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Article

Establishment of PEG-Mediated Transient Gene Expression in Protoplasts Isolated from the Callus of Cunninghamia lanceolata

by
Kaiyue Hong
1,†,
Zhenyu Chen
1,†,
Yasmina Radani
1,
Renhua Zheng
2,
Xueyan Zheng
3,
Yong Li
3,
Jinhui Chen
1,* and
Liming Yang
1,*
1
State Key Laboratory of Tree Genetics and Breeding, Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
2
The Key Laboratory of Timber Forest Breeding and Cultivation for Mountainous Areas in Southern China, Fujian Academy of Forestry, Fuzhou 350012, China
3
National Germplasm Bank of Chinese Fir, Fujian Yangkou Forest Farm, Shunchang 353211, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Forests 2023, 14(6), 1168; https://doi.org/10.3390/f14061168
Submission received: 22 April 2023 / Revised: 24 May 2023 / Accepted: 29 May 2023 / Published: 6 June 2023
(This article belongs to the Special Issue Advances in Plant Science Research: Celebrating Nanjing Forestry University’s 120th Anniversary)
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Abstract

:
Cunninghamia lanceolata (C. lanceolata) is an important timber tree species in southern China that requires gene function studies to understand its traits. In this study, we investigated the callus induction rates of immature zygotic embryos from reciprocal hybrids between genotypes B46 and B49. With zygotic embryo development, the callus induction rates showed an increasing trend, followed by a decreasing trend. Moreover, the rate of callus induction in genotype B46 × B49 immature zygotic embryos was greater than in genotype B49 × B46. Callus from C. lanceolata with genotype B46 × B49 was selected as the donor material for protoplast isolation. By using an enzymatic digestion solution containing cellulase, macerozyme, and pectinase, combined with an osmotic stabilizer, we obtained 9.76 × 106 protoplasts/mL with 92.7% viability. We subsequently transformed plasmids into C. lanceolata callus protoplasts and observed the location of the H2B-eYFP fusion protein in the nucleus. To achieve transient transfection of C. lanceolata callus protoplasts, we compared transfection efficiencies at different concentrations of PEG4000, PEG6000, or PEG8000 in a modified MMg solution. We found that 20% (w/v) PEG6000 mediated the transient transfection of C. lanceolata callus protoplasts with a 6.70% efficiency. This study provides a technical foundation for future research on transient transfection and functional analysis of C. lanceolata genes.

