The pine wilt disease (PWD), caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus), is a devastating forest disease, resulting in significant economic and ecological losses (Ye, 2019; Li et al., 2021). Currently, PWD is considered a quarantine disease in 52 countries worldwide (Wu et al., 2020). In China, this disease has rapidly spread to 19 provinces and 742 counties since its first report in Nanjing, Jiangsu Province, by the end of 2021 (Li et al., 2022b). PWN can naturally infest 51 pine species worldwide and at least 17 species in China, including Pinus massoniana Lamb. (Ye and Wu, 2022). P. massoniana is a conifer species native to southern China, and has both economic and ecological value. It is a major timber species and a pioneer species for afforestation of barren mountains (Fu et al., 1999). Currently, there is no economic viable method to control PWD in infested forests. Resistance breeding is an important way to prevent damage from pests and pathogens to forest trees (Xu et al., 2013; Zhu et al., 2019), and research into this approach is being carried out in China. In 2001, long-term resistance breeding of P. massoniana to pine wood nematodiasis was initialed in Anhui Province, China (Cai et al., 2005). A total of 1,201 seedlings from 251 families was selected from 324 healthy mother trees, and a resistance seed orchard was established (Xu et al., 2013). Additionally, artificial inoculation of 40 P. massonian accessions in South China was carried out, resulting in the selection of three resistant accessions (GX2, GX3 and GD5) (Xu et al., 2000). However, traditional breeding methods like these are time-consuming, limited by the season, and slow in terms of trait improvement speed.

Plant somatic embryogenesis (SE) is a powerful tool in plant biotechnology, providing a convenient way to achieve rapid propagation of elite genotypes (Pullman et al., 2015). Successful SE has been reported in at least 36 species of Pinus spp., with the majority being economically important timber species such as Pinus densiflora (Kim and Moon, 2014), Pinus elliottii (Yang et al., 2020), Pinus pinaster (Arrillaga et al., 2019), Pinus radiata (Montalban and Moncalean, 2019), Pinus taeda (Pullman et al., 2015) and Pinus thunbergii (Sun et al., 2019b). As one of the most important afforestation tree species in China, P. massoniana has received extensive attention in SE and plantlet regeneration studies The first SE system for P. massoniana was established using mature zygotic embryos as explants, resulting in the regeneration of three plantlets (Huang et al., 1995). Another system using immature zygotic embryos as explants was later established by Yang et al. (2011). In recent years, the embryonal mass initiation, proliferation, maturation and germination of somatic embryos from this species have been further optimized (Chen et al., 2019; Xia et al., 2021). However, despite the progress made in P. massoniana SE, several challenges still remain, including low frequency of embryonal mass initiation, low number and poor quality of mature embryos, and low plantlet regeneration rates (Xia et al., 2021). The maturation treatment has been found to affect somatic embryo germination (Salaj et al., 2019), making it important to investigate the effects of different maturation treatments on somatic embryo germination and subsequent plant growth (Jiang et al., 2021). Acclimatization stage is also considered a major obstacles to successful micropropagation of conifers (Mohammed and Vidaver, 1988). Regenerated plantlets grown in sterile tissue culture conditions lack interaction with microorganisms, making successful culture and subsequent acclimatization under non-sterile conditions difficult, leading to poor survival rates after transplantation (Zhu et al., 2010).

Mycorrhiza refers to a beneficial between plants and fungi that colonize their roots (Niemi et al., 2004). In nature, the roots of most terrestrial plants are colonized by mycorrhizal fungi, with pine species predominantly forming ectomycorrhizas (Niemi et al., 2004; Niemi et al., 2007; Zhu et al., 2010). Experimental studies have demonstrated that inoculating of roots with ectomycorrhizal fungi can promote soil water and nutrients absorption (Chen et al., 2016; Garcia et al., 2016), enhance plant tolerance to drought (Gehring et al., 2017), salinity (Guo et al., 2022) and heavy metal stress (Yang et al., 2022) and increase resistance to pests and diseases (Wang et al., 2022). Therefore, early inoculation of ectomycorrhizal fungi onto in vitro grown plantlets is an important method to obtain plantlets capable of withstanding various stresses. Previous studies have investigated the potential effects of ectomycorrhizal fungi on certain traits of tree species before and during the transplanting stage of regenerated plantlets (Cheng et al., 1995; Niemi and HäGgman, 2002; Zhu et al., 2010; Wang et al., 2015; Chen et al., 2022). However, the effect of ectomycorrhizal fungi on plantlets regenerated from somatic embryos of P. massoniana has not been reported to date.

