2.5. Effects of Tulasnella isolate on germination of D. officinale seeds
Tulasnella isolates were grown on PDA in darkness at 25 °C. After 10 days, five PDA agar plugs (diameter 2 mm) with active mycelium from the colony margin were inoculated to a petri dish (diameter 9 mm) containing 15 ml sterile oat meal agar (OMA: oat 2.5 g l−1 agar 12 g l−1, the pH measured at 5.2 prior to autoclaving) with five nylon clothes (1 cm × 1 cm), and grown at 25 °C in dark. After 1 week, approximately 150 axenic seeds of D. officinale ( Fig. 1d) were sown on the surface of each cloth. Each treatment was replicated on five plates. The treatment without fungi was used as a control. All treatments were placed in tissue culture chamber in 12 h light at 25 °C for 77 days. To assess and record the seed germination as well as protocorm development, a stereomicroscope (LAEICA S8APO) was used. Stewart et al. (2003) had divided the seed germination and protocorm development of orchids into 6 stages (0–5) just as Table 1 showing, which used as a reference for assessing the percentage of seed germination and protocorm development of D. officinale.