Agro-Morphological Traits and Molecular Diversity of Proso Millet (Panicum miliaceum L.) Affected by Various Colchicine Treatments

Abstract

Colchicine is a substance used to induce mutations in order to regulate important agronomic traits. The genotypes Pavlodarskoe 4, Quartet, and PI 289324, originating from Kazakhstan, the Russian Federation, and Hungary, respectively, were used as materials. The objective of this study was to investigate the effects of different colchicine concentrations (0.0, 0.04, 0.06, 0.08, and 0.1%) and treatment times (6, 12, and 24 h) on the agronomic traits of proso millet (Panicum miliaceum L.) and to assess the genetic diversity of the M₂ generation using inter simple sequence repeat (ISSR) markers. The experiment was conducted in 2021 for the M₁ generation and in 2022 for the M₂ generation, from May to September. The percentage of field germination decreased with increasing colchicine concentrations and exposure durations. The mean field germination percentages were 48.57% in Pavlodarskoe 4, 43.28% in Quartet, and 53.14% in PI 289324 under colchicine treatment. Chlorophyll-defective M₁-M₂ plants were obtained using various colchicine concentrations and exposure periods. The highest number of mutational modifications was attained with the 0.08–0.1% concentrations of colchicine. Based on the research results, a total of 248 plants with chlorophyll-defective mutations were selected from 2214 plants. The growing seasons of M₁ and M₂ plants were shortened by higher colchicine concentrations (0.08–0.1%) combined with soaking times of 12 and 24 h. Thus, the longest growing season (84 days) was observed with a 6 h treatment time for PI 289324, while the shortest (78 days) was recorded for 12 and 24 h treatments. The possibility of obtaining morphological mutations using colchicine has been confirmed. The ISSR primers amplified a total of 1333 fragments; 1281 bands were found to be polymorphic, and 52 bands were monomorphic. The percentage of polymorphism varied from 80 to 100%, with an average of 96.11%. Most of the different allelic bands were detected when applying the 0.08% colchicine concentration. These positive variations are a great opportunity to use colchicine as a tool for improving agronomic traits in plant breeding.

1. Introduction

Proso millet (Panicum miliaceum L.) is known as a tetraploid cereal (2n = 4× = 36) and is presumed to be an allotetraploid, as it has been found that exclusive bivalent formation occurs during meiosis [1,2]. Wild tetraploid ancestors of domesticated P. miliaceum have not yet been identified. Weedy forms, which may include a wild ancestor, are found throughout Eurasia, from Northeast China to the Aral–Caspian reservoir [3], in Central Europe [4], and in North America [5]. Its cultivation began 10,000 years ago in Northern China. The appearance of millet in Canada dates back to the 17th century [6]. Millet has a short growing season (60–90 days) and an exceptionally low need for water [7], requiring an average annual rainfall of less than 600 mm. For normal growth, it needs an average daily temperature above 17 °C during the growing season [3]. This species shows significant morphological variability, but the variability of isozymes or microsatellite molecular markers is low [6], which likely reflects the dual bottleneck of both polyploidization and domestication. Millet is a C₄ plant and can effectively fix carbon under conditions of drought, high temperatures, and limited amounts of nitrogen and carbon dioxide, and it is also one of the most resistant crops among spring cereals in terms of drought and heat tolerance, which is a very valuable trait for dry lands [8,9].

Mutation breeding is a comparatively quick method for the improvement of self-pollinated crops. It has been noticed that induced mutations in locally adapted genotypes are able to enhance agronomic performance, as well as other quantitative traits in plants [10]. Some mutations could induce new features that did not exist previously or that had been lost through long-term cultivation [11]. Chemical substances such as colchicine, nitrosomethyl urea, sodium azide, and many others are widely used as mutagenic agents [12].

Colchicine effectively functions as a “mitotic poison”, leading to noticeable mutagenic effects. Many studies have demonstrated the mutagenic effects of colchicine on plant performance [13]. According to these studies, a wide range of colchicine concentrations is used for the induction of polyploidy in different plant species, from the lowest concentration of 0.00001% in campion (Lychnic senno) to the extremely high concentration of 1.5% in maule’s quince (Chaenomeles japonica) [14]. Colchicine is used not only to double the set of plant chromosomes but also to induce mutations in plants. Plants that have been mutated through colchicine are known as colchi-mutants [15]. Colchicine has been used to induce some useful mutations in many plants, such as orchid (Dendrobium nobile) [16], chaste tree (Vitexagnus castus L.) [15], calendula (Calendula officinalis) [17], sultana (Impatiens walleriana) [18], gladiolus (Gladiolus grandiflorus) [19], and others [20]. Concerning millet, tetraploid foxtail millet was obtained using colchicine; the seeds were treated with 0.25% colchicine with an exposure period of four hours for the yellow sand variety [21]. Thanks to molecular markers, not only the agronomic traits of crops but also the mutagenic effects and the genetic diversity among various plant species can be evaluated more thoroughly [22]. Molecular studies of the DNA polymorphism of proso millet germplasm are mainly based on molecular markers such as RAPD, ISSR [23], AFLP [24], and SSR [25]. ISSR markers have been proven to be one of the most effective tools for genetic diversity analysis owing to their low cost, their simplicity, their reproducibility, and the lack of sequence knowledge required [26]. Due to their highly polymorphic nature, ISSR markers are extensively used [27]. Although colchicine-induced mutagenesis methods are applied to cultivated plants of various species all over the world, in the Republic of Kazakhstan, these studies have not been sufficiently conducted and have not been practically applied, especially in terms of proso millet at the molecular level [28]. The genetic diversity of a proso millet collection based on SSR markers in Kazakhstan was reported by Zargar et al. [29]. The purpose of the present study was (a) to investigate the effects of various colchicine concentrations and treatment periods on the agronomic traits of proso millet cultivated in field conditions and (b) to assess the genetic diversity resulting from the colchicine effect with the application of ISSR markers.

2. Materials and Methods

2.1. Plant Materials

Three genotypes of proso millet (Panicum miliaceum L.) were used as research materials: Pavlodarskoe 4 (Kazakhstan), Quartet (RF), and PI 289324 (Hungary).

2.2. Experimental Details and Treatments

The seeds were treated with C₂₂H₂₅NO₆ (95% (HPLC), powder, Sigma, Schnelldorf, Germany) in laboratory conditions, according to the protocol described by Swathi et al. [30]. Four different colchicine concentrations (0.04, 0.06, 0.08, and 1.0%) in combination with three seed-soaking durations (6, 12, and 24 h) were tested. Seeds amounting to 500 units were first immersed in a 12% hydrogen peroxide solution for 15 min to destroy harmful microflora on the grains, after which they were washed in distilled water three times. For seed treatments, 30 mL volumes of 0, 0.04, 0.06, 0.08, and 1.0% colchicine aqueous solutions were put in 50 mL conical tubes for 6, 12, and 24 h at room temperature, and the seeds were soaked in them. Following the treatment, the seeds were washed in distilled water and were sown in the soil in field conditions. Field experiments for the M₁ generation were carried out in the growing season of 2021 from May to September in the breeding nursery of the Baraev Scientific Production Center of Grain Farming (Shortandy village-1, Shortandy district, Akmola region, Kazakhstan) in the dry steppe zone of the Akmola region. The experiment was performed according to the All-Russian Institute of Plant Growing guidelines and the Field Experiment Methodology [31].

The total number of proso millet seeds used in the experiment was 6000 (12 treatments × 500 seeds for each treatment). Treated seeds were sown in May 2021, 250 seeds/m², with three replications. The row spacing was 20 cm, the distance between the plants was 5 cm, and the seeding depth was 3–4 cm. The ripened seeds of all selected and selfed M₁ plants from each mutagenic treatment were collected. The seeds obtained from the M₁ generation were replanted for the M₂ generation in May 2022.

