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Preparation and application of Dahlia protoplast

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Dahlia ( Dahlia sp. ), a species of the Asteraceae family, is widely cultivated in China and exhibits seasonal blooming. The main challenges associated with this species are low cross-breeding efficiency and slow breeding. In this study, dahlia leaves and petals were employed as test materials to ascertain the optimal conditions for protoplast isolation, to refine the critical factors for transient transformation, and to develop a system for the isolation, purification and utilisation of dahlia protoplasts. The best procedure for isolation of dahlia leaves protoplasts was 4°C dark pretreatment for 12 h + 1.0% cellulase + 0.5% macerozyme + 0.4% pectinase + enzyme digestion time for 4 h + 0.8 mol/L mannitol, with a maximum yield of 6.13 × 10 ⁶ protoplasts/mL and a maximum viability of 89.23%; and the best procedure for dahlia petal protoplasts was 1.0% cellulase + 0.5% macerozyme + 0.4% pectinase + enzyme digestion time 10 h + 1.0 mol/L mannitol, with a maximum yield of 5.46 × 10 ⁶ protoplasts/mL and a maximum viability of 88.83%. The pGBin-EGFP vector was used to assess transient transformation rates in leaves and petals protoplasts. The rates exhibited considerable variation across the samples, with values ranging from 32.57–60.67%. The optimal conditions for gene transfer in dahlia protoplast were identified as 50 ng/µL plasmid, 20% PEG, and a 20-minute transformation time.
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Preparation and application of Dahlia protoplast
Jing Zhou
Yunnan University
Dong Yang
Yunnan University
NingNing Zhou
Yunnan University
YangBo Tian
Yunnan University
Zhen Tian
Yunnan University
Qing Duan
Yunnan Academy of Agricultural Sciences
Mohamed A.A. Ahmed
Alexandria University Faculty of Agriculture
LiHua Wang
Yunnan Academy of Agricultural Sciences
Xuewei Wu
Yunnan University https://orcid.org/0000-0002-8560-1248
Research Article
Keywords: Dahlia sp. Protoplast preparation. PEG. Transient transformation. FDA
Posted Date: August 30th, 2024
DOI: https://doi.org/10.21203/rs.3.rs-4802700/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License. 
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Preparation and application of Dahlia protoplast 1
Ji ng Z ho ua , *, Dong Yanga,*, N ingNing Zhou a, Ya ng Bo Tia na, Zhen Tiana, Q in g Duan b, 2
Mo ha me d A. A. A hm ed a , c LiHua Wang b,* *, XueWei Wu a,** 3
a Agricultural College of Yunnan University, Kunming, 650504, China. 4
b Flower Research Institute, Yunnan Academy of Agricultural Sciences, Kunming, 650205, China. 5
c Plant Production Department (Horticulture-Medicinal and Aromatic Plants), Faculty of 6
Agriculture (Saba Basha), Alexandria University, Alexandria 21531, Egypt. 7
* Both authors contributed equally to the manuscript 8
(**) Corresponding author: wuxuewei@ynu.edu.cn (X.W. Wu); wanglihua2525@outlook.com (L.H. 9
Wang) 10
11
Abstract 12
Dahlia (Dahlia sp.), a species of the Asteraceae family, is widely cultivated in China and exhibits 13
seasonal blooming. The main challenges associated with this species are low cross-breeding efficiency 14
and slow breeding. In this study, dahlia leaves and petals were employed as test materials to ascertain the 15
optimal conditions for protoplast isolation, to refine the critical factors for transient transformation, and 16
to develop a system for the isolation, purification and utilisation of dahlia protoplasts. The best procedure 17
for isolation of dahlia leaves protoplasts was 4°C dark pretreatment for 12 h + 1.0% cellulase + 0.5% 18
macerozyme + 0.4% pectinase + enzyme digestion time for 4 h + 0.8 mol/L mannitol, with a maximum 19
yield of 6.13 × 106 protoplasts/mL and a maximum viability of 89.23%; and the best procedure for dahlia 20
petal protoplasts was 1.0% cellulase + 0.5% macerozyme + 0.4% pectinase + enzyme digestion time 10 h 21
+ 1.0 mol/L mannitol, with a maximum yield of 5.46 × 106 protoplasts/mL and a maximum viability of 22
88.83%. The pGBin-EGFP vector was used to assess transient transformation rates in leaves and petals 23
protoplasts. The rates exhibited considerable variation across the samples, with values ranging from 24
32.57% to 60.67%. The optimal conditions for gene transfer in dahlia protoplast were identified as 50 25
ng/µL plasmid, 20% PEG, and a 20-minute transformation time. 26
Key words: Dahlia sp . Protoplast preparation . PEG . Transient transformation. FDA 27
1. Introduction
28
Dahlia (Dahlia sp.) is a perennial bulbous flower of the dahlia genus of the Asteraceae family 29
(Compositae). It has its origins in Mexico, South America, and Colombia, and is most commonly found 30
at altitudes above 1500m in the plateau area. It has been demonstrated that it has the functions of 31
lowering blood lipids, lowering blood glucose, and promoting the absorption of minerals (Zhou et al. 32
2016). 33
Plants have a remarkable reprogramming potential, which facilitates plant regeneration, 34
especially from a single cell. Protoplasts have the ability to form a cell wall and undergo cell division, 35
allowing whole plant regeneration(Jeong et al. 2021). Plant leaves are the optimal material for 36
protoplast isolation due to their high cellularity. In their study of lily leaves, Esmaeil et al. (2012a) 37
identified the optimal isolation conditions as enzymatic digestion for 24 hours, using 4% cellulase and 1% 38
