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For better yield irrigation water must be free from soluble salts and from concentration of specific chemical substances that may hazard to soil with respect to salinity, sodicity, alkalinity and toxicity. Water analytical data which helpful to predict the magnitude of the effect of irrigation water on soil and plant growth under specific environmental circumstances so irrigation water must be analyzed for EC, pH, Na+, Ca⁺⁺, Mg⁺⁺, Cl^-, CO₃–, HCO₃-  and SO₄.^– 

6.1. Quality of irrigation water

Asia per capita water availability has fallen by around 80 per cent during last five decades.

Suitability of waters for a specific purpose depends on the types and amounts of dissolved salts.

Some of the dissolved ions such as NO₃ are useful for crops.

Water required for irrigation of cropped land is being degraded in terms of quantity and quality due to fast increasing demand for its use. Furthermore, the soil quality and crop productivity is linked to the quality of the water available for irrigation. In general, evaluation of irrigation water quality should focus on salt content, sodium concentration, the occurrence of nutrients and trace elements, alkalinity, acidity, and hardness of the water.

Introduction

Irrigated agriculture is dependent on an adequate water supply of usable quality. The water quality used for irrigation is essential for the yield of crops, maintenance of soil productivity, and protection of the environment as well. The quality of irrigation water is defined with respect to its effect on plant growth, soil properties, soil biological equilibrium, and irrigation technology. Conceptually, water quality refers to the characteristics of a water supply that will influence its suitability for a specific use, i.e. how well the quality meets the needs of the user. Specific uses have different quality needs and one water supply is considered more acceptable (of better quality) if it produces better results or causes fewer problems than an alternative water supply. For example, good quality river water which can be used successfully for irrigation may be unacceptable for municipal use without treatment to remove its sediment load. Similarly, snowmelt water of excellent quality for municipal use may be too corrosive for industrial use without treatment to reduce its corrosion potential.

1.1 Introduction

Water scarcity is seen as a major constraint to intensify agriculture in a sustainable manner as an attempt to meet the food requirements of a rapidly growing human population. The ever-increasing human population, climate change due to increased emissions of greenhouse gases (GHGs), and intensification of agriculture, are putting severe pressure on the world’s two major non-renewable resources of soil and water, and thus pose a big challenge to produce sufficient food to meet the current food demand. The present world population of 7.3 billion people is predicted to grow to over 9 billion by 2050, with the majority of this population increase occurring in developing countries, most of which already face food shortages. A 70% increase in current agricultural productivity will be required to produce sufficient food if these human population growth predictions prove to be correct. In this context, concerted efforts are being made globally to improve the effectiveness of water which will be used for enhancing the production of irrigated crops. Additionally, efforts are also being made to improve water harvesting and water conservation in rain-fed agriculture.

The injudicious use of saline/brackish water is all too often associated with the development of soil salinity, sodicity, ion toxicity, and groundwater pollution. Because of these negative effects, it is important to have a better understanding of exactly how the quality of water influences the management of irrigated agriculture, especially in arid and semi-arid regions.

Irrigation

Irrigation is defined as the artificial application of water to the soil for the purpose of crop production in supplement to rainfall and ground water contribution.

Time of Irrigation

The time schedule which indicates when irrigation water is to be applied and the quantity of water to be applied at that time is called irrigation schedule. There are several approaches for scheduling irrigation:

1. Soil moisture depletion approach based on the available soil moisture in the root zone.
2. Climatological approach based on the amount of water lost by evapotranspiration.
3. Combination approach based on soil moisture depletion and climatological approaches.
4. Critical stage approach which indicates the growth stages at which moisture stress leads to yield loss. These stages are known as critical period or moisture sensitive period. In cereals and millets panicle initiation and flowering stages are moisture sensitive stages. In pulses and oilseeds, flowering and pod development stages are critical stages. In sugarcane active tillering or formative stage is critical and in cotton boll development is the critical stage.

Technical Term            Definition

Infiltration                    Infiltration is movement of water from the surface into the soil or ground.

Introduction

Irrigation is the artificial application of water to soil or land to assist in the growth of crops, vegetation, or landscaping. It is a crucial agricultural practice used to supplement natural rainfall and provide controlled and consistent moisture to plants when needed. Irrigation systems can vary in complexity, from simple methods like watering cans and hoses to more advanced systems like drip irrigation, sprinklers, and flood irrigation.

