EFFECT OF PLANT GROWTH HORMONES, MUTAGENS AND DYE EFFLUENT ON SEED GERMINATION AND ON ENZYME ACTIVITY

CONTENT

Sr.No

1 } Abstract

2 } Introduction

3 } Aim & Objective

4 } Experimental

A} Instrumental
B} Materials / Chemicals
C} Methods
A} soaking & Germination process
B} Extraction of cotyledon arginase
C} Arginase activity assay
D} extraction of anthocyanin
E} paper chromatography
F} Thin layer chromatography
5 } Results and Discussion

6 } References

ABSTRACT
Most of the studies on plant arginase have focused on its role in mobilizing arginase during early seedling germination.

The arginase was isolated from vigna unguiculata. The effect of plant growth hormones , dye and mutagens on seed germination and arginase was studied.

Arginase activity increases sharply during germination in several species including cow pea.
Anthocyanin is one of the important pigment present in seed coat. This anthocyanin gives
Colors to the seeds and also present in cow pea and well studied by the paper chromatography and thin layer chromatography. The anthocyanin pigments are isolated from the different mutant varieties of cow pea and Rf Values are calculated by paper chromatography and thin layer chromatogarphy.
















INTRODUCTION

Enzymes are the most essential biomolecule ,that catalyses most of the metabolic processes
of the cell. They are proteins and have immense catalytic power and specificity.
Areginase is an important enzyme in urea cycle, which catalyses the conversion of arginine to urea and L- Ornithine and is one of the six members of the urea cycle enzymes, that convert ammonia to urea as the principle product of nitrogen excretion.
Arginase is a manganese containing enzyme. The reaction catalysed by this enzyme is

Arginine + H2O -------- L-Ornithine + urea .

The enzyme participates in krebs –Hensleit urea cycle, this enzyme is present in developing broad bean germinating seeds.
Arginase is most highly concentrated in mammalian liver. Arginase is also present in mammary glands where the urea cycle is not present .It has been suggested that,the ornithine is converted to proline.
Most of the study on plant arginase show its important role in mobilizing arginase during early seedling germination.( Chris kolloffel etal 1974)
There are several arginase isozymes ,a liver isozymes takes part in the final step of the urea cycle in ureotelic animals .Other isozymes take part in the frist step of arginine degredation in various cell types (the kindey ,small intestine and lactating mammary gland).

If this enzyme is deficient or not working properly, it causes the amino acid arginine (building bolck of proteins) and ammonia which is formed when proteins are broken down in the body ,is toxic if level become too high. The nervous system is specislly sensitive to effect of excess ammonia. Arginase deficiency is the common urea cycle disorder. (Masataka mori& Tomani Gotoh 2004).
Two isozymes of Arginse, arginase –I is found in the liver and contributes the vast majority of hepatic arginase activity whereas type – II is inducible and found in extra hepatic tissues.

Arginase deficiency show following symptomS :- (Karl S Roth)
• Heavy and rapid breathing
• Irritability
• Vomiting
• Poor growth may be observed
• Hyperammonemia
• Respiratory failure may occur in latter stage
• Coma
• Cerebral edema
• Death (if treatment is not effective)

The main treatment for arginase deficiency is a low - protein diet foods that need to be avoided:

• Milk ,cheese, and other dairy product
• Fish
• Eggs
• Butter
Eating these foods can cause ammonia and arginase to build up, resulting in the symptoms described above. So there is important mean to find out arginase source and its study from plant origin. Because our daily food content mainly coming from plants.










PLANT GROWTH HORMONE

The substances which are required in very small amounts for the growth and development of plants are known as growth regulating substances or plant growth hormones. These are the organic compounds other than nutrients, which in small amounts promote, inhibit or otherwise modify any physiological process in plants.’

Auxins i.e. Indol Acetic Acid (IAA), Indol Butyric acid (IBA) and Gibberellins (GA) inhibit the seed germination at high concentration.

Auxins and Gibberellins are one of the most important groups of plant growth hormones because they regulate many physiological processes in plants

Indol acetic acid and Gibberelic acid promotes the seed germination as concentration of growth hormone decreases and also the arginase activity also increases.

These plant hormones are generally applied in very low concentration, higher concentraition inhibit the germination and growth and exert toxic effects on plants
( Davies PT (1995)












DYE

Dyes are the most commonly used chemicals in textile, food, paper making, color – printing, leather and cosmetic industries. There are around 10.000 different dyes are available commercially. The textile industries daily discharge million liters of untreated effluents in the form of wastewater into public drains that eventually goes into rivers. This alters the biochemical oxygen demand and gives intense coloration to rivers.

