Understanding the Ecophysiology of Trophic Transition with Micropropagated Terrestrial Orchids for Improving Restoration Success

SHAHAB NIKABADI

Supervisors: Professor Kingsley Dixon Dr Eric Bunn Dr Jason Stevens Dr Shane Turner Dr Belinda Newman

This thesis is presented for the degree of Master of Philosophy School of Plant Biology

The University of Western Australia 2015

Abstract

Orchids are now one of the most threatened plant groups in the world; the result of both natural and anthropogenic causes (Kull et al., 2006). New integrated protocols to progress orchid conservation are desperately needed, with emphasis on both propagation of threatened species and successful reintroduction of vigorous and acclimatized seedlings. The need for application of ecophysiological principles to improve the ability of the prolific micropropagation system to deliver more robust and resilient orchid plants for restoration has never been more critical. This study set out to investigate abiotic (temperature and light) and biotic (fungal symbionts) influences on in vitro seed germination, seedling development and photosynthetic capacity of four terrestrial orchid species Caladenia latifolia, C. huegelii, Microtis media and Pterostylis sanguinea from south-west Western Australia. Germination was higher and development of seedlings faster overall in all test species in symbiotic compared with asymbiotic media treatments. Pterostylis sanguinea seeds demonstrated the best response (among species tested) to asymbiotic germination on ½ MS (Murashige and Skoog basal medium) with from 40 to 53% of germinated seeds reaching developmental stage 3 up to stage 5 in light or dark incubation (at 20 oC) respectively. Illumination had no effect on fungal symbiont growth across all species, however incubation temperature treatments (10, 15, 20 and 25 oC) affected fungal growth rate. Growth of the fungal symbionts of Caladenia huegelii, M. media and C. latifolia was lower at 10 oC, but the cumulative radial growth rate of the P. sanguinea fungal symbiont reached 64 cm2 after only two weeks at all temperatures tested, including 10 oC. The study highlights differences in symbiotic and asymbiotic germination and early protocorm development in vitro between co-occurring herbaceous terrestrial Australian orchid taxa in response to variations in basal media, temperature and light. Whole seedling CO2 exchange (i.e. photosynthetic activity) was analysed with Caladenia huegelii, Caladenia latifolia, Microtis media and Pterostylis sanguinea grown with and without fungal symbionts in response to both LED and fluorescent lamps, and with added nitrogen source (N). Results strongly indicated that photosynthesis at the early stages of orchid growth may be more affected by the presence and absence of fungus than by light source, light quantity or available N. This study demonstrates the difficulties in dealing with species that possess a complex life history and further studies will be needed to translate these findings into improving the survival of terrestrial orchid seedlings from in vitro propagation to the glasshouse environment. However, it is hoped that the study will be of benefit to the conservation of Australian terrestrial orchids specifically and terrestrial species generally.

Contents Abstract ...... 2 Acknowledgements...... 5 Statement of candidate contribution ...... 5 Chapter One ...... 6 Introduction ...... 6 Orchids and Symbiotic Associations ...... 6 Germination: the first obstacle in orchid propagation ...... 8 Temperature and Light: Two key environmental factors affect germination ...... 10 Temperature ...... 10 Light...... 10 Orchid and photosynthesis: ...... 11 Does photosynthesis drive orchids to become completely autotrophic? ...... 11 Thesis Aims and Outlines ...... 13 Chapter Two ...... 15 Introduction ...... 16 Materials and methods ...... 18 Seed collection ...... 18 Fungal isolation ...... 18 Seed germination ...... 19 Seed scoring ...... 20 Fungal growth test ...... 20 Data analysis ...... 21 Results ...... 21 Symbiotic germination under different incubation conditions ...... 21 Seedling development following asymbiotic germination ...... 24 Effects of temperature and light on fungal growth ...... 27 Discussion ...... 29 Symbiotic germination and fungal growth ...... 29 Asymbiotic germination ...... 31 Conclusion ...... 33 Chapter Three ...... 34 Abstract ...... 34 Introduction ...... 35 Rationale and objectives ...... 37

Materials and Methods ...... 37 Study species and plant material ...... 37 Experimental growth conditions ...... 38 Effect of light on photosynthesis ...... 38 Effect of nitrogen on photosynthesis and chlorophyll content ...... 39 Plant harvest ...... 39 Gas-exchange measurements ...... 39 Statistical analysis ...... 40 Results ...... 41 Plant growth ...... 41

Effect of absence and presence of fungus on CO2 assimilation and chlorophyll content ...... 41 Effect of light quantity and light type on photosynthesis and chlorophyll content ...... 43 Effect of nitrogen on photosynthesis and chlorophyll content ...... 50 Discussion ...... 54 Effects of mycosymbiont presence on leaf fresh weight, photosynthesis and chlorophyll ...... 54 Content ...... 54 Effect of nitrogen ...... 55 Effect of light and nitrogen on leaf fresh weight, photosynthesis and chlorophyll content ...... 56 Conclusions ...... 58 Chapter Four ...... 59 Optimizing Orchid Propagation ...... 59 Constraints in orchid propagation ...... 61 Key factors for the success of propagated orchids in restoration ...... 62 Seed germination ...... 63 Seedling growth and photosynthesis ...... 64 Key Findings from this study include ...... 66 References ...... 68

Acknowledgements

The author acknowledges the help of the following, who provided a great help in the thesis:

Emma Dalziell, Alison Ritchie, Anthea Challis, Benjamin Anderson who had a valuable assistance in writing.

Keran Keys and Tony Scalzo for their great laboratory techniques.

The Author is also indebted to Botanic Garden and Park Authority (Science) for their great support and outstanding laboratory facilities.

The author also owes special thanks the Botanic Garden and Park Authority (Science) for the provided funding.

Thanks also due to all my friends at Botanic Garden and Park Authority for their kindness and awesomeness. I also owe to all my supervisors; Professor Kingsley Dixon, Dr Eric Bunn, Dr Jason Stevens, Dr Shane Turner and Dr Belinda Newman great thanks for their outstanding supervision.

Statement of candidate contribution

This thesis contains a published paper that has been co-authored. The contribution of each authors are demonstrated below:

Nikabadi S, Bunn E, Stevens J, Turner SR, Newman B, Dixon KW (2014) Germination responses of four native terrestrial orchids from south-west South Australia to temperature and light treatments.

I contributed 60% to this paper including data collection, data analysis and manuscript preparation. My supervisors contributed 40% to initial the concept of idea and revision of the manuscript.

Chapter One General Introduction

Orchid propagation: Ecophysiological aspects of terrestrial orchids undergoing trophic transition

Introduction

Almost 12.5% of the world’s vascular plants are currently facing extinction as a part of the sixth global extinction event (Walter and Gillet, 1998) and the majority of these threatened species are located in biodiversity hotspots (Barthlott et al., 1996; Myers et al., 2000). The Orchidaceae, one of the most abundant and widespread plant families with almost 25,000 species, has been particularly affected by the latest extinction event (Swarts and Dixon 2009b) and orchids are now one of the most threatened plant groups in the world (IUCN, 2009). This decline to critical levels has been the result of both natural and anthropogenic causes (Kull et al., 2006). In particular, land-use management has impacted this species directly but also indirectly as it affects pollinators and mycorrhizal fungi that in many cases are species specific (Koh et al., 2004; Dunn et al., 2009). Orchids have also been affected by over-collection of wild plants for propagation and the illicit horticulture trade which is driven by their ornamental value (Li et al., 2002). Protection of key habitats (Ha´gsater and Dumont, 1996; Cribb et al., 2003) may be the solution to orchid conservation and while most conservation programs are willing to adopt new seed germination and plant growth protocols to progress orchid conservation, emphasis needs to be placed not only on the propagation of threatened species, but also the successful reintroduction of vigorous and acclimatized seedlings.

Orchids and Symbiotic Associations

Nutrient acquisition from nutrient-impoverished soils usually includes specialized root structures. Almost 90% of all plant species facilitate nutrient uptake of P, N and C with cluster roots, fungal symbiosis and root nodules (Lambers et al., 2008). In the highly nutrient deficient soils of Western Australia orchids extend from the tropical and subtropical north to the Mediterranean southwest corner and almost all species rely on the evolutionary adaptation of mycorrhizal associations to mobilize P, N and C (Lambers et al., 2006; Shane

6 and Lambers, 2005). This nutrient acquisition process occurs locally in the rhizosphere, as opposed to the long distance scavenging achieved by roots, root hairs and mycorrhizal fungi.

Orchids facilitate nutrient absorption by mycorrhizal association (Rasmussen, 1995). The orchid lifecycle begins with an association with appropriate fungi at the stage of seed germination (Mahendran and Bai, 2009). Terrestrial orchids have complex associations with mycorrhizal fungi that are vital for seed germination, seedling development, annual growth and maintenance (Warcup, 1973; Clements and Ellyard, 1979; Clements et al., 1986; Rasmussen, 1995). The orchids of south-west Western Australia have pronounced obligate mycorrhizal relationships, often involving only one fungal strain (Ramsay et al., 1986; Dixon, 1991).

Complete fungal obligation in orchids occurs at the achlorophyllous protocorm stage (Rasmussen and Rasmussen, 2009). A few weeks before shoot formation, mutualism starts with the production of trichomes by the protocorm. A few weeks following, the protocorm develops a leaf primordium which eventually develops into differentiated leaf shoots that become photosynthetic (Peterson et al., 1998; Ramsay and Dixon, 2003; Zettler et al., 2003). Following the first season of growth and senescence of the leaf shoot, the orchids can form dormant tubers, although Batty et al., 2002 has shown this can occur without the production of a photosynthetic leaf. As such, presence of appropriate fungi in any conservation program is necessary and suitable inoculums need to be introduced for survival to adulthood under field conditions (Masuhara and Katsuya, 1994; Zettler and Hofer, 1998; Batty et al., 2002; Ramsay and Dixon, 2003). Although orchid mycorrhizal fungi are usually located in the infected tissues of adult plants, the ability to inoculate the soil with the appropriate mycorrhizal fungus is difficult. Seed-baiting techniques are one of the most effective and low cost ways to test for the presence of fungal isolates in soil (Rasmussen and Whigham, 1993, 1998; Brundrett et al., 2003) however, as a bioassay they only represent points in space and time whereby a symbiosis can form (Phillips et al., 2011).

In the adult stage of the orchid life cycle, orchids are able to switch to partial autotrophic growth by increasing photosynthetic capacity. In this growth stage, the plants are able to fix C through photosynthesis and sequester minerals from the fungal symbiont (Smith and Read, 1997; Batty et al., 2002). The orchid may still gain C from its fungal partner despite becoming photosythetically active however, it is often assumed that the orchid has switched to gaining all assimilate via photosynthesis (Rasmussen and Rasmussen, 2009). These autotrophic and heterotrophic stages of the orchid lifestyle drive changes in mycorrhizal

7 dependency (Smith and Read, 1997; Batty et al., 2002). Although the degree of dependency of mature orchids on their mycorrhizal fungi is poorly understood, the obligation for specialized mycorrhizal fungi in terrestrial orchid at the stage of germination is well documented (Burgeff, 1909; Curtis, 1939; Warcup, 1971; Batty et al., 2002; Rasmussen, 2002).

Germination: the first obstacle in orchid propagation

Seed germination ecology has led to a better understanding of some seed characteristics such as timing of germination, maturation, dormancy and dispersal (Baskin and Baskin, 2001; Donohue, 2005). One characteristic that has a strong effect on seed dispersal ability is seed size (Venable and Brown, 1988; Westoby et al., 1990, 1992; Greene and Johnson, 1993). Seed size affects distribution abundance because; 1) small seeds are more easily dispersed than large seeds (Nelson and Chew, 1977), 2) longevity of small seeds is higher than large seeds which causes small seeds to germinate before predation (Harper et al., 1970; Silvertown, 1981), and 3) large seeds are more in danger of predation by mammals and birds compared to small seeds (Guo et al., 1995) and 4) species with larger seeds tend to travel shorter distances (Templeton and Levin, 1979). Large seeds are always rare and have low abundance. In contrast, whether small seeds are rare or common they are often distributed widely (Guo et al., 1998, 1999) and therefore it is often easy to collect a soil seed bank for aboveground plants with small seeds (Fenner, 1985; Thompson, 1987; Aguiar and Sala, 1997). In some plants such as orchids, small seeds and successful dispersal do not guarantee a seed bank or plant community. Instead, availability of appropriate mycorrhizal fungi may play a greater role in determining above-ground spatial distribution and persistence of orchid species (Swarts and Dixon, 2009b).

While some orchid species such as Sobralia macrantha and Bletilla hyacinthina have a differentiated embryo with a rudimentary cotyledon and germinate readily, most orchid species have an undifferentiated embryo without a cotyledon and endosperm and are difficult to germinate (Burgeff, 1936; Harley, 1951; Maheshwari and Narayanaswami, 1952). Terrestrial orchid seeds weigh from 0.3 to 14 µg (Burgeff 1936, Harley, 1951) and have suppressed endosperm hence, limited nutrient reserves and an obligate requirement from symbiotic fungi for nutritional maintenance during germination and growth (Dressler, 1981; Arditti, 1992).

Seed germination trials in the field are difficult and time consuming because orchid seeds are small, germination is often low (Brundrett et al., 2003; Zettler et al., 2005; Diez, 2007; Øien et al., 2007) and suitable fungi needs to be present for seedling development and establishment (Zelmer et al., 1996; Zettler and Hofer, 1998). As high specificity for fungal partners has been reported in natural habitats compared with laboratory conditions (Masuhara and Katsuya, 1994; Perkins and McGee, 1995) symbiotic and asymbiotic in vitro techniques have been developed to successfully germinate orchid seeds (Kauth et al., 2008).

The terms ‘Ecological specificity’ and ‘Potential specificity’ have been used to describe infection of orchids by mycorrhizal fungi under natural field conditions and laboratory conditions respectively (Harley and Smith, 1983; Masuhara and Katsuya, 1994). For some orchids, these specificities are different. For instance, Microtis parviflora is considered to have narrow ecological specificity, as it only forms mycorrhizal associations with two Epulorhiza species under field conditions. However it has high broad potential specificity as it has successful associations with nine different isolated fungi strains under laboratory conditions (Perkins and McGee, 1995). Narrow or broad potential specificity has been observed in other terrestrial orchids however, the specificity of these associations with endophytes in natural habitats is inadequately understood (Warcup, 1981; Alexander and Hadley, 1983; Muir, 1989). Zettler and McInnis (1993) found that high germination of orchid seeds is not always correlated with successful seedling establishment in soil. For instance, while 47% of Spiranthes cernua seeds germinated with one endophyte under ex situ conditions, only two of the seedlings established in soil. When Spiranthes cernua seeds inoculated with another endophyte, germination was lower (38%) but many of the seedlings survived the transfer to soil.

