International Journal of Agricultural Science and Research (IJASR) ISSN 2250-0057 Vol.

3, Issue 3, Aug 2013, 211-216 TJPRC Pvt. Ltd.

STUDYING GERMINATION, GROWTH AND TOLERANCE INDEX OF SUNFLOWER PLANTS UNDER HEXAVALENT CHROMIUM STRESS ALONG WITH ROLE OF SOIL NUTRIENTS
MUHAMMAD IMRAN ATTA1, TASVEER ZAHRA BOKHARI2, SAEED AHMAD MALIK3, ABDUL WAHID4 & SADIA SAEED5
1,3,4,5 2

Institute of Pure & Applied Biology, Bahauddin Zakariya University, Multan, Pakistan

Assistant Professor of Botany, Institute of Pure & Applied Biology, Bahauddin Zakariya University, Multan, Pakistan

ABSTRACT
Among heavy metals, chromium is important environmental pollutant. To check out various plant responses towards this pollutant, two commonly grown sunflower varieties (Hysun-33 & SF-5009) were treated with different Cr6+ doses. In Petri plate experiment, percent seed germination remained unpretentious but alteration in germination time was pronounced. Radicle length exhibited negative impact of nutrient free Cr solution. In pot experiment, various plant growth attributes were adversely affected by chromium use. In both types of Cr medium (solution &soil), reduction line was increased along Cr gradient. Chromium behavior at 50 mg/kg of soil was transformed phytoactive due to compensatory effect of soil characteristics as plant growth attributes were improved significantly (p0.05). Present study also predicts embryonic inactivity and abnormal functioning of growth hormones. Plants exposed to Cr metal slack their tolerance expressively. Although chromium stressed both test varieties, yet variety SF-5009 found to be more tolerant than variety Hysun-33 in having better growth pattern.

KEYWORDS: Chromium, Growth, Tolerance, Compensatory Effect, Minerals, Embryonic Inactivity INTRODUCTION
Due to thriving industrialization and urbanization in the world including Pakistan, release of heavy metals through effluents has turned soil and water unfit for crop growth and production (Wahid et al., 2000; Saeed et al., 2003). The heavy metals with density greater than 5g/cm3 are of essential and non-essential types are continuously increasing in soil due to unmanaged municipal wastes, mining, fertilizer and excessive use of pesticides (Alloway, 1995; Pandey & Pandey, 2008). Chromium has density 7.19g/cm3. It has two active states; Cr3+ and Cr6+ that show varying behavior chemically, epidemiologically and toxicologically (Avudainayagam et al., 2003; Kimbrough et al., 1999). Cr6+ is toxic to all living organisms, but Cr3+ is assumed as harmless micronutrient in humans (Shankar et al., 2005; ATSDR, 2000 and Brady et al., 2002). Seed germination aptitude and seedling growth is affected by Cr in melon (Akinci & Akinci 2010). Several other metabolic activities in plants are also interfered by chromium toxicity which causes reduction in root growth, photosynthesis and plant biomass following chlorosis. In plants, Cr induced oxidative stress reduces growth and yield attributes. Stunted plant growth and finally plant death is also instigated by Cr toxicity (Arun et al., 2005; McGrath, 1984; Satyakala and Jamil, 1992; Sharma et al., 1995). Sunflower is grown for its 40% edible oil which is good to heart patients for its high quality, low cholesterol profile and fat soluble vitamins A, B, E and K (Evertt et al., 1987, Goasal et al., 1988). In Pakistan, many researchers

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worked out heavy metal pollution; but in present study we have also explored some transformed effects of Cr heavy metal on various growth attributes in two sunflower varieties i.e. Hysun-33 & SF-5009.

