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1.1 STRESS IN PLANTS Any environmental factor that affects the normal growth and development of plants can be described as stress or disturbance. All plants are subjected to a number of stresses throughout their life. These can be broadly divided into biotic and abiotic stresses. Biotic stresses are caused by parasitic organisms that live off the plant while abiotic stresses originate from soil (e.g. nutrient supply, salinity), climate (e.g. cold, drought) and pollution (e.g. effluents). Depending on the species of plant and the source of stress, the plant will respond in different ways. It is observed that after a certain tolerance level is reached there is delay and a gradual decrease in the germination and plant growth (ElMonem, et al., 2008).

1.2 EFFECT OF SALT STRESS ON CROP PRODUCTIVITY Drought and salinity are the two major environmental factors determining plant productivity and plant distribution (Bartels, et al., 2005; Patade, et al., 2006). Salinity in soil or water is one of the major stresses that severely limit crop production (Yokoi, et al., 2002; Oo, et al., 2005; Ashraf, et al., 2004). Salinity is of two types: • Primary or natural salinity results from two natural processes. First is the weathering of rocks containing soluble salts (chlorides of calcium, sodium, magnesium) and second is the deposition of oceanic salt carried by wind and rain (Dr. Munns, 2010). • Secondary or human induced salinization results from human activities. The most common causes are (i) land clearing and the replacement of perennial vegetation with annual crops, and (ii) irrigation schemes using salt-rich irrigation water (water used for irrigation is not fresh hence it is not free of salts) or having insufficient drainage. Insufficient drainage raises water table and mobilizes salts previously stored in the subsoil and brings them up to the root zone. Plants use the water and leave the salt behind until the soil water becomes too salty for further water uptake by roots. The water table continues to rise, and when it comes close to the surface, water evaporates leaving salts behind on the surface and thus forming a ‘salt scald’. The mobilized salt can also move laterally to water courses and increase their salinity (Dr. Munns, 2010). 1

Figur 1. Global distribution of salt affe re l n ected soils (N National Resources Man nagement an nd Envir ronment Dep partment, 20 011)

Table 1. Global estimate of secondary salinisation in the wo y n orld's irrigate lands (D ed Dr. ns, Munn 2010) Total land area Country C Chin na India a Sovi Union iet Unit States ted Paki istan Iran Thai iland Egyp pt Aust tralia Arge entina Sout Africa th Subt total Wor rld cropp ped Mn ha h 97 169 9 233 3 190 0 21 15 20 3 47 36 13 843 3 1,47 74 Area irrigated Mn M ha 45 42 21 18 16 6 4 3 2 2 1 159 227 % 46 25 9 10 78 39 20 100 0 4 5 9 19 15 6. .7 7. .0 3. .7 4. .2 4. .2 1. .7 0. .4 0. .9 0. .2 0. .6 0. .1 29 9.6 45 5.4 Area of irrigated land that is f l salt-affect ted Mn ha n % 15 17 18 23 26 30 10 33 9 34 9 20 20 2

Large and increasing proportions of the world’s irrigated land are deleteriously affected by salinity (National Resources Management and Environment Department, 2011). While the exact area affected is not known, it is estimated that approximately 20 percent of the world's irrigated land is damaged by salinization (Dr. Munns, 2010; National Resources Management and Environment Department, 2011). The National Academy of Sciences of the USA includes salinization of soils as one of the leading processes contributing to a possible worldwide catastrophe (Teutonico, et al., 1985). An alternative to this problem is growing plants and crops suited to moderately saline conditions. Crop plants that belong to the genus Amaranthus have high nutritive value and a wide adaptability to diverse environments and inspite of this it has not been exploited. However, it has been considered as a promising crop for saline soils (Teutonico, et al., 1985). Introduction of under-exploited salt-tolerant minor crops is a logical alternative for many developing countries and thus Amaranthu tricolor has been selected for this study.

1.3 Amaranthus tricolor Amaranthus tricolor is a terrestrial, annual, erect herb which can grow upto 80 cm tall (PROTA, 2004). Synonyms: Amaranthus tristis L., Amaranthus gangeticus L. (Globinmed, 2010-2011) Common names: Amaranth, Joseph’s coat, Pigweed (PROTA, 2004) Local names: Amaranthus is locally known as Batu, Cholai, Ganhar, Harvae, Keere, Maarsu, Marsha, Pung-Keerai, Rajgeera, Ramdana, Sawal and Sil Origin and Distribution: Amaranthus tricolor originates from tropical Asia. In South and South-East Asia it is one of the major leaf vegetables and the most important Amaranthus species. In the local areas of the Himalayas this amaranth species now is an important crop. It has gained popularity in the northwestern plains of India as well as in the hills of southern India under the common names rajgira ("king seed"), ramdana ("seed sent by God"), and keerai. A number of domesticated forms are available, especially in Andhra Pradesh, Karnataka, Tamil Nadu, and Kerala. It occurs as a quite rare exotic vegetable in several African countries, apparently introduced by Indian immigrants and 3

occasionally cultivated around the big cities, especially in East and southern Africa. Its cultivation has been reported from Benin, Nigeria, Kenya and Tanzania (PROTA, 2004). Ecology: Weedy plants of Amaranthus tricolor can be found occasionally on cultivated land, flood plains, roadsides and wasteland. Vegetable amaranths, including Amaranthus tricolor, grow well at day temperatures above 25°C and night temperatures not lower than 15°C. Shade is disadvantageous except in cases of drought stress. Amaranthus tricolor is a vegetable suited for cultivation in the tropics from sea level up to 1000 m altitude and in subtropical areas and warm temperate areas during the summer. Amaranths like fertile, well-drained soils with a loose structure. The mineral uptake is very high (PROTA, 2004). Morphology: It has a solid stem, taproot which is white or brown and the leaves are elliptical to lance-shaped or broad-ovate, dark green, light green or red. Clusters of flowers are axillary, often spherical or slightly spherical, and with a reduced terminal spike, but occasionally the terminal spike is well-developed. There are 3 petals. The fruit is dehiscent with a dehiscing lid. The seeds are 1 to 1.25 mm in diameter, brown or black, shiny, slightly compressed, reticulate and with shallow out growths on the reticulum. There are about 1200-2900 seeds/g (PROTA, 2004).

Classification Kingdom : Plantae Subkingdom : Tracheobionta Superdivision : Spermatophyta Division Class Subclass Order Family Genus Species : Magnoliophyta : Magnoliopsida : Caryophyllidae : Caryophyllales : Amaranthaceae : Amaranthus L. : Amaranthus tricolor L.

