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.

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.

Earthen pots (12 in. diameter) were filled with five kg of soil per pot and arranged in a randomized block design (Kothari, 2004) with 5 pots for each treatment. 0 mM Cd(II) was added to the soil in form of anhydrous CdCl₂. The seeds of B. juncea var. RLC-1 (procured from Punjab Agricultural University, Ludhiana, India) were surface sterilized with 0.5% (v/v) sodium hypochlorite. These were then soaked in different concentration of castasterone (CS) (0 nM) for 8 h before sowing. After seven days of germination, 0 mM citric acid (CA) was supplemented to the soil. The plants were harvested after 30 days of sowing, and fresh mature top leaves were analysed for biochemical, physiological and molecular studies.

Castasterone (CS) and Citric Acid (CA) treatments also improved the osmolyte contents of the leaves which enabled the plants to tide over Cd (II) stress. Beta regression and multiple linear regression analysis showed that CS treatment has more positive effect on shoot height, fresh weight and photosynthetic pigments and efficiency than the CA treatment. The most effective concentration enhancing Cd(II) stress tolerance in B. juncea plants was seed soaking treatment with 100 nM CS and soil application of 0.6 mM/kg CA soil.

The present study found that relative expression of CHLASE gene was significantly increased by 3.57 folds under Cd(II) toxicity. Seed pre-sowing treatment of 100 nM CS and soil citric acid application significantly lowered expression of CHLASE in 30 day old B. juncea leaves. MLR analysis also revealed positive effect of Cd(II) and negative effects of CA and CS treatments on fold change of CHLASE expression. Maximum reduction in CHLASE expression of 1.19 fold was observed with binary combination of CS (100 nM) and CA (0.6 mM/kg) in the leaves of Cd(II) stressed B. juncea.Expression of PSY and CHS genes was significantly enhanced by binary combination of CS and CA treatment by 2.85 fold and 5.15 fold, respectively. MLR analysis also revealed that CS, CA and Cd(II) treatments positively regressed on fold change of PSY and CHS expressions.