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1. Introduction 1.

1 Germination and Ionizing radiation stress Plants and environmental stresses have been of most importance as a field of study and research throughout the ages. It was found that the different stresses that plants are exposed to are constantly increasing and detrimental in reducing the crop yield. Of these stresses, temperature, drought, salinity and microwaves are of major importance ( Reddy et al., !!"#. It has been long $nown that plants are sensitive to ioni%ing radiations such as beta particles and gamma rays. In recent research wor$s, seeds or unfertili%ed flower buds and whole plant or part of it, have been irradiated in order to investigate the effects of these radiations on plant. &tudies have shown that many plants or seeds can be exposed to radiation of different types at various intensities and specified that appreciable doses can either inhibit growth or $ill the plant completely. 'he radio sensitivities of different plants vary very greatly. (hile it re)uires a few million rads to $ill bacteria, more highly developed plans can be $illed by less than a thousand rads. *fter the end of the &econd (orld (ar, research on the effects of ioni%ing radiation on plants life began to attract general interest and systematic wor$ of botanists

+,ac$ey, !-./ *jayi and 0arsson( !! # have reported observations about the effects of nuclear radiation on seed germination and seedling growth in wheat, barley, etc., * general observation is that the seedling height decreases with increasing radiation dose. Other observations are that storage of seeds after being irradiated increases the radiation damage on seeds1 water absorbed by seeds immediately after irradiation could reduce the damaging effects of ioni%ing
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radiation +*jayi and 0arsson( !! #/. Presently, there is little research being done on effects of ioni%ing radiations on plants. In plant cells, the nucleus is considered the principal site of damage by ioni%ing radiation. 'he process leading to radiation damage may be summari%ed as2 the initial physical stage which lasts only a minute fraction of a second1 the physico3chemical stage lasting about 43 -s1 the chemical stage lasting a few seconds and the biological stage in which the time scale varies from tens of minutes to tens of years depending on the particular symptoms. Recent studies reported that there was a direct relationship between the radiosensitivity of a herbaceous plant and the average volume occupied by a chromosome in the cell nucleus. 'he larger the chromosome volume, the more sensitive is the plant and, hence, the smaller the radiation dose re)uired either to $ill the plant or to cause a certain degree of damage. * simple relationship has been found to exist for woody plants. Improved )uality and )uantity of crops have been achieved through radiation3induced mutation. *chieved important desirable properties include2 improved lodging resistance1 changed maturing times1 increased disease resistance1 increased yields1 improved agronomic characters1 and improved seed characteristics +I*5*, !!4/. 6owever, the harmful effects of deleterious mutation include death in the embryonic state, inability to reproduce, increased susceptibility to disease and decrease in life expectancy of a few months. *ll these observations prove that radiation may affect the 78* in an organism. 7ifferent types of radiation emit differing amounts of energy.
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,icrowaves have low levels, ultraviolet has a slightly greater amount than visible light, and x3rays have )uite a bit. 5xposure to these rays may result in mutations and growth problems. 1.2 Microwaves ,icrowaves are important because people use them in everyday life, but microwaves might also be harmful to living organisms. ,icrowaves have wavelength approximately in the range of 94 cm (fre)uency: ;6%# to mm (944 ;6%#. 'he microwave range includes ultra3high

fre)uency (<6=# (4.9349;6%#, super high fre)uency (&6=# (9394 ;6%# and extremely high fre)uency (56=# (943944;6%# signals. *bove 944 ;6%, the absorption of electromagnetic radiation by earth atmosphere is so great that it is effectively opa)ue, until the atmosphere becomes transparent again in the so3called infrared and optical window fre)uency ranges. ,icrowaves were found to affect plants and plant growth severally. 0ong exposure to microwaves affects the germination of seeds or plants. 6owever, wea$ microwaves don>t effect plant growth but as the microwave dose increases, the rate of germination decreases. ,icrowaves often coo$ seeds when microwave ovens heat up green beans, peas or other cold and sometimes raw fruits and vegetables. 'hese seeds, li$e pump$in seeds that have been in the oven, can no longer sprout, but have been essentially coo$ed and are only good for food stoc$. &eeds that have been subjected to microwave radiation, even when not microwave for food purposes, have a similar reaction. * study completed by the 5nvironmentors? Program irradiated wheatgrass seeds with microwaves to study their germination. 'he study found that germination
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Plate .1

Nature of Microwave radiation

itself was terminated in cases of heavy exposure to microwaves and sprouting of the seed mar$edly decreased in all cases where the seed had been directly irradiated with microwaves before planting. 1.2.1 Effect of radiation on Water *lthough radiation has a detrimental effect on seeds in a direct microwave application, integral partners in a seed?s eventual development can also be irradiated. (ater, for example, is as integral to a seed?s germination as the seed itself and has also been irradiated in experiments with very different results. Plants that ingested microwaved water grew consistently faster and taller than those that had no exposure to microwave radiation. 'he water was microwaved, allowed to cool and then fed to the seeds which sprouted faster and taller than the control which was not microwaved. 1.2.2 Effect of radiation on Soil <nless they have a hydroponic growing system, seeds need soil to sprout. 'he 5nvironmentors? experiment also irradiated soil and produced mixed results with a wide fluctuation regarding factors such as sprout emergence, general growth and plant development. 'he experiment found one general consistency in the length of time the soil was microwaved2 the longer a soil sample was microwaved, the shorter the plant that would develop in that soil environment. 'his would suggest a lac$ of nutrients in soil that has been exposed to high amounts of microwave radiation.

1.3. Pulses

*gricultural products grow to wider dimensions. 'he major *gro3products extend to crops li$e pulses, rice, wheat, barley etc. 'hey account for the average foodstuffs for the country and responsible for a healthy economy contributing for 9 @ of the countries ;7P and A.@ of India>s exports in agriculture. Pulses are basically grain legumes. 'hey occupy an important place in human nutrition due to their high protein content than cereal grains. In Indian dietary regime it occupies an important place. &ince majority of Indians are vegetarians, they depend largely on grain legumes (pulses# for their dietary protein. 0egumes contribute a major portion of lysine in the vegetarian diet. 'hey are also a fairly good source of vitamins li$e thiamine, niacin, riboflavin and much needed iron. 'hese crops have additional advantage for sustainable agriculture since they are nitrogen3fixing crops and provide an ecological alternative to chemical nitrogenous fertili%ers and also for its varied use as feed and fodder. 1. . Vigna radiate !".# Vigna radiate (L.) $nown as mung in Hindi, is the seed of Vigna radiate (0.# which is native to India. It is also $nown as golden gram (misleadingly# green so$. In the Philippines, it is called mungo or mongo. Binomial name % Vigna radiate !L.# Synonyms % Phaseolus aureus &o'(.

1. .1.Scientific classification
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Bingdom 7ivision Class Order =amily ;enus &pecies

Plantae

2 ,agnoliophyta 2 ,agnoliopsida 2 =abales 2 =abaceae 2 Vigna 2 Radiate

,ung beans are grown widely for use as a human food (as dry beans or fresh sprouts#, but can be used as a green manure crop and as feed for livestoc$. 1. .2. )*emical )onstituents
( A#

&eeds are high in carbohydrate (DE.@# and protein (DA @#1 fair source of calcium, iron, vitamins * and F. deficient in vitamin C. &prouts are a good source of vitamin F. &eeds are tonic and aperients.( Falatong,A44E# 1. .3. Medicinal +ro+erties

*nti scorbutic, diuretic, protective and curative. 1. . . Nutritional (enefits

Gegetable that is a source of iron and calcium, vitamins * and F. &prouted mongo (toge# is an excellent source of vitamin C, as well as vitamins * and F, calcium and iron .

1. .,.

Seed Pre+aration and Germination 'he major use of mung bean is for sprouts1 'herefore, excellent germination
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must be maintained by careful harvesting and storage systems. &eed is not generally treated with fungicides, insecticides or bactericides because of the possibility of ingestion of treated seed. *s the seed is small, careful handling and attention to planting machinery adjustments is necessary to ensure planting with little damage to the seed. 'he yields of mung beans depend largely on weather conditions, soil, cultural practices, and variety. Hields can range from 944 to over A,444 pounds per acre. Hields from second crop plantings are not as large as main crop yields. 'he food industry desires to obtain about ! or 4 grams of fresh sprouts for each gram of seed and larger seed with a glassy, green color seems to be preferred for the purpose.

