Storage of plant species with desiccation-sensitive germplasm in Compact Germplasm Bank.

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Paulo Hercilio Viegas Rodrigues, Guilherme Bovi Ambrosano This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7158126/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Plant species sensitive to desiccation are difficult to store and transport in the germplasm bank for space travel. Applying plant tissue culture can help to create a Plant Germplasm Bank for this species. For this purpose, the Compact Germplasm Bank (CGB) was created to store and transport in vitro explants, maintaining them for long periods in slow-grown storage. This study aimed to evaluate the CGB efficacy in the storage in vitro explant of Taioba ( Xanthosoma sagittifolium ). For this, three CGBs were connected, side by side, with different LED light spectra (CGB1: Red spectrum; CGB2: 50% Red + 50% Blue spectra; CGB3: Blue spectrum), containing each one nine test tubes with taioba explants (one per test tube), and LED lights intensity adjusted for 30 µmol m -2 s -1 . The CGBs were maintained for 120 days in the darkroom, at 25 o C temperature and 50-60% humidity. Explant storage in CGB3 showed lower root numbers and root lengths than in CGB1 and CGB2. The Blue spectrum in CGB3 reduced the root oxidation and browning, resulting in 100% live explants. The CGB fulfilled its proposed function of storing the in vitro explants. Microgravity Tissue culture LED light Clinostat Figures Figure 1 Figure 2 Figure 3 Key Message Compact chamber for transporting and storage of in vitro plant germplasm. Introduction Storage and transport of the Plant Germplasm Bank (PGB) is a challenge to maintaining plant-based bio-regenerative systems for the success of space trips (Wheeler 2010 ). Extremophile seeds, tolerant to desiccation, can be transported in dry environments through space. In contrast, numerous plant species produce seeds that are partially or fully sensitive to desiccation or propagated clonally and cannot be maintained in the dry state. Important species such as potato, yam, garlic, sweet potato, cassava, sugar cane, and banana are propagated through vegetative tissue. In addition, propagative explants, such as shoot tips, dormant buds, somatic embryos, or undifferentiated calluses, are highly sensitive to desiccation (Visscher et al. 2023 ). Therefore, to maintain the PGB during space travel, plant tissue culture techniques are the most suitable as they maintain plant health, reduce the volume transported, and require low inputs (Wheeler 2010 ). The storage of in vitro PGB requires periodic changes in the culture medium depending on the function of the laboratory. This operation involves procedures such as adequate structure, use of culture medium, and qualified labor (Tavazza et al. 2015 ). Applying the Slow-Grown Storage (SGS) technique reduces in vitro plant growth. In this way, SGS aims to reduce the plant's metabolism and consequently increase the in vitro period of the explant without altering the genetic characteristics and quality of the in vitro PGB (Kamińska et al. 2016 ). In in vitro growth incubation, the cultivation conditions can be changed, significantly reducing the plant's metabolism. Conditions such as temperature, light intensity, photoperiod, and components of the culture medium can be adjusted to maintain the plants in vitro in SGS (Gopal et al. 2010; Kaur et al. 2012 ; Carvalho et al. 2014 ; Thakur et al. 2015). In addition to these factors, the light spectra can be adjusted to reduce the metabolism of the plant and contribute to the SGS technique. The blue spectrum contributed significantly to the reduction of root growth and oxidative stress in the roots during in vitro storage of Heliconia champneiana for 12 weeks (Rodrigues et al. 2018 ). These results were corroborated using the same technique for 5 months in vitro SGS in banana cv. Prata Catarina (Rodrigues et al. 2022 ). To transport and maintain in vitro PGB, on Earth or in a Space Lab, the Compact Germplasm Bank (CGB) was created. This device has a small size, reduced weight, light intensity control, light spectra adjustment, and a photoperiodic system. In addition, the CGB hasn’t been screwed, it is mobile if powered by a 12V battery, and can be connected side by side to increase the useful area. Thus, the present work demonstrates how the CGB works with in vitro taioba explants in different light spectrums for long-term in vitro storage. Materials and Methods The plant material was in vitro explants of Taioba ( Xanthosoma sagittifolium ), from in vitro Germplasm Bank (GB) of Laboratory Tissue Culture Ornamental Plants (LTCOP), University of São Paulo. Taioba is propagated only from rhizomes, raw material for the in vitro GB. From this GB, aseptic taioba explants with 2.0 cm length x 0.5 cm Ø were selected and inoculated (one per tube) in the Pirex® test tubes (15.0 cm × 2.5 cm) with a cap, containing 7.0 mL of a semi-solid culture medium MS salts and vitamins (Murashige, T.; Skoog, F. 1962 ) plus 30 g L − 1 sucrose, and 2.0 g L − 1 Phytagel® (Sigma-Aldrich) – no Plant Grown Regulator (PGR); the pH of the medium was adjusted to 5.8 before autoclaving for 20 min at 121°C. The explants were introduced and incubated in three different CGBs, each one with a different LED light spectrum, CGB1 = 100% Red (660 nm), CGB2 = 50% Blue + 50% Red spectrum (considered control), and CGB3 = 100% Blue (450 nm), for 120 days in a dark room, maintained at 25°C ± 2°C and 50–60% humidity (similar ISS conditions). The CGB consists of one piece of aluminum cut and bent into a container measuring approximately 32 x 270 x 170 mm (A x B x C), comprising a frame with a lying “U”. In the upper inner portion (Fig. 1 a- 2 ) is placed a Photosynthetic Active Radiation (PAR) COB (Chip On Board) LED tape. On the side (Fig. 1 a- 3 ), fit the photosynthetic cycle system and the Photosynthetic Photon Flux (PPF) dimmer. The front cover (Fig. 1 a-5) and the back cover (Fig. 1 a-4) are fixed, wherein the frame and fitting portion have folded flaps and fastening hook and loop fasteners (3M Dual Lock™) disposed on the outer portion of the folded flaps for the firm and removable fit of the front and rear covers. The side fitting portion (Fig. 1 a-6) is fitted to the frame through a side opening, locking