1. { نَرْفَعُ دَرَجَاتٍ مِّن نَّشَاء وَفَوْقَ كُلِّ ذِي عِلْمٍ عَلِيمٌ } صَدَقَ الله العَليّ العَظِيم سورة يوسف الآية : 76 { نَرْفَعُ دَرَجَاتٍ مِّن نَّشَاء وَفَوْقَ كُلِّ ذِي عِلْمٍ عَلِيمٌ } صَدَقَ الله العَليّ العَظِيم سورة يوسف الآية : 76

2. The interaction in vitro study among salt stress with some Abiotic elicitors on active substances in Trigonella foenum graecum callus properties

4. The salinity The plants in outer surroundings expose to various biological and non- biological stress which showed adverse abnormal effects on plants. The salinity is the one of great problem limited the plant growth, development and production in aired semidried region in the world, the salinity has Primary effects in plant cell such as decrease the water potential, abnormal in ionic balance and Secondary effects, the Secondary effects include cell elongation and division, protein oxidation, enzymes inhibition and reactive oxygen Species (ROS production and the salt stress cause nuclear acid destroy DNA. The best solvent and cheap it is plants by mean improve its salt tolerant through plant tissue culture by enzymatic and non-enzymatic which are include Reactive Oxygen Species ( ROS) scavenge,gene activation and also signal transduction

5. Abiotic elicitors Plant secondary metabolites can be defined as the compounds that play an important role in the interaction of the plant with its environment such as osmotic regulation, therefore utilize elicitors to improve the synthesis of secondary metabolites in medicinal plants such of these elicitors are salicylic acid polyamines and phenylalanine Trigonella foenum-graecum (Fabaceae) was used to Treatment diabetes type I and II, inhibiting tumors and prevent gland, bladder, breast and stomach cancer because have many important secondary compounds especially alkaloids like trigonelline

7. For these reasons the study was Aimed the following : 1.Callus formation of Trigonella foenum-graecum (Fenugreek) by use the auxin NNA and cytokinin BA. 2. Choose the best accumulation media by use the elicitors are salicylic acid, polyamines and phenylalanine. 3.Which any elicitor may be give the best callus growth 4.Which any elicitor can be induction the callus to be tolerant salt stress. Use HPLC technique to determination trigonelline

9. First stage Sterilization of explant Seed germination Hypocotyl culturing in MS Subculture in accumulation media NaCl only Phe + NaCl Put + NaCl SA + NaCl Callus formation

10. Second stage Accumulation media Parameter determinations of accumulation medium culture Trigonelline by HPLC Primary secondary assay Fresh and Dry weight Ions assay Sodium 1- Protein 2-Charbohydrate SOD enzyme Secondary metabolism assay Calcium Phosphor Potassium Chloride 3-Proline Enzymatic assay

12. Table (1) showed the effect of salinity medium and salicylic acid concentrations on callus formation of Trigonella foenum-graecum SA Mean NaCl concentrations g\l SA* conc. mg\l 86420 67.803045809490 0 83.00576696 100 10 79.606379829480 20 87.80 7188100 80 30 55.20 4045507170 40 52.2064.6081.6091.0084.00 NaCl mean Table (2) showed the effect of salinity medium and concentration of salicylic acid on content of fresh weight of Trigonella foenum-graecum callus SA Mean NaCl concentrations g\l SA * conc. mg\l 86420 1.940.951.721.822.702.50 0 2.191.521.792.013.002.62 10 2.642.152.312.453.432.87 20 2.882.002.632.813.663.30 30 2.011.411.942.112.232.38 40 1.612.082.243.002.73 NaCl mean

