Propagation and reintroduction of Caladenia

Abstract

Many Caladenia species have been reduced to extremely small and/or fragmented populations, and reintroduction/translocation into natural or rehabilitated habitats, by using ex situ propagated plants or via direct seeding, represents an important adjunct in conservation planning. However, Caladenia species are some of the most difficult terrestrial orchid taxa to propagate, in part because of the specificity of the mycorrhizal associations and the need to provide growing conditions that suit both the mycorrhizal fungi and Caladenia plants. The present paper reviews recent advances in Caladenia propagation and reintroduction methods, including in vitro seed germination, transferral from in vitro to nursery environments, ex vitro symbiotic germination (germination in inoculated nursery media), nursery cultivation, the use of nurse plants and reintroduction of Caladenia into natural habitats by using seed, dormant tubers or growing plants. Techniques discussed in the present paper increase the options for future Caladenia conservation programs, especially for those species currently on the brink of extinction.

Full text

Propagation and reintroduction of Caladenia
Magali Wright
A,B,H
, Rob Cross
B
, Kingsley Dixon
C
, Tien Huynh
D
, Ann Lawrie
D
,
Les Nesbitt
E
, Andrew Pritchard
F
, Nigel Swarts
C
and Richard Thomson
B,G
A
Melbourne School of Land and Environment, Burnley Campus, The University of Melbourne,
500 Yarra Blvd, Richmond, Vic. 3121, Australia.
B
Royal Botanic Gardens Melbourne, Birdwood Avenue, South Yarra, Vic. 3141, Australia.
C
Kings Park and Botanic Garden, Fraser Avenue, West Perth, WA 6005, Australia.
D
Biotechnology & Environmental Biology, School of Applied Sciences, RMIT University,
Bundoora West Campus, Bundoora, Vic. 3083, Australia.
E
PO BOX 72, Walkerville, SA 5081, Australia.
F
Department of Sustainability and Environment, 78 Henna Street, Warrnambool, Vic. 3280, Australia.
G
Australasian Native Orchid Society (Victorian Group), PO Box 354, Glen Waverley, Vic. 3150, Australia.
H
Corresponding author. Email: mmwright@unimelb.edu.au
Abstract. Many Caladenia species have been reduced to extremely small and/or fragmented populations, and
reintroduction/translocation into natural or rehabilitated habitats, by using ex situ propagated plants or via direct
seeding, represents an important adjunct in conservation planning. However, Caladenia species are some of the most
difficult terrestrial orchid taxa to propagate, in part because of the specificity of the mycorrhizal associations and the need to
provide growing conditions that suit both the mycorrhizal fungi and Caladenia plants. The present paper reviews recent
advances in Caladenia propagation and reintroduction methods, including in vitro seed germination, transferral from in vitro
to nursery environments, ex vitro symbiotic germination (germination in inoculated nursery media), nursery cultivation, the
use of nurse plants and reintroduction of Caladenia into natural habitats by using seed, dormant tubers or growing plants.
Techniques discussed in the present paper increase the options for future Caladenia conservation programs, especially for
those species currently on the brink of extinction.
Introduction
Caladenia (synonym Arachnorchis) distribution is largely in the
disturbed natural environments of the south-east and south-west
of Australia. The populations of many species are in a fragile
condition, with seedling recruitment often low. In all, 43 taxa
are listed as Endangered or Critically Endangered and 15 as
Vulnerable (Department of the Environment Water Heritage
and the Arts 2008). Although in situ conservation management
of threatened orchids is of prime importance, at times, to guard
against extinction, the numbers of plants in existing populations
need to be augmented through direct seeding, by translocating
from other sites or by introducing/reintroducing ex situ-
propagated plants. Propagation also enables the establishment
of ex situ conservation collections as a further guarantee against
extinction and to act as seed orchards for further in situ
conservation work or for research.
Seed germination retains the broader genetic diversity needed
for conservation programs, and can potentially produce large
numbers of plants. Caladenia seed, like those of all orchids,
contains few nutrient reserves and mycorrhizal fungi are their
main source of nutrition during germination and early seedling
stages. Adult Caladenia plants have also evolved to rely heavily
on their mycorrhizal fungi for nutrition; they lack true roots
and have limited photosynthetic capacity (a single leaf).
Therefore, it is this orchid–fungi relationship that has been
central to the development of propagation, cultivation and
reintroduction techniques. An important research goal has
been to determine growth conditions that suit both the
Caladenia plants and their associated mycorrhizal fungi and so
maintain the balance in favour of the orchid.
There is currently a wide range of techniques employed in
Caladenia propagation, translocation and reintroduction across
Australia (Fig. 1). Each of these methods involves sowing
Caladenia seed in the presence of mycorrhizal fungi, either
under laboratory conditions (in vitro), into nursery growing
media (ex vitro) or soil (in situ ).
Reintroductions and translocations can be considered
successful only when new populations become self-sustaining
(Vallee et al. 2004). Caladenia species, like all orchids, rely on
other organisms to complete their life cycles. Not only do they
have specific mycorrhizal associations (Warcup 1971), but
they also have specific associations with their pollinators, with
many Caladenia taxa using deception for attracting them
(Peakall and Beattie 1996). Thus, these orchids often rely on a
specific insect species for seed production and a specific fungal
species for seedling recruitment and therefore the presence of
CSIRO PUBLISHING
www.publish.csiro.au/journals/ajb Australian Journal of Botany, 2009, 57, 373–387
 CSIRO 2009 10.1071/BT08137 0067-1924/09/040373

these organisms is required for translocated or reintroduced
populations to become self-sustaining.
The present paper outlines methods employed in Caladenia
propagation and translocation for conservation (Fig. 1), and
discusses the advantages and disadvantages of each. The
primary objective is to highlight recent advances and the key
knowledge gaps in maximising the success of these methods.
In vitro seed germination
Seed propagation is used in preference to clonal methods because
it provides the genetic diversity crucial for successful
conservation of the threatened terrestrial orchids (Fay and
Krauss 2003). Genetic diversity is thought to improve the
long-term viability of ex situ collections and translocated
populations. Tissue culture, long used for the commercial
production of epiphytic orchids (Chang and Chang 1998), is
less useful for conserving terrestrial orchids, although it may be
the preferred or only option for taxa with low seed set or
viability or when only a few individual plants remain. Tissue
culture has been used in the recovery of the Australian terrestrial
orchid, Diuris longifolia (Collins and Dixon 1992); however,
it has not been used for conserving Caladenia taxa where
hand-pollination produces adequate quantities of seed.
The following two types of in vitro seed germination are
used to propagate terrestrial orchid seed:
(1) symbiotic germination methods which involve inoculation
of seed with an appropriate mycorrhizal fungus; and
(2) asymbiotic methods in which seed is sown on specialised
high nutrient media without mycorrhizal fungi.
Asymbiotic techniques are often favoured by amateur
Caladenia growers because specialised mycological skills
such as fungal isolation and maintenance are not required.
Asymbiotic techniques have benefits in vitro as they can give
reliably high germination rates of >95% (Huynh et al. 2004)
and seedlings with long in vitro flask life. Low survival rates
are often encountered with asymbiotic propagation when
seedlings are transferred into soil (Anderson 1991; Oddie et al.
1994) and further development is severely delayed, with later
introduction of fungi unsuccessful in establishing symbiosis
(Raleigh 2005).
In vitro symbiotic seed germination (Fig. 1A) has been used in
conservation-management programs worldwide (Zettler and
McInnis 1993; Sharma et al. 2003; Stewart et al. 2003; Batty
et al. 2006a, 2006b; Brundrett 2007; Dearnaley 2007) and is
favoured over asymbiotic techniques as it allows introduction of
the appropriate mycorrhizal fungi with the orchid. Caladenia
species were originally thought to have extremely specific
mycorrhizal associations with the fungus Sebacina vermifera
(Warcup 1971, 1981, 1988). DNA sequencing has since shown
that Warcup’ s Sebacina vermifera isolates belong to a broad
Fig. 1. A flow diagram outlining the propagation, reintroduction and translocation methods used in Caladenia conservation programs. The desired endpoints
are indicated by the statements on the bottom row and include establishment of ex situ collections and reintroductions and/or translocations of plants into natural
or rehabilitated habitats. These endpoints are reached by collecting a variety of propagation material from in situ extant Caladenia plants, which can then be
treated in several ways. Caladenia seed and mycorrhizal fungi can be combined in (A) in vitro germination, (B) in situ germination or (C) ex vitro symbiotic
germination (in organic mulch or other growing media). Caladenia seed alone can be (D) sown around ‘nurse’ plants in potting media, or sown into field sites
that (E) contain adult plants or (F ) where mycorrhizal fungi have been located by fungal baiting techniques. Whole plants can be (G) translocated from
populations under direct threat of destruction.
374 Australian Journal of Botany M. Wright et al.

