Artificial seed technology modified for long-term preservation of Lemna aequinoctialis

Artificial seed technology modified for long-term preservation of Lemna aequinoctialis | 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 Artificial seed technology modified for long-term preservation of Lemna aequinoctialis PHUONG HOANG THI NHU, Truong Quy Phung This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5339618/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Apr, 2025 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted 4 You are reading this latest preprint version Abstract Duckweeds, the Lemnaceae family, are employed in a wide range of industries, including agriculture, animal feed, wastewater treatment, biofuel production, and human food. Duckweeds are therefore also of interest for researchers. However, because to their rapid growth and development and ease of contamination by bacteria and algae during the storage process, duckweed samples require a great deal of time and effort to be maintained. In order to overcome the difficulties in preserving duckweed samples for scientific research, we developed an approach for duckweed storage by modification the ‘artificial seed’ procedure. We found that encapsulation of entire fronds of Lemna aequinoctialis in sodium alginate (3, 4, and 5%) and calcium chloride (150, 200, and 250 mM) were appropriate for maintaining them in a metabolically reduced state. After being stored for two months, there were no obvious variations between artificial seeds having an endosperm composition of ½ DN, DN, and water. After two months, duckweed samples kept in seeds with DN endosperm medium containing 150 mM CaCl 2 and 3% sodium alginate recovered the best. Following a year of storage, a noticeable difference could be seen between the treatments with various endosperm medium compositionsThe optimal conditions were encapsulation in an aqueous medium containing 200 mM CaCl 2 and 3% sodium alginate. This procedure offers an effective, cost-saving long-term preservation strategy that may be adapted in future for further duckweed species. modified artificial seed technology duckweed cost-effective long-term preservation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Key Message Except for polar regions and deserts, duckweeds are found all over the world due to their excellent adaptability and rapid growth among flowering plants. Thus, it is of research interested in finding the most economical way to sustain the growth and development of duckweed in laboratory condition as well as its long-term preservation. Here we adapted a procedure, used to generate ‘artificial seeds’, for encapsulation and preservation of L. aequinoctialis fronds to be stored for more than a year with little efforts, without costly equipment or chemicals. Introduction The monocotyledonous aquatic plant family Lemnaceae includes 5 genera ( Spirodela, Landoltia, Lemna, Wolffiella and Wolffia ) comprising 35 species and two hybrid species (Braglia et al., 2024 ). Duckweeds grow and develop quickly and have high economic value (Acosta et al., 2021 , Lam et al., 2014 , Zhao et al., 2012 ). Depending on environmental factors and nutrient availability, duckweeds can quadruple their biomass in 16 to 48 hours (Bog et al., 2020 , Sree et al., 2015 , Ziegler et al., 2015 ). Except for Wolffiella , which is only distributed in the America and Africa, the remaining genera of duckweeds are distributed in many parts of the world. Duckweeds are a high-yield biomass source for energy generation. Therefore, they may help to replace fossil fuels. In addition, duckweed is also used to recycle urban and agricultural wastewater before being converted into biofuel (Cui and Cheng 2015 , Fujita et al., 2016 , Gatidou et al., 2017 ). Furthermore, duckweeds have been utilized as a supplemental feed in animal husbandry because of its high protein and amino acid content as well as the numerous macro and micronutrients, vitamins, and carotenoids. Utilizing duckweeds to moderate or partially substitute other feed ingredients high in protein, can boost cattle and poultry output and improve the quality of their meat and eggs (Basílico et al., 2016 , Cheng and Stomp 2009 , Flores-Miranda et al., 2015 ). Because of the potential applications the number of duckweed research groups in the world is increasing; and there is an increasing need to gather and preserve duckweed collections in laboratories. However, preserving and maintaining duckweed samples in the laboratory encounters many difficulties such as easy contamination of the cultures with algae and bacteria that inhibit duckweed growth. Due to their fast growth, duckweed needs to be subcultured frequently, which is costly and requires space and working time. Currently, up to thousands of duckweed samples are being preserved in several institutions world-wide. Cryopreservation has been applied to preserve duckweeds (Peterson et al., 2023 ); however, small-scale laboratories may find it challenging to use this technology due to its high equipment, technique, and cost requirements. Thus, alternative approaches for duckweed preservation that are low in cost and highly effective are desirable. Artificial seeds simulate natural seeds, consisting of a somatic embryo surrounded by a layer of Sodium alginate (SA) containing nutrients. Under unfavorable conditions, the sample will stop growing until it finds suitable ones to develop and sprout out of the synthetic ‘seed coat’ of sodium alginate. Artificial seeds have numerous benefits, including easy handling, extended storage possibility, minimal production expenses, genetic identity with the individual from which they are derived, and simpler control over development of artificial embryos (Ravi and Prakash 2012 ) When it was discovered that many plant species could produce somatic embryos in vitro , the demand for synthetic seed technology arose. Murashige (Murashige 1977 ) provided the first description of synthetic seeds, also referred to by various names like "synseeds." He defined a synthetic seed as “a single encapsulated somatic embryo”. A synthetic seed was later characterized as “a somatic embryo designed for practical use in commercial plant production” (Gray et al., 1991 ). Since then, a variety of plant components, such as shoot tips, auxillary buds, nodal segments, protocorm-like bodies (PLBs), microshoots, and embryogenic calluses, have been handled and stored, instead of somatic embryos, in a similar way as artificial seeds (Ahmad and Anis 2010 , Ara et al., 2000 , Danso and Ford-Lloyd 2003 , Rai et al., 2008 , Rihan et al., 2011 , Sharma and Shahzad 2012 ) for a variety of vegetables, medicinal plants, ornamentals, and forest trees (Bapat et al., 1987 , Rai et al., 2008 ). Artificial seeds or the corresponding technology has been applied in particular for plants of high commercial interest and those of suitable somatic embryo quality (Redenbaugh et al., 1991 ). Artificial seeds, or similarly encapsulated regenerative plant tissues, have the capacity for large-scale culture and for preservation of rare or endangered plant species. Their small size reduces transportation and culture costs. They can be used to maintain and propagate heterotic hybrids (Bekheet 2006 , Pond and Cameron 2003 ). The main steps of the artificial seed procedure are: (1) submerging isolated somatic embryos in a gelling agent solution before aspirating them with a micropipette to create a protective shell; (2) immersing the capsule shells in a CaCl 2 complex solution and rinsing them in sterile water in a laminar flow chamber under aseptic condition; (3) after storage germinating them in a Petri dish containing nutrient media (Ravi and Prakash 2012 ). Baskaran and colleagues applied a similar procedure to the shoot tips of Urginea altissima . The samples grown in MS medium, 3% SA and 100 mM CaCl 2 yielded > 60% of germinating shoots after storage. The rate of adventitious shoot regeneration of encapsulated shoot tips grown on semi-solid MS medium supplemented with 10 µM mT and 2 µM NAA after 15 days of storage at 4°C in the dark at 25°C ± 2°C reached 91% (Baskaran et al., 2018 ). Fonseka and colleagues used encapsulated nodal shoot explants of the Black Oil tree ( Celastrus paniculatus Willd) for regeneration. Under optimal conditions shoots regenerated at 94.43% after storage at 5°C for 8 weeks (Fonseka et al., 2019 ). In Citrus jambhiri , encapsulated dormant buds yielded after storage 96.67 to 100.00% of germination (Sharma and Roy 2020 ). Material and Methods Plant material The duckweed accession L. aequinoctialis used in this study was collected at Dalat City, Vietnam, sterilized and grown in liquid nutrient medium under 16 h white light of 100 µmol m -2 s -1 , at 24°C. Investigation of the effects of SA concentration, CaCl 2 and soaking time on the artificial seed production process - Firmness of capsules: different SA concentrations of 2; 3; 4 and 5% were used to encapsulate the fronds. If the SA concentration is too low, no capsule is formed, and if the SA concentration is too high, the capsule will be hard and hinder sprouting when