Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
Volume 16
Issue 2
Available Online: http://www.ejpau.media.pl/volume16/issue2/art-05.html


Mi這sz Smolik1, Patrycja Cieluch2, Kinga Mazurkiewicz-Zapa這wicz3
1 Department of Plant Genetics, Breeding and Biotechnology, West Pomeranian University of Technology in Szczecin
2 Division of Hydrobiology, Ichthyology and Biotechnology of Breeding, West Pomeranian University of Technology in Szczecin
3 Division of Hydrobiology, Ichthyology and Biotechnology of Biotechnology of Reproduction,
West Pomeranian University of Technology in Szczecin, Szczecin, Poland



The study presents the possibility of localization of reactive oxygen species (ROS) in cytological preparations of seminal roots and coleoptiles of eleven-day-old seedlings of recombinant inbred lines (RILs) of rye, subjected to the influence of abiotic stress caused by nitrogen and potassium deficiencies in a medium in in vitro cultures of mature embryos. Fifty-one recombinant inbred lines of rye (F9) considered as tolerant and susceptible to stress caused by nitrogen and potassium deficiencies in a medium were selected for the research. The plants were derived from the F2 generation, obtained from a combination of crossings of inbred lines of rye Ot0-6 and Ot1-3. The response of each line to stress was assessed under the conditions of high and decreased level of N and K in a medium. In the medium with high N and K content the seedlings developed on average longer coleoptiles, shorter the longest roots and less numerous roots in comparison with the medium with low content of N and K. Other described lines included lines which developed shorter coleoptiles and significantly shorter the longest roots on the medium with deficient nitrogen and potassium in comparison with the medium with a high content of nitrogen and potassium. Depending on individual morphological responses of the seedlings, the lines were clustered with the use of Ward's agglomerative method with regard to the response of three seedling traits: coleoptile length, the longest root length and root number. RILs with extreme responses to nutrient stress were identified in the outermost groups of Ward's dendrogram. ROS , mainly H2O2, were visualized in the epidermal part of the elongation zone of the seminal roots of seedlings subjected to the influence of nutrient stress. More intense fluorescence of the fluorescence dye (2',7'-dichlorofluorescein) was detected in the seedlings of the lines considered as susceptible to nutrient deficiencies as compared to tolerant lines. The generation of ROS , as important signal molecules provesfunctioning of the molecular mechanisms of sensing and plant cells response to nutrient stress.

Key words: rye, nitrogen-potassium starvation, ROS, dichlorofluorescein, inverted microscopy.


Plant response to different types of abiotic stress factors varies depending on the genus, species and cultivar of a given plant. For the purpose of cultivation of plants with greater tolerance to abiotic stresses, starting materials are searched for, i.e. genotypes tolerant to the stress of drought, salinity or nutrient deficiencies. The selection of genotypes tolerant to abiotic stresses, including nutrient stresses, is not easy (Rzepka-Plevneš et al. 1997; Ashraf and Harris 2005; Manschadi et al., 2008; Kell 2011; Messmer et al., 2011).

There is still a search for research methods which could be used to quickly and relatively easily determine genotype's tolerance to analyzed stress factor. Laboratory methods, such as hydroponics, aeroponics, pots or in vitro cultures, enable relatively cheap and effective assessment of response of a genotype to induced stress. Many researchers have demonstrated that plant response assessed at the seedling stage is correlated with response of a mature plant (Rzepka-Plevneš 1999; Liu et al. 2008; Kell 2011; Messmer et al., 2011). However, some authors have different opinions on the subject (Bolanõs et al., 1993; Bruce et al. 2002).

Rzepka-Plevneš et al., (1997) and Rzepka-Plevneš (1999) described the possibility of using mature embryos cultures in vitro to describe response to nutrient stress of different rye genotypes at the seedling stage, and to select genotypes tolerant and susceptible to stress caused by nitrogen and potassium deficiencies in a medium.

Rzepka-Plevneš and Kulpa (1999) have positively verified the usefulness of the method in a field experiment, thereby indicating the possibility of selection of tolerant genotypes on the basis of morphological traits of the seedlings, including of seminal roots. Nitrogen and potassium deficiencies result in different morphological responses of plants. Roots and root hairs are lengthened, although often also different reactions are observed. The molecular mechanism of plant response to stress caused both by nitrogen and potassium deficiencies may involve the same genes, and reactive oxygen species (ROS) act as signals influencing their expression (Shin and Schachtman 2004).

