Seed Germination and Stress Response of Mung Bean (1)

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Seed Germination and Stress Response of Mung Bean (Vignaradiata L.) on Various Abiotic Factors Custodio, SD. 1 De Vera, S. 1 Guillermo, KS. 1 Maramba, CN. 1 Salgado, CA. 1 1 Department of Biology, College of Science, University of the Philippines Baguio February 18, 2015 ABSTRACT Seeds undergo series of events during germination, wherein a radicle emerges through the seed coat. This experiment was conducted to understand the phenomenon on seed germination and seedling growth. Mung Bean, scientifically known as Vignaradiata L., was used as a test organism. It was exposed to different stresses pertaining to temperature, pH, osmotic concentration, light, and hormones. The effect on the rate of seed germination and growth of hypocotyl-root length of the germinated seeds were observed and recorded. Data gathered were analyzed using ONE-WAY ANOVA. On the one hand, based on the results obtained, seed germination did not occur under low temperature. On the other hand, increase seedling growth is high at increasing temperature, non-salty conditions, and with the presence of hormone Gibberellin. However, seedling growth varies in any pH and light conditions, yet, according to research, higher rate of growth is more preferable at neutral pH and with the presence of light. Therefore, based on the stress responses observed in the experiment, it is indicative that seed germination and seedling growth are possible at different conditions they have been exposed at. But under optimum conditions, they could grow as a healthy plant.

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Seed Germination and Stress Response of Mung Bean (1)

Transcript of Seed Germination and Stress Response of Mung Bean (1)

Seed Germination and Stress Response of Mung Bean (Vignaradiata L.) on Various Abiotic FactorsCustodio, SD.1 De Vera, S.1 Guillermo, KS.1 Maramba, CN.1 Salgado, CA.11Department of Biology, College of Science, University of the Philippines BaguioFebruary 18, 2015

ABSTRACTSeeds undergo series of events during germination, wherein a radicle emerges through the seed coat. This experiment was conducted to understand the phenomenon on seed germination and seedling growth. Mung Bean, scientifically known as Vignaradiata L., was used as a test organism. It was exposed to different stresses pertaining to temperature, pH, osmotic concentration, light, and hormones. The effect on the rate of seed germination and growth of hypocotyl-root length of the germinated seeds were observed and recorded. Data gathered were analyzed using ONE-WAY ANOVA. On the one hand, based on the results obtained, seed germination did not occur under low temperature. On the other hand, increase seedling growth is high at increasing temperature, non-salty conditions, and with the presence of hormone Gibberellin. However, seedling growth varies in any pH and light conditions, yet, according to research, higher rate of growth is more preferable at neutral pH and with the presence of light. Therefore, based on the stress responses observed in the experiment, it is indicative that seed germination and seedling growth are possible at different conditions they have been exposed at. But under optimum conditions, they could grow as a healthy plant.

INTRODUCTIONIn this experiment, it is necessary to comprehend how seed and seedling, germinate and grow, respectively. To observe the effects of different factors, such as temperature, pH, varying osmotic concentration, light, and hormones, on the developmental process.Seeds are embryonic plant in resting condition or seed dormancy. These remain dormant or inactive until conditions are right for germination. The resumption of growth of this embryonic plant is known to be the germination stage. Mung Bean (Vignaradiata L) belonging to the Legume Family, like other plant species, has to meet its optimum conditions, such as having enough sunlight and plenty of water, in order to germinate and grow as a healthy plant. (Leubner, 2000).Seed germination is a complex physiological process triggered by imbibitions, where the uptake of water by dry seed occur, after possible dormancy mechanisms have been released by a prompt. Germination depends on both external and internal conditions of the growing plant. It starts when a seed is provided with appropriate water and temperature. Seeds expand as they imbibe water, and their enzymes and food supplies become hydrated. Hydrated enzymes become active, thus the seed increases its metabolic activities to produce energy for the growth process. Moreover, the size of the cell is in proportion with the increase in pressure inside caused by the water. Under favorable conditions, rapid expansion on embryos growth culminates in the rupture of the covering layers and emergence of the radicle. The emergence of the radicle is considered as the completion of germination where the protrusion of the radicle tip is visible. According to seed physiologists, this transition point is also characterized by the loss of desiccation tolerance and is a molecular checkpoint a developmental molecular switch from the germination program to the seedling program. (Leubner, 2000).

