Asymbiotic and Symbiotic Seed Germination for Eulophia Alta

Plant Cell Tiss Organ Cult (2007) 90:313–323 DOI 10.1007/s11240-007-9270-z

ORIGINAL PAPER

Asymbiotic and symbiotic seed germination of Eulophia alta (Orchidaceae)—preliminary evidence for the symbiotic culture advantage

Timothy R. Johnson Æ Scott L. Stewart Æ Daniela Dutra Æ Michael E. Kane Æ Larry Richardson

Received: 29 March 2007 / Accepted: 7 July 2007 / Published online: 31 July 2007 Springer Science+Business Media B.V. 2007

Abstract Eulophia alta (Linnaeus) Fawcett & leaf-bearing protocorms were observed on Rendle seeds collected from the Florida Panther PhytoTechnology Orchid Seed Sowing Medium National Wildlife Refuge (Collier County, FL; (0.8%) and Vacin & Went Modified Orchid Medium FPNWR) were used in a screen of five asymbiotic (1.3%) than other media tested. Of the fungi tested, orchid seed germination media to determine their one fungal isolate (Ealt-396) promoted germination effectiveness in promoting germination and proto- to 69.0%, two isolates promoted germination to less corm development. In addition, 10 fungal isolates than 0.75% and did not support further protocorm collected from the roots of E. alta at sites in the development, and eight isolates did not support FPNWR, Highlands County (FL), and Goethe State germination. Seeds co-cultured in darkness with Forest (Levy County, FL; GSF), and a fungal isolate Ealt-396 grew more rapidly than asymbiotic seed- from the roots of Spiranthes brevilabris collected lings following germination. In addition, co-cultured from GSF were screened for their effectiveness at (=symbiotic) seedlings continued to develop more promoting in vitro symbiotic germination of E. alta rapidly than asymbiotic seedlings upon transfer to 16/ seeds. After 18 weeks asymbiotic culture, seeds sown 8 h light/dark photoperiod. Symbiotic seed culture of on PhytoTechnology Orchid Seed Sowing Medium E. alta may be a more desirable method of propa- germinated to a higher percentage (87.9%) and had a gation since protocorms develop more rapidly than higher percentage of protocorms with developing seeds sown on asymbiotic media. Symbiotic seed- protomeristems (32.7%) than seeds cultured on lings may be more appropriate for reintroduction to Knudson C, Malmgren Modified Terrestrial Orchid natural areas than asymbiotic seedlings since symbi- Medium, ½-strength Murashige & Skoog, or Vacin & otic seedlings could serve to inoculate soils with a Went Modified Orchid Medium. Significantly more germination promoting mycobiont.

Keywords Orchid Á Seed germination Á Native Á T. R. Johnson (&) Á S. L. Stewart Á D. Dutra Á Conservation Á Terrestrial Á Mycorrhizae Á Wild coco M. E. Kane Department of Environmental Horticulture, University of Florida, PO Box 110675, Gainesville, FL 32611, USA Abbreviations e-mail: timjohn@ufl.edu 1/5- 1/5-strength Potato dextrose agar PDA L. Richardson ½MS ½-strength Murashige & Skoog Florida Panther National Wildlife Refuge, U.S. Fish and Wildlife Service, 3860 Tollgate Blvd., Suite 300, Naples, CMA Corn meal agar FL 34114, USA FPNWR Florida Panther National Wildlife Refuge 123 314 Plant Cell Tiss Organ Cult (2007) 90:313–323 dd Distilled deionized situations, seedlings cultured symbiotically can serve KC Knudson C as both plant material and a source of mycobiont L/D Light/dark inoculum for reintroduction efforts. Introducing a MM Malmgren Modified Terrestrial Orchid compatible mycobiont into a site may facilitate the Medium establishment of self-sustaining populations. Addi- P723 PhytoTechnology Orchid Seed Sowing tionally, symbiotic seed germination may be a more Medium desirable means of producing orchids if symbiotic TZ Tetrazolium seedlings develop more rapidly than asymbiotic VW Vacin & Went Modified Orchid Medium seedlings. Eulophia is a pantropical genus with African affinities containing approximately 200 species worldwide (Brown 2005). Only one species, Eulo- Introduction phia alta (Linnaeus) Fawcett & Rendle (common name wild coco; Fig. 1), occurs in North America, Seed propagation represents the most efficient where it is found from southern Georgia to southern method of propagating native terrestrial orchids Florida. Eulophia alta populations are typically found (Stewart and Kane 2006a). Symbiotic seed germina- in moderately wet, grass-dominated roadsides or near tion can be a cumbersome process; root samples the edges of forested sites dominated by live oak must be collected from which many fungi are often (Quercus virginianus), saw palmetto (Serenoa re- isolated. Fungi must then be identified and screened pens), Sabal palmetto, and slash pine (Pinus eliottii). for growth promoting strains. Asymbiotic seed While not currently listed as a rare species, urban germination can be a more straight forward process development throughout its range is threatening the since mycobionts need not be isolated to germinate species’ current habitat. Because E. alta grows in seeds of orchid taxa. However, there are circum- concentrated populations (=locally abundant), habitat stances when symbiotically germinated seedlings are degradation at E. alta populated sites could have desired or necessary. Populations of orchids that are long-term ramifications for the future of this species. established with asymbiotic seedlings remain depen- Common orchid taxa may serve as models for dent on naturally occurring fungal symbionts for developing reintroduction programs, which can then seedling recruitment (Zettler 1997b). Due to possible be applied to threatened and endangered taxa. The ecological changes at historic orchid locales, a target first step in this process is establishing efficient orchid species’ mycobionts may not be present at a propagation protocols to produce plants for subse- site if the orchid itself is not present. In these quent experimentation. Little information is available