1. Introduction

Cunninghamia lanceolata is a valuable species found in the Yangtze River Basin and the south of the Qinling Mountains in China [1]. It is known for its rapid growth, wind resistance, soot tolerance, and abundant material, making it both economically and ecologically important [2]. However, C. lanceolata, like many other perennial woody gymnosperms, has a long sexual reproduction cycle, making it difficult to conserve its superior traits. Additionally, available regeneration systems are time-consuming, and obtaining transgenic plants is challenging. As a result, researchers often express C. lanceolata genes in the model plant Arabidopsis thaliana to explore their function; however, due to major genetic variations between the two species, this approach may not yield satisfactory results. They are members of different families and have diverged evolutionaryly over time. As a result, their genetic makeup and regulatory mechanisms may change dramatically, influencing the function of the expressed genes in the heterologous host. For example, in Arabidopsis thaliana, the regulatory elements that control gene expression in C. lanceolata may not function properly, resulting in incorrect or incomplete expression of the gene of interest. Alternatively, the protein products of the expressed genes may be incompatible with the cellular machinery of Arabidopsis thaliana, leading to protein misfolding or degradation. Overall, genetic variations between C. lanceolata and Arabidopsis thaliana can lead to significant differences in the function and behavior of expressed genes, limiting the utility of heterologous expression as a tool for studying gene function. To address this issue, transient transfection of protoplasts (TTPs) has been widely used for gene function identification, protein interaction analysis, subcellular localization, gene editing, plant regeneration, and vaccine preparation [3,4,5,6,7,8]. As a result, we investigated and established TTP technology in C. lanceolata to facilitate gene-function studies. Due to its speed and efficiency, TTP technology holds great potential for improving our understanding of the functional genomics of C. lanceolata.
Different transient transfection methods have been employed in gymnosperms, such as agrobacterium-mediated transient transfection systems in Pinus armandii, Pinus elliottii, Pinus taeda, Picea mariana, and Pinus sylvestris [9,10,11,12,13]. Gene gun-mediated transient transfection systems have been applied to P. armandii, Pinus radiate, and Picea glauca, whereas electroporation-mediated transient transfection systems have been established for P. taeda [14,15,16,17]. However, protoplasts are ideal recipients for genetic transfection since they lack a cell wall, making it easier for them to absorb exogenous genetic material. Polyethylene glycol (PEG)-mediated TTP has the advantage of being easy to operate as it achieves chemical binding by a simple mixing reaction at room temperature. This process does not require any special equipment and may be conducted with standard laboratory equipment. Therefore, PEG-mediated TTP technology is simple to use, does not require expensive or complex instrumentation, and can be conducted in a variety of laboratory and industrial settings [18,19]. Established systems for mediating TTP in mesophyll cells of Liriodendron chinense, Hevea brasiliensis, Populus L., and Elaeis guineensis make exploring the potential of PEG-mediated TTP for C. lanceolata extremely appealing [20,21,22,23].
Protocols for preparing protoplasts from secondary xylem, needle leaves [24], and suspension cells of C. lanceolate have been reported [25]. Callus cells are usually derived by taking a small piece of tissue from a plant body and culturing it in an appropriate medium. These tissues are typically cultured in a laboratory where environmental factors such as temperature, humidity, and light can be controlled. In contrast, other types of plant cells, such as leaf or root cells, must be collected from the natural environment and are thus subject to seasonal changes such as temperature and daylight hours. As a result, callus cells can be cultured at any time, regardless of the season. In particular, callus tissue inducted from immature zygotic embryos (IZEs) exhibits lower contamination rates than callus obtained from stem segments or needle leaves [26]. Previous studies have demonstrated that in gymnosperms, maternal genotypes have a stronger influence on callus induction than male parent genotypes, as seen in P. taeda [27]. Therefore, it is important to investigate the ability of C. lanceolata reciprocal hybrid IZEs to produce callus tissue. Exploring the use of IZE-induced callus for protoplast preparation could be an interesting avenue to pursue. However, there have been limited studies on the use of IZE-induced callus from C. lanceolata for protoplast preparation.
In this study, our primary objective was to compare the induction rate of embryogenic callus derived from IZE in reciprocal hybrids of C. lanceolata. Specifically, we focused on comparing the induction rates between genotypes B49 × B46 and B46 × B49 at various developmental phases. We also aimed to investigate the impact of varying mannitol concentrations on the degree of plasmolysis in C. lanceolata callus cells. Additionally, we analyzed the yield and viability of callus protoplasts (CPs) prepared with various enzymatic solutions containing mannitol as an osmotic stabilizer. Finally, we investigated the efficacy of PEG-mediated callus TTP at different PEG concentrations and molecular weights. By examining these various factors, our study provides a valuable technical platform for TTPs isolated from gymnosperm species, as well as functional analysis of C. lanceolata genes, and presents new ideas for their preparation.

2. Materials and Methods

2.1. Plant Materials and Plasmids

Cones from the superior genotypes B49×B46 and B46×B49 of the C. lanceolata species were carefully collected at different developmental phases in 2020. Based on previous research [28], the 24 June, 2 July, 12 July, 15 July, and 18 July 2020 sampling dates were classified as developmental phases 1, 2, 3, 4, and 5, respectively. The developmental phases of the collected seeds were categorized as cleavage polyembryony, dominant embryo, columnar embryo, or early cotyledonary. To ensure the materials’ sterility, immature seeds were gently removed from the cones and placed in conical flasks containing detergent. The flasks were carefully sealed with gauze and rinsed for 30 min with tap water.
Following that, the seeds were prepared for sterilization. The conical flask was drained of tap water, and then alcohol was added. The flasks were shaken for 30 s before removing the alcohol using a 150-mesh sieve (Sangon Biotech, Shanghai, China). After adding sodium hypochlorite, the flask was shaken for 8 min before removing sodium hypochlorite using a 150-mesh sieve (Sangon Biotech, Shanghai, China). Sterile water was then added, and the seeds were rinsed three times; the 150-mesh sieve was used to remove sterile water. Finally, the sterilized material was stored at 4 °C in a sterile conical flask until use.
Tiangen High Pure Maxi Plasmid Kit (DP116) (Tiangen, Beijing, China) was used to extract the plasmids, which were then diluted to 1.0 μg/μL in 20 μL aliquots for subsequent experiments. Figure 1 depicts the expression framework of the fusion protein reporter gene, comprising H2B and eYFP fluorescent proteins on the plasmid, as visualized using the ApE software (v3.1.3, 11 November 2022). The basic backbone of the plasmid is pjit166 [20], named pjit166-H2B-eYFP, which encompasses an expression framework that enables the synthesis of a fusion protein reporter gene comprising H2B and eYFP fluorescent proteins. This framework can be described as follows: A promoter sequence is present near the plasmid’s starting point (promoter region), which can be recognized by transcription factors within the cell, hence initiating gene transcription. The coding region of the H2B gene follows immediately downstream of the promoter, encompassing the DNA sequence responsible for encoding the histone H2B protein. A terminator codon is placed at the 3′ end of the H2B coding sequence, signaling the termination of the transcription process. Subsequent to the terminator, in the downstream region, lies the coding sequence of the eYFP (enhanced yellow fluorescent protein) gene, which encodes the fluorescent protein. In summary, the pjit166 plasmid contains a promoter for gene transcription, a coding sequence for the H2B protein, and a coding sequence for the eYFP fluorescent protein. This expression framework allows the synthesis of a fusion protein consisting of H2B and eYFP, thereby facilitating specific research or analysis purposes in experimental studies [29].