Particularly for small scale textile industries as they have poor working conditions and weak economic status, do not allow them to treat their wastewater before disposal. These textile dyes are very toxic and carcinogenic to all ecosystems. They affects plants, animals, soil, water and atmospheric environment by polluting them [ V.V.Dawkar et.at.(2007)]

The waste water which is not treated, when we use it in agriculture to plant or any crop to develop they can not grown, the growth is totally inhibited but when dyes are degraded by using some bacterial species, these dyes are decolorized and also show remarkable decrease in its toxicity.( V.V.Dawkar et al.2007) If this degraded dye – waste water we use to develop plant or crops they show growth in some extent and also prevent environment from pollution.












MUTAGENS

Mutations occur spontaneously by the environmental effect, they can be induced in the laboratory either by radiations, physical factors or chemicals these are called mutagens.
Mutations occur frequently in the nature and have been reported in many organism e.g. Drosophila, mice and other rodents, rats, rabbits, guinea pig and man. In rodents the mutations are responsible for black white and brown coats.
There is different type of mutagens which causes mutations in organisms, seeds, different animals etc.
1) Radiation: - The radiations which are important in mutagenesis are of two categories. Ionizing radiations such as X – rays, gamma rays, alpha & beta rays, electrons, neutrons, protons and other fast moving particles.
Non – Ionizing radiations such as ultraviolet and visible light.
2) Temperature: - The rates of all chemical reactions are influenced by temperature can be mutagenic.
It is not surprising that temprature can be mutagenic. It is reported that the rate of mutation is increased due to increase in temperature.
3) Chemical mutagens:- The ability of chemicals to induce mutations was first demonstrated by Auerabach and Robson in 1947. Using mustard gas related compounds as the nitrogen and sulphur.
All these mutagens, develops the new colored variety in seeds. These mutagens
Produces different colored pattern of seeds coats.
A Gustafsson has estimated that less than one in 1000 mutants produced may be useful in plant breeding. In India several useful mutations of various cereals and other crop plants have been developed.
This mutated variety can create harmful effect or some significant change in the seeds. The effect of the mutagens on enzyme level is an important point.




ANTHOCYANIN

Anthocyanin are the largest group of water soluble pigments in the plant kingdom, that are responsible for the blue, red, and purple colors of many fruits, vegetables, grains, flowers and leaves.
Over 300 structurally distinct anthocyanin have been identified in nature. Anthocyanins are one class of flavonoid compounds which are widely distributed plant polyphenols. " Eating healthy” has been a common slogan among educated consumers this means eating a balanced diet that provides sufficient proteins, carbohydrates, fats and vitamins.
With the discovery of health benefits of certain plant compounds that have antioxidant capabilities the term is changing and now includes such compounds as cartotenoids, flavonoids etc. which are beloved to posses tremendous health benefits such as antioxidant activity.
Cow pea seeds are one of the important, so many of the scientists are now focused on its study. Dietary antioxidant are the substance which are present in foods that significantly decreases the adverse effects of relative oxygen species, nitrogen species or both on normal physiological functions in humans.
Antioxidants are compounds that inhibit or delay the oxidation of other molecules by inhibiting the initiation or propagation of oxidizing chain reactions(Velioglu et al-(1998) )

Anthocyanin plays an important role in many diseases like
• Cancer
• Inflammation
• Diabetes
• Bacterial infections.

Its importance in health has focused its study from the different sources.



SOURCE:-

Botanical Name: - Vigna Unguiculata

Common Name: - Cow Pea.


Scientific Classification:-

Kingdom - Planate
Division - Magnoilphyta
Class - Magnoliopsida
Order - Fables
Family - Fabaceae
Sub family - Faboideae
Genus - Vigna
Species - Unguiculata


AIM AND OBJECTIVE

Arginase is the most important enzyme in the urea cycle. Arginase removing ammonia from the body which is a harmful substance. The heath depends on the concentration of arginase in the body.
Considering high importance and various applications of arginase it is essential to find out arginase from their sources. There are several different sources from which arginase can be extracted. The different treatments of plant growth hormone, dye and mutagens affect the arginase activity and seed germination which is essential thing in study of the enzyme level.
Another important part of seeds are the pigment, which give different colors to seeds. Seed coat contains different types of anthocyanins, which can be studied by paper chromatography or thin layer chromatography. These anthocyanin pigments are important in health, which show antioxidant property.

















