Materials and Reagents

Pipette tips (10, 100, and 1,000 μl)
50-ml tube
1.7-ml tubes with caps
2.0-ml tubes with caps
2.3-mm stainless steel beads (Bio Spec Products, catalog number: 11079123ss )
30-μmnylon meshes (Sysmex, CellTrics^®, catalog number: 04-0042-2316 )
20-μmnylon meshes (Sysmex, CellTrics^®, catalog number: 04-0042-2315 )
10-μm nylon meshes (Sysmex, CellTrics^®, catalog number: 04-0042-2314 )
Glass Petri dish (Corning, PYREX^®, catalog number: 423790 )
Leaf tissue of C-value standard (e.g., Nicotiana tabacum L. ‘Xanthi’; 2C = 10.04 pg, Johnston et al., 1999)
Spores of ferns or lycophytes (kept by dry storage and under < 4 °C)
PVP-40 (Sigma-Aldrich, catalog number: PVP40 )
2-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
RNaseA solution (10 mg/ml in ddH₂O) (Sigma-Aldrich, catalog number: R5000-100MG )
Triton X-100
Sodium sulfite (Na₂SO₃)
Tris-HCl (pH 7.5)
Propidium iodide
Backmen stock buffer (see Recipes)*
*Note: LB01 buffer (Doležel et al., 2007) or GPB buffer (Loureiro et al., 2007) can be alternatively used depending on plant material properties.
PI solution (see Recipes)

Equipment

Pipette (10, 100, and 1,000 μl)
Vortex (Scientific Industries, model: Vortex-Genie 2 )
Razors and razor pen
Flow cytometer (BD, BD Biosciences, model: FACScan )*
*Note: FACScan with a 15 microW blue argon ion laser of an emission wave length of 488 nm.

Software

BD FACSCan system (BD Biosciences, Franklin Lake, NJ, USA)

Procedure

Prepare buffer for use

Allocate appropriate amount of Backmen stock buffer to a 50-ml tube based on an estimation of 1-1.5 ml per sample.
Add 0.04 g PVP-40, 5 μl 2-mercaptoethanol, 1 μl RNase per ml of buffer.

Extract spore nuclei by bead-vortexing

For each sample, weigh ~0.007 g spores into a 1.7-ml tube. Green spores that usually have thin spore walls, 4 times the amount of spores are recommended (Kuo et al., 2017)*.
*Note: For bead-vortexing, the detailed process can be seen in the video supplied in Kuo et al. (2017): http://onlinelibrary.wiley.com/store/10.1111/nph.14291/asset/supinfo/nph14291-sup-0002-VideoS1.mov?v=1&s=dae6f0590d33413f1444bc0add857d285fb73cd6
Add 16 stainless steel beads into each 1.7-ml tube.
Add 250 μl of buffer into each 1.7-ml tube.
Vortex these tubes at a speed of 1,900 rpm for 1 min. For green spores, a speed of 3,200 rpm and a vortex duration of 0.5 min are recommended (Kuo et al., 2017).
Filter bead-vortexed samples into 2.0-ml tubes through nylon meshes. The size of nylon mesh is selected based on spore sizes to prevent spore being filtered through the mesh.
Add additional buffer to the samples, and ensure each of filtered spore nuclei solutions is greater than 500 μl in volume.

Extract standard nuclei by chopping leaf tissue

Add 500 μl of buffer to a glass Petri dish.
Add a (~400 mm²) piece of young leaf to the Petri dish, and chop it with a razor on ice until most tissue slices are less than 1 mm in size.
Filter the chopped sample into a 2.0-ml tube through a 30-μm nylon mesh.
Add additional buffer to the sample, and ensure that the filtered leaf nuclei solution is greater than 500 μl in volume or more depending on need.

Staining nuclei solutions

Mix spore nuclei and standard leaf nuclei solutions into a 500-μl volume in 2.0-ml tubes.
Add 10 μl PI solution into each of mixed nuclei solutions.
Incubate in the dark at 4 °C for 1 h for staining.

Data analysis

Set up a histogram plot of particle count vs. linear value of relative fluorescence in BD FACSCan system.
After PI staining, measure nuclear DNA content of the samples in BD FACSCan system, and adjust the fluorescence laser voltage to visualize the nuclei peaks on the histogram plot.
Measure > 1,300 particles for each peak, and the coefficient variation for each peak should be lower 5% as the quality criteria suggested by Greilhuber et al. (2007).
Genome size of spore nuclei (pg or Mbp) = x standard 2C value (Figure 1).

Figure 1. An example of flow cytometric result of spore and standard nuclei. These peaks are spore nuclei of Dryopteris varia and 2C and 4C leaf nuclei of Nicotiana tabacum L. ‘Xanthi’.

Notes

To dry fertile leaves to release and collect spores, air-drying process under room temperature for 2 to 3 days is recommended. For long-term storage, spore material is better stored in tubes without any solution at a temperature lower than 4 °C.

Recipes

Backmen stock buffer (Ebihara et al., 2005)
1.0% Triton X-100
50 mM Na₂SO₃
50 mM Tris-HCl (pH 7.5)
ddH₂O (the solvent)
Note: Store at 4 °C up to 1 year.
PI solution
2.04 mg/ml propidium iodide
ddH₂O (the solvent)
Note: Store in the dark at 4 °C for long-term storage.

Acknowledgments

The bead-vortexing condition to extract spore nuclei of seedless vascular plants is accessed and constructed by Kuo et al. (2017). We thank Fay-Wei Li and three anonymous reviewers for providing comments on the draft of this manuscript.

References

Doležel, J., Greilhuber, J. and Suda, J. (2007). Estimation of nuclear DNA content in plants using flow cytometry. Nat Protoc 2(9): 2233-2244.
Ebihara, A., Ishikawa, H., Matsumoto, S., Lin, S. J., Iwatsuki, K., Takamiya, M., Watano, Y. and Ito, M. (2005). Nuclear DNA, chloroplast DNA, and ploidy analysis clarified biological complexity of the Vandenboschia radicans complex (Hymenophyllaceae) in Japan and adjacent areas. Am J Bot 92(9): 1535-1547.
Greilhuber, J., Temsch, E. M. and Loureiro, J. C. M. (2007). Nuclear DNA Content Measurement. In: Doleel, J., Greilhuber, J. and Suda, J. (Eds.). Flow cytometry with plant cells: analysis of genes, chromosomes and genomes. Wiley, pp: 67-102.
Johnston, J., Bennett, M., Rayburn, A., Galbraith, D. and Price, H. (1999). Reference standards for determination of DNA content of plant nuclei. Am J Bot 86(5): 609-613.
Kuo, L. Y., Huang, Y. J., Chang, J., Chiou, W. L. and Huang, Y. M. (2017). Evaluating the spore genome sizes of ferns and lycophytes: a flow cytometry approach. New Phytol 213(4):1974-1983.
Loureiro, J., Rodriguez, E., Dolezel, J. and Santos, C. (2007). Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species. Ann Bot 100(4): 875-888.