Enhancing Regeneration in White Yam (Dioscorea rotundata) Through Friable Embryogenic Callus

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Syombua, Jaindra N. Tripathi, Leena Tripathi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6539506/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Sep, 2025 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted 4 You are reading this latest preprint version Abstract White yam ( Dioscorea rotundata ) is a critical staple food and an income generating crop in tropical regions. However, its improvement via classical breeding is challenging and time consuming due to an erratic flowering pattern, poor seed set, dioecious nature, low pollen fertility and low seed germination rates. These constraints limit the genetic gains achievable in each generation and prolong the breeding cycle to approximately 10 years, underscoring the need to implement faster biotechnological approaches. This study presents an optimized protocol for producing FECs (friable embryogenic callus) in yam, which serves as an ideal tissue type for transgene delivery and accelerated breeding by site specific nucleases. The various factors influencing FEC induction were optimised, including basal salt composition, tissue wounding, washing treatments, and medium supplementation with antioxidants. The results demonstrated that reduction in nitrogen supplements, along with 10 mg/L thiamine, 1000 mg/L proline and 600 mg/L casein hydrolysate, and enhanced callus fresh weight by up to 498.4 mg per explant and increased the embryogenic competence to 76.2%. Callus wounding by crushing through mesh further improved FEC induction and washing the crushed callus with 10 mg/L ascorbic acid reduced browning and necrosis, reducing recovery time from 25 to 13 days. The optimized FEC induction medium (FIM) induced somatic embryoes in over 77% of cultures. This protocol provides a robust platform for yam genetic improvement, offering an excellent starting material for protoplast isolation, regeneration and genome editing to enhance crop resilience and productivity. Yam (Dioscorea spp.) Friable Embryogenic Callus (FEC) Somatic embryogenesis Nitrogen Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key message Reducing nitrogen in culture media and mechanical wounding of callus significantly enhance friable embryogenic calli (FEC) induction and somatic embryo regeneration in white yam ( D. rotundata ), providing a robust platform for genetic transformation and accelerated breeding. Introduction Yams (genus Dioscorea ) are monocotyledonous vines that yield edible tubers and are widely grown in the tropics. It has about 600 species, of which only 11 species are widely cultivated as a staple food. Dioscorea cayenensis–D. rotundata (the Guinea yam complex) ranks among the world’s most vital tuber crop, providing both food sustainability and economic livelihoods across the subtropical and tropical (CropTrust, 2024 ). Other commercially important species include white, purple, yellow, aerial, Chinese yam and bitter yam, among which the white yam contributes to about 90% of global yam output (Darkwa et al., 2020 ). Yam productivity is greatly impacted by various pressures, including susceptibility to pests and diseases (nematodes, viruses, fungi, bacteria, etc.), drought, and low soil fertility, leading to poor quality produce and substantial yield losses. These constraints emphasize the need for integrated strategies to ensure sustained production and stable income for smallholder farmers (Kole, 2022). The development and deployment of diseases and pest resistant varieties remain the gold standard for boosting crop productivity. Current yam improvement initiatives rely on conventional breeding, which is not only labor intensive and time consuming, but is also highly constrained due to the crop’s complex genetic make-up (Kole, 2022a ). It is, therefore, essential to integrate breeding efforts with modern biotechnological approaches like in vitro culture systems and gene editing (Syombua, Zhang, et al., 2021 ). Successful genetic transformation requires introducing DNA into tissue capable of regenerating as a cohesive, uniform plant. However, existing yam protocols generate both somatic embryos and organogenic shoots in the same culture (Manoharan et al., 2016 ), making selection difficult and often producing chimeric plants. While true somatic embryos originating from single cells would solve this, isolating a homogeneous population of them in D. rotundata remains a major hurdle. Because somatic embryos originate from single cells, they avoid the chimerism often seen when shoots regenerate, so they are the preferred target for stable transformation (Gambino et al., 2021 ). To harness this pathway in D. rotundata , we, therefore, needed to refine our protocol to enrich exclusively for homogeneous embryogenic callus clusters. Friable embryogenic callus (FEC) meets those criteria; its loose, single-cell–derived clusters can be maintained in suspension, and nearly every cell remains totipotent (Taylor et al., 1996 ). FEC has proven effective not just in cassava (Syombua, Adero, et al., 2021 ) but also in litchi (Wang et al., 2023 ), and Alstroemeria (Lin et al., 2000 ), making it the gold standard tissue for direct gene transfer systems and downstream applications such as protoplast isolation and cell-level manipulation (Bull et al., 2009; Wen et al., 2020 ). Building on the earlier somatic embryogenesis system by Manoharan et al. ( 2016 ), this research aimed to optimize a system for generating FECs in Dioscorea rotundata. Through a series of carefully controlled experiments, the key parameters influencing secondary somatic embryogenesis, including basal salt composition, mechanical wounding, antioxidant treatment, and amino acid supplementation were evaluated. These modifications successfully mitigated tissue necrosis and encouraged cellular reprogramming and FEC regeneration. This study reports on the successive enrichment of primary somatic embryos, maintenance, proliferation and subsequent progression to whole plantlets. This system of generating totipotent callus tissue offers a foundation for advanced genetic studies to facilitate gene editing, trait improvement, and ultimately contributing to the development of resilient yam cultivars. Experimental Procedures Plant material and culture maintenance Sterile cultures of the yam ( Dioscorea rotundata ) accessions TDr 2579 and TDr 2436 were sourced from IITA’s germplasm collection (Ibadan, Nigeria) and maintained in vitro on the media YBM (Yam Basic Medium). YBM comprised Murashige & Skoog salts supplemented with 3% sucrose, 2 mg/L glycine, 100 mg/L myo-inositol, 0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxine-HCl, 0.1 mg/L thiamine-HCl, and solidified with 2.4 g/L Gelrite. The pH was adjusted to 5.8 before autoclaving for 15 min at 121°C. Cultures were sub cultured every 4–8 weeks and incubated at 25 ± 2°C under a 16 h light/8 h dark photoperiod (30 µmol m⁻² s⁻¹). Initiation of primary embryogenesis We cultured nodal segments following the protocol of Utsumi et al ( 2017 ) to initiate the formation of primary somatic embryos. Briefly, sterile single-node segments measuring approximately 15mm were excised from four to eight-week-old plantlets were placed on SBM (Shoot Bud Induction Medium) and maintained in the dark at 28°C for 72 hours. SBM comprised MS salts plus 2 µM CuSO₄, 1 mg/L 6-benzylaminopurine, 2% sucrose, 0.3% Gelrite, and the standard vitamin–glycine mix (0.5 mg/L each nicotinic acid and pyridoxine-HCl, 100 mg/L myo-inositol, 2 mg/L glycine, and 0.1 mg/L thiamine-HCl). Buds that formed were excised with a sterile syringe under magnification and moved to CIM (Callus Induction Medium), which had a similar composition to SBM except using 0.5 mg L⁻¹ picloram in place of BAP, and incubated in darkness at 25 ± 2°C for four weeks. Basal medium optimization for enhanced embryogenesis To identify the optimal nutrient regime for embryo induction, three basal media were compared: full-strength MS, half-strength MS, and a custom FEC Induction Medium (FIM; see Supplementary Table S1). FIM was formulated by reducing nitrate, phosphate, and potassium concentrations in MS salts and adding 10 mg L⁻¹ thiamine-HCl. For each medium, 100 explants (in triplicate) were scored after four weeks for the percentage forming direct somatic embryo-like structures (DSLS), the callus fresh weight (mg), and the quantity of DSLS in each explant. Secondary embryogenesis and wounding treatments Secondary somatic embryos were produced by subculturing primary embryos onto media containing 0.5 mg/L picloram. Four formulations of basal salt mixtures (Gresshoff & Doy [GD] premix, FIM, full- and half-strength MS) were tested. Each experiment consisted of ten plates per treatment, each with ten embryos, and were maintained at 26 ± 2°C in the dark for up to six months. To assess the impact of mechanical injury, callus was either passed through an approximately 2 mm stainless-steel mesh (Nyaboga et al., 2015 ) or gently teased apart into ~ 2–5 cm pieces. Control calli were transferred without wounding. All treatments were performed in triplicate. Callus washing and antioxidant/tyrosine treatments Meshed callus (1.5 g per plate) was collected into sterile tubes and rinsed three times in one of: (i) water; (ii) liquid MS; (iii) liquid MS + 10 mg/L ascorbic acid; or (iv) liquid MS + 20 mg/L ascorbic acid. After each rinse, callus was gently blotted dry on sterile tissue. Washed and unwashed calli were then plated onto GD media supplemented with 0.5 mg/L picloram; an additional set of washed calli was cultured on GD supplemented with 0.5 mg/L picloram plus 12 mg/L L-tyrosine. Two weeks later, tissue browning and necrosis were evaluated through visual observation on a 0–5 scale. Germination and acclimatization of FEC-derived embryos After FEC-derived embryos reached the torpedo or cotyledon stage, they were transferred to 1% charcoal-amended, MS medium without hormones and cultured for 45 days at 25 ± 2°C in a 16 h light/8 h growth chamber. Mature embryos were then shifted to MS supplemented with10 mg/L ascorbic acid and 0.4 mg/L BAP for final development, followed by rooting on YBM. Rooted plantlets were hardened in peat-filled cups covered with plastic bags; openings were introduced gradually over seven days before potting into a 1:1 peat:soil mix in the greenhouse. Histological analysis Samples of non-embryogenic callus, embryogenic calli, and FECs were fixed in Carnoy’s solution (60% ethanol, 30% chloroform, 10% acetic acid), dehydrated through a graded ethanol series (70–100%), then infiltrated with Technovit 7100 resin (2:1 → 1:1 → 1:2 ethanol:resin, 30 min each, vacuum). After an overnight pure-resin infiltration, tissues were embedded, sectioned at 5 µm (Leica RM 2155), stained with neutral red or 0.05% toluidine blue, and imaged on an Optika Vision Lite 2.1 microscope. Validation of the optimized protocol in yam variety TDr 2579 To validate the protocol with a different yam variety, the formation of somatic embryos in TDr 2579 was initiated using auxiliary buds from single nodal sections and cultured in CIM as detailed previously. Optimized concentrations of proline, casein hydrolysate, and thiamine were added, based on the protocol established for TDr 2436 and callus wounding was performed by meshing through a stainless-steel sieve with a pore size of 1–2 mm or detached using a pair of forceps. The crushed callus was washed with liquid CIM containing ascorbic acid (10 mg/L or 20 mg/L) and transferred to either GD or FIM media, following the optimized culture conditions. Regeneration was completed by transferring FECs to hormone-free MS medium supplemented with 1% activated charcoal for embryo germination, followed by embryo maturation in BAP medium, rooting in YBM, and gradual acclimatization in soil. Experimental design and statistics All experiments followed a completely randomized design with two biological repeats and three technical replicates per treatment (100 explants each). Data were analyzed by ANOVA with means compared using Duncan’s multiple range test (p < 0.05) in Minitab 17, and are presented as mean ± SE. Results Role of amino acids and vitamin additives in boosting embryogenesis and callus yield When cultured on SBM, nodes (Fig. 1 A) produced axillary buds within three days. After transferring these buds to CIM for two weeks, direct somatic embryos began to emerge from the explant surface. However, a portion of the explants instead generated non-embryogenic, white, and compact callus structures. Supplementating CIM with proline, thiamine, and casein hydrolysate significantly enhanced all metrics of somatic embryogenesis (Table 1 ). The fully supplemented treatment (casein hydrolysate + proline + thiamine) yielded a callus fresh weight of 498.4 mg, more than double the 221.3 mg obtained in the unsupplemented control and raised the proportion of embryogenic callus from 41.9–76.2%. Likewise, the mean number of embryos per callus jumped from 4.5 to 19.3. These enhancements were consistently superior to partial supplementations, so all subsequent CIM formulations were supplemented with these additives. Table 1 Impact of casein hydrolysate, proline and thiamine on somatic embryo induction and callus biomass CH (g L⁻¹) Proline (mg L⁻¹) Thiamine (mg L⁻¹) % Explants with DSE after 4 wk Mean DSE no per explant (4 wk) Callus fresh weight (mg) after 8 wk 0 0 0 41.9 ± 4.6e 4.5 ± 1.8e 221.3 ± 24.6d 0 600 0 52.4 ± 5.3d 7.3 ± 3.1de 314.8 ± 9.6c 1000 0 0 53.1 ± 3.1d 8.7 ± 2.5d 307.6 ± 14.6c 600 1000 0 64.2 ± 2.8c 12.3 ± 3.2c 368.7 ± 22.7b 0 0 10 65.4 ± 5.7c 14.8 ± 2.4bc 385.6 ± 19.5b 1000 0 10 68.6 ± 2.6bc 15.2 ± 1.6b 376.8 ± 28.8b 600 1000 10 76.2 ± 3.5a 19.3 ± 1.5a 498.4 ± 17.3 a Data represent the average ± standard error of three biological replicates (n = 3). Within each column, means labeled with different letters are statistically different (p ≤ 0.05, Duncan’s test). Influence of basal salt formulation on somatic embryo induction and callus proliferation Varying the basal mineral salt concentrations in the medium markedly affected explant embryogenic potential and callus proliferation (Table 2 ). The FIM medium achieved the highest embryo-induction rate, with 77.3% of explants producing somatic embryos and an average of 19.2 embryos per explant. Full‐strength MS yielded very similar results (76.5% responsiveness and 18.6 embryos per explant). In contrast, reducing the MS salts to half strength led to a marked drop in both induction frequency and mean embryo number: only 48.6% of explants produced embryos, averaging 4.7 embryos each. Callus biomass also tracked with salt strength; FIM and full‐strength MS substantially supported higher