CRYOPRESERVATION AND IN VITRO TECHNIQUES FOR THE CONSERVATION OF SELECTED FLORIDA NATIVE ORCHIDS

By

BENJAMIN A. HUGHES

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2017

© 2017 Benjamin A. Hughes

To Allison and Ronin

ACKNOWLEDGMENTS

I thank Dr. Michael Kane for his guidance, patience, and generosity in helping me develop into a competent researcher and scientist. You saw something in me years ago that I certainly didn’t. I thank Dr. Rebecca Darnell, Dr. Greg MacDonald, Dr. Carrie Reinhardt-

Adams, and Dr. Wagner Vendrame for serving on my supervisory committee and providing me necessary challenges to grow. I thank Dr. Héctor Pérez for acting as a sounding board since I was an undergraduate. There are too many other faculty and staff to name in the Environmental

Horticulture Department that have helped me but thank you as well.

I thank my peers that helped me throughout this process, beginning with my fellow graduate students: Paulina Quijia, Charles Stewart, Christian Christenson, Candice Prince,

Gabriel Campbell, Jameson Coopman, Tia Tyler, and Amber Gardner. Sharing ideas, problem- solving, and commiserating with you was essential to my graduate experience. I also thank my friends not in academia: Dom, Cory, Chris, Sam, Ethan, Johnathan, Adam, Mike, and Matt, amongst others, for all the good times that distracted me from work and helped keep me sane.

Finally, I thank my family, especially my parents, Neil and Jennie, supporting and encouraging me throughout this process even though they weren’t always sure what that entailed.

I thank my Aunt Rose for the family dinners and always asking how she could help me. Thanks to Charlie for the laughs, debates, and beers. Most importantly, I thank Allison Bechtloff for her love, support, and understanding. She has kept me grounded throughout this process and I doubt

I would have finished without her.

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TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 8

LIST OF FIGURES ...... 9

LIST OF ABBREVIATIONS ...... 11

ABSTRACT ...... 12

CHAPTER

1 LITERATURE REVIEW ...... 14

Introduction ...... 14 Orchid Pollinators ...... 16 Embryology ...... 17 Gametogenesis ...... 17 Fertilization and Embryo Development ...... 19 Orchid Seeds and Seedling Development ...... 21 Orchid Mycorrhizae ...... 23 Orchid Conservation ...... 24 In Vitro Orchid Propagation ...... 25 Vegetative Propagation ...... 25 Seed Propagation ...... 27 Liquid Media ...... 29 Bioreactors ...... 30 Orchid Germplasm Banking ...... 34 Low-Temperature Storage ...... 34 Cryopreservation ...... 36 Controlled rate cooling ...... 36 Vitrification without plant vitrification solution ...... 37 Vitrification with plant vitrification solution ...... 37 Direct Orchid Seed Cryopreservation ...... 38 Orchid Seed Cryopreservation by Vitrification ...... 42 Orchid Seed Cryopreservation by Encapsulation-Dehydration ...... 45 Protocorm Cryopreservation ...... 47 Florida Native Orchid Conservation ...... 47 Project Description ...... 49 Project Objectives ...... 49 Rationale ...... 49

2 DIRECT CRYOPRESERVATION OF SELECTED FLORIDA NATIVE ORCHID SEED ...... 51

5

Introduction ...... 51 Materials and Methods ...... 54 Plant Material ...... 54 Protocol Optimization for Direct Cryopreservation ...... 55 Direct Cryopreservation ...... 56 Statistical Analysis ...... 56 Results...... 57 Protocol Optimization ...... 57 Direct Cryopreservation ...... 58 Further Seedling Development ...... 58 Scanning Electron Microscopy ...... 59 Discussion ...... 59

3 EFFECTS OF WATER CONTENT AND LOW-TEMPERATURE STORAGE ON CRYOPRESERVATION OF TWO FLORIDA NATIVE ORCHIDS ...... 74

Introduction ...... 74 Materials and Methods ...... 76 Plant Material ...... 76 Experiment 1: Effect of Seed Moisture Content on Direct Cryopreservation ...... 77 Experiment 2: Effect of Low-Temperature Storage on Orchid Seed Cryopreservation ...... 78 Statistical Analysis ...... 79 Results...... 80 Moisture Content ...... 80 Low-Temperature Storage ...... 81 Discussion ...... 82 Moisture Content ...... 82 Low-Temperature Storage ...... 84 Conclusions ...... 85

4 MODIFICATION OF SPINNER FLASKS FOR ORCHID SEED BIOREACTOR CULTURE USING BLETIA PURPUREA ...... 97

Introduction ...... 97 Materials and Methods ...... 98 Bioreactor Modification ...... 98 Aeration Manifold ...... 99 Results and Discussion ...... 100 Bioreactor Inoculum ...... 101 Bioreactor Performance ...... 101

5 LIQUID FLASK AND BIOREACTOR CULTURE OF SELECTED FLORIDA NATIVE ORCHID SEED ...... 112

Introduction ...... 112 Materials and Methods ...... 114

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Plant Material ...... 114 Seedling Inoculum Culture Medium Optimization ...... 115 Comparison of Solid and Liquid Medium on Seed Germination and Subsequent Seedling Development ...... 116 Effect of Citric/Ascorbic Acid on Bioreactor Culture of Bletia purpurea ...... 117 Statistical Analysis ...... 118 Results...... 118 Seedling Inoculum Culture Medium Optimization ...... 118 Comparison of Solid and Liquid Medium on Seed Germination and Subsequent Seedling Development ...... 118 Effect of Citric/Ascorbic Acid on Bioreactor Culture of Bletia purpurea ...... 120 Discussion ...... 120 Seedling Inoculum Culture Medium Optimization ...... 121 Comparison of Solid and Liquid Medium on Seed Germination and Subsequent Seedling Development ...... 122 Effect of Citric/Ascorbic Acid on Bioreactor Culture of Bletia purpurea ...... 123 Conclusions ...... 125

6 CONCLUSIONS ...... 141

LIST OF REFERENCES ...... 144

BIOGRAPHICAL SKETCH ...... 165

7

LIST OF TABLES

Table page

2-1 Seed characteristics of nine Florida native orchid species...... 63

2-2 Orchid seed and seedling developmental stages...... 63

2-3 Effect of surface sterilization timing on germination of four directly cryopreserved Florida native orchid species ...... 64

2-4 Effect of direct cryopreservation on germination of five Florida native orchid species...... 64

3-1 Relative humidity produced from saturated salt solutions ...... 87

3-2 Orchid seed and seedling developmental stages...... 87

3-3 Effect of storage relative humidity prior to direct cryopreservation on seed moisture content and germination of two Florida native orchid species ...... 88

3-4 Effect of storage at -10 °C prior to direct cryopreservation on germination of two Florida native orchid species...... 88

4-1 Component list for construction of a single aerated bioreactor...... 104

4-2 Component list for construction of twenty-position aeration manifold...... 105

5-1 Seedling inoculum media treatment matrix...... 127

5-2 Orchid seed and seedling developmental stages...... 127

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LIST OF FIGURES

Figure page

2-1 Effect of surface sterilization (SS) timing on seed germination of four Florida native orchid species every 2 weeks ...... 65

2-2 Effect of surface sterilization timing on seedling developmental index of four Florida native orchid species ...... 66

2-3 Effect of direct cryopreservation on seed germination of five Florida native orchid species...... 67

2-4 Effect of direct cryopreservation on seedling developmental index of five Florida native orchid species ...... 68

2-5 Development of orchid seedlings after 1 year culture following experimental conditions...... 69

2-6 Further development of orchid seedlings ...... 70

2-7 Scanning electron micrographs of unfrozen and frozen seed...... 71

2-8 Scanning electron micrographs of unfrozen and frozen seed ...... 72

2-9 Scanning electron micrographs of unfrozen and frozen seed ...... 73

3-1 Effect of pre-cryopreservation equilibrium relative humidity on post- cryopreservation seed germination for two Florida native orchids...... 89

3-2 Effect of pre-cryopreservation equilibrium relative humidity on post- cryopreservation seedling developmental index for two Florida native orchids ...... 90

3-3 Effect of pre-cryopreservation storage at -10 °C on post-cryopreservation seed germination for two Florida native orchids...... 91

3-4 Effect of pre-cryopreservation storage at -10 °C on post-cryopreservation seedling developmental index for two Florida native orchids ...... 92

3-5 Polypropylene chamber used to adjust orchid seed water content ...... 93

3-6 Development of B. purpurea seedlings after 22 weeks culture following experimental conditions...... 94

3-7 Development of E. nocturnum seedlings after 22 weeks culture following experimental conditions ...... 95

3-8 B. purpurea seedlings derived from seed equilibrated to 76% relative humidity prior to cryopreservation that died after 8 weeks culture...... 96

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4-1 Spinner flask modifications...... 106

4-2 Bioreactor aeration components ...... 107

4-3 Aeration manifold components ...... 108

4-4 Effects of aeration methods on seedling cultures ...... 109

4-5 B. purpurea seedlings growing in impeller...... 110

4-6 Scanning electron micrographs showing B. purpurea seedling tissue damage ...... 111

5-1 Effect of four germination media on seed germination and subsequent seedling development of three Florida native orchid species following 4 weeks liquid suspension culture ...... 128

5-2 Effect of liquid and solid formulations of Knudson C medium on the germination of three Florida native orchid species...... 129

5-3 Effect of liquid and solid formulations of Knudson C medium on seedling developmental index of three Florida native orchid species ...... 130

5-4 Comparison of bioreactor culture of B. purpurea with and without 0.005% citric and ascorbic acid (CAA) ...... 131

5-5 Comparison of bioreactor culture on seedling survival and PLB formation of B. purpurea with and without 0.005% citric and ascorbic acid (CAA) 10 weeks following transfer to solid medium...... 132

5-6 Comparison of bioreactor culture on leaf production of Bletia purpurea with and without 0.005% citric and ascorbic acid (CAA) ...... 133

5-7 Bletia purpurea seedlings after 6 weeks culture ...... 134

5-8 Scanning electron micrographs of three Florida native orchid species after 4 weeks culture ...... 135

5-9 Development of B. purpurea seedlings following 8 weeks bioreactor culture ...... 136

5-10 Scanning electron micrographs showing damage to B. purpurea seedlings from bioreactor culture and evidence of early PLB proliferation ...... 137

5-11 Bioreactors showing different degrees of media browning ...... 138

5-12 Bioreactor with bacterial contamination ...... 139

5-13 B. purpurea seedlings following culture on solid P748 medium following bioreactor culture ...... 140

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LIST OF ABBREVIATIONS

CAM Crassulacean acid metabolism

CW Coconut water

DD Distilled deionized (water)

DI Developmental index

FAO Food and Agricultural Organization of the United Nations

FPNWR Florida Panther National Wildlife Refuge

GSF Goethe State Forest

KC Knudson C (medium)

LN Liquid nitrogen

MS Murashige and Skoog (medium)

PLB Protocorm-like body

PPM Plant preservative mixture

PVS Plant vitrification solution

RH Relative humidity

SEM Scanning electron micrograph

TCL Thin cell layer

TTC 2,3,5 Triphenyl-tetrazolium chloride

WC Water content

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

CRYOPRESERVATION AND IN VITRO TECHNIQUES FOR THE CONSERVATION OF SELECTED FLORIDA NATIVE ORCHIDS

By

Benjamin A. Hughes

December 2017

Chair: Michael Kane Major: Horticultural Sciences

Florida is a biodiversity hotspot for native orchids, containing half the species diversity in

North America. Unfortunately, many populations of Florida native orchids are imperiled, accentuating the need for germplasm conservation encompassing genetic diversity. Further, efficient propagation techniques must be identified for the reintroduction of plants to existing populations. We aimed to optimize cryopreservation and in vitro procedures for the conservation of Florida native orchids. We determined that direct cryopreservation of dried seed from nine

Florida native orchid species in liquid nitrogen did not reduce germination when compared to unfrozen seed. Additionally, Bletia purpurea and Epidendrum nocturnum seed could be equilibrated to relative humidity levels up to 55% prior to direct cryopreservation without negative impact. B. purpurea and E. nocturnum seed stored at -10 °C for up to six weeks prior to direct cryopreservation did not impact post-cryopreservation germination. A reduction in seedling vigor was observed in E. nocturnum after 28 days, but there is evidence this is related to seed aging prior to cryopreservation. Spinner flasks were modified in order to evaluate their potential as bioreactors for orchid seedling culture in a liquid medium. We determined that developing a seedling inoculum culture in small volumes of liquid medium prior to bioreactor culture and reduction of seedling and medium browning were required for efficient seedling

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production. We optimized seedling inoculum media for three Florida native orchid species and determined Knudson C medium most optimal. Germination and seedling development of these species in liquid and solid formulations of Knudson C was then compared. Germination did not differ between medium phases in any species, but the development of B. purpurea seedlings was slightly enhanced after 6 weeks liquid culture. The addition of citric and ascorbic acid supplements each at 0.005% to bioreactor medium did not reduce browning in B. purpurea, which resulted in high seedling mortality upon transfer to a solid medium. However, the seedlings that survived displayed more vigorous growth than ones cultured solely on a solid medium, therefore warranting further investigation into this production method. These studies represent initial procedures for the efficient conservation and production of Florida native orchids.

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CHAPTER 1 LITERATURE REVIEW

Introduction

The family Orchidaceae is the largest among flowering plants, consisting of over 28,000 species within over 730 genera (Chase et al. 2015; Christenhusz and Byng 2016). There are five subfamilies within the Orchidaceae: Apostasioideae, Cyorioedioideae, Vanilloideae,

Orchidoideae, and Epidendroideae, confirmed by analysis of rbcL nucleotide sequences

(Cameron et al. 1999). Orchids inhabit every continent with the exception of Antarctica, ranging latitudinally from northern Sweden to the southern tip of South America (Dressler 1990).

Orchids grow in or on a variety of different substrates and are commonly classified based on their life form. Terrestrial orchids root into soil, epiphytic orchids grow on trees, lithophytic orchids grow on rock faces in extreme environments (Koopowitz 2001), and those that reside in semi-aquatic habitats (Yoder et al. 2010). Phylogenetic analysis has established that epiphytes diverged from a common terrestrial ancestor (Garay 1972; Dressler 1990; Silvera et al. 2009;

Tsutsumi et al. 2011)

Like other monocots, orchids have parallel leaf venation, trimerous flower parts, and an inferior ovary (Dressler 1990). Orchids display either a monopodial or sympodial growth habit.

Monopodial orchids have indeterminate stems that grow continually each season and bear lateral inflorescences. Sympodial orchids have stems that grow for a determined period of time and cease to grow further, with new growth coming from lateral meristems and can have lateral or terminal inflorescences (Koopowitz 2001). The storage organs of terrestrial species tend to grow underground as tubers, rhizomes, or corms whereas epiphytic species use swollen internodes known as pseudobulbs for nutrient and water storage (Zimmerman 1990; Arditti 1992). The

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velamen, a spongy epidermal layer that covers the root and aids in water retention, is also much more pronounced on epiphytes than on terrestrial orchids (Dressler and Dressler 1993).

Though morphologically diverse, orchids can be distinguished by a number of common traits related to their flowers, pollen, and seeds. Orchid flowers often display bilateral symmetry but are highly variable (Dressler and Dressler 1993). Despite this fact, the most characteristic features of the Orchidaceae are related to their flowers. The stamens are arranged on the abaxial side of the flower, the pistil and stamen are either partially or fully fused into a structure known as the column, and a highly-modified petal (labellum) acts as a landing pad for pollinators

(Garay 1972; Dressler 1990; Arditti 1992; Roberts and Dixon 2008). The majority of orchid pollen aggregates to varying degrees, most commonly into a pair of structures known as pollinia

(Swamy 1949a; Dressler 1990; Freudenstein and Rasmussen 1997; Tremblay et al. 2005).

Following pollination, the ovary develops into a dehiscent fruit known as a capsule. Upon maturity, capsules split along their three carpels, releasing many dust-like seeds (Dressler 1990;

Arditti 1992; Arditti and Ghani 2000). Orchid seeds are primarily adapted for wind dispersal, facilitated by their reduced morphology (Arditti 1992; Arditti and Ghani 2000; Roberts and

Dixon 2008). Seed dispersal is often aided by spring hairs that also develop within the capsule

(Dressler and Dressler 1993). Seed capsules may contain as many as 4,000,000 seeds, as observed in Cynoches ventricosum var. chlorochilon or as few as 20-150, as seen in Rhizanthella gardneri (Arditti 1992).

A common physiological trait in epiphytic orchids is the utilization of the crassulacean acid metabolism (CAM) photosynthetic pathway (Arditti 1992; Kerbauy et al. 2012; Givnish et al. 2015). CAM has been linked to the evolution of epiphytism in orchids, arisen independently at least ten times in Orchidaceae and is likely due to the need for increased water efficiency

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(Silvera et al. 2009). CAM has been observed both in obligate and facultative (C3-CAM) forms, though a number of epiphytes solely rely on C3 photosynthesis (Zotz and Ziegler 1997;

Motomura et al. 2008; Silvera et al. 2010). Though it has been suggested that there is a positive correlation between leaf thickness and CAM expression (Neales and Hew 1975), exceptions have been found (Silvera et al. 2005). A more reliable relationship was found when comparing the thickness of leaf chlorenchyma cells with the strength of CAM (Zotz and Ziegler 1997). Hew and Khoo (1980) observed diurnal fluctuations in titratable acidity in the protocorms of CAM orchid species, however, acid accumulation is lower than in mature plants, suggesting an ontogenetic shift in photosynthetic pathways.

Orchid Pollinators

There is mounting evidence that the high level of diversity amongst orchids is due to their low reproductive success, leading to high levels of pollinator specificity (Tremblay et al. 2005).

The majority of orchids are pollinated by bees, butterflies, moths, flies, beetles, and birds

(Tremblay et al. 2005; Weston et al. 2005). Despite the number of total orchid pollinators, about

60% of orchids are pollinated by a single species (Tremblay 1992). Obligate autogamy is uncommon and is estimated to occur in the range of 3-20% of orchids, although many orchids are self-compatible (Tremblay et al. 2005). One pollination system used by orchids is reward- based. Nutrient rewards are usually nectar, but less frequently oil or pollen is used to attract a pollinator (Neiland and Wilcock 1998). Fragrance can also be used as a pollinator reward, as seen in Gongora orchids and euglossine bees (Hetherington-Rauth and Ramírez 2016).

A second pollination system, deceit, is used by approximately one-third of orchids to attract pollinators (Catling and Catling 1991; Jersáková et al. 2006). The most common form of pollinator deceit used by orchids is nutrient deception, wherein a flower gives the appearance of offering a nutrient reward when one doesn’t exist. This can be seen in Crytopodium punctatum

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and C. polyphyllum where aromatic compounds produced by the flower attract hymenopteran pollinators expecting an oil reward (Pemberton and Liu 2008; Pansarin et al. 2008; Dutra et al.

2009a). Another common form of deceit is sexual deception, where male insects attempt to mate with flowers due to the floral production of aromatic compounds. This form of deception is found in hundreds of Australian species, including Chiloglottis trapeziformis and a thynnine wasp (Schiestl et al. 2003; Weston et al. 2005). Less common forms of deceit are floral mimicry, brood site mimicry, and pseudo antagonism. Orchid flowers that employ floral mimicry are

Batesian mimics of a reward-offering flower, as seen in Disa nivea attracting the pollinator of

Zaluzianskya microsiphon (Anderson et al. 2005). Epipactis consimilis flowers give the appearance of aphid colonization, which attracts hover flies using brood mimicry (Ivri and Dafni

1977). Perhaps the most bizarre form of pollinator deceit is pseudo antagonism, which has been rarely described. The flowers of some Oncidium species imitate an intruder and are then attacked by Centris bees (Ackerman 1986).

Embryology

Gametogenesis

The orchid anther is composed of an epidermis, endothecium, two middle layers, and a tapetum. The tapetal cells are uninucleate, though binucleate tapetal cells can be found in more primitive orchids such as Paphiopedilum druryi and Spathoglottis plicata (Swamy 1949a; Sriyot et al. 2015). The primary archesporium divides by mitosis which forms the microspore mother cells. Microspore mother cells divide twice simultaneously by meiosis, forming an isobilateral or tetrahedral group of microspores (Swamy 1949a). The microspores divide mitotically to form a vegetative and a generative cell. The generative cell then becomes embedded in the vegetative cell, forming a pollen grain. (Swamy 1949a). In the majority of orchid species, pollen aggregates into large masses known as pollinia. Two pollinia are produced per flower and are held together

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with pollenkitt, elastoviscin, or callose (Pacini and Hesse 2002). Less pollen aggregation occurs in the more primitive orchids: some member of the tribes Cypripedilinae, including

Cephalanthera and Vanilla have free pollen; many species within Neottiinae, including Pogonia and Spiranthes, show limited aggregation and form compound grains; the entire Ophrydinae tribe and a portion of Neottiinae aggregate further to form massulae (Swamy 1949a). This trend indicates pollen aggregation has increased as orchid species have evolved.

Another unique trait in orchids is that most species will not produce ovules until after pollination, causing a lag between pollination and fertilization (Yeung and Law 1997). Ovule formation, along with other post-pollination syndromes are initiated by pollen-born auxin and ethylene, the production of which is initiated by auxin (Zhang and O’Neill 1993; Novak et al.

2014). Auxin alone triggers perianth death, stigmatal closure, and ovule enlargement and maturation (Novak et al. 2014). Ethylene alone causes petal senescence, pollen germination, and pollen tube growth. Ethylene in the presence of auxin enhances ovule development and differentiation (Zhang and O’Neill 1993). This phenomenon explains the lag between pollination and fertilization, which ranges from 4 days in Gastrodia elata to 60 days in Geodorum densiflorum, Bulbophyllum mysorense, and some Dendrobium species (Swamy 1943; Swamy

1949b). This period is shorter in more primitive orchids and a similar pattern can be seen in the time between fertilization and the first zygotic division (Swamy 1949b).

Prior to anthesis, the ovary possesses three longitudinal placental ridges that rapidly divide after pollination, forming dichotomously branched outgrowths. The branches terminate in a filamentous row of 6-7 nucellar cells, the last of which differentiates into an archesporial cell and the cells prior to the filament divide to form integuments (Swamy 1949a). The archesporial cell functions directly as the megaspore mother cell and the production of cytoplasm,

18

mitochondria, and plastids increase (Swamy 1949a; Yeung and Law 1997). After further growth and accumulation of starches and lipid droplets, the archesporial cell undergoes meiosis I to form dyad cells, after which the lower dyad cell undergoes meiosis II to produce two megaspore cells.

The functional megaspore cell divides three times to give rise to the embryo sac (Yeung and Law

1997). At this point, another event unique to orchids occurs known as the striking phenomenon, where a reduction of nuclei on the chalazal end of the embryo sac occurs (Swamy 1949a; Yeung and Law 1997).

