Polyacrylamide Hydrogels With Amber for Plants Micropropagation

Polyacrylamide Hydrogels With Amber for Plants Micropropagation | 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 Polyacrylamide Hydrogels With Amber for Plants Micropropagation Lyudmyla Kernosenko, Kateryna Samchenko, Olena Goncharuk, Natalya Pasmurtseva, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2085035/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Mar, 2023 Read the published version in Plants → Version 1 posted You are reading this latest preprint version Abstract The in vitro cultivation and reproduction of plants is one of the most modern and promising methods of cultivating valuable plants using artificial nutrient media. In this work, a new solid nutrient media for plant micropropagation based on highly dispersed polyacrylamide hydrogel (PAAG) with amber powder was synthesized and investigated. PAAG was synthesized by homophase radical polymerization with grounded amber addition. FTIR (Fourier transform infrared spectroscopy) and rheological studies were used to characterize structural properties of the materials. The synthesized hydrogel showed physicochemical and rheological parameters similar to the standard agar media. The estimation of acute toxicity of PAAG-amber was performed based on the influence of washing waters on the viability of the selected plant seeds (pea and chickpea) and animal ( Daphnia magna ). It proved its biosafety after four washes. The impact on plant rooting was studied using multiplication of Cannabis sativa on synthesized PAAG-amber saturated with Murashige-Skoog (MS) medium and compared with agar gel with MS. Developed substrate stimulated the rooting of the plants up to more than 98% in comparison to standard agar medium (95%). Also, PAAG-amber nutrient medium markedly enhanced metric indicators of seedling: root length increased by 28%, stem length – by 26.7%, root weight – by 167%, stem weight – by 67%, root and stem length – by 27%, root and stem weight – by 50%. This means that the developed hydrogel significantly accelerates reproduction and allows obtaining a larger amount of plant material within a shorter period than the standard agar medium. plants micropropagation polyacrylamide gel amber acrylamide toxicity biotesting Daphnia magna Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Key Message Polyacrylamide hydrogel with amber powder can be used as an agar-agar substitute for micropropagation of plants. Introduction Usually, the in vitro cultivation and reproduction of plants are carried out in solid nutrient media, and a high-cost agar substrate is the most used in this process (Kaçar et al. 2010 ; Adhikary et al. 2021 ; Lubell-Brand et al. 2021 ; Kaur and Mudgal 2021 ; Delgado-Aceves et al. 2022 ; Mood et al. 2022 ). This material is non-renewable, so for each subsequent passage of plants it must be used de novo (Espinosa-Leal et al. 2018 ). Because of agromarket’s needs to propagate plants commercially, the agar substrates are used in large amounts. To avoid such high costs, the search for alternative approaches is demanded. There is a great need to substitute the agar with cheaper disposable materials, such as starch (Henderson and Kinnersley 1988 ) or gum (Jain and Babbar 2002 ), or to develop an environmentally friendly solid substrate with optimal physicochemical and functional properties suitable for multiple uses. These requirements can be met by hydrogels based on spatially cross-linked hydrophilic polymers, which have the inherent ability to sorb and retain a significant amount of aqueous solutions due to adequate swelling properties. This process leads to an increase in hydrogel volume but its geometric shape remains unchanged. The so-called "smart hydrogels" are spatially cross-linked hydrophilic polymers with a three-dimensional structure capable of responding to various external physical factors, including minor changes in pH, temperature, ionic strength, etc., by changing the swelling degree, volume, sorption and diffusion characteristics (Schmaljohann 2006 ; Kocak et al. 2016 ; Samchenko et al. 2018 ; Goncharuk et al. 2020 ). They can be synthesized in different consistency and arbitrary shape, particularly in the form of granules of controlled sizes. Such hydrogels are characterized by high hydrophilicity and biological tolerance, as well as improved optical, sorption, and diffusion properties, which can be adjusted by varying the monomer composition and cross-linking density. The listed peculiarities make them suitable for immobilization of a wide range of both organic and inorganic compounds, as well as for use in novel technologies, namely water treatment(Sinha and Chakma 2019 ), manufacturing of selective membranes (Kosenko et al. 2006 ; Stadniy et al. 2011 ), cell cultivation (in particular stem ones) (Kosenko et al. 2006 ), targeted delivery, and controlled release of anticancer drugs (Schmaljohann 2006 ). In recent years, the substrates based on such hydrogels found application for plant vegetation under controlled conditions due to their ability to sorb and prolongedly release the necessary bioelements into the environment under the plant root exudates action (Milani et al. 2017 ; Ramli 2019 ; Singh et al. 2021 ; Khan et al. 2022 ). Some natural organic compounds with fungicidal and antibacterial properties can be included in hydrogels structure to enhance their functional properties. Primarily succinic acid and products of its transformation can be used as an additional source of microelements, endogenous physiologically active substances, growth or cell metabolism stimulators (Tumiłowicz et al. 2016 ; Levchyck et al. 2017 ; Mironov et al. 2018 ; Shimizu et al. 2020 ). It is known that amber, in particular, amber chips from the waste of various industries, is an environmentally safe and competitive product, so the inclusion of natural organic compounds in hydrogel substrates can be an effective and cost-effective way to increase the biological activity of the nutrient medium for plants micropropagation. In addition to the increasing bioactivity issue, the problem of ensuring biosafety is relevant for such materials. Since the monomers that compose the basic structural unit of polymers and, particularly hydrogels, are rather toxic materials and part of them always remains unreacted, the problem of synthesized hydrogels purification from residual microquantities of initial compounds is extremely important. The most affordable and effective way to purify hydrogels is to wash them repeatedly with distilled water. The risk control of toxic compounds in washing water is one of the most important steps in the preparation of synthesized material for further use. Today toxicology uses not only traditional spectrometric methods of analysis, but also a variety of biotesting methods that allow obtaining a comprehensive toxicological assessment of the aqueous environment using living test objects, including plants (cereals and legumes, some algae, etc.) and animals (unicellular, crustaceans, worms, etc.). Bioassays involving aquatic organisms, such as Daphnia magna , are common and widely used to study the toxicity of various chemicals (Kim et al. 2015 ; Olkova and Zimonina 2020 ). Biological tests on a biomodel D. magna are standardized in many countries (2004). Short-term testing allows determining the acute toxic effect of compounds in aqueous solution on the survival rate of branched crustaceans D. magna , which is one of the most sensitive biomarkers for determining the toxicity caused by different classes of chemical compounds (2004; Kim et al. 2015 ; Rand et al. 2020 ; Olkova and Zimonina 2020 ). The aim of the work is to develop the solid nutrient media for plants (namely, Cannabis sativa ) micropropagation based on highly dispersed polymer hydrogels with biologically active amber compounds, which will meet the requirements of high biocompatibility and suitable moisture content, as well as will demonstrate the environmental safety. Materials And Methods Materials Acrylamide (AA) (C 3 H 5 NO, "MERCK", Germany); N, N'-methylenebisacrylamide (MBA) (C 7 H 10 N 2 O 2 , "MERCK", Germany); potassium persulfate (K 2 S 2 O 8 , "SIGMA", USA); sodium metabisulfite (Na 2 S 2 O 5 ,"SIGMA", USA) were applied in the study without additional purification. Double-distilled water was used as a solvent in all experiments. For the synthesis of modified polyacrylamide gel (PAAG), the samples of Ukrainian raw amber from Zhytomyr, Olevsk and Rivne regions (Klesovo and Volodymyrets-Vostochny fields) were used. Methods Synthesis of PAAG The method of homophase radical polymerization in aqueous medium was used for synthesis of hydrogels based on acrylamide (Gertsiuk and Samchenko 2007 ). Polymerization was carried out at room temperature. The monomer solution was stirred on a laboratory magnetic stirrer (MM-5, 1200 rpm), then the red-ox initiation system (potassium persulfate–sodium metabisulfite) was added. The components' concentrations in the initiating mixture were chosen to provide the polymerization completion within an hour and to prevent excessive composition heating, which could adversely affect the hydrogels' properties. Cross-linking with the spatial network formation occurred due to copolymerization with a bifunctional monomer N,N'-methylene-bis-acrylamide (MBA). The gel was dispersed in a mortar to a granule size of Ø1–2 mm. The components used for PAAG synthesis are summarized in Table 1 . Table 1 Composition of reagents used for synthesis of PAAG samples Sample Water, g AA, g MBA, g Amber, % PAAG 13.6 32 0.04 - PAAG -A1 11.1 32 0.04 5.8 PAAG -A2 11.6 32 0.04 5.7 The synthesized PAAGs were repeatedly washed from unreacted components of the reaction mixture in distilled water in a ratio of 1 to 50 at a temperature of 45°C for 7 days. The washing process was monitored spectrophotometrically using a UV spectrophotometer SPECORD M40 (Carl Zeiss) (Gertsiuk and Samchenko 2007 ). The washing water was further analyzed for the presence of toxic impurities using test objects of plant and animal origin. Modification of PAAG with amber The raw amber was grounded using the laboratory mill Kinematica AG (Polymix® PX-MFC 90 D), and then it was divided into fractions sizes from 2–3 to15-20 mm. Milled samples of raw amber were pre-washed in running water and 10% NaCl. After that, the amber samples were cleaned from NaCl residues and dried at room temperature for several days or in a drying cabinet at 40-50 o C. The amber samples were differentiated by color into groups: opaque/transparent – the dark amber (samples A1); translucent/transparent – the light amber (samples A2). Amber samples were pre-cooled by liquid nitrogen to prevent the destruction of natural components during mechanical grinding due to overheating. Synthesis of polyacrylamide gels containing amber and cleaning them from unreacted components were performed according to the method described above. The fine amber A1 and A2 were added to the reaction mixture at a ratio of monomers and amber of 20 to 1 (Table 1 ). Biotesting of acrylamide using pea and chickpea seeds Biotesting of the toxicity of washing waters was performed by the Nelyubov method (1982), which bases on the fact that dyes stain only dead cell plasma (the plasma of a viable cell remains unstained). The viability was determined using pea and chickpea seeds. According to this technique, the seeds were soaked and, after 8 h, were released from the seed coat with a needle without damaging them. Ten peas were placed in the aqueous solutions of acrylamide with a concentration ranging from 0.001–10% and kept at a temperature of 30 o C for 3 h. Then the peas were transferred to 0.2% indigo carmine aqueous solution and kept for the next 3 h, after which the dye was drained, the peas were washed with distilled water, and their viability was determined according criteria of compliance with the conditions of viability shown in Table 2 . Biotesting of washing waters (from the 1st to the 7th washings) was carried out similarly. Table 2 Conditions for determining the seeds’ viability according to Nelyubov (1982): Viable seeds Non-viable seeds all parts are not stained all parts of the embryo are stained not stained root the tip of the root contains unstained spots not stained area around the root in the lower part of the cotyledon unstained spots root and cotyledon are completely/incompletely stained cotyledonous stained at the bottom cotyledonous is not painted Bioassay using Daphnia magna The acute toxicity of polyacrylamide hydrogel was also determined using the model of the hydrobiont D. magna (according to ISO 10706:2000([CSL STYLE ERROR: reference with no printed form.])). This method is based on estimating the influence