Plant materials and treatments
The seeds were obtained from Carthamus tinctorius L. cultivated in the medicinal botanical garden of Chengdu University of Traditional Chinese Medicine, Chengdu City, Sichuan Province, China, and stored at 4°C. The safflower seeds were rinsed under running water for 4 h, disinfected with 0.1% HgCl2 for 12 min, and rinsed 3 times with sterile water. The sterilized safflower seeds were sowed in MS medium (Table 1), under a 16/8 h (light/dark) photo-period at 25±2℃. After 7 days, sterile safflower seedlings were obtained.
callus was induced from the cotyledons of safflower sterile seedlings. In order to induce safflower callus, 6 induction medium containing different hormones were prepared in the study (Table 2). In addition to different hormones, these medias contained 4.43 g/L MS, 30 g/L sucrose and 8 g/L agar (Table 1). The medium was adjusted to pH 5.8-6.0 and sterilized at 121 ℃ for 20 minutes before being dispensed into petri dishes. Remove margins and midribs, wounds were made on the back, and then divided into 0.5 cm2, cotyledons were inoculated the back side down in the prepared medium. The inoculated cotyledons were cultured in the dark at 20 ℃ for 40d.
Agrobacterium‑mediated transient transformation
The binary vector pRI201 plasmid, harboring GUS and CaMV 35S promoter was selected in the experiment. The plasmids were induced into A. tumefaciens EHA105 competent (Tsingke, China) via the freeze/thaw method. The Agrobacterium spread on LB plates supplemented with 50 mg·L-1 Kana and 20 mg·L-1 Rif, and culture at 28 ℃ for 2-3 days in the dark.
Single clone of Agrobacterium was pick into LB liquid medium supplemented with the same antibiotics, and incubate at 28 ℃ for 3-4h at 180-220 rpm in dark. The bacterial liquid was inoculated into several medium containing antibiotics, and Agrobacterium cells were harvested when the density reached OD600 0.3, 0.4, 0.5, 0.6, and 0.7, respectively. The Agrobacterium were collected by centrifuging at 5000 rpm for 12 min, and then rinsed twice with suspension solution (Table 1). Finally, the Agrobacterium cells were suspended to reach the needed OD600.
The safflower callus were infiltrated with Agrobacterium solution, and the steps are as follows (The text in parentheses indicates the experimental parameters): (1) Put the safflower callus into a sterile centrifuge tube, and pour the Agrobacterium solution with original density of OD600 0.6 (OD600: 0.3, 0.4, 0.5, 0.6, 0.7) and concentration of OD600 0.4 (OD600: 0.2, 0.4, 0.6, 0.8, 1.0) into the tube to submerged the callus. (2) Gently upside down the centrifuge tube to make the callus fully contact with Agrobacterium. (3) 15 min (5, 10, 15, 20, 25 min) later, Agrobacterium liquid was removed. (4) The callus was moved in into the co-cultivation medium (Table 1), and then cultured at 28 ℃ for 2 days (1, 2, 3, 4, 5 d) in the dark. When studying the effect of AS concentration on the infecting efficiency, AS was added to make AS concentration reach 50, 100, 150, 200 and 250 mM·L-1.
The transformed callus were submerged in GUS staining solution (Coolaber. China) for 24h in the dark at 37 ℃. After removing the staining solution, samples were washed three times with 75% ethanol. The callus with the stained area more than 1/3 was regarded as positive.
Biolistics‑mediated transient transformation
Binary vector pBI221 was used to optimize parameters for biolistic-mediated transient transformation. The vector contains a GUS reporter gene and the promoter is CaMV 35S. This plasmids were transformed into E.coli-DH5α competent (Tsingke, China) to obtain sufficient plasmids for experiments. The transformed E. coli were propagated in dark at 37 °C and the plasmids were extracted.
The gold particle was prepared as follow[44]: (1) Take 50 mg of gold particles (1.0 μm, Bio-Rad) and wash them twice in 75% ethanol, then twice with sterile water, and finally 50% sterile glycerol was added to make the final concentration of gold particles at 50 mg·mL-1. (2) Vortex the gold particle solution to distribute the gold particles evenly in the solution, and quickly pipette 4 μL solution (when optimization the amount of gold particles, 2, 4, 6, and 8 μL solution were pipetted, respectively) into a sterile 1.5 ml centrifuge tube. (3) Keeping vortexing, 2 μL of plasmid (1000 ng·μL-1) (when optimization the amount of plasmid, 1, 2, 3, and 4 μL of the plasmid were pipetted respectively), 25 μL of 2.5 mol·L-1 CaCl2 solution, and 10 μL of 0.1 mol·L-1 spermidine solution were added into the centrifuge tube in sequence. (4) Centrifugation at 5000 rpm for 5 min to collect gold particles. And then, the gold particles were washed twice with 200 μL of 75% and 100% ethanol. (5) The microparticles which have been coated with plasmids were resuspended in 20 μl of 100 % ethanol, and temporarily place on ice. The microcarriers prepared according to the above steps were used for one bombardment.
