International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 2(9), 5-10, September (2013) Int. Res. J. Biological Sci. International Science Congress Association 5 Physiological behaviour of few Cultivars of Paddy (Oryza sativa L.) during Seed Germination and early Growth, subjecting to distillery Effluent StressMahesh K.S. 1*, Chandrashekara K.T.2,3, Rajashekar N. and Shobha Jagannath1 Department of Studies in botany, University of Mysore, Manasagangotri, Mysore – 570006, INDIA Department of Studies in Sericultural Science, University of Mysore, Manasagangotri, Mysore -570006, INDIA Department of Zoology, University of Delhi, Delhi- 110007, INDIA Available online at: www.isca.in Received 9th February 2013, revised 11th June 2013, accepted 27th July 2013Abstract An attempt was made to understand physiological behavior of few cultivars of paddy (Oryza sativa L.) subjecting to distillery effluent stress. Physio-chemical data of effluent reveals that it is rich in both organic and inorganic load. Subjecting five cultivars of paddy viz., Jaya, Jyothi, Mandya Vijay and MTU-1001, to different dilution of effluent (viz. 6%, 12%, 25%, 50% and 100%) preliminary germination was conducted. Percent germination, root length and shoot length was almost similar in control and 6% dilution of distillery effluent, with further increase in concentration, gradual inhibitory effect was noticed, whereas complete inhibition was observed at 100%. Physiological parameters such as total carbohydrate, total starch content was high, whereas reducing sugar was low in germinated seedlings treated at higher concentration of effluent. Total protein decreased at higher concentration in all cultivars of paddy. It was high at 6% and decreased gradually with increase in dilution concentration of effluent, when compared to control. These results reveal that degradation and mobilization of seed serves is impaired by cat ions and anions in the effluent by disturbing the activities of enzymes essential for it. Overall data of physiological parameter reveals that Jaya cultivar is sensitive among five cultivars of paddy, when subjected to distillery effluent stress. Keywords: Distillery effluent, Paddy cultivars, germination, total carbohydrate, total protein, total starch, reducing sugar. Introduction Paddy (Oryza sativa L.) is an important cereal crop in India. The crop serves as a staple food in most regions of the world. It constitutes about 53% of the total grain production and 55% of the total of total cereal production. The utilization of industrial effluent for irrigation of crop plant is one of the highly beneficial propositions of waste disposal. There may be two aspects of the use of effluent (Distillery) in agricultural, one is fertilizer value and the other is as a substitute for irrigation water. Distillery factory is one among major water consuming factory and play an important role in creating water pollution problems of considerable magnitude by spent wash. The Gemini Distilleries Pvt. Limited working since 1984 is one of the oldest distilleries in Mysore district, located at Nanjanagud industrial area. The industry produces 30 kilolitres of ethyl alcohol per