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Article

The Influence of X-ray Radiation on the Morphological, Biochemical, and Molecular Changes in Copiapoa tenuissima Seedlings

by
Piotr Licznerski
1,
Emilia Michałowska
2,
Alicja Tymoszuk
1,
Janusz Winiecki
3 and
Justyna Lema-Rumińska
4,*
1
Laboratory of Horticulture, Bydgoszcz University of Science and Technology, 6 Bernardyńska St., 85-029 Bydgoszcz, Poland
2
Institute of Forensic Genetics, Adam Mickiewicz Av. 3/5, 85-071 Bydgoszcz, Poland
3
Department of Medical Physics, Prof. Franciszek Łukaszczyk Memorial Oncology Center Bydgoszcz, 2 Romanowska St., 85-796 Bydgoszcz, Poland
4
Department of Environmental Biology, Kazimierz Wielki University in Bydgoszcz, 12 Ossoliński Av., 85-093 Bydgoszcz, Poland
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(9), 2155; https://doi.org/10.3390/agronomy14092155
Submission received: 21 August 2024 / Revised: 18 September 2024 / Accepted: 20 September 2024 / Published: 21 September 2024
(This article belongs to the Special Issue Plant Tissue Culture and Plant Somatic Embryogenesis–2nd Edition)
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Abstract

:
Cactaceae are a significant group of ornamental plants in the horticultural market. In the present study, X-rays were used for the first time to induce mutational changes in the cactus Copiapoa tenuissima. The aim of this study was to assess the genetic variability in seedlings exposed to in vitro X-ray irradiation at doses of 0, 15, 20, 25, and 50 Gy (radiation time from 5 min 13 s to 17 min 22 s) by morphological analysis, a spectrophotometric evaluation of plant pigment content, and the confirmation of changes at the genetic level using SCoT (start codon targeted) markers. The results showed that the percentage of colorful seedlings increased with the radiation dose and was the highest for the 50 Gy dose (4.89%). The radiation doses of 25 and 50 Gy generated seedlings with a new color (orange-brown), which had not yet been observed in C. tenuissima. With the increase in the radiation dose, as compared to control seedlings, brown seedlings showed an increase in the concentrations of carotenoids, chlorophyll a, and chlorophyll b, while green seedlings showed an increase in the concentrations of anthocyanins and chlorophyll b and a decrease in the concentrations of carotenoids and chlorophyll a. The unweighted pair group method analysis showed a very large genetic distance among the tested genotypes exposed to X-rays. The results of the present study provide a novel direction for using X-rays to breed new cultivars of C. tenuissima.

1. Introduction

Cacti (Cactaceae) are one of the most popular groups of horticultural plants in the world [1]. The genus Copiapoa originally included six species; however, as research progressed, their number increased remarkably. In the natural environment, most Copiapoa species are endemic, and some of them have a limited range of occurrence [2,3,4]. Approximately 50% of the species belonging to this genus are at risk of extinction. Today, Copiapoa species commonly occur in botanical gardens in the United States, Germany, and Great Britain [3,4].
Copiapoa tenuissima Ritt. (synonym: C. humilis var. tenuissima Ritt.) occurs in the extremely dry desert and stony regions of Chile. This species requires very strict protection against extinction; hence, it is included in Appendix II of the CITES convention. C. tenuissima Ritt. has long and tuberous roots because it grows in areas with scarce water and organic matter. This cactus has a spherical shape, slightly flattened, with a diameter of 2–5 cm. The epidermis of this cactus is dark in color, and depending on the environmental conditions, the epidermis color ranges from dark green and olive green to almost purple-black. The shoots have 13–16 ribs; they are often spiral and have abundantly wooly areoles ending in thin thorns. The flowers are light yellow and delicately scented; they bloom in spring and summer [5,6].
In vitro cultures can help maintain the genetic resources of this species and can be used to create new attractive cultivars for the horticultural market. The combination of breeding and selection enables them to meet the increasingly new market requirements. All ornamental plants have a long history of breeding and selection, which afforded them new morphological features and unprecedented properties. Breeders use various methods to change the morphological features of plants, often by interfering with their genes [7,8].
One of the attractive methods of breeding new cultivars is mutation breeding, which involves inducing mutations in the genetic material of the plant. These changes are permanent and inherited and are used at various stages of breeding programs [9,10,11,12,13].
Mutagenic factors can cause various mutations: from a single nucleotide to the duplication of the entire genome. Mutations can be broadly divided into point mutations (occurring within a gene in the DNA sequence), structural mutations within chromosomes (inversions, translocations, duplications, and deletions), and mutations leading to changes in the chromosome number (polyploidy, aneuploidy, and haploidy). Structural mutations are the result of chromosomal breaks and rearrangements. Ionizing radiation mainly causes such changes. There are four categories of such rearrangements: deficiencies, duplications, inversions, and translocations. The vast majority (about 90%) of radiation-induced chromosomal aberrations are deletions and are often lethal. However, some deletions can block biochemical pathways, and, for example, if this occurs in a pathway leading to the synthesis of a toxic metabolite, such a deletion can result in a useful, nontoxic plant product. Duplications are very important in mutation breeding, especially at the polyploid level. These mutations are divided into allopolyploidy, autopolyploidy, and aneuploidy. It is estimated that 50–70% of ornamental flowering plants are polyploids [14,15].
The use of radiomutation in ornamental plants has enabled us to obtain plants with a different color (within the gene pool of a given species) but with the same cultivation requirements; this approach is very valuable for plant producers and potential customers. The main goal of mutation breeding in ornamental plants is to obtain new flower colors, shapes, and sizes. Many ornamental crop cultivars with unique color characteristics have been developed as well using X-rays [14,16,17].
The radiomutation method produces mutants in the first mutational generation, thus enabling us to obtain a very large number of mutants within a short time [18].
Genetic changes induced by mutagenic agents can be detected using molecular markers. The most effective markers are SCoT (start codon targeted) polymorphism marker [19]. Because of their characteristics, these are dominant markers with several applications, as they demonstrate higher polymorphism and better detection ability [20,21,22,23]. These markers are reliable and economical, and the primers used are simple to design [21,24]. Moreover, SCoT markers are relatively closely associated with the target gene, thus providing more information correlated with the plant’s biological characteristics [24]. Based on their properties, SCoT markers are successfully used to generate genetic maps in plant genetic analyses through “DNA fingerprinting”, the mapping of quantitative trait loci (QTL), and phylogenetic investigations [25]. Biochemical changes are detected by analyzing metabolites, mainly plant pigments extracted from tissues, by using spectroscopic or chromatographic methods [26].
The research hypothesis assumes that X-rays at doses of 15, 20, 25, and 50 Gy may affect the seed germination rate, seedling color change, biochemical changes, and changes at the molecular level in Copiapoa tenuissima in comparison to the material not exposed to X-rays (as control).
To the best of our knowledge, in the present study, the effects of X-rays on the cactus C. tenuissima were studied for the first time. This study aimed to conduct a morphological assessment, spectrophotometric determination of plant pigment content in seedlings, and molecular analysis based on the SCoT marker for C. tenuissima seedlings obtained from seeds exposed to X-ray radiation at the 0, 15, 20, 25, and 50 Gy doses.

