A heavy metal is a member of a loosely defined subset of elements that exhibit metallic properties

A heavy metal is a member of a loosely-defined subset of elements that exhibit metallic properties

Introduction 1.1.1 Heavy metal A heavy metal is a member of a loosely-defined subset of elements that exhibit metallic properties (www.wikipedia.com, 15th December, 2010). It mainly includes the transition metals, some metalloids, lanthanides, and actinides. Many different definitions have been proposed—some based on density, some on atomic number or atomic weight, and some on chemical properties or toxicity.(John et al 2002) There is an alternative term toxic metal, for which no consensus of exact definition exists either. As discussed below, depending on context, heavy metal can include elements lighter than carbon and can exclude some of the heaviest metals. Heavy metals occur naturally in the ecosystem with large variations in concentration (www.wikipedia.com, 15th December, 2010). In modern times, anthropogenic sources of heavy metals, i.e. pollution, have been introduced to the ecosystem. Waste-derived fuels are especially prone to contain heavy metals, so heavy metals are a concern in consideration of waste as fuel. (Matlock et al, 1998)

1.1.2 Different types of Heavy metal Some of the heavy metals are dangerous to health or to the environment (e.g. mercury, cadmium, lead, chromium), some may cause corrosion (e.g. zinc, lead), some are harmful in other ways (e.g. arsenic may pollute catalysts). Within the European community the thirteen elements of highest concern are arsenic, cadmium, cobalt, chromium, copper, mercury, manganese, nickel, lead, tin, and thallium, the emissions of which are regulated in waste incinerators (www.wikipedia.com, 15th December, 2010). Some of these elements are actually necessary for humans in minute amounts (cobalt, copper, chromium, manganese, nickel) while others are carcinogenic or toxic, affecting, among others, the central nervous system (manganese, mercury, lead, arsenic), the kidneys or liver (mercury, lead, cadmium, copper) or skin, bones, or teeth (nickel, cadmium, copper, chromium).(Zevenhoven 2001)

Unlike organic pollutants, heavy metals do not decay and thus pose a different kind of challenge for remediation. Currently, plants or microorganisms are tentatively used to remove some heavy metals such as mercury (Hassinen 2009 ). Plants which exhibit hyper accumulation can be used to remove heavy metals from soils by concentrating them in their bio matter. Some treatment of mining tailings has occurred where the vegetation is then incinerated to recover the heavy metals (Hassinen 2009).

1.1.3 Environment and heavy metal Environment is defined as the totality of circumstances surrounding an organism or group of organisms especially, the combination of external physical conditions that affect and influence the growth, development and survival of organisms. It consists of the flora, fauna and the abiotic, and includes the aquatic, terrestrial and atmospheric habitats (Hassinen 2009). The environment is considered in terms of the most tangible aspects like air, water and food, and the less tangible, though no less important, the communities we live in. A pollutant is any substance in the environment, which causes objectionable effects, impairing the welfare of the environment, reducing the quality of life and may eventually cause death. Such a substance has to be present in the environment beyond a set or tolerance limit, which could be either a desirable or acceptable limit. Hence, environmental pollution is the presence of a pollutant in the environment; air, water and soil, which may be poisonous or toxic and will cause harm to living things in the polluted Environmental pollution by heavy metals is very prominent in areas of mining and old mine sites and pollution reduces with increasing distance away from mining sites. These metals are leached out and in sloppy areas, are carried by acid water downstream or run-off to the sea.

Through mining activities, water bodies are most emphatically polluted (Carr et al 2008). The potential for contamination is increased when mining exposes metal-bearing ores rather than natural exposure of ore bodies through erosion, and when mined ores are dumped on the earth surfaces in manual dressing processes. Through rivers and streams, the metals are transported as either dissolved species in water or as an integral part of suspended sediments, (dissolved species in water have the greatest potential of causing the most deleterious effects). They may then be stored in river bed sediments or seep into the underground water thereby contaminating water from underground sources, particularly wells; and the extent of

contamination will depend on the nearness of the well to the mining site. Wells located near mining sites have been reported to contain heavy metals at levels that exceed drinking water criteria.

1.1.4 Soil and Heavy metal Heavy metal pollution of surface and underground water sources results in considerable soil pollution and pollution increases when mined ores are dumped on the ground surface for manual dressing. Surface dumping exposes the metals to air and rain thereby generating much AMD. When agricultural soils are polluted, these metals are taken up by plants and consequently accumulate in their tissues .Animals that graze on such contaminated plants and drink from polluted waters, as well as marine lives that breed in heavy metal polluted waters also accumulate such metals in their tissues, and milk, if lactating .Humans are in turn exposed to heavy metals by consuming contaminated plants and animals, and this has been known to result in various biochemical disorders. In summary, all living organisms within a given ecosystem are variously contaminated along their cycles of food chain. (Bhuiyan et al 2010)

1.1.5 Toxic Effects of Different Heavy Metals Cadmium is toxic at extremely low levels (Duffus et al 2002). In humans, long term exposure results in renal dysfunction, characterized by tubular proteinuria (Hogan 2010). High exposure can lead to obstructive lung disease, cadmium pneumonitis, resulting from inhaled dusts and fumes. It is characterized by chest pain, cough with foamy and bloody sputum, and death of the lining of the lung tissues because of excessive accumulation of watery fluids (Duffus et al 2002). Cadmium is also associated with bone defects, viz; osteomalacia, osteoporosis and spontaneous fractures, increased blood pressure and myocardic dysfunctions. Depending on the severity of exposure, the symptoms of effects include nausea, vomiting, abdominal cramps, dyspnea and muscular weakness (Hogan 2010). Severe exposure may result in pulmonary odema and death. Pulmonary effects (emphysema, bronchiolitis and alveolitis) and renal effects may occur following subchronic inhalation exposure to cadmium and its compounds. Lead is the most significant toxin of the heavy metals, and the inorganic forms are absorbed through ingestion by food and water, and

inhalation. A notably serious effect of lead toxicity is its teratogenic effect. Lead poisoning also causes inhibition of the synthesis of haemoglobin; dysfunctions in the kidneys, joints and reproductive systems, cardiovascular system and acute and chronic damage to the central nervous system (CNS) and peripheral nervous system (PNS). Other effects include damage to the gastrointestinal tract (GIT) and urinary tract resulting in bloody urine, neurological disorder and can cause severe and permanent brain damage. While inorganic forms of lead, typically affect the CNS, PNS, GIT and other biosystems, organic forms predominantly affect the CNS. Lead affects children by leading to the poor development of the grey matter of the brain, thereby resulting in poor intelligence quotient (IQ). Its absorption in the body is enhanced by Ca and Zn deficiencies (Hogan 2010). Acute and chronic effects of lead result in psychosis.

Zinc has been reported to cause the same signs of illness as does lead, and can easily be mistakenly diagnosed as lead poisoning. Zinc is considered to be relatively non-toxic, especially if taken orally (Duffus et al 2002). However, excess amount can cause system dysfunctions that result in impairment of growth and reproduction. The clinical signs of zinc toxicosis have been reported as vomiting, diarrhea, bloody urine, icterus (yellow mucus membrane), liver failure, kidney failure and anemia.(Dhuruibe et al 2007)(Fig 1.1)

Fig1.1: Metabolism of heavy metal

1.1.6 Heavy metal contamination in water, soil, and vegetables of the industrial areas in Dhaka, Bangladesh Concentrations of Cu, Zn, Pb, Cr, Cd, Fe, and Ni have been estimated in soils and vegetables grown in and around an industrial area of Bangladesh. The order of metal contents was found to be Fe > Cu > Zn > Cr > Pb > Ni > Cd in contaminated irrigation water, and a similar pattern Fe > Zn > Ni > Cr > Pb > Cu > Cd was also observed in arable soils (Duffus et al 2002). Metal levels observed in different sources were compared with WHO, SEPA, and established permissible levels reported by different authors. Mean concentration of Cu, Fe, and Cd in irrigation water and Cd content in soil were much above the recommended level.

Accumulation of the heavy metals in vegetables studied was lower than the recommended maximum tolerable levels proposed by the Joint FAO/WHO Expert Committee on Food Additives (1999), with the exception of Cd which exhibited elevated content. Uptake and translocation pattern of metal from soil to edible parts of vegetables were quite distinguished for almost all the elements examined.(Fig 1.2,1.3)

Fig

1.2:

Particulate

matter

emission

(www.wikipedia.com, 15th December, 2010)

in

residential

area

of

Bangladesh

1.1.7 Conventional methods against heavy metal In order to be competitive economically, many of these chelating ligands are simple, easy to obtain, and, generally offer weak bonding for heavy metals. Poor and indiscriminant metal binding often lead to unstable metal ligand complexes. Laboratory testing of three commercial

reagents,

TMT

(Trimercaptotriazine),

Thio-Red

(potassium/sodium

thiocarbonate), and HMP-2000 (sodium dimethyldithiocarbamate), has shown that the investigated compounds were unable to reduce independent solutions of 50.00 ppm (parts per million) cadmium, copper, ferrous, lead, or mercury to meet EPA standards (CFR,

Fig 1.3: Indication of arsenic presence in drinking water of Bangladesh

1994). additionally the tested compounds displayed high leaching rates and in some cases decomposed to produce toxic substances. For this reason, a novel multidentate ligand has been developed for the safe and effective removal of heavy metal. (Matlock et al 1998).

