Effect of X-rays on Seedling Pigment, Biochemical Profile, and Molecular Variability in Astrophytum spp.

Abstract

Cacti are important in agricultural economies and one of the most popular horticultural plant groups. The genus Astrophytum is one of the most valuable and desirable cacti for growers and collectors around the world. By selecting the appropriate breeding methods to induce variations in combination with modern biotechnology tools for rapid change detection, it is possible to meet the challenges of the modern world in creating new variability in plants. However, there exists a lack of research concerning the impact of ionizing radiation on cacti. The aim of the study was to assess the effects of X-rays at different doses (0 Gy—control, 15, 20, 25, and 50 Gy) on the dynamics of seed germination in vitro, changes in the color of seedlings, biochemical changes in the content of metabolites and changes at the molecular level in Astrophytum spp. ‘Purple’. A significant effect of X-rays on the induction of genetic variation was observed. Remarkably high polymorphism rates were observed, ranging from 59.09% for primer S12 to a full 100.0% for S3 and S8, as determined by the SCoT (Start-Codon-Targeted) marker. In addition, a large variation in the content of plant pigments (anthocyanins, carotenoids, chlorophyll a, and chlorophyll b) was noted. Additionally, discernible alterations in the color of the tested cactus seedlings, assessed by the RHSCC catalog, were attributed to the impact of ionizing radiation. These findings hold promise for the application of radiomutation breeding in acquiring new cactus cultivars.

1. Introduction

The Cactaceae family is relatively large, consisting of 100 genera and approximately 2000 cactus species [1]. Most representatives of this family are found in the Americas, and the densest concentrations inhabit desert and arid regions, particularly in Mexico, the southwestern United States, and the southwestern part of the Andean region [2]. Around the world, cacti have been arousing great interest for years as ornamental plants that are particularly valued and desired by breeders and collectors. Modern breeding of ornamental plants is focused on the use of new, constantly improved, and advanced methods, such as the genetic modification of plants, and biotechnological tools, such as in vitro cultures and molecular markers [3]. By carefully choosing suitable breeding methods in conjunction with biotechnological tools, it is possible to fulfill the capacity to meet the market requirements posed not only by a wide range of consumers but also by a sublime group of collectors who pay special attention to new and competitive quality features in relation to previously known cultivars [4,5,6]. Although the most modern breeding methods are currently based on gene editing technology like CRISPR/Cas, mutagenic factors such as ionizing radiation (including X-rays), microwave radiation, high-energy photons, electrons, as well as gold or silver nanoparticles, and ethyl methanesulfonate (EMS) remain valuable tools for effecting genetic changes, especially in ornamental plants boasting large unknown genomes and in polyploid plants [7,8,9,10,11]. However, the literature lacks studies on the effect of ionizing radiation on cacti, which hold significant economic importance within the horticultural market [12].

The genus Astrophytum Lem. is one of the most valuable and relatively rare representatives of the Cactaceae family. This genus comprises six distinct species: Astrophytum asterias, Astrophytum capricorne, Astrophytum caput-medusae, Astrophytum coahuilense, Astrophytum myriostigma, and Astrophytum ornatum. This genus has a wide range of colors from green to brown. During periods of high humidity, Astrophytum specimens are green in color; however, during periods of drought, the cactus turns brown, merging with the ground. Flowers of this type are yellow (also with orange centers), oval, and fleshy (with lengths from 1.25 to 15 cm). The seeds are dark brown and shiny. Cacti of this type have spots covered with fine flocs [13].

In nature, species belonging to the genus Astrophytum can be found at low elevations, among shrubs and grasslands. This ecological niche aligns with the warm and subtropical steppe climate prevalent in regions such as Mexico and Texas [13].

A. asterias Lem. is one of the most desired species by producers and collectors around the world. It is also the most endangered cactus species in its native environment [14]. This species was first collected in 1843 by Baron von Karwinsky and described as Achinas echinocactus in 1845 by Józef Zuccarini [13]. Charles Lemaire in 1868 classified the A. asterias species in the genus Astrophytum. This species name for the plant is widely accepted by the scientific community up to this day [13].

The progressive degradation of the natural habitats of A. asterias due to competitive grasses has led to the full protection of this species [14]. Consequently, to protect the genetic resources of endangered species, including Astrophytum asterias, there arises a necessity for the development of new and effective methods of reproduction, including in vitro techniques [15]. Presently, numerous cultivars of this species have been meticulously bred for commercial purposes.

The use of modern breeding methods (such as the genetic modification of plants) and biotechnological tools (such as in vitro cultures and molecular markers) can be used to assess the quality of plant materials, particularly during the initial stages of breeding. This approach leads to the shortening of the breeding cycle and conscious and monitored gene transfer, increasing the efficiency of selection and, thus, significantly reducing the costs of obtaining new cultivars [16].

Morphological features, such as plant color, have proven effective both in exploring variability and in guiding plant breeding endeavors. Morphological markers, while cheap and easy due to their operation based on observation and visual analysis, have many limitations. The disadvantages of this marker system are associated with factors such as its dependence on environmental conditions, the limited number of markers, the late appearance of traits enabling assessment, or the dominant–recessive mode of inheritance of traits [17,18,19].

In contrast, molecular markers reveal differences at the DNA level, rendering them among the most reliable marker systems that have revolutionized biological sciences and fields related to forensic science [20,21,22].

One of the most innovative techniques is the SCoT (Start-Codon-Targeted) marker system, developed by the team of Collard and Mackill [23] and others [24,25]. This system is based on relatively short and conserved regions that surround the translation start codon—ATG (adenine–thymine–guanine)—in the plant genome. This marker system has extended to studies centered on genetic diversity in various plant species, including mango (Mangifera indica L.) [26], tomato (Lycopersicum esculentum Mill.) [27], peanut (Arachis hypogaea L.) [28], pistachio (Pistacia vera L.) [29], China grass (Boehmeria nivea L. Gaudich.) [30], quinoa (Chenopodium quinoa Willd) [31], maize (Zea mays L.) [32], and gerberas (Gerbera jamesonii) [33]. To the best of our knowledge, this is the first instance of research being conducted on the effects of ionizing radiation on the cactus species Astrophytum spp. ‘Purple’.

