CHAPTER 5 MORPHOLOGICAL AND BIOCHEMICAL CONSTITUENTS OF BLACK GRAM In olden days the use of chemical fertilizers caused soil pollution due to decrease in the amount of Nitrogen (N) in the soil. On repeated cultivation, the fertility of the soil is decreased. Hence to avoid this condition, people started using organic manure which is devoid of chemicals. This also helped in the improvement of soil quality (Hankin, 1998). 5.1 Morphology of Vigna mungo L. Black gram belongs to family Leguminoseae and sub family Papilionaceae. It is annual herb growing to a height of 30-100 cm. Stem is slightly ridged and covered with brown hairs. The leaves are large and trifoliate. The inflorescence consists of a cluster of 5-6 flowers at the top of a long hairy peduncle. The flower of black gram is bright yellow and the pod attaches upright to the peduncle. Black gram is a self fertilized crop. Black gram may be grown as pure crop in rice fallows after the harvest of the first or second crop of paddy. It can also be grown as pure or mixed crop during kharif season. The area of traditional cultivation of black gram is confined to South Asia and adjacent regions (India, Pakistan, Afghanistan, Bangladesh and Myanmar (Rajarathinam, 1999). 92
5.2 Biochemical constituents of black gram Black gram is a rich source of protein. It gives 340 calories per 100g dry weight. Black gram contains 20-25% that is double the amount of proteins compared to cereals. It also contains albumin and globulin. Black gram is a rich source of methionine, tryptophan and lysine. Black gram contains 55-60% soluble sugars, fibers, starch and unavailable carbohydrates. It contains minerals like calcium, magnesium, zinc, iron potassium and phosphorus. 80% phosphate is present as phytate phosphate, complexed with protein (Gopalan, 1991). Materials Black gram seeds (ATD mash-3) were collected from agricultural society of Tanjore. The experiment was carried out with two different types of soils. The fertile soil was collected from the village near Tanjore and the drought soil was collected from the village near Virudhunagar and kept in smallpots. Four different organic manures such as Seaweed, Vermicompost, Cow dung and Coir waste decomposed using Anabaena azollae were used for this experiment. Soils were plugged with organic manures before plantation of crop. Control plants were grown without adding manures. Seeds were sown and sprinkled with water at regular intervals. Germination of seeds was an index for growth of plants. The plant samples were taken at two stages of crop growth from all the treatments corresponding to 13 th and 36 th day to carryout the following assays. 93
Methods The morphological parameters like Plant height, Number of leaves and Leaf area were analyzed. Biochemical parameters like total chlorophyll, total carbohydrates, total proteins, methionine, ascorbic acid and iron were estimated. Plant height Plant height was recorded from the ground level to the growing point and expressed in cms. Number of leaves and branches The number of leaves and branches present in each plant at selected stages were counted and recorded. Leaf area as cm 2 per plant. Leaf area was assessed for the 3 rd leaf from the top and was reported Total chlorophyll and carbohydrate The chlorophyll and carbohydrate content of black gram leaves were estimated by acetone method as mentioned in chapter 4 and by anthrone method as explained in chapter 2. 94
Estimation of protein The protein was estimated by Lowry s method (Lowry etal. 1951). Estimation of methionine Methionine is one of the essential, sulphur containing amino acids. Although it is present in many food proteins, methionine is the limiting amino acid in most of the green legumes. Reagents a. 2N Hydrochloric acid b. 10N sodium hydroxide c. 10% sodium hydroxide d. 10% sodium nitroprusside3% glycine e. Orthophosphoric acid (Sp.gr.1.75) f. Standard methionine: Dissolve 100mg of DL-methionine in 4ml of 20% hydrochloric acid and dilute with water to 100ml. Methodology 0.5gm of plant sample was weighed into a 50ml conical flask. 6ml of 2N hydrochloric acid was added and autoclaved at 15lb pressure for 1hr. A pinch of activated charcoal was added to the hydrolysate (autoclaved sample) and heated. It was filtered when hot and the charcoal was washed with hot water. Then the filtrate was neutralized with 10N sodium hydroxide followed 95
by 0.15ml sodium nitroprusside. After 10min, 1 ml glycine solution was added and incubated for another 10min. 2ml of orthophosphoric acid was added and the contents were shaken vigorously. The intensity of red color was read after 10min at 520nm against a blank prepared in the same way but without nitroprusside. Estimation of ascorbic acid Reagents a. 4% Oxalic acid solution b. 0.5N Sulphuric acid c. 2% of 2, 4 Dinitrophenyl Hydrazine (DNPH) reagent d. 10% Thiourea solution e. 80% sulphuric acid f. Bromine water g. Ascorbic acid stock solution (100mg in 100ml oxalic acid and 1drop of bromine water) Methodology 500mg of sample was ground and 5ml of oxalic acid was added and centrifuged for 10min. The contents were filtered and the supernatant volume was measured. 3ml of the supernatant was taken in a test tube and 1drop of bromine water was added to it. The color was changed to orange yellow. The solution was made upto 10ml with oxalic acid. 0.5ml of solution was taken 96
