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Theses, Dissertations, and Student Research in Agronomy and Horticulture Agronomy and Horticulture Department

August 1997

ASYMBIOTIC in vitro SEED , MICROPROPAGATION AND SCANNING ELECTRON MICROSCOPY OF SEVERAL TEMPERATE TERRESTRIAL ORCHIDS ()

Erika Szendrák Hungarian Academy of Sciences, Secretariat General

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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 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 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 Cited ...... 81

CHAPTER III . Anatomical Observation of Some Temperate Terrestrial Orchids Using Scanning Electron Microscope with Special Reference to morio L.. Abstract ...... Introduction ...... Materials and Methods ...... Results and Discussion ...... Germination and early plant development ...... Roots and mycorrhiza ...... Underground storage organs ...... Stems and ...... Flowering and biting ...... Conclusions ...... Literature Cited ......

CHAPTER IV . Seed Morphology of Several Temperate Orchid Species with Special Emphasis on the Members of (Orchidaceae) ...... Abstract ...... Introduction ...... Materials and Methods ...... Results and Discussion ...... Overall observations ...... Detailed descriptions ...... Conclusions ...... TABLE OF CONTENTS (Continued)

Page Literature Cited ...... 181 Proposed Future Research ...... 202 Appendices ...... 206 A. Preliminary acclimatization attempts for in vitro derived temperate orchid plants ...... 206 I3 . Orchis rnorio L. f: albonivea Szendrik ...... 209 LIST OF FIGURES

Figure Page 1- 1 Asymbiotically germinating Orchis morio seeds cultured in vitro ...... 6 1 1-2 This young culture of Dactylorhim manrlata was transferred with the old layer of culture medium because of the very sensitive young protocorms. After transfer the protocorms continued to develop and became ready to individually transfer as plantlets...... 6 1 1-3 Cultures of Orchis morio from the same seed source under laboratory condition (16 hodday cool fluorescent light (32 pmol iLmW2 light intensity) and standard yearlong temperature) or under natural illumination with prewhg day-length (near a south-facing window) with seasonally changing temperature ...... 62 1-4 Young cuItures of Orchis morio £iom two different seed source under laboratory conditions (16 hourlday cool fluorescent light (32 pmol i' m-2 light intensity) and standard yearlong temperature) ...... 62 1-5 Spontaneously vegetatively proliferating isolated protoconns of Dactylorhim fuchsii ssp. ssooiana ...... 63 1-6 Three-year-old in vitro culture of Platanthera bzyolia with protocorm (1) originating fiom spontaneous vegetative proliferation. (2) new tuberoid, (3) old tuberoid ...... 63 1-7 Three-year-old in vitro derived culture of Himmtoglos~nhircimm ready for acclimatization ...... 64 1-8 Two-and-a half-year-old in vitro derived culture of Cypripdium cuiifomicum ready for acclirnathtion ...... 64 2-1 The effect of four different organic substances and medium consistency on the numbers of protocorms ofDactylorhizafuchii cultured in vifro(Three protocorms explanted/jar) ...... 87 LIST OF FIGURES (Continued)

Figure Page 2-2 The effect of four different organic substances and medium consistency on the final average number of protocorms of Dactylorhizc~fuchsiicultured in vitro (Three protocorms explantedfjar) ...... 2-3 The effect of four merent organic substances and medium consistency on the ha1total fiesh weight of protocorms of Dactylorhirofuchsii cultured in vitro (Three protocorms explantedjar) ...... 2-4 The effect of four different organic substances and medium consistency on the final average length of protocorms of DactyIothizafuchsii cultured in vitro (Three protocorms explanted/jar) ...... 2-5 The effea of four different organic substances and medium consistency on the numbers of protocorms of Dactylorhiiza maculata cultured in vitro (Three protocorms explanted/jar) ...... 2-6 The effect of four different organic substances and medium consistency on the final average number of protocoms of Dac2)1Iorhiza mamlata cultured in vitro (Three protocorms explanted/jar) ...... 2-7 The effect of four different organic substances and medium consistency on the find total Eesh weight of protocorms of Dactylorhim mamZuta cultured in vifro (Three protocorns explanted/jar) ...... 2-8 The effect of four different organic substances and medium consistency on the find average length of protocorms of DactyIorhim mac21Iata cultured in vitro (Three protoconns explantedljar) ...... 2-9 The effect of four different organic substances and medium consistency on the numbers of protocorms of Dactyorhiza majais (33j) cultured in vitro (Three protocorms explantedljar) ...... 2-10 The effect of four different organic substances and medium consistency on the final average number of protocorms of Dactylorhira majalis (33j) cultured in vitro (Three protocorms expIanted/jar) ...... xiii

LIST OF FIGURES (Continued)

Figure Page 2-1 1 The effect of four different organic substances and medium consistency on the find total fresh weight of protocorms of Dactylorhim majulis (33j) cultured in vitro (Three protocorms explanted/jar) ...... 97 2-12 The effect of four different organic substances and medium consistency on the final average length of protocorms ofDuctylorhiu~mqalis (33j) cultured in vizro (Three protocorms explanted/jar) ...... 98 2-13 The effect of four different organic substances and medium consistency on the numbers of protocorns of Dactylothiza majalis (33 k) cultured in vitro (Three protocorms expIanted/jar) ...... 99 2-14 The effect of four different organic substances and medium consistency on the final average number of protocorms ofDactylorhizu majalis (33 k) cultured in vitro (Three protocorms explantedljar) ...... 100 2- 15 The effect of four different organic substances and medium consistency on the final total fresh weight of protocorn of majalis (33k) cultured in vitru (Three protocorms explanted/jar) ...... 101 2- 16 The effect of four different organic substances and medium consistency on the final average length of protocorms of (33k) cultured in vitro (Three protocorms explanted/jar) ...... 102 2-17 The effectof four different organic substances and medium consistency on the numbers of protocorms of 2- cultured in M'tro (Three protocorms explanted/jar) ...... 103 2-1 8 The effect of four different organic substances and medium consistency on the final average number of protocorms of Ophrys lutea cultured in vifro (Three protocorms explantedljar) ...... 104 2-1 9 The effect of four Werent organic substances and medium consistency on the final total fkesh weight of protocorms of Ophrys lutea cultured in vim (Three protocorms expIanted/jar) ...... 105 LIST OF FIGURES (Continued)

Figure Page 2-20 The effect of four different organic substances and medium consistency on the final average length of protocorms of Ophrys lutea cultured in vitro (Three protocorms expIanted/jar) ...... 106 2-21 The effect of four different organic substances and medium consistency on the numbers of protocorms of Orchis morio cultured in vitro (Three protocorms explanted/jar) ...... 107 2-22 The effect of four different organic substances and medium consistency on the final average number of protocorms of Orchis morio cultured in vitro (Three protocorms exp1antedIja.r) ...... 108 2-23 The effect of four different organic substances and medium consistency on the final total fiesh weight of protocorms of Orchis morio cultured in vitro (Three protocorms explantecbjar) ...... 109 2-24 The effect of four different organic substances and medium consistency on the final average length of protoconns of Orchis morio cultured in vitro (Three protocorms explantecbjar) ...... 110 2-25 Protocorms of Dactylorhizafichsii at the end of the six-week-long experimental period from treatment. containing different organic substances (PE: peptone, C&L: casein-tlactalburnin, CW: coconut water, GL: glucose) in combination with liquid or gelled ("solid") media ...... 1 1 1 2-26 In vitro cultures of protocorns of Dactylorhiza maculata at the end of the six-week-long experimental period representing different treatments containing different organic substances (CW: coconut water, PE: peptone, C&L: casein+lactalbumin, GL: glucose) in combination with liquid or gelled ("solidn) media. Note the brown color of the liquid medium supplemented with glucose, probably caused by polyphenol exudation from stressed explants ...... 1 12 LIST OF FIGURES (Continued)

Figure Page 3-1 Swollen germinating Orchis morio seed. note the bulging embryo (SEM 20Ox) ...... 136 3-2 The embryo of Orchis morio rupturing the testa (SEM 265x) ...... 136 3-3 The fmt rhizoids (arrows) appear on the protocorm of Orchis morio (SEMlOOx)...... 136 3-4 The apical meristem (1) starting to develop on the protocorm of Orchis morio; (2) remnant seed-coat (SEM 100x) ...... 136 3-5 The conical meristematic dome (1) on the protocorm of Orchis morio . (2) round undifferentiated cells of the protocorm, (3) remnant seed-coat (SEMllOx) ...... 136 3-6 Sunken appical meristem (Orchis morio; SEM 1SOX) ...... 136 3-7 The first real appears (arrow) (Orchis morio; SEM 250~)...... 136 3-8 Structures of the protocoxm start to develop, the first stoma (arrow) appears (Orchis morio; SEM 1 18x) ...... 136 3-9 Young Orchis morio plantlet with photosynthetic ability (SEM 26.5~)...... 136 3-10 Cross section of the root of Orchis morio (SEM 55x) with the mycorrhizal pelotons in the cortex (SEM 55x) ...... 138 3-1 1 Fungal infection at the base of a rhizoid of the root of Orchis morio (SEM290x) ...... 138 3- 12 Orchid type mycorrhiza (Corallorhiza odontorhiza, no outer Hartnig-net on the outer surface of the root, the mycorrhiza only present inside the root in the cortex cells; SEM 145x) ...... 138 3- 13 Pelotons in the root cortex of Corallorhim odontorhiza (SEM 400x) ...... 138 3-14- 17 The stages of the mycorrhiza in the root of Orchis rnorio :...... 140 3-14 Anastomization (SEM 485x) ...... 140 3-15 Peloton (arrow) formation (SEM 500~);...... 140 LIST OF FIGURES (Continued)

Figure Page 3-16 Functioning peloton . (1) peloton, (2) starch grain, (3) microscopic ropes (SEM400x);...... 140 3- 17 Degenerated peloton (1). (2) microscopic ropes (SEM 320x) ...... 140 3- 18 Twin tuberoids of Orchis morio (magdication: 2.5~)...... 142 3- 19 The new bud in the neck of the younger tuberoid of Orchis morio (SEM 14x)...... 143 3-20 Cross section of the older tuberoid of Orchis morio . (1) stele, (2) vascular bundle, (3) rhizodermis (SEM 15x) ...... 143 3-2 1 Longitudinal section of the older tuberoid of Orchis rnorio . (1) stele, (2) rhizodermis, (3) stemheck of the tuberoid (SEM 13x)...... 143 3-22 Calcium oxalate needle crystals in the tuberoid of Orchis mo~o(SEM 400x) ... 143 3-23 Starch grains (1) and slime materials/salep (2) in the tuberoid of Orchismorio(SEM 1250x)...... 143 3-24 Starch aggregate ("pentamorion)in the tuberoid of Orchis morio (SEM 385x) ...... 143 3-25 Individual starch grains in the root of Spiranihes vedis(SEM 2500x) ...... 143 3-26 Individual starch grains in the root of C'ipedium ade(SEM 2000~)...... 143 3-17 Individual starch grains in the root of Galems qectabilis (SEM 1000~)...... 143 3-28 Starch aggregates in the root of Himanfoglosssu hircim (SEM 1000~)...... 145 3 -29 Starch aggregates in the root of C'ripedium califomicum (SEM 93 3x) ...... 145 3-3 0 Starch aggregates in the root of vemalis (SEM 2000~)...... 145 3-3 1 Bundle of calcium oxalate needle crystals (Platantheru hperborea; SEM 1130x) ...... 145 3-32 Complex calcium oxalate crystal near some needle crystals in the root of Cpripedium cuIifomicum (SEM 1440~)...... 145 3-33 Complex calcium oxalate crystal in the root of Cjpripedim acauie (SEM 1130~)...... 145 LIST OF FIGURES (Continued)

Figure Page 3-34 Complex calcium oxalate crystal in Platcmthera hyperborea (SEM 1370x). .. . 145 3-35 Higher magnification of the complex calcium oxalate crystal fiom Figure 3-34, Note the parallel needle crystals as they built up the complex form (SEM 9710x) ...... -...... --.. 145 3-36 Cross section of the root of C'ripedim acaule at the end of the vegetative season fiom its native habitat. No viewable mycorrhiza presenf and the

cortex is 111 of starch (arrow: rhizoid; SEM 33x)...... + ...... 145 3-37 Cross section of the stem of Orchis morio; epidermis (1); chlorenchyma (2); sclerenchyma (3); vascular bundle (4); parenchyma (5); central cavity or pith (6) (SEM 37x) ...... 147 3-38 Young stoma of Orchis morio with the remnant of the subsidi;uv/accessory

cells (arrow) (SEM 1400~)...... ,...... 147 3-39 Fuliy deveIoped stoma from the stern of Orchis morio (1 000~)...... 147 3-40 Degenerated stoma from the ieaf of Corullorhiza dontorhiza (SEM978x)...... I47 3-41 Trichomes with spherical head cells filled with oil fiom the surface of the stem of Cypripedium califomim (SEM 113x)...... 147 3-42 Wax structures firom the surface of the stem of Corallorhiza odontorhiza (SEM 5300~)...... -. 147 3-43 The structure of the flower of Orchis morio - generative parts: pollinium (I), rostelIum (2), column (3), entrance of the spur (4), staminodium (5)

(magnification: 8x). .. ., ...... 149 3-44 The structure of the flower of Orchis morio - vegetative parts: spur (I), bract (Z), hood formed fiom the and (3)

(magdication: 4x) ...... +...... 149

3-45 The upper surface of the tip of Orchis morio (SEM 250~)...... , . 1 5 1 LIST OF FIGURES (Continued)

Figure Page 346 The lower daceof the tip of Orchis rnorio with stomata and the braid-like edge (SEM 200x) ...... -...... 151 3-47 The upper surface of the tip of Spirmthes vernalis (arrow: mid-vein; sEM55.7~)...... 151 3-48 The head of the pollinarium (polhiurn) of Orchis morio (arrow: segments built up fiom pollen grains; SEM 67~)~...... 153 3-49 Viscidium fiom the poharium of Orchis morio (SEM 235x) ...... 153 3-50 A part of the caudicle and the upper surface of the viscidium of

Platanthera praeclara (SEM 59.1x) ...... , ,...... 153 3-5 1 The structure of the mature viscidium of Plaranlherapraeclara. (1) single basal epidermal layer, (2) sticky glue (SEM 687x)...... 153 3-52 Pollinarium of Orchis rnorzo. (1) pollinium; (2) caudicle; (3) viscidium (SEM 40x)...... - .. - - -.- - -. 155 3-53 PollinariumofSpiranthesvemIis(SEM3lx)...... 155 3-54 Pollinarium of Corallorhiza odontorhiza (SEM 95x)...... 15 5 3-55 The cross section of the twisted ovary of Orchis morio before (arrow: empty curves of the placenta; SEM 67x).,...... 157 3-56 The placenta with the anatropous ovules of Orchis morio before

pollination (SEM 200~)...... , ...... 157 3-57 Ovule of Orchis morio before pollination. (I) rnicropyle, (2) outer integument (SEM430x) ...... -...... -- 157 3-58 Longitudinal section of the ovule ofPhtantherapraeclara. (1) embryo, (2) micropyle, (3) integumental single ceU layer (SEM 450~)...... 157 3-59 The integument covering the embryo and the developing seed of Orchis morio (SEM 400x)...... -.. .. . 15 7 3-60 Fully developed seed before desiccation (Orchis morio; SEM 300x)...... 157 LIST OF FIGURES (Continued)

Figure Page 3-61 The untwisted ovarylyoung hit of Orchis morio after pollination, with the developing seeds (arrow: the curves of the placenta Ned with hygroscopic hairs; SEM 25x)...... 159 3-62 The outer surface of the ripe hit of Orchis morio with the desiccated epidermal cells and degenerated stoma (SEM 800~)...... 159 3-63 The placenta of Orchis morio with the ripening seeds (arrow: hygroscopic hairs; SEM 52x)...... 159 3-64 The curve of the placenta of Orchis morio filled with hygroscopic hairs (SEM 165x)...... 159 3-65 The placenta of spectabilis with the ripening seeds (arrow: hygroscopic hairs; SEM 30x)...... 159 3-66 The placenta of Platantherapraeclara with the ripening seeds (arrow: hygroscopic hairs; SEM 34x)...... 159 4-1 "Widow framen type surface pattern (arrows) formed from a membraneous zone attached to the anticlinal walls (Epipctis gigantea, SEM 1010x) ...... 184 4-2 The shorter anticlinal walls are raised (arrows) resembling an "arch-typen appearance (Phalaenopsi fmciata, SEM 1500x) ...... 184 4-3 "Grainn trpe surface pattern on the edges of the anticlinal walls (Dendrobium crumenatum, SEM 2000x)...... 185 4-4 Swirling lines on the periclinal wall as a surface pattern (Corallorhiza odontorhiza, SEM 2000x)...... 185 4-5 Parallel lines on the periclinal wall as a surface pattern. (a) Ophrysfirc1j70ra (SEM 1000~);(b) Orchis coriophora (SEM 500x); (c) Orchis morio (SEM 1000~); (d) Gennana diphylla (SEM 500x) ...... 186 4-6 Irregular (a) or longitudinal (b) ridges on the periclinal wall as a surface pattern (a: Cypripedium acaule, SEM 500x; b: Cephalanihera rubra, SEM 1000~)...... 187 LIST OF FIGURES (Continued)

Figure Page 4-7 No surface pattern on the periclinal walls of testa ceUs (Malmnspaludosa, SEM 2000~)...... 187 4-8 The seeds of Spiranthes dilwialis (a) SEM 250x; Spiranthes vemlis @) SEM 250x; Platanthera praeclara (c)SEM 250y Phkmfhera hyperborea (d) SEM 500x ...... 188 4-9 The seeds of Himantoglossurn hircimrm (a) SEM 250~;Chamorchis alpina (b) SEM 265x; Orchis purpurea (c) SEM 250x; Orchis militan's (d) SEM 260x...... 189 4- 10 The seeds of Ophrys sphegodes (a) SEM 250x; Orchis morio (b) SEM 175~;Dactylorhiza sambucinu (c) SEM 150x, Dactylorhiza fichsii ssp. sooiana (d) SEA4 170x ...... 190 4-1 1 The seeds of Orchis lux~jlora(a) SEM 125x; Orchzs coriophora ssp. ftagrans (b) SEM 150x; Dactylorhiza mumlata (c) SEM 125x; Dactylorhiur saccifea (d) SEM 1 3 5x...... 1 9 1 4- 12 The seeds of Dactylorhiza incanta (a) SEM 125x; Galearis spectabilis @) SEM 100x; Genmadiphyila (c) SEM 1 OOX, V& coenrlea (d) SEM 500x ...... 192 5-1 Orchis mono plantlets growing in their native habitat originated firom in vitro cultures ...... 2 1 1 5-2 Flowering Orchis morio plant acclimatized in autoclaved soil mixture ...... 2 12 5-3 Three-year-old in vitro culture of Orchis morio L.f: albonivea Szendrik ...... 2 13 LIST OF TABLES

Table Page 1-1 Basic culture medium for the asymbiotic culture of temperate terrestrial orchids: modified FAST (Fast, 1976; Szendrik and R, Esteki, 1993) ...... 57 1-2 Ingredients of POLYVITAPLEX@-8 vitamin complex (produced by CHINOIN Pharmaceutical Company, Hungary), used to replace vitamins in the modified FAST medium ...... 58 1-3 The list of orchid species in the in vitro germination experiment (in taxonomic order based on Dressier, 1993) ...... 59 4-1 Orchid species included in the scanning electron microscopy study (in taxonomic order based on Dressier, 1993)...... 193 4-2 Quantitative data table for the detailed description of orchid seeds ...... 199 Introduction

Orchids were among the first plants that were successllly cuItured in vitro. From the first attempts of in vitro seed germination (Knudson, 1921; Burgeff, 1936) and vegetative micropropagation (Rotor, 1949; Morel, 1960) numerous scientists worked to opdmize these procedures (Knudson, 1946; Reinert and Mob, 1967; Champagnat and Morel, 1972; Morel 1974; Arditti, 1977). This research, especially with tropicaI species, led to the development of one of the most profitable parts of the horticulture business (Arditti and Emst, 1993). Similar methods related to temperate terrestrial orchids were proven to be much more complicated and more specialized techniques became necessary as the interest (both consewation and commercial) grew to propagate these often endangered or rare species of the native flora (Morel, 1974; Ronse, 1989; Stewart, 1992; Arditti and Emst, 1993; Rasmussen, 1995). Although in recent years several pubIications became available about these propagation techniques @orris and Albrechf 1969; Eiberg, 1969; Stoutamire, 1974, 1983; Fast, 1976, 1978, 1982; Linden, 1980; He~chet al., 1981; Arditti et al., 19821, 1982b; Frosch, 1982; Hadley, 1982; Lucke, 1982, 1984; Myers and Ascher, 1982; Oliva and Arditti, 1984; Van Waes, 1984; Van Waes and Debergh, 1986; Muir, 1989; Srnreciu and Currah, 1989; Anderson, 1990; Barroso et a]., 1990), the results sometimes were not detailed enough or were even contradictory (Ardim, 1977; Arditti and Ernst, 1993). Also, numerous publications offer a wide range of media, but in most cases the effect of some of the important ingredients, such as defined and undefined organic compounds, was not tested specifically or scientifically compared with each other (Grditti and Emst, 1993). Only a few attempts were made to try an established method for severd different species, to provide information for direct comparison among the different species and overall observations and conclusions were prepared about the culture requirements and early development of these orchid species (Henrich, 1981; Arditti et al., 1982% 198%; Van Waes, 1984; Van Waes and Debergh, 1986). Similarly a procedure for the in vitro germination of temperate terrestrial orchids was proposed by Szendrik (1994b) based on previous work of R Eszeki and Szendrhk @ Eszeki and Szendrak, 1992; Szendrik and R Eszeki, 1993; Szendrik, 1994a). Additional research was necessary to try that procedure for several more different temperate species. Being one of the largest families of the plant kingdom, orchids have long been an important family for extended anatomical and taxonomical research (Van der Pijl and Dodson, 1966; Borsos, 1980, 1982, 1990; Whitner, 1985; Dressler, 1993). In recent decades several characteristics (such as embryo and pollinarium structure, Burns-Balogh, 1985; Veyret, 1985; Fredrikson, 1985) - which are only possible to observe in detail with a microscope with high magnification - became especially interesting for taxonomists. To observe additional characteristics (such as seed structure; Senghas a al., 1974; Barthlott, 1976; Arditti et al., 1979, 1980; Ziegler, 198 1; Healay, 2 980; Chase, 1988; Molvray and Kores, 1995) scientists applied modem equipmentlmethods such as transmission and scanning electron microscopy (SEM). In order to collect much needed additional data, extended research is necessary to obtain sufticient detailed information for the individual members of this huge plant family (Arditti and Ernst, 1993; Dressler, 1993). The data collected by using new equipment/techniques can be usefiI not only for classification purposes, but can also help to understand several developmental patterns and thus help to optimize culture requirements of these plants. The objectives of this research were (1) to test a previousiy proposed asymbiotic in vim germination procedure (Szendr% 1994b) for several different temperate orchids and folIow the development of these plants; (2) to develop a simple and efficient method to vegetatively multiply temperate orchid protocorms and compare the effects of different organic substances and medium consistency on their proliferation and development; (3) to provide a complete description of the life history of a temperate orchid species using SEM and (4) to collect detailed information about the seed morphology of numerous temperate terrestrial orchid species by SEM. Literature Cited

1. Anderson, A B. 1990. Improved gemination and growth of rare native Ontario orchid species. In: Allen, G. M., Eagles, G. M., Price, P. F. J. (ed.) Conserving Carolinian , pp.:65-73. University of Waterloo Press, WaterIoo, Canada. 2. Arditti, J. 1977. Clonal propagation of orchids by means of heculture - A manual. In: Arditti, J. (ed.) Orchid biology: Reviews and perspeclives I. pp.:203-293. Cornell University Press, Ithaca, New York. 3. Ardim, J., Emst, R 1993. Micropropagation of Orchids. John Wiey & Sons, Inc., New York. 4. Ardim, J., Michaud, J. D., Healay, P. L. 1979. Morphometry of orchid seeds. I. Paphiopedilum and native California and related species of Cypripedium. American Journal of Botany, 66(10): 1128- 113 7. 5. Arditti, J., Michaud, J. D., Healay, P. L. 1980. Morphometry of orchid seeds. 11. Native California and related species of Calypso, Cephalanthera, Corallorhiza and Epipactis. American Journal of Botany, 67(3):347-360. 6. Arditti, J., Michaud, J. D., Oliva, A P. 198% Practical germination of North American and related orchids. I. Epipactis utrombens and E. gigantea and E. helleborine. American Orchid Society Bulletin, 5 1: 162-17 1. 7. Arditti, J., Michaud, J. D., Oliva, A P. 1982b. Practical germination of North American and related orchids. II. Goorj.era oblong~oiiaand G. tesseluta American Orchid Society Bulletin, 5 1:394-397. 8. Barroso, J., Fevereiro, P., Oliveira, M. M., Pais, M-S. S. 1990. In Vitro Seed Germination, Differentation and Production of Minitubers fiom Ophrys lutea Cm., Ophrysfusca Link and Ophrys speculum Link. Scientia Horticulturae, 42:329-33 7. 9. Barthlott, W. 1976. Morphologie der Samen von Orchideen im Hinblick auf taxonomische und hnktionelle Aspecte. In Proceedings of the 8th World Orchid Conference, Frankfiuf pp:444-445. Deutsche Orchideen Gessellschaft, FrankfUrt. lO.Borris, H., Albrecht, L. 1969. Rationelle Samenvermehrung und Anzucht europaischer Erdorchideen. Gartenwelt, 69:5 11-5 13. 11. Borsos, 0.1980. Anatomy of Wild Orchids in Hungary. I. Tissue structure of leaf and floral axis. Acta Agronomica Academia Scientiarum Hungaricae, 29:369-390. 12. Borsos, 0. 1982. Anatomische untersuchung der Wildorchideen Ungarns 11. Stengelanatomie. Abstracts Botanica VII.: 1 17-129. 13. Borsos, 0. 1990. An anatomical-histochemical study of wild orchid tubers in Hungary. In: Proceedings of the Symposium on Biology and Ecology of European Orchids, pp. :25-32. 14.BurgeK H. 1936. Samenkeimung der Orchideen und Entwicklung ihrer Keirnpflanzen. Verlag von Gustav Fischer, Jena. 15. Burns-BaIogh, P. 1985. Orchid pollen/pollinaria atlas. IDC Microform, Zug, Switzerland. 16. Champagnat, M., Morel, G. 1972. La culture in vifro des tissus de tubercules d'0phv.s. C. R. Acad. Sci. Paris, 274:3379-3380. 17. Chase, M. W.,Pippen, J. S. 1988. Seed Morphology in the Oncidiinae and Related Subtribes (Orchidaceae). Systematic Botany, 13(3):3 13-323. 18. Dressier, R L. 1993. Phylogeny and Classification of the Orchid Family. Dioscorides Press, Portland, Oregon. 19. Eiberg, H. 1969. Keimung europaischer Erdorchideen. Die Orchidee, 20:266-270. 20. Fast, G. 1976. Moglichkeiten zur Massenvermehrung von Cypripedium cdceofus und anderen europaischen Widorchideen. Proc. 8th WorId Orchid Coif, Frankfbrt, pp.:359-363. 21. Fast, G. 1978. rjber das Keirnverhalten europaischer Erdorchideen bei asymbiotischer Aussat. Die Orchidee, 29270-274. 22. Fast, G. 1982. European terrestrial orchids (Symbiotic and asymbiotic methods). In: Arditti, J. (ed.) Orchid Biology. 11. Reviews and perspectives. Orchid seed germination and seedling culture - a manual, pp.:309-326. Cornell University Press, Ithaca, New York. 23. Fredrikson, M. 199 1. An embryological study of Platanthera bifoiicr (Orchidaceae). Plant Systematics and Evolution, 174:2 13-220. 24. Frednkson, M. 1992. The development of the female gamerophyte of Epipactis (Orchidaceae) and its inference for reproductive ecology. American Joumal of Botany, 79(1):63-68. 25. Frosch, W. 1982. Beseitigung der duch die innere Hiille bedingten Kermhemmung bei europaischen Orchideen. Die Orchidee, 3 3 :145- 146. 26. Hadley, G. 1982. European terrestrial orchids. In: Arditti, J. (ed.) Orchid Biology. II. Reviews and perspectives. Orchid seed germination and seedhg culture - a manual, pp.:32&329. Cornell University Press, Ithaca, New York. 37. Healay, P. L., Ardim, J., Michaud, J. D. 1980. Morphometry of orchid seeds. III. Native California and related species of Goodyera, Piperia, Platanthera and Spiranthes. American Journal of Botany, 67(4):508-5 18. 28. Henrich, J. E., Stimart, D. P., Ascher, P. D. 1981. Terrestrial Orchid Seed Germination in Yitro on a Defined Medium. J. Amer. Hort. Sci. 106(2): 193-196. 29. Knudson, L. 1921. La germination no simbiotica de las semillas de orquideas. Boll. Real. Soc. Espanola Hist. Nat. 21:250-260. 30. Knudson, L. 1946. A new nutrient solution for germination of orchid seed. American Orchid Society Bulletin, 15:214-217. 3 1. Linden, B. 1980. Aseptic germination of seeds of northern terrestrial orchids. Annales Botanici Fernici, 29:305-3 13. 32. Lucke, E. 1982. Samenstruktur und Samenkeimung europaischer Orchideen nach Veyret sowie weitere Untersuchungen. I. Die Orchidee, 32: 182- 188. 33. Lucke, E. 1984. Samenstructur und Samenkeimung europaischer Orchideen nach Veyret sowie weitere Untersuchungen. 1V.-V. Die Orchidee, 3 5: 13-20, 153-1 58. 34. Molvray, M., Kores, P.J. 1995. Character analysis of the seed coat in Spiranthoideae and , with special reference to the Diurideae (Orchidaceae). American Journal of Botany, 82(11): 1443-1454. 35. Morel, G. M. 1960. Producing virus-f?ee cymbidiums. American Orchid Society Bulletin, 29:495-497. 36. Morel, G. 1974. Clonal multiplication of orchids. In: Whitner, C. L. (ed.) The orchids. Scientific studies, pp.: 169-222. Wiley & Sons INC. New York. 37. Muir, H. J. 1989. Germination and mycomhizal hgus compatibility in European orchids. In: Pritchard, H. W. (ed.) Modem methods in orchid conservation, pp.:39-56. Cambridge University Press, Cambridge. 38. Myers, P. J., Ascher, P. D. 1982. Culture of North American orchids from seed. HortScience, 17:550. 39. Oliva, A. P., Arditti, J. 1984. Seed germination of North American orchids. II. Native California and related species of Aplecrrum, Cpn'pediium and Spirmhes. Botanical Gazette, 145:495-50 1. 40. Rasmussen, H. N. 1995. Terrestriai orchids from seeds to mycotrophic plant. Cambridge University Press, Cambridge. 41. Reinert, R. A, Mohr, H. C. 1967. Propagation of CurtIeya by tissue culture of lateral bud meristems. Proc. Amer. Soc. Hort. Sci. 9 1 :664-67 1. 42. R Eszeki, E., Szendrdc, E. 1992. Experiments to propagate native hardy orchids (Orchidaceae) in the ELTE Botanical Garden. Abstracts of 20th Cong. Hung. Biol. Soc., Kecskemkt, pp.:25. 43. Ronse, A. 1989. In Vitro Propagation of Orchids and Nature Conservation: Possibilities and Limitations. Mem. Soc. Roy. Bot. Belg., 11: 107-1 14. 44. Rotor, Jr. G. 1949. A method for vegetative propagation of PhaZaenopsis species and hybrids. American Orchid Society Bulletin, 18:738-739. 45. Senghas, K., Ehler, N., Schill, R, Barthlott, W. 1974. Neue Untersuchungen und Methoden zur Systematic und Morphologie der Orchideen. Die Orchide%25:157-168. 46. Smreciu, E. A, Currah, R S. 1989. Symbiotic germination of seeds of terrestrial orchids of North America and Europa. Lindleyana, 1:6- 15. 47. Stewart, J, 1992. The conservation of European orchids. Nature and environment, No. 57. Council of Europe Press, Strasbourg. 48. Stoutamire, W. P. 1974. Terrestrial orchid seedlings. In: Whitner, C. L. (ed.) The orchids. Scientific studies, pp.: 101-128. Wdey & Sons INC.New York. 49. Stoutamire, W. P. 1983. Early growth in North American terrestrial seedlings. In: Plaxton, E. H. (ed.) North American Terrestrial Orchids Symposium IT. Proceedings and lectures, pp.: 14-24. Michigan Orchid Society, Ann Arbor, USA. 50. Szendra E. 1994a. Az Orchis rnorio L. mikroszaporith 6s anatomiai vizsgdata. (Micropropagation and anatomical elaboration of Orchis mono L.) Proc. of XXTI. National Scientific Student Conference, Budapest. 51. Szen- E. 1994b. Az Orchis morio L. s6sEti lelbhelyinek florisztikai & c~jnoI6giai vizsgdata, 6s a faj disP16venytermesztbbe vonhak Iehetbdge. MSc. thesis, (The £loristical and phytocoenological measurements of the native habitat of Orchis morjo L. near 'Soskt' and the possibilities to use this species as a potential ornamental plant) Kertketi b klemiszeripari Egyetem, Kertbteti Kar, D~ov6nytermeszt~ Q Dendrologiai Tanszek N6venytani Tanszek, Budapest. 52. Szendrk E., R. Eszeki E. 1993. Hazai szabadfoldi kosborft51kk (Orchihceae) aszimbiotikus in vitro szaporitki. Publicationes Univ. Hen. Ind. Ali. Vol. Lm. pp.:66-70. 53. Van der Pijl, L., Dodson, C. H. 1966. Orchid flowers - Their Pollination and Evolution. University of Miami Press, Coral Gables, FIorida. 54. Van Waes, J. 1984. In vifru studie van de kiemingsfysiologie van Westeuropese orchideen. Thesis, Rijkuniversiteit Gent. 55. Van Was, J., Debergh, P. C. 1986. In vitro germination of some Western European orchids. Physiologia Plantarum, 67:253-261. 56. Veyret, Y. 1985. Development of the embryo and the young seedIing stages of orchids. In: The orchids - Scientific Studies. Ed.: Withner, C. L. pp.: 223-267. Robert E. Krieger PubIishing Company, Malabar, Florida. 57. Withner, C. L. (ed.) 1985. The orchids - Scientific Studies. Robert E. Krieger Publishing Company, Malabar, Florida. 58. Ziegler, B. 198 1. Mikrornorphologie der Orchideensamen unter Beriicksichtigung taxonomischer Aspecte. Ph.D. dissertation, Ruprecht Karls Universitat, Heidelberg. Literature Review

