New methods to improve symbiotic propagation of temperate terrestrial orchid seedlings from axenic culture to soil

2006 ◽  
Vol 54 (4) ◽  
pp. 367 ◽  
Author(s):  
A. L. Batty ◽  
M. C. Brundrett ◽  
K. W. Dixon ◽  
K. Sivasithamparam
Keyword(s):  
Mycorrhizal Fungi ◽  
Seedling Survival ◽  
Axenic Culture ◽  
Leaf Size ◽  
Petri Dish ◽  
Terrestrial Orchid ◽  
Symbiotic Germination ◽  
Root Tuber ◽  
Terrestrial Orchids ◽  
Summer Dormancy

This research aimed to improve the success of soil transfer of terrestrial orchid seedlings after symbiotic germination in the laboratory. Three native Western Australian terrestrial orchids (Caladenia arenicola Hopper & A.P.Brown, Diuris magnifica D.L.Jones and Thelymitra crinita Lindley) were used in this study. The key to improved seedling survival on transfer to soil was found to be the use of an intermediate stage between the Petri dish and soil where larger seedlings were grown in an axenic environment with controlled humidity. There was no apparent benefit of pre-inoculating potting medium with appropriate strains of mycorrhizal fungi for subsequent growth of symbiotic seedlings under glasshouse conditions. Initial survival of seedlings in the glasshouse was high. However, some seedlings failed to produce tubers (from modified roots or droppers) necessary for plant survival through the summer dormancy period, and this caused survival to decrease to 40–60% of the glasshouse-grown seedlings in the first year. The initiation of tubers on droppers by C. arenicola was inversely correlated with leaf size, with smaller plants more likely to form tubers. This suggests that leaves and tubers were competing for resources. However, larger seedlings that did tuberise had larger tubers that were more likely to survive summer dormancy. There was no correlation between leaf size and root tuber size in D. magnifica, but the number of tubers produced was greatest in larger plants. As with C. arenicola, plants of D. magnifica and T. crinita with larger tubers were more likely to survive summer dormancy. Methods developed in this study enable the production of both actively growing symbiotic seedlings and dormant tubers which improve the success of translocation of laboratory-grown terrestrial orchids to field sites.

In situ symbiotic seed germination and propagation of terrestrial orchid seedlings for establishment at field sites

2006 ◽  
Vol 54 (4) ◽  
pp. 375 ◽  
Author(s):  
A. L. Batty ◽  
M. C. Brundrett ◽  
K. W. Dixon ◽  
K. Sivasithamparam
Keyword(s):  
Mycorrhizal Fungi ◽  
Seedling Survival ◽  
Natural Habitat ◽  
Survival Rates ◽  
Growing Season ◽  
Individual Species ◽  
Symbiotic Germination ◽  
Terrestrial Orchids ◽  
Eucalypt Woodland

The establishment of five species of temperate terrestrial orchids (Caladenia arenicola Hopper & A.P.Brown, Diuris magnifica D.L.Jones, D. micrantha D.L.Jones, Pterostylis sanginea D.LJones & M.A.Clem. and Thelymitra manginiorum ms) in natural habitat through in situ seed sowing, or by planting of seedlings and dormant tubers, was evaluated. Seed of the Western Australian temperate terrestrial taxa, Caladenia arenicola and Pterostylis sanguinea germinated best when sown into soil inoculated with mycorrhizal fungi at field sites but failed to develop the tubers necessary for surviving summer dormancy. However, seedling survival improved when actively growing symbiotic seedlings were transferred to natural habitat during the growing season. Caladenia arenicola and P. sanguinea seedlings survived the initial transfer to field sites but only P. sanguinea survived into the second growing season. Highest survival was obtained by translocating dormant tubers of C. arenicola and Diuris magnifica, with D. magnifica persisting at the site 5 years after translocation. However, outplanted C. arenicola survived for only 2 years. In another trial, where seedlings and dormant tubers of a rare orchid Thelymitra manginiorum were translocated into eucalypt woodland, 18% persisted after 5 years. The rare orchid D. micrantha exhibited the highest survival rates, with greater than 80% of tubers surviving 5 years after transfer of mature dormant tubers to field sites. This study highlights the benefit of using optimised methods for seedling production by symbiotic germination and nursery growth to produce advanced seedlings or dormant tubers to maximise the survival of translocated plants. It also demonstrates the need to consider different strategies when dealing with individual species.

