For callus induction, embryogenesis and regeneration of Paulownia shantung species, the present study was carried out in the tissue culture laboratory of the college of Aburaihan, University of Tehran, Iran. Petiole explants were obtained from the petiole attachment to the leaf and the petiole attachment to the seedling hypocotyl (axil). The leaf and petiole explants were obtained from young and healthy six-month-old seedlings. Explants were selected from the top leaf of the mother plant grown in the greenhouse. In order to disinfect the explants, they were soaked in a solution of two-grams-per-liter captain fungicide containing four drops of Tween 60%. Then explants were washed in 70% alcohol for one minute and then washed with distilled water. The explants were then placed in a container of a solution of sodium hypochlorite 2% for 10 minutes and they were transferred to a laminar flow hood, afterwards. Then, the explants were placed on sterile filter paper. The Murashige-Skoog (MS) medium was used as a basal medium. Hormone compounds were prepared according to table 1 and the pH was set to 5.8. The media was sterilized in an autoclave at 121 °C and an atmospheric pressure of 1.5 bar. Vitamins and hormones were added to the media before autoclaving. A factorial experiment was conducted in a completely randomized design with two factors, explants (leaf and petiole) and eight media containing different hormone compounds in four replications. Cultivated explants were kept in a growth chamber with a temperature of 24-25 °C and a light intensity of 2720 lux. The explants and plantlets were subcultured every 4 weeks. After the formation of embryos on the culture media, young embryos and regenerated seedlings were selected for extraction of chlorophyll, carotenoids, soluble protein, anthocyanins, proline, and flavonoids. This experiment was performed in a completely randomized design with three replications.
Different stages of measuring embryogenic cell/callus induction formation and regeneration
In the first week, callus induction was observed in the media. This was more evident for culture media numbers four and eight. Nine days after placing explants (dap) in the culture medium, traits such as callus formation and callus percentage were measured. Notably, a stereomicroscope, 15 dap was used to detect stages of embryonic cells, especially the globular stage. Regeneration and subculture took place in the same culture media. During the heart-shaped stage, when embryonic cells were transferred to the hormone-free culture medium, there were no more stages and regeneration. This caused chlorosis and necrosis of the induced embryos. In addition, heart-shaped and torpedo-shaped stages, 20 dap were observed. On day 26, cotyledon and seedling stages were seen. Callus induction is difficult in paulownia. Moreover, seedling regeneration from calluses is a time-consuming process (six months) and due to the high production of phenol, most of the regenerated seedlings are destroyed. In addition, the application of PRs during organogenesis may cause somatic variations. To overcome the shortcomings, of proliferation (producing a large number of seedlings per explant) and the high rate of regeneration and growth of seedlings, somatic embryos were considered (Dumani et al., 2022).
Measurement of photosynthetic pigments content and anthocyanin content
The chlorophyll content and soluble carotenoids were determined by the method proposed by Lichtenthaler (1987). For this purpose, 0.2 gr of the plantlet regenerated from the embryogenic callus was excised and ground in 80% acetone solution for extraction (chlorophyll and carotenoids). Also, for the determination of anthocyanin concentration, 0.2 gr of the plantlet that was regenerated from the embryogenic callus was taken and extracted in 0.3% HCI in methanol at 25 °C and stored in the dark for one night. The amount of anthocyanin was calculated by extinction coefficient (ε = 33000 cm2 mol-1) (Wagner, 1979). All spectrophotometric measurements were carried out through OPTIZEN 3220UV spectrophotometer.
The concentration of chlorophylls a and b is obtained from the following formulas (Lichtenthaler, 1987):
= 12.7A663 - 2.69 A645 Chlorophyll a
= 22.9A645 - 4.68A663 b Chlorophyll
Total chlorophyll= 20.2 A645 - 8.02A663 l
Determination of Flavonoids content
Flavonoids of the plantlet regenerated from the embryogenic callus were estimated according to Krizek et al.’s method (1993). Tissues were homogenized in a mortar and pestle with 3 ml one percent acetic acid-ethanol solvent (1:99, v:v). The homogenate was centrifuged at 18000 rates per minute for 30 min, and then the supernatant was incubated in a water bath for 10 min at 80 °C and then allowed to cool to room temperature. The amount of flavonoids was determined from the absorbance at 270, 300 and 330 nm. Flavonoid content was expressed as µmol/g FW and the concentration of flavonoids was calculated using an extinction coefficient of flavonoids ε550 = 33000 cm2 mol
Measurement of protein content
Soluble protein content in leaf samples was determined through bovine serum albumin (BSA) as a standard by Bradford’s (1976) method. BSA standard curve was used for measuring the soluble protein concentration, was calculated and the values were expressed as mg/g-1 FW.
Two buffers were used to determine the concentration of proteins by Bradford method as follows.
Buffer No. 1: 0.01 gr of Coomassie Brilliant blue (R-250) was dissolved in 15 cm3 of 96% ethanol. Then, 10 cm3 of phosphoric acid (85%) was added to it. Following that, the resulting solution or distilled water reached a volume of 100 cc. The filter paper separated the Coomassie brilliant blue particles from the prepared solution.
Buffer No. 2: 1.77 gr of Na2HPO2 reached a volume of 400 cm3. Also, 1.95 gr of NaH2PO4 reached a volume of 400 cm3. The buffer of these two substances was prepared in an Erlenmeyer flask in a volume of 800. Then, the pH was adjusted to 6.8. After the buffers were prepared, 0.5 gr of the plantlet regenerated from the embryogenic callus was pounded with seven mg/L of buffer number two in a mortar. The samples were centrifuged at 12000 rpm for 15 min at 5 °C. After centrifugation, the supernatants were removed and placed in the refrigerator. Moreover, 100 cm3 of each sample was taken and one cc of buffer number one was added to them.Using a spectrophotometer (UPLAB model), absorbance was measured at 595 nm. The supernatants were analyzed in the same way as bovine serum albumin (BSN) samples (Dumani et al., 2022).
Measurement of proline content
The method introduced by Bates et al. (1973) was applied for the measurement of the amount of proline. For this reason, sulfosalicylic acid (3 % w/v) was used for extracting 0.2 gr of the plantlet regenerated from the embryogenic callus, and then the obtained solution was centrifuged at 12000 rates per minute for 15 min. Afterwards, 2 ml of acid ninhydrin solution and 2 ml of glacial acetic were added to the extract. The extract was transferred to a tube and heated in boiled water at 100 ˚C for 1 hour. When the tube cooled at room temperature, 4 cm3 toluene was added to the tube and mixed well. After the formation of two phases, the top phase was collected carefully by a sampler and the absorbance was measured at 520 nm by the spectrophotometer. The standard curve was consulted for calculating the proline content and was expressed as mg g-1 FW.
Cluster analysis and K-mean of culture media were performed to identify the highest and lowest similarity in the media with respect to all chlorophyll and antioxidant traits (Figure 9). After controlling data adequacy and ensuring the normality of the dates, statistical calculations of the data were performed by R software. The diagrams were plotted by R and Orgin.pro software. The mean of treatments was compared by Dunnett test at 1% Statistical significance level.