Resolving the degree of specificity in orchid–fungus interactions under laboratory and soil or field situations is, therefore, essential if methods are to be developed to optimise germination success, plant production and reintroduction into a soil environment (Masuhara and Katsuya, 1994; Perkins and McGee, 1995). Donohue (2005) reported that in vitro culture condition is the most appropriate method to simulate the effect of different temperature and light conditions on seed germination under field conditions. Seed responses to light and temperature play a critical role on the timing of germination in the field, and are often considered to be two of the most important environmental variables in creating conditions suitable for seedling establishment (Angevine and Chabot, 1979; Pons 2000). Although the Orchidaceae contribute to a large proportion of biodiversity in some ecosystems, our knowledge is low about the seasonal role of temperature on orchid seed

9 germination. Understanding of orchid physiology has not yet been widely applied to propagation and conservation practices. The first part of this study aims to clarify the effect of illumination and temperature on symbiotic and asymbiotic germination of the study species.

Temperature and Light: Two key environmental factors affect germination

Temperature

Temperature is considered one of the most critical factors in seed germination and the germination capacity of different species can vary according to temperature (Bewley and Black, 1994; Verma et al., 2010). In some species high temperatures can inhibit germination by inducing secondary dormancy (Nascimento et al., 2000), while in other species warm temperatures have been shown to break dormancy and promote germination (Baskin and Baskin, 2004; Leon and Owen, 2003; Turner et al., 2006). Cold (Baskin and Baskin, 2001; Moyo et al., 2009; Han and Long, 2010) or fluctuating temperatures (Baskin and Baskin, 2001, 2003) can also improve germination by breaking dormancy and oscillating temperatures can optimize germination by overcoming far-red light-induced inhibition (Benvenuti et al., 2001; Honda and Katoh, 2007). Interaction effects between temperature and light have also been found to regulate seasonal germination (Hilton 1984; Heschel et al., 2007). Seed germination inhibition factors can be suppressed by fluctuating temperatures and filtering by tree canopies. Bare soil surfaces are often less insulated from oscillating temperatures in comparison with soil surfaces covered with vegetation. However, seeds that disperse to safe and less competitive sites are exposed to far-red due to transmission of red light which is filtered by tree canopies, and fluctuations in temperature are still important in reducing the far-red light-induced inhibition effect (Benvenuti et al., 2001; Honda and Katoh, 2007).

Light

Soil moisture and the colour and size of sand grains are key factors of sandy soils that can influence photon flux density and light quality (Tester and Morris, 1987; Gutterman, 1994). Light may trigger germination of seeds in sandy soils (Gutterman, 1993; Rojas–Aréchiga et al., 1997; Flores et al., 2006). Soil depth and plant cover density affect the signal which seeds receive from light (Vázquez–Yañes and Orozco–Segovia, 1993). Seeds respond to light in three ways during germination. Some species are completely light obligated and

10 only germinate under light conditions (Baskin and Baskin, 1998). This is known as positive photoblasticism. Other species can germinate in light or the absence of light. This is known as neutral photoblasticism and germination rates in this group can be very different. Other species favour absolute darkness and can only germinate in the absence of light. This is known as negative photoblasticism (Baskin and Baskin, 1998). Positive photoblasticism is one of the functional physiological characteristics that support soil seed banks (Bowers, 2000). Neutral and negative photoblastic species are less likely to form seed banks because they require full or partial darkness to germinate. Light requirement specificity varies among different species, while some species germinate in response to brief exposure to light, others require exposure to long periods or sporadic lighting (Bewley and Black, 1994; Taiz and Zeiger, 2002). For many species red light (660–665nm wavelengths) has been shown to be very important in breaking seed dormancy. The level of red or far-red radiation in the light is critical in stimulate or inhibit germination through mediation of phytochrome activity (Gorski, 1975; Taiz and Zeiger, 2002).

Orchid and photosynthesis:

Does photosynthesis drive orchids to become completely autotrophic?

Myco-heterotrophic plants which comprise about 230 species are totally reliant on fungi because they lack chlorophyll (Leake, 1994). The achlorophyllus plants are partially or opportunistically parasitic in the mycorrhizal network. In many cases, this fungal dependency has resulted in loss or redundancy of the photosynthetic apparatus and potentially restricted the evolutionary potential of such species (Dressler, 2004). In orchids, reduction in photosynthesis and associated loss of chlorophyll is believed to have occurred about 20 times (Cameron et al., 1999; Molvary et al., 2000; Bateman et al., 2005). Most orchids produce chlorophyll as adults, and are only obligate myco-parasites at the early stages of protocorm development. By combining parasitism and photoassimilation as life style strategies, many orchids are considered mixotrophic (Gebauer and Meyer, 2003; Bidartondo et al., 2004; Selosse et al., 2004).

Only 170 orchid species (Leake, 1994), such as Rhizanthella gardeneri (the underground orchid) are known to lack chlorophyll and consequently remain completely parasitic on fungi (Brundrett, 2004). It is unknown whether species visibly low in chlorophyll are also unable to sufficiently photosynthesise to sustain themselves when conditions are difficult (Leake, 1994). The function of the photosynthetic apparatus can be affected by

11 environmental factors which can be variable or unpredictable, as they may be influenced by other factors such as species, growth stages, and supporting materials (Adelberg et al., 1992; Tanaka et al., 1992).

The low photosynthetic rate at the early stages of orchid life cycle under in vitro conditions is assumed to be the result of the absence of adequate photosynthetic surfaces, poor anabolism of essential substances such as amino acids (Rasmuassen, 1995), lack of CO2 in culture during the photoperiod and high relative humidity in enclosed culture (Fujiwara et al., 1987; Infante et al., 1989; Capellades, 1990). The rate of photosynthesis increases in vitro when light saturation levels are amplified and CO2 is supplied (Kitaya et al., 1995). However, Kozai et al., (1997) could not increase net photosynthetic rate when photosynthetic photon flux (PPFD) levels were high and CO2 supplies were low (Infante et al., 1989). This suggests that high levels of CO2 and PPFD are necessary for photosynthesis and these factors are interactive.

In in vitro studies, two types of leaves have been found in orchids. The first group are those with insufficient and incompetent photosynthetic apparatus function and the second group have leaves that are adapted to autotrophic conditions (Grout 1998). In some studies, in vitro leaves have been shown to act as storage organs to cover the metabolic demands of growing tissues during the early days of acclimatization (Capellades et al., 1991; Van Huylenbroeck and Debergh, 1996). In vitro propagated leaves also have a competent photosynthetic apparatus function (Van Huylenbroeck et al., 1998). Under ex vitro conditions in a glasshouse, successful acclimatization of orchid seedlings has been observed as a result of modulation of photosynthetic efficiency and pigmentation changes (Preece and Sutter, 1991). Failure to achieve these physiological changes during micropropagation and acclimation may lead to poor survival. Under in vitro conditions, exposure of seedlings to moderately high light intensity can switch relatively light-sensitive photosynthetic tissue to light-tolerant tissue, resulting in acclimation from in vitro to ex vitro conditions, with this mechanism possibly driven by photoinhibition of plantlets during acclimatization (Hanelt et al., 1997). Once the protocorm development stage has passed, the photosynthetic apparatus may then allow the orchids to be less dependent on the fungal symbiont (Rasmussen and Rasmussen, 2009).

Photosynthetic response in relation to biomass accumulation in the tuber and leaf under glasshouse and field conditions would help to elucidate the major stresses placed on seedling development during tuberisation (Swarts, 2007). Beyrle and Smith (1993) have found that

12 the growth rate of leafy seedlings of Cattleya species under even and constant irradiation (16 h, about 120 mmol m-2 s-1) was influenced by the available C resources of symbiotic saprotrophic fungus. This dependency caused the inhibition of photoassimilation when available organic materials in the substrate were plentiful and inhibition when organic materials were low. Wright (2009) observed that in the Caladenia genus functioning symbiotic associations appear to be a basic physiological requirement for the function of the photosynthetic apparatus. The implication for micropropagation programs involving Caladenia and other genera with similar fungal dependencies highlight that absence of the fungal symbiont will result in gradually poorer leaf production and ultimately plant death. Research is urgently required to find out why orchids exhibit such low post-in vitro survival characteristics. In the third chapter, we will focus on the ecophysiology of the transition phase from heterotrophic growth of orchid seedlings in vitro to full autotrophic growth as plants are moved to ex vitro conditions to try and find answers to the low survival rate problem.

Thesis Aims and Outlines

The aim of this research is to investigate the ecophysiology of trophic transition of the symbiotically and asymbiotically propagated C. huegelii, C. latifolia, P. sanguinea and M. media for improving restoration success. While the role and importance of mycosymbiont fungi in germination of terrestrial orchid seeds is relatively well understood, the effect of abiotic factors on the amount and rate of germination and growth and effectiveness of mycorrhizal fungi is not well studied with Australian terrestrial orchids. Terrestrial orchids are putatively mycotrophic during germination and early development stages but at later stages of development are presumed to become autotrophic or at least mixotrophic. This study addresses the following questions:

- Do temperature and light influence seed germination of the study species in the presence or absence of mycorrhizal fungus? - Can the fungal growth rate be affected by the temperature and light? - Which parameters influence symbiotically and asymbiotically propagated seedlings in the stage of trophic transition? - Is photosynthesis of early-stage propagated orchid seedlings affected by different light type, light quantity and nitrogen? - Does leaf chlorophyll content determine the photosynthetic rate of symbiotically or asymbiotically germinated orchid seedlings?

Chapter 2 investigates the potential vulnerability during germination of study species to abiotic environmental factors such as temperature and/or light. Germination and seedling development of the study species under in vitro conditions may be affected by temperature and light which consequently influence ex situ establishment of more robust plants. In this chapter, we also investigate the effect of temperature and light on growth and effectiveness of mycosymbiont fungi.

Chapter 3 investigates the effect of presence or absence of orchid mycosymbionts on photosynthetic capacity and chlorophyll content of terrestrial orchid seedlings under in vitro conditions. This chapter also aims to explore the dynamics of the transition from in vitro to ex vitro conditions and improve production and establishment of orchids for restoration of threatened taxa.

Chapter 4 The results from the previous chapters are integrated and discussed to develop an appropriate protocol for propagation of the study species from seed germination to autotrophic stage. Further research recommendations are made to understand and predict requirements for the propagation of the study species under glasshouse conditions.

Chapter Two

Germination responses of four native terrestrial orchids from south-west Western Australia to temperature and light treatments

Abstract

We report an investigation into the impact of temperature and illumination on in vitro symbiotic and asymbiotic germination of the threatened taxon Caladenia huegelii, and three other orchid species namely - Caladenia latifolia, Microtis media and Pterostylis sanguinea, all species from south-west Western Australia, a recognized biodiversity hotspot. High symbiotic germination on oatmeal agar (OMA+ fungal symbionts specific to each species) was recorded in three species in continuous dark incubation i.e. Caladenia huegelii seeds (98 % germination at 25 ⁰C), and M. media and P. sanguinea (93 and 98% respectively at 20 ⁰C). Highest symbiotic germination for C. latifolia (100%) was observed at 15 and 20 ⁰C under light treatment (12/12 hr light/dark). Low temperature incubation (10 ⁰C) significantly suppressed symbiotic germination/development of seedlings across all species. Asymbiotic media treatments assessed (OMA minus fungal symbionts, Pa5 and ½MS), failed to stimulate any germination of C. latifolia seeds at 20 oC in either light or dark treatments after an eight week incubation period. Seeds of M. media sown onto ½MS medium resulted in higher germination in all developmental stages (3-5) in dark treatment than OMA and Pa5. Seeds of P. sanguinea sown onto ½MS medium resulted in higher overall germination in all developmental stages (3-5) in light and dark incubation compared to OMA and Pa5. OMA supported the highest asymbiotic germination (100%) in both light and dark incubation with M. media (only to stage 3) but did not support germination and development with other species tested. Caladenia huegelii seeds reached developmental Stage 3 (i.e. germinated), but only on Pa5 medium and only at a relatively low rate in either light (2.6%) or dark (2.1%). Germination was higher and development of seedlings faster overall in all test species in symbiotic compared with asymbiotic media treatments. Pterostylis sanguinea seeds demonstrated the best response (among species tested) to asymbiotic germination on ½MS with 40-53% of germinated seeds spread over developmental stages 3-5 in light or dark incubation (at 20 oC) respectively. Illumination had no effect on fungal symbiont growth across all species, however incubation temperature treatments (10, 15, 20 and 25 ⁰C) affected fungal growth rate. Growth of the fungal symbionts of Caladenia huegelii, M.

15 media and C. latifolia demonstrated significantly lower activity at 10 ⁰C, but the cumulative radial growth rate of the P. sanguinea fungal symbiont reached 64 cm2 after only two weeks at all temperatures tested, including 10 ⁰C. The study highlights differences in symbiotic and aysmbiotic germination and early protocorm development in vitro between co-occurring herbaceous terrestrial Australian orchid taxa in response to variations in basal media, temperature and light.

Introduction

Although it is considered critically important to understand the effects of environmental factors on the recruitment of orchid seeds from the time of protocorm emergence from the testa to the seedling elongation stage (Baskin and Baskin, 2001; Donohue, 2005), in practise it is difficult and time consuming to clarify the environmental effects on orchid seed germination under in situ conditions in any detailed manner (Brundrett et al., 2003; Zettler et al., 2005; Diez 2007; Øien et al., 2007). Although in vitro symbiotic seed germination has been found to be an effective way to elucidate the effects of testable environmental factors on germination (Kauth et al., 2008), methods need improving, as germination efficiency is often low (Stewart et al., 2003; Zettler et al., 2005; Stewart and Kane, 2006). Ecotypic variation among orchid species can also add to the difficulties in achieving reliable propagation but this variation can be evaluated and quantified using established in vitro seed germination protocols as demonstrated by Probert et al., (1985) and Kauth et al., (2008). Although uncontrolled field conditions are likely to be very different compared to controlled laboratory conditions, in vitro seed germination is the best available technique for estimating and understanding the effects of environmental factors such as temperature and photoperiod on germination in orchid species (Donohue, 2005). Seed responses to light and temperature play a critical role in the timing of germination in the field, and are often considered to be two of the most important environmental variables in creating conditions suitable for seedling establishment (Pons 2000). Germination is further complicated by variation in environmental factors such as Photosynthetic Photon Flux Density (PPFD), spectral composition and different interactions between these factors and time (Pons, 2000; Probert, 2000). Additionally, unlike most conventional seeds many orchid taxa require species specific fungi to begin and sustain the seed germination process. As with seeds, soil temperature also plays a critical role in determining the fundamental physiological responses of terrestrial fungi, as has been demonstrated by Cooke and Whipps, (1993).

