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http://journals.tubitak.gov.tr/agriculture/

Turkish Journal of Agriculture and Forestry Turk J Agric For(2019) 43: 275-287© TÜBİTAKdoi:10.3906/tar-1805-62

Mesophyll protoplast isolation technique and flow cytometry analysis of ancient Platycladus orientalis (Cupressaceae)

Qianyi ZHOU1, Zhaohong JIANG2

, Yiming LI1, Tian ZHANG1

, Hailan ZHU1, Fei ZHAO3

, Zhong ZHAO1,*1Key Comprehensive Laboratory of Forestry, College of Forestry, Northwest A&F University, Yang Ling, Shaanxi, P.R. China

2College of Life Sciences, Northwest A&F University, Yang Ling, Shaanxi, P.R. China3Beijing Agricultural Technology Extension Station, Beijing, P.R. China

* Correspondence: zhaozhlunwen2010@126.com

1. IntroductionThe protoplast is a naked bioactive cell from which the cell wall has been removed, usually by enzyme digestion. Protoplasts can take up the source material of protoplasm such as DNA, plasmids, viruses, bacteria, and organelles in a variety of ways, including heat shock, electroporation, bacteria incubation, and high pH. These features make protoplasts a useful tool for genetic manipulation and gene transfer in plant cell engineering (Puite, 1992). Since the first isolation by Cocking in tomato root tips in 1960 (Cocking, 1960), additional progress in isolating the protoplasts of other horticultural plants, including wheat, barley, corn, soybean, rice, cotton, and potato, has been reported.

The isolation of woody plant protoplasts was started quite late, in 1987, by Ochatt et al. (Ochatt et al., 1987). Compared with the isolation of somatic cells, root cells, and callus cells from herbaceous plants, the isolation of protoplasts of gymnosperms lags far behind. Conifers are

the most important afforesting and greening gymnosperm species, representing 82% of terrestrial biomass (Neale and Kremer, 2011), but the progress in conifer protoplast isolation and in vitro culture is slow because of their complex cellular components and difficult regeneration. Even so, protoplasts have been isolated from a number of conifers and have produced promising results. Isolation of conifer protoplasts was first achieved in Pinus contorta in 1983 (Hakman and Arnold, 1983). Since then, protoplasts have been isolated successfully from several other conifer species. The starting materials and isolation techniques are shown in Table 1. Through their acquisition and cultivation, protoplasts are now used for rapid propagation (Fukumoto et al., 2005), cell development (Bornman et al., 2005), transgenics (Géomez-Maldonado et al., 2012), and combined plant hybridization and breeding improvement (Prange et al., 2010).

There are two main methods to isolate plant protoplasts: the mechanical method and the enzyme digestion method.

Abstract: Platycladus orientalis (Cupressaceae) is a native tree species used widely for landscaping and afforestation in arid and semiarid areas in Asia. Ancient P. orientalis trees not only have an important humanistic and historical value but also have a significant scientific value in aging mechanism research. Mesophyll protoplasts are an important material for studies of plant regeneration, transient gene expression, and senescence. Although an easy and effective technique of mesophyll protoplast isolation from mature trees is urgently needed, the isolation parameters have great material specificity. The young tissue of plants is an ideal material for protoplast preparation. In this study, we employed an orthogonal experiment and several single-variable experiments to determine the main factors influencing the successful isolation of mesophyll protoplasts and established an efficient technique for isolating mesophyll protoplasts from the young scale leaves of ancient P. orientalis. The optimal parameters for mesophyll protoplast isolation are as follows: mechanical homogenization of the leaf tissue, 1.5% Cellulase R-10, 0.4% Macerozyme R-10, 0.4% Pectolyase Y-23, 1.0% ligninase, 0.7 M mannitol (pH 5.8), and 16 h of incubation. Two centrifugations (100 × g for 3 min and 500 × g for 5 min) were repeated 2 times to obtain purified protoplast suspension. The yield and viability of protoplasts under optimal conditions were 4.8 × 106 g FW–1 (per gram fresh weight) and 82.5%, respectively. The results of flow cytometry analysis showed that the isolated protoplasts had ideal viability to meet the demands of further protoplast-based research.

Key words: Flow cytometry, mesophyll protoplast, orthogonal experiment, Platycladus orientalis, protoplast isolation

Received: 15.05.2018 Accepted/Published Online: 09.12.2018 Final Version: 11.06.2019

Research Article

This work is licensed under a Creative Commons Attribution 4.0 International License.

