Skip to main content

Isolation, purification and PEG-mediated transient expression of mesophyll protoplasts in Camellia oleifera

Abstract

Background

Camellia oleifera (C. oleifera) is a woody edible oil crop of great economic importance. Because of the lack of modern biotechnology research, C. oleifera faces huge challenges in both breeding and basic research. The protoplast and transient transformation system plays an important role in biological breeding, plant regeneration and somatic cell fusion. The objective of this present study was to develop a highly efficient protocol for isolating and purifying mesophyll protoplasts and transient transformation of C. oleifera. Several critical factors for mesophyll protoplast isolation from C. oleifera, including starting material (leaf age), pretreatment, enzymatic treatment (type of enzyme, concentration and digestion time), osmotic pressure and purification were optimized. Then the factors affecting the transient transformation rate of mesophyll protoplasts such as PEG molecular weights, PEG4000 concentration, plasmid concentration and incubation time were explored.

Results

The in vitro grown seedlings of C. oleifera ‘Huashuo’ were treated in the dark for 24 h, then the 1st to 2nd true leaves were picked and vacuumed at − 0.07 MPa for 20 min. The maximum yield (3.5 × 107/g·FW) and viability (90.9%) of protoplast were reached when the 1st to 2nd true leaves were digested in the enzymatic solution containing1.5% (w/v) Cellulase R-10, 0.5% (w/v) Macerozyme R-10 and 0.25% (w/v) Snailase and 0.4 M mannitol for 10 h. Moreover, the protoplast isolation method was also applicable to the other two cultivars, the protoplast yield for ‘TXP14’ and ‘DP47’ was 1.1 × 107/g·FW and 2.6 × 107/g·FW, the protoplast viability for ‘TXP14’ and ‘DP47’ was 90.0% and 88.2%. The purification effect was the best when using W buffer as a cleaning agent by centrifugal precipitation. The maximum transfection efficiency (70.6%) was obtained with the incubation of the protoplasts with 15 µg plasmid and 40% PEG4000 for 20 min.

Conclusion

In summary, a simple and efficient system for isolation and transient transformation of C. oleifera mesophyll protoplast is proposed, which is of great significance in various aspects of C. oleifera research, including the study of somatic cell fusion, genome editing, protein function, signal transduction, transcriptional regulation and multi-omics analyses.

Introduction

Camellia oleifera (C. oleifera) is a valuable oilseed crop belonging to the genus Camellia of the Theaceae family and is mainly distributed in many provinces in southern China and Southeast Asian countries such as Vietnam, India and Japan [1, 2]. Camellia seed oil, rich in unsaturated fatty acids, vitamins, minerals, and other bioactive compounds, is used extensively in China as high-quality edible oil and reputed as ‘Oriental Olive Oil’ [3]. Moreover, camellia seed oil not only can effectively prevent the development of cardiovascular diseases, but also has anti-inflammatory and antioxidant capabilities [4]. Hence, camellia seed oil has become increasingly popular. However, the market still lacks improved C. oleifera varieties due to the obstacles in conventional breeding. As a cross-pollinated plant, C. oleifera possesses a highly heterozygous state in the genetic background. Conventional breeding in C. oleifera bears a long breeding cycle and offspring with complex and genetically unstable trait. The application of modern biotechnology can help solve these problems. Somatic cell fusion technology breaks the barriers of hybridization between species in biology, enables two species that cannot be sexually hybridized to perform asexual hybridization, and creates new varieties with excellent traits of both species through screening and purification [5]. The protoplast culture and fusion technique enlighten the genetic improvement for new varieties of C. oleifera. The prerequisite for utilizing this technique is to obtain a large quantity of highly viable protoplasts.

Plant protoplasts were the living material of plant cells by removing the cell wall and including the protoplasm and plasma membrane [6]. Due to the absence of cell walls, plant protoplasts have been widely used for genetic transformation, cell fusion, and somatic mutation [7,8,9,10]. In addition, plant protoplasts have totipotency and can regenerate into new similar individuals under appropriate conditions [11]. Somatic hybridization with viable protoplasts can break the reproductive barriers in the process of sexual hybridization or distant hybridization, and create new germplasm or new varieties that cannot be obtained by conventional breeding [12]. At present, cell fusion technology has been successfully applied in citrus [12, 13], cotton [14], oilseed rape [15] and other plants.

In previous studies, protocols for protoplast isolation have been very successful in herbaceous plants such as wheat [6], maize [16], rice [17], carrot [10], Arabidopsis [18], and perennial ryegrass [19]. Nevertheless, in woody plants the development of protoplast isolation technology has only been reported in citrus [20, 21], apricot [22], peach [23], tea plants [24, 25], Populus [26] and Robinia pseudoacacia L. [27].

Protoplasts could be isolated from different plant organs by enzymatic digestion [28]. Many factors including the enzyme type and concentration, osmotic pressure, enzyme digestion time and purification method could affect the enzymatic digestion efficiency [29, 30]. Most research has shown that the isolation conditions for protoplasts vary greatly among different tissues of the same species [31, 32]. For example, the enzyme solution ratio and duration of enzyme application, were different among Robinia pseudoacacia L. mesophyll and callus [27]. Therefore, it is generally necessary to evaluate a protoplast isolation system separately of different organs in the same plant.

In recent years, due to the advantages of rapidity and high efficiency, the plant protoplast transient expression system has been widely used in all types of research such as subcellular localization of proteins, molecular interaction, and signal transduction [33,34,35]. There are several commonly used transient transformation methods, such as the polyethylene glycol (PEG)-mediated one. Due to the high transformation efficiency of PEG-mediated method, it is widely applied in molecular and cellular studies in plants [36, 37]. The protoplast transient expression system plays an increasingly important role in genomics and proteomics research [38]. At present, protoplast transient expression systems have been established for many plant species, such as Arabidopsis [39], rice [40], barley [41], grapevine [42], poplar [26], and tea plants [25], and are widely used in basic research. To date, there are no reports of transient expression system using mesophyll-derived protoplasts in C. oleifera. A rapid and convenient protoplast transient transformation technique would be particularly useful for testing gene function or exploring some new technologies, such as genome editing in C. oleifera.

Protocols for the isolation and purification of protoplasts from C. oleifera suspension have been reported [43]. In recent years, preliminary progress has been made in the application of biological techniques such as C. oleifera somatic embryogenesis [44]. However, only a few C. oleifera cultivars induced callus suitable for protoplast isolation, which limited the application of callus and suspension cell lines protoplast isolation system to other C. oleifera cultivars. Compared with callus and suspension cell lines, leaves of in vitro grown seedlings are easier to obtain and widely used for plant protoplast isolation [18]. Any efficient transient expression system using mesophyll protoplast in C. oleifera had not been reported yet.

In this study, a highly repeatable and efficient protocol for mesophyll protoplast isolation and PEG-mediated transient transformation system was developed using C. oleifera leaves as starting materials. This protocol will provide a facile tool for protein subcellular localization and bimolecular fluorescence complementation assays as well as other in vivo molecular studies.

