Genetic resistance among banana somaclones derived from the Grand Naine cultivar (Cavendish, AAA) to Fusarium oxysporum f. sp. cubense, subtropical race 4.

Genetic resistance among banana somaclones derived from the Grand Naine cultivar (Cavendish, AAA) to Fusarium oxysporum f. sp. cubense, subtropical race 4. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genetic resistance among banana somaclones derived from the Grand Naine cultivar (Cavendish, AAA) to Fusarium oxysporum f. sp. cubense, subtropical race 4. Tamyres Amorim Rebouças, Anelita de Jesus Rocha, Mileide dos Santos Ferreira, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6556573/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Feb, 2026 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted 4 You are reading this latest preprint version Abstract Banana production is affected by several pests and diseases, of which Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is notable. Despite the different control measures and management used to contain Fusarium wilt, the development of resistant cultivars has been the best approach to combating this disease. Therefore, this work uses biotechnology aimed to induce somaclonal variation using thidiazuron (TDZ) and Paclobutrazol® (PBZ) growth regulators banana cultivar ‘Grand Naine’ (Cavendish, AAA), and to identify somaclonal variants resistant to Fusarium wilt of the subtropical race 4 (ST4). Stem apices were cultured in vitro using medium MS medium supplemented with 1 mg L − 1 PBZ and 1 mg L − 1 TDZ. After a series of ten subcultures, the regenerated plants were planted in water tanks containing soil infested with the Foc in a greenhouse. At 90 days after inoculation, plants were evaluated for pathogen resistance using longitudinal sections near the rhizome as well as for symptoms using a graded scale. Thirteen somaclones of the cultivar ‘Grand Naine’ were selected as resistant to Foc. The resistant somaclones will be evaluated for agronomic and market potential, as well as for the validation of resistance stability, with the purpose of being recommended for farmers and future improvement studies. Musa spp. genetic improvement tissue culture somaclonal variation Figures Figure 1 Figure 2 Figure 3 1. Introduction The banana is the most consumed fresh fruit worldwide and represents one of the basic consumer products of the global population. Banana production is one of the most important sources of food and income, especially for small farmers (Castañeda et al., 2017; Thangavelu et al., 2021). The five largest world producers are India, with an annual production of 34.5 million tons, China with 11.7 million tons, Indonesia with 9.2 million tons, a Nigeria with 8.0 million tons, and Brazil, with approximately 7.0 million tons (FAOSTAT, 2024). One of the main limitations in banana production is the fungus Fusarium oxysporum f. sp. cubense (Foc) that causes Fusarium wilt. This disease threatens subsistence farming, local trade, and exports (Noar et al., 2022; Le Thi et al., 2022 Abubakara et al., 2023). Foc is a soil-dwelling necrotrophic pathogen that produces chlamydospores, which are survival structures that remain viable through periods of dormancy, allowing the disease’s persistence over long periods, even in the absence of the host (Dita et al., 2018, Ploetz, 2015). Banana plants are infected by the penetration of the fungus in the roots and colonization of the vascular system, resulting in the blockage of the xylem vessels, which causes wilting owing to the impediment of water and nutrient transport to the aerial part of the plant (Zheng et al., 2018; Lal et al., 2024). Significant damage caused by this pathogen began to appear in the 1900s and especially affected the cultivar ‘Gros Michel’ focused on the export market (Stover, 1962; Ploetz, 2005). Thus, Fusarium wilt became known as a disease of grave concern at a global scale, and the only effective control method was the replacement of ‘Gros Michel’ with resistant cultivars belonging to the subgroup Cavendish (Zheng et al., 2018). However, the disease occurred again in the 1980s with the detection of a highly virulent strain that was characterized as race 4, divided into subtropical (ST4) and tropical 4 (TR4) races. Foc (ST4) can cause infection in Cavendish cultivars when exposed to any antibiotic stressors; however, TR4 attacks under any environmental condition (Dita et al., 2018; Rocha et al., 2020). Despite different control measures used to contain Fusarium wilt, such as cultivation practices and biological control, developing resistant cultivars is the most efficient strategy to control this disease (Haddad et al., 2018; Thangavelu et al., 2019; Catambacan e Cumagun, 2021). However, some factors limit the improvement of the banana tree, of which the high sterility observed in some cultivars is notable, leading to a reduced number of seeds obtained from hybridization between commercial cultivars and improved diploids (Ghag et al., 2014; Alakonya et al., 2018). Thus, the genetic improvement of Musa sp. needs to use non-conventional improvement strategies, including the in vitro induction of somaclonal variation, focusing on selecting genotypes resistant to diseases and with superior agronomic characteristics (Chen et al., 2013; Ferreira et al. 2020; Rebouças, et al., 2021; Adly et al., 2023). Somaclonal variations are genetic or epigenetic changes induced in plant cells through tissue culture (Bairu et al., 2011; Azizi et al., 2020). This tool is undesirable in micropropagation when the objective is for all clones to faithfully preserve the characteristics of the parent plant, and for germplasm conservation (Bairu et al., 2011). However, somaclonal variation can be beneficial as it increases the genetic variability associated with desirable characteristics, contributing to improvement programs in developing new cultivars (Krishna et al., 2016; Zhang et al., 2022; Ferreira et al., 2023). Through this tool, variants with improved characteristics have been developed, including plant architecture, higher productivity, tolerance to abiotic stressors, and disease resistance (Krishna et al., 2016; Anil et al., 2018). In sugarcane cultivation, for example, the cultivar VSI 434 was developed through somaclonal variation induction using the plant growth regulator 2,4-D. Morphological variation in a number of characteristics was observed, besides moderate resistance to the fungus that causes red rot in sugarcane ( Colletotrichum falcatum ), when compared with the cultivar from which it was derived (CoC 671) (Tawar et al., 2016). Modgil et al. (2012) induced somaclonal variants from apple rootstocks using benzylpurine and thidiazuron (TDZ) plant regulators; in their study, five variants resistant to white root rot ( Rosellinia necatrix ) were selected after 60 days of evaluation in infested soil. In Musa sp., induction of somaclonal variation was successful in developing somaclones resistant to Fusarium wilt. Somaclonal variation in banana of the Cavendish subgroup was induced at the Taiwan Banana Research Institute and evaluated in an area infested by TR4. Among these, the GCTCV-218 somaclonal variant was selected and registered for commercial cultivation under the name ‘Formosana’ (Hwang; ko, 2004; Molina, 2016). At the Bhabha Atomic Research Centre (India), somatic banana embryos were subcultivated for 14 years, and four ‘Rasthali’ somaclones resistant to Foc race 1 were obtained (Ghag et al., 2014). Ferreira et al. (2020) and Ferreira et al. (2024) selected 15 somaclones derived from the Prata-Anã cultivar (Prata subgroup, AAB) with resistance to Foc ST4; and Rebouças et al. (2020) identified 02 somaclones of Grande Naine (Cavendish, AAA) resistant to the same race of Foc. In this context, the present study aimed to induce somaclonal variation using the growth regulators Paclobutrazol and Thidiazuron in the banana the cultivars ‘Grand Naine’ (Cavendish, AAA), and to identify somaclonal variants potentially resistant to Foc ST4. 