VEGETATIVE REPRODUCTION

Flowering plants reproduced by two methods; sexual and vegetative. By sexual method, seeds are produced which on germination form new plants. whereas vegetative reproduction is the formation of new plants from some vegetative part of plants like root, stem, leaf or bud. A vegetative part, capable of forming a new plant, always possesses a growing point or a bud. It also must have sufficient food for the early growth of new plants. Vegetative reproduction is seen in several plants. It is only method of reproduction in plants which do not flower and seed naturally, e.g., Pineapple, Banana, Sugarcane, etc. It is also being used by farmers to propagate the desired verieties quickly.
Types of vegetative reproductionI. Natural vegetative reproductionII. Artificial vegetative reproductionNatural vegetative reproductionVegetative reproduction is found mostly in the perennial plants. These plants propagate and reproduce naturally. Any part of the plants may accomplish vegetative reproduction. The various parts of the plants used for natural vegetative propagation are as follows:1. Root – Roots of some plants like Shisham, Guava, Poplar, Rose, etc. develop adventitious buds on them. On being separated from the parent plants or the removal of the aerial part, these roots develop into new plants. Some tuberous adventitious roots besides possessing adventitious buds contain sufficient quantities of food, e.g., Sweet potato, Dahlia, Asparagus. If sown in the soil these roots produce several leafy shoots which are known as slips. These slips develop their own roots. These roots are separated out into pieces and then planted in the soil.2. Underground stems – Underground stems are also capable of showing vegetative reproduction and form new plants. Vegetative propagation in some underground stems are discussed below.a) Suckers – A number of short underground stem branches known as suckers arise at the base of an aerial shoot. They grow into aerial branches which develop adventitious roots and new suckers at their bases. When these suckers are separated, a number of independent plants are developed, e.g., Mint, Chrysanthemum.b) Rhizomes – Rhizomes are the modified stems which have may buds and sufficient stored food. A piece of rhizomes containing a bud can give rise to a new plant. This method is adopted in agriculture in the propagation of plant like Banana, Ginger, Turmeric, etc. The rootstock rhizome of banana is very large and bears a number of buds. For vegetative propagation, the rhizome is cut into large pieces and planted into soil.c) Corms – Like rhizomes, the corms also have sufficient amount of stored food. They also bears many buds in the axils of scales present on the nodes. Under favourable conditions, all or several buds produce new shoots using the food stored in the corm. Each new shoot stores food at its base and produces a new corm. Examples are colocasia, crocus, freesia, etc.d) Bulb – A bulb is underground stem which has a number of buds. On being separated and planted, these buds give rise to new plants e.g., Garlic, Narcissus. Onion can also be propagated by bulb but multiplication by seeds is more economical.e) Tubers – A stem tuber is a swollen apical part of an underground stem branch which is known as sucker. It bears a number of nodes called eyes. Each eye possesses few buds. If the whole tuber is sown in the soil, only the terminal buds sprout because of apical dominance. Therefore, a tuber is cut into pieces, with each having one or more eyes. These pieces are then planted in the soil. New plants are produced from the buds presents on the eyes. Example – Potato

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Bacteriophage life cycle

Bacerophage, named T4- virus attacks Escherichia coli. It completes its lifecycle inside the bacterial cells. Virus exhibits two types of life cycles –

1. Lytic life cycle –

In lytic life cycle, virus multiplies in host cell, which stages:

a) Adsorption – The bacteriophage attaches itself over the surface of host cell (bacteria) by means of tail fibers.

b) Penetration (Injection) – Tip of tail possesses an enzyme called lysozyme which dissolved cell wall of the host. The nucleic acid of bateriophage passes into host through tube. However, capsid and tail sheath remain outside, as they have no role in multiplication.

c) Formative phase – After entering into bacterium, the viral nucleic acid takes over all the cellular activity of host. Viral genome replicates itself and codes for new types of proteins which are viral lysozyme, internal proteins and coat proteins.

d) Mutation – The coat proteins wrap itself over the viral genomes to produces a new virus. The period between the entry of phage genome inside bacterium upto formation of first new virus is called eclipse period. It is about 13 min in T4 bacteriophage.

e) Lysis – Soon after maturation, host cell wall ruptures, probably by the presence of some free lysozyme. The newly formed bacteriophages are released. They repeat the cycle again when they come in contact with another bacteria.

2. Lysogenic life cycle – In this cycle, the host does not undergo death and virus does not multiply in the hot cell. In case of some bacterio-phage, its genome does not take over the control of cellular machinery of host. Viral genome is integrated with host DNA. Stage is called prophage. Viral genime replicates along with host DNA.

When it breaks from host DNA, it may carry some host genes along with, which may be transferred to other cells on being infected. Such a mode of division of the prophage is called lysogey. The host bacterium is termed as lysogenic bacterium.

Retro-virus and Reverse transcription

Some DNA viruses (retroviruses) have a gene which codes for the enzyme reverse transcriptase. It helps in the synthesis of double stranded DNA from its original single RNA strand. This phenomenon is called reverse transcription.

RNA viruses capable of reverse transcription are called retrovirus. This phenomenon was described by D. Baltimore in 1970. Tumor causing virus and AIDS causing HIV are the example of retrovirus exhibiting reverse transcription.

About 20 viral gene have been identified which are responsible for triggering cancer cells which are known as oncogenes. Upon activation of oncogenes, cells divide abnormally and uncontrolled causing diseases cancer. However, the origin of cancer is a complex phenomenon.

GENETIC EXPRESSION AND ITS REGUALTION

Biochemically, a gene is a segment of DNA with a specific sequence of nitrogenous base. Functionally, genes are segment of DNA which controls the cellular functions by controlling the synthesis of a protein. Thus genes express itself in the form of a protein or enzyme that controls the development of specific character or a specific function.

In other way, expression refers to the molecular mechanism by which genes show its potential in the phenotype of an organism.

One gene one enzyme theory

Regarding the gene expression, the theory “one gene one enzyme” was proposed by Beadle and Tatum of California which working biochemical mutation on red mold Neurospora crassa. They were awarded Nobel Prize in 1958 for this work. Based on their work, they proposed a concept called “one gene one enzyme” hypothesis. It means that in a biosynthetic pathway several steps are involved each step is controlled by a specific enzyme which is synthesized under the control of specific gene. This hypothesis was later modified as one gene one polypeptide theory. Since it was found that the function unit at the genetic level is a polypeptide.

Viral gene expressions

Important characters of a virus

Virus (L. position) is a nucleoprotein entity which uses host machinery for its multiplication. The first virus to be discovered was Tobacco Mosaic Virus (TMV). The characteristic features of a virus are –

I. Virus is the smallest organism known so far.

II. It dose not have cellular structure.

III. It is obligate properties as it multiplies inside the living cells only.

IV. It exhibits properties of both living and non-living things. It has no metabolic activity of its own. It becomes active and multiply when it infects a living host cell.

V. Virus is capable of exhibiting mutation and recombination.

VI. It exhibits high degree of host specificity.

VII. It has very few enzymes like lysozyme, reverse transcriptase, etc.

Classification of viruses

Viruses are highly specific in nature and they have been classified into three categories on the hosts they live in –

I. Plant virus – virus that infects plants. e.g. Potato mosaic virus (PMV), Tobacco mosaic virus (TMV), etc.

II. Animal virus – virus that infects animals. e.g. Polio-myelitis virus, Infuenza virus. Small pox virus, Hepatitis virus, Mumps virus.

III. Bacterial viruses or bacteriophages – virus that infect bacteria.

There are different types of viruses containing DNA or RNA as a hereditary material. Depending on the type of the nucleic acid contained, viruses are placed in two groups –

I. Deoxyvira – virus that possesses DNA as the genetic material, also called as DNA virus. Majority of the animals viruses is DNA virus except polio virus, Rabies virus, Herps, etc.

