The seeds were surface sterilized and then soaked for two hours and sown in soil mixture having 3 parts of garden soil, 1 part of sand and 1 part of manure. The experiment was carried out in earthen pots of uniform size each containing 5 Kg of the soil mixture. Before sowing, the soil was amended with K₂CrO₄ for Cr treatments (0 µM/Kg), and Na₂SeO₄ for Se treatments (0 µM/Kg), both alone and in combinations. The concentration used for Cr was 50% inhibitory concentration (IC50), while for Se, the most stimulatory concentrations as observed from preliminary studies were used. The pots were kept in natural environmental conditions and were watered regularly. The experiment was conducted in triplicates. The harvesting of the plants was done after 30 days of sowing and stored at -20 ℃ . Some harvested plants were also dried by keeping them in hot air oven for 24 h at 80 ℃.

Se application aided in improving plant growth, reducing the oxidative damage and strengthening the antioxidative defence system in plants raised in soils with binary combinations of Cr and Se. Photosynthesis, which is one of the vital physiological processes, was positively influenced with application of Se. It helped in minimising the toxicity of Cr and enhanced the contents of pigments. The efficiency of photosynthetic machinery was further strengthened by the increase of net photosynthetic rate, transpiration rate, stomatal conductance and intercellular CO₂ concentration, and hence indicated its importance in combating stress. The study also highlighted the role of Se in enhancing the contents of secondary metabolites which play an important role in heavy metal chelation, complex formation and ROS scavenging, thereby reducing the chances of Cr to cause physiological damage.

A significant modulation in gene expression was observed in B. juncea in response to Cr and Se. The gene responsible for H₂O₂ production is respiratory burst oxidase (RBO) which showed a significant upregulation in its expression by 3.63 folds in response to Cr treatment. Se at 2 µM/Kg in combination with 300 µM/Kg Cr caused decrease by 1.62 folds in the expression of RBO gene.An increase in expression was observed SOD, CAT and GST-1 by 2.75 folds, 2.82 folds and 2.03 folds respectively in response to Cr. However, Cr treatment resulted in a reduction of relative expression of POD and GR genes by 0.54 and 0.61 folds respectively in leaves of B. juncea plants. The combined treatment of Se and Cr aided in reducing Cr toxicity by increasing the expression of genes coding all these enzymes. Maximum increase in expression in case of CAT (4.68 folds), GR (2.08 folds) and GST-1 (2.98 folds) was observed at binary combination of 4 µM/Kg Se and 300 µM/Kg Cr. For SOD, 4.25 folds increase in gene expression was observed at 6 µM/Kg Se and 300 µM/Kg Cr. The expression of POD enhanced by 1.75 folds at the concentration of 2 µM/Kg Se and 300 µM/Kg Cr. The genes coding forcholrophyllase (CHLASE) chalcone synthase (CHS) and phenylalanine ammonialyase (PAL) showed enhanced expression of 2.47 folds, 1.79 folds and 2.07 folds respectively in the plants raised in Cr spiked soils. The co-application of Se and Cr helped in increasing the expression of CHS and PAL, while aided in reducing the expression of CHLASE. The concentration of 4 µM/Kg for Se proved to be most beneficial for enhancing the gene expression of PAL by 3.92 folds, while the same concentration caused a decline in the expression of CHALSE by 1.65 folds. However, for CHS expression, 6 µM/Kg Se caused an increase by 2.52 folds. Statistical analysis by one-way ANOVA and MLR supported the observations. The values of beta-regression coefficients for Se indicated the stress alleviating effects of Se for all the genes.

The seeds of B. juncea(var. RLC 1) were sterilized with 0.01% mercuric chloride (HgCl₂), soaked in distilled water for 2h (h), and germinated in Petri dishes lined with Whatman No. 1 filter paper moistened with 3 ml solutions of sodium selenate (Na₂SeO₄) (2, 4, and 6µM) and potassium chromate (K₂CrO₄) (300µM), in unary and binary treatments. The concentrations of the elements were decided on the basis of preliminary experiments by obtaining the most stimulatory concentration of Se and 50% inhibitory concentration (IC50) of Cr. The treatment solutions were prepared in half strength Hoagland's nutrient medium and the experiment was conducted in triplicates with 16hphotoperiodand 25℃ temperature. The seedlings were harvested after 15days for the estimation of various parameters.

In conclusion, Cr negatively affects the metabolic activities of B. juncea, which were restored by Se application. Se application showed its protective effects on photosynthetic pigments and gene expression studies confirmed the spectrophotometric observations. The Scanning electron microscope (SEM) studies also clearly indicated the positive effects of Se against Cr toxicity on morphology and density of stomata, which further confirmed its imperative role in efficient gas exchange for both photosynthesis and respiration. Exogenous application of Se was also observed to enhance the lipid- and water-soluble antioxidants, which further indicated its role in alleviating oxidative damage. The positive effect of Se in maintaining the osmotic homeostasis as well as activation of thiols in Cr-stressed seedlings also showed its potential to enhance the ability of B. juncea seedlings to combat heavy metal stress effectively. The results, therefore, indicate that Se can be effectively used as a mitigating agent against Cr stress.