2.3. Data Collection

Data for the M₁ and M₂ generations were collected in the experimental seasons. Field germination percentages were determined at the early stage of ontogenesis. In M1 and M2 plants, the following phenological observations were carried out: the phases of germination, tillering, booting, heading, and early and full ripeness were identified; the modified forms in terms of plant productivity elements were compared with the original control variant. Phenological observations of plant growth and development, as well as the plant survival rate to harvest, were made during the growing season. Productive branches (piece), seed weight per panicle (g), 1000-seed weight (g), and grain yield (c/ha⁻¹) were measured to determine phenotypic differences after harvesting. The number of seeds was calculated using “DATA Count S-25” (DATA Detection Technologies Ltd., Tzora, Israel). The counting accuracy of “DATA Count S-25” is 99.8-100%, with a rate of up to 20,000 seeds per minute, automatic product setup, and a seed size range of 0.5mm-25mm. The 1000-seed weight was calculated as the average of 10 replicates containing 100 seeds, multiplied by 10. Differences in the 1000-seed weight were obtained by using a counter, up to the maximum level [32].

2.4. Analysis of Chlorophyll-Deficient Mutants

To determine chlorophyll-deficient foliar mutants in each plant exposed to different treatments, the number of mutant families was computed, and plants with chlorophyll-defective modifications were noted. The classification of chlorophyll mutations was used for the characterization of foliar mutants [33]. Plants with morphological modifications were labeled and harvested separately. The selected plants were grouped according to their trait modifications, and the frequencies of the modifications were determined. The frequency of putative foliar mutants was calculated as the ratio of the number of mutant families to their total number, according to the following formula [34]:

$$\mathrm{Mutation} \mathrm{frequency} \left(\%\right)=\frac{\mathrm{Number} \mathrm{of} \mathrm{mutant} \mathrm{plants}}{\mathrm{Total} \mathrm{number} \mathrm{of} \mathrm{plants}}\times 100$$

2.5. Molecular Analysis

DNA extraction was carried out using the modified CTAB method [35]. The concentration was calculated by measuring the absorbance of 1 µL of the sample at 260/280 nm using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA USA). The ISSR-PCR was performed according to the protocol described by Dvořákováa et al. [36]. PCR amplifications were performed in a total volume of 15 μL containing 8 μL of 2× Master Mix for PCR (BioRad, Hercules, CA, USA), 5.2 μL of ddH₂O, 1 μL of 10 μM primer (Lumiprobe Corporation, Cockeysville, MD, USA), and 100–150 ng of DNA template. PCR amplification was run using a VeritiPro™ Thermal Cycler (Applied Biosystems, Singapore) with the following program: denaturation at 94 °C for 5 min, then 40 cycles of 30 s at 94 °C, 30 s at the annealing temperature, and 30 s at 72 °C, followed by extension at 72 °C for 10 min. The PCR products were loaded into the 16-capillary system of the Fragment Analyzer™ Automated CE System (Advanced Analytical Techologies, Ankeny, IA, USA), and the results were converted into digital format with PROSize 3.0 software (Advanced Analytical Technologies, Ankeny, IA, USA). The fragment analysis was laid out for allele sizes in the range from 35 bp to 5000 bp. Sixteen ISSR markers were used to estimate genetic diversity among the tested proso millet genotypes with three replicates. The list of ISSR markers is presented in

Table 1. The list of ISSR primers used for the testing proso millet genotypes.

Primer Name	Primer Code	Sequence 5′→3′	Annealing Temperature, (°C)
ISSR 807	(AG)8T	AGAGAGAGAGAGAGAGT	42.0
ISSR 808	(AG)8C	AGAGAGAGAGAGAGAGC	47.4
ISSR 809	(AG)8G	AGAGAGAGAGAGAGAGG	46.3
ISSR 810	(GA)8T	GAGAGAGAGAGAGAGAT	42.0
ISSR 811	(GA)8C	GAGAGAGAGAGAGAGAC	43.2
ISSR 816	(CA)8T	CACACACACACACACAT	43.2
ISSR 817	(CA)8A	CACACACACACACACAA	53.0
ISSR 819	(GT)8A	GTGTGTGTGTGTGTGTA	47.4
ISSR 820	(GT)8C	GTGTGTGTGTGTGTGTC	50.1
ISSR 822	(TC)8A	TCTCTCTCTCTCTCTCA	45.8
ISSR 823	(TC)8C	TCTCTCTCTCTCTCTCC	47.4
ISSR 826	(AG)8C	ACACACACACACACACC	53.0
ISSR 834	(AG)8YT	AGAGAGAGAGAGAGAGYT	45.8
ISSR 835	(AG)8YC	AGAGAGAGAGAGAGAGYC	45.7
ISSR 840	(GA)8YT	GAGAGAGAGAGAGAGAYT	45.7
ISSR 841	(GA)8YC	GAGAGAGAGAGAGAGAYG	45.8

.

2.6. Statistical Analysis

The results were analyzed using Microsoft Excel 2.0 with the Student’s t-Test for Windows 10 software package. All the data are denoted as means and standard deviations. Statistically significant differences in agronomic traits between the various colchicine concentrations and exposure times were analyzed using a three-way analysis of variance (ANOVA), with the separation of means by the least significant difference test (p < 0.05). PIC stands for polymorphism information content, which provides information about the capacity of a molecular marker to detect polymorphism, so PIC measurements are extremely important for genetic studies. The formula described by Botstein et al. (1980) was used to calculate PIC [37]. To compute the number of different alleles (Na), the number of effective alleles (Ne), Shannon’s information index (I), the expected heterozygosity (He), the unbiased expected heterozygosity (uHe), and the percentage of polymorphic loci (P), the GenAlEx 6.503 package was used [38].

3. Results

3.1. Colchicine Effect on Field Germination

The effects of colchicine on field germination were assessed at the early stage. The germination percentage of seeds in the M₁ generation decreased with increasing colchicine concentrations and longer treatment periods. However, non-significant effects of the treatment concentration and duration in the second generation were observed. The mean germination percentages (%) of the M₁ and M₂ generations (2021–2022) of proso millet genotypes affected by different colchicine concentrations and treatment times are given in

Table 2. Germination (%) of proso millet genotypes affected by colchicine concentrations and soaking times for the M1 and M2 generations.

Pavlodarskoe 4
Treatment Times, h	M ± SD	Colchicine Concentrations, %
		0.0		0.04		0.06		0.08		0.1
		M1	M2	M1	M2	M1	M2	M1	M2	M1	M2
6	Mean	69.4	71.7	55.2	72.8	58.8	70.2	53.8	51.5	42.4	51.6
	SD	1.8	2.3	2.0	1.8	2.3	1.7	1.7	1.5	2.0	2.2
12	Mean	71.0	72.3	58.4	88.5	43.0	63.6	37.6	52.6	32.0	40.3
	SD	2.2	1.1	1.6	1.4	1.8	2.1	2.8	2.4	1.7	1.5
24	Mean	68.0	72.8	44.0	48.8	36.6	45.6	33.8	49.4	25.0	10.2
	SD	2.1	2.7	2.2	2.6	2.0	2.0	2.2	1.7	2.4	0.9
Quartet
6	Mean	70.6	75.1	53.2	69.6	50.6	68.4	43.2	57.2	20.0	32.0
	SD	2.2	2.7	2.4	1.9	2.2	1.2	1.9	2.4	2.6	1.9
12	Mean	69.2	74.0	58.0	69.6	59.0	54.0	43.0	50.7	26.0	32.6
	SD	2.1	1.9	1.8	1.1	2.2	3.1	1.9	1.7	1.5	1.9
24	Mean	69.0	78.0	43.0	32.3	35.0	29.4	30.0	30.3	30.0	21.6
	SD	1.6	1.2	2.2	2.4	1.9	1.2	1.7	2.1	2.0	1.1
PI289324
		M1	M2	M1	M2	M1	M2	M1	M2	M1	M2
6	Mean	68.8	78.9	57.0	70.8	61.0	68.7	56.2	64.4	33.4	52.5
	SD	2.2	2.5	1.8	1.0	2.4	4.0	2.3	1.5	1.6	1.0
12	Mean	73.0	81.5	65.6	70.2	61.0	64.8	65.4	52.3	42.0	27.3
	SD	1.9	1.6	2.2	1.6	2.0	1.0	1.7	2.7	1.5	1.1
24	Mean	68.0	73.6	47.0	49.4	43.0	32.9	37.0	34.0	18.0	27.0
	SD	1.6	1.6	1.7	1.3	2.0	1.7	1.8	1.2	2.3	1.6

.

