CHAPTER 1 INTRODUCTION

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 worlds 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, Josephs 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 25C and night temperatures not lower than 15C. 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)

Nutrition: Amaranthus is the worlds most nutritious pseudo-cereal grain with multiple uses. It is one of those rare plants whose leaves are eaten as a vegetable while the seeds are used as cereals (Mahajan, et al., 2002). The leaf protein levels of Amaranthus tricolor is 33% (National Research Council, 1984). With a protein content of 16%, Amaranthus seeds compare well with the conventional cultivars of wheat (12-14%), rice (7-10%), maize (910%) and other widely consumed cereals (Gorinstein, et al., 2002; Mahajan, et al., 2002; Pandey, et al., 2010). It has nearly twice the lysine content of wheat proteins, three times that of maize and as mush as that of milk, the standard of nutritional excellence (Mahajan, et al., 2002). They are good sources of minerals and vitamins and contain larger amounts than most of the common cereal grains. The nutritive value is about 75, compared with values of 44, 57 and 62 for maize, wheat and barley respectively. Furthermore pseudocereals contain relatively high amount of dietary fibre, which improves lipid metabolism and takes part in the prevention of LDL-C oxidation (Gorinstein, et al., 2002).

1.4 MECHANISM OF SALT TOLERANCE In plants the mechanisms of salt tolerance takes place at three levels of organization: whole plant, cellular and molecular. (Munns, et al., 2002) Control at whole plant level Salt tolerance depends on the ability of the plant to control the transport of salt. 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. (Munns, et al., 2002; Bohnert, et al., 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. 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. The organic solutes that most commonly accumulate under salinity are proline and glycine-betaine. Others include low molecular weight sugars, organic acids, polyols, amino acids, amides, imino acids, proteins, quaternary ammonium compounds (Ashraf, et al., 2004; Munns, et al., 2002). Control at molecular level 6

At the molecular level regulation of Na+ uptake across the plasmalemma would come from restricted uptake by selective cation transporters and channels, coupled with efflux by the antiporter (Munns, et al., 2002; Yokoi, et al., 2002). 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.

1.5 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. The introduction of genes from wild salt tolerant species has been explored for tomato (Rush, et al., 1981; Tal, et al., 1983; Saranga, et al., 1991) wheat (King, et al., 1997a; King, et al., 1997b) and pigeonpea (Subbarao, et al., 1990). However the approach has not led to the release of salt tolerant crops (Flowers, 2004). Use the variation already present in the existing crops. This methodology does not require a deep knowledge of the genetics of traits, merely that they display sufficient heritability and require suitable screening procedures to be developed (Flowers, 2004). Thus, conventional breeding programs can be employed to develop crop varieties resistant to salinity stress, but the progress is time consuming and tedious. Generate variation within existing crops by using recurrent selection, tissue culture and mutagenesis. Mutagenesis is an event capable of causing a mutation i.e. a genetic change. It can be broadly classified into random and site-directed mutagenesis. Random mutagenesis can be performed using either physical mutagens such as UV rays, gamma rays, etc. or chemical mutagens such as colchicine, EMS, etc. A combination of mutagenesis and in-vitro culture can speed up the breeding programs from generation of variability, through selection to multiplication of the new genotypes (El-Sayed, et al., 2007). Thus, 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, et al., 2006). Thus, the general objectives of this study were to better understand the tolerance of Amaranthus tricolor to salt stress by investigating:

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

CHAPETR 2

LITERATURE REVIEW Salinity is the major environmental stress that greatly affects plant productivity (ElSayed, et al., 2007; Patade, et al., 2006). According to FAO (2002) some 20 to 30 million hectares of irrigated land are currently seriously damaged by salinity and 0.25 to 0.5 million hectares are lost from production every year as a result of salt accumulation. Saline soils are characterized by presence of toxic levels of sodium and its chlorides and sulphates (Khan, et al., 2007). Salts in the soil or water may inhibit plant growth for two reasons. First, the presence of salt in the soil solution reduces the ability of the plant to take up water, and this leads to reductions in the growth rate. This is referred to as the osmotic or water-deficit effect of salinity. Second, 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. This is called the salt-specific or ion-excess effect of salinity (Dr. Munns, 2010; Bohnert, et al., 1999). Salinity becomes a problem when enough salts accumulate in the root zone to negatively affect plant growth, because it hinders plant roots from withdrawing water from the surrounding soil (Warrence, et al., 2003). Soil salinity may therefore have devastating effects on agriculture due to yield losses of crops. In an experiment with different wheat cultivars, Steppuhn and Wall (1997) showed that grain yields were reduced by more than 20% with root-zone salinity levels of less than 4.0 dS m-1. Thus, the increasing salinity in soils can lead to a worldwide catastrophe. An alternative to this is growing plants especially crop plants which are salt tolerant. The salt tolerance of a crop may be evaluated according to three criteria: (i) the ability of the crop to survive on saline soils, (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. Munns, 2010; US Salinity Laboratory Staff, 1954). Plants have their own mechanisms for fighting against any kind of stress. Salt tolerance in plants may be the result of: Control at the whole plant level 9