reactivation is aimed. - The durability of the capsule: different concentrations of CaCl 2 of 100; 150; 200 and 250 mM and soaking times of 5; 10 and 15 minutes were investigated to create the Ca-Alginate film. If the coat is too thin, it will break easily; if the coat is too hard, sprouting will be difficult. Investigation of the effects of mineral environment, SA and CaCl 2 concentrations on the formation and durability of L. aequinoctialis capsules The nutritional content of capsules plays an important role during reactivation of encapsulated fronds: if the environment does not provide enough nutrients, the vitality of the fronds will be weak, reducing the sprouting rate. Three different media were tested: (1) filtered tap water (Membrane water filtration system); (2) duckweed nutrient medium (denoted as DN): including KH 2 PO 4 (60 µM), Ca(NO 3 ) 2 (1 µM), KNO 3 (8 mM), MgSO 4 (1 mM), H 3 BO 3 (5 µM), MnCl 2 (13 µM), Na 2 MoO 4 (0.4 µM), FeEDTA (25 µM) (Appenroth et al., 2015); (3) half-concentrated duckweed nutrient medium (denoted as ½ DN). Each treatment consists of 40 samples/treatment. To maintain osmotic pressure balance, and to prevent leaking out of water from the capsules during their shelf life, the samples are kept in the same liquid medium as inside the capsule. Evaluation of the vitality of duckweed samples after 2 months and 1 year of storage in the capsules After 2 months or 1 year of storage, the encapsulated fronds were gently peeled off the shell to stimulate sprouting in duckweed nutrient medium at room temperature. The measurement of artificial seed firmness The firmness of L. aequinoctialis artificial seeds was assessed using the TA.XT plus texture analyser (Stable Micro System - UK) with probe P/5S (5mm Spherical Stainless), acquisition rate (200 point per second) at typical test time was 150 sec, pre-test speed (1 mm/sec), test speed (2 mm/sec) and post-test speed (10 mm/sec), distance was 1.5 mm and trigger force was 5g. Data were abtained by using Software Exponent Lite 6.1 Statistical analysis All experiments were repeated 3 times. Data were analyzed by Microsoft Excel 2019 software. Results Investigation of the effects of SA concentration, CaCl 2 and soaking time on the encapsulating process The quality of storage capsules depends on the nutritional content and the strength and firmness of the capsules. Thus, experiments were conducted to find a suitable combination of experimental factors for creating optimal capsules for duckweed. The main principle for the formation of the SA shell is the ion exchange between Na + in SA and Ca 2+ in CaCl 2 solution, this process takes place when SA droplets containing the fronds are dropped into CaCl 2 solution, creating capsule stability which depends on the concentration of two gelling agents (SA and CaCl 2 solution) as well as the time of soaking the capsules in CaCl 2 solution. Therefore, different combination between SA concentration (2%, 3%, 4% and 5%), CaCl 2 solution (100 mM, 150 mM, 200 mM and 250 mM) and soaking time (5 min, 10 min and 15 min) were tested. The results were shown in Table 1 . Table 1 Effects of SA, CaCl 2 concentrations and soaking time on the quality of artificial seeds SA (%) CaCl 2 (mM) Time (min) Endosperm formation Seed coat thickness Seed coat color Seed formation 2 100 5 None None - 10 Soft Thin Transparent + 15 Soft Thin Transparent + 150 5 None None - 10 Soft Thin Transparent + 15 Soft Thin Transparent + 200 5 Soft Thin Transparent + 10 Soft Thin Transparent + 15 Soft Thin Transparent + 250 5 Soft Thin Cloudy white + 10 Soft Thin Cloudy white + 15 Soft Thin Cloudy white + 3 100 5 None None - 10 Soft Thin Transparent + 15 Soft Thin Transparent + 150 5 None None - 10 Soft Thin Transparent + 15 Soft Thin Cloudy white ++ 200 5 Soft Thin Cloudy white ++ 10 Soft Thin Cloudy white ++ 15 Soft Thin Cloudy white ++ 250 5 Soft Thin Cloudy white +++ 10 Soft Thin Cloudy white +++ 15 Soft Thick Cloudy white +++ 4 100 5 Soft Thin Transparent + 10 Soft Thin Transparent + 15 Soft Thin Transparent + 150 5 Hard Thin Transparent ++ 10 Hard Thin Transparent ++ 15 Hard Thick Transparent ++ 200 5 Hard Thick Transparent ++ 10 Soft Thick Transparent +++ 15 Soft Thick Transparent +++ 250 5 Hard Thin Transparent ++ 10 Soft Thin Transparent ++ 15 Hard Thick Cloudy white ++ 5 100 5 Soft Thin Transparent + 10 Soft Thin Transparent + 15 Soft Thin Cloudy white ++ 150 5 Soft Thin Transparent + 10 Soft Thin Transparent ++ 15 Soft Thin Transparent ++ 200 5 Hard Thin Cloudy white + 10 Hard Thin Cloudy white +++ 15 Hard Thick Cloudy white +++ 250 5 Hard Thin Cloudy white + 10 Hard Thin Cloudy white ++ 15 Hard Thick Cloudy white ++ (-) No seed formation (+) Seed formation, didn’t not cover the entire sample (++) Seed formation, covering the entire sample (+++) Seed formation, covering the entire sample, good elasticity SA and CaCl 2 concentrations as well as seed soaking time mutually influence the quality of L. aequinoctialis encapsulation. In general, increasing SA and CaCl 2 concentration and soaking time, promote higher firmness and less capsule breakage during the operation (Fig. 1 ). In capsules produced in 2% SA at different CaCl 2 concentrations and soaking times, the medium inside the capsule did not completely cover the frond; the capsules were very soft and easily cracked during the manipulation. Thus, 2% SA was not optimal for duckweed encapsulation. For capsules produced in 100 mM CaCl 2 solution at different concentrations of SA and soaking times, the seed coat was very soft, even after 15 minutes. Thus, 100 mM CaCl 2 was not an optimal encapsulation condition for duckweed. Also, after soaking for 5 minutes at different concentrations of SA and CaCl 2 the capsules were weak and fragile. Capsules soaked for 10 minutes and 15 minutes were sufficiently durable. Thus, soaking time of 10 minutes was suitable for duckweed encapsulation, while longer soaking time in CaCl 2 solution increased the risk of infection of capsules. In summary, the concentration of SA (3%, 4% and 5%) as well as the concentration of CaCl 2 (150 mM, 200 mM and 250 mM) at a soaking time of 10 minutes were useful to encapsulate duckweed fronds. The impact of different CaCl 2 (150, 200, and 250 mM) and SA (3%, 4%, and 5%) concentrations on artificial seed diameter in millimeters was shown in Fig. 2 A. The smallest seed was 4.915 ± 0.26 mm at 150 mM of CaCl 2 and 3% SA. According to the data, the measured diameter typically rises slightly with increasing doses SA and CaCl 2 , with the effect being most noticeable at the maximum concentration of 5% and 250 mM, respectively. Overall, the data pointed to a trend in the seed diameter variable that was influenced by CaCl 2 and SA concentrations. Similar to the artificial seed diameter, the Fig. 2 B showed the effect of varied percentages of SA (3%, 4%, and 5%) and CaCl 2 concentrations (150 mM, 200 mM, and 250 mM) on artificial seed firmness. Higher amounts of SA and CaCl 2 were associated with increased seed hardness. Seed firmness increased from 53.3 ± 4.9 g at 150 mM of CaCl 2 and 3% SA to 126.1 ± 8.2 g at the highest concentration of CaCl 2 was 250 mM and SA was 5%. This showed that the firmness of the seeds and the CaCl 2 and SA concentrations were positively correlated. Investigation of the effects of mineral nutrients, SA and CaCl 2 concentrations on the formation and durability of encapsulated L. aequinoctialis fronds The results obtained in the above experiments showed that the chosen concentrations of SA, CaCl 2 at a soaking time of 10 minutes yielded suitable capsules. In this experiment, the effects of different mineral environments (water, ½ DN and DN) in combination with different concentrations of SA (3, 4 and 5%) and CaCl 2 (150, 200 and 250 mM) on the ability to create and preserve duckweed capsules were tested. To maintain osmotic balance, to prevent water from leaking out of the capsules, and to extend the capsules’ shelf life, the capsules were kept in the same liquid medium inside and outside. Capsules preserved for 2 months are shown in Fig. 3 . Because duckweed fronds are very thin and were easily to damage during manipulation, the operations must be performed quickly and gently to avoid damaging the sample. Although the capsule shape not uniform, their quality and the vitality of contained fronds were stable. After 2 months of storage, the fronds were still green, and the capsules were still elastic. During the experiment, the duckweed frond generated a small daughter frond. The mother frond faded over time, but the small frond remained green. Almost no differences between the treatments became obvious after 2 months. After storage up to 1 year in water endosperm, the fronds were still green and the capsules were still elastic. In ½ DN or DN L. aequinoctialis fronds turned white and died, some fronds turned brown and were in the process of decomposition. Thus, at increasing storage time the encapsulated L. aequinoctialis fronds showed a higher survival rate when kept in water than in nutrient solution (½ DN, DN) (Fig. 4 ). Evaluation of the vitality of encapsulated duckweed fronds after 2 months