Economic importance 4

Grain amaranth: In Mexico, grain amaranth is used chiefly for making alegria candies from popped seeds and molasses and for preparing atole, a drink from roasted and powdered seeds mixed with syrup and water. In Peru, seeds are popped and ground into flour or bound with syrup and made into belles. In India, the seeds are most commonly used in the form of candy known as laddoos, though the seeds are sometimes boiled with rice. Amaranth seeds are parched, ground into flour, and eaten as gruel (sattoo) in Nepal, while like chapattis in the Himalayas. It is also cultivated as a grain crop in Guatemala (Teutonico, et al., 1985). Vegetable amaranth: Many species of Amaranth are grown as vegetables throughout the tropics and Eastern Asia. It is used as an African leafy vegetable. It is also a popular pot herb. The leaves and the softest portions of the shoots are usually boiled in several changes of water and then separated from the cooking liquid, though they traditionally are steamed in Uganda. Amaranth leaves are combined with condiments to prepare soup in Nigeria, used in salad, boiled and mixed with a groundnut sauce in Mozambique, or pureed into a sauce and served over (farinaceous) vegetables in West Africa. The flavor of raw and cooked vegetable amaranth was reported as equal to or better than that of spinach or other similar greens (Teutonico, et al., 1985). Dye: Mature leaves of A. tricolor contain red-violet pigments - the betacyanins: amaranthin and isoamaranthin is used as dye. The red dye from Amaranthus leaves is used to colour alcoholic beverages in Bolivia and northwestern Argentina, to colour maize dough in Mexico and the southwestern United States, and to dye foods and beverages in Ecuador (Teutonico, et al., 1985; Xu, et al., 2001). Ornamental plant: Several Amaranthus species have been cultivated in the Old and New World since ancient times as ornamentals (Teutonico, et al., 1985). Medicine: Amaranthus tricolor is used externally to treat inflammations, and internally as a diuretic (PROTA, 2004). Other species are used against dysentery, as a cholagogue, abortifacient and to treat snake bite. (Oswald Asia, 2010)


The leaf protein levels of Amaranthus tricolor is 33% (National Research Council. et al.4 MECHANISM OF SALT TOLERANCE In plants the mechanisms of salt tolerance takes place at three levels of organization: whole plant. 1984). Pandey.. compared with values of 44. With a protein content of ≈ 16%. Mahajan. quaternary ammonium compounds (Ashraf.. et al. three times that of maize and as mush as that of milk. amino acids. (Munns. Furthermore pseudocereals contain relatively high amount of dietary fibre. 2004. imino acids.. Munns. 2010). et al. If the salt is sequestered in the vacuole then potassium and other organic solutes should accumulate in the cytoplasm and other organelles to balance the osmotic pressure of the ions in the vacuole.. et al. et al. The nutritive value is about 75. et al. et al. Amaranthus seeds compare well with the conventional cultivars of wheat (12-14%). 2002. maize (910%) and other widely consumed cereals (Gorinstein. 2002) • Control at whole plant level Salt tolerance depends on the ability of the plant to control the transport of salt. 2002). 1. organic acids. et al. the standard of nutritional excellence (Mahajan. In this the plant tries to restrict the uptake of salt at root level and exclude the salt that is taken up by the plant. amides. It is one of those rare plants whose leaves are eaten as a vegetable while the seeds are used as cereals (Mahajan.. proteins. Others include low molecular weight sugars. which improves lipid metabolism and takes part in the prevention of LDL-C oxidation (Gorinstein. The organic solutes that most commonly accumulate under salinity are proline and glycine-betaine. 57 and 62 for maize. Bohnert. et al.. 2002.. et al. It has nearly twice the lysine content of wheat proteins. 1999) • Control at the cellular level At the cellular level the plant keeps the salt out of the cytoplasm by sequestering it in the vacuole of the cell. et al. 2002). polyols.. rice (7-10%).. (Munns.Nutrition: Amaranthus is the world’s most nutritious pseudo-cereal grain with multiple uses. 2002.. wheat and barley respectively. cellular and molecular.. 2002). • Control at molecular level 6 . 2002). They are good sources of minerals and vitamins and contain larger amounts than most of the common cereal grains.

Mutagenesis is an event capable of causing a mutation i. EMS. or chemical mutagens such as colchicine. Saranga. conventional breeding programs can be employed to develop crop varieties resistant to salinity stress.e.. Random mutagenesis can be performed using either physical mutagens such as UV rays.. 1990).. et al. The introduction of genes from wild salt tolerant species has been explored for tomato (Rush. Tal. coupled with efflux by the antiporter (Munns. Yokoi. This methodology does not require a deep knowledge of the genetics of traits. • Use the variation already present in the existing crops.. etc. the intervention of mutagenesis and tissue culture can greatly facilitate the selection and isolation of useful tolerant lines which is the dire need of the hour (Patade. Thus. However the approach has not led to the release of salt tolerant crops (Flowers.. 1997a.5 APPROACHES FOR ENHANCING SALT TOLERANCE Salt tolerance can be developed by one of the following methods: • Use interspecific hybridization to raise the tolerance of current crops. et al.At the molecular level regulation of Na+ uptake across the plasmalemma would come from restricted uptake by selective cation transporters and channels. tissue culture and mutagenesis.. through selection to multiplication of the new genotypes (El-Sayed. • Generate variation within existing crops by using recurrent selection. 2004).. Thus. 2004). 2007). It can be broadly classified into random and site-directed mutagenesis. Thus. 1997b) and pigeonpea (Subbarao. et al. et al. 1981. et al. the general objectives of this study were to better understand the tolerance of Amaranthus tricolor to salt stress by investigating: 7 . A combination of mutagenesis and in-vitro culture can speed up the breeding programs from generation of variability. et al. 1983. Of all of these mechanisms the accumulation of proline and glycine-betaine can be studied rather easily and thus they serve as good biochemical markers of salinity stress. 2002). a genetic change. merely that they display sufficient heritability and require suitable screening procedures to be developed (Flowers. et al. gamma rays. King. 1. but the progress is time consuming and tedious. et al. et al. etc. 1991) wheat (King... 2002. 2006). et al..

The effect of different salt concentration and EMS treatment on protein expression. The effect of mutagenesis on seed germination and seedling growth in increasing salt concentrations. 8 . The effect of increasing salt concentrations on biochemical markers (proline and glycine-betaine).• • • • The effect of increasing salt concentrations on seed germination and seedling growth.

In an experiment with different wheat cultivars. because it hinders plant roots from withdrawing water from the surrounding soil (Warrence. 1954). Thus. According to FAO (2002) some 20 to 30 million hectares of irrigated land are currently seriously damaged by salinity and 0.. Plants have their own mechanisms for fighting against any kind of stress. Munns. and this leads to reductions in the growth rate. 2006).. This is called the salt-specific or ion-excess effect of salinity (Dr. 2007. Salts in the soil or water may inhibit plant growth for two reasons. if excessive amounts of salt enter the plant in the transpiration stream there will be injury to cells in the transpiring leaves and this may cause further reductions in growth. the presence of salt in the soil solution reduces the ability of the plant to take up water. An alternative to this is growing plants especially crop plants which are salt tolerant.. 2010.. (ii) the yield of the crop on saline soils and (iii) the relative yield of crop on saline soil as compared with its yield on non saline soils under similar growing conditions (Dr.. Salt tolerance in plants may be the result of: • Control at the whole plant level 9 . The salt tolerance of a crop may be evaluated according to three criteria: (i) the ability of the crop to survive on saline soils. et al.0 dS m-1. Bohnert.25 to 0. Salinity becomes a problem when enough salts accumulate in the root zone to negatively affect plant growth. et al. Steppuhn and Wall (1997) showed that grain yields were reduced by more than 20% with root-zone salinity levels of less than 4. Munns. 2007). et al. Second. This is referred to as the osmotic or water-deficit effect of salinity. 1999). 2003). the increasing salinity in soils can lead to a worldwide catastrophe. US Salinity Laboratory Staff.5 million hectares are lost from production every year as a result of salt accumulation. Patade. Soil salinity may therefore have devastating effects on agriculture due to yield losses of crops. et al. First. et al.CHAPETR 2 LITERATURE REVIEW Salinity is the major environmental stress that greatly affects plant productivity (ElSayed. 2010. Saline soils are characterized by presence of toxic levels of sodium and its chlorides and sulphates (Khan.