Our results indicated that germination decreased under colchicine treatment. The lowest germination percentages were detected at 0.1% with 12 and 24 h exposure durations in the M₁ and M₂ generations. The highest germination percentages were recorded when applying 0.04% colchicine with a 6 h exposure duration for the M₁ and M₂ generations. The mean germination in the M₂ generation was higher than in M₁ under colchicine treatment. In the M₁ generation, the mean germination was 69.6% for the control sample, 53.4% at 0.04% colchicine, 49.7% at 0.06% colchicine, 44.4% at 0.08% colchicine, and 29.8% at 0.1% colchicine, while in M₂, it was 75.3%, 63.5%, 55.2%, 49.1%, and 32.7%, respectively. Depending on the sensitivity to the colchicine treatment, the mean FG was 48.57% in the variety Pavlodarskoe 4, 43.28% in Quartet, and 53.14% in PI 289324. It was found that the long-term colchicine treatment of seeds leads to a decrease in germination compared to a short-term treatment.

3.2. Identification and Characterization of Chlorophyll-Deficient Foliar Mutants

The morphological analysis was conducted in 2021 for M₁ plants and in 2022 for M₂ plants. Seven different types of chlorophyll mutants (albina, chlorina, viridis, lutescent, corroded, maculata, and striata) were found in M₁ and M₂ generations when field seedlings were 10–20 days old.

Chlorophyll-deficient foliar mutants were determined according to the main clearly expressed mutant trait in comparison with the control sample (original genotype without treatment). The results showed that the high concentrations (0.08–0.1%) of colchicine induced the following types of mutations in M₁ plants at the germination-tillering stage: Albina (white seedlings) had weaker and smaller seedlings compared to normal ones. With sufficient humidity and a low temperature, 1–2 pairs of true white leaves managed to form. The plants wilted at the early stages of development (within two weeks) (Figure 1b). Chlorina plants had yellow seedlings; the true leaves were also yellow or yellow-green, and the plant did not change color as it grew. The plants lagged far behind both in growth and development and often wilted (Figure 1c). Viridis (viridis) had light-green shoots. True leaves and the whole plant were pale green and thin. There was a lag in growth (Figure 1d). In lutescent plants, the first leaves were green. As they grew, the upper part of the plant was clearly distinguished by a light-green color with a yellow tint, while the rest of the plant remained green. The “golden tip” type was the most suitable description for the change (Figure 1e). In corroded plants, the primary leaves were green. The real leaves were yellow-green in color and deformed, the edge of the leaf dried and curled up, and there were necrotic spots on the leaves (Figure 1f). Maculate seedlings showed whitish dots on the leaves or necrotic spots. The plants were vigorous, matured with a delay, and produced few seeds (Figure 1g). In striata plants, the first leaves were green. The plant was green, and the leaves had longitudinal stripes that were white or yellow (Figure 1h).

In the M₁ generation, a total of 2214 plants were studied. At all concentrations of the mutagen, except for 0.04%, chlorophyll mutations were detected. A total of 248 plants with modifications were selected in M₁, while in the M₂ generation, 500 plants for each mutation were analyzed (

Table 3. Frequency of chlorophyll-deficient foliar mutants in the M₁ and M₂ generations.

Types of Chlorophyll Changes	Analyzed Plants in M1	Frequency, %	Colchicine Concentrations, %			Analyzed Plants in M2	Frequency, %	Colchicine Concentrations, %
			0.06	0.08	0.1			0.06	0.08	0.1
albina	360	8.6	-	8	23	500	1.0	-	-	2
chlorina	348	10.0	5	12	18	500	1.0	-	1	1
viridis	270	17.4	6	14	27	500	5.5	2	4	5
lutescent	351	8.8	5	12	24	500	-	-	-	-
corroded	160	16.8	-	13	14	500	-	-	-	-
maculata	374	8.8	-	11	22	500	2.5	-	2	3
striata	351	9.6	-	13	21	500	-	-	-	-

).

Chlorophyll-deficient foliar mutants were detected in M₁-M₂ seedlings and adult plants, mostly those treated with high concentrations of colchicine (0.08 and 0.1%). These chlorophyll-deficient foliar mutants were found at a fairly high frequency, especially within the PI289324 genotype. In M₁ plants, at colchicine concentrations of 0.06–0.1%, the overall frequency of chlorophyll changes was 17.4%, generally for the viridis type. There were no changes at the 0.04% colchicine concentration in the M₁ and M₂ generations. Chlorophyll mutations with a lethal outcome were revealed in white seedlings of the albina type and yellow seedlings of the chlorina type. The death of these mutants occurred at the stage of the first leaves. Some mutants survived, but they were severely weakened and lagged behind in growth and development compared to the control plants. The albina type was observed at frequencies of 8.6% at the 0.08% and 0.1% colchicine concentrations in M₁ plants and 0.4% at the 0.1% colchicine concentration in M₂. In M₁ plants, the lutescent “golden top” type was observed with a sufficiently high frequency only at the 0.06–0.1% colchicine concentrations, with frequencies of 1.4, 3.4, and 6.8, respectively. The corroded type was observed in all genotypes at a relatively high frequency, and the maximum appearance was achieved at the 0.08% colchicine concentration, while in M₂, this type of mutation was not detected. In the maculata mutation type, seedlings showed whitish dots on leaves and necrotic spots, and M₁-M₂ plants were vigorous, matured with a delay, and produced few seeds; this mutation type was attained only at the highest (0.1%) concentration of colchicine. The striata mutation presented as white or yellow stripes on the leaves and was observed at higher colchicine concentrations (0.08 and 0.1%) at frequencies of 3.7 and 5.9% in M₁ and 0.4 and 0.6% in M₂, respectively.

3.3. Agronomic Trait Components

In total, seven agronomic traits associated with the vegetative period and yield elements were evaluated: plant survival (PS), growing season (GS), productive branches (PBs), seed weight per panicle (SWP), 1000-seed weight (TSW), and grain yield (GY). The data analysis indicated that there was a wide range of variation in responses among genotypes to various colchicine treatments (

Table 4. Agronomic traits of proso millet affected by colchicine concentrations and treatment times in the M₁ and M₂ generations.

