A pot experiment was carried out with three different percentage of dye from minimum concentration of 0.25%, 0.5%, 0.75% and maximum concentration upto 1% to categorize the growth level of green gram using a plant biomass Leucaena leucocephala (R1-remediator1), Albizia amara (R2- remediator 2) and combination of both Leucaena leucocephala and Albizia amara (R1 + R2).
Biometric Evaluation
Biometric evaluation of root length, shoot length, plant height and leaf area were evaluated in Vigna radiata (green gram) plant before and after remediation.
Figure 1 shows the developmental status of Vigna radiata, root length, shoot length, plant height and leaf area was reduced in dye simulated soil when compared to remediated and control soil. At all the four different concentrations of dye, the plant grown in remediated soil has better growth.
When comparison done within the plant biomass, the combined form of plant biomass (1:1) (R1 + R2) shows the better effect in the shoot length of plant Vigna radiata (green gram).Thus dye contaminated soil reduces the shoot growth in green gram due to their increase in toxicity and through the implement of phytoremediator in dye simulated soil shows good growth potential of plant Vigna radiata (green gram).
Biochemical estimation of Vigna radiata (green gram)
Since the maximum concentration of 1% is toxic upto the analysis of biometric evaluation, all the biochemical estimation carried out with 1% dye and followed. Plants are exposed to several environmental constrains generally attributed to abiotic and biotic stresses induced by pathogens. Abiotic stress factors belong to the main environmental factors affecting crop growth and productivity. Abiotic stress environment can induce a wide number of responses in plants ranging from readjustment of transport and metabolic processes leading to growth inhibition (Tardif et al. 2009).
Biochemical estimation of protein, amino acids, carbohydrate, DNA and amylase were carried out in Vigna radiata (green gram) root tissue after the 17th day of germination. A comparison between control, dye simulated and remediated roots were made. The root systems perform many essential adaptive functions including water and nutrient uptake, anchorage to the soil and the establishment of biotic interactions at the rhizosphere and so all the biochemical studies carried out in it. The biochemical changes observed for protein, amino acids, and total carbohydrates. Amylase is the first enzyme that gets activated on germination was also measured. The stress markers and antioxidant levels were studied for the abiotic stress induced by navy blue dye on Vigna radiata (green gram).
Table:1 results the biochemical estimation of protein, amino acids, total carbohydrates and amylase activity in all five groups of plant roots of Vigna radiata (green gram). The plant grown in dye simulated soil shows significant decrease of protein, free amino acids, DNA, total carbohydrates and amylase activity at p < 0.05 level. The plant biomass Leucaena leucocephala (R1), Albizia amara (R2) and combined plant biomass (R1 + R2) used for phytoremediation enhances the root growth and improved the biochemical activity in it shows significant increased in protein, free amino acids, DNA, total carbohydrates and amylase. On comparing the plant biomass Leucaena leucocephala and combined plant biomass (R1 + R2), Albizia amara shows statistical significance increase in protein, free amino acids, DNA, total carbohydrate and amylase of plant roots Vigna radiata at p < 0.05 level with standard control.
Table 2 explains the presence of stress markers such as phenol, salicylic acid, hydrogen peroxide and ascorbic acid level in plant roots of Vigna radiata (green gram) grown on before and after phytoremediation. The stress markers phenol salicylic acid and ascorbic acid level in plant roots of Vigna radiata (green gram) is significantly decreased in dye simulated soil whereas hydrogen peroxide level increases when compared to control and phytoremediator. The phytoremediator of plant biomass Leucaena leucocephala (R1), Albizia amara(R2) and combined form of plant biomass (R1 + R2) was used in this stressed soil. The phytoremediator used in this study enhances the plant growth and shows a significant increase in phenol content, salicylic acid, ascorbic acid and hydrogen peroxide shows significant decrease in the root of Vigna radiata (green gram).
Table 1
Biochemical analysis in green gram root
Groups
|
Total protein (mg/g of tissue)
|
Amino acids (mg/g of tissue)
|
DNA
(mg/g of tissue)
|
Total carbohydrates
(mg/g of tissue)
|
Amylase
(mg/g of tissue)
|
Control (G1)
|
28.3 ± 1.18
|
0.16 ± 0.01
|
1.91 ± 0.11
|
35.1 ± 1.41
|
59.7 ± 1.19
|
Test
(G2)
|
16.7 ± 0.97*a
|
0.34 ± 0.01*a
|
1.03 ± 0.07*a
|
10.8 ± 1.09*a
|
28.9 ± 0.80*a
|
AA(G3)
|
38.8 ± 1.57
a*/(ns)
|
0.18 ± 0.02 a*/ (ns)
|
2.78 ± 0.09
a*/ (ns)
|
30.1 ± 1.33
*a/(ns)
|
70.8 ± 1.29
*a/(ns)
|
LL(G4)
|
36.6 ± 1.39
a*/(ns)
|
0.14 ± 0.02 a*/(ns)
|
2.55 ± 0.08
*a/(ns)
|
29.5 ± 1.12
*a/(ns)
|
61.6 ± 1.13
a*/*ab
|
AA + LL
(G5)
|
39.5 ± 1.70
*a/(ns)
|
0.21 ± 0.02
*a/*ab
|
2.99 ± 0.12
*a/(ns)
|
34.5 ± 1.48
a*/(ns)
|
69.6 ± 1.20
*a/(ns)
|
1unit of amylase activity = µ moles of maltose released |
Values are mean ± SD of n = 6; Group comparison: G1 vs G2; G2 vs G3,G4, G3, G4 vs G5 |
Statistical significance- p < 0.05*a,*ab significant; b*no difference |
Table 2
Stress marker level in Vigna radiata (green gram) root before and after remediation
Groups
|
Phenol content
(µ moles/g tissue)
|
Salicyclic acid
content(µmoles/g tissue)
|
Hydrogen peroxide level
(µmoles/g tissue)
|
Ascorbic acid level (mg/g of tissue)
|
Control
|
98 ± 1.93
|
21.7 ± 0.46
|
53.9 ± 0.60
|
49.4 ± 0.79
|
Test
|
49.9 ± 0.70a
|
9.8 ± 0.49a
|
78.9 ± 0.80a
|
11.6 ± 0.40a
|
A.amara
|
1.22 ± 1.44ab
|
20.6 ± 0.48ab
|
44.5 ± 0.758ab
|
43 ± 0.72ab
|
L.leucocephala
|
1.20 ± 1.49ab
|
11.5 ± 0.47ab
|
40 ± 0.74ab
|
41 ± 0.78ab
|
A.amara+
L.leucocehala
|
1.09 ± 1.81ab
|
10.6 ± 0.43a
|
41.3 ± 0.76ab
|
42.4 ± 0.51ab
|
Values are mean ± SD of n = 6; |
Group comparison: G1 vs G2; G2 vs G3, G4, G3, G4 vs G5 |
Statistical significance- p < 0.05*a,*ab significant; b*no difference |
On comparing the plant biomass Leucaena leucocephala (R1) and combined form of plant biomass (R1 + R2) the plant biomass Albizia amara (R2) shows better improvement in stress markers level. Phenols are one of the important phytochemicals which generally thought to prevent oxidative damage by scavenging active oxygen species and by grating the radical chain reaction during lipid peroxidation (Sakihama and Yamasaki 2002).
Estimation of DNA
Figure 2 shows the presence of DNA content in root extract of Vigna radiata before and after remediation with AA, LL and AA + LL. It was observed that the DNA content was decreased in test roots of Vigna radiata (green gram) when compared with control and remediated roots. The DNA content in remediated roots was high when compared with dye simulated and control roots. The high amount of DNA was present in the remediated roots of AA, LL and AA + LL. When comparing within the remediator combined form of AA + LL (2.99 mg/g of root tissue) shows DNA content upto 2.99 mg/g of tissue.
b) DNA profiling
For quality and yield assessments, electrophoresis was done with all DNA samples in 0.8% agarose gel, stained with ethidium bromide and bands were observed in gel documentation system (Fisher 1968). DNA profiling before and after remediation shows a thick single band of high molecular weight confirmed the good quality of the extracted DNA. No DNA fragmentation due to shearing of DNA during extraction procedure was seen in any of samples and results were reproducible (Fig. 3). The DNA profile of the root tissue Vigna radiata (green gram) of control, test and remediated plants shows a band at 1500 Kbs. Thus, from this observation it explains that the DNA content was high in the entire root sample. DNA isolated produced good quality and high quantity of intact DNA. The intact double standard DNA forming