Since protoplasts are easy access to foreign molecules, they have been extensively developed and used for for gene function research recent years, such as cellular localization (Shen et al., 2017; Wang et al., 2022b), BiFC assays, Co-IP assay (Yu et al., 2017; Wang et al., 2021), gene editing (Gou et al., 2020; Huang et al., 2020; Poddar et al., 2020; Lin et al., 2018), and dual luciferase assay (Wang et al., 2022a) etc. The establishment of efficient protoplast separation and transient expression system provides the possibility for its application, such as Arabidopsis (Siemens et al., 1993; Yoo et al., 2007), tobacco (Locatelli et al., 2003), rice (Chen et al., 2006), soybean (Wu et al., 2018), cucumber (Huang et al., 2012), cassava (Wu et al., 2017), grape (Zhao et al., 2015), and tea plants (Xu et al., 2021) etc. However, efficient protoplast-based systems are still challenging for numerous horticultural plants.
It has been reported that kiwifruit protoplasts were successively isolated several decades ago from petiole (Oliveira and Pais, 1992), callus (Oliveira and Pais, 1991), and cell suspensions (Salomé et al., 1987), and even regenerated plants were cultured from protoplasts (Pedroso et al., 1988). However, due to complex operation and low efficiency, kiwifruit protoplasts have not been widely used up to now, and no an effective transient transformation system has been established. In this study, we obtained high-yield and high-quality protoplasts from the callus of kiwifruit deliciosa by systematically optimizing enzyme combination, mannitol concentration, enzymolysis time, cell filtration sieve size and callus subculture time, etc. The results showed that the maximum protoplast yield of 2% (w/v) cellulase and 0.5% (w/v) macerozyme was 2.83×106 /mL for 7 h with the highest activity of 86.94%, which was higher than that of Magnolias (Shen et al., 2017) and Carnations (Adedeji et al., 2022).
The main components of plant cell walls are cellulose, hemicellulose and pectin, so usually several enzymes were combined to use for cell walls removing (Tan et al., 2013). In this study, our results revealed that the combination of cellulase and macerozyme had the best effect on cell wall decomposition of kiwifruit callus, and the addition of pectinase dramatically reduced protoplast yield, which was consistent with the results in tea plant (Xu et al., 2021) and Arabidopsis (Yoo et al., 2007). While combination of cellulase and pectinase were commonly used to isolate protoplasts from suspension cells of sweet cherry (Yao et al., 2016). These results indicate that the appropriate enzyme combination varies with different genotypes or organs. For example, for cucumber, 1.5% cellulase and 1.5% pectinase had the best effect (Huang et al., 2012), while for kiwi callus, the combination of 1.5% cellulase and 0.5% enzyme solution had the best effect (Fig. 1). The protoplasts were sensitive to osmotic pressure. Here, our results showed 0.7 M mannitol had the best effect (Fig. 2), while 0.4 M mannitol was the optical value for cucumber (Huang et al., 2012). In addition, we found the enzyme solution containing MES (2-(N-morpholino) ethanesulfonic acid) buffer had better effect than CPW solution (data not shown), which were commonly used in the past decades for protoplast isolation. MES buffer has a better buffering effect on pH value, thus it is more commonly used in protoplast isolation in recent years.
The type of enzymolysis material is also one of the important factors affecting the protoplast separation effect. In previous studies, suspension culture cells of callus were used to kiwifruit protoplasts isolation (Oliveira et al., 1992), that is, loose callus should be obtained first, and then transferred into liquid media to suspension culture. This whole process of gaining enzymolysis material is not only time-consuming, but also affected by many factors. In this study, kiwifruit callus was directly used as the material for cell wall lysis in the enzymatic solution. Callus system has been established and preserved by subculture. Recently, an efficient protoplast separation and transformation system for callus was established in apple (Wang et al., 2021).
Protoplasts after cell wall removal are more likely to be transferred into foreign DNA, which is a good material for transient transformation. At present, PEG-mediated method is often used for transformation because of its convenience and simplicity. Although the protoplasmic conversion efficiency was higher than the stable transformation efficiency, up to 90% in Arabidopsis (Sheen, 2001), there are still great differences in different plants. For example, in citrus, a CaMV 35S promoter driven CRISPR/Cas9 vector carrying EGFP expression box was transformed into protoplasts by PEG-mediated plasmid transformation, achieving a transfection efficiency of approximately 66% (Huang et al., 2020). Transformation efficiency in mesophyll protoplast of Chinese kale was about 30% (Sun et al., 2019), while that reached only to 23.2% in Cannabis protoplasts (Matchett-Oates et al., 2021). The transformation efficiency can be improved by optimizing PEG concentration, plasmid DNA concentration, transfection time, etc. (Damm et al., 1989; Lee et al., 2001). The optimal Ca+ concentration in PEG solution may affect the ability of cells to absorb foreign substances, and as PEG concentration increases, DNA hydration decreases, resulting in structural changes that reduce transfection efficiency (Saenger et al., 1986). When the concentration of PEG4000 was 25%, transformation efficiency in the protoplasts from grape berry suspension cells reached the maximum level of approximately 62.1%, while a significant decrease was observed when PEG4000 concentration was increased to 30% (Wang et al., 2015). 50% PEG (w/v) leads to a decrease in protoplast viability, resulting in late protoplast agglomeration, resulting in difficulty in identifying individual protoplasts that produce GFP (Yao et al., 2016).
In conclusion, an efficient protoplast isolation and purifying procedure of kiwifruit callus was described, and parameter for transient transfection were optimized. To our knowledge, it is the first systematic and detailed report describing the PEG-mediated transformation of protoplasts derived from the kiwifruit callus. Although the maximum transfection frequency of this system (about 40%) need to be further improved, this method still provides a convenient instantaneous transformation option for kiwifruit to study subcellular localization of fusion proteins and functional analysis of transgenic products in the future.