Materials and methods

Plant material and growth conditions

C. oleifera ‘Huashuo’(HS), C. oleifera ‘TXP14’ and C. oleifera ‘DP47’ plant cultivars were obtained from the experimental base of Central South University of Forestry and Technology. In this study, the bud stems and seed embryos (Fig. 1A and C) for the three cultivars were used for culturing in vitro grown plantlets in MS (Murashige and Skoog) [45] medium for 40 days. When the bud stems and seed embryos were embryonic (Fig. 1B and D), they were kept on WPM (Woody Plant medium) [46] (pH 5.8) (Fig. 1E) containing 3.0% sucrose and 0.8% agar. Plants were kept at 25 ± 1 ℃, under a photocycle of 16 h/8 h (light (30 µmol·m− 2 ·s − 1) /dark) for 6–8 week to obtain fully expanded leaves (Fig. 1F–G). First, the protocol for isolating the mesophyll protoplasts of C. oleifera was explored through ‘HS’ cultivar, and then the protocol was applied to ‘TXP14’ and ‘DP47’cultivars to verify the general applicability for protoplasts isolation in different cultivars of C. oleifera.

Fig. 1
figure 1

Leaf selection and treatment of in vitro growth seedlings of C. oleifera. A Seed primary generation culture; B seeds sprout into seedlings; C primary culture of stem segments; D axillary bud germination of stem segment; E proliferation culture of tissue culture seedlings; F undeveloped leaf; G the 1st to 2nd true leaf; H C. oleifera leaves sliced into 0.5 – 1.0 mm strips with a fresh razor blade and placed in EME media

Protoplast isolation

Young leaves of C. oleifera in vitro grown seedlings (subcultured for 1–2 years on the medium) were used to isolate protoplasts at room temperature. The in vitro grown seedlings were treated with dark. Then leaves of in vitro grown seedlings of different leaf ages were collected on the ultra-clean workbench and transferred into a sterile culture flask containing EME solution consisting of MS, 0.5% ME (malt extract), and different concentrations (0.3, 0.4, 0.5 and 0.6 M) of sucrose. The main veins and leaf margins were cut off with a sterile sharp blade, and then the leaves were cut into 0.5–1.0 mm wide strips (Fig. 1H). The strips were immediately transferred into 10 ml sterile EME medium solutions. After all the leaves were cut, the bottle sealing film was capped and placed in a vacuum pump for vacuum pretreatment under negative pressure (− 0.07 MPa). Then 5 ml of EME was pipetted out and 5 ml of enzyme solution was added to form a 10 ml enzymatic hydrolysis system. Enzyme solutions consist of 5.63 mmol/l MES, 24.49 mmol/l CaCl2·2H2O, 7.05 mmol/l NaH2PO4·2H2O, different concentrations (0.3, 0.4, 0.5 and 0.6 M) of mannitol, Cellulase R-10 (Yakult, Japan), Macerozyme R-10 (Yakult, Japan), Pectolyase Y-23 (Shanghai yuan ye Bio-Technology Co., Ltd, China), Hemicellulase (Shanghai regal Biology Technology Co, Ltd, China) and Snailase (Shanghai regal Biology Technology Co, Ltd, China) as shown in Table 1. All enzyme solutions were adjusted to pH 5.8, filter-sterilized through a 0.22 μm syringe filter (Millex-GP, USA), and then stored at 4 ℃. The digestion was performed at 28 ℃ by gently shaking (40 rpm) in the dark. The key parameters affecting protoplast isolation were tested, including osmotic pressures (0.3, 0.4, 0.5, 0.6 M mannitol), dark pretreatment time of in vitro grown seedlings (0, 12, 24, 30, 36, 40 h), vacuum pretreatment time (0, 10, 20, 30, 60 min), leaf age (unexpanded leaves, the 1st to 2nd true leaves and the 3rd to 4th true leaves), and enzyme digestion time (2, 4, 6, 8, 10, 12, 14, 16 h).

Purification of protoplasts

After enzymatic digestion, the protoplasts were purified at room temperature by a combination of filtration, centrifugation and washing. The crude protoplast suspension was filtered through 200 mesh sterile steel sieve to exclude undigested tissues, cell clumps and cell wall debris. The filtrate was collected in a sterile centrifuge tube, and protoplasts were collected at low speed or natural rest. The mesophyll protoplasts of C. oleifera were purified by interfacial method and centrifugal precipitation. The interface method was similar to purifying protoplasts from C. oleifera suspension cells.

The protoplasts were resuspended in approximately 1:3 volumes with CPW14 salt solution (CPW14 salt solution contains 0.2 mmol/l KH2PO4, 1 mmol/l KNO3, 2.08 mmol/l MgSO4, 0.001205 mmol/l KI, 0.000012 mmol/l CuSO4·5H2O, 1.35 mmol/l CaCl2 and 400 mmol/l sucrose), CPW Ficoll 70 (Ficoll 70, Shanghai yuan ye Bio-Technology Co., Ltd, China) salt solution and CPW Ficoll 400 (Ficoll 400, Shanghai yuan ye Bio-Technology Co., Ltd, China) saline solution, respectively. Then CPW7 salt solution (CPW7 salt solution contains 0.2 mmol/l KH2PO4, 1 mmol/l KNO3, 2.08 mmol/l MgSO4, 0.001205 mmol/l KI, 0.000012 mmol/l CuSO4·5H2O, 1.35 mmol/l CaCl2 and 400 mmol/l mannitol) was gently added on top of it and centrifuged at 15 × g for 3 min to observe the purification effect.

Centrifuge precipitation was to gently add 4 ml W buffer (2 mmol/l MES, 125 mmol/l CaCl2, 5 mmol/l KCl, 154 mmol/l NaCl, 5 mmol/l glucose, pH 5.8) to the collected protoplasts, centrifuged at 15 ×g for 4 min, and then the supernatant was discarded. The pellet was resuspended with 4 ml of W buffer, and then the filtrate was centrifuged for 3 min at 50 ×g. After washing twice with the W buffer, the collected protoplasts were resuspended in 2 ml MMg solution (4 mmol/l MES, 0.4 mol/l mannitol, 15 mmol/l MgCl2, pH 5.8), incubated on ice for 15 min.

Protoplast yield and viability assessment

Purified protoplasts were counted using a blood cell count chamber under Olympus CX21 light microscope (Olympus, Japan). The yield was expressed as the number of protoplasts per gram fresh weight (g· FW). The viability was determined by fluorescein diacetate (FDA, Sigma-Aldrich, St. Louis, USA) staining according to Widholm [47]. The samples were incubated in dark for 5 min and then assessed under DMi8 inverted microscopy (Leica, Germany) with UV excitation light. Only viable protoplasts fluoresced bright green. The viability of the protoplasts was calculated by (viable protoplasts/total number of protoplasts) × 100%. For each sample, 3000 cells were analyzed in each replicate, and the counting was performed at least three times.

Protoplast transformation

The pCAMBIA1300-GFP vector (supplementary information, additional file [1]) was used to test the transformation efficiency of the C. oleifera mesophyll protoplasts. For each assay, different amounts of plasmid DNA (5, 10, 15, and 20 µg) were added to 100 µl prepared protoplast (about 2 × 106/g·FW protoplasts) and mixed gently. An equal volume of freshly prepared PEG solution (PEG, 0.3 M mannitol and 0.2 mol/l CaCl2) was immediately mixed with the protoplasts by shaking gently. PEG with different molecular weights (PEG3350, PEG4000, PEG6000, Sigma) and different final concentrations (20%, 30%, 40% and 50%) were tested. To optimize transfection duration, the mixture was incubated at room temperature for 10, 15, 20, and 25 min in the dark, respectively. After incubation, the transfection process was stopped by adding 200 µl W5 solution at room temperature. The mixture was centrifuged at 50 ×g for 1 min and the protoplasts were gently resuspended with 100 µl WI solution (4 mM MES, 0.4 M mannitol, 20 mmol/l KCl, pH 5.8). The transfected protoplasts were incubated at 25 °C in the dark for 12–16 h. The protoplasts expressing GFP-fusion were observed and images were captured using a confocal laser scanning microscope (Leica TCS SP8, Germany). The GFP fluorescence signals were acquired using 488 nm excitation wavelengths and 507 nm emission wavelengths. The exploration of each condition in GFP transformation experiment was performed at least three independent replicates. Transformation efficiency was calculated as follows: transformation efficiency (%) = (the number of bright green fluorescent protoplast in view/total number of protoplasts in view) × 100%.

