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Review

Somaclonal Variation and Clonal Fidelity in Commercial Micropropagation: Challenges and Perspectives

by
Sweety Majumder
1,2,
Abir U. Igamberdiev
2 and
Samir C. Debnath
1,*
1
St. John’s Research and Development Centre, Agriculture and Agri-Food Canada, St. John’s, NL A1E 0B2, Canada
2
Department of Biology, Memorial University of Newfoundland, St. John’s, NL A1C 5S7, Canada
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(6), 1489; https://doi.org/10.3390/agronomy15061489
Submission received: 30 April 2025 / Revised: 10 June 2025 / Accepted: 13 June 2025 / Published: 19 June 2025
(This article belongs to the Section Plant-Crop Biology and Biochemistry)
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Abstract

:
Plant tissue culture has been recognized as an essential technology in plant science research. This process is widely used to regenerate and conserve phenotypically and genetically identical plant resources. The advancements in tissue culture methods have become a feasible option for the micropropagation of plants at the commercial level. The success of commercial micropropagation necessitates genetic stability among regenerated plants. Sometimes, in vitro-grown plants show genetic and epigenetic alterations due to stressful artificial culture conditions, media compositions, and explant types. As a result, it is essential to ensure genetic stability among tissue culture-derived plantlets at a very early stage. Somaclonal variations can be detected by phenotypic assessment, cytogenetic, DNA-based molecular markers, bisulfite sequencing, and RNA sequencing. This review aims to describe the causes behind somaclonal variation, the selection of somaclonal variants, and their uses in crop and plant improvement at the commercial level. This study discusses the optimization processes of undesirable genetic and epigenetic variation among micropropagated plants and their application in global horticulture, agriculture, and forestry.

1. Introduction

The global demand for high-quality plant material for food and medicine is constantly rising owing to the growing global population [1]. Although conventional plant propagation methods, such as budding, grafting, cuttings, and seed propagation, act as the fundamental of the plant production system [1,2]. These methods often fail to fulfill the current and expected demands of efficient and cost-effective plant propagation systems due to insufficient agricultural land, requiring a long time to obtain progenies, dependence on seasons, and limitations in the consistency of the resulting plant material [3]. Nowadays, plant tissue culture is a powerful tool for reproducing identical plants of a genotype from a single plant part without dependence on geographical and climatic factors [4]. Thus, plant tissue culture is an alternative method of conventional vegetative propagation [4,5]. Micropropagation, initially applied to ornamental plants [5], has expanded to include various crops, fruit plants, vegetables, trees, and medicinal and aromatic plants, and is now a multibillion-dollar industry worldwide [6].
Tissue-based propagation, commonly known as micropropagation, is a rapid and efficient method that can be achieved in limited space [7]. In this process, a small segment of plant collected from selected plant species, known as explant, such as leaf sections, shoot tips, nodal segments, and root tips, are aseptically cultured on an artificial nutrient-rich medium. In the micropropagation process, plants can regenerate via organogenesis and somatic embryogenesis (SE) [8]. Organogenesis is generally classified into two distinct pathways: direct organogenesis, in which shoot buds are formed directly from the explant tissue, and indirect organogenesis, where shoot regeneration occurs through an intermediate callus formation phase [9]. Consequently, micropropagation has noticeably increased the mass production of genetically uniform and high-quality plant materials, contributing significantly to agriculture, horticulture, and forestry. However, despite these advancements, a critical drawback of in vitro propagation is the occurrence of somaclonal variation—heritable genetic and epigenetic modifications induced by the artificial conditions of tissue culture. Such variations can threaten clonal fidelity, deteriorate crop performance and quality, and complicate regulatory approval and market acceptance, especially in commercially sensitive crop systems [6,8].

2. Clonal Fidelity and Commercial Micropropagation

Micropropagation is a highly efficient method for producing genetically identical plant materials [9,10]. The genetically identical individuals ensure uniformity in yield, growth, and quality, which is essential for commercial consistency. This uniformity simplifies cultivation, reduces production costs, and enhances market appeal. Moreover, it allows rapid multiplication of elite genotypes while preserving desirable traits [10]. The first commercial micropropagation technology started with orchids in the 1970s [11]; it was later observed in numerous fruit plants, trees, spices, ornamentals, and crops [12]. Between the 1970s and 1980s, the commercial micropropagation process experienced significant growth due to the rise in commercial laboratories involved in these activities [12]. Micropropagation is an effective biotechnology technique used in the horticulture, forestry, and agriculture sectors that allows for the rapid and large-scale reproduction of traits that are true-to-type, disease-free, and genetically superior varieties [12,13]. Clonal fidelity—the capacity of a clone to maintain its genetic identity over time—is vital in in vitro propagation because the success of this process depends on producing large numbers of genetically identical, true-to-type plants from a small segment of a donor plant [14]. For example, in bananas, the clonal fidelity of micropropagated plants via axillary buds was observed when cultured under cytokinin-rich conditions [15]. Similarly, confirmed genetic uniformity in bioreactor-derived strawberry (Fragaria × ananassa) plants and correlated clonal fidelity with enhanced anthocyanin content and antioxidant activity compared to conventionally propagated plants [16]. Kundu et al. developed a high-fidelity somatic embryogenesis protocol in Vaccinium vitis-idaea (lingonberry). This study revealed that the regenerated plants retained genetic uniformity while also showing increased concentrations of flavonoids, anthocyanins, and antioxidant activity in comparison to the original donor plants [17]. In apple, 92–100% genetic similarity was observed in apple rootstock EMLA 111 [18]. Genetically unstable, off-type micropropagated plants are a primary cause of financial loss, especially in high-demand crops where uniformity and predictability are essential [19]. For example, variations in color, shape, and size can drastically reduce the market value of commercially important plant species. Growers face substantial losses when dealing with poor-quality, low-yield plants [19]. A further complication arises with perennial plants like woody ornamental plants, woody trees, medicinal plants, and fruit trees, where the effects of genetic instability may not become visible until several years after planting, leading to delayed losses after considerable resources have already been invested [19,20]. These regenerated plants may display a range of undesirable phenotypic variations, including sterility, precocious flowering, altered internodal length, leaf abnormalities such as albino or chlorotic leaves, increased salt resistance, and changes in fruit or flower color [21]. Additionally, somaclonal variation-induced genetic changes threaten plant health and the production of disease-resistant, uniform plant material [21]. This genetic instability can increase vulnerability to insect pests, pathogens, and environmental stresses, significantly reducing crop productivity and increasing disease outbreaks. To manage these risks, growers must invest heavily in advanced disease and pest management systems [22]. However, the costs associated with these disease control measures in affected micropropagated crops and plant species further diminish the economic advantages of micropropagation [23].

3. Somaclonal Variation in Micropropagated Plants

Although micropropagation offers numerous benefits, prolonged stress conditions during growth may sometimes lead to genetic instability in micropropagated plants [24]. Larkin and Scowcroft first introduced the term “Somaclonal variation” to describe the variation observed in plants regenerated through tissue or cell culture [25]. Somaclonal variation is unpredictable and uncontrollable, and most of the variation has no direct agronomic or commercial significance [26]. However, other names such as protoclonal, gametoclonal, and mericlonal variation are often used to describe variants from protoplast, anther, and meristem cultures, respectively [27]. Somaclonal variations result from the genetic and epigenetic changes that can occur in tissue culture-induced regenerated plants [28]. Genetic variation remains stable throughout the sexual cycle or repeated asexual propagation, whereas epigenetic variation may be unstable even during asexual propagation [29]. The presence of unexpected, uncontrolled, and spontaneous changes during the tissue culture is undesired in commercial micropropagation [30]. The types of explants, the number of subcultures, plant growth regulators, and the regeneration pathway are responsible for somaclonal variation [31,32,33,34]. However, somaclonal variation is considered as a limitation in micropropagation technique, it also offers valuable opportunities for producing novel disease-resistant and high-yielding variants, namely rice [35], tomatoes [36], eggplants [37], cucumber [38], carrot [39], bananas [40], and grape [41]. There are numerous techniques for determining and defining somaclonal variants, primarily based on various morphological features, numerical and structural variations in the chromosomes, and biochemical and molecular DNA markers. Understanding somaclonal variation is essential for optimizing micropropagation protocols and producing true-to-type plants. That can ensure the economic viability and quality of this valuable technology [42].

3.1. Mechanisms of Somaclonal Variation

Somaclonal variation is a complicated phenomenon, and it remains difficult to fully understand the ways in which genetic and epigenetic mechanisms interact within in vitro culture environments (Figure 1) [27,28]. A significant factor contributing to this variability is oxidative stress, which is frequently induced by different parameters such as the composition and pH of the medium, concentrations of plant growth regulators, hormonal imbalances, and fluctuations in temperature and humidity. These adverse conditions lead to the overproduction of reactive oxygen species (ROS), which are known to cause a variety of molecular disruptions, including alterations in DNA methylation patterns, chromosomal aberrations, strand breakages, and point mutations [43]. Consequently, the degree of somaclonal variation is closely associated with the specific conditions of the in vitro environment, emphasizing the necessity of developing and maintaining optimized, tightly controlled culture protocols to safeguard genetic integrity and clonal uniformity.

