Morphological Diversity of Chrysanthemum ( Dendranthema grandiflora Tzvelev) Genotypes induced by EMS ( Ethyl methane sulfonate )

. Ethyl methane sulfonate (EMS) is one of chemical mutagen which it known to cause point mutations in plants. The mutation was changing in one or some character based on Basa nucleotide. The changing shown by morphological characteristic to create novelties and superior characters. This study aim was to evaluate nine chrysanthemum genotypes of the MV4 generation by EMS treatment. The observation parameters were quantitative and qualitative characters. The quantitative charactera are number of flower buds, flower diameter (cm), flower stalk length (cm), leaf length (cm), leaf width (cm), plant height (cm), and stem diameter (cm). And the qualitative ones are stem, leaf, and ribbon flower colour. Morphological character analysis used the Statistical Tools for Agricultural Research (STAR) statistical program. One-way ANOVA was used to compare the means of different genotypes, and means were separated using Duncan’s Multiple Range Test (DMRT) at a significance level of 0.05. The highest plant, stem diameter, and flower stalk length were obtained genotype JE0 as a contol plant. Genotype JE4.8 as a selected plant showed the diameter of flower buds, number of flower buds, leaf length, leaf width and stem diameter. JE2.44 (Purple group 75 A) and JE5.31 (Red purple group 60 A) were significantly different of colour JE0 as control (Red purple group 70 B).


Introduction
Chrysanthemum (Dendranthema grandiflora Tzvelev) is a cut flowers from the Asteraceae family, originally from China and spread worldwide, including Indonesia.In Indonesia, chrysanthemum is one of the high-value ornamental plants, especially as a cut flower [1].
The highest commercial value of chrysanthemum lies in the diversity of flower colors and shapes [2].The high commercial value has driven the continuous development of this ornamental plant through plant breeding programs [3].Breeding on chrysanthemum plants has been carried out through conventional methods of crossing.Conventional breeding is the mainstay for producing high-yielding varieties, but this method has limitations that depend on weather conditions.Sunny weather and sufficient sunlight make applying stamens to the pistil head easier.Conversely, if the weather conditions are cloudy, the irradiation of the flowers becomes less.This condition makes the cider box difficult to break and the pollen difficult to obtain.As an alternative to conventional breeding, the method used is mutation breeding method.Through mutation, induction can increase the frequency of mutations.One of the methods used is mutation breeding which is highly effective for trait improvement [4].Mutations in plants can occur naturally, but mutation agents can accelerate the mutation process.Mutation agents can be physical or chemical mutagens.One of the chemicals commonly used as a mutation agent is Ethyl Methane Sulfonate (EMS) which is a commonly used chemical agent for mutations known to induce point mutations [5].Thus, it is used to alter one or several plant traits.
Mutations can occur in some parts of the plant (chimeras) or the entire plant [6].These changes can be visually observed by comparing the traits of the donor plant with its offspring genotype.Mutations can result in changes in both quantitative and qualitative plant traits.Quantitative trait changes include leaf length, leaf width and plant height, stem diameter.Qualitative trait changes include leaf color, flower color, flower shape, leaf shape, and stem shape.
The desired changes in traits are improvements or distinctive characteristics compared to the donor plant.Trait improvements include yield production, plant quality, biotic and abiotic stress resistance [7].The distinctive traits of the donor plant are more easily observed physically, such as flower color.Flower color is a key factor for consumers in determining their preferences [8].In chrysanthemum flowers, the chemical mutagen EMS has successfully resulted in color changes [9].Treating the chemical mutagen EMS is one step in producing superior chrysanthemum varieties.Despite the successful use of the chemical mutagen EMS to induce color changes in chrysanthemum flowers, there is a need to evaluate the genotype diversity of chrysanthemum callus regeneration resulting from EMS treatment.Therefore, this research aims to evaluate the genotype diversity of chrysanthemum callus regeneration resulting from the treatment of the chemical mutagen.