scholarly journals SEM and PCR study of mycorrhizal fungi isolated from the Australian terrestrial orchid: Prasophyllum

Lankesteriana ◽  
2015 ◽  
Vol 7 (1-2) ◽  
Author(s):  
Emily McQualter ◽  
Rob Cross ◽  
Cassandra McLean ◽  
Pauline Ladiges
Keyword(s):  
Mycorrhizal Fungi ◽  
Terrestrial Orchid ◽  
Current Conservation ◽  
Terrestrial Orchids

Most members of the genus Prasophyllum (Leek Orchids) are threatened and restricted in distribution in Australia. Prasophyllum species are obligate mycotrophic plants and current conservation proto- cols for terrestrial orchids in Australia require propa- gation with symbiotic mycorrhizal fungi. Unfortunately there is a paucity of knowledge regard- ing the mycosymbiont in this genus, hampering con- servation and re-introduction efforts.

scholarly journals Symbiotic germination of threatened Australian terrestrial orchids and the effect of nursery potting media on seedling survivals

Lankesteriana ◽  
2015 ◽  
Vol 7 (1-2) ◽  
Author(s):  
Magali Wright ◽  
Zoe Smith ◽  
Richard Thomson ◽  
Rob Cross
Keyword(s):  
Research And Development ◽  
Mycorrhizal Fungi ◽  
Ex Situ ◽  
Symbiotic Germination ◽  
Terrestrial Orchids ◽  
Potting Media ◽  
Victorian Department ◽  
The University

Since the early 1990s, the RBG has contributed to the conservation of Victoria’s Endangered orchids through its ex situ propagation program. Working cooperatively with the Victorian Department of Sustainability and Environment (DSE), the Melbourne Zoo, the Australasian Native Orchid Society, The University of Melbourne, RMIT University and Parks Victoria, research and development has lead to a greater understanding of Victoria’s terrestrial orchids and their associated mycorrhizal fungi, and assisted in the implementation of Recovery Plans. 

scholarly journals A Proposed Framework for Efficient and Cost-Effective Terrestrial Orchid Conservation

2020 ◽  
Author(s):  
Mark Brundrett
Keyword(s):  
Mycorrhizal Fungi ◽  
Habitat Management ◽  
Wild Plants ◽  
Sterile Culture ◽  
Seed Collection ◽  
Orchid Conservation ◽  
Symbiotic Germination ◽  
Terrestrial Orchids ◽  
Conservation Projects

This paper presents a comprehensive and adaptive framework for orchid conservation programs illustrated with data from published and unpublished case studies. There is a specific focus on West Australian terrestrial orchids, but many of the approaches have universal relevance. Aspects of the framework include (1) setting appropriate objectives, (2) establishing effective collaborations between scientists, volunteers and regulators to fill knowledge and funding gaps, (3) use of survey and demographics data to determine extinction risks and management requirements for species, (4) effective habitat management to overcome threats such as grazing, (5) finding potential new habitats by modelling climate and site data, (6) investigating the effectiveness of pollinators and (7) using seed baiting to detect mycorrhizal fungi. The relative cost and effectiveness of different methods used to propagate orchids for translocation are compared. Methods known to be successful, in order of complexity, include placement of seed in situ, vegetative propagation, symbiotic germination in non-sterile organic matter, symbiotic germination in sterile culture, asymbiotic sterile germination and clonal division in tissue culture. These form a continuum of complexity, cost, time required, faculties needed, as well as the capacity to maintain genetic diversity and produce seedlings preadapted to survive in situ. They all start with seed collection and lead to seed storage, living collections used as tuber banks and seed orchards, as well as translocation for conservation. They could also lead to commercial availability and sustainable ecotourism, both of which are needed to reduce pressure on wild plants. Overall, there has been a strong preference to use relatively complex, expensive and time-consuming methods for orchid conservation, despite evidence that simpler approaches have also been successful. These simpler methods, which include in situ seed placement and non-sterile germination on inorganic substrates, should be trialled in combination with more complex orchid propagation methods as part of an adaptive management framework. It is essential that orchid conservation projects harness the unique biological features of orchids, such as abundant seed production and mycorrhizal fungi which are far more widespread than their hosts. This is necessary to increase the efficiency and coverage of recovery actions for the largest and most threatened plant family.

scholarly journals Propagation and reintroduction of Caladenia

2009 ◽  
Vol 57 (4) ◽  
pp. 373 ◽  
Author(s):  
Magali Wright ◽  
Rob Cross ◽  
Kingsley Dixon ◽  
Tien Huynh ◽  
Ann Lawrie ◽  
...  
Keyword(s):  
Conservation Planning ◽  
Mycorrhizal Fungi ◽  
Ex Situ ◽  
Terrestrial Orchid ◽  
Natural Habitats ◽  
Nurse Plants ◽  
Mycorrhizal Associations ◽  
Symbiotic Germination ◽  
Growing Conditions

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.

