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Micropropagation and Genetic Transformation of Verticordia Grandis

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Micropropagation and Genetic Transformation of Verticordia Grandis vù TAMF tr*o r.d "S- ,+ MICROPROPAGATION AND GENETIC bfigg+ ' VERTICORDIA GRANDIS CIç ,""f" BY BELINDA E. STUMMER A thesis submitted for the Degree of Doctor of Philosophy in the Faculty of Agricultural and Natural Resource Sciences at The University of Adelaide. Deparunents of Plant and Soil Sciences Waite Agricultural Research Institute The University of Adelaide July 1993 Verticordín grandis i DECLARATION The work presenæd in this thesis contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institr¡tion and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference is made in tlre æxt. I give consent to this copy of my thesis, when deposited in the University Libnary, being available for photocopying and loan. B. E. Stummer July 1993 u ACKNOWLEDGMENTS I sincerely thank my supenrisors Dr Sally Smith and Dr Peær Langridge for their guidance, support and encouragement throughout my candidature. I also thank Ph¡oæch Aust. Pty. Ltd. (in particular Nick Cuthburtson and Ih Ma¡tin Barlass) and The National Teaching Company Scheme for their funding during the first two years of the project. The University of Adelaide awarded me a Postgraduaæ Scholarship for the following two years of my studies, for which I am grateful. Special thanks arc exprcssed to Mr and Mrs Moyle for supplyingtheVerticordiø stock material used in this study. Va¡ious staff members of the Waite Instituæ provided me with invaluable assistance during the course of this study. I specifically thank Carole Smith (glasshouse management), Jennie Groom and Andrew Dunbar þhoography), Lynne Giles (st¿tistical analyses), Angelo Ka¡akousis (molecular biology), Sandy Dickson (microscopy) and Jan Nield (laboratory organisation). I am also very graæful to my fellow postgraduate sn¡dents and friends, especially Paul Haney, Brett Inghem, Anne Tassie, David Hien (7æke), Jenny Guerin and everyone in the'Langridge laU for their friendship over the past few years. Finally, I thank my family and David Gillham for their encouragement and support throughout my studies. lu Summary Verticordiø grøndis (Scarlet feather flower) is the subject of this study. It is a member of the family Myrtaceae and has a brilliant display of red flowers on long upright stems. The horticultural potential of V. grøndis as a cut flower and for pot culture is recognised in the industry. However, supply is limited due to difficulties of propagating the plant. In particular, the inductíon of roots on explants invítro has limited the successful commercial micropropagation of this species. A system has been established for the micropropagation of V. grøndis. Protocols were developed for the initiation, shoot proliferation and root induction of V. grandis explants, in vitro. The sterilisation treatment successfully decontaminated 827o of ttre explants. Shoot multiplication rates of at least 3 fold were obtained on the multiplication medium. In addition, the location of merisæmatic cells at ttre leaf petiole region indicaæd that shoot proliferation from leaf discs was possible. The root induction medium consistently produced lOOTo rooting of explants. Anaomical investigations confirmed that roots were connected to stem vascula¡ elements. Therefore the methods developed were suitable for commercial production. These methods are essential requirements for nursery production and a¡e also important prerequisites for transformation studies. Rooted plantlets wero successfully transfered to glasshouse conditions. Sr¡rvival rates were influenced by both the plant genotype and the time of year of out-planting. Soil conditions critical for the successful pot culture of V. grandis included adequate drainage (air filled porosity of ?ßVo), slightly acidic medium (pH 5.5-6) and low supplementary nutrient application.V. grandís plants responded favourably to high illumination and low humidity levels. A foliar 6-benzyladenine (BA) spray (2LOmeI) in combination with apical bud removal increased the number of laæral shoots and were useful in improving the shape of the plants for commercial pot culture. lV The micropropagation and out-planting protocols developed for V. grurdis were utilised in Agrobacteriwt mediated transformation investigations. Initially, susceptibility trials using wild type A. rhizogen¿s strains were employed- Results obtained from these investigations indicated tlrat wild We A. rhizogencs does not induce hairy root formation rnV. grandis sæm explants. Howevën, V. grøndis