Utilization of Plant Bioassay System to Identify the Biological ‎Function of Various Auxin Hormones

To optimize the bioassay system of auxin hormones by identifying their effect on the growth of Arabidopsis plants under in vitro conditions, Arabidopsis seeds were germinated vertically in ½ MS medium. After 5 days, seedlings with the same root growth were transferred to ½MS medium culture containing IAA, IBA, NAA, and 2,4-D hormones with concentrations of 0, 0.01, 0.03, and 0.05 mg/ L. After 10 days, plant growth characteristics were measured using image analysis plus the fresh and dry weight of seedlings in each treatment. The different hormones had different effects on the growth. Increasing the concentration of IAA, IBA, and NAA hormones caused the reduction of the rootlets’ induction. The use of different concentrations of 2,4-D hormone prevented the growth of roots. Higher concentrations resulted in the production of callus in the roots. The concentration of 0.01 mg/L of the examined auxin hormones mostly had the highest rate of leaf formation and biomass production. This bioassay method can distinguish the effects of different auxin hormones on different aspects of plant growth. The results can help in selecting the required auxin hormone in various activities and make it possible to identify unknown auxin compounds. IntroductionPlant hormones perform different biological functions in different tissues according to the growth stage or in response to environmental conditions. Evidence shows that the response of the plant to different types of auxin is different from each other and the activity of each of these hormones causes the activation of a range of different physiological processes in the plant. On the other hand, all lateral root growth stages are dependent on the amount of intracellular auxin hormone accumulation. Despite the similarity, these hormones are functionally different due to differences in structure which affect their signal transmission and downstream physiological pathways. Therefore, it is necessary to optimize a bioassay system to identify and differentiate the auxin hormones based on their biological effects, so we tried measuring the performance of different concentrations of auxin hormones. Materials and MethodsThe effects of different levels of auxin on Arabidopsis thaliana (Columbia cultivars) seedlings were investigated and ½MS medium culture containing 1% sucrose and 0.75% agar. The pH of the medium culture was set at 5.8 ± 0.1 and autoclaved at 121°C for 20 minutes. The seeds were disinfected with 1% sodium hypochlorite and sterile distilled water for 10 minutes and were cultured in ½MS without hormones. The petri dishes were placed vertically in the growth room for 5 days. The seedlings with 1 cm roots length were selected and in the 4 hormonal treatments, medium cultures were sub-cultured including IBA, 2,4-D, IAA, and NAA in three concentrations of 0.01, 0.03, and 0.05 mg/L, as well as the control treatment without hormones with three replications. Hormones were added to the sterile medium culture after passing through a 2 μm filter. After 10 days, the number of leaves, root length, the number of lateral roots, and fresh weight were measured. The growth level of the shoot and root and the level of greenness of seedling leaves were performed using image analysis and ImageJ software, dry weight of the seedlings was measured after exposure to 72°C for 48 hours. All treatments were analyzed using one-way ANOVA. The mean comparison of treatments was performed by LSD test with a minimum significant difference at a 5% probability level using JMP software (version 8.0). Graphs were also drawn using Excel software.  Results and DiscussionThe presence of auxin in different concentrations inhibits the root length growth and the control seedlings showed the highest root length growth. Seedlings grown at the high concentrations of NAA and in the presence of 2,4-D had the lowest root length with an average of one cm. Seedlings grown in NAA hormone treatment with a concentration of 0.01 mg/L had the highest rootlets number. However, the roots formed in the presence of 2,4-D and high concentrations of NAA produced callus and did not grow normally. The root area in the presence of IBA was more than IAA. The number of seedling leaves grown at 0.01 mg/L concentration of hormones was equal to or more than the higher concentrations of these hormones. The lowest number of leaves belonged to the seedlings grown in 0.03 mg/L NAA and 0.05 mg/L 2,4-D treatments. IAA concentration had a direct relationship with leaf area and with the increase in the concentration of this hormone the leaf area increased, but the use of IBA had the opposite effect on leaf area and decreased with increasing its concentration. The highest leaf greenness was observed in three concentrations of 2,4-D and then in the control. The use of other auxin in the medium culture inhibited the production of chlorophyll in the cells. After the use of auxin in Arabidopsis plants, the fresh and dry weight of the seedlings grown in the presence of the concentration of 0.01 mg/L of NAA had the highest fresh and dry weight. ConclusionThe effect of auxin hormones on different aspects of plant growth was different from each other. IAA increased the number of rootlets and low concentration and formed many rootlets on the main root. High concentrations led to an increase in the number of lateral roots, but the root length significantly decreased. Also, 2,4-D stimulated callus production in the roots. The presence of IBA in the medium culture decreased the average root length. The difference in the morphological effects of different auxin hormones can be considered a road map that will provide the possibility of identifying the auxin hormone in unknown extracts in future research.