Effects of L-Methionine Essential Metals Complexes on Sorghum Crop
Irfan Ismail1, Mudassar Kamal1, Muhammad Abdul Qadir2,
ABSTRACT
In the present work, amino acids complexes with essential metals such as Ca, Mg, Cu, Co, Fe and
Zn were synthesized and characterized. Its metal to ligand ratio was determined by Atomic
Absorption Spectroscopy and it was observed that Metal:Ligand ratio was of the order of 1:1.
Further confirmation of the complexes was carried out by using FTIR, melting points and
solubility studies. The purification of these complexes was carried out by re-crystallization in
various solvents. In the present work we have achieved percentage yields in the range of 35- 70%.
Furthermore, confirmation of these complexes was proved by their melting points.
In order to study the effect of these complexes on plant growth and crop yields, these
complexes were applied on Sorghum (Jawar). An extra ordinary increase in crop yield up to 2025% was observed by using only 45gr/Acre of these complexes
Keywords: Micronutrients, Soil, Plants.
1.
Chemistry Department, Minhaj University, Township Lahore-Pakistan.
2.
Chemistry Department, Punjab University, Lahore-Pakistan.
INTRODUCTION
Carbon, hydrogen and oxygen are non-mineral nutrients. They are synthesized by plants by using
carbon dioxide and water. Nitrogen, phosphorous, potassium, sulfur, magnesium and calcium are
designated as micronutrients while iron, manganese, zinc, copper, boron, molybdenum and
chloride are designated as micronutrients because they are also required in small amounts for plant
growth. Deficiency of micronutrients is caused by growing repeated crops. Micronutrients play an
important role in activating enzymes for photosynthesis, lignin biosynthesis, carbohydrate
synthesis and transportation. Similarly synthesis of fatty acids and proteins are also regularized
through micronutrients. Calcium plays an important role in plant growth and nutrition, as well as
the cell wall deposition. The main roles of calcium as a soil amendment to help maintain the
chemical balance of the soil reduce soil salinity and improve water infiltration.[1] Calcium(Ca)
deficiency is a plant disease that may not be enough calcium in the growth medium, but more of a
low transpiration of the whole plant, or, more often, the affected tissue product . Iron is also an
essential metal which is needed for the synthesis of chlorophyll. It is also involved in the
activation of many enzymes which are used in photosynthesis and respiration. It is also involved
in compounds and physiological process in plants.[2] Deficiency of iron in crops harvested in
calcareous soil has been linked to higher concentration of carbonates and bicarbonates in the
soil(Coulombe et al,1984).Some scientists observed that main symptom of iron deficiency is
chlorosis or yellowing between the veins of new leaves. Zinc and Copper improve the roots of
sorghum plants (Fageria, 2002) which effect the overall growth of the sorghum plant.[3]
Micronutrients deficiencies also constitute malnutrition which is responsible for more deaths than
any other cause. It is involved in the formation of chlorophyll. It is also responsible for the organic
compounds that are useful plants, the synthesis of amino acids, resistance to adverse growth
factors and cellular protein synthesis.[4] Cobalt salts increase the number of growth processes,
such as stem and coleoptiles elongation, bud development.VitaminB12containing cobalt, which is
synthesized by Rhizobium.[5] Mg is involved in the formation of chlorophyll. It is also
responsible for the organic compounds that are useful plants, the synthesis of amino acids,
resistance to adverse growth factors and cellular protein synthesis. [6] Methionine is very
important in plants for source of dietary protein for humans and animals. It is also fundamental
metabolite n plant cell.[7] Zinc L-Methionine complex was found to be more effective for the
growth of the sorghum plant. Uptake of Zn with Zinc L-Methionine complex application was
superior to other sources (Shukla and Morris, 1967).[8] Nearly five million child deaths happen
because of micronutrient malnutrition every year. Malnutrition arising from dietary deficiency of
critically important mineral micronutrients such as Fe and Zn is a serious problem affecting nearly
half of `the world‘s population(WHO,2002).[9-10] The present paper is to study the effect of LMethionine chelated micronutrients on growth of Sorghum plant which intern enhance the crop
yields.
EXPERIMENTAL
Reagents
All the reagents were of analytical grade purchased from Sigma-Aldrich, china while distilled
water was prepared in our own laboratory using Milli-Q® direct water purification system.
