Biodegradable hydrogel microspheres from paper waste as a substrate for ex vitro adventitious rooting of Eucalyptus grandis x E. urophylla clonal plants

Biodegradable hydrogel microspheres from paper waste as a substrate for ex vitro adventitious rooting of Eucalyptus grandis x E. urophylla clonal plants | 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 Biodegradable hydrogel microspheres from paper waste as a substrate for ex vitro adventitious rooting of Eucalyptus grandis x E. urophylla clonal plants Cínthia Aparecida Silva, Evelize Aparecida Amaral Sashiki, Rafael Carvalho do Lago, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7546903/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Nov, 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 The use of hydrogels as support for plant rooting has been extensively studied. However, mineral substrates remain the most common choice despite their limitations in availability, cost, and environmental impact. In the context of plant biotechnology and sustainable clonal propagation systems, this study evaluated biodegradable hydrogel microspheres composed of cellulose microfibrils (CMF) and nanofibrils (CNF), derived from waste paper, as an alternative substrate for the ex vitro adventitious rooting and acclimatisation of Eucalyptus urophylla x E . grandis (urograndis eucalypt) clonal plants. The microspheres were subjected to alkaline pre-treatments: (1) sodium hydroxide (NaOH); (2) NaOH + hydrogen peroxide (Bleached); (3) calcium silicate (CaSiO₃); (4) magnesium silicate (MgSiO₃), and characterised using Fourier transform infrared spectroscopy (FTIR). Clonal plants' performance was assessed through morphological traits and the Dickson Quality Index (DQI). Following 30 days of observation, the Bleached, CaSiO₃, and MgSiO₃ treatments performed similarly to those of the vermiculite control in terms of rooting, vigour, and the absence of contamination. Following 90 days, favourable outcomes were maintained concerning height, stem diameter, and DQI. Notably, MgSiO₃-treated microspheres promoted greater leaf and shoot development, while Bleached microspheres enhanced leaf area. In contrast, NaOH-treated samples led to contamination and reduced performance. These findings demonstrate that CMF/CNF-based hydrogel microspheres, particularly those treated with MgSiO₃, represent a sustainable biotechnological innovation and effective alternative substrate for the large-scale clonal propagation of urograndis eucalypt. hydroretenter polymer cellulose nanofibrils sustainability acclimatisation clonal propagation Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Eucalyptus urophylla × E. grandis (urograndis eucalypt), one of the most widely propagated hybrids, stands out due to its rapid growth, adaptability to different environments, and superior wood quality (Souza et al. 2022 ; Griebeler et al. 2024a , b ). This makes it the predominant choice for commercial plantations in tropical and subtropical regions. The large-scale production of this hybrid's clonal plants relies on efficient micropropagation and rooting protocols (Esquivel et al. 2024 ; Wang et al. 2025 ). In the ex vitro rooting method, acclimatisation of the clonal plants occurs alongside root formation. This reduces the time and cost of micropropagation (Oakes et al. 2020 ; Nawandish et al. 2024 ; Souza et al. 2024 ). Adventitious rooting represents a critical stage in the micropropagation of woody species, particularly in Eucalyptus (de Oliveira et al. 2024 ; Griebeler et al. 2024a ). The success of this stage depends not only on the physiological competence of the explants but also on the use of efficient and economically sustainable substrates that support rooting, acclimatisation, and subsequent field establishment (Brondani et al. 2018 ; Sarma et al. 2024 ; Nawandish et al. 2024 ). Although mineral substrates are commonly used at this stage, they may be limited in terms of availability, cost, and environmental impact, as is the case with peat extraction. (Pirata et al. 2022 ; Madrid-Aispuro et al. 2020 ). In this context, biodegradable hydrogels emerge as a promising alternative. They provide physical support, as well as water, nutrients, and oxygen, to the roots. They also allow the study of root architecture (Patra et al. 2022 ). Numerous studies have demonstrated that integrating hydrogels into substrates, or utilising them as substitutes for conventional substrates, can enhance the rooting process of Eucalyptus clonal plants and other species (Azevedo et al. 2015 ; Neres et al. 2019 ; Ma et al. 2019 ; Kernosenko et al. 2023 ). Therefore, improving rooting efficiency through alternative substrates, such as hydrogels, is a key strategy for supporting the sustainable clonal micropropagation systems and expansion of urograndis eucalypt plantations. Hydrogels are three-dimensional polymers that possess a high capacity for water absorption and retention (Abdel-Raouf et al. 2018 ). Its absorption properties depend on factors such as composition, degree of cross-linking, pH, and the presence of mineral salts (Shaghaleh et al. 2022 ; Xu et al. 2019 ). Hydrogels based on natural polymers, particularly cellulose, have garnered increasing attention due to their biocompatibility, biodegradability, and renewable origin (Ahmed 2015 ; Nascimento et al. 2018 ; Saruchi et al. 2019 ; Akter et al. 2021 ). In addition to being sustainable, CMF/CNF-based hydrogels have good mechanical properties and are inexpensive (Le et al. 2023 ). Specific pre-treatments can be employed to isolate and modify cellulosic fibers for use in hydrogel formulations, promoting their dispersion and interaction within the polymeric matrix (Abe and Yano 2011 ). Among these, alkaline pre-treatments stand out, as they remove lignin and hemicellulose. This process increases the porosity and reactivity of the fibers, thereby enhancing the hydrogel's water absorption and mechanical resistance (Zhang et al. 2023 ; Nascimento et al. 2018 ). An innovative application of hydrogels involves the development of 'transparent soils' for root phenotyping. This medium enables the imaging of root systems in vivo , providing a more accurate representation of root development than traditional hydroponic systems. The transparent soil is made of hydrogel spheres that support root growth by providing air, water, and nutrients. This closely mimics real soil conditions without causing hypoxia in the roots (Ma et al. 2019 ). The hydrogel microspheres provide a favourable microenvironment for plant growth and root development (Karipçin 2023 ). However, it is essential to adjust hydrogel mechanical properties, including resistance and impedance, as they can negatively impact root growth (Wu et al. 2024a ,b). Therefore, this study evaluated the use of biodegradable hydrogel microspheres based on CMF/CNF, which had undergone different alkaline pre-treatments. The aim was to analyse the effect of these microspheres on the ex vitro rooting and acclimatisation of urograndis eucalypt clonal plants. Materials and Methods Source of plant material and study site The genetic material employed in this study to obtain explants was in vitro established from a small sample of the A211 hybrid clone of Eucalyptus grandis Hill ex Maiden x Eucalyptus urophylla S.T. Blake. The ministumps were established in a clonal minigarden under an automated semi-hydroponic system in a sand bed. They received a nutrient solution by means of dripping, which was distributed in four daily applications totalling 4 L m − 2 , according to Souza et al. ( 2022 ). The collection of shoots occurred 20 days after the pruning of the apex of the ministump. Shoots were maintained on Murashige and Skoog (MS) medium supplemented with indole-3-butyric acid (IBA), adjusted to pH 5.8 before autoclaving (121°C, 20 min), as described by Souza et al. ( 2024 ). The microcuttings obtained were standardised and allocated to different experimental treatments. The adventitious rooting and acclimatisation tests were conducted in the Laboratory of in vitro Culture of Forest Species and the Forest Nursery at the Department of Forestry Sciences, Federal University of Lavras (UFLA), Lavras, Minas Gerais, Brazil. Adventitious rooting and acclimatisation ex vitro From tufts of elongated shoots in vitro in medium MS supplemented with 2.68 µM α-naphthaleneacetic acid and 0.22 µM 6-benzylaminopurine (Molinari et al. 2021 ), microcuttings measuring 3 cm in length, were transplanted into microtubes and packaged in a mini-stufim system inside 250 ml bottles. They were kept under a photoperiod of 16 hours, with a luminous intensity of 40 µmol m⁻² s⁻¹. Irrigation was performed manually with a pipette every five days for 30 days (Brondani et al. 2018 ). At the end of this period, the following parameters were evaluated: shoot and leaf number, root presence and length, height (cm), vigour, and the occurrence of contamination. The rooted plants were then transferred to the nursery, where they remained for 90 days under an alternating irrigation system. After this period, their survival rate, height, stem diameter, leaf area, and Dickson Quality Index (DQI) were evaluated. The DQI is an integrative parameter that reflects the robustness and