The research aims to assess the impact of different doses of applied X-rays on several aspects of the cactus species Astrophytum spp. ‘Purple’. These aspects include the dynamics of seed germination in vitro, color changes, biochemical changes (including secondary metabolites such as carotenoids and anthocyanins), as well as the assessment of the content of chlorophyll a and chlorophyll b. Additionally, molecular changes will be investigated utilizing the SCoT marker in Astrophytum spp. ‘Purple’.

2. Materials and Methods

2.1. X-ray Treatment and the Scheme of Experiments

The research material comprised seeds of Astrophytum spp. ‘Purple’, obtained from our breeding efforts. These seeds underwent irradiation, utilizing X-rays with a nominal potential of 6 MV, administered through the Clinac 2300 CD accelerator situated at the Radiotherapy Department of the Oncology Center Prof. Franciszek Łukaszczyk in Bydgoszcz, Poland. The determination of the required exposure time was carried out in the Department of Medical Physics, using the Eclipse planning system by Varian.

The research encompassed radiation doses of 0 Gy (control), 15, 20, 25, and 50 Gy. During irradiation, 500 seeds for each combination (250 Petri dishes for each dose combination with 2 seeds) were contained within a 13.5 × 8 cm string bag (total 2500 seeds). These bags were situated atop solid water RW3 plates to enhance the secondary radiation interaction. Additionally, a bolus layer of known thickness, factored into the irradiation time calculations, was positioned atop the seeds.

After irradiation, the cactus seeds were subjected to presterilization procedures. This entailed a thorough rinsing under running water for 30 min, followed by rinsing with distilled water containing a drop of detergent for 5–10 min. Subsequently, the seeds were immersed in a 0.2% fungicide solution (62.5 WG Switch, Syngenta, Basel, Switzerland) to which detergent was added for around 17 h (on a shaker). A final soaking in a 0.5% fungicide solution, Amistar 250 S.C. (Syngenta, Basel, Switzerland), for 15 min was executed. After this regimen, the seeds were rinsed in distilled water for 5–10 min.

Continuing with the process, meticulous surface sterilization was performed under sterile conditions. The seeds underwent sequential treatments, including a 5–10 s exposure to 70% ethanol and a 30 min immersion in a 1.6% sodium hypochlorite solution; finally, they were rinsed three times for 10 min in sterile distilled water.

The subsequent stage entailed placing the prepared material, two seeds per specimen, onto MS medium [34]. This medium was solidified with agar (8 g dm⁻³, BioMaxima S.A., Lublin, Poland), and the pH was adjusted to 5.8 before autoclaving. The seeds were positioned within 5.5 cm diameter Petri dishes. The in vitro cultures, sealed with parafilm, were maintained within a growth room. The conditions in this environment were maintained at a temperature of 24 ± 2 °C under a 16 h photoperiod utilizing Philips TLD 54/34 W lighting and an average quantum irradiance of 47.84 µmol m⁻² s⁻¹.

Over the subsequent span of 8 weeks, daily observations were conducted to monitor the progression of the seed germination dynamics. The appearance of a germ root was considered a significant macroscopic symptom of seed germination. After 8 weeks, the seedlings obtained from the seeds were evaluated for color using the RHSCC color catalog [35]. In addition, measurements were taken to assess their size and weight, and the seedlings were set aside for further biochemical and molecular analyses. The experimental scheme is shown in Figure 1.

2.2. Biochemical Analyses

Seedlings randomly selected from each combination of radiation dose and color as determined by the RHSCC color catalog [35] were used for the biochemical tests. The tests were performed in triplicate for each combination, except for the seedlings without chlorophyll in Astrophytum spp. ‘Purple’. Due to the notably limited count of chlorophyll-free seedlings within Astrophytum spp. ‘Purple’, these were primarily reserved for genetic analyses.

In contrast, seedlings containing chlorophyll, categorized as green and brown seedlings, formed the primary focus of the biochemical testing. Additionally, creamy white seedlings (for doses of 0, 15, 20, and 25 Gy) or orange seedlings (for the 50 Gy dose) were included in the biochemical analysis.

2.3. Extraction of Plant Pigments from the Obtained Seedlings

The prepared plant material was carefully weighed using a precision analytical balance with an accuracy of 0.1 mg. Each seedling was subsequently crushed separately within a porcelain mortar, incorporating several dozen mg of quartz sand, which was ground alongside the seedling. For anthocyanin extraction, 3.5 mL of 1% HCl in methanol was added, while for the extraction of carotenoids, chlorophyll a, and chlorophyll b, 3.5 mL of 100% acetone was added.

The ensuing phase involved the quantitative filtration of the extracts, achieved through a funnel equipped with medium-grade qualitative filter paper, into 3.5 mL tubes. Absorption maxima were identified at wavelengths specific to each pigment (λ_max), and absorbance readings were taken at distinct wavelengths for the cumulative presence of the following: anthocyanins at 530 nm, carotenoids at 440 nm, and chlorophylls at 645 and 662 nm. The tests were carried out in triplicate for each combination of color and radiation dose, except in specified cases. The content of anthocyanins, carotenoids, and chlorophylls was assessed using the UV-VIS spectrophotometer (Shimadzu 1601PC, Kyoto, Japan) according to the modified procedure of Wettstein [36], Harborne [37], and Lichtenthaler and Buschmann [38], as referenced in Lema-Rumińska and Zalewska [39].

