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The results of the undertaken study indicate that herbicides depending on the chemical composition of the active substance, the application rate and their combination in mixtures with biologically active substances are able to influence significantly the formation of anatomical structure of leaf apparatus, in particular epidermis. Thus when applying higher rates of herbicides Granstar 75 (25g/ha), 2,4 DA – 500 (1,0 l/ha), Dicopur F 600 (1,5 l/ha), Lintur (140g/ha), Harmony (20g/ha) and Caliber (70/g/ha) to spring barley plantings the number of epidermic cells per 1mm2 of leaf increases. The increase in a number of epidermic cells is related to the formation of xeromorphic features which characterise the plants that grow and develop under unfavourable conditions. This is proved by the calculated coefficient of morphostructure of epidermis (the ratio of the number of epidermic cells per unit leaf area in the variant when preparation is used to the number of epidermic cells in the variant without preparation application).

Our calculations illustrate the following: the lower the value of the coefficient (lower than 1), the less is the number of cells formed per unit leaf area, though they have larger size and surface. This anatomical structure corresponds to mesomorphic type of leaf surface and characterises mesophytic plants that have high productivity.

At the same time the increase of the coefficient (up to 1 and more) indicates the increase of the number of epidermic cells per unit leaf area, though surface of cells in this case decreases and this corresponds to xeromorphic type of leaf apparatus which characterises xeromorphic plants. Thus in case of application of optimal rates of Каразинские естественнонаучные студии Каразінські природознавчі студії Karazin natural science studios herbicides in the mixture with biological preparations, the coefficient of morphostructure of epidermis was 0,– 0,90 and this corresponds to mesomorphic type of leaf area and conforms with the highest indices of plant productivity.

Generelized data of the research into morphostructure of leaf epidermis when applying herbicides of different chemical classes and their combinations with biologically active substances show that the leaf apparatus of spring barley with optimal structure is formed when the index of morphostructure is 0,7-0,9, its value is 0, -1,0 and higher. In this case leaf apparatus with xerophytic properties is formed which leads to the decrease of leaf area and its productivity.

Thus anatomical changes in the epidermis structure of the leaf apparatus under the application of phisiologically active substances is a direct reflection of the level of the preparations influence on metabolic processes in plants and may serve for disclosing of mode of action of investigated preparation on the plant body at different stages of its growth.

EFFECt SIZE oF SoME FaCtorS InFLUEnCInG proDUCtIVIty InDExES In DUNALIELLA SALINA tEoD. CULtUrE Mil’ko S.I. 1,komaristaya V.p. 1, rudas a.n. V.N. Karazin Kharkov national university, Kharkov ”Betacar-X” LLC, Kharkov The number of environmental factors induces b-carotene accumulation in the cells of microalga Dunaliella salina, which serves as a source of its industrial manufacturing. To so called carotenogenesis factors there are attributed elevated medium concentrations of osmotically active salts (medium density, salinity), elevated illuminance, elevated or reduced temperature, addition of carbon sources, deprivation of nutrients (nitrogen and phosphorus). b-carotene accumulation is thought to occur in the cu ltures, the growth in which is inhibited by unfavorable values of these factors. Therefore, culturing most often is performed in two stages: at the first stage cell concentration is grown, then the culture is exposed to carotenogenesis factors. Maximum intensity of carotenogenesis is thought to be achieved at simultaneous effect of all the factors [1]. Nevetherless, most researchers use in their work only some factors and their combinations, e.g. elevated salinity and illuminance, sometimes in combination with nitrogen deprivation. However, the input of each certain factor as well as their interactions into culture growth inhibition and carotenogenesis remains not investigated. And the two stage culturing scheme of D.

salina is rather time and labor consuming.

The goal of present research is to elucidate factors having the maximum effect on b-carotene accumulation in D. salina cells at the minimal culture growth inhibition for possible use in single stage culturing of D. salina.

For that, in multiple-factor experiment design the effects of medium density, illuminance, carbon, nitrogen and phosphorus sources availability on the yield of cells and b-carotene content in them were determined and the effect sizes of each factor on culture growth and carotenogenesis were calculated [2].

There were investigated 5 factors taken in 2 gradations selected on the basis of reference data and preliminary field and laboratory research, so that the culture preserved the ability to grow: medium density 1,10 and 1,15 g/cm3, illuminance 2 and 5 klux, carbon source being dissolved atmospheric CO2 only and with the addition of 100 mg/L NaHCO3, nitrogen and phosphorus deprived and minimal concentrations for growth support added.