Fertilization and Embryo Development

The pollen grain germinates and the pollen tube then grows along the funiculus and inner integument, during which the generative cell divides into two sperm cells. The pollen tube then enters the ovule through the micropyle, pierces the synergid, and releases the sperm cells

(Swamy 1949b; Swamy 1949a; Sriyot et al. 2015). Double fertilization does occur in most cases, but triple fusion either fails or when it occurs the endosperm nucleus degenerates shortly after.

This phenomenon is why endosperm is virtually nonexistent in orchid seed (Swamy 1949b; Yam et al. 2013).

Early embryo development is similar to other angiosperms, starting with a transverse division which forms a smaller terminal cell and a larger basal cell which further divides and forms the suspensor initial and a middle cell (Swamy 1949b; Yam et al. 2013). From here, orchid embryos take one of three forms. In the Asterad form, found in Spiranthes, Cypripedium reginae, and Listeria ovata, all cells become part of the embryo proper and no suspensor is formed. Most orchid embryos are of the Onagrad form, in which the suspensor initial becomes a distinct suspensor and the embryo is formed from the terminal and middle cells (Swamy 1949b). The

Cymbidium form, found in Cymbidium, Eulophia, Stanhopea, and Geodorum, undergo random divisions that result in an undifferentiated cell mass. All but one of the chalazal cells vacuolate

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and elongate, forming the suspensor. The remaining cell divides forming a filamentous proembryo that divides and forms a proper globular embryo (Swamy 1949b; Yeung 1996).

Suspensor development is also varied in orchids. Asterad-form embryos lack suspensors completely, Cymbidium-form suspensors are undifferentiated, while four main morphologies are found in Onagrad-type embryos (Swamy 1949b; Yam et al. 2013). The first suspensor type found in Onagrad-form embryos, found in Cypripedium, Dendrobium, and Spathoglottis, is single-celled, can remain the same size as the suspensor initial or elongate to a conical, tubular, or sac-shaped larger cell. The second suspensor type, found in Ophyrs, Habenaria, and Satyrium, is a filamentous row of five to ten cells that sends a haustorial process into nutritive tissue. This is the only aggressive suspensor, while the remainder are functionally absorptive. The third type of suspensor, found in Epidendrum and Sobralia, is the result of transverse followed by oblique and then vertical divisions, with the result looking similar to a bunch of grapes. The final suspensor type, found in Luisia, Cottonia, and Vanda, undergoes three vertical divisions that elongate and drape over the embryo proper (Swamy 1949b). Orchid suspensors have no cuticle and are highly vacuolated, which allow them to transfer and store both water and nutrients apoplastically from the maternal tissue (Lee et al. 2006; Yam et al. 2013).

Unlike the majority of angiosperms, orchid embryo development ceases at the globular stage (Swamy 1949b; Veyret 1974; Arditti 1992; Fang et al. 2016). Embryos begin to accumulate starch grains early in the globular stage. Starch is merely an intermediate storage product, as little to none are found once the embryo reaches its maturity. Instead, a large number of protein and lipid bodies can be found within mature embryo cells (Yeung 1996; Lee et al.

2005; Lee et al. 2006; Yam et al. 2013; Yang and Lee 2014). Other notable changes at embryo maturity include the breakdown of vacuoles, an increase in cytoplasmic density, and the

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deposition of cuticular and phenolic material to the embryo surface thought to prevent desiccation (Lee et al. 2006; Yam et al. 2013; Yang and Lee 2014). At maturity, embryos are elliptic or globular in shape and can range from eight cells in Epipogium aphyllum to seven hundred thirty-four in Bletilla striata (Yam et al. 2013). Another characteristic feature of embryos is that only the apical and protodermal cells are meristematic, as opposed to all cells in other angiosperms (Vinogradova and Andronova 2002). The testa develops from the integuments of the ovule and is hydrophobic due to the accumulation of cuticular substances, polyphenols, and lignin that accumulate in the integuments during development (Lee et al. 2005; Yam et al.

2013).

Orchid Seeds and Seedling Development

Orchid seeds are extremely small and light, ranging in length from 0.05 mm in

Anoectochilus imitans to 6.0 mm in Epidendrum secundum and in mass from 0.3-0.4 µg in

Anguloa to 14-17 µg in Galeola (Arditti and Ghani 2000). The seeds consist of an undifferentiated globular embryo contained within a thin testa. There is substantial airspace within the seed relative to the embryo. This allows seeds to float through the air for extended periods of time which provides a primary means of dispersal (anemochory) (Arditti 1992). The considerable airspace, along with the hydrophobic lipoid layer covering the testae results in seeds with a high buoyancy, which allows them to be transported by rivers and storm run-off

(hydrochory). Seeds can also be distributed by attaching to the feathers and fur of animals

(epizoochory) (Arditti and Ghani 2000).

All orchid seeds possess morphological dormancy due to their lack of a radicle and in most cases, a cotyledon (Baskin and Baskin 2014). Physiological dormancy has been identified in the seed of numerous orchid species. Cold stratification has improved germination rates in seeds of orchid species such as Calopogon tuberosus var. tuberosis (Kauth et al. 2011a),

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Platanthera praeclara (Sharma et al. 2003), Epipactis palustris (Rasmussen 1992), and P. leucophaea (Nuttall) Lindley (Zettler et al. 2001). Van der Kinderen (1987) found abscisic acid levels five times higher in seeds of a difficult to germinate orchid (Epipactis helleborine) than in an easy to germinate orchid (Dactylorhiza maculata). The addition of 6-benzylaminopurine (BA) to germination media promoted the germination of immature Pseudorchis albida seeds (50%) compared to a growth regulator-free control (4%) (Pierce and Cerabolini 2011). Zeatin and kinetin enhanced germination of Habenaria macroceratitis (Stewart and Kane 2006). Abscisic acid was identified as the germination-inhibiting compound in mature seeds of Cypripedium formosanum (Lee et al. 2015). Physical dormancy is not believed to occur in orchid seeds

(Baskin and Baskin 2014).

Orchid seeds imbibe water, and in some cases germinate independently, but without infection by a mycorrhizal fungi further development is impossible in nature (Dressler 1990;

Arditti 1992; Rasmussen 1995; Massey and Zettler 2007). Water uptake in seeds also appears to be affected by mycorrhizal fungi. Fungal hyphae penetrate the testa or widen seed micropores during infection, allowing for faster water imbibition (Yoder et al. 2000). Orchid seeds are slow to imbibe water and typically float on the surface for extended periods prior to imbibition

(Arditti and Ghani 2000). The seeds of epiphytic orchids are more porous than the seeds of terrestrial species, which allows for an increased rate of water uptake (Yoder et al. 2010).

When an orchid seed germinates, the embryo first develops into a spherical mass of undifferentiated cells known as a protocorm (Dressler 1990; Rasmussen 1995). Organogenesis pathways are activated upon the development of the protocorm, beginning with rhizoids and followed by roots and leaves as well as storage organs in some cases (Veyret 1974; Fang et al.

2016). Protocorms have a limited ability to photosynthesize prior to leaf development (Hew and

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Khoo 1980). Although protocorms are fairly uniform upon formation, their continued growth and development are diverse (Arditti 1992; Rasmussen 1995). In order to simplify documentation of orchid seedling development, it is often divided into a series of five to seven stages depending on the species (Rasmussen 1995; Stewart and Zettler 2002; Johnson et al. 2007; Dutra et al. 2009b).

For example, Dutra et al. (2009b) lists the stages of Cyrtopodium punctatum as 1-”Intact testa”,

2-”Embryo enlarged, testa ruptured (germination), 3-”Appearance of protomeristem”, 4-

”Emergence of two-first leaf primordia”, 5-”Elongation of shoot and further development”.

Orchid Mycorrhizae

Orchid seeds are unable to germinate naturally until infected with a compatible mycorrhizal fungus. Mycorrhizal fungi generally enter the seed through the suspensor and through root hairs in plants, after which they reside in cortical cells (Yoder et al. 2000;

Sathiyadash et al. 2012). The fungal hyphae develop round coiled forms known as pelotons, which are digested by the plant, a process known as myco-heterotrophy (Rasmussen and

Rasmussen 2007; Dearnaley et al. 2012). Non-pelotonic hyphae also reside within cortical cells and produce additional pelotons after existing pelotons begin breaking down due to digestion

(Senthilkumar and Krishnamurthy 1998). Although orchids depend on mycorrhizal associations primarily as a source of carbon, they can also receive nitrogen, phosphorus, and water from their host fungi (Yoder et al. 2000; Cameron et al. 2006; Cameron et al. 2007; Bougoure et al. 2010;

Liebel et al. 2015). Though not mycorrhizal, bacteria can also associate with orchids and promote germination due to their production of the auxin indole-3-acetic acid (Tsavkelova et al.

2007).

As orchids mature, they can reduce their dependence on mycoheterotrophy in favor of photoautotrophy (Dearnaley 2007; Rasmussen and Rasmussen 2007). This mixotrophic state has been proposed as an evolutionary shift towards a fully mycoheterotrophic state, which has been

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observed in albino individuals of the French terrestrial orchid Cephalanthera damasonium (Julou et al. 2005). Orchids appear to have the ability to control the degree of mycorrhizal infection through the biosynthesis of the phytoalexin orchinol (Rasmussen 2002).

The majority or orchid mycorrhizal fungi belong to three Basidiomycota groups:

Sebacinales, Ceratobasidiaceae, and Tulasnellaceae (Dearnaley et al. 2012). These fungi can be saprotrophic, pathogenic, or even ectomycorrhizal (Rasmussen 2002; Dearnaley et al. 2012).

There is evidence for fungal specificity in some orchid species (Otero et al. 2004; Irwin et al.

2007; Bougoure et al. 2010), though other species appear to be broad host generalists (Otero et al. 2004; Dearnaley et al. 2012; Rafter et al. 2016; Waud et al. 2017).

Orchid Conservation

Conservation biology is a field that was developed as a means to decrease the rapid loss of global biodiversity. It combines traditional biology with disciplines such as ecology, biogeography, population genetics, and environmental management, amongst others (Heywood and Iriondo 2003). There are two approaches to conservation: in situ, which is conservation within the natural area, and ex situ, which is any off-site form of conservation (Benson 2008a).

In situ conservation is the highest priority since it allows for the continuation of ecological functions, but ex situ methods can be a valuable supplement in the field. Li and Pritchard (2009) estimate that the cost of ex situ seed conservation can be as low as 1% of in situ conservation.

Most of the Orchidaceae family is listed in the International Union of Conservation of

Nature and Natural Resources Red Data Book, and the entire family is classified as Appendix-I or Appendix-II restriction under the Convention on International Trade in Endangered Species of

Wild Fauna and Flora (Chugh et al. 2009). Of the approximately two hundred fifty species of orchids native to North America, about one-hundred twenty of those inhabit Florida (Brown

2006). As of October 2015, seventy-seven of these species have been classified by the Florida

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Division of Plant Industry as endangered, threatened, or commercially exploitable

(http://www.freshfromflorida.com).

The threats facing wild orchids are numerous, including over-collection from the wild, pollinator and mycorrhizal specificity, inbreeding depression, and anthropogenic modification of the landscape, resulting in fire suppression, altered hydrology, and loss or fragmentation of land due to conversion to residential areas and agriculture (Koopowitz 2001; Wallace 2003; Stewart and Kane 2006; Johnson 2008; Hossain et al. 2013). Kautz et al. (2007) estimate that between

1989 and 2003 over 611,000 hectares of natural and semi-natural land in Florida were converted for urban use and over 700,000 hectares have been converted for agricultural use. Global climate change will have an unknown effect on overall orchid populations, but there is the risk of orchid flowering times and pollinator availability falling out of synchronization, and the effect of climate change is virtually unknown in regards to mycorrhizal fungi (Liu et al. 2010; Bartomeus et al. 2013).

In vitro applications towards plant conservation are a valuable resource in ex situ conservation. The most valuable tools offered are in vitro collections of genetically diverse propagules, both as a living collection but also the capability for large-scale propagation for genetic testing and reintroduction projects, and long-term genebanking using cryopreservation

(Sarasan 2010; Pence 2010). These tools offered through in vitro conservation are considered priorities outlined for ex situ conservation of orchids (Swarts and Dixon 2009; Seaton et al. 2010;

Krupnick et al. 2013).

In Vitro Orchid Propagation

Vegetative Propagation

In 1949 Gavino Rotor Jr used tissue culture to propagate Phalaenopsis from flower stalks, which was effectively the genesis of micropropagation (Hicks 1999). Georges Morel

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followed Rotor and documented the first successful Cymbidium shoot tip culture procedure

(Morel 1960). Most noteworthy in his publication was the first mention of protocorm-like bodies

(PLBs). PLBs are vegetative bodies that could be repeatedly subcultured. PLBs resemble protocorms, and until recently were considered somatic embryos (Lee et al. 2013). However, after monitoring gene expression in Phalaenopsis aphrodite, Fang et al. (2016) discovered PLBs do not follow the known embryogenesis molecular pathway. Instead, they follow a unique regeneration pathway with similarities to shoot organogenesis.

Another three years passed before the first true scientific paper describing a clonal method for propagating orchids was published: the shoot tip culture of Cymbidium (Wimber

1963). Wimber was also the first to publish a detailed report on the successful proliferation of

PLBs from orchid leaf segments for Cymbidium (Wimber 1965). This was a breakthrough because explants could be taken from leaves without endangering the plant as a whole. Floral bud and inflorescence culture was practiced since Rotor’s first report and has since played a major role in the commercial mass production of monopodial orchids such as Phalaenopsis and

Doritaenopsis (Griesbach 1983; Tokuhara and Mii 1993). Vegetative buds and PLBs have been induced from orchid roots, first reported in Catasetum fimbriatum (Kerbauy 1984) and more recently in Oncidium ‘Gower Ramsey’ and Vanda coerulea Griff. (Wu et al. 2004; Lang and

Hang 2006). Thin cell layer (TCL) culture is a relatively new orchid micropropagation technique, first described for Cymbidium by Begum et al. (1994) and later for other species such as Aranda Deborah, Spathoglottis plicata, and Dendrobium nobile Lindl. (Lakshmanan et al.

1995; Teng et al. 1997; Nayak et al. 2002). TCL involves taking thin (<1mm) transverse or longitudinal slices of tissue in order to proliferate PLBs (Teixeira da Silva and Tanaka 2006).

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The release of genetically identical (clonal) plants into natural areas will result in reduced biodiversity, and as such, they are not appropriate propagation methods from a reintroduction standpoint (Keller and Waller 2002; Reed and Frankham 2003). However, these methods offer potential conservation applications through horticultural production as a means to ease poaching pressure.

Seed Propagation

Orchids were the first plants to be propagated in vitro, both from seed and vegetative tissue. The first recorded description of orchid seed germination was by Jean-Henri Fabre in

1856 (Yam and Arditti 2009). Noel Bernard first proposed that germination of orchid seeds was dependent on infection by specific types of fungi in 1899. In 1911, Hans Burgeff successfully isolated a mycorrhizal fungus, which he named Orcheomyces (Yam and Arditti 2009).

Drawing from the research of Bernard and Burgeff, Lewis Knudson concluded that mycorrhizal fungi provided nutrients to infected seeds, causing them to germinate. Further experiments led him to believe orchid seeds could be germinated in the absence of mycorrhizal fungi if provided with the sugars glucose and sucrose. Two nutrient media named Knudson B and C, containing 2% sucrose and supplemented with macro- and micro-elements have been reported (Knudson 1922; Knudson 1924; Knudson 1946). The development of the Knudson media simplified orchid germination and vastly increased the number of orchid growers worldwide (Yam and Arditti 2009). Both Knudson media are used in asymbiotic germination today, along with basal salts and commercially premixed media such as Murashige and Skoog

(MS), Vacin and Went, Hyponex, PhytoTechnology Labs® Orchid Seed Sowing Medium

(P723), Malmgren Modified Terrestrial Orchid Medium, and New Dogashima, amongst others

(Hossain 2008; Dutra et al. 2008; Godo et al. 2010; Roy et al. 2011; Zeng et al. 2012; Abraham et al. 2012; Dowling and Jusaitis 2012).

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Asymbiotic germination media are appropriate for a wide range of species, but in some cases, the medium and environment must be optimized for a given species. Seed germination can be promoted or inhibited in some orchid species due to the form of nitrogen provided (Van Waes and Debergh 1986; Ponert et al. 2013). In general, sucrose is a sufficient carbohydrate source, however, in some species, other simple carbohydrates such as trehalose or mannitol can enhance or inhibit germination (Johnson and Kane 2012). The effects of light, especially photoperiod, are another well-studied variable in asymbiotic germination (Kauth et al. 2011a; Tsutsumi et al.

2011; Johnson and Kane 2012). A general trend is that light is required for epiphytic seed germination, but can inhibit germination in the seeds of many terrestrial species (Kauth et al.

2008a). Organic compounds are also added to media to enhance asymbiotic germination.

Activated charcoal is often used in orchid germination medium to bind phenols and other organic compounds that inhibit germination and growth (Thomas 2008). Coconut water (CW), due to in part to its cytokinin activity, is another common addition to orchid media to promote seed germination and growth (Yong et al. 2009).

Asymbiotic germination procedures have been described for orchid species worldwide, both for conservation and horticultural purposes. Over fifty publications describing asymbiotic seed germination exist for the slipper orchid genus Paphiopedilum alone (Zeng et al. 2016).

Symbiotic seed germination involves the germination of orchid seed in the presence of a compatible mycorrhizal fungus on oatmeal agar, which lacks any sugars that can be used by the orchid seed (Zettler 1997). This procedure requires the isolation of mycorrhizal fungi from the plant into pure culture (Zettler 1997). Fungi can be isolated from root tips of infected orchids or from germinating seeds and protocorms using a seed bait (Sharma et al. 2003; Rasmussen et al.

2015). A seed bait is a sealed packet with netting small enough to contain orchid seeds, but still

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allow fungal hyphae to penetrate. The packet is either buried near the root zone or placed in a tree depending on the target fungus (Brundrett et al. 2003; Zi et al. 2014; Cruz-Higareda et al.

2015). Sterile fungal hyphae tips from pure culture are then maintained on potato dextrose agar media at room or low temperature (Zettler 1997; Stewart and Kane 2006).

Symbiotic seed germination has a high conservation potential due to the production and reintroduction of genetically variable seedlings along with a compatible mycobiont together that allows for future seedling establishment (Massey and Zettler 2007). Symbiotic germination with compatible mycorrhizae resulted in faster protocorm development than the asymbiotic method in

Eulophia alta (Johnson et al. 2007), Dendrophylax lindenii (Hoang et al. 2017), Platanthera integrilabia and Epidendrum conopseum (Yoder et al. 2000).

Liquid Media

Liquid media has been used for in vitro plant production since the 1970s and has been applied to crops ranging from Chrysanthemum, Rhododendron, and Aechmea; all of which responded with higher multiplication rates than in agar-based media (Preil 2005). Liquid culture of orchids has resulted in higher growth rates compared to culture on agar-based medium. This includes seed germination and seedling growth in Doritaenopsis in static culture (Tsai and Chu

2008) and in Cypripedium calceolus var. pubescens agitated at 50 rpm on an orbital shaker (Chu and Mudge 1994). Interestingly, the use of liquid culture also eliminated the need for 8-week prechilling required for germination of C. calceolus var. pubescens on agar-solidified media.

Two novel approaches towards orchid seed germination that implement liquid medium have been reported. Flickingeria nodosa (Dalz.) Seidenf. seed germinated at 95% following 28 days culture in liquid Burgeff’s N3F medium in a matrix of sterilized brick pieces approximately

1 cm in size (Nagananda et al. 2012). Thompson et al. (2007) implemented a dual-phase culture system for the germination of ten African Disa species wherein a layer of solid medium is

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overlaid with an equal volume of liquid medium and agitated at 50 rpm on an orbital shaker. The solid layer was supplemented with activated charcoal to adsorb phenols released in liquid medium by the germinating seed. Seed of six species germinated at high percentages relative to a viability test using this method. Conversely, three species germinated at low percentages and one species did not germinate when sown using the system.

Liquid culture has also been implemented in vegetative propagation of orchids as well as a growth medium for plantlets. PLB multiplication of Phalaenopsis on cotton plates submerged in liquid medium exhibited higher multiplication and growth rates than those germinated on agar-solidified medium (Park et al. 1996). Cattleya plantlets were successfully cultured on liquid medium using microporous polypropylene membranes for 6-8 months (Adelberg et al. 1997).

Vanda tricolor shoots cultured in flasks filled with 5 mL liquid medium agitated at 120 rpm on a gyratory shaker exhibited high growth rates, but rarely survived acclimatization. Hyperhydricity, browning, and necrosis were common in these cultures (Esyanti et al. 2016). Using liquid culture can both save space and reduce the labor associated with using a solid medium, however, the risk of mass contamination, morpho-physiological disorders, and phytotoxicity is increased (Young et al. 2000). In some cases, the use of liquid medium in a bioreactor can reduce these risks.

Bioreactors

Bioreactors are sealed, sterile vessels used for mass culture of tissues or cells in a liquid nutrient media (Paek et al. 2005). Bioreactors have mainly been used for the culture of microorganisms and for the production of secondary metabolites isolated from suspensions of plant cells (Preil 2005). Bioreactors have a wide range of sizes (from 0.5L-500L) and designs that can broadly be defined as either continuous or temporary immersion (Curtis 2005; Paek et al. 2005; Eibl and Eibl 2008). The first group of continuous immersion systems are simple chambers that agitate and deliver oxygen to explants either exclusively with sparger-fed air

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bubbles in the case of bubble column and airlift reactors, or in conjunction with an impeller in the case of stirred reactors. The next group employs the use of a low-cost, disposable, gas- permeable plastic bag filled with media in conjunction with a rocker. The final type of continuous immersion bioreactors are the spray and mist bioreactors, where media is constantly released in either a fine spray or a mist and is generally only used in hairy root production (Eibl and Eibl 2008). Temporary immersion systems are reported to reduce hyperhydricity when compared to continuous immersion in crops such as banana, coffee, and citrus (Berthouly and

Etienne 2005). These systems include tilting/rocker systems, complete immersion chambers that use a system of pumps to drain and refresh the chamber with fresh media, a partial immersion ebb and flood system with explants supported on a membrane or screen, and sealed complete immersion chambers that have the same medium pneumatically pumped and drained from them.

Continuous immersion bioreactors appear to be an ideal culture method for propagation of numerous orchid species. Yang et al. (2010) compared continuous immersion to ebb and flood balloon-type air lift bioreactors using PLB sections of Oncidium ‘Sugar Sweet’ in a modified MS medium and found that the highest PLB growth ratio (10.9) was in the continuous immersion bioreactor after 40 days. Continuous immersion of Dendrobium ‘Zahra FR 62’ shoots in a 3 L bioreactor resulted in higher growth and PLB proliferation compared to culture in gelrite- solidified and liquid-flask cultures (Winarto et al. 2013). PLBs of Dendrobium candidum cultured in 3 L airlift bioreactors showed a fresh biomass increase 150% of those cultured on agar-solidified medium (Yang et al. 2015). Two studies comparing the effect of culture vessels on shoot multiplication of Anoectochilus formosanus both recommend the use of continuous immersion. Wu et al. (2007) found continuous immersion in 3 L balloon-type bubble bioreactors modified with a net to prevent total immersion of explants in the medium resulted in higher A.

