Eucommia ulmoides Oliver is in the family Eucommiaceae and is a relict plant of the tertiary period [1]. It is distributed in the subtropical and temperate areas of China (24°50${}^{\prime }$N-41°50${}^{\prime }$N, 76°00${}^{\prime }$E-126°00${}^{\prime }$E), and is also a high-quality natural rubber tree species and a rare medicinal tree species unique to China [2]. As a national strategic reverse resource, E. ulmoides gum in E. ulmoides has the characteristics of radar penetration, energy storage, energy absorption, energy conversion, shock absorption, and shape memory, etc. [3, 4, 5], and has potential value in the fields of national defense and military, aviation and aerospace, transportation, precision instruments, and medicine. In addition, E. ulmoides contain lignins, iridoids, phenylpropanoids, flavonoids, and other active ingredients [6], and has medicinal value in regulating blood pressure, lowering blood sugar, and enhancing immunity. Therefore, E. ulmoides has great development prospects in the international market.

Historically, due to technological limitations, researchers have primarily concentrated on pharmacology [7, 8] and resource collection [9, 10] when studying and utilizing E. ulmoides, but have not searched for functional genes [11], which has severely limited the research of molecular breeding and related biological characteristics of E. ulmoides. With the advancement of omics technology and the availability of E. ulmoides genome and transcriptome resources, there is a growing interest in identifying E. ulmoides functional genes.

However, genetic transformation technology still suffers from limitations of the cost of time, labor, and economy. For instance, a particle bombardment method and Agrobacterium-mediated method can establish a stable genetic transformation system in E. ulmoides, but they are limited by expensive equipment, low transformation efficiency, difficult genetic transformation, challenging in vitro regeneration, the long time required, and low functional expression efficiency of genes. These factors hinder the high-throughput analyses of functional genes.

Transient expression of protoplasts is a general and convenient technique for functional gene analysis. Protoplasts can be obtained by mechanical isolation or enzymolysis. Protoplasts can be used for high-throughput analysis of subcellular localization, gene function, promoter activity, protein interactions, hybridization, and gene expression. Currently, protoplast transformation systems have been established in a variety of commercial crops, including rice [12], sugarcane [13], perennial ryegrass [14], freesia [15], orchid [16], and soybean [17], of which some results have been achieved in the protoplast isolation of a few woody economic plants such as cassava [18], Magnolia [19], camellia oleifera [20], and poplar [21], indicating that the establishment of an efficient protoplast isolation method is feasible in the study of gene function of woody plants.

To the best of our knowledge, establishment of a protoplast transient expression system of E. ulmoides has not been reported. As the model plant of natural rubber and woody medicinal plants, it is necessary to establish a transient expression system, which is of great significance for the study of functional genes of E. ulmoides.

The aim of this study was to establish an efficient and simple protoplast isolation and transient transformation system for E. ulmoides. Efficient protoplasts were obtained by optimizing the factors that affect the yield and activity of protoplasts including the concentration of mannitol, enzymolysis time, and combination of enzymes. Furthermore, using green fluorescent protein (GFP) as the reporter gene, we verified the feasibility of the polyethylene glycol (PEG)-mediated protoplast transient expression system of E. ulmoides.

The results showed that the system is efficient and convenient, and can be used as a reference for future gene function studies of medicinal plants and hard rubber plants.

Plant protoplasts can be isolated from plants’ specific tissues or organs and can efficiently provide multiple independent single cell systems to solve specific problems because they retain their cellular properties [16]. It was once thought that the source tissues suitable for protoplast isolation were derived from non-seedling plants [22]. However, several studies in recent years have indicated that protoplasts can be isolated from young plant organs such as hypocotyls, leaves, roots, and root hairs [23], which provides a reference idea for the isolation of protoplasts from plants of different species.

