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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
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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.
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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
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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
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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.
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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
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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
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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).
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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
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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
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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
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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
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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?
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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.
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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.
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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).
17
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).
18
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
19
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.
20
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
21
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
22
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.
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
0
20
40
60
80
100
10⁰c 15⁰c 20⁰c 25⁰c
C. huegelii
0
20
40
60
80
100
10⁰c 15⁰c 20⁰c 25⁰c
M. media
0
20
40
60
80
100
10⁰c 15⁰c 20⁰c 25⁰c Temperature
P. sanguinea
0
20
40
60
80
100
10⁰c 15⁰c 20⁰c 25⁰c Temperature
C. latifolia
bc
a a
c c
c c c
b
Ger
min
atio
n (%
) d
c
b
c
a
b
b b
c
a a
b b
c
d
c c
a
bc
a
b
bc
bc
23
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).
0
20
40
60
80
100
4 8 11
C. huegelii
0
20
40
60
80
100
4 8 11
M. media 10 ° C
15 ° C
20 ° C
25 ° C
0
20
40
60
80
100
4 8 11 Week
P. sanguinea
0
20
40
60
80
100
4 8 11 Week
C. latifolia
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).
Ger
min
atio
n (%
)
24
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).
25
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
Species Culture medium
Light Dark Light Dark Light Dar
k
Caladenia huegelii
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
Pa5 2.6b 2.1b 0.0 a 0.0 a 0.0 a 0.0 a
OMA+Fungus 0.0 a 0.0 a 50.3b 45.1b 49.7b 54.9
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
Pa5 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a
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
Pa5 4.8b 0.0 a 4.8b 0.0 a 0.0 a 0.0 a
OMA+Fungus 18.5ab 21.1ab 32.1b 33.9b 5.9a 22.9
ab
Pterostylis sanguinea
OMA 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a 0.0 a
½MS 8.0b 12.4b 32.0c 28.8c 0.0 a 12.1
b
Pa5 9.9b 21.8c 6.9b 9.0b 0.0 a 0.0 a
OMA+Fungus 18.4c 4.3 b 15.8c 64.9e 0.0 a 28.1
d
26
Table 2. ANOVA results for the combined effects of temperature and illumination on symbiotic germination and fungal growth rate on four study species.
Species
Effect
d.f.
Germination Fungal Growth
F P F P
Caladenia huegelii
Temperature
Illumination
Interaction
3
1
3
13.1*
17.6*
6.1*
<.0001
0.0003
0.003
392.7**
1.09
1.98
<.0001
0.31
0.14
C. latifolia
Temperature
Illumination
Interaction
3
1
3
25.4*
36.4*
9.39*
<.0001
<.0001
<.0003
655.9**
0.19
0.24
<.0001
0.667
0.865
Microtis media
Temperature
Illumination
Interaction
3
1
3
31.1*
8.2*
4.3*
<.0001
0.0084
0.0146
292.7**
3.29
1.82
<.0001
0.082
0.171
Pterostylis sanguinea
Temperature
Illumination
Interaction
3
1
3
6.56
27.9*
10.52*
0.0021
<.0001
0.0001
0.51
0.45
0.74
0.001
0.51
0.54
* and ** = significant differences at 5 and 1% probability levels respectively.
27
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).
28
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 25⁰C (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).
0
10
20
30
40
50
60
70
0 2 4 6 8 0
10
20
30
40
50
60
70
0 2 4 6 8
10⁰C 15⁰C 20 ⁰C 25⁰C
0
10
20
30
40
50
60
70
0 2 4 6 8 0
10
20
30
40
50
60
70
0 2 4 6 8
Fun
gal G
row
th R
ate
(cm
2 )
Incubation time (weeks)
C. huegelii M. media
P. sanguinea C. latifolia
29
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
0
10
20
30
40
50
60
70
10 15 20 25
0
10
20
30
40
50
60
70
10 15 20 25
light
Dark
0
10
20
30
40
50
60
70
10⁰C 15⁰C 20⁰C 25⁰C 0
10
20
30
40
50
60
70
10⁰C 15⁰C 20⁰C 25⁰C
Temperature
Tot
al F
unga
l Gro
wth
(cm
2 ) M. media
a a a a a
b
bc
a C. huegelii
b b
a a a a a a
C. latifolia a a a a a a a a P. sanguinea
b b
a a a a a a
30
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.
31
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.
32
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
33
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.
34
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.
35
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
36
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.
37
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
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 South West Australian Floristic Region (SWAFR), displaying high levels of
fungal specificity (Myers et al., 2000; Batty et al., 2001b). Among the selected orchid
species for use in this study C. huegelii is declared Rare Flora based on IUCN criteria in a
revision by Hopper and Brown (2001), while other three taxa are not considered threatened.
Caladenia latifolia is found Australia-wide and in Western Australia it occurs in a variety of
habitats including dense coastal heath, woodlands, Karri forest, and in the central and
38
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%
39
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
40
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 = 15.65A666- 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.
41
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
a c b
g f e
d
h
42
fungus was significantly (P<0.05) higher (6.5 and 4 times respectively) compared with the
OMA without fungus (Fig. 2c).
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.
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
1 2 3 4
Leaf
fres
h m
ass
(g)
-2
-1
0
1
2
Phot
osyn
thes
is (µ
mol
CO
2 µg.