is susceptible to infection with A. rhizogenes as deærmined by gall formation and opine analysis. A system has been established for the genetic transformation of V. grønd¡s. This system utilises the method developed for plantlet regeneration from leaf discs. Leaf discs were inoculaæd with an Agrobacteriwnstatn containing a marker gene for antibiotic resistance (NPT II gene) and a reporter gene (GUS) for screening of transformants. The transformation results were confirmed by FCR and Southern hybridisation. These results represent ttre first example of genetic engineering of a plant from the Myrtaceae. This thesis describes, therefore, the establishment of in vitro organ cultr¡re æchniques for the propagation of V. grøndís and for the establishment and pot cultr¡re of plants in the glasshouse and demonstrates the application of the leaf disc regeneration method for the transformation and regeneration of V. grandis. v TABLE OF CONTENTS Page DECLARATION (Ð ACKNOWLEDGMENTS (ü) SI,JMMARY (üi) CHAPTER 1 LITERATIJRE REVIEW AI\D PROJECT AIMS 1 1.1 INTRODUCTION 1 1.2 MICROPROPAGATION: METHODS FOR THE 3 II\DUCTION OF ROOTS I.2.1 Introduction 3 1.2.2 Juvenility 7 1.2.3 Role of plant growth regulators 9 1.2.4 Effect of dark pre-treaünent 10 1.2.5 pHofthemedium 10 1.2.6 Length of time in culture 11 1.2.7 Genotypic Variability 11 1.3 OUT.PLAIYTING t2 1.4 POT CIJLTIJRE: II{DUCTION OF LATERAL SHOOTS 12 1.5 SI,JMMARY 13 1.6 AGRO BACT ERIU M II\FECTION AND GENETIC t4 TRANSFORMATION 1.6.1 Agrobacteriutn: Biology 15 1.6.2 Agrobacteriurn rhízogenes 16 1.6.3 The Ri phenotype 18 1.6.4 Genes involved in hairy root formation 18 1.6.5 Target cells for genetic transformation 20 1.6.6 Disarmed vector systems 2t L.6.7 Ma¡ker genes 2T vr I.7 APPLICATIONS 23 l.E PROJECT AIMS 25 CHAPTER 2 MATERHLS AI\D METHODS 26 2.1 TISSIJE CI]LTURE TECHNIQIJES 26 2.1.1 Plant material 26 2.1.2 Initiation of Verticordia grønd,is Clone 866 26 2.1.3 Media and tissue culnue conditions 28 2.1.4 Shoot multiplication media 28 2.L.5 Shoot regeneration from leaf discs 28 2.t.6 Root induction media 29 2.L.1 Pre-treaûnent 29 2.1.8 Darkpre-trrea ent 29 2.t.9 Auxin, pH and sucrose concentration 30 2.t.10 Genotype effects 30 2.l.Ll Explants from different sources 30 2.2 TRANSFER OF PLANTLETS TO SOIL 3l 2.2.L Glasshouseconditions 31 2.2.2 Hardening off conditions 3l 2.2.3 Effect of soil mix on plant growth 32 2.2.4 Methods of lateral shoot induction 34 2.2.5 Effect of light 35 2.3 TECHNTQIJES FOR ANATOMICAL STIIDTES OF V. GRANDIS 35 2.3.I Plantmaterial 35 2.3.2 Fixation and embedding 35 2.3.3 Sectioning 36 2.3.4 Staining 36 2.3.5 Photography 37 2.4 GENERAL TECHNIQUES AIIID MEDIA USED FOR AGRO BACT ERIA M CI]LTI,]RE 37 2.4.1 Strains and plasmids 37 vü 2.4.2 Bacterial cultue media 2.4.3 Intnoduction of plasmids ino wild-t¡'pe A. rhizogenes strains 2.4.4 Storage of isolates 2.5 TECHI{IQLJES FOR TNOCIJLATTON OF V. GRANDIS WITH STRAINS OF AGROBACTERII,JM 2.5.L Sæm inoculations with wild t¡'pe A. rhizogenes strains 2.5.2 Leaf disc inoculations 2.5.3 The oxicity of antibiotics to y. grøndß 2.5.4 Kanamycin selection 2.6 TECHNIQTJES TO TEST FOR TRANSFORMANTS 2.6.L Opine assay 2.6.2 ß-glucuronidase ar¡say (GUS and MUG assay) 2.6.3 Isolation of endoph¡ic bacæria 2.7 ISOLATION OF DNA TR,OM V. GRANDIS 2.1.1 Small scale isolation of otal DNA 2.7.2 Pu¡ification of V. grandß DNA 2.7.3 Digestion of DNA with restriction endonuclease 2.7.4 Elecrophoresis of DNA 2.8 SOUTHERN HYBRIDISATION PROCEDIJRE S 2.8.1 P¡eparation of membranes 2.8.2 Preparation of labelled probes 2.8.3 Hybridisation procedures 2.9 DNA AMPLIFICATION BY PCR 2.9.L Primers 2.9.2 PCRreaction 2.IO STATISTICALANALYS$ vru CHAPTER 3 MICROPROPAGATION OF VERTICORDIA GRAAIDIS 50 3.1 INTRODUCTION 50 3.2 EXPERIMENTAL PROCEDTJRES 52 3.3 RESULTS 53 3.3.1 Surface sterilisæion of V. grandß Clone 866 53 3.3.2 Effect of c¡okinin t¡rpe and concentration on shoot proliferation of V. grandß Clone 865 54 3.3.3 Root production: Effect of pre-treament 56 A Multiplication media 56 B Darkperiod 59 3.3.4 Effect of the followingvariables on tootproduction of V. grøndis Clone 865 explants, invitro 60 A pHof the medium 62 B Sucrose concentration 62 C Auxint¡'peandconcentration 62 D Genotype effects 63 3.3.5 Effect of different sources of explants (Ctone 866) 63 3.3.6 Shoot proliferation from merisæmatic regions of leaf discs & 3.3.7 Histological studies 65 A Location of meristematic regions 65 B Sæm/rootconnections 69 3.4 DTSCUSSTON 69 CHAPTER 4 OUT.PLAI{TING AND POT CIJLTT,'RE OF VERTICORDIA GRAI{DIS 74 4.1 INTRODUCTION 74 4.2 RESULTS 77 4.2.1 Transfer of plantlets to soil conditions 77 A Root initiation of V.
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