3.1.1. Genetic Mechanisms

One of the primary contributors to somaclonal variation involves genetic mechanisms, which include modifications at the DNA level that result in permanent genetic [43,44]. These genetic alterations can take several forms, including insertions, deletions, point mutations, chromosomal rearrangements, and ploidy level changes (Figure 2) [44]. Chromosomal rearrangements play a significant role in generating genetic variability. Point mutations alter individual nucleotides within gene coding sequences, which can subsequently change the structure and function of the proteins they encode [45]. Additionally, translocations, insertions, and deletions involving specific DNA regions can disrupt gene function and expression, further contributing to somaclonal variation [46]. Such chromosomal structural abnormalities have been widely reported in various plant species. In bread wheat, Karp et al. observed that its chromosome number increased when grown in high concentrations of 2,4-D (2,4-dichlorophenoxyacetic acid) [47]. In lily, chromosomal variation was induced during prolonged callus culture on MS medium with 1 mg/L picloram. The extended exposure to auxin led to polyploidization, resulting in tetraploid plantlets. Lily [48], demonstrating the prevalence and importance of these genetic mechanisms in plant tissue culture systems.

3.1.2. Epigenetic Mechanisms

Epigenetics involves studying heritable changes in gene activity that occur without alterations to the underlying DNA sequence [49]. In vitro conditions can regulate these epigenetic modifications in plant tissue culture systems, which profoundly influence plant phenotype and contribute to somaclonal variation [50]. Three major types of epigenetic changes have been identified: DNA methylation, histone modifications, and small RNA-mediated regulation. DNA methylation involves the covalent attachment of methyl groups to cytosine nucleotides, primarily resulting in three types of methylated bases: 5-methylcytosine (5 mC), N6-methyladenine (6 mA), and N4-methylcytosine (4 mC) [51]. The tissue culture environment influences methylation patterns, frequently causing hypermethylation or hypomethylation in specific genomic regions, which can result in somaclonal variation [52]. In addition to DNA methylation, histone modifications also play a significant role in regulating gene expression. Histone proteins, which help package and organize DNA within chromosomes, can undergo chemical modifications that alter chromatin structure and transcriptional activity, thereby contributing to phenotypic variability in micropropagated plants [52,53]. Histone modifications such as acetylation and methylation are dynamic and reversible processes, primarily controlled by distinct groups of enzymes, including families, namely histone acetyltransferases (HATs), histone deacetylases (HDACs), and histone lysine methyltransferases (HKMTs) [54]. In plants, HATs are organized into five structural subgroups, while HDACs are mainly grouped as HDA1 and HDA6—the latter being crucial for maintaining DNA methylation, suppressing transposable elements, and guiding development [55]. Two prominent histone H3 methylation marks, H3K4me3 and H3K27me3, are well-characterized for their opposing roles in gene regulation—H3K4me3 is linked to transcriptional activation, while H3K27me3 is associated with gene repression. The deposition of H3K4me3 at the 5′ regions of actively transcribed genes is facilitated by trithorax group (trxG) protein complexes in coordination with the initiating form of RNA polymerase [56,57]. Zhang et al. highlighted the widespread silencing role of H3K27me3, identifying its regulation of approximately 4400 genes in Arabidopsis, independent of small RNAs or DNA methylation pathways [58]. Another key epigenetic mechanism involves small RNA pathways, especially microRNAs (miRNAs) and small interfering RNAs (siRNAs), which play a vital role in regulating gene expression at both transcriptional and post-transcriptional levels, influencing essential processes such as cell fate in eukaryotes [59,60]. For example, in micropropagated strawberries, miR156 is notably upregulated, which facilitates juvenile leaf development and regulates developmental transitions [61]. Other studies have found that miR169a and miR169b are downregulated, whereas miR535 and miR390 are upregulated in micropropagated plants [62]. During micropropagation, these small RNA pathways can become disrupted, leading to the misregulation of critical genes and, consequently, the emergence of somaclonal [63] (Figure 3).

4. Factors Affecting Somaclonal Variation

Somaclonal variation in micropropagated plants arises from a complex interaction of genetic and epigenetic alterations, influenced by several key factors such as genotype, explant type, culture conditions, and medium compositions (Table 1) [64]. Understanding these factors is crucial for commercial micropropagation to reduce somaclonal variation and maintain genetic uniformity [64,65]. Notably, plant genotypes significantly influence this process, as their susceptibility to somaclonal variation varies [65,66]. Some genotypes possess greater genetic instability in tissue culture, mainly due to differences in genome size, ploidy levels, and genes involved in DNA repair and stress response mechanisms [67]. Therefore, selecting genetically stable genotypes—those less prone to genetic changes during tissue culture—can significantly reduce somaclonal variation [68]. Additionally, the choice of explant influences genetic stability, with meristematic tissues such as axillary buds [69], shoot tips [66], and root tips [22] generally exhibiting higher genetic stability among regenerated and donor plants compared to differentiated tissues like leaves [70] and stems [71], which are more susceptible to genetic alterations during culture. This is because meristematic cells, being less differentiated and divided more slowly, are less prone to genetic and epigenetic changes. However, Hesami et al. reported that in Cannabis sativa, somaclonal variation was more pronounced in meristematic regions compared to differentiated tissues, indicating a higher susceptibility of actively dividing cells to in vitro-induced genetic and epigenetic changes [72]. Culture conditions also profoundly impact, as in vitro environments differ significantly from natural conditions, subjecting plant cells to stresses that can alter their metabolism and trigger somaclonal variation [72,73]. Factors such as media composition, hormonal imbalances, culture cycles, and physical or chemical stressors collectively contribute to this. For example, nutrient imbalances in artificial media can cause cellular stress, affect metabolism, and increase mutation rates [73]. Likewise, hormonal imbalances caused by the frequent use of high concentrations of auxins and cytokinins, though vital for shoot initiation and proliferation, can destabilize the plant’s hormonal equilibrium and trigger genetic instability [73,74]. The age and frequency of culture also matter, with somaclonal variation increasing with prolonged subculturing. In bananas, for instance, somaclonal variants significantly increased after the eighth subculture while propagation rates declined [75], and similar patterns were noted in callus cultures [76,77]. Physical and chemical factors such as light intensity, temperature, and toxic substances can induce genetic changes [78]. Mechanical disruptions during subculturing also introduce stress and potential mutations. These interrelated factors dictate the frequency and type of somaclonal variation, emphasizing the need to carefully optimize genotype selection, explant sourcing, culture media, environmental controls, and subculture protocols to minimize variability in micropropagated plant populations [79,80].