morphological balance of the clonal plants. It is therefore a good indicator of the overall quality of the plant material (Dickson et al. 1960 ). The substrates tested in the acclimatisation were vermiculite, used as a control, and hydrogel microspheres formulated from paper waste, which had undergone various types of pre-treatments. Pre-treatment of waste paper The hydrogel microspheres were formulated from waste kraft paper tubes, which had an average grammage of 400 g/m². These tubes were supplied by Tubominas Indústria e Comércio LTDA. The waste material was shredded in a knife mill and then immersed in four distinct pre-treatment solutions: (1) NaOH 5% (w/w); (2) NaOH 5% + H₂O₂ 24% (w/w) - Bleached; (3) CaSiO₃ 10% (w/w); (4) MgSiO₃ 10% (w/w). The suspensions were maintained at 80 ± 2°C under constant stirring (400 rpm) for 2 hours. Following the administration of the treatments, the waste was subjected to a series of washes with deionised water until it reached a neutral pH level (Dias et al. 2019 ; Mascarenhas et al. 2022 ). The formulation of the microspheres included sodium alginate (CAS 9005-38-3) and calcium dihydrate chloride (CAS 10035-04-8). Obtaining cellulose micro/nanofibrils (CMF/CNF) The pre-treated fibers were suspended in deionised water at a concentration of 2% (w/w) and stirred at 500 rpm for 30 minutes to promote hydration and homogenisation. Fibrillation was performed using a Masuko Sangyo MKGA-80 Supermass-colloid defibrillator (Kawaguchi, Japan) operating at 1500 rpm with ten passes. The distance between the discs was initially set at 10 µm and increased to 100 µm as the viscosity of the suspension increased (Mascarenhas et al. 2022 ). Obtaining the hydrogel microspheres CMF/CNF suspensions were produced from waste paper tubes that underwent different alkaline pre-treatments, as mentioned above. The solids content of all suspensions was adjusted to 1% (w/w). In synthesizing the hydrogels, a 100:1 (w/w) ratio was employed, using a mixture of CMF/CNF suspension and sodium alginate. Subsequently, the mixtures were placed in an ultrasonic bath at 60°C for 30 minutes to ensure complete and uniform homogenisation of the solutions. The microspheres were formed by ionic gelification through dripping the suspensions into a calcium chloride (CaCl₂) solution using 1 mL Pasteur pipettes. The size of the obtained spheres was directly influenced by the diameter of the pipette nozzle. The hydrogel structure is formed through the interaction between alginate and the divalent calcium ions present in the solution. The formed microspheres were kept in a CaCl₂ solution for 24 hours to stabilise them. They were then washed with deionised water and left to soak in it until they were analyzed. Characterisation of hydrogel microspheres using Fourier transform infrared spectroscopy (FTIR) The pre-treated hydrogel microspheres were analysed using a Varian 600-IR FTIR spectrometer to examine their infrared vibrational spectroscopy. This device was coupled with a GladiATR accessory from Pike Technologies to enable attenuated total reflectance (ATR) measurements at a 45° angle using a zinc selenite crystal. The analysed spectral range was from 400 to 4000 cm − 1 , with a total of 32 scans and a resolution of 2 cm − 1 . Statistical analysis Ten repetitions were performed for each of the treatments that were tested. Data collection took place after 30 days of ex vitro culture in the laboratory and subsequently after 90 days in the nursery. The data for the response variables were verified for homoscedasticity and normal distribution using the Hartley and Shapiro–Wilk tests, respectively, with a 5% probability level. The results were evaluated using analysis of variance (ANOVA) (p < 0.05), followed by a Tukey's test (p < 0.05) to compare the means of the treatments. Results and Discussion The use of biodegradable hydrogel microspheres based on CMF/CNF as a substrate for the ex vitro adventitious rooting and acclimatisation of urograndis eucalypt clonal plants was evaluated. Figure 1 shows the effects of pre-treated hydrogel microspheres on the initial development of urograndis eucalypt clonal plants. The following variables were analysed after 30 days: leaf number (A), shoot number (B), shoot length (C), root presence (D), root length (E), vigour (F), and contamination (G). Figure 1 A shows that the leaf number was significantly higher in the MgSiO 3 treatment than in the vermiculite treatment. This increase may be related to the action of silicates as substrate conditioners, which improves the microsphere's physical and chemical properties, such as porosity, aeration, and water retention (Krahl et al. 2022 ). The greater number of leaves suggests an increase in photosynthetic area, which could encourage biomass accumulation in the initial growth stage, even without an external nutritional supply. Regarding shoot number (Fig. 1 B), only the MgSiO 3 treatment differed significantly from vermiculite. This trend was reinforced by the shoot length data (Fig. 1 C), which showed that the NaOH, bleached, and CaSiO 3 treatments performed better than vermiculite alone, although there were no significant differences between them. These results suggest that these treatments favour the formation and growth of shoots. This can be attributed to an improvement in the substrate's physical and chemical conditions, and a possible reduction in osmotic stress (Manning et al. 2017 ). In relation to adventitious rooting, there were no significant differences in root presence (Fig. 1 D) or length (Fig. 1 E) between treatments. These results indicate that the hydrogels performed as well as vermiculite, thus validating their effectiveness as a means of providing physical support for the root system during the initial stage of acclimatisation outside of the soil environment. Vigor (Fig. 1 F) was evaluated based on morphological parameters and was found to be significantly higher in the silicate treatments than in the NaOH treatment. This performance may be related to the balance between the growth of the aerial part and the root system, which highlights the importance of silicates in forming robust and well-structured clonal plants (Etesami and Jeong 2018 ). Regarding contamination (Fig. 1 G), there was a significant increase in the NaOH treatment, which negatively affected the overall performance of the plants in this group. This result can be attributed either to the residual presence of alkaline compounds in the material or to the incomplete degradation of organic components, which favour microbial proliferation. By contrast, treatments involving silicates and bleached produced low levels of contamination similar to those of vermiculite. This indicates that these substrates are non-toxic and safe for ex vitro cultivation. The effects of different pre-treatments on the morphology of urograndis eucalypt hybrid clonal plants transferred to the nursery after 90 days of acclimatisation are shown in Fig. 2 (A–H). The results showed that hydrogel microsphere substrates performed equally or better than vermiculite, particularly in silicate treatments. Regarding leaf area (Fig. 2A), the Bleached treatment showed higher average values than vermiculite. This may be due to an improvement in the substrate's physical and chemical properties, such as roughness, water retention, and aeration, resulting in greater leaf expansion and consequently a larger photosynthetic surface area (de Paula et al. 2024 ). Tomaszewska-Sowa ( 2020 ) also observed an increased number of leaves and leaf area under the influence of hydrogel during the acclimatisation of Lippia dulcis Trev. clonal plants. There were no statistically significant differences in plant height (Fig. 2B) or stem diameter (Fig. 2C) between treatments. However, the survival rate (Fig. 2D) was significantly lower only in the NaOH treatment (p < 0.05). The other treatments had higher values that were statistically similar to each other. This indicates that the modified substrates (Bleached, CaSiO 3 , and MgSiO 3 ) contributed to the establishment and viability of the clonal plants as much as the vermiculite. In terms of root dry mass (Fig. 2E), there was no statistically significant difference between the treatments and the vermiculite treatment. However, the NaOH treatment was significantly lower. A similar trend was observed for the shoot dry mass (Fig. 2F), although there was no statistical significance between treatments. The same pattern was observed for total dry mass (Fig. 2G). These results suggest that the silicates, particularly MgSiO 3 , promoted biomass accumulation by mitigating the effects of water stress, such as improved water retention and aeration. This benefited root development and consequently shoot growth. In addition, silicon (Si) is classified as a beneficial plant nutrient. It is commonly applied in the form of silicate and has been shown to induce tolerance to adverse conditions and contribute to plant growth under abiotic stress (Aras et al. 2020 ; Meng et al. 2021 ). Healthy clonal plants with a good stem diameter, well-formed roots, and an adequate root-to-aerial part ratio, as well as balanced nutrition, have a higher survival rate when planted, as well as greater resistance