The primary purpose of irrigation is to ensure that plants receive an adequate and consistent supply of water, especially in regions with insufficient or irregular rainfall. This helps promote healthy plant growth, increase crop yields, and prevent drought-related crop damage. Irrigation is not only used in agriculture but also for landscape maintenance, recreational fields, and other applications where controlled watering is essential.

OBSERVATION

The examination of the IRS - 1A imagery under raw and the digitally enhanced modes vividly display heavy concentration of suspended sediments in certain sectors along the east coast of Tamil Nadu. Such pockets of suspended sediments are seen as long and linear bodies trending parallel to the coast to the strike length of 20 - 30 km and width of 5 - 10 km. Such Phenomenon of suspended sediments concentration are strikingly observed in two sectors. One in between Tuticorin harbour and Vaippar river mouth and the other just at the mouth of Ponnaiyar river along Cuddalore-Pondicherry coast. Both of them are observed along conspicuous (Fig.1& 2) concave coasts.

Introduction

Agriculture as a primary sector in India is mostly production oriented. Global agriculture is experiencing significant changes in terms of technological and digital revolution, rapid urbanization, faster growth in middle income groups, changing food habits, market oscillations, and above all, climate change. During the last 50 years, India has recorded 4 to 10-fold increase in agriculture and food production(food grains-290 MT, horticultural produce -315 MT, milk production nearly 180 MT, and fish production approaching 12 MT), rendering the country as the second largest agrarian economy in the world (Singh, 2019). Despite the excellent progress, the country is home to over one third of the world’s small and marginal farm holders, with scattered land holding. Eighty f ive percent of the farmers are small and marginal. On the other hand, world’s population is growing and to meet the growing food demand there is a need to increase agricultural output by at least 60 percent globally, and 100 percent in developing countries (FAO, 2014a)

Introduction

Agriculture has historically been the primary source of income in India. It has the potential to alleviate poverty, increase income, and enhances food security for 80% of both the world’s poor and people who reside in rural areas primarily work in agriculture. One of the most effective strategies to eradicate extreme poverty, increase shared prosperity, and feed an estimated 9.7 billion people by 2050 is agricultural development. Compared to other modes, the agro - based sector’s progress is two to four times more successful in enhancing the earnings of the poorest people (www.worldbank.org).

Nearly 5 billion hectares, or 38% of the world’s land surface, is used for agriculture. Around one of this is devoted for farming, and the remaining two thirds are pastures and meadows for grazing animals. Over 10% of cropland is assigned to permanent crops like cocoa plantations, oil palm plantations, and fruit tree orchards. Yet another 21% of land is primed for irrigation, a crucial agricultural land management technique.

Introduction

Urbanization, a vital global phenomenon, refers to the process of city establishment and growth; the term commonly connotes population increase in the city from internal growth and immigration as well as their spatial expansion (Revi, 2007). Recent developments, uncontrolled population growth have been linked with the process of urbanization. Urban areas cover 3% of the global land but it gives shelter to nearly 26% of the global population (U.S. De, 2009). Increase in population growth and industrialization is posing threat to natural resources such as land air and water. Anthropogenic factors have led to change in land use pattern, high energy consumption, high level of generation of waste etc. As per UN survey, by 2050, approximately 70 percent of the global population will be living in cities (UN, World Urbanization Prospects, 2014). Unplanned growth is damaging the existing ecosystems along with that causing climate change and loss of biodiversity.

12.1. Origin and distribution

• Native of Persia and neighbouring tracts of Western and Southern Asia

• In India, North Guj arat, Raj asthan, Haryana and Bihar are the maj or states.

• Husk and peel - exported to USA, Germany, UK and France

12.2. Economic importance

• Psyllium from Greek meaning flea, in reference to colour, size and shape.

• Economic part - Seed coat, husk used in indigenous medicine. Seeds contain glucoside and tannin.

• Works as an anti-diarrhoea drug.

• Seed - Pentose, a sugar is isolated.

The cotton aphid: Aphis gossypii: Aphididae

Symptoms of Damage

Both nymphs and adults cause the damage by sucking the plant sap from shoot tips, pods and leaves. Severe infestation may result in complete drying of affected parts. Attacked leaves curl up, wither and fall. These are yellowish green or brown coloured aphids. The total life cycle from first instar nymph to adult occupies an average of 8-10days. It reproduces parthenogenetically and viviparously. The female may produce 10-30 young ones in a life span of 10-12 days. The nymphs transform into adult in 5-8 days after passing through four moults. 