MATERIALS AND METHODSMung bean seeds were immersed in water wherein the floating unhealthy seeds were discarded and the sunken viable seeds were used in the experiment. There were five different set-ups and each tested the effects of (a) temperature, (b) pH, (c) osmotic gradient, (d) light and of (e) hormones. Petri plates were cleaned, lined up with cotton and equally distributed with ten mung beans. For (a), three Petri plates were wet with distilled water, saturating the cotton. Each plate was put in a room, low (refrigerator) and high (incubator, 30) temperatures. For (b), three plates were wet with solutions of pH 3.0, pH 11.0 and distilled water. For (c), three plates were wet with NaCl solutions having varying concentrations of 0.5g NaCl/200ml, 2g NaCl/150ml, and of 5g NaCl/150ml. For (d), two plates were wet with distilled water. One plate was set on a normally lighted place while the other one was in a dark place. Lastly, for (e), three plates were wet with distilled water (control), 20ppm gibberellic acid (GA), and with 20ppm indole acetic acid (IAA) solution. The plates were placed in a dark area. All the plates in each set-up were labeled accordingly. After two days, percent germination of each set-up was observed then five days, the lengths of hypocotyl-root axis were measured using a ruler. The findings were analyzed using statistical methods.

RESULTS AND DISCUSSIONGermination entails a variety of biophysical and biochemical processes from the initial imbibitions of water and the re-establishment of membrane integrity to the activation of numerous enzymes and metabolic pathways and finally the elongation of the root which ruptures the testa(Simon et al, 1976). It is dependent on environmental factors, such as water, light, temperature and oxygen, thereby confirming our understanding of the ideal conditions required for the germination of seeds in a predetermined species (Oliveira et al., 2013).There were two separate data collections made in the experiment. The first data collection for every set-up was done two days after the start of the experiment and then followed by a second data collection which was done after five days.

Table 1. Raw data of the seed germination and seedling growth length.Set-upConditionPercent GerminationHypocotyls-Root Length (cm)

Effect of TemperatureRoom Temperature90%7.805.506.804.604.307.105.905.305.104.30

Oven100%10.713.19.212.713.588.27.37.37

Refrigerator0%No seed germinated

Effect of pHpH 7(distilled water)90%7.26.77.06.96.57.37.26.96.47.1

pH 3100%5.56.06.34.86.44.03.93.53.85.8

pH 1170%6.86.75.55.34.85.15.96.24.75.5

Effect of Varying Osmotic Concentration0.5 g NaCl/200 ml solution100 %4.003.904.404.705.105.004.604.105.304.80

2 g NaCl/ 150 ml solution40 %1.401.101.301.201.201.201.201.101.001.20

5 g NaCl/ 150 ml solution100 %1.201.301.101.101.101.201.101.201.101.20

Effect of LightLight100 %4.505.804.505.304.705.204.805.505.605.10

Dark100%6.606.805.406.005.806.205.506.605.605.50

Effect of TemperatureWater100%8.07.68.27.98.38.27.67.78.48.1

Indole Acetic Acid90%2.82.83.63.43.23.12.63.53.23.6

Gibberellic Acid100%7.507.206.807.807.106.907.506.807.407.20

Data recorded and used in statistical analysis.

Figure 1. Percent germination of the mung beans observed after 2 days.The data collected, two days after, showed great difference in the germination of mung beans subjected at different conditions. All of The mung beans placed inside the oven at 37C, pH 3, 0.5 g NaCl/200 ml solution, 5 g NaCl/ 150 ml solution, light conditions, dark conditions, water and gibberellic acid germinated which shows 100% germination after two days. The lowest number of seed germinated in the experiment was the mung beans placed inside the refrigerator where not a single mung bean germinated which shows 0% germination after two days.

TemperatureAfter five days, another set of data collection was done where the lengths of the newly sprouted seedlings were measured. Also, the current conditions of the mung bean seedlings during the time of data collection were as well noted. The first condition observed was the effects of temperature. Temperature is an environmental factor that significantly affects germination. However, there is no optimum and uniform temperature for all species (Bewley, D. et al., 2006). Germination occurs within a defined range and will not occur above or below these limits.

