Table 1.
List, genotype codes and numbers of plant materials applied with colchicine.
| Genotype | Genotype code | Plants used in colchicine application (number) |
|---|---|---|
| Calhoun Gray (Citrullus lanatus) | C | 180 |
| W1482 (Citrullus lanatus var. citroides) | N3 | 210 |
| W1832 (Citrullus lanatus var. citroides) | N5 | 210 |
| W2001 (Citrullus lanatus var. citroides) | N7 | 180 |
| CxW1482 | CxN3 | 210 |
| CxW1832 | CxN5 | 180 |
| CxW2001 | CxN7 | 210 |
| Total | 1,380 |
Table 2.
List of genotypes used in hydroponic culture.
| Genotype code | Ploidy level | Genotype code | Ploidy level |
|---|---|---|---|
| M1Calhon Gray | Tetraploid | M1CXN5-1 | Tetraploid |
| M1N3 | Tetraploid | M1CXN7-1 | Tetraploid |
| M1N5-1 | Tetraploid | M1CXN7-2 | Tetraploid |
| M1N5-2 | Tetraploid | M1CXN7-3 | Tetraploid |
| M1N5-3 | Tetraploid | M1CXN7-4 | Tetraploid |
| M1N5-4 | Tetraploid | M1CXN7-5 | Tetraploid |
| M1N5-5 | Tetraploid | Calhoun Gray | Diploid |
| M1N5-6 | Tetraploid | CXN3 | Diploid |
| M1N7-1 | Tetraploid | CXN5 | Diploid |
| M1N7-2 | Tetraploid | CXN7 | Diploid |
| M1N7-3 | Tetraploid | N3 | Diploid |
| M1N7-4 | Tetraploid | N5 | Diploid |
| M1CXN3-1 | Tetraploid | N7 | Diploid |
| M1CXN3-2 | Tetraploid | Crimson Tide | Diploid |
| M1CXN3-3 | Tetraploid | RS841 | Diploid |
| M1CXN3-4 | Tetraploid | Argentario | Diploid |
Table 3.
Number of plants applied, the number of surviving plants and their ratio (%).
| Genotype | Plants applied (number) | Surviving plants (number) | Surviving plants (%) |
|---|---|---|---|
| C | 180 | 120 | 66.67 |
| N3 | 210 | 119 | 56.67 |
| N5 | 210 | 110 | 52.38 |
| N7 | 180 | 158 | 87.78 |
| CXN3 | 210 | 116 | 55.24 |
| CXN5 | 180 | 119 | 66.11 |
| CXN7 | 210 | 116 | 55.24 |
| Total/mean | 1,380 | 858 | 62.17 |

Figure 1.
Stomal diameter and length in diploid (A) and tetraploid (B) plant leaves (magnified 400×).
Table 4.
Mean stomatal diameter, stomatal length, stomatal density and chloroplast number in presumed polyploid and diploid plants.
| Stomatal diameter (μm) | Stomatal length (μm) | Stomatal density (number · mm-2) | Chloroplast number (number) | |||||
|---|---|---|---|---|---|---|---|---|
| Ploidy level | Diploid | Tetraploid | Diploid | Tetraploid | Diploid | Tetraploid | Diploid | Tetraploid |
| Maximum | 16.09 | 20.56 | 23.30 | 31.42 | 627.30 | 341.75 | 10.18 | 16.41 |
| Minimum | 18.90 | 23.87 | 26.04 | 34.01 | 927.77 | 470.34 | 12.03 | 19.37 |
| Mean | 17.50 | 22.22 | 24.67 | 32.72 | 777.54 | 406.05 | 11.11 | 17.89 |
Table 5.
The number and percentage of tetraploid plants obtained as a result of microscope and flow cytometry analyses.
| Genotype | Number of tetraploid plants | Flow cytometry analysis percentage of tetraploid plants (%) | |
|---|---|---|---|
| Stomatal examinations (number) | Flow cytometry analysis | ||
| C | 8 | 1 | 12.50 |
| N3 | 15 | 1 | 6.67 |
| N5 | 11 | 6 | 54.55 |
| N7 | 12 | 4 | 33.33 |
| CXN3 | 14 | 4 | 28.57 |
| CXN5 | 11 | 1 | 9.09 |
| CXN7 | 13 | 5 | 38.46 |
| Total/mean | 84 | 22 | 26.19 |

Figure 2.