Statistical analysis

All data were performed with SPSS Version 18.0 (SPSS Inc. Chicago, IL, USA). One-way analysis of variance (one-way ANOVA) with a post hoc test of least significant difference (LSD) test was used for the statistical analysis. Data were presented as the mean value ± standard error (SE) from three independent experiments. P < 0.05 was considered to indicate a statistically significant difference.

Results

Effect of osmotic pressure on mesophyll protoplasts isolation in C. oleifera

The effects of different osmotic pressures on protoplast isolation of C. oleifera mesophyll were investigated using mannitol as an osmotic pressure regulator. The results showed that the isolation effect of C. oleifera mesophyll protoplasts increased initially and then decreased with the increase of osmotic pressure. Under the condition of low osmotic pressure (0.3 M mannitol), the protoplast yield and viability were low. When the osmotic pressure was 0.4 M and the enzyme concentration was 1.0% Cellulase R-10 and 1.0% Macerozyme R-10 for 14 h, the protoplast yield and viability reached the highest value, which was 2.0 × 105/g·FW and 90.3%, respectively. When the osmotic pressure reached 0.5 M and 0.6 M (Fig. 2A), we observed deformed cells and increased cell debris, as well as decreased yield and declined viability. Therefore, we concluded the optimal osmotic pressure for the protoplast isolation of C. oleifera mesophyll was 0.4 M.

Fig. 2
figure 2

Effects of mannitol concentration, pretreatment method and duration of enzyme application on mesophyll protoplasts isolation in C. oleifera. A Effects of mannitol concentration in enzyme solution (enzyme composition in 1.0% Cellulase R-10 and 1.0% Macerozyme R-10) on protoplast isolation; B effects of vacuum treatment on mesophyll protoplasts isolation in C. oleifera (Vacuum treatment 1 –5 indicates that the vacuum treatment time is 0, 10, 20, 30 and 60 min respectively); C effects of dark treatment on mesophyll protoplasts isolation in C. oleifera (Dark treatment 1–6 indicates that the dark treatment time is 0, 12, 24, 30, 36 and 40 h, respectively); D effects of duration in enzyme application (enzyme composition in 1.5% Cellulase R-10, 0.5% Macerozyme R-10 and 0.25% Snailase) on protoplast isolation. Different letters represent a statistically significant difference at P < 0.05, and bars represent standard errors

Effect of pretreatment method on mesophyll protoplasts isolation in C. oleifera

The pretreatment methods are extremely important for the efficient release of protoplasts from C. oleifera leaves. It was found that the yield and viability of C. oleifera mesophyll protoplasts were affected by vacuum and dark pretreatment.

First, vacuum pressure was applied to enhance the infiltration of the enzyme digestion solution into the leaf blades. The leaves of C. oleifera were pretreated with a vacuum (− 0.07 MPa) for different time lengths. The results showed that the yield and viability of protoplasts were increased after vacuum treatment compared with those without vacuum treatment (Fig. 2B), and the protoplasts were complete in morphology, with more inclusions and fewer impurities. The results showed that vacuum pretreatment effectively promoted the enzymatic hydrolysis of C. oleifera leaves and improved the isolation efficiency of mesophyll protoplasts. Within a certain range of negative pressure, the mesophyll protoplast yield and viability of C. oleifera increased first and then decreased with the extended time of vacuuming. When the vacuuming treatment lasted for 20 min, the protoplast yield of C. oleifera mesophyll cells reached 1.5 × 106/g·FW and the viability was 81.7%. Therefore, − 0.07 MPa vacuum pretreatment for 20 min is most suitable for the isolation of protoplasts from C. oleifera mesophyll.

In addition, protoplast yield and activity increased first and then decreased with the extended dark treatment (Fig. 2C). The protoplast yield reached 3.8 × 107/g·FW and the protoplast viability reached 90.6% when the in vitro grown seedlings were treated in dark for 24 h. After dark treatment for more than 24 h, protoplast yield and viability began to decrease. Therefore, 24 h dark treatment is optimal for the isolation of C. oleifera mesophyll protoplasts.

Effect of leaf age on mesophyll protoplasts isolation in C. oleifera

The effect of leaf age on protoplast yield and viability was investigated. The leaves at different stages of C. oleifera growth (undeveloped leaves, the 1st to 2nd true leaves and the 3rd to 4th true leaves) were used. The results indicated that the age of the leaf tissue greatly affected the protoplast releasing. The protoplasts isolated from in vitro grown seedlings without undeveloped leaves (Fig. 1F) had a very low yield and were relatively easy to be broken (Fig. 3A). When the 1st to 2nd true leaves (Fig. 1G) were used, the yield of isolated protoplasts could reach 8.1 × 106/g·FW (Fig. 3B), and the viability could reach 89.7% with other factors at optimal. However, the yield of protoplasts isolated from the 3rd to 4th true leaves was also low, accompanied by large amounts of debris and other irregular impurities (Fig. 3C). Compared with the 1st to 2nd true leaves, the yield and viability of protoplasts isolated from the 3rd to 4th true leaves were significantly reduced. Therefore, the 1st to 2nd true leaves of in vitro grown seedlings of C. oleifera should be the most appropriate for protoplast isolation.

Fig. 3
figure 3

Efficiency of mesophyll protoplast isolation from leaves of different age collected from in vitro grown seedlings of C. oleifera. A Undeveloped leaves; B the 1st to 2nd true leaves; C the 3rd to 4th true 1eaves. The scale bars = 50 μm

Effect of enzyme types, concentrations and digestion time on mesophyll protoplasts isolation in C. oleifera

The concentration and type of enzyme are critical for protoplast isolation. This study explored the effects of 10 enzyme combinations on the isolation of C. oleifera mesophyll protoplast (Table 1). In this study, the effects of 10 enzyme combinations on the isolation of C. oleifera mesophyll protoplasts (Table 1) were investigated when the osmotic pressure was 0.4 M and the enzyme digestion time was 14 h. We obtained the lowest yield of protoplasts when using 1.0% Cellulase R-10 and 1.0% Macerozyme R-10. In addition to the combination of Cellulase R-10 and Macerozyme R-10, a certain concentration of pectinase was added, then the yield of protoplasts was slightly increased. When the enzyme combination was 1.5% Cellulase R-10, 0.5% Macerozyme R-10 and 0.5% Hemicellulase, the protoplast yield was still low, along with the increase in cell debris. After many attempts, Hemicellulase was found not suitable for C. oleifera mesophyll protoplast isolation. Under the combination of Cellulase R-10, Macerozyme R-10 and Snailase, the yield of protoplasts was greatly increased. Further exploration of the optimal enzyme concentration of Cellulase R-10, Macerozyme R-10 and Snailase showed that treatment 7 had the best effect. The protoplast yield reached 3.5 × 107/g·FW, and viability reached 90.9%. Therefore, the optimal combination of enzyme concentration for the C. oleifera mesophyll protoplast isolation was 1.5% Cellulase R-10, 0.5% Macerozyme R-10 and 0.25% Snailase.

