Introduction

Flower color is important in ornamental plant breeding because of its effect on consumer demand (Su et al., 2019; Park and Jeong, 2019; Hoang et al., 2020). Chrysanthemum is one of the most important cultivated ornamental plants in South Korea due to its diverse flower colors, shapes, sizes, and long vase life which make it an exemplary plant to work with because it can be kept as a potted plant or used as a cut flower at any time of the year using photoperiod regulation (Poole, 1962; Sadhukhan et al., 2015; Lee et al., 2019). Developing new varieties of plants is a challenge in modern and industrialized horticulture. The potential for generating different forms and thereby remodeling chrysanthemum is boundless, which is a primary goal of most breeders (Momin et al., 2012).

Initially, breeders choose improved phenotypes of valuable quality without knowing the plants' genetic constitution. Mutation induction is a powerful tool for developing new and novel plant germplasms, and radiation is a remarkable mutation breeding method that yields superior cultivars in contrast to conventional breeding, which is time-consuming and laborious with inadequately induced genetic diversification (Beyaz and Yildiz, 2017; Anne and Lim, 2020). In ornamental horticulture, radiation has been used to alter flower color, shape, type of inflorescence, fertility, leaf color, or variegation (Brunner, 1995). Many flower cultivars have originated through spontaneous and induced mutations using gamma irradiation, ion beam irradiation, X-rays, and chemical mutagenesis to transform the color, shape, and petals of flower which is beneficial for mutation initiation in chrysanthemum (Yamaguchi et al., 2008; Matsumura et al., 2010). When chrysanthemum plants are exposed to different doses of gamma irradiation, the color of the inflorescence changes as a result of changes in the pigment content, and this increases the decorative value of cultivars (Datta and Gupta, 1981).

Mutation breeding has been used in vegetatively propagated ornamental plants to customize one or more traits in a superior cultivar without modifying its other phenotypic traits (Ibrahim et al., 2018; Su et al., 2019). In addition, in radiation technology, different dose rates are one of the most effective parameters by which the response rate can be measured by assessing the distinct physiological characteristics, including survivability, lethality, growth, reproduction, and yield. Likewise, the dose rate also controls radiation damage in chrysanthemums (Yamashita 1964; Killion and Constantin 1971; Killion et al., 1971; Broertijes et al., 1976; Yamaguchi et al., 2008; Anne and Lim, 2020). It has been reported that higher dose rates of gamma irradiation were more effective in inducing mutations than lower dose rates, high-yielding mutants with profitable traits (Nishiyama et al., 1966).

Among all physical mutagens, gamma radiation is more preferable because it is sparsely ionizing, deeply penetrating and non-particulate. The treatment is applied on seeds, seedlings, cuttings, any plant part, and also on in vitro plantlets (Patil and Patil, 2009). This study analyzed the effect of different doses of gamma irradiation on chrysanthemum by observing the morphological characteristics of treated cultivars and established high-yielding mutants with profitable traits.

Materials and Methods

The experiment was conducted with 11 chrysanthemum cultivars. The conventional method of propagation was used to gather terminal stem cuttings, about 7－8 cm in length, which were taken from healthy explants. Stem cuttings were exposed by 0, 10, 30, 50, 70, 100, and 150 Gy gamma irradiation at the Korean Atomic Energy Institute and were transplanted in the greenhouse of the Sejong University experimental center situated in Gwangju city, South Korea. Each treatment was replicated three times with 30 cuttings each. Data were recorded separately based on the different vegetative and floral characteristics. The morphological data were organized using Microsoft Office Excel (version 2013, Microsoft Co., Redmond, WA, USA). All data were analyzed using one-way analysis of variance (ANOVA) and Duncan’s multiple range test using SPSS software (version 20. Armonk, NY, USA). The data of survival (21 days after exposure to gamma irradiation) and flowering rates were transformed to percentages before statistical normality or variation in cultivars and mutants were observed in the M1V1 generation plants in different gamma-ray doses were also recorded. The results data were presented as the mean ± standard error (SE). Mutation frequency was calculated using the total number of mutants divided by investigated plants multiplied by 100.

Results

Effects of Gamma Radiation on Survival Percentage of Plants

A decrease in survival rate was detected with an increase in the gamma irradiation dose. The data presented in Fig. 1 reveal that the survival of irradiated shoots was affected by gamma-ray doses, where a significantly higher survival percentage was observed in the control (0 Gy) of all cultivars. The mean differences in the survival percentage between 10 and 150 Gy were statistically significant. A lethal effect of higher doses of 150 Gy was noted on the survival percentage. Less than 50 % of the population survived among ‘Raina’, ‘Olga pink’, ‘Sevda’, ‘Heimish’, ‘Rania’, ‘Attirance’, and ‘Leporem’ chrysanthemum cultivars.

Fig. 1.

Effect of gamma irradiation on survival percentage of chrysanthemum cultivars. Bars represent mean ± SE (standard error at p < 0.05).

