Chinese Bulletin of Botany ›› 2023, Vol. 58 ›› Issue (2): 285-297.DOI: 10.11983/CBB22034
• TECHNIQUES AND METHODS • Previous Articles Next Articles
Jindan Zhang, Min Feng(
)
HomeReceived:2022-02-23
Accepted:2022-09-07
Online:2023-03-01
Published:2023-03-15
Contact:
*E-mail: Jindan Zhang, Min Feng. A Plant Sample Optimal Pretreatment for Flow Cytometric Analysis[J]. Chinese Bulletin of Botany, 2023, 58(2): 285-297.
Figure1 Histograms of relative nuclear DNA content in soybean leaves nuclear suspensions treated with different concentration of tannic acid (TA) Samples were prepared with different isolation buffers adding different concentrations of tannic acid, and treated on ice for 15 min. After stained with Propidium Iodide (PI), relative nuclear DNA content of samples were measured with a LSRFortessa flow cytometer. (A)-(D) PVPK12-mGB2 (with 1% PVPK12); (E)-(H) PVPK17-mGB2; (I)-(L) PVPK30-mGB2; (M)-(P) PVPK12-mGB2 (with 2% PVPK12); (Q)-(T) WPB. PI-A: PI-area
| TA (mg?mL-1) | ||||||||
|---|---|---|---|---|---|---|---|---|
| 0 | 2 | 3 | 4 | |||||
| CV (%) | DF (%) | CV (%) | DF (%) | CV (%) | DF (%) | CV (%) | DF (%) | |
| PVPK12-mGB2 (with 1% PVPK12) | 3.70±0.17 | 27.37±1.76 | 4.37±0.46 | 30.63±4.40 | 4.47±0.25 | 52.43±10.00 | 6.23±0.15 | 79.37±5.56 |
| PVPK17-mGB2 | 3.30±0.10 | 30.67±2.12 | 3.90±0.35 | 51.77±6.20 | 5.10±0.17 | 70.80±1.04 | 7.70±0.70 | 87.33±0.71 |
| PVPK30-mGB2 | 3.47±0.32 | 27.93±1.77 | 3.63±0.25 | 41.00±8.66 | 5.70±0.46 | 82.73±3.07 | 7.30±0.17 | 88.93±3.90 |
| PVPK12-mGB2 (with 2% PVPK12) | 3.47±0.06 | 30.73±0.95 | 3.67±0.06 | 41.83±11.98 | 4.10±0.10 | 42.67±3.09 | 3.83±0.15 | 38.20±6.28 |
| WPB | 4.50±0.17 | 48.27±0.32 | 4.60±0.30 | 47.63±4.21 | 5.17±0.32 | 50.20±5.97 | 6.07±0.06 | 47.07±5.83 |
Table1 Effectiveness of relative nuclear DNA prepared from Glycine max leaf tissue with different isolation buffers exposed to different concentration of tannic acid (TA)
| TA (mg?mL-1) | ||||||||
|---|---|---|---|---|---|---|---|---|
| 0 | 2 | 3 | 4 | |||||
| CV (%) | DF (%) | CV (%) | DF (%) | CV (%) | DF (%) | CV (%) | DF (%) | |
| PVPK12-mGB2 (with 1% PVPK12) | 3.70±0.17 | 27.37±1.76 | 4.37±0.46 | 30.63±4.40 | 4.47±0.25 | 52.43±10.00 | 6.23±0.15 | 79.37±5.56 |
| PVPK17-mGB2 | 3.30±0.10 | 30.67±2.12 | 3.90±0.35 | 51.77±6.20 | 5.10±0.17 | 70.80±1.04 | 7.70±0.70 | 87.33±0.71 |
| PVPK30-mGB2 | 3.47±0.32 | 27.93±1.77 | 3.63±0.25 | 41.00±8.66 | 5.70±0.46 | 82.73±3.07 | 7.30±0.17 | 88.93±3.90 |
| PVPK12-mGB2 (with 2% PVPK12) | 3.47±0.06 | 30.73±0.95 | 3.67±0.06 | 41.83±11.98 | 4.10±0.10 | 42.67±3.09 | 3.83±0.15 | 38.20±6.28 |
| WPB | 4.50±0.17 | 48.27±0.32 | 4.60±0.30 | 47.63±4.21 | 5.17±0.32 | 50.20±5.97 | 6.07±0.06 | 47.07±5.83 |