fresh weights (504.8 mg and 486.7 mg, respectively) than half‐strength MS (236.9 mg). Despite this, both FIM and full‐strength MS yielded similar numbers of direct somatic embryo events per callus and comparable percentages of calli forming DSE. Given these outcomes and the straightforward preparation of standard MS, we chose full‐strength MS for all further embryo induction experiments. Table 2 Comparison of somatic embryo induction and callus biomass under different basal salt regimes Medium % DSE (4 wks) Mean DSE/explant (4 wks) Mean fresh weight (mg /explant, 8 wks FIM (reduced NPK + Thiamine) 77.3 ± 1.6a 19.2 ± 2.4a 504.8 ± 24.6a MS 76.5 ± 2.4 a 18.6 ± 1.7a 486.7 ± 13.5a Half strength MS 48.6 ± 3.9 b 4.7 ± 3.5b 236.9 ± 35.4b Mean ± SE (n = 3; 100 explants per replicate). Values sharing the same letter within a column are not significantly different (p < 0.05, Duncan’s multiple range test). Effect of callus wounding and washing meshed callus on FEC induction Three distinct calli morphologies were observed following the eight-week culture in CIM; the non-embryogenic type was discarded and only the embryogenic type was retained (Fig. 1 B). Crushing callus (Fig. 1 C) through the metal mesh to ~ 2 mm fragments and subsequent rinsing and blotting on sterile tissue (Fig. 1 D) eliminated the mucilaginous exudate and yielded clean, debris-free calli (Fig. 1 E). Within the first 14 days after culture in callus induction media 2, the callus edges subjected to wounding developed marked browning and necrotic zones. (Fig. 4.1F). However, washing and blotting significantly reduced tissue browning compared to unwashed callus and accelerated recovery (Table 3 ). Table 3 Browning severity and recovery time after different callus-washing treatments Washing treatment Recovery time (days Browning score No wash 25.4 ± 2.3d 5 Rinse in distilled water 21.3 ± 1.5c 4 Rinse in liquid MS 16.7 ± 1.8b 3 MS + 5mg/L ascorbic acid 13.5 ± 1.4a 1 MS + 10mg/L ascorbic acid 12.8 ± 2.1a 1 MS + 20mg/L ascorbic acid 17.6 ± 0.7b 2 MS + 12mg/L L-tyrosine 16.3 ± 0.2b 3 Browning was scored from 0 (no browning) to 5 (extensive browning). Results are expressed as the mean ± standard error from three independent biological replicates (n = 3 plates). Within each column, means bearing different superscript letters differ at p < 0.05 based on Duncan’s multiple range test. Supplementing the wash solution with 10 mg L⁻¹ ascorbic acid lowered browning to a score of 1 and shortened recovery to 12.8 days. However, doubling the ascorbic acid to 20 mg L⁻¹ backfired, increasing discoloration (score 2) and extending recovery to 17.6 days. In contrast, adding 12 mg/L L-tyrosine whether in liquid or solid CIM had had no measurable impact on either browning or recovery time. Starting in week 4, callus proliferation resumed visibly, and the browning diminished over time (Fig. 1 G). Influence of basal salt formulations on friable embryogenic callus induction Sub-culturing meshed calli through successive GD media stages boosted callus proliferation (Figs. 1 H & I), with FECs first appearing on GD 3 (Fig. 1 J), and expanded further when transferred to GD 4 (Figs. 1 K and L). In contrast, simply wounding callus in CIM 2 by removing non-embryogenic tissue and then sub-culturing them to GD failed to induce FECs and instead scarred and hardened. Among the basal salts tested, FIM medium demonstrated the highest FEC induction rate (11.78%), which is more than double that obtained in GD (4.1%). Meanwhile, neither half-strength nor full-strength MS medium supported FEC formation (Fig. 2 ). These results identify FIM as the optimal medium for FEC development. Subculturing FECs onto GD 5 (Fig. 3 A) triggered a burst of proliferation over 14 days, followed by a gradual purple coloration that marked the onset of maturation (Fig. 3 B). After 45 days on charcoal-amended medium, all embryos were uniformly purple (Fig. 3 C), and microscopic examination revealed distinct shoot and root meristem regions (Fig. 3 D). Upon transfer to a BAP-supplemented maturation medium, the embryos turned green and progressed to form single or fused cotyledons (Figs. 3 E, F, G, and H). Fully developed cotyledonary stage embryos were successfully rooted on YBM medium (Fig. 3 I). The regenerated plants exhibited normal growth, and no morphological variations were observed under green house evaluation (Fig. 3 J). Histological profile of callus tissues and FECs Histological analysis of callus and somatic embryos at the various stages of development was conducted to confirm the embryogenic origin of plantlets regenerated by the optimized regeneration system. Transverse sections of non-embryogenic cali generate following 2-week explant culture in CIM1 revealed irregularly shaped cells with many intercellular spaces, obscured nucleus, low cytoplasm density and large vacuoles (Fig. 4 A). On the contrary, embryonic regions were characterized by numerous starch storage grains and meristematic zones, evidenced by the presence of pro-embryonic masses (Fig. 4 B). Notably, the embryogenic sections predominantly originated from the tip of the auxiliary bud, whereas the base of the auxiliary bud formed watery or compact and hard non embryogenic tissues. Tissues from CIM2 were not analyzed histologically because this was a recovery phase consisting of small brown-coloured tissues. Initial signs of recovery were observed at the end of culture on GD1 medium, and analysis of the tissues at this stage revealed a highly organized structure with small compact cells, dense cytoplasm, prominent nucleus, and onset of protoderm formation (Fig. 4 C). During culture in GD2 medium, the cells underwent periclinal and anticlinal divisions to form globular shaped embryogenic structures that had small-sized cells with dense cytoplasm and small nuclei. These globular structures remained attached to the mother tissue (Fig. 4 D), and budding from these structures eventually generated multiple globular and heart shaped somatic embryos, many of which detached from the mother tissue to form mucilaginous clusters with high cytoplasmic content and prominent nuclei (Fig. 4 E). The clear bipolar structures of the somatic embryos were evident at the GD 3 stage (Fig. 4 E). GD4 marked a proliferation phase, during which the population of somatic embryos increased exponentially, and globular and heart-shaped forms (Figs. 4 F & G), maturing into torpedo-stage embryos by the end of this phase exhibited a mixture of globular and heart shaped embryos (Figs. 4 F & G), maturing into torpedo stage towards the end of culture in GD4 (Fig. 4 H). Upon transfer to hormone-free germination medium, embryos elongated to form a distinct shoot apical meristems (SAM) and root apical meristems (RAM). These structures showed a prominent scutella node, which aligns with characteristic features in the cotyledonary stage in monocots (Figs. 4 I & J). The embryos showed dense cell division between the two meristems and highly vacuolated cells on the outer section supporting further cell division to develop a cotyledonary embryo. A distinct connective tissue between the SAM and RAM was observed (Fig. 4 K) and the prominent lobe of the SAM elongated to form the single cotyledonary structure, typical of monocot embryo germination (Fig. 4 L). Unlike previous observations (Manoharan et al., 2016 ), the current histological analysis revealed that the regenerated shoots were of somatic origin, not adventitious. The full spectrum of embryogenic stages including pro-embryo, globular, heart, torpedo and cotyledonary, was observed confirming that the optimized protocol supports true somatic embryogenesis suitable for transgene integration and gene editing. Validation of the optimized protocol in yam variety TDr 2579 The optimized protocol was successfully validated in yam variety TDr 2579, yielding results comparable to those observed in TDr 2436. Nodal explants of TDr 2579 initiated axillary bud formation within three days on SBM medium. Callus proliferation and somatic embryogenesis were significantly enhanced by supplementing CIM with proline, casein hydrolysate, and thiamine. Measurements taken on callus weight, percentage of embryogenic callus, and embryo count per explant were either equivalent to or exceeded those obtained with TDr 2436, affirming the consistency of the optimized conditions. FIM medium led to a higher frequency of FEC formation in TDr 2579 (13.4%) compared to GD medium (6.1%), consistent with results in TDr 2436. The application of callus meshing through a small pore-size steel sieve facilitated FEC development in both varieties. The addition of 10 mg/L ascorbic acid significantly reduced callus browning, improved the recovery time and re-established callus proliferation, mirroring the effects seen in TDr 2436. The development of somatic embryos in TDr 2579 progressed through the globular, heart, torpedo, and cotyledonary stages, and mature embryos successfully rooted and acclimatized with 75% survival in soil. The regenerated plantlets exhibited healthy growth, and no signs of soma clonal variation, highlighting the stability and applicability of the optimized protocol across different yam varieties. This validation confirms that the regeneration system developed for TDr 2436 can reliably induce FEC formation, reduce callus browning, and support high rates of plant regeneration in diverse yam varieties. Discussion The production of FECs is a highly desirable tissue for genetic manipulations at the single cell level due to their totipotent nature, loose organization, small size and high numbers. FECs not only support the rapid establishment of stable, and well-dispersed cell suspension cultures but can also serve as good sources of totipotent protoplast isolation (Wen et al., 2020 ). While Manoharan et al. ( 2016 ) reported nodular, dense embryogenic calli, the protocol developed in the current study obtained friable and mucilaginous structures with high proliferative capacity. Similar to previous studies in Litchi chinensis (Wang et al., 2023 ), date palm (Zein El Din et al., 2022), cassava ( Utsumi et al., 2017 ; Syombua et al., 2019 ), and maize (Kang et al., 2022 ), this research found that media additives such as antioxidants, casein hydrolysate, and L-thiamine enhance the embryogenic competence of tissue culture explants. Plants cells in tissue culture environments are prone to oxidative stress, resulting in elevated levels of reactive oxygen species (ROS) that harm cellular components and suppress growth. Exogenous amino acids scavenge ROS, hence provide osmoprotection, and promote cellular proliferation and differentiation (Hosseinifard et al., 2022). For instance, supplementing culre media with proline enhances somatic embryogenesis in maize and groundnut. The exogenous proline alleviates stress in maize embryos by optimizing respiratory metabolism and regulating hormone levels, thereby supporting embryo development under adverse conditions (Garrocho-Villegas et al., 2012; Zuo et al., 2022; Truong et al., 2023). Similarly, Jain et al. ( 2001 ) demonstrated that exogenous proline alleviates oxidative harm to the cellular membranes of groundnut and prevents salt induced decline in biomass. Likewise, supplementing with thiamine supports somatic embryogenesis by catalyzing pyruvate conversion to acetyl-CoA, thereby supplying the energy and carbon skeletons required for rapid cell proliferation and embryo formation (Dhillon et al., 2011 ). Utsumi et al. ( 2017 ) reported that thiamine fortification drives carbon flux from glycolysis into the TCA cycle, which in turn elevates the frequency of FEC formation. In another study, s upplementing maize callus induction media with thiamine improved the embryogenic competence of immature embryos and promoted the progression of somatic embryos into regeneration-competent tissues (Vilaça Vasconcelos et al., 2018 ). Our findings are consistent with these reports as thiamine-enriched media enhanced the formation of FECs, potentially by supporting the increased energy demands during early embryogenic stages. L-tyrosine, a precursor for numerous metabolic pathways involved in antioxidative processes and cell differentiation, has potential to enhance somatic embryogenesis. For instance, it is a precursor for polyamines, such as spermidine and spermine, which could promote osmotic adjustment, division of cells and differentiation of plant cultures. Tyrosine is also a precursor for the synthesis of secondary metabolites like phenolics and flavonoids, some of which have antioxidant properties and can help in reducing oxidative stress in the culture environment. Studies in cassava and pine have shown that adding L-tyrosine to the medium enhances FEC induction ( Nyaboga et al., 2015 ; Castander-Olarieta et al., 2019 ). In contrast, our yam cultures did not respond to tyrosine supplementation. This species-specific discrepancy likely reflects differences in metabolic pathways and in vitro responsiveness, and may be further explained by yam’s high endogenous tyrosinase activity, which rapidly oxidizes L-tyrosine and limits its beneficial effects (Ilesanmi et al., 2014 ; Ilesanmi & Adewale, 2020 ; Mulla et al., 2018 ). As such, this enzymatic activity could, reduce its bioavailability and negate any growth-promoting effects. Moreover, the catalysis products include black to brown to melanin precursors that could result in callus darkening and negatively impact cell viability (Lu et al., 2021 ). Wounding is another critical factor influencing callus competence. Mechanical wounding of callus triggers the release of signaling molecules and transcription factors that stimulate dedifferentiation, repair mechanisms, reprogram gene expression patterns and enhance proliferation (Yang et al., 2024 ; Ikeuchi et al., 2017 ). This study demonstrated that callus wounding by mesh crushing promoted the development of FECs, which concurs with reported findings in cassava ( Taylor et al., 2012 ; Nyaboga et al., 2015 ). Similarly, Wang et al. ( 2023 ) demonstrated that blending maize leaf sheath in a food processor significantly enhanced the regeneration of transformed plants, relative to manually chopped leaf segments. One of the long-standing hypotheses is that wounding induces jasmonate-mediated wound signaling, which promotes auxin synthesis, and, subsequently, improves regeneration efficiency (Zhang et al., 2019 ). Similar to callus crushing through a stainless-steel mesh, mechanical wounding by blending homogenizes the explants and ensures uniform wounding, promoting more consistent regeneration outcomes, compared to manually chopped explants. The current findings also suggest that small explant sizes could enhance the regeneration competence by increasing the surface area-to-volume ratio in blended tissue