Table 1 Effect of different enzyme concentration combinations on mesophyll protoplast isolation in C. oleifera

To establish the optimal time for enzyme treatment, we digested leaves for 2–16 h. The results indicated enzyme digestion time has a significant influence on both the yield and viability of protoplasts isolated from leaves of C. oleifera. As enzyme digestion time increased from 2 to 10 h, protoplast yield increased gradually, peaked at 10 h and decreased significantly with further extension in enzyme digestion time (Fig. 2D). Based on these results we concluded that the optimal enzyme digestion time was 10 h for isolating C. oleifera mesophyll protoplasts.

Effects of purification method and CPW solution on protoplast purification

On the basis of protoplast purification from the cell suspension, the purification method of mesophyll protoplast of C. oleifera was explored. Firstly, the mesophyll protoplasts were purified by the interface method. CPW14, CPW Ficoll 70 and CPW Ficoll 400 buffer solutions were used to resuspend the mesophyll protoplasts, and then the CPW7 was added gently in a ratio of 3:1 to form stratification between the two liquids. After centrifugation, protoplasts are expected to accumulate at the interface in the form of clear strips (Fig. 4). Although the purification effect of CPW Ficoll 400 was better than that of CPW Ficoll 70 and CPW14, none of the three solutions was ideal due to the presence of a large number of cell debris and other impurities. The mesophyll protoplasts were further purified by centrifugal precipitation using W buffer as a cleaning agent, and relatively pure and highly active mesophyll protoplasts were obtained (Fig. 4H). The results showed that using W buffer as a cleaning agent by centrifugal precipitation was optimal, and the viability of purified protoplasts was as high as 90.9% (Fig. 5A, B).

Fig. 4
figure 4

Purification of C. oleifera mesophyll protoplasts. A, B CPW14 purification; C, D CPW Ficoll 400 purification; E, F CPW Ficoll 70 purification; G, H W buffer purification; A–F: the interface purification method; G–H: precipitation method. The scale bars = 50 μm

Fig. 5
figure 5

Determination of mesophyll protoplast viability in C. oleifera and the effects of different cultivars on mesophyll protoplast isolation of C. oleifera. A FDA dyed protoplast under the bright light; B FDA dyed protoplast under the ultraviolet light. C The effects of different cultivars on mesophyll protoplast isolation of C. oleifera. The scale bars = 50 μm

Effects of different C. oleifera cultivars on mesophyll protoplasts isolation

To verify the applicability of the present protocol, protoplasts were isolated from the 1st to 2nd true leaves of the other two C. oleifera cultivars (‘TXP14’ and ‘DP47’). The protoplast yield for ‘TXP14’ and ‘DP47’ was 1.1 × 107/g·FW and 2.6 × 107/g·FW, the protoplast viability for ‘TXP14’ and ‘DP47’ was 90.0% and 88.2%. Therefore, an effective protocol for isolating and purifying protoplasts from C. oleifera plants was established, and the effect of protoplast isolation in different C. oleifera cultivars was verified.

Transient transformation efficiency in C. oleifera mesophyll protoplasts

The effects of PEG4000 concentration and plasmid amount on transformation efficiency of C. oleifera mesophyll protoplasts were assessed using the pCAMBIA1300-GFP vector. To optimize PEG molecular weights, the effect of PEG molecular weights (PEG3350, PEG4000, PEG6000) on transformation efficiency was examined when the PEG concentration was 30%. The transformation efficiency was approximately 18% at PEG3350 (Fig. 6A), and the transformation efficiency improved with increasing PEG molecular weights. The transformation efficiency reached 58.2% at PEG4000. Then, as the PEG molecular weights continued to increase, the transformation efficiency dropped sharply. Thus, PEG4000 was regarded as the optimal PEG molecular weights for transient expression using C. oleifera mesophyll protoplasts. As shown in Fig. 6B, transformation efficiency first increased, then declined, along with increased PEG4000 concentration (20%, 30%, 40% and 50%, respectively). When PEG4000 was at concentration of 40%, transformation efficiency reached the maximum, approximately 73.07%. Subsequently, transformation efficiency reduced gradually. When PEG4000 concentration was 50% the transformation efficiency decreased to 11.11%, and the ratio of abnormal protoplasts rose and protoplast debris increased. In conclusion, 40% was the optimal concentration of PEG4000.

Fig. 6
figure 6

Efficient transfection of C. oleifera mesophyll protoplasts. Effects of PEG molecular weights (A), PEG4000 concentration (B), incubation time when plasmid amount was 10 µg (C), and plasmid amount (D) on C. oleifera protoplast transformation efficiency. Different letters represent a statistically significant difference at P < 0.05, and bars represent standard errors

The effects of PEG incubation time (10, 15, 20, and 25 min) on the transformation efficiency were analyzed (Fig. 6C). Increasing the transfection time from 10 to 20 min led to an increase in the transformation efficiency from 20.7 to 51.0%. However, the continued prolongation of transfection decreased the efficiency, indicating that the optimal incubation time for protoplast transient transformation was 20 min. To investigate the effect of plasmid amount on the transformation efficiency of C. oleifera mesophyll protoplasts, 5, 10, 15, and 20 µg of pCAMBIA1300-GFP vector were tested in 100 µl resuspended protoplasts in WI. The results showed that when plasmid concentration was 5 µg, the transformation efficiency was 42.37% (Fig. 6D). As plasmid amount increased, transformation efficiency increased as well and reached 70.66% at 15 µg. However, when plasmid amount was further increased to 20 µg, transformation efficiency decreased significantly to 42.7%. This indicated that the optimal plasmid amount for transient transformation was 15 µg.

Based on the obtained data, the optimal protocol of transformation in C. oleifera protoplast was found to be incubated with 40% PEG4000 and 15 µg plasmid for 20 min of transfection time. Using this method, a maximum transformation efficiency of approximately 70.6% (Fig. 7A1–A3) was obtained from C. oleifera mesophyll protoplasts. In addition, it was further found that the protoplasts transformed by the GFP vector under bright field were regular in shape, and the cell membrane was intact. At the excitation light of 488 nm, no auto-fluorescence signal could be observed in the untransformed protoplasts (Fig. 7B1–B3). The GFP-expressing region in the transformed protoplast showed obvious green fluorescence, indicating that the plasmid containing the gfp gene could be introduced into the C. oleifera mesophyll protoplasts and expressed transiently (Fig. 7C1–C3).

Fig. 7
figure 7

Transient expression of GFP in C. oleifera protoplasts. A1–A3 Efficiency transformation of C. oleifera mesophyll protoplasts with GFP plasmid (The scale bars = 100 μm); B1–B3 Unsuccessful transient expression of GFP in C. oleifera mesophyll protoplasts. C1–C3 Successful transient expression of GFP in C. oleifera mesophyll protoplasts. Bright: bright field image of protoplasts; GFP green fluorescent protein; Merged: GFP merged with chloroplast autofluorescence (The scale bars = 20 μm)

Discussion

C. oleifera is a widely distributed plant species in southern China [1], with a planting area of more than 4.5 million hectares [48]. The C. oleifera industry is rapidly expanding and developing, becoming one of the main industries for rural revitalization in China [4]. However, the lack of improved varieties and backward breeding technology limit the development of C. oleifera industry. Somatic hybridization is one of the promising technologies in advancing C. oleifera breeding. The protoplast system is the basis of somatic hybridization, and is also an important scientific research tool, which provides the possibility for molecular assisted breeding and molecular design breeding of C. oleifera. In this study, an optimal system for the isolation and purification of mesophyll protoplasts from C. oleifera in vitro grown seedlings was established.