5. Detection and Assessment of Somaclonal Variation in Micropropagated Plants

5.1. Detection of Genetic Variation in Micropropagated Plants

Selecting somaclonal variants early and excluding undesirable traits are crucial challenges in successful commercial micropropagation (Table 2). However, various methods are available to detect somaclonal variation and confirm clonal fidelity in progeny. Phenotypic observation is a traditional method for identifying somaclonal variations, focusing on external traits such as plant height, leaf shape, flower color, fruit size, and overall morphology [103]. This method, though widely used, is often influenced by environmental factors, such as light, temperature, and humidity, which may obscure genetic changes and lead to missed genetic alterations [103,104]. For example, in Solanum viarum [105], variations in flower color and fruit shape were observed through phenotypic observation, though some genetic changes were not detectable due to environmental influences. While phenotypic observation is an initial step in variant selection, it is insufficient for identifying genetic changes, especially in perennial crops where the plants must be observed until maturity [105,106]. The long maturation period required to detect somaclonal variations in perennial crops renders phenotypic observation a time-consuming and expensive method [106]. Conventional cytogenetic analysis, which involves light microscopy to observe stained chromosomes, is another widely used approach. However, this method is limited when plants have small chromosomes, as their structural or numerical variations are difficult to detect and require complex and time-consuming techniques [107]. For instance, in Citrus sp., chromosome analysis revealed variations, but this approach became problematic due to the small size of the chromosomes [108]. To overcome these limitations, more advanced techniques like flow cytometry have gained popularity. This method allows for precisely counting chromosomes and determining ploidy and nuclear DNA content, providing a more sensitive, rapid, and reliable approach to detecting genetic variation [109]. For instance, flow cytometry has been used to analyze the ploidy levels of Fragaria × ananassa, and no variation was observed between meristem-derived and conventionally propagated plants [110]. In Curcuma angustifolia, the mean 2C DNA content of conventionally propagated and micropropagated plants was estimated to be 2.26 pg and 2.31 pg, respectively. Somaclonal variations were absent in both the tissue-cultured plantlets and the donor plants [111]. Additionally, physiological and biochemical tests offer faster alternatives for identifying somaclonal variants, such as analyzing the plant’s response to hormones like gibberellic acid and evaluating pigment synthesis (e.g., chlorophyll and carotenoids [112]. Gibberellic acid, known for its role in plant growth regulation, has been shown to affect somaclonal variation in crops like bananas, making it a valuable marker for early detection [113]. The synthesis of pigments such as chlorophyll is also a key indicator of genetic stability, as variations in pigment levels are often linked to genetic changes [113]. Isozyme analysis has been another approach for detecting somaclonal variation. Isozymes are distinct forms of enzymes that regulate various metabolic activities, and changes in their activity can reflect genetic variation in the plant [111,113]. This method has been beneficial in soybeans [114], beans [115], and bananas [116], where specific isozymes, such as esterase or alcohol dehydrogenase, have been studied for their role in somaclonal variation. In addition to these traditional methods, molecular markers have become a cornerstone of somaclonal variation detection [114]. These markers offer greater precision and reliability, helping to identify even subtle genetic variations that may go unnoticed by phenotypic or cytogenetic methods [116]. Techniques such as RFLP (restriction fragment length polymorphism), RAPD (random amplified polymorphic DNA), AFLP (amplified fragment length polymorphism), ISSR (inter-simple sequence repeat), SSR (simple sequence repeat), and SCoT (Start Codon Targeted) markers are widely employed to detect somaclonal variations. RFLP detects polymorphisms in DNA by analyzing changes in the length of restriction enzyme-digested fragments, providing high-resolution detection of mutations [117]. For example, RFLP analysis revealed that soybean genetic variation mainly involves two alleles per locus. Tissue cultures generated new alleles, often matching those in other cultivars, suggesting stress-induced recombinational events [118]. Likewise, in rice, RFLP analysis of rice regenerants revealed DNA polymorphisms among plants from different callus cultures and siblings from a single callus. Longer incubation periods (67 days) led to higher genetic instability, with DNA rearrangements and methylation changes observed in some regenerants, affecting both structural and housekeeping genes [76]. However, the technique’s high cost, technical complexity, and requirement for large amounts of DNA have limited its widespread use [119]. RAPD is a more straightforward and affordable method that uses random primers to amplify DNA, quickly identifying somaclonal variation [120]. In orange, RAPD analysis showed that 99% of nucellar-derived plantlets were genetically identical to the mother plant, while 20–30% of ovary-derived plantlets exhibited somaclonal variation. The explant source and method influence genetic stability during in vitro regeneration [31]. Similarly, 38.33% polymorphism in Populus was revealed by using RAPD markers, demonstrating that alterations in tissue culture medium composition can induce genetic variation [32]. RAPD and ISSR analyses confirmed somaclonal variation during indirect organogenesis in sugarcane, with true-to-type rates above 86% in varieties (Co 86,032 and Q117) [77]. Moreover, orchids cultured in basal medium without phytohormones exhibited complete genetic uniformity (100% monomorphism), while regenerated in hormone-enriched medium showed low levels of genetic polymorphism, with 1.52% (RAPD), 1.19% (ISSR), and 3.97% (SCoT) variation [81]. In banana, the genetic uniformity of the micropropagated plantlets was assessed using RAPD and ISSR markers. A total of 50 RAPD and 12 ISSR primers produced 625 distinct bands, and all markers showed uniform patterns. The band intensity histograms confirmed the monomorphic nature of the plantlets, with no genetic variation detected [15]. RAPD analysis revealed genetic variation among three somaclones in chili pepper, with traits like early flowering and increased yield components. Tissue culture-induced variants in cultivars such as Shishitou and Takanotsume suggest potential for improvement through somaclonal variation [15]. Similarly, RAPD markers were used to screen genetic stability among regenerants in peach cultivars ‘Sunhigh’ and ‘Redhaven’, revealing polymorphism in some regenerants [121]. The in vitro raised plantlets of guava were assessed for genetic fidelity using RAPD, ISSR, and SSR markers, generating 2171 scorable bands. No polymorphism was observed between the somatic embryogenesis-derived plants and their respective donor mother plants, indicating genetic uniformity [122]. Despite its simplicity and low cost, RAPD can sometimes yield inconsistent results and may not be as reproducible as other methods, such as AFLP and SSR [121,122]. AFLP is another widely used technique that involves DNA digestion with restriction enzymes, ligation of adapters, PCR amplification, and gel electrophoresis. This method provides a detailed genetic fingerprint and can detect variations arising from mutations in restriction enzyme recognition sites [123]. ISSR markers amplify regions between microsatellites and offer a powerful method for detecting genetic variations. In Cucumis sativus, ISSR markers assessed somaclonal variation in three cultivars, revealing 75% genetic similarity between regenerated plantlets and in vivo plants of the Beith Alpha cultivar. Five primers produced 34 distinct band patterns, averaging 6.8 bands per primer [123]. Likewise, the genetic integrity of in vitro and field-grown plants of Curcuma angustifolia was revealed using ISSR primers. A total of 12 ISSR primers generated a total of 1260 well-resolved bands, showing monomorphic banding patterns across all plants analyzed [124]. Moreover, the plantlets regenerated from embryo axis, half-seed, axillary meristem, and cotyledonary node explants of Cicer arietinum were morphologically like the mother plants. Genetic fidelity, assessed using Start Codon Targeted and ISSR markers, confirmed that the in vitro regenerated plants were true to type with their mother plants [125], and 95.9% genetic similarity between the plantlets and the mother plant of Ficus palmata, confirming high uniformity and true-to-type regeneration [126]. SSR markers, also known as microsatellite markers, are highly informative and can detect variations at specific loci, providing precise genetic analysis [127]. The amplified fragment corresponds to the microsatellite itself, unlike the previously described ISSR, as the primers complement the repeated motif’s flanking regions. Because of the change in the number of motif repeats, the amplified fragments vary in size [126]. In Citrus madurensis, no variability was observed in BAP or hormone-free media using this marker, while phenylurea derivatives enhanced embryogenic potential and increased somaclonal variability [70]. Similarly, SSR marker assays generated a consistent amplification profile in both tissue culture and donor control plants, validating the clonal fidelity of bioreactor-derived micropropagated plants in the Latham, Heritage, Festival, and Nova cultivars of Rubus idaeus [82]. In Cannabis sativa, SSR analysis confirmed the genetic stability of micropropagated and regenerated plants, with monomorphic banding patterns matching the mother plants [128]. Moreover, Monomorphic bands in micropropagated shoots and plants of the lingonberry hybrid H1 and cultivar Erntedank were observed using SSR markers, confirming their genetic integrity [129]. In Hevea brasiliensis, the assessment of gene stability using SSR markers showed no variation between mother plants and in vitro plantlets [88], and no variation was observed in Magnolia dealbata using this technique [87]. SCoT (Start Codon Targeted) markers, which target conserved regions near the start codon, offer a cost-effective and efficient way to detect genetic consistency. Collard and McKeill first designed this technique for detecting variation in rice genotypes; recently, this technique has been used for various plant species, e.g., using SCoT primers targeting conserved regions [130]. The PCR amplification uses a single 18-mer primer to target the highly conserved region flanking the start codon ATG in plants [7]. Somaclonal variants were identified by this method in some plant species. For example, the genetic stability of in vitro-grown plants, assessed using SCoT primers, showed a close genetic relationship to the mother plant of Malus domestica [83]. Similarly, the genetic fidelity of in vitro regenerated plants was evaluated using 12 SCoT markers, revealing a genetic similarity of 0.987 between the tissue culture-derived plants and the mother plants of Coffea arabica [71]. In Tylophora indica, the SCoT marker produced a high percentage of monomorphic bands (94%), indicating genetic fidelity and similarity between regenerated and mother plants [131]. The application of SCoT primers to detect genetic consistency has grown dramatically due to their affordability, convenience, and effectiveness. The application of these molecular techniques has become increasingly essential in the micropropagation process, enabling early detection and precise elimination of somaclonal variants. Combined with traditional methods, these tools provide a comprehensive approach to enhancing clonal fidelity and ensuring the success of commercial micropropagation [129,131]. Moreover, Quantitative PCR (qPCR) is a valuable tool for detecting somaclonal variation by analyzing the expression of key genes in some plant species. For example, in Artemisia annua, qPCR was used to examine the expression of artemisinin biosynthetic genes (ADS, CYP71AV1, DBR2, and ALDH1) in somaclonal variants ASV1 to ASV13. Among these variants, ASV12 was found to produce significantly more artemisinin than the wild type, with an even greater increase when cultivated in saline soils [132]. Adly et al. [133] used qPCR to analyze starch-synthesis-related genes in the somaclonal variant Ros 119 of Solanum tuberosum, which showed a 42% increase in fresh tuber weight and a 75% increase in starch content compared to the parent cv Lady Rosetta. The enhanced starch accumulation was linked to the upregulated expression of AGPase, SSs, GBSS, and SBE genes. These findings demonstrate the potential of somaclonal variation and qPCR as effective tools for improving starch content and genetic diversity in potato breeding programs [133]. Similarly, this method was also employed to investigate somaclonal variation in Fragaria × ananassa between the parent cultivar ‘Benihoppe’ and its mutant ‘Xiaobai’ [134]. The results revealed elevated levels of volatile compounds such as ethyl isovalerate, ethyl hexanoate, and linalool in ‘Xiaobai’, which correlated with significantly increased expression of genes including FaLOX6, FaHPL, FaADH, FaAAT, FaAAT1, FaDXS, FaMCS, and FaHDR. In contrast, the content of eugenol was higher in ‘Benihoppe’, likely due to enhanced expression of FaEGS1a, highlighting the genetic basis of somaclonal variation in volatile compound profiles and offering potential for improving strawberry quality through genetic manipulation [134].