The plant tissue culture technique is a powerful biotechnological approach that can be used not only for rapid clonal propagation, but also for the study of various tree diseases, and can accelerate pest and disease resistance breeding (Fenning, 2019). This technique provides a relatively simple system that uses organs, tissues, or cells to study the interaction between the pathogen or pest and the host (Jang and Tainter, 1990). Fenning (2019) reviewed 66 publications reporting attempts to generate disease-resistant plantlets using tissue culture methods. These studies involved 30 forest and other tree species and approximately 31 different plant pathogens were identified.

Various studies have utilized plant tissue culture technique to study the interaction between plant pathogens or pests and their hosts. For instance, Jang and Tainter (1990) demonstrated differential resistance to Phytophthora cinnamomi, the little leaf disease pathogen, in calli obtained from Pinus species. Similarly, Terho et al. (2000) evaluated the growth reactions of 27 Scots pine embryogenic cell lines to Gremmeniella abietina, the Scleroderris canker fungus. Nagy et al. (2005) found that the embryogenic cell lines of Picea abies responded to Ceratocystis polonica, the blue-stain fungal pathogen, and Heterobasidion annosum, the butt rot pathogen, in a manner similar to that of trees with varying susceptibility to these diseases. Furthermore, Hashmi et al. (1993) investigated the response of peach cultures to Meloidogyne incognita, the root-knot nematode, and identified several that exhibited greater resistance than the parental cells. More recently, studies have confirmed the pathogenicity of aseptic PWN by inoculating them onto pine callus or micropropagated plantlets generated in vitro (Lin et al., 2017). In addition, Zhu et al. (2019) selected two clones with relatively high resistance levels to PWN by inoculating regenerated P. densiflora microshoots with PWN under aseptic conditions. However, there are no reports to date on the PWN resistance of regenerated plantlets of P. massoniana.

This study aimed to analyze the primary factors that influence somatic embryo development and maturation in P. massoniana as well as investigate the impact of maturation treatments on somatic embryo germination, survival, and root development. Furthermore, the formation of ectomycorrhizae in regenerated plantlets and its influence on PWN resistance in three embryonic cell lines of P. massoniana was evaluated in vitro.

In continuation of Xia et al. (2021), we successfully obtained embryogenic callus of P. massoniana. Our study revealed that different seed families exhibited significantly different capacities for embryonal mass initiation, ranging from 0% to 10%. Consistent with Xia et al. (2021), we found that families GX1 and GX4 were more responsive than families GX2 and GX3, highlighting the importance of selecting appropriate mother trees to increase the likelihood of obtaining desired genotypes (Carneros et al., 2009). Future studies could explore a wider range of mother tree types at the embryo initiation stage. Similar to previous reports by Kim and Moon (2007); Hosoi and Maruyama (2012), and Sun et al. (2019b), we found that not all callus could survive and proliferate on proliferation medium. In our experiments, few embryogenic calli survived and proliferated on proliferation medium. Multiple factors influence somatic embryos maturation, including genotype, PGRs, induction treatments, basal medium and medium supplements (Pullman and Skryabina, 2007; Vales et al., 2007; Pullman and Johnson, 2009; Pullman and Bucalo, 2014; Tang et al., 2020). In our study, we focused on four factors that potentially influence P. massoniana SE maturation, namely ABA, PEG 8000, AC, and maltose. We conducted multifactorial experiments on these factors to identify their primary and secondary relationships in the processes of SE maturation, germination, survival, and rooting.

Exogenous ABA is known to play a crucial role in regulating the formation and development of somatic embryos, as reported by several studies (Wang et al., 2002; Carneros et al., 2017; Zhang et al., 2019). PEG has been found to induce water stress that mimics drought conditions, thus reducing the water content of somatic embryos and promoting their maturation at an appropriate concentration (Shoji et al., 2006). AC, on the other hand, is believed to adsorb waste and toxic substances from the culture medium (Pullman et al., 2005). Apart from serving as a carbon source, maltose is also used as a penetrant in cell cultures (Xia et al., 2021). Our study used a multifactorial test, which revealed that ABA was the main factor affecting the maturation of somatic embryos in P. massoniana, while maltose was the secondary factor, similar to the findings of Liao and Amerson (1995). Addition of ABA has been reported to promote the maturation in somatic embryos in other pine species (Wang et al., 2002; Yang et al., 2020), but the required ABA concentration varies considerably between tree species. Sun et al. (2019a) found that 20 mg/L ABA maturation treatment increased the number of somatic embryos in P. thunbergii, while Yang et al. (2020) found that 5 mg/L ABA in suspension culture significantly increased somatic embryo production in P. elliottii. Furthermore, the response of SE to exogenous ABA is known to be genotype-dependent in some conifers (Kong and von Aderkas, 2007; Carneros et al., 2017). The promotion of SE formation by maltose has also been reported in other studies (Sun et al., 2019a; Xia et al., 2021).

