Nuclear DNA content as an indicator of inflorescence colour stability of in vitro propagated solid and chimera mutants of chrysanthemum

In chrysanthemum, breeders seek for desirable characteristics of the inflorescence, which can first be established once the plant is mature. The present study aims to determine whether measurement of DNA content can be useful in the detection of somaclonal variants and/or separation of chimera components in chrysanthemum at the early in vitro multiplication stage. Eleven Chrysanthemum × morifolium (Ramat.) Hemsl. cultivars of the Lady group (a mother cultivar and ten of its radiomutants obtained by X-ray- or γ-irradiation; solid and periclinal chimeras) were propagated in vitro. Single-node explants were cultured in Murashige and Skoog (MS) medium, either without plant growth regulators (PGRs) or supplemented with 6-benzyladenine (BA) and indole-3-acetic acid (IAA). The nuclear DNA content was measured by flow cytometry (FCM) in the shoots produced in vitro. After acclimatization and growth of the plants in a glasshouse, inflorescence colour was recorded. The addition of PGRs to the medium almost doubled the mean number of shoots produced in vitro per explant, but caused a change in inflorescence colour of all (‘Lady Apricot’; periclinal chimera) or part of the plants (‘Lady Amber’; solid mutant and ‘Lady Salmon’; periclinal chimera). All radiomutants contained less DNA than the mother cultivar ‘Richmond’. There were significant differences in DNA content between plants of the same cultivar grown in media with or without PGRs for ‘Lady Apricot’ and ‘Lady Salmon’, but no phenotype alternation occurred in chrysanthemums produced in PGR-free medium compared to the original cultivars. Conversely, in medium with PGRs, chimeras produced flowers different from the original colour. In all except one cultivar (‘Lady Amber’; solid mutant) a lack of differences in genome size between plants grown in either medium coincided with a stable inflorescence colour. The occurrence of some plants of ‘Lady Amber’ with different inflorescence colour may be due to small DNA changes, undetectable by FCM. It can be concluded that FCM analysis of DNA content in young plantlets can be indicative of the stability of inflorescence colour in chrysanthemum, especially chimeric cultivars, and for mutant detection.

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

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.