PeopIe have been fascinated by orchids for a long time, tracing back to the ancient Greek times. One myth claims that a young Greek prince called Orkhis fell in love with a priestess of the god BakWlos, but the wild animals who guarded the priestess tore him apart. From his blood-shed flowers grew which were named after him, orchises. This explained the origin of the name of one temperate (Orchis) and actually of the whole Orchidaceae family. The word "orchid" can be traced back to the works of Theophrastus between 370 and 285 BC. Probably because of the shape of the tuberoids of some of these plants, they were considered as aphrodisiacs and another myth indicates that the tuberoids were the favorite food of the satyrs (David, 1986). For several centuries the dried tuberoids of different temperate orchid species, especially of species fiom the Ophrys and Orchis genera, were a favorite and expensive item on the street markets of Europe. Although the habit of using these plants as aphrodisiacs or for flavoring foods vanished in most parts of the world, it is still an existing threat for the survival of these species, especially in Turkey, Greece and Crete (Stewart, 1992). In 1892, for example, almost 20 thousand kilograms of dried tuberoids were exported fiom Istanbul (Stewart, 1992) and Voth (1973) estimated that in the last decades of our century approximately 10 thousand kilograms of fksh orchid tuberoids were dug up each year in Turkey alone. Other serious global threats for temperate terrestrial orchids are the change and disappearance of native habitats, change of vegetation and exploitation of chemicals and introduction of other hazardous materials into the environment (Wochok, 1981). As a serious consequence, many of the temperate orchid species became rare, threatened or endangered and some of them are close to extinction (Beckner, 1979; Threatened Plants Committee, IIJCN, 1983; Ronse, 1989). Until the twentieth century, orchids were considered as one of the most difficult plants to propagate. Even the seeds were considered sterile until the beginning of the nineteenth century, when finally Salisbury (1804) and Moore (1849) proved that these seeds are able to germinate. Probably the difficult and slow proliferation techniques mdde these plants, especially the tropical orchids, the subject of one of the most expensive hobbies (Makara, 1982). Because of the high prices and Limited availability of these plants, the development of additional new propagation techniques became necessary to improve upon their traditional vegetative propagation or their in silu seed germination (Sessler, 1978; Sander, 1979; Makara, 1982). Although at the beginning of this century there was Little or no concern about conservation of temperate species and also there was less interest to produce them for sale to the public, scientists started to focus on the artificial propagation of temperate orchids. They believed that these plants could be a source of a successfil pharmaceutical drug against cholera, one of the most temfylng diseases of that age (Mindy, 1993). Based on the foundation prepared by Bernard (1889, 1909), Prof Hans Burgeff (1936) established the technique of symbiotic orchid seed germination. He tried to maintain a symbiotic relationship between specific hngi and orchids in viho, a symbiosis which is basic for orchids in their native environment. In this system, most of the nutrients which are necessary for germination are provided for the orchids fiom the mycorrhizal partner. In Burgees original work, the main steps of this method were: (1) isolation of mycorrhizal hgi from orchid root tissues; (2) inoculation of fbngal hyphae in a sterile medium; and (3) sowing of orchid seeds on medium which already contains the symbiotic partner. The most critical point of this method is to maintain the fragde balance between the mycorrhizal partner and the geminating orchids, because the highly active hngus can overgrow the cultures and destroy the young orchids. More recently developed techniques commonly provide two different media inside the same culture vessel, so there is a medium suitable for the growth of the mycorrhizal hyphae and another medium is included to provide a better environment for the orchid seedslplantlets but less suitable for the bgi(Clements and Ellyard, 1979; Clements et al., 1986; Breadmore and Pegg, 1987; Rasmussen, 1990; Anderson, 1991; Breddy, 1991). Parallel with the development of symbiotic methods Prof Lewis Knudson (1921, 1922% 1912b) established the methodology of asyrnbiotic orchid seed germination. With his discovery it became possible to germinate orchids on a simple nutrient medium containing sugars, and he concluded that hngus was not necessary for germination in this system. He successfully germinated the seeds and raised species fiom several tropical genera and he also proved that these plants are able to produce flowers (Knudson, 1930). In subsequent years, Knudson (1946) improved his initial medium, and this improved form - known as Knudson C - is widely used now throughout the world, especially for tropical orchids (Arditti, 1972). Although with this method the initial germination rate can be lower than in the case of symbiotic methods, the often higher survival rate in the cultures, the more simple medium preparation and the practically feasible culture techniques are some of the advantages of the asymbiotic method. From the 1960s-1970s the in vitro seed germination of orchids got an additional stimulus because of the growing public concern to save these plants for future generations. In vitro germinated plant material can be more suitable for reintroduction because of the higher genetic diversity. In addition, developmental information can be collected in the laboratory which can provide a great help to better understand the life of temperate orchid species in their native habitat (Ronse, 1989). Parallel with these changes a new method - the in vitro vegetative propagation of orchids - appeared as an alternative possibility for orchid proliferation, which was suitable to produce higher numbers of oapring, especially fiom rare and valuable tropical . Actually the very first attempt to in vitro asexually propagate any plant species was when Rotor (1949) successfblIy produced young Phahenopsis plantlets from disinfested flower stalk segments in a liquid medium. Almost all parts of an orchid plant such as roots (Beechey, 1970; Churchill et d., 1972; Tanaka et al., 1976; Street, 1973, 1977; Gautheret, 1983); leaves or Ieaf tips (Wirnber, 1965; Churchill et al. 1971, 1973); stems (Kunisaki et d., 1972; Teo, 1978; Emst, 1994) and flower buds and segments (Wither, 1955; Ayers, 1960; Fast, 1980) can be used as a stock plant material for in vitro vegetative propagation. In 1960 Morel established the first meristem culture for cymbidiums, and with this method the virus-fiee mass propagation of several tropical orchid species and cultivars became possible. He also discovered that protocorms (which were previously well known fiom the seed germination of orchids) appear during meristem propagation of these plants. He stated (Morel 1969): "The main difference between orchids and other pIants regenerating buds, like Begonia, gesneriads, lilies, etc., is that in orchids the regeneration starts by the formation of a very special organ - the protocorm, and it is very fortunate for the orchidist that the epidemic cells of this protocorm keep forever this faculty of regeneration." These vegetatively formed protocom are often referred to as protocorn like bodies or PLBs to distinguish them fi-om the ones developed fiom orchid embryos during germination (Arditti, 1977; Ardim and Emst, 1993). From these first fbndamentd methods for asymbiotic/symbiotic seed and vegetative (including meristem culture) in vitro propagation of orchids numerous attempts were made to improve these techniques for a wide range of different species. However the enormously broad spectrum of related literature will not be reviewed here because of the limited extent of this dissertation. Extended reviews are available for the reader compiled by Arditti (ed. 1967, 1977, 1982, 1984, 1987 and 1990), Comgan (1969), Makara (1982), Griesbach (1 986), Withner (1985), Pritchard (1 989), Singh (1992), Arditti and Ernst (1993). Additional information about related literature can be found in the introductions and discussions of the four chapters of this dissertation, and only the most significant or interesting references will be mentioned here to provide an overview about the different factors related to the in vitro culture of temperate orchids.

For storage of orchid seeds most references suggest low, approximately 24°C temperature. It is very important that the remnant hit tissues are removed and the seeds are dried before storage (Fast, 1982; Pritchard, 1984a; Seaton and Hailes, 1989; Paget, 1991; Mitchell, 1989). Pritchard (1984b) suggested as an alternative method for long term seed storage the use of liquid nitrogen and he noted that with the proper use of this technique no negative change was observed in the viability of orchid seeds from several species. Following the initial work of Singh (1981), Van Waes (1984) and Van Waes and Debergh (1986a) proposed a modified version of the classic tetrazolium method to measure seed viabiIity for several temperate orchid species. Using this method it can be known in advance that a later occuning lower germination rate is a consequence of the bad condition of the original seed material or caused by other factors during germination. For disinfestation of the seeds one of the most commonly used sterilizing agents is calcium hypochlorite, usually in 10% wlv concentration, dthough recently liquid bleach has become more fiequently used (Arditti, 1977; Bergman, 1996). Knudson (1948) and Sweet and Bolton (1979) reported that calcium hypochlorite is less damaging for the seeds. Van Waes and Debergh (1986a) emphasized that in addition to the disinfesting effa this chemical has a great importance in breaking the dormancy of the seeds thus assisting to achieve an earlier germination with higher germination rate. The optimum length of disinfestation varies by species (Linden, 1980; Van Waes and Debergh, 1986a) and especially some of the more difficult to germinate species, such as Cfln'pediurn qp. and Epipactis spp. require a much longer disinfesting time period (Van Waes, 1984; Chu and Mudge, 1994). Less fiequently used disinf-ts are ethanol, hydrogen peroxide and mercuric chloride according to the literature (Ardim, 1977). In some cases, disinfestants or some fUngicideshactericides are directly added to the medium, but the effects of these chemicals are often hdlor inhibitory for the geminating seeds (Brown et al., 1982; Yanagawa et al., 1995). Leaching or soaking in distilIed water or low concentrations of growth regulating chemicals can be helpfUl to promote germination but it has not been found to solve all difficulties related to breaking the dormancy of orchid seeds with problematic germination (Stoutamire, 1974; Linden, 1992). In addition to the traditional tissue culture media that are commonly used for tropical orchids, such as MS (Murashige and Skoog, 1962), Knudson C (Knudson, 1946) and RM (Reinert and Mohr, 1967), several media have been designed specifically for the asyrnbiotic in vitro germination and culture of temperate species. The most commonly used ones were developed by Curtis (1936), Norstog (1973), and Fast (1976, 1982) among others. The most obvious difference between these and other traditionally used media in plant tissue culture is that the concentrations of minerd ions are much lower, sometimes even ten times less in the media for temperate species, since these plants are much more sensitive to high salt concentration (Hamais, 1974; Fast, 1982; Van Waes, 1984). As in most media in plant tissue culture, sucrose is one of the most commonly used defined carbon sources for the in vitro cultures of orchids (Knudson, 1946; Reinert and Mob, 1967; Voth, 1976; Clements, 1982). Glucose (Curtis, 1936; Hadley, 1982) and hctose (Linden, 1980) are also commonly included in the medium, sometimes even in combination with each other or with sucrose (BurgefS 1936; Fast, 1976, 1978, 1982). From the earliest stage of seedling development the Werent members of the Vitamin B complex and as an antioxidant, Vitamin C, are important ingredients of the medium (Borris, 1969; Lucke, 1971; Szendrik and R Eszkki, 1993). Commonly the standard MS (Murashige and Skoog, 1962) or Nitsch (Nitsch and Nitsch, 1969) vitamin stock solution is added to the medium (Mhndy, 1993) or commercially available vitamin complexes are used (Szendrik and R Eszbki, 1993). Although the application of different plant growth regulators is basic in the in vim propagation for several tropical orchids, Van Waes (1984) based on an extended literature review emphasized that the use of these chemicals for temperate species, in most cases is not beneficial, and they do not help achieve the expected results. Similar negative results were collected by Szendriik (1994) using different plant growth regulators for an attempt to multiply young orchid seedlings. Probably the low success rate with plant growth regulators in the sterile culture of temperate orchids is one of the reasons why it is still so important to use different undefined organic compounds/complex additives in the media for these plants. Although the list of ingredients mies among these additives, most of them contain several different inorganic ions, nitrogenous compounds, amino acids and related substances, enzymes, vitamins, sugars and different natural plant hormones such as cytokinin and gibberellin (Arditti and Emst, 1993). The most commonly used complex additives are coconut milWwater (Borris and Albrecht, 1969; Boms, 1970; Lucke, 1971; Linden, 1980; Oliva and Aditti, 1984); peptone (Fast, 1974, 1976, 1982; Anderson, 1990; Riether, 1990; Szendrk and R Eszeki, 1993); casein and/or lactalbumin (Mead and Bulard, 1975; Van Waes, 1984; Van Waes and Debergh, 1986b; Hoppe and Hoppe, 1987% 1987b, 1980; R Esziki, personal communication). Other complex additives used in orchid tissue culture are yeast extract (Fast, 1974; Linden, 1980; Galunder, 1986); banana pulp (Graeflinger, 1950; Arditti, 1968; Oliva and Arditei, 1984); birch phloem sap Wether, 1990); fish emulsion (Chang, 1953; Harbeck, 1963); honey (Karasawa, 1966) and different hit juices (Arditti, 1966; Lucke, 1977; Ramin, 1983). The exact mechanisms of the effects of these compounds are still not known but they were proven to be highly beneficial for the asymbiotic in vih.0 germination and vegetative propagation of temperate orchids (Arditti and Emst, 1993). Liquid medium and suspension culture is commonly used in the mass propagation of tropical orchid species (Morel 1974; Arditti and Ernst, 1993) but only a few references offer this medium consistency as an alternative for the in vitro propagation of temperate orchids (Kano, 1965; Hoppe and Hoppe, 1987% 1987b, 1988). Generally, gelled medium is used in most cases (Arditti, 1977; Ardim and Ernst, 1993). From the initial attempts to germinate temperate orchid species, the importance of an initial lower temperature (ranging usually fiom 5OC to 20°C) and dark condition until germination has been weII known and tested several times (Burgeff, 1936; Arditti, 1979; Arditti et al., 1982% 1982b; Emst, 1982; Stoutamire, 1974; Harvais and Hadley, 1967; Fast, 1982; Rasmussen et al., I990a, 1990b; Szendrik and R Eszelu, 1993; Chu and Mudge, 1994). Initial experiments at the ELTE Botanical Garden, Budapest showed that seasonal temperature changes and different light sources have an important effect on the firther growth of germinated seedlings (Szendrik et al., 1994% 1994b). Henrich (1981) and Van Waes and Debergh (1986b) published the results of two extended research programs that provide information related to the culture requirements of not only one or a few, but a wide range of temperate orchid species and about their subsequent development in vitro. Similarly, based on several previous experiments an asymbiotic in vitro germination and culture method for different temperate species was proposed by Szendrik (1 994). One of the most common dficulties raised in using asymbiotic in vitro propagation of temperate orchids has been the problem of plantlet acclimatization at the end of the culture period, since the supporting mycorrhizal partner is necessary for the development of these plants in nature. As an alternative solution Bailes and Clements (1987) proposed the use of a special soil mixture for transfer of temperate orchid plantlets ex vitro. This mixture contains two parts of rough sand, two parts of fine pine bark, one part of compost and one part of soil (without sterilization) from a location where orchid plants grow wild. Anderson (1 99 1) reports the use of a half sand half Pro-mix BX soil mixture as an ex vizm medium for temperate orchid acclimatization. From the earliest attempts to culture temperate terrestrial orchids in M'tto, the reports have been accompanied by anatomical and morphological observations of these plants (Burgee 1936; Arditd, 1977). This is not surprising since these observations are very impomnt to optimize culture conditions for in Htro pIant materid. Another important source of anatomical and morphological information is the wide range of comprehensive works and monographs about the members of the Orchid family, such as those of the following authors: Danvin (1877), Arber, (1925), Fuchs and Ziegenspeck (1 925), Carnus (1928), So6 and KeIler (1930), Solereder and Meyer (1930), Ziegenspeck (1 936), Correll (1950), MetcaIfe (1960), Borsos (1952-1966), Van der Pijl and Dodson (19661, Arditti (ed. 1967, 1977, 1982, 1984, 1987 and 1990), Braun (1967), Magrath (1974), Luer (1975), Makara (1982), Draler (1981 and 1993a, 1993b), Withner (1985), Fanfani and Rossi (1988), Arditti (19921, Pridgeon (1992), Stewart and GdEths (1992) among others. Important anatomical and morphological information can also be obtained from symposium proceedings and journals of related associations (for example the American Orchid Society Bulletin (currently called Orchids), Lindleyana, Orquidea, and Die Orchid-) and also from field guides and flora works written by Britton and Brown (19701, Javorka and Csapody (1975), Williams et al. (1978), FiiIler (1983), Wiliams and Williams (1983), BrackIey (1985), Gupton and Swope (1986), Kaul (1986), Baurnan and KiinkeIe (1988), Uonczay (1990), Borsos (1992), Homoya (1993), Smith (1993). The above mentioned list of literature sources documents the extended volume of information, and to review aII of them can not be a realistic goal of this literature review. Rather than this, some of the most important references related to orchid anatomy and rnorphoIogy and to the applied methods wilI be reviewed here. Additionally, more detailed information based on the literature related to the topic will be presented in the introductions and results and discussions of chapters of this dissertation. One of the most comprehensive studies about the structure and development of orchid embryos was written by Veyret (1985). Also several orchid embryos have been described in the report by Wirth and Wither (1959), who based their classification system on the work of Johansen (1950). The embryos of different orchid species and the process of embryogenesis are essentially similar. Most orchids have an extremely simple, undifferentiated embryo formed fiom almost uniform cells and sometimes fiom a few other cells which form a suspensor region (Veyret, 1985). No cotyledon, endosperm, radicle and plumule can be found inside the testa except of a few species, such as Sobralia macrantha and Bletilla hyaqmthina, which have relatively Serentiated embryos including a rudimentary cotyledon (Arditti, 1967; Poddubnaya-Am014 1967). More recently Fredrikson (1990, 1991, 1992) published a series of publications about the detailed anatomical structure of the embryos of three European temperate orchid species. The appearance of potyembryony is reported &om the Orchidaceae several times. True polyembryony occurs mainly in the tribe of Cranicideae (Gwera,Spirunthes and other mostly tropical genera), and in the genus Ptewodium. Haploid parthenogenetic development has been discovered in a certain number of orchids, particularIy when the fertilization is late in occurring (BletiZIa, Garodia, EZeorchis). In some cases, although apomictic formation of the seeds and polyembryony have been recognized, it has not been determined by which process it takes place (Listera borealis. Zygoperalum mackqyi, Orchis IatiJolia, Orchis morio, Cypripdium cnlceolus) (Veyref 1985; Schmi th and Anthger, 1992; Dressier, 1993b). The development of germinating embryos and young plantlets are often included in reports about the symbiotic and asymbiotic gemination of temperate orchids (Stoutamire, 1963, 1964a, 1964b, 1983; Chu and Mudge, 1994; Zettler et al., 1995 among others), although they slightly differ in the segregation of different developmental stages. The phenomenon of mycotrophy is strongly connected with the germination of temperate orchid species and the root anatomy of these plants. To date, several species of kngi have been identified from the Rhizoctonia, Epulorhiza, Tulmella, Ceratobasi'dium and Tremella genera. Warcup (1966, 1971 and 1980) and more recentiy Currah and Zelmer (1992) provided an identification system for the different mycorrhizal fbng species. Gd and Halacsy (1994) described the appearance and seasonal changes of mycorrhyza in several European terrestrial species. A general appearance of a special type of endomycorrhyza is typical for the species of Orchidaceae and it is called "orchid-typen (Harley and Harley, 1987). The hyphae only can be found in the cortical cells of the roots; and these cortical cells are the location of the three dimensional hngal coil formation. These typical bodies fcrmed from the hyphae are called pelotons (Cumah, 1991). In nature the presence of mycorrhiza is essential for germination and may be required for plant development all year long or only during certain parts of the vegetative season (BurgefF, 1936). Starch is stored in large quantities in the root cortex before the appearance of mycorrhiza but this quickly disappears after infection purge% 1936). The different underground storage organs, such as tuberoids and rhizomes are also important locations for the storage of starch, calcium oxalate crystals and water. These stored materials have an important role to reserve energy and water for the less suitable parts of the vegetative season and prevent plant tissues fiom freezing and being damaged fiom ice crystals during the cold winter months. A detaiIed study of these storage organs of several European orchid species was published by Borsos (2990). With an extended light microscopy study, she provided information about the histological structure of orchid tuberoids and the location and accumulation of stored materials. Rhizomes and especially tuberoids provide a safe protected environment for the new buds during dormant periods until they become ready to grow and develop the new green vegetative surface of the plants (Burgee 1936; Borsos, 1990; Dressier, 1993b). The amount of literature is relatively limited about the stem and leaf anatomy of orchids, compared with the wide range of references deding with the mycorrh~zal connection or with flower structure and pollination. The leaves of orchids may be divided into three major types such as soft leathery, hard leathery and fleshy leaves (Withner, 1985). Most of the temperate orchids have thin soft leathery leaves, which bear continuous cuticle, generally thicker on the upper epidermis than the lower. Generally more stomata are present on the underside of the leaves, and they usudly lack subsidiary cells. Hairs may be present on either or both sides of the leaves (Withner, 1985). In her reports about the structure of the leaf and floral axis of different European orchid species, Borsos (1980 and 1982) emphasized the species speciflc presence of stoma index. Stoma index represents the ratio between stomata and other epidermal cells either on the upper or lower surface of the leaves. Orchid stems represent two major growth pattern types called sympodial and monopodial. The sympodial habit appears as successive growth, each originating fiom the base of the proceeding one. The monopodial type is characterized by a stem with the main growing axis producing leaves. Although tuberous temperate orchids appear to be monopodial, most of the temperate species belong to the sympodial type, because even in the case of tuberous species the new year's growth is initiated fkom the new tuberoid that develops from the basal part of the stem after flowering (Wither, 1985; Dressier, 1993b). Myc~rrhyzal&ngi have an important role in providing different nutrients, partly as an energy source, for the orchid plants through a symbiotic relationship. However, similarly to other plants the green leaves and also the stem are the most important source of energy for these orchid plants through photosynthesis. Other organs may have photosynthetic activity, too, such as roots of epiphytic species and different flower segments and hits following fertilization. Both Cs (Calvin-Benson pathway) and CAM (crassulacean acid metabolism) have been shown to operate in orchids and the possibility that C4 (Hatch and Slack pathway) exists in orchids has also been suggested (Avadhani et al., 1978 and 1982; Arditti, 1979). Thin-leaved temperate orchids are in the group of C3 plants, so they fix C02with ribulose 1,s-bisphosphate to form two molecules of 3-phosphoglyceric acid via the Calvin-Benson pathway of photosynthesis. This reaction is catalyzed by the enzyme ribulose bisphosphate carboxylase. Photorespiration is present in these plants, so they have low net photosynthetic rates and high C02 compensation points (Jones, 1985). The biggest diversity and evolutionarily speaking, one of the highest levels of specialization can be found among the structural features of orchid flowers. Basically it is almost impossible to summarize the related literature in a few paragraphs, so even with the best intention this review won't provide a fill overview but only will mention a few aspects of this topic. Orchids are generally considered to have long-lasting flowers; some of the tropical or subtropical species flowers last for several days or even weeks (Cymbidium, ). In other cases the individual flowen do not last very long, but the whole can continue to flower for a very long time (Epidehrn).But in both cases the flowers remain khuntil they are pollinated, afternard (or even with a similar effect to pollination Like a small physical damage of the column) the flowers become faded (Makara 1982; Dressier, 1993b). Another big part of the family (most of the temperate species) has a very short flowering time; possibly even only one day long or less (ephemeral); and the whole population can finish flowering in a very short period of time (Goh, Strauss, and Arditti, 1982). There is another very interesting group of these short-lived flowering plants, which has a synchronous flowering behavior (Palmorchis, PsiIochiIus, Sobralia). These species coordinate their flowering so that all plants in a population flower on the same day. The flower buds of these species develop to a ceriain stage and then stop. When a proper environmental sign is present (usually a cold rainstorm) all the buds in the population start to grow again. Afler a certain day following that effect the whole population starts flowering, giving a very effective stimulus to the pollinators (Dressler, 1993a). In every case, flowering happens in a certain part of the vegetative season; in the tropics usually a certain type of environmental &ect is the stimulus (low temperature, burning, etc.) (Goh, Strauss and Arditti, 1982); in other places that have a longer cold or dry period of the year, the plants become donnant and start to flower after a certain amount of time after the dormant period. It could be shortly after the end of dormancy, at the beginning of the vegetative season (Orchis, Dactylorhiza), or in the second part of the vegetative season in late summer or early fall (Spirimfhes auZzmznaZis) (Makara, 1982). Almost all the species have their own strategy for reaching the best results in generative propagation. Flowers are organs of , and both the time of the flowering and the biological structure of the inflorescence and flowers are developed to obtain the best result in attracting pollinators and finally with receiving the pollen fiom other flowers to combine the genetic materials f?om both parents. Most of the orchids are cross-poltinated, but in a few cases autogamy can occur. With Ophrys species, if insect polIination does not occur the position of the pofinarium changes, and finally autogarny occurs (Darwin, 1877; van der Pijl and Dodson, 1966). In a few other cases, obligate autogarny (or even cleistogamy) is the only way to achieve fertilization. This is the way of sexud reproduction of some very specialized, strange subterranean Australian species (Stoutamire, 1976). But as mentioned before, the basic strategy is cross-pollination. The features that distinguish orchids one fiom another and fiom other flowers are largely related to the pollination. Five main types of pollination occur in orchids. Usually the type of pollinator group determines the different flower characteristics. If the pollinators are the color of the flower is diverse, ultraviolet, but never pure red, and usually the flower has a sweet fi-agrance in daytime. With pollinators like birds (hummingbirds) or buttedies the color of the flower is vivid red or yellow, scent is not always present, and the form of the flower is generally tubular. In cases when moths are the pollinators the flower always has a strong sweet smell especially in the part of the day when the moths are active, the color is white, cream or pale green and the form is tubular. When nectar flies are delivering the pollen to the flower the odor is sweet and the color can vary fiom green, yellow, to brown and purple. In some cases the pollinators are ants or bats (Dressier, 1993a; Wiams, 1982). A highly specialized form of pollination can be observed in the group of Ophrys where the shape, color and pattern and sometimes even the odor of the flower mimic a female insect. The pollinators arrive at the flower to try to copulate with them and in the meantime perform the pollination (Van der Pijl and Dodson, 1966). The existence of fall-in (Coryanthes), fall-through (Stmhopea), and trap (Perisreria) flowers or the actively or passively moving lip (Acostea, Bulbophylum) and the different features on the surface of the lip (callus, wax, etc.) also show other very specialized strategies for the best result in fertilization (Van der Pijl and Dodson; 1966, Dressier, 1993a). Detailed descriptions of the anatomical and morphological structure of orchid flowers can be found in several publications and books which only focus on this huge and fascinating research area, including the structure of polIinia/pollinarium and the development of ovules/embryos before, during and after pollination. More detailed information will be presented related to this research area in the Results and Discussion section of Chapter III. Another evolutionary specialization of orchids is the extremely small, undifferentiated seeds fiom which several thousands can be found in a single orchid hit which is a dry capsule. Only in some cases the more primitive groups of the family have fleshy hits with pulp and a smaller amount of seeds (Vmilia, Palmorchis, Neznviedia) (Dressier, 1993b). The advantages of the extremely small size and light weight, which is typical for most orchid seeds, is that the buoyant seeds are able to disperse and traveI long distances by air or water currents. Thus orchids are commonly among the first pioneer plant species (Arditti, 1967; Healay et al., 1980). In recent years with the use of new techniques, such as scanning electron microscopy, it became apparent that the structural details of orchid seeds can be useM and important characteristics for classification. Using these methods some general and also some more detailed reports have been pubIished about the seed morphology of both tropical and temperate species by Clifford and Smith (1969), Barthlott (1976), Wiesmeyer and Hofkten (1976), Arditti et al. (1979, 1980), Heday et d. (1980), Ziegler (1981), Tohda (1983, 1985, 1986), Chase and Pippen (1988) and recently Molvray and Kores (1995). A major portion of the anatomical and morphological details presented in this dissertation (Chapters III and IV) was obtained using scanning electron microscopy. Detailed information related to the general techniques and use of equipment for SEM can be found in reports by Rosowski et al. (1 98 I), Lee (1984), Csillag (1 989) and Bozzola and Russell (1 99 1). Recently several more important and interesting reports were published although they were not available at the time when preparations were made to establish the research described in this dissertation. However, because of the very informative content of them, some of them should be mentioned in this literature review. An excellent book was published by Rasmussen (1995) which provides a detailed overview about several anatomical and propagation aspects of temperate terrestrial orchids. Also in 1996 the first IUCN action plan for plants was published, which was speci5cally focusing on orchids. Recently Chu and Mudge (1996) and Falem et al. (1997) reported new promising techniques for the in vitro propagation of Cypn'pedium species. New results and information were published in the Proceedings of the Conference for North American Native Terrestrial Orchids Propagation and Production (1996, ed.: Allen) including reports about ecology, propagation and reintroduction of these species. Literature Cited

1. Allen, C. (ed.) 1996. North American Native Terrestrial Orchids Propagation and Production Conference Proceedings. Published by: The North American Native Terrestrial Orchid Conference, Germantown, Maryland. 2. Anderson, A B. 1990. Improved germination and growth of rare native Ontario orchid species. In: Allen, G- M., Eagles, G. M., Price, P. F. J. (ed.) Conserving Carolinian Canada, pp.:65-73. University of Waterloo Press, Waterloo, Canada. 3. Anderson, A. B. 1991. Symbiotic and asymbiotic germination and growth of Spiranthes rnagnicamporum (Orchidaceae). Lindleyana, 6: 183- 186. 4. Arber, A 1925. : a morphological study. Cambridge University Press, Cambridge. 5. Arditti, J. 1966. The effect of tomato juice and its fractions on the gerrnination of orchid seeds and on seedling growth. American Orchid Society Bulletin, 35: 175-182. 6. Arditti, J. 1967. Factors affecting the germination of orchid seeds. Botanical Review, 33: 1-97. 7. Arditti, J. 1968. Germination and growth of orchids on banana hit time and some of its extracts. American Orchid Society Bulletin, 3 7: 112-1 16. 8. Arditti, J. 1972. Professor Lewis Knudson and the Asymbiotic Germination of Orchid Seeds: The Fiftieth Anniversary. American Orchid Society Bulletin, 41399-904. 9. Arditti, J. 1977. Clonal propagation of orchids by means of tissue cuIture - A manual. In: Arditti, J. (ed.) Orchid biology: Reviews and perspectives I. pp.:203-293. Cornell University Press, Ithaca, New York. 10. Arditti, J. (ed.) 1977. Orchid biology: Reviews and perspectives I. Cornell University Press, Ithaca, New York. 11. Arditti, J. 1979. Aspects of the physiology of orchids. In: H. W. Woolhouse (ed.) Advances in Botanical Research, pp.:421-655. Acad. Press, New York. 12. Arditti, J. (ed.) 1982. Orchid biology: Reviews and perspectives II. Cornell University Press, Ithaca, New York. 13. Arditti, J. (ed.) 1984. Orchid biology: Reviews and perspectives El. Cornell University Press, Ithaca, New York. 14. Arditti, J. (ed.) 1987. Orchid biology: Reviews and perspectives N. Cornell University Press, Ithaca, New York 15. Arditti, J. (ed.) 1990. Orchid biology: Reviews and perspectives V. Timber Press, Inc. Portland, Oregon. 16. Ardim J. 1992. Fundamental orchid biology. John Wiley&Sons, New York, NY. 17. Arditti, J., Emst, R 1993. Micropropagation of Orchids. John Wiley & Sons, Inc., New York. 18. Ardim, J., Michaud, J. D., Healay, P. L. 1979. Morphometry of orchid seeds. I. Paphiopedilum and native California and related species of Cypripedium. American Journal of Botany. 66(10): 1128- 1 137. 19. Arditti, J., Michaud, J. D., Healay, P. L. 1980. Morphometry of orchid seeds. II. Native California arid related species of Calypso, Cephalanthera, Corallorhiza and Epipactis. American Journal of Botany. 67(3):347-360. 20. Arditti, J., Michaud, J. D., Oliva, A P. 1982a. Practical germination of North American and related orchids. I. Epzpactis atrorubem and E. gigantea and E. hellebon'ne. American Orchid Society Bulletin, 5 1 :162- 171. 2 1. Arditti, J., Michaud, J. D., Oliva, k P. 1982b. Practical germination of North American and related orchids. II. G-era oblongifolia and G. tedlaia American Orchid Society Bulletin, 5 1:3 94-3 97. 22. Avadhani, P. N., Khan, I., Lee, Y. T. 1978. Pathways of carbon dioxide fixation in orchid leaves. In: Teoh, E. S. (ed.) Proc. Symp. on Orchidology, pp.:1-12. Orchid Society. S. E. Asia, Singapore. 23. Avadhari, P. N., Goh, C. J., Rao, A. N., Arditti, J. 1982. Carbon fixation in orchids. In: Arditti, J. (ed.) Orchid biology: Reviews and perspectives II. pp. : 173-1 94. Cornell University Press, Ithaca, New York. 24. Ayers, J. 1960. Embryo culture of Pha2aenopsi.s seedlings. American Orchid Society Bulletin, 29:s 18-5 19. 25. Bails, C., Clements, M. A, 1987. Propagation of European orchids. Orchid Review, 1: 19-24. 26. Barthlott, W. 1976. Morphologie der Samen von Orchideen im Hinblick auf taxonomische und finktionelle Aspecte. In Proceedings of the 8th World Orchid Conference, Frmpp.:444-455. Deutsche Orchideen Gessellschaft, Frankfurt. 27. Baumann, H., Kiinkele, S. 1988. Die Orchideen Europas. - Kosmos Naturfiihrer. Fmckh'sche Verlagshandlung, W. Keller & Co ., Stuttgart. 28. Beardmore, J., Pegg, G. F. 1981. A technique for the establishment of mywrrhizal infection in orchid tissue grown in aseptic culture. New Phytol., 87527-535. 29. Beckner, 1. 1979. Are Orchids Endangered? American Orchid Society Bulletin, 48:1010-1017, 30. Beechey, C. 1970. Propagation of orchids from areal roots? American Orchid Society Bulletin, 41:1085-1088. 31. Bergman, F. J. 1996. Disinfecting orchid seed. Orchids, October, 1996, pp.:1078- 1079.