the CGB (Fig. 1 a- 1 ). Each CGB weighs 592g (plus front and rear aluminum covers). Full of test tubes (culture media + explant), the CGB weighs 930 g. The CGBs Photosynthetic Cycle (PC) was adjusted with the mini digital-timer (THT 2401-0) for 12 h day/night, it was supplied by PAR-COB LED tape (CGB1, CGB2, and CGB3), at a photosynthetic photon flux density (PPFD) adjusted to 30 µmol m − 2 s − 1 (LI-250A, LI-COR) for all treatments. The treatments (CGBs) were connected side by side with fastening hook and loop fasteners (3M Dual Lock™) but separated by a black acrylic shell one each other (Fig. 1 b). The CGBs were connected to a power supply LSF-CB-12V60W5A (AC100-265V), and a Nobreak (SMS LITE600). The experiment was installed in a completely randomized design, with 9 replicates for each CGB. After 120 days, the CGBs were opened and the plants were taken from the test tubes, washed with tap water (to remove the culture media), and measured plantlet height (cm), number of roots, root length (cm), fresh mass (g), dry mass (g), and plantlet survived (%). For statistical analyses, initially, descriptive and exploratory analyses of the data were performed. A joint analysis of a group of two experiments was performed, and it was initially found that the variances obtained by the individual analyses were homogeneous. It was found that the data on plant length, root length, number of roots, fresh mass, and dry mass did not meet the assumptions for an analysis of variance (ANOVA). Therefore, it was necessary to apply the logarithmic transformation for the root length data and the number of roots data, and the square root transformation for the fresh mass data and the dry mass data, according to the Box-Cox methodology. In addition, for the fresh mass data, an outlier from the mixed light treatment of the second experiment was removed. For the plant length data, no transformation was found that would make the data meet the assumptions, so the non-parametric Kruskal-Wallis test was applied. After the transformations and removal of the outliers, the data were submitted to ANOVA. Cohen’s effect size was also calculated. The effect size was interpreted according to Table 1 , according to Cohen ( 2013 ). To verify the behavior of the variables studied together, multivariate analysis of variance (MANOVA) was applied, followed by linear discriminant analysis (LDA). For this, the variables that met the assumption of normality of the residuals after the Box-Cox transformation were used (logarithm of root length and number of roots, and square root of fresh and dry mass, removing an outlier from the mixed light treatment). It was verified that the MANOVA assumptions were met. The normality of the residuals within the light color levels was verified by the Shapiro-Wilk test, and the asymmetry and kurtosis by the Mardia tests (Mardia, 1970). Multiple comparisons were made using the Tukey test. All analyses were performed using R software (R Core Team, 2024 ) at a significant level of 5%. Table 1 Interpretation for Cohen's f effect size. Cohen's f Tamanho do efeito Less than 0.10 Very small Between 0.10 and 0.25 Small Between 0.25 and 0.40 Medium Greater than 0.40 Large Source: Cohen ( 2013 ) Results and Discussion The results of the joint analyses can be observed in Table 2 . For all variables, there was no significant effect of the experiment (p = 0.9833 for root length, p = 0.7384 for the number of roots, p = 0.8845 for fresh mass, and p = 0.9710 for dry mass) nor of the interaction of the experiment with treatment (p = 0.9783 for root length, p = 0.9535 for number of roots, p = 0.8182 for fresh mass and p = 0.9966 for dry mass; Table 2 ). No significant effects of the light colors were observed on the variables plant length (very small effect), fresh mass (medium effect), and dry mass (medium effect). However, a large and significant effect of light color on root length was observed (p = 0.0414; f = 0.3994). Furthermore, Table 2 shows that the effect of light color on root number was also large and significant (p = 0.0013; f = 0.6007). Plants from CGB1 had significantly greater root length and root number than those from CGB3. For this study, the CGB2 treatment was considered the control because it combines the two fundamental spectra required for in vitro cultivation. Table 2 Mean ± standard error of the variables studied as a function of the lights spectra and results of the joint analyses (p-value of the effect of the color of the lights and effect size f) and results of the MANOVA, including sources of variation, value of the Pillai statistic, value of the F statistic, degrees of freedom (DL) of the denominator and numerator for the F statistic and p-value. Variable Treatment (light spectra) p-value (Treatment) CGB1 CGB2 CGB3 Plant length (cm) 12.53 ± 0.63 a 10.78 ± 1.07 a 10.33 ± 0.70 a p = 0.1485; f = 0.2026 Root length (cm) 9.43 ± 1.09 a 7.78 ± 0.96 ab 6.08 ± 0.77 b p = 0.0414; f = 0.3994 Number of roots 8.80 ± 0.45 a 7.12 ± 0.60 ab 5.56 ± 0.88 b p = 0.0013; f = 0.6007 Fresh weight (g) 3.55 ± 0.32 a 3.34 ± 0.45 a 2.80 ± 0.29 a p = 0.2927; f = 0.2443 Dry weight (g) 2.42 ± 0.17 a 1.96 ± 0.28 a 1.69 ± 0.22 a p = 0.0732; f = 0.3595 Source of variation Pillai F DF (numerator/denominator) p-value Experiment 0.0059 0.0579 4 / 39 0.9935 Treatment 0.4590 2.9788 8 / 80 0.0057 Experiment×Treatment 0.0296 0.1503 8 / 80 0.9963 Table 2 shows, the MANOVA model obtained a significant Pillai trace for treatment and a large effect size (Pillai = 0.4590; p = 0.0057; partial f = 0.5458). Tukey's test indicated that plants under CGB3 differed significantly from those under CGB1, but plants under CGB2 did not differ from either of the other two treatments. By linear discriminant analysis (Fig. 2 ), a cloud of points formed by observations that received more scatter on CGB3 and a cloud of points that received more concentrated data on CGB1 can be observed. The samples on CGB2 cross both clouds of points. It was observed that 75% of the points classified as CGB3 were correct, as were 50% of the points classified as CGB2 and 60% of those classified as CGB1. Furthermore, 25% of the points classified as CGB2 had received CGB3, and another 25% had received CGB1. None of the points classified as CGB3 had received CGB1, while 15% of those that were classified as CGB1 had received treatment