Development of a protocol for the micropropagation of two forest species threatened with extinction in Ecuador

Development of a protocol for the micropropagation of two forest species threatened with extinction in Ecuador | 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 Development of a protocol for the micropropagation of two forest species threatened with extinction in Ecuador Eduardo Hector, Damaris Cevallos, Liliana Corozo, Fatima Macías, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4783431/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Sep, 2024 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted 4 You are reading this latest preprint version Abstract Handroanthus chrysanthus Jacq. S. O. Grose and Tabebuia rosea (Bertol.) Bertero ex A.DC are two forest species that grow in the coastal region of Ecuador and are threatened with extinction. A protocol for the mass multiplication of these species was developed using in vitro culture techniques. The cultures were initiated from seeds, and the effect of two culture media (WPM and MS), two concentrations of NaClO (0.5% and 1%), and two disinfection times (3 and 5 minutes) was studied. During multiplication, the effect of three concentrations of two cytokinins (6-BAP 4.4, 5.5, or 6.6 µM; KIN 4.6, 5.75, or 6.9 µM) on the number of shoots, their length, and diameter was analyzed. This phase of the experiment was carried out in two successive multiplications. For rooting, two concentrations of IBA (2.45 and 4.9 µM) were tested, and the number of roots formed and their length were determined. It was demonstrated that the WPM medium is the most suitable for the in vitro culture of both species and that disinfection time and NaClO concentration affect each species differently. For the multiplication of H. chrysanthus , the most suitable cytokinin was 6-BAP 6.6 µM; T. rosea performed better in the absence of cytokinins. IBA 2.45 µM produced the best results for the rooting of H. chrysanthus , while for T. rosea , IBA 4.9 µM was the most suitable. The acclimatized plants showed a high survival rate, demonstrating the feasibility of using this methodology for the accelerated propagation of these endangered species. tissue culture growth regulators woody plants micropropagation Figures Figure 1 Figure 2 Key Message In this research, the culture medium requirements and conditions for the micropropagation of H. chrysanthus and T. rosea were fine-tuned. This technique can be implemented to obtain plants for use in reforestation. Introduction The genera Handroanthus and Tabebuia belong to the family Bignoniaceae (Burley et al. 2021 ; El-Hawary et al. 2021 ), which includes more than 800 species (Olmstead et al. 2009 ). Ecuador is a megadiverse country, with around 750 forest species. In the coastal region of Ecuador, deciduous dry forests are threatened by overexploitation, exacerbated by the fact that there are few protected areas compared to green forests, which have received more attention (Rivas et al. 2020 ). Micropropagation through in vitro tissue culture is a biotechnological approach that allows the exponential increase in the number of plants to perpetuate individuals of particular value in a specific area, serving as a powerful tool for reforestation (Hazubska-Przybył 2019 ; Adugna et al. 2020 ; Morales et al. 2020). Handroanthus chrysanthus Jacq. S. O. Grose and Tabebuia rosea (Bertol.) Bertero ex A.DC are two forest species that grow in the dry forests of the Ecuadorian coast. Due to their high timber value, they have been subjected to indiscriminate logging, putting them at risk of extinction. Based on these circumstances, this research was developed with the aim of fine-tuning protocols for the micropropagation process of both species. Materials and methods The research was conducted at the Biotechnology Laboratory of the Faculty of Agronomic Engineering at the Technical University of Manabí, in Santa Ana, Manabí, Ecuador. Seed selection The seeds of the two species were obtained from fully mature fruits from well-developed and vigorous trees located in the University Botanical Garden of the Technical University of Manabí, Ecuador. Surface sterilization and in vitro seed establishment The seeds were washed with water and liquid soap; then they were transferred to the laminar flow cabinet, rinsed with sterile distilled water, and treated with sodium hypochlorite (NaClO) at 0.5% or 1% for two disinfection times (3 or 5 minutes). Finally, they were rinsed three times with sterile distilled water to remove excess disinfectant. The disinfected seeds were placed in test tubes containing WPM (McCown and Lloyd 1981) or MS (Murashige and Skoog 1962) culture medium, supplemented with 3% sucrose; 0.7% agar and 10 ppm cysteine were added. The pH of the media was adjusted to 5.7. The seeds were maintained at 24 ± 2 °C with a photoperiod of 8 hours of darkness and 16 hours of light. Each species underwent 8 treatments, consisting of combinations of culture medium, disinfectant concentration, and disinfection time. Each treatment comprised 50 repetitions (seeds), following a factorial design of 2x2x2 for each species. After 10 days, survival percentages were evaluated. In vitro multiplication For the multiplication of the species, apical buds from plants obtained from seeds established in vitro were used. In the laminar flow chamber, apices measuring 1 cm in length were cut according to the methodology of Delgado and Hoyos (2016). The apices were placed in culture tubes containing 10 ml of WPM medium supplemented with 3% sucrose, 0.7% agar, and adjusted to pH 5.7. Each species underwent 7 treatments, consisting of two growth regulators at three concentrations (6-benzylaminopurine 4.4, 5.5, 6.6 μM or kinetin 4.6, 5.75, 6.9 μM) and a control without growth regulators, in a factorial design (2x3+1) with 20 repetitions per treatment. The apices were maintained at 24 ± 2 °C with a photoperiod of 8 hours of darkness and 16 hours of light. The study was conducted during the first and second in vitro multiplication. After 8 weeks in each multiplication, the number of shoots produced per apex was counted, and their length and diameter were measured. In vitro rooting The plants obtained from each species during the multiplication stage were then subjected to rooting in WPM medium supplemented with 3% sucrose, 0.7% agar, and adjusted to pH 5.7. Three treatments were tested (control without growth regulators, indole-3-butyric acid 2.45 μM, índole-3-butyric acid 4.9 μM). The plants were maintained at 24 ± 2 °C with a photoperiod of 8 hours of darkness and 16 hours of light. Thirty plants were used per treatment in a completely randomized design. After four weeks, the number of roots was counted, and their length was measured. Acclimatization of plants The vitroplants obtained after 8 weeks in rooting medium (100 per species) were established in a mixture of 40% humus, 40% river sand, and 20% decomposed wood sawdust, previously sterilized by steam at 121°C for one hour. Plastic pots were used to protect the plants in micro-chambers for acclimatization. Irrigation was applied twice daily with a sprinkler for 15 days; thereafter, watering was reduced to once daily for another 15 days, and subsequently to three times per week. The survival rate of the plants was assessed 4 weeks after transplantation. At 60 days, the adapted plants were transferred to polyethylene sleeves for final hardening and eventual transplantation to the field. Statistical analysis Normality tests (Shapiro-Wilk) and homogeneity of variances tests (Levene) were conducted on the variable data. Once the statistical assumptions were confirmed, analysis of variance (ANOVA) were performed, and means were compared using Tukey's test (p ≤ 0.05). When the evaluated variables were expressed as percentages, the data were transformed prior to analysis to [2 arcsen(%)]. Results Surface sterilization and in vitro seed establishment The results obtained from surface sterilization and in vitro establishment of both species are shown in Table 1. In the Murashige and Skoog (MS) medium, seeds of H. chrysanthus did not thrive, thus only results from Woody Plant Medium (WPM) could be processed. In this species, no interaction effect between NaClO concentration and disinfection time was detected; however, there was an effect observed from each factor separately. The highest survival percentages (92.50%) were achieved with NaClO 1% and disinfection during 5 minutes. In T. rosea , differences were observed among the culture media used rather than among the concentrations of NaClO or disinfection times. In the MS medium, only 23.75% of the seeds survived, whereas in WPM, seed survival reached 86.25%. Table 1. Influence of NaClO concentration, disinfection time, and culture medium on the survival percentage (%) in the establishment of Handroanthus chrysanthus (Jacq.) S.O. Grose and Tabebuia rosea (Bertol.) Bertero ex A.DC Handroanthus chrysanthus NaClO concentration (%) Survival percentage 0,5 80.00 b 1,0 92.50 a Disinfection time (min) Survival percentage 3 80.00 b 5 92.50 a Tabebuia rosea Culture medium Survival percentage MS 23.75 b WPM 86.25 a Different letters indicate significant differences for Tukey's test (p ≤ 0.05) In vitro multiplication In the first multiplication of H. chrysanthus from shoots obtained in WPM establishment, treatment 4 (6-BAP 6.6 μM) resulted in the highest values for both shoot quantity and length, significantly differing from the others. Regarding shoot diameter, four treatments, including 6-BAP 6.6 μM, were significantly superior to the rest. Conversely, for T. rosea , the control without growth regulators was among the treatments that significantly outperformed the others across all three measured variables (Table 2). Table 2. Effect of growth regulators 6-benzylaminopurine (BAP) and kinetin (KIN) on the number, length, and diameter of shoots of Handroanthus chrysanthus (Jacq.) S.O. Grose and Tabebuia rosea (Bertol.) Bertero ex A.DC (first in vitro multiplication) Number of shoots Length of shoots Diameter of shoots Treatments H. chrysanthus T. rosea H. chrysanthus T. rosea H. chrysanthus T. rosea 1) Control 1.95±0.15 cd 2.90±0.19 a 1.84±0.07 c 2.80±0.08 a 2.65±0.20 bc 2.95±0.11 a 2) 6-BAP 4.4 μM 2.45±0.11 bc 2.20±0.17 bc 2.17±0.06 b 2.42±0.10 bc 3.20±0.16 ab 2.65±0.11 abcd 3) 6-BAP 5.5 μM 2.60±0.15 b 2.40±0.13 abc 1.97±0.05 bc 2.48±0.08 abc 3.00±0.14 abc 2.80±0.09 ab 4) 6-BAP 6.6 μM 3.35±0.15 a 2.60±0.11 ab 2.50±0.08 a 2.57±0.07 ab 3.35±0.11 a 2.75±0.10 abc 5) KIN 4.6 μM 1.60±0.13 d 1.80±0.12 c 1.87±0.05 c 2.17±0.097 c 2.55±0.114 c 2.35±0.109 d 6) KIN 5.75 μM 2.25±0.0993 bc 2.25±0.099 bc 2.00±0.03 bc 2.44±0,09 bc 2.60±0.11 c 2.45±0.11 cd 7) KIN 6.9 μM 1.95±0.11 cd 2.30±0.16 abc 2.00±0.04 bc 2.45±0.07 abc 2.90±0.12 abc 2.60±0.11 bcd Different letters in the same column indicate significant differences for Tukey's test (p ≤ 0.05) The second multiplication corroborated the results obtained in the first. In H. chrysanthus , the treatment with 6-BAP 6.6 μM consistently produced significantly higher results across all three measured variables compared to the other treatments. For T. rosea , the control without growth regulators showed overall superiority in shoot quantity and length, with no significant differences found among treatments for shoot diameter (Table 3). Table 3. Effect of growth regulators 6-benzylaminopurine (BAP) and kinetin (KIN) on the number, length, and diameter of shoots of Handroanthus chrysanthus (Jacq.) S.O. Grose and Tabebuia rosea (Bertol.) Bertero ex A.DC (second in vitro multiplication) Number of shoots Length of shoots Diameter of shoots Treatments H. chrysanthus T. rosea H. chrysanthus T. rosea H. chrysanthus T. rosea 1) Control 2.55±0.11 bc 3.20±0.09 a 2.08±0.08 bc 3.41±0.12 a 3.00±0.13 bc ns 2) 6-BAP 4.4 μM 2.55±0.13 bc 2.30±0.10 b 2.40±0.20 b 2.66±0.08 bc 3.05±0.11 b ns 3) 6-BAP 5.5 μM 2.70±0,15 b 2.35±0.11 b 2.07±0.85 bc 2.70±0.09 b 2.75±0.12 bcd ns 4) 6-BAP 6.6 μM 3.60±0.19 a 2.40±0.11 b 2.80±0.10 a 2.40±0.08 bc 3.45±0.11 a ns 5) KIN 4.6 μM 2.10±0.11 d 2.15±0.08 b 1.86±0.04 d 2.34±0.05 c 2.60±0.11 d ns 6) KIN 5.75 μM 2.25±0.01 cd 2.25±0.01 b 2.11±0.09 b 2.37±0.07 bc 2.70±0.10 cd ns 7) KIN 6.9 μM 2.70±0.13 b 2.30±0.10 b 2.07±0.07 bc 2.47±0.06 bc 2.65±0.13 cd ns Different letters in the same column indicate significant differences for Tukey's test (p ≤ 0.05). ns: no significant differences. In vitro rooting Both species responded positively to the inclusion of IBA in the culture medium. In H. chrysanthus , the addition of 2.45 μM IBA resulted in the highest values for both root number and length. Conversely, for T. rosea , the concentration of 4.9 μM produced the highest values (Table 4). Table 4. Effect of indole-3-butyric acid (IBA) on the in vitro rooting of Handroanthus chrysanthus (Jacq.) S.O. Grose and Tabebuia rosea (Bertol.) Bertero ex A.DC Number of roots Length of roots Treatments H. chrysanthus T. rosea H. chrysanthus T. rosea 1) Control 2.50±0.09 b 2.43±0.12 c 1.94±0.03 b 1.66±0.03 c 2) IBA 2.45 μM 5.17±0.13 a 4.93±0.15 b 2.73±0.04 a 2.57±0.04 b 3) IBA 4.9 μM 1.70±0.09 c 8.20±0.15 a 1.40±0.02 c 3.50±0.04 a Different letters in the same column indicate significant differences for Tukey's test (p ≤ 0.05) Acclimatization of plants Out of the total of 100 plants evaluated in H. chrysanthus , 96 successfully survived, indicating a very high survival rate in this species (96%). In T. rosea , 80 out of 100 vitroplants successfully adapted, resulting in a survival rate of 80%, which is considered high for a woody species. Fig 2 depicts the growth and development of the plants during the acclimatization stage. Discussion The establishment of explants is a crucial step in any in vitro culture protocol, influenced by the culture medium, disinfectant, and its concentration. The optimal combination of these factors aims to ensure that explants adapt to the culture medium with minimal contamination and minimal damage caused by the disinfectant. In woody plants, the exposure of explant materials to the environment typically results in high contamination rates, which is why various disinfectants and concentrations are tested, sometimes proving aggressive. In the establishment of nodal segments of Calophyllum brasiliense , Silveira et al. ( 2016 ) were compelled to use HgCl 2 in addition to 5% NaClO for disinfection. To achieve a 55% success rate in disinfecting nodal segments of Handroanthus heptaphyllus , Díaz et al. ( 2020 ) required 5% NaClO for 10 minutes. In the establishment of Myrcianthes ferreyrae , Araujo et al. ( 2023 ) employed more aggressive disinfectants and concentrations, such as 0.1% HgCl 2 and 15% NaClO for 10 and 15 minutes, respectively. Conversely, Kaviani et al. (2022) achieved successful disinfection of axillary buds of Piracantha angustifolia with 2% NaClO. In Pistacia vera , Gammoudi et al. ( 2022 ) observed that the effectiveness of disinfection with NaClO (0.54–1.26%) is lower than that of HgCl 2 and H 2 O 2 , but its toxicity to plant tissues is also much lower. Possibly due to the use of seeds rather than nodal segments to initiate the culture, our experiments achieved high survival rates with established and contamination-free seeds for both species using low concentrations of NaClO. Seeds typically have physical and chemical barriers that make them less penetrable by microorganisms, thereby resisting internal infection (Nallathambi et al. 2020 ), whereas axillary bud explants and nodal segments are more exposed. Based on the results observed in this study, the seeds of H. chrysanthus should be disinfected with 1% NaClO for 5 minutes, whereas 0.5% NaClO for 3 minutes is sufficient for T. rosea . The recalcitrance of numerous woody plant species to adapt to in vitro culture is a barrier to their propagation, leading to the development of the Woody Plant Medium (WPM) specifically tailored for them (McCown and Lloyd 1981 ). Generally, explants from trees and shrubs establish successfully in vitro with this culture medium (Chen et al. 2020 ; Song et al. 2021 ; Araujo et al. 2023 ). Therefore, it is not surprising that in our study, the establishment of H. chrysanthus was only achieved in WPM, and that for T. rosea , the survival rate in WPM was 86.25% compared to only 23.75% in MS medium. Based on these results, it is recommended to use WPM for the establishment and subsequent propagation of both species. The use of cytokinins in the culture medium is a common practice to promote shoot proliferation during the in vitro multiplication phase. In forest species, these growth regulators are generally essential to achieve this purpose. The type of cytokinin used and the required concentration can vary between species, as well as their combination with auxins. Adugna et al. ( 2020 ) achieved the maximum number of shoots per explant in Moringa stenopetala with 2.3 µM kinetin + 0.053 µM naphthalene acetic acid. De Oliveira et al. ( 2022 ) successfully promoted shoot regeneration in Eucalyptus cloeziana with 6-BAP 4.4 µM. In contrast, Kharel et al. ( 2022 ) needed to add between 17.6 and 35.2 µM of 6-BAP to successfully induce shoot formation in two cultivars of Vaccinium corymbosum L. Sözmen et al. ( 2024 ) achieved maximum results with 6-BAP 8.8 µM + 1.22 µM IBA in Lycium barbarum L. In our research, the best results in the in vitro multiplication of H. chrysanthus were achieved with the addition of 6-BAP 6.6 µM to the WPM culture medium, while in T. rosea , multiplication was successful without adding cytokinins to the medium. The explanation for this behavior could lie in: relatively high endogenous auxin concentrations in H. chrysanthus and low in T. rosea ; low endogenous cytokinin concentrations in H. chrysanthus and low in T. rosea , or a combination of both phenomena. In any case, the differentiation of shoots in vitro depends on the balance between the concentration of phytohormones existing in the plant tissues and the growth regulators applied in the culture medium, as noted by other authors (Isah and Umar 2018 ; Raspor et al. 2020 ). Root formation in woody species is typically enhanced by the use of auxins, particularly indole-3-butyric acid (IBA), which has been widely employed for this purpose (Vilasboa et al. 2018 ; Guo et al. 2019 ; Vielba et al. 2020 ). Once again, the balance between endogenous and exogenous concentrations of growth regulators plays a crucial role, though not fully understood (Zhao et al. 2022 ), and this could explain the different IBA concentrations that stimulated rooting in our research (2.45 µM for H. chrysanthus and 4.9 µM for T. rosea ). The successful acclimatization of the plants (96% in H. chrysanthus and 80% in T. rosea ) is a significant achievement that lends reliability to the set of procedures studied here, enabling their application in the micropropagation of these species. Conclusion Using in vitro culture techniques, a protocol has been established for the propagation of the forest species Handroanthus chrysanthus Jacq. S. O. Grose and Tabebuia rosea (Bertol.) Bertero ex A.DC. The procedures include surface disinfection and establishment of seeds, in vitro multiplication, and rooting. Growth regulator concentrations for these phases were determined. The vitroplants obtained were successfully acclimatized ex vitro , enabling the development of a methodology for the mass propagation of these endangered species. Declarations Data availability statement The raw data are available upon request from the corresponding author. Competing interests The authors have no relevant financial or non-financial interests to disclose. Authors Contribution EH : Conceptualization, methodology, data curation, review and editing; DC : Investigation, data curation, draft; LC : Conceptualization, methodology, review; FM : Methodology, investigation; OF : Formal analysis, review References Adugna AY, Feyissa T, Tasew FS (2020) Optimization of growth regulators on in vitro propagation of Moringa stenopetala from shoot explants. BMC Biotechnology 20: 60 https://doi.org/10.1186/s12896-020-00651-w Araujo AC, Medina FM, Flores PV, Rodriguez HL (2023). Micropropagation of Myrcianthes ferreyrae (McVaugh) “Arrayán”: An Endemic Species from Lomas De Atiquipa. In: Iano Y, Saotome O, Kemper Vásquez GL, Cotrim Pezzuto C, Arthur R, Gomes de Oliveira G (eds) Proceedings of the 7th Brazilian Technology Symposium (BTSym’21). BTSym 2021. Smart Innovation, Systems and Technologies, vol 207. Springer, Cham, pp 487-493 https://doi.org/10.1007/978-3-031-04435-9_50 Burley JT, Kellner JR, Hubbell SP, Faircloth BC (2021) Genome assemblies for two Neotropical trees: Jacaranda copaia and Handroanthus guayacan. G3: Genes, Genomes, Genetics 11(2) https://doi.org/10.1093/g3journal/jkab010 Chen S, Xion Y, Wu T, Wu K, Teixeira da Silva JA, Xiong Y, Zeng S, Ma G (2020) Axillary shoot proliferation and plant regeneration in Euryodendron excelsum H. T. Chang, a critically endangered species endemic to China. Sci Rep 10:14402 https://doi.org/10.1038/s41598-020-71360-9 Delgado LM, Hoyos RA (2016) Multiplicación clonal in vivo e in vitro de la especie forestal nativa Aniba perutilis Hemsl. Acta Agron 65(2):190-196 https://doi.org/10.15446/acag.v65n2.42808 De Oliveira LS, Brondani GE, Molinari LV, Dias RZ, Teixeira GL, Goncalves AN, De Almeida M (2022) Optimal cytokinin/auxin balance for indirect shoot organogenesis of Eucalyptus cloeziana and production of ex vitro rooted micro-cuttings. J For Res 33:1573-1584 https://doi.org/10.1007/s11676-022-01454-9 Díaz MI, Rodas JM, González LR, Vera M (2020) Establecimiento in vitro de segmentos nodales de Handroanthus heptaphyllus de flores blancas. Biot Veg 20(3): 203-210 El-Hawary SS, Taher MA, Amin E, AbouZid SF, Mohammed R (2021) Genus Tabebuia: A comprehensive review journey from past achievements to future perspectives. Arab J Chem 14 https://doi.org/10.1016/j.arabjc.2021.103046 Gammoudi N, Nagaz K, Ferchichi A (2022) Establishing of optimized in vitro disinfection protocol of Pistacia vera L. explants mediated a computational approach: multilayer perceptron-multi-objective genetic algorithm. BMC Plant Biol 22:324 https://doi.org/10.1186/s12870-022-03674-x Guo YX, Zhao YY, Zhang M, Zhang LY (2019). Development of a novel in vitro rooting culture system for the micropropagation of highbush blueberry ( Vaccinium corymbosum ) seedlings. PCTOC 139: 615-620 https://doi.org/10.1007/s11240-019-01702-7 Hazubska-Przybył T (2019) Propagation of Juniper Species by Plant Tissue Culture: A Mini-Review. Forests 10(11):1028 https://doi.org/10.3390/f10111028 Isah T, Umar S (2018) Influencing in vitro clonal propagation of Chonemorpha fragrans (moon) Alston by culture media strength, plant growth regulators, carbon source and photo periodic incubation. J For Res 31. https://doi.org/10.1007/s11676-018-0794-3 Kharel P, Creech MR, Nguyen CD, Vendrame WA, Munoz PR, Huo H (2022) Effect of explant type, culture medium, and BAP concentration on in vitro shoot development in highbush blueberry ( Vaccinium corymbosum L.) cultivars. In Vitro Cell Dev Biol-Plant 58:1057-1065 https://doi.org/10.1007/s11627-022-10299-0 McCown B, Lloyd G (1981) Woody Plant Medium (WPM)-A Mineral Nutrient Formulation for Microculture of Woody Plant Species. HortScience 16: 453 https://doi.org/10.21273/HORTSCI.16.3S.365 Morales JF, Sabás DC, Garcidueñas C, Pérez E (2019) Germination , in vitro propagation and soil acclimatization of Acacia farnesiana and Prosopis laevigata. South Afr J Bot 124:345-349 https://doi.org/10.1016/j.sajb.2019.05.034 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco cultures. Physiol Plant 15: 473-497 https://doi.org/10.1111/j.1399-3054.1962.tb08052.x Nallathambi P, Umamaheswari C, Lal SK, Manjunatha C, Berliner J (2020) Mechanism of Seed Transmission and Seed Infection in Major Agricultural Crops in India. In: Kumar R, Gupta A (eds) Seed-Borne Diseases of Agricultural Crops: Detection, Diagnosis & Management. Springer, Singapore https://doi.org/10.1007/978-981-32-9046-4_26 Olmstead RG, Zjhra ML, Lohmann LG, Grose SO, Eckert AJ (2009) A molecular phylogeny and classification of Bignoniaceae. Am J Botany 96:1731-1743 https://doi.org/10.3732/ajb.0900004 Raspor M, Motyka V, Ninković S, Dobrev PI, Malbeck J, Ćosić T, Cingel A, Savić J, Tadić V, Dragićević IČ (2020) Endogenous levels of cytokinins, indole-3-acetic acid and abscisic acid in in vitro grown potato: A contribution to potato hormonomics. Sci Rep 10:3437 https://doi.org/10.1038/s41598-020-60412-9 Rivas CA, Navarro-Cerillo RM, Johnston JC, Guerrero-Casado J (2020) Dry forest is more threatened but less protected than evergreen forest in Ecuador’s coastal region. Env Cons 1-5 https://doi.org/10.1017/s0376892920000077 Silveira SS, Cordeiro-Silva R, Degenhardt-Goldbach J, Quoirin M (2016) Micropropagation of Calophyllum brasiliense (Cambess.) from nodal segments. Braz J Biol 76(3): 656-663 https://doi.org/10.1590/1519-6984.23714 Song G, Chen Q, Callow P, Mandujano M, Han X, Cuenca B, Bonito G, Medina-Mora C, Fulbright DW, Guyer DE (2021) Efficient micropropagation of chestnut hybrids ( Castanea spp.) using modified Woody Plant Medium and zeatin riboside. Hortic Plant J 7(2):174-180 https://doi.org/10.1016/j.hpj.2020.09.006 Sözmen EU, Karaköy T, Aasim M (2024) Optimizing benzylaminopurine (BAP) and naphthalene acetic acid (NAA) concentration for in vitro micropropagation of Goji Berry ( Lycium barbarum L.) using various optimizing tools. Pak J Agri Sci 61(1):85-91 https://doi.org/10.21162/PAKJAS/24.95 Vielba JM, Vidal N, Ricci A, Castro R, Covelo P, San Jose, MC, Sanchez C (2020) Effects of auxin and urea derivatives on adventitious rooting in chestnut and oak microshoots. Isr J Plant Sci 671-2:52-68 https://dx.doi.org/10.1163/22238980-20191113 Vilasboa J, Da Costa CT, Fett-Neto AG (2018) Rooting of eucalypt cuttings as a problem-solving oriented model in plant biology. Prog Biophys Mol Biol 146:85-97 https://doi.org/10.1016/j.pbiomolbio.2018.12.007 Zhao Y, Chen Y, Jiang C, Lu MZ, Zhang J (2022) Exogenous hormone supplementation improve adventitious root formation in woody plants. Front Bioeng Biotechnol 10: 1009531 https://doi.org/10.3389/fbioe.2022.1009531 Cite Share Download PDF Status: Published Journal Publication published 19 Sep, 2024 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted Reviewers agreed at journal 28 Jul, 2024 Reviewers invited by journal 28 Jul, 2024 Editor assigned by journal 25 Jul, 2024 First submitted to journal 24 Jul, 2024 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-4783431","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":332662210,"identity":"83aea280-569f-499b-bcef-c3de123b4daa","order_by":0,"name":"Eduardo Hector","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAklEQVRIiWNgGAWjYFCCBCBmY2AwgPBsIBQPkVoYGxgY0iCqSdFymLAWfvYcw88VZTYM5uzNzx983HM+cb/YAcYHb9sY5PgbsGuR7HljLHnmXBqDZc8xw8YZz24n9kgnMBvObWMwljiAXYvBjRwDyca2wyAGYzPPAbAWNmneNobEDTgcZn8jx/gnWMv9NyAt50Ba2H8DtdTj0mIgkWMGtYUHpOUA2BZmoJYEAxxaJM48K7NsOJfGY3AmzXDmjAPJxj23E5sl55yTMJyBwy/87cmbbzaU2cgZHD/84MOHA3ay7bOTD354U2YjjyvEYAA5IkDxwyCBX/0oGAWjYBSMArwAAL9QWlcRdrRwAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-1371-7345","institution":"Universidad Técnica de Manabí: Universidad Tecnica de Manabi","correspondingAuthor":true,"prefix":"","firstName":"Eduardo","middleName":"","lastName":"Hector","suffix":""},{"id":332662211,"identity":"9b9f9d56-0354-4f1b-ba77-f92a90f30b3d","order_by":1,"name":"Damaris Cevallos","email":"","orcid":"","institution":"Universidad Tecnica de Manabi","correspondingAuthor":false,"prefix":"","firstName":"Damaris","middleName":"","lastName":"Cevallos","suffix":""},{"id":332662212,"identity":"3a53c8a8-3117-4877-8b30-2adf31d082ea","order_by":2,"name":"Liliana Corozo","email":"","orcid":"","institution":"Universidad Tecnica de Manabi","correspondingAuthor":false,"prefix":"","firstName":"Liliana","middleName":"","lastName":"Corozo","suffix":""},{"id":332662213,"identity":"09aaf723-b485-4a90-8226-cbf8614ba8ab","order_by":3,"name":"Fatima Macías","email":"","orcid":"","institution":"Universidad Tecnica de Manabi","correspondingAuthor":false,"prefix":"","firstName":"Fatima","middleName":"","lastName":"Macías","suffix":""},{"id":332662214,"identity":"c54485ff-8747-4864-808b-9f346c9612df","order_by":4,"name":"Osvaldo Fosado","email":"","orcid":"","institution":"Universidad Tecnica de Manabi","correspondingAuthor":false,"prefix":"","firstName":"Osvaldo","middleName":"","lastName":"Fosado","suffix":""}],"badges":[],"createdAt":"2024-07-22 16:50:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4783431/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4783431/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11240-024-02864-9","type":"published","date":"2024-09-19T15:57:33+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63100115,"identity":"9a7d4f4a-ab23-4965-bd00-91ed53407e17","added_by":"auto","created_at":"2024-08-23 06:48:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":641118,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of growth regulators on the development of vitroplantlets. \u003cstrong\u003eA:\u003c/strong\u003e \u003cem\u003eHandroanthus chrysanthus\u003c/em\u003e (Jacq.) S.O. Grose \u003cstrong\u003eB:\u003c/strong\u003e \u003cem\u003eTabebuia rosea \u003c/em\u003e(Bertol.) \u0026nbsp;\u0026nbsp;Bertero ex A.DC \u003cstrong\u003eT1:\u003c/strong\u003e control without growth \u0026nbsp;\u0026nbsp;regulators \u003cstrong\u003eT2:\u003c/strong\u003e 6-BAP 4.4 μM \u003cstrong\u003eT3:\u003c/strong\u003e 6-BAP 5.5 μM\u003cstrong\u003e T4:\u003c/strong\u003e 6-BAP 6.6 μM\u003cstrong\u003e T5:\u003c/strong\u003e KIN 4.6 μM \u003cstrong\u003eT6:\u003c/strong\u003e KIN 5.75 μM \u003cstrong\u003eT7:\u003c/strong\u003e KIN 6.9 μM\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4783431/v1/a592d1cd4288f2d8272d28c8.png"},{"id":63099638,"identity":"90427336-06d2-4a8d-af7e-ed3686c569d4","added_by":"auto","created_at":"2024-08-23 06:40:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1051901,"visible":true,"origin":"","legend":"\u003cp\u003eAcclimatization of plantlets \u003cstrong\u003ea.\u003c/strong\u003e Eight-week-old plantlets taken from the laboratory \u003cstrong\u003eb.\u003c/strong\u003e Plantlets transplanted to trays \u003cstrong\u003ec.\u003c/strong\u003eMicrochambers \u003cstrong\u003ed.\u003c/strong\u003e Plantlets after 15 days of acclimatization \u003cstrong\u003ee.\u003c/strong\u003e Plantlets after 60 days of acclimatization \u003cstrong\u003ef.\u003c/strong\u003e Plantlets transferred to polyethylene bags\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4783431/v1/0b681a5b6dfef0fac9542631.png"},{"id":65104212,"identity":"c779b3a2-1299-4337-aa41-28795ba2ad2e","added_by":"auto","created_at":"2024-09-23 16:12:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2768720,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4783431/v1/bb4083d3-e3cf-4443-ae18-41ef5c02f162.pdf"}],"financialInterests":"","formattedTitle":"Development of a protocol for the micropropagation of two forest species threatened with extinction in Ecuador","fulltext":[{"header":"Key Message","content":"\u003cp\u003eIn this research, the culture medium requirements and conditions for the micropropagation of \u003cem\u003eH. chrysanthus\u003c/em\u003e and \u003cem\u003eT. rosea\u003c/em\u003e were fine-tuned. This technique can be implemented to obtain plants for use in reforestation.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe genera \u003cem\u003eHandroanthus\u003c/em\u003e and \u003cem\u003eTabebuia\u003c/em\u003e belong to the family Bignoniaceae (Burley et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; El-Hawary et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which includes more than 800 species (Olmstead et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Ecuador is a megadiverse country, with around 750 forest species. In the coastal region of Ecuador, deciduous dry forests are threatened by overexploitation, exacerbated by the fact that there are few protected areas compared to green forests, which have received more attention (Rivas et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMicropropagation through \u003cem\u003ein vitro\u003c/em\u003e tissue culture is a biotechnological approach that allows the exponential increase in the number of plants to perpetuate individuals of particular value in a specific area, serving as a powerful tool for reforestation (Hazubska-Przybył \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Adugna et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Morales et al. 2020).\u003c/p\u003e \u003cp\u003e \u003cem\u003eHandroanthus chrysanthus\u003c/em\u003e Jacq. S. O. Grose and \u003cem\u003eTabebuia rosea\u003c/em\u003e (Bertol.) Bertero ex A.DC are two forest species that grow in the dry forests of the Ecuadorian coast. Due to their high timber value, they have been subjected to indiscriminate logging, putting them at risk of extinction. Based on these circumstances, this research was developed with the aim of fine-tuning protocols for the micropropagation process of both species.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eThe research was conducted at the Biotechnology Laboratory of the Faculty of Agronomic Engineering at the Technical University of Manab\u0026iacute;, in Santa Ana, Manab\u0026iacute;, Ecuador.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSeed selection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe seeds of the two species were obtained from fully mature fruits from well-developed and vigorous trees located in the University Botanical Garden of the Technical University of Manab\u0026iacute;, Ecuador.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurface sterilization and \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eseed establishment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe seeds were washed with water and liquid soap; then they were transferred to the laminar flow cabinet, rinsed with sterile distilled water, and treated with sodium hypochlorite (NaClO) at 0.5% or 1% for two disinfection times (3 or 5 minutes). Finally, they were rinsed three times with sterile distilled water to remove excess disinfectant.\u003c/p\u003e\n\u003cp\u003eThe disinfected seeds were placed in test tubes containing WPM (McCown and Lloyd 1981) or MS (Murashige and Skoog 1962) culture medium, supplemented with 3% sucrose; 0.7% agar and 10 ppm cysteine were added. The pH of the media was adjusted to 5.7. The seeds were maintained at 24 \u0026plusmn; 2 \u0026deg;C with a photoperiod of 8 hours of darkness and 16 hours of light. Each species underwent 8 treatments, consisting of combinations of culture medium, disinfectant concentration, and disinfection time. Each treatment comprised 50 repetitions (seeds), following a factorial design of 2x2x2 for each species. After 10 days, survival percentages were evaluated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003emultiplication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the multiplication of the species, apical buds from plants obtained from seeds established \u003cem\u003ein vitro\u003c/em\u003e were used. In the laminar flow chamber, apices measuring 1 cm in length were cut according to the methodology of Delgado and Hoyos (2016). The apices were placed in culture tubes containing 10 ml of WPM medium supplemented with 3% sucrose, 0.7% agar, and adjusted to pH 5.7. Each species underwent 7 treatments, consisting of two growth regulators at three concentrations (6-benzylaminopurine 4.4, 5.5, 6.6 \u0026mu;M or kinetin 4.6, 5.75, 6.9 \u0026mu;M) and a control without growth regulators, in a factorial design (2x3+1) with 20 repetitions per treatment. The apices were maintained at 24 \u0026plusmn; 2 \u0026deg;C with a photoperiod of 8 hours of darkness and 16 hours of light. The study was conducted during the first and second in vitro multiplication. After 8 weeks in each multiplication, the number of shoots produced per apex was counted, and their length and diameter were measured.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003erooting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe plants obtained from each species during the multiplication stage were then subjected to rooting in WPM medium supplemented with 3% sucrose, 0.7% agar, and adjusted to pH 5.7. Three treatments were tested (control without growth regulators, indole-3-butyric acid 2.45 \u0026mu;M, \u0026iacute;ndole-3-butyric acid 4.9 \u0026mu;M). The plants were maintained at 24 \u0026plusmn; 2 \u0026deg;C with a photoperiod of 8 hours of darkness and 16 hours of light. Thirty plants were used per treatment in a completely randomized design. After four weeks, the number of roots was counted, and their length was measured.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcclimatization of plants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe vitroplants obtained after 8 weeks in rooting medium (100 per species) were established in a mixture of 40% humus, 40% river sand, and 20% decomposed wood sawdust, previously sterilized by steam at 121\u0026deg;C for one hour. Plastic pots were used to protect the plants in micro-chambers for acclimatization. Irrigation was applied twice daily with a sprinkler for 15 days; thereafter, watering was reduced to once daily for another 15 days, and subsequently to three times per week. The survival rate of the plants was assessed 4 weeks after transplantation. At 60 days, the adapted plants were transferred to polyethylene sleeves for final hardening and eventual transplantation to the field.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNormality tests (Shapiro-Wilk) and homogeneity of variances tests (Levene) were conducted on the variable data. Once the statistical assumptions were confirmed, analysis of variance (ANOVA) were performed, and means were compared using Tukey\u0026apos;s test (p \u0026le; 0.05). When the evaluated variables were expressed as percentages, the data were transformed prior to analysis to [2 arcsen(%)].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSurface sterilization and \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eseed establishment\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results obtained from surface sterilization and \u003cem\u003ein vitro\u003c/em\u003e establishment of both species are shown in Table 1. In the Murashige and Skoog (MS) medium, seeds of \u003cem\u003eH. chrysanthus\u003c/em\u003e did not thrive, thus only results from Woody Plant Medium (WPM) could be processed. In this species, no interaction effect between NaClO concentration and disinfection time was detected; however, there was an effect observed from each factor separately. The highest survival percentages (92.50%) were achieved with NaClO 1% and disinfection during 5 minutes.\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003eT. rosea\u003c/em\u003e, differences were observed among the culture media used rather than among the concentrations of NaClO or disinfection times. In the MS medium, only 23.75% of the seeds survived, whereas in WPM, seed survival reached 86.25%.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Influence of NaClO concentration, disinfection time, and culture medium on the survival percentage (%) in the establishment of \u003cem\u003eHandroanthus chrysanthus\u003c/em\u003e (Jacq.) S.O. Grose and \u003cem\u003eTabebuia rosea\u003c/em\u003e (Bertol.) Bertero ex A.DC\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eHandroanthus chrysanthus\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"60%\"\u003e\n \u003cp\u003e\u003cstrong\u003eNaClO concentration (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSurvival percentage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"60%\"\u003e\n \u003cp\u003e0,5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40%\"\u003e\n \u003cp\u003e80.00 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"60%\"\u003e\n \u003cp\u003e1,0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40%\"\u003e\n \u003cp\u003e\u0026nbsp;92.50 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"60%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDisinfection time (min)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSurvival percentage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"60%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40%\"\u003e\n \u003cp\u003e80.00 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"60%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40%\"\u003e\n \u003cp\u003e\u0026nbsp;92.50 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eTabebuia rosea\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"60%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCulture medium\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSurvival percentage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"60%\"\u003e\n \u003cp\u003eMS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40%\"\u003e\n \u003cp\u003e23.75 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"60%\"\u003e\n \u003cp\u003eWPM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40%\"\u003e\n \u003cp\u003e86.25 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eDifferent letters indicate significant differences for Tukey\u0026apos;s test (p\u0026nbsp;\u0026le;\u0026nbsp;0.05)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003emultiplication\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the first multiplication of \u003cem\u003eH. chrysanthus\u003c/em\u003e from shoots obtained in WPM establishment, treatment 4 (6-BAP 6.6 \u0026mu;M) resulted in the highest values for both shoot quantity and length, significantly differing from the others. Regarding shoot diameter, four treatments, including 6-BAP 6.6 \u0026mu;M, were significantly superior to the rest. Conversely, for \u003cem\u003eT. rosea\u003c/em\u003e, the control without growth regulators was among the treatments that significantly outperformed the others across all three measured variables (Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Effect of growth regulators 6-benzylaminopurine (BAP) and kinetin (KIN) on the number, length, and diameter of shoots of \u003cem\u003eHandroanthus chrysanthus\u003c/em\u003e (Jacq.) S.O. Grose and \u003cem\u003eTabebuia rosea\u0026nbsp;\u003c/em\u003e(Bertol.) Bertero ex A.DC (first \u003cem\u003ein vitro\u003c/em\u003e multiplication)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"567\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.75485008818342%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.98236331569665%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of shoots\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.631393298059965%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eLength of shoots\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.631393298059965%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eDiameter of shoots\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.695957820738137%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatments\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.520210896309315%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eH. chrysanthus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eT. rosea\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eH. chrysanthus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eT. rosea\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eH. chrysanthus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eT. rosea\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.695957820738137%\"\u003e\n \u003cp\u003e1) Control\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.520210896309315%\"\u003e\n \u003cp\u003e1.95\u0026plusmn;0.15 \u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.90\u0026plusmn;0.19 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e1.84\u0026plusmn;0.07 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.80\u0026plusmn;0.08 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.65\u0026plusmn;0.20 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.95\u0026plusmn;0.11 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.695957820738137%\"\u003e\n \u003cp\u003e2) 6-BAP 4.4 \u0026mu;M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.520210896309315%\"\u003e\n \u003cp\u003e2.45\u0026plusmn;0.11 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.20\u0026plusmn;0.17 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.17\u0026plusmn;0.06 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.42\u0026plusmn;0.10 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e3.20\u0026plusmn;0.16 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.65\u0026plusmn;0.11 \u003csup\u003eabcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.695957820738137%\"\u003e\n \u003cp\u003e3) 6-BAP 5.5 \u0026mu;M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.520210896309315%\"\u003e\n \u003cp\u003e2.60\u0026plusmn;0.15 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.40\u0026plusmn;0.13 \u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e1.97\u0026plusmn;0.05 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.48\u0026plusmn;0.08 \u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e3.00\u0026plusmn;0.14 \u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.80\u0026plusmn;0.09 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.695957820738137%\"\u003e\n \u003cp\u003e4) 6-BAP 6.6 \u0026mu;M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.520210896309315%\"\u003e\n \u003cp\u003e3.35\u0026plusmn;0.15 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.60\u0026plusmn;0.11 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.50\u0026plusmn;0.08 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.57\u0026plusmn;0.07 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e3.35\u0026plusmn;0.11 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.75\u0026plusmn;0.10 \u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.695957820738137%\"\u003e\n \u003cp\u003e5)\u0026nbsp;KIN 4.6 \u0026mu;M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.520210896309315%\"\u003e\n \u003cp\u003e1.60\u0026plusmn;0.13 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e1.80\u0026plusmn;0.12 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e1.87\u0026plusmn;0.05 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.17\u0026plusmn;0.097 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.55\u0026plusmn;0.114 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.35\u0026plusmn;0.109 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.695957820738137%\"\u003e\n \u003cp\u003e6)\u0026nbsp;KIN 5.75 \u0026mu;M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.520210896309315%\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;2.25\u0026plusmn;0.0993 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.25\u0026plusmn;0.099 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.00\u0026plusmn;0.03 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.44\u0026plusmn;0,09 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.60\u0026plusmn;0.11 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.45\u0026plusmn;0.11 \u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.695957820738137%\"\u003e\n \u003cp\u003e7)\u0026nbsp;KIN 6.9 \u0026mu;M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.520210896309315%\"\u003e\n \u003cp\u003e1.95\u0026plusmn;0.11 \u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.30\u0026plusmn;0.16 \u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.00\u0026plusmn;0.04 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.45\u0026plusmn;0.07 \u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.90\u0026plusmn;0.12 \u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.35676625659051%\"\u003e\n \u003cp\u003e2.60\u0026plusmn;0.11 \u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eDifferent letters in the same column indicate significant differences for Tukey\u0026apos;s test (p\u0026nbsp;\u0026le;\u0026nbsp;0.05)\u003c/p\u003e\n\u003cp\u003eThe second multiplication corroborated the results obtained in the first. In \u003cem\u003eH. chrysanthus\u003c/em\u003e, the treatment with 6-BAP 6.6 \u0026mu;M consistently produced significantly higher results across all three measured variables compared to the other treatments. For \u003cem\u003eT. rosea\u003c/em\u003e, the control without growth regulators showed overall superiority in shoot quantity and length, with no significant differences found among treatments for shoot diameter (Table 3).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u003c/strong\u003e Effect of growth regulators 6-benzylaminopurine (BAP) and kinetin (KIN) on the number, length, and diameter of shoots of \u003cem\u003eHandroanthus chrysanthus\u003c/em\u003e (Jacq.) S.O. Grose and \u003cem\u003eTabebuia rosea\u0026nbsp;\u003c/em\u003e(Bertol.) Bertero ex A.DC (second \u003cem\u003ein vitro\u003c/em\u003e multiplication)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"557\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.63799283154122%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.060931899641577%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of shoots\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.85304659498208%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLength of shoots\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.448028673835125%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDiameter of shoots\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.63799283154122%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatments\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.440860215053764%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eH. chrysanthus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eT. rosea\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.232974910394265%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eH.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;chrysanthus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eT. rosea\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eH. chrysanthus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.827956989247312%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eT. rosea\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.63799283154122%\"\u003e\n \u003cp\u003e1) Control\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.440860215053764%\"\u003e\n \u003cp\u003e2.55\u0026plusmn;0.11 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e3.20\u0026plusmn;0.09 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.232974910394265%\"\u003e\n \u003cp\u003e2.08\u0026plusmn;0.08 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e3.41\u0026plusmn;0.12 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e3.00\u0026plusmn;0.13 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.827956989247312%\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.63799283154122%\"\u003e\n \u003cp\u003e2) 6-BAP 4.4 \u0026mu;M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.440860215053764%\"\u003e\n \u003cp\u003e2.55\u0026plusmn;0.13 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e2.30\u0026plusmn;0.10 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.232974910394265%\"\u003e\n \u003cp\u003e2.40\u0026plusmn;0.20 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e2.66\u0026plusmn;0.08 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e3.05\u0026plusmn;0.11 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.827956989247312%\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.63799283154122%\"\u003e\n \u003cp\u003e3) 6-BAP 5.5 \u0026mu;M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.440860215053764%\"\u003e\n \u003cp\u003e2.70\u0026plusmn;0,15 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e2.35\u0026plusmn;0.11 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.232974910394265%\"\u003e\n \u003cp\u003e2.07\u0026plusmn;0.85 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e2.70\u0026plusmn;0.09 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e2.75\u0026plusmn;0.12 \u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.827956989247312%\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.63799283154122%\"\u003e\n \u003cp\u003e4) 6-BAP 6.6 \u0026mu;M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.440860215053764%\"\u003e\n \u003cp\u003e3.60\u0026plusmn;0.19 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e2.40\u0026plusmn;0.11 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.232974910394265%\"\u003e\n \u003cp\u003e2.80\u0026plusmn;0.10 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e2.40\u0026plusmn;0.08 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e3.45\u0026plusmn;0.11 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.827956989247312%\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.63799283154122%\"\u003e\n \u003cp\u003e5)\u0026nbsp;KIN 4.6 \u0026mu;M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.440860215053764%\"\u003e\n \u003cp\u003e2.10\u0026plusmn;0.11 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e2.15\u0026plusmn;0.08 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.232974910394265%\"\u003e\n \u003cp\u003e1.86\u0026plusmn;0.04 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e2.34\u0026plusmn;0.05 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e2.60\u0026plusmn;0.11 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.827956989247312%\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.63799283154122%\"\u003e\n \u003cp\u003e6)\u0026nbsp;KIN 5.75 \u0026mu;M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.440860215053764%\"\u003e\n \u003cp\u003e2.25\u0026plusmn;0.01 \u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e2.25\u0026plusmn;0.01 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.232974910394265%\"\u003e\n \u003cp\u003e2.11\u0026plusmn;0.09 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e2.37\u0026plusmn;0.07 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e2.70\u0026plusmn;0.10 \u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.827956989247312%\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.63799283154122%\"\u003e\n \u003cp\u003e7)\u0026nbsp;KIN 6.9 \u0026mu;M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.440860215053764%\"\u003e\n \u003cp\u003e2.70\u0026plusmn;0.13 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e2.30\u0026plusmn;0.10 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.232974910394265%\"\u003e\n \u003cp\u003e2.07\u0026plusmn;0.07 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e2.47\u0026plusmn;0.06\u003csup\u003e\u0026nbsp;bc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.620071684587813%\"\u003e\n \u003cp\u003e2.65\u0026plusmn;0.13 \u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.827956989247312%\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eDifferent letters in the same column indicate significant differences for Tukey\u0026apos;s test (p\u0026nbsp;\u0026le;\u0026nbsp;0.05). ns: no significant differences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;rooting\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBoth species responded positively to the inclusion of IBA in the culture medium. In \u003cem\u003eH. chrysanthus\u003c/em\u003e, the addition of 2.45 \u0026mu;M IBA resulted in the highest values for both root number and length. Conversely, for \u003cem\u003eT. rosea\u003c/em\u003e, the concentration of 4.9 \u0026mu;M produced the highest values (Table 4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.\u003c/strong\u003e Effect of indole-3-butyric acid (IBA) on the \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003erooting of\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eHandroanthus chrysanthus\u003c/em\u003e (Jacq.) S.O. Grose and \u003cem\u003eTabebuia rosea\u0026nbsp;\u003c/em\u003e(Bertol.) Bertero ex A.DC\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.215686274509803%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.3921568627451%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of roots\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.3921568627451%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eLength of roots\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.215686274509803%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatments\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.764705882352942%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eH. chrysanthus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.627450980392158%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eT. rosea\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.764705882352942%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eH. chrysanthus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.627450980392158%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eT. rosea\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.215686274509803%\"\u003e\n \u003cp\u003e1) Control\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.764705882352942%\"\u003e\n \u003cp\u003e2.50\u0026plusmn;0.09 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.627450980392158%\"\u003e\n \u003cp\u003e2.43\u0026plusmn;0.12 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.764705882352942%\"\u003e\n \u003cp\u003e1.94\u0026plusmn;0.03 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.627450980392158%\"\u003e\n \u003cp\u003e1.66\u0026plusmn;0.03 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.215686274509803%\"\u003e\n \u003cp\u003e2)\u0026nbsp;IBA 2.45 \u0026mu;M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.764705882352942%\"\u003e\n \u003cp\u003e5.17\u0026plusmn;0.13 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.627450980392158%\"\u003e\n \u003cp\u003e4.93\u0026plusmn;0.15 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.764705882352942%\"\u003e\n \u003cp\u003e2.73\u0026plusmn;0.04 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.627450980392158%\"\u003e\n \u003cp\u003e2.57\u0026plusmn;0.04 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.215686274509803%\"\u003e\n \u003cp\u003e3)\u0026nbsp;IBA 4.9 \u0026mu;M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.764705882352942%\"\u003e\n \u003cp\u003e1.70\u0026plusmn;0.09 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.627450980392158%\"\u003e\n \u003cp\u003e8.20\u0026plusmn;0.15 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.764705882352942%\"\u003e\n \u003cp\u003e1.40\u0026plusmn;0.02 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.627450980392158%\"\u003e\n \u003cp\u003e3.50\u0026plusmn;0.04 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eDifferent letters in the same column indicate significant differences for Tukey\u0026apos;s test (p \u0026le; 0.05)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcclimatization of plants\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOut of the total of 100 plants evaluated in \u003cem\u003eH. chrysanthus\u003c/em\u003e, 96 successfully survived, indicating a very high survival rate in this species (96%). In \u003cem\u003eT. rosea\u003c/em\u003e, 80 out of 100 vitroplants successfully adapted, resulting in a survival rate of 80%, which is considered high for a woody species. Fig 2 depicts the growth and development of the plants during the acclimatization stage.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe establishment of explants is a crucial step in any \u003cem\u003ein vitro\u003c/em\u003e culture protocol, influenced by the culture medium, disinfectant, and its concentration. The optimal combination of these factors aims to ensure that explants adapt to the culture medium with minimal contamination and minimal damage caused by the disinfectant. In woody plants, the exposure of explant materials to the environment typically results in high contamination rates, which is why various disinfectants and concentrations are tested, sometimes proving aggressive.\u003c/p\u003e \u003cp\u003eIn the establishment of nodal segments of \u003cem\u003eCalophyllum brasiliense\u003c/em\u003e, Silveira et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) were compelled to use HgCl\u003csub\u003e2\u003c/sub\u003e in addition to 5% NaClO for disinfection. To achieve a 55% success rate in disinfecting nodal segments of \u003cem\u003eHandroanthus heptaphyllus\u003c/em\u003e, D\u0026iacute;az et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) required 5% NaClO for 10 minutes. In the establishment of \u003cem\u003eMyrcianthes ferreyrae\u003c/em\u003e, Araujo et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) employed more aggressive disinfectants and concentrations, such as 0.1% HgCl\u003csub\u003e2\u003c/sub\u003e and 15% NaClO for 10 and 15 minutes, respectively. Conversely, Kaviani et al. (2022) achieved successful disinfection of axillary buds of \u003cem\u003ePiracantha angustifolia\u003c/em\u003e with 2% NaClO. In \u003cem\u003ePistacia vera\u003c/em\u003e, Gammoudi et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) observed that the effectiveness of disinfection with NaClO (0.54\u0026ndash;1.26%) is lower than that of HgCl\u003csub\u003e2\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, but its toxicity to plant tissues is also much lower.\u003c/p\u003e \u003cp\u003ePossibly due to the use of seeds rather than nodal segments to initiate the culture, our experiments achieved high survival rates with established and contamination-free seeds for both species using low concentrations of NaClO. Seeds typically have physical and chemical barriers that make them less penetrable by microorganisms, thereby resisting internal infection (Nallathambi et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), whereas axillary bud explants and nodal segments are more exposed. Based on the results observed in this study, the seeds of \u003cem\u003eH. chrysanthus\u003c/em\u003e should be disinfected with 1% NaClO for 5 minutes, whereas 0.5% NaClO for 3 minutes is sufficient for \u003cem\u003eT. rosea\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe recalcitrance of numerous woody plant species to adapt to \u003cem\u003ein vitro\u003c/em\u003e culture is a barrier to their propagation, leading to the development of the Woody Plant Medium (WPM) specifically tailored for them (McCown and Lloyd \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). Generally, explants from trees and shrubs establish successfully \u003cem\u003ein vitro\u003c/em\u003e with this culture medium (Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Song et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Araujo et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, it is not surprising that in our study, the establishment of \u003cem\u003eH. chrysanthus\u003c/em\u003e was only achieved in WPM, and that for \u003cem\u003eT. rosea\u003c/em\u003e, the survival rate in WPM was 86.25% compared to only 23.75% in MS medium. Based on these results, it is recommended to use WPM for the establishment and subsequent propagation of both species.\u003c/p\u003e \u003cp\u003eThe use of cytokinins in the culture medium is a common practice to promote shoot proliferation during the \u003cem\u003ein vitro\u003c/em\u003e multiplication phase. In forest species, these growth regulators are generally essential to achieve this purpose. The type of cytokinin used and the required concentration can vary between species, as well as their combination with auxins. Adugna et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) achieved the maximum number of shoots per explant in \u003cem\u003eMoringa stenopetala\u003c/em\u003e with 2.3 \u0026micro;M kinetin\u0026thinsp;+\u0026thinsp;0.053 \u0026micro;M naphthalene acetic acid. De Oliveira et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) successfully promoted shoot regeneration in \u003cem\u003eEucalyptus cloeziana\u003c/em\u003e with 6-BAP 4.4 \u0026micro;M. In contrast, Kharel et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) needed to add between 17.6 and 35.2 \u0026micro;M of 6-BAP to successfully induce shoot formation in two cultivars of \u003cem\u003eVaccinium corymbosum\u003c/em\u003e L. S\u0026ouml;zmen et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) achieved maximum results with 6-BAP 8.8 \u0026micro;M\u0026thinsp;+\u0026thinsp;1.22 \u0026micro;M IBA in \u003cem\u003eLycium barbarum\u003c/em\u003e L.\u003c/p\u003e \u003cp\u003eIn our research, the best results in the \u003cem\u003ein vitro\u003c/em\u003e multiplication of \u003cem\u003eH. chrysanthus\u003c/em\u003e were achieved with the addition of 6-BAP 6.6 \u0026micro;M to the WPM culture medium, while in \u003cem\u003eT. rosea\u003c/em\u003e, multiplication was successful without adding cytokinins to the medium. The explanation for this behavior could lie in: relatively high endogenous auxin concentrations in \u003cem\u003eH. chrysanthus\u003c/em\u003e and low in \u003cem\u003eT. rosea\u003c/em\u003e; low endogenous cytokinin concentrations in \u003cem\u003eH. chrysanthus\u003c/em\u003e and low in \u003cem\u003eT. rosea\u003c/em\u003e, or a combination of both phenomena. In any case, the differentiation of shoots \u003cem\u003ein vitro\u003c/em\u003e depends on the balance between the concentration of phytohormones existing in the plant tissues and the growth regulators applied in the culture medium, as noted by other authors (Isah and Umar \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Raspor et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRoot formation in woody species is typically enhanced by the use of auxins, particularly indole-3-butyric acid (IBA), which has been widely employed for this purpose (Vilasboa et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Guo et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Vielba et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Once again, the balance between endogenous and exogenous concentrations of growth regulators plays a crucial role, though not fully understood (Zhao et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and this could explain the different IBA concentrations that stimulated rooting in our research (2.45 \u0026micro;M for \u003cem\u003eH. chrysanthus\u003c/em\u003e and 4.9 \u0026micro;M for \u003cem\u003eT. rosea\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eThe successful acclimatization of the plants (96% in \u003cem\u003eH. chrysanthus\u003c/em\u003e and 80% in \u003cem\u003eT. rosea\u003c/em\u003e) is a significant achievement that lends reliability to the set of procedures studied here, enabling their application in the micropropagation of these species.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eUsing \u003cem\u003ein vitro\u003c/em\u003e culture techniques, a protocol has been established for the propagation of the forest species \u003cem\u003eHandroanthus chrysanthus\u003c/em\u003e Jacq. S. O. Grose and \u003cem\u003eTabebuia rosea\u003c/em\u003e (Bertol.) Bertero ex A.DC. The procedures include surface disinfection and establishment of seeds, \u003cem\u003ein vitro\u003c/em\u003e multiplication, and rooting. Growth regulator concentrations for these phases were determined. The vitroplants obtained were successfully acclimatized \u003cem\u003eex vitro\u003c/em\u003e, enabling the development of a methodology for the mass propagation of these endangered species.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data are available upon request from the corresponding author.\u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthors Contribution\u003c/h2\u003e \u003cp\u003e \u003cb\u003eEH\u003c/b\u003e: Conceptualization, methodology, data curation, review and editing; \u003cb\u003eDC\u003c/b\u003e: Investigation, data curation, draft; \u003cb\u003eLC\u003c/b\u003e: Conceptualization, methodology, review; \u003cb\u003eFM\u003c/b\u003e: Methodology, investigation; \u003cb\u003eOF\u003c/b\u003e: Formal analysis, review\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdugna AY, Feyissa T, Tasew FS (2020) Optimization of growth regulators on \u003cem\u003ein vitro\u003c/em\u003e propagation of \u003cem\u003eMoringa stenopetala\u003c/em\u003e from shoot explants. BMC Biotechnology 20: 60 https://doi.org/10.1186/s12896-020-00651-w \u003c/li\u003e\n\u003cli\u003eAraujo AC, Medina FM, Flores PV, Rodriguez HL (2023). Micropropagation of \u003cem\u003eMyrcianthes ferreyrae\u003c/em\u003e (McVaugh) \u0026ldquo;Array\u0026aacute;n\u0026rdquo;: An Endemic Species from Lomas De Atiquipa. In: Iano Y, Saotome O, Kemper V\u0026aacute;squez GL, Cotrim Pezzuto C, Arthur R, Gomes de Oliveira G (eds) Proceedings of the 7th Brazilian Technology Symposium (BTSym\u0026rsquo;21). BTSym 2021. Smart Innovation, Systems and Technologies, vol 207. Springer, Cham, pp 487-493 https://doi.org/10.1007/978-3-031-04435-9_50 \u003c/li\u003e\n\u003cli\u003eBurley JT, Kellner JR, Hubbell SP, Faircloth BC (2021) Genome assemblies for two Neotropical trees: \u003cem\u003eJacaranda copaia\u003c/em\u003e and \u003cem\u003eHandroanthus guayacan. \u003c/em\u003eG3: Genes, Genomes, Genetics 11(2) https://doi.org/10.1093/g3journal/jkab010 \u003c/li\u003e\n\u003cli\u003eChen S, Xion Y, Wu T, Wu K, Teixeira da Silva JA, Xiong Y, Zeng S, Ma G (2020) Axillary shoot proliferation and plant regeneration in \u003cem\u003eEuryodendron excelsum\u003c/em\u003e H. T. Chang, a critically endangered species endemic to China. Sci Rep 10:14402 