Materials and methods
The research was conducted at the Indonesian Ornamental Plants Research Institute in Cianjur, West Java, Indonesia.The study took place at an altitude of 1100 m above sea level, from January 2020 to August 2021.Research uses callus as the basic material.The research stages included the formation of the donor plant population, callus initiation from leaf explants, callus induction using EMS solution, shoot initiation (early-generation vegetative mutant or MV0), subculture of chrysanthemum plantlets from MV1 to MV4 (Vegetative Mutants, 1st-4th generation), acclimatization, field planting, characterization, and evaluation.Chrysanthemum planting in the field followed the production cultivation standards of the Indonesian Ornamental Plants Research Institute with some modifications [10].
Callus is obtained from the formation of leaf explants of chrysanthemums of the Jaguar Pink variety (J).The study used a one-factor completely randomized design (CRD).Each treatment unit consists of 20 calluses repeated three times, so the total number of calluses required for treatment is 360.After the callus is formed, a soaking process is carried out using an EMS solution consisting of six levels of EMS dose: E0 (0%); E1 (0.2%); E2 (0.4%); E3 (0.6%); E4 (0.8%); and E5 (1%).
In the acclimatization phase, the plantlets are maintained for one month until the plants are ready to move to the field, characterized by root growth with a length of 1-2 cm.In the planting phase in the field, genotypes are selected based on the phenotypic appearance of plants, especially the appearance of flowers.In the planting phase in the field, genotypes are selected based on the phenotypic appearance of plants, especially the appearance of flowers.Nine genotypes were selected, namely JE2.34; JE2.44; JE2.62; JE3.21; JE4.8; JE4.10; JE4.23; JE5.31 and JE5.33 as well as the use of JE0 used as a control.The selected genotype and one comparison plant were observed with as many as three plants per test (three repeats), so that the total number of plants observed was 90.Complete information on the materials used in the study until selected genotypes are obtained is presented in Table 1.
The observed plant traits consisted of quantitative and qualitative characteristics.Quantitative traits included number of flower buds, flower diameter (cm), flower stalk length (cm), leaf length (cm), leaf width (cm), plant height (cm), and stem diameter (cm).Qualitative traits included stem color, leaf color, and ray flower color.Qualitative observations were conducted by comparing the observed plant parts with the RHS (Royal Horticultural Society) color chart.The morphological characterization data of the plants were collected using Microsoft Excel 2016 and further analyzed using the Statistical Tools for Agricultural Research (STAR) analysis program.A one-way ANOVA was utilized for contrasting various genotypes, followed by the execution of Duncan's Multiple Range Test (DMRT) as a subsequent test, with a statistical significance threshold set at 0.05.
Furthermore, cluster analysis was employed to group the chrysanthemum genotypes based on their similarity to the parent or donor plant.Cluster analysis is a statistical technique that identifies patterns and relationships within a dataset by grouping similar data points.It is beneficial for identifying clusters or subgroups within a larger population.This method helps identify distinct groups and can assist in selecting genotypes with desirable traits for further breeding or development purposes.Analysis of similarity between genotypes using Gower's dissimilarity method and clustering methods using average linkage (PBSTAT-CL 2.1.2)

Result and discussion
The EMS is known to be effective as a mutation agent [11].It has also been found to be effective in ornamental plants [12].The effectiveness of EMS on chrysanthemum callus can be observed morphologically in the regenerated genotypes.Morphological observations were conducted on chrysanthemum genotypes based on quantitative and qualitative traits.

Quantitative traits
Observations were carried out 6 months after planting when the chrysanthemum flowers had fully bloomed.The results of the quantitative traits are presented in Table 2. Observations on several chrysanthemum traits showed genetic diversity resulting from using EMS on the Jaguar Pink callus as the donor plant.Observations were conducted on nine genotypes derived from the donor plant and one genotype as the donor plant control (JE0).Based on the observations, JE0 had a plant height with an average of 89.53 cm, which was higher compared to its offspring genotypes.There was no significant difference in stem diameter between the control genotype (JE0) and some of its offspring genotypes, with an average diameter ranging from 0.50-0.64cm.However, a genotype, JE2.34, had a minor stem diameter with an average size of 0.35 cm.
Regarding leaf length and width, genotype JE4.8 showed superior measurements to other genotypes and the control plant (JE0).JE4.8 had a leaf length with an average of 9.40 cm and a leaf width of 6.17 cm.For the number of flower buds produced, genotypes JE2.62, JE4.8, JE4.10, and JE5.31 had the highest average number of flower buds, ranging from 13-14 buds.These four genotypes had a higher average number of flower buds than the control plant (JE0).Furthermore, genotype JE4.8 showed superior traits to the control plant and other genotypes regarding flower bud diameter.Genotype JE4.8 had an average diameter of 6.68 cm.Regarding flower stalk length, the control plant (JE0) had the most extended average flower stalk length, with an average of 5.73 cm, which was longer than the other genotypes.
The use of EMS on chrysanthemum resulted in genetic diversity based on quantitative traits such as number of flower buds, flower diameter (cm), flower stalk length (cm), leaf length (cm), leaf width (cm), plant height (cm), and stem diameter (cm).
The use of 0.77% EMS on chrysanthemum shoot tips led to diversity in quantitative traits like plant height [13].EMS application on Snapdragon plants (Antirrhinum majus) successfully increased genetic diversity [14].