5.2. Detection of Epigenetic Variation in Micropropagated Plants

In plant tissue culture, stress conditions can influence the epigenetic stability and genetic makeup of plant materials, often triggering epigenetic modifications that regulate plant responses to environmental cues [29,50]. Various molecular techniques have been developed to assess epigenetic stability under these conditions, with modified AFLP-based methods like methylation-sensitive amplified polymorphism (MSAP) and methylation-sensitive AFLP (metAFLP) being widely employed (Table 3). The MSAP technique is an adaptation of AFLP, where genomic DNA is digested using methylation-sensitive restriction enzymes. The DNA is divided into two aliquots, each digested with EcoRI, which targets the GAATTC sequence and is largely unaffected by cytosine methylation, and either MspI or HpaII, isoschizomers that both recognize the CCGG sequence but differ in sensitivity to methylation [145]. Following ligation to adapters and amplification with selective primers, the resulting fragments are separated and compared to reveal methylation changes [145]. For instance, epigenetic variation, including demethylation and CG methylation, was most pronounced in the first six months of micropropagation, with these changes stabilizing thereafter. MSAP analysis identified coding sequences and LTR Gypsy retrotransposons, indicating that epigenetic modifications contribute to somaclonal variation in Allium sativum [34]. In Vaccinium vitis-idaea, through MSAP analysis, the highest methylation was observed in leaf regenerants (LC1), which also showed the highest levels of secondary metabolites. Micropropagated plants (NC1, NC2, NC3, LC1) exhibited more methylation than cutting-propagated (ED) plants, but ED plants had higher levels of secondary metabolites, indicating a trade-off between methylation and metabolite production [93]. Similarly, AFLP and MSAP analyses of 145 three-year-old somatic embryogenesis-derived plants showed extremely low polymorphism between mother plants and embryos of Coffea arabica, with ranges of 0–0.003% and 0.07–0.18%, respectively. No significant differences were found between proliferation systems for the two hybrids, and no correlation was observed between variant phenotypes and specific MSAP patterns [94]. Moreover, global DNA methylation was assessed in lowbush blueberry (Vaccinium angustifolium) wild clone QB9C and cultivar Fundy, propagated via softwood cutting (SC) and tissue culture (TC) using MSAP. The results revealed higher DNA methylation in micropropagated plants (29% for QB9C and 20% for Fundy) compared to those propagated by SC (25% for QB9C and 19% for Fundy). These findings indicate that tissue culture promotes greater methylation events, suggesting that DNA methylation may play a role in regulating plant growth and development under different propagation methods [95]. Similarly, Epigenetic diversity in six sugarcane genotype groups (commercial cultivars, S. officinarum, S. spontaneum, S. robustum, S. barberi, and Erianthus sp.) was assessed using methylation-sensitive amplification polymorphism (MSAP). A total of 1341 MSAP loci were analyzed, with 83.29% showing cytosine methylation, contributing significantly to overall genetic diversity. The genotypes were divided into two subpopulations with high differentiation, and methylated fragments were found near CpG islands and regulatory sequences, suggesting their role in stress adaptation [146]. Moreover, a study on 21 maize progeny lines from tissue cultures of inbred strain A188 revealed 39% of families had altered methylation patterns, all showing a decrease in methylation. These changes were stably inherited across two generations, suggesting demethylation as a major contributor to tissue culture-induced variation, with certain probes detecting methylation changes more frequently [100]. Similarly, the metAFLP method involves DNA digestion with Acc65I/MseI or KpnI/MseI, enzymes with differing sensitivity to methylation, and follows a protocol comparable to MSAP [147]. In winter triticale, the impact of Cu2+ and Ag+ ions and incubation time on tissue culture-induced variation and the genetic homogeneity was examined. These results showed a 51% variation, including 43% sequence variation, 5% demethylation, and 3% de novo methylation using metAFLP. The findings suggest that modifications in the culture medium affect both DNA sequence changes and methylation, offering a way to control the level of variation in tissue culture-derived regenerants [79]. Beyond AFLP-based methods, high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis (HPCE) are highly sensitive techniques for quantifying global DNA methylation levels, especially by measuring 5-methylcytosine after enzymatic digestion of genomic DNA [146,147]. For example, DNA methylation levels varied across different tissues, cultivars, and growth stages in Camellia sinensis. Higher methylation levels were observed in tender leaves (38.34%) and pistils (38.19%), while lower levels were found in capillary roots (19.45%), stamens (19.61%), and old leaves (20.70%). The methylation levels followed a saddle curve during bud growth, with the lowest point at the one-leaf and bud stage. Additionally, DNA methylation levels in adventitious buds decreased as pruning intensity increased, suggesting a link between methylation and tea plant development [148]. Another advanced method is Chromatin Immunoprecipitation (ChIP), including ChIP-chip and ChIP-seq approaches, which map interactions between DNA and specific proteins to determine methylation patterns. However, the large volume of resulting data poses challenges for analysis [149]. In Arabidopsis, this method includes a nuclear isolation step before chromatin shearing, improving DNA yield (15 g from 0.2–0.4 g of tissue), sufficient for analyzing 25 or more genes. This cost-effective and efficient protocol has been successfully applied to Arabidopsis thaliana and can be adapted to other systems for epigenetic research [150]. Among the most precise techniques, Whole-Genome Bisulfite Sequencing (WGBS) enables base-pair resolution analysis of 5-methylcytosine by treating genomic DNA with sodium bisulfite, converting unmethylated cytosines to uracil while leaving methylated cytosines unchanged, followed by PCR amplification and NGS analysis [151]. For example, the study found that tissue culture induces consistent homozygous DNA methylation changes inherited across multiple independent lines. These changes, including both gains and losses of DNA methylation in CG and CHG contexts, suggest that specific loci may act as hotspots for epigenetic variation. While not all changes in callus plants are observed in primary regenerants, most methylation alterations in primary regenerants are passed onto subsequent generations. WGBS has revealed methylation changes in several micropropagated species of maize [152]. Moreover, WGBS was used to examine genomic DNA methylation changes in Fragaria nilgerrensis across six stages, from explants to out planting and acclimation. CG sites showed the highest methylation (49.5%), followed by CHG (33.2%) and CHH (12.4%). CHH methylation changes were most prominent, particularly in transposable element regions. Methylation levels fluctuated throughout the tissue culture process, with non-uniform distribution across different genetic regions [96]. Similarly, epigenetic variation examined in rice using WGBS exhibits distinct differences, particularly in chromosomal distribution, compared to variations that occur under natural conditions [80]. In pineapple, WGBS was used to analyze context-specific DNA methylation changes in the pineapple genome between the cutting seedlings and five somaclonal variation plants [97]. Similarly, in apple, the DNA methylome and transcriptome of rejuvenated apple rootstock M9T337 cuttings were induced by in vitro shoot culture. It is found that DNA hypomethylation enhances adventitious rooting by upregulating rooting-related genes, while DNA hypermethylation represses rooting-inhibiting genes. These results emphasize the significant role of DNA methylation changes in regulating rooting ability in apple rootstock [153]. Additionally, RNA sequencing has emerged as a practical approach for identifying tissue culture-induced epigenetic variations [154]. Recently, this method was used to detect altered methylation levels in strawberries’ long-term in vitro shoot cultures. The results showed increased DNA methylation in specific sequence contexts (CG, CHG, CHH), with changes linked to the expression of regeneration-related genes. Inhibition of DNA methylation with 5′-azacytidine (5′-Aza) disrupted callus formation and regeneration, highlighting the critical role of DNA methylation in these processes [155]. These molecular and biochemical techniques collectively provide powerful tools for assessing epigenetic variation in tissue-cultured plants, contributing to a better understanding of somaclonal variation and ensuring the epigenetic fidelity of regenerants.

