Uncaria tomentosa Willd. ex Roem. & Schult.: genetic and chemical diversity of accessions cultivated in vitro and their introduction into the native environment

Uncaria tomentosa Willd. ex Roem. & Schult.: genetic and chemical diversity of accessions cultivated in vitro and their introduction into the native environment | 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 Uncaria tomentosa Willd. ex Roem. & Schult.: genetic and chemical diversity of accessions cultivated in vitro and their introduction into the native environment Isabela Cristina Gomes Honório, Juliana Silva Coppede, Gustavo Henrique Teixeira Pinto, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9107751/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Uncaria tomentosa Willd. ex Roem. & Schult., known as cat's claw, is a medicinal plant that is native to the Amazon and used to treat inflammatory diseases. Currently plants are harvested in a predatory manner leading potentially to extinction of the species. The aims of this study were to develop an efficient protocol for the micropropagation of the species, to evaluate the genetic and chemical diversity of genotypes cultivated in vitro , and to determine the survival rate of plants introduced into the native environment. The optimised micropropagation protocol employed medium-sized flasks containing 20 mL of woody plant medium (WPM) supplemented with 5.0 µM 6-benzylaminopurine (BAP), with four explants per flask maintained under a 16 h photoperiod provided by light emitting diodes. Under these conditions, explants produced an average of 2.13 shoots bearing a total of 8.13 buds per explant. Twenty accessions of U. tomentosa , each originating from seeds of individual plants collected in three states of the Brazilian Amazon, were maintained in vitro and analyzed by start codon targeted (ScoT) molecular markers and chemically by liquid chromatography. These genotypes showed genetic diversity in vitro that was comparable with natural populations, and the majority of accessions accumulated mitraphylline and isomitraphylline. Plantlets cultivated in vitro were acclimatized and introduced into the native environment with a survival rate of 46 ± 18% after eighteen months. It is concluded that in vitro propagation of U. tomentosa is efficient for the production of vigorous plants capable of surviving in the natural conditions of the Amazon rainforest. Uncaria tomentosa micropropagation acclimatization genetic diversity alkaloid profile field establishment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key message An micropropagation protocol for enabled successful reintroduction of this medicinal species into the Amazon, supporting both its conservation and the development of sustainable production systems. Introduction Uncaria tomentosa Willd. ex Roem. & Schult., commonly known as cat's claw, is a medicinal plant from the Amazon region and used mainly in the treatment of inflammatory diseases (Arado et al. 2024 ). Based on its utilization in the traditional medicine of the indigenous peoples of the Amazon and its scientifically proven therapeutic efficacy, the species is included in monographs of selected medicinal plants by the World Health Organization (Blumenthal 1997 ), and in the National List of Essential Medicines of the Brazilian government (Ministério da Saúde 2013 ) and the United States Pharmacopeia and National Formulary (United States Pharmacopeial Convention 2022 ). Despite the popular use of U. tomentosa and the well-documented body of evidence regarding its pharmaceutical properties, exploitation of this valuable resource depends exclusively on predatory collection since efficient propagation of the species is limited by the scarcity of agronomic studies. As a consequence, natural populations of U. tomentosa are being compromised with increasing risk of genetic erosion (Honório et al. 2016 ). In fact, the vulnerability of U. tomentosa to extinction was first documented a decade earlier by Zavala and Zevallos ( 1996 ) and confirmed in a more recent study by Honório et al. ( 2017 ) involving the use of sequence-related amplified polymorphism (SRAP) markers. These authors reported the occurrence of low gene flow and an island-type genetic structure among the natural populations of U. tomentosa analyzed, and emphasized the importance of conserving accessions in in vitro germplasm banks. Moreover, field studies in the Amazon performed by members of our group have revealed further compounding factors that serve to increase the difficulty of collection from natural populations, including the minute size of the seeds ( 30 m). For these reasons it is likely that the in vitro culture of U. tomentosa will prove to be a sustainable solution for the large-scale production of phytomedicine. Although several researchers have reported the in vitro cultivation of U. tomentosa (Pereira et al. 2008 ; Venutolo 2010 ; Raposo and Teixeira 2011 ; Luna-Palencia et al. 2013 ), a protocol for the efficient micropropagation of the medicinal plant has yet to be developed. Moreover, the multiplication of U. tomentosa under in vitro conditions should be preceded by genetic analysis of introduced accessions in order to preserve genetic variation in the field and to conserve species diversity. Genetic stability and diversity in tissue culture-derived plants is typically assessed using molecular markers as, for example, the robust and cost-effective start codon targeted (SCoT) polymorphism technique (Sirikonda et al. 2023 ; Vrundha and Thomas 2023 ; Rai 2023 ). Thus, the aims of the present study were to establish protocols for the multiplication of U. tomentosa , to employ ScoT markers to verify genetic diversity among micropropagated plantlets maintained in vitro , to evaluate the chemical diversity of these accessions by high-performance liquid chromatography (HPLC), and to introduce the micropropagated plants into their natural environment in the Brazilian Amazon. Materials and methods Plant material Plants of U. tomentosa were identified by Dr. Piero Giuseppe Delprete (Herbier de Guyane, Institut de Recherche pour le Développement, Cayenne, French Guiana) and a specimen was deposited in the Medicinal Plant Herbarium of the Universidade de Ribeirão Preto (UNAERP) under accession number HJBOP 2723. All accessions used in the experiments were registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) under number AB94A2F. The accessions of U. tomentosa maintained in vitro at UNAERP were established from seeds of individual plants collected from the municipalities of Bannach (BN, State of Pará), Tarauacá (TA, State of Acre) and Boca do Acre (BC, State of Amazonas), all of which are located in the Brazilian Amazon rainforest as presented in Fig. 1 . Figure 1 here Seed sterilization and in vitro germination Seeds were sterilized by immersion in 1% w/v Cercobin® for 24 h under constant shaking, followed by treatment with 1% w/v sodium hypochlorite solution for 20 min under similar conditions. After surface asepsis, the seeds were washed with sterile distilled water and transferred to plates containing sucrose-free basal Murashige and Skoog (MS) medium (Murashige and Skoog 1962 ) solidified with 3 g·L − 1 of Gelzan™ CM (Sigma-Aldrich, St. Louis, MO, USA; # G1910) and adjusted to pH 6.0. Incubation was carried out in a growth chamber at 22 ± 2°C under a 16 h photoperiod provided by white fluorescent tubes (25 µmol·m − 2 ·s − 1 ). In order to test the influence of light on germination (photoblastism), batches of 50 seeds were incubated in the growth chamber under a 16 h photoperiod or dark conditions and the experiment repeated four times. The longevity of seeds was assessed by storing seeds at room temperature for one prior to germination. A total of 114 seedlings were established in vitro and analyzed by HPLC to determine the proportions of the alkaloids considered to be the chemical markers of the species, namely mitraphylline and isomitraphylline. Twenty seedlings derived from seeds originating from the three different areas of the Brazilian rainforest were selected for further investigation on the basis of vigor and alkaloid content. Optimization of culture conditions for in vitro micropropagation In order to determine the influence of culture conditions on the efficiency of in vitro micropropagation, explants comprising 1 cm nodal segments from the 20 selected seedlings were cultivated either individually or in groups on different culture media, with or without growth regulators, in diverse types of culture vessels and under various light sources. The experiment was of a completely randomized design and involved the following treatments: (i) Culture medium: individual explants were cultured in glass test tubes containing 5 mL of MS medium, woody plant medium (WPM; Lloyd and McCown 1980 ) or Schenck and Hildebrandt (SH; Schenck and Hildebrandt 1972 ) medium supplemented with 3% sucrose and solidified with 0.30% Gelzan CM at pH 6.0; (ii) Addition of growth regulators: individual explants were cultured in large jars containing 20 mL of WPM supplemented with cytokinins 6-benzylaminopurine (BAP), kinetin or zeatin at concentrations of 0, 0.5, 1.0, 2.5, 5.0 and 10.0 µM, and/or auxins naphthalene acetic acid (NAA) or indolebutyric acid (IBA) at concentrations of 0, 0.1 and 0.5 µM, or a combination of BAP (5.0 µM) and NAA or IBA (0, 0.1 and 0.5 µM). (iii) Size of glassware: individual explants were cultured in large jars (12 cm height x 5.4 cm diameter; 20 mL WPM), medium jars (8.5 cm height x 5.9 cm diameter; 20 mL WPM) or small test tubes(10 cm height x 2 cm diameter; 5 mL WPM) (Fig. 2 A); (iv) Light source: individual explants were cultured in small test tubes containing 5 mL WPM and incubated under a 16 h photoperiod provided by light emitting diode (LED) arrays or fluorescent tubes (25 µmol·m − 2 ·s − 1 ); and (v) Co-cultivation interaction: individual explants or groups of two to four were cultured in large jars containing 20 mL of WPM (Fig. 2 B). Figure 2 here Unless otherwise stated, all explants were cultured on WPM supplemented with 3% sucrose and solidified with 0.30% Gelzan CM at pH 6.0, and maintained in a growth chamber at 22 ± 2 ºC under a 16 h photoperiod provided by LED arrays (25 µmol·m − 2 ·s − 1 ). The variables of interest, namely height of plantlets, number of buds, number of shoots and number of roots), were measured 60 days after cultivation of explants. Each treatment was repeated 10 times and each repetition included 10 plantlets (replicates). Acclimatization of plantlets to greenhouse conditions and field cultivation Plantlets were transferred to 162-cell plastic trays (model 7100235; Nutriplan, Cascavel, PR, Brazil) containing commercial substrate (Carolina Soil, Santa Cruz, RS, Brazil) and maintained for 60 days in a climate-controlled greenhouse (Van der Hoeven, Delft, The Netherlands) with a relative humidity of 70% achieved by operating micro-sprinklers for 5 min, three times daily. Plants were subsequently relocated to 1 L plastic bags containing 900 g of a mixture of soil, manure and sand (3:2:1) and remained in the greenhouse for 10 months with micro-sprinkler irrigation twice a day. Acclimatized plants (40 ± 5 cm height) were transferred to forested areas in the municipality of Santarém, PA, Brazil, during the rainy season. Planting pits (20 cm x 20 cm x 20 cm) were dug near to native trees to provide support for the plants. The survival of the transplanted individuals was evaluated eighteen months after planting. Analysis of the genetic variability of the U. tomentosa accessions The genetic diversity of the 20 selected individuals maintained in vitro was determined by SCoT polymorphism analysis. Genomic DNA was extracted following the method of Doyle and Doyle ( 1987 ) as modified by Honório et al. ( 2017 ), and analyzed using the SCoT molecular marker technique following the method proposed by Collard and Mackill ( 2009 ). The 36 SCoT 18-mer primer sequences described by these authors were screened and eight (5, 8, 13, 15, 19, 24, 29 and 30) presenting the highest polymorphism with sharply resolved bands were selected for the full analysis. Polymerase chain reaction (PCR) amplifications were performed in a thermal cycler (Jingle Scientific, Shanghai, China) with a reaction mixture containing 1 µL of 10x reaction buffer, 1 µL of dNTP mix (2.5 mM), 1 µL of primer (8 µM), 0.1 µL of Taq DNA polymerase (5 U/µL), 2.5 µL of DNA (10 ng·µL − 1 ) and deionized water to final volume (10 µL). Amplifications were carried out under the following conditions: initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 50°C for 1 min and extension at 72°C for 2 min, with a final extension at 72°C for 5 min. The PCR products were separated by electrophoresis on 3% agarose gel prepared in 1x tris-borato-EDTA (TEB) buffer containing ethidium bromide (0.5 µg·mL − 1 ) and visualized under a Gel Logic 112 digital imaging system (Bruker BioSpin, Billerica, MA, USA) and analyzed using Carestream Molecular Imaging (Rochester, NY, USA) software. The bands produced were counted manually to generate a binary matrix (presence or absence of bands) for subsequent data analysis. Chemical analysis of the U. tomentosa accessions The alkaloid content of the U. tomentosa accessions was determined using a previously validated HPLC-diode array detector method (Honório et al. 2017 ) with external standards mitraphylline (LGC Standards CDx, Teddington, Middlesex, UK; # CDX-00013955–005) and isomitraphylline (Chromadex/Niagen Bioscience, Los Angeles, CA, USA; # ASB-00009417). The limits of detection (LOD) and quantification (LOQ) were: mitraphylline (0.22 and 0.75 µg·mL − 1 , respectively) and isomitraphylline (0.12 and 0.24 µg·mL − 1 , respectively). Validation of the analytical data (LOD, LOQ, linearity, precision and accuracy) was performed in accordance with the guidelines of the Agência Nacional de Vigilância Sanitária ( 2003 ). Calibration curves were constructed using triplicate injections of the standard alkaloids at concentrations of 500, 250, 125, 62.5, 31.2, 15.6, 7.8, 3.9, 1.9 µg·mL − 1 . The ratio of peak areas of the target alkaloids were plotted as a function of the corresponding concentration using linear regression of the standard curves as described by Honório et al. ( 2017 ). Data analysis Data relating to the micropropagation experiments were submitted to analysis of variance (ANOVA) and, when significant, means were compared using the Scott-Knott test at α = 0.05. Statistical analyses were performed with the aid of the program Sisvar (Ferreira 2011 ). Binary data (presence or absence of bands) obtained from the ScoT marker analysis were employed to estimate the genetic similarity between the 20 accessions of U. tomentosa using the Jaccard coefficient and unweighted