GENETIC ENGINEERING OF ANTHURIUM FOR BACTERIAL DISEASE RESISTANCE A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN HORTICULTURE MAY 1993 BY Fure-Chyi Chen Dissertation Committee: Adelheid R, KuehnIe, Chairperson Anne M. Alvarez Richard W. Hartmann Tadashi Higaki Richard M. Manshardt Samuel S. M, Sun We certify that we have read this dissertation and that, in our opinion, it is satisfactory in scope and quality as a dissertation for the degree of Doctor of Philosophy in Horticulture. DISSERTATION COMMITTEE Chairperson Q £ O ci^ %- i i ACKNOWLEDGEMENTS Special thanks go to my advisor Dr. Adelheid (Heidi) Kuehnie for financial support, continuous guidance, helpful criticisms and stimulating discussions during my study. I am very grateful to my dissertation committee members, Drs. Anne Alvarez, Tadashi Higaki, Richard Hartmann, Adelheid Kuehnie, Richard Manshardt and Samuel Sun, for critical comments, suggestions and advice. Thanks go to Dr. J. Jaynes (Biochem. Dept., Louisiana State Univ.) for providing antibacterial constructs, bacteria strains, antibacterial proteins and antibodies; to Drs. A. Roth and F. Dahlquist (Inst. Mol. Biol., Oregon State Univ.) for providing T4 lysozyme vector pHSeS. I would also like to thank Dr. Tessie Amore, Vicky Chao, Gillian Nan, Nellie Sugii, and Suwenza Lius for their helpful assistance. Dr. Anne Alvarez for providing lab space, David Norman for help with my bacteria challenge work, and Glenn Mochizuki for help with bactericidal assay. Thanks to Drs. Samuel Sun and Ming-Li Wang for their instructions and instruments. Dr. Marilyn Dunlap of the Uni. of Hawaii EM facility for her help with scanning electron microscopy. Drs. A. Marezsky and C. Nagai for kindly providing their particle gun for experiment. I thank my father (who passed away early 1992) and mother for their encouragement and support. I thank my brothers, brothers-in-law, and friends for financial help. Finally, I am very grateful to my wife Su-Fu for her understanding, encouragement, caring, and for taking care of our son Wei-An and daughter Grace. I l l ABSTRACT To aid transformation of anthurium, tissue culture and regeneration was achieved through either somatic embryogenesis or shoot regeneration using in vitro grown etiolated internodes, laminae, and root segments. Two gene transfer methods were used to transform anthurium tissues. Using bombardment of DNA-coated microprojectiles into anthurium calli and etiolated internodes, transient expression of S-glucuronidase (GUS) and neomycin phosphotransferase II (NPTII) was observed in these tissues. No transformed plants were recovered using this method. Antibacterial genes, including an insect attacin gene (Att), phage P22 gene P13, phage T4 lysozyme gene e, and a gene encoding an analog of insect cecropin B (Shiva-1) were driven by either double CaMV35S or potato wound inducible promoter, in the plant expression vector pBI121. Agrobacterium tumefaciens LBA4404 carrying either pCa2Att, pCa2P13, pCa2T4, pWlAtt or pWlShiva was used for cocultivation with internode or lamina explants of UH965 and UH1060. Following culture on selection media containing kanamycin and carbenicillin or cefotaxime, shoots regenerated from various calli pieces. Kanamycin-resistant plantlets were recovered from UH965 and UH1060 etiolated internode explants cocultivated with Agrobacterium with or without tobacco cell line 'Su' as nurse culture. GUS activity as determined by histochemical staining was absent in the kanamycin-resistant plants evaluated. Western blot analysis of total proteins from lamina calli formed de novo from kanamycin-resistant UH965 plants showed the presence of attacin protein. Polymerase chain reaction was used to amplify DNA fragments from the introduced genes. In six UH965 plants, A tt and nptii i v genes were amplified in the expected sizes of 546 and 1054 bp, respectively. In UH1060 plants, P13 and nptH genes were amplified. A GUS gene fragment was amplified in one of the UH965 plants. No amplification of the above-mentioned genes was observed in DNA samples of untransformed control plants. Southern hybridizations using amplified sequences from A tt, P I3 and NPTH all showed positive hybridization. Kanamycin-resistant UH965 plants with Ca2Att, Ca2P13 or Ca2T4 were challenged with the blight pathogen Xanthomonas campestris pv. dieffenbachiae strain D150 in the petiole end. The result of two challenge experiments indicated that most transgenic UH965 plants were partially resistant and a few were resistant to the blight bacteria. TABLE OF CONTENTS Acknowledgements ........................................................................ iii Abstract ............................................................................................ iv List of Tables .................................................................................... xii List of Figures ................................................................................... xiv 1. Introduction ............................................................................... 1 References..................................................................................... 4 2. Literature Review ...................................................................... 6 2.1 Anthurium .......................................................................... 6 2.1.1 Bacterial blight and its current control ........... 7 2.1.2 Breeding for disease resistance ....................... - 8 2.2 Genetic engineering for disease resistance ................. 10 2.2.1 Insect immune proteins ..................................... 12 2.2.2 Defense proteins from other animals .............. 16 2.2.3 Lysozymes ........................................................... 18 2.3 Genetic transformation .................................................... 21 2.3.1 Agrobacterium-me6\aX.B6 transformation ....... 22 2.3.2 Particle gun .......................................................... 29 2.3.3 Electroporation, PEG- and liposome-mediated transformation of protoplasts .............................. 31 2.3.4 Microinjection ...................................................... 33 2.3.5 Macroinjection ..................................................... 34 2.3.6 Pollen tube pathway ........................................... 34 2.3.7 Agroinfection and virus vectors ....................... 35 2.3.8 Other methods of gene transfer ...................... 36 v i 2.4 Tissue culture of anthurium and other aroids ............. 37 2.4.1 Callus culture ..................................................... 37 2.4.2 Axillary bud multiplication ............................... 39 2.4.3 Seed culture ......................................................... 39 2.4.4 Spadix culture ..................................................... 40 2.4.5 Liquid culture ...................................................... 40 2.5 References .............................................................. 41 3. Tissue Culture and Regeneration of Anthurium .................... 64 3.1 Introduction............. .............................................................. 64 3.2 Materials and methods .................................................... 65 3.2.1 Plant materials....................................................... 65 3.2.2 Media .................................................................. -70 3.2.3 Culture of internodes and laminae ................. 70 3.2.4 Effect of calcium concentration on somatic embryogenesis .................................................... 73 3.2.5 Effect of cytokinins on organogenesis and somatic embryogenesis ...................................... 74 3.2.6 Effect of 2iP and 2,4-D on the response of UH965 lamina culture ........................................ 74 3.2.7 Establishment of etiolated shoots from lamina culture .................................................................. 75 3.2.8 Root culture ........................................................ 75 3.3 Results ................................................................................ 76 3.3.1 Callus formation and somatic embryogenesis 76 3.3.2 Effect of calcium concentration on somatic embryogenesis ..................................................... 86 v i i 3.3.3 Cytokinins can induce shoots and somatic embryos on lamina cultures ................................................ 86 3.3.4 Response of UH965 lamina cultures to 2iP and 2.4-D 89 3.3.5 Formation of multiple etiolated shoots from lamina....................................................................... 92 3.3.6 Root culture ........................................................ 99 3.4 Discussion ......................................................................... 99 3.5 References ................................................................... 103 4. Asr/'o^acfe/'/t/m-mediated Transformation of >4/7f/7y/'/t//77 .... 106 4.1 Introduction ................................................................... 106 4.2 Materials and methods ..................................................... 1-08 4.2.1 Plant materials and media..................................... 108 4.2.2 Plasmids and bacteria .......................................... 108 4.2.3 Bactericidal assay of antibacterial proteins .... 110 4.2.4 Effect of kanamycin on formation of callus on lamina and internode explants.............................. 114 4.2.5 Cocultivation of explants and regeneration