*2.1. Plant Materials*

The 2x+4x ploidy chimera, induced by applying a colchicine treatment to nucellar embryos of the Meiwa kumquat [14], was used in the present study. The original diploid Meiwa kumquat, the tetraploid induced by treating nucellar embryos of the Meiwa kumquat with colchicine, and the ploidy periclinal chimera mutant Yubeni, which originated from the Meiwa kumquat and has diploids in L1 and tetraploids in L2 and L3, were used as the control. These plant materials were grafted onto the trifoliate orange and grown in 45 L containers for approximately five years in the greenhouse of the Faculty of Agriculture, Shizuoka University, Shizuoka, Japan.

### *2.2. Confirmation of Ploidy Level by Flow Cyotometry*

Approximately 50 mg segments of whole leaf, midrib, petal, filament, style, ovary, seed, juice sac, albedo, and flavedo were collected from each plant material. Their samples were chopped with a razor blade and blended for 5 min with a 2 mL bu ffer solution containing 1.0% (*v*/*v*) Triton X-100, 140 mM mercaptoethanol, 50 mM Na2SO3, and 50 mM Tris-HCl at pH 7.5, according to the preparation method of Yahata et al. [15]. An aliquot (550 μL) of each sample was filtered through Miracloth (Merck KGaA, Drarmstadt, Germany), and the filtrate was stained with 50 μL of 0.5 g L−<sup>1</sup> propidium iodide (PI). The relative fluorescence intensity of the nuclear DNA was measured with a flow cytometry system (FCM, EPICS XL; Beckman Coulter, Inc., Pasadena, CA, USA) equipped with an argon laser (488 nm, 15 mW).

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

The characteristics of fully expanded leaves (e.g., blade size, thickness, guard cell size, guard cell density, and cell sizes) and flowers just before bloom (e.g., the size of the flower bud, petal, pistil, ovary, and pollen, and the number of petals and stamens) were measured. Guard cells and pollen grains were observed using a scanning electron microscope (Miniscope ® TM3030Plus, Hitachi High-Technologies, Tokyo, Japan). The epidermises, palisade parenchymas, spongy parenchymas, vessels, and sieve tubes were used to measure the cell size. According to the methods of Nii et al. [16], their semi-ultra thin sections (1.5 μm) were cut using a glass knife before staining with methylene blue for histological examination under an optical microscope BX51 (Olympus Co., Ltd., Tokyo, Japan). Images were taken with a DP70 digital camera (Olympus). The weight, size, pericarp weight, the number of locules and seeds, soluble solids content (SSC), and titratable acidity (TA) of each kind of fruit were measured at the end of January. Each measurement used 20 samples.

Pollen fertility was evaluated by stainability and in vitro germination. Pollen stainability was estimated by staining the samples with 1% acetocarmine after squashing nearly mature anthers on a glass slide. In vitro germination of the pollen grains was performed on microscope slides covered with a 2 mm layer of 1% ( *w*/*v*) agar medium containing 10% sucrose. Five stamens, each from di fferent flowers, were rubbed on the agar medium, and the slides were then incubated for 10 h in a moistened chamber at 25 ◦C in the dark. Each test was evaluated from 500 grains with five repetitions.

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

The cross combinations are shown in Table 6. The flowers were pollinated immediately after emasculation and covered with para ffin paper bags. Seeds were collected from each mature fruit of all the crosses and were classified according to their size and shape into two groups: developed or undeveloped. After being numbered and weighed, both the developed and undeveloped seeds were cultured onMurashige and Skoog (MS) medium [17] containing 500 mg L−<sup>1</sup> malt extract, 30 g L−<sup>1</sup> sucrose, and 2 g L−<sup>1</sup> gellan gum at 25 ◦C under continuous illumination (38 μmol m<sup>−</sup><sup>2</sup> s<sup>−</sup>1). After germination, the seedlings were transplanted into vermiculite in pots and were transferred to a greenhouse. Ploidy analysis of the seedlings was performed by FCM using young leaves and chromosome observation using root tips according to the methods of Fukui [18] with some modifications.