of storage Encapsulated L. aequinoctialis fronds (at different concentrations of SA, CaCl 2 ) in nutrient medium were evaluated as to their vitality after 2 months of preservation. Because the L. aequinoctialis fronds are soft and unable to break the capsules, a razor blade was used to gently cut the capsules and the undamaged fronds were transfered seeds in DN solution to provide nutrients for growth and vegetative propagation. The development of 2-month-stored L. aequinoctialis fronds after 2 weeks of recovery on nutrient medium are shown in Fig. 5 . All stored fronds recovered and propagated rapidly after being delivered from the capsules, suggesting that encapsulation is a promising way to preserve L. aequinoctialis at the laboratory scale. In general, lower concentrations of SA and CaCl 2 yielded faster growth than higher ones. Figure 5 showed that all encapsulated fronds, independent of SA and CaCl 2 concentrations, recovered to 100%, whether maintained in water or ½ DN or. The concentrations of the SA solution, which created the liquid density inside the capsule, and the CaCl 2 solution, which made the thickness of the coat, both had an impact on the recovery of fronds kept in the three storage solutions. In water, the ability to fully recover was slower than in DN or ½ DN solutions (Fig. 4 ). Therefore, DN and ½ DN were more suitable than water for 2 months of storage of L. aequinoctialis fronds. The frond recovery rates progressively declined as the SA concentration rose, suggesting that the encapsulated fronds were still receiving nutrients to survive when the SA content was not higher than 3%. When the SA concentration reached 5%, a longer recovery time was required for the fronds to return to a healthy physiological state and continue growth and propagation after delivery from the capsule. When the concentration of CaCl 2 increased, the recovery rate dropped also. Therefore, the physiological state of the fronds is also impacted by the thickness of capsules. A thinner (but not fragile) capsule mediates a faster frond recovery. Evaluation of the vitality of of encapsulated duckweed fronds after 1 year of storage Similar to 2-month-stored encapsulated L. aequinoctialis fronds, the recovery declines as the SA content rises from 3–5%. Encapsulated L. aequinoctialis fronds, maintained for a year in water showed 100% recovery and propagation after being cultured on DN medium (Fig. 6 ), while no recovery was observed when capsules were kept in ½ DN or DN. Thus, encapsulated L. aequinoctialis fronds can be stored in water for a longer period than in DN or ½ DN, suggesting that nutrient supply enhances frond recovery in case of short-term storage (~ 2 months), while fronds after 1 year of storage in DN or ½ die (the entire sample turned white, see Figs. 3 and 5 ). This can be explained by a gradual water loss of encapsulated fronds due to reverse osmosis mediated by hypertonic DN or ½ DN solutions, while in water (as hypotonic solution), the fronds lose no water, stay green, and recover when delivered into favorable conditions. Thus, for a long-time preservation, storage in water is more suitable than in ½ DN or DN. Nevertheless, some encapsulated fronds, stored in DN or ½ DN, were able to generate new duckweed fronds, showing that even under steady supply of nutrients, recovery is possible. In few cases, the capsules can break up when the fronds reached a particular size and regenerate earlier than expected (Fig. 7 ). The aforementioned findings highlight a few important issues that should be considered for L. aequinoctialis preservation: (1) After storage, the medium inside and outside the capsules has an impact on the recovery and growth of the fronds. The fastest recovery was observed for samples preserved in DN for short-term preservation, while the slowest regeneration occurred when the capsules were stored in water. However, regeneration was better after long-term preservation in water; (2) Encapsulated fronds formed by the combination of SA (4%) with CaCl 2 (150, 200, 250 mM) or 200 mM CaCl2 with SA (3, 4, 5%) displayed the best recovery; (3) The optimal variant of for long-term preservation of L. aequinoctialis fronds is 3% SA, 200 mM CaCl 2 and storage in water. Figure 8 displayed a step-by-step summary diagram that included the following steps: (1) combining water, ½ DN, or DN solution with varying SA concentrations; (2) generating a homogenous solution by boiling in a water bath; (3) autoclaving to sterilize the SA solution; (4) submerging the L. aequinoctialis frond in the SA solution; (5) gradually incorporating the duckweed sample-containing SA solution drop by drop into the CaCl 2 solution at varying concentrations; (6) maintaining the artificial seeds in the endosperm-corresponding solution under sterile circumstances. Discussion Currently, there are many coagulants, studied for the production of artificial seeds or adequate capsules, such as Agar, SA, Carrageenan, Guar gum and Sodium pectate, among which SA is considered most suitable because it provides optimal capsule thickness, fast coagulation speed and low toxicity, and it suits for various plant samples. SA can be adjusted for the desired capsule structure and hardness, protecting samples against mechanical impacts (Fonseka et al., 2019 , Rai et al., 2008 , Sharma and Roy 2020 ). The ion exchange between Na + in SA and Ca + in CaCl 2 is the fundamental mechanism behind the production of artificial seeds. This process occurs when sodium alginate-encapsulated plant samples are dropped into a CaCl 2 solution, creating stable capsules. The gelling agent concentrations (SA and CaCl 2 ) and contact duration determine the firmness and hardness of capsules. We established suitable conditions of capsule formation for L. aequinoctialis fronds, which were similar to the results obtained for garlic ( Allium sativum L.) explants that after three weeks of storage were still green, firm, and able to sprout. The reported best combinations varied slightly for different plant samples (Ara et al., 2000 , Gantait et al., 2017 , La et al., 2021 , Sharma and Roy 2020 ). The concentration of the gelling agent and the duration of the mixing time were shown to influence hardness and durability of capsules, which in turn have an impact on storage capacity and seed recovery. Noteworthy, and in contrast to most published findings, encapsulated duckweed fronds yielded best recovery after two-month storage for when stored in 1/2 DN or DN, but died (Fig. 3 ) and were incapable to recover (Fig. 5 ) if the storage period was increased to one year. This was apparently due to osmotic loss of water from fronds into surrounding nutritional medium during longer storage. In water, however, the encapsulated fronds survived and recovered fully. Thus, the medium to store encapsulated duckweed frond should be chosen according to the intended storage time. Because of the similar anatomy and physiology of duckweeds, the presented storage approach of frond encapsulation should also fit for storage of other duckweed species. Substances will be transported through the cell membrane more easily in duckweed because its cells are different from those of terrestrial plants (contain less lignin and cellulose) (Pagliuso et al., 2021 , Yadav et al., 2017 ). The concentration of minerals entering the sample and the amount of water lost by the sample increase with the length of storage time. This could be the cause of the sample's declining vitality as the storage period is increased. In the meantime, the water endosperm cell does not lose water and its mineral content remains unchanged. As a result, although the growth rate has slowed, the sample is still green and in a dormant state. Conclusion This study provides a method to maintain duckweeds in vitro for at least one year at minimal space, low costs and efforts without sophisticated equipment or expensive chemical compounds. The observation of encapsulated samples is convenient, and stored duplicates prevent sample loss during collection maintenance. Abbreviations SA – Sodium alginate, DN – Duckweed nutrient solution Declarations Author contributions TQP and HTNP acquired data wrote the manuscript, participated in performing the experiments, interpreting of data and revision for intellectual content. HTNP wrote the manuscript. HTNP conceptualized and designed the study. All authors discussed the results and contributed to the final manuscript. Acknowledgements This work was supported by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) grant # 106.01-2020.33 for Hoang Thi Nhu Phuong. The authors would like to thank Prof. Ingo Schubert (IPK-Gatersleben) for critical reading of the manuscript, and Ms. Luong Vu Mai Quynh, Tran Ngoc Diep, Ca Kim Diem Quynh, Tran Nguyen Kim Ngan (Dalat University) for her assistance in measuring artificial seed firmness. Conflict of interest: The authors declare that they have no conflicts of interest. 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Duckweed in bloom: the 2nd International Conference on Duckweed Research and Applications heralds the return of a plant model for plant biology. Plant Mol Biol, 84 , 737-42. Murashige, T. Plant cell and organ cultures as horticultural practices. 1977. International Society for Horticultural Science (ISHS), Leuven, Belgium, 17-30. Pagliuso, D., Grandis, A., Lam, E. & Buckeridge, M. S. 2021. High Saccharification, Low Lignin, and High Sustainability Potential Make Duckweeds Adequate as Bioenergy Feedstocks. BioEnergy Research, 14 , 1082-1092. Peterson, A., Kishchenko, O., Kuhlmann, M., Tschiersch, H., Fuchs, J., Tikhenko, N., Schubert, I. & Nagel, M. 2023. Cryopreservation of Duckweed Genetic Diversity as Model for Long-Term Preservation of Aquatic Flowering Plants. Plants (Basel), 12. Pond, S. & Cameron, S. 2003. Tissue culture Artificial Seeds. In: THOMAS, B. (ed.) Encyclopedia of Applied Plant Sciences. Oxford: Elsevier. Rai, M. K., Jaiswal, V. S. & Jaiswal, U. 2008. Encapsulation of shoot tips of guava (Psidium guajava L.) for short-term storage and germplasm exchange. Scientia Horticulturae, 118 , 33-38. Ravi, D. & Prakash, A. 2012. Production and Applications of Artificial seeds: A Review. 74-78. Redenbaugh, K., Fujii, J., Slade, D., Viss, P. & Kossler, M. 1991. Artificial Seeds — Encapsulated Somatic Embryos. In: BAJAJ, Y. P. S. (ed.) High-Tech and Micropropagation I. Berlin, Heidelberg: Springer Berlin Heidelberg. Rihan, H., Al-Issawi, M., Burchett, S. & Fuller, M. 2011. Encapsulation of cauliflower (Brassica oleracea var botrytis) microshoots as artificial seeds and their conversion and growth in commercial substrates. Plant Cell, Tissue and Organ Culture (PCTOC), 107 , 243-250. Sharma, P. & Roy, B. 2020. Preparation of Synthetic Seeds of Citrus jambhiri Using in vitro Regenerated Multiple Plantlets. Biotechnology Journal International , 22-29. Sharma, S. & Shahzad, A. 2012. Encapsulation technology for short-term storage and conservation of a woody climber, Decalepis hamiltonii Wight and Arn. Plant Cell Tissue and Organ Culture, 111 , 191-198. Sree, K. S., Sudakaran, S. & Appenroth, K.-J. 2015. How fast can angiosperms grow? Species and clonal diversity of growth rates in the genus Wolffia (Lemnaceae). Acta Physiologiae Plantarum, 37. Yadav, D., Barbora, L., Bora, D., Mitra, S., Rangan, L. & Mahanta, P. 2017. An assessment of duckweed as a potential lignocellulosic feedstock for biogas production. International Biodeterioration & Biodegradation, 119 , 253-259. Zhao, H., Appenroth, K., Landesman, L., Salmeán, A. A. & Lam, E. 2012. Duckweed rising at Chengdu: summary of the 1st International Conference on Duckweed Application and Research. Plant Mol Biol, 78 , 627-32. Ziegler, P., Adelmann, K., Zimmer, S., Schmidt, C. & Appenroth, K. J. 2015. Relative in vitro growth rates of duckweeds (Lemnaceae) - the most rapidly growing higher plants. Plant Biol (Stuttg), 17 Suppl 1 , 33-41. Cite Share Download PDF Status: Published Journal Publication published 04 Apr, 2025 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted Reviewers agreed at journal 31 Oct, 2024 Reviewers invited by journal 30 Oct, 2024 Editor assigned by journal 30 Oct, 2024 First submitted to journal 26 Oct, 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-5339618","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":372434959,"identity":"9aaedeb7-77ea-4d27-9d9c-c325fa07fc81","order_by":0,"name":"PHUONG HOANG THI NHU","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYDACCTB5gIGBvf3gAyCLh49YLRIMPGeSDUBa2IjXIpFgBmYT1GIu3WP4uODXnTpzngNplV9z7GTYGJgfPrqBR4vlnDPGxjP7nklYtjceuy27LRnoMDZj4xw8Wgxu5JhJ8/YcljA4cyDttuQ2ZqAWHjZp4rTcSDArltxWT6QWnh8QLYwftx0mrMVyRlqxMW/DYckNZ84kSzNuO87DxkzAL+YSyRsf8/w5zG9wvP3gx5/bqu352ZsfPsbrMBDB2AbhMPOASTzK4VoY/kA4jD8IqB4Fo2AUjIKRCQCDRkqlhZnRpQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-7418-9091","institution":"University of DaLat","correspondingAuthor":true,"prefix":"","firstName":"PHUONG","middleName":"HOANG THI","lastName":"NHU","suffix":""},{"id":372434960,"identity":"9e6a371d-feb0-4116-aab0-c7fc318b636c","order_by":1,"name":"Truong Quy Phung","email":"","orcid":"","institution":"University of DaLat","correspondingAuthor":false,"prefix":"","firstName":"Truong","middleName":"Quy","lastName":"Phung","suffix":""}],"badges":[],"createdAt":"2024-10-27 04:06:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5339618/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5339618/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11240-025-03035-0","type":"published","date":"2025-04-04T15:57:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68753580,"identity":"0ddae102-28b9-4ce0-a5a4-d18874f1e55c","added_by":"auto","created_at":"2024-11-11 16:31:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":637665,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of SA, CaCl\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e concentrations, and soaking time on the quality of artificial seeds\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5339618/v1/78b1c23b28cc802477a375b0.png"},{"id":68754525,"identity":"06874fd9-87f5-4222-956b-23566d6a66f9","added_by":"auto","created_at":"2024-11-11 16:39:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":19078,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of SA and CaCl\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e concentrations on the diameter (A) and the firmness (B) of artificial seeds\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5339618/v1/2d72814833ed4d2a1e02fe5a.png"},{"id":68754527,"identity":"4244cdd2-3148-4ee8-af5e-5b9deb7835f5","added_by":"auto","created_at":"2024-11-11 16:39:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":831922,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDuckweed artificial seeds after 2 months of storage. Bar: 1cm\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5339618/v1/40a7685628f51697cc07cea6.png"},{"id":68753585,"identity":"a5ae6dde-bf71-4ae2-8f84-808944973929","added_by":"auto","created_at":"2024-11-11 16:31:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":938266,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDuckweed artificial seeds after 1 year of storage.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5339618/v1/0000f62f9e93d8b74e51aae0.png"},{"id":68753588,"identity":"08a8ec58-6d5e-4412-9773-ad14422dfb5d","added_by":"auto","created_at":"2024-11-11 16:31:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":834069,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe 2-month-old duckweed artificial seeds after 2 weeks of recovery on nutrient medium\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5339618/v1/d90157b644e08822f7ad888a.png"},{"id":68753584,"identity":"3f0ccb6c-5d50-4300-8ab4-3f4c2bfa91be","added_by":"auto","created_at":"2024-11-11 16:31:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":958269,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe 1-year-old duckweed artificial seeds after 2 weeks of recovery on nutrient medium\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5339618/v1/152584c36bcdb9a1ef73f096.png"},{"id":68753582,"identity":"6f295a1f-78ed-46db-a73e-99836b55d860","added_by":"auto","created_at":"2024-11-11 16:31:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":296687,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGermination of frond during storage\u003c/strong\u003e. Red arrows: dead frond (white) inside artificial seed; Black arrow: broken seed; Yellow arrow: new frond germinating from the broken seed\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5339618/v1/4452626d606ba411e5f897d9.png"},{"id":68754526,"identity":"8271b7c0-aa2a-4d32-b277-6df0d549b77c","added_by":"auto","created_at":"2024-11-11 16:39:41","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":531333,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eArtificial seed production process\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5339618/v1/2750b2e9c1e4822538ecd420.png"},{"id":80082013,"identity":"ee71d3c7-7fad-4c9a-8897-12f1a1306fd5","added_by":"auto","created_at":"2025-04-07 16:05:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7146024,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5339618/v1/2255a831-03c0-44f8-9c5e-eeffba0bb8be.pdf"}],"financialInterests":"","formattedTitle":"Artificial seed technology modified for long-term preservation of Lemna aequinoctialis","fulltext":[{"header":"Key Message","content":"\u003cp\u003eExcept for polar regions and deserts, duckweeds are found all over the world due to their excellent adaptability and rapid growth among flowering plants. Thus, it is of research interested in finding the most economical way to sustain the growth and development of duckweed in laboratory condition as well as its long-term preservation. Here we adapted a procedure, used to generate \u0026lsquo;artificial seeds\u0026rsquo;, for encapsulation and preservation of \u003cem\u003eL. aequinoctialis\u003c/em\u003e fronds to be stored for more than a year with little efforts, without costly equipment or chemicals.\u0026nbsp;\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe monocotyledonous aquatic plant family Lemnaceae includes 5 genera (\u003cem\u003eSpirodela, Landoltia, Lemna, Wolffiella and Wolffia\u003c/em\u003e) comprising 35 species and two hybrid species (Braglia et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Duckweeds grow and develop quickly and have high economic value (Acosta et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Lam et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Zhao et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Depending on environmental factors and nutrient availability, duckweeds can quadruple their biomass in 16 to 48 hours (Bog et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Sree et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Ziegler et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Except for \u003cem\u003eWolffiella\u003c/em\u003e, which is only distributed in the America and Africa, the remaining genera of duckweeds are distributed in many parts of the world.\u003c/p\u003e \u003cp\u003eDuckweeds are a high-yield biomass source for energy generation. Therefore, they may help to replace fossil fuels. In addition, duckweed is also used to recycle urban and agricultural wastewater before being converted into biofuel (Cui and Cheng \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Fujita et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Gatidou et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Furthermore, duckweeds have been utilized as a supplemental feed in animal husbandry because of its high protein and amino acid content as well as the numerous macro and micronutrients, vitamins, and carotenoids. Utilizing duckweeds to moderate or partially substitute other feed ingredients high in protein, can boost cattle and poultry output and improve the quality of their meat and eggs (Bas\u0026iacute;lico et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Cheng and Stomp \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Flores-Miranda et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBecause of the potential applications the number of duckweed research groups in the world is increasing; and there is an increasing need to gather and preserve duckweed collections in laboratories. However, preserving and maintaining duckweed samples in the laboratory encounters many difficulties such as easy contamination of the cultures with algae and bacteria that inhibit duckweed growth. Due to their fast growth, duckweed needs to be subcultured frequently, which is costly and requires space and working time. Currently, up to thousands of duckweed samples are being preserved in several institutions world-wide. Cryopreservation has been applied to preserve duckweeds (Peterson et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e); however, small-scale laboratories may find it challenging to use this technology due to its high equipment, technique, and cost requirements. Thus, alternative approaches for duckweed preservation that are low in cost and highly effective are desirable.\u003c/p\u003e \u003cp\u003eArtificial seeds simulate natural seeds, consisting of a somatic embryo surrounded by a layer of Sodium alginate (SA) containing nutrients. Under unfavorable conditions, the sample will stop growing until it finds suitable ones to develop and sprout out of the synthetic \u0026lsquo;seed coat\u0026rsquo; of sodium alginate. Artificial seeds have numerous benefits, including easy handling, extended storage possibility, minimal production expenses, genetic identity with the individual from which they are derived, and simpler control over development of artificial embryos (Ravi and Prakash \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eWhen it was discovered that many plant species could produce somatic embryos \u003cem\u003ein vitro\u003c/em\u003e, the demand for synthetic seed technology arose. Murashige (Murashige \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1977\u003c/span\u003e) provided the first description of synthetic seeds, also referred to by various names like \"synseeds.\" He defined a synthetic seed as \u0026ldquo;a single encapsulated somatic embryo\u0026rdquo;. A synthetic seed was later characterized as \u0026ldquo;a somatic embryo designed for practical use in commercial plant production\u0026rdquo; (Gray et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). Since then, a variety of plant components, such as shoot tips, auxillary buds, nodal segments, protocorm-like bodies (PLBs), microshoots, and embryogenic calluses, have been handled and stored, instead of somatic embryos, in a similar way as artificial seeds (Ahmad and Anis \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Ara et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Danso and Ford-Lloyd \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Rai et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Rihan et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Sharma and Shahzad \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) for a variety of vegetables, medicinal plants, ornamentals, and forest trees (Bapat et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1987\u003c/span\u003e, Rai et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Artificial seeds or the corresponding technology has been applied in particular for plants of high commercial interest and those of suitable somatic embryo quality (Redenbaugh et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1991\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eArtificial seeds, or similarly encapsulated regenerative plant tissues, have the capacity for large-scale culture and for preservation of rare or endangered plant species. Their small size reduces transportation and culture costs. They can be used to maintain and propagate heterotic hybrids (Bekheet \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, Pond and Cameron \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe main steps of the artificial seed procedure are: (1) submerging isolated somatic embryos in a gelling agent solution before aspirating them with a micropipette to create a protective shell; (2) immersing the capsule shells in a CaCl\u003csub\u003e2\u003c/sub\u003e complex solution and rinsing them in sterile water in a laminar flow chamber under aseptic condition; (3) after storage germinating them in a Petri dish containing nutrient media (Ravi and Prakash \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBaskaran and colleagues applied a similar procedure to the shoot tips of \u003cem\u003eUrginea altissima\u003c/em\u003e. The samples grown in MS medium, 3% SA and 100 mM CaCl\u003csub\u003e2\u003c/sub\u003e yielded\u0026thinsp;\u0026gt;\u0026thinsp;60% of germinating shoots after storage. The rate of adventitious shoot regeneration of encapsulated shoot tips grown on semi-solid MS medium supplemented with 10 \u0026micro;M mT and 2 \u0026micro;M NAA after 15 days of storage at 4\u0026deg;C in the dark at 25\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C reached 91% (Baskaran et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFonseka and colleagues used encapsulated nodal shoot explants of the Black Oil tree (\u003cem\u003eCelastrus paniculatus\u003c/em\u003e Willd) for regeneration. Under optimal conditions shoots regenerated at 94.43% after storage at 5\u0026deg;C for 8 weeks (Fonseka et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In \u003cem\u003eCitrus jambhiri\u003c/em\u003e, encapsulated dormant buds yielded after storage 96.67 to 100.00% of germination (Sharma and Roy \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePlant material\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe duckweed accession \u003cem\u003eL. aequinoctialis\u003c/em\u003e used in this study was collected at Dalat City, Vietnam, sterilized and grown in liquid nutrient medium under 16 h white light of 100 µmol m\u003csup\u003e-2\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e, at 24°C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eInvestigation of the effects of SA concentration, CaCl\u003csub\u003e2\u003c/sub\u003e and soaking time on the artificial seed production process\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e- Firmness of capsules: different SA concentrations of 2; 3; 4 and 5% were used to encapsulate the fronds. If the SA concentration is too low, no capsule is formed, and if the SA concentration is too high, the capsule will be hard and hinder sprouting when reactivation is aimed.\u003c/p\u003e\n\u003cp\u003e- The durability of the capsule: different concentrations of CaCl\u003csub\u003e2\u003c/sub\u003e of 100; 150; 200 and 250 mM and soaking times of 5; 10 and 15 minutes were investigated to create the Ca-Alginate film. If the coat is too thin, it will break easily; if the coat is too hard, sprouting will be difficult.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eInvestigation of the effects of mineral environment, SA and CaCl\u003csub\u003e2\u003c/sub\u003e concentrations on the formation and durability of L. aequinoctialis capsules\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe nutritional content of capsules plays an important role during reactivation of encapsulated fronds: if the environment does not provide enough nutrients, the vitality of the fronds will be weak, reducing the sprouting rate. Three different media were tested: (1) filtered tap water (Membrane water filtration system); (2) duckweed nutrient medium (denoted as DN): including KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (60 µM), Ca(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e (1 µM), KNO\u003csub\u003e3\u003c/sub\u003e (8 mM), MgSO\u003csub\u003e4\u003c/sub\u003e (1 mM), H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e (5 µM), MnCl\u003csub\u003e2\u003c/sub\u003e (13 µM), Na\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u0026nbsp;\u003c/sub\u003e(0.4 µM), FeEDTA (25 µM)\u0026nbsp;(Appenroth\u003cem\u003e\u0026nbsp;et al.,\u003c/em\u003e 2015); (3) half-concentrated duckweed nutrient medium (denoted as ½ DN). Each treatment consists of 40 samples/treatment. To maintain osmotic pressure balance, and to prevent leaking out of water from the capsules during their shelf life, the samples are kept in the same liquid medium as inside the capsule.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEvaluation of the vitality of duckweed samples after 2 months and 1 year of storage in the capsules\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 2 months or 1 year of storage, the encapsulated fronds were gently peeled off the shell to stimulate sprouting in duckweed nutrient medium at room temperature.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eThe measurement of artificial seed firmness\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe firmness of \u003cem\u003eL. aequinoctialis\u0026nbsp;\u003c/em\u003eartificial seeds was assessed using the TA.XT plus texture analyser (Stable Micro System - UK) with probe P/5S (5mm Spherical Stainless), acquisition rate (200 point per second) at typical test time was 150 sec, pre-test speed (1 mm/sec), test speed (2 mm/sec) and post-test speed (10 mm/sec), distance was 1.5 mm and \u0026nbsp;trigger force was 5g. Data were abtained by using Software Exponent Lite 6.1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were repeated 3 times. Data were analyzed by Microsoft Excel 2019 software.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eInvestigation of the effects of SA concentration, CaCl\u003csub\u003e2\u003c/sub\u003e and soaking time on the encapsulating process\u003c/h2\u003e \u003cp\u003eThe quality of storage capsules depends on the nutritional content and the strength and firmness of the capsules. Thus, experiments were conducted to find a suitable combination of experimental factors for creating optimal capsules for duckweed.\u003c/p\u003e \u003cp\u003eThe main principle for the formation of the SA shell is the ion exchange between Na\u003csup\u003e+\u003c/sup\u003e in SA and Ca\u003csup\u003e2+\u003c/sup\u003e in CaCl\u003csub\u003e2\u003c/sub\u003e solution, this process takes place when SA droplets containing the fronds are dropped into CaCl\u003csub\u003e2\u003c/sub\u003e solution, creating capsule stability which depends on the concentration of two gelling agents (SA and CaCl\u003csub\u003e2\u003c/sub\u003e solution) as well as the time of soaking the capsules in CaCl\u003csub\u003e2\u003c/sub\u003e solution. Therefore, different combination between SA concentration (2%, 3%, 4% and 5%), CaCl\u003csub\u003e2\u003c/sub\u003e solution (100 mM, 150 mM, 200 mM and 250 mM) and soaking time (5 min, 10 min and 15 min) were tested. The results were shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of SA, CaCl\u003csub\u003e2\u003c/sub\u003e concentrations and soaking time on the quality of artificial seeds\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSA\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCaCl\u003csub\u003e2\u003c/sub\u003e (mM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTime (min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEndosperm\u003c/p\u003e \u003cp\u003eformation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSeed coat thickness\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSeed coat color\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSeed formation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"11\" rowspan=\"12\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCloudy white\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCloudy white\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCloudy white\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"11\" rowspan=\"12\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCloudy white\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCloudy white\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCloudy white\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCloudy white\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCloudy white\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCloudy white\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThick\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCloudy white\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"11\" rowspan=\"12\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThick\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThick\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThick\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThick\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThick\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCloudy white\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"11\" rowspan=\"12\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCloudy white\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCloudy white\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCloudy white\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThick\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCloudy white\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCloudy white\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCloudy white\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThick\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCloudy white\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003cem\u003e(-) No seed formation\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003cem\u003e(+) Seed formation, didn\u0026rsquo;t not cover the entire sample\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003cem\u003e(++) Seed formation, covering the entire sample\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003cem\u003e(+++) Seed formation, covering the entire sample, good elasticity\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSA and CaCl\u003csub\u003e2\u003c/sub\u003e concentrations as well as seed soaking time mutually influence the quality of \u003cem\u003eL. aequinoctialis\u003c/em\u003e encapsulation. In general, increasing SA and CaCl\u003csub\u003e2\u003c/sub\u003e concentration and soaking time, promote higher firmness and less capsule breakage during the operation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn capsules produced in 2% SA at different CaCl\u003csub\u003e2\u003c/sub\u003e concentrations and soaking times, the medium inside the capsule did not completely cover the frond; the capsules were very soft and easily cracked during the manipulation. Thus, 2% SA was not optimal for duckweed encapsulation.\u003c/p\u003e \u003cp\u003eFor capsules produced in 100 mM CaCl\u003csub\u003e2\u003c/sub\u003e solution at different concentrations of SA and soaking times, the seed coat was very soft, even after 15 minutes. Thus, 100 mM CaCl\u003csub\u003e2\u003c/sub\u003e was not an optimal encapsulation condition for duckweed.\u003c/p\u003e \u003cp\u003eAlso, after soaking for 5 minutes at different concentrations of SA and CaCl\u003csub\u003e2\u003c/sub\u003e the capsules were weak and fragile. Capsules soaked for 10 minutes and 15 minutes were sufficiently durable. Thus, soaking time of 10 minutes was suitable for duckweed encapsulation, while longer soaking time in CaCl\u003csub\u003e2\u003c/sub\u003e solution increased the risk of infection of capsules.\u003c/p\u003e \u003cp\u003eIn summary, the concentration of SA (3%, 4% and 5%) as well as the concentration of CaCl\u003csub\u003e2\u003c/sub\u003e (150 mM, 200 mM and 250 mM) at a soaking time of 10 minutes were useful to encapsulate duckweed fronds. The impact of different CaCl\u003csub\u003e2\u003c/sub\u003e (150, 200, and 250 mM) and SA (3%, 4%, and 5%) concentrations on artificial seed diameter in millimeters was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. The smallest seed was 4.915\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 mm at 150 mM of CaCl\u003csub\u003e2\u003c/sub\u003e and 3% SA. According to the data, the measured diameter typically rises slightly with increasing doses SA and CaCl\u003csub\u003e2\u003c/sub\u003e, with the effect being most noticeable at the maximum concentration of 5% and 250 mM, respectively. Overall, the data pointed to a trend in the seed diameter variable that was influenced by CaCl\u003csub\u003e2\u003c/sub\u003e and SA concentrations. Similar to the artificial seed diameter, the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB showed the effect of varied percentages of SA (3%, 4%, and 5%) and CaCl\u003csub\u003e2\u003c/sub\u003e concentrations (150 mM, 200 mM, and 250 mM) on artificial seed firmness. Higher amounts of SA and CaCl\u003csub\u003e2\u003c/sub\u003e were associated with increased seed hardness. Seed firmness increased from 53.