Rejuvenation through Somatic Embryogenesis: Epigenetic and Telomeric Resetting in Melia volkensii

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Werbrouck This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7941105/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Feb, 2026 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted 4 You are reading this latest preprint version Abstract Melia volkensii is a drought-tolerant hardwood of high ecological and economic value in arid and semi-arid Africa, but conventional propagation is limited by poor seed viability and recalcitrance to regeneration. To explore molecular mechanisms of rejuvenation, we investigated telomere length and global DNA methylation in tissues derived from different propagation routes: adult micropropagation, juvenile seedlings, somatic embryogenesis, adventitious shoots, and root suckers. Telomere length, measured by Southern hybridization, varied significantly among tissue types. Adult micropropagated tissues showed the shortest telomeres (mean 5.0 kb), while juvenile seedlings (8.5 kb) and root suckers (8.9 kb) had markedly longer telomeres, suggesting a juvenile-like state. Somatic embryos (7.4 kb) and adventitious shoots (7.8 kb) exhibited intermediate lengths, indicating partial rejuvenation during regeneration. Global DNA methylation, quantified by ELISA, further distinguished somatic embryos, which showed the lowest absorbance (~ 2.0) and fold change (~ 0.88), significantly reduced compared to juvenile (~ 0.96), adventitious (~ 0.96), and adult tissues (~ 1.0). These findings demonstrate a strong link between telomere elongation, reduced 5-methylcytosine levels, and cellular rejuvenation in M. volkensii , offering valuable insights for optimizing clonal propagation and ensuring long-term genetic stability in forestry applications. DNA methylation In vitro rejuvenation Micropropagation Melia volkensii Somatic embryogenesis Telomere length Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction From the dawn of human civilization, the number of trees on Earth has declined by about 46% (Crowther et al 2015 ). The global reduction in forest cover and the loss of tree populations have raised serious concerns concerning ecosystem health and the ability to support human livelihoods and well-being (FAO 2020 ). Tree planting is increasingly being adopted as a strategy to combat global change, including mitigation, adaptation, and restoration (Clark et al 2023 ).The main challenges hampering tree planting programs are twofold: the limited availability of tree planting materials, which does not meet demand (Clark et al 2023 ), and the restriction of traditional propagation methods to only a few tree species. This reliance leads to monoculture plantations, significantly altering local biodiversity (Bennett 2015 ). To address these issues, micropropagation has emerged as a promising solution, allowing for the rapid multiplication of a wide variety of tree species in controlled environments. It is advantageous to select explants from elite trees, as they possess superior traits such as faster growth, disease resistance, drought tolerance and high-quality timber. Micropropagation enables the clonal propagation of these trees, preserving and multiplying their desirable genetic traits. However, as trees undergo ontogenic phase transitions and mature, their capacity for both micropropagation and rooting diminishes (von Adrekas 2000). While a successful plant regeneration pathway via somatic embryogenesis is considered an elegant tool for rejuvenation, the ability of leaf and shoot explants from many tree species to undergo somatic embryogenesis declines significantly with maturity (Mignon & Werbrouck 2018 ). This study investigated whether plants produced by somatic embryogenesis are rejuvenated in Melia volkensii , a tree native to Kenya, Somalia, and Tanzania, and a member of the Meliaceae family. It typically grows in dry bushland and wooded grasslands. Due to its rapid growth and notable drought resistance, M. volkensii is a valuable resource for reforestation initiatives in arid regions. However, the rising need for reforestation has not been sufficiently met by traditional seedling production. Given these challenges, plant tissue culture techniques have been explored for M. volkensii propagation over the past decade (Werbrouck 2024 ). Telomeres are nucleoprotein structures located at the termini of eukaryotic chromosomes that safeguard their structural integrity by preventing degradation and end-to-end fusion during replication. Without these protective caps, DNA damage could trigger programmed cell death or cellular senescence. The enzyme telomerase is a specialized reverse transcriptase that replicates telomeres, a function that cannot be performed by the cell's conventional DNA replication machinery. In the absence of telomerase activity, telomeres progressively shorten with each cell division, ultimately inducing senescence upon reaching a critically short length (Aronen et al., 2021 ). A consistent pattern has been observed where relative telomere length decreases with increasing ontogenic age, as documented in species such as almond trees (D’Amico-Willman et al., 2021 ). In addition, DNA methylation is a crucial epigenetic modification within eukaryotic genomes that involves the addition of a methyl group to the fifth carbon of a cytosine residue, thereby forming 5-methylcytosine (5mC). This process is fundamental for regulating gene expression, ensuring genome stability, and guiding developmental processes (Wyatt, 1951 ). DNA methylation plays a role in regulating plant maturity, particularly in trees (Thomas 2013 ). Multiple studies have shown that cytosine DNA methylation levels are higher in adult plant tissues compared to juvenile tissues and exhibit a positive correlation with ontogenic age (Huang et al., 2012 ; Yuan et al., 2014 ). Using human leukocyte DNA, Buxton et al. ( 2014 ) examined the relationship between telomere length, DNA methylation, and aging. Their study found that shorter telomeres and hypomethylation increase with age. Telomere shortening and DNA methylation reduction may be key indicators of human aging. While biomarkers like telomere length (D'Amico-Willman 2020) and DNA methylation (Dubrovina & Kiselev 2016 ), have been independently studied to understand ontogenetic aging in plants, an unambiguous relationship has not been established. To gain further insight, we measured telomere length and DNA methylation levels in the same tree materials to determine their roles in tree ontogenetic aging and rejuvenation. Telomere dynamics and cytosine DNA methylation both serve as key regulators of genome stability and cellular aging, with changes in these mechanisms often marking developmental phase transitions. 2. Materials and Methods 2.1 Plant material 2.1.1. Adult Mature Plant Elite Melia volkensii trees (~ 15 years old) were obtained from Better Globe Forestry (Kenya) and used as donor material. Young nodal segments bearing axillary buds were excised and used as explants. Explants were surface sterilized by rinsing in 70% ethanol for 30 s, followed by incubation in 10% (v/v) commercial bleach solution (8° active chlorine) for 15 min. The explants were then rinsed three times with sterile distilled water before being inoculated onto the culture medium. Sterilized explants were cultured on Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) supplemented with 20 g/L sucrose, 7 g/L Plant agar, and 10 µM meta-topolin riboside (mTR). The medium pH of the medium was adjusted to 5.4 prior to autoclave. The cultures were maintained in a growth chamber at 22°C under cool white fluorescent light (Philips) with a light intensity of 40 µmol m⁻² s⁻¹ and a 16-hour light/dark photoperiod. Resulting shoots were subcultured onto fresh medium every four weeks to sustain growth and multiplication. 2.1.2. Juvenile Plant Seeds from the same elite donor tree were surface sterilized by rinsing in 70% ethanol for 30 s, followed by incubation in a 10% (v/v) commercial bleach solution (8° active chlorine) for 15 min, and subsequently rinsed three times with sterile distilled water before inoculation onto culture medium. To enhance germination, the seed coat was carefully scarified under sterile conditions before inoculation. The sterilized seeds were germinated on MS medium supplemented with 20 g/L sucrose and 7 g/L Plant agar. The medium pH was adjusted to 5.4 prior to autoclaving. The healthiest seedlings were subsequently transferred to the same medium, with the addition of 10 µM meta-topolin riboside (mTR). All cultures were maintained under identical conditions: a temperature of 22°C, a light intensity of 40 µmol m⁻² s⁻¹, and a 16-hour photoperiod. 2.1.3. Plants from Adventitious Shoots Leaf explants from the same mature, elite plant were cultured in Petri dishes containing 10 mL MS medium supplemented with 20 g/L sucrose, 7 g/L Plant agar, and 10 µM thidiazuron (TDZ) to induce adventitious shoot formation. The cultures were incubated in a growth chamber at 22°C under a 16-hour photoperiod with cool white fluorescent light at an intensity of 40 µmol m⁻² s⁻¹. After six weeks, the developing shoots were subcultured onto fresh MS medium containing the same concentrations of sucrose and Plant agar, but with the addition of 10 µM meta-topolin riboside (mTR) instead of TDZ. These shoots were maintained for further growth under the same environmental conditions. 