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The latter method has recently been the most widely used (Jin and He, 2003). The main factors that affect enzymatic digestion efficiency are the plant material, plant tissue pretreatment method, enzyme type and concentration, enzymolysis time, concentration of osmotic stabilizer (a solution of high osmotic pressure that keeps the membrane from breaking and maintains the normal form of the protoplast), temperature, pH, etc.

At present, a relatively complete plant protoplast enzymatic isolation technology has been established worldwide. However, almost all studies have indicated that there are significant differences in the isolation conditions of protoplasts in different plant species and even in different tissues of the same plant. The young tissue of plants is the ideal material for protoplast preparation (Huang et al., 2013). Segments of the leaves, cotyledons, roots, and cotyledons are usually used to prepare protoplasts. There are few reports in the literature on successful isolation and culture of conifer protoplasts, and there are many restrictions on the materials that could be used. Moreover, the early attempts to isolate protoplasts from the leaves of conifers faced technical difficulties because of the special anatomical structure, the high levels of phenolic compounds, and secretion of cypress oils in the cells of these species.

P. orientalis (Cupressaceae) is a native tree species in Asia that is widely used for landscaping and afforestation in arid and semiarid areas (Wang et al., 2008). Furthermore, P. orientalis has an extremely long lifespan of several thousand years. The aging and senescence mechanisms of ancient trees have attracted interest from researchers (Zhu and Lou, 2013; Zhang et al., 2015; Chang et al., 2017a, 2017b). Ancient P. orientalis trees not only have important humanistic and historical value but also have significant scientific value in research on aging and senescence mechanisms (Zhang et al., 2015). However, the tough plant material of P. orientalis, which has a unique morphological structure and cellular components, makes mesophyll protoplast isolation difficult (Dörken, 2013). To date, no reports have been published on the isolation of the protoplasts of P. orientalis. The leaf is the primary photosynthetic organ and the most sensitive organ of a tree. Establishing an effective protocol for isolating protoplasts from the young leaves of P. orientalis would be important for the reproduction of precious genetic resources, establishment of an in vitro genetic transformation system, and characterization of mesophyll cells during tree aging.

Flow cytometry (FCM) is a current technique for rapid analysis and separation of multiple parameters of a cell or other particles in a fluid flow. The advantages of this technique include rapidity, sensitivity, and synchronization of multiparameter detection (Muirhead et al., 1985). Since the early 1980s, FCM technology has been applied in plants (Redenbaugh et al., 1982). After more than 30

years of development, this technology is widely used for the detection of DNA and RNA content and for cell membrane, protoplast, and cell wall regeneration research (Doležel et al., 1994), etc. Protoplasts are the only material available when FCM is used to detect vacuoles in plant cells in multinucleated cell research, or for selection of whole cells for further regeneration (Bergounioux et al., 2010). Moreover, the detection of protoplasts by FCM can reveal many cell-related characters such as chromatin compaction (Rustgi, 2013), DNA content abnormity (Carlberg et al., 1984), nuclear ploidy levels (Lindsay et al., 1994), and cell calcium influx (Maintz et al., 2014), which are significantly related to plant senescence. It is worth mentioning that, when using protoplasts as FCM samples, the cells remain alive and are conducive to dynamic detection of related changes (Lindsay, et al., 1994).

In this research, we designed single-factor experiments and an orthogonal experiment based on previous studies and references on protoplast isolation methods in other species of conifers to establish and optimize the parameters for isolation and purification of P. orientalis mesophyll protoplasts. The purpose of this study was to introduce a simple, effective, and reproducible method for the isolation of a large number of living mesophyll protoplasts from ancient P. orientalis. In addition, we used a freshly isolated protoplast suspension as the sample for FCM analysis to build a foundation for further research on cell sorting and fluorescence labeling of protoplasts.

2. Materials and methods2.1. Plant materialsYoung and fresh scale leaves (annual-growth leaves only, 2–3 cm in length from the tip) of healthy ancient P. orientalis (older than 2000 years) were collected in May 2016. The scale leaves were collected in petri dishes and stored in a 4 °C refrigerator before use.