2.2. Experimental Procedures

2.2.1. Callus Induction and Protoplast Preparation

C. lanceolata immature seeds were sterilized and placed on a bench. After cutting open the seed coats and removing the IZEs, a transverse incision was made across the endosperms, and the wound sites were pressed firmly against the modified basal medium DCR induction medium to induce callus formation [30]. The growth of the callus was observed using a microscope, and the material for protoplast preparation was chosen based on the genotype with the best growth rate. To prepare the protoplasts, we optimized the solutions and procedures based on a previously reported method for A. thaliana [31]. First, we treated the callus samples (1 g) for 6 h with a 0.3~0.6 M mannitol solution. Using a Zeiss Axio Imager D2 fluorescence microscope (Carl Zeiss, Jena, Germany), we then counted the number of cells in which the plasmalemma was effectively separated from the cell wall and measured the distances between them. After 4–5 h of enzymatic digestion, we obtained isolated protoplasts by centrifugation and calculated the yield with a 0.1 mm × 400 mm hemocytometer (Qiujing, Shanghai, China). The protoplast suspension was stained with fluorescein diacetate (FDA) (Sigma-Aldrich, St. Louis, MO, USA), and protoplasts with high green fluorescence were considered viable. Before transfection, the C. lanceolata protoplasts were placed in an ice bath with W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, and 2 mM MES at pH 5.7) [31]. We monitored the protoplast viability using the FDA stain protocol every 6 h from 0 to 24 h.

2.2.2. Mediation of TTP by PEG of Varying Molecular Weights Prepared at Different Concentrations

To prepare C. lanceolata protoplasts for transfection, a few modifications were made. Before transfection, the protoplasts were first incubated for 15 min in a modified MMg solution, followed by thorough mixing. This solution consisted of 0.5 M mannitol, 15 mM MgCl2, and 4 mM MES at pH 5.7. Different concentrations (10%, 15%, and 20% w/v) of PEG with varying molecular weights were prepared as storage solutions. After transfection, the protoplasts were centrifuged at 100× g for 2 min, and the supernatant was discarded. The pellets were resuspended in the modified MMg solution and diluted to a 1 × 105 cells/mL concentration. Transfection was carried out using the H2B-eYFP plasmid, a transient expression vector where the H2B gene is ligated with the enhanced yellow fluorescent protein (eYFP) tag gene [32]. The eYFP fluorescence signal was observed using a Zeiss Axio Imager D2 fluorescence microscope. To carry out transfection, protoplasts and PEG were mixed sequentially in a centrifuge tube and incubated at 23 °C for 15 min. After adding four times the volume of W5 for dilution, the protoplasts were collected by centrifugation at 100× g for 2 min. Finally, the protoplasts were resuspended with W5 and incubated in a 6-well cell culture plate for 16 h at 20~25 °C. Following that, the protoplasts were sampled and photographed for observation.

2.3. Data Analysis

C. lanceolata genotypes B46 × B49 and B49 × B46 were extracted from the same batch and inoculated using DCR media [33] (with five statistical replicates) at a rate of nine induced zygotic embryo explants per petri dish (with nine biological replicates). Table 1 presents the formulation of the callus induction medium for Cunninghamia lanceolata. Table 2 outlines the formulation of the subculture medium for Cunninghamia lanceolata callus. Table 3 provides the formula for the mother liquor of the DCR medium.
Equation (1) was then used to calculate the C. lanceolata-induced callus induction rate (CIR).
C I R = N u m b e r   o f   I Z E s   w i t h   i n d u c e d   c a l l u s T o t a l   n u m b e r   o f   I Z E s × 100 %
Statistical data processing was performed using Microsoft Office Excel, and the degree of C. lanceolata CP (protoplasts with cell walls removed) plasmolysis under 0.3~0.6 M mannitol solutions was analyzed using ANOVA in OriginPro2021 student version (OriginLab Corporation, Northampton, MA, USA). Box plots were generated to determine the appropriate mannitol concentration for plasmolysis of C. lanceolata callus cells. The viability of the obtained C. lanceolata CPs was calculated using Equation (2); the TTP (transformation and transfection protocol) efficiency of C. lanceolata callus was calculated using Equations (3) and (4) was used to calculate the yield of C. lanceolata CPs. C. lanceolata CP yield, viability, and TTP efficiency were measured three times, and the mean values were calculated.
P r o t o p l a s t s   v i a b i l i t y = N u m b e r   o f   v i a b l e   p r o t o p l a s t s T o t a l   n u m b e r   o f   p r o t o p l a s t s × 100 %
T T P   e f f i c i e n c y = T o t a l   n u m b e r   o f   f l u o r e s c e n t   p r o t o p l a s t s T o t a l   n u m b e r   o f   p r o t o p l a s t s × 100 %
P r o t o p l a s t   y i e l d = P r o t o p l a s t   d e n s i t y × t o t a l   v o l u m e   o f   s u s p e n s i o n T h e   f r e s h   w e i g h t   o f   t h e   m a t e r i a l   ( e . g . ,   p l a n t   t i s s u e )   u s e d   f o r   i s o l a t i n g   p r o t o p l a s t s . × 100 %