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formosanus growth compared to unmodified continuous immersion, temporary immersion, and agar-solidified culture systems. Alternately, Yoon et al. (2007) found continuous immersion of

A. formosanus without a net in 5 L balloon-type bioreactors resulted in four times the biomass accumulation compared to shoots cultured in a netted continuous immersion or temporary immersion system.

In other cases, temporary immersion bioreactors are optimal for orchid production. Rapid multiplication of Phalaenopsis PLB sections was reported in both continuous immersion bioreactors (air-lift column and air-lift balloon) and temporary immersion bioreactors (ebb and flood with or without activated charcoal filtration). The highest PLB proliferation (17/explant) after 8 weeks culture occurred in a temporary immersion bioreactor with activated charcoal filtration (Young et al. 2000). The greatest PLB proliferation rate for Doritaenopsis (6.5/explant) was observed in an air-driven period immersion bioreactor compared to liquid flask or agar- based culture (Liu et al. 2001). Hempfling and Preil (2005) reported multiplication of

Phaelanopsis using a dual 5L glass chamber sealed pneumatic temporary immersion system using shoot explants. The shoot multiplication rate and fresh weight were 2-8 times greater than those cultured on solid media, but the liquid culture period was 12 weeks and the solid culture period was only 8. Shoot multiplication of Vanilla planifolia Jacks. was highest when using a

RITA® brand temporary immersion system (14/explant) when compared to a partial immersion system (8.6/explant) and solid medium (5.8/explant) (Ramos-Castellá et al. 2014). In another study, it was reported that amongst three temporary immersion systems, shoot multiplication of

V. planifolia Jacks. was highest in BIT® brand (18/explant) when compared to RITA®

(12.8/explant) and BIG (6.8/explant) systems (Ramírez-Mosqueda and Iglesias-Andreu 2016).

The growth rate of Vanda tricolor was highest in a thin layer liquid system compared to a RITA®

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temporary immersion system. However, plantlets produced using RITA® systems showed no evidence of hyperhydricity, browning, or mortality during acclimation, a common occurrence in those produced using thin layer systems (Esyanti et al. 2016).

A unique system to multiply rhizomes of Cymbidium sinense was developed by Gao et al.

(2014). The first culture phase uses a continuous immersion bioreactor to induce tissue proliferation, after which the explants are transferred to a temporary immersion system which induces shoot formation followed by further plantlet development (Gao et al. 2014). Both culture phases used 3 L balloon-type bubble bioreactors, however, this methodology was not compared to any other system. Under optimal culture conditions, 27.6 rhizomes/explant were produced after 50 days continuous immersion and 7.2 shoots/rhizome were produced after 40 days temporary immersion.

Other factors affecting orchid bioreactor production include aeration, propagule density, and use of organic medium amendments. Most continuous immersion bioreactors were aerated at an optimal rate of 0.1 volume air per volume of media, but range from 0.06-5.0 (Wu et al. 2007;

Yoon et al. 2007; Yang et al. 2010; Winarto et al. 2013; Yang et al. 2015). The most common temporary immersion period was 2-5 min every 4 h (Liu et al. 2001; Ramos-Castellá et al. 2014;

Ramírez-Mosqueda and Iglesias-Andreu 2016), but range from 5 min every 2 h to 1 h on and 1 h off (Young et al. 2000; Gao et al. 2014). Propagule density was rarely optimized but resulted in significant propagule response when tested. A propagule density of 40 g/L resulted in the highest growth ratio in Oncidium ‘Sugar Sweet’ when compared to 20 or 60 g/L, whereas a density of 8 g/L of Anoectochilus formosanus shoots resulted in the greatest biomass increase compared to 4,

12, or 16 g/L (Yoon et al. 2007; Yang et al. 2010). The use of organic supplements in the culture medium such as activated charcoal and CW promoted tissue growth and proliferation. Three

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studies supplemented media with 0.2-0.5 g/L activated charcoal in continuous immersion bioreactors to mitigate phenolic accumulation (Wu et al. 2007; Yoon et al. 2007; Gao et al.

2014). CW was added to the media in six of the studies, ranging from 3-15% (v/v). Yoon et al.

(2007) found fresh and dry biomass significantly increased in Anoectochilus formsanus when 5%

CW was added to the media when compared to culture medium alone, whereas the effect of CW was not reported in any other published studies.

Orchid Germplasm Banking

Low-Temperature Storage

Seed banking is the most productive and efficient means of ex situ conservation (Pence

2010). Maintaining a seed bank can cost as little as 1% of in situ seed conservation, making it a more economically feasible option (Li and Pritchard 2009). Tissue culture, gene, and seed banks are used for many types of agricultural plants and wildflowers, however, these procedures have not been well developed for orchids (Koopowitz 2001). Orchid seeds are ideal candidates for seed banking due to their minuscule size and high number per capsule. Furthermore, many orchid seeds display orthodox storage behavior. Orthodox seeds are defined by their desiccation tolerance and a trend of increased seed longevity when temperature and or water content (WC) decreases, which contrasts with recalcitrant seeds which are short-lived and possess low desiccation tolerance (Pritchard et al. 2004). No orchid seeds have been described as recalcitrant, however, some species fall under a third storage class, intermediate. Intermediate seeds can withstand a reduction of temperature and desiccation, but not to the degree of orthodox seeds

(Pritchard and Seaton 1993). The Genebank Standards recommend low-temperature storage of orthodox seed at -18 °C whereas this will result in a loss of viability in intermediate seeds, with

15 °C reported as an ideal storage temperature (Ellis et al. 1990; Pritchard et al. 2004).

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A review by Pritchard and Seaton (1993) of research focused on orchid seed longevity under low-temperature conditions (ranging from +10 to -20 °C) indicate an extension in seed viability ranging from 2 weeks to 22 years. More recently, Shoushtari et al. (1994) tested the longevity of seeds of 25 orchid species stored over CaCl2 at ca. 4 °C for 10-20 years. Prior to storage, all seeds germinated to between 80-100%. The longest-lived seeds in this study,

Laeliocattleya ‘Hunter’s Gold’ and Cattleya trianae Linden & Rchb. F., germinated at 25% after

20.5 years storage and 0.21% after 19.2 years of storage, respectively.

Pritchard (1985) predicted an estimated 10 years of viability for seeds of many orchid species stored at 2 °C over desiccant. Thornhill and Koopowitz (1992) report that storage over desiccant and at -20 °C extended the viability of Disa uniflora over ambient conditions, but low- temperature storage is not ideal for long-term storage and suggested storage at -70 °C or lower would be an ideal temperature for long-term seed storage of D. uniflora. Another interesting observation from this study is that freeze-thaw cycles can significantly reduce seed viability.

Alvarez-Pardo and Ferreira (2006) reported that seeds of sixteen Brazilian species retained their viability after 1 year storage at 5 °C with a 6% WC, with the exception of Cattleya labiata which experienced an almost 50% reduction in viability. The results suggest that C. labiata may have intermediate storage characteristics. Seeds of Phaius tankervilleae stored at 4 °C with a 5% WC showed no loss of viability after 3 months, however, a 70% viability loss was observed after 6 months storage at these conditions (Hirano et al. 2009). Hay et al. (2010) used advanced aging techniques to determine that seeds of ten terrestrial Australian orchids were relatively short- lived, though they suggest storage at -18 °C is appropriate for the majority of species when equilibrated at 23% relative humidity.

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Cryopreservation

Although low-temperature storage can extend seed viability for years in some cases, immersion in liquid nitrogen (-196 °C) can theoretically halt biological time (Benson 2008b), extending viability indefinitely. Walters et al. (2004) estimated a half-life of 3,400 years for lettuce seeds stored in liquid nitrogen, whereas a half-life of up to 70 years was estimated for similar seeds stored at -18 °C, indicating that cryopreservation provides a clear advantage over low-temperature storage for long-term storage. Cryopreservation was more cost efficient than maintaining field-grown living collections as a long-term storage method of Coffea spp. (Dulloo et al. 2009). Successful cryopreservation of cells or tissues requires the prevention or control of intracellular ice nucleation during exposure to liquid nitrogen in order to prevent plant mortality

(Benson 2008b). There are two broad methods of cryopreservation: controlled rate cooling and vitrification-based. Vitrification is the transition of a liquid into an amorphous glass state that maintains the properties of a solid but lacking the crystalline structure (Benson 2008b). There are currently seven established vitrification-based procedures.

Controlled rate cooling

Controlled rate cooling is the oldest cryopreservation method, first described by Withers and King (1980). It begins with cold acclimation of donor plants, followed by preculture of tissues in media often supplemented with a cryoprotectant such as proline or dimethyl sulfoxide.

Explants are then loaded into cryovials with a cryoprotectant and held on ice for 30 minutes. The cryovials are then cooled at a controlled rate to a temperature of about -35 °C. This allows ice to form in the cryoprotectant, followed by the extracellular space of tissues. Water is drawn from within the cells of explants due to osmotic potential. Once the appropriate level of dehydration has been reached, cryovials are plunged into liquid nitrogen where tissues vitrify on contact

(Reed and Uchendu 2008).

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Vitrification without plant vitrification solution

Four vitrification methods do not require a vitrification solution. Dehydration, also described as the direct method, is the most basic protocol. Explants are dried in a laminar flow hood or by using compressed air or silica gel to a WC between 10-25% and plunged directly into liquid nitrogen (Engelmann 2011). Pritchard and Nadarajan (2008) suggest a WC between 8-

13% when directly cryopreserving orthodox seeds. Pregrowth procedures employ a defined preculture period in a medium containing a high sucrose concentration, followed by direct immersion in liquid nitrogen. Explants are warmed rapidly at 40 °C (Panis et al. 2002).

Pregrowth-dehydration is a combination of the previous two techniques and has been observed to improve recovery in crops such as asparagus and oil palm compared to standard pregrowth procedures (Uragami et al. 1990; Dumet et al. 1993). The encapsulation-dehydration technique involves suspending explants in sodium-alginate and dispensing the sodium-alginate suspension in a calcium chloride solution, forming a calcium alginate bead. This is followed by a subsequent period of pregrowth in a sucrose-enriched liquid medium ranging from a few hours to a few days. The beads are dehydrated to a WC of approximately 20% (Engelmann et al. 2008). The beads are then loaded into cryovials and rapidly plunged in liquid nitrogen, where the vitrification occurs. Thawing occurs slowly at room temperature (Gonzalez-Arnao and

Engelmann 2006).

Vitrification with plant vitrification solution

The final three vitrification techniques: vitrification, encapsulation-vitrification, and droplet-vitrification employ the use of various plant vitrification solutions (PVS). The most frequently used solutions are PVS2 (Sakai et al. 1990) and PVS3 (Nishizawa et al. 1993). PVS2 consists of 30% glycerol (w/v), 15% ethylene glycol (w/v), and 15% dimethyl sulfoxide (w/v) in growth regulator-free basal media with 0.4M sucrose and PVS3 is made up of 40% glycerol

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(w/v) and 40% sucrose (w/v) in growth regulator-free basal media. These solutions supercool upon rapid exposure to liquid nitrogen at approximately -100 °C and reach a glass phase at approximately -115 °C (Sakai and Engelmann 2007). The vitrification technique includes a preculture period: osmoprotection in a loading solution of 2M glycerol and 0.4M sucrose.

Tissues are then dehydrated using a vitrification solution. Cultures are both rapidly frozen and rewarmed, after which explants are placed in a recovery solution of basal media with 1.2M sucrose (Sakai et al. 2008). The encapsulation-vitrification protocol begins with encapsulation of explants as in the encapsulation-dehydration method, followed by the same steps as in the vitrification method (Sakai et al. 2008). Droplet-vitrification is the most recently developed cryopreservation method. Plant tissues are exposed to a loading solution, treated with a vitrification solution, and inserted into individual droplets of vitrification solution on a sheet of aluminum foil before being rapidly cooled in liquid nitrogen. (Sakai and Engelmann 2007).

Direct Orchid Seed Cryopreservation

The direct method has been employed for the majority of orchid seed cryopreservation studies. Pritchard (1984) first successfully cryopreserved orchid seeds using direct LN immersion. No significant difference in germination rate was observed between frozen and control seed of the terrestrial species Disa uniflora, Eulophia alta, E. stenophylla, E. streptopetala, Gymnadia conopsea, Orchis coriophora, Satyrium nepalense var. ciliatum, and S. napalense var. nepalanse, or in the epiphytic species Phalaenopsis equestris and Vanda pumilla.

Additionally, sequential LN freeze and thaw cycles resulted in a significant increase in germination of Orchis morio seeds when compared to an unfrozen control, likely due to increased water uptake by an ordinarily impermeable testa.

Nikishina et al. (2001) reported successful cryopreservation of six tropical orchid species using the direct method. There was a significant increase in germination between seeds directly

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plunged in LN and unfrozen controls of Calanthe Gorey (from 59% to 90%), C. vestita (from

56% to 69%), and Angraecum magdalenae (from 55% to 80%). There was no difference in germination between frozen and control seeds of Encyclia (=Prosthechea) cochleata, Miltonia flavescens x Brassia longissima, and Trichopilia tortilis Lindl.. Interestingly, frozen seeds of A. magdalenae and M. flavescens x B. longissima germinated between 7-10 days earlier than control seeds. This response may be attributed to enhanced water and nutrient uptake due to increased testa permeability after freezing. Further studies conducted on the post- cryopreservation seedling development of Miltonia flavescens x Bratonia longissima found that frozen seeds developed at a faster rate than controls for the first 45 days after germination.

However, after 639 days there were no differences between seedlings from either treatment with the exception of those germinated on ½ strength MS medium, where the development of frozen seeds was slightly higher (Popova et al. 2003; Popov et al. 2004).

Although considered successful, direct orchid seed cryopreservation can result in varied recovery, as evidenced by the results of Nikishina et al. (2007), Pirondini and Sgarbi (2014), He et al. (2010), and Wang et al. (2011). These researchers described the results of direct cryopreservation on five temperate species, eight Mediterranean species, Dendrobium crysanthum, and Doritis pulcherrima respectively. Cryopreserved seeds germinated at significantly higher rates than unfrozen controls in Dactylorhiza fuchsia (Druce) Soo (from 36% to 44%), D. incarnata (L.) Soo (from 14% to 27%), Ophrys sphegodes ssp. passionis (from 2% to 14%), and Orchis mascula (from 4% to 13%). There was no significant difference in germination between frozen and control seed of Dactylorhiza baltica (Klinge) Orlova, D. sambucina, Anacampis laxiflora, and A. pyramidalis. The germination rate was significantly lower in cryopreserved seeds than unfrozen controls in D. chrysanthum (from 89% to 52%), D.

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pulcherrima (from 96% to 25%), D. maculata (L.) Soo (from 71% to 42%), Anacampis coriophora (from 92% to 83%), Gymnadenia conopsea (from 15% to 6%), Ophrys x arachnitiformis (from 86% to 66%), and Platanthera bifolia (L.) Rich. (from 49% to 24%).

Despite the drop in germination rate of the negatively affected species, the fact that frozen seeds germinated at no less than 40% of the control seeds indicate the procedure was effective. Nikishina et al. (2007) speculated that super-optimal seed WC was the likely cause for the decreased germination rates in D. maculata (L.) Soo and P. bifolia, but were unable to determine WC due to lack of plant material. However, Pirondini and Sgarbi (2014) determined seed WC for A. coriophora (4.4%), G. conopsea (5.3%), and O. x arachnitiformis (5.6%), indicating a factor beyond WC has an effect on survival of directly cryopreserved seed.

A study by Jitsopakul et al. (2012) using immature and mature seeds of Vanda tricolor corroborates the previous report. In this case, immature seeds (45% WC) germinated at 10% after LN immersion compared to 26% in the unfrozen control, both of which were higher than mature seeds (40% WC) that germinated at 1% after LN immersion and 11% in the control. The author suggests dormancy developed in the mature seeds, which may explain the difference in germination rates amongst controls. Conversely, direct immersion was an unsuccessful technique for immature seeds of Bletilla striata, dropping viability of 2-month old seed from 78% to 0% and in 4-month old seed from 99% to 23% (Hirano et al. 2005a). These results were solely attributed to the high WC of immature seeds, which was 84% in 2-month old seed and 33% in 4- month old seed. Based on the few examples available, direct cryopreservation does not appear to be an ideal conservation method for immature seed.

The importance of seed WC in direct seed cryopreservation is further illustrated in a study by Batty et al. (2001) using 4 Australian terrestrial orchids: Caladena arenicola, Diuris

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manifica, Pterostylis sanguinea, and Thelymitra crinita. Seed from each species was either undried or held over silica desiccant for 24 h prior to LN immersion. In all species, the undried seed failed to germinate, whereas dried seed exhibited higher germination than unfrozen controls. Similar studies were conducted using Bletilla formosana and Dendrobium candidum seeds (Wang et al. 1998; Wu et al. 2013). Fresh seed of B. formosana (49.5% WC) and D. candidum (43% WC) that were directly plunged in LN showed a dramatic decline in germination when compared to unfrozen controls. B. formosana germinated at 1.8% compared to 77% in the control and D. candidum failed to germinate compared to 98% in the control. When B. formosana seeds were dried over silica desiccant (1.87% WC) or in an air-conditioned ambient lab setting (24.8% WC) for 24 h prior to LN immersion, the seed germinated at 68.5% and

68.6% respectively. After drying D. candidum seeds between 24 and 72 h prior to LN immersion, the germination rate increased to 95%.

Pre-equilibration of seeds to a set relative humidity prior to direct LN immersion has also been examined. Pritchard et al. (1999) compared germination of five orchid species stored at seven temperatures ranging from 6 °C to -196 °C over the course of a year. Prior to the experiment, all seed treatments were equilibrated in chambers with a 32% relative humidity.

Seed stored at 6 °C was used as the control. Post-storage germination was not significantly different for seeds stored at 6, -13, or -196 °C in four of five species. Dactlyorhiza fuschii seed stored at 6 °C germinated at a significantly lower rate (35%) than those stored at -13 (45%) and -

196 °C (51%). Cryopreserved seed from all species germinated at a lower rate when compared to fresh seed sown prior to equilibration. Four of the five species stored at -196 °C germinated between 50-70% of the fresh seed, except for Dendrobium anosmum (25%).

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Hay et al. (2010) stored the seeds of ten terrestrial orchid species pre-equilibrated to one of seven relative humidity levels between 5 and 92% at -196, -80, and -18 °C for 24 months. A reduction in germination was observed in all species following storage at all temperatures and relative humidity levels. Based on the results, the authors were unable to draw conclusions related to the interactions between WC and storage temperature. However, when comparing the ideal WC for each storage temperature, a higher percentage of cryopreserved seeds survived compared to those at -18 °C in the seeds of two species. Germination was unchanged between seeds stored at -196 °C and -18 °C in four species and was higher in seeds stored at -18 °C in the seeds of four species. Despite these results, cryostorage of orchid seeds was recommended and the authors further suggested that the short-lived behavior and poor-quality seed lots may have affected the results. Based on the results of these studies, it appears a period longer than 1-2 years is necessary to compare viability loss in orchid seeds at varying temperatures.

Orchid Seed Cryopreservation by Vitrification

Vitrification is another common method used for orchid seed cryopreservation ideal for seeds with high WC or intermediate storage characteristics. Seeds that were unable to survive direct cryopreservation and those with a WC as high as 57% have been successfully conserved using the vitrification method (Hirano et al. 2005a; Vendrame et al. 2007; Hu et al. 2013). PVS2 is the cryoprotectant of choice amongst the authors of every published study. The optimal PVS2 preculture time ranged from 15 min to 3 h, the most rudimentary of which do so without the standard preculture in a loading solution. Seed of Doritis (=Phalaenopsis) pulcherrima germinated at 63% following LN exposure compared to 91% in an unfrozen control and Vanda coerulea germinated at 67% compared to 0% in an unfrozen control when treated with PVS2 for

50 and 70 min, respectively (Thammasiri 2000; Thammasiri and Soamkul 2007). Thammasiri

(2008) reported seeds of six Thai orchid species treated with PVS2 for 30-70 min germinated

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between a range of 32-99%, however, no unfrozen control was used to evaluate the efficacy of the vitrification treatment.

Orchid seed is more commonly precultured for 15 or 30 min in a loading solution prior to an optimized PVS2 treatment and is the case for the remainder of seed vitrification studies. Seed of four Dendrobium hybrids was successfully cryopreserved when treated with PVS2 for 1-3 h prior to LN immersion. All cryopreserved seed germinated at a significantly lower rate (50-63%) than unfrozen controls (64-96%) while PVS2 exposure time only had an effect on germination of

‘Jaquelyn Thomas’, which decreased after 1 h PVS2 treatment (Vendrame et al. 2007). Similar results were reported for Bletilla formosana, Dendrobium chrysanthum, Dendrobium Swartz.

‘Dong Yai’, Doritis pulcherrima, and immature Vanda tricolor seed after 10 min, 45 min, 1h, 1-

3 h, and 3h PVS2 treatments respectively (He et al. 2010; Wang et al. 2011; Jitsopakul et al.

2012; Hu et al. 2013; Galdiano et al. 2014). Hirano et al. (2011) screened seven Cymbidium species and found an optimal PVS2 exposure time was 30 min for epiphytic species and 60 min for terrestrial species, which aligns with Yoder et al. (2000) which found epiphytic orchid seed had higher water permeability. There was no significant difference between the relative germination rates of frozen and unfrozen control seeds in these species.

A number of authors have reported that addition of a preculture step on a medium prior to the standard vitrification protocol or a supplement to the PVS2 solution can improve recovery after LN immersion. Preculture on New Dogashima medium with 0.2% gellan gum and 0.3 M sucrose for 3 days in constant light significantly improved germination of immature Bletilla striata seed when compared to the vitrification procedure alone and was later used to successfully cryopreserve mature B. striata and Phaius tankervilleae seed (Ishikawa et al. 1997;

Hirano et al. 2005a; Hirano et al. 2009). Preculture on identical medium for 24h in darkness had

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no significant effect on germination of cryopreserved immature seed of Ponerorchis graminifolia var. suzukiana (Hirano et al. 2005b). A 3 day preculture period in liquid MS medium with 0.3 M sucrose under an unspecified photoperiod was used in the successful cryopreservation of Bletilla formosana seed (Wu et al. 2013). Galdiano et al. (2012; 2013) evaluated the addition of 1% phloroglucinol and 1% Supercool X-1000® to the PVS2 solution for additional cryoprotection of

Dendrobium Swartz. ‘Dong Yai’ and Oncidium flexuosum Sims. Phloroglucinol was an effective supplement to PVS2, resulting in significantly higher germination rates than PVS2 treatments in both species. Supercool X-1000® added to PVS2 promoted germination in O. flexuosum seed when compared to PVS2 alone, but not as well as phloroglucinol. PVS2 supplemented with

Supercool X-1000® did not improve germination of Dendrobium ‘Dong Yai’ seed when compared to PVS2 treatment alone.