Mesophyll tissues of leaves are one of the most convenient sources for a large number of uniform cells for protoplast isolation [18, 28]. The yield of protoplasts was influenced by the physiological state and growth cycle of plant leaves. In woody plants, young tissues have consistently proved to be the best sources for protoplast isolation [49]. Protoplast yield drops sharply when isolated from old leaf tissue [50]. Thus, the age of tissue plays a critical role in the yield and viability of protoplasts. Furthermore, the suitable leaf age for mesophyll protoplast isolation was different among different plants. For example, the optimal leaf for wheat protoplast isolation is 7 days old [6], while for Arabidopsis is 3–4-week [18], and for cotton is 12 days [51]. Moreover, it has shown that the enzyme digestion time of unexpanded leaves was not easy to control, and also produced a large amount of cell debris [6], while the viability of protoplasts obtained in older leaves was lower [17]. Therefore, in this study, the 1st to 2nd true leaves of the in vitro grown seedlings of C. oleifera were the most suitable for the mesophyll protoplasts isolation.

Pretreatment of source tissue before enzymatic hydrolysis could change the physiological state of cells and cell walls and reduce the loss of protoplasm [52]. In the process of mesophyll protoplast isolation, pretreatment methods such as vacuuming, pre-plasmolysis, dark and low-temperature pretreatment were often used. Choury et al. found that vacuuming the leaves of Arbutus unedo for 30 min [53], and Rahmani et al. [54] treated Albizia julibrissin leaves or callus in 0.7 M sorbitol for 60 or 90 min could improve the isolation efficiency of the protoplast. Furthermore, Chang et al. [55] found that dark pretreatment was necessary for successful protoplast isolation from potato leaves. Liao et al. [56] found that at 4 °C low-temperature pretreatment could increase the viability of Arabidopsis mesophyll protoplasts. Previous studies have shown that vacuuming pretreatment was the most commonly used for the isolation of plant mesophyll protoplasts, such as sugarcane [57], Phaseolus vulgaris [37], and Brachypodium distachyon [58]. The pretreatment methods for the isolation of C. oleifera and tea (C. sinensis) protoplasts were not identical in Camellia genus. Xu et al. [59] reported that the efficiency of protoplast isolation in tea was improved by vacuuming treatment. Peng et al. [24] successfully obtained mesophyll protoplasts from tea seedlings grown in the dark. Previously, we have found that no pretreatment was required when isolating protoplasts from C. oleifera suspension cells [43]. In this study, the optimal pretreatment of C. oleifera leaves was dark treatment for 24 h and negative 0.07 MPa vacuum treatment for 20 min. It could be further speculated that different starting materials of the same genus and the same species of plants may need different pretreatments for protoplast isolation.

The concentration of osmotic stabilizers required for successful protoplasts isolation varied among the plant species and growing conditions [60]. For example, in barley, 0.3 M mannitol was found to be optimal for the high yield and viability [41]. Previous studies have shown that the optimum mannitol concentrations for Catalpa bungei, sorghum and Chinese kale for protoplast isolation are 0.4 M [61], 0.5 M [62] and 0.6 M [63], respectively. Furthermore, it was found that the optimal osmotic pressures for the isolation of Phalaenopsis aphrodite and bamboo mesophyll protoplasts were 0.7 M [64] and 0.8 M [65], respectively. In addition, studies have found that the osmotic pressure of different tissues of the same species could be the same or different. For example, the optimal osmotic pressure for grape mesophyll protoplast isolation is 0.6 M, while the optimal osmotic pressure for callus tissue is 0.5 M [66]. Peng et al. [24] found that the optimal osmotic pressure for protoplast isolation between young leaves and young radicles of tea plants is 0.4 M. In the study, it was found that 0.4 M mannitol was most suitable for mesophyll protoplast isolation of C. oleifera, which was consistent with the previous studies of C. oleifera suspension [43] and C. sinensis plant [24, 59]. In conclusion, 0.4 M may be a suitable osmotic pressure for Camellia plants (Additional file 1).

It has reported that appropriate enzyme digestion time and enzyme combination are crucial for protoplast isolation [67]. The composition of the enzyme solution and the enzymatic hydrolysis time required for protoplast isolation from different plants were generally different. Zhou et al. [25] found that the most mesophyll protoplasts were obtained from tea digested with 3% cellulase R-10 and 0.3% macerozyme R-10 for 12 h. While the optimal conditions for mesophyll protoplast isolation of Platycladus orientalis were 1.5% cellulase R-10, 0.4% macerozyme R-10, 0.4% pectolyase Y-23 and 1.0% ligninase for 16 h [68]. However, compared with woody plants, the enzyme concentration and enzyme time required for protoplast isolation of herbaceous plants were lower. Li et al. [69] found the highest yield and viability Phalaenopsis protoplasts were achieved with 1.0% Cellulase Onozuka R-10, 0.7% Macerozyme R-10 for 6 h. Adedeji et al. [70] found that a high Chrysanthemum protoplast yield was achieved using 1.5% cellulase, and a 4 h incubation period. It further suggested that the isolation of woody plant protoplasts required higher enzyme concentration, longer enzyme time and even some special enzymes. It was well known that protoplast isolation technology was underdeveloped in woody plants compared with herbaceous plants [25]. These differences might be due to the differences in cell wall composition and biological activity of cells, resulting from differences in the physiological characteristics of plants and growth environments [30].

In the process of protoplast isolation, no matter how efficient the enzymatic hydrolysis system was, a lot of impurities such as cell debris would always be produced. These impurities would have a negative impact on protoplast culture and transformation. Therefore, protoplasts must be purified to remove impurities. There are three commonly used methods for protoplast purification, namely centrifugal precipitation method, floating method and interface method. Different plant protoplast purification methods were different, the protoplasts of cucumber [71] and Catalpa bungee [61] were purified by centrifugal precipitation. Pisum and Lathyrus protoplasts were purified by floating method [72]. Mango protoplasts [73] and sweet cherry protoplasts [67] were purified by the interface method. In our study, it was found that the purification methods of mesophyll protoplasts and suspension cells protoplasts of C. oleifera were different, which might be due to the differences in contents, cell density and the other states of protoplasts isolated from the two explants. Generally, protoplast purification operation would reduce protoplast yield and viability, which was very important for subsequent protoplast culture, regeneration and genetic transformation. The mesophyll protoplast activity of C. oleifera isolated and purified by the method of this study reached 90.9%, which lays a good foundation for subsequent research such as somatic hybridization and gene editing.

PEG-mediated transient transformation of plant protoplasts is widely used in plants, but the transfection efficiency varies greatly among different plant species [17, 40]. Firstly, the effect of PEG molecular weights on transformation efficiency was explored. The result showed that the transformation efficiency is higher when PEG4000 was used. As PEG4000 concentrations increased, the transformation efficiency rose significantly, but impurities such as cell debris increased as well, which may inhibit the transformation efficiency [71]. For example, the optimum PEG4000 concentration for Populus and cassava have been reported to be 30% and 25%, respectively [26, 34]. We found that 40% PEG4000 is optimal for the transformation of protoplasts derived from C. oleifera. In addition, the optimum amount of plasmid for protoplast transient transformation is different in different species [42, 74]. Different amounts of plasmids, such as 20 µg for Brachypodium distachyon, and 10 µg for soybean, have been reported to be the optimal amounts of plasmid DNA in their established protocols respectively [58, 75]. Our assay demonstrated an increased transformation efficiency could be obtained with an increase in plasmid amount in C. oleifera, but it reached a plateau at 15 µg. Thus, 15 µg was considered to be the optimal amount of plasmid for the present C. oleifera protoplast transformation. The optimal incubation time for different species is different, such as 5 min for grapevine [42], 10 min for cassava [34], 15 min for Chinese kale [63], 20 min for barley [41], and 30 min for cucumber [71] protoplasts. The effect of incubation time on transformation efficiency was also explored in this study. The highest transformation efficiency was obtained when the C. oleifera protoplasts were incubated for 20 min.