species complex within the Sebacinaceae (order Sebacinales)
(Weiss et al. 2004). Molecular studies of mycorrhizal diversity
have revealed that Caladenia species associate with a range
of closely related fungi within this complex (Huynh 2003;
Bougoure et al. 2005; Raleigh 2005; Bonnardeaux et al. 2007;
Swarts 2007; Wright 2007). The majority of conservation
programs use a limited number of mycorrhizal isolates during
the propagation of terrestrial orchids (Zettler and McInnis 1993;
Sharma et al . 2003; Stewart et al. 2003; Batty et al. 2006a,
2006b; Wright et al. 2007a), which may not reflect the diversity
of mycorrhizal fungi occurring in natural populations.
Common Caladenia taxa appear to be able to take advantage
of a wider range of S. vermifera-like fungi than do endangered
taxa (Wright 2007), although there are exceptions to this
strategy, e.g. C. robinsonii (Raleigh 2005). C. tentaculata,a
relatively common species, was found to associate with
several S. vermifera-like taxa, whereas six endangered species
associated with a single fungal taxon across the same geographic
range (Wright 2007). Studies have revealed a similar situation
within Western Australian Caladenia (Hollick 2004; Swarts
et al. 2007). The endangered C. huegelii has been found to
associate with fungi with identical ITS sequences throughout
its entire distribution (Swarts et al. 2007), whereas the common
C. arenicola forms associations with a much wider range of
fungi (Hollick 2004). It has been suggested that the ability to
utilise a broad selection of fungi may render an orchid species
robust to environmental change (Rasmussen and Rasmussen
2007). The limited ability of endangered Caladenia taxa to
utilise a wide range of mycorrhizal fungi highlights the
importance of maintaining fungal diversity, as a progressive
reduction in mycorrhizal diversity may influence their rarity.
Understanding the specificity of Caladenia mycorrhizal
associations and their ecological implications could improve
conservation efforts by providing the appropriate mycorrhizal
fungi at recipient sites.
In Caladenia taxa, mycorrhizal colonisation is concentrated
in the stem-collar (Ramsay et al. 1986) and has also been located
in the tuber (Huynh et al. 2004). The stem-collar and its
mycorrhizal fungi are susceptible to seasonal influences
(Huynh et al. 2004), the collar region senescing with the leaf
during the summer dormancy, followed by the emergence of a
new collar and leaf from the tuber at the start of the next growing
season. Therefore, fungal isolation relies on the quality of
the stem-collar to obtain viable fungi for symbiotic
germination. Although there are instances where effective
fungi can be isolated from all actively growing stages from
leafi
ng to flowering (Huynh et al. 2004), this is not the case
for all species, with isolates from C. phaeoclavia and C. fulva
ranging widely in effectiveness throughout the growing
season (Raleigh 2005).
The symbiotic germination technique relies on mycorrhizal
fungi being isolated from the orchid tissue it colonises. In
Caladenia species, whole stem-collars are typically removed
for this purpose. A non-destructive method for collecting
mycorrhizal tissue for fungal isolation from terrestrial orchids
in situ involves removing a slice of tissue from organs
colonised by mycorrhizal fungi (K. W. Dixon and A. L. Batty,
pers. comm., 2004; S. L. Stewart, pers. comm., 2004). The ‘slice’
method is especially useful for Caladenia species (Fig. 2) because
their stem-collar is directly beneath the leaf and flower bud
which, when removed, stops photosynthesis and reproduction
in a given season. Taking a longitudinal collar slice makes it
possible to conduct both a fungal isolation and seed collection
from an individual plant in a single season, reducing the time
frame required for symbiotic propagation, and is particularly
useful for taxa with extremely small population sizes. A recent
study has shown that the ‘slice’ method can be used to culture
uncontaminated mycorrhizal fungi from both common and
endangered Caladenia species, without affecting plant survival
(Wright 2007).
Mycorrhizal isolates are collected from Caladenia species by
dissecting single pelotons (fungal coils) from stem-collar tissue
and culturing them on specialised Fungal Isolating Medium
(Huynh et al. 2004; Bougoure et al. 2005; Raleigh 2005; Batty
et al. 2006a, 2006b; Bonnardeaux et al. 2007; Wright 2007).
During in vitro symbiotic germination, potential mycorrhizal
isolates are introduced to surface-sterilised orchid seed under
aseptic conditions. Caladenia seed, with a compatible isolate,
germinates and starts to produce photosynthetic tissue within
4–12 weeks of inoculation (Huynh 2003; Raleigh 2005; Wright
et al. 2005; Swarts 2007).
Selection of appropriate mycorrhizal isolates and
pretreatment of orchid seed for symbiotic germination has
been described as a ‘hit or miss’ affair (Zettler 1997). The
pretreatment of orchid seed is important for optimising
germination and can vary depending on the resistance of the
testa and the quality of the seed. Caladenia seed requires no
specialised pretreatment; however, for aseptic cultures surface
sterilisation of the seed is essential. Fresh sodium hypochlorite
(NaOCl) is most commonly used as a surface sterilant. For
Fig. 2. The stem-collar of an in situ Caladenia tentaculata plant from
which a slice was taken for fungal isolation. Scale bar = 0.5 cm.
Caladenia propagation and reintroduction Australian Journal of Botany 375