Fig1.4: Polluted cities of the world (www.wikipedia.com, 15th December, 2010) 1.1.8 Phytoremediation The term phytoremediation ("phyto" meaning plant, and the Latin suffix "remedium" meaning to clean or restore) actually refers to a diverse collection of plant-based technologies that use either naturally occurring or genetically engineered plants for cleaning contaminated environments. The primary motivation behind the development of phytoremediative technologies is the potential for low-cost remediation. Although the term, phytoremediation,

is a relatively recent invention, its an age old practice. Research using semi-aquatic plants for treating radionuclide-contaminated waters existed in Russia at the dawn of the nuclear era.

Some plants which grow on metalliferous soils have developed the ability to accumulate massive amounts of the indigenous metals in their tissues without exhibiting symptoms of toxicity was the first to suggest using these "hyperaccumulators" for the phytoremediation of metal-polluted sites. However, hyperaccumulators were later believed to have limited potential in this area because of their small size and slow growth, which limit the speed of metal removal. By definition, a hyperaccumulator must accumulate at least 100 mg g -1 (0.01% dry wt.), Cd, As and some other trace metals, 1000 mg g -1 (0.1 dry wt.) Co, Cu, Cr, Ni and Pb and 10,000 mg g-1 (1 % dry wt.) Mn and Ni (Duffus et al 2002).(Fig 1.4) 1.2.1 Phytoremediation and jute Jute is highly popular in Bangladesh for its commercial applications .Beside this it has major role in environmental aspects. Its eco-friendly nature is the primary indication for its role in phytoremediation. The capacity of jute to grow in adverse condition and its stress tolerant behavior is the idea of this study. Brassica, Arabidopsis and Poplar are the principle phytoremediator plant charted till now (Hassinen 2009). Among them, Poplar is a good phytoremediator because of the endophytic bacteria living inside it (Hassinen 2009). There is no metabolic and genetic linkup of poplar with this. On the other hand, Arabidopsis and Brassica possess inherent molecular mechanisms for phytoremediation. And both of them have strong genetic similarity with jute varieties. This gives us the idea to launch an experiment on this capacity of jute.

1.2.2 Jute Jute is a natural fiber obtained as an extract from the bark of the jute plants that grows like any other organic crop. Jute has various inherent characteristics like, high tensile strength, low extensibility, long durability, fire and heat resistance, silkiness, luster and long staple length. Jute is the second most important natural fiber after cotton. It is the cheapest and mostly used bast fiber (fiber collected from bast or skin of the plant) in the world. Since jute fiber has a lustrous and shiny golden color, hence it is called "The Golden Fibre". Because of its high tensile strength and low extensive nature, jute fiber has been the best substitute for any other vegetable fibres and the best natural substitute for polypropylene. Jute was earlier

called by its Bengali name 'Pat' (Patta in Sanskrit). Depending on demand, price and climate, the annual production of jute and allied fibres in the world remains around 3 million tones.

Fig1.5: Jute Plant Jute is bio-degradable, strong, non-toxic, hygroscopic, less extensible, coarse and cheap as a fiber (Fig1.5). It is cultivated almost exclusively in developing countries of East Asia and in some parts of Latin America. Bangladesh, India and Thailand account for over 90 percent of world production. The fiber is processed mainly in the producing countries themselves and is used for the manufacturing of traditional products such as Hessian cloth, food grade bags, carpet backing and other floor covering. Diversified jute products, such as geo-textiles and composites are also manufactured in relatively small quantities. Jute constitutes a low proportion of the value of world trade, but its cultivation and processing is labour-intensive and therefore provides a livelihood and an important source of food security for many farmers and their families in Asia.

Jute is dicotyledenous fibre-yielding plant of the genus Corchorus, order Tiliaceae. The jute plant is an annual shrub which contains haploid number of 7 chromosomes and is a natural inhabitant of the tropical and subtropical regions of the world. In the trade there are usually

two names of jute, White (Corchorus capsularis) and Tossa (C. olitorius).. In India & Bangladesh Roselle is usually called Mesta. Jute fibres are finer and stronger than Mesta and are, therefore, better in quality. Genetic variability is limited in both species due to selfpollination. The cultivars of both species grow to a height of 9-10 feet and are sparsely branched.

The cultivar of C. olitorus (Tossa) is thought to have originated in many African countries for example, Mozambique, Tanzania, Zimbabwe, Zaire, Ethiopia, Somalia, Kenya, Uganda and Sudan. Evidence suggests it grows in Anatolia (Turkey), Socotra (Yemen-Aden) and Iraq. In the Middle East C. olitorius leaves have been in use as potherbs since Biblical times.

Cultivation in commercial scale is restricted to Bangladesh, India, Nepal, Taiwan and Brazil.

The cultivars of C. capsularis (white jute), on the other hand, occur in southern China, northern Burma including the Shan States, Malaysia, the Indonesian islands of Halmahera, Celebes and Timur, the northeastern hills of Meghalay, Mizoram, Nagaland and Tripura in India. Recently types have been collected in India from M.P., Mirzapur, A.P. and Eastern Ghats (JARI: collection). C. capsularis types have also been found in Sri Lanka, Philippines and Pakistan.

1.2.3 Taxonomical Characteristics The genus, Corchorus, belongs to the family, Tiliaceae. It includes about 40 species mostly distributed in the tropical regions. Fourteen of them including C. capsularis and C. olitorius are diploid (2n=14) and four are tetraploid (2n=28). C.capsularis and C. olitorius are both herbaceous annuals. The vegetative period of both is about 3-5 months. At the harvest stage varieties of C. capsularis attain a height of about 5-12 feet and those of C. olitorius 5-15 feet or more (Khan M.S. 1988). The stems of both are cylindrical. Leaves are glabrous. Flowers of both the species look yellow, are small in size and occur in condensed cymes. (Sarma.M.S. 1969). Flowers occur in the axel of' leaves, and composed of 4-5 sepals and petals, 5 to numerous stamens. Ovary is 2-6 locular. Seeds are small and numerous (Khan M.S. 1988).

Seeds of C. olitorius are smaller. C. capsularis seed is coppery in colour and weigh about 500-600 seed per 1 gram. C. olitorius seed is greyish in colour and weigh about 1000 seeds per 1 gram. Both the species are mostly self pollinated (Sarma.M.S. 1969).

1.2.4 Genetic Variability Each of the two jute species contains very limited genetic variability with respect to (i) adaptability to different agronomic environments (ii) fiber quality (iii) fiber yield and (iv) susceptibility to diseases and pests (Lafarge et al. 1997). C. capsularis is comparatively more resistant to flood and drought but slightly more susceptible to diseases and pests (Lafarge et al. 1997). It provides ‘white commercial fiber’ which is slightly weak. C. olitorius is relatively tolerant to diseases and pests and produces the stronger ‘Tossa commercial fiber’, the word ‘Tossa’ indicating a lustrous golden shine. A combination of the useful characters of the two species in a single genotype is desirable.

But unfortunately, these two species do not cross with each other, possibly because of the presence of a strong sexual incompatibility barrier between them (Patel and Datta, 1960; Swaminathan et al. 1961). Previous studies have revealed that the two jute species are distantly related. Presence of distinct patterns of diversity between the two species was also reported by Palit et al. (1996). Moreover, differences in geographical location of sources do not affect the genetic diversity as reported by (Palit et al. 1996). It is inferred that the two species are allopathic, sharing certain common alleles (Basu et al. 2004).

1.3.1 Jute and the Environment The worldwide awareness on environment and health is likely to provide new opportunities on jute, due to its environment-friendly characteristics. Jute has been employed for centuries as packaging materials. In recent times they are found to be a valuable aid to sound environmental management. As a natural fibre that can be used in many different areas, supplementing and/or replacing synthetics, has been receiving increasing attention from the industry. Their interests focus not only on the traditional uses of jute, but also on the production of other value-added products such as, pulp and paper, geotextiles, composites and home textiles etc.

Jute is an annually renewable energy source with a high biomass production per unit land area. It is biodegradable and its products can be easily disposed without causing environmental hazards (Grotz et al 2005). The roots of jute plants play a vital role in increasing the fertility of the soil. By rotating with other crops like rice and potatoes, jute acts as a barrier to pest and diseases for others crops and provides also a substantial amount of nutrients to other crops in the form of organic matter and micronutrients. Jute has ecological

adaptability, and can be grown on a range of soil types. They have a good tolerance to salinity, water stress and water logging. Agronomically, jute has advantages as regards their resistance to climatic extremes, pests and diseases.

Jute plants have high carbon dioxide (CO 2) assimilation rate (Basu et al 2005). Theoretically, one hectare of jute plants can consume about 15 tons of CO 2 from atmosphere and release about 11 tons of oxygen in the 100 days of the jute-growing season. Studies also show that the CO2 assimilation rate of jute is several times higher than that of other trees (Inagaki, 2000).

The defoliated jute leaves have fertilizer value and enriches the soil nutrients. Jute leaves are used as vegetables and have nutritional as well as medicinal values. Jute sticks are used for fuel and shelter in jute growing rural areas. This has helped reduce the use of wood in these applications. For instance, the total production of jute in the world is 3 million tons. This means that on an average 6 million tons of jute sticks are available to the rural people for use as firewood etc.