Vigna radiate (L.) Plant (Plate No:2)

Vigna radiate (L). Seeds (Plate No: 3)

2. &eview of "iterature ,icrowaves are electromagnetic waves with wave lengths ranging from 4 "3 4 6% (,oeller, !!A#. *ccording to (i$ipedia (A44!#, Ithe term microwave generally

refers to alternating current signals with fre)uencies. ,icrowaves have three ranges ultrahigh fre)uency, super3high fre)uency, and extremely Jhigh fre)uency.K * microwave oven is a $itchen supply. People use microwave oven to coo$ or hot food. People also use microwave to dry, and cure plywood, paint, in$s, and synthetic rubber, and also to reheat blood rapidly after certain types of surgery (Lohnson, !"A.#

,icrowaves have a similar radiation as mobile phone and people suggest it could cause cancer (6erberman, A44"#. Previous research has not been consistent. Reddy et al., ( !!"# investigated relationships between microwave operating conditions and resulting seed )uality in terms of germination and seeding vigour. 'hey microwaved the wheat seed for A4, 94, E4, and .4 seconds and let them grow for M days in glass jars. 'hen, the measured the plumule and the radicle length in cm. Infected seeds not treated with microwave had 44@ germination. Reduced germination and increased tissue damages also were observed by others with increased treatment duration. &$iles (A44-# exposed alfalfa plants to microwave. 'hey grow the plants indoor for E months at a AM degrees Celsius before exposure to microwave. 7uring the M wee$s of exposure to microwave, they measured the distance nodes of the leaves. *fter the M wee$s of exposure to microwave, test and control plants were harvest. 'he plants were dried at "4 degree Celsius for AEh and dry weight measured. 'hey also measured soil temperature. 'he results were that there were no difference between the fresh and the dry weights treatment and control. 'here was not no difference with the soil temperature either.
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;os et al., ( !!M# studied how microwave fre)uencies between E .-" and E .M ;6% affected the yeast &accharomyces cerevisiae. 'hey used a series of test chambers with a controlled exposure system and found no difference between cells exposed to microwaves and those that were not. ,agone ( !!-# studied duc$weed grown in flas$s outdoor, A$m away from a radio station transmitter. 'hey used a fre)uency and intensity of the radiation which was .-3 -A,6% and 4. 3A.-u(NcmA and observed an increase in vegetative

reduction rates, what they term Igrowth disturbanceK. ,ura$ami et al., (A44 # studied Frassica campestris ($nown as Frassica Rapa var. silvestris in the <&#. 'hey used an enclosure with the broadcast antenna supporting on a outdoor pole with a fre)uency of A.E.;6% with an intensity between .4 and ..4m(NcmA. 'hey observed slight growth acceleration at the lowest intensity which they attribute to a slight increase on the soil temperature. 'hey saw wilting of plants at the center of the enclosure, but were unable to determine if this was due to soil warming or to drought. <rech et al., ( !!-# studied the lichens Parmelia tiliacea and 6ypogymnia physodea in field experiments of duration ranging from to 9 years. 'hey found

reduced growth rate due to thermal effects. Pica%o et al., ( !!!# investigated the electromagnetic fields on thistles and lentils. 'hey found that the thistles decreased in both weight and length and the stem length of lentils increased over 9 wee$s. 'he effect of electromagnetic microwave irradiation on agricultural crops has scarcely been studied yet.

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6owever, some studies regarding the effect of electromagnetic microwave irradiation on wheat detected increased of the germination (Fhas$ara et al., !!"#.

*lso, Fhas$ara et al.,( !!.# used successfully electromagnetic irradiation of the radiofre)uency range and of the microwave range on seeds of charloc$, wheat, soybeans, peas and rice with the aim of prestorage destroying of the microorganisms. Hoshida et al. A444, treated soybean seeds with microwave rays for - to A minute for improving the triglycerides distribution in the seed coat. Hi3Ping Chen, A44- revealed in their results that microwave pretreatment of Isatis indigotica seeds enhanced <G3F stress resistance in the seedlings, increasing the activities of catalase, peroxidase, and superoxide dismutase. *ll these results suggest that microwave radiation enhances plant metabolism. 'he effect of microwave treatment on the seeds and their characteristics of vitality has not been fully explained (*ladjadjiyan ., A44A#. &ome authors have conducted studies regarding the effect of microwaves on seed rape to enhance oil production (GalentovO et al.,. A444, 8ovotnO et al., !!!, Oberndorfer et al., (olfgang, !!!#. 'he used of microwave on the germinations seeds may have other utili%ations, such as for example it was demonstrate that microwave induced stimulation of 037OP*, phenolics and antioxidant activity in fava bean ( Vicia faba# for Par$inson>s diet (Reena et Balidas, A44E#.

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3. -im and Sco+e Plants and their stresses have been of very great importance lately. ,icrowaves is the focus of the present study because of the drastic effects that they have, not only on humans and different mammals but on plants. In brief, we can say that microwaves are electromagnetic waves with wave length longer than those of terahert% ('6%# fre)uencies, but relatively short for radio waves. 'here are lots of sites contaminated by microwave radiation from cellular radiation, power plants and high voltage towers, etc., 'hese affect the plants development, which drew our attention towards this project. 'herefore the aim of the study is to assess the effect of microwaves in different doses on the germination, growth and biochemical changes of (,ung beans#. 'his is carried out by 5xposing microwave radiation on seeds, soil and water at different doses prior to germination of seedlings 5valuating the germination changes, morphological and biochemical changes for each conditions. 'o assess the stress level in the growing seedlings, induced if any due to microwave radiation exposure and *lso to ascertain which among the three condition is affected most due to radiation exposure. Vigna radiate (0.#

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. Materials and met*ods

.1 Seed Material &eeds of ,ung bean (Vigna radiate (0.# for the study was procured from 'amilnadu 6orticulture 7epartment, 5rode. A44 healthy seeds of uniform si%e were pic$ed for germination. .2 .ine )*emicals and &eagents 'he chemicals used were also of analytical grade and obtained from &igma and 6imedia Chemicals. Commercial 0; microwave oven with fre)uency ranging between A.E3A.. ;6% .was used as a source of microwaves in the current study. .3 Germination Procedure &eeds were surface sterilised in 4. @ (wNv# mercuric chloride for A min, rinsed with sterile water to avoid fungal contamination and germinated in pots. 5ach pot was filled with A$g of pre3sieved soil. =or soil analysis, the soil sample was tested and authenticated by the &oil 'esting Centre run by 'amilnadu *gricultural 7epartment, 5rode 7ivision. . E'+erimental design 'wenty five healthy seeds of uniform si%e were maintained in each pot and the seeds were dropped at a depth of -3M cm in the soil. *ll planted pots were $ept in the open garden and irrigated twice a day with .4ml (each time# of the respective solution. 'he planted pots are subdivided into various groups as follows and the experiment was conducted over a period of A days.

)ontrol 3

,ung bean seeds that were $ept irrigated with 7istilled water and sampled as control.

/est 1 3

,ung bean &eeds that were microwaved for 4sec , prior to soa$ing of chic$ pea seeds for germination and irrigated with 7istilled water.

/est 2 3

,ung bean &eeds that were microwaved for 4sec , prior to soa$ing of chic$ pea seeds for germination and irrigated with 7istilled water. 14

/est 3 %

,ung bean &eeds that were microwaved for 4sec , prior to soa$ing of chic$ pea seeds for germination and irrigated with 7istilled water.

/est

,ung bean &eeds sown in soil that were microwaved for 4sec, and irrigated with 7istilled water. ,ung bean &eeds sown in soil that were microwaved for 4sec, and irrigated with 7istilled water. ,ung bean &eeds sown in soil that were microwaved for 4sec, and irrigated with 7istilled water. ,ung bean &eeds sown in soil and irrigated with 7istilled water that was microwaved for 4 sec. ,ung bean &eeds sown in soil and irrigated with 7istilled water that was microwaved for A4 sec. ,ung bean &eeds sown in soil and irrigated with 7istilled water that was microwaved for 94 sec.

/est , % /est 0 % /est 1 % /est 2 % /est 3 %

., )ategories of Microwave /reatment 'here were three experimental categories2 microwaved water, seeds, and soil. 5ach category was treated with microwave radiation for 4 seconds (control#, seconds, 94 seconds. 5ach microwave condition was repeated three times with three different cups. 'hese experiments were organi%ed according to the following matrix I. Microwaving t*e seeds 'he microwave was set for the corresponding time (4 sec, . sec, 94 sec, #, and the seeds were microwaved in a petridish so they could get the same microwave radiation. 'he seeds were ta$en out after the time expired and allowed to cool to room temperature. (hen they finished cooling, they were planted in the soil. .

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Plate

4 )ommercial "G Microwave oven used for t*e stud$

II.