The encapsulation-vitrification and droplet-vitrification procedures have been applied to orchid seed once each. Thammasiri (2008) found that encapsulation-vitrification for 80 minutes was optimal for seed of Dendrobium hercoglossum when compared with the standard vitrification procedure, but no unfrozen control treatment was used. Jitsopakul et al. (2008a) used droplet-vitrification preceded by 3 hours preculture on liquid New Dogashima medium with

0.3M sucrose to cryopreserve both mature seed and 3-day germinating seed of Bletilla striata.

No reduction in germination was observed between cryopreserved and control seed.

Flow cytometry has been employed to compare the genetic stability of orchid seedlings developed from cryopreserved seed compared with seedlings derived from non-cryopreserved seed. No change in ploidy level was observed in Oncidium flexuosum or Dendrobium Swartz.

‘Dong Yai’, further confirming phenotypic evidence of genetic stability (Galdiano et al. 2013;

Galdiano et al. 2014). In addition to unchanged ploidy level, no significant differences in peaks

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in relative DNA content in seedlings from frozen and unfrozen immature Bletilla striata seed were reported (Hirano et al. 2005a).

Orchid Seed Cryopreservation by Encapsulation-Dehydration

Encapsulation-dehydration is the least published orchid seed cryopreservation method, though it has proven effective in all reports, including synchronous LN immersion of seed and mycobiont. Mature seeds of Oncidium bifolium Sims. and immature seeds of Cyrtopodium hatschbachii Pabst were successfully cryopreserved when encapsulated, pretreated in liquid medium containing increasing concentrations of sucrose up to 0.75M, and dehydrated using silica gel prior to LN immersion (Flachsland et al. 2006; Surenciski et al. 2012). Following cryopreservation, encapsulated O. bifolium seeds germinated at 67% (19.2% WC), significantly lower than unfrozen encapsulated control seeds that germinated at 90%, however, the number of beads that formed plantlets did not differ between cryopreserved (4.8%) and control beads

(5.7%). Frozen encapsulated C. hatschbachii seed germinated at 64% (18% WC), significantly higher than in unfrozen control beads in which germination was observed (50%). It was reported in both studies that encapsulation, sucrose pretreatment, and dehydration over silica gel had no effect on seed germination compared to unencapsulated control seed. A previous study by

Surenciski et al. (2007), which followed the same encapsulation-dehydration protocol as

Surenciski (2012), found stable ploidy levels (2n=46) and greater mean chromosome length in plantlets grown from cryopreserved seed when compared to unfrozen plants. Despite the discrepancies between chromosome lengths, which the authors suggested resulted from less condensation during mitotic metaphase, the overall chromosomal and phenotypic stability determined that the protocol was appropriate as a means of conservation of the species.

The encapsulation-dehydration technique has the added benefit of cryopreserving orchid seed as well as a mycorrhizal mycobiont. This method requires creating a sterile suspension of

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orchid seed and fungal hyphae in sodium alginate prior to dispensing beads in calcium chloride.

Seeds of Diuris arenaria, Pterostylis saxicola, Dactlyorhiza fuchsii, Anacamptis morio, and their respective mycobionts were successfully cryopreserved using encapsulation-dehydration with

0.75M sucrose pretreatment and 16-18h air drying by laminar flow (Wood et al. 2000;

Sommerville et al. 2008). Seed germination of D. arenaria (12%) and fungal recovery (16%) of its mycobiont Tulasnella calospora were not significantly different between beads stored at -196

°C for 6 months (11% WC) and control beads sown immediately after dehydration (22% germination and fungal recovery). Results were similar in P. saxicola (3.3% WC), seed germination (60%) and fungal recovery (100%) were not significantly different from control beads (68% and 100% respectively). Seeds of D. fuchsii and A. morio were both encapsulated

(20% WC) with Ceratobasidium cornigerum, a basidiomycete mycobiont compatible with both orchid species. After 30 days storage in LN, D. fuchsii germination (85%) and fungal recovery

(100%) was not significantly different than control beads (82% and 100%). No shoot emergence was observed in the cryopreserved or control alginate beads containing this species after 4 months, demonstrating one disadvantage of encapsulating orchid seed. Beads containing A. morio seed and C. cornigerum were stored in LN for 24 h prior to recovery and were negatively impacted by sucrose pretreatment. When the sucrose pretreatment was used in conjunction with air-drying, seed germination (68%) and fungus recovery (100%) was similar to that of the control (72% and 100%). Shoot emergence was significantly lower in this treatment (8%) compared to the control (22%). When the sucrose pretreatment was eliminated and the beads were only dried prior to LN immersion, fungus recovery dropped to 66%, but germination (95%) and shoot emergence (26%) were greater than in the control.

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Protocorm Cryopreservation

There are fewer reports of protocorm and PLB cryopreservation than of seed, but more techniques have been employed. These include dehydration, which resulted in about a 25% survival rate of Dendrobium candidum PLBs when desiccated to 11% WC (Bian et al. 2002).

Encapsulation-dehydration resulted in survival rates of 49% in Cleisostoma areitinum protocorms (Maneerattanarungroj et al. 2007) and 40% in Vanda coerulea protocorms

(Jitsopakul et al. 2008b).

Vitrification is the most published protocorm/PLB cryopreservation method, resulting in

68% survival in Dendrobium nobile protocorms (Vendrame and Faria 2011) and 40% in

Dendrobium ‘Bobby Messina’ PLBs (Antony et al. 2013). Watanawikkit et al. (2012) reported

Caladenia protocorms larger than 4 mm survive at 96%, significantly higher than 1-4 mm (76%) or less than 1 mm (8%) after 60 days cryopreservation. Bukhov et al. (2006) observed initial recovery of Bratonia hybrid protocorms following LN exposure, however, photosynthesis was strongly inhibited which led to the rapid death of the protocorms. Encapsulation-vitrification appears to be successful, resulting in a survival rate above 85% in Dendrobium candidum Wall. ex Lindl. (Yin and Hong 2009). Conversely, with 14% survival, droplet-vitrification was an ineffective procedure for cryopreserving Dendrobium protocorms (Galdiano et al. 2012).

Florida Native Orchid Conservation

Studies regarding developing in vitro germination and seedling development, as well as greenhouse acclimatization of Florida native orchids, are well documented. Asymbiotic seed germination protocols have been described for species including Encyclia boothiana var. erythronioides (Stenberg and Kane 1998), Bletia purpurea (Dutra et al. 2008), and Cyrtopodium punctatum (Dutra et al. 2009b). Symbiotic seed germination protocols have been developed for

Habenaria quinquiseta, H. macroceratitis, (Stewart and Zettler 2002; Stewart and Kane 2006),

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Epidendrum nocturnum (Zettler et al. 2007), and Spiranthes brevilabris (Stewart and Kane

2007). Both asymbiotic and symbiotic protocols exist for Habenaria macroceratitis (Stewart and

Zettler 2002; Stewart and Kane 2006), Eulophia alta (Johnson et al. 2007), and Dendrophylax lindenii (Hoang et al. 2017).

Successful orchid reintroduction studies in Florida have been reported for Spiranthes brevilabris and Epidendrum nocturnum (Stewart 2008). Preliminary studies were conducted for field establishment of Calopogon tuberosus which indicated planting in the early growing season led to highest propagule survival (Kauth et al. 2010).

Another area of research involving Florida native orchids is the ecophysiology of seeds and seedlings. Johnson et al. (2011) observed the effect of carbohydrates and light on Bletia purpurea seed germination and found that availability of compatible carbohydrates had a more significant role in the regulation of germination than photoperiod. An additional study on B. purpurea indicated that low temperature can inhibit germination regardless of light conditions

(Johnson and Kane 2012). Multiple seed germination studies conducted with ecotypes of

Calopogon tuberosus var. tuberosus from Florida, South Carolina, and Michigan indicate the presence of ecotypic variation in physiological dormancy, chilling response, seedling development rate, and corm formation (Kauth et al. 2008b; Kauth and Kane 2009; Kauth et al.

2011b).

The reproductive biology of Florida native orchids has also been investigated. Self- pollination of Eulophia alta resulted in only 7% capsule formation, indicating the reliance on an unobserved pollinator (Johnson et al. 2009). Cyrtopodium punctatum is incapable of self- fertilization and therefore requires a pollinator (Dutra et al. 2009b). Pemberton and Liu (2008) observed an 18-fold capsule formation increase in C. punctatum within Fairchild Botanic

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Gardens compared to Everglades National Park. This was attributed to the proximity of

Brysonima lucida, an oil reward member of Malpighaceae that attracted oil collecting Centris bees, indicating a form of oil reward deceit in C. punctatum.

Project Description

Project Objectives

 Determine the effect of direct cryopreservation on the seed germination and subsequent seedling development of Florida native orchids

 Determine whether the timing of surface sterilization has an effect on survival of directly cryopreserved Florida native orchid seed

 Determine the effect of seed water content on direct cryopreservation of Florida native orchid seed

 Determine the effect of low-temperature storage prior to direct cryopreservation of Florida native orchid seed

 Determine the efficacy of bioreactor culture on the asymbiotic germination of Florida native orchids

 Optimize liquid medium and culture period of flask cultures on orchid seed germination and subsequent development and evaluate their potential as bioreactor inoculum  Determine differences between orchid seed germination on liquid and agar-solidified medium  Determine the differences in further orchid seedling development between bioreactor and agar-solidified medium

Rationale

 Florida native orchids represent approximately one half of the species diversity of North American orchids and many are at risk of extinction due to anthropogenic forces and their complex biology.

 Expanding the knowledge base regarding storage characteristics of orchid seeds and developing standard methodologies for seed banking are necessary for developing a sustainable in vitro orchid conservation program. Defining long-term storage protocols on a species by species basis is the first logical step in an orchid seedbanking plan, as evidenced by the reports in the published literature.

 No studies exist on genebanking of Florida native orchids outside of seed cryopreservation reports on Eulophia alta and Prosthechea cochleata (Pritchard 1984; Nikishina et al. 2001).

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 Development of efficient methods to recover stored germplasm into plants suitable for field reintroduction is another crucial stage of an orchid conservation program. There is also potential to apply the horticultural practice of orchid PLB proliferation in bioreactor systems to seed production for orchid reintroductions.

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CHAPTER 2 DIRECT CRYOPRESERVATION OF SELECTED FLORIDA NATIVE ORCHID SEED

Introduction

With over 28,000 species, the Orchidaceae is the largest angiosperm family (Chase et al.

2015; Christenhusz and Byng 2016) and are found on all continents except Antarctica (Dressler

1990). Orchids also grow in terrestrial, epiphytic, lithophytic, and semi-aquatic habitats

(Koopowitz 2001; Stewart and Zettler 2002). Despite their apparent ubiquity, many orchid species are at risk of extinction due in part to anthropomorphic pressures such as over-collection of wild plants and habitat loss (Krupnick et al. 2013; Seaton et al. 2015). This is further exacerbated by their complex biology which includes producing seeds containing undeveloped embryos with essentially no energy reserves. As a result, the seeds form obligate associations with mycorrhizal fungi to stimulate germination, growth, and survival (Rasmussen 2002;

Dearnaley 2007; Rasmussen et al. 2015). Additionally, pollinator specificity is often limited to a single species (Tremblay 1992). The entire family is listed in either Appendix I or II under the

Convention on International Trade in Endangered Species of Wild Fauna and Flora

(http://www.cites.org). Florida is home to approximately one hundred twenty of the two hundred fifty species native to all of North America (Brown 2006). As of October 2015, seventy-seven of these species have been classified by the Florida Division of Plant Industry as endangered, threatened, or commercially exploitable (http://www.freshfromflorida.com).

Seed banking is a valuable tool for ex situ conservation; it allows for the collection of a large gene pool at as low as 1% of the cost of in situ seed conservation (Li and Pritchard 2009).

Orchid seeds possess a number of characteristics that make them ideal for seed banking. The first of which is that many species possess orthodox storage behavior, meaning their viability can be extended in response to a reduction in storage temperature and/or moisture content (Pritchard

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and Seaton 1993). Furthermore, a single seed capsule can contain hundreds to millions of dust- like seeds (Arditti 1992), ranging in length from 0.05 to 6.0 mm (Arditti and Ghani 2000).

Consequently, a genetically diverse seedbank containing every Florida native orchid could occupy as little as 2-3 square feet. A review by Pritchard and Seaton (1993) of studies focused on orchid seed longevity in low-temperature conditions (ranging from +10 to -20 °C) indicate an extension in seed viability ranging from 2 weeks to 22 years. More recently, Shoushtari et al.

(1994) tested the longevity of seeds of 25 orchid species stored over CaCl2 at ca. 4 °C for 10-20 years. Prior to storage, all seeds germinated between 80-100%. The longest-lived seeds in this study were Laeliocattleya ‘Hunter’s Gold’ and Cattleya trianae Linden & Rchb. F., which germinated at 25% after 20.5 years storage and 0.21% after 19.2 years of storage.

Although a limited extension of orchid seed viability has been observed using low temperature (-10 to -20 °C), storage at -196 °C in liquid nitrogen (LN) can theoretically maintain seed viability indefinitely (Benson 2008b; Kulus and Zalewska 2014). Most orchid seed ultra- low temperature (-196 °C) storage studies have been conducted using one of two methods: direct cryopreservation and vitrification. Direct cryopreservation, the simplest methodology, consists of the immersion of fresh or desiccated tissues, ideally between 8-13% water content (WC) in orthodox seeds, directly in LN followed by a brief recovery in 40 °C water or at ambient room temperature (Pritchard and Nadarajan 2008; Popova et al. 2016). Vitrification methodologies are based on infiltrating plant tissue with a concentrated cryoprotectant solution prior to LN immersion (Vendrame et al. 2014; Popova et al. 2016). Both methods serve to prevent lethal intracellular ice formation that would occur in untreated tissues by increasing the cellular solute concentration. This results in a phase change of water during LN immersion to a vitrified state

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that eliminates the crystalline structure of a solid but retains the remaining physical properties

(Benson 2008b).

Direct cryopreservation of dry seed has been successful in many cases. Pritchard (1984) reported no significant difference amongst germination rates of frozen or control seed in ten terrestrial and epiphytic orchid species. Nikishina (2001) observed only increased or unchanged germination rates in six tropical species compared to unfrozen controls. Temperate orchids appear to show a more variable response to the procedure. When five Russian orchid species were directly cryopreserved, germination was promoted in two species, inhibited in two species, and unaffected in one species when compared to unfrozen controls (Nikishina et al. 2007).

Similarly, when eight Mediterranean species were directly cryopreserved, germination was promoted in two species, inhibited in three species, and unaffected in three species when compared to unfrozen controls (Pirondini and Sgarbi 2014).

The vitrification method has also been generally successful and can serve as an alternative to direct cryopreservation. Vendrame et al. (2007) reported decreased germination of vitrified seeds compared to unfrozen controls of four Dendrobium hybrids, none of which survived direct cryopreservation. Hirano et al. (2011) reported variation in germination of frozen seeds of seven epiphytic and terrestrial Cymbidium species compared to unfrozen controls.

Germination increased in one species, was unchanged in three species, and decreased in three species. The decreased germination observed in the vitrified seeds in these studies remained above 50% of the control, supporting the utility of the procedure.

The interspecific variability reported in response to orchid seed cryopreservation methods indicates ultra-low temperature storage protocols should be defined on an individual species basis, which has been completed for approximately 0.31% of the family. Of the species studied,

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Eulophia alta and Prosthecea cochleata are the only Florida natives, accounting for 1.6% of the one hundred twenty species. By nature of its simple protocol, the direct cryopreservation method is more time and cost efficient than the vitrification method, making it a logical starting point for screening species. The objective of this study is to optimize the protocol of the direct cryopreservation method and analyze its potential for long-term conservation of Florida native orchid seed.

Materials and Methods

Plant Material

Mature seed capsules of the following species were collected from the Florida Panther

National Wildlife Refuge (FPNWR; Collier County, Florida) and Goethe State Forest (GSF;

Levy County, Florida) between 2006 and 2016: Bletia purpurea, Calapogon tuberosus,

Cyrtopodium punctatum, Encyclia tampensis, Epidendrum amphistomum, Epidendrum nocturnum, Epidendrum rigidum, Eulophia alta, and Prosthechea cochleata (Table 2-1). Seeds were removed from their capsules and stored in glass screw cap vials (Part No. 03-339-25C

Fisher Scientific, Pittsburg, PA) over calcium sulfate desiccant (WA Hammond Drierite Co Ltd,

Xenia, OH) for 2 weeks at 23 ±2 °C then transferred to a freezer at -10 °C in darkness until use.

Seeds stored in this manner will be referred to as desiccated seeds. Seed WC was determined using an ultra-microbalance (Model No. XP2U Mettler-Toledo LLC, Columbus, OH) by taking the initial weight of approximately 500 seeds and again after drying seeds in an oven at 80 °C for

24 hours or until a constant mass was reached. Seed viability was determined using a 2, 3, 5 triphenyl tetrazolium chloride (TTC) test adapted from Hosomi et al. (2012). Seeds were suspended in a 10% (w/v) sucrose solution for 24 hours in darkness at 23 ±2 °C. Seeds were then rinsed twice with distilled-deionized (DD) water before being replaced with a 1% (w/v) TTC solution. Seeds were incubated in the TTC solution for 24 hours in darkness at 40 °C. Following

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incubation, seeds were rinsed once with DD water and dispensed on steel blue seed germination paper (Product No. CDB3.5 Anchor Paper Co, St. Paul, MN). Seeds were observed with a stereoscope (Model No. SMZ-2T Nikon USA Inc, Melville, NY) and counted as viable if red staining of the embryo was observed.

Protocol Optimization for Direct Cryopreservation

The effects of dehydration pretreatment and timing of surface sterilization on the germination and subsequent seedling development of cryopreserved seeds of B. purpurea, C. punctatum, E. tampensis, and E. nocturnum were evaluated. Desiccated seeds that were either immediately plunged into LN then surface sterilized (post-sterilized seed) or surface sterilized prior to LN immersion (pre-sterilized seed) were compared with desiccated seed that was surface sterilized with no exposure to LN (unfrozen control seed).

All seed was surface sterilized using a solution of 5 mL absolute ethanol, 3.6 mL 8.25%

NaOCl, and 91.4 mL sterile deionized water for 1 minute followed by 3 rinses of sterile deionized water for 1 minute each. Post-sterilized seed was dispensed into sterile 1.2 mL cryovials (Product No. 10-500-25 Fisher Scientific, Pittsburg, PA). Cryovials were loaded into storage canes (Product No. 5015-0001 Thermo Fisher Scientific, Waltham, MA), inserted into a cryo sleeve (Product No. 4000218 Thermo Fisher Scientific, Waltham, MA), and plunged into

LN. After 1 hour cryovials were removed from LN and rewarmed for 2 minutes in a 40 °C water bath. Pre-sterilized seed was surface sterilized as described above, dispensed on sterile steel blue seed germination paper in an uncovered sterile Petri dish in a laminar air hood (0.46 m∙s-1 airflow) for a drying period of 1 hour before being transferred into cryovials and plunged in LN as described above.

Seed from all treatments were sown in Petri dishes filled with approximately 25 mL of

PhytoTechnology Orchid Seed Sowing Medium (Product No. P723 PhytoTechnology

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Laboratories, Shawnee Mission, KS). Petri dishes were sealed with a single layer of Parafilm M®

(Bemis Co Inc, Neenah, WI) and incubated at 23 ±2 °C in darkness for 2 weeks followed by a

16/8 photoperiod provided by cool white fluorescent bulbs (Product No. F96T12 General

Electric, East Cleveland, OH) at 40 µmol m²s-1. Germination and seedling development were observed and recorded every 2 weeks for 12 weeks. Five replicate dishes were used per treatment. An average of 169 seeds were dispensed in each Petri dish, divided into 4 subreplicates and subjected to aforementioned culture conditions. All experiments were repeated once.

Direct Cryopreservation

A second experiment was conducted using the seeds of C. tuberosus, E. amphistomum, E. rigidum, E. alta, and P. cochleata that eliminated the pre-sterilized seed treatment. Post- sterilized seed and unfrozen control seed were treated as described in the previous experiment.

This experiment was repeated once for all species.

Statistical Analysis

Germination percentages were determined by dividing the number of germinated seeds by the total number of seeds per subreplication. Developmental stage (Table 2-2) percentages were calculated by dividing the number of seeds or seedlings in each stage by the total number of seeds in each subreplicate. A developmental index (DI) was adapted from Kauth et al. (2011):

(N + N ∗ 2 + N ∗ 3 + N ∗ 4 + N ∗ 5 + N ∗ 6) (2-1) DI = 1 2 3 4 5 6 (N1 + N2 + N3 + N4 + N5 + N6) where N1 is the number of seeds in Stage 1, etc. Germination and developmental data were analyzed using generalized linear mixed model procedures (PROC GLIMMIX) and least square means (LSMEANS) at α=0.05 using SAS v9.4.

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Results

Protocol Optimization

In general, both seed germination and DI was greater in seeds surface sterilized after cryopreservation (post-sterilized) when compared to those surface sterilized prior to cryopreservation (pre-sterilized) (Table 2-3; Figure 2-2). Germination of post-sterilized B. purpurea, E. tampensis, and E. nocturnum seed was not significantly different from that of unfrozen controls at any observation period. Germination of post- and pre-sterilized C. punctatum seed was significantly lower than unfrozen seed at 2 weeks (Figure 2-1). For the following 10 weeks, post-sterilized seed germinated at significantly higher rates than pre- sterilized seed but significantly lower than unfrozen seed (Figure 2-1).

After 2 weeks culture, seeds had imbibed water and germination had occurred in every species subjected to each treatment, with the exception of pre-sterilized C. punctatum seeds which germinated after 4 weeks (Figure 2-1). First leaf emergence in post-sterilized B. purpurea plantlets was also observed after 2 weeks. After 4 weeks culture, both protomeristems and leaf primordia emerged in post-sterilized and unfrozen C. punctatum plantlets. Furthermore, first leaf emergence occurred in unfrozen B. purpurea and both unfrozen and post-sterilized E. tampensis plantlets.

After 8 weeks culture, first leaf emergence occurred in all E. nocturnum treatments, as did second leaf emergence in unfrozen B. purpurea and both unfrozen and post-sterilized E. tampensis plantlets. After 12 weeks culture, germination of post-sterilized seeds was not significantly different than unfrozen seeds in all species except for C. puncatum (Table 2-3). In contrast, pre-sterilized seeds germinated at a significantly lower rate than unfrozen or post- sterilized seeds.

