Low Temperature Treatment Affects Concentration and Distribution of Chrysanthemum Stunt Viroid in Argyranthemum

Abstract

Chrysanthemum stunt viroid (CSVd) can infect Argyranthemum and cause serious economic loss. Low temperature treatment combined with meristem culture has been applied to eradicate viroids from their hosts, but without success in eliminating CSVd from diseased Argyranthemum. The objectives of this work were to investigate (1) the effect of low temperature treatment combined with meristem culture on elimination of CSVd, (2) the effect of low temperature treatment on CSVd distribution pattern in shoot apical meristem (SAM), and (3) CSVd distribution in flowers and stems of two infected Argyranthemum cultivars. After treatment with low temperature combined with meristem tip culture, two CSVd-free plants were found in 'Border Dark Red', but none in 'Yellow Empire'. With the help of in situ hybridization, we found that CSVd distribution patterns in the SAM showed no changes in diseased 'Yellow Empire' following 5°C treatment, compared with non-treated plants. However, the CSVd-free area in SAM was enlarged in diseased 'Border Dark Red' following prolonged 5°C treatment. Localization of CSVd in the flowers and stems of infected 'Border Dark Red' and 'Yellow Empire' indicated that seeds could not transmit CSVd in these two cultivars, and CSVd existed in phloem. Results obtained in the study contributed to better understanding of the distribution of CSVd in systemically infected plants and the combination of low temperature treatment and meristem tip culture for production of viroid-free plants.

Keywords: CSVd; in situ hybridization; meristem culture; shoot apical meristem; viroid localization.

FIGURE 1

In situ localization of CSVd in infected in vitro culture of Argyranthemum ‘Border Dark Red’ and A. ‘Yellow Empire’ after different periods of 5°C treatment. (A) Shoot apical meristem (SAM) of ‘Border Dark Red’ at room temperature. (B–E) SAM of ‘Border Dark Red’ after 1, 2, 3, and 6 months 5°C treatment, respectively. (F) SAM of ‘Yellow Empire’ at room temperature. (G–J) SAM of ‘Yellow Empire’ after 1, 2, 3, and 6 months 5°C treatment, respectively. (K) SAM of healthy ‘Border Dark Red’. (L) Negative control with no probe. Inserts indicate higher magnification of AD of (A–K). Scale bars: (A–L) 200 μm, inserts 50 μm.

FIGURE 2

In situ hybridization of CSVd in flowers of Argyranthemum cultivars. (A) Cross-section of flower of CSVd-infected ‘Border Dark Red’. Insert indicates higher magnification of ovary. (B) Cross-section of flower of CSVd-infected ‘Yellow Empire’. Insert indicates higher magnification of ovary. Scale bars are 200 μm. Inserts: 50 μm.

FIGURE 3

In situ hybridization of CSVd in stems of Argyranthemum cultivars. (A,B) Cross sections of stems of CSVd-infected ‘Border Dark Red’ and ‘Yellow Empire’. Inserts indicate higher magnification. (C) Longitudinal sections of ‘Border Dark Red’ stems. (D) Longitudinal sections of ‘Yellow Empire’ stems. (E) Longitudinal sections of healthy ‘Border Dark Red’ stems. All are applied with anti-sense probe; Red arrows indicate CSVd localization. epi, epidermal cells; phl, phloem; xyl, xylem. Scale bars: (A,B) 310 μm, (C–E) 100 μm.