Microwaving t*e Soil =or soil category, the seeds were not microwaved. 'he soil is microwaved in

the same orientation as the seeds were in the previous experiment and for the same
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times. 'hen it allowed to stand at room temperature for sometime. 'hen, water is poured and planted the seeds and cover it with soil. III. Microwaving t*e water =or the water, it is microwaved in the same orientation as seedsNsoil for the same times. 'he microwaved water is allowed to cool to room temperature, then poured in the soil and the seeds are planted. .,.1. Irrigating t*e Plants =or the experiments, the seed and soil conditions are watered with distilled water at room temperature. =or the water condition, on each watering day, water is microwaved in the same way as described above, allowed water to cool to room temperature before watering plants. E'+erimental +rotocol .0. P*ase 5 I G&6W/7 P-&-ME/E&S .0.1 Germination 8 ;ermination percentage is an estimate of the viability of a population of seeds. 'he germination rate provides a measure of the time course of seed germination. 'he germination @ is evaluated by daily observations on radical emergence. &eed germination percentage was calculated using the following formula +I&'*, !!!/2 ;ermination @ :8umber of germinated seeds N 'otal number of seeds P 44

It s*ould (e mentioned *ere t*at t*e seedlings treated wit* microwave radiation for 29 sec and 39 sec +rior to germination failed to germinate and t*erefore rest of t*e growt* +arameters and mor+*ological anal$sis were not carried out for test II and test III seedlings. .0.2. .or &oots
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'he plants were smoothly uprooted from the pots, cleaned from soil residues and prepared for measurements. 'he length of the root of each germinated seeds in each group(pot# is measured with the thread and the scale from (ee$ to (ee$ E.

'he length of each germinated seed is recorded on the data sheet and the root length of the plant is calculated for ,ean length for each wee$. .0.3. .or S*oots 'he same process was repeated for obtaining plant shoots by ta$ing the shoot portion of the seeds using the same procedure. .0. . Num(er of lateral (ranc*es 'he above described process was repeated and number of lateral branches per plant were recorded.
.1. !:# P*ase 5 II ME/-:6"I/E -N-";SIS .1.1 E'traction Met*ods E'traction of )ar(o*$drates gram of plant tissue was ta$en and crushed thoroughly in mortar and pestle, the fine powder is dissolved in . ml of distilled water and centrifuged at .444 rpm for supernatant was then made up to 44 ml in a standard flas$ with distilled water. 4 minutes. 'he

E'traction of Proteins gram of the plant tissue is weighed accurately and crushed thoroughly in mortar and pestled with . ml of 4.. , 'ris 6Cl buffer p6 M.. (chilled condition#. 'he volume was made upto 4ml using 'ris 6Cl buffer and centrifuged at 4444 rpm for A4 minutes. 'he supernatant was collected and used for the estimation. E'traction wit* -lco*ol 18

.or estimation of .ree amino acids and /otal +*enol =or the estimation of free aminoacids and total phenol, the tissues were cut into pieces and plunged into boiling alcohol and allow to boil for 4 minutes. 93 4 ml alcohol was used over 4.. gram of tissue. 'he extraction was done on top of a steam bath. 5xtract was cooled and the tissues were crushed in a mortar and pestled to a fine paste and made up with alcohol. 'he extract was passed on a cheese cloth. 5xtraction was repeated twice and the filterates were pooled. 'he volume was raised by addition of the alcohol or reduced by evaporation of alcohol to represent .3 4 ml of extract for every 4.. gram of tissue used. E'traction wit* :uffer .or estimation of -m$lase =resh leaf samples + 44mg/ from . days old plants was ground in ..4ml of distilled water. Centrifuged at 9444rpm for 4 minutes and $ept at M4oC for . minutes to inactive Q3 amylase. 'his serves as the en%yme extract for the analysis. .or estimation of Pero'idase and )atalase *bout 4.. gm of tissue was suspended in ml of 4. , phosphate buffer (p6 M.A# and ground in a prechilled mortar. 'he extract after passing through A layers of cheese cloth was centrifuged at 9444 g for . minute and the pellet was reextracted with 4ml of the same buffer. 'he supernatants were pooled and used. &ince the en%yme activity in the discarded pellet was found to be less, it was rejected. .1.2 Estimation of /otal )ar(o*$drates 'otal carbohydrates were determined by anthrone method as reported by 6edge and 6ofreiter ( !-A#. Princi+le *nthrone reaction is the basis of a rapid and convenient method for the determination of hexoses, aldopentoses and hexuronic acids, either free or present in polysaccharides. Carbohydrates are dehydrated by concentrated sulphuric acid to form furfural. =urfural condenses with anthrone to form a blue colour complex which is measured colorimetrically. &eagents !-++endi'% I # . &toc$ standard glucose solution 19

A. (or$ing standard glucose solution 9. *nthrone Reagent Procedure Into a series of test tubes, pipetted out 4.A to .4 ml of the wor$ing standard solution corresponding to A4 to 44 Rg values respectively. .4 ml of the chic$ pea plant tissue samples were ta$en for the experiment. 'he volume in all the tubes were made up to .4 ml with distilled water. 'hen added E.4ml of anthrone solution and heated for 4 minutes in a boiling water bath. 'he tubes were cooled and the intensity of greenish blue color developed was read at -94 nm in a colorimeter. * standard graph was drawn by plotting the concentration of glucose in x3axis and the optical absorbance in H3axis. =rom the graph the amount of carbohydrates present in samples was calculated and expressed as RgNg of fresh weight. .1.3. Estimation of /otal Proteins 'otal protein was estimated by the method of 0owry et al.,( !. #. Princi+le 'he =olin 3 Ciocalteau reagent is )uite complex and contains phosphomolybdic acid and tungstate. 'he aromatic amino acids, tyrosine and tryptophan present in proteins react with these to give dar$ blue colour. 'he colour so formed is due to the reaction of al$aline copper with proteins and the reduction of phosphomolybdate by tyrosine, tryptophan present in proteins. 'he intensity of the colour depends on the amount of aromatic amino acids.

&eagents !-++endi'% II# . *l$aline copper reagent 2 (0owry>s Reagent# A. =olin>s phenol reagent 9. &tandard Fovine &erum *lbumin (F&*# 20

E. (or$ing &tandard &olution .. .4 @ 'C* Procedure Into a series of test tubes, pipetted out 4.A to .4 ml of the wor$ing standard F&*

solution corresponding to E4 to A44 Rg values respectively. .4 ml of the chic$ pea plant tissue samples were ta$en for the experiment. 'he volume in all the tubes were made up to .4 ml with distilled water. * blan$ was set by ta$ing .4 ml of distilled water and added E.. ml of al$aline copper sulphate reagent to all the tubes. *fter 4 minutes, 4.. ml of =olin phenol reagent was added to all the test tubes. 'he blue colour developed was read at -E4 nm after A4 minutes. 'he protein was calculated from the standard curve prepared from F&* and expressed as RgNg of fresh weight. .1. Estimation of /otal .ree -mino -cids 'he 'otal free aminoacid was estimated by the method of ,oore and &tein ( !E"#. Princi+le *mino acids are routinely estimated by 8inhydrin method. product which is colorimetrically measured at .M4nm. &eagents !-++endi'% III# . 4.A, Citrate buffer (p6 ..4# A. 8inhydrin reagent 9. 7iluent &olvent E. 0eucine3 (or$ing standard solution 8inhydrin, a powerful

oxidising agent, decarboxylates the Q aminoacids and yields an intensely colored bluish purple

Procedure 21

'o 4. ml of extract, ml of ninhydrin solution was added. 'he volume was made upto Aml with distilled water. 'he tubes were heated in a boiling water bath for A4 minutes. .ml of the diluents was added and the contents were mixed. *fter . minutes, the intensity of the purple colour developed was read against a reagent blan$ in a colorimeter at .M4nm. 'he color was stable for hr. 'he reagent blan$ was prepared as above by ta$ing 4. ml of "4@ ethanol 4. to ml of wor$ing standard solution giving a and 4Rg to 44 Rg was ta$en and treated as that of the sample. 'he instead of the extract. =or the standard, concentration range

aminoacid content was calculated from the standard curve prepared from 0eucine expressed as RgNg of fresh weight. .1., Estimation of Proline

Proline is a basic aminoacid and free proline is said to accumulate in plants under stress conditions. Proline was estimated by the method of Fates et al., ( !M9#. Princi+le Proline reacts with acid ninhydrin to form a red color to be read at .A4 nm. &eagents !-++endi'% I<# . *cid ninhydrin A. 9@ a)ueous sulphosalicylic acid 9. ;lacial acetic acid E. 'oluene .. &tandard Proline Procedure *pproximately 4.. gm of plant tissue was extracted by homogeni%ing it with 4 ml of 9@ a)ueous sulphosalicylic acid. 'he homogenate was filtered through whatman 8o3A filter paper. 'o A ml of filterate ta$en in a test tube, Aml of acid ninhydrin was added and heated in a boiling water bath for hour. 'he reaction was terminated by placing the tubes in an ice bath. E ml of toluene was added to the reaction mixture and the contents were stirred for A4394 seconds. 'he toluene layer was then separated and warmed to room temperature and the red color was read at .A4 nm. 'he proline was calculated from the standard curve and expressed as Rmoles Ng of fresh weight. .1.0. Estimation of /otal P*enol

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Proline was estimated by =olin>s ciocalteau method as reported by ,alic$ and &ingh ( !"4#. Princi+le 5stimation of total phenols was carried out with the =olin>cio calteau reagent . 'his method is based on the reaction between phenols and an oxidi%ing agent phosphomolybdic acid in =olin>ciocalteau reagent in al$aline medium which results in the formation of a blue coloured complex. 'he intensity of the coloured complex is measured at -.4 nm in a colorimeter. &eagents !-++endi'% <# . =olin>s ciocalteau reagent A. A4@ &odium carbonate 9. &tandard Catechol Procedure ml of the test solution obtained from plant extract in alcohol was pipetted into a graduated test tube. 'o this, ml of =olin>s ciocalteau reagent was added. 'his was followed by the addition of A ml of A4@ sodium carbonate and mixed thoroughly. water bath for exactly * blan$ containing all the reagents minus plant extract was ta$en. 'he test tubes were then placed in a boiling minute and cooled under a running tap. 'he blue colour developed was read at -.4 nm in a colorimeter. Catechol was ta$en as the standard. 'he total phenol was calculated from the standard curve and expressed as RgNg of fresh weight. .1.1. Enz$me Studies

.1.1.1. -SS-; 6. =%-M;"-SE &eagents !-++endi'% <I# . A. 9. E. .. . 23 &toc$ maltose solution (or$ing standard maltose solution 4. , citrate buffer of P63..4 A@ soluble &tarch 7initrosalicylic acid +78&/ reagent

Procedure 'a$en graded volumes of standard solution in the range of 4.A, 4.E, 4.-,4." and .4ml in the test tubes mar$ed as & to &.. *ll the tubes were made upto .4ml with distilled water. 'a$e .4ml of each test solution in test tubes mar$ed as ' and 'A respectively. 'hen all the tubes were added with .4ml of 4. , citrate buffer of P6 ..4 and 4..ml of A@ soluble starch and $ept at 94oC for 4 minutes. 'hen A.4ml of 78& reagent was added and $ept in a boiling water bath for . minutes. *fter cooling, the volume was made upto 4.4ml with distilled water. * blan$ was maintained stimultaneously with .4ml of distilled water and other reagents added above. 'he absorbance was read at .E4nm in spectrophotometer. -N/I6>I?-N/ S/@?; .1.2 ?etermination of Enz$mic antio'idants

.1.2.1 Estimation of Pero'idase activit$ AP6>B E) 1.11.1.1 C 'he en%yme was assayed according to the procedure described in *ddy and ;oodman ( !MA#. Peroxidase is and oxidative en%yme. It catalyses the following reaction. *6A S6AOA T * S A6AO where *6A may be a dye. Princi+le 'he dye Pyrogallol acts as a hydrogen donar. It is oxidi%ed to a colored product by peroxidase in presence of hydrogen peroxide. 'he amount of purpurogallol formed during the reaction can be estimated colorimetrically as EA4 nm. &eagents !-++endi'%<II# . 4.4. , Pyrogallol dissolved in 4. , Phosphate buffer (p6 M.A# A. @ 6AOA

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Procedure Pipetted 9ml of 4.4., Pyrogallol solution prepared in 4. , phosphate buffer p6 M.A

and 4. ml of the en%yme extract into a cuvette. *djusted the absorbance to 4 at EA4nm in the colorimeter. *dded 4..ml of @ 6AOA to the cuvette and inverted the cuvette immediately to mix the contents and replaced in the colorimeter. ,easured the changes in absorbance at A4 second intervals for 9 minutes. *verage changes in absorbance per A4 second between E4 and A4 seconds were used for calculation. =or blan$, substrate was omitted.

'he en%yme activity was measured as a change of O7 <nitsN minN mg of Protein.

.1.2.2 Estimation of )atalase A )-/B E) 1.11.1.0C 'he en%yme was assayed according to the method of Bar and ,ishra ( !M-#. Princi+le Catalase is an oxido reductase. It catalyses the brea$down of 6 AOA to water and ,olecular oxygen (catalatic# or peroxidatively, 6ydrogen peroxide is converted to water in the presence of hydrogen donor. 6AOA T * S U OA (Catalatic# 6AOA S *6A T * S A 6AO ( Peroxidative # 'he residual hydrogen peroxide in the reaction mixture is estimated by permanganate titration as a measure of catalase activity.

&eagents !-++endi'% <III# . 4. , Phosphate buffer p6 M.A A. 4. , 6AOA 9. A@ &ulphuric acid E. 4.4 8 Potassium Permanganate

Procedure 25

Pipetted 9 ml of 4. , Phosphate buffer p6 M.A into a porcelain crucible. 'hen 4. , 6ydrogen peroxide and incubated at A.V C for minute. 'he reaction was stopped by the addition of

ml of

ml of en%yme extract were added and this reaction mixture was 4 ml of A @

sulphuric acid. 'itrated the reaction mixture against 4.4 8 Potassium Permanganate to find out the residual hydrogen peroxide until a faint purple color persisted for atleast . seconds. &imilarly a control was maintained in which the en%yme activity had been stopped by sulphuric acid, prior for the addition of en%yme extract. One unit of activity was expressed as the amount of en%yme which brea$s down one Wmole of 6AOA N minuteN mg fresh weight tissue under assay conditions.

,. &esults and ?iscussion


26

In the present investigation an attempt has been made to study the influence of microwave radiation on Vigna radiate seeds at the time of germination. 'herefore the experiments in the present study were designed to see if microwaving water, seeds, or soil would affect the growth of the plant and conse)uently theyl were microwaved for different amounts of time starting at . seconds, 94 secs and E. secs. ,.1. P*ase 5 I Growt* +arameters 'he morphological observation of the Vigna radiate (0.# plants with treatment in different aspects is performed and duly analysed for our study.

,.1.1.

Percentage of germination
In =igure , this graph shows the average day that each seed emerged from the

soil. In the control for water, seed, and soil conditions, the result is the same because the data were combined. 'he control was not treated with microwave, and the result is it too$ A days to emerge from the soil. 'he graph in =igure shows the percentage of seeds that sprouted . 'he and 44@ from dayA.

percentage that grew in the control was !.@ on day

=or the seed condition, the seeds that were treated for 4 seconds had a .4@ chance of growing, and at A4 and 94 secs, the seeds did not grow at all. 5specially, the seeds that were 4 seconds microwaved too$ upto . days to emerge. =or the soil condition, the microwave treatment did not have a clear affect, as the soil were treated for A4 seconds or more, the seeds decreased in percentage of growth and for 94 secs treatment, it was .4@ on the A nd day and achieved germination rate almost on the fifth day. 44@

27

=or the water condition, all of the plants that were treated grew with not so much impact for 4 secs treatment. =or those with A4 and 94secs treatment , the germination rate pic$ed up from day A onwards. =or the water condition, it was also observed that the plants that were treated emerged more fast than the microved seed or soil conditions. 'hese results are in agreement with the findings of Chaomei and Hanlin ( !!9# on wheat (Triticum aestivum 0.#, who noticed that treating seeds with high rates of gamma radiation reduced germination with a corresponding decline in growth of plants. 'he symptoms fre)uently observed in the low or highdoseirradiated plants are enhancement or inhibition of germination, seedling growth, and other biological responses (Bim et al., A4441 (i et al., A44M#. *lthough, no certain explanations for the stimulatory effects of lowdose gamma radiation are available until now, in accordance to the results obtained by (i et al., (A44M#, there is a hypothesis that the low dose irradiation will induce the growth stimulation by changing the hormonal signaling networ$ in plant cells or by increasing the anti oxidative capacity of the cells to easily overcome daily stress factors such as fluctuations of light intensity and temperature in the growth condition ((i et al., A44M#. In contrast, the high dose irradiation that caused growth inhibition has been ascribed to the cell cycle arrest at ;AN, phase during somatic cell division andNor various damages in the entire Fritta, A449#. 'he results of Boing et al., (A44"# have shown that survival of plants to maturity depends on the nature and extent of chromosomal damage. Increasing fre)uency of chromosomal damage with increasing radiation dose may be responsible for less germinability and reduction in plant growth and survival. Changes in the germination percentage were found to attribute to gamma rays treatments. 'he stimulating causes
28

genome (Preussa and

of gamma ray on germination may be certified to the activation of R8* or protein synthesis, which occurred during the early stage of germination after seeds irradiated (*bdel6ady et al., A44"#. -s mentioned earlierB t*e seedlings treated wit* microwave radiation for 29 sec and 39 sec +rior to germination failed to germinate and t*erefore rest of t*e growt* +arameters and mor+*ological anal$sis were not carried out for test II and test III seedlings. ,.1.2 &oot and s*oot lengt* 'able A summari%es of the effect of microwave radiation on growth parameters (&hoot length, Root length and number of lateral roots # of treatment groups. 'he statistical analysis revealed that the plant growth in terms of root length and shoot length were found to be decreased significantly in comparison to the control plants and other plants groups underta$en for study. =or the seed condition, microwaving the seeds caused a notable reduction in the length of the plant .=igure Aa shows the length of the average plant that was treated with microwaved seeds . 'he 4 secs treated seedling showed a significantly slower root and shoot growth compared with the control. =or the soil condition, a considerable decrease in root and shoot length is observed for plant grown in microwave treated soil for 94 secs. =igure Ab shows the length of the average plant that was treated with microwaved soil . 'he plant in the soil that was treated for 4 and A4 secs also had a slower growth compared to the control but relatively better than 94secs treated plant groups.'he results clearly proved that more the soil were microwaved, the length of the plant decreased.

29

=or the water condition (=igure Ac#, all the plants grew and microwaving water had relatively less influence compared to microwaved seed or soil.'he plant that got microwaved water treatment for 4secs grew more than all of the other plants. 'hese results are in accordance with the findings of previous wor$ers (Fajaj et al., !M41 Bhanna, !"-1 ,andal and Fasu, !"-1 *I3&afadi and &irnon, !!-#, who recorded reduction in seedling growth with the increasing irradiation dose. Chaudhuri (A44A# reported that when radiation is sufficient to reduce the rooting percentages, then the root lengths do not exceed a few millimeters in length. &imilarly, Lac$s and 'hapa reported that in two pinus species, at early stage of growth, root and hypocotyls elongation were inhibition by gamma3rays exposures. 'his inhibition increased as the dose of exposure increased. *lso, Bon et al., reported that germination percentage, root and shoot length and root and shoot dry weight of long Fean seedling decreased with increasing dose of gamma ray. ,.1.3 Num(er of "ateral (ranc*es In our pot culture experiment, the number of lateral roots owing to the influence of in stressed and unstressed plants were measured at different stages of growth of ,ung bean and were presented in table 9. Our results indicated that the number of lateral roots per plant was noticeably lower in microwaved seeds, followed by micowaved soil and water compared to the control over the period of three wee$s.

30

Mor+*ological +arameters
Plate , 4 Effect of Microwave radiation Microwaved seedB soil and water conditions 5 .irst WeeD

31

Plate 0 4 Effect of Microwave radiation Microwaved seedB soil and water conditions 5 Second WeeD

Plate 1 4 Effect of Microwave radiation Microwaved seedB soil and 32

water conditions 5 /*ird WeeD

/a(le 1. Effect of Microwave radiation on Germination 8 in Microwaved seedB soil and water conditions 33

7ay 7ayA 7ay9 7ayE 7ay.

Control 4sec !.S ..A 44 44 44 44

4secs 4 4 94S ." E4S A.A E.S A."

&eed A4secs 4 4 4 4 4

94secs 4 4 4 4 4

4secs -4S9.. !.S-,A 44 44 44

&oil A4secs E.S A.M !4S.." 44 44 44

94secs 94S .A .4SA.M.SE.A ".SE." 44

4secs "4SE." !.S.." 44 44 44

(ater A4secs M4S9.. !.S-.44 44 44

94secs -4SA." !4S-.E 44 44 44

/a(le 2.Effect of Microwave radiation on &oot lengt* !cms# in Microwaved seedB soil and water conditions

&oot lengt* !cms#


Control 4sec 9..S 4.A -.ES 4.E ".!S 4.! 4secs ."S 4. 9.AS 4.A ..MS 4.A &eed A4secs 3 3 3 94secs 3 3 3 4secs 9.4S 4.9 ..AS 4.A -..S 4.E &oil A4secs A.MS 4. E.MS 4.A -.AS 4.. 94secs A..S 4.A E.ES 4.E E.MS 4.9 4secs 9.AS 4.9 ..-S 4.. M.ES 4.(ater A4secs 9.4S 4. ..AS 4.9 M.4S 4.M 94secs A.MS 4. E."S 4.. -.MS 4.M

Mth 7ay Eth 7ay A st 7ay

/a(le 3. Effect of Microwave radiation on S*oot lengt* in Microwaved seedB soil and water conditions

S*oot lengt* !cms#


Control 4sec Mth 7ay Eth 7ay
22.1E 2.

4secs
10.9E 1.

&eed A4secs 3 3

94secs 3 3

4secs
29.1E 1.0

&oil A4secs
12.2E 1.

94secs
12.9E 1.3

4secs
13.9E 2.1

(ater A4secs
13.9E 1.1

94secs
11.,E 1.,

2,.1E 3.2

12.2E 2.0

22. E 2.

29.1E 3.2

12.2E 1.0

23.2E 2.0

22.0E 2.2

21.3E 1.3

34

st

3 .2E 3.0

23.1E 2.

21.2E 2.

20.3E 2.3

2 .3E 2.,

23.0E 2.2

22.1E 3.2

21.3E 3.2

7ay

/a(le . Effect of Microwave radiation on lateral roots in Microwaved seedB soil and water conditions

"ateral roots4
Control 4sec ES A -S A 4S9 4secs AS 9S -S A &eed A4secs 3 3 3 94secs 3 3 3 4secs AS 9S MS A &oil A4secs AS 9S MS A 94secs AS 9S -S A 4secs 9S .S A !S 9 (ater A4secs AS ES "S 9 94secs AS ES "S 9

Mth 7ay Eth 7ay A st 7ay

.ig.1. Effect of Microwave radiation on Germination 8 in Microwaved seedB soil and water conditions

35

.ig.2. Effect of Microwave radiation on &oot lengt* in Microwaved seedB soil and water conditions

36

.ig.2. Effect of Microwave radiation on S*oot lengt* in Microwaved seedB soil and water conditions

37

.ig. . Effect of Microwave radiation on "ateral &oots in Microwaved seedB soil and water conditions

38

,.2. ,.2.1.

P*ase 5 II :I6)7EMI)-" -N-";SIS Effect on /otal Solu(le Sugars 'otal soluble sugar in the seeds is responsible for the growth and development

of

the crop. 'he sugars play major role in metabolic pathway as well as in the

transport of vitamins, mineral ions etc. 'he other possible role of sugar may be as a readily available energy source. 'hus in this study the important parameter was

signified by the analysis of the total soluble sugar content in the plant samples.

39

=igure . exemplified the effect of microwave radiation on total soluble sugars in microwaved seeds, soil and water condition during the germination and growing stages. In the present investigation, total soluble sugars were significantly increased in the control group and the decline trend of total soluble sugars was significant in the stressed plants especially grown in microwaved seed condition ('est II and 'est III#. 8oticable differences were also observed for 'est I, 'est GI and 'est IX plants for total sugar. 'hus it was found that the total sugar content is considerably lowered in the plants exposed to irradiation during the entire period of our study. &imilar to our results, Falauchi et al.,(A44!# reported that total soluble carbohydrates of flag leaf were significantly decreased, in plants exposed to <G3C radiation ,elevated COA and water stress than in plants grown under normal condition ,.2.2 Effect on Proteins 'he amount of protein present in seeds varies from Y 4@ (in cereals# to YE4@ (in legumes#, and thus contribute for a major source of dietary protein. 'he

mobili%ation of seed storage proteins represents one of the most important post3
.ig.,. Effect of Microwave radiation on /otal sugars in Microwaved seedB soil and water conditions

40

.ig.0. Effect of Microwave radiation on /otal +roteins in Microwaved seedB soil and water conditions

41

.ig.1. Effect of Microwave radiation on .ree -minoacids in Microwaved seedB soil and water conditions

42

.ig.2. Effect of Microwave radiation on Proline in Microwaved seedB soil and water conditions

43

germinative events in the growth and development of seedling. In this regard, proteolytic en%ymes have a central role to play in the biochemical mechanism of germination (Fewley and Flac$, !!E1 &hewry et al., !!.1 Gadde Rama$rishna, A44..
44

=igure - .of our study showed a remar$able decrease in protein content in the microwave treated mung bean seedling. In addition to that observation, present investigation confirmed that microwave treatment aggravated the condition by bringing down the protein levels in the mung bean seedling than for the seedlings grown in microwaved soil and microwaved water compared to control. 5arlier studies have reported that the effect of gamma radiation on protein can cause 78* damage caused losses and brea$ing hydrogen bonds in proteins (,aity, A44"#.It reduced the effect of increasing protein concentration due to salinity to prevent biosynthesis of proteins under salinity stress is performed because a noticeable change in the concentration of total nitrogen in plant leaves can be observed. ;amma radiation was reported to induce oxidative stress with overproduction of reactive oxygen species (RO&# such as superoxide radicals, hydroxyl radicals and hydrogen peroxide, which react rapidly with almost all structural and functional organic molecules, including proteins, lipids and nucleic acids causing disturbance of cellular metabolism (*lRumaih and *lRumaih, A44"1 *shraf, A44!1 8oreen and *shraf, A44!#.

