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After five rounds of non-selective subculture, the transformants were transferred to MEA plates containing 50μ.ml-1 hygromycin B. This experiment was repeated twice.

Individual transformants were transferred into PDA medium containing hygromycin B or Chlorimuron-ethy1and incubated until conidiogenesis.

Then, the obtained transformants were transferred onto media plates containing glucose as carbon source allowing only yeast cells with functional TPI to grow as well as onto plates containing 3% ethanol/0.1% glucose as control.

Rooted primary (T0) transformants were transferred into soil, flowering plants were self-pollinated and T1 seeds were collected.

When nodule formation was examined, the transformants were transferred to nitrogen-free Broughton and Dilworth medium and then inoculated with M. loti Tono.

For sporulation, the transformants were transferred to small PDA plates and purified by plating conidiospores onto PDA plates with 0.1% Triton X-100 as colony restrictor.

Single transformant colonies were transferred to liquid selective medium, grown to an OD600 of 0.4 and split into two tubes.

Positive transformant seedlings were transferred to soil.

Subsequently, the transformed cells were transferred to HB media containing 40 mg/L Basta for one month for selection of transformants.

Yeast transformants (Leu+, Trp+, and His+) were transferred onto nylon membranes, permeabilized in liquid nitrogen, and placed on Whatman 3 MM paper previously soaked in Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM MgCl2, 50 mM 2-mercaptoethanol, pH 7.0) containing 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal).

Transformants or wild type strains were transferred into glass tubes with increasing concentrations of paromomycin in JM.