The effect of several growth regulators and biostimulant on biochemical and physiological changes in acclimation of micropropagated Echinacea purpurea Moench. ‘Raspberry Truffle’

The effect of several growth regulators and biostimulant on biochemical and physiological changes in acclimation of micropropagated Echinacea purpurea Moench. ‘Raspberry Truffle’ | 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 The effect of several growth regulators and biostimulant on biochemical and physiological changes in acclimation of micropropagated Echinacea purpurea Moench. ‘Raspberry Truffle’ Monika Grzelak, Andrzej Pacholczak, Karolina Nowakowska This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4730353/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Oct, 2024 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted 4 You are reading this latest preprint version Abstract Micropropagation is currently one of the primary methods for plant propagation, known for its efficiency in producing disease-free and vigorous plants. However, the final stage of this - acclimatization, is critical due to the transfer from a controlled in vitro environment to external one. To reduce mortality and alleviate acclimation stress, plant growth regulators (PGRs) or biostimulants can be employed. This study investigated the effects of exogenously sprayed PGRs: 0.001 mg·L -1 abscisic acid (ABA), 0.001 mL·L -1 brassinolide (BL), 0.001 mL·L -1 24-epibrassinolide (24-epiBL), and 0.3 mL·L -1 biostimulant Goteo on the physiological and biochemical responses of Echinacea purpurea 'Raspberry Truffle' plantlets during the acclimation process. The effects of treatments at various acclimation stages on chlorophyll (chl) and carotenoids content, hydrogen peroxide (H 2 O 2 ), catalase (CAT), malondialdehyde (MDA), free amino acids, soluble proteins, total soluble sugars, and reduced soluble sugars were tested in this research. The results confirmed changes in biochemical parameters, including an increase in chlorophyll and carotenoids in the acclimatization period where the highest level obtained by BL spraying. A decline in stomatal conductance was also observed, where ABA influenced the most on drop. It was also recorded the decrease in H 2 O 2 and MDA concentration. CAT activity increased, especially by biostimulant treatment. We recorded an increase in total soluble proteins along acclimatization. Goteo affected the most on morphology parameters, ABA, BL and 24-epiBL increased acclimatization efficiency. Our studies indicate that potentially the most effective substances in the acclimatization of E. purpurea ‘Raspberry Truffle’ are brassinosteroids and ABA. abiotic stress in vitro brassinosteroids acclimatization plant hormones biostimulant Figures Figure 1 Figure 2 Figure 3 Key message Biochemical and physiological changes occur in plants during acclimation. We selected the most beneficial growth regulators alleviating acclimation stress caused by ex vitro transfer and increasing plant survival rate. Introduction Micropropagation is widely used method of vegetative plants multiplication. Thanks to the use of in vitro technology, a mass of genetically homogenous plantlets can be produced in a short time (Sharma et al. 2015 ). It was confirmed as cost-effective for multiplication of large amount species (Kikowska et al. 2014 ). Acclimatization is the last step of in vitro propagation. However, this is the most critical one, due to change of environment. In glass, microplants grow in favorable, disease-free conditions. Nevertheless, growing in vitro results in various range of abnormalities in plants on morphological, anatomical, physiological, biochemical and molecular levels. These are due to high air humidity, low irradiance, low CO 2 concentration during light period, supplementation of growing media with sugars and plant growth regulators (PGRs) (Baťková et al. 2008 ). Therefore, plantlets must be able to adapt to new, external conditions and correct the abnormal features. Additionally, ex vitro transfer of micropropagated plantlets is often accompanied by water stress and/or photoinhibition (Semorádová et al. 2002 , Carvalho et al. 2006 , Baťková et al. 2008 ). By transfer to external environment plants are exposed to stresses, which can lead to high mortality of plants. Transplantation to ex vitro conditions results in the increased rate of production of reactive oxygen species (ROS) like superoxide anions (O 2− ), hydroxyl radical (OH), singlet oxygen ( 1 O 2 ), and hydrogen peroxide (H 2 O 2 ) (Anjum et al. 2022 ). In normal conditions, ROS are byproducts of aerobic metabolism and can be signals that switch on developmental programs or regulate physiological processes as cell wall loosening necessary for cell elongation, modulation of cytosol Ca 2+ concentration, senescence, adaptation to abiotic stresses or resistance to pathogens (Papadakis and Roubelakis-Angelakis 2002 ; Gechev et al. 2005 ; Procházková and Wilhelmová 2007 ; Ślesak et al. 2007 ; Vilela et al. 2007 ; Baťková et al. 2008 ). However, excess of ROS can make harder the production of biomolecules such as lipids, proteins and nucleic acids and contribute to lipid peroxidation, protein degradation, enzyme inactivation and damage of DNA (Baťková et al. 2008 ; Gil and Tuteya 2010). For example, accumulation of H 2 O 2 can be detrimental for the plant cell, leading to cell death, a fine-tuning of this signaling molecule can result in stress management by coordinating intra-cellular and systemic signaling systems. ROS overproduction can be scavenged by the antioxidative systems which include low molecular mass antioxidants (ascorbate, glutathione, tocopherols, carotenoids, phenols) and antioxidative enzymes (superoxide dismutase, ascorbate peroxidase, catalase, glutathione reductase, monodehydroascorbate reductase, dehydroascorbate reductase) (Anjum et al. 2022 ). Peroxidation of membrane lipid is a main detrimental effect of ROS. Commonly, membrane lipid peroxidation in plant tissues is determined by measuring malondialdehyde (MDA). It is a frequently used marker of oxidative lipid injury caused by environmental stress (Amnan et al. 2022 ). A various range of studies have investigated MDA of plants under different stress conditions, including herbaceous plants as proved Kong at al. (2016) in oilseed rape or El-Batal et al. ( 2023 ) in carrot. Sugars can play as a signal molecule in various range of plant stresses. Soluble sugars gather while biotic and abiotic stress conditions apply to oxidative stress further pointing towards a relationship between sugars and stress-induced ROS accumulation in plants. It is observed a relationship between sugars and oxidative stress, as it was displayed that high sugar content can either promote or decrease ROS production in plants. Studies on in vitro revealed that the in vivo antioxidant capacity of sugars might be highly undervalued. Various range of sugars are proven to act as antioxidants (Couée et al. 2006 ; Keunen et al. 2013 ). Simple sugars and polysaccharides are known as a protection of cellular membranes, which is a premise for survival of plants on stress (Valluru and Van den Ende 2008 ). Chlorophyll (chl) is known as a pigment absorbing light which exploits sunlit energy for ATP synthesis (Coste et al. 2010 ). Determining chl content in leaves is one of the relevant parameter used to estimate the plants photosynthetic rate (Dalio et al. 2011 ). To alleviate stress associated with acclimatization, it is necessary to identify substances that mitigate adverse conditions. one of them can be ABA which is a stress-related signaling molecule in plants responding to plant stresses. It is present in most plant organs and is involved in stomatal closure, reduction of ROS, seed dormancy, and seedling development (Hauser et al. 2011 ). Hence the assumption that it may be effective in alleviating stress associated with acclimatization. Brassinosteroids (BRs) are relatively new group of hormone firstly isolated in rape. Various range of activities are regulated by them as seed germination, cell division, vascular differentiation, cell elongation, root development, stomata formation, flower development, male sterility, photomorphogenesis, and resistance to stresses. Moreover, they have a beneficial role in modulating plants from excess of ROS (Ranjan and Ranjan 2023). They are considered as short-distance signals, because they are not able to be transported within long distances, nonetheless they alter auxin transport by which act as an indirect long-distance signal molecule (Siddiqui et al. 2018 ). Its exogenous application of mentioned hormone affect on stress tolerance mechanisms, which was observed in cowpea bean (Sousa et al. 2022). BRs are able to increase the chl content as proved Alyemeni and Al-Quwaiz ( 2016 ). The using of plant biostimulants (PBs) is growing in plant production, by the increasing interest of farmers in natural materials and helpful microorganisms that can sustainably improve the productivity of crops and ornamentals. Many of them are widely used in plant cultivation, mainly owning to their beneficial effects on nutrient uptake, plant growth, yield, and fruit quality and the tolerance of plants to abiotic stressors (de Pascale et al. 2018 ; Kisvarga et al. 2022 ). PBs act on as a promising strategy to reduce the unfavorable effects of osmotic stress. It was proved their beneficial effect as activator the ROS scavenging enzyme system (Campobenedetto et al. 2020 ). One of plant PBs, containing brown seaweed - Ascophyllum nodosum can be implemented as nutrient supplements, biofertilizers in horticultural plant systems (Tavares et al. 2020 ). Echinacea purpurea (L.) Moench. it is a common long-blooming, herbaceous, perennial garden and cut plant called eastern purple coneflower (Armitage, 2008 ). E. purpurea ‘Raspberry Truffle’ is commercially important variety of this species bred by the plant hybridizer Arie Blom, registered in 2012. There are some reports concerning micropropagation of Echinacea . The study of Harbage ( 2001 ) with method allowing rapid multiplication withs selected PGRs or Chen et al. (2016) using supplementation of diethyl aminoethyl hexanoate (DA-6). However, the process of acclimation was not widely described so far. In this study there were used E. purpurea ‘Raspberry Truffle’ as an experimental plant. The aim of this work was to demonstrate the biochemical and physiological changes occurring in coneflower at different stages of acclimatization. We also would like to determine the effects of exogenous application of PGRs as ABA, BL, 24-epiBL, PB Goteo on biochemical parameters and stomatal conductance of Echinacea purpurea . We also assessed the impact of the mentioned applications on the morphology of acclimatized plants and how the specific treatments affects the number of acclimatized plants to ex vitro conditions, which translates into the profitability of this species in laboratory conditions. Materials and methods 1.1. Shoot Multiplication and Rooting The medium to multiplication phase was prepared using 0.35 mg·L − 1 BA and 0.35 mg·L − 1 IBA were added to the Murashige and Skoog ( 1962 ) basal media with 30 mg·L − 1 of sucrose, gelled with supplementation of 8 g·L − 1 agar, the pH was adjusted to 5.8 before autoclaving. Media with PGRs was autoclaved for 20 min. at 12 o C and 110 kPa. The microshoots produced in the multiplication stage were suitable for rooting. After 6 weeks multiplied shoots were transferred to the rooting MS medium supplemented with 0.5 mg·L − 1 IBA and 1 mg·L − 1 activated charcoal (Sigma Aldrich) with 30 mg·L − 1 of sucrose and 8 g·L − 1 agar, pH = 5.8 before autoclaving. Plantlets regenerated in the rooting medium for approx. 6 weeks (Fig. 1 ). During both phases, the glass jars containing explants were kept in a growth phytotron with 23 ± 1℃, 16 h day with light at 35 µmol m − 2 s − 1 photosynthetic photon flux density, provided by fluorescent lamps. 1.2. Acclimation phase experimental rationale and design After 6 weeks of regeneration in growth phytotron, the rooted plantlets were removed from the culture jars and the roots were washed using distilled water to remove traces of agar in the surface of roots. Next, microplants were planted in plastic boxes filled with peat (pH = 5.0–6.0) and perlite 1:1 (v/v). Then plantlets were sprayed separately with 200 mL water solution of: ABA in concentration 0.001 mL·L − 1 , 0.001 mL·L − 1 BL, 0.001 mL·L − 1 24-epiBL and 0.3 mL·L − 1 biostimulant Goteo (Table 1 ). The concentrations were selected after preliminary experiments. Control treatment was distilled water. Each treatment were tested in a thrice replicated experiment with 10 plantlets. After spraying, plants were covered with transparent lids and kept in growing room in temp. 25 o C, the photoperiod was established 16 h day (50 µmol·m 2 ·s 1 ). After 4 weeks of growing in phytotron, plants were transferred to the greenhouse (Fig. 1 ). Table 1 A list of treatments in the experiment. No. of treatment Treatment Concentration 1. Control “0” distilled water 2. ABA 0.001 ml·L − 1 3. BL 0.001 ml·L − 1 4. 24-epiBL 0.001 ml·L − 1 5. Goteo 0.3 ml·L − 1 1.3. Evaluation 1.3.1. Stomatal conductance measurements Stomatal conductance ( Gs ) of leaves in plantlets was measured from in vitro plants (0 term), at the beginning of ex vitro acclimation (1st term), at the end of growing in phytotrone (2nd term) and at the end of growing in the greenhouse (3rd term). During acclimation using a portable SC − 1 leaf porometer (Geomor Technik). Device consists in the principle of measuring the static change of humidity inside a closed measuring head with a leaf placed inside. Gs was expressed as mmol H 2 O m − 2 s − 1 . 1.3.2. Biochemical analyses The samples for biochemical analyses (leaves) were picked in the same terms as Gs was measured. The material was chopped, mixed, and samples were collected for each analysis. Three extractions were done for each analysis and 3 measurements were made for each extract. The absorbance was measured in spectrophotometer UV-1601 PC (Shimadzu, Columbia, MD, USA). 1.3.2.1. Total chlorophyll and carotenoids content The total contents of chl and carotenoids were assessed using the methods described by Lichtenthaler and Wellburn ( 1983 ). Plant material was ground in a mortar with a slight amount of quartz sand and 2.5 mL of 80% acetone. The extracts were subsequently filtered through filter paper into 25 mL volumetric flasks and filled up with acetone. The absorbance of filtrates was measured at: 470, 646, 652, and 663 nm. 1.3.2.2. Preparation of extracts to H 2 O 2 , catalase, soluble proteins 0.25 g samples were homogenized in 2.5 mL K-phosphate buffer. The extracts were centrifuged by 20 min. at 20.000 xg at 4°C. Next, the extract was transferred to tubes. 1.3.2.3. Hydrogen peroxide content H 2 O 2 content was measured using Pick and Keisari ( 1980 ) method. Reaction mixture was composed by 0.5 mL extract, 0.5 mL 0.1 M potassium phosphate buffer (pH = 7.0) and 1 mL of 1 M potassium iodide. Samples were incubated by 1 h in the darkness at room temperature and their absorbance were measured at 390 nm. 1.3.2.4. Catalase activity The analysis of catalase activity was conducted following the methodology outlined in the work by Goth (1991). The tubes were divided into two sets, each receiving 0.05 mL of the extract along with 0.45 ml of K-phosphate buffer. In tubes labeled 'A', 1 mL of 0.1 M K-phosphate buffer was added, while tubes labeled 'B' received 1 mL of H 2 O 2 in buffer (at a concentration of 65 µM). A control sample, denoted as 'K', was prepared by adding 1.5 mL of 0.1 M K-phosphate buffer, while sample 'C' consisted of 0.5 mL of 0.1 M K-phosphate buffer mixed with 1 mL of H 2 O 2 in buffer (at a concentration of 65 µM). All tubes were then placed in darkness for incubation for 10 minutes. Following incubation, 1 mL of 32.5 mM ammonium molybdate was added to each tube and thoroughly mixed. Subsequently, absorbance was measured at 405 nm. 1.3.2.5. MDA content MDA determination was based on the method described by Heath and Packer ( 1968 ). Leaf samples weighing 0.25 g were homogenized in 2.5 ml of trichloroacetic acid (TCA) (w/v). The resulting extract was centrifuged for 10 minutes at 10,000 xg and 4°C. The reaction mixture consisted of 1.0 mL of supernatant and 4.0 mL of 20% thiobarbituric acid (TBA) in 0.5% TCA (w/v). Subsequently, the samples were incubated for 30 minutes at 95°C, followed by stopping the reaction on ice for 10 minutes. Afterward, the samples underwent another centrifugation at 10,000 xg for 10 minutes at 4°C. Absorbance was measured at 532 nm and 600 nm to account for nonspecific absorbance using an extinction coefficient (ε) of 155 mM − 1 cm − 1 . 1.3.2.6. Free amino acids The procedure for determining the content of free amino acids was adapted from the Rosen ( 1957 ) method. Specifically, 0.2 ml of extract was combined with 0.5 mL of 0.2 mM sodium cyanide in acetate buffer (pH = 5.3–5.4) and 0.5 mL of ninhydrin. After thorough mixing, the solutions were incubated in a water bath at 100°C for 15 minutes. Subsequently, the tubes were taken out, and 5 mL of isopropyl alcohol was added to each warm tube. The mixtures were then allowed to cool. Absorbance readings were taken at 570 nm against a standard curve for leucine. 