Melatonin-primed seeds improve germination and enhance root system architecture in wheat (Triticum aestivum L.)

Introduction

Depending on the plant species and the experimental setup, melatonin has been documented to have both stimulatory and inhibitory effects on seed germination. Several studies have demonstrated that exogenous application of melatonin enhances seed germination. For instance, Liang et al. (2015) [7] found that melatonin treatment increased the germination rate, germination potential and seedling vigour of tomato seeds. On the other hand, melatonin can also exhibit inhibitory effects on seed germination. Exogenous melatonin application in lettuce seeds delayed germination and reduced germination percentage. These inhibitory effects may be attributed to the inhibitory action of melatonin on gibberellin synthesis or its interaction with other hormones involved in germination regulation. Melatonin controls primary growth, lateral root creation, and root hair elongation, all of which are essential for the development of root architecture. Exogenous melatonin treatment has been reported to promote root growth in various plant species. For example, Zhang et al. (2014) [14] demonstrated that melatonin treatment enhanced root length, surface area and volume in cucumber seedlings. Melatonin stimulates root growth by increasing cell division and elongation, enhancing auxin signalling and modulating the balance of reactive oxygen species (ROS) in roots. Melatonin also regulates lateral root formation. Zhang et al. (2016) [15] revealed that melatonin stimulates lateral root development in Arabidopsis thaliana by influencing auxin transport and distribution. Additionaly, melatonin can influence root hair elongation, a crucial factor for nutrient and water uptake. In maize, exogenous melatonin treatment increased root hair length and density (Wang et al., 2019) [13] . This effect is mediated by melatonin's ability to enhance auxin signalling and modulate the expression of genes involved in root hair elongation. To investigate the impact of different melatonin concentrations, we designed this study to first determine the optimal MT levels for improving wheat seed germination, seedling growth, germination indices, and overall root architecture. Materials and Methods Plant material, seed priming and growth conditions Wheat seeds (Triticum aestivum L., cv HD 2967) used in this study were acquired from the Division of Genetics at ICAR-Indian Agricultural Research Institute (IARI), New Delhi, India. Healthy seeds were selected and surface sterilized in a 1% sodium hypochlorite solution for 3 minutes. After rinsing with sterilized water, the seeds were air-dried on filter paper and treated with 1% Bavistin fungicide for 2 hours, then air-dried again. Seeds were primed by soaking them in MT solution of varying strength. Melatonin (Molecular weight: 232.28) was purchased from Sigma Aldrich with >99% purity. A suitable volume of anhydrous ethanol was used to dissolve 75 mg MT to create a stock solution. Next, deionized water was added to bring the total volume of the solution to 1 ml. Additional dilutions were prepared to obtain the melatonin concentrations: MT1 (15 mgl⁻¹), MT2 (30 mgl⁻¹), MT3 (50 mgl⁻¹), MT4 (75 mgl⁻¹), and NP (No Priming, just water). The seeds were then soaked in these solutions for 24 hours at 22 °C ± 2 °C in the dark. After this period, the seeds were thoroughly airdried at room temperature and placed in petri plates lined with moistened blotting paper for germination. Seeds were placed in pots with soilrite for additional growth in a growth chamber at 22 °C after radicle emergence began, which occurred within a day or two. Another set of seeds were germinated in germination papers and continued there for measurement of plant architecture and growth parameters.

Measurement of shoot-root, Fresh weight, Dry weight, Germination percentage

Daily measurements of the length of the roots and shoots were done according to the Association of Official Seed Analysts' (1983) manual. At the conclusion of the experiment, the final germination percentage (GP) was computed by multiplying the ratio of the total number of seeds to the number of seeds that germinated by 100. At the conclusion of each experiment, the final root and shoot lengths of the emerging seedlings were measured. Germination percentage (GP) = (Total number of normal seeds germinated/Total number of seeds sown initially) × 100. Fresh weight of samples was taken directly. For dry weight determination, plants were oven-dried at 80 °C for 1-2 days and weight measured.

Root Architecture System

After being carefully taken off germination sheets at the age of ten days, primed wheat seedlings were cleaned by running tap water over their roots and allowed to dry on blotting sheets. Following that, the seedlings were scanned at 300 dpi using a lit flatbed scanner. The scans were then analyzed using Win RHIZO software (Win RHIZO LA2400 Scanner, Regent Instruments Canada Inc.). Measurements included total root length, surface area, root volume, number of tips, number of forks, and average diameter.

Statistical Analysis:

The results are shown as means with standard error (S.E.). Analysis of variance (ANOVA) was utilized to evaluate the outcomes after Duncan's multiple range tests were employed to determine the significant difference (at p<0.05) between treatments for all morphological and physiological parameters. The ANOVA and critical difference value were computed using Microsoft Excel and SPSS 10.0 (SPSS Inc., Chicago, IL, USA).

Results and Discussion Melatonin Priming improves the growth parameters in wheat seedlings

For measurements of the root and shoot lengths, seedlings that were 10 days old were employed. The treatments differed significantly (p<0.05) when primed with various melatonin concentrations. Under MT3 and MT4 priming, the shoot length rose dramatically by 33.94% and 31.00%, respectively, while the root length increased by 98.44% and 93.25% compared to the comparable control. (Fig. 1a). As compared to controls, various melatonin doses enhanced the morphology and germination indices differently, which improved the overall physiological state of the plants. Furthermore, the germination potential increased by 7% in MT1, 10.4% in MT2, 18.7% in MT3, 17.9% in MT4 and 17.5% in MT5 respectively over the control (Fig. 1b). However, the fresh and dry biomass of germinating wheat seeds were dramatically enhanced by the optimised melatonin concentrations. The above study investigated the role of melatonin in seed germination and root architecture in plants regulating the basic plant growth processes under different melatonin concentration having MT3 and MT4 shows better result over other concentrations. These results are in line with Arnao et al. (2019) [1] and Fan et al. (2018) [10] who explained melatonin as a multi regulatory molecule with many diverse role in plants including plant growth, seed germination, root architecture, photosynthesis. Xiao et al.

(2019) [12] documented the promotional effects of melatonin at different concentration on cotton seed germination, germination potential, germination rate, fresh weight. These findings support our findings.

Conclusion

According to the results of our investigation, melatonin may have a function in the differential regulation and enhancement of germination indices and other physiological processes. The germination potential, branch length, radical length, fresh weight, and dry weight of wheat seed were all significantly improved by pre-treatment with melatonin at doses of 50 mgl-1 and 60 mgl-1. The results of this study imply that melatonin at these precise doses (50 mgl-1 and 60 mgl-1) can improve root architecture by increasing root length, root volume, and root surface area. Further investigations are needed to fully uncover the molecular mechanisms underlying theses effects and exploit the practical applications of melatonin in agriculture.

Acknowledgement

AcknowledgementsNRCPB-RPP 2012-2017-2021/02 of SB, an ICAR-NIPB inhouse initiative, served as the umbrella organization for the activity detailed below. In recognition of a research fellowship, SS thanks the PG School of the Indian Agricultural Research Institute (ICAR).

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