Figure 2 Histograms of relative nuclear DNA content in fresh herbaceous plant leaves Samples were prepared with isolation buffers PVPK12-mGB2 (with 1% PVPK12), LB01 and CyStain? PI Absolute P, and treated on ice for 15 min. After stained with Propidium Iodide (PI), relative nuclear DNA contents were measured using a LSRFortessa flow cytometer. (A) Capsicum annuum; (B) Oryza sativa; (C) Nicotiana tabacum; (D) Hosta plantaginea; (E) Salvia nemorosa; (F) Physostegia virginiana; (G) Ipomoea batatas; (H) Glycine max. PI-A: PI-area
| Species | PVPK12-mGB2 (with 1% PVPK12) | LB01 | CyStain? PI Absolute P | |||
|---|---|---|---|---|---|---|
| CV (%) | DF (%) | CV (%) | DF (%) | CV (%) | DF (%) | |
| Capsicum annuum | 2.47±0.15 | 28.13±6.05 | 2.93±0.15 | 28.60±2.33 | 2.53±0.23 | 25.07±5.35 |
| Oryza sativa | 3.13±0.21 | 21.47±3.46 | 4.03±0.15 | 31.33±6.56 | 2.97±0.35 | 24.40±4.34 |
| Nicotiana tabacum | 2.37±0.06 | 18.83±5.73 | 2.47±0.06 | 15.90±3.04 | 2.50±0.17 | 17.07±2.81 |
| Hosta plantaginea | 2.90±0.17 | 21.00±1.37 | 3.43±0.06 | 18.77±2.76 | 3.67±0.21 | 18.27±1.10 |
| Salvia nemorosa | 3.30±0.10 | 27.40±1.18 | 3.80±0.20 | 19.00±4.54 | 3.23±0.21 | 20.83±2.00 |
| Physostegia virginiana | 2.63±0.15 | 31.20±5.89 | 3.20±0.17 | 31.87±5.95 | 3.20±0.26 | 32.33±5.26 |
| Ipomoea batatas | 3.57±0.06 | 31.73±3.78 | 4.53±0.65 | 30.33±1.57 | 4.97±0.55 | 28.77±2.59 |
| Glycine max | 3.43±0.47 | 16.73±1.67 | 4.30±0.17 | 34.93±2.75 | 3.03±0.15 | 15.71±2.17 |
Table 2 Effectiveness of relative nuclear DNA of fresh herbaceous plant leaves with different isolation buffers
| Species | PVPK12-mGB2 (with 1% PVPK12) | LB01 | CyStain? PI Absolute P | |||
|---|---|---|---|---|---|---|
| CV (%) | DF (%) | CV (%) | DF (%) | CV (%) | DF (%) | |
| Capsicum annuum | 2.47±0.15 | 28.13±6.05 | 2.93±0.15 | 28.60±2.33 | 2.53±0.23 | 25.07±5.35 |
| Oryza sativa | 3.13±0.21 | 21.47±3.46 | 4.03±0.15 | 31.33±6.56 | 2.97±0.35 | 24.40±4.34 |
| Nicotiana tabacum | 2.37±0.06 | 18.83±5.73 | 2.47±0.06 | 15.90±3.04 | 2.50±0.17 | 17.07±2.81 |
| Hosta plantaginea | 2.90±0.17 | 21.00±1.37 | 3.43±0.06 | 18.77±2.76 | 3.67±0.21 | 18.27±1.10 |
| Salvia nemorosa | 3.30±0.10 | 27.40±1.18 | 3.80±0.20 | 19.00±4.54 | 3.23±0.21 | 20.83±2.00 |
| Physostegia virginiana | 2.63±0.15 | 31.20±5.89 | 3.20±0.17 | 31.87±5.95 | 3.20±0.26 | 32.33±5.26 |
| Ipomoea batatas | 3.57±0.06 | 31.73±3.78 | 4.53±0.65 | 30.33±1.57 | 4.97±0.55 | 28.77±2.59 |
| Glycine max | 3.43±0.47 | 16.73±1.67 | 4.30±0.17 | 34.93±2.75 | 3.03±0.15 | 15.71±2.17 |
Figure 3 Histograms of relative nuclear DNA content in fresh woody plant leaves Samples were prepared with isolation buffers PVPK12-mGB2 (with 1% PVPK12), LB01 and CyStain? PI Absolute P, then stained with Propidium Iodide (PI), relative nuclear DNA contents were measured using a LSRFortessa flow cytometer. (A) Eucommia ulmoides; (B) Viburnum rhytidophyllum; (C) Yulania denudata; (D) Prunus virginiana; (E) Actinidia chinensis; (F) Lonicera japonica; (G) Metasequoia glyptostroboides; (H) Ginkgo biloba. PI-A: PI-area