The geographical area of the union territory of Andaman and Nicobar Islands is 8, 24,900 ha with 86.9 % of it under reserve and protected forests. The humid tropical climate that prevails in the Andaman and Nicobar islands provides a unique opportunity to grow various crops. Owing to a number of factors, hardly 4.8% of the area is under cultivation. Maximizing the productivity and farm income mainly depends on effective management of natural resources like rain, soil, land as well as labour present in the Island ecosystem.  On an average, the Islands receive 3074 mm of rainfall distributed over 8 months. The rainfall is intense from June to September and may have even up to 30 rainy days per month sometimes, however, from January to April, the rainfall is scanty or absent, particularly in March. The undulating terrain results in severe water crisis during the period which is aggravated by higher evapotranspiration. The Islands receive south-west monsoon from May to September and north-east monsoon during October to January. The topography of A&N Island is undulating with a stiff terrain exposed to soil erosion due to heavy rainfall.  The crop growing season can be divided in to two major seasons viz., wet 
and dry seasons.

It 9001:2000 Means customer satisfaction and continuous improvement

ISO (International Organization for Standardization) is a worldwide federation of national Standards bodies from each country. The object of ISO is to promote the development of Standardization and related activities in the world with a view to facilitating international exchange of goods and services, and to developing cooperation in the spheres of intellectual, scientific, technological and economic activity. The results of ISO technical work are published as International Standards.

Quality management Systems (QMS) certification as per ISO 9001 : 2000 is an important opportunity that can enable the companies acquire the label of an enterprise caring for the customer needs and improve their economic performance at the same time. QMS provides an organization a systematic structured documented and effective approach to consistently maintain the product quality meeting customer requirements.

Hunter and Mohler discovered the isozymes. In 1959 Markert and Moler introduced the concept of isozymes, which they defined as the different molecular forms in which proteins may exist with the same enzymatic specificity (Buth 1984). This means that different variants on the same enzymes have identical or similar functions and are present in the same individual. As such, their importance for understanding gene action in development and differentiation was exploited during the1960s in both animals and plants. Nevertheless, isozymes played a minor role in research on plant biochemistry until 1966 when genetic polymorphism for isozymes within the same population was discovered (Stebbins 1989, Wendel 1989). That revealed the possibility for population genetics to make precise quantitative estimates of genetic variability based upon one parameter of the molecular structure of the primary products of the genes themselves.

Plant population genetics were not long in following their zoological colleagues and the investigation of both animals and plants increased explosively. This application, however, necessitated a partitioning of the isozyme concept, since the relevant isozyme subset, defined by Prakash (Buth 1984) as the variant proteins produced by allelic forms of the same locus, to avoid the now common confusion with isozymes which are the various polymer produced from monomers specified by different loci. The development of genetic distance or similarity coefficients (Lukasová 1985) allowed the summarization of isozyme data for intersample comparisons and so quantified isozyme data were soon applied to comparative studies of taxa.

Polysomes consist of two or more ribosome traversing a strand of mRNA, translating the nucleotide sequence into the corresponding amino acid sequence. Usually at any time, many ribosomes are translating an mRNA simultaneously forming a structure called a polyribosome or polysomes. One reason for isolating polyribosomes is to resolve them on sucrose gradients to determine the extent of ribosome loading. The second main reason is to provide a source of mRNA for in vitro translation.

Polyhydroxyalkanoates (PHAs) are diverse biopolyesters synthesized by many bacteria as carbon and energy reserved materials under unbalanced growth conditions. Sand-dunes have low nutrient contents especially nitrogen (N) and phosphorus (P) thus could harbor potential PHA accumulating bacteria. There is no report of isolation of PHA producing bacteria from the sand-dunes environment.  Sand samples of rhizosphere and non-rhizosphere regions of the coastal sand-dunes were processed on Nutrient Agar and TYG-Agar media for isolation of heterotrophic bacteria.

Introduction

Solanum trilobatum L., an important medicinal plant, belonging to Solanaceae is widely distributed in South Asia and has been used in Indian traditional medicinal systems of Siddha and Ayurveda. The members of Solanaceae accumulate steroidal alkaloids based on a C₂₇ cholestane skeleton, e.g. solasodine and tomatidine. Recently, mixtures of solasodine glycosides have been successfully used for the treatment of certain human skin carcinomas (Cham, 1994). Solasodine glycosides have antineoplastic activity in cell culture, animals and in humans (Cham and Daunter1990). It has been demonstrated that specific endogenous lectins which are present on the plasma membranes of susceptible cells recognize and bind the sugar moiety of the solasodine glycosides and the glycosides are subsequently internalized and cause cell death (Cham and Daunter1990). Hence the present study was undertaken to isolate the solasodine from S. trilobatum.