The sampling location is the Mausoleum of the Yellow Emperor, which is located on the Loess Plateau, Huangling County, Shaanxi Province, China (35°34′N, 109°15′E). The annual average temperature and precipitation are 9.4 °C and 596.3 mm. This area has a typical temperate continental climate with distinct seasonal features.2.2. Isolation of protoplastsThe isolation of protoplasts was performed by the enzyme digestion method (David et al., 1986). The collected young scale leaves were washed twice. First they were washed with distilled water to remove surface dust, and then washed with 0.53% sodium hypochlorite and 70% ethanol to make them surface-sterilized. After surface drying, 1 g of fresh leaves was weighed. Five pretreatment methods were tested separately with the scale leaves before enzyme incubation: blade shredding, shearing with scissors, gentle mechanical homogenization (300 rpm for 2 min

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ZHOU et al. / Turk J Agric ForTa

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ZHOU et al. / Turk J Agric For

by using a plant tissue homogenizer (Polytron PT 3000, Kinematica AG, Littau, Switzerland), hand lapping, and tearing the scale leaves. After pretreatment, leaves were incubated with 15 mL of a mixed-enzyme solution (0.1% BSA (bovine serum albumin), 10 mM CaCl2, 20 mM KCl, 20 mM MES (2-(N-morpholino) ethanesulfonic acid), different combinations of enzymes, at proper pH) in the dark at 25 °C and 40 rpm. Five enzymes including Cellulase R-10 (Kinki Yakult Co., Japan), Pectolyase Y-23 (Kinki Yakult Co.), Macerozyme R-10 (Kinki Yakult Co.), hemicellulase (Sigma H2125, USA), and ligninase (Sigma 42603) were used to explore the optimal combination for protoplast isolation. The different enzyme combinations and their effects on protoplast isolation are listed in Table 2. Different concentrations of enzymes were tested by an orthogonal experiment (the specific experimental design is shown in Table 3). Different concentrations of osmotic stabilizer from 0.55 M to 0.80 M were tested in the enzyme solution. The pH of the enzyme solution was adjusted from 5.0 to 6.0 by 0.1 M NaOH and 0.1 M HCl. The yield and viability were recorded from 0 to 22 h every 2 h. All chemicals were purchased from Solarbio Company (Solarbio Technology Co., Beijing, China) and were of analytical or biochemical grade.2.3. Orthogonal array experimental design The experiments were based on an orthogonal array experimental design (L9(34), OA9 matrix), as shown in Table 3 (Hedayat et al., 1999). Each trial group was triplicated.2.4. Analytical methodsData analysis was carried out through range analysis, analysis of variance (ANOVA), and multiple comparison analysis to examine the optimal reaction conditions and the magnitudes of their effects. The statistical analyses were performed using SPSS 21.0 (IBM Corp., Armonk, NY, USA).2.4.1. Range analysisThere are two important parameters in range analysis: Kij and Rj. Kij is the sum of the evaluation index of all levels (i indicates level 1, 2, or 3) in each factor (j indicates factor A, B, C, or D), and (the mean value of Kij) is used to determine the optimal combination of different levels of factors. For each factor, the optimal level is obtained when reaches its largest value. Rj is used for evaluating the rank importance of factors (Cui et al., 2010). Rj is defined as the range between the maximum and minimum value of .2.4.2. Multiple comparison analysisTests conducted on the subsets of data tested in a previous analysis are called post hoc tests. A class of post hoc tests that provide this type of detailed information for ANOVA results are called multiple comparison analysis tests (McHugh, 2011). The multiple comparison analysis in this study was performed using Duncan’s test.Pi

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g an

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, 198

9)

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n la

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(Lar

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lach

and

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laue

r, 19

95)

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s bun

gean

a &

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a w

ilson

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llens

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% su

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r the

mas

s pro

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n pr

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cc. e

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ast (

Fang

et a

l., 2

006)

Tabl

e 1.

Con

tinue

d.