The responses of mycorrhizal fungi associated with terrestrial Australian orchids to various in situ environmental factors and how these responses impact on seed germination remains understudied. Ensuring the conservation of the current 85 species of priority and endangered endemic terrestrial orchids of the South West Floristic Region (SWFR) of Western Australia will require an integrated approach involving multiple disciplines including reserve management, effective ex situ germplasm storage, and development of in situ restoration/translocation approaches (Swarts and Dixon, 2009a). Improving ex situ orchid conservation in a global sense, relies on greater understanding of seed propagation, in particular responses of seed-fungal symbiosis to significant ecological factors such as light and temperature that are likely to be impacted by climate change (Swarts and Dixon, 2009b). Although other studies on Australian terrestrial orchids species have investigated in vitro asymbiotic germination these have mainly focussed on comparing basal media (Dowling and Jusaitis, 2012) while direct comparisons of symbiotic with asymbiotic germination is rare (Oddie et al., 1994). To our knowledge, there are no prior studies that have investigated interactive effects of abiotic factors (temperature and light) on both symbiotic and asymbiotic germination with Australian terrestrial orchids.

Four species from three genera were selected for this study: Caladenia huegelii (Grand Spider Orchid) H.G. Reichb., Caladenia latifolia (Pink Fairy Orchid) R.Br., Microtis media (Common Mignonette Orchid) R.Br and Pterostylis sanguinea (Banded Greenhood) D.L.Jones & M.A.Clem. The genus Caladenia is found Australia wide, with 200 species endemic to the SWAFR, many of which display high levels of fungal specificity (Myers et al., 2000; Batty et al., 2001a). Among the selected orchid species utilised in this study C. huegelii is listed as Critically Endangered (CR) under IUCN criteria (IUCN, 1999; Hopper and Brown, 2001), while the other three taxa evaluated in this study are not considered threatened at present. Unlike the other species, Caladenia latifolia is found across southern Australia; from the south west of Western Australia, the south of South Australia, southern Victoria to Tasmania. In Western Australia it occurs in a variety of habitats including dense coastal heath, woodlands, Karri forest, and in the central and southern wheat belt is also seen on low rises near salt lakes. Microtis media is a weedy native species that is widespread in Western Australia extending from the south-west inland to Cue and Kalgoorlie, and eastwards along the south coast to the Great Australian Bight. This species is found in swampy habitat, shallow coastal heaths and in woodlands and forests (Hoffman and Brown, 1998). Pterostylis sanguinea is widespread between Mullewa and Ravensthorpe in Western Australia, Southern Victoria and Southern South Australia (Brown et al., 2008).

This study focuses on investigating abiotic factors on the in vitro symbiotic and asymbiotic germination behaviour and growth of four native terrestrial orchid species. Specifically, the aims of the study were to (i) investigate the effects of variation in temperature and illumination on both symbiotic and asymbiotic germination between species, ii) determine any interactive effects of illumination or temperature on seed germination responses for each of the study species and (iii) assess whether combinations of temperature and light affect symbiotic fungal growth and hence its effect on both germination and development of seedlings over time.

Materials and methods

Seed collection

Caladenia latifolia and M. media seeds were collected from plants with dehiscing capsules growing in remnant native bushland in Kings Park (31°1.6’0.48’’ S, 115°1.38’0.36’’ E) in October 2012. Seeds were stored in Eppendorf tubes with their lids partially unscrewed at 150C and 15% RH for two weeks to allow seeds to dry completely before the Eppendorf tubes were sealed in an airtight container. Seed lots from individual plants of C. latifolia and M. media were combined and mixed prior to experimental use. Seeds of C. huegelii were collected from Ken Hurst Park (32°0.6’1.58’’ S, 115°1.46’0.68’’ E) in October 2012, and Pterostylis sanguinea seeds were collected from multiple sites: Kings Park and Bold Park (31°56’0.96’’ S, 115°46’26.04’’ E); Koondoola Reserve (31°1.5’0.45’’ S, 115°13.3’6.55’’ E) and Warwick Open space (31°14.06’1.46’’ S, 115°13.66’13.66’’ E) in October 2012. Seed of C. huegelii and P. sanguinea were prepared and stored (for approx. 12 months) as previously described, then used in experiments. Prior assessment of Australian terrestrial orchid seeds (including C. huegelii and P. sanguinea) with similar preparation and storage conditions indicate viability and germination capabilities are retained following storage up to 24 months (Hay et al., 2010).

Fungal isolation

Infected sections of root, stem-collar and underground stem were utilized to obtain fungal isolates from all study species (Microtis media, Caladenia huegelii, C. latifolia and Pterostylis sanguinea), as per Ramsay et al., (1986). Samples were collected in situ during the growing season (Jun-Sep) in 2012 and placed in moist paper towels until laboratory processing. Pelotons were isolated from sterilised plant tissue according to the protocol of

Rasmussen (1995) and then plated onto fungal isolation media (FIM 0.3 g L-1 sodium nitrate, 0.2 g L-1 potassium dihydrogen orthophosphate, 0.1 g L-1 magnesium sulphate, 0.1 g L-1 potassium chloride, 0.1 g L-1 yeast extract, 2.5 g L-1 sucrose, 8 g L-1 agar, prepared to 1 L with deionized (DI) water, pH adjusted to 6.8 before autoclaving (20 min at 121 ⁰C, 105 kg cm-2). Ten ml L-1 of 1 mM filter sterilized streptomycin sulphate stock was added after autoclaving when media was cooled to approximately 50 ⁰C, for a final concentration of 10 µM (Bonnardeaux et al., 2007). After one week, fungal hyphae proliferated on FIM plates, with infected collar and root material producing sparse growing fungal cultures that allowed harvesting of singular hyphae growing tips, which were subcultured onto potato dextrose agar (PDA) to encourage fungal growth. Once fungal colonies reached optimal growth on PDA plates, two agar blocks were used to inoculate the symbiotic germination plates containing autoclaved oatmeal agar (OMA, with 2.5 gl-1 crushed oats, 8 g L-1 agar, pH 5.5) for subsequent seed germination trials.

Seed germination

Symbiotic germination

Seeds were enclosed in 3cm2 nylon mesh bags (0.45 µm mesh size) and surface sterilized for

10 min in a solution of 1% w/v calcium hypochlorite (3 g Ca(OCl)2 powder dissolved in 179 ml distilled deionised water + 21 ml of 1M hydrochloric acid). Mesh bags were rinsed twice in sterile DI water for 2 min. The bags were opened and the surface sterilized seeds diluted in 30 ml sterile distilled water, containing 0.05 % v/v surfactant (Tween 80) to eliminate seed clumping. The water-suspended seeds were spread onto 90 mm Petri plates containing 25 ml of autoclaved OMA. Once the water evaporated, seeds were inoculated with two 5 mm x 5 mm blocks of agar containing the appropriate symbiotic fungus. Plates were sealed with a single layer of thermoplastic film and randomly assigned to one of four temperature regimes (10 ⁰C, 15 ⁰C, 20 ⁰C and 25 ⁰C); and one of two light treatments (constant darkness or 12 hrs light/12 hrs darkness PPFD) administered at each incubation temperature. Four replicates for each treatment were implemented. Petri plates were wrapped in two layers of aluminium foil to provide constant dark conditions and were only opened and assessed for germination after 8 weeks incubation.

Asymbiotic germination

An asymbiotic seed germination trial was implemented to compare results with the symbiotic trial in order to clarify the role of the fungus and its effect in regulating the response of orchid seeds to different incubation temperatures. The asymbiotic trial was also used to determine the efficacy of different asymbiotic culture media. As such, seeds were sterilised (as previously described) and then spread onto 90 mm diameter Petri plates containing one of the following three different media types: 1) 25 ml OMA, 2) Pa5 (modified Burgeffs N3f, Collins and Dixon 1992), and 3) half-strength MS (½ MS macro- and micro-nutrients) (Murashige and Skoog, 1962) supplemented with 0.25 µM α- napthaleneacetic acid (NAA, from Sigma Chemical Co), 5 % v/v coconut water (CW) and 1.0 g L-1 activated charcoal (AC). Petri plates (plus seeds) were only assigned to one temperature regime (20 ⁰C) under light and dark conditions (as previously described). A completely randomized design (CRD) was used for all germination experiments with four replicate Petri plates for each treatment.

Seed scoring

Seeds in the light treatment were scored weekly starting from week four with final germination scored in week eight. Seeds incubated under dark conditions were only scored once at week eight. Seeds were scored in accordance with the method of Ramsay et al., (1986), where seeds were considered to have germinated when they had reached stage three of development, i.e. where the embryo ruptures the testa and trichomes (rhizoids) become visible. Germination in this study therefore represents the proportion (%) of seeds that passed stage 3 (and including germinants reaching stages 4 and 5). In Table 1 the percentages of germinating seeds are displayed according to the stage of development reached after eight weeks; stage 3 (protocorm + rhizoid), stage 4 (enlarged protocorm) and stage 5 (seedling elongation).

Fungal growth test

Fungal isolates used in this experiment were also grown on OMA without seeds using the same light and dark and temperature conditions used for the seed germination experiments to demonstrate the effect of these two environmental factors on fungal growth and development. The fungal growth rate under light treatment was measured weekly starting

20 from week one, with the final measurements recorded in week eight. Fungal radial growth was measured using root-scanning software (WinRHIZO, Regent Instruments Inc., Ottawa, ON, Canada). Fungal isolates incubated in dark conditions were only measured once at week eight with an average growth rate calculated from four Petri plates per isolate.

Data analysis

Statistical analysis was performed using SAS v 9.3 (SAS Institute, 2002). Data were subjected to Two-way ANOVA to test for significant and Fisher’s LSD was used to compare mean at P=0.05.

* and ** Indicate significant differences at 5 and 1% probability levels respectively.

Results

Symbiotic germination under different incubation conditions

Though the studied species showed different germination percentages across treatments, the overall germination response to both light and temperature between C. huegelii and M. media was similar (Fig. 1). While the germination response by P. sanguinea and C. latifolia seeds to temperature concurred, response to light/dark treatment was opposite (Fig. 1). After eight weeks of culture, seeds of C. huegelii exhibited a high germination percentage (nearly 100 %) at 25 ⁰C under dark conditions, the only study species to exhibit highest germination at this temperature. However, incubation at 10⁰C significantly suppressed germination/development across all species regardless of light or dark treatment (P<0.001) (Fig. 1). Illumination significantly suppressed the germination percentage of C. huegelii to 4 % and 33 % but only at 10 ⁰C and 25 ⁰C respectively; however no significant difference was found between dark and light germination of C. huegelii seeds at 15 ⁰C and 20 ⁰C. Microtis media revealed significantly high germination of 93 % and 88 % at 20 ⁰C under dark and light conditions respectively (P<0.0001), but M. media seeds grown under light at 10 ⁰C and 25 ⁰C responded with low germination of 4 % and 34 % (Fig. 1). Pterostylis sanguinea seed germination was significantly suppressed at 15-25 ⁰C under light conditions, however absence of light significantly enhanced germination with 89 % and 98 % of seed germinating at 15 ⁰C and 20 ⁰C respectively (Fig. 1). Among the studied species only C. latifolia exhibited a significantly higher germination percentage under light conditions, with seeds germinating up to 100 % at 15 ⁰C and 20 ⁰C in the presence of light, whereas absence

21 of light significantly suppressed germination at 15 ⁰C and 20 ⁰C (P<0.0001) (Fig. 1). The interaction between illumination and temperature was significant in all species.

a 100 a C. huegelii 100 M. media a

b b 80 b 80 b b b bc 60 60 c c c c 40 40

20 20

d d (%)

0 0 10⁰c 15⁰c 20⁰c 25⁰c 10⁰c 15⁰c 20⁰c 25⁰c a a a 100 P. sanguinea a 100 C. latifolia

80 80 Germination 60 60 b b

bc 40 c 40 c

bc bc c

20 20 c c c c 0 0 10⁰c 15⁰c 20⁰c 25⁰c 10⁰c 15⁰c 20⁰c 25⁰c Temperature Temperature

Fig. 1 Symbiotic germination of Caladenia huegelii, Microtis media, Pterostylis sanguinea and Caladenia latifolia after 8 weeks incubation under different light and temperature treatments. Light grey bars denote light treatment (12/12 light/dark, 15µmol m-2 s-1) and dark bars denote incubation without light (0/24 light/dark). Germination scored as % of seeds to reach stage 3 and above (stages 4-5) and denoted as means ± SE (n = 4). Different letters show significant differences between means within species at α=0.05 level based on LSD mean separation.

Extending the germination period from 8 to 11 weeks did not significantly alter the overall trends in symbiotic germination response under light conditions which still varied considerably among the study species; with Caladenia latifolia seed achieving 100 % germination at 15 ⁰C and 20 ⁰C after 8 weeks culture on OMA with fungus, while C. huegelii and P. sanguinea reached maximum germination (70 % and 25 % respectively) but

only after 11 weeks incubation (Fig. 2). While C. huegelii, P. sanguinea and C. latifolia showed less than 30 % germination at 20 ⁰C within 4 weeks, M. media seeds exhibited almost 90 % germination in the same period of time (Fig. 2). In general, the lowest and highest temperature treatments (10 and 25 oC) tended to have the most effect on slowing germination response in light for C. huegelii, M. media and C. latifolia whether at 8 or 11 weeks of incubation, while with P. sanguinea 10 and 15 oC reduced germination response overall in comparison with 20 and 25 oC (Fig. 2).

100 C. huegelii 10 ° C 100 M. media 15 ° C 80 80 20 ° C 25 ° C 60 60

40 40

20 20

0 (%) 0

4 8 11 4 8 11

100 100 C. latifolia P. sanguinea

80 80 Germination 60 60

20 20

0 0 4 8 11 4 8 11 Week Week

Fig. 2 The time dependence symbiotic seed germination trend of Caladenia huegelii, Microtis media, Pterostylis sanguinea and Caladenia latifolia cultured on oat meal agar (OMA) under 12:12 hrs light/dark photoperiod. Values are means ± SE (n = 4).