C. tentaculata, germination declined with increasing
concentration of NaOCl and the optimal exposure to avoid
contamination was 3 min with 0.5% NaOCl (Raleigh 2005).
This method has become commonly used (Huynh et al. 2004;
Wright et al. 2005; Wright 2007).
The effectiveness of mycorrhizal isolates can vary even
among those obtained from pelotons from the same Caladenia
plant, with variable germination percentages and developmental
stages achieved (Huynh 2003; Wright 2007). The variation in
the response of Caladenia seed to different fungal isolates
continues past the germination stage, affecting the leaf length
of the C. formosa seedling in vitro (Huynh 2003) and the
establishment of C. tentaculata ex vitro (Raleigh 2005; Wright
2007). There have been no studies to determine the ecological
importance of the functional variation observed in vitro;
however, it is possible that maintaining fungal diversity during
reintroductions will contribute substantially to the survival of
new populations.
An oatmeal-based agar medium described by Clements
et al. (1986) is the most commonly used for symbiotic
germination of terrestrial orchid seed (Ramsay and Dixon
2003). A variation of this medium was originally developed
for Caladenia species inoculated with S. vermifera (Warcup
1981) and is effective for a range of Australian (Clements
1981, 1988; Warcup 1981; Ramsay et al. 1986; Dixon 1987;
Huynh 2003; Raleigh 2005; Wright et al. 2005; Batty et al.
2006a), European (Clements et al. 1986) and American
(Zettler and McInnis 1993; Sharma et al. 2003) terrestrial
orchids. The addition of sucrose to this medium decreased the
germination of C. tentaculata seed (Wright 2007). However,
sucrose has been shown to enhance the growth of C. tentaculata
(Raleigh 2005) and Microtis parviflora (Perkins et al. 1995)
seedlings after symbiotic germination, even though it inhibited
tuberisation and seedling emergence (after transferral ex vitro)
of a close relative of Caladenia, Glossodia major (Raleigh
2005). Interestingly, of six C. tentaculata mycorrhizal isolates
tested only one was able to utilise sucrose as a sole carbon
source (Wright 2007), so it may be the growing seedlings and
not the mycorrhizal fungi that respond positively to the presence
of sucrose in the medium.
In vitro seed germination yields more seedlings than ex vitro
and in situ seed sowing. Preliminary comparisons of symbiotic
germination experiments with C. tentaculata seed reveal the
benefit of this method for endangered species where seed is
limited, with in vitro germination giving 44% germination,
ex vitro
0.26% (Fig. 1C) and in situ 0.02% (Fig. 1B) for the
same combination of seed and mycorrhizal isolates (Wright
2007). However, in vitro seed germination and transferral of
seedlings to soil requires specialised skills and equipment, and is
labour intensive (Scade et al. 2006), which can limit its use in
routine conservation efforts.
Transferral from in vitro to nursery environments
Establishing in vitro-propagated terrestrial orchid seedlings in
potting media or soil is problematic, with low survival rates
being observed for many species (Zettler and McInnis 1993;
Backhouse and Jeanes 1995; Batty 2001; Ramsay and Dixon
2003). The difficulty of establishing in vitro-grown Caladenia
seedlings ex vitro has meant that there are very few of these
plants in ex situ collections or available for reintroduction. Most
growers experience less than 10% survival by conventional
asymbiotic methods (R. Thomson, pers. obs.).
The importance of Caladenia mycorrhizal fungi in plant
growth and development under natural conditions suggests
that their use in propagation would increase successful nursery
establishment, by allowing them access to more nutrients.
Attempts to compare directly the effects of symbiotic and
asymbiotic methods of seed germination on nursery
establishment have not been successful, owing to the poor
seedling growth and contamination on asymbiotic media
(Batty et al. 2006a). However, indirect comparisons of
survival percentages reveal that symbiotically germinated
seedlings have much higher survival rates. These range from
37 to 80% under optimised cultural conditions (Raleigh
2005; Batty et al. 2006a ; Swarts 2007; Wright 2007) and
are considerably higher than the <10% survival experienced
by asymbiotic methods. Thus, propagation of Caladenia plants
by symbiotic methods is considered to be the most effective
means for maximising seedling germination, survival and
establishment.
The transferral of in vitro-grown orchid seedlings to nursery
conditions is often termed ‘deflasking’. Because Caladenia
species, like many Australian terrestrial orchids, are summer-
dormant, the timing of deflasking is critical to seedling survival
during the growing season and the development of a perennating
structure (tuber) to survive summer dormancy. Research has
shown that deflasking seedlings in April (early autumn), after
germination in the summer months, maximises the time available
for seedlings to establish mycorrhizal connections in the potting
medium and to initiate a dropper before tuber development
(Swarts 2007). Work completed on C. tentaculata seedlings
deflasked in April gave a near 20-fold increase in seedling
survival and re-emergence compared with those deflasked later
(June) in the growing season (Wright 2007).
Problems associated with deflasking into potting media or
field sites include high levels of initial mortality after removal
from Petri dishes or flasks, followed by an inability of the
seedlings to develop perennating structures (tubers) to survive
dormancy (Batty et al. 2006a). One of the causes of high initial
mortality and poor soil establishment is damage to the protocorms
and droppers when seedlings are removed from agar-based
media, breaking the hyphal connections to the food source
(Oddie et al. 1994). For this reason Raleigh (2005)
investigated a range of non-agar-based media for in vitro use
with older seedlings; of the media investigated, cellulose sponge
enhanced seedling growth in vitro but caused problems after
deflasking, with lower emergence after the summer dormancy.
Preliminary investigation into the in vitro use of a mixture of
scoria (macrovesicular volcanic rock) and rice hulls indicated
that this medium may be superior (Raleigh 2005), although
the variability in the quality of scoria makes it less desirable
for standardising media components for conservation
propagation programs.
Recent developments in in vitro support media include the
use of a sterile sand (Batty et al. 2006a) or vermiculite layer
(Dimech et al. 2008) over oatmeal agar in a plastic container,
acting as an intermediate incubation stage before transfer to soil.
376 Australian Journal of Botany M. Wright et al.

This method provides a substrate from which seedlings are
easily removed and seedlings from a wide range of Caladenia
species grown by this method have been successfully
established in potting media and habitat soil (Batty et al.
2006a, 2006b; Scade et al. 2006; Cross et al. 2007; Swarts
2007). The thickness of the layer appears to affect the quality
of seedling growth; however, further investigation is required to
determine why.
Another contributing factor to deflasking difficulties may
be the movement from high humidity under in vitro
conditions to ambient humidity in the nursery (Ramsay and
Dixon 2003; Threatened Orchid Recovery Team 2004; Batty
et al. 2006a). To mitigate these losses, this problem has
been addressed with the use of ‘fog tents’ for newly
deflasked seedlings (Wright et al. 2006) and loosening the
lids of flasks before deflasking, to start the acclimatisation
process earlier.
Aseptic in vitro environments limit exposure to pathogens,
can affect the normal metabolic pathways of plants, and can
alter the structure and functioning of stomata (Preece and Sutter
1991). These and other factors can influence the survival of
plants ex vitro, although exactly what is happening to newly
deflasked Caladenia seedlings is yet to be fully investigated.
Humidity in Caladenia flasks can be manipulated by aerating the
containers with filtered holes (Rossetto et al. 1992; Batty et al.
2006a). Decreasing the difference in humidity between the
in vitro container’s atmosphere and that of the nursery,
prepares the orchid seedlings for the nursery environment.
After 10-week incubation in these containers under laboratory
conditions, seedlings are significantly larger, can readily survive
the initial transfer into potting media under nursery conditions,
and are more likely to survive the growing season and produce
tubers that will survive summer dormancy (Batty et al. 2006a).
These methods were shown to be highly successful for the
propagation of C. huegelii seedlings after summer germination
(Swarts 2007). From the current data, it is clear that providing
optimal aeration/humidity conditions in vitro is essential in the
propagation of these species.
Summer dormancy has been identified as the point of highest
mortality of
in vitro-grown Caladenia seedlings transferred
into potting media (Raleigh 2005; Batty et al. 2006a; Wright
2007). For example, Raleigh (2005) achieved an average of
>80% survival 3 months after deflasking nine Caladenia
species, but an average of only 34% emergence the
following year. Survival of C. arenicola (Batty et al. 2006a)
and C. tentaculata (Wright 2007) seedlings also decreased by
up to 50% after their first summer dormancy. The success
of deflasking to the nursery environment, as indicated by
re-emergence the following year, may be linked closely to the
process of tuberisation. Batty et al. (2006 a) showed that this
decrease in survival was not only due to the failure to produce
tubers but could also be attributed to tuber mortality during
the summer dormancy, suggesting there may be a critical tuber
size necessary for survival. More research, spanning a wider
range of Caladenia taxa, is required to test this hypothesis.
There may be a critical size of plant for deflasking to obtain
maximum tuberisation, although this has not been critically
investigated, nor have the factors determining this vital
process for successful establishment.
Tuber mortality during the summer months may be attributed
to the moisture level of the potting medium. Waterlogged
conditions in the growth medium can be caused by over-
watering or the physical properties of the potting medium and
need to be avoided especially during the summer months when
high temperatures and excess water can cause tuber rotting
(Richards et al. 1984). It has been suggested that aeration is
amongst the most important characteristics of potting media
for Australian terrestrial orchids (Richards et al. 1984; Dixon
1987), with adequate aeration likely to avoid excess moisture
around the tuber. Interestingly, Raleigh (2005) found that there
was no significant difference in emergence among various
sand-potting mix-mulch mixtures for C. tentaculata, and
rotting of tubers did not appear to explain differences in
emergence the following year. Also, Wright (2007) found the
air-filled porosity of deflasking media had no effect on the
survival of C. tentaculata seedlings, suggesting that media
aeration alone does not improve survival. However,
components of the potting medium have been shown to
influence deflasking survival, with five endangered Caladenia
species showing higher survival in pine bark-based media
than in coarse sand–soil–leaf mould-based media (Wright
et al. 2007a).
The selection of mycorrhizal fungal isolates for in vitro seed
germination is likely to be important for orchid survival and
growth when deflasking into nursery conditions. Batty et al.
(2006
a) suggested that further research into the range of
fungal isolates able to germinate the seed of several Western
Australian orchid species may improve ex vitro survival rates.
On deflasking, the isolates used for initial in vitro seed
germination have been shown to influence seedling growth in
C. formosa (Huynh 2003), and growth and survival in
C. tentaculata (Wright 2007) and C. huegelii (Swarts 2007).
This mirrors the functional variation observed among Caladenia
mycorrhizal isolates in vitro (Huynh 2003; Wright 2007);
however, it is not necessarily the isolates that give the highest
percentage seed germination in vitro that lead to high ex vitro
establishment rates (Wright 2007). This has also been observed
during the deflasking of symbiotic Spiranthes cernua seedlings
(Zettler and McInnis 1993), and has important implications
on isolate selection for conservation programs. The selection
of isolates solely on their ability to germinate seeds in vitro
may lead to low survival rates after deflasking. As the
diversity of mycorrhizal fungi associating with Caladenia
plants (both at the seedling and adult stage) is still relatively
unknown, it is important to maintain possible functional
variation by the use of a range of isolates during symbiotic
seed germination.
The deflasking process and nursery establishment of in vitro-
grown Caladenia still requires further optimisation to increase
survival rates. However, transferring in vitro-grown seedlings
into the nursery environment produces much larger plants than
in situ and ex vitro seed sowing (Wright 2007), with 2-year-old
C. fulva plants in the nursery reaching a larger average size
than those observed in the field, even though the
environmental conditions were very different (Raleigh 2005).
These plants also flower years earlier than those germinating
ex vitro and in situ, i.e. 18–20 months after germination
(Dixon 1987; Raleigh 2005; Wright 2007; Fig. 3), compared
Caladenia propagation and reintroduction Australian Journal of Botany 377

