Induction and proliferation of calli from the culture of mature zygotic embryos of P. strobus
Mature zygotic embryos (Fig. 1a) were cultured on 1/2 LV medium with 1.0 mg/L 2,4-D. Calli were produced on the surfaces of zygotic embryos after 3 weeks of culture (Fig. 1b). The partially callus zygotic embryos were subcultured onto the same medium and turned to callus mass after three weeks of culture (Fig. 1c). Highly friable callus masses were obtained after consecutive subculture on the same medium with 1.0 mg/L 2,4-D five times at two-week intervals (Fig. 1d).
Calli were transferred onto various concentrations (0, 0.25, 0.5, 1.0, 2.0, and 4.0 mg/L) of 2,4-D to evaluate the optimal concentration of 2,4-D. After 3 weeks of culture, fresh weight and dry weight were calculated (Fig. 2a,b). Calli actively proliferated at 0.5 and 1.0 mg/L 2,4-D (Fig. 2a,b). Interestingly, combined treatment with BA and 2,4-D stimulated the growth of calli (fresh weight) compared to 2,4-D alone. However, there were no differences in the growth of calli induced by the different types of cytokinins (0.5 mg/L BA, zeatin, kinetin, TDZ, and 2-ip) with 1.0 mg/L 2,4-D (Fig. 2c). When calluses were cultured on medium with 1.0 mg/L 2,4-D and different concentrations of BA, 0.25 and 0.5 mg/L BA was better than 1.0 and 2.0 mg/L BA for callus production (Fig. 2d).
Enhanced DPME and PME accumulation by prolonged culture of calli
For proliferation of calli, P. strobus calluses were subcultured onto 1/2 LV medium with 1.0 mg/L 2,4-D and 0.5 mg/L BA at 3-week intervals. When the P. strobus callus was not subcultured until 3 months, the color of the callus turned pale yellow after one month of culture (Fig. 3a), turned brown after 2 months of culture (Fig. 3b), and turned dark brown after 3 months of culture (Fig. 3c). Calli with different colors were sampled and milled after drying. The color of the dried callus powders still had the same color as undried fresh callus masses (Fig. 3a-c). Microscopic observation of the callus revealed that a one-month-old faded yellow callus was composed of aggregated cells with a spherical structure with dense cytoplasm (Fig. 4a,b). However, three-month-old brown calli were composed of cells with a brown pigment near the cell walls and less dense cytoplasm than the cells of faded yellow calli (Fig. 4c,d).
Pinosylvin stilbene content in the MeOH extract of calli with different colors (faded yellow, brown, and dark brown) was analyzed by GC/MS. Interestingly, the accumulation of DPME and PME was highly increased by callus aging (Fig. 5). Only DPME was detected in a small amount, but PME was not at a detectable level in one-month-old calli with a faded yellow color (Fig. 5a). DPME and PME accumulations in calli were highly enhanced as the culture time proceeded to two and three months (Fig. 5b,c). The production of DPME and PME was confirmed by the retention times of authentic standards (Fig. 5d) and mass fraction pattern of the two compounds by GC/MS (Fig. 5e,f). Surprisingly, the total ion chromatogram over the full range of retention times revealed that the two DPME and PME peaks were the only major components in the extracts from dark brown calli (Fig. 5c). The amounts of DPME and PME in three-month-old calli with dark brown colors were 6.4 mg/g DW and 0.28 mg/g DW, respectively (Fig. 6).
Effect of MeJA treatment of callus on DPME accumulation
When P. strobus calli were transferred onto medium with MeJA at various concentrations (0, 10, 50, and 100 µM), callus growth after 2 weeks of culture was slightly suppressed by MeJA treatment, although there was no statistically significant difference (Fig. 7a). Pinosylvin synthase (STS) and pinosylvin O-methyltransferase (PMT) are key enzymes for pinosylvin stilbene biosynthesis in pine trees13, 35. Previously, we selected the best candidate genes of the STS and PMT genes involved in pinosylvin stilbene biosynthesis in P. strobus19. The effect of MeJA treatment on the expression levels of PsSTS and PsPMT genes in calli was analyzed by RT-PCR. The expression of PsSTS and PsPMT was weak without MeJA treatment but significantly enhanced by MeJA treatment (Fig. 7b). Among the different concentrations (zero, 10, 50, and 100 µM MeJA treatment), the highest accumulation of PsSTS and PsPMT mRNAs was detected in calli after 100 µM MeJA treatment (Fig. 7b). Gene expression of PsSTS and PsPMT in calli after 100 µM MeJA treatment was analyzed by qPCR analysis. Similar enhanced expression of PsSTS and PsPMT genes was achieved by MeJA treatment, and the expression of PsSTS gene was more strongly responded by MeJA treatment than the PsPMT (Fig. 7c,d).
Cell suspension culture was obtained by shake flask culture of P. strobus calluses in 1/2 LV liquid medium with 1.0 mg/L 2,4-D with 0.5 mg/L BA and with and without 100 µM MeJA. The accumulation of DPME and PME was monitored in the cell suspension culture during zero, 1, 3, and 7 days of culture. Without MeJA treatment, DPME accumulation was slightly increased as the culture period proceeded until 7 days (Fig. 7, Supplemental Fig. S1a). The accumulation of DPME was more conspicuous in 100 µM MeJA treatment after 7 days of culture (Fig. 8, Supplemental Fig. S1b). The amount of DPME in 100 µM MeJA treatment for 7 days was 0.358 mg/g DW (Fig. 8). However, PME was under the detectable levels in cell suspensions cultured with or without MeJA treatment (Supplemental Fig. S1b).
Nematicidal activity of crude extracts from P. strobus calluses
Crude extracts were obtained by 100% EtOH extraction from calli of different ages (faded yellow and dark brown). The crude extracts were dissolved in water containing 10 mg/L 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), which was used as an emulsifier of hydrophobic chemicals34. We previously demonstrated that the water soluble formulation of the pinosylvin stilbene:HP-β-CD complex was effective for the analysis of the nematocidal activity of DPME and PME compounds (Whang et al. 2021). The aqueous test solution of DPME and PME from extracts of dark brown callus was adjusted to the concentration of 120 µg/mL DPME (5.16 µg/mL PME) by dissolving in water containing HP-β-CD, and the same dilution was also applied for the crude extracts from faded yellow callus. The content of DPME and PME in faded yellow calli was nearly zero. These crude extracts were used to treat adult and juvenile PWNs. The extracts from dark brown calli showed strong nematicidal activity. More than 70% of adult PWNs lost their mobility with strait bodies after 3 h of callus extract treatment, and nearly all adult PWNs were immobilized after 24 h (Fig. 9a). However, the treated adult PWNs using the crude extracts from yellow calli did not exceed the immobilization of PWNs by less than 20% (Fig. 9a). In the control treatment (only water with HP-β-CD), less than 5% of PWNs showed immobilization of adult PWNs (Fig. 9a). When the crude extracts were treated with juvenile PWNs, the control water treatment and the extracts from faded yellow calli did not induce the immobilization of PWNs (Fig. 9b); only treatment with extracts from dark brown calli was effective for immobilization of PWNs at 67% after 24 h (Fig. 9b).
Photos of PWNs revealed that all the PWNs showed active pendulation of their bodies by the extract treatment from yellow calli both after 9 h (Fig. 10a) and 24 h (Fig. 10b). In contrast, many PWNs rapidly lost their mobility at 9 h (Fig. 10c) and became strait body shaped due to immobilization at 24 h (Fig. 10d).