Seedling development following asymbiotic germination

After 8 weeks of culture, germination and stage of development of germinated seeds varied between species and asymbiotic media, and the control treatment (OMA with mycosymbiots) (Table 1). Seeds of C. huegelii, P. sanguinea and C. latifolia did not germinate on OMA (minus fungal symbionts) by the conclusion of the incubation period (8 wk), while seeds of M. media all germinated on OMA by week 8 in light or dark incubation but only to stage 3. In contrast, the control treatment (OMA+fungal symbionts) showed comparatively high germination and also exhibited relatively high proportions of later stage (4-5) seedling development with all species (Table 1). Seed germination on ½ MS medium was zero with C. huegelii and C. latifolia after 8 wk in either light or dark incubation, but showed a low total germination response with M. media seeds of 5.1% and 11.3% in light and dark incubation respectively, but a better result with P. sanguinea with total germination of 40 and 53.3% in light and dark treatments respectively (Table 1). Microtis media and P. sanguinea seeds germinated on ½MS presented a higher proportion of seedlings attaining later development stages (4 and 5), with a mild bias towards dark incubation treatments, as indicated by significant differences between light and dark treatments at stages 4 and 5 germination with M. media and stage 5 with P. sanguinea (Table 1). Pterostylis sanguinea seeds responded to Pa5 medium with overall germination of 16.8 and 30.8 % in light and dark treatments respectively, but germinants only reached stages 3 and 4 at the conclusion of the experiment, with a significant difference between light and dark treatments at stage 3 germination but not stage 4 (Table 1). Caladenia huegelii only reached developmental stage

3 on Pa5 medium (in both light or dark incubation) while a small (but significant) proportion of M. media germinants reached stage 4 in Pa5 medium but only under light treatment (Table

1). Pa5 medium failed to stimulate any germination of C. latifolia seeds within the eight week incubation period (Table 1). Illumination affected germination overall, but varied with species and incubation temperature (Table 2, Column 4 - Germination,). Similarly, temperature also showed a significant effect on germination in all the study species (Table 2). The interaction between illumination and temperature was statistically significant across all species, but least significant with M. media compared to the other species examined (Table 2).

Table 1. Stage 3 and above germination and development of the four study species following eight weeks incubation under light and dark conditions at 200C. Media include: asymbiotic (oat meal agar

(OMA), ½MS and Pa5) and symbiotic (OMA + Fungus). Germination stages: 3; protocorm with rhizoid, 4; protocorm enlargement and 5; seedling elongation. Different letters show significant differences between means within species at α=0.05 level based on LSD mean separation.

Germination stage

Stage 3 Stage 4 Stage 5

Culture Dar Species Light Dark Light Dark Light medium k

Caladenia OMA 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a huegelii

½MS 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a

b b a a a a Pa5 2.6 2.1 0.0 0.0 0.0 0.0

54.9 OMA+Fungus 0.0 a 0.0 a 50.3b 45.1b 49.7b b

C. latifolia OMA 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a

½MS 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a

a a a a a a Pa5 0.0 0.0 0.0 0.0 0.0 0.0

OMA+Fungus 17.7b 36.2c 42.3c 19.7b 39.9c 2.1a

Microtis media OMA 100.0 b 100.0 b 0.0 a 0.0 a 0.0 a 0.0 a

½MS 1.1b 0.7b 4.05bc 8.3d 0.0 a 2.3bc

b a b a a a Pa5 4.8 0.0 4.8 0.0 0.0 0.0

22.9 OMA+Fungus 18.5ab 21.1ab 32.1b 33.9b 5.9a ab

Pterostylis OMA 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a sanguinea

12.1 ½MS 8.0b 12.4b 32.0c 28.8c 0.0 a b

b c b b a a Pa5 9.9 21.8 6.9 9.0 0.0 0.0

28.1 OMA+Fungus 18.4c 4.3 b 15.8c 64.9e 0.0 a d

Table 2. ANOVA results for the combined effects of temperature and illumination on symbiotic germination and fungal growth rate on four study species.

Germination Fungal Growth

Species Effect d.f. F P F P

Caladenia Temperature 3 13.1* <.0001 392.7** <.0001 huegelii Illumination 1 17.6* 0.0003 1.09 0.31

Interaction 3 6.1* 0.003 1.98 0.14

C. latifolia Temperature 3 25.4* <.0001 655.9** <.0001 Illumination 1 36.4* <.0001 0.19 0.667

Interaction 3 9.39* <.0003 0.24 0.865

Microtis Temperature 3 31.1* <.0001 292.7** <.0001 media Illumination 1 8.2* 0.0084 3.29 0.082

Interaction 3 4.3* 0.0146 1.82 0.171

Pterostylis Temperature 3 6.56 0.0021 0.51 0.001 sanguinea Illumination 1 27.9* <.0001 0.45 0.51

Interaction 3 10.52* 0.0001 0.74 0.54

* and ** = significant differences at 5 and 1% probability levels respectively.

Effects of temperature and light on fungal growth

While temperature had a significant effect on fungal symbiont growth for all species except P. sanguinea, symbionts showed no preference for light or dark incubation with growth almost identical with a 12/12 light-dark photoperiod treatment compared to total darkness over the whole incubation period (8 wk), with no significant effects across all species (Fig. 3; Table 2, Column 5 – Fungal Growth). At 10 ⁰C, cumulative fungal growth for Caladenia huegelii, M. media and C. latifolia was less than 35 cm2 after 8 weeks and showed markedly lower growth than at the other temperatures assessed (Fig. 3). In comparison, the fungal radial growth of P. sanguinea had reached 64 cm2 after only 2 weeks culture at the same temperature (Fig. 3). Caladenia huegelii, M. media and C. latifolia fungal isolates were also found to grow faster as incubation temperature increased though the rate of growth was quite variable (Fig. 3). Fungal hyphae of the C. huegelii isolate had covered the whole Petri plate after 4 weeks at 25 ⁰C, while for the mycosymbionts of M. media and C. latifolia the same extent of fungal growth occurred after only 2 weeks incubation at 20 ⁰C and 25 ⁰C respectively (Fig. 3).

70 C. huegelii 70 M. media 60 60 50 50

) 40 40 2 30 30 20 20 10⁰C 15⁰C 10 10 20 ⁰C 25⁰C 0 0 0 2 4 6 8 0 2 4 6 8

70 P. sanguinea 70 C. latifolia 60 60 50 50 Fungal Growth Rate (cm Rate Growth Fungal 40 40

30 30

20 20

10 10

0 0 0 2 4 6 8 0 2 4 6 8

Incubation time (weeks)

Fig. 3 Cumulative radial growth of fungal symbionts from Caladenia huegelii, Microtis media, Pterostylis sanguinea and Caladenia latifolia cultured under 12:12 hrs light/dark photoperiod on OMA. Values are means ± SE (n = 4).

For C. huegelii the earlier completion of fungal growth (covering whole the Petri plate) was observed at 25⁰C after four weeks, however, M. media and C. latifolia fungal growth completion simultaneously occurred only after two weeks at 20⁰C and 25 ⁰C (Fig 3). Among the studied species only P. sanguinea fungal isolate did not show a significant difference in growth rate among different temperatures (Fig 3 and 4), whereas the optimum temperature for fungal growth were found to be between 15 to 25⁰C for C. huegelii, M. media and C. latifolia (Fig 4). At these temperatures, values of fungal activity were almost 2 to 3 times higher than at 10⁰C. Absence of the light did not significantly influence the fungal growth among species at all temperatures (Fig.4).

C. huegelii M. media 70 a a a a a 70 a a light a a a a a 60 60 Dark

50 50 b 40 40 bc

30 ) b 30 2 b 20 20

10 10

0 0 10 15 20 25 10 15 20 25 P. sanguinea 70 a a a C. latifolia a a a a a 70 a a a a a a 60 60 50 50

40 40 Total Fungal Growth (cm Growth Total Fungal 30 30 b b 20

0 0 10⁰C 15⁰C 20⁰C 25⁰C 10⁰C 15⁰C 20⁰C 25⁰C

Temperature

Fig. 4. Effect of temperature and illumination on fungal radial growth from Caladenia huegelii, Microtis media, Pterostylis sanguinea and Caladenia latifolia after 8 weeks culture on OMA. Values are means ± SE (n = 4).

Discussion

Symbiotic germination and fungal growth

It was expected that the study species would show some evidence of species specific germination responses to different light and temperature conditions with at least some of this variation possibly due to provenance or subtle differences in habitat requirements as speculated for other Australian orchid taxa (Dowling and Jusaitis 2012). Indeed, seeds of all species showed variation in germination when exposed to different incubation temperatures (10, 15, 20 and 25 ⁰C) in both darkness and light. Unexpectedly, germination in all species was suppressed when seeds were cultured at 10 ⁰C indicating a common, low temperature threshold at which seed germination is significantly reduced. At the highest temperature

29 assessed (25 ⁰C) germination was also reduced under light (and dark) conditions for three of the four species and indicates that a possible upper temperature limit may exist for effective germination of these species, at least under in vitro conditions. A noticeably slower germination rate was clearly observed in C. huegelii, C. latifolia and P. sanguinea when compared to M. media (Fig. 2) at 15 ⁰C and 20 ⁰C which might reflect the intrinsic ‘weediness’ reported for M. media (De Long et al., 2013). Invasive orchids such as M. media support the concept that weedy plant species germinate more readily under a broad range of temperatures (Baskin and Baskin, 2001). However, to explore this concept further in future studies it will be necessary to assess additional sympatric orchid taxa as well as possible ecotypic effects of widely distributed taxa.

Pterostylis sanguinea and C. latifolia seeds varied significantly in their germination response to illumination over most of the temperature range tested, even though fungal symbiont growth of both species in response to light was similar at 20 and 25 oC. Van Waes and Debergh, (1986) reported that responses of terrestrial Western European orchid seeds to light are highly variable and low levels of light can greatly reduce germination. Among the studied species, only P. sanguinea was found to be negatively photoblastic as germination was observed to significantly improve under dark conditions. Conversely, C. latifolia seeds were positively photoblastic as the seeds displayed a consistent bias towards germination in light across all temperature treatments, with significant germination differences at 15 and 20 oC between light and dark treatments, suggesting that light could be a limiting factor for seed germination of C. latifolia, in some circumstances. Delgado-Sanchez et al., (2013) indicated that a positive response of inoculated orchid seeds to light can boost germination near the soil surface under natural conditions. In P. sanguinea, only low temperature (10 ⁰C) appears to suppress germination, overcoming the significantly positive germination effect of dark treatment. In terms of seed germination, Pterostylis sanguinea and C. latifolia seeds appear to be more affected by light (or its absence) rather than temperature at least at 15 and 20 ⁰C. In comparison, Caladenia huegelii and M. media were neutral photoblastic at 15 and 20 ⁰C however at low (15 ⁰C) or high (25 ⁰C) incubation temperatures the presence of light was found to have a significantly negative effect on germination. Even with the results from this study, generalizations regarding the effects of light on seed germination and seedling growth of Orchidaceae with wide geographic ranges are hard to achieve. It is likely that promotive and inhibitory effects of light on seed germination and seedling development are intrinsic characteristics for each species (Godo et al., 2011) and may assist species to co- exist in the same environment through niche partitioning.

Although illumination had no effect on fungal growth rate, the lowest incubation temperature tested (10 ⁰C) significantly suppressed fungal growth of all species except P. sanguinea. In the current study a low germination rate at 10 ⁰C for C. huegelii, C. latifolia and M. media could be due to the fact that the particular mycorrhizal fungi for those species do not grow well at temperatures below 15 ⁰C. Conversely, P. sanguinea showed poor germination at 10 ⁰C although the fungal growth rate at this temperature remained high. High seed germination may correlate with optimal fungal growth rate but it is also possible that low, suboptimal temperature might affect fungal ability to translocate nutrients through hyphae to the seed. Yet, it is crucial to emphasise that low germination at suboptimal temperatures could also be due to cryptic seed or fungal characteristics or fungal-seed interaction.

Asymbiotic germination

The asymbiotic media treatments were applied not only to examine the nutritional and cultural requirements of the study species, but also contribute to a better understanding of the role that fungi play in symbiotic orchid seed germination. To our knowledge, optimal asymbiotic media for the study species have not been previously reported. The ½ MS and

Pa5 media were selected according to available literature on asymbiotic germination of Australian and non-Australian orchids. OMA with non-soluble carbohydrate was applied as a nutrient-poor medium, whereas ½MS and Pa5 medium containing macro and microelements, vitamin and sugar (Murashige and Skoog 1962; Collins and Dixon 1992) were used in this study as nutrient rich media (in relative terms). Thompson et al., (2007); Mahendran and Bai (2009), Lee (2011) and Dowling and Jusaitis (2012) reported successful germination for South African, American, Asian and Australian orchid species by using basal ½MS medium. Dowling and Jusaitis (2012) also demonstrated that media formulations other than ½MS appeared to induce a higher proportion of advanced protocorm development stages over the same incubation period although this effect was species specific. The Pa5 formulation was also found to be successful for Australian terrestrial species such as Pterostylis arenicola (Jusaitis and Sorensen, 1993), Diuris longifolia, and Elythranthera brunonis (Collins and Dixon, 1992; Oddie et al., 1994) and Microtis arenaria (Dowling and Jusaitis, 2012). The study species simultaneously underwent culture on three asymbiotic media (OMA, ½MS and Pa5) and a symbiotic medium (OMA+Fungus) under light and dark conditions at 20 ⁰C. Both the germination rate and the protocorm development stage were affected by the different asymbiotic and symbiotic culture media as well as illumination.