32. Bernard, N. 1899. Sur lit germination du Neottia nidus-avis. Compt. Rend. Acad. Sci. Paris, 128:1253-1255. 33. Bernard, N. 1909. L'ewlution dans la symbiose, les orchidees et leurs champignons commensaux. Ann. Sci. Nat. Bot., Ser. 9., 9:l-196. 34. Bonis, H. 1969. Samenvermehrung und Anzucht europaisher Erdorchideen. Ber. 2. Europ. Orch~deencongress,pp.:74-78. Paris. 35. Borris, H. 1970. Vermehrung europaischer Orchideen aus Samen. Der Erwerbsgartner, 24:349-345. 36+Borris, H., Albrecht, L. 1969. RationelIe Samenvermehmng und Anzucht europaischer Erdorchideen. Gartenwelt, 69:s 1 1-5 13. 37. Borsos, 0. 1952. Magyarorszig 6 a Kiqat-medence orchideiinak geobotanikai monognifiaja. I. Annales Biol. Univ. Hung., 2: 183-192. 38. Borsos, 0. 1959. Geobotanische Monographie der Orchideen der pannonischen und karpatischen Flora 11. Annal. Univ. Budapest, Sect. Biol. 259-93. 39. Borsos, 0. 1960. Geobotanische Monographie der Orchideen der pannonischen und karpatischen FIora. TV, Annal. Univ. Budapest, Sect. BioI. 3 :93-129. 40. Borsos, 0. 1961. Geobotanische Monographie der Orchideen der pannonischen und karpatischen Flora. V. Annal. Univ. Budapest, Sect. Biol. 451-82. 41. Borsos, 0.1962. Geobotanische Monographie der Orchideen der pannonischen und karpatischen FIora. VI. Annal. Univ. Budapest, Sect. Biol. 5:27-61. 42. Borsos, 0. 1963. Geobotanische Monographie der Orchideen der pannonischen und karpatischen Flora. W.Annal. Univ. Budapest, Sect. Biol. 6:43-8 1. 43. Borsos, 0. 1964. Geobotanische Monographie der Orchideen der pannonischen und karpatischen FIora. Vm. Annal. Univ. Budapest, Sect. Biol. 7:45-71. 44. Borsos, 0. 1966, Geobotanische Monographie der Orchideen der pannonischen und karpatischen Flora. X Annal. Univ. Budapest, Sect. Biol. 8:71-86. 45. Borsos, O., Soo, R 1966. Geobotanische Monographie der Orchideen der pannonischen und karpatischen Flora. IX. Annal. Univ. Budapest, Sect. Biol. 8:3 15- 336. 46. Borsos, 0. 1980. Anatomy of Wid Orchids in Hungary. I. Tissue structure of leaf and floral axis. Acta Agronornica Academia Scientiarum Hungaricae, 29:369-390. 47. Borsos, 0. 1982. Anatornische untersuchung der Widorchideen Ungarns II. Stengelanatomie. Abstracts Botanica VII.: 1 17- 129. 48. Borsos, 0. 1990. An anatomical-histochemical study of wild orchid tubers in Hungary. In: Proceedings of the symposium on biology and ecology of European orchids, pp.:25-32. 49. Borsos, 0. 1992. Orchidaceae. In: Simon, T. A magyarorszigi flora edenyes ha~ozoja.Tankonyvkiado, Budapest. 50. Bozzola, J. J., Russell, L. D. 1992. Electron Microscopy Principles and Techniques for Biologists. Jones and Bartlett Publishers, Boston. 5 1. Brackley, F. E. 1985. The orchids of New Hampshire. Rhodora, 87: 1-1 17. 52. Braun, E. L. 1967. The Monocotyiedoneae, Cat-tails to Orchids. Ohio State University Press, Columbus. 53. Breddy, N. C. 1991. Orchid mycorrhiza and symbiotic raising techniques. American Orchid Society Bulletin, 60(6):556-569. 54. Britton, N. L., Brown, K A 1970. An Illustrated Flora of the Northen and Canada. I-lII. Dover Publications Inc., New York, NY. 55. Brown, D. M, Groom, C. L., Cvitanik, M., Brown, M, Cooper, J. L., Arditti, J. 1982. Effects of fungicides and bactericides on orchid seed gemination and shoot tip cultures in vitro. Plant Cell Tissue Organ Culture, 1: 165-1 80. 56.BurgeE H. 1936. Samenkeimung der Orchideen und Entwicklung ihrer Keimpflanzen. Verlag von Gustav Fiicher, Jena. 57. Camus, H. C. 1928. Iconographic des Orchidees dEurope. Lechevalier, Paris. 58. Chang, A C. 1953. Fish emulsion, a new orchid germinating medium. American Orchid Society Bulletin, 22:200-20 1. 59. Chase, M. W., Pippen, J. S. 1988. Seed Morphology in the Oncidiinae and Related Subtribes (Orchidaceae). Systematic Botany, 13(3):3 13-323. 60. Chy C. C., Mudge, K. W. 1994. Effects of prechilling and liquid suspension culture on seed germination of the yellow lady's slipper orchid (Cwripedirrm calceolus var. pubescens). Lindleyana 9(3): 153-159. 61. Chy C. C., Mudge, K. W. 1996. Propagation and Conservation of Native Lady's Slipper Orchids (Cpipedium acauIe, C. calceolus, and C. regime). Proc. of North American Terrestrial Orchid Conference (ed. : Carol Men), pp. :107- 11 2. 62. Churchill, M. E., Arditti, J., Ball, E. b 1971. Clonal propagation of orchids eom leaf' tips. American Orchid Society Bulletin, 42-109-1 13. 63. Churchill, M. E., Ball, E. A, Arditti, J. 1972. Tissue culture of orchids. 11. Methods for root tips. American Orchid Society Bulletin, 4 1: 726-73 0. 64. Churchill, M. E., Ball, E. A, Arditti, T. 1973. Tissue culture of orchids. I. Methods for leaf tips. New. Phytol., 72: 16 1-166. 65. Clements, M. A, Ellyard, R K. 1979. The symbiotic germination of Australian terrestrial orchids. American Orchid Society Bulletin, 48(8):8 10-8 16. 66. Clements, M. A 1982. Malian native orchids. In: Arditti, J. (ed.) Orchid Biology. II. Reviews and perspectives. Orchid seed germination and seedling culture - a manual, pp.:295-303. Cornell University Press, Ithaca, New York. 67. Clements, M. A, Muir, K, Cribb, P. J. 1986. A preliminary report on the symbiotic germination of European terrestrial orchids. Kew Bulletin, 41 :437-445. 68. Clifford, H. T., Smith, W. K. 1969. Seed morphology and classification of Orchidaceae. Phytomorphology, 19: 133- 139. 69. Correll, D. S. 1950. Native Orchids of North America North of Mexico. Stanford University Press. 70. Comgan, M (ed.) 1969. Proc. of 6th World Orchid Cod. Sydney. 71. CsiIlag, A 1989. Electron rnilcroszk6pia. Kert-ti t k~elmiszeri~ariEgyetem, Budapest. 72. Currah, R S. 1991. Taxonomic and developmental aspects of the hngal endophytes of terrestrial orchid mycorrhizae. Lindleyana, 6(4):2 11-2 13. 73. Currah, R S., Zelmer, C. 1992. A key and notes for tbe genera of fhgi mycorrhizal with orchids and a new species in the genus Eplrorhin. Reports of the Tottori Mycological Institute, 30:43-59. 74. Curtis, I. T. 1936. The germination of native orchid seeds. American Orchid Society Bulletin, 5:4247. 75. Darwin, C. 1877. The various contrivances by which orchids are fertilised by insects. University of Chicago Press, Chicago, London 76. David, M. 1986. Hazai orchideikrol. Orchidea - A Magyar Orchidea TMg TajCkoaatija, 9: 19. 77. Dressler, R L. 1981. The orchids - natural history and classification. Haward University Press, Cambridge, Massachusetts, London. 78. Dressler, R L. 1993a. Field guide to the orchids of Costa Rica and Panama. Cornstock Publishing Associates, Ithaca, London. 79. Dressler, R L. 1993b. Phylogeny and Classification of the Orchid Family. Dioscorides Press, Portland, Oregon. 80. Ernst, R 1982. Paphiopedilum. In: Arditti, I. (ed.) 1982. Orchid biology: Reviews and perspectives II. pp.: 350-353. Cornell University Press, Ithaca, New York. 81. Emst, R 1994. Effects of thidiazuron on in vitro propagation of Phalaenopsis and Doritaenopsis (Orchidaceae). Plant Cell, Tissue and Organ Culture, 39:273-275. 82. Faletra, P., Dovholuk, A, King, T., Sokols, K 1997. Saving Cypripedium reginae. Orchids, February, 1997, pp.: I38-143. 83. Fadhi, A, Rossi, W. 1988. Simon & Schuster's Guide to Orchids. Simon & Schuster Inc. New York, London, Toronto, Sydney, Tokyo, Singapore. 84. Fasf G. 1974. CTber eine Methode der kombinierten generativen-vegetativen Vermehrung von . Die Orchidee, 25: 125-1 29. 85. Fast, G. 1976. Moglichkeiten zur Massenvemehng von Cypripedium calceoIus und anderen europaischen Wildorchideen. Proc. 8th World Orchid Con-., FranHhn pp.:359-363. 86. Fast, G. 1978. ffber das Keirnverhaiten europaischer Erdorchideen bei asymbiotischer Aussat. Die Orchidee, 29:27&274. 87. Fast, G. 1980. Orchideen Kultur. Veriag Eugen Ulmer, Stuttgart. 88. Fast, G. 1982. European terrestrial orchids (Symbiotic and -biotic methods). In: Arditti, J. (ed.) Orchid Biology. II. Reviews and perspectives. Orchid seed germination and seedling culture - a manual, pp.:309-326. Cornell University Press, Ithaca, New York. 89. Fredrikson, M. 1990. An embryological study of Heminim munorchis (Orchidaceae) using confocal scanning laser microscopy. American JournaI of Botany, 77: 123-127. 90. Fredrikson, M. 1991. An embryoIogical study of Plafanihera bifolia (Orchidaceae). Plant Systematics and EvoIution, 174:2 13-220. 9 1. Fredrikson, M. 1992. The development of the female garnetophyte of Epipactis (Orchidaceae) and its inference for reproductive ecology. American Journal of Botany, 79(1):63-68. 92. Fuchs, A, Ziegenspeck, H- 1925. Bau und Form der Wuhrn der einheimischen Orchideen in Hinblick auf ihre Aufgabe. Bot. Archv 12. 93. Fiiller, F. 1983. Die Gattungen Orchis und Dactylorhiza. A Ziemsen Verlag, Wittenberg. 94. Gd, I., Halacsy, 1994. Termdhelyi Q mikorrhizavizsgdatok nehhy hazai orchidea fajnil. Diplomadolgozat. Eotvos Lorhd Tudominyegyetem, Term&ettudomhnyi Kar, Budapest. 95. Galunder, R 1986. Der Einfluss von Brauereihefe auf die Keimung von rnitteleuropaischen Erdorchideen. Die Orchidee, 37: 135-1 37. 96. Gautheret, R J. 1983. Plant tissue culture: A history. Bot. Mag. Tokyo, 96:393-410. 97. Goh, C. J., Strauss, M. S., Arditti, J. 1982. Flower induction and physiology in orchids. In: Orchid Biology, Reviews and perspectives, 11. Ed.: Arditti, J., pp.:213- 243. Cornstock Publishing Associates, Ithaca, London. 98. Graeflinger, B. 1950. Repicagem precore de orquideas sobre muscineas. Orquidea, 12: 13 1-134. 99. Griesbach, R 1. 1986. Orchid Tissue Culture. In: Zimmennann, R H., Hammerschlag, F. A, Lauson, R H. (eds.) Tissue culture as a plant production system for horticultural crops. Martinus Nijhoff Publishers, Boston. pp.: 343-349. lOO-Gupton, 0. W., Swope, C. F. 1987. Wid orchids of the middle Atlantic States. The University of Tennessee Press, Knoxville. lOl.Hadley, G. 1982. European terrestrial orchids. In: Arditti, J. (ed.) Orchid Biology. II. Reviews and perspectives. Orchid seed germination and seedling culture - a manual, pp.:326329. Cornell University Press, Ithaca, New York. 102.Harbeck, M. 1963. Einige Beobachtungen bei der Aussat verschiedener europaischer Erdorchideen auf sterilem Nahrboden. Die Orchidee, 1458-65. 103.Harley, J. L., Harley, E. L. 1987. Kinds of mycorrhiza. New Phytologist Suppl. to Vol. 105(2); pp.:2-9. 104.Harvais, G. 1974. Notes on the biology of some native orchids of Thunder Bay, their endophytes and symbionts. Canadian Journal of Botany, 52:45 1-460. 105.HarvaisYG. 1982. An improved culture medium for growing the orchid C'ripediurn reginae axenically. Canadian Journal of Botany, 60:2547-2555. 106.Harvais, G., Hadley, G. 1967. The relation between host and endophyte in orchid mycorrhiza. New Phytol., 66:205-2 15. 107.Healay, P. L., Arditti, J., Michaud, J. D. 1980. Morphometry of orchid seeds. III. Native California and related species of Goodyera, Piperia, Platanthera and Spirantha. American Journal of Botany, 67(4):508-5 18. lOS.Henrich, I. E., Stimart, D. P., Ascher, P. D. 1981. Terrestrial Orchid Seed Germination in yitro on a Defined Medium. J. Arner. Hort. Sci. 106(2): 193-196. 109.Homoya, M. A 1993. Orchids of Indiana, Indiana Academy of Science, Bloomington, Indiana. 1lO.Hoppe, E. G., Hoppe, H. J. 1987a Tissue culture of the European terrestrial orchid species Huds. In: Saito, K., Tanaka, R (eds.) Proc. 12th World Orchid Conf. Tokyo, pp.51-56. 11 1 .Hoppe, E. G., Hoppe, H, J. 1987b. Tissue culture of the European terrestrial orchid species Ophrys apifera Huds. In: Kondo, K., Hastirnoto, K. (eds.) Proc. World Orchid Hiroshima Symp. pp.: 104-108. Hiroshima Botanical Garden, Hiroshima, Japan. 112.Hoppe, E. G., Hoppe, H. J. 1988. Tissue culture of Ophrys apifera. Lindleyana, 3:190-194. I 13.IUCNISSC Orchid Specialist Group. 1996. Orchids - Status Swvey and Conservation Action Plan. WCN. (Compiled by: Pridgeon, k M.,Ed: Hagsater, E., Dumont, V.), Gland Switzerland and Cambridge, UK. 114.Javorka, S., Csapody, V. 1975. Iconographia florae partis Austro-Orientah Europae Centralis. Akaderniai Kiado, Budapest. 1lS.Johansen, D. A 1950. Plant embryology. Embryology of the Spermatophyta. Cron. Bot. Waltham, Mass. 116.Jones, C. A 1985. C4 grasses and cereals. John Wiey & Sons, Inc., New York. 117.Kan0, K. 1965. Studies on the media for orchid seed germination. Mem. Fac. Agric. Kagava. Univ. 20: 1-68. 118.Karasawa, K. 1966. On the media with banana and honey added for seed germination and subsequent growth of orchids. Orchid Review, 74:3 13-3 18. 1 19.Kau1, R B. 1986. Orchidaceae luss., the orchid family. In: Great Plains Flora Association, Flora of the Great Plains, pp.: 1268-1284. University Press of Kansas, Lawrence. 120.Knudso~L. 1921. La gerrninacion no simbiotica de las semillas de orquideas. Boll. Real. Soc. Espanola Hist. Nat. 2 1:250-260. lZl.Knudson, L. 1922a. Nonsyrnbiotic germination of orchid seeds. Botanical Gazette, 73:l-25. 122.Knudson, L. 1922b. Further observations on nonsymbiotic germination of orchid seeds. Botanical Gazette, 77:212-219. 123.hudson, L. 1930. Flower production by orchids grown non-symbiotically. Botanical Gazette, 89: 192-199. 124.Knudson, L. 1946. A new nutrient solution for germination of orchid seed. American Orchid Society Bulletin, 15:214-217. 125.Knudson, L. 1948. Clorox and calcium hypochlorite as disinfectants for orchid seed. American Orchid Society Bulletin, 17:347-3 53. 126.Kunisaki, J. T. Kim, K. K., Sagawa, Y. 1972. Shoot tip culture of Vanda. American Orchid Society Bulletin, 4 1:435-439. 127.Lee, K. W. 1984. Scanning electron microscopy of flowering apices. Proc. of the 42nd Annual Meeting of the Electron Microscopy Society of America, pp.:326-327. 128.Linden, B. 1980. Aseptic germination of seeds of northern terrestrial orchids. Annales Botanici Fernici, 17: 174-82. 129.Linden, B. 1992. Two methods for pretreatment of seeds of northern terrestrial orchids to improve germination in axenic culture. Annales Botanici Fernici, 29:305- 3 13. 130.Lucke, E. 1971. Zur Samenkeimung mediterraner Ophrys. Die Orchidee, 2262-65. 13 1.Lucke, E. 1977. Naturstohsatze bei der asymbiotischen Sarnenvermehrung der Orchideen in vitro. Die Orchidee, 28: 185- 191. 132.Luer, C. A 1975. The native orchids of the United States and Canada excluding Florida. The New York Botanical Garden, New York. 133.Makara, Gy. 1982. Orchideik 6 Bromeliik. Mezdgazdasagi Kiado, Budapest. 134.PviBndy (TiUyne), A 1993. Orchidea-felek. In: Jimbome BencAr, E. (ed.): t Disnovenyek mikroszporith Kertketi 6s Elelmiszeripari Egyetem, Budapet. 135.Mead, J. W., Bulard, C. 1975. Effects of vitamins and nitrogen sources on -biotic germination and development of Orchis lanflora and Oph~ysqhegcdes. New Phytologist, 74:3340. 136.Metcalfe, C. R 1960. Anatomy of the monocotyIedons. University Press, Oxford. 137.Mitchell, R B. 1989. Growing hardy orchids flom seeds at Kew. The Plantsrnan, I1:152-169. 138.Molvray1 M., Kores, P.J. 1995. Character analysis of the seed coat in Spiranthoideae and Orchidoideae, with specid reference to the Diurideae (Orchidaceae). American Journal of Botany, 82(11): 1443- 1454. 139.MooreYD. 1849. On growing orchids fiom seeds. Gard. Chon., 35549. 140.Morel, G. M. 1960. Producing virus-free cymbidiums. American Orchid Society Bulletin, 29:495-497. I4 1.Morel, G. M. 1969. The principles of clonal propagation of orchids. In: Corrigan, M. (ed.) Proc. of 6th WorId Orchid Conf. Sydney. Pp.: IOI- 106. 142.MoreJ G. 1974. Clonal multiplication of orchids. In: Whitner, C. L. (ed.) The orchids: Scientific studies. pp.:169-222. Wiey - Interscience, New York. 143.Murashige1 T., Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15:473497. 144.Nitsch, J. P., Nitsch, C. 1969. Haploid plants tiom pollen grains. Science, 163335-87. 145.Norstog K. 1973. New synthetic medium for the culture of premature barley embryos. In Vitro, 8:307-308. 146.OIiva, A P., Arditti, J. 1984. Seed germination ofNorth American orchids. II. Native California and related species of Aplecfrum, Cpn'pedium and Spiranthes. Botanical Gazette, 145:495-501. 147.Pagef A N. 199 1. Paget's Australian deciduous terrestrial orchid notes. Private circulation. 148.Poddubnaya-Arnoldi, V. A 1967. Comperative embryology of the Orchidaceae. Phytomorphoiogy, Panchanan Maheswari Memorial Volume, pp.:3 12-3 20. 149.Pridgeor1, A. H. (ed.) 1992. The Illustrated Encyclopedia of Orchids. Timber Press, Portland, Oregon. lSO.Pritchard, H. W. 1984a. Growth and storage of orchid seeds. Proc. of the 11 th World Orchid Conference, Miami, pp.:3-11. 151.Pritchard, H. W. 1984b. Liquid nitrogen preservation of terrestriaI and epiphytic orchid seed. Cryo-Letters, 5:295-300. 252.Pritchard, H. W. (ed.) 1989. Modem methods in orchid conservation. Cambridge University Press, Cambridge. 153.Rakonczay, Z. 1990. Vords kdnyv. Akadimiai Kiado, Budapest. 154.Rarnin, I. von 1983. Aussaten von Orchideen auf verschieden Agar-Nahrboden. Die Orchidee Sonderheft,Mart, 1983, pp.: 109- 111. 155.Rasmussen, H.N.,1 990. Cell diferentiation and mycorrM infection in Dac~Zorhiza majalis (Rchb. f ) Hunt & Surnrnerh. (Orchihceae) during germination in vitro. New Phytologtst, 1 16:I37-147. 156.Rasmussen, H. N. 1995. Terrestrial orchids fiom seeds to mycotrophic plant. Cambridge University Press, Cambridge. 157.Rasmussen, H. N., Andersen, T. F., Johansen, B. 1990a. Temperature sensitivity of in uitro germination and seedling development of Dactylorhiza mujalis (Orchidaceae) with and without a myco~hkdhngus. Plant, Cell and Environment, 13 :17 1- 177. 158.Rasmussen, H. N., Andersen, T. F., Johansen, B. 19906. Light stimulation and darkness requirement for the symbiotic germination of Dactylorhizu majalis in vitro. Physiologia Plantarum, 79:226-23 0. 159.Reinerf R A, Mohr, H. C. 1967. Propagation of CatlIeyu by tissue culture of lateral bud meristems. Proc. Amer. Soc. Hort. Sci. 9 1:664-671. 160.hether, W. 1990. Keirnverhalten tenesrischer Orchideen gemassigter Klimate. Die Orchidee, 41 :100-109. 161.Ronse, A 1989. In Vitro Propagation of Orchids and Nature Conservation: Possibilities and Limitations. Mem. Soc. Roy. Bot. Befg. 1 1: 107-1 14. 162.Rosowski, J. R,Hoagland, K. D., Roemer, S. C., Lee, K. W. 1981. Improving the Image of Delicate and Complex Biotogicd Surfaces. Scanning, 4: 18 I- 187. 163.RotorY Jr. G. 1949. A method for vegetative propagation of Phalaenopsis species and hybrids. American Orchid Society Bulletin, 18:73 8-739. 164.SaIisbury, R A 1804. On the gemination of the seeds of Orchideae. Trans. Linn. SOC.7:29-32. 165.Sander7D. 1979. Orchids and their cultivation. Blanford Press, Poole, Dorset. 166.Schmidt, J. M., Antlfinger, A E. 1992. The Ievel of agarnospermy in a Nebraska population of (Orchidaceae). American Journal of Botany, 79(5):501-507. 167.Seaton, P. T., Hailes, N. S. J. 1989. Effect of temperature and moisture content on the viabiIity of Cdeyaaurantiaca seed. In: Pritchard, H. W. (ed.) Modem Methods in Orchid Conservation, pp.:17-29. Cambridge University Press, Cambridge. 168.SesslerYG. J. 1978. Orchids & How to grow them. Prentice-Hall, Inc., Englewood cm, N. J. 169.Singh, F. 1981. Differential staining of orchid seeds for viability testing. American Orchid Society Bulletin, 50(4):416418. 170.Singh, F. 1992. In vitro orchid seed germination and cloning of orchids. A successful story. A publication of the Indian Institute of Horticulhxal Research, Bangalore, India. 171.Smith, W. R 1993. The orchids of . University of Minnesota Press. 172.SolerederYH., Meyer, F. J. 1930. Systernatische Anatomie der Monocotyledonen. VI. Scitaminea-Microspermae, pp.:92-242. Verlag von Gebriider Borntraeger, Berlin. 173.So0, R, Keller, G. 1930. Monographie und Iconographiie der Orchideen Europas und des Mittelmeergebites. II. Berlin-Dahlem. 174.Stewart, J. 1992. The conservation of European orchids. Nature and environmenf No. 57. Council of Europe Press, Strasbourg. 175.Stewart, J., Griffiths, M. 1992. ManuaI of Orchids. Timber Press, Portland, Oregon. 176. Stokes, M. J. 1974. The in-vitro propagation of Dactylorhirafichsii. Orchid Review, 82:62-65. 177.StoutamireY W. P. 1963. Terrestrial orchid seedlings. Australian Plants, 2: 119-122. 178.StoutamireYW. P. 1964a. TemestriaI orchid seedlings II. Australian Plants, 2:264-266. 179.StoutamireYW. P. 1964b. Seeds and seedlings of native orchids. Mich Bot., 3: 107- 119. 180.Stoutamire, W. P. 1976. Pollination straregies in orchids of Southern Australia. In: Szmant, W. J. (ed.) First Symposium on the scientific aspects of orchids. pp.:27-34. Chemistry Department, University of Detroit. 18 l.Stoutarnire, W. P. 1974. Terrestrial orchid seedlings. In: Whitner, C. L. (ed.) The orchids. Scientific studies, pp.: 10 1-128. Wiley & Sons INC. New York 182.Stoutamire, W. P. 2983. Early growth in North American terrestrial seedlings. In: Plaxton, E. H. (ed.) North American Terrestrial Orchids Symposium II. Proceedings and lectures, pp.: 14-24. Michigan Orchid Society, Ann Arbor, USA 183,Street, H. E. 1973. Roots. In: Kruse, P. I., Patterson, M. K. (eds.) Tissue culture: Methods and applications. Academic Press, New York. 184.Street, H. E. (ed.) 1977. Plant tissue and cell culture, 2nd ed. Botanical Monographs, Vol. 11. Univ. of California Press, Berkeley. 185.Szendr&, E. 1994. Az Orchis morio L. soskhti lel8heIyenek florisztikai 6 conol6giai vizsgdata, t% a faj disznovenytennesztt%be vonisinak lehetbsege. MSc thesis, (The floristical and phytocoenologicd measurements of the native habitat of Orchis morio L. near 'SosMt' and the possibilities to use this species as a potential ornamental plant.) Kertketi C 6le~ri~ariEgyetem, Kenketi Kar, Dkzndvinytermeszt6si Q Dendrologiai Tanszek, Novbnytani Tanszkk, Budapest. 186.Szendra E., Debergh, P. C., Jhbor-Bencnir, E. 1994a. Preliminary report on some environmental effects affecting the in vifro propagation of Orchis morio Lime (Orchidaceae). Med. Fac. Landbouww. Univ. Gent, 59/1, pp.: 81-83. 187.SzendrAk, E., Debergh, P. C., Jhbor-Bencair, E. 1994b. Report on some environmental effects on the in vim propagation and the scanning electron microscope studies of the development of germination of Orchis morio Linne (Orchidaceae). Kertheti Tudomiiny - Horticultural Science Vol. 26/2.pp.: 95-97. 188,Szendr&, E., R Eszelu E. 1993. Nazai szabadfoldi kosborfttlek (Orchidzceae) aszimbiotikus in vim szaporitha. Pubiicationes Univ. Hort. Ind. Ali. Vol. LUI. pp.:66-70. 189.Sweet, H. C., Bolton, W. E. 1979. The surface decontamination of seeds to produce axenic seedlings. American JournaI of Botany, 66:692-698. 190.Tanaka, M. Senda, Y, Hasegawa, A 1976. PlantIet formation by root-tip culture in Phalaenopsis. American Orchid Society Bulletin, 45: 1022-1024, 191.Te0, C. K. H. 1978. Clonal propagation of Haemarzh discolor by tissue culture. American Orchid Society Bulletin, 47: 1028-1030. 192.Threatened Plants Committee, IUCN. 1983. Liste des plantes ram, menacees et endemiques en Europe (edition 1982.). Council of Europe, Strasbourg. 193.Tohda, H. 1983. Seed morphology in Orchidaceae. I. Dact)Zorchis, Ponerorchis. Chondradenia, and Galeochis. Science Report Tohuku University (4th ser.) 38:253- 268. 194.Tohdq H. 1985. Seed morphology in Orchidaceae. IT. Tribe . Science Report Tohuku University (4th ser.) 39:21-43. 195.Tohdq H. 1986. Seed morphology in Orchidaceae. II. Tribe Neottieae. Science Report Tohuku University (4th ser.) 39: 103-1 09. 196.Van der Pijl, L., Dodson, C. H. 1966. Orchid flowers - Their pollination and evolution. University of Miami Press, Cord Gables, Florida. 197.Va.n Waes, J. 1984. In vitro studie van de kierningsfjlsiologie van Westeuropese orchideen. Thesis, Rijkuniversiteit Gent. 198.Van Waes, J., Debergh, P. C. 1986a. Adaptation of the tetrazolium method for testing the seed viability and scanning electron microscopy of some Western European orchids. Physiologia Plantarum, 66:435-442. 199.Van Waes, J., Debergh, P. C. 1986b. In vitro germination of some Western European orchids. Physiologia Plantarum, 67:253-26 1. 200.Veyret, Y. 1985. Development of the embryo and the young seedling stages of orchids. In: The orchids - Scientific Studies. Ed.: Withner, C. L. pp.: 223-267. Robert E. IGieger Publishing Company, Malabar, Florida. 20 1.Voth, W. 1973. Salep im Turkischen Speciseis. Die Orchidee, 24:29-3 1. 202.Voth, W. 1976. Aussat und Kultur von parviflora und S. orientalis. In: Senghas, K. (ed.) Proc. of the 8th World Orchid Conference, pp.:351-358. Frankhrt. 203.Warcup, J. H. 1966. Perfect states of Rhizoctonias associated with orchids. I. New Phytol., 66:63 1-64 1. 204.Warcupy J. H. 1971. Perfkt states of Rhizoctonias associated with orchids. I. New Phytol., 86:267-272. 2OS.WarcupY J. H. 1980. Specificity of mycorrhizal association in some Australian terrestrial orchids. New Phytol., 70:35-40. 206.WiesmeyerYH., Hofkten, A 1976. Electron Microscopy of Orchid Seedlings. Proc. of First Symposium on the Scientific Aspects of Orchids, pp.:1&26. Detroit, Michigan. 207.Williams, N. H. 1982. The biology of orchids and Euglossine bees. In: Orchid Biology, Reviews and perspectives, 11. Ed.: Arditti, J., pp.:119-173. Cornell University Press, Ithaca. 208.Wiams, J. G., Wiams, A E. 1983. Field guide to Orchids of North America. Universe Book, New York. 209.Wiax-n~~J.G., Williams, AE., Arlott, N. 1978. A field guide to the Orchids of Britain and Europe with North Afiica and the Middle East. W. Collins, Sons & CO., Ltd., London. 2 10.Wiber, D. E. 1965. Additional observations on clonal multiplication of cymbidiums through culture of shoot meristem. Cymbidium Soc. News, 20:7- 10. 211.Wi M. W., Whitner, C. L. 1959. Embryology and development in the Orchidaceae. In: The Orchids: A Scientific Survey, pp.:155-188. Ronald Press, New York. 2 12.Withner, C. L. 1955. Ovule culture and growth of Vanilla seedlings. American Orchid Society Bulletin, 24:3 80-3 82. 213.WithnerYC. L. 1985. Developments in orchid physiology. In: The orchids - Scientific Studies. Ed.: Withner, C. L. pp.: 129-169. Robert E. Krieger Publishing Company, Malabar, Florida. 214.WochoIq 2. S. 1981. The role of tissue culture in preserving threatened and endangered plant species. Biological Conservation, 20:83-89. 21 S.Yanagawa, T., Nagai, M., Ogino, T., Maeguchi, R 1995, Application of disinfectants to orchid seeds, plantlets and media as a means to prevent in vitro contamination. Lindleyana, 10(1):33-36. 216.Zettler, L. W., Barrington, F. V., McInnis, T. M. 1995. Developmental morphology of Spiranthes odoruta seedlings in symbiotic culture. Lindleyana, 10(3):2 1 1.216. 217.Ziegenspeck, H. 1936. Orchidaceae. In: Loew-Schrlitcr: Lebensgechichte der Bltitenpflanzen Mitteleuropas. 1./4., Stuttgart. 21 8-Ziegler, B. 198 1. Mikromorphologie der Orchideensamen unter Bedcksichtigung taxonomischer Aspecte. Ph.D. dissertation, Ruprecht Karls Universiq Heidelberg. Asymbiotic In Kfro Germination and Development of Several Temperate Terrestrial Orchids (Orchidaceae)

Abstract

Seeds from 52 different species of temperate terrestrial orchids were disinfested with a 10% Ca-hypochlorite solution for 10 min and sown on a modified FAST medium (Fast 1976; Szendriik and R EszClu, 1993). After sowing, the cultures were kept in the dark at 10-12°C for four weeks. The cultures then remained in the dark, but the temperature was raised to 25-26°C untiI germination occurred. Thereafter cultures required alternating seasonal temperatures: 25-26OC fiom the beginning of April to the end of September and 17-19°C from October to March. Growth stages, including the date of seed and embryo imbibing, opening of the seed coat and earliest protocorm stage, were recorded and compared among the different species. Seed germination was considered successfbl when small white protocorms covered with translucent hairs and possessing a definite apical dome were observed. This stage also varied si$ruficantlyamong species. The shortest time for complete germination was observed in the genus Orchis (-28 days); and the longest in the genus Cjprpedium (-250-300 days), but it generally ranged around 50 to 90 days. For the devetopment of the young plantlets natural dispersed light and prevailing day- length were favorable. Twenty-five different orchid species were successfblly grown with this method and after two to three years of aseptic culture they were suitable for transfer ex vitro. Introduction

In recent decades the rate of extinction of plant and animal species has been alarmingly high. The damaging effect of human activity on natural ecosystems, and particularly the rapid pace at which species have disappeared has become a growing public concern. The extinction of these species is a consequence of the accelerated spread of humans into remote habitats, the introduction of toxic chemicals into the environment and the exploitation of endemic animals and plants (Beckner, 1979; Wochok, 198 1; Ronse, 1989; R Eszeki and Szendrrik, 1992; Molter, 1997; ).

Most of the temperate native temestxial orchids are endangeredtfireatened species (Beckner, 1979; Arditti and Ernst, 1993; Stewart, 1992; IUCNISSC, 1996) and the propagation of these species Born seeds poses specific problems. Orchid seeds are extremely small and the embryo is in a rudimentary stage. In most cases the seed is actually devoid of endosperm and they need microscopic fbngi in a symbiotic relationship in nature for germination. Until 1804, when Salisbury found that orchid seeds could germinate, the seeds of these plants were considered sterile. Bernard in 1899 discovered the importance of mycorrhiza in the germination of orchid seeds, and his discovery opened the door to develop new techniques for orchid propagation (Bernard, 1899 and 1909). Following the work of Knudson (1921, 1922% 1922b, and 1946) and Clement (1924) who developed the basic techniques for asymbiotic orchid seed germination, and the work of Burgeff (1936) who introduced the techniques for symbiotic orchid seed germination, several attempts were made to improve the in vifro seed sowing methods of these species (Arditti, 1972). The germination and micropropagation of tropical orchids became one of the most profitable businesses in horticulture and similar propagation techniques for the relatively more difiicult temperate orchid species could provide a powefil tool for conservation purposes. Although the amount of literature available is much greater for the tropical species, there are still numerous references for the different techniques of symbiotic or asymbiotic germination of temperate orchids. Among these are some of the most important reports for the genera that were included in the research of this chapter and which previously were reported as relatively easy to germinate: Aceras (Eiberg, 1969; Van Waes and Debergh, 1986b), Barlia (Stoutamire, 1974; Rasmussen, 1995), Bletilla (Ichihashi and Yamashita, 1977; Ichihashi, 1979), Dactylorhim (Harbeck, 1968; Hadley, 1982; Van Waes, 1984; Galunder, 1986; Rasmussen et al., 1990a, 1990b; Rasmussen and Rasmussen, 1991), Gooclfyera (Hawak, 1974; Stoutamire, 1974; Arditti et al., 1982b; Van Waes, 1984; Riether, 1990; Zettler and McInnis, 1993), Herminiurn @asmussen, 1999, Himantoglossum (Lucke, 1976; Van Waes, 1984; Muir, 1989), NigriteIh (Veyref 1969), Ophrys (Fast, 1978; Eilhardf 1980; Frosch, 1982 and 1983; Van Waes and Debergh, 1986b; Clements et al., 1986; Muir, 1989; Barroso et d., 1990), Orchis (Eorris and Albrecht, 1969; Fast, 1978; Van Waes and Debergh, 1986b; Galunder, 1986; Muir, 1987, Smreciu and Currah, 1989), Spiranthes (Stoutamire, 1974; Lucke, 1982, 1984; Van Waes, 1984; Oliva and Arditti, 1984; Van Waes and Debergh, 1986b; Anderson, 1990, 199 1; Zettler and McInnis, 1993; Zettler et al., 1995) and Trmsteinera (Hadley, 1982; Van Waes and Debergh, 1986b; Galunder, 1986). The germination of different Cypripedium species takes a long time, sometimes more than 12 months, but it is still possible to achieve a good germination rate (Hamus, 1973, 1982; Fast, 1974 and 1976; Henrich et al., 1981; Frosch, 1986; Bdard, 1987; Butcher and Marlow, 1989; Ling et id., 1990; Stoutamire, 1983, 1990; Chu and Mudge, 1994). This is true for several PIatmthera species, also (Hkmus, 1974; Hadley, 1982; Anderson, 1990). On the other hand the members of Epipactis (Eiberg, 1969; Arditti et al., 1982a; Myers and Ascher, 1982; Van Waes and Debergh, 1986b; Riether, 19901, Cephalanthera (Fast, 1978; Van Waes, 1984; Van Waes and Debergh, 1986b) and Neoffia(Veyret, 1969; Smreciu and Currah, 1989; Riether, 1990) genera are considered difficult to geminate or the efforts to produce seedlings have totally failed. It is helpful to have these references related to the topic, but these researchers commonly used only some basic media with few modifications. Unfortunately the results were often contradictory, so it is very difficult to prepare a general foundation for hrther research. In 1995 an excellent work was published by Rasmussen in which several of the above mentioned references have been summarized and compared, which is of great assistance for the scientists involved in this research area. While many of the foregoing researchers reported on anly one or a very few species, two extensive research programs were published by Henrich et al. (1 98 1) and Van Waes and Debergh (1986b), which dealt with several different temperate orchid species and provided much more detailed information about their in vim development. Based on an extended literature review and on several previous experiments a successW medium for asymbiotic in vifro propagation was developed in the ELTE Botanical Garden, Budapest, Hungary (Szendrik and R Wki, 1993; Szendr* 1994% 19945; Szendrik et al., 1994% 1994b). Several traditionally commonly used media were compared (Curtis, 1936; Knudson, 1946; Reinert and Mohr, 1967; Borris and Albrecht, 1969; Fast, 1976 and 1982; Anderson, 1990) for in vitro germination and development of temperate orchid species. Based on these experiments a successfU1 asyrnbiotic in vifro germination procedure was developed and proposed including an effective disinfstation method, an optimal medium (a modsed version of Fast's, 1976) for seed germination and plant development, and basic environmental conditions (Szendrik, 1994% 1994b). The major goal of the work described in this chapter was to test this procedure for several: different temperate orchids (more than 50 different species fiom 24 different genera), observe the plant development and provide sufficient plant material for fUrther experiments.