II. Ribovira – virus that possesses RNA as the genetic material, also called as RNA virus. Majority of the plant viruses is RNA virus, Mumps, Influenza, and Rabies.

Structure of a virus

Structurally, a virus is made up of two components

a) Nucleoid – it is also called core. It is made up of a strand of highly coiled nucleic acid which is either DNA (in DNA virus) or RNA (in RNA virus).

b) Capsid – Capsid forms a covering around the nucleoid. It is made up of proteins or polypeptides. Its proteins are protective in function. They are resistant to proteolytic enzymes of the host. They have enzymatic properties. These also help the viruses in adsorption and penetration inside the host.

Some viruses like influenza or herpes have an additional lipoprotein membrane called enveloped made of lipoproteins.

Structure of bacteriophage

• A bacteriophage has two parts – head and a tail.

• Head is icosahedral and tail cylindrical.

• Head bears genetic material (DNA or RNA)

• Tail contains a hexagonal basal flat plate which bears six long tail fibres.

• These tail fibres remain coiled inside tail but spread out at the time of infection.

Genes expressions in eukaryotes

The genome of higher eukaryotes is very complex. Eukaryotes genome contains DNA many times as compared to prokaryotic genome. For example, drosophila has 5,000 to 10,000 genes. Human haploid genome seems to have at least 23,000 to 1,00,000 genes. In eukaryotes, most of the DNA is non functional or inactive and known as excess DNA or repetitive DNA. The diploid organism has two sets chromosomes. The genome in eukaryotes controls various function such as; growth and division of cells, differentiation and specialization of tissues such as muscles, liver, or heart in animals and parenchyma, chlorenchyma, Xylem and phloem in plants. As the eukaryotic genomes is very large, the genes expression and its regulation become very complex.
Genes regulation
In prokaryotes and eukaryotes, genes are regulated by various factors.
Following terms are used in genes regulation:
a) Exons and introns – In eukaryotes, some of the nitrogenous based do not code for amino acids. They are inserted between those segments of bases that normally code for amino acids. The coding segments of genes are called exons and non-coding segments are called introns.
b) Splicing – When the unwanted introns are removed and functional regions (exons), responsible for coding, are again joined, it is called as splicing.
c) Inducible genes and Inducer – All the genes present on the chromosome are not expressed simultaneously. The genes that remain inactive or repressed (i.e. an inducer) is present in the medium, are called inducible genes. The phenomenon of the action of these genes is called enzyme induction and substrate is called inducer.
For example, E. coli grown in a medium without lactose, does not produce enzymes required for lactose metabolism. But when the same bacteria is placed in a lactose supplemented medium, it starts producing enzyme like ß-galactosidase required for converting lactose to glucose and galactose. Therefore, since lactose is used to induce this enzyme, it is called inducer and this phenomenon is called enzyme induction.
d) Repressible genes and repression – When E. coli is supplied with certain metabolite more than required, the action of some genes, responsible for formation of some specific enzymes, can be inhibited or repressed. Repression may take place in the case even if the metabolite is being provided from outer source. As a result certain genes are repressed and do not produce enzymes. Such inactivated genes are known as repressible genes and phenomenon is called enzyme repression.
e) Co-repressor – Molecules that binds with the repressor protein to from a function repressor complex is called co-repressor.
In a tryptophan opero, tryptophan acts as a co-repressor by binding with the repressor protein to form a complex which on binding with the promoter switches it off and hence no transcription take place.
f) Constitutive genes – These refer to prokaryotic genes whose expression is not regulated. The products of these genes are produced at a constant, often low rate. Such genes are called constitutive genes and their expression is said to be constitutive. Such genes are involved in photosynthesis and respiration.
g) Structural genes – Genes that contains the information to determine the sequence of amino acid is called structural genes.
h) Regulatory genes – These genes codes for the product that regulates the level of expression of the structural genes. Although it is located at site away from the structural genes, it is called key element of operon. It forms repressor protein to make repressor complex.
i) Promoter genes – Genes that form the binding site of RNA polymerse is called promoter genes. Each genes may be regulated by a specific promoter.
j) Operator genes – Genes that operates the activity of structural genes, is called operator genes. It lies adjacent to the promoter site. Structural genes are expressed or not expressed depending upon whether the operator genes are on or off.
k) Operon genes – Genes are genetic unit consisting of an operator, a promoter and one or more structural genes whose activity is influenced by operator genes.

Operon concept
In order to study genes regulation or induction, Jacob and Monad (1961) proposed Operon concept in prokaryote (E. coli). An opron is a group of coordinately regulated genes, the products of which typically catalyze a multi-enzyme metabolic pathway and its controlling elements. Controlling elements include promoter genes, operator genes and regulatory genes.
Although there are many operons in bacterial cells, but the lactose or Lac operon discovered by Jacob Monad is classic example of all operons.

1. Structure of the lac operon – Two classes of genes are needed to named Z, Y, and A that code for three enzymes mentioned below.
• Z genes that codes for ß-galactosidase.
• Y genes that codes for galactoside permease.
• A genes that codes for thiogalactoside transacetylase.
These genes are located in a row adjacent to each other and hence they are called linked. They are known as polycistronic. Structural genes are regulated by operator genes and promoter genes.
a) Operator genes – Operator genes lies between the promoter genes and the structural genes. The operator genes act as a switch. Structural genes are expressed or not expressed depending upon whether operator genes are on or off. Single operator genes regulate all the three structural genes.
b) Promoter genes – Single promoter genes direct proper initiation of transcription. The lac Z, lac Y and lac A genes are expressed as a polycistronic message from a common promoter. The binding of DNA dependent RNA polymerase and promoter initiates the transcription of structural genes.
2. Regulatory genes – Regulatory genes are located away from structural genes. Hence regulatory genes are often not considered as part of operon. However, regulatory genes are the key element of operon. In codes for product that regulates the level of expression of structural gene. The regulatory genes constantly transcribe mRNA to produce the repressor protein.
Regulation of Lac operon expression
The absence and presence of lactose (inducer) switch on or off the transcription of mRNA and protein synthesis. This phenomenon can be described in following steps:
I. When E. coli is grown in a medium in absence of lactose, the regulator genes produce a repressor protein that bind the operator genes and block its activity. RNA polymerase can not move from promoter to structural genes. It stops the transcription of mRNA from structural genes and thus protein synthesis is switched off. Hence the enzyme is produced.
II. When the lactose is introduced in the medium, lactose binds to the repressor protein. In this way, repressor protein fails to bind to the operator genes. Then the operator genes remain active and hence switch is turned on. Operator genes induce RNA polymerase to bind to promoter mRNA corresponding to all three enzymes; Z genes code for ß-galactosidase, Y genes for galactoside permease and A genes for thiogalactosede transcetylase. With the expression of these three enzymes metabolism of lactose beings
Synthesis of enzymes is continued unless and until all the lactose molecules are consumed. When the last molecules of lactose bound to repressor is consumed, the inactive repressor becomes active and thus binds to operator site to switch off the operon as normal.

Role of repression and constitutive enzymes
When a substrate required by bacterium is supplied in excess amount from the outside, bacterium stops or inhibits the production of substance. In other way, we can say that the genes are being inactivated. These inactivated genes are thus called repressible genes and the phenomenon is called repression.
However, some of the cellular activities are functioning normally and constantly such glycolysis. The genes, that constantly expressed to take care of normal cellular activity such as glycolysis, are known as constitutive genes. The expressions of these genes are not regulated. The enzymes produced by bacterium for above function are known as constitutive enzymes. The constitutive enzymes are dehydrogenases.