Fig. 1 Eulophia alta. (a) Single flower (scale bar = 1.0 cm). (b) Vegetative plant of E. alta in native habitat. (c) Eulophia alta inflorescence (scale bar = 4.0 cm). (d) Typical habitat of E. alta on the Florida Panther National Wildlife Refuge (Collier County, FL)

123 Plant Cell Tiss Organ Cult (2007) 90:313–323 315 concerning seed germination of Eulophia species, Table 1 Nutrient composition of germination media used in and no information exists on the asymbiotic or the asymbiotic seed germination of Eulophia alta symbiotic seed germination requirements of E. alta. KC MM P723 ½MS VW The objectives of this research were (1) to evaluate the potential of asymbiotic and symbiotic seed Macronutrients (mM) propagation for the production of E. alta seedlings, Ammonium 13.82 – 5.15 10.31 7.57 (2) to identify germination-promoting mycobionts, Calcium 2.12 0.24 0.75 1.50 1.93 and (3) to document the germination and seedling Chlorine 3.35 – 1.50 1.50 – development of this species. The data collected Magnesium 1.01 0.81 0.62 0.75 1.01 from this study will be used to propagate plants for Nitrate 10.49 – 9.85 19.70 5.19 further investigations of orchid reintroduction Potassium 5.19 0.55 5.01 10.02 7.03 methods. Phosphate 1.84 0.71 0.31 0.63 3.13 Sulfate 4.91 0.92 0.71 0.86 4.92 Sodium – 0.20 0.10 1.51 0.20 Materials and methods Micronutrients (lM) Boron – – 30.00 50.00 – Seed source and sterilization Cobalt – – 0.03 0.11 – Copper – – 0.03 0.10 – Eulophia alta seeds were collected from the Florida Iron 90.00 100.00 50.00 50.00 100.00 Panther National Wildlife Refuge (FPNWR; Collier Iodine – – 1.20 2.50 County, FL) on 13 December 2005. Only seeds from Manganese 30.00 10.00 30.00 37.90 30.00 dehisced capsules were collected to ensure that they Molybdenum – – 26.00 0.52 – were mature. Seeds were stored at 23 ± 2C over Zinc – – 9.20 30.00 – silica gel desiccant until capsules ruptured, then Vitamins (mg/l) collected and stored at À10C for 15 weeks. Seeds Biotin – 0.05 – – – were transferred to a sterilized scintillation vial and Casein hydrolysate – 400.00 – – – surface sterilized for 45 s in a solution containing Folic acid – 0.50 – – – 5 ml absolute ethanol, 5 ml 6.0% NaOCl, and 90 ml Glycine – 2.00 – – – sterile distilled deionized (dd) water. Seeds were myo-Inositol – 100.00 100.00 – – rinsed three times with sterile dd water after surface Nicotinic acid – – 1.00 – – sterilization. Seeds were then suspended in sterile dd Peptone – – 2000.00 – – water. Solutions were removed from the vial with Pyridoxine – – 1.00 – – sterilized Pasture pipettes that were used only once. Thiamine – – 10.00 – – Total N (mM) 24.31 n/a unknown 30.01 12.76 Asymbiotic media survey NH4:NO3 1.32 n/a 0.52 0.52 1.46 Five nutrient media (Table 1) were assayed for their KC—Knudson C, MM—Malmgren Modified Terrestrial Orchid Medium, P723—PhytoTechnology Orchid Seed effectiveness in promoting germination and subse- Sowing Media, ½MS—½-strength Murashige & Skoog, quent development of E. alta seeds. All media were VW—Vacin & Went Orchid Medium prepared and modified by PhytoTechnology Labora- tories, Inc. (Shawnee Mission, KS): Knudson C (KC; basal media: 0.8% TC1 agar was added to KC, 2.0% Knudson, 1946), Malmgren Modified Terrestrial sucrose was added to both MM and ½MS. Media pH Orchid Medium (MM; Malmgren, 1996), PhytoTech- were adjusted to 5.8 using 0.1 N KOH prior to nology Orchid Seed Sowing Medium (P723), ½- autoclaving for 20 min at 121C and 117.7 kPa. strength Murashige & Skoog (½MS; Murashige and Sterilized media were dispensed as 30 ml aliquots Skoog, 1962), and Vacin & Went Modified Orchid into 9 cm diameter Petri plates (Fisher Scientific, Medium (VW; Vacin and Went, 1949). To standard- Pittsburg, PA). Surface sterilized seeds were then ize the sucrose and agar concentrations among media inoculated near the center of each plate using a sterile tested, the following modifications were made to bacterial inoculating loop before the plates were 123 316 Plant Cell Tiss Organ Cult (2007) 90:313–323 sealed with a single layer of Nescofilm (Karlan characteristics resembling previously described endo- Research Products, Santa Rosa, CA). Approximately phytic orchid mycobionts (Currah et al. 1987, 1997; 60 seeds were sown onto each plate (average seeds/ Richardson et al. 1993; Stewart et al. 2003; Zelmer plate: 59.4). Eight replicate plates were prepared for et al. 1996; Zettler 1997a, b). An additional isolate each treatment. Plates were stored at 22 ± 3Cin collected from the roots of Spiranthes brevilabris darkness for 18 weeks. Light was excluded by (Sbrev-266) in Levy County, FL was also used. wrapping plates in two layers of aluminum foil. Seeds Seeds were surface sterilized as previously de- were exposed to short periods of light (<20 min) scribed and sown onto the surface of a sterile during scoring. Plates were scored at two week 1 · 4 cm strip of Whatman No. 4 filter paper (W & intervals with the aid of a dissection stereoscope. R Balston Ltd., England) (average seeds/plate: 56.6) placed into Petri plates containing ca. 30 ml oat meal Fungal isolation and identification agar (OMA; Dixon 1987). Medium was then inocu- lated with a 1 cm3 block of 1/5PDA containing the The methods of Stewart and Zettler (2002) were actively-growing hyphae of one of the fungal isolates. modified for fungal isolation. Vegetative plants were Eight replicate plates were prepared for each myco- collected with their root systems intact, wrapped in biont treatment. Plates containing seeds but no fungus moist paper towel, and transported to the laboratory. served as the control. Root segments (ca. 5 cm in length) were rinsed in cold tap water for 10 min, then surface cleansed for one Germination scoring and statistical analysis min in a solution of 5 ml absolute ethanol, 5 ml 6.0% NaOCl, and 90 ml sterile dd water. Root segments Germination and seedling development for both the were macerated in Petri plates, then suspended in asymbiotic and symbiotic experiments were scored sterilized molten corn meal agar (CMA; Sigma- on a scale of 0–5 (Table 3; Stewart and Zettler 2002). Aldrich, St. Louis, MO) supplemented with 50 mg Percentage of seedlings in each stage was calculated lÀ1 novobiocin sodium salt (Sigma-Aldrich, St. Louis, for each treatment by dividing the number of seeds in MO). Plates were incubated in darkness for three days each stage by the total number of viable seeds in each at 25C. After incubation, hyphal tips from actively sample. Germination and developmental data were growing isolates were subcultured onto 1/5-strength analyzed with SAS v 9.1 (SAS 2003) using general potato dextrose agar (1/5PDA): 6.8 g PDA (BD linear model procedures and Waller mean separation Company, Sparks, MD), 6.0 g granulated agar (BD (a = 0.05). Data were arcsine transformed prior to Company, Sparks, MD), and 1 l dd water. analysis to normalize variability. Mycobiont characterization and tentative identifi- cation followed the methods outlined by Zelmer and Results Currah (1995), Currah et al. (1987, 1990, 1997), and Zelmer et al. (1996). Hyphal and cultural morpho- Asymbiotic germination logies were assessed visually and microscopically using a Nikon Labophat-2 light microscope (Nikon Tetrazolium (TZ) viability testing (Lakon 1949) USA, Melville, NY). Monilioid cells were surveyed completed prior to experimentation indicated that using the microscopic equipment mentioned previ- E. alta seeds collected from FPNWR were 59.7% ously. Fungal staining procedures followed those viable compared to observed seed germination per- described by Phillips and Hayman (1970), modified centages that ranged from 19.6% to 87.9% after by using acid fuchsin stain (Stevens 1974). 18 weeks culture (Fig. 2). Visible contamination was limited to 2.5% of all replicates. No fewer than 4 Symbiotic fungi survey replicates remained in all treatments. Seed germination was first scored 10 weeks after Eleven fungal isolates (Table 2) were screened for sowing (Fig. 3a). At this time, asymbiotic germina- their effectiveness at promoting E. alta symbiotic seed tion of E. alta seeds cultured in the dark (0/24 h L/D) germination in vitro. All E. alta isolates were col- on P723 (56.1%) was significantly greater than seeds lected from two sites in Florida, and all demonstrated cultured on KC (11.4%), MM (26.3%), ½MS (1.6%), 123 Plant Cell Tiss Organ Cult (2007) 90:313–323 317

Table 2 Fungi used in the symbiotic germination of Eulophia alta Isolate Host Identification Collection information