Effects of Gamma Radiation on the Vegetative Characteristics of Chrysanthemum

To assess the significance of morphology and mutations, such as plant height, stem diameter, leaf number, total number of leaves, leaf length and width, number of buds, leaf color variation, and leaf abnormality, among 11 chrysanthemum cultivars, we ascertained the growth and developmental characteristics as well as the induction of mutant plants of in vivo culture of the M1V1 generation. Plant height was significantly reduced in all the chrysanthemum cultivars compared to the control, and significant variation in plant height was also observed among the 11 chrysanthemum cultivars (Table 1). All cultivars showed similar results after low to high irradiation exposure. It was observed that plants were more sensitive to 100 and 150 Gy doses and showed dwarfism. Based on the data in Table 1, it can be assumed that all 11 cultivars showed dwarfism after high gamma irradiation (150 Gy) compared with the control. Data in Table 1 showed average stem diameter, which was observed to be insignificant among all the cultivars. Lower irradiation dose does not cause plants to produce more than one branch, but various numbers of branches were observed at 100 and 150 Gy in all cultivars. It can be assumed that gamma irradiation did not affect the stem diameter significantly. In addition, the average number of leaves, leaf length, and leaf width were also insignificant. Leaf number varied from cultivar to cultivar and also yielded different unstable results at different doses. After exposure to gamma irradiation, 100 and 150 Gy doses resulted in a smaller number of leaf initiations compared to the other doses. In addition, leaf length and leaf width did not vary among the cultivars in terms of irradiation. Leaf color variation was observed with increasing doses of gamma irradiation. Among all cultivars, no leaf color variation was observed in the control. The highest average leaf color variation was observed from those treated with 150 Gy irradiation. The amount of leaf abnormalities (deform shape) for each of the 11 cultivars at 100 and 150 Gy was noted. The highest average leaf abnormalities were observed at 150 Gy irradiation doses in all cultivars compared to the control and no leaf abnormalities were found among control cultivars. As shown in Table 1, every cultivar responded the same way after exposure to gamma irradiation, and no significant differences were observed at higher irradiation doses (100 and 150 Gy) but a significant difference was found between the control and irradiated cultivars.

Effects of Gamma Radiation on Flower Characteristics

Flower characteristics of in vivo culture plantlets are presented in Table 2, which demonstrated consequential differences concerning flower initiation days from planting to flowering, flower number, flower diameter, petal number, petal length, and width between the control and the treated cultivars. The higher radiation dose resulted in a significant reduction in the total number of petals, petal length, and petal width. Mutant flower color, shape, and size were detected as chimeras in the same cultivars within and between cultivars. Gamma rays produced mutants with various flower colors with individual doses in distinct cultivars, as shown in Table 3. Most of the mutants were taken from those induced at 30 and 70 Gy. However, only leaf cuttings treated with 150 Gy produced mutants for ‘Attirance’ cultivars. At the same time, the mutation frequency was lowest at 10 Gy and highest at 70 Gy. Table 3 highlights 61 mutants attained from 10, 30, 50, 70, and 150 Gy-treated cultivars. From cultivar ‘Aileen’ the control was observed to be light pink, while mutants were found to be light yellow, pink, and cream with spoon-shaped petals with the 30, 50, and 70 Gy treatments respectively. In ‘Attirances’ four different irradiations produced mutants that were half red and half pink, red and pink, dark pink with abnormal petal formation, and red at 30, 50, 70, and 150 Gy, respectively. In ‘Coral King’, the light pink petals in control plants were whitish pink and light-yellow, while the mutants were pink and white, pink with abnormal spoon-shaped petal formation, and white and red with petal deformation due to 30, 50, and 70 Gy, respectively. In ‘Eldora’, the orange color in the control was transformed to having flowers with color variations of half orange and half yellow, and orange and of yellow shade when treated with 10 and 30 Gy irradiation, respectively. In addition, ‘Leporem’ was dark yellow in control and light yellow and half yellow and half orange in mutants due to 10 and 30 Gy doses, respectively. ‘Olga Pink’ was light pink in control but half white and half pink and dark pink as a result of 30 and 70 Gy doses, respectively. ‘Raina’, light pink in the control, but white and orange as a result of 30 and 70 Gy doses, respectively. Also, in ‘Rania’, the control was pink but white and pink as a result of 30 and 70 Gy doses, respectively. ‘Heimish’ was greenish white in control but white (a smaller number of petals) after 30 Gy treatment. ‘Sevda’ was dark red in control, but dark orange as a result of 70 Gy doses while ‘White plume’ was white in control but pinkish (more petal formation) after 50 Gy treatment (Fig. 3). Non-significant delays in flowering over the control and treatments were found in the control (0 Gy) of cultivars (Table 2). However, ‘Raina’, ‘Aileen’, ‘Heimish’, ‘White Plume’, and ‘Leporem’ cultivars had no flowers after 100 and 150 Gy doses ‘Sevda’ and ‘Attirance’ did not flower after 100 Gy treatment and ‘Coral King’ did not flower after 150 Gy treatment. Fig. 2 presents the variation in flowering percentage in treated plants, which was more extensive than that of the control. The highest percentage of flowering was observed in the control (0 Gy) cultivars. The flower percentage was drastically decreased at higher irradiation, particularly at 100 and 150 Gy, although ‘Raina’, ‘Aileen’, ‘Heimish’, ‘White plume’, and ‘Leporem’ cultivars did not generate any flowers because apical bud initiation stopped after higher exposure to irradiation.

