Evaluation location: , China
To analyze chromosome number and ploidy level of the Thinopyrum
accessions, we prepared chromosome spreads following
the procedures of Kato et al. (2006) and Han and Lv (2013)
with some minor modification. The seeds were germinated on
moist filter paper at 23°C for 48 h. Root tips of 1 to 2 cm
length were collected and pretreated with nitrous oxide (N2O)
at 10 atm pressures for 2 h in a gas chamber, as described by
Kato et al. (2006). The root tips were then fixed in 90% acetic
acid for 10 min and washed three times with distilled water.
The apical meristem of the root tips were collected in 0.5 mL
microcentrifuge tube and treated with a mixture of 1% pectinase
(Yakult Pharmaceutical, Tokyo, Japan) and 2% cellulase
(Yakult Pharmaceutical, Tokyo, Japan) for 45 to 50 min. Then
the root sections were washed three times with 75% ethanol and
crushed by steel needles to form a suspension solution of single
cells. After centrifugation at 4000 × g for 3 min, the cell pellet
was washed with 75% ethanol and resuspended in 100% acetic
acid. About 8 μL of cell suspension solutions were dropped
onto a clean slide. After the slide was air dried, the root cells
were examined under an Olympus BX53 phase contrast microscope.
A minimum of 15 metaphase cells were observed for
each accession.