Jute contains cellulose like any other raw materials used for paper pulp. Experiments to convert jute fibre and whole jute plant into paper pulp have successfully produced good quality pulp and paper. The growing demand of pulp and paper worldwide on a continuous basis and increase of public awareness on environmental issues have created conditions to check depletion of forest resources through using jute for producing pulp and paper. This increasing demand for paper has led to excessive deforestation in both developed and developing countries. Jute, for its versatility, rightfully deserves to be branded as the “fiber for the future�. It is the natural option for a cleaner and healthier environment. Although some plants show promise for phytoextraction, there is no plant which possesses all of these desirable traits. Finding the perfect plant continues to be the focus of many plant-breeding and genetic-engineering research efforts.

Chapter 2 Objective of the study The objective of this study is to check the capacity of jute to uptake heavy metals from the soil and to find the molecular mechanism behind this. Pot analysis is the convenient method

for analyzing this type of experiment. On heavy metal rich soil (in different concentration), the growth of the jute plant will indicate the tolerable level. In addition, it will indicate the resistance and uptake level of heavy metal by its transport system. The molecular mechanism is also an important part of that study. The presence of the necessary genes responsible for its uptake will ensure the validity of the pot analysis experiment. The proof of the gene/genes at a time their expression level in stressed condition is very important for the establishment of hypothesis in the role of jute in phytoremediation. As industrial pollution is very common in Bangladesh, the wide cultivated crop like jute can be a very good way of phytoremediation.

Jute has quick degrading nature, either in the vegetative stage or in the form of products and bi-products. Therefore we can say it is eco-friendly. The success of this analysis can add another wing to the eco-friendly nature of jute.

•

Find a new tool for phytoremediation.

•

Analysis of jute characteristics on metal uptake.

•

Presence of necessary genes on jute

•

Bioinformatics analysis of genes.

•

Analysis of expression level of genes.

Thesis work plan

Metal uptake gene analysis

Plant (Corchorus olitorious)

Growth on pot (Heavy metal rich soil)

Gene Prediction

Gene identification

Spectrophotometer analysis

Sequence analysis

Heavy metal Uptake

Morphological Changes

Conclusion on Jute Phytoremediation Capacity

Expression analysis

Material and Methods Chapter 3 3.1 Pot analysis 3.1.1 Soil collection area •

Hazaribgah leather industry area: Contaminated with leather effluent. (Fig 3.1)

•

Dying industry area close to Buriganga River: Contain high level of dyeing industry waste rich in heavy metals. (Fig3.2.)

•

Dumping ground of Epz, Savar: Heavy industry effluents with huge chemical waste.

(Fig3.3).

Fig: 3.1 Hazaribgah leather industry areas

Fig: 3.2 Dying industry area close to Buriganga River

Fig: 3.3 Dumping ground of Epz, Savar

Fig: 3.4 Plants during Pot Analysis Period 3.1.2 Analyzed variety Corchorus olitorious (CVL-1)

3.1.3 Season followed Late harvesting period of jute (June-October) of 2010.

Pot analysis place: Bangladesh Jute research Institute, Manik Mia Avenue, Dhaka

3.1.4 Description of pot analysis 3.1.4.1 Materials & Equipment •

Seeds (beans work nicely)

•

Containers for plants (cups or potting containers)

•

Potting soil

•

Water

•

Ruler

•

Graduated cylinder/measuring cup

•

Growing lights (or well-lit area for plants)

3.1.4.2 Procedure 1. Each group has 2 pots.(FIG 3.4) 2. The pots are filled with an equal amount of potting soil.

3. Several hole in the soil.

4. One seed is placed into each hole, covered with the soil, and watered.

5. All the plants are placed in a well-lit area .

6. All the plants are measured each day and observations are noted.

7. At the end of the two weeks, conclusions are made.

3.1.4.3 Soil analysis The content of heavy metal in the soil was measured thrice.

•

At the starting of the experiment before harvesting.

•

At the middle of the study( after 2 month)

•

At the end of the study (after complete growth of the study, 4 month)

3.1.4.4Extraction procedure 3.1.4.4.1technical support 1. Atomic absorption spectrometry with electro thermal atomization. The measurements were performed with specter AA 220 varian atomic absorption spectrometer, equipped with a THGA graphite furnace and an AS-70 autosampler.

Background correction was performed by longitudinal Zeeman effect in a magnetic field with strength of approximately 0.9 Tesla. A hollow cathode lamp for mercury from Photron Pty. Ltd., Australia, operated at 3 mA, was used and a 0.7 nm spectral band width was selected to isolate the 253.7 nm resonance line. Platform atomization from transverse heated pyrolytically coated graphite tubes was used. Argon was used as purge gas. The measurements were done in the peak area mode Cold vapour atomic absorption spectrometry in flow-injection system .Two flow injection systems FIAS100 and FIMS, both from spectra AA 220 varian were used. The FIAS-100 was used in combination with spectra AA 220 varian atomic absorption spectrometer as described above. The volume of the sample loop was 500 gl and SnC12 was used as reducing agent. Mercury .vapours were stripped with argon (100 ml/min) and transferred to the absorption cell, 175 mm long, electrically heated to 140C . (Bulaska et al 1995)(Fig 3.5)

Fig3.5 : Spectra AA 220 Varian AAS

3.1.4.4.2Reagents •

Ultra pure HC1 and HNO 3 (J. T. Baker B. V., Holland),

•

SnC12 (POCH, Poland),

•

NaBH 4 (Aldrich, England),

•

PdC12 (Merek-Schuchardt, Germany),

•

H20 2 (BDH, England),

•

Hg(NO3) 2 stock standard solution containing 1 mg/ml Hg(II) (COBR-Wzormat, Poland and Cheman, Poland).

•

A standard solution of 10 mg/1 mercury(II) was prepared weekly from the stock solutions of 1.00 mg/ml by dilution with HC1 (1 mol/1).

3.1.4.5Forms of digestion In the digestion procedures four different types of acids have been used: A - 20 ml of 1 mol/1 HC1;B- 10 ml of conc. HNO 3 diluted 1:1; C- 5 ml of conc. HNO3; D 8 ml of aquilegia (3 vol. HC1 and 1 vol. HNO3).

I. Open vessel microwave digestion (Prolabo) About 1 g of the standard reference material was accurately weighed and rinsed with the digestion acid (as described above) into the digestion vessel. After 30 min the vessel was inserted into the microwave unit and the power/temperature program as given in Table 2 was run. After cooling down, the condenser at the top of the flask was rinsed with a small portion of water. The content of the vessel was filtered through a 5B-type filter and the residue was washed twice with water. The filtrates were diluted with water to give 50 g of solution140 E. (Day 1995) II. Closed vessel microwave digestion (CEM) About 1 g of the standard reference material was accurately weighed into the digestion vessel. The digestion acid was added to this vessel and in parallel to an empty digestion vessel. The digestion mixture was allowed to stand for at least half an hour. Both digestion vessels were inserted into the microwave unit and the power/temperature program as given in

Table 3 was run. After the digestion vessels had cooled down, they were opened carefully and the digestion solution was filtered and diluted as described in procedure I. III. Digestion on the heating block About 1 g of the standard reference material was accurately weighed and rinsed with the digestion acid into the digestion vessel, which was inserted in a hole of the cold heating block. Heating was turned on and the temperature of 95 ~ was reached with 15 min and held for 60 min. The digestion solution was filtered and diluted as described in the procedure I. IV. Digestion according to the standard method About 2.2 g of the standard reference material was accurately weighed in a 250 ml flask. The sample was moistened with water, and then 21 ml of conc. HC1 and 7 ml of conc. HNO 3 were added. Next 10 ml of 0.5 mol/L HNO 3 were added; the reflux condenser was connected with the absorption unit and allowed to stand.

3.1.4.6Procedure of digestion The digestion mixture was slowly heated and allowed to boil for 2 h. After the mixture had cooled down, the reflux condenser was rinsed with water into the digestion flask. The contents of the flask and the absorption acid were transferred quantitatively into 100ml calibrated flasks and diluted to volume. The digestion solution was analyzed after precipitate decantation (24 h).

3.1.4.6.1 for Soil •

Meshed Soil was taken for analysis

•

The soil was diluted with deionized water

•

pH maintained as soil: water=1:2.5

•

Aqua regia was mixed with the soil

•

The mixture was stored in 110•c for 5 hours

•

The solution was diluted up to 100 ml

•

Reading taken in Gydride generation AAS.

•

Quality control was ensured by taking reading of a certified reference material (CRM).

3.1.4.6.2 for Plant •

Plant Material was washed with deionized water.

•

4 ml HNO3 added to material and allowed to stand for 30 minute.

•

The mixture kept overnight for in 60۫c.

•

The dissolution was made in 140 •c

•

25 ml of the filtrate was taken for reading

(Jackson 1973)

3.1.4.6.3 Software used Microsoft Excel 2003 and MINITAB software used to analyze the data given by Spectra 220 Varian Atomic Absorption Spectrophotometer.