2. Material and Methods 2.1. In vitro induction of multi-sprouting from stem apices The experiments were conducted at the Tissue Culture Laboratory and a greenhouse at the Embrapa Mandioca e Fruticultura, Cruz das Almas, Bahia state, Brazil. To induce multi-sprouting, the stem apices of the, ‘Grand Naine’ cultivar were cultivated in vitro in MS medium supplemented with 1 mg L − 1 Paclobutrazol® (PBZ), and 1 mg L − 1 thidiazuron (TDZ), 1.6 mg L − 1 indole acetic acid, 80 mg L − 1 adenine hemisulfate, and 30 g L − 1 sucrose, with pH adjusted to 5.8, and solidified with 2.4 g L − 1 Phytagel®. The work was carried out according to the methodology described by Rebouças et al. (2021). Subcultivations was performed at intervals of 30 to 40 days by cleaning the explants, removing the oxidized parts and pieces of leaf sheaths to expose buds, and cutting longitudinally at the meristematic end to break apical dominance and stimulate multiplication. The experiment comprised five replicates, with each consisting of a plate containing ten multi-sprouting plants for ‘Grand Naine’ cultivar and subcultivated ten times. As a positive control, 208 plants of the Grande Naine cultivar without somaclonal variation induction were used. During the process of regeneration of multi-sprouting plants, they were transferred to the MS medium and kept in a growing room in the presence of light (light intensity of 36 µMol m − 2 s − 1 , 16 h light photoperiod), and temperature of 25 ± 2°C; subsequently, the regenerated plants were transplanted to tubes containing coconut fiber substrate (Plantmax®) and acclimatized for 60 days in the greenhouse. 2.2 Fusarium wilt resistance test The Foc isolate CNPMF 218A, belonging to the Plant Pathology Laboratory Biological Collection of Embrapa Mandioca e Fruticultura, was used for the resistance tests. This isolate is considered virulent and aggressive as it causes symptoms of Fusarium wilt in ‘Grand Naine’ (Cavendish), which is resistant to Foc race 1, after periods of stress caused by harsh winters or water deficit (Costa et al., 2015). This isolate was identified as Foc ST4 based on vegetative compatibility group (VCG) tests, and characterized as VCG 0120 (Rocha et al., 2020 and Rocha et al.,2022). The inoculum was produced from peaking the CNPMF 218A isolate in plates with potato dextrose agar culture medium to obtain an isolate suspension. Then, 10 mL of the suspension was inoculated into each rice bag at a temperature of 25°C with a 12 h photoperiod for 20 days. At the end of these 20 days, the colony-forming units were counted to ensure a concentration of 10 6 conidia per gram of inoculum, which is ideal for application in polyethylene boxes with soil. 2.2.1. First test of putative somaclones against Foc ST4 After 60 days of acclimatization, the somaclone seedlings were transferred to a greenhouse and planted in beds measuring 10 x 1 m, with soil infested with isolate CNPMF 218. 90 days after planting or at plant death, internal symptoms of rhizome discoloration were evaluated according to the graded scale of 1 to 5 described by Dita et al. (2014), where (1) indicates no symptoms, (2) early rhizome discoloration, (3) mild rhizome discoloration, (4) rhizome with most internal tissues showing necrosis, and (5) completely necrotic rhizome. The frequency of each grade of the internal symptom of the somaclones was calculated and converted to a percentage. 2.2.2. Second test of resistant somaclones against Foc ST4 The resistant somaclones from the first test were reintroduced in vitro and multiplied in MS medium + 2.5 mg L-1 of BAP to obtain a larger number of clone plants aiming for a new evaluation of resistance to Foc ST4. The same methodology from first test was used, differing in the use of 10 replicates for each somaclone to validate resistance to Foc. At 90 days after planting, the somaclones were evaluated for resistance level using the same internal symptom scale described in the first test battery. The internal symptom scores were transformed into a disease intensity index (DI) according to the formula described by McKinney (1923). The DI data were evaluated for normality of residuals using the Shapiro-Wilk test to ensure compliance with the analysis of variance, and the means were then grouped using the Scott-Knott test at 5% probability, with the Exp.Des.pt package in R software (R Core Development Team, 2017). 2.3 Histochemical analysis of resistant somaclones The analysis of root clarification and staining of fungal structures was conducted according to the method described by Phillips and Hayman. The roots were immersed in a 10% potassium hydroxide (KOH) solution at room temperature for 48 h, followed by immersion in a 1% HCl solution for 30 min. Trypan blue dye in a 0.05% solution (lactic ac-id:glycerol:water = 2:1:1) was applied for 1 h. After staining, the slides were prepared and fragments were microphotographed using an optical microscope (Olympus Latin Ameri-ca). 3. Results and Discussion Among the 852 somaclones evaluated 30 showed no disease symptoms (grade 1) and were thus selected as being resistant to Foc ST4. All plants used as the control of the evaluated cultivar showed reddish-brown rhizome dots (typical symptoms of the disease) visualized in the cross-section of the pseudostem and dead plants were also observed before the final evaluation period (Fig. 1 C). The other evaluated somaclonal variants showed Fusarium wilt symptoms varying from entre grade 2 to grade 5. Most of the somaclones were classified as grade 2, indicative of initial rhizome discoloration, which is a mild symptom of the disease. All 208 control plants of Grande Naine exhibited disease symptoms, with scores ranging from 2 to 5. The occurrence of genetic variability in resistant somaclones indicates that the combined action of the plant regulators paclobutrazol (PBZ) and thidiazuron (TDZ) in the culture medium associated with successive subcultivation series was effective in obtaining somaclonal variants. TDZ is a plant regulator of the cytokines group, responsible for the cell division process (Pelah et al., 2002; Sheibani et al., 2006; Ahmad et al., 2018). This regulator promotes high multiplication rates by triggering various functions in plant tissues, including increased formation of lateral buds, shoot formation, callus induction, and plant regeneration. These processes influence the induction of somaclonal variation, which can result in new genetic/epigenetic traits in plants (Ali et al., 2022; Ferreira et al., 2023). Studies conducted by Ferreira et al. (2020), Rebouças et al. (2020), and Ferreira et al. (2024) demonstrated the effectiveness of this regulator, highlighting its potential in generating banana somaclones resistant to Foc ST4. The study by Pop et al. (2023) on micropropagation employed different concentrations of TDZ (0.5, 1.0, and 2.0 mg/L) to evaluate its effectiveness in shoot regeneration from embryogenic calluses in five grapevine cultivars. The best results were observed in the Merlot variety, which exhibited the highest number of regenerated somaclones (5) under the influence of TDZ, indicating that this growth regulator is effective in inducing shoot regeneration in certain varieties. ‘Fetească albă’ and ‘Traminer roz’ responded less to TDZ treatment, each generating only one somaclone, suggesting variability in cultivar response to the regulator. Based on these results, the authors emphasize the importance of TDZ in inducing somaclonal variation, which can be explored for selecting superior genotypes in grapevine breeding programs. The paclobutrazol (PBZ) is a regulator that slows plant growth and acts by inhibiting gibberellin biosynthesis (Latimer; Scoggins, 2012). Besides, PBZ can act by interfering with the sterol biosynthesis, reducing the amount of abscisic acid, ethylene, and indole acetic acid, thereby increasing the number of cytokines (Razani et al., 2019). Thus, the simultaneous action of these two plant regulators promotes an increase