and 2.5 ml water was added to it. 1 ml of 2,4 dinitrophenyl hydrazine and 1drop of thiourea were added in all tubes. It was mixed well and incubated at 37 C for 1hr. Orangish red osazone crystals were formed. The crystals were dissolved in 7ml of 80% sulphuric acid. The orange color was read at 540nm spectrophotometerically. 5.3 Results and Discussion The various morphological parameters such as plant height, number of leaves, branches and leaf area were taken into account for analysis purpose. The analysis was made by estimation of biochemical constituents of black gram treated with four different manures in both fertile soil and drought soil. 97
(a) (b) (c) (d) (e) Figure 5.1 Morphological parameters of black gram in fertile soil: (a) Organic fertilizer (seaweed) (b) Cow dung manure (c) Vermicompost manure (d) Coir waste manure (e) Control plant without manure 98
(a) (b) (c) (d) (e) Figure 5.2 Morphological parameters of black gram in drought soil: (a) Seaweed manure (b) Cow dung manure (c) Vermicompost manure (d) Coir waste manure (e) Control plant without manure (died) 99
Table 5.1 Plant height (cm) of black gram supplied with different organic manures Fertile soil Drought soil 20 th Day 40 th Day 20 th Day 40 th Day Control 16 28 10 Died Vermicompost 17 30 15 27 Seaweed 15 29 13 26 Cow dung 26 30 15 28 Coir waste 18 33 17 31 Table 5.1 reveals the growth of the plant which was expressed in terms of height. The maximum height of the plant was observed using coir waste manure in both fertile soil and drought soil. Organic manures supply the nutrients for the growth of the plant. The poor growth was observed in control plant (without manures) in fertile soil. The plant died on 35 th day in drought soil (Figure 5.2.e). This was due to unavailability of nutrients needed for growth of the plants. Plants need nutrients which were absorbed from the soil, water and manures. Table 5.2 Number of leaves in black gram plant grown in fertile and drought soils supplemented with different organic manures 20 th Day 40 th Day 20 th Day 40 th Day Control 8 13 7 Died Vermicompost 8 14 7 10 Seaweed 7 13 6 11 Cow dung 7 12 5 13 Coir waste 8 14 6 14 100
Table 5.2 shows the health of a plant having maximum number of green leaves. This is due to the high content of chlorophyll pigments and enzymes which are supplied from the nutrients present in the soil and manures. The maximum numbers of leaves were observed in coir waste treated plant (Figure 5.1.d). Cowdung manure treated plants show less number of leaves in fertile soil. The leaf senesence was observed in control plant due to lack of nutrients. The numbers of leaves were decreased in plants using all manures except coir waste in drought condition, due to its water holding capacity. Table 5.3 Number of branches in black gram plant supplemented with different organic manures 20 th Day 40 th Day 20 th Day 40 th Day Control 4 7 3 Died Vermicompost 4 7 4 5 Seaweed 5 7 4 4 Cow dung 3 6 4 4 Coir waste 3 7 3 6 Branches in a plant are an index for the growth of plant. Table 5.3 states that the numbers of branches are gradually increased from 20 days to 40 days of growth in fertile soil. The branches with maximum number of leaves in drought soil were observed in coir waste manuring plant (Figure 5.2.d). This was due to storage of nutrients and water in the coir waste. It also enhances the water holding capacity of soil and increases the bulk density of soil. 101
Table 5.4 Leaf area (cm 2 ) of black gram plant supplemented with different organic manures 20 th Day 40 th Day 20 th Day 40 th Day Control 6.0 15.6 5.0 Died Vermicompost 6.6 16.5 6.0 10.2 Seaweed 2.2 10.0 4.0 9.8 Cow dung 6.8 19.3 6.5 17.4 Coir waste 8.1 23.0 7.2 18.1 Table 5.4 shows the leaf area of the black gram plant using different organic manures. The leaf area is calculated by multiplying the length and breadth of the leaves. The small size of leaf was observed in seaweed treated plants in both soils (Figure 5.1.a and 5.2.a). The leaf area was gradually increased from 20 days to 40 days. The maximum size of leaf was observed using coir waste treated plant in both fertile and drought soils. The morphological characteristics like height, number of leaves, and number of branches and leaf area of plant were shown in Figure 5.1 and Figure 5.2.The maximum growth was observed in coir waste treated plants. The poor growth was observed in untreated and drought plants due to poor translocation of nutrients in soil and low water holding capacity of the soil. Table 5.5 Total chlorophyll content (mg/g) of black gram supplemented different organic manures 0-20 Days 0-40 Days 0-20 Days 0-40 Days Control 78 178 70 Died Vermicompost 42 151 40 92 Seaweed 166 130 40 100 Cow dung 75 157 56 123 Coir waste 79 164 76 158 102