and ensuring better exposure to growth regulators and nutrients in media (Bennur et al., 2024 ). Besides mechanical damage, rinsing meshed calli in liquid CIM and gently blotting them on sterile tissue was key to reducing browning and necrosis. Supplementing the wash solution with ascorbic acid—a water-soluble antioxidant widely used to scavenge reactive oxygen species in vitro—further limited tissue discoloration (Hazubska-Przybył et al., 2024a ). Yam tissues release high levels of phenolic compounds (e.g., saponins, gracillin, diosgenin, dioscin, catechins) (Syombua et al., 2022 ) that, when leached into the medium, can darken it, inhibit growth regulators, and impede somatic embryogenesis. To remove these exudates, adsorbents like activated charcoal, polyvinylpyrrolidone, or ion-exchange resins can be added (Syombua et al., 2019 ).. Beyond its antioxidant role, ascorbic acid also promotes cell division and elongation, enhancing in vitro growth, a benefit documented in Norway spruce, faba bean, Musa, orchids, and Jatropha cultures (Hazubska-Przybył et al., 2024 Abdelwahd et al., 2008 ; Adero et al., 2023 ; Chugh et al., 2009 ; He et al., 2009 ). Our results clearly demonstrate that FIM medium outperforms GD, full-strength MS, and half-strength MS in driving FEC induction. FIM’s reduced nitrate, phosphate, and potassium levels combined with elevated thiamine create an environment uniquely favorable for embryogenic callus. Notably, neither half- nor full-strength MS supported any FEC formation, underscoring the critical roles that specific mineral ratios play in yam somatic embryo development. Furthermore, both GD and FIM media are richer in vitamins and amino acids than MS, suggesting that high levels of these supplements are also indispensable for generating friable, embryogenic callus. Moreover, because FIM (Utsumi et al., 2017 ) and GD (Gresshoff & Doy, 1974 ) both contain much greater concentrations of vitamins and casamino acids than MS, it suggests that abundant vitamin and amino acid supplementation is also essential for FEC development. The presence of excess thiamine in the medium further supports energy metabolism, which is crucial for the rapid cell proliferation needed for FEC formation and proliferation. Similar observations have been made in sorghum and Norway spruce, where the phosphorus and nitrogen levels in culture medium influenced morphogenesis and somatic embryogenesis (Elkonin & Pakhomova, 2000 ; Carlsson et al., 2017 ). Hence, fine-tuning the mineral and vitamin composition of the culture medium may be pivotal for enabling regeneration in recalcitrant species such as yam. Conclusion This article reports pioneering research for generation, proliferation and regeneration of FECs in the Dioscorea species. It presents a step-by‐step protocol covering the formation of organized primary embryogenic structures, FEC induction, embryo maturation, germination, rooting, shoot elongation, and final acclimatization. All the key steps in FEC induction, embryo maturation, and plantlet regeneration were optimized to enhance embryogenic competence and reduce tissue necrosis, notably through nutrient modifications and media supplementation with antioxidants. The findings underscore FIM's effectiveness in FEC formation compared to conventional media, likely due to reduced nitrate and phosphate levels. The study further introduces a novelty in yam FEC establishment, specifically callus meshing to induce tissue reprogramming and modify the tissue gene expression patterns. Importantly, FEC-derived plantlets remained genetically stable even after soil acclimatization and exhibited no somaclonal variation. This optimized protocol, therefore, provides a robust foundation for advanced genetic work in yam in yam and supports applications such as protoplast isolation and regeneration, as well as transgenic or genome-editing approaches. Declarations Author contributions L.T conceived the idea, E.S, J.T and L.T designed the study, E.S performed the experiments, J.T and L.T supervised the experiment, E.S and J.T analyzed the data, E.S wrote the manuscript, J.T and L.T revised the manuscript and L.T sourced the research funding. All authors have read and agreed to the published version of the manuscript. Data Availability Statement All study data that support the findings are included in the article or supplementary data. Acknowledgments The authors thank Professor Steven Runo and Dorothy Mbuvi at Kenyatta University for providing equipment and training on histological analysis of plant tissues. This project was supported by International Institute of Tropical Agriculture (IITA). Conflicts of Interest The authors declare no conflict of interest. References Abdelwahd, R., Hakam, N., Labhilili, M., & Udupa, S. M. (2008). Use of an adsorbent and antioxidants to reduce the effects of leached phenolics in in vitro plantlet regeneration of faba bean. African Journal of Biotechnology , 7 (8), Article 8. https://doi.org/10.4314/ajb.v7i8.58590 Adero, M., Tripathi, J. N., & Tripathi, L. (2023). Advances in Somatic Embryogenesis of Banana. International Journal of Molecular Sciences , 24 (13), 10999. https://doi.org/10.3390/ijms241310999 Bennur, P. L., O’Brien, M., Fernando, S. C., & Doblin, M. S. (2024). Improving transformation and regeneration efficiency in medicinal plants: Insights from other recalcitrant species. Journal of Experimental Botany , erae189. https://doi.org/10.1093/jxb/erae189 Carlsson, J., Svennerstam, H., Moritz, T., Egertsdotter, U., & Ganeteg, U. (2017). Nitrogen uptake and assimilation in proliferating embryogenic cultures of Norway spruce—Investigating the specific role of glutamine. PLOS ONE , 12 (8), e0181785. https://doi.org/10.1371/journal.pone.0181785 Castander-Olarieta, A., Montalbán, I. A., De Medeiros Oliveira, E., Dell’Aversana, E., D’Amelia, L., Carillo, P., Steiner, N., Fraga, H. P. D. F., Guerra, M. P., Goicoa, T., Ugarte, M. D., Pereira, C., & Moncaleán, P. (2019). Effect of Thermal Stress on Tissue Ultrastructure and Metabolite Profiles During Initiation of Radiata Pine Somatic Embryogenesis. Frontiers in Plant Science , 9 , 2004. https://doi.org/10.3389/fpls.2018.02004 Chugh, S., Guha, S., & Rao, I. U. (2009). Micropropagation of orchids: A review on the potential of different explants. Scientia Horticulturae , 122 (4), 507–520. https://doi.org/10.1016/j.scienta.2009.07.016 Croptrust. (2024). Plan for Bigger, Better Yams—Crop Trust . Darkwa, K., Agre, P., Olasanmi, B., Iseki, K., Matsumoto, R., Powell, A., Bauchet, G., De Koeyer, D., Muranaka, S., Adebola, P., Asiedu, R., Terauchi, R., & Asfaw, A. (2020). Comparative assessment of genetic diversity matrices and clustering methods in white Guinea yam ( Dioscorea rotundata ) based on morphological and molecular markers. Scientific Reports , 10 (1), Article 1. https://doi.org/10.1038/s41598-020-69925-9 Dhillon, R. S., Hooda, M. S., Pundeer, J. S., Ahlawat, K. S., & Chopra, I. (2011). Effects of auxins and thiamine on the efficacy of techniques of clonal propagation in Jatropha curcas L. Biomass and Bioenergy , 35 (4), 1502–1510. https://doi.org/10.1016/j.biombioe.2010.12.017 Elkonin, L. A., & Pakhomova, N. V. (2000). Influence of nitrogen and phosphorus on induction embryogenic callus of sorghum. Plant Cell, Tissue and Organ Culture , 61 (2), 115–123. https://doi.org/10.1023/A:1006472418218 Gambino, G., Moine, A., Boccacci, P., Perrone, I., & Pagliarani, C. (2021). Somatic embryogenesis is an effective strategy for dissecting chimerism phenomena in Vitis vinifera cv Nebbiolo. Plant Cell Reports , 40 (1), 205–211. https://doi.org/10.1007/s00299-020-02626-9 Gresshoff, P. M., & Doy, C. H. (1974). Derivation of a haploid cell line from Vitis vinifera and the importance of the stage of meiotic development of anthers for haploid culture of this and other genera. Zeitschrift Für Pflanzenphysiologie , 73 (2), 132–141. https://doi.org/10.1016/S0044-328X(74)80084-X Hazubska-Przybył, T., Obarska, A., Konecka, A., Kijowska-Oberc, J., Wawrzyniak, M. K., Piotrowska-Niczyporuk, A., Staszak, A. M., & Ratajczak, E. (2024a). Modulating ascorbic acid levels to optimize somatic embryogenesis in Picea abies (L.) H. Karst. Insights into oxidative stress and endogenous phytohormones regulation. Frontiers in Plant Science , 15 , 1372764. https://doi.org/10.3389/fpls.2024.1372764 Hazubska-Przybył, T., Obarska, A., Konecka, A., Kijowska-Oberc, J., Wawrzyniak, M. K., Piotrowska-Niczyporuk, A., Staszak, A. M., & Ratajczak, E. (2024b). Modulating ascorbic acid levels to optimize somatic embryogenesis in Picea abies (L.) H. Karst. Insights into oxidative stress and endogenous phytohormones regulation. Frontiers in Plant Science , 15 , 1372764. https://doi.org/10.3389/fpls.2024.1372764 He, Y., Guo, X., Lu, R., Niu, B., Pasapula, V., Hou, P., Cai, F., Xu, Y., & Chen, F. (2009). Changes in morphology and biochemical indices in browning callus derived from Jatropha curcas hypocotyls. Plant Cell, Tissue and Organ Culture (PCTOC) , 98 (1), 11–17. https://doi.org/10.1007/s11240-009-9533-y Hosseinifard, M., Stefaniak, S., Javid, M. G., Soltani, E., Wojtyla, Ł., & Garnczarska, M. (n.d.). Contribution of Exogenous Proline to Abiotic Stresses Tolerance in Plants: A Review . Ikeuchi, M., Iwase, A., Rymen, B., Lambolez, A., Kojima, M., Takebayashi, Y., Heyman, J., Watanabe, S., Seo, M., Veylder, L. D., Sakakibara, H., & Sugimoto, K. (2017). Wounding Triggers Callus Formation via Dynamic Hormonal and Transcriptional Changes. Plant Physiology , 175 (3), 1158–1174. https://doi.org/10.1104/pp.17.01035 Ilesanmi, O. S., & Adewale, I. O. (2020). Physicochemical properties of free and immobilized tyrosinase from different species of yam (Dioscorea spp). Biotechnology Reports , 27 , e00499. https://doi.org/10.1016/j.btre.2020.e00499 Ilesanmi, O. S., Ojopagogo, Y. A., & Adewale, I. O. (2014). Kinetic characteristics of purified tyrosinase from different species of Dioscorea (yam) in aqueous and non-aqueous systems. Journal of Molecular Catalysis B: Enzymatic , 108 , 111–117. https://doi.org/10.1016/j.molcatb.2014.07.009 Jain, M., Mathur, G., Koul, S., & Sarin, N. (2001). Ameliorative effects of proline on salt stress-induced lipid peroxidation in cell lines of groundnut (Arachis hypogaea L.). Plant Cell Reports , 20 (5), 463–468. https://doi.org/10.1007/s002990100353 Kang, M., Lee, K., Finley, T., Chappell, H., Veena, V., & Wang, K. (2022). An Improved Agrobacterium-Mediated Transformation and Genome-Editing Method for Maize Inbred B104 Using a Ternary Vector System and Immature Embryos. Frontiers in Plant Science , 13 , 860971. https://doi.org/10.3389/fpls.2022.860971 Kole, C. (Ed.). (2022a). Genomic Designing for Abiotic Stress Resistant Technical Crops . Springer International Publishing. https://doi.org/10.1007/978-3-031-05706-9 Kole, C. (Ed.). (2022b). Genomic Designing for Biotic Stress Resistant Technical Crops . Springer International Publishing. https://doi.org/10.1007/978-3-031-09293-0 Lin, H.-S., van der Toorn, C., Raemakers, K. J. J. M., Visser, R. G. F., De Jeu, M. J., & Jacobsen, E. (2000). Development of a plant regeneration system based on friable embryogenic callus in the ornamental Alstroemeria. Plant Cell Reports , 19 (5), 529–534. https://doi.org/10.1007/s002990050768 Lu, Y., Xu, Y., Song, M.-T., Qian, L.-L., Liu, X.-L., Gao, R.-Y., Han, R.-M., Skibsted, L. H., & Zhang, J.-P. (2021). Promotion effects of flavonoids on browning induced by enzymatic oxidation of tyrosinase: Structure–activity relationship. RSC Advances , 11 (23), 13769–13779. https://doi.org/10.1039/D1RA01369F Manoharan, R., Tripathi, J. N., & Tripathi, L. (2016). Plant regeneration from axillary bud derived callus in white yam (Dioscorea rotundata). Plant Cell, Tissue and Organ Culture (PCTOC) , 126 (3), 481–497. https://doi.org/10.1007/s11240-016-1017-2 Mulla, T., Patil, S., & Jadhav, J. (2018). Exploration of surface plasmon resonance for yam tyrosinase characterization. International Journal of Biological Macromolecules , 109 , 399–406. https://doi.org/10.1016/j.ijbiomac.2017.12.074 Nyaboga, E. N., Njiru, J. M., & Tripathi, L. (2015). Factors influencing somatic embryogenesis, regeneration, and Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) cultivar TME14. Frontiers in Plant Science , 6 . https://doi.org/10.3389/fpls.2015.00411 Syombua, E. D., Adero, M. O., Mbinda, W. M., Wanyonyi, C. N., Ngugi, M. P., & Oduor, R. O. (2021). A system for rapid gene introgression into cassava immature leaves and subsequent recovery of transformed lines. Plant Biotechnology Reports . https://doi.org/10.1007/s11816-020-00654-1 Syombua, E. D., Tripathi, J. N., Obiero, G. O., Nguu, E. K., Yang, B., Wang, K., & Tripathi, L. (2022). Potential applications of the CRISPR/Cas technology for genetic improvement of yam ( Dioscorea spp.). Food and Energy Security , 11 (1), e330. https://doi.org/10.1002/fes3.330 Syombua, E. D., Wanyonyi, C. N., Adero, M. O., Mbinda, W. M., Ngugi, M. P., Alakonya, A. E., & Oduor, R. O. (n.d.). Explant type and hormone regime influences somatic embryogenesis and regeneration in cassava . Syombua, E. D., Wanyonyi, C. N., Adero, M. O., Mbinda, W. M., Ngugi, M. P., Alakonya, A. E., & Oduor, R. O. (2019). Explant type and hormone regime influences somatic embryogenesis and regeneration of cassava. African Journal of Biotechnology , 18 (25), 532–539. https://doi.org/10.5897/AJB2019.16853 Syombua, E. D., Zhang, Z., Tripathi, J. N., Ntui, V. O., Kang, M., George, O. O., Edward, N. K., Wang, K., Yang, B., & Tripathi, L. (2021). A CRISPR/Cas9‐based genome‐editing system for yam ( Dioscorea spp.). Plant Biotechnology Journal , 19 (4), 645–647. https://doi.org/10.1111/pbi.13515 Taylor, N., Gaitán-Solís, E., Moll, T., Trauterman, B., Jones, T., Pranjal, A., Trembley, C., Abernathy, V., Corbin, D., & Fauquet, C. M. (2012). A High-throughput Platform for the Production and Analysis of Transgenic Cassava (Manihot esculenta) Plants. Tropical Plant Biology , 5 (1), 127–139. https://doi.org/10.1007/s12042-012-9099-4 Taylor, N. J., Edwards, M., Kiernan, R. J., Davey, C. D. M., Blakesley, D., & Henshaw, G. G. (1996). Development of friable embryogenic callus and embryogenic suspension culture systems in cassava ( Manihot esculenta Crantz). Nature Biotechnology , 14 (6), Article 6. https://doi.org/10.1038/nbt0696-726 Truong Thi Lan Anh a b c, , Nguyen Thi Nhu Mai a, , Hoang Thanh Tung d, , Hoang Dac Khai a, , Do Manh Cuong a, , Vu Quoc Luan a, , Hoang Thi Nhu Phuong c, , Nguyen Van Binh c, , Bui Van The Vinh e, , Nguyen Thi Thanh Thuy a, … Duong Tan Nhut a. (n.d.). Effect of spermidine, glutamine, and proline on somatic embryogenesis and silver nanoparticles supplied culture improved rhizome formation of Panax vietnamensis var. Langbianensis . Utsumi, Y., Utsumi, C., Tanaka, M., Ha, V. T., Matsui, A., Takahashi, S., & Seki, M. (2017). Formation of friable embryogenic callus in cassava is enhanced under conditions of reduced nitrate, potassium and phosphate. PLOS ONE , 12 (8), e0180736. https://doi.org/10.1371/journal.pone.0180736 Verónica Garrocho-Villegas 1, María Teresa de Jesús-Olivera, Estela Sánchez Quintanar. (n.d.). Maize somatic embryogenesis: Recent features to improve plant regeneration . Vilaça Vasconcelos, M. J., Antunes, M. S., De Oliveira, M. F., Lopes, M. A., & Fontes Figueiredo, J. E. (2018). CALLUS INDUCTION AND PLANT REGENERATION FROM IMMATURE EMBRYOS CULTURE OF TROPICAL MAIZE. Revista Brasileira de Milho e Sorgo , 17 (3), 359. https://doi.org/10.18512/1980-6477/rbms.v17n3p359-368 Wang, G., Liu, Y., Gao, Z., Li, H., & Wang, J. (2023). Effects of Amino Acids on Callus Proliferation and Somatic Embryogenesis in Litchi chinensis cv. ‘Feizixiao.’ Horticulturae , 9 (12), 1311. https://doi.org/10.3390/horticulturae9121311 Wang, N., Ryan, L., Sardesai, N., Wu, E., Lenderts, B., Lowe, K., Che, P., Anand, A., Worden, A., Van Dyk, D., Barone, P., Svitashev, S., Jones, T., & Gordon-Kamm, W. (2023). Leaf transformation for efficient random integration and targeted genome modification in maize and sorghum. Nature Plants . https://doi.org/10.1038/s41477-022-01338-0 Wang, S., Wang, G., Li, H., Li, F., & Wang, J. (2023). Agrobacterium tumefaciens-mediated transformation of embryogenic callus and CRISPR/Cas9-mediated genome editing in ‘Feizixiao’ litchi. Horticultural Plant Journal , 9 (5), 947–957. https://doi.org/10.1016/j.hpj.2023.01.011 Wen, F., Su, W., Zheng, H., Yu, B., Ma, Z., Zhang, P., & Guo, W. (2020). Plant regeneration via protoplast electrofusion in cassava. Journal of Integrative Agriculture , 19 (3), 632–642. https://doi.org/10.1016/S2095-3119(19)62711-5 Yang, W., Zhai, H., Wu, F., Deng, L., Chao, Y., Meng, X., Chen, Q., Liu, C., Bie, X., Sun, C., Yu, Y., Zhang, X., Zhang, X., Chang, Z., Xue, M., Zhao, Y., Meng, X., Li, B., Zhang, X., … Li, C. (2024). Peptide REF1 is a local wound signal promoting plant regeneration. Cell , 187 (12), 3024-3038.e14. https://doi.org/10.1016/j.cell.2024.04.040 Zein El Din, A. F. M., Darwesh, R. S. S., Ibrahim, M. F. M., Salama, G. M. Y., Shams El-Din, I. M., Abdelaal, W. B., Ali, G. A., Elsayed, M. S., Ismail, I. A., Dessoky, E. S., & Abdellatif, Y. M. R. (2022). Antioxidants Application Enhances Regeneration and Conversion of Date Palm (Phoenix dactylifera L.) Somatic Embryos. Plants , 11 (15), 2023. https://doi.org/10.3390/plants11152023 Zhang, G., Zhao, F., Chen, L., Pan, Y., Sun, L., Bao, N., Zhang, T., Cui, C.-X., Qiu, Z., Zhang, Y., Yang, L., & Xu, L. (2019). Jasmonate-mediated wound signalling promotes plant regeneration. Nature Plants , 5 (5), 491–497. https://doi.org/10.1038/s41477-019-0408-x Zuo, S., Li, J., Gu, W., & Wei, S. (n.d.). Exogenous Proline Alleviated Low Temperature Stress in Maize Embryos by Optimizing Seed Germination, Inner Proline Metabolism, Respiratory Metabolism and a Hormone Regulation Mechanism . Supplementary Files Supplementaryinformation27April2025.docx Supplementary information: Supporting information includes Figure S1 Schematic presentation of the direct embryogenesis protocol previously used in our lab and the timelines for each culture stage; Table S1: Media formulations used for FEC development and regeneration in yam; Table S2: Medium components used to optimize FEC regenerations. Cite Share Download PDF Status: Published Journal Publication published 24 Sep, 2025 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted Reviewers agreed at journal 17 May, 2025 Reviewers invited by journal 12 May, 2025 Editor assigned by journal 10 May, 2025 First submitted to journal 07 May, 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-6539506","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":455586677,"identity":"385502dd-6b3a-4452-b2b5-1104feef9cf6","order_by":0,"name":"Easter D. 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Tripathi","email":"","orcid":"","institution":"International Institute of Tropical Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Jaindra","middleName":"N.","lastName":"Tripathi","suffix":""},{"id":455586679,"identity":"9a6c5bfe-13e8-4715-af0e-b5cef1b8519d","order_by":2,"name":"Leena Tripathi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIie3RuwrCMBSA4VMOpEu0a6Q+xAHBy+KztAiuugteELq5+xiCII5CwC4FX0ERxLEFBwUHk9LBKdZNMD/kkCEfCQTAZvvBqnoEahHiTs0ShAHmRBCy4AsCmgAvcz4n3j49bbvjlsuz7DbsDsBdnK8fbsFlmPREZ15Z+3XqdaY8brY/EQgjFCQrGzWRQPSZ8YkFmSjCL4pMShOpCXNSkprg0UgQGxAmcW0lWcMHionxPTMJ8NzZ2XlsRx4d5Cm7P0fkuRGmRqO/xZkWe54/FZgwEngjzr2g5ltsNpvt33oBfP84w1Fnq+QAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-5723-4981","institution":"International Institute of Tropical Agriculture","correspondingAuthor":true,"prefix":"","firstName":"Leena","middleName":"","lastName":"Tripathi","suffix":""}],"badges":[],"createdAt":"2025-04-27 09:36:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6539506/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6539506/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11240-025-03194-0","type":"published","date":"2025-09-24T15:58:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82783846,"identity":"b1cf0c84-9ab8-40be-b82e-3674c86e37d1","added_by":"auto","created_at":"2025-05-15 08:52:12","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":114177,"visible":true,"origin":"","legend":"\u003cp\u003eSequential development of friable embryogenic callus (FEC) in yam accession TDr 2436. A: Nodal explants cultured in shoot bud induction medium (SBM). B: Callus derived from auxiliary buds on callus induction medium (CIM) 1. C: Meshing of callus through 1–2 mm size metal wire mesh. D: Blotting of meshed callus using sterile tissuee papers to remove excess moisture. E: Meshed callus cultured in CIM 2 medium for initial recovery. F: Appearance of meshed callus in E after one-week culture in CIM 2, showing browning, G: Meshed callus after eight weeks in CIM 2 with gradual recovery and proliferation. H: Callus transferred to Gresshoff and Doy (GD) 1 medium for further development, showing budding from the initial recovery callus formed in G. I: Callus in GD 2 after two weeks, showing organized cell structures and increased proliferation. J: Callus in GD 3 with visible FEC clusters. K: FEC clusters in GD 4 with pronounced proliferation and mucilaginous FEC characteristics. L: Fully developed FEC in GD 4 with an apparent friable texture. Scale bars; A, B, E and F 5 mm; C, D, G, H, I, J, K, and L 1 cm.\u003c/p\u003e","description":"","filename":"Figure127April2025.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6539506/v1/d8419f97187e7d8852829ec3.jpg"},{"id":82783849,"identity":"b2b1e795-9427-4039-87dd-097be725fb87","added_by":"auto","created_at":"2025-05-15 08:52:12","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":48870,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of basal salt composition on friable embryogenic callus (FEC) production. The frequency was determined by expressing the number of callus explants producing FEC clusters initiated in CIM2 (as shown in Fig. 1E) as a percentage of the total number of callus explants put on CIM2. FEC clusters were identified by their characteristic friable morphology, as illustrated in Figure 1K. Means with different letters are significantly different by Duncan’s multiple range test at P\u0026lt;0.05. Error bars represent standard error of the mean.\u003c/p\u003e","description":"","filename":"Figure227April2025.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6539506/v1/9313db1e874446558dd1ef27.jpg"},{"id":82784823,"identity":"eb756033-5196-42a9-b46d-9185c127c39e","added_by":"auto","created_at":"2025-05-15 09:00:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":123571,"visible":true,"origin":"","legend":"\u003cp\u003eProliferation, maturation, and regeneration of friable embryogenic callus (FEC). A:\u003cstrong\u003e \u003c/strong\u003eFECs proliferating in GD5 medium; B: Microscopic observation of FECs in GD5 beginning to mature, as indicated by the colour change. C: FECs in activated charcoal medium. D: Microscopic observation of a single embryo with a distinct root apical meristem (RAM) and shoot apical meristem (SAM). E: FEC maturation in BAP medium, arrows show green cotyledonary embryos. F: Green cotyledonary embryos sub-cultured onto maturation medium 2. G \u0026amp; H: Single and fused cotyledonary embryos; I: Cotyledonary embryos rooting in YBM medium. J: Plantlets regenerated from FECs well established in the soil. Scale bars; A, B, C, E, F and J 5 mm; D, G, H 50 µm, I 1cm.\u003c/p\u003e","description":"","filename":"Figure327April2025.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6539506/v1/42505ce5ae50c6da8cb49fa0.jpg"},{"id":82786238,"identity":"5a7e1c3d-2d9e-46b5-b29c-ef8929480429","added_by":"auto","created_at":"2025-05-15 09:16:12","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":256588,"visible":true,"origin":"","legend":"\u003cp\u003eHistological characteristic of Friable embryogenic callus (FEC) during induction, proliferation and germination in yam. A: Transverse section of non-embryogenic callus formed after two weeks in CIM1, showing loosely arranged cells with large vacuoles and irregular shapes. B: Embryogenic regions with prominent pro-embryonic masses (PEM) and starch grains (SG), originating mainly from the tip of the axillary bud. C: Organized cell structure in recovering tissues from GD1, with small, compact cells, dense cytoplasm, and beginning of protoderm formation. D: Globular embryogenic structures in GD2 attached to mother tissue. E: Proliferating globular embryo from GD2. F: Dividing embryogenic cells forming globular and heart-shaped structures. G \u0026amp; H: Advanced heart-shaped somatic embryos with densely cytoplasmic cells in GD3. I \u0026amp; J: torpedo stage embryos in GD4, showing increased cell density and organization to form shoot apical meristem (SAM) and root apical meristem (RAM). K: Development of a bipolar structure with distinct SAM and RAM, preparing for cotyledonary stage. L: Cotyledonary stage in monocots with single cotyledon structure forming, characterized by prominent SAM lobe and scutellar node.\u003c/p\u003e","description":"","filename":"Figure427April2025.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6539506/v1/0ac1476d299a8a1093c67d4e.jpg"},{"id":82783852,"identity":"4f4b52a5-dbef-467f-a837-77a2b7370d06","added_by":"auto","created_at":"2025-05-15 08:52:12","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":74053,"visible":true,"origin":"","legend":"\u003cp\u003eSynopsis of morphogenic pathways involved in yam regeneration by direct embryogenesis (Manoharan et al., 2016) and the FEC protocol optimized in the current study.\u003c/p\u003e","description":"","filename":"Figure527April2025.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6539506/v1/e5b723b89084c356652579b4.jpg"},{"id":92430582,"identity":"62bd39ab-99e2-4cbf-a499-f0c34b4f868d","added_by":"auto","created_at":"2025-09-29 16:06:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1625017,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6539506/v1/1429cbcc-1276-4aae-a913-1a2651a5dd42.pdf"},{"id":82785370,"identity":"68bcde09-2762-43a4-a21a-b74408dd9ab8","added_by":"auto","created_at":"2025-05-15 09:08:12","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":72926,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary information: \u003c/strong\u003eSupporting information includes Figure S1 Schematic presentation of the direct embryogenesis protocol previously used in our lab and the timelines for each culture stage; Table S1: Media formulations used for FEC development and regeneration in yam; Table S2: Medium components used to optimize FEC regenerations.\u003c/p\u003e","description":"","filename":"Supplementaryinformation27April2025.docx","url":"https://assets-eu.researchsquare.com/files/rs-6539506/v1/fa2c1c87b140a94caa1d0b52.docx"}],"financialInterests":"","formattedTitle":"Enhancing Regeneration in White Yam (Dioscorea rotundata) Through Friable Embryogenic Callus","fulltext":[{"header":"Key message","content":"\u003cp\u003eReducing nitrogen in culture media and mechanical wounding of callus significantly enhance friable embryogenic calli (FEC) induction and somatic embryo regeneration in white yam (\u003cem\u003eD. rotundata\u003c/em\u003e), providing a robust platform for genetic transformation and accelerated breeding.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eYams (genus \u003cem\u003eDioscorea\u003c/em\u003e) are monocotyledonous vines that yield edible tubers and are widely grown in the tropics. It has about 600 species, of which only 11 species are widely cultivated as a staple food. \u003cem\u003eDioscorea cayenensis\u0026ndash;D. rotundata\u003c/em\u003e (the Guinea yam complex) ranks among the world\u0026rsquo;s most vital tuber crop, providing both food sustainability and economic livelihoods across the subtropical and tropical (CropTrust, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Other commercially important species include white, purple, yellow, aerial, Chinese yam and bitter yam, among which the white yam contributes to about 90% of global yam output (Darkwa et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eYam productivity is greatly impacted by various pressures, including susceptibility to pests and diseases (nematodes, viruses, fungi, bacteria, etc.), drought, and low soil fertility, leading to poor quality produce and substantial yield losses. These constraints emphasize the need for integrated strategies to ensure sustained production and stable income for smallholder farmers (Kole, 2022). The development and deployment of diseases and pest resistant varieties remain the gold standard for boosting crop productivity. Current yam improvement initiatives rely on conventional breeding, which is not only labor intensive and time consuming, but is also highly constrained due to the crop\u0026rsquo;s complex genetic make-up (Kole, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). It is, therefore, essential to integrate breeding efforts with modern biotechnological approaches like \u003cem\u003ein vitro\u003c/em\u003e culture systems and gene editing (Syombua, Zhang, et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSuccessful genetic transformation requires introducing DNA into tissue capable of regenerating as a cohesive, uniform plant. However, existing yam protocols generate both somatic embryos and organogenic shoots in the same culture (Manoharan et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), making selection difficult and often producing chimeric plants. While true somatic embryos originating from single cells would solve this, isolating a homogeneous population of them in \u003cem\u003eD. rotundata\u003c/em\u003e remains a major hurdle. Because somatic embryos originate from single cells, they avoid the chimerism often seen when shoots regenerate, so they are the preferred target for stable transformation (Gambino et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To harness this pathway in \u003cem\u003eD. rotundata\u003c/em\u003e, we, therefore, needed to refine our protocol to enrich exclusively for homogeneous embryogenic callus clusters.