of aqueous solutions on the D. magna mortality rate (%). D. magna was kept in ventilated aquariums with carbon-filtered tap water (pH = 7.3 ± 0.3) at a temperature of 18–22 ºC and a dissolved oxygen concentration > 6.0 mg/L ([CSL STYLE ERROR: reference with no printed form.]). The illumination of the cultivation was 400–600 lux at a light period of 16 ± 1 h, and 8 ± 1 h of darkness. The water from the 1st to the 7th washings with an acrylamide concentration of 0.00125 to 0.00001 mmol/L were used for cultivation. Distilled water was used as a control. The experiment used newborns aged 12–24 h, obtained by cultivation. In each 50-ml glass container with 30 ml of the test solution, seven individuals of D. magna were placed. The newborn daphnias were fed using Chlorella vulgaris or a suspension of baker's yeast 2 h before the experiment and were not fed during the experiments. The mortality of individuals in each beaker was assessed within 24 and 48 h. Specimens that moved freely in the water column or floated to the tank surface no later than 15 sec after light shaking were considered alive ones. The experiments were performed in triplicate. The sensitivity of D. magna to the reference model toxicant, potassium dichromate (K 2 Cr 2 O 7 ), was also determined (for 24 h).This allowed to assess the suitability of D. magna culture for biotesting. Investigation of hydrogel structure and rheological properties Fourier transform infrared spectroscopy (FTIR) spectra of powdered samples over the 4000–400 cm − 1 range were recorded using a ThermoNicolet iS10 FTIR spectrometer with a diffuse reflectance mode. The rheological properties of PAAG were investigated by a rotary viscometer Rheotest 2.1 using a cylindrical system Z in the range of shear rates from 2.43 to 1073 с −1 at a temperature of 20°C. For rheological properties investigation, the synthesized chemically cross-linked PAAG were pre-treated in a ball mill with subsequent sieving to obtain a fraction of gel particles d < 1 mm in the non-swollen state. Then, the dry gel particles were mixed with a given amount of water, which corresponded to the conditions of their use for the microclonal propagation of plants. Micropropagation Glesia industrial hemp ( Cannabis sativa ) seeds were selected as test objects. The used variety of monoecious non-narcotic hemp with dense rhomboid inflorescences and seed productivity provides the ability to produce a seed yield of 2.0-2.2 t/ha. The period from emergence to the onset of the phase of technical maturity is 88–93 days, and to the onset of the phase of biological maturity – is 100–120 days. The seeds were obtained from the Institute of Bast Crops of the National Academy of Agrarian Science of Ukraine. The seeds and cuttings of juvenile plants were the primary materials for obtaining sterile (microorganism-free) hemp plants in vitro . The cuttings were pre-treated with 70% ethanol for 1 min with the following treatment with 0.5% thiomerosal (C 9 H 9 HgNaO 2 S) for 1.5 min. In turn, the seeds were pre-treated with 70% ethanol for 3 min with the following treatment with 0.5% thiomerosal for 5 min. Sterilized seeds and cuttings were transferred to a nutrient medium and germinated at 26°C. The duration of the period before the emergence of seedlings ranged from 14 to 20 days. Grafting of plants was performed in the presence of 3–5 internodes on plants. 1.0–1.5 cm parts of the stem (micropub) with two axillary buds basal part were placed vertically in agar nutrient medium to a depth of 0.3–0.5 cm. Cultivation was performed on solid substrates: agar-agar (control) and hydrogel substrates: PAAG and PAAG-A2, saturated with Murashige-Skuga (MS) culture medium with the concentration of macronutrients reduced by half (MS/2). This medium contains 0.5 doses of macro- and micronutrients with the addition of 30 g/L sucrose and has a slightly acidic reaction (pH 5.6-6.0). For comparison, the agar-based (7.45 g/L) medium with the same MS and sucrose content was used. The complete cultivation cycle was 60 days. The first stage of introduction to the culture took place at the air temperature of 26–28°C. The obtained specimens were subcultured on MS medium under illumination with fluorescent lamps (2000–2500 lux) with a 16-hour photoperiod at a temperature of 24–26°C and a humidity of 70%. In order to evaluate the allelopathic activity of aerial parts of hemp Cannabis sativa L. extracts, (Grodzinsky et al. 1987 ) the bioassays were employed. In this method, the one-day seedlings of cucumber Cucumis sativus L. cv. Konkurent were used as a test object (Bataineh et al. 2008 ). Results And Discussion FTIR The FTIR spectra of dried initial PAAG gel, initial amber, and PAAG -gels with amber are given in Fig. 1 . The most informative peaks of all studied materials are in the range of 1800–800 cm − 1 . The spectrum of PAAG shows two bands at 3436 and 2924 cm − 1 , which correspond with the N–H stretching vibration of the NH 2 group and C–H stretching vibrations. The bands at 1645 and 1465 cm − 1 are attributed to the stretching of the C = O group in amide and CH 2 scissoring, respectively (Nakanishi 1962 ). In the FTIR spectrum of initial amber, the wide shoulder of the 1160 cm − 1 peak stretching to 1260 cm − 1 (known as “Baltic shoulder”) corresponds to the high content of succinic acid and other succinate compounds. The intense peak at ~ 1700 cm − 1 is characteristic of the C = O group of carboxylic acids. The bands at 1445 and 1375 cm − 1 are attributed to C–H symmetric and asymmetric stretching vibrations. The 887 cm − 1 band could be assigned to the out-of-plane aromatic C–H bending.(Mänd et al. 2018 ; Karolina et al. 2022 ) In the spectra of polyacrylamide hydrogels with amber, the bands of amide I (1647 cm − 1 ) and amide II (1605 cm − 1 ) of acrylamide have the highest intensity. The bands in the range of 3000–3500 cm − 1 correspond with symmetric and asymmetric vibrations of amino groups ν (NH) of polyacrylamide. The maximum at 2938 cm − 1 is attributed to stretching vibrations of the methylene group. The intense maximum ν (C=O) at 1647 cm − 1 (Amide I) corresponds with the amide fragment, which overlaps with the bending vibration maximum δ (NH2) at 1605 cm − 1 (Amide II) with the formation of a broadened doublet. In the "fingerprint" region, a doublet at 1450–1410 cm − 1 caused by bending vibrations of the CH group, as well as a broadened band at ν (CN) = 1350 cm − 1 (Amide III) were noted.(Nakanishi 1962 ; Boldeskul et al. 2009 ) Acute toxicity Synthetic hydrogels, created by polymerization of highly toxic monomers such as acrylamide, contain unreacted residues that should be removed before using them for medical and biological purposes(Gertsiuk and Samchenko 2007 ). Acute toxicity of the studied compounds of different washes (from the 1th to the 7th) was determined based on the mortality rate (%) of D. magna. Data on the survival of individuals in each sample during 24 and 48 h of the exposure are presented in Fig. 2 , Table 3 . Table 3 Overall risk assessment of toxic compounds present in washing waters on the viability of biomarkers of plant (legume) and animal ( D. magna ) origin Washing / AA solution Conc. AA, mmol/l Death toll, % PAAG PAAG-A1 PAAG-A2 daphnia peas / chickpeas daphnia peas / chickpeas daphnia peas / chickpeas AA solution 1.4 100* 80/80 100* 80/80 100* 80/90 AA solution 0.14 100* 40/70 100* 40/80 100* 50/70 AA solution 0.014 100* 40/70 100* 40/80 100* 50/70 1 0.00125 100* 10/20 100* 10/20 100* 10/20 2 0.00096 100** 10 28.6** 10 28.6** 10 3 0.00054 42.8** 10 0 10 0 10 4 0.00007 0 0 0 0 0 0 5 0.00003 0 0 0 0 0 0 6 0.00002 0 0 0 0 0 0 7 0.00001 0 0 0 0 0 0 Control (DV) 0 0 0 0 0 0 * - died on the first day of the experiment ** - died on the second day of the experiment The performed experiments indicated that after the first wash of all tested samples, complete death of organisms (100%) was already recorded during 24 and 48 h of the experiment. However, the mortality decreased by 28.6% and 57.2% during 48 h in the second washing water for PAAG-A2 and PAAG-A1, respectively, (Fig. 2 ). In the third washing water, the mortality of D. magna individuals in the PAAG-A1 and PAAG-A2 samples was absent. No cases of mortality were recorded during 24 and 48 h of the experiment. Based on these results, D. magna was considered as an organism of high sensitivity to washing water composition, which can be used for diagnosis and risk assessment of the hydrogels and unreacted compounds. The results of biotesting of the toxicity of washing water by the Nelyubov method are depicted in Fig. 3 . It was found that 1–3 washes of hydrogels, in which the concentration of acrylamide was from 0.00054 mol/dm 3 to 0.00125 mol/dm 3 (according to the results of measurements using a UV spectrometer SPECORD M40), were the most toxic for all tested legumes. For peas, 10% of seeds was dead, while for chickpeas, 20%. The results of both biotesting experiments are presented in Table 3 . They indicated that the washing water from the 4th wash is safe for both D. magna and legumes. The hydrogels, washed in this way, can be safely used in practice. Tucson et al. also washed PAAG for three days (changing the water every 24 hours), after which the gel was safe for further use as substrates for the study of bacteria (Tuson et al. 2012 ). Rheological properties The structural and mechanical characteristics of hydrogel composites largely determine the application possibilities of these systems. They are related to bioavailability and release of biologically active components from hydrogel materials. Gels are structured systems that demonstrate the structural and mechanical properties of both liquids and solids. Hydrogel, as a dispersed system, acquires the properties of a solid body, that is, shear modulus and elasticity. The most important rheological characteristics of hydrogels include shear stress and viscosity. Effective viscosity is a characteristic of the equilibrium state between the processes of destruction and recovery. Its fluctuation causes a change in the coagulation-crystallization structure of the hydrogel, affecting its performance characteristics. The spatial structure in the hydrogel is determined by measuring the mechanical properties and, in particular, shear deformation under the constant stress. Solids are characterized by a sharp change in the pattern of shear deformation ε depending on the magnitude of the shear stress Р . At rather low stresses (less than the yield strength P k ), a free flow with constant and extremely high viscosity η 1 is observed. In this case, the coagulation structure is destroyed, but has time to recover. As the shear stress increases to the yield strength P k , the viscosity decreases significantly, down to the lowest limit value η m . Rheological behaviors observed for agar gel and PAAG are presented in Fig. 4 . Non-linear change in effective viscosity values indicates non-Newtonian system properties of studied gels. During the measurement, the destruction of interparticle bonds progresses as the shear rate ( γ ) increases, which is manifested in the peculiarities of the shape of the flow curves, causing deviations from straight lines. Measuring in the reverse mode indicates recovery of the effective viscosity due to the restoration of the system structure, but the effective viscosity values remain lower than the initial ones. Both dispersed PAAG and agar gel showed thixotropic properties, as evidenced by the hysteresis loops of the dependence of the effective viscosity of gels on the shear rate (Fig. 4 a,b). Table 4 and Fig. 4 show the initial and final values of the effective viscosity at the minimum and maximum shear rates for homopolyacrylamide and agar gels. The initial effective viscosity values are close for both gels. During the measurement. The viscosity values at a shear rate of 2.45 s − 1 at the end of the measurement in the reverse shear rate reduction mode for PAAG and agar are 156.398 and 200.32 Pa∙s, which is 37.0 and 40.2% of the initial value, respectively. This means that PAAG had rheological properties and effective viscosity values similar to agar-agar gel. Table 4 Effective viscosity at minimum (γ = 2.45 s − 1 ) and maximum shear rates (γ = 1073 s − 1 ) Sample η ( at γ = 2,45 s − 1 ), Pа∙s η ( at γ = 1073 s − 1 ), Pа∙s initial final initial final Agar 498.519 200.32 0.501 0.306 PAAG 422.986 156.398 0.471 0.410 Micropropagation For micropropagation of plants in vitro , all synthesized hydrogels were saturated with a culture medium with a complex of micro- and macronutrients, vitamins, amino acids, growth hormones, etc. This process provided the formation of hydrogel composites with sparingly soluble bioelements, which are localized in the hydrogel pores and can