Molecular Soil Biology 2025, Vol.16, No.4, 188-198 http://bioscipublisher.com/index.php/msb 1 92 3.2.3 Inoculation methods The inoculation method of rhizobia directly affects its colonization efficiency and growth promoting effect. Common inoculation methods include seed coating, root soaking in seedbed and soil perfusion. The seed coating method is simple and suitable for large-scale field application; The seedbed root soaking method is suitable for greenhouse or plot experiments, which can ensure that the root system is in full contact with the bacterial solution (Alam et al., 2015). The inoculum dose and bacterial solution concentration should be reasonably set according to the characteristics of the strain and the test scale, so as to ensure that each soybean plant can obtain enough effective bacteria. In order to improve the effect of inoculation, some studies also adopted the joint inoculation strategy, using rhizobia, growth promoting bacteria, phosphorus solubilizing bacteria and other cooperative applications to improve the nutrient absorption and stress resistance of Soybean (Shome et al., 2022; Zhang et al., 2023). After inoculation, the formation of root nodules and plant growth should be closely monitored, and the management measures should be adjusted in time. 3.3 Analytical techniques 3.3.1 Nodulation measurement Roots are washed to remove soil, nodules are counted, and their fresh and dry weights are recorded (Maier and Brill, 1978; Alam et al., 2015). Nodule size, shape, and distribution are also noted. Microscopy is sometimes used to study colonization. 3.3.2 Acetylene reduction assay (ARA) The acetylene reduction assay (ARA) is used to measure nitrogenase activity. In this method, acetylene is added to plant or soil samples, and the amount of ethylene formed is determined with a gas chromatograph (Maier and Brill, 1978; Alam et al., 2015; Hu et al., 2023). 3.3.3 Biomass and yield At harvest, shoot and root biomass are recorded. In field trials, pod number, seed number, 100-seed weight, and total yield are measured (Maier and Brill, 1978; Alam et al., 2015). 3.3.4 Molecular confirmation PCR, qPCR, and RT-PCR are used to confirm gene presence and expression (Igiehon et al., 2019). Protein and enzyme assays such as Western blot or ELISA are also used. In some cases, genome or transcriptome sequencing is carried out. 4 Results 4.1 Genetic modification and molecular confirmation Selected rhizobium strains were engineered by adding or enhancing genes for nitrogenase (nif), nodulation regulation (nod), and stress resistance (exoX, htrA). CRISPR/Cas editing was used to change specific genes, and plasmid transfer was applied to introduce new sequences. The nif and nod genes were inserted or increased in activity, while exoX and htrA were modified to improve survival under heat, drought, or other stress. The modified strains stayed genetically stable during lab culture, with no sign of losing genes or gaining mutations. PCR tests gave clear, strain-specific bands for all target genes, confirming that the DNA was successfully integrated. Sanger sequencing showed the inserted sequences were identical to the design, with no unexpected changes (Igiehon et al., 2019). Gene expression measured by qPCR indicated that the engineered strains had higher levels of nif and nod transcripts than the controls. The stress-resistance genes exoX and htrA were also expressed at higher levels in several modified strains. 4.2 Nodulation performance Greenhouse and field trials compared nodulation between engineered strains and control strains (wild type or
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