Improvement of German Chamomile (Matricaria recutita L.) for Mechanical Harvesting, High Flower Yield and Essential Oil Content Using Physical and Chemical Mutagenesis

Chamomile (Matricariarecutita L.) is one of the most important medicinal plants with various applications. The flowers and flower heads are the main organs inthe production of essential oil. The essential improvement goals of chamomile are considered to be high flower yield and oil content, as well asthe suitability for mechanical harvesting. The present study aimed to improve the flower yield, oil content and mechanical harvestability of German chamomile via chemical and physical mutagens. Three German chamomile populations (Fayum, Benysuif and Menia) were irradiated with 100, 200, 300 and 400 Gray doses of gamma rays, as well as chemically mutagenized using 0.001, 0.002 and 0.003 mol/mL of sodium azide for 4 h. The two mutagens produced a wide range of changes in the flowers’ shape and size. At M3 generation, 18 mutants (11 from gamma irradiation and 7 from sodium azide mutagenization) were selected and morphologically characterized. Five out of eighteen mutants were selected for morphological and chemical characterization for oil content, oil composition and oil quality in M4 generation. Two promising mutants, F/LF5-2-1 and B/HNOF 8-4-2, were selected based on their performance in most studied traits during three generations, as well as the high percentage of cut efficiency and a homogenous flower horizon, which qualify them as suitable candidates for mechanical harvesting. The two mutants are late flowering elite mutants; the F/LF5-2-1 mutant possessed the highest oil content (1.77%) and number of flowers/plant (1595), while the second promising B/HNOF 8-4-2 mutant hada high oil content (1.29%) and chamazulene percentage (13.98%) compared to control plants. These results suggest that the B/HNOF 8-4-2 and F/LF5-2-1 mutants could be integrated as potential parents into breeding programs for a high number of flowers, high oil content, oil composition and oil color traits for German chamomile improvement.


Introduction
Matricaria L., a member of the Asteraceae family, is considered one of the most widely used medicinal plants around the world. Its origins can be traced back to the near east as

Characterization of M 2 Populations
The air-dried seeds of the three populations were irradiated with 100 Gy, 200 Gy, 300 Gy and 400 Gy of gamma rays. The dose rate was 5.6 Gy/minute. In a separate experiment the seeds were treated with sodium azide (NaN 3 ) with 0.001, 0.002 and 0.003 mol/mL of SA for four hours. In the M 2 population, 1180 plants were screened for the mutagenicity effects of gamma rays and 810 1180 plants were screened for the mutagenicity effects of sodium azide. M 2 populations that contained various morphological mutants were obtained from the three populations of German chamomile. These mutants involved traits affecting plant height traits (tall, dwarf, semi-dwarf), large stem diameter, the high number of branches, early flowering, late flowering and the high number of flowers (Table 1). Treatments with gamma rays and sodium azide mutagens produced a varied number of mutations; 37 mutants were produced for plant height (dwarf, semi-dwarf and tall), 38 mutants involved traits affecting early and late flowering, in addition to 25 mutants with a high number of flowers. Results showed that low doses of gamma rays (100 Gy and 200 Gy) produced higher mutation frequency than higher doses (300 Gy and 400 Gy). In contrast, higher concentrations of sodium azide (0.003 mol/mL and 0.002 mol/mL) gave higher mutation frequency while the lower concentration (0.001 mol/mL) produced lower mutation frequency. We also found that gamma rays had a lower mutation frequency (15.94%) than the mutation frequency scored by sodium azide (16.2%).
To reduce the blossoming stages for increasing mechanical harvesting efficiency, the selection of mutations suitable for mechanical harvesting using cut efficiency percentage was applied on M 2 populations (Table S2). The results recorded on M 2 plants (Table S2) indicated that mutations obtained from Benysuif populations were more suitable for mechanical harvesting than the Menia and Fayoum populations because of late flowering compared to Menia and Fayoum populations. The best kind of mutations suitable for mechanical harvesting were late flowering mutants followed by the high number of flowers followed by tall plants and the high number of buds, due to the sum of both blossoming stages (three and four) making up more than 95% of total flower yield. Dwarf, semi-dwarf and early flowering mutants were not suitable for mechanical harvesting.

