eChapters

Breeding methods for vegetable crops are basically similar to those adopted in others like cereals, oilseeds or pulses. Certain aspects, peculiar to vegetables, however, require specific attention: (i) In many cases, the edible part of the plant is not the seed (as cauliflower, cabbage, carrot and onion) and hence while breeding for improved plant part that is used as vegetable, its seed production capacity needs due consideration, (ii) Vegetables like tomato, garden pea and onion are consumed in different forms (fresh or processed) and the end use of the product (like processing quality) has to be taken into account in breeding programmes, (iii) Consumer preferences (as for example in eggplant) size, colour and shape of the vegetable has to be given due emphasis, (iv) Being perishable in nature, keeping quality and suitability for long distance transportation are important attributes in vegetable crops unlike grain crops where bulk handling and haulage can be mechanised, (v) Another peculiarity with vegetable crops is that they vary widely in modes of reproduction necessitating appropriate choice of breeding methods. In addition to vegetatively propagated crops like potato, sweet potato, cassava (tapioca), garlic, pointed gourd or “Parwal (Trichosanthes dioica L.) and ivy gourd or ‘Kundru’ (Coccinia cordifolia L.), there are many seed propagated vegetable crops which are auto- or allogamous or intermediate (Table 1).

Within these broad groups, variations in the extent of cross- or self-pollination, namely, 15-20 per cent natural cross-pollination in chillies and 20-25 per cent natural self-pollination in muskmelon have been recorded.

Forage crops are plant species used to feed domestic animals (such as cattle, sheep, goat, pig, and poultry). Both cultivated and wild plants can serve as forage. The domestic animals feed on forages either by grazing or stall feeding (cut fodder eaten in stalls).

Grazing, that is pasturing of animals, is cheaper and allows production of both livestock and dairy produce at a lower cost because no expense is involved in growing, cutting, transporting or feeding. In North and South America, Australia, New Zealand, and in many other temperate countries pastures are widespread and popular because sufficient land is available. While land is cheap and plentiful, labour is scarce and expensive; hence management of natural, and in recent years sown pastures has received much attention.

Until about fifty years ago, the natural grasslands in the Western countries met the minimum needs of the farmers and grassland management was operated at a level comparable to subsistence agriculture of many developing countries. In other words, emphasis was laid on exploitation of natural productivity of the grassland, using a low level of management based on minimum use of inputs like chemical fertilizers. However, it was soon realized that productivity of native grasslands will have to be greatly improved if the high demands for meat and milk were to be adequately met.

In India, however, out of a total cultivable area of about 131.11 million ha less than 2 per cent is utilized for forage crops because feeding the large human population takes the first preference for land use to produce grain and cash crops. It is, therefore, necessary to intensify efforts, particularly in over-populated countries, for a more efficient production of high yielding forage crops to get maximum return out of minimum land.

Introduction

Tree improvement through the application of genetic principles is directed towards modifying the heredity of tree populations so that they are able to meet the needs of the forester and the wood based industry. The breeder of annual crop plants resorts to hybridisation, selection, testing of the improved strains, multiplication of improved seeds and finally their release for commercial planting in that logical sequence. But the tree breeder, whose career may be shorter than the life cycle of the tree species taken up for improvement, tends to carry out the above operations in a modified form and often some of the operations are carried out concurrently.

Many of our tree species are wild unselected populations. They are, therefore, expected to exhibit considerable natural variability for many characters and also to respond well to selection for further improvement. Further, altitudinal and latitudinal variation in many characters between populations are expected for those tree species which have a wide geographical distribution. So, the selection of desirable seed sources or provenances is an essential step that is basic to all tree improvement programmes. The right provenance is identified through well laid out provenance trials at a number of locations in the area where the improved selection is to be grown. Further improvement is then planned and executed for exploiting the naturally available intrapopulation variation. The variation normally measured is the phenotypic variation which is the result of interaction of the genotype of the individual with the environment. However, what is essential for selection to be effective is genetic variation for the character under consideration. Genetic variation is assessed by appropriate progeny trials. Since many of the economically valuable characters of tree species are polygenically controlled and exbibit continuous variation, measurements have to be in metric units. The use of biometrical techniques for assessing and analysing this variation, therefore, becomes an essential requirement.

Most varieties of fruit plants have originated as chance seedlings or through somatic mutation and have been maintained and multiplied through vegetative propagation by fruit growers. The improvement of fruit plants through breeding has not yet attracted the serious attention of the research workers. Unlike field crops, fruit plant breeding is time consuming and laborious. Studies on the mode of inheritance of important economic characters of fruit plants are difficult because of the heterozygous nature of the plants. Moreover, varying degrees of sexual incompatibility have complicated the situation. Due to a long juvenile phase, it takes a long time to identify a useful hybrid. This factor has become a serious handicap because most research workers leave or shift from the programme before tangible results are obtained.

During the last two decades, considerable useful information has been generated on breeding methodology for the improvement of some of the important fruit crops. In this chapter fruits like mango, banana and papaya have been included.

The immediate and long-term objective of modern plant breeding in all countries irrespective of the state of their advancement is increasing the availability of food to meet the calorie-protein requirement of their people. In the past, breeders have concentrated their efforts on increasing the yield of the important crop species. Better adaptation to local environmental conditions, high production and better resistance to the major pests and diseases have been among the priority goals of genetic plant improvement. Yielding ability and stability undoubtedly deserve high priority but enough attention also needs to be paid to improving nutritional characteristics of the plant produce for their maximum utilisation. Awareness of nutritional requirements and the optimal characteristics of crop products to be utilised as food and feed, therefore, becomes an important parameter of breeding effort.