Salt tolerance depends on the ability of the plant to control the transport of salt at five sites (Figure 3), 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. The initial uptake of Na+ and Cl could occur at the epidermis, at the exodermis, or if soil solution flows apoplastically across the root cortex, it would occur at the endodermis. Loading of the xylem: There is evidence for a preferential loading of K+ rather than Na+ by the cells of the stele. Removal of salt from the xylem in the upper part of the roots, the stem, petiole or leaf sheaths. In many species, Na+ is retained in the upper part of the root system and in the lower part of the shoot, 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. Loading of the phloem: There is little re translocation of Na+ or Cl in the phloem, particularly in the more tolerant species. This ensures that salt is not exported to growing tissues of the shoot. Excretion through salt glands or bladders: Only halophytes have these specialized cell types. All halophytes have well-developed mechanisms to control the uptake, transport and excretion of salt. Glycophytes rely on the first three mechanisms, and exhibit these mechanisms to various degrees (Munns, et al., 2002).

Figure 3. Control points at which salt transport is regulated. These are: (1) selectivity of uptake from the soil solution; (2) loading of the xylem; (3) removal of salt from the xylem in the upper part of the plant; (4) loading of the phloem; and (5) excretion through salt glands or bladders. 10

Production of osmolytes and organic solutes Na+ and Cl- are toxic at high levels. The plant removes Na+ and Cl- from the cytoplasm by sequestering it in vacuoles. To maintain an osmotic balance the plant should produce organic solutes to maintain the osmotic pressure of the ions in the vacuole (Munns, et al., 2002). Proline and glycinebetaine are the most commonly produced osmolytes under salinity stress. Although proline can be synthesized either from glutamate or ornithine, glutamate is the primary precursor in osmotically stressed cells. The biosynthetic pathway consists of two important enzymes viz. pyrroline carboxylic acid synthetase and pyrroline carboxylic acid reductase. The molecular weight of pyrroline carboxylic acid reductase is 320 kDa whereas the polypeptide has a molecular mass of 28.145 kDa (from nucleotide sequence) and 26.5 kDa (experimentally) (Caspi, et al., 2010). The molecular weight of pyrroline carboxylic acid synthetase is 78 kDa. With increasing salt concentrations, increase was reported in proline accumulation in Cajanus cajan (Jaleel, et al., 2008) in rice (Thach, et al., 1999) in wheat (El-Sayed, et al., 2007) in lentils (El-Monem, et al., 2008) and in Amaranthus tricolor (Wang, et al., 2000). Chen, et al. (2011) reported that glycinebetaine in plants is synthesized from choline by a two step oxidation reaction. 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. The amaranth CMO polypeptide has a molecular mass of 45 kDa same as that of spinach and sugar beet (Russell, et al., 1998). BADH in its native state has a molecular mass of 125 kDa. It is formed by two sub units with molecular masses of 63 and 70 kDa as determined by SDS PAGE (Figueroa-Soto, et al., 2001). An increase was also observed in the glycine betaine levels in Cajanus cajan (Jaleel, et al., 2008) in sorghum (Mickelbart, et al., 2003) in Amaranthus tricolor (Wang, et al., 2000; Wang, et al., 2004).

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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, et al., 1999; Blumwald, 2000; Schachtman, et al., 1999). There is no specific Na+ transporter. 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, et al., 1999). Na+ can be effluxed from the cytoplasm through Na+/H+ antiporters, driven by the pH gradient across the plasmalemma (Blumwald, 2000). Intracellular compartmentation is by a vacuolar Na+/H+ antiporter, driven by a pH gradient across the tonoplast (Blumwald, 2000). The transporters that maintain low Na+ concentrations in organelles such as chloroplasts and mitochondria are not known.

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, et al., 1998).Salt are usually most damaging to young plants, particularly cereal crops. Rice for example is tolerant during germination becomes sensitive during early seedling growth, and more tolerant as it matures (Miloslav, 1982). 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. 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, et al., 1998). Gradual decrease in rate of germination and inhibition of germination with increasing salt concentrations was reported in Jute (Abbas, et al., 2005) in five lentil cultivars (ElMonem, et al., 2008) in cabbage, sugarbeet, amaranth and pak-choi (Jamil, et al., 2006). Morphological and physiological responses of plants to salt stress may also vary. Plants affected by excessive soluble salt concentrations may appear normal but a general stunting of growth is observed. Besides there may be difference in the foliage colour than normal plants and sometimes the leaves are thicker and succulent. Jamil, et al. (2006), Abbas, et al. (2005) and El-Monem, et al. (2008) reported a reduction in root and shoot 12

