Hydropriming and Biopriming Improve Medicago truncatula Seed Germination and Upregulate DNA Repair and Antioxidant Genes

Abstract

Seed germination is a critical parameter for the successful development of sustainable agricultural practices. While seed germination is impaired by environmental constraints emerging from the climate change scenario, several types of simple procedures, known as priming, can be used to enhance it. Seed priming is defined as the process of regulating seed germination by managing a series of parameters during the initial stages of germination. Hydropriming is a highly accessible and economic technique that involves soaking of seeds in water followed by drying. Biopriming refers to the inoculation of seeds with beneficial microorganism. The present study aims to investigate whether hydropriming and biopriming could enhance seed germination. Thereby, the germination of Medicago truncatula seeds exposed to hydropriming and/or Bacillus spp. isolates was monitored for two-weeks. The seeds were sown in trays containing two types of in situ agricultural soils collected from Northern India (Karsara, Varanasi). This region is believed to be contaminated by solid waste from a nearby power plant. Phenotypic parameters had been monitored and compared to find the most appropriate combination of treatments. Additionally, qRT-PCR was used to evaluate the expression levels of specific genes used as molecular indicators of seed quality. The results show that, while hydropriming significantly enhanced seed germination percentage, biopriming resulted in improved seedling development, represented by increased biomass rather than seedling length. At a molecular level, this is reflected by the upregulation of genes involved in DNA damage repair and antioxidant defence. In conclusion, hydropriming and biopriming are efficient to improve seed germination and seedling establishment in soils collected from damaged sites of Northern India; this is reflected by morphological parameters and molecular hallmarks of seed quality.

Keywords: Bacillus spp.; Medicago truncatula; abiotic stress; biopriming; hydropriming; qRT-PCR; seed germination.

Figure 1

Experimental design. (a) Hydropriming treatment of Medicago truncatula seeds. (b) Germination tray; each row (R1–R5) is considered as a replicate of 21 seeds each. (c) Treatment labels.

Figure 2

Germination of primed and non-primed Medicago truncatula seeds grown on two types of agricultural soils (Soil_A, TDS 82.9; Soil_B, TDS 103) collected from Northern India (Karsara, Varanasi). (a) Germination percentage (%). (b) The time required for 50% of seeds to germinate (T₅₀). Data are represented as means ± SD of five replicates. Significant differences are shown with lowercase letters and the P-values are included in the upper panel of each graphic (P, priming; S, soil, PxS, interaction between priming and soil). HP0, non-primed control; HP2, 2 h hydropriming; HP4, 4 h hydropriming; BP1, Bacillus strain isolated from mustard rhizosphere; BP2, Bacillus strain isolated from and linseed rhizosphere; BP1HP4, 4 h of hydropriming and BP1 strain.

Figure 3

Medicago truncatula seedling growth on two types of agricultural soils (Soil_A, TDS 82.9; Soil_B, TDS 103) collected from Northern India (Karsara, Varanasi). (a) Seedling length (mm). (b) Measurement of fresh weight (FW, g). (c) Measurement of dry weight (DW, g). Data are represented as means ± SD of five replicates collected at 14-days after sowing. Significant differences are shown with lowercase letters and the P-values are included in the upper panel of each graphic (P, priming; S, soil, PxS, interaction between priming and soil). HP0, non-primed control; HP2, 2 h hydropriming; HP4, 4 h hydropriming; BP1, Bacillus strain isolated from mustard rhizosphere; BP2, Bacillus strain isolated from and linseed rhizosphere; BP1HP4, 4 h of hydropriming and BP1 strain.

Figure 4

Gene expression patterns in Medicago truncatula seeds and seedlings grown from primed and non-primed seeds on two types of agricultural soils (Soil_A, TDS 82.9; Soil_B, TDS 103) collected from Northern India (Karsara, Varanasi). (a) Relative expression of OGG1, FPG, MT2, APX, and SOD genes in dry seeds (DS) and seeds treated with hydropriming for 2 h (HP2) and 4 h (HP4). Asterisks ‘*’ show statistical significance (p < 0.05) compared to dry seeds (DS). (b) Relative expression of OGG1 gene in 14-days-old seedlings. (c) Relative expression of FPG gene in 14-days-old seedlings. (d) Relative expression of MT2 gene in 14-days-old seedlings. (e) Relative expression of APX gene in 14-days-old seedlings. (f) Relative expression of SOD gene in 14-days-old seedlings. Data are represented as means ± SD of five replicates. Significant differences are shown with lowercase letters and the P-values are included in the upper panel of each graphic (P, priming; S, soil, PxS, interaction between priming and soil). HP0, non-primed control; HP2, 2 h hydropriming; HP4, 4 h hydropriming; BP1, Bacillus strain isolated from mustard rhizosphere; BP2, Bacillus strain isolated from and linseed rhizosphere; BP1HP4, 4 h of hydropriming and BP1 strain.

Figure 5

Principal component analysis (PCA). (a) Loading plot explaining the distribution of samples focusing on priming treatments. (b) Loading plot explaining the distribution of samples focusing on soil type. (c) Loading plot explaining the contribution of each measured variable (germination, T50, seedling length, FW, DW, SOD, APX, OGG1, MT, FPG). HP0, non-primed control; HP2, 2h hydropriming; HP4, 4 h hydropriming; BP1, Bacillus strain isolated from mustard rhizosphere; BP2, Bacillus strain isolated from and linseed rhizosphere; BP1HP4, 4 h of hydropriming and BP1 strain.