Ploidy level of diploid (A) and tetraploid watermelon (B).

Figure 3.
Seedling of diploid (A) CXN5 and tetraploid (B) M1CXN5-1 genotypes, leaf of diploid (C) CXN5 and tetraploid (D) M1CXN5-1 genotypes, male flower of diploid (E) N5 and tetraploid (F) M1N5-1 genotypes and seeds of diploid (G) CXN3 and tetraploid (H) M1CXN3-1 genotypes.
Table 6.
Thousand seed weights, germination rate, cotyledon width, cotyledon length, stem diameter and % change in genome size and 1,000 seed weights.
| Ploidy level | % Change | ||
|---|---|---|---|
| Tetraploid | Diploid | ||
| 1,000 seed weight (g) | 162.67 | 144.12 | 11.40 |
| Germination rate (%) | 65.8 | 94.8 | -30.59 |
| Cotyledon width (mm) | 27.92 | 25.89 | 7.76 |
| Cotyledon length (mm) | 39.99 | 35.53 | 12.58 |
| Hypocotyl diameter (mm) | 3.14 | 2.57 | 20.62 |

Figure 4.
Diploid N7 (A) and tetraploid M1N7-1 (B) genotypes and diploid CXN3 (C) and tetraploid M1-CXN3-1 (D) genotype plants grown in hydroponic culture for 21 days after transplanting.
Table 7.
PSD, plant height, SFW and SDW of the plants grown in hydroponic culture.
| Genotype code | Ploidy level | PSD (mm) | PL (cm) | SFW (g · plant-1) | SDW (g · plant-1) |
|---|---|---|---|---|---|
| M1Calhon Gray | Tetraploid | 5.82 b–d | 30.83 mn | 12.58 no | 1.42 op |
| M1N3 | Tetraploid | 5.61 b–d | 35.53 k–m | 21.37 k–m | 2.30 l–n |
| M1N5-1 | Tetraploid | 5.53 b–d | 67.40 c–f | 49.14 de | 4.96 d–f |
| M1N5-2 | Tetraploid | 5.57 b–d | 67.67 c–f | 37.50 f–i | 3.92 g–j |
| M1N5-3 | Tetraploid | 5.17 b–d | 73.67 cd | 39.17 f–h | 4.08 g–i |
| M1N5-4 | Tetraploid | 5.23 b–d | 70.33 cd | 39.17 f–h | 3.97 g–j |
| M1N5-5 | Tetraploid | 5.26 b–d | 54.00 e–j | 30.33 h–j | 3.21 i–k |
| M1N5-6 | Tetraploid | 4.94 b–d | 51.12 f–k | 30.83 h–j | 3.16 jl |
| M1N7-1 | Tetraploid | 5.32 b–d | 49.70 g–l | 23.33 j–m | 2.45 k–n |
| M1N7-2 | Tetraploid | 4.88 cd | 65.05 c–g | 25.00 j–m | 2.68 k–m |
| M1N7-3 | Tetraploid | 5.19 b–d | 64.25 c–h | 40.83 e–g | 4.21 f–h |
| M1N7-4 | Tetraploid | 5.38 b–d | 75.65 c | 43.33 e–g | 4.51 e–h |
| M1CXN3-1 | Tetraploid | 6.05 b–d | 46.67 i–m | 30.83 h–j | 3.19 jk |
| M1CXN3-2 | Tetraploid | 5.90 b–d | 48.18 h–l | 58.33 c | 5.93 c |
| M1CXN3-3 | Tetraploid | 5.77 b–d | 67.67 c–f | 45.00 e–g | 4.68 e–h |
| M1CXN3-4 | Tetraploid | 6.13 b–d | 52.02 f–k | 25.83 j–l | 2.69 k–m |
| M1CXN5-1 | Tetraploid | 6.48 b–d | 45.47 j–m | 46.33 ef | 4.75 e–g |
| M1CXN7-1 | Tetraploid | 6.12 b–d | 64.00 c–h | 44.67 e–g | 4.62 e–h |