1.3.2.7. Total soluble proteins The concentration of total soluble proteins was measured using Bradford's method (1976). The reaction mixture comprised 0.1 mL of the extract and 5.0 mL of Bradford's reagent. The mixture was incubated in the dark for 5 minutes, and the absorbance was then measured at 595 nm. 1.3.2.8. Total sugars content Total sugars were measured by the colorimetric method of Dubois et al. ( 1956 ). The samples were homogenized in hot 80% ethanol. The extract was centrifuged for 20 minutes at 20.000 xg at 4°C. Then, the supernatant was transferred into resealable tubes and adjust to 25 mL with 80% ethanol. The reaction mixture comprised 100 µl of extract, 1 mL of 5% phenol, and 5 ml of sulphuric acid (H 2 SO 4 , 96%) and mixed thoughtfully. The solutions incubated for 20 minutes. The absorbance was measured against a standard curve for glucose at 490 nm. 1.3.2.9. Reduced sugars content Reduced sugars content was determined by Nelson ( 1944 ) method. The samples were homogenized in hot 80% ethanol. The extract was centrifuged for 20 minutes at 20.000 xg at 4°C. Then, the supernatant was transferred into resealable tubes and adjust to 25 mL with 80% ethanol. 100 µL of extract were taken. Then, 1 mL of copper reagent was added. The mixture was incubated for 20 minutes in a water bath at 100°C. Afterward, it was cooled in cold water. 1 mL of molybdenum-arsenic reagent was added and mixed. Finally, distilled water was added to make up the volume to 5 mL. The absorbance was measured against a standard curve for glucose at 520 nm. 1.3.3. Morphological parameters Biometric data (plant height, number of leaves, average length and width of 3 leaves, number of roots, length of roots) were recorded at the 4th week of acclimation stage. Mortality of the plantlets were calculated using following equation: $$\:mortality\:\left[\%\right]=\frac{number\:of\:dead\:plants}{total\:number\:of\:plantlets}*100$$ 1.4. Data analysis Statistical analysis, data and graphical presentation were performed with Statistica Version 13.1 (STATSoft, Tulsa, OK, USA) and Microsoft Excel 365 (Version 2023). Experimental data were subjected to one way and two-way analysis of variance and then to Tukey’s multiple range test to separate the means at the significance level of p ≤ 0.05. Results 2.1. Stomatal conductance Initially (at date 0), stomatal conductance was 192.9, which is similarly high as on the 1st date. The greatest drop in Gs relative to the initial value occurred with 24-epiBL spraying. When considering treatments separately on the 1st date, the values were significantly the highest with BL and Goteo treatments. A marked decrease was noticed on the 2nd date, while values did not differ from each other except for untreated plants with the highest result. Plants treated with ABA on the 3rd date remained at the same low level as before. However, Gs with the other applications slightly increased. Overall, summarizing the average values of treatments, the values did not differ from each other (Table 2 ). Table 2 Changes in contents of stomatal conductance ( Gs) [mmol H 2 O m − 2 s − 1 ] during acclimation of Echinacea purpurea Moench. ‘Raspberry Truffle’ depending on date and treatments. Parameter Treatment Mean (for terms) Gs [mmol H 2 O m − 2 s − 1 ] Term Control ABA BL 24-epiBL Goteo 0 192.9 1 186.20 bcd 195.26 bcd 212.33 d 174.33 b 217.43 d 197.16 c 2 175.53 bc 86.86 a 112.70 a 103.2 a 90.70 a 113.70 a 3 164.10 b 83.83 a 194.03 bcd 210.83 cd 182.57 bcd 167.35 b Mean (for treatments) 175.28 a 122.00 a 173.02 a 163.02 a 163.57 a * the same letter in the lines indicates no difference between the means at a significance level of α = 0.05 2.2. Photosynthetic pigments The content of chl in in vitro growing plants was 7.80 and was higher compared to acclimatized plants. Overall, the content of its gradually grows within acclimation process (Fig. 2 ). Taking into account the averages of the treatments the highest values were recorded in 24-epiBL and BL treatments. Significantly lower values were recorded in remaining treatments. Regarding terms, the lowest chl content was observed at the beginning of acclimation (4.69) whereas application of 24-epiBL and BL resulted in the increase of chl on that date. On the 2nd term the chl content was at a similar level, besides BL and control plantlets. On the 3rd date the highest values was obtained by ABA, BL and 24-epiBL treatments (Table 3). Initially ( in vitro ) the carotenoids content was 2.11. In the initial observation period, fewer carotenoids were recorded. There was observed significant increase in its content among acclimatization (1.32 in 1st period, 1.71 in 2nd ). Regarding treatments in 1st period the highest values were obtained by 24-epiBL spraying. In the 2nd period the content was the highest by 24-epiBL and Goteo spraying. In the 3rd term, the content varied depending on the treatment, with statistically the highest content observed in BL, followed by Goteo and 24-epiBL (Table 4 ). Table 4 Changes in contents of chlorophyll [mg·g − 1 d.w.] and carotenoids [mg·g − 1 d.w.] during acclimation of Echinacea purpurea Moench. ‘Raspberry Truffle’ depending on date and treatments. Data is represented using 3 replicates. Parameter Treatment Mean (for terms) chlorophyll [mg·g − 1 d.w.] Term Control ABA BL 24-epiBL Goteo 0 7.80 1 4.24 ab 4.69 bc 4.88 c 5.58 d 4.04 a 4.69 a 2 4.38 ab 5.50 d 6.33 e 5.63 d 5.66 d 5.50 b 3 5.44 d 6.51 ef 6.90 f 6.45 ef 5.68 d 6.19 c Mean (for treatments) 4.69 a 5.57 bc 6.04 d 5.88 cd 5.12 ab carotenoids [mg·g − 1 d.w.] 0 2.11 1 1.29 a-d 1.21 abc 1.08 a 1.72 ef 1.33 bcd 1.32 a 2 1.09 ab 1.49 de 2.01 gh 1.77 f 1.86 fg 1.64 b 3 1.42 cd 1.51 de 2.15 h 1.70 ef 1.77 f 1.71 b Mean (for treatments) 1.27 a 1.40 a 1.74 b 1.73 b 1.65 b *the same letter in the lines indicates no difference between the means at a significance level of α = 0.05 2.3. Stress biomarkers and antioxidant activity Acclimation and treatments affects significantly on H 2 O 2 content. In vitro growing plantlets, similarity to control group on the 1st date accumulated the highest content of H 2 O 2 (Fig. 3 ). Spraying with PGRs and biostimulant influenced the decline values. Similarly high values were on the 2nd term. On the 3rd date its contents generally fell down. The highest H 2 O 2 content was on untreated plants, but there was no significant differences. The average of MDA was significantly higher on the 1st and 2nd term. In other treatments the values slightly dropped. The MDA concentration significantly decreased on the 3rd date. In terms of the treatments, Goteo affected on fall the values in all treatments. In the 3rd term, the lowest value was recorded by 24-epiBL application. Conversely, the increase of CAT activity was recorded during acclimation period. Plantlets on the 0 day had the lowest CAT activity, the highest values observed in 3rd term. It gradually grows in all treatments, however overall the highest values was recorded after Goteo, ABA and both, BRs treatments in comparison to control. In spite of total sugars content, treatment and acclimation period affected the values. On the term 0 and 1st were recorded the lowest contents, spraying of ABA and BL affected on significant decreasing of the content. All other values in this term did not differ significantly from one another, nor did they differ from the respective results in the control treatment. On the 2nd term were recorded the lowest values in the control treatment, within remaining treatments were different values as well. Goteo treatment values were higher in comparison to control plants. Both, ABA and 24-epiBL were higher than above-mentioned treatments and did not differ significantly from one to another. BL affected on the highest values in 2nd term. On the 3rd term were observed decrease of total sugars content compared with previously described. The lowest value was recorded in BL treatment. ABA and 24-epiBL values were higher than BL and similar to each other. On the control and Goteo treatment were observed the highest levels in comparison to remaining groups. Overall, considering all treatments in all terms there were no significant differences between the values. In the case of reduced sugars content it was observed gradual decrease of the values as with CAT activity. Control, Goteo and 24-epiBL treatments on the 1st term were significantly higher than two other treatments. There were no significant differences between ABA and BL values. In spite of 2nd term distinctly the highest value was recorded only with 24-epiBL spraying. BL and Goteo treatment affected on the highest reduced sugars content in comparison to all other groups. To consider the means in the treatments overall, the values were not differ significantly from another, but the highest reduced sugars content were recorded 24-epiBL (Table 5 ). Table 5 Changes in contents of H 2 O 2 [µg·g − 1 d.w.], MDA [µmol·g d.w.] and CAT [mkat g − 1 d.w.] activity during acclimation of Echinacea purpurea Moench. ‘Raspberry Truffle’ depending on date and treatments. Data is represented using 3 replicates. Parameter Treatment Mean (for terms) H 2 O 2 [µg·g − 1 d.w.] Term Control ABA BL 24-epiBL Goteo 0 154.46 1 154.46 f 123.90 de 135.51 ef 103.3 c 114.14 cd 126.26 b 2 113.89 cd 131.18 ef 143.03 fg 132.98 ef 145.08 fg 133.23 b 3 88.1 b 78.13 ab 63.89 a 77.12 ab 76.65 ab 76.78 a Mean (for treatments) 118.82 ab 111.07 ab 114.14 ab 104.47 a 122.64 b CAT [mkat g − 1 d.w.] 0 42.97 1 138.83 a 240.39 b 239.64 b 352.79 cd 394.31 ef 273.19 a 2 390.70 ef 479.36 g 327.85 c 327.13 c 413.58 f 387.72 b 3 366.117 de 484.48 g 489.69 g 534.51 h 553.51 h 485.66 c Mean (for treatments) 298.55 a 401.41 bc 352.39 b 404.81 bc 453.80 c MDA [µmol·g d.w.] 0 0.61 1 0.81 cd 0.97 gh 0.99 h 0.97 gh 0.52 b 0.86 b 2 1.02 h 1.45 i 0.87 ef 0.70 c 0.30 a 0.87 b 3 0.51 b 0.47 b 0.43 b 0,31 a 0.51 b 0.45 a Mean (for treatments) 0.78 bc 0.91 c 0.78 bc 0.70 b 0.44 a *the same letter in the lines indicates no difference between the means at a significance level of α = 0.05. 2.4. Free amino acids and soluble proteins content Free amino acids values in on day 0 (488.00) and 1st term (439.20) were higher than remaining ones. Within the treatments on 1st term the largest amount of free amino acids were detected by application of 24-epiBL, lower than above-mentioned were by ABA and BL application, but were comparable to each other. Significantly lower free amino acids content were recorded in biostimulant, whereas control plantlets had distinctly the lowest content. On 2nd term, the content dropped sixfold in case average of all treatments. There were observed almost tenfold drop to compare with 1st term in case of spraying with BL, 24-epiBL and Goteo. Thereby these values were the lowest concerning 2nd term. Significantly higher contents recorded by application of distilled water and ABA, the values was differ from ich other. On the 3rd term overall free amino acids content were still lower than on the 1st term, but higher than on the 2nd term. Goteo treatment was not differ than on the 2nd date. Generally considering average of treatments, there were no significant differences between groups, but an increased content of free amino acids was observed in ABA and 24-epiBL application. The accumulation of proteins increased in acclimation stage. Initially ( in vitro ), the content was 5.17 to drop in 1st date by almost 25%. Taking into account treatments separately on 1st date, the values were not differ from each other, however the higher value recorded by 24-epiBL spraying. On the 2nd date was observed an increase of soluble proteins content. Similarly high values were observed by application of ABA, BL and Goteo. Distinctly lower contents observed in case of control and 24-epiBL sprayed plantlets. On the 3rd date the content was by almost 24% higher and differ in comparison with previously described one. There were no differences between treatments on this date. Focusing on an average soluble proteins content for all treatments, the lowest values were recorded in control plantlets, there were no significant differences between remaining treatments (Table 6 ). Table 6 Changes in contents of free amino acids [mmol leucine g − 1 d.w.] and total soluble proteins [mg g − 1 d.w.] during acclimation of Echinacea purpurea Moench. ‘Raspberry Truffle’ depending on date and treatments. Data is represented using 3 replicates. Parameter Treatment Mean (for terms) free amino acids [mmol leucine g − 1 d.w.] Term Control ABA BL 24-epiBL Goteo 0 488.00 1 331.93 e 468.05 g 465.39 g 517.33 i 413.31 f 439.20 c 2 136.64 c 103.91 b 46.36 a 46.91 a 40.30 a 74.82 a 3 164.29 d 146.17 cd 89.86 b 131.77 c 47.85 a 115.99 b Mean (for treatments) 210.95 ab 239.38 b 200.54 ab 232.01 b 167.15 a total soluble proteins [mg g − 1 d.w.] 0 5.17 1 3.73 a 3.54 a 3.94 ab 5.12 b 3.16 a 3.90 a 2 10.55 c 15.80 ef 16.52 fg 13.96 d 14.96 de 14.36 b 3 17.01 e-h 17.80 gh 18.11 h 16.56 efg 17.61 fgh 17.42 c Mean (for treatments) 10.43 a 12.37 b 12.86 b 11.88 b 11.91 b *the same letter in the lines indicates no difference between the means at a significance level of α = 0.05 Initially, (day 0), the total sugars content was 68.66. On the 1st date, the average value was similar (61.04). Within the term, the highest value was recorded by Goteo spraying (78.14) the lowest content was determined in ABA and BL treated plants. On the 2nd term there was observed significant rise of reduced sugars compared to previous date. Among this term the lowest values were recorded in control plants (66.94), in treated ones, the content were above 110, to be the highest in BL treatment – 197.04. On the 3rd date there has been decrease in the values. An average did not differ than on the 1st term. Among the treatments, the highest value was determined in Goteo treated plants, which was comparable to control. Lower values was noted by ABA and 24-epiBL treatments. The lowest content were observed in BL treatment (Table 6 ). In case od total sugars content on the 0 day the content was 51.60. The average value of that on the 1st term was 53.46. Among the treatments similar lower values were observed in ABA and BL, whereas significantly higher content were recorded in remaining treatments. On the next terms there was observed significant drop of the values (46.34). Least sugars content was determined in Goteo treatment, similarly to control. Higher, but not significantly values were recorded in ABA and BL sprayed plants, to be significantly the highest by 24-epiBL treatment. An average sugars content was not differ from the value on the 2nd date (43.25). significantly the highest content was observed by Goteo treatment (Table 7 ). Table 7 Changes in contents of total [mg glucose · g d.w. −1 ] and reduced sugars [mg glucose · g d.w. −1 ] content during acclimation of Echinacea purpurea Moench. ‘Raspberry Truffle’ depending on date and treatments. Data is represented using 3 replicates. Parameter Treatment Mean (for terms) total sugars [mg glucose · g d.w. −1 ] Term Control ABA BL 24-epiBL Goteo 0 68.66 1 69.71 cde 38.66 a 49.30 b 69.39 cde 78.14 e 61.04 a 2 66.94 cd 132.48 h 197.04 i 132.49 h 117.66 g 129.32 b 3 95.40 f 72.49 de 60.10 c 74.82 de 95.40 f 79.04 a Mean (for treatments) 77.36 a 81.21 a 102.1115 a 92.23 a 96.05 a reduced sugars [mg glucose · g d.w. −1 ] 0 51.60 1 60.45 fg 44.35 bcd 43.72 bcd 54.42 fg 64.38 g 53.46 b 2 34.33 a 47.41 cde 45.19 bcd 66.16 g 38.64 abc 46.34 a 3 41.25 a-d 38.20 ab 46.64 b-e 41.17 a-d 49.02 ef 43.25 a Mean (for treatments) 45.34 ab 43.32 a 45.18 ab 53.92 b 50.68 ab *the same letter in the lines indicates no difference between the means at a significance level of α = 0.05 2.5. Biometric parameters and mortality of plants The highest plants were observed by Goteo treatment (16.00). The ones treated with ABA were significantly shorter (14.00). However, the shortest plants were recorded in the remaining treatments. Goteo had the most significant impact on root length (average 23.90). Similarly, Goteo resulted in the highest number of leaves, while ABA treatment resulted in the fewest. Leaf blades (both length and width) were the largest when using the PB. The highest number of roots was produced by plants treated with ABA, 24-epiBL, and Goteo (5, 5, and 4 respectively). In terms of mortality, the highest percentage of plants survived in untreated group (40%), while plants treated with Goteo had a mortality rate of 20%. For the other groups, mortality rates were 4% (ABA, BL) and 7% (24-epiBL) (Table 8 ). Table 8 Biometric parameters of Echinacea purpurea ‘Raspberry Truffle’ at 4th week of acclimatization. Parameter Treatment Control ABA BL 24-epiBL Goteo Height of plants [cm] 12.10 b 14.00 c 11.90 b 12.10 b 16.00 d Number of leaves 6.60 c 4.00 a 4.70 b 4.80 b 6.23 c Length of leaf blade [cm] 4.20 b 3.90 b 4.10 b 3.80 b 5.70 c Width of leaf blade [cm] 2.60 c 2.50 bc 2.40 b 2.40 b 3.70 d Number of roots 4.10 c 5.00 d 3.10 b 5.00 d 4.00 c Length of roots [cm] 15.00 b 12.90 a 14.70 b 17.10 c 23.90 e Mortality [%] 40.00 a 4.00 b 4.00 b 7.00 b 20.00 ab *the same letter in the lines indicates no difference between the means at a significance level of α = 0.05 Discussion The use of PGRs or biostimulant can facilitate the condition of Echinacea purpurea ‘Raspberry Truffle’. To study changes in the state and activity of the stomata it is commonly using stomatal conductance measurements