For each treatment, ca. 300 seeds were cultured in a 500-ml culture
flask containing 90 ml of medium. All experiments consisted
of three independent replicates with 10 culture flasks per replicate.
Cultures were observed every 30 days for signs of germination
and subsequent protocorm development using a Zeiss stereomicroscope.
Developmental stages (Table 1, Fig. 1a–f) were adapted
from Stewart and Zettler (2002) and Johnson and Kane (2007). The
percentage of seed/protocorms at each developmental stage was
calculated by dividing the number of seed/protocorms in each stage
by the total number of cultured seeds (including viable and nonviable
seeds) in each flask. Germination was considered to have
occurred only if a swollen embryo was present and if the testa had
ruptured (Stage 1). The start of germination was calculated when
round or ovoid hyaline embryos (viable embryos) were present.