| M1CXN7-2 | Tetraploid | 5.93 b–d | 34.55 l–n | 40.17 fg | 4.07 g–i |
| M1CXN7-3 | Tetraploid | 5.82 b–d | 27.67 no | 36.35 g–i | 3.81 h–j |
| M1CXN7-4 | Tetraploid | 6.05 b–d | 60.62 c–j | 55.00 cd | 5.65 cd |
| M1CXN7-5 | Tetraploid | 6.53 b–d | 62.17 c–i | 49.33 de | 5.10 d–f |
| Calhoun Gray | Diploid | 6.07 b–d | 17.83 o–r | 5.08 op | 0.67 pr |
| CXN3 | Diploid | 3.86 d | 15.55 o–r | 3.60 p | 0.41 r |
| CXN5 | Diploid | 5.48 b–d | 44.67 j–m | 30.83 h–j | 3.25 i–k |
| CXN7 | Diploid | 5.07 b–d | 58.52 d–j | 29.67 k | 3.13 jl |
| N3 | Diploid | 4.76 d | 26.22 n–p | 16.52 mn | 1.70 no |
| N5 | Diploid | 4.96 b-d | 37.23 k–n | 20.82 mn | 2.26 mn |
| N7 | Diploid | 4.11 d | 30.83 mn | 17.33 l–n | 1.81 no |
| C. tide | Citrullus lanatus | 2.79 d | 11.00 p–r | 2.72 p | 0.39 r |
| RS841 | C.maxima × C. moschata | 9.73 a | 95.83 b | 84.00 a | 8.58 a |
| Argentario | Lagenaria siceraria | 9.62 ab | 115.83 a | 73.33 b | 7.43 b |
Table 8.
RFW, RDW, RL, RV and RD of the plants grown in hydroponic culture for 21 days after transplanting.
| Genotype code | Ploidy level | RFW (g · plant-1) | RDW (g · plant-1) | RL (cm) | RV (cm3 · plant-1) | RD (mm) |
|---|---|---|---|---|---|---|
| M1Calhon Gray | Tetraploid | 9.81 mn | 1.15 pr | 1,319.16 m | 3.31 l–n | 0.40 g |
| M1N3 | Tetraploid | 10.50 l–n | 1.22 o–r | 1,702.86 m | 1.82 o | 0.37 i |
| M1N5-1 | Tetraploid | 22.13 fg | 2.26 h–k | 4,818.78 ef | 3.52 k–m | 0.44 f |
| M1N5-2 | Tetraploid | 22.94 d–f | 2.46 f–i | 3,570.95 g–k | 3.85 kl | 0.38 i |
| M1N5-3 | Tetraploid | 23.61 d–f | 2.53 e–h | 5,175.32 de | 5.92 gh | 0.53 c |
| M1N5-4 | Tetraploid | 26.78 c–e | 2.73 d–g | 3,470.66 h–l | 5.25 h–j | 0.44 f |
| M1N5-5 | Tetraploid | 20.26 f–h | 2.20 h–k | 3,514.55 h–k | 4.32 l | 0.40 g |
| M1N5-6 | Tetraploid | 23.48 d–f | 2.43 f–i | 3,660.05 g–j | 4.48 i–k | 0.58 a |
| M1N7-1 | Tetraploid | 13.97 k–m | 1.52 n–p | 4,046.80 f–i | 4.44 i–k | 0.37 i |
| M1N7-2 | Tetraploid | 14.51 j–l | 1.63 m–o | 2,764.20 kl | 3.95 kl | 0.55 b |
| M1N7-3 | Tetraploid | 20.65 f–h | 2.19 h–k | 3,722.38 g–j | 5.38 h–j | 0.44 f |
| M1N7-4 | Tetraploid | 21.48 fg | 2.33 g–j | 6,083.45 c | 2.81 mn | 0.37 i |
| M1CXN3-1 | Tetraploid | 15.73 i–k | 1.68 l–n | 2,658.33 l | 4.35 j–l | 0.46 e |
| M1CXN3-2 | Tetraploid | 28.37 c | 2.93 de | 4,223.48 f–h | 6.53 fg | 0.46 e |
| M1CXN3-3 | Tetraploid | 22.50 fg | 2.43 f–i | 3,333.78 i–l | 6.67 fg | 0.51 d |
| M1CXN3-4 | Tetraploid | 18.24 g–j | 1.93 j–n | 2,905.59 j–l | 3.79 k–m | 0.40 g |