Genes expression in prokaryotes

J. Lederberg and E. I. Tatum (1946) demonstrated sexuality in bacteria for the first time and this opened a new era of research. Most of the important work on genetics of bacteria was initially done on the colon Bacillus bacteria named Escherichia coli.
Bacteria genome is represented by a circular double stranded DNA. Its DNA is associated with few proteins. E. coli contains 2000-3000 genes. Bacterium can survive on glucose diet giving the idea that these genes have information for synthesis of all organic compounds it needed.
Modes of genetic transfer in bacteria
Bacteria mainly reproduce asexually by binary fission. Meiosis is lacking. They do not show sexual reproduction like eukaryotes. However, some bacteria show primitive form of sexual reproduction to exchange genetic material between two cells. There are three modes of exchange of genetic materials or genetic recombination. They are:
a) Transformation
b) Transduction
c) Conjugation
a) Transformation – A short fragment of naked DNA isolated from one type of bacteria cell is incorporated into other type of bacterial cell. A recombinant or hybrid DNA is, thus, formed. This phenomenon is called as transformation. In this way, DNA of donor cell expresses some of its properties into the recipient cell. Griffith confirmed it with experiment on Diplococcus pheumonia. Normally E. coli does not pick up foreign DNA but can be done in the presence of calcium chloride.
b) Transduction – This is similar to the transformation but transfer of DNA from one bacterium to another is mediated through a vector. This process is called transduction. A vector may be a bacteriophage (virus) or a plasmid or a cosmid.
During this process, bacteriophage is used as a plasmid to transfer a small piece of double stranded DNA from one bacterial cell (so called donor cell) to another cell (so called recipient cell). A recombinant or hybrid DNA is, thus formed which consists of genome of both bacteria and thus expresses both of the properties.
c) Conjugation – Transfer of DNA from one cell to another through a sexual mating is called conjugation. It is similar to sexual mating in eukaryotes. Lederberg and Tatum first demonstrated it in E. coli.
In this process, male bacterium makes sex pili which enables cell to cell contact. The male bacterium is represented as a donor (F?). The female bacterium (recipient F?) lacks pili and receives the DNA from the male bacterium. This result to new genetic recombination. The progeny of the recipient then expresses some of the donor’s traits due to recombination.
Fertility factor and Hfr strain
The ability of transferring genetic material from male is regulated by sex or fertility factor (F genes) present in a plasmid. The bacteria with F genes are said to be F positive (F?) and another F negative (F?). F genes code for producing sex pili and other functions required for transferring DNA. At times F factor integrates into the bacterial chromosome
Such bacteria can transfer their genetic material into female with high frequency (Hfr) in a particular sequence. The are called as Hfr strains. Frequency of recombination was very low in Lederberg’s experiments. Hayes (1952) found a strain of E. coli in which the frequency of recombination was as high as 100 to 1000 times as reported by Lederberg. The strain was called as high frequency recombinant (Hfr) strain.

Other fermented alcoholic beverages

Fermented alcoholic beverages are consumed all over the world. In some country the use of a particular beverage has been passed down from ancient times, some of the alcoholic beverages is explained here:

1. Wine: wine is the product made by the normal alcoholic fermentation of the juice of ripe grapes (vitis vinifera ). Relatively small enounce of wine are made from apples, raisins, black berries, peaches, cherries, orange, currents, apricots, grape fruit, pomegranates, raspberries, pears, honey and straw berries. The wine made from the fruits is named after the fruits, for example apple wine.

2. Whiskey: Whiskey is an alcoholic distillate from the fermented mash of grains. Whiskey is obtained from a fermented mash. After several distillation of mash the low wines are resulted. Further distillation straight whisky. At first several principles are present, wines makes whiskey harsh and unpalatable. The whiskey is aged in charred oak containers. At first whisky is colourless , the colour develops during the aging process. A continued distillation of high wines results in the fermentation of neutral spinets, which are used in blended whiskies and cordials.

3. Rum: Rum is an alcohol distillation from the fermented juice of sugarcane syrup, sugarcane molasses or other sugarcane byproduct. Rum is manufactured and used in general in those countries which grew sugarcane or export molasses or other sugarcane products. It possesses a characteristic flavor, aroma and colour. The flavor and aroma improve with aging. Rum contains about 41% alcohol. Rum is usually aged in charred white oak barrels. Rum may be used in the perspiration of ice-cream, candies in the curing of tobacco, as a beverage and as medicinal.

4. Brandy: Brandy is distillate form of wine. It is also distillate from the remounted juice of various fruit. The best brandy is made in France known as cognac. The other French brandies are known as annoyance. The finest grades of brandy are made from white wines. The brown colour of brandy is coloured with caramel. It contains about 65 to 70 percent alcohol. Apple brandy is known as apple jack.

5. Gin: It is obtained by distillation from a fermented mash of malt or raw grain. The finest gin is distillated from a malt of barley and rye. It requires several distillation. The flavor of gin and any medicinal value are due to oil of jumper.

Application of fermentation in industries
Beside alcohol, other products are also formed by process of fermentation. The product produced depends upon the nutrient medium. Main products are n-propanol, butanol, phenyl ethanol, amyl alcohol, glycerol, lactic acid, acetic acid, pyruvic acid, succinic acid, ethyl acetate, caporic acid, etc. Yeast is also used as animal feed, yeast extract, food supplements and vitamins. Some of important industrial use of fermentation are:
1. Organic acid
a) Citric acid produced in commercial scale from fermenting molasses or purified glucose syrup from maize using Aspergillus niger as microorganism. This fungus also produces gluconic acid.
b) Lactic acid is produced using thermotolerant or thermophilic lactic acid bacteria.
c) Acetic acid most important acid, is produced by the fermentation of carbohydrates

2. Amino acids
Commercial production of number of amino acids is being done using fermentation technology. Out of 20 naturally occurring amino acids, twelve are produced by industrial fermentation or enzyme conversion.

3. Vitamins
Vitamins can be synthesized by chemical processes or biological means. Riboflavin is produced from the fungus Eremothecium ashbyii where vitamin B12 is chiefly synthesized from Streptomyces griseus. Streptomyces also produces antibodies. During fermentation process, change in the operational conditions may lead to the production of vitamin rather than antibiotics.

4. Enzymes
Several thousand enzymes possessing different substrate specificities are now known. The bulks of enzymes used commercially are obtained from microbial, sources, and are produced by fermentation processes. Enzymes from microbial, rather than plant or animal, sources are usually used because of their availability, grater stability and their variety and case of genetic manipulation. The industrial enzymes are produced from different species of bacteria and fungi.

Fermentation process

Fermentation is done with one of the following method

a) Batch process – in this process, nutrient medium and fermenting organism like yeast are allowed to remain in the bioreactor (fermentation tank) till the maximum fermentation product is produced. The entire medium with organism and the product is removed after each charge. Then the bioreactor is cleaned and a fresh batch is stored.

b) Continuous process – In the process, product is drawn off at regular intervals and fresh medium is introduced into bioreactor

c) Immobilized fermentation process – Living yeast cells are immobilized in calcium alginate beads. The beads with living cells are placed in the nutrient medium in fermentation tank. This technique allows quick fermentation about 20 times faster than the batch process.
CO2 is a bye product of alcohol fermentation. It is collected separately. Yeast cells are also isolated from fermentation product. A part of yeast is kept further inoculation. The remaining part of the yeast is washed, dried and employed as animal feed.

Fermentation process involves two broad groups –
I. Upstream process – All the operations before starting the fermenter are collectively called upstream process such as sterilization of the fermenter, preparation and sterilization of culture medium and the preparation and growth of a suitable inoculums of microbial strain.

II. Downstream process – All the operations after the fermentation are known as downstream process. It includes the purification of fermented products from fermentation broth. Methods commonly used are distillation, centrifugation, filtration, and solvent extaction.

