MUTATION TYPES AND FREQUENCY IN NIGELLA DAMASCENA L. IN THE М 2 AND M 3 GENERATION, USING ETHYL METHANESULFONATE, NITROSOMETHYLUREA AND A NEW DERIVATIVE OF DIMETHYLSULFATE, DG-2

Aim . To identify mutations and evaluate the mutation frequency in Nigella damascena L. cultivars (cvs) Bereginya and Charivnytsya (M 2 and M 3 generation), following treatment of their seeds with ethyl methanesulfonate (EMS), nitrosomethylurea (NMU) and a new derivative of dimethyl sulfate, DG-2. Methods . Treated Nigella seeds of two cvs with the mutagens for 6 and 16 h and in concentrations of 0.01 and 0.5 % for EMS and NMU and 0.05 and 0.5 % for DG-2. Results. A wide range of mutations (59 types) was obtained, that was divided into six groups: ﬁ ve groups with changes in the morphological type and one group with changes in the physiological type. Among the detected mutations, there were both previously known mutations and those obtained in this culture for the ﬁ rst time. The highest mutation frequency (30 %) affecting synthesis of chlorophyll and structure of stem, shoots and leaves and 20 % for physiological features, was registered for NMU at 16 h and 0.05 % in cv. Bereginya. However, this NMU concentration appeared to be lethal for cv. Charivnytsya. Conclusions. The new mutagen DG-2 proved to be most effective for inducing mutations in the corolla petal color of nigella, namely 4.0 at a 0.5 % concentration of the mutagen and 16h exposure for cv. Bereginya and 4.0 % at the same concentration and exposure for cv. Charivnytsya. DG-2 caused a substantial number of mutations in all six mutation groups affecting morphological and physiological traits. The classic mutagen EMS was also effective across the spectrum of mutation groups in our study; however, it caused mutations at a lower frequency. The maximum mutation frequency under in ﬂ uence of EMC at a concentration of 0.05 % and an exposure of 16 h in cv. Bereginya was 11.0 %, and in cv. Charivnytsya 8.0 %. For all three mutagens used, an increase in the concentration of the active substance and of exposure time led to an increase in the mutation frequency in N. damascena plants. We will select mutants with economically valuable traits, such as tall, lodging-resistant plants and early maturing ones, for further work on the development of new cultivars of N. damascena for industrial cultivation.


INTRODUCTION
, which is valued for anti-microbial activity. Furthermore, damascenine, an alkaloid known for antipyretic, analgesic, and anti-edematous properties. In addition to the medicinal value, seeds of N. damascena are also used as a food preservative and spice, due to their strawberry-like scent that is determined by caproic and butyric esters (Wais et al, 2009). Presently, plants are widely used in industry for the production of different kinds of primary and secondary metabolites, which are used as dyes, fragrances, food additives, insecticides, or drugs (Hussain et al, 2012;Klimek-Chodacka et al, 2020). It was demonstrated that N. sativa has some anticancer properties due to thymoquinone, the main component of its ether oil. The second sesquiterpene, β-element, which, in its turn, is intensely biosynthesized in N. damascena, also has therapeutic potential (Helvacıoğlu et al, 2021). N. damascena seeds are still used for example in Sicilian folk medicine as a galactagogue (Geraci et al, 2018). Other uses are as emmenagogue, vermifuge, and disinfectant (Heiss and Oeggl, 2005). This plant was also used as an anthelmintic and to treat hematuria, and skin diseases in the Serbian medieval medicine (Jarić et al, 2014). The former use has also been reported in Epirus, northwestern Greece (Vokou et al, 1993). Traditionally, N. damascena is used for treating trachoma in Tunisia and Italy (Leporatti and Ghedira, 2009). Apart from its use as herbal remedy, N. damascena is used as a condiment in several regions (Heiss and Oeggl, 2005), including in Morocco (Khabbach et al, 2011;Salehi et al, 2021). A rich source of β-elemene is the essential oil of N. damascena, in which β-elemene accounts for 47 %. Antimicrobial activity of this essential oil and β-elemene (against Mycobacterium tuberculosis strain H37Ra) was established by Sieniawska et al (2019).