Chl
-1s-1
)
0
500
1000
1500
2000
2500
P.sanguinea M. media P. sanguinea M. media
Chl
orop
hyll
cont
ent (
µ g
g-1 F
W)
OMA+Fungus OMA
a
b
a
c
(c)
a
b b
a
(b)
(a)
b
a
ab
ab
43
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).
44
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.
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
1 2 3
Leaf
fre
sh m
ass
(g)
-6
-5
-4
-3
-2
-1
0
Phot
osyn
thes
is (µ
mol
CO
2 µg
Chl
-1 s
-1)
0
500
1000
1500
2000
LED 65 LED 110 LED 160
Chl
orop
hyll
cont
ent (
µ g
g-1
FW)
Irradiance ( µmol m-2 s-1)
a
a
a
(c)
(b)
a
a
a (a)
a
a
a
45
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).
46
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.
0
0.01
0.02
0.03
0.04
0.05
0.06
1 2 3 4
Leaf
fres
h m
ass
(g)
-6
-5
-4
-3
-2
-1
0
1
2
3
4
5
Phot
osyn
thes
is (µ
mol
CO
2 µg
Chl
.-1s-1
)
0
500
1000
1500
2000
2500
3000
3500
4000
4500
C. latifolia M. media C. huegelii P. sanguinea
Chl
orop
hyll
cont
ent (
µg g
-1FW
)
Fluorescent 35 (µ mol m-2 s-1)
LED 65 (µmol m-2 s-1)
(a)
bc
a
c
bc
c
ab
bc
a
(b)
a
de cd
bc ab
d
ab
e
b
(c)
b b
a
ab
a
b
b
47
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).
48
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.
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
1 2
Leaf
fres
h m
ass
(g)
-0.4
-0.35
-0.3
-0.25
-0.2
-0.15
-0.1
-0.05
0
0.05
0.1
0.15
Phot
osyn
thes
is (µ
mol
CO
2 µg
Chl
-1s-1
)
0
100
200
300
400
500
600
700
800
1 2
Chl
orop
hyll
cont
ent (
µg g
-1FW
)
M.media C. huegelii
Fluorescent Fluorescent+LED
(a)
(b)
c
a
c
b
a ab
c
b
b
a
a
a (c)
49
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).
50
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
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0.045
0.05
1 2
Leaf
fres
h m
ass
(g)
M. media P. sanguinea 0
100
200
300
400
500
600
1 2
Chl
orop
hyll
cont
ent (
µg g
-1FW
)
M. media P. sanguinea
-0.06
-0.04
-0.02
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
35 Fluorescence 80 Fluorescence 65 LED 400 LED
Phot
osyn
thes
is (
µ m
ol C
O2 µ
g C
hl -1
s-1
)
Irradiance (µmol m-2 s-1)
M. media
P. sanguinea
b
a (a)
a a
(b)
ab
b
ab ab
ab
ab
a
a
(c)
51
presence and absence of fungus (Fig. 7b). There was no interaction effect observed between
species, N and irradiance (Table 2).
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
-2
-1.5
-1
-0.5
0
0.5 Ph
otos
ynth
esis
( µm
ol C
O2 µg
Chl
-1s-1
)
0
500
1000
1500
2000
2500
3000
3500
P. sanguinea M. media P. sanguinea M. media
Chl
orop
hyll
cont
ent (
µg/F
W)
OMA+Fungus OMA
0 N (µM)
360 N (µM)
(a)
a
a a a
b
b
c
d
(b) a
a
a
a
b b b b
52
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).
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.
0
0.05
0.1
0.15
0.2
0.25
0.3
0 360
Phot
osyn
thes
is (µ
mol
CO
2 µg
Chl
-1 s
-1)
-0.2
-0.15
-0.1
-0.05
0
0.05
0.1
0.15
0.2
0.25
0.3
0 360
Phot
osyn
thes
is (µ
mol
CO
2 µg
Chl
-1 s
-1)
Nitrogen (µ M)
35 Fluorescent (µ mol m-2 s-1)
80 Fluorescent (µ mol m-2 s-1)
400 LED (µ mol m-2 s-1)
a a a
a a a
M. media
b bc
e
cde de
a P. sanguinea
53
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.
Species
Effect
d.f.
Photosynthesis
F P
M. media & P. sanguinea
M. media
P. sanguinea
species Light
Species* Light
Light Nitrogen
Light*Nitrogen
Light Nitrogen
Light*Nitrogen
1 3 3
2 2 4
2 2 4
0.63* 2.72* 2.11*
1.12*
0.24 0.54
3.31* 17.54**
1.13*
0.4302 0.0462 0.1012
0.33 0.78 0.71
0.044 <.0001 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.
Species
Effect
d.f.
Photosynthesis Chlorophyll content
F P P F
C. latifolia M. media
C. huegelii P. sanguinea
Species Nitrogen Species*Nitrogen Light Species*Light Nitrogen*Light Species*Nitrogen*Light
3 1 3 1 3 1 3
2.24* 2.98* 0.80
70.3** 3.87* 0.38* 0.49
0.0919 0.0893 0.4971 <.0001 0.0131 0.5379 0.6920
3.67* 1.75* 0.97* 4.25* 3.43* 0.36 2.12*
0.015 0.191 0.414 0.043 0.022 0.549 0.106
54
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
55
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
56
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
57
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
58
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.
59
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
60
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.
61
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
62
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).
63
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 10⁰C. Temperature optima for germination were
found to be 15⁰C 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
64
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
65
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.
66
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.
67
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.
68
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