pair-group method with arithmetic mean (UPGMA) cluster analysis. Phylogentic trees were constructed and analysed with the aid of Free Tree software (Hampl et al. 2001 ). Genetic variability was investigated using principal coordinates analysis (PCoA) with the help of GenAlEx 6.5 software (Peakall and Smouse 2012 ). Bayesian assignment analysis was conducted using STRUCTURE (Pritchard et al. 2000 ) to infer the genetic structure of the accessions, grouping individuals into K distinct clusters (K = 1 to 20) based on genetic similarities. Analyses were run under the correlated allele frequencies and no admixture model, which assumes that each individual originated from a single cluster, with a burn-in of 300,000 steps followed by 500,000 Markov chain Monte Carlo (MCmC) iterations. The most likely number of clusters (K) was determined using the ΔK method of Evanno et al. ( 2005 ). Results and discussion Germination and establishment of seedlings in vitro Seeds of U. tomentosa showed positive photoblastism. Germination under a 16 h photoperiod occurred between 10 and 15 days after inoculation onto the medium, with a germination rate of 92 ± 3.4%, while no seeds germinated during this time period under dark conditions. Seeds exhibited low longevity since the germination rate declined to 10 ± 0.5% after one year of storage under the experimental conditions, and no germination was observed following storage for two years. The 114 seedlings established in vitro were assessed for vigor and alkaloid content and 20 were assigned for the micropropagation study. The selected accessions comprised eight originating from TA (UTTA1, UTTA2, UTTA5, UTTA9, UTTA10, UTTA19, UTTA20 and UTTA30), 11 from BN (UTBN207, UTBN233, UTBN332, UTBN354, UTBN358, UTBN499, UTBN750, UTBN767, UTBN847, UTBN902 and UTBN985) and one from BC (UTBC1). Optimization of micropropagation conditions The effects of culture conditions on the development of U. tomentosa explants are presented in Table 1 . Regarding culture medium, WPM favored increased plantlet height and number of shoots compared with MS and SH media. This finding may be related to the lower concentration of mineral salts in WPM relative to MS and SH media, a factor frequently associated with superior responses in woody species and plants native to oligotrophic environments. Considering that U. tomentosa plants occur naturally in nutrient-poor Amazonian soils, it is reasonable to assume that the physiology of the species is better adapted to low nutrient concentrations as already discussed for tropical forest species (Lloyd and McCown 1980 ; Ferreira et al. 2006 ). Table 1 Effects of diverse culture conditions on the development of Uncaria tomentosa explants in vitro Treatments Variables evaluated Height of plantlets (cm) No. of shoots # No. of buds # No. of roots Culture medium MS 2.80b 1.28b 4.48a - SH 1.29b 1.05b 3.52b - WPM 3.58a 1.81a 4.28a - Addition of growth regulators BAP (µM) 0 2.37a 1.33b 6.47b 3.93a 0.5 1.50b 1.33b 5.93b 2.00a 1.0 1.25b 0.93b 9.67a 1.87a 2.5 1.16b 1.80a 7.27b 0.40b 5.0 1.16b 2.13a 8.13a 1.60a 10.0 1.21b 2.06a 6.40b 0.80b Kinetin (µM) 0 2.07a 1.40a 5.93a 4.27a 0.5 1.33a 1.53a 3.80b 2.27b 1.0 1.98a 1.00a 3.00b 1.93b 2.5 1.75a 1.33a 2.73b 1.53b 5.0 1.96a 1.27a 2.96b 1.67b 10.0 1.57a 1.47a 2.57b 0.47b Zeatin (µM) 0 1.96a 1.40a 5.93a 2.73a 0.5 1.86a 1.53a 4.27a 1.20a 1.0 2.34a 1.00a 3.33b 1.27a 2.5 1.45b 1.33a 2.47b 0.20b 5.0 1.43b 1.27a 2.43b 0.33b 10.0 1.20b 1.47a 2.23b 0.13b NAA (µM) 0 1.46b 1.37b 3.27b 1.20c 0.1 1.78a 1.40b 4.37a 2.73b 0.5 2.10a 1.76a 4.63a 3.97a IBA (µM) 0 1.46b 1.37b 3.27b 1.20c 0.1 1.04c 1.27b 2.57b 1.90b 0.5 2.12a 1.77a 5.90a 3.30a Cytokinin (µM) + auxin (µM) 0 2.50a 1.28b 3.85b 0.21a BAP (5) + NAA (0.5) 2.52a 2.06a 5.13a 0.40a BAP (5) + IBA (0.5) 2.08a 1.20b 3.92b 0.34a Size of glassware Large jar 1.90a 1.86a 5.96b 2.46a Medium jar 2.35a 1.86a 7.80a 3.03a Small test tube 2.31a 1.53a 5.29b 2.90a Light source LED arrays 2.14a 1.32a 6.60a 3.66a Fluorescent tubes 1.90a 1.50a 6.14a 2.30b Co-cultivation of explants 1 explant 2.38a 1.53a 7.33a 3.13a 2 explants 2.69a 1.23a 7,35a 3.13a 3 explants 1.77b 1.42a 4.93b 2.27b 4 explants 2.25a 1.57a 6.68a 2.95a BAP - benzylaminopurine, IBA - indolebutyric acid, MS - Murashige and Skoog, NAA - naphthalene acetic acid, SH - Schenck and Hildebrandt, WPM - woody plant medium, LED - light emitting diode Within each treatment, mean values in the columns followed by dissimilar lowercase letters are significantly different according to the Scott-Knot test at 5% probability # Variables submitted to \(\:\sqrt{x}+0.5\) transformation Table 1 here Supplementation of WPM with cytokinins BAP, kinetin or zeatin in the concentration range 0.5 to 10.0 µM revealed that 5 µM BAP was most efficient in breaking apical dominance and stimulating the production of buds and shoots in U. tomentosa. Similar results have been reported for this species and other woody medicinal plants (Luna-Palencia et al. 2013 ; Okao et al. 2025 ). Addition of auxins to tissue culture medium may also assist in the production of multiple shoots, and in the present study, a concentration of 0.5 µM of NAA or IBA induced significantly higher numbers of shoots. WPM supplemented with a combination of 5 µM BAP and 0.5 µM NAA produced a statistically greater number of buds (5.13 ± 0.43) and shoots (2.06 ± 0.36) compared with 5 µM BAP + 0.5 µM IBA. However, the combination treatment also gave rise to some undesirable effects such as swelling of the base and reduced vigor of plantlets when maintained for prolonged periods. These effects have been associated with cytokinin/auxin imbalance, which causes morphophysiological changes and reduction in shoot quality (Manchanda et al. 2025 ). Interestingly, no significant differences were observed regarding plantlet height or number of roots in any of the cytokinin-auxin combinations. The in vitro cultivation of U. tomentosa in WPM, MS and quarter strength MS media supplemented with BAP at concentrations ranging from 2.2 to 44.4 µM has been described previously, with reported multiplication rates ranging between 1.5 and 5 ± 2 shoots per plantlet (Pereira et al. 2008 ; Venutolo 2010 ; Raposo and Teixeira 2011 ; Luna-Palencia et al. 2013 ). Although these studies demonstrated the feasibility of the in vitro cultivation of U. tomentosa , none of them established an optimized protocol that integrates multiplication efficiency, maintenance of genetic diversity and post-acclimatization performance. As shown in Table 1 , medium sized glass jars afforded the best conditions for the multiplication of U. tomentosa since the number of buds was significantly higher than those produced in the larger or smaller vessels. In tissue culture laboratories, it is common practice to evaluate different types of vessels to determine the most suitable growing conditions. . Whilst the application of different sources of light exerted no significant influence on the height of plantlets or the numbers of shoots or buds, exposure to LED arrays induced a higher number of roots compared with fluorescent tubes (3.66 and 2.30 per plantlet, respectively). The use of fluorescent tubes in growth chambers has always been very common, although the tendency is to replace them by LED arrays owing to their lower energy consumption. The use of LEDs as an alternative to conventional lighting in plant tissue culture installations reportedly gives satisfactory results regarding rhizogenesis, stem elongation, axillary shoot formation and increased photosynthetic capacity, all of which can be regulated by adjusting the spectral properties of the source (Gupta and Jatothu 2013 ; Marino et al. 2025 ). The reported advantages of LED arrays, such as greater durability, lower operating cost and higher energy efficiency, will serve as a stimulus to upgrade the lighting systems in growth chambers. There were no significant differences in the height of plantlets or the number of shoots, buds or roots of explants cultivated in isolation or in co-cultivation with two or four explants per vessel. However, the height of plantlets and the number of buds and roots were reduced significantly when three explants were cultivated in the same vessel. Thus, co-cultivation of four explants is recommended for U. tomentosa to maximize the use of space and economize culture medium without detriment to the growth of plantlets. Co-cultivation of explants can be a cost-effective technique for the large-scale micropropagation of species whose development is not impaired under such conditions. In the case of U. tomentosa , large-scale micropropagation with two or four explants per vessel proved to be a productive approach since it optimizes the use of physical space, culture medium and labor without impairing in vitro morphogenesis. The assessment of co-cultivation on solid medium is particularly relevant since liquid or temporary immersion bioreactors, such as the RITA® system developed by CIRAD, although efficient, can cause physiological disturbances, including hypoxia and hyperhydricity, especially in woody species (Bethge et al., 2023 ; Ersali, 2024 ). The optimised micropropagation protocol developed in this study employed medium-sized flasks containing 20 mL of woody plant medium (WPM) supplemented with 5.0 µM BAP, with four explants per flask maintained under a 16 h photoperiod provided by LED arrays. Under these conditions, explants produced an average of 2.13 shoots bearing a total of 8.13 buds per explant. Acclimatization of plantlets and survival in forested areas The acclimatization of micropropagated U. tomentosa plantlets was considered excellent considering that the species is restricted climatically to tropical humid environments. The survival rate of the plantlets after two months under ex vitro conditions was 100%, while that of plants transferred to their natural habitat in the Amazonian forest was 46 ± 18% after eighteen months of planting (Fig. 3 ). Figure 3 here Genetic diversity of U. tomentosa accessions maintained in vitro Genomic DNA was extracted efficiently from the 20 selected U. tomentosa genotypes maintained in vitro . The eight primers employed in the ScoT marker analysis of genetic diversity amplified between 23 and 40 bands per primer, totaling 150 bands and exhibiting an average polymorphism of 88%. This high level of polymorphism was in accord with the value (92.22%) reported by Pinto et al. ( 2022 ) for accessions of U. guianensis mantained in an in vitro germplasm bank. Such notable degree of polymorphism within the genus Uncaria is significant and essential for conservation of the species. The UPGMA dendrogram (Fig. 4 ) and the dispersion (variance) PCoA plot (Fig. 5 ) revealed genetic structuring and variability among the 20 individuals cultivated in vitro . Cluster analysis indicated grouping patterns among the 20 accessions that mirrored their geographic origin in which the genetic similarity among genotypes from TA was higher than among those from BN, whilst the accession from BC (UTBC1) formed an independent branch signifying comparatively greater genetic divergence. Is it worth noting that while UTBC stands out as genetically dissimilar from the UTTA and UTBN accessions (Figs. 4 & 5 ), it is UTTA1 that is assigned entirely to one cluster whilst UTBC has mixed ancestry like all of the other accessions (Fig. 6 ). Bayesian model-based clustering performed using the software STRUCTURE revealed the presence of two genetic groups, as represented by the red and green bands in Fig. 6 . The bars of all accessions, with the single exception of UTTA1, showed both red and green segments indicating that the vast majority of individuals were of mixed ancestry with different proportions of membership in both clusters. In contrast, the uniform red bar presented by UTTA1 suggests that it is genetically assigned entirely to one cluster with no admixture. The results of the present study are analogous to those reported by Honório et al. ( 2017 ) in which the existence of genetic structuring and low gene flow was demonstrated in eight natural populations of U. tomentosa . In particular, the conclusions of these authors regarding the ancestry of the natural populations align with the inference from STRUCTURE analysis of the presence of two gene groups within the 20 in vitro accessions. This result is significant because it implies that the in vitro germplasm bank of U. tomentosa is representative of the genetic groups found in natural populations. Maintaining this genetic diversity in accessions cultivated in vitro is especially relevant from a conservationist point of view, since it reduces the risks of genetic erosion during clonal propagation and allows the selection of elite genotypes for the pharmaceutical industry without compromising natural populations. Few actively managed germplasm collections have been assessed for the genetic diversity of their accessions. Knowledge of genetic variation is particularly important in the case of in vitro collections since they maintain accessions free from disease and in their specific allelic state, thereby assisting in the identification of useful traits that can be used in genetic improvement programs (Anglin et al., 2025 ). Chemical characteristics of the selected individuals HPLC analysis revealed significant quantitative and qualitative variability in alkaloid content among the 20 accessions of U. tomentosa (Table 2 ). Thus, UTBN233 presented the highest concentration of mitraphylline (3.329 mg·g − 1 fresh weight), while UTTA5 exhibited the highest concentration of isomitraphylline (1.422 mg·g − 1 fresh weight). Although the majority of accessions produced both alkaloids, two individuals from BN (UTBN358 and UTBN750) contained no mitraphylline and two (UTBN233 and UTBN332) contained no isomitraphylline, whereas UTBN207 accumulated neither of these alkaloids. Table 2 Alkaloid content of the 20 selected Uncaria tomentosa individuals Indivíduals Mitraphylline (mg·g − 1 fresh weight) Isomitraphylline (mg·g − 1 fresh weight) UTTA 1 0.08 m 0.00 j UTTA 2 2.52 c 0.53 f UTTA 5 2.24 d 1.43 a UTTA 9 1.63 g 1.14 b UTTA 10 1.87 f 1.00 c UTTA 19 2.80 b 1.17 b UTTA 20 2.07 e 0.69 e UTTA 30 2.27 d 0.94 c UTBN 207 0.00 m 0.00 j UTBN 233 3.32 a 0.00 j UTBN 332 1.04 i 0.00 j UTBN 354 0.90 j 0.25 h UTBN 358 0.00 m 0.32 g UTBN 499 0.88 j 0.20 h UTBN 750 0.00 m 0.31 g UTBN 767 1.04 i 0.12 i UTBN 847 0.73 k 0.83 d UTBN 902 1.01 i 0.30 g UTBN 985 1.35 h 0.00 j UTBC 1 0.21 l 0.04 j Mean values in each column followed by dissimilar lowercase letters are significantly different according to the Scott-Knot test at 5% probability. This variation in alkaloid constituents appears to be a normal pattern in U. tomentosa since it has been described for natural populations of the species in previous studies (Peñaloza et al. 2015 ; Torrejón