considering its precision and non-invasiveness. At the initial stages (0 and 1 st date) was observed the highest values of Gs . Results indicates on reaction of plant to stress by stomata closuring. ABA controls stomatal conductance and hence CO 2 transport into leaf mesophyll (Pospíšilová et al. 2009). This phenomenon is likely associated with the extent of stomata. Indeed, several authors have noted abnormalities in these structures under in vitro conditions, as well as the lack of stomatal function during in vitro cultivation. Początek formularza(Chaari-Rkhis et al. 2015). The decline of Gs during acclimation was noticeable by Fila et al. (2006) on grape. On the other hand, the results of Pospíšilová at al. (2009) on tobacco shoved significant drop of Gs in the 1 st date of acclimation to be slightly higher on the 7 th day. Our results are consistent with Chaari-Rkhis et al. (2015), where a significant increase in the values of Gs just after transfer in micropopagated olive plants to gradual decline in the acclimation period. ABA application after transfer to external environment strongly reduced water loss even when stomata are still non-functional. Foliar application microalgae biostimulant to grape plants increased Gs under drought stress (Mancuso et al. 2006). Application of ABA on tobacco affected on the lower Gs values on the 7 th day of acclimation in contrast to control (Pospíšilová et al. 2009). It is observed upgrade of photosynthetic pigments during acclimation and in the duration of the process, drop od ROS and other stress markers level and launching of antioxidant system. The present study confirms that acclimation period is critical during micropropagation at least on physiological and biochemical levels. Our study revealed that both treatments and term significantly affected photosynthetic pigments. The results indicate an increase in chl and carotenoids content along the acclimation period. It was recorded higher chl content in ABA treatment compared to control. Study of Siddique and Anis (2008) also revealed the increasing chl a, chl b and carotenoids content in acclimatization period of Ocimum basilicum. In turn, Al-Deeb et al. (2023) though it was not significant, ABA appears to mitigate the negative effect of higher temperature and water stress on the total chl content on tomato seedlings. ABA application increased chl and carotenoids contents in wheat genotypes under moderate water stress (Agarwal et al. 2005). Results of Pospíšilová et al. (2009) also proved the beneficial impact of ABA on chl a+b and carotenoids content. BRs affect defense mechanisms in plants by increasing their tolerance to stresses. According to Anwar et al. (2018) the application of BRs increase chl content which improves the photosynthesis rate. Indeed, in this study we recorded both, higher chl and carotenoids content in comparison to control. BRs caused an increase in total chl and carotenoids, with their levels being the highest among all treatments. Similar results were reported by Asha and Lingakumar (2015), who observed an increase in this parameters as well as soluble proteins and proline in Vigna seedlings. Higher, though not significant, chl content results were obtained using Goteo in our experiment. The results of Carillo et al. (2019) results indicates that application of biostimulant in jute strongly affected chl b increase. As it is known that under critical conditions, including ex vitro acclimation, the level of ROS increases. The most common is H 2 O 2 , the concentration of which is regulated due to the enzymatic activity of CAT, which decay H 2 O 2 into water and oxygen (Sofo et al. 2015). In our study initially, we observed raised content of H 2 O 2 (0, 1 st and 2 nd terms) and MDA (1 st and 2 nd terms). Considering the average H 2 O 2 levels, they were not significantly lower in treated plants compared to the control. However, at the first time point, the application of ABA, 24-epiBL, and Goteo significantly reduced these values, which may have contributed to alleviating stress. In contrast Dias et al. (2014) did not observed significant differences in the content of H 2 O 2 in control and ABA treated plants in acclimated Ulmus minor . The results corresponds with Liu et al. (2009) where chilling stress in Chorispora bungeana caused increases in H 2 O 2 and OH − contents, which were inhibited by 24-epiBL treatment. Seyed Hajizadeh et al. (2023) reported the impact of Acsophyllum nodosum with nano-silicone on drop of H 2 O 2 and MDA content in Rosa damascena under salinity stress. In our study MDA content decreased, suggesting that exogenous treatments can improve the photosynthesis and antioxidant properties of coneflower and thus alleviate the acclimation stress. Our experiment revealed that Goteo affected mostly on drop of MDA in each term. In contrast ABA treatment did not affected on its drop, furthermore we observed more MDA than in control plants. However, drop of MDA by ABA application obtained Li et al. (2014). In study of Kumar et al. (2012) ABA dosing on stressed chickpea also caused decreasing of MDA and H 2 O 2 . We did not obtained lower values in BRs treatment compared to control, although although the application of 24-epiBL at 3 rd term of observations resulted in a reduction of the marker which, considering the averages, gives a significant result Results corresponds with rice growing in Cd stress whereas 0.1 mM brassinolide application affected on drop of MDA and CAT activity (Li et al. 2023). To tolerate acclimation stresses, plants have adopted biochemical mechanisms including CAT production. It is beneficial enzyme for the removal and control of high H 2 O 2 levels, but they are less suited for a fine tuning of sensitive redox balances with low H 2 O 2 concentrations that may be important for regulatory mechanisms. Our research proved a positive correlation between CAT accumulation and H 2 O 2 drop in 3 rd term which indicate adaptation to ex vitro conditions (Fig. 2). Several abiotic stresses resulted in the accumulation of CAT which has many functions like maintenance of different metabolites and redox balance. Hameed et al. (2013) revealed that permanent oxidative stress caused by H 2 O 2 grew up the CAT activity. Oxidative stress launched by H 2 O 2 can modulate the antioxidant defense system in wheat leaves. Regarding this, H 2 O 2 has been referred both as a signaling molecule and a regulator of the expression of some cell genes such as encoding antioxidants, cell rescue/defense proteins (Hung et al. 2005, Hameed et al. 2013). In addition, CAT is distinguished from numerous other peroxide-metabolizing enzymes by its high specificity for H 2 O 2 and weak activity against organic peroxides (Mhamdi et al. 2010). A study conducted on maize indicated that CAT exhibited the greatest responsiveness to H 2 O 2 , displaying heightened activity within the initial 48 hours. In terms of H 2 O 2 elimination rates, CAT activities were found to be, on average, 21 and 99 times higher compared to GPX and APX activities, respectively (Gondim et al. 2012). ABA in ex vitro acclimatization prevents water loss and improves the activity of antioxidant enzymes needed to alleviation the ex vitro stresses (Aguilar et al. 2000). In conducted experiment we obtained increased CAT activity in all treatments in comparison to untreated plants. Also, stressed sugar cane sprayed with ABA showed higher CAT activity (Li et al. 2014). Exogenous application of 24-epiBL increased the activity of antioxidant enzymes such as CAT to overcome the toxic effects caused by Pb of Brassica juncea seedlings (Santos Neri Soares et al. 2020). Rice growing in Cd stress whereas application of BR resulted in growing CAT activity (Li et al. 2023). Application of PBs caused a slight improvement in antioxidant activities in maize and soybean plants (De Vasconcelos et al. 2009). As well foliar application of biostimulant based on propolis and maize improved the antioxidant mechanism on by higher activity of SOD, CAT, POX, APX in faba exposed to various stresses (Desoky et al. 2021). The raised content of sugars can act as a signal molecule in plants stress. Our research display the altered sugars content along acclimation. The slight increase of reduced sugars in the 1 st term indicates plant stress. Its content drop on the acclimation period. Sperdouli and Moustakas (2012) announced an accumulation and correlation of increased anthocyanins, soluble sugars and free proline maintaining an antioxidant protection in Arabidopsis thaliana leaves affected drought stress. Our results seem to confirm this studies. On the other hand, increased content of sugars was noticed on by application 24-epiBL by BR biosynthesis inhibitor application. (Bajguz and Asami, 2005). Sorghum seedlings treated with ABA produced higher level of soluble sugars which indicates that under less germination increased levels of sugars along with enzymes might be involved in acclimation (Thakur and Sharma, 2005). ABA foliar application significantly increased glucose and fructose concentrations in tomato fruits (Casey Barickman et al. 2017). Our study shows high reduced sugars content in the 2 nd and 3 rd terms than in 1 st term by Goteo spraying. In work of Babazadeh et al. (2023) the algal extract on barley further increased the concentration of soluble carbohydrates in cold-stressed plants, resulting in the highest accumulation of carbohydrates. The expression of plant response on acclimation was also exemplified in the increasing level of soluble proteins, where it increased substantially along this period. The average level, except of control was not significantly differ in the treatments. Our results points that decreasing of protein content of in vitro, increasing thereafter. The data support that initial acclimatization is stressful, and part of the degraded protein is probably rubisco (Carvalho et al. 2005). Brito et al. (2009) also determined soluble proteins content as stress indicator in acclimated plants where they observed higher content in field than in vitro conditions. Free amino acids are known as essential in forming young tissues as they are responsible for the regeneration of tissues exposed to stresses (Rai, 2002; Vardhini, 2014). According to this, plantlets were under the strongest stress on the first period of acclimation, or also at the same time they formed new tissues as at 0 term. At the remaining dates, the level of free amino acids dramatically dropped. What is similar results were revealed by Pustovoitova et al. (2001) in cucumber. In our study, on average, the lowest level of amino acids was obtained by Goteo treatment. However, interestingly at the 2 nd timepoint we found drop of its by BRs spraying in contrast to remaining treatments. Pacholczak et. al (2021) proved application of BRs with the auxin affected in high content of free amino acids. Our research has shown that the application of growth regulators has a direct impact on plant survival, which can influence increased profitability in production. The least acclimatized plants were observed in the control group (up to 60%) which corresponds with Ożarowski et al. (2023) where they obtained 70% plant survival. Goteo results in a higher percentage of acclimatised plants, which is 80%. A positive impact was particularly noted with ABA and BL on the acclimatization of coneflower ‘Rapberry Truffle’ (average mortality was 4%) and the both brassinosteroids (7% mortality). Application of BRs did not affected significantly on biometric parameters of coneflower. de Souza Arantes et al. (2020) reported the positive impact of BR analogue on larger leaf area, sprout diameter number of leaves. Our study shoved PB Goteo have a positive effect on stem, leaves and root growth, promoting cell proliferation, however the mortality was quite high and was 20%. The study on stressed strawberry also revealed the impact on improving the growth parameters by PBs based on Ascophyllum nodosum (Shakya et al. 2023). Conclusions This experiment on the chemistry and slight physiology of acclimation period on Echinacea purpurea by application of selected PGRs and PB provides a convincing evidence that and we can regulate the critical acclimation period by dozing substances, what probably provides high quality and limit the mortality of acclimatized plants. In the acclimatization period it was observed the increase of chl, carotenoids, CAT, soluble proteins, drop of MDA, H 2 O 2 , free amino acids content and altered sugars content. Applied substances affected significantly on levels of biochemical parameters. Moreover, the results concerning BRs provide evidence for the effectiveness of these hormones in the acclimatization of E. purpurea . The application of BRs or ABA in E. purpurea ‘Raspberry Truffle’ has the most beneficial impact reduce mortality of acclimatized plants, which is crucial in plant production. The effectiveness of these rarely used hormones may also be efficient in other species. We believe that our experiment contributes to the advancement of knowledge regarding the acclimatization process of herbaceous plants to ex vitro conditions and serves as a guide for identifying substances that may better alleviate plant stress and enhance their growth during acclimatization. Abbreviations ABA – abscisic acid APX - ascorbate peroxidase BA - 6-Benzylaminopurine BR – brassinolide CAT – catalase chl - chlorophyll GPX - glutathione peroxidase Gs - stomatal conductance H 2 O 2 – hydrogen peroxide IBA - indole-3-butyric acid MDA – malondialdehyde PBs – plant biostimulants PGRs – plant growth regulators POX – peroxidases ROS – reactive oxygen species SOD - superoxide dismutase 24-epiBL - 24-epibrassinolide Declarations Data availability The data supporting the findings are available from the corresponding author upon request. Funding This research received no external funding. Competing Interests The authors have no relevant financial or non-financial interests to disclose. 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Rosen H (1957) A modified ninhydrin colorimetric analysis for amino acids. Archives of Biochemistry and Biophysics 67(1):10–15. https://doi.org/10.1016/0003-9861(57)90241-2. Santos Neri Soares TF, Fernandes dos Santos Dias DC, Santos Oliveira AM, Mendes Ribeiro D, dos Santos Dias LA (2020) Exogenous brassinosteroids increase lead stress tolerance in seed germination and seedling growth of Brassica juncea L. Ecotoxicology and Environmental Safety 193. 110296. https://doi.org/10.1016/j.ecoenv.2020.110296. Semorádová Š, Synková H, Pospíšilová J (2002) Responses of tobacco plantlets to changes of irradiance during transfer from in vitro to ex vitro conditions. – Photosynthetica 40:605-614. https://doi.org/10.1023/A:1024316405903. Seyed Hajizadeh H, Azizi S, Aghaee Karakus S, Kaya O (2023) Nano-silicone and Ascophyllum nodosum-based biostimulant down-regulates the negative effect of in vitro induced-salinity in Rosa damascena . BMC Plant Biology 23:560. https://doi.org/10.1186/s12870-023-04584-2. Shakya R, Capilla E, Torres-Pagán N, Muñoz M, Boscaiu M, Lupuţ I, Vicente O, Verdeguer M (2023) Effect of two biostimulants, based on Ascophyllum nodosum extracts, on strawberry performance under mild drought stress. Agriculture 13:2108. https://doi.org/10.3390/agriculture13112108. Sharma GK, Jagetiya S, Dashora R (2015) General Techniques of Plant Tissue Culture. North Carolina, United States. Siddique I, Anis M (2008) An improved plant regeneration system and ex vitro acclimatization of Ocimum basilicum L. Acta Physiologiae Plantarum 30:493–499. https://doi.org/10.1007/s11738-008-0146-6. Siddiqui H, Yusuf M, Faraz A, Faizan M, Sami F, Hayat S (2018) 24-Epibrassinolide supplemented with silicon enhances the photosynthetic efficiency of Brassica juncea under salt stress. South African Journal of Botany 118:120-128. https://doi.org/10.1016/j.sajb.2018.07.009. Sofo A, Scopa A, Nuzzaci M, Vitti A (2015) Ascorbate peroxidase and catalase activities and their genetic regulation in plants subjected to drought and salinity stresses. International Journal of Molecular Sciences 16(6): 13561-13578. https://doi.org/10.3390/ijms160613561. Sousaa DJP, Nogueiraa GAS, Teixeiraa KBS, Monteiroa GGTN, Britoa AEA, Nascimentoa VR, Albuquerquea GDP, Oliveiraa TJM, Souzaa LC, Freitasa JMN, Oliveira Netoa CF, Okumurab RS (2022) Mitigation of the effects of salt stress in cowpea bean through the exogenous aplication of brassinosteroid. Brazilian Journal of Biology 82:260818 7/7. https://DOI:10.1590/1519-6984.260818. Sperdouli I, Moustakas M (2012) Interaction of proline, sugars, and anthocyanins during photosynthetic acclimation of Arabidopsis thaliana to drought stress. Journal of Plant Physiology 169:577–585. https://doi.org/10.1016/j.jplph.2011.12.015. Ślesak I, Libik M, Karpinska B, Karpinski S, Miszalski Z (2007) The role of hydrogen peroxide in regulation of plant metabolism and cellular signalling in response to environmental stresses. Acta Biochimica Polonica 54(1), 39-50. Tavares AR, dos Santos PLF, Zabotto AR, do Nascimento MVL, Jordão HWC, Boas RLV, Broetto F (2020) Seaweed Extract to Enhance Marigold Seed Germination and Seedling Establishment. SN Applied Sciences 2:1792. https://doi.org/10.1007/s42452-020-03603-3. Thakur M, Sharma AD (2005) Salt stress and phytohormone (ABA)-induced changes in germination, sugars and