| Summary: | Screening and identification of tolerant genotypes using osmotic materials under in vitro culture could be rapid, easy, and even accurate. In this research, three Iranian landraces of <i>Cucurbita</i> sp. included Tanbal Ajili (<i>Cucurbita maxima</i> Duch.), Ajili Razan (<i>Cucurbita pepo</i> L.), and Balghabakhi (<i>Cucurbita moschata</i> Duch.) seeds were cultured in ¼ MS medium. After germination, plantlets were transferred to MS media containing mannitol and PEG 6000. Mannitol and PEG at three concentrations of 0.1, 0.2, and 0.4 M and 0.009, 0.012, and 0.015 M, respectively, were added into the MS medium, while the MS medium without any adding was used as control. Our findings revealed that osmotic treatments significantly increased shoot and root dry weight (DW), malondialdehyde (MDA), and proline content, but significantly reduced coleoptile length, shoot and root fresh weight, and photosynthesis pigments content. Protein content, phenols, and flavonoids content, enzymatic and non-enzymatic antioxidant including ascorbate peroxidase (APX), guaiacol peroxidase (GPX), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) activity, reduced ascorbate (AsA), reduced ascorbate/dehydroascorbic acid (AsA/DHA), reduced glutathione (GSH), dehydroascorbic acid (DHA) and oxidized glutathione (GSSG), and reduced glutathione/oxidized glutathione (GSH/GSSG) were significantly increased at moderate osmotic stress induced by mannitol and PEG. In contrast, the previous physiological parameters were significantly reduced at higher water deficit conditions. With respect to most attributes and concentrations, mannitol simulated osmotic stress better than PEG. Our results revealed that applying PEG and mannitol under in vitro conditions could be an efficient way to evaluate and screen cucurbit genotypes for future breeding programs.
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