Due to its many benefi cial properties, the area of N. damascena cultivation has extended and exceeds at present the area of its natural occurrence considerably. Since Nigella damascena was previously used more often as an ornamental plant, and now it is gaining increasing interest as an oilseed and essential oil crop, breeding of new cultivars is necessary. To enlarge the sowing areas of nigella, it is necessary to create new highly performing cultivars with valuable traits, meeting the requirements for production by the processing industry, namely, increased performance, standing ability, early ripeness and high content of useful compounds. In order to obtain such cultivars mutagenesis, in particular induced mutagenesis, could be a solution (Lomtatidze et al, 2009).
It is important to fi nd mutagens that will have the least effect on the survival of treated plants. Along with the application of classic chemical mutagens, there are studies on new compounds capable of inducing mutations, since there is a need to induce further specifi c changes and preferably with higher frequency. In addition, the need to protect the environment determined the search for compounds with high mutagenic effects, but with lower toxicity. Since mutagens are toxic to humans, because they are teratogenic, it is necessary to adhere to safety rules when working with these substances, see for example EU Chemical Abstract Service (CAS) documentation EINECS No: 201-058-1 (https:// echa.europa.eu/documents/10162/8fcaf1f0-ea6d-4ec8-a129-f0405def7c33).
Mutations are a main cause of hereditary variation of all living organisms. Changes in the genetic material may be caused by physical (ultraviolet radiation, shortwave radiation, etc.), biological (viruses, bacteria), and chemical mutagens with much higher frequency than spontaneous mutations (Holme et al, 2019). Induced mutagenesis greatly facilitates obtaining valuable breeding material with hereditary properties and characteristics (Chaudhary et al, 2019). The creation of mutant cultivars is based on a high frequency of benefi cial mutations, mobilizing the traits unattainable for other breeding methods (Ke et al, 2019). Mutagenesis is a relevant instrument of improving resistance, yield and quality characters. Mutagenesis is an important tool in crop improvement and is free of the regulatory restrictions imposed on genetically modifi ed organisms (Kolakar et al, 2018). A direct genetic approach allows for the identifi cation of improved or new phenotypes which can be used in traditional breeding programs. Powerful reverse genetic strategies that allow the detection of induced point mutations in individuals of the mutagenized populations can address the major challenge of linking sequence information to the biological function of genes and can also identify novel variation for plant breeding (Parry et al, 2009). N-Nitroso-N-methylurea (NMU) and N-nitrosoethyl urea (NEU) supermutagens are used in selection to get enhanced induced mutation. In small concentrations these substances are used as growth stimulators. The above-mentioned super mutagens have an ability to enter the cell and cause such reconstruction of the genetic material when gene mutations are generated primarily and chromosome aberrations are insignifi cant. They frequently cause systemic mutations which serve as a basis for taxonomic differentiation. It is believed that the high genetic ability of super mutagens is caused by the sum action of such compounds on the DNA molecules. Interaction of NMU and NEU with DNA molecules, followed by regrouping of pairs of nitrogenous bases, may occur in both ways: transition and transversion. These super mutagens induce more useful mutations in agricultural plants than the known earlier mutagen agents (Lomtatidze et al, 2009).
Ethyl methanesulfonate is a chemical mutagen, which is currently being used in plant breeding, to increase genetic variability in genes of agronomic interest, of species useful in agriculture. It primarily causes single base point mutations by inducing guanine alkylation, resulting in GC to AT transitions. Its effect is different between clones of a genotype and between genotypes of the same species. (Joya-Dávila and Gutiérrez-Miceli, 2020).
New mutagens of the DG series were fi rst used in studies on fl ax by Tigova and Soroka (2018a and b;. Mutagens of the DG series (DG-2, DG-6, DG-7, and DG-9) are new chemical compounds, derivatives of dimethyl sulfate, synthesized at the Institute of Bioorganic Chemistry and Petrochemistry of the National Academy of Sciences of the Ukraine and given to us by Ph.D. Dulnev. The essence of the mutagenic action of DMS is alkylation of the DNA molecule by the incorporation of ethyl or methyl groups. It is also known from the scientifi c literature that DMS often causes chromosomal ruptures and that most of the reunions are intrachromosomal, which leads to the formation of a large number of chromosomal inversions. The mutagen DG-2, which we chose for our study, was effective in inducing fl ower and seed color mutations in a study on fl ax. Moreover, mutagen DG-2 was effective for mutations to physiological attributes of growth and development and it was most effective in changing biochemical indexes of oil in seeds (Tigova et al, 2022).