with four or more years for ex situ plants (L. Nesbitt, pers. obs.)
and 5–7 years for in situ plants (G. French, pers. comm.).
The faster rate of development of deflasked in vitro-grown
Caladenia plants potentially increases the speed of recovery
for threatened Caladenia populations.
Ex vitro symbiotic germination
The intriguing and complex association of terrestrial orchids
with endophytic fungi is an interaction that can be exploited
for the purposes of efficient propagation of Caladenia
plants ex vitro. Ex vitro symbiotic seed germination (Fig. 1C)
describes the germination of orchid seed in nursery media,
organic mulch or site soil inoculated with mycorrhizal fungi
in the nursery environment. It has been shown to be successful
for Western Australian (Ramsay and Dixon 2003) and Victorian
(Wright 2007) Caladenia taxa, resulting in an increase in plants
in cultivation.
Given the complex methods and resources required to
produce and propagate seedlings in vitro, facilitating
propagation ex vitro provides useful benefits in terms of high-
health and vigour in seedlings, improved development of
tubers and higher survival rates when plants are transferred
from nursery to field conditions (Dixon and Buirchell 1986;
Oddie et al. 1994; Quay et al. 1995; Batty et al. 2006b;
Wright 2007).
Experimental work needs to evaluate the method that results
in the highest numbers of healthy seedlings able to survive
summer dormancy for the least seed and resources expended
(Batty et al. 2006b). One of the ways this can be achieved is
through providing an artificial inoculum of a compatible and
germination-efficacious endophyte to soil before orchid seed is
sown. A specialised soil for orchid-seed germination can be
prepared by adding inoculum sourced from endophyte cultured
on potato dextrose agar (PDA) or, alternatively, inoculating
sterile millet seed with mycorrhizal fungi and then using
the seed to inoculate soil before sowing of orchid seed (Quay
et al. 1995).
Generation of millet seed-based mycorrhizal inoculum
involves the addition of a fungus to sterilised, moistened millet
seed, followed by incubation for 2–3 months, with agitation
on a weekly basis to ensure even distribution of hyphae
throughout the millet seed. The inoculated millet seed can then
be air-dried and stored before use. Amending soil with millet
inoculum involves addition of quantities of millet seed to
the top 10 mm of soil (usually enriched with a layer of
pasteurised leaf mulch). After 1–3 weeks of incubation under
greenhouse conditions (15–25

C), orchid seed mixed with clean
dry sand is carefully incorporated into the inoculated surface
layer of organic material and watered in. An additional
light application of fine mulch over the surface following
seed sowing can assist in maintaining soil moisture during
seedling germination (Hollick et al. 2007). The success of
ex vitro propagation methods has resulted in the commercial
application of the technology for horticultural production of
terrestrial orchid seedlings (Ramsay and Dixon 2003; Fig. 4).
Although little published data are available on the
effectiveness of ex vitro methods, recent findings by Swarts
(2007) and Wright (2007) suggest this method may be
particularly advantageous for generation of healthy seedlings
of both common and threatened Caladenia species. An
advantage of ex vitro seed sowing over in vitro culture
methods is the potential for seedlings to form associations
with other beneficial organisms such as orchid-associated
bacteria (OAB) that have been shown to provide growth and
Fig. 3. Caladenia ameona plants in the ex situ collection at the Royal
Botanic Gardens, Melbourne, flowering 18 months after germination
(with mycorrhizal fungi) in vitro. Scale bar = 1 cm.
Fig. 4. Orchid seed kit developed by Kings Park and Botanic Gardens in
Perth, which uses ex vitro methods to generate orchid seedlings where
mycorrhiza-inoculated millet and orchid seed dispersed in an inert dry
sand are incorporated into a pasteurised mulch of Allocasuarina needles.
Scale bar = 1.5 cm.
378 Australian Journal of Botany M. Wright et al.