The main source of food in the developing world is cereals which provides 45 to 85 per cent of the total calories and 50 to 80 per cent of the total protein requirements. The consumption of animal proteins, which are nutritionally well balanced, is low and costly and beyond the reach of the low income groups. On the other hand, plant proteins are cheaper to produce and easier to store and transport. All these factors go in favour of increasing the quantity and quality of cereal proteins in order to improve the dietary and nutritional status of the masses in the developing world.

Proteins are made up of about twenty different amino acids. For man, eight of these amino acids, valine, methionine, leucine, isoleucine, threonine, phenylalanine, lysine and tryptophan, are essential (EAA) for constituting a well balanced diet. In impioving the grain quality of cereals consideration should, therefore, be given apart from the content of carbohydrates, fats, minerals and vitamins in the grains, to improving the protein content as also the amino acid composition of the protein.

Plant diseases, in spite of the various measures adopted to control them, continue to be a major cause of crop losses. At times these losses have been so heavy that they have caused severe famines. Diseases are caused by a wide variety of organisms such as fungi, bacteria, viruses, mycoplasma and nematodes. The diseases may be partially or completely controlled by chemical, cultural and genetic methods. When no single method is effective in controlling a disease, integrated control constituting a combination of the three methods of disease control (chemical, cultural, genetic) may be effective. If host resistance alone is sufficient to check a disease, then it is to be preferred to other methods because it is the least expensive, has no adverse environmental effects and involves no specific action on the part of the farmer, even when only partial, genetic resistance supplements chemical control and makes it more effective in comparison with chemical control on a completely susceptible cultivar.

Many plant diseases have been successfully controlled by exploiting genetic resistance especially where differentiation in the pathogen with respect to resistance genes in the host is lacking. In some cases, even when pathotype differentiation occurs in the pathogen, genetic resistance in combination with other methods has given adequate control. Breeding for resistance to highly variable air-borne fungal pathogens has claimed much time and effort of plant breeders and success has often been transient. The reasons for the shortlived success have become clear from research during the last quarter of a century.

A close relationship between the mode of reproduction of a cultivated plant and the methods to be employed for its improvement is now well accepted. Crop plants are broadly placed in two groups according to the mode of their reproduction: sexually reproduced and asexually propagated or reproduced plants. Asexual reproduction does not involve the union of gametes and/or reduction in chromosome numbers.

Plants may reproduce asexually in a variety of ways. In 1980, Mayo has identified the following methods of asexual reproduction on the basis of the classification made earlier by Stebbins in 1941 and Brown in 1972.

1) Origin and distribution

Pearl millet (Pennisetum americanum (L) Leeke, 2n-14) originated in west Africa from where it was introduced into India. Large variability is still found in Africa. Pearl millet is a dual purpose crop; its grain is used for human consumption and its fodder as cattle feed. It is cultivated mainly in Africa and Asia, primarily as a grain crop in an area of 26 million ha with a production of 13 million tonnes. As a poor man’s bread, it sustains a large population of Africa and Asia. It also contributes to the economy of countries like the U.S.A. where it is grown as a forage crop.

In India, Pennisetum americanum (bajra) is cultivated on 11 to 12 million ha land with a production of about 6 million tonnes. It is grown on marginal lands and in areas of scanty rainfall. Rajasthan, Gujarat, Maharashtra, Uttar Pradesh and Haryana account for about 90 per cent of the total area under this crop.

Cross-pollinated species, or outbreeders, are highly heterozygous, heterogeneous and carry a higher mutation load than the inbreeders. Their mode of reproduction bestows upon them considerable heterozygous balance under panmitic population structure. Inbreeding such species disturbs the natural rhythm and leads to inbreeding depression. It is, therefore, essential that if selfing has created any disturbance in outbreeders, the heterozygous structure be restored. Since artificial selfing and outcrossing can be carried out in such crops at will, all breeding methodologies, even those meant for self-pollinated crops, can be used for their improvement. In fact, allogamous crops have provided opportunities for innovating breeding procedures to achieve the maximum efficiency by combining selfing and outcrossing cycles.

Thus, the classical separation of breeding procedures for self- and cross-pollinated crops has become unnecessary, particularly, for outbreeders which are amenable to selfing. Exploitation of heterosis has been the major objective in cross-pollinated crops and most breeding procedures developed earlier aimed at maximising heterosis. In later years, with better understanding of genetic structure of populations and availability of efficient biometrical methods of estimating genetic parameters, newer procedures of accumulating desirable genes have been developed. The presently available breeding procedures for cross-pollinated crops can be broadly classified under two heads: (1) Hybrid development, and (2) Population improvement. Both these methodologies, though independent, are not mutually exclusive. The steps for operating the two procedures, however, have contrasting requirements. For hybrid development, fixation of genes through cycles of selfing is necessary whereas such fixation is avoided while developing populations. As can be seen from the scheme given below, in both cases the homozygosity attained through initial selfing is converted to heterozygosity at the end of breeding programme. Irrespective of whether hybrids, synthetics or composites are being developed, great emphasis is placed on the attainment of homogeneity in the end products.

Man-directed evolution of distinct types and cultivars (cultivated populations) of self-fertilised crops had their beginning about 10,000 to 12,000 years ago. As a breeding system, self-fertilisation favoured constancy and fitness (Stebbins, 1950) of the genotypes to the immediate environment. This provided an alternative to the slash and burn migrant agriculture of the Neolithic farmers (7,000-6,000 B.C.) and to stabilisation of production.