2. Materials and Methods

2.1. Plant Material and X-ray Treatment

The research material comprised C. tenuissima seeds obtained from our breeding. These seeds were irradiated with X-rays at 6 MV potential by using the Clinac 2300 CD accelerator at the Radiotherapy Department of Prof. Franciszek Łukaszczyk Oncology Center, Bydgoszcz, Poland. The exposure time was calculated at the Department of Medical Physics by using the EclipseTM treatment planning system from Varian. The radiation doses used were as follows: 0 (control), 15, 20, 25, and 50 Gy. The radiation time was from 5 min 13 s to deliver 15 Gy to 17 min 22 s to deliver 50 Gy. During irradiation, the seeds in a given combination were placed in string bags of 13.5 × 8 cm (500 seeds per bag). The bags were placed on solid water RW3 plates and covered with a bolus layer of known thickness. After irradiation, the seeds were subjected to initial and proper sterilization according to a previously described procedure [27] and placed (2 seeds each) on Murashige and Skoog 1962 medium [28] with agar (8 g dm−3) solidified in 5.5 cm diameter Petri dishes, with a pH set to 5.8 before autoclaving. In vitro cultures (sealed with parafilm) were maintained in a growth room at 24 ± 2 °C, with a 16 h photoperiod (Philips TLD 54/34 W), with an average quantum irradiance of 47.84 µmol m−2 s−1.
After 8 weeks, the seedlings obtained from the seeds were assessed for color by using The Royal Horticultural Color Chart Catalog [29], measured, weighed, and used for further biochemical and molecular analyses. Because of their very small number, colorful seedlings were mainly used for genetic analyses. Furthermore, apart from seedlings containing chlorophyll (green and brown seedlings), yellow-cream seedlings (for 15, 20, and 25 Gy doses) or orange-brown seedlings (for 50 Gy dose) were used for pigment tests.

2.2. Biochemical Analyses

Randomly selected seedlings from each combination of radiation dose and color were selected for biochemical tests. The tests were performed in triplicate for each combination.
The irradiated seedlings were weighed on an analytical balance. Each seedling was crushed individually in a porcelain mortar by adding a few mg of quartz sand, and the mixture was grounded. To extract anthocyanins, 3.5 mL of 1% HCl in methanol p.a. was added to the mixture. To extract carotenoids and chlorophyll a and chlorophyll b, 3.5 mL of 100% acetone p.a. was added to the mixture. The extracts were quantitatively filtered through a funnel with filter paper (medium quality) into 3.5 mL tubes. The absorption maxima at a wavelength characteristic of a given pigment (λmax) were determined. Absorbance measurements were conducted at specific wavelengths for anthocyanins at 530 nm, carotenoids at 440 nm, and chlorophylls at 645 and 662 nm. The tests were conducted in triplicate for each combination of seedling color and radiation dose. The contents of anthocyanins, carotenoids, and chlorophylls were determined using a Shimadzu 1601PC spectrophotometer according to the modified procedure of [30,31,32] described by Licznerski et al. [27].