In our earlier study on N. damascena plants of the M 1 generation, it was shown that the classical mutagen nitrosomethylurea strongly affects the survival of nigella plants, while the new mutagen DG-2 had the least effect on the survival of treated plants. (Gubanova and Soroka, 2019) Aim: The study was aimed at detecting mutations and evaluating the mutation frequency in Nigella damascena L. of М 2 generation under the impact of the chemical mutagens EMS, NMU, and a new chemical mutagen DG-2, (a complex of 3-N,N dimethylaminosulfolane with dimethyl sulfate, mutagen DG-2 differs from the original compound of DMC by an additional group of sulfolane with dimethylamine, see Tigova et al, 2022) Our study intended to demonstrate the effi ciency of the mutagens studied for N. damascena, and when so them in our further research and selection.

MATERIALS AND METHODS
Air-dried seeds of Nigella damascena, cvs Bereginya and Charivnytsya, were treated with chemical mutagens -ethyl methanesulfonate (EMS), nitrosomethylurea (NMU), and a new chemical mutagen, DG-2 (a complex of 3-N,N-dimethyl aminosulfolane with dimethylsulfate) (Tigova et al, 2022). In each variant, 350 seeds were treated at one time. The seeds were placed in cotton bags and soaked in 0.01 and 0.05 % aqueous solutions of mutagens EMS and NMU, and 0.05 and 0.5 % aqueous solutions of DG-2. The use of the 10-fold higher concentration of DG-2 (which was used only once before this study) was based on our team's experience with the compound (Tigova et al, 2022). Seeds of the corresponding cultivar, soaked in distilled water, were used as the control. The exposure lasted 6 and 16 h. After the treatment, the seeds of each variant were washed for one hour in the running water and sown in rows of 2.5 m with an interrow spacing of 20 cm and a 50 cm distance between plots on the same day in an open fi eld. Prior to blossoming, the nigella plants were isolated with separate micro-perforated polypropylene bags for pollination. The plants in the experimental and control groups were observed on the experimental plots of the Institute of Oil Crops, the NAAS, in 2019-2021. The scheme of nigella sowing in the second mutant generation was as follows: row length 1.5 m, interrow distance 0.3 m, and the distance plots 0.5 m. The scheme of sowing in the third mutant generation: the row length 1 m, interrow distance 0.4 m and distance between plots 0.5 m.
The experiment was randomized and performed in triplicate. Each time numbering of seed samples before each new sowing was changed, genotypes were swapped and order of sowing changed. Only the control plot remained unchanged. The M1, M2 and M3 generation was planted in three consecutive years. The number of families in the samples corresponded to the rules of classical mutagenesis and was quite large, see Tables 1 to 4). The generations М 2 and М 3 were sown in fi eld conditions by families: family in М 2 -generation of one plant from generation М 1 (all the capsules, each capsule 5 fused true seed pods, from one isolator from one plant after self-pollination); family in М 3 -generation of one family from М 2 (after self-pollination in the isolators). As for generation М 2 , 100 families of each treatment variant were sown, except the ones which GUBANOVA had been treated with nitrosomethylurea, because it affected vitality too much. The data about the number of families in the variants of the second mutant generation are indicated in Tables 1-4. The families of М 2 , in which a percentage of mutations were lethal, were collected completely to detect again lethality in generation М 3 . During the vegetation period, there were phenological observations, the plants with changed morphologi-     cal and physiological traits were noted, the following generation was checked for the inheritance of the isolated changes. All kinds of mutations were registered at each stage of plant growth and development. Only the changes in the traits of plants, which were inherited in subsequent generations, were considered to be mutations. The mutations were isolated based on the absence of these changes in the control group plants of the corresponding cultivars in all the observed generations of the untreated plants.