in cell division by increasing the amounts of cytokine, which may have triggered changes in DNA, causing some type of variation. According to Alvares et al. (2012), the action of plant regulators on the culture medium promotes an increase in the cell division process capable of generating extensive cell reprogramming during regeneration cycles, which can cause loss of genes or their function, or even the activation of muted genes. In addition, the increase in the activity of transposable elements which occurs during in vitro cultivation conditions, may result in changes in the plant genome (Ghag et al., 2014) as its mobility near or within the gene regions may modify the expression of a given gene causing both genetic and epigenetic phenotypic changes (Kashkush et al., 2003; Galindo-González et al., 2018). Viljoen et al. (2020) evaluated four banana somaclones developed in Taiwan (TBRI) and a Cavendish mutant, in an area naturally infested with Foc TR4 in Mozambique, for resistance to the pathogen. The DPM-25 mutant showed greater susceptibility to Foc TR4, when compared to GCTCV’s. This study found that the GCTCV-119 somaclone was the most resistant to Foc TR4 among all genotypes; however, the GCTCV-218 somaclone, considered moderately resistant, showed better bunch conformation and productivity. Another determining factor for the induction of somaclonal variation is the number of subcultures (Ferreira et al., 2023; Ferreira et al., 2024). In this study, we performed 10 subcultures and obtained 13 resistant somaclones. Ferreira et al. (2020) induced somaclonal variation in 2,520 plants of the banana cultivar ‘Prata-Anã’, subcultivated 12 times using the plant regulators Thidiazuron. They identified two somaclones resistant to the isolate CNPMF 218A after two resistance validation tests by inoculation in a greenhouse. Rebouças et al. (2020) selected somaclones of the Grand Naine cultivar (Cavendish, AAA) resistant to Foc ST4. These studies demonstrated that it is possible to select somaclones resistant to Fusarium wilt, making this method a promising approach for banana genetic improvement targeting resistance to Foc TR4. Ghag et al. (2014) achieved somaclonal variation in banana embryos subcultivated for 14 years, under tissue culture conditions, by regenerating twenty-six somaclones of ‘Rasthali’, four of which were considered resistant to Fusarium wilt. The 30 resistant somaclones of the Grand Naine cultivar identified in the initial evaluation against Foc ST4 were subjected to a new resistance assessment using 10 replicates (plants) per genotype in a completely randomized design. In the second round of tests, variance analysis revealed statistically significant differences among the 30 somaclones evaluated. The Scott-Knott test identified the formation of distinct groups related to disease symptomatology. Of the 30 somaclones, 13 were classified as highly resistant to Foc ST4: S5, S6, S7, S9, S13, S14, S15, S17, S20, S21, S22, S23, and S28. Somaclones S1, S16, and S30, which exhibited symptom levels below 30%, were classified as resistant (Fig. 2 and Figure S1). Somaclones S2, S24, S26, S10, S19, S29, S25, S18, S27, and S11 were classified as moderately resistant, with disease indices of up to 40% symptoms. Somaclones S3, S12, S4, and S8 exhibited indices ranging from 50–60% and were evaluated as susceptible, while the control showed highly susceptible behavior, with disease indices of 90% (Fig. 2 and Figure S1). These results indicate genetic or epigenetic variability in the response to Fusarium wilt among the somaclones of the 'Grand Naine' cultivar, as they demonstrated distinct responses to the pathogen. The observed variation highlights the complexity of plant-pathogen interactions and the importance of understanding the underlying mechanisms of resistance, which can guide future strategies for genetic improvement and disease management. In Fig. 3 A, no pathogen structures were identified in the roots of the 13 somaclones classified as highly resistant. In Fig. 3 B, representing resistant or moderately resistant somaclones, only chlamydospores were observed. In Fig. 3 C, both hyphae and chlamydospores of the pathogen were detected in the roots of somaclones in the susceptible groups. These results may reflect the different levels of resistance among the somaclones. In the positive control, represented in Fig. 3 D, a greater quantity of hyphae and chlamydospores was observed. The highly resistant somaclones, represented in Fig. 3 A, may exhibit monogenic resistance to the fungus, suggesting the expression of a single gene, as they showed no disease symptoms or presence of the pathogen within their tissues, which may indicate that hyphal penetration through the roots did not occur. On the other hand, in the resistant and moderately resistant somaclones, it can be inferred that resistance is polygenic, involving the expression of multiple genes in defense against the pathogen. This is evidenced by the penetration and colonization of tissues with sporulation presence, although symptoms were below 20% (Figs. 3 B and 3 C). Both types of resistance are valuable in combating Foc; however, somaclones with polygenic resistance tend to be more stable over time, as the fungus would need to overcome multiple genes to surpass this resistance. The resistant somaclones, may have developed physical and chemical barriers to prevent the entry of pathogens, including lignification, which makes the cell wall more resistant; formation of papillae at the penetration sites; and accumulation of gels and tyloses, which are produced in the xylem in response to infection. Resistant cultivars produce these compounds earlier, blocking systemic infection owing to the obstruction of the pathogen’s action and the production of antifungal compounds, a mechanism possibly present in the resistant somaclones identified in this study (Ploetz, 2015; Bani et al., 2018). An example is the ‘Formosana’ somaclone (GCTCV-218), developed by the Taiwan Banana Research Institute, which showed early biochemical defense induction against an isolate of Foc ST4 (VCG 0120), through the positive regulation of cell wall strengthening enzymes such as peroxidase and phenylalanine ammonia-lyase and by the accumulation of phenolic content linked to the cell wall (Hwang; Ko, 2004; Van den Berg et al., 2007). As a defense response to Foc, the activation of reactive oxygen species (ROS) and antioxidant enzymes may also have occurred, acting as an important signal molecule in the banana’s defense response against Foc (Li et al., 2013; Fung et al., 2019; Liu et al., 2020). In the study by Ming et al. (2019), ROS such as superoxide anion (O 2 - ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (OH) were among the first responses observed in the banana cultivar 'Berangan' after pathogen invasion. The authors reported that the ROS were able to delay the pathogen advance in this cultivar and that production of ROS is important to ensure that the hypersensitivity response works properly. Further evaluations should be conducted to identify the type of resistance of the selected somaclones and whether this resistance has been preserved. It is assumed that the type of resistance, primarily associated with somaclones showing low levels of symptoms, such as somaclones S1 and S16 (Fig. 2 ), is quantitative. This is related to the contribution of several genes with small and moderate effects and, therefore, facilitates the use of the somaclonal variation induction tool on a specific target, causing modifications that result in a defense response in the plant (Clair et al., 2010; Mundt et al., 2014; Corwin & Kliebenstein, 2017). According to Haddad et al. (2018), quantitative resistance associated with cultivation practices and biological control can effectively control the disease by inhibiting focus infection, suppressing the inoculum, decreasing pathogenic effects, or even increasing plant defenses. Considering the difficulty in developing Cavendish-type hybrids through hybridization, the induction of somaclonal variation in vitro proves to be a promising tool, especially since highly resistant genotypes were selected in our study. These somaclones are currently undergoing agronomic field evaluations to assess their agronomic potential and marketability, as well as the stability of their resistance. If the agronomic traits are preserved and the stability of acquired resistance is confirmed, 'Grand Naine' somaclones may have great potential for use not only in Brazil but also in other countries facing the presence of Foc ST4, given the importance of Cavendish cultivars in the global banana market. 