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Direct Cryopreservation

After 12 weeks culture, there was no significant difference in germination and developmental index between cryopreserved and unfrozen treatments from all species, with the exception of germination in C. tuberosus where cryopreserved seed germinated at a significantly higher rate than control seed (Table 2-4; Figure 2-4).

Seeds from all species had imbibed water after 2 weeks culture. Germination had also occurred in all species except for E. alta, ranging from 13.7% in control C. tuberosus seed to

93.3% in cryopreserved P. cochleata seed (Figure 2-3). After 4 weeks culture, germination was observed in E. alta, albeit at less than 1%, in both cryopreserved and unfrozen seed.

Furthermore, first leaf emergence was observed in C. tuberosus. After 6 weeks culture, second leaf emergence was observed in C. tuberosus. First leaf emergence occurred in less than 0.1% of cryopreserved E. amphistomum plantlets after 8 weeks, followed by E. rigidum and control E. amphistomum plantlets after 10 weeks culture. Plantlets of P. cochleata and E. alta from both treatments did not form leaves after 12 weeks culture (Figure 2-4).

The germination and DI of cryopreserved E. rigidum seed and DI only of cryopreserved

P. cochleata seed significantly lagged that of the unfrozen controls at 2 weeks culture, but at no other time interval. A similar effect was observed in germination and DI of E. alta at 8 weeks.

Alternately, control seed lagged cryopreserved seed in germination of E. amphistomum and E. alta after 6 weeks culture and germination of C. tuberosus from 8-12 weeks culture (Figure 2-3).

Further Seedling Development

No morphological differences were observed between seedlings from cryopreserved and unfrozen seeds (Figure 2-5). Further seedling growth in either the greenhouse or on

PhytoTechnology Orchid Maintenance/Replate Medium (Product No. P748 PhytoTechnology

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Laboratories, Shawnee Mission, KS) continued to show normal growth when compared to unfrozen controls (Figure 2-6).

Scanning Electron Microscopy

It appears that LN immersion does not cause physical damage to the testa of any of the nine species studied. Ruptures in the testa were observed in both the frozen and unfrozen seed of

B. purpurea, C. tuberosus, E. alta, and P. cochleata (Figure 2-7, 9), whereas no testa damage was observed in the remaining species.

Discussion

Our results confirm that germination was unaffected by direct immersion in LN and post- surface sterilized in eight of nine native orchid species, and although C. punctatum germination was inhibited, this represents a viable long-term orchid seed banking protocol. Conversely, germination of pre-sterilized seed was greatly inhibited by LN immersion and requires more time and materials compared to post-sterilizing seed, so this treatment was eliminated after the first four species were tested. The early seedling development of germinated seeds appears unaffected following cryopreservation.

Water content is a major factor in plant tissues surviving LN immersion. Ensuring seed

WC is below a critical level will prevent intracellular ice formation and instead result in a phase change of the remaining water into a vitrified state (Benson 2008b). The seeds used in this study had a WC ranging from 2-5% (Table 2-1), which falls well below the recommended levels of 8-

13% for direct cryopreservation (Pritchard and Nadarajan 2008; Popova et al. 2016). It is probable that an increase in seed WC can be attributed to the inhibited germination of pre- sterilized seeds. Our results align with a number of previous studies that reported high germination rates following direct cryopreservation of orchid seed (Pritchard 1984; Nikishina et al. 2001; Nikishina et al. 2007; Pirondini and Sgarbi 2014). A study by Wu et al. (2013) further

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demonstrates the importance of seed WC: desiccation of Bletilla formosana seed by silica gel to a WC of 1.87% or using laminar air flow to a WC of 24.8% prior to LN immersion resulted in germination rates of ~69% compared to 1.8% in fresh seed with a WC of 49.5%.

We observed interspecific variation within in the species studied: LN immersion decreased germination in C. punctatum and increased germination in C. tuberosus, though the reasons for these responses are unclear. The significant decrease in post-sterilized C. punctatum seed germination compared to the unfrozen control is similar to reports regarding seeds of other orchid species, such as Oncidium flexuosum and four Dendrobium hybrids, which required a vitrification pretreatment prior to LN immersion in order to germinate at a high rate (Vendrame et al. 2007; Galdiano et al. 2013). Despite the reduction in germination, post-sterilized C. punctatum seed germinated at approximately 75% of control seed, high enough that a vitrification pretreatment is unwarranted. The seed WC of these species was within the range of the others used in the study, therefore other orchid seed properties must play a role in LN freeze tolerance.

Orchid embryos are extremely reduced, comprised solely of an undifferentiated mass of cells surrounded by a thin seed coat (Swamy 1949b; Yam et al. 2013). They lack cotyledons and endosperm commonly found in seeds, and instead utilize a high concentration of lipid and protein bodies within the cells as food reserves (Lee et al. 2006; Yam et al. 2013; Yang and Lee

2014). Crystallization of lipids, particularly triacylglycerols, has been attributed to poor survival of other desiccated oil-rich seeds such as Cuphea and soybean after storage at subfreezing temperatures (Vertucci 1989; Crane et al. 2003; Crane et al. 2006). Cattleya aurantiaca, Microtis media, Pterostylis recurva, and Caladenia flava are orchid species that can tolerate desiccation at temperatures above freezing, but exhibit high mortality when stored at subfreezing temperatures

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(Seaton and Hailes 1989; Hay et al. 2010). Differential scanning calorimetric thermographs of dried seeds of these species showed similar peaks to those of Cuphea and soybean (Pritchard and

Seaton 1993; Merritt et al. 2014). Pritchard and Seaton (1993) suggest the damage to C. aurantiaca at -18 °C is not due to a phase change in the lipids, but instead a conformational change. Lipid composition could explain the effect of cryopreservation on C. punctatum seed, although further research is needed for confirmation.

Similar to the results observed in C. tuberosus, an increase in germination following LN immersion was reported for seeds of Angraecum magdalenae, Calanthe vestita, C. Gorey

(Nikishina et al. 2001), and Orchis morio (Pritchard 1984). Pritchard (1984) suggested this response was not related to increased permeability of the seed coat, but instead due to lipid dissociation within the embryo that then could be used as an immediately available food source.

While this may be the case, immediately available food sources should result in a lag in initial germination of control seed but not final germination as there are sufficient nutrients to support germination in the culture medium.

While an increase in germination following cryopreservation has been attributed to testa damage in orthodox seed from families outside the Orchidaceae, these species had hardened endocarps and in some cases, physical dormancy (Salomão 2002; Tarré et al. 2007). Damage to the seed coat varied amongst species in this study but appears to have no relation to LN immersion since any testa damage found in a given species was observed in both the treatment and control seed. E. alta and C. tuberosus were the most damaged and had been in storage for 8 and 10 years respectively, whereas all other seed had been stored for 2 years or less. This suggests testa damage may be a natural process or a result of mechanical disturbance due to handling. These results align with reports by Pirondini and Sgarbi (2014), where small testa

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breaks were seen in both cryopreserved and unfrozen seed of eight Mediterranean orchid species and Batty et al. (2001), which reported minor pitting in the seed coats of all treatments of four

Australian orchid species.

We recommend direct cryopreservation of desiccated orchid seed as a long-term gene banking method for the conservation of all nine species screened in our study. Desiccation may be an ideal pretreatment for the cryopreservation of orchid seed from many species due to its ease, low cost, and lack of phytotoxicity. However, it is clear that seed response to direct cryopreservation does vary amongst, and perhaps within, species. Further research is required to determine both the ideal and maximum seed WC for cryopreservation of these and other orchid species. Additionally, understanding the role of seed lipid content and composition in LN tolerance could be useful when determining the feasibility of cryopreserving other threatened orchid species.

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Table 2-1. Seed characteristics of nine Florida native orchid species. Species Date Collected Source Viability Mass Water Content (%)* (µg) (% fresh weight) B. purpurea 27 June 2015 FPNWR 84.47 1.498 3.61 C. tuberosus 01 June 2006 GSF 81.29 1.938 4.00 C. punctatum 07 March 2014 FPNWR 97.45 2.419 3.98 E. tampensis 03 March 2014 FPNWR 97.49 1.174 2.22 E. amphistomum 17 March 2016 FPNWR 89.17 0.455 5.33 E. nocturnum 27 June 2015 FPNWR 48.63 0.570 4.79 E. rigidum 17 March 2016 FPNWR 93.99 0.579 4.49 E. alta 20 January 2008 FPNWR 88.37 1.499 4.70 P. cochleata 17 March 2016 FPNWR 97.99** 0.503 4.68 * Determined using TTC test adapted from Hosomi et al. (2012) **Unresponsive to TTC, result of 10-day germination test on P723

Table 2-2. Orchid seed and seedling developmental stages. Stage Description for C. punctatum Description for all other species used 0 Ungerminated seed, no embryo growth Ungerminated seed, no embryo growth 1 Enlarged embryo, intact testa Enlarged embryo, intact testa 2 Enlarged embryo, ruptured testa Enlarged embryo, ruptured testa (=germination) (=germination) 3 Appearance of protomeristem Emergence of first leaf 4 Emergence of two-first leaf primordia Emergence of second leaf 5 Elongation of shoot and further Elongation of second leaf and further development development Adapted from Dutra et al. (2009) and Stewart and Zettler (2002).

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Table 2-3. Effect of surface sterilization timing on germination of four directly cryopreserved Florida native orchid species. Means with the same letter within species are not significantly different by Tukey-Kramer grouping for least square means (α=0.05). All values are percent germination at 12 weeks culture following immersion in liquid nitrogen. Treatment Germination (%) B. purpurea C. punctatum E. tampensis E. nocturnum Surface sterilize after 85.79a 56.75b 91.52a 85.62a cryopreservation Surface sterilize before 39.98b 0.49c 0.27b 40.28b cryopreservation Unfrozen control 83.41a 76.03a 87.94a 94.80a

Table 2-4. Effect of direct cryopreservation on germination of five Florida native orchid species. Means with the same letter within species are not significantly different by Tukey-Kramer grouping for least square means (α=0.05). All values are percent germination at 12 weeks culture following immersion in liquid nitrogen. Treatment Germination (%) C. tuberosus E. amphistomum E. rigidum E. alta P. cochleata Cryopreserved 40.67a 95.30a 97.33a 93.92a 97.11a Unfrozen control 35.43b 93.98a 96.97a 95.86a 95.57a

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Figure 2-1. Effect of surface sterilization (SS) timing on seed germination of four Florida native orchid species every 2 weeks for 12 weeks culture. Values represent mean response of the experiment repeated once. Bars represent ± standard error.

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Figure 2-2. Effect of surface sterilization timing on seedling developmental index of four Florida native orchid species following 12 weeks culture. Values represent mean response of the experiment repeated once. Means with the same letter within species are not significantly different by Tukey-Kramer grouping for least square means (α=0.05).

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Figure 2-3. Effect of direct cryopreservation on seed germination of five Florida native orchid species every 2 weeks for 12 weeks culture. Values represent mean response of the experiment repeated once. Bars represent ± standard error.

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Figure 2-4. Effect of direct cryopreservation on seedling developmental index of five Florida native orchid species following 12 weeks culture. Values represent mean response of the experiment repeated once. Means with the same letter within species are not significantly different by Tukey-Kramer grouping for least square means (α=0.05).

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Figure 2-5. Development of orchid seedlings after 1 year culture following experimental conditions. A) Unfrozen B. purpurea seedlings, B) Post-sterilized B. purpurea seedlings, C) Pre-sterilized B. purpurea seedlings. D) Unfrozen C. punctatum seedlings, E) Post-sterilized C. punctatum seedlings, F) Pre-sterilized C. punctatum seedlings. Scale bar = 1 cm. Photo courtesy of author.

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Figure 2-6. Further development of orchid seedlings. A) B. purpurea seedlings after 30 weeks in greenhouse, B) C. punctatum seedlings after 1 year on PhytoTechnology Orchid Maintenance/Replate Medium (P748). Scale bars = 1 cm. Photo courtesy of author.

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Figure 2-7. Scanning electron micrographs of unfrozen and frozen seed. Arrows indicate seed coat damage. A) Unfrozen B. purpurea, B) Frozen B. purpurea, C) Unfrozen C. tuberosus, D) Frozen C. tuberosus, E) Unfrozen C. punctatum, F) Frozen C. punctatum. Scale bar = 100 µM. Photo courtesy of author.

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Figure 2-8. Scanning electron micrographs of unfrozen and frozen seed. A) Unfrozen E. tampensis, B) Frozen E. tampensis, C) Unfrozen E. amphistomum, D) Frozen E. amphistomum, E) Unfrozen E. nocturnum, F) Frozen E. nocturnum. Scale bar = 100 µM. Photo courtesy of author.

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Figure 2-9. Scanning electron micrographs of unfrozen and frozen seed. Arrows indicate seed coat damage. A) Unfrozen E. rigidum, B) Frozen E. rigidum, C) Unfrozen E. alta, D) Frozen E. alta, E) Unfrozen P. cochleata, F) Frozen P. cochleata. Scale bar = 100 µM. Photo courtesy of author.

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CHAPTER 3 EFFECTS OF WATER CONTENT AND LOW-TEMPERATURE STORAGE ON CRYOPRESERVATION OF TWO FLORIDA NATIVE ORCHIDS

Introduction

The Orchidaceae is the largest plant family and among the most diverse with approximately 28,000 species within 736 genera (Chase et al. 2015; Christenhusz and Byng

2016). However, many orchid species are endangered or threatened in the wild due to various factors including loss or alteration of habitats, over-collection, pollinator specificity, and inbreeding depression (Koopowitz 2001; Wallace 2003). Seed banking is a well-established method of ex situ plant conservation used for long-term storage of genetically diverse propagules, often at subzero temperatures (Linington and Pritchard 2001; Neto and Custódio

2005).

Orchids are ideal candidates for conservation through seed banking. They produce seed capsules that contain thousands to millions of extremely small (0.05-6.0 mm) seeds (Arditti and

Ghani 2000), which allows for a diverse genepool to be stored in a small volume. Additionally, many orchid seeds display orthodox seed behavior, which is characterized by high desiccation tolerance and an extension of viability when water content (WC) and storage temperature decrease (Pritchard et al. 2004; Pritchard and Nadarajan 2008). Furthermore, orchid seeds are most efficiently germinated using in vitro techniques, where nutrients are provided from the medium or in co-culture with isolated mycorrhizal fungi that are normally provided through mycorrhizal associations in nature. Although many seeds germinate readily in response to a broad range of media or mycorrhizal fungi, others have specific germination requirements

(Rasmussen 2002; Johnson and Kane 2012; Dowling and Jusaitis 2012; Ponert et al. 2013;

Rasmussen et al. 2015). Therefore, seed banking can provide an extension of seed viability until optimal germination protocols can be established.

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Conventional low-temperature seed storage procedures are generally followed in accordance with genebank standards defined by the Food and Agricultural Organization of the

United Nations (FAO) (Linington and Pritchard 2001; Hay and Probert 2013), which recommends storage of orthodox seeds at -18 °C following desiccation (FAO 2014). Hay et al.

(2010) suggest orchids produce relatively short-lived seeds, which also appears to be reflected in low-temperature storage studies. The longest successful orchid seed storage reported is

Laeliocattleya ‘Hunter’s Gold’, which retained viability after 20.5 years storage at 4 °C

(Shoushtari et al. 1994), relatively shorter than the majority of angiosperm species (Walters et al.

2005). Seed banking through ultra-low temperature storage (cryopreservation) at -196 °C in liquid nitrogen (LN) halts biological aging, which can theoretically extend viability indefinitely

(Walters 2004; Walters et al. 2004; Benson 2008b). Direct immersion in LN, also known as direct cryopreservation, was a successful storage method for desiccated seed of nine Florida native species (see Chapter 2). Similar findings have been reported in a diverse range of orchid species (Pritchard 1984; Nikishina et al. 2007; Pirondini and Sgarbi 2014).

A critical component of successful orchid seed cryopreservation is seed WC. A superoptimal WC can lead to lethal intracellular ice formation upon freezing (Benson 2008b).

Batty et al. (2001) reported 0% germination in cryopreserved fresh seed of four Australian orchids, however, cryopreservation of seed dried over silica desiccant for 24 hours resulted in seed germination similar to that of unfrozen controls. Similarly, Wu et al. (2013) reported an increase in germination of Bletilla formosana following cryopreservation from 2% in fresh seed to 69% in seed dried using silica desiccant or laminar air flow for 24 hours. Additionally, differences in germination of orchid seed that has been equilibrated to specific relative humidity

(RH) levels prior to cryopreservation suggests that interspecific variability exists in optimal seed

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WC. Pritchard et al. (1999) reported that seed of five orchid species equilibrated to 32% RH prior to cryopreservation for 1 year germinated at a rate between 25-70% of unfrozen controls.

Hay et al. (2010) also reported interspecific variation amongst ten Australian orchid species equilibrated to one of seven RH levels between 5-92% prior to cryopreservation. Although drying of orchid seeds is essential to tolerating cryopreservation, Pritchard et al. (1999) found over-drying seeds can result in reduced longevity.

The effect of storage temperature prior to immersion in liquid nitrogen is an unexplored facet of direct cryopreservation of orchid seed. Although FAO genebank standards recommend storage of orthodox seeds at -18 °C following desiccation (FAO 2014), no published reports exist on direct cryopreservation of orchid seeds previously stored at sub-zero temperatures. Instead, orchid seeds are typically stored at temperatures between 2-20 °C following initial seed desiccation (Pritchard 1984; Pritchard et al. 1999; Popova et al. 2003; Hay et al. 2010; Pirondini and Sgarbi 2014).

The purpose of this study was to optimize seed moisture content and determine the effects of storage time at -10 °C prior to direct cryopreservation on post-cryopreservation germination and subsequent seedling development of Florida native orchid seed using Bletia purpurea and Epidendrum nocturnum.

Materials and Methods

Plant Material

Mature seed capsules of Bletia purpurea and Epidendrum nocturnum were collected from the Florida Panther National Wildlife Refuge (Collier County, Florida) in June 2015. Seeds were removed from their capsules and stored in glass screw cap vials (Part No. 03-339-25C Fisher

Scientific, Pittsburg, PA) over calcium sulfate desiccant (WA Hammond Drierite Co Ltd, Xenia,

OH) for 2 weeks at 23 ±2 °C. Unless otherwise stated, seeds were then transferred to a freezer at

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-10 °C in darkness until use. Seed WC was determined using an ultra-microbalance (Model No.

XP2U Mettler-Toledo LLC, Columbus, OH) by taking the initial weight of approximately 500 seeds and again after drying seeds in an oven at 80 °C for 24 hours until a constant mass was reached. Seed viability was determined using a 2, 3, 5 triphenyl tetrazolium chloride (TTC) test adapted from Hosomi et al. (2012). Seeds were suspended in a 10% (w/v) sucrose solution for 24 hours in darkness at 23 ±2 °C. Seeds were then rinsed twice with distilled-deionized (DD) water before being replaced with a 1% (w/v) TTC solution. Seeds were incubated in the TTC solution for 24 hours in darkness at 40 °C. Following incubation, seeds were rinsed once with DD water and dispensed on steel blue seed germination paper (Product No. CDB3.5 Anchor Paper Co, St.

Paul, MN). Seeds were observed with a stereoscope (Model No. SMZ-2T Nikon USA Inc,

Melville, NY) and counted as viable if red staining of the embryo was observed. Seed viability was determined to be 81.62% in B. purpurea and 53.06% in E. nocturnum.

Experiment 1: Effect of Seed Moisture Content on Direct Cryopreservation

In order to adjust seed WC, seeds were equilibrated to one of seven relative humidity

(RH) levels using saturated salt solutions (Table 3-1). Saturated salt solutions were prepared using boiling DD water. Once cooled, saturated salt solutions were then transferred into airtight

0.6 L polypropylene containers (Product No. 0411 Sterilite Co, Townsend, MA; Figure 3-5).

Saturated salt solutions were maintained as a slurry and covered in 2-3 mm of DD water.

Containers were stored at 25 °C with saturated salt solutions for 2 weeks to allow for RH stabilization.

Seeds were placed in three micro aluminum weigh dishes (Product No. EW-01019-07

Cole-Parmer Instrument Co, Vernon Hills, IL) per treatment, one containing approximately 1000 seeds designated for cryopreservation and two containing approximately 500 seeds each used to measure seed WC. Weigh boats were held above saturated salt solutions in polypropylene

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enclosures at 25 °C in the dark and weighed weekly using the ultra-microbalance until seed weights in all treatments equalized.

Following seed weight equilibration, seeds designated for WC determination were placed in a drying oven at 80 °C for 48 hours until a constant weight was reached. Seeds designated for cryopreservation were loaded into 1.2 mL cryovials and directly plunged in LN. After 1 hour cryovials, were removed from LN and rewarmed for 2 minutes in a 40 °C water bath. Seed was then surface sterilized for 1 minute using a solution of 5 mL absolute ethanol, 3.6 mL 8.25%

NaOCl, and 91.4 mL sterile deionized water, followed by 3 rinses of sterile deionized water for 1 minute each. Following sterilization, seeds from each treatment were sown in Petri dishes containing approximately 25 mL of PhytoTechnology Orchid Seed Sowing Medium (Product

No. P723 PhytoTechnology Laboratories, Shawnee Mission, KS). An average of 138 seeds were dispensed in each Petri dish, divided into 4 subreplicates and subjected to aforementioned culture conditions. Five replicate dishes were used per treatment. Petri dishes were sealed with a single layer of Parafilm M® (Bemis Co Inc, Neenah, WI) and incubated at 23 ±2 °C in darkness for 2 weeks followed by a 16/8 photoperiod provided by cool white fluorescent bulbs (Product No.

F96T12 General Electric, East Cleveland, OH) at 40 µmol m²s-1. Germination and seedling development were recorded every 2 weeks for 12 weeks. This experiment was repeated once.

Experiment 2: Effect of Low-Temperature Storage on Orchid Seed Cryopreservation

A second experiment was conducted to determine if seed storage at -10 °C had an effect on post-cryopreservation response. Seeds were stored at -10 °C for 1, 7, 14, 28, and 42 days prior to direct cryopreservation. Seeds were also directly cryopreserved at 0 days (immediately after 2 weeks desiccation at 23 ±2 °C). Non-cryopreserved seeds from 0, 28, and 42 days of -10 °C storage were used as controls.

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Following storage, seeds were dispensed into sterile 1.2 mL cryovials (Product No. 10-

500-25 Fisher Scientific, Pittsburg, PA). Cryovials were loaded into storage canes (Product No.

5015-0001 Thermo Fisher Scientific, Waltham, MA), inserted into a cryo sleeve (Product No.

4000218 Thermo Fisher Scientific, Waltham, MA), and plunged into liquid nitrogen (LN). After

1 hour, cryovials were removed from LN and rewarmed for 2 minutes in a 40 °C water bath.