Figure 2. Lengths of the mung bean seedlings at varying temperature.The effects of temperature to the mung beans were observed in the experiment. All of the mung beans germinated at the three varying temperature setting after five days. The mung bean seedlings placed at room temperature had lengths ranging from 4.3cm to 7.80cm. The mung bean seedlings placed inside the oven where the recorded temperature was 37C, had lengths raging from 7.0cm to 13.5cm which shows a faster seedling growth. The mung bean seedlings placed inside the refrigerator had lengths ranging from 0.80cm to 1.3cm which shows poor seedling growth. (Figure 2)According to the study of Simon et al. (1979), low temperature may not only reduce percentage of germination but also delay germination. Still on the study of Simon et al. (1979) on mung beans, the lowest temperature at which 50% of the seeds would germinate was about 11.5oC and at temperatures a little below this, some seeds will germinate but even after prolonged periods the majority still fails to germinate. Temperature affects the speed and percentage of germination, primarily influencing water uptake and impacting the biochemical reactions and physiological processes that determine germination (Taiz and Zeiger, 2009).The measured lengths of the mung bean seedlings were also subjected to statistical analysis (table 2 and 3) to see if there are significant differences among the varying temperature conditions.Table 2. The mean, standard deviation and 95% confidence intervals for the dependent variable (Length) for each separate group (Room Temperature, Oven Temperature and Refrigerator Temperature), as well as when all groups are combined

Descriptives

LENGTHNMeanStd. DeviationStd. Error95% Confidence Interval for MeanMinimumMaximum

Lower BoundUpper Bound

room105.67001.21568.384434.80046.53964.307.80

oven109.68002.59991.822177.820111.53997.0013.50

ref101.1200.13984.044221.02001.2200.801.30

Total305.49003.90034.712104.03366.9464.8013.50

Table 3. The table that shows the output of the ANOVA analysis of the mung bean seedling length subjected to varying temperaturesANOVA

LENGTHSum of SquaresdfMean SquareFSig.

Between Groups366.8542183.42766.644.000

Within Groups74.313272.752

Total441.16729

We can see that the significance level is below 0.05 (table 3). Therefore, there is a statistically significant difference in the mean length of the mung beans subjected to varying temperatures (see appendix to see post-hoc results).

pH

Figure 3. Lengths of the mung bean seedlings at different pH.Next, the effects of pH to the mung beans were observed in the experiment. All of the mung beans germinated at the three varying pH setting after five days. The mung bean seedlings added with water which is neutral had lengths ranging from 6.4cm to 7.3cm. The mung bean seedlings added with a solution at pH 11 which is basic had lengths raging from 4.7cm to 6.8cm. The mung bean seedlings added with a solution at pH 3 which is acidic had lengths ranging from 3.5cm to 6.3cm (Figure 3).Neutral pH is the most preferable pH for seed to germinate. Some enzymes may be inactivated by the very acidic environment. Sometimes, the presence of H+ ions only has a negative effect on plant development (Chodura, 2004).In the case of some plants their growth in acid soil is possible but seed germination must take place in less acidified environment because of the need for maintenance of the appropriate pH of the soil solution for amylolytic enzymes initiating germination (Lee, 1998). However, acid soil pH stimulates initial development phases of some species. These species include plants with thick seed coats. The effect of acid pH may be direct, manifesting itself in dissolution of the seed coat or indirect which involves the stimulation of conditions for development of some species of fungi whose action causes perforation of the seed coat (Vleeshouwers et al., 1995)A study conducted by Bukvic et al. (2007) on the germination of Pisum sativum showed a higher seed germination affected by lowering pH to 5.0, yet a further development of seedlings was better at a higher pH.The measured lengths of the mung bean seedlings were also subjected to statistical analysis (table 4 and 5) to see if there are significant differences among the different pH levels.

Table 4. The mean, standard deviation and 95% confidence intervals for the dependent variable (Length) for each separate group (water, pH11, pH3), as well as when all groups are combined.