development benefits in the early stages of seedling
establishment (Wilkinson et al. 1989).
Future research into ex vitro methods of propagation should
focus on the quantity of seed used in such trials to produce
plants to evaluate the effectiveness of this method relative to
in vitro symbiotic germination methods. As discussed previously,
a preliminary comparison has shown that ex vitro symbiotic
seed germination utilises much more of the seed resource than
in vitro symbiotic seed germination, making it less appropriate
for endangered taxa with scarce seed resources. Interestingly,
seedlings resulting from ex vitro seed germination have much
higher emergence percentages after the first summer dormancy
than do in vitro-germinated seedlings transferred into potting
media (Wright 2007). However, differences in emergence
between C. tentaculata seedlings germinated with the same
mycorrhizal isolate in vitro (12%) and ex vitro (94%) did not
make up for the comparatively low germination rates observed
ex vitro. So, although ex vitro symbiotic germination may not
be as appropriate for endangered taxa, the fact that it gives
higher survival rates and is less time consuming makes it
useful for propagating Caladenia taxa with high seed
production. Clearly, ex vitro technologies offer significant
opportunities for orchid translocation programs and
horticultural development, particularly where community-
based conservation programs are involved or there is limited
access to greenhouse or laboratory facilities.
Nursery cultivation
The need to provide for the growth of the orchid and also for its
associated fungi can make it difficult to keep Caladenia species
long term in nursery pot collections. Caladenia plants will
commonly perish after a progressive loss in size and leaf
pigmentation during successive seasons. This loss in vigour
may indicate a failure of the mycorrhizal association. Adult
Caladenia plants may survive without nutrients from their
mycorrhizal fungi for a short time, by drawing on food
reserves from their tubers. However, these food reserves must
be replaced to ensure long-term survival.
Specific growth requirements to maintain a healthy
mycorrhizal association must be considered when designing
growth houses, potting media, repotting timelines and seasonal
watering regimes.
This section summarises growth conditions used successfully
by Caladenia growers across Australia. As techniques employed
in the nursery cultivation of Caladenia species have been well
documented during the past 30 years (Richards et al. 1984;
Threatened Orchid Recovery Team 2004; Nesbitt 2006), the
main focus of this section are recent advances and the key
knowledge gaps.
It is difficult to ascertain the mycorrhizal status of Caladenia
plants without disturbing them. A healthy leaf, with no signs of
chlorosis, is a good indicator of the presence of a strong
mycorrhizal association. The appearance of germinating
seedlings within pots, and the use of the ex situ baiting
technique developed by Brundrett et al. (2003) also indicate
the presence of healthy mycorrhizal fungi within the potting
medium, with these methods also providing increased plant
numbers. If some disturbance is acceptable, plants with
healthy mycorrrhizal associations will often have potting
medium clinging to their stem-collar when removed from the
potting medium, which is held in place by external mycelium.
Adefinitive method for ascertaining mycorrhizal presence is
examining a stem-collar slice for the presence of pelotons by
light microscopy. This method has been used on several
C. tentaculata plants (Wright 2007), all of which emerged
after the summer dormancy.
Removing tissue slices from the collar of cultivated Caladenia
plants can also be used to isolate fungi for use in symbiotic
propagation, avoiding the need to collect mycorrhizal tissue
from wild plants. As the collection of orchid material from
natural habitat requires special permits, this method could
potentially allow Caladenia growers to incorporate symbiotic
methods, thus increasing their deflasking survival rates.
Collection of mycorrhizal material (roots) from cultivated
plants has been successfully employed in the recovery of
Diuris fragrantissima (Smith 2006) and D. dendrobioides
(Cross et al. 2005), with plants grown symbiotically with the
resulting fungal isolates. This method has been successfully
applied to the common C. tentaculata (M. Wright, unpubl.
data) and C. robinsonii (Raleigh 2005) and has the potential
to radically increase the use of symbiotic propagation of
Caladenia species. If propagated plants are grown for
reintroduction into natural habitats, provenance records need
to be very accurate and the cultivation in the nursery such
that there is little chance of cross-contamination of fungi from
different provenances.
Even though a pine bark-based medium is advised for use
during deflasking Caladenia seedlings (Threatened Orchid
Recovery Team 2004) and increases seedling survival in
comparison to coarse sand–soil–leaf mould media (Wright
et al. 2007a), it is the latter that is commonly used by most
growers of adult Caladenia plants. The medium used for
Caladenia species is a drier, more freely draining medium
than that used for other Australian terrestrial orchid genera
(Richards et al. 1984). Growers generally use variations of the
Australasian Native Orchid Society (ANOS) medium, which
contains soil, wood chips, coarse sand and leaf mould
(Richards et al. 1984), and is modified to suit local climatic
conditions. This medium is topped with a mulch of Casuarina/
Allocasuarina needles cut into 20-mm lengths. The ANOS
medium varies among growers, depending on local sources
and availability of the components. This variability and the
hygiene issues related to some ingredients sourced from the
natural environment mean that the ANOS medium, although
successfully used by amateur growers, is less suitable for
conservation programs (Wright et al. 2007a).
Anecdotal evidence suggests that the addition of fertilisers
is detrimental to the health of Caladenia species. Blood and
bone is added in very small quantities to the ANOS medium
during preparation (one tablespoon to 9 L of media) (Richards
et al. 1984). However, the use of additional fertiliser in the
cultivation of Caladenia plants is not advised (Threatened
Orchid Recovery Team 2004), with some growers even
excluding fertilisers from their potting medium. Nutrient
concentration, especially nitrogen, can affect the function of
orchid mycorrhizal associations. Dijk and Eck (1995) found
that an increase in ammonium availability caused the fungal
Caladenia propagation and reintroduction Australian Journal of Botany 379

associate, Ceratorhiza, to become parasitic on Dactylorhiza
orchid species, resulting in the death of the orchid seedlings.
Walsh (2003) found that fertiliser application increased the
growth of Pterostylis plants in cultivation. However, more
plants rotted in the presence of fertiliser than in its absence,
indicating increased nutrient availability may increase parasitism
of these orchid plants by their mycorrhizal fungi. Studies of this
type have not been attempted with Caladenia species.
Repotting is undertaken during the summer dormancy
(Richards et al. 1984), although avoided until tubers reach the
bottom of the pot. Reduction in plant numbers is often observed
after repotting and may be due to a failure of the mycorrhizal
association in the new growing medium. A proportion of the
original growing medium is incorporated into the fresh medium
(Richards et al. 1984) and several growers have emphasised
the importance of including the old collar with repotted tubers;
this may act as a source of fungal inoculum, as suggested
by Huynh et al. (2004).
Balancing seasonal water availability is critical to the
successful cultivation of summer-dormant Caladenia species.
The potting medium must be kept moist during the active
growth season (autumn–spring) and fairly dry throughout the
summer. As discussed previously, moist potting medium in
summer temperatures can lead to the rotting of dormant tubers.
From late spring, watering is limited to a light application once
a fortnight to prevent tuber desiccation (Richards et al. 1984).
Some growers place pots in polystyrene boxes covered with
damp hessian to decrease the temperatures plants are exposed
to during the summer months. Heavier watering generally
resumes from early to mid-autumn, with some pots requiring
soaking to overcome hydrophobicity in the potting medium
(Richards et al. 1984).
Monitoring the moisture levels in individual pots during
the active growing season (autumn–spring) is important. The
aim is to keep the medium moist, not wet, as over-watering can
be detrimental to plant health even during the growing season.
Pots are watered individually and must be checked at regular
intervals, especially during the changeable weather of spring.
The pot size and shape, and the structure of the potting
medium all contribute to the proportion of medium in a pot
that has the right conditions for growth (Handreck and Black
2001). Even a well-designed medium with good drainage has
a layer at the base of the pot that remains saturated. The proportion
of saturated medium is smaller in taller pots, and in pots that
taper inwards towards the base rather than being vertically sided.
Further studies into the design of potting media to minimise
the saturated bottom layer may lead to increased plant survival,
and simplified watering regimes and monitoring.
Growers exclude natural rainfall from their growth houses,
either all year or for specific seasons, to help control the moisture
levels in their Caladenia pots. Air circulation is important in
the growth-house design for optimal growth from autumn to
spring. It reduces the time potting media remains saturated,
allows plant leaves to dry in the afternoon and provides a less
favourable environment for insect pests (e.g. fungus gnats).
Growth houses are often sealed on the sides with shade cloth
to prevent birds and animals gaining access because they will
often consume plants and their tubers. Shade cloth also allows
good air circulation and meets the light requirements of
Caladenia species (Richards et al. 1984). The use of mesh
benching ~750 mm high deters slugs and snails and promotes
air circulation. For a more complete review of growth-house
design see Richards et al. (1984), Threatened Orchid Recovery
Team (2004) and Nesbitt (2006).
The main purpose of nursery cultivation of Caladenia species
for conservation planning is to provide ex situ collections as a
guarantee against extinction, as seed orchards, and to provide
plants for reintroduction into natural and rehabilitated habitats.
The last purpose can be difficult to achieve, as discussed later
in this paper, and survival rates may be improved by increased
understanding of the phenology of Caladenia plants ex situ.
Initiation of tuber formation is an important stage within the
annual cycle of a
Caladenia plant, and if it is disturbed by
translocation too early in its development, plants may die,
because they require at least 3 months to produce a new
dropper (L. Nesbitt, pers. obs.). Observations during
reintroductions of Caladenia plants in Victoria indicate that
the majority have started to form a new tuber in July (mid-
winter); however, tuber formation has been observed before
this time (R. Thomson, pers. obs.). As earlier reintroductions
would give plants longer time to become established before
the summer dormancy, clarifying the timing of tuber formation
more accurately and determining the most appropriate stage
to reintroduce would be beneficial. Research is needed on the
key factors triggering dropper and tuber formation, and whether
they are triggered only by plant size, by environmental conditions
or by a mixture of both.
The use of nurse plants
An additional ex vitro propagation method for Caladenia taxa is
sowing seed adjacent to mother plants of the same species
(Fig. 1D). This technique maximises the opportunity for seed
to form a mycorrhizal association with a compatible endophyte
and has been a long-standing method used from the earliest days
of orchid propagation (Rasmussen 1995).
Caladenia species can be propagated in the nursery by
germinating seedlings around adult (nurse) Caladenia plants.
Seed is sown on the surface of the potting medium in autumn,
just before the seasonal rains (generally April). Mixing
Caladenia seed with fine sand, a method also utilised during
in situ seed sowing (Batty et al. 2006b; Wright et al. 2007b),
ensures even sowing. Seedling leaves appear 4–5 months
after sowing and can continue to emerge up to 7 months later
(Fig. 5). The majority of seedlings grow within 20 mm of the
nurse plant (L. Nesbitt, pers. obs.), which is likely to indicate
where most of the external mycelium capable of stimulating
germinating seed is located in the medium. This method
generally results in 75% of new seedlings surviving the
first summer dormancy (L. Nesbitt, pers. obs.). Plants
propagated in this fashion take 4 or more years to flower
(L. Nesbitt, pers. obs.).
The nurse plant can be used only across consecutive seasons
if it is appropriately maintained. When Caladenia plants
become overcrowded, it is possibly the increased competition
for nutrients that causes leaves to undergo chlorosis and then
necrosis. By withholding water, seedlings that appear late in
the season are allowed to die to maintain the health of the nurse
380 Australian Journal of Botany M. Wright et al.