,.2.3 Effect of .ree -minoacids *mino acids is a putative osmoprotective solute leading to lowering osmotic potential in several tissues exposed to stress.
45

In seeds, proteins accumulate in

organelles called protein3bodies. <pon germination, by the action of endo and exoproteinases, small peptides and amino acids generated by cleavage of the storage proteins may remain in the storage tissue or translocated to the developing plant (Callis, !!.#. Fecause they are destined for food use most of the studies concerning protein composition have been carried out in seeds of cereals and legumes, such as wheat, mai%e, soybean, rice and beans (Payne, !"-#. 5arlier wor$s suggested that amino acids accumulation seemed to have been directly related to their non3conversion into proteins, causing a decline in the protein content. =igure M showed the levels of free aminoacids in the plants grown under conditions of microwaved seed, soil and water for varying period of secs. Our results indicated that microwave treated plants had a significantly higher 4, A4 and 94

values of total free amino acids than control plants . ,ung bean seeds exposed to microwave treatment ('est I # recorded higher amounts of total free aminoacids followed by microwaved soil and water treated plants. 'he exposure of ,ung bean seedlings to radiation stress prior to germination induced an accumulation of free amino acids which attained -3M fold increase over the control value. =urther, in the present investigation, in the microwave treatment resulted in aminoacid accumulation invariably in all the plant groups except for plants grown in microwaved water at 4 and A4 secs. ,.2. . Effect of Proline

46

Proline seems to have diverse roles under osmotic stress conditions, such as stabili%ation of proteins, membranes and subcellular structures, and protecting cellular functions by scavenging reactive oxygen species. Proline is an osmoregulation in abiotic stress in plants (Fates et al., !M9#. It acts as an intracellular osmotic solute for maintenance of osmotic balance between cytoplasm and vacuole (=lowers et al., !MM#. Proline also acts as a reserve source of carbon, nitrogen and energy during recovery from stress (Zhang et al. 1997). *mong various compatible solutes, proline is the only molecule that has been shown to protect plants against singlet oxygen and free radical induced damages (*lia, Pardha &aradhi, !!M# by acting as scavenger of

O63radicals and also stabili%e proteins, 78* as well as membranes (&mirnoff. and Cumbes, !"!#. 'hus, proline is not only an important molecule in redox signalling, but also an effective )uencher of reactive oxygen species formed under salt, metal and dehydration stress conditions in all plants, including algae (*lia and Pardha &aradhi, !! # . Proline is not only an important molecule in redox signalling, but also an

effective )uencher of reactive oxygen species formed under salt, metal and dehydration stress conditions in all plants, including algae (*lia and Pardha &aradhi, !! # . Proline seems to have diverse roles under osmotic stress conditions, such as stabili%ation of proteins, membranes and subcellular structures, and protecting cellular functions by scavenging reactive oxygen species. (Fates et al., !M9#. In the present investigation, figure " revealed that proline content was significantly increased in the microwave treated plants ('est I# than in the control. 'here was also a raise in the proline content in the chic$ pea plants subjected to microwave exposure in soil and water prior to germination.'he elevation of proline level was more prominent , when duration of radiation exposure is increased in all the
47

category of plants.It suggests that microwave radiation3induced proline accumulation protects plants against radiation promoted peroxidation processes. 'here was a convincing evidence which showed that the osmolyte synthesis such as proline involved in protective mechanisms were altered with several environmental stresses, including gamma irradiation (*l Rumaih and *lRumaih, A44"#. Proline is a compatible osmolyte and it may interact with en%ymes to preserve en%yme structure and activities. Indeed, proline has been shown In vitro to reduce en%yme denaturations caused due to heat, 8aCl stress, gamma stress, etc., (Bavi Bishor et al., A44.1 *shraf and =oolad, A44M#. 'he present increase in proline content was reported to cope with the problem of oxidative reagents (=alahati et al., A44M#. In this study the proline contents of gamma irradiated seedlings showed a increase as the gamma doses increased. 6owever, =alahti et al., (A44M# contradicted this statement by proposing that the radiation may have promoted the level of antioxidants and conse)uently there would be no need for extra amount of proline to cope with the same problem of oxidative reagents. Enz$me Studies

,.2.,. Effect on = -m$lase 'he seeds treated with microwave radiation ( 4secs# showed a significant reduction in Q3amylase activity than the untreated seeds. &ince decrease in the number of parenchyma cells led to decreased rate of cell division, the lesser number of root hairs cause better water absorption.'he root growth
48

may be simply compared with the growth of stem. *uxin fail to accelerate very early growth of the root tip. 'he activity of Q 3 amylase is directly related to auxin

activity.'his has been proved indirectly by experiments that radiations decreases the hydrolysation of starch during germination, due to the lowered activity of Q3amylase. On the other hand , mung pea plants that were treated with microwaved soil and water exhibited a considerable reduction in the amylase activity but comparatively better than that of microwaved seed treated plants. (=ig. !# Effect on Enz$mic antio'idant s$stem Effect of micrrowave on Pero'idase and )atalase activities Catalase and peroxidase en%ymes are involved in various physiological processes by protecting the bodies of free radicals but in conditions of stress is found modifying of their activities. (hile catalase acting on the large )uantities of 6AOA present in tissues (vegetal or animal# that occurs as a result of metabolic changes, peroxidase cataboli%ed small )uantities of 6AOA remaining undecomposed by catalase. 'o investigate the antioxidant defense system, microwave induced changes of antioxidant en%ymes were examined in the mung bean arietinum plants pretreated with microwave in the seedling, soil and water. ,.2.0. Effect on Pero'idase activit$ Peroxidase en%ymes are $nown to decompose 6 AOA by oxidation of phenolic compounds and prevent lipid peroxidation of membranes. In effect, 6 AOA is an essential substrate for the cell wall stiffening process cataly%ed by peroxidase in the cell wall which is attributed to be one of the mechanisms leading to growth inhibition (&chopfer, !!-#. It is also believed that environment stress generally cause reduction
49

on the growth of the roots due to production of free radicals and lead to increased peroxidase activity. =igure 4 revealed the response of the activity of peroxidase in the ,ung bean plants exposed to radiation stress. In the present study, a notably higher peroxidase activity was observed for microwave treated seeds ('est I # than all other treatments . Peroxidase activity is stimulated when the plants are exposed to stress and the activity was more in the microwaved seeds than microwaved soil and water. ,.2.1.Effect on )atalase activit$ Catalase, which is involved in the degradation of 6 AOA into water and oxygen, is the major 6AOA scavenging en%yme in all3aerobic organisms. In the present investigation, Catalase activity revealed a significant increasing trend in the microwave treated plants('est I# . 'he increase is more significant when compared to the control. 'he results showed that the microwaves determined variations of catalase and peroxidase activities depending on the time of exposure to microwave action and state of seeds (germinated and non germinated# exposed to microwave. In the second and third wee$ after effect of microwaves on the germinated seed there is a remar$able stimulation of catalase activity than the peroxidase activity.'he study also present a similar behavior with regard to the wor$ of the two en%ymes in the microwaved soil and water for 94 secs period. (=ig. #

'hus in our study , both C*' and POX showed enhanced activity under microwave treatment ,supporting the findings of earlier studies on various plants (Hannarelli et al., A44-1 Costa et al., A44A1 Lain et al., A44E1 Xu et al., A44"#.
50

Other earlier wor$s had also reported that the microwave pre treatment in wheat seedlings induces the en%yme activities of catalase andperoxidase, (Chen et al., A44"#. 'he increase in peroxidase activity might be due to an increase in 6AOA production probably as a result of induced &O7 activity during the <G3F radiation (&harma et al., !!"#. =urther, peroxidases are believed to utilise phenolic compounds as co3substrate (Otter and Polle, !!E#.

.ig.3. Effect of Microwave radiation on -m$lase activit$ in Microwaved seedB soil and water conditions

51

.ig.19. Effect of Microwave radiation on Pero'idase activit$ in Microwaved seedB soil and water conditions 52

.ig.11. Effect of Microwave radiation on )atalase activit$ in Microwaved seedB soil and water conditions

53

0. )onclusion

'he main purpose of investigating the effects of microwave radiation on the germination of mung bean seeds was to determine whether intense microwave
54

radiation can affect the rate at which plants grow and whether or not seeds will sprout. 'he study was conducted by microwaving mung bean seeds for 4, 4, A4 and 94 seconds and monitoring their growth over the course of three wee$s. On the basis of the results obtained in the present investigation, the following conclusions can be formulated for the effect of microwave treatment on seed development,2 . ,ung bean seedlings grown by microwaving seeds has the most inhibitory effect compared to seedlings grown under microwaved soil and water conditions. A. Inhibition is stronger for the treatment of seeds for A4secs and 94 secs than 4secs.. 9. In the case of soil condition, microwaving the soil probably eradicated some of the soil microflora necessary for promoting growth and thus resulted in slower growth of the seedlings. E. 'he inhibitory effect was further proved by the growth parameters such as germination percentage, root and shoot length, number of lateral roots and also with biochemical changes by determination of total soluble sugars, total proteins, =ree aminoacids, 'otal phenol and en%yme activities of amylase, Catalase and Peroxidase. 'hus, microwave radiation affects seeds differently when it irradiates the seed itself and also when it irradiates the water or soil , the seed uses to grow and the results in the present investigation clearly proved that different microwave doses do affect the germination and growth of Vigna radiate (L.) seeds. In future, analysis can be done with different types of microwave radiation li$e microwaves from cell phones, 'G remote controls and antennas, computer signals on plants. If signals from these things does affect plants, they could also affect any living
55

things li$e humans, animals, etc., then it is high time that we may have to use them less and protect ourselves from on towards radiation.