with CGB3. The linear discriminant function 1, which explains 87.4% of the discriminatory variance, indicates that the cloud on CGB3 differs from the others by the difference between fresh mass and dry mass (the CGB3 has a lower dry mass content), by the smaller number of roots, and shorter root length. These analyses show that the plants under CGB3 had a reduced metabolism. They also showed that the results were consistent, so few plants that received blue spectrum (CGB3) had characteristics similar to those that received red spectrum (CGB1). In addition, plants that received blue spectrum treatment showed characteristics that were not observed in plants under the red spectrum (CGB1). Figure 3 a, c, and e show the CGB1, CGB2, and CGB3 with in vitro taioba plant test tubes, after 120 days on SGS. The CGBs operated stably and without failures after 120 days of the experiment. In day PC operating, the maximum temperature near the electronic components and COB LEDs (35 W/m) was 26°C (± 1°C), and on the external surface of the CGB (aluminum structure) was 27°C (± 1°C), for the adjusted PPF of 30 µmol m − 2 s − 1 . The test tubes' temperature (with culture medium and explant) was 25°C, the same as room temperature. During the night PC operating, the temperature of the entire system and test tubes was constant at 25°C, the same temperature as the dark room. The relative humidity recorded in the dark room varied between 50 and 60%, which did not interfere with the performance of the CGBs or the development of the in vitro explants. The in vitro relative humidity (inside the test tubes) has a range between 90 and 97% (Chen, 2004 ). In this way, the test tube caps were suitable for maintaining the environmental conditions within the test tube. The ISS temperature should range from 18–25ºC; in the same way, relative humidity ranges from 25–75% (Laurentini et al. 1999). During the experiment, the temperature range in the in vitro explant environment was maintained at 25°C (± 1°C), with a relative humidity of 50–60%, similar to the ISS environment. Figure 3 b, d, and f show the taioba plantlets after 120 days. In all treatments, there was a lack of uniformity in the final size of the plantlets. Even though the explants were selected at the beginning of the trial, this variation in plantlet size may occur at the end of 120 days, mainly because auxin was not added to the culture medium. Thus, the explants used the natural hormonal balance for elongation and rooting, as observed in the explants of Satoimo taro ( Colocasia esculenta (L.) (Karyanti et al. 2025). Although the CGB is isolated from external light, gas exchange is facilitated using Velcro and the corners that are not welded. This configuration helps with the gas exchange of test tubes. Even so, the accumulation of ethylene in the test tubes can be a determining factor in the success of the SGS (Rodrigues et al. 2022 ). In the CGB1 and CGB2 treatments, greater rooting occurred than in CGB3. This rooting process induces more ethylene production, oxidation, and consequent browning and plant death. The yellow circle in Fig. 3 b and d shows this effect, resulting in 88% survival. On the other hand, in CGB3 all plantlets survived (100%) and showed less yellowing in the leaves and oxidized roots (Fig. 3 f). These results were similar to the in vitro SGS of Heliconia champneiana and Banana cv. Prata Catarina (Rodrigues et al. 2018 ; Rodrigues et al. 2022 ). In these works, the authors suggest that the use of the blue spectrum in vitro can help reduce oxidation, browning, rooting, and yellowing of leaves. To date, there are no reports of compact and mobile tools for the storage and transportation of in vitro explants for use on Earth or in space. The CGB described in this work may be an option for studies on the storage and transportation of plant germplasm for long periods. The development of this type of equipment helps in the maintenance of food production systems and techniques for astronauts and in the improvement of bioregenerative technologies for life support systems in space. The authors of the present work address the same question as Visscher et al. 2023 : ``How do we transport plant species with desiccation-sensitive germplasm in space?'' The CGB is small, light, easy to operate, and resistant. Inside, it can be adapted that cloud-based sensor networks can continuously measure temperature, light spectra and intensity, growth metrics, and a subset of platforms can even implement AI-based alerts or adaptive controls. Thus, the CGB, applying SGS techniques, is ready to operate on Earth, connected to a clinostat for microgravity studies of in vitro plants. Its application on Earth could reduce costs and anticipate results in the storage of in vitro plants. In conclusion, the CGB fulfilled its proposed function of storing the explants in vitro , and the blue spectrum kept the taioba explants in the best condition. Abbreviations MS - Murashige and Skoog medium, PGB - Plant Germplasm Bank, SGS - Slow-Grown Storage, GB - Germplasm Bank, LS – Light Spectra, ISS - International Space Station, PC - Photosynthetic Cycle Declarations This work was financially supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Project number:406999/2022-8). Author G.A. has received research support from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). The authors have no relevant financial or non-financial interests to disclose. Conflict of interest : The authors have not disclosed any competing interests. Paulo Hercílio Viegas Rodrigues: Conceptualization, Methodology, Formal analysis and investigation, Writing - original draft preparation, Writing - review and editing, Funding acquisition. Guilherme Bovi Ambrosano: Methodology, Formal analysis and investigation, Writing - original draft preparation, Writing - review and editing. The authors agree to the availability of the manuscript data. Acknowledgments We appreciate AEB (Brazilian Space Agency) for the support and João Geraldo Brancalion for the figures and pictures. References Carvalho V, dos Santos DS, Nievola CC (2014) In vitro storage under slow growth and ex vitro acclimatization of the ornamental bromeliad Acanthostachys strobilacea. S Afr J Bot 92:39–43. https://doi.org/10.1016/j.sajb.2014.01.011 Chen C (2004) Humidity in plant tissue culture vessels. Biosyst Eng 88:231–241. http://doi.org/10.1016/j.biosystemseng.2004.02.007 Cohen J (2013) Statistical power analysis for the behavioral sciences. p. 567. New York, NY Routledge Gopal J, Chauhan NS (2010) Slow growth in vitro conservation of potato germplasm at low temperature. Potato Res 53:141–149. https://doi.org/10.1007/s11540-010-9158-x Kamińska M, Skrzypek E, Wilmowicz E et al (2016) Effect of light conditions and ABA on cold storage and post-storage propagation of Taraxacum pieninicum . 