Bioreactor and its type
A bioreactor or fementer is a container designed to provide an optimum environment in which microorganisms or their enzymes can interact with a substrate and form a desired products.

Bioreactors are of two types:-
a) Open type: It allows continuous processing with substance entering at one end and products leaving at the another end.

b) Closed type: In this type, the fermentation is done in batches. Microbes are grown on nutrients placed in vessel at the start of fermentation. The stirred-tank fermenter is a versatile design and is used in a range of sizes from one litre laboratory unit to production-scale vessels of typically 100-ton capacity. Its vessel is cooled by a water jacket. Air is pumped into bottom of the liquid, and acid or alkali added as necessary. A stirrer keeps the contents well mixed. Steam lines are provided so that the vessel can be sterilized after each fermentation batch.

During fermentation it is necessary to regulate many factors within predetermined valves. These O2 and CO2, pH, temperature and media concentration, etc. It is also essential to maintain high degree of sterility within bioreactor. It should be made of stainless steel or copper because such bioreactor is resistant to steam sterilization.

Fermentation Technologies in biology

Fermentation is a process of incomplete oxidation of sugar, especially glucose, into alcohol and CO2. It is generally an anaerobic process. By this process, glucose is splitted up by glycolysis into pyruvic acid which is further degraded enzymatically to alcohol and CO2. Several species of yeast, bacteria and other fungi are essential as they contain many enzymes that are used to carry out fermentation.

Fermentation was discovered by ancient people by prolonged soaking (steeping) of grains before cooking or storing juices of fruits. Alcohol was the first product of biotechnology. Now a day a modern technology developed for the production of alcohol on industrial scale is the fermentation.

Yeast and its types
The organism presently used in alcoholic fermentation is the yeast. It is a microscopic fungus Saccharomyces cerevisae. Louis Pasture in the middle of nineteenth century first reported the role of yeast in fermentation process. Presently, yeast products for human and animal consumption are products in large scale in many countries.

There are basically two types of yeast:-
1. Baker’s yeast – These include the selected strains of Saccharomyces cerevisae and Torulopsis grown on molasses. These are used to:
I. Flavor the food
II. Supplement nutrient ingredient as it is rich in proteins, vitamins, etc and
III. Ferment and rise dough in bread making (leaving agent). Leavening is caused by three enzymes secreted by yeast. These three enzymes are amylase, maltase and zymase. Amylase hydrolyses starch into maltose, maltase degrades maltose into glucose and zymase causes fermentation of glucose in anaerobic and produces mainly ethyl alcohol and CO2. Both CO2 and ethyl alcohol evaporate and make the bread porous and soft.

2. Alcohol (Brewer) yeast – These yeast are grown on carbohydrate, sucrose for the production of several types of alcoholic beverages. Majorities of alcoholic beverages are manufactured by metabolic activity of Saccharomyces cerevisae and other yeast.
How and why do microorganisms make alcohol?

Like other organisms, yeast has property to maintain the stock of ATP, which possible due to the consumption of sugars like glucose and fructose. Sucrose is the main component of sugarcane juice which consists of one glucose molecule attached to one molecule of fructose.

If the yeast is grown in oxygenated medium, the sugar will be broken down step by step, into smaller and smaller molecules and at the end, CO2 and energy are liberated. If yeast is grown in anaerobic medium, a series of chemical breakdown processes can be completed and sugar is broken down into ethanol or fuel alcohol.

Glucose is splitted up into two molecules of pyruvic acids via glycolysis. During fermentation pyruvic acids are degraed enzymatically into alcohol and CO2.

Yeast, which carries out alcoholic fermentation, contain two important enzymes –
(1) Pyruvate decarboxylase (2)Alcohol dehydrogenase. The pyruvate decarboxylase catalyzer decarboxylation of pyruvic acid to acetaldehyde which is further reduced to ethanol by NADH in presence of alcohol dehydrogenase.

Process of alcohol manufacture in industries
The raw material used for preparation of ethyl alcohol is molasses. It is thick, dark syrup drained from raw sugar during the refining process. It is also called the products of sugar (in sugar industry). Molasses mash (mixture of raw sugar and hot water) is adjusted to the desired sugar concentration and temperature by addition of water and to the desired pH by addition of a measured quantity of acid or base. Molasses is kept in a bioreactor (fermentation tank). Yeast (Saccharomyces), so called starter, is added and mixed uniformly with molasses mash in the same bioreactor. The mash and starter become well mixed as they spatter and fall to the bottom of the tank. Fermentation soon becomes vigorous with evolution of large quantities of CO2. Fermentation is completed within fifty hours or less. Then the fermented molasses is distilled and separated the alcohol and other volatile constituents from mash. The purification of alcohol is made by means of rectifying columns and then stored in a container.

Applications of recombinant DNA technology

Recombinant DNA technology has opened up new opportunities for highly specific manipulation of the genetic materials. This technology broadens the possibilities of gene transferring gene between unrelated organisms and creating novel genetic information by specific alteration of cloned genes. Recombinant DNA has wide applications in the fields of agriculture and medicine and human health which are discussed herewith.

A. Application of genetic engineering in the field of medicine and human health
The recent results of recombinant DNA technology have revolutionized the field of clinical medicine. This has greatly developed the preparation of wide range of vaccines, development of highly specific diagnostic laboratory tests and prenatal diagnosis of human genetic diseases. Some of the products synthesized using genetic engineering are given below:

1. Vaccines – vaccine is preparation, which contains an antigen composed of whole disease causing organisms. Vaccines are used to confer immunity against antigens.
Recombinant DNA technology can be used to clone the gene for the protective antigen protein. A number of vaccines against virus have been developed using this technology. These vaccines are – Hepatitis B, influenza, HIV (AIDS), Herpes, Foot and Mouth Disease, etc.

2. Human growth hormones – Various human growth hormones have been synthesized commercially using Recombinant DNA Technology. This hormone is used in treatment of dwarfism.

3. Insulin – Insulin, a small protein hormone, is used for controlling diabetes. Bacterially produced human insulin using recombinant DNA technology is now available in market. Products of microbial origin are often safer than those derived from traditional sources. Insulin, produced by microbes does not cause allergic reaction.

4. Interferons – interferons are the glycoproteins which have an inhibitory action upon the multiplication of viruses in cells more or less adjacent to the affected ones. They are being used for the treatment of several diseases including a rare form of cancer called hairy cell leukemia. Various interferon are formed such as – a – interferon, ß – interferon and ? – interferon. The bulk of a – interferon is produced by buffy coats (leukocyte pellets), ß – interferon by fibroblasts and ? – interferon by B or T lymphocytes. Japanese companies have succeeded in producing cloned ? – interferon. Interferon are being used for the treatment of several diseases.

5. Monoclonal antibodies – Antibodies are specific proteins produced by the immune system in response to presence of a specific antigen. Monoclonal antibodies can be produced using hybridoma technology.

Hybridoma technology is used to fuse a normal antibody producing lymphocytes (B-cells or plasma cells) with myelonema cells (a kind of tumor cells) giving a hybridoma. Hybridoma has the potential to grow indefinitely in culture and hence can be a source of unending supply of antibody of choice. Since antibody produced by a hybridoma is biochemically pure, it is called monoclonal antibody. Monoclonal antibodies are used to develop effective vaccines against human, animal and plant diseases.

6. Antibiotics – Antibiotics are the chemical substances, produced both by microorganisms and synthetically. They can inhibit the growth of bacteria and others microorganisms and even destroy them. Penicillin is the first antibiotic discovered by Alexander Flemming in 1928 using recombinant technology. Some useful antibiotics being produced on the commercial scale employing biotechnology technique.