2.1.4. Plants from Somatic Embryogenesis Somatic embryogenesis was induced following a modified protocol based on Mulanda et al. ( 2012 ). Leaf explants from the same adult plants were incubated in complete darkness on 10 mL MS medium supplemented with 20 g/L sucrose, 7 g/L Plant agar, and 10 µM TDZ to promote embryogenic callus formation. Emerging somatic embryos were then germinated on MS medium containing 20 g/L sucrose, 10 µM meta-topolin riboside (mTR), 5 µM indole-3-butyric acid (IBA), and 7 g/L Plant agar, with pH adjusted to 5.4. The cultures were maintained at 22°C under 40 µmol m⁻² s⁻¹ cool white fluorescent light and a 16-hour photoperiod. 2.1.5. Plants from Root Suckers Root suckers were collected from the same elite tree at Better Globe plantation, Kenya. The leaves were dried and used for DNA extraction and Southern blot analysis of telomeric repeats 2.2 Sampling for DNA Isolation Young, fully expanded leaves were sampled from (i) micropropagated shoots from elite adult trees, (ii) seed-derived juvenile plants, (iii) plants regenerated from adventitious shoots, and (iv) plants regenerated via somatic embryogenesis. For all material types, the same developmental stage of leaf tissue was chosen to minimize variability. Leaves were harvested in the morning, immediately frozen in liquid nitrogen, and stored at − 80°C until DNA extraction. Dried leaves from root sucker sample were also used for DNA extraction. 2.3 DNA extraction and Southern blot analysis of telomeric repeats Total genomic DNA was extracted using a modified protocol originally developed by Lodhi et al. ( 1994 ), as outlined by Valjakka et al. ( 2000 ) and Aronen et al. ( 2003 ). Southern blot hybridization was carried out following the procedure of Kilian et al. ( 1995 ), incorporating modifications described by Aronen and Ryynänen ( 2012 ). A synthetic telomeric sequence was amplified via PCR using primers T1 (5′-TTTAGGG-3′) and T2 (5′-CCCTAAA-3′), as described by Cox et al. ( 1993 ), and served as a positive control. Hybridization signals were detected via chemiluminescence using the manufacturer's protocol (Roche Diagnostics GmbH), scanned with an AlphaImager Imaging System (Alpha Innotech Co./ProteinSimple, CA, USA), and analysed with AlphaEase®FC software, using a digoxigenin-labelled molecular weight marker. To confirm the terminal location of the detected telomeric sequences, genomic DNA was treated with BAL-31 exonuclease as described by Aronen and Ryynänen ( 2012 ), followed by Southern blot hybridization analysis. 2.4 Quantification of global DNA methylation Young leaves were harvested from four plant sources: (i) micropropagated shoots derived from elite adult Melia volkensii trees (~ 15 years old), (ii) seed-derived juvenile plants, (iii) adventitious shoot-derived plants, and (iv) plants regenerated via somatic embryogenesis. Total genomic DNA was extracted using the Invisorb Spin Plant Mini Kit (Stratec) according to the manufacturer’s protocol. DNA concentration was performed using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). MethylFlash™ Global DNA Methylation (5-mC) ELISA Easy Kit (Colorimetric) (Epigentek, NY) was used according to the manufacturer’s instructions. One hundred nanograms of purified DNA from each sample were added to an ELISA plate where the methylated fraction of DNA was detected using capturing and detecting antibodies. Optical density (OD) intensity at 450 nm was read with a microplate spectrophotometer and this was proportional to the amount of methylated DNA. For quantification of global DNA methylation levels, a standard curve was generated using methylated DNA standards provided in the kit. The value for each sample was calculated as a ratio of the OD of the sample relative to the OD of the standard, after subtracting the negative control readings. The assay was performed in duplicate per biological replicate, meaning that per accession and condition, six samples were analyzed. 2.5 Statistics Analysis For statistical analysis, mean telomere length values from biological replicates were subjected to one-way analysis of variance (ANOVA) using Python 3.11 (SciPy v1.10.1 and Seaborn v0.12.2). ANOVA tested the null hypothesis that there were no differences in mean telomere length across tissue types. The significance level was set at α = 0.05. Visualizations were created using Seaborn and Matplotlib libraries. Post-hoc tests were not applied; thus, the ANOVA indicates only whether a significant difference exists among groups, not between specific pairs. The methylation experiment was carried out with 11 explants for different distinct tissue types. Data was analyzed using SPSS 25.0 (SPSS Inc. USA). The independent sample t-test was used. The other data were analyzed by analysis of variance (ANOVA) followed by Duncan's new multiple range (DMR) test to separate the mean differences (Tallarida and Murray 1987 ). Comparisons of each parameter were conducted by Dunnetz’s t-tests (P < 0.05) (Zar 1984 ). 3. Results 3.1 Telomere Length Variation Across Tissue Types Telomere length was quantified in various tissue types, including adult leaves (Adult 1 and Adult 2), juvenile seedlings (Juvenile 1 and Juvenile 2), somatic embryos (SE 1 and SE 2), adventitious shoots (Adv Shoot 1 and Adv Shoot 2), and root suckers. Southern hybridization analysis revealed distinct differences in telomere length among these distinct tissue types. Figure 1 displays typical Southern hybridization images used to assess and quantify telomeric length. Juvenile micropropagated tissues (lanes 3, 4) and somatic embryogenesis-derived tissues (lanes 5, 6) exhibited longer telomeres compared to adult micropropagated tissues (lanes 1, 2). Adventitious shoot tissues (lanes 7, 8) showed moderate telomere lengths, while root sucker tissues (lane 9) displayed telomere length patterns like those of juvenile tissues, suggesting a partial reversion to a juvenile-like state. Comparison of telomeric lengths for different tissues (Fig. 2 ) revealed a tendency toward variation in these tissues. Quantitative analysis of mean telomere length in different Melia volkensii tissue types revealed notable variation associated with developmental stage and propagation method. Adult micropropagated tissues exhibited the shortest telomeres, with a mean length of 5.1 kb, indicating a more aged or mature state. In contrast, juvenile micropropagated tissues showed significantly longer telomeres (8.5 kb), suggesting a more youthful genomic profile. Tissues derived from somatic embryogenesis (7.4 kb) and adventitious shoots (7.8 kb) also displayed relatively long telomeres, indicating partial rejuvenation during the regeneration process. Root sucker tissues had the longest telomeres (8.9 kb), further supporting the hypothesis that naturally regenerated tissues may retain or regain juvenile characteristics. These results suggest a strong association between telomere length and rejuvenation potential in clonally propagated Melia volkensii tissues. A one-way analysis of variance (ANOVA) was conducted to assess statistical differences in telomere lengths among tissue types. The results indicated a significant effect of tissue type on telomere length (F (4, 8) = 28.03, p = 0.0035). This suggests that telomere length is significantly influenced by developmental stage and regeneration methods (Fig. 3 ). Notably, tissues derived from somatic embryos and adventitious shoots exhibited intermediate telomere lengths, suggesting a partial rejuvenation effect compared to adult tissues. These findings align with the hypothesis that in vitro regeneration methods may restore telomere length, potentially reversing age-related genomic erosion. 3.2 Global DNA methylation The ELISA-based quantification of global DNA methylation, assessed through absorbance at 410 nm and fold change in 5-methylcytosine (5-mC) levels, reveals significant epigenetic variation between SE, juvenile shoots, adventitious shoots, and mature tissues. In the graph measuring absorbance at 410 nm (Fig. 4 ), which correlates directly with the amount of methylated cytosine, SE tissues display the lowest absorbance (~ 2.0), significantly lower than the values observed in the other tissues (all ~ 2.2 or higher). This trend is further confirmed in Fig. 4 , which presents the fold change in global methylation. Here, plants from somatic embryo (SE) again show the lowest value (~ 0.88), while juvenile and adventitious shoots display a fold change around 0.96, and mature tissues reach a baseline level of 1.0. The statistical annotation reinforces this distinction, as SEs are again significantly different from the other tissues. Taken together, both graphs consistently demonstrate that somatic embryos undergo a substantial reduction in global DNA methylation, while tissues that are either naturally developed (juvenile, mature) or regenerated through organogenesis (adventitious shoots) maintain higher methylation levels. 