INFLUENCE OF POLYETHYLENE GLYCOL (PEG) CONCENTRATIONS ON WATER STRESS INDUCING

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1 INFLUENCE OF POLYETHYLENE GLYCOL (PEG) CONCENTRATIONS ON WATER STRESS INDUCING ANDREEA NISTOR 1, GH. CÂMPEANU 2, N. ATANASIU 2 NICOLETA CHIRU 1, DIANA KARACSONYI 1 1 INCDCSZ, Bra ov, tican_andreea@yahoo.com 2 USAMV, Bucure ti Keywords: plantlets, concentration, growth, height, leaves ABSTRACT Drought causes changes in growth of plants. Tissue culture is useful in studying the mechanism of tolerance to water stress in vitro conditions. Culture technique in vitro reduced variation due to medium (the nutria medium, controlled conditions). Polyethylene glycol is used in simulating water stress for plants and as such, and the influence of PEG was studied to NIRDPSB Brasov. It had been experienced over the plantlets in vitro, on Romanian cultivars. Influence of modified media supplemented with polyethylene glycol on growth in height of plantlets and number of leaves/plantlets obtained in vitro was different in the two cultivars tested. INTRODUCTION Drought is one of the most common environmental stresses affecting plant growth and productivity (Boyer, 1982). Plant cell and tissue culture has been an useful tool to study stress tolerance mechanisms under in vitro conditions (Bajji et al., 2000). Water deficit is a common stress in potato production, which leads to decrease in tuber quality and yield. Because of potato susceptibility to drought (Hassanpanah et al, 2008), preparing sufficient water is very important for increasing potato quality and quantity. It is very important to study about tolerance of different potato cultivars against water deficit stress. Water deficit decreased number of leaves, plant water potential (Frensch, 1997), leaf area, stem height, ground coverage, tuber number, growth and yield. In this study, our aim is to determine the potential reactions of plants to drought in vitro by inducing water stress using polyethylene glycol (PEG). MATERIAL AND METHOD In Plant Tissue Culture Laboratory of NIRDPSB Brasov, in 2009, was fitted the experience that followed the influence of polyethylene glycol PEG's on the plantlets, experience bifactorial, with 10 options, divided into three repetitions. To achieve the experience, have been studied experimental variants presented in next table: 61

2 Table 1. Experimental variants depending of PEG concentration (NIRDPSB Brasov, 2009) Var. Cultivar (a) Basic medium used PEG concentration Component of layers (b) Number of layers V 1 - MS (b 1) 1 V 2 0,006 M PEG MS M PEG (b 2) 1 V 3 - bottom layer: MS M Christian MS+ sucrose 0,006 M PEG PEG - upper layer: MS (b 3) 2 (a 1) (20g/l)+ Phyto V 4 - Agar (8 g/l) M PEG MS M PEG (b 4) 1 V M PEG - bottom layer: MS M PEG - upper layer: MS (b 5) V 6 - MS (b 1) 1 V 7 V 8 V 9 V 10 Roclas (a 2) MS+ sucrose (20g/l)+ Phyto - Agar (8 g/l) 0,006 M PEG 0,006 M PEG M PEG MS M PEG (b 2) - bottom layer: MS M PEG - upper layer: MS (b 3) MS M PEG (b 4) - bottom layer: MS M PEG - upper layer MS (b 5) Bifactorial experience axb (5x2) 10 variants was performed using the following graduations: - experimental factor a, cultivar, with 2 graduations: a 1 - Christian a 2 - Roclas - experimental factor b, nutrition medium, with 5 graduation: b 1 - M1 medium witness, Murashige Skoog, enriched with vitamins, 20g/l sucrose, 8g/l agar; b 2 - M2 contains a single layer: Murashige - Skoog, enriched vitamins 20 g/l sucrose, 8g / l agar, M PEG b 3 -M3- contains double layer: bottom layer medium was Murashige - Skoog, enriched with 20 g / l sucrose, 8g / l agar, M PEG, and the upper layer was without PEG (which is poured after the first layer was solidifiat) - b 4 -M4- contains a single layer - b5-m5- - contains double layer: bottom layer medium was Murashige - Skoog,

