woody Plant Media (WPM) were used as the basal media for shoot multiplication. All E. grandis and E. amplifolia clones were initiated by soaking explants in 10% bleach for 20 minutes. Shoot induction was achieved on MS without PGRs, and they were transferred to fresh media every 3 days. Then shoot multiplication was achieved using basal media containing 0.4 mg/l BAP. DKW was the most efficient basal medium for clones, especially for G1and G2. E. grandis shoot elongation was best on DKW containing 0.1-0.5 mg/l BAP combined with 0.1mg/l NAA. Rooting was best on DKW containing 0.5 mg/l NAA. PVP were added to media in every step to prevent phenolic compound oxidation. Media were changed at 3-week intervals to prevent vitrification, phenolic exudation and callusing. Eight microsatellite loci were chosen for the estimation of the 60 E. grandis clones? allelic diversity. The result showed that E. grandis clones originated from 4 subpopulations. Kinship coefficients were used to estimate the genetic relatedness between pairs of these clones and the low kinship coefficient explained the genotypic response in tissue culture to some degree.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Zhi Yang.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Rockwood, Donald L.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024073:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024073/00001

Material Information

Title: Vegetative Propagation and Genetic Finger Printing for Eucalyptus grandis and Eucalyptus amplifolia
Physical Description: 1 online resource (97 p.)
Language: english
Creator: Yang, Zhi
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: cutting, eucalyptus, finterprinting, micropropagation, multiplication, rooting
Forest Resources and Conservation -- Dissertations, Academic -- UF
Genre: Forest Resources and Conservation thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Eucalyptus grandis and Eucalyptus amplifolia clones may be grown as short rotation woody crops in Florida. Several propagation methods were evaluated for 4 well-known and 6 new E. grandis as well as 9 new E. amplifolia clones. The propagation methods included traditional cutting (macrocutting), mini-cutting, and tissue culture. Healthy branches were cut into 40 mm long macrocuttings with two-half leaves. NAA commercial powder was added to stimulate rooting. Juvenile sprouts from felled trees were rooting better than the branches from the crown of the trees. Rooting response from cuttings made in fall was higher than that in spring. After successful propagation by cuttings, some healthy plants served as stock plants for mini-cutting. Three-month-old new shoots on the stock plants were collected for mini-cutting, and the method for mini-cutting was just like the traditional cutting. Shoots from different parts of the stock plants influenced the rooting percentage; as the basal part from the lateral branches was optimal material for mini-cutting. Although the rooting percentage for mini-cuttings was not as high as for traditional cuttings, the mini-cutting could provide consistent supply of plant material. Macrocuttings and mini-cuttings worked as stock plants for tissue culture, and nodes from 6-week-old branches worked well as explants. Driver Kuniyuki Walnut (DKW), Murashige and Skoog (MS), and Woody Plant Media (WPM) were used as the basal media for shoot multiplication. All E. grandis and E. amplifolia clones were initiated by soaking explants in 10% bleach for 20 minutes. Shoot induction was achieved on MS without PGRs, and they were transferred to fresh media every 3 days. Then shoot multiplication was achieved using basal media containing 0.4 mg/l BAP. DKW was the most efficient basal medium for clones, especially for G1and G2. E. grandis shoot elongation was best on DKW containing 0.1-0.5 mg/l BAP combined with 0.1mg/l NAA. Rooting was best on DKW containing 0.5 mg/l NAA. PVP were added to media in every step to prevent phenolic compound oxidation. Media were changed at 3-week intervals to prevent vitrification, phenolic exudation and callusing. Eight microsatellite loci were chosen for the estimation of the 60 E. grandis clones? allelic diversity. The result showed that E. grandis clones originated from 4 subpopulations. Kinship coefficients were used to estimate the genetic relatedness between pairs of these clones and the low kinship coefficient explained the genotypic response in tissue culture to some degree.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Zhi Yang.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Rockwood, Donald L.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024073:00001


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1 VEGETATIVE P ROPAGATION AND GENETIC FINGERPRINTING OF EUCALYPTUS GRANDIS AND EUCALYPTUS AMPLIFOLIA By ZHI YANG A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009

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2 Zhi Yang

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3 To my parents Hanlin Yang and Chuanxia Zhu

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4 ACKNOWLEDGMENTS I would like to sincerely thank my graduate committee chairman, Dr. Donald Rockwood, for his patient mentoring for 3 years. I deeply appreciate his help. Then, I would like to thank Dr. Jude Grosser for his advice and for the use of his lab and facility for my research. My thanks also go to Dr. Matias Kirst for his microsatellite marker research and Dr. Gary Peter for his informative lecture in forestry theory course. I would like to thank all the people in Dr. Rockwoods research group, Br ian Becker, Bijay Tamang, Paul Pro c tor, and William McKinstry who helped me a lot in propagation in greenhouse during these 2 years. Thanks also goes to Milica Calovic, research assistant in Dr. Grosser lab, who taught me a lot about tissue culture, and Carolina Boaventura in Dr. Kirsts lab who ran all the marker analysis. Acknowledgement also goes to the College of Agricultural and Life Sciences and Dr. Rockwoods program for providing me a graduate assistantship to support my research. Thanks are extended to all the faculty members, supporting staff and graduate students in the School of Forest Resources and Conservation and the Citrus Re search and Education Center at University of Florida.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF TABLES ................................................................................................................................ 7 LIST OF FIGURES .............................................................................................................................. 8 LIST OF OBJECTIVES ..................................................................................................................... 10 LIST OF ABBREVIATIONS ............................................................................................................ 11 ABSTRACT ........................................................................................................................................ 12 CH A P T E R 1 GENERAL INTRODUCTION .................................................................................................. 14 Eucalyptus Genus ........................................................................................................................ 14 Propagation Method .................................................................................................................... 15 Objectives .................................................................................................................................... 16 2 LITERATURE REVIEW ........................................................................................................... 17 Ease of Vegetative Propagation ................................................................................................. 17 Cutting Method ............................................................................................................................ 17 Mini Cutting Method .................................................................................................................. 18 Plant Tissue Culture Media ........................................................................................................ 18 Micropropagation of Eucalyptus ................................................................................................ 20 Explant and Culture Ste rilization ........................................................................................ 20 Direct Shoot Culture ............................................................................................................ 21 Organogenesis ...................................................................................................................... 21 Somatic Embryogenesis ...................................................................................................... 22 Genetic Variation ........................................................................................................................ 23 DNA Marker ........................................................................................................................ 23 Data Analysis for Markers .................................................................................................. 24 3 MATERIALS AND METHODS ............................................................................................... 27 Cutting Method ............................................................................................................................ 27 Plant Material ....................................................................................................................... 27 Method.................................................................................................................................. 27 Mini Cutting Method .................................................................................................................. 30 Plant Material ....................................................................................................................... 30 Method.................................................................................................................................. 30 Micropropagation ........................................................................................................................ 30

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6 Plant Material and Sterilization .......................................................................................... 30 Establishment of Cultures ................................................................................................... 31 Shoot Multiplication ............................................................................................................ 31 Shoot Elongation and Rooting ............................................................................................ 32 Acclimatization .................................................................................................................... 32 Data Collection and Statistical Analy sis ............................................................................ 33 Genetic Fingerprinting ................................................................................................................ 33 Plant Material ....................................................................................................................... 33 Polymorphism of Microsatellite Loci ................................................................................. 33 Population Structure ............................................................................................................ 33 SPAGeDi .............................................................................................................................. 34 4 RESULTS AND DISCUSSION ................................................................................................ 36 Propagation by Cuttings .............................................................................................................. 36 Propagation by Mini Cuttings .................................................................................................... 41 Micropropagation ........................................................................................................................ 44 Explant and Sterilization ..................................................................................................... 44 Shoot Induction .................................................................................................................... 47 Shoot Multiplication ............................................................................................................ 50 Elongation ............................................................................................................................ 54 Rooting and Acclimation .................................................................................................... 57 Cost Reduction ..................................................................................................................... 63 Genetic Fingerprinting ................................................................................................................ 64 Pedigree and Genetic Relatedness ...................................................................................... 64 Marker .................................................................................................................................. 64 Propagation and Fingerprinting .......................................................................................... 71 5 GENERAL CONCLUSION ....................................................................................................... 72 6 FURTHER RESEARCH NEEDED ........................................................................................... 76 APPEN D I X A BASAL MEDIA COMPONENT ............................................................................................... 78 B PROTOCOL FOR AMPLIFICATION OF SSR ....................................................................... 80 C SIXTY E. GRANDIS CL ONES GROUPING BY STRUCTURE ........................................... 81 D KINSHIP COEFFICIENTS FOR PAIRS OF SIXTY E. GRANDIS CLONES ...................... 84 REFERENCES ................................................................................................................................... 87 BIOGRAPHICAL SKETCH ............................................................................................................. 97

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7 LIST OF TABLES Table page 3 1 Location, age (years) and clone numbers by 3 methods (cutting, minicutting, and microcutting) for E. grandis E. amplifolia and C. torelliana clones ................................. 28 3 2 Relationship and kinship coefficient (Weir et al., 2005) ..................................................... 35 4 1 Effect of shoot age (3, 6, and 9 -m onth) on mini -cutting rooting percent for 4 E. grandis clones (G1, G2, G3, and G4) ................................................................................... 44 4 2 Decontamination rate of one -month old shoo ts from stock plants and from mature trees of E. grandis and E. amplifolia .................................................................................... 44 4 3 E. grandis and E. amplifolia shoot survival rate with pre -soaking, addition of charcoal, and addition of PVP with change medium interval of 2, 5, or 14 days. ............. 47 4 4 E. grandis and E. amplifolia shoot induction in media with different PGR combinations ........................................................................................................................... 49 4 5 Multiplication rate on 12 multip lication media (4 levels of PGR: 0.1, 0.4, and 1mg/l BAP combined with 0 or 0.1mg/l NAA on 3 basal media: MS, DKW, and WPM) across 5 E. grandis clones (G1, G2, G3, G4, and G34). ...................................................... 51 4 6 Component (Zn(SO4)2 Zn(NO3)2, K2SO4, KNO3, Mg(SO4)2 ,Ca(NO3)2, CaCl2) comparison between DKW, MS, and WPM basal medium. ............................................... 53 4 7 Shoot multiplication rate in DKW plus NAA (0.1, 0.5, or 1mg/l) across 5 E. grandis clones (G1, G2, G3, G4, and G34). ....................................................................................... 55 4 8 Effect of NAA on rooting number and length in medium MS across 5 clones (G1, G2, G3, G4, and G34) ............................................................................................................ 58 4 9 E. grandis clones originating from common 3GM or 4GM (Clone 295, 293, 294, 298, and 92). ........................................................................................................................... 64 4 10 Allele number for 8 loci in the study .................................................................................... 65 4 11 Pairs of E. grandis clones with kinship coefficient between 0.2 and 0.5 ........................... 70 4 12 The kinship coefficient for E. grandis clones involved in propagation ( G1, G2, G3, G4, G34, G34, G37, and G43). ............................................................................................. 71

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8 LIST OF FIGURES Figure page 4 1 Cutting rooting percent from 2 sources (crown and stump sprouts) for 4 well -tested E. grandis clones (G1, G2, G3, and G4).. ............................................................................ 36 4 2 Cutting rooting percent in 2 different seasons (fall and spring) for 4 well -tested E. grandis clones (G1, G2, G3, and G4) .................................................................................. 37 4 3 Cutting rooting percent for 10 E. grandis clones (G1, G2, G3, G4, G34, G36, G37, G43, G48, and G51) in fall.. .................................................................................................. 38 4 4 Cutting rooting percent for nine E. amplifolia clones (A1, A2, A3, A4, A5, A6, A7, A8, and A9).. .......................................................................................................................... 39 4 5 Mini -cutting rooting percent for 6 E. grandis clones (G1, G2, G3, G4, G34 and G37) from 3 sources (stem, top of branches and base of branches). ............................................ 42 4 6 Comparison of rooting percent for macrocutting and mini -cutting for 6 E. grandis clones (G1, G2, G3, G4, G34, and G37). ............................................................................ 43 4 7 Shoot multiplication rate on media (DKW+0.4mg/l BAP or MS+0.4mg/l BAP) for 5 clones (G1, G2, G3, G4, and G34) respectively.. ................................................................ 51 4 8 Multiplication rate (how many shoots longer than 2 mm produced on single shoot per 3 weeks) for 8 subculture generations on DKW with 0.4 mg/l media for 5 E. grandis clones (G1, G2, G3, G4, and G34).. ...................................................................................... 52 4 9 Elongation in DKW plus NAA (0.1, 0.5, or 1mg/l) across 5 E. grandis clones (G1, G2, G3, G4, and G34).. .......................................................................................................... 55 4 10 Shoot elongation in DKW plus 0.1 mg/l NAA with 0.5 mg/l BAP or 0.1 mg/l NAA with 0.1mg/ l BAP for 5 E. grandis clones (G1, G2, G3, G4, and G34), respectively.. ..... 56 4 11 Effect of MS concentration with 0.5 mg/l IBA or NAA on rooting percent across 4 clones (G1, G2, and G3, and G4). ......................................................................................... 58 4 12 Propagation of E. grandis clones: A -sprouts from felled tree crown; B -cuttings in the greenhouse; C -sterilization of stems; D -shoot multiplication stage; E,F -shoot elongation stage; G -rooting stage; H acclimatized plantlets from in vitro to greenhouse .............................................................................................................................. 61 4 13 Allele number and frequency for 8 microsatellite loci (EMBRA2, EMBRA28, EMBRA10, ES76, EMBRA37, EMBRA63, EG62, and EG65) in the population of 60 Florida E. grandis clones. ................................................................................................. 68

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9 4 14 Population structure for of 60 Florida E. grandis clones (Appendix C). Each clone was represented by vertical line, which was partitioned i nto 4 colored segments that represent that clones estimated membership fraction in each of the K inferred clusters. ................................................................................................................................... 69 4 15 Distribution of pairwise relative kinship estimates in 60 E. grandis clones; 0.5 identical twins; 0.25 offspring within a family, parent offspring; 0.125 half -siblings. ..... 69 5 1 Propagation cycle for Eucalytus ............................................................................................ 74

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10 LIST OF OBJECTIVE S Objective page 1 Develop vegetative propagation protocols for Eucalyptus grandis and Eucalyptus amplifolia clones by cutting, mini -cutting, and micropropagation. .................................... 16 2 Genetically fingerprint the clones to find the genetic relatedness ...................................... 16

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11 LIST OF ABBREVIATIONS BAP Benzylaminopurine DKW Driver Kuniyuki Walnut media GA3 Gibberellic acid IAA Indoleacetic acid IBA Indole 3 -butyric acid NAA 1 naphthalene acetic acid MS Murashige and Skoog media PGR Plant growth regulator PVP Polyvinylpyrolidone WPM Woody Plant Media 2 iP 2 Isopentenyl adenine

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12 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science VEGETATIVE P ROPAGATION AND GENETIC FINGERPRINTING OF EUCALYPTUS GRANDIS AND EUCALYPTUS AMPLIFOLIA By ZHI YANG August 2009 Chair: Donald Rockwood Major: Forest Resource and Conservation Eucalyp tus grandis and Eucalyptus amplifolia clones may be grown as short rotation woody crop s in Florida. Several p ropagation method s were evaluated for 4 well known and 6 new E grandis as well as 9 new E. amplifolia clones. The propagation method s incl uded traditional cutting (macro cutting), mini -cutting and tissue culture. Healthy branches were cut into 40 mm long macrocuttings with two -half leaves NAA commercial powder was added to stimulate rooting. J uvenile sprouts from f e lle d trees were root ing better than the branches from the crown of the trees. R ooting response from cuttings made in fall was higher than that in spring. A fter successful propagat ion by cuttings, some healthy plants served as stock plants for mini -cutting. Three -month -old new shoots on the stock plants were collected for mini -cutting and the method for mini -cutting was just like the traditional cutting. Shoots from different parts of the stock plants influenced the rooting percent age ; as the basal part from the lateral branches was o ptimal material for mini -cutting Although the rooting percent age for mini -cutting s was not as high as for traditional cuttings, the mini -cutting could provide consistent supply of plant material.