4. Discussion 4.1 Telomere Length as an Indicator of Developmental Stage and Rejuvenation Capacity Somatic embryogenesis (SE) is recognized as the most effective approach for rejuvenating plants, especially mature trees, as it produces plantlets with juvenile characteristics by effectively resetting the embryonic developmental program, even when starting from mature donor tissues (Shmakov et al., 2024 ). Our findings demonstrate a clear relationship between telomere length and the ontogenic or regenerative origin of the tissue. As anticipated, micropropagated adult tissues had the shortest telomeres, reflecting cellular aging and extensive mitotic divisions. In contrast, juvenile tissues and root suckers possessed notably longer telomeres, indicating a more juvenile genomic state. Tissues regenerated through somatic embryogenesis and adventitious shoots from elite adult trees showed intermediate telomere lengths, suggesting partial rejuvenation during invitro regeneration. Plant roots are generally considered as juvenile (Crawford et al., 2015 ), so shoots originating from root suckers are juvenile as expected. Our data confirm this, as root sucker tissues from mature trees exhibited longer telomere lengths compared to those derived directly from the shoots. Previous studies have reported mixed results: Moriguchi et al. ( 2005 ) found no significant telomere length differences across tissues in apple and cherry trees over several years, while Song et al. ( 2010 ) and Aronen & Ryynänen ( 2014 ) observed stable telomere lengths regardless of age in ginkgo and silver birch, respectively. Conversely, Zhang et al. ( 2025 ) documented a decline in telomere length with age in leaves of younger Platycladus orientalis . Our study similarly shows shorter telomeres in adult Melia volkensii tissues compared to juvenile and root sucker tissues, implying ontogenic related telomere shortening. This effect is well documented in species like Larix decidua (Kretzschmar & Ewald, 1994 ), Sequoia (Arnaud, 1993), Pinus (Prehn et al., 2003 ), and Picea (Zarei et al., 2020 ). Correspondingly, adventitious shoots from adult tissues in our study showed increased telomere length. On the other hand, prolonged duration of micropropagation or somatic embryogenesis, or even stressful conditions during micropropagation, have shown to shorten telomeres in silver birch (Aronen & Ryynänen 2014 ) and Norway spruce (Aronen et al 2021 ), and should thus be avoided. So, in addition to developmental stage, both stress exposure and duration in culture can influence telomere dynamics, and these factors should be considered in the interpretation of the present Melia volkensii material. Furthermore, tree genotype has been shown to affect telomere length (Aronen & Ryynänen, 2012 ; Aronen & Ryynänen, 2014 ; Aronen et al., 2021 ). However, in the present study the same (related) genotypes were used when comparing different tissue materials, indicating that the observed differences more likely reflect tissue type–specific or developmental effects rather than genotypic variation. 4.2 Decreased Global DNA Methylation in Somatic Embryogenesis-Derived Tissues Alongside telomere analysis, ELISA-based quantification of 5-methylcytosine (5-mC) revealed a marked decrease in global DNA methylation in somatic embryo-derived tissues compared to juvenile, adventitious, and mature tissues. This suggests a unique epigenetic state associated with somatic embryogenesis, likely reflecting extensive reprogramming during this developmental route. The significant reduction in methylation in somatic embryo-derived tissues aligns with findings in other plants where somatic embryogenesis is linked to methylation changes. For instance, oil palm somatic embryos with the mantled phenotype show hypomethylation compared to normal embryos (Jaligot et al., 2000 ). Similarly, a transient methylation decrease is critical to initiating somatic embryogenesis in chestnut (Viejo et al., 2010 ), with dynamic methylation fluctuations observed in early stages of somatic embryogenesis in Coffea canephora (Nic-Can et al., 2013 ). Maritime pine also exhibits developmental hypomethylation during somatic embryogenesis (Trontin et al., 2025 ). Juvenile and mature tissues, representing stable developmental stages, typically maintain higher and more stable global methylation important for tissue identity and gene silencing (Li et al 2023 ). Adventitious tissues, although also cultured in vitro, may follow distinct epigenetic pathways compared to somatic embryos, lacking the same level of epigenetic resetting. This methylation decrease could have several consequences: it might contribute to phenotypic variation often seen in somatic embryogenesis-derived plants, including somaclonal variation, by altering gene expression and affecting growth, development, and stress responses (Phillips et al., 1994 ). Additionally, this hypomethylated state might increase sensitivity to environmental stress since DNA methylation is crucial for genome stability and defense (Boyko & Igor, 2008). No significant methylation differences were observed between adult, juvenile, and adventitious tissues. Previous reports indicate higher cytosine methylation in adult tissues increasing with age (Huang et al., 2012 ; Yuan et al., 2014 ). Micropropagation often elevates methylation levels, contributing to epigenetic variation (Ghosh et al., 2021 ), which may explain our observations. Moreover, methylation patterns vary by species, tissue type, organelle, and age (Vanyushin, 2006 ), so species-specific factors might influence differences between adult and seed-derived juvenile tissues. High-resolution methods such as whole-genome bisulfite sequencing (WGBS) are recommended for future studies to pinpoint specific differentially methylated regions (DMRs) and their associated genes, enhancing understanding of how methylation changes relate to gene expression and somatic embryo development. Examining methylation stability during plant acclimatization and maturation will also be important to assess the long-term epigenetic fidelity of somatic embryogenesis-derived plants. In summary, our data reveals a significant reduction in global DNA methylation in somatic embryo-derived tissues, indicating extensive epigenetic reprogramming during somatic embryogenesis. This epigenetic signature may be critical for developmental plasticity but also calls for further study of its long-term developmental effects. 4.3 Combined Telomere and Methylation Profiles The presence of elongated telomeres alongside hypomethylation in regenerated tissues—especially those from somatic embryogenesis—points to a coordinated molecular rejuvenation process. Although telomeres themselves are usually not methylated, DNA methylation in sub telomeric regions influences telomere length and stability, thereby affecting cell development and differentiation (Vega-Vaquero et al., 2016 ). It is plausible that hypomethylation in somatic embryogenesis tissues loosens chromatin near telomeres, allowing partial telomere extension. Interestingly, adventitious shoots exhibited both longer telomeres and higher methylation compared to somatic embryos, possibly reflecting differences in their cellular origin or regeneration mechanisms (dedifferentiation versus transdifferentiation) that influence telomere and epigenetic resetting. 4.4 Implications for Clonal Propagation and Conservation Practically, these findings have important implications for clonal propagation of Melia volkensii . Tissues regenerated through somatic embryogenesis and adventitious shoots regain juvenile molecular features that may enhance rooting, growth, and field adaptability. Root suckers, with the longest telomeres, represent promising explants for producing rejuvenated clones. Moreover, the observed epigenetic flexibility highlights the need to monitor methylation and telomere dynamics during tissue culture, incorporating these molecular markers into quality control for elite clone selection in forestry biotechnology and conservation efforts. 4.5 Limitations and Future Perspectives While this study establishes links between telomere length, methylation status, and tissue origin in Melia volkensii , limitations include the focus on global methylation and average telomere length without locus- or cell-type-specific resolution, and a small sample number. Future research with more extensive materials or by employing high-resolution bisulfite sequencing and single-cell telomere length assays could provide deeper mechanistic insight. Additionally, investigating telomerase activity and telomere-associated proteins (e.g., TERT, TRF-like proteins) would clarify pathways involved in telomere resetting. Long-term studies tracking telomere dynamics through propagation cycles and field growth are also warranted. 