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9 g at 150 mM of CaCl\u003csub\u003e2\u003c/sub\u003e and 3% SA to 126.1\u0026thinsp;\u0026plusmn;\u0026thinsp;8.2 g at the highest concentration of CaCl\u003csub\u003e2\u003c/sub\u003e was 250 mM and SA was 5%. This showed that the firmness of the seeds and the CaCl\u003csub\u003e2\u003c/sub\u003e and SA concentrations were positively correlated.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eInvestigation of the effects of mineral nutrients, SA and CaCl\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003econcentrations on the formation and durability of encapsulated L. aequinoctialis fronds\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe results obtained in the above experiments showed that the chosen concentrations of SA, CaCl\u003csub\u003e2\u003c/sub\u003e at a soaking time of 10 minutes yielded suitable capsules. In this experiment, the effects of different mineral environments (water, \u0026frac12; DN and DN) in combination with different concentrations of SA (3, 4 and 5%) and CaCl\u003csub\u003e2\u003c/sub\u003e (150, 200 and 250 mM) on the ability to create and preserve duckweed capsules were tested.\u003c/p\u003e \u003cp\u003eTo maintain osmotic balance, to prevent water from leaking out of the capsules, and to extend the capsules\u0026rsquo; shelf life, the capsules were kept in the same liquid medium inside and outside. Capsules preserved for 2 months are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBecause duckweed fronds are very thin and were easily to damage during manipulation, the operations must be performed quickly and gently to avoid damaging the sample. Although the capsule shape not uniform, their quality and the vitality of contained fronds were stable. After 2 months of storage, the fronds were still green, and the capsules were still elastic. During the experiment, the duckweed frond generated a small daughter frond. The mother frond faded over time, but the small frond remained green. Almost no differences between the treatments became obvious after 2 months.\u003c/p\u003e \u003cp\u003eAfter storage up to 1 year in water endosperm, the fronds were still green and the capsules were still elastic. In \u0026frac12; DN or DN \u003cem\u003eL. aequinoctialis\u003c/em\u003e fronds turned white and died, some fronds turned brown and were in the process of decomposition. Thus, at increasing storage time the encapsulated \u003cem\u003eL. aequinoctialis\u003c/em\u003e fronds showed a higher survival rate when kept in water than in nutrient solution (\u0026frac12; DN, DN) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of the vitality of encapsulated duckweed fronds after 2 months of storage\u003c/h2\u003e \u003cp\u003eEncapsulated \u003cem\u003eL. aequinoctialis\u003c/em\u003e fronds (at different concentrations of SA, CaCl\u003csub\u003e2\u003c/sub\u003e) in nutrient medium were evaluated as to their vitality after 2 months of preservation. Because the \u003cem\u003eL. aequinoctialis\u003c/em\u003e fronds are soft and unable to break the capsules, a razor blade was used to gently cut the capsules and the undamaged fronds were transfered seeds in DN solution to provide nutrients for growth and vegetative propagation. The development of 2-month-stored \u003cem\u003eL. aequinoctialis\u003c/em\u003e fronds after 2 weeks of recovery on nutrient medium are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. All stored fronds recovered and propagated rapidly after being delivered from the capsules, suggesting that encapsulation is a promising way to preserve \u003cem\u003eL. aequinoctialis\u003c/em\u003e at the laboratory scale.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn general, lower concentrations of SA and CaCl\u003csub\u003e2\u003c/sub\u003e yielded faster growth than higher ones.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e showed that all encapsulated fronds, independent of SA and CaCl\u003csub\u003e2\u003c/sub\u003e concentrations, recovered to 100%, whether maintained in water or \u0026frac12; DN or. The concentrations of the SA solution, which created the liquid density inside the capsule, and the CaCl\u003csub\u003e2\u003c/sub\u003e solution, which made the thickness of the coat, both had an impact on the recovery of fronds kept in the three storage solutions. In water, the ability to fully recover was slower than in DN or \u0026frac12; DN solutions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Therefore, DN and \u0026frac12; DN were more suitable than water for 2 months of storage of \u003cem\u003eL. aequinoctialis\u003c/em\u003e fronds.\u003c/p\u003e \u003cp\u003eThe frond recovery rates progressively declined as the SA concentration rose, suggesting that the encapsulated fronds were still receiving nutrients to survive when the SA content was not higher than 3%. When the SA concentration reached 5%, a longer recovery time was required for the fronds to return to a healthy physiological state and continue growth and propagation after delivery from the capsule.\u003c/p\u003e \u003cp\u003eWhen the concentration of CaCl\u003csub\u003e2\u003c/sub\u003e increased, the recovery rate dropped also. Therefore, the physiological state of the fronds is also impacted by the thickness of capsules. A thinner (but not fragile) capsule mediates a faster frond recovery.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEvaluation of the vitality of of encapsulated duckweed fronds after 1 year of storage\u003c/h3\u003e\n\u003cp\u003eSimilar to 2-month-stored encapsulated \u003cem\u003eL. aequinoctialis\u003c/em\u003e fronds, the recovery declines as the SA content rises from 3\u0026ndash;5%.\u003c/p\u003e \u003cp\u003eEncapsulated \u003cem\u003eL. aequinoctialis\u003c/em\u003e fronds, maintained for a year in water showed 100% recovery and propagation after being cultured on DN medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), while no recovery was observed when capsules were kept in \u0026frac12; DN or DN. Thus, encapsulated \u003cem\u003eL. aequinoctialis\u003c/em\u003e fronds can be stored in water for a longer period than in DN or \u0026frac12; DN, suggesting that nutrient supply enhances frond recovery in case of short-term storage (~\u0026thinsp;2 months), while fronds after 1 year of storage in DN or \u0026frac12; die (the entire sample turned white, see Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This can be explained by a gradual water loss of encapsulated fronds due to reverse osmosis mediated by hypertonic DN or \u0026frac12; DN solutions, while in water (as hypotonic solution), the fronds lose no water, stay green, and recover when delivered into favorable conditions. Thus, for a long-time preservation, storage in water is more suitable than in \u0026frac12; DN or DN.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNevertheless, some encapsulated fronds, stored in DN or \u0026frac12; DN, were able to generate new duckweed fronds, showing that even under steady supply of nutrients, recovery is possible. In few cases, the capsules can break up when the fronds reached a particular size and regenerate earlier than expected (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe aforementioned findings highlight a few important issues that should be considered for \u003cem\u003eL. aequinoctialis\u003c/em\u003e preservation: (1) After storage, the medium inside and outside the capsules has an impact on the recovery and growth of the fronds. The fastest recovery was observed for samples preserved in DN for short-term preservation, while the slowest regeneration occurred when the capsules were stored in water. However, regeneration was better after long-term preservation in water; (2) Encapsulated fronds formed by the combination of SA (4%) with CaCl\u003csub\u003e2\u003c/sub\u003e (150, 200, 250 mM) or 200 mM CaCl2 with SA (3, 4, 5%) displayed the best recovery; (3) The optimal variant of for long-term preservation of \u003cem\u003eL. aequinoctialis\u003c/em\u003e fronds is 3% SA, 200 mM CaCl\u003csub\u003e2\u003c/sub\u003e and storage in water.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e displayed a step-by-step summary diagram that included the following steps: (1) combining water, \u0026frac12; DN, or DN solution with varying SA concentrations; (2) generating a homogenous solution by boiling in a water bath; (3) autoclaving to sterilize the SA solution; (4) submerging the \u003cem\u003eL. aequinoctialis\u003c/em\u003e frond in the SA solution; (5) gradually incorporating the duckweed sample-containing SA solution drop by drop into the CaCl\u003csub\u003e2\u003c/sub\u003e solution at varying concentrations; (6) maintaining the artificial seeds in the endosperm-corresponding solution under sterile circumstances.