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ZHOU et al. / Turk J Agric For

2.5. Protoplast purificationBecause of the high levels of secreted cypress oil in the cells of P. orientalis, the purification of coarse protoplast suspension is indispensable. After leaf tissue was digested, the suspension was collected and filtered through a 55-µm nylon sieve (commercial product, Huarun Company, Anhui, China). The filtrate was collected and centrifuged at a lower speed (100 × g) for 3 min to remove undigested leaf debris. The precipitate was removed, and the supernatant was collected and recentrifuged at a higher speed of 500 × g for 5 min. The supernatant was discarded to remove organelles and clots, and the precipitate was resuspended in 15 mL of CPW buffer (Nassour et al., 2003) (27.2 mg/L KH2PO4, 101.0 mg/L KNO3, 1480.0 mg/L CaCl2·2H2O, 240.0 mg/L MgSO4·7H2O, 0.16 mg/L KI, 0.025 mg/L CuSO4·5H2O, pH 5.8, osmotic stabilizer added). The two centrifuge-wash steps were repeated one time.2.6. Yield and viability of mesophyll protoplastsThe final precipitate was resuspended gently in 2 mL of prechilled W5 solution (2 mM MES, 154 mM NaCl,

125 mM CaCl2, 5 mM KCl, pH 5.8). Protoplasts were counted on a hemocytometer (Géomez-Maldonado et al., 2012) under a UOP UB200i light microscope (UOP Photoelectric Technology Company, Chongqing, China). Viability was tested with 0.2% fluorescein diacetate (FDA) (Sigma F7378) staining and a UOP UB200i fluorescence microscope with a 488-nm laser. Images were taken and measured with a Tucsen image analysis system (Tucsen Photonics, Fuzhou, China). Protoplast viability was calculated using the following equation: protoplast viability (%) = (fluorescent protoplast number in view/total protoplast number in view) × 100%.2.7. FCM and data analysisA total of 0.2 mL of the abovementioned FDA-stained protoplast suspension containing no more than 2 × 105 cells was then transferred to a glass tube. The protoplasts were then diluted with 2 mL of MMG solution (0.7 M mannitol, 4 mM MES, 15 mM MgCl2, pH 5.8). No more than 5 min after staining with the FDA solution, protoplast fluorescence was immediately measured through FCM

Table 2. Effects of different type of enzymes on protoplast isolation.

Types of enzymeComposition of enzyme solutionE1 E2 E3 E4 E5 E6

Cellulase R-10 + – + + + +Pectolyase Y-23 + + – + + +Hemicellulase + + + – + +Ligninase + + + + – +Macerozyme R-10 + + + + + –Protoplast production Not good No No Yes No YesYield (g FW–1) 6.41 × 103 \ \ 8.22 × 103 \ 4.95 × 103

Viability (%) 10.5 \ \ 23.5 \ 26

“+” and “–” mean addition and no addition of corresponding enzymes, respectively. Enzyme concentration (W/V): 1% Cellulase R-10, 0.4% Pectolyase Y-23, 1% Rhozyme HP-150, 0.5% ligninase, 0.4% Macerozyme R-10. pH 5.5, time = 8 h, osmotic stabilizer 0.7 M mannitol, tissue homogenate method.

Table 3. Levels and factors affecting the yield and viability of protoplast [design of L9(34) orthogonal test].

LevelFactorsCellulase R-10 concentration (%), A

Macerozyme R-10 concentration (%), B

Pectolyase Y-23 concentration (%), C

Ligninase concentration (%), D

1 1.0 0.4 0.2 0.52 1.5 0.5 0.3 1.03 2.0 0.6 0.4 1.5

Enzymolysis condition: pH 5.5, time = 8 h, osmotic stabilizer 0.7 M mannitol, tissue homogenate method.

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(BD FACSAria, BD Biosciences Company, USA) at a wavelength of 488 nm. For each sample, 20,000 cells were analyzed by using BD FACSDiva Software (BD Biosciences Company, USA).

3. Results3.1. The effects of different enzyme combinations and enzyme concentrations on protoplast isolationWe chose five enzymes according to the published literature (Table 1) and the results of preliminary experiments. The different enzyme combinations and their effects on the protoplast isolation are listed in Table 2. For the protoplast isolation results, experiments E2, E3, and E5 indicated that Cellulase R-10, Pectolyase Y-23, and ligninase are indispensable for ensuring that protoplasts can be isolated. Groups E4 and E6 yielded viable protoplasts, and the yield in E4 was greater than that in E6, which indicated that the combination of Pectolyase Y-23 and Macerozyme R-10 can achieve better results. Although the E1 group yielded protoplasts, microscopic observation showed that the suspension contained a large number of enzyme crystals and impurities. The poor isolation result in E1 led us to abandon the use of hemicellulase. Finally, four enzymes (Cellulase R-10 (A), Macerozyme R-10 (B), Pectolyase Y-23 (C), and ligninase (D)) were selected for further concentration optimization for protoplast isolation. The yield and viability of mesophyll protoplasts of P. orientalis were examined by using four enzymes at three levels in an orthogonal experiment (Table 3), and the results are shown in Table 4. According to the OA9 matrix, the range of protoplast yield varied from 2.74 × 103 to 1.52 × 105

g FW–1 (per gram fresh weight) and the viability ranged from 12.3% to 52.4%. These original data were used in the range analysis and ANOVA analysis.