4. Conclusions In this study, the use of plant regulators PBZ and TDZ, along with an increased number of subcultures, is associated with the induction of somaclonal variants resistant to Fusarium wilt. It was possible to select 13 somaclones from the Cavendish subgroup resistant to Foc ST4. The results of this study may provide a foundation for future research on somaclone induction in Musa and support new studies to understand the type of resistance involved at the molecular level. Declarations Acknowledgements The authors thank CNPq for the research productivity grant to Amorim EP, to Fapesb, for the Ph.D. scholarship granted to the first author, and the “ Breeding Better Bananas/ IITA/Bill and Melinda Gates Foundation” project for funding the research. Author contributions TAR performed the experiments. AdJRC, MdSF, WDdSO, FdSN, TSC, and VBdOA assisted TAR in performing the experiments. Analyses and interpretations were performed by TAR and AdJRC. The manuscript was written by TAR, AdJRC, FdSN, and WDdSO. WSS, JPFLdJ, VBdOA, MdSF, EPA, CFF, JAdS-S, and FH critically reviewed the paper. All authors commented on earlier versions of the manuscript. EPA administered the project, facilitated, and supervised the research. Data availability All data generated or analysed during this study are included in this published article Conflict of interest The authors declare no conflicts of interest. References Abubakar, A. I., Khairulmazmi, A., Yasmeen, S., Muhammad,A.,Abdul, W., Abdulaziz, B. K., Adamu, A., Syazwan, A., M., Z., Arifin, A., Siti N., A., A., 2023. Fusarium wilt of banana: Current update and sustainable disease control using classical and essential oils approaches. Hortic. Plant J. 9, 1-28. https://doi.org/10.1016/j.hpj.2022.02.004. Adly, W.M.R.M., Niedbała, G., EL-Denary, M.E., Mohamed, M.A., Piekutowska, M., Wojciechowski, T., Abd El-Salam, E.-S.T., Fouad, A.S., 2023. 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12:45:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6556573/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6556573/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11240-026-03354-w","type":"published","date":"2026-02-21T15:59:40+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82069195,"identity":"bfd5f1e9-16d1-4fae-bf5b-399444d4423b","added_by":"auto","created_at":"2025-05-06 13:07:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3175182,"visible":true,"origin":"","legend":"\u003cp\u003eThe evaluation of internal symptoms indicative of the presence of \u003cem\u003eFusarium oxysporum \u003c/em\u003ef. sp. \u003cem\u003ecubense \u003c/em\u003eat 90 days after inoculation in the somaclonal variants of the cultivars Grand Naine. A: Greenhouse test, B: resistant somaclone; C: control.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6556573/v1/9090c6da3685f5b1128d6553.png"},{"id":82069194,"identity":"4a287432-88c9-4f3f-848b-27af9c6fa97b","added_by":"auto","created_at":"2025-05-06 13:07:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":360434,"visible":true,"origin":"","legend":"\u003cp\u003eResponse of somaclones to \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003ecubense\u003c/em\u003esubtropical race 4 under greenhouse conditions. Data are presented as mean ± SD (standard deviation) from three replicates. HR: highly resistant; R: resistant; MR: moderately resistant; S: susceptible; HS: highly susceptible.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6556573/v1/a381efa0d481771dc9fa642b.png"},{"id":82068849,"identity":"fe9297f6-aaa3-4a33-97fa-24ae81e20dbe","added_by":"auto","created_at":"2025-05-06 12:59:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2792832,"visible":true,"origin":"","legend":"\u003cp\u003eMicrographs of root fragments from somaclones of the Grande Naine cultivar, showing resistance levels to infection by \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003ecubense\u003c/em\u003esubtropical race 4. (A) Highly resistant somaclone without the presence of pathogen structures in root tissues. (B) Resistant somaclone with the presence of pathogen structures. (C) Moderately resistant somaclone with the presence of pathogen structures (hyphae and chlamydospores) in the roots. (D) Positive control showing pathogen structures (Grande Naine plants not subjected to the somaclonal induction process). Arrows indicate chlamydospores (Chl) and fungal hyphae (Hyp).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6556573/v1/811cd66b4d8619e71b929b87.png"},{"id":103251583,"identity":"30e28b4a-f055-439a-8ade-516ed553aa1f","added_by":"auto","created_at":"2026-02-23 16:10:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9683745,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6556573/v1/3f417400-69b1-492f-b7d8-ee35d213585e.pdf"}],"financialInterests":"","formattedTitle":"Genetic resistance among banana somaclones derived from the Grand Naine cultivar (Cavendish, AAA) to Fusarium oxysporum f. sp. cubense, subtropical race 4.","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe banana is the most consumed fresh fruit worldwide and represents one of the basic consumer products of the global population. Banana production is one of the most important sources of food and income, especially for small farmers (Casta\u0026ntilde;eda et al., 2017; Thangavelu et al., 2021). The five largest world producers are India, with an annual production of 34.5\u0026nbsp;million tons, China with 11.7\u0026nbsp;million tons, Indonesia with 9.2\u0026nbsp;million tons, a Nigeria with 8.0\u0026nbsp;million tons, and Brazil, with approximately 7.0\u0026nbsp;million tons (FAOSTAT, 2024).\u003c/p\u003e \u003cp\u003eOne of the main limitations in banana production is the fungus \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003ecubense\u003c/em\u003e (Foc) that causes \u003cem\u003eFusarium\u003c/em\u003e wilt. This disease threatens subsistence farming, local trade, and exports (Noar et al., 2022; Le Thi et al., 2022 Abubakara et al., 2023). Foc is a soil-dwelling necrotrophic pathogen that produces chlamydospores, which are survival structures that remain viable through periods of dormancy, allowing the disease\u0026rsquo;s persistence over long periods, even in the absence of the host (Dita et al., 2018, Ploetz, 2015). Banana plants are infected by the penetration of the fungus in the roots and colonization of the vascular system, resulting in the blockage of the xylem vessels, which causes wilting owing to the impediment of water and nutrient transport to the aerial part of the plant (Zheng et al., 2018; Lal et al., 2024).\u003c/p\u003e \u003cp\u003eSignificant damage caused by this pathogen began to appear in the 1900s and especially affected the cultivar \u0026lsquo;Gros Michel\u0026rsquo; focused on the export market (Stover, 1962; Ploetz, 2005). Thus, Fusarium wilt became known as a disease of grave concern at a global scale, and the only effective control method was the replacement of \u0026lsquo;Gros Michel\u0026rsquo; with resistant cultivars belonging to the subgroup Cavendish (Zheng et al., 2018). However, the disease occurred again in the 1980s with the detection of a highly virulent strain that was characterized as race 4, divided into subtropical (ST4) and tropical 4 (TR4) races. Foc (ST4) can cause infection in Cavendish cultivars when exposed to any antibiotic stressors; however, TR4 attacks under any environmental condition (Dita et al., 2018; Rocha et al., 2020).