Characterization of M 3 Mutants
The performance stability of selected M 3 mutants from different classes of the populations were evaluated for numerous morphological features, oil content and oil colors ( Table 2). The results showed that the late flowering mutants possessed the highest values for flower dry weight and number of flowers in all three populations. The results also showed that the mutants of the high number of flowers possessed high values for flowers' dry weight and the number of flowers in the two populations Benysuif and Menia (Table 2).
In the Fayoum population, the F/LF 5-2 mutant showed the highest performance in morphological features and oil content (1.77%) and the oil colors were very light blue, so it is considered a desirable mutant ( Table 2). In the same context, in the Benysuif population, the B/HNOF8-4 mutant possessed the highest performance in all studied traits except the number of branches and days to flowering, and the oil color was very light blue. Meanwhile, in the Menia population, M/HNOF 4-1 presented the highest performance in all studied traits except stem diameter, the number of branches and days to flowering. The results of M 3 generation also confirmed that early flowering mutants showed stability in contrast to days to flowering, which showed low stability in Benysuif and Menia populations.
Economically, the traits of the number of flowers and oil content are of great importance to be selected during mutation breeding of German chamomile populations. In the Fayoum population, the F/LF 5-2 mutant possessed the highest values of those characteristics. In the Benysuif population, B/HNOF 4-3 and B/HNOF 8-4 mutants were the best, while in the Menia population, the early flowering mutants M/EF 4-1 and M/EF 5-2 performed the same.

Characterization of M 4 Mutants
Five promising M 4 mutants, F/LF 5-2-1, F/HNOF3-1-1, B/HNOF 4-3-1, B/HNOF8-4-2 and M/HNOF 4-1-1 (Figure 1), were selected from M 3 populations to evaluate their stability and their homogeneity regarding morphological characteristics, oil content, oil colors (Table 3) and cut efficiency percentage ( Figure 2). The results showed that the five promising mutants in M 4 generation presented close values to M 3 generation, especially B/HNOF 8-4-2, M/HNOF4-1-1, F/LF5-2-1 and B/HNOF4-3-1, respectively. Notes: Values (mean ± SE) with different letters in the same column are significantly different at p < 0.05 and vice versa. Different letters in the same column refer to the significant difference among genotypes at p < 0.05. Green color refers to the two promising mutants that could be integrated as potential parents into breeding programs.   The results also showed that the five promising mutants gave a similar cut efficiency percentage at M 3 and M 4 generations and were suitable for mechanical harvesting in three stages, in other words, reducing blossoming stages from four to three stages ( Figure 2). B/HNOF 8-4-2 and F/LF5-2-1, out of the five promising mutants evaluated in M4, possessed the highest values of flower fresh weight (g), flower dry weight (g), number of flowers, plant height (cm), stem diameter (mm), number of branches, days to flowering, oil content (%) and oil colors.

Essential Oil Composition Analysis of M 4 Selected Mutants
Gamma irradiation and sodium azide not only affected the oil content but also the oil composition of chamomile essential oil in the five M 4 promising mutants. The basic composition of chamomile essential oil was recorded in Table 4. A total of 46 compounds were identified, which accounted for 95.46-100% of the total amount of oil. The main constituents found in the essential oils as detected by GC-MS were bisabolol oxide A (33.19-47.32%), bisabolone oxide A (1.34-12.36%), bisabolol oxide B (1.2-20.62%) and chamazulene (1.58-13.98%). The oil composition of the studied mutants was quite different, whereas other components appeared in amounts less than 2%. Generally, the examined mutants were clustered in two main groups: one concerning chamazulene and the second one concerning high α-bisabolol oxide A, B and chamazulene content. The analysis of the essential oil constituents revealed a low content of bisabolol and chamazulene in the M/HNOF 4-1-1 mutant where the chamazulene was absent, while in the F/LF 5-2-1mutant, the yield of bisabolol and chamazulene was equal or even surpassed the respective of the control. Concerning the percentage of bisabolol, mutant B/HNOF 4-3-1 outyielded all other mutants, estimated at 53.81%, followed by F/HNOF 3-1-1 (52.58). For chamazulene, the B/HNOF 8-4-2 mutant exhibited the greatest value (13.93%), followed by the F/HNOF 3-1-1 mutant (2.74%). The control presented 47.05% and 1.58% for bisabolol and chamazulene, respectively. It is evident that the highest chamazulene percentage, 13.