Descriptives

LENGTHNMeanStd. DeviationStd. Error95% Confidence Interval for MeanMinimumMaximum

Lower BoundUpper Bound

water106.9200.30478.096386.70207.13806.407.30

ph11105.6500.73673.232985.12306.17704.706.80

ph3105.00001.12940.357154.19215.80793.506.40

Total305.85671.11840.204195.43906.27433.507.30

Table 5. The table that shows the output of the ANOVA analysis of the mung bean seedling length subjected to pH levelsANOVA

LENGTHSum of SquaresdfMean SquareFSig.

Between Groups19.07329.53614.969.000

Within Groups17.20127.637

Total36.27429

We can see that the significance level is below 0.05 (table 5). Therefore, there is a statistically significant difference in the mean length of the mung beans subjected to different pH level (see appendix to see post-hoc results).

Osmotic Concentration

Figure 4. Lengths of the mung bean seedlings at different osmotic concentrations.After that, the effects of varying Osmotic Concentration to the mung beans were observed in the experiment. All of the mung beans germinated at the three varying osmotic concentration setting after five days. The mung bean seedlings added with 0.5 g NaCl/ 200 ml solution had lengths ranging from 3.9cm to 5.3cm. The mung bean seedlings added with 2 g NaCl/ 150 ml solution had lengths raging from 1.0cm to 1.4cm. The mung bean seedlings added with 5g NaCl/ 150 ml solution had lengths ranging from 1.1cm to 1.3cm (Figure 4).In many plant type, germination and seedling growing phase is very sensitive to salt stress. In general, the highest germination percentage occurs in non-salty conditions and it decreases depending on the ascending salt concentrations (Khan et al,2009). Seeds germination begins with water intake but it decreased by the salt (Othman, 2005). The decrease in water intake of the seed in salty conditions, osmotically and by the ion toxicity with accumulation of Na and Cl ions highly around the seeds, prevents the seed germination. In the experiment, low solute concentration showed the highest hypocotyl-root axis length and the high solute concentration showed low or none hypocotyls-root axis length. The higher the salt concentration, the lower is the hypocotyls-root axis length. Higher solute inside the seed, then there is lower solute concentration in the outside environment. As the solute moves out, water goes inside the seed.Strong delay of germination was observed mainly at the higher level of salt concentration. A study by Jamil et al. (2005) reported that germination of Brassica species ( cabbage, cauliflower, canola) decreased as the salinity concentration increased. According to the study conducted by Rahman et al(2009), ascending salt concentrations not only prevent the germination of the seeds but also extend the germination time by delaying the start of germination. High salt concentration decreases germination percentage. Salt tolerance studies which have done during germination time are important for determining the plants salt tolerance in the early and late growing phases (Zapata et al., 2003).Seeds require higher amount of water uptake during the germination under the salt stress due to the accumulation of the soluble solutes around the seeds which increases the osmotic pressure. This causes excessive uptake of the ions which results in toxicity in plants (Jones, 1986). Moreover, water potential gradient between the external environment and the seeds also inhibits the primary root emergence (Eneas Filho et al., 1995).The measured lengths of the mung bean seedlings were also subjected to statistical analysis (table 6 and 7) to see if there are significant differences among the different osmotic concentrations.Table 6. The mean, standard deviation and 95% confidence intervals for the dependent variable (Length) for each separate group (0.5g NaCl/ 200 ml solution, 2 g NaCl/ 150 ml solution, 5g NaCl/ 150 ml solution ), as well as when all groups are combined

Descriptives

LENGTHNMeanStd. DeviationStd. Error95% Confidence Interval for MeanMinimumMaximum

Lower BoundUpper Bound

0.5g104.5900.48178.152354.24544.93463.905.30

2g101.1900.11005.034801.11131.26871.001.40

5g101.1600.06992.022111.11001.21001.101.30

Total302.31331.66085.303231.69322.93351.005.30

Table 7. The table that shows the output of the ANOVA analysis of the mung bean seedling length subjected to varying temperatures

ANOVA

LENGTHSum of SquaresdfMean SquareFSig.

