Harvest and Phenotyping
Plants were harvested prior to panicle emergence after six weeks of
growth over a five-day period. Individual plants were extracted from
pots by pulling the plastic bag to prevent root damage and plants with
attached roots were removed from the soil by shaking over wire mesh.
Soil rhizosphere samples for parents were collected by dipping root
systems into sterilized 50 ml tubes filled with 1X phosphate buffered
saline. Plants were then hung by the shoot base on a clamping apparatus
and soil particles were removed from the root system with a spray of
UV-sterilized water and roots were separated from shoots and preserved
in 90% ethanol for future phenotyping. Tiller number and flag leaf area
of the main tiller was measured. Shoot and leaf tissue were dried at
55°C and weighed separately to obtain aboveground biomass and to
calculate specific leaf area (SLA; fresh leaf area / dry mass of the
leaf (cm2 g-1)).
Root systems were phenotyped by
scanning the entire intact root system and one representative nodal root
with attached lateral roots on an EPSON 12000XL flatbed scanner (Epson
America, Inc., San Jose, CA, USA) calibrated for use with WinRhizo Pro
2019 root image analysis software (Regent Instruments Inc., Canada)
(full description in Appendix S1). For parents, a small portion of the
central root system was frozen for DNA extraction and PCR amplification
to determine root endosphere microbial community composition. The
remaining roots were collected and dried for 96 hours at 55°C, and
weighed.