Orchidaceous rhizoctonias in pot cultures of vesicular–arbuscular mycorrhizal fungi

1985 ◽  
Vol 63 (7) ◽  
pp. 1329-1333 ◽  
Author(s):  
P. G. Williams
Keyword(s):  
Mycorrhizal Fungi ◽  
Arbuscular Mycorrhizal ◽  
Pasture Legumes ◽  
Affected Plant ◽  
Symbiotic Germination ◽  
Terrestrial Orchids ◽  
Vesicular Arbuscular ◽  
Orchid Seed ◽  
Sebacina Vermifera ◽  
Pot Culture

The paper reports that previously undescribed, sterile, septate fungi (Rhizoctonia) with affinity to and attributes of orchid mycorrhizal fungi, commonly occur in pot cultures of vesicular–arbuscular (V–A) mycorrhizal fungi. Seventeen pot cultures of V–A endophytes from several sources were studied. The endophytes included unidentified organisms as well as species of Glomus, Acaulospora, and Gigaspora. A Rhizoctonia was present in every pot culture. In different cases, Rhizoctonia isolates were obtained from sporelike cells in intramatrical vesicles, extramatrical hyphae, and chlamydospores or roots of pot culture plants. In pure culture, the rhizoctonias formed pale or yellow–brown, submerged colonies composed of narrow, irregularly septate hyphae. Monilioid hyphae and terminal or intercalary, spherical chlamydospores about 12 μm in diameter developed in older mycelia. Fruiting experiments by J. H. Warcup indicated that the teleomorph of three Rhizoctonia isolates is related to Sebacina vermifera Oberwinkler, a mycorrhizal endophyte of certain Australian terrestrial orchids. Positive tests for symbiotic germination of orchid seed with one isolate are described. Pasture legumes and ryegrass plants were inoculated with mycelia of Rhizoctonia strains in the presence or absence of V–A mycorrhizal fungi. Inoculation affected plant growth only when V–A mycorrhizal fungi were present: in steamed soil containing residual inoculum of a V–A endophyte, the growth response following infection by the V–A endophyte occurred in inoculated plants several weeks earlier than in uninoculated plants; in different natural soils, inoculation increased, decreased, or had no effect on growth, depending on the strain of Rhizoctonia used.

scholarly journals Methods for ex Vitro Germination of Australian Terrestrial Orchids

HortScience ◽  
1995 ◽  
Vol 30 (7) ◽  
pp. 1445-1446 ◽  
Author(s):  
Letitia Quay ◽  
Jen A. McComb ◽  
Kingsley W. Dixon
Keyword(s):  
Mycorrhizal Fungi ◽  
Mycorrhizal Fungus ◽  
Ex Vitro ◽  
Terrestrial Orchid ◽  
Orchid Species ◽  
Terrestrial Orchids ◽  
Leaf Mulch ◽  
Seed Sowing ◽  
Protocorm Formation

Seeds of two Australian terrestrial orchid species (Caladenia latifolia R.Br. and Diuris magnifica D. Jones) were germinated in a potting mix of Allocasuarina fraseriana (Miq.) L. Johnson leaf mulch and perlite (1:1). The potting mix was irradiated (7 Gy for 14 hours), steam pasteurized (70C for 30 minutes) or nontreated, and inoculated with the appropriate mycorrhizal fungus for each species, a sterile red fungus (SRF), or both. Protocorm formation and green shoots were evident at 8 and 10 weeks, respectively, after seed sowing. The highest mean number of seedlings was 84 for C. latifolia and 234 for D. magnifica per 270-ml container in pasteurized potting mix inoculated with mycorrhizal fungi and SRF. Shoots were longest after 20 weeks (28 mm for C. latifolia and 52 mm for D. magnifica, respectively) in pasteurized potting mix inoculated with mycorrhizal fungi only. Germination was absent in control treatments without mycorrhizal fungi; with SRF only; or in nonsterile potting mix with mycorrhizal fungi, SRF, or both.

