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Effects of Plant Growth Regulators on Seed Establishment of Lolium perenne in Saline Soil Conditions

Introduction
Salinity is one of the major environmental factors limiting plant growth and productivity of both irrigated and non-irrigated lands in many areas of the world (Uddin. et al. 2011). Soils directly affect all stages of plant growth and development in vegetative growth prior to reproductive stage (Uddin. et al. 2011) Salinity is a general term used to describe the presence of elevated levels of different salts, such as chlorides, sulfates and bicarbonates of sodium, magnesium, and calcium in soil and water, and it usually results from water tables rising to, or close to, the ground surface (Munns. 2002) Water related problems are on the increase in turfgrass sites because of using recycled water. Managers for perennial turfgrass must deal with problems of reduced growth, tissue dehydration, nutritional imbalances and specific ion toxicities, slow recovery from injury, and poor long term persistence that can be caused by salinity stress (Carrow and Duncan. 1998). High levels of sodium also supress essential ions such as K, Ca, and Mg in certain turfgrass species, which are essential to sustainable growth. (Zhang et al.2012) To achieve salt tolerance, three important, interconnected aspects of plant activities need to occur: damage must be prevented or alleviated, homeostatic conditions must be re-established in the new stressful environment, and growth must resume, albeit at a reduced rate (Flowers and Colmer. 2008). In plants grown under salinity, epidermal cells became thickened, reduction occurred in the number of vascular bundles, and metaxylem and protoxylem elements became disorganized and deformed in shape (Rashid. 1999; Dolatabadian et al. 2011). Salinity also reduced the final germination rate and daily germination rate. Similarly, shoot dry weight, longest root length, and percentage of green tissue of mature grasses declined with increasing salinity levels. However, root dry weight was not significantly affected by salinity (Zhang et al.2012). Studies have shown that the use of plant growth regulators (PGRs) has an ameliorative effect on plants grown under saline conditions (Ratnakar and Rai. 2013). There are a wide variety of PGRs that control different aspects of growth, flowering and root elongation in plants. Plant growth regulators have been reported to increase metabolic reaction of seed germination (Jamil and Rha. 2007) With increasing agricultural land and more golf courses using effluent water, it is important that we study other methods to aid in establishing seed with saline water. In the present investigation the

effect of giberellic acid (GA), kinetin and ethephon on Lolium perenne, cultivated under saline soil conditions is assesed.

Materials and Methods
Perennial Ryegrass (Lolium perenne) seeds were obtained from the Prairie Turfgrass Research Centre (PTRC) at Olds College. The seeds were treated with one of five treatments: saline and one of the three PGRs, saline, and distilled water. The independent variables were the PGR’s used in the experiment along with the use of saline water and the dependant variables are the total germination and the time to germination. The remaining four treatments were sterilized using 10% bleach for 30 seconds then triple rinsed in distilled water and treated with one of the following PGR’s, Kinetin (0.05 mmol/L), Ethephon (10.0 mmol/L) and Giberellic acid (3.0 mmol/L). 25 seeds each were placed into disposable petri dishes using sterilized forceps for a total of 125 seeds for each replicate, and then each dish was covered throughout the experiment. The first four treatments were given 2ml of saline water using a syringe to maintain moisture. Treatment five received 2ml of distilled water instead of saline. The petri dishes were labelled with the respective treatments, stacked in a Ziploc bag in random order and placed in an incubator at 20-25⁰C. Germinated seeds were recorded in a Microsoft Excel spreadsheet and removed with sterile forceps. The seeds were considered germinated when a >2mm radical had emerged. There were seven replicates for a total of 35 experimental units. The seeds were checked every Monday, Wednesday and Friday over a 31 day period. The data was analysed using ANOVA.

Results and Analysis
The null hypothesis of this experiment was to conclude that PGRs would have no effect in ryegrass germination while being irrigated with NaCl concentrated water. Data was interpreted using the program Costat and Excel, data was compiled using ANOVA test as seen in figure 1.3. The LSD was entered at 0.05. Figure 1.4 shows that there was no significant difference in percent germination among the five treatments when P= 0.631. The graph shows in figure1.1 that treatment five (the control) had the best success of total germination with an average of 22.14 seeds with an incorporated 14.9% error.

Total Germination
23.00 22.00 Seeds Germinated 21.00 20.00 19.00 18.00 1 2 3 Treatment 4 5

(Figure 1.1)
The second part of the experiment was to record the time to 50% germination of each treatment. This was deduced through Costat and Excel as well and interpreted using the ANOVA test as seen in figure 1.5. There is no significant difference among treatments at P= 0.457. The graph shows in figure 1.2 that treatment five (the control) also took the shortest amount of days to reach 50% germination with an average of 5.86 days with an incorporated error of 2.77%.