Materials and Methods

Dry ripe seeds from dehisced capsules of more than 50 orchid species (representing 24 different genera) were legally field collected, received from botanical gardens and other institutions (see also Chapter IV, Table 4-1) and stored in small paper envelopes at 4OC. The seeds were surface disinfested for 10 minutes in a 10% (wlv) calcium-hypochlorite (Ca(OC1)2) solution without wetting agent, shook several times and then separated fiom the soIution and debris with the help of a small glass tube and rubber stopper. If a test tube with the disinfesting solution and seeds is stoppered and then inverted, the stoppers are at the bottom of the tubes, the seeds will float on the upper surface of the solution. With the very slow removd of the rubber stoppers a small vacuum is created inside the top portion of the tubes. As the liquid level slowly subsides, a ring of seeds stays attached to the inner glass surface of the tubes. Following removal of the stoppers, the sterilizing solution and debris can be discarded and afker flaming the opening of the tubes, the seeds can be collected with a sieriIe blade and deposited onto the culture medium. With the help of one drop of sterile distilled water and a flamed glass rod the seeds easily can be spread on the surface of the medium. The advantages of this simple method are that much less direct handling of the seeds is necessary (no atering, etc.), which greatly reduces the risk of contamination; it requires less time, and also fewer seeds become lost during disinfkstation, which could be critical when stock plant material is hmited. From the first step of disinfestation all manipulations were performed in a laminar-flow cabinet. The medium used for both germination and plant development was a modified Fast medium (Fast 1976; Szendrdc and R Eszeki, 1993) as shown in Table 1-1. To make the preparation of the medium easier the vitamin stock solution was replaced with 1 tablet of POLYWTAPLEX?-8 vitamin complex (produced by CHINOIN Pharmaceutical Company, Hungary) per liter and no dfierences were observed due to this change (tested in preliminary experiments). The composition of this vitamin complex is shown in Table 1- 2. The pH of the medium was adjusted to 5.5 with citric acid before autoclaving. The medium was divided into baby food jars or small (125 mI) Erlenmeyer flasks (50 mVjar) and was autoclaved for 30 min at 121°C and 1.2 Kg cme2. Depending on the amount of seeds available, one to five vessels of each species were sown. After sowing., the cultures were kept in the dark at 10-12°C for four weeks. The cultures were than placed under dark conditions in a laboratory where the prevailing temperature was 2&1°C until germination occurred. Following germination and initial protocom development the cultures were maintained at 2€&1°C, at alternating seasonal temperatures (25-26°C from the beginning of April to the end of September and 17-19°C fiom October to March) or in a simulated dormant period by storage at 4°C and under 16 hour/day cool fluorescent light (32 pmol S-' m-2 light intensity at the tops of the vessels) or under natural illumination with prevahg day-length (near a south-facing window). Cdtures were observed at weekly intends with a stereo-microscope to determine the time of germination and the appearance of early developmental stages. Post-germination changes were recorded and photographs of representative cultures were taken several times during development. Also small samples were removed for scanning electron microscope observation (see Chapter ID). Because the original stock seed material was extremely Limited, no statistical design or data analysis were conducted, but replications were performed and compared in all cases when it was possible. The development of the plantlets was followed for three years, starting from the date of seed sowing.

Experiments were conducted in the Micropropagation Laboratory of the Botanical Garden, ELTE Scientific University, Budapest, at the Department of Floriculture and Dendrology, University of Horticulture and Food, Budapest, Hungary and at the Department of Horticulture, University of Nebraska-Lincoln.

Results and Discussion

The fim stage of germination was the imbibition of water by the seed and the embryo. Soon after imbibition the embryos became white and spherical, but they were still covered with the testa. The embryos continued to grow and hdy ruptured the testa. The appearance of the apical rneristem signaled the beginning of the protocom stage. The progress of seed germination was called successftl when small white protocorms covered with translucent hain and possessing a definite apical dome were observed. Mer the apical dome subsided into the body of the protocom the £httrue leaves appeared and a small stem developed. Until that stage all of the germinated protocorms from each species required dark. (Ifthey were moved into light too early the protocorms turned brown and died.) After the development of the small stems the little plantlets were suitable to move into light and soon, usually within two weeks, the apical region turned green. Orchids have no primary roots, and the first secondary roots appeared after the first leaves had already developed and were fbnctioning (see Figures 3-1 to 3-9 in Chapter lII). Imbibition by the seeds and the white embryos were observed in about the same time interval if there was any sign of germination (usually during the first two to four weeks following sowing) but the appearance of protocorms varied greatly among the different species. Of the species sown, 25 species germinated and continued to grow into plantlets and an additional six species developed small protocoms, but were not abIe to pass that stage. The rest of the species sown did not show any sign of germination, or in several cases the development of the embryos was observed as small imbibed or a little bigger spherical ball inside the seed coat, but they either remained in that stage for several months or turned brown and died (Table 1-3). Unfortunately due to the limited seed material it was not possible to test the viability of the seeds prior to seed sowing as it was suggested by Van Waes and Debergh (1986a and personal communication) which could have been helpfbl to determine whether the ungerminated seeds were originally not viable or if the germination technique was not suitable for the seeds. Based on the literature several of these ungerminated species belong to genera that are among the group of "orchids with problematic germination" as noted in the introduction.

The earliest germinating species were Orchis morio and , where the protocoms appeared -25-30 days after sowing (Figure 1-1). The rest of the Orchis species, all the DacNorhira, Ophrys species and Himuntoglossurn hircirmm germinated approxirnatety 50 days after sowing (also the protocorms of dserent Epipactis, Cephafanthera,Gymrzadenia and Trausteinera species appeared for that time but did not continue to deveIop). The germination of the seeds of Barlia robertba and Bletilla sn?bta took around 60 days and Disa uniflora required approximately 85-90 days to germinate. The sIowest germinating species were Plafmfhera bijalia (-200 days) and different Cjpripediurn species (-250-300 days). After the appearance of the first protocorms the germination often continued for several weeks or months. For species with the longest germination it became necessary to provide additional nutrients for the seeds, because the original medium was depleted and started to dry out. In that case, the entire older layer of the medium with the seeds/protocorms was transferred into another jar of eesh medium (Figure 1-2). Most of the protocorms from different species were ready to be placed in light after an additional 40-50 days after germination and soon after that they turned green. The first roots started to develop three to six weeks after the apical region turned green. It happened considerably later in the case of Barlia robertiana, where the protocorms developed leaves and the basal part of the plant originating fiom the spherical protocorrn hnctioned as a bigger pretuberoid for almost a year without any root development. The first roots appeared when the plants were considerably bigger than any other plantlets firom different species in the same stage. When the plants developed to about 5-10 mrn in length, they could be transferred individually (without the old medium) into fresh medium. The optimal transfer interval for most orchid species was 2-3 months, which is considerably longer than the usual 4-5 week for most plant species. By the end of the first year most of the tuberous orchid species developed the first small tuberoid (e. g. Himantoglossum, Ophrys, Orchis), or the other species without tuberoids developed several roots (C'ripedium, Disa). The plants were very sensitive to transfer during the tuberoid development, so it was more suitable to move them right before tuberoid development or after they entered into their first dormant period. All the species observed showed seasonal changes even under continuous yearlong laboratory conditions. At the end of the normal vegetative season (usually in late fad) they became dormant, most of the leaves dried out and almost no plant activity was observed untd late spring, when new leavedstems developed from the buds on the tuberoiddrhizomes. Until the end of the first year the cultures developed satisfactorily under the standard laboratory environment but after their htdormant period they performed much better if they were grown near a window which provided seasonally chanpng temperature (25-26°C fiom the beginning of April to the end of September and 17-19°C fiom October to March) and natural illumination with prevailing day-length. In that case the plants grew much faster and they developed bigger and stronger organs, with no stress signs such as exudation of purported polyphenol-like substances observed (Figure 1-3). Cypripedium acaule, C. calceolus, C. califoricum and PIatanthera bijolia required an additional three to five week long cold treatment (4°C) at the end of the dormant period, without which the development of the new buds into leaves and stems was abnormaI or absent. Another interesting observation of this experiment was that cultures fiom the same species (the seeds originated from the same geographical location, but fiom different populations) responded somehow differently for the same treatment. Plants fiom one seed- lot of Orchis morio developed satisfactorily in the laboratory, but polyphenol exudation was observed on the same medium under the same condition for another seed source (Figure 1-4). This suggests that not only do different species require somewhat different culture conditions, but it can vary even within the species and among different populations fiom the same geographical area. This is probably one reason why the available references often contradict each other. However, it is stiIl possible to suggest a general procedure, with the understanding that some plants will respond better than others and that upon repeating experiments, the results can be difEerent because of only slightly different original plant material. Spontaneous vegetative proliferation was observed with several different species (Table 1-3) - this was especially important if the original germination rate was low -, which appeared in basically three different ways: a.) some of the young protocorms did not continue to grow into small plantlets if they were left in the dark, but spontaneously started to divide and produce additionaI protocorms (actually these should be called "protocorm like bodies" (PLB), because of the vegetative origin, but the general "protocom" tern is used here for them too). These new protocorms developed into normal plantlets if they were moved into light. The proliferation stopped or slowed down (Dactylorhiza, Uphrys. Orchis) if placed in light but continued under dark condition. No differences were observed, other than origin, between the protocorms developed £torn the embryos or PLBs (Figure 1-5). b.) Additional small protocom deveioped vegetatively on the olderlmature plant parts, most commonly on the roots, tuberoids or rhizomes (Cpripedium, Dactylorhiza, Disa, Himantoglossurn, Orchzs, Platanthera) (Figure 1-6) and sometimes on the stems (Dac@lorhiza, Orchis). c.) New small plantlets developed without the protocorm stage on the body of the original plant (Bletilla, Cyprrpediurn, Disa, Orchis) or with the division of it (Bletilla, Barlia. C~ripedium,Dim, Goodyera, Hirnantoglosmm, Ophrys, Orchis). It is relatively common to find vegetatively proliferating orchid plants by division or direct organogenesis in nature. In some cases, small developing protocorms were found on the underground organs of mature plants in nature, but they usually were considered seedling protocorns, where the original seeds supposiveIy landed near the mother plant and started to germinate. These in vitro observations showed that vegetative development of protocorms can take place spontaneously on mature organs. Twenty-five different orchid species were succdyraised with this method. Some of them even reached the flowering stage in vitro, such as Orchis coriophora, 0. mcula, 0. mono, and and after two to three years of aseptic culture they were suitable for transfer ex vitro (Table 1-3; Figures 1-6, 1-7 and 1-8). The most suitable developmental stage for this and the initial attempts for acclimatization will be described in the Appendix.

The asymbiotic in vitro seed germination method used for the experiments descnibed above is similar to the ones published by Henrich et al., 1981; Van Waes and Debergh, 1986b; Zettler et al., 1995 in that darkness was the most suitable condition to initiate germination and early plant development and also the medium used in all cases had relatively low nutrient concentration. Controversy exists in the available literature about the temperature requirements of germinating seeds of temperate orchid species. According to Borris and Albrecht (1969) and Stoutamire (1974), a few weeks of cold treatment are necessary for germination but Fast (1982), Harvais (1982), Henrich et al. (198 1) and Van Waes and Debergh (1986b) doubt the importance of this. Furthermore Van Waes and Debergh (1986b) found that initial cold treatment was inhibitory for satisfactory temperate orchid seed germination. The preliminary experiments mentioned earlier (Szendrik, 1994% 1994b) and the experiments described in this chapter showed that the initial cold treatment for the asymbiotic cultures helped to induce germination and no negative effect was found the temperate orchid species tested. Henrich et al. (1981) reported sporadic germination, where after several weeks of the appearance of the first geminating protocorn, different developmental stages, such as germinating seeds, protocorms and young plantiets were present in the same culture vessel. The same phenomenon was also observed in the experiments described above. Zettler and McInnis (1993) and Zettler et d. (1995) reported similar developmental stages during in vilro germination and culture as it is reported in this chapter, although in less detail. No exact information was found in the available literature about the effect of different light sources and about the developmental differences among piantlets derived from different seed lots of the same species. Also no written references were found about the length of the optimal in vipo transfer period, the appearance of seasonal changes during in virro culture and spontaneous in vino vegetative proliferation, but personal communication with several scientists fiom this research area (Esaer R Eszeki - ELTE Botanical Garden, Budape* Hungary; Anne Ronse and Johan Van Waes - National Botanical Garden, Meise, Belgium and Margaret Ramsay - Royal Botanic Gardens, Kew, United Kingdom) reported similar obsemtions to those found during this research.

Conclusions

Several different temperate orchid species were germinated and grown with the above described method. Therefore the modified Fast medium and the proposed in vitro culture conditions appear to be suitable for the asymbiotic germination and development of a range of terrestrial orchid species. Several important observations were made during these plant development studies which can be very helpful to optimize the in vifro culture of these plants after gemination. Based on these observations, more detailed and extended experiments can be conducted. Although differences were observed among the species, all small plantlets £kom the completely germinated species reached the mature stage. Further experiments are necessary to find the optimal individual in vitro and acclimatization conditions for each species, to fully understand the effects of several environmental factors during culture (such as cold treatment, natural and laboratory illumination) and to develop an even more advanced seed-sowing technique to enhance germination responses for additional more recalcitrant species.

13. Borris, H., Albrecht, L. 1969. Rationelle Sarnenvermehrung und Anzucht europaischer Erdorchideen. Gartenwelt, 6951 1-5 13. 14. Burgeq H. 1936. Samenkeimung der Orchideen und Entwicklung ihrer Keimpflanzen. Verlag von Gustav Fischer, Jena. 15. Buthcher, D., Marlow, S. A 2989. Asymbiotic gemination of epiphytic and terrestrial orchids. In: Pritchard, K W. (ed.) Modem methods in orchid conservation, pp.:31-38. Cambridge University Press, Cambridge. 16. Chu, C. C., Mudge, K W. 1994. Effects of prechilling and liquid suspension culture on seed germination of the yellow lady's slipper orchid (C'ripedium calceolus var. pubescens). Lindleyana, 9(3): 153- 159. 17. Clement, E. 1924. The non-symbiotic germination of orchid seeds. Orchid Review, 32:359-365. 18. Clements, M. A, Muir, H, Cribb, P. J. 1986. A preliminary report on the symbiotic germination of European terrestrial orchids. Kew Bulletin, 4 1:43 7-445. 19. Curtis, J. T. 1936. The germination of native orchid seeds. American Orchid Society Bulletin, 5:42-47. 20. Dressier, R L. 1993. Phylogeny and Classification of the Orchid Family. Dioscorides Press, Portland, Oregon. 21. Eiberg, H. 1969. Keimung europaischer Erdorchideen. Die Orchidee, 20:266-270. 22. Eilhardt, K. H. 1980. Einige Beobachtungen bei der asymbiotischen Aussat von Orchideensamen. Die Orchidee, 3 1: 183-1 85,260. 23. Fast, G. 1974. ijber eine Methode der kombinierten generativen-vegetativen Vermehrung von Cypripedium calceoius. Die Orchidee, 25: 125- 129. 24. Fast, G. 1976. Moglichkeiten zur Massenvennehrung von Cypripedium calceolus und anderen europaischen Widorchideen. Proc. 8th World Orchid Conf., FrankfUrt, pp.:359-363. 25. Fast, G. 1978. ijber das Keimverhalten europaischer Erdorchideen bei asymbiotischer Aussat. Die Orchidee, 29:270-274. 26. Fast, G. 1982. European tenestrial orchids (Symbiotic and asymbiotic methods). In: Arditti, J. (ed.) Orchid Biology. 11. Reviews and perspectives. Orchid seed germination and seedling culture - a manual, pp.:309-326.Cornell University Press, Ithaca, New York. 27. Frosch, W. 1982. Beseitigung der duch die innere Hiille bedingten Keimhemmung bei europaischen Orchideen. Die Orchidee, 33 :145- 146. 28. Frosch, W. 1983. Asymbiotische Vennehg von Ophrys holosericea mit Bliiten nach 22 Monaten. Die Orchidee, 3458-61. 29. Frosch, W. 1986. Miiglichkeiten und Grenzen einer Langzeit-lagexng von Saatgut europaischer Orchideen. Die Orchidee, 37:239-240. 30. Galunder, R 1986. Der Eiuss von Brauereihefe auf die Keimung von mitteleuropaischen Erdorchideen. Die Orchidee, 37: 135- 137. 3 1. Hadley, G. 1982. European terrestrial orchids. In: Arditti, J. (ed.) Orchid Biology. II. Reviews and perspectives. Orchid seed gemination and seedling culture - a manual, pp.:326-329.CorneU University Press, Ithaca, New York. 32. Harbeck, M. 1968. Versuche zur Samenvermehrung einiger Dactylorhiza-Arten. Jahresbericht der Naturwissenschaftlichen Vereins in Wuppertal, 2 1/22:1 12- 1 18. 33. Harvais, G. 1973. Growth requirements and development of Cjpripedium reginae in axenic culture. Canadian Journal ofBotany, 51~327-332. 34. Harvais, G. 1974. Notes on the biology of some native orchids of Thunder Bay, their endophytes and symbionts. Canadian Journal of Botany, 52:45 1-460. 35. Harvais, G. 1982. An improved cuIture medium for growing the orchid C'ripedium reginae axenically. Canadian Journal of Botany, 60:2547-2555. 36. Henrich, J. E., Stimart, D. f., Ascher, P. D. 1981. Terrestrial Orchid Seed Germination in Vitro on a Defined Medium. J. her. Hort. Sci. 106(2): 193-196. 37. Ichihashi, S. 1979. Studies on the media for orchid seed germii-ation. 111. The effect of total ionic concentration, catiodanion ratio, NHJN03 ratio and minor elements on the growth of BletilZa sfriara seedlings. Journal of the Japanese Society for Horticultural Science, 47524-536. 38. Ichihashb S., Yarnashita, M. 1977. Studies on the media for orchid seed gemination. I. The effects of balances inside each cation and anion group for the germination and seedling development of Bletilla mata seedlings. Journal of the Japanese Society for Horticultural Science, 45:407-4 13. 39, NCN/SSC Orchid Specialist Group. 1996. Orchids - Status Survey and Conservation Action Plan. IUCN. (Compiled by: Pridgeon, A M., Ed: Hagsater, E., Dumont, V.), Gland Switzerland and Cambridge, UK. 40. Knudson, L. 192 1. La germination no simbiotica de las sernillas de orquideas. Boll. Real. Soc. Espanola Hist. Nat. 21:250-260. 41. Knudson, L. 2922a. Nonsyrnbiotic germination of orchid seeds. Botanical Gazette, 73:l-25. 42. Knudson, L. 1922b. Further observations on nonsymbiotic germination of orchid seeds. Botanic' Gazette, 77:2 12-2 19. 43. Knudson, L. 1946. A new nutrient solution for germination of orchid seed. American Orchid Society Bulletin, 15:214-2 17. 44. Ling H., Mowen, P., Reilly, B. 1990. Propagation of Cjpripediunt acaule: Tissue culture, greenhouse, and garden trials. In: Sawyers, C. E. North American native terrestrial orchid propagation and production, pp.:93-94. Brandywine Conservancy, Chadds Ford, Pennsylvania. 45. Lucke, E. 1976. Erste Ergebnisse zur asymbiotische Sarnenlceirnung von Himantoglossurn hircinurn. Die Orchidee, 27:60-6 1. 46. Lucke, E. 1982. Samenstruktur und Samenkeimung europaischer Orchideen nach Veyret sowie weitere Untersuchungen. I. Die Orchidee, 32: 182-188. 47. Lucke, E. 1984. Sarnenstructur und Samenkeimung europaischer Orchideen nach Veyret sowie weitere Untersuchungen. IV.-V. Die Orchidee, 35: 13-20, 153-1 58. 48. Muir, H. J. 1987. Symbiotic micropropagation of Orchis lm7rora. Orchid review, 95 :27-29. 49. Muir, H. J. 1989. Germination and mycorrhizal fingus compatibility in European orchids. In: Pritchard, H. W. (ed.) Modem methods in orchid conservation, pp.:39-56. Cambridge University Press, Cambridge. 50. Myers, P. J., Ascher, P. D. 1982. Culture of North American orchids &om seed. HortScience, 17550. 5 1. Oliva, A. P., Arditfi, J. 1984. Seed germination of North American orchids. II. Native California and related species of Aplechum, Cypripdium and Spiranthes. Botanical Gazette, 145:495-501. 52. Rasrnussen, H. N. 1995. Terrestrial orchids from seeds to mycotrophic plant. Cambridge University Press, Cambridge. 53. Rasmussen, H. N., Andersen, T. F., Johansen, B. 1990a. Temperature sensitivity of in vitro germination and seedling development of Dactylorhim majalis (Orchidaceae) with and without a mywrrhizal hgus. Plant, Cell and Environment, 13: 171-177. 54. Rasmussen, H. N., Andersen, T. F., Johansen, B. 1990b. Light stimulation and darkness requirement for the symbiotic germination of Dactylorhim majulis zn vim. Physiologia PIantarum, 79:226-23 0. 55. Rasmussen, H. N., Rasmussen, F. N. 1991. Climatic and seasonal regulation of seed plant establishment in Dactylorhiza majatis inferred fiom symbiotic experiments in vitro. Lindleyana, 6:22 1-227. 56. Reinert, R A, Mohr, H. C. 1967. Propagation of CatfIeyaby tissue culture of lateral bud meristems. Proc. Amer. Soc. Hort. Sci. 91 1664-671. 57. R Eszeki, E., Szendrik, E. 1992. Experiments to propagate native hardy orchids (Orchidaceae) in the ELTE Botanical Garden. Abstracts of 20th Cong. Hung. Biol. Soc., KecskemCt, pp.:25. 58. Riether, W. 1990. Keimverhalten terresrischer Orchideen gemassigter Klimate. Die Orchidee, 4 1: 100-1 09. 59. Ronse, A 1989. In Vitro Propagation of Orchids and Nature Conservation: PossibiIities and Limitations. Mem. Soc. Roy. Bot. Belg. 11: 107-1 14. 60. Salisbury, R A 1804. On the germination of the seeds of Orchideae. Trans. Lim. SOC.7:29-32. 61. Smreciu, E. A, Currah, R S. 1989. Symbiotic gemination of seeds of terrestrial orchids of North America and Europa. Lindleyana, 1515. 62. Stewart, J. 1992. The conservation of European orchids. Nature and environment, No. 57. Council of Europe Press, Strasbourg. 63. Stoutamire, W. P. 1974. Terrestrial orchid seedlings. In: Whitner, C. L. (ed.) The orchids. Scientific studies, pp.:101-128. Wiey & Sons Inc., New York. 64. Stoutamire, W. P. 1983. Early growth in North American terrestrial seedlings. In: Plaxton, E. H. (ed.) North American Terrestrial Orchids Symposium IZ. Proceedings and lectures, pp.: 14-24. Michigan Orchid Society, Ann Arbor, USA. 65. Stoutamire, W. P. 1990. Eastern American Cypnpedium species and the biology of Cypripedium canddm. In: Sawyers, C. E. North American native terrestrial orchid propagation and production, pp.:40-48. Brandywine Conservancy, Chadds Ford, Pennsylvania. 66. Szendr& E. 1994a Az Orchis morio L. mikroszaporitha b anatomiai vizsgilata. (Micropropagation and anatomical elaboration of Orchis morio L.) Proc. of XXII. National Scientific Student Conference, Budapest. 67. Szendr* E. 1994b. Az Orchis morio L. sosbti leldhelyenek florisztikai 6 ciinoIogiai vizsgidata, 6 a faj dhovt5nytermesztbbe vonhshak lehet6sige. MSc thesis, (The floristicd and phytocoenological measurements of the native habitat of Orchis morio L. near 'Sosktit' and the possibilities to use this species as a potential ornamental # plant,) Kertketi Q Elelrniszeripari Egyetem, Kertketi Kar, Diszn6venytermesztQi 6s Dendrologiai Tanszek, Novtnytani Tanszkk, Budapest. 68. Szendr* E., Debergh, P. C., Jirnbor-Bencnjr, E. 1994a. Preliminary report on some environmentat effi affecting the in vitto propagation of Orchis rnorio Lime (Orchidaceae). Med. Fac. Landbouww. Univ. Gent, 5911, pp. : 8 1-83. 69. Szendrik, E., Debergh, P. C., Jbbor-Benczlir, E. 1994b. Report on some environmental effects on the in vitro propagation and the scanning electron microscope studies of the development of germination of Orchis morio Lime (Orchidaceae).Kertketi Tudornkny - Horticultural Science Vol. 26/2. pp.: 95-97. 70. Szendrhk, E., R Eszeki E. 1993. Hazai szabadfdldi kosborf21ek (Orchiubceue) aszimbiotikus in vitro szaporitisa. Publicationes Univ. Hort. Ind. AIi. Vol. LJJI. pp.:66-70. 71. Van Waes, J. 1984. In vitro studie van de kiemingsfysiologie van Westeuropese orchideen. Thesis, Rijkuniversiteit Gent. 72. Van Waes, J., Debergh, P. C. 1986a. Adaptation of the tetrazolium method for testing the seed viability and scanning electron microscopy of some Western European orchids. Physiologia Plantarum, 66:43 5-442. 73. Van Waes, J., Debergh, P. C. 1986b. In vitro germination of some Western European orchids. Physiologia Plantarum, 67:253-26 I. 74. Veyret, Y. 1969. La structure des semences des orchidaceae et leur aptitude a Ia germination in vitro en cultures pures. Musee d'Histoire Naturelle de Paris, Travaux du Laboratoire La Jaysinia, 3:89-98. 75. Wochok, 2. S. 1981. The role of tissue culture in preserving threatened and endangered plant species. Biological Conservation, 20:83-89. 76. Zettler, L. W., McInnis, T. M. 1993. Symbiotic seed germination and development of Spiranthes cemua and Goodjera pubescens (Orchidaceae: Spiranthoideae). Lindleyana, 8(3): 155-162. 77. Zettler, L. W., Banington, F. V., McInnis, T. M. 1995. Developmental morphology of Spiranthes odorata seedlings in symbiotic culture. Lindleyana, 10(3):2 1 1.2 16. Table 1-1. Basic culture medium for the asyrnbiotic culture of temperate ternstrial orchids: modified FAST (Fast 1976; Szendrdc and R Eszekr, 1993)

Amount (mg11000 ml medium)

Macroelemenis.. Ca(N03h .4H*O NH$Jo3 Kbp04 KC1 MgSOd .m20 Na-Fe EDTA

vitamins: ascorbic acid nicotinic acid thiamine hydrochloride pyridoxine gl ycine

Sucrose fiuctose peptone agar (Sigma) pH= 5.5 (adjusted with citric acid) Table 1-2. Ingredients of POLYVITAPLEX@-8 vitamin complex (produced by CHINOIN Pharmaceutical Company, Hungary), used to replace vitamins in the modified FAST medium.

Inmedients Amount (per tablet) ascorbic acid (Vitamin C) 50 mg nicotinic acid (Vitamin PP) 10 mg thiamine hydrochloride (Vitamin BI) 5 mg pyridoxine hydrochloride (Vitamin Bs) 0.5 mg riboflavin (Vitamin Bz) 0.5 mg cyanocobalamin (Vitamin B 12) 1 mg retinol (Vitamin A) 3000 I. U.* ergocalciferol (Vitamin D2) 1000 1. u.

*I. U.= Lnternational Unit Table 1-3. The list of orchid species in the in vim germination experiment (in taxonomic order based on Dressier, 1993)

Taxonomic unit Taxonomic unit (oanL)

CHAPTER I1

The Effects of Medium Consistency and Organic Substances on the Protocorm Proliferation of Some Temperate Terrestrial Orchids (Orchidaceae) Cultured in Vitro

Abstract

The effects of organic compounds most commonly used for orchid micropropagation and the physical condition of the medium were investigated for the development of young temperate orchid protocorms. Separate experiments were conducted with five different temperate orchid species: Soo, Dactylorhiza maculafa (L.) Soo, Dactylorhiza majalis (RCHB.),Ophrys lutea Cav., Orchis morio L. Small (2-4 rnm diarn.) protocorms were placed in baby food jars (3 per jar) containing 50 ml modified FAST medium (Fast 1976; Szendra and R Eszek~,1993) supplemented with one of 8 treatments in a split-plot design with 5 replications. Both the liquid medium (gyrotary shaker, 125 rpm) and the gelled medium (8 g agarfl) were each supplemented with one of the following compounds: 2 g peptonefl, 100 rnl coconut waterA, 1 g casein+I g IactalburninA, or 10 g glucosd as a treatment with a defined compound. AlI treatments were kept in the dark at 25°C. The number of protocorms/jar were counted weekly over a six-week-long period and the length and fresh weight of protocorms were measured at the end of the sixth week. The medium consistency had an important role in protocorm proliferation and development, but the effect varied among species (in most cases the liquid medium increased proliferation and the size of the protocorms). Although there were not always sigdicant differences among organic compounds, the media supplemented with the undefined organic compounds showed a much better effect than the medium supplemented with glucose. Generally, when dl responses were considered peptone and coconut water led to the best development of protocorns, but this varied with species. The development of protocorms into plantlets was normal in all cases. Introduction

There is a worldwide growing interest in nature conservation and the obvious need to conserve and save plant species close to extinction. Therefore the IUCN (International Union for Conservation of Nature and Natural Resources/ The WorId Conservation Union) stated in its Conservation Strategy as some of the most important goals for botanical gardens, universities and other institutions related to the area, the conservation of rare (sometimes threatened or endangered) plant species, the preservation of their native habitats, development of new proliferation/propagation techniques and reestablishment of the species into previous native habitats or into similar protected environments. The first Action Plan by IUCN for plants was prepared especially for orchids (IUCNISSC Orchid Specialist Group, 1996). New propagation techniques serve as a tool not only for producing plant material for reintroduction but could provide pIants available for the public by lessening collectors' temptation to gather them in the wild (Ronse, 1989; Stewart, 1992). In recent years a new popular tendency in horticulture is to use native or wild species in the landscape. This requires development of new propagation techniques especially for plants that are hard to propagate. Such techniques include new methods to propagate several endangered plants (among them orchids) and select desirable cultivars for the landscape, which not only provide a uniform quality for ornamental horticulture, but help to distinguish the plants for sale from the illegally collected ones (Ronse, 1989; IUCNISSC Orchid Specialist Group, 1996). Temperate terrestrial orchids have great potential as high value ornamental perennial plants, especially for shade and rock gardens and wet areas, depending on origin. For these purposes it is necessary to develop vegetative propagation techniques for several temperate orchid species. Although for reintroduction the plant material derived fkom seed is preferable because of the higher genetic diversity, in cases when the species have almost totally disappeared, vegetatively propagated material can provide one of the last resorts to prevent extinction (Ronse, 1989; Arditti and Emst, 1993). Also in the regulations of CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) the only "non permit required" method to exchange, seIl and transport any member of the orchid family is through tissue culture propagated material and seeds (nrCN/SSC Orchid Specialist Group, 1996; USDA Central Management Authority, personal communication 1997). The traditiond way of orchid propagation which was the only way to produce new plants until the beginning of this century required much more time and provided fewer oEpring. Dividing a mature plant that has many pseudobulbs (sympodial type of growth habit) and growths or side branches (monopodial type of growth habit) under greenhouse conditions is one of the most common ways of increasing the number of orchids. Some orchid species (for example Phalaenopsis lueallelmaniana) are able to form small new plantlets with roots on a pseudobulb or stem and sometimes even on the flower stalk. These little pIants are called "keiki". Removing such piantlets and planting them into small pots can provide an easy method to propagate these orchids (Sessler, 1978; Sander, 1979; Makara, 1982).

The vegetative micropropagation of orchids in tissue culture made a revolutionary change in orchid propagation and commerce at the middle of the twentieth century (Ardim and Emst, 1993). Since then there have been numerous different methods developed for the vegetative in vim propagation of tropical orchids, starting fiom different explants and a huge number of different species and hybrids. There are two main groups: the first starts the culture from the very small growing shoot tip, or meristern; and the second more diverse group initiates the culture fiom different organs of the plant. In rneristem culture (in orchids often referred to as "mericloningn) the small apical meristem (sometimes with some leaf primordia) is excised and placed on a special in vitro medium after disinfestation. The small explant can be excised fiom the actively growing stem or shoot-tip, but dormant buds on the pseudobulbs or at the base of the leaves have also been used. This method made it possible to make virus-fiee cultures of several species, and because of the high propagation rate a lot of previously very rare tropical cultivars became available. The first successfid meristem culture was established for Cymbidiums by Morel in 1960 (Arditti and Ernst, 1993). Other plant parts are commonly used for cuIture initiation, such as roots, leaves or leaf tips (this method is not very common), flower buds and segments, stems and floral axis (Arditti and Emst, 1993). Rotor, as a graduate student in 1949, inserted surface-disinfested Phalaenopsis flower stalk nodes into sterile medium, and some of these expIants formed little plantlets. According to Arditti and Ernst (1993) a vegetative part of an orchid had never been cultured before, and no other plant was succ&lly asexually propagated in vitro before that. It is unfortunate, that his name is usually no; mentioned in the literature on orchid propagation. From these first attempts several methods were published and used for the vegetative propagation of different tropical orchxd species and this established a multt-nillion dollar business in horticuIture. Excelrent reviews of these methods were published by Ardim (1977) and by Arditti and Ernst (1993).

The available literature for in vitro vegetative propagation of temperate orchid species is considerably less than for tropical orchids, and unfortunately some of the available references do not provide sufficient detaiIs of their techniques (also noted by Arditti, 1977; and Arditti and Ern% 1993). Most commonly they initiated the cultures fiom tuberoids or small tuberoid segments as in the case of Nigritella (Haas, 1977) and Ophrys (Champagnat and Morel, 1972; Morel, 1974), dormant buds or stems of Cypripedium (Chu and Mudge, 1996) and Dactylorhiza (Stokes, 1974), meristems of Anacamptis (MoreI, 1970) and Orchis (Allenberg, 1976) and in virro derived plantlets of Cyprpedium (Fast, 1974; Faletra et al., 1997) and Ophrys (Hoppe and Hoppe, 1987% 1987b, 1988; Barroso et al., 1990). In a few cases the initial pIant material was root pieces of BZetilla (Yam and Weatherhead, 199 1b) and Neottia (Champagnaf 197I) or leaf tips of Ble~illa were used as stock material (Yam and Weatherhead, 1991a). Generally, protoconn like bodies PLBs or sometimes simply calIed protocorms, as for those derived fiom seeds) formed fiom the explant with or without an intermediate callus stage, and later the protocorms were regenerated into small plantlet.. Only a few references report direct plantIet deveIopment from the excised plant pieces (Cjpripedium: FaIetra et d., 1997; Chu and Mudge, 1996). Based on the literature review and dso on the work of Arditti and Ernst (1993) only Gruenschneider (1973) used seedling protocorms for the vegetative propagation of a temperate orchid (Dacfylorhizumamlata). His methods and the insuficient details in that publication were criticized several times (Arditti, 1977; Arditti and Ernst, 1993), but it is still surprising that the in vi~oprotocorm proliferation method which is one of the most important proliferating techniques for tropical orchids was not accepted and tried more commonly for temperate species.

