**2. Materials and Methods**

#### *2.1. Plant Materials, Growth Conditions, and Cold Stress Treatment*

Seeds of *Corchorus capularis* (Y49, Y38, and Y1) and *Corchorus olitorius* (T8, W57, M33, M18) varieties were collected from the Institute of Bast Fiber Crops (IBFC), Chinese Academy of Agricultural Sciences (CAAS) Changsha, Hunan (Table 1). These varieties were previously screened out from large populations by studying low-temperature stress based

on their germination rate, survival rate, visual scoring under cold stress and physiological, and biochemical parameters. The selected jute varieties were distinguished as tolerant (Y49 and M33), intermediate (W57 and M18), and sensitive (Y38, T8 and Y1) to low temperature and used in this present study. Seeds of all varieties were carefully rinsed with sterile deionized water after 10 min of surface sterilization with 10% NaClO. Then seeds were placed in a 19 × 14.5 × 6 cm germination box with three layers of sterile filter paper. The box was placed in a brightly illuminated incubator set at 25 ◦C with daily water top ups for germination. After three days of sprouting, seedlings were moved to quarterstrength Hoagland nutritional solution containing 5.79 mmol L−<sup>1</sup> calcium and placed in culture pots (40 × 20 cm) (NO3)2, 8.02 <sup>μ</sup>mmol L−<sup>1</sup> KNO3, 1.35 mmol L−<sup>1</sup> NH4H2PO4, 4.17 mmol L−<sup>1</sup> MgSO4, 8.90 μmol L−<sup>1</sup> MnSO4, 48.3 μmol L−<sup>1</sup> H3BO3, 0.94 μmol L−<sup>1</sup> ZnSO4, 0.20 μmol L−<sup>1</sup> CuSO4, 0.015 μmol L−<sup>1</sup> (NH4)2MoO4, and 72.6 μmol L−<sup>1</sup> Fe-EDTA for subsequent growth [19]. Seedlings were grown in a culture room with a day/night temperature regime of 28/16 ◦C, a photoperiod of 16 h/8 h (light/dark), relative humidity of 60%, and a light intensity of 300 μmol m−2s−1. Every other day, the nutrition solution was replenished.

**Table 1.** Origin of 7 varieties of *C. capsularis* and *C. olitorius*.


Five-week-old morphologically uniform seedlings were selected for treatment and transferred to another chamber for low-temperature stress. The treatment chamber's temperature was set at 5 ◦C to simulate the low-temperature condition, and the plant maintained an optimal condition (28 ◦C) that was regarded as control. After 24 h of low-temperature stress, three to four fully expanded leaves were collected. Each sample contained a minimum of three individual plants of similar varieties mixed to form one sample. After collection, each sample tube was immediately steeped in liquid nitrogen and stored at −80 ◦C until analysis.