Preparation of Metal Complexes
Different metal- L-methionine complexes were synthesized by preparing different metal salt
solutions and L-methionine solutions of equal molar concentration and made the volume of each
solution up to the specified mark by adding distilled water. Metal salt solution and L-methionine
solution was mixed up and then adjusted the pH by pH meter by adding 1M NaOH or 0.1M HCl.
Solution was then placed on hot plate after adjusting temperature between 70-90°C. When volume
remained to about 15-20ml then solution was cooled in the refrigerator and crystals were obtained.
Best yield of Calcium and magnesium L-Methionine complexes were achieved at pH 8-10 by
adding few drops of 1M NaOH and that of copper, zinc ,cobalt and iron L-Methionine complexes
were achieved at pH 4-6 by adding few drops of 0.1 HCl.
In the present work, complexes have been prepared by using concentrated reactants and
modifying the procedure to get pure crystals of these complexes. 0.5M solution of CaCl 2.2H2O
was prepared by dissolving 3.675g of calcium chloride in 50ml of water in 100ml measuring
flask.0.5M solution of L-Methionine was prepared by dissolving 3.725g L-Methionine in 50ml of
water. These two solutions were mixed up and pH was maintained at 8-10 by pH meter by using
few drops of NaOH solution. This solution was then refluxed for an hour and the change in the
color was noticed. This refluxed solution was heated in china dish to reduce the volume to 1520ml. The dish was then cooled at room temperature and was then kept in refrigerator for
crystallization of complex. The lumps of crystals were observed. The crystals were washed with
organic solvent i.e. 9ml acetone with 1ml of water. The solvent was pouring off and crystals were
dried in open air and kept in sample voils. Similarly 0.5M solution of FeSO4.2H2O was prepared
by dissolving 9.392g of iron sulphate in 50ml of water in 100ml measuring flask. 0.5M solution of
L-Methionine was prepared by dissolving 3.725g L-Methionine in 50ml of water. These two
solutions were mixed up and pH was maintained at 4-6 by pH meter by using few drops of HCl
solution. This solution was then refluxed for an hour and the change in the color was noticed. The
same process was repeated as above. In the same way, 0.5 solution of MgCl2.6H2O was prepared
by dissolving 3.285g of magnesium chloride in 50ml of water in 100ml measuring flask. This was
0.5M solution of MgCl2.6H2O. 0.5M solution of L-Methionine was prepared by dissolving 3.725g
L-Methionine in 50ml of water. These two solutions were mixed up and pH was maintained at 8-9
by pH meter by using few drops of NaOH solution. This solution was then refluxed for an hour
and the change in the color was noticed. This refluxed solution was heated in china dish to reduce
the volume to 15-20ml. The dish was then cooled at room temperature and was then kept in
refrigerator for crystallization of complex. The lumps of crystals were observed. The same process
was repeated once again as above. 0.5M solution of CuCl2.2H2O was prepared by dissolving 4.25g
of copper chloride in 100ml measuring flask. 0.5M solution of L-Methionine was prepared by
dissolving 3.725g L-Methionine in 50ml of water. These two solutions were mixed up and pH was
maintained at 4-6 by pH meter by using few drops of HCl solution. This solution was then
refluxed for an hour and the change in the color was noticed. This refluxed solution was heated in
china dish to reduce the volume to 15-20ml. The dish was then cooled at room temperature and
was then kept in refrigerator for crystallization of complex. The lumps of crystals were observed.
The same process was repeated once again. 0.5M solution of ZnSO4.7H2O was prepared by
dissolving 6.55g of zinc sulphate in 50ml measuring flask. 0.5M solution of L-Methionine was
prepared by dissolving 3.725g L-Methionine in 50ml of water. These two solutions were mixed up
and pH was maintained at 4-6 by pH meter by using few drops of HCl solution. This solution was
then refluxed for an hour and the change in the color was noticed. The same process was repeated
once again as above. 0.5M solution of CoCl2.6H2O was prepared by dissolving 5.947g of cobalt
chloride in 100ml measuring flask. 0.5M solution of L-Methionine was prepared by dissolving
3.725g L-Methionine in 50ml of water. These two solutions were mixed up and pH was
maintained at 4-6 by pH meter by using few drops of HCl solution. This solution was then
refluxed for an hour and the change in the color was noticed. The same process was repeated once
again.
Analysis of Soil and Water Samples
Soil test was performed to estimate the nutrient supplying power of soil before the crop was
planted. 15 samples were taken randomly and composite sample was prepared for final analysis.
