eChapters

Immunoassays rely on the ability of an antibody to recognize and bind a specific macromolecule in what might be a complex mixture of macromolecules. In immunology the particular macromolecule bound by an antibody is referred to as an antigen and the area on an antigen to which the antibody binds is called an epitope.

In some cases an immunoassay may use an antigen to detect for the presence of antibodies, which recognize that antigen, in a solution. In other words, in some immunoassays, the analyte may be an antibody rather than an antigen.

Gravimetric analysis describes a set of methods in analytical chemistry for the quantitative determination of an analyte based on the mass of a solid. A simple example is the measurement of solids suspended in a water sample. A known volume of water is filtered, and the collected solids are weighed.
 
In gravimetry, the analyte needs to be first converted to a solid by precipitation with an appropriate reagent. The precipitate can then be collected by filtration, washed, dried to remove traces of moisture from the solution, and weighed. The amount of analyte in the original sample can then be calculated from the mass of the precipitate and its chemical composition.

In some cases, it is easier to remove the analyte by evaporation. The analyte might be collected—perhaps in a cryogenic trap or on some adsorbent material such as activated carbon—and measured directly. Or, the sample can be weighed before and after it is dried; the difference between the two masses gives the mass of analyte lost. This is especially useful in determining the water content of complex materials such as foodstuffs.

Titration, also known as titrimetry, is a common laboratory method of quantitative chemical analysis that is used to determine the unknown concentration of an identified analyte.  Since volume measurements play a key role in titration, it is also known as volumetric analysis. A reagent, called the titrant or titrator is prepared as a standard solution. A known concentration and volume of titrant reacts with a solution of analyte or titrand  to determine concentration. The volume of titrant reacted is called titre. Volumetric analysis originated in late 18^th-century in France.

DNA and RNA are the carriers of genetic information in all the living organisms. The study and analysis of the genetic code can provide information that can be used in clinical diagnostics, drug discovery, genetic engineering and forensic sciences. Hence there is need to understand the techniques in isolation of DNA and RNA followed by their amplification and sequencing.

Organic material especially, seeds, grains, leaves, fruits and nuts contain prominently three groups of compounds. They are carbohydrates, proteins and fats/oils, mostly in that order. They also contain 3-5% minerals and smaller quantities of vitamins and other minor constituents. Cereal grains contain more of carbohydrates while oilseeds and nuts contain more oil. Tree nuts and even oilseeds may contain 30-50% protein while cereal grains contain 5-8% protein only. The starch and sugar contents of oilseeds may be less than 10% while the oil content of cereal grains ranges around 5%. The actual content and the composition of these groups of compounds really account for their quality as well as commercial value.

Carbohydrates are the most abundant biomolecules on earth. They occur in nature as glucose, starch and cellulose etc. Each year, photosynthesis converts more than 100 billion metric tonnes of carbon dioxide and water into starch, cellulose and other plant products. Carbohydrates are polyhydroxy aldehydes or ketones and their derivatives. They provide 4K calories of energy per gram. They promote utilization of lipids and reduce wastage of proteins. They are readily available in roots, tubers, cereal grains, sugarcane and sugar beet.

Lipids are a diverse group of chemical molecules. They are generally insoluble in water but soluble in organic solvents. Lipids include long-chain hydrocarbons, alcohols, aldehydes, fatty acids, and their derivatives, especially, glycerides, phospholipids, glycolipids and sulfolipids. Other compounds like sterols, fatty acid esters of sterols, vitamins A, D, E, and K, carotenoids, and other poly-isoprenoids also are part of the lipids. Oils and fats are mainly triglycerides and neutral molecules. Those in the liquid state at ambient temperatures are called oils while those in the solid state are called fats. Lipids and lipid components have varied uses in the food and non food sectors. Vegetable and animal matter contain a variety of these constituents in different proportions. It is neccessry to evaluate the composition and quality of these constituents from the point of view of their utility.

The word protein was first coined in 1838 to emphasize the importance of this class of molecules. The word is derived from the Greek word proteios which means “of the first - rank”. Proteins are polymers/macro molecules made up of several amino acids. When there are more than 50 amino acids in a chain, the polypeptide is normally called a protein. Each protein in the cells of living beings has a unique sequence of amino acids that determines its biological function. The proteins are folded into specific defined structures, which are maintained by large number of relatively weak bonds. The three dimensional structure is well defined and suited to it’s specific function.