Manu Smriti (700–500 B.C.) proclaimed Subeejam Sukshetre Jayate Sampadyate meaning that good seed in a good field yields abundant produce. The knowledge that good seed is essential for a good harvest, led to conscious efforts for selecting types which provided a dependable source of food. Besides their utility as food, choice of plants and their seeds as progenitors of the next generation depended on personal fancy, observation and experience. As farmers learnt their crop plants better, they sorted them out into similar types (species). While migrating from the centres of origin they carried seeds of mixed populations.

Self-pollinated cereal crops of barley, wheat, oats and important grain legumes such as lentil, peas and chickpea spread from the Near East Centre, first to far and remote areas of Asia, Europe and Africa; and subsequently in comparatively recent times, to the Americas and Australia. In new environments, the introduced mixed populations diversified into well adapted identities of land races. These land races continued to be the main basis of cereal production till early 1900. Improvement in crop plants made by some early civilisations was mainly through introduction and mass selection, based on the natural tendency of preserving the seeds of preferred colour and size from vigorous, healthy and more productive plants.

It was only after the rediscovery of Mendel’s laws in 1900 that breeding procedures based on the knowledge of the genetic principles and the inheritance pattern of different characteristics were developed. This permitted the improvement of crops with objectivity and predictability.

Plant breeding is essentially a technology. Any technology will stand the test of time if it is based on sound concepts. In the case of plant breeding, the concepts are provided by the science of genetics.

Plant productivity in its broad sense requires consideration of not only yield but also of a number of its direct and indirect components. For example in groundnut, besides pod yield, shelling percentage, 100-kernel weight and oil content are components relevant to defining productivity. A number of physiological (like biomass, leaf area, specific leaf weight), morphological (like days to flowering, number of branches, number of mature seeds), nitrogen-fixing, (where relevant, for example, number of nodules, nodule mass, nitrogenase activity) and such other characters influence yield directly or indirectly.

Yield and most yield contributing characters are quantitative in nature. A quantitative character is one which shows continuous variation. In contrast, qualitative characters like flower colour and seed coat colour show, in general, discrete variation and are controlled by fewer genes with major effects. A quantitative trait, however, is governed by many genes. The range of values of a quantitative character is described by a continuous distribution, usually assumed to be a normal distribution. The distribution is specified by the two parameters: mean and variance.

NTSYSpc 2.0 software is used for various types of analysis including calculation of similarity index values and construction of dendrogram. Construction of dendrogram using gel data in the form of bands. This software accepts zero-one data.

Metagenomics is a relatively new field that combines molecular biology and genetics in an attempt to identify, and characterize the genetic material from environmental samples and apply that knowledge. The genetic diversity is assessed by isolation of DNA followed by direct cloning of functional genes from the environmental sample. But more importantly, it is extremely beneficial in the study of synthetically made genomes. Metagenomics is described as “the comprehensive study of nucleotide sequence, structure, regulation, and function”. Scientists can study the smallest component of an environmental system by extracting DNA from organisms in the system and inserting it into a synthetic model organism. The model organism then expresses this DNA where it can be studied using standard laboratory techniques. The development of metagenomics stemmed from the ineluctable evidence that as-yet-uncultured microorganisms represent thevast majority of organisms in most environments on earth. This evidence was derived from analyses of 16S rRNA gene sequences amplified directly from the environment, an approach that avoided the bias imposed by culturing and led to the discovery of vast new lineages of microbial life.

Metagenomics is employed as a means of systematically investigating, classifying, and manipulating the entire genetic material isolated from environmental samples. This is a multi-step process that relies on the efficiency of four general steps as shown in the diagram. The procedure consists of: the isolation of genetic material, the manipulation of the genetic material, library construction, and the analysis of genetic material in the metagenomic library. Metagenomics provides a second tier of technical innovation that facilitates study of the physiology and ecology of environmental microorganisms. Novel genes and gene products discovered through metagenomics include the first bacteriorhodopsin of bacterial origin; novel small molecules with antimicrobial activity; and new members of families of known proteins, such as an Na⁺(Li⁺)/H⁺ ant porter, RecA, DNA polymerase, and antibiotic resistance determinants. Reassemblyof multiple genomes has provided insight into energy and nutrient cycling within the community, genome structure, gene function, population genetics and micro heterogeneity, and lateral gene transfer among members of an uncultured community. The application of metagenomic sequence information will facilitate the design of better culturing strategies to link genomic analysis with pure culture studies.

Sanger Method for DNA Sequencing

One necessity first know a little about the structure of DNA:

1. A segment of DNA, which is ordinarily double stranded, has a specific orientation, as it has a 5' (read as “5 prime”) and a 3' (“3 prime”) end. This can be simply thought of as a front and tail end to the DNA segment.
2. When DNA is synthesized in the lab, the two strands are separated and new bases are added to the 3' end-thus DNA is assembled from the 5' to 3' end.
3. DNA cannot be synthesized from scratch. A short piece of DNA, called a primer, is required for the reaction to begin.
4. Primers are designed such that they are able to bind to the target DNA, the binding of which is the initiator for DNA synthesis.

