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 & Rendle seeds collected from the Florida Panther National Wildlife Refuge (Collier County, FL; FPNWR) were used in a screen of five asymbiotic orchid seed germination media to determine their effectiveness in promoting germination and protocorm development. In addition, 10 fungal isolates collected from the roots of E. alta at sites in the FPNWR, Highlands County (FL), and Goethe State Forest (Levy County, FL; GSF), and a fungal isolate from the roots of Spiranthes brevilabris collected from GSF were screened for their effectiveness at promoting in vitro symbiotic germination of E. alta seeds. After 18 weeks asymbiotic culture, seeds sown on PhytoTechnology Orchid Seed Sowing Medium germinated to a higher percentage (87.9%) and had a higher percentage of protocorms with developing protomeristems (32.7%) than seeds cultured on Knudson C, Malmgren Modified Terrestrial Orchid Medium, ½-strength Murashige & Skoog, or Vacin & Went Modified Orchid Medium. Significantly more

T. R. Johnson (&)
S. L. Stewart
D. Dutra
M. E. Kane Department of Environmental Horticulture, University of Florida, PO Box 110675, Gainesville, FL 32611, USA e-mail: [email protected] L. Richardson Florida Panther National Wildlife Refuge, U.S. Fish and Wildlife Service, 3860 Tollgate Blvd., Suite 300, Naples, FL 34114, USA

leaf-bearing protocorms were observed on PhytoTechnology Orchid Seed Sowing Medium (0.8%) and Vacin & Went Modified Orchid Medium (1.3%) than other media tested. Of the fungi tested, one fungal isolate (Ealt-396) promoted germination to 69.0%, two isolates promoted germination to less than 0.75% and did not support further protocorm development, and eight isolates did not support germination. Seeds co-cultured in darkness with Ealt-396 grew more rapidly than asymbiotic seedlings following germination. In addition, co-cultured (=symbiotic) seedlings continued to develop more rapidly than asymbiotic seedlings upon transfer to 16/ 8 h light/dark photoperiod. Symbiotic seed culture of E. alta may be a more desirable method of propagation since protocorms develop more rapidly than seeds sown on asymbiotic media. Symbiotic seedlings may be more appropriate for reintroduction to natural areas than asymbiotic seedlings since symbiotic seedlings could serve to inoculate soils with a germination promoting mycobiont. Keywords Orchid
Seed germination
Native
Conservation
Terrestrial
Mycorrhizae
Wild coco Abbreviations 1/51/5-strength Potato dextrose agar PDA ½MS ½-strength Murashige & Skoog CMA Corn meal agar FPNWR Florida Panther National Wildlife Refuge

123

314

dd KC L/D MM P723 TZ VW

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

Distilled deionized Knudson C Light/dark Malmgren Modified Terrestrial Orchid Medium PhytoTechnology Orchid Seed Sowing Medium Tetrazolium Vacin & Went Modified Orchid Medium

Introduction Seed propagation represents the most efficient method of propagating native terrestrial orchids (Stewart and Kane 2006a). Symbiotic seed germination can be a cumbersome process; root samples must be collected from which many fungi are often isolated. Fungi must then be identified and screened for growth promoting strains. Asymbiotic seed germination can be a more straight forward process since mycobionts need not be isolated to germinate seeds of orchid taxa. However, there are circumstances when symbiotically germinated seedlings are desired or necessary. Populations of orchids that are established with asymbiotic seedlings remain dependent on naturally occurring fungal symbionts for seedling recruitment (Zettler 1997b). Due to possible ecological changes at historic orchid locales, a target orchid species’ mycobionts may not be present at a site if the orchid itself is not present. In these 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

situations, seedlings cultured symbiotically can serve as both plant material and a source of mycobiont inoculum for reintroduction efforts. Introducing a compatible mycobiont into a site may facilitate the establishment of self-sustaining populations. Additionally, symbiotic seed germination may be a more desirable means of producing orchids if symbiotic seedlings develop more rapidly than asymbiotic seedlings. Eulophia is a pantropical genus with African affinities containing approximately 200 species worldwide (Brown 2005). Only one species, Eulophia alta (Linnaeus) Fawcett & Rendle (common name wild coco; Fig. 1), occurs in North America, where it is found from southern Georgia to southern Florida. Eulophia alta populations are typically found in moderately wet, grass-dominated roadsides or near the edges of forested sites dominated by live oak (Quercus virginianus), saw palmetto (Serenoa repens), Sabal palmetto, and slash pine (Pinus eliottii). While not currently listed as a rare species, urban development throughout its range is threatening the species’ current habitat. Because E. alta grows in concentrated populations (=locally abundant), habitat degradation at E. alta populated sites could have long-term ramifications for the future of this species. Common orchid taxa may serve as models for developing reintroduction programs, which can then be applied to threatened and endangered taxa. The first step in this process is establishing efficient propagation protocols to produce plants for subsequent experimentation. Little information is available

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

concerning seed germination of Eulophia species, and no information exists on the asymbiotic or symbiotic seed germination requirements of E. alta. The objectives of this research were (1) to evaluate the potential of asymbiotic and symbiotic seed propagation for the production of E. alta seedlings, (2) to identify germination-promoting mycobionts, and (3) to document the germination and seedling development of this species. The data collected from this study will be used to propagate plants for further investigations of orchid reintroduction methods.