Enzymes are organic catalysts produced by living organisms. They make possible biochemical reactions. Hence they are called biocatalysts. They are mostly a specialized class of proteins. However, there are some non-protein enzymes like ribozymes, which are RNAses. Enzymes speed up reactions at ambient temperatures. This is in contrast to chemical reactions like saponification of lipids which take place when lipids are boiled for a few hours with concentrated alkali. Enzymes, namely, lipases, hydrolyze lipids at body temperatures in minutes. They are superior to chemical catalysts by a factor of 10⁷ to 10¹⁴ . An enzyme may be a simple protein or a complex protein. They are produced by cells and are mostly globular proteins. Each cell contains an estimated 3000 different enzymes.

Nucleic acids were first discovered in 1868 by Friedrich Meischer. Nucleic acids are high molecular weight polymers that are present in our cells. They store and transfer genetic material from generation to generation. Knowledge of how genes are expressed and how they can be manipulated is becoming increasingly important for understanding of nearly every aspect of biochemistry. These macromolecules p ass on information for cellular growth and reproduction. All the genetic information in the cells is called the genome. Every time a cell divides, the information is copied and passed on to the new cells.

Vitamins are essential organic nutrients required by an organism in small quantities. They are essential since they cannot be synthesized in sufficient quantities by an organism, and must be obtained from the diet. The term vitamin does not include other essential nutrients like dietary minerals, essential fatty acids, or essential amino acids which are needed in larger quantities than vitamins. The term vitamin is derived from “vitamine,” coined in 1912 by the Polish biochemist Funk. The name is from vital and amine, meaning amine of life. Thirteen vitamins are generally recognized presently. Vitamins are used in small quantities by an organism to keep it in a healthy condition.

Phenol is normally a benzene ring containing an –OH group, and hence an aromatic alcohol. The term “phenolic” refers to similar aromatic compounds with one or more -OH groups. Phenolics constitute one of the most widespread classes of secondary metabolites. Commonly talked about class of compounds like anthocyanins, flavonoids, lignins, tannins, coumarins and chalcones are all phenolic substances. Phenolics are water insoluble substances. However, the water solubility increases with the increase in the number of -OH groups. Phenolics absorb electromagnetic radiation strongly. Many of the phenolics absorb in the visible part of the spectrum (Ex. anthocyanins) and are coloured. Phenolics are produced in plants by 1. Shikimate pathway through phenylalanine (C₆-C3) and 2. Acetate/malonate pathway.

Certain constituents associated with plants are toxic. When such items are consumed by men and animals, they become hazardous. Mold growth on food items may be the cause of toxicity. Some of them are called mycotoxins. Aflatoxins are the best examples of such a group of compounds. Sometimes processing also produces some toxins. Fumigants, smoke and some metals (mercury, lead, cadmium, arsenic etc.) used for such purposes are some such toxic constituents. Pesticides used in protecting the crops and grains during storage remain on the items and the residues are hazardous.

Alkaloids are of plant origin and are alkaline in reaction. They contain N as part of a hetercyclic ring. They exhibit pharmacological activity. Presently more than 2000 alkaloids are known. They have a lot of uses in medicine and as  flavoring agents, psychoactive agents, pain killers and as pesticides.

Sixteen chemical elements are known to be important to a plant’s growth and survival. The sixteen chemical elements are divided into two main groups: non-mineral and mineral.  The Non-Mineral Nutrients are hydrogen (H), oxygen (O), and carbon (C). These nutrients are found in the air and water.
  
In a process called photosynthesis, plants use energy from the sun to change carbon dioxide (CO₂ - carbon and oxygen) and water (H₂O- hydrogen and oxygen) into starches and sugars. These starches and sugars are the plant’s food. Since plants get carbon, hydrogen, and oxygen from the air and water, there is little the farmers and gardeners can do to control  how much of these nutrients a plant can use.

Plant pigments impart colour to the different components like leaves, flowers, fruits etc. They in turn are responsible for the colour of raw food, we eat. Pigments like chlorophyll, anthocyanins, flavonoids, tannins, xanthones etc. are the different pigments that are present in plants. The primary function of pigments in plants is photosynthesis, which uses the green pigment chlorophyll along with several red and yellow pigments that help to capture as much light energy as possible. Chlorophyll is green in colour. Carotenoids like a, b, and g, carotenoids are yellow in colour. Lycopene is red in colour. Flavones and falvonols are also yellow in colour. Anthocyanins are purple and magenta in colour. Other functions of pigments in plants include attracting insects to flowers to encourage pollination. The assay of pigments is mostly based on their native colour.