enzymes of carbohydrate metabolism in Sorghum bicolor (L.) Moench Seeds. Journal of Agriculture & Social Sciences 1(2):90-93. Valluru R, Van den Ende W (2008) Plant fructans in stress environments: emerging concepts and future prospects.. Journal of Experimental Botany 59(11):2905–2916. https://doi.org/10.1093/jxb/ern164. Vardhini BV (2014) Brassinosteroids’ role for amino acids, peptides and amines modulation in stressed plants - A review. Plant Adaptation to Environmental Change: Significance of Amino Acids and their Derivatives; CABI: Wallingford, UK, 300–316. Vilela BJ, Carvalho LC, Ferreira J, Amâcio S (2007) Gain of function of stomatal movements in rooting Vitis vinifera L. plants: regulation by H 2 O 2 is independent of ABA before the protruding of roots. Plant Cell Reports 26:2149-2157. https://doi.org/10.1007/s00299-007-0427-3. Cite Share Download PDF Status: Published Journal Publication published 10 Oct, 2024 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted Reviewers agreed at journal 16 Jul, 2024 Reviewers invited by journal 16 Jul, 2024 Editor assigned by journal 15 Jul, 2024 First submitted to journal 12 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4730353","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":327971977,"identity":"e06cd666-7f6b-4702-80aa-f211cbfc5372","order_by":0,"name":"Monika Grzelak","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-8925-4557","institution":"Warsaw University of Life Sciences: Szkola Glowna Gospodarstwa Wiejskiego w Warszawie","correspondingAuthor":true,"prefix":"","firstName":"Monika","middleName":"","lastName":"Grzelak","suffix":""},{"id":327971978,"identity":"e4cba958-713c-4281-8e4a-f9ca3d3b1ec4","order_by":1,"name":"Andrzej Pacholczak","email":"","orcid":"","institution":"Warsaw University of Life Sciences: Szkola Glowna Gospodarstwa Wiejskiego w Warszawie","correspondingAuthor":false,"prefix":"","firstName":"Andrzej","middleName":"","lastName":"Pacholczak","suffix":""},{"id":327971979,"identity":"adf32164-98e0-4993-bf59-065fc15972fa","order_by":2,"name":"Karolina Nowakowska","email":"","orcid":"","institution":"Warsaw University of Life Sciences: Szkola Glowna Gospodarstwa Wiejskiego w Warszawie","correspondingAuthor":false,"prefix":"","firstName":"Karolina","middleName":"","lastName":"Nowakowska","suffix":""}],"badges":[],"createdAt":"2024-07-12 12:43:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4730353/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4730353/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11240-024-02869-4","type":"published","date":"2024-10-10T15:57:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62097036,"identity":"cd1121ce-4f74-4f80-a8fe-20e44cd4f35b","added_by":"auto","created_at":"2024-08-09 08:55:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4334964,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e regenerated \u003cem\u003eEchinacea purpurea\u003c/em\u003e ‘Raspberry Truffle’ Moench. Microplants (a). Plantlets from \u003cem\u003ein vitro\u003c/em\u003e conditions ready to acclimatization (b), plantlets being acclimated (c,d). Micropropagated plantlets in the 4\u003csup\u003eth\u003c/sup\u003e week of acclimatization prior transferring to the greenhouse (e). Leftwards: control, ABA, BL, 24-epiBL, PB Goteo (e). Plants which had grown in the greenhouse (f).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4730353/v1/af12412d5d860fd1737a3994.png"},{"id":62097035,"identity":"338aa7b0-60ed-4458-8326-205aa33df245","added_by":"auto","created_at":"2024-08-09 08:55:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":36448,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in contents of total chlorophyll\u003csub\u003e \u003c/sub\u003e(A), and carotenoids (B) during acclimation of \u003cem\u003eEchinacea purpurea \u003c/em\u003eMoench. ‘Raspberry Truffle’ depending on date and treatments. *The same letter in the lines indicates no difference between the means at a significance level of α = 0.05.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4730353/v1/95ad1e3a491a0bba12b6d98d.png"},{"id":62097037,"identity":"9ebc51be-8a1d-438d-b9cd-89de0e4b5f9a","added_by":"auto","created_at":"2024-08-09 08:55:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":136009,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in contents of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003e(A), MDA (C) and CAT activity (B) during acclimation of \u003cem\u003eEchinacea purpurea \u003c/em\u003eMoench. ‘Raspberry Truffle’ depending on date and treatments. *The same letter in the lines indicates no difference between the means at a significance level of α = 0.05.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4730353/v1/9f889c3182f2409c452a8f78.png"},{"id":66597545,"identity":"91c729ce-61fe-49d2-a664-e34fdca27b3c","added_by":"auto","created_at":"2024-10-14 16:10:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7927322,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4730353/v1/81c0c3b2-26fa-41f2-b5a5-3bea987e4fe6.pdf"}],"financialInterests":"","formattedTitle":"The effect of several growth regulators and biostimulant on biochemical and physiological changes in acclimation of micropropagated Echinacea purpurea Moench. ‘Raspberry Truffle’","fulltext":[{"header":"Key message","content":"\u003cp\u003eBiochemical and physiological changes occur in plants during acclimation. We selected the most beneficial growth regulators alleviating acclimation stress caused \u003cem\u003eby ex vitro\u003c/em\u003e transfer and increasing plant survival rate.\u003c/p\u003e\n"},{"header":"Introduction","content":"\u003cp\u003eMicropropagation is widely used method of vegetative plants multiplication. Thanks to the use of \u003cem\u003ein vitro\u003c/em\u003e technology, a mass of genetically homogenous plantlets can be produced in a short time (Sharma et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). It was confirmed as cost-effective for multiplication of large amount species (Kikowska et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Acclimatization is the last step of \u003cem\u003ein vitro\u003c/em\u003e propagation. However, this is the most critical one, due to change of environment. In glass, microplants grow in favorable, disease-free conditions. Nevertheless, growing \u003cem\u003ein vitro\u003c/em\u003e results in various range of abnormalities in plants on morphological, anatomical, physiological, biochemical and molecular levels. These are due to high air humidity, low irradiance, low CO\u003csub\u003e2\u003c/sub\u003e concentration during light period, supplementation of growing media with sugars and plant growth regulators (PGRs) (Baťková et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Therefore, plantlets must be able to adapt to new, external conditions and correct the abnormal features. Additionally, \u003cem\u003eex vitro\u003c/em\u003e transfer of micropropagated plantlets is often accompanied by water stress and/or photoinhibition (Semorádová et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Carvalho et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, Baťková et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). By transfer to external environment plants are exposed to stresses, which can lead to high mortality of plants. Transplantation to \u003cem\u003eex vitro\u003c/em\u003e conditions results in the increased rate of production of reactive oxygen species (ROS) like superoxide anions (O\u003csup\u003e2−\u003c/sup\u003e), hydroxyl radical (OH), singlet oxygen (\u003csub\u003e1\u003c/sub\u003eO\u003csup\u003e2\u003c/sup\u003e ), and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) (Anjum et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In normal conditions, ROS are byproducts of aerobic metabolism and can be signals that switch on developmental programs or regulate physiological processes as cell wall loosening necessary for cell elongation, modulation of cytosol Ca\u003csup\u003e2+\u003c/sup\u003e concentration, senescence, adaptation to abiotic stresses or resistance to pathogens (Papadakis and Roubelakis-Angelakis \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Gechev et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Procházková and Wilhelmová \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Ślesak et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Vilela et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Baťková et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). However, excess of ROS can make harder the production of biomolecules such as lipids, proteins and nucleic acids and contribute to lipid peroxidation, protein degradation, enzyme inactivation and damage of DNA (Baťková et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Gil and Tuteya 2010). For example, accumulation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e can be detrimental for the plant cell, leading to cell death, a fine-tuning of this signaling molecule can result in stress management by coordinating intra-cellular and systemic signaling systems. ROS overproduction can be scavenged by the antioxidative systems which include low molecular mass antioxidants (ascorbate, glutathione, tocopherols, carotenoids, phenols) and antioxidative enzymes (superoxide dismutase, ascorbate peroxidase, catalase, glutathione reductase, monodehydroascorbate reductase, dehydroascorbate reductase) (Anjum et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Peroxidation of membrane lipid is a main detrimental effect of ROS. Commonly, membrane lipid peroxidation in plant tissues is determined by measuring malondialdehyde (MDA). It is a frequently used marker of oxidative lipid injury caused by environmental stress (Amnan et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A various range of studies have investigated MDA of plants under different stress conditions, including herbaceous plants as proved Kong at al. (2016) in oilseed rape or El-Batal et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) in carrot. Sugars can play as a signal molecule in various range of plant stresses. Soluble sugars gather while biotic and abiotic stress conditions apply to oxidative stress further pointing towards a relationship between sugars and stress-induced ROS accumulation in plants. It is observed a relationship between sugars and oxidative stress, as it was displayed that high sugar content can either promote or decrease ROS production in plants. Studies on \u003cem\u003ein vitro\u003c/em\u003e revealed that the \u003cem\u003ein vivo\u003c/em\u003e antioxidant capacity of sugars might be highly undervalued. Various range of sugars are proven to act as antioxidants (Couée et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Keunen et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Simple sugars and polysaccharides are known as a protection of cellular membranes, which is a premise for survival of plants on stress (Valluru and Van den Ende \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Chlorophyll (chl) is known as a pigment absorbing light which exploits sunlit energy for ATP synthesis (Coste et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Determining chl content in leaves is one of the relevant parameter used to estimate the plants photosynthetic rate (Dalio et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo alleviate stress associated with acclimatization, it is necessary to identify substances that mitigate adverse conditions. one of them can be ABA which is a stress-related signaling molecule in plants responding to plant stresses. It is present in most plant organs and is involved in stomatal closure, reduction of ROS, seed dormancy, and seedling development (Hauser et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Hence the assumption that it may be effective in alleviating stress associated with acclimatization. Brassinosteroids (BRs) are relatively new group of hormone firstly isolated in rape. Various range of activities are regulated by them as seed germination, cell division, vascular differentiation, cell elongation, root development, stomata formation, flower development, male sterility, photomorphogenesis, and resistance to stresses. Moreover, they have a beneficial role in modulating plants from excess of ROS (Ranjan and Ranjan 2023). They are considered as short-distance signals, because they are not able to be transported within long distances, nonetheless they alter auxin transport by which act as an indirect long-distance signal molecule (Siddiqui et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Its exogenous application of mentioned hormone affect on stress tolerance mechanisms, which was observed in cowpea bean (Sousa et al. 2022). BRs are able to increase the chl content as proved Alyemeni and Al-Quwaiz (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The using of plant biostimulants (PBs) is growing in plant production, by the increasing interest of farmers in natural materials and helpful microorganisms that can sustainably improve the productivity of crops and ornamentals. Many of them are widely used in plant cultivation, mainly owning to their beneficial effects on nutrient uptake, plant growth, yield, and fruit quality and the tolerance of plants to abiotic stressors (de Pascale et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kisvarga et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). PBs act on as a promising strategy to reduce the unfavorable effects of osmotic stress. It was proved their beneficial effect as activator the ROS scavenging enzyme system (Campobenedetto et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). One of plant PBs, containing brown seaweed - \u003cem\u003eAscophyllum nodosum\u003c/em\u003e can be implemented as nutrient supplements, biofertilizers in horticultural plant systems (Tavares et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eEchinacea purpurea\u003c/em\u003e (L.) Moench. it is a common long-blooming, herbaceous, perennial garden and cut plant called eastern purple coneflower (Armitage, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eE. purpurea\u003c/em\u003e ‘Raspberry Truffle’ is commercially important variety of this species bred by the plant hybridizer Arie Blom, registered in 2012. There are some reports concerning micropropagation of \u003cem\u003eEchinacea\u003c/em\u003e. The study of Harbage (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) with method allowing rapid multiplication withs selected PGRs or Chen et al. (2016) using supplementation of diethyl aminoethyl hexanoate (DA-6). However, the process of acclimation was not widely described so far. In this study there were used \u003cem\u003eE. purpurea\u003c/em\u003e ‘Raspberry Truffle’ as an experimental plant.\u003c/p\u003e \u003cp\u003eThe aim of this work was to demonstrate the biochemical and physiological changes occurring in coneflower at different stages of acclimatization. We also would like to determine the effects of exogenous application of PGRs as ABA, BL, 24-epiBL, PB Goteo on biochemical parameters and stomatal conductance of \u003cem\u003eEchinacea purpurea\u003c/em\u003e. We also assessed the impact of the mentioned applications on the morphology of acclimatized plants and how the specific treatments affects the number of acclimatized plants to \u003cem\u003eex vitro\u003c/em\u003e conditions, which translates into the profitability of this species in laboratory conditions.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003cdiv id=\"Sec7\" class=\"Section4\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section4\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section4\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section4\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section4\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section4\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section4\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section4\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section4\"\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Materials and methods","content":"\u003ch2\u003e1.1. Shoot Multiplication and Rooting\u003c/h2\u003e\u003cp\u003eThe medium to multiplication phase was prepared using 0.35 mg·L\u003csup\u003e− 1\u003c/sup\u003e BA and 0.35 mg·L\u003csup\u003e− 1\u003c/sup\u003e IBA were added to the Murashige and Skoog (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1962\u003c/span\u003e) basal media with 30 mg·L\u003csup\u003e− 1\u003c/sup\u003e of sucrose, gelled with supplementation of 8 g·L\u003csup\u003e− 1\u003c/sup\u003e agar, the pH was adjusted to 5.8 before autoclaving. Media with PGRs was autoclaved for 20 min. at 12\u003csup\u003eo\u003c/sup\u003eC and 110 kPa. The microshoots produced in the multiplication stage were suitable for rooting. After 6 weeks multiplied shoots were transferred to the rooting MS medium supplemented with 0.5 mg·L\u003csup\u003e− 1\u003c/sup\u003e IBA and 1 mg·L\u003csup\u003e− 1\u003c/sup\u003e activated charcoal (Sigma Aldrich) with 30 mg·L\u003csup\u003e− 1\u003c/sup\u003e of sucrose and 8 g·L\u003csup\u003e− 1\u003c/sup\u003e agar, pH = 5.8 before autoclaving. Plantlets regenerated in the rooting medium for approx. 