\u003c/p\u003e \u003cp\u003eFriable embryogenic callus (FEC) meets those criteria; its loose, single-cell\u0026ndash;derived clusters can be maintained in suspension, and nearly every cell remains totipotent (Taylor et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). FEC has proven effective not just in cassava (Syombua, Adero, et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) but also in litchi (Wang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and Alstroemeria (Lin et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), making it the gold standard tissue for direct gene transfer systems and downstream applications such as protoplast isolation and cell-level manipulation (Bull et al., 2009; Wen et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBuilding on the earlier somatic embryogenesis system by Manoharan et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), this research aimed to optimize a system for generating FECs in \u003cem\u003eDioscorea rotundata.\u003c/em\u003e Through a series of carefully controlled experiments, the key parameters influencing secondary somatic embryogenesis, including basal salt composition, mechanical wounding, antioxidant treatment, and amino acid supplementation were evaluated. These modifications successfully mitigated tissue necrosis and encouraged cellular reprogramming and FEC regeneration. This study reports on the successive enrichment of primary somatic embryos, maintenance, proliferation and subsequent progression to whole plantlets. This system of generating totipotent callus tissue offers a foundation for advanced genetic studies to facilitate gene editing, trait improvement, and ultimately contributing to the development of resilient yam cultivars.\u003c/p\u003e"},{"header":"Experimental Procedures","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material and culture maintenance\u003c/h2\u003e \u003cp\u003eSterile cultures of the yam (\u003cem\u003eDioscorea rotundata\u003c/em\u003e) accessions TDr 2579 and TDr 2436 were sourced from IITA\u0026rsquo;s germplasm collection (Ibadan, Nigeria) and maintained \u003cem\u003ein vitro\u003c/em\u003e on the media YBM (Yam Basic Medium). YBM comprised Murashige \u0026amp; Skoog salts supplemented with 3% sucrose, 2 mg/L glycine, 100 mg/L myo-inositol, 0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxine-HCl, 0.1 mg/L thiamine-HCl, and solidified with 2.4 g/L Gelrite. The pH was adjusted to 5.8 before autoclaving for 15 min at 121\u0026deg;C. Cultures were sub cultured every 4\u0026ndash;8 weeks and incubated at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C under a 16 h light/8 h dark photoperiod (30 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInitiation of primary embryogenesis\u003c/h3\u003e\n\u003cp\u003eWe cultured nodal segments following the protocol of Utsumi et al (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) to initiate the formation of primary somatic embryos. Briefly, sterile single-node segments measuring approximately 15mm were excised from four to eight-week-old plantlets were placed on SBM (Shoot Bud Induction Medium) and maintained in the dark at 28\u0026deg;C for 72 hours.\u003c/p\u003e \u003cp\u003eSBM comprised MS salts plus 2 \u0026micro;M CuSO₄, 1 mg/L 6-benzylaminopurine, 2% sucrose, 0.3% Gelrite, and the standard vitamin\u0026ndash;glycine mix (0.5 mg/L each nicotinic acid and pyridoxine-HCl, 100 mg/L myo-inositol, 2 mg/L glycine, and 0.1 mg/L thiamine-HCl). Buds that formed were excised with a sterile syringe under magnification and moved to CIM (Callus Induction Medium), which had a similar composition to SBM except using 0.5 mg L⁻\u0026sup1; picloram in place of BAP, and incubated in darkness at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for four weeks.\u003c/p\u003e\n\u003ch3\u003eBasal medium optimization for enhanced embryogenesis\u003c/h3\u003e\n\u003cp\u003eTo identify the optimal nutrient regime for embryo induction, three basal media were compared: full-strength MS, half-strength MS, and a custom FEC Induction Medium (FIM; see Supplementary Table S1). FIM was formulated by reducing nitrate, phosphate, and potassium concentrations in MS salts and adding 10 mg L⁻\u0026sup1; thiamine-HCl. For each medium, 100 explants (in triplicate) were scored after four weeks for the percentage forming direct somatic embryo-like structures (DSLS), the callus fresh weight (mg), and the quantity of DSLS in each explant.\u003c/p\u003e\n\u003ch3\u003eSecondary embryogenesis and wounding treatments\u003c/h3\u003e\n\u003cp\u003eSecondary somatic embryos were produced by subculturing primary embryos onto media containing 0.5 mg/L picloram. Four formulations of basal salt mixtures (Gresshoff \u0026amp; Doy [GD] premix, FIM, full- and half-strength MS) were tested. Each experiment consisted of ten plates per treatment, each with ten embryos, and were maintained at 26\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C in the dark for up to six months. To assess the impact of mechanical injury, callus was either passed through an approximately 2 mm stainless-steel mesh (Nyaboga et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) or gently teased apart into ~\u0026thinsp;2\u0026ndash;5 cm pieces. Control calli were transferred without wounding. All treatments were performed in triplicate.\u003c/p\u003e\n\u003ch3\u003eCallus washing and antioxidant/tyrosine treatments\u003c/h3\u003e\n\u003cp\u003eMeshed callus (1.5 g per plate) was collected into sterile tubes and rinsed three times in one of: (i) water; (ii) liquid MS; (iii) liquid MS\u0026thinsp;+\u0026thinsp;10 mg/L ascorbic acid; or (iv) liquid MS\u0026thinsp;+\u0026thinsp;20 mg/L ascorbic acid. After each rinse, callus was gently blotted dry on sterile tissue. Washed and unwashed calli were then plated onto GD media supplemented with 0.5 mg/L picloram; an additional set of washed calli was cultured on GD supplemented with 0.5 mg/L picloram plus 12 mg/L L-tyrosine. Two weeks later, tissue browning and necrosis were evaluated through visual observation on a 0\u0026ndash;5 scale.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGermination and acclimatization of FEC-derived embryos\u003c/h2\u003e \u003cp\u003eAfter FEC-derived embryos reached the torpedo or cotyledon stage, they were transferred to 1% charcoal-amended, MS medium without hormones and cultured for 45 days at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C in a 16 h light/8 h growth chamber. Mature embryos were then shifted to MS supplemented with10 mg/L ascorbic acid and 0.4 mg/L BAP for final development, followed by rooting on YBM. Rooted plantlets were hardened in peat-filled cups covered with plastic bags; openings were introduced gradually over seven days before potting into a 1:1 peat:soil mix in the greenhouse.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistological analysis\u003c/h3\u003e\n\u003cp\u003eSamples of non-embryogenic callus, embryogenic calli, and FECs were fixed in Carnoy\u0026rsquo;s solution (60% ethanol, 30% chloroform, 10% acetic acid), dehydrated through a graded ethanol series (70\u0026ndash;100%), then infiltrated with Technovit 7100 resin (2:1 \u0026rarr; 1:1 \u0026rarr; 1:2 ethanol:resin, 30 min each, vacuum). After an overnight pure-resin infiltration, tissues were embedded, sectioned at 5 \u0026micro;m (Leica RM 2155), stained with neutral red or 0.05% toluidine blue, and imaged on an Optika Vision Lite 2.1 microscope.\u003c/p\u003e\n\u003ch3\u003eValidation of the optimized protocol in yam variety TDr 2579\u003c/h3\u003e\n\u003cp\u003eTo validate the protocol with a different yam variety, the formation of somatic embryos in TDr 2579 was initiated using auxiliary buds from single nodal sections and cultured in CIM as detailed previously. Optimized concentrations of proline, casein hydrolysate, and thiamine were added, based on the protocol established for TDr 2436 and callus wounding was performed by meshing through a stainless-steel sieve with a pore size of 1\u0026ndash;2 mm or detached using a pair of forceps. The crushed callus was washed with liquid CIM containing ascorbic acid (10 mg/L or 20 mg/L) and transferred to either GD or FIM media, following the optimized culture conditions. Regeneration was completed by transferring FECs to hormone-free MS medium supplemented with 1% activated charcoal for embryo germination, followed by embryo maturation in BAP medium, rooting in YBM, and gradual acclimatization in soil.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design and statistics\u003c/h2\u003e \u003cp\u003eAll experiments followed a completely randomized design with two biological repeats and three technical replicates per treatment (100 explants each). Data were analyzed by ANOVA with means compared using Duncan\u0026rsquo;s multiple range test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in Minitab 17, and are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRole of amino acids and vitamin additives in boosting embryogenesis and callus yield\u003c/h2\u003e \u003cp\u003eWhen cultured on SBM, nodes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) produced axillary buds within three days. After transferring these buds to CIM for two weeks, direct somatic embryos began to emerge from the explant surface. However, a portion of the explants instead generated non-embryogenic, white, and compact callus structures. Supplementating CIM with proline, thiamine, and casein hydrolysate significantly enhanced all metrics of somatic embryogenesis (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The fully supplemented treatment (casein hydrolysate\u0026thinsp;+\u0026thinsp;proline\u0026thinsp;+\u0026thinsp;thiamine) yielded a callus fresh weight of 498.4 mg, more than double the 221.3 mg obtained in the unsupplemented control and raised the proportion of embryogenic callus from 41.9\u0026ndash;76.2%. Likewise, the mean number of embryos per callus jumped from 4.5 to 19.3. These enhancements were consistently superior to partial supplementations, so all subsequent CIM formulations were supplemented with these additives.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eImpact of casein hydrolysate, proline and thiamine on somatic embryo induction and callus biomass\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCH (g L⁻\u0026sup1;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProline (mg L⁻\u0026sup1;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThiamine (mg L⁻\u0026sup1;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e% Explants with DSE after 4 wk\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMean DSE no per explant (4 wk)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCallus fresh weight (mg) after 8 wk\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e221.3\u0026thinsp;\u0026plusmn;\u0026thinsp;24.6d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1de\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e314.8\u0026thinsp;\u0026plusmn;\u0026thinsp;9.6c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e53.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e307.6\u0026thinsp;\u0026plusmn;\u0026thinsp;14.6c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e368.7\u0026thinsp;\u0026plusmn;\u0026thinsp;22.7b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e65.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.7c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e385.6\u0026thinsp;\u0026plusmn;\u0026thinsp;19.5b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e68.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e376.8\u0026thinsp;\u0026plusmn;\u0026thinsp;28.8b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e76.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e498.4\u0026thinsp;\u0026plusmn;\u0026thinsp;17.3 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eData represent the average\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of three biological replicates (n\u0026thinsp;=\u0026thinsp;3). Within each column, means labeled with different letters are statistically different (p\u0026thinsp;\u0026le;\u0026thinsp;0.05, Duncan\u0026rsquo;s test).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eInfluence of basal salt formulation on somatic embryo induction and callus proliferation\u003c/h2\u003e \u003cp\u003eVarying the basal mineral salt concentrations in the medium markedly affected explant embryogenic potential and callus proliferation (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The FIM medium achieved the highest embryo-induction rate, with 77.3% of explants producing somatic embryos and an average of 19.2 embryos per explant. Full‐strength MS yielded very similar results (76.5% responsiveness and 18.6 embryos per explant). In contrast, reducing the MS salts to half strength led to a marked drop in both induction frequency and mean embryo number: only 48.6% of explants produced embryos, averaging 4.7 embryos each. Callus biomass also tracked with salt strength; FIM and full‐strength MS substantially supported higher fresh weights (504.8 mg and 486.7 mg, respectively) than half‐strength MS (236.9 mg). Despite this, both FIM and full‐strength MS yielded similar numbers of direct somatic embryo events per callus and comparable percentages of calli forming DSE. Given these outcomes and the straightforward preparation of standard MS, we chose full‐strength MS for all further embryo induction experiments.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of somatic embryo induction and callus biomass under different basal salt regimes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e% DSE (4\u0026nbsp;wks)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean DSE/explant (4\u0026nbsp;wks)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean fresh weight (mg /explant, 8\u0026nbsp;wks\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFIM (reduced NPK\u0026thinsp;+\u0026thinsp;Thiamine)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e77.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e504.8\u0026thinsp;\u0026plusmn;\u0026thinsp;24.6a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e76.