Preexisting meristematic tissue is the prerequisite for GGB formation in D. roosii
The maintenance of SAM organization in plants results from a balance between cell division and differentiation in this region. Cell fate in SAM is regulated spatially and temporally; cells in the central zone remain undifferentiated, while cells gradually acquire differentiation ability when recruited into organs in the peripheral zone [28]. SAM of ferns was once thought to be composed of a single apical cell, which would be the main source of the entire plant body [29–32]. However, cytohistochemical observations have shown that fern meristems are multicellular with specific zonation [6]. Recently, some regulatory factors have been identified for the maintenance of SAM in ferns [6, 30]. These results suggest that the organization of SAM in ferns may also be balanced by cell division and differentiation. Our results suggest that the addition of 6-BA in the induction medium altered the organization of SAM in D. roosii by reverting the cell fate from differentiation to division, or by enhancing the division ability of those undifferentiated cells, which resulted in unscheduled cell proliferation. Then, an excessive cell division produced GGB with a large number of meristematic cells, suggesting that the meristematic cells originated GGB in D. roosii. Although GGB can be successfully induced on different explants in several fern species [9, 11, 14, 15], the precise cell origin of GGB has not been addressed. We believe this cognition was developed largely since the explants used for GGB induction already had meristematic cells.
In an in vitro culture system, plant regeneration largely depends on the ability to respecify cell fate [2]. For plant tissue culture, explants from juvenile parts usually exhibit more regenerative capacity than those from mature parts, because plant cells in immature or juvenile bodies tend to have a high potential to redirect the cellular fate [2]. The most frequently used explants for fern GGB induction are shoot tips and young leaves [8]. Under our inductive conditions, GGBs of D. roosii were generated on rhizomes, young leaves and petioles of young sporophytes. For rhizome explants, the GGB formed directly from cell proliferation of the preexisting SAM in the rhizome tip region, and indirectly from the newly developed shoot that transformed from LAM. In leaf and petiole explants, GGBs were formed by the meristematic cell proliferation of SEs, which were produced on the epidermis of both explants. These results suggest that the preexisting meristem is a prerequisite for the formation of GGB. In addition, under our induction conditions, the cells of both LAM and SAM can proliferate to form GGB, while the cells of root apical meristem failed to form GGB, which indicated that the meristematic cells from LAM resembled those from the SAM in that they were more easily proliferative. These results corroborate studies that reported that fern leaves are developmentally equivalent to shoots [33–35].
Plant regeneration from GGB underwent organogenesis in D. roosii
Fern regeneration from GGBs is highly efficient, as GGB differentiation gives rise to a large number of shoots, which then develop into juvenile sporophytes when inoculated on the proper medium. In an in vitro culture system, the dividing cells follow three developmental pathways: organogenesis, somatic embryogenesis, or disordered division [36]. However, the classification of the regeneration pathways of ferns via GGB induction and differentiation is still debated. This is probably because of overlapping of the two developmental states, proliferation and differentiation. Here, the induction and proliferation processes of GGB in D. roosii were analyzed. We found that the response of explants to 6-BA in the medium led to the formation of GGB, and the GGBs developed into shoots rapidly through the differentiation of epidermal cells into apical cells after they were inoculated into PGR-free medium, indicating the competency of GGB to recover the SAM organization. In addition, when GGB began to differentiate, the epidermal cells gave rise to the shoot primordia, leaf primordia, or even leafy shoots. We also verified the development of root primordia in the inner face of GGBs. Furthermore, the micro X-ray computed tomography analysis demonstrated that there were developing vascular tissues inside the GGB connecting all these primordia or organs. Indeed, the somatic embryos were observed to be involved in the process of GGB induction from the leaf and petiole explants, while in the differentiation process, no intermediate state resembling the somatic embryogenesis was detected. Collectively, these results suggest that the regeneration of D. roosii initiated by GGB differentiation could be considered as organogenesis pathway.
Similarities and distinctions between GGB and other propagules
GGB vs. SE
Somatic embryogenesis is characterized by the establishment of polarity [37, 38] and the absence of vascular tissue connections from the maternal tissues [38]. Polarity is not necessarily established during the embryogenesis of ferns, that is, the shoot meristem and the root meristem do not develop as opposite to each other. In addition, the embryonic root only exists for a short time, and the young embryo produces foot cells that play an important role in fixing itself on the gametophyte before the root system is formed and of absorbing nutrients until the developing embryonic organs could be fully autotrophic [5, 7, 39]. The observation of somatic embryogenesis of ferns demonstrated that the developmental process of SE is similar to that of zygote embryogenesis, with no connection between SE and explant via vascular tissue [40]. Here, we found that although there was no obvious embryonic root formed in SE produced on leaf and petiole explants in D. roosii, the vascular tissue of SE was isolated from those of the explants. Moreover, the cells at the touching area between SE and explant exhibited similar characteristics to the foot cells of the zygotic embryo. However, none of these results were found during the process of GGB formation on rhizome explants, suggesting that the formation of GGB on rhizome explants in D. roosii under our induction condition did not involve the somatic embryogenesis pathway. Recently, the marker gene that is expressed during plant somatic embryogenesis, SERK, was identified in GGBs of the fern Adiantum capillus-veneris [15, 41], suggesting that the development of GGB shares similar characteristics of that of SE. However, the SERK gene is not unique to the somatic embryogenesis pathway, and it was also expressed during the organogenesis pathway in some plant species [42–45]. Considering that, in our study, all the SE derived from the leaf and petiole explants subsequently formed GGB in the induction medium, we speculate that the GGBs produced via SE formation on the leaf and petiole explants in D. roosii would probably be embryogenic. However, the somatic embryogenesis that occurred in this process might have occurred because the explants need to establish meristematic tissue for regeneration. Furthermore, even though somatic embryogenesis pathway was involved in the GGB formation process, we believe SE does not essentially characterize the generation of GGB.
GGB vs. callus
Here, shoots and roots were regenerated by GGB in a way that resembles the regeneration of callus via de novo organogenesis. However, recent studies have revealed that callus formation on callus-induced medium followed a lateral root development pathway, and the callus was initiated from xylem-pole pericycle cells of root explants and pericycle-like cells of aerial organs [46–48]. In contrast to callus, GGB was initiated from meristematic cells in SAM and/or LAM, as discussed above. Callus formation and shoot regeneration are two different stages in plant tissue culture, and callus formation alone is not sufficient for shoot regeneration [49, 50]. However, the regeneration in GGB differs from that in callus, because shoot regeneration can be easily obtained when GGBs are transplanted into PGR-free media. Nevertheless, the formation of both callus and GGB requires an important process to establish meristematic tissues. A callus is a disorganized cell mass formed by the cell proliferation process after meristematic cells are generated by the explants [51], rather than a process of reprogramming to an embryonic state [48, 52]. As to GGB, they are formed directly by meristematic cells in the SAM of rhizome explants. The formation of GGB on leaf and petiole explants was indirect and needed a process that established the meristematic tissue via somatic embryogenesis. Therefore, we speculate that during the induction period, the dividing state of cells might be essential for promoting the proliferation capability to respond to the induction factors, both in callus and GGB.
GGB vs. protocorm/PLBs
It has been reported that GGB of ferns is a structure reminiscent of the PLB of orchids [11, 14]. In orchids, protocorm typically describes an indispensable stage after seed germination and before the formation of the first shoot or root, while PLBs appear in plant in vitro culture systems [53]. In these plants, seeds lack embryos or meristem and the protocorm is responsible for the formation of the meristem [54]. In contrast, our analysis indicated that GGB in D. roosii was succeeded by preexisting or reestablished meristematic tissues. Furthermore, protocorms grow spontaneously, whereas GGBs in D. roosii could only be generated under proper inductive conditions, as corroborated by Fernández [8]. Nevertheless, GGBs in D. roosii and PLBs share similarities as both produce multiple shoot meristems when transferred to differentiation media, showing high plant regeneration efficiency.