3.2 Molecular Analysis 3.2.1DNA Isolation from Jute Plant The primary objective of the isolation process was to recover the maximum yield of high molecular weight DNA devoid of protein and other restriction enzymes (Sam brook et al.

1989).

3.2.1.1 Materials Liquid Nitrogen: Liquid Nitrogen was collected from Bangladesh Oxygen Company (BOC).

1. Preparation of 1M of Tris.HCl (stock solution): Tris-HCl (MW. 121.14) •

1M Tris-HCl was prepared by dissolving 12.11 of Tris in 80 ml of water with the help of magnetic stirrer.

•

The pH was adjusted to 8.0 by concentrated HCl. Final volume was made 100 ml and sterilized by autoclaving.

2. Preparation of 0.5M EDTA (stock solution): EDTA (MW. 372.2) 40 ml of did water was taken and 18.61g of EDTA was dissolved with the help of magnetic stirrer.

The pH was adjusted to 8.0 by Noah pellet. The volume was then adjusted to 100 ml and sterilized by autoclaving. All the solutions (as mentioned below) were made with deionized, sterile water and autoclaved except phenol.

3. 5M NaCl (MW. 58.44) •

29.22g of NaCl were dissolved in 40 ml of dad water.

•

The volume was then made 100 ml by dd water and the solution was autoclaved.

4. Chloroform: Isoamyl alcohol (24:1, v/v) 5. RNase solution (DNase free) (stock 10mg/ml) •

Pancreatic RNase (RNase A) was dissolved at a concentration of 10 mg/ml in 0.01M sodium acetate (pH 5.2).

•

It was allowed to cool slowly at room temperature after heating to 1000C for 15 minute.

•

Then pH was adjusted by adding 0.1 volume of 1M Tris-HCl (pH- 7.4). This RNase solution was dispensed into aliquots and stored at -200C.

All the materials were added in a conical flask and heated at 600C in the water bath until all the CTAB melted. Then the volume was adjusted to 100ml with autoclaved water and stored at room temperature.

3.1.1.1.1 Preparation of Chemicals /Reagents * 2 -Mercaptoethanol * CTAB-Extraction Buffer * CTAB-NaCl Buffer * CTAB-Precipitation Buffer * 24:1(v/v) Chloroform-Isoamyl alcohol * High Salt TE buffer

* Isopropanol

(Ice cold)

* 80% Ethanol (Ice cold) * PCR TE buffer 1. CTAB Extraction Buffer (100ml): CTAB 2% (w/v) 2g Tris-HCl (1M) 100mM 10ml EDTA (0.5M) 20mM 4ml NaCl (5M) 1.4M 28ml Add dd water up to ~ 98ml Before use the buffer, 2 ml of Mercapto Ethanol (2ME) was added in the solution.

2. CTAB-NaCl Buffer (100ml): NaCl dd water CTAB

4.1g 80ml 10g

If necessary, it can be heated at 65°C in water bath for 3-5 hour to dissolve.

The volume was then made up to 100ml with dd water.

3. CTAB Precipitation Buffer (100ml): CTAB Tris-HCl (1M) EDTA (0.5M)

1% (w/v) 50mM 10mM

1g 5ml 2ml

The volume was then made up to 100ml with dd water.

4. High Salt TE Buffer (100ml): Tris-HCl (1M) EDTA (0.5M) NaCl (5M)

10mM 0.1mM 1M

1ml 20µl 20ml

The volume was made up to 100ml with dd water.

3.2.1.2 Method imam •

2-ME was added to the required amount of CTAB extraction solution to give a final concentration of 2% (v/v). This solution and CTAB/NaCl solution was heated to 65ºC. (Approximately 6 ml of 2ME/CTAB extraction solution and 0.4 to 0.5 ml CTAB-NaCl solution are required for each gram of fresh leaf tissue).

•

A homogenizer with liquid nitrogen (-196ºC) was chilled. Plant tissue was homogenized to a fine powder and the frozen tissue was transferred to an organic solvent-resistant test tube or beaker.

•

2ME/CTAB extraction solution was heated and added to the homogenized tissue.

Then it was mixed thoroughly. This was incubated 10 to 60 min at 65ºC with occasional mixing.

•

Homogenate was extracted with an equal volume of 24:1 chloroform/isoamyl alcohol, mixed well by inversion, centrifuged 5 min at 10000 rpm in a micro centrifuge at 4ºC. The aqueous phase was recovered (2 times).

•

1/10 vol CTAB-NaCl solution (65ºC) was added to the recovered aqueous phase and mixed well by inversion.

•

Extracted with an equal volume of chloroform/isoamyl alcohol, mixed, centrifuged and recovered the aqueous phase.

•

Added exactly 1 vol CTAB precipitation solution and mixed well by inversion.

•

Centrifuged 5 min at 27000 rpm in microcentrifuge at 4ºC.

•

Removed supernatant and resuspended pellet in high-salt TE buffer (0.5 to 1 ml per g of starting matrial).

•

The nucleic acid was precipitated by adding 0.6 vol isopropanol. Then mixed well and centrifuged 15 min at 10000 rpm at 4ºC.

•

The DNA was analyzed by electrophoresis in 0.8% neutral agarose gel and molecular weight marker determined the size of DNA. The DNA was preserved at

-20°C.

3.2.1.3 Quantification of DNA Two methods were followed for quantification of DNA. These were: (a) Spectrophotometric method: The optical density (OD) of the isolated DNA was measured at 260 and 280 nm wavelength (Specord 50, Analytikjena) to assess the quality and quantity. (b) Gel analysis: The genomic DNAs along with λ DNA were run in a 0.8% agarose gel, the gel was stained by ethidium bromide solution and visualized using UV illumination and documented by Kodak Bio doc system. The DNAs were quantitated by comparing with florescent of known concentration of λ DNA.

3.2.2Polymerase Chain Reaction (PCR).

3.2.2.1Materials The Following components were required for the polymerase chain reaction.

1. 10X PCR Reaction Buffer, which contains

i.

500mM KCl

ii.

100mM Tris.HCl (pH 8.3)

iii.

0.1% gelatin

2. MgCl2 (25mM) in water for PCR 3. Sodium salt of deoxyadenosine-5/-triphosphate (10mM aqueous solution, pH 7.35) 4. Sodium salt of deoxy-thymidine -5/- triphosphate (10mM aqueous solution, pH 7.35)

5. Sodium salt of deoxyguanosine-5/-triphosphate (10mM aqueous solution, pH 7.35) 6. Sodium salt of deoxycytidine-5/-triphosphate (10mM aqueous solution, pH 7.35) 7. Isolated Taq DNA polymerase

8. PCR molecular markers.

9. DNA template, the sample DNA was collected from jute plant.

10. SSR Primers (Vizon,CANADA)

11. Autoclaved ultra-pure water 12. TE solution for the dilution of the template DNA i.

10mM Tris.HCl (pH 8.0)

ii. 0.1mM EDTA (pH 8.0)

Primer No F

Primer Name R

01

ZIP 1

02

ZIP 3

ZIP 1 ZIP 3

Forward

Reverse

GMTGCATMWCHCWGG

GGATTCATAAAATCA

CAGGTWTTGGAGKTG

CCAGCACCAAGAAGG

Fig3.6: Used Primer

3.2.2.2 Preparation of dNTPs Mixtures 100µl of (their concentrations being 10mM each) were mixed in fresh, autoclaved expender tube and the final volume was made 1000µl by adding 600µl of dd water and dispensed as aliquots in tubes and stored at –20ºC. The concentration of each of the nucleotide in the above mixture was 1.0mM.

3.2.2.3Dilution of the DNA Template Since the isolated DNA was highly concentrated and unsuitable for using in PCR, it was diluted with TE solution (10mM Tris.HCl, 0.1mM EDTA, pH 8.0) before use. Thus, the working concentration of the template DNA was made 10ng/µl.

3.2.2.4Dilution of Primer For the dilution of the primer TE was added to the tube to make 100 stock solutions as its molecular weight. Then 10µl of the primer was added to 90µl of TE. So, the final concentration of the primer was 10ng/µl. (Fig 3.6)

3.2.2.5 Preparation of the Master Mix Master mix was prepared by mixing all the components of PCR except the component against which the optimization strategy was intended. In each reaction, the volume of PCR buffer used was 1/10th of the total volume that was 25µl. After thorough mixing and momentary spin of the master mix, it was transferred to different microphage tubes. The PCR component in question was then added. The volume was made 25µl by adding varying amounts of sterilized ultra-pure water. Taq DNA polymerase was added just before the start of the reaction. Finally, the tubes were subjected to momentary spin and transferred to thermocycles for the amplification reaction.(Fig3.8)

3.2.2.6 Thermal Cycling Profile Used in PCR The thermal cycling profiles that were programmed to amplify the gene by Polymerase Chain Reaction (PCR) for 35 cycles are as on the following page:

3.2.2.7 Thermal Cycling Profile

Steps Initial denaturation Denaturation Annealing Elongation Final elongation

Temperature 95ºC 95ºC (52-57)ºC 72ºC 72ºC

Time 5 minutes 40seconds 50 seconds 50 seconds 5 minutes

No. of Cycles 1 (First) 35 35 35 1 (Last)

Fig 3.7: Thermal Cycling Profile For different primers different annealing temperatures were employed. For degenerate primers the optimum annealing temperature used was between 52-57°C. (Fig 3.7)

Fig3.8 : How Primer works

3.2.2.8 Visualizing the PCR Product 4µl of DNA dye was added to the PCR amplified DNA. After a momentary spin the PCR products were loaded in wells of 1.5% agarose gel. Electrophoresis was accomplished at 60 volts. Then the gel was stained in ethidium bromide (0.5µg / ml) and de-stained in water, followed by visualization under UV Tran illuminator (Sam brook et al. 1989).