Liquid suspension culture is commonly used in the mass propagation of tropical orchid species (Morel, 1974; Arditti and Emst, 1993). Although gelled medium was used for most of the experiments described in the references related to temperate species, promising results were published with the use of Liquid medium in the case of micropropagation of Ophrys (Hoppe and Hoppe, 1987% 1987b, 1988) and C~ripedium(Chu and Mudge, 1994, 1996; Fdetra et d., 1997). Complex additives have been commonly used in the medium, which actually was general for plant tissue culture in the early yeam (it was originally suggested by Haberlandt (1902) with the use of embryo sac fluids), but later the use of defined media with exactly known constituents became preferred for most plant species. The use of complex additives became even more important for most of the temperate terrestrial orchid species because the use of conventional plant growth regulators did not show the expected results (Van Waes, 1984; Van Waes and Debergh, 1986; Arditti and Ernst, 1993; Szendrilq 1994% 1994b). From the middle of this century coconut milk (or coconut water, the liquid endosperm of coconut) became one of the most often used undefined complex additives in tissue culture (van Overbeek et al., 1941). It contains several inorganic ions, nitrogenous compounds, amino acids and related substances, enzymes, vitamins, sugars, and different plant hormones such as cytokinin and gibberellin, but the exact amount of them can easily change with the ripening stage and age of coconut (a partial composition list of coconut water was published by Arditti and Ernst, 1993). Coconut water is commonly used in orchid tissue culture and it was reported as a component for several different media for temperate terrestrid orchid micropropagation and in vitro seed germination (Boms and Albrecht, 1969; Bomis, 1970; Lucke, 1971; Linden, 1980; Oliva and Arditti, 1984; among others). Another commonly used complex additive is peptone, which is a byproduct of the digestion of meat protein. It aIso consists of several different inorganic ions, nitrogenous compounds, amino acids and related substances, and vitamins (Arditti and Emsf 1993). It is widely used in several media for temperate terrestrial orchids, too (Fast, 1974, 1976, 1982; Anderson, 1990; Riether, 1990; Szendriik and R Eszeki, 1993). Casein (casamino acids) and lactalbumin are similar complex additives derived from bovine milk, and they include 18 or 17 different amino acids, respectively, in different concentrations (Fluka Chemika-BioChemika Catalog, 1995196) and several inorganic ions, nitrogenous compounds, and vitamins, but in lower concentration and in less complexity as in coconut milk or peptone (Arditti and Ernst, 1993). The use of them with temperate orchids was reported by Mead and Bulard, 1975, Van Waes (1984), Van Waes and Debergh (1 986), Hoppe and Hoppe (1987a, 1987b, 1980) and R Eszkki @ersonal communication). Other complex additives used in orchid tissue culture include yeast extract (Fast, 1974; Linden, 1980), banana pulp (Graeflinger, 1950; Arditti, 1968; Oliva and Arditti, 1984), birch phloem sap (Riether, 1990), fish emulsion (Harbeck, 1963) and honey (Karasawa, 1966), among others. Several attempts were made to use a purely defined medium for orchid tissue culture without the above mentioned additives, most commonly employing inorganic nitrogen sources and different carbon sources. The most commonly used carbon sources in orchid tissue culture have been sucrose (Knudson, 1946; Reinert and Mohr, 1967; Voth, 1976; Clements, 1982), glucose (Curtis, 1936; Hadley, 1982) and fructose (Linden, 1980). Sometimes the combinations of these carbon sources were added into both defined or undefined media (Burgee 1936; Fast, 1976, 1978, 1982). It is quite surprising that among the numerous references related to culture media for temperate terrestrial orchids, none were found that compared the effectiveness of different undehed media components. Even in the in vitro culture of tropical orchids only a Lmited number of these additives and organic sources have been compared according to Arditti and Emst (1993) (Emst, 1967% 1967b; Kano, 1965). During the experiments to germinate and raise several temperate terrestrial orchids in vitro (described in Chapter I), some of the young orchid protocorms showed signs of spontaneous vegetative protiferation. The appearance of this phenomenon and the curiosity to answer some of the above mentioned problems related to the constituents of temperate orchid tissue culture media led to the experiments described in this chapter.

Materials and Methods

The temperate terrestrial orchid species chosen for the experiments are native in Europe and potentially can be grown in a wide range of temperate areas. Dactylorhim fuchsii So6 spontaneously appeared in North America in 1960 near Timmins, Ontario (Luer, 1975) and from that time this population produced flowers each year. AlI the five species are relatively hardy and with their attractive flowers they could be used as excellent potential perennial ornamental species (the descriptions are based on the works of Borsos (1992) and Williams et al. (1985)). The in vitro derived protocorms that were used as explants were cultured as described in Chapter I in baby food jars containing 50 ml gelled (0.8% agar) modified Fast medium (Table 1-1, Chapter I; Fast, 1976; Szendrik and R Eszeki, 1993) supplemented with 2 g of peptone per 1 in the dark at 26 *l°C prior to the experiments of this chapter. Dacrylorhb fuchsii So6 has pale pink flowers with deeply three-lobed lips in a dense inflorescence, and has attractive purple spotted leaves. It mainly occurs on calcareous soils in grasslands, open scrub or woodland margins. Spontaneously proliferating protocorms were isolated f?om cultures described in Chapter I and raised in the above described way until (over 10 months) a sufficient amount of protocorms were available for the experiments. Dactylurhiza maculnia (L.) So6 has variable pink to reddish flowers with a darker margin in a dense inflorescence and the leaves are usually spotted. In its native habitat it grows in acid soils, on wet meadows, heaths, bogs and lightly wooded areas. Protocorms with the same origin as Dactylorhiza&chsii were used for stock plant material for the experiments. Dactyforhiza majalis (RCHB.) (codes: 33j and 33k) has very attractive purple spotted wide [eaves and a many-flowered dense inflorescence with deep magenta to purplish lilac flowers. It usually appears in wet meadows, swamps and marshy ground. Protocorms with the same origin as the previously mentioned two species were used for the protocorm proliferation experiments. The experiments with this species were repeated with protocorms derived from two different original seed-lots (seeds &om two different locations). Ophrys lutea Cav. The inflorescence of this species contains two to seven greenish flowers with a fascinating yellow, reddish-brown and blue lip that resembles a female . In its warmer Mediterranean native habitat it grows in grassy areas. The protocorms used for the protocorm proliferation experiments originated &om the isolated culture of a single germinated protocorm, which spontaneously started and continued to proliferate under conditions as described above. Orchis morio L. has a loose inflorescence of five to twelve flowers that have an attractively broad and often spotted lip. The color of the flowers is variable, ranging fiom the rare white to the more common carmine-pink and deep purple (see Figures 3-43 and 3-44 in Chapter III). It grows on a wide range of soils (fiom acid to calcareous) in pasture-lands, grasslands and open woodlands. Two-month-old seedling protocorms were used as stock material for the protocorm proliferation experiments. They were germinated with the same method as described in Chapter I and until the protoconn proliferation experiments they were kept in the dark so they stayed in their protoconn stage.

Small 2-4 mm diameter protocorms isolated &om the above described stock plant material were placed in baby food jars (3 per jar) containing 50 ml modified FAST medium supplemented with one of 8 experimental treatments. The four organic substances were peptone at 2 gA (code: PE), coconut water at 100 mM (code: CW), casein and lactalburnin at lg+lgA (code:C&L) and glucose (as a defined component) at 10 gil (code: GL) (In the latter case, three different sugars were added to the medium, because the basic medium already included sucrose and fnrctose). The organic compound concentrations were determined based on the most cornmody used concentrations in the available literature. AU test compounds were added to the media prior to autoclaving. Two different medium consistencies were used with the combination of the above mentioned organic substances. The gelled ("solid") medium was gelled with the addition of 8 g agar (Sigma) per liter, and the jars containing the gelIed media with the protocorms were kept in the dark in a cardboard box. The cuIture vessels containing the liquid media (no solidiflmg agent added) containing the experimental pht materiai, were maintained on a gyrotary shaker on 125 rpm, kept in the dark by covering them with one layer of aluminum foil and placing in small paper boxes on the shaker (due to the speed of rotation no damage of the protocorn was observed). The temperature for all the cultures was maintained at 26 *l°C. The dark condition was determined to be necessary in preliminary experiments on in vitro protocorm proliferation of the five different orchid species on the basic modified Fast medium supplemented with 2g peptone per liter. A split-plot experirnentaI design with different medium consistency as a whoIe-plot and the organic substances as the sub-plot treatment factors with 5 replications was used. Separate experiments were conducted with the above described different temperate orchid species. The number of protocoms/jar were counted weekly over a six-week-long period and the size and fresh weight of protocorms were measured at the end of the sixxh week. Data were analyzed using analysis of variance and means were separated using least si&cant difTerence (LSD) (with the use of procedures and statistical computer program of SAS Institute Inc., 1988). Results and Discussion

In general (based on the results of six separate experiments) the number of protocorms increased until the end of the fourth week and for most species it continued until the end of the sixth week, although there were not always sigdicant difference (PM.05) among the different organic substances and between the two medium consistencies in the final number of protocoms (sigdicant difference (Pc0.05) occurred among the organic compounds for three species). Significant differences (P<0.05) were found for the effect of the organic substances on the final total fish weight of all five species and in most cases between the medium consistencies. In three cases significant interaction (PC0.05) was observed between the different organic substances and medium consistencies in the effect on the final total fiesh weight of protocorms. Although there were not always simcant differences present among organic compounds and between medium consistencies in the effect on the final average length of protocorms, in three species significant differences (P<0.05) were found among organic compounds and in two species between the medium consistencies for protocorm length. The fresh weight and the length of the protocorms of different orchid species increased primarily because of active growth and proliferation of the protocorms, and imbibing water fiom the culture medium, which was especially noticeable with protocorms developing in liquid medium.

The change in Dactylorhim fuchsii protocorm number during the six-week-long experimental period is shown in Figure 2-1, continuously increasing with the use of most medium combinations, except for the liquid media supplemented with casein+lactalbumin or with glucose. There was no significant difference (P>0.05) in the final number of protocorms in the liquid or on the solid media. More protocorms were produced in the media supplemented with peptone than the media supplemented with coconut water (P>O.OS), casein+lactalbumin (P0.05) was observed for the final number of protocoms among coconut water, casein+lactalbumin or glucose (Figure 2-2). The final total fresh weight was significantly (WO.05) higher in liquid medium and also peptone significantly (P

The number of protocorms of Ductjdorhi~macuIafu increased until the end of the experimental period idon all media supplemented with undefined organic compounds but showed almost no changes idon media supplemented with glucose (Figure 2-5). Although there were no sidcant differences (P>0.05) observed among the organic compounds and between medium consistencies, the liquid medium supplemented with coconut water and the solid medium supplemented with casein+lactalbumin resulted in higher final protocorm number (Figure 2-6). Liquid medium led to significantly (P<0.05) greater final total eesh weight. Media supplemented with coconut water produced a higher find total &esh weight and longer average final total length than media supplemented with casein+lactalburnin (P>0.05), peptone (PX.05) or glucose (P<0.05). No significant difference was observed among media supplemented with casein+lactalbumin, peptone or glucose (Figures 2-7 and 2-8). At the end of the experimental period strong purportedly phenolic exudation was observed in the liquid medium supplemented with glucose, which appeared as a much darker brownish coloration of the medium, probably because of the more stressed protocorms (Figure 2-26). The best overall appearance of the protocorms was observed in the liquid medium supplemented with coconut water and the worst in liquid medium supplemented with glucose. The number of protocorms increased until the end of the fifth week in the case of both Dactylorhiza majalis lots, except Won the liquid or solid media supplemented with glucose (Figures 2-9 and 2-13). In the rest of the experimental data the observations were different for the protocorms from the two different lots although some similarities were present. This agrees with the preliminary observation of Chapter I, that the optimum requirements for in vitro culture can be different not only for different temperate orchid species but also for different populations fiom the same species. More protocorms of Dacfylorhiza majah (code: 33j) were produced in liquid media, but the difference was not significant (P>0.05) based on the statistical analysis among organic compounds whether liquid or gelled (Figure 2-10). The same was observed in the effects on the ha1average length of protocorms Figure 2-12). Medium consistency and organic compounds signtticantly interacted (P<0.05) on the find total fiesh weight of protocoms of DactyZorhiza mujalis (code: 33j), where liquid media supplemented with peptone, casein+lactalbumin or with coconut water gave significantly (PxO.05)better results than any other combination. No simcant differences p0.05) were observed among gelled media supplemented with any of the four organic compounds and liquid medium supplemented with glucose (Figure 2-1 1). The best overall appearance of protoconns was observed in the liquid medium supplemented with peptone, but liquid media suppIemented with casein+lactalburnin or with coconut water also gave excellent results. The number of Dnctylorhiia majalis (code: 33k) protoconns increased until the end of the fifth week, but unlike the other lot (33j) it did not continue with dl the combinations, but started to decrease in liquid media suppIemented with coconut water or peptone. This shows that probably an earlier transfer into fresh medium could help to obtain continuous development (Figure 2-13). After six weeks no significant difference (P>0.05)was observed between the two medium consistencies for ha1 average number and length of protocoms. The explants produced significantly more (F'c0.05) protocorms infon media supplemented with casein+lactalbumin than idon media supplemented with peptone or with giucose. Also media supplemented with coconut water resulted in siguficantiy higher protocorm number (Pc0.05) than media supplemented with glucose (Figure 2-14). The ha1 average length of the protocorms was longer on the media supplemented with peptone (P<0.05),coconut water (P>0.05) or with casein+lactalbumin (P>0.05)than with gIucose (Figure 2-16). Final total fresh weight yieId was sigruficantly (P<0.05)affected by medium consistency and organic compounds interaction (Figure 2- 15). Gelled media supplemented with casein+lactalbumin or with peptone and liquid medium supplemented with peptone was favorable (PcO.05) for gain in fresh weight, with fiesh weight production approximately 3 times higher than in the liquid medium supplemented with glucose. The use of media supplemented with coconut water and liquid medium with casein+lactalbumin or gelled medium supplemented with glucose resuIted (P

As noted in the Materials and Methods section, the protocorms of Orchis rnorio were not already spontaneously proliferating protocorms, but were seed-derived protocorms and used for the experiments two months after germination. Although the number of protocorms did not change so dramaticaIly as in the case of the other species, it was a very important observation that protocorm proliferation occurred (especially between the second and fourth week in liquid medium supplemented with peptone) even fiom these direct seed origin protocorms (Figure 2-22 and 2-22). After the third week the speed of protocorm proliferation slowed down, and in the last two weeks of the experiment the number of viable protocorms decreased to the original number. After six weeks of culture, no significant differences (P>0.05) were observed between medium consistencies and among organic compounds in the effect on the find number of protoconns, although the number of protocorms appeared to be slightly higher on media supplemented with peptone and coconut water (Figure 2-22). The final total fresh weight was affected by the sigrdicant interaction (P<0.01) between medium consistency and organic compound treatments. Liquid media supplemented with coconut water, peptone or with casein+lactalbumin resulted in at least two to four times higher (Px0.05) final fresh weight yields than any other treatment combination (Figure 2-23). In liquid medium, sigdcantly (P<0.01) longer protocorms were produced and the media supplemented with peptone, coconut water or with casein+Iactalburnin signScantly increased the length of the protocorms over than the media supplemented with glucose (Figure 2-24). The best overall appearance was observed in the liquid medium supplemented with peptone and then in the liquid medium supplemented with coconut water.

Following the six-week-long experimental period and data collection, all living protocorms derived fiom the experiments were transferred onto a gelled (8 g agar per 1) modified Fast medium supplemented with 2 g peptone and 100 ml coconut water per 1 and half of them were cultured in dark, the other half of them under 16 hour/day cool fluorescent light (32 pmol s" m-' light intensity) at 26 *l°C. After six weeks protocorms fiom each species regenerated into normal plantlets if placed under light condition and others continued to produce additional protocorms in the dark, as it was observed previously (Chapter I) in the case of spontaneously proliferating protocorms before the protocorm proliferation experiment. The above described observations agree with the related references (Chu and Mudge, 1994, 1996; FaIetra et al., 1997) that liquid medium can result in a higher proliferation rate and greater plant growth, although these experiments did not always show signrficant differences between the liquid and gelled media, and the results also varied among the different species tested. These experiments also corroborate the proposal by Gruenschneider (1973) that proliferation of seedling or seedling derived protocorms can be a successll method for the in vitro mass vegetative propagation of temperate terrestrial orchids. In spite of the many effort. to establish defined media for the in vim culture of temperate terrestrial orchids (Clements, 1982; Curtis, 1936; Hadley, 1982; Linden, 1980; Henrich et al., 1981; Yam and Weatherhead 1991% 1991b) these current experiments suggest that with the use of undefined organic compounds (or complex additives) much better plant development can be achieved. Based on these observations the best choice for organic compound would be peptone or coconut water, rather than casein and lactalbumin, but their effect changes with species. This also supports the fact that most of the media for the in virro culture of temperate species are using either coconut milk @oms and Albrecht, 1969; Boms, 1970; Lucke, 197 1; Linden, 1980; Oliva and Arditti, 1984) or peptone (Fast, 1974, 1976, 1982; Anderson, 1 990; Riether, 1 990; Szendrik and R Eszeki, 1993;). In several cases it is difficult to find sufficient amount and quality of coconut milklwater at the market, but peptone is more easily available and it provided sufficient if not the best results for the above described experiments. Conclusions

Based on the results of this experiment it can be concluded that the medium consistency had an important role in protocorm proliferation and development, but the effect varied among species (in most cases the liquid medium increased proliferation and the size of protocorms). Although there were not always sigdicant differences among organic compounds, the media supplemented with the undefined organic compounds showed a much better growth effect than the medium suppiemented with glucose. Generafly peptone and coconut water led to the best development of protoconns, but this also varied with species. Protocorm proliferation was achieved in d cases using compIex additives without the addition of pure plant growth regulators. However, additional experiments will be required to determine the optimal concentration of each organic compound and to test their effect in combination with each other. Further experiments are also necessary to find the length of the optimal cuIture period between transfers for different species. Literature Cited

1. Anderson, A B. 1990. Improved germination and growth of rare native Ontario orchid species. In: Allen, G. M., Eagles, G. M., Price, P. F. J. (ed.) Conserving Carolinian Canada, pp. :65-73. University of Waterloo Press, Waterloo, Canada. 2. Allenberg, H. 1976. Notizen zur Keirnung, Meristemkultur und Regeneration von Erdorchideen. Die Orchidee, 27:28-3 1. 3. Arditti, J. 1968. Germination and growth of orchids on banana fruit tissue and some of its extracts. American Orchid Society Bulletin, 37: 112-1 16. 4. Arditti, J. 1977. Clonal propagation of orchids by means of tissue culture - A manual. In: Arditti, J. (ed.) Orchid biology: Reviews and perspectives I. pp.:203-293. Cornell University Press, Ithaca, New York. 5. Arditti, I., Ernst, R 1993. Micropropagation of Orchids. John Wiley & Sons, Inc., New York. 6. Barroso, J., Fevereiro, P., Oliveira, M. M., Pais, M. S. S. 1990. In Vitro Seed Gemination, Differentation and Production of Minitubers &om Ophrys lutea Cav.. Ophrysfusca Link and Ophrys speculum Link. Scientia Horticulturae, 42329-3 37. 7. Boms, H. 1970. Vermehrung europaischer Orchideen aus Samen. Der Erwerbsgaxtner, 24:3 49-345. 8. Boms, H., Albrecht, L. 1969. Rationelle Samenvermehrung und hcht europaischer Erdorchideen. Gartenwelt, 69:s 11-5 13. 9. Borsos, 0. 2992. Orchidaceae. In: Simon, T. A magyaro~gifl6ra edenyes hatirozoja. Tankonyvkiad6, Budapest. 10. Burgeff, H. 1936. Samenkeimung der Orchideen und Entwicklung ihrer Keimpflanzen. Verlag von Gustav Fischer, Jena. 1I. Champagnat, M. 1971. Resherches sur la multiplication vegetative de Neottia nidus- avis Rich. Ann. Sci. Nat. Bot. Biol. Veg. Ser. 12, 12209-247. 12. Champagnat, M., Morel, G. 1972. La culture in vitro des tissus de tubercules d'Ophr-s. C. R Acad. Sci. Paris, 274:3379-3380. 13. Chu, C. C., Mudge, K. W. 1994. Effects of prechilling and liquid suspension culture on seed germination of the yellow lady's slipper orchid (Cypriipedium calceolus var. pubescem). Lindleyana, 9(3): 153-1 59. 14. Chu, C. C., Mudge, K. W. 1996. Propagation and Conservation of Native Lady's Slipper Orchids (C'ripdium acaule, C. caiceolus, d C. reginae). Proc. of North American TerrestriaI Orchid Conference (ed.: Carol Men), pp.: 107-1 12. 15. Clements, M. A 1982. Australian native orchids. In: Arditti, J. (ed.) Orchid Biology. XI. Reviews and perspectives. Orchid seed germination and seedling culture - a manual, pp.:295-303. Cornell University Press, Itham, New York. 16. Curtis, J. T. 1936. The germination of native orchid seeds. American Orchid Society Bulletin, 5:42-47. 17. Ernsf R 1967a. Effect of carbohydrate selection on the growth rate of Phalaenopsis and Dendrobium seed. American Orchid Society Bulletin, 36: 1068- 1073. 18. Ernsf R 1967b. Effect of setect organic nutrient additives on growth in vitro of Phalaenopsis seedlings. American Orchid Society Bulletin, 3 6:3 86-394. 19. Faletra, P., Dovholuk, A, King, T., Sokols, K. 1997. Saving Cypripediurn reginae. Orchids, February, 1997, pp.: 138-143. 20. Fast, G. 1974. aer eine Methode der kombinierten generativen-vegetativen Vermehrung von Cypripedium calceolw. Die Orchidee, 25 :125- 129. 21. Fast, G. 1976. MogLichkeiten zur Massenvermehrung von Cypripedium calceolus und anderen europaischen Wildorchideen. Proc. 8th World Orchid Conf., FrankfUrt, pp.:359-363. 22. Fast, G. 1978. ner das Keimverhalten europaischer Erdorchideen bei asymbiotischer Aussat. Die Orchidee, 29:270-274. 23. Fast, G. 1982. European terrestrial orchids (Symbiotic and asymbiotic methods). In: Arditti, J. (4.)Orchid BioIogy. II. Reviews and perspectives. Orchid seed germination and seedling culture - a manual, pp.:309-326. Cornell University Press, Ithaca, New York. 24. Fluka Chernika-BioChernika Catalog 1995/96. pp.:334, 869. Fluka Chemie AG. 25. Grae£linger, B. 1950. Repicagem precore de orquideas sobre muscineas. Orquidea, 12:131-134. 26. Gruenschneider, A 1973. Protokormproliferation von Dactylorhiza maculata - eine Mijglichkeit zur Massenvermehrung und rationellen Anzucht. Die Orchidee, 24:249- 250. 27. Haas, N. F. 1977. Erste Ergebnisse zur Meristemvermehrung von Nigritella nigra (L.) Rcb. F. und Nigritella miniata (Cr.) Janchen. Die Orchidee, 28: 153-1 55. 28. Haberlandt, G. 1902. Kulturversuche mit isolierten Pflanzenzellen. Sitzundber. Akad. Wi.Wien Math. Naturwiss. KI. Abt. I/111:69-92. 29. Hadley, G. 1982. European terrestrial orchids. In: Arditti., J. (ed.) Orchid Biology. D. Reviews and perspectives. Orchid seed germination and seedling culture - a manual, pp.:32&329. Cornell University Press, Ithaca, New York 30. Harbeck, M. 1963. Einige Beobachtungen bei der Aussat verschiedener wropaischer Erdorchideen auf sterilem Nahrboden. Die Orchidee, 1458-65. 3 1. Hoppe, E. G., Hoppe, H. J. 1987a. Tissue culture of the European terrestrial orchid species Ophrys apifera Huds. In: Saito, K., Tanaka, R (eds.) Proc. 12th World Orchid Conf. Tokyo, pp.51-56. 32. Hoppe, E. G., Hoppe, H. J. 1987b. Tissue culture of the European terrestrial orchid species Ophrys apifera Huds. In: Kondo, K, Hashimoto, K. (eds.) Proc. World Orchid Hiroshima Symp. pp.: 104-108. Hiroshima Botanical Garden, Hiroshima, Japan. 33. Hoppe, E. G., Hoppe, H. J. 1988. Tissue culture of Ophrys apifera. Lindleyana, 3 :190- 194. 34. IUCNISSC Orchid Specialist Group. 1996. Orchids - Status Survey and Conservation Action Plan. TUCN. (Compiled by: Pridgeon, A M.,Ed: Hagsater, E., Dumont, V.), Gland Switzerland and Cambridge, UK 35. Kano, K. 1965. Studies on the media for orchid seed germination. Mem. Fac. Agnc. Kagava. Univ. 20: 1-68. 36. Karasawa, K. 1966. On the media with banana and honey added for seed germination and subsequent growth of orchids. Orchid Review, 74:3 13-3 18. 37. Knudson, L. 1946. A new nutrient solution for germination of orchid seed. American Orchid Society Bulletin, 15:214-2 17. 38. Linden, B. 1980. Aseptic germination of seeds of northern terrestrial orchids. Annales Botanici Fernici, 29:3 05-3 13. 39. Lucke, E. 1971. Zur Samenkeimung mediterraner Om.Die Orchidee, 2262-65- 40. Luer, C. A 1975. The native orchids of the United States and Canada excluding Florida. The New York Botanical Garden, New York. 4 1. Makara, Gy. 1982. Orchid* 6s BromCli&. Mezagazddgi Kiado, Budapest. 42. Mead, I. W.,Bdard, C. 1975. Effects of vitamins and nitrogen sources on asymbiotic germination and development of Orchis Imyora and Oms qhegodes. New Phytolocrisf 74:33-40. 43. Morel, G. M. 1960. Producing virus-fiee cymbidiums. Am. Orchid Soc. Bd.29:495- 497. 44. Morel, G. 1970. Neues ouf dem gebiet der Meristem-Forschung. Die Orchidee, 20:433443. 45. Morel G. 1974. Clonal multiplication of orchids. In: Whitner, C. L. (ed.) The orchids: Scientific studies. pp. :169-222. Wiley - Interscience, New York. 46. Oliva, A P., Arditti, J. 1984. Seed germination ofNorth American orchids. 11. Native California and related species of Apiecfrum, C'ripedium and Spi~hes.Botanical Gazette, 145:495-50 1. 47. Reinert, R A, Mohr, H. C. 1967. Propagation of CattZeya by tissue culture of lateral bud meristems. Proc. Amer. Soc. Hort. Sci. 91 :664-671. 48. R. Eszeki, E., Szendriik, E. 1992. Experiments to propagate native hardy orchids (Orchidhceae) in the ELTE Botanical Garden. Abstracts of 20th Cong. Hung. Biol. Soc., Kecskemet, pp.:25. 49. Riether, W. 1990. Keimverhdten terresrischer Orchideen gemassigter Klimate. Die Orchidee, 4 1: 100-1 09. SO. Ronse, A 1989. In Vitro Propagation of Orchids and Nature Conservation: Possibilities and Limitations. Mem. Soc. Roy. Bot. Belg. 11 : 107-1 14. 5 1. Rotor, Jr. G. 1949. A method for vegetative propagation of Phalaenopsis species and hybrids. Am. Orchid Soc. Bull. 18:738-739. 52. Sander, D. 1979. Orchids and their cultivation. Bladord Press, Poole, Dorset. 53. SAS Institute Inc. 1988. SASlStat usets guide. Release 6.03. SAS Inst., Inc., Cary, NC. 54. Sessler, G. J. 1978. Orchids & How to grow them. Prentice-Hall, Inc., Englewood Cliffs, N. J. 55. Stewart, J. 1992. The conservation ofEuropean orchids. Nature and environment, No. 57. Council of Europe Press, Strasbourg. 56. Stokes, M. J. 1974. The in-vitro propagation of DactyorhiraficIrsii. Orchid Review, 82:62-65. 57. Szendra E. 1994a. Az Orchis morio L. mikroszaporitisa 6s anatbmiai vizsgilata. wcropropagation and anatomical elaboration of Orchis morio L.) Proc. of XW. National Scientific Student Conference, Budapest. 58. SzendrCik, E. 1994b. Az Orchis morio L. sosfiti leldhelyenek florisaikai C c6nol6giai vizsgilata, Q a faj dkznov6nytemeszt&be vonhsbak lehet6sege. MSc thesis, (The floristical and ph ytocoenological measurements of the native habitat of Orchis morio L. near 'Sdskiit' and the possibilities to use this species as a potential ornamental plant.) Kertbzeti C 61elmiszeripari Egyetem, Kenheti Kar, Dkn6v6nytermeszt&i C Dendrologiai Tanszkk, Novinytani Tanszek, Budapest 59. Szendriik, E., R Eszkki EE.1993. Hazai uabadfoldi kosborfkldk (Orchidaceae) aszimbiotikus in vitro szaporitika. Publicationes Univ. Hort. Ind. Ali. Vol. LXII. pp. :66-70. 60. van Overbeek, J., Conklin, M. E., Blakeslee, A F. 1941. Factors in coconut milk essential for growth and development of very young Datura embryos. Science, 94:350-351. 61. Van Waes, J. 1984. In vitro studie van de kiemingsfysiologie van Westeuropese orchideen. Thesis, Rijkuniversiteit Gent- 62. Van Waes, J., Debergh, P. C. 1986. In vitro germination of some Western European orchids. Physiologia Plantarum, 67:253-261. 63. Viith, W. 1976. Aussat und Kultur von Serapias pamiflora und S. orientalis. In: Senghas, K. (ed.) Proc. of the 8th World Orchid Conference, pp.:351-358. FrankfUrt. 64. Williams, J.G., Williams, AE., Arlott, N. 1978. A field guide to the Orchids of Britain and Europe with North Af5ca and the Middle East. W. Collins, Sons & CO., Ltd., London. 65. Yam, T. W.,Weatherhead, M. A 1991a. Leaf-tip culture of several native orchids of Hong Kong. Lindleyana, 6: 147-1 50. 66. Yam, T. W., Weatherhead, M. A. 1991b. Root-tip culture of several native orchids of Hong Kong. Lindleyana, 6:15 1- 153.

Liquid 1 .Gelled 1

Coconut water Peptone Casein&Lactalb. Glucose -2 1 Organic substances Figure 2-2. The effect of four different organic substances and medium consistency on the final average number of protocorms of Dactylorhiza fuchsii cultured in vitro (Three protocorms expfantedl jar)

I 1 1 initiation week1 week2 week3 week4 week5 week6 Weeks in culture Figure 2-5. The effect of four different organic substances and medium consistency on the numbers of protocorms of Dactylorhiza maculata cultured in vitro (Three protocorms explantedl jar) Liquid III Gelled

Coconut water Peptone Casein&Lactalb. Glucose Organic substances Figure 2-6. The effect of four different organic substances and medium consistency on the final average number of protocorms of Dactylarhiza maculata cultured in vitro (Three protocorms explantedl jar) Liquid 1 .Gelled 1

Coconut water Peptone CaseinBtactalb. Glucose Organic substances Figure 2-7. The effect of four different organic substances and medium consistency on the final total fresh weight of protocorms of Daclylorhiza maculata cultured in vitro (Three protocorms explantedl jar)

Ll Liquid II Gelled

8 --

6 --

4 --

2 --

ow- Coconut water Peptone CaseintkLactalb. Glucose Organic substances Figure 2-10. The effect of four different organic substances and medium consistency on the final average number of protocorms of Daclylorhiza majalis (33j) cultured in vitro (Three protocorms explantedl jar) El Liquid IGelled

Coconut water Peptone CaseinBLactalb. Glucose Organic substances Figure 2-11. The effect of four different organic substances and medium consistency on the final total fresh weight of protocorms of Dactylorhiza majaljs (33j) cultured in vjtro (Three protocorms explantedl jar) El Liquid Gelled

Coconut water Peptone CaseinBLactalb. Glucose Organic substances Figure 2-12. The effect of four different organic substances and medium consistency on the final average length of protocorms of Dactylorhixa majalis (33j) cultured in vifro (Three protocorms explantedl jar)

Liquid Gelled

Coconut water Peptone Glucose

Organic substances Figure 2-14. The effect of four different organic substances and medium consistency on the final average number of protocorms of Dactylorhiza majalis (33k) cultured in vitro (Three protocorms explantedl jar) Coconut water Peptone Casein&Lactalb. Glucose Organic substances Figure 2-1 5. The effect of four different organic substances and medium consistency on the final total fresh weight of protoconns of Daclylorhixa majalis (33k) cultured in vitro (Three protocorms explantedl jar) El Liquid H Gelled

Coconut water Peptone Casein&tactalb. Glucose Organic substances Figure 2-16. The effect of four different organic substances and medium consistency on the final average length of protocorms of Dacfylorhiza majalis (33k) cultured in vitro (Three protocorms explantedl jar) initiation wee kl wee k2 week3 week4 wee k5 week6 Weeks in culture Figure 2-17. The effect of four different organic substances and medium consistency on the numbers of protocorms of Ophrys lulea cultured in vitro (Three protocorms explantedl jar) I3 Liquid 1 .Gelled

Coconut water Peptone CaseinBLactalb.

Organic substances Figure 2-18. The effect of four different organic substances and medium consistency on the final average number of protocorms of Ophrys lutea cultured h vitro (Three protocorms explantedl jar)

Liquid 1 .Gelled 1 T

Coconut water Peptone Casein&Lactalb. Glucose

Organic substances Figure 2-23. The effect of four different organic substances and medium consistency on the final total fresh weight of protocorms of Orchis morio cultured in vitro (Three protocorms explantedl jar) LI Liquid I .Gelled 1

Coconut water Peptone CaseinBLactalb. Glucose Organic substances Figure 2-24. The effect of four different organic substances and medium consistency on the final average length of protocorms of Orchis morio cultured in vitro (Three protocorms explanted1jar) 111 112 Anatomical Observations of Some Temperate Terrestrial Orchids Using Scanning Electron Microscopy with Special Reference to Orchis morio L.

Abstract

The fascinating anatomical structures and developmentaI patterns of severai temperate orchid species were observed. Samples of CoralIorhin odontorhiza (Wid.) Nut., Cypripedium acaule Aiton, Cypripedium calceolus L., Cypn'pdium caIifomicflm A Gray, Dactylorhim fuchrii Soo, Dac@lorhizu maculata (L.) Sob, Dacfylorhiza majalis (RCHB.),Guiems spectubifis (L.) Rafin., HimantogIosnrm hircim 6.)Spreng., Ophrys lutea Cav., Orchis mmla L., Orchis mono L., Platmthera bifolia (L.) Rich., Platanthera lgperborea &.) Lindley, Platanthera praeclara Shev. & Bow., Spiranthes cema (L.) L. C. Rich., Spirunthes vernalis Eng. & Gray were legally field collected, received from botanical gardens, taken fiom herbarium specimens or were harvested fiom in vifro cdtures initiated from seeds. Plant material samples fiom several different organs were observed macroscopically and also by scanning electron microscopy (SEM). The first visible step of germination was the opening of the seed coat, when the first few white cells became visible. After a few weeks the apical meristem appeared. The young protocorm was covered with numerous translucent rhizoids. In the last stage of the germination the first true leaf and the first root started to develop. Structure of the mature organs and tissues were also examined. New information was found and is reported in this chapter about the structural details of roots with orchid-type Iny~~~hizaand stored materids (calcium oxdate crystals and starch), Idstem stornatal structure and anatomical details of the generative organs and ovulefseed development. Introduction

Being one of the most specialized groups of the plant world, the members of the Orchidaceae provide fascinating phenological, anatomical and histological facts to scientists. This family is among the most highly evolved plant families in the Monocotyledonopsida which is notable for its unique flower structure, specialized life cycles and modes of proliferation, among others. The number of species in the fkndy is about 20,000, and even in this number there is a great variability among the different species and a huge diversity in mctural details. For several decades the most commonly used taxonomic system of the orchid family was based on the work of Schlechter (1926) and after several revisions and development of other classification schemes (Garay, 1972; Dressler, 1981; Burnst-Balogh and Funk, 1986), the most recent and widely accepted system is based on Dressier's work (1993b). Although the published literature about the family and the structure of its members is quite large, several authors pointed out that additional work and research are necessary, especially for the Iess intensively observed temperate group, to reveal more detailed information (Dressler, 1993b; Arditti and Ernst, 1993). The three most recent and comprehensive overviews of the family are Withner's (ed. 1985), Dresslets (1993b) and Rasmussen's (1 995) works, in which the latter especially focuses on the temperate species. Transmission (TEPUT) and scanning electron microscopy (SEM) are relatively new tools for the study of plant anatomy and classification, and most of the available information derived with these techniques is about tropical orchid species (Ernst et al., 1970; Senghas et al., 1974; Wiesmeyer and Hofiten, 1976; Williams and Broome, 1976; Lin, 1987; Steinhart et al., 1980; Yukawa et al., 1992) and only a few focussed on temperate orchids (Holman et al., 198 1; Frednkson, 1992; Szendrik et al., 1994). In this chapter observations are presented depicting the detailed anatomical structure and plant development from germination until hiting of several temperate terrestrial orchids with the use of SEN and Orchis mono was selected to represent the observations. This species is a very good general example of the group of temperate terrestrial orchids, widespread in different habitats thorough Europe to North Africa and the Middle East. The author of this chapter had the opportunity to follow the life of this species in great detail in both its native habitat and in tissue culture. The major goal of this chapter was to use macroscopic and SEM observations to provide a detailed description of the life history of a typical temperate orchid species. This description includes some new anatomical morphological and developmental information which hopehlly will cover some of the "blank areasn of orchid biology.