particularly in the more tolerant species. petiole or leaf sheaths. it would occur at the endodermis. the stem. transport and excretion of salt. This ensures that salt is not exported to growing tissues of the shoot. and (5) excretion through salt glands or bladders. Loading of the phloem: There is little re translocation of Na+ or Cl− in the phloem. Excretion through salt glands or bladders: Only halophytes have these specialized cell types. Removal of salt from the xylem in the upper part of the roots. These are: (1) selectivity of uptake from the soil solution. 10 . Glycophytes rely on the first three mechanisms. and exhibit these mechanisms to various degrees (Munns. (4) loading of the phloem. Figure 3. indicating an exchange of K+ for Na+ by the cells in the stele of the roots or in the vascular bundles in stems and petioles. at the exodermis.Salt tolerance depends on the ability of the plant to control the transport of salt at five sites (Figure 3). The initial uptake of Na+ and Cl− could occur at the epidermis. 2002). Loading of the xylem: There is evidence for a preferential loading of K+ rather than Na+ by the cells of the stele. (3) removal of salt from the xylem in the upper part of the plant. or if soil solution flows apoplastically across the root cortex. In many species. as summarised below: Selectivity of uptake by root cells: It is still unclear which cell types control the selectivity of ions from the soil solution. (2) loading of the xylem. Na+ is retained in the upper part of the root system and in the lower part of the shoot. Control points at which salt transport is regulated. All halophytes have well-developed mechanisms to control the uptake.. et al.

2004). et al. et al.. pyrroline carboxylic acid synthetase and pyrroline carboxylic acid reductase. Wang. 2008) in sorghum (Mickelbart. et al... 2008) and in Amaranthus tricolor (Wang...• Production of osmolytes and organic solutes Na+ and Cl. BADH in its native state has a molecular mass of 125 kDa. 2000. The amaranth CMO polypeptide has a molecular mass of 45 kDa same as that of spinach and sugar beet (Russell. (2011) reported that glycinebetaine in plants is synthesized from choline by a two step oxidation reaction. Although proline can be synthesized either from glutamate or ornithine.. 2007) in lentils (El-Monem. The biosynthetic pathway consists of two important enzymes viz. An increase was also observed in the glycine betaine levels in Cajanus cajan (Jaleel. et al. glutamate is the primary precursor in osmotically stressed cells. Chen. With increasing salt concentrations. 2001). et al.. et al..from the cytoplasm by sequestering it in vacuoles.. 11 . increase was reported in proline accumulation in Cajanus cajan (Jaleel. Proline and glycinebetaine are the most commonly produced osmolytes under salinity stress. 1998). 2002). et al. et al. et al.. The molecular weight of pyrroline carboxylic acid synthetase is 78 kDa. 2003) in Amaranthus tricolor (Wang.are toxic at high levels. et al. 2010). The molecular weight of pyrroline carboxylic acid reductase is 320 kDa whereas the polypeptide has a molecular mass of 28. et al.145 kDa (from nucleotide sequence) and 26. 1999) in wheat (El-Sayed. et al. et al. It is formed by two sub units with molecular masses of 63 and 70 kDa as determined by SDS PAGE (Figueroa-Soto. 2008) in rice (Thach. The first step is catalyzed by choline monooxygenase (CMO) which converts choline to betaine aldehyde and the second step is catalyzed by betaine aldehyde dehydrogenase (BADH) which gives glycine betaine as the end product. 2000).... To maintain an osmotic balance the plant should produce organic solutes to maintain the osmotic pressure of the ions in the vacuole (Munns. The plant removes Na+ and Cl. et al.5 kDa (experimentally) (Caspi.

Besides there may be difference in the foliage colour than normal plants and sometimes the leaves are thicker and succulent. et al. et al. 2005) in five lentil cultivars (ElMonem. Gradual decrease in rate of germination and inhibition of germination with increasing salt concentrations was reported in Jute (Abbas. Intracellular compartmentation is by a vacuolar Na+/H+ antiporter. Blumwald.• Control at the molecular level: ion transporters The ion channels and transporters that regulate the net movement of salt across cell membranes have been recently reviewed (Amtmann. Soil salinity may affect the germination of seeds by creating an osmotic potential to prevent water uptake and/or by providing conditions for the entry of ions which may be toxic to the embryo or developing seedling. 1982). driven by the pH gradient across the plasmalemma (Blumwald. et al. 1999. 1999).. (2006).. driven by a pH gradient across the tonoplast (Blumwald. et al. 1999).Salt are usually most damaging to young plants. Schachtman. et al. Na+ could enter the cell through high affinity K+ carriers or through low affinity channels called nonselective cation channels that are strongly influenced by Ca2+.. These cation channels could allow entry of large amounts of Na+ from a highly saline soil if not adequately regulated (Amtmann. Morphological and physiological responses of plants to salt stress may also vary. There is no specific Na+ transporter. et al. et al. Different growth stages of plant species may have variable tolerances to salt in soils and the effects on different parts of a plant may also vary (Miller. 2000). Plants affected by excessive soluble salt concentrations may appear normal but a general stunting of growth is observed. (2008) reported a reduction in root and shoot 12 . sugarbeet. et al.. Jamil.. amaranth and pak-choi (Jamil. et al. 1998). 1998). particularly cereal crops. and more tolerant as it matures (Miloslav. 2008) in cabbage.. Rice for example is tolerant during germination becomes sensitive during early seedling growth.. 2000.. 2000). Na+ can be effluxed from the cytoplasm through Na+/H+ antiporters. et al. Abbas. et al. 2006). Although plants are in general more tolerant to saline conditions during germination high salt concentrations of salts may reduce the rate of germination or even inhibit the rate of germination (Miller. (2005) and El-Monem. The transporters that maintain low Na+ concentrations in organelles such as chloroplasts and mitochondria are not known.

They exposed the seeds to 1. sugarbeet.2 and 1. cv. gamma rays) and certain chemicals (colchicines. amaranth and pak-choi). Conventional breeding procedures which involve. then can be used to induce mutations and generate genetic variations from which desired mutants may be selected (Novak. 2005). Percentage of mortality increased while plant height and internode length decreased with increasing gamma ray doses (Tah... 1992). (2005) collected three high yielding and early maturing mutants by treating seeds of Brassica juncea L. et al. jute and five cultivars of lentil respectively. 2010). Mutagenic agents.0.. 13 . Shah. ABASIN-95 by induced mutation. et al. S-9 with gamma rays (750 -1000 KGy) and EMS.length. In-vitro culture techniques offer many advantages such as expression of recessive genes that may be immediately recognized in haploid genotypes derived from the in-vitro culture of anthers.4 KGy gamma rays and the resulting new variety was high yielding. 1990)... 2006). The use of mutagens in combination with in-vitro culture has created great interest in plant breeders to create genetic variation. The germination percentage of somatic embryos and development of such embryos into plant with leaf and root reduced with increasing EMS concentrations (Novak. 2010). and fresh and dry weight of Lepidium sativum L (Majeed. Atrazine resistant plants were selected using EMS (Venkataiah. resistant to Alternaria blight and white rust. et al. such as radiation (x rays. the production of new genetic combinations from already existing parental genes is rather time consuming and tedious (Majeed. fresh and dry weight with increasing salt concentrations in four vegetable species (cabbage.. Khatri. cv. (2001) developed a new oil seed Brassica Napus L. et al. EMS).. et al. Moreover very large population of plant cells or plants can be handled relatively easily. 1. With increase in dose of gamma rays a decrease was observed in the root and shoot length. et al. et al.