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Vienna, Austria: R Foundation for Statistical Computing. Available at: https://www.R-project.org/ Rodrigues PHV, Arruda F, Forti VA (2018) Slow-grown in vitro conservation of Heliconia champneiana cv. Splash under different light spectra. Sci Agric 75:163–166. http://dx.doi.org/10.1590/1678-992X-2016-0394 Rodrigues PHV, Oliveira EL, Demetrio CA, Ambrosano GB, Piedade SMS (2022) Effects of different light spectra on the slow-grown in vitro storage and quality of banana plantlets cv. Prata Catarina (AAB). Plant Cell Tiss Organ Cult 150:479–485. https://doi.org/10.1007/s11240-022-02280-x Tavazza R, Rey AN, Papacchioli V, Pagnotta MA (2015) A validated slow-growth in vitro conservation protocol for globe artichoke germplasm: a cost-effective tool to preserve from wild to elite genotypes. Sci Hort 197:135–143. https://doi.org/10.1016/j.scienta.2015.09.024 Visscher AM, Pritchard HW, Neri G, Ballesteros D (2023) How do we transport plant species with desiccation-sensitive germplasm in space? Life Sciences in Space Research. 36:135–137. https://doi.org/10.1016/j.lssr.2022.09.008 Wheeler RM (2010) Plants for human life support in space: from Myers to Mars Gravitational and Space Biology. Gravitational Space Biology 23:25–36 Cite Share Download PDF Status: Posted Version 1 posted 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-7158126","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":490725090,"identity":"d009b9ff-6e29-48d6-b00e-f8ce5fbbaf3c","order_by":0,"name":"Paulo Hercilio Viegas Rodrigues","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYDACCSBOADHYG0DkAVK08BwgRQuEkUCkFnPp3mcSDypq5eVnvjH+XMBwJ5+gFss5x80kEs4cN9xwO8dMegbDM8sGQloMbqQxGyS2HWPcIJ1jxszDcNiAoC0QLf+O2c+fecb4M7FaGB8kNtQkNtzgMZAmSovlDKCWhGMHkjecSSuTnmHwjLAWc4k0hoM/aups57cf3vy5oOIOEQ6DUIfBJDMDYQ1wLXVQLaNgFIyCUTAKsAAAFY0+T2lDVWMAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7099-0087","institution":"Universidade de Sao Paulo Escola Superior de Agricultura Luiz de Queiroz","correspondingAuthor":true,"prefix":"","firstName":"Paulo","middleName":"Hercilio Viegas","lastName":"Rodrigues","suffix":""},{"id":490725091,"identity":"e0319335-8ac8-4d29-b77f-417cd81680d9","order_by":1,"name":"Guilherme Bovi Ambrosano","email":"","orcid":"","institution":"Universidade de São Paulo Escola Superior de Agricultura Luiz de Queiroz: Universidade de Sao Paulo Escola Superior de Agricultura Luiz de Queiroz","correspondingAuthor":false,"prefix":"","firstName":"Guilherme","middleName":"Bovi","lastName":"Ambrosano","suffix":""}],"badges":[],"createdAt":"2025-07-18 13:24:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7158126/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7158126/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87854842,"identity":"8d8d76b2-b8b1-4b44-830d-9f063def31b9","added_by":"auto","created_at":"2025-07-29 16:32:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":234455,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003eSchematic drawing of CGB. \u003cstrong\u003eb\u003c/strong\u003eCGB3, CGB2, and CGB1 are connected side by side.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7158126/v1/cd06a4d5e22308ccc6912378.png"},{"id":87854844,"identity":"9420dfba-f04a-4f7e-a989-c41d42e3260a","added_by":"auto","created_at":"2025-07-29 16:32:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":48307,"visible":true,"origin":"","legend":"\u003cp\u003eProjection of observations from treatments CGB1, CGB2, and CGB3 onto the first two linear discriminant functions. Arrows represent the standardized discriminant coefficients for the dry mass (g), root length (cm), number of roots, and fresh mass (g).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7158126/v1/76d9b97388cf373b48eec313.png"},{"id":87854847,"identity":"031aa1df-4da3-4e4b-a299-b4ee7f5943a5","added_by":"auto","created_at":"2025-07-29 16:32:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":454269,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea, c, and e\u003c/strong\u003e CGBs working test in different LS. \u003cstrong\u003eb\u003c/strong\u003e In vitro Taioba plants from CGB1 (yellow circle shows plant died). \u003cstrong\u003ed\u003c/strong\u003e In vitro Taioba plants from CGB2 (yellow circle shows plant died). \u003cstrong\u003ef\u003c/strong\u003e In vitro Taioba plants from CGB3 (100% of plants live). Barr: 3.0 cm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7158126/v1/d812e0aa32a3306675480b1b.png"},{"id":89501713,"identity":"6a7d1f75-a4fd-4f22-8726-29debc1547c9","added_by":"auto","created_at":"2025-08-20 16:13:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1265788,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7158126/v1/cad8fbed-935e-474a-89ac-644114d3f1e4.pdf"}],"financialInterests":"","formattedTitle":"Storage of plant species with desiccation-sensitive germplasm in Compact Germplasm Bank.","fulltext":[{"header":"Key Message","content":"\u003cp\u003eCompact chamber for transporting and storage of \u003cem\u003ein vitro\u003c/em\u003e plant germplasm.