The noted mutations included 1) mutations ones related to the disrupted chlorophyll synthesis, 2) mutations in the structure of stem, shoots, and leaves, the mutations in the fl ower (changes in the corolla petal color, the form of petals, and buds), 3) mutations in the structure of seed capsules and 4) mutations related to the physiological traits of growth and development. Therefore, each variant considered all the types of mutational variation of nigella and the number of plants of each type. Each plant with the mutant trait in М 2 was taken into account once.
The frequency of mutant changes was determined in per cent as the ratio between the number of mutant families and their total number in generation М 2 . The fi nal conclusion about the presence of mutations in М 2 was made after their confi rmation in generation М 3 .
The results of the observations were calculated using standard mathematical and statistical methods (Wasserman, 2004). The main statistical characteristics of the quantitative change in the investigated indices were dispersion (s2), standard deviation (s), standard error of the mean (sx), and criterion χ 2 (Wasserman, 2004).
The application of the χ 2 criterion allowed us to obtain a good approximation of the binomial criterion for all values of the manifestation of р and its absence q for tables of the 2 × 2 type, where the sample size n exceeded 50. In the case of a two-digit population, the test statistic χ 2 was defi ned as follows (1.1) where P i is the actual (empirical) frequency in the cell of the conjugation table; Q i are expected, theoretically calculated frequencies in the same cell.
The cv. Bereginya is a morph with single simple blue fl owers, cv. Charivnytsya a morph with double blue fl owers RESULTS After the nigella seeds were treated with chemical mutagens EMS and NMU in the concentrations of 0.01 and 0.05 % and mutagen DG-2 in the concentrations of 0.05 and 0.5 %, we obtained a wide spectrum of mutations in M 2 generation which was represented by 59 types of changes, divided into six groups -fi ve groups with morphological and one group with physiological changes (Tables 1-4).
To classify the mutants in our study, we used the classifi cation, developed by Morgun and Logvinenko 1995), amended by Tigova and Soroka (2019). We amended the classifi cation of Tigova and Soroka as follows: from the classifi cation, developed by Tigova and Soroka, we used the following of their mutation groups: a) disrupted chlorophyll synthesis, b) structure of stem, shoots, and leaves, and c) physiological traits of growth and development. We divided Tigova and Soroka's group of fl ower mutations into  Note. Сonc. -mutagen concentration; I -VI group -groups of mutations in M 2 generation; * -statistically signifi cant differences from control at a signifi cance level of the sum of mutations р < 0.05; ** -this concentration proved to be lethal for the seeds, treated for 16 h two groups: 1) mutations in the fl ower structure and 2) those in the corolla petal color. We also found a group of mutations not described by Tigova and Soroka (2019), namely mutations in the structure of the seed capsule.
Mostly we took the terms for the names in the classifi cation of chlorophyll mutations from the classification of Morgun and Logvinenko (1995), Holm G. (1954). Therefore, the types of mutations with similar phenotypical manifestations have Latin names in our study. Some mutations were fi rst revealed by us, so we amended the original classifi cation. For instance, we were the fi rst to detect the so-called light green margin mutation (Fig. 1) -these are the plants with fi rst white leaves and actual leaves with a light green margin at the edge. The plants were fertile and progeny was obtained from the mutants. We also obtained for the fi rst time mutants with a phenotype showing white parts randomly distributed on the stems (Fig. 2). The mutants with this trait are fertile. This trait is passed on to the offspring of mutants, but splits. The trait is also inherited by some plants from the mutant family that did not have the trait in the phenotype.
In our experiment, we discovered, as far as we know, for the fi rst time, hereditary changes such as witches' broom-like leaf bushes (Fig. 3), wid ely spaced internodes (Fig. 4, a). We discovered, to our knowledge, for the fi rst time the hereditary change in the color of apetalous morph (so-called double fl ower) of Nigella damascena -a mutation of a fl ower with a blue brim (Fig. 4, b and Fig. 5).
In the control plants of the cv. Charivnytsya (a cultivar with apetalous morph -so-called double fl ower) the fl ower, after blooming, fi lls with color as the gynoecium and androecium develop. At fi rst, the fl owers are light, gradually they turn blue and by the time the petal-like sepals fall off, they turn dark blue. In mutants with the trait fl ower with a blue margin, the light ring in the color of the fl ower is retained at the  In our experiment, we also discovered, as far as we know, for the fi rst time fl eshy petioles mutation (Fig. 6,  a, b). The fl ower of Nigella damascena is different from the fl ower of black cumin, but Datta, Biswas (1985) also noted fl ower deformations in the latter. The mutation which we called fl ower rotated 90 degrees was fi rst discovered for this species in our study (Fig. 7).
