Somaclonal Variation in Callus Cultures of Rose Periwinkle, Catharanthus Roseus L. Under Induced Salt and Osmotic Stresses
The Second Faculty of Agriculture, Damascus University, Damascus, Syrian Arab Republic
Researcher in The National Commission for Biotechnology, Damascus, Syrian Arab Republic
Academic Editor: Mohamed Farag Mohamed Ibrahim
Special Issue: Molecular Plant Physiology under Abiotic Stress Conditions
Received: July 29, 2023 | Accepted: October 20, 2023 | Published: October 26, 2023
OBM Genetics 2023, Volume 7, Issue 4, doi:10.21926/obm.genet.2304200
Recommended citation: Alaakel S, AL-Ammouri Y. Somaclonal Variation in Callus Cultures of Rose Periwinkle, Catharanthus Roseus L. Under Induced Salt and Osmotic Stresses. OBM Genetics 2023; 7(4): 200; doi:10.21926/obm.genet.2304200.
© 2023 by the authors. This is an open access article distributed under the conditions of the Creative Commons by Attribution License, which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is correctly cited.
Rose periwinkle, Catharanthus roseus L. It is one of the most important medicinal plants recognized in the international pharmacopoeia. This plant belongs to the Apocynaceae family , which includes a large number of genera, and the genus Catharanthus is the most important, as this genus consists of eight species C. coriaceus, C. longifolius, C. lanceus, C. ovalis, C. pusillus, C. trichophyllus, C. scitulus, in addition to C. roseus. It is widespread in warm regions of the world, characterized by the ability to withstand high temperatures, and grown as an ornamental plant in gardens and parks due to the variety of flower colors (white, pink, and purple) [2,3].
Rose periwinkle, Catharanthus roseus L. plant contains more than 130 indole alkaloids [4,5]. Recent studies have been able to identify the active components of C. roseus, such as Vincristine (VCR) and Vinblastine (VLB), which have anti-cell division properties [6,7]. Due to the low concentration of alkaloids in cultivated plants, the extraction process of (VLB) and (VCR) is usually costly and exhausting . This prompted researchers to work on extracting these medically valuable compounds in larger quantities through modern biotechnology methods, such as tissue culture , which is based on the Totipotency theory, which is the ability of any living cell to form a complete plant, and the genetic information required for the manufacture of secondary metabolites is present in the callus cells. By activating these genes, the required secondary metabolites can be produced .
The application of biotechnology in the C. roseus plant, especially callus cultures, will lead to new genotypes with good characteristics based on the somaclonal genetic variation in In vitro cell populations . Moreover, applying these technologies in the presence of the stress factor in the nutrient medium will improve tolerance to abiotic stresses, with an increase in the production efficiency of secondary metabolites. Callus culture technology is considered one of the important technologies as a new source of secondary metabolites , and to obtain the necessary somaclonal variation (genetic variation) in breeding programs . The findings of  showed that somaclonal variation through In vitro regeneration might be used to create new phenotypes from Rose Periwinkle. Many studies, such as  showed that somaclonal variation may be exploited for C. roseus callus improvement, which is used as an alternative source of bioactive metabolites.
The term somaclonal variation was used as an indicator to denote the changes that occur in regenerative plants from tissue culture and plant cells, and it is known that such changes occur frequently when protoplasts are isolated also in callus culture . Somaclonal variation describes the genetic and non-genetic changes in plants (the variation in physiological, biochemical, and genetic characteristics among In vitro cells). It may appear during or after the In vitro culture of plant cells, organs, or calluses . These variations can be detected using one of the molecular markers methods. The Inter Simple Sequence Repeats technique (ISSR) is an effective method for detecting the somaclonal variation expected to occur in calluses in the presence or absence of the stress factor, which is a helpful tool in detecting such variations . It has been used to detect genetic variation in many crops, such as potato, Solanum tuberosum , and barley, Hordeum vulgare , as well as to reveal the somaclonal variation among In vitro callus cultures in comparison with the mother plant (In vitro) of many medicinal plants, such as Golden Henbane, Hyoscyamus aureus .
This research aimed to study the somaclonal variations in callus cultures of Rose periwinkle, C. roseus plant at different ages (14, 21, and 30 days) and compare them with the introduced species and In vitro plants. In addition, determine the effect of induced osmotic stress and salt stress on causing genetic changes by using the ISSR technique.
2. Materials and Methods
2.1 Location of Research
This research was conducted at the Biotechnology of Medicinal Plants Department laboratories, the National Commission for Biotechnology, Damascus, Syria, from 2020-2022.