Although we expected to observe higher germination on Pa5 medium (as previously reported for Australian orchids) ½MS medium supported the most advanced plantlet development, but only with P. sanguinea. Germinated seeds in ½MS and Pa5 medium after 8 weeks were observed for M. media and P. sanguinea, yet both media recorded zero germination for C. latifolia, while C. huegelii seeds also did not germinate on ½MS. Stewart and Kane (2010) and Johnson et al., (2011) revealed that the presence of carbohydrates in the medium is a key factor in protocorm development of Habenaria macroceratitis (Florida terrestrial orchid) and Bletia purpurea (an Australian terrestrial orchid) respectively. In this study, although ½MS and Pa5 were supplied with 20 g/L sucrose, C. huegelii and C. latifolia failed to germinate on one or both (Table 1). The reason for this result could be due to: a) inability of these two species to assimilate carbohydrate and nitrogen from the medium or, b) C. huegelii and C. latifolia preferentially utilise other sources of carbohydrate (or nitrogen) than that provided by ½MS or Pa5 medium. Dowling and Jusaitis, (2012) reported reduced protocorm development of Prasophyllum pruinosum and Thelymitra pauciflora on media containing only organic sources of nitrogen whereas, Pterostylis nutans and Thelymitra pauciflora showed the most advanced protocorm development on the same media. Stewart and Kane, (2006) and Johnson and Kane, (2007) also stimulated significant growth of protocorms by replacing inorganic nitrogen with amino acids supplements for germination in Habenaria macroceratitis (a terrestrial orchid species from Florida, USA) and some hybrid orchids respectively. Australian orchid fungal isolates of Caladenia flava, Diuris corymbosa and Drakaea recurva were able to utilise inorganic nitrogen in the form of ammonium, whereas Pterostylis recurva utilised both ammonium and nitrate (Nurfadilah et al., 2013). These fungal isolates also greatly utilized nitrogen in the form of aspartic acid and glutamic acid (Nurfadilah et al., 2013). In soil, ammonium is derived from organic matter through mineralization and is subsequently nitrified via nitrite to nitrate (Connell et al., 1995). The type of assimilated nitrogen varies among species and is highly dependent on the available forms in the habitat soil (Lambers et al., 2008). In this study, the Pa5 and ½MS media nitrogen source appeared to be ineffective on C. huegelii and C. latifolia seeds which suggest that the seeds of these species may be unable to allocate enough carbon to utilise inorganic nitrogen at least under the in vitro conditions in this study. This is supported by the increased germination observed when C. huegelii and C. latifolia seeds were inoculated with fungus, suggesting a nutritional benefit was derived from the symbiotic fungal partner (Table 1). A possible explanation is that inadequate carbon allocation and its related effects on inorganic nitrogen absorption when seeds were placed in asymbiotic culture, can be compensated by the presence of fungus in the symbiotic culture conditions. The carbon cost

32 of nitrogen acquisition is quite high and follows nitrate> ammonium> amino acid, with nitrate being the most carbon-costly to acquire from the substrate and amino acids the least

(Clarkson, 1985). As a final point, the inclusion of activated charcoal (AC) in the Pa5 and ½ MS asymbiotic media as used in this study may have altered the availability of a range of compounds including plant growth regulators present in CW (Chugh et al., 2009) through its adsorption characteristics, as well as possibly changing the degree of hydrolysis of sucrose during autoclaving (Hossain et al., 2010; Pan and Van Staden, 1999). Activated charcoal may have beneficial (or potentially harmful) effects depending on the amount used, composition of basal medium and variation in species response (Pan and Van Staden, 1999). In future studies it may be prudent to test the effects of AC and CW separately in basal media before combining them, especially with asymbiotic germination, as nutrient/hormonal requirements may be highly specific at critical stages of germination (in the absence of a fungal chaperone) for some species.

Conclusion

The study indicates significant but generally species specific effects of temperature, light and dark incubation on in vitro germination and development stages in both symbiotic and asymbiotic culture with Caladenia huegelii, C. latifolia, Microtus media and Pterosylis sanguinea. Interactive effects between temperature and light on germination were observed in all species and growth of fungal symbionts was significantly temperature sensitive in three species (C. huegelii, C. latifolia and M. media) but not P. sanguinea. The results of this study also demonstrate that the obligation of C. huegelii and C. latifolia seeds to their fungal associates in order to utilize substrate (germination media) nutrients may be more acute than for M. media and P. sanguinea. Mycosymbiont ability to utilize a variety of nutrients is considered a significant ecological factor in orchid biodiversity that is thought to minimize species-species competition. However if fungal symbiont growth becomes limited by any environmental factors (e.g. temperature) this could mean that successful germination becomes more challenging for some species. For future research, it may be beneficial to quantify the impact of temperature on nutrient translocation from mycorrhizal fungi to orchid seed and its effect on germination of Australian terrestrial orchid species.

Chapter Three

Photosynthesis in in vitro cultured terrestrial orchids is regulated by symbiotic fungus rather than light and nitrogen supply

Abstract

We investigated the photosynthetic function of Caladenia huegelii, Caladenia latifolia, Microtis media and Pterostylis sanguinea in the presence and absence of fungus in response to LED and fluorescent light as well as absence and presence of nitrogen. Photosynthesis (A) of P. sanguinea and M. media was negative in the presence of fungus, however A significantly increased with the absence of fungus. Conversely with A, chlorophyll content of P. sanguinea and M. media grown in the oat meal agar (OMA) inoculated with fungus was significantly higher (6.5 and 4 times) compared with the OMA without fungus. A was negative in all symbiotically propagated species exposed to both fluorescent and LED light, however LED showed a significantly more negative rate of A. Among the study species, total chlorophyll content of P. sanguinea and C. latifolia did not responds to either fluorescent or LED light or presence/absence of N. In contrast, total chlorophyll contents of C. huegelii (4105 µgg-1 FW with zero N added to the medium) and M. media (3670 µgg-1 FW with 360 µM N added to medium) exhibited significantly higher concentrations under LED light. A considerably less negative A was observed for C. huegelii exposed to 5 weeks continues fluorescent light (35 µmolm-2s-1) followed by 4 weeks continues LED light (65 µmolm-2s-1) in comparison with 9 weeks continuous fluorescent light only. A was also higher where M. media seedlings were grown under fluorescent and LED light compared with fluorescent light alone, however this was not significant. Our results suggest that photosynthesis at the early stages of orchid growth may be more affected by the presence and absence of fungus compared with light type, light quantity and N concentration.

Introduction

The family Orchidaceae, with an estimated 20 to 35,000 species (Cribb et al., 2003), has a high potential for extinction which has prompted conservation research into this, the largest plant family (Koopowitz, 2001; Dixon et al., 2003; Swart and Dixon, 2009b). Effective conservation of critically endangered orchid species (with few plants remaining in the wild) may ultimately depends on the ability to produce robust seedlings capable of reintroduction to field sites (Ramsay, 1998 and Batty et al., 2001b).

Terrestrial orchids, particularly rare taxa, are often hard to propagate under laboratory conditions and establish in soil in sufficient quantities to achieve a sustainable conservation outcome (Zelmer and Currah, 1997; Stewart and zettler, 2002). Orchid in vitro propagation is limited by initial mortality among germinating seeds from the Petri dish and is subject to further mortality on transfer of seedlings to sand over agar substrate (Batty et al., 2006). Mortality at these stages might be due to the stresses imposed during the trophic transition of orchid seedlings. Orchid trophic transition is variously classified as mycoheterotrophic (completely fungus dependent) (Leake, 1994), mixotrophic (Heifetz et al., 2000; Selosse et al., 2004) and fully autotrophic. The degree of orchid specificity to mycorrhizal fungus is correlated with the degree of heterotrophy which implies that fully mycoheterotrophic orchids are more specific in their requirement than photoautotrophic orchids (Girland et al., 2006). Low specificity of an orchid to its mycorrhizal fungi may allow increased C acquisition, possibly through either surrogate fungal symbionts or photosynthesis (Francis and Read, 1984; Simard et al., 1997). The correlation between fungal obligation rate and reduced photosynthetic efficiency can represent an excellent system with which to study the evolution of orchid mycoheterotrophy and photoautotrophy (Bidartondo et al., 2004; Selosse et al., 2004), and may also infer the propensity to either absorb or fix C by different orchid species. The absence of photosynthesis during the early phases of the orchid life cycle invoke an interesting nutrient acquisition strategy whereby mycoheterotrophy dominates initially, then may be replaced by autotrophy as the plant matures (Leake, 1994). However, this strategy may result in a decrease in survival rate of in vitro propagated seedlings by excluding or delaying mixotrophy and autotrophy at crucial stages e.g. the transition from in vitro to ex vitro conditions.

Detailed examination of the heterotrophic phase in orchid growth reveals the extent to which photosynthetic capability has been curtailed as a result of increasing dependence on mycorrhizal symbiosis during orchid evolution (dePamphilis, 1995). The process of decline

35 in photosynthesis and chlorophyll content during the life of an orchid due to contact with a fungus is believed to have arisen at least 20 times during the evolution of orchids (Cameron et al., 1999; Molvary et al., 2000; Bateman et al., 2005). Light is one of the most critical environmental factors, affecting plant growth and development not only as the sole source of energy, but also acting as an external signal (Terfa et al., 2013). Spectral variation in illumination can affect photosynthetic capacity and trigger morphogenetic responses, which in turn can vary among different plant species. The red and blue wavelengths of the spectrum are particularly important in plant growth. Blue light drives formation of chlorophyll, stomatal opening, leaf photosynthetic function, phototropism and photomorphogenesis (Senger, 1982, Schuerger et al., 1997; Dougher and Bugbee, 1998; Heo et al., 2002, Whitelam and Halliday, 2007), while red light is important for the development of the photosynthetic apparatus and many other photomorphogenic processes (Saebo et al., 1995). Consequently, in plant production systems light-emitting diodes (LED) that emit wavelength in red (600-700 nm) and blue (400-500 nm) while minimizing or removing wavelengths in other spectral regions (Voskresenskaya et al., 1977) have been developed to increase plant performance. Matsuda et al., (2004, 2008) observed an increased photosynthetic capacity of rice plant and spinach seedling when grown under a combination of blue and red LED compared to filtered blue light. Blue light with combination of red light was also found to increase photosynthesis in Atriplex triangularis compared to plants grown under red light alone (Leong and Anderson, 1984). However, red LED light significantly increased the number of leaves and stem length of lettuce plants compared with plants grown under blue LEDs (Yanagi et al. ., 1996)

The function of the photosynthetic apparatus and its components such as light harvesting centre, electron transport components and enzymes strongly relies on leaf nitrogen concentration (Lambers et al., 2008). Nitrogen deficiency suppresses the activity of photosynthetic components such as Rubisco, chlorophyll, and stomatal conductance activity as a result of diffusion and biochemical limitations (Rundel and Sharifi, 1994). Because diffusion and biochemical components of photosynthesis are limited under light stress, it is important to study the interaction between light and nitrogen (Lambers et al., 2008). Nitrogen might indirectly suppress photosynthetic function of symbiotically propagated orchids through increased density of mycorrhizal fungus which consequently may facilitate C acquisition from media rather than fixing through photosynthesis. Nurfadilah et al., (2013) reported an increased biomass of orchid mycorrhizal fungus grown under different sources of N and C.

Rationale and objectives

Understanding how altering light quantity, light type and nitrogen concentration can affect plant photosynthetic responses and chlorophyll content is crucial to elucidate whether orchids can increase photosynthetic performance and therefore increase their resilience to transplanting from in vitro to glasshouse and field environments. The goal of this study is to manipulate the in vitro orchid growth environment to enable examination of the photosynthetic efficiency of symbiotically and asymbiotically propagated seedlings to determine the presence and extent of photosynthetic plasticity during the critical growth phase of deflasking.

Specifically, the aim was to determine the effect of light quantity (photosynthetic photon flux density, PPFD), light type (fluorescent or light-emitting diode i.e. LED) and irradiance period variation on shoot fresh weight, photosynthetic activity (via CO2 uptake) and chlorophyll content of the study species. To further explore possible substrate nutrient interactive factors, the absence and presence of nitrogen on photosynthetic function of the study species was also investigated. We also hypothesised that photosynthetic activity of the in vitro propagated study species may differ under fluorescent light compared to a combination of fluorescent and LED light. A separate experiment examined the effect of short duration high illumination (both fluorescent and LED) on photosynthetic activity in order to ascertain the basic integrity of the photosynthetic apparatus.

Materials and Methods

Study species and plant material

37 southern wheat belt also occurs on low rises near salt lakes. Microtis media is similarly widespread in Western Australia, extending from the south-west inland to Cue and Kalgoorlie and eastwards along the south coast to the Great Australian Bight where it is found in several different habitats, including swamps, shallow costal heaths, woodlands and forests, and they have a weedy habit and can often be found growing in irrigated garden beds (Hoffman and Brown, 1998). Pterostylis sanguinea is widespread in drier inland areas of south-west WA between Mullewa and Ravensthorpe (Brown et al., 2008). Leaf type among the study species was variable, with Pterostylis sanguinea producing a rosette of short wide leaves, while C. latifolia, M. media and C. huegelii all produce single narrow leaves.

Experimental growth conditions

Seeds of C. huegelii, C. latifolia, P. sanguinea and M. media were previously symbiotically (oat meal agar + fungus) and asymbiotically (½MS) Murashige and Skoog (1962) sown and incubated for 10 weeks, with C. huegelii, C. latifolia and M. media incubated under 12 h light/12 dark at 15⁰C, while P. sanguinea was incubated under constant darkness according to optimal conditions as outlined in Chapter 2. After ten weeks, symbiotically and asymbiotically propagated protocorms at the stage of seedling elongation (stage 5) (Ramsay et al., 1986) were transferred to 70 ml (screw cap) heat-resistance flat bottom containers (Techno Plas, Rowe scientific, Western Australia). Ventilation of each container occurred by placing a 5mm diam. hole in the lid covered with an adhesive Millipore disc (0.5 µm, FWMS 01800, Nihon Millipore Ltd, Yonezawa, Japan) followed by autoclaving for 20 min at 121⁰C. 30 ml oat meal agar (OMA) with 0.25% w/v crushed oats was added to a 70 ml container and then separately autoclaved for 20 min at 121⁰C. Once the OMA had set in the container, a 10 mm layer of double autoclaved (40 min at 121⁰C) white acid-washed silica sand was added under sterile conditions and left for 24hr on OMA to absorb moisture before seedlings were transferred.

Effect of light on photosynthesis

To evaluate the effect of light quantity on photosynthetic capacity, symbiotically germinated seedlings of P. sanguinea were exposed for 5 weeks to LED light for 12 h day-1 at different PPFD levels of 65, 110 and 160 µmol m-2 s-1. LED lighting was supplied by an LED-light set with five clusters (Fig. 1f), from Grow Master Global co, Orlando, USA) containing 80%

38 red (R; peak wavelength at 660 nm and 20% blue (B; peak wavelength at 465nm). To evaluate the effect of light type, symbiotically propagated seedlings of C. huegelii, C. latifolia, P. sanguinea and M. media were exposed to 65 µmolm-2s-1 and 34 µmolm-2s-1 for 12 h day-1 by LED lamps and fluorescent lamps respectively. Fluorescent lighting was supplied by two fluorescent lamps (Sylvania SLT5-21W) per shelf, with output of approximately 34 µmolm-2s-1 measured at plant level using Li-cor light meter (model Li-250, USA). Propagated seedlings of C. huegelii and M. media were grown 9 weeks under fluorescent light (34 µmolm-2s-1) and compared with seedlings grown firstly for 5 weeks under fluorescent light (34 µmolm-2s-1) following by 4 weeks under LED light (65 µmol m-2 s-1). To test the effect of an instant jump in light intensity on photosynthesis, asymbiotically propagated seedlings of M. media and P. sanguinea were grown for 5 weeks under fluorescent light (34 µmolm-2s-1) then abruptly exposed to 80 µmolm-2s-1 of fluorescent light, or 65 and 400 µmol m-2 s-1 of LED light for 5 minutes.