Cryopreserved seeds and non-cryopreserved control seeds were surface sterilized using a solution of 5 mL absolute ethanol, 3.6 mL 8.25% NaOCl, and 91.4 mL sterile deionized water for 1 minute followed by 3 rinses of sterile deionized water for 1 minute each. Seeds from both treatments were sown in Petri dishes containing approximately 25 mL of P723. An average of

102 seeds were dispensed in each Petri dish, divided into 4 subreplicates and subjected to aforementioned culture conditions. Petri dishes were sealed with a single layer of Parafilm M®

(Bemis Co Inc, Neenah, WI) and incubated at 23 ±2 °C in darkness for 2 weeks followed by a

16/8 photoperiod provided by cool white fluorescent bulbs (Product No. F96T12 General

Electric, East Cleveland, OH) at 40 µmol m²s-1. Germination and seedling development were recorded every 2 weeks for 12 weeks. Five replicate dishes were used per treatment. This experiment was not repeated due to lack of plant material.

Statistical Analysis

Germination percentages were determined by dividing the number of germinated seeds by the total number of seeds per subreplication. Developmental stage (Table 3-2) percentages were calculated by dividing the number of seeds or seedlings in each stage by the total number of seeds in each subreplicate. A developmental index (DI) was adapted from Kauth et al. (2011):

(N + N ∗ 2 + N ∗ 3 + N ∗ 4 + N ∗ 5 + N ∗ 6) (3-1) DI = 1 2 3 4 5 6 (N1 + N2 + N3 + N4 + N5 + N6)

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where N1 is the number of seeds in Stage 1, etc. Germination and developmental data were analyzed using generalized linear mixed model procedures (PROC GLIMMIX) and least square means (LSMEANS) at α=0.05 using SAS v9.4.

Results

Moisture Content

We observed interspecific variation in equilibrium WC, B. purpurea seed consistently equilibrated to lower WC than E. nocturnum at each RH level (Table 3-3). Seed moisture content prior to direct LN immersion had a significant effect on germination and DI of both B. purpurea and E. nocturnum. Germination and DI of B. purpurea seed equilibrated to 6, 13, and 33% RH prior to cryopreservation were not significantly different than unfrozen controls at any observation period (Table 3-3, Figure 3-1, 3-2). Germination of E. nocturnum seed equilibrated to 6, 13, 33, and 55% RH prior to cryopreservation was not significantly different from unfrozen controls at any observation period (Table 3-3, Figure 3-1). The DI of E. nocturnum seed from all cryopreserved treatments was lower than in the unfrozen control, but amongst cryopreserved seed was highest in those equilibrated to 13 and 55% RH (Figure 3-2). All seed equilibrated to

90% RH was completely contaminated by fungi after 2 weeks. As such, it was not included as a treatment when the experiment was repeated.

After 2 weeks culture, B. purpurea seed subject to all treatments had imbibed water and germinated. At this time, only seed equilibrated to 76% RH germinated at a significantly lower rate than the unfrozen control (Figure 3-1). First leaf emergence was observed in all B. purpurea treatments after 4 weeks except for those equilibrated to 0% RH, which occurred after 6 weeks.

Second leaf emergence was also observed after 6 weeks in treatments equilibrated to 6, 13, 55, and 76% RH. After 8 weeks, second leaf emergence was observed in treatments equilibrated to 0 and 33% RH. Interestingly, second leaf emergence occurred in unfrozen control seedlings after

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all other treatments at 10 weeks. Approximately 4% of the seeds equilibrated to 76% RH that germinated, turned pink-orange in color and began dying after 8 weeks culture (Figure 3-7).

After 12 weeks culture, seed germination when equilibrated to 6, 13, and 33% RH was not significantly different from the control, seed equilibrated to 0 and 55% RH germinated to a slightly lower level, and those equilibrated to 76% RH was drastically lower, at half of the unfrozen control.

Similar to B. purpurea, E. nocturnum seed equilibrated to all RH levels had imbibed water and germinated at 2 weeks. Only seed equilibrated to 76% RH did so at a lower rate than unfrozen control seed. First leaf emergence was observed at 8 weeks in treatments equilibrated up to 55% RH and at 10 weeks in those equilibrated to 76% RH. After 12 weeks culture germination was not significantly different from the control except for seed equilibrated to 0 and

76% RH, which germinated at about 89% and 4% of the control respectively. Additionally, second leaf emergence was observed in up to 2% of seedlings except those equilibrated to 76%

RH.

Low-Temperature Storage

Freezer storage at -10 °C for up to 6 weeks appears to have little effect on the germination of B. purpurea or E. nocturnum seed regardless if they were cryopreserved (Figure

3-3). While the subsequent development of B. purpurea seedlings was similarly unaffected by any freezer storage treatment, a clear reduction in DI can be observed in E. nocturnum seedlings following 28 and 42 days freezer storage (Figure 3-4).

After 2 weeks culture, B. purpurea seed subject to all treatments had imbibed water and germinated. First leaf emergence was observed after 4 weeks at no more than 1% in control seedlings stored for 28 days and both cryopreserved and control seedlings stored for 42 days which increased to 2-5% in all treatments after 6 weeks culture. Second leaf emergence was first

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observed after 8 weeks culture in less than 0.3% of cryopreserved seedlings stored for 42 days.

After 10 weeks culture second leaf emergence had occurred in 2% of cryopreserved and control seedlings stored for 42 days and in less than 1% of control seedlings that weren’t freezer stored and those that were cryopreserved after 28 days freezer storage. After 12 weeks culture, there were no differences in germination amongst the treatments (Table 3-4). By this time second leaf emergence was observed in all treatments, with an overall increase in seedlings with two leaves after 28 and 42 days of freezer storage (Figure 3-4).

E. nocturnum seed subject to all treatments had also imbibed water and germinated after

2 weeks culture. After 8 weeks culture, first leaf emergence was observed in 0.4-3.5% of seed subject from 0 to 14 days freezer storage. After 10 weeks culture first leaf emergence was observed in all treatments, ranging from 3.5% to 24% in seed cryopreserved after 42 days and 1 day freezer storage respectively. After 12 weeks culture, the only significant reduction in germination was between seed cryopreserved without prior freezer storage and control seeds subject to 0 and 28 days freezer storage. Additionally, second leaf emergence was observed in less than 1% of seedlings cryopreserved after 7 and 14 days freezer storage and control seed that had not been exposed to freezer storage. Counter to what was observed in B. purpurea, there was a trend of reduced leaf emergence with increased storage time (Figure 3-4).

Discussion

Moisture Content

Despite the variation amongst treatment responses, the final germination of both B. purpurea and E. nocturnum equilibrated to RH levels up to 55% are acceptable from the perspective of long-term seed banking. Although storage at 76% RH was deleterious to the germination of both species used, E. nocturnum was more sensitive to extreme RH. We also

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observed interspecific variation in equilibrium WC and post-cryopreservation seedling development.

Seed WC is perhaps the most important factor in surviving direct cryopreservation, and an ideal range for orthodox seed falls between 8-13% (Pritchard and Nadarajan 2008; Popova et al. 2016). Only one treatment, E. nocturnum seed equilibrated to 55% RH, fell within this range

(9.7%). The only other seeds within the range were those from both species equilibrated to 76%, which we did not consider an ideal treatment. This could suggest that a lower range of WC is ideal for orthodox orchid seed, which would explain our results and those such as seed of a

Dendrobium hybrid dried to ~10% WC failing to germinate following direct cryopreservation

(Vendrame et al. 2007). Another possibility raised by Hay et al. (2010) is that although many orchid seeds share some characteristics with orthodox seed, they cannot be classified as such due to their unpredictable storage behavior. While the storage behavior of many more orchid species is required to make a definitive statement either way, the latter suggestion certainly explains the report of Prosthechea cochleata seed germinating at 100% following direct cryopreservation despite a WC of 24% (Nikishina et al. 2001).

It is notable that of the acceptable treatments, equilibration to 0% RH prior to cryopreservation resulted in the lowest germination (approximately 89% of the control) in both

B. purpurea and E. nocturnum. Over-drying of orchid seed has been attributed to reduced longevity for Dactylorhiza fuchsia, Dendrobium anosmum, Eulophia gonychila, and Cattleya aurantiaca (Seaton and Hailes 1989; Pritchard et al. 1999). Therefore, the reduction in viability we observed could be an effect of dry storage prior to LN immersion.

The two responses that we observed that differed between B. purpurea and E. nocturnum were the equilibrium WC at each RH level and seedling DI after germination. A negative

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correlation between seed WC and lipid content has been reported for seeds of multiple species

(Vertucci 1989; Sacandé et al. 2000; Hor et al. 2005), and has also been applied to orchid seed

(Pritchard and Seaton 1993; Pritchard et al. 1999). If we assume these species follow the same general trend, the differences in WC can be attributed to B. purpurea having a higher seed lipid content than E. nocturnum.

The interspecific variability in DI is less easily explained. While B. purpurea seedling development was uniform amongst treatments, that of E. nocturnum was significantly less so. A reduction in seedling vigor with an increasing seed storage period was reported for another orchid, Disa uniflora, which was attributed to a tradeoff between viability and vigor (Thornhill and Koopowitz 1992). The variability within E. nocturnum treatments may be further evidence of unpredictable orchid seed storage behavior.

Although the slight increase in germination after hydrating seeds to optimal WCs may not warrant using saturated salt solutions, equilibration to ambient indoor conditions prior to cryopreservation may have a similar effect. This procedure could also prove valuable in cryopreservation of intermediate-type orchid seeds that rapidly lose viability when stored at subzero temperatures or in species otherwise recalcitrant to direct cryopreservation after desiccation such as such as C. aurantiaca (Seaton and Hailes 1989) or Cyrtopodium punctatum

(Chapter 2).

Low-Temperature Storage

We found that freezer storage does not appear to offer any benefit prior to direct cryopreservation of desiccated B. purpurea or E. nocturnum seed. However, we can recommend storage at -10 °C to extend seed viability prior to cryopreservation of these species.

Although there was no significant difference in DI amongst treatments for B. purpurea, more seedlings developed a second leaf when seed had been stored for 28-42 days regardless of

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cryopreservation. There was a lag in germination after 2 weeks for seed stored 0-1 day (24-32%) compared to those stored 7 days or more (58-64%). This suggests there was an after-ripening effect, or relief of non-deep physiological dormancy after dry storage (Finch-Savage and

Leubner-Metzger 2006), that reduced time until germination rather than more rapid seedling development following germination. This aligns with a reported increase in seed germination for seven Australian orchid seeds that was attributed to an after-ripening period ranging from 15-26 days (Hay et al. 2010).

In contrast with B. purpurea, E. nocturnum seed stored for 28-42 days exhibited decreased seedling vigor. This response is similar to what we observed in the moisture content experiment, although a clearer trend between treatment and response exists. As previously mentioned, a similar decrease in seedling vigor for Disa uniflora with increasing storage has been reported (Thornhill and Koopowitz 1992).

Shoushtari et al. (1994) concluded that maintaining orchid seed over desiccant at refrigerator temperatures (4 °C) was a simple and inexpensive storage method. We do not fully dispute this assertion, as we reached a similar conclusion for the species we used, albeit at a lower temperature. However, it is clear through our study and others (Pritchard and Seaton 1993;

Hay et al. 2010) that there is interspecific variation in seed storage response within the

Orchidaceae which should be considered prior to storage at any single temperature.

Conclusions

Based on the post-cryopreservation responses observed in this study, we can recommend the direct cryopreservation of B. purpurea and E. nocturnum seed equilibrated to an RH ranging from 0-55%. Additionally, the WC threshold for direct cryopreservation of orthodox orchid seed appears to be lower than that of other orthodox seed. Optimizing seed WC prior to direct

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cryopreservation could provide value to seed that previously required vitrification treatments prior to cryopreservation or are otherwise recalcitrant to direct cryopreservation.

We can also recommend the storage of B. purpurea and E. nocturnum seed at -10 °C to temporarily extend seed viability prior to direct cryopreservation. Further studies should examine seed response to long-term storage at room temperature, freezer temperature, and liquid nitrogen temperature to further elucidate the storage behavior of these and other Florida native orchid species.

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Table 3-1. Relative humidity values generated by saturated salt solutions at 25 °C. Salt Relative humidity

CaSO4 0%

ZnCl2 6% LiCl 13%

MgCl2 33% NaBr 55%

NH4Cl 76%

KNO3 90% Adapted from Vertucci and Roos (1993).

Table 3-2. Orchid seed and seedling developmental stages. Stage Description 0 Ungerminated seed, no embryo growth 1 Enlarged embryo, intact testa 2 Enlarged embryo, ruptured testa (=germination) 3 Emergence of first leaf 4 Emergence of second leaf 5 Elongation of second leaf and further development Adapted from Dutra et al. (2009b) and Stewart and Zettler (2002).

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Table 3-3. Effect of storage relative humidity prior to direct cryopreservation on seed moisture content and germination of two Florida native orchid species. Means with the same letter within species are not significantly different by Tukey-Kramer grouping for least square means (α=0.05). Moisture content values are percent seed moisture content prior to direct cryopreservation. Germination values are percent germination at 12 weeks culture following immersion in liquid nitrogen. B. purpurea E. nocturnum Moisture Moisture Relative Humidity (%) Content (%) Germination (%) Content (%) Germination (%) 0 3.25 83.37b 4.53 81.23c 6 3.79 93.39a 5.42 87.30bc 13 4.67 90.82ab 6.45 95.45a 33 5.61 88.97ab 7.64 92.05ab 55 6.77 83.61b 9.70 95.02a 76 11.46 47.25c 14.25 3.89d Unfrozen control 3.74 93.29a 4.86 90.79ab

Table 3-4. Effect of storage at -10 °C prior to direct cryopreservation on germination of two Florida native orchid species. Means with the same letter within species are not significantly different by Tukey-Kramer grouping for least square means (α=0.05). All values are percent germination at 12 weeks culture following immersion in liquid nitrogen. Days stored at -10 °C Germination (%) B. purpurea E. nocturnum 0 88.27a 81.62b 1 81.64a 84.25ab 7 85.76a 88.88ab 14 81.83a 84.18ab 28 84.50a 85.84ab 42 81.51a 86.29ab Unfrozen control 0d 88.33a 89.95a Unfrozen control 28d 82.26a 89.34a Unfrozen control 42d 81.06a 87.27ab

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Figure 3-1. Effect of pre-cryopreservation equilibrium relative humidity on post-cryopreservation seed germination for two Florida native orchids every 2 weeks for 12 weeks culture. Values represent mean response of the experiment repeated once. Bars represent ± standard error.

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Figure 3-2. Effect of pre-cryopreservation equilibrium relative humidity on post- cryopreservation seedling developmental index for two Florida native orchids following 12 weeks culture. Values represent mean response of the experiment repeated once. Means with the same letter within species are not significantly different by Tukey-Kramer grouping for least square means (α=0.05).

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Figure 3-3. Effect of pre-cryopreservation storage at -10 °C on post-cryopreservation seed germination for two Florida native orchids every 2 weeks for 12 weeks culture. Bars represent ± standard error.

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Figure 3-4. Effect of pre-cryopreservation storage at -10 °C on post-cryopreservation seedling developmental index for two Florida native orchids following 12 weeks culture. Means with the same letter within species are not significantly different by Tukey- Kramer grouping for least square means (α=0.05).

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Figure 3-5. Polypropylene chamber with sealable cover (right) used to adjust water content of orchid seed held in micro weigh dishes (arrow). Photo courtesy of author.

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Figure 3-6. Development of B. purpurea seedlings after 22 weeks culture following direct cryopreservation. A) Unfrozen control seedlings, B) Seedlings derived from seed equilibrated to 0% RH, C) Seedlings derived from seed equilibrated to 6% RH, D) Seedlings derived from seed equilibrated to 13% RH, E) Seedlings derived from seed equilibrated to 33% RH, F) Seedlings derived from seed equilibrated to 55% RH, G) Seedlings derived from seed equilibrated to 76% RH. Scale bars = 1 cm. Photo courtesy of author.

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Figure 3-7. Development of E. nocturnum seedlings after 22 weeks culture following direct cryopreservation. A) Unfrozen control seedlings, B) Seedlings derived from seed equilibrated to 0% RH, C) Seedlings derived from seed equilibrated to 6% RH, D) Seedlings derived from seed equilibrated to 13% RH, E) Seedlings derived from seed equilibrated to 33% RH, F) Seedlings derived from seed equilibrated to 55% RH, G) Seedlings derived from seed equilibrated to 76% RH. Scale bars = 1 cm. Photo courtesy of author.

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Figure 3-8. B. purpurea seedlings derived from seed equilibrated to 76% relative humidity prior to cryopreservation that died after 8 weeks culture. A) Standard micrograph of two dead seedling next to a living seedling, B) Scanning electron micrograph at 180x magnification. Scale bars = 100 µM. Photo courtesy of author.

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CHAPTER 4 MODIFICATION OF SPINNER FLASKS FOR ORCHID SEED BIOREACTOR CULTURE USING BLETIA PURPUREA

Introduction

Bioreactors are sealed sterile vessels used to produce biological cultures in a liquid medium. These vessels were originally developed for microorganisms and later for secondary metabolite production. Takayama and Misawa (1981) were first to report the application of bioreactors for in vitro plant production using Begonia plantlets. In the following years, plant bioreactor culture has expanded to the production of shoots, bulbs, corms, tubers, and embryos of many food and ornamental species (Preil 2005; Takayama and Akita 2005). These systems allow for greater contact between the medium and plant surfaces which can result in greater proliferation and increased growth rates compared to conventional tissue culture on agar- solidified media (Ziv 1999; Preil 2005). Further advantages include reduction of space and labor costs, as well as the ability to precisely control culture conditions such as pH, dissolved oxygen, and agitation rate (Chu 1995; Paek et al. 2005). These advantages also come with risks such as hyperhydricity, oxidative stress, and mass contamination (Aitken-Christie et al. 1995; Takayama and Akita 1998).

Bioreactors used for in vitro plant culture can be broadly classified into two groups: continuous immersion and temporary immersion. Continuous immersion bioreactors, in which plant material is in contact with liquid medium for the entirety of the culture period, are agitated either mechanically by impellers or pneumatically using air spargers (Takayama and Akita 1994;

Eibl and Eibl 2008). Temporary immersion systems are a more recent development in which plant material is exposed to liquid culture media intermittently using ebb and flow or rocker systems (Berthouly and Etienne 2005; Georgiev et al. 2014). Temporary immersion systems have been reported to reduce hyperhydricity and mechanical damage when compared with

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continuous immersion systems (Yoeup and Chakrabarty 2003; Ziv et al. 2003; Georgiev et al.

2014).

Various bioreactor systems have been applied to the clonal propagation of orchid shoots and protocorm-like bodies (PLBs). Successful production of species such as Phalaenopsis,

Oncidium, Dendrobium, and Anoectochilus has been reported using continuous immersion

(Young et al. 2000; Wu et al. 2007; Yang et al. 2010; Winarto et al. 2013; Yang et al. 2015), as well as Vanilla, Phalaenopsis, and Doritaenopsis using temporary immersion (Liu et al. 2001;

Hempfling and Preil 2005; Ramírez-Mosqueda and Iglesias-Andreu 2016). Orchid seed culture using liquid medium has been reported (Chu and Mudge 1994; Tsai and Chu 2008), but to our knowledge, there have been no reports of bioreactors used for seed propagation of orchids.

Orchid seed culture using bioreactors could result in more uniform seed germination and subsequent seedling development compared to agar-solidified media.

The objective of this study was to develop an aerated continuous-immersion bioreactor optimized for orchid seed culture using Bletia purpurea as a model species.

Materials and Methods

Bioreactor Modification

The bioreactor was constructed by modifying a Bellco® 250 mL spinner flask for aeration (Figure 4-1). The spinner flasks were primarily chosen because they were small enough to allow for sufficient experimental replications in a limited space. Further, the sidearm allowed for the addition of aeration assemblies without permanently altering the vessels. Refer to Table

4-1 for full bioreactor construction parts descriptions. To allow for impeller rotation, the airstone must be ground or filed to 1½ in x ½ in x ⅜ in. The pre-installed airstone connector was removed because it was constructed of non-autoclavable plastic. It was replaced with a 3 cm section of serological pipette and secured 1.3 cm above the airstone using silicone sealant (Figure 4-2B).

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The airstone was then attached to the spinner flask using silicone sealant. The airstone was parallel with the flask sidearm, with the airstone bottom 1.6 cm from the flask bottom (Figure 4-

1B).

A 7.6 cm section of ⅛ in silicone tubing was attached to the airstone. Central holes with a diameter of 1 cm for supply and 1.3 cm for exhaust were cut in the septa (Figure 4-2C). The ⅛ in male hose connector was inserted through the supply septum and screwed into the ⅛ in female hose connector (Figure 4-2E). The supply septum assembly was placed into the supply sidearm and the ⅛ in silicone tubing was attached to the ⅛ in male hose connector using forceps. The ¼ in hex nut edges were filed down to a circular shape 1.7 cm in diameter so that it could fit within a flask sidearm (Figure 4-2D). The ¼ in male hose connector was inserted through the exhaust septa and screwed into the ¼ in hex nut. The exhaust septum assembly was then placed into the exhaust sidearm. Both septa assemblies were held in place by screwing open-top caps onto each sidearm. A 9 cm section of ¼ in silicone tubing was attached to both supply and exhaust hose connector. To prevent microbial contamination, bacterial air vents were attached to the ends of the silicone tubing, with the inlet facing outwards on the supply end and inwards on the exhaust end (Figure 4-2F, G).

A second aeration system was tested following initial trials using the airstone (Figure 4-

1C). A 6 cm segment of a Pasteur pipette, cut at the tapered section so the end had a 3 mm diameter, was attached to a 7 cm section of ⅛ in silicone tubing using silicone sealant (Figure 4-

2A). The other end of the silicone tubing was then attached to the ⅛ in male hose connector of the supply septum assembly.

Aeration Manifold

Aeration was provided by an aquarium pump and distributed using a PVC manifold that allows for aeration of up to twenty bioreactors. Refer to Table 4-2 for full aeration manifold parts

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descriptions. The manifold consists of a main manifold which connects to the air pump and four sub-manifolds. The main manifold (Figure 4-3A) was constructed by cutting six 10 cm segments of 1 in PVC and connecting them to the reducing tees, with the 1 in to ¾ in reducing tee in the center, and 1 in PVC caps attached at both ends. The ¾ in hose barb was screwed into the central

¾ in reducing tee and four ¼ in hose barbs were screwed into the remaining ½ in reducing tees.

All slip fittings were sealed using PVC cement.

Each of the sub-manifolds (Figure 4-3B) were constructed using six 5 cm segments of ½ in PVC connected to five ½ in tee fittings. A ½ in PVC cap was attached to one end and a ½ in

PVC threaded adapter was attached to the other end. Ball valves were screwed into each ½ in tee fitting using a PVC nipple. The ¼ in hose barbs were screwed into each ball valve, as well as into the ½ in PVC threaded adapter. All slip fittings were sealed using PVC cement.