Treatments of Se and Cr aided in modulating the gene expression of CHLASE, PSY and CHS. CHLASE showed a significant upregulation of 366.3% in its expression in the Cr treated seedlings with respect to untreated control seedlings. The expression of PSY, however, was observed to be downregulated with Cr application by 28.6%, while CHS expression showed an upregulation by 73.13%. Se application at 4 µM in combination with 300 µM Cr downregulated its expression by 46.92% when compared to Cr-treated seedlings. On the other hand, the same concentration of Se caused an increase of PSY and CHS expression by 425.2 and 209.4%, respectively.

The seeds of B. juncea(var. RLC 1) were sterilized with 0.01% mercuric chloride (HgCl₂), soaked in distilled water for 2h (h), and germinated in Petri dishes lined with Whatman No. 1 filter paper moistened with 3 ml solutions of sodium selenate (Na₂SeO₄) (2, 4, and 6µM) and potassium chromate (K₂CrO₄) (300µM), in unary and binary treatments. The concentrations of the elements were decided on the basis of preliminary experiments by obtaining the most stimulatory concentration of Se and 50% inhibitory concentration (IC50) of Cr. The treatment solutions were prepared in half strength Hoagland's nutrient medium and the experiment was conducted in triplicates with 16hphotoperiodand 25℃ temperature. The seedlings were harvested after 15days for the estimation of various parameters.

In conclusion, Cr negatively affects the metabolic activities of B. juncea, which were restored by Se application. Se application showed its protective effects on photosynthetic pigments and gene expression studies confirmed the spectrophotometric observations. The Scanning electron microscope (SEM) studies also clearly indicated the positive effects of Se against Cr toxicity on morphology and density of stomata, which further confirmed its imperative role in efficient gas exchange for both photosynthesis and respiration. Exogenous application of Se was also observed to enhance the lipid- and water-soluble antioxidants, which further indicated its role in alleviating oxidative damage. The positive effect of Se in maintaining the osmotic homeostasis as well as activation of thiols in Cr-stressed seedlings also showed its potential to enhance the ability of B. juncea seedlings to combat heavy metal stress effectively. The results, therefore, indicate that Se can be effectively used as a mitigating agent against Cr stress.

Treatments of Se and Cr aided in modulating the gene expression of CHLASE, PSY and CHS. CHLASE showed a significant upregulation of 366.3% in its expression in the Cr treated seedlings with respect to untreated control seedlings. The expression of PSY, however, was observed to be downregulated with Cr application by 28.6%, while CHS expression showed an upregulation by 73.13%. Se application at 4 µM in combination with 300 µM Cr downregulated its expression by 46.92% when compared to Cr-treated seedlings. On the other hand, the same concentration of Se caused an increase of PSY and CHS expression by 425.2 and 209.4%, respectively.

To observe the effect of earthworms in Cd-polluted soils, a pot experiment was conducted. The seeds were washed with Tween 20 and surface sterilized before sowing. The pots were filled with soil and cattle dung (partially decomposed) in the ratio of 2:1. The pots were inoculated with earthworms (5 earthworms Kg -1 soil) 7 days prior to sowing, and seeds were then uniformly sown in pots. Plants were allowed to grow under natural conditions and then harvested after 30 and 60 days after sowing (DAS).Different concentrations of Cd (0.50, 0.75, 1.00 and 1.25 mM) were prepared using CdCl₂ (anhydrous) obtained from HiMedia. The various concentrations were prepared by dissolving CdCl₂ in double-distilled water. The soil was spiked with the different concentrations of Cd and analysed for Cd concentration before experimental set-up using atomic absorption spectrophotometer (AAS). The Cd content was found to be approximately 56 mg (0.5 mM Cd), 84 mg (0.75 mM Cd), 112 mg (1.00 mM Cd) and 140 mg (1.25 mM) in respective concentrations per kilogram of soil. All metal-treated soils were supplemented with fixed number of earthworms. Two sets were maintained which included one without earthworms (WTE) and other supplemented with earthworms (WE).

Earthworms help to mitigate the toxic effects produced by Cd on plant growth and photosynthetic efficiency along with enhanced phytoremediation capacity when co-inoculated with Cd in soil.

In the present study, the expression of genes CHLASE, PSY, CHS and PAL was enhanced in plants grown in Cd-treated soils supplemented with earthworms. Significant differences in expression of CHLASE, PSY, CHS and PAL were observed after the analysis of data using two-way ANOVA and Tukey's HSD test. The expression of gene CHLASE was enhanced by 1.56-fold in Cd-treated plants which was further enhanced to 3.63-fold when earthworms were co-inoculated along with Cd-treated soils. In comparison to control plants, the expression of PSY and CHS was significantly enhanced by 1.43-fold and 2.07-fold when plants were grown in 1.25 mM Cd treatment. The expression was further enhanced to 3.32-fold (PSY) and 3.37-fold (CHS) when earthworms were supplemented along with Cd treatment (1.25 mM). Similarly, significant increase in the expression of PAL was also observed in Cd-treated plants by 1.64-fold in Cd-treated soil which was enhanced to 2.74-fold in the presence of earthworms.

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) .