6 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). During both phases, the glass jars containing explants were kept in a growth phytotron with 23 ± 1℃, 16 h day with light at 35 µmol m\u003csup\u003e− 2\u003c/sup\u003e s\u003csup\u003e− 1\u003c/sup\u003e photosynthetic photon flux density, provided by fluorescent lamps.\u003c/p\u003e\u003ch2\u003e1.2. Acclimation phase experimental rationale and design\u003c/h2\u003e\u003cp\u003eAfter 6 weeks of regeneration in growth phytotron, the rooted plantlets were removed from the culture jars and the roots were washed using distilled water to remove traces of agar in the surface of roots. Next, microplants were planted in plastic boxes filled with peat (pH = 5.0–6.0) and perlite 1:1 (v/v). Then plantlets were sprayed separately with 200 mL water solution of: ABA in concentration 0.001 mL·L\u003csup\u003e− 1\u003c/sup\u003e, 0.001 mL·L\u003csup\u003e− 1\u003c/sup\u003e BL, 0.001 mL·L\u003csup\u003e− 1\u003c/sup\u003e 24-epiBL and 0.3 mL·L\u003csup\u003e− 1\u003c/sup\u003e biostimulant Goteo (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The concentrations were selected after preliminary experiments. Control treatment was distilled water. Each treatment were tested in a thrice replicated experiment with 10 plantlets. After spraying, plants were covered with transparent lids and kept in growing room in temp. 25\u003csup\u003eo\u003c/sup\u003eC, the photoperiod was established 16 h day (50 µmol·m\u003csup\u003e2\u003c/sup\u003e·s\u003csup\u003e1\u003c/sup\u003e). After 4 weeks of growing in phytotron, plants were transferred to the greenhouse (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cdiv class=\"gridtable\"\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\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\u003eA list of treatments in the experiment.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo. of treatment\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl “0”\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edistilled water\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=\"left\" colname=\"c2\"\u003e \u003cp\u003eABA\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.001 ml·L\u003csup\u003e− 1\u003c/sup\u003e\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=\"left\" colname=\"c2\"\u003e \u003cp\u003eBL\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.001 ml·L\u003csup\u003e− 1\u003c/sup\u003e\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=\"left\" colname=\"c2\"\u003e \u003cp\u003e24-epiBL\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.001 ml·L\u003csup\u003e− 1\u003c/sup\u003e\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=\"left\" colname=\"c2\"\u003e \u003cp\u003eGoteo\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.3 ml·L\u003csup\u003e− 1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003ch2\u003e1.3. Evaluation\u003c/h2\u003e\u003ch2\u003e1.3.1. Stomatal conductance measurements\u003c/h2\u003e\u003cp\u003eStomatal conductance (\u003cem\u003eGs\u003c/em\u003e) of leaves in plantlets was measured from \u003cem\u003ein vitro\u003c/em\u003e plants (0 term), at the beginning of \u003cem\u003eex vitro\u003c/em\u003e acclimation (1st term), at the end of growing in phytotrone (2nd term) and at the end of growing in the greenhouse (3rd term). During acclimation using a portable SC − 1 leaf porometer (Geomor Technik). Device consists in the principle of measuring the static change of humidity inside a closed measuring head with a leaf placed inside. \u003cem\u003eGs\u003c/em\u003e was expressed as mmol H\u003csub\u003e2\u003c/sub\u003eO m\u003csup\u003e− 2\u003c/sup\u003es\u003csup\u003e− 1\u003c/sup\u003e.\u003c/p\u003e\u003ch2\u003e1.3.2. Biochemical analyses\u003c/h2\u003e\u003cp\u003eThe samples for biochemical analyses (leaves) were picked in the same terms as \u003cem\u003eGs\u003c/em\u003e was measured. The material was chopped, mixed, and samples were collected for each analysis. Three extractions were done for each analysis and 3 measurements were made for each extract. The absorbance was measured in spectrophotometer UV-1601 PC (Shimadzu, Columbia, MD, USA).\u003c/p\u003e\u003ch2\u003e1.3.2.1. Total chlorophyll and carotenoids content\u003c/h2\u003e\u003cp\u003eThe total contents of chl and carotenoids were assessed using the methods described by Lichtenthaler and Wellburn (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). Plant material was ground in a mortar with a slight amount of quartz sand and 2.5 mL of 80% acetone. The extracts were subsequently filtered through filter paper into 25 mL volumetric flasks and filled up with acetone. The absorbance of filtrates was measured at: 470, 646, 652, and 663 nm.\u003c/p\u003e\u003ch2\u003e1.3.2.2. Preparation of extracts to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, catalase, soluble proteins\u003c/h2\u003e\u003cp\u003e0.25 g samples were homogenized in 2.5 mL K-phosphate buffer. The extracts were centrifuged by 20 min. at 20.000 xg at 4°C. Next, the extract was transferred to tubes.\u003c/p\u003e\u003ch2\u003e1.3.2.3. Hydrogen peroxide content\u003c/h2\u003e\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content was measured using Pick and Keisari (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1980\u003c/span\u003e) method. Reaction mixture was composed by 0.5 mL extract, 0.5 mL 0.1 M potassium phosphate buffer (pH = 7.0) and 1 mL of 1 M potassium iodide. Samples were incubated by 1 h in the darkness at room temperature and their absorbance were measured at 390 nm.\u003c/p\u003e\u003ch2\u003e1.3.2.4. Catalase activity\u003c/h2\u003e\u003cp\u003eThe analysis of catalase activity was conducted following the methodology outlined in the work by Goth (1991). The tubes were divided into two sets, each receiving 0.05 mL of the extract along with 0.45 ml of K-phosphate buffer. In tubes labeled 'A', 1 mL of 0.1 M K-phosphate buffer was added, while tubes labeled 'B' received 1 mL of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in buffer (at a concentration of 65 µM). A control sample, denoted as 'K', was prepared by adding 1.5 mL of 0.1 M K-phosphate buffer, while sample 'C' consisted of 0.5 mL of 0.1 M K-phosphate buffer mixed with 1 mL of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in buffer (at a concentration of 65 µM). All tubes were then placed in darkness for incubation for 10 minutes. Following incubation, 1 mL of 32.5 mM ammonium molybdate was added to each tube and thoroughly mixed. Subsequently, absorbance was measured at 405 nm.\u003c/p\u003e\u003ch2\u003e1.3.2.5. MDA content\u003c/h2\u003e\u003cp\u003eMDA determination was based on the method described by Heath and Packer (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1968\u003c/span\u003e). Leaf samples weighing 0.25 g were homogenized in 2.5 ml of trichloroacetic acid (TCA) (w/v). The resulting extract was centrifuged for 10 minutes at 10,000 xg and 4°C. The reaction mixture consisted of 1.0 mL of supernatant and 4.0 mL of 20% thiobarbituric acid (TBA) in 0.5% TCA (w/v). Subsequently, the samples were incubated for 30 minutes at 95°C, followed by stopping the reaction on ice for 10 minutes. Afterward, the samples underwent another centrifugation at 10,000 xg for 10 minutes at 4°C. Absorbance was measured at 532 nm and 600 nm to account for nonspecific absorbance using an extinction coefficient (ε) of 155 mM\u003csup\u003e− 1\u003c/sup\u003ecm\u003csup\u003e− 1\u003c/sup\u003e.\u003c/p\u003e\u003ch2\u003e1.3.2.6. Free amino acids\u003c/h2\u003e\u003cp\u003eThe procedure for determining the content of free amino acids was adapted from the Rosen (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1957\u003c/span\u003e) method. Specifically, 0.2 ml of extract was combined with 0.5 mL of 0.2 mM sodium cyanide in acetate buffer (pH = 5.3–5.4) and 0.5 mL of ninhydrin. After thorough mixing, the solutions were incubated in a water bath at 100°C for 15 minutes. Subsequently, the tubes were taken out, and 5 mL of isopropyl alcohol was added to each warm tube. The mixtures were then allowed to cool. Absorbance readings were taken at 570 nm against a standard curve for leucine.\u003c/p\u003e\u003ch2\u003e1.3.2.7. Total soluble proteins\u003c/h2\u003e\u003cp\u003eThe concentration of total soluble proteins was measured using Bradford's method (1976). The reaction mixture comprised 0.1 mL of the extract and 5.0 mL of Bradford's reagent. The mixture was incubated in the dark for 5 minutes, and the absorbance was then measured at 595 nm.\u003c/p\u003e\u003ch2\u003e1.3.2.8. Total sugars content\u003c/h2\u003e\u003cp\u003eTotal sugars were measured by the colorimetric method of Dubois et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1956\u003c/span\u003e). The samples were homogenized in hot 80% ethanol. The extract was centrifuged for 20 minutes at 20.000 xg at 4°C. Then, the supernatant was transferred into resealable tubes and adjust to 25 mL with 80% ethanol. The reaction mixture comprised 100 µl of extract, 1 mL of 5% phenol, and 5 ml of sulphuric acid (H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 96%) and mixed thoughtfully. The solutions incubated for 20 minutes. The absorbance was measured against a standard curve for glucose at 490 nm.\u003c/p\u003e\u003ch2\u003e1.3.2.9. Reduced sugars content\u003c/h2\u003e\u003cp\u003eReduced sugars content was determined by Nelson (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1944\u003c/span\u003e) method. The samples were homogenized in hot 80% ethanol. The extract was centrifuged for 20 minutes at 20.000 xg at 4°C. Then, the supernatant was transferred into resealable tubes and adjust to 25 mL with 80% ethanol. 100 µL of extract were taken. Then, 1 mL of copper reagent was added. The mixture was incubated for 20 minutes in a water bath at 100°C. Afterward, it was cooled in cold water. 1 mL of molybdenum-arsenic reagent was added and mixed. Finally, distilled water was added to make up the volume to 5 mL. The absorbance was measured against a standard curve for glucose at 520 nm.\u003c/p\u003e\u003ch2\u003e1.3.3. Morphological parameters\u003c/h2\u003e\u003cp\u003eBiometric data (plant height, number of leaves, average length and width of 3 leaves, number of roots, length of roots) were recorded at the 4th week of acclimation stage. Mortality of the plantlets were calculated using following equation:\u003c/p\u003e\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:mortality\\:\\left[\\%\\right]=\\frac{number\\:of\\:dead\\:plants}{total\\:number\\:of\\:plantlets}*100$$\u003c/div\u003e\u003c/div\u003e\u003ch2\u003e1.4. Data analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis, data and graphical presentation were performed with Statistica Version 13.1 (STATSoft, Tulsa, OK, USA) and Microsoft Excel 365 (Version 2023). Experimental data were subjected to one way and two-way analysis of variance and then to Tukey’s multiple range test to separate the means at the significance level of p ≤ 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003e2.1. Stomatal conductance\u003c/h2\u003e\n \u003cp\u003eInitially (at date 0), stomatal conductance was 192.9, which is similarly high as on the 1st date. The greatest drop in \u003cem\u003eGs\u003c/em\u003e relative to the initial value occurred with 24-epiBL spraying. When considering treatments separately on the 1st date, the values were significantly the highest with BL and Goteo treatments. A marked decrease was noticed on the 2nd date, while values did not differ from each other except for untreated plants with the highest result. Plants treated with ABA on the 3rd date remained at the same low level as before. However, \u003cem\u003eGs\u003c/em\u003e with the other applications slightly increased. Overall, summarizing the average values of treatments, the values did not differ from each other (Table \u003cspan\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eChanges in contents of stomatal conductance (\u003cem\u003eGs)\u003c/em\u003e [mmol H\u003csub\u003e2\u003c/sub\u003eO m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e] during acclimation of \u003cem\u003eEchinacea purpurea\u003c/em\u003e Moench. \u0026lsquo;Raspberry Truffle\u0026rsquo; depending on date and treatments.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"8\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean (for terms)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e\u003cem\u003eGs\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e[mmol H\u003csub\u003e2\u003c/sub\u003eO m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTerm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24-epiBL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGoteo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003e192.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e186.20 bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e195.26 bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e212.33 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e174.33 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e217.43 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e197.16 c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e175.53 bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e86.86 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e112.70 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e103.2 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90.70 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e113.70 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e164.10 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e83.83 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e194.03 bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e210.83 cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e182.57 bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e167.35 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean (for treatments)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e175.28 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e122.00 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e173.02 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e163.02 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e163.57 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\"\u003e\u003cem\u003e*\u003c/em\u003ethe same letter in the lines indicates no difference between the means at a significance level of \u0026alpha;\u0026thinsp;=\u0026thinsp;0.05\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\"\u003e\n \u003ch2\u003e2.2. Photosynthetic pigments\u003c/h2\u003e\n \u003cp\u003eThe content of chl in \u003cem\u003ein vitro growing plants\u003c/em\u003e was 7.80 and was higher compared to acclimatized plants. Overall, the content of its gradually grows within acclimation process (Fig. \u003cspan\u003e2\u003c/span\u003e). Taking into account the averages of the treatments the highest values were recorded in 24-epiBL and BL treatments. Significantly lower values were recorded in remaining treatments. Regarding terms, the lowest chl content was observed at the beginning of acclimation (4.69) whereas application of 24-epiBL and BL resulted in the increase of chl on that date. On the 2nd term the chl content was at a similar level, besides BL and control plantlets. On the 3rd date the highest values was obtained by ABA, BL and 24-epiBL treatments (Table\u0026nbsp;3). Initially (\u003cem\u003ein vitro\u003c/em\u003e) the carotenoids content was 2.11. In the initial observation period, fewer carotenoids were recorded. There was observed significant increase in its content among acclimatization (1.32 in 1st period, 1.71 in 2nd ). Regarding treatments in 1st period the highest values were obtained by 24-epiBL spraying. In the 2nd period the content was the highest by 24-epiBL and Goteo spraying. In the 3rd term, the content varied depending on the treatment, with statistically the highest content observed in BL, followed by Goteo and 24-epiBL (Table \u003cspan\u003e4\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 4\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eChanges in contents of chlorophyll [mg\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d.w.] and carotenoids [mg\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d.w.] during acclimation of \u003cem\u003eEchinacea purpurea\u003c/em\u003e Moench. \u0026lsquo;Raspberry Truffle\u0026rsquo; depending on date and treatments. Data is represented using 3 replicates.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"8\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean (for terms)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003echlorophyll\u003c/p\u003e\n \u003cp\u003e[mg\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d.w.]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTerm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24-epiBL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGoteo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003e7.