Introduction

Peripheral blood mononuclear cells (PBMCs) consist of chiefly of lymphocytes and monocytes. Purified PBMCs are used in vitro to evaluate a variety of functions of lymphocytes viz; a) proliferation to mitogenic (PHA, Con-A) stimulation, b) monitoring of prior sensitization in antigen recall assays by scoring lymphocyte proliferation, c) immunophenotyping for surface markers as well as intracellular molecules in monocytes and lymphocytes etc. Activation of monocytes/macrophages by small molecules, cytokines and pathogen components can also be monitored. PBMCs can also be used for a variety of structural and functional studies for addressing issues in human immunology such as scoring for apoptosis and production of cytokines as well as other mediators in vitro.

Background information

Blood is a liquid mixture comprised of plasma, cells and proteins. The liquid portion, plasma, makes up half of the blood volume, and cells the other half. Isolation of lymphocytes is a common procedure in immunology and cell biology that involves separating and collecting lymphocytes.

Introduction

Photosynthetic bacteria are autotrophs and anaerobes. These bacteria produce Hydrogen sulphide. These are predominant in the bottom of stagnant water enriched with mud.

Requirements

1. Mud sample
2. Enrichment medium of Photosynthetic bacteria or salt solution (1.0g/lt. of (NH⁴)₂SO⁴ 1 g; K²HPO⁴, 1 g; MgSO⁴,0.2g; NaCl, 5g; pH 7.2)
3. Sterilized bottle with plug
4. Fluorescent tube

Isolation of Protoplasts

Protoplast is the living material of the cell however an isolated protoplast is the cell from which the  cell wall is removed. In plant breeding programme many desirable combination of characters could not be transmitted through the conventional method of genetic manipulation. Higher plants that could lead to the genetic process involving fusion between the subsequent developments of a product to a hybrid plant is known as somatic hybridization. Plant protoplasts can be isolated from cells by two methods:

Introduction

The actinomycetes are gram positive, non-motile, non-sporulating and nonacid fast bacteria. Sometimes these are called ray fungi and are connecting link between the bacteria and the true fungi. They possess a very fine vegetative mycelium that does not fragment into bacillary or coccoid forms and therefore their colonies on agar are commonly cohesive or tough in consistency. In many soils, the actinomycetes accounts from 10 to 25% of the total count on agar plates. Their number is higher in soils of low moisture content than in wet soils. They are responsible for degrading more resistant carbon material in soil like humus. They are also important flora of manure heaps. Most of these, particularly Streptomyces, produce antibiotics and so are of a great value

Introduction

Soil is the natural habitat of a number of microbes viz. bacteria, fungi, actinomycetes, algae and many viruses. Out of these, bacteria are morphologically very simple as made up of just one cell of small size, therefore can multiply rapidly. Each viable bacterium at appropriate dilution can develop into a colony. Hence by counting the number of these colonies which develop on a selective growth medium, the number of viable cells of bacteria present in soil can be ascertained. Since the number of the bacteria present in soil is very high, the soil suspension is to be sufficiently diluted to reduce the number of colonies and to prevent their overlapping. The number of colonies formed from a diluted sample multiplied by the extent of dilution (dilution factor) gives the number of viable cells present

Introduction

Fungi constitute an important part of microflora of normal soil. In soils, fungal propagules vary from 2 x 10⁴ to 1 x 10⁶ per gram soil. The propagule being considered as fungal hyphae, spore, and hyphal fragments which is capable of giving colony on the plate. Most soil forms have a filamentous organisation capable of reproduction by means of spores. Their development is especially favoured by soils having acidic reactions. Soil fungi has significant role in organic matter decomposition, as a plant pathogens and in the interaction with other soil microorganisms. Agar plate count method is commonly used as a measure of fungal population in soil.

1. Deoxyribo Nucleic Acid (DNA)

DNA is a genetic material, originally confined to nucleus of a cell. In plants, some DNAs are also present in mitochondria and chloroplasts. DNAs are vital macro molecules having high molecular weight.