Introduction

The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation, morphogenesis, as well as abnormal or diseased conditions. Northern blotting involves the use of electrophoresis to separate RNA samples by size and detection with a hybridization probe complementary to part of or the entire target sequence. The term ‘northern blot’ actually refers specifically to the capillary transfer of RNA from the electrophoresis gel to the blotting membrane; however the entire process is commonly referred to as northern blotting. The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University. Northern blotting takes its name from its similarity to the first blotting technique, the Southern blot, named for biologist Edwin Southern. The major difference is that RNA, rather than DNA, is analyzed in the northern blot.

Introduction

Western blot analysis can detect one protein in a mixture of any number of proteins while giving you information about the size of the protein. It does not matter whether the protein has been synthesized in vivo or in vitro. This method is, however, dependent on the use of a high-quality antibody directed against a desired protein. So you must be able to produce at least a small portion of the protein from a cloned DNA fragment. You will use this antibody as a probe to detect the protein of interest.

Western blotting tells you how much protein has accumulated in cells. If you are interested in the rate of synthesis of a protein, Radio-Immune Precipitation (RIP) may be the best assay for you. Also, if a protein is degraded quickly, Western blotting won’t detect it well; you’ll need to use (RIP). See the section on RIP for more information, as well as a helpful comparative chart that illustrates the differences between these two techniques.

Limitations of Bacteria

• Normally genetically engineered bacteria are very useful for the large scale production of valuable eukaryotic proteins.
• Bacteria grow rapidly, They grow on relatively simple media, they are easy to handle, they are easily transformed, they can grow on wide range of substrates, they multiply, fast, they contain simple and well characterized genome, they can be easily modified but even though, they have their own problems…

1. Bacteria do not have intrudes and so they do not have any system or machinery to remove introns.
2. Many eukaryotic proteins undergo different types of modifications after they are produced. These modifications are known as “post translation al modifications”.
3. Each bacterial cell usually contains many copies of the plasmid but there is no mechanism to ensure the equal distribution of plasmids in daughter cells.

To isolate a streptomycin resistant mutant of E.coli by Indirect selection using Replica Plate Method.

Introduction

J. Lederberg & his wife E. M. Lederberg devised this technique in 1952.

U.V. light coming from the sun emits strong radiation to extent the antibacterial effect in the microbial population. However it has a very short penetration power, UV light is used as a mutagenic agent & was used by Beedle & Tatum to obtain biochemical mutation. The mutation rate is only once in 10^-9 cells per population. Then hence using mutagens like UV light by 10 to 1000 folds will increase this mutation rate.

In isolation of the mutant using UV light the dose of UV light is measured by its units/nm² organisms. The time selected for the mutation is calculated on the basis of 0.1 to 1% survival & a suitable biochemical character is used to evaluate the mutation.

To isolate a streptomycin resistant mutant of E.coli by Direct selection using Gradient Plate Method.

Introduction

Zylbalskii devised this technique.

A mutation is any change in the nucleotide sequence of DNA. These changes are heritable. Although most mutations are harmful to the organism, a small percentage is beneficial under certain environmental conditions. Mutation can occur during chromosome duplication and they may be spontaneous of they may be induced by mutagenic agent. In this experiment, we select for a mutation causing streptomycin resistance in E. coli. Resistant mutants will be detected directly using thy gradient plate method.

Principle

Microbiologist developed a highly efficient method for isolation of antibiotic resistant mutant. The gradient is said as gradient of antibiotic formed on an agar plate. A gene imparting resistant toward an antibiotic is present in plasmid or chromosome. The occurrence of mutant with different level of streptomycin resistant involves mutation of gene with unequal potential. This means that under certain cells degree of mutation is more while few shows less mutation. Some cells show intermediate resistant while others show completes resistant. The resistant in cell against streptomycin of this type, while in case of penicillin resistant increases step arise & so resistant mutant of high concentration of penicillin cannot be isolated/ obtained. While it is possible with streptomycin obtaining high resistance to penicillin stepwise & so this stepwise increase in resistance involves mutation of gene with equal potential. This theory of mutation through multi step procedure. The gene direct the organism to produce an enzyme, which inactivates an antibiotic & thus resistance to antibiotic, is observed in case of cells. Resistance of antibiotic is a plasmid-mediated resistance.

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.

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

Genetic variability is essential for the evolutionary success of all organisms. In diploid eukaryotic the processes of crossing over exchange of genetic material between homologous chromosomes and meiosis contributes to his variability. In haploid, asexually reproducing prokaryotic organisms, genetic recombination may occur by conjugation transduction, and transformation. In this experiment only the process of conjugation is considered.

Conjugation is a matting process during which a one direction transfer of genetic material occurs at physical contact between two sexually differentiated cell types. This differentiation or existence of different mating strains in some bacteria, is determined by the presence of a fertility factor of F factor, within the cell. Cells that lack the F factor are recipients (females) of the genetic material during conjugation and are designated as F^- . cells possessing the F factor have ability to act as genetic donors (males)during the mating. If this F factor is extra chromosomal (a Plasmid or episome), the cells are designated as F⁺; most commonly only the F factor is transferred during conjugation. If this factor becomes incorporated into the bacterial chromosome there is a transfer of chromosomal genes, although generally not involving the entire chromosome or the F factor. The resulting cells are designated as Hfr, for high frequency recombinants.

Introduction

The most common method of bacterial transformation utilizes high levels of calcium chloride to facilitate the entry of plasmid DNA vectors into E.coli in conjunction with structural alteration of the bacterial cell wall. In general, there are three basic steps in the introduction of plasmid DNA unto cells.

1. Preparation of “competent” cells to enable cells to accept DNA.
2. Transformation of these competent cells, and
3. Selection of transformants.