The algebraic method was used to quantify the concentration of total anthocyanins, using the following formula:

$$C_{\mathrm{A}}=\frac{A_{530}}{h·k}[\mathrm{g}·{\mathrm{d}\mathrm{m}}^{-3}]$$

where k = 61.7 (extinction coefficient for 3-cyanidine glycoside) and h = 1 cm (layer thickness).

The concentration of carotenoids was calculated according to the formula:

$$C_{\mathrm{K}}=4.695·A_{440}[\mathrm{m}\mathrm{g}·{\mathrm{d}\mathrm{m}}^{-3}]$$

Chlorophyll a was calculated according to the formula:

$$C_{\mathrm{a}}=11.24·A_{662}-2.04·A_{645}[\mathrm{\mu }\mathrm{g}·\mathrm{m}\mathrm{L}]$$

Chlorophyll b was calculated according to the formula:

$$C_{\mathrm{b}}=20.13·A_{645}-4.19·A_{662}[\mathrm{\mu }\mathrm{g}·\mathrm{m}\mathrm{L}]$$

2.4. Molecular Analyses

DNA was isolated from the obtained 2-month-old seedlings using a ready-made DNA isolation kit—Genomic Mini AX Plant (Spin) from A&A Biotechnology (Gdańsk, Poland). For each combination of radiation dose and color as identified by the RHSCC color catalog, DNA isolation was performed according to the Genomic Mini AX Plant (Spin) protocol. Approximately 100 mg of fresh seedling weight was used for the extraction process. This plant material was placed within a 1.5 mL tube and homogenized with the FastPrep^®-24 device (MP Biomedicals, Irvine, CA, USA). Subsequently, 900 µL of LS lysing suspension and 20 µL of Proteinase K solution were added. The whole sample was mixed and incubated at 50 °C for 10 min using a Biosan TS-100C thermoshaker (Biosan Medical-Biological Research and Technologies, Riga, Latvia) with continuous mixing at 1400 RPM. To ensure the thorough elimination of RNA, 2 µL of RNAse (10 mg/mL), sourced from A&A Biotechnology, was added. After incubation, the samples were vortexed for 2 min at 1000–1400 rpm. The samples were then subjected to centrifugation at 14,000× g for 5 min using a MPW-260R centrifuge (MPW Med. Instruments, Warsaw, Poland).

After centrifugation, 600 µL of the supernatant was collected and applied to a 2 mL Mini AX Spin column. These columns were spun for 30–60 s at 8000× g. After the used 2 mL tube was removed, the Mini AX Spin column was placed within a fresh 2 mL tube. Washing was initiated using the first wash buffer (W1) at a volume of 600 µL. The whole sample was subjected to centrifugation at 8000× g for 30–60 s. Similar steps were taken using the second wash buffer (W2) at a volume of 500 µL (A&A Biotechnology, Gdańsk, Poland).

The subsequent addition of an N-neutralizing buffer was omitted since the isolated DNA was not subjected to freezing. Following centrifugation, the utilized 2 mL tube was carefully removed, and the Mini AX Spin column was transferred to an elution tube. Subsequently, 150 µL of elution buffer E was loaded, and this mixture was allowed to incubate at room temperature for 5 min. After the incubation period, the system was centrifuged for 30–60 s at 8000× g. The column was then removed, and the tube containing the purified DNA was capped. DNA purity was measured using the NanoPhotometer^® NP 80 (Implen GmbH, Germany). The isolated DNA was stored in a refrigerator at 6 °C.

For molecular analyses, seven SCoT primers were employed twice. The attributes of these primers are outlined in

Table 1. Sequences of the SCoT primers used in the molecular analysis.

Primer	Sequence 5′-3′
S3	CAACAATGGCTACCACCG
S4	CAACAATGGCTACCACCT
S8	CAACAATGGCTACCACGT
S12	ACGACATGGCGACCAACG
S13	ACGACATGGCGACCATCG
S25	ACCATGGCTACCACCGGG
S33	CCATGGCTACCACCGCAG

.

PCR reactions were performed within a Thermal Cycler (BIO-RAD, model C1000 Touch^TM, Bio-Rad Laboratories, Hercules, CA, USA), employing a total volume of 25 µL. The final volume consisted of 1 µM of the single primer, 12.5 µL of 2×PCR MIX Plus kit (A&A Biotechnology, Gdańsk, Poland), which included 0.1 U/µL of Taq DNA polymerase, 4 mM MgCl₂, 0.5 mM of each dNTPs, 0.8 ng/µL of template DNA, and sterile water. The PCR reaction sequence was initiated with an initial denaturation step at 94 °C for 4 min. This was followed by 45 cycles: each cycle involved denaturation at 94 °C for 1 min, annealing at 50 °C for 1 min, and extension at 72 °C for 2 min. The last cycle was followed by the final extension step of 4 min at 72 °C. After the amplification process, 10 µL of the PCR reaction product was subjected to separation on a 1.5% agarose gel. The gel was stained with ethidium bromide in 1×TBE buffer.

Electrophoresis took place at 90 V for 20 min, followed by a subsequent run at 110 V for 90 min within the Standard Power Pack25 chamber (Biometra, Göttingen, Germany). The loading process involved the utilization of 6× TriTrack DNA Loading Dye (Thermo Fisher Scientific, Waltham, MA, USA) and the GenRuler Express DNA Ladder ready-to-use band size marker (Thermo Fisher Scientific).

After the procedure, the acquired products were archived utilizing the Gel Doc™XR+ archiving system (Bio-Rad Laboratories, Hercules, CA, USA). The labeling of the gel samples can be referenced in

Table 2. Genotype designation based on molecular analysis.