Qualitative traits
The qualitative traits of the ten chrysanthemum genotypes resulting from the EMS chemical mutagen treatment, along with one genotype as the control plant, were observed based on stem color, leaf color, and ray flower color (Table 2).The stem color varied from the yellow-green group to the green group.The yellow-green stem color accounted for 80% of the genotypes, while the green stem color accounted for 20%.For leaf color, it also varied from the yellow-green group to the green group.The yellow-green leaf color constituted 20% of the genotypes, while the green leaf color accounted for 80%.As for the ray flower color, it resulted in shades of reddish-purple and purple (Figure 1).The reddish-purple ray flower color made up 90% of the genotypes, while the purple ray flower color accounted for 10%.The use of EMS solution on chrysanthemum callus has generated genotypes with variation in ray flower color.In line with these results, the use of 0.77% EMS concentration resulted in ray flower colors such as yellow, white, light-pink, pink-salmon, salmon and malachite [9].In China aster plants (Callistephus chinensis L.), EMS has produced mutants with darker flower colors compared to the donor plants at a concentration of 0.75% EMS [15].JE3.21 (EMS 0.6%); f.JE4.8 (EMS 0.8%); g.JE4.10 (EMS 0.8%); h.JE4.23 (EMS 0.8%); i. JE5.31 (EMS 1%); j.JE5.5.33 (EMS 1%).

Analysis clusters based on quantitative and qualitative characters
The next analysis is to cluster the genotypes based on the similarity of each genotype to the parent or donor plant.Cluster analysis is a method of grouping data based on their similarity or the small differences among the data [16].In other words, cluster analysis is an analysis to group data based on similarity levels [17].The cluster analysis results based on the quantitative and qualitative traits of chrysanthemum divided the 10 genotypes into three clusters.The cluster analysis results showed a dissimilarity coefficient of 38% among the plants (Figure 2).

Fig. 2. Cluster analysis of ten chrysanthemum genotypes based on quantitative and qualitative characters
The first cluster combines four mutant genotypes with the donor or control plant (JE0).The members of this cluster are JE2.44,JE3.21, JE4.8, and JE4.10.The dissimilarity coefficient within the first cluster is 28.These four genotypes were generated from different EMS soaking treatments.JE2.44 is a genotype resulting from callus regeneration after soaking in a 0.4% EMS solution.JE3.21 is a genotype resulting from callus regeneration after soaking in a 0.6% EMS solution.Genotypes JE4.8 and JE4.10 are genotypes resulting from callus regeneration after soaking in an 0.8% EMS solution.
The second cluster combines three genotypes, namely JE2.62, JE3.34, and JE4.23.The dissimilarity coefficient within the second cluster is 26%.The genotypes in the second cluster were generated from different EMS soaking treatments.Genotype JE2.62 is a result of callus regeneration after soaking in a 0.4% EMS solution.JE3.34 is a result of callus regeneration after soaking in a 0.6% EMS solution.Genotype JE4.23 is a result of callus regeneration after soaking in an 0.8% EMS solution.The third cluster combines two genotypes, JE5.31 and JE5.33.This cluster has a dissimilarity coefficient of 32%.Genotypes JE5.31 and JE5.33 are genotypes resulting from callus regeneration after soaking in an 0.8% EMS solution.
The diversity of genotypes resulting from multiple EMS concentration levels proves that mutational traits are random.However, using EMS can produce high-intensity mutants [18].In vegetatively propagated plants, mutation induction effectively produces mutant plants.In ornamental crops, mutations increase novelty values such as changes in color, shape and size of flowers [19].Moreover, to obtain stable mutants, the multiplication of shoots up to the fourth generation (MV4) [20].

Conclusion
The evaluation results of the ten genotypes obtained from chrysanthemum callus regeneration after EMS soaking treatment showed diversity in both quantitative and qualitative traits.Based on the quantitative morphological measurements, the JE0 genotype as the control plant excelled in plant height, stem diameter, and flower stalk length.Genotype JE4.8 as a selected plant showed superior traits in number of lower buds, flower bud diameter, stem diameter, leaf length, and leaf width.In terms of qualitative traits, genotypes JE2.44 (Purple group 75 A) and JE5.31 (Red purple group 60 A) exhibited distinct colors compared to the control plant (JE0), which belongs to the Red purple group 70 B.
The author would like to express gratitude to the late Ir.Kurnia Yuniarto, MP, and colleagues at the Ministry of Agriculture for their assistance, facilities, and support throughout the research process.

Table 1 .
Information on the use of materials used in research until selected genotypes are obtained.

Table 2 .
Quantitative character of the chrysanthemum genotype resulting from callus regeneration after EMS treatment (leaf length); LW (leaf width); PH (plant height); SD (stem diameter).Any numbers in the same column that share a common letter do not display significant differences per the Duncan Multiple Range Test (DMRT) at a 5% level.

Table 3 .
Qualitative character of the chrysanthemum genotype resulting from callus regeneration after EMS treatment