Treatment Times, h	Traits	Colchicine Concentrations, %
		0.0%		0.04%		0.06%		0.08%		0.1%
		M1	M2	M1	M2	M1	M2	M1	M2	M1	M2
Pavlodarskoe 4
6	PS (%)	65.6 ± 1.1	60.8 ± 3.4	50.8 ± 0.8	63.2 ± 3.5	53.6 ± 2.1	62.0 ± 3.1	49.2 ± 1.0	44.0 ± 2.5	38.0 ± 0.7	44.8 ± 2.1
	GS (days)	92.0 ± 1.4	92.0 ± 1.6	92.0 ± 1.4	92.0 ± 1.6	92.0 ± 1.6	91.0 ± 1.7	91.0 ± 1.8	91.0 ± 1.7	91.0 ± 2.8	90.0 ± 1.7
	PBs (piece)	1.6 ± 0.1	1.4 ± 0.07	2.0 ± 0.3	1.3 ± 0.06	2.1 ± 0.1	2.0 ± 0.09	2.3 ± 0.2	1.8 ± 0.1	2.3 ± 0.1	2.3 ± 0.12
	SWP (g)	1.33 ± 0.3	2.0 ± 0.1	1.4 ± 0.1	1.2 ± 0.06	1.55 ± 0.2	1.5 ± 0.1	1.35 ± 0.1	1.1 ± 0.06	1.25 ± 0.2	1.6 ± 0.1
	TSW (g)	4.9 ± 0.4	6.46 ± 0.31	4.57 ± 0.1	5.50 ± 0.2	4.35 ± 0.1	5.75 ± 0.2	4.81 ± 0.3	6.25 ± 0.3	4.5 ± 0.1	6.52 ± 0.3
	GY (c/ha)	34.9 ± 2.7	42.6 ± 0.6	35.6 ± 0.7	24.6 ± 0.6	43.6 ± 1.6	46.5 ± 0.7	38.2 ± 0.8	21.8 ± 0.4	27.3 ± 2.7	41.2 ± 0.7
12	PS (%)	64.8 ± 2.0	58.4 ± 3.5	54.0 ± 1.1	74.8 ± 4.1	41.2 ± 1.7	52.0 ± 2.8	32.8 ± 2.7	46.4 ± 3.0	28.0 ± 2.4	49.6 ± 3.1
	GS (days)	92.0 ± 1.7	92.0 ± 1.8	89.0 ± 2.9	90.0 ± 1.7	88.0 ± 3.1	89.0 ± 1.7	88.0 ± 2.8	88.0 ± 1.6	88.0 ± 2.5	88.0 ± 1.6
	PBs (piece)	1.3 ± 0.1	1.4 ± 0.06	2.0 ± 0.2	1.5 ± 0.07	2.3 ± 0.1	2.3 ± 0.04	2.4 ± 0.3	1.8 ± 0.06	2.4 ± 0.1	2.5 ± 0.07
	SWP (g)	2.0 ± 0.3	2.4 ± 0.09	1.62 ± 0.1	1.6 ± 0.06	1.87 ± 0.1	2.2 ± 0.07	2.1 ± 0.1	2.8 ± 0.1	2.9 ± 0.4	1.8 ± 0.06
	TSW (g)	4.76 ± 0.4	5.5 ± 0.25	4.28 ± 0.1	5.75 ± 0.25	4.65 ± 0.1	5.75 ± 0.3	4.7 ± 0.3	6.45 ± 0.3	6.49 ± 0.3	7.1 ± 0.4
	GY (c/ha)	42.1 ± 1.6	49.1 ± 0.7	43.7 ± 1.8	44.9 ± 0.7	44.3 ± 2.3	65.8 ± 1.2	41.3 ± 1.6	58.5 ± 1.1	48.7 ± 2.1	55.8 ± 1.0
24	PS (%)	63.2 ± 2.0	60.0 ± 1.6	38.8 ± 2.4	33.6 ± 1.8	32.0 ± 1.6	34.0 ± 1.0	30.4 ± 1.1	40.4 ± 1.6	21.2 ± 1.4	30.4 ± 1.0
	GS (days)	92.0 ± 1.4	92.0 ± 2.8	87.0 ± 2.1	88.0 ± 1.7	87.0 ± 3.5	88.0 ± 1.8	86.0 ± 1.8	86.0 ± 1.4	86.0 ± 1.4	86.0 ± 1.4
	PBs (piece)	1.4 ± 0.1	1.8 ± 0.1	2.0 ± 0.3	1.1 ± 0.3	2.3 ± 0.1	1.5 ± 0.1	3.0 ± 0.3	2.2 ± 0.3	1.6 ± 0.1	1.5 ± 0.1
	SWP (g)	1.73 ± 0.2	1.9 ± 0.3	1.77 ± 0.1	1.9 ± 0.1	1.63 ± 0.3	1.6 ± 0.1	2.14 ± 0.2	3.1 ± 0.1	2.0 ± 0.4	3.2 ± 0.3
	TSW (g)	4.85 ± 0.3	6.91 ± 0.4	4.4 ± 0.4	6.15 ± 0.3	4.45 ± 0.1	6.34 ± 0.7	4.72 ± 0.2	6.80 ± 0.1	5.1 ± 0.3	7.20 ± 0.6
	GY (c/ha)	38.3 ± 2.2	51.3 ± 1.8	34.3 ± 1.5	17.6 ± 2.4	30.0 ± 1.6	20.4 ± 2.3	48.8 ± 1.3	68.9 ± 3.8	17.0 ± 1.3	36.5 ± 2.7
PI289324
6	PS (%)	62.8 ± 1.7	78.4 ± 3.4	52.0 ± 2.7	60.8 ± 2.8	55.6 ± 3.5	60.4 ± 3.3	49.6 ± 2.4	58.4 ± 1.7	29.6 ± 1.1	46.0 ± 1.7
	GS (days)	81.0 ± 1.6	81.0 ± 1.4	79.0 ± 2.8	80.0 ± 1.4	78.0 ± 1.4	80.0 ± 1.6	78.0 ± 1.8	80.0 ± 1.8	78.0 ± 1.4	79.0 ± 2.8
	PBs (piece)	1.5 ± 0.1	1.2 ± 0.3	1.6 ± 0.3	1.8 ± 0.4	1.8 ± 0.06	1.8 ± 0.1	1.7 ± 0.1	2.1 ± 0.3	2.4 ± 0.2	2.2 ± 0.1
	SWP (g)	1.49 ± 0.1	2.5 ± 0.3	1.8 ± 0.1	2.0 ± 0.1	1.74 ± 0.09	2.3 ± 0.3	1.9 ± 0.4	1.9 ± 0.3	2.1 ± 0.1	2.4 ± 0.1
	TSW (g)	5.36 ± 0.1	6.1 ± 0.1	5.29 ± 0.3	5.85 ± 0.3	4.88 ± 0.25	6.4 ± 0.4	5.04 ± 0.3	5.6 ± 0.1	5.23 ± 0.1	5.8 ± 0.4
	GY (c/ha)	35.1 ± 2.3	58.8 ± 1.8	37.4 ± 1.6	54.7 ± 2.1	43.5 ± 0.7	62.5 ± 2.1	40.1 ± 2.1	58.3 ± 2.2	37.3 ± 1.8	60.7 ± 3.3
12	PS (%)	65.6 ± 2.1	64.4 ± 1.1	58.0 ± 0.7	62.0 ± 0.8	54.8 ± 1.1	60.0 ± 2.1	56.0 ± 1.0	42.8 ± 1.0	37.2 ± 0.8	8.4 ± 0.7
	GS (days)	81.0 ± 1.6	81.0 ± 1.4	79.0 ± 2.8	80.0 ± 1.4	78.0 ± 1.4	79.0 ± 1.6	78.0 ± 1.8	78.0 ± 1.8	78.0 ± 1.4	78.0 ± 2.8