Materials and methods Seed source and sterilization Eulophia alta seeds were collected from the Florida Panther National Wildlife Refuge (FPNWR; Collier County, FL) on 13 December 2005. Only seeds from dehisced capsules were collected to ensure that they were mature. Seeds were stored at 23 ± 2C over silica gel desiccant until capsules ruptured, then collected and stored at 10C for 15 weeks. Seeds were transferred to a sterilized scintillation vial and surface sterilized for 45 s in a solution containing 5 ml absolute ethanol, 5 ml 6.0% NaOCl, and 90 ml sterile distilled deionized (dd) water. Seeds were rinsed three times with sterile dd water after surface sterilization. Seeds were then suspended in sterile dd water. Solutions were removed from the vial with sterilized Pasture pipettes that were used only once. Asymbiotic media survey Five nutrient media (Table 1) were assayed for their effectiveness in promoting germination and subsequent development of E. alta seeds. All media were prepared and modified by PhytoTechnology Laboratories, Inc. (Shawnee Mission, KS): Knudson C (KC; Knudson, 1946), Malmgren Modified Terrestrial Orchid Medium (MM; Malmgren, 1996), PhytoTechnology Orchid Seed Sowing Medium (P723), ½strength Murashige & Skoog (½MS; Murashige and Skoog, 1962), and Vacin & Went Modified Orchid Medium (VW; Vacin and Went, 1949). To standardize the sucrose and agar concentrations among media tested, the following modifications were made to

315 Table 1 Nutrient composition of germination media used in the asymbiotic seed germination of Eulophia alta KC

MM

P723

½MS VW

10.31 7.57

Macronutrients (mM) Ammonium

13.82 –

5.15

Calcium

2.12

0.24

0.75

1.50

1.93

Chlorine

3.35

–

1.50

1.50

–

Magnesium

1.01

0.81

0.62

0.75

1.01

Nitrate

10.49 –

9.85

19.70 5.19

Potassium

5.19

0.55

5.01

10.02 7.03

Phosphate

1.84

0.71

0.31

0.63

Sulfate

4.91

0.92

0.71

0.86

4.92

Sodium

–

0.20

0.10

1.51

0.20

Boron

–

–

30.00

50.00 –

Cobalt

–

–

0.03

0.11

–

Copper

–

–

0.03

0.10

–

Iron Iodine

90.00 100.00 50.00 – – 1.20

Manganese

30.00 10.00

30.00

37.90 30.00

Molybdenum

–

–

26.00

0.52

Zinc

–

–

9.20

30.00 –

–

0.05

–

–

3.13

Micronutrients (lM)

50.00 100.00 2.50 –

Vitamins (mg/l) Biotin

–

Casein hydrolysate –

400.00 –

–

–

Folic acid

–

0.50

–

–

–

Glycine

–

2.00

–

–

–

myo-Inositol

–

100.00 100.00

–

–

Nicotinic acid

–

–

1.00

–

–

Peptone

–

–

2000.00

–

–

Pyridoxine

–

–

1.00

–

–

Thiamine

–

–

10.00

–

–

Total N (mM) NH4:NO3

24.31 n/a

unknown 30.01 12.76

1.32

0.52

n/a

0.52

1.46

KC—Knudson C, MM—Malmgren Modified Terrestrial Orchid Medium, P723—PhytoTechnology Orchid Seed Sowing Media, ½MS—½-strength Murashige & Skoog, VW—Vacin & Went Orchid Medium

basal media: 0.8% TC1 agar was added to KC, 2.0% sucrose was added to both MM and ½MS. Media pH were adjusted to 5.8 using 0.1 N KOH prior to autoclaving for 20 min at 121C and 117.7 kPa. Sterilized media were dispensed as 30 ml aliquots into 9 cm diameter Petri plates (Fisher Scientific, Pittsburg, PA). Surface sterilized seeds were then inoculated near the center of each plate using a sterile bacterial inoculating loop before the plates were

123

316

sealed with a single layer of Nescofilm (Karlan Research Products, Santa Rosa, CA). Approximately 60 seeds were sown onto each plate (average seeds/ plate: 59.4). Eight replicate plates were prepared for each treatment. Plates were stored at 22 ± 3C in darkness for 18 weeks. Light was excluded by wrapping plates in two layers of aluminum foil. Seeds were exposed to short periods of light (