Materials and Methods
Plant
materials and growth conditions
The mycorrhizal tubers of G. elata were developed from the
symbiotic culture with Armillaria strain 2, according to a
standard protocol (Yeh, Liao, Huang, Miyajima & Lee 2017). In brief,
timber blocks of Liquidambar formosana were inoculated with PDA
cubes containing Armillaria. After one month of culture, the
protocorm-like bodies (PLBs) derived from G. elata callus were
placed on the colonized timber blocks and grown until they formed
juvenile tubers. During 18 months of symbiotic cultivation,
G. elata tubers were harvested at seven stages of development for
analysis (Fig. S1). Mature tubers were collected from about 1.5 years of
symbiotic cultivation. Tubers were frozen at -80 oC before analysis.
Arabidopsis thaliana and transgenic Arabidopsis plants used in
this study were all Columbia ecotype (Col-0). For all experiments, seeds
were surface-sterilized (30% CLOROX bleach and 0.1% Triton X100, v/v)
and then stratified at 4 oC for three days.
Seeds were germinated and grown on one-half-strength Murashige and Skoog
basal salt mixture agar media (½MS) or potting soils in a growth chamber
(22 °C and 16/8 h light/dark regime, with ~80 μmol m-2
s-1 illumination) for most experiments (except where indicated). For
root growth analysis, seeds were germinated on half-strength MS medium
supplemented with 0%, 1%, 2%, 5% or 7% sucrose, and grown for 7 d
before imaging. For leaf area measurement, seeds were sown and grown on
half-strength MS medium containing 2 µg ml-1 hygromycin for 7 d under
short-day conditions (10/14 h light/dark regime). Seedlings of similar
sizes were then transferred to soil and grown for another 3 wks. All
leaves were imaged by a scanner. Primary root length and leaf area were
quantified using ImageJ.