day and also produces 12-15 litres of spent wash per litre of ethyl alcohol, amounting to 45000 litres of effluent per day. Continuous discharge of distillery effluent to paddy field has made the land uncultivable and decline in the yield. Toxicity is a complex biological phenomenon, only biological tests can fully distinguish between what is or is not harmful for living organism. Studies on physiological and biochemical investigation are of great importance in understanding various metabolic processes of plant system. Pollen fertility and germinations are varied with different media invitro. Heavy metals from industrial effluent may also impact Hence present investigation aims to assess physiological behaviour of few cultivars varieties of paddy during seed germination and early growth, subjecting to abiotic (Distillery effluent) stress. Material and MethodsThe seed samples of certified varieties of paddy viz., Jaya, Jyothi, Mangala, Mandya Vijaya and MTU 1001 were procured from Regional Institute of Agricultural University, V.C. Farm, Mandya, Karnataka. The effluent sample was collected at the point of discharge, in plastic container from Gemini Distillery, Nanjangud. The effluent sample was brought to the laboratory for the analysis of physicochemical parameters. The effluent samples were stored at 4C before chemical analysis was done, for every three days. The collected effluent was analyzed for the physio-chemical parameters by adapting standard procedures from the American Public Health Association4,5. The effluent was then used for the experimental purpose. The certified seeds of paddy cultivars were surface sterilized with 2% sodium hypochloride for 5 min. and rinsed with distilled water for 10-12 times to remove excess of chloride. Five hundred comparably sized paddy seeds were selected and arranged at equal distance on germination sheets – between paper method, with thee replicates supplemented with different International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(9), 5-10, September (2013) Int. Res. J. Biological Sci. International Science Congress Association 6 dilution of distillery effluent viz., 6, 12, 25, 50 and 100% along with control, at room temperature 28 ± 2° C in dark. The germination percent, root length and shoot length were recorded as per ISTA standard. The biochemical experiments were conducted for total starch, total carbohydrate, total protein, and reducing sugar10, analysis from 7th, 10thand 14th day old seedling and estimated according to standard protocol. The obtained data were subjected to analysis of variance and the means were compared with Tukey’s studentized range tests, using SPSS 10.1 version package11. Results and Discussion The physio-chemical analysis of distillery effluent is presented in table-1. pH of the distillery effluent is acidic in nature and it contains high amount of calcium (3000mg/L), chlorides (8500mg/L), sulphates (6745mg/L), nitrogen (1579mg/L), and potassium (14800mg/L). The effluent is also rich in BOD and COD. The physico-chemical analysis data of distillery effluent clearly shows that it is very rich in organic and inorganic load. To minimize the deleterious effects before the waste is discharged to land, further treatment is necessary to reduce the load. This is in accordance with the findings on different distillery effluents12-16. Table-1 Characteristics of raw and treated spent wash Sl. No. Parameters Units Raw Spent wash Treated Spent wash 1 pH - 3.5 - 4.5 7.1 - 7.9 2 Color - Dark brown Faint brown 3 Temp. Degree C 35-38 30-32 4 BOD mg/lt. 63040 5000 5 COD mg/lt. 128900 35000 6 Total solids mg/lt. 167400 145500 7 Suspended solids mg/lt. 13900 23900 8 Total dissolved solids mg/lt. 153500 121600 9 Sulphate as SOmg/lt. 6745 6745 10 Chlorides mg/lt. 8500 8500 11 Sodium mg/lt. 400 400 12 Potassium mg/lt. 14800 14800 13 Magnesium mg/lt. 1600 1600 14 Calcium mg/lt. 3000 3000 15 Nitrogen as Nitrite mg/lt. 1579 1579 16 Phosphorus mg/lt. 244 244 The preliminary germination studies were conducted, where the seed germination and early seedling growth affected due to water pollution stress (table- 2). Hence, further investigations on some biochemical changes at seedling stage under distillery effluent stress were conducted. Enzymes are activated, synthesized and stored, insoluble foods degraded, soluble foods translocated, nutrients mobilized and synthetic reaction related to growth takes place are some of the events takes place during germination. And also, the metabolism increases rapidly and the substrate for this is derived from the storage reserves. The hydrolytic enzymes, particularly amylase, proteases, ribonuclease, acid phosphatase and many other enzymes are under the control of gibberllin (GA). During inhibition of the seed, synthesis of polyribosome, endoplasmic reticulum and proteins, followed latter by hydrolytic enzymes were induced by the supply of the hormone GA-3 from the embryo to the aleurone. If any one of these events or even more is affected, that would consequently affect the other events and eventually hinders the germination of the seeds 17, 18, 19. Probably cations and anions of effluents inhibit enzymes necessary of degradation and mobilization of seed reserve, which are essential of growth and development. During seed germination the seed reserve – carbohydrate i.e. principally the starch are degraded to provide sugars by amylolytic or phosphorolytic pathways20. Total carbohydrates responded significantly (F= 2581.543; P .000) to the treatment of effluents. It was significant high at 6% treatment and a gradual decrease towards higher concentrations, when compared to control in all paddy cultivars (table-3). Increase in carbohydrate content at lower concentrations21-23 was mainly due to the availability of more nutrients in the effluent or due to innate tendency to accumulate inorganic nutrients in organic form24. The effect of the treatment of effluent concentration on the quantity of starch (F= 1271.785; P .000), showed a gradual decrease from 6% to 50% treatment in comparison to control, in all five cultivars of paddy (Table-4). Some workers observed lower starch content at higher effluent concentration25-27. The amount of reducing sugars was least in the 7th day seedling and the quantity significantly increases in 14th day seedling (table-5). No significant effect on reducing sugar content in Zea mays grown in solid and distillery effluent supplement with different concentrations of nitrogen14. The common trend was true in case of total protein also, that higher the concentration greater the impact. The concentration of protein between treatments was found to be significant (F= 347.595; P .000, table-6). Decrease in the protein content at higher concentrations has been reported28-31. These results reveal that degradation and mobilization of seed serves is impaired by cations and anions in the effluent by disturbing the activities of enzymes essential for it. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(9), 5-10, September (2013) Int. Res. J. Biological Sci. International Science Congress Association 7 Table-2 Germination percentage (Germn); Root length (R Length); Shoot length (S Length); of five paddy cultivars with different dilution of distillery effluent Cultivar Concentration Germn R Length S Length Jaya Control 6% 12% 25% 50% Overall 99.25±0.96 89.00±2.58 81.25±2.22 70.00±2.83 42.75±4.57 76.45±20.05 23.43±0.56 19.58±0.51 14.08±0.60 4.40±0.36 1.43±0.33 12.58±8.72 16.40±0.50 14.65±0.39 12.47±0.65 3.70±0.36 2.20±0.94 9.88±5.99 Jyothii Control 6% 12% 25% 50% Overall 99.25±1.50 85.75±5.32 76.00±4.32 67.75±5.80 41.50±4.93 74.05±20.29 22.83±0.97 17.60±0.63 15.35±0.83 9.23±0.59 0.93±0.21 13.19±7.74 16.08±0.75 14.45±0.19 13.50±0.56 10.10±1.07 2.93±0.49 11.41±4.83 Mangala Control 6% 12% 25% 50% Overall 100.00±0.00 88.50±3.32 80.50±2.52 70.75±1.50 41.25±2.99 76.20±20.56 22.90±0.90 17.58±0.56 15.15±0.40 9.30±0.34 0.90±0.22 13.17±7.74 15.73±0.89 14.10±0.61 13.48±0.57 10.78±0.61 3.73±0.59 11.56±4.38 Mandya vijaya Control 6% 12% 25% 50% Overall 99.25±0.96 88.75±1.71 73.25±2.99 55.00±4.76 41.50±6.95 71.55±22.03 21.60±0.70 15.53±0.56 11.25±0.66 8.03±0.72 1.50±0.37 11.58±6.98 18.00±0.61 16.43±1.26 13.63±0.79 8.78±0.60 3.38±0.83 12.04±5.54 MTU 1001 Control 6% 12% 25% 50% Overall 100.00±0.00 90.75±2.22 77.75±1.71 66.00±6.06 45.75±2.06 76.05±19.73 21.55±0.62 18.50±0.57 12.63±0.75 5.30±0.42 1.58±0.46 11.91±7.80 17.10±1.41 17.63±0.48 12.40±0.51 8.68±0.61 3.85±0.80 11.93±5.39 OverallControl 6% 12% 25% 50% 99.55±0.89 e 88.55±3.35d 77.75±3.96c 65.90±7.11b 42.55±4.45a 22.46±1.03 e 17.76±1.46d 13.69±1.70c 7.25±2.13b 1.27±0.42a 16.66±1.15 e 15.45±1.52d 13.09±0.78c 8.41±2.62b 3.22±0.91a F-Value 7.008 30.744 27.954 Note: Overall means followed by the same letter within a column are not significantly when subjected to Tukey’s mean range test (P0.05). ConclusionAccording to our observation we may conclude that industrial effluents are rich in organic and inorganic materials and some toxic substances, which may cause impairment in ionic mobilization and variation in the enzymatic activity. Jaya cultivar is sensitive among five cultivars of paddy, when subjected to stress of distillery effluent. References 1.Anonymous, The Hindu-Survey of Indian Agriculture, Kasturi Publishers, Chennai, 27 (2002)2.Sugar India, Anekant Prakashan, Jayasingapur, Kolhapur (2005) 3.Cavusoglu A. and Sulusoglu M., In vitro Pollen Viability and Pollen Germination in Medlar (Mespilus germanica L.), Int. Res. J. Biological Sci.,2(5), 49-53 (2013) 4.A.P.H.A. Standard methods for the examination of water and wastewater, American Public Health Association,(1985) 5.Trivedi R.K. and Goel P.K., Chemical and biological methods for water pollution studies, Environment publication, Karad, (1986) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(9), 5-10, September (2013) Int. Res. J. Biological Sci. International Science Congress Association 8 Table-3 Total Carbohydrate content of five paddy cultivars with different dilutions of distillery effluent at different time intervals of germinating seedlings Concentration Duration Jaya Jyothi Mangala Mandya vijaya MTU 1001 Overall Control th Day 10thDay 14th Day Overall 29.33±1.15 45.33±4.04 60.33±5.51 45.00±13.87 36.67±3.06 46.67±4.16 60.67±7.02 48.00±11.31 50.33±3.51 55.33±2.52 66.00±5.20 57.22±7.71 43.00±7.00 54.67±6.66 64.67±7.23 54.11±11.16 49.33±9.012 59.00±9.00 69.50±3.54 58.00±10.89 41.73±9.43 52.20±7.32 63.85±6.06 52.34±11.83a 6% th Day 10thDay 14th Day Overall 38.33±2.52 50.00±8.89 67.67±3.06 52.00±13.68 47.33±3.51 58.00±2.00 70.33±2.08 58.56±10.22 52.67±2.52 64.00±4.00 74.33±6.03 63.67±10.14 57.00±6.56 65.00±6.25 64.00±16.46 62.00±10.12 60.33±7.77 71.00±5.29 67.33±20.817 66.22±12.35 51.13±9.09 61.60±8.83 68.73±10.98 60.49±11.95b 12% th Day 10thDay 14th Day Overall 50.67±2.08 62.33±3.79 76.33±5.69 63.11±11.69 56.00±2.65 64.00±3.00 79.00±9.00 66.33±11.25 66.33±1.53 76.33±3.21 89.33±2.08 77.33±10.20 64.00±10.44 77.67±10.97 74.67±18.82 72.11±13.59 68.33±8.50 74.33±5.51 67.33±16.92 70.00±10.39 61.07±8.70 70.93±8.42 77.33±12.77 69.78±12.01c 25% th Day 10thDay 14th Day Overall 60.00±2.00 74.33±3.79 91.67±10.21 75.33±14.81 67.00±6.08 78.33±3.06 97.33±10.07 80.89±14.60 72.67±3.51 88.00±6.25 102.67±2.52 87.78±13.53 74.33±11.93 91.33±6.11 87.67±23.63 84.44±15.64 76.67±9.87 88.67±8.08 82.67±18.58 82.67±12.41 70.13±8.96 84.13±8.37 92.40±14.57 82.22±14.21d 50% th Day 10thDay 14th Day Overall 61.00±3.61 77.33±2.08 101.67±7.37 80.00±18.22 66.33±6.11 81.67±1.53 97.67±3.06 81.89±14.01 78.33±2.52 93.33±4.51 114.67±5.03 95.44±16.22 74.67±7.02 87.67±10.02 93.67±16.26 85.33±13.20 77.00±8.54 92.00±8.00 96.33±19.86 88.44±14.49 71.47±8.58 86.40±8.19 100.80±12.86 86.22±15.62d Overall th Day 10thDay 14th Day Overall 47.87±12.94 61.87±13.85 79.53±16.76 63.09±19.37a 54.67±12.56 65.73±13.63 81.00±16.30 67.13±17.69ab 64.07±11.60 75.40±15.18 89.40±18.83 76.29±18.41c 62.60±14.36 75.27±15.87 76.93±19.19 71.60±17.46bc 66.33±13.14 77.00±13.94 77.14±19.26 73.41±16.07c -- Note: F=;50.667; P0.005 Variance between cultivar, F=278.851; P0.005 Variance between concentration, 2-way interaction, F=2.933; P0.005.Variance between different concentration and cultivars. Overall means followed by the same letter within a columns and rows of overall are not significantly different when subjected to Tukey’s mean range test (P0.05). Table-4 Starch content of five paddy cultivars with different dilutions of distillery effluent at different time intervals of germinating seedlings Concentration Duration Jaya Jyothi Mangala Mandya vijaya MTU 1001 Overall Control th Day 10thDay 14th Day Overall 15.67±1.53 21.33±5.86 21.67±1.53 19.56±4.28 16.33±3.21 23.33±6.11 26.00±3.61 21.89±5.82 20.67±4.16 20.33±3.51 27.33±3.79 22.78±4.76 20.00±1.00 22.67±2.52 25.00±1.00 22.56±2.60 19.67±3.06 19.67±1.53 26.00±3.00 21.78±3.90 18.47±3.20 21.47±3.91 25.20±3.10 21.71±4.35a 6% th Day 10thDay 14th Day Overall 15.00±1.00 24.00±2.00 27.33±2.52 22.11±5.78 18.00±1.00 27.67±2.52 28.67±3.51 24.78±5.56 24.33±4.04 27.33±2.89 31.00±3.00 27.56±4.10 28.00±2.65 27.00±2.65 29.33±1.53 28.11±2.26 20.33±1.53 25.67±2.08 31.33±1.53 25.78±4.99 21.13±5.15 26.33±2.50 29.53±2.64 25.67±4.99b 12% th Day 10thDay 14th Day Overall 19.00±1.00 29.67±2.08 32.00±2.00 26.89±6.19 21.67±1.53 28.00±2.65 34.33±3.21 28.00±5.92 27.00±3.00 31.00±1.73 35.00±0.00 31.00±3.87 27.00±2.00 32.00±2.00 37.00±2.00 32.00±4.66 25.67±1.53 28.33±2.52 32.67±4.16 28.89±3.98 24.07±3.69 29.80±2.45 34.20±2.91 29.36±5.15c 25% th Day 10thDay 14th Day Overall 24.33±2.08 36.33±5.51 38.67±1.53 33.11±7.32 26.33±2.08 34.33±4.51 37.33±2.08 32.67±5.61 29.33±2.08 35.67±1.53 40.33±1.53 35.11±5.01 30.67±2.52 37.67±3.21 41.33±2.08 36.56±5.22 32.00±2.00 34.00±2.00 39.33±1.53 35.11±3.66 28.53±3.44 35.60±3.40 39.40±2.06 34.51±5.43d 50% th Day 10thDay 14th Day Overall 25.33±2.08 38.67±2.08 45.33±4.16 36.44±9.18 31.00±1.00 40.00±2.00 43.00±3.00 38.00±5.72 32.67±4.16 41.33±2.08 50.00±4.00 41.33±8.12 30.33±2.52 37.67±3.06 41.67±2.08 36.56±5.46 29.33±2.52 35.33±2.52 39.33±3.79 34.67±5.07 29.73±3.39 38.60±2.92 43.87±4.78 37.40±6.96e Overall th Day 10thDay 14th Day Overall 19.87±4.64 30.00±7.72 33.00±8.87 27.62±9.12a 22.67±5.83 30.67±6.87 33.87±6.82 29.07±7.96ab 26.80±5.23 31.13±7.69 36.73±8.55 31.56±8.23c 27.20±4.41 31.40±6.53 34.87±7.04 31.16±6.75c 25.40±5.36 28.60±6.19 33.73±5.85 29.24±6.65b -- Note: F=15.446; P0.005 Variance between cultivar, F=239.631; P0.005 Variance between concentration, 2-way interaction, F=2.220; P0.005.Variance between different concentration and cultivars. Overall means followed by the same letter within a columns and rows of overall are not significantly different when subjected to Tukey’s mean range test (P0.05) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(9), 5-10, September (2013) Int. Res. J. Biological Sci. International Science Congress Association 9 Table-5 Reducing sugar of five paddy cultivars with different dilutions of distillery effluent at different time intervals of germinating seedlingsConcentration Duration Jaya Jyothi Mangala Mandya vijaya MTU 1001 Overall Control th Day 10thDay 14th Day Overall 29.67±1.53 34.33±1.53 40.00±1.00 34.67±4.64 26.00±2.00 34.00±1.73 42.67±3.06 34.22±7.50 30.33±1.53 40.33±2.52 45.33±2.08 38.67±6.86 25.67±2.08 31.33±2.08 39.00±1.00 32.00±6.00 33.67±2.08 40.00±2.65 48.67±4.51 40.78±7.10 29.06±3.45 36.00±4.11 43.13±4.31 36.07±6.99e 6%thDay 10thDay 14th Day Overall   \n \n \n\r\n\n\n \n\r   \n       \n\n \n\r\r      \n   \n\r\n     \r  \r 12% th Day 10thDay 14th Day Overall 24.33±1.54 28.67±2.52 37.00±3.00 30.00±5.96 20.33±1.53 26.00±2.00 34.67±0.58 27.00±6.38 26.00±2.00 31.00±2.00 36.00±2.00 31.00±4.66 20.33±2.52 24.67±2.52 25.67±6.11 23.56±4.30 25.67±2.08 31.67±1.15 39.67±7.23 32.33±7.18 23.33±3.09 28.40±3.33 34.60±6.25 28.78±6.38c 25% th Day 10thDay 14th Day Overall 19.00±1.00 22.67±2.08 28.00±1.73 23.22±4.18 18.67±1.53 23.00±1.00 35.67±5.51 25.78±8.18 18.33±2.08 22.67±3.06 