	PBs (piece)	1.3 ± 0.1	1.4 ± 0.1	1.7 ± 0.1	1.6 ± 0.3	1.8 ± 0.1	1.8 ± 0.1	1.6 ± 0.2	1.7 ± 0.2	1.8 ± 0.3	1.4 ± 0.1
	SWP (g)	1.73 ± 0.2	2.6 ± 0.3	2.0 ± 0.2	2.5 ± 0.1	1.95 ± 0.3	1.9 ± 0.2	1.85 ± 0.1	1.7 ± 0.1	1.72 ± 0.1	1.1 ± 0.2
	TSW (g)	5.75 ± 0.1	6.7 ± 0.4	4.75 ± 0.1	6.32 ± 0.1	4.44 ± 0.4	6.45 ± 0.1	4.52 ± 0.3	5.55 ± 0.3	5.41 ± 0.1	5.4 ± 0.1
	GY (c/ha)	36.9 ± 1.6	58.6 ± 2.7	49.3 ± 2.7	62.0 ± 0.7	48.1 ± 2.7	51.3 ± 1.6	41.4 ± 0.8	30.9 ± 0.8	28.8 ± 0.7	3.2 ± 2.7
24	PS (%)	60.8 ± 1.1	64.8 ± 3.0	40.4 ± 1.8	37.2 ± 2.5	38.0 ± 1.0	23.6 ± 3.5	30.0 ± 1.0	24.0 ± 3.1	14.8 ± 1.6	18.8 ± 2.0
	GS (days)	81.0 ± 1.8	81.0 ± 1.4	76.0 ± 1.7	78.0 ± 2.8	75.0 ± 1.4	78.0 ± 1.4	75.0 ± 1.8	75.0 ± 1.4	75.0 ± 2.8	75.0 ± 2.8
	PBs (piece)	1.1 ± 0.3	1.5 ± 0.3	1.9 ± 0.3	1.6 ± 0.1	1.8 ± 0.1	3.2 ± 0.3	2.1 ± 0.1	2.0 ± 0.1	2.0 ± 0.1	2.0 ± 0.3
	SWP (g)	2.2 ± 0.2	2.2 ± 0.3	1.8 ± 0.1	0.7 ± 0.1	1.92 ± 0.3	1.4 ± 0.1	2.46 ± 0.1	1.3 ± 0.2	2.2 ± 0.3	0.8 ± 0.1
	TSW (g)	5.93 ± 0.2	6.0 ± 0.3	6.6 ± 0.3	5.9 ± 0.1	5.58 ± 0.6	5.15 ± 0.3	5.39 ± 0.7	6.0 ± 0.4	5.7 ± 0.4	5.5 ± 0.1
	GY (c/ha)	36.8 ± 1.3	53.5 ± 2.3	34.5 ± 2.4	10.4 ± 2.3	32.8 ± 2.7	26.4 ± 1.6	38.7 ± 2.3	15.6 ± 4.5	16.3 ± 1.8	7.5 ± 1.6
Quartet
6	PS (%)	64.0 ± 1.3	69.6 ± 2.0	49.6 ± 1.1	56.0 ± 2.4	46.4 ± 1.1	48.0 ± 1.6	38.8 ± 2.0	52.4 ± 1.1	16.8 ± 2.1	25.2 ± 1.4
	GS (days)	89.0 ± 1.8	89.0 ± 1.4	89.0 ± 1.4	89.0 ± 2.1	89.0 ± 2.3	89.0 ± 3.5	88.0 ± 1.8	88.0 ± 1.8	88.0 ± 2.0	87.0 ± 1.4
	PBs (piece)	1.5 ± 0.1	1.4 ± 0.1	2.2 ± 0.1	1.6 ± 0.3	2.0 ± 0.3	1.9 ± 0.1	2.4 ± 0.1	1.8 ± 0.3	2.6 ± 0.2	2.0 ± 0.1
	SWP (g)	1.2 ± 0.3	2.1 ± 0.2	1.35 ± 0.1	2.5 ± 0.1	1.32 ± 0.1	2.4 ± 0.3	1.0 ± 0.3	2.6 ± 0.2	1.28 ± 0.4	2.7 ± 0.4
	TSW (g)	4.8 ± 0.4	6.2 ± 0.3	4.87 ± 0.1	6.41 ± 0.4	4.67 ± 0.3	6.6 ± 0.1	4.51 ± 0.4	6.2 ± 0.2	4.22 ± 0.2	6.15 ± 0.3
	GY (c/ha)	28.8 ± 1.3	51.2 ± 2.2	36.8 ± 2.4	56.0 ± 1.5	30.6 ± 2.0	54.7 ± 1.6	23.3 ± 1.7	61.3 ± 1.3	14.0 ± 0.11	34.0 ± 1.3
12	PS (%)	62.0 ± 2.8	63.2 ± 2.0	53.2 ± 3.4	56.0 ± 1.1	52.8 ± 1.7	47.2 ± 1.7	38.4 ± 3.3	62.4 ± 2.7	22.4 ± 1.7	27.2 ± 2.4
	GS (days)	89.0 ± 2.8	89.0 ± 1.7	88.0 ± 1.4	89.0 ± 2.9	88.0 ± 3.2	87.0 ± 3.1	86.0 ± 1.4	87.0 ± 2.8	86.0 ± 2.5	86.0 ± 2.5
	PBs (piece)	1.6 ± 0.4	1.5 ± 0.1	1.9 ± 0.3	2.3 ± 0.2	2.1 ± 0.3	1.4 ± 0.1	2.1 ± 0.1	2.0 ± 0.3	2.3 ± 0.1	2.1 ± 0.1
	SWP (g)	1.36 ± 0.1	1.8 ± 0.3	1.2 ± 0.3	2.1 ± 0.1	1.49 ± 0.3	2.4 ± 0.1	1.08 ± 0.3	1.8 ± 0.1	2.84 ± 0.1	3.3 ± 0.4
	TSW (g)	4.77 ± 0.3	6.85 ± 0.4	4.41 ± 0.1	6.26 ± 0.1	3.48 ± 0.1	5.67 ± 0.1	4.1 ± 0.4	6.0 ± 0.3	5.21 ± 0.4	6.8 ± 0.3
	GY (c/ha)	33.7 ± 2.1	42.7 ± 1.6	30.3 ± 1.8	67.6 ± 1.8	41.3 ± 2.2	39.6 ± 2.3	21.8 ± 2.1	56.2 ± 1.6	36.6 ± 3.3	47.1 ± 2.1
24	PS (%)	61.2 ± 2.8	66.0 ± 1.3	37.6 ± 3.1	24.4 ± 1.1	32.0 ± 3.0	22.0 ± 1.1	25.6 ± 3.5	23.2 ± 2.0	24.0 ± 4.1	16.0 ± 2.1
	GS (days)	89.0 ± 1.7	89.0 ± 1.8	83.0 ± 1.6	87.0 ± 1.4	83.0 ± 1.6	87.0 ± 2.3	83.0 ± 1.8	84.0 ± 1.8	82.0 ± 1.7	84.0 ± 2.0
	PBs (piece)	1.2 ± 0.04	1.6 ± 0.1	2.1 ± 0.07	2.4 ± 0.1	2.1 ± 0.06	2.2 ± 0.3	2.1 ± 0.06	2.0 ± 0.1	2.0 ± 0.07	1.7 ± 0.2
	SWP (g)	2.05 ± 0.07	1.7 ± 0.3	1.83 ± 0.06	1.7 ± 0.1	1.7 ± 0.1	1.3 ± 0.1	1.78 ± 0.09	1.7 ± 0.3	1.5 ± 0.06	2.3 ± 0.4
	TSW (g)	4.6 ± 0.3	6.0 ± 0.4	4.51 ± 0.4	6.65 ± 0.1	4.76 ± 0.3	7.25 ± 0.3	4.6 ± 0.25	6.2 ± 0.4	4.3 ± 0.25	6.2 ± 0.2
	GY (c/ha)	37.6 ± 1.2	44.9 ± 1.3	36.1 ± 1.0	24.9 ± 2.4	28.6 ± 1.1	15.7 ± 2.0	23.9 ± 0.7	19.7 ± 1.7	18.0 ± 0.7	15.6 ± 0.11

).

Table 4 shows that colchicine treatments resulted in a lower PS rate than controls. Colchicine treatments resulted in a lower PS rate than controls. The lowest PS rate was observed in the case of a 24 h treatment at the 0.08–0.1% colchicine concentrations for M₁ and M₂ plants. Longer soaking times at the highest colchicine concentrations (0.08–0.1%) had negative effects on this trait for both the M₁ and M₂ generations. For example, the PS rates in the Quartet variety were 61 and 66% in M₁ and M₂ control plants, while at 0.1% with a 24 h treatment time, they were 24 and 16%, respectively. The greatest colchicine concentration (0.1%), combined with the longest soaking time (24 h), generated the lowest PS rate for M₁ and M₂ plants.

The GS in both generations varied with different mutagenic concentrations. The results obtained from the M₁ and M₂ generations demonstrated significant mutagen effects on the GS for all genotypes. With increasing mutagen concentrations, the GS decreased by 4–5 days (Figure 2a). In both the M₁ and M₂ generations, the GS was the shortest for plants exposed to 0.08–0.1% with 12 and 24 h soaking times (Figure 2b). The average mean of the GS of the PI289324 (80 days) genotype was lower than those of the Pavlodarskoe 4 (88 days) and Quartet (87 days) cultivars. The trait values ranged from 78 to 92 days in the M₁ and M₂ generations. Figure 2 indicates that the growing season of proso millet for the control plants was delayed compared with the colchicine-treated plants.

The figures clearly present the following results: For the control, most of the plants were still green in the early phase of ripening; for the mutants, almost 60–70% of the plants fully ripened, and the ripeness depended on the concentration. A 24 h (Figure 2b) exposure period to colchicine significantly impacted the maturation rate of the plants. In the M₁ and M₂ generations, variation was observed in some agronomic traits, such as the 1000-seed weight, seed weight per panicle, and productive branches. The increase in colchicine concentration led to a rise in the number of PBs per plant in the M₁ and M₂ generations. The 0.08–0.1% colchicine concentrations combined with exposure durations of 6, 12, and 24 h significantly increased the observed number of PBs per plant by approximately 25% compared with the control. The highest concentration of colchicine combined with 6, 12, and 24 h exposure durations resulted in the highest number of PBs.

Noticeable differences were detected in SWP for both the M₁ and M₂ generations. The maximum values of SWP were 3.3 g for M₂ Quartet plants from the 0.1% colchicine concentration treatment, 3.2 g for the Pavlodarskoe 4 cultivar at 0.1% colchicine with 24 h exposure, and 2.5 g for the PI289324 variety at 0.04% colchicine with 12 h exposure. The lowest values of SWP were at the 0.06% concentration with 24 h exposure for the Quartet genotype (1.3 g), at the 0.1% concentration with 24 h exposure for the PI289324 variety, and at 0.08% concentration with 6 h exposure for the Pavlodarskoe 4 cultivar (1.1 g). In general, the treated M₂ plants prevailed over the controls for the M₂ Pavlodarskoe 4 genotype after the 24 h treatment and for the Quartet variety at all concentrations and exposure durations, except for the 0.06% concentration with the 24 h treatment. Concerning M₂ PI289324, the SWP for the control variant was higher than that for the treated sample.

We evaluated the GY performance of M₁ and M₂ plants exposed to various colchicine concentrations combined with different soaking times. The data presented in Table 4 suggest that the GY in the M₁ generation was reduced with increasing colchicine doses (0.08–0.1%), whereas the opposite was observed at concentrations of 0.04–0.06% combined with a 12 h soaking time. As for the GY, the highest values were obtained in the M₂ plants of Quartet (67.6) at the 0.04% concentration, Pavlodarskoe 4 (65.8) at the 0.06% concentration, and PI289324 (62.0) at the 0.04% concentration with a 12 h treatment time. The lowest GY values were in the M₂ plants of Quartet (15.6) at 0.1% and Pavlodarskoe 4 (17.6) at 0.04% with a 24 h treatment time and PI289324 (3.2) at the 0.1% concentration with 12 h exposure.

There were noticeable differences in the TSW between the M₁ and M₂ generations. The TSW was higher in the M₂ generation than in M₁, regardless of the colchicine concentration and exposure duration. The mean value of the TSW (5.1–5.8 g) was slightly lower in the M₁ plants of the PI289324 genotype than in control plants (6.0–6.7 g). The highest 1000-grain weight was observed in the M₁ plants of the Quartet variety (7.2 g) at the 0.06% colchicine treatment with 24 h exposure.

A factorial analysis of variance was performed to confirm the effects of the treatment duration and mutagen concentration on the studied agronomic traits of M₁ and M₂ plants. The results of ANOVA for the FG, PS, GS, PB, SWP, TSW, and GY traits are depicted in

Table 5. ANOVA test for agronomic traits of M₁ generation.

Agronomic Traits	Treatment Times, h			Colchicine Concentrations, %
	6	12	24	0.04	0.06	0.08	0.1
FG	0.894 ns	0.048 *	0.044 *	9.87 × 10−6 ***	4.37 × 10−5 ***	8.57 × 10−6 ***	3.38 × 10−10 ***
PS	0.987 ns	0.031 *	0.036 *	2.12 × 10−5 ***	4.39 × 10−5 ***	3.76 × 10−6 ***	3.90 × 10−10 ***
GS	0.494 ns	0.222 ns	0.082 ns	0.298 ns	0.225 ns	0.155 ns	0.145 ns
PB	0.489 ns	0.574 ns	0.965 ns	9.59 × 10−6 ***	1.94 × 10−6 ***	6.65 × 10−5 ***	1.20 × 10−5 ***
SWP	0.019 *	0.0001 ***	0.655 ns	0.813 ns	0.949 ns	0.759 ns	0.219 ns
TSW	0.940 ns	0.225 ns	0.322 ns	0.444 ns	0.058 ns	0.083 ns	0.867 ns
GY	0.088 ns	0.029 *	0.414 ns	0.580 ns	0.584 ns	0.373 ns	0.013 *

Note: *** = Significant at 0.001 significance level; * = significant at 0.05 significance level; ns: p > 0.05.

and

Table 6. ANOVA test for agronomic traits of M₂ generation.

Agronomic Traits	Treatment Times, h			Colchicine Concentrations, %
	6	12	24	0.04	0.06	0.08	0.1
FG	0.408 ns	0.018 *	0.001 ***	0.073 ns	0.002 **	3.57 × 10−6 ***	1.25 × 10−7 ***
PS	0.497 ns	0.031 *	0.004 **	0.109 ns	0.005 **	0.0006 ***	1.46 × 10−5 ***
GS	0.297 ns	0.359 ns	0.437 ns	0.136 ns	0.538 ns	0.037 *	0.025 *
PB	1.000 ns	0.595 ns	0.599 ns	0.403 ns	0.029 *	0.0003 ***	0.012 *
SWP	0.602 ns	0.157 ns	0.299 ns	0.338 ns	0.373 ns	0.810 ns	0.866 ns
TSW	0.814 ns	0.550 ns	0.352 ns	0.297 ns	0.609 ns	0.148 ns	0.422 ns
GY	0.502 ns	0.001 ***	0.007 **	0.601 ns	0.795 ns	0.508 ns	0.450 ns

Note: *** = Significant at 0.001 significance level; ** = significant at 0.01 significance level; * = significant at 0.05 significance level; ns: p > 0.05.

for the M₁ and M₂ generations, respectively.

The results for FG and PS with 12 h and 24 h treatment durations in M₁ plants were observed to be statistically significant (p < 0.05). FG and PS were also significantly affected (p < 0.001) by all colchicine concentrations (0.04, 0.06, 0.08, and 0.1%) in the M₁ generation (Table 5). There were no significant correlations between the GS and TSW and the colchicine concentration and treatment duration in M₁ plants. The SWP was also related to 12 and 24 h treatment times, with 0.031 and 0.004, respectively (p< 0.05 and p < 0.001). There was a relationship between FG and PS and treatment durations of 12 and 24 h in M₂ plants, and the p-values were <0.05 and <0.01–0.001, respectively (Table 6).

In addition, it was found that the GY was also influenced by 12 and 24 h treatment durations, with p-values < 0.001 and <0.01, respectively. The analysis of variance suggested that FG and PS were highly related to colchicine concentrations of 0.06 (p < 0.01), 0.08, and 0.1% (p < 0.001). Concentrations of 0.08 and 0.1% showed a significant relationship with the GS (p < 0.05). The concentrations of 0.06, 0.08, and 0.1% were related to PBs (p < 0.05, p < 0.001, and <0.01, respectively). The ANOVA showed that the treatment duration and the concentration of the mutagen significantly affected the variation in the FG, PS, GS, PB, and GY traits, where the p-values ranged from <0.001 to <0.05.

3.4. Molecular Analysis

In the present study, 16 ISSR markers were tested in the M₂ plants of three genotypes: Pavlodarskoe 4, Quartet, and PI 289324. The total number of bands produced by ISSR markers varied from 4 to 53, with an average of 20.82 amplicons per primer. The size of the amplified fragments ranged from 39 bp to 4827 bp, as shown in

Table 7. Details of ISSR markers used during the present study.

Primer Name	Colchicine Concentrations, %	Total Loci	Size Range (bp)	Polymorphic Loci	Polymorphism, %	PIC
ISSR 807	0.00	12	229–3268	12	100	0.233
	0.04	15	210–848	15	100
	0.06	27	206–1301	25	92.5
	0.08	33	172–3950	33	100
ISSR 808	0.00	28	208–3778	28	100	0.249
	0.04	16	213–1840	15	90.9
	0.06	15	211–4689	15	100
	0.08	23	206–1732	23	100
ISSR 809	0.00	19	48–1055	19	100	0.217
	0.04	16	218–1486	15	90.9
	0.06	14	233–4313	14	100
	0.08	25	214–2479	23	92.0
ISSR 810	0.00	5	336–1069	5	100	0.219
	0.04	8	170–4334	8	100
	0.06	28	210–1400	26	92.8
	0.08	21	218–1179	18	75.0
ISSR 811	0.00	4	975–1068	4	100	0.228
	0.04	5	457–1520	5	100
	0.06	7	539–1724	7	100
	0.08	14	492–1626	14	100
ISSR 816	0.00	5	542–3954	5	100	0.226
	0.04	4	3326–3466	4	100
	0.06	9	536–4186	9	100
	0.08	17	379–3489	16	94.1
ISSR 817	0.00	11	278–3094	10	90.9	0.203
	0.04	6	42–2769	6	100
	0.06	12	39–3405	12	100
	0.08	10	41–1630	10	100
ISSR 819	0.00	11	224–3258	8	72.7	0.202
	0.04	8	42–4525	8	100
	0.06	8	42–2347	7	87.5
	0.08	5	42–3417	4	80.0
ISSR 820	0.00	53	49–2922	53	100	0.219
	0.04	27	202–2769	27	100
	0.06	6	205–1697	6	100
	0.08	8	44–2014	8	100
ISSR 822	0.00	28	156–3845	26	92.8	0.214
	0.04	22	162–3415	22	100
	0.06	24	155–4293	23	95.8
	0.08	12	157–4449	11	91.6
ISSR 823	0.00	21	248–1568	19	90.4	0.226
	0.04	22	247–1590	22	100
	0.06	19	252–4661	19	100
	0.08	14	255–1568	14	100
ISSR 826	0.00	23	181–1396	23	100	0.237
	0.04	18	52–4827	18	100
	0.06	18	52–1775	18	100
	0.08	23	51–1884	23	100
ISSR 834	0.00	36	146–4051	33	91.6	0.293
	0.04	38	187–4798	38	100
	0.06	37	193–2849	27	72.9
	0.08	34	189–4091	33	97.0
ISSR 835	0.00	38	108–4320	34	89.4	0.311
	0.04	35	134–4382	34	97.1
	0.06	38	160–4093	35	92.1
	0.08	43	134–4279	43	100
ISSR 840	0.00	41	138–4000	38	92.6	0.322
	0.04	45	144–4625	45	100
	0.06	45	138–4417	45	100
	0.08	32	49–4688	32	100
ISSR 841	0.00	24	141–3964	24	100	0.266
	0.04	18	155–2164	18	100
	0.06	27	164–4442	24	88.8
	0.08	23	158–4061	23	100
Mean	-	20.82	-	20.01	96.11	0.241

.

The 16 ISSR primers produced a total of 1333 fragments, among which 1281 were polymorphic, with a mean polymorphic percentage of 96.11%. The maximum levels of polymorphism were produced by the markers ISSR 811, ISSR 820, and ISSR 826 (100%), followed by ISSR 840, ISSR 816, and ISSR 807 98 (98%); the markers ISSR 808, ISSR 817, ISSR 841, and ISSR 823 (97%); and the markers ISSR 809, ISSR 822, and ISSR 835 (95). The markers ISSR 810, ISSR 834, and ISSR 819 showed lower levels of polymorphism of 91, 90, and 85%, respectively. The primers ISSR 807, ISSR 810, ISSR 811, ISSR 816, ISSR 835, and ISSR 840 amplified more bands with the 0.08% colchicine treatment. The polymorphism information content (PIC) of markers varied from 0.202 to 0.322, with an average of 0.241. The maximum PIC was recorded for ISSR 840 (0.322), followed by ISSR 835 (0.311), while the lowest (0.202) was for ISSR 819. The ISSR 840 marker amplification profile of the capillary system is illustrated in Figure 3 as an example.

According to the original capillary electrophoresis data, different allelic bands were detected depending on the mutagen concentration among different experimental variants. Different genetic diversity estimates for the three concentrations of colchicine were calculated, where the maximum Na was recorded for the control (0.913 ± 0.023), followed by the 0.08% (0.801 ± 0.023), 0.04% (0.676 ± 0.022), and 0.06% (0.499 ± 0.020) colchicine concentrations, with an average of 0.722 ± 0.022 (

Table 8. Genetic diversity information of proso millet depending on different concentrations of colchicine provided by ISSR markers.

Colchicine Concentrations	Mean/SE	Na	Ne	I	He	uHe	P
Control	Mean	0.913	1.224	0.228	0.147	0.176	45.34
	SE	0.023	0.006	0.006	0.004	0.005	2.561
0.04%	Mean	0.676	1.232	0.199	0.136	0.181	32.85
	SE	0.022	0.008	0.007	0.005	0.006	1.784
0.06%	Mean	0.499	1.169	0.145	0.099	0.132	23.92
	SE	0.020	0.007	0.006	0.004	0.006	1.483
0.08%	Mean	0.801	1.194	0.199	0.128	0.153	39.84
	SE	0.023	0.006	0.006	0.004	0.005	2.457

Note: Na—number of different alleles; Ne—number of effective alleles; I—Shannon’s information index; He—expected heterozygosity; uHe—unbiased expected heterozygosity; P—percentage of polymorphic loci.

).

The Ne was higher at 0.04% (1.232) and lower at 0.06% (1.169), with an average of 1.205. I ranged from 0.145 to 0.228, with an average of 0.192. The He ranged from 0.099 to 0.147, with a mean value of 0.127. The average value of uHe was 0.380, with values of 0.181 at 0.04%, 0.176 in the control, 0.153 at 0.08%, and 0.132 at 0.06%. The average percentage of polymorphic loci was lower at the 0.06% concentration (23.92%) and higher in the control (45.34%), with a mean of 35.49%.