Effect of nitrogen on photosynthesis and chlorophyll content

The effect of absence and presence of nitrogen on photosynthesis and chlorophyll content of the study species was evaluated using L-Glutamic acid (Sigma Aldrich) as the sole nitrogen source at 360 µmol L-1. As a precaution, in case of organic N instability at high temperatures during autoclaving, L-Glutamic acid was sterilized using a 0.22-µm Millipore syringe- driven filtration unit and added directly to OMA, and the pH adjusted to 5 (Nurfadilah Pers. Comm).

Plant harvest

After five weeks and nine weeks (depending on the treatment) seedlings were harvested for analysis: removed from sand and green shoots separated from protocorms. The green shoots were weighed fresh and the fresh weight was used to calibrate photosynthesis and chlorophyll content on a mass basis.

Gas-exchange measurements

Photosynthesis and stomatal conductivity were measured after five and nine weeks of light and nitrogen treatment using a portable gas exchange system (LI-6400, Li-Cor, Lincoln, NE, USA) and exposed ambient light conditions. In order to measure gas exchange of the tiny leaves of study orchid species we manipulated the Li-cor chamber in a way to capture the

39 lowest amount of exchanged CO2. To do this, two holes in the lid of the culture container (70 ml flat bottom container) were created and connected to tubing for gas exchange

(Fig.1d). During all measurements, CO2 concentration in the leaf chamber was 400 µmol mol-1, the air flow was 100 µmol s-1 and leaf chamber temperature 25⁰C.

For chlorophyll analysis, whole shoots of each species were cut fresh and weighed. Samples of leaves were placed in 2 ml tubes and immediately put in ice and kept in dark at 4⁰C overnight, then methanol added at 1.5 ml to each tube with glass beads then placed on an orbital shaker for 20 seconds to break up the samples. Each sample was then centrifuged at 13000 g for 2 min and 1 ml of the supernatant transferred to a 96 well plate. Absorbance was measured with Multiskan Spectrum v1.2 (Thermo Electron Corporation) and chlorophyll concentration was calculated according to the method of Wellburn (1994): where A653 and A666 are absorbance at 653and 666 nm respectively and chlorophyll content is based on µg ml-1 in methanol.

Chla = 15.65 A666- 7.34 A653 Chlb = 27.05A653- 11.2 A666

Statistical analysis

The experiment was implemented in the culture room and each experiment consists of 5-10 individual plants. The effect of presence and absence of fungus, light quantity, light type and nitrogen on photosynthesis and chlorophyll content were assessed by one-way ANOVA. The interaction effect between light, fungus and nitrogen on photosynthesis and chlorophyll content was evaluated by two-way ANOVA. All statistical analysis was carried out using SAS v 9.3 (SAS Institute 2002). Fisher’s LSD was used to compare mean at P=0.05. * indicates significant values.

a b c d

e f g h

Figure 1. Experimental growth conditions. Protocorms of C. latifolia (a) and P. sanguinea (b) at the stage of seedling elongation (stage 5) ready for transferring from Petri plate to container (C). Manipulated Li-cor chamber (d) and seedlings growing under fluorescent (35 µmol m-2 s-1) and LED (65 µmol m-2 s-1) light (e and f). Caladenia latifolia (g) and P. sanguinea (h) before harvest, grown 5 weeks under fluorescent and LED light respectively.

Results

Plant growth

Leaf fresh weight of P. sanguinea and M. media was not affected by the presence or absence of fungus when grown for 5 weeks under 35 µmolm-2s-1 of fluorescent light (Fig. 2a). Different irradiances of LED light also did not influence leaf fresh weight of symbiotically propagated P. sanguinea (Fig. 3a). Leaf fresh weight of C. latifolia and M. media was significantly (P<0.05) higher when grown symbiotically 5 weeks under 35 µmolm-2s-1 fluorescent light compared with 65 µmolm-2s-1 of LED light (Fig. 4a). The effect of irradiance period variation on leaf fresh weight of symbiotically propagated M. media and C. huegelii was also tested. Microtis media leaf fresh weight was significantly (P<0.05) higher (1.5 fold) when grown for 9 weeks under 35 µmolm-2s-1 of fluorescent light compared to 5 weeks fluorescent light (35 µmolm-2s-1) followed by 4 weeks LED light (65 µmolm-2s-1) (Fig. 5a).

Effect of absence and presence of fungus on CO2 assimilation and chlorophyll content

Photosynthesis of P. sanguinea and M. media was negative in the presence of fungus, however photosynthesis significantly increased with the absence of fungus (Fig. 2b). However, chlorophyll content of P. sanguinea and M. media grown in OMA inoculated with

41 fungus was significantly (P<0.05) higher (6.5 and 4 times respectively) compared with the OMA without fungus (Fig. 2c).

0.07 (a) a 0.06

0.05 ab

0.04 ab

0.03 b

0.02 Leaf fresh mass (g) mass fresh Leaf

0.01

0 1 2 3 4

(b) ) 1 - s 1 -

1 µg.Chl

0 a a

b -1

Photosynthesis (µ mol CO mol (µ Photosynthesis c

-2 (c) 2500 a

2000 FW) 1 - a 1500

1000 b 500 b Chlorophyll content (µ g g (µ content Chlorophyll 0 P.sanguinea M. media P. sanguinea M. media OMA+Fungus OMA Figure 2. Leaf fresh mass, photosynthesis and chlorophyll content of M. media and P.sanguinea in response to presence and absence of fungus under 35 µmolm-2s-1 irradiance of fluorescent light (a, b and c). Values are means ± SE (n = 5), different letters indicate significant differences between treatments at α=0.05 level based on LSD mean separation.

Effect of light quantity and light type on photosynthesis and chlorophyll content

In order to determine whether the photosynthetic capacity of C. huegelii, C. latifolia, P. sanguinea and M. media was affected by testing light quantity and light type, the study species were exposed to different PPFD treatments. Symbiotically propagated P. sanguinea were 5 weeks exposed to 65, 110 and 160 µmolm-2s-1 of LED light and there was no significant effect of different light quantity on A and chlorophyll content (Fig 3 b and c).

0.018 (a) a a 0.016 0.014

0.012 a 0.01 0.008 0.006 0.004 Leaf fresh mass (g) mass fresh Leaf 0.002 0 1 2 3

(b) 0

) 1 - s 1 - -1

µg Chl µg -2

-3 a -4 a -5 a -6 Photosynthesis (µmol CO (µmol Photosynthesis

2000 (c) a a FW) 1 - 1500 a

1000

500 Chlorophyll content ( µ g g g ( µ content Chlorophyll 0 LED 65 LED 110 LED 160 Irradiance ( µmol m-2 s-1)

Figure 3. Leaf fresh mass, photosynthesis and chlorophyll content of symbiotically propagated P. sanguinea in response to 65,110 and 160 µmolm-2s-1 irradiance of LED light (a,b and c). Values are means ± SE (n = 5), different letters indicate significant differences between treatments at α=0.05 level based on LSD mean separation.

Symbiotically propagated study species for 5 weeks were exposed to 35 and 65 µmolm-2s-1 of fluorescent and LED light respectively. A was negative in all species exposed to both fluorescent and LED light however, LED showed a significantly higher negative A (Fig. 4b). Chlorophyll content of M. media grown under LED light was nearly 2-fold more than fluorescent light however this variation was not significant (P<0.05) for other species (Fig. 4c).

(a) 0.06 a

0.05

a 0.04 ab 0.03 bc bc

0.02 bc Leaf fresh mass (g) mass fresh Leaf

c 0.01 c

0 1 2 3 4

5 (b)

) 1

- 4 s 1 - . 3 2 µg Chl µg

2 1 0 -1 a -2 ab ab -3 bc -4 cd d de -5 e Photosynthesis (µmol CO (µmol Photosynthesis -6

4000 a a

3500 FW) 1 - 3000 ab 2500 b 2000 b b b b 1500

1000

500 Chlorophyll content (µg g (µg content Chlorophyll

0 C. latifolia M. media C. huegelii P. sanguinea

Figure 4. The effect of light type on leaf fresh mass, photosynthesis and chlorophyll content of symbiotically propagated study species (a, b and c). Values are means ± SE (n = 5-10), different letters indicate significant differences between treatments at α=0.05 level based on LSD mean separation.

The irradiance period variation was also tested to evaluate the effects of continuous fluorescent light and combined exposure of fluorescent and LED light on A and chlorophyll content of symbiotically propagated M. media and C. huegelii. A considerably less negative A (four times) was observed for C. huegelii exposed to 5 weeks fluorescent light (35 µmolm- 2s-1) followed by 4 weeks LED light (65 µmol m-2 s-1) in comparison with 9 weeks continuous fluorescent light (Fig. 5b ). There was not a significant (P<0.05) difference in A between M. media and C. huegelii exposed to irradiance period variation (Fig. 5b). Additionally, there was not a significant (P<0.05) difference in chlorophyll content of C. huegelii exposed to irradiance period variation however, M. media grown under combined exposure of fluorescent and LED light had 3.5-fold more chlorophyll content compared to fluorescent light only (Fig. 5c).

(a) 0.18 a

0.16

0.14

0.12 b

0.1

0.08

Leaf fresh mass (g) mass fresh Leaf 0.06 c c 0.04

0.02

0 1 2

0.15 (b)

) a 1 0.1 - ab s 1 - 0.05 0 µg Chl µg

2 -0.05 -0.1 -0.15 b -0.2 -0.25 -0.3

Photosynthesis (µ mol CO mol (µ Photosynthesis -0.35 -0.4 c

800 (c) Fluorescent a Fluorescent+LED a 700 a FW) 1 - 600

500

400

300

200 b

Chlorophyll content (µg g (µg content Chlorophyll 100

0 1 2 M.media C. huegelii Figure 5. The effect of irradiance period variation on leaf fresh mass, photosynthesis and chlorophyll content of symbiotically propagated M. media and C. huegelii (a, b and c). Values are means ± SE (n = 5-10), different letters indicate significant differences between treatments at α=0.05 level based on LSD mean separation.

We also tested the effect of instant exposure of different light quantity and light type on A of asymbiotically propagated M. media and P. sanguinea grown 5 weeks under fluorescent light (35 µmol m-2 s-1). A, as measured for P. sanguinea exhibited a negative rate at 35 µmol m-2 s-1 of fluorescent light and this significantly increased by exposing to 80 µmol m-2 s-1 of fluorescent light. No significant difference was observed by exposing both M. media and P. sanguinea seedlings to 65 and 400 µmol m-2 s-1 of LED light (Fig. 6c). There was no interaction effect between species and different irradiance (Table 1).

(a) 600 (b) 0.05 a

a 0.045 500

FW) a 1

0.04 -

0.035 400 0.03 300 0.025 b 0.02 200 0.015 Leaf fresh mass (g) mass fresh Leaf 0.01 100 Chlorophyll content (µg g (µg content Chlorophyll 0.005 0 0 M. media1 P.2 sanguinea M. media1 P. 2sanguinea

0.14 (c) M. media a P. sanguinea 0.12 ab

) 1 -

s 0.1 1 - a 0.08 µg Chl µg

2 ab ab 0.06 ab ab 0.04

0.02

0 35 Fluorescence 80 Fluorescence 65 LED 400 LED -0.02 Photosynthesis (µ mol CO (µ mol Photosynthesis Irradiance (µmol m-2 s-1) -0.04 b -0.06

Figure 6. Leaf fresh mass (a) and chlorophyll content (b) of asymbiotically propagated M. media and P. sanguinea grown 5 weeks under fluorescent light (35 µmolm-2s-1). Photosynthesis of asymbiotically propagated M. media and P. sanguinea grown 5 weeks under fluorescent light (35 µmolm-2s-1) followed by instant exposure to different light quantity and light type (c). Values are means ± SE (n = 10), different letters indicate significant differences between treatments at α=0.05 level based on LSD mean separation.

Effect of nitrogen on photosynthesis and chlorophyll content

Except symbiotically propagated P. sanguinea that exhibited significantly (P<0.05) less negative photosynthesis in the absence of N, A of symbiotically propagated M. media as well as asymbiotically propagated P. sanguinea and M. media were not significantly affected by different nitrogen concentrations (Fig. 7a). Also there was not a pronounced effect of nitrogen concentration on chlorophyll content of the study species grown in

50 presence and absence of fungus (Fig. 7b). There was no interaction effect observed between species, N and irradiance (Table 2).

0.5 (a)

) 1 - a a a s 1 - 0 a µg Chl µg 2 -0.5 b -1 b

c -1.5 Photosynthesis ( µmol CO ( µmol Photosynthesis d -2

3500 (b) a 3000 a 0 N (µM) 2500 360 N (µM) a a 2000

1500

1000

Chlorophyll content (µg/FW) content Chlorophyll b b b 500 b

0 P. sanguinea M. media P. sanguinea M. media OMA+Fungus OMA

Figure 7. Effect of nitrogen on photosynthesis (a) and chlorophyll content (b) in presence and absence of fungus. Values are means ± SE (n = 5-10), different letters indicate significant differences between treatments at α=0.05 level based on LSD mean separation.

The effect of light quantity and type and its interaction with (360 µM) or without (zero) N were also tested by exposing asymbiotically propagated M. media and P. sanguinea to fluorescent and LED lights of differing intensities. A of M. media was not affected by addition of nitrogen (Fig. 8 and Table 2). The highest significant values of A observed with P. sanguinea seedlings occurred in the treatment combining both 360 µM N and PPFD of 80 µmolm-2s-1 (supplied by fluorescent light). Pterostylis sanguinea seedlings grown in the

51 absence of N showed a significant suppression of A at all irradiance levels, compared with seedlings supplied with 360 µM N (Fig 8 and Table 2).

0.3 M. media

) 1 - s 1 - 0.25 µg Chl µg

2 0.2

0.15

0.1 a a

a a a a 0.05 Photosynthesis (µmol CO (µmol Photosynthesis

0 0 360

0.3 35 Fluorescent (µ mol m-2 s-1) P. sanguinea 80 Fluorescent (µ mol m-2 s-1) a

0.25 ) 1 - 400 LED (µ mol m-2 s-1) s 1 - 0.2

0.15 b µg Chl µg bc 2 0.1

0.05

0 0 360 -0.05 cde -0.1

Photosynthesis (µ mol CO mol (µ Photosynthesis de -0.15 e Nitrogen (µ M) -0.2

Figure 8. Effect of nitrogen on photosynthesis of asymbiotically propagated M. media and P. sanguinea exposed 5 weeks to 35 µmol m-2 s-1 of fluorescent light followed by 5 minutes instant exposure to different light quantity and light type. Values are means ± SE (n = 6), different letters indicate significant differences between treatments at α=0.05 level based on LSD mean separation.

