The experiments were conducted under controlled conditions using plastic pots having lower diameter of 7.8 cm, upper diameter of 13.5 cm and 12 cm in height. The soil was collected from the top layer (0-20 cm) from the Botanical Garden of the university. Soil was air dried crushed and sieved through 2 mm filter autoclaved at 121 ℃ for 2 h. The soil was autoclaved to exclude soil pathogens and other microorganisms if any. The autoclaved soil was poured in pots and kept in the growth chamber. The pots were filled with 500 g uncontaminated soil and partially decayed compost (cow manure) (2:1) and was used as growing medium. The cow dung was added into the soil for better performance of earthworms. A subsample of the study soil before mixing with compost was analyzed for its physicochemical characteristics. The soil used for the experiment was sandy loam soil having pH 7.8 , EC (Electrical conductivity) (µS/cm) =184.25 , TDS (Total Dissolved Solids) (mg/kg) = 130 , N (Nitrogen) (mg/kg) = 103 , P (Phosphorus) (mg/kg) = 10.6 , K (Potassium) (mg/kg) = 0.343 , %OC = 0.894, Cd (mg/kg) = ND (not detected by AAS).The Cd treatment was given by using anhydrous CdCl₂ (Minimum assay: 95.0%) procured from Hi-Media laboratories. The CdCl₂ anhydrous was added to the soil to make different concentrations of Cd 0.50 mM, 0.75 mM, 1.00 mM, and 1.25 mM (i.e. 56 mg/Kg , 84 mg/Kg , 112 mg/Kg and 140 mg/Kg respectively). The various treatments given are as shown below:(1)C0 (Control): (Cadmium absence);(2)C1: (0.5 mM Cd);(3)C2: (0.75 mM Cd);(4)C3: (1.00 mM Cd);(5)C4: (1.25 mM Cd).Each Cd treatment was given in soils without as well as with earthworms (WTE = without, WE = with earthworms). Earthworms (3 earthworms per pot) were inoculated after seven days of Cd treatment and incubated for 7 d in soil with earthworms. The seeds after surface sterilization were sown in soil containing different concentration of Cd and earthworms in plastic pots. These pots were kept in seed germinator under controlled conditions i.e. 25 ℃ temperature and 16:8 h dark: light photoperiod (1700 lx) for 15 d. Seedlings were harvested after 15 d followed by washing with distilled water. The growth and biochemical analysis was done on these seedlings.

Increased Cd uptake in plants in presence of earthworms enhances the total antioxidative capacity, metal chelating compounds and content of other antioxidants in plants grown under metal polluted soils. Earthworms can improve plant growth by improving nutrient availability to plants through their vermicasting activity. Their role in modifying soil pH and increasing metal phytoavailability made their use ideal in phytoremediation of polluted soils. Increased uptake and accumulation of Cd in plants activates the antioxidative system of plants takes place by addition of earthworms to soil.

The gene expression for the key enzymes involved in organic acid metabolism was studied to understand the role of earthworms in organic acid metabolism in plants under Cd metal stress. It was observed that in comparison to control (C0) seedlings the expression of CS, SUCLG1, SDH and FH was enhanced 1.72, 1.58, 1.65 and 1.88 folds in seedlings given C4 treatment with 1.25 mM dose of Cd respectively . However, after supplementation of earthworms to Cd treated soils given C4 treatment resulted in further enhancement in expression of CS (2.53 fold), SUCLG1 (2.35 fold), SDH (2.13 fold) and FH (3.06 fold) .