Wild type and mutant EiGS1 (EiGS1-1-WT and EiGS1-1-R59, respectively) coding sequences were amplified using the primer pair pOX-GS1-F and pOX-GS1-R (Table 2). The PCR conditions were 35 cycles at 98°C for 10 s, 55°C for 15 s and 72°C for 30 s, followed by 72°C for 7 min. The amplicons were sub-cloned into the pOX vector using the In-fusion HD Cloning kit (Takara). The recombinant vectors were introduced into Agrobacterium tumefaciens strain EHA105, which was then used to transform Nipponbare rice.
Hygromycin-resistant (50 mg L -1 ) and proliferating calli were transferred onto fresh N6D plates containing glufosinate at 0, 50, 100, 200 or 400 μM. For each glufosinate concentration, 10 transformed calli were used, and growth response to glufosinate compared between WT and R59 transformants 18 days after treatment. Hygromycin-resistant and glufosinate-untreated calli were transferred to differentiation and rooting medium to obtain transgenic rice seedlings. The introduction of the transgene into rice calli and seedlings was confirmed by PCR using the primer pair HygF/HygR (Table 2) amplifying the hygromycin phosphotransferase (hpt) gene.
Seedlings of five T1 WT and five mutant R59 transgenic lines were tested for response to glufosinate (990 g ha -1 ). Surviving plants from each line were grown to maturity to obtain T2 seeds.
The EiGS1-1 gene copy number in T2 rice plants was estimated by qPCR (Ding et al., 2004) with the sucrose phosphate synthase (SPS) gene used as the endogenous reference gene. Primers for EiGS1-1 and SPS gene copy number detection are listed in Table2. Three WT and three R59 T2 lines with a single copy of EiGS1-1 were used for the glufosinate sensitivity test. These T2 lines were foliar sprayed with glufosinate (0, 248, 495, 990, 1480 and 1980 g ha -1 for WT and 0, 495, 990, 1480 WT and 0, 495, 990, , 1980 WT and 0, 495, 990, and 2970 g ha -1 for R59), and plant survival was determined three weeks after treatment. The experiment was conducted in a glasshouse during the normal warm rice growing season at Guangdong Academy of Agricultural Sciences, China. There were three replicate pots per treatment each containing eight seedlings.
Hygromycin-resistant (50 mg L -1 ) and proliferating calli were transferred onto fresh N6D plates containing glufosinate at 0, 50, 100, 200 or 400 μM. For each glufosinate concentration, 10 transformed calli were used, and growth response to glufosinate compared between WT and R59 transformants 18 days after treatment. Hygromycin-resistant and glufosinate-untreated calli were transferred to differentiation and rooting medium to obtain transgenic rice seedlings. The introduction of the transgene into rice calli and seedlings was confirmed by PCR using the primer pair HygF/HygR (Table 2) amplifying the hygromycin phosphotransferase (hpt) gene.
Seedlings of five T1 WT and five mutant R59 transgenic lines were tested for response to glufosinate (990 g ha -1 ). Surviving plants from each line were grown to maturity to obtain T2 seeds.
The EiGS1-1 gene copy number in T2 rice plants was estimated by qPCR (Ding et al., 2004) with the sucrose phosphate synthase (SPS) gene used as the endogenous reference gene. Primers for EiGS1-1 and SPS gene copy number detection are listed in Table2. Three WT and three R59 T2 lines with a single copy of EiGS1-1 were used for the glufosinate sensitivity test. These T2 lines were foliar sprayed with glufosinate (0, 248, 495, 990, 1480 and 1980 g ha -1 for WT and 0, 495, 990, 1480 WT and 0, 495, 990, , 1980 WT and 0, 495, 990, and 2970 g ha -1 for R59), and plant survival was determined three weeks after treatment. The experiment was conducted in a glasshouse during the normal warm rice growing season at Guangdong Academy of Agricultural Sciences, China. There were three replicate pots per treatment each containing eight seedlings.