The aquarium pump was connected to the central ¾ in hose barb of the main manifold using the hose and hose clamps provided with the pump (Figure 4-3C, D). The ¼ in silicone tubing was used to connect the sub-manifolds to the main manifold and the bioreactors to the sub-manifolds.

Results and Discussion

Initial tests demonstrated that the bioreactors were fully autoclavable. However, we found that when bioreactors containing 250 mL medium were autoclaved, the medium would obstruct the septa. Thus, we decided to autoclave media separately from bioreactors and add to bioreactors once media had cooled. We connected bioreactors containing autoclaved 250 mL medium without plant material to the aeration manifold and confirmed the aerated medium remained sterile.

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Bioreactor Inoculum

In a pilot study, sowing B. purpurea seed directly in bioreactors resulted in a complete inhibition of germination. Instead, we determined implementing a seedling inoculum culture was necessary. Seed was germinated in 250 mL Erlenmeyer flasks containing 10 mL medium agitated on a New Brunswick G10 gyratory shaker (see Chapter 5), and early-stage protocorms were transferred from the flasks into the bioreactors. The use of an inoculum culture prior to bioreactor culture is a common practice (Takayama and Akita 1998; Takayama and Akita 2005), and has been reported for small explants such as non-zygotic embryos of banana and Siberian ginseng and nodules of Charybdis (Wawrosch et al. 2005; Yang et al. 2012; Chin et al. 2014).

Orchid seed germination in small volumes of liquid media have been reported for Doritaenopsis in a static Petri dish culture and Cypripedium calceolus var. pubescens in agitated flask culture

(Chu and Mudge 1994; Tsai and Chu 2008), though not for the purposes of an inoculum for bioreactor culture.

Bioreactor Performance

Using an airstone provided aeration to the B. purpurea seedling cultures, however, due to its attachment to the bioreactor sidewall and the impeller-stirred agitation of the medium, a large number of seedlings were removed from suspension and instead grew attached to the airstone and sidewall (Figure 4-4A). Due to the impeller design, seedlings also grew between the baffles

(Figure 4-5A, B) and central impeller shaft as well as inside of the impeller shaft itself (Figure 4-

5C, D). Sorvari et al. (2005) reported adherence of non-zygotic embryos to the nylon screen of a cell-lift impeller and reported a reduction in adherence from 25% to 10-11% after replacing the nylon screen with either a silicone membrane or nuclear track membrane. The Bellco® impellers are made of polytetrafluoroethylene, an adhesion-resistant material, however, sealing them with a similar membrane could prevent seedlings from becoming caught within the impeller or

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between the central column and the paddle. Another downside of an impeller-stirred culture system is mechanical damage to seedlings due to collisions with the impeller blades, sidewall baffles, and other seedlings (Figure 4-6) which appears to have led to excessive medium browning (Figure 4-4D).

Medium aeration also resulted in foaming, which displaced seedlings to the bioreactor sidewall above the surface of the medium. This was so detrimental we tested a second type of aerator, composed of a glass Pasteur pipette section (Figure 4-1C, 4-2A) which produced fewer bubbles and allowed for space between the aerator and bioreactor sidewall. Fewer seedlings became trapped against this type of aerator. However, using this aerator did not decrease medium foaming and seedlings continued to be displaced above the medium (Figure 4-4B, C). Foaming is common in aerated plant bioreactors. It can be associated with polysaccharides released by plant tissues, aeration rate, and bioreactors with a uniform diameter (Ziv 1999; Abdullah et al. 2000;

Paek et al. 2001). The use of antifoaming agents containing propylene glycol or silicone have been applied to plant cell bioreactor cultures. Though effective, these products greatly reduce oxygen mass-transfer rates in liquid medium, which has an inhibitory effect on maximum biomass production (Wongsamuth and Doran 1994; Li et al. 1995; Abdullah et al. 2000).

We determined that active aeration was not feasible when using plant material as small as

B. purpurea protocorms in this culture vessel. Instead, we adopted a passive aeration method where no aerator was used, but the septa assemblies were still in place (Figure 4-1D). This culture system allowed for the majority of seedlings to remain in suspension (Figure 4-4D), though seedling collision damage and seedling adherence to the impeller were not remedied.

Other bioreactor types may reduce a number of the drawbacks observed in this study, making for more efficient orchid seedling culture. Of the continuous immersion types, balloon-type bubble

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bioreactors have a rounded shape designed to reduce foaming and lack an impeller (Paek et al.

2001). This design could decrease the overall seedling loss observed in our modified bioreactor designs.

Temporary immersion systems also offer the potential for efficient production of orchids from seed. Ebb and flow systems such as RITA® (Vitropic, France) appear to be an ideal alternative due to the elimination of shear forces, mechanical damage from seedling collisions, and low-oxygen conditions (Berthouly and Etienne 2005; Georgiev et al. 2014). Additionally, the system could be used for all in vitro stages of orchid seed propagation because of the ease of media renewal and the screens or baskets used to contain plant material, although they may have to be modified with a finer screen to prevent plant loss at early stages. This could possibly eliminate the need for an inoculum culture and transfer to agar-solidified media prior to greenhouse acclimatization. Another alternative temporary immersion system is gas-permeable disposable bag-type bioreactors agitated by rocker machines, but would not eliminate the need for the seedling inoculum and agar-solidified culture steps potentially offered by ebb and flow systems.

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Table 4-1. Component list for construction of a single aerated bioreactor. Part Full Description Part Number Quantity Spinner Flask Bellco® Bell-Flo™ Spinner Flasks with 1965-80255 1 Impeller Assembly, 250 mL Digital Stirrer Bellco® Bell-Ennium™ Digital Display 7785-D2005 1 Stirrer, 5 Position Bacterial Air Vents Pall® Bacterial Air Vent, 1µm Pore Size 4308 2

Open-top Caps Corning® GL32 Red Open Top High 1395-32HTSC 2 Temperature PBT Screw Cap Septa Corning® Silicone Septa for GL32 Open 1395-32SS 2 Top PBT Screw Cap ⅛ in Silicone Thermo Scientific™ Nalgene™ 50 8060-3016 1 Tubing Platinum-Cured Silicone Tubing Size 16 ¼ in Silicone Thermo Scientific™ Nalgene™ 50 8060-3017 1 Tubing Platinum-Cured Silicone Tubing Size 17 Airstone Diffuser Sweetwater® Silica Airstone, 1½ in x ½ 10-AS-1 1 in x ½ in Serological Pipette Corning® PYREX® 1x0.01mL Sterile 7077-1N 1 Glass Serological Pipette ⅛ in Male Hose Industrial Specialties Mfg. ⅛ in Hose BFA-18-2-N 1 Connector Barb x ⅛ in Male NPT, Nylon ¼ in Male Hose Industrial Specialties Mfg. ¼ in Hose BFA-14-4-N 1 Connector Barb x ¼ in Male NPT, Nylon ⅛ in Female Hose Industrial Specialties Mfg. ¼ in Hose FCB-14-2F-N 1 Connector Barb x ⅛ in Female NPT, Nylon ¼ in Hex Nut Industrial Specialties Mfg. ¼ in NPSM NN-14NPSF-N 1 Straight Thread Hex Nut, Nylon Silicone Sealant Aqueon Silicone Aquarium Sealant, 3 oz 65003 1

Pasteur Pipette Fisherbrand® 9 in Borosilicate Pasteur 13-678-8B 1 Pipettes

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Table 4-2. Component list for construction of twenty-position aeration manifold. Part Full Description Part Number Quantity Aquarium Pump Pondmaster Air Pump AP-40 04540 1

1 in PVC Charlotte Pipe and Foundry Company® PVC 4010 1 Schedule 40 PVC Pipe – Plain End 1 in x 10 ft ½ in PVC Charlotte Pipe and Foundry Company® PVC 4005 1 Schedule 40 PVC Pipe – Plain End ½ in x 10 ft 1 in to ¾ in LASCO Fittings Schedule 40 PVC 1 in x 1 402131 1 Reducing Tee in x ¾ in Reducing Tee, Slip x Slip x FPT 1 in to ½ in LASCO Fittings Schedule 40 PVC 1 in x 1 402130 4 Reducing Tee in x ½ in Reducing Tee, Slip x Slip x FPT ½ in Tee Fitting LASCO Fittings Schedule 40 PVC ½ in 402005 20 Tee, Slip x Slip x FPT ¾ in Hose Barb B&K™ ProLine Series™ ¾ in x ¾ in Nylon NHB-590 1 Hose Barb x MIP ¼ in Hose Barb B&K™ ProLine Series™ ¼ in x ½ in Nylon NHB-193 28 Hose Barb x MIP 1 in PVC Cap LASCO Fittings Schedule 40 1 in Slip Cap 447010 2

½ in PVC Cap LASCO Fittings Schedule 40 ½ in Slip Cap 447005 4

½ in PVC LASCO Fittings Schedule 40 PVC ½ in 435005 4 Threaded Adapter Female Adapter, Slip x FPT Ball Valve American Valve® ½ in Schedule 40 PVC P200 20 Molded-in-Place One Piece Ball Valve PVC Nipple LASCO Fittings Schedule 80 PVC 205020 20 Threaded Both Ends Nipple PVC Cement Oatey® All Purpose Cement 4 oz 30818 1

¼ in Silicone Thermo Scientific™ Nalgene™ 50 8060-3017 1 Tubing Platinum-Cured Silicone Tubing Size 17

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Figure 4-1. Spinner flask modifications. A) Unmodified Bellco® spinner flask, B) Airstone aeration modification, C) Pasteur pipette aeration modification, D) Passive aeration modification. Scale bars = 5 cm. Photo courtesy of author.

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Figure 4-2. Bioreactor aeration components. A) Modified Pasteur pipette aerator, B) Airstone aerator: unmodified (left), modified (right), C) Septa: supply (left), exhaust (right), D) Exhaust connectors, E) Supply connectors, F) Complete exhaust assembly with bacterial air vent, G) Complete supply assembly with bacterial air vent. Scale bars = 1 cm. Photo courtesy of author.

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Figure 4-3. Aeration manifold components. A) Main manifold, B) Sub-manifold, C) Aquarium pump with ¾ in hose barb, D) Aquarium pump attached to main manifold. Scale bars = 5 cm. Photo courtesy of author.

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Figure 4-4. Effects of aeration methods on B. purpurea seedling cultures. A) Seedlings attached to airstone, B) Seedlings displaced by and trapped against pipette aerator, C) Excessive foaming, D) Seedlings in suspension in passively aerated bioreactor with browning in medium. Photo courtesy of author.

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Figure 4-5. B. purpurea seedlings growing in impeller. A) Seedlings growing inside impeller baffle, B) Seedlings removed from impeller baffle, C) Seedlings growing within the impeller shaft, D) Seedlings removed from within impeller shaft showing inhibited growth. Scale bar = 1 cm. Photo courtesy of author.

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Figure 4-6. Scanning electron micrographs showing B. purpurea seedling tissue damage (arrows) due to mechanical impacts at: A) 20x magnification, B) 60x magnification. Scale bar = 500 µm. Photo courtesy of author.

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CHAPTER 5 LIQUID FLASK AND BIOREACTOR CULTURE OF SELECTED FLORIDA NATIVE ORCHID SEED

Introduction

Approximately half of the two hundred fifty orchids native to North America can be found in Florida (Brown 2006), making it a hotspot of orchid diversity. However, many wild orchid populations have decreased in Florida due to the negative impacts of over-collection, habitat loss or fragmentation, and invasive species (Brown 2006; Kautz et al. 2007; Stewart and

Hicks 2010). As a result, seventy-seven of these species have been classified as endangered, threatened, or commercially exploitable by Florida’s Regulated Plant Index (Florida

Administrative Code Rule 5B-40.0055). Orchids’ reliance on complex symbioses with mycorrhizal fungi and often species-specific pollinators (Tremblay 1992; Rasmussen 1995), further reduces the chances of natural recruitment. Thus, ex situ seed propagation is an important means to supplement existing populations.

In vitro seed germination is an efficient method for producing genetically diverse native orchids for reintroduction (Kauth et al. 2006; Johnson et al. 2007; Dutra et al. 2008). Orchid seeds germinate in nature after infection by a compatible mycorrhizal fungus, which solely provides carbohydrates and nutrients to the orchid until it becomes autotrophic (Liebel et al.

2015; Rasmussen et al. 2015). Asymbiotic orchid seed germination bypasses the need for a compatible mycorrhizal fungus by providing the necessary carbohydrates and nutrients directly to the seed via a sterile tissue culture medium (Stewart and Kane 2006; Kauth et al. 2008a), resulting in higher germination rates and more rapid seedling growth than occurring in nature.

Conventionally, asymbiotic seed germination media are agar-solidified. Though efficient, orchid seeds are minute and are often sown in close proximity to one another, leading to nutrient competition amongst seedlings. Additionally, the fusion of seedlings and entanglement of roots

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can result in increased labor and damage to plantlets upon subculture or transfer to an ex vitro environment. In contrast, orchid seeds have been successfully germinated in liquid media which can alleviate the aforementioned limitations of agar-solidified media. For example, Cypripedium calceoulus var. pubescens seedlings germinated in agitated liquid medium flask culture developed more rapidly than those on agar-solidified medium and also eliminated the need for an

8-week pre-chilling period (Chu and Mudge 1994). Further, Doritaenopsis seedlings germinated in an unagitated liquid medium grew at a higher rate than those germinated on agar-solidified medium (Tsai and Chu 2008). Although liquid culture media offer certain advantages over agar- solidified media, there are risks such as mass contamination, hyperhydricity, and browning due to the accumulation of phenolic exudates (Aitken-Christie et al. 1995; Paek et al. 2005; Winarto et al. 2013).

Bioreactors are vessels used for sterile, automated mass culture of plant tissues. Although no published research exists on orchid seed/seedling bioreactor culture, there are numerous reports of bioreactor culture used in the clonal propagation of orchids. Oncidium ‘Sugar

Sweet’and Dendrobium candidum protocorm-like bodies, as well as Anoectochilus formosanus shoots, have been successfully multiplied using continuous immersion bioreactor culture (Wu et al. 2007; Yoon et al. 2007; Yang et al. 2010; Yang et al. 2015). Temporary immersion bioreactors have also been used in the multiplication of Phalaenopsis and Doritaenopsis protocorm-like bodies and Vanilla planifolia and Vanda tricolor shoots (Young et al. 2000; Liu et al. 2001; Ramos-Castellá et al. 2014; Esyanti et al. 2016). In order to maintain high genetic diversity, clonal production is unsuitable for the purpose of native orchid reintroduction (Keller and Waller 2002; Reed and Frankham 2003).

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The objectives of this study were to: 1) Optimize liquid media for producing a seedling inoculum for bioreactor culture for the Florida native orchids Bletia purpurea, Epidendrum nocturnum, and Prosthecea cochleata; 2) Compare seed germination and seedling development rates between liquid or agar-solidified culture medium for B. purpurea, E. nocturnum, and P. cochleata; and 3) Determine the feasibility of bioreactor culture as a means of mass-producing seedlings for reintroduction using B. purpurea.

Materials and Methods

Plant Material

Seed of Bletia purpurea, Epidendrum nocturnum, and Prosthechea cochleata was collected from mature capsules from the Florida Panther National Wildlife Refuge (Collier

County, Florida) between 2015 and 2016. Seeds were removed from their capsules and stored in glass screw cap vials (Part No. 03-339-25C Fisher Scientific, Pittsburg, PA) over calcium sulfate desiccant (WA Hammond Drierite Co Ltd, Xenia, OH) for 2 weeks at 23 ±2 °C then transferred to a freezer at -10 °C in darkness until use. Seed viability was determined using a 2, 3, 5 triphenyl tetrazolium chloride (TTC) test adapted from Hosomi et al. (2012). Seeds were suspended in a 10% (w/v) sucrose solution for 24 hours in darkness at 23 ±2 °C. Seeds were then rinsed twice with distilled-deionized (DD) water before being replaced with a 1% (w/v) TTC solution. Seeds were incubated in the TTC solution for 24 hours in darkness at 40 °C. Following incubation, seeds were rinsed once with DD water and dispensed on steel blue seed germination paper (Product No. CDB3.5 Anchor Paper Co, St. Paul, MN). Seeds were observed with a stereoscope (Model No. SMZ-2T Nikon USA Inc, Melville, NY) and counted as viable if red staining of the embryo was observed. Seed viability was determined to be 90.63% in B. purpurea, 65.20% in E. nocturnum, and 0.00% in P. cochleata, though a 10-day-test on Orchid

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Seed Sowing Medium (P723; Product No. P723 PhytoTechnology Laboratories, Shawnee

Mission, KS) resulted in a germination of 97.99%.

Seedling Inoculum Culture Medium Optimization

The effects of four media treatments were evaluated for all three species (Table 5-1).

Seeds were surface sterilized using a solution of 5 mL absolute ethanol, 3.6 mL 8.25% NaOCl, and 91.4 mL sterile deionized water for 1 minute followed by 3 rinses of sterile deionized water for 1 minute each. Sterilized seed was sown in 125 mL Erlenmeyer flasks containing 10 mL of treatment media. At the onset of the experiment, two liquid formulations of low-salt media were compared: Knudson C Orchid Medium (KC; Product No. K400 PhytoTechnology Laboratories,

Shawnee Mission, KS) and P723. P723 was formulated using concentrated stock solutions in order to exclude agar and activated charcoal included in the commercially prepared medium and supplemented with 2% (w/v) sucrose. Both media were supplemented with 10% (v/v) coconut water (CW; Product No. C195 PhytoTechnology Laboratories, Shawnee Mission, KS) and adjusted to pH 5.7 prior to autoclaving.

Flasks were placed on a New Brunswick G10 gyratory shaker (New Brunswick Scientific

Co, Edison, NJ) at 80 rpm under a photoperiod of 16/8 provided by cool white fluorescent bulbs

(Product No. F96T12 General Electric, East Cleveland, OH), at 30 µmol m2s-1 and a temperature of 23±2 °C. In order to support seedling development, an additional 10 mL of treatment media were added to each flask after 2 weeks culture. This was either the low-salt medium used at the onset of the experiment or Murashige and Skoog Medium (MS; Product No. M524

PhytoTechnology Laboratories, Shawnee Mission, KS), a high-salt medium, supplemented with

3% (w/v) sucrose and 10% (v/v) CW. MS medium was also adjusted to pH 5.7 prior to autoclaving. Five replicate flasks were used per treatment with an average of 700 seeds

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dispensed per flask. Germination and seedling development was observed and recorded after 4 weeks culture. This experiment was repeated once for each species.

Comparison of Solid and Liquid Medium on Seed Germination and Subsequent Seedling Development

After an optimal seedling inoculum medium was determined from the first experiment, a second experiment compared the effect of liquid and agar-solidified phases on seed germination and subsequent seedling development for all three species. Seeds were surface sterilized using a solution of 5 mL absolute ethanol, 3.6 mL 8.25% NaOCl, and 91.4 mL sterile deionized water for 1 minute followed by 3 rinses of sterile deionized water for 1 minute each. Sterilized seed was sown in one of two culture systems: 125 mL Erlenmeyer flasks and containing 10 mL of

Knudson C (KC) medium supplemented with 10% (v/v) coconut water (CW) and Petri dishes filled with approximately 25 mL KC medium supplemented with 10% (v/v) CW, 0.1 % (w/v) activated charcoal, and 0.7% (w/v) purified grade TC agar (Product No. A175 PhytoTechnology

Laboratories, Shawnee Mission, KS). Both media treatments were adjusted to pH 5.7 prior to autoclaving.

Flasks were placed on the gyratory shaker at 80 rpm and both treatments were maintained under a photoperiod of 16/8 provided by cool white fluorescent bulbs (Product No. F96T12

General Electric, East Cleveland, OH), at 30 µmol m2s-1 and a temperature of 23±2 °C. An additional 10 mL of liquid medium was added to flask cultures after 2 weeks. Fifteen replicate culture vessels were used per treatment with an average of 530 seeds dispensed per flask and an average of 170 seeds dispensed per dish, divided into 4 subreplicates. Germination and seedling development were observed and recorded from five randomly selected culture vessels from each treatment every 2 weeks for 6 weeks. This experiment was repeated once for each species.

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Effect of Citric/Ascorbic Acid on Bioreactor Culture of Bletia purpurea

After pilot studies resulted in excessive seedling browning (see Chapter 4), the effects of citric and ascorbic acid on bioreactor culture of B. purpurea were observed. Twenty inoculum cultures of B. purpurea seedlings were grown in 125 mL Erlenmeyer flasks in KC medium for 6 weeks on the gyratory shaker at 80 rpm under a photoperiod of 16/8 and a temperature of 23 °C.

Seedling cultures were randomly selected and transferred into sterilized 250 mL Bellco® Bell-

Flo™ spinner flasks (Product No. 1965-80255, Bellco Glass Inc., Vineland, NJ) modified for passive aeration (as described in Chapter 4). Inoculum medium was removed with a sterile pipette and treatment media was dispensed in 250 mL aliquots into 7 spinner flasks per treatment. Treatment media used was liquid 2x strength P723 salts supplemented with 2% (w/v) sucrose and 10% (v/v) CW, either with or without 0.005% (w/v) of both citric and ascorbic acids.

Spinner flasks were placed on Bellco® Bell-Ennium™ digital display stirrers (Product No.

7785-D2005, Bellco Glass Inc., Vineland, NJ) set at 60 rpm. All treatments were maintained under a photoperiod of 16/8 and a temperature of 23 °C. A complete renewal of culture medium occurred after 4 weeks. After 8 weeks culture, the experiment was terminated and seedling development stage was recorded. In order to evaluate post-bioreactor culture survival and regrowth, approximately 120 seedlings per bioreactor were transferred in four equal subreplicates to four Phytatrays™ (Product No. P1552, Sigma-Aldrich, St. Louis, MO) containing

Orchid Maintenance/Replate Medium (P748; Product No. P748 PhytoTechnology Laboratories,

Shawnee Mission, KS). Phytatrays™ were sealed with a single layer of Parafilm M® (Bemis Co

Inc, Neenah, WI) and incubated at 23 ±2 °C under a 16/8 photoperiod provided by cool white fluorescent bulbs (Product No. F96T12 General Electric, East Cleveland, OH) at 40 µmol m²s-1.

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Seedling recovery and regrowth data were recorded after 10 weeks culture. This experiment was repeated once.

Statistical Analysis

Germination percentages were determined by dividing the number of germinated seeds by the total number of seeds per subreplication. Developmental stage (Table 4-2) percentages were calculated by dividing the number of seeds or seedlings in each stage by the total number of seeds in each subreplicate. A developmental index (DI) was adapted from Kauth et al. (2011):

(N + N ∗ 2 + N ∗ 3 + N ∗ 4 + N ∗ 5 + N ∗ 6) (5-1) DI = 1 2 3 4 5 6 (N1 + N2 + N3 + N4 + N5 + N6) where N1 is the number of seeds in Stage 1, etc. All data were analyzed using generalized linear mixed model procedures (PROC GLIMMIX) and least square means (LSMEANS) at α=0.05 using SAS v9.4.