Chemicals in the form of insecticides ,fungicides and herbicides are extensively used in large numbers in agricultural production. Commercial production of some crops would be impossible if chemicals are not used. Small quantities of chemical residues often remain in such crops and the harvested grains and other products. The residues of pesticides  have been observed in small quantities (ppm level) in soil, water, agricultural produce  and even in animal tissues and their products like meat and  milk. They finally reach the consumer, namely, man (and even animals). Even the environment  is  now-a-days polluted with pesticides and other chemicals harmful to the humans. In view of the importance and seriousness of the problem the residues need to be estimated in the food and non-food products to assess their safety. Analytical details of a few pesticide residue procedures are presented below. Specialized texts may be consulted for  more extensive information.

A disease is characterized by the sum total of symptoms as expressed by the host plant and the signs of the pathogen involved. As stated earlier, it is the core part of disease diagnosis because in every diseased plant there are some symptoms/signs of the problem regardless of the ease in their visibility. Even in latent infections there are certain clues or at least presence of the pathogen inside the host serves as sign of the pathogen. However, the term, ‘disease’ itself refers to an uncomfortable situation of the concerned host. It reflects in the form of reduction in the vitality and performance of the host to varying degrees depending on the quality and quantity of the factors involved in production of the disease. Study of the characteristics of disease is, therefore, important for its quick and accurate identification.

2.1 Loose Smut of Wheat

The disease is prevalent in all the wheat growing areas in the world. However, it is a serious problem in humid and sub-humid areas. The incidence of the disease is comparatively more in cooler and humid parts of northern India as compared to the drier and tropical parts of the country. Being a simple interest disease epidemiologically, this is a threat to the cultivation of wheat for seeds. The disease is therefore, dreaded by seed growing agencies who have to treat the seeds with costly chemicals.

3.1 Damping-off Disease of Seedlings

The disease is quite common in nurseries or seedbeds all over the world. It is virtually a serious threat to almost all the nursery crops of vegetables and other crops including tomato, chillies, cauliflower, cabbage, brinjal, cucurbits, tobacco and a number of other crops. The economic importance of the disease is more because it takes the toll of precious seedlings in the very initial stages of the crop cultivation. The seed can become infected with a fungus which may cause darkening and softening of the seed leading to its decay. Similarly, seedlings may also be infected while sprouting or even after emergence. The disease is more serious in heavy soils with poor drainage. Abundant soil moisture with 90-100% humidity and temperature around 200C is favourable for the disease.

In this section we shall discuss diseases that do not involve any parasitic etiology. Such diseases are now more appropriately called ‘Disorders’. The disorder may be of many kinds but all of them have adverse effects on the normal physiology and total performance of the crop host. They are therefore, more often called ‘Physiological disorders’. Lack of proper soil and environmental conditions, nutrient status, irrigation factors and other parameters for successful crop cultivation may result in such disorders.

Abstract

Nitrate uptake from the soil is the first step in nitrate acquisition and utilization by plants. Nitrate uptake and its release into cells is mediated by nitrate transporter(s) located into the plasma membrane of the root. Three transporters have been identified by kinetic measurements in plant roots. These are constitutive high affinity nitrate transporter (cHATS), inducible high affinity nitrate transporter (iHATS) and low affinity nitrate transporter (LATS). Two of these display saturable kinetics; a low capacity constitutive system and a high capacity inducible system. In addition, a non-saturating low affinity, high capacity system becomes apparent only at higher external NO^-₃ concentration. Genes that encode representatives of each class of transport system have been identified and fall into two families: NRT1 and NRT2. Members of these families are induced in response to nitrate in the environment and are regulated by internal signals including nitrogen metabolites and shoot demand for nitrogen. This chapter deals with the physiology and molecular biology of nitrate transporters in higher plants.