The peculiarity of this experimental design was that nitrogen and phosphorus concentrations in the medium were kept at relatively constant level by the periodical additions of suitable quantities of nutrient salts [3]. Full experimental design consisted of 25=32 variants. For statistical processing of the results nonparametric tests were used, as preliminary research had shown that cell concentrations and b-carotene content in D. salina did not fit normal distribution [3].

Каразинские естественнонаучные студии Каразінські природознавчі студії Karazin natural science studios Fig. 1 and 2 represent respectively the values of b-carotene content in the algal cells and cell concentrations on 28th day of growth (stationary phase) in all 32 variants of the experiment. The maximum b-carotene accumulation was observed not at simultaneous effect of all the factor values, which were anticipated to promote carotenogenesis (medium density 1,15 g/cm3, illuminance 5 klux, 100 mg/L NaHCO3, nitrogen and phosphorus deprivation), but at lower medium density (1,10 g/cm3) (fig. 1). Stimulating effect of nutrient deprivation on carotenogenesis not always occurred at NaHCO3 additions (fig. 1), though concentrated stock solution of NaHCO3 was shown not to contain nitrogen or phosphorus traces.

Nitrogen deprivation substantially inhibited culture growth (fig. 2). Growth inhibition by deprivation of phosphorus manifested to greater extent at the higher medium density (fig. 2). It is likely that high salinity of medium solution decreased solubility and availability of phosphate traces to the cells. The higher illuminance promoted both more intensive culture growth and b-carotene accumulation in the cells (fig. 1 and 2).

Kruskal-Wallis test showed that only illuminance, nitrogen and phosphorus availability influenced statistically significantly both cell concentration in stationary phase and cell b-carotene content (table 1). The effect of culture medium density and carbon source in the range of the factors values studied appeared insignificant.

Table 1. Values of Kruskal-Wallis test H for investigated factors and variables (df=1; p0,05; 2 =3,84) tabl.

Variable Cell concentration Cell b-carotene content Factor Каразинские естественнонаучные студии Каразінські природознавчі студії Karazin natural science studios Medium density 0,51 0,Illuminance 4,07* 4,14* NaHCO3 addition 0,26 0,Nitrogen availability 13,09* 9,32* Phosphorus availability 4,30* 7,36* Note: * - H tabl.

For studied factor value gradations the maximum effect size on culture growth and carotenogenesis was caused by nitrogen availability, and the minimum – by illuminance (table 2). Phosphorus deprivation caused almost the same little effect on culture growth as lowered illuminance and practically the same effect as nitrogen deprivation on carotenogenesis (table 2).

Table 2. Influence power 2 of carotenogenesis factors on cell concentration and b-carotene content in D.

salina Teod. culture (df=1; p0,05; 2 =3,84) tabl.

Variable Cell b-carotene Cell concentration Factor content 2±s 0,02±0,03 0,01±0,Medium density 2 0,62 0,2±s 0,13±0,03 0,13±0,Illuminance 2 4,03* 4,03* 2±s 0,01±0,03 0,02±0,NaHCO3 addition 2 0,31 0,2±s 0,42±0,03 0,30±0,Nitrogen availability 2 13,02* 9,3* 2±s 0,14±0,03 0,24±0,Phosphorus availability 2 4,34* 7,44* Note: * - 2 tabl.

That means, that phosphorus deprivation to lesser extent than nitrogen deprivation inhibited culture growth, but to greater extent stimulated carotenogenesis, and therefore phosphorus deprivation can be considered to be promising method for carotenogenesis induction in single-stage D. salina culture.

1. Масюк Н.П. Морфология, систематика, экология, географическое распространение рода Dunaliella Teod. и перспективы его практического использования. – Киев: Наук. думка, 1973. – 244 с.

2. Атраментова Л.О., Утєвська О.М. Статистичні методи в біології: підручник для вузів.- Х.: Вид-во ХНУ, 2007. - 286 с.

3. Комаристая В.П., Антоненко С.П., Рудась А.Н. Культивирование Dunaliella salina Teod. при субоптимальных концентрациях азота и фосфора и исключении их из среды // Альгология. – 2010. – Т. 20, № 1. – С. 42-55.

thE IntEraCtIonS oF LEaD anD SaLICyLIC aCID on GroWth anD aMoUntS oF ChLorophyLL In root anD Shoot oF tWo CULtIVarS oF BRASSICA NAPUS L. UnDEr hyDroponIC CULtUrE ranjbar M.1, Boroumand-Jazi Sh.1, Department of Biology, Islamic Azad University, Falavarjan Branch, Esfahan, Iran.