3 enriched with 20 g / l sucrose, 8g / l agar, M PEG, and the upper layer was without PEG. In his experience was working with Romanian varieties: Christian and Roclas. Polyethylene glycol (PEG) with molecular weight of 6000, was used as a simulator of water stress in vitro. Have been used two PEG concentrations (0.006 M, M). The investigations were made both on a single layer of polyethylene glycol, and dual-layer (bottom layer: MS and PEG, and the upper layer: MS). There have been measurements of the height of plantlets, number of leaves, at six to eight weeks from inoculation to determine the response to drought. Fig.1. Plantlets with control medium Fig.2. Plantlets with PEG concentrations, reduced height and reduced number of leaves RESULTS AND DISCUSSION Results on the influence of modified media supplemented with polyethylene glycol on the height of plantlets obtained in vitro, for the varieties tested Analyzing the growth of plantlets (Table 2) for Christian cultivar on the five culture media is observed that the presence of PEG-site to varying degrees of water stress simulated the growth of plantlets was inhibited. These results are shown in fig 3, where V1 is apparent that the control has not hampered the growth of plants, resulting from experiments height is cm. The lowest height of the plantlets is observed for the variant with addition of M PEG which strongly inhibited growth, the resulting plantlets height was 2.27 cm. If we examine the variations in terms of number of layers used in culture media may indicate that variants 2 and 4 containing a single layer of culture medium enriched with M, M PEG of PEG that have directly 63

4 influenced the plant height resulting in the lowest values that differ from the control of 12cm and cm respectively. Table 2. Plantlets height variation in the number of leaves in the varieties tested in vitro experimental variants Var. Cultivar, a Plantlets height (cm) Number of leaves/ plantlets 1 12,53 15,27 2 2,53 3,53 3 Christian 4,43 8,33 4 a1 2,27 2,27 5 3,60 5, ,33 13,40 7 2,27 3,33 8 Roclas 4,27 6,47 9 a2 1,53 2, ,43 4,47 In case of Roclas cultivars, fig.., it also notes that the version control, V1 has obtained the highest plants, with an average height of cm and the smallest heights were obtained in variant 4 with an average height of 1.53 cm. We can see that the presence of PEG-site at a rate of 0,012 m of water stress inhibited growth ensuring. Fig.3. Variation of plantlets height for the cultivars tested(nirdpsb Brasov, 2009) 64

5 If we compare the two varieties Roclas and Christian, the height of plantlets (Fig..) for Christian cultivar can say that has responded better than getting Roclas potato plantlets (plantlets height was bigger in all cases). The statistical interpretation of results of the plantlets height From assessment results, it is noted that the Christian cultivar exceeded the average value of Roclas cultivar, the difference was not statistically, insignificant, negative, -0,661 for Roclas cultivar (Table 3). Table 3. Influence of variety on growth in height of plantlets (NIRDPSB Brasov, 2009) Cultivar Height Dif. Significance (cm) (%) (cm) Christian (a 1 ) (Ct) 5, , Roclas (a 2 ) 4,366 86,85-0,661 Insignificant DL 5% = 2,7663 cm DL 1% = 4,1841 cm DL 0,1% = 6,7216 cm Overview of the plantlets height (Table 4) of the two cultivar have very significant negative results in the same variety, but insignificant between the two varieties, which shows that the culture medium influenced the very significant growth in both cultivars but plantlets results were not significantly influenced by self-chosen variety. Table 4. Combined influence of cultivar and culture medium on the growth of plantlets height (NIRDPSB Brasov, 2009) Cultivar (a) Medium (b) Christian (a 1 ) Roclas (a 2 ) Dif. between cultivars (cm) Dif. (cm) Signif. (cm) Dif. (cm) Signif. a 2 -a 1 (cm) b 1 ( Ct) 12, , ,20 insignif. b 2 2,53-12,00 ooo 2,27-8,06 ooo 0,26 insignif b 3 4,43-8,10 ooo 4,27-6,06 ooo 0,16 insignif b 4 2,27-10,26 ooo 1,53-8,80 ooo 0,74 insignif b 5 3,60-8,93 ooo 3,43-6,90 ooo 0,17 insignif DL 5% = 3,13 cm DL 5% = 4,144 cm DL 1% = 4,24 cm DL 1% = 5,231 cm DL 0,1% = 5,65 cm DL 0,1% =6,012 cm Looking at plant height using regression (Fig. 4) between the concentration of PEG and increasing the height of plantlets can be said that the use of polynomial regression coefficients of two terms, show highly significant correlations of and for the Roclas cultivar and Christian cultivar. 65