Materials and Methods

Samples of seventeen different European or North American species were legally field collected, received fiom botanical gardens, taken fiom herbarium specimens or were harvested from in vifro cultures initiated fiom seeds. Plant materials originated fiom in vitro cultures were produced by asymbiotically germinating and culturing seeds, described in Chapter I. Plant samples after receipt from different sources or harvested fiom in vitro cultures were stored at 4°C and as soon as possible (generally after one or two days in the case of fiesh, still living pIant material) were fixed and prepared for scanning electron microscopy (SEM). All of the specimens were examined macroscopically, under stereo microscope and in more detail with SEM. The samples collected from in vitro cultures included germinating seeds, young protocorms and plantlets and different organs and tissues fkom older plants. Additional samples originated from specimens collected in their native habitat, and they represented only plant parts of older pIants, but no germinating materials (seeds, protocorms and young plantlets) were obtained in silu. The following samples were used for the observations by SEM: Coru~lorhizaodontorhiza (Wid.) Nut. (Samples were received fiom Dr. Robert B. Kaul, School of Biological Sciences, University of Nebraska-Lincoln and fkom the Charles E. Bessey Herbarium, University of Nebraska State Museum): rhizome, stem, leaf, flower, young and mature hit; C'ripedirrm amle Aiton (Samples were received 6om Dr. Zsolt Debreczy, International Denrological Institute, Boston): root, rhizome, leaf; C'ripedium calceolus L. (Samples were hmested fiom in vim culture and also received from the Charles E. Bessey Herbarium, University of Nebraska State Museum): rhizome, stem, leaf; mature fit; Cjpri+umcaliforniicum A Gray (Samples were harvested from in vim culture and also received &om the Charles E. Bessey Herbarium, University of Nebraska State Museum): rhizome, stem, leaf, Daclyorhirafuchsiz So6 (Samples were harvested from in vitro culture): protocorms and young plantlets from several different developmental stages, root, tuberoid, leaf, D~ctylorhiznmamIata (L.) So6 (Samples were harvested fkom in vitro culture): protocorms and young plantlets fiom several different developmental stages, root, tuberoid, le&, Dactyforhizz majalis (RCHB.) (Samples were harvested from in vitro culture): protocorms and young plantlets £tom several diaerent developmental stages, root, tuberoid, led, Galemis spectabifis (L.) Rafin. (Samples were received from the Charles E. Bessey Herbarium, University of Nebraska State Museum): roof rhizome, stem, l& flower, young and mature hit; Himuntoglosssum hircimrm (L.) Spreng. (Samples were harvested from in vi~oculture): root, tuberoid, leaf; Ophrys lutea Cav. (Samples were hwested ffom in vifro culture): protocorms and young plantlets fiom several different developmental stages, root, tuberoid, leaf, Orchis marnrla L. (Samples were harvested from in vitro culture): root, stem, leaf, inflorescence; Orchis rnorio L. (Samples were harvested from in vitro culture and also legally field collected near Sosktit, Hungary): protocorms and young piantlets from several different developmental stages, roof tuberoid, leaf, stem, inflorescence, young and mature fruit; Plaianfhera bifolia (L.) Rich. (Samples were harvested fiom in vitro culture and also received fiom Dr. Olga Borsos, UTE Scientific University of Budapest, Hungary): root, stem, leaf, inflorescence; Phthera hperborea (L.) Lindley (Samples were received fiom the Nebraska Game and Parks Commission and fiom the Charles E. Bessey Herbarium, University of Nebraska State Museum): stem, le& flower, young and mature hit; Plafanfhera praeclara Shev. & Bow. (Samples were received fiom the Nebraska Statewide Arboretum and fiom the CharIes E. Bessey Herbarium, University of Nebraska State Museum): stem, leaf, flower, young and mature hif seeds; Spiranrhes cemua (L.) L. C. Rich. (Samples were received £?om Dr. Ann Antlfinger, Department of Biology, University of Nebraska at Omaha and from the Charles E. Bessey Herbarium, University of Nebraska State Museum): root, stem, leaf, inflorescence, young and mature hit; Spirm~hesvernalis Eng. & Gray (Samples were received from Dr. Ann Antlfinger, Department of Biology, University of Nebraska at Omaha and fiom the Nebraska Game and Parks Commission): root, stem, leaf, flower, young and mature hit;

Plant material samples for SEM were fixed in 5% glutaraldehyde for 3 hours, and kept in 1% osmium tetroxide for 2 hours, in pH 7.4 phosphate buffer for 2 hours and washed three times in the same buffer. Then they were dehydrated in an ethanol series and critical- point-dried through C02 before being mounted on aluminum stubs and sputter-coated with 30 nrn of gold using a Denton Vacuum Desk II Cool Sputter Coater (in US) or an SCD 040 Balzers Sputter Coater (in Hungary). Samples were observed with Cambridge Stereoscan 90 (in US) or with Tesla BS 300 (in Hungary) scanning electron microscopes at 15 or 20kV respectively. No differences were observed related to the two different equipment setups. Each sample was examined at different magnifications and photographs were taken in each case. The observations took place at the Central Research Laboratory of the University of Horticulture and Food, Budapest, Hungary and at the Laboratory For Electron Microscopy, School of Biologrcal Sciences, University of Nebraska-Lincoln, USA Further observations by SEM are currently under investigation- Because of the reasons described in the introduction and also because the available plant material sarnpIes covered the most detail of the life history, studies will focus on the green-winged orchid (Orchis morio L.) and wilI be described in detail in this chapter. Data will be provided about the other species only if they show additional details or different information than was observed in the case of Orchis rnorio.

Results and discussion

Germination and earlv plant development As the initial step of the germination, the microscopic orchid seeds imbibe water. The median sector of the seed widened because the embryo swelled and later the embryonic cells started to divide (Figure 3-1). The distinct spherical embryo in the middle of the testa continued to grow and finalIy ruptured the seed-coat, usually dong one of the longitudinal ridges formed fiom the longitudinal anticlinal walls of the testa (Figure 3-2). At this point the white embryo was still rather undifferentiated, all the round cells were uniformly about the same size and there was no sign of cotyledons, primary root or plumule. Among the outer surface cells of the embryo, rhizoid hairs started to develop (Figure 3-3), then soon after this the apical meristem appeared. The cells of the apical meristem were much smalIer than the original outer cells of the embryo; they initially appeared as a round superficial disc built up fiom small cells (Figure 3-4), which later grew into a conical meristematic dome (Figure 3-5). Gradually the apicd meristem sank down into the inner body of the young protocorn, first appearing only as a shallow depression then it totally disappeared fiom the surface (Figure 3-6). The leftover hmel-like structure appeared to hntion as the first initial scale leaf, and f?om the safe deep environment of this crater the first true leaf appeared (Figure 3-7). Pardel with these changes the histological arrangement of the new shoot started to deveIop, appearing in dense parallel lines built up fiom small epidermal cells incIuding among them the first stomata (Figure 3-8). The basal portion of the protocorm gradually started to elongate, and was covered with numerous translucent rhizoids. In nature, this part of the protocorm is believed to be responsible for maintaining the symbiotic relationship with the mycorrhizal fungi (Rasmussen, 1995), but because the samples originated &om in vifro asymbiotic cultures, it was not possible to observe the steps of the initial hngal infection and symbiosis. This basal part of the protocorm in hnction resembled the later developing tuberoids, serving as an absorbing and nutritional underground support of the little plantlet. From the appearance of the 6rst true leaf, the plants started photosynthesis, gradually becoming autotrophic and the elongated apical shoot turned green (Figure 3-9). Orchids have no primary roots at all; and the first adventitious roots developed a few weeks after the appearance of the little shoot. With this step the protocorm stage was finished. The little plantlets continued to develop one or two more roots and a few more leaves, and at the end of the first vegetative season the first tuberoid developed fiom a basal axiilary bud of the stem. From that point the new plant entered the usual year-long developmental cycle, and showed typical seasonal changes.

In several orchid species the apical and basal (suspensor) regions of the embryo show some differences in anatomical structure and even the inner cells are different f?om the outer epidermal cells, whereas in the case of some other species the cells of the embryo are almost uniform (Hamson, 1977; Manning and Van Saden, 1987; Rasmussen, 1990, 1995). Although orchids have no primary root, the basal suspensor end of the protocorm forms a mycotrophic tissue, which is nutritionally supporting the rest of the body with the help of symbiotic hngi (Burgee 1936; Rasmussen, 1995). The seeds lack endosperm, but there are some nutrients stored in the cells of the embryo, mainly as proteins and lipids and sometimes simple soluble sugars, which provide initial support for germination until the mycorhizal infection takes place (Carlson, 1940; Harvais, 1974; Rasmussen, 1990, 1995). For successfi1 germination, several species depend on an external sucrose source, which becomes available for the germinating embryo through the rnycorrhiza or in asymbiotic tissue culture fiom the medium (Emst et al., 1971; Rasmussen and Whigham, 1993; Rasmussen, 1995). Orchid seedlings are initially heterotrophic and later with the development of photosynthetic tissues they slowIy and gradually become autotrophic, but sometimes even in that stage they require some additional amino acids and other nutrients from an external source (Harvais and Raitsakas, 1975; Rasmussen, 1995). The length of the protocorm stage can vary greatly. Although there is limited information available about the duration of this time in nature, Rasmussen and Whigharn (1993) found that protocorms of Go&verapubescens were still in that developmental stage six months after germination. It makes this situation more confusing that after a longer time period it is hard to discern differences between the older protocorms and the newly formed smaI1 dormant tuberoids or rhizomes (Rasmussen, 1995).

Roots and rnvcorrhiza The roots of Orchis morio were shown to be monostelic in these observations, with only one vascular bundle in the middle of this organ. Beneath a thin layer of rhizodennal cells, the cortex of the root was the location of the mycorrhizaI peloton formation (Figure 3-10). The cells of the cortex were full of starch before the kngal infection, but later the starch content became lower because the hyphae used it for their own development (Burgee 1936; Hadley, 1980; Rasmussen, 1995). Generally the phloem appeared to be more dominant in the central cylinder than the xylem because of the presence of hngi in the roots. The terminal end of the root (except the actively growing and dividing root tip) was covered with transparent root-hair-like structures, termed rhizoids. Rhizoids had a major role in nutrient and water absorption, and making the connection between the plant and the mycorrhizal kngi in the soil (Figure 3-1 1). Orchids have a special type of mycorrhiz. called an "orchid-type" mycorrhiza, which is specific for the family (Harley and Harley, 1987), so the hngi were present only in the cortical cells of the root, but there was no sigmficant fbngal growth and hyphae development on the outer daceof the root (shown in Corallorhim odontorhiza, Figure 3-12). Following infection, anastomization occurred then the hyphae formed a three dimensional body, called a peloton. The pelotons were stabilized in the middle of the cortical cek by microscopic "ropes". Afterward the fingi lost their function and enter a dormant stage (Figures 3-14,3-15, 3-16 and 3-17). The presence of mycorrhiza was seasonal in several temperate species. MycorrW hngi had an important role at the beginning of the vegetative season, but later when the hUy developed green vegetative body provided enough foodlenergy for the plant through photosynthesis, it was almost impossible to find any visible sign of the symbiotic fungi in the roots of Orchis morio. Galearis qec&ilis, Cypripedium acaule, Spitmthes vernalis and Spirmtthes cema. In the case of CoruZIorhiza odontorhira the mycorhiza was present and functional all year long because this saprophytic species obligately relies on the hngi throughout its whole life (Figure 3-13).

Terrestrial orchids have an extremely simple root system and contain only a few unbranched fleshy adventitious roots, because orchids have no primary root. The first roots develop close to the bad end of the protocorm; later in the mature plant the roots form adventitiously from the basal part of the stem, just above the tuberoid. The roots are the organs of absorption and storage, and most importantly they are the location of the mycorrhlza, the symbiotic relationship built up between the Living cells of the plant and microscopic fungi. This is a special case of endomycorrhiza, where following germination no hyphae (Hartig net) are found on the outer surface of the underground parts of the plant. The fbngus is present only in the protocorms after gemination, and only in the cortical cells of the root in the mature plant. One of the identified symbiotic partners of Orchis rnorio is Ceratobasr'dium comigenim (Burgeq 1936; Hadley, 1980; Giil and Halacsy, 1994; Rasmussen, 1995). There is a very fragile balance in this relationship; the plant is obligate for the fungus for its survival but with a stronger infection the presence of the fbngus could be lethal. The orchid plant is known to have a protective mechanism producing different phytoalexins (orchinol, hircinol) that control the growth of the symbiotic partner (Harley and Smith, 1983). The fbngi provide inorganic nitrogen and phosphorous substances (Alexander, 1987), and organic substances and vitamins (Harley, 1969; Purves and Hadley, 1976) to the plant. The plant also produces certain chemical compounds and vitamins that are important for the development of the hngi (Harvais and Pekkala, 1975; Purves and Hadley, 1976; Hadley and Ong, 1978; Hadley, 1980). Undermound storage organs As in many ternstrial orchids, including the green-winged orchid (Orchis morio), there was a very special storage organ formed from the roots and from the stem, called a tuberoid (this special name also shows the origin of this organ, and that is the reason for this separate name originating from the word, tuber). This root-stem tuberoid was mainly a storage root, but the basal portion had a sheath of root structure around a centered stem structure witch consisted of the new bud (Figure 3-19). The structure of the tuberoid was polystelic, so the vascular-system consisted of scattered steles, with the number of steles in Orchis moria between twelve to fifty-four. The whole tuberoid was covered with a 2-4 cell thick exodermis (rhizodennis). Under it there was a layer of parenchymatous cortex called ground tissue. This was the place of storage of different materials. Every individual stele was surrounded by endodermis of cortical origin. The vascular bundle itself was collaterally closed. In older tubers, the parenchymatous tissue became disorganized and the tuberoid fell apart into individual steles (Figures 3-20,3-21) The twin-tuberoid was the organ for storage of different materials such as water, starch, mucilage and other organic materials ("slime materials" or sometimes called as salep (Borsos, 1990)), and long needle- like calcium oxalate crystals (Figure 3-22). The size and the shape of the starch grains were specific for the species, in Orchis morio they were spherical and about 80 mm in diameter (Figures 3-23). Sometimes a more aggregated body formed fiom the starch, mucilage and other "slime materials", which in this species was a polyhedronlike body called "pentamorio" by Szendrik (Szendr* 1994; Szendrik et al., 1994) (Figure 3-24). The morphology of the starch grains and the aggregated irregular or spherical bodies formed from the grains were also observed in several other species with SEM which showed a specific appearance of these structures among the different species or genera (Figures 3-25, 3-26, 3-27, 3-28, 3-29, 3-30). In Cypripedium acaule and Platmthera Merborea, more complex forms of the calcium oxalate crystals were observed near the usual individual needles and bigger bundles of long crystals (Figures 3-3 1, 3-32, 3-33, 3- 34 and 3-35). In the Orchis genus usually two tuberoids can be found on the plant (Figure 3-18). One developed in the previous year and is directly connected to the leaves and the floral axis (old or mother-tuber). The second is a younger one which developed from the bad part of the stem after flowering, and consists of storage materials and a new bud for the next year. (The twin-tuberoids also are found when these species are cultured in virro). At the end of the vegetative season the plant becomes dormant, and in spring the tuberoid quickly provides utilizable energy for the young plant as it develops fiom the bud (Borsos, 1990; Dressier, I 993b; Rasmussen, 1995). In other temperate species which do not form tuberoids, other storage organs such as the rhizome (Corallorhiza, Cypripedium, Gaieuris), fleshy roots (Platanthera, Spiranthes) or even simple roots (C'ripedium) may accumulate starch (Figure 3-36). Limited somation has been published about the size arid appearance of starch grains of some temperate terrestrial orchids using light microscopy (Borsos, 1990). However, no detailed observations or data have been found that describe the structures of temperate orchid starch grains and aggregates. Dr. David S. Jackson, Department of Food Science and Technology, UNL has also indicated that there is little to no information available about starch structures in orchids (personal communication).

Stems and leaves The cross-section of the stem of Orchis morio was circular in shape and the stem was never branched. A one cell-row thick epidermal layer with cuticle and wax covered the stem at the outside surface. There were a few stomata found between the epidermal cells. Beneath the epidermal layer there was a 68 cell-row thick chlorenchyma. Inside the chlorenchyma layers there was a continuous circle of sclerenchyma. Directly under this layer there were several closed collateral vascular bundles arranged in a ring embedded in the parenchyma. The parenchyma ceiis of the medulla were large with big intercellular spaces between them and in the middle of the stem there were breaks where a large cavity (pith) was formed (Figure 3-37). The leaves were covered on both sides with a prominent wax layer. The cells of the upper epidermis were large, elongated and had a small number of stomata between them. The young stomata were round (sometimes with small remnant subsidiary cells) and later became oval and had no accessory cells (Figures 3-38, 3-39). The cells of the lower epidermis were highly elongated parallel to the longitudinal axis of the leaf. The mesophyll which was rich in chloroplasts, was composed of spongy parenchyma tissue and generally was 6-8 cells thick in the middle of the leaf near the main vein. There were large intercellular air spaces in the mesophyll. The vascular bundles, which run parallel along the length of the leaf, were of a collateral closed type, in which the phloem was adjacent to the lower epidermis, and the xylem was adjacent to the upper epidermis. The bundles were surrounded by a sheath of parenchyma cells and the biggest bundles were found at the middle of each leaf.

Stems and leaves of temperate orchid species are essentially much less specialized than other organs in the family, so in structure they are quite similar to an ordinary monocot stem or leaf. In Daci'yZorhizu, OOrch, Ophrys, PZatanfhera and Spiranthes the internodes are very close to each other at the base of the stem, and the lanceolate leaves are in a spiral arrangement, making a rosette of leaves. In contrast, the floral axis has only a few or no internodes and leaves between the leaf-rosette and the inflorescence. (Borsos 1980, 1982; Dressler 1993b). The leaves of these plants, are of a typical monocot type, isolateral homogenous with many parallel veins and almost indiscernible connections between them (Dressler, 1993 b) . In the saprophyte CoralZorhiza odotorhira, the leaves appear only as bladeless sheaths on the stem with no chlorophyll in the tissues of the stem and leaves, but apparently some degenerated stoma are still visible (Figure 340). The surface of the leaves can be glabrous without any prominent structure (Orchis, Galearis), glabrous but with some exuded waxy structures (CoraZZorhiza, Figure 3-42) or can bear hairs or trichomes (Cpripediurn, Figure 3-41) which sometimes serve as an insect repellent and produce lipids in the terminal head cells (Holman et al., 1981). If the surface of the leaf is pubescent, there are more epidermal hairs on the upper surface than the lower surface of the leaf (Luer, 1975; Borsos, 1980). After flowering, the green-winged orchid goes into dormancy and has almost no activity during the dry summer months. At the beginning of the fall, a few leaves of the new leaf rosette develop. Then the plant stays dormant during the winter and resumes growth in early spring, terminating in the inflorescence (Baumann and Kiinkele, 1988; Williams et al., 1985).

Flowering and fruiting In every case a bract can be found at the base of the flowers of Orchis morio. It was usually smaller in length than the spur, and green and purple-colored, making the flower even more striking (Figure 3-44). The anatomical structure of the bract was sLnilar to that of normal leaves. The sepals and petals were also similar to normal leaves in structure, aithough they had somewhat more rounded cushion-like epidermal cells. The cells on the upper surtace of the lip flabellurn) were large and had a sparkling appearance because of the high water content, making the appearance velvet-like (Figure 3-45). There were no stomata on the upper surface of the lip, but the lower surface had several stomata and the anatomical pattern was very similar to a normal led. A1 the perianth segments were edged with a delicate braid-type trimming (Figure 3-46). In Spircmthes cema and Spzrcmthes vernalis the upper surface of the labellum was even more specialized, where three different epidermal cell types directed the insects near the mid-vein in the direction of the gynostemium (Figure 3-47). The inner epidermis of the spur was glabrous without papilla or secretory cells, but the large elongated balloon shaped papilla cells at the apex of the labellum served as osmophores. The pedicel was totally united with the ovary, with no distinct border between them and they were twisted because of the resupination. The ovary was built up from three carpels, and division was lacking between carpels. There were numerous (several thousands) ovules in the ovary and their position was anatrop (Figures 3-55, 3-56). The ovary usually was not fully developed at the time of flowering, but finished its differentiation following fertilization. Inside the head of the pollinarium (called pollinium) the pollen grains united to form smaller segments. A sticky material (derived From the tapeturn), called elatoviscin, held the individual pollen grains together. The segments bdt up from the individual pollen grains were partially embedded in a sponge-like material at the apical end of the caudicles (Figure 3-48). The caudicles were buiIt up fkom sterile pollen grains and elatoviscin; and usually were lacking in any ceIlular detail. At the beginning of its development, the viscidium (or sticky pad (Dressier, 1993b)) was built up from rostellar cells, but later the majority of these cells were broken down, making a sticky glue, with just one cell layer remaining as the connection between the glue and the pollinium (Figure 3-49). This was even more clearly observed in the case of Platmtherapraeclara (Figures 3-50 and 3-5 1). In the case of Orchis rnorio the flowering starts about eight weeks after the end of dormancy, from the end of March through the beginning of June, depending upon latitude (Wiiiams et al., 1978). After pollination both color and morphological changes were detectable in the flowers. The position of the flowers changed, and usually the flower became nonresupinate. GeneralIy the untwisted form of the ovary was a very good sign that pollination has occurred (Figure 3-61). The perianth became faded, and the petals and sepals became couapsed over the lip. The strumre of the ovary was not fully developed during the time of flowering, and it continued its differentiation after fertilization occurred. The individual ovaries were undifferentiated. They consisted of onIy the female archesporiai cell and a few other cells (Figures 3-57 and 3-58). Even the fertilization was very unusual, totally different fiom other plants, because of the rudimentary stage of the ovary. When the hit was ready to dehisce, the different integument layers of the ovule degenerated (the internal integument of the ovule as well as the deepest layers of the externd integument degenerated). The outer cell layer of the external integument graduaIly covered the embryo and this constituted the cover of the seed (testa) (Figures 3-59 and 3-60). By the end of the seed development, the suspensor of the embryo had degenerated, and the embryo did not have any cotyledon, primary root or plumule. The cells of the seed-coat of the ripe seeds were dead, empty, and generally transparent. The color of the ovary and later the young hit (partly covered with the bract) after fertilization was green and actively involved in photosynthesis, providing more energy for the developing seeds. Later the stomata of the fiuit lost their structure (Figure 3-62) and the hits and the surrounding bracts dried out. There were special hygroscopic hairs at the top and basal portion of the fruit and in the curves of the placenta, a phenomenon observed in all species examined (Figures 3-63, 3-64, 3-65 and 3-66). When the fitwas totally ripe and the moisture of the air was suitable for the seeds, these hygroscopic hairs helped to open the fiuit and disperse the seeds. The epidermal ceUs of the hit also changed their structure, becoming organized into thread-like lines that dry ouf thus also helping the hit to open (Figure 3-62). The ripe dry capsular hits usually spIit at the midline (mid-vein) of each carpel.

The inflorescence of Orchis morio is not branched and there are approximately 6 to 15 flowers on the floral axis. The flowers in the inflorescence appear simultaneously and open almost at the same time or within a short the interval. The flowers have a bilateral symmetry (zygomorphic flowers). All of the individual flowers are perfect flowers, so they include both of the male and female organs (Figure 3-43). The rachis of the inflorescence is erect, so the fp of the small undeveloped flower faces up in the flower bud. Later this orientation changes because of the 180' twisting of the pedicel and the flower becomes resupinate (Baumann, Kiinkele, 1988; Williams et al., 1985; Dressier, 1993b). The color and texture of the individual perianth segments vary in orchids. The color of the green- winged orchid flower is variable fiom dark-purpIe and carmine-pink to white. The segments of the outer whorl of the flower are called the sepals. They have a protective finction while the young flower is developing and are less colorful compared with the other segments of the perianth. The edges meet, but do not overlap in the flower bud. The lateral sepds are streaked green with some purple. The two lateral segments of the inner flower whorl are called petals; they are usually thinner and have a more striking color and pattern. The sepals and petals are incurved forming a Ioose, rounded hood. The third segment of the inner whorl is caIled the lip or labellurn. This is the largest and most prominent of the segments of the penanth and gives the orchid flower its colorful and exotic appearance (Figure 344). All these characteristics aid in the main function of the lip, which is to provide a tempting landing sufiace for the insects involved in the pollination (Darwin, 1877; Van der Pijl and Dodson, 1966; Luer, 1975; Holman et al., 1981; Williams, 1982). An additional part of the perianth is the stubby spur, which is formed by the lateral sepals and the basal part of the lip. The main knction of the spur is to provide nectar for the pollinators, but in the case of the green-winged orchid it only mimics that fbnction. Many orchid flowers have an exceptional scent. Special floral glands, or osmophores, on the sepals, petals or the Lip secrete pefimes that entice pollinators to the flowers. (Dressler, 1981, 1993% 1993b; Goh, Strauss and Ardim, 1982). In the majority of orchids there is a union between the style and the stamina1 filaments that is called the column or gynostemium. The anther is usually found in a clearly defined area close to the end of the column. The column is the functional center of the orchid flower. Generally it has lateral wings, called staminodia, which are actually the sterile stamens. Except for the ancient groups (Apostasioideae) of the orchid family and the groups of lady's slippers (Cyprypedioideae), all other members of the family normally have only the median anther, with the two lateral stamens being sterile or absent (Dressler, 1993b). In Orchis morio the anther opens toward the surface of the column (Makara, 1982). One of the most important steps in the evolution of orchids is that the individual pollen grains are united to become pollinia. In the ancient groups of orchids the pollen remained powdery, as is the case for most other flowering plants, but in the majority of the family the pollen grains became aggregated. This characteristic is adaptively related to the huge numbers of ovules waiting for pollination in the orchid ovary. When the pollen grains united to form this bigger unit the surface of the individual grains changed; the exine is usually thick on the outer surface of the pollinia, but missing between the grains. With this type of sedile pollinia a single pollinium could be enough to fertilize several stigmas. The original four pollinia are united into two distinct parts (Burns-Balogh, 1985; Dressler, 1993b). When the pollinia is aggregated in a small head, usually this segment has a handle or stalk, called a caudicle. The hnction of the caudicle is primarily to hold the pollinium, but it could be broken following the pollination, so the pollinium can be separated from the insect and remain in the flower. In many orchids there is another attachment to the pollinium, called the viscidium (or sticky pad), which can be found at the end of the stalk. This sticky end is involved in attaching the mass of pollen grains to the body of the pollinator (Dressier, 198 1, 1993% 1993b; Goh, Strauss and Arditti, 1982). The pollinium, its handle and the viscidium together are called the pollinarium. The poharium is usually the whole unit that is removed from the flower by the pollinator, thus facilitating pollination. The pollinarium of each taxonomic unit is very typical (Figures 3-52, 3-53, 3-54) and has a great role in classification (Burns-Balogh, 1985; Burns-Balogh and Funk, 1986). Following pollination the bright, vivid color of the flowers becomes more paler or brownish. The wilting of the sepals and petals after pollination is the result of different hormonal changes in the flower, especially in relation to different auxins and ethylene (Withner, 1985). The faded perianth and the column fall off after fertilization occurs, or remain for a while on the top of the fiuit as a dried leftover from the flower. Movement of amino acids and other nitrogenous compounds fiom the sepals, petals, and lip into the column and ovary has also been observed (Withner, 1985). Embryogenesis is a relatively short stage compared with the length of fiuit formation; it starts around the middle of the time period between the pollination and the dehiscence of the ovary and lasts an average of two weeks. The development of the embryo continues after germination, when the meristematic apex of the embryo forms an apical bud, and a special stage develops, called a protocorm (Veyret, 1985; Fredrikson, 1991, 1992). Conclusions

Most of the obsemtions presented here were consistent with the information available in the related Literature about the anatomical structure and development of temperate terrestrial orchids. Several new details were observed of the germination (especially of the development of the apical meristem and young leaves); root and storage organs, including the structure of starch grains and calcium oxalate crystals in different species; and the development of the ovaries following fertilization with structural changes of ovules and developing seeds. Malguth (1901) found that the hygroscopic hairs in the hit are specific characteristic of epiphytic orchids, which was also mentioned in Dresslds (1993b) work. The obsenmtions presented in this chapter do not support this statement, since more or less hygroscopic hairs were found in each fblly developed hit of the temperate species examined, mostly in the curves of the placenta and sometimes at the two ends of the hit. The most important result of this work is that the complete detailed Life history of a temperate species (Orchis nrorio) was followed fkom germination through the development of the seeds in the fruits. It is proposed that this approach could be used as a basic protocol for firther observations with other species. Literature Cited

1. Alexander, C. E. 1991. Mycorrhizal infection in adult orchids. In: Mycorrhizae in the next decade, pp.:324-327. 2. Arditti, J., Emst, R 1993. Micropropagation of Orchids. John Wdey and Sons, Inc., New York, Chishester, Brisbane, Toronto, Singapore. 3. Baumann, H., Kiinkele, S. 1988. Die Orchideen Europas. - Kosmos Naturfiihrer. Franckh'sche Verlagshandlung, W. Keller & Co., Stuttgart. 4. Borsos, 0. 1980. Anatomy of Wid Orchids in Hungary. I. Tissue structure of leaf and floral axis. Acta Agronomica Academia Scientiarum Hungaricae, 29:369-390. 5. Borsos, 0. 1982. Anatomische untersuchung der Widorchideen Ungarns 11. Stengelanatornie. Abstracts Botanica VII. :1 17- 129. 6. Borsos, 0.1990. An anatomical-histochemical study of wild orchid tubers in Hungary. In: Proceedings of the symposium on biology and ecology of European orchids, pp.:25-32. 7. Burgeff, H. 1936. Samenkeimung der Orchideen und Entwicklung ihrer Keimpflanzen. Verlag von Gustrav Fischer, Jena. 8. Burns-Balogh, P. 1985. Orchid pollen/pollinaria atlas. IDC Mcroform, Zug Switzerland. 9. Burns-BaIogh, P., Funk, V. A. 1986. A phyiogenetic analysis of the Orchidaceae. Srnitsonian Contributions to Botany, 61 :1-79. 10. Carbon, M. C. 1940. Formation of the seed of Cypripedium pmj'7orum. Botanical Gazette, 102:295-30 1. 11. Darwin, C. 1877. The various contrivances by which orchids are fertilised by insects. University of Chicago Press, Chicago, London 12. Dressier, R L. 1981. The orchids - natural history and classification. Harvard University Press, Cambridge, Massachusetts, London. 13. Dressier, R L. 1993a. Field guide to the orchids of Costa Rica and Panama. Cornstock Publishing Associates, Ithaca, London. 14. Dressier, R L. 1993b. Phylogeny and Classification of the Orchid Family. Dioscorides Press, Portland, Oregon. 15. Ernst, R, Arditti, J., Healey, P.L., 1970. The nutrition of orchid seedlings. American Orchid Society Bulletin, 39:559-565,69 1-700. 16. Emst, R, Arditii, J,, Healey, P.L., 1971. Carbohydrate physiology of orchid dings. American Journal of Botany, 58:827-83 5. 17. Fredrikson, M. 199 1. An embryological study of Platkmthera bifolia (Orchihceae). PIant Systematics and Evolution, 174:2 13-220. 18. Fredrikson, M. 1992. The development of the female gametophyte of Epipactis (Orchidaceae) and its inference for reproductive ecology. American Journal of Botany, 79(1):63-68. 19. a,I., Halicsy, A. 1994. Temehelyi 6 mikorrhizavizsg~atokntihiny bazai orchidea fajng. Diptomadolgozat. Eijtv(is Lorind Tudomhyegyetem, Termkettudominyi Kar, Budapest. 20. Gamy, L. A 1972. On the origin of the Orchidaceae, II. Journal of the Arnold Arboretum, 53:202-215. 21. Goh, C. J., Strauss, M. S., Arditti, J. 1982. Flower induction and physiology in orchids. In: Orchid Biology, Reviews and perspectives, II. Ed.: Arditii, J., pp.213- 243. Cornstock Publishing Associates, Ithaca, London. 22. Hadley, G. 1980. Orchid mycorrhiza. In: Orchid Biology Reviews and Perspectives 11. (ed.: Arditti, J.) pp. 83-1 18. Cornell University Press, Ithaca. 23. Hadley, G., Ong, S. H. 1978. Nutritional requirements of orchid endophytes. New Phyt010gisf 81 :561-569. 24. Harley, J. L. 1969. Biology of mycorrhiza. 2nd edition. Leonard Hill, London. 25. Jkley, J. L., Smith, S. E. 1983. Mycorrhizal symbiosis. 26. Harley, J. L., HarIey, E. L. 1987. Kinds of mycorrhiza. New Phytologist Suppl. to Vol. 2 05(2); pp. 2-9. 27. Harnson, C. R 1977. Ultrastructural and histochemical changes during the gemination of Cdtleya mrantiaca (Orchidaceae). Botanical Gazette, 138:4 1-45. 28. Hanmis, G. 1974. Notes on the biology of some native orchids of Thunder Bay, their endophytes and symbionts. Canadian Journal of Botany, 52:45 1460. 29. Hanmis, G., Pekkda, D., 1975. Vitamin production by a kngus symbiotic with orchids. Canadian loumal of Botany, 53 :1 56163. 30. Harvais, G., Raitsakas, A, 1975. On the physiology of a hgus symbiotic with orchids. Canadian Journal of Botany, 53 :144-1 55. 3 1. Holman, R T., Cunningham, W. P., Swanson, E. S. 1981. A closer look at the glandular hairs on the ovaries of Cypripediums. American Orchid Society Bulletin, 50(6):683-687. 32. Lin, C. 1987. Histological observations on in-vitro formation of protocorm-like bodies fkom flower stalk internodes of Phalaenopsis. Lindleyana, 2(1): 58-65. 33. Luer, C. A. 1975. The native orchids of the United States and Canada excluding Florida. The New York Botanical Garden, New York. 34. Makara, Gy. 1982.Orchidedc C Bromki&. Mdgazddgi Kiadb, Budapest. 35. Malguth, R 1901. Biologische Eigenthiimlichkeiten der Friichte epiphytischer Orchideen. PhD. Thesis, Breslau. 36. Manning, J. C., Van Staden, J. 1987. The development and mobilisation of seed reserves in some *can orchids. Australian Journal of Botany, 35:324-377. 37. Purves, S., Hadley, G. 1976. The physiology of symbiosis in Gmdyera repens. New Phytolo- 77:689-696. 38. Rasmussen, H.N.,1990. Cell diferentiation and mycorrhizal infection in Ductylorhiira mujulris (Rchb. f)Hunt & Summerh. (Orchidacae) during germination in vitro. New Phytobgkt, 116:137-147. 39. Rasmussen, H. N. 1995. Terrestrial orchids fiom seed to mycotrophic plant. Cambridge University Press, Cambridge. 40. Rasmussen, H.N.,Whigham, D., 1993. Seed ecology of dust seeds in situ: a new study technique and its application in terrestrial orchids. American Journal of Botany, 80: 1374-1378. 4 1. Schlechter, R 1926. Das System der Orchidaceen. Notizblatt des Botanischen Gartens und Museums zu BerIin-Dahfem, 9563-59 1. 42. Senghas, K., Ehler, N., Schill, R, Barthlotf W. 1974. Neue Untersuchungen und Methoden zur Systematic und Morphologie der Orchideen. Die Orchidee, 25: 157- 168. 43. Steinhart, W. L., Ammen, I. S., Nash, I. A, Howland, J. L. 1980. Surface Contours of Orchid Columns: A Scanning Electron Microscope Study. American Orchid Society Bulletin, 49(5):49 1494. 44. Szendrdc, E. 2994. Az Orchis morio L. sbskti IelBhelyCnek florisztikai Q dnologiai vizsgilata, 6 a faj diszn6venytermeszt4sbe von&inak lehetaskge. Diplomadolgozat. (The floristical and phytocoenological measurements of the native habitat of Orchis morio L. near 'Sos~t'and the possibilities to use this species as a potential ornamental t phnt.) Kertketi Q Elelmisreripari Egyetem, Kertketi Kar, Disznovknytermeszt6i is Dendrologiai Tanszek, Novenytani Tanszek, Budapest. 45. Szendrilq E., Debergh, P. C., Jbbor-Bencztir, E. 1994. Report on some environmental effects on the in vitro propagation and the scanning electron microscope studies of the development of germination of Orchis morio Linne (Orchidbceae).Kertketi Tudominy - Horticultural Science, Vol. 2612. pp.: 95-97. 46. Van der Pijl, L., Dodson, C. H. 1966. Orchid flowers - Their pollination and evolution. University of Miami Press, Coral Gables, Florida. 47. Veyret, Y. 1985. Development of the embryo and the young seedling stages of orchids. In: The orchids - Sciendfic Studies. Ed.: Withner, C. L. pp.: 223-267. Robert E. Krieger Publishing Company, Malabar, Florida. 48. Wiesmeyer, H., Hohen. A 1976. Electron Microscopy of Orchid Seedlings. Proc. of First Symposium on the Scientific Aspects of Orchids pp.: 16-26. University of Detroit., Michigan. 49. Wdliams, N. H. 1982. The biology of orchids and Euglossine bees. In: Orchid Biology, Reviews and perspectives, 11. Ed.: Arditti, J., pp.: 119-173. Cornell University Press, Ithaca. SO. Williams, N. H., Broome, C. R 1976. Scanning electron microscope studies of orchid pollen. American Orchid Society Bulletin, 45:699-707. 5 1. Williams, J.G.,Wiams, AE., Arlo& N. 1978. A field guide to the Orchids of Britain and Europe with North Africa and the Middle East W. Collins, Sons & CO., Ltd., London. 52. Withner, C. L. 1985. Developments in orchid physiology. In: The orchids - Scientific Studies. Ed.: Withner, C. L. pp.: 129-169. Robert E. Krieger Publishing Company, Mabar, Florida. 53. Withner, C. L. (ed.) 1985. The orchids - Scientific Studies. Robert E. Krieger Publishing Company, Malabar, Florida. 54. Yukawa, T., Ando, T., Karasawa, K, Hashimoto, K. 1992. Existence of two stomatd shapes in the genus Dendrobium (Orchidaceae) and its systematic significance. American Journal of Boa379(8):946-952. Figure 3-1. Swollen germinating Orchis morio seed, note the bulging embryo (SEM 200x) Figure 3-2. The embryo of Orchis morio rupturing the testa (SEM 265x) Figure 3-3. The first rhizoids (mows) appear on the protocorm of Orchis morio (SEM 1OOX) Figure 34. The apical meristem (1) starting to develop on the protocorm of Orchis morio; (2) remnant seed-coat (SEM 100x) Figure 3-5. The conical meristematic dome (I) on the protocorm of Orchis morio. (2) round undifferentiated cells of the protocop (3) remnant seed-coat (SEM 1lox) Figure 3-6. Sunken appical meristem (Orchis mom; SEM 1SOX) Figure 3-7.The first red leaf appears (arrow) (Orchis morio; SEM 250x) Figure 3-8. Structures of the protocorm start to develop, the first stoma (arrow) appears (Orchis morio; SEM 1 18x) Figure 3-9. Young Orchis murio plantlet with photosynthetic ability (SEM 26.5~)