The mean values of Kij () from the range analysis of four factors with three levels are shown in Table 5. As described in the method above, a higher signifies that the corresponding level has a larger effect on the results. In Table 5, the highest of each level for each factor was clearly distinguished, and the best combination for protoplast yield was 1.5% Cellulase R-10, 0.4% Macerozyme R-10, 0.4% Pectolyase Y-23, and 1.0% ligninase (A2B1C3D2; refer to Table 3). The best combination for protoplast viability was A2B1C1D1. The range value (R) indicates the significance of the effect of each factor, with a larger R indicating a greater impact of a given factor on the results. Therefore, the ranked significance of the effect of each factor on yield was A > D > B > C, and that on viability was A > D > C > B. The results presented above showed that the protoplast yield and viability were primarily affected by cellulase and ligninase and secondarily affected by macerozyme and pectolyase. Ta

ble

4. Y

ield

and

via

bilit

y of

pro

topl

ast i

n O

A9 m

atrix

.

Tria

l no.

Fact

ors

Resu

lts

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ZHOU et al. / Turk J Agric For

To further analyze the results and determine the optimal enzyme concentrations, ANOVA and multiple comparison analysis were conducted. The results of ANOVA in Table 6 indicate that each of the four enzymes had an extremely significant effect on protoplast yield and viability. In other words, there were no unnecessary enzymes used in the optimization process. Multiple comparison analysis can reveal significant differences between the three levels of each factor. The results of these comparisons (Table 7) showed that the optimal enzyme combination for the protoplast yield was A2B1C3D2. The best combination for protoplast viability was A2B1C1D1. These results are the same as those from the range analysis. In both the range analysis and ANOVA results, cellulase concentration level 2 and macerozyme concentration level 1 were the best choices for both protoplast yield and viability. Levels 1 and 3 had almost the same influence on yield and viability. Based on the economic principles and causing less damage to protoplasts, pectolyase had the weakest effect on viability. The optimal level of pectolyase was level 1 (0.2%). For ligninase concentration, level 1 had a strong negative effect on yield but not on viability; thus, the optimal level of ligninase was level 1 (1.0%).

In sum, combining economic principles and the optimization results, the optimal combination for enzyme digestion was as follows: 1.5% Cellulase R-10, 0.4% Macerozyme R-10, 0.4% Pectolyase Y-23, and 1.0% ligninase (A2B1C2D1). This optimal combination was used for subsequent experiments.3.2. Optimization of osmotic stabilizer concentration, scale leaf treatment method, pH, and incubation timeTo optimize the other factors affecting protoplast isolation, single-variable experiments were conducted under the

optimal enzyme combination and concentrations. The concentration of the osmotic stabilizer was between 0.55 M and 0.80 M (Figure 1A). When the osmotic stabilizer was at 0.70 M, the yield and viability of protoplasts both clearly reached their highest peaks. Five different pretreatment methods of scale leaves before enzyme incubation were tested (Figure 1B). Mechanical homogenization clearly produced the maximum yield. For protoplast viability, the method of tearing scale leaves caused the least damage to the protoplast cells. However, considering both the yield (9.4 × 106 g FW–1) and viability (60.4%) results, the gentle mechanical homogenization method seemed to be the best way to pretreat the leaf tissue before enzyme digestion.

To better investigate the effect of pH, a single-variable experiment was designed under the optimal conditions above, and the result is shown in Figure 1C. The variation in yield and viability show volcano-type relationships with pH. During the promotion stage, ranging from pH 5.0 to 5.8, an increase in pH is beneficial to protoplast yield and viability. Yield and viability can be up to 8.7 × 106 g FW–1 and 73.8% when the pH is 5.8. At higher pH values, both yield and viability decreased sharply.