Sbrev-266 Spiranthes brevilabris Epulorhiza repens Collected 30 April 1999 from GSF Ealt-385 Eulophia alta Epulorhiza sp. Collected 24 July 2005 from FPNWR Ealt-386 Eulophia alta Sclerotinia sp Collected 24 July 2005 from FPNWR Ealt-387 Eulophia alta Epulorhiza sp. Collected 24 July 2005 from FPNWR Ealt-389 Eulophia alta Epulorhiza sp. Collected 24 July 2005 from FPNWR Ealt-390 Eulophia alta Fusarium sp. Collected 24 July 2005 from FPNWR Ealt-391 Eulophia alta Fusarium sp. Collected 24 July 2005 from FPNWR Ealt-392 Eulophia alta Fusarium sp. Collected 24 July 2005 from FPNWR Ealt-395 Eulophia alta Epulorhiza sp. Collected 24 July 2005 from AVON Ealt-396 Eulophia alta Armillaria sp. Collected 24 July 2005 from AVON Ealt-397 Eulophia alta Epulorhiza sp. Collected 24 July 2005 from AVON GSF—Goethe State Forest (Levy County, FL), FPNWR—Florida Panther National Wildlife Refuge (Collier County, FL), AVON— Avon Park (Highlands County, FL)

Table 3 Developmental stages of asymbiotically and symbi- higher percentage of seeds cultured on MM devel- otically cultured Eulophia alta seeds and seedlings oped to Stage 2 protocorms (85.3%) than seeds Stage Description cultured on all other media, very few protocorms cultured on MM developed beyond Stage 3. A limited 0 Hyaline embryo, testa intact number of protocorms developed to Stage 4 (true leaf 1 Embryo swollen, rhizoids present (=germination) present) on P723 (0.8%), ½MS (0.2%), and VW 2 Continued embryo enlargement, testa ruptured (1.3%) by week 18. 3 Appearance of protomeristem 4 Emergence of first leaf Fungal identification 5 Elongation of first leaf and further development Ten mycobionts were recovered from the roots of and VW (11.1%; Fig. 2). Seeds sown on P723 also vegetative plants of E. alta (Table 2). Six mycobionts exhibited a significantly higher percentage of Stage 2 were identified as Basidiomycotina species, while the (54.7%) and Stage 3 (2.1%) protocorms than other remaining E. alta mycobionts were identified as treatments. Ascomycotina species. Isolates Ealt-385, 387, 389, After 14 weeks culture, Stage 3 protocorms were 395, and 397 were assigned to the anamorphic genus present in all treatments; however, a significantly Epulorhiza Moore (Moore 1987), while isolate Ealt- higher percentage of Stage 3 protocorms (7.1%) and 386 was identified as a species of Sclerotinia Fckl. germinated seeds (93.8%) were observed on P723 and isolates Ealt-390, 391, and 392 were identified as (Fig. 2). Seeds cultured on MM exhibited a signifi- species of Fusarium Link ex Gray. Eulophia alta cantly higher germination percentage (81.5%) and isolate Ealt-396 was identified as a species of percentage of Stage 2 seedlings (80.9%) than seeds Armillaria (Fr:Fr) Staude and accessioned into the cultured on KC, ½MS, or VW. No significant differ- University of Alberta Microfungus Herbarium as ence in the percentage of Stage 3 protocorms was found UAMH 10807. Isolate Sbrev-266 (UAMH 9824), among KC, MM, or ½MS treatments. A significantly originating from the roots of the Florida terrestrial higher percentage of Stage 3 protocorms were observed orchid Spiranthes brevilabris, was previously identi- on VW (4.5%) compared to KC, MM, and ½MS fied as a strain of Epulorhiza repens (Bernard) Moore treatments (all less than 0.5%), although more Stage 2 (Moore 1987; Stewart et al. 2003). This isolate has protocorms were observed in MM treatments than in been shown to support the germination of epiphytic VW treatments (80.9% and 52.3%, respectively). (Zettler et al. 2007) and terrestrial orchids (Stewart By week 18, total germination ranged from 19.6% and Kane 2006b; Stewart and Zettler 2002) native to (½MS) to 87.9% (P723; Fig. 2). While a significantly Florida. 123 318 Plant Cell Tiss Organ Cult (2007) 90:313–323

Fig. 2 Effects of culture 100 (18 Weeks) media on the asymbiotic P723 D germination and seedling KC D development of Eulophia 80 MM alta after 10, 14, and 1/2 MS 18 weeks in vitro culture in ) VW

% CD dark conditions. Histobars (

60 n

in each stage with the same o C i

t C letter are not significantly a n

i B

different (a = 0.05). KC— m r 40 B Knudson C, MM— e G D Malmgren Modified Terrestrial Orchid Medium, Phyto 20 A P723— Technology A A Orchid Seed Sowing Media, C AB AB ½MS—½-strength A A AB A B Murashige & Skoog, VW— 0 Vacin & Went Orchid 100 (14 Weeks) Medium E D D 80 ) %

( D 60 n o i C t a

n C i m

r B

e 40 G

20 B A

A C A A A B 0

100 D (10 Weeks) C C

80 B ) % ( 60 n o

i D t a n i A m r

e 40 G C

20 B B

A A B A A 0 Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 Stage 5 Developmental Stage

Symbiotic germination (Stage 1; 0.35%), but did not germinate. A limited number of seeds cultured in the presence of Ealt-386 Three fungal isolates tested (Ealt-386, Ealt-388 and and Ealt-388 germinated after 11 weeks of culture Ealt-396) supported seed germination (Stage 2), but (0.47% and 0.20%, respectively). Final germination only Ealt-396 supported further protocorm develop- of seeds cultured in the presence of Ealt-388 ment. The embryos in control treatments swelled (0.20%) was not significantly different than 0%. Final 123 Plant Cell Tiss Organ Cult (2007) 90:313–323 319