| Species | PVPK12-mGB2 (with 1% PVPK12) | LB01 | CyStain? PI Absolute P | |||
|---|---|---|---|---|---|---|
| CV (%) | DF (%) | CV (%) | DF (%) | CV (%) | DF (%) | |
| Eucommia ulmoides | 3.57±0.25 | 60.93±5.85 | 5.53±0.15 | 68.40±2.75 | 3.57±0.40 | 68.70±4.56 |
| Viburnum rhytidophyllum | 4.70±0.46 | 49.10±2.86 | 4.20±0.17 | 48.13±3.52 | 3.77±0.55 | 47.37±2.66 |
| Yulania denudata | 2.93±0.32 | 28.60±0.57 | 4.57±0.90 | 47.90±4.45 | 4.10±0.57 | 27.77±2.90 |
| Prunus virginiana | 3.40±0.30 | 49.80±4.17 | 3.80±0.35 | 49.53±6.08 | 4.23±0.45 | 44.77±3.93 |
| Actinidia chinensis | 3.30±0.10 | 41.30±3.24 | 4.47±0.15 | 48.67±2.89 | 4.80±0.10 | 47.23±4.45 |
| Lonicera japonica | 2.70±0.10 | 20.03±0.92 | 3.40±0.20 | 23.53±6.90 | 2.70±0.17 | 23.13±5.07 |
| Metasequoia glyptostroboides | 2.87±0.31 | 27.83±1.59 | 5.37±0.50 | 29.40±1.76 | 3.53±0.21 | 26.20±3.22 |
| Ginkgo biloba | 3.30±0.10 | 29.40±3.30 | 4.53±0.21 | 39.16±3.56 | 3.73±0.31 | 26.47±1.50 |
Table 3 Effectiveness of relative nuclear DNA of fresh woody plant leaves with different isolation buffers
| Species | PVPK12-mGB2 (with 1% PVPK12) | LB01 | CyStain? PI Absolute P | |||
|---|---|---|---|---|---|---|
| CV (%) | DF (%) | CV (%) | DF (%) | CV (%) | DF (%) | |
| Eucommia ulmoides | 3.57±0.25 | 60.93±5.85 | 5.53±0.15 | 68.40±2.75 | 3.57±0.40 | 68.70±4.56 |
| Viburnum rhytidophyllum | 4.70±0.46 | 49.10±2.86 | 4.20±0.17 | 48.13±3.52 | 3.77±0.55 | 47.37±2.66 |
| Yulania denudata | 2.93±0.32 | 28.60±0.57 | 4.57±0.90 | 47.90±4.45 | 4.10±0.57 | 27.77±2.90 |
| Prunus virginiana | 3.40±0.30 | 49.80±4.17 | 3.80±0.35 | 49.53±6.08 | 4.23±0.45 | 44.77±3.93 |
| Actinidia chinensis | 3.30±0.10 | 41.30±3.24 | 4.47±0.15 | 48.67±2.89 | 4.80±0.10 | 47.23±4.45 |
| Lonicera japonica | 2.70±0.10 | 20.03±0.92 | 3.40±0.20 | 23.53±6.90 | 2.70±0.17 | 23.13±5.07 |
| Metasequoia glyptostroboides | 2.87±0.31 | 27.83±1.59 | 5.37±0.50 | 29.40±1.76 | 3.53±0.21 | 26.20±3.22 |
| Ginkgo biloba | 3.30±0.10 | 29.40±3.30 | 4.53±0.21 | 39.16±3.56 | 3.73±0.31 | 26.47±1.50 |
Figure 4 Histograms of relative nuclear DNA content of silica gel-desiccated plant leaves Samples were prepared with different isolation buffers PVPK12-mGB2 (with 1% PVPK12), LB01 and CyStain? PI Absolute P, then stained with Propidium Iodide (PI), and relative nuclear DNA contents were measured using a LSRFortessa flow cytometer. (A) Eucommia ulmoides; (B) Viburnum rhytidophyllum; (C) Yulania denudata; (D) Prunus virginiana; (E) Actinidia chinensis; (F) Lonicera japonica; (G) Metasequoia glyptostroboides; (H) Ginkgo biloba. PI-A: PI-area
| Species | PVPK12-mGB2 (with 1% PVPK12) | LB01 | CyStain? PI Absolute P | |||
|---|---|---|---|---|---|---|
| CV (%) | DF (%) | CV (%) | DF (%) | CV (%) | DF (%) | |
| Eucommia ulmoides | 4.40±0.17 | 63.30±2.41 | 6.70±0.26 | 45.77±6.25 | 4.23±0.55 | 44.13±7.17 |
