18 Journal homepage: www.fia.usv.ro/fiajournal Journal of Faculty of Food Engineering, Ştefan cel Mare University of Suceava, Romania Volume XVIII, Issue 1 - 2019, pag. 18 - 26 STUDY OF HEAVY METALS EFFECTS ON IN VITRO CULTURES OF SEDUM TELEPHIUM SSP.MAXIMUM L. *Mirela ARDELEAN 1 , Andrei LOBIUC 2,3, Marian BURDUCEA 3 , Ciprian MIHALI 4 , Daniela-Teodora MARȚI 4 1 „Vasile Goldiş” Western University from Arad, Plant Biotechnology, Institute of Life Science, Romania *corresponding author e-mail: mirela.ardelean1@yahoo.com 2 ”Stefan cel Mare” University, Faculty of Food Engineering, Universitatii Str. 13, Suceava, Romania 3 „Alexandru Ioan Cuza” University of Iasi, Romania, Faculty of Biology, Carol I Bd., Iasi, Romania 4 „Vasile Goldiş” Western University from Arad, Romania Abstract: In this study we aim by using plant biotechnology to study the effect of heavy metals Cd and Pb on the regeneration capacity of Sedum telephium ssp. maximum L. plant species that grows spontaneously in our country and can also be cultivated for ornamental purposes. The results obtained at different concentrations of each metal will be compared to establish, from a morphological point of view, the existence of a dose-response relationship. According to the rules of the USA Energy Department, the hyperaccumulator plants have to fulfill the following characteristics: rapid growth and large biomass; pest and disease resistance; inedible for humans and animals; easy to harvest; very branched root system; accumulation of different types of heavy metals. Following preliminary results (in vitro and ex vitro) we considered that this species fulfils the above conditions. Keywords: heavy metals, Sedum, in vitro, phytoremediation. 1. Introduction Plant tissue culture is a convenient laboratory instrument for phytoremediation studies. The most used forms of tissue culture are cell suspensions, calluses and organogenesis [1]. Once established, these in vitro cultures can be propagated indefinitely and are available on request [2]. Whole plants however, are cultivated in soil or grown in hydroponics, the systems have a limited life span and each individual plant has to be replaced and re- established after each experiment. Thus, the time for investigations can be substantially reduced using tissue culture instead of whole plants [3]. Therefore, in this study we aim by using plant biotechnology to study the effect of heavy metals Cd and Pb on the regeneration capacity of Sedum telephium ssp. maximum L. Sedum gender belongs to the Crassulaceae family [4]; [5] and consists of almost 400 species with succulent leaves. Sedum telephium ssp. maximum (L.) Krock is frequently spread in the Romanian flora as a spontaneous species, as well as an ornamentally cultivated species. More than that, the Romanian traditional medicine considers that this plant might have therapeutic (vulnerary, antiseptic, wounds) effects. In the middle of the sixteenth century, Hieronymus Bock reported that extracts of Sedum telephium ssp. maximum were used in the Rhine valley to treat internal injuries like lung ulcers [6]. Now today, medical researchers isolate the active ingredients from those traditional medicine plants and test their efficacy [13]. In the early 1990's, some researchers in Munich have identified two polysaccharides in Sedum telephium ssp. maximum that were anti-inflammatory [7]. A few years later, some italian scientists observed the ways in which the polysaccharides and flavonols operated on http://www.fia.usv.ro/fiajournal Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVIII, Issue 1 – 2019 Mirela ARDELEAN, Andrei LOBIUC, Marian BURDUCEA, Ciprian MIHALI, Daniela-Teodora MARȚI, Study of heavy metals effects on in vitro cultures of Sedum Telephium Ssp.Maximum L, Food and Environment Safety, Volume XVIII, Issue 1 – 2019, pag. 18 – 26 19 cells during wound healing [14]. The vegetable from the in vitro culture is part of modern biotechnology industry that focuses on various areas, including a special interest in plant biotechnology presents, that in vitro cultivation of physiotherapeutic interest [8]. In general, many herbs micro propagated in vitro were used as starting material in the popular culture media that are filled with bioreactors, and the biomass collected from a number of days in vitro culture pass extraction and condition the compounds of pharmaceutical interest [9]. 