Discussion
It has been established that both radiation and chemical mutagens (gamma rays and sodium azide) play a role in enhancing genetic variations of studied characters in German chamomile. Total mutations, also known as heritable changes to the genetic material, can occur at the chromosomal level or as point mutations [19]. It has been found that several chemical and physical mutagens cause variability for economic features in various crops [14,20]. In programs to improve various crop plants through mutation breeding, useful morphological mutations are crucial. Numerous researchers have concluded that gene mutations and chromosomal aberrations are the causes of morphological mutations [21].The morphological mutations concerning flowers' fresh weight, flowers' dry weight, number of flowers/plant, plant height, stem diameter, number of branches, days to flowering, oil content % and oil colors were noticed in the M 2 generation of the three populations of German chamomile. Even though the majority of morphological mutants are not economically viable, many of them can be useful and utilized in crossbreeding programs or to improve quantitative traits, track crop evolution and conduct gene mapping investigations [22][23][24][25]. The frequency of various mutant types may be caused by various mutagens and treatment procedures [26]. The pleiotropic effects of the defective genes lead to morphological mutations [27]. The three populations of German chamomile varied in the frequency of morphological mutations. In contrast to Benysuef and Menia populations, the Fayoum population displayed a higher frequency of morphological mutants, demonstrating the inter-ecotype response to various doses and concentrations of physical and chemical mutagens. A report on the inter-varietal response to mutagen treatments was made by Gottschalk [27] in barley. The results of the current investigation showed that the M 2 populations of chamomile contained tall mutants. Additionally, tall mutants were noted by Solanki, et al. [28] in Lens culinaris Medik. and Kumar, et al. [29] in Mungo L. using different mutagens. Dwarf mutants observed in the present study had short internodes, which could be due to a reduction in cell number and cell length. These dwarf mutants have also been reported in Vigna mungo L. Hepper [30], grasspea (Lathyrus sativus L.) [31] and barley (Hordeum vulgare L.) [32]. The dwarf mutants resulted due to a decrease in internode number or internode length. A decline in plant height may be caused by altered gibberellic acid or uneven mitotic divisions, according to several studies [33,34]. In wheat, the semi-dwarf mutant character is controlled by polygenes [35]. According to numerous researchers, many morphological mutations, such as tallness and dwarfism, are monogenic and recessive [36][37][38].
Based on our results, it is clear that gamma rays and sodium azide mutagens induced high variations in morphological features, essential oil content, fresh and dry flower yield and flowering periods depending upon the two mutagen types. Additionally, it was noted that the two mutagens also caused changes in oil composition and oil color in several mutants, including dark blue, light blue, dark yellow and light brown.
The cut efficiency percentage at M 3 and M 4 generations for all mutants also confirms the previous results that the late flowering mutants promise great results in the third and fourth cuts. These results are confirmed by the work of Albrecht et al. [39] who studied the breeding of a new variety of chamomile to increase the blossom product yield in up to three harvest stages through a homogenous flower horizon (pick height) and even flowering time.
We conclude from the previous results that most late flowering and the high number of flower mutants at the M 4 generation possess high performance in addition to high oil content. These results are confirmed by Lal, et al. [14] who produced a new variety of chamomile to increase flower yield and essential oil content.
The results showed that the five promising mutants were found to be similar in cut efficiency percentage at M 3 and M 4 generations. Thus, they are suitable for mechanical harvesting in three stages.
Interestingly, as a result of greater mutagenic efficacy and massive screening in field trials especially, the high percentage of cut efficiency and a homogenous flower horizon for the selected mutants led to reducing the significant loss in flower yield, which makes them suitable for mechanical harvesting, especially the late flowering elite mutant (F/LF5-2-1) that possesses the highest oil content and number of flowers, and the B/HNOF 8-4-2 mutant that possesses high oil content as well as high chamazulene percentage.
Despite the possibility that this study will support traditional breeding programs [40] for the genetic improvement of German chamomile, more recent genetic approaches such as genetic engineering [41], genome editing approaches [42,43], molecular markers [44][45][46][47] and phylogeny analysis [48][49][50][51] are important to determine the genetic diversity among different species, which could effectively be utilized to breed and develop chamomile for many desirable traits.

Mutagen Agents
The seeds of the different populations of chamomile were treated with gamma radiation and sodium azide (NaN 3 ) as follows:

Physical Mutagen
The air-dried seeds of each population were irradiated with 100 Gy, 200 Gy, 300 Gy and 400 Gy of gamma rays with a radioisotope Co 60 source (Gamma chamber Model-900 supplied by Nuclear Research Center, Inshas, Egypt). The dose rate was 5.6 Gy/minute.

Chemical Mutagen
Separately, the seeds were treated with sodium azide (NaN 3 ); one gram of seeds of each population was treated with 0.001, 0.002 and 0.003 mol/mL of SA for four hours with intermittent shaking at room temperature, washed for an hour under running water, then immediately sown.

