2.5 Seed symbiotic and asymbiotic cultures
Seed symbiotic and asymbiotic cultures were conducted according to the
methods described by Zhou and Gao (Zhou & Gao, 2016). Mixed seeds of
each treatment were removed from storage at -20℃ and kept at ambient
temperature for 10 h. Appropriate amount of seeds of each treatment were
surface-sterilized using sodium hypochlorite solution containing 0.1%
available chlorine for 3 min followed by washing with
ddH2O for three times. Each sterilized circular nylon
cloth with a diameter of 2.6 cm and spores of 45 µm was inoculated with
50-100 surface-sterilized seeds using a pipette. Then it was transferred
individually to a cylindrical glass bottle (height 9 cm, diameter 6.5
cm) containing 35 mL of AGS (MS 0.9 L/L, natural mature coconut juice
0.1 L/L, carbon powder 1 g/L, sucrose 20 g/L, and agar 6 g/L) or
OMA+Epa-01 medium (OMA: oat 4 g/L, agar 8 g/L, pH =
5.8; Epa-01: a highly compatible fungus, which promotes seed germination
and protocorm development of P. teres ) such that its surface was
completely covered with the Epa-01 strain colony. Each treatment had at
least 60 replicates.
Thereafter, all glass bottles containing OMA+Epa-01and AGS medium were incubated at 26.0 ± 0.5℃ and 14 / 10-h light/dark
cycle for 60 and 70 days, respectively. Developmental stages of P.
teres seeds were determined according to the methods described by
Stewart and Kane (Stewart, 2008). P. teres seeds that reached
developmental stage ≥2 were considered as germinated. P. teresseedlings with roots were considered as developmental stage 6. Seed
states, including developmental stage, germination rate, and fresh
weight of germinated seeds, were recorded after incubation. Electronic
balance with high accuracy (one-millionth) was used to measure fresh
weight of germinated seeds. During incubation, glass bottles
contaminated with other fungi were discarded. The calculation of average
developmental stage for each treatment was based on the methods
described by Nontachaiyapoom et al (Nontachaiyapoom et al., 2011).