Detection and identification of ROS is technically difficult since ROS are characterized by high reactivity with other molecules and short life time. ROS level can be analyzed by measurement of by-products, such as H2O2. Methods the most often used for ROS detection include methods employing chemiluminescence (Cash et al. 2007), fluorescence and enzymatic analyses (Gill and Tuteja 2010) as well as electron spin trapping methods (Warwar et al. 2011). It is noted that the methods are not a perfect approach to the needs of research as well as visualization of ROS in single cells. Hence researches on abiotic stress in plants more and more often use confocal laser scanning microscopy (CLSM) (Shin and Schachtman 2004; Sandalio et al. 2008). Owing to the use of appropriate fluorescence dyes, such as 2'7'-dichlorofluorescein diacetate (DCF-DA) – among other things used as an indicator of H2O2 presence, or dihydroethidium (DHE), used as a marker identifying presence of O2, it is possible to identify the topological location of specific fluorescence of the used dyes in cytological preparations, and thereby to assess the level of stress already at the level of single cells depending on used factor (Sandalio et al. 2008).

The aim of the present study was to put the selected recombinant inbred lines of rye into the state of nutrient stress caused by nitrogen and potassium deficiencies, and to describe and verify the morphological response of the seedlings of recombinant inbred lines of rye considered as tolerant and susceptible to nitrogen and potassium deficiencies in a medium, and to make an attempt to visualize and localize reactive oxygen species in cytological preparations of seminal roots and coleoptiles generated as a result of stress caused by the state of nutrient deficiency.


Plant material included fifty-one recombinant inbred lines (RILs) (F9) of rye (Secale cereale L.) considered as tolerant or susceptible to N and K deficiencies in a medium. They were derived from the F2 population, obtained by selfing F1 plants as results of crossing two inbred lines Ot0-6 and Ot1-3 with different response to nutrient deprivation stress assessed at the seedling stage in a mature embryos culture (Rzepka-Plevneš et al., 1997 and Rzepka-Plevneš 1999). The recombinant inbred lines were selected for the research on the basis of previous results of morphological, physiological and biochemical analyses of eleven-day-old seedlings of the population of 191 RILs of rye (F7) (unpublished results).

Plant mature embryos culture conditions was established according to Rzepka-Plevneš et al. (1997); Rzepka-Plevneš and Kulpa (1999) protocols. Rye seeds were surface-sterilized by soaking in 1% and 7% solution of H2SO4 and NaOCl for 15 min each, respectively. Then seeds were washed three times with distilled water for 10 min, and kept in sterile water for 24 h. The mature embryos were prepared with a preparation needle from endosperm and sterilized for 10 min in 10% solution of NaOCl, washed three times in sterile water and transferred into proper medium. Four mature embryos were placed in each glass tube (9 × 3.5 cm) containing 30 ml of the medium. Tubes, closed with aluminium foil and parafilm, were placed for 11 days in a phytotron at 250C under cool white light with 16-hour photoperiod (36 μmol·m-2·s-1) and 75% RH. The treatments were composed of MS mediums (Murashige and Skoog 1962). High-nitrogen-potassium (HNK) medium consisted of 6.003 mM N, 2.005 mM K, whereas low nitrogen-potassium (LNK) medium consisted of 0.334 mM N and 0.332 mM K (Rzepka-Plevneš 1999). The pH of the nutrient mediums was adjusted to 5.7. Each RILs were represented by 60-80 embryos.

Seedling's development observation. The seedlings were harvested after 11 days when they had one (two) visible leaves, and biometric measurements, such as: CL - coleoptyle length (cm), the longest root length - LRL (cm), and root number - RN, were carried out.

