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.