5. Conclusion In conclusion, telomere length and global DNA methylation in Melia volkensii are influenced by tissue ontogenic stage and regeneration method. These molecular markers provide critical insights into rejuvenation potential and can guide clonal propagation strategies aimed at sustainable forestry and conservation of this ecologically vital species. Declarations Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethics declaration Not Applicable. Funding This study was funded by Erasmus + Swagata program of European Union and the Transnational Access to Research Infrastructures Activity in the 7th Framework Programme of the European Union under the Trees 4 Future project no. 284181 for conducting the research (at Punkaharju Research Unit, Finnish Forest Research Institute Punkaharju, Finland). Author Contributions NB: Writing – original draft, Conceptualization, Methodology; AV: Methodology TJ: Writing – review & editing, Conceptualization, Supervision; SPOW: Writing – original draft, Conceptualization, Supervision; TM: Supervision Acknowledgments The authors acknowledge Mrs. Aila Viinanen for valuable technical assistance in realization of Southern analyses for telomere length measurement Data Availability Statement The data supporting the findings of this study are available within the manuscript and its supplementary materials. 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New Phytol 197:696–711. https://doi.org/10.1111/nph.12047 Trontin JF, Sow MD, Delaunay A, Modesto I, Teyssier C, Reymond I, Canlet F, Boizot N, Le Metté C, Gibert A, Chaparro C, Daviaud C, Tost J, Miguel C, Lelu-Walter MA, Maury S (2025) Epigenetic memory of temperature sensed during somatic embryo maturation in 2-yr-old maritime pine trees. Plant Physiol 197:kiae600. https://doi.org/10.1093/plphys/kiae600 Valjakka M, Aronen T, Kangasjärvi J, Vapaavuori E, Häggman H (2000) Genetic transformation of silver birch ( Betula pendula ) by particle bombardment. Tree Physiol 20:607–613. https://doi.org/10.1093/treephys/20.9.607 Vanyushin BF (2006) DNA methylation in plants. In: Doerfler W, Böhm P (eds) DNA Methylation: Basic Mechanisms. Curr Top Microbiol Immunol 301:67–122. Springer, Berlin. https://doi.org/10.1007/3-540-31390-7_4 Vega-Vaquero A, Bonora G, Morselli M, Vaquero-Sedas MI, Rubbi L, Pellegrini M, Vega-Palas MA (2016) Novel features of telomere biology revealed by the absence of telomeric DNA methylation. Genome Res 26:1047–1056. https://doi.org/10.1101/gr.202465.115 Viejo M, Rodríguez R, Valledor L, Pérez M, Cañal MJ, Hasbún R (2010) DNA methylation during sexual embryogenesis and implications on the induction of somatic embryogenesis in Castanea sativa Miller. Sex Plant Reprod 23:315–323. https://doi.org/10.1007/s00497-010-0145-9 von Aderkas P, Bonga JM (2000) Influencing micropropagation and somatic embryogenesis in mature trees by manipulation of phase change, stress and culture environment. Tree Physiol 20:921–928. https://doi.org/10.1093/treephys/20.14.921 Werbrouck SPO (2024) Biotechnological advances for Melia volkensii , a climate-resilient tree for reforestation in East Africa. Book of Proceedings 20 Wyatt GR (1951) Recognition and estimation of 5-methylcytosine in nucleic acids. Biochem J 48:581–584. https://doi.org/10.1042/bj0480581 Yuan JL, Sun HM, Guo GP, Yue JJ, Gu XP (2014) Correlation between DNA methylation and chronological age of moso bamboo ( Phyllostachys heterocycla var. pubescens ). Bot Stud 55:4. https://doi.org/10.1186/1999-3110-55-4 Zar JH (1984) Biostatistical Analysis. Prentice-Hall International, Sydney Zarei M, Salehi H, Jowkar A (2020) Controlling the barriers of cloning mature Picea abies (L.) H. Karst. via tissue culture and co-cultivation with Agrobacterium rhizogenes. Trees 34:637–648. https://doi.org/10.1007/s00468-019-01945-z Zhang Y, Yu X, Zhang S (2025) Analysis of the relationships among telomere-associated protein-encoding gene expression, tree age and telomere length in Platycladus orientalis (L.) Franco. Tree Genet Genomes 21:23. https://doi.org/10.1007/s11295-025-01758-y Cite Share Download PDF Status: Published Journal Publication published 14 Feb, 2026 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted Reviewers agreed at journal 11 Nov, 2025 Reviewers invited by journal 31 Oct, 2025 Editor assigned by journal 29 Oct, 2025 First submitted to journal 27 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7941105","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":538182412,"identity":"cb9fa0da-86ff-425b-9202-e96094f0cc15","order_by":0,"name":"Nandini Bhogar Suresh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYDACCR4wJQciDjwgRYsxWEsCKVoSG0AkUVrMpXsPPvxScy99ftjhh0Bb7OR0GwhosZxzLtlY5lhx7sbbaQZALcnGZgcIaDG4kWMmLcGWkLtxdgJIy4HEbcRp+ZeQbjg7/QPxWiQ/tiUkyEvnEGvLnTPGxox9CYYbpHMKDiQYEOOX2z2GD398S5CXn52++cOHCjs5glpAgBkUNQZglQZEKAcBxh9AQr6BSNWjYBSMglEw8gAApbtHAgYMQNUAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-4331-5128","institution":"Ghent University: Universiteit Gent","correspondingAuthor":true,"prefix":"","firstName":"Nandini","middleName":"Bhogar","lastName":"Suresh","suffix":""},{"id":538182413,"identity":"b58d680c-6d1e-4688-8b72-cea0c1c70ae1","order_by":1,"name":"Tuija Aronen","email":"","orcid":"","institution":"Natural Resources Institute Finland Savonlinna: Luonnonvarakeskus Savonlinna","correspondingAuthor":false,"prefix":"","firstName":"Tuija","middleName":"","lastName":"Aronen","suffix":""},{"id":538182414,"identity":"39faf09a-6e42-4298-bf29-fbfac0f24342","order_by":2,"name":"Titus Magomere","email":"","orcid":"","institution":"Kenyatta University","correspondingAuthor":false,"prefix":"","firstName":"Titus","middleName":"","lastName":"Magomere","suffix":""},{"id":538182415,"identity":"52716a0a-6394-4827-a302-6fdb8b7be243","order_by":3,"name":"Stefaan P.O. 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17:49:33","extension":"xml","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":109392,"visible":true,"origin":"","legend":"","description":"","filename":"PCTOD25007950structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7941105/v1/fbf79d7f20ff9e25e7156716.xml"},{"id":95760488,"identity":"7e7ad2e3-d32b-4652-b825-88bd333a9a90","added_by":"auto","created_at":"2025-11-12 17:49:33","extension":"html","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":117545,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7941105/v1/9f7395498002f6a95f451f74.html"},{"id":95760473,"identity":"d82257e6-d693-40f8-b618-e97dff4ce8e7","added_by":"auto","created_at":"2025-11-12 17:49:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":292330,"visible":true,"origin":"","legend":"\u003cp\u003eSouthern hybridization shows telomere length in various types of \u003cem\u003eMelia volkensii\u003c/em\u003eclones. 1. 2. Adult micropropagated tissue 3. 4. Juvenile micropropagated tissue 5.6. Somatic embryogenesis tissue 7. 8. Adventitious shoot tissue 9. Root sucker. The unnamed well did not run properly.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7941105/v1/8bcfa8326c6b7a162d46f8f1.png"},{"id":95760475,"identity":"0b7d41bc-3cf7-4eaa-a879-4daafa1415dd","added_by":"auto","created_at":"2025-11-12 17:49:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":82314,"visible":true,"origin":"","legend":"\u003cp\u003eLength of telomeric repeats of \u003cem\u003eMelia volkensii \u003c/em\u003etissue types, expressed as molecular weight (bp). The minimum, maximum, and average size of telomere lengths are shown. \"Adult\" refers to tissue from adult micropropagated plants, \"Juvenile\" to tissue from juvenile seedlings, \"SE\" to tissue from plants generated by somatic embryogenesis, \"Adv shoot\" to tissue from plants generated by adventitious shooting, and \"Root sucker\" to tissue from plants that have outgrown from the roots of an adult tree\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7941105/v1/390e8e37dfaab73061bc57df.png"},{"id":95801945,"identity":"2dbbb119-f8f8-4299-961f-46300ae2dd84","added_by":"auto","created_at":"2025-11-13 08:26:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":82255,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eTelomere length variation across tissue types.\u003c/em\u003e Boxplots show the distribution of mean telomere lengths (in base pairs) for nine different sample types: mature adult leaves (Adult 1 \u0026amp; 2), juvenile seedlings (Juvenile 1 \u0026amp; 2), somatic embryos (SE 1 \u0026amp; 2), adventitious shoots (Adv Shoot 1 \u0026amp; 2), and root suckers. Individual biological replicate means are overlaid as black dots\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7941105/v1/80cd83f5eca08a82c5653748.png"},{"id":95760477,"identity":"d0646e05-7a7c-4711-bbf6-99faa0aae447","added_by":"auto","created_at":"2025-11-12 17:49:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":23547,"visible":true,"origin":"","legend":"\u003cp\u003eFold change in global DNA Methylation in different tissue of \u003cem\u003eMelia volkensii\u003c/em\u003e (bars represent standard error of the mean). Means with the same letter are not significantly different according to the independent samples t-test (p0,01).