Reactive oxygen species (ROS) visualization and localization was carried out according to Shin and Schachtman (2004) protocol. The seedlings grown in the HNK and LNK treatments were taken out of the agar medium, washed in distilled water and transferred to Corning tubes (50 ml), to which the liquid media (HNK and LNK) were added. Corning tubes were transferred back to a phytotron for 30 h. After 30 h the seedlings were taken out, the seminal roots were washed and placed in a set of newly-prepared tubes containing fresh solutions of the HNK and LNK media. From 3 to 4 seedlings among the RILs considered as tolerant and susceptible to nutrient deficiencies in a medium were analysed. Then to the Corning tubes: 30 ml of the HNK medium + 50 μM of CM-H2DCFDA (5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate) solution diluted in dimethyl sulfoxide (DMSO) to the concentration of 0.0025%; 30 ml of the LNK medium + 50 μM of CM-H2DCFDA solution, and as the positive control – the HNK + 50 μM of CM-H2DCFDA solution + 30 μl of H2O2 were added. After half an hour the seedling roots were washed with medium solutions not containing CM-H2DCFDA and cytological observations were made. 2',7'-dichlorofluorescein diacetate (DCFH-DA) undergoes deacetylation by intracellular esterase to polar non-fluorescent compound DCFH (2'7' dichlorofluorescein). Then is fast oxidized into highly fluorescent compound DCF (2'7' dichlorofluorescein) as a result of cell activation. Upon reaction with H2O2 or hydroperoxides, a fluorescent DCF-derived compound is formed that can be detected by monitoring the fluorescence at excitation and emission wavelengths of 480 and 530 nm, respectively. DCF emits a fluorescent signal with green and green-yellow colour. The elongation zone of the seminal roots and part of the coleoptyle of the seedlings growing in the HNK and LNK media were subjected to cytological analysis. The pictures were taken with the use of an inverted microscope – Nikon (ECLIPSE TE 2000-S) in a laboratory of Division of Hydrobiology; Ichthyology and Biotechnology of Breeding - West Pomeranian University of Technology in Szczecin (Poland).

Statistical analysis. Results of biometric measurements were statistically analysed with the use of t-test Student's and two-way analysis of variance. Plant response presented as morphological changes in CL, LRL and RN of the seedlings of each analysed RIL were expressed as the value of difference (d‗) between means of traits assessed under the HNK and LNK treatments. The significance of the obtained differences were analyzed using t-test Student's. This response was also presented as percentage of the HNK, and were used as main variables to the clustering of RILs into groups of lines tolerant and susceptible to nutrient deficiency stress using Ward's agglomerative method. Mean values of the examined traits of RILs were used to calculate simple correlation coefficients. All calculations have been made with the use of Statistica 9 software package (StatSoft Poland).


Considerable morphological variability in terms of response of the analyzed lines to stress caused by nitrogen and potassium deficiencies in a medium was demonstrated (Table 1). In the HNK medium the lines developed coleoptiles of the mean length of 6.46 cm, and of 4.83 cm in the LNK. The analyzed traits were significantly affected by both genotype, medium type and interaction. The examined lines developed on average longer the longest roots in the LNK in comparison with the HNK medium. Under the conditions of decreased N and K level, the mean length of the longest roots was 3.97 cm, and under the conditions of HNK – 3.16 cm (Table 1). The trait was highly significantly affected by genotype and medium type. Also a highly significant interaction was noted (Table 1). A significant influence of medium type as well as highly significant influence of genotype and interaction were found for the trait of RN under the conditions of the experiment. The RILs developed on average 3.88 roots in the LNK medium, while in the HNK – 3.05 (Table 1). The range of the observed variability and the value of standard deviation for the analyzed traits are presented in Table 1.

Table 1. The mean squares of two-way variance analysis for analyzed traits of the RILs seedlings grown at HNK and LNK treatments and mean values and range variability
N-K level
Analysis of variance
(NK × RIL)
CL (cm)


















LRL (cm)




































*P < 0.05. **P < 0.01 – significant differences between RILs; SD – standard deviation; CV – coefficient of variability

Responses of the analyzed lines to stress caused by nitrogen and potassium deficiencies in a medium were varied (Table 1). Generally in the HNK the lines developed longer coleoptiles when compared to the LNK. Also lines that developed significantly longer the longest roots in the HNK in comparison with the LNK were described, or the other way round – shorter and less numerous roots in the HNK when compared to the LNK medium. The responses are presented as differences between the means for the seedlings grown in the HNK medium in comparison to those from the LNK, while the effect of clustering of the analyzed RILs has been showed in a dendrogram (Table 1, Fig. 1).

Fig. 1. Clustering of the selected RILs (F9) of rye. The cuts-off used to form groups ('a'-'f') according to the qi value was depicted as vertical lines.

The analysis of topology of the Ward's dendrogram distinguished six separate groups on it ('a'-'f') (Fig. 1). Seven lines were described in group 'a', in 'b' – 16, 'c' – 4, 'd' – 17, 'e' – 2, and in 'f' – 9. The lines differed in the mean coleoptile length, longest root length and root number (Fig. 2). These lines developed on average shorter coleoptiles in the LNK medium in comparison with the HNK medium (from 54 to 98.95% of the HNK) (Fig. 2). The lines which developed on average the shortest LRL (60.44% of the HNK) in the LNK medium when compared to the HNK were identified in group 'a', and in group 'b' – 75.1%, in groups 'c' and 'd' – respectively 64.9 and 157.5%, while in groups 'e' and 'f' – respectively 282.28 and 166.54% of the HNK (Fig. 2). Similar responses were described for root number (Fig. 2). In group 'a' the lines developed on average 85.3% of the HNK root number, and in group 'b' – 79.1%, 'c' – 70%, in groups 'd'-'f' – respectively from 122.4 to 109% (Fig. 2). Hence it was possible to divide the analyzed lines into lines developing longer the longest roots and more numerous roots in comparison with the HNK and this lines were described as tolerant, whereas lines that developed shorter and less numerous roots when compared to the HNK, as susceptible (Fig. 1).

Fig. 2. Coleoptile length (CL – A), the longest root length (LRL – B), root number (RN – C) of rye seedlings grown in the HNK treatment when compared to the LNK treatment. The response to induced stress was presented as percentage of the control (HNK) and as values of differences (d‗) between the means for each trait and for each group ('a'-'f') as presented in figure 1 (D – F); SD - standard deviation.

Highly significant positive correlations were found for the morphological traits of the analyzed RILs between CL and LRL (r = 0.45, r = 0.58; p < 0.01), CL and RN (r = 0.46, r = 0.66; p < 0.01) and between LRL and RN (r = 0.52, r = 0.75; p < 0.01), respectively in the HNK and LNK treatments.

ROS visualization. Localization of reactive oxygen species was carried out in the selected cytological preparations of rye seedlings. The analysis included fragments of the elongation zone of seminal roots (Fig. 3A-F) and fragments of coleoptiles (Fig. 3G, H). More intense fluorescence of dichlorofluorescein (DCF) in the preparations of seminal roots was observed in the epidermal zone of seminal roots than in the cortex-endodermis zone (Fig. 3A). After adding 30 μl of H2O2 to a medium, control overfluorescence of dichlorofluorescein (DCF) was provoked to check for the possibility of causing fluorescence, and the effect is presented in figure 3B. Lower fluorescence of dichlorofluorescein (DCF) was observed in the lines considered as tolerant to N and K deficiencies in the elongation zone of seminal roots in the LNK treatment when compared to the HNK treatment (Fig. 3C), while the fluorescence was higher in the lines considered as susceptible in the LNK treatment when compared to the HNK treatment (Fig. 3D). Microscopic preparations of analyzed roots of rye seedlings informatively show root cells during intensive elongation (Fig. 3E, F) as well as stomata lines in a coleoptile fragment (Fig. 3G). Also distinct fluorescence of DCF was found in a cytological preparation in the place of coleoptile partly tearing off (Fig. H). Also distinct fluorescence of DCF was found in a cytological preparation in the place of coleoptile partly tearing off (Fig. H). Intracellular ROS exhibit high reactivity and short life cycle so their measurement is extremely difficult. DCF assay was used as a qualitative marker of oxidative stress, not as a precise indicator of the rate of H2O2 generation in biological systems.

Fig. 3. Images of the selected fragments of seminal roots (A, B, C, D, E, F) and coleoptiles (G, H) of the chosen RIL's seedlings differing in response to stress caused by nutrient deprivation in the test under in vitro conditions made by a inverted microscope. Scale bars = 8 – 80 μm.


Breeding for crop genotypes with enhanced soil resource acquisition will be an important strategy for reducing environmental pollution and decreasing agricultural reliance on fertilizer inputs (Cakmak et al. 1997; Rzepka-Plevenš et al., 1997; Kell 2011; Messmer et al., 2011). Genotypic specificity (plasticity) in root response, as revealed in some oat and barley cultivars with deeper penetration and more vigorous root growth under low N-supply, has been recognized to be a valuable adaptive characteristic for breeding plants on light-textured soils (G鏎ny 1993).

The present study shows the possibility of application of a modified laboratory test developed by Rzepka-Plevneš et al., (1997), Rzepka-Plevneš (1999) and used for the assessment of response of different genotypes of rye and triticale to nutrient stress caused by nitrogen and potassium deficiencies in mature embryoscultures in vitro .

The results of the research on coleoptile length, length and number of seminal roots, regenerated from mature embryos, are in accordance with the results presented by Rzepka-Plevneš and Kulpa (1999), Rzepka-Plevneš (1999) for open-pollinated cultivars, inbred lines, breeding strains or selected populations (S1-S3) analyzed in media with high and low content of nitrogen and potassium.

Bürün and Poyrazoğlu (2002) for seedlings regenerated in in vitro cultures of mature barley embryos for plant length, root number or root length, did not find any differences in morphological response of seedlings regenerated in MS, 1MS, RC or B5 media in terms of root and plant length. The regenerated barley seedlings developed on average 4.3 roots in MS and 1MS media, while in RC and B5 mediums they developed 3.4 roots. The difference was statistically significant. Bürün and Poyrazoğlu (2002) emphasize the influence of selection of effective methods of disinfection of barley embryos on the efficiency of seedling regeneration, which ranged from 91.3 (MS medium) to 85.7% (B5 medium). Similar observations were presented in studies by Rakoczy-Trojanowska and Malepszy (1995); Rzepka-Plevneš et al. (1997) who took into account also the cleanliness (lack of symptoms of fungal diseases) and equalization of embryos collected for the research.

The research revealed that under the conditions of nutrient stress the analyzed lines generally developed longer and more numerous roots in comparison with the control, and these differences, similarly as in the case of coleoptile length, depended on RILs' genotype. Such a type of response to nutrient stress caused by nitrogen and potassium deficiencies is typical (Høgh-Jensen and Pedersen 2003) and has been described in many different experiments for oat (G鏎ny and Szo趾owska 1996), rye (Paponow et al., 1999) or for maize (Mi et al., 2007).

Lambers and Poorter (1992), Reynolds and Antonio (1996), Anandacoomaraswamy et al. (2002) and Poorter et al. (2011) state that in the case of nitrogen deficit, this type of response may be explained by increasing dry matter allocation to the roots. Plants allocated more biomass to roots at low nitrogen, whereas more biomass was allocated to the shoot at high nitrogen. This general pattern of plant response is called, according to Brouwer (1963), as the concept of a 'functional equilibrium'. The molecular mechanism of biomass allocation is intricate and depends on many factors, including analyzed genotype (Poorter et al. 2011). Paponov et al., (1999) reckon that dry matter allocation cannot explain interspecific differences in adaptation to low N levels. The authors attribute the significant role in plant adaptation to the growth under the conditions of low level of nutrients to the trait of root length. They indicate then that increasing root length may be important for nutrient acquisition. Other authors have similar opinions (G鏎ny 1993; G鏎ny and Szo趾owska 1996; Tuberosa and Salvi 2007; Liu et al., 2008).

Apart from the above-mentioned response of the analyzed rye lines to stress, also lines which had shorter and less numerous roots in the LNK medium in comparison with the HNK were described. According to Chun et al. (2005) and Guo et al. (2005), these responses may result from perception of available nitrogen level as low by plant sensing mechanisms.

In the research on species as well as cultivars of cereals, it is possible to describe the 'potential' of individual genotypes to grow under the conditions of nutrient deficiencies, salinity or drought under laboratory conditions, and the response is reflected in field conditions at the mature plant stage. It is an extremely vital statement, supported by numerous research results, which confirms the legitimacy of laboratory analyses of plant response to factors inducing e.g. abiotic stress (Guo et al., 2005; Tuberosa and Salvi 2007; Kell 2011; Messmer et al., 2011).