VI. Mutations in the physiological traits of growth and development (4 types): early ripe plants, sterile plants, plants resistant to lodging, and plants lodging strongly (control plants of both cultivars have medium resistance to lodging; mutants either have strong resistance to lodging, or, conversely, are lodging strongly).
Similar to the classic mutagens EMS and NMU, the mutagen DG-2 induced hereditary morphological and physiological changes with different frequencies in different groups. The effi ciency of each mutagen is in direct proportion to the frequency of the mutation induced by it. Yet the studied compounds were considerably different in the spectrum of the mutations. For instance, mutagen DG-2 was found to be most effective in obtaining the group of mutations in fl ower, including the change in the color of the corolla petals (group V) for both cv. Bereginya and cv. Charivnytsya after the longer 16h exposure (Table 2).
For the exposure of 16 h to the mutagens we obtained the following results (also see Table 2 and 4): Among all the investigated mutagens, DG-2 at 0.5 % initiated the maximum mutation frequency (causing change in the color of the corolla petals for both cultivars used at 16h exposure), namely 4 % (group V, Table 2). The frequency of the classic mutagen EMS at 0.05 % in this group was c. 2.0 % for Bereginya, and zero for the cv. Charivnytsya. It is noteworthy that EMS caused mutations in all the six groups isolated in our study at 0.05 %. In addition to fl ower mutations DG-2 also caused mutations in all other groups at 0.5 %. Mutations, affecting the change in the seed capsule structure (group IV) and disrupted chlorophyl synthesis (group I), were most frequent for DG-2 (10.0 % in both cultivars and 5 and 10 % in cv. Bereginya and cv. Charivnytsya respectively). The percentage mutations in group II (stems, shoots, leaves) were also high for DG-2, namely 8 and 13 % respectively.
Using NMU at 0.05 % and 16 h exposure, cv. Bereginya had a higher survival rate than cv. Charivnytsya. NMU had highest mutation frequency for several groups of traits, namely, for the disrupted chlorophyll synthesis (group I), 30.0 %, for the physiological traits of growth and development (group VI), 20.0 %. For group IV (capsule structure) NMU had a similar mutation frequency as DG-2 at 0.5 %, namely 10 % (Table 2).
EMS was also effective in all the groups of traits. The highest frequency of mutagens was induced by EMS at 0.05 % and 16 h exposure, variety in the structure of stem, shoots and leaves (group II 11.0 % in cv. Bereginya and 6 % in cv. Charivnytsya. In group I (disrupted chlorophyll synthesis) it was 4.0 and 8 % for the respective cultivars.

DISCUSSION
Based on the study of Datta and Biswas (Datta, Biswas, 1985) on black cumin, a close relative of Nigella damascena, we can conclude that some mutations found in our experiment, are similar to those discovered by Datta and Biswas. For instance, these were: cotyledon, divided into several parts, deformed leaves, convolute (crooked) fl ower spike and stem (Fig. 3), abnormal branching, extended leaf blade, absence of petioles on the lower leaves, multifl orants (branched, high plants, low plants, dwarfs. These are mostly mutations in the structure of the stem, shoots, and leaves. Ethyl methanesulfonate (EMS) is a classic mutagen that proved itself in the breeding practice to obtain breeding material with different characteristics. EMS was effectively used to obtain plants of the genus Nigella L. with changed morphological and physiological traits (Gilot et al, 1967;Phai, 1976;Biswas and Datta, 1983;Datta and Biswas, 1985;Asif and Ansari, 2019;Gubanova and Soroka, 2021). In our study, EMS was also found to be effi cient evoking mutations, in all six groups of traits for the cv. Bereginya, but not in group V (fl ower mutations) in cv. Charivnytsya. In our hands EMS had a lower mutation frequency than NMU or DG-2, but it has been shown that EMS in combination with X-ray treatment (Datta and Biswas, 1985, using N. damascena) or gamma-radiation (Gosh and Datta, 2005, using N. damascena;Kaul andBahn, 1977, using rice andTamilzharasi et al, 2022, using black mung bean) can enhance mutation frequency (Datta and Biswas, 1985). This could be a direction of our future research. Amin et al (2019) using N. sativa found that the frequency of morphological variants increased when increasing the mutagenic dose. The maximum frequency was observed in a combination of EMS and gamma rays. In our study, lower concentrations of this substance were used without radiation, but, as in the study of Amin et al (2019), with increasing dose, the number of mutations also increased.