3. Results

3.1. Callus Induction from Immature Embryos of Different Developmental Phases

Reciprocal hybrids of C. lanceolata genotypes B46 and B49 were subjected to callus induction from IZEs. One week following preparation, the resultant embryos became swollen and enlarged, and calluses formed after two weeks. Although the callus induction rate (CIR) varied, the appearance of the callus induced by the reciprocal hybrid IZE hydration was similar across all samples, with a loose, white texture. (Figure 2). The callus obtained from reciprocal hybrids requires three consecutive passages. The CIR for C. lanceolata genotype B46 × B49 IZE was greater than for genotype B49 × B46 IZE. The CIR of genotype B49 × B46 IZEs at developmental phase 1 was 30%, but those at developmental phases 2–5 did not induce callus tissue. Conversely, callus formation was seen in genotype B46 × B49 IZEs harvested during developmental phases 1–3. Specifically, genotype B46 × B49 IZEs obtained during developmental phase 1 exhibited a 20% CIR, while those obtained at developmental phase 2 showed the highest CIR (30%). However, genotype B46 × B49 IZEs collected at developmental phase 4 resulted in the formation of roots or shoots, with only a 15% rate of induced callus cells. At developmental phase 5, genotype B46 × B49 IZEs did not induce any callus but only roots or shoots. Overall, the CIR tended to increase and then decrease with the IZE developmental phase (Figure 2).

3.2. Preparation of Protoplasts

3.2.1. Mannitol Concentration Screening

Plasmolysis levels were clearly visible in callus cells induced by IZE treatment at mannitol concentrations of 0.4 M and 0.5 M, but not at 0.3 M (Figure 3 and Figure 4). The degree of plasmolysis in callus cells of genotype B49 × B46 varied with increasing mannitol concentration. Our data analysis revealed that the impact of 0.3 M to 0.6 M mannitol concentrations on the level of plasmolysis in callus cells of genotype B49 × B46 was statistically significant at a probability level of 0.01 (Figure 3), with the highest level of significance observed in the 0.6 M mannitol treatment group. As such, plasmolysis in callus cells was not immediately apparent at low mannitol concentrations; conversely, at high mannitol concentrations, the protoplasts shrank, and clumped together, making recovery difficult. Based on the degree and distribution of plasmolysis across the different mannitol concentrations, we selected 0.4 M mannitol as the optimal concentration for osmotic stabilization during CP preparation.

3.2.2. Screening of Enzymatic Digestion Conditions

Upon subculturing the C. lanceolata callus in solid medium, a bright translucent structure with a white appearance was observed, and the cells appeared to be rich in water (Figure 5a). Microscopic analysis revealed that the embryoid pedicel mass was composed of the embryo head and long rod-shaped embryo stem cells, with elongated cell clusters on the cell surface, corresponding to embryoid callus. (Figure 6a). Following the preparation of the C. lanceolata callus protoplasts, three types were identified: those with few inclusions (Figure 6b), those with a small number of conspicuous inclusions (Figure 6c), and those with full-filled conspicuous inclusions (Figure 6d).
Among the four enzymatic solutions tested for protoplast preparation, enzymatic combination C, which consisted of 2% (w/v) Cellulase R-10, 0.2% (w/v) Macerozyme R-10, 0.1% (w/v) Pectolyase Y-23, and 5 mM mercaptoethanol, yielded the highest number of protoplasts with 9.76 × 106 cells/mL (Figure 7 and Table 4). Therefore, this combination was chosen for the preparation of C. lanceolata CPs. During enzymolysis of the C. lanceolata callus, the enzymolysis liquid gradually changed from clear to milky, and complete digestion of the callus to obtain the protoplast crude extract was achieved in 3 to 4 h (Figure 5b,c). The protoplasts obtained from enzymatic digestion with 0.4 M mannitol as the osmotic stabilizer exhibited similar shape and size, clear edges, more inclusions, and fewer fragments (Figure 5d). The crude extract of protoplasts was then filtered using a 0.7-micrometer cell strainer to effectively remove the incompletely dissociated C. lanceolata callus. After centrifugation, collection, and washing with W5 solution (Figure 5e,f), purified protoplasts from C. lanceolata calluses were obtained (Figure 5g).
After purification, the viability of C. lanceolata’s CPs was originally 92.7%, but it gradually decreased with increasing duration in ice incubation, particularly after 12 h (Figure 6e,f and Figure S1). After 24 h, there was an increase in contamination and fragments within the microscope field, which led to an irregular morphology of the protoplasts. As a result, the viability of CPs was reduced to 79.0%.