Figure 3- 10. Cross section of the root of Orchis morio with the mycorrhizal peiotons in the cortex (SEM 55x) Figure 3-1 1. Fungal infection at the base of a rhizoid of the root of Orchis morio (SEM 290x) Figure 3-12. Orchid type mycorrhiza (Corallorhim aioniorhiza, no outer Hartnig-net on the outer daceof the root, the mywrrhiza only present inside the root in the cortex cells; SEM 145x) Figure 3- 13. Pelotons in the root cortex of Corallorhim dontorhim (SEM 400x) 139 Figure 3- 14.- 17. The stages of the mycorrhiza in the root of Orchis morio : Figure3-14. Anastomization (SEM 485x); Figure 3-15. Peloton (arrow) formation (SEM 500~); Figure 3-16.Functioning peloton. (1) peloton, (2) starch grain, (3) microscopic ropes (SEM 400x); Figure 3-17.Degenerated peloton (1). (2) microscopic ropes (SEM 320x) 141 142 Figure 3-19. The new bud in the neck of the younger tuberoid of Orchis morio (SEM 14x) Figure 3-20. Cross section of the older tuberoid of Orchis morio. (1) stele, (2) vascular bundle, (3) rhizodemis (SEM 15x) Figure 3-21. Longitudinal section of the older tuberoid of Orchis morio. (1) stele, (2) rhizodermis (SEM 13x) Figure 3-22. Calcium oxalate needle crystals in the tuberoid of Orchis morio (SEM 400x) Figure 3-23. Starch grains (1) and slime materiaWsalep (2) in the tuberoid of Orchis morio (SEM 1250~) Figure 3-24. Starch aggregate ("pentamorio")in the tuberoid of Orchis mwio (SEM 385x) Figure 3-25. Individual starch grains in the root of Spimthes vemlis (SEM 2500~) Figure 3 -26. Individual starch grains in the root of C'ripedium amIe (SEM 2000x) Figure 3-27. Individual starch grains in the root of Gaiems qectdilis (SEM 1000~) 144 Figure 3-28. Starch aggregates in the root of HimantogIossum hircimm (SEM 1000~) Figure 3-29. Starch aggregates in the root of C)pripdium caIi$omim (SEM 933x) Figure 3-30. Starch aggregates in the root of Spiranihes vernalis (SEM 2000x) Figure 3-3 1. Bundle of calcium oxalate needIe crystals (Plaanthera hyperborea; SEM 1130x) Figure 3-32. Complex calcium oxalate crystal near some needle crystals in the root of Cypripedium caI~ontimm(SEM 1440~) Figure 3-33. Complex calcium oxalate crystal in the root of Cypripedium ade(SEA4 1130x) Figure 3-34. Complex calcium oxalate crystal in Piatanthertz hyperborea (SEM 1370~) Figure 3-35. Higher magnification of the complex calcium oxalate crystal fiom Figure 3- 34. Note the pardel needle crystals as they built up the complex form (SEM 9710~) Figure 3-36. Cross section of the root of C~npediumamle at the end of the vegetative season fiom its native habitat. No visible mycofthiza present, and the cortex is full of starch (arrow: rhoid; SEM 33x) 146 Figure 3-37. Cross section of the stem of Orchis morio; epidermis (1); chlorenchyma (2); sclerenchyma (3); vascdar bundle (4); parenchyma (5); central cavity or pith (6) (SEM 37x). Figure 3-38. Young stoma of Orchis morio with the remnant of the subsidiary/accessory cells (arrow) (SEM 1400~) Figure 3-39. Fully developed stoma fkorn the stem of Orchis morio (SEM 1000~) Figure 3-40. Degenerated stoma &om the leaf of Corallorhim dontorhiza (SEM 978x) Figure 3-41. Trichomes with spherical head cells fUed with oil &om the dace of the stem of C'ripedium ~4Iifornicum(SEM 1 13x) Figure 3-42. Wax structures from the surface of the stem of Corallorhiza dontorhim (SEM 5300~) 148 Figure 3-43. The structure of the flower of Orchis morio - generative parts: pollinium (I), rostellum (2), column (3), entrance of the spur (4), staminodium (5) (magnification: 8x) Figure 3-44. The structure of the flower of Orchis morio - vegetative parts: spur (I), bract (2), hood formed fiom the sepaIs and petals (3), lipflabellurn (4) (magnification: 4x) 150 Figure 3-45. The upper surface of the lip of Orchis morio (SEM 250x) Figure 3-46. The lower surface of the lip of Orchis morio with stomata and the braid-like edge (SEM 200x) Figure 3-47. The upper surface of the lip of Spiranthes vedis(arrow: mid-vein; SEM 55.7~) 152 Figure 3-48. The head of the pollinarium @oIlinium) of Orchis morio (arrow: segments built up &om pollen grains; SEM 67x) Figure 3-49. Viscidium fiom the pollinarium of Orchis morio (SEM 235x) Figure 3-50. A part of the caudicle and the upper daceof the viscidium of PZatanthera praeclara (SEM 59.1x) Figure 3-5 1. The structure of the mature viscidiurn of Platanthera praeclara. (1) single basal epidermal layer, (2) sticky glue (SEM 687x) 154 Figure 3-52. Pollinarium of Orchis morio. (1) poliinium; (2) caudicle; (3) viscidium (SEM 40x) Figure 3-53. PoUinarium of Spirmthes vedis(SEM 3 lx) Figure 3-54. PoUinarium of CoralZorhira odonforhiza (SEM 95x) 156 Figure 3-55. The cross section of the twisted ovary of Orchis morio before pollination (mow: empty curves of the placenta; SEM 67x) Figure 3-56. The placenta with the anatropous ovules of Orchis morio before pollination (SEM 200x) Figure 3-57. Ovule of Orchis morio before pollination. (1) micropyle, (2) outer integument (SEM 43 Ox) Figure 3-58. Longitudinal section of the ovule of Plafanthera praeclara. (1) embryo, (2) micropyle, (3) integurnentai single cell layer (SEM 450x) Figure 3-59. The integument covering the embryo and the developing elongated seed of Orchis morio (SEM 400x) Figure 3-60. Fully developed seed before desiccation (Orchis morio; SEM 300x) 158 Figure 3-61. The untwisted ovary/young fruit of Orchis morio after pollination, with developing seeds (arrow: the curves of the placenta filled with hygroscopic hairs; SEM 25x) Figure 3-62. The outer surface of the ripe fiuit of Orchis morio with the desiccated epidermal cells and degenerated stoma (SEM 800x) Figure 3-63. The placenta of Orchis morio with ripening seeds (arrow: hygroscopic hairs; SEA4 52x) Figure 3-64. The curve of the placenta of Orchis morio 6lled with hygroscopic hairs (arrow) (SEM 165x) Figure 3-65. The pIacenta of Galearis spectabilis with ripening seeds (arrow: hygroscopic hairs; SEM 30x) Figure 3-66. The placenta of Plafmihera praeclara with ripening seeds (arrow: hygroscopic hairs; SEM 34x) 160 Seed Morphology of Several Temperate Orchid Species with Special Emphasis on the Members of Orchideae (Orchidaceae)

Abstract

There is only a limited amount of detailed information available on the structure and fluface pattern of orchid seeds. A better knowledge of the structure of these microscopic seeds can help identify new taxonomic evidence as well as help us understand several phenomena that occur during germination. In this study extensive scanning electron microscope (Sw observations were conducted on the seeds of more than 120 temperate orchid species (fiom 46 different genera). The seeds were legally field collected, received from botanical gardens and other institutions or taken fiom herbarium specimens. Dry, ripe and properly stored orchid seeds were directly mounted on aluminum stubs and sputter-coated with 30 nrn of gold, since they did not require iixation, dehydration and critical point drying processes. The samples were observed with SEM at 15 or 20 kV. The size of even these microscopic seeds varied widely, with the length of the smallest ones -200 prn (Spiranthes qp.) and the largest ones sampled reached -1700 pm (Cypripedium, Epipacts spp.) in length. Both quantitative and qualitative data were collected and based on these characteristics, detailed descriptions of the seeds of the different orchids were prepared. Several varied fascinating surface patterns and shapes were found among the dserent species. These differences were specific not only on the level of higher taxonomic units, but sometimes were distinctive even for species or subspecies. Introduction

Orchid seeds are among the smallest seeds of the plant kingdom so it is very dif5cult to study their detaded anatomical structure with the naked eye. Throughout the orchid Literature there are several illustrations of seeds (Beer, 1863; Burgee 1936; Ziegenspeck, 1936; Stoutamire, 1963; Wiesmeyer and Hofkten, 1976), but these usually do not provide enough detailed information to be usefid either for identification or other taxonomic purposes. Some detailed information has been published by Clifford and Smith (1969), Barthlott (1976), Ardim, Michaud and Heday (1979, 1980), Healay, Michaud and Arditti (1980), Ziegler (1981), Tohda (1983, 1985, 1986), Chase and Pippen (1988), Cameron (1995) and Molvray and Kores (I995), and one of their most important conclusions is that the seed characteristics of orchids can be a great taxonomic help for classification. Dressler (1993) in his most recent general overview of the orchid family briefly summarized a descriptive system of orchid seeds throughout the family based on Ziegler (1981) and Ziegler and Barthlott's (unpublished) research. To date this work is the most comprehensive work about the seed morphology of the family. They differentiate 20 different seed types based on five main characteristics: overall seed shape, relative elongation of some cells in the testa, height and sculptwing of cell walls and features of cell wall adhesion (mentioned in Molvray and Kores, 1995). Nevertheless most of the Literature available describing orchid seeds on the species level deals only with pIants fiorn the tropics or fkom the temperate zones of America and Australia. From the above mentioned references only Ziegler (1981) and Barthlotr and Ziegler (1981 and unpublished) worked with temperate orchid species fiom Europe in more detad, but unfortunately it is very hard to access these references, and according to Molvray and Kores (1995) many of the results were not published. No literature has been found on the structural details of at least half of the seeds of the -120 orchid species discussed in this chapter (Table 41). Two basic approaches are prevalent in the orchid seed literature. The majority of the available references prepared their descriptions based on qualitative data and supplemented them with more or less quantitative data (Clifford and Smith, 1969; Barthlott, 1976; Arditti, Michaud and Healay, 1979, 1980; Healay, Michaud and Arditti, 1980; Ziegler, 1981; Tohda, 1983, 1985, 1986;) On the other hand more recently Molvray and Kores (1995) emphasized the importance of pure quantitative methods. This may provide less subjective analysis, but it would appear to be only useli for wider taxonomic purposes and not for identification on the species level. The most unique characteristics of orchid seeds, such as the surface pattern of the testa cells and seed shape, are qualitative characteristics and even if their definition is subjective because of the use of descriptive terms, these characteristics are indispensable for 111 determination. To obtain detailed information about the morphological features of temperate orchid seeds it is therefore important to prepare both a detailed qualitative and quantitative database. The typical mature orchid hit is a dry capsule with millions of dry seeds. The seeds are usually extremely small, light-weight and lack endosperm. They consist of only an undifferentiated embryo and a seed coat which develops from the degenerated integumental Iayers (Veyret, 1985). The number and size of the cells of the testa are very typical for each species and the surface pattern of each cell of the seed coat has a significant importance in classification (Dressier, 1993). The major goal of the research in this chapter was, therefore, to provide specific information about the seeds of different temperate orchid species that could be used for identification purposes on the species level, which of course can be useful for evaluating higher taxonomic relationships, too. The major difference between the work described in this chapter and the majority of other available references (except Arditti et al., 1979, 1980; and Healay et al., 1980), is that the work described in this chapter mainly focuses on the seed structure of the individual species while others do not emphasize the species- speczc features and only concentrate on the characteristics useful for higher classification. Materials and Methods

Seeds of more than I20 orchid species (representing 46 different genera) were legally field collected, received fiom botanical gardens and other institutions or taken from herbarium specimens (Table 4-1) and stored in small paper envelopes at 4OC. Dry ripe and properly stored orchid seeds did not require fixation, dehydration and critical point drying processes so they were directly mounted on aluminum stubs and sputter coated with 30 am of gold using a Denton Vacuum Desk II Cool Sputter Coater (in US) or an SCD 040 Balzers Sputter Coater (in Hungary). Samples were observed with Cambridge Stereoscan 90 (in US) or Tesla BS 300 (in Hungary) scanning electron microscopes (SEM) at 15 or 20kV respectively. No differences related to the two different equipment setups were noted. Each seed sample was examined at a minimum of six or seven different magdications (3k 60% loox, 250~500% 1000x, 2000~)and photographs were taken in each case. The observations took place at the Central Research Laboratory of the University of Horticulture and Food, Budapest, Hungary and at the Laboratory For Electron Microscopy, School of Biological Sciences, University of Nebraska-Lincoln, USA

The following observations or measurements were made for each sample (minimum 5 to 10 seeds per sample): - quantitative characters: average seed width; average seed length; average median cell length; the ratio of average seed length to average median cell length; testa cell number/seed. Testa cell numberlseed estimate was obtained by counting the visible seed coat celIs of individual seeds on the SEM images, then multiplying that number by two. - qualitative characters: presence of intercelluIar gaps; arrangement of testa cells; seed overall shape; testa cell shape; pattern on perichal walls of the testa cells; special characteristics of the anticlinal walls of the testa ceUs (such as relative size of ridging, presence of arching). Because of the limited length of this chapter, detaded descriptions and quantitative data (Table 4-2) are included only for the species successfblly germinated in vitro (Chapter I), and some closely related species. The seed morphology of these dzerent species is described in taxonomic order based on Dresslets (1993) system. Further observations by SEM are currently under investigation.

Results and Discussion

Overall observations

- Size: Even the size of these miniature orchid seeds varied greatly. The length of the smallest seeds was around 200 pm (Spiranthes dilwialis, Figure 48a; Via& coenrlea, Figure 4-12d) and the length of the biggest ones ranged between 1600-1700 pm (Epipactis thunbergii, Cpripedium acauie).

- Shape: The shape of the observed orchid seeds can be separated into two major categories. The first category was the group of seeds with an irregular amorphous seed, in which case the shape could not be defined as a distinct geometrical shape. The second major category was when the seeds have a distinct defite geometric shape. There were several different types inside the definite shape category: a) spherical b) Worm c) fbsiforrn (the medial section was wider than the part of the seed close to the apical and chalazal regions), and the variations of this, such as oval, elongated balloon, and flask shape (with a distinctly narrower neck at the chalazal end). CWord and Smith (1969) proposed five different basic seed shapes in orchids, but they do not use verbal terms, only provided schematic drawings to introduce them. Three of these groups are basically the same as it is observed in this work, resembling the Worm, fbsiform and flask shape. Working with oncidioid seeds, Chase and Pippen (1988) found that the basic seed shape is fbsiform, and they considered this as a hndamental characteristic. They used the size of the seeds and special surface characteristics for hrther identification purposes, but these characteristics were not applicable to the species examined in the research reported here because of the extreme taxonomic distance inside the family.

- Special characteristics of the anticlinal walls of the testa cells: a) "Widow framen type surface pattern which appeared as a membraneous zone attached to the vertical side of the anticlinal walls (Figure 4-1). b) The shorter anticlinal walls were raised resembling an "arch-typen appearance (Figure 4-1)

C) "Grainn type surface pattern on the edges of the anticlinal walls (Figure 4-3) d) Relative height of anticlinal walls: 1. High: It was relatively easy to measure the height of the antichal walls. The vertical side of the anticlinal walls was usually more than one fourth and less than one third of the width of the periclinal walls. 2. Medium high: It was easy to see but difficult to make exact measurements of the vertical side of the anticlinal cell walls (the height of them was less than a quarter of the width of the periclinal walls). 3. Low: The vertical and the horizontal sides of the anticlinal walls were almost the same height; the anticlinal walk appear as distinct reticular lines on the surface of the seed. The relative height of the anticlind walls of the testa cells was a characteristic which was very hard to use consistently because of varying subjective evaluation by different observers, but it still may be helpful, especially when comparing different species, or based on some very characteristic reference specimens.

- Surface patterns on the periclinal walls of the testa cells: a) swirling lines (Figure 44) b) dots c) parallel lines (which can run diagonal or parallel with either the shorter or longer anticlinal walls and sometimes there are some additional shorter dividing lines between them) (Figures 4-5 a, b, c and d) d) irregular or longitudinal ridges (Figures 4-6 a and b) e) no sudace pattern on the perictinal walls of testa cells (Figure 4-7) Similar terms to the above mentioned types of surface patterns have been suggested by Ziegler (1981) for the relative height of the anticlinal walls without def*ming the appearance of them. Molvray and Kores (1995) listed and described several different qualitative characters, such as membrane-covered adjacent walls (one version of this is the "window framewtype pattern (Figure 4-1) described in this current research), wall height, wall sculpturing (in ths work calIed "surface pattern"), hardness of testa They emphasize that these characteristics should be used with caution and they try to build their evaluation mainly on qualitative characteristics. As an opposite opinion Healay et al. (1980) stated that "the presence and nature of reticulation (of testa cells) are good taxonomic markers, at least at the generic level". Averyanov (1990) had similar conclusions, finding sigdicant relationships between the appearance of four main surface pattern types and the taxonomic groups within the genus Dactjdorhiza. The surface patterns of the pericIinaI wails and the features of the anticlinal walls of the testa cells were called "wall thickening types" in the work of Clifford and Smith (1969). In that category they found "thickening the vertical walIs of the testa", "discontinuous thickening of the horizontal walk" and "beaded Iongitudind walls" as important characteristics of the testa, but these terms are too general to use in detaiIed seed morphology work Chase and Pippen (1988) and Averyanov (1990) died the above mentioned characteristics as "sculpturing of the testa1'. Where possible the morphological terms which were previously used by Arditti et aI. (1979, 1980), Healay (1980) and Ziegler and Barthlott (described in Dressier, I993 and in Molvray and Kores, 1995) were used in this work. Because in severaI cases there were no previously existing suitable terms availabIe for description, new terms were used such as the "window fiarne" type surface pattern attached to the anticlinal walk, and several surface pattern types such as swirling or pdlel lines on the periclinal walls of the testa cells. Detailed descriptions

The most prominent difFerence observed between the temperate and tropical orchids was the different relative appearance of the anticlinal and periclinal walls of the testa cells. In the case of tropical orchids (five representative genera) the anticha1 walls of the testa cells were dominant in appearance, they were raised and very dense so the periclinal walls were almost invisible. In contrast in temperate species the periclinal walls were the dominant visible part of the outer surface of the seed coat.

Subfamily: Cypripedioideae C'ipedium seeds (figures not shown) were among the largest seeds observed, ranging between 800-1700 pm in length. The testa consisted of more than 100 cells, commonly more than 200 cells (Table 4-2), and the median testa cells were only slightly or not longer than the apical or chalaza1 cells. The individual testa cells were rectangular shaped with more or less sharply angled comers and the anticlinal walls were low or medium high. The average length of the seeds of Cypripedium candidurn was around 800 pm, which was noticeably shorter than in the case of any other C'n'pedium species observed. C'ripedium candidurn seed shape was fksiform, the testa cells were arranged into longitudinal files, the ridges formed fiom the anticlinal walls were low to medium high, and there was no surface pattern on the periclinal walls. The shape of the seeds of Cypripedium cdceolus were similarly firsifom, but they were a little more eIongated. There were distinct differences between the length of the seeds among the dflerent varieties, ranging between 1600-1700 pm in the case of C'ripediurn calceolus var. pubescens, 1 150- 1300 pm in the case of Cypn'pedium calceolus var. parwJorurn and around 1100 pm in the case of the European Cypripedium caZceoIus var. acalceolus (Table 4-2). In other characteristics the three varieties were quite similar, with the testa cells more or less arranged into longitudinal files, the ridges fiom the anticlinal walls low to medium high, and there was no surface pattern on the periclinal wds. The appearance of the seeds of Cypripdium calvornicum was very similar to the seeds of Cypripedium cdceolus, with the length around 1500 pm, but the seeds were a little wider and the arrangement of the testa cells was not so obviously longitudinal. In the case of the last two observed Cypripedium species, the shape of the seeds was somewhat different fiom the previously mentioned ones. One or both ends of the seeds tapered to be thinner than the median part. In the case of Cyprrpedium reginae only the chalazal end of the seed tapered from the medial part. The seed length ranged between 1300-1400 prn and the seeds were flask shaped with a long neck at the chalazd end. There was no surface pattern on the periclinal walls of the testa but there was some "window fiarne" type pattern attached to the anticlinal walls. In the case of Cypripedium acuule both the suspensor (chalazal) and the apical end of the seeds gradually became thinner, the seed shape close to filiform. The length of the seeds ranged between 1600-1700 prn and there were irregular ridges on the periclinal walls of the testa cells and the anticlinal walk were longitudinally folded (see again Figure 4-6a).

Arditti et al. (1979) presented detailed observations about all the above described Cpripedium species including SEM images. The details of that work are mostly consistent with these observations, although their quantitative characteristics (especially seed length) of Cypripdium calceolus vur. pupubescens and Cypripeedium californic11m are significantly different fiom data presented in this chapter. Also they found that Cypripediurn ucuule was distinctly separated from the other Cypripedium species in seed characteristics, but in work presented here C'pediumacarle and Cypripedium reginae appeared to be together in a separate group than the other species observed in the genus. Other authors do not mention individual descriptions, even if they mention a specific C'ripedium species in their work (Clifford and Smith, 1969). Subfamily: Spiranthoideae The seeds of Gwdyera repens (tribe: Cranichideae, subtribe: Goodyerinae) (figures not shown) had a very distinct appearance because there were rounded triangular gaps at the locations where the comers of the anticlind walls are joined. The median testa cells were only slightly or not longer than the apical or chald cells and the testa consisted of around 200 cells. The individual testa ceUs were irregularly rectangular or polyangular shaped with more or less rounded comers. The shape of the seed was elongated balloon with a length of 700-800 pm and there was no surface pattern present on the periclinal walls. Similar observations were published by Heday et al. (1980) and Molvray and Kores (1995) about this species but in a less detailed way.

The seeds of different Spirmthes (tribe: Cranichideae, subtribe: Spiranthinae) species had some similarity to the previously mentioned C'ripedizim and Goeera genera because the median testa cells were only slightly or not longer than the apical or chalazal cells. However, Spiranthes spp. seeds were much smaller, with an average seed length (distance between the chalazal and apical ends) that was always less than 500 pm. The smallest seeds in the genus were those of Spiranthes diIuviaZis, with an average length between 200-250 pm (which is one of the smallest among all of the orchid seeds observed, Table 4-2) (Figure 4-8a). The slightly elongated seeds of this species had some surface pattern on the periclinal walls of the testa cells, with parallel lines on the periclinal walls which were also parallel with the shorter anticlinal walls. In the case of both Spiranlhes cenruo and Spiranthes vemlis (Figure 4-8b) the seed length ranged between 300-400 pm and shape of the seeds was close to hiform. Although the seeds were very similar, there were some differences between these two species in the surface pattern of the periclinal walls of the testa cells. Both species had parallel lines on the periclinal walls which were almost parallel with the shorter anticlind walls, but in the case of Spiranthes cemua these lines were more dense. Also there were some smd dots on the periclinal walls, which were not seen on the surface of Spimhes vemlis. Another typical feature of Spiranthes cemwas that multiple embryos were sometimes present in the seed (as was also previously reported by Schmidt and Antfiger, 1992), making the shape of the seed more irregular. Details about one or more of Spirmthes species were published by Healay et al. (1980), Molvray and Kores (1995) and about the genus (Tohda, 1985; Dressier, 1993), and they are consistent with observations reported here. However, this is the first description of Spiranthes dilwialis seeds. This species was recently separated fiom the Spiranihes cenrua complex (Sheviak, 1982, 1984) and the distinct seed morphology described here supports its new taxonomic position.

Subfamily: Orchidoideae The tribe Orchideae typically had median testa cells that were more elongated than the apical or chalaza1 cells. Also the testa consisted of less than 200 cells, commonly less than 100 celIs. Based on the average length of the seeds, there were some more elongated seeds with an average length of more than 500 pm, and others had a more compact shape when the length of the seeds was less than 500 pm. These characteristics are probably not enough definitive, but help organize the genera observed into a more convenient system.

The seeds of Barlia r~be~una(tribe: Orchideae, subtribe: Orchidinae) were hiform with somewhat oval shape and the ratio between the length and the medial width of the seed was approximately 3.0. No surface pattern was present on the anticlinal walls but there was a distinct surface pattern present on the periclinal walls of the testa cells. This pattern had parallel lines that ran close to a diagonal direction. The seed only had a few testa cells (226) and the length of the seeds ranged between 460-520 pm (Table 4-2).

Chamorchis alpina (tribe: Orchideae, subtribe: Orchidinae) had short, small oval seeds, with a ratio between the Iength and the medial width of approximately 1.6 (length/width), and the length of the seeds ranged between 250-300 pm (Figure 4-9b). The testa consisted of more than 60 cells, generally around 80 (Table 4-2). This species had no distinct surface pattern present on the periclinal walls. The oval seeds of Himantoglossum hircimm (tribe: Orchideae, subtribe: Orchidinae) were also rather smalI with a lengthlwidth ratio of around 2.5. The length of the seeds ranged between 300-400 pm. The testa had a conspicuously small amount of cells, usually around 38 (Figure 4-9a). There were only one ring of 69elongated cells in the median section and rest of the cells were much smaller and located close to the two polar regions (Table 4-2). The periclinal walls of the testa cells had surface pattern of parallel Lines, but this pattern was less noticeabie than the medium high to high ridges formed fiom the pcriclinal walls, which were the most dominant surface feature of these seeds.

The seeds of Nign'ella nigm (tribe: Orchideae, subtribe: Orchidinae) were among the smallest seeds in the tribe of Orchideae. The distinct squat vase shaped seeds were very short, and had a length/width ratio of %l.4.The testa consisted of no more than 50-60 cells per seed. The fen& of the seeds ranged between 300-350 prn (Table 4-2), with no distinct surface pattern on the periclinal walls.

All seeds of the species examined of the Ophrys genus (tribe: Orchideae, subtribe: Orchidinae) were relatively small and more or less flask shaped with a distinct narrower neck to the direction of the chalaza1 end but there were distinct differences in the size of the seeds and the in the surface pattern on the periclinal walls of the testa.

Ophrys qhegodes had the smallest seeds observed in this genus, with a length of around 380 pm. The shape of the seeds was more compact because the neck of the seed was shorter than in the other two species, and there was no surface pattern present on the periclinal walls of the testa cells (Figure 4- 1Oa).

The periclinal walls of the testa cells of Ophrys lutea exhibited surface pattern of faintly dense parallel lines which also were parallel to the shorter anticlinal walls. The average length of the seeds was 400 pm and the testa consisted of around 40 cells. Ophrys ficlj7ora seeds had very definite, strong parallel lines present as a surface pattern on the periclinal walls, which also were more or less parallel to the shorter anticIinaI walls (Figure 4-5a). The average seed length was 500 pm and the testa was made of around 60 cells (Table 4-2). There were some cracks and intercellular gaps present on the seed-coat.

The seeds in the genus DactyIorhiza (tribe: Orchideae, subtribe: Orchidinae) were larger than the foregoing in this subfamily with an average length of more than 600 pm. The seeds had typical elongated flask shape and the cells were often arranged into longitudinal files. The majority of the seeds were very similar to the ones in the Orchid genus but the seeds were never twisted and the number of cells along the longitudinal axis of the seed was more than six (in the Orchid genus this number is always less than five); (also see Tohda, 1983 and Averyanov, 1990). The length of the seed neck and the surface pattern on the periclinal walls helped distinguish the seeds of different species in the genus. The seeds of Dactylorhiza fuchsii ssp. sooiana, Dactylorhiza sambucina, Dactylorhiza maculata and Dactylorhiza saccifera showed quite simiIar surface patterns of swiriing limes on the surface of the periclinaI walls of the testa cells. The seeds of Dactylorhizafirchsii ssp. sooiana were typically flask shaped, with a distinctly narrow neck (Figure 4-10d). There were faint swirling lines on the periclinal walls of the testa, resulting in a slightly reticulate appearance of each testa cell. The length of the seeds ranged between 600-700 pm and the testa had around 76 cells (Table 4-2). The anticlinal walls were distinct, but low. The seeds of Dactylorhim sumbucina were flask shaped, but the neck of the seed was onIy slightly elongated and relatively wide (Figure 4-10c). The swirling lines were very dominant on the periclinal walls, resulting in a very distinct reticular appearance of the testa cells. The average length of the seed length was 650 pm and the testa had around 90- 95 celfs (Table 4-2). The anticlinal walls were low to medium in height. Dactylorhiza maculata had long seeds, ranging between 800-900 pm, and the shape was elongated flask (Figure 4-1 lc). The testa consisted of about 95 cells. There were distinct swirhng lines on the periclinal walls of the testa cells, some of them somewhat parallel with the shorter anticlinal walls. The anticlinal walls were distinct, but low. The seeds of DuctyIorhiza succifera were very similar in overalI appearance (shape and sufiace pattern) to the seeds of Dactyorhiw maculata but the swirling lines on the periclinal walls of the testa cells were only slightly raised and the seeds were much smaller. The seed length ranged between 650-750 pm and the testa had around 120 cells (Figure 4-1 Id). The next two species &om the Dactylorhim genus (D.in-a and D. majdlis) were very different in appearance from the others, and they were clearly quite different fkom the general type in the Orchideae because there was a less distinct difference in shape between the cells at the median and at the polar regions which were more rounded rather than elongated. Also the testa cell number was higher, ranging between 180-200, and the cells were more disorganized. The shape of the seeds were still typically flask shaped as in the other seeds in the genus. The seeds of both DactyIorhiiz~inamata (Figure 4-12a) and Dactyllohim majalis had a similar size, with lengths ranging between 650-700 pm, and there was no significant surface pattern on the periclinal walls of the testa celIs. The seeds of Dactylohizcl majaiis were a little narrower at the median section, and there were some "window hentype patterns attached to the periclinal walls of the testa cells.

The seeds of Galemis ~pectabilis(tribe: Orchideae, subtribe: Orchidinae) were also similar to the seeds of the Orchis genus, but they were more elongated flask shaped, sometimes close to fiiiform (Figure 4-12b). The embryos were readily visible in the middle of the seed. The length of the seeds ranged between 750-800 prn and the testa had around 85-90 cells (Table 4-2). No surface pattern was present on the periclinal walls of the testa cells and the anticlind cell walls were thin but distinct and medium high to high.