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eStorage and transport of the Plant Germplasm Bank (PGB) is a challenge to maintaining plant-based bio-regenerative systems for the success of space trips (Wheeler \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Extremophile seeds, tolerant to desiccation, can be transported in dry environments through space. In contrast, numerous plant species produce seeds that are partially or fully sensitive to desiccation or propagated clonally and cannot be maintained in the dry state. Important species such as potato, yam, garlic, sweet potato, cassava, sugar cane, and banana are propagated through vegetative tissue. In addition, propagative explants, such as shoot tips, dormant buds, somatic embryos, or undifferentiated calluses, are highly sensitive to desiccation (Visscher et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTherefore, to maintain the PGB during space travel, plant tissue culture techniques are the most suitable as they maintain plant health, reduce the volume transported, and require low inputs (Wheeler \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The storage of \u003cem\u003ein vitro\u003c/em\u003e PGB requires periodic changes in the culture medium depending on the function of the laboratory. This operation involves procedures such as adequate structure, use of culture medium, and qualified labor (Tavazza et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eApplying the Slow-Grown Storage (SGS) technique reduces \u003cem\u003ein vitro\u003c/em\u003e plant growth. In this way, SGS aims to reduce the plant's metabolism and consequently increase the \u003cem\u003ein vitro\u003c/em\u003e period of the explant without altering the genetic characteristics and quality of the \u003cem\u003ein vitro\u003c/em\u003e PGB (Kamińska et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In \u003cem\u003ein vitro\u003c/em\u003e growth incubation, the cultivation conditions can be changed, significantly reducing the plant's metabolism. Conditions such as temperature, light intensity, photoperiod, and components of the culture medium can be adjusted to maintain the plants \u003cem\u003ein vitro\u003c/em\u003e in SGS (Gopal et al. 2010; Kaur et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Carvalho et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Thakur et al. 2015). In addition to these factors, the light spectra can be adjusted to reduce the metabolism of the plant and contribute to the SGS technique. The blue spectrum contributed significantly to the reduction of root growth and oxidative stress in the roots during \u003cem\u003ein vitro\u003c/em\u003e storage of \u003cem\u003eHeliconia champneiana\u003c/em\u003e for 12 weeks (Rodrigues et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These results were corroborated using the same technique for 5 months \u003cem\u003ein vitro\u003c/em\u003e SGS in banana cv. Prata Catarina (Rodrigues et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo transport and maintain \u003cem\u003ein vitro\u003c/em\u003e PGB, on Earth or in a Space Lab, the Compact Germplasm Bank (CGB) was created. This device has a small size, reduced weight, light intensity control, light spectra adjustment, and a photoperiodic system. In addition, the CGB hasn\u0026rsquo;t been screwed, it is mobile if powered by a 12V battery, and can be connected side by side to increase the useful area. Thus, the present work demonstrates how the CGB works with \u003cem\u003ein vitro\u003c/em\u003e taioba explants in different light spectrums for long-term \u003cem\u003ein vitro\u003c/em\u003e storage.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThe plant material was \u003cem\u003ein vitro\u003c/em\u003e explants of Taioba (\u003cem\u003eXanthosoma sagittifolium\u003c/em\u003e), from \u003cem\u003ein vitro\u003c/em\u003e Germplasm Bank (GB) of Laboratory Tissue Culture Ornamental Plants (LTCOP), University of S\u0026atilde;o Paulo. Taioba is propagated only from rhizomes, raw material for the \u003cem\u003ein vitro\u003c/em\u003e GB. From this GB, aseptic taioba explants with 2.0 cm length x 0.5 cm \u0026Oslash; were selected and inoculated (one per tube) in the Pirex\u0026reg; test tubes (15.0 cm \u0026times; 2.5 cm) with a cap, containing 7.0 mL of a semi-solid culture medium MS salts and vitamins (Murashige, T.; Skoog, F. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1962\u003c/span\u003e) plus 30 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e sucrose, and 2.0 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Phytagel\u0026reg; (Sigma-Aldrich) \u0026ndash; no Plant Grown Regulator (PGR); the pH of the medium was adjusted to 5.8 before autoclaving for 20 min at 121\u0026deg;C. The explants were introduced and incubated in three different CGBs, each one with a different LED light spectrum, CGB1\u0026thinsp;=\u0026thinsp;100% Red (660 nm), CGB2\u0026thinsp;=\u0026thinsp;50% Blue\u0026thinsp;+\u0026thinsp;50% Red spectrum (considered control), and CGB3\u0026thinsp;=\u0026thinsp;100% Blue (450 nm), for 120 days in a dark room, maintained at 25\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 50\u0026ndash;60% humidity (similar ISS conditions). The CGB consists of one piece of aluminum cut and bent into a container measuring approximately 32 x 270 x 170 mm (A x B x C), comprising a frame with a lying \u0026ldquo;U\u0026rdquo;. In the upper inner portion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) is placed a Photosynthetic Active Radiation (PAR) COB (Chip On Board) LED tape. On the side (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), fit the photosynthetic cycle system and the Photosynthetic Photon Flux (PPF) dimmer. The front cover (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-5) and the back cover (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-4) are fixed, wherein the frame and fitting portion have folded flaps and fastening hook and loop fasteners (3M Dual Lock\u0026trade;) disposed on the outer portion of the folded flaps for the firm and removable fit of the front and rear covers. The side fitting portion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-6) is fitted to the frame through a side opening, locking the CGB (Fig.\u0026nbsp;\u0026lt;link rid=\"fig1\"\u0026gt;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u0026lt;/link\u0026gt;\u003c/span\u003ea-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Each CGB weighs 592g (plus front and rear aluminum covers). Full of test tubes (culture media\u0026thinsp;+\u0026thinsp;explant), the CGB weighs 930 g.\u003c/p\u003e\u003cp\u003eThe CGBs Photosynthetic Cycle (PC) was adjusted with the mini digital-timer (THT 2401-0) for 12 h day/night, it was supplied by PAR-COB LED tape (CGB1, CGB2, and CGB3), at a photosynthetic photon flux density (PPFD) adjusted to 30 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (LI-250A, LI-COR) for all treatments. The treatments (CGBs) were connected side by side with fastening hook and loop fasteners (3M Dual Lock\u0026trade;) but separated by a black acrylic shell one each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The CGBs were connected to a power supply LSF-CB-12V60W5A (AC100-265V), and a Nobreak (SMS LITE600). The experiment was installed in a completely randomized design, with 9 replicates for each CGB. After 120 days, the CGBs were opened and the plants were taken from the test tubes, washed with tap water (to remove the culture media), and measured plantlet height (cm), number of roots, root length (cm), fresh mass (g), dry mass (g), and plantlet survived (%).