Abstract

Nitrate reductase (NR) catalyzes the reduction of nitrate to nitrite, which is the first step in the nitrate assimilation pathway, but can also reduce nitrite to nitric oxide (NO), an important signaling molecule that is thought to mediate a wide array of developmental and physiological processes in plants. Accordingly, the amount of NR protein and its catalytic activity are closely controlled in the cell. This chapter focuses on the posttranslational regulation of NR activity, which has been known for some time to involve reversible protein phosphorylation. The hinge 1 region of NR contains the serine-534 regulatory site, which when phosphorylated can bind a 14-3-3 protein resulting in inhibition of NR activity. This fundamental mechanism underlies the rapid modulation of NR activity in leaves in response to light/dark transitions, and the activation of NR that occurs in response to hypoxia (e.g., as with soil flooding). Recent results suggest that the basis for hypoxia-induced NR activation, which is thought to be essential for plant survival, may involve reactive oxygen species (ROS), such as H₂O₂, that are produced in response to many plant stressors including hypoxia.

Abstract

Nitrogen is the most important element in terms of nutrition for plants, nitrate being the most preferred form. Nitrate not only acts as nutritional element but also as a signal to modulate metabolism and plant architecture. Nitrate sensing mostly happens in the root tip through a hitherto unknown mechanism, but its response can be demonstrated on an organism-wide scale. Functional genomic studies revealed over a thousand nitrate-responsive genes, involved not only in N and C metabolism, but also in various other physio-logical processes. Identification of nitrate response element/s common to all these genes could pave the way to unravel the mechanism of nitrate signaling, but findings in this direction have remained inconclusive so far. Several trans-acting factors have been implicated in N signaling and response, but none of these have been convincingly demonstrated to be specific to this response. Nitrate uptake, which is sometimes associated with nitrate sensing, is also highly regulated process and involves multiple transport systems like HATS, LATS and dual affinity transporters.

Abstract

Nitrogen is the most essential nutrient for all plants and is also the major limiting factor in plant productivity. High yielding varieties of all crops are responsive to nitrogen but its ever-increasing use has shown detrimental impact on the environment along with extremely low use efficiency. Nitrogen use efficiency (NUE) in crop plants depends on various internal and external factors, which are dealt in detail. There has been a significant interest in genetic engineering of the crops to improve NUE. The use of biotechnological interventions by manipulating genes of the nitrogen utilization pathway to improve NUE has not been very successful. But transgenics/mutants with modified capacities for nitrate uptake, assimilation and remobilization have enhanced our understanding of the genetic control of NUE. In both cellular, and at whole plant level the mechanisms involved in N remobilization from the senescing organs towards the grain have recently gained importance but their understanding is still preliminary. Recent evidences have shown that grain filling during later stages of crop growth is supported by N recycling.

Abstract

The abiotic stresses viz. drought, high temperature, water stress, salinity, heavy metal contaminations etc. cause variable effect on nitrogen (N) mineralization, its uptake and transport, assimilation and remobilization, and subsequently the growth and productivity of plants. The affect is generally negative, however, the magnitude of decrease in various processes is dependent on genotype, age of the plant, tissue under investigation, type and extent of the stress and other agro-climatic conditions. Biological nitrogen fixation and nitrate assimilation appear to be more sensitive than the ammonium assimilation possibly because re-assimilation of ammonia released in the tissue due to catabolism of N molecules is essential for the survival of plants. In addition, synthesis of stress metabolites (proline, glycine betaine and poly amines) is directly dependent on the availability of primary amino acid, glutamic acid. There are few well coordinated and detailed studies, determining the key factors involved in regulating NUE under abiotic stresses.

Abstract

Intensification of agriculture during the last few decades has resulted in considerable depletion of the productivity of Indian soils and the balance sheet of potassium (K) is highly negative due to very low level of K fertilization. Although the need for balanced fertilization is known for many years, it is not always observed in practice by farmers. Since the effects of nitrogen are easily seen and crops look more vigorous and yield better, excessive use of nitrogenous fertilizers, accompanied by an inadequate supply of other nutrients, is considered by farmers as a reasonable insurance against yield losses. Often, the lack of K restricts nitrogen uptake and utilization, and hence plant growth. When K is limiting, nitrogen fertilizers are used inefficiently by the plants. This nutrient imbalance is thus closely associated with low nitrogen-use-efficiency (NUE) of crop plants, which is detrimental to crop production and environment – because of the consequences of N unused by the crop.

Abstract

During the past five decades, the role of nitrogen (N) fertilizer in augmenting foodgrain production has widely been recognized the world over. In Indian soils, N deficiency is recognized as a universal problem and its consumption has increased by more than 300 times during the last 60 years.

Abstract

Increases in atmospheric concentrations of CO₂ are likely to have major physiological consequences for plants, due to the major effects of atmospheric CO₂ on photosynthetic rates and the centrality of photosynthesis to plant metabolism. We review what is known about the effects of growth at elevated levels of CO₂ on the protein and N concentrations of plants in general, and food crops in particular. Growth at elevated CO₂ typically reduces plant tissue N concentrations by 10-15%. The physiological mechanisms behind this decrease in tissue protein and N are likely complex, and include both dilution from increased tissue concentrations of non-structural carbohydrates and decreased root uptake of N.

Abstract

Nitrogen is the most limiting nutrient controlling production of all agricultural systems. Application of N fertilizer has increased food production tremendously all over the world. However, it also contributes towards global warming and ozone layer depletion through emission of nitrous oxide, a greenhouse gas (GHG). Therefore, there is a need to assess the possibilities of N management for climate change abatement and food security enhancement, while minimizing environmental and human health impacts. In this chapter, role of gaseous-N emission on N use and various mitigation options for Indian agriculture have been discussed. For the base year 2007, the direct and indirect N₂O emissions from Indian agricultural soils was estimated to be 136.29 Gg (35.3 Tg CO₂ equivalent) and 30.61 Gg (5.8 Tg CO₂ equivalent), respectively.

Pigeonpea (Cajanus cajan L. Millsp.) is crowned as poor man’s crop which provides much needed protein to farming families with least inputs. It is a versatile plant species which can grow successfully in a range of soil types, temperatures and photoperiods. Its deep root system enables it to overcome intermittent drought and other stresses. In India, the crop is grown annually on about 4.04 m ha (FAO, 2012) and the major pigeonpea growing states are Maharashtra, Andhra Pradesh, Karnataka, Madhya Pradesh, Uttar Pradesh, Bihar and Gujarat (Table 1). The high protein pigeonpea dal (decorticated dry splits) is widely consumed across the country in various cuisines. The annual national production of pigeonpea is about 2.56 m tones. This produce, however, is not sufficient to feed the ever growing population of the country, and hence, necessitates huge (500,000 tons) imports of this pulse. In spite of its importance in food security and sincere research efforts, its national productivity could never cross the bar of 800 kg/ha (Fig 1) since independence. Since last 50 years, ICAR has been very seriously pursuing the issue of genetic enhancement with huge investments and some out-standing pure line varieties with about 10% more grains over the best local varieties were developed.

Male sterility is a situation where the male reproductive parts of a plant are either absent, aborted, or non-functional; and therefore, they fail to participate in the process of natural sexual reproduction. This situation generally arises due to some developmental defect that occurs at any stage of microsporogenesis (process of development or release of pollen grains) in the plants. Since male sterility is the manifestation of abnormal growth and development and can occur at any point of time, the action of genes controlling male sterility is also be variable and inconsistent across the genotypes. Based on the type of malfunctioning of androecium, the male sterility systems have been classified as structural (absence or deformity of anthers), sporogenous (defective microsporogenesis), and functional (failure of mature pollen to germinate). In addition, on the basis of genetic control mechanism of the male sterility, it has also been classified as genetic, cytoplasmic, and cytoplasmic nuclear (or genetic) male sterility. With respect to utilization of male sterility in crop breeding, it is essential that individuals with altered male fertility keep their female fertility intact. The fertilization of such plants takes place with pollen grains from other plant that is transferred with the help of external agencies such as wind, insects, or human beings.

3.1. Natural cross-pollination

Breeding procedures in any plant species of economic importance are guided by its reproductive system particularly, pollination mechanism. In the cereal crops such as maize, pearl millet etc., where cross-pollination is facilitated by wind, the breeders have developed schemes for commercial exploitation of dominance and epistatic genetic variation through hybrid and population breeding. In contrast in self-pollinated crops, the efforts are always made to exploit additive genetic variation primarily through pedigree and mass selection.
 
In pulses, self-pollination is a rule and pigeonpea is no exception. However under field conditions, occurrence of hybrid plants in the populations is not uncommon; and it happens due to some degree of natural out-crossing that took place in the preceding generation. This type of partial natural out-crossing in pigeonpea is affected by a variety of insects (Fig 10) which forage on its large flowers to collect the nectar. These minor incidences of out-crossing are the primary source of varietal deterioration. Recently, the pigeonpea breeders converted this constraint into an opportunity for genetic enhancement of yield through exploitation of hybrid vigor and developing commercial hybrids.