Fig. 3 Protocorms and developing seedlings of Eulophia alta. Symbiotic protocorms of E. alta cultured 8 weeks in dark on (a) Asymbiotic protocorms of E. alta cultured 8 weeks in dark oat meal agar (OMA) with mycobiont Ealt-396 (scale on Vacin & Went Modified Orchid Medium (VW; scale bar = 0.5 cm). (d) Seedlings of E. alta cultured symbiotically bar = 0.5 cm). (b) Asymbiotic seedlings of E. alta after on OMA with mycobionts Ealt-396 after 18 weeks dark 28 weeks culture (18 weeks in dark, followed by 10 weeks incubation followed by 10 weeks under a 16/8 L/D photope- under a 16/8 h L/D photoperiod; scale bar = 1.0 cm). (c) riod (scale bars = 1.0 cm) germination of seeds cultured with Ealt-386 was seedlings continued to develop and produced elon- 0.75% after 18 weeks culture. Germination of seeds gated leaves by week 28 (Fig. 3d), while asymbiotic cultured with Ealt-396 initiated six weeks after seeds protocorms did not progress as rapidly. After were sown. Maximum percent germination was 28 weeks culture, asymbiotic protocorms developed reached at this time (70.1 ± 2.6%) with 44.3 ± 2.8% into rhizominous masses (Fig. 3b). Leaves were of the protocorms developing to Stage 3. By week 11, observed on very few asymbiotic seedlings and, 57.2 ± 3.0% of protocorms had reached Stage 3 when present, were much shorter than those observed (Fig. 3b). Additional protocorm development was on symbiotic seedling cultures. not observed beyond week 11 until protocorms were transferred to fresh media (Fig. 3d). Less than 1% of E. alta seeds were observed in Stage 1 since seeds Discussion rarely produced rhizoids before the testa was ruptured. The number of reports on the successful in vitro Comparison of asymbiotic and symbiotic production of North American terrestrial orchids has germination methods been increasing (Kauth et al. 2006; Stewart and Kane 2006a, b; Stewart and Zettler 2002; Stewart et al. Seeds co-cultured with Ealt-396 had a higher percent 2003; Zettler 1997a, b; Zettler and McInnis 1994; germination and more advanced development than Zettler et al. 2007). This trend may result from a seeds cultured on asymbiotic media after 18 weeks growing concern among conservationists that many culture (compare Fig. 3a and 3c). Upon transfer to habitats which harbor terrestrial orchids are being fresh medium and lighted conditions, symbiotic converted to residential and commercial land uses. A 123 320 Plant Cell Tiss Organ Cult (2007) 90:313–323 number of Eulophia species have been successfully nitrogen source, nitrogen availability, or enzyme germinated using asymbiotic methods. Eulophia synthesis or activation within developing protocorms. cucullata, E. petersii, and E. streptopetala were all Other studies on amino acid utilization by terres- cultured on MS, but only germinated after at least trial orchid seeds and seedlings indicate that species three months culture (McAlister and Van Staden vary in their ability to utilize different amino acids 1998). Seeds of Eulophia yushuiana were germinated (Curtis 1947; Spoerl 1948; Malmgren 1996). Organic on various formulations of KC and MS (Weatherhead nitrogen may be more readily utilized by young et al. 1986). Neither of these previous reports protocorms as available amino acids may by-pass included precise data on the germination or subse- certain steps in the nitrogen assimilation process quent growth of protocorms as is included in the (Malmgren, 1992; 1996). Alternatively, development current study. This is the first report of asymbiotic of protocorms cultured in the presence of inorganic and symbiotic seed propagation of E. alta, the only nitrogen may be postponed due to a delay in the North American species of the genus. production of nitrate reductase until several months TZ staining indicated that E. alta seed viability after imbibition (ca. 60 days for Cattleya; Raghavan was lower than observed during germination exper- and Torrey 1964). Currently, the optimal asymbiotic iments. The opposite scenario has been documented nitrogen source and concentration during seed ger- with hard-seeded orchids such as Cypripedium where mination has not been determined for any orchid TZ staining grossly overestimated germinability species. Further investigation into the effects of (Lauzer et al. 1994; Vujanovic et al. 2000). Differ- nitrogen source may be useful in improving the ences in estimated viability and observed germina- asymbiotic culture of E. alta. bility of E. alta may be due to less than optimal Both Sclerotinia and Fusarium are members of the pretreatment or staining methods. These results Ascomycotina and typically not associated with exemplify the importance of testing germinability of endophytic, root-inhabiting orchid mycorrhizae. orchid seeds and not relying on TZ staining alone as However, Fusarium oxysporum has been isolated an estimate of viability. from a number of other terrestrial and epiphytic Asymbiotic orchid seed germination represents an orchid taxa, including Bletia purpurea, Dendrophylax efficient means to culture a wide range of orchid taxa. lindenii, Encyclia tampensis, Epidendrum nocturnum, Most asymbiotic germination media contain similar E. stangeanum, and Platanthera praeclara (Richard- components—sugars, mineral salts, and agars. The son 1993; Zelmer 1994; SL Stewart, personal com- asymbiotic media used in this study varied greatly in munication). Armillaria species have previously been mineral salt, nitrogen, organic additives, and vitamin isolated from the achlorophyllus terrestrial orchid compositions. Several researchers have reported that Galeola septentrionalis (Terashita and Chuman nitrogen type and concentration can play an impor- 1989). However, the current isolation of an Armil- tant role during in vitro asymbiotic orchid seed laria species was from the heteromycotrophic orchid germination (Curtis 1947; Kauth et al. 2006; Malm- E. alta. Interestingly, Armillaria species have been gren 1992, 1996; Raghavan and Torrey 1964; Spoerl shown to be effective mycobionts of both G. septen- 1948; Stewart and Kane 2006a). Curtis (1947) trionalis (Marxmu¨ller 1992) and E. alta (present reported that media containing peptone better sup- study) by supporting the in vitro symbiotic seed ported protocorm development in Spathoglottis