13. Table (3): the effect of salinity medium and concentration of salicylic acid on content dry weight (g) of Trigonella foenum-graecum callus. SA Mean NaCl concentrations g\l SA * conc. mg\l 86420 0.1230.0910.1150.1290.1280.150 0 0.1570.1030.1410.1580.2100.172 10 0.1740.1140.1530.1760.2340.193 20 0.1930.1310.1690.2050.2520.210 30 0.1250.0820.1250.1350.1420.140 40 0.1040.1410.1610.1930.173 NaCl mean Table (4 ): the effect of salinity medium And concentration of salicylic acid in content of proline mg. g -1 fresh weight. Trigonella foenum-graecum callus SA Mean NaCl concentrations g\l SA * conc. mg\l 86420 2.543.713.002.412.011.56 0 2.303.152.812.331.861.37 10 2.092.922.512.251.731.02 20 1.742.672.171.901.210.77 30 2.302.642.402.252.202.00 40 3.022.582.231.801.34 NaCl mean

14. Table (5): the effect of salinity medium And concentrations salicylic acid in content of Trigonelline mg. g -1 dry weight Trigonella foenum-graecum callus SA Mean NaCl concentrations g\l SA * conc. mg\l 86420 0.2340.3960.2300.2200.2140.111 0 0.2290.3900.2380.2110.1870.121 10 0.2300.3580.2550.2020.1930.143 20 0.2490.3320.2980.2000.2290.187 30 0.2470.3090.2360.2230.2350.231 40 0.3570.2510.2110.2120.159 NaCl mean Table (6): the effect of salinity medium and salicylic acid concentrations on content of phosphorus mg.g -1 dry weight of Trigonella foenum-graecum callus. SA Mean NaCl concentrations g\l SA * conc. mg\l 86420 3.591.933.194.024.524.27 0 3.852.213.714.084.764.47 10 4.042.843.804.244.814.50 20 4.192.963.894.364.964.77 30 3.531.913.254.144.204.16 40 2.373.574.174.654.43 NaCl mean

15. Table (6): showed the effect of salinity medium concentrations salicylic acid on content of potassium mg.g -1 dry weight of Trigonella foenum-graecum callus SA Mean NaCl concentrations g\l SA * conc. mg\l 86420 5.6872.534.3935.5418.8357.135 0 10.0176.218.859.4715.639.925 10 10.8225.2511.1010.7516.7010.31 20 12.7189.289.4711.517.5915.75 30 9.016.867.038.2510.9411.96 40 6.038.179.1013. 9411.016 NaCl mean Table ( ): the effect of salinity medium salicylic acid concentrations in content sodium mg. g -1 dry weight of Trigonella foenum-graecum callus SA Mean NaCl concentrations g\l SA * conc. mg\l 86420 1.902.101.971.911.811.73 0 1.852.061.921.821.761.70 10 1.802.041.871.741.701.65 20 1.802.101.811.791.701.61 30 1.871.971.901.891.861.71 40 2.051.891.831.771.68 NaCl mean

16. SA Mean NaCl concentrations g\l SA * conc. mg\l 86420 3.0681.2052.2292.9014.8814.124 0 5.5093.0144.6095.2038.8805.838 10 6.1512.5735.9356.1789.8236.248 20 7.2924.6165.2326.42410.3479.843 30 5.2563.5783.9734.2487.496.99 40 2.9974.3964.9918.2846.609 NaCl mean Table (7): the effect of salinity medium and SA in content of Ca ++ mg. g -1 dry weight in Trigonella foenum-graecum callus of calcium. SA Mean NaCl concentrations g\l SA * conc. mg\l 86420 2.681.512.022.683.174.02 0 3.391.872.433.604.504.57 10 3.802.122.924.004.935.04 20 4.172.503.304.705.125.25 30 2.661.702.232.463.413.50 40 1.942.583.494.234.48 NaCl mean

17. Table (8): the effect of salinity medium and salicylic acid concentration on content of Chloride mg.g -1 dry weight of Trigonella foenum-graecum callus SA Mean NaCl concentrations g\l SA * conc. mg\l 86420 12.5320.4115.0713.318.545.325 0 11.6118.3813.4711.178.276.76 10 10.0714.5012.008.418.127.31 20 8.7411.2110.058.147.267.06 30 10.7515.0011.299.319.119.05 40 15.9012.3810.078.267.10 NaCl mean