to environmental stresses and better growth (Nicoletti et al. 2014 ; Silva et al. 2015 ). The evaluated DQI values were good for all the hydrogel treatments and were similar to those for vermiculite. Treatment with MgSiO₃ produced a higher DQI value than treatment with NaOH (Fig. 2H), demonstrating the positive impact of silicates on the formation of more uniform and robust clonal plants. According to Tomaszewska-Sowa ( 2020 ), hydrogels are effective in micropropagation and acclimatisation of plants. Using them can increase the efficiency of rooting and clonal plant growth during the transition to ex vitro conditions. Figure 3 (A–E) shows the acclimatisation and rooting stages ex vitro of urograndis eucalypt clonal plants. The hydrogels promote rooting by providing physical support to plants and maintaining moisture levels, which relates to the hydrogel's physical properties of porosity, roughness, and water retention capacity. These properties are closely linked and have a critical influence on the performance of the hydrogel. Good crosslinking in the matrix contributes to mechanical and biological compatibility between the root system and the hydrogel, optimising the substrate's water-air balance (Qin et al. 2025 ). Conversely, in substrates with low porosity, water accumulation can hinder aeration, resulting in hypoxic conditions that are detrimental to rhizogenesis (Schafer and Lerner 2022 ). Porosity directly influences the swelling and biological performance of hydrogels, which vary according to cellulose concentration and crosslinking agent type (Akalin and Pulat 2018 ). The degree of crosslinking and the synthesis method can modulate the surface roughness, which can increase the swelling rate (Kabiri et al. 2003 ; Xu et al. 2016 ; Womack et al. 2022 ). Increasing the amount of crosslinker intensifies this roughness, providing greater structural stability and resistance to degradation. (Wanat et al. 2025 ). Thus, it is possible that the CMF/CNF hydrogel microspheres, cross-linked with CaCl 2 , became more stable and humid, favouring the extension and formation of the urograndis eucalypt root system branches. Figure 4 shows the FTIR spectra of hydrogels with different pre-treatments, confirming the presence of functional groups that are characteristic of interactions between MFC/NFCs, alginate, and CaCl₂. Bleached and NaOH treatments showed an increase in the intensity of the bands at ~ 3335 and ~ 3313 cm⁻¹, respectively. This is attributed to axial stretching of the hydroxyl groups (-OH), which indicates greater exposure of free hydroxyls and favours interaction with alginate through hydrogen bonds. (Goncharuk et al. 2024 ; Shen et al. 2017 ). In addition, it increases water retention capacity, a property that is relevant for plant rooting applications. The absence of bands at ~ 1730 cm⁻¹, which are associated with the C = O stretching of oxidised carboxyl and carbonyl groups, suggests low surface functionalisation (Rzayev et al. 2016 ). This condition may be associated with the maintenance of a more alkaline residual pH and a lower antimicrobial barrier capacity, thereby corroborating the increased incidence of contamination observed in treatment with NaOH, which in turn compromises the performance of the clonal plants. The presence of the band at ~ 1602 cm⁻¹ was observed in all treatments analysed, which corresponds to the asymmetric elongation of the carboxylate group (–COO⁻) (Bajestani et al. 2025 ). The different intensities are attributed to the complexation between the carboxylate groups (–COO⁻) and the calcium íons (Ca²⁺), which are derived from crosslinking with CaCl₂. (Samchenko et al. 2024 ). This complexation reduces the vibrational mobility of the carboxylate groups, resulting in a decrease in band intensity, as observed in silicate treatments. In the Bleached treatment, the intensity of the 1602 cm⁻¹ band was higher, suggesting a greater formation of carboxylate groups resulting from the oxidative process (Huang et al. 2025 ). The band at ~ 1419 cm⁻¹ corresponds to the symmetric stretching of the carboxylate group (–COO⁻), which was evident in all treatments. This corroborates the formation of ionic bonds between Ca²⁺ and the functional groups present in alginate and CMF/CNF (Bajestani et al. 2025 ; Goncharuk et al. 2024 ). This ionic crosslinking was fundamental to forming a stable three-dimensional network. It also allowed for the controlled release of Ca²⁺ ions, which are essential for root development due to their role in regulating cell division, root elongation, and signalling responses to stress. The CaSiO₃ and MgSiO₃ treatments exhibited spectral patterns that were similar to those of the other treatments, but with more pronounced bands in the region of ~ 1021 e ~ 999 cm⁻¹. These bands are attributed to asymmetric stretching of Si–O–Si bonds (Chen et al. 2021 ; Han et al. 2022 ), as well as possible contributions from C–O–C bonds in glycoside bridges of CMF/CNFs. The presence of these bands indicates that there are surface interactions between the ions released by the silicates (Ca²⁺, Mg²⁺, and Si) and the functional groups of the polymer matrix. This results in structural alterations to the hydrophilic network. These modifications result in the gradual release of ions, which may have been beneficial for rooting since silicon is associated with increased resistance to abiotic stress, such as drought and salinity, as well as cell wall strengthening and root growth stimulation (Santos et al. 2021 ). Root penetration and elongation are directly affected by hydrogel impedance. Different gel substrate compositions promote phenotypic variations in root development. When gel hardness exceeds a certain threshold, root penetration is significantly hindered (Wu et al. 2024a ,b). Making adjustments to the pre-treatment of cellulose fibers and the reticulation solutions of the hydrogel matrix can effectively meet the needs of plant roots (Ma et al. 2023 ). The results showed that the hydrogel microspheres formulated with 1% alginate provided adequate physical structure, allowing penetration and root development, regardless of the pre-treatment applied. In addition to the growth of the main root, significant formation of lateral roots was observed (Fig. 3 E). These lateral roots play a fundamental role in the absorption of water and nutrients, making the plant more efficient at acquiring resources from the medium. Previous studies on the optimisation of sodium alginate concentrations added to MFC/NFC showed that a concentration of 1% produced greater hardness, resilience, and water retention. These mechanical properties are favourable for rooting, since the microspheres need to be strong enough not to collapse under their own weight (data not shown). In addition, alginate-based hydrogels are particularly promising due to their ability to promote plant growth. Sodium alginate, a polysaccharide derived from brown algae, is composed of β-D-manuronic and α-L-guluronic acids. The enzymatic degradation of these monomers produces oligosaccharides that improve germination, shoot elongation, and root growth (Pettinelli et al. 2024 ). Hydrogels have the potential to enhance the rooting process in vegetative propagation through cuttings, particularly for forest species such as Eucalyptus, by providing the necessary moisture and structural conditions for the development of adventitious roots. When choosing a hydrogel, it is important to consider not only its water retention function but also how it interacts directly with the roots, influencing the supply of water, nutrients, and aeration (Agbna and Zaidi 2025 ). Conclusion This study demonstrated that CMF/CNF-based hydrogel microspheres, particularly those pre-treated with MgSiO₃, improved rooting, leaf development, and overall plant quality more effectively than vermiculite. Treatments with Bleached, CaSiO₃, and MgSiO₃ maintained low contamination levels, thereby reinforcing their suitability for use in controlled micropropagation systems. These biodegradable hydrogel microspheres, derived from paper waste, represent a sustainable biotechnological alternative to non-renewable mineral substrates. They contribute to the efficient clonal propagation of urograndis eucalypt and support more environmentally responsible forestry practices. Declarations Founding sources This work was supported by the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) (Code Project RED 180/2023 and Code Project RED-00225-23). Declaration of Competing Interest All the authors of this manuscript state that they have no conflicts of interest. Authors Contribution Statement Cínthia Aparecida Silva: conceptualisation, data curation, formal analysis, investigation, methodology, writing – original draft, writing – review and editing. Evelize Aparecida Amaral Sashiki: Conceptualisation, data curation, formal analysis, investigation, writing – original draft. Rafael Carvalho do Lago: conceptualisation, data curation, formal analysis, project administration. Júlia Naves Teixeira: Conceptualisation, investigation, methodology. Douglas Machado Leite: Conceptualisation, data curation, formal analysis, investigation, methodology, writing – original draft. Gilvano Ebling Brondani: conceptualisation, funding acquisition, supervision. Gustavo Henrique Denzin Tonoli: conceptualisation, funding acquisition, supervision. Lourival Marin Mendes: conceptualisation, funding acquisition, supervision, project administration. Acknowledgments The authors gratefully acknowledge the financial support provided by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). Data Availability All data generated or analyzed during this study are included in this published article or can be obtained from the corresponding author on request. References Abdel-Raouf ME, El-Saeed SM, Zaki EG, Al-Sabagh AM (2018) Green chemistry approach for preparation of hydrogels for agriculture applications through modification of natural polymers and investigating their swelling properties. Egyptian J Petroleum 27:1345–1355. https://doi.org/10.1016/j.ejpe.2018.09.002 Abe K, Yano H (2011) Formation of hydrogels from cellulose nanofibers. 