https://doi.org/10.1038/s41598-020-71360-9\u003c/li\u003e\n\u003cli\u003eDelgado LM, Hoyos RA (2016) Multiplicaci\u0026oacute;n clonal in vivo e in vitro de la especie forestal nativa \u003cem\u003eAniba perutilis\u003c/em\u003e Hemsl. Acta Agron 65(2):190-196 https://doi.org/10.15446/acag.v65n2.42808\u003c/li\u003e\n\u003cli\u003eDe Oliveira LS, Brondani GE, Molinari LV, Dias RZ, Teixeira GL, Goncalves AN, De Almeida M (2022) Optimal cytokinin/auxin balance for indirect shoot organogenesis of \u003cem\u003eEucalyptus cloeziana\u003c/em\u003e and production of \u003cem\u003eex vitro\u003c/em\u003e rooted micro-cuttings. \u003cem\u003eJ For Res\u003c/em\u003e 33:1573-1584 https://doi.org/10.1007/s11676-022-01454-9\u003c/li\u003e\n\u003cli\u003eD\u0026iacute;az MI, Rodas JM, Gonz\u0026aacute;lez LR, Vera M (2020) Establecimiento \u003cem\u003ein vitro\u003c/em\u003e de segmentos nodales de \u003cem\u003eHandroanthus heptaphyllus\u003c/em\u003e de flores blancas. Biot Veg 20(3): 203-210\u003c/li\u003e\n\u003cli\u003eEl-Hawary SS, Taher MA, Amin E, AbouZid SF, Mohammed R (2021) Genus Tabebuia: A comprehensive review journey from past achievements to future perspectives. Arab J Chem 14 https://doi.org/10.1016/j.arabjc.2021.103046 \u003c/li\u003e\n\u003cli\u003eGammoudi N, Nagaz K, Ferchichi A (2022) Establishing of optimized \u003cem\u003ein vitro \u003c/em\u003edisinfection protocol of Pistacia vera L. explants mediated a computational approach: multilayer perceptron-multi-objective genetic algorithm. BMC Plant Biol 22:324 https://doi.org/10.1186/s12870-022-03674-x\u003c/li\u003e\n\u003cli\u003eGuo YX, Zhao YY, Zhang M, Zhang LY (2019). Development of a novel in vitro rooting culture system for the micropropagation of highbush blueberry (\u003cem\u003eVaccinium corymbosum\u003c/em\u003e) seedlings. PCTOC 139: 615-620 https://doi.org/10.1007/s11240-019-01702-7\u003c/li\u003e\n\u003cli\u003eHazubska-Przybył T (2019) Propagation of Juniper Species by Plant Tissue Culture: A Mini-Review. Forests 10(11):1028 https://doi.org/10.3390/f10111028\u003c/li\u003e\n\u003cli\u003eIsah T, Umar S (2018) Influencing in vitro clonal propagation of \u003cem\u003eChonemorpha fragrans\u003c/em\u003e (moon) Alston by culture media strength, plant growth regulators, carbon source and photo periodic incubation. J For Res 31. https://doi.org/10.1007/s11676-018-0794-3\u003c/li\u003e\n\u003cli\u003eKharel P, Creech MR, Nguyen CD, Vendrame WA, Munoz PR, Huo H (2022) Effect of explant type, culture medium, and BAP concentration on \u003cem\u003ein vitro\u003c/em\u003e shoot development in highbush blueberry (\u003cem\u003eVaccinium corymbosum\u003c/em\u003e L.) cultivars. In Vitro Cell Dev Biol-Plant 58:1057-1065 https://doi.org/10.1007/s11627-022-10299-0\u003c/li\u003e\n\u003cli\u003eMcCown B, Lloyd G (1981) Woody Plant Medium (WPM)-A Mineral Nutrient Formulation for Microculture of Woody Plant Species. HortScience 16: 453 https://doi.org/10.21273/HORTSCI.16.3S.365 \u003c/li\u003e\n\u003cli\u003eMorales JF, Sab\u0026aacute;s DC, Garcidue\u0026ntilde;as C, P\u0026eacute;rez E (2019) Germination\u003cem\u003e, in vitro\u003c/em\u003e propagation and soil acclimatization of \u003cem\u003eAcacia farnesiana\u003c/em\u003e and \u003cem\u003eProsopis laevigata. South Afr J Bot 124:345-349 \u003c/em\u003ehttps://doi.org/10.1016/j.sajb.2019.05.034\u003cem\u003e \u003c/em\u003e\u003c/li\u003e\n\u003cli\u003eMurashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco cultures. Physiol Plant 15: 473-497 https://doi.org/10.1111/j.1399-3054.1962.tb08052.x\u003c/li\u003e\n\u003cli\u003eNallathambi P, Umamaheswari C, Lal SK, Manjunatha C, Berliner J (2020) Mechanism of Seed Transmission and Seed Infection in Major Agricultural Crops in India. In: Kumar R, Gupta A (eds) Seed-Borne Diseases of Agricultural Crops: Detection, Diagnosis \u0026amp; Management. Springer, Singapore https://doi.org/10.1007/978-981-32-9046-4_26 \u003c/li\u003e\n\u003cli\u003eOlmstead RG, Zjhra ML, Lohmann LG, Grose SO, Eckert AJ (2009) A molecular phylogeny and classification of Bignoniaceae. Am J Botany 96:1731-1743 https://doi.org/10.3732/ajb.0900004 \u003c/li\u003e\n\u003cli\u003eRaspor M, Motyka V, Ninković S, Dobrev PI, Malbeck J, Ćosić T, Cingel A, Savić J, Tadić V, Dragićević IČ (2020) Endogenous levels of cytokinins, indole-3-acetic acid and abscisic acid in \u003cem\u003ein vitro\u003c/em\u003e grown potato: A contribution to potato hormonomics. Sci Rep 10:3437 https://doi.org/10.1038/s41598-020-60412-9\u003c/li\u003e\n\u003cli\u003eRivas CA, Navarro-Cerillo RM, Johnston JC, Guerrero-Casado J (2020) Dry forest is more threatened but less protected than evergreen forest in Ecuador\u0026rsquo;s coastal region. Env Cons 1-5 https://doi.org/10.1017/s0376892920000077 \u003c/li\u003e\n\u003cli\u003eSilveira SS, Cordeiro-Silva R, Degenhardt-Goldbach J, Quoirin M (2016) Micropropagation of \u003cem\u003eCalophyllum brasiliense \u003c/em\u003e(Cambess.) from nodal segments. Braz J Biol 76(3): 656-663 https://doi.org/10.1590/1519-6984.23714 \u003c/li\u003e\n\u003cli\u003eSong G, Chen Q, Callow P, Mandujano M, Han X, Cuenca B, Bonito G, Medina-Mora C, Fulbright DW, Guyer DE (2021) Efficient micropropagation of chestnut hybrids (\u003cem\u003eCastanea\u003c/em\u003e spp.) using modified Woody Plant Medium and zeatin riboside. Hortic Plant J 7(2):174-180 https://doi.org/10.1016/j.hpj.2020.09.006 \u003c/li\u003e\n\u003cli\u003eS\u0026ouml;zmen EU, Karak\u0026ouml;y T, Aasim M (2024) Optimizing benzylaminopurine (BAP) and naphthalene acetic acid (NAA) concentration for in vitro micropropagation of Goji Berry (\u003cem\u003eLycium barbarum\u003c/em\u003e L.) using various optimizing tools. Pak J Agri Sci 61(1):85-91 https://doi.org/10.21162/PAKJAS/24.95\u003c/li\u003e\n\u003cli\u003eVielba JM, Vidal N, Ricci A, Castro R, Covelo P, San Jose, MC, Sanchez C (2020) Effects of auxin and urea derivatives on adventitious rooting in chestnut and oak microshoots. Isr J Plant Sci 671-2:52-68 https://dx.doi.org/10.1163/22238980-20191113 \u003c/li\u003e\n\u003cli\u003eVilasboa J, Da Costa CT, Fett-Neto AG (2018) Rooting of eucalypt cuttings as a problem-solving oriented model in plant biology. Prog Biophys Mol Biol 146:85-97 https://doi.org/10.1016/j.pbiomolbio.2018.12.007\u003c/li\u003e\n\u003cli\u003eZhao Y, Chen Y, Jiang C, Lu MZ, Zhang J (2022) Exogenous hormone supplementation improve adventitious root formation in woody plants. Front Bioeng Biotechnol 10: 1009531 https://doi.org/10.3389/fbioe.2022.1009531\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"tissue culture, growth regulators, woody plants, micropropagation","lastPublishedDoi":"10.21203/rs.3.rs-4783431/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4783431/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eHandroanthus chrysanthus\u003c/em\u003e Jacq. S. O. Grose and \u003cem\u003eTabebuia rosea\u003c/em\u003e (Bertol.) Bertero ex A.DC are two forest species that grow in the coastal region of Ecuador and are threatened with extinction. A protocol for the mass multiplication of these species was developed using \u003cem\u003ein vitro\u003c/em\u003e culture techniques. The cultures were initiated from seeds, and the effect of two culture media (WPM and MS), two concentrations of NaClO (0.5% and 1%), and two disinfection times (3 and 5 minutes) was studied. During multiplication, the effect of three concentrations of two cytokinins (6-BAP 4.4, 5.5, or 6.6 \u0026micro;M; KIN 4.6, 5.75, or 6.9 \u0026micro;M) on the number of shoots, their length, and diameter was analyzed. This phase of the experiment was carried out in two successive multiplications. For rooting, two concentrations of IBA (2.45 and 4.9 \u0026micro;M) were tested, and the number of roots formed and their length were determined. It was demonstrated that the WPM medium is the most suitable for the \u003cem\u003ein vitro\u003c/em\u003e culture of both species and that disinfection time and NaClO concentration affect each species differently. For the multiplication of \u003cem\u003eH. chrysanthus\u003c/em\u003e, the most suitable cytokinin was 6-BAP 6.6 \u0026micro;M; \u003cem\u003eT. rosea\u003c/em\u003e performed better in the absence of cytokinins. IBA 2.45 \u0026micro;M produced the best results for the rooting of \u003cem\u003eH. chrysanthus\u003c/em\u003e, while for \u003cem\u003eT. rosea\u003c/em\u003e, IBA 4.9 \u0026micro;M was the most suitable. The acclimatized plants showed a high survival rate, demonstrating the feasibility of using this methodology for the accelerated propagation of these endangered species.\u003c/p\u003e","manuscriptTitle":"Development of a protocol for the micropropagation of two forest species threatened with extinction in Ecuador","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-23 06:40:00","doi":"10.21203/rs.3.rs-4783431/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-07-28T04:55:56+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-28T04:35:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-26T02:18:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2024-07-24T10:42:21+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":"cc4afca3-eabd-4d27-9bed-c2c26e92fb12","owner":[],"postedDate":"August 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-23T16:07:45+00:00","versionOfRecord":{"articleIdentity":"rs-4783431","link":"https://doi.org/10.1007/s11240-024-02864-9","journal":{"identity":"plant-cell-tissue-and-organ-culture-pctoc","isVorOnly":false,"title":"Plant Cell, Tissue and Organ Culture (PCTOC)"},"publishedOn":"2024-09-19 15:57:33","publishedOnDateReadable":"September 19th, 2024"},"versionCreatedAt":"2024-08-23 06:40:00","video":"","vorDoi":"10.1007/s11240-024-02864-9","vorDoiUrl":"https://doi.org/10.1007/s11240-024-02864-9","workflowStages":[]},"version":"v1","identity":"rs-4783431","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4783431","identity":"rs-4783431","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}