No. of Samples	Dose of X-Radiation [Gy]	Color of Seedling
1	0	brown
2	0	green
3	0	creamy white
4	15	brown
5	15	green
6	15	creamy white
7	15	orange
8	15	red
9	20	brown
10	20	green
11	20	creamy white
12	20	orange
13	25	brown
14	25	green
15	25	creamy white
16	25	orange
17	50	brown
18	50	green
19	50	creamy white
20	50	orange
21	50	red

.

2.5. Statistical Analyses

The obtained results from both biochemical analyses and the morphological parameters were statistically analyzed using the analysis of variance at a significance level of p ≤ 0.05 and Fisher’s F test using the Statistica 13.3 software (StatSoft Polska, Cracow, Poland). Molecular analyses were performed using GelAnalyzier 19.1 by Istvan Lazar Jr., and Istvan Lazar Sr. [40]. Within the molecular analyses, the SCoT marker loci for each genotype were counted using a binary system. In this system, the presence of a band was denoted as (1), while the absence of a band was represented as (0). The resultant matrix served as the basis for subsequent statistical calculations.

The dendrograms were created based on 0–1 binary matrices using agglomerative hierarchical clustering (AHC) with the unweighted pair–group average method (UPGMA) (Statistica 13.3 software, StatSoft, Cracow, Poland). Population groups were distinguished based on the Huff et al. [41] genetic distance calculation, analysis of molecular variance (AMOVA) and principle cluster analysis (PCoA) estimates using GeneAlEx 6.5 software [42].

3. Results

3.1. In Vitro Seed Germination Dynamics following X-ray Exposure

The progression of seed germination in Astrophytum spp. ‘Purple’ subjected to X-ray treatments at doses of 0 Gy (control), 15, 20, 25, and 50 Gy was closely observed over the ensuing 8 weeks following the initial sterile sowing on MS medium [34]. During the first 3 days of the in vitro experiment, no observable macroscopic indicators of seed germination (germ roots) were evident in the Astrophytum spp. ‘Purple’ cacti that had been exposed to X-rays as well as in the control seeds (Figure 2).

On the fourth day, the onset of germination was detected, marked by the emergence of a germ root in a single seedling from the pool that had previously been irradiated with a dose of 20 Gy. However, the commencement of germination for nonirradiated seeds and those exposed to higher doses (25 and 50 Gy) were notably delayed by approximately 3–4 days in comparison with the 15–20 Gy doses.

The subsequent days, spanning from the sixth to the nineteenth day, emerged as particularly fruitful in terms of seed germination for those subjected to X-rays at doses of 15 and 20 Gy. The number of germinated seeds for these two doses (15 and 20 Gy) was significantly higher (p ≤ 0.05) than that of the nonirradiated seeds (0 Gy control) and the seeds treated with the 25 and 50 Gy doses. It was only on the 19th day of observation that the number of germinating nonirradiated seeds equaled that of the seeds treated with 20 Gy radiation. Subsequently, from the 22nd day onwards, the number of germinating control seeds began to exceed all other combinations, a trend that persisted throughout the remaining observation period. Between the 14th and 18th days of culture, as well as from the 36th day until the end of the observation period, the significantly (p ≤ 0.05) least germinating seeds were identified among those irradiated at a dose of 50 Gy. In comparison with the control group, the difference in germinating seed count for those treated with X-rays at a 50 Gy dose was calculated to be 39.23%.

3.2. Evaluation of the Color of Seedlings and the Concentration of Plant Pigments after Seed Exposure to X-rays

Eight weeks after the irradiation of Astrophytum spp. ‘Purple’ cactus seeds with doses of 0 Gy (control), 15, 20, 25, and 50 Gy, and after initiating an in vitro culture, an evaluation of the seedlings’ color was assessed (according to the RHSCC catalog). Additionally, the concentrations of plant pigments, including anthocyanins, carotenoids, chlorophyll a, and chlorophyll b, were determined. Conversely, the implementation of X-rays in Astrophytum spp. ‘Purple’ resulted in the manifestation of a newfound red and orange color in the seedlings (Figure 3).

The in vitro cultivation of Astrophytum spp. ‘Purple’ seeds resulted in the generation of 1869 seedlings, with 1425 originating from seeds exposed to varying X-rays doses (Figure 4). Among these, the highest number of chlorophyll-free seedlings was obtained from seeds previously exposed to a radiation dose of 50 Gy (5.12%), whereas the lowest number was obtained from control seedlings not treated with the mutagenic factor (1.33%).

Given the limited number of nonchlorophyll seedlings (as tabulated in Figure 4), these were primarily designated for genetic analyses. Apart from these, among the seedlings containing chlorophyll (either green or brown), only creamy white seedlings were observed for doses of 0, 15, 20, and 25 Gy, while orange ones were noted for the 50 Gy dosage.

Concerning Astrophytum spp. ‘Purple’, all the creamy white seedlings, regardless of the radiation dose applied, exhibited an absence of anthocyanins, carotenoids, chlorophyll a, and chlorophyll b (

Table 3. Seedling color (according to the RHSCC catalog) and the concentration of the sum of anthocyanins, carotenoids, and chlorophyll a and b per 1 g of fresh weight of the seedling in relation to the X-ray dose in Astrophytum spp. ‘Purple’.