The Orchis genus (tribe: Orchideae, subtribe: Orchidinae) had the typical seed type of the Orchideae because the median testa cells were more elongated than the apical or chalaza1 cells and the number of cells was always under 100. The number of testa cells along the longitudinal axis of the seed was always less than five. The seeds in each case were more or less flask shaped with a distinctly narrow neck at the chalazal end and the cells of the testa were often arranged into longitudinal files. In several cases the neck of the seed was twisted. The seed size varied greatly in the genus, from an average length of 360 pm to an average length of 850 pm, depending on species (Table 4-2). The periclind walls of Orchis pupurea were so thick that it was dficult to observe the embryo through them. The antichal walls were low or medium high but they showed a very distinct definite line. The length of the seed ranged between 400-450 prn and the testa consisted of 45-50 cells (Figure 4-9c). The rest of the species observed in the genus had seeds with more or less translucent periclinal walls, so it was easier to see the embryo. The typical flask shaped seeds of Orchis mililmis were relatively short, with an average length of 360 pm and the testa consisted of only approximately 40 cells (Figure 4- 9d). There was no surface pattern present on the periclinal walls of the testa. The anticlinal walls were medium high. Orchis morio had pronounced parallel lines on the periclinal walls of the testa cells which were running diagonally into the longer anticlinal walls (see again Figure 4-5c). The elongated typical flask shaped seeds were 550-600 pm Long, with a testa of around 55-60 cells (Table 4-2) and the anticha1 walls of the testa were medium high (Figure 4-10b). The seeds of Orchis rnascula. Orchis coriophora, and Orchis ZuxiZflora were distinctly elongated flask shaped and the entire testa or the neck of the seeds was often twisted. Among these three species, Orchis mmczila had the longest seeds, ranging between 750- 850 pm, with a testa around 40-45 cells. There was a surface pattern of parallel lines on the periclind walls of the testa cells, which were somewhat diagonal to the anticlinal walls. The anticlinal walls ranged from medium high to high. The length of the seeds of Orchis coriophora ranged between only 600-650 pm and the testa had around 60 cells. There were dense parallel lines on the periclinal walls of the testa, almost parallel with the longer anticlinal walls (see again Figure 4-5b). The subspecies of the species, Orchis coriophora ssp. fragm had distinctly longer seeds (approximately 750 pm) but the testa consisted of only around 40 cells (Figure 4-1 lb). The surface pattern was the same as in the case of the basic species. The third species with twisted seed neck was Orchis Imrrflora, which had the most eIongated thin seeds, but they still resembled a flask shape. The length of the seeds was quite long, ranging between 800-850 pm and the testa had only around 36 cells (Figure 4- 1la). There were parallel lines on the periclinal walls of the testa, diagonal to the longer anticlinal walls, but this surEdce pattern was not always noticeable. The seeds of Orchis lmnflora s~p.pZusb73 were quite different, not twisted, with a typical flask shape and only 450-550 pm in length. The surface pattern was similar to the basic species.

The most comprehensive work about orchid seed morphology by Ziegler and BarthIott could provide more detaiied information as a basic reference, but the results have not been pubIished yet and only limited information is available about it through the writings of other authors @ressler, 1993; Molvray and Kores, 1995). Tohda (1983) and Molvray and Kores (1995) published some species-specific information about the seeds of Galems spectubilis which is consistent with the description above. Tohda (1983) found that the appearance of the surface pattern of the testa cells of different Dactylorhim species can vary geographicalIy, but it still is considered consistent enough to use it for classification. Averyanov (1990) specifically reviewed the Dactylorhira genus and in his work he included several stmctural and morphological details of the seeds in the genus. He concluded that the specific morphological characters of the periclinal walls of the testa were significantIy connected with the different taxonomic groups with the genus. Based on these characteristics he separated four basic seed type groups in the genus: the Dactylorhiza incmta, the Dactylorhiza maculuta, the Dactylorhim romana, and the Dc~ctyIorhiraarisata types. Although this grouping fits observations reported here, there are some other important and distinct characteristics (such as the shape of the testa cells, number of testa cells) of this genus which are important when dividing the seeds into separate types.

The genus of Platanthera (tribe: Orchideae, submbe: Orchidinae) had very diverse seed types. Among the described species in this work only the European Platmthera bifolia showed the typical seed form of the tribe; the seeds of the other American species examined were quite different. This difference may be explained by the relatively big taxonomic distance among the three species described here, or because the exact classification of several species in the genus is still not as clear as it is for other previously described species @ressler, 1993). The elongated flask shaped seeds of Platanthero bifoliu had distinct parallel lines on the periclinal wds of the testa cells as a surface pattern. These parallel lines were strictly paralIel with the shorter anticlinal walls of the testa cells. The length of the seeds ranged between 600-700 prn and the testa included around 60 cells (Table 4-2). The anticlind walls were medium high, The seeds of Platanthera praeclura were fusiform with a somewhat ovaI shape. Although there was no surface pattern present on the periclinal walls, this species was the only species among all of the observed temperate species which had a very distinct surface pattern on the relatively high anticlinal walls of the testa cells (Figure 4-8c). This special character appears as unique luminescent, translucent spots on the ridges formed £iom the anticlinal cell walls. The length of the seeds was quite small (around 300 pm) and the testa consisted of approximately 60 cells (Table 4-2). The third species, Platanrhera hyperborea also displayed very different seed characteristics. The testa consisted of around 140 cells and these cells were apprordmately the same size over the entire surface. The oval, short balloon shaped seeds ranged between 700-750 pm in length. There was absolutely no surface pattern present on the periclinal walls of the tern cells and the anticlinal walls were very low and longitudinally folded, giving a net-lke appearance to the seed surface (Figure 4-8d). Healay et aI. (1980) provided some generalized information about the seed morphology of the temperate group of the PIutunthera genus. However, they based their conclusions on observations fiom three relatively close species (PIatanthera diIalufa, P. hperborea, and P- saccuta), so their general statements are inadequate to cover the diversity present in the genus, as shown clearly in these representative species (Plutmthera bifolia, P. berborea and P. praeclara). Genm-udip&fIa (tribe: Orchideae, subtribe: Habenariinae) has very unusual seeds, especially because of the intensely eminent surface pattern. Parallel lines on the periclinal walls make a dominant reticular surface pattern on each seed-coat cell. The paralIel lines are more or less parallel with the shorter anticha1 walls and they often have some additional shorter dividing lines between them (see again Figure 4-56). Cracks on the periclinal walk were very common, especially near the surface pattern lines and near the anticha1 walls of the testa cells. This species had relatively large seeds for this subfamily (Orchidoideae). The seed length ranged between 900-1000 pm and they were elongated fusiform to elongated flask shaped. The testa consisted of around 60 celIs (Figure 4-12c).

In the Orchidoideae and Spiranthoideae, Ziegler (1981, based on the report by Molvray and Kores, 1995) described three different seed types: the "Orchis typen the "Disa-Diuris type" and the "Goodyera type". A1I of the species described herewith, except Goorjera repens, belong to the "Orchid type". This grouping appears to be too general, especially because of the differences between Spiranthes seeds and the other seeds of the Orchideae. Molvray and Kores (1995) revised this system and defined four basic groups in these two subfamilies, the "orchidoid", "diuroid", "spiranthoid" and "goodyeroid" types. Although this grouping is closer to describing groups observed here, the definition of the groups is not always consistent with observations in this work. They define the "orchidoid" seeds as aiways having 20 or fewer testa cells, and most of the seeds observed here as typicaI orchid type seeds won't fit into their category, but rather into their "diuroidn type. Probably this difference is because only a few among the 95 species by MoIvray and Kores (1995) are common with the more than 120 species in this study. On the other hand the description of their spiranthoid seed group is basically the same as observed here and this is probably because that the few mutually observed species almost aU belong in this tatter group. Because the primary goal of their paper was to analyze systematic relations in the two subfamilies, they unfortunately did not publish individual observations about seeds that they observed. Subfamily: Epidendrioideae As a point of reference the seeds of some tropical species fiom the subfamily of Epidendrioideae were also observed. Of course this examination did not give a fU picture about the seed structure of the huge and diverse group of tropical (commonly epiphytic) orchids, but provided an overalI view which will present several difEerences compared to the temperate terrestrial species. As mentioned at the beginning of the detailed seed descriptions, the most obvious distinction between tropical and temperate species was shown in the more dominant and dense appearance of the anticiinal walls of the testa cells of tropical orchids, which in severaI cases made the periclinal walls a minor factor in the description of the seed. The seeds of CattIeya aclandue, Dendrobiurn cnmrenahrm and Vanda coerulea had a more compact fixsiform shape (the medial section wider than the the apical and chald regions). Cattleya uclandae seed length ranged between 450-500 pm (Table 4-2). The apical end of the seed was eIongated, narrow and terminated in a sharp point. The shorter anticlinal walls of the testa cells were raised, resembling an arch in appearance. The anticlinal walls were very dense, which made it diEcult to observe the periclinal walis. The average length of the Dendrobium crumenahrm seeds was 300 pm (TabIe 4-2). The testa cells were arranged in longitudinal files. The apical end of the seed was twisted, elongated and much narrower than the rest of the seed, resembling a little tail-like structure. The longitudinal anticha1 walls of the testa cells were very dense and they had some "grain" type surface pattern (see again Figure 4-3). No surface pattern was present on the periclinal walls of the seed-coat cells. The seeds of V& coenrlea ranged between 180-210 pm in length (Table 4-2) and the shape of them was short fusifom. The testa was twisted throughout the length of the seed. The anticlinal walIs were very densely arranged and had longitudinal ridges. The periclinal walls were mostly invisible (Figure 4-12d). Epidendnrm nocfurnum seeds had an average length of 600 pm (Table 4-2). The Iong Worm shaped seeds were often siightly twisted. The shorter anticlinal walIs were a IittIe raised, resulting in an arch type appearance. The periclinal walls were densely patterned with small spots. Phafaenopsisf&ciuta had relatively small seeds, averaging 450 pm in Iength (Table 4- 2), and they had elongated with iong filiform shape. The shorter anticlinal walls of the testa were raised, resulting in a very distinct looping arch type appearance (see again Figure 4-2). The longitudind anticha1 walls were very dense and the periclinal walls sometimes invisible. In cases where the perichal walls were visible on the surface, they were patterned with short parallel iines resembling a ladder in appearance. Wiesmeyer and Hofiten (1976) published SEM images of the testa of a Phalaenopsis , but other than this they gave no description of the seeds and gave no specific terms and data that couId be usehl for identification purposes.

Conclusions

Observations of several of the orchid seeds described in this chapter have provided new information about these species. In most cases, the orchid testa showed such specific characteristics that the species can be identified by seeds alone, even without other plant samples. Several important characteristics can be identified with a good quality stereo or light microscope after obtaining the detaded standard information by SEM. From the practical horticultural viewpoint, this could be especially usefil when seeds provided for propagation purposes have a questionable identity, because the time between seed sowing and the mature stage (when the plant is suitable again for identScation) can be several years. Knowing the correct identity of the species can help researchers provide species- or genus-specific optimal conditions during culture. Of course these characteristics have an even more important taxonomic value for each individual species. Usually one given genus shows overall uniform seed structure, especially in quantitative characters and these characteristics are relatively homogeneous within a single subtribe. As was previously also suggested by other authors (Clifford and Smith, 1969; Chase and Pippen, 1988; Mofmay and Kores, 1995), seed morphology can be a helpfbl tool for classification purposes in the Orchidaceae, because of the conservative and phylogenetically informative characteristics of orchid testa. Literature Cited

1. Arditti, J. 1980. Scanning Electron Microscope Photographs of Orchid Seeds. American Orchid Society Bulletin, 49(8):868. 2. Arditti, J., Michaud, J. D., Healay, P. L. 1979. Morphometry of orchid seeds. I. PaphiopediIurn and native California and reIated species of Cypripedium. American Journal of Botany, 66(10): 1128-1 137. 3. Arditti, J., Michaud, J. D., Healay, P. L. 1980. Morphometry of orchid seeds. II. Native California and related species of Calypso, CephaIanthera, Corallorhiza and Epipads. American Journal of Botany, 67(3):347-360. 4. Averyanov, L. V. 1990. A review of the Genus DactyZurhiur. In: Arditti, J. (ed.) Orchid Biology - Reviews and Perspectives, V. pp.: 159-206. Timber Press, Portland, Oregon. 5. Averyanov, L. V. 1983. Genus Dactylorchiza Nevski (Orchidaceae) in the USSR. Dissertation. Bot. Inst. Ac. Sci. USSR Leningrad. 6. Barthlott, W. 1976. MorphoIogie der Samen von Orchideen im Hinblick auf taxonomische und hnktionelle Aspecte. In Proceedings of the 8th World Orchid Conference, Fradcfbrt, pp.:444455. Deutsche Orchideen Gessellsch& Frank&rt. 7. Barthlott, W., Ziegler, B. 1981. Mikromorphologie der Samenschden aIs systematische Merkmal bei Orchideen. Beric hte Deutsche Botanische GesseIlschafl, 94:267-273. 8. Beer, 1. G. 1863. BeItrage zur Morphologie und Biologie der Fade der Orchdeen. Cart Gerold's Soh, Vienna, Austria 9. Burgee K. 1936. Samenkeimung der Orchideen und Entwicklung ihrer Keimpflanzen, mit Anhang tiber praktische Orchideenanzucht. Veriag von Gustav Fischer, Jena. 10. Cameron, K. M., 1995. Seed morphology of vanilloid orchids. American Journal of Botany (Abstracts), 82(6) : 15. I 1. Chase, M. W., Pippen, J. S. 1988. Seed Morphology in the Oncidiinae and Related Subtribes (Orchidaceae). Systematic Botany, 13(3):3 13-323. 12. Clifford, H. T., Smith, W, K. 1969. Seed morphology and classfication of Orchidaceae. Phytomorphology, 19:133- 139. 13. Dressier, R L. 1993. Phylogeny and Classification of the Orchid Family. Dioscorides Press, Portland, Oregon. 14. Healay, P. L., Ardim, J., Michaud, J. D. 1980. Morphornetry of orchid seeds. III. Native CaKornia and related species of Goodyera, Piperia, Platanthera and Spiranthes. American Journal of Botany, 67(4):508-5 18. 15. MoIvray, M., Kores, P.J. 1995. Character anaIysis of the seed coat in Spiranthoideae and Orchidoideae, with special reference to the Diurideae (Orchidaceae). American Journal of Botany, 82(11): 1443-1454. 16. Schmidt, J. M., Antbger, A. E. 1992. The leveI of agarnospermy in a Nebraska population of Spiranthes cernua (Orchidaceae). American Journal of Botany, 79(5):501-507. 17. Sheviak, C. J. 1982. Biosystematic study of Spiranthes cemua complex. Bulletin No. 448. New York State Museum, Albany, NY. 18. Sheviak, C. J. 1984. Spiranthes diluvialis (Orchidaceae), a new species from the Western United States. Brittonia, 36(1):8-14. 19. Stoutamire, W,P. 1983. Early growth in North American terrestrial orchid seedlings. In: Plaxton, E. H. (ed.), North American Terrestrial Orchids Symposium H. Proceeding and lectures, pp: 14-24. Michigan Orchid Society, Livoniq MI. 20. Tohda, H. 1983. Seed morphology in Orchidaceae. I. Dac~lorchis,Ponerorchis. Chondradenia, and Galeochis. Science Report Tohuku University (4th ser.) 3 8:253- 268, 2 1. Tohda, H. 1985. Seed morphology in Orchidaceae. II. Tribe Cranichideae. Science Report Tohuku University (4th ser.) 39:21-43. 22. Tohda, H. 1986. Seed morphology in Orchidaceae. 11. Tribe Neomeae. Science Report Tohuku University (4th ser.) 39: 103-109. 23. Veyref Y. 1985. Development of the embryo and the young seedling stages of orchids. In: The orchids - Scienac Studies. Ed.: Withner, C. L. pp.: 223-267. Robert E. Krieger Publishing Company, Malabar, Florida. 24. Wiesmeyer, H., HoMen, A 1976. EIectron Microscopy of Orchid Seedlings. Proc. of First Symposium on the Scientific Aspects of Orchids, pp.: 16-26. Detroit, Michigan. 25. Ziegenspeclc, H. 1936. Orchidaceae. In: Loew-Schrbter: Lebensgechichte der Bliitenpflanzen Mitteleuropas. L/4., Stuttgart. 26. Ziegler, B. 1981. Mikromorphologie der Orchideensamen unter Beriicksichtigung taxonomischer Aspecte. Ph.D. dissertation, Ruprecht Kark Universita~Heidelberg. 184 185 186 187

189 190 191 192 Table 4-1. Orchid species included in the scanning electron microscopy study (in taxonomic order based on Dressler, 1993)

I Molvray and Kores, 1995 Goodyera repem ELTE 8olanial hdapeac Healey et al.. 1980 Cbula E Bescy Hakium UNL Molvray and Kores, 1995 Gtxidyera tesselata ~EB~+~~~H~u~.uNLHealey et al.. 1980 Spiranthinae Spiranthes CliEord and Smith, 1969 Ziegler and Barthlott (according to Dressler, 1 993) Spiranthes cemua ELTE Bol.llial G.nlm, Bud.pas Schmidt and Antlfkger. 1992 Dr. Arm Adfhgcr, UNO Spiranthes dilwialis I)~.R~~~~B.K.uLUNL Spiranthes lacera I CbarksE&acyH~tkh~UNL Spiranthes lucida QarIaE~HerbariqUNL Spiranthes rnagnicamponun Q~~~E~HU~~~QUNL Spiranthes rornamffiana Q~~I~E&~~~HCT~U~~UNLHealey et al., 1980 Spiranthes sinensis ELTE Bot.niaI Gda~ CWord and Smith, 1969 Molvray and Kores, I995 ELTE ~btmial~udes Spiranthes vernalis Dr. AWDhlbgcr, UNo. Molvray and Kores, 1995 Nebruk.GMeurdParhCamniplioa Spiranthe tuberosa QuluEmHehrium,UNL

L Taxonomic unit (Cont.) Source References Gast rodieae I Epipoginae Epiwgj,! CWord and Smith, 1969 ELTE Botanical U Malaxidae Lids CWord and Smith, 1969 liliifolia ~~d~~~~rrscy~absriun~,~~~ Liparis l0edi.i ELTEmmicd Charles E -7s CbrrlaE~H~UNL Liparis hawaiiensis I

Malaxis I Midaxis paludosa ELTE B&nial Gar&rs Wldapert Caiypsoae

-SO boredis ELTEB~tanicli~Ebhput ~barl~~E ~essy ~akri~m, UNL Calypso bubosa v. americana-- - . Arditti et al., 1980 Coralforhiza Corallorhiza maculata cbrkr~~cr#y~erberium,~m Arditfi et al., 1980 Corallorhiza odontorhiza m. ~obatR Kd,UNL; CharlesE.BascyHabarium,UNL Corallorhiza mta ChrlaEBersyHerhium,UNL Arditti et al., 1980 Corallorhiza wisteriana CbulcrEBerryHahri~UNL Curallorhiza trifida ELTE RotmidG.rdas Barwott (wto hfer, ~csE&aKyHabui~WNL 1993); Arditti et ale, 1980 Cymbidieae Cvmbidium Clifford and Smith, 1%9 Barthlott (according to Dressler, 1993) Cymbidium pumilum ELTE Gu&IS Budq~S Arethuseae BIetiinae BIetilla CWord and Smith, 1969 Watt (eccarding to Dressler, 1993)

, Bletilla striata ~~'f~~crrl~uder~~~dapm Hexalectris Hexalectris spicata MCI E B- ~akrium,UNL Swth~d~tti~ Barthlott (aamding to I)ressler, 1993) Spathoglottis plicata ELTE BO~UI~~Iu ~udspea; McmknoftbcOrutaOmrhordlid

sociely I Epidend reae Lneliinae I CanhB Balwott (- ta Dressl~, 1993); Wiesmeyer and Hofsten, 1976 , CattIeya aclandiae EL= ~al~arderr,~udapert Taxonomic unit (Cont.) Source References E~idendnun Clifford and Smith, 1%9 Barthlott (according to Dreasler. 1993) Epidendnun nmum ELTE BotrniaI Garth. Budrplt Dendmbieae Den& biinae Dendrobium Clifford and Smith, 1%9 Barthlott (according to mer, 1993) Dendrobium crumenatum UTE~alOardes~ Vandac Aeridinae Phalaend Barthlott (eccording to Dressier. 1993); Wiesmeyer and Hofsteq 1976 Phalaenopsis fasciata ELTEBo(mialGudcsBudapat Vanda Bartblott (acEording to Dressier, 1993) Van& cowulea ~~~~~t~ulial~Budapat Mzsms: Pqgoniinue Cameron, 1995 Po~onia Pogonia ophiogIossoides ~E.~H~~~~u~UNL Table 4-2. Quantitative data table for the detailed description of orchid seeds

Seed leneth: Testacell

Seed width Seed ien& Median cell median cell mberwr S~ecies luml luml 14r~ml ka& -sced , Cypripedium acaule 250 1600.1700 150 10.67 120

Cypripedium calceolus 250 1100 150 7.33 130

var. calceolus

Cypripedium calceolus 270 1150-1300 180 6.67 140

var. parYlfl0l~m

Cypripedium cnlceolus 250 1600-1700 150 10.67 140

var. pubescens

Cypripedium 400 1500 150 10 180

californicum

Cypripedium condidurn 200 800 100 8 120

Cypripedium reginae 300 1300-1400 100 13 170

Coodyern repens 190 700-800 70 10 200

Spirnnthes cernua 150 350-500 70 5.7 90

Spirnnthes diluvialis 120 200-250 65 3.85 60

Spirnnthes vernalis 100 350-400 8&100 4 58

Barlia robertiana 160 460-520 150 3.3 26

Chamorchis alpina 170 250-300 80 3.75 82

? Seedlength: Testacell

S~ecies(Cont.) Seed width Seed lennth Median cell median ceU eumberriq IkEI bI!l lenzth Iuml Ienath -seed

DactylorhhP fucbii ssp. 300 600-700 100 6.5 76 miann

Dactylorhiza incarnata 280-300 650-700 70 10 180

Dactylorhiza maculata 200 800-900 130 6.92 %

Dactylorhizn majalis 250-300 700 90 7.78 200

Dactylorhim saccifern 200 650-750 85 8.8 120

Dactylorhim sambudna 240 650 100 6.5 92

Galearis spectnbilis 150 750-800 200 3.75 88

tiimantoglossurn 140 3 00-400 140 2.86 38 hircinum

Nigritelln algrn 240 300-350 100 3.5 50

Ophiys fudflora 150 500 200 25 60

Ophiys lutea 140 400 110 3.6 40

Ophw sphegodes 170 380 100 3.8 70

Orchis coriophorn 170 600650 180 3.33 60

Orchis coriophor~ssp. 160 750 200 3.75 40

frpgr-

Orchis ladnorp 200 800-850 280 3.04 36

Orchis laxiflorn spp. 170-190 450-550 150 3.5 32 palustris

Orchis moscula 170 750-850 300 2.5 42

Orchis militaris 150 360 100 3.5 40 , Seed lend: Testacell

S~ecies(Cont) Sefd width Seed lenh Median cell median cell number IXT luml luml length rum1 !!21?Jh seed

I Orchir morio 150-200 550-600 150 4 56

Orchis purpum 150 400-450 110 4 46

Phtanthera bifolia 140 600-700 200 3 60

PIatantbern hyptrborea 300 750 95 7.89 140

PIntanthera praeclara 130 300 70 4.28 60

Genoaria dipbylln 250 900-1000 250 4 60

Cattlyea aclandiae 100 450-500 130 3.5 difIicdt to

count

Epidendrum noctumum 87 600 170 3.53 difficult to

count

Dendrobium crumenalum 85 300 300 1 I6

Phnlnenopsis fasciato 60 450 85 5.29 adtto

mt

Vanda rwrulea 70 180-210 difficunto - difficult ta

meaLurc count Proposed Future Research

I. In vi~ogermination and micropropagation of different temperate orchid species. I. Develop new methods for disinfestation of seeds in an attempt to effectively overcome seed dormancy: - changing the length of disinfiition treatment and concentration of sterilizing solution using alternative disinfesting agents. - leaching or soaking the seeds before seed sowing; comparing the effect of different soaking solutions supplemented with different concentration of gibberellic acid or natural substances such as coconut mWwater or phloem sap of merent woody species (as was previously successhlly used for in GPO germination of recalcitrant Paeonia vp. (Jhbor-Bencnir, personal communication)). 2. Optimization of medium and environmental factors for asymbiotic seed germination of recalcitrant orchid species that did not show germination results in previous experiments. 3. Preliminary experiments showed very promising results for geminating temperate orchid species in liquid medium (gyro- shaker, 125 rpm). The modified Fast medium (see again Chapter I, Table 1-2) was supplemented with different concentrations of peptone, coconut water or both. Seeds of different Cyprtpedium, Dactylorhim and Orchis species in the liquid medium germinated in one-third the time of those germinated on gelled medium under the same conditions. After germination and transfer onto gelled media the seedlings continued to develop as others germinated later on the gelled medium (showing developmental stages as was described in Chapter I). Additional experiments are necessary to obtain sufficient amount of data for statistical analysis and to try this potential method for additional orchid species. 4. Parallel experiments to compare developmental stages and their time of appearance of different temperate orchid species germinated and cultured asymbiotically and symbiotically in vifro. A preliminary effort was initiated by the author working together with Dr. Ann Antlfinger, Department of Biology, UNO, for such a research project with Spiranthes cerma and Spiranlhes vernalis. It is still too early to present any experimental results of this project in this dissertation. 5. Observe the effect of different light conditions on the development of orchid seedlings and plantlets (natural illumination with prevailing day-length and 16 houriday artificial illumination provided fiom different light sources of different wavelength). 6. Optimization of temperature for the in vifro germination and culture of different temperate orchids, including seasonal temperature changes and simulated cold dormant period. 7. Continue experiments to compare the effect of different undefined organic compounds (complex additives) in different concentrations alone and in combination with each other in liquid and gelled medium on the in vitro vegetative multiplication of several different temperate orchid species. 8. Develop and optimize acclimatization techniques for in vitro derived temperate orchid plantlets from several different species with and without the use of soil from their native habitat. 9. Initial in silu reintroduction studies for temperate orchids using in vitro derived plant material samples.

II. Anatomical studies 1. Cluster analysis and dendrogram preparation based on quantitative data collected (Chapter IV) to ilIustrate reiationships among different temperate orchid genera and species based on seed morphology. 2. Observe the seed structure of several other temperate species by SEM and also by TEM. 3. Observe the anatomy and rnorphoIogy of several other temperate species by SEM and TEM with special focus on the structural features of calcium oxalate and starch in the storage organs of different species. 4. Use SEM to compare the anatomy and development of protocorms derived Erom seeds or vegetatively. Examine differences and similarities between in vitro orchid protocorm development and in vitro somatic embryogenesis of other monocot species, such as different grassa cultured in vim.

IU. In situ experiments related to in vii'ro culture of temperate orchid species. I. Hand pollination studies to obtain higher seed set, which also can be used for in vim germination studies of temperate orchid species. Initial experiments by the author showed very promising results with some temperate orchid species, achieving greater seed production by plants that were pollinated by hand, especialiy in cases where the naturaI pollinator population had almost disappeared because of environmental problems. 2, Record developmental stages and growth patterns of different orchid species growing in their native habitat. Such data could be extremely useful to determine appropriate environmental factors for in vitro germination and culture. 3. During the observation of different native habitats of CoraIIorhiza odonrorhzza (Willd.) Nutt. (late coral-root) an interesting relationship was discovered between this orchid and another plant species, Monotrop unryora L. (Indian pipe). Monotropa uniflora always accompanied Corallorhim odontorhiza, with Indian pipe plants found in a11 cases only a few inches away. The appearance of the strikrng white plant body of Indian pipe was a helphl signal for a possible habitat of CoruiZorhim odontorhiza and made it much easier to locate the smd, brownish orchid plants which almost disappeared in the s~rnilarcolored environment. Although the two species taxonomicalIy are very different, they exhibited several similar convergent anatomical features (for example seed structure and mycutrophic underground organs), and probably they appeared in the same locations because of a mutual mycorrhizaI fingal partner. To corroborate this interesting phenomenon, more observations and research, inchding anatomical and habitat are required.

Some of these proposed research projects, other than those already started by this author, were initiated by other students at the Department of Horticulture, University of Nebraska-Lincoln, Department of Biology, University of Nebraska at Omaha and at the ELTE Botanical Garden, Budapest following initial training, discussion and advice from the author. The author of this dissertation deeply hopes that these research projects will lead to vduabIe and exciting results, for which she wishes lots of success and persistence. Appendices

A Preliminary acclimatization attempts for in viiro derived temperate orchid plants.

Some preliminary experiments were set up using the first available plant material with suitable size to observe some potential methods for acclimatization of in vifro derived temperate orchid plants. Two and a half to three-year-old in vifro cultured (described in Chapter I) orchid plants (Bletilla strina, Dactyorhiza manrlata, Orchis rnorio) were removed from the culture medium and the underground plant parts were washed with sterile distilled water until all the remnant culture medium was removed. The plant material was in the following developmental stages: - tuberoids/pseudobulbs at the end of the donnant period showing small green buds as the beginning of the new year's growth; - tuberoids/pseudobulbs with developed leaves but the root development had not yet started or only small, short roots were visible at the beginning of their development; - plantlets with fresh new leaves and roots in an advanced developmental stage but before the development of the new hlberoids or pseudobulbs. The plant materials then were transferred into daerent soil conditions: 1. Dactylorhiiza mamZata and Orchis morio plantlets were transferred into baby food jars containing a soil mixture (1:1:1=peat:perlite:vermicuIite) which was previously moistened with modified Fast medium without gelling agent and sugar and autoclaved for 30 min at 12 1 OC and 1.2 Kg AU manipuiations were perfomed in a sterile laminar hood and the cultures were kept in the laboratory under the same conditions as the in vitro cultures on gelled medium described in Chapter I. 2 Bletilla striafa plantlets were transferred into small plastic pots containing sphagnum moss which previously was soaked in modified Fast medium without gelling agent and sugar. The cultures than were sprayed with 1% captan solution and kept under glass at 2M 1°C under 16 hour/day cool fluorescent light (32 pmol s" mJ light intensity). 3. Orchis morio plantlets were transferred back at the beginning of May into locations fiom where the initial seed material was collected, at the Papphills site near Sosk13, Hungary. After transfer the surrounding soil was soaked with water and the location of the plantlets were marked. Following transfer the location was visited every two to four weeks to follow plant development and additional water was applied when it was necessary.

Results and Discussion

The majority of the plantlets (approximately 90%) survived the transfer fiom the in vitro culture medium into soil. However the plant material transferred without developed new roots adapted much easier and grew much healthier than the plantlets with previously developed roots. Most of the pIants that did survive in the first few months following transfer originated fiom this latter type of stock material. Orchis morio plantlets developed small leaf rosettes both in the sterile soil mixture or in their native habitat (Figure 5-1). Some of these Orchis mono plantlets transferred into the sterile soil mixture even developed in the summer of the year of transfer (Figure 5-2). It was one of the most important observations of these preliminary acclimatization attempts that Dactylorhiza rnaculata and Orchis morio plantlets survived and continued to develop in sterile soil, where no hngi were present to establish a symbiotic relationship to support further plant development. Plantlets transferred back into their native habitat were likely provided with the mycorrhizal hngi which were present in the local soil and in other wild grown Orchzs morio plants (as it was observed by SEM and described in Chapter m). On the other hand in nature the young transferred plantlets did not have such a protected and controlled environment as was available for plantlets growing in sterile soil in the laboratory. Bletilla *ata plantlets transferred as dormant pseudobulbs acclimatized without hrther problems in sphagnum moss and continued to develop new leaves, roots and pseudobulbs through the vegetative season. These preliminary experiments showed that the most convenient developmental stage to initiate acclimatization in soil was the stage when the plants had finished the dormant period but had not yet developed new root systems. Additional years are necessary to see how these in vifro derived orchid plants will develop compared with plants that originally germinated in their native habitat. Also additional experiments are necessary to obtain mflicient data for stadstical analysis and to develop acclimatization methods for additional orchid species. B. Orchis morio L. f. albonivea Szendrik

Albinism is one of the most common genetic disorders found among humans and throughout the plant and animal world. Albinism in plants is characterized by the absence of chlorophyll, and albino plants generally die at a very early stage of plant development. In the orchid family there are a few exceptions because the usually lethal achlorophyllism is accompanied with a heterotrophic mechanism such as saprophytism or rnycotrophy (Light and MacConaill, 1989). There are only a few species of orchids that are true 1ea.Q albino plants in nature, that don't have any type of pigmentation, and the plants are totalIy white. One of these species is Cephalanfheraaustinae which is native to the Pacific Northwest (Luer, 1975) and there are reports of albino variants of a few other orchid species, such as Epi@s helleborine _f: mono fropides (Gnesbach, 1979), Platanthera werborea var. hperborea f: alba (Light and MacConailI, 1989), Platanfhera ~parsiioravur. brevifolia (Luer, 1979, and Triphora trianthophora (Keenan, 1 988). The totally white, albino orchid, which was developed under heterotrophic tissue culture conditions is an achlorophyllus form of the Green-winged Orchid (Orchis morio L.). This form has been proposed by the author of this dissertation as Orchis morio L. f: albonivea Szendrik. No reports were found about specimens of this form in nature or under culture conditions. The seeds used as stock material for the asymbiotic in vitro culture originated &om several different populations of this species at the Papphills near Soskt, Hungary. These populations were phytocoenologically and floristically observed for several yem by the author (Szendrilq 1994), but no albino forms were found. However there were numerous albino plants among the in vitro germinated plants fiom every seed source, so probably the genetic background of albinism is present in the native habitat, but lethal fiorn a very early developmental stage. Albino Orchis morio L. f: albonivea Szendrik plants were germinated and grown on a modifmi FAST medium (Fast, 1976; Szendr& and R. Eszeki, 1993) using culture methods as described in Chapter I. They exhibited the same characteristics typical of the normal green ones except that they developed at a more rapid rate than the green plants (fonna albonivea insignis; folia alba; aliter velut in qecie). These in vim grown plants are now almost three years old and they are being continued in culture under in vitro conditions. Spontaneous vegetative proliferation was observed in the case of some specimens of this plant, similarly to the other orchid species reported in Chapter I. However the aIbino plants did not require dark for spontaneous protocorm proliferation and they have continued to rapidly develop new additional protocorn under 16 hodday cool fluorescent Light in the laboratory.

Literature Cited

1. Fast, G. 1976. Moglichkeiten zur Massenvermehrung von Cypripedium calceolus und anderen europaischen Wildorchideen. Proc. 8th WorId Orchid Cod., Frankfbrt, pp.:359-363. 2. Griesbach, R J. 1979. The albino form of Epipacris helleborine. American Orchid Society Bulletin, 48: 808-809. 3. Keenan, P. E. 1988. Three-birds orchids at GoIden Pond. American Orchid Society Bulletin, 57:25-27. 4. Light, M. H. S., MacConaiII, M. 1989. Albinism in Platanfhera hyperborea. Lindleyana, 4(3): 158- 160. 5. Luer, C. A 1975. The native orchids of the United States and Canada excluding Florida. The New York BotanicaI Garden, New York. 6. Stendre E. 1994. Az Orchis nrorio L. soskiiti lel8helyknek florisztikai is Mnologiai vizsgilata, 6s a faj disn6vinytermeszt&be vonikhak lehetesbge. MSc. thesis, (The floristicaI and phytocoenological measurements of the native habitat of Orchis rnorio L. near 'SosMt' and the possibiiities to use this species as a potential ornamental I plant.) Kertketi ds Elelmiszeripari Egyetem, Kertketi Kar, Disznovknytermesztbi 4s Dendrologiai Tanszik, NZivenytani Tanszdc, Budapest. 7. Szendriik, E., R. Eszeki E. 1993. Hazai szabadfofdi kosbodelek (Orchihceae) aszimbiotikus in vitro szaporitk. PubIicationes Univ. Hort. Ind. ALi. Vol. Lm. pp.:6&70. 211 212 213