Mosses are increasingly being used in the development of greenery in city buildings and indoor environments. The green landscape in cities has beneficial impact on psychological well-being because lacking of vegetation in cities may lead to lower social and mental health issues (Kuo and Sullivan, 2001). In addition to these benefits, mosses do not need fertilizer or pesticides compared with the flowering or vascular plants, making them easy to maintain (Julinova and Beckovsky, 2019). Furthermore, mosses can survive long periods of drought stress and can regenerate once they get moist again. Giving these advantages, mosses are becoming more and more popular for use in designing natural living landscapes in urban buildings and indoor environments. This includes the use of moss in vertical gardens, moss walls (Udawattha et al., 2018; Perini et al., 2022), green roofs (Anderson et al., 2010; Cruz de Carvalho et al., 2019), and bio-walls. Mosses growing on flexible materials can also be cut into customized shapes, logos, or art ornaments for indoor decoration or micro-landscape design (Julinova and Beckovsky, 2019).

Although the utilization of mosses in the greenery establishment was widely reported (Gunawardena and Steemers, 2019; Perini et al., 2022), the source of moss materials remains to be a challenge. The selected moss species collected from the wild may not be homogeneous and may contain other unknown organisms that could be harmful to humans, because moss layers are important habitats for periphyton and invertebrates (Wulf and Pearson, 2017). Furthermore, a vast amount of disruption of the wild moss layer may lead to land degradation (Adessi et al., 2021). Thus, artificial planting is the most sustainable way to obtain moss materials. Previous studies have reported on the axenic cultivation of various moss species for research purposes (Collier and Hughes, 1982; Cove et al., 2009b), commercial applications (Zhao et al., 2019; Heck et al., 2021) and rare taxa conservation (Sabovljević et al., 2012b; Sabovljević et al., 2022). However, to date, there is no report on the cultivation of V. montagnei.

These applications of mosses for establishing greenery requires a strong root-like system that can attach to the supporting surfaces. However, unlike vascular plants with a root system, mosses only possess root-like structures called rhizoids, which provide less adhesive ability. This limitation restricts their use in urban greenery establishment, particularly in the vertical applications. To overcome this issue, promising solutions include screening and selecting specific moss species suitable for this application or breeding new moss lines using biotechnological approaches.

Polyploidy refers to the presence of more than two haploid genomes in a single nucleus of an organism. This phenomenon is considered a potent evolutionary force that drives genetic variation and functional novelty (Paun et al., 2007; Hegarty and Hiscock, 2008). Polyploidization has been proven to be a useful biotechnology and is wildly used to improve agronomic traits of horticultural crops, such as Citrus (Aleza et al., 2009), Lycium (Rao et al., 2020), and Populus (Xu et al., 2016) plants. Phenotypic changes that occur after polyploidization include increases in leaf size, plant height, stomatal length and stress resistance (Eng and Ho, 2019). However, the induction of polyploidy in mosses for improved horticultural applications is still rare.

Jasmonic acid (JA) is a lipid-derived plant hormone that is generated from trienoic fatty acid through the octadecanoid pathway. JA is involved in regulating various aspects of plant development, including root architecture (Wasternack and Hause, 2013; Wasternack and Strnad, 2018). In Arabidopsis, the JA signaling pathway was found to inhibit root growth and the formation of lateral and adventitious roots (Wasternack and Hause, 2013). However, JA was shown to promote lateral root formation in rice (Wang et al., 2002). Compared with that in vascular plants, the effects of JA in early landed plants seem to be more complex. A study in a fern plant Platycerium bifurcatum revealed that low concentrations of JA (0.01-1 μM) promoted the formation and elongation of primary rhizoids, whereas concentrations exceeding 1 μM had an inhibitory effect (Camloh et al., 1996). Both cyclopentenone (+)-cis-12-oxo-phytodienoicacid (OPDA, the precursor of JA) and methyl jasmonate (JA-Me) at the concentration of 5-50 μM significantly inhibited the growth of rhizoids. However, only OPDA but not JA or its derivative JA-Ile, could be detected in the moss P. patens (Stumpe et al., 2010; Ponce De León et al., 2012). Nevertheless, JA has been detected in several other moss species using phytohormone profiling (Záveská Drábková et al., 2015).

Mosses are increasingly popular in establishing natural living landscapes in urban buildings or indoor environments. The stable and sustainable way of obtaining mosses for these applications demands a standardized in vitro cultivation system. The previous study reported that the development of cultivation systems for different peat moss species is also important in mitigating climate change (Zhao et al., 2019; Heck et al., 2021). In our study, the moss V. montagnei that can adapt to different climate environments was chosen to establish an in vitro cultivation system.

Our results showed that adding sucrose to the BCDAT medium promotes both germination and the growth state of V. montagnei, which differs from P. patens that grows well without external carbon source (Cove et al., 2009b). It was also reported that external sugars had a negative impact on protonemal development and shoot multiplication of the moss Atrichum undulatum (Sabovljevic et al., 2005). Thus, different mosses may respond differently to exogenous sugars. However, in different species from the same genus of Sphagnum, sucrose- or glucose-addition has been consistently found to boost growth (Simola, 1969; Graham et al., 2010; Beike et al., 2015). Despite carbon source, nitrogen source has also been reported to be critical for determining growth in diverse bryophytes (Basile, 1975). In our study, simply increasing the concentration of KNO₃ did not enhance the growth of V. montagnei ( Figure 1B-H ). An appropriate ratio between sucrose and ammonium nitrate is critical for the growth of protonema of the moss Polytrichum commune (Yu et al., 2008). The addition of sucrose and ammonium was able to increase the biomass of Sphagnum palustre (Beike et al., 2015). Moreover, different nitrogen sources also affected the growth rates, bud production rates, and plant morphology in the cultivation of Splachnum ampullaceum (Gonzalez et al., 2006). Our optimized medium may provide valuable information for the studying other Vesicularia species. In addition to sucrose and nitrogen sources, plant hormones were used to adjust the growth of mosses. The combinations of BA and IBA with different concentrations were used to obtain of moss Bruchia vogesiaca in different developmental phases (Sabovljević et al., 2012b). It will be interesting to investigate the effect of plant hormones on the growth of V. montagnei in the future.

Various media types, such as BCD, Murashige and Skoog (MS), and Knop, have been used for growing different species of mosses (Reski and Abel, 1985; Cove et al., 2009b; Sabovljević et al., 2012a; Pandey et al., 2014). Previous studies compared the effectiveness of BCD and half-strength MS (MS/2) media in cultivating Bruchia vogesiaca (Bruchiaceae) and Entosthodon hungaricus (Funariaceae) (Sabovljević et al., 2012a; Sabovljević et al., 2012b). It was found that MS/2 medium was the most suitable for the propagation and subculturing of E. hungaricus (Sabovljević et al., 2012a). However, B. vogesiaca had better growth in terms of protonema diameter and bud initiation when cultivated in the BCD medium (Sabovljević et al., 2012b). In this study, the successful axenic cultivation of V. montagnei for its entire life cycle was achieved on the BCDAT solid medium with added sucrose ( Figure 2 ). It appears that different species of moss may have varying preferences for the types of media. Our development of this axenic cultivation system has enabled the propagation and multiplication of homogeneous and insect-free V. montagnei plant materials. And most importantly, this system allows for the establishment of several lines of diploid V. montagnei plants with an improved rhizoid system ( Figures 4A–C ). These lines may hold promising potential for greenery applications. It has been reported that cultivation of Sphagnum mosses in the liquid Knop medium yielded high amounts of biomass (Beike et al., 2015; Zhao et al., 2019; Heck et al., 2021). Conducting further investigations to test or modify the liquid medium for the purpose of increasing the biomass of V. montagnei moss can be helpful for its application in greenery establishment.

Artificial-induced polyploidization is a commonly used technique in horticultural plant breeding, which usually produces larger organs such as flowers, fruits, seeds, leaves, stems, and roots in vascular plants (Eng and Ho, 2019). This phenomenon is termed as “giga” effect. Our study found that the diploidization of the early landed moss V. montagnei also resulted in enhanced growth of rhizoids ( Figures 4A, B ) and slightly larger leaf-like phyloid ( Figures 4G, H, I, J ). These results provided an additional example of the “giga” effects of polyploidization in early landed plants. However, a previous study in P. patens showed that diploidization significantly decreased plant growth rate and dry weight (Gabriele et al., 2005). These different phenotype changes indicated the divergence of the genome structure between different moss species. Our results indicated that artificial-induced polyploidization has great potential for developing diploid moss lines with improved growth performances.

In this study, JA was detected in the fresh samples of both the WT and diploid lines of V. montagnei ( Figure 6H ). Consistently, several putative homologs of the JA synthesis pathway were detected in the transcriptomic analysis ( Figure 6A ), which may form a complete synthesis pathway in the V. montagnei. Similarly, previous phytohormone profiling of several bryophytes (including 6 and 24 species of liverworts and mosses, respectively) successfully detected JA with the concentration range from 1.20 pmol/g FW (Sphagnum sp.) to 78.80 pmol/g FW (Pogonatum urnigerum) (Záveská Drábková et al., 2015). In our study, JA-Ile is not detectable in V. montagnei, which may be caused by the extremely low level or the inactive JAR1-like enzyme. The former reason is more likely because JA-Ile is far less abundant than JA and occurs at very low amounts (almost close to the limit of detection) in several moss species (Pellia endiviifolia, Sphagnum compactum, Sphagnum sp., and Polytrichum commune) (Záveská Drábková et al., 2015). However, studies in the P. patents and the liverwort Marchantia polymorpha could only detect the precursor OPDA but not JA or the conjugate JA-Ile (Stumpe et al., 2010; Ponce De León et al., 2012; Yamamoto et al., 2015), even though the P. patens genome contains several OPR-like genes (Breithaupt et al., 2009; Li et al., 2009). These results suggested that there may be evolutionary divergences in the JA biosynthesis pathway among the bryophytes. Further investigation of the enzymes responsible for the metabolism downstream of OPDA (such as OPR-like and OPCL) may help to clarify this.

The JA-Ile is the bio-active form of JA in the vascular plants and is able to induce the expression of several JA signaling genes through the negative feedback loop (Chico et al., 2008). Interestingly, JA-Ile was not detected in the moss V. montagnei, but the signaling homologs VmJAZ1 and VmNINJA showed increased expression levels in the diploid lines with elevated JA content ( Figures 6K, M ). This suggests that the bio-active jasmonate in the moss V. montagnei is likely not JA-Ile. Notably, a study in Nicotiana attenuata found that JA-Ile is not the only active oxylipin signal molecule (Wang et al., 2008). While the JA-Ile application fully restored the resistance to Manduca sexta feeding in JAR4/6-silenced plants (impaired in JA-Ile synthesis), it only partially restored resistance in LOX3-silenced plants (deficient in JA–Ile and other oxylipins). Further search for the bio-active jasmonate besides JA-Ile will be very interesting. Our investigation provided evolutionary evidence for the existence of other bio-active jasmonate besides JA-Ile and improved our understanding of jasmonate biosynthesis.

It was proposed that rhizoid in moss gametophytes and root hair on the roots of vascular plant sporophytes were close related in the evolutionary perspective (Jones and Dolan, 2012). Because a similar gene regulatory network controls the development of rhizoids and root hairs. In the vascular plant Arabidopsis, JA has been shown to have a pronounced effect on promoting root hair formation (Zhu et al., 2006; Han et al., 2020). Consistently, our study found a significant increased rhizoid density associated with the accumulation of JA content in the diploid lines of V. montagnei ( Figures 4A–C ). These results suggested that JA has a conserved function in regulating the development of both rhizoids and root hairs. Based on our findings, it is promising to enhance the rhizoid growth through the direct application of JA in vitro, which may improve the ability of the plants to adhere to the surfaces for greenery decoration purposes.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://ngdc.cncb.ac.cn/gsa, GSA: CRA009372.

LL designed and supervised the study. QL and YH performed most of the experiments and analyzed the data. ZC contributed in the diploid induction. ZX analyzed the RNA-seq data. ZC, HL, and CW contributed in the preparing of mosses. YH and QL drafted the manuscript. LL, LD, and HY revised the manuscript. All authors contributed to the article and approved the submitted version.