Dose of X-Radiation [Gy]	Color of Seedling (RHSCC)	Concentration [mg dm−3]
		Anthocyanins	Carotenoids	Chlorophyll a	Chlorophyll b
0	176 B, C	23.64 ± 0.00 c *	40.91 ± 0.03 d	41.53 ± 0.02 g	35.81 ± 0.19 a
	158 B	-	-	-	-
	144 A, C	30.47 ± 0.15 a	38.82 ± 0.00 f	45.58 ± 0.10 d	26.12 ± 0.01 e
15	176 B, C	17.42 ± 0.00 e	57.41 ± 0.05 a	75.21 ± 0.37 a	33.50 ± 0.19 b
	158 B	-	-	-	-
	144 A, C	13.56 ± 0.12 i	41.70 ± 0.01 d	51.01 ± 0.00 c	30.10 ± 0.02 c
20	176 B, C	15.45 ± 0.05 f	45.09 ± 0.00 c	43.06 ± 0.03 f	21.41 ± 0.20 f
	158 B	-	-	-	-
	144 A, C	24.37 ± 0.05 b	47.85 ± 0.02 b	66.50 ± 0.00 b	27.12 ± 0.12 d
25	176 B, C	22.88 ± 0.11 d	29.85 ± 0.02 f	35.76 ± 0.00 h	13.51 ± 0.05 h
	158 B	-	-	-	-
	144 A, C	23.20 ± 0.09 d	33.33 ± 0.00	44.57 ± 0.13 e	19.83 ± 0.14 g
50	176 B, C	14.18 ± 0.13 g,h	17.97 ± 0.02 g	24.74 ± 0.00 k	8.28 ± 0.62 i
	144 A, C	12.40 ± 0.04 j	8.33 ± 0.01 i	11.15 ± 0.00 i	4.06 ± 0.00 j
	28 C	14.68 ± 0.03 g	11.49 ± 0.11 h	7.88 ± 0.15 j	7.70 ± 1.96 i

* Means ± standard deviations within a column marked with the same letter do not differ significantly, with p ≤ 0.05.

). For the orange-colored, nonchlorophyll seedlings, the concentration of plant pigments was assessed due to their presence in these tested specimens.

Among the various seedlings, the highest concentration of anthocyanins and chlorophyll b was detected in the control seedlings (0 Gy dose), while the carotenoid and chlorophyll a concentrations were highest for seedlings resulting from prior irradiation of seeds with a dose of 15 Gy. On the contrary, the lowest levels of anthocyanins, carotenoids, and chlorophyll a were recorded from seedlings previously treated with a radiation dose of 50 Gy. The lowest concentration of chlorophyll b was observed in seedlings stemming from seeds subjected to 25 Gy radiation.

Across all the seedlings, irrespective of their color or the previous radiation dosage administered to the seeds, a reduced concentration of anthocyanins was evident in comparison with the control sample. The highest concentration of these pigments was present in a green seedling originating from a nonirradiated seed (30.47 mg dm⁻³), while the lowest concentration (12.40 mg dm⁻³) was identified in a green seedling derived from a seed previously exposed to X-rays at a dose of 50 Gy. A similar level of anthocyanin concentration to the control (brown seedling: 23.64 mg dm⁻³; green seedling: 30.47 mg dm⁻³) was apparent in material obtained from seeds treated with X-ray radiation of 25 Gy (brown seedling: 22.88 mg dm⁻³; green seedling: 23.20 mg dm⁻³).

The most substantial carotenoid content was identified in the seedling stemming from a seed exposed to a radiation dose of 15 Gy, which displayed a brown color (57.41 mg dm⁻³). In contrast, the lowest carotenoid levels were observed in the seedling originating from a seed treated with 50 Gy radiation, which exhibited an orange color (11.49 mg dm⁻³). In comparison with the brown control sample (40.91 mg dm⁻³), seedlings of the same brown color obtained from seeds treated with the mutagenic agent at 15 and 20 Gy doses exhibited higher concentrations of anthocyanins (57.41 and 45.09 mg dm⁻³, respectively).

Considering the green control sample (38.82 mg dm⁻³), seedlings sharing the same green color, obtained from seeds exposed to X-rays at 15 and 20 Gy doses, exhibited a higher concentration of carotenoids (41.70 and 47.85 mg dm⁻³, respectively).

The highest chlorophyll a concentration (75.21 mg·dm⁻³) was detected in samples extracted from the brown seedling whose seeds were subjected to X-ray irradiation at a dose of 15 Gy. Conversely, the lowest chlorophyll a concentration (7.88 mg dm⁻³) was observed in the sample extracted from the orange seedling resulting from a seed treated with the mutagenic agent at 50 Gy.

Relative to the brown control seedling (41.53 mg dm⁻³), samples derived from seeds exposed to radiation doses of 15 Gy (75.21 mg dm⁻³) and 20 Gy (43.06 mg dm⁻³) showcased increased chlorophyll a concentrations. Contrasting with the control sample that was characterized by a green color, extracts from seedlings of the same hue (resulting from seeds irradiated at 15 and 20 Gy doses) demonstrated amplified chlorophyll a levels (51.01 and 66.50 mg dm⁻³, respectively).

Chlorophyll b displayed its highest concentration (35.81 mg dm⁻³) in the extract from the brown control seedling, while the lowest concentration (13.51 mg dm⁻³) was noted in the extract from the seedling originating from a seed subjected to a 25 Gy radiation dose.

The extract derived from the green seedling, stemming from a seed exposed to X-rays (15 Gy dose), displayed a higher anthocyanin concentration (30.10 mg dm⁻³) compared with the control of the same color (26.12 mg dm⁻³). A slightly lower chlorophyll b concentration was observed in the brown seedling resulting from a seed exposed to a radiation dose of 15 Gy (33.50 mg dm⁻³) in comparison with the control seedling of the same color (35.18 mg dm⁻³) that had not been exposed to the mutagenic agent.

3.3. Molecular Analysis of Cactus Seedlings Obtained In Vitro from Seeds Exposed to X-rays

The outcomes of the molecular investigation conducted using the SCoT marker are presented in

Table 4. Number of products, band size range, number of loci, and polymorphisms obtained by molecular analysis using the SCoT marker in Astrophytum spp. ‘Purple’.