*Corresponding Author: ranjbar@iaufala.ac.ir Lead is a danger heavy metal, which plotted the environment. Toxicity of lead on plant caused inhibition of root growth and the amount of chlorophyll (Mathe-Gaspar, 2002). It has been proposed that salicylic acid acts as an endogenous signal molecule responsible for inducing abiotic stress tolerance in plants (Gunes, 2007). In this research the effect of lead poison on some parameters of growth and amount of chlorophyll on 20 day old seedlings Каразинские естественнонаучные студии Каразінські природознавчі студії Karazin natural science studios of two cultivars of Brassica napus L. (Okapi & Opera) were investigated.

The seeds after sterilization and 7 days grow move to plastic container with 450 ml of hogland’s nutrient solution and treated with different concentration of lead (Pb (No3)2) (0.25, 0.5, 0.75, 1, 1.5 & 2 mol) and salicylic acid 5 &10 µM with three repeations. The growing period was 20 days and after it root and shoot length (Cm), leaf area (Cm2), fresh and dry weight (gr) of root and shoot of both cultivars were measured and amount of photosynthesis pigments including chlorophyll a, b & a+b were measured by Arnon’ method (Arnon, 1957).

Statistical analyses with SPSS statistical soft ware and test of Duncan performed and all graphs were drawn by SPSS saft ware too.

Lead caused a decrease of root and shoot length, leaf area, fresh and dry weight of root and shoot in two cultivars (P<0.01).The result was shown that 20 day old seedlings of Okapi cannot tolerance versa high concentration of lead (1.5 & 2 mol) and dide. Percentage of reduction root and shoot length, leaf area, dry and fresh weight of root and shoot on Okapi was higher than the Opera, but percentage reduction shoot length on Opera was higher than the Okapi. The result was shown with increasing the concentration of Pb (No3)2, the pigments of photosynthesis including chlorophyll a, b and a+b in leaves of both cultivars were significantly decreased (P<0.01).Decreasing amount of chlorophyll a in all treatment higher than chlorophyll b. Percentage of reduction of amount of chlorophyll a, b and a+b in Okapi were higher than the Opera.

Under Lead and salicylic acid amount of chlorophyll a in Okapi and chlorophyll b in Opera were significantly increased but no effect on amount of chlorophyll a in Opera and b in Okapi and a+b in both cultivars were not seen (P<0.05).Also salicylic acid has no effect on shoot length and fresh weight of shoot in okapi and dry weight of shoot in Opera but in other parameters cause to increased significantly (P<0.05) (P<0.01).

In presence of lead (10-3 M concentration) the reduction of root and shoot growth in Brassica Juncea has been suggested by Liu et al (2000). Mathe-Gaspar and Anton (2002) have been reported the effect of lead on two cultivars of radish shown reduction percentage in dry weight of shoot and root. Salicylic acid increased root and shoot length and fresh and dry weight in the presence of cadmium (Metwally, 2003).

Zawoznik reported that salicylic acid under Cadmium stress case to increased amount of chlorophyll and biosynthesis in Arabidopsis thaliana (Zawoznik, 2007).

Reference 1. Arnon D. I. Copper enzymes in isolated chloroplasts, polyphenol oxidaes in Beta vulgaris // Plant Physiology. – 1957. – 24. – P.115.

2. Gunes A., Inal A., Alpaslan M., Eraslan F., Guneri Bagci, E., Cicek, N, Salicylic acid induced changes on some physiological parameters symptomatic for oxidative stress and mineral nutrition in maize (Zea mays L.) grown under salinity // Journal of Plant Physiology. – 2007. – 164. – P.728-736.

3. Liu, D., Jiang, C., Xin, C, Uptake and accumulation of lead by roots, hypocotyls and shoots of indian mustared Brassica Guncea L // Bioresour, Technol. – 2000. – 71. – P.273-277.

4. Mathe-Gaspar G., Anton A, Heavy metal uptake by two radish varieties // Acta Biology, Szegediensis. – 2002. – 46. – P. 113-114.

5. Metwally A., Finkemeier I., Georgi M., Dietz K. J, Salicylic acid alleviates the cadmium toxicity in Barly seedlings // Plant Physiology. – 2003. – Vol 132. – P. 272-281.

6. Zawoznik, M. S. M., Gropp, M. D., Tomaro, M. L., Benavides, M. P, Endogenous salicylic acid potentates cadmium induced oxidative stress in Arabidopsis thaliana // Plant Science. – 2007. – 173. – P.190-197.


rastegari a. a.

Department of Molecular and Cell Biochemistry, Islamic Azad University, Falavarjan Branch, Esfahan, Iran Email: rastegari@iaufala.ac.ir Dunaliella is an unicellular halotolerant microalga with a great potential as transgenic bioreactor, and more significantly, food source and pharmaceuticals due to its ability to accumulate large amounts of carotenoids.

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