3.5. 150mM. The media used was autoclaved at 121oC for 20 minutes at 15 lb/in2 pressure and used for seed germination after it had attained room temperature. 1/2 MS. the seeds were grown in 1/4 MS media containing 3% sucrose. 1/4 MS and 1/8 MS media containing 3% sucrose. The seeds were then transferred into a petriplate containing blotting paper and allowed to dry before inoculating.7 . 0. The seeds were then treated with 70% ethanol for 30 seconds and rinsed thrice with autoclaved distilled water.7 . 3.8.CHAPTER 3 MATERIALS AND METHODS 3. pH maintained between 5. The media used was autoclaved at 121oC for 20 minutes at 15 lb/in2 pressure and used for seed germination after it had attained room temperature.1 Plant material collection Seeds of Amaranthus tricolor (variety Pusa Kirti) were procured from IARI. 0.8% agar and pH maintained between 5. 200mM and 250mM).8% agar.4 In-vitro screening of salt tolerance For screening of salt tolerance in-vitro.1% HgCl2 for 7 minutes and rinsed twice with autoclaved distilled water.2 Seed Sterilization • • • The seeds were treated with 0. New Delhi. 14 .5. 3.3 In-vitro seed germination and seedling growth studies in different strength of MS media The optimum concentration of MS media for in-vitro growth was selected after studying the seed germination percentage and seedling growth in MS. 100mM.8 and different concentrations of NaCl (50mM.

15 .0..5%) of EMS for 4 hours. 0. 3.3. 0.2%. Standard BSA Method (A) Sample extraction: • • • Samples were harvested and homogenized in 1(sample):1(extraction buffer) using a mortar and pestle. et al.6. Seeds were treated with varying concentrations (0. 1951) Material HEPES-KOH extraction buffer.... Seeds were then sterilized as given in 3...000 rpm for 10 minutes at 4oC.7-5.2. Seeds were then washed twice with 0. The samples were mixed well and incubated at room temperature for 10 minutes. The supernatant was then used for estimation of total protein content and protein profiling studies (B) Protein estimation: • • • • 1 ml of sample solution was taken.. 5 ml of Reagent C was added.. Reagent C.1 Estimation of protein by Lowry’s method (Lowry. NOTE: EMS is carcinogenic and volatile. 0/3%. FC reagent (2N).6 Biochemical Studies 3..1M sodium thiosulphate for 15 minutes each.. Seeds were then washed twice with distilled water for 15 minutes each.5 Chemical mutagenesis Determination of LD50 of EMS on seeds of Amaranthus tricolor (variety Pusa Kirti) • • • • • Seeds were presoaked in distilled water for 6 hours.1%.8% agar and pH maintained between 5. Refer to handling instructions given in Appendix B and C before using EMS... 2.8.. The homogenate was centrifuged at 14.5 ml of FC reagent (1N) was added... inoculated in 1/4MS containing 3%sucrose.

02M EDTA.8). A standard graph is prepared in the same way using standard BSA. The gel was treated with AgNO3 solution in dark for 15 minutes followed by two washings with distilled water for 30 seconds each. β mercaptoethanol. Agar. India. 50% ethanol.5M Tris – HCl pH 8. A linear relationship between concentration and absorbance is observed in the range of 10 µg/ml – 100 µg/ml of protein. et al. (B) SDS-PAGE: Equal concentrations of proteins were loaded and were separated using 8% resolving gel and 4% stacking gel.8.02% Na2S2O3. AgNO3 solution. ) Material Acrylamide – bisacrylamide stock (30 : 0.8. The gel was then rinsed with distilled water twice for 30 seconds each. Bromophenol blue. 0.8.02% Na2S2O3. 10%APS. Developing solution was added and gel was gently rocked till bands develop.02M EDTA Method (A) Sample preparation: The protein sample was mixed with equal volume of sample buffer 2X. (C) AgNO3 staining: • • • • • • The gel was rinsed with 50% ethanol twice for 30 minutes each. 0. 16 .5M Tris –HCl pH 6. A protein molecular weight marker was loaded in one well. TEMED. 1. The mixture was boiled for 3 minutes in a boiling water bath and cooled to room temperature.6. 1. Glycerol. Glycine. 3.• • • It was mixed well and incubated at room temperature in dark for 30 minutes and absorbance was read at 660nm. The protein molecular weight marker (205 kDa – 3 kDa) used was from Bangalore Genei. The gel was then rinsed with 0.5M Tris – HCl pH 6. distilled water. 10%SDS.. Reaction was stopped by adding 0.5H2O for 1 minute.5H2O.2 Protein profiling using SDS – PAGE (Sambrook. Developing solution and 0.

2ml of cold potassium triiodide reagent was added and the mixture was gently mixed with vortex mixture and stored at 0 .6. The homogenate was filtered through Whatmann No. et al. Standard Proline Method • • • • • • • • • 0.2 – dichloroethane Method • Extract was prepared by finely ground plant material (0.4 Estimation of glycine-betaine (Greive. 1983) Material Distilled water. linear relationship between concentration and absorbance is observed in the range of 0. et al.6. 1 filter paper.1mM per ml of proline. glacial acetic acid. The red colour intensity was measure at 520 nm. samples were transferred to centrifuge tubes and then centrifuged at 10. After the expiring of the period.5ml) was measured into test tube and cooled in ice water for 1 hour. 3% aqueous sulphosalicylic acid. 2N Sulphuric acid and 1.4oC for 16 hours. 3.1 gm) which was mechanically shaken with 4ml of distilled water for 48 hours at 25oC.02mM – 0.000g for 15 minutes at 0oC.1 gm of plant material was extracted by homogenizing in 2 ml of 3% aqueous sulphosalicylic acid. 0. The reaction was terminated by placing the tubes in ice bath.3. Potassium tri-iodide reagent. 2 ml of filtrate was taken in a test tube. It was heated in a boiling water bath for 1 hour. 1973) Material Acid ninhydrin. toluene. To which 2 ml of glacial acetic acid and 2 ml of acid ninhydrin was added. The samples were then filtered and the filtrate was diluted 1:1 with 2N sulphuric acid. • • • Aliquot (0.. 17 .. The toluene layer was separated and warmed to room temperature. 4 ml toluene was added to the reaction mixture and stirred well for 20-30 seconds. For the standards.3 Estimation of Proline (Bates.

18 . linear relationship between concentration and absorbance is observed in the range of 50µg/ml . After 2 – 2. Vigorous vortex mixing was done to effect complete solubility in developing solvent.200 µg/ml of glycine-betaine. The periodite crystals were dissolved in 9 ml of 1.• • • • • The supernatant was carefully aspirated with 1 ml micropipette. For the standards.2 – dichloroethane.5 hours the absorbance was measured at 365 nm with UV spectrophotometer.