Most E.coli strains exhibit transformation efficiency (measured as transformants per microgram plasmid DNA) in the range of 10⁵ to 10⁸.

The factors influencing this efficiency depends upon the condition that render cells competent.

To Prepare the Competent Cell and Carry Out Transformation Introduction

Transformation results from the uptake of purified DNA by bacterial cells and is the most frequently used procedure for introducing recombinant DNA molecules into bacteria. It is now widely used to transfer small plasmids from one bacterial strain to another. DNA is extracted from donor cell and used to treat the recipient cells that have been rendered more susceptible to DNA uptake. These competent cells allow DNA to enter through pores or channels in the cell membrane and in the case of plasmids, permit subsequent plasmid replication.

Introduction

Ligation of DNA fragment is one prime important in genetic engineering because without ligation we could not transfer the interest gene.DNA cloning requires the DNA sequence of interest to be inserted in a vector DNA molecule. For this, both the vector as well as insert DNA is prepare by digestion with compatible restriction enzymes, so that the ends produced during digestion is complementary in both. When setting up ligation, it is important to consider the permutations that can occur and bias the relative concentration of DNA accordingly. Usually a 5- to 10-fold excess of insert over the vector DNA is the norm. This ensures that enough ligated product will be produced in the right orientation.

Introduction

Southern blotting 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. For example, Southern Blotting could be used to locate a particular gene within an entire genome. The amount of DNA needed for this technique is dependent on the size and specific activity of the probe. Short probes tend to be more specific. Under optimal conditions, you can expect to detect 0.1 pg of the DNA for which you are probing.

This diagram shows the basic steps involved in a Southern blot.

Introduction

The AFLP amplified fragment polymorphism also known as selective restriction fragment amplification (SRFA) technique is used to visualize hundreds of amplified DNA restriction fragments simultaneously. The AFLP band patterns, or fingerprints, can be used for many purposes, such as monitoring the identity of an isolate or the degree of similarity among isolates. Polymorphisms in band patterns map to specific loci, allowing the individuals to be genotyped or differentiated based on the alleles they carry. produces highly complex DNA profiles by arbitrary amplification of restriction fragments ligated to double-stranded adaptors with hemi-specific primers harboring adaptor-complementary 5' termini.The technique has been widely used in the construction of genetic maps containing high densities of DNA marker loci.

The AFLP protocol amplifies restriction fragments obtained by endonuclease digestion of target DNA using “universal” AFLP primers complementary to the restriction site and adapter sequence. However, not all restriction fragments are amplified because AFLP primers also contain selective nucleotides at the 3' termini that extend into the amplified restriction fragments. These arbitrary terminal sequences result in the amplification of only a small subset of possible restriction fragments. The number of amplified fragments can therefore be “tailored” by extending the number of arbitrary nucleotides added to the primer termini. Alternatively, the use of endonuclease combinations that vary in their restriction frequency can also be used to tune the number of amplicons. Generally, the abundant restriction fragments produced from complex genomes require of AFLP primers with longer selective regions. Conversely, analysis of small genomes require of only few arbitrary nucleotides added at the primer 3' termini. The resulting AFLP fingerprints are usually a rich source of DNA polymorphisms that can be used in mapping and general fingerprinting endeavors.

RAPD stands for Random Amplification of Polymorphic DNA. It is a type of PCR reaction, but the segments of DNA that are amplified are random. The scientist performing RAPD creates several arbitrary, short primers (8-12 nucleotides), then proceeds with the PCR using a large template of genomic DNA, hoping that fragments will amplify. By resolving the resulting patterns, a semi-unique profile can be gleaned from a RAPD reaction.

No knowledge of the DNA sequence for the targeted gene is required, as the primers will bind somewhere in the sequence, but it is not certain exactly where. This makes the method popular for comparing the DNA of biological systems that have not had the attention of the scientific community, or in a system in which relatively few DNA sequences are compared (it is not suitable for forming a DNA databank). Because it relies on a large, intact DNA template sequence, it has some limitations in the use of degraded DNA samples. Its resolving power is much lower than targeted, species specific DNA comparison methods, such as short tandem repeats. In recent years, RAPD is used to characterize, and trace, the phylogeny of diverse plant and animal species.

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.

Introduction

This method can be scaled to yield amounts of DNA ranging from less than ten to more than hundreds of micrograms of DNA. However, shearing forces are generated at every step, with the result that the DNA molecules in the final preparation rarely exceed 100-150 kb in length. DNA of this size is adequate for Southern analysis on standard agarose gels, as a template in Polymerase chain reactions (PCRs), and the construction of genomic DNA libraries in bacteriophage Lambda vectors.

Principle

The method described in protocol involves digesting eukaryotic cells or tissues with proteinase K in the presence of EDTA (to sequester divalent cations and thereby inhibit DNases) and solubilizing membranes and denaturing proteins with a detergent such as SDS. The nucleic acids are then purified by phase extractions (isopropanol). Contaminating RNA is eliminated by digestion with an RNase, and low molecular-weight substances are removed by dialysis (or centrifugation).

DNA can be easily isolated and purified after size selection on an agarose gel. The fragment of interest is simply cut out of the gel with a razor blade and purified by a number of different methods.The easiest is to use a method that involves first dissolving the agarose slice in a solution at 50°C, then binding the DNA from the melted agarose to a silica-gel membrane.

Principle

In this method following gel electrophoresis using low gelling/ melting temperature agarose, the band of interest is removed from the gel. The agarose slice is then melted and subjected to phenol extraction.