Samples from 15-25 cm, 15-30 cm and 60-100 cm depth were obtained from Sorghum field
respectively in a grid pattern separately by stainless steel soil auger and stored in plastic (HDPE)
bags until analysis. 500 mL water sample from area located for plant cultivation was taken and
stored in plastic bottles which were soaked with concentrated hydrochloric acid overnight then
washed with distilled water. The water samples were preserved by adding 2 drops of concentrated
nitric acid. All the soil samples were crushed and ground in wooden pestle mortar and sieved
through 2 mm plastic sieve. A saturation paste of soil was made by taking 300 g dry mass in
enameled cup and stayed overnight to ensure the saturation then saturation extract was obtained.
The pH and electrical conductivity (salinity) bicarbonates (HCO3-), carbonates (CO32-) and
chlorides (Cl-) of soil was determined using saturated extract. The metals were determined by
digesting the 1.0 g dried soil in 4 mL in aqua regia (Issam and Antoine, 2007; Mwegoha and
Kihampa, 2010).
Instrumentation
The soil and water pH and electrical conductivity (ECe) was determined by Orion 5 star multimeter (Thermo scientific, UK), sodium (Na) and potassium (K) were analyzed by Flame
photometer (Sherwood, UK), essential metals like calcium (Ca), magnesium (Mg) and trace
metals (micronutrients) like zinc (Zn), iron (Fe), copper (Cu), cobalt (Co) and manganese (Mn)
were estimated by Atomic absorption spectrophotometer (PG-990, PG-Instruments, UK), HCO3-,
CO32- and Cl- were determined titrimetrically. The elemental analysis carbon (C), nitrogen (N),
sulphur (S) and hydrogen (H)) of prepared complexes were performed by Flash HT Plus elemental
analyzer, Thermo, UK.
Application of Micronutrients to Sorghum
In order to test the effects of micronutrients on plant growth, two plots of 36000 feet2 (1 Acre) were
selected for each of the plant species. One plot was designated as controlled whiles the other as
sample. After 30 days of sowing the crop, the chelated micronutrients were applied to the crops.
Foliar application was chosen for these experiments. Foliar application was chosen for these
experiments.
Different amounts of micronutrients were added in the formulations for different crops as
reported in table-3, by using the rough estimates of micronutrient demand for specific crop through
literature (Mengel, 1990), different formulations were tested, but only which produce best results are
reported. About 60gram of Amino Acids complexes were dissolved in 2Liter of water. The 500 mL
of each formulation was further diluted in eighty liter water. In this way, 1.5L of micronutrient was
sprayer per 36000 feet2 of the cultivated land in three successive sprays. Second spray was
conducted after 27-30 days, the growth of plant in sample and control was monitored. The third
spray was conducted before the ripening of the crop. At the end, the % increase in yield of crop of
Sorghum was calculated.
RESULTS AND DISCUSSION
Before application of micronutrients to selected fields the respective field soil and water samples
were tested. Physiochemical characteristics of soil and water before the application of
micronutrients are presented in table 1.
Table 1: Physiochemical Characteristics of Soil and Water
Test
Soil
Water
ECe(dSm-1)
1.35
0.81
pH
7.67
7.37
Ca2+ (mmol/L)
4.91
3.61
Mg2+(mmol/L)
1.98
1.52
Na+(mmol/L)
5.31
2.63
K+ (mmol/L)
1.65
0.73
CO32- (mmol/L)
0.56
0.04
HCO3- (mmol/L)
1.57
0.71
Cl- (mmol/L)
2.25
1.41
Zn2+ (ppm)
18.90
1.32
Fe2+ (ppm)
31.56
1.02
Co2+(ppm)
2.17
0.23
Cu2+(ppm)
5.93
1.45
Mn2+(ppm)
16.89
1.27
Electrical conductance of soil is 1.35dSm-1 while water having 0.81 dSm-1, pH of soil is 7.67 while
water having 7.37. Ca2+, Mg2+,Na+ , K+, CO32-, HCO3- and Cl- of soil are 4.91, 1.98 ,5.31, 1.65,
0.56, 1.57 and 2.25(mmol/L) while water having 3.61, 1.52, 2.63, 0.73, 0.04, 0.71 and
1.41(mmol/L), respectively. The concentrations of Zn2+ , Fe2+ , Co2+, Cu2+ and Mn2+ in soil are
18.90,31.56, 2.17, 5.93and 16.89ppm while water having 1.32, 1.02, 0.23, 1.45 and 1.27ppm
respectively.