19 1/2 MS media 92 ± 4.17 4.17 3. ± SE Figure 4.045 ± 0.813 ± 0.809 ± 0.22 3.18 1/8 MS media 92 ± 7.17 Table 2.254 ± 0.66 2.013 ± 0.16 3. Pusa Kirti) grown in-vitro in different strength of MS media.CHAPTER 4 RESULTS AND DISCUSSION 4.605 ± 0.1 In-vitro seed germination and seedling growth in different strength of MS media MS media Germination (%) Root length (cm) Shoot length (cm) 80 ± 5.27 4. Germination percentage of seeds with root and shoot length of Amaranthus tricolor (var.926 ± 0.2 1/4 MS media 100 ± 0 3.982 ± 0.38 3. Germination percentage of seeds of Amaranthus tricolor (var. Pusa Kirti) grown in different strength of MS media 19 .

Shoot length of seedlings of Amaranthus tricolor (var.Figure 5. Pusa Kirti) grown in different strength of MS media 20 . Root length of seedlings of Amaranthus tricolor (var. Pusa Kirti) grown in different strength of MS media Figure 6.

Thus.Discussion: Seed germination was initiated after 48 hours of inoculation. in all further studies the seeds were inoculated in the same amount of media (10 ml per test tube) and the samples for further analysis were harvested on the 12th day. 21 . 1/4 MS media was selected for further studies. Higher germination percentage. Therefore. It did not change after 72 hours. All seeds inoculated in 1/4 MS media had germinated. Pusa Kirti) was observed in 1/4 MS media as compared to MS. root and shoot length of Amaranthus tricolor (var. 1/2 MS and 1/8 MS media. Seedlings started to wither after 12 days due to depletion of nutrients.

22 .434 ± 0.69 3. ± SE Figure 8.633 ± 0.18 100 mM 76 ± 6.2 In-vitro seed germination and seedling growth in 1/4 MS media in different concentrations of NaCl Control Germination (%) Root (cm) Shoot length (cm) length 92 ± 4.96 ± 0.93 ± 0.25 3. Germination percentage of seeds of Amaranthus tricolor (var.4.18 0.2 50 mM 84 ± 6.08 200 mM 250 mM 0 0 0 0 0 0 Table 3. Pusa Kirti) grown in 1/4 MS media containing different NaCl concentrations. Pusa Kirti) grown in 1/4 MS media containing different NaCl concentrations.26 3.1 150 mM 24 ± 3.65 ± 0.25 ± 0.69 4.2 3.38 4.514 ± 0.58 0. Germination percentage with root and shoot length of Amaranthus tricolor (var.156 ± 0.

Shoot length of seedlings of Amaranthus tricolor (var. Pusa Kirti) grown in 1/4 MS media containing different NaCl concentrations. Root length of seedlings of Amaranthus tricolor (var. 23 .Figure 9. Figure 10. Pusa Kirti) grown in 1/4 MS media containing different NaCl concentrations.

100 and 150 mM NaCl germinated within 48 – 72 hours. after the 5th and 7th day respectively. A gradual delay and decrease in germination percentage was observed with increasing salt concentrations. Pusa Kirti) can tolerate salt concentrations up to 100 mM. It can therefore be concluded from the present study that Amaranthus tricolor (var. 24 .Discussion: In control germination was observed after 48 hours and did not change after 72 hours. Seeds inoculated in media containing 50 mM NaCl did not show delay in germination but showed decrease in germination percentage. Seeds subjected to salt stress germinated at different times depending on the concentration. Germination percentage decreases significantly in media containing 150 mM NaCl. Seeds inoculated in media containing 50. Improved morphology of the seedlings (maximum root and shoot length) was observed in media containing 50mM NaCl which indicates that NaCl in small amounts may enhance the growth of the plant. Seeds did not germinate in media containing 200 and 250 mM NaCl.

33 50 46.33 13.2 1. Pusa Kirti) Table 4. the lethal dose on the basis of in-vitro germination of seeds and survival of seedlings of Amaranthus tricolor (var.3 0. Seeds treated with 2% EMS did not germinate.33 53.5 0.6 0.67 63. 25 .67 56.33 63.2 0.67 53.3 1. Pusa Kirti) was adjudged as 1%.33 60 56.4 0.1 1.8 0.5 2 Germination (%) 66.4 1.4.3 Determination of LD50 of EMS on seeds of Amaranthus tricolor (var.9 1 1.33 53. Thus. Based on the germination percentage LD50 was revealed to be 1%.67 43. Germination percentage of seeds treated with different concentrations of EMS Concentration of EMS Control 0.33 10 0 Discussion: 50% germination was observed in seeds treated with 1% EMS.7 0. This further confirmed that LD50 is 1%.33 33.

69 4.965 ± 0.08 1.02 100 mM 14.953 ± 0.67 ± 0.29 ± 0.91 ± 0.91 ± 0.47 1.3 ± 0.09 100 mM 0 0 0 150 mM 0 0 0 200 mM 250 mM 0 0 0 0 0 0 Table 5.9 ± 0.67 ± 0.27 0.633 ± 0.19 50 mM 84 ± 6.4 Effect of mutagenesis on seed germination.93 ± 0.06 1.86 ± 0.27 2.31 ± 0.06 3.25 2.1 50 mM 46.67 ± 0.03 150 mM 0 0 0 200mM 0 0 0 250mM 0 0 0 Seeds treated with 1.1 ± 0.2 0.69 3.7% EMS 0 mM Germination (%) Root length (cm) Shoot length (cm) 63.27 2.27 3.05 1.65 ± 0.2 3.5% EMS 0 mM Germination (%) Root length (cm) Shoot length (cm) 70 ± 0.26 3.72 ± 0. seedling growth and salt tolerance Seeds treated with 0% EMS (Control) 0 mM Germination (%) Root length (cm) Shoot length (cm) 93 ± 4.82 ± 0. Germination percentage with root and shoot length of Amaranthus tricolor (var.45 ± 0.06 100 mM 16.88 ± 0.38 4.514 ± 0.87 ± 0.4. Pusa Kirti) treated with different concentrations of EMS and grown in 1/4 MS media containing different NaCl concentrations.1 150 mM 0 0 0 200 mM 250 mM 0 0 0 0 0 0 Seeds treated with 0.33 ± 0.9 ± 0.1 150 mM 26 ± 3.11 1.05 2.6 0.05 0.1 ± 0.94 ± 0. ± SE 26 .157 ± 0.1 2.1 200 mM 0 0 0 250mM 0 0 0 Seeds treated with 0.178 100 mM 75 ± 6.27 0.11 50 mM 56.27 1.12 50 mM 13.55 ± 0.0% EMS 0 mM Germination (%) Root length (cm) Shoot length (cm) 50 ± 0 1.294 ± 0.29 ± 0.

Root length of seedlings of Amaranthus tricolor (var. Figure 13.Figure 12. Germination percentage of seeds of Amaranthus tricolor (var. Pusa Kirti) treated with different concentrations of EMS and grown in 1/4 MS media containing different NaCl concentrations. 27 . Pusa Kirti) grown in 1/4 MS media containing different NaCl concentrations after seeds were treated with different concentrations of EMS.

