**The Confirmation of a Ploidy Periclinal Chimera of the Meiwa Kumquat (***Fortunella crassifolia* **Swingle) Induced by Colchicine Treatment to Nucellar Embryos and Its Morphological Characteristics**

**Tsunaki Nukaya 1,**†**, Miki Sudo 1,**†**, Masaki Yahata 1,\*, Tomohiro Ohta 1, Akiyoshi Tominaga 1, Hiroo Mukai 1, Kiichi Yasuda 2 and Hisato Kunitake 3**

1 Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan; t\_nukaya54@yahoo.co.jp (T.N.); sudo.miki@shizuoka.ac.jp (M.S.); tomohiro1\_ota@pref.shizuoka.lg.jp (T.O.); tominaga.akiyoshi@shizuoka.ac.jp(A.T.);mukai.hiroo@shizuoka.ac.jp(H.M.)


Received: 26 August 2019; Accepted: 12 September 2019; Published: 18 September 2019

**Abstract:** A ploidy chimera of the Meiwa kumquat (*Fortunella crassifolia* Swingle), which had been induced by treating the nucellar embryos with colchicine, and had diploid (2n = 2x = 18) and tetraploid (2n = 4x = 36) cells, was examined for its ploidy level, morphological characteristics, and sizes of its cells in its leaves, flowers, and fruits to reveal the ploidy level of each histogenic layer. Furthermore, the chimera was crossed with the diploid kumquat to evaluate the ploidy level of its reproductive organs. The morphological characteristics and the sizes of the cells in the leaves, flowers, and fruits of the chimera were similar to those of the tetraploid Meiwa kumquat and the ploidy periclinal chimera known as "Yubeni," with diploids in the histogenic layer I (L1) and tetraploids in the histogenic layer II (L2) and III (L3). However, the epidermis derived from the L1 of the chimera showed the same result as the diploid Meiwa kumquat in all organs and cells. The sexual organs derived from the L2 of the chimera were significantly larger than those of the diploid. Moreover, the ploidy level of the seedlings obtained from the chimera was mostly tetraploid. In the midrib derived from the L3, the chimera displayed the fluorescence intensity of a tetraploid by flow cytometric analysis and had the same size of the cells as the tetraploid and the Yubeni. According to these results, the chimera is thought to be a ploidy periclinal chimera with diploid cells in the outermost layer (L1) and tetraploid cells in the inner layers (L2 and L3) of the shoot apical meristem. The chimera had desirable fruit traits for a kumquat such as a thick pericarp, a high sugar content, and a small number of developed seeds. Furthermore, triploid progenies were obtained from reciprocal crosses between the chimera and diploid kumquat.

**Keywords:** anatomy; citrus; flow cytometry; histogenic layer; polyploidy breeding