pectinase. Zhao (2009) demonstrated that sterile seedling leaves of the edible chrysanthemum cultivar 39
'Yinlongshu' were more suitable for protoplast isolation. The utilisation of oil tea petals for protoplast 40
isolation yielded remarkable results, with approximately 1.42 × 107 cells obtained for each gram of petals 41
and a cell viability of 89% (Lin et al. 2023). In their study, Pan et al. (2022) demonstrated that a higher 42
yield of petal protoplast could be obtained by enzymatic digestion for a period of six hours at a 43
concentration of 1.5% cellulase and 0.4% macerozyme. 44
To date, researchers have successfully established an efficient protoplast isolation system for a 45
variety of ornamental plants. These plants include Phalaenopsis (Xia et al. 2022), chrysanthemum (Zhou 46
et al. 2005), petunia (Kang et al. 2020), and others. The most commonly used enzyme-liquid 47
combinations for plant protoplast isolation include the combination of cellulase and pectinase or 48
cellulase and macerozyme. The researchers aimed to optimize the isolation of protoplasts from 49
chrysanthemums by regulating the levels of mannitol and cellulase, the incubation time, and the 50
purification method, leading to the efficient transformation of callus and buds (Adedeji et al. 2020). The 51
leaves of pearl bud konjac were enzymatically digested at 30°C for 4 h in the dark, resulting in the 52
production of high-quality protoplasts (Wang 2023). The combination of an enzyme solution comprising 53
1.0% cellulase R10 and 0.7% macerozyme R10 yielded the highest yield of protoplasts with the least 54
cellular debris, with an average of 5.944 × 10⁶ protoplasts/g, after 6 h of enzymatic digestion (Li 2019). 55
Mannitol and sorbitol are frequently employed as osmotic pressure stabilisers during protoplast isolation. 56
To ensure the stability and viability of protoplast membranes, the isolation process typically involves the 57
addition of calcium chloride (CaCl₂), potassium dihydrogen phosphate (KH₂PO₄), 58
2-(N-morpholino)ethanesulfonic acid (MES), potassium chloride (KCl), and bovine serum albumin 59
(BSA). 60
In terms of research, He et al. (2024) found that the concentration of mannitol contributed most to 61
the viability of pineapple leaves-based protoplasts, as analysed by a three-factor, three-level orthogonal 62
test. At a mannitol concentration of 0.6 mol/L, the yield of pineapple leaves-based protoplasts reached 63
1.96 × 106 protoplasts/g with an 85.54% viability. The isolation of protoplasts from the perennial betel 64
palm revealed that the concentration of mannitol (optimum: 0.7 M) plays a pivotal role in the isolation of 65
betel protoplasts(Wang et al. 2023). 66
The purification of protoplast can be achieved through a number of methods, including filtration, 67
centrifugation, suspension, sedimentation, and interfacial techniques. These methods used the differing 68
specific gravities of protoplast and impurities in a mixed solution to facilitate stratification. In recent 69
years, the technology of transient transformation of protoplasts has been used extensively in the research 70
of garden ornamental plants. The protoplast transient transformation system was successfully 71
constructed and the technology was successfully applied in chrysanthemum (Ding et al. 2023), zoysia 72
(Liu et al. 2023), jasmine (Zhang et al. 2019a), moso bamboo (Ye 2022) among other plants. 73
2. Materials and methods
74
2.1 Plant material 75
The petals and leaves of 'D. imperialis', and the petals of 'Black Beauty' and 'Lotus Fairy' were used 76
as the materials for the experiments, respectively. Protoplasts of leaves were prepared from fully 77
expanded young leaves (1 × 3 cm) of 'D. imperialis'. Protoplasts of petals were prepared from peripheral 78
ligulate petals (2 × 5 cm) and intermediate petals (1.5 × 3 cm) at the beginning of the first flowering stage 79
of 'Black Beauty' and 'Lotus Fairy'. 80
2.2 Protoplast isolation
81
The material treatment methods of Arabidopsis thaliana (Yoo et al. 2007) and jasmine (Zhang et al. 82
2019b) were employed in the treatment of dahlia petals and leaves, respectively. The crushing, shredding, 83
and cutting methods were utilised, with fresh dahlia leaves and petals subsequently weighed according to 84
the ratio of plant material: enzyme solution 1:10. For shredding, newly expanded true leaves were cut 85
into 1 mm strips using a surgical blade along the direction parallel to the midvein. Chunking method, the 86
leaves and petals are cut into 5 mm squares with a razor blade. Crushing method, two blades are used to 87
cut the material simultaneously to make it as small as possible, and 2-3 drops of sterile water are added to 88
the petri dish to avoid cell inactivation. 89
The treated experimental materials were loaded into 60 mm disposable cell culture dishes, and 5 mL 90
of protoplast enzymatic solution was added. The dishes were then sealed with a sealing film and placed 91
in a vacuum drying oven to evacuate for 30 minutes. The plant tissues were enzymatically digested by 92
low-speed shaking (30 r/min) at room temperature under dark conditions. The enzymatic solution 93
contained 0.2 mol/L MES (pH 5.8), 1.0 mol/L CaCl2, 0.2 mol/L KCl, 1.0 mol/L NaH2PO4, 0.1% BSA. 94
Mannitol concentration were 0.4, 0.6, 0.8, 1.0 and 1.2 mol/L to optimize the protoplast isolation and 95
enzyme digestion time for 2, 4, 6, 8, 10, 12 h in the enzyme solution. Based on the optimal treatment, 96
mannitol concentration and digestion time, a three-factor, three-level orthogonal experimental design 97