Pusa Kirti) grown in 1/4 MS media containing different NaCl concentrations after seeds were treated with different concentrations of EMS.7 and 1.0%.7% EMS germinated in NaCl concentrations upto 100 mM. Seeds treated with 0. 28 .Figure 14.5% EMS show high germination percentage compared to 0.5 and 1.0%. Discussion: Seeds treated with 0. However. root length of seeds treated with 0. Root length in seeds treated with 0. Shoot length of seedlings of Amaranthus tricolor (var.7% was greater than those treated with 0. The germination percentage of seeds at each concentration of EMS decreased with increasing NaCl concentration.5% EMS and grown in 1/4 MS media containing different NaCl concentrations show germination pattern similar to seeds treated with 0% (Control). Increasing concentrations of EMS resulted in decrease of germination percentage.5 and 0. Those treated with 1% EMS germinated in NaCl concentrations upto 50 mM.

5% and 0. 29 . Moreover. On comparing with control it can be inferred that.Shoot length of seeds treated with 1% was greater than those treated with 0. EMS may not be causing any beneficial mutation for Amaranthus tricolor (var. So. the concentration of EMS may be hampering the seed germination and seedling growth.7%. further studies using still lower concentrations of EMS can be done in order to confirm the observations of the present study. Also shoot length in each concentration of EMS decreased with increasing NaCl concentrations. Pusa Kirti) so as to bring about an increase in its salt tolerance.

112 0.040 0. et al. Standard graph of BSA 30 . Absorbance of different concentrations of BSA (µg/ml) at 660 nm Figure 16.5 Protein estimation by Lowry’s method (Lowry.170 0. 1951) Concentration of BSA (µg/ml) 0 10 20 40 60 80 100 Absorbance at 660nm 0.223 0.4.061 0..000 0.261 Table 6.

3 7.2 2.552 Table 7.440 0.07 6.53 3.1 gm of fresh tissue (µg) 140 220 240 253 306 306 630 733 775 Concentration of protein (mg/gm of fresh tissue) 1.0% EMS 0.5% EMS + 50 mM NaCl 0.07 3. Pusa Kirti) grown in 1/4 MS media containing different NaCl concentrations. Concentration of protein in seedlings of EMS treated seeds of Amaranthus tricolor (var. 31 .261 1. Pusa Kirti) grown in 1/4 MS media containing different NaCl concentrations.4 2.483 0.33 7. Figure 17.75 Control 50 mM NaCl 100 mM NaCl 150 mM NaCl 0.4 2.613 0.5% EMS + 100 mM NaCl 0.613 1.5% EMS 0.Samples Absorbance at 660 nm Concentration of protein in 0.466 1. Concentration of protein in seedlings of EMS treated seeds of Amaranthus tricolor (var.7% EMS 1.506 0.282 0.

32 . A significant increase was observed in the protein concentration in the EMS treated samples indicating that certain proteins may have been over expressed or new proteins may have been expressed. Protein estimation in 0. The over expression of proteins was confirmed by increase in intensity of protein bands as seen in SDS PAGE.7% EMS treated seeds grown in 50 and 100 mM and 1.Discussion: In control (0% EMS) with increasing NaCl concentrations an increase was observed in the protein concentration per gm fresh tissue.0% EMS treated seeds grown in 50 mM could not be performed since sufficient sample was not available for analysis.

33 .000 0.331 0.6 Estimation of proline (Bates.06 0.04 0. Standard graph of proline. Figure 18.211 0.1 Absorbance at 520nm 0.08 0.096 0.4. et al.504 0.. Absorbance of different concentrations of proline (mM) at 520 nm.597 Table 8. 1973) Concentration of proline (mM) 0 0.02 0.01 0.062 0.

034 0.261 Concentration of proline (mM) 0.200 0. at higher salt 34 . Pusa Kirti) grown in 1/4 MS media containing different NaCl concentrations.010 0.011 0.060 0. Discussion: On comparison with control it was observed that at 50 mM seedlings accumulated proline at concentrations almost similar to control but.044 Table 9. Pusa Kirti) grown in 1/4 MS media containing different NaCl concentrations.065 0. Concentration of proline (mM) in seedlings of Amaranthus tricolor (var. Figure 19. Concentration of proline (mM) in seedlings of Amaranthus tricolor (var.Absorbance at 520 nm Control 50 mM NaCl 100 mM NaCl 150 mM NaCl 0.

35 .concentrations there is a significant increase in proline concentration indicating that plants may not be stressed at 50 mM. Estimation of proline in EMS treated samples could not be performed due to non availability of sufficient sample.

. et al.4.26 0.7 Estimation of glycine-betaine (Greive. 36 . Standard graph of glycine-betaine.10 0. Absorbance of different concentrations of betaine (µg/ml) at 365 nm.32 0. Figure 20.38 0.42 Table 10.00 0. 1983) Concentration of glycine-betaine (µg/ml) 0 50 75 100 125 150 175 200 Absorbance at 365 nm 0.16 0.21 0.

Discussion: Very small difference was observed in the concentration of glycine-betaine that accumulated in seedlings at 0 and 50 mM but.317 (µg) 118 129 576 658. at higher salt concentrations there is a significant increase in glycine-betaine concentration indicating that plants may not be stressed at 50 mM.Absorbance at 365 Concentration of glycine. Estimation of glycine-betaine in EMS treated samples could not be performed due to non availability of sufficient sample.258 1. Pusa Kirti) grown in 1/4 MS media containing different NaCl concentrations.betaine nm Control 50 mM NaCl 100 mM NaCl 150 mM NaCl 0.152 1.236 0. Pusa Kirti) grown in 1/4 MS media containing different NaCl concentration.6 Table 11. Figure 21. Concentration of glycine-betaine (µg) in seedlings of Amaranthus tricolor (var. Concentration of glycine-betaine (µg) in seedlings of Amaranthus tricolor (var. 37 .

Though. 70kDa and 78 kDa band in lane no. For better separation of protein bands and identification of protein bands of interest the low molecular weight proteins were allowed to run out of the gel.4. The background of the gel in Figure 23 is not clear due to late development of protein bands. 3 can be due to 2 reasons. Absence of the 63 kDa. The protein bands in Figure 22 are too close to one another. 38 . Either the proteins are not produced or the protein bands have not been properly developed. On comparison with control the EMS treated samples showed variability and increase in intensity of bands. protein bands have been properly separated.8 Protein profiling using SDS PAGE Intensity of protein bands increased with increase in NaCl concentrations indicating increased protein expression under salt stress.

Protein profiling studies only showed increased expression of proteins. (var. This research work has been extended to further analyze the salt tolerance by using lower concentrations of EMS. Salt tolerant lines will be analyzed for genetic variation using RAPD. Pusa Kirti) and can tolerate salt concentrations upto 100 mM. Protein profiling studies revealed increased expression of proteins with increase in NaCl concentration.CONCLUSION From the present study of screening for salt tolerant mutants in Amaranthus tricolor L. Germination percentage decreased significantly at 150 mM salt stress. From this it can be inferred that no beneficial mutation has occurred and high concentrations of EMS hampered the growth and germination of seedlings. Various biochemical analysis will be performed to see how the nutritive value and other metabolites and vitamins changes. No variability was seen in protein bands. 0. The seedling showed enhanced growth and accumulated proline and glycine-betaine at levels almost similar to control when subjected to 50 mM salt stress.5 and 0. Plants will also be analyzed for oxalic acid content which accumulates in plants in salt stress since ingestion of plants containing high amounts of oxalic acid would be harmful. Salt tolerance would also be analysed using gamma radiations. From this it can be concluded that small amounts of salt enhances the plant growth of Amaranthus tricolor L. 39 .7 % EMS treated seeds which were grown in media containing different NaCl concentrations germinated upto 100 mM salt stress while 1% EMS treated seeds germinated only upto 50 mM salt stress. Increasing NaCl concentration resulted in accumulation of proline and glycine-betaine. Pusa Kirti) it was found that increasing NaCl concentrations delayed germination and reduced the germination percentage and seedling growth. (var. The different growth parameters studied in seedlings of EMS treated seeds showed variable results.