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13 M acrocuttings and mini -cuttings worked as stock plants for ti ssue culture, and nodes from 6 -week -old branches worked well as explants. Driver Kuniyuki Walnut ( DKW ), Murashige and Skoog (MS), and Woody Plant Media ( WPM ) were used as the basal medi a for shoot multiplication. All E. grandis and E. amplifolia clones were initiated by soaking explants in 1 0% bleach for 20 minutes. Shoot induction was achieved on MS without PGR s and they were transferred to fresh media every 3 days. Then shoot multiplication was achieved using basal medi a containing 0.4 mg/l BAP. DKW w as the most efficient basal medium for clone s especially for G1and G2. E. grandis s hoot elongation was best on DKW containing 0.1 0. 5 mg/l BAP combined with 0.1 mg/l NAA. Rooting was best on DKW containing 0. 5 mg/l NAA. PVP were added to medi a in every step to prevent phenolic compound oxidation. Media were changed at 3 -week interval s to prevent vitrification, phenolic exudation and callus ing Eight microsatellite loci were chosen for the estimation of the 60 E. grandis clones allel ic diversity. The result showed that E grandis clones originated from 4 subpopulations. Kinship coefficients were used to estimate the genetic relatedness between pairs of these clones and the low kinship coefficient explained the genotypic response in tiss ue culture to some degree.

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14 CHAPTER 1 GENERAL INTRODUCTION Eucalyptus Genus Eucalyptus mostl y originating in Australia, is a widely cultivated genus in th e world, particular ly in tropical and subtropical region s It is a member of the family Myrtaceae and now comprises about 800 species, in number second only to Acacia in Australia (Brooker, 2002), ranging from straight -trunked forest trees up to 90 m tall, to multiple -stemmed, shrubby mallees (Chippendale, 1976). The popular conception of the discovery of Eucalyptus is the voyages of Captain James Cook in the Endeavour in the 1770s (Brooker, 2002). Because of their high productivity in short rotation plantations, easy management, and adaptation to a wide range of environment s Eucalyptus clon es have attracted world attention. They have multiple uses, including firewood, windbreaks, shade, pole timber, and paper pulp; species used for these purpose are limited to a small number, such as E. grandis, E. saligna, E. globulus, E. tereticornis, E. n itens and E. dunnii Most of them are tall trees and naturally originated from wet forests in Australia. Some other smaller trees in the genus are grown in drier and less fertile regions. Most Eucalyptus species of the arid interior Australia are usually f ound in the regions along the seasonal streams and in the rocky hills (Brooker, 2002). In addition, one of the well known characteristics is that some Eucalyptus leaves have oil glands, which can provide industry oil. Eucalyptus plantation have under went rapid expansion around the world (Edgrand Campinhos, 1999) ; demand for Eucalyptus market pulp has been growing at 11.2 percent/yr since 1980 ( Wilson et al., 1995) Eucalypts were first planted in Florida in 1878, but industrial plantations were not establ ished until 1972. E. grandis was found through species trials to be a suitable s pecies for

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15 south Florida (Geary et al., 1983). F reeze hardy E. amplifolia was suited to good sites in northeastern Florida and E. camaldulensis was suitable for a wide range of sites in central and southern Florida (Rockw ood et al. 2006). They are highly productive in Florida. Propagation Method There are two traditional ways to propagate Eucalyptus sexual propagation by seed and asexual propagation (vegetative propagation) Propagation by seed is the major method by which plants reproduce in nature, and is one of the most common ly used methods for culture. Since sexual propagation can combine the characteristics of parents during the cross fertilization to produce some new genotypes, it is the basis for breeding. From the point of view of breeding and seed production of E ucalyptus for wood production, the distinctive characteristics of sexual reproduction of E ucalyptus are: F lower is herma phrodite (male and female in one flower); P ollination is by animals, mainly insects and birds, not by the wind; O utcrossing is favored by various mechanisms, including protandry, which means that the stigma is not receptive until some days after the pollen has started shedding from the a nthers; F ruits are dry woody capsules and seeds are very small in the species used for fast growing plantations; S eed production varies enormously due to non -genetic factors such as spacing, site and seasonal conditions. Vegetative propagation differ s from seedling propagation in that all members of population have originated from a single plant and are expected to possess the same genotype. Vegetative propagation is very important when it is essential to maintain the same characteristics of the genotype. This practice is possible because of adventitious shoots and roots. Vegetative propagation methods include cutting, grafting and lay er ing, among which cutting is the most efficient and widely used method for many species.

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16 Micropropagation, also called tissue culture, develops whole plant s from plant cells or organs in an aseptic in vitro environment. The successful plant tissue culture was first accomplished by Gottlieb Haberlandt in the 19th century ( Caponetti et al. 2005). Common steps of the microp ropagation procedure include donor plant selection, establishment, shoot multiplication, pre -transplant and acclimatization. Objective s Object ive 1. D evelop vegetative propagation protocol s for Eucalyptus grandis and Eucalyptus amplifolia clones by cutting, mini -cutting and micropropagation. Object ive 2. Genetically fingerprint the clones to find the genetic relatedness

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17 CHAPTER 2 L ITERATURE REVIEW Ease of Vegetative Propagation In tree breeding, vegetative propagation is commonly used for the establishment of clone banks and clonal seed orchards, and can be utilized for the production (Doran, 2002) Mass propagation by stem cutting is the common method to establish clonal forestr y because it is less expensive Micropropagation is an alternative method of propagation. Cutting Method Cutting propagation is the most important means for clonal regeneration, and adventitious root formation is prerequisite to successful cutting propagation. Propagation by stem cutting requires th e formation of new adventitious root system s since a potential shoot system (a bud) is already present. Cuttings include 3 kinds in general: hardwood cuttings, semi -hardwood cuttings and soft wood cuttings (Har tmann 2002) Cutting is the preferred method of conventional vegetative propagation, but cuttings from most mature E ucalyptus are not able to root (Paton et al. 1970) However cuttings from seedlings and juvenile trees did root well and manipulation of the state of juvenility of the shoots had allowed cuttings to be used with great success in plantations of E. grandis and various other species. Shoots which sprouted fro m the stump when a tree was felled or girdled had juvenile characteristics including the ability to produce adventitious roots. To develop the desired genotypes, trees with the superior characteristics were felled or girdled. Cuttings were taken from the c oppice shoots, and further selection was made for individuals with high rooting ability. Rooting p ercent of 80% or more were reported for E. grandis and the method allowed production of millions of trees per year (Campinhos and Ikemori, 1977). The method c ould be extended to the other species, but it was not always possible to fell the superior

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18 trees, because this species did not sprout from stumps. In such cases auxin application to the stump promoted sprouting (Davidson, 1977). Damage to the base of a sta nding tree induced epicormic shoots and Mazalewsky and Hackett (1979) promoted basal sprouts of a 2 meter high E. ficifolia using cytokinin, but it was not known if this method worked well with mature trees in the forest. Management of stock plants to maximize rooting begins with the selection and maintenance of source material that is easy to root, rejuvenation of stock plant materials and selection of cuttings from stock plants. Mini -C utting Method Mini -cuttings are similar to traditional rooted cutting s except that they are based on rooting of axillary sprouts from rooted stem -cutting s (or mini hedges) (Romero 2004). The advantage of mini -cutting systems over conventional rooted cutting programs is that re -propagation of clones could be extremely fast and could reduce the conventional clonal deployment cycle by 23 years (which is needed to produce enough cuttings to establish an outdoor clonal garden with traditional rooted cuttings) (Romero 2004). Plant T issue C ulture M edia The tot i potency of plant cell s was the basis for plant tissue culture. The first successful plant tissue and cell culture was created by Haberlandt in 1902 (Krikorian and Berquam, 1969). By the 1970s, the role of five major classes of plant growth regulator s in tissue culture had been recognized and investigated T hey are auxins, cytokinins, gibberellins, ethylene and abscisic acid (Caponetti et al., 2005) .Murashige and Skoogs (MS) (1962) medium, developed originally for tobacco, by Murashige in Skoogs lab is now the most popula r basic media for various species with high salt media for its K and N content (Beyl, 2005). WPM developed by Lloyd and McCown (1980) is very commonly used for woody trees.

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19 Sugar i s a necessary component in medium because explants in vitro are unable to photosynthesize effectively. Twenty to 60g/l sucrose is most commonly used concentrations A lternative sugar s could be glucose, maltose, or lactose (Coffin et al., 1976). Vitamins are o rganic substances that function as metabolic substances and enzymes. Of the vitamin s only vitamin B1 (at 1 5 mg/l) was essential in culture because it was involved in carbohydrate metabolism and the biosynthesis of some amino acids (Beyl, 2005). Activated charcoal is useful for absorpt ion of brown pigments and oxidized phenolic compounds. 0.2 3% (w/v) concentration proved feasible and absorbed some PGR and vitamins (Nissen and Sutter, 1990). In addition, polyvinylpyrrolidone (PVP, 2501000mg/ l) and antioxidants, such as citric acid, asc orbic acid or thiourea reduced the inhibitory effects of polyphones (Beyl, 2005) Agar is the most commonly used gelling agent to solidify the culture media. It physically support s plants to contact with medium while allowing aeration Generally, researc her s use agar at concentrations between 0.5% and 1.0%. Too high concentrations l ea d to poor growth; too low concentrations of agar causes a layer of liquid to form on the top of the gelled medium and results in hyperhydric plants (Singha, 1982) Agarose an d g elrite are alternative gelling agar. Adventitious shoot formation generally requires auxin and cytokinin. Auxin is involved in cell elongation. IAA is a natural auxin, but it is destroyed by light or autoclave ; o ther auxins are preferred, like IBA and NAA, which are light and heat stable. 2, 4 D is a strong promoter of callus induction and growth but it is not common for tree tissue culture. Cytokinins main function is to promote cell division and stimulate initiation of shoots, including BA, kinetin, 2 iP and Z eatin (Beyl, 2005).

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20 Micropropagation of Eucalyptus Explant and C ulture Sterilization Roots, lignotubers, anthers, shoot tips and microcuttings were used as the explants to establish micropropagation for Eucalyptus (Leroux and Vanstaden, 1991). The surface sterilization of E ucalyptus explants for in vitro culture was relatively eas y if juvenile material was available The sterilization of mature, field grown material proved difficult because of endogenous microbial contamination. Seedling and juvenile plants growing in glasshouses were eas ier to sterilize, especially if care. T aking appropriate care of stock plants was the major method to reduce the contamination (Leroux and Vanstaden, 1991). Eucalyptus tissue culture initially concentrated on callus culture from young seedlings (Sussex, 1965; Jacquiot 1964; Bachelard and Stowe 1963). Use of the nodes as microcuttings without multiplication was the subsequent objective and was achieved with E. grandis (Cresswell and de Fossard, 1974; Cresswell and Nitsch, 1975). Hartney and Barker (1980) reported media for multiplication and rooting for seedling nodal segments of 12 Eucalyptus species. G erminated seedlings of E. erythronema E. stricklandi hybrid (g em) from sterilized seeds were used as the explants and seeds were sterilized in 70% ethanol for 30 second and then 3% NaOCl for 20 min (Glocke et al. 2006) A s imilar method was reported for culture of E. regnans (Mountain ash) (Blomstedt et al., 1991), but the main difference was that the explant for former was the germinated shoots but for latter were seedlings nodes Sterilization efforts were unsuccessful in destroying the surface contaminants while keep ing the buds viable at the same time. Eu calyptus tissue was often killed by sterilizing solutions (Cresswell and d e Fossard, 1974). A severe sterilization treatment reported by Holden and Paton (1981) include d 75 min in saturated calcium hypochlorite, followed by 4h of UV

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21 irradiation. Using the field grown E. grandis they obtained minimal contamination and 50% survival of explants; 5% of these eventually produced shoots. When HgC l2 was used for E. marginata, all shoots were dead Comparisons among sodium hypochlorite, calcium hypochlorite, mercu ric chloride and zephiran (benzalkonium chloride) had shown that zephiran resulted in comparable levels of contamination, but improved levels of survival compared with the other sterilization methods Treatment of 15 -second in 1% zephiran, followed by 10% alcohol result ed in 5090% clean explants and 10 70% survived to produce shoots, whereas using sodium hypochlorite was common for all clean shoots to die (Durand -Cresswell et al. 1982). Direct S hoot C ulture There were many reports concerning direct shoot cultures for commercially important Eucalyptus species, including E. grandis (Sita and Rani, 1985; Wachira, 1997), E. niten (Gomes and Canhoto, 2003), E. globules (Salinero, 1983; Trindade et al., 1990; Benne tt et al., 1994; Schwambach et al., 2005) E. tereticornis (Das and Mitra, 1990; Sharma and Ramanurthy, 2000; Rao, 1988), and E. torelliana (Gupta, 1983) BAP alone or in combination with NAA were used to induce and multiply shoots in most of the reports, and IBA or NAA were used for rooting. Sometimes GA was used for elongation before rooting as the PGR. Some pretreatments were used to prevent the oxidation of phenolic compounds produced by Eucalyptu s. Organogenesis Significant progress ha s been made with regeneration of some Eucalyptus species via organogenesis, in species such as E. camaldulensis (Muralidharan and Mascarenhas, 1987), E. grandis (Warrag et al., 1991) and E. globulus although there are some limitations. Eucalyptus hypocotyls were reported to be more responsive to culture than other type s of explants (Kithara and Caldas, 1975). The type of explants of E. grandis used for callus inducti on

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22 was investigated by Hajari et al. ( 2006) and the shoot s stem s and leaves did not show significant diffe rence s With the exception of studies of Warrag (1991) who employed NAA and k inetin and Cid et al. (1999) who used TDZ, all reported organogenic callus induction in Eucalyptus has been accomplished with the combination of NAA and BAP (Muralidharan and Masc arenhas, 1987; Laine and David, 1994; Tibok, et al. 1995; Azmi, et al. 1997; and Mullins et al., 1997). T he organogenesis could provide a large number of plantlets for general vegetative propagation; h owever it may not be optimal for genetic engineering purposes. Somaclonal variation was possible through this organogenesis ( Warrag, 1991). Somatic E mbryogenesis Callus cultures ha ve been established from a number of Eucalyptus species explants from both juvenile a nd adult plant materials (Durnad -Cresswell et al. 1982). The most used hormones were the auxins IAA, NAA and IBA alone, or in combination with the zeatin or BAP to induce callus (Kitahara and Caldas, 1975; Bennett and McComb, 1982; DurnadCresswell et al., 1982). Enhanced somatic embryogenesis was achieved on B5 medium supplemented with 5mg/l NAA for E. citriodora (Muralidharan and Mascarenhas 1987) Eucalyptus somatic embryogenesis was re p orted by Sita (1986) and Ouyang et al. (1981), where they did not defin itely identify somatic embryos. Sita (1986) described formation of embryo-like structures, which did not develop into plantlets. More recently, somatic embryogenesis was reported for E. nitens and E. globulus (Bandyo padhyay and Hamill, 2000); but plantlet regeneration was not achieved. Pinto et al. (2004) reported the regeneration of somatic embryos from juvenile explants of E. globulus for the first time.