Between Groups77.753238.876468.181.000

Within Groups2.24227.083

Total79.99529

We can see that the significance level is below 0.05 (table 7). Therefore, there is a statistically significant difference in the mean length of the mung beans subjected to varying osmotic concentrations (see appendix to see post-hoc results).Light

Figure 5. Lengths of the mung bean seedlings at differently lighted areas

Then, the effects of light exposure to the mung beans were observed in the experiment. All of the mung beans germinated at the two different light setting, lighted place and dark place, setting after five days. The mung bean seedlings placed under the lighted place had lengths ranging from 4.5cm to 5.6cm. The mung bean seedlings placed at the dark area had lengths raging from 5.4cm to 6.8cm (Figure 5).Mung bean germinates even without exposure to light. Large seed with thin coat that does not need light to start germination. Both the amount of light which includes length of exposure and photosynthetic photon flux density and quality of light are environmental cues that signal conditions potentially suitable for seedling establishment and survival (Pons, 2000).According to the study conducted by Serrano-Bernardo et al (2007), light is important for seed germination and that many species respond to the environment with the optimal growth and development according to the light they receive. In accordance with the result of our study, some seeds germinate similarly in light and darkness while others do it more readily either under light or darkness conditions. Also, light requirements for germination can vary with temperature (Serrano-Bernardo et al., 2007). Knowledge of species-specific light requirements for germination could indicate whether restoration practitioners should time seed sowing efforts to plant canopy development or whether excess sedimentation common in new restorations in agricultural landscapes reduced seed germination of plants on wetland areas (Jurik et al., 1994).The measured lengths of the mung bean seedlings were also subjected to statistical analysis (table 8 and 9) to see if there are significant differences between the availability of light.

Table 8. The mean, standard deviation and 95% confidence intervals for the dependent variable (Length) for each separate group (lighted place, dark place), as well as when all groups are combined

Descriptives

LENGTHNMeanStd. DeviationStd. Error95% Confidence Interval for MeanMinimumMaximum

Lower BoundUpper Bound

light105.1000.46188.146064.76965.43044.505.80

dark106.0000.52281.165335.62606.37405.406.80

Total205.5500.66610.148945.23835.86174.506.80

Table 9. The table that shows the output of the ANOVA analysis of the mung bean seedling length subjected to varying temperatures

ANOVA

LENGTHSum of SquaresdfMean SquareFSig.

Between Groups4.05014.05016.644.001

Within Groups4.38018.243

Total8.43019

We can see that the significance level is below 0.05 (table 9). Therefore, there is a statistically significant difference in the mean length of the mung beans subjected different light availability.Hormones

Figure 6. Lengths of the mung bean seedlings added with different hormonesLastly, the effects of hormones to the mung beans were observed in the experiment. All of the mung beans germinated at the three different hormone setting after five days. The mung bean seedlings added with Gibberellic Acid had lengths ranging from 6.8cm to 7.5cm. The mung bean seedlings added with Indole Acetic Acid had lengths raging from 2.6cm to 3.6cm. The mung bean seedlings added with water had lengths ranging from 2.6cm to 3.6cm (Figure 6).The evidence for hormone participation comes from the association of hormone concentration with specific development stages, effects of applied hormones and the relationship of hormones to metabolic activities. The applications of gibberellins increases the seed germination percentage by adding the fact that gibberellins also increases the amino acid content in embryo and cause release of hydrolytic enzyme required for digestion of endospermic starch when seeds renew growth at germination (Chauhan, J. S. et al., 2009).The measured lengths of the mung bean seedlings were also subjected to statistical analysis (table 10 and 11) to see if there are significant differences among the different hormones.Table 10. The mean, standard deviation and 95% confidence intervals for the dependent variable (Length) for each separate group (GA, IAA, water), as well as when all groups are combined

Descriptives

LENGTHNMeanStd. DeviationStd. Error95% Confidence Interval for MeanMinimumMaximum

Lower BoundUpper Bound

GA107.2200.33267.105206.98207.45806.807.80

IAA103.1800.35528.112352.92583.43422.603.60

water108.0000.29059.091897.79218.20797.608.40

Total306.13332.17166.396495.32246.94422.608.40

Table 11. The table that shows the output of the ANOVA analysis of the mung bean seedling length subjected to varying temperatures

ANOVA

LENGTHSum of SquaresdfMean SquareFSig.

Between Groups133.875266.937624.934.000

Within Groups2.89227.107

Total136.76729

We can see that the significance level is below 0.05 (table 11). Therefore, there is a statistically significant difference in the mean length of the mung beans subjected to different hormones (see appendix to see post-hoc results).The experiment shows the different evidences about the contributing factors which governs the overall development of plant. Plant growth is not only regulated by their genes but also regulated by the growth hormones, nutrient and environmental factors.

LITERATURE CITEDBernardo-Serrano, F., Rosua, JL., & Diaz-Miguel, M. (2007). Light and temperature effects on seed germination of four native species of Mediterranean high mountains (Spain). International Journal of Experimental Botany, 76, 27-38.Bukvic G., Grljusic S., Rozman V., Lukic D., Lackovic R., Novoselovic D.(2007). Seed age and pH water solution effects on field pea (Pisum sativum L.) germination. Not. Bot. Hort. Agrobot. Cluj 35(1), 20-26.Chauhan, J.S.,Tomar, Y.K., Indrakumar Singh, N., Ali,Seema and Debarati. (2009). Effect Of Growth Hormones On Seed Germination And Seedling Growth Of Black Gram And Horse Gram. Journal of American Science, 5(5):79-84.Chodura P., Komosa A., Koota T.(2004). Effect of pH of media on dynamics of macroelement content in leaves of greenhouse tomato grown on mineral wool. Rocz. ARw Poznaniu CCCCLVI, 29-35 [in Polish].De Oliveira, AK, Ribeiro, JW,Pereira, KC. & Silva, CA. (2013). Effects of Temperature on the germination of Diptychandra aurantiaca( Fabaceae) seeds. Acta Scientiarum, 35 (2), 203-208.Eneas, Filho, J., Oliveira Neto. O. B., Prisco, J. T., Gomes Filho, E., Monteiro C. (1995), Effects of salinity in vivo and in vitro on cotyledonary galactosidases from Vigna unguiculata (L.) Walp. during seed germination and seedling stablishment. Revista Brasileira de Fisiologia Vegetal, 7 (2), 135-142.Jamil, M., C.C. Lee, S.U. Rehman, D.B. Lee and M. Ashraf. 2005. Salinity (NaCl) tolerance of Brassica species at germination and early seedling growth. Electron. J. Environ. Agric. Food Chem. 4, 970976.Jones, R. A. (1986). High salt tolerance potential in Lycopersicon species during germination. Euphytica, 35, 575-582.Jurik TW, Wang S-C, van der Valk AG. (1994). Effects of sediment load on seedling emergence from wetland seed banks. Wetlands 14,159165.Khan, H.A., C.M. Ayub, M.A. Pervez, R.M. Bilal, M.A. Shahid and K. Ziaf. (2009). Effect of seed priming with NaCl on salinity tolerance of hot pepper (Capsicum annuum L.) at seedling stage. Soil & Environ. 28(1): 8187.Lee J.A.(1998). Plant-soil interactions at low pH: principles and management. R.A. Date, N.J.Grundon, G.E. Rayment, M.E. Probert (eds.), Vegetatio ,136(2), 249-250.Leubner, G. (2000) Seed germination. The Seed Biology Place. Retrieved from http://www.seedbiology.de/germination.aspOthman, Y.( 2005). Evaluation of Barley Cultivars Grown in Jordan for Salt Tolerance. Ph.D Thesis, Jordan University of Science and Technology, Jordan.Pons TL. 2000. Seed responses to light. In: Fenner M, ed. Seeds: the ecology of regeneration in plant communities. New York, NY: CAB International.Rahman, M., U.A. Soomro, M. Zahoor-ul-Haq and S. Gul. (2008.) Effects of NaCl salinity on wheat(Triticum aestivum L.) cultivars. World Journal of Agricultural Sciences. 4(3), 398403.Simon, E, Minchin, A, Mcmenahin, M, & Smith, JM.(1976) The low Temperature limit for Seed Germination. New Phytologist, 77, 301-311.Taiz, L, & Zeiger, E.(2009). Plant Physiology 4 ed. Porto Alegre: Artmed.Vleeshouwers L.M., Bowmeester H.J., Karssen C.M.(1995). Redefining seed dormancy: an attempt to integrate physiology and ecology. J. Ecol., 83, 1031-1037.Zapata, P.J., M., Serrano, M.S. Pretel, A. Amoros and M.A. Botella. (2003). Changes in ethylene evolution and polyamine profiles of seedlings of nine cultivars of Lactuca sativa L. in response to salt stress during germination. Plant Science. 164:557563.APPENDIXEFFECT OF TEMPERATURE