Introduction

The primary tools used by the molecular biologists in manipulating DNA are restriction enzymes and other DNA/RNA modifying enzymes. Over the years, the number and uses of these enzymes have increased as new molecules have been discovered. Restriction endonucleases are bacterial enzymes that cleave double-stranded DNA. They are present in bacteria presumably to destroy DNA from foreign sources. By cleaving the foreign DNA at specific sites. The host bacterial DNA os protected from cleavage because specific recognition sites are modified, usually by methylation form methylase at one of the bases in the site, making the site no longer a substrate for RE cleavage. Host bacteria used to propagate cloned DNA in the laboratory arte usually mutant in the host restriction genes, thus their intracellular enzyme activities will not destroy the foreign recombinant sequences. The endonuclaase with its accompanying methylase is called a restriction modification (R-M) system.

At least four different kinds of R-M systems exist characterized on the basis of the subunit composition, cofactor requirements and type of DNA cleavage. Most characterized enzymes belong to type II class, together with type IIS class. They comprise the commercially available restriction enzymes used for DNA analysis and manipulation. Type I and Type III enzymes are relatively uncommon and a few additional enzymes fit none of the classes.

Among more than 3000 different enzymes isolated from bacterial strains many share common specificities. Restriction enzymes that recognize identical sequences have been called as isoschizomers.

History of PCR

A 1971 Kleppe and co-workers first described a method using an enzymatic assay to replicate a short DNA template with primers in vitro. However, this early manifestation of the basic PCR principle did not receive much attention, and the invention of the polymerase chain reaction in 1983 is generally credited to Kary Mullis.

At the core of the PCR method is the use of a suitable DNA polymerase able to withstand the high temperatures of >90°C (>195°F) required for separation of the two DNA strands in the DNA double helix after each replication cycle. The DNA polymerases initially employed for in vitro experiments presaging PCR were unable to withstand these high temperatures. So the early procedures for DNA replication were very inefficient, time consuming, and required large amounts of DNA polymerase and continual handling throughout the process.

The discovery in 1976 of Taq polymerase (a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally occurs in hot (50 to 80 °C (120 to 175 °F) environments) paved the way for dramatic improvements of the PCR method. The DNA polymerase isolated from T. aquaticus is stable at high temperatures remaining active even after DNA denaturation, thus obviating the need to add new DNA polymerase after each cycle. This allowed an automated thermocycler-based process for DNA amplification.

Developed the PCR in 1984 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. In 1993 Mullis was awarded the Nobel Prize in Chemistry for his work on PCR.

Introduction

Electrophoresis is the migration of charged molecules in solution in response to an electric field. Their rate of migration depends on the strength of the field; on the net charge, size and shape of the molecules and also on the ionic strength, viscosity and temperature of the medium in which the molecules are moving. As an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used analytically to study the properties of a single charged species, and as a separation technique. SDS-PAGE is the most widely used method for qualitatively analyzing any protein mixtures. The method is based on the separation of proteins according to their size and then locating by binding them to a dye. It is particularly useful for monitoring protein purity and determines their relative mass. The SDS-PAGE enables the students to understand the theory behind the techniques in checking the purity of the protein and identifying the protein in a test sample.

Separation of Proteins

Proteins are amphoteric compounds; their net charge therefore is determined by the pH of the medium in which they are suspended. In a solution with a pH above its isoelectric point, a protein has a net negative charge and migrates towards the anode in an electrical field. Below its isoelectric point, the protein is positively charged and migrates towards the cathode. The net charge carried by a protein is in addition independent of its size - i.e.: the charge carried per unit mass (or length, given proteins and nucleic acids are linear macromolecules) of molecule differs from protein to protein. At a given pH therefore, and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both size and charge of the molecules.

Electrophoresis is a method of separating out large molecule ( DNA or protein). An electric current is passed through a medium containing the molecules, and each molecule travels at a different rate depending on its electrical charge, size and shape. Separation by electrophoresis is based on these differences. In electrophoresis, agarose and acrylamide gels are used for electrophoresis of nucleic acids and proteins.

What is Gel Electrophoresis?

A gel is a solid form. The term electrophoresis describes the migration of charged particle under the influence of an electric field. Electro refers to the energy of electricity. Phoresis, from the Greek verb phoros, means “to carry across.” Thus, gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. Activated electrodes at either end of the gel provide the driving force. A molecule’s properties determine how rapidly an electric field can move the molecule through a gelatinous medium.

Principle

In this method the estimation of pentose sugar is carried out. Acid hydrolysis of RNA releases ribose (a pentose sugar) which in the presence of strong acid undergoes dehydration to yield furfural. Orcinol, in the presence of ferric chloride as a catalyst, reacts with furfural producing a green colored compound which can be quantitatively estimated at A_{665 nm}. DNA gives a limited positive reaction with Orcinol test.

Materials and Reagents

1. Spectrophotometer
2. Boiling water bath
3. 5% chloroform (HClO₄)
4. Standard RNA solution : Dissolve yeast RNA (500ìg/ml) in 5% HClO₄ . make different dilutions to obtain solutions containing 100 – 500ìg RNA/ml with 5% HClO₄ .
5. Orcinol reagent : Dissolve 100mg of ferric chloride (FeCl₃.6H₂O) in 100 ml of concentration HCl and then add 3.5 ml of 6% solution of Orcinol prepared in alcohol.