pli- germination of both species. The isolation of several cata than did media containing asparagine. Similarly, strains of Epulorhiza from the roots of E. alta was Kauth et al. (2006) hypothesized that peptone in not surprising, especially given the ubiquitous distri- P723, as used in the present study, likely helped bution of Epulorhiza species throughout orchid support the rapid germination and advanced proto- habitats worldwide (Zelmer 2001). corm development in the North American terrestrial The mycobiont Epulorhiza repens (Sbrev-266), orchid Calopogon tuberoses. Interestingly, VW and collected from Spiranthes brevilabris in Levy P723 supported similar percentages of Stage 2 and County, FL, failed to promote germination. The Stage 4 protocorms, while considerably fewer Stage 3 inability of Sbrev-266 to support germination may protocorms were observed on VW than P723. The point to a degree of fungal preference in E. alta since reason for this is unclear, but may be linked to this strain has been useful in germinating several 123 Plant Cell Tiss Organ Cult (2007) 90:313–323 321 terrestrial and epiphytic orchid taxa (Stewart and morphology between asymbiotic and symbiotic seed Kane 2006b; Stewart and Zettler 2002; Zettler et al. cultures has not been previously documented and 2007). Furthermore, a number of Epulorhiza isolates warrants further investigation. Advanced in vitro were obtained from the roots of E. alta and none of seedling development and plant formation appears to these mycobionts resembled E. repens or supported be reliant upon digestion of a compatible mycobiont or the in vitro symbiotic seed germination of this uptake of a growth promoting substance provided by species as well as isolate Ealt-396. Ealt-396 (Armil- mycobiont digestion (such as a plant growth regulator; laria sp.) collected from a vegetative E. alta plant Rasmussen 1995) that is not present in the asymbiotic was found to be more effective in promoting in vitro orchid seed media screened in this study. However, it symbiotic germination and further development than can not be discounted that a fully optimized asymbiotic control or other isolates tested. This may be further seed germination protocol could begin to parallel the evidence that germination of E. alta is dependent efficient in vitro seed germination of E. alta. Symbi- upon infection by a preferred mycobiont or group of otic seed germination proved to be a more efficient mycobionts. This type of preference is not uncom- method of germinating and supporting early develop- mon in the Orchidaceae and can be genus, species, or ment of E. alta than asymbiotic germination. Although site specific (McCormick et al. 2006; McKendrick relatively few North American orchid species have et al. 2002; Stewart and Kane 2006b, 2007; Taylor been successfully germinated in vitro using symbiotic and Bruns 1997). germination, our results indicate that time spent Seeds co-cultured with mycobiont Ealt-396 ceased collecting, isolating, and culturing mycobionts, as well developing after 11 weeks of culture. At this time, as persistence in attempting to successfully co-culture necrosis became apparent in some cultures. After native orchids may prove more efficient than asymbi- 18 weeks of culture in the dark, many protocorms otic culture methods. became too brittle to transfer to fresh medium. Since Zettler (1997a) concluded that if an orchid is symbiotic protocorms continued to develop into plants critically dependent on a compatible mycorrhiza for when transferred to fresh medium and a 16/8 h L/D germination, the loss of that fungus in situ will photoperiod, the protocorm death observed earlier may ultimately result in the inability of that species to be attributed to (1) mycobiont nutrient stress resulting establish new stands. An additional benefit of cultur- in fungal pathenogenicity upon the germinated ing orchid seeds symbiotically is that the resulting seeds or (2) a lack of light and the protocorms’ seedlings can serve as both plant material and inability to become heteromycotrophic. The line inoculum for conservation efforts (Batty et al. between orchid–fungal association and fungal parasit- 2006). The isolation of a suitable mycobiont for ism has been shown to be in part controlled by fungal E. alta is a promising step forward in ongoing efforts nutrient availability (Beyrle et al. 1991) and temper- to develop reintroduction and conservation protocols ature (Rasmussen et al. 1990). The role of light on for this species, as well as other endangered and symbiosis is less well understood. Transferring seed- threatened orchid species. Continued research should lings to light and fresh medium earlier may increase the focus on improving the efficiency of E. alta symbi- efficiency of the present symbiotic protocol. otic seed germination, acclimatization, and in situ In nature, endophytic orchid mycobionts presum- establishment methodologies to further progress in ably provide the essential nutrients germinating seeds rare orchid conservation techniques. require (Cameron et al. 2006; Hadley and Purves 1974; Rasmussen 1995). Mimicking this system in vitro Acknowledgements The authors thank the Florida Panther (=symbiotic seed germination) has been shown to National Wildlife Refuge—US Fish and Wildlife Service for providing financial and logistical support for this project. effectively enhance germination and protocorm devel- Appreciation is also extended to Dr. Carrie Reinhardt Adams opment of E. alta compared to asymbiotic germination (University of Florida) for the use of microscopic equipment, 18 weeks after seeds were sown. In addition, symbiotic and Dr. James Kimbrough (University of Florida) for protocorms rapidly produced elongated leaves follow- assistance in fungal identification. The authors would also like to thank Philip Kauth (University of Florida) and Nancy ing transfer to fresh medium while asymbiotic proto- Philman (University of Florida) for help constructing and corms formed rhizominous masses with only a few revising this paper. Brand names are provided for references; short leaves (see Fig. 3b). Such a distinct difference in the authors do not solely endorse these particular products. 123 322 Plant Cell Tiss Organ Cult (2007) 90:313–323