Table 12. Multiple Comparisons at the effects of temperatures

Dependent Variable: LENGTH Tukey HSD (I) TEMP(J) TEMPMean Difference (I-J)Std. ErrorSig.95% Confidence Interval

Lower BoundUpper Bound

roomoven-4.0100(*).74193.000-5.8496-2.1704

ref4.5500(*).74193.0002.71046.3896

ovenroom4.0100(*).74193.0002.17045.8496

ref8.5600(*).74193.0006.720410.3996

refroom-4.5500(*).74193.000-6.3896-2.7104

oven-8.5600(*).74193.000-10.3996-6.7204

* The mean difference is significant at the .05 level.

LENGTH

Tukey HSD TEMPNSubset for alpha = .05

123

ref101.1200

room10 5.6700

oven10 9.6800

Sig. 1.0001.0001.000

Means for groups in homogeneous subsets are displayed.a Uses Harmonic Mean Sample Size = 10.00

EFFECT OF PH

Table 13. Multiple Comparisons at the effects of pH

Dependent Variable: LENGTH Tukey HSD (I) PH(J) PHMean Difference (I-J)Std. ErrorSig.95% Confidence Interval

Lower BoundUpper Bound

waterph111.2700(*).35695.004.38502.1550

ph31.9200(*).35695.0001.03502.8050

ph11water-1.2700(*).35695.004-2.1550-.3850

ph3.6500.35695.182-.23501.5350

ph3water-1.9200(*).35695.000-2.8050-1.0350

ph11-.6500.35695.182-1.5350.2350

* The mean difference is significant at the .05 level.

LENGTH

Tukey HSD PHNSubset for alpha = .05

12

ph3105.0000

ph11105.6500

water10 6.9200

Sig. .1821.000

Means for groups in homogeneous subsets are displayed.a Uses Harmonic Mean Sample Size = 10.000.

EFFECT OF OSMOTIC CONCENTRATION

Table 14. Multiple Comparisons at the effects of osmotic concentration

Dependent Variable: LENGTH Tukey HSD (I) OSMOTIC(J) OSMOTICMean Difference (I-J)Std. ErrorSig.95% Confidence Interval

Lower BoundUpper Bound

0.5g2g3.4000(*).12887.0003.08053.7195

5g3.4300(*).12887.0003.11053.7495

2g0.5g-3.4000(*).12887.000-3.7195-3.0805

5g.0300.12887.971-.2895.3495

5g0.5g-3.4300(*).12887.000-3.7495-3.1105

2g-.0300.12887.971-.3495.2895

* The mean difference is significant at the .05 level.

LENGTH

Tukey HSD OSMOTICNSubset for alpha = .05

12

5g101.1600

2g101.1900

0.5g10 4.5900

Sig. .9711.000

Means for groups in homogeneous subsets are displayed.a Uses Harmonic Mean Sample Size = 10.000.

EFFECTS OF HORMONES

Table 15. Multiple Comparisons at the Effects of Hormones

Dependent Variable: LENGTH Tukey HSD (I) HORMONE(J) HORMONEMean Difference (I-J)Std. ErrorSig.95% Confidence Interval

Lower BoundUpper Bound

GAIAA4.0400(*).14636.0003.67714.4029

water-.7800(*).14636.000-1.1429-.4171

IAAGA-4.0400(*).14636.000-4.4029-3.6771

water-4.8200(*).14636.000-5.1829-4.4571

WaterGA.7800(*).14636.000.41711.1429

IAA4.8200(*).14636.0004.45715.1829

* The mean difference is significant at the .05 level.

LENGTH

Tukey HSD HORMONENSubset for alpha = .05

123

IAA103.1800

GA10 7.2200

water10 8.0000

Sig. 1.0001.0001.000

Means for groups in homogeneous subsets are displayed.a Uses Harmonic Mean Sample Size = 10.000.