was set up for the three enzyme reagents, with nine different combinations of cellulase (1.0%, 1.5%, 98
2.0%), macerozyme (0.5%, 0.75%, 1.0%), and pectinase (0.4%, 0.6%, 0.8%). The aforementioned 99
solutions should be prepared in advance with sterile water and filtered through 0.45 μm disposable 100
sterilised filters. 101
2.3 Purification method of the protoplast
102
The protoplast wash solution W5 and protoplast suspension solution K3 were prepared in advance. 103
At the conclusion of the enzymatic digestion, the enzyme solution was filtered twice with 100 µm and 70 104
µm sterile cell filters and collected into 50 mL sterile centrifuge tubes. The centrifuge tube and cell 105
strainer were lubricated with 5 mL of W5 solution, and the residual protoplast cells were collected into a 106
50 mL centrifuge tube, gently mixed, and then centrifuged at 4°C for 5 minutes at 2000r/min. The 107
supernatant was discarded, and the precipitate was collected. A volume of 5 mL of W5 solution should be 108
added carefully along the wall of the tube to a 50 mL centrifuge tube, after which the protoplast cells 109
should be resuspended. The solution should then be centrifuged at 4°C for 5 minutes, after which the 110
supernatant should be discarded and the precipitate taken. The precipitate should then be resuspended in 111
2 mL of W5 solution. A new 10 mL round-bottomed clear centrifuge tube should be taken, 2 mL of 112
protoplast purification solution(13% sucrose solution and K3 solution) added along the wall of the tube, 113
and the resuspended protoplast cells carefully transferred to the centrifuge tube containing the protoplast 114
purification solution. Centrifugation should be repeated for 8 min. The protoplast suspension in the 115
middle layer was collected and transferred to a new centrifuge tube, which was then set aside on ice. 116
Plant cells were counted with a haemocytometer under the microscope. The protoplast yield was 117
calculated with the following formula: 118
Cell concentration (cells / mL)=dilution ratio×
 119
The activity size of protoplasts was determined by fluorescent diacetate FDA staining, viable pro- 120
toplasts absorb FDA dye and emit bright-green fuorescence, while non-viable cells do not. FDA was 121
added to the proto- plast solution at 0.01% and then incubated in darkness for 10 min at room 122
temperature. The protoplast viability was calculated as follows: 123
Protoplasts activity (%) = 
 00 124
2.4 Transient transformation of the protoplast
125
The PEG-mediated method was employed for the transient transformation of protoplasts. The 126
transformation plasmid was pGBin-EGFP, which was to be amplified by E. coli and subsequently 127
subjected to PCR to ascertain the purity and concentration of the plasmid. Thereafter, the plasmid was to 128
be extracted and stored at -20°C for subsequent use. The protoplast density was adjusted to a range of 1.0 129
× 10⁵ to 1.0 × 10⁶ protoplasts per millilitre using a solution of mannitol, magnesium chloride and glucose 130
(MMG). For each transformation, 10 µL of various plasmid DNA concentrations and 100 µL of 131
protoplast solution were combined in a 2 mL centrifuge tube. Subsequently, 110 µL of a freshly prepared 132
PEG-CaCl₂ solution was added and gently mixed. Subsequently, the reaction was terminated at the 133
conclusion of the transformation by the addition of two times the volume of W5 solution. This was 134
followed by gentle flicking of the tube to ensure thorough mixing. The reaction was then centrifuged at 135
1000 r/min for two minutes, after which the supernatant was discarded. The protoplast solution was 136
resuspended with W1 solution, which contained 20 mmol/L KCl, 0.5 mol/L mannitol, 4 mmol/L MES, 137
pH 5.7, and incubated in a dark incubator at 22°C for approximately 12 hours. The transformation 138
efficiency was evaluated by observing the luminescence of green fluorescent protein (GFP) by laser 139
confocal microscopy. Successful transformation of the protoplasts would result in the display of green 140
fluorescence with excitation and emission wavelengths of 488 nm and 505-535 nm, respectively. The 141
formula is as follows: 142
Protoplasts transformation efficiency(%)= 
 00 143
2.5 Histological observations of plant materials
144
In order to investigate the reasons for the differences in the preparation of protoplasts from different 145
source materials of dahlias, we chose young leaves and the middle part of petals for our study. Fix plant 146
tissues in penicillin vials, add FAA fixative and then vacuum dry for more than 30 minutes until the plant 147
material stops settling. Place in a refrigerator at 4°C for more than 24 hours of fixation. Remove the 148
fixative and wash twice with 50% anhydrous ethanol solution, then use 50%, 70%, 85%, 95% anhydrous 149
ethanol in sequence to dehydrate for 1 hour each, and finally 100% anhydrous ethanol for 30 minutes. 150
Transparent treatment using different ratios of anhydrous ethanol and xylene mixture for 1 hour each in 151
the ratio of 2:1, 1:1, 2:1 in turn. Followed by transparent treatment with xylene solution for two times in 152
the ratio of 30 minutes and 15 minutes respectively. Penicillin vials were filled with paraffin shavings, 153
treated in an oven to evaporate xylene and then dipped in a mixture of paraffin and beeswax, with several 154
changes of wax to ensure complete saturation of the material. Put the wax liquid in a box made from tin 155
foil. Add the penicillin vial material and liquid to the box. Move the material around, then cool in ice. 156
Remove the tin foil, cut the wax block into trapezoids and stored at 4°C were frozen for at least 20 157
minutes, removed and melted at the bottom. They were then glued to a small wooden block. Cut the wax 158
into 5 μm slices with a Leica RM2255 and Leica 818 blade to make sure the slices are not curled and the 159
material is intact. The cut sheets are spread in a spreader and then dried in a dryer until completely dry. 160
The wax discs were sequentially dewaxed, rehydrated, stained (sapphire red and solid green stains) and 161
finally sealed with a mixture of Canada gum and xylene. Finally, the histomorphological characteristics 162
of the plant material were observed under the microscope and photographed. 163
2.6 Statistical analysis
164
All experiments were replicated three times, and the results are the mean ± standard deviation of the 165
three replications. All statistical analyses of data were performed using SPSS Statistics 25 at the P < 0.05 166
level, and image processing was performed using Origin 2022, Adobe Photoshop CC 2019 and ZEN 167
software. 168
3. Results
169
3.1 Preparation of dahlia leaves protoplasts
170
Leaves were selected as the isolated starting material for the preparation of dahlia protoplast. Fresh 171
dahlia leaves were treated by the shredding, blocking, and crushing methods, respectively, before 172
enzymatic digestion. The amount of enzyme solution was uniformly set at 5 mL, and the enzymatic 173
digestion time was set at 4 h. The results indicate that the shredding method yielded the highest 174
protoplast yield, with a value of 3.25 × 10⁵ protoplasts/mL, which was 2-8 times higher than that of the 175
blocking and crushing methods (Fig. 1a). The block-cutting method yielded the second-highest 176
protoplast yield, at approximately 1.20 × 10⁵ protoplasts/mL, while the crushing method yielded the 177
lowest yield, at only 0.42 × 10⁵ protoplasts/mL. With regard to protoplast viability, the shredding method 178
yielded 70.00% viability, the blocking method yielded 64.25%, and the crushing method yielded only 179
25.00%. Accordingly, the subsequent experiments were conducted using the filament cutting method for 180
the isolation of protoplasts from dahlia leaves. 181
The study was conducted under otherwise constant conditions, with enzyme digestion times ranging 182
from 2 to 10 hours. The results demonstrated that the yield and viability of protoplasts exhibited a 183
tendency to increase and then decrease with increasing enzyme digestion time. The maximum yield of 184
8.75 × 10⁵ protoplasts per millilitre and viability of 89.23% were achieved at 4 hours. The isolation effect 185
at 6 hours was similar to that at 4 hours, but then the yield and viability decreased sharply ( Fig. 1b). 186
Consequently, the optimal duration for enzymatic digestion of dahlia leaves protoplasts was identified as 187
4 hours, which yielded the most favourable isolation outcomes. 188
189
Fig. 1 Optimization of dahlia leaves protoplast yield and viability. a Effect of treatments on protoplast production in 190
dahlia leaves. b Effect of enzyme digestion time on the separation of protoplast from dahlia leaves 191
The viability of the dahlia leaves protoplasts was found to exceed 80% at enzyme digestion times of 192
less than 6 hours (Fig. 2). The morphology of the protoplasts was complete and almost round, and they 193
exhibited a strong green fluorescence after FDA staining. 194
195
Fig. 2 FDA staining of dahlia leaves protoplast 196
Note: A - C and D - F are the field of view of leaves protoplast under 20x objective and 10x objective, respectively; A 197
and D are bright-field; B and E are fluorescence-field; and C and F are superimposed-field. Bar= 100 µm. 198
The optimal enzyme digestion time (4 h) was used as a basis for the experiment, which was 199
conducted in accordance with the designed 3 factors, 3 levels and 9 treatment combinations. The results 200
of the experiment, which examined the effect of different enzyme combinations on the separation of 201
protoplasts from dahlia leaves, are presented in Table 1. The minimum leaves protoplast yield was 3.50 × 202
10⁵ protoplasts/mL, while the maximum was 2.28 × 10⁶ protoplasts/mL. The minimum viability was 203
69.72%, while the maximum was 87.01%. The results demonstrated that the optimal method for 204
protoplast separation from dahlia leaves was achieved through the use of a solution comprising 1.0% 205
cellulase, 0.5% macerozyme, and 0.4% pectinase. This approach yielded a protoplast concentration of 206
2.28 × 10⁶ protoplasts/mL, with a protoplast viability of 87.01%. 207
Table 1 Effect of enzyme combination on the isolation of protoplast from dahlia leaves 208
Number
Cellulase(%)
Macerozyme(%)
Pectolase(%)
Yield105
protoplasts/mL)
M1
1.00
0.50
0.40
22.83 ± 0.95 a
M2
1.00
0.75
0.60
8.58 ± 0.85 b
M3
1.00
1.00
0.80
9.17 ± 1.60 b
M4
1.50
0.50
0.60
8.75 ± 0.38 b
M5
1.50
0.75
0.80
7.25 ± 0.54 bc
M6
1.50
1.00
0.40
4.08 ± 0.30 d
M7
2.00
0.50
0.80
3.50 ± 0.37 d
M8
2.00
0.75
0.40
4.33 ± 0.36 d
M9
2.00
1.00
0.60
4.75 ± 0.84 cd
K1 yield
40.58
35.08
31.25
K2 yield
20.08
20.17
22.08
K3 yield
12.58
18.00
19.92
X1 yield
13.53
11.69
10.42
X2 yield
6.69
6.72
7.36
X3 yield
4.19
6.00
6.64
R yield
9.33
5.69
3.78
K1 vigour
249.05
239.54
239.89
K2 vigour
235. 14
228.77
243.04
K3 vigour
225.94
241.82
227.20
X1 vigour
83.02
79.85
79.96
X2 vigour
78.38
76.26
81.01
X3 vigour
75.31
80.61
75.73
R vigour
7.70
4.35
5.28
In the optimal enzymatic conditions (shredding method + enzymatic digestion for 4 h + 1.0% 209
cellulase + 0.5% macerozyme + 0.4% pectinase), the pretreatment (R1) by running water rinsing and 210
dark treatment at 4℃ increased the protoplast yield of dahlia leaves protoplasts to 6.13 × 106 211
protoplasts/mL with a 10.37% increase in viability compared with the untreated control (R0) (Table 2). 212
The isolated protoplasts exhibited a round, green morphology, closely resembling the surface coloration 213
of the leaves, and were observed to be rich in chloroplasts (Fig. 3). 214
215
Fig. 3 Pretreated dahlia leaves protoplast 216
Note: A. Leaves protoplast without pretreatment; B. Pretreated leaves protoplast; C. Pretreated leaves protoplast 217
under optical microscope; D. Pretreated leaves protoplast stained by FDA under fluorescence microscope. Bar = 100 µm. 218
Table 2 Effect of pre-treatment on the isolation of dahlia leaves protoplast 219
Number
Protoplast yield( × 105 protoplasts /mL)
Protoplast viability(%)
R0
19.58 ± 1.26 b
65.52 ± 0.98 b
R1
61.25 ± 2.70 a
75.89 ± 2.03 a
The impact of five distinct concentrations on the isolation of protoplasts from dahlia leaves was 220
examined utilising mannitol as an osmotic pressure regulator in accordance with optimal enzymatic 221
conditions. The results demonstrated that under 0.8 mol/L mannitol, the protoplast yield was 222
significantly higher than that of the other four groups, reaching 6.07 × 106 protoplasts/mL. In the range of 223
0.4 to 0.8 mol/L, the majority of the protoplasts exhibited a spherical morphology, with a few displaying 224
an ellipsoidal or elongated shape. At 1.0 mol/L mannitol, the spherical protoplasts accounted for 225
approximately half of the total number of cells, while the remainder exhibited oval, elongated, or 226
irregular shapes (Fig. 4). The yield and viability were significantly reduced at 1.2 mol/L mannitol, with a 227
yield of 10.42 × 10⁵ protoplasts/mL and a viability of 57.38%, respectively. Consequently, the 0.8 mol/L 228
mannitol concentration was identified as the optimal osmotic pressure concentration for protoplast 229
isolation from dahlia leaves. 230
231
Fig. 4 Dahlia leaves protoplast at different mannitol concentrations 232
Note: A. 0.4 mol/LD - mannitol; B. 0.6 mol/LD - mannitol; C. 0.6 mol/LD - mannitol; D. 0.8 mol/LD - mannitol; E. 1.2 233
mol/LD - mannitol. Bar = 100 µm. 234
3.2 Preparation of dahlia petals protoplasts
235
Tissue isolation of Dahlia petals was conducted and observed under a microscope, resulting in the 236
classification of protoplast cells extracted from dahlia petals into four species (Fig. 5), all of which were 237
basically spherical or ellipsoidal, and the colour was transparent, brown, pinkish-purple, and light pink, 238
with the pinkish-purple and light-pink cells accounting for approximately 80% of the cells (Fig. 6). 239
240
Fig. 5 Petals protoplast of different morphologies
241
Note: A. Clear-coloured globular petals protoplast; B. Brown globular petals protoplast; C. Pink-purple globular 242
petals protoplast; D. Light pink globular petals protoplast. Bar = 50 µm. 243
244
Fig. 6 'D. imperialis' petals and isolated protoplast 245
Note: A. Flowers of 'D. imperialis'; B. Petals of 'D. imperialis'; C. Protoplast isolated from petals of 'D. imperialis'. 246
Bar= 100 µm. 247
Following the treatment of dahlia petal tissues by shredding, blocking, and crushing methods, the 248
number of protoplasts and their viability were counted after 4 h of enzyme digestion. The shredding 249
method yielded the highest number of protoplasts, at 3.67 × 10⁵ per millilitre, which was 2 to 11 times 250
higher than that of the crushing and blocking methods(Fig. 7a). The viability of dahlia protoplasts was 251
found to be greater than 80%, although the yield was lower at 2 and 4 hours. The viability was 252
approximately 65% at 8 and 10 hours. The yield increased by 9.73 × 10⁵ protoplasts/mL at 10 h compared 253
to 8 h, indicating that 10 h was the optimal digestion time. The digestion time significantly affected both 254
the yield and viability (P < 0.01)(Fig. 7b). 255
256
Fig. 7 Optimization of dahlia petals protoplast yield and viability. a Effect of treatments on the separation of dahlia 257
petals protoplast. b Effect of enzyme digestion time on the separation of protoplast from dahlia petals. 258
Mannitol was used as a regulatory agent for osmotic pressure in the study, with the objective of 259
investigating its impact on the extraction of protoplasts from dahlia petals within a concentration range of 260
0.4 to 1.2 mol/L. The results demonstrated that the yield of protoplasts increased with the increase of 261
mannitol concentration from 0.4 to 1.0 mol/L. Conversely, the yield was lower at lower than 1.0 mol/L, 262
reaching approximately 105 protoplasts/mL. The yield reached a maximum value of 2.78 × 10⁶ 263
protoplasts/mL at 1.0 mol/L, after which the concentration was increased to 1.2 mol/L. At this point, the 264
protoplasts began to crumple, resulting in a drop in yield to 1.2 mol/L due to the high osmotic pressure. In 265
comparison to the yield, the viability of protoplasts exhibited a fluctuating range of 75% to 85%. This 266
indicated that the concentration of mannitol had a minimal impact on the viability. 267
Based on the optimal enzyme digestion time (10 h), experiments were conducted using the designed 268
3-factor, 3-level and 9-treatment combinations. The results of the effect of different enzyme solution 269
combinations on the isolation of petal protoplasts of dahlia are presented in Table 3. Specifically, the 270
