Asymbiotic Culture Research Articles

Phytohormone pre-treatment for the enhancement of seed germination and protocorm formation by the terrestrial orchid, Calanthe discolor (Orchidaceae), in asymbiotic culture

Pre-soaking the seeds of Calanthe discolor Lindl. for 7 days in solutions containing α-naphthaleneacetic acid (NAA) at 1–100 mg l −1, 2-chloroethane phosphonic acid (Ethephon) at 1000 mg l −1 or 6-benzylaminopurine (BA) at 10, and 100 mg l −1 prior to germination in a growth-regulator-free medium resulted in overall 1.5–2.9 fold increases in protocorm formation compared with control cultures. Gibberellic acid (GA 3) exerted no stimulatory effect on seed germination and protocorm formation. The possible mechanisms of seed germination and protocorm formation of this species are discussed.

Light enhancement of symbiotic seed germination and development of an endangered terrestrial orchid ( Platanthera integrilabia)

Seeds of an endangared orchid, Platanthera integrilabia, were exposed to one of three light treatments: 7 days initial darkness followed by 16 h per day photoperiods, 16 h per day photoperiods for 7 days followed by darkness, and 16 h per day photoperiods. Alternatively, a continuous dark treatment in symbiotic and asymbiotic culture was provided. Seeds exposed to 16 h per day photoperiods or an initial 7 days of darkness followed by 16 h per day photoperiods were largely inhibited from germinating. Seeds exposed to 16 h per day photoperiods during the photoperiods during the first 7 days after fungal inoculations followed by darkness had a significantly higher percent germination than the other treatments, including those in continuous darkness. Seedling (protocorm) development (i.e., formation of leaf primordia) was also enhanced by initial light compared to seedlings under continuous darkness. We speculate that light exposure followed by darkness occurs naturally starting with the shedding of seeds from capsules to their immersion in a substrate where germination occurs. The beneficial effects of light in this study argues in support of light usage to stimulate germination and seedling development of temperate terrestrial orchids.

Temperature sensitivity of in vitro germination and seedling development of Dactylorhiza majalis (Orchidaceae) with and without a mycorrhizal fungus

Abstract. Symbiotic germination and development in vitro of Dactylorhiza majalis seeds with a strain of Rhizoctonia is very temperature dependent. Above an optimum at 23–25 °C there is a marked decline in germination percentage. Seeds that did germinate at higher temperatures had only little or no development of mycorrhiza, and developed few or no rhizoids compared with seedlings raised at optimal or lower temperatures. Six‐week‐old seedlings grown for additional 4 weeks on a range of temperatures had an optimal length increase at 23–24.5 °C mean temperature. At superoptimal temperatures (26 °C), the seedlings contained smaller starch reserves than those at lower temperatures and increased about as much in length as seedlings grown at 13 °C but much less than those grown at optimum. Temperature also influenced the differentiation of the leafy shoot, seedlings growing to a larger size before shoot initiation in the temperature range of optimal growth. Because of the small span between optimal and too‐high temperatures, a careful assessment of temperature optimum will be necessary in any orchid/fungus relationship before judging the success of symbiosis. At optimal temperature, symbiotic germination gave a germination percentage about twice that using a good asymbiotic method. The increase in seedling length was about 45% per week in symbiotic culture compared with less than 30% in the asymbiotic culture.

Ultrasonic treatment for enhancing seed germination of terrestrial orchid, Calanthe discolor, in asymbiotic culture

Mature seeds of Calanthe discolor were sterilized with 1% sodium hypochlorite solution and sown in liquid media after ultrasonic treatment. The seed coat was removed from almost all the seeds after 4 min of sonication treatment. However, prolonged treatment of more than 8 min increased the percentage of destroyed embryos. The rate of seed germination was significantly increased by the sonication treatment. Less than 10% of the seeds germinated in the control cultures, whereas 60% germinated in the cultures with sonication treatment for 4–16 min.

Mycorrhizal Associations and Seedling Development in Australian Lobelioideae (Campanulaceae)

The mycorrhizal associations of Australian Lobelioideae (Lobelia, Pratia and Isotoma) were found to be complex. Perennial species of these genera were solely VA mycorrhizal, whereas annual species were both VA and ectomycorrhizal. Lobelia gibbosa, L. simplicicaulis and L. rhomblfolia had unusual inter- cellular mycorrhizas formed with ectomycorrhizal ascomycetes. While all annual Australian Lobelioideae formed associations with both VA and ectomycorrhizal fungi, five species of Lobelia from South Africa or North America were solely VA mycorrhizal, whether annual or perennial. Seed of the Australian Lobelioideae examined required a ripening period of several months or longer before germination. Seed of L. gibbosa, L. simplicicaulis and L. rhombifolia on germination produced only a short radicle that did not develop further unless invaded by a mycorrhizal fungus. Seed of L. gibbosa and L. simplicicaulis placed beside growing mycorrhizas of certain ascomycetes with young plants of Melaleuca uncinata germinated, and the seedlings became mycorrhizal, grew, and flowered. The subterranean stage dependent on mycorrhizal association with a companion plant lasted 3-4 months. Germinated seed of L. gibbosa on simple agar media failed to become mycorrhizal with appropriate ascomycetes or to develop further. In asymbiotic culture on media incorporating sucrose seedling growth was slight but, in the presence of sucrose and kinetin or N-benzylaminopurine, L. gibbosa developed shoots and often formed small plants that flowered. Root development, however, was scanty or absent even in the presence of the growth substances α-naphthaleneacetic acid or 2,4-Dichlorophenoxyacetic acid. Young plants of L. gibbosa from the field transplanted to U.C. mix or sterilised forest soil failed to produce new roots. The plants grew slowly but remained small and produced few flowers, suggesting that mycorrhizal companion plants may also contribute to their growth besides being necessary in seedling development.

THE PHYSIOLOGY OF SYMBIOSIS IN GOODYERA REPENS

SummaryIn asymbiotic culture, Goodyera repens germinated and grew well in the presence of 1% glucose and potato extract. Growth on trehalose, a fungal sugar, was comparable with that on glucose. Infected protocorms which grew best on a cellulose carbon source were larger than asymbiotic ones on all media tested.Asymbiotic protocorms grew slowly and acquired high levels of starch while infected material grew rapidly without accumulating starch. When starch‐rich protocorms were infected, their starch became depleted while their growth rate increased, indicating that the fungus suppressed starch synthesis by its host.When green plantlets were fed with 14CO2, infected plantlets showed greater incorporation into insoluble substances than did asymbiotic ones.