| Viburnum rhytidophyllum | 3.70±0.17 | 48.97±4.50 | 5.13±0.31 | 37.67±9.35 | 4.47±0.67 | 22.60±1.84 |
| Yulania denudata | 3.50±0.10 | 33.70±3.87 | 3.80±0.36 | 29.47±3.86 | 5.70±0.82 | 16.70±3.58 |
| Prunus virginiana | 3.30±0.10 | 23.97±3.07 | 4.40±0.10 | 32.13±1.23 | 5.07±0.55 | 25.97±4.05 |
| Actinidia chinensis | 3.50±0.10 | 33.73±4.04 | 5.13±0.12 | 36.23±5.81 | 5.00±0.30 | 33.00±4.43 |
| Lonicera japonica | 4.10±0.10 | 24.20±3.81 | 3.57±0.15 | 23.33±1.10 | 3.67±0.21 | 15.23±0.23 |
| Metasequoia glyptostroboides | 7.20±1.14 | 46.57±2.87 | 19.27±0.68 | 37.97±3.04 | 8.93±1.33 | 29.63±2.70 |
| Ginkgo biloba | 6.47±0.23 | 19.03±0.40 | 9.67±0.25 | 45.60±1.73 | 22.87±0.90 | 48.70±0.69 |
Table 4 Effectiveness of relative nuclear DNA of silica gel-desiccated woody plant leaves with different isolation buffers
| Species | PVPK12-mGB2 (with 1% PVPK12) | LB01 | CyStain? PI Absolute P | |||
|---|---|---|---|---|---|---|
| CV (%) | DF (%) | CV (%) | DF (%) | CV (%) | DF (%) | |
| Eucommia ulmoides | 4.40±0.17 | 63.30±2.41 | 6.70±0.26 | 45.77±6.25 | 4.23±0.55 | 44.13±7.17 |
| Viburnum rhytidophyllum | 3.70±0.17 | 48.97±4.50 | 5.13±0.31 | 37.67±9.35 | 4.47±0.67 | 22.60±1.84 |
| Yulania denudata | 3.50±0.10 | 33.70±3.87 | 3.80±0.36 | 29.47±3.86 | 5.70±0.82 | 16.70±3.58 |
| Prunus virginiana | 3.30±0.10 | 23.97±3.07 | 4.40±0.10 | 32.13±1.23 | 5.07±0.55 | 25.97±4.05 |
| Actinidia chinensis | 3.50±0.10 | 33.73±4.04 | 5.13±0.12 | 36.23±5.81 | 5.00±0.30 | 33.00±4.43 |
| Lonicera japonica | 4.10±0.10 | 24.20±3.81 | 3.57±0.15 | 23.33±1.10 | 3.67±0.21 | 15.23±0.23 |
| Metasequoia glyptostroboides | 7.20±1.14 | 46.57±2.87 | 19.27±0.68 | 37.97±3.04 | 8.93±1.33 | 29.63±2.70 |
| Ginkgo biloba | 6.47±0.23 | 19.03±0.40 | 9.67±0.25 | 45.60±1.73 | 22.87±0.90 | 48.70±0.69 |
| Materials | PVPK12-mGB2 (with 1% PVPK12) | CyStain? PI Absolute P | ||
|---|---|---|---|---|
| Genome size (Mb) | CV (%) | Genome size (Mb) | CV (%) | |
| Fresh leaf | 276.82 | 2.83±0.74 | 281.80 | 3.43±0.32 |
| Dry leaf | 280.54 | 3.80±0.10 | 282.54 | 4.00±0.30 |
Table 5 Estimation of genome size of Ficus carica leaf nuclear DNA with different nuclear isolation buffers
| Materials | PVPK12-mGB2 (with 1% PVPK12) | CyStain? PI Absolute P | ||
|---|---|---|---|---|
| Genome size (Mb) | CV (%) | Genome size (Mb) | CV (%) | |
| Fresh leaf | 276.82 | 2.83±0.74 | 281.80 | 3.43±0.32 |
| Dry leaf | 280.54 | 3.80±0.10 | 282.54 | 4.00±0.30 |
Figure 5 Determination of Ephedra intermedia DNA ploidy Samples were prepared with different isolation buffers, then stained with Propidium Iodide (PI) and measured using a LSRFortessa flow cytometer (a: Ephedra equisetina; b: E. intermedia). (A) PVPK12-mGB2 (with 1% PVPK12); (B) LB01; (C) CyStain? PI Absolute P; (D) WPB. DI: DNA index; PI-A: PI-area
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