Conclusion

In summary, a highly efficient protocol for C. oleifera mesophyll protoplast isolation and PEG-mediated transient expression was developed. To our knowledge, this is the first report describing the isolation of mesophyll protoplasts from the C. oleifera and of the PEG-mediated protoplast transfection. The developed method could be a convenient technique for protein subcellular localization, promoter function validation, and many other molecular biology studies in C. oleifera.

Availability of data and materials

The datasets supporting the conclusions of this article are included in the article.

References

  1. Luan F, Zeng J, Yang Y, He X, Wang B, Gao Y, et al. Recent advances in Camellia oleifera Abel: a review of nutritional constituents, biofunctional properties, and potential industrial applications. J Funct Foods. 2020;75:104242.

    Article  CAS  Google Scholar 

  2. Zhang SY, Pan YG, Zheng LL, Yang Y, Zheng XY, Ai BL, et al. Application of steam explosion in oil extraction of camellia seed (Camellia oleifera Abel.) And evaluation of its physicochemical properties, fatty acid, and antioxidant activities. Food Sci Nutr. 2018;7:1004–16.

    Article  Google Scholar 

  3. Ye ZC, Yu J, Yan WP, Zhang JF, Yang DM, Yao GL, et al. Integrative iTRAQ-based proteomic and transcriptomic analysis reveals the accumulation patterns of key metabolites associated with oil quality during seed ripening of Camellia oleifera. Hortic Res. 2021;8(1):157.

    Article  CAS  Google Scholar 

  4. He JH, Wu XH, Yu ZL. Microwave pretreatment of camellia (Camellia oleifera Abel.) Seeds: Effect on oil flavor. Food Chem. 2021;364:130388.

    Article  CAS  Google Scholar 

  5. Kang L, Li PF, Wang AF, Ge XH, Li ZY. A novel cytoplasmic male sterility in Brassica napus (inap CMS) with Carpelloid Stamens via Protoplast Fusion with Chinese Woad. Front Plant Sci. 2017;8:529.

    Article  Google Scholar 

  6. Jia XY, Zhang XH, Qu JM, Han R. Optimization conditions of wheat mesophyll protoplast isolation. Agric Sci. 2016;7:850–8.

    CAS  Google Scholar 

  7. Karamian R, Sharifzadeh A, Ranjbar M. Evidence of somatic embryogenesis and plantlet regeneration from protoplast culture of Muscari neglectum Guss. Afr J Agric Res. 2011;6(14):3247–51.

    Google Scholar 

  8. Aoyagi H. Application of Plant Protoplasts for the production of useful metabolites. Biochem Eng J. 2011;56:1–8.

    Article  CAS  Google Scholar 

  9. Kiełkowska A, Adamus A. An alginate-layer technique for culture of Brassica oleracea L. Protoplasts. 2012;48:265–73.

    Google Scholar 

  10. Gieniec M, Siwek J, Oleszkiewicz T, Maćkowska K, Klimek–Chodacka M, Grzebelus E, et al. Real-time detection of somatic hybrid cells during electrofusion of carrot protoplasts with stably labelled mitochondria. Sci Rep. 2020;10:18811.

    Article  CAS  Google Scholar 

  11. Lin CS, Hsu CT, Yuan YH, Zheng PX, Wu FH, Cheng QW, et al. DNA-free CRISPR-Cas9 gene editing of wild tetraploid tomato Solanum peruvianum using protoplast regeneration. Plant Physiol. 2022;188(4):1917–30.

    Article  CAS  Google Scholar 

  12. Grosser JW, Gmitter FG. Protoplast fusion for production of tetraploids and triploids: applications for scion and rootstock breeding in citrus. Plant Cell Tissue Organ Cult. 2011;104(3):343–57.

    Article  CAS  Google Scholar 

  13. Xiao SX, Biswas MK, Li MY, Deng XX, Xu Q, Guo WW. Production and molecular characterization of diploid and tetraploid somatic cybrid plants between male sterile Satsuma mandarin and seedy sweet orange cultivars. Plant Cell Tissue Organ Cult. 2014;116(1):81–8.

    Article  CAS  Google Scholar 

  14. Fu LL, Yang XY, Zhang XL, Wang ZW, Feng CH, Liu CX, et al. Regeneration and identification of interspecific asymmetric somatic hybrids obtained by donor-recipient fusion in cotton. Chin Sci Bull. 2009;54(17):3035–44.

    Article  CAS  Google Scholar 

  15. Zhao ZG, Hu TT, Ge XH, Du XZ, Ding L, Li ZY. Production and characterization of intergeneric somatic hybrids between Brassica napus and Orychophragmus violaceus and their backcrossing progenies. Plant Cell Rep. 2008;27(10):1611–21.

    Article  CAS  Google Scholar 

  16. Chen J, Yi Q, Song Q, Gu Y, Zhang J, Hu Y. A highly efficient maize nucellus protoplast system for transient gene expression and studying programmed cell death-related processes. Plant Cell Rep. 2015;34(7):1239–51.

    Article  CAS  Google Scholar 

  17. Zhang Y, Su JB, Duan S, Ao Y, Dai JR, Liu J, et al. A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes. Plant Methods. 2011;7(1):30.

    Article  CAS  Google Scholar 

  18. Yoo SD, Cho YH, Sheen J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc. 2007;2(7):1565–72.

    Article  CAS  Google Scholar 

  19. Yu GH, Cheng Q, Xie ZN, Xu B, Huang BR, Zhao BY. An efficient protocol for perennial ryegrass mesophyll protoplast isolation and transformation, and its application on interaction study between LpNOL and LpNYC1. Plant Methods. 2017;13:46.

    Article  Google Scholar 

  20. Guo WW, Deng XX, Yi HL. Somatic hybrids between navel orange (Citrus sinensis) and grapefruit (C. paradisi) for seedless triploid breeding. Euphytica. 2000;116(3):281–5.

    Article  CAS  Google Scholar 

  21. Cai X, Fu J, Guo W. Mitochondrial genome of callus protoplast has a role in mesophyll protoplast regeneration in Citrus: evidence from transgenic GFP somatic Homo-Fusion. Hortic. Plant J. 2017;3(05):177–82.

    Google Scholar 

  22. Ortín-Párraga F, Burgos L. Isolation and culture of mesophyll protoplast from apricot. J Hortic Sci Biotechnol. 2003;78(5):624–8.

    Article  Google Scholar 

  23. Honda C, Moriguchi T. High GUS expression in protoplasts isolated from immature peach fruits. Sci Hortic. 2006;109(3):0–247.

    Article  CAS  Google Scholar 

  24. Peng Z, Tong HR, Liang GL, Shi YQ, Yuan LY. Protoplast isolation and fusion induced by PEG with leaves and roots of tea plant (Camellia sinensis L. O. Kuntze). Acta Agron Sin. 2018;44(03):463–70.

    Article  Google Scholar 

  25. Zhou Y, Deng RF, Xu XL, Yang ZY. Isolation of mesophyll protoplasts from tea (Camellia sinensis) and localization analysis of enzymes involved in the biosynthesis of specialized metabolites. Beverage Plant Res. 2021;1(1):1–9.

    Article  Google Scholar 

  26. Tan BY, Xu M, Chen Y, Huang MR. Transient expression for functional gene analysis using Populus protoplasts. Plant Cell Tissue Organ Cult. 2013;114(01):11–8.

    Article  CAS  Google Scholar 

  27. Kanwar K, Bhardwaj A, Deepika R. Efficient regeneration of plantlets from callus and mesophyll derived protoplasts of Robinia pseudoacacia L. Plant Cell Tissue Organ Cul. 2009;96(1):95–103.

    Article  Google Scholar 

  28. Mukhtar I, Bajwa R, Nasim G. Isolation of mesophyll protoplasts from leaves of Dalbergia sissoo Roxb. J Appl Sci Environ Manage. 2012;6(1):11–5.

    Google Scholar 

  29. Shen YM, Meng D, McGrouther K, Zhang JH, Cheng LL. Efficient isolation of Magnolia protoplasts and the application to subcellular localization of MdeHSF1. Plant Methods. 2017;13:44.

    Article  Google Scholar 

  30. Lai Q, Wang YL, Zhou QY, Zhao Z. Isolation and purification of mesophyll protoplasts from Ginkgo biloba L. Cytologia. 2020;85(1):27–32.

    Article  CAS  Google Scholar 

  31. Jones A, Chattopadhyay A, Shukla M, Zoń J, Saxena PK. Inhibition of phenylpropanoid biosynthesis increases cell wall digestibility, protoplast isolation, and facilitates sustained cell division in american elm (Ulmus americana). BMC Plant Biol. 2012;12(1):75–5.

    Article  CAS  Google Scholar 

  32. Ren R, Gao J, Yin DM, Li K, Lu CQ, Ahmad S, et al. Highly efficient leaf base protoplast isolation and transient expression systems for orchids and other important monocot crops. Front Plant Sci. 2021;12:626015.

    Article  Google Scholar 

  33. Ren R, Gao J, Lu C, Wei Y, Jin J, Wong SM, et al. Highly efficient Protoplast isolation and transient expression system for functional characterization of flowering related genes in Cymbidium Orchids. Int J Mol Sci. 2020;21(7):2264.

    Article  CAS  Google Scholar 

  34. Wu JZ, Liu Q, Geng XS, Li KM, Luo LJ, Liu JP. Highly efficient mesophyll protoplast isolation and PEG-mediated transient gene expression for rapid and large-scale gene characterization in cassava (Manihot esculenta Crantz). BMC Biotechnol. 2017;17(1):29.

    Article  Google Scholar 

  35. Mittelberger C, Stellmach H, Hause B, Kerschbamer C, Schlink K, Letschka T, et al. A novel effector protein of apple proliferation phytoplasma disrupts cell integrity of Nicotiana spp. protoplasts. Int J Mol Sci. 2019;20(18):4613.

    Article  CAS  Google Scholar 

  36. Wang HL, Wang W, Zhan JC, Huang WD, Xu HY. An efficient PEG-mediated transient gene expression system in grape protoplasts and its application in subcellular localization studies of flavonoids biosynthesis enzymes. Sci Hortic. 2015;191:82–9.

    Article  CAS  Google Scholar 

  37. Nanjareddy K, Arthikala MK, Blanco L, Arellano ES, Lara M. Protoplast isolation, transient transformation of leaf mesophyll protoplasts and improved Aobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid gene exgrpression analysis. BMC Biotechnol. 2016;16(1):53.

    Article  Google Scholar 

  38. Gou YJ, Li YL, Bi PP, Wang DJ, Ma YY, Hu Y, et al. Optimization of the protoplast transient expression system for gene functional studies in strawberry (Fragaria vesca). Plant Cell Tissue and Organ Cult. 2020;141(1):41–53.

    Article  CAS  Google Scholar 

  39. Martinho C, Confraria A, Elias CA, Crozet P, Rubio-Somoza I, Weigel D, et al. Dissection of miRNA pathways using Arabidopsis mesophyll protoplasts. Mol Plant. 2015;8(2):261–75.

    Article  CAS  Google Scholar 

  40. Yang JW, Fu JX, Li J, Cheng XL, Li F, Dong JF, et al. A novel co-immunoprecipitation protocol based on protoplast transient gene expression for studying protein-protein interactions in rice. Plant Mol Biol Rep. 2014;32(1):153–61.

    Article  CAS  Google Scholar 

  41. Bai Y, Han N, Wu J, Yang Y, Wang J, Zhu M. A transient gene expression system using barley protoplasts to evaluate microRNAs for post-transcriptional regulation of their target genes. Plant Cell Tissue Organ Cult. 2014;119(1):211–9.

    Article  CAS  Google Scholar 

  42. Zhao FL, Li YJ, Hu Y, Gao YR, Zang XW, Ding Q, et al. A highly efficient grapevine mesophyll protoplast system for transient gene expression and the study of disease resistance proteins. Plant Cell Tissue Organ Cult. 2016;125(1):43–57.

    Article  CAS  Google Scholar 

  43. Li SF, Ye TW, Xu X, Yuan DY, Xiao SX. Callus induction, suspension culture and protoplast isolation in Camellia oleifera. Sci Hortic. 2021;286(1):110193.

    Article  CAS  Google Scholar 

  44. Zhang M, Wang AB, Qin M, Qin XJ, Yang SW, Su SC, et al. Direct and indirect somatic embryogenesis induction in Camellia oleifera Abel. Front Plant Sci. 2021;12:644389.

    Article  Google Scholar 

  45. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 1962;15:473–97.

    Article  CAS  Google Scholar 

  46. Salih AM, Al-Qurainy F, Khan S, Tarroum M, Nadeem M, Shaikhaldein HO, et al. Mass propagation of Juniperus procera Hoechst. Ex Endl. From seedling and screening of bioactive compounds in shoot and callus extract. BMC Plant Biol. 2021;21(1):192.

    Article  CAS  Google Scholar 

  47. Widholm JM. The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technol. 1972;47(4):189–94.

    Article  CAS  Google Scholar 

  48. Hu Y, Gao C, Deng QE, Qiu J, Wei HL, Yang L, et al. Anatomical characteristics of petalized anther abortion in male sterile Camellia oleifera plants. J Am Soc Hortic Sci. 2021;146(6):411–23.

    Article  CAS  Google Scholar 

  49. Wakita Y, Sasamoto H, Yokota S, Yoshizawa N. Plantlet regeneration from the mesophyll protoplast of Betula platyphylla var. japonica. Plant Cell Rep. 1996;16(1–2):50–3.

    Article  CAS  Google Scholar 

  50. Raikar SV, Braun RH, Bryant C, Conner AJ, Christey MC. Efficient isolation, culture and regeneration of Lotus corniculatus protoplasts. Plant Biotechnol Rep. 2008;2(3):171–7.

    Article  Google Scholar 

  51. Li NN, Ding LY, Zhang ZY, Guo WZ. Isolation of Mesophyll Protoplast and Establishment of Gene transient expression system in cotton. Acta Agron Sin. 2014;40(2):231–9.

    Article  CAS  Google Scholar 

  52. Ahuja PS, Hadiuzzaman S, Davey MR, Cocking EC. Prolific plant regeneration from protoplast-derived tissues of Lotus corniculatus L. (birdsfoot trefoil). Plant Cell Rep. 1983;2(2):101–4.