Agronomic Practices and Data Collection
As presented in Figure 3, the irradiated, as well as sodium azide, treated seeds of each population with control seeds were sown individually in rows in an incubator in the middle of August. Forty-five-day-old seedlings were transplanted in the open field.Surface irrigation was supplied every 3 weeks and after each cut. Routine agricultural practices were carried out as usually practiced in chamomile cultivation. During the seedling stage and before the flowering stages, weeds were pull out by hand. German chamomile is not a demanding crop in terms of fertilization. The plants were harvested separately in each treatment. M 3 seeds from M 2 selected mutants were grown in three rows with three replications for M 3 generation, as well as the control of each population. In the 2021/2022 season, the seeds of M 4 plants from selected mutants were sown in three replications with their parents to evaluate mutants.

Statistical Analyses
The data recorded on different traits resulting from mutagens treatments were subjected to statistical analysis to find the individual and comparative effects of different mutagens. The mean, standard deviation and the LSD0.05were calculated using IBM SPSS statistics software version 22.

Conclusions
Chamomile is in high demand on the global market because of its vast medical uses and excellent pharmacological characteristics. Additionally, the usage of natural compounds rather than synthetic chemicals has increased. The present study was conducted to induce mutations suitable for mechanical harvesting, high flower productivity, oil content and oil quality in three German chamomile populations using sodium azide and gamma radiation treatments. Different morphological mutants of the three populations' M2 population were isolated. These mutants involved traits affecting the flowers' fresh weight, flowers' dry weight, number of flowers, plant height, stem diameter, number of branches, days to flowering, oil content % and oil colors. Most of the useful mutations obtained in the M3 generation resulted from using gamma rays (11 mutants) instead of the 7 mutants obtained using sodium azide. Five promising mutants were selected in the M4 generation based on their characteristics, especially flower yield, oil content and oil quality. Two out of five mutants (F/LF5-2-1 and B/HNOF 8-4-2) could be integrated as

Essential Oil Analyses
The essential oil content of air-dried flowers was determined using Clavenger's apparatus for determining oil quality in M 3 and M 4 selected mutants according to the method described in Santich [52]. Hydro distillation of 20 g of dried inflorescences takes four hours. The percentage of oil content was calculated as the average value of three measures. The essential oils were stored at 4 • C until analysis. Essential oil samples produced from M 4 selected mutants (F/LF 5-2-1, F/HNOF 3-1-1, B/HNOF 4-3-1, B/HNO F 8-4-2 and M/HNOF 4-1-1) were subjected to GC-MS investigation to identify their oil composition. A percentage of the total chromatographic area was used to compute the relative content of each component. The compounds were identified based on a comparison of retention indices (RI) concerning n-alkanes (C7-C22), with relevant literature data and by matching their spectra with those of MS libraries (NIST 98, Willey, DuPage, USA) [53].

Statistical Analyses
The data recorded on different traits resulting from mutagens treatments were subjected to statistical analysis to find the individual and comparative effects of different mutagens. The mean, standard deviation and the LSD 0 . 05 were calculated using IBM SPSS statistics software version 22.

Conclusions
Chamomile is in high demand on the global market because of its vast medical uses and excellent pharmacological characteristics. Additionally, the usage of natural compounds rather than synthetic chemicals has increased. The present study was conducted to induce mutations suitable for mechanical harvesting, high flower productivity, oil content and oil quality in three German chamomile populations using sodium azide and gamma radiation treatments. Different morphological mutants of the three populations' M 2 population were isolated. These mutants involved traits affecting the flowers' fresh weight, flowers' dry weight, number of flowers, plant height, stem diameter, number of branches, days to flowering, oil content % and oil colors. Most of the useful mutations obtained in the M 3 generation resulted from using gamma rays (11 mutants) instead of the 7 mutants obtained using sodium azide. Five promising mutants were selected in the M 4 generation based on their characteristics, especially flower yield, oil content and oil quality. Two out of five mutants (F/LF5-2-1 and B/HNOF 8-4-2) could be integrated as potential parents into breeding programs for a high number of flowers, high oil content and oil quality traits. The selected desirable mutants will directly be used for commercial production after registration.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants11212940/s1, Table S1: The characterization of morphological characteristics for selected mutants, i.e., flowers' fresh weight, flowers' dry weight, number of flowers, plant height, stem diameter, number of branches and days to flowering in M 2 generation of Egyptian chamomile ecotypes; Table S2: Flowers' cut efficiency percentage of dwarf, semi-dwarf, tall, big stemdiameter, high number of branches, early flowers, late flowers and high number of flowers isolated mutants of M 2 generation of German chamomile ecotypes.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding authors.