The germination of somatic embryos can be influenced by various factors, including the maturation treatment (Salaj et al., 2019). The results of the current study indicate that the primary factor affecting the subsequent germination of somatic embryos in P. massoniana was ABA, followed by AC. The maximum germination rate was observed to be 87.3 ± 9.1%. The combination of ABA and AC was found to further promote the formation of cotyledon embryos, which is consistent with previous studies (Huang et al., 1995). Yao and Wang (2020) have reported that AC supplementation in the medium is important for the germination of mature somatic embryos, but excessive amounts of AC can have an inhibitory effect on germination. In our study, we used different amounts of AC in the maturation medium, which could have contributed to the observed variation in the germination rate of subsequent somatic embryos.

Shoji et al. (2006) investigated the effect of osmotic pressure (as influenced by ABA, PEG and maltose) on somatic embryo maturation in P. densiflora when the somatic embryos were transferred to a hormone-free medium, the cotyledons developed normally, and the hypocotyls elongated, but rooting was suppressed in all embryos tested. Therefore, we investigated the effects of maturation treatments on embryo survival and rooting. The primary factor affecting the subsequent survival of somatic embryos was PEG, followed by ABA, with a survival rate up to 59.6 ± 6.8%. ABA was the primary factor affecting the subsequent rooting of somatic embryos, with maltose being the secondary factor. The rooting rate reached up to 55.2 ± 29.3%. The low rooting rate was similar to that identified by Salaj et al. (2019), which showed that maturation treatment affected somatic embryo germination. Based on the above analysis, we found that the concentration of ABA added in the maturation treatment had a continuing impact on the subsequent growth of P. massoniana embryos, which may be associated with changes in the expression of some specific genes (Song et al., 2018). In summary, the best effects on subsequent SE of P. massoniana were obtained with 2 - 3 mg/L ABA, 130 - 150 g/L PEG 8000, 1 - 1.5 g/L AC and 20 g/L maltose added to the maturation medium. The optimum composition of this medium differed from that of Xia et al. (2021), reflecting differences in SE capacity between genotypes (Diaz-Sala, 2019). Somatic embryo development was dependent on the cell line (Salajova et al., 1999).

The somatic embryo germination of P. massoniana in the multifactorial test was predominantly low and survival was also low, Therefore, we conducted a single-factor test on PEG, which is the main factor affecting the survival of somatic embryos. As PEG concentration increased, the number of mature cotyledonary embryos initially increased and then decreased. These results were consistent with those reported Zhou et al. (2017); Li et al. (2022a). Higher stable osmolarity and the appropriate molecular weight of PEG are key factors for the production of high-quality somatic embryos (Shoji et al., 2006). In our study, the germination rate of somatic embryos tended to increase and then decrease in response to the increase in PEG concentration in the maturation medium. The highest germination rate of somatic embryos (75.3 ± 21.6%) was achieved when 110 g/L PEG was added. Interestingly, somatic embryo plantlets treated with low concentrations of PEG in the maturation medium exhibited better survival rates, whereas those treated with high concentrations of PEG rooted better. This phenomenon had not been reported previously, as earlier studies primarily focused on the impact of PEG on maturity (Jiang et al., 2021). It should be noted that the results from one cell line tested may not be generalizable, and more cell lines need to be studied in subsequent experimentation. However, adding different concentrations of PEG during somatic embryo maturation and observation of the subsequent growth may be a viable method for directly screening the best culture protocol of SE for experimental purposes.