2.3. Molecular Analyses

DNA was isolated from seedlings from each combination of radiation dose and seedling color in accordance with the Genomic Mini AX Plant (Spin) protocol from A&A Biotechnology (Gdańsk, Poland). Approximately 100 mg of fresh seedlings was placed in a 1.5 mL tube and homogenized using the FastPrep®-24 device (MP Biomedicals, Irvine, CA, USA). The procedure was described in detail by Licznerski et al. [27]. Molecular analysis was performed using seven SCoT primers (Genomed S.A., Warszawa, Poland) [19]; the characteristics of these primers are presented in Table 1. The obtained products were archived using the Gel Doc™ XR+ gel archiving system (Bio-Rad Laboratories, Hercules, CA, USA). Table 2 shows the labeling of the samples in the molecular analyses.

2.4. Statistical Analyses

Statistica 13.3 (StatSoft, Krakow, Poland) was used for the statistical analysis of the obtained results of biochemical analysis and morphological parameters by using an analysis of variance, with a significance level of p < 0.05. Molecular analyses were performed using the Gel Analyzer 19.1 software (www.gelanalyzer.com, accessed on 14 March 2023). All SCoT marker loci, indicated for each genotype, were counted using a binary system, where the presence or absence of a band was represented by 1 or 0, respectively. The resulting matrix was used as a basis for statistical calculations. Cluster analysis using the unweighted pair group method (UPGMA) analysis was used to develop the results with the Statistica 13.3 program. Population groups were differentiated based on Huff et al. [33], molecular variance analysis (AMOVA), and principal coordinate analysis (PCoA) estimates using GenAlEx 6.5 software [34].

3. Results

3.1. Assessment of Seedling Color and Morphological Characteristics of Seedlings Obtained from Seeds Exposed to X-ray Radiation

Eight weeks after the irradiation of C. tenuissima seeds with X-rays at doses of 0, 15, 20, 25, and 50 Gy and the establishment of an in vitro culture, the color of the obtained seedlings was assessed according to the RHSCC catalog (Figure 1). The lowest number of colorful seedlings was detected in control seedlings (0.00%), while the highest number was found in the seedlings obtained from seeds irradiated at the 50 Gy dose (4.89%; Figure 2). The significantly greatest number of germinating seeds (GR 43.2 ± 4.26%) was obtained for seeds irradiated with the 25 Gy dose as compared to non-irradiated seeds (GR 28.2 ± 1.28; Table 3). Following seed irradiation with the 15 to 50 Gy doses, a total of 11.07% colorful seedlings were obtained, which exhibited orange-brown and yellow-cream colors.
In the morphological characterization of the seedlings obtained from seeds irradiated with X-rays at various doses (from 0 to 50 Gy), in addition to color, the fresh weight, length, and width of the seedling and the length of its longest root were determined (Table 3). The lowest average fresh weight parameters, hypocotyl and epicotyl lengths, epicotyl width, and the longest root length were recorded for seedlings obtained from seeds treated with the 20 Gy radiation dose, but the seedling length and epicotyl width did not differ significantly for the seeds irradiated at the 50 Gy dose.
However, the seedlings obtained from seeds irradiated with the 25 Gy dose had the significantly highest average value of parameters such as fresh weight, the length of the entire seedling (epicotyl and hypocotyl), and epicotyl width, but the epicotyl and hypocotyl length and the longest root length did not differ significantly from those of the control. The difference between the fresh weight of the control and treatment 25 Gy was 19.99%. The average longest root length was recorded for a seedling obtained from seeds treated with 50 Gy X-ray irradiation.