3.2.2.8.1Agarose Gel Electrophoresis of DNA The standard method was used to separate and identify DNA fragments through agarose gel electrophoresis (Sambrook et al. 1989).

3.2.2.8.1.1Materials Ultra pure agarose, TAE buffer, gel loading dye and gel electrophoresis apparatus were used.

Preparation of stock solution (1 liter) of 50X TAE buffer Tris base

242.0g

Glacial acetic acid

57.1 ml

0.5M EDTA (pH 8.0)

100 ml

This solution was made up to 1000ml with ddH2O.

Preparation of 1.5% agarose gel (100 ml) •

To prepare 25ml of 1.5% agarose gel, 0.375g of agarose powder was weighed in conical flask.

•

0.5ml of 50X TAE was taken in measuring cylinder and the volume was made up to 25ml with dd water.

•

The mixture of 50X TAE and H20 was poured into flask containing agarose and then melted in microwave oven at 600C for 2-3 minutes.

Composition of DNA dye i. Xylem canal : 0.25%

ii. Bromophenol blue

: 0.25%

iii.Glycerol

: 30%

Preparation of 1X TAE buffer used in gel electrophoresis For 250ml of TAE 50X TAE

5ml

dd H2O

245ml

Electrophoresis was carried out on a horizontal slab gel apparatus (Bio-Rad, DNA sub Cell).

0.8% gel was prepared by boiling 0.8gm agarose in 98.0ml of dd water and 2ml of 50X TAE buffer.

The gel was poured in the gel case (in which the comb was assembled for wells) and allowed to solidify. DNA samples were loaded in wells and the electrophoresis was carried out at 60 volts. Then the DNA bands were visualized under ultraviolet trans-illuminator (Kodak EDAS, Japan).

3.2.2.9Gel Electrophoresis For sequencing 3.2.2.9.1 DNA recovery

Materials a) Elution buffer i.

0.5M EDTA pH 8.0

ii.

1M ammonium acetate

b) TE buffer i.

1M Tris-HCl pH 8.0

ii.

0.5M EDTA pH 8.0

c) EB buffer

3.2.2.9.2 Method • PCR-products (4 μl of PCR products + 3 μl of dye) and ladder (3μl) were run on the 1.5% agarose gel. Then it was stained with Ethidium bromide containing solution and the desired band was excised under UV light from the multiple non specific band (if any) from the agarose gel with a clean, sharp scalpel. The size of the gel slice was minimized by removing extra agarose.

• The gel slice was weighed in a colorless tube. 3 volumes of Buffer QG was added to 1 volume of gel (100 mg or approximately 100 μl).

• The tube was incubated at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, It was mixed by vortexing the tube every 2–3 min during the incubation.

• After the gel slice has dissolved completely, the color of the mixture was checked whether it was yellow (similar to Buffer QG without dissolved agarose).

• 1 gel volume of is propane was added to the sample and mixed by inverting the tube several times.

• A Mine lute column was placed in a provided 2 ml collection tube in a suitable rack.

• To bind DNA, the sample was applied to the Mine lute column, and centrifuged for 1 min at 10,000 x g.

• The flow-through was discarded and the Min Elute column was placed back in the same collection tube • 500 μl of Buffer QG was added to the spin column and centrifuged for 1 min • The flow-through was discarded again and the Min Elute column was placed back in the same collection tube • To wash, 750 μl of Buffer PE was added to the Min Elute column and centrifuge for 1 min. The column was let to stand 2–5 min after addition of Buffer PE, before centrifuging.

Fig3.9: Gel Extraction Process

• The flow-through was discarded and the Min Elute column was centrifuged for an additional1 min at 10,000 x g.

• The Mine lute column was then placed into a clean 1.5 ml micro centrifuge tube • To elute DNA, 20 μl of Buffer EB (10 mM Tris•Cl, pH 8.5) or water was added to the center of the membrane, the column was let to stand for 1 min, and then centrifuged for 1 min. the elution buffer was dispensed directly onto the center of the membrane for complete elution of bound DNA.(Fig3.9) • Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5.

• The purified DNA was analyzed on a gel, 3 volume of Loading Dye was added to1

volumes of purified DNA and the solution was mixed by pipetting up and down before loading the gel. 2 ul of lambda DNA was also run with the sample for comparing the concentration of purified DNA with known concentration with lambda DNA.

3.2.2.9.3 Re-amplification of isolated band 1. The isolated products were reamplified using their corresponding primers.

2. The PCR (25 µl) was carried out with 10X reaction buffer (2.5µl), 10mM dNTPs (0.5µl), 50.0mM MgCl2 (0.8µl), water (17.4µl), Taq polymerase (0.2µl), T 12AG Anchored dT (0.8µl), Arbitary primer(0.8µl) and 2µl of template DNA. 3. Template DNA was amplified in a thermal cycler programmed as follows: after preheating for 5 minutes at 940C; 40 cycles of 40 s at 940C (denaturation), 1:20 min at 380C (annealing) and 40 s at 720C (extension); and a final extension at 72 0C for 20 minutes followed by cooling to 40C. 4. The amplification products were checked by running on a 1.5% agarose gel.

3.2.2.10 Isolation of total RNA from Jute seedling: Total RNA was isolated from both the low temperature sensitive and tolerant plant seedlings.

The plant samples are: •

Seedling Stressed under Zinc

•

Seedling stressed under Chromium

•

Seedling Stressed under Lead.

3.2.2.10.1Handling RNA The precautions are followed listed below to avoid contaminating sample with RNases: • Always disposable gloves used and change frequently • Good microbiological technique is used to avoid contamination • Pipettes are specially reserved for RNA work •Aerosol-resistant pipette tips are used to reduce sample-to-sample contamination or reagent contamination.

• Non-disposable items are treated with RNase AWAY™ or similar product to remove

RNase contamination • RNase-free water was prepared by drawing the water into RNase-free containers, adding diethylpyrocarbonate (DEPC) to a final concentration of 0.01% (v/v).

Let stand overnight and autoclave. Use this water to prepare RNase-free solutions.

3.2.2.10.2RNA extraction protocol (GNTC/Phenol method) (Modification of Akop Seksanyan’s protocol, which in itself is a modification of Chomczynski and Sacchi’s protocol)

3.2.2.10.2.1Reagents (Sufficient volumes of all reagents are prepared in advance): -

GNTC (NB: 8µl of β-mercaptoethanol was added to every 1ml of GNTC solution before use!)

-

2M Sodium acetate, pH 4

-

DEPC-phenol (RNase-free phenol equilibrated in DEPC-water)

-

Chloroform: so-amyl alcohol (49:1)

GNTC Buffer (weights and volumes for 150ml given below) 4M Guanidine thiocyanate (70.9g / 150ml) 25mM Sodium citrate (1.1g / 150ml) 0.5% Sodium lauryl sarcosinate (2.5ml / 150ml) -pH to 7.0, filter-sterilize and store at 4ºC.

3.2.2.10.2.2Procedure Day One 1.

After homogenizing the tissue in the appropriate amount of GNTC/βmercaptoethanol solution, the following reagents are added (in the order written) and vortexes between each addition: a. 0.1V of 2M Sodium Acetate, pH 4.0 b. 1V of DECP-phenol c. 0.3V of Chloroform:Iso-amyl alcohol (the solution should turn milky by the end) 2.

The mix was incubated on ice for 20 minutes; two layers formed.

3.

While waiting for the layers to separate, the centrifuge is prepared for spin with ‘fast cool’.

4.

The mixture is Centrifuged at 10,000 rpm for 25-30 minutes at 4ºC.

5.

The top (aqueous) layer is carefully transferred into the new tubes.

6.

1V of isopropanol (2-propanol) and 3M Sodium Acetate (pH 5.2) are added to each tube.

7.

Solution incubated for overnight at -20ºC to precipitate.

Day Two 8.

Again, the centrifugation is done at 4ºC by doing a “fast-spin”.

9.

Samples are centrifuged at 10,000 rpm for 25-30 minutes.

10.

The supernatant is discarded very carefully. Add 1 ml 70% ethanol.

11.

Centrifuge at 10,000 rpm for 25-30 minutes at 4ºC.

12.

Supernatant is removed quickly.

13.

100µl of nuclease-free water is added to it..

14.

¼ volume of 10M LiCl2 is added and mix by tapping.

15.

The samples are dipped in Liq. Nitrogen for 2min.

16.

Then allowed the samples to thaw.

17.

Samples are centrifuged at 10000 rpm for 20min.

18.

the pellet is washed with 70% ethanol.

19.

Pellet is dried and dissolved in 30µl of DEPC treated water.

Sufficient care was taken during pipetting of the aqueous phase, and the interphase was completely avoided.