Time to 50% Germination
8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 1 2 3 Treatment 4 5

(Figure 1.2)

Days

Groups Kinetin Giberellic Acid Ethephon Saline Distilled

Count 7 7 7 7 7

Sum 137 150 140 149 155

Average Variance Std.Dev. 19.57143 5.952381 9.66 21.42857 12.95238 9.31 20 34 9.89 21.28571 6.904762 10.20 22.14286 1.47619 10.20

Std. Error 3.651701 3.518883 3.739323 3.854702 3.854702

ANOVA Source of Variation Between Groups Within Groups Total Kinetin 23 19 17 20 21 16 21 Mean 19.57

SS 31.82857143 367.7142857 399.5428571 Giberellic Acid 23 25 15 22 24 23 18 Mean 21.43

df

MS F P-value F crit 4 7.957143 0.649184 0.631872 2.689628 30 12.25714 34

Ethephon 19 25 5 20 25 23 20 Mean 19.57

Saline 21 21 23 24 23 16 21 Mean 21.29

Distilled 23 23 23 21 22 20 23 Mean 22.14

(Figure 1.3)

Rank Mean Name Mean n Non-significant ranges ----- --------- ------------- ------- --------------------------------------15 22.1428571429 7a 23 21.4285714286 7a 34 21.2857142857 7a 41 19.5714285714 7a 52 19.5714285714 7a

(Figure 1.4)

SUMMARY Groups Kinetin Giberellic Acid Ethephon Saline Distilled Count 7 7 7 7 7 Sum 50 41 50 52 39 Average 7.142857 5.857143 7.142857 7.428571 5.571429 Variance 5.142857 2.142857 8.142857 8.285714 3.285714
Std. Dev.

2.27 2.95 1.46 2.88 1.81

Std. Error 0.857143 1.114835 0.553283 1.087968 0.685119 0.85966

ANOVA Source of Variation Between Groups Within Groups Total Kinetin 8 8 8 5 5 11 5 Mean 7.14

SS 20.17142857 162 182.1714286

df

MS F 4 5.042857 0.933862 30 5.4 34 Saline 8 8 5 5 5 13 8 Mean 7.43 Distilled 8 5 8 5 3 5 5 Mean 5.57

P-value F crit 0.45767 2.689628

Giberellic Acid Ethephon 8 11 8 5 5 5 5 8 5 5 5 11 5 5 Mean Mean 5.86 7.14

(Figure 1.5)

Discussion
It was concluded that PGR’s did not have a significant effect on seed germination while being irrigated with salt water at a concentration of 18dS/m. Besides the control, treatment 2 (GA) had the best germination rate at 86% and was the quickest to germinate (besides the control) at 5.86 days. Treatment 2 (GA) showed the best overall results when it came to a resistance of salt water at 18dS/m. Treatment 1 (SA) and treatment 3 (Kin) had the worst overall results with total germination reaching 78%, which is a total difference of nearly two days compared to the control, and T50 of both treatments being 7.14 days. Our group’s speculation of the results came out virtually as expected. Distilled being the best and saline being the worst as a result of the thickened epidermal cells, along with the suppression of essential ions such as K, Ca and Mg, as mentioned in the introduction. I also mentioned that past research had discussed the ameliorative effects of PGR’s on plants grown under saline conditions but no such results were found in this experiment. Depending on the site and water quality, specific environmental factors on certain PGR’s should have been researched. The PGR’s used in this experiment might have reacted better if in their proper environment. Another considerable factor would be the falsifying of data within certain groups. Since three groups confirmed not conducting any data recording, the odds of groups forging their numbers is very high. The final experimental error would be the accidental irrigation with the incorrect water source. This would change the chemical reaction within the plant and produce significantly different results. As previously stated, for future research suggestions, the placement of the PGR’s in their best suited environment should be studied and irrigation should be the same for every treatment (not just when it “looks dry”).

Literature Cited
1. Jamil, M., & Rha, E.S. (2007). Gibberellic Acid (GA3) Enhanced Seed Water Uptake, Germination and Early Seeling Growth in Sugar Beet under Salt Stress. Pakistan Journal of Biological Sciences, 10(4), 654-658.pdf 2. Qi Zhang1, q., Kevin, R., & Sheng, W. (2012). Salinity Effect on Seed Germination and Growth of Two Warm-season Native Grass Species.Hortscience, 47(4), 527-530. 3. Ratnakar, A., & Rai, A. (2013). Alleviation of the Effects of NaCl Salinity in Spinach (Spinacia oleracea L. var. All Green) Using Plant Growth Regulators. Journal Of Stress Physiology & Biochemistry, 9(3), 122-128. 4. Uddin, M., Juraimi, A., Ismail, M., Hossain, A., Othman, R., & Rahim, A. (2011). Effect of salinity stress on nutrient uptake and chlorophyll content of tropical turfgrass species. Australian Journal Of Crop Science, 5(6), 620-629. 5. Uddin, M., Juraimi, A., Ismail, M., Othman, R., & Rahim, A. (2011). Relative salinity tolerance of warm season turfgrass species. Journal of Environmental Biology, 32(3), 309-312. 6. Younis, A., Riaz, A., Ikram, S., Nawaz, T., Hameed, M., Fatima, S., & Ahmad, F. (2013). Salinityinduced structural and functional changes in 3 cultivars of Alternanthera bettzickiana (Regel) G.Nicholson. Turkish Journal of Agriculture & Forestry, 37(6), 674-687. doi:10.3906/tar-1301-78