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7941105/v1/1e8849237521c02ccbc4abd9.png"},{"id":102785743,"identity":"4b3c9a2f-f9a7-4a69-abec-1b0b5aca1cd1","added_by":"auto","created_at":"2026-02-16 16:09:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1237590,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7941105/v1/2d295ccd-ffe5-4242-892d-af44a7c7542a.pdf"}],"financialInterests":"","formattedTitle":"Rejuvenation through Somatic Embryogenesis: Epigenetic and Telomeric Resetting in Melia volkensii","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eFrom the dawn of human civilization, the number of trees on Earth has declined by about 46% (Crowther et al \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The global reduction in forest cover and the loss of tree populations have raised serious concerns concerning ecosystem health and the ability to support human livelihoods and well-being (FAO \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Tree planting is increasingly being adopted as a strategy to combat global change, including mitigation, adaptation, and restoration (Clark et al \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).The main challenges hampering tree planting programs are twofold: the limited availability of tree planting materials, which does not meet demand (Clark et al \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and the restriction of traditional propagation methods to only a few tree species. This reliance leads to monoculture plantations, significantly altering local biodiversity (Bennett \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). To address these issues, micropropagation has emerged as a promising solution, allowing for the rapid multiplication of a wide variety of tree species in controlled environments. It is advantageous to select explants from elite trees, as they possess superior traits such as faster growth, disease resistance, drought tolerance and high-quality timber. Micropropagation enables the clonal propagation of these trees, preserving and multiplying their desirable genetic traits. However, as trees undergo ontogenic phase transitions and mature, their capacity for both micropropagation and rooting diminishes (von Adrekas 2000). While a successful plant regeneration pathway via somatic embryogenesis is considered an elegant tool for rejuvenation, the ability of leaf and shoot explants from many tree species to undergo somatic embryogenesis declines significantly with maturity (Mignon \u0026amp; Werbrouck \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis study investigated whether plants produced by somatic embryogenesis are rejuvenated in \u003cem\u003eMelia volkensii\u003c/em\u003e, a tree native to Kenya, Somalia, and Tanzania, and a member of the Meliaceae family. It typically grows in dry bushland and wooded grasslands. Due to its rapid growth and notable drought resistance, \u003cem\u003eM. volkensii\u003c/em\u003e is a valuable resource for reforestation initiatives in arid regions. However, the rising need for reforestation has not been sufficiently met by traditional seedling production. Given these challenges, plant tissue culture techniques have been explored for \u003cem\u003eM. volkensii\u003c/em\u003e propagation over the past decade (Werbrouck \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTelomeres are nucleoprotein structures located at the termini of eukaryotic chromosomes that safeguard their structural integrity by preventing degradation and end-to-end fusion during replication. Without these protective caps, DNA damage could trigger programmed cell death or cellular senescence. The enzyme telomerase is a specialized reverse transcriptase that replicates telomeres, a function that cannot be performed by the cell's conventional DNA replication machinery. In the absence of telomerase activity, telomeres progressively shorten with each cell division, ultimately inducing senescence upon reaching a critically short length (Aronen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). A consistent pattern has been observed where relative telomere length decreases with increasing ontogenic age, as documented in species such as almond trees (D\u0026rsquo;Amico-Willman et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn addition, DNA methylation is a crucial epigenetic modification within eukaryotic genomes that involves the addition of a methyl group to the fifth carbon of a cytosine residue, thereby forming 5-methylcytosine (5mC). This process is fundamental for regulating gene expression, ensuring genome stability, and guiding developmental processes (Wyatt, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1951\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDNA methylation plays a role in regulating plant maturity, particularly in trees (Thomas \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Multiple studies have shown that cytosine DNA methylation levels are higher in adult plant tissues compared to juvenile tissues and exhibit a positive correlation with ontogenic age (Huang et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yuan et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Using human leukocyte DNA, Buxton et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) examined the relationship between telomere length, DNA methylation, and aging. Their study found that shorter telomeres and hypomethylation increase with age. Telomere shortening and DNA methylation reduction may be key indicators of human aging. While biomarkers like telomere length (D'Amico-Willman 2020) and DNA methylation (Dubrovina \u0026amp; Kiselev \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), have been independently studied to understand ontogenetic aging in plants, an unambiguous relationship has not been established. To gain further insight, we measured telomere length and DNA methylation levels in the same tree materials to determine their roles in tree ontogenetic aging and rejuvenation.\u003c/p\u003e\u003cp\u003eTelomere dynamics and cytosine DNA methylation both serve as key regulators of genome stability and cellular aging, with changes in these mechanisms often marking developmental phase transitions.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Plant material\u003c/h2\u003e\u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\u003ch2\u003e2.1.1. Adult Mature Plant\u003c/h2\u003e\u003cp\u003eElite \u003cem\u003eMelia volkensii\u003c/em\u003e trees (~\u0026thinsp;15 years old) were obtained from Better Globe Forestry (Kenya) and used as donor material. Young nodal segments bearing axillary buds were excised and used as explants. Explants were surface sterilized by rinsing in 70% ethanol for 30 s, followed by incubation in 10% (v/v) commercial bleach solution (8\u0026deg; active chlorine) for 15 min. The explants were then rinsed three times with sterile distilled water before being inoculated onto the culture medium.\u003c/p\u003e\u003cp\u003eSterilized explants were cultured on Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) supplemented with 20 g/L sucrose, 7 g/L Plant agar, and 10 \u0026micro;M meta-topolin riboside (mTR). The medium pH of the medium was adjusted to 5.4 prior to autoclave. The cultures were maintained in a growth chamber at 22\u0026deg;C under cool white fluorescent light (Philips) with a light intensity of 40 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1; and a 16-hour light/dark photoperiod. Resulting shoots were subcultured onto fresh medium every four weeks to sustain growth and multiplication.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.1.2. Juvenile Plant\u003c/h2\u003e\u003cp\u003eSeeds from the same elite donor tree were surface sterilized by rinsing in 70% ethanol for 30 s, followed by incubation in a 10% (v/v) commercial bleach solution (8\u0026deg; active chlorine) for 15 min, and subsequently rinsed three times with sterile distilled water before inoculation onto culture medium. To enhance germination, the seed coat was carefully scarified under sterile conditions before inoculation.\u003c/p\u003e\u003cp\u003eThe sterilized seeds were germinated on MS medium supplemented with 20 g/L sucrose and 7 g/L Plant agar. The medium pH was adjusted to 5.4 prior to autoclaving. The healthiest seedlings were subsequently transferred to the same medium, with the addition of 10 \u0026micro;M meta-topolin riboside (mTR). All cultures were maintained under identical conditions: a temperature of 22\u0026deg;C, a light intensity of 40 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;, and a 16-hour photoperiod.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.1.3. Plants from Adventitious Shoots\u003c/h2\u003e\u003cp\u003eLeaf explants from the same mature, elite plant were cultured in Petri dishes containing 10 mL MS medium supplemented with 20 g/L sucrose, 7 g/L Plant agar, and 10 \u0026micro;M thidiazuron (TDZ) to induce adventitious shoot formation. The cultures were incubated in a growth chamber at 22\u0026deg;C under a 16-hour photoperiod with cool white fluorescent light at an intensity of 40 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;. After six weeks, the developing shoots were subcultured onto fresh MS medium containing the same concentrations of sucrose and Plant agar, but with the addition of 10 \u0026micro;M meta-topolin riboside (mTR) instead of TDZ. These shoots were maintained for further growth under the same environmental conditions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.1.4. Plants from Somatic Embryogenesis\u003c/h2\u003e\u003cp\u003eSomatic embryogenesis was induced following a modified protocol based on Mulanda et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Leaf explants from the same adult plants were incubated in complete darkness on 10 mL MS medium supplemented with 20 g/L sucrose, 7 g/L Plant agar, and 10 \u0026micro;M TDZ to promote embryogenic callus formation. Emerging somatic embryos were then germinated on MS medium containing 20 g/L sucrose, 10 \u0026micro;M meta-topolin riboside (mTR), 5 \u0026micro;M indole-3-butyric acid (IBA), and 7 g/L Plant agar, with pH adjusted to 5.4. The cultures were maintained at 22\u0026deg;C under 40 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1; cool white fluorescent light and a 16-hour photoperiod.