| M1CXN5-1 | Tetraploid | 33.02 b | 3.41 bc | 7,678.59 b | 11.09 b | 0.44 f |
| M1CXN7-1 | Tetraploid | 27.09 cd | 2.86 d–f | 6,001.24 c | 7.40 d–f | 0.40 g |
| M1CXN7-2 | Tetraploid | 19.91 f–i | 2.05 i–m | 4,416.07 e–g | 4.50 i–k | 0.35 j |
| M1CXN7-3 | Tetraploid | 20.60 f–h | 2.23 h–k | 3,658.58 g–j | 7.28 ef | 0.51 d |
| M1CXN7-4 | Tetraploid | 28.94 c | 3.04 c–e | 4,104.75 f–i | 8.21 de | 0.51 d |
| M1CXN7-5 | Tetraploid | 22.82 d–f | 2.45 f–i | 5,825.66 cd | 8.29 d | 0.44 f |
| Calhoun Gray | Diploid | 3.16 o | 0.48 s | 344.85 n | 0.78 p | 0.44 f |
| CXN3 | Diploid | 1.70 o | 0.22 s | 173.10 n | 0.27 p | 0.46 e |
| CXN5 | Diploid | 19.29 f–i | 2.09 h–l | 3,645.02 g–j | 4.22 l | 0.40 g |
| CXN7 | Diploid | 16.70 h–k | 1.83 k–n | 4,428.47 e–g | 5.51 hi | 0.40 g |
| N3 | Diploid | 8.37 n | 0.89 r | 1,454.43 m | 1.82 o | 0.40 g |
| N5 | Diploid | 13.90 k–n | 1.57 n–p | 3,591.94 g–k | 4.31 l | 0.40 g |
| N7 | Diploid | 10.77 n | 1.16 pr | 1,719.21 m | 2.44 no | 0.44 f |
| C. tide | Citrullus lanatus | 1.19 o | 0.24 s | 148.66 n | 0.32 p | 0.37 i |
| RS841 | C. maxima × C. moschata | 38.17 a | 4.00 a | 11,098.84 a | 13.04 a | 0.39 h |
| Argentario | Lagenaria siceraria | 34.00 b | 3.50 b | 11,000.44 a | 9.70 c | 0.34 k |

Figure 5.
PCA of diploid (D) and tetraploid (T) watermelon genotypes based on hydroponic culture morphological features. PCA, principal component analysis; PL, plant length; PSD, plant stem diameter; RD, root diameter; RDW, root dry weight; RFW, root fresh weight; RL, root length; RV, root volume; SDW, shoot dry weight; SFW, shoot fresh weight.
Table 9.
Pearson’s correlation coefficients (r values) between plant growth parameters and direct (DNA content) and indirect (stomatal measurement) ploidy determination methods in 22 tetraploid and seven diploid watermelon genotypes.
| PSD | PL | SFW | SDW | RFW | RDW | RL | RV | RD | |
|---|---|---|---|---|---|---|---|---|---|
| DNA content | 0.33* | 0.48* | 0.60** | 0.60** | 0.60** | 0.61** | 0.45* | 0.47* | -0.02ns |
| Stomatal diameter | –0.15ns | –0.49* | –0.56** | –0.56** | -0.58** | -0.58** | -0.53** | -0.44* | 0.26ns |
| Stomatal length | 0.26ns | 0.50** | 0.49* | 0.49* | 0.52* | 0.53* | 0.38* | 0.32* | -0.15ns |
| Stomatal density | 0.30ns | 0.50** | 0.55** | 0.55** | 0.59** | 0.59** | 0.43* | 0.40* | -0.12ns |
| Chloroplast number | –0.19ns | 0.19ns | –0.10 ns | –0.10 ns | 0.02ns | 0.01ns | -0.05ns | -0.24ns | -0.26ns |