**1. Introduction**

The shoot apical meristem of higher plants consists of three histogenic layers [1]. This is known as the "Tunica-Corpus" theory; i.e., the shoot apical meristem consists of L1 (Tunica) and L2/L3 (Corpus). In the genus *Citrus* and its related genera, the histogenic layers are di fferentiated by their parts: the dermal system (guard cell and juice sac) in L1, parenchyma and reproductive organs (mesophyll cell, pollen and seed) in L2, and the vascular bundle (cambium and pith) in L3, respectively [2]. A plant is considered to be a chimera if it has two or more genetic constitutions in its shoot apical meristem,

and chimeras are classified into three types: sectorial, periclinal, and mericlinal [3,4]. Sectorial chimeras have a sector of all cell layers that is genetically different. Periclinal chimeras are chimeras in which one or more entire cell layer(s) is genetically distinct from another cell layer. Mericlinal chimeras have part of one or more layers that is genetically different.

In the genus *Citrus*, spontaneously arising chimeras have been reported previously; i.e., autogenous chimeras such as the "Suzuki wase" satsuma mandarin (*C. unshiu* Marcow.) and the "Thompson" grapefruit (*C. paradisi* Macfad.) [5,6] and graft chimeras such as the "Kobayashi mikan" [Natsudaidai (*C. natsudaidai* Hayata) + Satsuma mandarin], the "Kinkoji unshiu" [Kinkoji (*C. obovoidea* hort. ex Takahashi) + Satsuma mandarin], and the "Zaohong" navel orange ["Robertson" navel orange (*C. sinensis* Osbeck var. *Brasiliensis* Tanaka) + Satsuma mandarin] [7,8] are well known. All of these cultivars are periclinal chimeras. In recent years, production of synthetic graft chimeras has been successful [9,10], and many periclinal graft chimera cultivars have been registered in Japan. Furthermore, 2x+4x ploidy chimeras were produced by treating the apical meristems, undeveloped ovules, calluses, and protoplasts with antimitotic agents [11]. These were then used as breeding materials for triploid production in the genus *Citrus*.

In kumquats (*Fortunella* spp.), Yubeni which have a large fruit size and a high sugar content was discovered to be a bud mutation of the Meiwa kumquat. Yasuda et al. [12] demonstrated that Yubeni was 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, and showed that the Yubeni increase in fruit quality was due to the tetraploidization in L2 and L3. Furthermore, this result showed that the 2x–4x–4x ploidy periclinal chimera kumquat could be used both for the parental line in triploid breeding and for direct domestication. However, since the ploidy chimera kumquat has rarely been reported [12], information on their characteristics is lacking. To prove the future usefulness of the ploidy chimera kumquat, it is necessary to evaluate more kinds and collect information.

Yahata et al. [13] produced many tetraploid Meiwa kumquats by applying a colchicine treatment to nucellar embryos. After they were grafted onto the trifoliate oranges [*Poncirus trifoliata* (L.) Raf.], they showed vigorous growth and they flowered and fruited for the first time, five years after budding. Nukaya et al. [14] examined whether these plants maintained tetraploidy, and found a 2x+4x ploidy chimera among their tetraploids. If this ploidy chimera is a ploidy peripheral chimera with the same histogenic layer as the Yubeni (layer constitution: L1–L2–L3 = 2x–4x–4x), it would be very useful in future kumquat breeding.

To clarify the ploidy level of each histogenic layer in the 2x+4x ploidy chimera, in the present study, we performed ploidy analysis by flow cytometry, cell observation using histological techniques and morphological characteristics in several tissues and organs of this ploidy chimera, and an evaluation of the reproductive organs of the chimera by crossing it with the diploid kumquat.