\u003c/p\u003e \u003cp\u003eDespite different control measures used to contain Fusarium wilt, such as cultivation practices and biological control, developing resistant cultivars is the most efficient strategy to control this disease (Haddad et al., 2018; Thangavelu et al., 2019; Catambacan e Cumagun, 2021).\u003c/p\u003e \u003cp\u003eHowever, some factors limit the improvement of the banana tree, of which the high sterility observed in some cultivars is notable, leading to a reduced number of seeds obtained from hybridization between commercial cultivars and improved diploids (Ghag et al., 2014; Alakonya et al., 2018). Thus, the genetic improvement of \u003cem\u003eMusa\u003c/em\u003e sp. needs to use non-conventional improvement strategies, including the \u003cem\u003ein vitro\u003c/em\u003e induction of somaclonal variation, focusing on selecting genotypes resistant to diseases and with superior agronomic characteristics (Chen et al., 2013; Ferreira et al. 2020; Rebou\u0026ccedil;as, et al., 2021; Adly et al., 2023).\u003c/p\u003e \u003cp\u003eSomaclonal variations are genetic or epigenetic changes induced in plant cells through tissue culture (Bairu et al., 2011; Azizi et al., 2020). This tool is undesirable in micropropagation when the objective is for all clones to faithfully preserve the characteristics of the parent plant, and for germplasm conservation (Bairu et al., 2011). However, somaclonal variation can be beneficial as it increases the genetic variability associated with desirable characteristics, contributing to improvement programs in developing new cultivars (Krishna et al., 2016; Zhang et al., 2022; Ferreira et al., 2023). Through this tool, variants with improved characteristics have been developed, including plant architecture, higher productivity, tolerance to abiotic stressors, and disease resistance (Krishna et al., 2016; Anil et al., 2018).\u003c/p\u003e \u003cp\u003eIn sugarcane cultivation, for example, the cultivar VSI 434 was developed through somaclonal variation induction using the plant growth regulator 2,4-D. Morphological variation in a number of characteristics was observed, besides moderate resistance to the fungus that causes red rot in sugarcane (\u003cem\u003eColletotrichum falcatum\u003c/em\u003e), when compared with the cultivar from which it was derived (CoC 671) (Tawar et al., 2016). Modgil et al. (2012) induced somaclonal variants from apple rootstocks using benzylpurine and thidiazuron (TDZ) plant regulators; in their study, five variants resistant to white root rot (\u003cem\u003eRosellinia necatrix\u003c/em\u003e\u003cb\u003e)\u003c/b\u003e were selected after 60 days of evaluation in infested soil.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eMusa\u003c/em\u003e sp., induction of somaclonal variation was successful in developing somaclones resistant to Fusarium wilt. Somaclonal variation in banana of the Cavendish subgroup was induced at the Taiwan Banana Research Institute and evaluated in an area infested by TR4. Among these, the GCTCV-218 somaclonal variant was selected and registered for commercial cultivation under the name \u0026lsquo;Formosana\u0026rsquo; (Hwang; ko, 2004; Molina, 2016). At the Bhabha Atomic Research Centre (India), somatic banana embryos were subcultivated for 14 years, and four \u0026lsquo;Rasthali\u0026rsquo; somaclones resistant to Foc race 1 were obtained (Ghag et al., 2014). Ferreira et al. (2020) and Ferreira et al. (2024) selected 15 somaclones derived from the Prata-An\u0026atilde; cultivar (Prata subgroup, AAB) with resistance to Foc ST4; and Rebou\u0026ccedil;as et al. (2020) identified 02 somaclones of Grande Naine (Cavendish, AAA) resistant to the same race of Foc.\u003c/p\u003e \u003cp\u003eIn this context, the present study aimed to induce somaclonal variation using the growth regulators Paclobutrazol and Thidiazuron in the banana the cultivars \u0026lsquo;Grand Naine\u0026rsquo; (Cavendish, AAA), and to identify somaclonal variants potentially resistant to Foc ST4.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. \u003cem\u003eIn vitro\u003c/em\u003e induction of multi-sprouting from stem apices\u003c/h2\u003e \u003cp\u003eThe experiments were conducted at the Tissue Culture Laboratory and a greenhouse at the Embrapa Mandioca e Fruticultura, Cruz das Almas, Bahia state, Brazil.\u003c/p\u003e \u003cp\u003eTo induce multi-sprouting, the stem apices of the, \u0026lsquo;Grand Naine\u0026rsquo; cultivar were cultivated \u003cem\u003ein vitro\u003c/em\u003e in MS medium supplemented with 1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Paclobutrazol\u0026reg; (PBZ), and 1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e thidiazuron (TDZ), 1.6 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indole acetic acid, 80 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e adenine hemisulfate, and 30 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e sucrose, with pH adjusted to 5.8, and solidified with 2.4 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Phytagel\u0026reg;. The work was carried out according to the methodology described by Rebou\u0026ccedil;as et al. (2021). Subcultivations was performed at intervals of 30 to 40 days by cleaning the explants, removing the oxidized parts and pieces of leaf sheaths to expose buds, and cutting longitudinally at the meristematic end to break apical dominance and stimulate multiplication.\u003c/p\u003e \u003cp\u003eThe experiment comprised five replicates, with each consisting of a plate containing ten multi-sprouting plants for \u0026lsquo;Grand Naine\u0026rsquo; cultivar and subcultivated ten times. As a positive control, 208 plants of the Grande Naine cultivar without somaclonal variation induction were used. During the process of regeneration of multi-sprouting plants, they were transferred to the MS medium and kept in a growing room in the presence of light (light intensity of 36 \u0026micro;Mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 16 h light photoperiod), and temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C; subsequently, the regenerated plants were transplanted to tubes containing coconut fiber substrate (Plantmax\u0026reg;) and acclimatized for 60 days in the greenhouse.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Fusarium wilt resistance test\u003c/h2\u003e \u003cp\u003eThe Foc isolate CNPMF 218A, belonging to the Plant Pathology Laboratory Biological Collection of Embrapa Mandioca e Fruticultura, was used for the resistance tests. This isolate is considered virulent and aggressive as it causes symptoms of Fusarium wilt in \u0026lsquo;Grand Naine\u0026rsquo; (Cavendish), which is resistant to Foc race 1, after periods of stress caused by harsh winters or water deficit (Costa et al., 2015). This isolate was identified as Foc ST4 based on vegetative compatibility group (VCG) tests, and characterized as VCG 0120 (Rocha et al., 2020 and Rocha et al.,2022).\u003c/p\u003e \u003cp\u003eThe inoculum was produced from peaking the CNPMF 218A isolate in plates with potato dextrose agar culture medium to obtain an isolate suspension. Then, 10 mL of the suspension was inoculated into each rice bag at a temperature of 25\u0026deg;C with a 12 h photoperiod for 20 days. At the end of these 20 days, the colony-forming units were counted to ensure a concentration of 10\u003csup\u003e6\u003c/sup\u003e conidia per gram of inoculum, which is ideal for application in polyethylene boxes with soil.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. First test of putative somaclones against Foc ST4\u003c/h2\u003e \u003cp\u003eAfter 60 days of acclimatization, the somaclone seedlings were transferred to a greenhouse and planted in beds measuring 10 x 1 m, with soil infested with isolate CNPMF 218. 