Table 5.5 shows the increase in chlorophyll content of black gram up to the maturity of the plant using fertile soil and drought soil. The seaweed treated plant showed high amount of chlorophyll up to 20 th day of growth, but generally reduces in the maturity of plant. The chlorophyll content of black gram was rapidly increased from 30 days onwards. The high content of chlorophyll (123mg/g) was observed in cow dung plant on the flowering of the plant in drought soil (Figure 5.2.b). The coir waste treated plant showed the high amount of chlorophyll up to 40 th day on drought soil. The yellow color appearance of leaves on 26 th day of drought soil which was due to poor availability of chlorophyll content of plant. Table 5.6 Total carbohydrate content (mg/g) of black gram using different organic manures 0-20 Days 0-40 Days 0-20 Days 0-40 Days Control 0.33 1.26 0.30 Died Vermicompost 0.67 1.09 0.30 0.72 Seaweed 0.49 0.84 0.35 0.81 Cow dung 0.36 1.3 0.24 0.71 Coir waste 0.61 1.44 0.60 0.89 Table 5.6 shows high amount of carbohydrate was observed in coir waste treated plants. Blue green algae such as Anabaena azollae treated coir waste supply nitrogen to the soil via symbiotic nitrogen fixation. It enhances the carbohydrate contents of leaves and delays leaf senescence. This would be supported by Handerson (2004) who observed organic manures supply nitrogen and carbon skeleton, which serve as raw material for the production 103
of carbohydrates. The carbohydrate was found to be higher in organic manure treated plants in both fertile soil and drought soil when compared with control (untreated plant). This finding was supported by Mishra (2001) in sugar beet plant. Table 5.7 Protein content (mg/g) of black gram supplemented with different organic manures 0-20 Days 0-40 Days 0-20 Days 0-40 Days Control 15 3.9 10 Died Vermicompost 22 51 14 28 Seaweed 21 52 13 34 Cow dung 21 57 14 51 Coir waste 22 53 20 35 Table 5.7 highlights the gradual increase in protein content up to the 36 th day of plants grown in fertile and drought soil using different organic manures. The high protein content was observed in vermicompost and coir waste treated plants at initial stage but gradually declined in the maturity stage. The high content of protein was observed at maturity stage in cow dung treated plants in both fertile and drought soil. This was due to sudden decline in the ph of the soil, which enhanced the nitrogen content of soil. This nitrogen serves as a raw material for the production of protein. This finding was supported by Gerald (2004) in pea plant. 104
Table 5.8 Iron content (mg/g) of black gram supplemented with different organic manures 0-20 Days 0-40 Days 0-20 Days 0-40Days Control 1.2 3.5 1.0 Died Vermicompost 1.1 3.5 0.8 3.6 Seaweed 2.6 4.5 2.3 4.0 Cow dung 1.0 3.0 0.5 1.4 Coir waste 2.5 4.5 2.6 4.3 Table shows 5.8 showed the gradual increase of iron content of black gram upto 40 th day of growth. The seaweed and coir waste treated plants showed the maximum amount of iron in fertile soil as well as drought soil. The poor iron content was observed in cow dung treated plants. Table 5.9 Ascorbic acid content (mg/g) of black gram supplemented different organic manures 0-20 Days 0-40 Days 0-20 Days 0-40 Days Control 18.75 25.27 7.23 Died Vermicompost 16.6 19.72 13.4 16.5 Seaweed 17.64 20.90 8.40 10.7 Cow dung 16.25 25.27 9.2 13.8 Coir waste 11.76 10.38 11.3 12.8 Table 5.9 elucidates the ascorbic acid content of black gram which gradually increased up to 40 th day of maturation. The high amount of ascorbic 105
acid was observed in untreated plant and cow dung treated plants. Coir waste treated plants contained less amount of ascorbic acid. The ascorbic acid content rapidly decreased in seaweed treated plant in drought soil. Organic manures supply the nutrients for the growth of the plants. The control plant was died in drought soil without adding any manure. This was due to poor absorption of nutrients by the plants and poor water holding capacity in soil. Table 5.10 Methionine content (mg/g) of black gram supplemented different organic manures 0-20 Days 0-40 Days 0-20 Days 0-40 Days Control 12 10 11 Died Vermicompost 12 8 9 7 Seaweed 10 16 9 13 Cow dung 22 11 14 10 Coir waste 13 8 12 10 Methionine is one of the essential sulphur containing amino acids. It is found mostly in legumes. Table 5.10 showed the methionine content slowly increased up to 24 days of growth in both drought and fertile soil in plants treated with different organic manures. But it was suddenly reduced on the flowering stage of all plants except in seaweed treated plant. The methionine content of black gram reduced rapidly in plants treated with cow dung in drought soil. 106