Seeds of okra (Abelmoschus esculentus L.) “xian zhi” were purchased from Vegetable Research Institute of Zhejiang Academy of Agricultural Sciences. The seeds were germinated in 3-cm-diameter black plastic pots filled with a mixture of vermiculite, peat, and perlite (1:2:1, v:v:v) and grown in a growth chamber with a 14/10-h photoperiod under a light intensity of 300 μmol m− 2 s− 1 at 28/24 °C day/night temperatures, and at 60% RH. All seedlings were uniformly watered every 2 days and fertilized weekly with 1/2 strength Hoagland’s solution [50]. When the two cotyledons were fully expanded, seedlings of uniform size were transferred to larger plastic pots (7-cm-diameter).

To determine the effects of exogenous application of melatonin in salt stress tolerance of plant, seedlings (with a true leaf and a newly unfolded young leaf) were first irrigated with 0, 50, or 100 μM melatonin (50 mL per plant) on roots for a total of three times (once every other day). Melatonin was dissolved by alcohol, and then diluted with Milli-Q water. Six days later, the plants were irrigated with 300 mM NaCl (50 mL per plant). Seven days later, samples of leaf (the second true leaf beneath the growing point) were harvested for biochemical assay after measuring physiological parameters. Harvested samples were rapidly frozen in liquid nitrogen and stored at − 80 °C until the biochemical measurements [50]. A 50 μM concentration of melatonin was used in the following experiments.

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