The Bio-Rad 1000/He PDS particle delivery system (Bio-Rad, USA) were used, and all items used in the experiment were sterilized. Safflower callus should be pre-cultured on hypertonic medium (Table 1) for 4 hours before bombardment. The safflower callus were transformed under different parameters, including the amount of gold particles (100, 200, 300, and 400 μg·shot-1), the amount of plasmids （1, 2, 3, and 4 μg·shot-1), helium pressure (1100psi and 1350psi), vacuum (-0.2, -0.4, -0.6, -0.8, and -1.0 bar), flight distance (6.5, 8.0, 9.5, and 11.0 cm) and number of bombardments (1, 2 and 3 times). The transformed callus was cultured in the dark at 28 ℃ in hypertonic medium for 24 h.
The transformation efficiency of callus was represented by GUS enzymatic activity. Successfully transformed callus can produce GUS protease, which can catalyze 4-methylumbelliferone glucuronide (4-MUG) into fluorescent 4-methylumbelliferone (4-MU). By measuring the fluorescence, the GUS (β-glucuronidase) enzymatic activity can be quantitatively detected. According to the method of the kit (Coolaber, China), transformation efficiencies under different experimental variables were detected.
Agrobacterium- and biolistic-mediated overexpression of CtCHS1 in safflower callus
The flower of safflower was gathered from medical botanical garden of Chengdu University of Traditional Chinese Medicine too. RNA was extracted from flowers and reverse transcribed into cDNA. The sequence of CtCHS1 is derived from a reported study, and the study showed that CtCHS1 has a significant effect on the accumulation of some flavonoids [15]. The specific primer, CtCHS1-F and CtCHS1-R (Table 3), was designed with Primer Premier 5.0. PCR was carried out as follows: initial denaturation at 98 ℃ for 3 min; 34 cycles of 98 ℃ for 30 s, 56 ℃ for 30 s, and 72 ℃ for 2 min; and a final extension at 72 ℃ for 8 min. The PCR products were cloned into a pMD19-T vector (Tiangen, China) for sequencing (Tsingke, China).
The pRI201 plasmid was used for Agrobacterium-mediated transient expression, and the restriction enzyme site, Nde I (Takara, Japan), was selected in the experiment. The pBI221 plasmid was used for biolistic-mediated transient expression, and the restriction enzyme site was BamH I (Takara, Japan). On the basis of the ORF of the CtCHS1 gene and restriction enzyme sites, gene-specific primers were designed (Table 2). The CtCHS1 fragments were cloned into pRI201 and pBI221 respectively, and constructed plasmids verified by sequencing (Tsingke, China).
The constructed pRI201-CtCHS1 plasmid was extracted from E. coli, and then transformed into EHA105 Agrobacterium-competent, finally, the safflower callus was infiltrated under the optimized experimental condition. After multiplying in E. coli, constructed pRI201-CtCHS1 plasmid was extracted. And then, plasmid was loaded on gold particles, and delivered into the safflower callus under optimized conditions.
The transformed safflower callus were divided into three parts. One was used to measure the transformation efficiency. One was used to extract RNA which would be reverse transcribed for real time-PCR (RT-PCR) (Bio-Red, USA). The RT-PCR was employed to detect the expression level of CtCHS1 gene. The 25S rRNA gene, which obtained from Carthamus tinctorius L., was used as the internal reference to identify differences. The specific primers were designed with Primer Premier 5. software too. The RT-PCR cycling were carried out as follows: 95°C for 3 min, followed by 40 cycles of 95 ℃ for 10 s and 58.5 ℃ for 30 s [45]. The remaining samples was freeze-dried, and then were ultrasonically extracted in water twice to get total flavonoids. The extracts were finally dissolved in methanol, and analyzed by LC-MS system (Synapt, Waters, USA). The experimental methods referred to reference [19]. Safflower callus transformed with empty vector was used as control.
Statistical analysis
Statistical analysis of the callus induction rate, GUS strain frequency of Agrobacterium-transformed callus, GUS enzymatic activity in biolistic-transformed callus, the expression level of CtCHS1 gene, and relative flavonoid content were performed by Student’s t-test (p<0.01). The callus induction rate (%) (number of successfully induced callus/total number of unstained explants ×100%), Agrobacterium-transformation efficiency (%) (number of positive callus/total number of infected callus×100%), biolistic-transformation GUS enzymatic activity (calculated from established standard curve) and relative flavonoid content (the peak areas of experimental group/ the peak areas of control group) were calculated. Each experiment was repeated three times independently.