plants. Unlike field plants that have greater access to nutrients
via mycorrhizal connections, nursery plants have restricted
access to nutrients because the fungal mycelium is limited in
volume by the restraints imposed by the pot. Interestingly
though, competition among C. hastata seedlings has been
observed under field conditions when seedlings emerge at
high densities (A. Pritchard, pers. obs.). Similar competition
has been observed in vitro with symbiotic Dactylorhiza
majalis seedlings which have slower growth rates when plated
at higher densities (Rasmussen et al. 1989), suggesting that
high seedling density is detrimental in all growth environments.
New nurse pots can be set up to limit competition in the
nursery. Two successful methods are the following:
(1) Seedling clumps. Clumps (1 cm
2
) of 1-year-old seedlings
are pricked out with the adhering surface layer from old
nurse pots in mid-winter (July) and transferred into new pots
with fresh medium. Transferring in July ensures that the
collar has developed and is colonised by mycorrhizal fungi
and it also allows sufficient time (3 months) for a dropper
and tuber to form on the transplanted seedlings. Small
seedlings are chosen because their tiny tubers are near the
surface and the entire seedlings are easily removed, without
damaging larger, adjacent plants. The small holes in the
medium of old nurse pots are filled with fresh potting
medium. The pots with the newly transferred young plants
are more successful as nurse pots in their second growing
season.
(2) The ‘cake slice’ method. Crowded nurse pots are knocked
out during the dormant season and split into two or more
cake-like slices. Each slice containing plants and seedlings is
then placed in the centre of a new pot and filled with fresh
medium (Fig. 6). The orientation of the slice in the new pot
is important to ensure that mycorrhizal fungi remain in the
surface layers and for the correct direction of growth of the
Caladenia plants. When a new shoot emerges from the tuber
it will grow up through the old sheath, which must be
positioned to face the surface of the pot. Any tubers that
have reached the bottom of the pot are pulled off and
replanted ~30 mm deep to prevent them from rotting the
following winter. Caladenia tubers grow deeper each
year, until an optimum depth is reached; however,
standard 15-cm-diameter pots are not of sufficient depth and
the largest tubers reach the bottom of the pot 4–5 years
after germination. Allocasuarina needle mulch is applied
and the pot is watered before going back into the growth
house, although the benefits of this mulch have not been
critically assessed.
The higher plant species found in Caladenia habitat may
also associate with orchid fungi. Warcup (1981) showed that
fungi trapped by Caladenia seeds from sandy soil around
ectomycorrhizal roots of a Melaleuca (paperbark) species were
effective in germination and continued growth. This raises the
possibility that associated higher plants may ‘nurse’ the orchid
with photosynthates via shared fungal linkages. The only direct
evidence for this assumption in Caladenia comes from in vitro
experiments in which C. fulva was grown in tripartite culture
with an effective fungal isolate and eucalypt seedlings; the
eucalypts weighed less (P = 0.08) and their roots were
surrounded by hyphae after only 3 weeks when the orchids
were present in comparison to the control in which the orchids
were absent (Ong 2005). As other members of the fungal order
Sebacinales form tripartite associations with orchids and
neighbouring tree species (Selosse et al. 2002), it would not be
surprising if further evidence of S. vermifera forming such
associations with Caladenia species was found.
Direct seeding, translocation and reintroduction
of Caladenia into natural and rehabilitated habitats
Translocation and reintroduction are not simple procedures and
often fail because the biological and ecological requirements
of taxa are poorly understood (Burgman and Lindenmayer
1998). There has been limited research into factors influencing
successful translocation of terrestrial orchids (Batty 2001).
This section summarises important findings about the
Fig. 5. Caladenia seedlings growing around a ‘nurse’ plant as a result of
seed sowing. Scale bar = 2 cm.
Fig. 6. The ‘cake slice’ method of producing new nurse pots for Caladenia
seed sowing. Slices ofexisting nurse pot are placed in new pots and surrounded
with fresh potting medium. Scale bar = 5 cm.
Caladenia propagation and reintroduction Australian Journal of Botany 381

