University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Theses, Dissertations, and Student Research in Agronomy and Horticulture Agronomy and Horticulture Department August 1997 ASYMBIOTIC in vitro SEED GERMINATION, MICROPROPAGATION AND SCANNING ELECTRON MICROSCOPY OF SEVERAL TEMPERATE TERRESTRIAL ORCHIDS (ORCHIDACEAE) Erika Szendrák Hungarian Academy of Sciences, Secretariat General Follow this and additional works at: https://digitalcommons.unl.edu/agronhortdiss Part of the Plant Sciences Commons Szendrák, Erika, "ASYMBIOTIC in vitro SEED GERMINATION, MICROPROPAGATION AND SCANNING ELECTRON MICROSCOPY OF SEVERAL TEMPERATE TERRESTRIAL ORCHIDS (ORCHIDACEAE)" (1997). Theses, Dissertations, and Student Research in Agronomy and Horticulture. 1. https://digitalcommons.unl.edu/agronhortdiss/1 This Article is brought to you for free and open access by the Agronomy and Horticulture Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Theses, Dissertations, and Student Research in Agronomy and Horticulture by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. ASYMBIOTIC IIV nTRO SEED GERMINATION,MICROPROPAGATION AND SCANNING ELECTRON MICROSCOPY OF SEVERAL TEMPERATE TERRESTRTAL ORCHIDS (ORCHIDACEAE) by Erika Szendriik A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska in Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy Interdepartnental Area of Major: Horticulture and Forestry Under the Supervision of Professor PzuI E, Read Lincoln, Nebraska August, 1997 ASYMBIOTIC l7V MTRO SEED GERMINATION, MICROPROPAGATION AND SCANNING ELECTRON MICROSCOPY OF SEVERAL TEMPERGTE TERRESTRlAL ORCHIDS (ORCHIDACEAE) Erika Szend* Ph.D. University of Nebraska, 1997 Advisor: Paul E. Read Twenty-five different orchid species were success~yasymbioticdy germinated and raised on a modified FAST medium (Fasf 1976; Szendrhk and R Eszelq 1993). The development of geminating protocorms and young plantlets were recorded and compared among species. Spontaneous vegetative proliferation was also observed. Natural dispersed daylight and prevailing day-length were more favorable than ldhour cool white fluorescent light for plantlet development. After two to three years of culture, the young plants were suitable for transfer ex vipo. The effects of organic compounds most commonly used for orchid micropropagation (peptone, coconut water, casein+lactalburnin and glucose) and medium consistencies were investigated for the development of temperate orchid protocorms fiom five species. Medium consistency had an important role in protocorm proliferation and development. In most cases, the liquid medium increased proliferation and the size of the protocorms. The media supplemented with the undefined organic compounds led to a greater proliferation rate and larger protocotms than the medium supplemented with glucose. In general, peptone and coconut water resulted in best development and proliferation of protocorms, but this varied with species. Anatomical structures and developmental patterns of seventeen temperate orchid species were observed by scanning electron microscopy (SEM). New information was recorded about the early stages of seed germination and protocum development, the structural details of roots with mycomhm, stored materials (calcium oxalate crystals and starch), leaflstem stomatd structure and anatomical details of the generative organs and ovule/seed development. Extensive SEM observations were also conducted on the seeds of more than 120 temperate orchid species. The length of the smallest seeds was -200 mm (Spiranthesvp.) and the largest ones reached -1700 mm (Cypripedium, Epipctis spp) in length. Both quantitative and qualitative data were collected and detailed descriptions of the seeds of the different orchids were prepared. Several fascinating surface patterns and shapes were found, which were specific not only on the level of higher taxonomic units, but sometimes were distinctive even for the species or subspecies. This dissertation is dedicaled to my mother Piroska Nyolczas d to my dear grandfather Ignciz Nyolczas whose infinite love, inspiration, encouragement rmd support enabled me to achieve my g0c1Is. "As Orchids are universally c~cknowiedgedto rank amongst the most singular and most mod~j?edforms in the vegetable kingdom, I huve thought that the facts to be given might lead some observers to look more euriotcsi) into the habits of our several nutive species. .... In my emination of Orchids, hardly any fact has struck me sa much as the endless diversities of structure, - the prodigality of resources, - for gaining the wry same end, namely, the fertilisution of one jlower by pollen fiom another plant." Charles Darwin, 1877 vii ACKNOWLEDGEMENTS The