18. Table (17): the effect of salinity medium And phenylalanine in callus formation of Trigonella foenum-graecum callus. Phe Mean NaCl concentrations g\l Phe * conc. mg\l 86420 73.404558809490 0 80.006066859792 10 85.40677090100 20 91.20788593100 30 89.80738096100 40 64.6071.8088.8098.2096.40 NaCl mean Table (18): the effect of salinity medium And phenylalanine in content of fresh weight Trigonella foenum-graecum callus Phe Mean NaCl concentrations g\l Phe * conc. mg\l 86420 1.940.951.721.822.702.50 0 2.161.011.882.113.202.62 10 2.371.151.932.633.322.81 20 2.581.242.082.813.812.98 30 2.851.622.253.194.023.15 40 1.191.972.513.412.81 NaCl mean

19. Table (19): the effect of salinity medium And phenylalanine in content of dry weight Trigonella foenum-graecum callus. Phe Mean NaCl concentrations g\l Phe * conc. mg\l 86420 0.117 0.0910.1000.1150.1290.150 0 0.180 0.1510.1600.1930.2140.183 10 0.219 0.1810.1990.2 400.2500.236 20 0.275 0.2110.2600. 4020.3280.274 30 0. 403 0.2200.2910.3200.3690.315 40 0.1710.2020.2320.2580.232 NaCl mean Table (20): the effect of salinity medium And phenylalanine in content of proline mg. g -1 fresh weight in Trigonella foenum-graecum callus. Phe Mean NaCl concentrations g\l Phe * conc. mg\l 86420 2.543.713.002.412.011.56 0 2.103.222.712.001. 401.25 10 1.632.282.171.251.231.20 20 1.171.371.181.171.001.14 30 1.101.401.161.140.90 40 2.402.041.591.291.21 NaCl mean

20. Table (21): the effect of salinity medium And phenylalanine in content of phosphorous ions mg. g -1 dry weight in Trigonella foenum-graecum callus Phe Mean NaCl concentrations g\l Phe * conc. mg\l 86420 3.59 1.933.194.024.524.27 0 4.04 2.414.004.414.754.62 10 4.31 2.714.454.594.944.84 20 4.54 2.994.614.705.444.97 30 4.69 3.254.694.815.595.09 40 2.664.194.515.054.76 NaCl mean Table (22): the effect of salinity medium And phenylalanine in content of potassium K + mg. g -1 dry weight in Trigonella foenum-graecum callus Phe Mean NaCl concentrations g\l Phe * conc. mg\l 86420 5.69 2.534.3935.5418.8357.135 0 13.17 8.5210.7712.0317.8716.68 10 15.19 9.4312.6616.6819.0418.13 20 16.71 11.1013.9517.4020.4420.66 30 18.55 13.0715.7118.0023.9522.02 40 8.9311.5013.9318.0316.93 NaCl mean

21. Table (22): the effect of salinity medium And phenylalanine in content of Sodium Na + mg. g -1 dry weight in Trigonella foenum-graecum callus PheMean NaCl concentrations g\l Phe * conc. mg\l 86420 2.101.971.911.811.732.10 0 2.031.941.851.761.692.03 10 1.901.871.801.701.651.90 20 1.831.771.731.621.611.83 30 1.751.671.601.551.431.75 40 1.921.841.781.691.621.92 NaCl mean Phe Mean NaCl concentrations g\l Phe * conc. mg\l 86420 3.068 1.2052.2292.9014.8814.124 0 7.254 4.1975.5516.50210.1539.869 10 8.631 4.9636.7379.26611.20010.987 20 9.906 6.0657.88110.05712.61712.912 30 11.600 7.4689.40711.2514.47715.398 40 4.7806.3617.99510.66610.658 NaCl mean