et al. 2010 ). The existence of chemical heterogeneity reinforces the importance of conserving multiple genotypes, especially considering the interest of the phytotherapeutic industry in plant materials with high levels of these bioactive compounds. In this context, the introduction of genetically diverse and chemically distinct accessions into in vitro germplasm banks is a pertinent strategy for both the conservation and sustainable use of the species. Tissue culture has been widely recognized as an important conservation strategy for threatened medicinal species that are subject to predatory exploitation (Moraes et al. 2021 ). Furthermore, micropropagation and in vitro germplasm maintenance allow not only the rapid multiplication of selected genotypes but also the preservation of genetic variability, especially for species with difficult sexual propagation or low seed viability (Singh and Chokheli 2025 ). Conclusions Genetic analysis by SCoT markers of 20 accessions of U. tomentosa cultivated in vitro indicated that they descended from two inferred ancestral genetic clusters, a situation similar to that established for natural populations. Fifteen of the 20 genotypes accumulated both mitraphylline and isomitraphylline, albeit in varying concentrations, which is typical of the species. In contrast, four of the genotypes accumulated only one of the alkaloids and one genotype contained neither. The survival rate of the plants introduced into the natural environment was highly satisfactory considering the environmental and abiotic constraints, such as nutrient and light limitation and high humidity and temperature, posed by the Amazon rainforest. The results obtained reinforce the potential of in vitro culture as a complementary approach to the in situ conservation of U. tomentosa , since both contribute to the protection of natural populations and the sustainable supply of plant material. Declarations Ethical approval Not applicable. Conflict of interest The authors declare that they have no conflicts of interest Funding This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grant no. 440537/2022-3) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; grant no. 23/05517-3). Author Contribution Conceptualization: I.C.G.H., J.S.C and A.M.S.P.; Methodology: I.C.G.H., J.S.C., G.H.T.P., B.S.R., R.N.S.M., E.F.S and W.S.; Data curation: I.C.G.H., G.H.T.P. and B.W.B.; Formal analysis: I.C.G.H., J.S.C. and B.W.B; Investigation: I.C.G.H., J.S.C. and B.W.B; Visualization: I.C.G.H. and S.C.F.; Writing - Original draft: I.C.G.H., B.S.R., S.C.F and B.W.B.; Validation: A.M.S.P; Writing – review & editing: A.M.S.P; Supervision: A.M.S.P Funding This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grant no. 440537/2022-3) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; grant no. 23/05517-3). Data availability Data is available upon reasonable request. References Agência Nacional de Vigilância Sanitária (2003) Resolução-RE n. 899, de 29 de maio de 2003. Dispõe sobre o guia para validação de métodos analíticos e bioanalíticos. ANVISA. https://bvsms.saude.gov.br/bvs/saudelegis/anvisa/2003/res0899_29_05_2003 Accessed 6 February 2026 Anglin NL, Wenzl P, Azevedo V, Lusty C, Ellis D, Gao D (2025) Genotyping genebank collections: Strategic approaches and considerations for optimal collection management. Plants 14(2):252. https://doi.org/10.3390/plants14020252 Arado GM, Amatto PPG, Marins M, Rizzi ES, França SC, Coppede JS, Carmona F, Pereira MAS (2024) Anti-inflammatory and/or immunomodulatory activities of Uncaria tomentosa (cat's claw) extracts: A systematic review and meta-analysis of in vivo studies. Front Pharmacol 15:1378408. https://doi.org/10.3389/fphar.2024.1378408 Bethge H, Mohammadi Nakhjiri Z, Rath T, Winkelmann T (2023) Towards automated detection of hyperhydricity in plant in vitro culture. Plant Cell Tiss Organ Cult 154:551–573. https://doi.org/10.1007/s11240-023-02528-0 Blumenthal M (ed) (1997) World Health Organization Medicinal Plants Monographs. HerbalGram 40(38) Collard BCY, Mackill DJ (2009) Start codon targeted (SCoT) polymorphism: A simple, novel DNA marker technique for generating gene-targeted markers in plants. Plant Mol Biol Rep 27:86–93. https://doi.org/10.1007/s11105-008-0060-5 Doyle JJ, Doyle JL (1987) Isolation of plant DNA from fresh tissue. Focus (Rockville) 12(1):13–15 Ersali Y (2024) Control of hyperhydricity of Pistacia khinjuk stocks in vitro shoots. BMC Biotechnol 24:97. https://doi.org/10.1186/s12896-024-00929-3 Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol Ecol 14(8):2611–2620. https://doi.org/10.1111/j.1365-294X.2005.02553.x Ferreira DF (2011) Sisvar: a computer statistical analysis system. Cienc Agrotec 35(6):1039–1042. https://doi.org/10.1590/S1413-70542011000600001 Ferreira SJF, Luizão FJ, Miranda SAF, Silva MSR, Vital ART (2006) Nutrients in soil solution in an upland forest submitted to selective logging in central Amazonia. Acta Amaz 36(1):59–68. https://doi.org/10.1590/S0044-59672006000100008 Gupta SD, Jatothu B (2013) Fundamentals and applications of light-emitting diodes (LEDs) in in vitro plant growth and morphogenesis. Plant Biotechnol Rep 7:211–220. https://doi.org/10.1007/s11816-013-0277-0 Hampl V, Pavlíček A, Flegr J (2001) Construction and bootstrap analysis of DNA fingerprinting-based phylogenetic trees with the freeware program FreeTree: application to trichomonad parasites. Int J Syst Evol Microbiol 51:731–735. https://doi.org/10.1099/00207713-51-3-731 Honório ICG, Bertoni BW, Pereira AMS (2016) Uncaria tomentosa and Uncaria guianensis an agronomic history to be written. Cienc Rural 46(8):1401–1410. https://doi.org/10.1590/0103-8478cr20150138 Honório ICG, Bertoni BW, Telles MPC, Braga RS, França SC, Coppede JS, Correa VSC, Diniz Filho JAF, Pereira MAS (2017) Genetic and chemical diversity of Uncaria tomentosa (Willd. ex. Schult.) DC. in the Brazilian Amazon. PLoS ONE 12(5):e0177103. https://doi.org/10.1371/journal.pone.0177103 Lloyd G, McCown BH (1980) Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia , by use of shoot-tip culture. Combined Proc Intl Plant Prop Soc 30:421–427 Luna-Palencia GR, Huerta-Heredia AA, Cerda-García-Rojas CM, Ramos-Valdivia AC (2013) Differential alkaloid profile in Uncaria tomentosa micropropagated plantlets and root cultures. Biotechnol Lett 35:791–797. https://doi.org/10.1007/s10529-012-1128-8 Manchanda P, Sharma D, Kaur G, Kaur H, Vanshika (2025) Exploring the significance of somaclonal variations in horticultural crops. Mol Biotechnol 67:2185–2203. https://doi.org/10.1007/s12033-024-01214-6 Marino LA, Ruffa P, Mozzanini E, Patono DL, Sereno A, Pavese V (2025) LEDs in plant tissue culture: Boosting micropropagation of Castanea sativa cultivars. J Plant Growth Regul 44:6046–6060. https://doi.org/10.1007/s00344-025-11812-6 Ministério da Saúde (2013) Relação nacional de medicamentos essenciais (RENAME). Sistema Único de Saúde (SUS). https://www.gov.br/saude/pt-br/composicao/sectics/rename Accessed 6 Feb 2026 Moraes RM, Cerdeira AL, Lourenço MV (2021) Using micropropagation to develop medicinal plants into crops. Molecules 26(6):1752. https://doi.org/10.3390/molecules26061752 Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x Okao M, Bharati R, Fernández-Cusimamani E (2025) Effect of explant physiology and media composition on callogenesis of Vitellaria paradoxa leaf explants. Horticulturae 11(9):1127. https://doi.org/10.3390/horticulturae11091127 Peakall R, Smouse PE (2012) GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research - an update. Bioinformatics 28(19):2537–2539. https://doi.org/10.1093/bioinformatics/bts460 Peñaloza EMC, Kaiser S, Resende PE, Pittol V, Carvalho ÂR, Ortega GG (2015) Chemical composition variability in the Uncaria tomentosa (cat’s claw) wild population Quim Nova. 38(3):378–386. https://doi.org/10.5935/0100-4042.20150007 Pereira RCA, Valente LMM, Pinto JEBP, Bertolucci SKV, Bezerra GM, Alves FF, Santos PFP, Benevides PJC, Siani AC, Rosario SL, Mazzei JL, d’Avila LA, Gomes LNF, Aquino-Neto FR, Emmerick ICM, Carvalhaes SF (2008) In vitro cultivated Uncaria tomentosa and Uncaria guianensis with determination of the pentacyclic oxindole alkaloid contents and profiles. J Braz Chem Soc 19(6):1193–1200. https://doi.org/10.1590/S0103-50532008000600021 Pinto GHT, Lopes AA, Morel LJF, Crevelin EJ, Miranda CES, Contini SHT, França SC, Bertoni BW, Pereira AMS (2022) Genetic diversity among genotypes of Uncaria guianensis (Aubl.) J.F. Gmel. maintained in an in vitro germplasm bank. 3 Biotech 12:8. https://doi.org/10.1007/s13205-021-03016-y Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155(2):945–959. https://doi.org/10.1093/genetics/155.2.945 Rai MK (2023) Start codon targeted (SCoT) polymorphism marker in plant genome analysis: current status and prospects. Planta 257:34. https://doi.org/10.1007/s00425-023-04067-6 Raposo A, Teixeira RB (2011) Metodologia científica: cultivo in vitro de Unha-de-gato. Circular Técnica 57. Embrapa Acre, Rio Branco, Brazil. https://www.infoteca.cnptia.embrapa.br/bitstream/doc/912085/1/24058.PDF Accessed 06 February 2026 Schenck RU, Hildebrandt AC (1972) Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 50(1):199–204. https://doi.org/10.1139/b72-026 Singh RK, Chokheli VA (2025) Plant biotechnology: Applications in in vitro plant conservation and micropropagation. Horticulturae 11(4):358. https://doi.org/10.3390/horticulturae11040358 Sirikonda A, Jogam P, Mood K, Allini VR (2023) Micropropagation of 2-methoxy-4-vinyl phenol rich Flemingia strobilifera and assessment of genetic and biochemical fidelity by SCoT and GC-MS analysis. Plant Cell Tiss Organ Cult 154:541–550. https://doi.org/10.1007/s11240-023-02526-2 Torrejón GD, Martín JJG, Loayza DG, Alanoca R (2010) Contenido de alcaloides en corteza de Uncaria tomentosa (Wild.) DC procedente de diferentes hábitats de la región Ucayali-Perú. Rev Soc Quim Peru 76(3):271–278 United States Pharmacopeial Convention (2022) Cat’s Claw ( Uncaria tomentosa (Willd.) DC.). In: United States Pharmacopeia and National Formulary (USP–NF). United States Pharmacopeial Convention, Rockville Venutolo AS (2010) Establecimiento in vitro y cultivo de células de la Uña de gato ( Uncaria tomentosa ). Tecnol Marcha 23(5):24–33 Vrundha CPK, Thomas TD (2023) Indirect shoot regeneration from root explants, assessment of clonal fidelity of regenerated plants using SCoT primers and antioxidant analysis in Thottea siliquosa (Lamk.) Ding Hou. Plant Cell Tiss Organ Cult. 155:255–266. https://doi.org/10.1007/s11240-023-02578-4 Zavala A, Zevallos PP (1996) Taxonomía, distribución geográfica y status del género Uncaria en el Perú. Facultad de Ciencias Forestales, Universidad Nacional Agraria La Molina, Lima, Peru. 103 pp Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 21 Apr, 2026 Reviews received at journal 09 Apr, 2026 Reviewers agreed at journal 01 Apr, 2026 Reviewers agreed at journal 01 Apr, 2026 Reviewers agreed at journal 31 Mar, 2026 Reviewers invited by journal 30 Mar, 2026 Editor assigned by journal 23 Mar, 2026 Submission checks completed at journal 23 Mar, 2026 First submitted to journal 12 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9107751","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":615450225,"identity":"9ab41720-0ab7-4774-be78-6b8dbe4a474c","order_by":0,"name":"Isabela Cristina Gomes Honório","email":"","orcid":"","institution":"Universidade do Estado de Minas Gerais (UEMG)","correspondingAuthor":false,"prefix":"","firstName":"Isabela","middleName":"Cristina Gomes","lastName":"Honório","suffix":""},{"id":615450226,"identity":"bd931fd6-04db-4c9d-83c9-e6546dd03a8e","order_by":1,"name":"Juliana Silva Coppede","email":"","orcid":"","institution":"Universidade de Ribeirão Preto (UNAERP)","correspondingAuthor":false,"prefix":"","firstName":"Juliana","middleName":"Silva","lastName":"Coppede","suffix":""},{"id":615450227,"identity":"57439b00-51fc-4fea-8f59-326f3a295df8","order_by":2,"name":"Gustavo Henrique Teixeira Pinto","email":"","orcid":"","institution":"Universidade de Ribeirão Preto (UNAERP)","correspondingAuthor":false,"prefix":"","firstName":"Gustavo","middleName":"Henrique Teixeira","lastName":"Pinto","suffix":""},{"id":615450228,"identity":"18181d14-1d39-48a4-b21c-ebb25a594c07","order_by":3,"name":"Beatriz Souza Rodrigues","email":"","orcid":"","institution":"Universidade de Ribeirão Preto (UNAERP)","correspondingAuthor":false,"prefix":"","firstName":"Beatriz","middleName":"Souza","lastName":"Rodrigues","suffix":""},{"id":615450229,"identity":"b39ef454-675e-437a-865c-bddc17924ce6","order_by":4,"name":"Suzelei Castro França","email":"","orcid":"","institution":"Universidade de Ribeirão Preto (UNAERP)","correspondingAuthor":false,"prefix":"","firstName":"Suzelei","middleName":"Castro","lastName":"França","suffix":""},{"id":615450230,"identity":"6fece1ff-40a3-49a6-b717-56f7585924c5","order_by":5,"name":"Renata Naomi Sato Mendonça","email":"","orcid":"","institution":"Universidade Federal do Oeste do Pará (UFOPA)","correspondingAuthor":false,"prefix":"","firstName":"Renata","middleName":"Naomi Sato","lastName":"Mendonça","suffix":""},{"id":615450231,"identity":"41bd0607-56ff-43ef-8b67-82c8de41f784","order_by":6,"name":"Eliandra Freitas Sia","email":"","orcid":"","institution":"Universidade Federal do Oeste do Pará (UFOPA)","correspondingAuthor":false,"prefix":"","firstName":"Eliandra","middleName":"Freitas","lastName":"Sia","suffix":""},{"id":615450240,"identity":"1352e066-043e-48ac-9503-0d7ed7afd0a8","order_by":7,"name":"Wilson Sabino","email":"","orcid":"","institution":"Universidade Federal do Oeste do Pará (UFOPA)","correspondingAuthor":false,"prefix":"","firstName":"Wilson","middleName":"","lastName":"Sabino","suffix":""},{"id":615450242,"identity":"afbd8ee3-1fe8-423b-b239-fc16100e57a1","order_by":8,"name":"Bianca Waléria Bertoni","email":"","orcid":"","institution":"Universidade de Ribeirão Preto (UNAERP)","correspondingAuthor":false,"prefix":"","firstName":"Bianca","middleName":"Waléria","lastName":"Bertoni","suffix":""},{"id":615450243,"identity":"e0c6bfbd-2dfb-4be5-aef8-ce30fdfae6ef","order_by":9,"name":"Ana Maria Soares Pereira","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYNACAwYGfiQ2kVokG0jTAlJ5gFgt/LObj334UHBP3vhG7sEPH/fcYTCXPoBfi8SdY8kzZxgUG267kZcsOePZMwbLvgQC1tzIMWbmMUhg3HYjx0Ca58BhBoMzBHTI38j/zPzHIMF+84wc499/iNFicCOHmZnBICFxg0SOmTQDMVoM7xwzZuwxSEieceaNmWXPgWc8lj0EtMjdbn7M8ONPgm1/e47xjR8H7siZ8xDQwiCByj1AUAOmFsI6RsEoGAWjYMQBAH59RgU/RmrTAAAAAElFTkSuQmCC","orcid":"","institution":"Universidade de Ribeirão Preto (UNAERP)","correspondingAuthor":true,"prefix":"","firstName":"Ana","middleName":"Maria Soares","lastName":"Pereira","suffix":""}],"badges":[],"createdAt":"2026-03-12 18:54:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9107751/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9107751/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105909926,"identity":"6bce04c8-af77-497c-9eb4-831c9f480872","added_by":"auto","created_at":"2026-04-01 10:45:51","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":135045,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eUncaria tomentosa\u003c/em\u003e: \u003cstrong\u003eA\u003c/strong\u003e - Aerial parts, \u003cstrong\u003eB\u003c/strong\u003e – Flowers, and \u003cstrong\u003eC\u003c/strong\u003e - Plantlet grown \u003cem\u003ein vitro\u003c/em\u003e;. Panel \u003cstrong\u003eD\u003c/strong\u003e shows the collection location of individuals in the Brazilian Amazon\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9107751/v1/b49749cf715e438880a23427.jpg"},{"id":105909928,"identity":"775ee6fe-6e19-4fe3-8fe4-faf0ca4a47ed","added_by":"auto","created_at":"2026-04-01 10:45:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":103868,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent treatments employed in optimizing the micropropagation protocol of \u003cem\u003eUncaria tomentosa\u003c/em\u003e showing: \u003cstrong\u003eA\u003c/strong\u003e - Size of glassware (\u003cstrong\u003ea1\u003c/strong\u003e) large jar, (\u003cstrong\u003ea2\u003c/strong\u003e) medium jar, (\u003cstrong\u003ea3\u003c/strong\u003e) small test tube; and \u003cstrong\u003eB\u003c/strong\u003e - Co-cultivation of explants: (\u003cstrong\u003eb1\u003c/strong\u003e) one explant/flask, (\u003cstrong\u003eb2\u003c/strong\u003e) two explants/flask, (\u003cstrong\u003eb3\u003c/strong\u003e) three explants/flask, (\u003cstrong\u003eb4\u003c/strong\u003e) four explants/flask\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9107751/v1/6b06c3c21fc74ad00eccb269.jpg"},{"id":105909929,"identity":"866f5f0e-3c8b-48a2-8c40-f9e35e7f06da","added_by":"auto","created_at":"2026-04-01 10:45:52","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":163700,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eUncaria tomentosa\u003c/em\u003e: \u003cstrong\u003eA\u003c/strong\u003e - plantlets \u003cem\u003ein vitro\u003c/em\u003e, \u003cstrong\u003eB\u003c/strong\u003e - plants acclimatized in the greenhouse, \u003cstrong\u003eC\u003c/strong\u003e– plants transferred to a natural environment, \u003cstrong\u003eD \u003c/strong\u003e- plant after eighteen months cultivation in the Amazon rainforest\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9107751/v1/72729fece02c8fad2f1f5315.jpg"},{"id":105910087,"identity":"6f7de5bc-6e52-4fa7-a159-3b983f8beccc","added_by":"auto","created_at":"2026-04-01 10:46:47","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":49464,"visible":true,"origin":"","legend":"\u003cp\u003eUPGMA dendrogram of 20 selected \u003cem\u003eUncaria tomentosa\u003c/em\u003e individuals generated using SCoT markers\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9107751/v1/82ab3c95fa8a20b445da086a.jpg"},{"id":105909935,"identity":"75cf916a-0f8f-48eb-b405-652b25d67be6","added_by":"auto","created_at":"2026-04-01 10:45:59","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":66328,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal coordinates analysis (PCoA) plot obtained from the Jaccard similarity matrix, generated using SCoT markers, showing dispersion among the 20 selected \u003cem\u003eUncaria tomentosa\u003c/em\u003e accessions\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9107751/v1/353fd68e80c3eafa1a87e418.jpg"},{"id":105910089,"identity":"7b5b2e4e-cf1b-4f1b-8953-7bed2302d161","added_by":"auto","created_at":"2026-04-01 10:46:49","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":92517,"visible":true,"origin":"","legend":"\u003cp\u003eBayesian clustering analysis of 20 \u003cem\u003eUncaria tomentosa \u003c/em\u003eaccessions with bar plot constructed using software STRUCTURE. The red and green bars correspond to the proportion of individuals assigned to a hypothetical cluster.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9107751/v1/16e10cb7155439ad5af169e2.jpg"},{"id":105912003,"identity":"cfc61057-3e65-4ad1-b12f-b008ce812be5","added_by":"auto","created_at":"2026-04-01 10:56:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1843621,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9107751/v1/362f978c-1c17-4a27-b3cb-dcf45f254f08.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Uncaria tomentosa Willd. ex Roem. \u0026 Schult.: genetic and chemical diversity of accessions cultivated in vitro and their introduction into the native environment","fulltext":[{"header":"Key message","content":"\u003cp\u003eAn micropropagation protocol for enabled successful reintroduction of this medicinal species into the Amazon, supporting both its conservation and the development of sustainable production systems.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eUncaria tomentosa\u003c/em\u003e Willd. ex Roem. \u0026amp; Schult., commonly known as cat's claw, is a medicinal plant from the Amazon region and used mainly in the treatment of inflammatory diseases (Arado et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Based on its utilization in the traditional medicine of the indigenous peoples of the Amazon and its scientifically proven therapeutic efficacy, the species is included in monographs of selected medicinal plants by the World Health Organization (Blumenthal \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), and in the National List of Essential Medicines of the Brazilian government (Minist\u0026eacute;rio da Sa\u0026uacute;de \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and the United States Pharmacopeia and National Formulary (United States Pharmacopeial Convention \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite the popular use of \u003cem\u003eU. tomentosa\u003c/em\u003e and the well-documented body of evidence regarding its pharmaceutical properties, exploitation of this valuable resource depends exclusively on predatory collection since efficient propagation of the species is limited by the scarcity of agronomic studies. As a consequence, natural populations of \u003cem\u003eU. tomentosa\u003c/em\u003e are being compromised with increasing risk of genetic erosion (Hon\u0026oacute;rio et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In fact, the vulnerability of \u003cem\u003eU. tomentosa\u003c/em\u003e to extinction was first documented a decade earlier by Zavala and Zevallos (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) and confirmed in a more recent study by Hon\u0026oacute;rio et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) involving the use of sequence-related amplified polymorphism (SRAP) markers. These authors reported the occurrence of low gene flow and an island-type genetic structure among the natural populations of \u003cem\u003eU. tomentosa\u003c/em\u003e analyzed, and emphasized the importance of conserving accessions in \u003cem\u003ein vitro\u003c/em\u003e germplasm banks.\u003c/p\u003e \u003cp\u003eMoreover, field studies in the Amazon performed by members of our group have revealed further compounding factors that serve to increase the difficulty of collection from natural populations, including the minute size of the seeds (\u0026lt;\u0026thinsp;1 mm) and the height of the lianas (\u0026gt;\u0026thinsp;30 m). For these reasons it is likely that the \u003cem\u003ein vitro\u003c/em\u003e culture of \u003cem\u003eU. tomentosa\u003c/em\u003e will prove to be a sustainable solution for the large-scale production of phytomedicine.\u003c/p\u003e \u003cp\u003eAlthough several researchers have reported the \u003cem\u003ein vitro\u003c/em\u003e cultivation of \u003cem\u003eU. tomentosa\u003c/em\u003e (Pereira et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Venutolo \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Raposo and Teixeira \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Luna-Palencia et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), a protocol for the efficient micropropagation of the medicinal plant has yet to be developed. Moreover, the multiplication of \u003cem\u003eU. tomentosa\u003c/em\u003e under \u003cem\u003ein vitro\u003c/em\u003e conditions should be preceded by genetic analysis of introduced accessions in order to preserve genetic variation in the field and to conserve species diversity.\u003c/p\u003e \u003cp\u003eGenetic stability and diversity in tissue culture-derived plants is typically assessed using molecular markers as, for example, the robust and cost-effective start codon targeted (SCoT) polymorphism technique (Sirikonda et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Vrundha and Thomas \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Rai \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Thus, the aims of the present study were to establish protocols for the multiplication of \u003cem\u003eU. tomentosa\u003c/em\u003e, to employ ScoT markers to verify genetic diversity among micropropagated plantlets maintained \u003cem\u003ein vitro\u003c/em\u003e, to evaluate the chemical diversity of these accessions by high-performance liquid chromatography (HPLC), and to introduce the micropropagated plants into their natural environment in the Brazilian Amazon.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material\u003c/h2\u003e \u003cp\u003ePlants of \u003cem\u003eU. tomentosa\u003c/em\u003e were identified by Dr. Piero Giuseppe Delprete (Herbier de Guyane, Institut de Recherche pour le D\u0026eacute;veloppement, Cayenne, French Guiana) and a specimen was deposited in the Medicinal Plant Herbarium of the Universidade de Ribeir\u0026atilde;o Preto (UNAERP) under accession number HJBOP 2723. All accessions used in the experiments were registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) under number AB94A2F.\u003c/p\u003e \u003cp\u003eThe accessions of \u003cem\u003eU. tomentosa\u003c/em\u003e maintained \u003cem\u003ein vitro\u003c/em\u003e at UNAERP were established from seeds of individual plants collected from the municipalities of Bannach (BN, State of Par\u0026aacute;), Tarauac\u0026aacute; (TA, State of Acre) and Boca do Acre (BC, State of Amazonas), all of which are located in the Brazilian Amazon rainforest as presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e here\u003c/p\u003e \u003cp\u003e \u003cb\u003eSeed sterilization and\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003egermination\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSeeds were sterilized by immersion in 1% w/v Cercobin\u0026reg; for 24 h under constant shaking, followed by treatment with 1% w/v sodium hypochlorite solution for 20 min under similar conditions. After surface asepsis, the seeds were washed with sterile distilled water and transferred to plates containing sucrose-free basal Murashige and Skoog (MS) medium (Murashige and Skoog \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1962\u003c/span\u003e) solidified with 3 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of Gelzan\u0026trade; CM (Sigma-Aldrich, St. Louis, MO, USA; # G1910) and adjusted to pH 6.0. Incubation was carried out in a growth chamber at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C under a 16 h photoperiod provided by white fluorescent tubes (25 \u0026micro;mol\u0026middot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e \u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003eIn order to test the influence of light on germination (photoblastism), batches of 50 seeds were incubated in the growth chamber under a 16 h photoperiod or dark conditions and the experiment repeated four times. The longevity of seeds was assessed by storing seeds at room temperature for one prior to germination.\u003c/p\u003e \u003cp\u003eA total of 114 seedlings were established \u003cem\u003ein vitro\u003c/em\u003e and analyzed by HPLC to determine the proportions of the alkaloids considered to be the chemical markers of the species, namely mitraphylline and isomitraphylline. Twenty seedlings derived from seeds originating from the three different areas of the Brazilian rainforest were selected for further investigation on the basis of vigor and alkaloid content.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOptimization of culture conditions for\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003emicropropagation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn order to determine the influence of culture conditions on the efficiency of \u003cem\u003ein vitro\u003c/em\u003e micropropagation, explants comprising 1 cm nodal segments from the 20 selected seedlings were cultivated either individually or in groups on different culture media, with or without growth regulators, in diverse types of culture vessels and under various light sources.\u003c/p\u003e \u003cp\u003eThe experiment was of a completely randomized design and involved the following treatments: (i) Culture medium: individual explants were cultured in glass test tubes containing 5 mL of MS medium, woody plant medium (WPM; Lloyd and McCown \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1980\u003c/span\u003e) or Schenck and Hildebrandt (SH; Schenck and Hildebrandt \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1972\u003c/span\u003e) medium supplemented with 3% sucrose and solidified with 0.30% Gelzan CM at pH 6.0; (ii) Addition of growth regulators: individual explants were cultured in large jars containing 20 mL of WPM supplemented with cytokinins 6-benzylaminopurine (BAP), kinetin or zeatin at concentrations of 0, 0.5, 1.0, 2.5, 5.0 and 10.0 \u0026micro;M, and/or auxins naphthalene acetic acid (NAA) or indolebutyric acid (IBA) at concentrations of 0, 0.1 and 0.5 \u0026micro;M, or a combination of BAP (5.0 \u0026micro;M) and NAA or IBA (0, 0.1 and 0.5 \u0026micro;M). (iii) Size of glassware: individual explants were cultured in large jars (12 cm height x 5.4 cm diameter; 20 mL WPM), medium jars (8.5 cm height x 5.9 cm diameter; 20 mL WPM) or small test tubes(10 cm height x 2 cm diameter; 5 mL WPM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA); (iv) Light source: individual explants were cultured in small test tubes containing 5 mL WPM and incubated under a 16 h photoperiod provided by light emitting diode (LED) arrays or fluorescent tubes (25 \u0026micro;mol\u0026middot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e \u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e); and (v) Co-cultivation interaction: individual explants or groups of two to four were cultured in large jars containing 20 mL of WPM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e here\u003c/p\u003e \u003cp\u003eUnless otherwise stated, all explants were cultured on WPM supplemented with 3% sucrose and solidified with 0.30% Gelzan CM at pH 6.0, and maintained in a growth chamber at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026ordm;C under a 16 h photoperiod provided by LED arrays (25 \u0026micro;mol\u0026middot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e \u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The variables of interest, namely height of plantlets, number of buds, number of shoots and number of roots), were measured 60 days after cultivation of explants. Each treatment was repeated 10 times and each repetition included 10 plantlets (replicates).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAcclimatization of plantlets to greenhouse conditions and field cultivation\u003c/h3\u003e\n\u003cp\u003ePlantlets were transferred to 162-cell plastic trays (model 7100235; Nutriplan, Cascavel, PR, Brazil) containing commercial substrate (Carolina Soil, Santa Cruz, RS, Brazil) and maintained for 60 days in a climate-controlled greenhouse (Van der Hoeven, Delft, The Netherlands) with a relative humidity of 70% achieved by operating micro-sprinklers for 5 min, three times daily. Plants were subsequently relocated to 1 L plastic bags containing 900 g of a mixture of soil, manure and sand (3:2:1) and remained in the greenhouse for 10 months with micro-sprinkler irrigation twice a day.\u003c/p\u003e \u003cp\u003eAcclimatized plants (40\u0026thinsp;\u0026plusmn;\u0026thinsp;5 cm height) were transferred to forested areas in the municipality of Santar\u0026eacute;m, PA, Brazil, during the rainy season. Planting pits (20 cm x 20 cm x 20 cm) were dug near to native trees to provide support for the plants. The survival of the transplanted individuals was evaluated eighteen months after planting.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of the genetic variability of the\u003c/b\u003e \u003cb\u003eU. tomentosa\u003c/b\u003e \u003cb\u003eaccessions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe genetic diversity of the 20 selected individuals maintained \u003cem\u003ein vitro\u003c/em\u003e was determined by SCoT polymorphism analysis. Genomic DNA was extracted following the method of Doyle and Doyle (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1987\u003c/span\u003e) as modified by Hon\u0026oacute;rio et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and analyzed using the SCoT molecular marker technique following the method proposed by Collard and Mackill (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The 36 SCoT 18-mer primer sequences described by these authors were screened and eight (5, 8, 13, 15, 19, 24, 29 and 30) presenting the highest polymorphism with sharply resolved bands were selected for the full analysis.\u003c/p\u003e \u003cp\u003ePolymerase chain reaction (PCR) amplifications were performed in a thermal cycler (Jingle Scientific, Shanghai, China) with a reaction mixture containing 1 \u0026micro;L of 10x reaction buffer, 1 \u0026micro;L of dNTP mix (2.5 mM), 1 \u0026micro;L of primer (8 \u0026micro;M), 0.1 \u0026micro;L of Taq DNA polymerase (5 U/\u0026micro;L), 2.5 \u0026micro;L of DNA (10 ng\u0026middot;\u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and deionized water to final volume (10 \u0026micro;L). Amplifications were carried out under the following conditions: initial denaturation at 94\u0026deg;C for 3 min, followed by 35 cycles of denaturation at 94\u0026deg;C for 1 min, annealing at 50\u0026deg;C for 1 min and extension at 72\u0026deg;C for 2 min, with a final extension at 72\u0026deg;C for 5 min. The PCR products were separated by electrophoresis on 3% agarose gel prepared in 1x tris-borato-EDTA (TEB) buffer containing ethidium bromide (0.5 \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and visualized under a Gel Logic 112 digital imaging system (Bruker BioSpin, Billerica, MA, USA) and analyzed using Carestream Molecular Imaging (Rochester, NY, USA) software. The bands produced were counted manually to generate a binary matrix (presence or absence of bands) for subsequent data analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eChemical analysis of the\u003c/b\u003e \u003cb\u003eU. tomentosa\u003c/b\u003e \u003cb\u003eaccessions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe alkaloid content of the \u003cem\u003eU. tomentosa\u003c/em\u003e accessions was determined using a previously validated HPLC-diode array detector method (Hon\u0026oacute;rio et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) with external standards mitraphylline (LGC Standards CDx, Teddington, Middlesex, UK; # CDX-00013955\u0026ndash;005) and isomitraphylline (Chromadex/Niagen Bioscience, Los Angeles, CA, USA; # ASB-00009417). The limits of detection (LOD) and quantification (LOQ) were: mitraphylline (0.22 and 0.75 \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively) and isomitraphylline (0.12 and 0.24 \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively). Validation of the analytical data (LOD, LOQ, linearity, precision and accuracy) was performed in accordance with the guidelines of the Ag\u0026ecirc;ncia Nacional de Vigil\u0026acirc;ncia Sanit\u0026aacute;ria (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Calibration curves were constructed using triplicate injections of the standard alkaloids at concentrations of 500, 250, 125, 62.5, 31.2, 15.6, 7.8, 3.9, 1.9 \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The ratio of peak areas of the target alkaloids were plotted as a function of the corresponding concentration using linear regression of the standard curves as described by Hon\u0026oacute;rio et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eData relating to the micropropagation experiments were submitted to analysis of variance (ANOVA) and, when significant, means were compared using the Scott-Knott test at α\u0026thinsp;=\u0026thinsp;0.05. Statistical analyses were performed with the aid of the program Sisvar (Ferreira \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBinary data (presence or absence of bands) obtained from the ScoT marker analysis were employed to estimate the genetic similarity between the 20 accessions of \u003cem\u003eU. tomentosa\u003c/em\u003e using the Jaccard coefficient and unweighted pair-group method with arithmetic mean (UPGMA) cluster analysis. Phylogentic trees were constructed and analysed with the aid of Free Tree software (Hampl et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Genetic variability was investigated using principal coordinates analysis (PCoA) with the help of GenAlEx 6.5 software (Peakall and Smouse \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBayesian assignment analysis was conducted using STRUCTURE (Pritchard et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) to infer the genetic structure of the accessions, grouping individuals into K distinct clusters (K\u0026thinsp;=\u0026thinsp;1 to 20) based on genetic similarities. Analyses were run under the correlated allele frequencies and no admixture model, which assumes that each individual originated from a single cluster, with a burn-in of 300,000 steps followed by 500,000 Markov chain Monte Carlo (MCmC) iterations. The most likely number of clusters (K) was determined using the ΔK method of Evanno et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e \u003cb\u003eGermination and establishment of seedlings\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSeeds of \u003cem\u003eU. tomentosa\u003c/em\u003e showed positive photoblastism. Germination under a 16 h photoperiod occurred between 10 and 15 days after inoculation onto the medium, with a germination rate of 92\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4%, while no seeds germinated during this time period under dark conditions. Seeds exhibited low longevity since the germination rate declined to 10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5% after one year of storage under the experimental conditions, and no germination was observed following storage for two years.\u003c/p\u003e \u003cp\u003eThe 114 seedlings established \u003cem\u003ein vitro\u003c/em\u003e were assessed for vigor and alkaloid content and 20 were assigned for the micropropagation study. The selected accessions comprised eight originating from TA (UTTA1, UTTA2, UTTA5, UTTA9, UTTA10, UTTA19, UTTA20 and UTTA30), 11 from BN (UTBN207, UTBN233, UTBN332, UTBN354, UTBN358, UTBN499, UTBN750, UTBN767, UTBN847, UTBN902 and UTBN985) and one from BC (UTBC1).\u003c/p\u003e\n\u003ch3\u003eOptimization of micropropagation conditions\u003c/h3\u003e\n\u003cp\u003eThe effects of culture conditions on the development of \u003cem\u003eU. tomentosa\u003c/em\u003e explants are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Regarding culture medium, WPM favored increased plantlet height and number of shoots compared with MS and SH media. This finding may be related to the lower concentration of mineral salts in WPM relative to MS and SH media, a factor frequently associated with superior responses in woody species and plants native to oligotrophic environments. Considering that \u003cem\u003eU. tomentosa\u003c/em\u003e plants occur naturally in nutrient-poor Amazonian soils, it is reasonable to assume that the physiology of the species is better adapted to low nutrient concentrations as already discussed for tropical forest species (Lloyd and McCown \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Ferreira et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of diverse culture conditions on the development of \u003cem\u003eUncaria tomentosa\u003c/em\u003e explants \u003cem\u003ein vitro\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eVariables evaluated\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHeight of plantlets (cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo. of shoots\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo. of buds\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo. of roots\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCulture medium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.80b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.28b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.48a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.29b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.05b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.52b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWPM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.58a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.81a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.28a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAddition of growth regulators\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBAP (\u0026micro;M)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.37a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.33b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.47b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.93a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.50b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.33b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.93b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.00a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.25b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.93b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.67a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.87a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.16b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.80a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.27b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.40b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.16b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.13a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.13a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.60a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.21b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.06a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.40b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.80b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKinetin (\u0026micro;M)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.07a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.40a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.93a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.27a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.33a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.53a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.80b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.27b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.98a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.00a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.00b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.93b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.75a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.33a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.73b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.53b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.96a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.27a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.96b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.67b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.57a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.47a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.57b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.47b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZeatin (\u0026micro;M)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.96a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.40a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.93a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.73a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.86a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.53a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.27a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.20a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.34a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.00a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.33b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.27a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.45b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.33a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.47b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.20b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.43b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.27a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.43b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.33b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.20b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.47a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.23b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.13b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNAA (\u0026micro;M)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.46b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.37b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.27b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.20c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.78a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.40b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.37a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.73b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.10a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.76a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.63a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.97a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIBA (\u0026micro;M)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.46b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.37b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.27b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.20c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.04c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.27b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.57b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.90b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.12a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.77a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.90a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.30a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eCytokinin (\u0026micro;M) + auxin (\u0026micro;M)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.50a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.28b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.85b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.21a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBAP (5)\u0026thinsp;+\u0026thinsp;NAA (0.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.52a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.06a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.13a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.40a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBAP (5)\u0026thinsp;+\u0026thinsp;IBA (0.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.08a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.20b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.92b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.34a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSize of glassware\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLarge jar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.90a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.86a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.96b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.46a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedium jar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.35a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.86a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.80a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.03a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSmall test tube\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.31a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.53a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.29b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.90a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLight source\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLED arrays\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.14a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.32a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.60a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.66a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFluorescent tubes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.90a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.50a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.14a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.30b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCo-cultivation of explants\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1 explant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.38a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.53a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.33a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.13a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2 explants\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.69a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.23a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7,35a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.13a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3 explants\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.77b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.42a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.93b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.27b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4 explants\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.25a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.57a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.68a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.95a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eBAP - benzylaminopurine, IBA - indolebutyric acid, MS - Murashige and Skoog, NAA - naphthalene acetic acid, SH - Schenck and Hildebrandt, WPM - woody plant medium, LED - light emitting diode\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eWithin each treatment, mean values in the columns followed by dissimilar lowercase letters are significantly different according to the Scott-Knot test at 5% probability\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003e#\u003c/sup\u003e Variables submitted to \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\sqrt{x}+0.5\\)\u003c/span\u003e\u003c/span\u003e transformation\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e here\u003c/p\u003e \u003cp\u003eSupplementation of WPM with cytokinins BAP, kinetin or zeatin in the concentration range 0.5 to 10.0 \u0026micro;M revealed that 5 \u0026micro;M BAP was most efficient in breaking apical dominance and stimulating the production of buds and shoots in \u003cem\u003eU. tomentosa.\u003c/em\u003e Similar results have been reported for this species and other woody medicinal plants (Luna-Palencia et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Okao et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Addition of auxins to tissue culture medium may also assist in the production of multiple shoots, and in the present study, a concentration of 0.5 \u0026micro;M of NAA or IBA induced significantly higher numbers of shoots. WPM supplemented with a combination of 5 \u0026micro;M BAP and 0.5 \u0026micro;M NAA produced a statistically greater number of buds (5.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43) and shoots (2.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36) compared with 5 \u0026micro;M BAP\u0026thinsp;+\u0026thinsp;0.5 \u0026micro;M IBA. However, the combination treatment also gave rise to some undesirable effects such as swelling of the base and reduced vigor of plantlets when maintained for prolonged periods. These effects have been associated with cytokinin/auxin imbalance, which causes morphophysiological changes and reduction in shoot quality (Manchanda et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Interestingly, no significant differences were observed regarding plantlet height or number of roots in any of the cytokinin-auxin combinations.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e cultivation of \u003cem\u003eU. tomentosa\u003c/em\u003e in WPM, MS and quarter strength MS media supplemented with BAP at concentrations ranging from 2.2 to 44.4 \u0026micro;M has been described previously, with reported multiplication rates ranging between 1.5 and 5\u0026thinsp;\u0026plusmn;\u0026thinsp;2 shoots per plantlet (Pereira et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Venutolo \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Raposo and Teixeira \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Luna-Palencia et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Although these studies demonstrated the feasibility of the \u003cem\u003ein vitro\u003c/em\u003e cultivation of \u003cem\u003eU. tomentosa\u003c/em\u003e, none of them established an optimized protocol that integrates multiplication efficiency, maintenance of genetic diversity and post-acclimatization performance.\u003c/p\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, medium sized glass jars afforded the best conditions for the multiplication of \u003cem\u003eU. tomentosa\u003c/em\u003e since the number of buds was significantly higher than those produced in the larger or smaller vessels. In tissue culture laboratories, it is common practice to evaluate different types of vessels to determine the most suitable growing conditions. .\u003c/p\u003e \u003cp\u003eWhilst the application of different sources of light exerted no significant influence on the height of plantlets or the numbers of shoots or buds, exposure to LED arrays induced a higher number of roots compared with fluorescent tubes (3.66 and 2.30 per plantlet, respectively). The use of fluorescent tubes in growth chambers has always been very common, although the tendency is to replace them by LED arrays owing to their lower energy consumption.\u003c/p\u003e \u003cp\u003eThe use of LEDs as an alternative to conventional lighting in plant tissue culture installations reportedly gives satisfactory results regarding rhizogenesis, stem elongation, axillary shoot formation and increased photosynthetic capacity, all of which can be regulated by adjusting the spectral properties of the source (Gupta and Jatothu \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Marino et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The reported advantages of LED arrays, such as greater durability, lower operating cost and higher energy efficiency, will serve as a stimulus to upgrade the lighting systems in growth chambers.\u003c/p\u003e \u003cp\u003eThere were no significant differences in the height of plantlets or the number of shoots, buds or roots of explants cultivated in isolation or in co-cultivation with two or four explants per vessel. However, the height of plantlets and the number of buds and roots were reduced significantly when three explants were cultivated in the same vessel. Thus, co-cultivation of four explants is recommended for \u003cem\u003eU. tomentosa\u003c/em\u003e to maximize the use of space and economize culture medium without detriment to the growth of plantlets. Co-cultivation of explants can be a cost-effective technique for the large-scale micropropagation of species whose development is not impaired under such conditions. In the case of \u003cem\u003eU. tomentosa\u003c/em\u003e, large-scale micropropagation with two or four explants per vessel proved to be a productive approach since it optimizes the use of physical space, culture medium and labor without impairing \u003cem\u003ein vitro\u003c/em\u003e morphogenesis. The assessment of co-cultivation on solid medium is particularly relevant since liquid or temporary immersion bioreactors, such as the RITA\u0026reg; system developed by CIRAD, although efficient, can cause physiological disturbances, including hypoxia and hyperhydricity, especially in woody species (Bethge et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Ersali, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe optimised micropropagation protocol developed in this study employed medium-sized flasks containing 20 mL of woody plant medium (WPM) supplemented with 5.0 \u0026micro;M BAP, with four explants per flask maintained under a 16 h photoperiod provided by LED arrays. Under these conditions, explants produced an average of 2.13 shoots bearing a total of 8.13 buds per explant.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAcclimatization of plantlets and survival in forested areas\u003c/h2\u003e \u003cp\u003eThe acclimatization of micropropagated \u003cem\u003eU. tomentosa\u003c/em\u003e plantlets was considered excellent considering that the species is restricted climatically to tropical humid environments. The survival rate of the plantlets after two months under \u003cem\u003eex vitro\u003c/em\u003e conditions was 100%, while that of plants transferred to their natural habitat in the Amazonian forest was 46\u0026thinsp;\u0026plusmn;\u0026thinsp;18% after eighteen months of planting (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e here\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenetic diversity of\u003c/b\u003e \u003cb\u003eU. tomentosa\u003c/b\u003e \u003cb\u003eaccessions maintained\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGenomic DNA was extracted efficiently from the 20 selected \u003cem\u003eU. tomentosa\u003c/em\u003e genotypes maintained \u003cem\u003ein vitro\u003c/em\u003e. The eight primers employed in the ScoT marker analysis of genetic diversity amplified between 23 and 40 bands per primer, totaling 150 bands and exhibiting an average polymorphism of 88%. This high level of polymorphism was in accord with the value (92.22%) reported by Pinto et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) for accessions of \u003cem\u003eU. guianensis\u003c/em\u003e mantained in an \u003cem\u003ein vitro\u003c/em\u003e germplasm bank. Such notable degree of polymorphism within the genus \u003cem\u003eUncaria\u003c/em\u003e is significant and essential for conservation of the species.\u003c/p\u003e \u003cp\u003eThe UPGMA dendrogram (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and the dispersion (variance) PCoA plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) revealed genetic structuring and variability among the 20 individuals cultivated \u003cem\u003ein vitro\u003c/em\u003e. Cluster analysis indicated grouping patterns among the 20 accessions that mirrored their geographic origin in which the genetic similarity among genotypes from TA was higher than among those from BN, whilst the accession from BC (UTBC1) formed an independent branch signifying comparatively greater genetic divergence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIs it worth noting that while UTBC stands out as genetically dissimilar from the UTTA and UTBN accessions (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), it is UTTA1 that is assigned entirely to one cluster whilst UTBC has mixed ancestry like all of the other accessions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBayesian model-based clustering performed using the software STRUCTURE revealed the presence of two genetic groups, as represented by the red and green bands in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. The bars of all accessions, with the single exception of UTTA1, showed both red and green segments indicating that the vast majority of individuals were of mixed ancestry with different proportions of membership in both clusters. In contrast, the uniform red bar presented by UTTA1 suggests that it is genetically assigned entirely to one cluster with no admixture.