\u003c/p\u003e\u003cp\u003eFor statistical analyses, initially, descriptive and exploratory analyses of the data were performed. A joint analysis of a group of two experiments was performed, and it was initially found that the variances obtained by the individual analyses were homogeneous. It was found that the data on plant length, root length, number of roots, fresh mass, and dry mass did not meet the assumptions for an analysis of variance (ANOVA). Therefore, it was necessary to apply the logarithmic transformation for the root length data and the number of roots data, and the square root transformation for the fresh mass data and the dry mass data, according to the Box-Cox methodology. In addition, for the fresh mass data, an outlier from the mixed light treatment of the second experiment was removed. For the plant length data, no transformation was found that would make the data meet the assumptions, so the non-parametric Kruskal-Wallis test was applied. After the transformations and removal of the outliers, the data were submitted to ANOVA. Cohen\u0026rsquo;s effect size was also calculated. The effect size was interpreted according to Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, according to Cohen (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). To verify the behavior of the variables studied together, multivariate analysis of variance (MANOVA) was applied, followed by linear discriminant analysis (LDA). For this, the variables that met the assumption of normality of the residuals after the Box-Cox transformation were used (logarithm of root length and number of roots, and square root of fresh and dry mass, removing an outlier from the mixed light treatment). It was verified that the MANOVA assumptions were met. The normality of the residuals within the light color levels was verified by the Shapiro-Wilk test, and the asymmetry and kurtosis by the Mardia tests (Mardia, 1970). Multiple comparisons were made using the Tukey test. All analyses were performed using R software (R Core Team, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) at a significant level of 5%.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInterpretation for Cohen's f effect size.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCohen's f\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTamanho do efeito\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLess than 0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVery small\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBetween 0.10 and 0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSmall\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBetween 0.25 and 0.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMedium\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGreater than 0.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLarge\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"2\"\u003eSource: Cohen (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e)\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eThe results of the joint analyses can be observed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. For all variables, there was no significant effect of the experiment (p\u0026thinsp;=\u0026thinsp;0.9833 for root length, p\u0026thinsp;=\u0026thinsp;0.7384 for the number of roots, p\u0026thinsp;=\u0026thinsp;0.8845 for fresh mass, and p\u0026thinsp;=\u0026thinsp;0.9710 for dry mass) nor of the interaction of the experiment with treatment (p\u0026thinsp;=\u0026thinsp;0.9783 for root length, p\u0026thinsp;=\u0026thinsp;0.9535 for number of roots, p\u0026thinsp;=\u0026thinsp;0.8182 for fresh mass and p\u0026thinsp;=\u0026thinsp;0.9966 for dry mass; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). No significant effects of the light colors were observed on the variables plant length (very small effect), fresh mass (medium effect), and dry mass (medium effect). However, a large and significant effect of light color on root length was observed (p\u0026thinsp;=\u0026thinsp;0.0414; f\u0026thinsp;=\u0026thinsp;0.3994). Furthermore, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows that the effect of light color on root number was also large and significant (p\u0026thinsp;=\u0026thinsp;0.0013; f\u0026thinsp;=\u0026thinsp;0.6007). Plants from CGB1 had significantly greater root length and root number than those from CGB3. For this study, the CGB2 treatment was considered the control because it combines the two fundamental spectra required for \u003cem\u003ein vitro\u003c/em\u003e cultivation.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the variables studied as a function of the lights spectra and results of the joint analyses (p-value of the effect of the color of the lights and effect size f) and results of the MANOVA, including sources of variation, value of the Pillai statistic, value of the F statistic, degrees of freedom (DL) of the denominator and numerator for the F statistic and p-value.