Nitrosomethylurea (NMU) gave the highest percentage of mutations, but had a strong negative effect on the survival of nigella plants, that was also observed in our earlier study using M1 plants (Gubanova and Soroka, 2019). Usatov et al (2019) using similar concentrations of NMU induced effectively plastid mutations in sunfl ower. Despite its high lethality NMU induced mutations with very high frequency, up to 30 % in a considerable part of the groups of detected traits. In the study by (Phai, 1976), nitrosomethylurea proved to be one of the most effective mutagens, second only to ethyleneimine. They also used a concentration of nitrosomethylurea of 0.01 % like us, the rest of their concentrations were lower than ours. The nitrosomethylurea concentration of 0.05 % seems to be too high as it greatly affects the survival of the treated Nigella plants.
In the study of Phai (1976), nitrosomethylurea was also effective in inducing chlorophyll mutations.
DG-2 was again the most optimal mutagen, inducing mutations of morphological and physiological nature in all the groups detected by us with low lethality and this was also established in our research on the M1 generation (Gubanova and Soroka, 2019). Similar to the study of Tigova, in which mutagen DG-2 was used for the fi rst time (Tigova, Soroka, 2018a, mutagen DG-2 was found especially effective in inducing chlorophyll and fl ower mutations (color of corolla petals). Ke et al (2019) showed that non-presoaked seeds are more sensitive to EMS treatment than presoaked seeds. This is consistent with the experience that presoaking of seeds can reduce injury caused by chemical mutagens. In our study, we did not use pre-soaking the seeds. We washed the seeds in running water after treatment with mutagens.

CONCLUSIONS
A wide spectrum of inheritable mutations in Nigella damascena cvs Bereginya and Charivnytsya of the M2 and M3 generation (59 types), (divided into six groups according to the phenological manifestations at different stages of development), were obtained using three different mutagens, namely EMS, NMU and GG-2. These mutations included both well-known types and a few new types which were not previously known for this species.
The highest mutation frequency in most groups was induced by NMU. However, this mutagen at 0.05 % and 16 h exposure was found to be lethal for cv. Charivnytsya.
EMS was also found effective in our study, inducing mutations with a frequency, lower than that for NMU and the spectrum fairly similar to that of DG-2, It did not impact the change in the color of corolla petals in cv. Charivnytsya.
Noteworthy was the wide spectrum of mutations invoked by the relatively new mutant DG-2, a derivative of dimethylsulfate. It proved to be most effective in inducing the mutations of the change in the color of the fl ower corolla petals (4.0 % at a concentration of 0.5 % and 16 h exposure). The overall mutation frequency for DG-2 was 1.0-30.0 % in cv. Bereginya and 1.0-16.0 % in cv. Charivnytsya. For all three mutagens it was established that an increase in their concentration and exposure time led to increase in mutation frequency.
The mutated N. damascena material obtained in the M2 and M3 will be used in our future research where we will investigate mutants with economically useful traits. We are going to select mutants with economically valuable traits, such as tall, lodging-resistant plants and early maturing ones, in order to develop new cultivars of N. damascena for industrial cultivation.
Adherence to ethical standards. This study was performed by the author in compliance with the ethics requirements and did not envisage any research involving the participation of animals or people. Confl ict of interests. The author declared the absence of confl ict of interest and fi nancial obligations. Financing. This study was fi nanced according to the following programs of the National Academy of Agrarian Sciences of Ukraine: 15.02.01.08.PSh, Genetic variation of Nigella damascena and 18.00.00.12.PK, Genetic variation of Nigella damascena under the impact of chemical mutagenesis.