To Estimate the DNA Using Diphenylamine

Introduction

DNA estimation is helpful to know about the total amount of DNA of prokaryotic as well as eukaryotic system. One method used for DNA estimation is Diphenylamine assay, is that of Burton who modified the original procedure of Dische. This procedure is based on treatment of nucleic acid with hot perchloric acid to hydrolyze DNA nucleotides. Diphenylamine reacts specifically with deoxyribose in DNA to form a blue-colored complex. Since diphenylamine does not react with ribose, one can assay for DNA in the presence of contaminating RNA.

Principle

The DPA method is based on the colorimetry measurement of products formed by the reaction of ù- hydroxylevulinaldehyde (from the deoxyribose released after depurination of DNA) with diphenylamine in 2.5 N perchloric acid.

When DNA is treated with diphenylamine under acidic condition (PCA) a blue complex is formed with a sharp absorption maximum at 595 nm (600 nm). In acid solution, the straight chain form of the deoxypentose is converted to highly reactive ω – hydroxylevulinaldehyde that reacts with diphenylamine to give blue complex. The reaction yields two mixture of product which together have absorbance maximum at 595 nm (600 nm). Interferences include proteins, lipids, saccharides and RNA. In DNA the only deoxyribose of the purine nucleotides reacts, so that the value obtained represents half of the total deoxyribose present.

Introduction

DNA denaturation, also called DNA melting, is the process by which double-stranded deoxyribonucleic acid unwinds and separates into single-stranded strands during the breaking of hydrogen bonding between the bases. Both terms are used to refer to the process as it occurs when a mixture is heated, although “denaturation” can also refer to the separation of DNA strands induced by chemicals like urea. For multiple copies of DNA molecules, the melting temperature (T_m) is defined as the temperature at which half of the DNA strands are in the double-helical state and half are in the “random-coil” states. The melting temperature depends on both the length of the molecule, and the specific nucleotide sequence composition of that molecule.

Applications of DNA Denaturation

The process of DNA denaturation can be used to analyze some aspects of DNA. Because cytosine / guanine base-pairing is generally stronger than adenosine / thymine base-pairing, the amount of cytosine and guanine in a genome (called the “GC content”) can be estimated by measuring the temperature at which the genomic DNA melts. Elevated temperatures are associated with high GC content. GC ratios within a genome are found to be markedly variable. These variations in GC ratio within a genome of higher organisms results in a mosaic like formation with islet regions called isochores. This results in the variations in staining intensity in the chromosomes. The isochores include in them essential protein coding genes, termed housekeeping genes and thus determination of ratio of these specific regions contributes in mapping these essential genesbacterial systematics has recommended use of GC ratios in higher level hierarchical classification. For example, the Actinobacteria are characterised as “high GC-content bacteria”. In Streptomyces coelicolor A3(2), GC content is 72%. The GC-content of Yeast (Saccharomyces cerevisiae) is 38%, and that of another common model organism Thale Cress is 36%. Because of the nature of the genetic code, it is virtually impossible for an organism to have a genome with a GC-content approaching either 0% or 100%. A species with an extremely low GC-content is Plasmodium falciparum (GC% = ~20%), and it is usually common to refer to such examples as being AT-rich instead of GC-poor.

Procedure

Place a portion of your RNA sample (approximately 40 mg, hydrated) into a heavy walled Pyrex test tube. Add 1.0 ml of 1 N HCl and seal the tube.

Heat the tube in a boiling water bath for 1 hour.

Cool the tube, open it and place the contents into a centrifuge tube. Centrifuge the contents at 2,000 RPM in a clinical centrifuge to remove any insoluble residue. The supernatant contains your hydrolyzed RNA.

Prepare Whatman filter paper No. 1 for standard one-dimensional chromatography.

Using a micropipette, spot 20 µl of your hydrolyzate onto the paper, being careful to keep the spots as small as possible (repeated small drops are better than one large drop). Allow the spots to completely dry before proceeding.

Place the paper chromatogram into your chromatography tank and add the solvent (acetic acid:butanol:water). Allow the system to function for an appropriate time (approximately 36 hours for a 20 cm descending strip of Whatman #1). Remove the paper and dry it in a circulating air oven at 40° C for about 2 hours.

Locate the spots of nucleotides by their fluorescence under an ultra-violet light source. Expose the paper chromatogram to a UV light source and outline the spots using a light pencil. The order of migration from the point of origin is guanine (light blue fluorescence), adenine, cytilic acid and finally, uridylic acid.

After cafefully marking the spots, cut them out with cutters and place the paper cut outs into separately labeled 15 ml conical centrifuge tubes. Add 5.0 ml of 0.1 N HCl to each tube and allow the tubes to sit for several hours to elute the nucleotides from the paper.

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

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).

DNA Migration in Agarose and Polyacrylamide Gels

Recommended Gel Percentages for Separation of Linear DNA

Mole (mol)

1 mole of any substance contains 6.02 x 10²³ units. The number of moles in a given weight of substance can be calculated using following equation.

1. pH Meter

pH meter/electrode systems are the most common method of measuring pH in the biology laboratory. Although more expensive and more complicated to use than chemical indicators, pH meters provide greater accuracy, sensitivity, and flexibility. When used properly, pH meters can measure the pH of a solution to the nearest 0.1 pH unit or better, and they can be used with a variety of samples.

2. UV Spectrophotometer

Perhaps there is no other technique except UV-visible spectroscopy, which has been so extensively employed and has such a profound impact on progress in biochemical investigations. UV-visible spectroscopy has undoubtedly become an indispensable tool and is routinely used for identification and quantitative estimation of most of the biomolecules. Besides other applications, most of the enzyme assays and the screening of biomolecules recovered in different fractions by separation processes such as column chromatography, density gradient centrifugation etc, rely heavily on spectrophotometer or colorimetric technique.