4. Discussion

Chemical mutagenesis plays an important role in the breeding improvement of crops. The application of chemical mutagens for plant improvement has been developed for many crops. Colchicine is highly toxic to plants, and therefore, low concentrations with prolonged exposure durations are considered reliable for reducing its toxic effects and increasing the polyploid production rate [39]. In our study, seed soaking in colchicine solutions with various concentrations (0.04% to 0.1%) for 6, 12, and 24 h affected the percentage of seed germination in field conditions. All colchicine-treated plants were significantly different from the control plants in terms of germination. The colchicine treatment duration was positively correlated with seedling mortality in many plant species [39,40]. Our results showed that the increasing concentration of colchicine and its treatment duration significantly decreased the field germination of proso millet genotypes in the M₁-M₂ generations. Notably, germination was reduced by more than 50% by the 0.08–0.1% colchicine treatments with 12–24 h exposure times. It seems that soaking seeds in a colchicine solution for 12–24 h inhibited field germination. There was a decrease in the percentage of seed germination with higher colchicine concentrations and longer exposure durations. Sasiree et al. [41] suggested that the reduction in the germination rate with the increase in the concentration of colchicine may be due to the occurrence of tissue necrosis when the seeds are exposed to different concentrations of colchicine. Reduced plant growth at higher colchicine concentrations might be due to sudden changes in the metabolic status of the seeds at certain levels of the mutagen, along with growth inhibitor destruction, an increase in growth promoters, and a decline in the assimilation mechanism [42]. Also, some studies demonstrated enhanced lethality in the obtained plants after treatment with high concentrations of colchicine; it was explained by the highly toxic effect of colchicine on the mitotic spindle (blocking spindle microtubule production) [43].

Chlorophyll mutations are a common test in experimental plant mutagenesis studies, mainly because they are relatively easy to observe for a large number of studied plants. The chlorophyll mutation frequency is useful for the evaluation of the potency of a mutagen and its genetic effects and for the estimation of mutational events due to the simplicity of identification. However, to achieve the full characterization of the action of a mutagen, a complete analysis of all emerging mutations is required. Accounting for chlorophyll mutations is only a preliminary estimate of the intensity of the mutation process. In the case of our studies on experimental mutagenesis in proso, we have isolated several types of chlorophyll mutations. The frequency of chlorophyll mutations and their spectra are used to assess the effectiveness and specificity of the mutagen action and the mutability of varieties [44]. Death and reduced viability might be the reasons for the poor genetic knowledge of most chlorophyll mutants [45]. Chlorophyll mutations, despite the complexity of the mechanism of their manifestation, serve as important elements for assessing the activity of the mutagen and the resistance of the plant genotype to mutagenic factors. Mutants with complete or partial chlorophyll deficiency are usually determined by recessive genes: al (albina), y (yellow seedling), lu (lutescent), v (virescent), fs, and z (leaf striping (zebra) or variegation).

According to the literature, it is known that the chlorophyll mutation of the albina type is the result of point recessive mutations and is inherited in a monogenic manner [46]. Different types of chlorophyll mutations, such as albina, xantha, albo-xantha, xanthalba, alboviridis, virescence, chlorina, albescence, tigrina, and maculata were identified in six varieties of Lathyrus sativus L. by Prasad and Das [47]. Moreover, the chlorophyll mutations in flax reported by researchers can be interesting from the point of view of the intensity of the mutation process and also as a source of new marker traits. For example, the yellow seedlings of the chlorina-type mutation are easily identified from the seedling stage to plant maturation and can be of significant importance for genetic and cytogenetic studies as markers of individual chromosomes and their regions [12,48].

The results of our study showed that the maximum frequency of putative viridis mutations was attained at a mutagen concentration of 0.1%, and the minimum was obtained at 0.06%. The viridis type includes light-green mutants. Chlorophyll-deficient mutants of this type had reduced viability and were slightly lacking in growth and development. Plants with this type of chlorophyll modification were weak and less productive. In the variant with seeds soaked in 0.08 and 0.1% colchicine solutions in M₁, 31 plants with the albina-type mutation were found in total. Among all of the treatments, the highest mutation frequency was found at the 0.1% colchicine concentration in M₁ and M₂ proso millet plants. For the M₁ and M₂ plants, the average frequencies of chlorophyll-deficient foliar mutants were 11.2 and 2.5%, respectively. The comparison of the chlorophyll mutations by type generally indicated that the mutation rate was enhanced with an increase in the concentration and exposure duration; however, the frequency of chlorophyll-deficient foliar mutants was higher in the M₁ population than in M₂. All identified types of chlorophyll mutants among the crops are directly related to mutations in chloroplast biosynthesis, the further degradation of chlorophyll, and bleaching due to the deficiency of carotenoids and may be associated with their preferential action on chlorophyll development genes [49,50,51].

We observed that the number of PBs rose with increasing colchicine concentrations and treatment durations. An increase in PBs was also reported with colchicine treatment by Hewawasam et al. [52]. An increased number of productive branches provides an increase in the number of panicles and the seed weight per panicle. We can also report that the most promising results (branch production and early ripening) were observed when applying the following treatment conditions: a colchicine concentration varying from 0.08 to 0.1% with a soaking duration varying from 12 h to 24 h. This study highlights a significant improvement in grain yield and the production of early-ripening varieties as effects of colchicine treatments for future proso millet breeding processes. At all colchicine concentrations and exposure periods, the agronomic traits SWP and TSW barely differed from the control values in M₁ and M₂ plants. At the higher colchicine concentrations of 0.08–0.1% combined with the longer soaking periods, the GSs of M₁ and M₂ plants decreased. In particular, it was shown that M₁-M₂ generations ripened nearly 4–7 days earlier than the control plants.

In this study, we used the ISSR marker method to evaluate the genetic changes at the molecular level after colchicine treatment. ISSR markers have proven to be suitable markers for genetic diversity analysis due to their high polymorphism and repeatability across the entire genome. For their use, no previous genomic information is required, and small amounts of DNA are needed [53,54]. The molecular data analysis using ISSR markers showed the existence of significant genetic diversity across proso millet exposed to various concentrations of colchicine. The number of genetic loci detected by the ISSR markers ranged from 4 to 53, with a total of 1333 loci across primers. The PIC was higher for the primer ISSR 840 (0.322) and lower for ISSR 819 (0.202). Of these 1333, 1281 loci were polymorphic, with a polymorphism percentage that ranged from 80 to 100%. Our results highlight that more bands were amplified at the 0.08% colchicine treatment, which indicates the usefulness of ISSR markers in detecting the mutagenic effects of colchicine.

5. Conclusions

The effects of colchicine on plant agronomic traits were assessed in the M1–M2 generations. At 0.06–0.08% colchicine concentrations with 12 and 24 h soaking durations, an increased number of productive branches was observed in both studied generations. In contrast, these 0.06–0.08% colchicine concentrations and 12 and 24 h treatment durations had adverse effects on the FG and PS parameters of M₁ and M₂ plants. The concentrations of 0.08–0.1% with a soaking duration of 24 h were found to be excessive, because they had a negative impact on the plants, particularly the inhibition of PG and PS. ISSR markers were used to confirm the existence of genetic diversity at the molecular level in response to the different mutagen concentrations. The effectiveness of colchicine as a mutagenic factor in the creation of new source material for proso millet breeding has been established. We identified the optimal colchicine concentration and treatment duration that positively affect the growing season longevity, productive branches, and grain yield. The obtained data revealed that the growing season length was reduced by colchicine treatment. Our studies will aid in creating varieties with features such as early ripening and higher yield in the proso millet breeding process.