For incubation times of 0 to 22 h, yield first increased with increasing incubation time and then decreased after 18 h of incubation. In contrast, viability first decreased, then increased slowly, and finally decreased after 16 h of incubation. To obtain as many viable protoplasts as possible, 16 h of incubation should be used, as this incubation time produced the highest yield (4.8 × 106 g FW–1) and viability (82.5%).

Therefore, the optimal enzymatic digestion conditions were 0.7 M mannitol, gentle mechanical homogenization of the leaf tissue, pH 5.8, and 16 h of incubation time.

Table 5. Range analysis data of the protoplast yield and viability.

Results grouping Value name Cellulase R-10 concentration A

Macerozyme R-10 concentration B

Pectolyase Y-23 concentration C

Ligninase concentration D

Yield (g FW–1)

1.00 × 104 6..87 × 104 4.99 × 104 3.96 × 104

1.23 × 105 4.47 × 104 5.13 × 104 8.27 × 104

3.22 × 104 5.19 × 104 6.40 × 104 4.29 × 104

Ry 1.13 × 105 2.41 × 104 1.41 × 104 4.31 × 104

Viability (%)

25.8 40.6 42.7 43.345.8 35.0 33.9 35.838.4 34.4 33.4 30.9

Rv 19.9 6.19 9.2 12.4

, , are the mean values of protoplast yield at corresponding enzyme concentration levels 1, 2, and 3, respectively. Ry is the range of , , . Likewise, , , are the mean values of protoplast viability at corresponding enzyme concentration levels 1, 2, and 3, respectively. Rv is the range of , , .

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3.3. Ancient P. orientalis mesophyll protoplast isolation and purification Based on the above optimization of each factor involved in protoplast isolation, a diagram is shown in Figure 2. The purification process requires being repeated twice,

containing two centrifugations at a low speed (100 × g for 3 min) and a high speed (500 × g for 5 min).The growth conditions of ancient P. orientalis were quite good even though the tree is more than 2000 years old (Figure 3A). An annual young scale leaf is shown in

Table 6. Analysis of variance (ANOVA) of protoplast yield and viability in the OA9 matrix.a,b

Results grouping Source SS df Mean square F Fa Sig.Yield Corrected model 7.88 × 1010 c 8 9.85 × 109 627.3** F0.05(8, 18) = 2.5 0.000

A 6.46 × 1010 2 3.23 × 1010 2057.7** F0.05(2, 18) = 3.55 0.000B 2.75 × 109 2 1.37 × 109 87.5** F0.01(8, 18) = 3.71 0.000C 1.03 × 1010 2 5.17 × 109 329.5** F0.01(2, 18) = 6.01 0.000D 1.09 × 109 2 5.43 × 108 34.6** 0.000Error 2.83 × 108 18 15.57 × 107

Total 1.61 × 1011 27Viability Corrected model 3231.1d 8 403.9 35.5** 0.000

A 1831.0 2 915.5 80.6** 0.000B 212.4 2 106.2 9.3** 0.002C 700.6 2 350.3 30.8** 0.000D 486.9 2 243.5 21.4** 0.000Error 204.6 18 11.4Total 39757.7 27

a Tests of between-subjects effects. b Dependent variable: yield, viability. c R-squared = 0.996 (adjusted R-square = 0.995). d R-squared = 0.996 (adjusted R-square = 0.995). SS: Sum of squares of deviation from mean. F: Ratio of the sum of the square of each factor’s mean deviations to that of the experimental error. Fa: Critical value of F-value for different inspection levels. **: Extremely significant (P < 0.01).

Table 7. Multiple comparisons of the levels of each factor in the protoplast isolation test.

Results grouping FactorsLevels1 2 3

Yield Cellulase R-10 concentration A c a bMacerozyme R-10 concentration B a c bPectolyase Y-23 concentration C b b aLigninase concentration D b a b

Viability Cellulase R-10 concentration A c a bMacerozyme R-10 concentration B a b bPectolyase Y-23 concentration C a b bLigninase concentration D a b c

Confidence interval: 99%. Different lowercase letters for the same enzyme denote the least significant difference by Duncan’s test at P < 0.01 comparing the data at different concentration levels.