Materials:-

A] Collection of samples :- Samples were collected from the local market and also brought from prof.M.T.Patil .(department of Botany)

B] Chemicals:-
1) Anhydrous Sodium Carbonate
2) Sodium Carbonate
3) 2 Mm mncl2
4) 130 Mm L- Arginine
5) 10 % TCA
6) Acetic acid
7) Ninhydrin
8) Tris-hcl
9) 10 % glycerol
10) Potassium chloride
11) O – Phosphoric acid
12) Indol Acetic acid ( IAA)
13) Indol Butyric acid (IBA)
14) Gibberelic acid
15) Dye (Malachite green)
16) Degreded dye
17) n- butanol
18) methanol
19) HCL
20) Chlorofrom
21) silica gel
22) Acetone



C ) Instruments / Apparatus :-
1) Centrifuge machine
2) Centrifuge tube
3) Colorimeter
4) Boiling water both
5) Mortar – pestle
6) Petri plates
7) Test tubes
8) Beakers
9) Pipettes , micro pipette
10) Thermometer
11) Glass plate for etc.
12) Whatman No.1 Filter paper
13) Iodine chamber.

Methods : -

A) Soaking and germination process:-
Different concentration of Growth hormone gibberelic acid & indol acetic acid (1000ppm,100ppm,10ppm,1ppm,and 0.1ppm) was prepared and seeds of cow pea was kept for soaking and also seeds of cow pea was kept for soaking in distilled water ,degraded dye and dye solutions for 4 hours .After that all seeds were kept for germination in Petri plates .

B) Extraction of cotyledon arginase :-
Extraction of enzyme was carried out at 0-4oc .The 1gm of germinated cotyledons were homogenized in a mortar –pestle with 3 ml of 10mM tris-cl (pH7.6) containing 0.6 mm Mncl2 100mM KCL and 10 % glycerol along with sand .Then ,this homogenate was clarified by centrifugation at 10.000 rpm for 5 minute

C) Arginase activity assay:-
Briefly the reaction mixture consisting of 10mm carbonate bicarbonate buffer (PH9.2), 2mM Mncl2,130 mM L-arginine and enzyme solution in a total volume 3.0ml was incubated for 30 minute at 37o c. The reaction was terminated by adding 10 % TCA .Then 1 ml from above mixture was mixed with 1ml of glacial acetic acid and 1 ml Ninhydrin reagent .The mixture was kept in boiling water bath for 20 minute .The color developed was measured at 520 nm on colorimeter (Roman& Ruys method )

D) Extraction of anthocyanin :-
Seeds of different mutant varieties of cow pea was soaked over night in water. Then next day seed coats were separated from the seed. 200 mg of fresh weight of seed coat was then ground in a mortar – pestle with 5ml of 1 % HCL in methanol .The extract was filtered and washed with 5ml of petroleum ether to remove lipids and polyphenols. This partially purified extract was concentrated by evaporating overnight at room temperature and used for paper chromatography and thin layer chromatography .(R.N.Pandey& S.E. pawar ,2000)

E ) Paper chromatography : -
The compounds were separated on what man no.1 filter paper. The n- butanol: Acetic acid: Water system was used. Firstly, what man filter paper was run in solvent system and dried completely. After the first run, sample spots are given on what man filter paper and again run in same solvent system .Paper was removed and dried completely and kept in iodine chamber.
The mobility (Rf values) of the bands of the individual anthocyanin was noted down then , the bands of the chromatogram were eluted with 0.1 % methanolic- Hcl and absorption maxima was measured on spectrophotometer .
(R.N. Pandy & S.E.pawar 2000, J.Raman 1999)



F) Thin – Layer Chromatography:-
Plate for TLC was prepared. After that sample spots were given on the plate and dried completely .The plate were run in n- butanol : Acetic acid : water system .After running ,the plate was removed and dried ,and kept in iodine chamber for visualize spots. Then the mobility (Rf) value of each sample spot were noted down. (J.Raman 1999)





















RESULT & DISCUSSION

1) Effect of plant growth hormone :-
Indol acetic acid (IAA)


IAA 1 IAA 2 IAA 3 IAA 4

Observation table :-

Conc. Of hormone IAA 1
(100 ppm) IAA 2
(10 ppm) IAA 3
(1 ppm) IAA 4
(0.1 ppm)
Length of
Seedling in cm 0.97 2.33 2.96 3.35
Optical density
At 520 nm 1.49 1.55 1.58 1.28

Plant growth hormone, Indol Acetic Acid increases the seed germination as well as arganase activity. But arginase activity is maximum in one ppm concentration. The arginase activity increases during the seed germination.