All the metals form complexes with L-Methionine in 1:1 which were confirmed by AAS and
elemental analysis. The metal and elemental confirmatory results are summarized in table-2.
Table 2: Analytical data of complexes
Complexes
Calc.(Found)%
S
C
N
Theo.
Exp.
Theo.
Exp.
Values
Value
Values
Values Value
s
Theo.
Metal
Exp.
Theo.
Exp.
Value
Value
Value
s
s
s
s
C5H10NO2SCaCl[223.5]
14.31
14.25
26.84
26.82
6.26
6.12
17.89
17.84
C5H10NO2SMgCl[183.5]
17.43
17.37
32.96
32.78
7.62
7.69)
13.07
13.01
(C5H10NO2SFe)2SO4[50
19.05
18.98
23.82
23.75
5.55
5.47
22.17
12.16
C5H10NO2SCoCl [242.4]
13.20
13.32
24.75
24.67
5.77
5.74
24.29
24.39
C5H10NO2SCuCl [247]
12.95
12.81
24.29
24.25
5.66
5.73
25.70
25.59
C5H10NO2SZnCl
12.85
12.88
24.10
24.17
5.62
5.70
26.27
26.24
3.68]
[248.89]
Different amounts of micronutrients were added in the formulations for different crops as reported
in table 3, by using the rough estimates of micronutrient demand for specific crop through
literature (Mengel, 1990), different formulations were tested, but only which produce best results
are reported. Soil fertility increases with addition of micronutrients to soil. When these
micronutrients are added in soil or given to plant directly, the plant shows a remarkable growth
which increases the crop yield. Similar effects have been observed in the present work. With the
addition of micronutrients the growth of the plant increases and the sample plants show
remarkable difference as compared to control crop.
Table-3
Amount required for micronutrients for SORGHUM (jawar) plant for 1 Acre
Complex
Amount in
Gram/2L
Zn-L-methionine
24.8
Mg-L-methionine
16
Co-L-methionine
0.17
Ca-L-methionine
7.11
Cu-L-methionine
0.44
Fe-L-methionine
10.22
. Soil fertility increases with addition of micronutrients to soil. When these micronutrients are
added in soil or given to plant directly, the plant shows a remarkable growth which increases the
crop yield. Similar effects have been observed in the present work. With the addition of
micronutrients the growth of the plant increases and the sample plants show remarkable difference
as compared to control crop and are reported in table 4-5 respectively.
Statistical data of crops
Table-4
Height of Plants (Average of 10 Plants)
Plant height
First
Spray
after Second Spray after Third Spray after
four Weeks in Inches four Weeks in Inches four Weeks in Inches
Sample
34
83
124
Control
26
51
97
Increase in Height
8
32
27
Table-5
Crop
Plant mass in Mass of seed Diameter
of Length
of
kg
in kg
stem in mm
root in Inches
Sample
12816
2.205
21
12
Control
10348.8
1.845
17
17
%age
23.8%
19.5%
23.5%
increase
After four week of first spray, the height of control plant was 26 inches while the height of
sample plant was 34 inches .Increase in plant height was found to be 8. After four weeks of 2 nd
spray, the height of control plant was 51 inches while the height of sample plant was 83 inches.
The increase in height was found to be 32. After four weeks of third spray, height of control plant
was 97 inches while the height of sample plant was 124 inches .Increase in height was found to be
27 inches. The mass of control plant was 10348.8 kg whereas mass of sample plant was 12816kg.
The increase in mass was found to be 23.8% at the end of ripening of the crop. The mass of seeds
of control plant was 1.845 kg whereas mass of seed of sample plant was 2.205kg. The increase in
mass was found to be 19.5% at the end of ripening of the crop. The length of root of control was
17 inches while the length of root of sample plant was 12 inches with greater thickness of root.
CONCLUSION
In the present work, amino acids complexes with essential metals such as Ca, Mg, Cu, Co, Fe and
Zn were synthesized and characterized by Atomic Absorption Spectroscopy, FTIR and melting
points and solubility studies. The present methodology gives approximately 47.25%Zn-Lmethionine complex, 35.33% Mg-L-methionine complex, 42.64% Co-L-methionine complex,
56.86% Ca-L-methionine complex, 68.41% Cu-L-methionine complex and 61.72% Fe-Lmethionine complex yield respectively.
From these complexes, an extra ordinary increase in crop yield up to 20-25% was observed by
using only 45g/Acre of these complexes.
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