scholarly journals Tulasnella calospora (UAMH 9824) retains its effectiveness at facilitating orchid symbiotic germination in vitro after two decades of subculturing

2021 ◽  
Vol 62 (1) ◽  
Author(s):  
Lawrence W. Zettler ◽  
Caleb J. Dvorak
Keyword(s):  
Mycorrhizal Fungi ◽  
Terrestrial Orchid ◽  
Orchid Species ◽  
The North ◽  
Symbiotic Germination ◽  
Germinating Seeds ◽  
Tulasnella Calospora

Abstract Background The technique of symbiotic germination—using mycorrhizal fungi to propagate orchids from seed in vitro—has been used as one method to cultivate orchids in North America and abroad for > 30 years. A long-held assumption is that mycorrhizal fungi used for this purpose lose their effectiveness at germinating seeds over time with repeated subculturing. Results We provide evidence for the lingering efficacy of one particular strain of Tulasnella calospora (266; UAMH 9824) to stimulate seed germination exemplified by the North American terrestrial orchid, Spiranthes cernua, as a case study. This fungus was originally acquired from roots from Spiranthes brevilabris in 1999 and sub-cultured during the two decades since. Seeds inoculated with the fungus in vitro developed to an advanced protocorm stage after 16 days, and leaf elongation was pronounced after 42 days. In a pilot study, seedlings co-cultured with Tulasnella calospora 266 were deflasked after 331 days and later transferred to soil under greenhouse conditions where they eventually initiated anthesis. During the course of two decades, seeds of 39 orchid species, cultivars and hybrids spanning 21 genera, germinated in vitro co-cultured with Tulasnella calospora 266. These orchids included temperate terrestrials and tropical epiphytes alike. Conclusions The sustained effectiveness of this fungus is noteworthy because it argues against the concept of mycorrhizal fungi losing their symbiotic capability through prolonged subculturing. This study serves as an example of why in situ habitat preservation is essential for the conservation of orchids as a source of potentially useful mycorrhizal fungi.

scholarly journals Finding a mycorrhizal fungus for reintroductions of the threatened terrestrial orchid Diuris fragrantissima

Lankesteriana ◽  
2015 ◽  
Vol 7 (1-2) ◽  
Author(s):  
Zoe Smith ◽  
Elizabeth James ◽  
Cassandra McLean
Keyword(s):  
Mycorrhizal Fungi ◽  
Mycorrhizal Fungus ◽  
Single Site ◽  
Critically Endangered ◽  
Terrestrial Orchid ◽  
Important Prerequisite ◽  
Terrestrial Orchids

Australian terrestrial orchids rely on associations with suitable mycorrhizal fungi for in situ seed germi- nation and establishment, an important prerequisite for self sustaining populations. Finding an appropri- ate mycorrhizal fungus is therefore imperative to suc- cessful reintroductions. Reintroductions have been planned to conserve the terrestrial orchid Diuris fra- grantissima, which is Critically Endangered in Victoria, Australia, having been reduced to less than 25 plants at a single site. 

A “paper bridge” system to improve in-vitro propagation of arbuscular mycorrhizal fungi

Botany ◽  
2010 ◽  
Vol 88 (6) ◽  
pp. 617-620 ◽  
Author(s):  
Yolande Dalpé ◽  
Sylvie Seguin
Keyword(s):  
Arbuscular Mycorrhizal Fungi ◽  
Mycorrhizal Fungi ◽  
Arbuscular Mycorrhizal ◽  
Petri Dish ◽  
Efficient System ◽  
Arbuscular Mycorrhizal Fungal ◽  
Slow Growing ◽  
Paper Bridge ◽  
Bridge System

The in-vitro culture of arbuscular mycorrhizal fungi on excised roots, especially when performed on bi-compartmented Petri dishes, has proven to be an efficient system for the production of root-free fungal material. However, even after the contact between fungal hyphae and the excised roots in the proximal root compartment has occurred, up to several weeks may be required for the fungal runner hyphae to cross the median Petri dish wall and reach the distal fungal compartment. This delay is particularly long for the cultivation of slow-growing strains that usually colonize the substrate less aggressively. The delay is due to the difficulty the runner hyphae have in crossing the median Petri dish wall that separates compartments. To facilitate the passage of the fungus across the median wall, a “paper bridge” system has been devised and tested with a number of arbuscular mycorrhizal fungal strains. This method substantially accelerated fungal propagation and simplified the manipulations necessary. The proposed paper-bridge system is described and its advantages discussed.

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