3.3. Different Concentrations and Molecular Weights of PEG Were Used to Mediate the Transient Gene Expression from C. lanceolata Callus

Our results indicate that the H2B-eYFP fusion protein is targeted to the nucleus of C. lanceolata CPs (Figure 6), which is consistent with prior studies on Arabidopsis thaliana [34]. Moreover, the findings demonstrate that PEG at various concentrations and molecular weights can facilitate transient gene expression in C. lanceolata calluses and allow for the localization of H2B-eYFP to the nucleus (Figure 8). Specifically, the highest efficiencies were observed with 20% (w/v) PEG6000-mediated transient transfection efficiency, which peaked at 6.70%, and 15% (w/v) PEG8000-mediated transient transfection efficiency, which peaked at 8.99%. Considering the potential adhesions and breakage of transfected protoplasts, we recommend the use of 20% (w/v) PEG6000 for mediating TTP of C. lanceolata callus (Figure 9).

4. Conclusions

One of the critical steps in plant TTP studies is the preparation of high-quality protoplasts. Gymnosperm protoplasts can be derived from various sources such as callus (Picea abies [35], Akjesfabri, Pinus lambertiana [36]), needle leaves (C. lanceolata, Platycladus orientalis [37]), and suspension cells (P. taeda L., C. lanceolata, P. lambertiana, P. mariana, Larix gmelinii [38]). Among these sources, enzymatic digestion of C. lanceolata callus is comparatively easier than needle leaves [39]. The suspension cell system requires a long experiment cycle, and stem segments have a high contamination rate; therefore, C. lanceolata callus was selected to prepare protoplasts in our study. To obtain abundant protoplasts, the first step is to optimize the conditions for callus induction. Callus induction is known to be affected by the degree of IZE development and genotype [40]. Our study found that the CIR of C. lanceolata IZE increased and then decreased with the developmental phase, which is consistent with previous observations on Pinus koraiensis [41,42], P. elliottii [43], Pinus thunbergii [44], and P. taeda L. [45]. Furthermore, we found that genotype has a major influence on callus induction [46], with significant differences observed in the CIR of IZE of C. lanceolata reciprocal hybrids between genotypes B46 and B49 collected at the same developmental phase. Studies on P. aspoerata, P. sylvestris, and P. taeda L. have also revealed that the maternal genotype has a greater effect on embryonic callus induction than the paternal genotype [47].
Currently, mannitol is widely used as an osmotic stabilizer during protoplast preparation [48]. However, the osmotic stabilizer concentration must be carefully controlled to avoid damaging the protoplasts. The protoplasts can break apart if the concentration is too low, and they can shrink or contract if the concentration is too high. Previous studies have indicated that 0.4 M mannitol is the optimal concentration for protoplast isolation from various plant species, including C. lanceolata callus, Populus L., Quercus palustris, and Camellia sinensis leaves [49]. Enzymes such as Cellulase R-10, Macerozyme R-10, and Pectolyase Y-23 are commonly used in protoplast preparation protocols; however, the optimal combination of enzymes varies depending on the plant species. In Onobrychis taneitica, for example, viable protoplasts were obtained using only Cellulase R-10 and Pectolyase Y-23 [50], while in Lotus corniculatus L., increasing the concentration of Macerozyme R-10 improved protoplast production [51]. In this study, we found that increasing the concentration of Cellulase R-10 alone did not significantly improve the yield or viability of C. lanceolata protoplasts. However, increasing both Cellulase R-10 and Macerozyme R-10 concentrations led to a significant increase in protoplast yield. After screening various enzyme combinations, we identified a final enzymatic cocktail containing Cellulase R-10, Macerozyme R-10, Pectolyase Y-23, and 5 mM mercaptoethanol, which enabled us to obtain a high yield of C. lanceolata protoplasts with minimal cell debris. TTP efficiency is influenced by several factors. A high protoplast concentration can make cells sticky and unfavorable for observation; conversely, a low concentration can lead to transient transfection failure. To ensure high transfection efficiency, the protoplast concentration should be controlled within the range of 0.5 to 2 × 105 cells/mL [51]. In our study, we used CPs of C. lanceolata genotype B49×B46 at a concentration of 1 × 105 cells/mL as the recipient for PEG-mediated transient transfection. The highest efficiency was obtained with 25% (w/v) PEG-mediated TTP in Triticum aestivum L. [52]; while in Zea mays and Sorghum bicolor, 30% (w/v) PEG-mediated leaf TTP yielded the best results [53,54]. However, we found that the TTP efficiency of C. lanceolata callus initially increased and then decreased as the PEG concentration increased. Moreover, TTP efficiency is also affected by PEG molecular weight [55]. For example, in Citrus reticulata, Phalaenopsis aphrodite, and Carica papaya L., the highest mesophyll TTP efficiency was achieved with PEG4000, PEG6000, and PEG8000, respectively [56,57]. In our study, we found that 15% PEG8000 mediated the highest TTP efficiency in C. lanceolata callus, although this resulted in severe damage to some protoplasts. As a result, we selected 20% (w/v) PEG6000 as the optimal medium for TTP transfection in C. lanceolata calluses. Additionally, in Manihot esculenta, longer transfection periods were associated with higher protoplast damage by PEG [58]. In Chenopodium quinoa, the highest protoplast transfection efficiency was observed at a transfection duration of 15 min [59]. Similarly, we observed that the TTP efficiency of C. lanceolata callus initially improved and then dropped as transfection time increased, with many protoplasts fragmenting after 30 min. Therefore, we selected 15 min as the optimal transfection duration.
Here, IZEs from the C. lanceolata genotype B49×B46 were utilized to induce callus, and a C. lanceolata CP transient transfection system was established. These significant findings lay a solid foundation for gene localization studies, functional analysis, and germplasm innovation related to this important species.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f14061168/s1. Figure S1: Viability test of protoplasts of C. lanceolata callus from 0 to 24 h.

