Protoplasts that share a similar phenotype at the beginning of culture follow a series of fates, such as live, dead, and dividing cells, with different fates occurring throughout the culture period. The tracking of protoplasts during the early stage of culture can help identify some of the aspects of protoplasts with similar phenotypes (Xu et al. 2021). The characteristics of protoplasts identified through tracking largely include (1) changes in the number and size of vacuoles and (2) an increase in cell size. Prior to cell expansion, we confirmed an increase in the number and size of vacuoles. This is consistent with the results of previous studies showing that an increase in vacuole pressure is preceded by biosynthesis of endogenous auxin. As a result, cells expand (Sakamoto et al. 2022) and provide mechanisms that contribute to the maintenance of homeostasis (Löfke et al. 2015; Scheuring et al. 2016; Kaiser and Scheuring 2020). Cell expansion also occurred in protoplasts, and an increase in their size as the culture period progresses was reported in tobacco leaf-derived protoplasts (Wu et al. 2011). Kaiser and Scheuring (2020) showed that a certain amount of cytoplasm can be maintained by acidifying the apoplast and loosening the cell wall, causing the cell to expand and allowing the vacuole to occupy this space. Thus, the limited amount of cytoplasm may not affect cell growth as a limiting factor (Kaiser and Scheuring 2020). Although protoplasts are not completely identical after the cell wall is removed, it can be inferred that they may be involved in cell expansion through a similar mechanism. In addition, expansion of protoplasts is a process that precedes cell division (Coutts and Grout 1975), and this cell expansion is achieved by stimulation of intracellular auxin (Steffens and Lüthen 2000; Steffens et al. 2001), which is a result of cell division.
Protoplasts isolated from plants exist in a more fragile form compared with that of general plant cells whose cell walls are removed (Lei et al. 2015). Some protoplasts among the isolated ones can be regenerated into plants through a series of processes. However, in general, most protoplasts exhibit a low cell proliferation rate (Suzuki et al. 1998; Chupeau et al. 2013). Thus, various protoplast culture methods have been studied (Davey et al. 2005a), and one must be established for stable regeneration experiments. The liquid culture method is conventionally used for culturing protoplasts and has been used for various plants as well (Pongchawee et al. 2006; Ali et al. 2018; Sangra et al. 2019). Liquid culture methods can also be used for gene expression analysis, such as RNA-seq (Yang et al. 2008; Chupeau et al. 2013). Although this method is a relatively easy technique when compared with other culture methods, it exhibits apoptosis and low cell division activity due to cell aggregation, resulting in a low cell regeneration efficiency (Hall et al. 1993; Davey et al. 2005a; Kiełkowska and Adamus et al. 2012; Jeong et al. 2021). Therefore, a sieve culture method was used to compensate for the disadvantages of liquid culture. In sieve culture, a sieve support with micro-pore sizes was added to the existing liquid culture method. Protoplasts can be cultured evenly on sieves; it has an advantage in that the medium composition can be changed without having a considerable physical impact on protoplasts (Lei et al. 2015). In addition, the protoplasts share a narrow area in the sieve and thus have a high plating density. Culture density is an important determinant of the frequency of early cell division. Panis (1993) reported that, in the case of banana leaf-derived protoplasts, a high plating density is important regardless of the protocol used. In contrast, the frequency of protoplasts that did not divide while surviving in the three culture methods was the highest in the culture of EC-derived protoplasts. These results are consistent with those of previous studies showing that cell density above a certain level inhibits the proliferation of protoplasts (Damm and Willmitzer 1988; Gandhi and Khurana 2001). In addition, protoplasts derived from ECs have a lower regeneration capacity in a high-density environment. The TAL culture method is a solid culture method devised by Davey (2005a). Alginate forms a hydrogel through cross-linking with divalent alkali metal ions (Ca2+ and Ba2+) (Mørch et al. 2006) and helps to stabilize the culture without cell aggregation, which is one of the drawbacks of using liquid culture for fixing protoplasts. Thus far, the alginate-embedding method has been widely employed for all types of plant species, such as the model plants, A. thaliana and Nicotiana tabacum, including Petunia and Phalaenopsis, as a means for stable protoplast regeneration (Shrestha et al. 2007; Meyer et al. 2009; Lei et al. 2015; Klimek-Chodacka et al. 2020; Jeong et al. 2021). Furthermore, the best plating efficiency was reported for TAL among the other culturing methods tested (Damm and Willmitzer 1988; Gandhi and Khurana 2001). The TAL culture method also showed the highest frequency of multi-cell formation in protoplasts derived from EC, which was the experimental material used in this study. This suggests that TAL can have the highest regeneration ability among the culture methods tested.
