**3. Results**

### *3.1. Confirmation of the Ploidy Level by FCM*

The fluorescence intensities of all the examined tissues and organs of the diploid and the tetraploid Meiwa kumquats showed only diploid and tetraploid DNA values, respectively. In the 2x+4x ploidy chimera (Table 1, Figure 1), the whole leaf, petal, filament, style, ovary, and flavedo had both a diploid peak and a tetraploid peak, although the juice sac was diploid, and the midrib, seed, and albedo were tetraploid, respectively. Furthermore, the chimera showed the same fluorescence intensities as the Yubeni.


**Table 1.** Flow cytometric analysis of each organ and tissue in the diploid, the tetraploid, the Yubeni, and the chimera of the Meiwa kumquat.

**Figure 1.** Flow cytometric analysis of each organ and tissue in leaves, flowers, seeds, and fruits of the chimera of the Meiwa kumquat. **A**: Leaf, **B**: Midrib, **C**: Petal, **D**: Filament, **E**: Style, **F**: Ovary, **G**: Seed, **H**: Juice sac, **I**: Albedo, **J**: Flavedo.

### *3.2. The Characteristics of Leaves, Flowers, Pollen, and Fruits in the Chimera*

The morphological characteristics of the chimera were compared with those of the diploid, the tetraploid, and the Yubeni. The chimera had significantly rounder and thicker leaves as compared to those of the diploid, and it was mostly equal to those of the tetraploid and the Yubeni (Table 2, Figure 2A, Figure 3). On the other hand, the size and density of guard cells in the chimera was mostly equal to those of the diploid and the Yubeni (Figure 4). The epidermic cell size of the chimera showed the same value as the diploid and the Yubeni (Table 3, Figure 3). On the other hand, the palisade parenchyma and spongy parenchyma cells of the chimera were shown to be mostly the same sizes as the tetraploid and the Yubeni. Additionally, there were no differences in the cell sizes of the vessels and sieve tubes in midribs among the tetraploid, the Yubeni, and the chimera (Figure 5).

**Table 2.** Comparison of morphological characteristics of leaves in the diploid, the tetraploid, the Yubeni, and the chimera of the Meiwa kumquat.


y Length of leaf blade/width of leaf blade; z different letters represent significant differences in Tukey's multiple test, 1% level.

**Figure 2.** Comparison of the morphological characteristics of leaves (**A**, bar = 5 cm), flowers (**B**, bar = 1 cm), and fruits (**C**, bar = 3 cm) in the diploid, the tetraploid, the Yubeni, and the chimera of the Meiwa kumquat.

**Figure 3.** Transversal sections of leaves in the diploid (**A**), the tetraploid (**B**), the Yubeni (**C**), and the chimera (**D**) of the Meiwa kumquat. Bar = 100 μm. EP: Epidermis, PP: Palisade parenchyma, SP: Spongy parenchyma.

**Figure 4.** Scanning electron micrographs of guard cells in the diploid (**A**), the tetraploid (**B**), the Yubeni (**C**), and the chimera (**D**) of the Meiwa kumquat. Bars = 30 μm.

**Figure 5.** Transversal sections of midribs in the diploid (**A**), the tetraploid (**B**), the Yubeni (**C**), and the chimera (**D**) of the Meiwa kumquat. Bars = 100 μm. VE: Vessel, ST: Sieve tube.


**Table 3.** Comparison of the cell sizes of leaves in the diploid, the tetraploid, the Yubeni, and the chimera of the Meiwa kumquat.

z Different letters represent significant differences in Tukey's multiple test, 1% level.

The chimera had significantly larger flower buds and ovaries as compared to those of the diploid (Table 4, Figure 2B). No di fference in flower morphology was observed among the tetraploid, the Yubeni, and the chimera. The average size of the pollen grains from the chimera was larger than that of the grains from the diploid (Table 4, Figure 6). The pollen fertility of the chimera was significantly lower than that of the diploid and was about the same as those of the tetraploid and the Yubeni.

**Figure 6.** Scanning electron micrographs of pollen grains in the diploid ( **A**), the tetraploid (**B**), the Yubeni ( **C**), and the chimera ( **D**) of the Meiwa kumquat. Bars = 30 μm.

There was no significant di fference in the size of the fruits (Table 5, Figure 2C). However, the percentage of the pericarp weight per fruit in the tetraploid, the Yubeni, and the chimera was significantly higher in comparison to that of the diploid. The average number of seeds per fruit obtained from the diploid was 5.7, whereas that of the chimera was significantly lesser at 2.6. SSC in the pericarp and juice sac of the chimera was significantly higher than that of the diploid.

### *3.3. Crossing for the Evaluation of the Reproductive Organs of the Chimera*

In order to evaluate the ploidy level in the reproductive organs of the chimera, crossing with the diploid Meiwa kumquat was carried out (Table 6). When the diploid was pollinated with pollen of the chimera, the frequency of developed seeds (23.1%) was lower than that of the self-pollinated fruit (86.8%). On the other hand, the frequency of developed seeds in the chimera was 86.7% when reverse-crossed, whereas that in the self-pollinated fruit was 89.2%. These developed seeds cultured on an MS medium germinated normally. The ploidy levels of these seedlings were confirmed by FCM analysis and chromosome observation (Table 6, Figure 7). Consequently, 8 and 12 triploid seedlings were obtained from crosses between the diploid and the chimera and from the reverse cross, respectively (Figure 8). Moreover, when the chimera was used as a seed parent, most of the seedlings were tetraploids.



z (Diameter of fruit/height of fruit) × 100. y Different letters represent significant differences in Tukey's multiple test, 1% level. Wt.: weight.


z NS: no significant difference. \* mean is significantly different at 1% levels by *t*-test. y (No. of developed seeds/No. of seeds) × 100. Av.: average.