90 days after planting or at plant death, internal symptoms of rhizome discoloration were evaluated according to the graded scale of 1 to 5 described by Dita et al. (2014), where (1) indicates no symptoms, (2) early rhizome discoloration, (3) mild rhizome discoloration, (4) rhizome with most internal tissues showing necrosis, and (5) completely necrotic rhizome. The frequency of each grade of the internal symptom of the somaclones was calculated and converted to a percentage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2. Second test of resistant somaclones against Foc ST4\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe resistant somaclones from the first test were reintroduced in vitro and multiplied in MS medium\u0026thinsp;+\u0026thinsp;2.5 mg L-1 of BAP to obtain a larger number of clone plants aiming for a new evaluation of resistance to Foc ST4. The same methodology from first test was used, differing in the use of 10 replicates for each somaclone to validate resistance to Foc.\u003c/p\u003e \u003cp\u003eAt 90 days after planting, the somaclones were evaluated for resistance level using the same internal symptom scale described in the first test battery. The internal symptom scores were transformed into a disease intensity index (DI) according to the formula described by McKinney (1923). The DI data were evaluated for normality of residuals using the Shapiro-Wilk test to ensure compliance with the analysis of variance, and the means were then grouped using the Scott-Knott test at 5% probability, with the Exp.Des.pt package in R software (R Core Development Team, 2017).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Histochemical analysis of resistant somaclones\u003c/h2\u003e \u003cp\u003e The analysis of root clarification and staining of fungal structures was conducted according to the method described by Phillips and Hayman. The roots were immersed in a 10% potassium hydroxide (KOH) solution at room temperature for 48 h, followed by immersion in a 1% HCl solution for 30 min. Trypan blue dye in a 0.05% solution (lactic ac-id:glycerol:water\u0026thinsp;=\u0026thinsp;2:1:1) was applied for 1 h. After staining, the slides were prepared and fragments were microphotographed using an optical microscope (Olympus Latin Ameri-ca).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003eAmong the 852 somaclones evaluated 30 showed no disease symptoms (grade 1) and were thus selected as being resistant to Foc ST4. All plants used as the control of the evaluated cultivar showed reddish-brown rhizome dots (typical symptoms of the disease) visualized in the cross-section of the pseudostem and dead plants were also observed before the final evaluation period (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe other evaluated somaclonal variants showed Fusarium wilt symptoms varying from entre grade 2 to grade 5. Most of the somaclones were classified as grade 2, indicative of initial rhizome discoloration, which is a mild symptom of the disease. All 208 control plants of Grande Naine exhibited disease symptoms, with scores ranging from 2 to 5.\u003c/p\u003e \u003cp\u003eThe occurrence of genetic variability in resistant somaclones indicates that the combined action of the plant regulators paclobutrazol (PBZ) and thidiazuron (TDZ) in the culture medium associated with successive subcultivation series was effective in obtaining somaclonal variants. TDZ is a plant regulator of the cytokines group, responsible for the cell division process (Pelah et al., 2002; Sheibani et al., 2006; Ahmad et al., 2018). This regulator promotes high multiplication rates by triggering various functions in plant tissues, including increased formation of lateral buds, shoot formation, callus induction, and plant regeneration. These processes influence the induction of somaclonal variation, which can result in new genetic/epigenetic traits in plants (Ali et al., 2022; Ferreira et al., 2023). Studies conducted by Ferreira et al. (2020), Rebou\u0026ccedil;as et al. (2020), and Ferreira et al. (2024) demonstrated the effectiveness of this regulator, highlighting its potential in generating banana somaclones resistant to Foc ST4.\u003c/p\u003e \u003cp\u003eThe study by Pop et al. (2023) on micropropagation employed different concentrations of TDZ (0.5, 1.0, and 2.0 mg/L) to evaluate its effectiveness in shoot regeneration from embryogenic calluses in five grapevine cultivars. The best results were observed in the Merlot variety, which exhibited the highest number of regenerated somaclones (5) under the influence of TDZ, indicating that this growth regulator is effective in inducing shoot regeneration in certain varieties. \u0026lsquo;Fetească albă\u0026rsquo; and \u0026lsquo;Traminer roz\u0026rsquo; responded less to TDZ treatment, each generating only one somaclone, suggesting variability in cultivar response to the regulator. Based on these results, the authors emphasize the importance of TDZ in inducing somaclonal variation, which can be explored for selecting superior genotypes in grapevine breeding programs.\u003c/p\u003e \u003cp\u003eThe paclobutrazol (PBZ) is a regulator that slows plant growth and acts by inhibiting gibberellin biosynthesis (Latimer; Scoggins, 2012). Besides, PBZ can act by interfering with the sterol biosynthesis, reducing the amount of abscisic acid, ethylene, and indole acetic acid, thereby increasing the number of cytokines (Razani et al., 2019). Thus, the simultaneous action of these two plant regulators promotes an increase in cell division by increasing the amounts of cytokine, which may have triggered changes in DNA, causing some type of variation.\u003c/p\u003e \u003cp\u003eAccording to Alvares et al. (2012), the action of plant regulators on the culture medium promotes an increase in the cell division process capable of generating extensive cell reprogramming during regeneration cycles, which can cause loss of genes or their function, or even the activation of muted genes. In addition, the increase in the activity of transposable elements which occurs during \u003cem\u003ein vitro\u003c/em\u003e cultivation conditions, may result in changes in the plant genome (Ghag et al., 2014) as its mobility near or within the gene regions may modify the expression of a given gene causing both genetic and epigenetic phenotypic changes (Kashkush et al., 2003; Galindo-Gonz\u0026aacute;lez et al., 2018).\u003c/p\u003e \u003cp\u003eViljoen et al. (2020) evaluated four banana somaclones developed in Taiwan (TBRI) and a Cavendish mutant, in an area naturally infested with Foc TR4 in Mozambique, for resistance to the pathogen. The DPM-25 mutant showed greater susceptibility to Foc TR4, when compared to GCTCV\u0026rsquo;s. This study found that the GCTCV-119 somaclone was the most resistant to Foc TR4 among all genotypes; however, the GCTCV-218 somaclone, considered moderately resistant, showed better bunch conformation and productivity.\u003c/p\u003e \u003cp\u003eAnother determining factor for the induction of somaclonal variation is the number of subcultures (Ferreira et al., 2023; Ferreira et al., 2024). In this study, we performed 10 subcultures and obtained 13 resistant somaclones. Ferreira et al. (2020) induced somaclonal variation in 2,520 plants of the banana cultivar \u0026lsquo;Prata-An\u0026atilde;\u0026rsquo;, subcultivated 12 times using the plant regulators Thidiazuron. They identified two somaclones resistant to the isolate CNPMF 218A after two resistance validation tests by inoculation in a greenhouse. Rebou\u0026ccedil;as et al. (2020) selected somaclones of the Grand Naine cultivar (Cavendish, AAA) resistant to Foc ST4. These studies demonstrated that it is possible to select somaclones resistant to Fusarium wilt, making this method a promising approach for banana genetic improvement targeting resistance to Foc TR4. Ghag et al. (2014) achieved somaclonal variation in banana embryos subcultivated for 14 years, under tissue culture conditions, by regenerating twenty-six somaclones of \u0026lsquo;Rasthali\u0026rsquo;, four of which were considered resistant to Fusarium wilt.