1. :i(liogra+*$ -(del%7ad$B M.S.B E.M. 6Das*aB S.S.-. Soliman and M. /alaat. A44". 5ffect of ;amma radiation and gibberellic acid on germination and al$aloid production in Atro a belladonna. Aust. !."asic A l., A(9#2 E4 3E4.. -dd$B S. F. and GoodmanB &. N.,( !MA#. Indian #$%to at$ol., A.B.M.J.M!.
56

-Ga$i and :. "arson, 5ffects of gamma radiation on seed germination and seedling growth in wheat. 8ig. Lour. of Phy. 9( !! # 443 4.. -lia and Pard*a Sarad*iB P., ( !! # Proline accumulation under heavy metal stress. !. #lant #$%siol., 9", ..EJ..". -liaB Pard*a Sarad*iB P. and Mo*ant$B P.B ( !!M# Involvement of proline in protecting thyla$oid membranes against free radical3induced photodamage. !. #$otoc$em. #$otobiol., 9", A.9JA.M. -l &umai*B M. M. and M. M. -l &umai*. A44". ?Influence of ioni%ing radiation on antioxidant en%ymes in three species of Trigonella?. Am. !.&nviron. 'ci., E (A#2 . .-. -l%SafadiB :. and P. W. Simon. !!-. ;amma irradiation induced variation in carrots ((aucus carota 0.#.L. Amer.'oc. )ort. 'ci., A (E#2 .!!3-49. -s*rafB M. A44!. ?Fiotechnological approach of improving plant salt tolerance using antioxidants as mar$ers?. "iotec$nol. Adv. AM2"E!9. -s*rafB M. and M. &. .oolad. A44M. ?Roles of glycine, betaine and proline in improving plant abiotic stress resistance?. &nviron. &* . "ot. .! (A#2 A4-A -. :aGaGB ;.P.S.B -.W. Saettler and M.W. -dams. !M4.;amma irradiation studies on seeds, seedlings and callus tissue cultures of #$aseolus vulgaris 0. Radiat."ot, 42 !3 AE. :alatong3I0ist of herbal medicinal plantsK.(A44E#w.w.w.tuartxchange.org :ates ".B Waldern &.P. and /eare I.?. , ( !M9#. Rapid determination of free proline for water stress. &tud. Plant and &oil, PP 2 A4. J A4M. :ates ".B Waldern &.P. and /eare I.?. , ( !M9#. Rapid determination of free proline for water stress. &tud. Plant and &oil, PP 2 A4. J A4M. :ewle$ H.?., :lacD. M.B ( !!E#. &eeds2 Physiology of development and germination, A 5d. 8ew Hor$, 0ondon, Plenum Press. :*asDara &edd$ M. <.B G. S. <. &ag*avanB -. ). Fus*ala++aB /. ). Paulitz . I5ffect of ,icrowave 'reatment on [uality of (heat &eeds Infected with +usarium :*asDara &edd$B M.<. -.).Fus*ala++aB G.S.<. &ag*avan and M.M.P.StevensonB !!.. --th *nnual meeting of the Canadian Phytopathological &ociety, Lune A.3 A", 'oronto. )allis H. ( !!.#. Regulation of protein degradation. #lant ,ell., M, "E.3".M. )*aud*uriB F.S. A44A. * simple and reliable method to detect gamma irradiated lentil (Lens culinaris ,edi$.# seeds by germination efficiency and seedling growth test. Radiat. #$%s.,$em., -E2 9 3 9-.
57

)*enB ;.P.B H... Hia and >.". 7an, A44". (ea$ microwave can alleviate water deficit induced by osmotic stress in wheat seedlings. Planta, AA!(A#2 A! 3A!". )*risto+* 6(erndorferB Wolfgang "IcDeB !!!. Part I2 Influence on mechanical oil extraction, vol. 4 (.#, pp. -E3 -M. .ala*atiB -.B S. F. Fazemita(arB -. &. :a*ramiB M. "a*outi and M. .. &a*imi. A44M. ?'he study of gamma irradiation effects on drought tolerance in rice (-r%.a sativa 0.#?. Indian !.,ro 'ci. A ( #2 .. .". .lowers /.H.B /roDe P... and ;eo -.&. ( !MM#. 'he ,echanism of &alt 'olerance in 6alophytes. Annu. Rev. #lant #$%siol., A"2 "!3 A . GosB P.:. et al. I5xtremely high fre)uency electromagnetic fields at low power density do not affect the division of exponential phase &accharomyces cerevisiae cellsK graminearum./ L. agric. 5ngng Res. M ( !!"#2 90 M 7edge H. E and 7ofreiter :./ ( !-A# In2 Carbohydrate Chemistry M ( 5ds (histler R 0 and Fe ,iler , L 8 # *cademic Press 8ew Hor$. 7er(ermanB &onald.K Researcher sees cancer ris$ from mobiles.K International 6erald 'ribune AE Luly A44". International *stronautically congress, 'oulouse, =rance 3. October, A44 . International -tomic Energ$ -genc$ !I-E- #. Isotopes in everyday life. ( !!4# E3 4. HacDsB ). /*a+a, -ur 1ature, 299 , A, 93 M. HainB F.B S. FatariaB F.N. Guru+rasad4 5ffect of <G3F radiation on antioxidant en%ymes and its modulation by ben%o)uinone and 3tocopherol in cucumber cotyledons. Curr. &ci., "M, "M3!4 (A44E#. Far M. and ?.Mis*ra. ( !M-#. Catalase,peroxidase and polyphenol3oxidase activities during rice leaf senescence. Plant Physiol..M2 9 .39 !. Favi Fis*orB P. :.B S. Sangam and &. N. -mrut*a .A44.. ?Regulation of proline biosynthesis,degradation, upta$e and transport in higher plants2 Its implications in plant growth and abiotic stress tolerance?. ,urr. 'ci. ""2 EAEE9". FimB H.S.B E.F. "eeB M.7. :acDB ?.7. Fim and ;.:. "ee. A444. Influence of 0ow dose % radiation on the physiology of germinative seed of vegetable crops. 2orean !. &nv. Agric., !2 ."3- . FiongB -.B -.B "ing PicDB S.7. Grace "ai and -.&. 7arun. A44". Physiological responses of -rt$osi $on stamineus plantlets to gamma irradiation. Am0 &urasian !. 'ustain. Agric., A(A#2 9.3 E!. FonB 6. -*medB S. SaainB N. MaGidB Amer. J. Appli. Sci., 2991, E( A#, 4!43 4!9.
58

lichensK Fioelectromagnetics M ( !!-#2 9AM399E. "owr$ 6.7B &ose(roug* N.HB .arr -." and &andall &.H ( !. # L Fiol Chem !93A-.. MacDe$B ,utation breeding in 5urope. Froo$haven &ymposia in Fiology. !( !-.# E 3 .-. MagoneB I. I'he effects of electromagnetic radiation from the &$runda radio location MalicD ).P and Sing*B M.: ( !"4# In1 Plant 5n%ymology and 6isto 5n%ymology Balyani Publishers, 8ew 7elhi p3A"-. MandalB F. and &.N. :asu. !"-. Control of age3 and irradiation3induced seed deterioration in rice (-r%33asativa L.) by hydration3dehydration treatments. 'eed Res. E2 !M3A4.. MelDiB M. and -. Marouani. A44!. 5ffects of gamma rays irradiation on seed germination and growth of hard wheat. &nviron ,$em Lett., 7oi2 4. 44MNs 49 344!34AAA3 . MoeollerB ?ade W. 5nvironmental 6ealth. Cambridge, ,*26arvard <niversity Press, !!A Moore S. and Stein W.7 ( !E"#. ln1 ,ethods in 5n%ymol(5ds.Colowic$,& P and Baplan, 8 7# *cademic Press 8ew Hor$ 9 E-". MuraDamiB 7. et al., IRecent progress in long3duration microwave exposureK, in .And NoreenB J. and M. -s*raf. A44!. ?Changes in antioxidant en%ymes and some $ey metabolites in some genetically diverse cultivars of radish ( Ra $anus sativus 0.#?.&nviron. &* . "ot. -M2 9!.E4A. NovotnK J.B <alentovK 6.B Svo(oda J.B Sc*warz W.B FKL HB &ci., M (A#, MM3"4. !!!. C%ech L. =ood

