Plant materials
Arabidopsis pumila seeds were preserved in our laboratory. The seeds were sterilized by rinsing with absolute ethanol for 1 min, and then with 20% sodium hypochlorite for 10 min, and finally with sterile water six times. Sterilized seeds were sown on Murashige and Skoog (MS) agar medium with 3.0% (w/v) sucrose and 0.8% (w/v) plant agar added. Seedlings were grown in climate-controlled conditions at 22 ± 1 °C, 300 μmol m−2 S−1 of light intensity, 16 hours light/8 hours dark, and 70% relative humidity. Pieces of young root (1 cm long, Fig.1a, c), hypocotyl (1 cm long, Fig.1a, b), leaf (1 cm2, Fig.1d, e) and petiole (1 cm long, Fig.1d, e) tissue were collected from 8-day-old and 30-day-old seedlings to serve as explants (Fig. 1).
Adventitious shoot induction
Adventitious shoots were induced under conditions of 22 ± 1 °C, 300 μmol m−2 S−1 light intensity and 70% relative humidity. Explants of young root, hypocotyl, leaf, and petiole were cultivated on induction medium containing 0.5 mg/L 6-Benzylaminopurine (6-BA) and 0.1 mg/L α-Naphthalene acetic acid (NAA).
The induction medium was supplemented with 3.0% (w/v) sucrose and 0.8% (w/v) agar powder, and the pH was adjusted to 5.8. The medium was autoclaved at 121 °C for 20 min and then cooled to 55 °C before adding the plant hormones, and was finally placed into 150 mL sterilized flasks (about 25 mL per flask). The basic MS medium and hormones (6-BA and NAA) used in this study were purchased from Beijing Boao Tuoda Biotechnology Co., Ltd. (Boao Tuoda, Beijing, China).
Root induction and plant domestication
The elongated adventitious shoots (1–2 cm) were transferred for cultivation to MS medium (pH 5.8) containing 0.1 mg/L NAA and 500 mg/L carbenicillin (Carbenicillin, Carb) at 22 ± 1 °C with a 16/8 h (light/dark) photoperiod with 300 μmol m−2 S−1 light intensity until roots were induced. In order to adapt the cuttings to the growth environment, the flasks containing the test tube plantlets were moved from the culture room to a greenhouse for 4–5 d. The seedlings were then carefully removed from the flasks, rinsed with tap water, and transferred to a culture pot containing 1:1 (w/w) nutrient soil to vermiculite.
Agrobacterium culture and transformation process
Based on previous studies of the full-length transcriptome of A. pumila under salt stress, it was found that two P5CS homologous genes exhibited elevated expression characteristics while under salt stress (Yang et al. 2018) (Supplementary Fig. 1). We cloned the P5CS1.1 gene, and after sequencing (the length of ORF is 2154 bp), and then connected it to the hygromycin-resistant pCAMBIA1301 vector. After double digestion with Nco I and Bst EII to verify the plasmid, the correctly constructed 35S:ApP5CS1.1 vector plasmid (Supplementary Fig. 2) was transferred into Agrobacterium tumefaciens GV3101. A single colony of Agrobacterium tumefaciens was cultured at 28 ℃ in Luria-Bertani liquid medium containing 50 mg/L kanamycin, 25 mg/L rifampicin, and 50 mg/L gentamicin until the culture density reached an OD600 of 0.8. Agrobacterium tumefaciens cells were obtained by centrifugation at 5000 rpm for 10 min, and the cells were suspended in a 1/2 MS solution (pH 5.8) containing 3.0% (w/v) sucrose and 20 mg/L acetosyringone.
The petiole and leaf explants were cut on an ultra-clean workbench and infected in infection solution of OD600 = 0.6 for 5 min and 10 min, and an infection solution with OD600 = 1.0 for 5 min and 10 min, respectively. The experimental design comprised a total of four treatments, each with three replicates. Sterile filter paper was used to absorb excess bacteria, and the samples were incubated on symbiotic medium (MS basal medium containing 0.5 mg/L 6-BA, 0.1 mg/L NAA, and 20 mg/L AS; pH 5.8) for 2 d in the dark. The petiole and leaf explants were then transferred to selection medium (MS basal medium containing 0.5 mg/L 6-BA, 0.1 mg/L NAA, 10 mg/L Hygromycin B, and 500 mg/L Carb; pH 5.8) for shoot induction. The samples were transferred to fresh selection medium once a week. The induction status of the calluses and adventitious shoots were recorded every 7 d during the culture period. The shoot samples (1–2 cm long) were then cut and transferred to MS agar medium (pH 5.8) and supplemented with 0.1 mg/L NAA and 500 mg/L Carb for root induction.
Genomic DNA extraction and PCR identification
The genomic DNA was isolated from leaves of transgenic and wild-type A. pumila plants via the Fastpure plant DNA isolation Mini Kit (Vazyme, Nanjing, China). For PCR analyses, the forward primer 5´-ATGGAGGAGCTAGATCGTTCA-3´, reverse primer 5´-TTAGGCTTGGATGGGAATGTC-3´ were designed according to the CDS of ApP5CS1.1 (GenBank accession number: MW227238). The final reaction mixture (20 μL) contained 2 μL 10 × Taq buffer, 1.5 μL 2.5 mmol/L dNTP, 0.5 μL forward and reverse primers respectively, 0.5 μL Taq DNA polymerase (Takara, Dalian, China), and ddH2O supplement. The reaction conditions were 94 °C 2 min, followed by 35 cycles of 30s at 94 °C, 30s at 56 °C, 2 min 20s at 72 °C, and 72 °C 10 min for extension.
Quantitative real-time PCR (qRT-PCR) assays
The seedlings of wild type and transgenic A. pumila plants were collected and immediately stored in liquid nitrogen. Total RNA was extracted using RNAprep Pure Plant Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. After purification using DNase without RNase (Tiangen, Beijing, China), the cDNA synthesis reactions were performed using the PrimeScript II first Strand cDNA Synthesis Kit MIX (Bioteke, Beijing, China) following the manufacturer’s instructions with 300 ng of total RNA per reaction. qRT-PCR was carried out on an Applied Biosystems 7500/7500-Fast Real-Time PCR system (ABI, Foster City, CA, United States) with the 2 × ChamQ Universal SYBR Green qPCR Master Mix (Vazyme, Nanjing, China). The conditions of PCR were performed according the methods by Huang et al. (2017). The gene-specific forward primer of ApP5CS1.1 was 5´-GGAAGAATCGTTGGTGGCTC-3´, and the reverse primer was 5´-ACAAGTGCATCAGGTCGAGA-3´. Glyceraldehyde-3-phosphate dehydrogenase gene (MW227237) was used as an internal reference control (Jin et al. 2019). Three biological replicates were performed with RNA isolated independently and each RT reaction had three replicates. The initial denaturation time was 94 °C 30s, followed by 40 cycles at 95 °C 15s, 60 °C 15s, 72 °C 15s, and finally extended at 72 °C for 10 min. The relative gene expression levels were calculated by 2–ΔΔCt method (livak and schmittgen 2001).