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.24 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.69 bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.88 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.58 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.04 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.69 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.38 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.50 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.33 e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.63 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.66 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.50 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.44 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.51 ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.90 f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.45 ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.68 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.19 c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean (for treatments)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.69 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.57 bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.04 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.88 cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.12 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003ecarotenoids [mg\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d.w.]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003e2.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.29 a-d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.21 abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.08 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.72 ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.33 bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.32 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.09 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.49 de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.01 gh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.77 f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.86 fg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.64 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.42 cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.51 de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.15 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.70 ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.77 f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.71 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean (for treatments)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.27 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.40 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.74 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.73 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.65 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\"\u003e*the same letter in the lines indicates no difference between the means at a significance level of \u0026alpha;\u0026thinsp;=\u0026thinsp;0.05\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\"\u003e\n \u003ch2\u003e2.3. Stress biomarkers and antioxidant activity\u003c/h2\u003e\n \u003cp\u003eAcclimation and treatments affects significantly on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content. \u003cem\u003eIn vitro\u003c/em\u003e growing plantlets, similarity to control group on the 1st date accumulated the highest content of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Fig. \u003cspan\u003e3\u003c/span\u003e). Spraying with PGRs and biostimulant influenced the decline values. Similarly high values were on the 2nd term. On the 3rd date its contents generally fell down. The highest H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content was on untreated plants, but there was no significant differences. The average of MDA was significantly higher on the 1st and 2nd term. In other treatments the values slightly dropped. The MDA concentration significantly decreased on the 3rd date. In terms of the treatments, Goteo affected on fall the values in all treatments. In the 3rd term, the lowest value was recorded by 24-epiBL application. Conversely, the increase of CAT activity was recorded during acclimation period. Plantlets on the 0 day had the lowest CAT activity, the highest values observed in 3rd term. It gradually grows in all treatments, however overall the highest values was recorded after Goteo, ABA and both, BRs treatments in comparison to control. In spite of total sugars content, treatment and acclimation period affected the values. On the term 0 and 1st were recorded the lowest contents, spraying of ABA and BL affected on significant decreasing of the content. All other values in this term did not differ significantly from one another, nor did they differ from the respective results in the control treatment. On the 2nd term were recorded the lowest values in the control treatment, within remaining treatments were different values as well. Goteo treatment values were higher in comparison to control plants. Both, ABA and 24-epiBL were higher than above-mentioned treatments and did not differ significantly from one to another. BL affected on the highest values in 2nd term. On the 3rd term were observed decrease of total sugars content compared with previously described. The lowest value was recorded in BL treatment. ABA and 24-epiBL values were higher than BL and similar to each other. On the control and Goteo treatment were observed the highest levels in comparison to remaining groups. Overall, considering all treatments in all terms there were no significant differences between the values. In the case of reduced sugars content it was observed gradual decrease of the values as with CAT activity. Control, Goteo and 24-epiBL treatments on the 1st term were significantly higher than two other treatments. There were no significant differences between ABA and BL values. In spite of 2nd term distinctly the highest value was recorded only with 24-epiBL spraying. BL and Goteo treatment affected on the highest reduced sugars content in comparison to all other groups. To consider the means in the treatments overall, the values were not differ significantly from another, but the highest reduced sugars content were recorded 24-epiBL (Table \u003cspan\u003e5\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 5\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eChanges in contents of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e [\u0026micro;g\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d.w.], MDA [\u0026micro;mol\u0026middot;g d.w.] and CAT [mkat g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d.w.] activity during acclimation of \u003cem\u003eEchinacea purpurea\u003c/em\u003e Moench. \u0026lsquo;Raspberry Truffle\u0026rsquo; depending on date and treatments. Data is represented using 3 replicates.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"8\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean (for terms)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e [\u0026micro;g\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d.w.]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTerm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24-epiBL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGoteo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003e154.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e154.46 f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e123.90 de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e135.51 ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e103.3 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e114.14 cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e126.26 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e113.89 cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e131.18 ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e143.03 fg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e132.98 ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e145.08 fg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e133.23 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88.1 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e78.13 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e63.89 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77.12 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e76.65 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e76.78 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean (for treatments)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e118.82 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e111.07 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e114.14 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e104.47 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e122.64 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003eCAT [mkat g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d.w.]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003e42.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e138.83 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e240.39 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e239.64 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e352.79 cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e394.31 ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e273.19 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e390.70 ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e479.36 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e327.85 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e327.13 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e413.58 f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e387.72 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e366.117 de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e484.48 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e489.69 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e534.51 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e553.51 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e485.66 c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean (for treatments)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e298.55 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e401.41 bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e352.39 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e404.81 bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e453.80 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003eMDA [\u0026micro;mol\u0026middot;g d.w.]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003e0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.81 cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.97 gh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.97 gh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.52 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.86 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.02 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.45 i\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.87 ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.70 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.30 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.87 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.51 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.47 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.43 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,31 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.51 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.45 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean (for treatments)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.78 bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.91 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.78 bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.70 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.44 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e*the same letter in the lines indicates no difference between the means at a significance level of \u0026alpha;\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\"\u003e\n \u003ch2\u003e2.4. Free amino acids and soluble proteins content\u003c/h2\u003e\n \u003cp\u003eFree amino acids values in on day 0 (488.00) and 1st term (439.20) were higher than remaining ones. Within the treatments on 1st term the largest amount of free amino acids were detected by application of 24-epiBL, lower than above-mentioned were by ABA and BL application, but were comparable to each other. Significantly lower free amino acids content were recorded in biostimulant, whereas control plantlets had distinctly the lowest content. On 2nd term, the content dropped sixfold in case average of all treatments. There were observed almost tenfold drop to compare with 1st term in case of spraying with BL, 24-epiBL and Goteo. Thereby these values were the lowest concerning 2nd term. Significantly higher contents recorded by application of distilled water and ABA, the values was differ from ich other. On the 3rd term overall free amino acids content were still lower than on the 1st term, but higher than on the 2nd term. Goteo treatment was not differ than on the 2nd date. Generally considering average of treatments, there were no significant differences between groups, but an increased content of free amino acids was observed in ABA and 24-epiBL application. The accumulation of proteins increased in acclimation stage. Initially (\u003cem\u003ein vitro\u003c/em\u003e), the content was 5.17 to drop in 1st date by almost 25%. Taking into account treatments separately on 1st date, the values were not differ from each other, however the higher value recorded by 24-epiBL spraying. On the 2nd date was observed an increase of soluble proteins content. Similarly high values were observed by application of ABA, BL and Goteo. Distinctly lower contents observed in case of control and 24-epiBL sprayed plantlets. On the 3rd date the content was by almost 24% higher and differ in comparison with previously described one. There were no differences between treatments on this date. Focusing on an average soluble proteins content for all treatments, the lowest values were recorded in control plantlets, there were no significant differences between remaining treatments (Table \u003cspan\u003e6\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 6\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eChanges in contents of free amino acids [mmol leucine g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d.w.] and total soluble proteins [mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d.w.] during acclimation of \u003cem\u003eEchinacea purpurea\u003c/em\u003e Moench. \u0026lsquo;Raspberry Truffle\u0026rsquo; depending on date and treatments. Data is represented using 3 replicates.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"8\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean (for terms)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003efree amino acids [mmol leucine g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d.w.]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTerm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24-epiBL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGoteo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003e488.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e331.93 e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e468.05 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e465.39 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e517.33 i\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e413.31 f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e439.20 c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e136.64 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e103.91 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46.36 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46.91 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.30 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e74.82 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e164.29 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e146.17 cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.86 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e131.77 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47.85 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e115.99 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean (for treatments)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e210.95 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e239.38 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200.54 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e232.01 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e167.15 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003etotal soluble proteins [mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d.w.]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003e5.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.73 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.54 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.94 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.12 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.16 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.90 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.55 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.80 ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.52 fg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.96 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.96 de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.36 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.01 e-h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.80 gh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.11 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.56 efg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.61 fgh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.42 c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean (for treatments)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.43 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.37 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.86 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.88 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.91 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\"\u003e*the same letter in the lines indicates no difference between the means at a significance level of \u0026alpha;\u0026thinsp;=\u0026thinsp;0.05\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eInitially, (day 0), the total sugars content was 68.66. On the 1st date, the average value was similar (61.04). Within the term, the highest value was recorded by Goteo spraying (78.14) the lowest content was determined in ABA and BL treated plants. On the 2nd term there was observed significant rise of reduced sugars compared to previous date. Among this term the lowest values were recorded in control plants (66.94), in treated ones, the content were above 110, to be the highest in BL treatment \u0026ndash; 197.04. On the 3rd date there has been decrease in the values. An average did not differ than on the 1st term. Among the treatments, the highest value was determined in Goteo treated plants, which was comparable to control. Lower values was noted by ABA and 24-epiBL treatments. The lowest content were observed in BL treatment (Table \u003cspan\u003e6\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eIn case od total sugars content on the 0 day the content was 51.60. The average value of that on the 1st term was 53.46. Among the treatments similar lower values were observed in ABA and BL, whereas significantly higher content were recorded in remaining treatments. On the next terms there was observed significant drop of the values (46.34). Least sugars content was determined in Goteo treatment, similarly to control. Higher, but not significantly values were recorded in ABA and BL sprayed plants, to be significantly the highest by 24-epiBL treatment. An average sugars content was not differ from the value on the 2nd date (43.25). significantly the highest content was observed by Goteo treatment (Table \u003cspan\u003e7\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab6\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 7\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eChanges in contents of total [mg glucose \u0026middot; g d.w.\u003csup\u003e\u0026minus;1\u003c/sup\u003e] and reduced sugars [mg glucose \u0026middot; g d.w.\u003csup\u003e\u0026minus;1\u003c/sup\u003e] content during acclimation of \u003cem\u003eEchinacea purpurea\u003c/em\u003e Moench. \u0026lsquo;Raspberry Truffle\u0026rsquo; depending on date and treatments. Data is represented using 3 replicates.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"8\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean (for terms)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003etotal sugars [mg glucose \u0026middot; g d.w.\u003csup\u003e\u0026minus;1\u003c/sup\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTerm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24-epiBL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGoteo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003e68.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e69.71 cde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.66 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49.30 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e69.39 cde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e78.14 e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e61.04 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66.94 cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e132.48 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e197.04 i\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e132.49 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e117.66 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e129.32 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95.40 f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72.49 de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60.10 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e74.82 de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95.40 f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79.04 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean (for treatments)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77.36 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e81.21 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e102.1115 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.23 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96.05 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003ereduced sugars [mg glucose \u0026middot; g d.w.\u003csup\u003e\u0026minus;1\u003c/sup\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003e51.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60.45 fg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44.35 bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.72 bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54.42 fg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64.38 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.46 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.33 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47.41 cde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45.19 bcd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66.16 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.64 abc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46.34 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.25 a-d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.20 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46.64 b-e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.17 a-d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49.02 ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.25 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean (for treatments)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45.34 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.32 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45.18 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.92 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.68 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\"\u003e*the same letter in the lines indicates no difference between the means at a significance level of \u0026alpha;\u0026thinsp;=\u0026thinsp;0.05\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\"\u003e\n \u003ch2\u003e2.5. Biometric parameters and mortality of plants\u003c/h2\u003e\n \u003cp\u003eThe highest plants were observed by Goteo treatment (16.00). The ones treated with ABA were significantly shorter (14.00). However, the shortest plants were recorded in the remaining treatments. Goteo had the most significant impact on root length (average 23.90). Similarly, Goteo resulted in the highest number of leaves, while ABA treatment resulted in the fewest. Leaf blades (both length and width) were the largest when using the PB. The highest number of roots was produced by plants treated with ABA, 24-epiBL, and Goteo (5, 5, and 4 respectively). In terms of mortality, the highest percentage of plants survived in untreated group (40%), while plants treated with Goteo had a mortality rate of 20%. For the other groups, mortality rates were 4% (ABA, BL) and 7% (24-epiBL) (Table \u003cspan\u003e8\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab7\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 8\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eBiometric parameters of \u003cem\u003eEchinacea purpurea\u003c/em\u003e \u0026lsquo;Raspberry Truffle\u0026rsquo; at 4th week of acclimatization.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24-epiBL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGoteo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHeight of plants [cm]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.10 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.00 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.90 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.10 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.00 d\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNumber of leaves\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.60 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.00 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.70 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.80 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.23 c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLength of leaf blade [cm]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.20 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.90 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.10 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.80 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.70 c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWidth of leaf blade [cm]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.60 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.50 bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.40 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.40 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.70 d\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNumber of roots\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.10 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.00 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.10 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.00 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.00 c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLength of roots [cm]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.00 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.90 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.70 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.10 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.90 e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMortality [%]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.00 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.00 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.00 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.00 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.00 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003e*the same letter in the lines indicates no difference between the means at a significance level of \u0026alpha;\u0026thinsp;=\u0026thinsp;0.05\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe use of PGRs or biostimulant can facilitate the condition of \u003cem\u003eEchinacea purpurea\u003c/em\u003e \u0026lsquo;Raspberry Truffle\u0026rsquo;.\u0026nbsp;To study changes in the state and activity of the stomata it is commonly using stomatal conductance measurements considering its precision and non-invasiveness. At the initial stages (0 and 1\u003csup\u003est\u003c/sup\u003e date) was observed the highest values of \u003cem\u003eGs\u003c/em\u003e. Results indicates on reaction of plant to stress by stomata closuring. ABA controls stomatal conductance and hence CO\u003csub\u003e2\u003c/sub\u003e transport into leaf mesophyll (Posp\u0026iacute;\u0026scaron;ilov\u0026aacute; et al. 2009). This phenomenon is likely associated with the extent of stomata. Indeed, several authors have noted abnormalities in these structures under \u003cem\u003ein vitro\u003c/em\u003e conditions, as well as the lack of stomatal function during \u003cem\u003ein vitro\u003c/em\u003e cultivation. Początek formularza(Chaari-Rkhis et al. 2015). The decline of \u003cem\u003eGs\u0026nbsp;\u003c/em\u003eduring acclimation was\u003cem\u003e\u0026nbsp;\u003c/em\u003enoticeable by Fila et al. (2006) on grape. On the other hand, the results of Posp\u0026iacute;\u0026scaron;ilov\u0026aacute; at al. (2009) on tobacco shoved significant drop of Gs in the 1\u003csup\u003est\u003c/sup\u003e date of acclimation to be slightly higher on the 7\u003csup\u003eth\u003c/sup\u003e day. Our results are consistent \u0026nbsp;with Chaari-Rkhis et al. (2015), where a significant increase in the values of \u003cem\u003eGs\u003c/em\u003e just after transfer in micropopagated olive plants to gradual decline in the acclimation period. ABA application after transfer to external environment strongly reduced water loss even when stomata are still non-functional. Foliar application microalgae biostimulant to grape plants increased \u003cem\u003eGs\u003c/em\u003e under drought stress (Mancuso et al. 2006). Application of ABA on tobacco affected on the lower \u003cem\u003eGs\u003c/em\u003e values on the 7\u003csup\u003eth\u003c/sup\u003e day of acclimation in contrast to control (Posp\u0026iacute;\u0026scaron;ilov\u0026aacute; et al. 2009).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is observed upgrade of photosynthetic pigments during acclimation and in the duration of the process, drop od ROS and other stress markers level and launching of antioxidant system. The present study confirms that acclimation period is critical during micropropagation at least on physiological and biochemical levels. Our study revealed that both treatments and term significantly affected photosynthetic pigments. The results indicate an increase in chl and carotenoids content along the acclimation period. It was recorded higher chl content in\u0026nbsp;ABA treatment compared to control.\u0026nbsp;Study of Siddique and Anis (2008) also revealed the increasing chl a, chl b and carotenoids content in acclimatization period of \u003cem\u003eOcimum basilicum.\u0026nbsp;\u003c/em\u003eIn turn, Al-Deeb et al. (2023) though it was not significant, ABA appears to mitigate the negative effect of higher temperature and water stress on the total chl content on tomato seedlings. ABA application increased chl and carotenoids contents in wheat genotypes under moderate water stress (Agarwal et al. 2005). Results of \u0026nbsp; Posp\u0026iacute;\u0026scaron;ilov\u0026aacute; et al. (2009) also proved the beneficial impact of ABA on chl a+b and carotenoids content. BRs affect defense mechanisms in plants by increasing their tolerance to stresses. According to Anwar et al. (2018) the application of BRs increase chl content which improves the photosynthesis rate. Indeed, in this study we recorded both, higher chl and carotenoids content in comparison to control.\u0026nbsp;BRs caused an increase in total chl and carotenoids, with their levels being the highest among all treatments. Similar results were reported by Asha and Lingakumar (2015), who observed an increase in this parameters as well as soluble proteins and proline in \u003cem\u003eVigna\u0026nbsp;\u003c/em\u003eseedlings. Higher, though not significant, chl content results were obtained using Goteo in our experiment. The results of Carillo et al. (2019) results indicates that application of biostimulant in jute strongly affected chl b increase.\u003c/p\u003e\n\u003cp\u003eAs it is known that under critical conditions, including \u003cem\u003eex vitro\u003c/em\u003e acclimation, the level of ROS increases. The most common is H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, the concentration of which is regulated due to the enzymatic activity of CAT, which decay H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into water and oxygen (Sofo et al. 2015).\u0026nbsp;In our study initially, we observed raised content of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (0, 1\u003csup\u003est\u003c/sup\u003e and 2\u003csup\u003end\u003c/sup\u003e terms) and MDA (1\u003csup\u003est\u003c/sup\u003e and 2\u003csup\u003end\u003c/sup\u003e terms). Considering the average H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels, they were not significantly lower in treated plants compared to the control. However, at the first time point, the application of ABA, 24-epiBL, and Goteo significantly reduced these values, which may have contributed to alleviating stress. \u0026nbsp;In contrast Dias et al. (2014) did not observed significant differences in the content of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in control and ABA treated plants in acclimated \u003cem\u003eUlmus minor\u003c/em\u003e.\u0026nbsp;The results corresponds with Liu et al. (2009) where chilling stress in \u003cem\u003eChorispora bungeana\u003c/em\u003e caused increases in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and OH\u003csup\u003e\u0026minus;\u003c/sup\u003e contents, which were inhibited by 24-epiBL treatment.\u0026nbsp;Seyed Hajizadeh et al.\u0026nbsp;(2023) reported the impact of \u003cem\u003eAcsophyllum nodosum\u003c/em\u003e with nano-silicone on drop of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eand MDA content in \u003cem\u003eRosa damascena\u003c/em\u003e under salinity stress.