translocation of Caladenia taxa, much of it originally
unpublished.
The genus Caladenia is among the more difficult to
translocate and reintroduce into natural habitats. In vitro,
C. arenicola seedlings had the poorest tuberisation rates
when transferred into natural habitat, in comparison with
orchids from four other genera (Scade et al. 2006). Batty et al.
(2006b) was successful in reintroducing in vitro seedlings
of the threatened orchid, Thelymitra manginiorum, which have
survived for 5 years after transferral. This success was not
repeated for work with seedlings of C. arenicola, which did
not emerge after the first summer dormancy (Batty et al. 2006b).
The difficulty in translocating Caladenia species may be due
to their specific mycorrhizal associations. Conditions in the
new environment may not be appropriate for the fungi that
were used to germinate seed in vitro .
Selecting an appropriate recipient site for orchid
translocations and reintroductions has been shown to influence
the success of these processes for several terrestrial orchid
species (McKendrick 1995; Scade et al. 2006; Rangel-
Villafranco and Ortega-Larrocea 2007). The environmental
factors that should be considered when selecting orchid
translocation/reintroduction sites include vegetation type and
condition, soil type, condition and leaf litter, climate, aspect,
presence of appropriate pollinator species and presence of
appropriate mycorrhizal fungi. Matching vegetation type, soil
type and aspect are important steps in site selection and are
routinely used in translocation/reintroduction plans for
Caladenia taxa (French 2007; Vlcek 2007a, 2007b, 2007c;
Nevill 2007).
The presence of both appropriate mycorrhizal fungi and
pollinator species is essential for the ultimate success of an
orchid translocation/reintroduction, because these organisms
are required for orchids to naturally complete their life cycles.
Fungal-baiting techniques (Rasmussen and Whigham 1993;
Brundrett et al. 2003) are frequently used to help identify
potential translocation/reintroduction sites (French 2004;
Swarts 2007). There has been a recent increase in the use of
pollinator baiting as described by Phillips et al. (2009) in the
selection of translocation/reintroduction sites (Nevill 2007;
Vlcek 2007b). Site selection for Caladenia translocation/
reintroduction requires careful planning and is likely to
influence the success of these endeavours considerably.
Direct seeding of reintroduction sites, if successful, is
the most cost- and time-effective way of reintroducing an
orchid to natural habitats. With the transfer of seed directly
into the natural environment, the resulting plants will have
formed mycorrhizal associations in situ and may have a
higher likelihood of survival than plants transferred from
artificial conditions (i.e. in vitro seedlings or
ex situ plants).
However, this process consumes a large amount of seed and,
thus, is not the ideal for critically endangered species where
seed is limited.
Caladenia seed has been directly introduced into field sites in
the following three ways:
(1) seed sown directly around the base of an extant adult plant in
much the same fashion as the use of potted ‘nurse’ plants of
Caladenia described earlier (Fig. 1E);
(2) seed sown into sites where mycorrhizal activity has
previously been located using either in situ (Rasmussen
and Whigham 1993) or ex situ baiting (Brundrett et al.
2003) methods (Fig. 1F); and
(3) seed sown into soil inoculated with mycorrhizal fungi
(Fig. 1B).
All three methods have been used with varying success
in Australia.
Sowing seed directly around in situ adult plants has been
highly successful for the Victorian species C. rosella
(C. Beardsell, pers. comm.), C. hastata (Hill and Pritchard
2002) and C. amoena (Sullivan and French 2002), its use
resulting in increases in plant numbers. Intensive site
management, including the preparation of scarified seedbeds,
addition of organic matter to site soil, soil disturbance and
supplementary watering, has been employed and the successes
attributed to these measures. However, because of the
species in these cases being endangered, little seed has been
available for scientific experimentation; it has been difficult to
determine which site-management measures – singly or in
combination – provide optimum conditions for in situ seed
germination and seedling survival. Interestingly, the
application of these methods to a wider range of Victorian
species has not always been successful, with seedlings failing
to emerge. Thus, this method has led to an increase in
plant numbers for several taxa; however, it is difficult to apply
to other Caladenia reintroductions.
Sowing seed where mycorrhizal fungi have been previously
located by in situ fungal-baiting methods has been successful
with C. hastata (A. Pritchard, pers. obs.). In situ fungal baiting
has been employed to aid in selection of seed sowing; however,
for >10 Victorian Caladenia species at reintroduction sites,
limited success has been achieved. The usefulness of in situ
baiting, which utilises retrievable packets to determine the
whereabouts of fungi (Rasmussen and Whigham 1993), has
been questioned in moisture-limited Australian environments
(Huynh 2003; Raleigh 2005; Smith 2006). It is not clear
whether low success rates reflect the extremely limited
distribution of mycorrhizal fungi in Australian orchid habitats
or whether the conditions for orchid-seed germination are
rarely met in these environments. Either would explain the low
or absent recruitment observed in many threatened Caladenia
populations. When in situ baiting has been successful in
Caladenia habitats, the distribution of naturally occurring
mycorrhizal fungi was shown to be patchy and transitory
(Batty et al. 2001; Scade et al. 2006; A. Pritchard, pers. obs.).
The transitory nature of Caladenia mycorrhizal fungi indicates
that seed sowing into sites where fungi have previously been
located may fail as the fungi may not be present at the time
of sowing.
The development of direct sowing of orchid seed into field
sites presents useful opportunities for more cost-effective
translocation of orchid plants, particularly rare and threatened
taxa (Batty et al. 2006b). With the use of millet inoculum, an
endophyte of Caladenia arenicola was effectively introduced
into field sites, with the fungus growing up to 50 cm from
the inoculum source and persisting at the site for the
subsequent three seasons (limit of the study period) (Hollick
382 Australian Journal of Botany M. Wright et al.

et al. 2007). The Hollick study also found that the success of
direct seed-sowing methods is affected by soil moisture, with
supplemental watering increasing seed germination and
seedling survival (Hollick et al. 2007) and highlighting the
critical role of continuous soil moisture for ensuring success
of orchid seed germination under field conditions. Equally, the
role of reliable soil moisture in enabling seedling establishment
is of particular concern in reintroduction programs under
climate-change scenarios of temperate Australia, where
predictions of lower winter rainfall could result in greatly
reduced opportunities for recruitment of Caladenia species.
Studies that have involved sowing Caladenia seed into
soil inoculated with mycorrhizal fungi have met with variable
success, possibly owing to a failure in maintaining continuous
soil moisture (Batty et al. 2006b; Wright 2007). C. arenicola
seed sown into soil inoculated with a mycorrhizal fungus
resulted in densely germinating seedlings; however, these
failed to survive the summer dormancy (Batty et al. 2006b).
A similar study involving sowing C. tentaculata seed into
inoculated soil prepared with a variety of site-management
treatments resulted in much lower germination; however,
seedlings survived the summer dormancy (Wright et al.
2007b). The objective of the latter study was to identify the
most successful site-management technique for enhancing
germination of Caladenia seed. Soil disturbance and the
addition of organic material were shown to have a positive
effect on seed germination; however, a combination of soil
disturbance, addition of organic material and supplementary
watering was the most successful treatment (Wright et al.
2007b).
Seedling overcrowding at seed-sowing sites has led to
several highly successful translocations of C. hastata during
the past 6 years (Table 1). The decision to move these
plants followed observations of loss of vigour (leaf size and
flowering) across several seasons (A. Pritchard, per. obs.),
similar, although not as extreme, as those mentioned above in
crowded ‘nurse’ pots. Again, it is assumed that this reduction
in vigour was due to increased competition by the mycorrhizal
fungi for nutrients. Once the crowed seedlings were separated
and translocated to an alternative site nearby, increases in
average leaf length between 9–17 mm were documented for
two 2006 translocations (K. Vlcek, unpubl. data).
Translocations have also been conducted for Caladenia
populations under threat from development (Fig. 1G). A group
of C. hastata plants were moved to accommodate an aluminium
smelter, with the 10 surviving plants making up a quarter of
the known extant plants before intensive in situ management
(Hill and Pritchard 2002), much of which is detailed in this
section. In all, 22 C. huegelii plants were translocated from the
site of a proposed freeway into areas baited to confirm
the presence of mycorrhizal fungi (Swarts 2007). In both
cases, the plants were moved to sites selected after careful
consideration, and the translocations were conducted as a last
resort to safeguard the plants from destruction. The re-emergence
of the C. huegelii plants has decreased from 77% in the
fi
rst year after translocation to 45% in the third (Swarts 2007).
It is clear that moving extant plants of endangered species
to make way for development should be a last resort. Not only
is the planning and implementation expensive and time
consuming, but also the risk to plant survival is high.
Most researchers have experienced plant losses when
transferring orchids that have been propagated ex situ into
natural habitats (Zettler and McInnis 1993; Ramsay 1994).
Caladenia plants have been transferred both directly from
in vitro containers into field sites (Batty et al. 2006b;
Scade et al. 2006; Swarts 2007) and after being transferred
and grown on in potting media (French 2007; Nevill 2007;
Swarts 2007; Vlcek 2007a, 2007b, 2007c). The transferral of
in vitro symbiotic seedling and/or dormant tubers directly into
habitat has been employed in Western Australian studies
(Batty et al . 2006b; Scade et al. 2006). Batty et al. (2006b)
found that transferring dormant C. arenicola tubers produced
in vitro was more successful than the transferral of actively
growing symbiotic seedlings; however, the plants survived
for two growing seasons only. Transferred Caladenia plants
that were established in potting media have been shown to
survive for much longer periods after transferring to in situ
locations. C. hastata plants treated in this fashion have
survived for up to 6 years after transferral into natural habitat
(Table 2). The high emergence and flowering percentages and
the observation of natural pollination (Table 2) suggest that
these plants are successfully established and have the potential
to recruit naturally in the future.
The successes with C. hastata have led to the preparation
and application of this technique with several other endangered
Victorian Caladenia taxa, including C. amoena, C. cruciformis,
C. calcicola, C. versicola and C. xanthochila (Table 3). Seedlings
have been propagated symbiotically in vitro and then transferred
into potting media in the nursery. The majority were grown for
two seasons (two summer dormancies) in the nursery before
transferral to in situ sites, with the exception of 27 of the
C. ameona plants that had been grown for one season only.
The plants were reintroduced to natural habitat in winter
(June–August). Many of the plants flowered, and natural
pollination was observed for three species (Table 3). However,
the C. xanthochila plants entered dormancy 2 months after they
Table 1. Emergence percentages from five translocations of Caladenia
hastata seedlings sown in situ performed across the past 6 years
Translocation Years since
translocation
No. of
plants
Flowering
% in 2007
Emergence
% in 2008
1. 2002 6 13 46 100
2. 2005 3 22 27 77
3. 2005 3 15 27 73
4. 2006 2 55 2 85
5. 2006 2 13 0 54
Table 2. Emergence percentages from three reintroductions of ex situ
Caladenia hastata plants (in vitro propagated) performed across the past
6 years
Reintroduction Years
since
reintroduction
No.
of
plants
Flowering
%in
2007
% natural
pollination
in 2007
Emergence
% in 2008
1. 2002 6 9 56 0 89
2. 2003 5 14 57 0 71
3. 2004 4 11 64 9 91
Caladenia propagation and reintroduction Australian Journal of Botany 383