Primer	No. of Products	Band Size Range [bp]	No. of loci			Total loci	Polymorphism [%]
			Monomorphic	Polymorphic	Specific
S3	137	370–2830	0	21	1	22	100.00
S4	179	516–3613	1	22	1	24	95.83
S8	99	379–2338	0	22	3	25	100.00
S12	278	361–4878	9	11	2	22	59.09
S13	246	353–1900	3	12	4	19	84.21
S25	307	370–3040	3	27	1	31	90.32
S33	298	372–2776	5	18	3	26	80.77
Total	1544	-	21	133	15	169	-

. The cumulative count of the obtained products for Astrophytum spp. ‘Purple’ totaled 1544, averaging around 220.57 per primer. Notably, the highest number of products for Astrophytum spp. ‘Purple’ was observed with the S25 primer, while the lowest count was linked to S8. Band sizes exhibited a range spanning 353 to 4878 base pairs. In particular, a maximum of 31 loci were identified with primer S25. Remarkably high levels of polymorphism (ranging from 59.09% for primer S12 to 100.0% for S3 and S8) were ascertained following X-ray exposure.

The results of the UPGMA cluster analysis conducted on the examined genotypes within Astrophytum spp. ‘Purple’ is presented in Figure 5. Notably, the most significant genetic distance was observed with genotype 11 (creamy white seedling, resulting from a 20 Gy X-ray dose), which formed a distinct cluster apart from the rest. The correlation analysis between pigmentation and the molecular test results (UPGMA) showed that the creamy white color of seedlings was the most genetically distant from the other colors (genotypes 11, 3, 15, and 19), except for genotype 6, which was genetically close to the genotype 5 green seedling color (both obtained as a result of a radiation dose of 15 Gy). However, close genetic relatedness was noted for the orange and red genotypes (genotypes 7 and 8 and genotypes 20 and 21, respectively). Within the two subclusters, genotype 2—a green seedling that was not subjected to X-rays (control)—emerged as a separate cluster. Conversely, the smallest genetic distance was noted between genotypes 7 (orange) and 8 (red), both irradiated with a 15 Gy dose, as well as between genotypes 5 (green seedling) and 6 (creamy white seedling), both subjected to a 15 Gy dose. Similarly, a minimal genetic distance was recorded between genotype 20 (orange) and 21 (red), both exposed to a 50 Gy radiation dose.

A slightly different interpretation of the data was provided by the PCoA analysis of the Astrophytum spp. ‘Purple’ genotypes (1–21) (Figure 6). Among the tested plants, a distinct group was formed by genotypes 1, 2, and 4. High distinctiveness from other tested plants was revealed also for genotypes 9, 10, 13, and 14. The majority of the tested genotypes (3, 5, 6, 7, 8, 11, 12, 15, 16, 17, 18, 19, 20, 21) were arranged in the third uniform group. The smallest genetic distance was found for genotypes 7 and 8 and for genotypes 5 and 6.

The AMOVA analysis confirmed the occurrence of interspecific genetic variation. Molecular variance amounted to 100% among the tested Astrophytum spp. ‘Purple’ genotypes, according to the SCoT analysis (

Table 5. Analysis of molecular variance (AMOVA) in the studied Astrophytum spp. ‘Purple’ genotypes (1–21) based on SCoT analysis.

Summary of AMOVA
Source of variation	df	SS	MS	Est. Var.	%
Among populations	20	1381.43	69.07	23.02	100%
Within populations	42	0.000	0.00	0.00	0%
Total	62	1381.43		23.02	100%

).

4. Discussion

Inducing mutations into genes with ionizing radiation is a well-known method of increasing genetic diversity in crop breeding. X-rays have proven effective in inducing phenotypic changes in many ornamental plant species, including achimenes, alstroemeria, azalea, begonia, bougainvillea, chrysanthemum, dahlia, carnation, hibiscus, lily, calla, rose, and tulip [11,43,44,45,46,47,48,49,50]. However, the literature lacks studies on the effect of ionizing radiation on cacti.

Mutations can occur spontaneously but at a very low frequency, or they can be artificially induced through physical or chemical treatments [51]. Among the commonly used physical agents for inducing mutations, ionizing radiation holds a significant place. This radiation, due to its high energy, can penetrate deeply into tissues. A crucial consideration in researching the influence of radiation on mutation formation is the appropriate selection of plant tissue to be subjected to the mutagenic factor. It is important to note that irradiating nonmeristematic tissues increases the chance of obtaining genetically altered mutants that are homogeneous and genetically stable. This phenomenon occurs as these mutants come from single mutated cells, and the new color may cover the entire plant organ, such as the inflorescence [52]. Conversely, when dealing with meristematic tissues, genetically heterogeneous chimeras often emerge, as frequently observed in chrysanthemums [47].

Numerous research teams have delved into the effects of radiation on seeds. Generally, a high dose of X- or γ-irradiation is understood to impede plant development, thereby retarding growth [53,54,55,56]. Kumar et al. [57] indicated that radiation significantly inhibits the germination of seeds exposed to higher doses. However, scientists also indicate that a properly selected small dose can benefit plants [56,57,58,59,60]. Consequently, preliminary studies are imperative to ascertain the optimal radiation doses. In the context of cacti, these ideal doses remain unknown [51].

The only study concerning the effect of radiation on cactus seeds, specifically γ-radiation exposure, was reported by Boujghagh et al. [51]. Prickly pear seeds (Opuntia ficus-indica) originating from two regions, Aït Baamrane (southern Morocco) and SkhourRhamna (Marrakech), were exposed to gamma irradiation at varying doses: 50, 100, 150, and 200 Gy. Germinated seeds were counted every 2 weeks for a period of 4 months. Notably, the control group displayed a much higher germination rate (97%, equivalent to 0.75 seeds daily) than the seeds previously exposed to radiation. In contrast, the irradiated seeds showed a reduction in germination proportionate to the increase in radiation dose.