Isolation of plant DNA is cumbersome or difficult primarily because of the presence of a number of secondary plant products. Secondly, a protocol, which is found suitable for isolation of DNA in one plant, may not work in other. The efficiency of DNA extraction depends on many factors, such as sample material, ionic conditions of the extraction medium and type of lysing agent used etc.

There are two methods, one developed by Dellaporta et al. (1983) and the other by Murray and Thompson (1980) which are most commonly used for extraction of DNA in a number of plant genera.

Murray and Thompson’s (1980) method is described here below with minor modifications.

Cell culture is the most convenient and widely used system for the isolation and propagation of many viruses. Either primary cell culture or established cell lines can be used to isolate the suspected viruses. One can normally get 10-100 virus particles per cell inoculated. Many details of the infection process are controllable to a high degree (including cell density, age, multiplicity of infection (M.O.I.), medium composition, cell type and length and temperature of incubation). Many cell types from different species can be susceptible to a particular virus and vice versa. For replication to occur, the cell type used must be permissive for a particular virus under study.

Protoplasts are cells which have had their cell wall removed, usually by digestion with enzymes. Cellulase enzymes digest the cellulose in plant cell walls while pectinase enzymes break down the pectin holding cells together. Once the cell wall has been removed the resulting protoplast is spherical in shape. Digestion is usually carried out after incubation in an osmoticum (a solution of higher concentration than the cell contents which causes the cells to plasmolyse). This makes the cell walls easier to digest. Debris is filtered and/or centrifuged out of the suspension and the protoplasts are then centrifuged to form a pellet. On resuspension the protoplasts can be cultured on media which induce cell division and differentiation.

Viruses are smaller than bacteria and require living cell for their growth. They can be detected through the bacteria to which they parasitise. Viruses are associated with both eukaryotes as well as prokaryotes and contain either DNA or RNA; only few have both DNA and RNA.

Viruses that infect bacteria are called bacteriophage. They consist of head or capsid and a tail. The head may be round, oval, polyhedral and is made up of proteins while tail is hollow and provides an exit for nucleic acid to enter in bacterial cell. The tail also helps in attachment to the cell wall surface of bacteria. After injection of the nucleic acid into bacteria they multiply and form large number of bacteriophage particles which cause lysis of the bacterial cell wall releasing many bacteriophages. Phages that cause lysis are known as lytic phages and that do not cause lysis are called temperate phage. The most common lytic phage is E.coli – a coliphage.

1. VARIETAL SEED PRODUCTION

3 m : Rice, soybean

5 m : Blackgram, greengram, cowpea, field lab lab lab lab

5-10 m : Groundnut

25 m : Tomato

Alkaloids are a class of basic, naturally occurring organic compounds that contain at least one nitrogen atom. Alkaloids showed quite diverse medicinal properties (Rates, 2001). Many of them possess local anesthetic properties, but their practical use is limited for clinical purposes (Hesse, 2002). Morphine is one of the most known alkaloids isolated from Papaver somniferum (opium poppy), which had been used and still is for medical purposes. This alkaloid is a powerful narcotic which is used for the relief of pain, but its usefulness is limited because of its addictive properties.

The isolation of microbes is commonly done using serial dilution technique.

1. Trichoderma

Trichoderma is one of the important biocontrol agents. The colonies first produce mycelial growth and then turn yellow and finally produce a dark green color mat.

Anthocyanins are members of the flavonoid group of phytochemicals, a group predominant in teas, honey, wines, fruits, vegetables, nuts, olive oil, cocoa and cereals. The primary players in this group include the anthocyanins (eg, cyanidin, pelargonidin, petunidin), flavonols (quercetin, kaempferol), flavones (luteolin, apigenin), flavanones (myricetin, naringin, hesperetin, naringenin), flavan-3-ols (catechin, epicatechin, gallocatechin), and although sometimes classified separately, the isoflavones (genistein, daidzein) (Skibola and Smith, 2000).

Introduction

The isolation of mutants is often critical to the study of cellular process, especially when such studies are performed at the molecular level. The phenotype of a particular mutant is attributable to the loss of a particular cellular function; the loss of that function is actually attributable to a mutation in a gene necessary for that function. In this experiment we will isolate auxotrophic mutant which can grow only if provided with specific amino acid. For example arg^- mutant have lost the catalytic function of an enzyme involved in arginine biosynthesis due to mutation in the structural gene that encodes the enzyme.