were reintroduced, failing to remain above ground until the
flowering season, a phenomena that was also observed in the
natural population (G. Pollard, pers. comm.). Nevertheless,
60% of the translocated C. xanthochila plants emerged after
their first summer dormancy in the recipient site
(Table 3). One year after reintroduction, the initial emergence
of the reintroduced C. cruciformis, C. amoena (2-year olds) and
C. versicolor plants is very encouraging, with the emergence
>70% for all (Table 3). Although a progressive decrease in
re-emergence rate of translocated Caladenia plants after each
summer dormancy has been observed (Batty et al. 2006b),
experiences with C. hastata (Table 2) suggest that survival
rates with plants established in potting media before
translocation can remain high.
Swarts (2007) showed that the length of time Caladenia
plants are grown in the nursery before reintroduction into
natural habitats affects their survival. He found that plants
transferred directly from in vitro containers had 45%
emergence after the first summer dormancy, whereas plants
transferred into potting media for 1 year before transferral in situ
had 65% emergence. Those grown on for an additional year
under nursery conditions had 80% emergence. The majority of
the C. amoena plants that emerged after the summer dormancy
(Table 3) were those that were grown for 2 years under nursery
conditions. It is clear that establishing Caladenia plants in
potting media before transferral into bushland environments
increases their survival rates. Although growing plants for
longer periods under nursery conditions does add to the cost
of their recovery, it remains an important consideration for
endangered taxa.
Conclusions
Propagation, reintroductions and translocations are currently
an important and effective adjunct to conservation planning
for Caladenia taxa. There is a wide range of processes
undertaken to return Caladenia plants into natural and
rehabilitated habitats and to increase their numbers in ex situ
collections. Of these processes, in vitro symbiotic seed
germination, followed by seedling transferral into potting
media is still one of the most effective techniques for
endangered species with limited seed reserves. Not only does
the use of in vitro techniques result in the germination of a
large proportion of seed, but also growing these plants under
nursery conditions for at least 2 years increases their chance of
survival once transferred into natural habitats. Although not
as efficient in their use of seed, ex vitro symbiotic seed
germination, the use of nurse plants and direct seed sowing
into natural habitats provide effective means of increasing the
numbers of Caladenia plants without the use of specialised
equipment and can result in high survival rates. Translocation
of extant adult plants is clearly a last resort, although careful
planning is likely to increase the success of this process.
Recent advances in translocation and propagation techniques
and improvement of existing techniques have increased
plant survival rates and helped to consolidate the recovery of
many endangered Caladenia taxa. Non-agar in vitro substrates
and aerated in vitro containers have increased deflasking
survival rates. The use of cultivated adult Caladenia as
‘nurse’
plants has been successful in increasing the size of ex situ
collections. The application of the ‘slice’ technique of
harvesting mycorrhizal fungi from cultivated plants increases
the potential for using mycorrhizal fungi in Caladenia
propagation. The success of selecting reintroduction/
translocation sites that can support self-sustaining Caladenia
populations can potentially be improved by using pollinator
baiting and baiting for the appropriate mycorrhizal fungi.
Several key areas requiring additional investigation may
further improve the success of the propagation, reintroduction
and translocation of Caladenia taxa. Whether or not
mycorrhizal specificity influences the rarity of Caladenia
needs to be confirmed across a wider range of Caladenia
taxa, and the implications of the present work on the viability
of reintroduced populations of endangered taxa need to be
considered when a single mycorrhizal isolate is used. Studies
are also warranted to investigate whether the functional
variation observed among Caladenia mycorrhizal isolates
in vitro and during nursery establishment also influence the
survival of reintroduced plants. Such information would
help conservation planners determine the level of mycorrhizal
diversity necessary for Caladenia reintroductions.
The number of Caladenia plants available for reintroduction
and ex situ collections could be increased by further optimising
the deflasking process. Studies may identify improved in vitro
support media, flask design, flask gaseous composition, ex vitro
deflasking potting media and nursery environment. Elucidating
the factors that influence tuber development of seedlings
transferred into potting media could potentially increase
deflasking survival rates up to 50%; determining whether there
is a critical tuber size for survival during the summer dormancy
also requires further study.
As preliminary comparisons indicate that ex vitro and in situ
seed-germination methods use considerably more seed than
in vitro methods, improving germination percentages and
seedling survival with these methods could increase their
usefulness in Caladenia conservation programs, and
potentially provide efficient methods requiring less time and
equipment.
The poorer health observed in overcrowded seedlings, both
in the nursery and the field, needs further attention. Is it due to
an exhaustion of nutrients affecting mycorrhizal growth? Can
this be avoided by manipulating fungal growth conditions,
Table 3. Emergence percentages from reintroductions of five
Caladenia species (in vitro propagated plants grown in nursery media)
performed in 2007
Species No. of
plants
Flowering
%in
2007
Natural
pollination
in 2007
Emergence
% in 2008
Caladenia
amoena
93 23 0 69 (81% of
2-year olds)
Caladenia
cruciformis
212 8 1 90
Caladenia
calcicola
66 34 8 77
Caladenia
versicolor
80 19 4 88
Caladenia
xanthochila
240 0 0 60
384 Australian Journal of Botany M. Wright et al.

so that more nutrients are available for it to support increased
numbers of Caladenia plants?
All of the techniques discussed in the present paper exploit
the natural interaction between Caladenia and their mycorrhizal
fungi in the propagation, translocation and reintroduction of
these orchids. It is clear that furthering the understanding
of the growth requirements of the mycorrhizal fungi of
Caladenia, especially how best they can be manipulated to
benefit the growth of Caladenia plants, will considerably
enhance the recovery of many endangered taxa.
Acknowledgements
We acknowledge the following people for information for recent Caladenia
reintroductions and translocations: Kate Vlcek, Gary French, Geoff Nevill,
Pauline Rudolph and David Pitts. We also thank Dr Hanne Rasmussen for her
comments on the manuscript.
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Manuscript received 31 July 2008, accepted 12 January 2009
Caladenia propagation and reintroduction Australian Journal of Botany 387
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