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23 Genetic Variation DNA Marker Genetic variation of trees was traditionally studied by progeny tests and provenance trials although the methods are expensive and laborious Genetic markers were introduced after 1980s to make the genetic variation analysis easier. Genetic marker is a known DNA sequence, which could be described as a variation. It could range from a single base -pair change, to a long one. There are commonly used types of genetic markers including Restriction fragment length polymorphism markers (RFLP), Random amplified polymorphic DNA (RAPD), A mplifi ed fragment length polymorphism (AFLP), M icrosatellite (SSR), and SNP (Single nucleotide polymorphism ). These markers could be further categorized as dominant and co -dominant markers Dominant markers allow for analyzing many loci at one time, e.g. RAPD and it is fast and efficient But, in diploids, a band obtained if either or both of the homologous chromo somes contain an amplifiable sequence which underlie the genotypes ( Falush et al., 2007) On the contrary, co -dominant could discriminate heterozygous from homozygous state in diploid organism (Weising et al., 2002) When a genetic marker is co -dominant all genotypes are distinguishable from one another (Holsinger et al., 2002). For m ost plant s, more than 60% genomes are believed to be c omposed of repetitive sequences (Kubis et al., 1998). Microsatellites, or simple sequence repeats (SSRs), are 1 6 bp tan dem repeated DNA motifs which may vary in the number of repeats at a given locus, and most tandem repeats are non -coding DNA. The number of copies of repeats commonly varied in different individual organisms. Microsatellite could be detected by PCR because DNA with more repeats had longer amplified fragment after synthesize in PCR reaction. Amplified fragments

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24 were separated with the use of gel electrophoresis to produce a serious of bands, which quantify the population genetic difference (Pierce, 2005) T he SSR a ssay is easy to perform and often prod uces very polymorphic markers, and it is a highly attractive tool for studies at the population level (Echt et al. 1996) mainly because they are typically co -dominant and display many alleles per locus (Hardy, 2002). In addition, they are abundant dispersed in plant genomes and highly polymorphic allowing precise discrimination even of closely related individuals (Brondani et al., 1998). The disadvantage of SSR is that it is time -consuming to identify th e single -locus marker from genomic library for new species Thus the use of this marker has some limitation. Another disadvantage is the null allele by SSR, resulting in the underestimates of heterozygosity (Wang and Szmidt, 2001) The presence of microsa tellites in plant genomes was first reported in forest trees (Conditt and Hubbell, 1991). Then microsatellites have been reported in several forest -tree species such as Pinus radiata (Smith and Devey 1994) Pinus stro b u s (Echt et al. 1996) and Swietenia humilis (White and Powell, 1997) In Eucalyptus nitens 4 microsatellites were reported (Byrne et al. 1996) Data Analysis for Markers Population genetics focus on the genetic variation in terms of origin and distribution in populations of organism at or below species level (Templeton, 2006) Normally, mutation, nonrandom mating, natural selection, genetic drift and finite population size result in gene freq uency change, which lead to population structure. Population structure always includes system of mating for population, size of population and genetic change with other populations (Templeton, 2006) He (Heterozigosity) and Fst (fixation index) are often u sed to measure

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25 structure at population and subpopulation level while different methods are used to quantify genetic distance between subpopulation or individual s How the different markers were applied to detect population structure was addressed by Pritchard and Roseberg (1999). The model assumed that there were K populations characterized with different allele frequency at each locus and individuals were classifi ed into different populations on the basis of their genotypes (Pritchard et al., 2000) Broadly speaking, there are two ways to cluster genetically similar individual s : distancebased methods and model -based methods. Distance -based methods estimate gene tic distance between every pair of individuals, thus they are easy way; model -based method are run on the assumption that individuals from clusters are randomly taken from parametric model, which are finished by statistical method (Pritchard et al., 2000) Model -based clustering methods were developed by Pritchard. The program structure cluster s the individual organisms into different populations on the basis of genotype data from SSR (Pritchard et al., 2000) The model assumed that markers are not in link age disequilibrium within subpopulations. The version 2.0 allows the use of null alleles which offset the disadvantage of SSR In the data file, genotype data and missing genotype data are input Some modeling are needed to decide by users by structure, including how long to run the program, what the ancestry model is ( no admixture or admixture ), and allele frequency models (independent or correlated). In the estimation of K, different values of K are run and the output of Estimated Ln Prob of Data is t he result of Pr(X/K). Many runs should be needed to verify the consistency of the result. If the variability in different run is high, longer runs are needed (Pritchard et al., 2000)

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26 If ancestry model i s admixture, the vector Q is used to represent proportion of individual originated from population K (Pritchard et al., 2000) Different forms of plots are shown in the output to reflect Q and Fst. In the text output for each individual inferred clusters, estimated membership in each cluster, and alle le frequency are showed. Spatial Pattern Analysis of Genetic Diversity ( SPAGeDi ) is a computer package, designed to analyze the spatial genetic structure of mapped individuals and populations using genotype data of ploidy level (Hardy and Vekemans, 2002) The program requires the information for each individual: one to three spatial coordinates, value of a categorical variable and genotype at each locus of a codominant marker (missing data are allowed). The program first computes pairwise statistics show ing genetic relatedness for all pairs of individuals. Then by averaging the pairwise statistics and regressing them on spatial distance and on logarithm, t he association between these values and pairwise spatial distances are obtained (Hardy and Vekemans, 2002) At the individual levels, m any different statistics were calculated in the program, including: kinship coefficient (Loiselle et al., 1995; Ritland, 1996), relationship coefficient by Queller and Goodnight (1989) Hardy and Vekemans (1999), Lynch and Ritland (1999), and Wang (2002) ; fraternity coefficient described by Lynch and Ritland (1999) and Wang (2002); and genetic distant measure by Rousset (2000) At population level, Fst, Rst (an Fst analogue but based on microsatellite allele size, Slatkin 1995), and Ds (Neis 1978 standard genetic distance) are provided (Hardy and Vekemans, 2002)

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27 CHAPTER 3 MATERIALS AND METHOD S Twenty t hree clones were propagated by traditional cutting, mini -cutting, and / or micropropagation (Table 3 1). C utting Method Plant Material Four well tested E. grandi s clones ( G1 G2 G3 and G4 ) wer e initially selected. Thirteen year -old trees with the required superior growth condition were felled in Tampa Port Authority, Tampa, FL, USA in 2005. New clones were also t ried, including 6 E. grandis clones ( G34 G36 G37 G43 G48 and G51 ) from Southwest Ra nche s 9 E. amplifolia clones ( A1 A2 A3 A4 A5 A6 A7 A8 and A9 ), and 4 C torelliana clones ( T1 T2 T3 and T4 ) from C&B Farms, Clewiston (Table 3 1) Method Four well tested E. grandis clones ( G1 G2 G3 and G4 ) were taken by 3 stocks to test the factors affecting the cuttings, including the origin of the cuttings and the season of the cutting. First stock of material s was taken in August, 2006 from the crow n of some 13year -old trees. After that, the old trees were cut down to induce new sprouts. The second and third stocks of materials were taken from the new sprouts of the felled trees in November 2006 and June 2007, respectively. The comparison between first and second stock indicated which origin of cuttings was better for propagation. The difference between the second and third stock showed the differen t seasons effect on the efficiency of rooting the cuttings.

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28 Table 3 1. Location, age ( year s) and clone numbers by 3 methods (cutting, minicutting and microcutting) for E. grandis E. amplifolia and C. torelliana clones clone information propagation method and time Aug,06 Nov,06 May,07 Jan,08 Jun,07 Sep,08 Species clone Location Age cutting cutting cutting mini micro micro E.g G1 TPA 13 42 44 44 75 135 G2 TPA 13 92 61 61 78 85 G3 TPA 7 50 89 89 98 69 G4 TPA 13 84 229 229 109 90 G34 SRWC 100 4 36 146 280 G36 SRWC 100 4 49 48 G37 SRWC 100 4 58 79 147 10 G43 SRWC 100 4 38 67 12 G48 SRWC 100 4 35 57 G5 1 FC 2 90 46 E.a A1 SRWC 80 8 14 A2 SRWC 80 8 14 A3 SRWC 80 8 11 A4 SRWC 80 8 6 A5 SRWC 80 8 17 A6 SRWC 80 8 58 20 A7 SRWC 80 8 16 15 A8 SRWC 80 8 32 18 A9 SRWC 80 8 26 18 C.t T1 C&B 15 16 T2 C&B 15 19 T3 C&B 15 20 T4 C&B 15 27

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29 New clones of E. grandis (G34, G36, G37, G43, G48, and G51), and E. amplifolia ( A1 A2 A3 A4 A5 A6 A7 A8 and A9 ) were tried to determine the most efficient method for propagation. Healthy branches wi th leaves were selected and cut into 30 40 cm sections as the starting materials. The branches were covered with wet paper towel, placed into the polyethylene bags in a container filled with ice and transported back to greenhouse. Collect ion date and clo ne number were recorded on the bag. As possible, all the cuttings were made as soon as they got in greenhouse. If not, branches in polyethylene bags were placed in cooler for up to 3 weeks. All plant materials involved in the experiments were made cutting very soon. Cuttings were made by re -cutting the branches into 10 12cm long sections, with 2 nodes each one near the upper segments and the other near the base. Two half leaves were left on the cutting They were put in the 20% Scotts Banrot Broad Spectrum fungicide (4% wettable powder) for 5 seconds and then 2 cm base of the segments were treated by dipping into Green light F Rooting Hormone (active NAA 0. 1%) powder to induce rooting. C uttin gs were inserted vertically 5cm to 7 cm in to deepots containing moist medi um consisting of 1 peat: 1 perlite: 1 vermiculite (v:v:v) Fifty cuttings for each treatment with 2 replications were made. After 8 weeks, rooting percentages were recorded. Rooted cuttings were transplanted into de e pots containing the same medium and retained under intermittent for 4 weeks. Then they were transp lanted into 15 cm diameter big pots and moved out of the greenhouse to the shed outside and misted ev ery 30-min. After another 8 weeks, they grew up to 1 m high and were taken to the field.

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30 Mini -C utting Method Plant M aterial The healthy macrocuttings could work as the stock plants for mini -cuttings after they were successfully propagated. Clones of E. grandis G1 G2 G3 G4 G34 and G37 were tried. Method Twenty weeks after the macrocuttings rooted the mini -cuttings were made by cutting the macrocutting branches into 7 8 cm long sections, with 1 node near the upper segments and the other near the bas e. Cuttings from the main stem, the top part of the lateral branches and the base part of the lateral branches were 3 treatments to determine the optimum part of the stock plants for most efficient rooting All the cuttings were left with 2 half leaves. M ini -cutting were dipped into Rootone F BrandRooting Hormone (0.01% NAA) powder and then put into the soils in the tubes. Twenty -five cuttings for each treatment with 2 replications were made. After 8 weeks, rooting percentage was recorded In addition, in order to see how long the macrocutting s worked as stock plants, mini cuttings were collected after stock plants surviving 3, 6 and 9 months Also mini -c utting and traditional cutting rooting efficiency were compared Micropropagation Plant Material and Sterilization Stem cuttings and mini -cuttings which survive successfully may s e r ve as donor plant s for micropropagation. E. grandis (G1 G2 G3 G4, and G34 ) and E. amplifolia (A6 ) clones which propagate d successfully as cuttings and mini -cuttings were used as the source of explants in tissue culture. Healthy, vigorous and green branches were collected and immediately placed into water containing 2 drop s of Tween 20. All the adhering dust was removed by washing with tap w ater

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31 for 1 hour. Then cuttings were cut to smaller sizes and under went sterilization The small cuttings were immers ed into a beaker with or without 70% ethanol for 30 seconds, followed by soaking in 10% to 20% bleach solution with 2 drops of 2 tween for 20 minutes to find best sterilization method Constant agitation carried out in a laminar transfer. Then cuttings were rinsed in sterilized water fo r 3 times to remove the chlorine All these p rocedures were performed in the laminar flow hood under aseptic condition s The following procedures and conditions were shared in all the steps. The basal media of MS or DKW was used. The other components in the media include d 8g/L agar, 20g/L sucrose and 1g/L PVP. All media were adjusted to p H between 5.6 and 5.7 a nd autoclaved at 121oC for 30 min. Establishment of Culture s E xplants were cultured in Petri dish es (100*25 mm) containing pre initia tion medium MS to screen for contaminated explants. Soaking, charcoal, and PVP in media were tested to find optimal phenolic chemicals prevention method. The contaminated and phenols -producing explants were thrown away. After 7 days, decontaminated explants were cultured on MS with 0.5mg/l BAP, Kinetin, 2 iP (no PGR as control) to induce shoot regeneration. Shoot Multi plication MS, DKW and WPM were used as the basal media. Basal media supplemented with different concentration s of BAP (0.1, 0.4, and 1 mg/L) individually and in combinations with NAA (0.1mg/l) were u sed for shoot multiplication. Fifteen s hoot s per clone f rom the initiation step were assigned randomly to each treatment. Five explants were placed in one Petri dish for one treatment with 5 replications. Shoot multiplication was assessed after 3 weeks in culture by counting clearly visible shoot s approximately 2 mm or longer.

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32 After the first round, explants continued to generate new shoots, and subculturing provided further multiplication. S hoots that formed were subdivided and subculture d in fresh multiplication medium every three weeks. Eight generation s of subculture w ere continued and subculture multiplication rate s, i.e, how many shoots longer than 2 mm were produced per shoot were recorded. Samples used to get data were the third generation shoots. Shoot Elongation and Rooting DKW w as used as the bas al media. Various combinations of NAA and BAP were tested to optimize the propagation system. NAA and BAP combinations test were: 0. 1 /0, 0. 1 /0.1, 0.1 /0.5, 0.5/ 0 0.5 /0 .1 0.5 /0. 5 1/ 0 1/ 0.1 and 1 /0 .5 (mg/l) Shoot length (average length for longest 3 sho ots for one cluster ) and shoot multiplication ra te for each subculture w as recorded after 3 weeks F ive explants were placed in one Magenta vessel (Magenta, Chicago, Il, USA) with 3 replications for one treatment Samples used to get data were the third generation shoots. The individual shoots longer than 30 mm were picked and the basal leaves were discarded to insert vertically into the Magenta vessels containing MS with NAA (0, 0.5, 1, or 2 mg/l) Different concentrations of bas al medium (1/2 MS and MS ) were t ried and 0.1 2 mg/l NAA or IBA was added; rooting percent rooting length and rooting number were recorded after 3 weeks Five replications for every treatment for rooting were recorded. Acclimatization Plantlets with roots no longer than 10 mm w ere transferred to a greenhouse. Agar was removed from p ro pagules without root damage and roots were washed in a solution of fungicide to prevent contamination. Plantlets were planted in trays containing a potting mixture (vermiculite: perlite = 1:1 ( v : v) ) and maintained un der the plastic lids for 1 week. Plantlets were then maintained under mist for 4 weeks. The mist lasted 30 seconds every 2 minutes.

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33 Data Collect ion and Statistical Analysis The design of all experiments was a complete randomized block For cutting, each treatment involved 5 to 10 plants with 3 time replications F or multiplication and elongation, 15 replications were used. Rooting treatment was replicated for 5 times. Graph Prism 4.0 was used for data ANOVA. Genetic Fingerprinting Plant Material Sixty E. grandis clones (Appendix C) in Florida were obtained as the plant material for DNA extraction Total genomic DNA was extracted from adult lea ves The protocol was described earlier (Brondani et al. 1998). Polymorphism of Microsatellite Loci Eight microsatellite l oci (EMBRA2, EMBRA28, EMBRA10, ES76, EMBRA37, EMBRA63, EG62 and EG65) were selected for genetic information These loci were developed earlier of Brondani et al (2002). PCR amplification was performed and products were separated and sized according to method by Brondani et al. (1998). The allele size and frequency for each locus were recorded. Expected heterozigosity (He) were estimated for each locus by He =1 (Pi)2 where Pi is the frequency for i th allele. Population Structure We use d Structure 2.0 to analyze the population structure. This is a model based clustering method for inferring population structure using genotype information obtained from markers. The model assumes that there are K populations which are cha racterized with different allele frequencies at each locus. H ow long to run the program is one of major issues to consider and two factors requiring deci sion : the burnin length (run the simulation before collecting data to minimize the effect of the starti ng configuration) and how long to run the simulation after the

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34 burnin g to obtain an accurate parameter estimate. Normally, a burning of 10,000100,000 is preferred The number of individuals (60), the number of loci (8) and value of missing data ( 9) wer e entered in data input. When setting the parameter, the run length burnin g was 30 000 and the number of MCMC steps after burnin g was 50 000. The a ncestry model selected w as Admixture with population data and allele frequency model was selected as Allele frequency independent. The model was run after K value (the number of populations) was set from 1 to 6 Then the result s were compared. S PAG e D i SPAGeDi estimate d genetic distances between populations or relatedness coefficients between individuals using genotype data from markers (Hardy and Vekemans, 2002). During loading the datafile, name of the desired SPAGeD i file was entered and analysis level selected: population (F statistics) or individual (kinship coefficient). The obser ved allele number and fr equency for 8 loci were used to generate the 3600 kinship coefficient s (Loiselle et al., 1995) reflecting genetic distance between each pair of 60 individual clones.

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35 Spatial genetic structure was analyzed using kinship coefficients estimated relative to a sample of genotyped individuals between 2 individuals (Table 3 2) Table 3 2 Relationship and kinship coefficient (Weir et al., 200 5 ) k 2 k 1 k 0 k 1 /4+ k 2 /2 Twins 1 0 0 0.50 Full siblings 0.25 0.5 0.25 0.25 Parent child 0 1 0 0.25 Double -first cousins 0.125 0.375 0.5625 0.125 Half siblings 0 0.5 0.5 0.125 First cousins 0 0.25 0.75 0.0625 Unrelated 0 0 1 0 k0 = the probability of two individuals sharing 0 alleles k1 = the probability of two individuals sharing 1 allele k2 = the probability of two individuals sharing 2 allele e fficient

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36 CHAPTER 4 RESULT S AND DISCUSSION Propagation by Cutting s All the plant materials involved in the experiments below were made cutting very soon after collection. Collected plant materials kept in cooler up to 3 weeks exhibited drying resulting in reduced rooting efficiency Different genotypes influenced the rooting percent no matter what methods were used. S ource of plant materials affect ed the rooting percent s ignificant ly Control treatment (no auxin added) for 2 sources were both 0 (data not showed). The rooting percent (24%52%) from stump sprout s was significantly higher (p<0.001) than that from top of the crown (0 to 2%) for all genotypes The crown shoots induced at most 2% rooting and was therefore not a good source for cutting material. Although the top crown produced more juvenile materials compared with the other parts of the crown, it was not juvenile enough. The stump sprouts were more juvenile lead ing to higher rooting efficiency (Figure 4 1). Figure 4 1. Cutting rooting percent from 2 sources (crown and stump sprouts) for 4 well tested E. grandis clones ( G1 G2 G3 and G4 ). Ten plants were assigned to each treatment with 3 replica tion s for each clone. Within clones, means for a given origin with different lower case letters indicate significant differences according to Bonferronis HSD Multiple Range Test at p < 0.05 level. b b b b a a a a 0 10 20 30 40 50 60 G1 G2 G3 G4 Rooting percent (%)E. grandis clone crown sprout

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37 Rooting success from top crown cuttings did not vary much b y ge notypes. The lowest percentage was 0 for G1 and G2 and the highest percent was 2.3% for G4 T here was significant difference among genotypes from stump sprouts with 52% rooting for G2 followed by G1 with 32% The rooting percent for clone s G3 and G4 was only 24 25% (Figure 4 -1). Cutting season also a ffect ed r ooting efficiency. Generally, b ranches from stump sprouts in fall rooted better than in spring. Clone G4 rooted better in spring than in fall, but the difference was not significant. The season, genotype and interaction of the season and genotype were significant ly different ( Figure 4 2). Figure 4 2 Cut ting rooting percent in 2 different season s (fall and spring) for 4 well tested E. g randis clones ( G1 G2 G3 and G4 ) Ten plants were assigned to each treatment with 3 replication s for each clone. Within clones, means for a given time with different lower case letters indicate significant differences according to Bonferronis HSD Multiple Range Test at p < 0.05 level. a a a a b b b a 0 10 20 30 40 50 60 G1 G2 G3 G4 Rooting percent (%)E. grandis clone fall spring

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38 Cuttings from stump sprouts were made for 6 new and 4 old clones of E. grandis in fall. All the clones root ed but the rooting percent varied greatly among different clones, from 3% for G37 to 95% for G34 New clones G37 G48 and G51 rooted poor ly with only 3% to 6%. New clo ne G36 had similar rooting to old clones, including G1 and G4 New clone G34 achieved 95% rooting and was easier to root than the old well tested clones (Figure 4 3). Figure 4 3 Cutting r ooting percent for 10 E. grandis clones ( G1 G2 G3 G4 G34 G36 G37 G43 G48 and G51 ) in fall. Ten plants were assigned to each clone with 3 replication s Means with same letters are not significant different according to Tukeys HSD Multiple Range Test at p < 0.05 level Cuttings for nine E. amplifolia clones had lower rooting effi ci ency than E. grandis Clones A1 A2 A3 and A4 failed to root, while clones A9 A5 and A7 achieved only 3.85%, 5.88% and 6.25% rooting rate respectively. Clone s A6 and A8 had the highest rooting percent ages of 17% and 25% respe ctively (Figure 4 4) b c a b e b a d a a 0 10 20 30 40 50 60 70 80 90 100 G1 G2 G3 G4 G34 G36 G37 G43 G48 G51Rooting percent (%)E. grandis clones

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39 Figure 4 4 Cutting r ooting percent for nine E. amplifolia clones ( A1 A2 A3 A4 A5 A6 A7 A8 and A9 ). Five plants were assigned to each treatment with 3 replication s for each clone. Means with same letters are not significant different according to Tukeys HSD Multiple Range Test at p < 0.05 level. Woundingrelated compounds (WRC) were produced at the surface of the cut ting place and resulted in destruction of cell compartment, which, t hereafter, released catabolic enzymes. WRC improved rooting, by increasing auxin uptake or reducing conjugation or oxidation of auxin (Klerk et al., 1999) The traditional methods of vegetative propagation were not successful when used on adult phase trees for many Eucalyptus species (Cresswell and Nitsch 1975) Eucalyptus s pecies rooted easily from stem tissue if leafy cuttings were taken from young seedlings (Pryor and Willing, 1963). E. grandis had also been successfully propagated by stem cutting s by Paton et al. (1970), but there were no previous reports regarding rooting of E. amplifolia P revious reports indicated that potential rooting ability decline d with the age of donor plants for many tree species (Hackett, 1988) One of possible reasons for Eucalyptus species was that they produced lignotubers containing some buds and tissue In the mature shoots, lignotubers developed into mass structure ; in ju venile shoots it remained as before and shoots originated from lignotubers regenerated some juvenile leaf which had the potential to form a a a a b c b d b 0 5 10 15 20 25 30 A1 A2 A3 A4 A5 A6 A7 A8 A9Rooting percent (%)E. amplifolia clones

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40 adventitious roots (Hartney, 1980). In our study, shoots from the crown h aving no potential to root was possibly becau se lignotubers in mature cuttings were unable to produce shoots anymore thus prevented further root formation. There were also other factors contributing to the declin ation of rooting potential, including: auxin, other plant growth substances (endogenous ABA, GA, and ethylene), rooting cofactors and promoters, and rooting inhibitors (Hackett, 1988) Rooting cofactors, promoters and inhibitors influenced rooting ability in Camellia species (Richards, 1964) and in pear cultivars (Fadl and Hartmann 1967) E. grandis cuttings from both sources increased rooting percentage due to the treatment of exogenous IBA, reflecting that a uxin played an essential role in determination of rooting ability (Fogaca and Fett Neto, 2 005) Previous reports indicated that auxin enhanced rooting for Eucalyptus especially for difficult to root species, e.g., E. globulus (Fogaca and Fett Neto, 2005) By contrast, difficult -to root species E. amplifolia were not improved better by treatmen t of auxin than E. grandis indicating that there were also other factors inhibiting rooting for E. amplifolia In our study, mature plant material from crown still did not improve rooting efficiency with IBA treatment as much as juvenile material from sp rout. In this case, absence of exogenous auxin was not a key factor to inhibit rooting efficiency for E. grandis cuttings from mature materials. Paton et al. (1970) had isolated from adult tissue of E. grandis rooting inhibitors, the concentration of which increased with the decreased rooting ability of stem cutting. He indicated that adult E. grandis had high rooting inhibitor content and was difficult to root, while the juvenile E. grandis was still classifie d as easy to root.

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41 Harrison Murray and Thompson (1988) showe d that seasonal tim e when cuttings were taken play ed an important role in rooting. Some easy -to root species could be rooted readily throughout the whole year, while for difficult -to -root species could only be rooted during the optimal season (Hartmann, 2002) E xo genous IAA was the essential factor for easy -to -root plants, but not for difficult to root cuttings (Porlingis and Therios 1976) because treatment of plants with auxin enhance root initi altion for juvenile cuttings, however they promote cell division without root initiation for mature or difficult to root cuttings (Hackett, 1988) More IAA w as found in juvenile material s than in mature ones and they also varied in different time. Wu and B arnes (1981) measured highest concentration of IAA in summer for Rhododendronponticum and Rhododendron Britannia. In this case, possible reason for cutting response with different time was that endogenous IAA lev el s were varied when the season changed. In conclusion, E. amplifolia a difficult-to -root plant may be inhibited by some rooting inhibit ors and treatment of exog enous auxin cannot significantly overcome low rooting efficiency Cuttings for E. grandis from juvenile materials rooted better than th at from mature materials probably due to the absence of lignotubers and some rooting inhibitors produced in mature cuttings. Propagation by Mini -C utting s Clones and origin w ere very important factor s affecting the E. grandis mini -cutting rooting efficien cy T he cuttings from the bas al part of the lateral branches had the highest rooting percent age comp ared with stem terminals and the top part of branches despite genotypes regardless of which clone it was (Figure 4 5) The cuttings from stem terminals rooted less efficiently than cuttings from the other two origins. There w as no interaction of origin and genotype and all the genotype responded similarly. Hartmann (2002) indicated that semi -

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42 hardwood cuttings root better from lateral branches than termi nal branches for many species and this supported by our result. Figure 4 5 Mini -cutting rooting percent for 6 E. grandis clones ( G1 G2 G3 G4 G34 and G37 ) from 3 sources (stem, top of branches and base of branches) ; five plants were assigned to each treatment with 3 replication s for each clone. W ithin clones, means for a given time with different lower case letters indicate significant differences according to Bonferronis HSD Multiple Range Test at p < 0.05 level. A possible reason for this was that endogenous hormones developed in the plant influenced the mini -cuttings rooting efficiency. Endogenous IAA was synthesized at the top of the plants and different parts of the plants have different IAA level, which influence d the rooting efficiency. The abi lity to form adventitious root was restricted to the stem between the cotyledons and the 15th nodes and Eucalyptus showed a close link between rooting ability and the juvenility of the mother plant (Muckadell, 1959). In the mini -cutting experiment, our res ults regard ing explant origin were similar. New C lone G34 had the highest m ini -cutting rooting efficiency followed by G1 G2 G3 and G4 Clone G37 was not good compared with the well -tested clones (Figure 4 5 ). a a a a a a b b b a b a c c c b c b 0 5 10 15 20 25 30 35 40 45 G1 G2 G3 G4 G34 G37Rooting percent (%)E. grandis clones stem top of branch base of branch

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43 Comparing the difference between mini -cutting and macrocutting rooting for E. grandis the ranking of the rooting percent ages among different clones did not change by altering the cutting method. But rooting percent age by macrocutting was significantly higher (p<0.01) than that of mini cutting especially for clone s G34 and G2 In summary, the mini -cutting method did not improve the rooting efficiency but the method was feasible for propagation (Figure 4 6) Figure 4 6 Comparison of rooting percent for macro cutting and mini -cutting for 6 E. grandis clones ( G1 G2 G3 G4 G34 and G37 ). Each treatment was replicated 15 times for each clone. W ithin clones, means for a given time with different lower case letters indicate significant differences according to Tukeys HSD Multiple Range Test a t p Mini -cuttings were also tried for E. amplifolia and because of the low rooting rate from cuttings o nly clones A6 and A8 were involved and the rooting percentages were 5% and 4%, respectively (not showed in figure). This rate was even lower than the most difficult to root E. grandis clones. E. amplifolia was more difficult to root than E. grandis Although our study showed that mini -cutting had lower rooting efficiency than macrocutting, Stape et al. (2001) indicated that macrocutting system was replaced by mini cutting system for Eucalyptus plantations in Brazil due to its lower production costs, reduced branching, and improved root system (Higashi et al., 2000). a a a a a a b b a b b a 0 20 40 60 80 100 G1 G2 G3 G4 G34 G37Rooting (%)E. grandis clone mini macro

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44 Stock plant age for mi ni -cutting, just like with macrocutting also affected rooting efficiency Six month old shoot as the cutting material was best compared with 3 month and 9 month old material (Table 4 1 ). Three month old branches were too y oung and the shoot was too soft. The mist in the green house may have been too heavy for the young shoots and there was no success for these shoots. W h ereas 9 month old shoots were too old and the rooting percent declined compared with the 6 -month old branches In this case, the rejuvenation was necessary of the stock plants for successful rooting of mini -cuttings. Table 4 1 Effect of shoot age (3, 6, and 9 -month) on mini cutting rooting percent for 4 E. grandis clones ( G1 G2 G3 and G4 ) shoot age clone 3 month 6 month 9 month G1 0 18% 10% G2 0 15% 8% G3 0 14% 5% G4 0 13% 5% Each treatment involved 10 explants with 3 replicat ion s for each clone Micropropagation Explant and Sterilization Table 4 2 Decontamination rate of one -month -old shoots from stock plants and from mature trees of E. grandis and E. amplifolia Material source 70% etha nol no etha nol 10% bleach 20% bleach 10% bleach 20% bleach Shoots from stock plants 10% 6% 80% 20% Shoots from mature trees 0% 0% 0% 0% Each treatment involved 10 explants with 3 replicat ion s for each clone Ethanol and bleach concentration were 2 factors influencing the decontamination rate of the explants. It was difficult to sterilize mature shoots from field -grown trees without damaging the tissues (de Fossard et al., 1978; DurandCresswell et al., 1982), and our experiment confirmed this. Shoots collected directly from trees in field were all contaminated in culture

