A Flow Cytometry Protocol for Measurement of Plant Genome Size Using Frozen Material
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Material
2.2. Pretreatment of Leaf Material
2.3. Reagents and Buffers
- Woody plant buffer (WPB) [as per 25,34]: 0.2 M Trizma hydrochloride (Sigma, Merck Life Science Pty Ltd., Bayswater, VIC, Australia, Product No. 93363-50G), 0.04 M Magnesium chloride hexahydrate (Sigma, Merck Life Science Pty Ltd., Bayswater, VIC, Australia, M2670-100G), 0.02 M ethylene diamine tetra acetic acid disodium salt dihydrate (EDTA.Na2, Chem Supply Australia, Gillman, SA, Australia, Product code: Product code: EA023-500G), 86 mM Sodium chloride (Sigma, Merck Life Science Pty Ltd., Bayswater, VIC, Australia, Product No. 71380-500G), 10 mM Sodium metabisulfite (Sigma, Merck Life Science Pty Ltd., Bayswater, VIC, Australia, S9000-500G), 1% Triton X-100 (Chem Supply Australia, Gillman, SA, Australia, Product code: TL125-P), UltraPure DNase/RNase free distilled water (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA, Cat. No. 10977-015, 300 mL), 3% Polyvinylpyrrolidone-10 (Sigma, Merck Life Science Pty Ltd., Bayswater, VIC, Australia, Product No. PVP10).
- Staining buffer (20 µL per 400 µL of sample): 100 µL propidium iodide (PI, 1 mg/mL, Sigma, Merck Life Science Pty Ltd., Bayswater, VIC, Australia, Product ID P4864-10ML), 1 µL of RNase 10 mg/mL (Stock Solution 20mg/mL, Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA, Cat. No. 12091021). The buffer was kept on ice and covered with aluminum foil due to the light-sensitive nature of PI.
- 10× Homogenization buffer (HB) for frozen preparations: 0.1 M Trizma Base (Sigma, Merck Life Science Pty Ltd., Bayswater, VIC, Australia, Product No. 93362-500G), 0.8 M Potassium chloride (Chem Supply Australia, Gillman, SA, Australia, Product code: PA054-500G), 0.1 M ethylene diamine tetra acetic acid (Sigma, Merck Life Science Pty Ltd., Bayswater, VIC, Australia, Product No. E6758-500G), 17 mM spermidine (Sigma, Merck Life Science Pty Ltd., Bayswater, VIC, Australia, Product No. 85558-5G), 17 mM spermine (Sigma, Merck Life Science Pty Ltd., Bayswater, VIC, Australia, Product No. 85590-5G), 10 M NaOH (Chem Supply Australia, Gillman, SA, Australia, Product code: SA178) to adjust pH to 9. The solution can be stored in a glass bottle at 4 °C for up to one year.
- 100 mL Triton sucrose buffer (TSB) for frozen preparations: Triton X-100 (20%), 10× HB (10%), 0.5 M sucrose (Sigma, Merck Life Science Pty Ltd., Bayswater, VIC, Australia, Product No. S0389-500G), volume was made up to 100 mL with distilled water. The solution can be stored in a glass bottle at 4 °C for up to one year.
- 1000 mL 1× Homogenization buffer (HB) for frozen preparations: 10× HB (10%), Sucrose (0.5 M). Volume was made up to 1 L with distilled water.
- 50 mL/sample Nuclei Isolation Buffer (NIB) for frozen preparations: 1× HB (48.75 mL), TSB (1.25 mL), 0.5gm polyvinylpyrrolidone-360 (Sigma, Merck Life Science Pty Ltd., Bayswater, VIC, Australia, Product No. PVP360-100G). Add 125 µL of 2-mercaptoethanol (Sigma, Merck Life Science Pty Ltd., Bayswater, VIC, Australia, Product No. 63689-100ML-F) before use and keep NIB on ice.