Author Contributions

J.C. and L.Y. conceived, planned, coordinated the project, and finalized the manuscript. K.H. performed the experiments, performed the data analysis, and wrote the draft. Z.C. validated and contributed to data analysis and curation and revised the manuscript. Y.R. has made edits and polished the language of this text. R.Z., X.Z. and Y.L. reviewed and edited the manuscript. J.C. coordinated, contributed to data curation, finalized, and funded this research. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Seed Industry Innovation and Industrialization Project of Fujian Province, grant number ZYCX-LY-202101, the Research Startup Fund for High-Level and High-Educated Talents of Nanjing Forestry University, and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Data Availability Statement

Not applicable.

Acknowledgments

We thank Wei Jianhua for supplying PtoMYB148-eYFP. Transient expression vectors of eYFP-tagged fusions of PtoMYB148-eYFP were provided by Jianhua Wei, Beijing Agro-Biotechnology Research Center. We recombined the H2B-eYFP fusion protein into the instantaneous protein expression plasmid vector PJIT-166 preserved in our laboratory. It was used to assess the ability of the expression system to target the product of an exogenously introduced gene to the nucleus.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Gene expression framework of the H2B-eYFP fusion protein. 2 × 35s: duplication of the cauliflower mosaic virus (CaMV) 35S promoter. H2B: histone H2B protein; eYFP: enhanced yellow fluorescent protein. NOS: NOS terminator. *: Stop codon. Green triangle: universal sequencing primers m13 rev.
Figure 1. Gene expression framework of the H2B-eYFP fusion protein. 2 × 35s: duplication of the cauliflower mosaic virus (CaMV) 35S promoter. H2B: histone H2B protein; eYFP: enhanced yellow fluorescent protein. NOS: NOS terminator. *: Stop codon. Green triangle: universal sequencing primers m13 rev.
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Figure 2. Callus induction of IZEs from the reciprocal hybrid of C. lanceolata at different developmental phases. B49 × B46 and B46 × B49: collection of fruits from reciprocal crosses of C. lanceolata. IZEs: immature zygotic embryos. CIR: callus induction rate. DP: developmental phase. (1-a–e,2-a–e): IZEs were cleaned, sterilized, and placed on DCR induction medium. (1-f–j,2-f–j): images of callus induced from IZEs. (2-i): only buds were induced without callus. (1-g–j): no callus was induced. Bar = 0.5 cm.
Figure 2. Callus induction of IZEs from the reciprocal hybrid of C. lanceolata at different developmental phases. B49 × B46 and B46 × B49: collection of fruits from reciprocal crosses of C. lanceolata. IZEs: immature zygotic embryos. CIR: callus induction rate. DP: developmental phase. (1-a–e,2-a–e): IZEs were cleaned, sterilized, and placed on DCR induction medium. (1-f–j,2-f–j): images of callus induced from IZEs. (2-i): only buds were induced without callus. (1-g–j): no callus was induced. Bar = 0.5 cm.
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Figure 3. Plasmolysis of C. lanceolata callus at different concentrations of mannitol solutions. The callus samples were treated with a 0.3~0.6 M mannitol solution for 6 h, and the distance of plasmolysis was measured by the software of the Zeiss microscope after taking photos. ** above the box of the 0.6 M mannitol treatment group indicates significant differences between treatments on any other variable at p < 0.01.
Figure 3. Plasmolysis of C. lanceolata callus at different concentrations of mannitol solutions. The callus samples were treated with a 0.3~0.6 M mannitol solution for 6 h, and the distance of plasmolysis was measured by the software of the Zeiss microscope after taking photos. ** above the box of the 0.6 M mannitol treatment group indicates significant differences between treatments on any other variable at p < 0.01.