3.2. Biochemical Analyses of Plant Pigments of Seedlings Obtained from Seeds Depending on X-ray Radiation

The significantly lowest concentrations of anthocyanins were noted for orange-brown seedlings (RHSSC code 163 A) obtained from seeds exposed to X-ray irradiation at 50 Gy, while the significantly highest concentrations at the 15 Gy dose were noted for green seedlings (RHSCC code 144 A, C; Table 4). The control seedlings showed the significantly lowest concentrations of carotenoids, chlorophyll a, and chlorophyll b. The remaining green seedlings (RHSCC code 144 A, C) obtained from seeds treated with X-rays at the 20, 25, and 50 Gy doses also had significantly higher anthocyanin concentrations than that of the control sample (2.83 mg dm−3). Samples extracted from brown seedlings (RHSCC code 176 B, C, obtained from seeds treated with 15, 20, 25, and 50 Gy irradiation doses) had a significantly lower concentration of anthocyanins than that of samples obtained from control seedlings with the same color. Yellow-cream seedlings (RHSCC 158 B, C) had a significantly lower concentration of anthocyanins than brown control seedlings but showed a higher concentration of anthocyanins than green seedlings. The significantly lowest and highest concentrations of carotenoids were detected for control seedlings, i.e., for green seedlings (52.40 mg dm−3) and brown seedlings (5.28 mg dm−3), respectively. In green seedlings, the concentration of carotenoids decreased with the increase in the applied radiation dose (from 39.38 mg dm−3 for 15 Gy dose to 32.84 mg dm−3 for 25 Gy dose and 33.21 mg dm−3 for 50 Gy dose). Regardless of the applied radiation doses, extracts from brown and yellow-cream seedlings contained significantly higher concentrations of carotenoids than those from control samples without irradiation.
The significantly lowest (7.32 mg dm−3) and highest (87.32 mg dm−3) concentrations of chlorophyll a were detected for samples extracted from control seedlings. X-ray irradiation significantly reduced chlorophyll concentration in green seedlings; however, brown seedlings from seeds exposed to X-rays showed a significant increase in chlorophyll a concentration. The significantly lowest concentration was detected for brown seedlings from the control sample (3.30 mg dm−3), while the highest concentration (36.97 mg dm−3) of chlorophyll b was found in the extracts from a green seedling obtained from a seed exposed to the 15 Gy irradiation dose. Brown, orange-brown, and yellow-cream seedlings from seeds treated with X-rays exhibited higher anthocyanin concentrations than control seedlings of the same color but showed lower anthocyanin concentrations than green control seedlings (Table 4).

3.3. Molecular Analysis of Seedlings Obtained from Seeds Following In Vitro Exposure to X-ray Irradiation

For C. tenuissima, the total number of products for all primers was 926, with an average of 132.28 products per primer (Table 5). Primer S13 generated the greatest number of products, while primer S8 generated the lowest number of products. The band sizes ranged from 319 to 3733 base pairs. The largest number of loci (29) was generated by primer S25. The polymorphism percentage ranged from 53.33% for primer S13 to 100% for primer S8. Cluster analysis revealed two separate clusters for C. tenuissima (Figure 3). There were only four genotypes in one cluster, three of which formed a separate subcluster (brown seedlings from seeds exposed to 15 Gy radiation and genotypes 9 and 10 (brown and green seedlings, respectively, from seeds exposed to 25 Gy dose)). The second subcluster was independently formed of 13 genotypes (brown seedling from seeds treated with 50 Gy dose). In the second cluster, however, all other genotypes were clustered. The smallest genetic distance was found between genotypes 11 and 12 exposed to the 25 Gy irradiation dose and between genotypes 6 and 7 exposed to the 20 Gy irradiation dose. The PCoA categorized the studied Copiapoa genotypes (1–16) into four groups: the first group included genotypes 2, 5, 6, 7, and 8; the second group included genotypes 11, 12, 14, 15, and 16; the third group included genotypes 1, 3, and 4; and the fourth group included genotypes 9, 10, and 13 (Figure 4). AMOVA confirmed the occurrence of interspecific genetic variation. The molecular variance was 100% among the tested Copiapoa genotypes according to the SCoT marker analysis (Table 6).

