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Bushiness in Micropropagated Zantedeschia: an Investigation at the Environmental, Physiological and Molecular Levels

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Bushiness in Micropropagated Zantedeschia: an Investigation at the Environmental, Physiological and Molecular Levels Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. Bushiness in micropropagated Zantedeschia: An investigation at the environmental, physiological and molecular levels A thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy In Plant Biology at Massey University, Palmerston North Suzanne Maree D' Arth 2003 Instituteof Molecular o UniversityCOllEGE OF SCIENCES BloScIences Massey Private Bag 11 222, Palmerston Narth. New Zealand Telephone: 6463569099 Facsimile: 646350 5688 Email: [email protected] CERTIFICATE OF REGULATORY COMPLIANCE This is to certify that the research carried out in the Doctoral Thesis entitled "Bushiness in micropropagated Zantedeschia: An investigation at the enviromental, physiological and molecular levels" in the Institute of Molecular BioSciences at Massey University, New Zealand: (a) is the original work of the candidate, except as indicated by appropriate attribution in the text and/or in the acknowledgements; (b) that the text, excluding appendices/annexes, does not exceed 100,000 words: (c) all the ethical requirements applicable to this study have been complied with as required by Massey University, other organizations and/or committees which had a particular association with this study, and relevant legislation. Please insert Ethical Authorisation code(s) here: (if applicable) Candidate's Name: Suzanne D'Arth Supervisor's Name: Paula Jameson Signature: Signature: () la, lt1 p<; � D r- Date: 411/1 Date: 1 0'blfS/o2-/0:.x Te Kunenga ki Purehuroa Inception to Infinity: Massey University's commiunent to learning as a life-long journey Institute of Molecular o UniversityCOUEGE OF SCIENCES BloSclences Massey Private Bag 11 222. Palmerston North. New Zealand Telephone: 64 6 3569099 Facsimile: 646 350 5688 Email: [email protected] CANDIDATE'S DECLARATION This is to certify that the research carried out for my Doctoral thesis entitled "Bushiness in micropropagated Zantedeschia: An investigation at the enviromental, physiological and molecular levels" in the Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand is my own work and that the thesis material has not been used in part or in whole for any other qualification. Candidate's Name Suzanne Maree D'Arth Signature 20 Te Kunenga ki Purehuroa Inception to Infinity: Massey University's commitment to learning as a life-Iongjoumey Institute ofMolecular o COUEGE OF SCIENCES Massey University BloScIe� Private Bag 11 222. Palmerston North. NewZealand Telephone: 64 6 3569099 Facsimile: 64 6 350 5688 Email: [email protected] TO WHOM IT MAY CONCERN This is to state that the research carried out for the Doctoral thesis" Bushiness in micropropagated Zantedeschia: An investigation at the enviromental, physiological and molecular levels" was done by Suzanne D'Arth in the Institute of Molecular BioSciences at Massey University, Palmerston North, New Zealand. This thesis material has not been used in part or in whole for any other qualification, and I confirm that the candidate has pursued the course of study in accordance with the requirements of the Massey Univeristy regulations. Supervisor's Name Paula Jameson Signature Te Kunenga ki Pilrehuroa Inception to Infinity: Massey University's commitment to learning as a life-longjourney Abstract The commercial Zantedeschia industry fac ed a potentially serious problem with the cultivar Florex Gold periodically producing 'bushy' plants with several short non­ flowering shoots and squat tubers with mUltiple nodes. These plants, derived from tissue culture, resulted in a reduced financial returnto the Zantedeschia industry. A number of potentially causative factors were investigated in this project but no single factor emerged as the cause of the bushy syndrome. The selection of tubers with multiple eyes for initiation into tissue culture, or tubers themselves recently derived from tissue culture, may be a contributing factor to bushiness. A variable growing environment (screen house), shortened storage time (11 weeks) and plant position were also shown to accentuate individual bushy characteristics. Endogenous cytokinin concentration and profile, determined by high performance liquid chromatography and radioimmunoassay, in tubers at three stages in the Zantedeschia life cycle were similar between bushy and non-bushy plants and cannot explain the bushy phenotype. Micropropagation of Florex Gold on Murashige and Skoog medium supplemented with low cytokinin concentration (4.4 IlM 6-benzylaminopurine, 6-BAP) did not produce bushy plants or