3.2.2.10.4Quantitation of RNA The following reagents were required for spectro-photometric determination of the concentration of RNA. 1µl of the purified RNA were added in 499µl of DEPC treated water in eppendorf tube. The reading was taken at the wavelengths of 260nm and 280nm. The ratio between readings of 260nm (OD260/OD280) was calculated.

Calculation: If the O.D is 1 then DNA concentration is 40µg/ml If the O.D is X then RNA concentration is 40xX µg/ml.

Dilution factor = 500/1 (say)

Concentration of RNA = O.D. x 40 x dilution factor µg/ml = Xx40x500 µg/ml = Xx40x500/1000 µg/µl =Xx20 µg/µ

3.2.2.10.5 Purity of RNA The ratio of the readings at 260 nm and 280 nm (A260/A280) provides an estimate of the purity of RNA with respect to contaminants that absorb in the UV, such as protein. Pure RNA has an A260/A280 ratio of 1.9–2.1. The spectrophotometer calibrate with the same solution.

3.2.2.10.6 Agarose Gel Electrophoresis of RNA The standard method was used to separate and identify RNA fragments through agarose gel electrophoresis.

•

Ultra pure agarose

•

MOPS buffer

•

RNA gel loading dye

•

Gel electrophoresis kit Preparation of Stock Solution (1 liter) of 10X MOPS Buffer:

MOPS (Mw=209.27) Na Acetate 0.5M EDTA (pH 8.0) pH to adjusted to 7.0 with NaOH This solution was made up to 1000ml with ddH2O

41.86g 4.1g 20.0 ml

Fig 3.10: Preparation of Stock Solution (1 liter) of 10X MOPS Buffer

3.2.2.10.7Preparation of 1.3% Agarose Gel (50ml) DEPC-treated water 10X MOPS Agarose

Formaldehyde To prepare 50 ml of 1.3% gel, 0.65g of agarose powder was weighed in conical flask, 5.0 ml of 10x MOPS (3-Morpholinopropane sulfanic acid) Buffer and 21.25 ml of DEPC-water was melted in microoven at 600C for 1.5-2 minutes. When the mixture was cooled at 40-45ºC temperature, 2.5 ml formaldehyde was added.

Sample preparation and gel electrophoresis:

3.2.2.10.7.1Materials RNA 1×MOPS (Electrophoresis buffer) Thermal cycler RNA-loading buffer (dye)

3.2.2.10.7.2Methods 1.0 µl of RNA sample was added to 3.0µl of dye and the samples were denatured

in a

thermal cycler at 650C for 20 min.

The gel was assembled in the electrophoresis system and samples were applied to the wells and electrophoresis was carried out at 60V for 45min.

Afterwards, photograph of the gel was taken.

Composition of RNA loading buffer: 10×MOPS

40µl

Form amide

200µl

37% Formaldehyde

70µl

Glycerol

20µl

DEPC Water

20µl

0.5 M EDTA

0.5µl

10 mg/ml Ethidium bromide

2.0µl

Add pinch of Bromophenol Blue

3.2.2.10.8Measurement of RNA Quality

Intact total RNA run on a denaturing gel will have sharp 28S and 18S rRNA bands (eukaryotic samples). The 28S RNA band should be approximately twice as intense as the 18S rRNA band (Figure 1, lane 3). This 2:1 ratio (28S:18S) is a good indication that the RNA is intact. Partially degraded RNA will have a smeared appearance, will lack the sharp rRNA bands, or will not exhibit a 2:1 ratio. Completely degraded RNA will appear as a very low molecular weight smear (Figure 1, lane 2). Inclusion of RNA size markers on the gel will allow the size of any bands or smears to be determined and will also serve as a good control to ensure the gel was run properly

3.2.2.11cDNA Preparation Following steps were followed for DNA preparation.

1. 350 ng of RNA was taken in PCR tube.

2. 1 all of 10 mM dot was added.

3. 0.2 all of primer was added.

4. DEPC treated H2O was added to make the final volume 13 ÎźL. 5. The mix was heated to 65oC and incubated on ice for 2 minutes 6. Following reagents were added afterwards: a. 5x 1st strand buffer 4 all b. 0.1 M DTT 1 all c. RNase In 1 all d. RT Enzyme 1 all The mix was subjected to following thermal profiling

3.2.3Semi Quantitative PCR

3.2.3.1Preparation of the Master Mix For semi quantitative Reverse Transcription PCR, PCR was carried out using prepared Can as templates for 30 cycles. For RT-PCR following reaction mix composition was used.

3.2.3.2Procedure: •

After combining cane and master mix, mix thoroughly, then take 15 ul out of mix and

put into each of two tubes. Now you should have three tubes per sample, two with 15 ul, and one with 20 ul. Label them somehow to differentiate.

•

The standard program with temperatures and times specific for those primers was used.

And the same master mixture was used 30 cycle amplification. It is important to select the appropriate number of cycles so that the amplification product is clearly visible on an agarose gel and can be quantified, but also so that amplification is in the exponential range and has not reached a plateau yet.

•

3ul of the 30 cycle samples were run on a 1.5% agarose gel and then stained with

ethidium bromide and checked on UV light to see if reactions worked and difference in expression level was observed by the difference intensity of the band.

•

A control PCR with acting primers that is a housekeeping gene was performed with all

the sample DNA to check for equal DNA synthesis and equal PCR amplification among samples. It leads to a prettier result.

3.2.4Bioinformatics Analysis tools Bioinformatics analysis was used in several purposes in this study including; •

Primer design

•

Alignment of sequences

•

Assembly of sequences

•

Determination of restriction cutting site on the sequence

•

To reverse transcribe nucleic acid sequence

3.2.4.1Primer 3 Primer3 is a widely used program for designing PCR primers. Primer3 can also design hybridization probes and sequencing primers. Consequently, primer3 has many different input parameters that you control and that tell primer3 exactly what characteristics make good primers for your goals. This program also offers the user to observe the criteria like primer size, melting temperature(Tm), self complementarily between primers, primer GC content, product size, etc.

3.2.4.2OligoAnalyzer Oligo Analyzer is a tool for oligonucleotide analysis in case of PCR and primer design. This programme includes analysis of hairpins, self-dimerization, hetero-dimerization and other parameter

3.2.4.3BLAST (Basic Local Alignment Search Tool) This tool finds similarity between biological sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches.

BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families. Blast include

o Nucleotide blast searches a nucleotide database using a nucleotide query.

Algorithms: blast, mega blast, discontinuous mega blast.

•

Protein blast searches a protein database using a protein query Algorithms: blast, psi-blast, phi-blast.

 blast searches protein database using a translated nucleotide query.  Tblastn searches translated nucleotide database using a protein query.  Tblastx searches translated nucleotide database using a translated nucleotide query.

3.2.4.4CAP 3 ASSEMBLY This tool is usually used to assemble the sequences to form contigs.

3.2.4.5Expasy Translate tool The ExPASy (Expert Protein Analysis System) is a proteomics server of the Swiss Institute of Bioinformatics (SIB) which analyzes protein sequences and structures. The server functions in collaboration with the European Bioinformatics Institute. Translate is a tool which allows the translation of a nucleotide (DNA/RNA) sequence to a protein sequence

3.2.4.6ClustalW Cluster is a general purpose multiple sequence alignment program for DNA or proteins. It produces biologically meaningful multiple sequence alignments of divergent sequences. It calculates the best match for the selected sequences, and lines them up so that the identities, similarities and differences can be seen. Evolutionary relationships can be seen via viewing Clad grams or Holograms.

3.2.4.7NEB cutter 2.0 NEB cutter is a program to cut DNA with any of the known restriction enzymes. It takes a linear or circular DNA sequence and finds large (>100 amino acids), non-overlapping open reading frames and the sites for all restriction enzymes that cut the sequence just once. It also shows the enzymes which could be used in a complete digest to excise each open reading frame that it finds. NEB cutter incorporates everything that is known about the methylation

sensitivity of any of the enzymes displayed when they overlap Dam or Dam sites as well as CpG, EcoK and Echo sites.

3.2.4.8Nucleic acid massager To reverse transcribe the nucleic acid sequence this tool was used.

Results Chapter 4 4.1 Soil Reading before pot analysis Table 4.1: Initial soil heavy metal reading

Sample

Parameter

Concentration (mgkg-1)

Leather industry

Chromium

280.6

Dyeing industry, buriganga

Nickel

35.1

Epz ,Savar

Lead

91.9

Shuvadda ,keraniganj

arsenic

1.3

Concentration (mgkg-1) 300 250 200 150

Concentration (mgkg-1)

100 50 0 Chromium Leather industry

Nickle

Lead

arsenic

Dyeing Epz ,Savar Shuvadda industry, ,keraniganj

Chart 4.1: Initial soil heavy metal reading The initial measurements of soil heavy metal concentration values are shown. The Hazaribagh soil was collected from the Leather tanning industry area. As expected, the chromium concentration is very high here. The dyeing industry soil is rich in nickel as it is an important ingredient of dye preparation. The EPZ dumping ground soil, on the other hand, has a high content of lead.