References McKendrick SL, Leake JR, Taylor DL, Read DJ (2002) Symbiotic germination and development of the myco- Batty AL, Brundrett MC, Dixon KW, Sivasithamparam K heterotrophic orchid Neottia nidus-avis in nature and its (2006) In situ symbiotic seed germination and propagation requirement for locally distributed Sebacina spp. New of terrestrial orchid seedlings for establishment at field Phytol 154:233–247 sites. Aust J Bot 54:375–381 Moore RT (1987) The genera of Rhizoctonia-like fungi: Beyrle H, Penningsfeld F, Hock B (1991) The role of nitrogen Ascorhizoctonia, Ceratorhiza gen. nov., Epulorhiza gen. concentration in determining the outcome of the interac- nov., Moniliopsis, and Rhizoctonia. Mycotaxon 39: tion between Dactylorhiza incarnate (L.) Soo´ and Rhi- 91–99 zoctonia sp. New Phytol 117:665–672 Murashige T, Skoog F (1962) A revised medium for rapid Brown PM (2005) Wild orchids of Florida. University Press of growth and bio assays with tobacco tissue cultures. Florida, Florida Physiol Plant 15:473–497 Cameron DD, Leake JR, Read DJ (2006) Mutualistic mycor- Phillips JM, Hayman DS (1970) Improved procedures for rhiza in orchids: evidence from plant–fungus carbon and clearing roots and staining parasitic and vesicular–arbus- nitrogen transfers in the green-leaved terrestrial orchid cular mycorrhizal fungi for rapid assessment of infection. Goodyera repens. New Phytol 171:405–416 Tran Br Mycol Soc 55:158–160 Currah RS, Sigler L, Hambleton S (1987) New records and Raghavan V, Torrey JG (1964) Inorganic nitrogen nutrition of new taxa of fungi from the mycorrhizae of terrestrial the seedlings of the orchid, Cattleya. Am J Bot 51:264– orchid of Alberta. Can J Bot 62:2473–2482 274 Currah RS, Smreciu EA, Hambleton S (1990) Mycorrhizae and Rasmussen HN (1995) Terrestrial orchid: from seed to myco- mycorrhizal fungi of boreal species of Platanthera and trophic plant. Cambridge University Press, United King- Coeloglossum (Orchidaceae). Can J Bot 68:1171–1181 dom Currah RS, Zelmer CD, Hambleton S, Richardson KA (1997) Rasmussen HN, Andersen TF, Johansen B (1990) Temperature Fungi from orchid mycorrhizas. In: Arditti J, Pridgeon sensitivity of in vitro germination and seedling develop- AM (eds) Orchid biology: reviews and perspectives, VII. ment of Dactylorhiza majalis (Orchidaceae) with and Kluwer Academic Publishing, Great Britain, pp 117–170 without a mycorhizal fungus. Plant Cell Environ 13:171– Curtis JT (1947) Studies on the nitrogen nutrition of orchid 177 embryos. AOS Bull 16:654–660 Richardson KA (1993) Endophytic fungi from Costa Rican Dixon K (1987) Raising terrestrial orchid from seed. In: Harris orchids. University of Albert, Edmonton, MSc Thesis WK (ed) Modern orchid growing for pleasure and profit. Richardson KA, Currah RS, Hambleton S (1993) Basidiomy- Orchid Club of S. Australia, Inc., Adelaide, AU, pp 47– cetous endophytes from the roots of neotropical epiphytic 100 Orchidaceae. Lindleyana 8:127–137 Hadley G, Purves S (1974) Movement of 14carbon from host to SAS Institute Inc. (2003) SAS version 9.1. SAS Institute, North fungus in orchid mycorrhiza. New Phytol 73:475–482 Carolina Kauth PJ, Vendrame WA, Kane ME (2006) In vitro seed cul- Spoerl E (1948) Amino acids as a source of nitrogen for orchid ture and seedling development of Calopogon tuberosus. embryos. Am J Bot 35:88–95 Plant Cell Tiss Org Cult 85:91–102 Stevens RB (ed) (1974) Mycology guidebook. University of Knudson L (1946) A new nutrient solution for the germination Washington Press, Seattle, p 703 of orchid seeds. Bot Gaz 73:1–25 Stewart SL, Kane ME (2006a) Asymbiotic germination and Lakon G (1949) The topographical tetrazolium method for in vitro seedling development of Habenaria macrocera- determining the germination capacity of seeds. Plant titis (Orchidaceae), a rare Florida terrestrial orchid. Plant Physiol 24:389–394 Cell Tiss Org Cult 86:147–158 Lauzer D, St-Arnaud M, Barabe´ D (1994) Tetrazolium staining Stewart SL, Kane ME (2006b) Symbiotic germination of and in vitro germination of mature seeds of Cypripedium Habenaria macroceratitis (Orchidaceae), a rare Florida acaule (Orchidaceae). Lindleyana 9:197–204 terrestrial orchid. Plant Cell Tiss Org Cult 86:159–167 Malmgren S (1992) Large-scale asymbiotic propagation of Stewart SL, Kane ME (2007) Symbiotic seed germination and Cypripedium calceolus-plant physiology from a surgeon’s evidence for in vitro mycobionts specificity in Spiranthes point of view. Bot Gard Microprop News 1:59–63 brevilabris (Orchidaceae), and its implications for spe- Malmgren S (1996) Orchid propagation: theory and practice. cies-level conservation. In Vitro Cell Dev Biol Plant In: Allen C (ed) North American native orchids: propa- 43:178–186 gation and production. North American Native Terrestrial Stewart SL, Zettler LW (2002) Symbiotic germination of three Orchid Conference, Germantown, Maryland, pp 63–71 aquatic rein orchids (Habenaria repens, H. quinqueseta, Marxmu¨ller H (1992) Some notes on the taxonomy and H. macroceratitis) from Florida. Aquat Bot 72:25–35 nomenclature of five European Armillaria species. My- Stewart SL, Zettler LW, Minso J, Brown PM (2003) Symbiotic cotaxon 44:267–274 germination and reintroduction of Spiranthes brevilabris McAlister BG, Van Staden J (1998) In vitro culture of Eulo- Lindley, an endangered orchid native to Florida. Selbyana phia species. S Afr J Bot 64:264–266 26:64–70 McCormick MK, Whigham DF, Sloan D, O’Malley K, Hod- Taylor DL, Bruns TD (1997) Independent, specialized invasion kinson B (2006) Orchid-fungus fidelity: a marriage meant of ectomycorrhizal mutualism by two nonphotosynthetic to last? Ecol 87:903–911 orchids. Proc Natl Acad Sci USA 94:5410–5415