The establishment of a stable protoplast isolation system is a prerequisite for generating a large number of protoplasts with high viability. The isolation of protoplasts is typically influenced by several factors including material selection, enzyme type and concentration, osmotic pressure, and enzymatic digestion time. Although efficient protoplast isolation systems have been reported in several herbaceous plants in recent years, isolation of sufficient amounts of live protoplasts in woody plants is still a challenge. This is especially true for Eucommia, which has great potential for exploitation because the whole plant, except the xylem, is rich in Eucommia-rubber (hard rubber), which is a major challenge for the isolation of E. ulmoides protoplasts.

Experiments have been reported on the selection of materials from plants from the outdoors that can affect the efficiency of protoplast isolation due to their thick cuticle and thick cell walls [24], but the preparation and supply of test tube seedlings often require skillful aseptic manipulation, economic cost, and time cost. With the aim of convenience, economy, and efficiency, we used stems from 1-week-old live seedlings of E. ulmoides cultured at a simulated room temperature, which were not lignified and contained less Eucommia rubber to conduct the study.

Free protoplasts are fragile when they have no cell wall to maintain their shape, and this fragility is reflected in the inability of protoplasts to remain isotonic with the external environment, resulting in swell, burst, or shrinkage of protoplasts. The addition of an appropriate concentration of osmotic stabilizer to the enzymatic solution can maintain the equilibrium of osmotic pressure in and out of the protoplasts. Most current protoplast isolation systems add mannitol and sorbitol to maintain the osmotic pressure of free protoplasts. The appropriate concentration of the osmotic stabilizer depends on the properties of the material itself. The results of several studies showed that appropriate addition of mannitol in the range of 0.4 mol/L to –0.8 mol/L can maintain the shape of protoplasts for a long time, depending on the material. Our results indicated that both hypoosmolarity and hyperosmolarity were detrimental to isolation of protoplasts from the stems of E. ulmoides seedlings. When the mannitol concentration was 0.4 mol/L to –0.5 mol/L, most of the protoplasts swelled and ruptured. When the mannitol concentration was greater than 0.6 mol/L, some of the protoplasts shrunk, which was not conducive to the progress of transient transformation. Therefore, the addition of 0.6 mol/L mannitol was determined to be the optimum concentration for protoplast isolation from the stems of E. ulmoides seedlings.

Enzymatic digestion time is one of the important factors in obtaining protoplast yield and viability, which varies according to different tissues and organs. When protoplasts were isolated from the leaf mesophyll of Catalpa bungei, the enzymatic digestion time was too short, resulting in inadequate enzymatic digestion of protoplasts, and the yield could not meet the need of instantaneous transformation, whereas excessive enzymatic digestion time could lead to a decrease of protoplast activity [23]. The experimental results suggested that the protoplast total yield and live protoplast yield showed a stepwise increase within 10 h, and peaked at 10 h. Therefore, enzymatic digestion time of 10 h is appropriate. Unexpectedly, we found that even though there was a decreasing trend in protoplast total yield and live protoplast yield after 10 h, the decrease in viability was not significant. Based on the comparison of previous methods with this study, it was speculated that the survival rate of protoplasts might be mainly influenced by the following factors: (i) the oscillation rate during enzymatic digestion; (ii) the state of the experimental material; and (iii) the composition of inorganic salts in the enzymatic solution.

The type and concentration of enzymes directly affect the preparation of protoplasts. Cellulases, pectinases, macerozymes, and hemicellulases are commonly used for the degradation of cell walls, and the chemical composition of cell walls varies depending on the species and material sources, so the appropriate enzyme combinations and enzyme concentrations for efficient preparation of protoplasts is not uniform. Protoplast yields of 6.5 $\times$ 10${}^5$ protoplasts/g FW and 1.0 $\times$ 10${}^5$ protoplasts/g FW were obtained in the preparation of Pineapple leaves and Skunk cabbage leaves protoplasts, respectively [25, 26]. Isolated different amounts of protoplasts from different organs of Cymbidium orchids tender leaf base, young leave, flower pedicels, root tips, and flower petals were 2.50 $\times$ 10${}^7$/g FW, 3.22 $\times$ 10${}^6$/g FW, 5.26 $\times$ 10${}^6$/g FW, 7.66 $\times$ 10${}^5$/g FW, and 3.3 $\times$ 10${}^7$/g FW, respectively [16]. These previous contributions indicate that increasing enzyme concentration resulted in increased protoplast production but reduced protoplast viability [24]. It was thought that this might be due to high concentrations of enzymes that disrupted the integrity of cell membrane, thus affecting the physiological activity of protoplasts. This study showed that the viability of prepared protoplasts from the stems of E. ulmoides seedlings did not significantly change after reaching a certain concentration, but the total protoplast yield and live protoplast yield showed a decreasing trend. We obtained the highest total protoplast yield (12.00 $\times$ 10${}^5$/g FW) and also the highest live protoplast yield (11.33 $\times$ 10${}^5$/g FW) using medium concentrations of enzymes, that is, 2.5% Cellulase R-10, 2.5% pectinase, 0.5% hemicellulase, and 0.6% Macerozyme R-10.

Establishing a stable genetic transformation system is an important constraint for genetic improvement of woody plants. Protoplast-based transient expression is a rapid, economical, and efficient alternative system compared to the traditional stable expression of transgenes, and is important for the analysis of plant gene function. This system has proven to be a powerful experimental tool in molecular biology for a wide range of applications such as subcellular localization [27], promoter activity analysis [28], protein–protein interactions [29], protein–DNA interactions [30], protein-based biochemical assays [31] and quantitative real-time polymerase chain reaction (QRT-PCR) for gene expression analysis.

In this study, a PEG-mediated transient protoplast transformation method based on the protoplast isolation system was established for stems of E. ulmoides seedlings, using mGFP as a reporter gene, and the feasibility of the system was verified. At the same time, the effect of transformation time on transformation efficiency was initially investigated by taking the transformation time as an independent variable. Notably, even though 15 min in 35% PEG solution was beneficial for transformation efficiency (up to 35.01%), similar to the results of other studies, there are still optimization factors in our transformation system and the efficiency may be further improved.

In this study, we utilized the stems of E. ulmoides seedlings as the material and systematically investigated 9 enzyme digestion solutions, 5 mannitol concentrations, and 14 durations, leading to the development of an optimized protocol for efficient isolation of E. ulmoides protoplasts. The optimized enzyme solution, composed of 2.5% Cellulase R-10 (w/v), 0.6% Macerozyme R-10 (w/v), 2.5% pectinase (w/v), 0.5% hemicellulase (w/v), and 0.6 mol/L mannitol, enabled the generation of 1.13 $\times$ 10${}^6$ active protoplasts/g FW within a 10 h enzymatic digestion with a viability up to 94.84%. Regarding PEG-mediated expression of GFP, among the four different transfection time periods tested, a 15 min duration yielded optimum transfection efficiency of 35.01%. This study not only established the optimal protocol for E. ulmoides protoplasts isolation but also confirmed the feasibility of a PEG-mediated transient transformation system for E. ulmoides.

All data points generated or analyzed during this study are included in this article and there are no further underlying data necessary to reproduce the results. The raw data are available from the corresponding author on reasonable request.

Conceptualization—BH, MD and QZ; Data acquision—BH, RL, WS and JP; Formal analysis—YW, LM and YD; Methodology—BH, CW and QZ; Writing original draft—BH; Corresponding—QZ. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Eucommia ulmoides (Jishou University, Hunan, China) plants were used in this study. The plant materials were provided by Prof. Qiang Zhou (Jishou University, Hunan, China).

We are grateful to the Institute of Botany, the Chinese Academy of Sciences for supporting this work.

This research was funded by National Natural Science Foundation of China under Grant [32160388].

The authors declare no conflict of interest.