6tterB /. and PolleB -. 133 . 'he influence of apoplastic ascorbate on the activities of cellwall associated peroxidase and 8*76oxidases in needless of 8orway &pruce (#iceaabies 0.#. #lant ,ell #$%siolog% 9.2 A9 3 A9". Pa$ne P.I. ( !"-#. 5ndosperm proteins. In3 A 4enetic A roac$ to #lant "ioc$emistr% . eds. *.7. Flonstein, P.L. Bing. &pringer3Gerlag, 8ew Hor$, <&*, pp A4.3A9 . PicazoB M.". et al IInhibition in the growth of thistles (Cynara cardunculus 0.# and lentils (0ens culinaris 0.# due to chronic exposure to .436% .3u' electromagnetc fieldsK5lectro and ,agnetobiology " ( !!!#2 EM3 .-. P"-N/ S6I" EN<I&6N., 55, A44! ( 4#2 EE9JE.9
59

PreussaB S.:.B -.:. :ritta. A449. * 78*3damage3induced cell cycle chec$point in Arabido sis.4enetics, -E2 9A9399E. &eena &and*ir and Falidas S*ett$, A44E. Process Fiochemistry, Golume 9! ( pp. MM.3 M"E. #, 94

Sc*o+ferB P. ( !!-#. 6ydrogen peroxide3mediated cell3wall stiffening in vitro in mai%e coleoptile. Planta !!4 E93E!. S*armaB P.F.B -nandB P. and SanD*alDarB S. 1332. Oxidative damage and changes in activities of antioxidant en%ymes in wheat seedlings exposed to <ltraviolet3F radiation. ,urrent 'cience M.29.!39-.. S*ewr$ P.&, Na+ier H.-B /at*am -.S ( !!.#. &eed storage proteins2&tructures and biosynthesis, 'he Plant Cell M2 !E.3!.-. SDilesB H.W. IPlant response to microwaves at A.E.;6%I *cta *stronautica ." (A44-#2A."3A-9. Smirnoff NB )um(es M.H. ( !"!#. 6ydroxyl radical scavenging activity in compatible solutes. Phytochem A"2 4.M3 4-4. /oDerB ).B :. @zunB 7. )anciB .. 6ncu )e$lan. A44.. 5ffects of gamma irradiation on the shoot length of ,icer seeds. Radiat. #$%s. ,$em., M92 9-.39-M. @rec*B M. et al I5ffects of microwave and radio fre)uency electromagnetic fields on <adde &amaDris*na and &amaDris*na &ao P. ! A44.#. Purification of acidic protease from the cotyledons of germinating Indian bean ( (olic$os lablab L. var lignosus# seeds. *frican Lournal of Fiotechnology Gol. E (M#, pp. M493M4M. <alentovK 6.B NovotnK J.B Svo(oda J.B Sc*warz W.B FKL HB A444. L. =ood 0ipids, M, pp. A9M3AE.. WiB S.G.B :.;. )*ungB H.S. Fim and et al. 2991. 5ffects of gamma irradiation on morphological changes and biological responses in plants. 6icron, 9"2 ..93.-E. ;annarelliB G.G.B S.M. Gallego and M.". /omaro 2 5ffect of <G3F radiation on the activity and isoforms of en%ymes with peroxidase activity in sunflower cotyledons. 5nviron. 5xp. Fot., .-, ME3 " (A44-#. ;i%Ping )*enB 2990. Photochemistry and photobiology, vol. "A (A#, pp. .493.4M. ;os*ida 7.B S. /aDagiB ;. 7iraDawa B 2999. =ood Chemistry, M4, pp. -93-!.

60

J*ang ).S.B "u M. and <erma ?.P.S.B ( !!M#. Characteri%ation of 3 pyrroline3.3 carboxylate synthetase gene promoter in transgenic Arabido sis t$aliana subjected to water stress. #lant 'ci., A!, " 3"!.

2. -++endices

-PPEN?I>5 I ES/IM-/I6N 6. /6/-" S@G-&S 1. StocD standard glucose solution A44 mg of glucose was accurately weighed and made up to 44 ml in a standard flas$ with distilled water. (Concentration : A mg N ml.#

1.

WorDing standard glucose solution


61

4 ml of the stoc$ was diluted to 44 ml with distilled water in a 44 ml standard flas$. (A4RgNml# 3. -nt*rone &eagent 4.A @ anthrone (A44mg N concentrated &ulphuric acid. 44ml of concentrated &ulphuric acid# solution in ice cold

-PPEN?I> 5 II ES/IM-/I6N 6. /6/-" P&6/EINS 1. -lDaline co++er reagent !"owr$Ns &eagent#

a. A@ 7isodium carbonate (8aACO9# in 4. sodium hydroxide (8aO6# b. 4..@ Copper sulphate (Cu&OE# in water. c. @ &odium potassium tartarate (Rochell>s salt#

d. (ater
.4 ml of 7isodium carbonate, 4..ml of Copper sulphate, .4 ml of &odium potassium tartarate were mixed when necessary. 2. .olinNs +*enol reagent 'o .4 ml of commercially available reagent ,A.4ml of distilled water was added. 3. Standard :ovine Serum -l(umin A:S-C 44 mg of F&* was dissolved in 44 ml of distilled water (Conc 3 . WorDing Standard Solution 4 ml of the stoc$ was diluted to 44ml with distilled water (Concentration : 4. mg N ml.# mg N ml.#

-PPEN?I>5 III ES/IM-/I6N 6. /6/-" .&EE -MIN6 -)I?S

1.

Nin*$drin
62

4."g stannous chloride (&nClA.A6AO# was dissolved in .44ml of 4.A, citrate buffer (p6 ..4#. *dd this solution to A4g of ninhydrin in .44ml of methoxyethanol. 2. 9.2M )itrate (uffer. +7 ,.9 *2 E.A g of citric acid in 44ml water F2 .."g of sodium citrate in 44ml water A4..ml of * \ A!..ml of F were ta$en and made upto 44ml and p6 chec$ed for ..4. 3. ?iluent solvent 5)ual volumes of water and n3propanol were mixed and used. . Standard .4mg leucine was dissolved in .4ml of distilled water. ,. WorDing standard 4ml of the stoc$ standard was ta$en and diluted to 44ml.

-PPEN?I>5 I< ES/IM-/I6N 6. P&6"INE

1.

-cid Nin*$drin
.A.g ninhydrin was warmed in 94ml glacial acetic acid and A4ml -, phosphoric acid,with agitation until dissolved. &tored at EOC and used within AE hrs.

2.

38 -Oueous Sul+*osalic$lic acid


9g of sulpho salicylic acid dissolved in 44ml of water.

3.

Glacial -cetic acid

-PPEN?I>5 <

ES/IM-/I6N 6. /6/-" P7EN6" 1. 298 Et*anol


63

2.

.olinNs ciocalteau reagent


'o .4 ml of commercially available reagent added A.4ml of distilled water.

3.

298 Sodium )ar(onate A4gm of &odium carbonate was dissolved in 44ml water .

Standard )atec*ol 44mg Catechol dissolved in 44ml water. 7iluted 4 times for wor$ing standard.

-PPEN?I>5 <I -SS-; 6. P%-M;"-SE !0,B 00# &eagent Pre+aration 1. StocD maltose solution 44mg of maltose was dissolved in 44ml of distilled water. 2. WorDing standard maltose solution 'a$e .4ml of stoc$ solution and made up to 4ml with distilled water. 3. 9.1M citrate (uffer of P7%,.9 *dd .!A grams of Citric acid +an hydrous/ and 4."" grams of &odium hydroxide pellets to distilled water to a final volume of 44ml and adjust p6 with store at room temperature. . 28 solu(le starc* A grams of soluble starch was dissolved in 44 ml of distilled water. ,. ?initrosalic$lic acid A?NSC reagent gram of 9..ml 78& is A4ml of A8 &odium hydroxide and 94 gram of &odium potassium tartarate and ma$e up to 44ml with distilled water. -PPEN?I>5 <II ES/IM-/I6N 6. PE&6>I?-SE 64 4 8 8a O6. *uto clave and

1.

9.9, M P$rogallol 9."mg of pyrogallol was dissolved in . ml of 4. , Phosphate buffer (p6 M.A#.

2.

18 7262 ml of 94@ 6AOA was diluted to 44ml with water. prepared freshly.

-PPEN?I>5 <III ES/IM-/I6N 6. )-/-"-SE

1.

9.1M P*os+*ate (uffer +7 1.2 * 2 .9! g of sodium dihydrogen Phosphate in 44 ml water F 2 A.-"g of 7isodium 6ydrogen Phosphate in 44 ml water. Eml of * \ 9-ml of F solution diluted -4 44ml and p6 chec$ed for M.A.

2.

9.1 M 7262 ml of 6ydrogen peroxide was mixed with ""ml of water.

3.

28 Sul+*uric acid Aml of sulphuric acid mixed with 44ml of water.

9.91N Potassium Permanganate 4.9 -g of Potassium permanganate dissolved in litre water.

65