Asymbiotic in vitro germination and seed quality assessment of Australian terrestrial orchids

Abstract

Determining the seed quality and germination requirements for threatened orchid species in storage is vital for future conservation efforts. Seeds of many Australian terrestrial orchid species are held in conservation collections around the country, but few have been germinated in vitro, fuelling concerns over their long-term viability. This study tested three methods of assessing orchid seed quality; asymbiotic germination was compared with vital staining using triphenyltetrazolium chloride or fluorescein diacetate. Six culture media were examined for efficacy in promoting asymbiotic seed germination of four Australian terrestrial orchid species (Pterostylis nutans, Microtis arenaria, Thelymitra pauciflora and Prasophyllum pruinosum). Germination occurred on all media but germination rates were consistently highest on BM1 and development was most advanced on BM1, P723 and Malmgren media. Subsequent trials tested the efficacy of BM1 for asymbiotic germination of additional genera (Caladenia, Calochilus and Diuris), several congeneric species, and two species collected from several different provenances within each of their ranges. The results indicate that asymbiotic germination on BM1 medium is an effective technique for testing the performance of Australian terrestrial orchid seeds. The efficacy of vital stains to determine seed viability, however, remains uncertain, as significant disagreement between degree of staining and germinability was observed for some species.

Ex situ Conservation and Cryopreservation of Orchid Germplasm

“…For example, seeds of three widely disjunct terrestrial orchids-Dactylorhiza fuchsii (a European forest species), Dendrobium anosmum (a lowland tropical forest species), and Eulophia gonychila (an African woodland species)-all survived drying to water contents in equilibrium with 5% relative humidity (RH; Pritchard et al 1999). Similarly, seeds of 10 terrestrial orchid species from Mediterranean and temperate climatic regions of Australia survived drying to equilibrium with either 5% or 23% RH without any detrimental effects on germination , and 60%-100% germination of a further 10 terrestrial Australian orchids was achieved following drying to 15% RH (Dowling and Jusaitis 2012). For seeds of epiphytic orchids, data reviewed by Pritchard and Seaton (1993) provide examples of desiccation tolerance, with 160% germination recorded for species including Cattleya aurantiaca, Dendrobium phalaenopsis, and Phalaenopsis amabilis after storage at 5Њ-8ЊC for varying periods at ∼4%-7% water content, or water contents in equilibrium with anhydrous calcium chloride (∼30% RH).…”

“…But indiscriminate use of asymbiotic media can be problematic and inferior to symbiotic media (Johnson et al 2007), and inappropriate culture media can confuse the interpretation of seed viability. The variation in germination that can occur among different asymbiotic media has been recently illustrated in a study of four Australian orchids (Dowling and Jusaitis 2012). Germination, when defined as the early stages of testa rupturing and enlargement of the protocorm, varied significantly between six germination media, differing by as much as 50% for seeds of Thelymitra pauciflora.…”

“…Germination, when defined as the early stages of testa rupturing and enlargement of the protocorm, varied significantly between six germination media, differing by as much as 50% for seeds of Thelymitra pauciflora. Further, when considering those seeds that progressed through to the final stage of germination (i.e., shoot differentiation), for two species (T. pauciflora and Prasophyllum pruinosum), none of the media were suitable (Dowling and Jusaitis 2012).…”

Refining the tetrazolium test for evaluation of Cattleya labiata and C. tigrina seeds viability

“…Salazar-Mercado (2012) points out that the use of 1.0% sodium hypochlorite was important to tegument scarification in Cattleya mendelli, which allowed better tetrazolium salt penetration, the same being true for Thelymitra pauciflora and Prasophyllum pruinosum (Dowling and Jusaitis, 2012). Ideally, tetrazolium solution pH should be between 6 and 8, which fundamentally depends on the water quality used in its preparation.…”

Handling and propagation of Dendrobium ‘Iriana Jokowi’ in Bogor Botanic Gardens, Indonesia

“…The method seed for seed viability testing is through in vitro germination, either symbiotic or asymbiotic; germination in the wild, seed coloring with triphenyltetrazolium chloride or fluorescein diacetate (Nontachaiyapoom et al 2011;Dowling and Jusaitis 2012;Lemay et al 2015;Seaton et al 2015). Viable seed will grow and develop into protocorms.…”