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.1.5. Plants from Root Suckers\u003c/h2\u003e\u003cp\u003eRoot suckers were collected from the same elite tree at Better Globe plantation, Kenya. The leaves were dried and used for DNA extraction and Southern blot analysis of telomeric repeats\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Sampling for DNA Isolation\u003c/h2\u003e\u003cp\u003eYoung, fully expanded leaves were sampled from (i) micropropagated shoots from elite adult trees, (ii) seed-derived juvenile plants, (iii) plants regenerated from adventitious shoots, and (iv) plants regenerated via somatic embryogenesis. For all material types, the same developmental stage of leaf tissue was chosen to minimize variability. Leaves were harvested in the morning, immediately frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until DNA extraction. Dried leaves from root sucker sample were also used for DNA extraction.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.3 DNA extraction and Southern blot analysis of telomeric repeats\u003c/h2\u003e\u003cp\u003eTotal genomic DNA was extracted using a modified protocol originally developed by Lodhi et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), as outlined by Valjakka et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) and Aronen et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Southern blot hybridization was carried out following the procedure of Kilian et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1995\u003c/span\u003e), incorporating modifications described by Aronen and Ryyn\u0026auml;nen (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). A synthetic telomeric sequence was amplified via PCR using primers T1 (5\u0026prime;-TTTAGGG-3\u0026prime;) and T2 (5\u0026prime;-CCCTAAA-3\u0026prime;), as described by Cox et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1993\u003c/span\u003e), and served as a positive control. Hybridization signals were detected via chemiluminescence using the manufacturer's protocol (Roche Diagnostics GmbH), scanned with an AlphaImager Imaging System (Alpha Innotech Co./ProteinSimple, CA, USA), and analysed with AlphaEase\u0026reg;FC software, using a digoxigenin-labelled molecular weight marker. To confirm the terminal location of the detected telomeric sequences, genomic DNA was treated with BAL-31 exonuclease as described by Aronen and Ryyn\u0026auml;nen (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), followed by Southern blot hybridization analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Quantification of global DNA methylation\u003c/h2\u003e\u003cp\u003eYoung leaves were harvested from four plant sources: (i) micropropagated shoots derived from elite adult \u003cem\u003eMelia volkensii\u003c/em\u003e trees (~\u0026thinsp;15 years old), (ii) seed-derived juvenile plants, (iii) adventitious shoot-derived plants, and (iv) plants regenerated via somatic embryogenesis. Total genomic DNA was extracted using the Invisorb Spin Plant Mini Kit (Stratec) according to the manufacturer\u0026rsquo;s protocol. DNA concentration was performed using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). MethylFlash\u0026trade; Global DNA Methylation (5-mC) ELISA Easy Kit (Colorimetric) (Epigentek, NY) was used according to the manufacturer\u0026rsquo;s instructions. One hundred nanograms of purified DNA from each sample were added to an ELISA plate where the methylated fraction of DNA was detected using capturing and detecting antibodies. Optical density (OD) intensity at 450 nm was read with a microplate spectrophotometer and this was proportional to the amount of methylated DNA. For quantification of global DNA methylation levels, a standard curve was generated using methylated DNA standards provided in the kit. The value for each sample was calculated as a ratio of the OD of the sample relative to the OD of the standard, after subtracting the negative control readings. The assay was performed in duplicate per biological replicate, meaning that per accession and condition, six samples were analyzed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Statistics Analysis\u003c/h2\u003e\u003cp\u003eFor statistical analysis, mean telomere length values from biological replicates were subjected to one-way analysis of variance (ANOVA) using Python 3.11 (SciPy v1.10.1 and Seaborn v0.12.2). ANOVA tested the null hypothesis that there were no differences in mean telomere length across tissue types. The significance level was set at α\u0026thinsp;=\u0026thinsp;0.05. Visualizations were created using Seaborn and Matplotlib libraries. Post-hoc tests were not applied; thus, the ANOVA indicates only whether a significant difference exists among groups, not between specific pairs. The methylation experiment was carried out with 11 explants for different distinct tissue types. Data was analyzed using SPSS 25.0 (SPSS Inc. USA). The independent sample t-test was used. The other data were analyzed by analysis of variance (ANOVA) followed by Duncan's new multiple range (DMR) test to separate the mean differences (Tallarida and Murray \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). Comparisons of each parameter were conducted by Dunnetz\u0026rsquo;s t-tests (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Zar \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1984\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Telomere Length Variation Across Tissue Types\u003c/h2\u003e\u003cp\u003eTelomere length was quantified in various tissue types, including adult leaves (Adult 1 and Adult 2), juvenile seedlings (Juvenile 1 and Juvenile 2), somatic embryos (SE 1 and SE 2), adventitious shoots (Adv Shoot 1 and Adv Shoot 2), and root suckers. Southern hybridization analysis revealed distinct differences in telomere length among these distinct tissue types. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e displays typical Southern hybridization images used to assess and quantify telomeric length.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eJuvenile micropropagated tissues (lanes 3, 4) and somatic embryogenesis-derived tissues (lanes 5, 6) exhibited longer telomeres compared to adult micropropagated tissues (lanes 1, 2). Adventitious shoot tissues (lanes 7, 8) showed moderate telomere lengths, while root sucker tissues (lane 9) displayed telomere length patterns like those of juvenile tissues, suggesting a partial reversion to a juvenile-like state. Comparison of telomeric lengths for different tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) revealed a tendency toward variation in these tissues.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eQuantitative analysis of mean telomere length in different \u003cem\u003eMelia volkensii\u003c/em\u003e tissue types revealed notable variation associated with developmental stage and propagation method. Adult micropropagated tissues exhibited the shortest telomeres, with a mean length of 5.1 kb, indicating a more aged or mature state. In contrast, juvenile micropropagated tissues showed significantly longer telomeres (8.5 kb), suggesting a more youthful genomic profile. Tissues derived from somatic embryogenesis (7.4 kb) and adventitious shoots (7.8 kb) also displayed relatively long telomeres, indicating partial rejuvenation during the regeneration process. Root sucker tissues had the longest telomeres (8.9 kb), further supporting the hypothesis that naturally regenerated tissues may retain or regain juvenile characteristics. These results suggest a strong association between telomere length and rejuvenation potential in clonally propagated \u003cem\u003eMelia volkensii\u003c/em\u003e tissues. A one-way analysis of variance (ANOVA) was conducted to assess statistical differences in telomere lengths among tissue types. The results indicated a significant effect of tissue type on telomere length (F (4, 8)\u0026thinsp;=\u0026thinsp;28.03, p\u0026thinsp;=\u0026thinsp;0.0035). This suggests that telomere length is significantly influenced by developmental stage and regeneration methods (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Notably, tissues derived from somatic embryos and adventitious shoots exhibited intermediate telomere lengths, suggesting a partial rejuvenation effect compared to adult tissues. These findings align with the hypothesis that in vitro regeneration methods may restore telomere length, potentially reversing age-related genomic erosion.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.2 Global DNA methylation\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eThe ELISA-based quantification of global DNA methylation, assessed through absorbance at 410 nm and fold change in 5-methylcytosine (5-mC) levels, reveals significant epigenetic variation between SE, juvenile shoots, adventitious shoots, and mature tissues. In the graph measuring absorbance at 410 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), which correlates directly with the amount of methylated cytosine, SE tissues display the lowest absorbance (~\u0026thinsp;2.0), significantly lower than the values observed in the other tissues (all ~\u0026thinsp;2.2 or higher).