Plants materials and growth conditions
Winter wheat (Triticum aestivum ) cultivar Dongnongdongmai1 (Dn1)
was obtained from the Wheat Breeding Institue of Northeast Agricultural
University, Harbin, China. The field trial referred to previous study
(Tian et al., 2021),was carried out in autumn, September 8th, 2018, at
Northeast Agricultural University Experimental Site (45°7’N, 126°6’E) in
Harbin, China. Then the tillering nodes were collected at 5℃ (October
15th, 2018), 0℃ (November 2nd, 2018), -10℃ (November 23rd, 2018) and
-25℃ (January 14th, 2019) (average of minimum temperature for ten
consecutive days) and store at -80°C. The indoor trail was carried out
as follow: Dn1 seeds were sowed in plastic pots (5 plants/ pot)
containing soil and vermiculite (1:1, v/v) and grown in green house for
15 days at 22℃ in a 16 h photoperiod under 160-180 μmol
m-2 s-1 light. Then seedlings were
transferred to 4℃ for cold acclimation. After 28 d, the Dn1 seedlings
were frozen at -10℃ for 2 h. the tillering nodes were pooled from each
seedlings and the tissue immediately frozen in liquid nitrogen and
stored at -80℃
Arabidopsis thaliana accession Columbia (Col-0) was used for gene
transformation and as a phenotypic control. The stratification and
planting conditions of Arabidopsis referred to previous study (J. Wang,
Ma, Kojima, Sakakibara, & Hou, 2013). Its stratification condition was
4°C for 3 d in darkness and culturing condition was as follow: 24°C,
16/8 photoperiod, light intensity of 120-150 μmol m-2s-1, and 60% relative humidity. During freezing
treatment, the soil culture one-month old Arabidopsis seedlings were
treated in low-temperature incubator at 4℃ for 3 d as cold acclimation
and then the temperature dropped to -10℃ for 2 h. After freezing
treatment, the plants were recovered for 7 d under normal condition. The
Arabidopsis survival rate was counted after the recovery phase. Plants
that turned yellow and could not be regenerated were defined as death.
Survival rates were calculated from the results of three repeated
experiments, and the amount of each plant was approximately 80 in one
experiment.