    Article  CAS  Google Scholar 

  53. Choury Z, Meschini R, Dell’Orso A, Fardusi MJ, Mugnozza GS, Kuzminsky E. Optimized conditions for the isolation of mesophyll protoplasts along the growing season from Arbutus unedo and their use in single cell gel electrophoresis. Plant Cell Tissue Organ Cult. 2017;132:535–43.

    Article  Google Scholar 

  54. Rahmani MS, Pijut PM, Shabanian N. Protoplast isolation and genetically true-to-type plant regeneration from leaf- and callus-derived protoplasts of Albizia julibrissin. Plant Cell Tissue Organ Cult. 2016;127(2):1–14.

    Article  Google Scholar 

  55. Chang MM, Loescher WH. Effects of preconditioning and isolation conditions on potato (Solanum tuberosum L. cv. Russet Burbank) protoplast yield for shoot regeneration and electroporation. Plant Sci. 1991;73(1):103–9.

    Article  Google Scholar 

  56. Liao JM, Wang BC, Wang YC, Tan JQ. Optimization conditions of Arabidopsis mesophyll protoplast isolation. Acta Bot Boreali-Occident Sin. 2010;30(6):1271–6.

    CAS  Google Scholar 

  57. Wang QL, Yu GY, Chen ZY, Han JL, Hu YF, Wang K. Optimization of protoplast isolation, transformation and its application in sugarcane (Saccharum spontaneum L.). Crop J. 2021;9(1):133–42.

    Article  Google Scholar 

  58. Hong SY, Seo PJ, Cho SH, Park CM. Preparation of leaf mesophyll protoplasts for transient gene expression in brachypodium distachyon. J Plant Biol. 2012;55(5):390–7.

    Article  CAS  Google Scholar 

  59. Xu XF, Zhu HY, Ren YF, Feng C, Ye ZH, Cai HM, et al. Efficient isolation and purification of tissue-specific protoplasts from tea plants (Camellia sinensis (L.) O. Kuntze). Plant Methods. 2021;17(1):84.

    Article  CAS  Google Scholar 

  60. Ling A, Phua G, Tee C, Hussein S. Optimization of protoplast isolation protocols from callus of Eurycoma longifolia. J Med Plants Res. 2010;4(17):1778–85.

    Google Scholar 

  61. Ma WJ, Yi F, Xiao Y, Yang GJ, Chen FJ, Wang JH. Isolation of leaf mesophyll protoplasts optimized by orthogonal design for transient gene expression in Catalpa bungei. Sci Hortic. 2020;274:109684.

    Article  CAS  Google Scholar 

  62. Meng R, Wang C, Wang L, Liu Y, Zhan Q, Zheng J, et al. An efficient sorghum protoplast assay for transient gene expression and gene editing by CRISPR/Cas9. PeerJ. 2020;8(1):e10077.

    Article  Google Scholar 

  63. Sun B, Zhang F, Xiao N, Jiang M, Yuan Q, Xue SL, et al. An efficient mesophyll protoplast isolation, purification and PEG-mediated transient gene expression for subcellular localization in chinese kale. Sci Hortic. 2018;241:187–93.

    Article  CAS  Google Scholar 

  64. Lin HY, Chen JC, Fang SC. A protoplast transient expression system to enable molecular, cellular, and functional studies in Phalaenopsis orchids. Front Plant Sci. 2018;9:843.

    Article  Google Scholar 

  65. Hisamoto Y, Kobayashi M. Protoplast isolation from bamboo leaves. Plant Biotechnol. 2010;27(4):353–8.

    Article  Google Scholar 

  66. Shu XJ, Wen TJ, Xing JY, Lu L, Hu JF. Isolation of protoplast and establishment of transient expression system in grapevine (Vitis vinifera L.). Acta Bot Boreali-Occident Sin. 2015;35(6):1262–8.

    CAS  Google Scholar 

  67. Yao LP, Liao X, Gan ZZ, Peng X, Wang P, Li SJ, et al. Protoplast isolation and development of a transient expression system for sweet cherry (Prunus avium L.). Sci Hortic. 2016;209:14–21.

    Article  CAS  Google Scholar 

  68. Zhou QY, Jiang ZH, Li YM, Zhang T, Zhao Z. Mesophyll protoplast isolation technique and flow cytometry analysis of ancient Platycladus orientalis (Cupressaceae). Turk J Agric For. 2019;43(3):275–87.

    Article  CAS  Google Scholar 

  69. Li J, Liao X, Zhou S, Liu S, Jiang L, Wang G. Efficient protoplast isolation and transient gene expression system for Phalaenopsis hybrid cultivar ‘Ruili Beauty’. In Vitro Cell Dev Biol: Plant. 2018;54(1):87–93.

    Article  CAS  Google Scholar 

  70. Adedeji OS, Naing AH, Kim KC. Protoplast isolation and shoot regeneration from protoplast-derived calli of Chrysanthemum cv. White ND. Plant Cell Tissue and Organ Cult. 2020;141(3):571–81.

    Article  Google Scholar 

  71. Huang HY, Wang ZY, Cheng JT, Zhao WC, Li X, Wang HY, et al. An efficient cucumber (Cucumis sativus L.) protoplast isolation and transient expression system. Sci Hortic. 2013;150:206–12.

    Article  CAS  Google Scholar 

  72. Durieu P, Ochatt SJ. Efficient intergeneric fusion of pea (Pisum sativum L.) and grass pea (Lathyrus sativus L.) protoplasts. J Exp Bot. 2000;51:1237–42.

    CAS  Google Scholar 

  73. Rezazadeh R, Williams RR, Harrison DK. Factors affecting mango (Mangifera indica L.) protoplast isolation and culture. Sci Hortic. 2011;130(1):214–21.

    Article  Google Scholar 

  74. Cao J, Yao D, Lin F, Jiang M. PEG-mediated transient gene expression and silencing system in maize mesophyll protoplasts: a valuable tool for signal transduction study in maize. Acta Physiol Plant. 2014;36(5):1281.

    Article  Google Scholar 

  75. Xiong L, Li C, Li H, Lyu X, Zhao T, Liu J, et al. A transient expression system in soybean mesophyll protoplasts reveals the formation of cytoplasmic GmCRY1 photobody-like structures. Sci China Life Sci. 2019;62(8):1070–7.

    Article  CAS  Google Scholar 

Download references

Funding

This work was supported by the Special Funds for Construction of Innovative Provinces in Hunan Province (2021NK1007), the National Natural Science Foundation of China (31500553), the National Key R&D Program of China (2018YFD1000603).

Author information

Authors and Affiliations

Authors

Contributions

SL performed experiments, analyzed the data and wrote the manuscript. RZ performed experiments, analyzed the data and wrote the manuscript. TY performed experiments and analyzed the data. RG revised and checked the manuscript. LX performed experiment. XM and JZ involved in plasmid construction. SX and DY carried out conception of the research and guided the entire study, revised the manuscript and provided valuable comments and suggestions. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Shixin Xiao or Deyi Yuan.

Ethics declarations

Ethics approval and consent to participate

All authors read and approved the manuscript.

Consent for publication

All authors agreed to publish this manuscript.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1. 

The schematic representation of the T-DNA region of pCAMBIA1300-GFP vector.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, S., Zhao, R., Ye, T. et al. Isolation, purification and PEG-mediated transient expression of mesophyll protoplasts in Camellia oleifera. Plant Methods 18, 141 (2022). https://doi.org/10.1186/s13007-022-00972-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13007-022-00972-1

Keywords