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eVariable\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eTreatment (light spectra)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ep-value (Treatment)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCGB1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCGB2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCGB3\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePlant length (cm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.1485; f\u0026thinsp;=\u0026thinsp;0.2026\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRoot length (cm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0414; f\u0026thinsp;=\u0026thinsp;0.3994\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of roots\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0013; f\u0026thinsp;=\u0026thinsp;0.6007\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFresh weight (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.2927; f\u0026thinsp;=\u0026thinsp;0.2443\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDry weight (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0732; f\u0026thinsp;=\u0026thinsp;0.3595\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSource of variation\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePillai\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eF\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eDF (numerator/denominator)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003ep-value\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExperiment\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0059\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0579\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4 / 39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.9935\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.4590\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.9788\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8 / 80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0057\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExperiment\u0026times;Treatment\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.0296\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.1503\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8 / 80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.9963\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows, the MANOVA model obtained a significant Pillai trace for treatment and a large effect size (Pillai\u0026thinsp;=\u0026thinsp;0.4590; p\u0026thinsp;=\u0026thinsp;0.0057; partial f\u0026thinsp;=\u0026thinsp;0.5458). Tukey's test indicated that plants under CGB3 differed significantly from those under CGB1, but plants under CGB2 did not differ from either of the other two treatments.\u003c/p\u003e\u003cp\u003eBy linear discriminant analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), a cloud of points formed by observations that received more scatter on CGB3 and a cloud of points that received more concentrated data on CGB1 can be observed. The samples on CGB2 cross both clouds of points. It was observed that 75% of the points classified as CGB3 were correct, as were 50% of the points classified as CGB2 and 60% of those classified as CGB1. Furthermore, 25% of the points classified as CGB2 had received CGB3, and another 25% had received CGB1. None of the points classified as CGB3 had received CGB1, while 15% of those that were classified as CGB1 had received treatment with CGB3. The linear discriminant function 1, which explains 87.4% of the discriminatory variance, indicates that the cloud on CGB3 differs from the others by the difference between fresh mass and dry mass (the CGB3 has a lower dry mass content), by the smaller number of roots, and shorter root length.\u003c/p\u003e\u003cp\u003eThese analyses show that the plants under CGB3 had a reduced metabolism. They also showed that the results were consistent, so few plants that received blue spectrum (CGB3) had characteristics similar to those that received red spectrum (CGB1). In addition, plants that received blue spectrum treatment showed characteristics that were not observed in plants under the red spectrum (CGB1).\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, c, and \u003cb\u003ee\u003c/b\u003e show the CGB1, CGB2, and CGB3 with \u003cem\u003ein vitro\u003c/em\u003e taioba plant test tubes, after 120 days on SGS. The CGBs operated stably and without failures after 120 days of the experiment. In day PC operating, the maximum temperature near the electronic components and COB LEDs (35 W/m) was 26\u0026deg;C (\u0026plusmn;\u0026thinsp;1\u0026deg;C), and on the external surface of the CGB (aluminum structure) was 27\u0026deg;C (\u0026plusmn;\u0026thinsp;1\u0026deg;C), for the adjusted PPF of 30 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The test tubes' temperature (with culture medium and explant) was 25\u0026deg;C, the same as room temperature. During the night PC operating, the temperature of the entire system and test tubes was constant at 25\u0026deg;C, the same temperature as the dark room. The relative humidity recorded in the dark room varied between 50 and 60%, which did not interfere with the performance of the CGBs or the development of the \u003cem\u003ein vitro\u003c/em\u003e explants. The \u003cem\u003ein vitro\u003c/em\u003e relative humidity (inside the test tubes) has a range between 90 and 97% (Chen, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In this way, the test tube caps were suitable for maintaining the environmental conditions within the test tube. The ISS temperature should range from 18\u0026ndash;25\u0026ordm;C; in the same way, relative humidity ranges from 25\u0026ndash;75% (Laurentini et al. 1999). During the experiment, the temperature range in the \u003cem\u003ein vitro\u003c/em\u003e explant environment was maintained at 25\u0026deg;C (\u0026plusmn;\u0026thinsp;1\u0026deg;C), with a relative humidity of 50\u0026ndash;60%, similar to the ISS environment.\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, d, and \u003cb\u003ef\u003c/b\u003e show the taioba plantlets after 120 days. In all treatments, there was a lack of uniformity in the final size of the plantlets. Even though the explants were selected at the beginning of the trial, this variation in plantlet size may occur at the end of 120 days, mainly because auxin was not added to the culture medium. Thus, the explants used the natural hormonal balance for elongation and rooting, as observed in the explants of Satoimo taro (\u003cem\u003eColocasia esculenta\u003c/em\u003e (L.) (Karyanti et al. 2025).