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Figure 3B. Two main scales and two lateral scales that were connected to the main stem in opposite directions are enlarged in the inset of Figure 3B. The leaf mesophyll protoplasts showed uniform spheroidicity, had intact cell membranes, and contained many green chloroplasts (Figure 3C–3E). Protoplast viability was tested by the fluorescent dye FDA and the protoplast in Figure 3F exhibited green fluorescence. At the same time, the chloroplast contained in the protoplast exhibited red fluorescence under 488-nm excitation light; thus, the overall fluorescence of the mesophyll protoplast was yellow-green. The fluorescence in Figure 3F indicated that the mesophyll protoplast had good viability.3.4. FCM and data analysis of P. orientalis mesophyll protoplastsA dot plot of mesophyll protoplasts obtained through FCM is shown in Figure 4A. To avoid the analysis of cell

debris and adhesion cells, the gate was determined by the forward scatter channel (FSC) and the side scatter channel (SSC). Cells in the gate were intact single protoplasts. Only the fluorescence of single cells was recorded and analyzed. In Figures 4B and 4C, the cell count is represented on the ordinate axis and the FDA fluorescence intensity is represented on the abscissa axis. The median of the fluorescence of the stained and unstained protoplasts was used for data analysis, and the results are shown in Figure 4D. Based on measurements of the fluorescence of FDA (green light, 530-nm detection channel), protoplasts with and without FDA staining were significantly different. The stained protoplasts exhibited a strong green fluorescence, while the unstained ones produced only a faint fluorescence. The fluorescence of unstained protoplasts was mainly caused by debris and organelles. The FCM data analysis indicated that the mesophyll protoplasts isolated from ancient trees had high viability.

0.55 0.60 0.65 0.70 0.75 0.800.000.05

5

10

15

20

25

30B

Osmotic stabilizer concentration (M)

Yiel

d (x

104 g

FW

-1)

A

0

20

40

60

80

100

Via

bilit

y (%

)

Blade shredding

Scissors shears

Mechanical homogenate

Hand lapping

Tearing scale leaves

0.000.010.02

0.60.890

100

110

120 Yield Viability

Scale leaves treatment

Yiel

d (x

105 g

FW

-1)

0

20

40

60

80

100

5.0 5.2 5.4 5.6 5.8 6.00

20

40

60

80

100

pH

Yiel

d (x

105

g FW

-1)

C

0

20

40

60

80

100 Yield Viability

Via

bilit

y (%

) V

iabi

lity

(%)

Via

bilit

y (%

)

0 2 4 6 8 10 12 14 16 18 20 220.00.20.4

10

60

90

120

150

Yield Viability

Yield Viability

Incubation time (h)

Yiel

d (x

105 g

FW

-1)

D

0

20

40

60

80

100

Figure 1. The effects of osmotic stabilizer concentration, scale leaf treatment method, pH, and incubation time on protoplast yield and viability. Relationships between the protoplast yield or viability and (A) concentration of the osmotic stabilizer mannitol, (B) scale leaf treatment method used before enzyme digestion, (C) pH, and (D) incubation time for enzyme digestion. The enzyme digestion conditions were as follows: (A) mechanical homogenization method, pH 5.6, time = 8 h; (B) osmotic stabilizer concentration = 0.7 M, pH 5.6, time = 8 h. All four experimental groups were subjected to the enzyme combination of 1.5% Cellulase R-10, 0.4% Macerozyme R-10, 0.4% Pectolyase Y-23, and 1.0% ligninase.

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4. DiscussionThe plants’ cell wall composition and cell activity changed in different growth stages. The developmental stage of the leaf had a major effect on the yield, viability, and mitotic activity of protoplasts (Frearson et al., 1973). In this study, young annual-growth leaves were selected as the original

material for protoplast isolation. Because of the unique characteristics of the scale leaves of P. orientalis, especially the main stem surrounded by scale leaves and the resin compounds contained in the cells, special attention must be given to the pretreatment of leaf tissue before incubation and to the purification of raw suspension of

clotsclots

Figure 2. The diagram of mesophyll protoplast isolation and purification technique of ancient P. orientalis protoplasts. The enzyme digestion conditions were as follows: gentle mechanical homogenate pretreatment of the leaf tissue; the enzyme digested in a mixed CPW buffer contained 1.5% Cellulase R-10, 0.4% Macerozyme R-10, 0.4% Pectolyase Y-23, 1.0% ligninase, and 0.7 M mannitol at pH 5.8; incubated 16 h before purification.