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Ornam Hortic 28:181–192. https://doi.org/10.1590/2447-536X.v28i2.2496 Shaghaleh H, Alhaj Hamoud Y, Xu X et al (2022) A pH-responsive/sustained release nitrogen fertilizer hydrogel based on aminated cellulose nanofiber/cationic copolymer for application in irrigated neutral soils. J Clean Prod 368:133098. https://doi.org/10.1016/j.jclepro.2022.133098 Shen X-J, Huang P-L, Chen J-H et al (2017) Comparison of Acid-hydrolyzed and TEMPO-oxidized Nanocellulose for Reinforcing Alginate Fibers. BioResources 12:8180–8198 Silva CRA, Ribeiro A, de Oliveira AS et al (2015) Desenvolvimento biométrico de mudas de eucalipto sob diferentes lâminas de irrigação na fase de crescimento. Pesquisa Florestal Brasileira 35:381–390. https://doi.org/10.4336/2015.pfb.35.84.897 Souza DMCS, Martins AR, Fernandes SB et al (2022) In Vitro Multiplication of Eucalyptus pilularis and Eucalyptus grandis x E. urophylla (Urograndis Eucalypt): Effect of Light Quality in Temporary Immersion Bioreactor. 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Planr Cell Tissue Organ Cult 160:1–11. https://doi.org/10.1007/s11240-025-02967-x Womack NC, Piccoli I, Camarotto C et al (2022) Hydrogel application for improving soil pore network in agroecosystems. Preliminary results on three different soils. CATENA 208:105759. https://doi.org/10.1016/j.catena.2021.105759 Wu Q, Xie J, Li J et al (2024a) Engineering Rapeseed Germination and Root Growth with Mechanical Strength of Polysaccharide Hydrogel. ACS Appl Bio Mater 7:3496–3505. https://doi.org/10.1021/acsabm.4c00416 Wu Q, Xie J, Li J, Men Y (2024b) Engineering living root with mechanical stimulation derived from reciprocating compression in a double network hydrogel as elastic soil. Adv Agrochem 1–9. https://doi.org/10.1016/j.aac.2024.10.001 Xu H, Liu Y, Xie Y et al (2019) Doubly cross-linked nanocellulose hydrogels with excellent mechanical properties. Cellulose 26:8645–8654. https://doi.org/10.1007/s10570-019-02689-2 Xu Z, Li J, Zhou H et al (2016) Morphological and swelling behavior of cellulose nanofiber (CNF)/poly(vinyl alcohol) (PVA) hydrogels: Poly(ethylene glycol) (PEG) as porogen. RSC Adv 6:43626–43633. https://doi.org/10.1039/c6ra03620a Zhang Z, Abidi N, Lucia L et al (2023) Cellulose/nanocellulose superabsorbent hydrogels as a sustainable platform for materials applications: A mini-review and perspective. Carbohydr Polym 299:120140. https://doi.org/10.1016/j.carbpol.2022.120140 Cite Share Download PDF Status: Published Journal Publication published 26 Nov, 2025 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted Reviewers agreed at journal 10 Sep, 2025 Reviewers invited by journal 10 Sep, 2025 Editor assigned by journal 06 Sep, 2025 First submitted to journal 05 Sep, 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. 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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-7546903","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":512963578,"identity":"809004d5-7b60-404d-ad6e-148be38c97a1","order_by":0,"name":"Cínthia Aparecida Silva","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIiWNgGAWjYDACZhAyALEYGx8wMByAiR/AoR6shbEZooW52YA4LQwgLWDA3iZBlBb+dt7jjwsK7PL4pRvbqnlq7sjxMzA/fHSD4U4+Li0Sh/kSm2cYJBdLzjnYdpvn2DNjyQY2Y+MchmeWDTi0GDDzGDbzGDAnbriRCNTCdjhxwwEeNukchsMGuGyBaqlP3A/UUszzj3gtQJUSiW3MvG1EaJE4zGM4m8fgeOKMOwebJef2HTaWbAb5xeAZTi38/WcMPvP8qU7sn93+8MObb4fl+NmbHz7OqbiDUwuSfQwMTDwgBjPYwYQ1gLUw/iBG4SgYBaNgFIw4AACFvlYMDHfBzQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-5021-1840","institution":"Federal University of Lavras: Universidade Federal de Lavras","correspondingAuthor":true,"prefix":"","firstName":"Cínthia","middleName":"Aparecida","lastName":"Silva","suffix":""},{"id":512963579,"identity":"970ad5ad-52ad-4d1a-b8c3-eae297496621","order_by":1,"name":"Evelize Aparecida Amaral Sashiki","email":"","orcid":"","institution":"Federal University of Lavras: Universidade Federal de Lavras","correspondingAuthor":false,"prefix":"","firstName":"Evelize","middleName":"Aparecida Amaral","lastName":"Sashiki","suffix":""},{"id":512963580,"identity":"d741f423-505d-4904-be5a-89e5df044adf","order_by":2,"name":"Rafael Carvalho do Lago","email":"","orcid":"","institution":"Federal University of Lavras: Universidade Federal de Lavras","correspondingAuthor":false,"prefix":"","firstName":"Rafael","middleName":"Carvalho do","lastName":"Lago","suffix":""},{"id":512963581,"identity":"09b09d79-e6f3-467e-8751-db30a09c24fe","order_by":3,"name":"Júlia Naves Teixeira","email":"","orcid":"","institution":"Federal University of Lavras: Universidade Federal de Lavras","correspondingAuthor":false,"prefix":"","firstName":"Júlia","middleName":"Naves","lastName":"Teixeira","suffix":""},{"id":512963582,"identity":"fc0d5c57-f714-46e3-b8f8-8008290ce4e4","order_by":4,"name":"Douglas Machado Leite","email":"","orcid":"","institution":"Federal University of Lavras: Universidade Federal de Lavras","correspondingAuthor":false,"prefix":"","firstName":"Douglas","middleName":"Machado","lastName":"Leite","suffix":""},{"id":512963583,"identity":"458210d6-2b4c-42a3-868f-5f7397e7654e","order_by":5,"name":"Gilvano Ebling Brondani","email":"","orcid":"","institution":"Federal University of Lavras: Universidade Federal de Lavras","correspondingAuthor":false,"prefix":"","firstName":"Gilvano","middleName":"Ebling","lastName":"Brondani","suffix":""},{"id":512963584,"identity":"28cdd0c6-be60-4393-89c7-d928262af12f","order_by":6,"name":"Gustavo Henrique Denzin Tonoli","email":"","orcid":"","institution":"Federal University of Lavras: Universidade Federal de Lavras","correspondingAuthor":false,"prefix":"","firstName":"Gustavo","middleName":"Henrique Denzin","lastName":"Tonoli","suffix":""},{"id":512963585,"identity":"c1437451-5e2f-483b-a2cf-daf143adc07e","order_by":7,"name":"Lourival Marin Mendes","email":"","orcid":"","institution":"Federal University of Lavras: Universidade Federal de Lavras","correspondingAuthor":false,"prefix":"","firstName":"Lourival","middleName":"Marin","lastName":"Mendes","suffix":""}],"badges":[],"createdAt":"2025-09-05 19:57:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7546903/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7546903/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11240-025-03308-8","type":"published","date":"2025-11-26T15:56:52+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91526872,"identity":"7516eda7-ecc6-4547-b70b-77a3f4094089","added_by":"auto","created_at":"2025-09-17 11:12:02","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":203437,"visible":true,"origin":"","legend":"\u003cp\u003eVariables were evaluated during acclimatisation of urograndis eucalypt microcuttings after 30 days of \u003cem\u003eex vitro\u003c/em\u003e culture: A) Leaf number; B) Shoot number; C) Shoot length; D) Root presence; E) Root length; F) Vigour; G) Contamination. Different letters indicate a significant difference between the treatments (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7546903/v1/4b14c6a13e7d454bc31d2717.jpeg"},{"id":91524932,"identity":"b18f3ad6-0fce-4643-a033-b94711de7557","added_by":"auto","created_at":"2025-09-17 11:04:02","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":247214,"visible":true,"origin":"","legend":"\u003cp\u003eVariables evaluated during acclimatisation of urograndis eucalypt microcuttings in the nursery, after 90 days: A) leaf area; B) height, C) stem diameter; D) survival; E) root dry mass, F) shoot dry mass, G) total dry mass; H) DQI. Different letters indicate a significant difference between the treatments (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7546903/v1/9d1ab20fd45971205bd18b6a.jpeg"},{"id":91524935,"identity":"88e2e62c-6936-4f77-88bd-872028af6903","added_by":"auto","created_at":"2025-09-17 11:04:02","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":230975,"visible":true,"origin":"","legend":"\u003cp\u003eElongation, acclimatisation and adventitious rooting of urograndis eucalypt microcuttings: A) Microcuttings in the elongation stage in the culture laboratory; B) Clonal plants transferred to hydrogel microspheres in microtubes; C) Clonal plants in microtubes in a mini-stufim system with hydrogel microspheres after 30 days; D) Clonal plants transferred to the nursery; E) Roots evaluated after 90 days.