Fig. 2.

Effect of gamma irradiation on flowering percentage of chrysanthemum cultivars. Bars represent mean ± SE (standard error at p < 0.05)

Fig. 3.

Mutant plants affected by different doses of gamma rays on flower color in the M1V1 generation. (a) In ‘Aileen’, light pink control plants and light yellow, pink, and light yellow generated by 30, 50, and 70 Gy, respectively. (b) In ‘Attirance’, the dark pink control where half pink and red; pink and red; pink, red, and white; and red generated by 30, 50, 70, and 150 Gy. C) In ‘Coral King’, light pink in control and whitish pink and light-yellow; pink and white, and pink, white, and red as a result of 30, 50, and 70 Gy. D) In ‘Eldora’, orange in control and half orange and half yellow, and orange and yellow generated by 10 and 30 Gy, respectively. E) In ‘Leporem’, dark yellow in control and light yellow, half yellow and half orange mutant color generated by 10 and 30 Gy, respectively. F) In ‘Olga Pink’, light pink in control and half white and half pink and dark pink as a result of 30 and 70 Gy, respectively. G) In ‘Raina’, light pink in control and white and orange color mutants generated by 30 and 70 Gy respectively. H) In ‘Rania’, pink in control, and white and pink generated by 30 and 70 Gy, respectively I) In ‘Heimish’, greenish white in control, and white (abnormal petal formation) as a result of 30 Gy. J) In ‘Sevda’, dark red in control and dark orange from 70 Gy. K) In ‘White plume’, white in control and pinkish mutant flower (more petal formation) initiated by 50 Gy.

Discussion

The comparable growth and development performance of the vegetative and flower characteristics of the M1V1 generation are shown in Tables 1 and 2. The lower dose rate of radiation generated limited damage to the plant's growth and development (Neary et al., 1957, Datta, 1990). Fig. 1 shows that the survival rates of irradiated cultivars noticeably decreased with increased exposure to doses of gamma irradiation. Patil et al. (2017), Sadhukhan et al. (2015), and Patil et al. (2015) observed a similar reduction in survival percentage with increasing doses of gamma irradiation in chrysanthemum cultivars. A significant reduction in plant height was observed with an increase in the irradiation dose. High gamma irradiation doses inactivate auxin, thereby shortening plants (Banerji and Datta, 1992; Banerji and Datta, 2002; Kapoor et al., 2014). The delay in bud initiation was eventually derived during late blooming. Datta et al. (2001) reported that the delay in flowering might be due to disturbances in the biochemical pathway, which assists in the synthesis of flower-inducing chemical substances. The irradiation alters many biochemical pathways, which may be associated with the flower orientation. These outcomes were also reported by Dilta et al. (2003) and Soliman et al. (2014). Mutants produced by higher dose rates demonstrated more flower color mutations than the control cultivars. Flower color changes may be due to either quantitative or qualitative changes in flower pigments as a result of gamma-ray-induced mutations in the biosynthesis pathways (Datta, 1990). Gamma ray-induced flower color and shape mutations in chrysanthemum have already been reported (Datta et al., 1985; Broertjes and van Harten, 1988; Datta, 1988; Datta, 1990; Datta et al., 2001). In this study, changes in petal development during flowering may be attributed to alteration in the distribution of cell division, differentiation, and elongation (van der Krol et al., 1993; Sangeeta et al., 2019). Dilta et al. (2003) also reported a considerable reduction in the petal length of chrysanthemum plants after exposure to certain doses of gamma radiation. In the M1V1 generation, 30 and 70 Gy irradiation levels developed more flower color mutants (Fig. 3). The maximum mutation frequency was obtained at 70 Gy, and the lowest frequency was recorded as 10 Gy in the first gamma-ray induced generation (Table 3). A high dosage of gamma radiation might cause plant sensitivity by impacting the number of endogenous growth regulators as a result of separation or lack of synthesis of chemical properties (Ambavane et al., 2015). This study demonstrated that conventional in vivo culture techniques can be used to customize one or two traits with gamma rays and that flower color mutants could be segregated and amplified throughout shoot cuttings to establish improved varieties of chrysanthemum. ‘Aileen’, ‘Eldora’, ‘Leporem’, ‘Olga pink’, ‘Raina’, and ‘Rania’ produced good flower quality with better shape, size, and color after exposure to various gamma irradiations. It was observed that mutants produced cultivars upon exposure with lower irradiation doses, and it has less effect on physiological developments. After observing and evaluating 61 mutants in comparison with the original varieties, we selected three cultivars, ‘Attirance’, ‘Olga pink’, and ‘Aileen’ for further study because they produced good color mutant flowers. This study successfully induced mutations in plants exposed to gamma rays. The applied dosage could be used to produce mutant plants in the future as well as to develop a variety of diverse cultivars.