Mutagenic Agent

Mutation is an alteration in the coding sequence of a gene. Mutation occur spontaneously at a frequency of about 10^-8 per bacterial cell per generation. The rate of mutation can be increased with mutagenic agents. Mutants are strains with any structural alteration of their DNA that gives rise to a mutant phenotype. Mutagens are chemical agents (e.g. nitorsoguanidine, 5 bromouracil), physical (e.g.UV) and biological agents (e.g. trnasposons) that cause mutation to occur.

1. Bacterial (Prokaryote) Genome

The chromosome of most bacteria is a single super coiled double stranded circular DNA molecule. A complex packaging mechanism is necessary in order to ensure that the entire DNA is folded within the bacterial cell in a manner, which will not inhibit transcription nor allow entanglement of the two daughter strands to occur during replication (Highly condensed >10 mg/ml in region less than l ìm in diameter). This is achieved by two main mechanisms.

• First the DNA is folded into between 40-100 loops
• Second each of the quasicircles is it self super coiled independently of the others each loop are abolished by Ribonuclease.

Deoxyribonucleic remove the super helical nature of the loops, though limited treatment with this enzyme will attack only a small number of the loops and therefore leads to removal of only some of the super coiling. The intact folded chromosome or nucleoid can be isolated in the presence of non ionic detergents and high molarities of salt in two possible forms free and membrane associated. The free form which sediments at 1600-1700s consists of about 60%DNA, 30%RNA and 10%protein the bulk of which is the enzyme RNA polymerase. The membrane bound form contains an additional 20% of membrane-attached protein. Bacterial genome size ranges from 580 to 9900kbp, & the average gene size is constant from 900 to 1100bp of which 87-94% are protein coding (Rickettisa’s 76% and Borelia Plasmid’s 41% are exceptions).

ABSTRACT

A plant grows in a highly competitive environment. It is continually being threatened by other plants encroaching upon the space from which it draws its sustenance, by microorganisms, by insects, avian, reptilian or mammalian herbivores. In order to survive, each plant must draw upon a complex of defenses, which may be physical, such as Spines or Leathery Leaves or Chemical. These defenses are passive, coming into action only when the plant is attacked which repel insects or the herbivores that attempt to feed on them. Plants protect themselves from insect attack by providing a multitude of repellent chemicals including toxic and bitter alkaloids and tannins. Non-specialist insects and other animals that attempt to feed on the plant may be repelled, injured through severe gastrointestinal malfunction or even killed. The cabbage, spinach, mustard etc. are well known for their mustard pungent flavor that deters most animals from consuming it. Even most medicinal plants like Cynanchum collialatum of the family Asclepediaceae have tannins or alkaloids like “cardenolides” that repel most animals and insects. All these plants have a basic enzyme “Myrosinase” which react with these specific tannins to produce the toxic metabolite, which repels insects and animals. But caterpillars of few insects are known to tolerate these toxic alkaloids (ex: caterpillar of Striped Tiger Butterfly-Danaus genutia) and in turn sequester the toxic alkaloids into their tissues making themselves non-palatable to their enemies. We hypothesize the analysis of possible roles of toxic alkaloids isolated from these butterflies as bio-insecticides

1. Mitochondria

Mitochondria are known as the “Power House” of a cell because they are the site of ATP production. In addition, they are also involved with photorespiration and oxidation of fatty acids and work in association with peroxisomes, glyoxysomes and chloroplasts.

Isolation of intact mitochondria is rather difficult. This is because the cell walls of plant tissue are rigid as compared to the single-layered wall surrounding the mitochondria. A significant fraction of mitochondria suffer structural damage during the process of tissue homogenization. Therefore, the pelleted mitochondria need further purification by sucrose gradient centrifugation.

Mitochondria can be isolated from green as well as etiolated leaf; however, the latter is preferred to obtain higher yield of mitochondria (Moore and Proudlove, 1983).

12.1. Routine Media for Fungal Isolation

In recent years commercially available readymade culture media have been used in research laboratories for the isolation of fungal plant pathogens. These culture media are standardized and they are in different types. Some media are used for general purposes while some others are used for the isolation and growth of specific pathogens e.g.

Transfer 1.5 mL overnight culture to a 1.5 mL microfuge tube, centrifuge for 30 sec, and decant supernatant.

Resuspend cells in 400 µL TE by vortexing, add 50 µL 10% SDS, 50 µL proteinase K (20 mg/mL in TE). Incubate for 1 hour at 37^oC.