6 Fig. 4. Influence of the addition of PEG on the plantlets height Results on the influence of modified media supplemented with polyethylene glycol on the number of leaves of plantlets obtained in vitro, for the varieties tested For Christian cultivar, unmodified culture medium without addition of PEG, determining obtaining an average number of leaves on plantlets of On versions with modified culture media decreased the number of leaves, the lowest number being recorded in for variant 4 with m with an average PEG of Fig.5. Variation of number of leaves on different culture medium to experimental varieties (NIRDPSB Bra ov, 2009) 66

7 For Roclas cultivar, the highest number of leaves on the environment has been medioum control, medium without added PEG, the value being 13.4; the lowest number of leaves on plantlets was obtained variant 4 of medium which had a PEG concentration of 0,012 M (fig. 5). Statistical interpretation of results obtained by the number of leaves on plantlets of varieties tested In the case of the average values obtained from the analysis of cultivar, it is noted that the differences are small by leaves and were statistically significant as distinct, but negative (Table 5). Table 5. Influence of variety on the average number of leaves per plantlets for the two varieties (NIRDPSB Bra ov, 2009) Var. Cultivar Number of leaves/ plantlet Dif. Significance Nr. % A1 (Mt) Christian (a 1 ) 6, , A2 Roclas (a 2 ) 6,028 87,36-0,872 oo The statistical interpretation of the combined influence of these two factors the cultivar and the culture medium (PEG's presence in the substrate) shows that differences between cultivars are statistically distinct and significant for the control variant 3 and are not statistically the other variants (Table 6). Table 6. Combined influence of variety and culture medium over emergence of leaves on plantlets (NIRDPSB Bra ov, 2009) Cultivar, a/ Christian (a 1 ) Roclas (a 2 ) Dif. Medium Medium, b Nr. Dif. Signif. Nr. Dif. Signif. a 2 -a 1 b 1 ( Mt) 15, , ,87 ** b 2 3,53-11,74 ooo 3,33-10,07 ooo +0,20 ns b 3 8,33-6,94 ooo 6,47-6,93 ooo +1,86 ** b 4 2,27-13,00 ooo 2,47-10,93 ooo - 0,20 ns b 5 5,13-10,14 ooo 4,47-8,93 ooo + 0,66 ns DL 5%= 0,3534 (leaves /plantlets) DL 5%= 0,8390 (leaves /plantlets) DL 1% = 0,4867 (leaves /plantlets) DL 1% = 1,6198 (leaves /plantlets) DL 0,1% =0,6701 (leaves /plantlets) DL 0,1% =1,9466 (leaves /plantlets) Regression statistical analysis based on the two cultivars have different coefficients. Thus Christian cultivar is highly significant regression coefficient, statistically, so there is a very significant correlation between the concentration of PEG and the number of leaves produced. In case of Roclas cultivar is only 67

8 distinctly significant regression coefficient, means that the concentration of PEG in the substrate influenced the number of leaves beside the cultivar factor (fig. 6). Fig.6. Influence of PEG addition on leaves numbers per plantlets (NIRDPSB Bra ov, 2009) CONCLUSIONS - control V1 (constitution without the addition of PEG) has not hampered the growth of plants, height resulting from experiments was cm and cm of the Christian and Roclas cultivars; the lowest height of plantlets is observed in variant with the addition of M PEG which strongly inhibited height growth, the resulting plantlets were 2.27 and 1.53 cm for the Christian and Roclas cultivars. - Christian and Roclas cultivars differ in the height of plantlets obtained from the Roclas cultivar, with an average difference of cm from the Christian variety. - results on the number of leaves produced on substrates, indicating that variants 2 and 4 with the substrates to which PEG was added directly, the number of leaves were decreased for both cultivars, with values of 3.53 and 2.27 for the Christian cultivar and 3.33 and 2.47 for the Roclas cultivar. REFERENCES 1. Bajji, M., S. Lutts, J. M. Kinet, Physiological changes after exposure to and recovery from polyethylene glycol-induced water deficit in callus culture issued from durum wheat (Triticum durum) cultivars differing in drought resistance. J. Plant Physiol., 156, Boyer, J.S., Plant productivity and environment. Science 218, Frensch, J., Primary response of root and leaf elongation to water deficits in the atmosphere and soil solution. J.Exp. Bot., 48: Hassanpanah, D, E. Gurbanov, A. Gadimov and R. Shahriari, Determination of yield stability in advanced potato cultivars asa affected by water deficit and potassium hum,tate in ardabial region, Iran, Pak., J. Biol. Sci., 15: