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Introduction: Tissue culture is an effective technique for mass propagation of walnut that has many advantages. Plants were obtainedby in vitro techniques in comparison with in vivotechniques areableto producet fruitearlier. However one of the major problems in walnut micropropagation is the difficulty of rooting. Auxin protection against auxin-oxidase system can make a major contribution to rooting. Among all the compounds that can play the synergistic role with auxin, they will probably have the ability of auxin protection against enzymes. In this experiment, the effect of lignosulfunate on rooting of micropropagated walnut was investigated for the first time. Materials and Methods: In this experiment, Hartley cultivar of walnut was used. At first, explants were washed under running water for 1 hour then explants were placed in 70% alcohol for 1 minute and after that in 10% bleach for 10 minutes. After sterilization, under laminar air flow hood, explants were washed three times with distilled water and werecultured on Driverand Kuniyuki, 1984(DKW) medium supplemented with 2.2 g l-1phytagel, 2 mg l-1 BA, 0.01 mg l-1 IBA and 30 gl-1 sucrose (establishment stage). In multiplication stage, plantlets were subcultured every 25 days. All of the plantlets were placed in jars and were kept inside a growth chamber in photoperiod of 16 hours of light. All the multiplicated shoots were used as explants for the trails. Twodifferenttestswere usedto induceroot in explants. At the first trial, explants were transferred to induction medium containing IBA (3, 5, 7 and 10 mg l-1) and treatmentswere placedin thedark for 3, 5and7 days. Treatments related to theconcentrations of5and7 mg l-1IBAand7 daysof darknesshadthe highestpercentage ofrooting. In the next experiment, thecombination ofthree levels oflignosulfunate (1, 2 and 3 g l-1), and two concentration of 5and7mg l-1IBAwere used. Treatmentswere placedindarknessfor 7 days. After root induction,shootlets were transferred to root development medium. Rootdevelopmentmedium includesa quarteroftheDKWand vermiculite. Results and Discussion:The aim of the first trial was to determine the concentration of IBA which produced the highest percentage of rooting. Among all the auxins, it was shown in other experiments that IBA has the best results in rooting of walnut. Due to this, we chose IBA as root induction hormone. With increasing of IBA concentrations and the induction period, rooting increased. Because the higher amount of exogenous auxin will induce the higher amount of endogenous auxin (IAAsp). However, rooting increased to a certain level and then began to decrease. With increasing concentrations above 10 mgl-1 IBA rooting reduced and formation of callus in the shoot end increased which is not good for rooting because callus would not let the cells form roots. It seems that accumulation of IAAsp induces self-productive cells in root area to grow and duplicate abnormally and maybe root formation stops because of this accumulation and also because of the inadequacy of the IAAsp to transfer to neighboring cells. Also with increasing concentration, defoliation and wilting happens. A lower concentration of IBA (about3 mgl-1) caused loss of rooting. The highest percentage of rooting for the first treatment with IBA was with 5 mgl-1 IBA and 7 days of darkness and 7 mgl-1 IBA and 7 days of darkness. The treatments were placed in darkness due to degradation of auxin under light condition. The induction time was related to auxin concentration. If the auxin concentration is less, the exposure time in the dark will be more. Root induction in the dark had better results than induction in light. The capacity of rooting in walnut is related to the amount of endogenous (IAAsp) and exogenous auxin. The amount of endogenous auxin is completely related to the cultivar and thatis why some cultivars respond really well to the amount of exogenous hormones in rooting stage. Exogenous auxin induces the production of endogenous auxin (IAAsp). These two concentrations were chosen for next treatment with lignosulfunate. Rootingratedecreaseswith increasinglignosulfunate. However, the highest root induction among all the treatments wasachieved on medium containing 1 g l-1lignosulfunate. The reason of transferring all explants after root induction to root development medium was changing the hormone and salts concentrations. At this stage, the ¼ DKW was used as a medium. This is due to the reduction of salts, root induction and rooting accelerate. Conclusions: In this study, the effect of lignosulfunate (auxin synergist) on rooting stage of Hartley cultivar of walnut was investigated. For this goal, two trials were done. The first trial was to determine the best concentration of IBA for rooting. Two concentrations were chosen and another trial was the effect of the combination of lignosulfunate with IBA on rooting. For the first time in this study, we showed that lignosulfunatecan improve rooting of walnut.