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45 (Table 4 2 ), so mature plants were always made cutting first. After 3 month, the vigourously grown cuttings worked as the stock plants to provide material for micropropagation. Although the period was longer, the sterilization result was much better. One -month -old fresh shoots from stock plants in greenhouse were preferred in our experiment. Ten percent bleach without etha n o l pretreatment resulted in 80% decontaminated healthy shoots, which was optimal, compared with treatment with eth a n o l and h igh level bleach, which burn the juvenile plants and inhibited the new shoot induction (Table 4 2 ). There was no significant difference between two species in this stage. Double sterilization with an interval of 48 hours between treatments reduced the contamination to some degree, but the effect was not very significant (data not shown) Ikemori (1987) artificially induced epicormic shoots on severed branches of E. grandis by placing the shoot in a greenhouse under intermittent mist irrigation. After 2 to 3 weeks, epicormic shoots approximately 30 mm in length were harvested from branches and used as explants. But in our study, this method did not work out. Leaves and nodes were used as the explant s to initialize the experiment. Leaves, whatever their age did not produce any shoots during 2 months in culture However, nodes without contamination induced some shoots in 2 to 3 weeks on MS basal media, with or without PGR. It was virtually impossible to sterilize mature, field -grown shoots without damaging the tissues. The ideal material should have many of the qualities of young juvenile shoots ( d e Fossard et al., 1978) Before collecting the materials, i t was necessary to keep t he stock plants dry and to spray them with fungicide several days before taking the required material (Durand Cresswell et al., 1982) Nodes from upper and lower branches of the main trunk and the coppiced shoots were used as the explants to see which source was suitable ; c oppiced shoots were considered as the

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46 desirable explant for this genus as they demonstrated higher survival. Coppiced shoots or branches collected from old trees were the main source for the explants in some reports, but in our study because of the limited plant material s and the contamination situation the shoots from coppiced trees were used as the plant material s for cuttings. Then, grown cutting worked as the stock plants to provide the node explants for tissue culture, wh ich made explants easier to sterilize and available all the time. Because even the sprouts from field trees were 100% contaminated. Many different explants were used to successfully initiate culture for E. grandis (Leroux & Vanstaden, 1991) Aseptic shoot s and flora buds were used to establish the culture from explants. Aseptic cultures had been established from stump coppice shoots (Furze and Cresswell, 1985) scion shoots (Franclet and Boulay, 1982 ), epicormic shoots (Ikemori, 1987) and from young and vi gorously growing shoots on mature trees (d e Fossard et al., 1977 ; Rao, 1988) In addition, both seedlings and juvenile plants were good source for shoot induction. G erminated seedlings from sterilized seeds were good source s of e xplants (Glocke et al., 2006) B ut to keep the genotypic characteristic in our study germinated seedlings were not suitable and the nodes or shoots from certain clone were used as the explants. In addition, shoot tips showed practically no contamination whereas nodal segments h a d levels of contamination higher than 50% (Gomes, 2003). Although the shoot tips were more difficult to obtain, the ir success rate in establishing aseptic cultures was much higher. However, nodes were widely used as explants due to availability Age and season of material s were also important factors determining the success in establishing the aseptic cultures (Grewal et al. 1980) T he optimum time was November to

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47 March and trees of the different genotypes g a ve widely different results (McComb and Bennett 1986) Our result indicated that age of the branches was very significant (p<0.001). Our sterilization method was similar to the other reports. A ll the papers shared the similar sterilization method by soaking the explants in the 70% ethanol for 30 to 60 seconds, followed by treatment in 0.05 0.1% (w/v) HgC l2 for 5 to 15 min, or 1 to 10 NaOCl or Ca(OCl)2 for 10 to 30 min ute s, followed by several rinses in sterile distilled water. Our study indicated that ethanol would burn the explants even though it ca n kill the contaminants. Shoot Induction In addition to de contamination, the survival rate of the explants in the initial stage was associated with oxidation of phenolic exudation released from the cut end of the stem. After 1 or 2 days of inoculation, browning of explants result ing from oxidation of phenolic compounds would kill the explants. PVP improve d the situation although it could not prevent it completely. Soaking of the explants in water was not good because the long time of exposure to water made the explants turn yellow. The possible reason for this was that the explants were too young and soft, and the soaking was not good for them. Charcoal did not have signif icant effect on preventing exudation of phenolic compound during the initial stage in these experiments Our results showed that combination of pretreatment of antioxidant PVP and high frequency changing media was effective to reduce the browning and death of explants due to phenolic compounds, although the successful rate was not high (Table 4 3). Table 4 3 E. grandis and E. amplifolia s hoot survival rate with pre -soaking, addition of charcoal and addition of PVP with change medium interval of 2, 5 or 14 days C hange of treatment T reatment 2 days 5 days 14 days pre -soaking 8% 5% 0 Charcoal 10% 7% 0 PVP 20% 10% 2% Each treatment involved 10 explants with 3 replicat ion s for each clone

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48 Reduction of oxidation of phenolic compounds was essential for successful explant culture. Explants tended to produce a brown exudate which prevented growth of shoots that survived. Before inoculating explants on culture media, some extra treatments were made to reduce oxidation of phenolic compounds because the phenolic substances could turn explant brown and even be lethal sometimes. Cresswell and Nitsch (1975) overcame the problem for E. grandis by soaking the sterilized explants in sterilized water for 2 hours and keeping in culture in darkness for first 7 days Similarly, Wachira (1997) indicated that initial soaking of explants of E grandis in distilled water, coupled with pretreatment in PVP and initial incubation at 5oC in darkness was necessary to reduce the accumulation of oxidized polyphenolic compounds which were associated with media browning and subsequent explant death. Das and Mitra (1990) reported that to prevent the death of buds because of exudation of phenolic substances, the inoculated media had to be changed 3 4 times at 2 3 days interval from the beginning of the culture to overcome this although the effect was little. Sharma and Ramamurthy (2002) also attempted to prevent this problem by 2 treatments, incubation under darkness or antioxidant pretreatment. The result was that the darkness treat ment indeed had some positive effects on some genotypes while the antioxidant treatment did no good. Joshi et al. (2003) reported that to overcome the problem, media should be changed frequently at short intervals and PVP, ABA and charcoal failed to get de sirable results. Although the previous efforts were tried, the results were still not good. When m edi a supplemented with P GR were compared with the media free of PGR, PGR free media were optimal for shoot induction. Any basal media with 0.5mg/L and higher concentration BAP, Kinetin 2 iP or BA restricted the shoot development. The explants on

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49 media with BAP and Kinetin only produced 1 or 2 very short, small, and compact shoots ; the media supplemented with BA or 2 -iP could not induce any shoots (Table 4 -4 ). Compared with media supplemented with PGR, PGR -free media was optimal for adventitious shoots induction, and after 7 days healthy and long shoots without any callus appeared at the node s. The response of E. amplifolia and E. grandis had no difference in this stage. Table 4 4 E. grandis and E. amplifolia shoot induction in medi a with different PGR combination s Plant growth regulation combination (mg/l) Shoot and callus observation BAP Kinetin 2iP IAA shoot number shoot length(mm) callus 0.5 0.1 0 + 0.5 0.1 12 1 + 0.5 0.1 0 + 0.5 12 1 / 12 5 / + indicate there is some callus; / means there is no callus. Each treatment was replicated 15 times for each clone. Although the induction stage was very necessary, the regeneration frequency of primary explants had no effect on the further success of the micropropagation if a few new clean shoots was obtained ( Sharma and Ramamurthy, 2000). In study good quality shoots were obtained from hormone free media although the shoot number was only 1 or 2. After this, the shoots were transferred to multiplication medium for further culture. MS was used in m ost previous reports as the basal medi um for micropropagation Twenty to 30 g/L sucrose and 7g/L agar were used for initializing and maintaining explants in most of the literatures. Sharma and Ramamurthy (2000) reported that agar was more desirable since phytagel would lead to some vitrification of plants. Higher concentrat ions of cytokinin would make the shoot s small and compact which were not good. Our observation also confirmed this point.

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50 Vitrification, also named as tissue waterlogging or hyperhydric transformation, was the phenomenon that shoots become glassy and transparent, with swollen, brittle leaves and stems (George and Sherrington 1984) Boulay (1983) found that when vitrification occurred in shoot multiplication cultures, usually only a few buds were affected. He also indicated th at vitrified buds were difficult to multiply and almost impossible to root. DurandCresswell et al. (1982) recommended a reduction in cytokinin concentrations to reduce this problem M odification of growth medium (Zimmerman and Cobb, 1989) and adding of Ba cto -peptone fractions (Sato et al., 1993) were also employed although they reduced multiplication rate simultaneously (Zemmerman et al., 1995) Eucalyptus shoot cultures frequently produce d a white callus, sugary in appearance on leaf surfaces, stem nodes and in the axils of leaves of E. dalrympleana, E. ficifolia E. grandis E. regnans and E. gunnii ( de Foss ard and Bourne, 1976; Durand-Cresswell et al., 1982). Boulay (1983) recommended frequent subculturing every 15 days to prevent this problem. Our stu dy confirmed the need for frequent media transfering to prevent vitrification. Shoot M ultiplication Mean multiplication rate in DKW medium was significantly higher than the rate in MS or WPM for all the clones (p<0.0001). H ighly significant variation was also obs erved in different level of PGRs (p<0.0001) and interaction of basal media and PGR (p<0.0001). No matter what the basal media and genotype were, 0.4mg/l BAP was optimal for the shoot multiplication, while DKW with 0.4mg/l BAP was optimal medium and the multiplication rate was 5.7 When the BAP concentration increased to 1.0mg/l, more callus were produced The addition of NAA did not improve the multiplication rate, compared with 0.1 mg/l BAP, but it did improve elongation of the shoot s (Table 4 5 ).

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51 Table 4 5 Multiplication rate on 12 multiplication medi a (4 levels of PGR: 0.1, 0.4, and 1mg/l BAP combined with 0 or 0.1mg/l N AA on 3 basal medi a: MS, DKW, and WPM) across 5 E. grandis clones ( G1 G2 G3 G4 and G34 ). PGR Multiplication rate NAA(mg/l) BAP(mg/l) MS DKW WPM 0 0 1a 1a 1a 0 0.1 1.5b 1.5b 1.4a 0 0.4 3. 8 c 5. 7 c 3.3 c 0 1 3.2c 4 .2 c 3c 0.1 0.1 1.2ab 2b 1.2a The third generation shoots were used in this experiment. Each treatment was replicated 15 times for each clone. W ithin column means for a given time with different lower case letters indicate significant differences according to Tukeys HSD Multiple Range Test at p Multiplication rate means how many shoots longer than 2 mm were produced on single shoot. Genotypic effect was very strong. The difference of multiplication rate between shoots in DKW with 0.4 mg/l BAP and MS with 0.4 mg/l BAP was very significant for C lones G1 and G2 (<0.0001), and for clones G3 and G34 the difference was also significant (p<0.001) (Figure 4 7) In addition, the multiplication rate varied greatly for different clones on the same media. The highest rate was 8 for Clone G34 while the lowest rate was only 3 .5 for clone G4 on DKW with 0.4 mg/l BAP. In this case, the optimal medium for multiplication was DKW with 0.4 mg/l BAP Figure 4 7 Shoot multiplication rate on media ( DKW+0.4mg/l BAP or MS+0.4mg/l BAP ) for 5 clones ( G1 G2 G3 G4 and G34 ) respectively Experimental material was used from th ird generation explant. Each treatment was replicated 15 times for each clone. W ithin clones means for a given time with indicate significant differences according to Tukeys HSD Multiple Range Test at p 01 level ** indicate significant differences according to Tukeys HSD Multiple Range Test at p 001 level Multiplication rate means how many shoots longer than 2 mm were produced on single shoot. ** ** ** 0 2 4 6 8 10 G1 G2 G3 G4 G34Multiplication rateClone DKW+0.4mg/l BAP MS+0.4mg/l BAP WPM+0.4mg/l BAP *

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52 Subculture s were made at 3 week interval s T he shoot number (> 2 mm) produced by each generation of each clone was quite different (Figure 4 8). Commonly, generations 5 and 6 produced more shoots. The multiplication rate of G1 and G34 reached 1 2 and 8.7 per explants at the generation 5 respectively. Comparatively, Clone s G3 and G4 only produce d 5 and 4 shoots respectively. After 5 to 6 generation s the multiplication rate began to decline gradually. At the 8 th generation, the multiplication rate was as slow as the initial rate. Figure 4 8 Multiplication rate ( how many shoots longer than 2 mm produced on single shoot per 3 weeks) for 8 subculture generations on DKW with 0.4 mg/l media for 5 E. grandis clones (G1 G2 G3 G4 and G34 ). Each treatment was replicated 15 times for each clone. For all the E. grandis clones, mean multiplication rate on DKW medium was significantly higher than the rate on MS or WPM for all the clones (p<0.0001). There were no significant difference between WPM and MS (Table 4). Although MS was used as the basal media for majorit y of the Eucalyptus culture, suitability of DKW for other woody plants, like Ulmus procera has been indicated by for the healthier shoots produced on this (Fenning et al., 1993). Glocke et al. (2006) used MS, B5, WPM, AP, TK and QL as the basal media and found that WPM was most successful and better than MS because shoots were healthier. Gomes and Canhoto (2003) also used different basal media, including MS or FSB to culture E nitens and 0 2 4 6 8 10 12 14 1 2 3 4 5 6 7 8Multiplication rateGeneration G34 G1 G2 G3 G4

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53 observed that the best multiplication rate was obtained when they w ere cultured on medium containing the major nutrients and minor elements of MS medium, t he organics of DF medium. Mg2+, K+, Ca2+, NO3, SO4 2were higher in D K W than in MS ; t hese inorganic salts were lowest in WMP (Table 4 6). In this case, for some specif ic clones of E. grandis further investigation is needed to see which mineral salts are more needed for the clones. Table 4 6 Component ( Zn (SO4)2 Zn(NO3)2, K2SO4, KNO3, Mg(SO4)2 ,Ca(NO3)2, CaCl2) comparison between DKW MS and WPM basal medium Component KDW MS WPM CaCl2 112.5 332.2 72.5 Ca(NO3)2 1367 0 386 Mg(SO4)2 360 180 180.7 KNO3 0 1900 0 K2SO4 1600 0 990 Zn(NO3)2 17 0 0 Zn(SO4)2 0 8 8.6 PGR s w ere very important in multiplication stage, and BAP was the most common ly used cytokinin for Eucalyptus Warrag (1989) reported that MS medi um supplemented with BAP alone in the range of 0.4mg/L to 0.8mg/L was optimal for shoot multiplication of E. grandis Wachira (1997) initiated the experiment by using nodal explants of E grandis and found that shoot regeneration was efficient from 0 to at least 3 mg/L BAP. Bunn (2005) reported that BAP produced the highest shoot multiplication rates when compared to equivalent concentrations of other cytokinins ( K inetin or Z eatin), but high conce ntration of BAP would produce hyperhydric shoots. Our finding also showed that for the E. grandis BAP at 0.4 mg/l w as optimal. In other papers, combination s of cytokinin and auxin were used in this stage. The optimum shoot regeneration was achieved at 0. 2mg/L BAP combined with 0.4 mg/L or 1mg/L NAA. In culturing Eucalyptus hybrids of E. tereticornis E. grandis (Joshi et al 2003 ), single shoot were multiplied in media with 1.0mg/L BAP and 1.0/L NAA. After this stage, multiplication rate

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54 increased with every subculture, while the bud clumps were more compact and rather smaller in size. To overcome this, subculturing was carried out in MS+ 1.0 mg/l BAP without NAA. The similar find ing was also reported by Sharma and Ramamurthy (2000) culturing E. teretico rnis A higher multiplication rate in some Eucalyptus was obtained on MS with BAP alone at 0.4 mg/L to 0.8 mg/L or with 0.1mg/L NAA. O ther reports indicated that cultures were good at the beginning on the MS with the combination of 0.41mg/L BAP with 1mg/L NAA. High NAA made the culture compact and smaller, and in this case, after some generations of subculture, only 0.5 1 mg/L BAP in the medi um were effective for multiplication. Elongation The BAP and NAA interaction effect was high (p< 0.01). When BAP was 0.1 or 0.5 mg/l, shoot length decreased with increasing NAA concentration s But when there was no BAP, the shoot elongation increased with NAA concentration (Figure 4 9). Elongation of shoots was best on 0.1mg/l NAA with 0.1 or 0.5mg/l BAP. This combination increased shoot length to 2.5 cm after 3 weeks.

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55 Figure 4 9 Elongation in DKW plus NAA (0.1, 0.5 or 1mg/l) across 5 E. grandis clones ( G1 G2 G3, G 4 and G34). Each treatment was replicated 15 times for each clone. D ifferent lower case letters indicate significant differences according to Tukeys HSD Multiple Range Test at p 1 level. The third generation shoots were used for this experiment. In addition, BAP multiplied the shoots at the same time. Multiplication ra te was higher at 0.5 mg/l BAP than at 0.1 BAP no matter what the NAA concentration was (Table 4 7 ). In this case, before rooting, the plantlets could be transferred by clusters. GA was also tried in our experiment, but elongation was not good, and there were callus on the shoots. Table 4 7 Shoot multiplication rate in DKW plus NAA (0.1, 0 .5 or 1mg/l) across 5 E. grandis clones ( G1 G2 G3, G 4 and G34). NAA(mg/l) BAP(mg/l) Shoot number 0.1 0 1 a 0.1 0.1 2 b 0.1 0.5 5 e 0.5 0 1 a 0.5 0.1 3 c 0.5 0.5 4 d 1 0 1 a 1 0.1 2 b 1 0.5 2 b The third generation shoots were used for this experiment. Each treatment was replicated 15 times for each clone; different lower case letters indicate significant differences according to Tukeys HSD Multiple Range Test at p a ac ac c c c bc 0 0.5 1 1.5 2 2.5 3 0.1 0.5 1shoot length (cm)NAA (mg/l) BAP=0 mg/l BAP=0.1 mg/l BAP=0.5 mg/l

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56 There was no significant difference among four clones (Figure 4 10). There were no significant (p>0.05) difference for the shoot length in the 2 best media (0.1mg/l NAA with 0.5mg/l BAP; 0.1mg/l NAA with 0.1mg/l BAP). The suggestion was that media with 0.1mg/l NAA and 0.5mg/l BAP were used first to multiply the elongated shoots and then transferred to media with 0.1mg/l NAA and 0. 1 mg/l BAP for further elongation. Figure 4 10. Shoot elongation in DKW plus 0.1 mg/l NAA with 0.5 mg/l BAP or 0.1 mg/l NAA w ith 0.1mg/l BAP for 5 E. grandis clones ( G1 G2 G3, G 4 and G34), respectively Each treatment was replicated 1 5 times for each clone. The third generation shoots were used for this experiment. Quoirin et al. (1974) reported that a period of elongation following multiplication predisposes the shoots to root in response to rooting treatments. Although GA3 was not effective in our study, other papers had some contrast ing results. Shoots of 2 or 3 cm in le ngth were cultured on MS with 1mg/L GA3 for elongation for E. grandis (Sita and Rani 1985) WPM with GA3 w as chosen for elongation for E grandis according to Glocke et al. (2006) while 1/2 MS without any hormone gave good shoot elongation response for hy brid of E. tereticornis and E. grandis (Joshi et al., 2003 ). G ibberellins were tried by Franclet and Boulay (1982) for E. gunnii where they suggest ed the use of 0.1 mg/L of gibberellic acid with activated charcoal was very efficient Higher concentration of gibberellins tended to produce abnormal leav es and stems. The 0 5 10 15 20 25 30 35G1 G2 G3 G4 G34Shoot lenghth (mm)Clone 0.1 mg/l NAA+0.5mg/l BAP 0.1mg/l NAA+0.1mg/l BAP

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57 reason may be that the different species had different response to the GA (Gomes and Canhoto, 2003) In addition, combination s of auxin and cytokinin were also used for elongation. 2.5 mg/ l IBA and 1.0 to 1.5 mg/l Zeatin in MS were the best elongation media for hybrid of E grandis according to Warrag (1991). Because Zeatin was more expensive than NAA, the replacement of Zeatin with NAA was necessary. Auxins were suggested to act directly i n the induction of cytodifferentiation, elongation and cell division (Fukuda and Komamine 1985). The p ossible advantage of using high concentrations of auxin at the elongation stage was the improvement of subsequent rooting. McComb and Bennett (1986) stre ssed the carry -over effect from multiplication or elongation media on subsequent rooting ability. They mentioned that use of gibberellins for elongation seem ed to greatly reduce rooting. Rooting and Acclimation Different levels of NAA (0.1 to 2 mg/l ) induced rooting and the optimal medium was 0.5mg/l NAA because it produced higher root number s and shortest lengths P roducts of one subculture w ere good to go to greenhouse (Table 4 8 ). NAA and IBA did not differ significant ly for rooting percent but IB A induced slightly higher rooting percent ; MS and 1/2MS did not significantly influence the rooting percent but strength MS improved a little ; all the treatments achieved as high as about 95% rooting (Figure 4 11). Actually, rooting percent age was appro ximately 90% no matter what level of auxin was used, and auxin was only influence the shoot length and shoot number.

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58 Table 4 8 Effect of NAA on r ooting number and length in medium MS across 5 clones (G1 G2 G3, G4 and G34) NAA (mg/l) root number root length (mm) 0 1.8 a 6 a 0.1 2 b 6 a 0.5 3.5 c 8 ab 1 3 a 12 b 2 3 a 12 b Each treatment was replicated 5 times for each clone. D ifferent lower case letters indicate significant differences according to Tukeys HSD Multiple Range Test at p The third generation shoots were used for this experiment. Figure 4 11. Effect of MS concentration with 0.5 mg/l IBA or NAA on rooting percent across 4 clones ( G1 G2 and G3 and G4 ). Each treatment was repli cated 15 times for each clone. Rooting phase for many E ucalyptus species was the most critical and limiting step Hartney (1980) reported a rooting protocol involving two serial subcultures, while in the protocol reported by Rao (1988) and Das and Mitra (1990), three serial subcultures were used Moreover, three auxins together for the process of root induction were used ( Gupta and Mascarenhas,1987; Rao, 1988). In comparison, single subculture with only one hormone IBA was successful for E. tereticornis (Sharma and Ramamurthy 2000) Warrag (1991) also reported that rooting was better in a medium with only auxin and basal nutrients of reduced concentration for E. grandis 95% 95% 96% 96% 97% 97% 98% 1/2MS MS Rooting percent NAA IBA

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59 The combination of 2 mg/l of IBA and 1/4 MS with a p H in the range of 5 to 5.5 gave the best results. Increasing IBA to more than 2mg/l tended to produce more callus. Addition of BAP at 0.2 mg/l inhi bited rooting completely. In our study, rooting for the E. grandis clones w as not difficult to root and this was consistent with the other reports. Durand Cresswell et al. (1982) recommended that transfer from rooting media should be done as soon as a few roots were produced. Blomstedt et al (1991) reported that a ctivated charcoal had been used for elongation and successfully enhanced rooting in other Eucalyptus species. Also, basal medium formulation was also an important factor affecting rooting. Although strength MS and IBA slightly improved rooting efficiency it was not significant in our study Auxin has role in stimulating ce ll elongation, cell division, producing roots, and extending stems. Cytokinins main function is to cause cell division and release shoot apical dominance, thus promote growth of lateral buds (Gaba 2002) The different ratios of auxin to cytokinin have di fferent effects: high ratio induces rooting; intermediate ratio induces callus; and low ratio stimulates adventitious shoots. In our study, BAP alone at intermediate level had good effects on shoots multiplication because it promoted cell division; NAA at 0.1 2 mg/l promoted rooting. The combination of BAP and NAA could elongate stems and multiply elongated shoots simultaneously, especially when ratio of BAP to NAA was 1 to 5. PGR carry over effect was one issue needed to be considered for the micropropag ation (Bennett et al., 1994) The cytokinin added in multiplication medium had effect on subsequent rooting induction and degree was varied among different in Eucalyptus species; the reason was that rooting response was associated with the production of fl avonoids influenced by cytokinin (Bennett et al., 1992) Because E. grandis was easy to root, the influence was not obvious. On

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60 the contrary, auxin treatment for the rooting had an adverse effect on shoots sometimes, and needed to be avoided (Klerk et al., 1999) The reduced multiplication rate and leave abnormalities were present in culture of E. globules after three periods (Bennett et al. 1994). The combination of BAP with kinetin were tried and improved the response. In our study, declining multiplica tion rate with time also need s to be overcome and combination of different cytokinin s require additional test ing

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61 A B C D E F G H Figure 4 1 2 Propagation of E. grandis clones : A -sprouts from felled tree crown; B cuttings in the green house; C -sterilization of stems ; D -shoot multiplication stage; E ,F -shoot elongation stage; G -rooting stage ; H acclimatized plantlets from in vitro to greenhouse

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62 There were 3 physiological phases in adventitious rooting process, including induc tion, initiation and expression (Kevers et al., 1997) and success of rooting depended on the whole 3 phases. Auxin was essential for inducing roots; however it inhibited root formation (De Klerk et al. 1990) On the contrary, Bellamine et al. (1998) add antiauxins into medium after cuttings were treated with auxin previously and the result indicated that auxin was required even in expression phase. According to this study, there was no need to remove auxin from medium in later rooting stage. IBA was more stable than IAA and NAA (Fogaca and Fett Neto 2005) IBA was better than NAA in E. globules and E. saligna and was possibl y associated with NAAs longer persistence remaining in tissue (Fogaca and Fett Neto 2005), which inhibited rooting Our result s wer e in agreement with this statement. Quantitative trait locus (QTLs) controlling vegetative propagation in E. grandis have been identified though g enetic mapping by Grattapaglia et al. ( 1995). E. grandis clone was crossed with E. urophylla clone to produce F1 individuals, which were used for construction of genetic maps by RAPD markers. QTL map related to traits of vegetative propagation response of E. grandis clones were made. Therefore phenotype of fresh weight of micropropagated shoots, stump sprout cuttings, and % rooting cuttings were measured respectively ; result s showed that there were 10 QTL detected for micropropagation, 6 for stump sprouting ability, and 4 for rooting ability. There were almost no overlap positions among 3 differe nt propagation traits. It indicated that cutting and micropropagation method were not controlled by the same locus. However, the plantlets responded very similar in 2 methods, which was contrast to the research above.

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63 Different genotype response in cutting and micropropagation could be explained by genetic diversity in the following parts. Clones that were easier to propagate were chosen for mass propagation. Cost Reduction The greatest operational cost of propagule production was labor. No reliable figures were available, but it was estimated to account for 40% to 60% of the total cost. Franclet and Boulay (1982) estimated the cost of E ucalyptus propagules was three times that of seedlings. Most of the cost consiste d of manual cutting and transferring of cultured materials. Automation of some or all parts of the system could greatly reduce this cost. Because of clump transfer, and the uniformity and even age of shoots in this study at the multiplication and elongatio n phases, this might be an excellent system for automation. Bioprocessor as was described by Levin et al. (1988) could be used for separation, sizing, and dis tribution to culture vessels. Another area of cost reduction cou ld be through by -passing the in -vitro rooting step. The elongation medium produced shoots longer than 30mm, which could be sliced and rooted under mist conditions directly. Three genotypes of E. grandis were studied by Warrag (1989), who reported no significant variation between them Th is may suggest that the system developed in the study could be used at least in a broader sense. In some species in this genus, the genotypic variation in ability of cuttings to root had been exploited in conventional propagation as in the successful sele ction work carried out in the Congo and Brazil (Hartmann 2002) The genotype also strongly influenced rooting ability in vitro

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64 Genetic Fingerprinting Pedigree and Genetic Relatedness Florida E. grandis clones ha ve known maternal pedigree s extending up to four ge nerations. M any clones originated from some common 3GM or 4GM trees, like 295 and 293, and should be genetically related. Ancestor 2 95 was common to the most clones; for example, G1 G2 and G43 which could be expected to be closely related ( Table 4 9). Table 4 9 E. grandis clones originat ing from common 3GM or 4GM ( C lone 295, 293, 294, 298, and 92). Origin Related Clones 295 G1 G2 3127 3159 G8 G12 G15 G18 4340 G27 G33 G35 G43 G48 G56 G59 293 G37 G32 G41 G47 G52 294 G49 G10 G16 298 G5 G30 G55 92 G23 G24 G26 Marker Missing data rate was not very high here. T otal number of alleles observed per locus for 60 clones ranged from 7 to 26, with an overall total of 157 allele s across 8 loci Expected heterozigosity (He) varied at different loci from 0.63 to 0.91(Table 4 10). In our study, only two loci (EMBRA63 and EG62) had He lower than 0.7 and others were higher than 0.85 (Table 4 10). Previous reports indicated that SSRs were efficient markers with He higher than 0.8 at locus EMBRA 4, EMBRA 5, EMBRA 10, EMBRA 11, EMBRA 15, and EMBRA 16 (Kirst, et al. 2005) Brondali et al. (1998) also reported all He for 20 loci higher than 0.7 except EMBRA13.

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65 Table 4 1 0 Allele number for 8 loci in the study Locus Marker Missing data Number of alleles He Locus 1 EMBRA2 2.50% 22 0.89 Locus 2 EMBRA28 5.00% 26 0.9096 Locus 3 EMBRA10 8.30% 22 0.889 Locus 4 ES76 9.20% 25 0.9139 Locus 5 EMBRA37 3.30% 25 0.8608 Locus 6 EMBRA63 0.80% 9 0.6569 Locus 7 EG62 11.70% 7 0.6344 Locus 8 EG65 8.30% 21 0.8676 Total 157 Microsatellite s locus EG 62 ha d only 7 alleles and most frequent allele peaked as high as 54%. Similarly, EMBRA 63 had 9 alleles with one domina ting at 52% frequency. Expected heterozygosit ies (He) for these 2 markers were the lowest (0.6344 and 0.6569 respectively), compared with others. Because E xpected heterozygosity reflected the probability of the heterozygous individual in the population EG62s relatively low He could be explained by that the alle le with 54% frequency at EG62 could generate a high probability of the homozygous individuals, thus reduce the He correspondingly. The previous reports by Brondani et al. (1998) indicated that frequencies of all alleles at all loci (EMBRA 1 to EMBRA 19) we re less than 30%, resulting in high He. Different results showed that different locus have impact on the He estimation. On the contrary, there were no predominant alleles at loci EMBRA2, EMBRA28, EMBRA10, and EMBRA76, because the highest allele frequencies for these loci were lower than 20%, thus all allele s distributed similarly (Figure 4 1 3) The high He (0.89, 0.9096, 0.889, and 0.9139 respectively ) for these markers also indicated this. For loci EMBRA37 and EG65, some alleles had relatively high frequency (28% and 21%), although they were not very

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66 dominant, their frequencies were higher than other alleles and also to reduce the He 0.86. Rare allel es (<0.1%) were about 30% and common alleles (>0.5%) were about 30% for 15 populations for E. marginata (Wheeler et al., 2003) The cluster result indicated that 60 E. grandis clones were originated from 4 populations represented by 4 different colors (red green, blue and yellow) (Figure4 14). The individuals were sort by Q, which made it easier to see which clones were more related. The individual label and corresponding clone number were showed in Appendix C. Sixty clones can also be divided into 4 grou ps depending on which original population accounted for most proportion in individual. The first 24 clones in plot were 40%60% originated from red cluster, while next 12 clones were largely (60 100%) from green cluster; 14 clones followed were mainly originated from blue cluster and last 10 clones shared 3 cluster (red, blue and yellow) equally (Figure4 14). The result of structure was very consistent with kinship coefficient for individual clones. For example, identical clones G20 G21 and G4 with high coefficient was sort together in plot which reflected their high similarity in origin. All the pairs with high kinship coefficient (Table 4 11) were classified in the same group and sort closely. This was because both the result from the Structure and SPA GeDi were originated from the genotype data from 8 microsatellite loci. The real reason why these kinship coefficients were high and why some individual were originated from the same clusters with similar proportion was that alleles in 8 loci for clones w ere very similar. For example, clone G4 and G20 (kinship coefficient =0.51) were identical clones because all 16 alleles for 8 SSR loci were almost the same, except for one missing data (Appendix C). Clone G1 and G45 (kinship coefficient =0.26) were full -s iblings because 4 of 8 loci shared common alleles. Clone G1 and G2 were not genetic

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67 related (kinship coefficient =0.06) because only one of two alleles in EG65 and EMBRA2 were shared by 2 clones while there were no common in other 6 loci.

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68 Figure 4 1 3 Allele number and frequency for 8 microsatellite loci (EMBRA2, EMBRA28, EMBRA10, ES76, EMBRA37, EMBRA63, EG62, and EG65) in the population of 60 Florida E. grandis clones

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69 Figure 4 1 4 P opulation s tructure for of 60 Florida E. grandis clones (Appendix C). Each clone was represented by vertical line, which was partitioned into 4 colored segments that represent that clones estimated membership fraction in each of the K inferred clusters. Figure 4 1 5 Distribution of pairwise relative kinship estimates in 60 E. grandis clones; 0.5 identical twins; 0.25 offspring within a family, parent offspring; 0.125 half -siblings. In addition, 60 clones were also summarized by the kinship coefficient (Appendix D) to analyze the genetic dist ance between each pair of two individuals. The kinship estimated the approximate IBD (identity by descent) by adjusting the probability of identity by state between two individuals with the average probability of identity by state between random individual s (Yu et al., 2006) Three thousand and six hundred coefficients were distributed approximately normal,

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70 and 90% of the pairwise kinship coefficients were close to 0, ranging from 0 to 0.25. Very f ew of them were distributed around 0.35 and 0.5, reflecting the highly genetic relatedness (Figure 4 15). Clones originat ing from the same GM were compared by the kinship coefficient. For example clones originat ing from 295 which were also involved in the genetic fingerprinting were G1 G2 G8 G12 G15 G18 G33 G35 and G43 K inship coefficient s for G1 with others were 0.07 (G2 ), 0.17 (G12 ), 0.04 (G15 ), 0.05 (G18 ), and 0.02 (G35 ). Except for G12 all the coefficients for others were below 0.1, which mean that they were almost not genetically related to each oth er (Table 4 9; Appendix D). The highest coefficients among the pairs of clones were 0.512 ( G20 and G4 ), 0.5586 ( G21 and G4 ), and 0.4172 ( G23 and G4 ); according to the definition of coefficient (Table 3 2) they were identical clones. Besides, there were s ome pairs of clones with coefficient between 0.2 and 0.5, which were considered full -siblings (Table 4 11). Table 4 11. Pairs of E. grandis clones with kinship coefficient between 0 .2 and 0.5 clone 1 clone 2 coefficient clone 1 clone 2 coefficient G20 G4 0.5127 G10 G27 0.3009 G21 G4 0.5586 G1 G45 0.2649 G9 G46 0.4638 G14 G43 0.2323 G23 G4 0.4172 G11 G45 0.2312 G24 G25 0.3874 G29 G55 0.2196 G9 G56 0.3732 G33 G56 0.2150 G44 G50 0.3700 G15 G11 0.2117 G13 G47 0.3698 G26 G12 0.2116 G41 G47 0.3241 G37 G1 0.2095 G30 G33 0.3127 G42 G56 0.2025 G53 G57 0.3098

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71 Propagation and Fingerprinting Ten E.grandis clones used in propagation were not genetic related because the kinship coefficients were too low (Table 4 12). Clone G34 was the most efficient both in cutting and micropropagation, and it was not genetic ally related to any other clones. Clone G2 and G1 could be multiplied in DKW media better than other clones and they were cultured well in the same media component but t he coefficient for the G1 and G2 w as 0.06 which indicated no relatedness. A ll the coefficients for other pairs of clones involved in micropropagation were 0. Table 4 12. The kinship coefficient for E. grandis clones involved in propagation ( G1 G2 G3 G4 G34 G34 G37 and G43 ). Clones G1 G2 G4 G34 G36 G37 G43 G1 0.0678 0.0242 0.0019 0.047 0.2095 0.002 G2 0.0551 0.0448 0.0173 0.0213 0.1003 G4 0.0885 0.048 0.0224 0.0523 G34 0.0507 0.0605 0.0057 G36 0.0572 0.0218 G37 0.0241 G43 Clone G3 G48, and G51 were not analyzed in the fingerprinting study.

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72 CHAPTER 5 GENERAL CONCLUSION Vegetative propagation of E ucalyptus clones is a useful tool for developing short rotation woody crop systems. To develop optimum propagation system s traditional cutting, mini -cutting and micropropagation were tested Traditional cutting was feasible for propagation of E. grandis and E. amplifolia but the rooting percent varied among different clones. The key factor for the success of this method w as the source of the plant material for cuttings. Juvenile shoots were more suitable material and relatively juvenile sprouts from girdling or fallen trees were better than that from the branches of the mature trees top crown. In addition, seasonal timing wa s also an important factor influencing the rooting efficiency ; fall was better than spring. When E. grandis sprouts were not available for the traditional cutting, mini -cutting from the stock plants was a successful alternative although the rooting effic iency was not as high as the traditional cuttings for the same clones. All the successfully rooted traditional cuttings worked as stock plants. Rejuvenation of the stock plants was necessary and 6 month old branches were optimal for mini -cutting s In addi tion, the basal part from the lateral branches was better than terminal branches as the plant material for the mini -cuttings. Direct micropropagation was successful for E. grandis but there was a very strong genotypic response. Successful rooted cuttings worked as the stock plants to provide explants for the micropropagation. Leaves were not successful for shoot induction, but nodes were optimal explants. Six -week new shoots from stock plants result ed in highest living shoot percent after sterilization. O ne time 1 0% bleach for 20 minutes without ethanol pretreatment was good for the sterilization.

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73 To prevent phenolic exudation, PVP was supplemented in the medi a and explants were subcultured every 3 days with good effect. After that, initial s hoot induction was conducted. The addition of PGR s inhibited the shoot induction and induced callus. Best performance was on the basal medium without any PGR and there was no difference for shoot induction among these basal medium when no PGR was in the medium DKW w as the best basal medium for all c lones and DKW with 0.4 mg/l BAP was o ptimal multiplication medium Three -week subculture was needed because it reduced the vitrification and prevented the phenolic exudation. Multiplication rate of each generation varied, and generations 6 to 7 provided the highest levels of shoot production. Shoot elongation was performed with the same basal medium as the multiplication medium for each clone. Best elongation occurred on the DKW medi a with 0. 1 0.5 mg/l BAP and 0. 1 mg/l NAA no matter which clones Rooting on DKW medium with 0.1 1 mg/l NAA and 0. 5 mg/l was optimal. The genotype effect was very obvious and rooting efficiency per macro cutting varied greatly Clone G 34 had the highest rooting percent age and was easiest to propagate by both macrocutting and minicutting. In tissue culture, G 34 could be multiplied by both MS and DKW and both rate was high, w hile for clones G1 and G2 DKW were significant better for multiplication than MS In addition, clone G2 and G1 showed similar performance. The recommended steps for plantlet production are : Girdle or f ell trees to induce sprouts for cuttings. In late fall, t he healthy branches are selected and 10-cm stem cuttings were cut with 2 half leave remaining. The base of the apical cutting is dipped in IBA commercial powder and inserted into the tube containing the soil Surviving cuttings are transplanted to the depot after 2 months, and then transplanted to 30 cm -diameter pot after another 2 months. Healthy plants were m aintained as the stock plants in a green house.

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74 Six -month old base apical shoots are selected from stock plants and processed for rooting The method is the same with the method for macrocutting. Use s uccessfully rooted macrocuttings or mini -cuttings as the stock plants maintained in a greenhouse to provide the material plants for in vitro culture establishment Collect nodal segments from 2 -month -old sprouts and steriliz e with 1 0% bleach for 20 minutes. A dd PVP to the medium in every stage to prevent phenolic compounds. Establish culture on hormone -free media (MS, DKW or WPM) to induce shoots from the de contaminated explants. C hange the medium every 3 days for the first month to prevent the exudated phenolic from killing the explants. Transfer shoots to multiplication medium. Excise shoots longer than 10 mm and place vertically into elongation medium. Remove the lower part shoots and put single plantlet into rooting medium. Remove the plantlets from the rooting media and wash agar from the roots and transfer them to mist conditions for 2 weeks and then normal greenhouse. Figure 5 1. Propagation cycle for Eucalytus Shoots sprouts from felled trees to make cuttings, and minicuttings (3 moonth) Cuttings as stock plants to provide explants Explants were cultured in MS PGR free medium for regeneration (3 week) DKW+0.4mg/l BAP were used for multiplication (3 week) DKW+0.1 0.5mg/l BAP+0.1mg/l NAA for elongation (6 week) 0.5 mg/l NAA or IBA for rooting and then acclimation (8 week)

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75 It took 3 months from sprouts in field trees to stock plan ts. Then the whole micropropagation took another 5 months from shoots induction to plants in greenhouse (Figure 5 1). In our study, traditional c utting and mini -cutting were used, especially in clean environment, to provide plant material for tissue culture. Tissue culture was most efficient means for mass production, although cost was more expensive. Eight SSR loci were chosen as the genotype data to es timat e the genetic variation in the studied clones High He and polymorphism of alleles demonstrated population structure from population level. Sixty E. grandis clones were originated from 4 subpopulations with admixture ancestors model Kinship coeffici ents were used to estimate the genetic distance between each pair of these clones. T he highest coefficient among the pairs of the clones were 0.512 (G20 and G4 ), 0.5586 ( G21 and G4 ) and 0.4172 ( G23 and G4 ), suggesting that these clones were very identical. 10% of the kinship coefficients for pairs of the clones were among 0 to 0.5, which showed more or less genetic relatedness.

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76 CHAPTER 6 FURTHER RESEARCH NEE DED For success in rooting cuttings, different substrate s and environmental condition in the greenhouse are important factors influencing the rooting result. M ore work on these parameters is recommended to improve the rooting response To establish in vitro culture c ontamination is still an issue. It is more difficult to obtain clean and viable material for shoot cultures from field -grown forest trees. The sterilization of mature, field grown material proved difficult because of endogenous microbial contamination. Juvenile stock plants growing in a greenhouse for explants were relatively easy to sterilize, but the contaminated rate was still high. To improve decontamination special care is needed to prevent insect infestation and splashing from the soil surface during watering. Strict insect and disease control conducted on stock plants i s impor tant to reduce the contamination. Rooting of E. amplifolia cuttings was more difficult than from E. grandis ; the suggestion is to see if different substrate works better. A cleaner condition in the greenhouse is also needed to prevent fungi or bacterial a ttack on these plants. Vitrification occurred in shoot multiplication cultures, although usually only a few buds were affected. A reduction in cytokinin concentrations could reduce this problem, but lead to a lower multiplication rate. More frequent subc ulturing was effective to minimize this problem. However more methods are needed to prevent the vitrification. Eucalyptus shoot cultures frequently produce a white callus, with a sugary appearance on leaf surfaces, and stem nodes. Frequent subculturing w as attempted to reduce this problem but with no success More efforts were needed to prevent the callus development on the shoots or stems.

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77 In our study, only 8 generations were recorded for different clones. More generation s are needed to see when the regeneration rate would stop completely. In this way, more exact calculation s could be made to estimate the long term efficiency of micropropagation. Clone s G34 G1 and G2 were successfully multiplied on DKW medium with a rapid rate For E. grandis Clone s G3 and G4 the multiplication rate was still very low on WPM, MS or DKW Better medi a need to be develop ed for these 2 clones. Further experiments are required to determine why DKW works better for some clones. More research about organogenesis and somatic embryogenesis are needed to see if these 2 methods could feasible be used to develop a mass production system Further research is needed to reduce the cost and make production more efficient. Direct rooting of elongated shoots under mist in the greenhouse would eliminate the in vitro rooting step and this could improve the economics of system. In addition, because we did not test the genetic variation in plantlets produced from micropropagation, an analysis of somaclonal variati on in recovered plants over the 6 subculture periods would be beneficial.

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78 APPENDIX A BASAL MEDIA COMPONENT

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79 component (mg/l) ChuN6 DKW GamborgB5 Horgland WPM MS QL SH White Ammonium nitrate 1416 400 1650 400 Ammonium phosphate monobasic 115.03 300 Ammonium sulfate 463 134 Boric acid 1.6 4.8 3 2.86 6.2 6.2 6.2 5 1.5 Calcium chloride anhydrous 125.33 112.5 113.24 72.5 332.2 151 Calcium nitrate 1367 656.4 386 833.77 200 Cobalt chloride 6H2O 0.025 0.025 0.025 0.1 Cupric sulfate 5H2O 0.25 0.025 0.08 0.25 0.025 0.025 0.2 Na2 EDTA 37.25 45.4 37.3 37.3 37.26 37.3 20 Ferric sulfate 2.5 Ferric tartrate 2H 2 O 5.32 Ferrous sulfate 7H2O 27.85 33.8 27.8 27.8 27.8 15 Magnesium sulfate 90.37 361.49 122.09 240.76 180.7 180.7 175.79 195.4 360 Manganese chloride 4H2O 1.81 Manganese sulfate H2O 3.33 33.5 10 22.3 16.9 0.76 10 5.04 Molybdenum trioxide 0.016 Molybdic acid (sModium salt) 2H2O 0.39 0.25 0.25 0.25 0.25 0.1 Potassium chloride 65 Potassium iodide 0.8 0.75 0.83 0.08 1 0.75 Potassium nitrate 2830 2500 606.6 1900 1800 2500 80 Potassium phosphate 400 265 170 170 270 Potassium sulfate 1559 990 Sodium phosphate monobasic 130.5 16.5 Sodium sulfate 200

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80 APPENDIX B PROTOCO L FOR AMPLIFICATION OF SSR Microsatellite -marker amplifi cation was performed in 96 -well V bottom plates in a 13 ul reaction volume containing 0.3 u M of each primer, 1 unit of T aq DNA polymerase, 0.2 m M of each dNTP, 10mM Tris HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, DMSO (50%), and 7.5 ng of template DNA. Ampli fi cations were performed using a MJ Research PT 100 thermal controller with the following conditions: 96C for 2 min, then 29 cycles of 94C for 1 min, 56C for 1 min and 72C for 1 min; and a fi nal elongation step at 72C for 7 min. Inheritance, segregation and mapping was carried out in a 3.5% Metaphor agarose (FMC Bioproducts) gel containing 0.1 ug/ml of ethidium bromide in* 1] TBE buffer (89mM Tris borate,2mM EDT A pH 8.3). Submarine electrophoresis was carried out at 120V for 2 h in custom -made gel boxes that contained 96 samples per gel.