123 Plant Cell Tiss Organ Cult (2007) 90:313–323 323

Terashita T, Chuman S (1989) Armillaria species isolated Zelmer CD, Currah RS (1995) Ceratorhiza pernacatena and from the wild orchid Galeola septentrionalis. Proc 7th Epulorhiza calendulina spp. nov.: mycorrhizal fungi of Int Conf Root Butt Rots, Vernon and Victoria Canada, terrestrial orchids. Can J Bot 73:1981–1985 pp 364–370 Zelmer CD, Cuthburtson L, Currah RS (1996) Fungi associated Vacin EF, Went FW (1949) Some pH changes in nutrient with terrestrial orchid mycorrhizas, seeds and protocorms. solutions. Bot Gaz 110:605–613 Mycoscience 37:439–4448 Vujanovic V, St-Arnaud M, Barabe´ D, Thibeault G (2000) Zettler LW (1997a) Orchid–fungal symbiosis and its value in Viability testing of orchid seed and the promotion of conservation. McIlvainea 13:40–45 coloration and germination. Ann Bot 86:79–86 Zettler LW (1997b) Terrestrial orchid conservation by symbi- Weatherhead MA, Zee SY, Battetto G (1986) Some obser- otic seed germination: techniques and perspectives. vations on the early stages of development of Eulophia Selbyana 18:188–194 yushuiana. Memoirs Hong Kong Nat Hist Soc 17: Zettler LW, McInnis TM (1994) Light enhancement of sym- 85–90 biotic seed germination and development of an endan- Zelmer CD (1994) Interactions between northern terrestrial gered terrestrial orchid (Platanthera integrilabia). Plant orchids and fungi in nature. University of Alberta, MSc Sci 102:133–138 thesis Zettler LW, Poulter SB, McDonald KI, Stewart SL (2007) Zelmer CD (2001) Root-associated organisms of the Conservation-driven propagation of Epidendrum noctur- Cypripedioideae (Orchidaceae). University of Guelph, num (Orchidaceae) with a mycorrhizal fungi. HortScience Ontario, PhD thesis 42:135–139

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