4.2 Metal Concentration on plant leaf after growth (two months) Table 4.2: Plant leaf heavy metal reading (two months)

Sample

Parameter

Concentration (mgkg-1)

Leather industry

Chromium

In plant leaf 30.18

Dyeing industry, buriganga

Nickel

35.1

Epz ,Savar

Lead

41.24

Shuvadda ,keraniganj

arsenic

0.2

Concentration (mgkg-1) 45 40 35 30 25 20 15 10 5 0

Concentration (mgkg-1)

Chromium

Nickel

Leather industry

Dyeing industry,

Lead

arsenic

Epz ,Savar Shuvadda ,keraniganj

Chart 4.2: Plant leaf heavy metal reading (two months)

4.3 Metal Concentration after plant growth (four months) Table 4.3: Final reading after plant growth for chromium Sample

Parameter

Leather industry

Chromium

Organ

Concentration

Leaf Stem Root

(mgkg-1) 25.3 20.7 29.4

Concentration (mgkg-1) 35 30 25 20

Concentration (mgkg-1)

15 10 5 0 Leaf

Stem

Root

Chromium Leather industry

Chart 4.3: Final reading after plant growth for chromium Table 4.4: Final reading after plant growth for nickel Sample Dyeing buriganga

Parameter industry, Nickel

Organ

Concentration

Leaf Stem Root

(mgkg-1) 13.8 9.15 19.4

Concentration (mgkg-1) 25 20 15

Concentration (mgkg-1)

10 5 0 Leaf

Stem

Root

Nickel Dyeing industry, buriganga

Chart 4.4: Final reading after plant growth for nickel Table 4.5: Final reading after plant growth for lead

Sample

Parameter

Epz ,Savar

Lead

Organ

Concentration

Leaf Stem Root

(mgkg-1) 28.25 16.45 12.6

Concentration (mgkg-1) 30 25 20 15

Concentration (mgkg-1)

10 5 0 Leaf

Stem

Root

Lead Epz ,Savar

Chart 4.5: Final reading after plant growth for lead

The uptake of chromium nickel and lead by the plants are shown respectively here. The content in root, leaf and stem are shown separately. The accumulation is maximum in root for chromium and nickel. Whereas the accumulation of lead is maximum in leaf.

4.4 Final reduction of heavy metal in soil Table 4.6: Total reduction of heavy metals after plant growth Sample

Chromium nickel Lead

Initial value

Final value

Reduction

Reduction

(mgkg-1)

(mgkg-1)

(mgkg-1)

%

280.6 35.1 91.6

189.6 16.35 51.6

91.0 18.75 40.0

33% 54% 44%

by

300 250 200 Initial value (mgkg-1)

150

Final value (mgkg-1)

100 50 0 Chromium

nickel

Lead

Chart4.6: Total reduction of heavy metals after plant growth The reduction of chromium, nickel and lead are given discretely here. The rate of reduction is different for these three. Among them the rate of reduction is maximum for Nickel whereas according to quantity the maximum reduction is found here for chromium (91Mg/kg)

Reduction by %

Chromium, 33% Lead, 44%

Chromium nickel Lead nickel, 54%

Chart 4.7: Total reduction of heavy metals (%)

4.5Regression analysis of content reduction Formula: Y=a+b1x Regression analysis is done through the use of software SPSS 12.0 for windows XP. As here is one Independent and one Dependent variable thus linear regression model is used.

Here the independent variable is initial soil heavy metal content and the dependent variable is final soil heavy metal content.

Variables Entered/Removed(b)

Table 4.8: All requested variables entered.

Model 1

Variables Entered Initial(a)

Variables Removed .

Method Enter

Model Summary

Table 4.9: Predictors: (Constant), Initial Model 1

R 1.000(a)

R Square .999

Adjusted R Square .998

Std. Error of the Estimate 3.60313

ANOVA(b)

Table 4.10: Predictors: (Constant)

Model 1

Regressio n Residual Total

Sum of Squares

df

Mean Square

F

Sig.

16754.392

1

16754.392

1290.529

.018(a)

12.983

1

12.983

16767.375

2

t

Sig.

-3.175

.194

35.924

.018

Coefficients (a)

Table 4.11: Dependent Variable: Final

Model 1

(Constant ) Initial

Unstandardized Coefficients

Standardized Coefficients

B

Std. Error

Beta

-10.799

3.401

.712

.020

1.000

4.6 Physiological Properties of jute plant on heavy metal treatment •

Normal stem and leaf structure.

•

Regular behavior to abiotic stress.

•

Regular behavior to plant disease.

•

Normal watering needed.

4.6.1Significant changes in properties.

•

Low Diameter of stem.

•

Delayed flowering Period. (5 month)

•

Low Height(4.5-5 fit, average 5.5-6 fit)

Fig4.1: Control Plant

Subject plant

4.7 PCR with ZIP 1 and ZIP 3 gene

Primers for ZIP 1 gene Forward 5’ GMTGCATMWCHCWGG3’ Reverse 5’ GGATTCATAAAATCA3’ 450 bp

Fig 4.2: PCR for ZIP 1 gene

PCR with Primer for ZIP 3 Gene

Primer for ZIP 3 gene Forward 5’ CAGGTWTTGGAGKTG 3’ Reverse 5’ CCAGCACCAAGAAGG 3’

600 bp band

Fig 4.3: PCR for ZIP 3 gene

Fig: 4.2 and 4.3 expresses the PCR result ZIP 1 and ZIP 3 gene with degenerate primer. The desired band size for ZIP1 primer is 450 bp whereas For ZIP 3 it is around 600 bp. The desired band size is calculated from the ‘Primer Blast’ tool of NCBI.

4.8 Semi Quantitative PCR Preparation 4.8.1 Plant growth on plate • Chromium(Cr) Stress • Lead(Pb) Stress • Zinc(Zn) Stress • Control

Fig4.4: Lead Stress Stress Concentration 100 农g/kg

Fig4.5: chromium Stress Stress concentration: 100 农g/kg

Fig4.6: Zinc Stress Stress Concentration 100 农g/kg

Fig4.7: Control Plant (without Stress) 4.9 RNA Isolation 4.9.1Gel Analysis

28s RNA 18s RNA

Fig 4.8: RNA quality analysis

4.10 Semi Q PCR for ZIP 1 primer 4.10.1 Time Interval •

After 16 hours.

•

After 24 hours.

•

After 36 hours.

Table 4.12: Different samples for gene expression study Species

C.olitorius

Sample

Age of Seedling + time before RNA isolation

Pb16

4 days uninfected + 16 hours after infection

Pb 24

4 days uninfected + 24 hours after infection

Pb 36

4 days uninfected + 36 hours after infection

Cr 16

4 days uninfected + 16 hours after infection

Cr 24

4 days uninfected + 24 hours after infection

Cr 36

4 days uninfected + 36hours after infection

Zn 16

4 days uninfected + 16hours after infection

Zn 24 Zn 36

4 days uninfected + 24hours after infection 4 days uninfected + 36 hours after infection

Control

Pb

Pb

Pb

Cr

16 h

24h

36h

16h

Ladder

Cr

Cr

Zn

Zn

Zn

24h

36h

16h

24h

36h

Fig4.9: Semi Q PCR for ZIP 1 In the Semi quantitative PCR for ZIP 1 (Fig4.9) the result found positive for chromium stress (36 hour) and zinc stress (16, 24 and 36 hour).For control and other stress conditions, no PCR band is found.

4.11Semi Q PCR for ZIP 3 primer 4.11.1Time Interval •

After 16 hours.

•

After 24 hours.

•

After 36 hours.

Control

Pb

Pb

Pb

Cr

Cr

Cr

16 h

24h

36h

16h

24h

36h

Ladder

Zn

Zn

Zn

16h

24h

36h

Fig4.10: Semi Q PCR for ZIP 3

In the Semi quantitative PCR for ZIP 3 (Fig4.10) the result found positive for chromium stress (36 hour) and zinc stress (16, 24 and 36 hour).For control and other stress conditions, no PCR band is found.

4.12 Confirmation of semi Q PCR with Actin primer The result of actin PCR with cDNA found positive for all stressed condition (Fig: 4.11).

This is a house keeping gene primer and authenticates the PCR result of Semi quantitative PCR. Control

Pb

Pb

Pb

Cr

Cr

Cr

Zn

Zn

Zn

16 h

24h

36h

16h

24h

36h

16h

24h

36h

Fig4.11: PCR with actin primer

Discussion Chapter 5

The current study can be divided into two parts (i) Pot Analysis (ii) Molecular Biology Analysis.

The vision of this study was to analyze jute characteristics to use it as a tool to reduce environmental pollution. In case of unplanned industrial areas, heavy metal pollution is very acute. There are some plants that can uptake these heavy metals from the soil (Hassinen 2009). Thlaspi caerulescens is a plant like this (Hassinen 2009). Besides, there are several other plants that have shown the same type of activity e. g., Brassica and Arabidopsis. From that point of view this experiment is an approach to find similar activity in jute, the most common and popular commercial plant in Bangladesh.

5.1 Pot analysis The Pot analysis is a Part of the practical study to check the capacity of jute. The Work area of this was Bangladesh Jute Research Institute. Soil samples were collected from the three industrially polluted areas of Dhaka.

•

Hazaribagh leather industry area: Chromium rich soil because of effluent dumping.

•

Dying industry area close to Buriganga River: Rich in nickel that is ingredient of dye.