4. Discussion

The latest breeding methods are currently based on gene editing technology (CRISPR/Cas9) by Saini et al. [35]. However, inducing mutations through mutagenic factors remains an important tool for genetic changes in plants [27,36,37,38,39]. X-rays have been successfully used to induce phenotypic changes in many species of ornamental plants [12,39,40,41,42,43,44,45,46]. However, thus far, very few studies have reported on the effect of ionizing radiation on cacti. The first extensive study on the influence of X-rays on Astrophytum spp. ‘Purple’ was conducted by Licznerski et al. [27]. The authors stated that X-rays induced the formation of non-chlorophyll seedlings in Astrophytum spp. ‘Purple’ with new colors (orange and red), which were not previously detected in this species. Moreover, the authors found that most of the non-chlorophyll seedlings were formed at the 50 Gy dose of X-ray radiation.
Studies on the effects of radiation on the seeds of cacti C. tenuissima have not been conducted thus far. Our present study showed a significant effect of X-ray radiation on the increase in the number of colorful seedlings containing chlorophyll in C. tenuissima. In C. tenuissima, an increased dose of X-ray radiation increased the percentage of colorful seedlings with the orange-brown and yellow-cream colors (from 2.11% for 15 Gy dose to 4.90% for 50 Gy dose). No colorful seedlings were found for control seeds that were not subjected to radiation. X-ray radiation also influenced other parameters such as fresh weight, epicotyl and hypocotyl length, epicotyl width, and the longest root length. The seedlings obtained from seeds irradiated with the 25 Gy dose exhibited the highest average values of the following parameters: fresh weight (41.7 mg), epicotyl and hypocotyl length (4.42 mm), and epicotyl width (3.04 mm). The longest root (10.16 mm) was observed for a seedling obtained from seeds treated with the 50 Gy dose. Seeds irradiated with the 20 Gy dose showed the lowest average values of fresh weight (16.0 mg), hypocotyl and epicotyl length (3.52 mm), epicotyl width (2.20 mm), and the longest root length (6.14 mm). Thus, the results clearly indicate that X-ray radiation may lead to higher values of morphological parameters in irradiated seeds than in non-irradiated control seeds. Reznik et al. [45] obtained mutants from Ethiopian calla (Zantedeschia aethiopica) seeds previously treated with X-rays at different doses. Based on several studies, the authors found that the 50 Gy dose was best suited for the plant survival rate. However, the 100 Gy dose showed the greatest phenotypic variability. The experiments yielded plants with a different shape, colors of leaves, and number of stems. During the flowering period, further differences were found, including flowering time; the number of flowers obtained; and the size, shape, color, and scent of flowers. In C. tenuissima, the percentage of colorful seedlings increased with the increase in the radiation dose used and was the highest for the 50 Gy dose. Irradiation with the 25 and 50 Gy doses led to the development of a new color of seedlings; this phenotypic change was previously undetected in this cactus species. Thus, the present study confirmed that an increase in phenotypic changes depends on an increase in the dose of X-rays.
Miler et al. [38] recommended using high-energy photons at a dose of 10 Gy per ovary in chrysanthemum (Chrysanthemum × grandiflorum Ramat./Kitam.) to induce mutations. This dose was the most effective one in inducing stable mutations in the color and shape of the inflorescence without undesirable side effects such as a delay or extension of cultivation time. Increasing the radiation dose led to a decrease in the ability to regenerate explants in chrysanthemums. In C. tenuissima, the percentage of colorful seedlings was the highest for the 50 Gy dose. The use of the 25 and 50 Gy doses produced a new color of seedlings, which is unusual for this cactus species. Thus, the present study confirmed the relationship between an increase in the applied radiation dose and an increase in phenotypic changes.
In the date palm (Phoenix dactylifera), at 7 days after the application of X-ray radiation, an increase was observed in the morphological parameters, i.e., root length ranged from 3.7% (0.05 Gy dose) to 8.05% (15 Gy dose) as compared to the non-irradiated control. Within 2 weeks, the highest root growth was achieved for the 15 Gy dose (10.94%), which increased to 15.19% after 4 weeks. Moreover, the length of date palm leaves significantly increased at doses ranging from 0.05 Gy to 0.25 Gy [47,48]. In the present study, we observed a significant effect of X-ray radiation on the morphological parameters of C. tenuissima, such as fresh weight, epicotyl and hypocotyl length, epicotyl width, and the longest root length. The highest values for these parameters were obtained for seeds treated with the 25 Gy dose, whereas the lowest values were obtained for seeds treated with the 20 and 50 Gy doses. The color of plant flowers depends on the presence and amount of plant pigments, including anthocyanins, carotenoids, flavones, and flavonols [49]. Chlorophyll plays an effective role in photosynthesis, and its content provides information regarding the efficiency of plant metabolism and health. Carotenoids play a supporting role in photosynthesis and protect cells against oxidative stress [50,51,52,53]. New colors can be created because of the modification of the biosynthetic pathways of plant pigments [54]. The flavonoid biosynthesis pathway is particularly important because a mutation in just one gene is enough to produce an inactive protein (enzyme), the lack of which will cause the accumulation of intermediate metabolites and, consequently, a change in the plant’s color [16,55]