\u0026nbsp;In our study MDA\u0026nbsp;content decreased, suggesting that exogenous treatments can improve the photosynthesis and antioxidant properties of coneflower and thus alleviate the acclimation stress. Our experiment revealed that\u0026nbsp;Goteo affected mostly on drop of MDA in each term.\u0026nbsp;In contrast ABA treatment did not affected on its drop, furthermore we observed more MDA than in control plants. \u0026nbsp;However, drop of MDA\u0026nbsp;by ABA application\u0026nbsp;obtained Li et al. (2014).\u0026nbsp;In study of Kumar et al. (2012) ABA dosing on stressed chickpea also caused decreasing of MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. We did not obtained lower values in BRs treatment compared to control, although although the application of 24-epiBL at 3\u003csup\u003erd\u003c/sup\u003e term of observations resulted in a reduction of the marker which, considering the averages, gives a significant result\u0026nbsp;Results corresponds with rice growing in Cd stress whereas 0.1 mM brassinolide application affected on drop of MDA and CAT activity (Li et al. 2023). To tolerate acclimation stresses, plants have adopted biochemical mechanisms including CAT production. It is beneficial enzyme for the removal and control of high H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003elevels, but they are less suited for a fine tuning of sensitive redox balances with low H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations that may be important for regulatory mechanisms.\u0026nbsp;Our research proved a positive correlation between CAT accumulation and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003edrop in 3\u003csup\u003erd\u003c/sup\u003e term which indicate adaptation to \u003cem\u003eex vitro\u003c/em\u003e conditions\u0026nbsp;(Fig. 2).\u0026nbsp;Several abiotic stresses resulted in the accumulation of CAT which has many functions like maintenance of different metabolites and redox balance. Hameed et al. (2013)\u0026nbsp;revealed that permanent oxidative stress caused by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e grew up the CAT activity.\u0026nbsp;Oxidative stress launched by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e can modulate the antioxidant defense system in wheat leaves.\u0026nbsp;Regarding this, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e has been referred both as a signaling molecule and a regulator of the expression of some cell genes such as encoding antioxidants, cell rescue/defense proteins (Hung et al. 2005, Hameed et al. 2013). In addition, CAT is distinguished from numerous other peroxide-metabolizing enzymes by its high specificity for H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and weak activity against organic peroxides (Mhamdi et al. 2010).\u0026nbsp;A study conducted on maize indicated that CAT exhibited the greatest responsiveness to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, displaying heightened activity within the initial 48 hours. In terms of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e elimination rates, CAT activities were found to be, on average, 21 and 99 times higher compared to GPX and APX activities, respectively (Gondim et al. 2012).\u0026nbsp;ABA in \u003cem\u003eex vitro\u003c/em\u003e acclimatization prevents water loss and improves the activity of antioxidant enzymes needed to alleviation the \u003cem\u003eex vitro\u003c/em\u003e stresses (Aguilar et al. 2000). In conducted experiment we obtained increased CAT activity in all treatments in comparison to untreated plants. Also, stressed sugar cane sprayed with ABA showed higher CAT activity (Li et al. 2014). Exogenous application of 24-epiBL increased the activity of antioxidant enzymes such as CAT to overcome the toxic effects caused by Pb of \u003cem\u003eBrassica juncea\u003c/em\u003e seedlings (Santos Neri Soares et al. 2020). Rice growing in Cd stress whereas application of BR resulted in growing CAT activity (Li et al. 2023). Application of PBs caused a slight improvement in antioxidant activities in maize and soybean plants (De Vasconcelos et al. 2009). \u0026nbsp;As well foliar application of biostimulant based on propolis and maize improved the antioxidant mechanism on by higher activity of SOD, CAT, POX, APX in faba exposed to various stresses (Desoky et al. 2021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe raised content of sugars can act as a signal molecule in plants stress. Our research display the altered sugars content along acclimation. The slight increase of reduced sugars in the 1\u003csup\u003est\u003c/sup\u003e term indicates plant stress. \u0026nbsp;Its content drop on the acclimation period.\u0026nbsp;Sperdouli and Moustakas (2012) announced an accumulation and correlation of increased anthocyanins, soluble sugars and free proline maintaining an antioxidant protection in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e leaves affected drought stress. Our results seem to confirm this studies.\u0026nbsp;On the other hand, increased content of sugars was noticed on by application 24-epiBL by BR biosynthesis inhibitor application. (Bajguz and Asami, 2005). Sorghum seedlings treated with ABA produced higher level of soluble sugars which indicates\u0026nbsp;that under less germination increased levels of sugars along with enzymes might be involved in acclimation (Thakur and Sharma, 2005). ABA foliar application significantly increased glucose and fructose concentrations in tomato fruits (Casey\u0026nbsp;Barickman et al. 2017). Our study shows high reduced sugars content in the 2\u003csup\u003end\u003c/sup\u003e and 3\u003csup\u003erd\u003c/sup\u003e terms than in 1\u003csup\u003est\u003c/sup\u003e term by Goteo spraying.\u0026nbsp;\u0026nbsp;In work of\u0026nbsp;Babazadeh et al. (2023) the algal extract on barley further increased the concentration of soluble carbohydrates in cold-stressed plants, resulting in the highest accumulation of carbohydrates.\u003c/p\u003e\n\u003cp\u003eThe expression of plant response on acclimation was also exemplified in the increasing level of soluble proteins, where it increased substantially along this period. The average level, except of control was not significantly differ in the treatments. Our results points that decreasing of protein content of \u003cem\u003ein vitro,\u0026nbsp;\u003c/em\u003eincreasing thereafter.\u0026nbsp;The data support that initial acclimatization is stressful, and part of the degraded protein is probably rubisco\u0026nbsp;(Carvalho et al. 2005). Brito et al. (2009) also determined soluble proteins content as stress indicator in acclimated plants where they observed higher content in field than \u003cem\u003ein vitro\u003c/em\u003e conditions. Free amino acids are known as essential in forming young tissues as they are responsible for the regeneration of tissues exposed to stresses (Rai, 2002; Vardhini, 2014). According to this, plantlets were under the strongest stress on the first period of acclimation, or also at the same time they formed new tissues as at 0 term. At the remaining dates, the level of free amino acids dramatically dropped. What is similar results were revealed by\u0026nbsp;Pustovoitova et al. (2001) in cucumber. In our study, on average, the lowest level of amino acids was obtained by Goteo treatment. However, interestingly at the 2\u003csup\u003end\u0026nbsp;\u003c/sup\u003etimepoint we found drop of \u0026nbsp;its by BRs spraying in contrast to remaining treatments. Pacholczak et. al (2021) proved application of BRs with the auxin affected in high content of free amino acids.\u003c/p\u003e\n\u003cp\u003eOur research has shown that the application of growth regulators has a direct impact on plant survival, which can influence increased profitability in production. The least acclimatized plants were observed in the control group (up to 60%) which corresponds with Ożarowski et al. (2023) where they obtained 70% plant survival. Goteo results in a higher percentage of acclimatised plants, which is 80%. A positive impact was particularly noted with ABA and BL on the acclimatization of coneflower \u0026lsquo;Rapberry Truffle\u0026rsquo; (average mortality was 4%) and the both brassinosteroids (7% mortality). Application of BRs did not affected significantly on biometric parameters of coneflower. de Souza Arantes et al. (2020) reported the positive impact of BR analogue on larger leaf area, sprout diameter number of leaves. Our study shoved PB Goteo have a positive effect on stem, leaves and root growth, promoting cell proliferation, however the mortality was quite high \u0026nbsp;and was 20%. The study on stressed strawberry also revealed the impact on improving the growth parameters by PBs based on \u003cem\u003eAscophyllum nodosum\u003c/em\u003e (Shakya et al. 2023).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis experiment on the chemistry and slight physiology of acclimation period on \u003cem\u003eEchinacea purpurea\u003c/em\u003e by application of selected PGRs and PB provides a convincing evidence that and we can regulate the critical acclimation period by dozing substances, what probably provides high quality and limit the mortality of acclimatized plants. In the acclimatization period it was observed the increase of chl, carotenoids, CAT, soluble proteins, drop of MDA, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, free amino acids content and altered sugars content. Applied substances affected significantly on levels of biochemical parameters. Moreover, the results concerning BRs provide evidence for the effectiveness of these hormones in the acclimatization of \u003cem\u003eE. purpurea\u003c/em\u003e.\u0026nbsp;The application of BRs or ABA in \u003cem\u003eE. purpurea\u003c/em\u003e \u0026lsquo;Raspberry Truffle\u0026rsquo; has the most beneficial impact reduce mortality of acclimatized plants, which is crucial in plant production.\u0026nbsp;The effectiveness of these rarely used hormones may also be efficient in other species. We believe that our experiment contributes to the advancement of knowledge regarding the acclimatization process of herbaceous plants to \u003cem\u003eex vitro\u003c/em\u003e conditions and serves as a guide for identifying substances that may better alleviate plant stress and enhance their growth during acclimatization.\u003c/p\u003e\n"},{"header":"Abbreviations","content":"\u003cp\u003eABA \u0026ndash; abscisic acid\u003c/p\u003e\n\u003cp\u003eAPX - ascorbate peroxidase\u003c/p\u003e\n\u003cp\u003eBA - 6-Benzylaminopurine\u003c/p\u003e\n\u003cp\u003eBR \u0026ndash; brassinolide\u003c/p\u003e\n\u003cp\u003eCAT \u0026ndash; catalase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003echl - chlorophyll\u003c/p\u003e\n\u003cp\u003eGPX - glutathione peroxidase\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGs\u0026nbsp;\u003c/em\u003e- stomatal conductance\u003c/p\u003e\n\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e\u0026ndash; hydrogen peroxide\u003c/p\u003e\n\u003cp\u003eIBA - indole-3-butyric acid\u003c/p\u003e\n\u003cp\u003eMDA \u0026ndash; malondialdehyde\u003c/p\u003e\n\u003cp\u003ePBs \u0026ndash; plant biostimulants\u003c/p\u003e\n\u003cp\u003ePGRs \u0026ndash; plant growth regulators\u003c/p\u003e\n\u003cp\u003ePOX \u0026ndash; peroxidases\u003c/p\u003e\n\u003cp\u003eROS \u0026ndash; reactive oxygen species\u003c/p\u003e\n\u003cp\u003eSOD - superoxide dismutase\u003c/p\u003e\n\u003cp\u003e24-epiBL - 24-epibrassinolide\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no external funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAutor\u0026rsquo;s contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to this study\u0026rsquo;s design and conception MG collected the data and performed the experiments and laboratory work. AP conducted statistical analyses. AP and KN assisted analysis of stomatal parameters and anatomical structures. MG wrote the manuscript. AP revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAgarwal S, Sairam RK, Srivastava GC, Tyagi A, Meena RC (2005) Role of ABA, salicylic acid, calcium and hydrogen peroxide on antioxidant enzymes induction in wheat seedlings. Plant Science 169(3):559-570. https://doi.org/10.1016/j.plantsci.2005.05.004.\u003c/li\u003e\n\u003cli\u003eAguilar ML, Espadas FL, Coello J, Maust BE, Trejo C, Robert ML, Santamaria JM (2000) The role of abscisic acid in controlling leaf water loss, survival and growth of micropropagated Tagetes erecta plants when transferred directly to the field. 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Journal of Agriculture \u0026amp; Social Sciences 1(2):90-93.\u003c/li\u003e\n\u003cli\u003eValluru R, Van den Ende W (2008) Plant fructans in stress environments: emerging concepts and future prospects.. Journal of Experimental Botany 59(11):2905\u0026ndash;2916. https://doi.org/10.1093/jxb/ern164.\u003c/li\u003e\n\u003cli\u003eVardhini BV (2014) Brassinosteroids\u0026rsquo; role for amino acids, peptides and amines modulation in stressed plants - A review. Plant Adaptation to Environmental Change: Significance of Amino Acids and their Derivatives; CABI: Wallingford, UK, 300\u0026ndash;316.\u003c/li\u003e\n\u003cli\u003eVilela BJ, Carvalho LC, Ferreira J, Am\u0026acirc;cio S (2007) Gain of function of stomatal movements in rooting \u003cem\u003eVitis vinifera\u003c/em\u003e L. plants: regulation by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is independent of ABA before the protruding of roots. Plant Cell Reports 26:2149-2157. https://doi.org/10.1007/s00299-007-0427-3.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"abiotic stress, in vitro, brassinosteroids, acclimatization, plant hormones, biostimulant","lastPublishedDoi":"10.21203/rs.3.rs-4730353/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4730353/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicropropagation is currently one of the primary methods for plant propagation, known for its efficiency in producing disease-free and vigorous plants. However, the final stage of this - acclimatization, is critical due to the transfer from a controlled \u003cem\u003ein vitro\u003c/em\u003e environment to external one. To reduce mortality and alleviate acclimation stress, plant growth regulators (PGRs) or biostimulants can be employed. This study investigated the effects of exogenously sprayed PGRs: 0.001 mg·L\u003csup\u003e-1\u003c/sup\u003e abscisic acid (ABA), 0.001 mL·L\u003csup\u003e-1\u003c/sup\u003e brassinolide (BL), 0.001 mL·L\u003csup\u003e-1\u003c/sup\u003e 24-epibrassinolide (24-epiBL), and 0.3 mL·L\u003csup\u003e-1\u003c/sup\u003e biostimulant Goteo on the physiological and biochemical responses of \u003cem\u003eEchinacea purpurea\u003c/em\u003e 'Raspberry Truffle' plantlets during the acclimation process. The effects of treatments at various acclimation stages on chlorophyll (chl) and carotenoids content, hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), catalase (CAT), \u0026nbsp;malondialdehyde (MDA), free amino acids, soluble proteins, total soluble sugars, and reduced soluble sugars were tested in this research. The results confirmed changes in biochemical parameters, including an increase in chlorophyll and carotenoids in the acclimatization period where the highest level obtained by BL spraying. A decline in stomatal conductance was also observed, where ABA influenced the most on drop. It was also recorded the decrease in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and MDA concentration. CAT activity increased, especially by biostimulant treatment. We recorded an increase in total soluble proteins along acclimatization. Goteo affected the most on morphology parameters, ABA, BL and 24-epiBL increased acclimatization efficiency. Our studies indicate that potentially the most effective substances in the acclimatization of \u003cem\u003eE. purpurea \u003c/em\u003e‘Raspberry Truffle’ are brassinosteroids and ABA.\u003c/p\u003e","manuscriptTitle":"The effect of several growth regulators and biostimulant on biochemical and physiological changes in acclimation of micropropagated Echinacea purpurea Moench. ‘Raspberry Truffle’","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-09 08:55:24","doi":"10.21203/rs.3.rs-4730353/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-07-17T03:43:47+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-17T03:42:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-15T11:56:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2024-07-12T06:28:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0c8c22fe-0bf0-4102-a512-4a1eb1421776","owner":[],"postedDate":"August 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-14T16:06:42+00:00","versionOfRecord":{"articleIdentity":"rs-4730353","link":"https://doi.org/10.1007/s11240-024-02869-4","journal":{"identity":"plant-cell-tissue-and-organ-culture-pctoc","isVorOnly":false,"title":"Plant Cell, Tissue and Organ Culture (PCTOC)"},"publishedOn":"2024-10-10 15:57:08","publishedOnDateReadable":"October 10th, 2024"},"versionCreatedAt":"2024-08-09 08:55:24","video":"","vorDoi":"10.1007/s11240-024-02869-4","vorDoiUrl":"https://doi.org/10.1007/s11240-024-02869-4","workflowStages":[]},"version":"v1","identity":"rs-4730353","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4730353","identity":"rs-4730353","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}