1445 HORTSCIENCE, VOL. 30(7), DECEMBER 1995 HORTSCIENCE 30(7):1445–1446. 1995. Methods for ex Vitro Germination of Australian Terrestrial Orchids Letitia Quay and Jen A. McComb School of Biological and Environmental Sciences, Murdoch University, Murdoch, WA 6150, Australia Kingsley W. Dixon Kings Park and Botanic Garden, West Perth, WA 6005, Australia Additional index words. seed germination, mycorrhizal fungi, propagation, Caladenia latifolia, Diuris magnifica Abstract. 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. Received for publication 16 Feb. 1995. Accepted for publication 28 Aug. 1995. This research was funded by Biotech International through the Cooperative Education for Enterprise Development program. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact. Seed also was collected from C. latifolia plants that had set seed naturally. The ripe pods were placed in paper envelopes and allowed to dry before being stored in a desiccator at room temperature. The nonsterile potting mix was prepared from a 1 sheoak mulch (partially mulched leaves from around the base of Allocasuarina fraseriana trees located in Kings Park) : 1 perlite (v/v) mixture. Gamma-irradi- ated potting mix was prepared by mixing sheoak mulch (sterilized at 70 Gy for 14 h) with perlite that had been autoclaved at 121C and 98 kPa for 15 min. The mixture was moistened with sterile distilled water. The study included mycorrhizal fungi from C. latifolia and D. magnifica and also a sterile red fungus (SRF) that acts as a biological control agent against various diseases and stimulates plant growth (Dewan and Sivasithamparam, 1988, 1989a, 1989b). The appropriate mycorrhizal fungus for each orchid species was obtained from parent plants using the methods of Ramsay et al. (1986); its symbiotic potential was confirmed because it stimulated the germination of seeds of the appropriate orchid species (preliminary studies, in vitro). Mycorrhizal fungi were grown on potato dextrose agar in petri dishes and incubated at 20C. The mycelium and some agar of a 3-week-old culture of mycorrhizal fungus was roughly chopped using a scalpel, and applied to the soil at 6% (v/v) using a 10- ml sterile syringe. SRF was prepared by Biotech International and provided as mycelium on sterilized perlite, which was inoculated into the potting mix at 2% (v/v) and distributed evenly within the mixture. Pasteurized potting mix was made by steam-pasteurizing the sheoak mulch at 70C for 30 min and adding perlite (autoclaved as before) and using sterile distilled water to moisten the mixture, if nec- essary. Fungal inoculation was carried out as described. The potting mixes inoculated with mycor- rhizal fungi were placed in 250-ml polycar- bonate tubs and incubated at 23C for 1 to 2 weeks before being dispersed into containers ready for seed sowing. Potting mix was placed into containers (12 × 7 × 4 cm), eight of which were fitted into a plastic tray. Each container represented one treatment replicate. Each con- tainer had at the bottom a 1-cm layer of noninoculated sheoak mulch and perlite (1:1), followed by a 2-cm layer of sheoak mulch and perlite (1:1) inoculated with mycorrhizal fun- gus, SRF, or both; then the sand and seed mixture (described below) was sprinkled over it. This mixture was covered further with a 1- cm layer of sheoak mulch containing orchid mycorrhizal fungus, SRF, or both. To produce the sand/seed mixture, seed from eight pods of C. latifolia or 10 pods of D. magnifica was mixed with 80 ml white sand. Ten milliliters of this orchid seed/sand mixture was sprinkled evenly into each container. Due to limited seed quantities available, gamma-irradiated potting mix was not used with D. magnifica, and no control was set up with C. latifolia, since the gamma irradiation would have killed all microorganisms, includ- ing mycorrhizal fungi, which are necessary for germination. Containers were placed under a fogging system (nonsterile) in a greenhouse at ambient temperatures (≈8 to ≈26C) at the end of May, and monitored until the end of Octo- ber. The number of shoots visible above the surface of the sheoak mulch (an estimate of germination) was scored periodically, and in the final assessment (after 20 weeks), shoot lengths were measured and the number of “elite” individuals (seedlings with shoot lengths >50 mm for C. latifolia and >100 mm for D. magnifica) scored. Statistical analysis of data was carried out using the SPSSX subprograms ANOVA and MANOVA (SPSS, Chicago). No seedlings from either orchid species emerged in potting mixes that were not gamma- irradiated or pasteurized (data not shown). Germination of seeds of both species and the formation of protocorms were evident after 8 weeks, and healthy, green shoots were visible 10 weeks after seed sowing. The maximum mean number of seedlings of C. latifolia and D. magnifica per container after 20 weeks was 84 and 234, with seedlings having a mean shoot length of 28 and 52 mm, respectively (Fig. 1A–D). The seedling production levels are a marked improvement on results from the in situ sowing methods currently used, in which seeds sown around a growing plant produce only a dozen or so plants (K.W. Dixon, personal communication). Germination was absent in containers with- out mycorrhizal fungi, or where SRF was the only fungal inoculant. Germination also was absent in nonsterile potting mix with mycor- rhizal fungus, SRF, or both. More than twice the number of C. latifolia seedlings were produced in pasteurized pot- ting mix than in gamma-irradiated potting mix (Fig. 1A), although there was little effect on Western Australian is renowned for its wildflowers, particularly its terrestrial orchids. Many of these orchids are horticulturally de- sirable, but are more difficult to propagate than epiphytic species due to their greater dependence upon symbiotic (mycorrhizal) fungi for germination and development. Na- tive orchids have been successfully raised from seed using in vitro germination tech- niques, symbiotically (fungal assisted) and asymbiotically (without fungal assistance) (Arditti, 1982; Bunn, 1988; Dixon, 1987). However, these methods may not be economi- cal for mass propagation or horticultural pur- poses. Ex vitro germination (in situ seed sowing) of orchid seeds has been carried out by sowing around established parent plants, but little work has been done on seed germination in soil preinoculated with the appropriate mycor- rhizal fungus (Dixon, 1987). The aim of this study was to develop ex vitro germination techniques for two species of terrestrial or- chids, Caladenia latifolia R.Br. and Diuris magnifica D. Jones. Plants of C. latifolia and D. magnifica located in the bushland of Kings Park, Perth, Western Australia, were cross-pollinated by hand to ensure production of abundant seed.