6. Methods for Optimizing Somaclonal Variation

Several strategies have been developed and successfully applied in various plant systems to optimize somaclonal variation control and maintain clonal fidelity during micropropagation. One of the most critical factors is explant selection, as explants derived from meristematic tissues such as axillary buds, shoot tips, and root tips are less prone to somaclonal variation than highly differentiated tissues like roots, leaves, and stems [162]. By carefully choosing appropriate explants, it is possible to reduce the incidence of unwanted variation. In addition, optimization of culture conditions plays a vital role in minimizing somaclonal variation. This involves adjusting the nutrient composition of culture media, fine-tuning the ratios of plant growth regulators (PGRs), and limiting the number of subcultures, as excessive subculturing increases the chance of genetic and epigenetic alterations, particularly in callus and cell suspension cultures [79,106]. Rodrigues et al. showed that somaclonal variants appeared from the fifth subculture (1.3%) onwards and increased to 3.8% after 11 subcultures in Musa spp. [163]. Studies have shown that reducing PGR concentrations can significantly decrease the likelihood of somaclonal variation [68,162], while optimizing environmental conditions such as temperature and light intensity can further stabilize cultures by avoiding stress-induced changes [68]. Bioreactor systems have also demonstrated potential in reducing the frequency of somaclonal variation by providing uniform and controlled culture environments [16]. For example, in Uncaria guianensis, the RITA (Recipient for Automated Temporary Immersion) system has been identified as the most effective bioreactor design. These micropropagation systems ensure efficient propagation and genome stability in Uncaria guianensis [164]. Another crucial approach involves favoring direct organogenesis over indirect methods, as indirect organogenesis typically consists of a callus phase associated with prolonged cell proliferation and extended culture exposure, increasing the risk of genetic instability [158]. In contrast, direct organogenesis produces shoots or roots directly from explants without intermediate callus formation, thus minimizing variation and preserving genetic fidelity [165]. Furthermore, cryopreservation has proven to be an effective long-term strategy for preserving plant tissues, embryogenic lines, and somatic embryos at ultra-low temperatures (−196 °C) without compromising their genetic integrity [69]. This technique reduces maintenance time and costs and minimizes the accumulation of somaclonal variation or contamination risks over time [166]. Finally, Molecular Marker-Assisted Selection (MAS) is a powerful tool for early detection and elimination of somaclonal variants during micropropagation [26]. Various molecular markers such as RAPD, ISSR, AFLP, SSR, and SNPs have been successfully applied for screening and identifying somaclonal variants in different plant species [11,26], allowing for the maintenance of genetic uniformity and selection of true-to-type regenerants. Together, these complementary strategies provide a comprehensive framework for minimizing somaclonal variation and enhancing the reliability of plant micropropagation systems.

7. Application of Somaclonal Variation as a Crop Improvement Tool

However, somaclonal variation is generally considered undesirable in clonal propagation. It can be a valuable tool for crop improvement [45]. The induced variation can be screened for desired traits to create new cultivars with desirable quality, known as somaclonal selection [68]. This process successfully selects elite genotypes in crops and plant species with improved quality, such as insect, pest, disease resistance, stress tolerance, or increased production rate [167]. However, selecting new variants requires careful consideration and ensuring that only desirable characteristics are selected, and harmful variants are removed [168]; using molecular markers is one of the reliable methods to identify and characterize desirable variants that prevent the propagation of deleterious variants [169]. In recent years, somaclonal variation has been used to improve agricultural crops, primarily for drought tolerance. Some desirable somaclonal variants used in plant breeding programs are presented in Table 4.

8. Future Prospects

The integration of high-throughput molecular techniques such as MASP, WGBS, RNA sequencing, and CRISPR/Cas9 is revolutionizing the detection and minimizing of somaclonal variation in the agriculture, horticulture, and forest sectors [177]. Although CRISPR/Cas9 introduces targeted mutations, it actually supports the maintenance of clonal fidelity by offering precise and controlled gene editing, unlike the random and widespread genetic alterations associated with somaclonal variation [178]. Commercial micropropagation companies frequently employ these revolutionary techniques to validate planting materials, especially for high-value crops and nursery stocks intended for export. However, some challenges remain in making these molecular assays routine and affordable for experimental and commercial uses. To overcome these obstacles, it needs to optimize culture protocols, minimize the subculture cycle, select genotypes, and use meristematic explants with direct organogenesis [26]. The bioreactor system provides a controlled environment that provides consistent growth conditions for plant tissues, thereby minimizing somaclonal variation [16,82]. Moreover, cryopreservation techniques, which store plant tissues at extremely low temperatures, help maintain genetic stability over time [179]. While unstable epigenetic changes may have limited and reversible effects on plant phenotype, the integration of genetic and epigenetic screening in the micropropagation process is essential for reliably maintaining clonal fidelity for harnessing beneficial somaclonal variation in commercial micropropagation.

9. Conclusions

Somaclonal variation is significantly influenced by micropropagation techniques, with direct organogenesis generally producing more genetically stable plants compared to indirect organogenesis. Key factors contributing to this variation include the composition of the culture medium, the ratio and type of plant growth regulators (PGRs), explant source, frequency and type of subcultures, as well as environmental conditions such as light intensity and temperature. To optimize economic loss in commercial micropropagation, it is essential to identify and eradicate variants at the very early stages of their growth. Different approaches, like morphological analysis, cytological analysis, and molecular markers, are used to detect genetic and epigenetic variations. However, a few somaclonal variants are used as a new agronomic trait in crop improvement; at the same time, it is also a crucial problem in micropropagated plants. Numerous studies are being conducted on somaclonal variations during tissue culture, but a comprehensive understanding remains elusive.

Author Contributions

S.M., conceptualization, writing—original draft, and writing—review and editing. S.C.D., conceptualization, writing—original draft, writing—review and editing, and funding acquisition. A.U.I., conceptualization, writing—original draft, writing—review and editing, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

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Figure 1. Schematic overview of tissue-culture induced somaclonal variations.
Figure 1. Schematic overview of tissue-culture induced somaclonal variations.
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Figure 2. Mechanisms of genetic modification during the micropropagation process.
Figure 2. Mechanisms of genetic modification during the micropropagation process.
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Figure 3. Mechanism of epigenetic modification in somaclonal variation. (‘X’—Gene A, B, C, D stopped expression, and Gene E was normally expressed).
Figure 3. Mechanism of epigenetic modification in somaclonal variation. (‘X’—Gene A, B, C, D stopped expression, and Gene E was normally expressed).
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Table 1. Summary of factors affecting somaclonal variation during the micropropagation of some essential crops and plant species.
Table 1. Summary of factors affecting somaclonal variation during the micropropagation of some essential crops and plant species.
Plant NameExplantsMedia Composition and PGRsGenotypeSubcultures CyclesEnvironmental FactorsKey FindingsReference
Populus alba (White Poplar)Shoot tips, leaf explants, or nodal segmentsMS medium with sucrose (PGRs inferred)Not specifiedNot
specified		Standard in vitro conditionsSignificant variation was observed in morphological, physiological, and molecular traits.[32]
Citrus jambhiri
(Orange)		Leaf, nodal, root segments, stigmas, styles, ovaries, nucellar tissues, cotyledons, juice vesiclesMS medium with BAP, KN, ME, 2,4-D for callus induction; BA, 2,4-D, ABA for somatic embryo induction, sucrose for germinationNot specifiedNot
specified		Controlled
Environment		Callus was induced from most explants except styles and juice vesicles; somatic embryos formed from ovary and nucellar tissues with BA and ME, and 20–30% variation in ovary-derived plantlets.[31]
Vaccinium vitis-idaea (Lingonberry)Juvenile leaf explants from two genotypesBerry basal medium + 5.5 µM thidiazuron for callus induction; 4.0 µM zeatin for plantlet regenerationNot specifiedNot
specified		Standard in vitro conditions92% embryogenic callus induction with thidiazuron, enhanced plantlet regeneration with 4.0 µM zeatin, confirmed somatic embryo development, and demonstrated increased flavonoids, anthocyanins, antioxidants, stress resistance.[17]
Musa acuminata (Banana)Leaf base explantsMS medium with high levels of BAP (up to 53.28 µM) and kinetin (55.80 µM) for shoot proliferationNanjanagudu RasabaleNot
specified		Standard in vitro conditionsHigh cytokinin levels (BAP and kinetin) produced up to 80 shoots per segment and ensuring genetic stability for conserving the endangered NR cultivar.[33]
Saccharum officinarum (Sugarcane)Callus cultures derived from embryogenic tissueCallus induction medium supplemented with PVP, casein hydrolysate, MES buffer, and PEG 8000 for somatic embryogenesisCo 86,032 and Q117Not specifiedStandard in vitro conditionsMedium VI with PVP, casein hydrolysate, and MES buffer induced 79.66% callus in Co 86,032 and 82.83% in Q117, with PEG 8000 enhancing somatic embryogenesis and minimal somaclonal variation.[77]
Dendrobium fimbriatum (Orchid)Not
specified		Mitra medium supplemented with KN (0.8–4.8 mg/L), IBA, NAA, or BAP for regenerationNot
specified		Not
specified		Standard in vitro conditionsEffective shoot formation was achieved with KN, and root development with IBA and BAP/KN combinations, while low genetic polymorphism was observed.[81]
Oryza sativa (Rice)Callus cultures from various explantsNot
specified		Not
specified		More prolonged incubation (67 days) and shorter (28 days)Standard in vitro conditionsRegenerants from more extended callus incubation periods (67 days) exhibited higher genetic instability, with DNA polymorphisms and methylation changes linked to somaclonal variation in both structural and housekeeping genes.[76]
Rubus idaeus (Red Raspberry)Leaf
segments		Liquid medium + 2.3–9.0 μM thidiazuron (TDZ) for regeneration, 4.4 μM BA for shoot elongation, PGRs for rooting‘Latham’, ‘Heritage’, ‘Festival’, ‘Nova’Not specifiedBioreactor system, controlled field
conditions		Tissue culture plants of ‘Latham’ and ‘Festival’ outperformed root cutting (RC) plants in cane and berry yields, with clonal fidelity observed. In contrast, bioreactor culture influenced juvenile branching, promoting growth and berry production.[82]
Malus domestica (Apple Rootstock MM 104)Axillary budsMS basal medium, 3.0% sucrose, 0.8% agar, BA (5.0 µM), NAA (1.0 µM) for bud establishment, PGRs BA (1.0 µM) + NAA (1.0 µM) for shoot multiplication, IBA (0.1 µM) for rootingMM 104Not
specified		5-day dark period for rooting, acclimatization in vermiculite: perlite: sand: soil (2:2:1:1) mixHigh shoot multiplication (100%) with 9.8 shoots/explant and 76% survival under field conditions.[83]
Citrus mitis (Calamondin)Style-stigma explantsBAP (6 µM), 4-CPPU, PBU, 2,3-MDPU (12 µM each), hormone-free (HF) conditionsNot specifiedNot specifiedIn vitro culture conditionsPhenylurea derivatives (4-CPPU, PBU, 2,3-MDPU) enhanced embryogenic potential, with 2,3-MDPU and PBU causing 3.7% somaclonal variation, while BAP/HF medium showed no variability[70]
Coffea arabica (Coffee)Foliar explantsMS + 0.4–0.6 mg/L 2,4-D for callus induction; IAA and Kn also testedS.4202, S.4932Not specifiedIn vitro culture followed by field testingEfficient somatic embryogenesis was achieved in both hybrids, with SRAP and SCoT marker analyses showing high genetic similarity to mother plants.[71]
Aloe vera (Aloe vera)Axillary buds (direct); Inflorescence base (indirect via callus)Not specifiedSweet genotypeNot
specified		In vitro regeneration with and without callusDirect regeneration from axillary buds showed 0% polymorphism (true to type), while indirect regeneration via callus from the inflorescence base exhibited 80% polymorphism.[84]
Ficus palmata
(Fig)		Leaves and stemsMS basal medium, 2,4-D (2.0 mg/L) + Kin (0.2 mg/L)Not specifically namedNot mentionedGamma radiation at 0, 20, 30, 40 GyHighest callus induction at 30–40 Gy. A total of 99.58% polymorphism due to gamma treatment, yet tissue culture plants remained genetically similar to mother plants. GC-MS revealed increased secondary metabolites at 30 and 40 Gy.[78]
Carica papaya (Papaya)Apical, nodal, petiole, leaf, and root segmentsMS basal medium, BAP, Zeatin, NAA (direct); MS + BAP (1.0 mg/L), TDZ (0.3 mg/L), NAA (0.10 mg/L), 30 g/L sucrose (indirect)Red Lady 786Not specifiedStandard in vitro conditionsDirect regeneration with 65–88% success; indirect regeneration with 75–85% somatic embryogenesis; efficient medium for somatic embryos with TDZ, BAP, NAA, and sucrose.[85]
Stevia rebaudiana
(Candy leaf)		Not
specified		MS + BAP (4.0 mg/L), NAA (2.0 mg/L), 2,4-D (2.0 mg/L)Not specified6 (Six)
subcultures		Standard in vitro conditionsIndirect organogenesis induced somaclonal variation, improving rebaudioside A content and Rebaudioside A/stevioside ratio, with SCoT analysis showing genetic variation, while subculturing had no effect on genotype or glycoside profile.[86]
Magnolia dealbata
(Cloudforest magnolia)		Not specified (embryogenic tissues)MS medium + 2,4-D (2.26 or 4.52 μM)Not specified2 cycles of secondary somatic embryogenesisStandard in vitro conditionsHighest embryo yield at 4.52 μM 2,4-D. Genetic similarity between donor and regenerants was 0.90, indicating low somaclonal variation. [87]
Hevea brasiliensis (Rubber tree)Mixed floral explantMS + 2.0 mg/L BA + 1.5 mg/L 2,4-D; for germination: MS + 0.25 mg/L GA3Early introduced clone (white root disease resistant)3 passages (4 weeks each)Standard in vitro conditionsHighest SE induction (39.84%) and 3.25 cotyledonary embryos per callus. A total of 50% of somatic embryos developed embryonic axes, 25% formed shoots, and confirmed genetic stability.[88]
Alhagi maurorum
(Camelthorn)		Leaf (direct), hypocotyl, cotyledon, root (indirect)MS + BA, IBA or NAA (0.2 or 0.5 mg/L); rooting on ½ MS ± IBA (0.2–2.0 mg/L)Not specifiedNot
specified		Standard in vitro conditionsDirect organogenesis produced high chlorogenic acid, while indirect regeneration via callus yielded higher 20-HE content, with genetic similarity ranging from 0.765–0.947.[89]
Musa acuminata
(Banana)		Individual shoot apex; transverse sectionsMS + 10, 20, or 30 µM BAP + 1.0 µM NAA (proliferation); MS + 6 µM IBA (rooting)‘Grand Naine’Monthly subcultures for 6 monthsStandard in vitro conditionsTransverse sectioning of shoot apex improved shoot production (up to 32.77 shoots/section in 6 months). No genetic variation in plants from transverse sections; 23.46% polymorphism detected in plants from whole shoot apex.[90]
Vanilla planifolia
(Vanilla)		Immature seedsMS + 2.27 µM TDZ (callus induction); MS + 8.88 µM BA (shoot regeneration); MS without PGRs (rooting)‘Vanilla planifolia’Indirect organogenesis (not specified)Standard in vitro conditionsTransverse sectioning of the shoot apex improved shoot production (up to 32.77 shoots/section in 6 months), with no genetic variation in plants from transverse sections.[91]