APPENDIX A: Composition of media Table . disodium Ferrous sulphate mg/L 7460 5560 IV Stock D ( 5 ml/L ) Meso-inositol Nicotinic acid Pyridoxine HCl Thiamine HCl Glycine mg/L 20000 100 100 20 400 40 . Composition of MS media I Stock A ( 50 ml/L ) Ammonium nitrate Potassium nitrate Calcium chloride Magnesium sulphate Potassium dihydrogen orthophosphate mg/L 33000 38000 8800 7400 3400 II Stock B ( 5 ml/L ) Potassium iodide Boric acid Mangnese sulphate Zinc sulphate Sodium molybdate Copper sulphate Cobalt chloride mg/L 166 1240 4460 1720 50 5 5 III ` Stock C ( 5 ml/L ) EDTA.

EMS treatment should be performed in well ventilated laboratory ideally under a fume hood Discard washes containing EMS in a beaker containing 0. labcoat. gloves.1 M sodium thiosulphate Decontaminate all material that has come in contact with EMS by treatment with 10% sodium thiosulphate. All glassware coming in contact with EMS should be immersed in 1N NaOH or laboratory bleach before recycling or disposal. and closed footwear. Dry discard including tissue. should be sealed in plastic bags under the fume hood. 41 . gloves. facemask. etc.APPENDIX B: Handling instructions for EMS • • • • • • Personnel safety: Safety goggles. vials.

com. of eye contact (irritant). Potential Chronic Health Effects: CARCINOGENIC EFFECTS: Classified 2B (Possible for human. Houston. Slightly hazardous in case of skin contact (irritant. 14025 Smith Rd. TERATOGENIC EFFECTS: Not available. Ethyl ester of methylsulfonic acid. permeator). 42 . Ethyl ester of methanesulpnonic CHEMTREC (24HR Emergency Telephone). call: 1-281-441-4400 Section 2: Composition and Information on Ingredients Composition: Name Methanesulfonic Acid Ethyl Ester CAS # 62-50-0 % by Weight 100 Toxicological Data on Ingredients: Methanesulfonic Acid Ethyl Ester: ORAL (LD50): Acute: 470 mg/kg [Mouse]. call: 1-703-527-3887 For non-emergency assistance. Ethyl ester of methanesulfonic acid. MUTAGENIC EFFECTS: Mutagenic for mammalian somatic cells.Appendix C: Material Safety Data Sheet for EMS Section 1: Chemical Product and Company Identification Product Name: Methanesulfonic Acid Ethyl Ester Catalog Codes: SLM2476 CAS#: 62-50-0 RTECS: PB2100000 TSCA: TSCA 8(b) inventory: Methanesulfonic Acid Ethyl Ester CI#: Not available. DEVELOPMENTAL TOXICITY: Classified Reproductive system/toxin/male [POSSIBLE]. of inhalation. Section 3: Hazards Identification Potential Acute Health Effects: Hazardous in case of ingestion. Ethyl ester of methylsulphonic acid. The substance may be toxic to the reproductive system. Synonym: Ethyl Methanesulfonate. Texas 77396 US Sales: 1-800-901-7247 International Sales: 1-281-441-4400 Order Online: ScienceLab. Repeated or prolonged exposure to the substance can produce target organs damage. Methylsulfonic acid. Ethyl methanesulphonate. call: 1-800-424-9300 International CHEMTREC.) by IARC. ethyl ester Chemical Name: Methanesulfonic Acid Ethyl Ester Chemical Formula: C3-H8-O3-S Contact Information: Sciencelab. Mutagenic for bacteria and/or yeast. Inc.

give artificial respiration.Section 4: First Aid Measures Eye Contact: Check for and remove any contact lenses. Flammable Limits: Not available. Special Remarks on Explosion Hazards: Not available. CO2). Ingestion: Do NOT induce vomiting unless directed to do so by medical personnel. Products of Combustion: These products are carbon oxides (CO. Section 7: Handling and Storage Precautions: 43 . Serious Ingestion: Not available. Section 5: Fire and Explosion Data Flammability of the Product: May be combustible at high temperature. Get medical attention if irritation develops. belt or waistband. Fire Fighting Media and Instructions: SMALL FIRE: Use DRY chemical powder. If not breathing. If the victim is not breathing. of heat. Get medical attention. perform mouth-to-mouth resuscitation. Inhalation: If inhaled. fog or foam. If large quantities of this material are swallowed. immediately flush eyes with plenty of water for at least 15 minutes. call a physician immediately. LARGE FIRE: Use water spray. Large Spill: Absorb with an inert material and put the spilled material in an appropriate waste disposal. Serious Skin Contact: Not available. Skin Contact: Wash with soap and water. Slightly explosive in presence of heat. tie. Fire Hazards in Presence of Various Substances: Slightly flammable to flammable in presence of open flames and sparks. Nonexplosive in presence of shocks. give oxygen. Never give anything by mouth to an unconscious person. Non-flammable in presence of shocks. belt or waistband. Cover the irritated skin with an emollient. remove to fresh air. Explosion Hazards in Presence of Various Substances: Risks of explosion of the product in presence of static discharge: Not available. Auto-Ignition Temperature: Not available. In case of contact. Get medical attention if irritation occurs. If breathing is difficult. Special Remarks on Fire Hazards: Not available. tie. Do not use water jet. If breathing is difficult. Section 6: Accidental Release Measures Small Spill: Absorb with an inert material and put the spilled material in an appropriate waste disposal. Seek medical attention. administer oxygen. Serious Inhalation: Evacuate the victim to a safe area as soon as possible. Flash Points: CLOSED CUP: 100°C (212°F). Loosen tight clothing such as a collar. Loosen tight clothing such as a collar.

Solubility: Not available. Do not breathe gas/fumes/ vapor/spray. Ensure that eyewash stations and safety showers are proximal to the work-station location. Critical Temperature: Not available. pH (1% soln/water): Not available. well-ventilated area. consult a specialist BEFORE handling this product. Suggested protective clothing might not be sufficient. Boiling Point: 213°C (415. Specific Gravity: 1. Water/Oil Dist. Volatility: Not available. Personal Protection: Safety glasses. Odor Threshold: Not available. Boots. 44 . Gloves. Odor: Not available. log(oil/water) = 0. Full suit. Section 10: Stability and Reactivity Data Stability: The product is stable. Taste: Not available. Instability Temperature: Not available. Storage: Keep container tightly closed. evaporate the residue under a fume hood. Lab coat.2 g/mole Color: Colorless. alkalis. Exposure Limits: Not available. Empty containers pose a fire risk. Ground all equipment containing material.1452 @ 22 deg..Keep locked up. Keep away from sources of ignition. Keep away from incompatibles such as oxidizing agents. Section 9: Physical and Chemical Properties Physical state and appearance: Liquid. Section 8: Exposure Controls/Personal Protection Engineering Controls: Provide exhaust ventilation or other engineering controls to keep the airborne concentrations of vapors below their respective threshold limit value. Keep away from heat.1 Ionicity (in Water): Not available. Dispersion Properties: Not available. Molecular Weight: 124. Wear suitable protective clothing. If ingested. Do not ingest.: The product is equally soluble in oil and water. seek medical advice immediately and show the container or the label. C (Water = 1) Vapor Pressure: 0 kPa (@ 25°C) Vapor Density: Not available.4°F) Melting Point: Not available. Keep container in a cool. Coeff. Personal Protection in Case of a Large Spill: Splash goggles.