Shear DNA by 3-5 passages through a 26 G needle.
Extract twice with 500 µL phenol: chloroform (1:1), and twice with 500 µL chloroform. 
Precipitate nucleic acids by adding 25 µL 5 M NaCl and 1 mL 95% EtOH, vortex, and centrifuge for 10 min, decant supernatant. Dry pellet.

Introduction

Mutation refers to any heritable change in the base sequence of a DNA molecule. A mutagen is a physical of chemical agent that causes mutations to occur (or increase the frequency of their occurrence). Mutagenesis is the process of producing a mutation. If the mutation occurs in nature without the addition of a mutagen, it is referred as a spontaneous mutation, and the resulting mutants are spontaneous mutants. If it caused by a mutagen, the process is induced mutagenesis.

Ultraviolet light is a fairly potent mutagen. The main DNA lesions are two chemically different types of covalent dimers: cyclobutane-pyrimidine dimers and pyrimidine dimers. The pyrimidine dimers only account for 20% of the total dimers but are correspondingly more mutagenic. In E. coli the number of mutants induced by ultraviolet (UV) light can be reduced by exposure to visible light (photoreactivation), which implicates cyclobutane pyrimidine dimers since dimers can not be photoreactivated.

Aromatic amino acid of proteins, and purine as well as and pyrimidine bases of nucleic acid absorb ultra violet radiation. Pyridine bases, particularly thymine of DNA is the main target compound involved in bactericidal action of ultra violet radiation. The energy absorbed by thymine causes it to under go a photochemical reaction with adjacent ( adjoining) thymine molecule in same DNA strand. DNA replication is blocked because the formation of thymine: thymine (T:T) dimmers. As the dosage increase, cytosine-thymine (C:T) to (C:C) cytosine-cytosine dimmers can be found to add the lethality of ultra violate radiation.

Introduction

Biological nitrification is a two-phase process, which consists of the oxidation of ammonia-nitrogen to nitrite-nitrogen by species of genera Nitrosomonas, Nitrosospira and Nitrosogloea, and the subsequent oxidation of nitrite-nitrogen to nitrate-nitrogen by species of the genera Nitrobacter and Nitrocystis. The following equations explain the reaction.The importance of nitrifying organisms in soil is based on their capacity to produce nitrate, the major source of nitrogen assimilated by higher plants. The rate of nitrification is dependent on such factors as concentration of NH₄+, temperature, moisture, aeration and pH. Bacteria have their optimal activity at an alkaline reaction. Below pH 7.0 the process declines.

By Alkaline Lysis Method

Introduction

DNA of prokaryotic cells is relatively simple in comparison to that of eukaryotic cells. In addition to chromosomal DNA, a bacterium may also carry an additional piece of DNA called a plasmid. Bacterial plasmids are double stranded closed circular DNA molecules that range in size from 1 kb to more than 200kb. Plasmids are useful to bacterial cells since they carry genes for antibody resistance, RM system etc that allow bacteria to survive in non-ideal conditions.

Plasmids constructed in the laboratory are used as cloning vehicles. These synthetic plasmids contain only essential features such as origin of replication, antibiotic resistance, multiple cloning sites etc. various copy number of plasmid For e.g. pBR322 20 copy/cell – low copy number, pUC 18, pUC 18- 800 copy/cell – high copy number.

Alkaline lysis, in combination with the detergent sds, has been used for more than 20 years to isolate plasmid DNA from E.coli (Birnboim and Doly 1979). This experiment describes how to extract plasmid from bacteria – which involves growth of bacterial culture, harvesting and lysis of bacteria and isolation of plasmid DNA from the lysate using alkaline lysis method. The DNA is analysed on agarose gel.

What is dermal fibroblast?

Skin fibroblasts are a type of cell found in the dermis, the middle layer of the skin. They are the most common cells in connective tissue and play a crucial role in maintaining the structural integrity of the skin. Fibroblasts are responsible for synthesizing and secreting extracellular matrix proteins such as collagen, elastin, and fibronectin, which provide support and elasticity to the skin. Additionally, fibroblasts play a role in wound healing by proliferating and migrating to the site of injury, where they aid in tissue repair and scar formation.

Aim: To prepare the primary cell culture of fibroblasts from canine skin.

Tools and instrument required for the procedure

1. Tissue collection box with ice cubes - To prevent contamination and maintain the stable temperature

2. Surgical Gloves - To prevent contamination

3. Tissue collection tube with 1X PBS - To collect sample

4. Laminar flow hood - To work in a sterile environment and prevent contamination

5. Cell culture dish - To digest the skin

6. Sterile 1000 μL and 200 μL pipette and tips-For accurate measurement and transfer of cell suspension and culture media.

7. Forceps - Fine-tipped forceps for handling and manipulating tissue.

8. Scalpel and blade holder - Scalpel for the dissection of skin tissue.

9. Surgical scissors - Chopping of the skin tissue

10. Falcon 70 μm cell strainer - To filter out undigested tissue debris and obtain a homogeneous cell suspension.

11. 15 ml and 50 ml Centrifuge tubes - To collect and centrifuge the digested cell suspension.

What is keratinocyte?

The epidermal layer consists of keratinocytes, melanocytes, Langerhans cells, and Merkel cells. Their major function is to produce Keratinocytes which are the most abundant cells in the epidermis, keratin protein. Keratinocyte synthesizes major structural components of the epidermal barrier through a programmed process of differentiation. They are present in the innermost layer of the epidermis (Eckert et al?, 1989). The epidermis is composed of several layers, stratum corneum (horny layer), stratum lucidum (clear layer), stratum granulosum (granular layer), stratum spinosum (prickle cell layer), and stratum basale (basal layer), from the outermost to the deepest layer respectively. The process of maturation and movement of the keratinocytes from the stratum basale to the stratum corneum is what we call epidermal differentiation.

Procedures for isolation of canine karatinocytes

When studying skin cells, isolating keratinocytes from tissue samples, such as skin biopsies, is a critical initial step. Here is a general guide on how to isolate keratinocytes:

What is primary cell culture?

In vitro culture of cells freshly obtained from the tissue is called primary cell culture. Unlike that of cell line, primary cell culture grows for a limited period of time. Examples, are primary mammary epithelial cells, primary hepatocyte culture.

Aim: To prepare the primary cell culture from chicken embryos.

Materials required for the primary cell culture

• 70% alcohol

• Cotton swabs

• Small beaker 20-50 ml

• Forceps

• Straight and required curved,

• 9 cm Petri dishes

• 11 day embryonated eggs

• Balanced Salt solution (BSS)

• 250 ml conical flask

• Magnetic centrifuge tubes

• Haemocytometer

• Growth medium with serum

• Culture flasks

4.1 From Soil

Soil contains varied group of microorganisms such as fungi, bacteria, actinomycetes, algae and protozoa. The type of nutrients available, moisture, temperature and pH of the soil determine the nature of microbial population in soil. Different laboratory techniques for isolation of microorganisms have been detailed in chapter-III. These are used in particular for the isolation of specific type of microorganisms. All the techniques adopted are just to segregate the pathogens/ microorganisms from the mixture of microbial population present in nature.

(a) Isolation of Fungi

Soil contains large number of fungi and constitutes a major place of microbial population. They can be isolated in pure form by following the methods given below:

Introduction

Yeast contain a hard cell wall. Spheroplasts are yeast cells where the cell wall has been enzymatically degraded. These spheroplasts are very fragile so we must be careful with them. Once we have spheroplasts it will be very easy to lyse the yeast cells and quickly release the RNA and proteins in the cell into a controlled environment where we can temporarly keep the cellular RNases from degrading the RNA. We don’t want to lyse the cells before they are in this controlled environment.

Growth of Yeast Cultures

Cultures of wild-type and yeast containing various mutations will be grown up the day of the experiment. Each section will be given one wild type yeast culture and a culture containing one of the mutant varieties that we have discussed in class.

Yeast are grown very similarly to bacteria. Like bacteria, they have different stages of growth. Scientists divide these stages of growth into early-log phase, mid log-phase, late log-phase, and the stationary phase. These phases are based on the amount of yeast that are around in the broth and how quickly they are able to grow and divide based on nutrient use. Reading the absorbance of the sample at 600 nm is a way in which you can determine what phase of the yeast growth series you are in and how many cells you have.

Haploid yeast cells contain approximatley 1.2 pg of RNA per cell

Predict approximatly how much RNA you should isolate

Introduction

Studies on biocontrol agents have revealed the presence of a diverse range of antifungal metabolites derived from amino acid, polyketones and isoprenoid biosynthetic pathways, each of which has distinctive regulatory points and is subjected to different stimuli within organisms. In this experiment, the extraction of secondary metabolites from the culture of T. viride and Chaetomium globosum will be demonstrated.