To avoid interference from plant size and reproduction stage, all plants were chosen from 2-month-old hardened transgenic Cavendish banana, Cavendish banana WT and ‘Dajiao’ plants, which were grown in plastic pots filled with a mixture of vermiculite and soil (1:1) under a photoperiod of 16 h/8 h (light/dark) at 25 °C. At this stage, there were no apparent differences in plant size and growth between the transgenic and Cavendish banana WT plants. To assess their cold resistance, transgenic lines and WT plants were directly exposed to 10 °C for 48 h without pre-acclimation, followed by recovery at an ambient environment for 5 days [41, 42]. Cold injury and recovery of the plants were recorded and photographed. The degree of cold damage was assessed after recovery as previously described [41].

Two transgenic lines (#11 and #13), Cavendish banana WT and ‘Dajiao’ plants, which were subjected to 10 °C for 48 h and subsequent recovery at normal conditions, were adopted in the present study. The leaves were collected before and/or after the cold stress to analyze the electrolyte leakage, Pro content, and MDA level. The leaves used for measurements of electrolyte leakage were cut into 1-cm segments and washed three times with ultrapure water. The segments were placed in tubes containing 5 mL of ultrapure water and incubated at 25 °C. After 2 h, the electrical conductivity of the bathing solution (L1) was measured. Then the tubes were incubated at 100 °C for 20 min and subsequently at 25 °C for 1 h, and the electrical conductivity (L2) was measured again. The relative electrolyte leakage was calculated by the formula (L1-L0)/(L2-L0) × 100 (L0, the conductivity of ultrapure water) [53]. MDA level and the Pro content were determined using the commercially available kits (Nanjing Jiancheng Bioengineering Institute, China). Five replicates were performed for each sample. Three independent experiments were performed as biological replicates.

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