29.00±1.00 23.33±5.02 17.67±1.53 16.67±5.13 23.67±0.58 19.33±4.24 20.67±0.58 26.33±1.53 30.33±2.52 25.78±4.47 18.87±1.60 22.27±4.08 29.33±4.69 23.49±5.70b 50% th Day 10thDay 14th Day Overall 16.67±1.15 21.00±2.65 25.00±1.00 20.89±3.92 16.67±1.53 21.00±3.00 28.33±2.08 22.00±5.48 19.67±2.52 23.00±3.00 25.00±1.00 22.56±3.0* 15.00±1.00 19.00±1.00 22.00±2.00 18.67±3.28 21.33±2.52 23.00±3.00 27.33±1.53 23.89±3.41 17.87±2.85 21.40±2.72 25.53±2.64 21.60±4.15a Overall th Day 10thDay 14th Day Overall 24.00±5.81 28.00±6.29 34.27±7.04 28.76±7.57b 21.67±4.39 27.00±5.36 36.07±5.56 28.24±7.83b 24.80±5.41 31.73±8.71 36.00±8.67 30.84±8.90c 20.80±4.63 24.13±6.28 29.87±8.27 24.93±7.45a 27.13±6.39 31.60±6.87 38.13±9.28 32.28±8.74c -- Note: F=; 50.667; P0.005 Variance between cultivar, F=278.851; P0.005 Variance between concentration, 2-way interaction, F=2.933; P0.005.Variance between different concentration and cultivars. Overall means followed by the same letter within a columns and rows of overall are not significantly different when subjected to Tukey’s mean range test (P0.05) Table-6 Total protein content of five paddy cultivars with different dilutions of distillery effluent at different time intervals of germinating seedlings Concentration Duration Jaya Jyothi Mangala Mandya vijaya MTU 1001 Overall Control th Day 10thDay 14th Day Overall 7.33±.58 10.67±.58 13.33±.58 10.44±2.65 9.00±1.00 11.33±1.53 13.33±0.58 11.22±2.11 8.67±1.53 10.00±2.00 11.67±2.52 10.11±2.20 6.00±2.00 7.33±2.08 10.00±2.00 7.78±2.49 5.50±0.50 8.00±1.00 9.00±1.00 7.50±1.73 7.30±1.79 9.47±2.07 11.47±2.23 9.41±2.63d 6% th Day 10thDay 14th Day Overall 8.00±.50 11.67±.58 12.00±1.00 10.56±2.02 10.00±1.00 11.17±1.04 12.67±1.15 11.28±1.48 8.33±2.52 9.67±3.06 10.50±1.32 9.50±2.29 6.33±1.15 7.67±2.25 8.00±3.61 7.33±2.33 6.00±1.00 7.33±0.76 9.00±1.00 7.44±1.53 7.73±1.92 9.50±2.38 10.43±2.43 9.22±2.48d 12% th Day 10thDay 14th Day Overall 7.33±.29 8.50±.50 9.67±.58 8.50±1.09 7.67±1.04 8.83±0.76 10.67±1.15 9.06±1.57 9.00±1.00 9.67±1.53 12.00±1.00 10.22±1.72 7.00±1.73 7.17±2.02 8.50±1.80 7.56±1.75 5.50±0.50 6.50±0.50 7.67±0.58 6.56±1.04 7.30±1.46 8.13±1.58 9.70±1.85 8.38±1.89c 25% th Day 10thDay 14th Day Overall 5.83±.29 7.47±.45 8.67±.58 7.32±1.29 6.00±1.00 5.83±1.44 8.00±2.00 6.61±1.69 7.67±0.58 8.33±2.31 9.17±.76 8.39±1.41 5.00±1.00 6.50±1.32 7.83±1.89 6.44±1.76 5.33±1.53 6.83±1.76 7.67±1.53 6.61±1.73 5.97±1.26 6.99±1.60 8.27±1.37 7.07±1.68b 50% th Day 10thDay 14th Day Overall 4.33±.58 6.50±.50 7.67±.58 6.17±1.54 6.00±2.00 6.17±1.04 6.00±1.00 6.06±1.24 5.67±1.53 7.00±1.73 8.67±1.53 7.11±1.90 4.50±0.87 5.83±1.04 7.17±0.76 5.83±1.39 4.33±0.58 5.67±1.53 6.00±1.00 5.33±1.22 4.97±1.29 6.23±1.16 7.10±1.37 6.10±1.53a Overall th Day 10thDay 14th Day Overall 6.57±1.43 8.96±2.05 10.27±2.25 8.60±2.45b 7.73±1.97 8.67±2.64 10.13±3.07 8.84±2.73b 7.87±1.81 8.93±2.19 10.40±1.89 9.07±2.19b 5.77±1.52 6.90±1.67 8.30±2.11 6.99±2.04a 5.33±0.96 6.87±1.30 7.87±1.46 6.69±1.62a -- Note: F=28.903; P0.005 Variance between cultivar, F=47.281; P0.005 Variance between concentration, 2-way interaction, F=3.342; P0.005.Variance between different concentration and cultivars. 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