22. Table (23): the effect of salinity medium And phenylalanine in content of calcium Ca ++ mg. g -1 dry weight Trigonella foenum-graecum callus Phe Mean NaCl concentrations g\l Phe * conc. mg\l 86420 2.68 1.512.022.683.174.02 0 3.82 2.113.464.004.714.80 10 4.23 3.003.714.494.954.98 20 4.72 3.504.105.005.50 30 5.43 3.814.905.766.506.20 40 2.793.644.394.975.10 NaCl mean Table (24): the effect of salinity medium and phenylalanine in content of chloride ions mg. g -1 dry weight Trigonella foenum-graecum callus Phe Mean NaCl concentrations g\l Phe * conc. mg\l 86420 12.53 20.4115.0713.318.545.325 0 9.47 18.649.277.826.754.86 10 7.76 13.508.247.005.644.42 20 6.95 13.506.936.114.373.82 30 5.64 10.105.755.294.053.00 40 15.239.057.915.874.29 NaCl mean

23. Table (24): the effect of salinity medium And phenylalanine in content of Trigonella foenum-graecum callus of Trigonelline Phe Mean NaCl concentrations g\l Phe * conc. mg\l 86420 0.2340.3960.2300.2200.2140.111 0 0.2460.3500.2330.2170.2100.218 10 0.2350.3000.2190.2090.2250.223 20 0.2010.2500.2110.1960.2200.128 30 0.1830.2370.2020.1820.2020.090 40 0.3070.2190.2050.2140.154 NaCl mean

24. Table (9): the effect of salinity medium and putrescine concentrations in content of callus formation percentage in Trigonella foenum-graecum callus Put Mean NaCl concentrations g\l Put * conc. mg\l 86420 67.80 3045809490 0 83.80 8068849592 10 87.00 8075889795 20 88.80 638794100 30 71.20 4870808870 40 60.2069.2085.2094.8089.40 NaCl mean Table (10 the effect of salinity medium and putrescine concentrations in content of fresh weight(g) in Trigonella foenum-graecum callus. Put Mean NaCl concentrations g\l Put * conc. mg\l 86420 1.94 0.951.721.822.702.50 0 2.20 1.361.872.412.832.52 10 2.41 1.581.982.772.962.75 20 2.65 1.782.153.013.432.90 30 1.86 1.101.492.052.292.35 40 1.351.842.412.842.60 NaCl mean

25. Table (11): the effect of salinity medium and putrescine concentrations in content of dry weight (g) in Trigonella foenum-graecum callus Put Mean NaCl concentrations g\l Put * conc. mg\l 86420 0.123 0.0910.1150.1290.1280.150 0 0.141 0.1020.1270.1400.1790.159 10 0.159 0.1150.1430.1670.1950.173 20 0.193 0.1300.1980.2210.2270.191 30 0.133 0.1000.1210.1310.1570.155 40 0.1080.1410.1580.1770.166 NaCl mean Table (12): the effect of salinity medium and putrescine concentrations in content of proline mg. g -1 fresh weight. in Trigonella foenum-graecum callus Put Mean NaCl concentrations g\l Put * conc. mg\l 86420 2.54 3.713.002.412.011.56 0 2.36 3.422.952.211.901.34 10 2.24 3.052.832.111.731.49 20 2.11 2.902.561.841.651.60 30 2.66 3.032.742.602.532.42 40 3.222.822.231.961.68 NaCl mean

26. Table ( ): the effect of salinity medium and putrescine Conc. in culture medium in content of trigonelline mg. g -1 dry weight in Trigonella foenum-graecum callus. Put Mean NaCl concentrations g\l Put * conc. mg\l 86420 0.234 0.3960.2300.2200.2140.111 0 0.285 0.3110.2730.2910.2710.279 10 0.299 0.2700.3380.3040.2830.300 20 0.322 0.2140.3920.3190.2990.386 30 0.404 0.1830.4310.4630.4430.500 40 0.2750.3330.3190.3020.315 NaCl mean. Table (13 the effect of salinity medium and putrescine concentrations in content of phosphorus ions mg. g -1 dry weight in Trigonella foenum-graecum callus Put Mean NaCl concentrations g\l Put * conc. mg\l 86420 3.59 1.933.194.024.524.27 0 3.90 2.423.734.164.634.58 10 4.09 2.683.904.344.874.67 20 4.26 2.904.054.614.934.81 30 3.63 1.803.234.294.444.40 40 2.353.624.284.684.55 NaCl mean