For seeds from the Aït Baamrane region, the germination rate was lower, with figures of 37% for a 200 Gy dose, 39% for 150 Gy, and 49% for 100 Gy. Likewise, for the SkhourRhamna region, the germination rates stood at 37% and 42% for a 200 Gy dose, 39% for 150 Gy, and 52% for 100 Gy. This irradiation resulted not only in a decrease in germination capacity but also extended the time frame for germination. This phenomenon could potentially be attributed to the damage incurred by the seeds, which, by nature, had an early germination tendency and relatively thin seed coats. In addition, the irradiated seeds exhibited reduced water content in comparison with those unaffected by mutagenic agents [58]. As the experiment concluded, the percentage of nongerminating seeds escalated as the applied radiation dose increased.

Based on those outcomes, it was determined that the optimal radiation dose for Opuntia seeds should fall within the range of 50–125 Gy. Within this range, germination capacities of 70% (for Aït Baamrane seeds) and 50% (for SkhourRhamna seeds) could be achieved. Unlike this previous study, no research has been conducted thus far on the effect of radiation on the seeds of the Astrophytum spp. ‘Purple’ cacti. In these studies, the varying radiation doses led to fluctuations in the number of seedlings observed, along with variations in the acceleration or deceleration of seed germination.

Similar to the study by Boujghagh et al. [51], our research also showed a significant effect of X-rays on seed germination in the tested Astrophytum spp. ‘Purple’. Exposure of seeds to X-rays resulted in a lower percentage of germinating seeds compared with the control (0 Gy). The highest dose of 50 Gy resulted in the lowest percentage of germinating seeds. However, in contrast to the research on prickly pear, despite the adverse effect of X-rays on the number of seedlings obtained, the use of low doses of radiation had the beneficial effect of shortening the germination time. Doses of 15 and 20 Gy accelerated the formation of seedlings by 3–4 days compared with the control (0 Gy). Similarly, in line with the experiment by Boujghagh et al. [51], an increase in the radiation dose used (25–50 Gy) delayed germination by 1–2 days compared with the control.

Research by Boujghagh et al. [51] on the germination of Opuntia ficus-indica seeds irradiated with γ-radiation in vivo suggests that for cacti, the optimal dose should range from 50 to 125 Gy, yielding 70% and 50% germination, respectively. It is worth noting that such high doses of ionizing radiation are lethal for most plant species; for example, in the case of large-flowered chrysanthemums, a dose of 25 Gy is already sublethal [61].

In addition to the selection of the appropriate plant material for irradiation, the selection of the appropriate radiation dose also assumes significance in the context of X-ray applications. Suyitno et al. [62] obtained five groups of mutants resulting from the treatment of orchid seeds (Spathoglottis plicata) with X-rays at dosages of 0 rad, 6 rad, 12 rad, 18 rad, and 24 rad (1 rad = 0.01 Gy). Mutations encompassed variations in the leaf structure (thick, unopened leaves), root system (a proliferation of lateral roots), and shoot development (feeble, swollen shoots), pigmentation shifts (yellow leaf coloration, white leaf spots), and accelerated flowering. Their findings demonstrated that doses between 12 rad and 18 rad had the capacity to spur morphological diversity, particularly pertaining to roots, leaves, and shoots.

Our investigation into cacti showed a pronounced influence of X-rays in increasing the number of nonchlorophyll-bearing seedlings in Astrophytum spp. ‘Purple’. In this species, the percentage of nonchlorophyll seedlings displaying orange, creamy white, and red colorations surged in tandem with escalating X-ray dosages (ranging from 1.44% at 15 Gy to 5.12% at 50 Gy). In the case of our research involving Astrophytum spp. ‘Purple’, the percentage of nonchlorophyll seedlings was amplified proportionally with the dosage of radiation administered, with the highest recorded at the 50 Gy threshold.

The study conducted by Miler et al. [9] equally underscored the significance of selecting an appropriate radiation dose. Miler et al. [9] studied the effect of irradiation at different levels (5, 10, and 15 Gy) on the ovules of the large-flowered chrysanthemum cultivars ‘Professor Jerzy’ and ‘Karolina’.

The assessment encompassed growth and flowering parameters, wherein the most satisfactory results were achieved at a dosage of 10 Gy. This particular dose exhibited the most effectiveness in eliciting stable mutations in inflorescence color and form, devoid of undesirable side effects, such as a delay or extension of cultivation time. In the context of the ‘Professor Jerzy’ cultivar, the new phenotypes of inflorescences emerged as dark yellow and pinkish, while in the case of ‘Karolina’, the mutation led to orange–red inflorescences. Elevating the radiation dosage demonstrated a negative correlation with the ability to regenerate the explants.

Findings from the investigation involving Astrophytum spp. ‘Purple’ cacti showed that the percentage of nonchlorophyll seedlings was most pronounced at the 50 Gy dose. Our research aligns with this, affirming the relationship between escalated dosage and heightened phenotypic changes.

In ornamental plants like chrysanthemums, the color of their inflorescence hinges on the presence and quantity of various pigments, including anthocyanins, carotenoids, flavones, and flavonols, predominantly found in the L1 and L2 layers [47,63]. Anthocyanins are responsible for generating the blue, red, and violet colors, while flavonols and flavones contribute to the white, cream, and yellow colors. Carotenoids influence colors ranging from yellow to red and orange [39]. Chlorophyll assumes a crucial role in converting light energy into chemical energy, serving as an indicator of plant metabolism efficiency and health. Carotenoids, conversely, play a major role in photoprotection and defense against oxidative stress in plant cells [64,65,66,67]. The emergence of new inflorescence colors is often attributed to modifications in the biosynthetic pathways of plant pigments [68].