MATERIALS AND METHODS

Petri Plate Experiment An 8-10 days experimental evaluation was steered to study seed germination and radicle length. Selected seeds of test varieties Hysun-33(A) & SF-5009 (B) were arranged on Petri plates evenly and K2Cr2O7 (Merck-Germany) was used as Cr metal source. For moisture absorption, two filter papers were fitted out in each Petri plate. Nutrient free Cr solutions (50, 100, 150, 250, 350, 400, 500 mg/L) were prepared with distilled water succeeding two replicates treatment-1 and seed germination analysis was made following Akinci &Akinci, 2010. For germination time, the day number was counted at which all seeds in a respective treatment were germinated. At day 10, radicle length was measured and data from Cr treatments was compared with control. Pot Experimental Trial Soil Preparation & Analysis Fine canal sand& garden clay soil was sieved and mixed in 3:1 ratio. The soil was humified by 3:1 ratio of soil & humus, filled in empty, prepared earthen pots of 16 inch diameter. Cr doses (50, 100, 150, 250, 350, 400, 500 mg/kg of soil) were carefully weighed and mixed with pot soil. Six uniform sized seeds from each variety-A&B were sown in each respective pot treatment-1. Irrigation started with tap water and 50% thinning was implemented at day 20 when seedlings attained approximate height of six inch. Each treatment followed by seven replicates and a total of 112 pots were arranged in a randomized block design. Analysis of Cr free soil samples was executed for pH 7.21, EC 1.74 ds cm-1, organic matter 7.12%, C 5.85%, N 1.10%, Si (silica sand) 67.55%, Al (clay) 19%, Fe 3.95%, Ca 3.01%, Mg 2.13%, K 2.42%, Na 1.95% and S 0.26% used for plant growth study. Soil organic matter & carbon was projected by loss on ignition method (Goldin, 1987&Konare et al., 2010) using following formula of sample weight difference. O.M & Carbon (%) = Oven dried sample weight - weight after ignition 100 Oven dried sample weight The oxides of soil minerals (Si, Al, Fe, Ca, Mg, K, Na, S) were determined using software based XRF spectrometer (PW-1660, Philips-Holland) following standard operation procedure. Soil textural classes were determined from international soil textural triangle (USDA-USA & UK-ADAS). Biochemical Analysis of Plants For Cr metal uptake (ppm), plant organs were analyzed following combustion and digestion method using PerkinElmer atomic absorption spectrometer (Panichev et al., 2005). Analysis of Plant Growth and Development Different growth parameters i.e. total plant height, root & shoot length, shoot girth, number of leaves plant -1, leaf area (De Swart et al., 2004) and heavy metal tolerance index (Iqbal and Rahmati, 1992.) were measured in selected plants treatment-1.The difference of treatment means was determined by standard deviation, one way ANO VA and DMRT at 0.05% using statistical software suit SPSS-17.0 and MS-Excel-2003 program.

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RESULTS AND DISCUSSIONS

Effect on Seed Germination, Germination Time and Radicle Length In test varieties Hysun-33(A) & SF-5009(B), seed germination remained unpretentious till day 8 along all treatments. However, a significant effect (p0.05) was noticed on mean germination time along increasing Cr 6+ concentration. Germination time for variety-Awas 6.25 days and for variety-Bit was 5.75 days(Table 1).Seed germination is a physiological activity and is switched with water imbibition under enzymatic activity strictly inhibited in plants at 10 mM Cr metal solution(Peralta et al., 2001; Chen et al., 2001). In melon plants, mean seed germination time was adversely affected up to 300mg/L Cr solution (Akinci & Akinci, 2010).In present study, alteration in seed germination not only predicts affected imbibition process, but also indicates embryonic inactivity to germinate under Cr toxicity. Thus sunflower seeds took a long time to germinate as compared to control. Radicle length for germinated seeds treatment-1 was measured and found decreased along Cr treatments. Mean reduction in radicle length from 50-500mg Cr/L was 35-45% (Figure 1). There was a less reduction up to 150mg/L Cr which increased up to 500mg/L Cr solution. Reduced radicle length also predicts embryonic inactivity under Cr toxicity. Effect on Plant Height; Root-Shoot Length& Shoot Girth In both sunflower varieties total plant height, root and shoot lengths were decreased. By comparing with control, there was a gradual decrease in these plant organs from 100-150 mg/kg Cr (0.26-3.2%, 1.5-4.9% and 0.1-2.5%) while plants acquired a rapid decline at 250-500 mg/kg Cr i.e. 4.9-12.7%, 7.9-23.3% and 2.8-10.5%. Shoot girth was declined 2.46-7.60% at 100-150mg/kg and 16.7-29.23% at 250-500mg/kg. Mean reduction values also interpret phytotoxic effect of Cr (Figure 1). Rout (1977) explored contrary effects of Cr both on plant height and shoots length. Shankar et al.,(2005) found that decreased root length in plants was due to Cr heavy metals stress and inhibited root cell division unable roots to absorb water (Bracelo, 1986). Present findings also agreed these documentations following inhibition in plant height comprising root & shoot length as Cr was more in roots (up to 189.6 ppm) than shoot (up to 37.9 ppm) and leaves ( up to 7 ppm). It predicts stressed efficiency of plant growth hormones and abnormal longitudinal cell division due to increased osmotic pressure along Cr accumulation. Similarly at Cr concentration 40-80 ppm, root length and plant growth was affected significantly (Srivastava et al., 2011). In present study, at 50 mg/kg Cr dose plant growth (root & shoot length) improved likewise control revealing Cr to be phytoactive. At the same Cr application (mg/L), radicle length decreased in petri experiment. The difference in growth pattern in two Cr mediums shows compensatory effect of soil pH, organic matter and soil minerals on growth mechanism by transformed Cr behavior. Orhue (2010) reported stem girth reduction due to Cr doses (50, 100, 200mg/kg) but in present study, we observed an increase in stem girth at 50mg/kg Cr dose. It might be due to hormonal controlled active transverse cell divisions. Effect on Leaf Number Plant-1 and Leaf Area Plant-1 Application of Cr (100-500 mg/kg) left negative impacts both on leaf number and area. In Cr treated plants, number of leaves and leaf area decreased1.5-34%&0.5-5.2% as compared to control. Number of leaves plant-1 and leaf area reduced in canola plants was due to inhibitory effect of wastewater having Pb, Cd and Cr (Ahmad et al., 2011).Less leaf number predicts exhibition of growth inhibiting hormones to cause senescence in affected plants while less leaf area allied with abnormal leaf cell division and expansion. Both parameters remained unaffected like control at 50mg Cr/kg predicting this dose valuable for plants.

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Muhammad Imran Atta, Tasveer Zahra Bokhari, Saeed Ahmad Malik, Abdul Wahid & Sadia Saeed

Effect on Heavy Metal Tolerance Index Tolerance index (T.I %)in Cr affected plants was varying for both varieties. The reduction in Cr tolerance at 100500mg/kg was 0.5-12.8%. At 50mg Cr /kg, tolerance index improved significantly (Table 1). Khan et al.,(2003) correlated varying metal tolerance response with physiological variations during growth activities in wheat cultivars. In present study, a significant difference among means for tolerance index has also interpreted genetic inheritability of two cultivars against Cr. The growth responses of both test varieties compared by mean reduction values have also concluded Cr phytotoxicity more for Hysun-33 than SF-5009 (Figure 1).

CONCLUSIONS
Seed germination time, seedling length (root & shoot length), leaves plant-1, leaf area plant-1 found to be good criteria to assess Cr toxicity on growth pattern of sunflower test varieties. Plant tolerance index limitation showed sunflower viability against Cr metal. Growth responses less harmed at lower Cr doses (100-150mg/kg) than at higher doses (250-500mg/kg). Soil minerals contributed positive effect on plant growth and transformed Cr behavior at 50mg/kg. Sunflower variety SF-5009 found to be Cr tolerant more than variety Hysun-33 and could be recommended to grow in Cr affected soils.