27. Table (14): the effect of salinity medium and putrescine concentrations in content of potassium ions mg. g -1 dry weight in Trigonella foenum-graecum callus. Put Mean NaCl concentrations g\l Put * conc. mg\l 86420 5.69 2.5314.3935.5418.8357.135 0 9.91 4.329.9111.4012.8311.09 10 11.56 6.718.6313.3415.1214.00 20 14.15 6.7511.0315.9419.5317.52 30 10.44 4.006.269.1617.0115.76 40 4.868.0411.0814.6713.10 NaCl mean the effect of salinity medium and putrescine concentrations in content of Sodium ions mg. g -1 dry weight in Trigonella foenum-graecum callus. Put Mean NaCl concentrations g\l Put * conc. mg\l 86420 1.90 2.101.971.911.811.73 0 1.78 2.001.881.751.661.61 10 1.70 1.881.671.681.601.58 20 1.61 1.721.661.61.561.51 30 1.55 1.701.601.551.481.43 40 1.881.771.701.621.57 NaCl mean

28. Table ( ): the effect of salinity medium and putrescine concentrations in content of ratios of potassium to sodium in Trigonella foenum-graecum callus Put Mean NaCl concentrations g\l Put * conc. mg\l 86420 3.068 1.2052.2292.9014.8814.124 0 5.287 2.163.1436.5147.7286.888 10 6.944 3.5694.9037.9409.458.860 20 8.894 3.926.6449.96212.51911.423 30 6.937 2.353.9125.90911.49311.020 40 2.6414.1666.6459.2148.463 NaCl mean Table (15 ): the effect of salinity medium and putrescine concentrations in content of calcium ions mg. g -1 dry weight in Trigonella foenum-graecum callus Put Mean NaCl concentrations g\l Put * conc. mg\l 86420 2.68 1.512.022.683.174.02 0 2.88 1.662.192.763.724.09 10 3.35 1.842.553.704.254.40 20 3.78 2.502.744.304.504.88 30 3.03 1.602.203.613.813.93 40 1.822.343.413.894.26 NaCl mean

29. Table (16 ): the effect of salinity medium and the putrescine in content in chloride ions mg. g -1 dry weight in Trigonella foenum-graecum callus Put Mean NaCl concentrations g\l Put * conc. mg\l 86420 12.53 20.4115.0713.318.545.325 0 11.01 18.5412.0012.188.014.335 10 10.07 15.1611.8911.367.824.12 20 8.72 12.0010.5010.147.043.91 30 7.72 10.019.208.156.115.11 40 15.2211.7311.037.504.56 NaCl mean

37. The high concentrations of salt NaCl inhibition growth Stimulates growth of callus at the concentration of 2 g/l of salt NaCl in nutrient medium MS Callus growth differed among the stimulators concentrations, which give the callus developed with top continue to grow well planted in the MS medium the Phenylalanine the development in down town SA and Put 4- Phenylalanine has hormonal activates characters

39. 4- Effect salicylic acid and salts stress on Ascorbic acid oxidase ، catalase (CAT) ، Phenylalanine amenolyase. 2- Study the effect of elicitors on genes expression 1-The study suggests determine the stress effect genetically by RAPD Assay, study the specific genes that responsible of salts tolerance 3-Effect elicitor on alkaloid pathways 5- Agriculture Trigonella foenum-graecum in arid and semi-arid regions 4-chemical study of the active groups (OH, NH3, and OH and NH3) of salicylic acid, peutericin and phenylalanine respectively to scavenge free radicals