production of protoplasts from dahlia petals was optimised using an enzyme combination of 1.0% 271
cellulase, 0.5% macerozyme and 0.4% pectinase, resulting in a yield of 5.46 × 10⁶ protoplasts/mL and a 272
protoplast viability of 68.69%. 273
Table 3 Effect of enzyme combination on the isolation of protoplast from dahlia petals 274
Number
Cellulase(%)
Macerozym(%)
Pectolase(%)
Yield(×105
protoplasts/mL)
Vigour(%)
E1
1.00
0.50
0.40
54.60 ± 4.31 a
68.69 ± 1.85 bc
E2
1.00
0.75
0.60
36.83 ± 2.54 bc
66.32 ± 2.68 cd
E3
1.00
1.00
0.80
21.90 ± 2.04 d
62.39 ± 1.99 e
E4
1.50
0.50
0.60
38.83 ± 1.26 b
65.70 ± 1.86 cde
E5
1.50
0.75
0.80
32.40 ± 5.56 c
71.75 ± 1.42 ab
E6
1.50
1.00
0.40
20.80 ± 2.22 de
63.77 ± 1.91 de
E7
2.00
0.50
0.80
15.20 ± 1.52 e
72.71 ± 2.13 a
E8
2.00
0.75
0.40
25.33 ± 3.01 d
68.18 ± 2.31 bc
E9
2.00
1.00
0.60
37.80 ± 4.19 bc
58.09 ± 2.50 f
K1 yield
113.33
108.63
100.73
K2 yield
92.03
94.57
113.47
K3 yield
78.33
80.50
69.50
X 1 yield
37.78
36.21
33.58
X2 yield
30.68
31.52
37.82
X3 yield
26. 11
26.83
23.17
R yield
11.67
9.38
14.66
K1 vigour
197.40
207.10
200.64
K2 vigour
201.22
206.24
190.10
K3 vigour
198.98
184.25
206.85
X 1 vigour
65.80
69.03
66.88
X2 vigour
67.07
68.75
63.37
X3 vigour
66.33
61.42
68.95
R vigour
1.27
7.61
5.58
The study used two solutions, K3 and 13% sucrose, to treat dahlia petal protoplasts. The K3 solution 275
suspended approximately 1.6 times as many protoplasts as the sucrose solution, but had little effect on 276
protoplast viability (Table 4). 277
Table 4 Effect of purification solution on the isolation of dahlia petals protoplast 278
Number
Solution type
Protoplast production( × 105
protoplasts /mL)
Protoplast viability(%)
C1
K3 solution
3.00 ± 0.20 a
65.88 ± 0.83 a
C2
13%Sucrose solution
1.88 ± 0.24 b
69.25 ± 1.25 a
3.3 General system of dahlia petals protoplast system
279
In view of the successful establishment of a programme for the isolation of dahlia petal protoplasts 280
and to investigate its applicability to other dahlia species. In this study, the petal tissues of dahlia 'Black 281
Beauty' and dahlia 'Lotus Fairy' were used as test materials based on the optimal conditions (shredding 282
method + 1.0 mol/L mannitol + 1.0% cellulase + 0.5% macerozyme + 0.4% pectinase). The isolation of 283
protoplasts was successfully achieved. The results demonstrated that protoplasts with nearly round 284
morphology could be successfully isolated from the petals of both varieties of dahlia. The enzyme 285
digestion time was varied between 4, 6, 8 and 10 h, while all other enzyme digestion conditions were kept 286
constant. The enzyme digestion time of 6 hours yielded the highest protoplast yield of dahlia 'Black 287
Beauty', reaching 2.40 × 106 protoplasts/mL. In contrast, the enzyme digestion time of 8 hours yielded 288
the highest protoplast yield of dahlia 'Lotus Fairy', reaching 1.06 × 106 protoplasts/mL. The viability of 289
protoplasts from the dahlia cultivar 'Black Beauty' was found to range from 30.37% to 41.19%. The 290
viability of the protoplasts of the Fairy variety ranged from 61.73% to 76.42%. 291
3.4 The protoplast transient transformation system 292
In this study, the factors influencing the PEG-mediated transformation of 'D. imperialis' protoplasts 293
were optimised, including plasmid concentration (50 ng/µL, 100 ng/µL, 150 ng/µL), PEG concentration 294
(20%, 30%, 40%) and transformation time (10 min, 20 min, 30 min). The results demonstrated that the 295
pGBin-EGFP fluorescent vector was successfully transformed into 'D. imperialis' leaves protoplasts(Fig. 296
8). The one-way analysis indicated that the leaves protoplasts were transformed most efficiently at 50 297
ng/µL plasmid, 20% PEG, and 10 min transformation time. The R-value comparison analysis revealed 298
that the optimal transformation condition was 50 ng/µL plasmid, 40% PEG, and 20 min transformation 299
time, with a transformation efficiency of 57.80%. The pGBin-EGFP fluorescent vector was successfully 300
transformed into protoplasts isolated from dahlia petals. The conversion is illustrated in Fig. 9. 301
302
Fig. 8 Transformation of dahlia leaves protoplast
303
304
Fig. 9 Transformation of dahlia petals protoplast 305
Note: A is bright-field; B is fluorescence-field; C is superimposed-field. Bar = 100 µm. 306
The optimal transformation conditions for dahlia petal protoplasts were identified as 100 ng/µL 307
plasmid, 30% PEG, and a transformation time of 20 minutes, resulting in a transformation efficiency of 308
56.33%. The optimal transformation conditions for ‘Black Beauty’ and ‘Lotus Fairy’ petal protoplasts 309
were determined to be 50 ng/µL plasmid + 40% PEG + 20 min transformation time, resulting in an 310
efficiency of protoplast transformation of 47.04% and 60.67%, respectively. With regard to the 311
individual factors, it was found that a 50 ng/µL plasmid, 20% PEG, and 10 min transformation time 312
could enhance the transformation efficiency of dahlia protoplast. However, when considering the 313
interaction effect among the three factors, the experimental cost, and the transformation time, it was 314
determined that these factors had minimal impact on the transformation efficiency. Consequently, the 315
optimal system for the transient transformation of dahlia protoplast was determined to be 50 ng/µL 316
plasmid + 20% PEG + 20 min transformation time (Fig.10). 317
318
Fig. 10 Effect of different transformation conditions on protoplast transformation efficiency of dahlia 319
3.5 Histological characterization of dahlia