2.2 Plant Material
C. roseus seeds were obtained from the Dutch company Syngenta Flowers (goldsmith seeds) and germinated In vitro. As for the introduced species, the plant was presented to Syria and classified by Damascus University. It is adapted to the Syrian environment and is widespread as an ornamental plant.
2.3 Seeds Germination
The seeds were sterilized with ethanol (from SIGMA) and then with 0.5% sodium hypochlorite NaOCl solution (from DTEC) for 5 minutes, then washed with sterile distilled water three times at a rate of 5 minutes each time. Seeds germinated using pyrex® tubes (25 × 150 mm) in the MS primary culture medium  without growth regulators. The boxes were incubated at 24 ± 2°C, 16 hours of light, and 8 hours of darkness alternately until the seed germinated. After 45 days, they were re-planted on MS medium supplemented with two hormones, 6-benzyl adenine (BA) (2 mg L-1) and Naphthalene Acetic Acid (NAA) (1 mg L-1) (from MERCK) , to obtain sufficient plant material needed to carry out the callus development experiments.
2.4 Callus Development
Callus was developed from the leaves of C. roseus after 90 days on 5C01 nutrient medium  by using pyrex® tubes (25 × 150 mm), and fortified with a group of vitamins (from MERCK), such as Myo-inositol (0.08 g), and casein (0.5 g), in addition to some plant growth regulators NAA (1 mg L-1) and Kinetin (Kin) (2 mg L-1). The samples were taken at 14, 21, and 30 days before applying stress factors.
2.5 Stress Applied to Callus Cultures
Calluses were cultured in tubes with the same 5C01 media and supplementary hormones, with the addition of progressively increasing levels of a stress factor (NaCl) (0, 30, 60, 90, and 120 mM) and polyethylene glycol PEG-6000-induced osmotic stress (0, -0.2, -0.4, and -0.6 Mpa). Calluses were transferred to a higher level every 30 days.
2.6 Study of Somaclonal Variations
Somaclonal variations were studied in all samples: the introduced species, the In vitro plant, the unstressed callus at ages (14 days, 21 days, and 30 days), the salt-stressed callus, and the osmotic stress callus, the ISSR technique was used. Studying these variations was carried out by the following steps:
2.6.1 DNA Isolation by Cetyltrimethylammonium Bromide (CTAB) Method 
Approximately 200 mg of the frozen and ground plant material, in liquid nitrogen, for all the studied samples was placed in a 2 ml Eppendorf tube, then 750 μl of preheated CTAB extraction solution at 65°C was added, and the mixture was stirred well. The extraction solution consisted of the following materials:
(2% w/v) CTAB (Vivantis) + 100 mM Tris-Hcl (pH = 8) (Vivantis) + 1.4 M NaCl (ScP) + 20 mM EDTA (pH = 8) (AVONCHEM) + 0.2% mercaptoethanol (v/v).
The tubes were placed in a water bath at 65°C for an hour. 750 µL of chloroform: isoamyl alcohol (24:1) (from Scharlau) was added to remove proteins and lipids. The tubes were centrifuged with a Heraeus centrifuge at a speed of (12000 rpm) for 10 minutes at a temperature of 4°C. At this stage, the mixture was separated into three phases. Isopropanol from (Merck) was added to the resulting aqueous phase by stirring gently until DNA precipitated. The next day, the tubes were centrifuged with cold washing buffer (70% ethanol stored at -20°C), then the DNA samples were dissolved in 100 µl of TE solution.
2.6.2 Quantitative and Qualitative Determination of DNA
A Hitachi U2900 UV Spectrophotometer was used to quantify DNA and determine its purity at wavelengths of 260 and 280 nm , and the DNA concentration was calculated from the following mathematical equation :
DNA concentration = 50 μg/mL × OD260 × dilution factor
Then, the DNA samples were diluted to obtain a concentration of 50 ng µl-1, and the qualitative assessment was done on an agarose gel (0.8%) (From SIGMA), as the excellent quality DNA appeared in the form of a band. In contrast, the lousy-quality DNA was smeared.
2.6.3 DNA Amplification
The DNA was amplified by polymerase chain reaction (PCR) according to the protocol of Williams et al , with some modifications so that the final reaction volume was (25 µl), consisting of 2 µl of DNA at a concentration of (50 ng µl-1) per sample, and 12.5 µl Master Mix (Kapa) and 1.25 µl of the primer at a concentration of (10 pmol µl-1), then complete the volume to 25 µl with sterile distilled water. The PCR reaction was performed using the PCR kit (Kapa 2G fast-ready Mix) according to the manufacturer's instructions. The DNA bands were separated by electrophoresis on an agarose gel (2%) in a 1X TBE solution.