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81 APPENDIX C SIXTY E. GRANDIS CLONES GROUPING BY S TRUCTURE

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82 group # Clone EMBRA2 EMBRA28 EMBRA10 ES76 EMBRA37 EMBRA63 EG62 EG65 1 51 G54 123 134 211 224 126 135 119 157 120 145 172 9 189 189 254 285 38 G39 128 132 185 9 108 118 120 137 110 123 174 180 189 189 263 278 60 T6 127 127 199 9 114 124 116 133 130 143 174 184 195 205 242 242 37 G38 109 128 180 205 126 140 119 119 106 129 182 182 189 198 238 269 35 G36 128 137 209 209 118 137 136 136 120 127 174 180 189 9 269 273 57 3486B 107 117 201 201 9 9 9 9 123 123 174 174 189 189 263 267 5 G6 117 117 181 205 114 120 137 153 118 130 174 174 195 195 257 269 39 G40 111 117 205 205 120 135 121 121 114 127 170 170 189 195 270 270 52 G55 124 132 201 201 126 139 137 142 116 126 174 174 189 189 263 269 55 G58 105 115 206 216 118 126 125 9 127 147 170 182 9 9 260 269 28 G29 132 132 201 201 126 135 117 117 112 131 174 184 195 195 263 288 47 G49 106 134 181 181 126 130 118 118 145 149 182 182 189 195 245 279 59 Lykes 117 124 183 215 118 118 125 125 118 127 170 170 189 198 260 269 42 G43 125 129 207 207 118 134 123 123 112 127 174 174 189 195 251 254 36 G37 132 145 197 217 129 139 122 122 121 129 170 174 189 195 251 257 56 G59 125 128 195 208 134 139 120 137 118 131 174 174 189 9 238 257 13 G14 128 9 207 9 118 132 125 125 124 127 174 180 189 204 254 254 34 G35 131 131 211 215 119 141 117 121 110 120 170 174 195 201 251 254 33 G34 106 132 195 203 132 139 157 157 118 122 170 174 189 217 242 254 2 G2 115 131 220 220 126 134 133 133 127 155 174 176 189 195 239 251 30 G31 129 138 209 209 124 124 153 153 112 129 174 174 189 9 239 248 16 G17 115 129 193 211 130 141 139 171 120 9 174 174 189 201 254 269 12 G13 111 117 216 222 130 130 119 124 118 122 176 182 189 204 257 263 58 G48B 118 127 194 199 136 142 139 153 126 130 174 182 189 189 9 9 20 G21 128 128 193 218 118 126 129 129 120 9 168 174 201 204 229 257 19 G20 128 128 193 218 118 126 119 129 120 120 168 174 201 204 229 257 3 G4 128 128 193 217 118 126 118 129 120 9 168 174 201 204 229 257 22 G23 128 128 193 218 118 129 119 129 120 120 168 174 201 205 230 257 23 G24 106 128 209 216 120 134 9 9 118 126 174 174 189 204 257 257 26 G27 128 132 209 218 130 134 141 141 128 128 174 182 9 9 239 269 9 G10 128 128 209 217 124 130 141 141 127 145 174 182 189 204 239 260 21 G22 109 128 193 205 116 134 119 137 126 126 174 182 201 204 239 254 17 G18 106 106 218 218 126 134 119 141 118 145 176 176 189 195 269 269 24 G25 106 128 209 215 120 134 147 147 118 126 174 174 189 205 257 257 18 G19 106 131 193 193 134 144 119 9 128 9 172 176 195 204 245 245

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83 31 G32 109 128 216 216 114 9 119 119 122 127 174 176 189 195 239 9 3 44 G45 115 118 193 217 124 142 122 122 120 129 174 184 201 201 239 257 53 G56 118 131 199 199 124 124 119 119 118 129 170 176 189 201 254 257 10 G11 115 131 193 193 124 9 121 121 126 129 170 174 9 9 239 257 1 G1 106 132 217 217 134 144 122 122 124 127 180 186 195 201 239 263 14 G15 111 127 213 217 124 124 121 121 118 125 174 180 189 201 269 269 48 G56 127 9 217 9 118 130 119 119 125 125 170 174 189 201 282 288 41 G42 106 118 199 215 124 142 141 9 118 153 176 182 189 9 254 263 43 G44 106 128 217 217 132 141 119 119 120 125 170 174 189 195 288 9 49 G52 129 9 199 217 126 9 121 121 118 122 174 180 195 198 9 9 6 G7 106 134 195 205 134 134 126 126 118 124 170 184 195 217 254 263 46 G47 111 111 203 203 130 130 126 141 118 133 176 182 9 9 257 263 27 G28 143 143 193 203 124 136 119 119 118 118 168 174 189 195 257 263 25 G26 106 117 209 209 124 139 137 9 126 147 180 184 189 189 257 269 32 G33 125 134 215 215 128 133 137 137 118 147 174 174 189 189 251 251 4 50 G53 125 132 209 209 118 9 132 139 118 118 170 174 189 189 251 9 45 G46 106 125 205 205 120 139 117 155 118 118 176 180 189 195 251 260 4 G5 125 125 203 203 124 140 155 155 120 126 174 184 189 189 269 269 8 G9 106 132 205 205 130 139 128 155 118 118 180 180 189 195 251 251 54 G57 115 131 195 195 9 9 9 9 118 118 180 186 189 189 251 251 29 G30 127 134 195 226 118 126 137 9 118 126 174 174 189 189 251 254 7 G8 125 125 205 205 118 130 121 137 120 133 174 174 189 201 251 269 11 G12 106 117 217 9 9 9 117 117 128 128 180 180 189 189 257 257 15 G16 106 134 211 9 124 134 123 155 118 127 174 184 189 9 9 9 40 G41 111 119 203 209 126 130 123 123 118 125 176 184 189 9 257 9

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84 APPENDIX D KINSHIP COEFFICIENTS FOR PAIRS OF SIXTY E. GRANDIS CLONES G1 G45 G37 G7 G12 G29 G19 G44 G52 0.2649 0.2095 0.1807 0.1723 0.136 0.1111 0.1061 0.0975 G2 G32 G57 G43 G35 G18 G58 G19 G1 0.1154 0.1135 0.1003 0.0878 0.0754 0.0702 0.0682 0.0678 G4 G21 G20 G23 G45 G17 G14 G10 G22 0.5586 0.5127 0.4172 0.1783 0.1302 0.1132 0.0998 0.0973 G5 G8 G16 G15 G26 G55 G17 G36 G31 0.1832 0.1515 0.1323 0.1272 0.1092 0.0953 0.0857 0.0756 G6 G40 G29 G24 G46 G8 G25 G52 G28 0.1375 0.1073 0.1016 0.1002 0.0945 0.0906 0.0777 0.0654 G7 G1 G34 G40 G46 G9 G18 G16 G19 0.1807 0.1407 0.1271 0.1262 0.1161 0.1156 0.1137 0.1084 G8 G33 G5 G17 G43 G53 G30 G40 G46 0.196 0.1832 0.1565 0.1193 0.114 0.1068 0.1059 0.1049 G9 G46 G57 G53 G7 G40 G12 G8 G33 0.4638 0.3732 0.1823 0.1161 0.1016 0.1008 0.0948 0.0828 G10 G27 G14 G31 G42 G36 G4 G47 G32 0.3009 0.1302 0.1275 0.1237 0.1013 0.0998 0.0922 0.0887 G11 G45 G15 G56 G31 G40 G35 G19 G22 0.2312 0.2117 0.1842 0.1821 0.1601 0.1281 0.1028 0.0979 G12 G26 G1 G41 G46 G9 G19 G44 G27 0.2116 0.1723 0.1373 0.1073 0.1008 0.089 0.0767 0.0737 G13 G47 G41 G32 G49 G28 G42 G56 G38 0.3698 0.1817 0.122 0.103 0.0941 0.0844 0.0836 0.0832 G14 G43 G58 G10 G21 G36 G4 G20 G39 0.2323 0.1673 0.1302 0.1168 0.1147 0.1132 0.1073 0.0976 G15 G11 G52 G50 G5 G40 G31 G8 G56 0.2117 0.1871 0.1544 0.1323 0.0912 0.0859 0.0845 0.0813 G16 G5 G41 G43 G7 G54 G57 G42 G31 0.1515 0.1309 0.1161 0.1137 0.1134 0.0874 0.0865 0.0828 G17 G35 G8 G45 G23 G21 G4 G20 G5 0.1642 0.1565 0.1531 0.1377 0.1338 0.1302 0.1242 0.0953 G18 G19 G42 G27 G46 G7 G49 G38 G2 0.1914 0.1646 0.154 0.1365 0.1156 0.1106 0.0908 0.0754 G19 G18 G22 G28 G32 G56 G1 G27 G7 0.1914 0.1479 0.1308 0.1224 0.1203 0.1111 0.1095 0.1084 G20 G21 G4 G23 G45 G22 G17 G14 G19 0.5889 0.5127 0.4839 0.136 0.1276 0.1242 0.1073 0.0912

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85 G21 G20 G4 G23 G45 G17 G14 G22 G24 0.5889 0.5586 0.4934 0.1456 0.1338 0.1168 0.1008 0.0763 G22 G38 G19 G20 G32 G24 G23 G21 G11 0.1563 0.1479 0.1276 0.1225 0.1051 0.1047 0.1008 0.0979 G23 G21 G20 G4 G45 G17 G22 G44 G14 0.4934 0.4839 0.4172 0.1495 0.1377 0.1047 0.0996 0.0844 G24 G25 G59 G22 G6 G26 G41 G20 G21 0.3874 0.1671 0.1051 0.1016 0.0902 0.0863 0.0763 0.0763 G25 G24 G59 G6 G33 G41 G23 G26 G31 0.3874 0.1485 0.0906 0.0832 0.0812 0.0764 0.0756 0.0603 G26 G12 G36 G31 G41 G5 G55 G33 G24 0.2116 0.1636 0.1535 0.138 0.1272 0.1145 0.1037 0.0902 G27 G10 G18 G19 G42 G47 G36 G12 G31 0.3009 0.154 0.1095 0.1042 0.1012 0.0795 0.0737 0.0683 G28 G56 G19 G47 G13 G11 G57 G32 G6 0.1759 0.1308 0.1191 0.0941 0.0804 0.0711 0.069 0.0654 G29 G55 G1 G52 G35 G6 G7 G37 G43 0.2196 0.136 0.1221 0.1196 0.1073 0.0969 0.0894 0.0595 G30 G33 G57 G55 G59 G53 G8 G39 G43 0.3127 0.2176 0.1781 0.1601 0.158 0.1068 0.1012 0.0907 G31 G11 G26 G36 G10 G53 G15 G43 G16 0.1821 0.1535 0.1484 0.1275 0.1154 0.0859 0.0844 0.0828 G32 G38 G22 G19 G13 G44 G2 G10 G28 0.1308 0.1225 0.1224 0.122 0.1174 0.1154 0.0887 0.069 G33 G30 G57 G53 G8 G59 G55 G43 G26 0.3127 0.215 0.2109 0.196 0.1766 0.122 0.1072 0.1037 G34 G7 G57 G9 G54 G59 G53 G37 G30 0.1407 0.1019 0.0788 0.0735 0.0637 0.0616 0.0605 0.0545 G35 G17 G11 G29 G40 G2 G52 G8 G56 0.1642 0.1281 0.1196 0.1135 0.0878 0.0838 0.0705 0.0672 G36 G26 G53 G31 G14 G39 G10 G5 G27 0.1636 0.1618 0.1484 0.1147 0.1051 0.1013 0.0857 0.0795 G37 G1 G45 G29 G12 G9 G34 G44 G53 0.2095 0.1821 0.0894 0.0725 0.0723 0.0605 0.0596 0.0551 G38 G22 G49 G32 G58 G56 G18 G54 G19 0.1563 0.1481 0.1308 0.1145 0.0924 0.0908 0.086 0.0836 G39 G55 G59 G36 G30 G14 G33 G26 G53 0.1285 0.1105 0.1051 0.1012 0.0976 0.0815 0.0739 0.0721

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86 G40 G11 G46 G6 G7 G35 G52 G8 G9 0.1601 0.148 0.1375 0.1271 0.1135 0.1077 0.1059 0.1016 G41 G47 G13 G26 G12 G16 G24 G25 G57 0.3241 0.1817 0.138 0.1373 0.1309 0.0863 0.0812 0.0732 G42 G56 G47 G18 G10 G27 G57 G16 G13 0.2025 0.1765 0.1646 0.1237 0.1042 0.0874 0.0865 0.0844 G43 G14 G8 G16 G33 G2 G53 G30 G31 0.2323 0.1193 0.1161 0.1072 0.1003 0.0978 0.0907 0.0844 G44 G50 G32 G19 G1 G23 G4 G20 G38 0.37 0.1174 0.1065 0.1061 0.0996 0.0922 0.0862 0.0786 G45 G1 G11 G37 G4 G17 G23 G21 G20 0.2649 0.2312 0.1821 0.1783 0.1531 0.1495 0.1456 0.136 G46 G9 G57 G40 G18 G7 G53 G12 G8 0.4638 0.2367 0.148 0.1365 0.1262 0.1198 0.1073 0.1049 G47 G13 G41 G42 G49 G28 G27 G7 G10 0.3698 0.3241 0.1765 0.129 0.1191 0.1012 0.093 0.0922 G49 G38 G47 G18 G58 G54 G19 G13 G42 0.1481 0.129 0.1106 0.1091 0.1058 0.1034 0.103 0.0766 G50 G44 G15 G56 G1 G12 G32 G45 G38 0.37 0.1544 0.0932 0.084 0.0674 0.059 0.0566 0.0565 G52 G15 G29 G40 G1 G11 G35 G6 G9 0.1871 0.1221 0.1077 0.0975 0.091 0.0838 0.0777 0.0723 G53 G57 G33 G9 G36 G30 G46 G31 G8 0.3098 0.2109 0.1823 0.1618 0.158 0.1198 0.1154 0.114 G54 G16 G49 G38 G19 G17 G34 G30 G42 0.1134 0.1058 0.086 0.0776 0.0743 0.0735 0.0609 0.0508 G55 G29 G30 G39 G33 G59 G26 G5 G31 0.2196 0.1781 0.1285 0.122 0.1147 0.1145 0.1092 0.0629 G56 G42 G11 G28 G45 G19 G50 G38 G13 0.2025 0.1842 0.1759 0.1288 0.1203 0.0932 0.0924 0.0836 G58 G14 G38 G49 G40 G2 G36 G32 G18 0.1673 0.1145 0.1091 0.0986 0.0702 0.0515 0.0498 0.0458 G59 G33 G24 G30 G25 G55 G57 G39 G8 0.1766 0.1671 0.1601 0.1485 0.1147 0.1129 0.1105 0.0797

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97 BIOGRAPHICAL SKETCH Zhi Yang was born in Wuhan, China. She has been living in her hometown for 22 years. In 2001 she began her undergraduate studies in Huazhong Agricultural University and received b achelor degree of a griculture in June, 2005 with major in l andscape a rchitecture and e nvironmental h orticulture. Then she worked as the h orticulture technician in New Art Garden Cooperati ve Wuhan, China She began her graduate career at the University of Florida in August, 2006, under the direction of Dr. Donald Rockwood In August 2009 she was awarded a Master of Science degree in Forestry with an emphasis on p lant propagation and genetics.