\u003c/p\u003e \u003cp\u003eThe 30 resistant somaclones of the Grand Naine cultivar identified in the initial evaluation against Foc ST4 were subjected to a new resistance assessment using 10 replicates (plants) per genotype in a completely randomized design.\u003c/p\u003e \u003cp\u003eIn the second round of tests, variance analysis revealed statistically significant differences among the 30 somaclones evaluated. The Scott-Knott test identified the formation of distinct groups related to disease symptomatology. Of the 30 somaclones, 13 were classified as highly resistant to Foc ST4: S5, S6, S7, S9, S13, S14, S15, S17, S20, S21, S22, S23, and S28. Somaclones S1, S16, and S30, which exhibited symptom levels below 30%, were classified as resistant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Figure S1).\u003c/p\u003e \u003cp\u003eSomaclones S2, S24, S26, S10, S19, S29, S25, S18, S27, and S11 were classified as moderately resistant, with disease indices of up to 40% symptoms. Somaclones S3, S12, S4, and S8 exhibited indices ranging from 50\u0026ndash;60% and were evaluated as susceptible, while the control showed highly susceptible behavior, with disease indices of 90% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Figure S1).\u003c/p\u003e \u003cp\u003eThese results indicate genetic or epigenetic variability in the response to Fusarium wilt among the somaclones of the 'Grand Naine' cultivar, as they demonstrated distinct responses to the pathogen.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe observed variation highlights the complexity of plant-pathogen interactions and the importance of understanding the underlying mechanisms of resistance, which can guide future strategies for genetic improvement and disease management.\u003c/p\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, no pathogen structures were identified in the roots of the 13 somaclones classified as highly resistant. In Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, representing resistant or moderately resistant somaclones, only chlamydospores were observed. In Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, both hyphae and chlamydospores of the pathogen were detected in the roots of somaclones in the susceptible groups. These results may reflect the different levels of resistance among the somaclones. In the positive control, represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, a greater quantity of hyphae and chlamydospores was observed.\u003c/p\u003e \u003cp\u003eThe highly resistant somaclones, represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, may exhibit monogenic resistance to the fungus, suggesting the expression of a single gene, as they showed no disease symptoms or presence of the pathogen within their tissues, which may indicate that hyphal penetration through the roots did not occur. On the other hand, in the resistant and moderately resistant somaclones, it can be inferred that resistance is polygenic, involving the expression of multiple genes in defense against the pathogen. This is evidenced by the penetration and colonization of tissues with sporulation presence, although symptoms were below 20% (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eBoth types of resistance are valuable in combating Foc; however, somaclones with polygenic resistance tend to be more stable over time, as the fungus would need to overcome multiple genes to surpass this resistance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe resistant somaclones, may have developed physical and chemical barriers to prevent the entry of pathogens, including lignification, which makes the cell wall more resistant; formation of papillae at the penetration sites; and accumulation of gels and tyloses, which are produced in the xylem in response to infection. Resistant cultivars produce these compounds earlier, blocking systemic infection owing to the obstruction of the pathogen\u0026rsquo;s action and the production of antifungal compounds, a mechanism possibly present in the resistant somaclones identified in this study (Ploetz, 2015; Bani et al., 2018). An example is the \u0026lsquo;Formosana\u0026rsquo; somaclone (GCTCV-218), developed by the Taiwan Banana Research Institute, which showed early biochemical defense induction against an isolate of Foc ST4 (VCG 0120), through the positive regulation of cell wall strengthening enzymes such as peroxidase and phenylalanine ammonia-lyase and by the accumulation of phenolic content linked to the cell wall (Hwang; Ko, 2004; Van den Berg et al., 2007).\u003c/p\u003e \u003cp\u003eAs a defense response to Foc, the activation of reactive oxygen species (ROS) and antioxidant enzymes may also have occurred, acting as an important signal molecule in the banana\u0026rsquo;s defense response against Foc (Li et al., 2013; Fung et al., 2019; Liu et al., 2020). In the study by Ming et al. (2019), ROS such as superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), and hydroxyl radical (OH) were among the first responses observed in the banana cultivar 'Berangan' after pathogen invasion. The authors reported that the ROS were able to delay the pathogen advance in this cultivar and that production of ROS is important to ensure that the hypersensitivity response works properly.\u003c/p\u003e \u003cp\u003eFurther evaluations should be conducted to identify the type of resistance of the selected somaclones and whether this resistance has been preserved. It is assumed that the type of resistance, primarily associated with somaclones showing low levels of symptoms, such as somaclones S1 and S16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), is quantitative. This is related to the contribution of several genes with small and moderate effects and, therefore, facilitates the use of the somaclonal variation induction tool on a specific target, causing modifications that result in a defense response in the plant (Clair et al., 2010; Mundt et al., 2014; Corwin \u0026amp; Kliebenstein, 2017). According to Haddad et al. (2018), quantitative resistance associated with cultivation practices and biological control can effectively control the disease by inhibiting focus infection, suppressing the inoculum, decreasing pathogenic effects, or even increasing plant defenses.\u003c/p\u003e \u003cp\u003eConsidering the difficulty in developing Cavendish-type hybrids through hybridization, the induction of somaclonal variation in vitro proves to be a promising tool, especially since highly resistant genotypes were selected in our study. These somaclones are currently undergoing agronomic field evaluations to assess their agronomic potential and marketability, as well as the stability of their resistance. If the agronomic traits are preserved and the stability of acquired resistance is confirmed, 'Grand Naine' somaclones may have great potential for use not only in Brazil but also in other countries facing the presence of Foc ST4, given the importance of Cavendish cultivars in the global banana market.\u003c/p\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eIn this study, the use of plant regulators PBZ and TDZ, along with an increased number of subcultures, is associated with the induction of somaclonal variants resistant to Fusarium wilt. It was possible to select 13 somaclones from the Cavendish subgroup resistant to Foc ST4. The results of this study may provide a foundation for future research on somaclone induction in \u003cem\u003eMusa\u003c/em\u003e and support new studies to understand the type of resistance involved at the molecular level.