•

These

Dumping ground of Epz, Savar: Soil containing high amount of lead.

soils

were

subjected

to

heavy

metal

analysis

with

atomic

absorption

spectrophotometer. Chromium, Nickel and Lead were found in significant amount compared to general soil of Bangladesh (Table 4.1). These three soil samples were placed in six different pots for duplication. Seeds were then sowed in equal amount in each pot. Growth was analyzed with proper monitoring. The growth details were noted every week.

During the middle of the growth period of jute (After two months) the plant samples from the pot were analyzed in AAS to check whether they were up-taking heavy metal. The result indicated that jute plants are able to uptake heavy metals present in the soil samples (table 4.2). The same procedure was continued until the end of the growth period of jute. The atomic absorption analysis was done again on the soil and different plant organs. The objective was to find the rate and amount of uptake in different organs of plant as well as the reduction of content of heavy metal in the soil. The result indicated that in case of chromium and nickel, the accumulation was maximum in roots (table 4.3, 4.4). But in case of lead the accumulation was highest in the leaf (table 4.5). As most of the chelator molecules are in the root and root is the primary organ for the absorption of metal from the soil thus metal accumulation is expected to be maximum in the roots (Hassinen 2009). In case of lead it is possible that the metal transporters are abundant in this particular tissue (root) and the chelator concentration could be less compared to other tissues.

However, when reduction is considered, all the (three) samples showed significant reduction (more than 30%) and in terms of percentage of reduction, the prizewinner is nickel followed by 44% reduction (for Pb) followed by 33% for chromium. Similar studies on Populus and Brassica (Taghavi et al 2005) showed similar pattern and the prizewinner in this case, was cadmium. It seems puzzling that in the current study, jute plants did not show any significant reduction in cadmium concentration (data not shown).This may be because of its low content in sample soil comparative to others (9.1 mg/kg) (Saunders 1983). Another possibility is the cation exchange capacity (CEC) of plants. Perhaps the transporter molecule needed for exchange is not present in jute. Thus successful uptake of cadmium is not possible for jute.

5.2 Statistical analysis To address whether the reduction of heavy metals in the subject sample soil is significant or not, linear regression analysis was performed that explains the relationship between the independent and dependent variable. Here, the initial values were considered as independent variables.

The R square value is 0.999 which indicates that the equation express 99% of the relationship between the independent and dependent variables (Table 4.7). The F value of the test is 1290.529, which is greater than the table value (table 4.8). It interprets that the null hypothesis should be rejected and the alternative accepted. According to standard regression model the null hypothesis indicates weak or no correlation between the two variables.. So the alternative hypothesis is, these two are correlated. As the alternative one is accepted, thus it can be predicted that there is a strong correlation between this two variables. The level of correlation is expressed in table 4.9. The B value is 0.712, so for 100% change in the independent variable, there will be 71.2% change in the dependent one. Standard error expresses the chance of error here which is 2 %( 0.2), which is negligible. From this statistical analysis the amount of reduction is significant.

5.3 Physiological property analysis

During the pot analysis study, some physiological changes of jute have been monitored.

Although there is no certain standard set of properties that can be universally applied to estimate the changes, some irregular changes have been monitored (Fig 4.1). The diameter of the stem was found to be comparatively small with respect to control plants. The flowering

time was also found to have been delayed by one-month compared to plants growing under normal conditions. Besides these, other characteristics of normal jute plant were present in the subject plants (Fig 4.1).

5.4 Molecular analysis 5.4.1 PCR analysis The major question that needs to be addressed is the molecular mechanism behind the phytoremediation property. Previous studies showed that, most common family of genes that are responsible for heavy metal uptake is ZIP family genes (Grotz et al 1998). There are six of them found in Arabidopsis that which are associated with uptake and transport of zinc and other heavy metals (Grotz et al 1998). From the available sequences in the NCBI database, degenerate primers were designed to amplify ZIP1 and ZIP3 genes that are associated with Zn and other metals (Hassinen et al 2009). PCR was carried out with these primers using conventional PCR protocol with template DNA isolated from the subject plants Gel electrophoresis gave the desired band size of DNA (Fig 4.2, 4.3).

5.4.2 Semi quantitative PCR analysis

For Semi quantitative PCR analysis RNA was isolated by GNTC method .Although it takes more time compared to conventional TRIZOL method, this technique is more efficient in terms of RNA quality and quantity. After the isolation of RNA the concentration was analyzed through the use of nanodrop spectrophotometer and gel electrophoresis was also used (FIG: 4.8). The ratio of absorbance at 260 to 280 nanometer ranged between 1.674 to 1.863, which indicated good quality of RNA (for good quality of RNA the range should be 1.8-2.0). However, as degraded but highly purified RNA may also give such good value; the

quality of isolated RNA was further checked by electrophoresis. Distinct band of ribosomal RNAs (FIG: 4.8) indicated presence of un-degraded RNA in samples.

False positive result found from a number of intrinsic and extrinsic factors are intimately linked to the technique. Factors which influence are the systems being compared, experimental designs, appropriate internal controls, criteria for picking bands, reaction setup, type of pcr tube, type of thermocycler, variation in the quality of RNA samples, primers.

Superscript Reverse Transcriptase, Taq polymerase and of course the training and experience of a researcher, all can contribute greatly to the rate of false positives (Liang P., 1998).

Therefore, all the reactions parameters were strictly kept the same. Particularly, each of the isolated RNA samples were aliquoted to a number of micro-centrifuge tubes to avoid repeated thawing before use, as this severely damages RNA. The same thermocycler was used for all RT-PCRs in this experiment.

Later for the three different samples with three time intervals, semi quantitative PCR was performed. The motive of this operation is to check the expression level of ZIP 1 and ZIP 3 gene under the heavy metal stressed condition. For this PCR the cycle number was thirty. The same procedure was applied for ZIP 1 and ZIP 3 genes with two different primer pairs and both of these primer pairs showed same expression pattern. In case of chromium, ZIP 1 and ZIP 3 showed expression only at 36 hrs.

The size of the amplified product found from zinc stressed samples was as expected (600 and 450 bp ). The ZIP family proteins are accumulators and transporters of zinc as well as for other heavy metals (Taghavi et al 2005). For zinc, the band found in the 16 hours time interval is very faint. The intensity of band increased with 24 and 36 hours time interval. But for the lead stressed sample, no band was found. It could be possible if there are other accumulator or transporter molecules present to uptake lead. Moreover, ZIP protein family may be a transporter protein for lead but it may be less sensitive to it than zinc. This hypothesis is more reliable because in case of chromium no band was found for 16 and 24 hours time interval. But after 36 hrs of incubation, expression was detected. It is likely that Zn and Cr induces ZIP genes through different regulatory circuits and as Cr induced expression takes longer period and as the level of expression is less compared to Zn-36 sample, it is likely that ZIP genes are major components for Zn phytoremediation pathway for jute. And for Cr, ZIP genes may have a minor role to play in this pathway.

To authenticate the result the actin gene was amplified as a control. This is a primer of house keeping genes and is always in the expressed form. The result of the PCR is also positive for all three samples (Fig: 4.11) which confirm the value obtained for the semi quantitative PCR for ZIP 1 and ZIP 3.

5.5 Conclusion This study gave an insight into the physical property as well as molecular biology of jute to find its behavior as a phytoremediator.

The results indicate that jute has a good

phytoremediation property. Further study in this area may unravel new information as well as may establish jute as a very good phytoremediator plant.

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Appendix Chapter 7 APPENDIX COMMON LABORATORY BIOCHEMICAL RESEARCH

APPARATUS

USED

IN

NAME

DESCRIPTION

SOURCE

Eppendorf tubes

Size 1.5 ml, colorless

Fisher Scientific, USA.

Micropipette tips PCR tubes Gloves Glass wares Micropipettes Gel kit and power Autoclave machine Micro-centrifuge DNA thermal cycler

Vol. range 0.1- 10µl, 0.5-

Labsystems, Finland.

200µl and 200-1000µl Size 0.5 ml Perkin Elmer Cetus,USA. Disposable Vinyl medical gloves,USA. Pyrex brand USA 0.5-10 , 0.5-10µl,5-20µl, Labsystems, Finland.

10-100µl, 50-1000µl BRL, Life technologies Inc Model H5 Horizontal USA. E Hirayama Mfg Corp.

Model HL-42A Japan.

Eppendorf centrifuge5415C Germany GeneAmpPCR System

Vortex Reciprocal shaking bath Circulating water bath

Model 1190-1 Model 25 Model260

UV transilluminator

Model T2201

H

P meter Eppendorf tube rack Balance machine Incubator shaker

THE

Innova 4300

Centrifuge tube Refrigerated super speed Model RC 5B

9700, Applied Bio-systems.

Labline Instruments,USA. Precision Scientific.

Precision Scientific.

SIGMA Chemical CO., USA. Orion Research, USA. SIGMA, USA. Mettler AE 100, Switzerland New Brunswick Scientific Nalgene.

USA. Sorvall

centrifuge Freeze drier Refrigerator Microwave Biodoc System Spectrophotometer Nanodrop

Model 12525 -80째C Kodak EDAS 290 Analytikjena Specord 50 Nanodrop 100 spectrophotometer

VirTis Comp.USA. Barka Profiline Emerson, Korea.

Japan Germany