320
Paraffin sections of dahlia leaves, and petals were stained and observed, and it was found that the 321
cellular structure of the two types of dahlia materials exhibited notable differences in arrangement. The 322
leaves were composed of a compact arrangement of epidermal cells, tubular leaves vein cells, and 323
chloroplasts with visible chloroplasts and thin cell walls. The petals were peripherally composed of 324
epidermal cells, with uniformly arranged, oval-shaped central cells and thin cell walls (Fig. 11). The 325
structural features of the plant tissue in question influence the difficulty of isolating protoplasts. The 326
provenance of protoplasts is intimately linked to the configuration of cells, their morphological 327
characteristics and the thickness of their cell walls. 328
329
330
Fig. 11 Histological features of three dahlia materials
331
Note: A-C. Leaves; B-D.Petals; A and B are the of view under 10x objective; E and F are the of view under 40x 332
objective. Bar = 100 µm. 333
4. Discussion
334
4.1 Factors affecting the preparation of protoplast
335
It is well-known that plant materials of diferent origins might be the most crucial factor in protoplast 336
preparation. The utilisation of distinct plant materials, including leaves, and petals, for isolation can 337
demonstrably influence the yield and viability of protoplasts. The majority of isolation materials are 338
selected from young leaves (Ma et al. 2024). For instance, protoplasts were successfully isolated from 339
leaves induced by axillary buds of Felicia bergeriana and Brachycome iberidifolia (Malaure et al. 1990). 340
The present study used 'D. imperialis' leaves and petals, and petals of Dahlia 'Black Beauty' and 341
'Lotus Fairy' as materials to isolate protoplast. The results demonstrated that the yield and vigor of 342
protoplasts isolated from leaves were as high as 6.13 × 10⁶ protoplasts/mL and 89.23%, respectively. In 343
comparison, the yield and vigor of protoplasts isolated from petals were the next highest, at 5.46 × 10⁶ 344
protoplasts/mL and 88.83%, respectively. 345
Protoplasts were isolated from oil tea petals, with a yield of approximately 1.42 × 107 346
protoplasts/mL of petal material. The cell viability was found to be as high as 89% (Lin et al. 2023). The 347
isolation of chrysanthemum protoplasts from 'Chrystal Regal', 'Zihong Tuogui', and 'Zi Fengche' led to 348
the conclusion that the number and vigor of protoplasts extracted from petals were higher than those 349
extracted from leaves (Li et al. 2023). In contrast, the yield and viability of protoplasts extracted from 350
dahlia leaves were found to be higher than those of petal protoplasts. This discrepancy may be attributed 351
to the difference in tissue composition between dahlia and chrysanthemum plant materials. In 352
comparison to petals, leaves are the primary site of photosynthesis. The surface of the leaves contains 353
cells that absorb phytase activity, resulting in a relatively high level of cellular activity. This explains 354
why leaves have the highest viability of all protoplasts. 355
In terms of material processing methods, the results indicated that the yield and viability of 356
protoplasts obtained by the crushing method were lower than those obtained by the shredding method. 357
This may be attributed to the inactivation of cells and the mixing of impurities as a consequence of an 358
excessive operation time or excessive crushing of tissues. Conversely, the shredding method facilitated 359
greater contact between the enzyme solution and the cells, thereby enhancing the efficiency of enzymatic 360
digestion. The current research on the enzymatic digestion of protoplasts indicates that the most 361
commonly used enzyme solution combinations for the separation of plant protoplasts are cellulase + 362
pectinase or cellulase + catalase. For instance, a highly efficient protoplast separation protocol for orchid 363
petals was established by optimising the enzyme digestion conditions. In a study by Ren et al. (2020), the 364
highest yield of protoplasts from orchid petals was achieved under the following conditions: 0.5 mol/L 365
mannitol, 1.2% cellulase, 0.6% macerozyme and 6 h of enzymatic digestion. The optimal conditions for 366
the isolation of protoplasts from lily leaves as explants were determined to be 24 hours of digestion, 4% 367
cellulase, and 1% pectinase (Esmaeil et al. 2012b). However, when the two enzymes do not perform well 368
together, the results of protoplast preparation can be improved by adding hemicellulase. This was 369
demonstrated by Zhou et al. (2005) in the isolation of chrysanthemum protoplasts, where the addition of 370
hemicellulase was more effective, and the yield reached 4.86 × 105 protoplasts/g. In this study, three 371
different enzyme combinations were selected for the preparation of protoplast: cellulase, macerozyme 372
and pectinase. The cellulase is of the RS type, exhibiting three times the enzyme activity of cellulase R10 373
and demonstrating superior separation efficacy. The pectinase is a liquid enzyme, analogous to the 374
enzyme combinations used in cabbage (Sivanandhan et al. 2021). 375
In this study, protoplasts were purified by a combination of suspension, centrifugation and filtration. 376
The protoplast mixture was filtered through a cell strainer, and then centrifuged twice at 2000 r/min for 5 377
min each time. The protoplasts were then suspended in K3 solution, which resulted in the most efficient 378