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThis trend is further confirmed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, which presents the fold change in global methylation. Here, plants from somatic embryo (SE) again show the lowest value (~\u0026thinsp;0.88), while juvenile and adventitious shoots display a fold change around 0.96, and mature tissues reach a baseline level of 1.0. The statistical annotation reinforces this distinction, as SEs are again significantly different from the other tissues. Taken together, both graphs consistently demonstrate that somatic embryos undergo a substantial reduction in global DNA methylation, while tissues that are either naturally developed (juvenile, mature) or regenerated through organogenesis (adventitious shoots) maintain higher methylation levels.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Telomere Length as an Indicator of Developmental Stage and Rejuvenation Capacity\u003c/h2\u003e\u003cp\u003eSomatic embryogenesis (SE) is recognized as the most effective approach for rejuvenating plants, especially mature trees, as it produces plantlets with juvenile characteristics by effectively resetting the embryonic developmental program, even when starting from mature donor tissues (Shmakov et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Our findings demonstrate a clear relationship between telomere length and the ontogenic or regenerative origin of the tissue. As anticipated, micropropagated adult tissues had the shortest telomeres, reflecting cellular aging and extensive mitotic divisions. In contrast, juvenile tissues and root suckers possessed notably longer telomeres, indicating a more juvenile genomic state. Tissues regenerated through somatic embryogenesis and adventitious shoots from elite adult trees showed intermediate telomere lengths, suggesting partial rejuvenation during invitro regeneration. Plant roots are generally considered as juvenile (Crawford et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), so shoots originating from root suckers are juvenile as expected. Our data confirm this, as root sucker tissues from mature trees exhibited longer telomere lengths compared to those derived directly from the shoots.\u003c/p\u003e\u003cp\u003ePrevious studies have reported mixed results: Moriguchi et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) found no significant telomere length differences across tissues in apple and cherry trees over several years, while Song et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and Aronen \u0026amp; Ryyn\u0026auml;nen (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) observed stable telomere lengths regardless of age in ginkgo and silver birch, respectively. Conversely, Zhang et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) documented a decline in telomere length with age in leaves of younger \u003cem\u003ePlatycladus orientalis\u003c/em\u003e. Our study similarly shows shorter telomeres in adult \u003cem\u003eMelia volkensii\u003c/em\u003e tissues compared to juvenile and root sucker tissues, implying ontogenic related telomere shortening. This effect is well documented in species like \u003cem\u003eLarix decidua\u003c/em\u003e (Kretzschmar \u0026amp; Ewald, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), \u003cem\u003eSequoia\u003c/em\u003e (Arnaud, 1993), \u003cem\u003ePinus\u003c/em\u003e (Prehn et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), and \u003cem\u003ePicea\u003c/em\u003e (Zarei et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Correspondingly, adventitious shoots from adult tissues in our study showed increased telomere length. On the other hand, prolonged duration of micropropagation or somatic embryogenesis, or even stressful conditions during micropropagation, have shown to shorten telomeres in silver birch (Aronen \u0026amp; Ryyn\u0026auml;nen \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and Norway spruce (Aronen et al \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and should thus be avoided. So, in addition to developmental stage, both stress exposure and duration in culture can influence telomere dynamics, and these factors should be considered in the interpretation of the present \u003cem\u003eMelia volkensii\u003c/em\u003e material. Furthermore, tree genotype has been shown to affect telomere length (Aronen \u0026amp; Ryyn\u0026auml;nen, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Aronen \u0026amp; Ryyn\u0026auml;nen, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Aronen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, in the present study the same (related) genotypes were used when comparing different tissue materials, indicating that the observed differences more likely reflect tissue type\u0026ndash;specific or developmental effects rather than genotypic variation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e4.2 Decreased Global DNA Methylation in Somatic Embryogenesis-Derived Tissues\u003c/h2\u003e\u003cp\u003eAlongside telomere analysis, ELISA-based quantification of 5-methylcytosine (5-mC) revealed a marked decrease in global DNA methylation in somatic embryo-derived tissues compared to juvenile, adventitious, and mature tissues. This suggests a unique epigenetic state associated with somatic embryogenesis, likely reflecting extensive reprogramming during this developmental route. The significant reduction in methylation in somatic embryo-derived tissues aligns with findings in other plants where somatic embryogenesis is linked to methylation changes. For instance, oil palm somatic embryos with the mantled phenotype show hypomethylation compared to normal embryos (Jaligot et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Similarly, a transient methylation decrease is critical to initiating somatic embryogenesis in chestnut (Viejo et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), with dynamic methylation fluctuations observed in early stages of somatic embryogenesis in \u003cem\u003eCoffea canephora\u003c/em\u003e (Nic-Can et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Maritime pine also exhibits developmental hypomethylation during somatic embryogenesis (Trontin et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Juvenile and mature tissues, representing stable developmental stages, typically maintain higher and more stable global methylation important for tissue identity and gene silencing (Li et al \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Adventitious tissues, although also cultured in vitro, may follow distinct epigenetic pathways compared to somatic embryos, lacking the same level of epigenetic resetting. This methylation decrease could have several consequences: it might contribute to phenotypic variation often seen in somatic embryogenesis-derived plants, including somaclonal variation, by altering gene expression and affecting growth, development, and stress responses (Phillips et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Additionally, this hypomethylated state might increase sensitivity to environmental stress since DNA methylation is crucial for genome stability and defense (Boyko \u0026amp; Igor, 2008). No significant methylation differences were observed between adult, juvenile, and adventitious tissues. Previous reports indicate higher cytosine methylation in adult tissues increasing with age (Huang et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yuan et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Micropropagation often elevates methylation levels, contributing to epigenetic variation (Ghosh et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which may explain our observations. Moreover, methylation patterns vary by species, tissue type, organelle, and age (Vanyushin, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), so species-specific factors might influence differences between adult and seed-derived juvenile tissues. High-resolution methods such as whole-genome bisulfite sequencing (WGBS) are recommended for future studies to pinpoint specific differentially methylated regions (DMRs) and their associated genes, enhancing understanding of how methylation changes relate to gene expression and somatic embryo development. Examining methylation stability during plant acclimatization and maturation will also be important to assess the long-term epigenetic fidelity of somatic embryogenesis-derived plants.\u003c/p\u003e\u003cp\u003eIn summary, our data reveals a significant reduction in global DNA methylation in somatic embryo-derived tissues, indicating extensive epigenetic reprogramming during somatic embryogenesis. This epigenetic signature may be critical for developmental plasticity but also calls for further study of its long-term developmental effects.