Gelrite

Rice (Oryza sativa L. cv. Nipponbare) was used as the WT control. Seeds from T1 to T4 of the P_OsUbi7-BSR1 and P_PR1b-BSR1 lines were sown on half-strength MS medium (Wako, Osaka, Japan) containing 3% sucrose, 0.4% Gelrite (Wako), and hygromycin B (30 μg/mL; Wako), and the hygromycin-resistant seedlings were selected on this medium. WT seeds were sown and grown on the same medium without hygromycin B. WT and hygromycin-resistant transgenic seedlings were then transplanted into pots containing soil (Bonsol no. 2; Sumitomo Kagaku Kougyo, Osaka, Japan) and grown in a greenhouse at 27–30 °C, as previously described [19 (link)].

Agrobacterium-mediated transformation of rice was performed as described previously (Toki et al., 2006 (link)). Rice (Nipponbare) was used for GT transformation following a method described previously (Saika et al., 2011 (link)). Calli transformed with Agrobacterium harboring pKOD4/mALS were selected on callus induction (N6D) medium solidified with 0.4% gelrite (Wako Pure Chemical Industries) containing 50 mg/L hygromycin and 25 mg/L meropenem (Wako Pure Chemical Industries, http://www.wako-chem.co.jp/). GT candidate calli were transferred to N6D medium without hygromycin and meropenem and cultured for 1 month. For marker excision, GT candidate calli were transformed with Agrobacterium to introduce an expression vector encoding hyPBase, and were selected on N6D medium with 35 mg/L geneticin (Nacalai tesque, https://www.nacalai.co.jp) and 25 mg/L meropenem. For regeneration, transgenic calli were transferred to regeneration medium with 25 mg/L meropenem, and shoots arising from callus were transferred to Murashige and Skoog (MS) medium (Murashige and Skoog, 1962 ) without phytohormones. The hyPBase-expressing regenerated plants were subjected to marker excision analysis. The T₁ progeny plants were obtained from self-pollinating marker-free T₀ plants containing two point mutations, W548L and S627I, in the ALS locus and were subjected to further analysis.

Hybrid seeds of N. suaveolens × N. tabacum were obtained according to the method of Yamada et al.^{39 (link)}. The seeds were surface-sterilized by immersion in 70% ethanol for 30 s, followed by immersion in 5% sodium hypochlorite for 20 min. After washing three times with sterile water on a clean bench, seeds were sown in half-concentration MS medium⁴⁰ (0.5 × MS) that had been adjusted to pH 5.8 and supplemented with 1% sucrose and a gelling agent (0.2% Gelrite (FUJIFILM Wako) or 0.3% Phytagel (Sigma-Aldrich)). The medium containing the seeds then was incubated at 28 °C in an incubator with a photoperiod of 24 h. The number of seedlings that remained viable 20 days after germination was determined.

Escherichia coli and T. kodakarensis strains and plasmids used in this study are listed in Supplementary Table S1. Escheichia coli strains were cultivated at 37°C in Lysogeny broth (LB) medium. Ampicillin and gentamycin were added at a final concentration of 100 μg/ml and 30 μg/ml, respectively, when needed. Thermococcus kodakarensis strains were grown anaerobically at 85°C or 93°C in ASW-YT medium composed of 0.8-fold concentration of artificial seawater (ASW) (17 (link)), 10 g/l yeast extract and 5.0 g/l tryptone with either 2.0 g/l elemental sulfur (S⁰) (ASW-YT-S⁰) or 5.0 g/l sodium pyruvate (ASW-YT-Pyr), or in MA-YT-Pyr medium composed of 3.04 g/l Marine Art SF-1 (Osaka Yakken, Osaka, Japan), 10 g/l yeast extract, 5 g/l tryptone and 5 g/l sodium pyruvate (37 (link)). Plate media were solidified with Gelrite (Wako Pure Chemical Industries, Osaka, Japan) (17 (link)). All the anaerobic manipulations were done within the COY anaerobic chamber (COY Lab Products, Grass Lake, MI, USA).

The Solanum tuberosum L. cv. Sayaka was used. Potato plants were cultured on medium containing an MS basal salt mixture (Fuji Film Wako, Tokyo, Japan) (Murashige and Skoog 1962 ), 3% sucrose, and 0.3% Gelrite (Fuji Film Wako) with the pH adjusted to 6.0. Plants were grown under long-day conditions with 16 h light and 8 h dark at 23°C in a growth chamber. The regeneration of potato plants was performed as described previously (Ohnuma et al. 2020 ). Regenerated potato plants were cultivated using a growth room. They were grown under 16 h light (200–400 µmol m⁻² s⁻¹) and 8 h dark conditions at 23°C in a growth chamber. Tubers were harvested from wild-type and transformed potatoes, which were grown at 22°C under 16 h light and 8 h dark conditions in a growth chamber.