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7546903/v1/3f59994c3294b64cd6e94f9e.jpeg"},{"id":91527552,"identity":"c0f50ce3-20dd-4082-8aed-7dc4f0cd75b4","added_by":"auto","created_at":"2025-09-17 11:20:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":16931,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of hydrogel microspheres subjected to different pre-treatments: NaOH, Bleached; CasiO\u003csub\u003e3\u003c/sub\u003e; MgSiO\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7546903/v1/f00ce8beb7936a7e5cde5fbf.png"},{"id":97178232,"identity":"f15dfacc-24b6-4e63-b55d-39bc6cef3108","added_by":"auto","created_at":"2025-12-01 16:03:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1395899,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7546903/v1/dc7846ca-7fc5-4902-a34a-0ca718eb8623.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eBiodegradable hydrogel microspheres from paper waste as a substrate for \u003cem\u003eex vitro \u003c/em\u003eadventitious rooting of \u003cem\u003eEucalyptus grandis\u003c/em\u003e x \u003cem\u003eE. urophylla \u003c/em\u003eclonal plants\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eEucalyptus urophylla\u003c/em\u003e \u0026times; \u003cem\u003eE. grandis\u003c/em\u003e (urograndis eucalypt), one of the most widely propagated hybrids, stands out due to its rapid growth, adaptability to different environments, and superior wood quality (Souza et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Griebeler et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003eb\u003c/span\u003e). This makes it the predominant choice for commercial plantations in tropical and subtropical regions. The large-scale production of this hybrid's clonal plants relies on efficient micropropagation and rooting protocols (Esquivel et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In the \u003cem\u003eex vitro\u003c/em\u003e rooting method, acclimatisation of the clonal plants occurs alongside root formation. This reduces the time and cost of micropropagation (Oakes et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Nawandish et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Souza et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAdventitious rooting represents a critical stage in the micropropagation of woody species, particularly in Eucalyptus (de Oliveira et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Griebeler et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e). The success of this stage depends not only on the physiological competence of the explants but also on the use of efficient and economically sustainable substrates that support rooting, acclimatisation, and subsequent field establishment (Brondani et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sarma et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Nawandish et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough mineral substrates are commonly used at this stage, they may be limited in terms of availability, cost, and environmental impact, as is the case with peat extraction. (Pirata et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Madrid-Aispuro et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this context, biodegradable hydrogels emerge as a promising alternative. They provide physical support, as well as water, nutrients, and oxygen, to the roots. They also allow the study of root architecture (Patra et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eNumerous studies have demonstrated that integrating hydrogels into substrates, or utilising them as substitutes for conventional substrates, can enhance the rooting process of Eucalyptus clonal plants and other species (Azevedo et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Neres et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ma et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kernosenko et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, improving rooting efficiency through alternative substrates, such as hydrogels, is a key strategy for supporting the sustainable clonal micropropagation systems and expansion of urograndis eucalypt plantations.\u003c/p\u003e\u003cp\u003eHydrogels are three-dimensional polymers that possess a high capacity for water absorption and retention (Abdel-Raouf et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Its absorption properties depend on factors such as composition, degree of cross-linking, pH, and the presence of mineral salts (Shaghaleh et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Hydrogels based on natural polymers, particularly cellulose, have garnered increasing attention due to their biocompatibility, biodegradability, and renewable origin (Ahmed \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Nascimento et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Saruchi et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Akter et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition to being sustainable, CMF/CNF-based hydrogels have good mechanical properties and are inexpensive (Le et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSpecific pre-treatments can be employed to isolate and modify cellulosic fibers for use in hydrogel formulations, promoting their dispersion and interaction within the polymeric matrix (Abe and Yano \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Among these, alkaline pre-treatments stand out, as they remove lignin and hemicellulose. This process increases the porosity and reactivity of the fibers, thereby enhancing the hydrogel's water absorption and mechanical resistance (Zhang et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Nascimento et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAn innovative application of hydrogels involves the development of 'transparent soils' for root phenotyping. This medium enables the imaging of root systems \u003cem\u003ein vivo\u003c/em\u003e, providing a more accurate representation of root development than traditional hydroponic systems. The transparent soil is made of hydrogel spheres that support root growth by providing air, water, and nutrients. This closely mimics real soil conditions without causing hypoxia in the roots (Ma et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe hydrogel microspheres provide a favourable microenvironment for plant growth and root development (Karip\u0026ccedil;in \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, it is essential to adjust hydrogel mechanical properties, including resistance and impedance, as they can negatively impact root growth (Wu et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e,b). Therefore, this study evaluated the use of biodegradable hydrogel microspheres based on CMF/CNF, which had undergone different alkaline pre-treatments. The aim was to analyse the effect of these microspheres on the \u003cem\u003eex vitro\u003c/em\u003e rooting and acclimatisation of urograndis eucalypt clonal plants.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSource of plant material and study site\u003c/h2\u003e\u003cp\u003eThe genetic material employed in this study to obtain explants was \u003cem\u003ein vitro\u003c/em\u003e established from a small sample of the A211 hybrid clone of \u003cem\u003eEucalyptus grandis\u003c/em\u003e Hill ex Maiden x \u003cem\u003eEucalyptus urophylla\u003c/em\u003e S.T. Blake. The ministumps were established in a clonal minigarden under an automated semi-hydroponic system in a sand bed. They received a nutrient solution by means of dripping, which was distributed in four daily applications totalling 4 L m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, according to Souza et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The collection of shoots occurred 20 days after the pruning of the apex of the ministump. Shoots were maintained on Murashige and Skoog (MS) medium supplemented with indole-3-butyric acid (IBA), adjusted to pH 5.8 before autoclaving (121\u0026deg;C, 20 min), as described by Souza et al. (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe microcuttings obtained were standardised and allocated to different experimental treatments. The adventitious rooting and acclimatisation tests were conducted in the Laboratory of \u003cem\u003ein vitro\u003c/em\u003e Culture of Forest Species and the Forest Nursery at the Department of Forestry Sciences, Federal University of Lavras (UFLA), Lavras, Minas Gerais, Brazil.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAdventitious rooting and acclimatisation\u003c/b\u003e \u003cb\u003eex vitro\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFrom tufts of elongated shoots in vitro in medium MS supplemented with 2.68 \u0026micro;M α-naphthaleneacetic acid and 0.22 \u0026micro;M 6-benzylaminopurine (Molinari et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), microcuttings measuring 3 cm in length, were transplanted into microtubes and packaged in a mini-stufim system inside 250 ml bottles. They were kept under a photoperiod of 16 hours, with a luminous intensity of 40 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;. Irrigation was performed manually with a pipette every five days for 30 days (Brondani et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). At the end of this period, the following parameters were evaluated: shoot and leaf number, root presence and length, height (cm), vigour, and the occurrence of contamination. The rooted plants were then transferred to the nursery, where they remained for 90 days under an alternating irrigation system. After this period, their survival rate, height, stem diameter, leaf area, and Dickson Quality Index (DQI) were evaluated. The DQI is an integrative parameter that reflects the robustness and morphological balance of the clonal plants. It is therefore a good indicator of the overall quality of the plant material (Dickson et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1960\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe substrates tested in the acclimatisation were vermiculite, used as a control, and hydrogel microspheres formulated from paper waste, which had undergone various types of pre-treatments.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePre-treatment of waste paper\u003c/h3\u003e\n\u003cp\u003eThe hydrogel microspheres were formulated from waste kraft paper tubes, which had an average grammage of 400 g/m\u0026sup2;. These tubes were supplied by Tubominas Ind\u0026uacute;stria e Com\u0026eacute;rcio LTDA. The waste material was shredded in a knife mill and then immersed in four distinct pre-treatment solutions: (1) NaOH 5% (w/w); (2) NaOH 5% + H₂O₂ 24% (w/w) - Bleached; (3) CaSiO₃ 10% (w/w); (4) MgSiO₃ 10% (w/w). The suspensions were maintained at 80\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C under constant stirring (400 rpm) for 2 hours. Following the administration of the treatments, the waste was subjected to a series of washes with deionised water until it reached a neutral pH level (Dias et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mascarenhas et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe formulation of the microspheres included sodium alginate (CAS 9005-38-3) and calcium dihydrate chloride (CAS 10035-04-8).\u003c/p\u003e\n\u003ch3\u003eObtaining cellulose micro/nanofibrils (CMF/CNF)\u003c/h3\u003e\n\u003cp\u003eThe pre-treated fibers were suspended in deionised water at a concentration of 2% (w/w) and stirred at 500 rpm for 30 minutes to promote hydration and homogenisation. Fibrillation was performed using a Masuko Sangyo MKGA-80 Supermass-colloid defibrillator (Kawaguchi, Japan) operating at 1500 rpm with ten passes. The distance between the discs was initially set at 10 \u0026micro;m and increased to 100 \u0026micro;m as the viscosity of the suspension increased (Mascarenhas et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eObtaining the hydrogel microspheres\u003c/h3\u003e\n\u003cp\u003eCMF/CNF suspensions were produced from waste paper tubes that underwent different alkaline pre-treatments, as mentioned above. The solids content of all suspensions was adjusted to 1% (w/w).\u003c/p\u003e\u003cp\u003eIn synthesizing the hydrogels, a 100:1 (w/w) ratio was employed, using a mixture of CMF/CNF suspension and sodium alginate. Subsequently, the mixtures were placed in an ultrasonic bath at 60\u0026deg;C for 30 minutes to ensure complete and uniform homogenisation of the solutions.\u003c/p\u003e\u003cp\u003eThe microspheres were formed by ionic gelification through dripping the suspensions into a calcium chloride (CaCl₂) solution using 1 mL Pasteur pipettes. The size of the obtained spheres was directly influenced by the diameter of the pipette nozzle. The hydrogel structure is formed through the interaction between alginate and the divalent calcium ions present in the solution. The formed microspheres were kept in a CaCl₂ solution for 24 hours to stabilise them. They were then washed with deionised water and left to soak in it until they were analyzed.\u003c/p\u003e\n\u003ch3\u003eCharacterisation of hydrogel microspheres using Fourier transform infrared spectroscopy (FTIR)\u003c/h3\u003e\n\u003cp\u003eThe pre-treated hydrogel microspheres were analysed using a Varian 600-IR FTIR spectrometer to examine their infrared vibrational spectroscopy. This device was coupled with a GladiATR accessory from Pike Technologies to enable attenuated total reflectance (ATR) measurements at a 45\u0026deg; angle using a zinc selenite crystal. The analysed spectral range was from 400 to 4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, with a total of 32 scans and a resolution of 2 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eTen repetitions were performed for each of the treatments that were tested. Data collection took place after 30 days of \u003cem\u003eex vitro\u003c/em\u003e culture in the laboratory and subsequently after 90 days in the nursery. The data for the response variables were verified for homoscedasticity and normal distribution using the Hartley and Shapiro\u0026ndash;Wilk tests, respectively, with a 5% probability level. The results were evaluated using analysis of variance (ANOVA) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), followed by a Tukey's test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) to compare the means of the treatments.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eThe use of biodegradable hydrogel microspheres based on CMF/CNF as a substrate for the \u003cem\u003eex vitro\u003c/em\u003e adventitious rooting and acclimatisation of urograndis eucalypt clonal plants was evaluated. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the effects of pre-treated hydrogel microspheres on the initial development of urograndis eucalypt clonal plants. The following variables were analysed after 30 days: leaf number (A), shoot number (B), shoot length (C), root presence (D), root length (E), vigour (F), and contamination (G).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA shows that the leaf number was significantly higher in the MgSiO\u003csub\u003e3\u003c/sub\u003e treatment than in the vermiculite treatment. This increase may be related to the action of silicates as substrate conditioners, which improves the microsphere's physical and chemical properties, such as porosity, aeration, and water retention (Krahl et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The greater number of leaves suggests an increase in photosynthetic area, which could encourage biomass accumulation in the initial growth stage, even without an external nutritional supply.\u003c/p\u003e\u003cp\u003eRegarding shoot number (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), only the MgSiO\u003csub\u003e3\u003c/sub\u003e treatment differed significantly from vermiculite. This trend was reinforced by the shoot length data (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), which showed that the NaOH, bleached, and CaSiO\u003csub\u003e3\u003c/sub\u003e treatments performed better than vermiculite alone, although there were no significant differences between them. These results suggest that these treatments favour the formation and growth of shoots. This can be attributed to an improvement in the substrate's physical and chemical conditions, and a possible reduction in osmotic stress (Manning et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn relation to adventitious rooting, there were no significant differences in root presence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) or length (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) between treatments. These results indicate that the hydrogels performed as well as vermiculite, thus validating their effectiveness as a means of providing physical support for the root system during the initial stage of acclimatisation outside of the soil environment.\u003c/p\u003e\u003cp\u003eVigor (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) was evaluated based on morphological parameters and was found to be significantly higher in the silicate treatments than in the NaOH treatment. This performance may be related to the balance between the growth of the aerial part and the root system, which highlights the importance of silicates in forming robust and well-structured clonal plants (Etesami and Jeong \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRegarding contamination (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), there was a significant increase in the NaOH treatment, which negatively affected the overall performance of the plants in this group. This result can be attributed either to the residual presence of alkaline compounds in the material or to the incomplete degradation of organic components, which favour microbial proliferation. By contrast, treatments involving silicates and bleached produced low levels of contamination similar to those of vermiculite. This indicates that these substrates are non-toxic and safe for \u003cem\u003eex vitro\u003c/em\u003e cultivation.\u003c/p\u003e\u003cp\u003eThe effects of different pre-treatments on the morphology of urograndis eucalypt hybrid clonal plants transferred to the nursery after 90 days of acclimatisation are shown in Fig.\u0026nbsp;2 (A\u0026ndash;H). The results showed that hydrogel microsphere substrates performed equally or better than vermiculite, particularly in silicate treatments. Regarding leaf area (Fig.