\u003c/p\u003e \u003cp\u003eThe results of the present study are analogous to those reported by Hon\u0026oacute;rio et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) in which the existence of genetic structuring and low gene flow was demonstrated in eight natural populations of \u003cem\u003eU. tomentosa\u003c/em\u003e. In particular, the conclusions of these authors regarding the ancestry of the natural populations align with the inference from STRUCTURE analysis of the presence of two gene groups within the 20 \u003cem\u003ein vitro\u003c/em\u003e accessions. This result is significant because it implies that the \u003cem\u003ein vitro\u003c/em\u003e germplasm bank of \u003cem\u003eU. tomentosa\u003c/em\u003e is representative of the genetic groups found in natural populations. Maintaining this genetic diversity in accessions cultivated \u003cem\u003ein vitro\u003c/em\u003e is especially relevant from a conservationist point of view, since it reduces the risks of genetic erosion during clonal propagation and allows the selection of elite genotypes for the pharmaceutical industry without compromising natural populations.\u003c/p\u003e \u003cp\u003eFew actively managed germplasm collections have been assessed for the genetic diversity of their accessions. Knowledge of genetic variation is particularly important in the case of \u003cem\u003ein vitro\u003c/em\u003e collections since they maintain accessions free from disease and in their specific allelic state, thereby assisting in the identification of useful traits that can be used in genetic improvement programs (Anglin et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChemical characteristics of the selected individuals\u003c/h3\u003e\n\u003cp\u003eHPLC analysis revealed significant quantitative and qualitative variability in alkaloid content among the 20 accessions of \u003cem\u003eU. tomentosa\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Thus, UTBN233 presented the highest concentration of mitraphylline (3.329 mg\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e fresh weight), while UTTA5 exhibited the highest concentration of isomitraphylline (1.422 mg\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e fresh weight). Although the majority of accessions produced both alkaloids, two individuals from BN (UTBN358 and UTBN750) contained no mitraphylline and two (UTBN233 and UTBN332) contained no isomitraphylline, whereas UTBN207 accumulated neither of these alkaloids.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAlkaloid content of the 20 selected \u003cem\u003eUncaria tomentosa\u003c/em\u003e individuals\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIndiv\u0026iacute;duals\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMitraphylline\u003c/p\u003e \u003cp\u003e(mg\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e fresh weight)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIsomitraphylline\u003c/p\u003e \u003cp\u003e(mg\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e fresh weight)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUTTA 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.08 m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.00 j\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUTTA 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.52 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0.53 f\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUTTA 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e2.24 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.43 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUTTA 9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1.63 g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.14 b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUTTA 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1.87 f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.00 c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUTTA 19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e2.80 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.17 b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUTTA 20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e2.07 e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.69 e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUTTA 30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e2.27 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.94 c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUTBN 207\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.00 m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.00 j\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUTBN 233\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e3.32 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.00 j\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUTBN 332\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1.04 i\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.00 j\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUTBN 354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.90 j\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.25 h\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUTBN 358\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.00 m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.32 g\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUTBN 499\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.88 j\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.20 h\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUTBN 750\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.00 m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.31 g\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUTBN 767\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1.04 i\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.12 i\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUTBN 847\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.73 k\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.83 d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUTBN 902\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1.01 i\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.30 g\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUTBN 985\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1.35 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.00 j\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUTBC 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.21 l\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.04 j\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eMean values in each column followed by dissimilar lowercase letters are significantly different according to the Scott-Knot test at 5% probability.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThis variation in alkaloid constituents appears to be a normal pattern in \u003cem\u003eU. tomentosa\u003c/em\u003e since it has been described for natural populations of the species in previous studies (Pe\u0026ntilde;aloza et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Torrej\u0026oacute;n et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The existence of chemical heterogeneity reinforces the importance of conserving multiple genotypes, especially considering the interest of the phytotherapeutic industry in plant materials with high levels of these bioactive compounds. In this context, the introduction of genetically diverse and chemically distinct accessions into \u003cem\u003ein vitro\u003c/em\u003e germplasm banks is a pertinent strategy for both the conservation and sustainable use of the species.\u003c/p\u003e \u003cp\u003eTissue culture has been widely recognized as an important conservation strategy for threatened medicinal species that are subject to predatory exploitation (Moraes et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, micropropagation and \u003cem\u003ein vitro\u003c/em\u003e germplasm maintenance allow not only the rapid multiplication of selected genotypes but also the preservation of genetic variability, especially for species with difficult sexual propagation or low seed viability (Singh and Chokheli \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eGenetic analysis by SCoT markers of 20 accessions of \u003cem\u003eU. tomentosa\u003c/em\u003e cultivated \u003cem\u003ein vitro\u003c/em\u003e indicated that they descended from two inferred ancestral genetic clusters, a situation similar to that established for natural populations. Fifteen of the 20 genotypes accumulated both mitraphylline and isomitraphylline, albeit in varying concentrations, which is typical of the species. In contrast, four of the genotypes accumulated only one of the alkaloids and one genotype contained neither. The survival rate of the plants introduced into the natural environment was highly satisfactory considering the environmental and abiotic constraints, such as nutrient and light limitation and high humidity and temperature, posed by the Amazon rainforest. The results obtained reinforce the potential of \u003cem\u003ein vitro\u003c/em\u003e culture as a complementary approach to the \u003cem\u003ein situ\u003c/em\u003e conservation of \u003cem\u003eU. tomentosa\u003c/em\u003e, since both contribute to the protection of natural populations and the sustainable supply of plant material.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthical approval\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConflict of interest\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no conflicts of interest\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq; grant no. 440537/2022-3) and Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de S\u0026atilde;o Paulo (FAPESP; grant no. 23/05517-3).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: I.C.G.H., J.S.C and A.M.S.P.; Methodology: I.C.G.H., J.S.C., G.H.T.P., B.S.R., R.N.S.M., E.F.S and W.S.; Data curation: I.C.G.H., G.H.T.P. and B.W.B.; Formal analysis: I.C.G.H., J.S.C. and B.W.B; Investigation: I.C.G.H., J.S.C. and B.W.B; Visualization: I.C.G.H. and S.C.F.; Writing - Original draft: I.C.G.H., B.S.R., S.C.F and B.W.B.; Validation: A.M.S.P; Writing \u0026ndash; review \u0026amp; editing: A.M.S.P; Supervision: A.M.S.P Funding This work was supported by Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq; grant no. 440537/2022-3) and Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de S\u0026atilde;o Paulo (FAPESP; grant no. 23/05517-3).\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eData is available upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAg\u0026ecirc;ncia Nacional de Vigil\u0026acirc;ncia Sanit\u0026aacute;ria (2003) Resolu\u0026ccedil;\u0026atilde;o-RE n. 899, de 29 de maio de 2003. Disp\u0026otilde;e sobre o guia para valida\u0026ccedil;\u0026atilde;o de m\u0026eacute;todos anal\u0026iacute;ticos e bioanal\u0026iacute;ticos. ANVISA. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bvsms.saude.gov.br/bvs/saudelegis/anvisa/2003/res0899_29_05_2003\u003c/span\u003e\u003cspan address=\"https://bvsms.saude.gov.br/bvs/saudelegis/anvisa/2003/res0899_29_05_2003\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e Accessed 6 February 2026\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnglin NL, Wenzl P, Azevedo V, Lusty C, Ellis D, Gao D (2025) Genotyping genebank collections: Strategic approaches and considerations for optimal collection management. Plants 14(2):252. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/plants14020252\u003c/span\u003e\u003cspan address=\"10.3390/plants14020252\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArado GM, Amatto PPG, Marins M, Rizzi ES, Fran\u0026ccedil;a SC, Coppede JS, Carmona F, Pereira MAS (2024) Anti-inflammatory and/or immunomodulatory activities of \u003cem\u003eUncaria tomentosa\u003c/em\u003e (cat's claw) extracts: A systematic review and meta-analysis of \u003cem\u003ein vivo\u003c/em\u003e studies. Front Pharmacol 15:1378408. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphar.2024.1378408\u003c/span\u003e\u003cspan address=\"10.3389/fphar.2024.1378408\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBethge H, Mohammadi Nakhjiri Z, Rath T, Winkelmann T (2023) Towards automated detection of hyperhydricity in plant \u003cem\u003ein vitro\u003c/em\u003e culture. Plant Cell Tiss Organ Cult 154:551\u0026ndash;573. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11240-023-02528-0\u003c/span\u003e\u003cspan address=\"10.1007/s11240-023-02528-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlumenthal M (ed) (1997) World Health Organization Medicinal Plants Monographs. HerbalGram 40(38)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCollard BCY, Mackill DJ (2009) Start codon targeted (SCoT) polymorphism: A simple, novel DNA marker technique for generating gene-targeted markers in plants. Plant Mol Biol Rep 27:86\u0026ndash;93. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11105-008-0060-5\u003c/span\u003e\u003cspan address=\"10.1007/s11105-008-0060-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDoyle JJ, Doyle JL (1987) Isolation of plant DNA from fresh tissue. Focus (Rockville) 12(1):13\u0026ndash;15\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eErsali Y (2024) Control of hyperhydricity of \u003cem\u003ePistacia khinjuk\u003c/em\u003e stocks \u003cem\u003ein vitro\u003c/em\u003e shoots. BMC Biotechnol 24:97. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12896-024-00929-3\u003c/span\u003e\u003cspan address=\"10.1186/s12896-024-00929-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEvanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol Ecol 14(8):2611\u0026ndash;2620. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-294X.2005.02553.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-294X.2005.02553.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerreira DF (2011) Sisvar: a computer statistical analysis system. Cienc Agrotec 35(6):1039\u0026ndash;1042. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/S1413-70542011000600001\u003c/span\u003e\u003cspan address=\"10.1590/S1413-70542011000600001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerreira SJF, Luiz\u0026atilde;o FJ, Miranda SAF, Silva MSR, Vital ART (2006) Nutrients in soil solution in an upland forest submitted to selective logging in central Amazonia. Acta Amaz 36(1):59\u0026ndash;68. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/S0044-59672006000100008\u003c/span\u003e\u003cspan address=\"10.1590/S0044-59672006000100008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupta SD, Jatothu B (2013) Fundamentals and applications of light-emitting diodes (LEDs) in \u003cem\u003ein vitro\u003c/em\u003e plant growth and morphogenesis. Plant Biotechnol Rep 7:211\u0026ndash;220. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11816-013-0277-0\u003c/span\u003e\u003cspan address=\"10.1007/s11816-013-0277-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHampl V, Pavl\u0026iacute;ček A, Flegr J (2001) Construction and bootstrap analysis of DNA fingerprinting-based phylogenetic trees with the freeware program FreeTree: application to trichomonad parasites. Int J Syst Evol Microbiol 51:731\u0026ndash;735. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1099/00207713-51-3-731\u003c/span\u003e\u003cspan address=\"10.1099/00207713-51-3-731\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHon\u0026oacute;rio ICG, Bertoni BW, Pereira AMS (2016) \u003cem\u003eUncaria tomentosa\u003c/em\u003e and \u003cem\u003eUncaria guianensis\u003c/em\u003e an agronomic history to be written. Cienc Rural 46(8):1401\u0026ndash;1410. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/0103-8478cr20150138\u003c/span\u003e\u003cspan address=\"10.1590/0103-8478cr20150138\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHon\u0026oacute;rio ICG, Bertoni BW, Telles MPC, Braga RS, Fran\u0026ccedil;a SC, Coppede JS, Correa VSC, Diniz Filho JAF, Pereira MAS (2017) Genetic and chemical diversity of \u003cem\u003eUncaria tomentosa\u003c/em\u003e (Willd. ex. Schult.) DC. in the Brazilian Amazon. PLoS ONE 12(5):e0177103. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0177103\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0177103\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLloyd G, McCown BH (1980) Commercially-feasible micropropagation of mountain laurel, \u003cem\u003eKalmia latifolia\u003c/em\u003e, by use of shoot-tip culture. Combined Proc Intl Plant Prop Soc 30:421\u0026ndash;427\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuna-Palencia GR, Huerta-Heredia AA, Cerda-Garc\u0026iacute;a-Rojas CM, Ramos-Valdivia AC (2013) Differential alkaloid profile in \u003cem\u003eUncaria tomentosa\u003c/em\u003e micropropagated plantlets and root cultures. Biotechnol Lett 35:791\u0026ndash;797. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10529-012-1128-8\u003c/span\u003e\u003cspan address=\"10.1007/s10529-012-1128-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManchanda P, Sharma D, Kaur G, Kaur H, Vanshika (2025) Exploring the significance of somaclonal variations in horticultural crops. Mol Biotechnol 67:2185\u0026ndash;2203. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12033-024-01214-6\u003c/span\u003e\u003cspan address=\"10.1007/s12033-024-01214-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarino LA, Ruffa P, Mozzanini E, Patono DL, Sereno A, Pavese V (2025) LEDs in plant tissue culture: Boosting micropropagation of \u003cem\u003eCastanea sativa\u003c/em\u003e cultivars. J Plant Growth Regul 44:6046\u0026ndash;6060. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00344-025-11812-6\u003c/span\u003e\u003cspan address=\"10.1007/s00344-025-11812-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMinist\u0026eacute;rio da Sa\u0026uacute;de (2013) Rela\u0026ccedil;\u0026atilde;o nacional de medicamentos essenciais (RENAME). Sistema \u0026Uacute;nico de Sa\u0026uacute;de (SUS). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.gov.br/saude/pt-br/composicao/sectics/rename\u003c/span\u003e\u003cspan address=\"https://www.gov.br/saude/pt-br/composicao/sectics/rename\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e Accessed 6 Feb 2026\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoraes RM, Cerdeira AL, Louren\u0026ccedil;o MV (2021) Using micropropagation to develop medicinal plants into crops. Molecules 26(6):1752. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules26061752\u003c/span\u003e\u003cspan address=\"10.3390/molecules26061752\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473\u0026ndash;497. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1399-3054.1962.tb08052.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1399-3054.1962.tb08052.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOkao M, Bharati R, Fern\u0026aacute;ndez-Cusimamani E (2025) Effect of explant physiology and media composition on callogenesis of \u003cem\u003eVitellaria paradoxa\u003c/em\u003e leaf explants. Horticulturae 11(9):1127. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/horticulturae11091127\u003c/span\u003e\u003cspan address=\"10.3390/horticulturae11091127\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeakall R, Smouse PE (2012) GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research - an update. Bioinformatics 28(19):2537\u0026ndash;2539. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/bioinformatics/bts460\u003c/span\u003e\u003cspan address=\"10.1093/bioinformatics/bts460\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePe\u0026ntilde;aloza EMC, Kaiser S, Resende PE, Pittol V, Carvalho \u0026Acirc;R, Ortega GG (2015) Chemical composition variability in the \u003cem\u003eUncaria tomentosa\u003c/em\u003e (cat\u0026rsquo;s claw) wild population Quim Nova. 38(3):378\u0026ndash;386. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5935/0100-4042.20150007\u003c/span\u003e\u003cspan address=\"10.5935/0100-4042.20150007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePereira RCA, Valente LMM, Pinto JEBP, Bertolucci SKV, Bezerra GM, Alves FF, Santos PFP, Benevides PJC, Siani AC, Rosario SL, Mazzei JL, d\u0026rsquo;Avila LA, Gomes LNF, Aquino-Neto FR, Emmerick ICM, Carvalhaes SF (2008) \u003cem\u003eIn vitro\u003c/em\u003e cultivated \u003cem\u003eUncaria tomentosa\u003c/em\u003e and \u003cem\u003eUncaria guianensis\u003c/em\u003e with determination of the pentacyclic oxindole alkaloid contents and profiles. J Braz Chem Soc 19(6):1193\u0026ndash;1200. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/S0103-50532008000600021\u003c/span\u003e\u003cspan address=\"10.1590/S0103-50532008000600021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePinto GHT, Lopes AA, Morel LJF, Crevelin EJ, Miranda CES, Contini SHT, Fran\u0026ccedil;a SC, Bertoni BW, Pereira AMS (2022) Genetic diversity among genotypes of \u003cem\u003eUncaria guianensis\u003c/em\u003e (Aubl.) J.F. Gmel. maintained in an \u003cem\u003ein vitro\u003c/em\u003e germplasm bank. 3 Biotech 12:8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s13205-021-03016-y\u003c/span\u003e\u003cspan address=\"10.1007/s13205-021-03016-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155(2):945\u0026ndash;959. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/genetics/155.2.945\u003c/span\u003e\u003cspan address=\"10.1093/genetics/155.2.945\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRai MK (2023) Start codon targeted (SCoT) polymorphism marker in plant genome analysis: current status and prospects. Planta 257:34. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00425-023-04067-6\u003c/span\u003e\u003cspan address=\"10.1007/s00425-023-04067-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaposo A, Teixeira RB (2011) Metodologia cient\u0026iacute;fica: cultivo \u003cem\u003ein vitro\u003c/em\u003e de Unha-de-gato. Circular T\u0026eacute;cnica 57. Embrapa Acre, Rio Branco, Brazil. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.infoteca.cnptia.embrapa.br/bitstream/doc/912085/1/24058.PDF\u003c/span\u003e\u003cspan address=\"https://www.infoteca.cnptia.embrapa.br/bitstream/doc/912085/1/24058.PDF\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e Accessed 06 February 2026\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchenck RU, Hildebrandt AC (1972) Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 50(1):199\u0026ndash;204. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1139/b72-026\u003c/span\u003e\u003cspan address=\"10.1139/b72-026\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh RK, Chokheli VA (2025) Plant biotechnology: Applications in \u003cem\u003ein vitro\u003c/em\u003e plant conservation and micropropagation. Horticulturae 11(4):358. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/horticulturae11040358\u003c/span\u003e\u003cspan address=\"10.3390/horticulturae11040358\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSirikonda A, Jogam P, Mood K, Allini VR (2023) Micropropagation of 2-methoxy-4-vinyl phenol rich \u003cem\u003eFlemingia strobilifera\u003c/em\u003e and assessment of genetic and biochemical fidelity by SCoT and GC-MS analysis. Plant Cell Tiss Organ Cult 154:541\u0026ndash;550. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11240-023-02526-2\u003c/span\u003e\u003cspan address=\"10.1007/s11240-023-02526-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTorrej\u0026oacute;n GD, Mart\u0026iacute;n JJG, Loayza DG, Alanoca R (2010) Contenido de alcaloides en corteza de \u003cem\u003eUncaria tomentosa\u003c/em\u003e (Wild.) DC procedente de diferentes h\u0026aacute;bitats de la regi\u0026oacute;n Ucayali-Per\u0026uacute;. Rev Soc Quim Peru 76(3):271\u0026ndash;278\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUnited States Pharmacopeial Convention (2022) Cat\u0026rsquo;s Claw (\u003cem\u003eUncaria tomentosa\u003c/em\u003e (Willd.) DC.). In: \u003cem\u003eUnited States Pharmacopeia and National Formulary (USP\u0026ndash;NF).\u003c/em\u003e United States Pharmacopeial Convention, Rockville\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVenutolo AS (2010) Establecimiento \u003cem\u003ein vitro\u003c/em\u003e y cultivo de c\u0026eacute;lulas de la U\u0026ntilde;a de gato (\u003cem\u003eUncaria tomentosa\u003c/em\u003e). Tecnol Marcha 23(5):24\u0026ndash;33\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVrundha CPK, Thomas TD (2023) Indirect shoot regeneration from root explants, assessment of clonal fidelity of regenerated plants using SCoT primers and antioxidant analysis in \u003cem\u003eThottea siliquosa\u003c/em\u003e (Lamk.) Ding Hou. Plant Cell Tiss Organ Cult. 155:255\u0026ndash;266. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11240-023-02578-4\u003c/span\u003e\u003cspan address=\"10.1007/s11240-023-02578-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZavala A, Zevallos PP (1996) Taxonom\u0026iacute;a, distribuci\u0026oacute;n geogr\u0026aacute;fica y status del g\u0026eacute;nero \u003cem\u003eUncaria\u003c/em\u003e en el Per\u0026uacute;. Facultad de Ciencias Forestales, Universidad Nacional Agraria La Molina, Lima, Peru. 103 pp\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":false,"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":"Uncaria tomentosa, micropropagation, acclimatization, genetic diversity, alkaloid profile, field establishment","lastPublishedDoi":"10.21203/rs.3.rs-9107751/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9107751/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eUncaria tomentosa\u003c/em\u003e Willd. ex Roem. \u0026amp; Schult., known as cat's claw, is a medicinal plant that is native to the Amazon and used to treat inflammatory diseases. Currently plants are harvested in a predatory manner leading potentially to extinction of the species. The aims of this study were to develop an efficient protocol for the micropropagation of the species, to evaluate the genetic and chemical diversity of genotypes cultivated \u003cem\u003ein vitro\u003c/em\u003e, and to determine the survival rate of plants introduced into the native environment. The optimised micropropagation protocol employed medium-sized flasks containing 20 mL of woody plant medium (WPM) supplemented with 5.0 \u0026micro;M 6-benzylaminopurine (BAP), with four explants per flask maintained under a 16 h photoperiod provided by light emitting diodes. Under these conditions, explants produced an average of 2.13 shoots bearing a total of 8.13 buds per explant. Twenty accessions of \u003cem\u003eU. tomentosa\u003c/em\u003e, each originating from seeds of individual plants collected in three states of the Brazilian Amazon, were maintained \u003cem\u003ein vitro\u003c/em\u003e and analyzed by start codon targeted (ScoT) molecular markers and chemically by liquid chromatography. These genotypes showed genetic diversity \u003cem\u003ein vitro\u003c/em\u003e that was comparable with natural populations, and the majority of accessions accumulated mitraphylline and isomitraphylline. Plantlets cultivated \u003cem\u003ein vitro\u003c/em\u003e were acclimatized and introduced into the native environment with a survival rate of 46\u0026thinsp;\u0026plusmn;\u0026thinsp;18% after eighteen months. It is concluded that \u003cem\u003ein vitro\u003c/em\u003e propagation of \u003cem\u003eU. tomentosa\u003c/em\u003e is efficient for the production of vigorous plants capable of surviving in the natural conditions of the Amazon rainforest.\u003c/p\u003e","manuscriptTitle":"Uncaria tomentosa Willd. ex Roem. \u0026amp; Schult.: genetic and chemical diversity of accessions cultivated in vitro and their introduction into the native environment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-01 10:27:09","doi":"10.21203/rs.3.rs-9107751/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-21T21:08:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-09T20:59:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"185694879859691173272179562104588219919","date":"2026-04-01T13:33:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"283562455958361891776172894668679643851","date":"2026-04-01T10:53:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"27579181393554313551768285052298026180","date":"2026-03-31T17:25:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-30T09:22:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-24T01:08:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-24T01:07:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2026-03-12T18:49:20+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":"0b978256-d060-4efe-80d3-3781606616ee","owner":[],"postedDate":"April 1st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-01T10:27:10+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-01 10:27:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9107751","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9107751","identity":"rs-9107751","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}