2.4. Equipment
2.4.1. For Fresh Preparations
2.4.2. For Frozen Preparations
2.5. Nuclei Isolation
2.6. Flow Cytometry
2.7. Conventional Histogram Analysis
2.8. Histogram Modeling and Debris Compensation-Based Analysis
2.9. Experimental Setup
2.10. Statistical Analysis
3. Results
3.1. Genome Size Estimates for Adenanthos sericeus
3.2. Genome Size Estimates for Hollandaea sayeriana
3.3. Genome Size Estimates for Macadamia tetraphylla
3.4. GS Estimates for Macadamia jansenii
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
FCM | Flow cytometry |
GS | Genome size |
pg | Picograms |
CV | Coefficient of variance |
ANOVA | Analysis of variance |
nls | Non-linear least squares |
RCS | Residual chi-square |
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Species | Interaction | Df | Mean Sq. | Residuals Df | Residuals Mean Sq. | F Value | Pr (>F) |
---|---|---|---|---|---|---|---|
A. sericeus | Nuclei preparation: data analysis | 3 | 4.5 × 10−5 | 16 | 4.75 × 10−5 | 0.947 | 0.35 |
H. sayeriana | Nuclei preparation: data analysis | 3 | 3.2 × 10−4 | 16 | 1.38 × 10−4 | 2.32 | 0.15 |
M. tetraphylla | Nuclei preparation: data analysis | 3 | 0.000 | 16 | 3.5 × 10−4 | 0.000 | 0.99 |
M. jansenii | Nuclei preparation: data analysis | 3 | 3.2 × 10−4 | 16 | 4.4 × 10−4 | 0.725 | 0.40 |
Species | Nuclei Prep + Data Analysis | Avg. GS (pg ± SE) | Avg. Events in Sample ± SE | Avg. Events in Std. ± SE | Avg. Sample CV% ± SE | Avg. Std. CV% ± SE |
---|---|---|---|---|---|---|
A. sericeus | fresh + non-compensated | 0.47 ± 0.005 | 812 ± 183 | 1471 ± 258 | 3.28 ± 0.21 | 3.32 ± 0.09 |
fresh + compensated | 0.46 ± 0.003 | 982 ± 176 | 1180 ± 142 | 4.09 ± 0.25 | 3.11 ± 0.15 | |
frozen + non-compensated | 0.47 ± 0.001 | 9050 ± 393 | 11,520 ± 862 | 3.16 ± 0.09 | 4.14 ± 0.14 | |
frozen + compensated | 0.47 ± 0.000 | 3600 ± 273 | 3240 ± 186 | 4.59 ± 0.11 | 4.14 ± 0.07 | |
H. sayeriana | fresh + non-compensated | 1.1 ± 0.009 | 693 ± 89 | 1350 ± 191 | 3.76 ± 0.21 | 4.56 ± 0.12 |
fresh + compensated | 1.04 ± 0.003 | 522 ± 96 | 530 ± 165 | 3.69 ± 0.24 | 3.16 ± 0.15 | |
frozen + non-compensated | 1.08 ± 0.001 | 2130 ± 250 | 5810 ± 608 | 3.58 ± 0.23 | 4.22 ± 0.04 | |
frozen + compensated | 1.04 ± 0.001 | 1450 ± 180 | 3288 ± 429 | 3.58 ± 0.14 | 3.89 ± 0.09 | |
M. tetraphylla | fresh + non-compensated | 0.86 ± 0.012 | 512 ± 105 | 1150 ± 92 | 3.74 ± 0.50 | 4.20 ± 0.13 |
fresh + compensated | 0.82 ± 0.011 | 147 ± 142 | 522 ± 37 | 4.26 ± 0.48 | 2.70 ± 0.20 | |
frozen + non-compensated | 0.88 ± 0.003 | 1120 ± 121 | 1937 ± 99 | 3.82 ± 0.16 | 4.16 ± 0.14 | |
frozen + compensated | 0.84 ± 0.000 | 490 ± 73 | 561 ± 30 | 4.40 ± 0.21 | 4.38 ± 0.08 | |
M. jansenii | fresh + non-compensated | 0.82 ± 0.017 | 176 ± 21 | 1010 ± 179 | 3.94 ± 0.30 | 4.42 ± 0.15 |
fresh + compensated | 0.78 ± 0.007 | 62 ± 21 | 540 ± 129 | 4.09 ± 0.64 | 3.23 ± 0.24 | |
frozen + non-compensated | 0.86 ± 0.005 | 1480 ± 166 | 2880 ± 449 | 3.10 ± 0.35 | 4.02 ± 0.16 | |
frozen + compensated | 0.82 ± 0.003 | 372 ± 83 | 630 ± 68 | 3.94 ± 0.3 | 5.30 ± 0.12 |
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Soni, A.; Constantin, L.; Furtado, A.; Henry, R.J. A Flow Cytometry Protocol for Measurement of Plant Genome Size Using Frozen Material. Appl. Biosci. 2025, 4, 28. https://doi.org/10.3390/applbiosci4020028
Soni A, Constantin L, Furtado A, Henry RJ. A Flow Cytometry Protocol for Measurement of Plant Genome Size Using Frozen Material. Applied Biosciences. 2025; 4(2):28. https://doi.org/10.3390/applbiosci4020028
Chicago/Turabian StyleSoni, Abhishek, Lena Constantin, Agnelo Furtado, and Robert J Henry. 2025. "A Flow Cytometry Protocol for Measurement of Plant Genome Size Using Frozen Material" Applied Biosciences 4, no. 2: 28. https://doi.org/10.3390/applbiosci4020028
APA StyleSoni, A., Constantin, L., Furtado, A., & Henry, R. J. (2025). A Flow Cytometry Protocol for Measurement of Plant Genome Size Using Frozen Material. Applied Biosciences, 4(2), 28. https://doi.org/10.3390/applbiosci4020028