4.1. Flower and flowering in pigeonpea

Growth habit: Primarily, there are two major growth types in pigeonpea. These are determinate and non-determinate. The determinate growth in the pigeonpea plants is characterized by flowers in bunches at the top of the canopy (Fig 17a). In this case the epical growing points are transformed in to reproductive buds and bunches of flowers appear on the top of the canopy. Such cultivars are not popular as the management of insects such as Helicoverpa and Maruca becomes very difficult. Most of the cultivated types belong to another growth category called as non-determinate. In such genotypes the apical growing points remain vegetative and the plants keep growing under favorable conditions and it results in tall canopy with many spreading or compact branches. The inflorescence in this case emerges from the axil of secondary and tertiary branches (Fig 17b); and hang sideways. These types show less susceptibility to insects due to non-clustering nature of flowers.

5.1 Seed back up programs

In hybrid breeding program, testing of new hybrid combinations is a continuous process and in most cases, the availability of crossed (hybrid) seed for their annual evaluation of selected hybrids is an important issue. Since, the selection of hybrids for advancement is based on yield data, one year will be required to produce seed for testing in the subsequent season. This means for each cycle of advancement, one valuable season will be consumed in their seed production. To avoid this situation and enhance the testing program at a fast pace, an aggressive hybrid seed production program should be followed. This can be achieved by visually selecting relatively more number of hybrids based on their flowering time, plant vigor, disease resistance, and early pod load about 4-5 weeks before harvesting. During this period, the new seed of the selected hybrids can be produced manually. This operation is facilitated by the perennial nature of pigeonpea plant, which can flower for extended periods of 40-50 days; that allows completion of desired number of pollinations of the target crosses in the same cropping season. One trained person can pollinate about 300 buds in 6-7 hours; and with 20% success, about 150 seeds can be produced. Experience has shown that, with good planning and crop management, this program can be achieved with ease.

Fig. 25: ICPH 2671 in field

To overcome the challenge of popularizing pigeonpea hybrids in the country, it is considered fair enough to compare the hybrid rice with respect to their seed production technology, yield advantages and profitability to the farmers and seed producers. Since both pigeonpea and rice are grouped under self-pollinated crops, this comparison, in the views of authors, may help in encouraging hybrid pigeonpea seed producers and those involved in marketing of hybrid seed.
  
Rice is a highly self-pollinated crop and earlier hybrids were not possible because it was essential that large quantities of hybrid seeds are produced easily and economically. In China, the birth place of hybrid rice, this impossible looking task was accomplished. To launch this program, the Chinese scientists decided to alter the pollination system of rice.

With the advent of hybrid technology, it is natural to assess its potential benefits at the national level. To meet the domestic requirement of pigeonpea, about 50.000–60,000 metric tons of pigeonpea is imported annually from Myanmar and Africa. In this communication, an attempt has been made to look into the possibility if we can meet the entire domestic needs of pigeonpea through the adoption of hybrid technology. It is a big question but the goal is not impossible. The estimates shown in Table 18 show that if only 2% of total pigeonpea area can be brought under hybrids, the target can be met with ease. This will require seed production in only 700-800 hectares.

The hybrid breeding program in pigeonpea is unique and the first among the food legume. The concept of hybrid in this crop was conceived in 1974; and it is based on a small window of natural out-crossing present in the crop. This natural out-crossing in pigeonpea is mediated by a range of flying insects, which forage on large bright colored flowers of the crop in search of nectar and in the process affect the cross-fertilization. In the last 40 years a lot of basic and applied research has been successfully completed and over 50 research papers have been published in national and international journals of repute. A coordinated effort of scientists of different institutions has led to the release of two hybrids, one for central and another for peninsular zone of India.

Molecular biology labo-ratories are becoming very common in every University and research institutes and a good laboratory manners is extremely important as many of the equipments are very expensive and sensitive. Hence familiarity with equipments becomes very crucial before starting any experiment.

Introduction

This section of the manual contains instructions for preparing stock solutions that are commonly used in laboratory protocols for the analysis of DNA, RNA and proteins and visualization of gene or protein expression. Each of these stock solutions is provided as either the most convenient concentration or the most frequently used stock concentrations to be used in the protocols of an individual laboratory.

All chemicals must be reagent grade or molecular biology grade, and the water used in the preparation of all solutions must be the highest quality available. Use sterile-glass distilled deionised water whenever possible. Unless otherwise stated, most solutions required sterilization either by filtration or autoclaving.