author wishes to express her thanks and appreciation to her advisor. Dr. Paul E. Read for his guidance, support, encouragement and inspiration, and also to her graduate committee, Dr. Robert B. Kaul, Dr. Dale T. Lindgren, Dr. Ellen T. Paparazzi and Dr. Donald H. Steinegger for their useM inputs and advice. Special thanks to Mis. Emebet Tirnok, Eszter R Edki MS, Dr. Elizabeth Jiimbor- Bencziir, Dr. Olga Borsos and Dr. Giibor Schmidt for the basic training in the area and for their encouragement to work on this project; to Dr. Aniko Csillag and Dr. Kit W. Lee for their essential technical help in scanning electron microscopy and to Dr. Pierre C. Debergh for his valuable help to obtain several diflicult to reach references for the literature review. This program has been possible with the financial help of the University of Nebraska- Lincoln, the Hungarian State E6mos Scholarship Board, the University of Horticulture and Food, Budapest and the Omaha Rotary Club. Their support is greatly appreciated. The author is also indebted to all who provided the valuable seed- and plant material for this research, especially to the Botanical Garden and Orchid Herbarium of the E6tviis Lorind Scientific University (EL=) of Budapest, the Charles E. Bessey Herbarium, UNL, the members of the Greater Omaha Orchid Society, the Nebraska Statewide Moreturn, the Nebraska Game and Parks Commission, to Dr. Ann AntJfinger, Dr. Robert B. Kaul Dr. Zsolt Debreczy and Mr. Chin C. Chu. Among many others to acknowledge are: Dr. Servet Kefi for her willingness to discuss statistical approaches, Ruth Hansen, Melissa Maurer, Lisa Sutton, Handan Biiyiikdemirci, Cindy Loope, Waynetta Morningstar, Rosemary Neemann and especially the Eisloeffel and Read families for their fiendship and support. The author deeply bows before the memory of the late Dr. Mka Sulyok, who several years ago gave her the first professional possibility to work with orchids, these beautfil and exciting plants. Loving gratitude is extended to the authoh mother, grandfather, sister Agnes and her dearest nieces Agnes and Katalin for their love and encouragement throughout her doctoral program which were essential to the klfillment of this extraordinary opportunity. TABLE OF CONTENTS Page Title ............................................................................................... 1 ... Abstract .......................................................................................... UI Dedication ....................................................................................... v Acknowledgements ............................................................................. vii -.. Table of Contents ............................................................................... VIU List of Figures ................................................................................... xi List of Tables ................................................................................... xxi Introduction ..................................................................................... 1 Literature Review .............................................................................. 8 CHAPTER I. Asymbiotic In Ktro Germination and Development of Severat Temperate Terrestrial Orchids (Orchidaceae) ......................................... 39 Abstract .......................................................................................... 39 Introduction ..................................................................................... 40 Materials and Methods ......................................................................... 42 Results and Discussion ......................................................................... 44 Conclusions ...................................................................................... 49 Literature Cited ................................................................................. 50 CHAPTER II. The Effects of Medium Consistency and Organic Substances on the Protocorm Proliferation of Some Temperate Terrestrial Orchids (Orchidaceae) Cultured in Vi~o........................................................... 65 Abstract .......................................................................................... 65 Introduction ..................................................................................... 66 Materials and Methods ......................................................................... 7 1 TABLE OF CONTENTS (Continued) Page Rdtsand Discussion ......................................................................... 74 Conclusions ...................................................................................... 80 Literature