7. Diagnosis of infection disease – Modern medical practice depends on laboratory tests for the specific and correct diagnosis of many diseases. Recombinant DNA technology allows for the production of highly specific diagnosis tests.

Some of the common infectious diseases are cholera, small pox, measles meningitis, hepatitis, etc. These diseases lead the serious damage to the human health. Infectious diseases diagnosis mainly depends upon isolation and identification of pathogens, which may take several days. Development of diagnostic kits to identify pathogenic organisms by knowing the organism-specific DNA sequence has provided rapid, specific and correct diagnosis.

In this way, advancement in biotechnology has made easy early, correct and quick diagnosis of infectious diseases. Various diagnostic kits have been developed for AIDS, cancer, foot and mouth diseases, tuberculosis, etc. Different biotechnological tools used in diagnosis of infectious diseases biotechnology tools used in diagnosis of infectious diseases and prenatal diseases are ELISA, PCR based technique, RIA Essays, etc.

Application of genetic engineering in the field of agriculture

During the last decade, tremendous progress has been made in the area of genetic engineering towards agriculture. Recombinant DNA technology has opened up new opportunities for highly specific manipulation of the genetic material. Once applied and developed to a sufficient degree, it promises ultimately to provide a powerful additional tool to the plant breeders. This technology broadens the possibilities of transferring genes between unrelated organisms and creating novel genetic information by specific alternation of cloned genes. Application of genetic engineering in field of agriculture is:

1. Creation of resistance varieties of plants – Transfer of specific genes from one species to another may be of great significance in exploiting diseases, insects and pest resistance mechanisms more efficiently. Various genes responsible for resistance to diseases have been identified, cloned and incorporated or manipulated into another species. For example, Bacillus thuringiensis is a bacterium that has ability to destroy stem borer pest in rice and maize. Keeping this view in mind, scientists isolated Bt gene from a bacterium Bacillus thuringiensis and cloned and then incorporated it into rice or maize genome. As a result rice or maize shows the resistance against stem-borer due to presence of Bt gene in rice genome. Various novel plants with resistance to various diseases pests and stresses have been created using recombinant DNA technique.

2. Bio-fertilization – Molecular nitrogen in the atmosphere is converted into biologically usable form by nitrogen fixing micro-organisms e.g. Rhizobium. The most sophisticated approach to bio-fertilization is to create plants that possess genetic capacity for nitrogen fixation. Attempts are being made to transfer genes for nitrogen fixation gene called nif gene from bacteria to rice or other non-leguminous crops.

3. Increase the protein content – One of the major source of protein for human and animals consumption is constituted by the proteins contained in seeds of many plant species. The cereals and legumes which are major sources of storage proteins, contain limited amount of certain amino acids which are essentials for human beings. Majorities of these cereals are deficient in lysine whereas legumes are deficient in sulphur amino acids.

The genes which code for a number of storage proteins have been cloned. For example, the gene encoding the French bean protein, phaseolein, has been expressed in sunflower.

4. Creation of transgenic animals – animals that have foreign genes inserted into their germ lines are called transgenic. They can pass the gene on to their offspring, and it can be inherited as a Mendalian trait.

Possible dangers of genetic engineering
Genetic engineering has numerous potential benefits, some of which have been discussed above. However any new scientific discoveries offer the possibility of both beneficial and destructive effects. Some of the possible dangers due to genetic engineering might be as follows:

1. Due to manipulation of genes might, by accident, result in the origin of various new kinds of diseases or organisms containing fatal genetic element.

2. There is a risk of creation of drug resistance germs and out-break diseases against which there is no known prevention.

3. Any accidental escape of laboratory strains may create havoc on earth. It may contaminate a large population.

4. Introduction of gene like that of viral cancer into bacteria through plasmids may involve the risk of introducing these harmful genes into man when these bacteria infect human.

5. Hybrid genomes may create some serious ecological problems, the nature of which is still unknown. Naturally all these harmful hybrids possibly raise many moral, ethical and legal questions.

Releasing the risk involved in genetic engineering by a group of scientists working on recombinant DNA technology, the US National Institute of Health (NIH) established an advisory committee in 1976. Its main objectives are to evaluate the potential of biological and ecological hazards of recombination DNA molecules and develop the procedures which will minimize hazards and devise guidelines for the investigators in the research line.

Genetic Engineering

Genetic engineering can be defined as the science of adding, removing or replacing genetic units in order to achieve permanent and heritable changes in plants and animals for the benefit of mankind. Terms such as “gene surgery”, “gene therapy”, “gene manipulation”, “gene transplantation”, etc. have been synonymously used with genetic engineering. Basically the term genetic engineering refers to techniques that are used to manipulate, move, recombine and propagate DNA.

A detailed knowledge of the molecular nature of the gene and ability to manipulate cells of higher organisms as well as bacteria may eventually allow the possibility of genetic engineering. It has been very useful in plants and animals and has brought biological revolution. Luciferase gene from firefly has been introduced in tobacco plant enabling scientists to study the activation of genes of genes of their choice by measuring the light emitted by the plants. Introduction of nitrogen fixation gene (NIF gene) and Bt gene in cereals are really a boon in agriculture. In human being, genetic engineering is being used for diagnosis of hereditary diseases and thus has bright prospects to be used for gene therapy.

Genetic engineering provides great promises for the improvement of crop plant. Genetically engineered crops with better nutritional status, resistance to insects, pets and herbicides, resistance to fungal, bacterial and viral diseases and resistance to environmental stresses have shown a great potential. The techniques of biotechnology have already increased the capacity to enhance plant productivity. Similarly it has tremendous impact on medicine for diagnosis and treatment of many diseases.
Cloning of DNA
In gene cloning, firstly the DNA of an organism containing the gene of interest in cut into smaller pieces. This gene is called target gene or foreign DNA.

Secondly, the target DNA is joined (in vitro) to a second piece of DNA that can replicate itself and attach any target DNA. This second DNA is often called vector or cloning vehicle. The result of the joining is a hybrid molecule, a hybrid or recombinant DNA
Thirdly, the jointed target and vector is then introduced into a living cell. The cell serves as a biological copying machine, making many exact copies of recombinant molecule. This all process is called molecular cloning.

Tools of gene cloning
Cloning is a basic step in recombinant DNA technology. It involves incorporation of a piece of foreign DNA into a vector. Following tools are needed for cloning of DNA:
a) Restriction endonucleases or enzymes – These enzymes cleave double stranded DNA into smaller fragments. They cut the DNA at specific sites. They are popularly known as molecular scissors.
Restriction enzymes are isolated from bacteria and are named for the bacteria from which they are derived. About hundreds of different restriction enzymes have been isolated. The best known example of a restriction enzyme is EcoR1. These enzymes are highly specific and recognized specific sequences in double-stranded DNA and make two sequence-specific cuts, one in each strand. The most commonly used restriction endonucleases.
b) DNA ligase – DNA ligase is used to covalently link the 3’ – hydroxyl end of one strand of DNA to the 5’ – phosphate ends of second strand. Therefore, DNA segments that are cut by restriction endonucleases can be ligated or joined by DNA ligas enzyme.
c) Vectors – for cloning, a vector is an essential tool in which the foreign gene is inserted. They are also known as cloning vehicles. Vectors must have the following features:
i. They must be able to replicate within the host cell.
ii. They must be capable of insertion into the host cell.
iii. They must have a selectable marker.
iv. They must contain a site for insertion of foreign DNA. In order to carry foreign DNA into cell, the vector must be linked to the foreign DNA.