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Figure 4. Microscopic observations of C. Lanceolata callus at different mannitol concentrations. (a): 0.3 M, (b): 0.4 M, (c): 0.5 M, and (d): 0.6 M. Scale = 100 µm. Red arrows indicate excessive separation of the mass walls. Blue arrows indicate moderate separation of the bulk walls. Yellow lines show the measurement method for the mass wall separation distance.
Figure 4. Microscopic observations of C. Lanceolata callus at different mannitol concentrations. (a): 0.3 M, (b): 0.4 M, (c): 0.5 M, and (d): 0.6 M. Scale = 100 µm. Red arrows indicate excessive separation of the mass walls. Blue arrows indicate moderate separation of the bulk walls. Yellow lines show the measurement method for the mass wall separation distance.
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Figure 5. Purification of protoplasts from C. Lanceolata calluses. (a): Callus on solid medium; the small picture is a close-up of a single piece of callus. (b): Initiation of enzymatic digestion of C. lanceolata callus. (c): Enzymatic digestion after 3~4 h. (d): Protoplasts before purification. (e): Protoplasts being washed with W5 solution for purification. (f): Protoplasts collected by centrifugation after washing. (g): Protoplasts after purification. Scale without labeled distance is 1 cm.
Figure 5. Purification of protoplasts from C. Lanceolata calluses. (a): Callus on solid medium; the small picture is a close-up of a single piece of callus. (b): Initiation of enzymatic digestion of C. lanceolata callus. (c): Enzymatic digestion after 3~4 h. (d): Protoplasts before purification. (e): Protoplasts being washed with W5 solution for purification. (f): Protoplasts collected by centrifugation after washing. (g): Protoplasts after purification. Scale without labeled distance is 1 cm.
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Figure 6. Microscopic observation and viability assessment of C. Lanceolata protoplasts. (a,c): C. Lanceolata callus, scale = 100 µm. (b–d): Protoplast type, scale = 5 µm. (e): FDA staining to detect the viability of protoplasts by FITC channel, scale = 50 µm. (f): Percentages of viable protoplasts after standing on ice for different periods of time. Means sharing with ±SD, and common letter do not differ significantly at p ≤ 0.05 according to ANOVA with the Duncan test.
Figure 6. Microscopic observation and viability assessment of C. Lanceolata protoplasts. (a,c): C. Lanceolata callus, scale = 100 µm. (b–d): Protoplast type, scale = 5 µm. (e): FDA staining to detect the viability of protoplasts by FITC channel, scale = 50 µm. (f): Percentages of viable protoplasts after standing on ice for different periods of time. Means sharing with ±SD, and common letter do not differ significantly at p ≤ 0.05 according to ANOVA with the Duncan test.
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Figure 7. Visual representation of enzymatic solutions for protoplast isolation. (a–d) represent formulas a–d of enzymatic solutions, respectively, in Table 4. Scale = 100 µm.
Figure 7. Visual representation of enzymatic solutions for protoplast isolation. (a–d) represent formulas a–d of enzymatic solutions, respectively, in Table 4. Scale = 100 µm.
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Figure 8. Subcellular location of the H2B-eYFP recombinant protein in protoplasts of C. lanceolata callus. (a) The image represents the YFP channel fluorescence. (b) The image is captured in bright-field illumination. (c) The image is a merged composite, combining different channels or modalities. YFP-Channel: the photo was taken using Filter 46-YFP of the Zeiss fluorescence microscope with excitation wavelength λex = 426–446 nm and emission wavelength λem = 520–550 nm.
Figure 8. Subcellular location of the H2B-eYFP recombinant protein in protoplasts of C. lanceolata callus. (a) The image represents the YFP channel fluorescence. (b) The image is captured in bright-field illumination. (c) The image is a merged composite, combining different channels or modalities. YFP-Channel: the photo was taken using Filter 46-YFP of the Zeiss fluorescence microscope with excitation wavelength λex = 426–446 nm and emission wavelength λem = 520–550 nm.