2.6.4 ISSR Technique Application
A group of primers (Table 1) from BioNeer Company was used to study the genetic and somaclonal variations occurring in callus cultures. After that the polymorphic ratio of the used primers was calculated, and a dendrogram of the genetic relationship was drawn.
2.6.5 Electrophoresis, Staining and Imaging
The migration was carried out on a 2% agarose gel (Agarose Ultrapure Fingerprinting USA) in 1X TBE solution and with red-safe dye (from intron). An electric field of 90 volts was used for two hours to separate the DNA bands resulting from the amplification, and a (DNA marker -100 bp from JenaBioscience) to determine the molecular size of the resulting bands. After that, the gel was imaged with the Imag Analyzer (Agle Eye II teratogen).
2.7 Statistical Analysis
The Total Lab Program was used to analyze the results of the molecular study. The results of the amplification process were collected in tables based on comparing the presence or absence of DNA bands between samples. The somaclonal variations between the samples were studied using the Jaccard coefficient, and then the Dendogram of the genetic relationship was drawn by applying the UPGMA (Unweighted Pair Group Method with Arithmetic Averaging) using the Power Marker Program.
3.1 Polymorphism Resulting from the Application of the ISSR Technique
The molecular study included exploiting 21 primers, all of which proved effective in exhibiting polymorphism among the studied samples (Table 2) (Figure 1), and resulted in 141 bands. The number of bands ranged between a minimum 3 bands for primers (I9 and Q16), and 11 bands as a maximum in the primers (T19), with an average of 6.71 bands per primer. The number of polymorphic bands was 128, and ranged between 3 bands for the primers (I9 and Q16) as a minimum and 10 bands for the primers (T19 and A1), with an average of 6.10 for each primer. The standard of polymorphic ratio was 92.16%, the lowest in the primer H8 (57.14%), and the highest in the primers A1, B2, D4, I9, J10, L12, M13, N14, P15, Q16, S18, and U20, reaching 100%.
Figure 1 Migration images of ISSR-PCR for studied samples (Primers: V21, t19, R17, Q16, S18, A1, J10, P15). A: introduced species, B: in vitro plants, C: 14-day-old callus, D: 21-day-old callus, E: 30-day-old callus, F: salt stressed callus, G: induced osmotic stressed callus.
The total number of bands was (93, 99, 102, 87, 100, 90, and 29 bands). The number of polymorphic bands was (91, 95, 100, 85, 97, 87, and 22 bands) for introduced species, In vitro plant, 14-day-old callus, 21-day-old callus, 30-day-old callus, salt-stressed calluses, and osmotically stressed calluses, respectively. Moreover, it was noted that the highest polymorphism percentage was in the 14-day-old callus (98.04), followed directly by the introduced species (97.85%). In comparison, the polymorphism percentage in salt-stressed calluses was approximately 96.67%, and in osmotic-stressed calluses, 75.86%, it was about 95.96% for In vitro plants.
When applying the Percent Disagreement Values (PDV) (Table 3), where the high values indicated the presence of genetic variation and this genetic variation increased with the increase of these values, the lowest value of Jaccard coefficient was 0.1121 for 14-day-old calluses and 21-day-old calluses, and this indicated that they were the most similar genetically, while the highest value was approximately 0.8364 for the In vitro plant and the osmotic stressed callus, which stated that they were the farthest genetically.
3.2 Cluster Analysis of Cellular Lines of C. Roseus Callus, the Introduced Species Plant and the In Vitro Plant:
Cluster analysis (Figure 2) showed the separation of the studied samples according to the degree of genetic similarity, as the models were separated into two main clusters. The first cluster contained the osmotic-stressed callus in a separate collection and was genetically the farthest from the other samples. In contrast, the second cluster was separated into two sub-clusters. Under the first sub-cluster, the salt-stressed callus and the 30-day-old callus were included, while the second sub-cluster was divided into two parts. The first part contained the introduced species, while the second part contained the In vitro mother plant in the glass, the 14-day-old callus, and the 21-day-old callus, as these treatments were genetically closest.
Figure 2 The cluster analysis of the studied samples.
The somaclonal variation primarily results from mutations that take place during tissue culture. Variable stress conditions such as culture media (hormonal imbalance), tissue exposure to chemicals during surface sterilization, and wounding can lead to mutations. Oxidative stress was discovered to be the primary source of somaclonal variation in tissue culture plants in many instances .