Al-Enezi and Al-Khayri [47,48] reported that X-ray radiation significantly influenced the concentration of plant pigments (such as chlorophyll a, chlorophyll b, and carotenoids) in date palms. Compared to the non-irradiated control seedlings (0 Gy), seedlings exposed to X-ray radiation at doses of 5–1500 rad (0.05–15 Gy) showed a reduced concentration of the tested plant pigments. An inverse relationship was observed between the applied radiation dose and the content of photosynthetic pigments. With the increase in the radiation dose, the content of chlorophyll a, chlorophyll b, and carotenoids decreased. The chlorophyll a content decreased at the 5 rad (0.05 Gy) dose (11.43 µg/g) and peaked at the 1500 rad (15 Gy) dose (7.49 µg/g). The chlorophyll b content showed increasingly lower concentrations (4.31 µg/g) after the dose of 10 rad. The lowest concentration of chlorophyll b (2.55 µg/g) was recorded for the dose of 1500 rad (15 Gy). The decrease in carotenoid concentration was similar to that for the other plant pigments. The carotenoid concentration was significantly reduced at the dose of 5 rad (0.05 Gy) (2.66 µg/g) and reached the lowest level at the dose of 1500 rad (15 Gy) (2.94 µg/g). The content of plant pigments subsequently significantly influenced the color of the plants. In the present study, the relationship between the radiation dose used and the pigment content varied in C. tenuissima. The X-ray irradiation of the seeds showed a diverse effect on the obtained concentration of plant pigments, which eventually influenced the plant color. Following the increase in the X-ray radiation dose, the concentrations of carotenoids, chlorophyll a, and chlorophyll b were increased in brown seedlings as compared to those in non-irradiated control seedlings. However, in green seedlings, the concentrations of anthocyanins and chlorophyll b increased, while those of chlorophyll a and carotenoids decreased. Licznerski et al. [27] reported that, in Astrophytum spp. ‘Purple’, the concentration of plant pigments decreased with an increase in the applied dose of X-rays. An exception was the 15 Gy dose, which led to an increased concentration of carotenoids and chlorophyll in the brown seedlings of this species. The 50 Gy dose resulted in the lowest concentration of anthocyanins, chlorophyll b, and carotenoids in green seedlings and chlorophyll a in orange seedlings.
Dhawi et al. [56] showed that low radiation doses had a beneficial effect on the photosynthetic pigments of date palms, whereas high doses had a negative effect. The authors also observed that chlorophyll a and carotenoids are more sensitive to magnetic fields than chlorophyll b. Pick Kiong Ling et al. [57] found that sweet orange (Citrus sinensis) seedlings irradiated with γ radiation showed a lower chlorophyll content than non-irradiated seedlings. The chlorophyll content, however, was virtually insensitive to low doses of γ radiation. In contrast, Abu et al. [58] found an increase in the levels of chlorophyll a, chlorophyll b, and total chlorophyll in cowpea (Vigna unguiculata) seeds exposed to γ radiation [56,57,58]. In C. tenuissima, the pigment concentrations varied according to the radiation doses and seedling color.
The creation of a new cultivar (genotype) is confirmed based on morphological features, a biochemical analysis of the composition of plant pigments (qualitative and quantitative), and the use of various molecular markers. The present study used SCoT markers and confirmed very high genetic diversity in X-ray-irradiated C. tenuissima seedlings. In a previous study, X-rays were found to be a more effective mutagen in heartwort (Lamprocapnos spectabilis) as compared to gold nanoparticles and microwaves [40]. In particular, a radiation dose of 20 Gy was the most effective one in inducing mutations in heartwort; however, the number of plants with genetic changes was relatively small, as only 3.3% of the plants had changes in their DNA content after X-ray irradiation. The polymorphism percentage (depending on the SCoT primer) ranged from 0% to 6.25%, and the average number of polymorphic loci was 2.4. In the present study, a large genetic distance was observed in C. tenuissima seedlings obtained from seeds irradiated with the 50 Gy dose. Depending on the SCoT primer used, the seedlings exhibited a considerably higher level of polymorphism, which ranged from 53.33% to 100%. Licznerski et al. [27] also confirmed their very high genetic diversity using SCoT markers in seedlings obtained from X-ray-irradiated seeds of Astrophytum spp. ‘Purple’. Similar to the polymorphism level in irradiated C. tenuissima seedlings, a high level of polymorphism was detected in the irradiated seedlings of Astrophytum spp. ‘Purple’ depending on the SCoT primer used (59.09% to 100%). Lema-Rumińska et al. [59] also found a similarly high genetic distance (>78%) for separate species belonging to the genera Astrophytum and Frailea based on the SCoT marker. This finding confirmed that X-rays induce changes in cacti at the genetic level that are comparable in both new cultivars and separate species. The next very interesting step will be to check how the acquired features are passed on to subsequent generations. However, further long-term studies are necessary to understand the mechanism of passing on features. In addition, non-chlorophyll forms require grafting onto rootstocks or in vitro cloning, because they are unable to survive on their own. However, from the point of view of cactus breeding, these forms are particularly valuable for cactus growers and collectors. Currently, however, micropropagation methods are already available that allow for in vitro cacti cloning in laboratory conditions, which can significantly accelerate the vegetative reproduction of such valuable colorful genotypes.

