Discussion
The results indicated that the liquid medium (shoot p < 0.001, root p < 0.001) conveyed the greatest effect on the growth of shoots and roots in P. thunbergii SRPs compared to sucrose (shoot p < 0.001, roots p = 0.002) and the transplantation stroma (shoot p = 0.03, root p = 0.09). For nematode-resistantP. thunbergii SRPs, the optimal liquid medium was 1/2 WPM followed by the GD medium. However, the DCR medium was obviously not suitable for the growth of P. thunbergii SRPs. Similarly, the slash pine plantlet showed a better growth performance in the GD medium than DCR medium (Zhu and Wu 2005). Further analysis revealed that the NO3-, NH4+, PO43-, Mg2+, and Ca2+ contents in the DCR medium were not significantly different compared to the 1/2 WPM medium; however, the K+ content was only half that of the 1/2 WPM medium (Table S2). It is well known that plant growth is modulated by K+ (Claussen et al. 1997). Tode and Luthen (2001) reported that the enhancement of plant growth via the fungal toxin fusicoccin and auxin both required K+ uptake in maize coleoptiles. However, the K+ content of the DCR medium was lowest of all the media. Thus, we speculated that the poor growth performance of SRPs under the DCR medium might have been related to a lower K+ content. Additionally, the 1/2 WPM medium contained sufficient minor elements (e.g., Mn, Zn, and Ni) in contrast to the other media. The Mn and Zn ions improved chlorophyll synthesis and photosynthesis, which resulted in the modulation of plant growth, such as tomato (Shenker et al., 2004; Hansch and Mendel, 2009). As a component of urease, Ni promoted plant growth of Caryophyllaceae and Cruciferae, by modulating the transport of nitrogen from the roots to the leaves (Aller et al. 2010). This indicated that the improved performance of SRPs in the 1/2 WPM medium might be correlated with the concentration of trace elements.
In this experiment, the mixture of perlite and vermiculite at a 1:1 ratio as transplantation stroma was superior to the perlite and vermiculite only, for improving the growth of P. thunbergii SRPs. The pH of the transplantation stroma affected the root differentiation, whereas its porosity was positively correlated with the plant height (Gao et al. 1992; Wang et al. 2014). Thus, the transplantation stroma mixture improved plant growth through the regulation of these elements. Similarly, the P. elliottii root length was improved by the mixture of perlite and vermiculite compared to perlite only (An et al., 2011). As for carbohydrates, the quality of P. thunbergii SRPs were improved under sucrose concentrations of from 20 to 30 g/L. Moreover, the color of the SRPs needles was significantly darkened. Bhattacharyya et al. (2006) reported that 20 g/L sucrose efficiently enhanced the vertical growth and rooting of Dendrobium nobileplantlets. Further, Zhu et al. (2018) reported that photoassimilates were continuously increased when the Hevea leaf color changed from light to dark green, which was consistent with our research. Although sugar is essential for plant growth, in excess it can be detrimental. We found that the shoot and root lengths of P. thunbergii SRPs were inhibited when the sucrose concentration reached 40 g/L, as high concentrations of sucrose hindered photosynthesis (Hdider and Desjardins, 1994). This was evidenced by the restricted growth of rice and maize when they accumulated higher concentrations of sugars in their leaves (Eom et al. 2011). Appropriate sucrose concentrations, which promoted the growth of P. thunbergii SRPs, might have been related to the regulation of photosynthesis. Akin to the sucrose treatment, the 1 µg/L BR root treatment showed the similarly enhancement for SRPs (Fig. 3B). BR mutants of Arabidopsis display a stronger dwarf phenotype and de-etiolated growth in the dark (Tanaka et al., 2003), and the application of BR increased the plant height of papaya (de Assis-Gomes et al. 2018). Further, Gao et al. (2017) reported the enhancement of photosynthesis in maize by foliar spraying with BR. The P. thunbergii SRPs enhancement by the BR might relate to the regulation of photosynthesis through the photosynthesis (the needles green to darker in 1 µg/L BR). However, the growth of P. thunbergii SRPs was inhibited when the BR concentration reached 10 µg/L; thus, the promotion of somatic plantlets using exogenous BR was optimized at lower concentrations. The application of BR for roots at lower concentrations was more effective for plantlet growth (Arteca et al., 2001; Pandey et al., 2020).