Amniocentesis & test-tube Baby

Amniocentesis
Amniocentesis is the technique of obtaining amniotic fluid from the womb of a pregnant woman for prenatal detection of foetal disorders. More than 30 genetic diseases have been detected in the growing foetus by means of this technique. Information about sex of the unborn child, which is of immense importance to the genetic counselor, can also be obtained through amniocentesis.
In this process sample of amniotic fluid surrounding the foetus is obtained with the help of hypodermic needle passed through abdominal wall from a pregnant woman. The foetal cells which are shed normally into this amniotic fluid can be finally cultured under test-tube conditions. This facilitates to process or analyze the foetal cells in two ways:

1. Karyotype analysis – This cultured foetal cells is useful for the detection of chromosomal abnormalities in growing embryo. It is also helpful in determination of sex on the basis of presence or absence of sex chromatin.

2. Biochemical test – It also detects the presence or absence of certain enzymes or other metabolic features. It is useful for the detection of prenatal diseases.
Amniocentesis is usually employed when the foetus has a high risk of genetic disease or when pregnant woman is over 35 years of age. This technique is, however, being misused even to abort normal female foetus. Therefore it has been now banned.

Test-tube babies
In some rare cases, women are unable to conceive and give birth to a child normally. In such women the fertilization is not possible in the uterus. Due to remarkable advances in medical science, for such cases, unfertilized ovum is taken out kept under sterile or aseptic conditions in a test-tube. The fertilization process is completed with a sperm taken from her husband outside the body. The zygote thus formed is allowed to developed in vitro upto 32 cells stage and then it is put into the female reproduction tract for implantation which undergoes further development in the womb till birth. Such babies are called test tube babies.
The first attempt to produce a test tube baby was named by the Italian scientist Dr. Petrucci in 1959 when he began his epoch-making experiment. He removed an ovum from a patient and put it a glass tube among swimming million of spermatozoa, one of which met and fertilized the egg under rigidly monitored condition in a glass dome. The embryo grew. Although the embryo survived for just 29 days, Dr. Petrucci’s experiment opened up a new vista that had the potential to revolutionize the future of mankind.
The first complete successful experiment through in vitro (test tube) embryology is, however, credited to the Gynecologist Patric Steptoe and research Physiologists Robert Edwards of England who had been working for over a decade to perfect a technique for fertilizing human eggs outside the human body. On July 25, 1978, the world’s first test tube baby’ – a baby girl named Louise Joy Brown, was born. The Browns had been trying to have a child for some nine years. The baby, Mrs. Brown eventually had, was conceived in laboratory culture disc.

The method used involved removing a ripe egg from the wife’s ovary and placing it in a culture disc together with sperm taken from her husband. The embryo was allowed to develop for two and a half days. Then embryo was placed in the uterus of a woman, where it become implanted and grew like any other foetus. The birth of an absolutely normal baby by caesarian section took place 28 weeks and 5 days after Mrs. Brown’s menstrual period.

This method has been employed to some of women who are not able to have a normal conception. This has opened a wide biomedical application but raised several ethical and legal problems, such as right over the child.

Bio-fertilizers

Biofertilizers are the living organism which bring about nutrient enrichment of soil. Biofertilizers are bacteria, cynobacteria and fungi. These organisms improve the soil fertility by adding nutrient due to their biological activity.
1) Bacteria as biofertilizers –
Some bacteria enrich the soil by fixing free atmospheric nitrogen into nitrogen compounds. These bacteria and their roles are as follows –
i. Rhizobium – rhizobium is a symbiotic bacterium which is found in soil and forms nodules in the roots of legume plants cannot use free nitrogen from atmospheric but can use in form of nitrates, therefore, bacteria fix free atmosphere nitrogen into nitrates. The leguminous plants utilize this nitrogen in form of nitrates.
ii. Azotobacter – Azotobater is a free living and non-shmbiotic bacteria found in soil. It can fix atmospheric nitrogen is utilized by crops like cereals (rice, maize), millets, fruits, vegetables, etc.
2) Cyanobacteria as biofertilizers
Certain cyanobateria that fix the free nitrogen of the air are –
I. Anabaena azolla – Anabaena azolla is a symbiotic cyanobacteria found in the cavities of leaves of fast growing and floating fern, Azolla. This cynobacterium fixes free atmospheric nitrogen and exudates nitrogenous compounds into the cavities of leaves. Such leaves can be an excellent biofertilizer.
II. Anabaena, nostoc, Spirulina – These are free living cynobacteria (green algal bacteria) which fix the nitrogen through photosynthesis. These organisms have been used as bio-fertilizers in India.
3) Fungi as bio-fertilizers
Certain fungi make a symbiotic association with the roots of certain seeds bearing plants. This association is called mycorrhyzal. Mycorrhyzal associated converts a marginal land into fertile land and reduces the dependency on irrigation and fertilizers. Mycorrhyzal are of two types –
I. Ectomycorrhyza – It is an association in which fungus lives outside of the roots of oak, pine, peach, Eucalyptus,etc. These absorb and store nitrogen, calcium, potassium and phosphorus in the fungal mantle. This result is greater plant vigour, growth and yield.
II. Endomycorrhyza – It is an association in which fungus lives between and within the cells of cortex. Ti is found in many herbaceous species like orchids and some woody plants. In some endomycorrhyzae fungal hypae penetrate into the cortical cells, these are called vesicular arbuscular mycorrhyza (VAM). VAM are important in the phosphate nutrition.

Nitrogen fixation
Nitrogen fixation may be defined as the phenomenon of the conversion of free nitrogen of the atmosphere into nitrogenous salts (in form of nitrate) which are readily absorbed by the plants.
Certain symbiotic bacteria like Rhizobium and Bacillus radiciola live in the nodule of the roots of the leguminous plants. These bacteria on entering the roots through the root hairs from the soil form the root nodule appears pinkish due to the presence of a pigment, leghaemoglobin. This pigment is related to haemoglobin, the red pigment of human blood. Like haemoglobin, leghaemoglobin also absorbs oxygen. Enzyme nitrogenase catalyses the fixation of nitrogen under anaerobic conditions.

Certain free-living microorganisms like cyanobacteria and photosynthetic bacteria also fix nitrogen. Certain cyanobateria live in symbiotic association with lichens Anhoceros (a liverwort), fronds of Azolla (a water fern) and roots of Cycas.

TISSUE AND ORGANS TRANSPLANTATION

Generally transplantation is defined as the replacement of diseased tissue or organs of an individual by healthy ones. Transplantation of healthy tissues or organs is achieved by the process of surgery. During transplantation, recipient (the individual in or onto which new healthy tissues or organs are tansplanted tissue or organs are tansplanted) develops some antibodies. Therefore, new transplanted tissues or organs are not easily accepted by the recipient which are regarded as antigens. The antibodies developed inside the body of recipient act against the new transplanted part and cause destruction.

Sometime tissues from one area are transplanted to another area of the same individual. For example skin from thigh is called autograft. If the tissues are transplanted from the identical twins such a process is called isograft e.g. kidney of one of the twins when transplanted to another one. Isograft generally involves the transplantation of the body parts in between individuals of the same species which are also identical genetically (e.g. twins).

If the transplantation is carried out between genetically different individuals of the same species, it is called allograft. Blood transfusion from one person to another person is an example of allograft which is very common now-a-days. But, if the tissue or organs are transplanted from individual of one species to the individual of other, it is regarded as xenograft.

We have already discussed that the recipient develops antibodies against the transplanted tissue or organs. This is the immune power of the body. But the production of antibodies by the recipient can be suppressed by utilizing different methods which is called immunosupperssion. The agent that causes immunosuppression is called immunosuppressant.

Immunosuppressant causes the inhibition of body’s normal immune power so that the recipient is succeptible to all other kinds of infections. A special kind of immune suppressant should be developed that shows effects on the specific immune response, not to the whole immnune system of the body.

Such immune response to destroy transplanted organs or tissues can also be reduced by the exposure of bone marrow and lymph tissue to x-irradiation.

