Plants materials, cold treatment, and freezing tolerance assay
The wild-type B. rapa variety ‘Bre’ was grown under a 16-h
light/8-h dark (22 °C) photoperiod for 3 weeks. To analyze the miRNAs
abundance in ‘Bre’ under cold stress, some ‘Bre’ plants were moved to 4
°C condition for 7 days cold treatment and some were grown at 22 °C for
additional 7 days as control. The aboveground parts of treated and
untreated were then harvested, frozen in liquid nitrogen, and stored at
−80 °C until extraction of total RNA. Small RNA sequencing (one
biological replicate) was performed using the Illumina GAII sequencer at
BIG (ShenZhen, China).
The wild-type B. napus variety ‘K407’, a Semi-winter
ecotype, was grown under a 16-h light/8-h dark (22 °C) photoperiod with
light intensity of 250 μmol m−2s−1. For the cold resistance assay, the plants were
grown under standard conditions (22 °C) for 6 weeks before transferring
to a cold environment (4 °C or 2 °C, 16-h/8-h light/dark photoperiod).
The similar methods for studying cold stress in Brassica crops have been
used in previous reports (Raza et al., 2021; Hussain et al., 2022).
After cold treatment, the cold resistance of plants was determined. The
freezing tolerance assay was performed in a freezing chamber, after cold
acclimation for 24 h, in which wild-type and transgenic lines were
subjected to a −10 °C treatment for 2.5 h. The plants were then allowed
to recover at 22 °C for 7 days before determining the survival rate. The
Pro content was measured immediately after the freezing treatment using
a commercial assay kit (A107-1-1, Nanjing JianCheng Bioengineering
Institute, Nanjing, China), according to the manufacturer’s
instructions.
The wild-type B. rapa cultivar “Bre”, B. napus cultivar
‘K407’ are all inbred lines.