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Figure 9. Optimizing transfection efficiency in C. lanceolata callus protoplasts by PEG solutions with varying molecular weights and concentrations. Working concentrations of weight/volume (w/v): 10%/15%/20%. PEG-4k/6k/8k: polyethylene glycol (PEG) with a molecular weights of 4000/6000/8000. Bar = 50 µm.
Figure 9. Optimizing transfection efficiency in C. lanceolata callus protoplasts by PEG solutions with varying molecular weights and concentrations. Working concentrations of weight/volume (w/v): 10%/15%/20%. PEG-4k/6k/8k: polyethylene glycol (PEG) with a molecular weights of 4000/6000/8000. Bar = 50 µm.
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Table
Table 1. Formulation of callus induction medium for Cunninghamia lanceolata.
Table 1. Formulation of callus induction medium for Cunninghamia lanceolata.
PharmaceuticalCulture Medium Number 01Culture Medium Number 02
Quantity of Ingredients Used for Preparing 1 LQuantity of Ingredients Used for Preparing 1 L
Mother liquor with a high concentration of elements50 mL50 mL
Mother liquor containing micronutrition10 mL10 mL
Organic10 mL10 mL
Iron salt10 mL10 mL
2,4-D20 mL10 mL, 20 mL, 30 mL
6-BA5 mL5 mL
KT5 mL5 mL
VC10 mL10 mL
Casein acid hydrolysate0.5 g/L0.5 g/L
Sucrose20 g/L20 g/L
Active charcoal3 g/L3 g/L
Glutamine0.45 g/L0.45 g/L
Inositol0.1 g/L0.1 g/L
Table
Table 2. Formulation of the subculture medium for the callus of Cunninghamia lanceolata.
Table 2. Formulation of the subculture medium for the callus of Cunninghamia lanceolata.
PharmaceuticalCulture Medium Number 03
Quantity of Ingredients Used for Preparing 1 L
Mother liquor with a high concentration of elements50 mL
Mother liquor containing micronutrition10 mL
Organic10 mL
Iron salt10 mL
2,4-D10 mL
6-BA5 mL
KT5 mL
VC10 mL
Casein acid hydrolysate0.5 g/L
Sucrose20 g/L
Active charcoal3 g/L
Glutamine0.45 g/L
Inositol0.1 /L
Table
Table 3. Formula of the mother liquor of DCR medium.
Table 3. Formula of the mother liquor of DCR medium.
PharmaceuticalElementsg/L
Mother liquor with a high concentration of elementsKNO30.34
Ca(NO3)2·4H2O0.556
MgSO4·7H2O0.37
NH4NO30.4
KH2PO40.17
CaCl20.064/0.085
Mother liquor containing micronutritionKI0.00083
H3BO30.0062
MnSO4·4H2O0.0294
ZnSO4·7H2O0.0086
Na2MoO40.00025
CuSO4·5H2O0.00025
CoCl2·6H2O0.000025
NiCl20.000025
Iron saltFeSO4·7H2O0.0278
EDTA0.0373
OrganicInositol0.2
Glycine0.002
Niacin0.0005
VB1 (Thiamine hydrochloride)0.001
VB6 (Pyridoxine HCL)0.0005
VC 1
2,4-D 0.1
6-BA 0.1
KT 0.1
Table
Table 4. Protoplasts prepared under different enzymatic solutions from C. lanceolata callus.
Table 4. Protoplasts prepared under different enzymatic solutions from C. lanceolata callus.
N.Cellulase R-10Macerozyme R-10Pectolyase Y-23Yield (Cells/mL)Whether to Add Mercaptoethanol
a1.5%0.1%0.1%2.47 × 106yes
b2%0.2%0.1%5.24 × 105no
c2%0.2%0.1%9.76 × 106yes
d3%0.1%0.2%1.38 × 106yes
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Hong, K.; Chen, Z.; Radani, Y.; Zheng, R.; Zheng, X.; Li, Y.; Chen, J.; Yang, L. Establishment of PEG-Mediated Transient Gene Expression in Protoplasts Isolated from the Callus of Cunninghamia lanceolata. Forests 2023, 14, 1168. https://doi.org/10.3390/f14061168

AMA Style

Hong K, Chen Z, Radani Y, Zheng R, Zheng X, Li Y, Chen J, Yang L. Establishment of PEG-Mediated Transient Gene Expression in Protoplasts Isolated from the Callus of Cunninghamia lanceolata. Forests. 2023; 14(6):1168. https://doi.org/10.3390/f14061168

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Hong, Kaiyue, Zhenyu Chen, Yasmina Radani, Renhua Zheng, Xueyan Zheng, Yong Li, Jinhui Chen, and Liming Yang. 2023. "Establishment of PEG-Mediated Transient Gene Expression in Protoplasts Isolated from the Callus of Cunninghamia lanceolata" Forests 14, no. 6: 1168. https://doi.org/10.3390/f14061168

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