Overexpressing Soybean Salt Tolerance Gene GmCHX1

The full-length coding sequence of GmCHX1 from W05 was cloned into the binary vector V7 (ref. 56 (link)) between XbaI and XhoI sites downstream of the constitutive Cauliflower Mosaic Virus 35S promoter. As a negative control, the gene for the GFP was cloned instead of GmCHX1 using the same vector and promoter. Both constructs were then transformed into the salt-sensitive parent C08. The soybean hairy root transformation and salt treatments were performed as previously described57 with some modifications. Surface-sterilized soybean seeds were germinated on germination medium (15 mg l⁻¹ NaH₂PO₄·H₂O, 1 mM CaCl₂·2H₂O, 25 mM KNO₃, 1 mM (NH₄)₂SO₄, 1 mM MgSO₄·7H₂O, 0.1 mM Na₂EDTA·2H₂O, 0.1 mM FeSO₄·7H₂O, 10 mg l⁻¹ MnSO₄·2H₂O, 2 mg l⁻¹ ZnSO₄·7H₂O, 3 mg l⁻¹ H₃BO₃, 0.25 mg l⁻¹ Na₂MoSO₄·2H₂O, 0.025 mg l⁻¹ CuSO₄·5H₂O, 0.025 mg l⁻¹ CoCl₂·6H₂O, 0.75 mg l⁻¹ KI, 1 × B5 vitamin (10 mg l⁻¹ thiamine, 1 mg l⁻¹ pyridoxal phosphate, 1 mg l⁻¹ nicotinic acid and 100 mg l⁻¹ myo-inositol), 2% sucrose, 0.6% agar, pH 5.8) for 4 days (16 h light/8 h dark). Agrobacterium rhizogenes strain K599 containing the recombinant constructs was grown in yeast extract peptone medium containing 50 mg l⁻¹ kanamycin and 200 μM acetosyringone at 28 °C for 16 h. It was then used to infect the cotyledons through scalpel incisions. The cotyledons were co-cultivated with A. rhizogenes in the dark for 5 days on moist filter paper. After that, the infected cotyledons were transferred to root-inducing medium (4.3 g l⁻¹ Murashige and Skoog (MS) medium, 1 × B5 vitamin, 3% sucrose, 250 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin). After 2 weeks, cotyledons with roots emerging from the incision sites were transferred to new root-inducing medium with 100 mM NaCl or medium without NaCl as untreated control. Root mass was weighed about 2 weeks after treatment.