1 } Gibberelic Acid (GA) :-


GA 1 GA 2 GA 3 GA 4

Observation Table:-

Concentration of
Hormone GA 1 GA 2 GA 3 GA 4
Length of
Seeding in cm 2.85 3.43 3.48 3.70
Optical density
At 520 nm 1.54 1.62 1.65 1.55


Gibberelic acid increases the germination of seeds and increases the arginase activity when counterattraction of growth hormone decreases. But arginase activity was more in 1 ppm concentration.
High concentration of growth hormone inhibits the seed germination but low concentration of growth hormone promotes the seed germination.


2 } Effect of Dye degraded Dye :-


Distilled Water Degraded Dye Dye



Observation Table :-

Distilled water Degraded Dye Dye
Length of seeding
in cm 4.45 1.95 0.02
Optical density
At 520 nm 1.50 1.39 1.39

The germination in distilled water is more than the degreded dye and dye solution. Dye solution totally inhibits the seed germination but degraded dye show the germination but less than distilled water. This suggest that, if we degraded the dye before disposal it does not affect the maximum seed germination and also neutralizes its harmful effect.
Arginase activity is more in distilled water than the degraded dye & dye. It shows that the dye when degraded it does not affect arginase activity.


3 } Effet of mutagens

Observation Table:-

Sr. No Variety of cow
pea (mutated) Length of Seedling
in cm after Optical density at
520 nm after

12 hrs 36 hrs 12 hrs 36 hrs
1} Control 0.00 0.55 1.17 1.68
2} E- 28 0.00 0.00 1.24 1.69
3} B- 3 0.55 0.88 1.30 1.68
4} B- 123 0.85 2.28 1.35 1.68
5} G- 69 0.60 0.70 1.27 1.67
6} H- 98 0.00 0.00 1.29 1.67
7} D- 29 0.00 0.00 1.41 1.67
8} 0.05% 0.90 1.9 1.36 1.79
9) I -50 0.65 1.32 1.22 1.45
10} C-22 0.90 0.90 1.20 1.20
11} F-100 0.35 083 1.17 1.65
12} F-1 1.25 1.25 1.25 1.74
13} A-68 0.17 1.08 1.30 1.68
14} J-36 0.70 0.98 1.48 1.76

The above results show that, there is no effect of mutagens on araginase enzyme. The gene which is responsible for arginase is not mutated so there is not remarkable change in enzyme activity. Seed germination is also not fast mutated variety.
Paper chromatography profile of anthocyanin from cowpea mutants:-

Sr.No Variety Rf Values
Band –1 Band –2 Band-3
1} Control 0.19 0.26 0.69
2} Black - Red 0.19 0.26 0.69
3} Black – Eyed bold 0.19 0.26 0.69
4} Dark – Pink 0.19 0.26 0.69
5} Dark – Red 0.19 0.26 0.69
6} Faint – Red 0.19 0.26 0.69
7} Pink 0.19 0.26 0.69
8} B & W Hybrid 0.19 0.26 0.69
9} Medium – Red 0.19 0.26 0.69
10} Reddish Black 0.19 0.26 0.69
11} Black 0.19 0.26 0.69
12} Bold Red 0.19 0.26 0.69

The mobility of anthocyanins, Rf values by paper chromatography is observed. In all varieties three bands are same Rf values in band – 1, band –2 and band –3. There are no changes in Rf values, it suggests that, the mobility of the anthocyanins are same.
Thin layer chromatography profile of anthocyanins from cowpea mutants :-


Sr. No. Variety Rf Vlues
Band – 1 Band – 2 Band – 3
1} Control 0.45 0.59 0.93
2} Black – Red 0.45 0.62 0.93
3} Black – eyed bold 0.53 0.78 0.93
4} Dark – Pink 0.53 0.79 0.93
5} Dark – Red 0.52 0.81 0.93
6} Faint – Red 0.52 0.82 0.93
7} Pink 0.52 0.77 0.93
8} B &W Hybrid 0.52 0.77 0.93
9} Medium – Red 0.54 0.83 0.93
10} Reddish – Black 0.52 0.77 0.93
11} Black 0.52 0.78 0.93
12} Bold - Red 0.53 0.79 0.93

The Rf values of anthocyanin observed in twelve different varieties of cow pea seeds show different Rf values. In all varieties, first two bands show different values, but Band-3 Show same Rf values observed by paper chromatography and Thin – layer chromatography.
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STUDIES ON EFFECT OF DIFFERENT AGENTS ON SEED GERMINATION ALONG WITH ARGINASE ACTIVITY AND CHARACTERISATION OF ANTHOCYANIN PIGMENT FROM Vigna Unguiculata.