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Combined Telomere and Methylation Profiles\u003c/h2\u003e\u003cp\u003eThe presence of elongated telomeres alongside hypomethylation in regenerated tissues\u0026mdash;especially those from somatic embryogenesis\u0026mdash;points to a coordinated molecular rejuvenation process. Although telomeres themselves are usually not methylated, DNA methylation in sub telomeric regions influences telomere length and stability, thereby affecting cell development and differentiation (Vega-Vaquero et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). It is plausible that hypomethylation in somatic embryogenesis tissues loosens chromatin near telomeres, allowing partial telomere extension. Interestingly, adventitious shoots exhibited both longer telomeres and higher methylation compared to somatic embryos, possibly reflecting differences in their cellular origin or regeneration mechanisms (dedifferentiation versus transdifferentiation) that influence telomere and epigenetic resetting.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e4.4 Implications for Clonal Propagation and Conservation\u003c/h2\u003e\u003cp\u003ePractically, these findings have important implications for clonal propagation of \u003cem\u003eMelia volkensii\u003c/em\u003e. Tissues regenerated through somatic embryogenesis and adventitious shoots regain juvenile molecular features that may enhance rooting, growth, and field adaptability. Root suckers, with the longest telomeres, represent promising explants for producing rejuvenated clones. Moreover, the observed epigenetic flexibility highlights the need to monitor methylation and telomere dynamics during tissue culture, incorporating these molecular markers into quality control for elite clone selection in forestry biotechnology and conservation efforts.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e4.5 Limitations and Future Perspectives\u003c/h2\u003e\u003cp\u003eWhile this study establishes links between telomere length, methylation status, and tissue origin in \u003cem\u003eMelia volkensii\u003c/em\u003e, limitations include the focus on global methylation and average telomere length without locus- or cell-type-specific resolution, and a small sample number. Future research with more extensive materials or by employing high-resolution bisulfite sequencing and single-cell telomere length assays could provide deeper mechanistic insight. Additionally, investigating telomerase activity and telomere-associated proteins (e.g., TERT, TRF-like proteins) would clarify pathways involved in telomere resetting. Long-term studies tracking telomere dynamics through propagation cycles and field growth are also warranted.\u003c/p\u003e\u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, telomere length and global DNA methylation in \u003cem\u003eMelia volkensii\u003c/em\u003e are influenced by tissue ontogenic stage and regeneration method. These molecular markers provide critical insights into rejuvenation potential and can guide clonal propagation strategies aimed at sustainable forestry and conservation of this ecologically vital species.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of Interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003ch2\u003eEthics declaration\u003c/h2\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis study was funded by Erasmus\u0026thinsp;+\u0026thinsp;Swagata program of European Union and the Transnational Access to Research Infrastructures Activity in the 7th Framework Programme of the European Union under the Trees 4 Future project no. 284181 for conducting the research (at Punkaharju Research Unit, Finnish Forest Research Institute Punkaharju, Finland).\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eNB: Writing \u0026ndash; original draft, Conceptualization, Methodology; AV: Methodology TJ: Writing \u0026ndash; review \u0026amp; editing, Conceptualization, Supervision; SPOW: Writing \u0026ndash; original draft, Conceptualization, Supervision; TM: Supervision\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eThe authors acknowledge Mrs. Aila Viinanen for valuable technical assistance in realization of Southern analyses for telomere length measurement\u003c/p\u003e\n\u003ch2\u003eData Availability Statement\u003c/h2\u003e\n\u003cp\u003eThe data supporting the findings of this study are available within the manuscript and its supplementary materials. Any additional datasets generated and analysed during the current study are available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eArnaud Y, Franclet A, Tranvan H, Jacques M (1993) Micropropagation and rejuvenation of \u003cem\u003eSequoia sempervirens\u003c/em\u003e (Lamb) Endl: A review. \u003cem\u003eAnn Sci For\u003c/em\u003e 50:273\u0026ndash;295. https://doi.org/10.1051/forest:19930305\u003c/li\u003e\n \u003cli\u003eAronen T, Ryyn\u0026auml;nen L (2012) Variation in telomeric repeats of Scots pine (\u003cem\u003ePinus sylvestris\u003c/em\u003e L.). \u003cem\u003eTree Genet Genomes\u003c/em\u003e 8:267\u0026ndash;275. https://doi.org/10.1007/s11295-011-0438-7\u003c/li\u003e\n \u003cli\u003eAronen T, Ryyn\u0026auml;nen L (2014) Silver birch telomeres shorten in tissue culture. \u003cem\u003eTree Genet Genomes\u003c/em\u003e 10:67\u0026ndash;74. https://doi.org/10.1007/s11295-013-0663-7\u003c/li\u003e\n \u003cli\u003eAronen T, Tiimonen H, Tsai CJ, Jokipii S, Chen X, Chiang V, H\u0026auml;ggman H (2003) Altered lignin in transgenic silver birch (\u003cem\u003eBetula pendula\u003c/em\u003e) expressing PtCOMT gene. 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H. Karst. via tissue culture and co-cultivation with \u003cem\u003eAgrobacterium rhizogenes.\u003c/em\u003e \u003cem\u003eTrees\u003c/em\u003e 34:637\u0026ndash;648. https://doi.org/10.1007/s00468-019-01945-z\u003c/li\u003e\n \u003cli\u003eZhang Y, Yu X, Zhang S (2025) Analysis of the relationships among telomere-associated protein-encoding gene expression, tree age and telomere length in \u003cem\u003ePlatycladus orientalis\u003c/em\u003e (L.) Franco. \u003cem\u003eTree Genet Genomes\u003c/em\u003e 21:23. https://doi.org/10.1007/s11295-025-01758-y\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"DNA methylation, In vitro rejuvenation, Micropropagation, Melia volkensii, Somatic embryogenesis, Telomere length","lastPublishedDoi":"10.21203/rs.3.rs-7941105/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7941105/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eMelia volkensii\u003c/em\u003e is a drought-tolerant hardwood of high ecological and economic value in arid and semi-arid Africa, but conventional propagation is limited by poor seed viability and recalcitrance to regeneration. To explore molecular mechanisms of rejuvenation, we investigated telomere length and global DNA methylation in tissues derived from different propagation routes: adult micropropagation, juvenile seedlings, somatic embryogenesis, adventitious shoots, and root suckers. Telomere length, measured by Southern hybridization, varied significantly among tissue types. Adult micropropagated tissues showed the shortest telomeres (mean 5.0 kb), while juvenile seedlings (8.5 kb) and root suckers (8.9 kb) had markedly longer telomeres, suggesting a juvenile-like state. Somatic embryos (7.4 kb) and adventitious shoots (7.8 kb) exhibited intermediate lengths, indicating partial rejuvenation during regeneration. Global DNA methylation, quantified by ELISA, further distinguished somatic embryos, which showed the lowest absorbance (~\u0026thinsp;2.0) and fold change (~\u0026thinsp;0.88), significantly reduced compared to juvenile (~\u0026thinsp;0.96), adventitious (~\u0026thinsp;0.96), and adult tissues (~\u0026thinsp;1.0). These findings demonstrate a strong link between telomere elongation, reduced 5-methylcytosine levels, and cellular rejuvenation in \u003cem\u003eM. volkensii\u003c/em\u003e, offering valuable insights for optimizing clonal propagation and ensuring long-term genetic stability in forestry applications.\u003c/p\u003e","manuscriptTitle":"Rejuvenation through Somatic Embryogenesis: Epigenetic and Telomeric Resetting in Melia volkensii","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-12 17:49:28","doi":"10.21203/rs.3.rs-7941105/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-11-12T01:36:19+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-31T16:51:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-29T17:09:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2025-10-27T05:03:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e1caff3b-5de3-4c87-954d-30d5d07644e4","owner":[],"postedDate":"November 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-16T16:06:55+00:00","versionOfRecord":{"articleIdentity":"rs-7941105","link":"https://doi.org/10.1007/s11240-026-03382-6","journal":{"identity":"plant-cell-tissue-and-organ-culture-pctoc","isVorOnly":false,"title":"Plant Cell, Tissue and Organ Culture (PCTOC)"},"publishedOn":"2026-02-14 15:58:09","publishedOnDateReadable":"February 14th, 2026"},"versionCreatedAt":"2025-11-12 17:49:28","video":"","vorDoi":"10.1007/s11240-026-03382-6","vorDoiUrl":"https://doi.org/10.1007/s11240-026-03382-6","workflowStages":[]},"version":"v1","identity":"rs-7941105","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7941105","identity":"rs-7941105","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}