ANTIBIOTICS INFORMATION TO HUMAN

The term ‘antibiotics’ was coined by Selman in 1942. He defined antibiotic to be a substance produced by a micro-organism and which in low concentration is antagonistic to the growth of other micro-organism. Elymologically, antibiotic means ‘against life’. Clinically, antibiotics are organic products which in low concentration are able to inhibit the metabolic activities of pathogenic organisms without harming the host.

Antibiosis was first demonstrated by Babes in 1855. He found that micro-organisms might produce substance that could inhibit growth of other organisms. In 1877 pasteur and joubet found inhibitation of growth of Bacillus anthracis (anthran baclesia) by certain bacteria. Paul vuilenin proposed the concept to antibiosis of antagonism between two living organisms. In 1928 Felming found that destructutions of cultures of staphylococcus aereus in the region of untamination by pemicillium notatum. Florey etal (1939) discovered the chemotherapeutic value of chemical produced by Penillium notatum and commercialised the product pencillin, which was first usedclinically during the second world war. Waks man and Schater found actimycetes (mycelia becteria)to be potencial produces of antibiotics. Waksman isolated actinomyces is streptomycin in 1949 both from the genus streptomyces.A single species of streptomyces is known to from more than 40 antibiotics. Antibiotics are produced by lichens, finger, actinomyces and eubecteria.

The lichens cantibioticladonia and Usnea produce Ushic acid. Similarly, finger Penicillion produces penicillin, griseofulion, patulin, Emericellopsis prodncescephalosporins, antiamoebin. Eubacteri like bacillus and pesuclomones produce eubacteriates.

Depending upon their effect, antibiotics are of two types-broad spectrum and specific. Broad spectrum antibiotics is able to destroy a large number of pathogens in structure and composition of their walls. The specific antibiotics have a limited spectrum. They act on a few similar type of a pathogens. However all antibiotics inhibits growth or destroy a number of bacteria and fungi. Modern medicine has specific antibiotic for every bacterial pathogen. Antibiotics are relatively inexpensive, safe and sure than other durgs.

Fertilizers of biological origin

Soil contains various nutrients that are essential for growth and development of plants. Constant use of leads to the loss of its important nutrient and ultimately its fertility. Deficiency of any one or more nutrients ultimately affects the yield of crop plants.

There are the several method to increase the soil fertility. Farmers usually apply chemical fertilizers to enhance soil fertility. However, frequent application of chemical fertilizer is equally harmful to the soil as they affect the soil composition and nutrients. Therefore, in order to reduce the hazards caused by chemical fertilizers, the recent trend to make use of the fertilizers which are environment friendly and are of biological origin. Materials of biological origin which are commonly used to maintain and improve soil fertility are called biofertilizers. There have been grouped into two main categories-
1. Manures
2. Biofertilisers

Both when added to soil increase in nutrient value especially the nitrogen content and thus enhance the crop productivity.

Manures
Manures are the organic materials when added to soil increases its nutrients value especially the nitrogen content and thus enhance the crop productivity. The manures are of three types:
• Farmyard manure – It is the most valuable organic matter commonly applied to the soil. It consists of mixture of cattle dung and crop residues like remnants of the straw and plant stalks fed to cattle. The cattle dung is to properly stored in a pit. The pit of one meter depth is to be dug under the shade and the dung is kept moist. The surface is plastered with mud. The manure becomes ready for use after 2-3 months.
• Composite manure – It consist of rottened vegetables and animal-shed wastes, refuse, farm weeds and other substances. These are properly mixed and used after rottening or decomposition.
• Green manures – Green manures are the green crops which are the back under the soil. Thus, green manuring can be defined as the practices of growing, plugging under and mixing of green crops with soil to Improve soil fertility and ultimately crop productivity. It also provides a protection action against soil erosion and leaching. There is 30-50% increase in the crops yield by using green manure. Some of the commonly used green manures are mainly leguminous and non-leguminous.
Cowpea, sun hemp, dhaincha, berseen, lentil (Mansur)

Important of green manure in agriculture
1. Green manures are the cheapest and easily available source or fertilizers.
2. They provide the additional nitrogen as well as other organic matters to soil.
3. They supply practically all the nutrient required by the crops.
4. They check soil erosion, leaching and percolation.
5. They increase the crop yield from 30% to 50%.
6. They improve soil aeration and drainage condition.


Prospectus of green manure in Nepal
Since there is no fertilizer factory in Nepal, the use of green manures can be the only supplemental source of fertilizer especially for nitrogen fertilizers. Dr. Bhola Man Singh Basnet of Nepal Agriculture Research Council (NARC) khumaltar has extensively carried out his research on green manure using Sesbania which is called locally as Dhanicha.

Sesbania can be successfully grown and incorporated in the soil. It has been estimated that by the use of sesbania, the yield of paddy has increased from 21 to25%. Therefore, green manure can very good source of fertilizer in Nepal.

Types of genetic resistance

The two types of genetic resistance to pathogen in plants are monogenic and polygenic. While monogenic characters are stable over a wide range of conditions, polygenic resistance is highly variable and influenced by environmental conditions. Polygenic resistance is also influenced by host nutrition, while monogenic resistance is completely stable. Repeated selections through breeding or resistance in a crop result in accumulation of more genes for resistance in the new varieties, which is the case in most of the varieties of crop plants which are widely cultivated over the world.

Another recent finding is that in a crop variety there may be a gene which inhibits completely or partially the expression of resistance to a diseases. Similarly, an inhibitor gene may suppress the expression of susceptibility in some varieties. It has been shown in some varieties that resistance may fluctuate within certain limits. There are also reports showing that some crop varieties are resistant to a pathogen at certain temperature, but May completely breakdown and become susceptible at a different temperature. If the pathogen is capable of producing many races, the monogenic resistance in the host may result in its becoming susceptible to one or more of the newer races to the gungus. This is what happens in wheat varieties which breakdown due to infection by newer rust races. If large numbers of genes for resistance are accumulated in one variety by repeated breeding and selection, then the possibilities of its sustained resistances to most races of rust are improved. Host resistance may inherited as a qualitative character, and its expression is influenced by environment by environment. In polygenic a virulent recombination in the pathogen, durability of resistance is prolonged.

A thorough knowledge of the variability and genetic characters of the pathogen as well as the inheritance and expression of resistance of susceptibility in the host is essential in a breeding programme for diseases resistance. Plant improvements in agriculture have been achieved through several means, the chief ones being introduction of new varieties of crops into a locality, hybridization and selection, and induced mutation. A variety resistant to a given diseases can be obtained either by selecting lines which already have genes for resistance or by hybridization to combine resistance. To obtain the desired variety through selection, we must have (i) resistant lines or biotypes in the population we are working with, (ii) reliable technique for screening the varieties for resistance, and (iii) the selected variety should combine other desirable properties, including good agronomic qualities. The variety must be tested under optimum condition for resistance to the diseases, to avoid diseases escape leading to erroneous conclusions.

The breeding programme is a continuing process. Plant breeders, pathologists and agronomists should work hand in hand to reach the goal. Worldwide search for genetic material in different crops plants has been going on and extensive collection of germ-plasma are available at many institutions. These wild and cultivated plants carrying resistance genes for various plant diseases. It is for the plant breeders, agronomists and pathologists to make best use of them and evolve resistant varieties with combined agronomic qualities.

