{ 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

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

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

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

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

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

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

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 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 NaCl mean

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 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 NaCl mean

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 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 NaCl mean

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 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 NaCl mean

SA Mean NaCl concentrations g\l SA * conc. mg\l 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 NaCl mean

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 NaCl mean

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 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 NaCl mean

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 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 NaCl mean

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 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 NaCl mean

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 NaCl mean Phe Mean NaCl concentrations g\l Phe * conc. mg\l NaCl mean

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 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 NaCl mean

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 NaCl mean

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 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 NaCl mean

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 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 NaCl mean

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 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 NaCl mean

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 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 NaCl mean

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 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 NaCl mean

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 NaCl mean

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

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