length, fresh and dry weight with increasing salt concentrations in four vegetable species (cabbage, sugarbeet, amaranth and pak-choi), jute and five cultivars of lentil respectively. Conventional breeding procedures which involve, the production of new genetic combinations from already existing parental genes is rather time consuming and tedious (Majeed, et al., 2010). The use of mutagens in combination with in-vitro culture has created great interest in plant breeders to create genetic variation. 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. Moreover very large population of plant cells or plants can be handled relatively easily. Mutagenic agents, such as radiation (x rays, gamma rays) and certain chemicals (colchicines, EMS), then can be used to induce mutations and generate genetic variations from which desired mutants may be selected (Novak, et al., 1992). Khatri, et al., (2005) collected three high yielding and early maturing mutants by treating seeds of Brassica juncea L. cv. S-9 with gamma rays (750 -1000 KGy) and EMS. Shah, et al., (2001) developed a new oil seed Brassica Napus L. cv. ABASIN-95 by induced mutation. They exposed the seeds to 1.0, 1.2 and 1.4 KGy gamma rays and the resulting new variety was high yielding, resistant to Alternaria blight and white rust. Atrazine resistant plants were selected using EMS (Venkataiah, et al., 2005). With increase in dose of gamma rays a decrease was observed in the root and shoot length, and fresh and dry weight of Lepidium sativum L (Majeed, et al., 2010). Percentage of mortality increased while plant height and internode length decreased with increasing gamma ray doses (Tah, 2006). The germination percentage of somatic embryos and development of such embryos into plant with leaf and root reduced with increasing EMS concentrations (Novak, et al., 1990).

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CHAPTER 3

MATERIALS AND METHODS

3.1

Plant material collection Seeds of Amaranthus tricolor (variety Pusa Kirti) were procured from IARI, New Delhi.

3.2

Seed Sterilization The seeds were treated with 0.1% HgCl2 for 7 minutes and rinsed twice with autoclaved distilled water. The seeds were then treated with 70% ethanol for 30 seconds and rinsed thrice with autoclaved distilled water. The seeds were then transferred into a petriplate containing blotting paper and allowed to dry before inoculating.

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, 1/2 MS, 1/4 MS and 1/8 MS media containing 3% sucrose, 0.8% agar and pH maintained between 5.7 - 5.8. 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.

3.4

In-vitro screening of salt tolerance For screening of salt tolerance in-vitro, the seeds were grown in 1/4 MS media containing 3% sucrose, 0.8% agar, pH maintained between 5.7 - 5.8 and different concentrations of NaCl (50mM, 100mM, 150mM, 200mM and 250mM). 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.

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3.5

Chemical mutagenesis Determination of LD50 of EMS on seeds of Amaranthus tricolor (variety Pusa Kirti) Seeds were presoaked in distilled water for 6 hours. Seeds were treated with varying concentrations (0.1%,0.2%, 0/3%............... 2.5%) of EMS for 4 hours. Seeds were then washed twice with 0.1M sodium thiosulphate for 15 minutes each. Seeds were then washed twice with distilled water for 15 minutes each. Seeds were then sterilized as given in 3.2, inoculated in 1/4MS containing 3%sucrose, 0.8% agar and pH maintained between 5.7-5.8.

NOTE: EMS is carcinogenic and volatile. Refer to handling instructions given in Appendix B and C before using EMS.

3.6

Biochemical Studies

3.6.1 Estimation of protein by Lowrys method (Lowry, et al., 1951) Material HEPES-KOH extraction buffer, Reagent C, FC reagent (2N), Standard BSA Method (A) Sample extraction: Samples were harvested and homogenized in 1(sample):1(extraction buffer) using a mortar and pestle. The homogenate was centrifuged at 14,000 rpm for 10 minutes at 4oC. 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. The samples were mixed well and incubated at room temperature for 10 minutes. 0.5 ml of FC reagent (1N) was added.

15

It was mixed well and incubated at room temperature in dark for 30 minutes and absorbance was read at 660nm. A standard graph is prepared in the same way using standard BSA. A linear relationship between concentration and absorbance is observed in the range of 10 g/ml 100 g/ml of protein.

3.6.2 Protein profiling using SDS PAGE (Sambrook, et al., ) Material Acrylamide bisacrylamide stock (30 : 0.8), 0.5M Tris HCl pH 6.8, 1.5M Tris HCl pH 8.8, 1.5M Tris HCl pH 6.8, 10%SDS, 10%APS, TEMED, mercaptoethanol, Glycerol, Bromophenol blue, Glycine, Agar, 50% ethanol, 0.02% Na2S2O3.5H2O, distilled water, AgNO3 solution, Developing solution and 0.02M EDTA Method (A) Sample preparation: The protein sample was mixed with equal volume of sample buffer 2X. The mixture was boiled for 3 minutes in a boiling water bath and cooled to room temperature. (B) SDS-PAGE: Equal concentrations of proteins were loaded and were separated using 8% resolving gel and 4% stacking gel. A protein molecular weight marker was loaded in one well. The protein molecular weight marker (205 kDa 3 kDa) used was from Bangalore Genei, India.

(C) AgNO3 staining: The gel was rinsed with 50% ethanol twice for 30 minutes each. The gel was then rinsed with 0.02% Na2S2O3.5H2O for 1 minute. The gel was then rinsed with distilled water twice for 30 seconds each. The gel was treated with AgNO3 solution in dark for 15 minutes followed by two washings with distilled water for 30 seconds each. Developing solution was added and gel was gently rocked till bands develop. Reaction was stopped by adding 0.02M EDTA. 16

3.6.3 Estimation of Proline (Bates, et al., 1973) Material Acid ninhydrin, 3% aqueous sulphosalicylic acid, glacial acetic acid, toluene, Standard Proline Method 0.1 gm of plant material was extracted by homogenizing in 2 ml of 3% aqueous sulphosalicylic acid. The homogenate was filtered through Whatmann No. 1 filter paper. 2 ml of filtrate was taken in a test tube. To which 2 ml of glacial acetic acid and 2 ml of acid ninhydrin was added. It was heated in a boiling water bath for 1 hour. The reaction was terminated by placing the tubes in ice bath. 4 ml toluene was added to the reaction mixture and stirred well for 20-30 seconds. The toluene layer was separated and warmed to room temperature. The red colour intensity was measure at 520 nm. For the standards, linear relationship between concentration and absorbance is observed in the range of 0.02mM 0.1mM per ml of proline. 3.6.4 Estimation of glycine-betaine (Greive, et al., 1983) Material Distilled water, Potassium tri-iodide reagent, 2N Sulphuric acid and 1,2 dichloroethane Method Extract was prepared by finely ground plant material (0.1 gm) which was mechanically shaken with 4ml of distilled water for 48 hours at 25oC. The samples were then filtered and the filtrate was diluted 1:1 with 2N sulphuric acid. Aliquot (0.5ml) was measured into test tube and cooled in ice water for 1 hour. 0.2ml of cold potassium triiodide reagent was added and the mixture was gently mixed with vortex mixture and stored at 0 - 4oC for 16 hours. After the expiring of the period, samples were transferred to centrifuge tubes and then centrifuged at 10,000g for 15 minutes at 0oC. 17

The supernatant was carefully aspirated with 1 ml micropipette. The periodite crystals were dissolved in 9 ml of 1,2 dichloroethane. Vigorous vortex mixing was done to effect complete solubility in developing solvent. After 2 2.5 hours the absorbance was measured at 365 nm with UV spectrophotometer. For the standards, linear relationship between concentration and absorbance is observed in the range of 50g/ml - 200 g/ml of glycine-betaine.

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CHAPTER 4

RESULTS AND DISCUSSION

4.1 In-vitro seed germination and seedling growth in different strength of MS media

MS media Germination (%) Root length (cm) Shoot length (cm) 80 5.66 2.605 0.22 3.045 0.19

1/2 MS media 92 4.38 3.813 0.17 3.926 0.2

1/4 MS media 100 0 3.982 0.17 4.254 0.18

1/8 MS media 92 7.16 3.809 0.27 4.013 0.17

Table 2. Germination percentage of seeds with root and shoot length of Amaranthus tricolor (var. Pusa Kirti) grown in-vitro in different strength of MS media. SE

Figure 4. Germination percentage of seeds of Amaranthus tricolor (var. Pusa Kirti) grown in different strength of MS media 19

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

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

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Discussion: Seed germination was initiated after 48 hours of inoculation. It did not change after 72 hours. All seeds inoculated in 1/4 MS media had germinated. Seedlings started to wither after 12 days due to depletion of nutrients. Thus, 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. Higher germination percentage, root and shoot length of Amaranthus tricolor (var. Pusa Kirti) was observed in 1/4 MS media as compared to MS, 1/2 MS and 1/8 MS media. Therefore, 1/4 MS media was selected for further studies.

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4.2 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 4.38 4.25 0.26 3.434 0.2

50 mM 84 6.69 4.514 0.2 3.633 0.18

100 mM 76 6.69 3.96 0.25 3.156 0.1

150 mM 24 3.58 0.93 0.18 0.65 0.08

200 mM 250 mM 0 0 0 0 0 0

Table 3. Germination percentage with root and shoot length of Amaranthus tricolor (var. Pusa Kirti) grown in 1/4 MS media containing different NaCl concentrations. SE

Figure 8. Germination percentage of seeds of Amaranthus tricolor (var. Pusa Kirti) grown in 1/4 MS media containing different NaCl concentrations. 22

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

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

23

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

24

4.3 Determination of LD50 of EMS on seeds of Amaranthus tricolor (var. Pusa Kirti) Table 4. Germination percentage of seeds treated with different concentrations of EMS Concentration of EMS Control 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 2 Germination (%) 66.67 63.33 63.33 60 56.67 56.67 53.33 53.33 53.33 50 46.67 43.33 33.33 13.33 10 0

Discussion: 50% germination was observed in seeds treated with 1% EMS. Based on the germination percentage LD50 was revealed to be 1%. Seeds treated with 2% EMS did not germinate. This further confirmed that LD50 is 1%. Thus, the lethal dose on the basis of in-vitro germination of seeds and survival of seedlings of Amaranthus tricolor (var. Pusa Kirti) was adjudged as 1%.

25

4.4 Effect of mutagenesis on seed germination, seedling growth and salt tolerance Seeds treated with 0% EMS (Control) 0 mM Germination (%) Root length (cm) Shoot length (cm) 93 4.38 4.157 0.26 3.294 0.19 50 mM 84 6.69 4.514 0.2 3.633 0.178 100 mM 75 6.69 3.965 0.25 2.953 0.1 150 mM 26 3.6 0.93 0.2 0.65 0.1 200 mM 0 0 0 250mM 0 0 0

Seeds treated with 0.5% EMS 0 mM Germination (%) Root length (cm) Shoot length (cm) 70 0.47 1.72 0.11 1.86 0.11 50 mM 56.67 0.27 2.94 0.06 1.45 0.06 100 mM 16.67 0.27 0.82 0.05 1.1 0.1 150 mM 0 0 0 200 mM 250 mM 0 0 0 0 0 0

Seeds treated with 0.7% EMS 0 mM Germination (%) Root length (cm) Shoot length (cm) 63.3 0.27 3.29 0.1 2.29 0.1 50 mM 46.67 0.27 2.88 0.08 1.87 0.02 100 mM 14.55 0.27 1.91 0.05 0.9 0.03 150 mM 0 0 0 200mM 0 0 0 250mM 0 0 0

Seeds treated with 1.0% EMS 0 mM Germination (%) Root length (cm) Shoot length (cm) 50 0 1.9 0.06 3.1 0.12 50 mM 13.33 0.27 0.91 0.05 2.31 0.09 100 mM 0 0 0 150 mM 0 0 0 200 mM 250 mM 0 0 0 0 0 0

Table 5. Germination percentage with root and shoot length of Amaranthus tricolor (var. Pusa Kirti) treated with different concentrations of EMS and grown in 1/4 MS media containing different NaCl concentrations. SE 26

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.

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

Figure 14. Shoot length of seedlings of Amaranthus tricolor (var. Pusa Kirti) grown in 1/4 MS media containing different NaCl concentrations after seeds were treated with different concentrations of EMS. Discussion: Seeds treated with 0.5 and 0.7% EMS germinated in NaCl concentrations upto 100 mM. Those treated with 1% EMS germinated in NaCl concentrations upto 50 mM. Increasing concentrations of EMS resulted in decrease of germination percentage. The germination percentage of seeds at each concentration of EMS decreased with increasing NaCl concentration. Seeds treated with 0.5% EMS show high germination percentage compared to 0.7 and 1.0%. Root length in seeds treated with 0.5% EMS and grown in 1/4 MS media containing different NaCl concentrations show germination pattern similar to seeds treated with 0% (Control). However, root length of seeds treated with 0.7% was greater than those treated with 0.5 and 1.0%.

28

Shoot length of seeds treated with 1% was greater than those treated with 0.5% and 0.7%. Also shoot length in each concentration of EMS decreased with increasing NaCl concentrations. On comparing with control it can be inferred that, EMS may not be causing any beneficial mutation for Amaranthus tricolor (var. Pusa Kirti) so as to bring about an increase in its salt tolerance. Moreover, the concentration of EMS may be hampering the seed germination and seedling growth. So, further studies using still lower concentrations of EMS can be done in order to confirm the observations of the present study.

29

4.5 Protein estimation by Lowrys method (Lowry, et al., 1951)

Concentration of BSA (g/ml) 0 10 20 40 60 80 100

Absorbance at 660nm 0.000 0.040 0.061 0.112 0.170 0.223 0.261

Table 6. Absorbance of different concentrations of BSA (g/ml) at 660 nm

Figure 16. Standard graph of BSA

30

Samples

Absorbance at 660 nm

Concentration of protein in 0.1 gm of fresh tissue (g) 140 220 240 253 306 306 630 733 775

Concentration of protein (mg/gm of fresh tissue) 1.4 2.2 2.4 2.53 3.07 3.07 6.3 7.33 7.75

Control 50 mM NaCl 100 mM NaCl 150 mM NaCl 0.5% EMS 0.5% EMS + 50 mM NaCl 0.5% EMS + 100 mM NaCl 0.7% EMS 1.0% EMS

0.282 0.440 0.483 0.506 0.613 0.613 1.261 1.466 1.552

Table 7. Concentration of protein in seedlings of EMS treated seeds of Amaranthus tricolor (var. Pusa Kirti) grown in 1/4 MS media containing different NaCl concentrations.

Figure 17. Concentration of protein in seedlings of EMS treated seeds of Amaranthus tricolor (var. Pusa Kirti) grown in 1/4 MS media containing different NaCl concentrations.

31

Discussion: In control (0% EMS) with increasing NaCl concentrations an increase was observed in the protein concentration per gm fresh tissue. 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. The over expression of proteins was confirmed by increase in intensity of protein bands as seen in SDS PAGE. Protein estimation in 0.7% EMS treated seeds grown in 50 and 100 mM and 1.0% EMS treated seeds grown in 50 mM could not be performed since sufficient sample was not available for analysis.