Table 1. A two-way ANOVA is used to test for the effect of light and nitrogen on photosynthesis of study species. * and ** means significant and highly significant at 5 and 1% respectively.

Photosynthesis Species Effect d.f. F P

species 1 0.63* 0.4302 M. media & Light 3 2.72* 0.0462 P. sanguinea Species* Light 3 2.11* 0.1012

Light 2 1.12* 0.33

Nitrogen 2 0.24 0.78 M. media Light*Nitrogen 4 0.54 0.71

Light 2 3.31* 0.044

Nitrogen 2 17.54** <.0001 P. sanguinea Light*Nitrogen 4 1.13* 0.35

Table 2. A two-way ANOVA is used to test for the effect of species, light and nitrogen and their interactions on photosynthesis and chlorophyll content. * and ** means significant and highly significant at 5 and 1% respectively.

Photosynthesis Chlorophyll Species Effect d.f. content F P P F

C. latifolia Species 3 2.24* 0.0919 3.67* 0.015 M. media Nitrogen 1 2.98* 0.0893 1.75* 0.191 C. huegelii Species*Nitrogen 3 0.80 0.4971 0.97* 0.414 P. sanguinea Light 1 70.3** <.0001 4.25* 0.043 Species*Light 3 3.87* 0.0131 3.43* 0.022 Nitrogen*Light 1 0.38* 0.5379 0.36 0.549 Species*Nitrogen*Light 3 0.49 0.6920 2.12* 0.106

Discussion

Effects of mycosymbiont presence on leaf fresh weight, photosynthesis and chlorophyll

Content

In this study the presence of the symbiont fungi resulted in a significant suppression of photosynthesis (P<0.05) in P. sanguinea and M. media, and no difference in leaf fresh weight. The specific reason(s) for photosynthesis suppression in the presence of fungus are unknown and requires additional investigation, but could be attributed to a variety of possible causes. In orchids, the energy cost for carbon acquisition through symbiotic fungus is putatively lower compared when orchids fix C through photosynthesis (Cameron et al., 2006). Emerging leaves of orchids are considered to have potential for photosynthesis (Smith 1966; McKendrick et al., 2000), however in the present study, it was observed that with in vitro symbiotically grown seedlings with green leaves, evidence for photosynthetic activity (as indicated by positive photosynthesis values) was lacking.

Deleterious effects of parasitic species on their host plants photosynthetic capabilities have been previously reported by Watling and Press (2001). Significant decreases in photosynthesis have been observed and this has been attributed to the direct effect of resource abstraction and indirect effect of non-source-sink interaction (Watling and Press, 2001; Cameron et al., 2005). The mechanism by which the orchid mycorrhizal association suppresses photosynthesis at the early stages of growth is not fully understood because this is a mechanism that has evolved specifically with orchids and may be difficult to manipulate effectively under ex situ conditions. In contrast to orchid mycorrhizal associations, Daughtridge et al., (1986), Wright et al., (1998) and Birhane et al., (2010) reported arbuscular mycorrhizal fungi (provided water is not limiting) significantly increased photosynthesis and growth of Boswellia seedlings. This variation between orchid mycorrhizal associations and arbuscular mycorrhizal symbioses could be due to a high dependency of the orchid on its symbiotic association at the early stages of growth. It is also possible that absence of photosynthesis in the presence of the symbiont fungus might be due to a lack of sufficient chlorophyll production. However in this study in vitro grown symbiotic P. sanguinea and M. media seedlings showed significantly higher leaf chlorophyll contents compared to asymbiotic seedlings, but showed negative photosynthesis readings compared with asymbiotic seedlings (that mostly exhibited positive photosynthesis). This

54 intrinsic result demonstrates leaf chlorophyll content may not necessarily predict photosynthetic capacity (or activity) of micropropagated orchids at least during the early stages of growth. Cameron et al., (2008) observed a significant decrease in total chlorophyll content of Phleum bertolonii parasitized by Rhinanthus minor compared with the uninfected host. This decrease in chlorophyll content was reportedly due to the lack of vascular connectivity between host and parasite (Cameron et al., 2006; Cameron and Seel, 2007). In this study, significantly higher chlorophyll content of symbiotically propagated P. sanguinea and M. media compared to asymbiotically grown plants could be result of a high dependency of orchid seeds on their respective fungal symbioses and consequently higher vascular connectivity. In Corallorhiza gagnebin (Barrett and Freudenstein, 2008), and Corallorhiza trifida (Zimmer et al., 2008; Cameron et al., 2009) a high concentration of chlorophyll was noted as a potential identifying trait for autotrophy in these orchids. Weedy orchids species such as M. media (Ackerman, 2007) that do not appear to have any specific obligate fungal relationship (De long et al., 2013) can potentially switch from heterotrophy to autotrophy and fix C through photosynthesis. This might support the idea that orchids which are obligate to only one or a limited number of mycosymbionts may exhibit a lower potential to become autotrophic compared with those which are capable of utilizing a broader range of symbiotic partners and thus may display more weed-like attributes.

Effect of nitrogen

It has been reported that photosynthesis is strongly affected by nitrogen availability as the photosynthetic apparatus includes more than half of the leaf nitrogen content (Lambers et al., 2008). The nitrogen source (L-Glutamic acid) applied in this study was previously reported as a preferred nitrogen source for growth of the mycorrhizal symbiont of the genera Caladenia, Diuris, Drakaea and Pterostylis (Nurfadilah et al., 2013). In symbiotically propagated seedlings, therefore, the presence of a readily assimilated nitrogen source (e.g. L- Glutamic acid) should promote fungal growth and consequently suppress photosynthetic rate compared with absence of such an N source, and conversely the availability of nitrogen to asymbiotically propagated seedlings should increase the rate of photosynthesis. However, in this study it was found that absence and presence of N did not appear to have a straightforward influence on either photosynthesis or chlorophyll content of P. sanguinea and M. media. Other research has confirmed the greater effectiveness of nitrogen (as

Ca(NO3)2) on photosynthesis and chlorophyll content of barley leaves compared with urea and (NH4)2 SO4, while Ca(NO3)2 compared to urea and (NH4)2SO4 had a significant

55 influence on the suppression of oxidative stress caused by heavy metals which consequently creates higher photosynthetic rates and chlorophyll contents (Ali et al., 2013). A reduction effect of low N on photosynthesis and chlorophyll content was also reported in olive (Boussadia et al., 2010) and Arabidopsis (Martin et al., 2002). These examples indicate that while direct beneficial effects of N on photosynthesis and chlorophyll content can be readily demonstrated in a number of species, such effects could not be defined with the orchid species in this study.

Effect of light and nitrogen on leaf fresh weight, photosynthesis and chlorophyll content

Symbiotically propagated P. sanguinea seedlings were not able to photosynthesize under LED light treatments (65, 110 and 160 µmol m-2 s-1). We also found that species grown symbiotically under LED lighting showed higher negative photosynthesis than those grown under fluorescent light. The results suggest that in early-stage propagation of orchid seedlings it may be better to use fluorescent light (35 µmol m-2 s-1) rather than LED light (≥65 µmol m-2 s-1). Conversely, a significant positive effect of LED light on photosynthesis was previously reported for Phaseolus vulgaris (Barreiro et al., 1992), wheat (Fukuda et al., 2011) and rose plant (Terfa et al., 2013). In the current study photosynthesis was affected by light type when measured at different PPFD, however negative photosynthetic rates further support the idea that light treatment alone did not affect photosynthetic rate of the symbiotically propagated orchid seedlings at the early stages of growth. Total chlorophyll content of only one of the study species (M. media) significantly increased by exposure to 65 µmolm-2s-1 from LED’s, however there was no significant difference observed in chlorophyll content of other study species exposed to LED light compared with 35 µmol m-2 s-1 fluorescent light.

In the present study, no correlation between the total chlorophyll content, irradiance and addition of N supplement could be found, however the presence/absence of symbiont fungi had a pronounced effect on leaf chlorophyll content on the study species. Studies on non- orchid species demonstrated a significant increase of total chlorophyll in response to an increase of total leaf nitrogen, even under variable light conditions. Kitajima and Hogan, (2003) reported a nearly 2-fold increase in chlorophyll content of Tabebuia rosea leaves containing a total leaf nitrogen concentration of 45 mmol m-2, compared with leaves containing 28 mmol m-2 N, at both low (0.29 mol m-2 d-1) or high (6.77 mol m-2 d-1) light intensity. Higher total chlorophyll content was reported for rose plants grown under 100 µmolm-2s-1 LED light compared with 100 µmolm-2s-1 HPS (Terfa et al., 2013). However, in

56 this study, it was found that light and supplemental nitrogen (as L-glutamine) did not affect chlorophyll content of either symbiotically or asymbiotically propagated orchid seedlings. Although it was originally thought that chlorophyll content should increase under optimum light conditions, it was the presence of symbiont fungi that appeared to have the most influence in enhancing chlorophyll content of the study species, even when photosynthesis was suppressed.

There was no correlation between leaf fresh mass, photosynthesis and chlorophyll content of symbiotically propagated M. media and C. huegelii subject to irradiance variation. Although chlorophyll content of M. media seedlings grown 5 weeks on fluorescent light followed by 4 weeks LED light was significantly higher compared to those seedlings subject to irradiance by only fluorescent light for 9 weeks, no significant photosynthesis was observed. Chlorophyll content of C. huegelii grown under both irradiance types was close to that of M. media grown for 5 weeks under fluorescent lights followed by 4 weeks LED light. However, photosynthesis of C. huegelii and M. media was negative and positive respectively. Different responses of M. media and C. huegelii to the same irradiance are considered to be more likely due to their differing dependency on their respective fungal symbionts. Caladenia huegelii reportedly has a highly specific and range limited fungal partner (Swarts et al., 2010) that could make it relatively difficult to facilitate C through surrogate fungal symbionts or alternative strategies such as photosynthesis. Conversely, weedy orchids species such as M. media (Ackerman, 2007) that do not appear to have any specific obligate fungal relationship (De long et al., 2013) can potentially switch from heterotrophy to autotrophy more easily and fix carbon through photosynthesis. This might support the idea that those orchids which are obligate to only one or a limited number of mycosymbionts may possess a lower potential to become autotrophic compared with those which are capable of utilizing a broader range of symbiotic partners.

The reason for the slow response of M. media to the instant exposure of different light quantity might be related to its intrinsic weedy characteristics that have adapted to allow it to grow in what might be considered inappropriate environments. Instant exposure of low and high irradiance of fluorescent and LED light as well as different N concentrations did not appear to influence A of M. media. De long et al., (2013) reported M. media formed symbiotic associations with a broad taxonomic and geographic range of mycorrhizal fungi which demonstrate capability of M. media to germinate and grow in a wide variety of edaphic habitats. Conversely, in P. sanguinea, a negative A response to a PPFD of 35 µmolm-2s-1 from fluorescent light switched to a significantly positive A value under 80

µmolm-2s-1 from fluorescent lamps. A significantly higher A value for P. sanguinea under 80 µmol m-2 s-1 of fluorescent light and 360 µmol l-1 N, might be related to an interaction effect between light and nitrogen as A was significantly decreased by exposing P. sanguinea seedlings to 35 and 400 µmol m-2 s-1 fluorescent and LED light respectively.

Conclusions

We show here for the first time that presence of symbiont fungi have a pronounced effect on photosynthesis in the early stages of propagation of the study orchid species, compared to light and nitrogen. The results of this study also demonstrated that although fluorescent light (80 µmol m-2 s-1) was found to be the more effective light source for the growth of orchid seedlings compared to LED light, growth of the early in vitro propagated seedlings appears to be more dependent on the fungal association rather than light, whether the source is fluorescent or LED.

Chapter Four

General Discussion and Future Research

Optimizing Orchid Propagation

Ex situ conservation programs are valuable for endangered species due to ability for mass propagation via in vitro culture, which can ultimately lead to translocation of plants to the field (Cribb et al., 2003;). In a restoration context, the main aim of any orchid conservation and propagation is to produce a self-sustaining population in the field. Although the regeneration and multiplication of herbaceous terrestrial orchids has been difficult in the past, the development of robust and useful methodologies has been essential for the overall success of several reintroduction programs (Batty et al., 2006). Propagation through in vitro symbiotic seed germination is considered an appropriate method in orchid conservation (Arditti, 2008). Ex situ approaches are especially useful for orchid seeds that have a suppressed endosperm and requirement of fungal partner (Rubluo et al., 1993; Buyun et al., 2004; Lo et al., 2004; Santos-Herna´ndez et al., 2005). Terrestrial orchids contain 1,300 to 4,000,000 seeds per capsule (Darwin, 1888; Anonymous, 1890; Harley, 1951; Tournay, 1960; Arditti, 1961) but less than 1% of these may successfully germinate and pass the critical summer dormancy period once they have produced their first tuber following winter germination (Batty et al., 2001a).

In vitro propagation techniques can be readily utilized for conservation of threatened plant species, as it is possible to produce large numbers of plantlets relatively quickly compared to conventional propagation methods (Engelmann, 2011). Appropriate protocols for in vitro germination of orchid seed is species-specific and varies depending on several factors such as capsule (seed) maturity, culture media components, photoperiod, and temperature conditions during germination (Arditti, 1982; Kauth et al., 2011). In vitro propagation of orchids can produce an abundance of robust orchid seedlings, but it can be a slow process, because their seeds lack endosperm and require mycorrhizal-assisted germination. Therefore there is a need to develop commercial-scale micropropagation techniques to generate quality orchid plantlets suitable for planting in situ (Pence, 2011).

In temperate terrestrial orchid propagation programs, the establishment of actively growing seedlings or dormant tubers (produced in vitro) under in situ conditions was more successful

59 compared to when seeds were directly sown in situ (Batty et al., 2006). However, the variable responses among genera suggests that further trials should be undertaken to optimise both in vitro propagation and direct seeding methods when performing translocations on untested orchid taxa as both approaches are likely to convey various advantages under different circumstances (Batty et al., 2006). Seedlings produced using such different approaches should be planted into a range of different standard and improvised substrates to begin the acclimation process for deflasking and the re- establishment back into a soil based substrate under glasshouse and later field conditions.

If transfer of in vitro propagated seedlings directly to the glasshouse fails, the most likely reasons for this include unsuitable moisture and/or temperature conditions in the glasshouse compared with the in vitro environment, Transferral from the high humidity environment of the Petri dish to larger containers with increased permeability/venting has typically been used as in intermediate step to glasshouse or field transfer, and increases survival by allowing the seedling to acclimatise to soil conditions and a lower humidity environment. An optimal humidity in containers can be implemented as long as careful management and evaluation of the compromise between adequate ventilation and excessive water loss loading is undertaken (Hahn and Paek, 2001). Higher survival following transfer of seedlings to intermediate growth containers has resolved some of the earlier problems associated with the transfer of protocorms from Petri dishes directly to soil and glasshouse conditions.