tubers. The use of elevated cytokinin during micropropagation did not produce plants with a range of bushy symptoms, although elevated thidiazuron (13.6 IlM), but not 6-BAP, was shown to affect flowerspathe length and plant shoot number. Florex Gold, when compared to a range of non-bushy Zanledeschia cultivars, displayed greater sensitivity to cytokinin during micropropagation assessed by a root length bioassay. Increased sensitivity to 6-BAP, during micropropagation may indicate a predisposition to bushiness. Differences in the DNA methylation profileof bushy and non-bushy plants were fo und in DNA using DNA Amplified Fragment Length Polymorphism. However, the isolated sequences were not similar to any gene(s) currently listed in public databases. Diffe rential gene expression levels, determined by cDNA Amplified Fragment Length Polymorphism, fo und 60S and 18S ribosomal RNA down-regulated in bushy Florex Gold. These two genes may play a role in the expression of the bushy phenotype. Changes in DNA methylation and diffe rential gene expression patterns and the results from the micropropagation experiments, provide evidence that Zantedeschia Florex Gold tubers selected fo r commercial micropropagation should be deep and apicaUy dominant with fe w nodes and buds, and not themselves recently derived from tissue culture. 11 Acknowledgemen ts I would like to thank my supervisors Professor Paula Jameson (Institute of Molecular BioSciences), Sandra Simpson (Multi flora Laboratories Limited, Auckland) and John Seelye (Crop & Food Research, PalrnerstonNorth), fo r their support, advice and help throughout my PhD. I wish to express my appreciation fo r the financial support provided by Technology New Zealand. I would like to thank all the past and present staff at MultifloraLaboratori es fo r their support and friendshipthroughout my PhD. I am very grateful to the staff at Crop & Food Research, especially, Bruce Dobson, Bev Hoffman, Andrew Mullan, and Garry Burge. I would like to thank all those people in the Institute of Molecular Biosciences, especially Anya Lambert, Trish Murray, Richard Scott, Trish McLenachan, Andrew Clarke, Angie Richards, Nigel Gapper, and Jason Song. My thanks to Dr Jean Finnegan (CSIRO, Canberra) fo r help with DNA methylation research and Sheree Cato and Lisa McMillian (Forest Research, Rotorua) fo r help with the cDNA AFLP technique. I would like to thank Andrew Wearmouth and Tina and Rex Reed, both Zantedeschia growers, fo r supplying tubers and infonnation fo r my research. Also D. Harrison and J. Kapityen fo r supplying Zantedeschia tubers. My thanks to my family, especially my sister Adrienne fo r her support. I would like to thank my inlaws, Syd and Shirley, fo r letting me stay with them during my studies in Palmerston North. Finally a special thanks to my husband Coos, who didn't see me very oftenbut was always there fo r me. 111 Table of Contents Abstract ................. ......... ....... ......... ........ ....... ..................... ......................... ......... ........... ... i Acknowledgements .......................................................................................................... iii List of Figures .................................................................................................................. xi List of Tables ................................................................................................................. xvi Chapter One: Introduction ............................................................................................ 1 1.1 Overview .................. ........ ... .................................. ............. ...... ... ..... ................... 1 1.1.1 The genus Zantedeschia .................. ................ ...................................... ...... 2 1.1.1.1 Botanical classification and morphology ...... .......... ................ ..... ..... 2 1.1.1.2 Commercial life cycle of Zantedeschia ..... ......... ................. ...... ........ 3 1.1.2 Bushy syndrome in Florex Gold ........... .. ................ ........... ... .... .... .... .......... 5 1.1.2.1 First reports of bushiness in New Zealand Florex Gold ......... ... ........ 5 1.1.2.2 Suggestions fo r the bushy syndrome ......... ..... ....... .... ...... ...... ...... .... 6 1.1.2.3 New Zealand commercial Zantedeschia growing conditions and bushiness ... ............. .. .... ............. .............. ......... .. ............. ................ 8 1.2 Cytokinins and bushiness................. ...................... .............. ......................... ....... 9 1.2.1 Cytokinin structure .......... ....... ................................ .......................... ........... 9 1.2.2 Cytokinin biosynthesis ............ ................ .......
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