Diseases Resistance varieties of plants

In many cases, growing resistant crop varieties id the only method to control diseases, and perhaps is the ideal one. In an ecological set-up equilibrium is maintained. If the equilibrium is altered in any one respect then several changes take place. If the plant has been growing wild in a locality, it is exposed to plant pathogens. Due to continued exposure to pathogenic invasions, the host might develop resistance to infection. Likewise the plant may alter several of its morphological and physiological characters. The Darwinian principles of variation, struggle for existence and survival of the fittest play under these circumstance and we get a resistant plant containing to grow and perpetuate while the susceptible ones may disappear from the scene. This has been going on for hundreds of thousands of years. When man cultivates the plant in bulk in field, he is upsetting the natural ecological balance and exposing the plants to a newer environment. Under the influence of cultural practices and other factors it may become susceptible to the diseases. Hence, he has to select and choose crop varieties which are resistant or tolerant to higher doses of fertilizers, high quality of the grains or other plant products, and resistance to pets and diseases. The pets and diseases of important or major crops plants like wheat, rice, sorghum and other millets are many. To obtained a variety resistant to all the diseases of a crops and to combine in it the best of agronomic qualities is an impossible task. Added to this, the pathogens are constantly changing, evolving new races of pathogens arise to survive. Hence, it is a never-ending struggle for man to go on evolving newer crops varieties not only to secure better agronomic qualities and increases production, but to combat newer and more virulent pathogens and pests. In a way, the breeder encourages the development of new races!

With the rediscovery of Gregor Mendel’s finding by de Vries in Holland, Correns in Germany and Tschermak in Austria almost simultaneously in 1900, the science of genetics had a rebirth. Though there are earlier reports on the possibilities of obtaining varieties resistant to diseases, systematic studies to select varieties of disease resistance started only during 1900, and the credit for this goes to W.A. Orton of the United States Department of Agriculture, who selected cotton varieties resistance to Fusarium wilt. The breeding work for resistant varieties against bunt and smut of wheat started in Australia by the turn of the century and later various workers including Stakman and his associates worked on resistance to rust and smuts. They evolved several techniques for testing crop varieties for disease resistance and also to differentiate the varieties and races of fungal species, based on host reactions under a given set of environment conditions. These results revealed that new resistant varieties should not be expected to solve the problem permanently, since races arise in nature by more than one method. Along with this information, data on the heterothallic nature and genetics of other fungi, such as Neuospora, were worked out. Starting from 1946, the basis for the occurrence of newer races among fungi came to be better understood. Expansion of our knowledge on the physiology and biochemistry of different living systems has helped us to understand many aspects of host-pathogen relationships, as also the genetic basis of diseases resistance in plants.

SUPERIOR VARIETIES OF PLANTS

Superior varieties of crop plants are obtained from outside and accilimitised to local environment. Before introducing a planhttp://technologyofbiology.blogspot.com/2010/03/5-induced-mutations-mutations-are-new.htmlt, study of its growth patterns, soil and climatic conditions in the original habitat is carried out. Choice is then made of equivalent soil and climatic conditions. A number of plants having all type of variations in the original habitat are planted in the new habitat. The plants which show good performance in the new habitat are picked up for further propagation.
Hybridization – Hybridization is the obtaining of progeny after crossing two or more types of plants which differ genetically one another in one or more traits. Depending upon the traits involved, the cross is called monohybrid (single trait), dihybrid (double trait) or polyhybrid. Similarly, hybridization may be performed between two plants (single cross) or more than two plants (multicross). Hybridization may be intravarietal, intervariental, interspecific and intergenetic. Hybridization is performed for two reasons-
1. Development of hybrid vigour or heterosis and
2. Bringing together desirable characters present in different races, varieties etc. into the individual or hybrid.

Polyploid breeding – An organism having number of complete chromosome sets higher than diploid number is called polyploid. The polyploid is known as triploid, tetraploid, pentaploid etc. when it contains 3, 4 and 5 sets of chromosomes respectively. Polyploidy occurs in mature due to failure of chromosomes to separate at the time of anaphase either due to nondisjunction or nonformation of spindle, failure of meiosis during sporogenesis or gametogenesis fertilization of an egg with more than one sperm. Polyploidy can be induced artificially by application of colchicines and granosan. Polyploidy is the three types – autopolyploidy, allopolyploidy and autoallopolyploidy.
Autopolyploidy occurs within a species and improves the numerical increase in the same chromosome set e.g. autotriploid (AAA) autotetraploid (AAAA). Allopolyploidy develops due to hybridization between two species followed by doubling of chromosomes. Allotetraploid (AABB) is the common type. Autoalloploidy develops both by numerical increase of chromosome etc. within a species as well as hybridization between two species followed by chromosome dounling (AAAABB) is common type of autoallopoloidy.
Polyplioids are also called euploids because they possess exact multiple of haploid chromosome set. The plants having few or extra chromosome than the multiple of haploid set are called aneuploids. The condition of having few or extra chromosomes than the multiple of haploid genome is termed as aneuploidy or homologons pairs so that one type of gametes come to have extra chromosome (N + 1) while the others become deficient in one chromosome (N – 1 ). Fusion with similar or normal gametes give rise to aneuploids.

5 Induced Mutations

are new sudden stable inheritable discontinuous variations which appeat in organism due to permanent change in their genotypes. Mutations can occur naturally and automatically in organisms without apparent reasons. They are termed as spontaneous mutations or spots. Useful spontaneous mutation occurring in osmotic cells of vegetatively propagated plants can be easily picked up and multiplied, e.g. colour sports in apple varieties, seedless grapes, navel orange etc. in others somatic variations vanish with the death of the organisms. Mutations which occur in the germ cells and can be transferred to progeny are germinal mutations. They may not express their effect immediately because most of the mutations are recessive.
Recessive mutations would produce their effect only in the homozygous state. This is mainly in self pollinated and vegetatively propagated plants. In many crop plants, genetic improvement is made through sexual reproduction which is then maintained through cloning or vegetative multiplication by means of tubers (potato), cutting (apple, sugarcane), runners or stolons (strawberry) etc. An important mutations occurring in the sexually reproducing plants is the stiff ears (nonshattering quality) in wheat.
The frequency of spontaneous mutation is very low. Therefore they cannot be relied upon for rapid improvement of crop plants. Plants breeders employ induced mutations are those mutations which develop in response specific factors or chemicals called mutagens.
6 Tissue culture and Genetic Engineering – Plant tissue culture is the technique of maintaining and growing plant cells tissues or organs on artificial medium in suitable containers under controlled environment conditions. The part which is cultured is called explants. It has to be first disinfected with clorax water, dilute hypochlorite or methiolate. The explants may be root, stem shoot tip, leaf petiole, embryo etc. It may be grown directly or sectioned into this discs, plates etc. The culture medium can be liquid, semi solid or solid. The nutrition solution contains source of energy (2-4% sucrose), vitamins, amino acid, minerals etc. The growth regulators can be replaced by organic complex like coconut water or milk, yeast extract, banana pulp etc.
The explants can produce the whole plant or specific organ like fruit from a pollinated pistil or ovary. Tissues, rections or cells usually produce first an irregular, undifferentiated, unorganized but actively dividing mass called callus. Darkness favours callus formation. The callus can divided and subcultured. Differentiation of organogenesis occurs when callus is exposed to light or provided with a proper dosage of auxin and cytokinins. Therefore, conditions in the culture room and composition of culture medium. Tissue culture can help improvement of crop plants by the following techniques:
1. Mircopropagation
2. Production of diseases free plants
3. Haploid
4. Embryo rescue
5. Induced mutation etc.
Genetic engineering or recombinant DNA is the most recent mechanism of providing superior heredity in crop plants. The technique is useful in deleting undesirable genes and introduction of useful or desirable genes. The most different job in genetic engineering is to locate and isolate fragment of DNA having the desirable gene or genes. For this the chromosome mapping or genome study of all crop pant and their wild relatives would genes in its genome. After obtaining the desirable DNA segment, the same is introduced in the cells through vector (virus, plasmid etc.) microinjection electroporation etc. The transformed cells are then allowed to multiply and form a whole plant.