Under aseptic conditions, the survival rate of in vitro grown plantlets was low due to the absence of ectomycorrhizal fungi (Wang et al., 2015). Inoculating mycorrhizal fungi can promote the growth of in vitro grown seedlings, and improve the adaptability of the in vitro regenerated plantlets to the transplant environment (Niemi and HäGgman, 2002; Zhu et al., 2010). Wang et al. (2015) reported that the mycorrhizal fungi Rhizopogen luteous, Pisolithus tinctorius and Boletus edulis were effective in improving the survival rate of transplanted cultured plantlets of P. densiflora. Niemi et al. (2007) showed that using specific ectomycorrhizal fungi can promote plant development, but not root formation. In our study, we also observed that inoculation with an ectomycorrhizal fungus promoted plant development. Ectomycorrhizal inoculation increased the height of regenerated plantlets of cell line 20-1-7, but this effect was not evident in the other two cell lines. The effects of inoculation on the growth of cell lines varied from cell line to cell line, as reported by Vaario et al. (2015). Yamanaka et al. (2012) demonstrated that growth did not change immediately after mycorrhizal inoculation, but positive effects on root-to-crown ratio and root density were found at five months after inoculation. Only one of the three cell lines responded positively to growth after mycorrhizal inoculation in the current study. The reason for this may be the short growth period before transplanting. All ectomycorrhizal plantlets showed lush growth of needles and leaves after transplanting, and mycorrhization also improved the survival rate of transplants. This finding was consistent with the research results of Oliveira et al. (2003); Wang et al. (2015).

PWD is a very complex disease, and in-vitro inoculation using aseptic nematodes and host plantlets grown aseptically has been proven suitable for accurate study of this plant-pathogen interaction (Zhu et al., 2012). Zhu et al. (2012) demonstrated that inoculation with aseptic PWNs caused wilting of young pine microcuttings and seedlings, and Faria et al. (2015); Lin et al. (2017) and Zhu et al. (2020) verified that aseptic PWNs are pathogenic. In this study, inoculation with aseptic PWNs caused wilting of both non-mycorrhizal and mycorrhizal plantlets, confirming that aseptic PWNs demonstrate pathogenicity. However, the results of this study differed from those of other studies (Han et al., 2003; Wang et al., 2018). The resistance of non-mycorrhizal and mycorrhizal plantlets of P. massoniana to PWN was studied in this experiment. One cell line (20-1-7) showed relatively high resistance levels to PWN. Both of the wilting rate and the number of nematodes recovered from ECL 20-1-7 were significantly lower than those from ECLs 20-1-4 and 20-1-16.

Ectomycorrhizal fungi are known to form symbioses relationships with plant roots to promote nutrient uptake, but it remains unclear whether they also induce disease resistance in plants (Wang et al., 2022). Some studies, including Wu et al. (2007) and Deng et al. (2017), have suggested that ectomycorrhizas may improve the resistance of plants to disease, while Wang et al. (2022) suggested that ectomycorrhizal colonization triggers a systemic defense response, enhancing the resistance of pine seedlings to Sphaeropsis shoot blight. In the current study, we found that the wilting ratios of non-mycorrhizal regenerated plantlets in all cell lines were significantly higher than those of mycorrhizal regenerated plantlets, indicating that ectomycorrhizas can improve resistance against PWD and delay the death of regenerated pine plantlets inoculated with B. xylophilus. This result suggests that ectomycorrhizal fungi may induce disease resistance. However, to obtain more detailed information, stronger supporting evidence using modern molecular biology methods is needed. Currently, the reason for the inhibition of nematode propagation in cell line 20-1-7 and in mycorrhized plantlets in this study is unclear, and further research is required.

In this study, we investigated the primary and secondary factors affecting the maturation of P. massoniana somatic embryos. Additionally, we examined the effects of supplements added to the maturation medium on the subsequent germination, survival and rooting of P. massoniana somatic embryos. The regenerated plantlets were then inoculated with ectomycorrhizal fungi and acclimated for transplanting. Our results showed that inoculation with aseptic PWNs caused wilting of non-mycorrhizal and mycorrhizal plantlets of P. massoniana. However, the proportion of wilting plants from mycorrhizal regenerated plantlets in all cell lines was lower than that from non-mycorrhizal regenerated plantlets. This plantlet regeneration system and mycorrhization method developed in this study could be applied to produce nematode-resistant plantlets on a large-scale and to further study the interaction between nematode, pines, and mycorrhizal fungi.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

Y-MC, L-HZ and J-RY conceived and designed the study. Y-MC, QF, X-RX, and XK performed the experiments and carried out the statistical analysis. Y-MC wrote the manuscript. All authors contributed to manuscript revision and have read and approved the submitted version. All authors contributed to the article and approved the submitted version.