Symbiotic propagation protocols used in orchid propagation programs are capable of rapid production of large numbers of healthy seedlings in a short period of time (Clements and Ellyard, 1979; Warcup, 1973). In contrast, asymbiotic protocols using more developed tissue culture methodologies such as organogenesis were found to be inefficient for the production of robust protocorms that are transferable to soil (Batty et al., 2006). Ramsay (1998) reported that in the absence of suitable fungal isolates for symbiotic propagation, Lady’s Slipper Orchid (Cypripedium calceolus) was successfully re-established from asymbiotically germinated seedlings. Although symbiotic seed germination typically creates a greater number of more robust protocorms either in the laboratory or in natural recruitment situations, refining of growth media in conjunction with asymbiotic propagation can also be successful (Ramsay, 1998). Successful reintroduction programs will still rely on optimising the production of robust seedlings whether from symbiotic or asymbiotic germination. Apart from optimising initial propagation of orchid protocorms, translocation survival as a fully autotrophic seedling also needs to be refined.

Constraints in orchid propagation

Conservation of endangered species has improved due to identification of establishment constraints. Recruitment in the field can fail due to: (1) seed dispersal to an unsuitable site (Murren and Ellison, 1998; Diez, 2007; Jersáková and Malinová , 2007); (2) development of an abnormal embryo as a result of genetic constitution (Wallace, 2003) insufficient nutrient supply (Lee et al., 2006); (3) low seed longevity (Baskin and Baskin, 2001; Batty et al., 2000; Whigham et al., 2006); (4) seedling competition with surrounding plants and desiccation (Fenner, 1985) and (5) in the case of terrestrial orchids, the absence of an appropriate fungal symbiont (Batty et al., 2000; McKendrick et al., 2000; Diez, 2007; Bidartondo and Read, 2008) and/or inappropriate soil nutrients, pH and moisture (Rasmussen, 1995; Batty et al., 2001a; Diez, 2007). Propagated orchid seedlings transferred to the field may fail to establish and this could be due to patchy distribution of mycorrhizal fungi in soil and the scarcity of suitable habitats in the landscape (Batty et al., 2002). Therefore, the successful reintroduction of native orchids into natural bushland areas will be limited by a complex interplay between the presences of compatible fungi, key habitat requirements and other factors that affect orchid survival (Pate and Hopper, 1993).

In the areas with poor soils, seedling desiccation and nutrient limitation can play a critical role in seedling recruitment (Grime and Curtis 1976; de Jong and Klinkhamer, 1988), however efficacy of the nominated factors varies depending on biotic and abiotic condition of the site. Silvertown and Tremlett (1989), and Edwards and Crawley (1999) reported that the success of seedling establishment also depends on the species-specific recruitment techniques and the interactions between species and their biotic factors. In natural habitats, seed dispersal patterns, recruitment and establishment can all influence fine scale gene flow and population genetic structure (Chung et al., 2004; Jacquemyn et al., 2009). Thus, understanding patterns of seed dispersal and recruitment is critical to understanding population dynamics such as population growth rates (Silvertown et al., 1993; Clark et al., 1999; Munzbergova and Herben, 2005; Uriarte et al., 2005). Many orchids in the south-west Western Australian urban bushland are significantly declining in numbers due to unscrupulous collection for commercial uses (Swarts and Dixon, 2009b), land clearing (ANPC 1997). Altered fires regimes (Batty et al., 2002), degradation of bush fragments due to weed invasion or illegal rubbish dumping and recruitment failure due to the loss of

61 pollinators (Dixon et al., 2003) are also affecting the orchids in the south West of Western Australia.

The translocation of plants into natural or restored bushland areas has become an increasingly favoured strategy for the reintroduction of species that are locally extinct (Scade et al., 2006). Unfortunately, translocations are typically not cost-effective procedures due to the high inputs required and the high losses presently experienced, largely because the ecophysiological aspects of in situ recruitment are presently poorly understood (ANPC, 1997). Once our understanding of the drivers of seedling establishment are better understood, the management and translocation practices can be utilised to ensure more successful and sustainable translocations and bolstering of wild populations (Scade et al., 2006). The Botanic Gardens and Parks Authority (BGPA) and the University of Western Australia (UWA) are involved in a number of world-class terrestrial orchid research and conservation programs. Their integrated research has resulted in advances in both in situ and ex situ conservation (Dixon et al., 2003, Batty et al., 2006, Hollick et al., 2001). Some of the methods that have been employed in the conservation of rare orchids include cryo-storage (Thoma, 1982; Hay et al., 2010) of critical seed and fungal material, identification of genetically important individuals (Caladenia huegelii) using DNA fingerprinting (Anderson, 1989) and improved population techniques. In vitro conservation programs can facilitate propagation and maintenance of healthy adult plants in relatively short periods of time (Arditti and Krikorian, 1996) with the aim to preserve threatened species outside their natural habitats through the long-term storage of seed or tissue cultured plant material (Cochrane et al., 2007).

Key factors for the success of propagated orchids in restoration

Reintroduction of self-sustaining orchid populations under in situ conditions has been attempted on several occasions, with varying degrees of success (Haeggstrom, 1992; Stewart, 1993; Warren and Miller, 1994). In a study by Swarts (2007), failure of Caladenia huegelii to tuberize at the end of the growing season, or failure of the tuber to survive and grow following summer dormancy resulted in high seedling mortality. Environmental factors including nutrient availability, water content, and gas exchange play a significant role in regulation survival and growth of the orchid seedling and also influence tuber development (O’ Brian et al., 1998). Swarts (2007) reported that pots containing 1/3 sand and 2/3 soil mix (sand layer under the OMA/sand topsoil) reduced tuber development and seedling re-emergence following summer dormancy compared to the control (only soil mix).

By highlighting the role of the tuber in facilitating seedling establishment, it can be concluded that many different factors determine the success or failure of seedling establishment under glasshouse and field conditions. Prevailing climatic conditions, such as high soil temperatures (≥30◦C) and 4–5 months without rainfall (therefore low soil moisture) play a key role in regulating the physiology of dormant tubers (Tieu et al., 2001). In Western Australia, natural recruitment of terrestrial orchids may occur only in years when a longer growing season results from above-average spring rainfall before the beginning of summer dormancy. Such seasons would expose juvenile plants in a Mediterranean climate to conditions more conducive to the formation of tubers. Occasional summer rainfall, which is more common in some years than others, may also help provide a less stressful environment for juvenile tuber survival during the aestivation period. In successive growing seasons, larger replacement tubers are produced deeper in the soil profile, which in Caladenia species are less vulnerable to summer drought stress or predation by digging/foraging animals (Batty et al., 2006a). In the absence of mycorrhizal fungi, temperate terrestrial orchids may survive on tuber reserves or photosynthetic activity for at least two years before a decline in fitness is observed (Dixon, 1991). So, plants produced through in vitro propagation often fail to make the transition from heterotrophic growth in tissue culture to autotrophic growth in situ. There is an almost 90% loss of tissue cultured plants in this transition phase, which requires significant over-production of plants in the culture system to cope with poor deflasking survival (Batty et al., 2001a).

The need for application of ecophysiological principles to improve the ability of the prolific micropropagation system to deliver more robust and resilient orchid plants for restoration has therefore never been more critical and the following paragraphs summarise the outcomes of this study as they impact on achieving this objective.

Seed germination

In chapter 2, seeds of C. huegelii, C. latifolia, P. sanguinea and M. media showed variation in germination when incubated at different temperature (10, 15, 20 and 25⁰C) in both darkness and light. Study species showed a low temperature threshold as germination was suppressed when seeds were cultured at 10⁰ C. Temperature optima for germination were found to be 15 ⁰C and 20⁰C however germination also corresponded to presence and absence of light. Under natural conditions, some seeds are buried and exposed to darkness which requires a large energy store to emerge from the soil (Plummer and Bell, 1995; Grant et al., 1996; Plummer et al., 1997; Bell, 1999). Lack of energy as a result of undifferentiated

63 endosperm makes orchid seeds hard to germinate (Burgeff, 1936; Harley, 1951; Maheshwari and Narayanaswami, 1952). Therefore, seeds with light requirements must remain at the soil surface (Plummer and Bell, 1995). Positively photoblastic response of C. latifolia is a typical characteristic of smaller seeded species as the energy required to fuel further growth needs to be obtained through photosynthesis (Rokich and Bell, 1995). Being situated on the soil surface subjects seeds to the desiccation risk caused by drought that regularly occurs in south Western Australia soils (Bell et al., 1995). The negatively photoblastic response of P. sanguinea might be an adaptation that is species specific and correlates with its symbiotic fungi. Conversely, the species of this current study were found to be largely unaffected by light. The main aim of applying asymbiotic culture in this study was to clarify the role that fungi are playing in symbiotic culture. This study also aimed to determine an appropriate protocol to propagate orchid seedlings under in vitro conditions. The presence of carbohydrate in asymbiotic media was previously reported as a key factor for protocorm development and seedling growth (Stewart and Kane, 2010; and Johnson et al., 2011). In this study however, C. huegelii and C. latifolia failed to germinate and grow on media with a high carbohydrate supply (½MS and Pa5). Variation in nutrient assimilation among orchid species demonstrates a diverse ability of orchids and their fungal symbionts to utilise food sources under different natural conditions. Future orchid propagation programs need to take into account orchid-fungal ecology as the environmental requirements of the fungi may need to be replicated in symbiotic media under in vitro conditions.

Seedling growth and photosynthesis

In chapter 3, the effect of symbiont partners on photosynthesis of the study orchid species was observed. Presence of mycosymbionts in the media suppressed photosynthesis in all species. Significant decreases in the photosynthesis of host plant due to nutrient abstraction in other symbiotic relationships has been shown (Watling and Press, 2001; Cameron et al., 2005) however, the suppression of photosynthesis by orchid mycorrhizae at the early stages of growth of orchid plants is not fully understood. Variation in the partial or complete suppression of photosynthesis in orchids may be due to the high dependency of orchids on their symbiotic associations at the early stages of growth. In chapter 3, we sought to test the premise that becoming autotrophic may aid in the survival of orchid seedlings through the critical transferral from in vitro laboratory growth to growth in a soil medium in the glasshouse. To achieve this aim, we manipulated growing conditions to evaluate seedling photosynthetic capacity of the study species. Different types of light, light quantity and

64 nitrogen were applied to test whether or not the orchid plants were capable of becoming autotrophic. The results obtained showed that presence of a symbiotic fungus appears to have a pronounced effect on photosynthesis at the early seedling stage when compared to concurrent variations in light and nitrogen. In asymbiotically grown orchids in the current study, only M.media seedlings were found to become photosynthetically active. It could be due to this species known intrinsic weedy characteristics that allow it to recruit and establish in a wide variety of environments. Different responses displayed by M. media to light type, light quantity and nitrogen compared to other study species might be due to variation in fungal dependency (Swarts and Dixon, 2009b). For instance, C. huegelii is known to have a highly specific and range limited fungal partner (Swarts et al., 2010) that would tend to make it difficult to facilitate C through other fungal symbionts or even other strategies such as photosynthesis. Alternatively, weedy species that do not have specific obligate relationships, such as M. media may be able to switch from the early myco-heterotrophic stage to becoming fully autotrophic much earlier in development, and thus obtain carbon through photosynthesis rather than incur the nutritional cost of C acquisition from a fungal symbiont (Ackerman, 2007; De long et al., 2013). We concluded that the species that did not become autotrophic under optimum in vitro conditions may be photosynthetic once the seedlings have developed further and are transferred to glasshouse soil. The scope of this study needs to be further expanded to include a preliminary protocol for propagation of the study species under glasshouse conditions. It is considered that the impact of the particular fungal symbiont as well as light and nitrogen on photosynthetic capacity of orchid seedlings is likely to be different under glasshouse conditions, which will consequently impact on the survival rate of the study species. The inherent ‘weediness’ of M. media would be expected to translate to better ex vitro survival under glasshouse conditions than the other study species. Based on the results and observations in this study it would appear that this is the most likely outcome as M. media did not display any apparent sensitivities to optimum germination temperatures, germinated in light or dark but most importantly asymbiotically germinated seedlings exhibited a readiness to switch to photoautotrophy at the late development stage, but this is offset by the relatively low germination and seedling development on asymbiotic media. Of the other study species it is less clear which would be likely to perform best in glasshouse conditions without follow-up laboratory and glasshouse trials.

Key Findings from this study include

1. Though the studied species showed different germination percentages across treatments the overall germination response to both light and temperature between C. huegelii and M. media was similar, while the germination response by P. sanguinea and C. latifolia seeds to temperature concurred, response to light/dark treatment was opposite.

2. High symbiotic germination on oatmeal agar (OMA+ fungal symbionts specific to each species) was recorded in C. huegelii, M. media and P. sanguinea in continuous dark incubation.

3. Germination was higher and development of seedlings faster overall in all test species in symbiotic compared with asymbiotic media treatments.

4. Illumination had no effect on fungal symbiont growth across all species, however incubation temperature treatments (10, 15, 20 and 25 ⁰C) affected fungal growth rate. Growth of the fungal symbionts of Caladenia huegelii, M. media and C. latifolia demonstrated significantly lower activity at 10 ⁰C, but the cumulative radial growth rate of the P. sanguinea fungal symbiont reached 64 cm2 after only two weeks at all temperatures tested, including 10 ⁰C.

5. Photosynthesis of P. sanguinea and M. media was negative in the presence of fungus, however A significantly increased with the absence of fungus.

6. Chlorophyll content of P. sanguinea and M. media seedlings grown in OMA inoculated with fungus was significantly higher (6.5 and 4 times) compared to seedlings on OMA without fungus.

7. Among the study species, total chlorophyll content of P. sanguinea and C. latifolia seedlings did not responds to either fluorescent or LED light or presence/absence of N. In contrast, total chlorophyll contents of C. huegelii (4105 µg g-1 FW without N added to the medium) and M. media (3670 µg g-1 FW with 360 µM N added to medium) exhibited significantly higher concentrations under LED light.

8. Our results suggest that photosynthesis at the early stages of orchid growth may be more affected by the presence and absence of mychorrhizal fungus compared with light type, light quantity and N concentration.

This study demonstrates the difficulties in dealing with species that possess a complex life history and further studies will be needed to translate these findings into improving the survival of terrestrial orchid seedlings from in vitro propagation to the glasshouse environment. However, it is hoped that the study will be of benefit to the conservation of Australian terrestrial orchids specifically and terrestrial species generally.

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