\u0026nbsp;2A), the Bleached treatment showed higher average values than vermiculite. This may be due to an improvement in the substrate's physical and chemical properties, such as roughness, water retention, and aeration, resulting in greater leaf expansion and consequently a larger photosynthetic surface area (de Paula et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Tomaszewska-Sowa (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) also observed an increased number of leaves and leaf area under the influence of hydrogel during the acclimatisation of \u003cem\u003eLippia dulcis\u003c/em\u003e Trev. clonal plants.\u003c/p\u003e\u003cp\u003eThere were no statistically significant differences in plant height (Fig.\u0026nbsp;2B) or stem diameter (Fig.\u0026nbsp;2C) between treatments. However, the survival rate (Fig.\u0026nbsp;2D) was significantly lower only in the NaOH treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The other treatments had higher values that were statistically similar to each other. This indicates that the modified substrates (Bleached, CaSiO\u003csub\u003e3\u003c/sub\u003e, and MgSiO\u003csub\u003e3\u003c/sub\u003e) contributed to the establishment and viability of the clonal plants as much as the vermiculite.\u003c/p\u003e\u003cp\u003eIn terms of root dry mass (Fig.\u0026nbsp;2E), there was no statistically significant difference between the treatments and the vermiculite treatment. However, the NaOH treatment was significantly lower. A similar trend was observed for the shoot dry mass (Fig.\u0026nbsp;2F), although there was no statistical significance between treatments. The same pattern was observed for total dry mass (Fig.\u0026nbsp;2G). These results suggest that the silicates, particularly MgSiO\u003csub\u003e3\u003c/sub\u003e, promoted biomass accumulation by mitigating the effects of water stress, such as improved water retention and aeration. This benefited root development and consequently shoot growth. In addition, silicon (Si) is classified as a beneficial plant nutrient. It is commonly applied in the form of silicate and has been shown to induce tolerance to adverse conditions and contribute to plant growth under abiotic stress (Aras et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Meng et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHealthy clonal plants with a good stem diameter, well-formed roots, and an adequate root-to-aerial part ratio, as well as balanced nutrition, have a higher survival rate when planted, as well as greater resistance to environmental stresses and better growth (Nicoletti et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Silva et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The evaluated DQI values were good for all the hydrogel treatments and were similar to those for vermiculite. Treatment with MgSiO₃ produced a higher DQI value than treatment with NaOH (Fig.\u0026nbsp;2H), demonstrating the positive impact of silicates on the formation of more uniform and robust clonal plants.\u003c/p\u003e\u003cp\u003eAccording to Tomaszewska-Sowa (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), hydrogels are effective in micropropagation and acclimatisation of plants. Using them can increase the efficiency of rooting and clonal plant growth during the transition to \u003cem\u003eex vitro\u003c/em\u003e conditions. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e (A\u0026ndash;E) shows the acclimatisation and rooting stages \u003cem\u003eex vitro\u003c/em\u003e of urograndis eucalypt clonal plants.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe hydrogels promote rooting by providing physical support to plants and maintaining moisture levels, which relates to the hydrogel's physical properties of porosity, roughness, and water retention capacity. These properties are closely linked and have a critical influence on the performance of the hydrogel. Good crosslinking in the matrix contributes to mechanical and biological compatibility between the root system and the hydrogel, optimising the substrate's water-air balance (Qin et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Conversely, in substrates with low porosity, water accumulation can hinder aeration, resulting in hypoxic conditions that are detrimental to rhizogenesis (Schafer and Lerner \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePorosity directly influences the swelling and biological performance of hydrogels, which vary according to cellulose concentration and crosslinking agent type (Akalin and Pulat \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The degree of crosslinking and the synthesis method can modulate the surface roughness, which can increase the swelling rate (Kabiri et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Womack et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Increasing the amount of crosslinker intensifies this roughness, providing greater structural stability and resistance to degradation. (Wanat et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThus, it is possible that the CMF/CNF hydrogel microspheres, cross-linked with CaCl\u003csub\u003e2\u003c/sub\u003e, became more stable and humid, favouring the extension and formation of the urograndis eucalypt root system branches. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the FTIR spectra of hydrogels with different pre-treatments, confirming the presence of functional groups that are characteristic of interactions between MFC/NFCs, alginate, and CaCl₂.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBleached and NaOH treatments showed an increase in the intensity of the bands at ~\u0026thinsp;3335 and ~\u0026thinsp;3313 cm⁻\u0026sup1;, respectively. This is attributed to axial stretching of the hydroxyl groups (-OH), which indicates greater exposure of free hydroxyls and favours interaction with alginate through hydrogen bonds. (Goncharuk et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Shen et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In addition, it increases water retention capacity, a property that is relevant for plant rooting applications. The absence of bands at ~\u0026thinsp;1730 cm⁻\u0026sup1;, which are associated with the C\u0026thinsp;=\u0026thinsp;O stretching of oxidised carboxyl and carbonyl groups, suggests low surface functionalisation (Rzayev et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This condition may be associated with the maintenance of a more alkaline residual pH and a lower antimicrobial barrier capacity, thereby corroborating the increased incidence of contamination observed in treatment with NaOH, which in turn compromises the performance of the clonal plants.\u003c/p\u003e\u003cp\u003eThe presence of the band at ~\u0026thinsp;1602 cm⁻\u0026sup1; was observed in all treatments analysed, which corresponds to the asymmetric elongation of the carboxylate group (\u0026ndash;COO⁻) (Bajestani et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The different intensities are attributed to the complexation between the carboxylate groups (\u0026ndash;COO⁻) and the calcium \u0026iacute;ons (Ca\u0026sup2;⁺), which are derived from crosslinking with CaCl₂. (Samchenko et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This complexation reduces the vibrational mobility of the carboxylate groups, resulting in a decrease in band intensity, as observed in silicate treatments. In the Bleached treatment, the intensity of the 1602 cm⁻\u0026sup1; band was higher, suggesting a greater formation of carboxylate groups resulting from the oxidative process (Huang et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe band at ~\u0026thinsp;1419 cm⁻\u0026sup1; corresponds to the symmetric stretching of the carboxylate group (\u0026ndash;COO⁻), which was evident in all treatments. This corroborates the formation of ionic bonds between Ca\u0026sup2;⁺ and the functional groups present in alginate and CMF/CNF (Bajestani et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Goncharuk et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This ionic crosslinking was fundamental to forming a stable three-dimensional network. It also allowed for the controlled release of Ca\u0026sup2;⁺ ions, which are essential for root development due to their role in regulating cell division, root elongation, and signalling responses to stress.