Figure 3. Leaf structure of ancient P. orientalis and microscopic observation of mesophyll protoplasts. Image of ancient P. orientalis (A) and its annual scale leaves (B), with the image in the inset in (B) showing an enlargement of the scale leaf. (C) and (D) are different mesophyll protoplasts under a light microscope. (E) is a bright-field image of a protoplast; (F) is a viability test of the protoplast in (E) stained with FDA. (G) is a merged image of (E) and (F). Scale bars in A, B, and C–G are 1 m, 1 cm, and 10 µm, respectively.

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enzyme-digested protoplasts. Compared to the other tested methods, gentle mechanical homogenization was found to be an effective and convenient pretreatment for exposing mesophyll to the enzyme solution.

The most influential factors that affect the isolation of protoplasts are enzyme type and concentration (Wenck and Márton, 1995). Tests should be conducted to determine the type and concentration of suitable enzymes when a different material is used. The suitable combination of enzymes for protoplast isolation can be chosen as that which damages the exposed protoplasts the least (Evans and Bravo, 1983).

The appropriate stabilizer concentration, enzymatic incubation time, and pH for protoplast isolation may be

related to the type of original plant material and to the types and concentrations of enzymes. The concentration for conifers was 0.4–0.9 M mannitol (Winton et al., 1975). In this experiment, we added 0.8 M mannitol as an osmotic stabilizer. The yield and viability of protoplasts were greatly influenced by the time of enzymatic digestion. The protoplasts of Pseudotsuga menziesii cotyledons were incubated for only 3.5 h (Kirby, 1980), but the protoplasts of young leaves of P. orientalis should undergo 16 h of enzymatic incubation. Usually, during the process of enzyme digestion for protoplast isolation, the mixed-enzyme solution contains different types and concentrations of enzymes, and the optimal pH of these enzymes is often unknown. For example, the optimum pH

Figure 4. P. orientalis mesophyll protoplasts analyzed by FCM. (A) FSC-SSC plot of mesophyll protoplasts, with the black circle in the plot showing the gate of FCM analysis; 20,000 cells were analyzed; (B) FDA fluorescence intensity plot of unstained protoplasts; (C) FDA fluorescence intensity plot of FDA-stained protoplasts; (D) FDA fluorescence comparison by data analysis, with ** indicating an extremely significant difference (P < 0.01).

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of cellulase is generally 4.5–6.5, while the optimum pH of pectinase is 3.0 (Duquenne et al., 2007). Based on the yield and viability of isolated protoplasts, the optimal pH for the protocol used in this study is 5.8.

FCM, which requires individualized or single-cell preparations, is particularly amenable to studies of protoplast biology (Bergounioux et al., 2010). However, all the follow-up high-flux studies are based on the efficient isolation of protoplasts. In the present study, large numbers of viable protoplasts were obtained from the young leaves of ancient P. orientalis. The membrane integrity of isolated protoplasts was verified by the accumulation of FDA. FCM was used to analyze the total fluorescence of protoplasts, which indicated that a large number of protoplasts were viable. The data can also be used to compare the viability differences of large groups of specific types of protoplasts.

The viable protoplasts isolated from the ancient trees provide the possibility for cell or seedling regeneration of valuable ancient trees. These cells or seedlings form a specific aging-related cell system for transient gene expression. In addition, the FCM detection system of protoplasts provides technical support for the detection and sorting of specific groups of protoplasts when combined with different types of fluorescent labeling.

Further research may focus on the rapid analysis of aging-related cellular characteristics. These characteristics may reveal tree aging mechanisms at the cellular or organelle level.

This study established an efficient technique for isolating mesophyll protoplasts from the young scale leaves of ancient P. orientalis. It may provide some experimental basis for isolating the protoplasts of other Cupressaceae species, and it provides the opportunity for further protoplast-based research on cell sorting and fluorescence labeling in the future.

AcknowledgmentsThis work was financially supported by the National Forestry Industry Research Special Funds for Public Welfare Projects (China) (201404302). It is highly appreciated that the Key Laboratory of Agriculture in Arid Areas of Northwest A&F University (China, Shaanxi, Yangling) and the Key Comprehensive Laboratory of Forestry of Northwest A&F University (China, Shaanxi, Yangling) provided the experiment platform for us. We are also grateful to the staff of the management department of the Mausoleum of Emperor Huang and the Forestry Bureau of Huangling County.

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