REFERENCES
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APPENDICES
50 40
% Reduction in plant growth & tolera nce

45

Hysun-33(A) 35

30 20

17.7 15.4 16.8 16.5 6 5.7 3 2.5 Sh. G Lf. P L.A 6.11 5.8

10 0 Rd. L

TPH

T.I

Figure 1: Comparison of Mean Reduction for Plant Growth & Tolerance against Cr Metal in Sunflower Variety A&B Table 1: Effect of Chromium Metal on Seed Germination, Plant Growth & Tolerance Index in Two Sunflower Varieties
Cr mg/L Var Tg Rd.L(mm) A 3 20.260.7 B 3 20.60.4 50 A 4 19.80.72 B 3 20.00.5 100 A 6 16.50.5 B 5 18.40.53 150 A 7 12.01.48 B 6 16.630.55 250 A 7 10.50.95 B 7 13.230.3 350 A 7 7.030.5 B 7 10.90.17 400 A 8 6.430.51 B 7 8.51.32 500 A 8 5.70.3 B 8 6.10.2 *Tg (A) = 6.25 days, Tg (B) = 5.75 days A 23 23 DF B 23 23 F- valueA 10.2*** 170*** B 12.26*** 248*** F- valueCr6+ 0 RL(cm) + 19.00.82 20.10.29 19.40.48 20.51.13 18.70.65 19.80.84 18.10.55 19.10.71 17.50.57 18.40.59 17.00.38 17.60.48 16.30.58 16.70.47 15.70.64 15.40.61 55 55 34.5*** 50.3** Sh. L(cm) = 129.50.67 129.02.13 130.40.95 128.83.24 129.40.96 126.72 128.61.06 125.22.28 122.51.03 123.21.65 120.10.72 121.91.1 120.00.89 119.80.99 117.90.91 118.60.78 55 55 263.4** 43.4*** TPH 148.51.25 149.03.46 150.10.94 149.31.86 148.11.19 146.41.83 146.51.13 144.33.3 140.00.33 141.63.7 137.10.57 139.33.14 136.30.87 136.52.12 129.60.79 134.15.6 55 55 388*** 27.8** Cr mg/kg of Soil Sh. G(cm) Lf. P 6.50.32 200.58 6.70.65 21.11.34 6.60.39 20.61.1 6.80.38 21.61.13 6.30.38 19.71.1 6.30.45 20.31.4 6.00.17 190.82 6.10.34 19.41.27 5.20.17 16.60.82 5.50.53 18.11.72 5.10.24 161.13 5.20.4 17.41.81 4.80.17 15.50.96 5.30.44 161.82 4.60.34 0 13.30.74 5.10.35 0 14.41.72 55 55 53.85*** 15.0** 55 55 55.5*** 19.0*** L.A(cm2)T.I % 61.60.4 1001.29 61.80.14 1001.6 61.50.35 1011.3 61.90.09 100.22 61.30.5 99.61.3 61.50.38 98.21.0 60.90.86 98.61.7 61.10.18 96.50.8 59.40.77 94.30.7 59.80.18 95.03.7 59.30.57 92.31.9 59.90.17 93.51.7 590.61 91.80.21 59.80.42 91.61.44 58.30.45 87.22.1 59.10.2 90.01.73 55 55 33.5*** 133.1*** 55 55 183.4*** 20.1***

Var = Variety, Rd. L = Radicle Length, Sh. L= Shoot Length, TPH= Total Plant Height, Sh. G= Shoot Girth, Lf. P= No. of Leaves Plant-1, LA = Leaf Area, T.I= Tolerance Index, Standard Deviation, *Tg= Mean Germination Time for Variety A&B, Sig.= ***Highly Significant, **Less Significant, Means Taken by DMRT at -0.05 level