2. Materials and methods 2.1 Plant Material, composition of the growth medium and the vitro culture The growth substrate used for all vitro culture experiments was made from agarised Murashige - Skoog (1962) (MS) base medium [10] consisting of macroelements, Fe EDTA and microelements, mineral blend according to the original formulation with the addition of 100 mg/l m-inositol, 30 g/l sucrose and 10 g/l agar-agar [11]. There was no addition of growth regulators (cytokinins or auxins) to this base medium. The added heavy metals were CdSO4 and PbCl2. The growth medium variants made during the experiments and presented in the order in which they were performed as well as the concentration of the heavy metals added to the culture medium are shown in table 1. Prior to the sterilisation of the growth medium it’s pH was adjusted to 5.5 with hydrochloric acid or sodium hydroxide depending on the basicity or acidity of the final medium [12]. 15 ml of medium were introduced in culture containers made of colorless and thermoresistant glass, 8 cm high and 4 cm in diameter. For autoclaving the containers used in all experiments, after portioning the growth medium, were coverd in aluminium foil. The sterilisation of the containers with the growth media was performed in an autoclave for 30 minutes at 121°C and 1 atm [15]. The plant material used to initiate the vitro cultures was represented by 2 cm long side shoots with 1-2 nodes plus and apical bud, taken from Sedum telephium ssp. maximum L plantlets regenerated from the zygotic embryos of seeds germinated for 30 days on Murashige - Skoog (1962) growth medium without growth regulators. The seeds from which the explants germinated were sterilised in a 0.1 % sodium hypochlorite solution diluted with sterile water (1:2) with 2 – 3 drops of Tween 20 added to 150 ml disinfectant solution [15]. The containers with the inocula were transfered in the growth chamber, placed on shelves and exposed to a temperature between 230C ± 20C during the light period and 200C ± 20C during dark and a photoperiod of 16 h light/24 h. The light intensity was 1700 lux (Osram white fluorescent tubes; wavelength 590nm; dimensions: Lx590 mm Øx26 mm) [15]. 2.2 Statistical Methods applied in the interpretation of the results The results were expressed in averages ± standard error. To evaluate the statistically significant differences between the treatments, the averages were compared by variance analysis (ANOVA). The data was tested for the normality and homogeneity of variance using the Levene test. When the results were statistically significant, a multiple comparison post-hoc Tukey (p ≤0.05) test was used. The software used for statistical analysis was IBM SPSS v20. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVIII, Issue 1 – 2019 Mirela ARDELEAN, Andrei LOBIUC, Marian BURDUCEA, Ciprian MIHALI, Daniela-Teodora MARȚI, Study of heavy metals effects on in vitro cultures of Sedum Telephium Ssp.Maximum L, Food and Environment Safety, Volume XVIII, Issue 1 – 2019, pag. 18 – 26 20 Table 1. General scheme for the organisation of vitro culture experiments N r. cr t. Experiment type Experimental variants code Compozițion of MURASHIGE – SKOOG (1962) growth media and the type of heavy metal Heavy metal concentration Vitro culture duration 1. Germination of Sedum telephium ssp. maximum L. seeds in aseptic conditions on Murashige - Skoog (1962) culture medium without growth regulators. - Murashige – Skoog (1962) Base medium No growth regulators - 30 days 2. Initiation of Sedum telephium ssp. maximum L. vitro cultures from apical cuttings from regenerated plantlets from zygotic embryos on the 30th day of vitro germination on Murashige - Skoog (1962) culture medium. V0 (control) Murashige – Skoog (1962) Base medium No heavy metals - 30 days V1 Murashige – Skoog (1962) Base medium with CdSO4 50 ppm V2 Murashige – Skoog (1962) Base medium with PbCl2 50 ppm V3 Murashige – Skoog (1962) Base medium with CdSO4 25 ppm V4 Murashige – Skoog (1962) Base medium with PbCl2 25 ppm 3. Results and discussion The cultivated vitro plantlets were monitored every 7 days for 4 weeks. The biometrization of the qualitative and quantitative characters of vitro plantlets from this study consisted in making the following observations: • measurements on the aerial system (vegetative) of plants made with a ruler (in cm) - average stalklet length; - number of branches at the base; - average length of the branches; - number of leaflets; - average length of the leaflets; - average width of the leaflets. • measurements on the root system of plants made with a ruler (in cm): - number of rootlets; - total length of the rootlets. The observations made during the 4 weeks of in vitro cultivation on the Murashige - Skoog (1962) (MS) growth medium allowed us to assert that the development of Sedum plantlets was positively influenced by the presence of the heavy metals Pb (PbCl2) and Cd (CdSO4) in the growth medium. The average length of the stalklet at 28 days was significantly greater compared to the control (V0) especially for the plantlets grown on Murashige - Skoog (1962) (MS) growth medium supplemented with Pb, variant V4 (Fig. 1). Regarding plant growth through formation of lateral branches, the plants grown on V4 – PbCl2 variant, the concentration of 25 ppm has recorded higher values compared to the V3 – CdSO4 variant of the same concentration with both variants having significantly smaller branches compared to the control. However, after 28 days the Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVIII, Issue 1 – 2019 Mirela ARDELEAN, Andrei LOBIUC, Marian BURDUCEA, Ciprian MIHALI, Daniela-Teodora MARȚI, Study of heavy metals effects on in vitro cultures of Sedum Telephium Ssp.Maximum L, Food and Environment Safety, Volume XVIII, Issue 1 – 2019, pag. 18 – 26 21 vitro plantlets grown on Murashige - Skoog (1962) (MS) medium supplemented with 25 ppm CdSO4 (V3) had a greater number of grown leaflets compared to the V4 – PbCl2 variant but significanlty smaller when compared to V0 (Fig. 5). The average length of the leaflets for V4 variant has reached the highest value compared to the rest of the experimental variants followed by V2, being able to notice the fact that the addition of 25 ppm PbCl2 to the growth medium has stimulated the formation and development of the leaves. Thus, it can be observed that the accumulation of metal stimulates the growth in length of the plantlets, the length and width of the leaves and especially the rhisogenesis (Figs. 2, 3, 5 and 8). Fig. 1. Graphic representation of the average values corresponding to the average length of the stalklet of the Sedum telephium ssp. maximum L. vitro-cultures grown for 7 – 28 days on Murashige – Skoog (1962) base medium with no added growth regulators (V0 – control) and of those grown on Murashige – Skoog (1962) base medium supplemented with CdSO4 and PbCl2. Note: the values represent the average ± standard error (n = 10). The different letters represent significant differences (p<0,05). Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVIII, Issue 1 – 2019 Mirela ARDELEAN, Andrei LOBIUC, Marian BURDUCEA, Ciprian MIHALI, Daniela-Teodora MARȚI, Study of heavy metals effects on in vitro cultures of Sedum Telephium Ssp.Maximum L, Food and Environment Safety, Volume XVIII, Issue 1 – 2019, pag. 18 – 26 22 Fig. 2. Morphological aspects after 28 days of in vitro culture of plantlets grown on culture media without heavy metals (V0 – control) and of those grown on media with heavy metals (V1 – V4). Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVIII, Issue 1 – 2019 Mirela ARDELEAN, Andrei LOBIUC, Marian BURDUCEA, Ciprian MIHALI, Daniela-Teodora MARȚI, Study of heavy metals effects on in vitro cultures of Sedum Telephium Ssp.Maximum L, Food and Environment Safety, Volume XVIII, Issue 1 – 2019, pag. 18 – 26 23 Note: the values represent the average ± standard error (n = 10). The different letters represent significant differences (p<0.05). Fig. 3. Graphic representation of the average values corresponding to the number of branches for the Sedum telephium ssp. maximum L. vitro-cultures grown for 7 – 28 days on Murashige – Skoog (1962) base medium without growth regulators (V0 – control) and of those grown on Murashige - Skoog (1962) (MS) base medium supplemented with CdSO4 and PbCl2. Note: the values represent the average ± standard error (n = 10). The different letters represent significant differences (p<0.05). Fig. 4. Graphic representation of the average values corresponding to the average length of branches for the Sedum telephium ssp. maximum L. vitro-cultures grown for 7 – 28 days on Murashige – Skoog (1962) base medium without growth regulators (V0 – control) and of those grown on Murashige - Skoog (1962) (MS) base medium supplemented with CdSO4 and PbCl2. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVIII, Issue 1 – 2019 Mirela ARDELEAN, Andrei LOBIUC, Marian BURDUCEA, Ciprian MIHALI, Daniela-Teodora MARȚI, Study of heavy metals effects on in vitro cultures of Sedum Telephium Ssp.Maximum L, Food and Environment Safety, Volume XVIII, Issue 1 – 2019, pag. 18 – 26 24 Note: the values represent the average ± standard error (n = 10). The different letters represent significant differences (p<0.05). Fig. 5. Graphic representation of the average values corresponding to the newly formed leaflets for the Sedum telephium ssp. maximum L. vitro-cultures grown for 7 – 28 days on Murashige – Skoog (1962) base medium without growth regulators (V0 – control) and of those grown on Murashige - Skoog (1962) (MS) base medium supplemented with CdSO4 and PbCl2. The values represent the average ± standard error (n = 10). The different letters represent significant differences (p<0,05). Fig. 6. Graphic representation of the average values corresponding to the average length of the leaflet for the Sedum telephium ssp. maximum L. vitro-cultures grown for 7 – 28 days on Murashige – Skoog (1962) base medium without growth regulators (V0 – control) and of those grown on Murashige - Skoog (1962) (MS) base medium supplemented with CdSO4 and PbCl2. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVIII, Issue 1 – 2019 Mirela ARDELEAN, Andrei LOBIUC, Marian BURDUCEA, Ciprian MIHALI, Daniela-Teodora MARȚI, Study of heavy metals effects on in vitro cultures of Sedum Telephium Ssp.Maximum L, Food and Environment Safety, Volume XVIII, Issue 1 – 2019, pag. 18 – 26 25 Note: the values represent the average ± standard error (n = 10). The different letters represent significant differences (p<0,05). Fig.7. Graphic representation of the average values corresponding to the average width of the leaflets for the Sedum telephium ssp. maximum L. vitro-cultures grown for 7 – 28 days on Murashige – Skoog (1962) base medium without growth regulators (V0 – control) and of those grown on Murashige - Skoog (1962) (MS) base medium supplemented with CdSO4 and PbCl2. Note: the values represent the average ± standard error (n = 10). The different letters represent significant differences (p<0,05). Fig.8. Graphic representation of the average values corresponding to the number of rootlets for the Sedum telephium ssp. maximum L. vitro-cultures grown for 7 – 28 days on Murashige – Skoog (1962) base medium without growth regulators (V0 – control) and of those grown on Murashige - Skoog (1962) (MS) base medium supplemented with CdSO4 and PbCl2. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVIII, Issue 1 – 2019 Mirela ARDELEAN, Andrei LOBIUC, Marian BURDUCEA, Ciprian MIHALI, Daniela-Teodora MARȚI, Study of heavy metals effects on in vitro cultures of Sedum Telephium Ssp.Maximum L, Food and Environment Safety, Volume XVIII, Issue 1 – 2019, pag. 18 – 26 26 4. Conclusions We consider that our results are in agreement with the data presented in the literature stating that heavy metals can induce the reduction of certain morphological features in cultivated plants such as, in our case, the reduction of the number of leaflets and branches. However, the growth was not slowed down, but on the contrary, the growth medium supplemented with the higher concentration of PbCl2 (V4) has stimulated the plant biomass and the vitro plantlets have not been affected morphologically. This means that the plant has the capacity to tolerate the heavy metal concentrations added to the growth medium and also to accumulate them. 5. Acknowledgements This work was supported by the grant “Elimination of toxic compounds (pesticides, heavy metals, etc.) from soils through phytoremediation” co -financed by the Academy of Romanian Scientists. 6. References [1]. 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