purification of protoplast. In instances where the production of protoplasts is limited and collection is 379
challenging, the floating purification method may be used. The enzymatic treatment of lily leaves 380
protoplasts was purified by the addition of a 23% sucrose solution following filtration and centrifugation 381
operations. The results demonstrated a reduction in the number of impurities observed under the 382
microscope (Liu 2006). The purification solution used in this study demonstrated enhanced efficacy 383
through the utilisation of a K3 suspension (comprising a 13% sucrose solution and a range of inorganic 384
compounds) in lieu of a pure sucrose solution. This approach proved to be more effective in the 385
purification of protoplasts, with an approximately 1.6-fold increase in yield observed relative to the 13% 386
sucrose solution. 387
4.2 Transient transformation of dahlia protoplast
388
The study utilised PEG-mediated transformation of dahlia protoplast, the results demonstrated that 389
the transient transformation rate of dahlia protoplast ranged from 32.57% to 60.67%, which is 390
comparable to the findings of Hu et al. (2022), who also used chrysanthemum petal protoplasts as the 391
receptor material. The transient transformation efficiency of protoplasts was found to be significantly 392
influenced by the plasmid concentration, PEG species and concentration, and transformation time. The 393
most commonly used PEG species is 40% PEG4000. Furthermore, the transformation efficiency could 394
be up to 70% when the concentration of plasmid was 20 µg (Wang et al. 2022). In the castor protoplast 395
transformation study, a transformation efficiency of 50.7% was achieved by transforming for 15 minutes 396
under dark room temperature conditions, followed by dark culture for 72 hours, using the pGH00.0126 397
plasmid with a concentration of 10 µ g and a 40% PEG solution (Paula et al. 2023). 398
4.3 Enhancing genetic transformation in dahlia protoplast
399
The subsequent stage of this study will be to conduct in-depth genetic engineering and cell 400
engineering in order to facilitate the application of dahlia protoplast. The majority of genetic studies 401
conducted on dahlia species have focused on the regulation of genes associated with petal senescence. 402
These include the XTH (Wang et al. 2013) and XET (Kan et al. 2011) genes. In ethylene-insensitive 403
dahlias, the growth and development of petal cells are closely related to the DpXTH1 and DpXTH2 genes 404
of the XTH gene family, particularly during petal senescence. Furthermore, the expression and regulation 405
of the DpXTH1 gene plays a pivotal role in the physiological metabolism associated with the growth and 406
development of dahlia petals (Zhang et al. 2015). In the present study, the dahlia DpXTH gene or other 407
functional genes of Asteraceae can be introduced into dahlia protoplast to observe their expression, thus 408
facilitating the rapid transformation of exogenous genes. Furthermore, cell fusion of dahlia protoplast 409
from different cultivars can be conducted on the basis of a successfully established protoplast isolation 410
and transformation system. The regeneration of protoplasts into plants can be carried out according to the 411
method of Sauvadet MA (1990), thus enabling the cultivation of new dahlia varieties. These will serve as 412
a reference for the research on molecular biology and cell breeding of dahlia. 413
5. Conclusions 414
In this study, dahlia leaves, and petals were used as test materials to investigate the effects of dahlia 415
species, isolated plant material, enzyme solution type and concentration, enzyme digestion time, 416
mannitol concentration, purification method, and other factors on the yield and viability of dahlia 417
protoplasts. In accordance with the findings of this study, a plasmid containing green fluorescent protein 418
(GFP) was introduced into protoplasts extracted from dahlia petals via a PEG-mediated transformation. 419
In order to identify the optimal transformation conditions for dahlia protoplasts, the impact of key 420
parameters, including the type of polyethylene glycol (PEG) used, the concentration of PEG, and the 421
duration of the transformation process, was evaluated. 422
423
Acknowledgements 424
This work is funded by Major science and technology programme project of Science and 425
Technology Department of Yunnan Province project (Grant Number 202102AE090052). 426
Author contributions 427
Jing Zhou: Conceptualization, Methodology, Software, Formal analysis, Investigation, Data 428
curation, Writingoriginal draft, Writingreview & editing. Dong Yang: Conceptualization, 429
Methodology, Software, Investigation, Formal analysis, Data curation, Writingreview. NingNing 430
Zhou: Conceptualization, Methodology,Supervision. YangBo Tian: Conceptualization, Methodology, 431
Investigation. Zhen Tian: Conceptualization, Methodology, Investigation, Formal analysis. Qing Duan: 432
Conceptualization, Software, Methodology. Mohamed A.A. Ahmed: Investigation, Formal analysis, 433
Data curation, Writingreview. LiHua Wang: Supervision, Funding acquisition. XueWei Wu: 434
Conceptualization, Supervision, review & editing, Funding acquisition. 435
Data availability
436
Data supporting this study are included within the article and/or supporting materials. 437
Compliance with ethical standards 438
Declarations 439
The authors declare that they have no conflict of interest. 440
441
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