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e486.7\u0026thinsp;\u0026plusmn;\u0026thinsp;13.5a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHalf strength MS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e236.9\u0026thinsp;\u0026plusmn;\u0026thinsp;35.4b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE (n\u0026thinsp;=\u0026thinsp;3; 100 explants per replicate). Values sharing the same letter within a column are not significantly different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Duncan\u0026rsquo;s multiple range test).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffect of callus wounding and washing meshed callus on FEC induction\u003c/h2\u003e \u003cp\u003eThree distinct calli morphologies were observed following the eight-week culture in CIM; the non-embryogenic type was discarded and only the embryogenic type was retained (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Crushing callus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) through the metal mesh to ~\u0026thinsp;2 mm fragments and subsequent rinsing and blotting on sterile tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) eliminated the mucilaginous exudate and yielded clean, debris-free calli (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eWithin the first 14 days after culture in callus induction media 2, the callus edges subjected to wounding developed marked browning and necrotic zones. (Fig.\u0026nbsp;4.1F). However, washing and blotting significantly reduced tissue browning compared to unwashed callus and accelerated recovery (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBrowning severity and recovery time after different callus-washing treatments\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWashing treatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRecovery time (days\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBrowning score\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo wash\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRinse in distilled water\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRinse in liquid MS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS\u0026thinsp;+\u0026thinsp;5mg/L ascorbic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS\u0026thinsp;+\u0026thinsp;10mg/L ascorbic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS\u0026thinsp;+\u0026thinsp;20mg/L ascorbic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS\u0026thinsp;+\u0026thinsp;12mg/L L-tyrosine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eBrowning was scored from 0 (no browning) to 5 (extensive browning). Results are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error from three independent biological replicates (n\u0026thinsp;=\u0026thinsp;3 plates). Within each column, means bearing different superscript letters differ at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 based on Duncan\u0026rsquo;s multiple range test.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSupplementing the wash solution with 10 mg L⁻\u0026sup1; ascorbic acid lowered browning to a score of 1 and shortened recovery to 12.8 days. However, doubling the ascorbic acid to 20 mg L⁻\u0026sup1; backfired, increasing discoloration (score 2) and extending recovery to 17.6 days. In contrast, adding 12 mg/L L-tyrosine whether in liquid or solid CIM had had no measurable impact on either browning or recovery time. Starting in week 4, callus proliferation resumed visibly, and the browning diminished over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eInfluence of basal salt formulations on friable embryogenic callus induction\u003c/h2\u003e \u003cp\u003eSub-culturing meshed calli through successive GD media stages boosted callus proliferation (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eH \u0026amp; I), with FECs first appearing on GD 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ), and expanded further when transferred to GD 4 (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eK and L). In contrast, simply wounding callus in CIM 2 by removing non-embryogenic tissue and then sub-culturing them to GD failed to induce FECs and instead scarred and hardened. Among the basal salts tested, FIM medium demonstrated the highest FEC induction rate (11.78%), which is more than double that obtained in GD (4.1%). Meanwhile, neither half-strength nor full-strength MS medium supported FEC formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results identify FIM as the optimal medium for FEC development.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSubculturing FECs onto GD 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) triggered a burst of proliferation over 14 days, followed by a gradual purple coloration that marked the onset of maturation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). After 45 days on charcoal-amended medium, all embryos were uniformly purple (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), and microscopic examination revealed distinct shoot and root meristem regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Upon transfer to a BAP-supplemented maturation medium, the embryos turned green and progressed to form single or fused cotyledons (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F, G, and H). Fully developed cotyledonary stage embryos were successfully rooted on YBM medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). The regenerated plants exhibited normal growth, and no morphological variations were observed under green house evaluation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHistological profile of callus tissues and FECs\u003c/h2\u003e \u003cp\u003eHistological analysis of callus and somatic embryos at the various stages of development was conducted to confirm the embryogenic origin of plantlets regenerated by the optimized regeneration system. Transverse sections of non-embryogenic cali generate following 2-week explant culture in CIM1 revealed irregularly shaped cells with many intercellular spaces, obscured nucleus, low cytoplasm density and large vacuoles (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). On the contrary, embryonic regions were characterized by numerous starch storage grains and meristematic zones, evidenced by the presence of pro-embryonic masses (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Notably, the embryogenic sections predominantly originated from the tip of the auxiliary bud, whereas the base of the auxiliary bud formed watery or compact and hard non embryogenic tissues.\u003c/p\u003e \u003cp\u003eTissues from CIM2 were not analyzed histologically because this was a recovery phase consisting of small brown-coloured tissues. Initial signs of recovery were observed at the end of culture on GD1 medium, and analysis of the tissues at this stage revealed a highly organized structure with small compact cells, dense cytoplasm, prominent nucleus, and onset of protoderm formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). During culture in GD2 medium, the cells underwent periclinal and anticlinal divisions to form globular shaped embryogenic structures that had small-sized cells with dense cytoplasm and small nuclei. These globular structures remained attached to the mother tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), and budding from these structures eventually generated multiple globular and heart shaped somatic embryos, many of which detached from the mother tissue to form mucilaginous clusters with high cytoplasmic content and prominent nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). The clear bipolar structures of the somatic embryos were evident at the GD 3 stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eGD4 marked a proliferation phase, during which the population of somatic embryos increased exponentially, and globular and heart-shaped forms (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eF \u0026amp; G), maturing into torpedo-stage embryos by the end of this phase exhibited a mixture of globular and heart shaped embryos (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eF \u0026amp; G), maturing into torpedo stage towards the end of culture in GD4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Upon transfer to hormone-free germination medium, embryos elongated to form a distinct shoot apical meristems (SAM) and root apical meristems (RAM). These structures showed a prominent scutella node, which aligns with characteristic features in the cotyledonary stage in monocots (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eI \u0026amp; J). The embryos showed dense cell division between the two meristems and highly vacuolated cells on the outer section supporting further cell division to develop a cotyledonary embryo. A distinct connective tissue between the SAM and RAM was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eK) and the prominent lobe of the SAM elongated to form the single cotyledonary structure, typical of monocot embryo germination (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eL).\u003c/p\u003e \u003cp\u003eUnlike previous observations (Manoharan et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), the current histological analysis revealed that the regenerated shoots were of somatic origin, not adventitious. The full spectrum of embryogenic stages including pro-embryo, globular, heart, torpedo and cotyledonary, was observed confirming that the optimized protocol supports true somatic embryogenesis suitable for transgene integration and gene editing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eValidation of the optimized protocol in yam variety TDr 2579\u003c/h2\u003e \u003cp\u003eThe optimized protocol was successfully validated in yam variety TDr 2579, yielding results comparable to those observed in TDr 2436. Nodal explants of TDr 2579 initiated axillary bud formation within three days on SBM medium. Callus proliferation and somatic embryogenesis were significantly enhanced by supplementing CIM with proline, casein hydrolysate, and thiamine. Measurements taken on callus weight, percentage of embryogenic callus, and embryo count per explant were either equivalent to or exceeded those obtained with TDr 2436, affirming the consistency of the optimized conditions. FIM medium led to a higher frequency of FEC formation in TDr 2579 (13.4%) compared to GD medium (6.1%), consistent with results in TDr 2436. The application of callus meshing through a small pore-size steel sieve facilitated FEC development in both varieties.\u003c/p\u003e \u003cp\u003eThe addition of 10 mg/L ascorbic acid significantly reduced callus browning, improved the recovery time and re-established callus proliferation, mirroring the effects seen in TDr 2436. The development of somatic embryos in TDr 2579 progressed through the globular, heart, torpedo, and cotyledonary stages, and mature embryos successfully rooted and acclimatized with 75% survival in soil. The regenerated plantlets exhibited healthy growth, and no signs of soma clonal variation, highlighting the stability and applicability of the optimized protocol across different yam varieties. This validation confirms that the regeneration system developed for TDr 2436 can reliably induce FEC formation, reduce callus browning, and support high rates of plant regeneration in diverse yam varieties.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe production of FECs is a highly desirable tissue for genetic manipulations at the single cell level due to their totipotent nature, loose organization, small size and high numbers. FECs not only support the rapid establishment of stable, and well-dispersed cell suspension cultures but can also serve as good sources of totipotent protoplast isolation (Wen et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). While Manoharan et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ereported\u003c/span\u003e nodular, dense embryogenic calli, the protocol developed in the current study obtained friable and mucilaginous structures with high proliferative capacity.\u003c/p\u003e \u003cp\u003eSimilar to previous studies in \u003cem\u003eLitchi chinensis\u003c/em\u003e (Wang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), date palm (Zein El Din et al., 2022), cassava ( Utsumi et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Syombua et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and maize (Kang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), this research found that media additives such as antioxidants, casein hydrolysate, and L-thiamine enhance the embryogenic competence of tissue culture explants. Plants cells in tissue culture environments are prone to oxidative stress, resulting in elevated levels of reactive oxygen species (ROS) that harm cellular components and suppress growth. Exogenous amino acids scavenge ROS, hence provide osmoprotection, and promote cellular proliferation and differentiation (Hosseinifard et al., 2022). For instance, supplementing culre media with proline enhances somatic embryogenesis in maize and groundnut. The exogenous proline alleviates stress in maize embryos by optimizing respiratory metabolism and regulating hormone levels, thereby supporting embryo development under adverse conditions (Garrocho-Villegas et al., 2012; Zuo et al., 2022; Truong et al., 2023). Similarly, Jain et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) demonstrated that exogenous proline alleviates oxidative harm to the cellular membranes of groundnut and prevents salt induced decline in biomass. Likewise, supplementing with thiamine supports somatic embryogenesis by catalyzing pyruvate conversion to acetyl-CoA, thereby supplying the energy and carbon skeletons required for rapid cell proliferation and embryo formation (Dhillon et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUtsumi et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) reported that thiamine fortification drives carbon flux from glycolysis into the TCA cycle, which in turn elevates the frequency of FEC formation. In another study, \u003cb\u003es\u003c/b\u003eupplementing maize callus induction media with thiamine improved the embryogenic competence of immature embryos and promoted the progression of somatic embryos into regeneration-competent tissues (Vila\u0026ccedil;a Vasconcelos et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Our findings are consistent with these reports as thiamine-enriched media enhanced the formation of FECs, potentially by supporting the increased energy demands during early embryogenic stages.