We cut 1 cm long segments of leaves, stems, and roots of P. praeruptorum and treated them with a 75% ethanol solution for 30 s followed by a 0.1% HgCl₂ disinfection solution for 10 min. Plant material was then inoculated within Murashige and Skoog (MS) medium supplemented at 5 different concentrations of 2,4-D (0, 0.5, 1.0 mg/L) and 6-BA (0, 0.5, 1.0 mg/L). Each dish was inoculated with 7 explants, including 5 dishes in each group. After 14 d, the callus induction was observed and the callus induction rate for each group was calculated.

The callus of P. praeruptorum was cut into blocks of approximately 0.3 cubic cm and inoculated on an MS medium supplemented with 5 different concentrations of IBA (0, 0.5, 1.0 mg/L) and 6-BA (0, 0.5, 1.0 mg/L). Each bottle was inoculated with 6 callus clumps, 8 bottles in each group. After 28 d, the growth status of callus, the color and texture of callus, the number and morphology of adventitious buds, and the proliferation rate were observed and recorded. The differentiation rate and browning rate of each bottle were also calculated.

Adventitious buds of P. praeruptorum approximately 3 cm in length and 2–3 leaves were inoculated on MS medium containing 5 different concentrations of IBA (0.2, 0.4, 0.6, 0.8, 1.0 mg/L). Each bottle was inoculated with 6 adventitious buds, 8 bottles in each group. After 14 d, the growth status of adventitious buds, the number and morphology of rooting of multiple buds, and the browning rate were observed and recorded. The rooting rate and average rooting number of each group were also calculated.

The cluster seedlings of P. praeruptorum with root length of more than 3 cm were selected and acclimated for 14 d on the filter paper bridge of MS liquid medium and the mixed nursery soil of humus soil: vermiculite: perlite = 2: 1: 1. 3 cluster seedlings were inoculated in each bottle/pot, with a total of 16 bottles/pot in each group. After 14 d, the growth status of tissue culture seedlings, the morphology, and the color of leaves and roots were observed and recorded. The survival rate of each group was calculated.

The sample solution was created by using 0.1 g of sample powder mixed with 5 ml of chloroform. The solution was exposed to ultrasonic treatment for 30 min and followed by extraction of 4 mL of continuous filtrate. The resulting material was dried using a nitrogen blowing instrument, subsequently dissolved using 1 mL of methanol, and then filtered (0.22 μm) before analysis. The chromatographic column used was a Symmetry C18 (4.6 * 250 mm, 5 μm). The mobile phase was methanol-water (80∶ 20). The injection volume was 10 μL, the detection wavelength was set at 321 nm, the detection time was set at 15 min, the flow rate was set at 1.0 mL/min, and the column temperature was set at 30°C. Praeruptorin A was quantified at 60, 80, 100, 200, 400 μg/mL, Praeruptorin B and Praeruptorin E were quantified at 10, 20, 40, 60, 80 μg/mL. The standard curves were Y = 23.805X-451.350 (R² = 0.9994), Y = 21.244X-43.189 (R² = 0.9995), Y = 19.886X-36.769 (R² = 0.9994).

The universal premix of SYBR Green I chimeric fluorescence method (Vazyme, Nanjing, China) was used. The final concentration of each primer was 0.2 μM ( Supplementary Table S1 ). The cDNA of 10 ng total RNA was used as the template, and the total system was 20 uL. Real Time PCR reaction was performed on the ABI 7500 machine with a two-step method. The reaction procedure was 95°C/10 s, and 40 cycles of 95°C/10 s and 60°C/30 s. The data were exported, and the 2^^-(ΔCt) value was calculated.

Explants of P. praeruptorum could successfully induce callus in the medium with different ratios of 2,4-D to 6-BA, however, the ability of callus induction in leaves, stems, and roots were significantly different ( Supplementary Table S2 ). In addition, the morphological characteristics of callus induced by different explants of P. praeruptorum also varied. Among them, leaf calluses were mostly light white or yellow-green and comprised of loose and fragile callus blocks gathered in granular shape. Callus derived from stem segments was mostly yellow-green and composed of dumbbell-shaped, compact structures. The root callus was light yellow or brown forming a loose and tight callus block. At a 2,4-D/6-BA concentration ratio of 1 ( Figure 1 ), the leaves, stems and roots of different explants of P. praeruptorum were inoculated on the medium. After two weeks of dark culture, callus began to form around the wound. The induction rate was the highest compared with other groups, which were 85.71%, 51.43% and 20.00%, respectively. Overall, leaves were determined to be the best explant selection for P. praeruptorum and MS + 2,4-D 0.5 mg/L + 6-BA 0.5 mg/L was the optimal induction medium for callus of P. praeruptorum.