32

4.6 Estimation of proline (Bates, et al., 1973) Concentration of proline (mM) 0 0.01 0.02 0.04 0.06 0.08 0.1 Absorbance at 520nm 0.000 0.062 0.096 0.211 0.331 0.504 0.597

Table 8. Absorbance of different concentrations of proline (mM) at 520 nm.

Figure 18. Standard graph of proline.

33

Absorbance at 520 nm Control 50 mM NaCl 100 mM NaCl 150 mM NaCl 0.060 0.065 0.200 0.261

Concentration of proline (mM) 0.010 0.011 0.034 0.044

Table 9. Concentration of proline (mM) in seedlings of Amaranthus tricolor (var. Pusa Kirti) grown in 1/4 MS media containing different NaCl concentrations.

Figure 19. Concentration of proline (mM) in seedlings of Amaranthus tricolor (var. Pusa Kirti) grown in 1/4 MS media containing different NaCl concentrations. Discussion: On comparison with control it was observed that at 50 mM seedlings accumulated proline at concentrations almost similar to control but, at higher salt 34

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.

35

4.7 Estimation of glycine-betaine (Greive, et al., 1983) Concentration of glycine-betaine (g/ml) 0 50 75 100 125 150 175 200 Absorbance at 365 nm 0.00 0.10 0.16 0.21 0.26 0.32 0.38 0.42

Table 10. Absorbance of different concentrations of betaine (g/ml) at 365 nm.

Figure 20. Standard graph of glycine-betaine.

36

Absorbance at 365 Concentration of glycine- betaine nm Control 50 mM NaCl 100 mM NaCl 150 mM NaCl 0.236 0.258 1.152 1.317 (g) 118 129 576 658.6

Table 11. Concentration of glycine-betaine (g) in seedlings of Amaranthus tricolor (var. Pusa Kirti) grown in 1/4 MS media containing different NaCl concentration.

Figure 21. Concentration of glycine-betaine (g) in seedlings of Amaranthus tricolor (var. Pusa Kirti) grown in 1/4 MS media containing different NaCl concentrations. Discussion: Very small difference was observed in the concentration of glycine-betaine that accumulated in seedlings at 0 and 50 mM but, at higher salt concentrations there is a significant increase in glycine-betaine concentration indicating that plants may not be stressed at 50 mM. Estimation of glycine-betaine in EMS treated samples could not be performed due to non availability of sufficient sample. 37

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

38

CONCLUSION From the present study of screening for salt tolerant mutants in Amaranthus tricolor L. (var. Pusa Kirti) it was found that increasing NaCl concentrations delayed germination and reduced the germination percentage and seedling growth. Germination percentage decreased significantly at 150 mM salt stress. Increasing NaCl concentration resulted in accumulation of proline and glycine-betaine. Protein profiling studies revealed increased expression of proteins with increase in NaCl concentration. The seedling showed enhanced growth and accumulated proline and glycine-betaine at levels almost similar to control when subjected to 50 mM salt stress. From this it can be concluded that small amounts of salt enhances the plant growth of Amaranthus tricolor L. (var. Pusa Kirti) and can tolerate salt concentrations upto 100 mM. The different growth parameters studied in seedlings of EMS treated seeds showed variable results. 0.5 and 0.7 % 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. Protein profiling studies only showed increased expression of proteins. No variability was seen in protein bands. From this it can be inferred that no beneficial mutation has occurred and high concentrations of EMS hampered the growth and germination of seedlings. This research work has been extended to further analyze the salt tolerance by using lower concentrations of EMS. Salt tolerance would also be analysed using gamma radiations. 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. Various biochemical analysis will be performed to see how the nutritive value and other metabolites and vitamins changes. Salt tolerant lines will be analyzed for genetic variation using RAPD.

39

APPENDIX A: Composition of media

Table . 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, 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

APPENDIX B: Handling instructions for EMS Personnel safety: Safety goggles, facemask, gloves, labcoat, and closed footwear. EMS treatment should be performed in well ventilated laboratory ideally under a fume hood Discard washes containing EMS in a beaker containing 0.1 M sodium thiosulphate Decontaminate all material that has come in contact with EMS by treatment with 10% sodium thiosulphate. Dry discard including tissue, vials, gloves, etc. should be sealed in plastic bags under the fume hood. All glassware coming in contact with EMS should be immersed in 1N NaOH or laboratory bleach before recycling or disposal.

41

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. Synonym: Ethyl Methanesulfonate; Ethyl ester of methanesulfonic acid; Ethyl ester of methanesulpnonic acid; Ethyl ester of methylsulfonic acid; Ethyl ester of methylsulphonic acid; Ethyl methanesulphonate; Methylsulfonic acid, ethyl ester Chemical Name: Methanesulfonic Acid Ethyl Ester Chemical Formula: C3-H8-O3-S Contact Information: Sciencelab.com, Inc. 14025 Smith Rd. Houston, Texas 77396 US Sales: 1-800-901-7247 International Sales: 1-281-441-4400 Order Online: ScienceLab.com CHEMTREC (24HR Emergency Telephone), call: 1-800-424-9300 International CHEMTREC, call: 1-703-527-3887 For non-emergency assistance, 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].

Section 3: Hazards Identification

Potential Acute Health Effects: Hazardous in case of ingestion. Slightly hazardous in case of skin contact (irritant, permeator), of eye contact (irritant), of inhalation. Potential Chronic Health Effects: CARCINOGENIC EFFECTS: Classified 2B (Possible for human.) by IARC. MUTAGENIC EFFECTS: Mutagenic for mammalian somatic cells. Mutagenic for bacteria and/or yeast. TERATOGENIC EFFECTS: Not available. DEVELOPMENTAL TOXICITY: Classified Reproductive system/toxin/male [POSSIBLE]. The substance may be toxic to the reproductive system. Repeated or prolonged exposure to the substance can produce target organs damage.

42

Section 4: First Aid Measures

Eye Contact: Check for and remove any contact lenses. In case of contact, immediately flush eyes with plenty of water for at least 15 minutes. Get medical attention if irritation occurs. Skin Contact: Wash with soap and water. Cover the irritated skin with an emollient. Get medical attention if irritation develops. Serious Skin Contact: Not available. Inhalation: If inhaled, remove to fresh air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. Get medical attention. Serious Inhalation: Evacuate the victim to a safe area as soon as possible. Loosen tight clothing such as a collar, tie, belt or waistband. If breathing is difficult, administer oxygen. If the victim is not breathing, perform mouth-to-mouth resuscitation. Seek medical attention. Ingestion: Do NOT induce vomiting unless directed to do so by medical personnel. Never give anything by mouth to an unconscious person. If large quantities of this material are swallowed, call a physician immediately. Loosen tight clothing such as a collar, tie, belt or waistband. Serious Ingestion: Not available.

Section 5: Fire and Explosion Data

Flammability of the Product: May be combustible at high temperature. Auto-Ignition Temperature: Not available. Flash Points: CLOSED CUP: 100C (212F). Flammable Limits: Not available. Products of Combustion: These products are carbon oxides (CO, CO2). Fire Hazards in Presence of Various Substances: Slightly flammable to flammable in presence of open flames and sparks, of heat. Non-flammable in presence of shocks. Explosion Hazards in Presence of Various Substances: Risks of explosion of the product in presence of static discharge: Not available. Slightly explosive in presence of heat. Nonexplosive in presence of shocks. Fire Fighting Media and Instructions: SMALL FIRE: Use DRY chemical powder. LARGE FIRE: Use water spray, fog or foam. Do not use water jet. Special Remarks on Fire Hazards: Not available. Special Remarks on Explosion Hazards: Not available.

Section 6: Accidental Release Measures

Small Spill: Absorb with an inert material and put the spilled material in an appropriate waste disposal. Large Spill: Absorb with an inert material and put the spilled material in an appropriate waste disposal.

Section 7: Handling and Storage

Precautions: 43

Keep locked up.. Keep away from heat. Keep away from sources of ignition. Empty containers pose a fire risk, evaporate the residue under a fume hood. Ground all equipment containing material. Do not ingest. Do not breathe gas/fumes/ vapor/spray. Wear suitable protective clothing. If ingested, seek medical advice immediately and show the container or the label. Keep away from incompatibles such as oxidizing agents, alkalis. Storage: Keep container tightly closed. Keep container in a cool, well-ventilated area.

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. Ensure that eyewash stations and safety showers are proximal to the work-station location. Personal Protection: Safety glasses. Lab coat. Personal Protection in Case of a Large Spill: Splash goggles. Full suit. Boots. Gloves. Suggested protective clothing might not be sufficient; consult a specialist BEFORE handling this product. Exposure Limits: Not available.

Section 9: Physical and Chemical Properties

Physical state and appearance: Liquid. Odor: Not available. Taste: Not available. Molecular Weight: 124.2 g/mole Color: Colorless. pH (1% soln/water): Not available. Boiling Point: 213C (415.4F) Melting Point: Not available. Critical Temperature: Not available. Specific Gravity: 1.1452 @ 22 deg. C (Water = 1) Vapor Pressure: 0 kPa (@ 25C) Vapor Density: Not available. Volatility: Not available. Odor Threshold: Not available. Water/Oil Dist. Coeff.: The product is equally soluble in oil and water; log(oil/water) = 0.1 Ionicity (in Water): Not available. Dispersion Properties: Not available. Solubility: Not available.

Section 10: Stability and Reactivity Data

Stability: The product is stable. Instability Temperature: Not available. 44

Conditions of Instability: Excess heat, igniton sources, incompatible materials Incompatibility with various substances: Reactive with oxidizing agents, alkalis. Corrosivity: Not available. Special Remarks on Reactivity: Not available. Special Remarks on Corrosivity: Not available. Polymerization: Will not occur.