Results

Seedling Inoculum Culture Medium Optimization

The media treatments had a significant effect on the germination of B. purpurea, but not

E. nocturnum or P. cochleata seed (Figure 5-1). B. purpurea germination in KC and KC-MS was approximately 1.3 times greater than those in P723 or P723-MS. B. purpurea DI was greatest in

KC, but KC-MS was still greater than in P723 or P723-MS. E. nocturnum DI was lower in P723 than KC or KC-MS, whereas P723-MS did not differ from any media treatment. P. cochleata DI was unaffected by media treatments. Notably, B. purpurea was the only species in this study to produce leaves while in liquid culture (Figures 5-1, 5-7B).

Comparison of Solid and Liquid Medium on Seed Germination and Subsequent Seedling Development

Seed germination did not differ between liquid and solid medium at any single time point in any of the species studied (Figure 5-2). P. cochleata seed germination was rapid: after 2 weeks

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culture it was not significantly different than after 4 or 6 week culture periods. In contrast, B. purpurea and E. nocturnum germination were lower after a 2 week culture period than after 4 or

6 week culture periods. B. purpurea cultured on solid medium for 4 weeks germinated to an equivalent rate to either media treatment after a 6 week culture period, though seeds cultured in liquid medium for 4 weeks germinated at a lower rate. (Figure 5-2).

For the most part, there was no difference in DI amongst media treatments at any given culture period (Figure 5-3). The sole exception was B. purpurea, which had the greatest DI after

6 weeks culture in liquid medium, whereas the solid medium treatment for this culture period did not differ from either media treatments after a 4 week culture period (Figure 5-3). E. nocturnum seedlings did not develop further after a 6 week culture period than after 4 weeks. Similar to the results of the first experiment, there was no difference in DI for P. cochleata between any media treatment or culture time (Figure 5-3).

Approximately 1.3% of B. purpurea seedlings cultured on solid medium and 1.4% of those cultured in liquid medium developed a first leaf after 4 weeks culture. After 6 weeks culture seedlings with one leaf increased to 7% cultured on solid media and 32% cultured in liquid medium. Further, a second leaf was produced in 3.7% of seedlings cultured in liquid culture, none were produced on solid culture. Just over 0.1% of E. nocturnum seedlings cultured in liquid medium developed a single leaf. No P. cochleata seedlings developed leaves in this study. Despite this lack of leaf formation, shoot apex and rhizoid formation were observed in many E. nocturnum and P. cochleata seedlings (Figure 5-8). We observed a degree of hyperhydricity in B. purpurea seedlings after 6 weeks liquid culture (Figure 5-7B). SEM analysis revealed no notable morphological differences between seedlings produced in liquid or on solid media (Figure 5-8).

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Effect of Citric/Ascorbic Acid on Bioreactor Culture of Bletia purpurea

The addition of 0.005% citric and ascorbic acid had no discernable effect on bioreactor culture of B. purpurea compared to the control. Seed germination was just below 100% in both treatments (Figure 5-4A), and though more seedlings reached both stage 4 and 5 in bioreactors supplemented with citric and ascorbic acid than without, there was no significant difference in

DI between the treatments (Figure 5-4B). Seedlings from the majority of both bioreactor treatments were green with a small degree of browning at their bases (Figure 5-9A, C). Some variation was observed within treatments: mass seedling death slowly occurred in one control bioreactor (Figure 5-9B), whereas reduced and asynchronous seedling growth was observed in one of the bioreactors with added citric and ascorbic acid (Figure 5-9D).

After 10 weeks culture on solid medium following bioreactor culture, the results were much less promising as seedling survival was low: about 26% and 19% respectively for the control and with citric and ascorbic acid supplemented treatments (Figure 5-5). Of the surviving seedlings, an additional 21% and 41% had produced protocorms-like bodies (PLBs; Figure 5-5).

If the seedlings did survive after transfer to solid medium, their growth was more vigorous than when cultured solely on solid medium. When transferred to solid medium the average leaf number was slightly below two for both treatments, and after 10 weeks, had increased to about three and a half leaves (Figure 5-6A), representing an increase of between 170-190% (Figure 5-

6B). There was no significant difference between treatments in seedling survival, percent forming PLBs, or average percent leaf increase after 10 weeks culture on solid medium.

Discussion

This study clearly shows that seed of the native orchids used in this study germinate readily in liquid suspension culture and make ideal bioreactor inoculum. The bioreactor portion of the study shows promise based on the high level of seedling development coupled with rapid

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further development, but because of the low survival upon transfer to a solid medium, major protocol refinements are needed before the system can be implemented efficiently.

Seedling Inoculum Culture Medium Optimization

B. purpurea was the only species in which we observed significant differences in germination and DI amongst media. This was unexpected, as B. purpurea has been reported to germinate readily, even germinating at low levels in the absence of sucrose and light (Johnson et al. 2011). Our results also counter the results of a media screen for B. purpurea using six agar- solidified orchid germination media (including P723 and KC), where no differences in germination were observed after 5 weeks (Dutra et al. 2008). There are a number of differences in macronutrient concentrations between KC and P723, most notably that KC has over twelve times the sulfate, about six times the phosphate, and a total salt concentration almost twice that of P723. In contrast, P723 contains a broad range of micronutrients and organics whereas KC only contains iron and manganese. Dutra et al. (2008) suggested phosphate may be the limiting factor in advanced seedling development of B. purpurea, which aligns with our DI results.

Interestingly, the addition of a high-salt media with increased sucrose content after two weeks culture had no promotive effect on seedling development, and only inhibited development in B. purpurea. Thus we can conclude that germination and subsequent seedling development of

E. nocturnum and P. cochleata can be supported by many media compositions. Although no single germination medium was ideal for E. nocturnum and P. cochleata, we used KC for the following comparison of liquid and solid media in order to better compare the results amongst species.

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Comparison of Solid and Liquid Medium on Seed Germination and Subsequent Seedling Development

Germination was unaffected between liquid and solid medium at any given time point for all three species, which aligns with reports of static liquid culture of Doritaenopsis (Tsai and Chu

2008). The only developmental advantage we observed in liquid culture was in B. purpurea after

6 weeks: there were significantly more seedlings that had progressed to stage 3 and 4 than on solid medium. We observed a degree of hyperhydricity in these seedlings (Figure 5-7), however, it was not observed in seedlings produced in bioreactors (Figure 5-13). Although it appears liquid medium offers no clear advantages from a developmental standpoint in the other two species, it is still the preferable method for preparing a bioreactor inoculum. A higher seed density can be sown in a single culture vessel without the fusing or entanglement of seedlings seen in agar-solidified media. Additionally, transferring a seedling suspension can be accomplished by simply pouring the suspension from one vessel to another, which reduces the labor and potential for contamination compared with transferring single seedlings with forceps from a solid medium.

The ideal culture period prior to bioreactor inoculation differs amongst all three species we used. P. cochleata germinated so rapidly that they would be appropriate for transfer to a bioreactor in 2 weeks, E. nocturnum after 4 weeks, and B. purpurea after 6 weeks. There was also an apparent developmental bottleneck related to leaf emergence. With the exception of four

E. nocturnum seedlings observed from a single flask after 6 weeks culture, leaf emergence was only observed in B. purpurea. The vast majority of orchid species do not produce seeds with cotyledons, but B. purpurea is one of the approximately ten species that does (Nishimura 1991), which may explain its relatively high production of leaves while in liquid suspension. Neither E. nocturnum nor P. cochleata seeds, germinated on solid KC medium, produced leaves after 6

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weeks culture, so this experiment would need to be extended in length to determine if liquid suspension culture inhibits the production of leaves.

Effect of Citric/Ascorbic Acid on Bioreactor Culture of Bletia purpurea

It is clear that further optimization of the bioreactor culture system is needed to efficiently produce B. purpurea seedlings, but the addition of citric and ascorbic acid at 0.005% does not appear to have an effect of any kind on seedling survival or development. The seedlings appeared healthy upon 8 weeks bioreactor culture and transfer to solid P748 medium with no observation of hyperhydric characteristics seen in the previous experiment (Figure 5-9A, C), but it appears that the slight browning at the bases of the seedlings was indicative of a larger problem, as the majority of seedlings rapidly browned and died upon transfer to solid medium.

The seedlings that did survive after 10 weeks culture on solid P748 medium showed vigorous growth (Figure 5-13A), however, 20-40% of these seedlings also proliferated PLBs from the base (Figure 5-13B). PLBs and other clonal propagules are not ideal when producing plants for reintroduction purposes, as reduced genetic variation can lead to inbreeding depression and reduce population fitness (Keller and Waller 2002; Reed and Frankham 2003).

Browning is a well-reported problem in orchid tissue culture, the damage can range from inhibited plant growth to death (Gow et al. 2009; Gow et al. 2010; Tao et al. 2011; Winarto et al.

2013; Gao et al. 2014; Chuanjun et al. 2015). Plants release phenols as a wound response

(Saltveit 2016), which form quinones that when oxidized that cause browning in media and plant tissue (Laukkanen et al. 1999; Ndakidemi et al. 2014). The low oxygen levels in the bioreactors could have prevented phenolic oxidation and masked the full degree of damage to the seedlings.

We observed browning in pilot studies (See Chapter 4) and attempted to mitigate the problem by implementing media renewal after 4 weeks culture and including citric and ascorbic acid

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supplements as a treatment. Clearly, neither of these attempts were successful and other methods to prevent browning need to be further addressed.

We observed substantial mechanical damage in SEMs of seedlings from both treatments

(Figure 5-10). This is a downside of using a stirred bioreactor system in which the seedlings collide with the impeller blades, sidewall baffles, and other seedlings. Reducing the impeller speed from 60 rpm to the minimum speed that ensures seedlings remain in suspension could reduce seedling damage and subsequent leaching of phenols. Increasing the frequency of media renewal could also reduce the detrimental effect caused by browning. The modification of a stirred bioreactor culture system that provided continuous medium renewal eliminated browning in Glycyrrhiza inflata (Wang et al. 2010). While this is not a feasible option in most situations, renewing the medium every one or two weeks may be a simple solution. The mechanical damage to the seedlings from the impeller rotation also appears to be the cause of PLB proliferation

(Figure 5-13B). This is not surprising, as wounding orchid tissue as a means of inducing PLBs is a common practice (Chen and Chang 2000; Huang et al. 2014). A more dramatic alternative would be changing from a stirred bioreactor system to an airlift bioreactor, balloon-type bubble bioreactor, or even a temporary immersion bioreactor system, all of which would reduce mechanical damage by impeller rotation (Paek et al. 2001; Berthouly and Etienne 2005).

Further media supplementation could also reduce browning. Although the citric and ascorbic acid addition at 0.005% was ineffective at preventing browning, increasing the concentration could be effective. The addition of ascorbic acid at 0.005% had little effect on the browning of Brahylaena huillensis cultures, but increased concentrations up to 0.02% reduced browning by 77% (Ndakidemi et al. 2014). Alternately, the rapid degradation of ascorbic acid in liquid tissue culture media by autoclaving and flask shaking has been reported (Elmore et al.

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1990), so filter sterilization of the compounds should also be considered. Another amendment that should be tested is activated charcoal, it is added to many orchid media for the purpose of adsorbing phenolic compounds (Thomas 2008). A reduction in browning and an increase in biomass was reported in bioreactor culture of Cymbidium sinense when activated charcoal was added (Gao et al. 2014). Similar results were reported in Anoectochilus formosanus but only when activated charcoal and CW were used in combination.

Contamination is another issue when producing plants in liquid culture (Aitken-Christie et al. 1995; Takayama and Akita 1998) which we encountered (Figure 5-12). The risk of losing large numbers of propagules and time invested due to contamination leads us to recommend the addition of plant preservative mixture (PPM; Plant Cell Technology Inc., Washington D.C.) when producing orchids using bioreactor culture, especially if there is a limited amount of seed.

PPM is a commercially prepared isothiazolone biocide that can prevent microbial contamination in plant tissue culture (Niedz 1998). Additionally, PPM has been reported to decrease phenolic browning in liquid culture of cauliflower microshoots (Rihan et al. 2012), though this appears to be the only reported occurrence.

Conclusions

We have developed an efficient system for the germination of three native orchid species using liquid suspension culture and determined ideal culture periods for using these species as bioreactor inoculum. Moreover, we have determined little differences exist in the development of these species between liquid and solid culture though liquid culture provides a degree of operational efficiency. Further studies on seedling inoculum culture should focus on determining optimal seed density and hyperhydric seedling recovery following liquid culture.

We have also outlined the potential for using bioreactors for the production of orchid seedlings although much of the system still requires optimization before it could be considered a

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viable seedling production method. The most important issue to resolve is browning due to phenolic exudation that leads to high mortality upon transfer to solid medium, by either reducing impeller speed, screening medium supplements at varying concentrations, changing the bioreactor type, or a combination of these variables. Once this issue has been resolved, studies regarding seedling density, medium optimization, system aeration, and direct comparison with multiple bioreactor systems and traditional asymbiotic orchid seed production on solid medium.

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Table 5-1. Seedling inoculum media treatment matrix. Treatment Initial Media Media Added After 2 Weeks P723 P723, 2% sucrose, 10% CW P723, 2% sucrose, 10% CW P723-MS P723, 2% sucrose, 10% CW MS, 3% sucrose, 10% CW KC KC, 2% sucrose, 10% CW KC, 2% sucrose, 10% CW KC-MS KC, 2% sucrose, 10% CW MS, 3% sucrose, 10% CW

Table 5-2. Orchid seed and seedling developmental stages. Stage Description 0 Ungerminated seed, no embryo growth 1 Enlarged embryo, intact testa 2 Enlarged embryo, ruptured testa (=germination) 3 Emergence of first leaf 4 Emergence of second leaf 5 Elongation of second leaf and further development Adapted from Dutra et al. (2009) and Stewart and Zettler (2002).

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Figure 5-1. Effect of four germination media on seed germination and subsequent seedling development of three Florida native orchid species following 4 weeks liquid suspension culture. Values represent mean response of the experiment repeated once. Means with the same letter within species are not significantly different by Tukey- Kramer grouping for least square means (α=0.05). Bars represent ± standard error.

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Figure 5-2. Effect of liquid and solid formulations of Knudson C medium on the germination of three Florida native orchid species every 2 weeks for 6 weeks culture. Values represent mean response of the experiment repeated once. Means with the same letter within species are not significantly different by Tukey-Kramer grouping for least square means (α=0.05). Bars represent ± standard error.

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Figure 5-3. Effect of liquid and solid formulations of Knudson C medium on seedling developmental index of three Florida native orchid species following 6 weeks culture. Values represent mean response of the experiment repeated once. Means with the same letter within species are not significantly different by Tukey-Kramer grouping for least square means (α=0.05).

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Figure 5-4. Comparison of bioreactor culture of B. purpurea with and without 0.005% citric and ascorbic acid (CAA) on A) seed germination and B) seedling developmental index after 6 weeks inoculum culture followed by 8 weeks bioreactor culture. Values represent mean response of the experiment repeated once. Means with the same letter are not significantly different by Tukey-Kramer grouping for least square means (α=0.05).

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Figure 5-5. Comparison of bioreactor culture on seedling survival and PLB formation of B. purpurea with and without 0.005% citric and ascorbic acid (CAA) 10 weeks following transfer to solid medium. Values represent mean response of the experiment repeated once. Means with the same letter are not significantly different by Tukey-Kramer grouping for least square means (α=0.05).

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Figure 5-6. Comparison of bioreactor culture on leaf production of B. purpurea with and without 0.005% citric and ascorbic acid (CAA) 10 weeks following transfer to solid medium. A) Average leaf number upon transfer to solid medium and after 10 weeks, B) Average percent leaf increase from 0 weeks to 10 weeks culture on solid medium. Values represent mean response of the experiment repeated once. Means with the same letter are not significantly different by Tukey-Kramer grouping for least square means (α=0.05).

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Figure 5-7. B. purpurea seedlings after 6 weeks culture. A) Seedlings cultured on solid medium, B) Seedlings cultured in liquid medium in flasks with glassy appearance characteristic of hyperhydricity. Scale bars = 1 mm. Photo courtesy of author.

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Figure 5-8. Scanning electron micrographs of three Florida native orchid species after 4 weeks culture. A) B. purpurea cultured in liquid medium, B) B. purpurea cultured on solid medium, C) E. nocturnum cultured in liquid medium, D) E. nocturnum cultured on solid medium, E) P. cochleata cultured in liquid medium, F) P. cochleata cultured on solid medium. Scale bars = 100 µm. Photo courtesy of author.

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Figure 5-9. Development of B. purpurea seedlings following 8 weeks bioreactor culture. A) Representative sample of seedlings produced in control treatment bioreactor, B) Seedlings from control bioreactor where mass mortality occurred, C) Representative sample of seedlings produced in bioreactor with citric and ascorbic acid supplements at 0.005%, D) Seedlings from 0.005% citric and ascorbic acid supplemented bioreactor showing asynchronous development. Scale bars = 1 cm. Photo courtesy of author.

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Figure 5-10. Scanning electron micrographs showing damage to B. purpurea seedlings from bioreactor culture and evidence of early PLB proliferation. Scale bars = 500 µm. Photo courtesy of author.

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Figure 5-11. Bioreactors showing different degrees of media browning following 8 weeks seedling culture. Scale bar = 5cm. Photo courtesy of author.

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Figure 5-12. Bioreactor with bacterial contamination. Scale bar = 2 cm. Photo courtesy of author.

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Figure 5-13. B. purpurea seedlings following culture on solid P748 medium following bioreactor culture. A) Seedlings without proliferation (dead seedling far right), B) Seedlings with PLB proliferation (arrows). Scale bars = 1 cm. Photo courtesy of author.

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CHAPTER 6 CONCLUSIONS

Orchids are a diverse plant family and a high concentration of those native to North

America are native to Florida. Many Florida native orchids are endangered or threatened due to anthropomorphic threats combined with their narrow range of pollinator and mycorrhizal symbiont compatibility. In vitro methods have been used to propagate orchids, both clonally and from seed, since the early 1900s. Efficient methods for long-term storage are critical for conservation, and cryopreservation is a valuable tool used for the long-term conservation of both wild and cultivated plant species. Asymbiotic seed germination is an in vitro approach essential for orchid conservation as it allows for orchid seed germination more rapidly and in a higher percentage than occurs in nature. The aim of this study was to develop and evaluate these procedures in order to conserve and produce genetically diverse Florida native orchids.

We determined that cryopreservation of desiccated seed in liquid nitrogen (direct cryopreservation) was an effective conservation method for nine Florida native orchid species.

Post-cryopreservation germination response varied amongst the species, ranging from 75-115% of their unfrozen controls. Direct cryopreservation is the simplest method of storing plant tissues in liquid nitrogen, which also reduces time and cost associated with pretreatments used in other cryopreservation protocols. These advantages lead us to recommend screening orchid species and evaluating their response using this technique before attempting more complicated cryopreservation methods.

Little has been published regarding the effect of various storage conditions prior to orchid seed cryopreservation. We determined that equilibrating Bletia purpurea and Epidendrum nocturnum seed to relative humidity (RH) levels up to 55% prior to direct cryopreservation had no negative impact on germination, whereas higher RH levels would result in significantly

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reduced germination or fungal contamination. Additionally, storage of seed from these two species at -10 °C for up to 6 weeks prior to direct cryopreservation did not reduce post- cryopreservation germination. A reduction in seedling vigor for E. nocturnum was observed in both experiments, but there is evidence this related to seed aging prior to cryopreservation.

Bioreactors are used for the efficient propagation of many plant species in a liquid medium. While these are commonly used for clonal orchid propagation in the commercial industry, little has been reported on their application towards orchid seed propagation. We modified spinner flasks and evaluated their potential for in vitro orchid seedling production.

Although early studies using B. purpurea revealed the potential of this bioreactor system, much of the protocol required further refinement. We identified two crucial elements to focus on: the need for pre-germination of seeds in small volumes of media (inoculum cultures) and reduction of seedling and medium browning, which was considered a result of mechanical damage seen using scanning electron microscopy.

In order to first develop seedling inoculum cultures, we evaluated four liquid media treatments for three orchid species: B. purpurea, E. nocturnum, and Prosthechea cochleata. Seed germination only differed amongst media for B. purpurea, ranging from 55-76% after 4 weeks culture, with Knudson C medium (KC) giving the best results. Seed germination did not significantly differ for the other species, ranging from 74-77% and 81-88% for E. nocturnum and

P. cochleata respectively. Comparative seed germination and subsequent seedling development between liquid and solid KC for the three species were then evaluated. No differences in germination were observed for any of the three species, but more advanced seedling development was observed in B. purpurea produced in liquid medium (36% of seedlings formed at least one leaf in liquid medium, compared to 7% on solid medium). We further screened the

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seedling development in bioreactors using B. purpurea seedling inoculum cultures and found that citric and ascorbic acid supplements at 0.005% did not reduce seedling browning, resulting in high mortality (75-80%) of seedlings following transfer from bioreactors to a solid medium.

Mechanical damage of seedlings also appeared to cause clonal proliferation in 20-40% of seedlings that survived. Further refinement of this system was warranted, as seedlings that did survive following transfer to solid medium demonstrated more vigorous growth than those produced on solid medium.

Orchids have a unique physiology that requires in vitro seed propagation for efficient production in the absence of a mycorrhizal mycobiont. Using in vitro techniques such as cryopreservation, asymbiotic seed germination, and liquid culture systems offer great promise for both the long-term storage and production of genetically diverse plants that can be reintroduced to their native habitats. The protocols used in this study can be applied to other

Florida native orchid species as well as many other imperiled orchid species worldwide in order to optimize conservation procedures.

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BIOGRAPHICAL SKETCH

Ben Hughes was born in England and raised in Florida, Minnesota, and Colorado. After serving in the Coast Guard, he moved to Gainesville to pursue his college career. He enrolled at

Santa Fe College where his interest in biology and plants began. He toured a University of

Florida micropropagation lab for a botany class, which convinced him to transfer into the school as an environmental horticulture major. Little did he know how much time he would spend in that lab. Shortly after taking Dr. Michael Kane’s micropropagation class, he volunteered as an undergraduate researcher working with seed of various Florida native orchids. He graduated with his B.S. in 2013 and returned to the lab once again, only this time his research was in pursuit of a

Doctor of Philosophy. Upon completion of his PhD in December 2017, Ben plans to pursue a position that will broaden his experience in micropropagation or seed biology. Ben enjoys spending time with his girlfriend, dog, and friends. He distracts himself from work by reading, cooking, playing the guitar (poorly), and following the NBA.

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