Plant growth regulators are considered one of the main contributors to somaclonal variations. According to studies, tissue culture conditions with an imbalanced hormone ratio can increase polyploid plants. In contrast, their absence or insufficient use might lead to the production of typical plants [29,30]. In addition to the hormone concentration, the type of hormone utilized in culture media can also result in somaclonal variation . For instance, increasing the rate of DNA methylation by adding auxins such as 2,4-Dichlorophenoxyacetic acid (2, 4D) and NAA to the growth conditions of callus or cell suspension .
Depending on the source, explants have different levels of genetic stability . Meristematic or undifferentiated tissues like cambium and procambium explants minimize variations. However, highly differentiated tissues, such as stems, roots, and leaves, result in more variations .
It was noted that there were genetic variations between the introduced species, In vitro growing plants, and the resulting callus. The callus cells lost the differentiation and returned to the meristematic division state, thus becoming morphologically, physiologically, and genetically different from the In vitro growing plants . These results were consistent with the results of Ikeuchi et al. , which confirmed that the types of callus cultures that belong to the same plant were genetically different. These differences are because the process of callus development includes massive changes in gene expression due to changes in the level of cell differentiation . This confirms that the interaction between environment and genotype limits the characteristic of genetic variations in callus cultures. These differences are attributed to a group of mechanisms, such as changes in the methylation of DNA, the release of the genetic material outside the cell membrane or what is known as Exocytosis, the exchange of the genetic material between cells, and the loss of a large amount of DNA especially in polyploid cells .
Through cluster analysis, it was noted that there were genetic variations between the Rose Periwinkle C. roseus introduced species, which is widely cultivated and adapted to the Syrian environment, and the In vitro plant, whose source was the seeds imported from Syngenta Flowers Company, and this confirmed the need to use seeds from a reliable source to ensure the genetic purity of the variety on which studies were required. Moreover, it was noted that there were genetic variations between the In vitro and the resulting callus, in addition to the occurrence of genetic variations during the growth stages of the callus. This confirmed the importance of callus cultures as an essential source of genetic variations and the effectiveness of the ISSR technique in detecting these variations. Furthermore, callus culture technology is helpful in cellular selection processes to reach cell strains that can be highly productive of biologically active substances. These results were consistent with the results of  in their study on genetic variations induced by tissue culture of Gentiana spp., and the effects of  in their reflections on the Golden Henbane, Hyoscyamus aureus who, showed that genetic variations occurred between the In vitro plant and the resulting callus, where the callus separated by a distance of 0.33 from the In vitro plant.  Stated that abiotic stresses affected morphological, physiological, chemical, and molecular traits and negatively affected growth and productivity. In addition to many studies that proved the damage caused by free radicals resulting from abiotic stresses at the cellular level [40,41,42], free radicals attacked all cell components, such as proteins, enzymes, and nucleic acids, especially when the enzymatic defense systems were ineffective, and caused mutations at the DNA level, represented by a change in the chromosome number, aneuploidy as a result of a break in the DNA chain, in addition to deletions and replacements in the nitrogenous bases involved in the structure of DNA . This study also agreed with the results of , who showed the effect of the salt medium in causing genetic variations in callus developed from the leaves of corn, Zea mays L.
Callus culture technology was an essential source of genetic variations that can be exploited in selecting cell lines with specific and targeted traits, and the ISSR technique was effective in detecting these genetic variations that appeared between the callus and the In vitro plant. Moreover, instability was observed in the genetic structure of the callus during its growth period, as the genetic structure of the callus changed between the ages of 14 days and 30 days, and the addition of the stress factor to the culture medium resulted in genetic variations between the unstressed callus and the salt and osmotic stressed callus. On the other hand, cluster analysis and the dendrogram of genetic relationship showed the separation of the studied samples into two main clusters, and the PEG-induced osmotic stress was the most isolated from other calluses, as it lacked most of the DNA bands that appeared in the different samples.
All thanks and appreciation to Dr. Shaza Besher, Prof. Fahed Albiski, and Eng. Buthina Al-Salama from the National Commission for Biotechnology, Department of Molecular Biology, Damascus, Syria, for their support in implementing this research.
All authors participate in the research concept and design. The first draft of this manuscript, material preparation, and data collection were performed by the first author; data analysis and manuscript proofreading were performed by the second author.
The study has been supported by The National Commission for Biotechnology, Ministry of Higher Education and Scientific Research, Damascus, Syria.
The authors have declared that no competing interests exist.
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