Plant cell culture
Introduction

As the human population is expanding there has been increasing concern over whether current rates of food productions can support the growing population. In response to the increasing demand, scientists and farmers have been working hard to come up with new ideas to meet these needs. These ideas range from simple procedures including selection of the best seeds to more complex procedures requiring the transplantation of genes into different plant species. This chapter focuses on one of the important aspects of plant science which is the growth of plant cells in an artificial media. This process is known as plant cell culture and can be applied in many different ways. 

DNA Isolation Protocol
Introduction

Most of the all procedures involved in molecular biology experiments require the purified form of nucleic acid. It forms a fundamental requirements for genome characterization, gene mapping and for identification and isolation of gene for genetic engineering. The most important step involved in removal of proteins which is carried out in different ways. The following protocol is one of the most commonly used protocols in animal, plant and microbial samples.

Introduction

In order to use a fine quality of DNA for various molecular biological work like sequencing, cDNA synthesis and cloning, RNA transcription, transfection, nucleic acid labeling (random primer labeling), etc., quality and quantity of DNA is extremely important to have the defined template concentration. Failure to produce results from the above techniques can sometimes be attributed to an incorrect estimate of the DNA template used.

Introduction

This chapter gives first hand information on PCR for beginners. PCR is used to amplify a short, well-defined part of a DNA strand. This can be a single gene, or just a part of a gene. As opposed to living organisms, the PCR process can copy only short DNA fragments; usually up to 10 kb (kb stands for kilo base pairs). Certain methods can copy fragments up to 40 kb in size, which is still much less than the chromosomal DNA of a eukaryotic cell-for example, a human cell contains about three billion base pairs (Kary Mullis, 1983).

Introduction

Gel electrophoresis is a technique for separating charged molecules with different sizes. Two kinds of gels are commonly used: agarose and polyacrylamide. Agarose gels can be applied to a wider range of sizes than polyacrylamide gels.  By using standard agarose electrophoresis, nuclei acids up to 50 kb may be separated. If Pulsed Field Gel Electrophoresis is used, the upper limit can be extended to 10 Mb. Polyacrylamide gels may separate nucleic acids that differ in length by only 1 nucleotide if their length is less than 500 bp (Sambrook et al., 1989).

Introduction

Restriction enzymes, recognizes specific base sequences in double-helical DNA and cleaves at specific places, both strands of a duplex containing the recognized sequences. To biochemists, these exquisitely precise scalpels are marvelous gifts of nature. They are indispensable for analyzing chromosome structure, sequencing very long DNA molecules, isolating genes, and creating new DNA molecules that can be cloned. Werner Arber and Hamilton Smith discovered restriction enzymes, and Daniel Nathans pioneered their use in the late 1960s.

Southern Blotting
Introduction

Southern hybridization was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s. To oversimplify, DNA molecules are transferred from an agarose gel onto a membrane. Southern blotting is designed to locate a particular sequence of DNA within a complex mixture. It is also called Southern blotting since the procedure for transfer of DNA from the gel to the nitrocellulose filter resembles blotting. In Southern hybridization, a sample of DNA containing fragments of different sizes is subjected to electrophoresis using either polyacrylamide or agarose gel.

Introduction

Only after the discovery of restriction endonucleases it became possible to digest any DNA molecule at a specific site and any two pieces of DNA could be joined together with the help of another modifying enzyme, the DNA ligase. Consequently, a new branch of molecular biology, namely genetic engineering has been developed. With the help of genetic engineering techniques, it is now possible to identify and isolate the gene (for a protein of interest and insert it into the genome of another organism with the help of a suitable vehicle. The foreign gene thus cloned, can be amplified to produce millions of its copies. The insert gene can be expressed by the host to synthesize the gene product. The technique can also be used for understanding the regulation of expression of the cloned gene. The possibilities are endless and probably sky is the only limit. In this chapter we will briefly discuss the basic principles and protocol involved in gene cloning.

Introduction

DNA sequencing is the process of determining the nucleotide order of a given DNA fragment. The most commonly used sequencing method is the dideoxy method. This method uses dideoxynucleotide triphosphates (ddNTPs) which have an H on the 3’ carbon of the ribose sugar instead of the normal OH found in deoxynucleotide triphosphates (dNTPs). Dideoxynucleotides are chain terminators. In a synthesis reaction, if a dideoxynucleotide is added instead of the normal deoxynucleotide, the synthesis stops at that point because the 3’OH necessary for the addition of the next nucleotide is absent (Sanger et al., 1977).