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank CNPq for the research productivity grant to Amorim EP, to Fapesb, for the Ph.D. scholarship granted to the first author, and the \u0026ldquo;\u003cem\u003eBreeding Better Bananas/ IITA/Bill and Melinda Gates Foundation\u0026rdquo;\u0026nbsp;\u003c/em\u003eproject for funding the research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTAR performed the experiments. AdJRC, MdSF, WDdSO, FdSN, TSC, and VBdOA assisted TAR in performing the experiments. Analyses and interpretations were performed by TAR and AdJRC. The manuscript was written by TAR, AdJRC, FdSN, and WDdSO. WSS, JPFLdJ, VBdOA, MdSF, EPA, CFF, JAdS-S, and FH critically reviewed the paper. All authors commented on earlier versions of the manuscript. EPA administered the project, facilitated, and supervised the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbubakar, A. I., Khairulmazmi, A., Yasmeen, S., Muhammad,A.,Abdul, W., Abdulaziz, B. K., Adamu, A., Syazwan, A., M., Z., Arifin, A., Siti N., A., A., 2023. 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S., et al. 2022. Molecular, histological and histochemical responses of banana cultivars challenged with Fusarium oxysporum f. sp. cubense with mpacto f levels of virulence.\u0026nbsp;\u003cem\u003ePlants\u003c/em\u003e. 11, 1-23. doi: 10.3390/plants11182339.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSantana. W.S., Rocha. A.D.J., Silva.W.S Oliveira., S., Ramos A.P.S., A Haddad., F Amorim. E.P., 2024. Selection of Improved Banana Diploid Resistant to \u003cem\u003eFusarium oxysporum\u003c/em\u003e f.sp. \u003cem\u003ecubense\u003c/em\u003e Races1andSubtropical4, Agronomy 14,1277; https://doi.org/10.3390/agronomy14061277.\u003c/li\u003e\n \u003cli\u003eShalaby, T. A., Taha, N. A., Taher, D. I., Metwaly, M. M., El-Beltagi, H. S., Rezk, A. A., ... \u0026amp; Bayoumi, Y. A., 2022. Paclobutrazol improves the quality of tomato seedlings to be resistant to Alternaria solani Blight disease: Biochemical and histological perspectives. Plants, 11, 1-15. https://doi.org/10.3390/plants11030425\u003c/li\u003e\n \u003cli\u003eStover, R.H., 1962. Fusarium wilt (Panama disease) of bananas and other \u003cem\u003eMusa\u003c/em\u003e species. Common wealth Mycological Inst., Kew, UK.\u003c/li\u003e\n \u003cli\u003eTawar, P.N., Sawant, R. A., Sushir, K.V., Devarumath, R. M., Hapase, R.S., Meti, N.T., 2016. VSI 434: new sugarcane variety obtained through somaclonal variation. Agric. Resv. 5, 127-136. https://doi.org/10.1007/s40003-016-0203-0.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eThangavelu, R., Mostert, D., Gopi, M., Devi, P.G., 2019. Padmanaban, B., Molina, A. B., Viljoen, A. First detection of Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) on Cavendish banana in India. \u0026nbsp;Eur. J. Plant Pathol. 154, 777-786. \u0026nbsp; https://doi.org/10.1007/s10658-019-01701-6.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eVan Den Berg, N., Berger, D.K., Hein, I., Birch, P.R.J., Wingfield, M.J., Voljoen, A., 2007. Tolerance in banana to Fusarium wilt is associated with early up-regulated ofcell wall-strengthening genes in the roots. Mol. Plant Pathol. 8, 333-341. https://doi.org/10.1111/j.1364-3703.2007.00389.x.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWarman, N. M., Aitken, E. A. B., 2018. The movement of \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003ecubense\u003c/em\u003e (sub-tropical race 4) in susceptible cultivars of banana. \u0026nbsp;Front. Plant Sci. 9, 1748-1769. https://doi.org/10.3389/fpls.2018.01748/full.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eZheng, S. J., Garc\u0026iacute;a-Bastidas, F. A., LI, X. D., Zeng, L., Bai, T. T., Xu, S. T., Yin, K. S., Li, H. X. X., Fu, G., Yu, Y.C., Yang, L., NGUYEN, H.C., Douangboupha, B., Khaing, A. A., Drenth, A., Seidl, M. F., Meijer, Zuo, C., Deng, G., Li, B., Huo, H., LI, C., Hu, C., Kuang, R., Yang, Q., Dong, T., Sheng, O., Yi, G., 2018.Germplasm screening of Musa spp. for resistance to \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003ecubense\u003c/em\u003e tropical race 4 (Foc TR4). Eur. J. Plant Pathol.151, 723-734. https://dx.doi.org/10.3389/fpls.2018.00457.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eViljoen, A., Mostert, D., Chiconela, T., Beukes, I., Fraser, C., Dwyer, J., et al., 2020. Occurrence and spread of the banana fungus \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003ecubense\u003c/em\u003e TR4 in Mozambique. S Afr J Sci. 116 (11/12), Art. 8608, 11. https://doi.org/10.17159/sajs.2020/8608.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWhitehouse, AB., Johnson, A.W., Passey, A.J, McLeary, K.J., Simpson, D.W., 2014. Serenity\u0026rsquo;: a paler skin-coloured somaclonal variant of the short-day cultivar \u0026lsquo;Florence. Acta Hortic. 1049, 819-821. https://doi.org/10.17660/ActaHortic.2014.1049.130.\u003c/li\u003e\n \u003cli\u003eZhang, Y., Xu, Z.; Xie, T., Zhang, W.; He, Y., \u0026amp; Liu., C. 2022. In vitro selection and identification of a cold-tolerant variant in pineapple (\u003cem\u003eAnanas comosus\u003c/em\u003e). Horticulture, Environment, and Biotechnology. 63, 275-286. https://doi.org/10.1007/s13580-021-00396-1.\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":"Musa spp., genetic improvement, tissue culture, somaclonal variation","lastPublishedDoi":"10.21203/rs.3.rs-6556573/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6556573/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBanana production is affected by several pests and diseases, of which Fusarium wilt, caused by \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003ecubense\u003c/em\u003e (Foc), is notable. Despite the different control measures and management used to contain Fusarium wilt, the development of resistant cultivars has been the best approach to combating this disease. Therefore, this work uses biotechnology aimed to induce somaclonal variation using thidiazuron (TDZ) and Paclobutrazol\u0026reg; (PBZ) growth regulators banana cultivar \u0026lsquo;Grand Naine\u0026rsquo; (Cavendish, AAA), and to identify somaclonal variants resistant to Fusarium wilt of the subtropical race 4 (ST4). Stem apices were cultured \u003cem\u003ein vitro\u003c/em\u003e using medium MS medium supplemented with 1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e PBZ and 1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TDZ. After a series of ten subcultures, the regenerated plants were planted in water tanks containing soil infested with the Foc in a greenhouse. At 90 days after inoculation, plants were evaluated for pathogen resistance using longitudinal sections near the rhizome as well as for symptoms using a graded scale. Thirteen somaclones of the cultivar \u0026lsquo;Grand Naine\u0026rsquo; were selected as resistant to Foc. The resistant somaclones will be evaluated for agronomic and market potential, as well as for the validation of resistance stability, with the purpose of being recommended for farmers and future improvement studies.\u003c/p\u003e","manuscriptTitle":"Genetic resistance among banana somaclones derived from the Grand Naine cultivar (Cavendish, AAA) to Fusarium oxysporum f. sp. cubense, subtropical race 4.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 12:59:42","doi":"10.21203/rs.3.rs-6556573/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-05-01T04:41:30+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-30T14:20:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-30T10:02:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2025-04-29T08:45:03+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":"27794ded-ee8b-49fc-8b11-26fa31df007a","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T16:08:09+00:00","versionOfRecord":{"articleIdentity":"rs-6556573","link":"https://doi.org/10.1007/s11240-026-03354-w","journal":{"identity":"plant-cell-tissue-and-organ-culture-pctoc","isVorOnly":false,"title":"Plant Cell, Tissue and Organ Culture (PCTOC)"},"publishedOn":"2026-02-21 15:59:40","publishedOnDateReadable":"February 21st, 2026"},"versionCreatedAt":"2025-05-06 12:59:42","video":"","vorDoi":"10.1007/s11240-026-03354-w","vorDoiUrl":"https://doi.org/10.1007/s11240-026-03354-w","workflowStages":[]},"version":"v1","identity":"rs-6556573","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6556573","identity":"rs-6556573","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}