Moreover, in our study, monochromatic red light promoted the elongation of taproots, while monochromatic blue light promoted shoot elongation, the optimal combination of spectra for P. thunbergii SRPs growth was the red and blue at 8R:2B ratio. Kvaalen (1999) reported that red light wavelengths inhibited the elongation of Norway spruce (Picea abies ) shoots compared to blue light and cool white fluorescent light. However, for lettuce, shoot growth resulted from red light exposure rather than blue light (Zhao et al., 2007), which indicated that light spectrum treatments on plant growth was variable between species. As is known, the photoreceptors for red light are the phytochromes (PhyA and PhyB), while the blue light photoreceptors are phototropins (Briggs et al., 2001; Rockwell et al., 2007). In terms of light regulators, phytochrome interacting factors (PIFs) played a key role in the modulation of plant growth. In green seedlings, elongation is primarily mediated by PIF4 and PIF5 (Lorrain et al., 2008; Hornitschek et al., 2012). In an Arabidopsis thaliana study, blue light stimulated the expression of phytochrome interacting factor4 (PIF4) and PIF5 in seedlings, whereas the PIF4 and PIF5 negatively modulated auxin signaling. For example, PIF4 and PIF5 repressed the expression of auxin-responsive marker genes IAA5 and GH3-LIKE (Sun et al., 2013). This suggested that blue light promoted stem elongation of Arabidopsis thaliana by suppressing the expression of auxin genes. The results of our light spectrum treatments were consistent with this study, which indicated that the blue light promotion of shoot elongation in conifers might be associated with low concentrations of auxin. Further, our results indicated that although the growth of the aboveground portions of the plantlets was suppressed, root development was promoted by red light. Adjusting the red to blue light ratio combined the advantages of monochromatic light; for example, the higher number of root tips, root volume, root length, and survival rate of nematode-resistant P. thunbergii were obtained under the 8R2B treatment. The promotion of root development by red light has been reported (Casal, 2000, 2013; Li et al., 2021). Ranade et al. (2016) suggested that combined red and blue light treatments enhanced the biomass as well as fiber size, resulting in stable tree structures. In general, light spectrum treatments have been recognized as an important factor for improving plant production and quality, which is extensively used in horticulture (Li et al., 2010). Conifer seedlings ware also known to respond to light spectrum treatment (Ranade SS and Gil MRG., 2016). A novel study was conducted regarding the effects of red and blue LED combinations, as well as white light treatments during post-germination and root development in nematode-resistant P. thunbergii plantlets. In this report, we discussed the effects of red and blue light, and white light treatments during root and shoot development of nematode-resistant P. thunbergii plantlets following germination, which influenced root development, shoot elongation, and the survival rate of plantlets after transplantation. Our studies on post-gemination plantlets revealed that the light quality could be manipulated to obtain high quality plantlets (improved shoot and root growth).
All treatments to enhance the growth of P. thunbergii SRPs contributed to its survival rate, which indicated that it is necessary to promote plant growth and improve the quality of plantlets to enhance the survival rate. Luis et al. (2009) considered that larger seedlings may augment transplantation performance in contrast to smaller seedlings. Further, although no statistically significant correlation was identified between the rootstock ratio and survival rate in our study, the high survival rate was focused on a rootstock ratio of about 2 and 10. Interestingly, at a rootstock ratio of ~10, and a root length of from between 10 and 30 cm, the survival rate was >60 %. However, at a rootstock ratio of 2 and root length less than 5 cm, the survival rate was also >60 % in half of the plantlets, which may have been related to their high quality. The correlation between root development and the survival rate showed that root tips had the greatest impact on the survival rate, followed by root surface area, root volume, and root diameter, with the root length having the lowest. Thus, for evaluating plantlet quality, the root tips, root surface area, and volume were the main criteria. In short, this study showed that enhancing SRPs quality, especially, promoting the plant root development is an effective strategy for improving the SRPs survival rate. This study makes a big step forward for pines afforestation strategy using SRPs. This study had made pines breeding progress in afforestation strategy using somatic embryo seedlings closer to success.