While anthocyanins are commonly found in higher plants, in cacti and many other species within the Caryophyllales order, they are replaced by betalains. The biosynthesis of anthocyanins can be blocked at a late stage during the transition from dihydroflavonols to anthocyanins. Sakuta et al.’s [69] research on dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) isolation and functional characterization revealed that the absence of anthocyanins is attributed to the suppression of these enzymes. However, in the transgenic cultivar Astrophytum myriostigma, the possibility of anthocyanin biosynthesis, instead of betalains, in flower petals was confirmed [69].

The content of plant pigments holds significant sway over the resulting coloration of plants. Our studies have demonstrated that the relationship between the radiation dose used and the pigment content differs in the case of Astrophytum spp. ‘Purple’, where an inverse correlation was observed between increasing X-ray doses and the concentration of plant pigments. A noteworthy exception was observed at the 15 Gy dose, which resulted in an increased concentration of carotenoids and chlorophyll a in brown seedlings. Conversely, the 50 Gy dose yielded the lowest concentrations of anthocyanins, chlorophyll b, and carotenoids in green seedlings, as well as chlorophyll a in orange seedlings.

Dhawi et al. [70] established that low radiation doses had a positive impact on photosynthetic pigments in date palms (Phoenix dactylifera), while higher doses exerted a negative effect. Their study also indicated that chlorophyll a and carotenoids were more susceptible to magnetic fields than chlorophyll b. Similarly, Pick Kiong Ling et al. [71] found lower chlorophyll levels in seedlings exposed to γ-radiation, as compared to nonirradiated sweet orange (Citrus sinensis) seedlings. Nonetheless, the chlorophyll content appeared to be largely unaffected by low doses of γ-radiation. Conversely, Abu et al. [72] showed an increase in chlorophyll a, chlorophyll b, and total chlorophyll levels in γ-irradiated Vigna unguiculata [70,71,72]. In the context of Astrophytum spp. ‘Purple’ our studies noted a decrease in plant pigment concentration with escalating X-ray doses, excluding the 15 Gy dosage.

The determination and differentiation of newly acquired cultivars from their mother plants often rely on multiple approaches. Morphological traits are analyzed to establish distinctness, which is often accompanied by an assessment of the qualitative and quantitative composition of the plant pigments [39]. Furthermore, biotechnological tools, such as molecular markers, are employed to enhance objectivity and reliability [73]. Genetic markers play a pivotal role in determining genetic distance in mutation breeding.

Our research validated a remarkably high genetic diversity using SCoT markers in X-irradiated Astrophytum spp. ‘Purple’ seedlings. In parallel studies, X-rays have emerged as the most effective mutagen against gold nanoparticles and microwaves in L. spectabilis ‘Valentine’ [11]. In particular, the 20 Gy radiation dose demonstrated remarkable efficacy in inducing mutations in the golden heart. Nonetheless, the number of plants exhibiting genetic changes remained relatively modest, with only 3.3% displaying alterations in DNA content post-X-ray irradiation. The number of polymorphisms ranged from 0% to 6.25%, depending on the SCoT primer, with an average of 2.4 polymorphic loci.

In our research, a notably extensive genetic distance was observed among cactus seedlings irradiated with a 50 Gy dose. Within the examined cacti, considerably higher levels of polymorphism in seedlings were discerned, ranging from 59.09% to 100% in the case of Astrophytum spp. ‘Purple’. It is worth noting that a similar genetic distance was generated by the SCoT marker for separate species belonging to the genus Astrophytum and Frailea [74]. Similar levels of polymorphism (PPB%), ranging from 75% to 100%, were demonstrated by Nasri et al. [10] in chrysanthemum mutants generated through the action of the chemical mutagen EMS using the IRAP (Inter-Retrotransposon Amplified Polymorphism) marker. It also turned out that the largest genetic distances between seedlings exposed to X-rays in Astrophytum spp. ‘Purple’ occurred for creamy white genotypes, which may be the result of differences related to the pigment biosynthesis pathways.

5. Conclusions

X-rays had a noticeable impact on the germination dynamics of Astrophytum spp. ‘Purple’; doses of 15 and 20 Gy accelerated the onset of seed germination by 3–4 days in comparison with nonirradiated (control) seeds. Conversely, seeds exposed to higher doses (25–50 Gy) germinated 1–2 days later than the control seeds. The maximum count of germinating seeds was observed in the nontreated seeds (control—0 Gy), while the 50 Gy radiation dose exhibited the most pronounced influence in diminishing the number of germinating seeds relative to the control (by 39.23%) and other radiation doses.

The application of X-rays influenced the percentage of nonchlorophyll seedlings in Astrophytum spp. ‘Purple’, with the percentage of nonchlorophyll seedlings increasing in correspondence with the radiation dose, peaking at the 50 Gy dose. The dose of 20 Gy is optimal for Astrophytum spp. ‘Purple’ due to the significantly higher percentage of nonchlorophyll seedlings with a simultaneous high percentage of germinating seedlings. The application of X-rays introduced a novel seedling color (red and orange), a phenomenon previously unobserved in Astrophytum spp. ‘Purple’. In addition, the effect of X-rays on seedling color and the varied concentration of plant pigments in seedlings was visible. As the X-ray dose escalated, a decrease in plant pigment concentration was discerned, even within a specific color category. An exception was the 15 Gy irradiation, which amplified the carotenoid and chlorophyll a content in brown seedlings. Notably, the utilization of 50 Gy irradiation resulted in the lowest concentration of anthocyanins, chlorophyll b, and carotenoids in green seedlings, as well as chlorophyll a in orange seedlings. Creamy white seedlings exhibited no presence of anthocyanins or carotenoids and chlorophylls.

The significant influence of X-rays on the genetic diversity of seedlings was established through the application of the SCoT marker, with polymorphism levels proving remarkably high. In addition, the UPGMA analysis showcased substantial genetic divergence among the examined genotypes exposed to X-rays. Thus, X-rays can be a valuable tool for breeding new cultivars in Astrophytum spp.