Zea mays (Maize)Callus cultureMS mediumMultiple independent linesMultiple cyclesStandard in vitro conditions DNA methylation changes were observed at specific loci in tissue culture, including gains and losses of methylation, primarily affecting CG and CHG contexts, with some methylation changes being heritable.[92]
Vaccinium vitis-idaea
(Lingonberry)		Leaf, Node, and Cutting explantsZeatin-induced media; Liquid medium (NC1), Semi-solid medium (NC2), Node culture-derived (NC3), Leaf culture-derived (LC1)Erntedank cultivarMultiple subcultures, both in vitro and ex vitroTissue culture conditions Highest methylation was observed in culture-derived plants) with 108 methylation bands, NC1, NC2, NC3, and LC1 showed higher methylation than cutting-propagated (ED) plants (79 bands), with higher secondary metabolites than micropropagated shoots and plants.[93]
Coffea arabica
(Coffee)		Embryogenic suspensionsLow 2,4-D concentrations, short proliferation periodsHybrids6 months of cultureField plots for phenotypic assessmentSomaclonal variation was very low (0.74%), with minimal genetic polymorphism, chromosome loss in rare variants, and no phenotype-MSAP pattern correlation.[94]
X Triticosecale spp.
(Winter triticale)		Explants from cultured tissuesCu2+ (0.1, 5, 10 µM), Ag+ (0, 2, 10 µM), MS medium modificationsNot specified9 different conditions testedIncubation time: 35, 42, 49 days51% tissue culture-induced variation was observed, and variation was influenced by culture medium composition.[79]
Lowbush Blueberry
(Blue berry)		Softwood cutting (SC), Tissue culture (TC)Standard tissue culture mediumWild clone QB9C, cultivar FundyNot specifiedConventional propagation for SC, in vitro culture for TCTissue culture plants showed higher DNA methylation (29% in TC QB9C, 20% in TC Fundy) than SC plants (25% and 19%, respectively), with polymorphism detected only in TC plants.[95]
Fragaria x ananassa
(Strawberry)		Shoot tipsStandard tissue culture mediumFragaria nilgerrensis6 stages (explants to acclimation)Tissue culture conditions Dedifferentiation/redifferentiation stages affecting genes in hormone metabolism, development, and stress response, are linked to epigenetic variation.[96]
Oryza sativa
(Rice)		Somaclonal line (TC-reg-2008)Standard tissue culture mediumOryza sativa (cv. Hitomebore)Extensive selfing of somaclonal lineNormal growing conditions and abiotic stress conditionsTissue culture extensively induced heritable genomic variation, non-randomly distributed across 12 chromosomes, affecting functional genes with stress-responsive phenotypic effects, linked to transposable element mobilization and DNA methylation changes.[80]
Ananas comosus
(Pineapple)		Callus culture-derived plantsStandard tissue culture mediumAnanas comosusNot specifiedControlled in vitro conditionsDNA methylation changes observed in somaclonal variation (SV) plants, with significant differences in methylation patterns between SV plants and cutting seedlings (CK).[97]
Hordeum vulgare
(Barley)		Somatic embryosTissue culture media with copper and silver ionsHordeum vulgareNot specifiedIn vitro conditions DNA methylation changes observed in barley regenerants. The study optimized ion concentrations and culture duration to minimize or maximize tissue culture-induced variation.[98]
Dendrocalamus asper
(Giant Bamboo)		Shoot tips, plantletsMS, MSR media with modified phosphorus, sucrose, salts, and mycorrhizal inoculation with Rhizoglomus clarumDendrocalamus asper3 subculturesStandard in vitro conditionsBAP promoted shoot multiplication with decreased DNA methylation in the third subculture, while mycorrhization occurred only in MSR and MS/2 media, offering insights into methylation.[99]
Zea mays (Maize)Embryo tissuesStandard maize tissue culture media, no specific PGRs mentionedZea mays A188Not
specified		Standard in vitro conditions Altered DNA methylation patterns were found in 39% of progeny lines, primarily due to stable, heritable demethylation.[100]
Lactuca sativa
(Lettuce)		Various accessions (major cultivars and wild relatives)Not specifiedMajor lettuce cultivars, wild relativesNot
specified		Controlled environmentLettuce domestication led to a significant increase in DNA methylation, with epigenetic variations linked to leafy and stem types, influencing gene expression and chromatin accessibility and contributing to their divergence.[101]
Coffea canephora
(Robusta coffee)		Somatic tissues for Somatic Embryo (SE)Not specified; pharmacological inhibitor (5-Azacytidine) usedNot specifiedNot specifiedSE induction and maturation phases; use of epigenetic inhibitorDynamic changes in DNA methylation and histone modifications accompany SE progression.[102]
MS medium—Murashige and Skoog medium, BAP/BA—6-benzylaminopurine (also written as BA for benzyladenine), KN—Kinetin, 2,4-D—2,4-dichlorophenoxyacetic acid, ABA—abscisic acid, PVP—polyvinylpyrrolidone, MES buffer—2-(N-morpholino) ethanesulfonic acid, PEG—polyethylene glycol, NAA—α-naphthaleneacetic acid, CPPU-N—(2-chloro-4-pyridyl)-N’-phenylurea, PBU-1—phenyl-3-(1,2,3-thiadiazol-5-yl)urea, 2,3-MDPU—N-phenyl-N′-[6-(2,3-dimethylphenyl) pyridazin-3-yl]urea, and GA3—gibberellic acid.
Table 2. Compilation of genetic variation reports, their causes, and detection methods in important agricultural and horticultural species.
Table 2. Compilation of genetic variation reports, their causes, and detection methods in important agricultural and horticultural species.
Plant SpeciesTissue Culture
Method/Explants		Detection MethodKey FindingsReference
Citrus jambhiri (Rough lemon)Somatic embryogenesisRAPD
markers		Plantlets regenerated from nucellar tissues showed no variation, while those raised from ovaries showed variation in 20–30%.[31]
Populus alba (White Poplar)OrganogenesisRAPD markersHighlighting 38.33% genetic instability in tissue culture-derived plants.[32]
Saccharum officinarum (Sugarcane)Callus and shoot cultureISSR and RAPD markersFound genetic variation in sugarcane callus cultures[33]
Dendrobium fimbriatum (Orchid)MicropropagationRAPD, ISSR, and SCoT markersOrchids grown in basal medium without phytohormones showed 100% monomorphism, while those in hormone-enriched media showed low genetic polymorphism.[81]
Vaccinium vitis-idaea (Lingonberry)Somatic embryogenesis/leaf explantsEST-SSR and GSSR markersThe genetic stability observed[17]
Musa spp. (Banana) MicropropagationISSR and RAPD markersDetected somaclonal variation among regenerants emphasized the need for genetic fidelity assessment in micropropagation.[33]
Artemisia annua (Sweet wormwood)Callus culture/apical shoots, nodal segmentsqPCR, SCAR, and RAPD markersIdentified genetic differences between in vitro and field-grown plants, affecting high artemisinin production.[132]
Solanum tuberosum
(Potato)		Callus culture/Internode explantsqPCREnhanced starch accumulation[133]
Glycine max (Soybean)Root tissue callus cultureRFLP markersTissue cultures developed RFLP allelic differences; new alleles matched those in other cultivars, suggesting recombinational events.[118]
Oryza sativa (Rice)Callus culture/
leaf explants		RFLP markersHigher genetic instability with more extended callus incubation periods; correlation with methylation changes.[76]
Malus domestica (Apple)OrganogenesisISSR and SCoT markersHigh genetic fidelity with 98.43% monomorphic bands; minimal somaclonal variation detected.[83]
Citrus madurensis
(Calamondin)		Callus cultureSSR, RAPD markersDiphenylurea derivatives enhance somatic embryogenesis in Citrus madurensis but compromise genetic stability, unlike BAP or Hormone Free media, which maintain clonal fidelity.[70]
Prunus persica
(Peach)		Callus culture/
cotyledon		RAPD markersHigh somaclonal variation was observed in regenerants.[121]
Fragaria × ananassa
(Strawberry)		Micropropagation/shoot tipsqPCRSomaclonal variation was observed[134]
Ficus carica
(Fig)		Callus culture/leaf and stem explantsISSR markersHigh genetic variation observed[78]
Elaeis guineensis
(African oil palm)		Callus cultureSSR and RAPD markersGenetic variation found in oil palm regenerants. SSR and RAPD markers detected polymorphisms linked to culture conditions and subculturing practices.[135]
Bixa Orellana
(lipstick tree)		Organogenesis/
nodal explants		ISSR and RAPD markersThe results of the RAPD marker system revealed the genetic stability among the micropropagated plants.[136]
Rubus ideals
(Raspberry)		Direct organogenesis through
bioreactor
leaf segments		SSR markersGenetically stable.[82]
Aloe vera
(Aloe)		Organogenesis/axillary shoot buds
callus culture/inflorescence axis		ISSR markers, and RAPDPlantlets produced through indirect organogenesis exhibited considerable variation, while those generated via direct organogenesis showed complete uniformity.[84]
Coffea arabica
(Coffee)		Somatic embryogenesisSRAP and SCoT markers98% and 99% genetic similarities, respectively, between the regenerated and mother plants.[94]
Phoenix dactylifera
(Date palm)		Callus culture/
shoot tips and axillary shoot meristems		RAPD markersNo variation observed.[44]
Corylus avellana (Hazel)EmbryogenesisISSR and RAPD markersIdentified genetic differences between in vitro and field-grown hazelnuts, affecting nut quality and tree height.[137]
Caladium × hortulanum
(Caladium)		Callus culture/leaf cultureSSR markers, and