Fluorescence of DCF registered for selected RILs of rye with an inverted microscope was localized as described by Shin et al. (2005) in the epidermis zone of 11-day-old rye seminal roots. ROS play a role wall loosening (Liszkay et al. 2004 – Fig. 3E, F), such as in the regulation of gene expression in response to the many stressful factors (Kreslavski et al., 2012), for instance to the deficiency of several macronutrients, including potassium, nitrogen and phosphorus, whereas changes in ROS concentrations may suggest that root hair cells are important for response to nitrogen and potassium deprivation (Shin and Schachtman 2004; Shin et al., 2005, Kreslavski et al., 2012).

In the experiment ROS accumulation in potassium- and nitrogen-deprived Arabidopsis roots was localized more in the epidermis than in the cortex, whereas when the roots were deprived of phosphorus – more in the cortical tissue (Shin and Schachtman 2004; Shin et al., 2005). This fact may signify that the test used for the evaluation of the response of rye RILs to nutrient stress was able to express the differences in this response in an appropriate manner.

Similar results of research were presented by Hernandez et al. (2012). Under the conditions of potassium deficiency in the root elongation zone of Solanum lycopersicum L., with the use of laser confocal microscopy, H2O2 accumulation was observed mainly in the epidermal cells in the elongation zone and meristematic cells of the root tip and the epidermal cells of the mature zones of potassium starved roots (Hernandez et al. 2012).

Tyburski et al. (2009) showed ROS accumulation in the roots of A. thaliana in case of stress caused by phosphorus deficiency. The authors observed root cytological preparations with a confocal microscope and DCFH, and demonstrated that while ROS were identified in the root elongation zone and quiescent center at high P, they were not found at low P. Tyburski et al. (2009) suggest that P deficiency affects location (place of synthesis) of ROS in the distal parts of Arabidopsis roots. Under the conditions of deficiency, greater ROS synthesis was noted in the cortical tissue than in the epidermis. Similar observations were previously made by Shin et al. (2005) for P deprived roots of Arabidopsis.

The possibilities of three dimensional imagining of ROS synthesis in plant cells with an inverted microscope under the conditions of stress caused by toxicity of Cd or Al were presented by Rodr璲uez-Serrano et al. (2009) for pea plants; and by Silva et al. (2000) for soybean genotypes.

In the present research more intense fluorescence of DCF was observed in the lines considered as susceptible to nutrient deficiencies (Fig. 3C, D), which may be explained by less effective mechanism of neutralization of reactive oxygen species when compared to the tolerant line. Shin et al. (2005) demonstrated that ROS generated in Arabidopsis in the same epidermal part of roots deficient in both N and K, may prove the presence and functioning of similar mechanisms recognizing the state of both N and K deficiency in rye. .


The laboratory test used in the research enabled to reveal individual responses of RILs to stress caused by nitrogen and potassium deficiencies. The lines responding to stress in various ways when compared to the control were clustered using Ward's agglomerative method. Cytological images of seminal roots registered with an inverted microscope showed less intense fluorescence of 2'7' dichlofluorescein (DCF) in these roots as opposed to the lines considered as susceptible. The status confirms existence of definite selection pressure exerted on the examined RILs, which enabled describing different responses of the examined genotypes to N and K deficiencies. They may be used for further research as initial material or in researches on mechanisms determining molecular response of rye to nutrient stress.


This work was supported by the Polish Ministry of Science and Higher Education under grant No. N N302 281936.


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Accepted for print: 19.06.2013

Mi這sz Smolik
Department of Plant Genetics, Breeding and Biotechnology, West Pomeranian University of Technology in Szczecin
S這wackiego St. 17,
71-434 Szczecin, Poland
Phone: +48 91 449 61 95
email: msmolik@zut.edu.pl

Patrycja Cieluch
Division of Hydrobiology, Ichthyology and Biotechnology of Breeding, West Pomeranian University of Technology in Szczecin
S這wackiego St. 17,
71-434 Szczecin, Poland

Kinga Mazurkiewicz-Zapa這wicz
Division of Hydrobiology, Ichthyology and Biotechnology of Biotechnology of Reproduction,

West Pomeranian University of Technology in Szczecin, Szczecin, Poland
K. Kr鏊ewicza 4
71-550 Szczecin
email: Kinga.Mazurkiewicz-zapalowicz@zut.edu.pl

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