Ingestion: Harmful (toxic) if swallowed. Slightly hazardous in case of skin contact (irritant. DEVELOPMENTAL TOXICITY: Classified Reproductive system/toxin/male [POSSIBLE].s. alkalis. permeator).1: Poisonous material. Chronic Effects on Humans: CARCINOGENIC EFFECTS: Classified 2B (Possible for human. long term degradation products may arise. Section 11: Toxicological Information Routes of Entry: Absorbed through skin. Section 13: Disposal Considerations Waste Disposal: Waste must be disposed of in accordance with federal. BOD5 and COD: Not available. Products of Biodegradation: Possibly hazardous short term degradation products are not likely. MUTAGENIC EFFECTS: Mutagenic for mammalian somatic cells. incompatible materials Incompatibility with various substances: Reactive with oxidizing agents. Special Remarks on Toxicity to Animals: Not available.Conditions of Instability: Excess heat. Special Remarks on Reactivity: Not available. brain tumors) Special Remarks on other Toxic Effects on Humans: Acute Potential Health Effects: Skin: May cause skin irritation. May cause cancer (kidney tumors. Mutagenic for bacteria and/or yeast. Toxicity to Animals: Acute oral toxicity (LD50): 470 mg/kg [Mouse]. However. Special Remarks on Corrosivity: Not available. Identification: : Toxic Liquid. igniton sources. Corrosivity: Not available. Other Toxic Effects on Humans: Hazardous in case of ingestion. n. lung tumors. Inhalation: May cause respiratory tract irritation. May cause digestive tract irritation. Section 14: Transport Information DOT Classification: CLASS 6. May cause damage to the following organs: the reproductive system. Polymerization: Will not occur.) by IARC. (Methanesulfonic Acid Ethyl Ester) UNNA: 2810 PG: III 45 . Section 12: Ecological Information Ecotoxicity: Not available. May affect genetic material (mutagenic). Special Remarks on Chronic Effects on Humans: May cause adverse reproductive effects and birth defects(teratogenic). Eye contact. Special Remarks on the Products of Biodegradation: Not available. Eyes: May causes eye irritation.o. of inhalation. Toxicity of the Products of Biodegradation: The products of degradation are less toxic than the product itself. state and local environmental control regulations. Organic.

birth defects or other reproductive harm.: Methanesulfonic Acid Ethyl Ester: 1 lbs. CLASS D-2B: Material causing other toxic effects (TOXIC).In case of accident or if you feel unwell. Lab coat.A. S45. 65: This product contains the following ingredients for which the State of California has found to cause cancer.S.obtain special instructions before use. Other Special Considerations: Not available. Section 16: Other Information References: Not available. Safety glasses.May cause cancer. Other Classifications: WHMIS (Canada): CLASS D-1B: Material causing immediate and serious toxic effects (TOXIC). HMIS (U.May cause heritable genetic damage. R45. S24/25.Harmful if swallowed. seek medical advice immediately (show the label where possible).Special Provisions for Transport: Not available. Section 15: Other Regulatory Information Federal and State Regulations: California prop. S36/37/39.Avoid contact with skin and eyes. which would require a warning under the statute: Methanesulfonic Acid Ethyl Ester California prop. EINECS: This product is on the European Inventory of Existing Commercial Chemical Substances.Possible risk of impaired fertility.): Health: 2 Flammability: 1 Reactivity: 0 Specific hazard: Protective Equipment: Not applicable.A.: Methanesulfonic Acid Ethyl Ester Illinois toxic substances disclosure to employee act: Methanesulfonic Acid Ethyl Ester Illinois chemical safety act: Methanesulfonic Acid Ethyl Ester New York release reporting list: Methanesulfonic Acid Ethyl Ester Pennsylvania RTK: Methanesulfonic Acid Ethyl Ester Minnesota: Methanesulfonic Acid Ethyl Ester Massachusetts RTK: Methanesulfonic Acid Ethyl Ester Massachusetts spill list: Methanesulfonic Acid Ethyl Ester New Jersey: Methanesulfonic Acid Ethyl Ester New Jersey spill list: Methanesulfonic Acid Ethyl Ester Louisiana spill reporting: Methanesulfonic Acid Ethyl Ester California Director's List of Hazardous Substances: Methanesulfonic Acid Ethyl Ester TSCA 8(b) inventory: Methanesulfonic Acid Ethyl Ester TSCA 5(a)2 final significant rules: Methanesulfonic Acid Ethyl Ester TSCA 8(a) IUR: Methanesulfonic Acid Ethyl Ester TSCA 12(b) annual export notification: Methanesulfonic Acid Ethyl Ester CERCLA: Hazardous substances. DSCL (EEC): R22. 65: This product contains the following ingredients for which the State of California has found to cause cancer which would require a warning under the statute: Methanesulfonic Acid Ethyl Ester Connecticut hazardous material survey.1200). R46.S.4536 kg) Other Regulations: OSHA: Hazardous by definition of Hazard Communication Standard (29 CFR 1910.Avoid exposure . (0.): Health Hazard: 2 Fire Hazard: 1 Reactivity: 0 Personal Protection: a National Fire Protection Association (U.Wear suitable protective clothing. S53. 46 . R62. Wear appropriate respirator when ventilation is inadequate. gloves and eye/face protection.

1N NaOH) Reagent B (0.01 ml 0.CHOH.02M) Glycerol D/W TOTAL 0.APPENDIX D: Composition of buffers.5) β – mercaptoethanol EDTA (0.0 ml 7.9 ml 8.5 ml 0. reagents and solutions used Table 13.005 ml 1 ml 8.050 ml 0. Composition of resolving and stacking gels for SDS – PAGE RESOLVING GEL (8%) D/W Acrylamide-bisacrylamide 1.66 ml 1.COONa) 50 ml 1 ml Table 15.3 ml 0.005 ml 0. Composition of AgNO3 solution AgNO3 Formaldehyde D/W 0. HEPES KOH extraction buffer composition HEPES buffer (1 M pH 7.983 ml 10 ml Table 14.5M Tris HCl pH 8.CHOH.3 ml 0.3 gm 75 µl 100 ml 47 . Composition of Reagent C for Folin-Lowry method Reagent C Reagent A (2% Na2CO3 in 0.002 ml 0.8 10% SDS 10% APS TEMED TOTAL 13.5M Tris HCl pH 6.5% CuSO4 in 1 % COOK.050 ml 0.8 0.25 ml 0.018 ml 30 ml STACKING GEL (5%) 0.018 ml 5 ml Table 16.

Composition of developing solution Na2CO3 Na2S2O3.5H2O Formaldehyde D/W 6 gm 1 mg 50 µl 100 ml Table 18.Table 17.7 gm 20 gm 100 ml 48 . Composition of potassium tri-iodide reagent Iodine Potassium iodide D/W 15.25 gm 30 ml 20 ml Table 19. Composition of Acid ninhydrin Ninhydrin Glacial acetic acid Phosphoric acid 6M 1.

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