flow cytometry analysis		Somaclonal variation was high in in vitro-cultured caladium aneuploids, with tetraploid aneuploid caladium exhibiting the greatest variability[138]
Carica papaya
(Papaya)		Somatic embryogenesisRAPD and ISSR markersRAPD and ISSR markers for detecting genetic fidelity[85]
Tylophora indica
(Indian Ipecac)		indirect, direct and somatic embryoSCoT marker, and
flow cytometry		Determination of 2C DNA content and verification of genetic uniformity using SCoT molecular markers.[131]
Cannabis sativa
(Hemp)		Callus cultureSSR markersNo variation observed.[128]
Musa rubra
(Bronze Banana)		Micropropagation/shoot tipsISSR markersGenetic stability observes.[139]
Vaccinium vitis-idaea
(Lingonberry)		Micropropagation/shoot tipsEST and SSR primersGenetic equality observed.[129]
Hevea brasiliensis
(Rubber)		Somatic embryogenesis/
flower explants		RAPD and SSR markersNo genetic variation was detected between the mother and in vitro plantlets, as indicated by RAPD and SSR marker analysis.[88]
Psidium guajava (guava)Somatic embryogenesis/zygotic embryoRAPD, ISSR or SSR markersApproximately 99% of bands were monomorphic.[122]
Vanilla planifolia
(Vanilla)		Callus culture/immature capsulesISSR marker and Phenotypic observationMolecular analysis of regenerated plantlets revealed 71.66% genetic polymorphism.[91]
Musa acuminata
(Banana)		Organogenesis/shoot apicesRAPD markersMolecular analysis showed 23.46% polymorphism, whereas transverse sections confirmed genetic uniformity with the parent plants.[90]
Hypericum gaitii
(St. John’s wort)		Organogenesis/apical and axillary meristemsISSR markersNo polymorphism among the micropropagated plants and mother plants.[140]
Saccharum officinarum
(Sugarcane)		Organogenesis/shoot tipsSCoT markersGenetic stability observed.[141]
Rhaponticum carthamoides
(Maral root)		Direct organogenesis/leaf explantsflow cytometry, RAPD, and ISSR markersGenetic stability observed.[89]
Curcuma angustifolia
(Indian Arrowroot)		Organogenesis/shoot tipsEST-ISSR markersNo variation observed.[124]
Artemisia vulgaris (Mugwort)Organogenesis/
nodal explants		SCoT, ISSR markers and DNA barcodingGenetic stability observed.[142]
Cicer arietinum
(Chickpea)		Organogenesis/
embryo axis, half-seed, axillary meristem, and cotyledonary node explant		SCoT and ISSR markersNo polymorphism was detected.[125]
Magnolia dealbataSomatic embryogenesis from zygotic embryoSSRGenetic integrity 90%.[87]
Cymbidium aloifolium
(Cymbidium)		Organogenesis/seedsSCoT, DAMD, and ISSR markersThe analysis of the in vitro-derived plantlets showed 86.87% genetic monomorphism and 13.13% polymorphism.[143]
Rhynchostylis retusa
(Punjab fig)		Organogenesis/
Capsule		RAPDHigh uniformity was observed among regenerated plants and mother plants.[144]
RAPD—random amplified polymorphic DNA, RFLP—restriction fragment length polymorphism, SSR—simple sequence repeat, DAMD—Directed Amplification of Minisatellite-region DNA, sequence-related amplified polymorphism (SRAP) markers, SCoT (Start Codon Targeted) markers, and SCAR—sequence characterized amplified region.
Table 3. List of reported epigenetic variations, their causes, and detection methods in major crops, fruit plants, and trees.
Table 3. List of reported epigenetic variations, their causes, and detection methods in major crops, fruit plants, and trees.
Plant SpeciesTissue Culture MethodDNA Methylation DetectionVariation ObservedReference
Zea mays
(Maize)		Embryogenic callus cultureBisulfite sequencingVariation observed[92]
Allium sativum
(Garlic)		Direct
Organogenesis, meristem tissue		AFLP, and
MASP		Genetic and epigenetic polymorphism under field growing conditions[34]
Vaccinium vitis-idaea
(Lingonberry)		Organogenesis/
Stem and leaf explants		MSAPMore methylation events are observed in vitro-derived plants than in those derived
from cuttings of plants		[93]
Coffea arabica
(Coffee)		Somatic embryogenesis regeneration/
Nodal segments		MASPGenetically stable[94]
X Triticosecale spp.
(Winter triticale)		Another Culture,
Anther		metAFLP51% of tissue culture-induced variation[156]
Fragaria x ananassa
(Strawberry)		Indirect organogenesis,
runner tips		WGSVariation observed[96]
Oryza sativa
(Rice)		Callus cultureBisulfite sequencingVariation observed[80]
Ananas comosus
(Pineapple)		Callus cultureWGBSVariation observed[97]
Malus domestica
(Apple)		Shoot cultureBisulfite sequencingVariation observed[153]
Humulus lupulus
(Hop)		Direct organogenesisMASPNo variation observed[157]
Hordeum vulgare
(Barley)		Somatic embryogenesisMet-AFLPPolymorphism observed[98]
Manihot esculenta
(Cassava)		Meristem micropropagationMASPVariation observed[158]
Hordeum vulgare
(Barley)		MicropropagationHPLCNo variation observed [159]
Cannabis sativaOrganogenesisGlobal epigenetic analysisDNA hypomethylation progressively occurred in micropropagated shoots compared to plants grown in the greenhouse, with the level of hypomethylation rising over extended periods of in vitro culture, potentially impacting gene expression and plant development[160]
Vaccinium angustifolium
(Lowbush Blueberry)		Micropropagation via softwood cuttings and tissue cultureMSAPVariation observed[95]
Saccharum spp.
(Sugarcane)		MicropropagationMSAPVariation observed[146]
Salix purpurea
(Basket willow)		Regenerated shoots (in vitro)WGBSExtensive methylation reprogramming observed during regeneration[161]
Dendrocalamus asper
(Giant Bamboo)		In vitro shoots (multiple passages)ELISA (5-mC quantification)Methylation decreased with subculturing, linked to somaclonal variation[99]
Zea mays (Maize)Callus cultureMSAP and HPLCNo variation observed[100]
Camellia sinensis (Tea)Various tissues (leaves, roots, etc.)HPLC with UV detectionObserved tissue-specific variations in DNA methylation levels across different tea plant tissues[148]
Lactuca sativa
(lettuce)		Callus cultureGlobal epigenetic analysisPolymorphism observed[101]
× Triticosecale
(Triticale)		Anther culturemetAFLPVariation observed[162]
MASP—methylation-sensitive amplified polymorphism, met-AFLP—methylation-sensitive AFLP, HPLC—high-performance liquid chromatography, WGBS—Whole-Genome Bisulfite Sequencing, and ELISA—Enzyme-Linked Immunosorbent Assay.
Table 4. List of some desirable somaclonal variants used in plant breeding programs.
Table 4. List of some desirable somaclonal variants used in plant breeding programs.
Common NameScientific NameMode of RegenerationSomaclonal VariantsReferences
RiceOryza sativaIndirect organogenesisDrought resistant (cv PR113)[35]
TomatoSolanum lycopersicumIndirect organogenesisHigh-yielding variety
(SE10, SE1, SS5)		[36]
EggplantSolanum melongenaIndirect organogenesisSalt stress-tolerant variant[37]
CucumberCucumis sativusIndirect organogenesisMore number of lateral shoots and the highest main shoot length (MSC 28)[38]
CarrotDaucus carotaIndirect organogenesisDrought resistant[39]
PomegranatePunica granatumIndirect organogenesis
(Callus formation)		Better fruit quality[170]
Garden TulipTulipa suaveolensDirect organogenesisDisease resistant[169]
BananaMusa sp.Indirect organogenesis
Callus formation (shoot tips)		Fusarium wilt-resistant variety[40]
Japanese MintMentha arvensisIndirect organogenesisIncreased quality[167]
BananaMusa sp.Direct organogenesisImproved resistance to Fusarium wilt and better bunch quality[171]
CottonGossypium hirsutumDirect organogenesisDrought-tolerant variety[172]
PotatoSolanum tuberosumDirect organogenesisIncreased resistance to viral diseases and improved tuber quality[168]
TomatoSolanum lycopersicumIndirect organogenesisImproved fruit size, shape, and resistance to Fusarium wilt[173]
RiceOryza sativaDirect organogenesisEnhanced resistance to rice blast and better grain quality[35]
SugarcaneSaccharum officinarumIndirect organogenesisIncreased sugar yield, fiber quality, stalk length, internode length, and disease resistance[165]
GrapeVitis viniferaDirect
Organogenesis		Seedless fruit variety[41]
PineappleAnanas comosusSomatic embryogenesisImproved fruit size and resistance to pests[174]
WheatTriticum aestivumSomatic embryogenesisIncreased resistance to wheat rust and spot blotch disease, enhanced grain size[175]
CarnationDianthus caryophyllusIndirect organogenesisResistant to Fusarium oxysporum[176]
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Majumder, S.; Igamberdiev, A.U.; Debnath, S.C. Somaclonal Variation and Clonal Fidelity in Commercial Micropropagation: Challenges and Perspectives. Agronomy 2025, 15, 1489. https://doi.org/10.3390/agronomy15061489

AMA Style

Majumder S, Igamberdiev AU, Debnath SC. Somaclonal Variation and Clonal Fidelity in Commercial Micropropagation: Challenges and Perspectives. Agronomy. 2025; 15(6):1489. https://doi.org/10.3390/agronomy15061489

Chicago/Turabian Style

Majumder, Sweety, Abir U. Igamberdiev, and Samir C. Debnath. 2025. "Somaclonal Variation and Clonal Fidelity in Commercial Micropropagation: Challenges and Perspectives" Agronomy 15, no. 6: 1489. https://doi.org/10.3390/agronomy15061489

APA Style

Majumder, S., Igamberdiev, A. U., & Debnath, S. C. (2025). Somaclonal Variation and Clonal Fidelity in Commercial Micropropagation: Challenges and Perspectives. Agronomy, 15(6), 1489. https://doi.org/10.3390/agronomy15061489

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