\u003c/p\u003e \u003cp\u003eL-tyrosine, a precursor for numerous metabolic pathways involved in antioxidative processes and cell differentiation, has potential to enhance somatic embryogenesis. For instance, it is a precursor for polyamines, such as spermidine and spermine, which could promote osmotic adjustment, division of cells and differentiation of plant cultures. Tyrosine is also a precursor for the synthesis of secondary metabolites like phenolics and flavonoids, some of which have antioxidant properties and can help in reducing oxidative stress in the culture environment. Studies in cassava and pine have shown that adding L-tyrosine to the medium enhances FEC induction \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(\u003c/span\u003eNyaboga et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Castander-Olarieta et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In contrast, our yam cultures did not respond to tyrosine supplementation. This species-specific discrepancy likely reflects differences in metabolic pathways and \u003cem\u003ein vitro\u003c/em\u003e responsiveness, and may be further explained by yam\u0026rsquo;s high endogenous tyrosinase activity, which rapidly oxidizes L-tyrosine and limits its beneficial effects (Ilesanmi et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ilesanmi \u0026amp; Adewale, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mulla et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). As such, this enzymatic activity could, reduce its bioavailability and negate any growth-promoting effects. Moreover, the catalysis products include black to brown to melanin precursors that could result in callus darkening and negatively impact cell viability (Lu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWounding is another critical factor influencing callus competence. Mechanical wounding of callus triggers the release of signaling molecules and transcription factors that stimulate dedifferentiation, repair mechanisms, reprogram gene expression patterns and enhance proliferation (Yang et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ikeuchi et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This study demonstrated that callus wounding by mesh crushing promoted the development of FECs, which concurs with reported findings in cassava \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e(\u003c/span\u003eTaylor et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Nyaboga et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Similarly, Wang et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) demonstrated that blending maize leaf sheath in a food processor significantly enhanced the regeneration of transformed plants, relative to manually chopped leaf segments. One of the long-standing hypotheses is that wounding induces jasmonate-mediated wound signaling, which promotes auxin synthesis, and, subsequently, improves regeneration efficiency (Zhang et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Similar to callus crushing through a stainless-steel mesh, mechanical wounding by blending homogenizes the explants and ensures uniform wounding, promoting more consistent regeneration outcomes, compared to manually chopped explants. The current findings also suggest that small explant sizes could enhance the regeneration competence by increasing the surface area-to-volume ratio in blended tissue and ensuring better exposure to growth regulators and nutrients in media (Bennur et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBesides mechanical damage, rinsing meshed calli in liquid CIM and gently blotting them on sterile tissue was key to reducing browning and necrosis. Supplementing the wash solution with ascorbic acid\u0026mdash;a water-soluble antioxidant widely used to scavenge reactive oxygen species in vitro\u0026mdash;further limited tissue discoloration (Hazubska-Przybył et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e). Yam tissues release high levels of phenolic compounds (e.g., saponins, gracillin, diosgenin, dioscin, catechins) (Syombua et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) that, when leached into the medium, can darken it, inhibit growth regulators, and impede somatic embryogenesis. To remove these exudates, adsorbents like activated charcoal, polyvinylpyrrolidone, or ion-exchange resins can be added (Syombua et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).. Beyond its antioxidant role, ascorbic acid also promotes cell division and elongation, enhancing in vitro growth, a benefit documented in Norway spruce, faba bean, Musa, orchids, and Jatropha cultures (Hazubska-Przybył et al., 2024 Abdelwahd et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Adero et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Chugh et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; He et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur results clearly demonstrate that FIM medium outperforms GD, full-strength MS, and half-strength MS in driving FEC induction. FIM\u0026rsquo;s reduced nitrate, phosphate, and potassium levels combined with elevated thiamine create an environment uniquely favorable for embryogenic callus. Notably, neither half- nor full-strength MS supported any FEC formation, underscoring the critical roles that specific mineral ratios play in yam somatic embryo development. Furthermore, both GD and FIM media are richer in vitamins and amino acids than MS, suggesting that high levels of these supplements are also indispensable for generating friable, embryogenic callus. Moreover, because FIM (Utsumi et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and GD (Gresshoff \u0026amp; Doy, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1974\u003c/span\u003e) both contain much greater concentrations of vitamins and casamino acids than MS, it suggests that abundant vitamin and amino acid supplementation is also essential for FEC development. The presence of excess thiamine in the medium further supports energy metabolism, which is crucial for the rapid cell proliferation needed for FEC formation and proliferation. Similar observations have been made in sorghum and Norway spruce, where the phosphorus and nitrogen levels in culture medium influenced morphogenesis and somatic embryogenesis (Elkonin \u0026amp; Pakhomova, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Carlsson et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Hence, fine-tuning the mineral and vitamin composition of the culture medium may be pivotal for enabling regeneration in recalcitrant species such as yam.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis article reports pioneering research for generation, proliferation and regeneration of FECs in the \u003cem\u003eDioscorea\u003c/em\u003e species. It presents a step-by‐step protocol covering the formation of organized primary embryogenic structures, FEC induction, embryo maturation, germination, rooting, shoot elongation, and final acclimatization. All the key steps in FEC induction, embryo maturation, and plantlet regeneration were optimized to enhance embryogenic competence and reduce tissue necrosis, notably through nutrient modifications and media supplementation with antioxidants. The findings underscore FIM's effectiveness in FEC formation compared to conventional media, likely due to reduced nitrate and phosphate levels. The study further introduces a novelty in yam FEC establishment, specifically callus meshing to induce tissue reprogramming and modify the tissue gene expression patterns. Importantly, FEC-derived plantlets remained genetically stable even after soil acclimatization and exhibited no somaclonal variation. This optimized protocol, therefore, provides a robust foundation for advanced genetic work in yam in yam and supports applications such as protoplast isolation and regeneration, as well as transgenic or genome-editing approaches.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.T conceived the idea, E.S, J.T and L.T designed the study, E.S performed the experiments, J.T and L.T supervised the experiment, E.S and J.T analyzed the data, E.S wrote the manuscript, J.T and L.T revised the manuscript and L.T sourced the research funding. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll study data that support the findings are included in the article or\u0026nbsp;supplementary data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Professor Steven Runo and Dorothy Mbuvi at Kenyatta University for providing equipment and training on histological analysis of plant tissues. This project was supported by International Institute of Tropical Agriculture (IITA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbdelwahd, R., Hakam, N., Labhilili, M., \u0026amp; Udupa, S. M. (2008). Use of an adsorbent and antioxidants to reduce the effects of leached phenolics in in vitro plantlet regeneration of faba bean. \u003cem\u003eAfrican Journal of Biotechnology\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e(8), Article 8. https://doi.org/10.4314/ajb.v7i8.58590\u003c/li\u003e\n \u003cli\u003eAdero, M., Tripathi, J. N., \u0026amp; Tripathi, L. (2023). Advances in Somatic Embryogenesis of Banana. \u003cem\u003eInternational Journal of Molecular Sciences\u003c/em\u003e, \u003cem\u003e24\u003c/em\u003e(13), 10999. https://doi.org/10.3390/ijms241310999\u003c/li\u003e\n \u003cli\u003eBennur, P. L., O\u0026rsquo;Brien, M., Fernando, S. C., \u0026amp; Doblin, M. S. (2024). Improving transformation and regeneration efficiency in medicinal plants: Insights from other recalcitrant species. \u003cem\u003eJournal of Experimental Botany\u003c/em\u003e, erae189. https://doi.org/10.1093/jxb/erae189\u003c/li\u003e\n \u003cli\u003eCarlsson, J., Svennerstam, H., Moritz, T., Egertsdotter, U., \u0026amp; Ganeteg, U. (2017). Nitrogen uptake and assimilation in proliferating embryogenic cultures of Norway spruce\u0026mdash;Investigating the specific role of glutamine. \u003cem\u003ePLOS ONE\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e(8), e0181785. https://doi.org/10.1371/journal.pone.0181785\u003c/li\u003e\n \u003cli\u003eCastander-Olarieta, A., Montalb\u0026aacute;n, I. A., De Medeiros Oliveira, E., Dell\u0026rsquo;Aversana, E., D\u0026rsquo;Amelia, L., Carillo, P., Steiner, N., Fraga, H. P. D. F., Guerra, M. P., Goicoa, T., Ugarte, M. D., Pereira, C., \u0026amp; Moncale\u0026aacute;n, P. (2019). Effect of Thermal Stress on Tissue Ultrastructure and Metabolite Profiles During Initiation of Radiata Pine Somatic Embryogenesis. \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e, 2004. https://doi.org/10.3389/fpls.2018.02004\u003c/li\u003e\n \u003cli\u003eChugh, S., Guha, S., \u0026amp; Rao, I. U. (2009). Micropropagation of orchids: A review on the potential of different explants. \u003cem\u003eScientia Horticulturae\u003c/em\u003e, \u003cem\u003e122\u003c/em\u003e(4), 507\u0026ndash;520. https://doi.org/10.1016/j.scienta.2009.07.016\u003c/li\u003e\n \u003cli\u003eCroptrust. (2024). \u003cem\u003ePlan for Bigger, Better Yams\u0026mdash;Crop Trust\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eDarkwa, K., Agre, P., Olasanmi, B., Iseki, K., Matsumoto, R., Powell, A., Bauchet, G., De Koeyer, D., Muranaka, S., Adebola, P., Asiedu, R., Terauchi, R., \u0026amp; Asfaw, A. (2020). Comparative assessment of genetic diversity matrices and clustering methods in white Guinea yam ( Dioscorea rotundata ) based on morphological and molecular markers. \u003cem\u003eScientific Reports\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e(1), Article 1. https://doi.org/10.1038/s41598-020-69925-9\u003c/li\u003e\n \u003cli\u003eDhillon, R. S., Hooda, M. S., Pundeer, J. S., Ahlawat, K. S., \u0026amp; Chopra, I. (2011). Effects of auxins and thiamine on the efficacy of techniques of clonal propagation in Jatropha curcas L. \u003cem\u003eBiomass and Bioenergy\u003c/em\u003e, \u003cem\u003e35\u003c/em\u003e(4), 1502\u0026ndash;1510. https://doi.org/10.1016/j.biombioe.2010.12.017\u003c/li\u003e\n \u003cli\u003eElkonin, L. A., \u0026amp; Pakhomova, N. V. (2000). Influence of nitrogen and phosphorus on induction embryogenic callus of sorghum. \u003cem\u003ePlant Cell, Tissue and Organ Culture\u003c/em\u003e, \u003cem\u003e61\u003c/em\u003e(2), 115\u0026ndash;123. https://doi.org/10.1023/A:1006472418218\u003c/li\u003e\n \u003cli\u003eGambino, G., Moine, A., Boccacci, P., Perrone, I., \u0026amp; Pagliarani, C. (2021). Somatic embryogenesis is an effective strategy for dissecting chimerism phenomena in Vitis vinifera cv Nebbiolo. \u003cem\u003ePlant Cell Reports\u003c/em\u003e, \u003cem\u003e40\u003c/em\u003e(1), 205\u0026ndash;211. https://doi.org/10.1007/s00299-020-02626-9\u003c/li\u003e\n \u003cli\u003eGresshoff, P. M., \u0026amp; Doy, C. H. (1974). Derivation of a haploid cell line from Vitis vinifera and the importance of the stage of meiotic development of anthers for haploid culture of this and other genera. \u003cem\u003eZeitschrift F\u0026uuml;r Pflanzenphysiologie\u003c/em\u003e, \u003cem\u003e73\u003c/em\u003e(2), 132\u0026ndash;141. https://doi.org/10.1016/S0044-328X(74)80084-X\u003c/li\u003e\n \u003cli\u003eHazubska-Przybył, T., Obarska, A., Konecka, A., Kijowska-Oberc, J., Wawrzyniak, M. K., Piotrowska-Niczyporuk, A., Staszak, A. M., \u0026amp; Ratajczak, E. (2024a). Modulating ascorbic acid levels to optimize somatic embryogenesis in Picea abies (L.) H. Karst. Insights into oxidative stress and endogenous phytohormones regulation. \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e, 1372764. https://doi.org/10.3389/fpls.2024.1372764\u003c/li\u003e\n \u003cli\u003eHazubska-Przybył, T., Obarska, A., Konecka, A., Kijowska-Oberc, J., Wawrzyniak, M. K., Piotrowska-Niczyporuk, A., Staszak, A. M., \u0026amp; Ratajczak, E. (2024b). Modulating ascorbic acid levels to optimize somatic embryogenesis in Picea abies (L.) H. Karst. Insights into oxidative stress and endogenous phytohormones regulation. \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e, 1372764. https://doi.org/10.3389/fpls.2024.1372764\u003c/li\u003e\n \u003cli\u003eHe, Y., Guo, X., Lu, R., Niu, B., Pasapula, V., Hou, P., Cai, F., Xu, Y., \u0026amp; Chen, F. (2009). Changes in morphology and biochemical indices in browning callus derived from Jatropha curcas hypocotyls. \u003cem\u003ePlant Cell, Tissue and Organ Culture (PCTOC)\u003c/em\u003e, \u003cem\u003e98\u003c/em\u003e(1), 11\u0026ndash;17. https://doi.org/10.1007/s11240-009-9533-y\u003c/li\u003e\n \u003cli\u003eHosseinifard, M., Stefaniak, S., Javid, M. G., Soltani, E., Wojtyla, Ł., \u0026amp; Garnczarska, M. (n.d.). \u003cem\u003eContribution of Exogenous Proline to Abiotic Stresses Tolerance in Plants: A Review\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eIkeuchi, M., Iwase, A., Rymen, B., Lambolez, A., Kojima, M., Takebayashi, Y., Heyman, J., Watanabe, S., Seo, M., Veylder, L. D., Sakakibara, H., \u0026amp; Sugimoto, K. (2017). Wounding Triggers Callus Formation via Dynamic Hormonal and Transcriptional Changes. \u003cem\u003ePlant Physiology\u003c/em\u003e, \u003cem\u003e175\u003c/em\u003e(3), 1158\u0026ndash;1174. https://doi.org/10.1104/pp.17.01035\u003c/li\u003e\n \u003cli\u003eIlesanmi, O. S., \u0026amp; Adewale, I. O. (2020). Physicochemical properties of free and immobilized tyrosinase from different species of yam (Dioscorea spp). \u003cem\u003eBiotechnology Reports\u003c/em\u003e, \u003cem\u003e27\u003c/em\u003e, e00499. https://doi.org/10.1016/j.btre.2020.e00499\u003c/li\u003e\n \u003cli\u003eIlesanmi, O. S., Ojopagogo, Y. A., \u0026amp; Adewale, I. O. (2014). Kinetic characteristics of purified tyrosinase from different species of Dioscorea (yam) in aqueous and non-aqueous systems. \u003cem\u003eJournal of Molecular Catalysis B: Enzymatic\u003c/em\u003e, \u003cem\u003e108\u003c/em\u003e, 111\u0026ndash;117. https://doi.org/10.1016/j.molcatb.2014.07.009\u003c/li\u003e\n \u003cli\u003eJain, M., Mathur, G., Koul, S., \u0026amp; Sarin, N. (2001). Ameliorative effects of proline on salt stress-induced lipid peroxidation in cell lines of groundnut (Arachis hypogaea L.). \u003cem\u003ePlant Cell Reports\u003c/em\u003e, \u003cem\u003e20\u003c/em\u003e(5), 463\u0026ndash;468. https://doi.org/10.1007/s002990100353\u003c/li\u003e\n \u003cli\u003eKang, M., Lee, K., Finley, T., Chappell, H., Veena, V., \u0026amp; Wang, K. (2022). An Improved Agrobacterium-Mediated Transformation and Genome-Editing Method for Maize Inbred B104 Using a Ternary Vector System and Immature Embryos. \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e, 860971. https://doi.org/10.3389/fpls.2022.860971\u003c/li\u003e\n \u003cli\u003eKole, C. (Ed.). (2022a). \u003cem\u003eGenomic Designing for Abiotic Stress Resistant Technical Crops\u003c/em\u003e. Springer International Publishing. https://doi.org/10.1007/978-3-031-05706-9\u003c/li\u003e\n \u003cli\u003eKole, C. (Ed.). (2022b). \u003cem\u003eGenomic Designing for Biotic Stress Resistant Technical Crops\u003c/em\u003e. Springer International Publishing. https://doi.org/10.1007/978-3-031-09293-0\u003c/li\u003e\n \u003cli\u003eLin, H.