Optimization of plant regeneration efficiency conditions
To increase plant regeneration efficiency, the following single factors were optimized based on a preset general regeneration media (MS + 30 g/L sucrose + 7 g/L agarose + 0.5 mg/L NAA + 4 mg/L 6-BA + 4 mg/L AgNO3, pH 5.8), explant type (hypocotyl, cotyledon-petiole, and root), sucrose concentration (0, 10, 20, 30, 40, and 50 g/L), 6-BA concentration (0, 2, 4, 6, 8, and 10 mg/L), and AgNO3 concentration (0, 2, 4, 6, and 8 mg/L). Thirty-six explants were used for each treatment with three replicates. The rate of adventitious shoots was evaluated after cultivation for 20 days. The optimal conditions with the appropriated factor combination were selected for subsequent transformation experiments.
Optimization of Agrobacterium-mediated transformation factors
To improve genetic transformation efficiency, single-factor experiments were designed to screen optimal conditions. The effect of Hyg was first evaluated because it is critical for resistant shoot screening. Based on the previous studies and the plant regeneration conditions we established, the preset transformation conditions were as follows: pre-cultivation for one day, OD600 value = 0.5, infection 15 min, and co-cultivation for two days. The selection medium was preset as MS + 30 g/L sucrose + 7 g/L agarose + 0.5 mg/L NAA + 4 mg/L 6-BA + 4 mg/L AgNO3 + 200 mg/L TMT + 20 mg/L Hyg (pH 5.8, 25 ± 2℃, 16 h/d). The root induction medium was preset as MS + 1.5 mg/L IBA + 1.5 mg/L NAA + 200 mg/L TMT.
Different concentrations of Hyg (0, 5, 10, and 15 mg/L) were first screened to determine the threshold concentration for untransformed explant elimination. Two replicates with 100 untransformed explants each were used. After that, Hyg concentrations (10, 15, 20, and 25 mg/L) were evaluated for resistant shoot differentiation using 50 explants with two replicates. Other factors, including TMT concentration (0, 100, 150, and 200 mg/L), AS concentration (50, 100, 150, and 200 µM), pre-culture duration (0, 1, 2, and 3 days), Agrobacterium strains (GV3101, LB4404, EHA105, and GA101), and suitable hormone proportion for root induction were optimized.
Agrobacterium -mediated plant transformation
The plasmid of pCAMBIA1305.1-35S-BraA1000785 was transformed into the Agrobacterium strains and cultured in 1 mL of LB medium containing 50 mg/L kanamycin and 50 mg/L gentamicin at 28ºC overnight. Positive transformation was confirmed by PCR using BraA1000785-F/R primers (Additional file 1 Table S1). A transformant was picked and inoculated in LB liquid medium with an agitation of 220 rpm for 12 h until OD600 was 0.5. Then, a 2-mL aliquot was collected and centrifuged at 6,000 rpm for 10 min. The bacterial precipitate was suspended in an equal volume of DM medium (MS medium with 100 µm/L AS, Table 1) and repeated twice. Finally, the bacterial solution was diluted 10-fold with DM medium for explant infection.
The explants were immersed in an infection solution of Agrobacterium for 15 min (gently shaking at 5-min interval). After infection, the bacterial suspensions were removed, and the explants were blotted dry on sterilized paper towels and subsequently cultured on co-cultivation medium (Table 1) in a controlled growth room at dark conditions. After two days of co-cultivation, the explants were transferred onto a selection medium (Table 1) for shoot induction at 14-day intervals until resistant shoots developed. Hygromycin-resistant shoots 2–3 cm in length were cut and transferred to a root-induction medium (Table 1). Regenerated plantlets with well-developed roots were thoroughly washed in tap water to remove Phytagar™ and then transferred to pots and grown for 25 days in the same environmentally-controlled growth chamber as described above. The acclimatized plantlets were then transplanted to the greenhouse.
The optimal conditions and the entire procedure for transformation are presented in Table 1 and Fig. 1. To test the stability of the system, three replicate experiments were conducted using the established optimal conditions.