A reduction in the concentration ratio of IBA to 6-BA in the medium resulted in a decrease in the proliferation rate of callus, which subsequently increased. In contrast, the differentiation rate of callus exhibited an inverse relationship ( Supplementary Table S3 ). At a IBA/6-BA concentration ratio of 2 ( Figure 2A ), the callus tended to proliferate, exhibiting the highest growth state and the most advanced browning situation among all groups. When the IBA/6-BA concentration ratio was 1 ( Figure 2C ), the callus tended to differentiate, the number of cluster buds were relatively large, and the size was suitable and not intertwined. Furthermore, the color of the leaves was dark green, the growth was consistent, and the differentiation rate was 68.75%. At a IBA/6-BA concentration ratio of 0.5 ( Figure 2E ), the callus exhibited a bias towards proliferation, with an overall green color, a relatively compact structure, and the production of embryonic cells, and the proliferation rate was 66.67%. Furthermore, when the MS medium contained solely IBA ( Figure 2B ), the bud points exhibited differentiation, browning, and a generalized growth state. In contrast, when the MS medium contained solely 6-BA ( Figure 2D ), the overall growth state of the callus was poor, and browning was pronounced. Overall, MS + 6-BA 1.0 mg/L + IBA 0.5 mg/L was determined as the optimal proliferation medium for callus and MS + 6-BA 0.5 mg/L + IBA 0.5 mg/L was the most suitable differentiation medium for cluster buds of P. praeruptorum.

As the concentration of IBA in the medium increased gradually, the rooting rate of adventitious buds initially increased and then subsequently decreased ( Supplementary Table S4 ). At a concentration of 0.2 mg/L ( Figure 3A ), the number of rooting of cluster buds was found to be the lowest among all groups, with the majority exhibiting fibrous roots, which were fine and short in shape. At a concentration of 0.8 mg/L ( Figure 3D ), the growth state was relatively poor and exhibited a limited number of rooting buds. However, the main root was larger than the fibrous root. Upon reaching a concentration of 1.0 mg/L ( Figure 3E ), the growth state was found to be the most adverse among all groups. This was accompanied by a notable inhibition of rooting of cluster buds and a significant browning phenomenon. Furthermore, when the IBA concentration was between 0.4 and 0.6 mg/L ( Figures 3B, C ), the growth state of cluster buds was the most optimal among all groups. The rooting rate was also the highest among groups, reaching 69.44% and 66.67%, respectively. The rooting rate, average rooting number, and growth state of multiple shoots were analyzed to determine the optimal rooting medium for the tissue culture seedlings. The results indicated that the IBA concentration of 0.5 mg/L, equivalent to MS + IBA 0.5 mg/L, was the most effective for rooting the seedlings.

The results demonstrated that the survival rate of seedlings grown in humus soil, vermiculite, and perlite in a 2: 1: 1 ratio was 79.16%. The survival rate of seedlings grown in MS liquid medium using the filter paper bridge method was higher, reaching 91.67% ( Supplementary Table S5 ). However, when considering the growth status of the tissue culture seedlings, it can be observed that those grown in the nursery soil ( Figures 4A, B ) exhibited relatively good growth, with the leaves stretched and a dark green color. In contrast, those grown on the filter paper bridge ( Figures 4C, D ) exhibited relatively poor growth, with the leaf shape being shrunken and the color being light green. However, subsequent observation of root growth revealed that the root expansion and growth rate of tissue culture seedlings on the filter paper bridge were significantly greater than those observed in the seedling soil.

The purpose of this experiment was to determine the contents of three representative coumarins (Praeruptorin A, Praeruptorin B and Praeruptorin E) in different tissue culture materials (callus, adventitious buds and cluster seedlings). Additionally, the contents of these three coumarins in different parts (roots, stems and leaves) of tissue culture seedlings were also determined ( Figure 5 ). The total content of coumarin in different materials was analyzed. We found that the total coumarin content, from highest to lowest, was as follows: adventitious buds > cluster seedlings > callus ( Figure 6 ). Furthermore, the total coumarin content in adventitious buds was 3.67 times that of callus. The total content of coumarin was highest in the roots of the tissue culture seedlings and 1.67 times more than in the leaves ( Figure 7 ). Moreover, the content of coumarins in different tissue culture materials was as follows: Praeruptorin A > B > E. Furthermore, the content of Praeruptorin A was found to be an important factor in determining the total content of coumarins. In different parts of tissue culture seedlings, the content of Praeruptorin A was found to be higher in stems and leaves, whereas the contents of Praeruptorin B and Praeruptorin E were found to be higher in roots than in stems and leaves.

To reveal the differences of coumarin secondary metabolites in different materials of P. praeruptorum at the molecular level, we determined the expression levels of key enzyme genes PpPAL, PpC4H, PpC2’H in different tissue culture materials (i.e., embryonic callus, adventitious buds, cluster seedlings) and different parts (i.e., roots, stems, leaves) of tissue culture seedlings ( Figure 8 ). The results showed that the expression level of PpC2’H was higher than that of PpC4H and PpPAL in different experimental materials or different tissues ( Figure 9 ). The expression level of PpC2’H in adventitious buds was much higher than that in cluster seedlings and callus. This observation was posited as one of the reasons why the total coumarin content was higher than the other two groups. In addition, the expression trend of the three key enzyme genes in different parts of the tissue culture seedlings was also PpC2’H > PpC4H > PpPAL ( Figure 10 ). This was consistent with the result that the total content of coumarin in roots was higher, indicating that the expression of PpC2’H was positively correlated with the total content of coumarin in P. praeruptorum.