Nitrogen responsiveness of three-finger millet genotypes (differing in their seed coat colour) PRM-1 (brown), PRM-701 (golden), and PRM-801 (white) grown under different nitrogen doses was determined by analyzing the growth, yield parameters and activities of nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase; GOGAT, and glutamate dehydrogenase (GDH) at different developmental stages. High nitrogen use efficiency and nitrogen utilization efficiency were observed in PRM-1 genotype, whereas high nitrogen uptake efficiency was observed in PRM-801 genotype. At grain filling nitrogen uptake efficiency in PRM-1 negatively correlated with NR, GS, GOGAT activities whereas it was positively correlated in PRM-701 and PRM-801, however, GDH showed a negative correlation. Growth and yield parameters indicated that PRM-1 responds well at high nitrogen conditions while PRM-701 and PRM-801 respond well at normal and low nitrogen conditions respectively. The study indicates that PRM-1 is high nitrogen responsive and has high nitrogen use efficiency, whereas golden PRM-701 and white PRM-801 are low nitrogen responsive genotypes and have low nitrogen use efficiency. However, the crude grain protein content was higher in PRM-801 genotype followed by PRM-701 and PRM-1, indicating negative correlation of nitrogen use efficiency with source to sink relationship in terms of seed protein content.
Cereal grains are considered to be one of the most important sources of dietary proteins, carbohydrates, vitamins, minerals, and fiber for people all over the world. Finger millet commonly referred as ragi or mandua ranks fourth in importance among millets in the world after sorghum (
Nitrogen use efficiency (NUE) at the plant level is its ability to utilize the available nitrogen (N) resources to optimize its productivity. This includes nitrogen uptake and assimilatory processes, redistribution within the cell and balance between storage and current use at the cellular and whole plant level [
Since, from both economical and ecological point of view, agricultural practices are going towards extensive systems using lower N fertilizers, a better knowledge of physiological basis of nitrogen use efficiency (NUE) in economically important crop such as finger millet is required. Although finger millet is highly nitrogen use efficient crop yet, there is a wide variation across the genotypic level. Thus, the development of finger millet that can make the best use of N in low-nitrogen soils is essential for the sustainability of agriculture [
Three finger millet (
The plant height and leaf area (LICOR-3000 leaf area meter) were measured at the vegetative stage (40 days after sowing) and the flowering stage. SPAD value was noted by chlorophyll meter SPAD-502 at vegetative stage (40 days after sowing), the flowering stage, and grain filling stages. The dry matter and grain yield were noted at the time of harvest. Heading date was determined by counting the number of days from sowing to 50% of spikes fully emerged from the boot.
The four enzymes, namely, NR, GS, GOGAT, and GDH, were assayed in freshly harvested flag leaf at three different developmental stages of finger millet genotypes. The protein was determined from all of the enzyme extracts [
The nitrate reductase (NR) activity was estimated by using the method described by Hageman and Hucklesby, 1971 [
The extraction buffer included, 10 mM-Tris HCl (pH 7.6), 1 mM-MgCl2, 1 mM-EDTA, and 1 mM-2 mercaptoethanol. Leaves (2 g) were grinded using liquid N2 in the presence of cover slips followed by centrifugation at 12,000 xg for 30 min at 4°C [
Activity of GOGAT was determined in enzyme preparation described for GS. Standard assay mixture contained 40 mM potassium phosphate buffer (pH 7.5), 10 mM L-glutamine, 10 mM 2-oxoglutarate, 0.14 mM NADH, and crude enzyme (final volume 3 m1). Increase in absorbance at 340 nm for 3-4 min at room temperature (25°C) was recorded. Absorbance (340 nm/min) was calculated from initial linear portion of the curve.
Extraction buffer (pH 7.9) consisted of 0.05 M imidazole, 5 mM DTT. Leaves (1 g) were grinded using liquid N2 in the presence of cover slips in chilled mortar and pestle and were centrifuged at 12,000 xg for 40 min at 4°C. Supernatant was collected and stored at −20°C. The assays were carried out by continuous spectrophotometric rate determination method.
Nitrogen content in grains and straw was determined by micro-Kjeldhal method [
Random sample of the grains from individual genotypes was obtained. These grain samples were dried at room temperature (30°C) to minimize intrinsic moisture content uniformity. Then, these dried grain samples were weighed by electronic weighing balance to detect grain weight per plant.
A complete factorial arrangement of treatments was used (soil condition × genotype) as a complete randomized design with three replications. Mean ± standard error mean (SEM) and critical difference at 5% (CD at 5%) values were calculated for statistical analysis. Correlation coefficients were also measured for various physiological and biochemical parameters.