5. Conclusions

The irradiation of C. tenuissima seeds with X-rays influenced the percentage of colorful seedlings. Moreover, X-ray irradiation significantly affected the morphological parameters of C. tenuissima seedlings. The percentage of colorful seedlings increased with the radiation dose and was the highest for the 50 Gy dose. The application of radiation doses of 25 and 50 Gy generated seedlings with a new color (orange-brown), which had not yet been observed in C. tenuissima.
The morphological parameters of the obtained seedlings were the highest at the 25 Gy dose and the lowest at the 20 and 50 Gy doses. X-ray irradiation significantly influenced the seedling color and concentrations of different plant pigments in seedlings. With an increase in the radiation dose, the concentration of carotenoids, chlorophyll a, and chlorophyll b increased in brown seedlings as compared to those in control seedlings; however, in green seedlings, the concentrations of anthocyanins and chlorophyll b increased, and those of carotenoids and chlorophyll a decreased.
Based on the SCoT marker, X-rays led to a significantly high level of polymorphisms in C. tenuissima seedlings. Both UPGMA analysis and PCoA confirmed a very large genetic distance in the tested genotypes exposed to X-rays. Based on the study findings, X-rays can be used in radiomutation breeding to obtain new cultivars of cacti in C. tenuissima.

Author Contributions

Conceptualization, J.L.-R. and P.L.; methodology, J.L.-R., P.L. and J.W.; validation, J.L.-R., P.L. and J.W.; formal analysis, J.L.-R., P.L., E.M. and A.T.; investigation, P.L. and J.L.-R.; resources, P.L.; data curation, P.L. and J.L.-R.; writing—original draft preparation, P.L. and J.L.-R.; writing—review and editing, P.L. and J.L.-R.; visualization, J.L.-R. and P.L.; supervision, J.L.-R. All authors have read and agreed to the published version of the manuscript.