-S., van der Toorn, C., Raemakers, K. J. J. M., Visser, R. G. F., De Jeu, M. J., \u0026amp; Jacobsen, E. (2000). Development of a plant regeneration system based on friable embryogenic callus in the ornamental Alstroemeria. \u003cem\u003ePlant Cell Reports\u003c/em\u003e, \u003cem\u003e19\u003c/em\u003e(5), 529\u0026ndash;534. https://doi.org/10.1007/s002990050768\u003c/li\u003e\n \u003cli\u003eLu, Y., Xu, Y., Song, M.-T., Qian, L.-L., Liu, X.-L., Gao, R.-Y., Han, R.-M., Skibsted, L. H., \u0026amp; Zhang, J.-P. (2021). Promotion effects of flavonoids on browning induced by enzymatic oxidation of tyrosinase: Structure\u0026ndash;activity relationship. \u003cem\u003eRSC Advances\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(23), 13769\u0026ndash;13779. https://doi.org/10.1039/D1RA01369F\u003c/li\u003e\n \u003cli\u003eManoharan, R., Tripathi, J. N., \u0026amp; Tripathi, L. (2016). Plant regeneration from axillary bud derived callus in white yam (Dioscorea rotundata). \u003cem\u003ePlant Cell, Tissue and Organ Culture (PCTOC)\u003c/em\u003e, \u003cem\u003e126\u003c/em\u003e(3), 481\u0026ndash;497. https://doi.org/10.1007/s11240-016-1017-2\u003c/li\u003e\n \u003cli\u003eMulla, T., Patil, S., \u0026amp; Jadhav, J. (2018). Exploration of surface plasmon resonance for yam tyrosinase characterization. \u003cem\u003eInternational Journal of Biological Macromolecules\u003c/em\u003e, \u003cem\u003e109\u003c/em\u003e, 399\u0026ndash;406. https://doi.org/10.1016/j.ijbiomac.2017.12.074\u003c/li\u003e\n \u003cli\u003eNyaboga, E. N., Njiru, J. M., \u0026amp; Tripathi, L. (2015). Factors influencing somatic embryogenesis, regeneration, and Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) cultivar TME14. \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e, \u003cem\u003e6\u003c/em\u003e. https://doi.org/10.3389/fpls.2015.00411\u003c/li\u003e\n \u003cli\u003eSyombua, E. D., Adero, M. O., Mbinda, W. M., Wanyonyi, C. N., Ngugi, M. P., \u0026amp; Oduor, R. O. (2021). A system for rapid gene introgression into cassava immature leaves and subsequent recovery of transformed lines. \u003cem\u003ePlant Biotechnology Reports\u003c/em\u003e. https://doi.org/10.1007/s11816-020-00654-1\u003c/li\u003e\n \u003cli\u003eSyombua, E. D., Tripathi, J. N., Obiero, G. O., Nguu, E. K., Yang, B., Wang, K., \u0026amp; Tripathi, L. (2022). Potential applications of the CRISPR/Cas technology for genetic improvement of yam ( \u003cem\u003eDioscorea\u003c/em\u003e spp.). \u003cem\u003eFood and Energy Security\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(1), e330. https://doi.org/10.1002/fes3.330\u003c/li\u003e\n \u003cli\u003eSyombua, E. D., Wanyonyi, C. N., Adero, M. O., Mbinda, W. M., Ngugi, M. P., Alakonya, A. E., \u0026amp; Oduor, R. O. (n.d.). \u003cem\u003eExplant type and hormone regime influences somatic embryogenesis and regeneration in cassava\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eSyombua, E. D., Wanyonyi, C. N., Adero, M. O., Mbinda, W. M., Ngugi, M. P., Alakonya, A. E., \u0026amp; Oduor, R. O. (2019). Explant type and hormone regime influences somatic embryogenesis and regeneration of cassava. \u003cem\u003eAfrican Journal of Biotechnology\u003c/em\u003e, \u003cem\u003e18\u003c/em\u003e(25), 532\u0026ndash;539. https://doi.org/10.5897/AJB2019.16853\u003c/li\u003e\n \u003cli\u003eSyombua, E. D., Zhang, Z., Tripathi, J. N., Ntui, V. O., Kang, M., George, O. O., Edward, N. K., Wang, K., Yang, B., \u0026amp; Tripathi, L. (2021). A CRISPR/Cas9‐based genome‐editing system for yam ( \u003cem\u003eDioscorea\u003c/em\u003e spp.). \u003cem\u003ePlant Biotechnology Journal\u003c/em\u003e, \u003cem\u003e19\u003c/em\u003e(4), 645\u0026ndash;647. https://doi.org/10.1111/pbi.13515\u003c/li\u003e\n \u003cli\u003eTaylor, N., Gait\u0026aacute;n-Sol\u0026iacute;s, E., Moll, T., Trauterman, B., Jones, T., Pranjal, A., Trembley, C., Abernathy, V., Corbin, D., \u0026amp; Fauquet, C. M. (2012). A High-throughput Platform for the Production and Analysis of Transgenic Cassava (Manihot esculenta) Plants. \u003cem\u003eTropical Plant Biology\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e(1), 127\u0026ndash;139. https://doi.org/10.1007/s12042-012-9099-4\u003c/li\u003e\n \u003cli\u003eTaylor, N. J., Edwards, M., Kiernan, R. J., Davey, C. D. M., Blakesley, D., \u0026amp; Henshaw, G. G. (1996). Development of friable embryogenic callus and embryogenic suspension culture systems in cassava ( Manihot esculenta Crantz). \u003cem\u003eNature Biotechnology\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e(6), Article 6. https://doi.org/10.1038/nbt0696-726\u003c/li\u003e\n \u003cli\u003eTruong Thi Lan Anh a b c, , Nguyen Thi Nhu Mai a, , Hoang Thanh Tung d, , Hoang Dac Khai a, , Do Manh Cuong a, , Vu Quoc Luan a, , Hoang Thi Nhu Phuong c, , Nguyen Van Binh c, , Bui Van The Vinh e, , Nguyen Thi Thanh Thuy a, \u0026hellip; Duong Tan Nhut a. (n.d.). \u003cem\u003eEffect of spermidine, glutamine, and proline on somatic embryogenesis and silver nanoparticles supplied culture improved rhizome formation of Panax vietnamensis var. Langbianensis\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eUtsumi, Y., Utsumi, C., Tanaka, M., Ha, V. T., Matsui, A., Takahashi, S., \u0026amp; Seki, M. (2017). Formation of friable embryogenic callus in cassava is enhanced under conditions of reduced nitrate, potassium and phosphate. \u003cem\u003ePLOS ONE\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e(8), e0180736. https://doi.org/10.1371/journal.pone.0180736\u003c/li\u003e\n \u003cli\u003eVer\u0026oacute;nica Garrocho-Villegas 1, Mar\u0026iacute;a Teresa de Jes\u0026uacute;s-Olivera, Estela S\u0026aacute;nchez Quintanar. (n.d.). \u003cem\u003eMaize somatic embryogenesis: Recent features to improve plant regeneration\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eVila\u0026ccedil;a Vasconcelos, M. J., Antunes, M. S., De Oliveira, M. F., Lopes, M. A., \u0026amp; Fontes Figueiredo, J. E. (2018). CALLUS INDUCTION AND PLANT REGENERATION FROM IMMATURE EMBRYOS CULTURE OF TROPICAL MAIZE. \u003cem\u003eRevista Brasileira de Milho e Sorgo\u003c/em\u003e, \u003cem\u003e17\u003c/em\u003e(3), 359. https://doi.org/10.18512/1980-6477/rbms.v17n3p359-368\u003c/li\u003e\n \u003cli\u003eWang, G., Liu, Y., Gao, Z., Li, H., \u0026amp; Wang, J. (2023). Effects of Amino Acids on Callus Proliferation and Somatic Embryogenesis in Litchi chinensis cv. \u0026lsquo;Feizixiao.\u0026rsquo; \u003cem\u003eHorticulturae\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e(12), 1311. https://doi.org/10.3390/horticulturae9121311\u003c/li\u003e\n \u003cli\u003eWang, N., Ryan, L., Sardesai, N., Wu, E., Lenderts, B., Lowe, K., Che, P., Anand, A., Worden, A., Van Dyk, D., Barone, P., Svitashev, S., Jones, T., \u0026amp; Gordon-Kamm, W. (2023). Leaf transformation for efficient random integration and targeted genome modification in maize and sorghum. \u003cem\u003eNature Plants\u003c/em\u003e. https://doi.org/10.1038/s41477-022-01338-0\u003c/li\u003e\n \u003cli\u003eWang, S., Wang, G., Li, H., Li, F., \u0026amp; Wang, J. (2023). Agrobacterium tumefaciens-mediated transformation of embryogenic callus and CRISPR/Cas9-mediated genome editing in \u0026lsquo;Feizixiao\u0026rsquo; litchi. \u003cem\u003eHorticultural Plant Journal\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e(5), 947\u0026ndash;957. https://doi.org/10.1016/j.hpj.2023.01.011\u003c/li\u003e\n \u003cli\u003eWen, F., Su, W., Zheng, H., Yu, B., Ma, Z., Zhang, P., \u0026amp; Guo, W. (2020). Plant regeneration via protoplast electrofusion in cassava. \u003cem\u003eJournal of Integrative Agriculture\u003c/em\u003e, \u003cem\u003e19\u003c/em\u003e(3), 632\u0026ndash;642. https://doi.org/10.1016/S2095-3119(19)62711-5\u003c/li\u003e\n \u003cli\u003eYang, W., Zhai, H., Wu, F., Deng, L., Chao, Y., Meng, X., Chen, Q., Liu, C., Bie, X., Sun, C., Yu, Y., Zhang, X., Zhang, X., Chang, Z., Xue, M., Zhao, Y., Meng, X., Li, B., Zhang, X., \u0026hellip; Li, C. (2024). Peptide REF1 is a local wound signal promoting plant regeneration. \u003cem\u003eCell\u003c/em\u003e, \u003cem\u003e187\u003c/em\u003e(12), 3024-3038.e14. https://doi.org/10.1016/j.cell.2024.04.040\u003c/li\u003e\n \u003cli\u003eZein El Din, A. F. M., Darwesh, R. S. S., Ibrahim, M. F. M., Salama, G. M. Y., Shams El-Din, I. M., Abdelaal, W. B., Ali, G. A., Elsayed, M. S., Ismail, I. A., Dessoky, E. S., \u0026amp; Abdellatif, Y. M. R. (2022). Antioxidants Application Enhances Regeneration and Conversion of Date Palm (Phoenix dactylifera L.) Somatic Embryos. \u003cem\u003ePlants\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(15), 2023. https://doi.org/10.3390/plants11152023\u003c/li\u003e\n \u003cli\u003eZhang, G., Zhao, F., Chen, L., Pan, Y., Sun, L., Bao, N., Zhang, T., Cui, C.-X., Qiu, Z., Zhang, Y., Yang, L., \u0026amp; Xu, L. (2019). Jasmonate-mediated wound signalling promotes plant regeneration. \u003cem\u003eNature Plants\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e(5), 491\u0026ndash;497. https://doi.org/10.1038/s41477-019-0408-x\u003c/li\u003e\n \u003cli\u003eZuo, S., Li, J., Gu, W., \u0026amp; Wei, S. (n.d.). \u003cem\u003eExogenous Proline Alleviated Low Temperature Stress in Maize Embryos by Optimizing Seed Germination, Inner Proline Metabolism, Respiratory Metabolism and a Hormone Regulation Mechanism\u003c/em\u003e.\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":"Yam (Dioscorea spp.), Friable Embryogenic Callus (FEC), Somatic embryogenesis, Nitrogen","lastPublishedDoi":"10.21203/rs.3.rs-6539506/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6539506/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWhite yam (\u003cem\u003eDioscorea rotundata\u003c/em\u003e) is a critical staple food and an income generating crop in tropical regions. However, its improvement via classical breeding is challenging and time consuming due to an erratic flowering pattern, poor seed set, dioecious nature, low pollen fertility and low seed germination rates. These constraints limit the genetic gains achievable in each generation and prolong the breeding cycle to approximately 10 years, underscoring the need to implement faster biotechnological approaches. This study presents an optimized protocol for producing FECs (friable embryogenic callus) in yam, which serves as an ideal tissue type for transgene delivery and accelerated breeding by site specific nucleases. The various factors influencing FEC induction were optimised, including basal salt composition, tissue wounding, washing treatments, and medium supplementation with antioxidants. The results demonstrated that reduction in nitrogen supplements, along with 10 mg/L thiamine, 1000 mg/L proline and 600 mg/L casein hydrolysate, and enhanced callus fresh weight by up to 498.4 mg per explant and increased the embryogenic competence to 76.2%. Callus wounding by crushing through mesh further improved FEC induction and washing the crushed callus with 10 mg/L ascorbic acid reduced browning and necrosis, reducing recovery time from 25 to 13 days. The optimized FEC induction medium (FIM) induced \u0026nbsp;somatic embryoes in over 77% of cultures. This protocol provides a robust platform for yam genetic improvement, offering an excellent starting material for protoplast isolation, regeneration and genome editing to enhance crop resilience and productivity.\u003c/p\u003e","manuscriptTitle":"Enhancing Regeneration in White Yam (Dioscorea rotundata) Through Friable Embryogenic Callus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-15 08:52:07","doi":"10.21203/rs.3.rs-6539506/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-05-17T05:44:29+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-12T21:29:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-10T14:12:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2025-05-07T14:01:19+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":"2d1ff163-da6c-4361-bce4-38d230a322b5","owner":[],"postedDate":"May 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-29T16:01:52+00:00","versionOfRecord":{"articleIdentity":"rs-6539506","link":"https://doi.org/10.1007/s11240-025-03194-0","journal":{"identity":"plant-cell-tissue-and-organ-culture-pctoc","isVorOnly":false,"title":"Plant Cell, Tissue and Organ Culture (PCTOC)"},"publishedOn":"2025-09-24 15:58:02","publishedOnDateReadable":"September 24th, 2025"},"versionCreatedAt":"2025-05-15 08:52:07","video":"","vorDoi":"10.1007/s11240-025-03194-0","vorDoiUrl":"https://doi.org/10.1007/s11240-025-03194-0","workflowStages":[]},"version":"v1","identity":"rs-6539506","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6539506","identity":"rs-6539506","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}