\u003c/p\u003e\u003cp\u003eAlthough the CGB is isolated from external light, gas exchange is facilitated using Velcro and the corners that are not welded. This configuration helps with the gas exchange of test tubes. Even so, the accumulation of ethylene in the test tubes can be a determining factor in the success of the SGS (Rodrigues et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the CGB1 and CGB2 treatments, greater rooting occurred than in CGB3. This rooting process induces more ethylene production, oxidation, and consequent browning and plant death. The yellow circle in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and \u003cb\u003ed\u003c/b\u003e shows this effect, resulting in 88% survival. On the other hand, in CGB3 all plantlets survived (100%) and showed less yellowing in the leaves and oxidized roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). These results were similar to the \u003cem\u003ein vitro\u003c/em\u003e SGS of \u003cem\u003eHeliconia champneiana\u003c/em\u003e and Banana cv. Prata Catarina (Rodrigues et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Rodrigues et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In these works, the authors suggest that the use of the blue spectrum \u003cem\u003ein vitro\u003c/em\u003e can help reduce oxidation, browning, rooting, and yellowing of leaves.\u003c/p\u003e\u003cp\u003eTo date, there are no reports of compact and mobile tools for the storage and transportation of \u003cem\u003ein vitro\u003c/em\u003e explants for use on Earth or in space. The CGB described in this work may be an option for studies on the storage and transportation of plant germplasm for long periods. The development of this type of equipment helps in the maintenance of food production systems and techniques for astronauts and in the improvement of bioregenerative technologies for life support systems in space. The authors of the present work address the same question as Visscher et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e: ``How do we transport plant species with desiccation-sensitive germplasm in space?''\u003c/p\u003e\u003cp\u003eThe CGB is small, light, easy to operate, and resistant. Inside, it can be adapted that cloud-based sensor networks can continuously measure temperature, light spectra and intensity, growth metrics, and a subset of platforms can even implement AI-based alerts or adaptive controls. Thus, the CGB, applying SGS techniques, is ready to operate on Earth, connected to a clinostat for microgravity studies of \u003cem\u003ein vitro\u003c/em\u003e plants. Its application on Earth could reduce costs and anticipate results in the storage of \u003cem\u003ein vitro\u003c/em\u003e plants.\u003c/p\u003e\u003cp\u003eIn conclusion, the CGB fulfilled its proposed function of storing the explants \u003cem\u003ein vitro\u003c/em\u003e, and the blue spectrum kept the taioba explants in the best condition.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMS - Murashige and Skoog medium, PGB - Plant Germplasm Bank, SGS - Slow-Grown Storage, GB - Germplasm Bank, LS \u0026ndash; Light Spectra, ISS - International Space Station, PC - \u0026nbsp; Photosynthetic Cycle\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThis work was financially supported by CNPq (Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico, Project number:406999/2022-8). Author G.A. has received research support from CAPES (Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior).\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e: The authors have not disclosed any competing interests.\u003c/p\u003e\n\u003cp\u003ePaulo Herc\u0026iacute;lio Viegas Rodrigues: Conceptualization, Methodology, Formal analysis and investigation, Writing - original draft preparation, Writing - review and editing, Funding acquisition.\u003c/p\u003e\n\u003cp\u003eGuilherme Bovi Ambrosano: Methodology, Formal analysis and investigation, Writing - original draft preparation, Writing - review and editing.\u003c/p\u003e\n\u003cp\u003eThe authors agree to the availability of the manuscript data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe appreciate AEB (Brazilian Space Agency) for the support and \u003cem\u003eJo\u0026atilde;o Geraldo Brancalion\u003c/em\u003e for the figures and pictures.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCarvalho V, dos Santos DS, Nievola CC (2014) In vitro storage under slow growth and ex vitro acclimatization of the ornamental bromeliad Acanthostachys strobilacea. 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Gravitational Space Biology 23:25\u0026ndash;36\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Microgravity, Tissue culture, LED light, Clinostat","lastPublishedDoi":"10.21203/rs.3.rs-7158126/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7158126/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlant species sensitive to desiccation are difficult to store and transport in the germplasm bank for space travel. Applying plant tissue culture can help to create a Plant Germplasm Bank for this species. For this purpose, the Compact Germplasm Bank (CGB) was created to store and transport \u003cem\u003ein vitro\u003c/em\u003e explants, maintaining them for long periods in slow-grown storage. This study aimed to evaluate the CGB efficacy in the storage \u003cem\u003ein vitro\u003c/em\u003e explant of Taioba (\u003cem\u003eXanthosoma sagittifolium\u003c/em\u003e). For this, three CGBs were connected, side by side, with different LED light spectra (CGB1: Red spectrum; CGB2: 50% Red + 50% Blue spectra; CGB3: Blue spectrum), containing each one nine test tubes with taioba explants (one per test tube), and LED lights intensity adjusted for 30 µmol m\u003csup\u003e-2\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e. The CGBs were maintained for 120 days in the darkroom, at 25\u003csup\u003eo\u003c/sup\u003eC temperature and 50-60% humidity. Explant storage in CGB3 showed lower root numbers and root lengths than in CGB1 and CGB2. The Blue spectrum in CGB3 reduced the root oxidation and browning, resulting in 100% live explants. The CGB fulfilled its proposed function of storing the \u003cem\u003ein vitro\u003c/em\u003e explants.\u003c/p\u003e","manuscriptTitle":"Storage of plant species with desiccation-sensitive germplasm in Compact Germplasm Bank.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-29 16:32:52","doi":"10.21203/rs.3.rs-7158126/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"66b1189f-6f97-4408-9d21-597ef35647bb","owner":[],"postedDate":"July 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-20T16:05:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-29 16:32:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7158126","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7158126","identity":"rs-7158126","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}