Section 11: Toxicological Information

Routes of Entry: Absorbed through skin. Eye contact. Toxicity to Animals: Acute oral toxicity (LD50): 470 mg/kg [Mouse]. Chronic Effects on Humans: CARCINOGENIC EFFECTS: Classified 2B (Possible for human.) by IARC. MUTAGENIC EFFECTS: Mutagenic for mammalian somatic cells. Mutagenic for bacteria and/or yeast. DEVELOPMENTAL TOXICITY: Classified Reproductive system/toxin/male [POSSIBLE]. May cause damage to the following organs: the reproductive system. Other Toxic Effects on Humans: Hazardous in case of ingestion. Slightly hazardous in case of skin contact (irritant, permeator), of inhalation. Special Remarks on Toxicity to Animals: Not available. Special Remarks on Chronic Effects on Humans: May cause adverse reproductive effects and birth defects(teratogenic). May affect genetic material (mutagenic). May cause cancer (kidney tumors, lung tumors, brain tumors) Special Remarks on other Toxic Effects on Humans: Acute Potential Health Effects: Skin: May cause skin irritation. Eyes: May causes eye irritation. Inhalation: May cause respiratory tract irritation. Ingestion: Harmful (toxic) if swallowed. May cause digestive tract irritation.

Section 12: Ecological Information

Ecotoxicity: Not available. BOD5 and COD: Not available. Products of Biodegradation: Possibly hazardous short term degradation products are not likely. However, long term degradation products may arise. Toxicity of the Products of Biodegradation: The products of degradation are less toxic than the product itself. Special Remarks on the Products of Biodegradation: Not available.

Section 13: Disposal Considerations

Waste Disposal: Waste must be disposed of in accordance with federal, state and local environmental control regulations.

Section 14: Transport Information

DOT Classification: CLASS 6.1: Poisonous material. Identification: : Toxic Liquid, Organic, n.o.s. (Methanesulfonic Acid Ethyl Ester) UNNA: 2810 PG: III 45

Special Provisions for Transport: Not available.

Section 15: Other Regulatory Information

Federal and State Regulations: California prop. 65: This product contains the following ingredients for which the State of California has found to cause cancer, birth defects or other reproductive harm, which would require a warning under the statute: Methanesulfonic Acid Ethyl Ester California prop. 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.: 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.: Methanesulfonic Acid Ethyl Ester: 1 lbs. (0.4536 kg) Other Regulations: OSHA: Hazardous by definition of Hazard Communication Standard (29 CFR 1910.1200). EINECS: This product is on the European Inventory of Existing Commercial Chemical Substances. Other Classifications: WHMIS (Canada): CLASS D-1B: Material causing immediate and serious toxic effects (TOXIC). CLASS D-2B: Material causing other toxic effects (TOXIC). DSCL (EEC): R22- Harmful if swallowed. R45- May cause cancer. R46- May cause heritable genetic damage. R62- Possible risk of impaired fertility. S24/25- Avoid contact with skin and eyes. S36/37/39- Wear suitable protective clothing, gloves and eye/face protection. S45- In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible). S53- Avoid exposure - obtain special instructions before use. HMIS (U.S.A.): Health Hazard: 2 Fire Hazard: 1 Reactivity: 0 Personal Protection: a National Fire Protection Association (U.S.A.): Health: 2 Flammability: 1 Reactivity: 0 Specific hazard: Protective Equipment: Not applicable. Lab coat. Wear appropriate respirator when ventilation is inadequate. Safety glasses.

Section 16: Other Information

References: Not available. Other Special Considerations: Not available. 46

APPENDIX D: Composition of buffers, reagents and solutions used Table 13. HEPES KOH extraction buffer composition HEPES buffer (1 M pH 7.5) mercaptoethanol EDTA (0.02M) Glycerol D/W TOTAL 0.01 ml 0.002 ml 0.005 ml 1 ml 8.983 ml 10 ml

Table 14. Composition of Reagent C for Folin-Lowry method Reagent C Reagent A (2% Na2CO3 in 0.1N NaOH) Reagent B (0.5% CuSO4 in 1 % COOK.CHOH.CHOH.COONa) 50 ml 1 ml

Table 15. Composition of resolving and stacking gels for SDS PAGE RESOLVING GEL (8%) D/W Acrylamide-bisacrylamide 1.5M Tris HCl pH 8.8 0.5M Tris HCl pH 6.8 10% SDS 10% APS TEMED TOTAL 13.9 ml 8.0 ml 7.5 ml 0.3 ml 0.3 ml 0.018 ml 30 ml STACKING GEL (5%) 0.66 ml 1.25 ml 0.050 ml 0.050 ml 0.005 ml 0.018 ml 5 ml

Table 16. Composition of AgNO3 solution AgNO3 Formaldehyde D/W 0.3 gm 75 l 100 ml

47

Table 17. Composition of developing solution Na2CO3 Na2S2O3.5H2O Formaldehyde D/W 6 gm 1 mg 50 l 100 ml

Table 18. Composition of Acid ninhydrin Ninhydrin Glacial acetic acid Phosphoric acid 6M 1.25 gm 30 ml 20 ml

Table 19. Composition of potassium tri-iodide reagent Iodine Potassium iodide D/W 15.7 gm 20 gm 100 ml

48

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