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCurrently, there are many coagulants, studied for the production of artificial seeds or adequate capsules, such as Agar, SA, Carrageenan, Guar gum and Sodium pectate, among which SA is considered most suitable because it provides optimal capsule thickness, fast coagulation speed and low toxicity, and it suits for various plant samples. SA can be adjusted for the desired capsule structure and hardness, protecting samples against mechanical impacts (Fonseka et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Rai et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Sharma and Roy \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The ion exchange between Na\u003csup\u003e+\u003c/sup\u003e in SA and Ca\u003csup\u003e+\u003c/sup\u003e in CaCl\u003csub\u003e2\u003c/sub\u003e is the fundamental mechanism behind the production of artificial seeds. This process occurs when sodium alginate-encapsulated plant samples are dropped into a CaCl\u003csub\u003e2\u003c/sub\u003e solution, creating stable capsules. The gelling agent concentrations (SA and CaCl\u003csub\u003e2\u003c/sub\u003e) and contact duration determine the firmness and hardness of capsules.\u003c/p\u003e \u003cp\u003eWe established suitable conditions of capsule formation for \u003cem\u003eL. aequinoctialis\u003c/em\u003e fronds, which were similar to the results obtained for garlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.) explants that after three weeks of storage were still green, firm, and able to sprout. The reported best combinations varied slightly for different plant samples (Ara et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Gantait et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, La et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Sharma and Roy \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The concentration of the gelling agent and the duration of the mixing time were shown to influence hardness and durability of capsules, which in turn have an impact on storage capacity and seed recovery.\u003c/p\u003e \u003cp\u003eNoteworthy, and in contrast to most published findings, encapsulated duckweed fronds yielded best recovery after two-month storage for when stored in 1/2 DN or DN, but died (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and were incapable to recover (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) if the storage period was increased to one year. This was apparently due to osmotic loss of water from fronds into surrounding nutritional medium during longer storage. In water, however, the encapsulated fronds survived and recovered fully. Thus, the medium to store encapsulated duckweed frond should be chosen according to the intended storage time.\u003c/p\u003e \u003cp\u003eBecause of the similar anatomy and physiology of duckweeds, the presented storage approach of frond encapsulation should also fit for storage of other duckweed species. Substances will be transported through the cell membrane more easily in duckweed because its cells are different from those of terrestrial plants (contain less lignin and cellulose) (Pagliuso et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Yadav et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The concentration of minerals entering the sample and the amount of water lost by the sample increase with the length of storage time. This could be the cause of the sample's declining vitality as the storage period is increased. In the meantime, the water endosperm cell does not lose water and its mineral content remains unchanged. As a result, although the growth rate has slowed, the sample is still green and in a dormant state.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides a method to maintain duckweeds \u003cem\u003ein vitro\u003c/em\u003e for at least one year at minimal space, low costs and efforts without sophisticated equipment or expensive chemical compounds. The observation of encapsulated samples is convenient, and stored duplicates prevent sample loss during collection maintenance.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eSA\u003c/strong\u003e \u0026ndash; Sodium alginate, \u003cstrong\u003eDN\u003c/strong\u003e \u0026ndash; Duckweed nutrient solution\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTQP and HTNP acquired data wrote the manuscript, participated in performing the experiments, interpreting of data and revision for intellectual content. HTNP wrote the manuscript. HTNP conceptualized and designed the study. All authors discussed the results and contributed to the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) grant # 106.01-2020.33 for Hoang Thi Nhu Phuong.\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Prof. Ingo Schubert (IPK-Gatersleben) for critical reading of the manuscript, and Ms. Luong Vu Mai Quynh, Tran Ngoc Diep, Ca Kim Diem Quynh, Tran Nguyen Kim Ngan (Dalat University) for her assistance in measuring artificial seed firmness.\u003c/p\u003e\n\u003cp\u003eConflict of interest: The authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003eEthical approval: This article does not contain any studies with human participants or animals performed by any of the authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResearch involved human and animal rights: This research did not involve experiments with human or animal participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData sharing does not apply to this article as no datasets were generated during the current study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAcosta, K., Appenroth, K. 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Relative in vitro growth rates of duckweeds (Lemnaceae) - the most rapidly growing higher plants. \u003cem\u003ePlant Biol (Stuttg),\u003c/em\u003e 17 Suppl 1\u003cstrong\u003e,\u003c/strong\u003e 33-41.\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":"modified artificial seed technology, duckweed, cost-effective long-term preservation, ","lastPublishedDoi":"10.21203/rs.3.rs-5339618/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5339618/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDuckweeds, the Lemnaceae family, are employed in a wide range of industries, including agriculture, animal feed, wastewater treatment, biofuel production, and human food. Duckweeds are therefore also of interest for researchers. However, because to their rapid growth and development and ease of contamination by bacteria and algae during the storage process, duckweed samples require a great deal of time and effort to be maintained. In order to overcome the difficulties in preserving duckweed samples for scientific research, we developed an approach for duckweed storage by modification the \u0026lsquo;artificial seed\u0026rsquo; procedure. We found that encapsulation of entire fronds of \u003cem\u003eLemna aequinoctialis\u003c/em\u003e in sodium alginate (3, 4, and 5%) and calcium chloride (150, 200, and 250 mM) were appropriate for maintaining them in a metabolically reduced state. After being stored for two months, there were no obvious variations between artificial seeds having an endosperm composition of \u0026frac12; DN, DN, and water. After two months, duckweed samples kept in seeds with DN endosperm medium containing 150 mM CaCl\u003csub\u003e2\u003c/sub\u003e and 3% sodium alginate recovered the best. Following a year of storage, a noticeable difference could be seen between the treatments with various endosperm medium compositionsThe optimal conditions were encapsulation in an aqueous medium containing 200 mM CaCl\u003csub\u003e2\u003c/sub\u003e and 3% sodium alginate. This procedure offers an effective, cost-saving long-term preservation strategy that may be adapted in future for further duckweed species.\u003c/p\u003e","manuscriptTitle":"Artificial seed technology modified for long-term preservation of Lemna aequinoctialis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-11 16:31:36","doi":"10.21203/rs.3.rs-5339618/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-10-31T14:14:45+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-31T00:47:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-30T04:13:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2024-10-27T00:05:31+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":"ec99c855-2dc0-455d-8212-b8240a13d1f4","owner":[],"postedDate":"November 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-07T16:00:20+00:00","versionOfRecord":{"articleIdentity":"rs-5339618","link":"https://doi.org/10.1007/s11240-025-03035-0","journal":{"identity":"plant-cell-tissue-and-organ-culture-pctoc","isVorOnly":false,"title":"Plant Cell, Tissue and Organ Culture (PCTOC)"},"publishedOn":"2025-04-04 15:57:22","publishedOnDateReadable":"April 4th, 2025"},"versionCreatedAt":"2024-11-11 16:31:36","video":"","vorDoi":"10.1007/s11240-025-03035-0","vorDoiUrl":"https://doi.org/10.1007/s11240-025-03035-0","workflowStages":[]},"version":"v1","identity":"rs-5339618","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5339618","identity":"rs-5339618","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}