There was variation in the heading dates within these finger millet genotypes, that is, the heading date for brown (PRM-1) genotype ranged from 77 to 85 days, whereas for golden (PRM-701) and white (PRM-801) genotypes ranged from 119 to 130 days. This indicates that brown (PRM-1) genotype is early flowering and golden (PRM-701) and white (PRM-801) are late flowering genotypes. Nitrogen fertilization significantly increased plant height and leaf area (Table
Influence of genotype and soil condition on growth parameters.
Plant height (cm) | Leaf area (cm2) | |||
---|---|---|---|---|
Vegetative | Flowering | Vegetative | Flowering | |
Brown (PRM-1) | ||||
High nitrogen | 11 | 78.25 | 16.75 | 28 |
Normal nitrogen | 9.1 | 57.4 | 13.3 | 26.3 |
Low nitrogen | 7.6 | 56.5 | 12.03 | 24.1 |
FYM | 8.8 | 78.73 | 15.1 | 29 |
Control | 8.2 | 41.7 | 9.4 | 24 |
| ||||
Golden (PRM-701) | ||||
High nitrogen | 11.1 | 73.3 | 18 | 27 |
Normal nitrogen | 10.3 | 77.9 | 12 | 36 |
Low nitrogen | 7.7 | 72.3 | 16 | 27.4 |
FYM | 11.6 | 75 | 15.3 | 27.6 |
Control | 9.5 | 60 | 10.7 | 25 |
| ||||
White (PRM-801) | ||||
High nitrogen | 14.3 | 68.99 | 18.6 | 32.5 |
Normal nitrogen | 13.1 | 76 | 14.6 | 28 |
Low nitrogen | 13.6 | 70 | 17 | 32 |
FYM | 17.6 | 97 | 19.7 | 37.8 |
Control | 11.1 | 67 | 13 | 26 |
| ||||
SEm± | 1.10 | 2.35 | 0.93 | 1.87 |
CD at 5% | ||||
Genotype | 1.43 | 3.04 | 1.19 | 2.42 |
Soil condition | 1.84 | 3.93 | 1.54 | 3.12 |
Genotype X soil condition | 3.19 | 6.81 | 2.67 | 5.40 |
Influence of nitrogen fertilization on SPAD value at various developmental stages of finger millet genotypes (a) brown PRM-1, (b) golden PRM-701, (c) white PRM-801.
Nitrogen use efficiency and nitrogen utilization efficiency were significantly affected by different genotypes and soil conditions. The interaction between genotype and soil condition for nitrogen use efficiency and nitrogen utilization efficiency was significant. However, this was in contrast to nitrogen uptake efficiency. The highest nitrogen use efficiency was observed in brown (PRM-1) genotype (5.1 g g−1) followed by golden (PRM-701) genotype (4.1 g g−1) and white (PRM-801) genotype (2.4 g g−1) relative to control. Similarly, the highest nitrogen utilization efficiency was observed in brown (PRM-1) genotype (2.7 g g−1) followed by golden genotype (PRM-701) (2.2 g g−1) and white genotype (PRM-801) (1.2 g g−1) relative to control. However, the highest nitrogen uptake efficiency was observed in white genotype (PRM-801) (3.0 g g−1) followed by golden genotype (PRM-701) (2.7 g g−1) and brown genotype (PRM-1) (2.2 g g−1) with respect to control. The highest nitrogen use efficiency was observed (Table
Influence of genotype and soil condition on yield parameters.
Dry matter (g) | 1000 grain weight (g) | Number of grains per spike | Crude grain protein content |
Nitrogen use efficiency |
Nitrogen utilization efficiency |
Nitrogen uptake efficiency | |
---|---|---|---|---|---|---|---|
Brown (PRM-1) | |||||||
High nitrogen | 7.0 (180) | 2.7 (17.4) | 680 (36) | 8.1 (8) | 6.7 (31.8) | 3.4 (25.9) | 2.2 (0) |
Normal nitrogen | 5.8 (132) | 2.4 (4.3) | 665 (33) | 7.6 (1.6) | 6.0 (18.1) | 3.6 (33.7) | 2.0 (−9.09) |
Low nitrogen | 3.5 (40) | 2.7 (17.4) | 600 (20 | 7.9 (4.9) | 5.5 (8.9) | 3.1 (12.9) | 2.2 (0.0) |
FYM | 5.6 (124) | 2.5 (8.7) | 570 (14) | 8.0 (6.7) | 6.4 (25.5) | 3.4 (27.0) | 2.2 (0.0) |
Control | 2.5 | 2.3 | 500 | 7.5 | 5.1 | 2.7 | 2.2 |
| |||||||
Golden (PRM-701) | |||||||
High nitrogen | 3.3 (65) | 3.1 (55.0) | 530 (51) | 9.1 (1.3) | 6.1 (48.3) | 3.3 (50.0) | 2.4 (−11.1) |
Normal nitrogen | 3.9 (95) | 3.2 (60.0) | 480 (37) | 9.6 (6.5) | 6.0 (45.9) | 3.3 (50.0) | 2.7 (0.0) |
Low nitrogen | 3.6 (80) | 3.0 (50.0) | 600 (71) | 9.8 (8.3) | 7.4 (81.5) | 3.4 (54.5) | 2.9 (7.4) |
FYM | 7.0 (250) | 3.0 (50.0) | 450 (29) | 9.1 (1.1) | 4.0 (−3.7) | 2.2 (0.0) | 2.4 (−11.1) |
Control | 2.0 | 2.0 | 350 | 9.0 | 4.1 | 2.2 | 2.7 |
| |||||||
White (PRM-801) | |||||||
High nitrogen | 2.5 (25) | 3.0 (36.4) | 420 (40) | 9.8 (1.6) | 3.1 (29.2) | 1.5 (25.0) | 2.6 (−13.3) |
Normal nitrogen | 2.6 (30) | 2.8 (27.3) | 400 (33) | 9.9 (2.9) | 4.3 (79.2) | 2.1 (75.0) | 2.9 (−3.3) |
Low nitrogen | 4.2 (110) | 3.6 (63.6) | 370 (23) | 10 (4.2) | 4.5 (87.5) | 1.9 (58.3) | 3.1 (3.3) |
FYM | 6.0 (200) | 3.3 (50.0) | 360 (20) | 10 (4.2) | 4.4 (83.3) | 2.3 (91.7) | 2.7 (−10.0) |
Control | 2.0 | 2.2 | 300 | 9.6 | 2.4 | 1.2 | 3.0 |
| |||||||
SEm± | 0.46 | 0.15 | 37.88 | 0.35 | 0.37 | 0.11 | 0.17 |
CD at 5% | |||||||
Genotype | 0.59 | 0.18 | 48.93 | 0.45 | 0.47 | 0.14 | 0.22 |
Soil condition | 0.76 | 0.24 | 63.17 | NS | 0.61 | 0.18 | NS |
Genotype X soil condition | 1.31 | 0.42 | 109.41 | NS | 1.06 | 0.32 | NS |
Parenthesis: % increase with respect to control of particular genotype.
From the Figure
Influence of nitrogen fertilization on nitrate reductase (NR) enzyme activity (specific activity) at various developmental stages of finger millet genotypes (a) brown PRM-1, (b) golden PRM-701, (c) white PRM-801.
The study of ammonium assimilating enzymes (Figures
Influence of nitrogen fertilization on glutamine synthetase (GS) enzyme activity (specific activity) at various developmental stages of finger millet genotypes (a) brown PRM-1, (b) golden PRM-701, (c) white PRM-801
Influence of nitrogen fertilization on glutamate synthase (GOGAT) enzyme activity (specific activity) at various developmental stages of finger millet genotypes (a) brown PRM-1, (b) golden PRM-701, (c) white PRM-801.
Influence of nitrogen fertilization on glutamate dehydrogenase (GDH) activity (specific activity) at various developmental stages of finger millet genotypes (a) brown PRM-1, (b) golden PRM-701, (c) white PRM-801.
In the present study, GDH activity was found to be highest in brown genotype (0.73 Units/mg protein) (Figure
Therefore, brown PRM-1 genotype is high nitrogen responsive and has high nitrogen use efficiency, whereas golden PRM-701 and white PRM-801 are low nitrogen responsive genotypes and have low nitrogen use efficiency. However, the crude grain protein content was higher in white PRM-801 genotype followed by golden PRM-701 and brown PRM-1 genotypes. From this study, it is inferred that study of biochemical parameter with physiological parameter will enable us to identify genotypes that are beneficial from the agricultural point of view.
Nitrate reductase
Glutamine synthetase
Glutamate synthase
Glutamate dehydrogenase
Nitrogen use efficiency
Nitrogen utilization efficiency
Nitrogen uptake efficiency.
The authors wish to acknowledge the Department of Biotechnology, Government of India for providing financial support in the form of Programme Support for research and development in Agricultural Biotechnology at G.B. Pant University of Agriculture and Technology, Pantnagar (Grant no. BT/PR7849/AGR/02/374/2006). N. Gupta, V. S. Gaur and A. Kumar Gupta’s work were supported by Junior Research Fellowship from the DBT and DST. The support provided by Dean, College of Basic Sciences and Humanities and Director, Experiment Station, G.B. Pant University of Agriculture and Technology, Pantnagar is also thankfully acknowledged.