Based on Design-Expert software, the widely applied central composite design (CCD) model was used for model design. This model provides an independent estimate of the experimental error and is expressed through first-order and second-order models. The advantage of this model is that it does not require a three-step factorial experiment to build a second-order model (Bhattacharya et al. 2021). In this study, the medium conditions were optimized by designing an RSM model using the three independent factors, 2,4-D, kinetin, and PSK, for the developmental steps up to the regeneration of EC-derived protoplasts. For the regeneration of protoplasts, different media compositions are required for each plant species and culture stage. In a study on the regeneration of protoplasts of various plants, such as A. thaliana, Chrysanthemum cv. White ND, and Kalanchoë aromatica, for each developmental stage, including protoplast induction, callus formation, and pro-EC formation, different media compositions were used (Monteiro et al. 2003; Cui et al. 2019; Adedeji et al. 2020; Jeong et al. 2021). As such, to form an appropriate medium for each developmental period, various concentrations of plant growth regulators and medium salt compositions must be considered, which have mainly been studied in model plants (Damm and Willmitzer et al. 1988; Díaz et al. 2011). Recently, the medium composition according to each developmental stage was established for the cultivation of protoplasts from various plant species (Shi et al. 2016; Cui et al. 2019; Li et al. 2021). It may be time-intensive to set up an appropriate culture medium because the culture conditions must first be investigated (Jeong et al. 2021). Therefore, in this study, by using RSM based on the CCD method, the optimal culture conditions were established while the time and cost for setting up the culture medium were reduced. Various stress factors, such as immersion and washing of cell wall digestion enzymes in the process of isolating protoplasts, and oxidative stress reactions, such as the release of H2O2 during culture (Pasternak et al. 2007), act on protoplasts. Therefore, the already dedifferentiated cells can undergo cell cycle re-entry and cell division. Only cytokinin (0.5 mg∙L− 1 kinetin) was required at the time of the first division of protoplasts derived from the EC of A. gigas. Although there are differences depending on the plant species, cytokinins are responsible for cell cycle re-entry because they upregulate transcription of the D-type cyclin cell cycle regulator, CYCLIN D3 (CYCD3), one of the reporters related to cell proliferation among genes involved in cell cycle re-entry. Thus, cytokinin has been reported to promote proliferation (Riou-Khamlichi et al. 1999). Ikeuchi (2017) reported in a callus formation study via a wound-induced stress response in A. thaliana that the expression of CYCD3 is cytokinin-dependent and involved in callus formation. After evaluating the interaction between 2,4-D, kinetin, and PSK, which were all involved in the first cell division used in this experiment, the highest F value was obtained for the linear term of 2,4-D. 2,4-D had the greatest influence on the frequency of the first cell division and a negative correlation when referring to the regression equation. In addition, increasing the concentration of kinetin through a negative interaction in the square root of kinetin had a negative effect on cell division. PSK did not have a positive effect on the first cell division of isolated protoplasts. This is similar to the results of previous studies in which the effect of PSK was not significant on carrots and cabbages, which have a genotype that induces cell division well (Godel-Jędrychowska et al. 2019; Kiełkowska and Adamus 2019).
After optimization for multi-cell formation, increased concentrations of 2,4-D and kinetin were required compared with those in the first cell division. The interaction with other linear terms or an increase in the concentration of kinetin as the second term had the greatest negative effect on multi-cell formation. The combinations of auxins and cytokinins differ depending on the genotype of isolated protoplasts and source type (Reed and Bargmann 2021). In the culture of protoplasts derived from the cell suspension of Populus beijingensis, a relatively high auxin-to-cytokinin ratio was effective for micro-calli formation (Cai and Kang 2014).
In contrast, in Kalanchoe leaf-derived protoplasts, the formation of micro-calli was increased at a higher cytokinin-to-auxin ratio. In another study, when only cytokinin was used, micro-calli formation decreased and the cells eventually died (Castelblanque et al. 2010). Based on these results, we suggest that a high auxin concentration and relatively low concentration of cytokinin could help in the process of multi-cell formation. These positive results are consistent with the research results related to the promotion of cell division previously described (Grzebelus et al. 2012; Maćkowska et al. 2014). The mechanism related to the promotion of protoplast cell division should be characterized in future studies.
PSK helped to form multi-cells of EC-derived protoplasts from A. gigas but did not significantly aid in growth at the time of the first cell division and EC stage (Table 4, Fig. 4, E and F). Godel-Jędrychowska et al. (2019) reported that in leaf-derived protoplasts of several Daucus genera, PSK promoted multi-cell formation of D. montevidensis, a wild species in which protoplast regeneration was challenging; however, an EC was not formed. As such, the effects of PSK may differ depending on the plant species and genotype.
When the CCD-based RSM method used in this experiment is employed, the optimal medium composition can be confirmed at each stage, including the points where tests were not performed within the experimental range, and time and cost can be reduced by optimizing the experiment within a relatively short period of time. Thus, this study is important as it characterizes a predictive method that can be used to maximize the formation of ECs that can be regenerated into whole plants through parameter optimization.