gradually diffuse into the external environment. The experiment showed the acceleration of the rooting time of cuttings on PAAG-A2 gel, which was less than two weeks of incubation. On agar-agar this time equaled three weeks. In addition, an intensification of growth and development of the main shoot was observed on hydrogels: 10 and almost 30% higher on PAAG and PAAG-A2, respectively, compared to the samples grown on agar-agar. The rooting of the plants on agar medium was 95%, while on hydrogel substrates – higher than 98%. On hydrogel substrates with the addition of amber, there was a 1.7-fold increase in shoot growth intensity, and on substrates without amber – 1.5 times. When the plants remained for 10–15 days in a tall vessel, their height reached 110–130 mm (Fig. 5 ). In vitro deposition was carried out with periodic transplantations to the new nutrient medium to avoid drying out and changes in the composition of the environment due to the effect of the products of plant metabolism. After the first cycle, the spent hydrogel material was regenerated – washed in distilled water, sterilized, and used in repeated cultivation cycles. The obtained in vitro rooted plants Cánnabis satíva are suitable for conversion in vivo (Fig. 6 ). The increase in the number of the metric indicators of leaf blades were also found. During transferring plants from іn vitro to іn vivo it was found that the dose-dependent effect of plant knees on test subjects persists ( Fig. 7 , Table 5 ). In general, using a hydrogel instead of agar stimulated the growth of Cánnabis satíva . Use of hydrogel-amber substrate increased metric indicators of seedling (in comparison to agar): the root length increased by 28%, stem length – by 26.7%, root weight – by 167%, stem weight – by 67%, root and stem length – by 27%, and root and stem weight – by 50%. Table 5 Metric indicators of Cánnabis satíva seedlings Experiment techniques Cánnabis satíva seedlings Root length (cm) Stem length (cm) Root weight (g) Stem weight (g) Root and stem lengths (cm) Root and stem weight (g) In vitro Agar 3.5 7.5 0.3 0.6 11 0.9 PAAG 4 8 0.6 0.7 12 1.3 PAAG + A2 4.5 9.5 0.8 1 14 1.8 In vivo Sоil 15.2 21.8 1.3 1.4 37 2.7 After hydrogel recycling (ten-fold washing with distilled water), all nutrients, succinic acid and products of its transformation were much better absorbed by the plant and stimulated the growth and development of seedlings after transfer in vivo . In addition, the allelopathic activity of extract from biological material in 1:10, 1:50, and 1:100 dilutions was studied. It has been shown that allelopathic stress is dose-dependent when used a typical MS medium. Under 1:10 dilution in vitro , Cucumis sativus L . growth inhibition was observed up to 53%; however growth inhibition was reached 87% (Table 6 ). Under 1:100 dilution іn vivo "Suppressive" effect almost didn’t fall (95%), which indicates the successful transfer of plants from іn vitro to іn vivo. This pattern can be traced on the example of morphometric parameters of Cánnabis satíva seedlings (Table 6 ). Table 6 Allelopathic effect of aqueous extract of aboveground parts of Cánnabis satíva on seedlings of Cucumis sativus L . Dilution of the extract In vivo , (%) In vitro , (%) 1:10 67 53 1:50 75 60 1:100 95 87 Thus, both in vivo and in vitro a positive effect of amber in the composition of the hydrogel substrate on the main parameters of germination and development of the studied plants was revealed. According to the possible influence mechanism the biologically active components of highly dispersed amber powder affect biological objects at the cellular level, increasing the efficiency of processes in plants, and also take part in forming the microelement balance, i.e., they are bioactive. Highly dispersed amber powder embedded in the copolymer matrix is non-toxic, released gradually, its ionic form quickly includes in biochemical reactions, and therefore, when washing it with MS culture medium, the biologically active amber acid is washed out prolonged and dosed. This fact explains the high bioavailability and biocompatibility of the synthesized nutrient substrate, the possibility of obtaining planting material in a shorter time, accelerated transition of plants from the juvenile to reproductive phase of development, and increased intensity of growth and development of the main shoot. Conclusions A spatially cross-linked polyacrylamide hydrogel with immobilized amber was synthesized. It has been demonstrated that in terms of its physicochemical and rheological properties, the obtained material is similar to agar-agar and can be used as its inexpensive substitute for micropropagation of plants. It is biosafe after four washings. During them, all unreacted toxic monomers and initiator residues were removed from the hydrogel structure, that is, it was purified effectively. The biosafety of new hydrogel was confirmed by the experiments performed using both biological objects, such as pea/chickpea seeds and D. magna , and traditional UV spectroscopic method. There was no mortality for both legume seeds and D. magna after application of water from 4th washing. The use of new hydrogel, instead of agar, stimulated the growth of Cánnabis satíva . For example, the root weight increased by 167%, whilst the stem weight, by 67%. Thus, the described materials should be considered as novel effective nutrient media, which can accelerate reproduction and allow obtaining a higher amount of plant material within a short period of time in comparison with the standard agar medium. Declarations Author contributions LK, OD and OM conceived and designed the study. LK, KS, OG, NP, TP and OD performed the experiments. LK, OG, OS and KSK analyzed the data, contributed inputs, wrote and critically reviewed the manuscript. Funding: The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Conflict of interest The authors declare that they have no conflict of interest. Financial interests: The authors have no relevant financial or non-financial interests to disclose. References Adhikary D, Kulkarni M, El-Mezawy A et al (2021) Medical Cannabis and Industrial Hemp Tissue Culture: Present Status and Future Potential. Front Plant Sci 12:275. https://doi.org/10.3389/FPLS.2021.627240/BIBTEX Bataineh SM, Saadoun I, Hameed KM, Ababneh Q (2008) Screening for soil streptomycetes from North Jordan that can produce herbicidal compounds. Pol J Microbiol 57:297–305 Boldeskul IE, Samchenko YM, Sukhodub LB et al (2009) Application of vibrational spectroscopy in express analysis of acrylic hydrogel and hydroxyapatite based composite materials. Methods objects Chem Anal 4:92–96 Delgado-Aceves L, Portillo L, Folgado R et al (2022) New approaches for micropropagation and cryopreservation of Agave peacockii, an endangered species. Plant Cell Tissue Organ Cult 150:85–95. https://doi.org/10.1007/S11240-022-02246-Z/TABLES/4 Espinosa-Leal CA, Puente-Garza CA, García-Lara S (2018) In vitro plant tissue culture: means for production of biological active compounds. Planta 2018 2481 248:1–18. https://doi.org/10.1007/S00425-018-2910-1 Gertsiuk M, Samchenko YM(2007) Separation of nonreacted acrylamide from polyacrylamide gel for endoprothesing.Ars Separatoria Acta98–101 Goncharuk O, Samchenko Y, Kernosenko L et al (2020) Thermoresponsive hydrogels physically crosslinked with magnetically modified LAPONITE® nanoparticles. Soft Matter 16:5689–5701. https://doi.org/10.1039/D0SM00929F Grodzinsky AM, Golovko EA, Gorobets SA (1987) Experimental allelopathy. Naukova Dumka, Kyiv Henderson WE, Kinnersley AM (1988) Corn starch as an alternative gelling agent for plant tissue culture. Plant Cell Tissue Organ Cult 1988 151 15:17–22. https://doi.org/10.1007/BF00039885 Jain N, Babbar SB (2002) Gum katira – a cheap gelling agent for plant tissue culture media. Plant Cell, Tissue Organ Cult 2002 713 71:223–229. https://doi.org/10.1023/A:1020379024347 Kaçar YA, Biçen B, Varol et al (2010) Gelling agents and culture vessels affect in vitro multiplication of banana plantlets. Genet Mol Res 9:416–424. https://doi.org/10.4238/VOL9-1GMR744 Karolina D, Maja MS, Magdalena DS, Grażyna Ż (2022) Identification of treated Baltic amber by FTIR and FT-Raman - A feasibility study. Spectrochim Acta A Mol Biomol Spectrosc 279:121404. https://doi.org/10.1016/J.SAA.2022.121404 Kaur J, Mudgal G (2021) An efficient and quick protocol for in vitro multiplication of snake plant, Sansevieria trifasciata var. Laurentii [Prain]. Plant Cell Tissue Organ Cult 147:405–411. https://doi.org/10.1007/S11240-021-02132-0/FIGURES/2 Khan F, Atif M, Haseen M et al (2022) Synthesis, classification and properties of hydrogels: their applications in drug delivery and agriculture. J Mater Chem B 10:170–203. https://doi.org/10.1039/D1TB01345A Kim HJ, Koedrith P, Seo YR (2015) Ecotoxicogenomic Approaches for Understanding Molecular Mechanisms of Environmental Chemical Toxicity Using Aquatic Invertebrate, Daphnia Model Organism. Int J Mol Sci 16:12261. https://doi.org/10.3390/IJMS160612261 Kocak G, Tuncer C, Bütün V (2016) pH-Responsive polymers. Polym Chem 8:144–176. https://doi.org/10.1039/C6PY01872F Kosenko OO, Lukash LL, Samchenko YM et al (2006) Copolymeric hydrogel membranes for immobilization and cultivation of human stem cells. Biopolym Cell 22:143–148. https://doi.org/10.7124/BC.000729 Levchyck NY, Liubinska AV, Skrypchenko NV et al (2017) Biological activity of aqueous solution of amber. Biotechnol Acta 10:53–60. https://doi.org/10.15407/BIOTECH10.06.053 Lubell-Brand JD, Kurtz LE, Brand MH (2021) An In Vitro–Ex Vitro Micropropagation System for Hemp. Horttechnology 31:199–207. https://doi.org/10.21273/HORTTECH04779-20 Mänd K, Muehlenbachs K, McKellar RC et al (2018) Distinct origins for Rovno and Baltic ambers: Evidence from carbon and hydrogen stable isotopes. Palaeogeogr Palaeoclimatol Palaeoecol 505:265–273. https://doi.org/10.1016/J.PALAEO.2018.06.004 Milani P, França D, Balieiro AG, Faez R (2017) Polymers and its applications in agriculture. Polimeros 27:256–266. https://doi.org/10.1590/0104-1428.09316 Mironov OL, Kachalova NM, Dzyuba OI et al (2018) Research of biological activity of Ukrainian amber. In: Popov A (ed) Physico-organic chemistry, pharmacology and pharmaceutical technology of biologically active substances: coll. science. work. KNUTD, Kyiv, pp 100–120 Mood K, Jogam P, Sirikonda A et al (2022) Micropropagation, morpho-anatomical characterization, and genetic stability studies in Lippia javanica (Burm.f.) Spreng: a multipurpose medicinal plant. Plant Cell Tissue Organ Cult 150:427–437. https://doi.org/10.1007/S11240-022-02294-5/FIGURES/4 Nakanishi K (1962) Infrared Absorption Spectroscopy, Practical. Nankodo, Tokyo, Japan; Holden-Day, San Francisco, Calif Olkova A, Zimonina N (2020) Assessment of the Toxicity of the Natural and Technogenic Environment for Motor Activity of Daphnia magna. J Ecol Eng 21:11–16. https://doi.org/10.12911/22998993/125459 Ramli RA (2019) Slow release fertilizer hydrogels: A review. Polym Chem 10:6073–6090 Rand GM, Wells PG, McCarty LS (2020) Introduction to Aquatic Toxicology. Fundam Aquat Toxicol 3–67. https://doi.org/10.1201/9781003075363-2 Samchenko Y, Korotych O, Kernosenko L et al (2018) Stimuli-responsive hybrid porous polymers based on acetals of polyvinyl alcohol and acrylic hydrogels. Colloids Surf Physicochem Eng Asp 544:91–104. https://doi.org/10.1016/J.COLSURFA.2018.02.015 Schmaljohann D (2006) Thermo- and pH-responsive polymers in drug delivery. Adv Drug Deliv Rev 58:1655–1670. https://doi.org/10.1016/J.ADDR.2006.09.020 Shimizu E, Shimoda N, Kawamura T et al (2020) Comparison of the Biological Activity and Constituents in Japanese Ambers. Adv Biol Chem 10:99–112. https://doi.org/10.4236/ABC.2020.103008 Singh N, Agarwal S, Jain A, Khan S (2021) 3-Dimensional cross linked hydrophilic polymeric network “hydrogels”: An agriculture boom. Agric Water Manag 253:106939. https://doi.org/10.1016/j.agwat.2021.106939 Sinha V, Chakma S (2019) Advances in the preparation of hydrogel for wastewater treatment: A concise review. J Environ Chem Eng 7:103295. https://doi.org/10.1016/J.JECE.2019.103295 Stadniy IA, Konovalova VV, Samchenko YM et al (2011) Development of Hydrogel Polyelectrolyte Membranes with Fixed Sulpho-Groups via Radical Copolymerization of Acrylic Monomers. Mater Sci Appl 2:270–275. https://doi.org/10.4236/MSA.2011.24035 Tumiłowicz P, Synoradzki L, Sobiecka A et al (2016) Bioactivity of Baltic amber – fossil resin. Polimery 61:347–356. https://doi.org/10.14314/POLIMERY.2016.347 Tuson HH, Renner LD, Weibel DB (2012) Polyacrylamide hydrogels as substrates for studying bacteria. Chem Commun 48:1595–1597. https://doi.org/10.1039/C1CC14705F (2004) Test No. 202: Daphnia sp.Acute Immobilisation Test (1982) Seeds of farm crops. Methods for determination of viability.USSR State Stand ISO - ISO 10706:2000 - Water quality — Determination of long term toxicity of substances to Daphnia magna Straus (Cladocera, Crustacea). https://www.iso.org/standard/18795.html . 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2085035","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":144383649,"identity":"d0973b77-1936-46d1-96fa-6536f53028f5","order_by":0,"name":"Lyudmyla Kernosenko","email":"","orcid":"","institution":"National Academy of Sciences of Ukraine Department of Chemistry: Nacional'na akademia nauk Ukraini Viddilenna 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himii","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Oleg","middleName":"","lastName":"Mironov","suffix":""},{"id":144383657,"identity":"af9511fe-e50d-4891-9ff3-efdbecebe453","order_by":8,"name":"Katarzyna Szewczuk-Karpisz","email":"","orcid":"","institution":"Institute of Agrophysics Polish Academy of Sciences: Instytut Agrofizyki im Bohdana Dobrzanskiego Polskiej Akademii Nauk","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Katarzyna","middleName":"","lastName":"Szewczuk-Karpisz","suffix":""}],"badges":[],"createdAt":"2022-09-20 13:03:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2085035/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2085035/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.3390/plants12051196","type":"published","date":"2023-03-06T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":27955910,"identity":"4e3a1788-43a6-4919-9d26-8088f654b344","added_by":"auto","created_at":"2022-10-18 19:40:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":130827,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of amber, polyacrylamide hydrogel and polyacrylamide hydrogel with amber\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2085035/v1/80ae3cfa85db4175e0169f70.png"},{"id":27955911,"identity":"e4860189-54bc-425a-91ab-b5b56863d8d9","added_by":"auto","created_at":"2022-10-18 19:40:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":16477,"visible":true,"origin":"","legend":"\u003cp\u003eDynamics of mortality of individuals \u003cem\u003eD.magna\u003c/em\u003e depending on the number of washings: PAAG – the homopolyacrylamide gel; PAAG-A1 – the homopolyacrylamide gel containing 5.8 % dark amber; PAAG-A2 – the homopolyacrylamide gel containing 5.7 % light amber\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2085035/v1/78e8ba4dfc0e3d60c19777d1.png"},{"id":27955915,"identity":"77defb6f-a5cd-4ed6-ac5b-d4eb27c779e9","added_by":"auto","created_at":"2022-10-18 19:40:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":557789,"visible":true,"origin":"","legend":"\u003cp\u003eVisualization of Nelyubov method used for determining the viability of peas (b,d) and chickpeas (c,e): a – the control (distilled water), b,c – the first washing, d,e – the second washing. The incubation time was 90 min.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2085035/v1/4304dbce0daa69ed4390d74c.png"},{"id":27956053,"identity":"5ba38bee-0eab-4152-8b71-aebf73cd3fce","added_by":"auto","created_at":"2022-10-18 19:45:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":28422,"visible":true,"origin":"","legend":"\u003cp\u003eDependence of effective viscosity (η, mPa ∙ s) of polyacrylamide gel (PAAG) (b,d) and agar gel (a, c); a,b - on the shear rate (γ, s\u003csup\u003e-1\u003c/sup\u003e); c,d - on the shear rate (γ, s\u003csup\u003e-1\u003c/sup\u003e) over time (t, min)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2085035/v1/006fa323798ba7d38932f7cf.png"},{"id":27955912,"identity":"6482fd96-baeb-4b5f-ae51-293eb2e20efa","added_by":"auto","created_at":"2022-10-18 19:40:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1972144,"visible":true,"origin":"","legend":"\u003cp\u003eVisualization of the first cultivation cycle of \u003cem\u003eCánnabis satíva\u003c/em\u003e (14 days) on agar substrate (a), polyacrylamide (PAAG) (b), and polyacrylamide substrate with the addition of amber (PAAG-A2) (c)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2085035/v1/3d48bb45c25e27880ab888fc.png"},{"id":27956580,"identity":"cf5a5a07-31dc-46ae-818d-7033eb0feb5a","added_by":"auto","created_at":"2022-10-18 19:50:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":448774,"visible":true,"origin":"","legend":"\u003cp\u003eSeedlings of \u003cem\u003eCánnabis satíva\u003c/em\u003e after transferfrom \u003cem\u003ein vivo \u003c/em\u003eto the phytotron – the glasshouse of the M. M. Hryshko National Botanical Garden of the National Academy of Sciences of Ukraine, Allelopathy Department\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2085035/v1/0967b339f431f2e83b90e5e7.png"},{"id":27956051,"identity":"554418a3-5c44-4480-9852-bf636b1737b3","added_by":"auto","created_at":"2022-10-18 19:45:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":251101,"visible":true,"origin":"","legend":"\u003cp\u003eMetric indicators visualization of seedling growth \u003cem\u003eCánnabis satíva\u003c/em\u003e, transferred \u003cem\u003ein vivo: \u003c/em\u003eon agar substrate (1), on polyacrylamide substrate (PAAG) (2),and on polyacrylamide substrate with amber addition (PAAG-A2) (3)\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-2085035/v1/fcc955b9989730274ae32264.png"},{"id":36337603,"identity":"08580828-3ec2-41c1-886c-c3a1f9932de0","added_by":"auto","created_at":"2023-04-26 17:11:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3100004,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2085035/v1/b1108fca-6750-4904-a58c-94d7863d789a.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003ePolyacrylamide Hydrogels With Amber for Plants Micropropagation\u003c/p\u003e","fulltext":[{"header":"Key Message","content":"\u003cp\u003ePolyacrylamide hydrogel with amber powder can be used as an agar-agar substitute for micropropagation of plants.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eUsually, the \u003cem\u003ein vitro\u003c/em\u003e cultivation and reproduction of plants are carried out in solid nutrient media, and a high-cost agar substrate is the most used in this process (Ka\u0026ccedil;ar et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Adhikary et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lubell-Brand et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kaur and Mudgal \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Delgado-Aceves et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mood et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This material is non-renewable, so for each subsequent passage of plants it must be used \u003cem\u003ede novo\u003c/em\u003e(Espinosa-Leal et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Because of agromarket\u0026rsquo;s needs to propagate plants commercially, the agar substrates are used in large amounts. To avoid such high costs, the search for alternative approaches is demanded. There is a great need to substitute the agar with cheaper disposable materials, such as starch (Henderson and Kinnersley \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1988\u003c/span\u003e) or gum (Jain and Babbar \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), or to develop an environmentally friendly solid substrate with optimal physicochemical and functional properties suitable for multiple uses.\u003c/p\u003e \u003cp\u003eThese requirements can be met by hydrogels based on spatially cross-linked hydrophilic polymers, which have the inherent ability to sorb and retain a significant amount of aqueous solutions due to adequate swelling properties. This process leads to an increase in hydrogel volume but its geometric shape remains unchanged. The so-called \"smart hydrogels\" are spatially cross-linked hydrophilic polymers with a three-dimensional structure capable of responding to various external physical factors, including minor changes in pH, temperature, ionic strength, etc., by changing the swelling degree, volume, sorption and diffusion characteristics (Schmaljohann \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Kocak et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Samchenko et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Goncharuk et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). They can be synthesized in different consistency and arbitrary shape, particularly in the form of granules of controlled sizes. Such hydrogels are characterized by high hydrophilicity and biological tolerance, as well as improved optical, sorption, and diffusion properties, which can be adjusted by varying the monomer composition and cross-linking density. The listed peculiarities make them suitable for immobilization of a wide range of both organic and inorganic compounds, as well as for use in novel technologies, namely water treatment(Sinha and Chakma \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), manufacturing of selective membranes (Kosenko et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Stadniy et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), cell cultivation (in particular stem ones) (Kosenko et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), targeted delivery, and controlled release of anticancer drugs (Schmaljohann \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In recent years, the substrates based on such hydrogels found application for plant vegetation under controlled conditions due to their ability to sorb and prolongedly release the necessary bioelements into the environment under the plant root exudates action (Milani et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ramli \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Singh et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Khan et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSome natural organic compounds with fungicidal and antibacterial properties can be included in hydrogels structure to enhance their functional properties. Primarily succinic acid and products of its transformation can be used as an additional source of microelements, endogenous physiologically active substances, growth or cell metabolism stimulators (Tumiłowicz et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Levchyck et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mironov et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Shimizu et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It is known that amber, in particular, amber chips from the waste of various industries, is an environmentally safe and competitive product, so the inclusion of natural organic compounds in hydrogel substrates can be an effective and cost-effective way to increase the biological activity of the nutrient medium for plants micropropagation. In addition to the increasing bioactivity issue, the problem of ensuring biosafety is relevant for such materials. Since the monomers that compose the basic structural unit of polymers and, particularly hydrogels, are rather toxic materials and part of them always remains unreacted, the problem of synthesized hydrogels purification from residual microquantities of initial compounds is extremely important. The most affordable and effective way to purify hydrogels is to wash them repeatedly with distilled water. The risk control of toxic compounds in washing water is one of the most important steps in the preparation of synthesized material for further use.\u003c/p\u003e \u003cp\u003eToday toxicology uses not only traditional spectrometric methods of analysis, but also a variety of biotesting methods that allow obtaining a comprehensive toxicological assessment of the aqueous environment using living test objects, including plants (cereals and legumes, some algae, etc.) and animals (unicellular, crustaceans, worms, etc.). Bioassays involving aquatic organisms, such as \u003cem\u003eDaphnia magna\u003c/em\u003e, are common and widely used to study the toxicity of various chemicals (Kim et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Olkova and Zimonina \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Biological tests on a biomodel \u003cem\u003eD. magna\u003c/em\u003e are standardized in many countries (2004). Short-term testing allows determining the acute toxic effect of compounds in aqueous solution on the survival rate of branched crustaceans \u003cem\u003eD. magna\u003c/em\u003e, which is one of the most sensitive biomarkers for determining the toxicity caused by different classes of chemical compounds (2004; Kim et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Rand et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Olkova and Zimonina \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe aim of the work is to develop the solid nutrient media for plants (namely, \u003cem\u003eCannabis sativa\u003c/em\u003e) micropropagation based on highly dispersed polymer hydrogels with biologically active amber compounds, which will meet the requirements of high biocompatibility and suitable moisture content, as well as will demonstrate the environmental safety.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eAcrylamide (AA) (C\u003csub\u003e3\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eNO, \"MERCK\", Germany); N, N'-methylenebisacrylamide (MBA) (C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, \"MERCK\", Germany); potassium persulfate (K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e, \"SIGMA\", USA); sodium metabisulfite (Na\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e,\"SIGMA\", USA) were applied in the study without additional purification. Double-distilled water was used as a solvent in all experiments.\u003c/p\u003e \u003cp\u003eFor the synthesis of modified polyacrylamide gel (PAAG), the samples of Ukrainian raw amber from Zhytomyr, Olevsk and Rivne regions (Klesovo and Volodymyrets-Vostochny fields) were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMethods\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eSynthesis of PAAG\u003c/h2\u003e \u003cp\u003eThe method of homophase radical polymerization in aqueous medium was used for synthesis of hydrogels based on acrylamide (Gertsiuk and Samchenko \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Polymerization was carried out at room temperature. The monomer solution was stirred on a laboratory magnetic stirrer (MM-5, 1200 rpm), then the red-ox initiation system (potassium persulfate\u0026ndash;sodium metabisulfite) was added. The components' concentrations in the initiating mixture were chosen to provide the polymerization completion within an hour and to prevent excessive composition heating, which could adversely affect the hydrogels' properties. Cross-linking with the spatial network formation occurred due to copolymerization with a bifunctional monomer N,N'-methylene-bis-acrylamide (MBA). The gel was dispersed in a mortar to a granule size of \u0026Oslash;1\u0026ndash;2 mm. The components used for PAAG synthesis are summarized 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\u003eComposition of reagents used for synthesis of PAAG samples\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"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=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater, g\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAA, g\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMBA, g\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAmber, %\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAAG -A1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAAG -A2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe synthesized PAAGs were repeatedly washed from unreacted components of the reaction mixture in distilled water in a ratio of 1 to 50 at a temperature of 45\u0026deg;C for 7 days. The washing process was monitored spectrophotometrically using a UV spectrophotometer SPECORD M40 (Carl Zeiss) (Gertsiuk and Samchenko \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The washing water was further analyzed for the presence of toxic impurities using test objects of plant and animal origin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eModification of PAAG with amber\u003c/h2\u003e \u003cp\u003eThe raw amber was grounded using the laboratory mill Kinematica AG (Polymix\u0026reg; PX-MFC 90 D), and then it was divided into fractions sizes from 2\u0026ndash;3 to15-20 mm. Milled samples of raw amber were pre-washed in running water and 10% NaCl. After that, the amber samples were cleaned from NaCl residues and dried at room temperature for several days or in a drying cabinet at 40-50\u003csup\u003eo\u003c/sup\u003eC. The amber samples were differentiated by color into groups: opaque/transparent \u0026ndash; the dark amber (samples A1); translucent/transparent \u0026ndash; the light amber (samples A2). Amber samples were pre-cooled by liquid nitrogen to prevent the destruction of natural components during mechanical grinding due to overheating.\u003c/p\u003e \u003cp\u003eSynthesis of polyacrylamide gels containing amber and cleaning them from unreacted components were performed according to the method described above. The fine amber A1 and A2 were added to the reaction mixture at a ratio of monomers and amber of 20 to 1 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003eBiotesting of acrylamide using pea and chickpea seeds\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eBiotesting of the toxicity of washing waters was performed by the Nelyubov method (1982), which bases on the fact that dyes stain only dead cell plasma (the plasma of a viable cell remains unstained). The viability was determined using pea and chickpea seeds. According to this technique, the seeds were soaked and, after 8 h, were released from the seed coat with a needle without damaging them. Ten peas were placed in the aqueous solutions of acrylamide with a concentration ranging from 0.001\u0026ndash;10% and kept at a temperature of 30 \u003csup\u003eo\u003c/sup\u003eC for 3 h. Then the peas were transferred to 0.2% indigo carmine aqueous solution and kept for the next 3 h, after which the dye was drained, the peas were washed with distilled water, and their viability was determined according criteria of compliance with the conditions of viability shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Biotesting of washing waters (from the 1st to the 7th washings) was carried out similarly.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eConditions for determining the seeds\u0026rsquo; viability according to Nelyubov (1982):\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eViable seeds\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNon-viable seeds\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eall parts are not stained\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eall parts of the embryo are stained\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003enot stained root\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ethe tip of the root contains unstained spots\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003enot stained area around the root\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ein the lower part of the cotyledon unstained spots\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eroot and cotyledon are completely/incompletely stained\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecotyledonous stained at the bottom\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecotyledonous is not painted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBioassay using\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eDaphnia magna\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe acute toxicity of polyacrylamide hydrogel was also determined using the model of the hydrobiont \u003cem\u003eD. magna\u003c/em\u003e (according to ISO 10706:2000([CSL STYLE ERROR: reference with no printed form.])). This method is based on estimating the influence of aqueous solutions on the \u003cem\u003eD. magna\u003c/em\u003e mortality rate (%). \u003cem\u003eD. magna\u003c/em\u003e was kept in ventilated aquariums with carbon-filtered tap water (pH\u0026thinsp;=\u0026thinsp;7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3) at a temperature of 18\u0026ndash;22 \u0026ordm;C and a dissolved oxygen concentration\u0026thinsp;\u0026gt;\u0026thinsp;6.0 mg/L ([CSL STYLE ERROR: reference with no printed form.]). The illumination of the cultivation was 400\u0026ndash;600 lux at a light period of 16\u0026thinsp;\u0026plusmn;\u0026thinsp;1 h, and 8\u0026thinsp;\u0026plusmn;\u0026thinsp;1 h of darkness. The water from the 1st to the 7th washings with an acrylamide concentration of 0.00125 to 0.00001 mmol/L were used for cultivation. Distilled water was used as a control. The experiment used newborns aged 12\u0026ndash;24 h, obtained by cultivation. In each 50-ml glass container with 30 ml of the test solution, seven individuals of \u003cem\u003eD. magna\u003c/em\u003e were placed. The newborn daphnias were fed using \u003cem\u003eChlorella vulgaris\u003c/em\u003e or a suspension of baker's yeast 2 h before the experiment and were not fed during the experiments. The mortality of individuals in each beaker was assessed within 24 and 48 h. Specimens that moved freely in the water column or floated to the tank surface no later than 15 sec after light shaking were considered alive ones. The experiments were performed in triplicate. The sensitivity of \u003cem\u003eD. magna\u003c/em\u003e to the reference model toxicant, potassium dichromate (K\u003csub\u003e2\u003c/sub\u003eCr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e), was also determined (for 24 h).This allowed to assess the suitability of \u003cem\u003eD. magna\u003c/em\u003e culture for biotesting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eInvestigation of hydrogel structure and rheological properties\u003c/h2\u003e \u003cp\u003eFourier transform infrared spectroscopy (FTIR) spectra of powdered samples over the 4000\u0026ndash;400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range were recorded using a ThermoNicolet iS10 FTIR spectrometer with a diffuse reflectance mode.\u003c/p\u003e \u003cp\u003eThe rheological properties of PAAG were investigated by a rotary viscometer Rheotest 2.1 using a cylindrical system Z in the range of shear rates from 2.43 to 1073 с\u003csup\u003e\u0026minus;1\u003c/sup\u003e at a temperature of 20\u0026deg;C. For rheological properties investigation, the synthesized chemically cross-linked PAAG were pre-treated in a ball mill with subsequent sieving to obtain a fraction of gel particles d\u0026thinsp;\u0026lt;\u0026thinsp;1 mm in the non-swollen state. Then, the dry gel particles were mixed with a given amount of water, which corresponded to the conditions of their use for the microclonal propagation of plants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eMicropropagation\u003c/h2\u003e \u003cp\u003eGlesia industrial hemp (\u003cem\u003eCannabis sativa\u003c/em\u003e) seeds were selected as test objects. The used variety of monoecious non-narcotic hemp with dense rhomboid inflorescences and seed productivity provides the ability to produce a seed yield of 2.0-2.2 t/ha. The period from emergence to the onset of the phase of technical maturity is 88\u0026ndash;93 days, and to the onset of the phase of biological maturity \u0026ndash; is 100\u0026ndash;120 days. The seeds were obtained from the Institute of Bast Crops of the National Academy of Agrarian Science of Ukraine. The seeds and cuttings of juvenile plants were the primary materials for obtaining sterile (microorganism-free) hemp plants \u003cem\u003ein vitro\u003c/em\u003e. The cuttings were pre-treated with 70% ethanol for 1 min with the following treatment with 0.5% thiomerosal (C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e9\u003c/sub\u003eHgNaO\u003csub\u003e2\u003c/sub\u003eS) for 1.5 min. In turn, the seeds were pre-treated with 70% ethanol for 3 min with the following treatment with 0.5% thiomerosal for 5 min. Sterilized seeds and cuttings were transferred to a nutrient medium and germinated at 26\u0026deg;C. The duration of the period before the emergence of seedlings ranged from 14 to 20 days. Grafting of plants was performed in the presence of 3\u0026ndash;5 internodes on plants. 1.0\u0026ndash;1.5 cm parts of the stem (micropub) with two axillary buds basal part were placed vertically in agar nutrient medium to a depth of 0.3\u0026ndash;0.5 cm.\u003c/p\u003e \u003cp\u003eCultivation was performed on solid substrates: agar-agar (control) and hydrogel substrates: PAAG and PAAG-A2, saturated with Murashige-Skuga (MS) culture medium with the concentration of macronutrients reduced by half (MS/2). This medium contains 0.5 doses of macro- and micronutrients with the addition of 30 g/L sucrose and has a slightly acidic reaction (pH 5.6-6.0). For comparison, the agar-based (7.45 g/L) medium with the same MS and sucrose content was used. The complete cultivation cycle was 60 days. The first stage of introduction to the culture took place at the air temperature of 26\u0026ndash;28\u0026deg;C. The obtained specimens were subcultured on MS medium under illumination with fluorescent lamps (2000\u0026ndash;2500 lux) with a 16-hour photoperiod at a temperature of 24\u0026ndash;26\u0026deg;C and a humidity of 70%.\u003c/p\u003e \u003cp\u003eIn order to evaluate the allelopathic activity of aerial parts of hemp \u003cem\u003eCannabis sativa L.\u003c/em\u003e extracts, (Grodzinsky et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1987\u003c/span\u003e) the bioassays were employed. In this method, the one-day seedlings of cucumber \u003cem\u003eCucumis sativus L.\u003c/em\u003e cv. Konkurent were used as a test object (Bataineh et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Results And Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFTIR\u003c/h2\u003e \u003cp\u003eThe FTIR spectra of dried initial PAAG gel, initial amber, and PAAG -gels with amber are given in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The most informative peaks of all studied materials are in the range of 1800\u0026ndash;800 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The spectrum of PAAG shows two bands at 3436 and 2924 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which correspond with the N\u0026ndash;H stretching vibration of the NH\u003csub\u003e2\u003c/sub\u003e group and C\u0026ndash;H stretching vibrations. The bands at 1645 and 1465 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are attributed to the stretching of the C\u0026thinsp;=\u0026thinsp;O group in amide and CH\u003csub\u003e2\u003c/sub\u003e scissoring, respectively (Nakanishi \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1962\u003c/span\u003e). In the FTIR spectrum of initial amber, the wide shoulder of the 1160 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e peak stretching to 1260 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (known as \u0026ldquo;Baltic shoulder\u0026rdquo;) corresponds to the high content of succinic acid and other succinate compounds. The intense peak at ~\u0026thinsp;1700 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is characteristic of the C\u0026thinsp;=\u0026thinsp;O group of carboxylic acids. The bands at 1445 and 1375 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are attributed to C\u0026ndash;H symmetric and asymmetric stretching vibrations. The 887 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e band could be assigned to the out-of-plane aromatic C\u0026ndash;H bending.(M\u0026auml;nd et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Karolina et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the spectra of polyacrylamide hydrogels with amber, the bands of amide I (1647 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and amide II (1605 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of acrylamide have the highest intensity. The bands in the range of 3000\u0026ndash;3500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e correspond with symmetric and asymmetric vibrations of amino groups ν\u003csub\u003e(NH)\u003c/sub\u003e of polyacrylamide. The maximum at 2938 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is attributed to stretching vibrations of the methylene group. The intense maximum ν\u003csub\u003e(C=O)\u003c/sub\u003e at 1647 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Amide I) corresponds with the amide fragment, which overlaps with the bending vibration maximum δ\u003csub\u003e(NH2)\u003c/sub\u003e at 1605 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Amide II) with the formation of a broadened doublet. In the \"fingerprint\" region, a doublet at 1450\u0026ndash;1410 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e caused by bending vibrations of the CH group, as well as a broadened band at ν\u003csub\u003e(CN)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1350 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Amide III) were noted.(Nakanishi \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1962\u003c/span\u003e; Boldeskul et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAcute toxicity\u003c/h2\u003e \u003cp\u003eSynthetic hydrogels, created by polymerization of highly toxic monomers such as acrylamide, contain unreacted residues that should be removed before using them for medical and biological purposes(Gertsiuk and Samchenko \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Acute toxicity of the studied compounds of different washes (from the 1th to the 7th) was determined based on the mortality rate (%) of \u003cem\u003eD. magna.\u003c/em\u003e Data on the survival of individuals in each sample during 24 and 48 h of the exposure are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOverall risk assessment of toxic compounds present in washing waters on the viability of biomarkers of plant (legume) and animal (\u003cem\u003eD. magna\u003c/em\u003e) origin\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\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=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eWashing / AA solution\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eConc. AA,\u003c/p\u003e \u003cp\u003emmol/l\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c8\" namest=\"c3\"\u003e \u003cp\u003eDeath toll, %\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u003cb\u003ePAAG\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e\u003cb\u003ePAAG-A1\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e\u003cb\u003ePAAG-A2\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003edaphnia\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003epeas / chickpeas\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003edaphnia\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003epeas / chickpeas\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003edaphnia\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003epeas / chickpeas\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAA solution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80/80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e80/80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e80/90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAA solution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40/70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e40/80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e50/70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAA solution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40/70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e40/80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e50/70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.00125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10/20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10/20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10/20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.00096\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e28.6**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e28.6**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.00054\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42.8**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.00007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.00003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.00002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.00001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl (DV)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003e* - died on the first day of the experiment\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003e** - died on the second day of the experiment\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe performed experiments indicated that after the first wash of all tested samples, complete death of organisms (100%) was already recorded during 24 and 48 h of the experiment. However, the mortality decreased by 28.6% and 57.2% during 48 h in the second washing water for PAAG-A2 and PAAG-A1, respectively, (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In the third washing water, the mortality of \u003cem\u003eD. magna\u003c/em\u003e individuals in the PAAG-A1 and PAAG-A2 samples was absent. No cases of mortality were recorded during 24 and 48 h of the experiment. Based on these results, \u003cem\u003eD. magna\u003c/em\u003e was considered as an organism of high sensitivity to washing water composition, which can be used for diagnosis and risk assessment of the hydrogels and unreacted compounds.\u003c/p\u003e \u003cp\u003eThe results of biotesting of the toxicity of washing water by the Nelyubov method are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. It was found that 1\u0026ndash;3 washes of hydrogels, in which the concentration of acrylamide was from 0.00054 mol/dm\u003csup\u003e3\u003c/sup\u003e to 0.00125 mol/dm\u003csup\u003e3\u003c/sup\u003e (according to the results of measurements using a UV spectrometer SPECORD M40), were the most toxic for all tested legumes. For peas, 10% of seeds was dead, while for chickpeas, 20%. The results of both biotesting experiments are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. They indicated that the washing water from the 4th wash is safe for both \u003cem\u003eD. magna\u003c/em\u003e and legumes. The hydrogels, washed in this way, can be safely used in practice. Tucson et al. also washed PAAG for three days (changing the water every 24 hours), after which the gel was safe for further use as substrates for the study of bacteria (Tuson et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRheological properties\u003c/h2\u003e \u003cp\u003eThe structural and mechanical characteristics of hydrogel composites largely determine the application possibilities of these systems. They are related to bioavailability and release of biologically active components from hydrogel materials. Gels are structured systems that demonstrate the structural and mechanical properties of both liquids and solids. Hydrogel, as a dispersed system, acquires the properties of a solid body, that is, shear modulus and elasticity. The most important rheological characteristics of hydrogels include shear stress and viscosity. Effective viscosity is a characteristic of the equilibrium state between the processes of destruction and recovery. Its fluctuation causes a change in the coagulation-crystallization structure of the hydrogel, affecting its performance characteristics. The spatial structure in the hydrogel is determined by measuring the mechanical properties and, in particular, shear deformation under the constant stress. Solids are characterized by a sharp change in the pattern of shear deformation \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eε\u003c/span\u003e depending on the magnitude of the shear stress \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eР\u003c/span\u003e. At rather low stresses (less than the yield strength \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eP\u003c/span\u003e\u003csub\u003ek\u003c/sub\u003e), a free flow with constant and extremely high viscosity \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eη\u003c/span\u003e\u003csub\u003e1\u003c/sub\u003e is observed. In this case, the coagulation structure is destroyed, but has time to recover. As the shear stress increases to the yield strength \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eP\u003c/span\u003e\u003csub\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ek\u003c/span\u003e\u003c/sub\u003e, the viscosity decreases significantly, down to the lowest limit value \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eη\u003c/span\u003e\u003csub\u003em\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eRheological behaviors observed for agar gel and PAAG are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Non-linear change in effective viscosity values indicates non-Newtonian system properties of studied gels. During the measurement, the destruction of interparticle bonds progresses as the shear rate (\u003cb\u003eγ\u003c/b\u003e) increases, which is manifested in the peculiarities of the shape of the flow curves, causing deviations from straight lines. Measuring in the reverse mode indicates recovery of the effective viscosity due to the restoration of the system structure, but the effective viscosity values remain lower than the initial ones. Both dispersed PAAG and agar gel showed thixotropic properties, as evidenced by the hysteresis loops of the dependence of the effective viscosity of gels on the shear rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e show the initial and final values of the effective viscosity at the minimum and maximum shear rates for homopolyacrylamide and agar gels. The initial effective viscosity values are close for both gels. During the measurement. The viscosity values at a shear rate of 2.45 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at the end of the measurement in the reverse shear rate reduction mode for PAAG and agar are 156.398 and 200.32 Pa∙s, which is 37.0 and 40.2% of the initial value, respectively. This means that PAAG had rheological properties and effective viscosity values similar to agar-agar gel.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffective viscosity at minimum (γ\u0026thinsp;=\u0026thinsp;2.45 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and maximum shear rates (γ\u0026thinsp;=\u0026thinsp;1073 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"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=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cem\u003eη\u003c/em\u003e ( at γ\u0026thinsp;=\u0026thinsp;2,45 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), Pа∙s\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cem\u003eη\u003c/em\u003e ( at γ\u0026thinsp;=\u0026thinsp;1073 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), Pа∙s\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003einitial\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003efinal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003einitial\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003efinal\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAgar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e498.519\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e200.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.501\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.306\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e422.986\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e156.398\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.471\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.410\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMicropropagation\u003c/h2\u003e \u003cp\u003eFor micropropagation of plants \u003cem\u003ein vitro\u003c/em\u003e, all synthesized hydrogels were saturated with a culture medium with a complex of micro- and macronutrients, vitamins, amino acids, growth hormones, etc. This process provided the formation of hydrogel composites with sparingly soluble bioelements, which are localized in the hydrogel pores and can gradually diffuse into the external environment. The experiment showed the acceleration of the rooting time of cuttings on PAAG-A2 gel, which was less than two weeks of incubation. On agar-agar this time equaled three weeks. In addition, an intensification of growth and development of the main shoot was observed on hydrogels: 10 and almost 30% higher on PAAG and PAAG-A2, respectively, compared to the samples grown on agar-agar.\u003c/p\u003e \u003cp\u003eThe rooting of the plants on agar medium was 95%, while on hydrogel substrates \u0026ndash; higher than 98%. On hydrogel substrates with the addition of amber, there was a 1.7-fold increase in shoot growth intensity, and on substrates without amber \u0026ndash; 1.5 times. When the plants remained for 10\u0026ndash;15 days in a tall vessel, their height reached 110\u0026ndash;130 mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e deposition was carried out with periodic transplantations to the new nutrient medium to avoid drying out and changes in the composition of the environment due to the effect of the products of plant metabolism. After the first cycle, the spent hydrogel material was regenerated \u0026ndash; washed in distilled water, sterilized, and used in repeated cultivation cycles. The obtained \u003cem\u003ein vitro\u003c/em\u003e rooted plants \u003cem\u003eC\u0026aacute;nnabis sat\u0026iacute;va\u003c/em\u003e are suitable for conversion \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe increase in the number of the metric indicators of leaf blades were also found. During transferring plants from \u003cem\u003eіn vitro\u003c/em\u003e to \u003cem\u003eіn vivo\u003c/em\u003e it was found that the dose-dependent effect of plant knees on test subjects persists \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In general, using a hydrogel instead of agar stimulated the growth of \u003cem\u003eC\u0026aacute;nnabis sat\u0026iacute;va\u003c/em\u003e. Use of hydrogel-amber substrate increased metric indicators of seedling (in comparison to agar): the root length increased by 28%, stem length \u0026ndash; by 26.7%, root weight \u0026ndash; by 167%, stem weight \u0026ndash; by 67%, root and stem length \u0026ndash; by 27%, and root and stem weight \u0026ndash; by 50%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMetric indicators of \u003cem\u003eC\u0026aacute;nnabis sat\u0026iacute;va\u003c/em\u003e seedlings\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperiment techniques\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eC\u0026aacute;nnabis sat\u0026iacute;va\u003c/em\u003e seedlings\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoot length\u003c/p\u003e \u003cp\u003e(cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStem length\u003c/p\u003e \u003cp\u003e(cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRoot weight\u003c/p\u003e \u003cp\u003e(g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eStem weight\u003c/p\u003e \u003cp\u003e(g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRoot and stem lengths (cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eRoot and stem weight (g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAgar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePAAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePAAG\u0026thinsp;+\u0026thinsp;A2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSоil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAfter hydrogel recycling (ten-fold washing with distilled water), all nutrients, succinic acid and products of its transformation were much better absorbed by the plant and stimulated the growth and development of seedlings after transfer \u003cem\u003ein vivo\u003c/em\u003e. In addition, the allelopathic activity of extract from biological material in 1:10, 1:50, and 1:100 dilutions was studied. It has been shown that allelopathic stress is dose-dependent when used a typical MS medium. Under 1:10 dilution \u003cem\u003ein vitro\u003c/em\u003e, \u003cem\u003eCucumis sativus L\u003c/em\u003e. growth inhibition was observed up to 53%; however growth inhibition was reached 87% (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Under 1:100 dilution \u003cem\u003eіn vivo\u003c/em\u003e \"Suppressive\" effect almost didn\u0026rsquo;t fall (95%), which indicates the successful transfer of plants from \u003cem\u003eіn vitro\u003c/em\u003e to \u003cem\u003eіn vivo.\u003c/em\u003e This pattern can be traced on the example of morphometric parameters of \u003cem\u003eC\u0026aacute;nnabis sat\u0026iacute;va\u003c/em\u003e seedlings (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAllelopathic effect of aqueous extract of aboveground parts of \u003cem\u003eC\u0026aacute;nnabis sat\u0026iacute;va\u003c/em\u003e on seedlings of \u003cem\u003eCucumis sativus L\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDilution of the extract\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e,\u0026nbsp;(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e,\u0026nbsp;(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1:10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1:50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1:100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThus, both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e a positive effect of amber in the composition of the hydrogel substrate on the main parameters of germination and development of the studied plants was revealed. According to the possible influence mechanism the biologically active components of highly dispersed amber powder affect biological objects at the cellular level, increasing the efficiency of processes in plants, and also take part in forming the microelement balance, i.e., they are bioactive. Highly dispersed amber powder embedded in the copolymer matrix is non-toxic, released gradually, its ionic form quickly includes in biochemical reactions, and therefore, when washing it with MS culture medium, the biologically active amber acid is washed out prolonged and dosed. This fact explains the high bioavailability and biocompatibility of the synthesized nutrient substrate, the possibility of obtaining planting material in a shorter time, accelerated transition of plants from the juvenile to reproductive phase of development, and increased intensity of growth and development of the main shoot.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eA spatially cross-linked polyacrylamide hydrogel with immobilized amber was synthesized. It has been demonstrated that in terms of its physicochemical and rheological properties, the obtained material is similar to agar-agar and can be used as its inexpensive substitute for micropropagation of plants. It is biosafe after four washings. During them, all unreacted toxic monomers and initiator residues were removed from the hydrogel structure, that is, it was purified effectively. The biosafety of new hydrogel was confirmed by the experiments performed using both biological objects, such as pea/chickpea seeds and \u003cem\u003eD. magna\u003c/em\u003e, and traditional UV spectroscopic method. There was no mortality for both legume seeds and \u003cem\u003eD. magna\u003c/em\u003e after application of water from 4th washing. The use of new hydrogel, instead of agar, stimulated the growth of \u003cem\u003eC\u0026aacute;nnabis sat\u0026iacute;va\u003c/em\u003e. For example, the root weight increased by 167%, whilst the stem weight, by 67%.\u003c/p\u003e \u003cp\u003eThus, the described materials should be considered as novel effective nutrient media, which can accelerate reproduction and allow obtaining a higher amount of plant material within a short period of time in comparison with the standard agar medium.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e LK, OD and OM conceived and designed the study. LK, KS, OG, NP, TP and OD performed the experiments. LK, OG, OS and KSK analyzed the data, contributed inputs, wrote and critically reviewed the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict \u0026nbsp; of \u0026nbsp;interest\u003c/strong\u003e\u0026nbsp; The \u0026nbsp;authors \u0026nbsp; declare \u0026nbsp;that \u0026nbsp;they \u0026nbsp; have \u0026nbsp;no \u0026nbsp;conflict \u0026nbsp; of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinancial interests:\u003c/strong\u003e The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdhikary D, Kulkarni M, El-Mezawy A et al (2021) Medical Cannabis and Industrial Hemp Tissue Culture: Present Status and Future Potential. Front Plant Sci 12:275. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/FPLS.2021.627240/BIBTEX\u003c/span\u003e\u003cspan address=\"10.3389/FPLS.2021.627240/BIBTEX\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBataineh SM, Saadoun I, Hameed KM, Ababneh Q (2008) Screening for soil streptomycetes from North Jordan that can produce herbicidal compounds. Pol J Microbiol 57:297\u0026ndash;305\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoldeskul IE, Samchenko YM, Sukhodub LB et al (2009) Application of vibrational spectroscopy in express analysis of acrylic hydrogel and hydroxyapatite based composite materials. Methods objects Chem Anal 4:92\u0026ndash;96\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDelgado-Aceves L, Portillo L, Folgado R et al (2022) New approaches for micropropagation and cryopreservation of Agave peacockii, an endangered species. Plant Cell Tissue Organ Cult 150:85\u0026ndash;95. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/S11240-022-02246-Z/TABLES/4\u003c/span\u003e\u003cspan address=\"10.1007/S11240-022-02246-Z/TABLES/4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEspinosa-Leal CA, Puente-Garza CA, Garc\u0026iacute;a-Lara S (2018) In vitro plant tissue culture: means for production of biological active compounds. Planta 2018 2481 248:1\u0026ndash;18. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/S00425-018-2910-1\u003c/span\u003e\u003cspan address=\"10.1007/S00425-018-2910-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGertsiuk M, Samchenko YM(2007) Separation of nonreacted acrylamide from polyacrylamide gel for endoprothesing.Ars Separatoria Acta98\u0026ndash;101\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoncharuk O, Samchenko Y, Kernosenko L et al (2020) Thermoresponsive hydrogels physically crosslinked with magnetically modified LAPONITE\u0026reg; nanoparticles. Soft Matter 16:5689\u0026ndash;5701. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/D0SM00929F\u003c/span\u003e\u003cspan address=\"10.1039/D0SM00929F\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrodzinsky AM, Golovko EA, Gorobets SA (1987) Experimental allelopathy. Naukova Dumka, Kyiv\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHenderson WE, Kinnersley AM (1988) Corn starch as an alternative gelling agent for plant tissue culture. Plant Cell Tissue Organ Cult 1988 151 15:17\u0026ndash;22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF00039885\u003c/span\u003e\u003cspan address=\"10.1007/BF00039885\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJain N, Babbar SB (2002) Gum katira \u0026ndash; a cheap gelling agent for plant tissue culture media. Plant Cell, Tissue Organ Cult 2002 713 71:223\u0026ndash;229. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1023/A:1020379024347\u003c/span\u003e\u003cspan address=\"10.1023/A:1020379024347\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKa\u0026ccedil;ar YA, Bi\u0026ccedil;en B, Varol et al (2010) Gelling agents and culture vessels affect in vitro multiplication of banana plantlets. Genet Mol Res 9:416\u0026ndash;424. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4238/VOL9-1GMR744\u003c/span\u003e\u003cspan address=\"10.4238/VOL9-1GMR744\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarolina D, Maja MS, Magdalena DS, Grażyna Ż (2022) Identification of treated Baltic amber by FTIR and FT-Raman - A feasibility study. Spectrochim Acta A Mol Biomol Spectrosc 279:121404. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.SAA.2022.121404\u003c/span\u003e\u003cspan address=\"10.1016/J.SAA.2022.121404\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaur J, Mudgal G (2021) An efficient and quick protocol for in vitro multiplication of snake plant, Sansevieria trifasciata var. Laurentii [Prain]. Plant Cell Tissue Organ Cult 147:405\u0026ndash;411. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/S11240-021-02132-0/FIGURES/2\u003c/span\u003e\u003cspan address=\"10.1007/S11240-021-02132-0/FIGURES/2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan F, Atif M, Haseen M et al (2022) Synthesis, classification and properties of hydrogels: their applications in drug delivery and agriculture. J Mater Chem B 10:170\u0026ndash;203. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/D1TB01345A\u003c/span\u003e\u003cspan address=\"10.1039/D1TB01345A\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim HJ, Koedrith P, Seo YR (2015) Ecotoxicogenomic Approaches for Understanding Molecular Mechanisms of Environmental Chemical Toxicity Using Aquatic Invertebrate, Daphnia Model Organism. Int J Mol Sci 16:12261. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/IJMS160612261\u003c/span\u003e\u003cspan address=\"10.3390/IJMS160612261\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKocak G, Tuncer C, B\u0026uuml;t\u0026uuml;n V (2016) pH-Responsive polymers. Polym Chem 8:144\u0026ndash;176. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/C6PY01872F\u003c/span\u003e\u003cspan address=\"10.1039/C6PY01872F\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKosenko OO, Lukash LL, Samchenko YM et al (2006) Copolymeric hydrogel membranes for immobilization and cultivation of human stem cells. Biopolym Cell 22:143\u0026ndash;148. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7124/BC.000729\u003c/span\u003e\u003cspan address=\"10.7124/BC.000729\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLevchyck NY, Liubinska AV, Skrypchenko NV et al (2017) Biological activity of aqueous solution of amber. Biotechnol Acta 10:53\u0026ndash;60. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.15407/BIOTECH10.06.053\u003c/span\u003e\u003cspan address=\"10.15407/BIOTECH10.06.053\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLubell-Brand JD, Kurtz LE, Brand MH (2021) An In Vitro\u0026ndash;Ex Vitro Micropropagation System for Hemp. Horttechnology 31:199\u0026ndash;207. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21273/HORTTECH04779-20\u003c/span\u003e\u003cspan address=\"10.21273/HORTTECH04779-20\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM\u0026auml;nd K, Muehlenbachs K, McKellar RC et al (2018) Distinct origins for Rovno and Baltic ambers: Evidence from carbon and hydrogen stable isotopes. Palaeogeogr Palaeoclimatol Palaeoecol 505:265\u0026ndash;273. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.PALAEO.2018.06.004\u003c/span\u003e\u003cspan address=\"10.1016/J.PALAEO.2018.06.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMilani P, Fran\u0026ccedil;a D, Balieiro AG, Faez R (2017) Polymers and its applications in agriculture. Polimeros 27:256\u0026ndash;266. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/0104-1428.09316\u003c/span\u003e\u003cspan address=\"10.1590/0104-1428.09316\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMironov OL, Kachalova NM, Dzyuba OI et al (2018) Research of biological activity of Ukrainian amber. In: Popov A (ed) Physico-organic chemistry, pharmacology and pharmaceutical technology of biologically active substances: coll. science. work. KNUTD, Kyiv, pp 100\u0026ndash;120\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMood K, Jogam P, Sirikonda A et al (2022) Micropropagation, morpho-anatomical characterization, and genetic stability studies in Lippia javanica (Burm.f.) Spreng: a multipurpose medicinal plant. Plant Cell Tissue Organ Cult 150:427\u0026ndash;437. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/S11240-022-02294-5/FIGURES/4\u003c/span\u003e\u003cspan address=\"10.1007/S11240-022-02294-5/FIGURES/4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakanishi K (1962) Infrared Absorption Spectroscopy, Practical. Nankodo, Tokyo, Japan; Holden-Day, San Francisco, Calif\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOlkova A, Zimonina N (2020) Assessment of the Toxicity of the Natural and Technogenic Environment for Motor Activity of Daphnia magna. J Ecol Eng 21:11\u0026ndash;16. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.12911/22998993/125459\u003c/span\u003e\u003cspan address=\"10.12911/22998993/125459\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRamli RA (2019) Slow release fertilizer hydrogels: A review. Polym Chem 10:6073\u0026ndash;6090\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRand GM, Wells PG, McCarty LS (2020) Introduction to Aquatic Toxicology. Fundam Aquat Toxicol 3\u0026ndash;67. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1201/9781003075363-2\u003c/span\u003e\u003cspan address=\"10.1201/9781003075363-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSamchenko Y, Korotych O, Kernosenko L et al (2018) Stimuli-responsive hybrid porous polymers based on acetals of polyvinyl alcohol and acrylic hydrogels. Colloids Surf Physicochem Eng Asp 544:91\u0026ndash;104. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.COLSURFA.2018.02.015\u003c/span\u003e\u003cspan address=\"10.1016/J.COLSURFA.2018.02.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchmaljohann D (2006) Thermo- and pH-responsive polymers in drug delivery. Adv Drug Deliv Rev 58:1655\u0026ndash;1670. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.ADDR.2006.09.020\u003c/span\u003e\u003cspan address=\"10.1016/J.ADDR.2006.09.020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShimizu E, Shimoda N, Kawamura T et al (2020) Comparison of the Biological Activity and Constituents in Japanese Ambers. Adv Biol Chem 10:99\u0026ndash;112. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4236/ABC.2020.103008\u003c/span\u003e\u003cspan address=\"10.4236/ABC.2020.103008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh N, Agarwal S, Jain A, Khan S (2021) 3-Dimensional cross linked hydrophilic polymeric network \u0026ldquo;hydrogels\u0026rdquo;: An agriculture boom. Agric Water Manag 253:106939. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.agwat.2021.106939\u003c/span\u003e\u003cspan address=\"10.1016/j.agwat.2021.106939\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSinha V, Chakma S (2019) Advances in the preparation of hydrogel for wastewater treatment: A concise review. J Environ Chem Eng 7:103295. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.JECE.2019.103295\u003c/span\u003e\u003cspan address=\"10.1016/J.JECE.2019.103295\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStadniy IA, Konovalova VV, Samchenko YM et al (2011) Development of Hydrogel Polyelectrolyte Membranes with Fixed Sulpho-Groups via Radical Copolymerization of Acrylic Monomers. Mater Sci Appl 2:270\u0026ndash;275. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4236/MSA.2011.24035\u003c/span\u003e\u003cspan address=\"10.4236/MSA.2011.24035\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTumiłowicz P, Synoradzki L, Sobiecka A et al (2016) Bioactivity of Baltic amber \u0026ndash; fossil resin. Polimery 61:347\u0026ndash;356. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.14314/POLIMERY.2016.347\u003c/span\u003e\u003cspan address=\"10.14314/POLIMERY.2016.347\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTuson HH, Renner LD, Weibel DB (2012) Polyacrylamide hydrogels as substrates for studying bacteria. Chem Commun 48:1595\u0026ndash;1597. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/C1CC14705F\u003c/span\u003e\u003cspan address=\"10.1039/C1CC14705F\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e(2004) Test No. 202: Daphnia sp.Acute Immobilisation Test\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e(1982) Seeds of farm crops. Methods for determination of viability.USSR State Stand\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eISO - ISO 10706:2000 - Water quality \u0026mdash; Determination of long term toxicity of substances to Daphnia magna Straus (Cladocera, Crustacea). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.iso.org/standard/18795.html\u003c/span\u003e\u003cspan address=\"https://www.iso.org/standard/18795.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Accessed 12 Aug 2022\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"plants micropropagation, polyacrylamide gel, amber, acrylamide toxicity, biotesting, Daphnia magna","lastPublishedDoi":"10.21203/rs.3.rs-2085035/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2085035/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e cultivation and reproduction of plants is one of the most modern and promising methods of cultivating valuable plants using artificial nutrient media. In this work, a new solid nutrient media for plant micropropagation based on highly dispersed polyacrylamide hydrogel (PAAG) with amber powder was synthesized and investigated. PAAG was synthesized by homophase radical polymerization with grounded amber addition. FTIR (Fourier transform infrared spectroscopy) and rheological studies were used to characterize structural properties of the materials. The synthesized hydrogel showed physicochemical and rheological parameters similar to the standard agar media. The estimation of acute toxicity of PAAG-amber was performed based on the influence of washing waters on the viability of the selected plant seeds (pea and chickpea) and animal (\u003cem\u003eDaphnia magna\u003c/em\u003e). It proved its biosafety after four washes. The impact on plant rooting was studied using multiplication of \u003cem\u003eCannabis sativa\u003c/em\u003e on synthesized PAAG-amber saturated with Murashige-Skoog (MS) medium and compared with agar gel with MS. Developed substrate stimulated the rooting of the plants up to more than 98% in comparison to standard agar medium (95%). Also, PAAG-amber nutrient medium markedly enhanced metric indicators of seedling: root length increased by 28%, stem length \u0026ndash; by 26.7%, root weight \u0026ndash; by 167%, stem weight \u0026ndash; by 67%, root and stem length \u0026ndash; by 27%, root and stem weight \u0026ndash; by 50%. This means that the developed hydrogel significantly accelerates reproduction and allows obtaining a larger amount of plant material within a shorter period than the standard agar medium.\u003c/p\u003e","manuscriptTitle":"Polyacrylamide Hydrogels With Amber for Plants Micropropagation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-10-18 19:40:41","doi":"10.21203/rs.3.rs-2085035/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"13b38964-dfa3-4c38-bf65-56a3aacfad79","owner":[],"postedDate":"October 18th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-04-26T17:11:18+00:00","versionOfRecord":{"articleIdentity":"rs-2085035","link":"https://doi.org/10.3390/plants12051196","journal":{"identity":"plants","isVorOnly":true,"title":"Plants"},"publishedOn":"2023-03-06 00:00:00","publishedOnDateReadable":"March 6th, 2023"},"versionCreatedAt":"2022-10-18 19:40:41","video":"","vorDoi":"10.3390/plants12051196","vorDoiUrl":"https://doi.org/10.3390/plants12051196","workflowStages":[]},"version":"v1","identity":"rs-2085035","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2085035","identity":"rs-2085035","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}