Cold tolerance test
To conduct the cold tolerance test, germinating seeds of OE, knockout,
and NIP were seeded in a cup 10 cm in diameter; 16 seedlings remained
before cold stress treatment. The three-leaved seedlings (approximately
15 days old) were moved to a growth chamber at a temperature of 5 °C and
relative humidity of 80±5%. After cold treatment for 5 days, the
temperature in the growth chamber was gradually increased to 25 °C
within 24 h, at a rate of 0.5 °C/h. The seedlings were then transferred
to ambient conditions in a greenhouse for recovery. The number of
surviving seedlings in each cup was recorded on the 7th day after
recovery. The plant survival rate was used to evaluate the cold
tolerance of the tested lines. Three cups were used for each line and
the test was repeated twice.