\u003c/p\u003e\u003cp\u003eThe CaSiO₃ and MgSiO₃ treatments exhibited spectral patterns that were similar to those of the other treatments, but with more pronounced bands in the region of ~\u0026thinsp;1021 e\u0026thinsp;~\u0026thinsp;999 cm⁻\u0026sup1;. These bands are attributed to asymmetric stretching of Si\u0026ndash;O\u0026ndash;Si bonds (Chen et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Han et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), as well as possible contributions from C\u0026ndash;O\u0026ndash;C bonds in glycoside bridges of CMF/CNFs. The presence of these bands indicates that there are surface interactions between the ions released by the silicates (Ca\u0026sup2;⁺, Mg\u0026sup2;⁺, and Si) and the functional groups of the polymer matrix. This results in structural alterations to the hydrophilic network. These modifications result in the gradual release of ions, which may have been beneficial for rooting since silicon is associated with increased resistance to abiotic stress, such as drought and salinity, as well as cell wall strengthening and root growth stimulation (Santos et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRoot penetration and elongation are directly affected by hydrogel impedance. Different gel substrate compositions promote phenotypic variations in root development. When gel hardness exceeds a certain threshold, root penetration is significantly hindered (Wu et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e,b). Making adjustments to the pre-treatment of cellulose fibers and the reticulation solutions of the hydrogel matrix can effectively meet the needs of plant roots (Ma et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The results showed that the hydrogel microspheres formulated with 1% alginate provided adequate physical structure, allowing penetration and root development, regardless of the pre-treatment applied. In addition to the growth of the main root, significant formation of lateral roots was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). These lateral roots play a fundamental role in the absorption of water and nutrients, making the plant more efficient at acquiring resources from the medium.\u003c/p\u003e\u003cp\u003ePrevious studies on the optimisation of sodium alginate concentrations added to MFC/NFC showed that a concentration of 1% produced greater hardness, resilience, and water retention. These mechanical properties are favourable for rooting, since the microspheres need to be strong enough not to collapse under their own weight (data not shown). In addition, alginate-based hydrogels are particularly promising due to their ability to promote plant growth. Sodium alginate, a polysaccharide derived from brown algae, is composed of β-D-manuronic and α-L-guluronic acids. The enzymatic degradation of these monomers produces oligosaccharides that improve germination, shoot elongation, and root growth (Pettinelli et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHydrogels have the potential to enhance the rooting process in vegetative propagation through cuttings, particularly for forest species such as Eucalyptus, by providing the necessary moisture and structural conditions for the development of adventitious roots. When choosing a hydrogel, it is important to consider not only its water retention function but also how it interacts directly with the roots, influencing the supply of water, nutrients, and aeration (Agbna and Zaidi \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrated that CMF/CNF-based hydrogel microspheres, particularly those pre-treated with MgSiO₃, improved rooting, leaf development, and overall plant quality more effectively than vermiculite. Treatments with Bleached, CaSiO₃, and MgSiO₃ maintained low contamination levels, thereby reinforcing their suitability for use in controlled micropropagation systems. These biodegradable hydrogel microspheres, derived from paper waste, represent a sustainable biotechnological alternative to non-renewable mineral substrates. They contribute to the efficient clonal propagation of urograndis eucalypt and support more environmentally responsible forestry practices.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eFounding sources\u003c/h2\u003e\u003cp\u003eThis work was supported by the Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de Minas Gerais (FAPEMIG) (Code Project RED 180/2023 and Code Project RED-00225-23).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e\u003cp\u003eAll the authors of this manuscript state that they have no conflicts of interest.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eAuthors Contribution Statement\u003c/h2\u003e\u003cp\u003eC\u0026iacute;nthia Aparecida Silva: conceptualisation, data curation, formal analysis, investigation, methodology, writing \u0026ndash; original draft, writing \u0026ndash; review and editing. Evelize Aparecida Amaral Sashiki: Conceptualisation, data curation, formal analysis, investigation, writing \u0026ndash; original draft. Rafael Carvalho do Lago: conceptualisation, data curation, formal analysis, project administration. J\u0026uacute;lia Naves Teixeira: Conceptualisation, investigation, methodology. Douglas Machado Leite: Conceptualisation, data curation, formal analysis, investigation, methodology, writing \u0026ndash; original draft. Gilvano Ebling Brondani: conceptualisation, funding acquisition, supervision. Gustavo Henrique Denzin Tonoli: conceptualisation, funding acquisition, supervision. Lourival Marin Mendes: conceptualisation, funding acquisition, supervision, project administration.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eThe authors gratefully acknowledge the financial support provided by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES), Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq), and Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de Minas Gerais (FAPEMIG).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this published article or can be obtained from the corresponding author on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdel-Raouf ME, El-Saeed SM, Zaki EG, Al-Sabagh AM (2018) Green chemistry approach for preparation of hydrogels for agriculture applications through modification of natural polymers and investigating their swelling properties. 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Carbohydr Polym 299:120140. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.carbpol.2022.120140\u003c/span\u003e\u003cspan address=\"10.1016/j.carbpol.2022.120140\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\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":"hydroretenter polymer, cellulose nanofibrils, sustainability, acclimatisation, clonal propagation","lastPublishedDoi":"10.21203/rs.3.rs-7546903/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7546903/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe use of hydrogels as support for plant rooting has been extensively studied. However, mineral substrates remain the most common choice despite their limitations in availability, cost, and environmental impact. In the context of plant biotechnology and sustainable clonal propagation systems, this study evaluated biodegradable hydrogel microspheres composed of cellulose microfibrils (CMF) and nanofibrils (CNF), derived from waste paper, as an alternative substrate for the \u003cem\u003eex vitro\u003c/em\u003e adventitious rooting and acclimatisation of \u003cem\u003eEucalyptus urophylla\u003c/em\u003e x \u003cem\u003eE\u003c/em\u003e. \u003cem\u003egrandis\u003c/em\u003e (urograndis eucalypt) clonal plants. The microspheres were subjected to alkaline pre-treatments: (1) sodium hydroxide (NaOH); (2) NaOH\u0026thinsp;+\u0026thinsp;hydrogen peroxide (Bleached); (3) calcium silicate (CaSiO₃); (4) magnesium silicate (MgSiO₃), and characterised using Fourier transform infrared spectroscopy (FTIR). Clonal plants' performance was assessed through morphological traits and the Dickson Quality Index (DQI). Following 30 days of observation, the Bleached, CaSiO₃, and MgSiO₃ treatments performed similarly to those of the vermiculite control in terms of rooting, vigour, and the absence of contamination. Following 90 days, favourable outcomes were maintained concerning height, stem diameter, and DQI. Notably, MgSiO₃-treated microspheres promoted greater leaf and shoot development, while Bleached microspheres enhanced leaf area. In contrast, NaOH-treated samples led to contamination and reduced performance. These findings demonstrate that CMF/CNF-based hydrogel microspheres, particularly those treated with MgSiO₃, represent a sustainable biotechnological innovation and effective alternative substrate for the large-scale clonal propagation of urograndis eucalypt.\u003c/p\u003e","manuscriptTitle":"Biodegradable hydrogel microspheres from paper waste as a substrate for ex vitro adventitious rooting of Eucalyptus grandis x E. urophylla clonal plants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 11:03:57","doi":"10.21203/rs.3.rs-7546903/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-09-10T12:12:01+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-10T08:40:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-06T06:36:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2025-09-05T15:56:56+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":"ac01c106-e222-4b25-8e6d-d7fdc5330130","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T15:59:26+00:00","versionOfRecord":{"articleIdentity":"rs-7546903","link":"https://doi.org/10.1007/s11240-025-03308-8","journal":{"identity":"plant-cell-tissue-and-organ-culture-pctoc","isVorOnly":false,"title":"Plant Cell, Tissue and Organ Culture (PCTOC)"},"publishedOn":"2025-11-26 15:56:52","publishedOnDateReadable":"November 26th, 2025"},"versionCreatedAt":"2025-09-17 11:03:57","video":"","vorDoi":"10.1007/s11240-025-03308-8","vorDoiUrl":"https://doi.org/10.1007/s11240-025-03308-8","workflowStages":[]},"version":"v1","identity":"rs-7546903","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7546903","identity":"rs-7546903","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}