Abstract
Effects of various factors on seed germination and subsequent protocorm growth of Eulophia graminea, an endangered terrestrial orchid species native to Japan, were examined using New Dogashima medium without applying plant growth regulators and natural ingredients. For the culture, aseptic mature seeds collected from undehiscent pods were used throughout of the study, and New Dogashima medium supplemented with 29.2 mM sucrose and 8 g/L agar was used as standard medium. For examining the effect of calcium hypochlorite on seed germination, the seeds were pre-treated for 0–60 min with the solution containing 1% available chlorine and cultured on standard medium. The effects of other factors were examined using the seeds without calcium hypochlorite pretreatment. The effects of culture temperature on seed germination and subsequent growth were examined at the temperature range of 15–35°C. Optimum sucrose concentration and suitable kind of carbohydrate were examined using 0-175.3 mM of sucrose and 29.2 mM of sucrose, maltose, trehalose and glucose, respectively. As the results, germination rate increased with increase in the treatment duration of calcium hypochlorite solution and the maximum germination rate was 85.8% at 60 min treatment. Optimum sucrose concentrations for seed germination were 29.2–58.4 mM, which gave low germination rates (7–8%). Among the four kinds of carbohydrates tested, trehalose gave the highest germination rate (26.8%). On the effect of temperatures, both the highest germination rate and subsequent growth with shoot development were obtained at 30°C. Direct shoot development from protocorm without forming rhizome was shown at this high temperature.