Funding

The linguistic proofreading of this work was supported by the Polish Minister of Science and Higher Education under the “Regional Initiative of Excellence” (Grant No. 008/RID/2018/19).

Data Availability Statement

Data will be made available on reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agronomy 14 02155 g001
Figure 1. Colors of seedlings obtained following X-ray irradiation of C. tenuissima seeds: (a) brown seedling 0 Gy (176 C); (b) green seedling 0 Gy (144 B); (c) brown seedling 15 Gy (176 B); (d) yellow-cream seedling 15 Gy (158 C); (e) green seedling 20 Gy (144 C); (f) orange-brown 50 Gy (163 A) (bar = 1 cm; color code in brackets according to RHSCC catalog).
Figure 1. Colors of seedlings obtained following X-ray irradiation of C. tenuissima seeds: (a) brown seedling 0 Gy (176 C); (b) green seedling 0 Gy (144 B); (c) brown seedling 15 Gy (176 B); (d) yellow-cream seedling 15 Gy (158 C); (e) green seedling 20 Gy (144 C); (f) orange-brown 50 Gy (163 A) (bar = 1 cm; color code in brackets according to RHSCC catalog).
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Figure 2. The number and percentage of colorful seedlings (yellow-cream 158 B, C, and orange-brown 163 A, according to the RHSCC catalog; mean ± SD) and the number of total germinating seeds in Copiapoa tenuissima after X-ray irradiation.
Figure 2. The number and percentage of colorful seedlings (yellow-cream 158 B, C, and orange-brown 163 A, according to the RHSCC catalog; mean ± SD) and the number of total germinating seeds in Copiapoa tenuissima after X-ray irradiation.
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Figure 3. A dendrogram based on the estimation of the genetic distance coefficient and UPGMA clustering for Copiapoa tenuissima genotypes after X-ray irradiation revealed by the start codon targeted (SCoT) polymorphism marker (for genotype destination, see Table 2).
Figure 3. A dendrogram based on the estimation of the genetic distance coefficient and UPGMA clustering for Copiapoa tenuissima genotypes after X-ray irradiation revealed by the start codon targeted (SCoT) polymorphism marker (for genotype destination, see Table 2).
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Figure 4. Graph of principal coordinate analysis (PCoA) of Copiapoa genotypes (1–16) based on start codon targeted polymorphism analysis (for genotype destination, see Table 2).
Figure 4. Graph of principal coordinate analysis (PCoA) of Copiapoa genotypes (1–16) based on start codon targeted polymorphism analysis (for genotype destination, see Table 2).
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Table 1. Characteristics of primers.
Table 1. Characteristics of primers.
PrimerSequence 5′-3′
S3CAACAATGGCTACCACCG
S4CAACAATGGCTACCACCT
S8CAACAATGGCTACCACGT
S12ACGACATGGCGACCAACG
S13ACGACATGGCGACCATCG
S25ACCATGGCTACCACCGGG
S33CCATGGCTACCACCGCAG
Table 2. The labeling of the samples in the molecular analyses.
Table 2. The labeling of the samples in the molecular analyses.
No. of SamplesX-ray Dose
[Gy]		Seedling’s Color/RHSCC Code
10brown/176 B
20green/144 A
315brown/176 B
415green/144 A
515yellow-cream/158 C
620brown/176 B
720green/144 C
820yellow-cream/158 B
925brown/176 B
1025green/144 A
1125orange-brown/163 A
1225yellow-cream/158 B
1350brown/176 B
1450green/144 A
1550orange-brown/163 A
1650yellow-cream/158 B
Table 3. Morphological characteristics of Copiapoa tenuissima seedlings obtained from seeds exposed to X-rays and total germination rate (GR) based on statistical data.
Table 3. Morphological characteristics of Copiapoa tenuissima seedlings obtained from seeds exposed to X-rays and total germination rate (GR) based on statistical data.
X-ray Dose
[Gy]		Seedlings
Fresh Weight
[mg]		Epicotyl and Hypocotyl Length [mm]Epicotyl Width [mm]Longest Root Length
[mm]		GR
[%]
0 (control)33.4 ± 2.99 b4.34 ± 1.08 a3.04 ± 1.47 a8.90 ± 3.86 a28.2 ± 1.28 b
1532.5 ± 4.73 b4.00 ± 1.80 b2.84 ± 1.39 ab7.38 ± 4.57 b28.4 ± 1.46 b
2016.0 ± 1.64 d3.52 ± 0.74 c2.20 ± 0.53 c6.14 ± 3.94 c27.0 ± 1.60 b
2541.7 ± 4.45 a4.42 ± 1.60 a3.04 ± 1.03 a9.92 ± 4.53 a43.2 ± 4.26 a
5020.0 ± 2.65 c3.44 ± 1.23 c2.46 ± 0.86 c10.46 ± 4.91 a *28.6 ± 2.33 b
* The mean ± SD within a column marked with the same letter does not differ significantly, with p ≤ 0.05.
Table 4. The seedling color (according to the RHSCC catalog) and concentration of anthocyanins, carotenoids, and chlorophyll a and chlorophyll b per gram of fresh seedling weight of Copiapoa tenuissima seedlings depending on the X-ray dose.
Table 4. The seedling color (according to the RHSCC catalog) and concentration of anthocyanins, carotenoids, and chlorophyll a and chlorophyll b per gram of fresh seedling weight of Copiapoa tenuissima seedlings depending on the X-ray dose.
X-ray Dose (Gy)Color of Seedling
(RHSCC Code)		Concentration of Pigments [mg dm−3]
AnthocyaninsCarotenoidsChlorophyll aChlorophyll b
0176 B, C26.22 ± 0.02 c *5.28 ± 0.00 k7.32 ± 0.01 m3.30 ± 0.03 k
144 A, C2.83 ± 0.01 l52.40 ± 0.08 a87.32 ± 0.25 a29.73 ± 0.16 d
15176 B, C17.75 ± 0.08 h23.10 ± 0.04 e31.23 ± 0.02 f16.32 ± 0.02 g
144 A, C46.76 ± 0.23 a39.38 ± 0.07 b36.74 ± 0.00 d36.97 ± 0.00 a
158 B, C24.18 ± 0.92 d21.20 ± 0.00 f17.47 ± 0.11 j24.00 ± 0.69 e
20176 B, C14.36 ± 0.07 j13.22 ± 0.02 i20.38 ± 0.03 h26.84 ± 0.16 a
144 A, C6.06 ± 0.02 k37.87 ± 0.05 c43.81 ± 0.05 b35.04 ± 0.04 b
158 B, C11.29 ± 0.00 i22.78 ± 0.48 e19.85 ± 0.17 i23.84 ± 0.15 e
25176 B, C21.72 ± 0.04 f19.06 ± 0.04 g29.95 ± 0.03 g13.23 ± 0.05 h
144 A, C22.46 ± 0.10 e32.84 ± 0.08 d36.36 ± 0.17 e30.09 ± 0.28 d
158 B, C7.61 ± 0.00 i18.00 ± 0.00 f16.60 ± 0.12 k20.83 ± 0.00 f
50176 B,C29.20 ± 0.10 b13.64 ± 0.06 h15.99 ± 0.11 l7.61 ± 0.15 j
144 A, C19.27 ± 0.07 g33.21 ± 0.27 d40.03 ± 0.42 c33.63 ± 0.82 c
163 A0.44 ± 0.00 ł8.45 ± 0.07 j8.37 ± 0.19 ł10.53 ± 0.43 i
* The mean ± SD within a column marked with the same letter does not differ significantly, with p ≤ 0.05.
Table 5. Number of products, range of band sizes, and number of loci and polymorphisms obtained in Copiapoa tenuissima seedlings following molecular analysis using SCoT marker.
Table 5. Number of products, range of band sizes, and number of loci and polymorphisms obtained in Copiapoa tenuissima seedlings following molecular analysis using SCoT marker.
PrimerNo. of ProductsBand Size Range (bp)No. of lociTotal lociPolymorphism
(%)
MonomorphicPolymorphicSpecific
S3143366–280611441994.74
S478455–278711101291.67
S849956–21230729100.0
S12158319–300911531994.74
S13177365–19497801553.33
S25175362–373322162993.10
S33146873–254621431989.47
Total926-149018122-
Table 6. Analysis of molecular variance in studied Copiapoa genotypes (1–16) based on start codon targeted polymorphism analysis.
Table 6. Analysis of molecular variance in studied Copiapoa genotypes (1–16) based on start codon targeted polymorphism analysis.
Summary AMOVA
Source of VariationdfSSMSEst. Var.%
Among Populations15850.6956.7118.90100%
Within Populations320.000.000.000%
Total47850.69 18.90100%
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Licznerski, P.; Michałowska, E.; Tymoszuk, A.; Winiecki, J.; Lema-Rumińska, J. The Influence of X-ray Radiation on the Morphological, Biochemical, and Molecular Changes in Copiapoa tenuissima Seedlings. Agronomy 2024, 14, 2155. https://doi.org/10.3390/agronomy14092155

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Licznerski P, Michałowska E, Tymoszuk A, Winiecki J, Lema-Rumińska J. The Influence of X-ray Radiation on the Morphological, Biochemical, and Molecular Changes in Copiapoa tenuissima Seedlings. Agronomy. 2024; 14(9):2155. https://doi.org/10.3390/agronomy14092155

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Licznerski, Piotr, Emilia Michałowska, Alicja Tymoszuk, Janusz Winiecki, and Justyna Lema-Rumińska. 2024. "The Influence of X-ray Radiation on the Morphological, Biochemical, and Molecular Changes in Copiapoa tenuissima Seedlings" Agronomy 14, no. 9: 2155. https://doi.org/10.3390/agronomy14092155

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