BIOTECHNOLOGICAL APPROACHES TO ROSE BREEDING. by Robert Marchant, B.Sc. (Hons) Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy, July 1994. ACKNOWLEDGEMENTS. I wish to thank Drs. J. B. Power and M. R. Davey for their help and supervision, S.E.R.C. and David Austin Roses, Albrighton, UK for funding this research and Dr. J. M. Chartier-Hollis for useful discussions. I also wish to thank all of my research colleagues for their help and friendship. My family and friends deserve special thanks for the patience, inspiration, practical help, encouragement and loving support without which I would not have been able to complete my studies. The photographic skills of Brian Case, and the technical assistance provided by Dave Wilson, John Gaskin and Ian Gilder are also gratefully acknowledged. CONTENTS. ABSTRACT..................................................................................... ix ABBREVIATIONS ............................................................................ xi CHAPTER 1. Gene.'al Introduction. 1.1 Tissue culture of woody species an update .................................................................................. 1 1.2 Tissue culture for genetic engt'neering of woodv sl)ecies ................................ 3 ~ ~. 1.2.1 Somatic hybridisation ............................................................ 3 1.2.2 Genetic transfonnation ............................... , ........................ '" 5 1.2.3 Tissue culture and genetic engineering of woody ornamental species ..................................................................................... 8 1.3 Classification and history of Roses ........................................................ 11 1.3.1 Introductory remarks ............................................................. 11 1.3.2 Biological and field classification ............................................... 11 1.3.3 The history of the cultivation and breeding of roses .......................... 13 1.4 Breeding objecti ves .......................................................................... 17 1.5 General background to rose biotechnology and genetic engineering .................. 18 1.5.1 Micropropagation of rose: its development and commercial uses ........... 18 1.5.2 Plant regeneration from cultured rose tissues .................................. 21 1.5.3 Ploidy manipulation and mutagenesis .......................................... 24 1.5.4 Isolation and culture of protoplasts and subsequent plant regeneration ............................................................................... 26 1.5.5 The role of somatic hybridisation in rose improvement. ..................... 27 1.5.6 Transfonnation technologies as applied to rose ............................... 28 1.7 Thesis objectives ............................................................................. 31 CHAPTER 2. Sexual hybridisation, embryo rescue and the generation of intervarietal rose hybrids. 2.1 Introduction ........................................................................ " ......... 32 2.2 Materials and Methods ....................................................................... 34 2.2.1 Embryo rescue techniques ....................................................... 34 2.2.1.1 Source of material ..................................................... 34 2.2.1.2 Conventional gennination of rose seed ............................. 35 2.2.1.3 Procedure for excising embryos ..................................... 35 2.2.2 Investigations into the requirements for the culture of excised rose embryos ................................................................................... 36 - 1 - 2.2.2.1 The effect of culture medium composition on excised rose embryo germination ....................................................... 36 2.2.2.2 Effects of light and temperature on the germination of rescued embryos ................................................................ 38 2.2.2.3 Transfer and acclimation of embryo rescue derived seedlings to glasshouse conditions, and the use of Sorbarods®. ........ 40 2.2.2.4 Investigations into the failure of conventional achene germination ...................................................................... 41 2.2.3 Determination of sexual incompatibility in intercrossed English rose cultivars .............................................................................. 42 2.2.3.1 Assessment of pollen viability ....................................... 43 2.2.3.2 Methods of observing of pollen germination and pollen tube growth on and within stylar tissues ..................................... 43 2.2.4 Statistical analysis ................................................................. 44 2.3 Results ......................................................................................... 44 2.3.1 Conventional gennination of rose seed ......................................... 44 2.3.2 Recovery and culture of embryos ............................................... 44 2.3.2.1 Effects of culture media composition on the germination of excised rose embryos ........................................................ 44 2.3.2.2 Effects of light and temperature on the germination of excised rose embryos ........................................................... 46 2.3.2.3 Transfer of seedlings to ex vitro (glasshouse) conditions ........................................................................ 48 2.3.3 Investigations into the failure of conventional achene germination ......... 49 2.3.4 Possible causes of sexual incompatibility in English Rose crosses ..................................................................................... 50 2.3.4.1 Assessment of pollen viability ...................................... 50 2.3.4.2 Observation of pollen germination and growth on/within the style ........................................................................... 50 2.4 Discussion ..................................................................................... 53 2.4.1 Embryo rescue .................................................................... 53 2.4.2 Possible causes of sexual incompatibility ..................................... 60 2.5 Summary ...................................................................................... 64 CHAPTER 3. Cryopreservation of rose pollen. 3.1 Introduction ................................................................................... 66 3.2 Materials and Methods ....................................................................... 68 3.2.1 Assessment of different methods for determining pollen viability ......... , 68 3.2.2 Collection and dehiscence of anthers - II - the effects of dehiscence treatment and degree of flower opening on pre-storage pollen viability ................................................. 69 3.2.3 Cryopreservation of rose pollen ................................................. 70 3.2.4 Morphological examination of pollen prior to and post- cryopreservation .......................................................................... 71 3.2.5 Use of cryopreserved pollen to effect fertilisation of rose flow'ers .......... 72 3.2.6 Efficacy of pollen cryopreservation in comparison to other techniques ................................................................................. 72 3.2.7 Statistical analyses ............................................................... 73 3.3 Results ...................................................................................... '" 73 3.3.1 Methods for assessing pollen viabili1)l ......................................... 73 3.3.2 Effects of dehiscing treatments and degree of flower opening on pre-storage rose pollen viability ........................................................ 73 3.3.3 Cryopreservation .................................................................. 74 3.3.3.1 Assessment of different methods of cryopreservation............ 74 3.3.3.2 Effects of dehiscing treatments on post-cryopreservation pollen viability ................................................................... 74 3.3.4 Morphological examination of pre- and post-cryopreserved pollen using scanning electron microscopy (SEM) .......................................... 74 3.3.5 Cross pollination using cryopreserved pollen ................................. 75 3.3.6 Comparison of cryopreservation for long-term storage of rose pollen, compared to other methods. .................................................. 75 3.4 Discussion ..................................................................................... 81 3.4.1 Methods for assessing pollen viability ......................................... 81 3.4.2 The long-term storage of rose pollen .......................................... 82 3.4.3 Efficacy of cryopreservation, for the long tern} storage of rose pollen, in comparison to other techniques ............................................. 86 3.5 Sununary ...................................................................................... 86 CHAPTER 4. Development of tissue culture systems fOi' rose. 4.1 Introduction ................................................................................... 88 4.2 Materials and Methods ............................................................ , .......... 90 4.2.1 Plant material ori gin and sources ............................................................... 90 4.2.2 Establishment of axenic shoot cultures ......................................... 91 4.2.2.1 Assessment