| 生长方案: |
Undifferentiated wild type (WT) friable calluses were induced from leaves of cardoon plants (“Spagnolo” genotype), following the method described in (Figueiredo et al., 1987), and gently provided for this study from Dr. M. De Palma (CNR-IBBR). Calluses were grown on Gamborg B5 agar medium including vitamins (Duchefa Biochemie, #G0209, www.duchefa-biochemie.com), supplied with 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mg/L adenine, 0.1 mg/L kinetin, 3% (w/v) sucrose, 7,5% (w/v) agar, adjusted to pH = 5.8. WT calluses were grown in the dark at 25°C, sub-cultured approximately every 30 days. Cell suspension cultures were started by inoculating 1.5 g of friable callus in a 250 mL Erlenmeyer flask containing 50 mL of liquid medium and kept on a gyratory shaker at 100 rpm in the dark at 25°C. To determine the cell growth, CVS was measured using 250 mL Erlenmeyer flasks with a graduated beak. This method has been shown to be a rapid and non-destructive one for the routine estimation of cell biomass, given how CVS is highly correlated with the fresh weight of cells (Blom et al., 1992). All measures were performed on biological triplicates. |
| 处理方案: |
Cell suspensions of WT cardoon were set in 250 mL sterile flasks and kept in the dark at 25°C in gentle agitation, until the start of exponential growth (5 days); next, 5 mL of cell suspension and 10 mL of fresh liquid medium were added to a 100 mL sterile flask, and agitated for 5 additional days. Meanwhile, a single colony of recombinant Agrobacterium was used to start a 50 mL inoculum in LB medium, which was grown at 28°C with 200 rpm shaking until OD600 = 0.70 – 0.90 (approximately 48 h), then pelleted (15 min at 2000 x g) and re-suspended in Gamborg B5 liquid medium to a final OD600 = 0.80-0.85. Co-cultivation was performed adding 1 mL of Agrobacterium cells to each cell suspension in presence of 400 M acetosyringone (Sigma Aldrich, #D134406, www.sigmaaldrich.com), leaving flasks in the dark at 25°C in gentle agitation for 48 h. Afterwards, pelleted cells were washed three times with fresh Gamborg B5 medium supplied with 200 mg/L CFX and 10 mg/L PPT before spreading them on sterile filter paper to remove the liquid excess; cells were then moved to solid selective medium containing CFX and PPT. After 30 days, emerging resistant calluses were individually transferred to new plates and separately sub-cultured approximately every 21 days to fresh selective medium. PCR was used to confirm the presence of the transgene and to evaluate residual Agrobacterium contamination using AGL1 and EHA105 colonies as positive controls (Haas et al., 1995). |
| 提取方案: |
For RNA extraction, 200 mg of callus were grinded under liquid nitrogen, lysed/homogenized with 1 mL of TRIzol (Thermo Fisher Scientific, #15596026) and, after addition of 0.2 mL of Phenol:Chloroform:Isoamyl-alcohol (25:24:1) and centrifugation (10 min, 13000 x g), the RNA was precipitated from the aqueous phase with 1 volume of isopropanol. Total RNA was treated using the Ambion TURBO DNA-free DNase (Thermo Fisher Scientific, #AM1907) and then reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, #4368814) according to the manufacturers’ instructions. |
| 建库方案: |
Sequencing libraries were prepared according to the manufacturer’s instructions using the Illumina TruSeq RNA Sample Prep kit and sequenced on NovaSeq 6000 Sequencing System (Illumina, www.illumina.com) to obtain 15-22M 150 bp paired-end reads per sample.
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