Search for genetic material—nucleic acid or protein/DNA or RNA?

• Griffith’s Transformation Experiment
• Avery’s Transformation Experiment
• Hershey-Chase Bacteriophage Experiment
• Tobacco Mosaic Virus (TMV) Experiment

Timeline of Events

• 1890 Weismann - substance in the cell nuclei controls development.
• 1900 Chromosomes shown to contain hereditary information, later shown to be composed of protein & nucleic acids.
• 1928 Griffith’s Transformation Experiment
• 1944 Avery’s Transformation Experiment
• 1953 Hershey-Chase Bacteriophage Experiment
• 1953 Watson & Crick propose double-helix model of DNA
• 1956 Gierer & Schramm/Fraenkel-Conrat & Singer Demonstrate RNA is viral genetic material

BC

1750 The Sumerians brew beer.

500 The Chinese use moldy soybean curds as an antibiotic to treat boils.

250 The Greeks practice crop rotation to maximize soil fertility.

100 Powdered chrysanthemum is used in China as an insecticide.

AD: Before the 20^th Century

1590 The microscope is invented by Janssen.

1663 Cells are first described by Robert Hooke.

1675 Leeuwenhoek discovers protozoa and bacteria.

1745 Maupertuis proposes an adaptationist account of organic design

1797 Jenner inoculates a child with a viral vaccine to protect him from smallpox.

1802 The word “biology” first appears.

The Laboratory Worker is Surrounded by Many Dangers such as from..........

Handling of infectious materials.

Handling of broken glassware.

Accidental spill of corrosive reagents.

Swallowing of corrosive reagents such as concentrated Sulphuric acid,

Hydrochloric acid, sodium hydroxide, trichloroacetic acid etc.

Swallowing of infectious specimen.

Inhalation of poisonous fumes.

Infection in the conduct of necropsies.

Potential hazards in the form of inflammable chemicals and gas leakages.

Due to increase in population, rapid urbanization, industrialization and shrinking land holdings, there is tremendous pressure on availability of land resources for agriculture, which results in decreased arable land area year after year. The huge gap between present production and future requirements necessitates intensive vegetable production and availability at affordable prices. Vegetable production can be enhanced either by increasing productivity, use of improved cultivars and ago-techniques, bringing additional land under vegetable crops or by promotion of protected cultivation. By adopting protected cultivation, year round availability of quality produces both for domestic use and export can be assured.

Several biotic and abiotic stresses are the major factors responsible for low productivity and poor quality in large number of vegetable crops under open field cultivation more specifically during rainy and post rainy season. The increasing demand of off-season and high quality vegetables in various markets of the big cities had call the attention of the vegetable growers for diversification from traditional ways of vegetable cultivation to modern methods of vegetables cultivation in an agri-business entrepreneurial models. Under the new era of FDI (Foreign Direct Investment) in retail these kinds of models are having high potential for betterment of the farmers opting for quality and offseason vegetable cultivation through protected cultivation. Protected cultivation structures are useful for combating both biotic and abiotic stresses that limits the productivity and quality of vegetable crops. This requires careful planning, attention and details about timing of production and moreover, harvest time to coincide with high market prices, choice of varieties adopted to the off season environments, and able to produce economical yields of high quality produce etc.

Introduction

Organic farming per se is not a new concept rather is an age old practice of cultivation crops without synthetics. However, in modern usage it is a type of farming without synthetics but at the same time taking advantage of all the modern agriculture technologies like soil testing, nutrient management, ecofriendly pest management, etc. developing and developed world have different incentives for taking up organic farming. Growing health consciousness among the people of developed world is the prime motive behind interest in organic farming. However, conversion of “by default” organic agriculture into “certified organic” is perspective in the developing world so that farmers get price premium. Whatever the case may be, the area under organic farming as well as demand for organic food/product is increasing both in developing as well as in the developed world.

Organic production is a systems approach. Although many aspects of vegetable production remain the same in both “organic” and “non-organic” or “conventional” systems, there are differences. The major components of production which differ between these two systems include transplant production, soil fertility management, and insect, disease, and weed management. “Organic” usually refers to a crop management system that promotes biodiversity, biological cycles, and soil biological activity. Organic production concentrates on natural processes and how to manage them. Other materials and products are additions to, not replacements for, management. An organic system takes time to develop. It may be achieved in stages, for example by starting with organic soil amendments and other soil improving procedures. Then, try biological control of pests, some companion plants, etc. Eventually, the entire system will be changed. It will take years to convert to a healthy organic system.

A successful vegetable production industry is very dependent on a sustainable supply of seeds to the growers. At the present time, the seed industry plays an important role in both production and distribution of high quality vegetable seeds.

Successful seed production of a crop depends on a thorough knowledge of the reproductive process of a particular crop. The techniques of seed production must take into account several features of reproduction: whether it is sexual, asexual, or a combination of the both, the nature of floral structures, the amount of pollen transfer, the degree and means of self-incompatibility and the effect of inbreeding on vigour. For most purposes the important consideration to the seed producers is the extent of cross-pollination. It is, therefore, necessary that before engaging himself in seed production, a producer must acquaint himself with the following details of reproduction in the particular crop:

Methods of Reproduction

The method of reproduction in vegetable crop plants may be broadly grouped in to two categories, asexual/vegetative and sexual: