Biology

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Advancements In Modern Biotechnology

Modern biotechnology embraces all the genetic manipulations, protoplasmic fusion techniques and the improvements made in the old biotechnological processes. Some of the major advancements in modern biotechnology are described below.

Genetic Engineering

Genetic engineering or recombinant DNA technology or gene cloning is a collective term that includes different experimental protocols resulting in the modifiation and transfer of DNA from one organism to another.

The definition for conventional recombination was already given in Unit II. Conventional recombination involves exchange or recombination of genes between homologous chromosomes during meiosis. Recombination carried out artificially using modern technology is called recombinant DNA technology (r-DNA technology). It is also known as gene manipulation technique.

This technique involves the transfer of DNA coding for a specific gene from one organism into another organism using specific agents like vectors or using instruments like electroporation, gene gun, liposome mediated, chemical mediated transfers and microinjection.

Steps involved in Recombinant DNA Technology

The steps involved in recombinant DNA technology are:

• Isolation of a DNA fragment containing a gene of interest that needs to be cloned. This is called an insert.
• Generation of recombinant DNA (rDNA) molecule by insertion of the DNA fragment into a carrier molecule called a vector that can self-replicate within the host cell.
• Selection of the transformed host cells is carrying the rDNA and allowing them to multiply thereby multiplying the rDNA molecule.
• The entire process thus generates either a large amount of rDNA or a large amount of protein expressed by the insert.
• Wherever vectors are not involved the desired gene is multiplied by PCR technique. The multiple copies are injected into the host cell protoplast or it is shot into the host cell protoplast by shot gun method.

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Methods Of Biotechnology

Fermentation

The word fermentation is derived from the Latin verb ‘fervere’ which means ‘to boil’. Fermentation refers to the metabolic process in which organic molecules (normally glucose) are converted into acids, gases, or alcohol in the absence of oxygen or any electron transport chain.

The study of fermentation, its practical uses is called zymology and originated in 1856, when French chemist Louis Pasteur demonstrated that fermentation was caused by yeast.

Fermentation occurs in certain types of bacteria and fungi that require an oxygenfree environment to live. The processes of fermentation are valuable to the food and beverage industries, with the conversion of sugar into ethanol to produce alcoholic beverages, the release of CO₂ by yeast used in the leavening of bread, and with the production of organic acids to preserve and flavour vegetables and dairy products.

Bioreactor (Fermentor)

Bioreactor (Fermentor) is a vessel or a container that is designed in such a way that it can provide an optimum environment in which microorganisms or their enzymes interact with a substrate to produce the required product. In the bioreactor aeration, agitation, temperature and pH are controlled. Fermentation involves two process namely upstream and downstream process.

(i) Upstream process

All the process before starting of the fermenter such as sterilization of the fermenter, preparation and sterilization of culture medium and growth of the suitable inoculum are called upstream process.

(ii) Downstream process

All the process after the fermentation process is known as the downstream process. This process includes distillation, centrifuging filtration and solvent extraction. Mostly this process involves the purification of the desired product.

Procedure of Fermentation

• Depending upon the type of product, bioreactor is selected.
• A suitable substrate in liquid medicine is added at a specific temperature, pH and then diluted.
• The organism (microbe, animal/plant cell, sub-cellular organelle or enzyme) is added to it.
• Then it is incubated at a specific temperature for the specified time.
• The incubation may either be aerobic or anaerobic.
• Withdrawal of product using downstream processing methods.

Application of fermentation in industries

Fermentation has industrial application such as:

1. Microbial biomass production

Microbial cells (biomass) like algae, bacteria, yeast, fungi are grown, dried and used as source of a complete protein called ‘single cell protein (SCP)’ which serves as human food or animal feed.

2. Microbial metabolites

Microbes produce compounds that are very useful to man and animals. These compounds called metabolites, can be grouped into two categories:

a. Primary metabolites:

Metabolites produced for the maintenance of life process of microbes are known as primary metabolites Eg. Ethanol, citric, acid, lactic acid, acetic acid.

b. Secondary metabolites:

Secondary metabolites are those which are not required for the vital life process ofmicrobes, but have value added nature, this includes antibiotics e.g – Amphotericin – B (Streptomyces nodosus), Penicillin (Penicillium chryosogenum) Streptomycin (S. grises), Tetracycline (S. aureofacins), alkaloids, toxic pigments, vitamins etc.

3. Microbial enzymes

When microbes are cultured, they secrete some enzymes into the growth media. These enzymes are industrially used in detergents, food processing, brewing and pharmaceuticals. Eg. protease, amylase, isomerase, and lipase.

4. Bioconversion, biotransformation or modification of the substrate

The fermenting microbes have the capacity to produce valuable products, eg. conversion of ethanol to acetic acid (vinegar), isopropanol to acetone, sorbitol to sorbose (this is used in the manufacture of vitamin C), sterols to steroids.

Single Cell Protein (SCP)

Single cell proteins are dried cells of microorganism that are used as protein supplement in human foods or animal feeds. Single Cell Protein (SCP) offers an unconventional but plausible solution to protein deficiency faced by the entire humanity.

Although single cell protein has high nutritive value due to their higher protein, vitamin, essential amino acids and lipid content, there are doubts on whether it could replace conventional protein sources due to its high nucleic acid content and slower in digestibility. Microorganisms used for the production of Single Cell Protein are as follows:

• Bacteria – Methylophilus methylotrophus, Cellulomonas, Alcaligenes
• Fungi – Agaricus campestris, Saccharomyces cerevisiae (yeast), Candida utilis
• Algae – Spirulina, Chlorella, Chlamydomonas

The single cell protein forms an important source of food because of their protein content, carbohydrates, fats, vitamins and minerals. It is used by Astronauts and Antarctica expedition scientists.

Spirulina can be grown easily on materials like waste water from potato processing plants (containing starch), straw, molasses, animal manure and even sewage, to produce large quantities and can serve as food rich in protein, minerals, fats, carbohydrate and vitamins. Such utilization also reduces environmental pollution. 250 g of Methylophilus methylotrophus, with a high rate of biomass production and growth, can
be expected to produce 25 tonnes of protein.

Applications of Single-Cell Protein

• It is used as protein supplement
• It is used in cosmetics products for healthy hair and skin
• It is used as the excellent source of protein for feeding cattle, birds, fihes etc.
• It is used in food industry as aroma carriers, vitamin carrier, emulsifying agents to improve the nutritive value of baked products, in soups, in ready-to-serve-meals, in diet recipes.
• It is used in industries like paper processing, leather processing as foam stabilizers.

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Development Of Biotechnology

Biotechnology has developed by leaps and bounds during the past century and its development can be well understood under two main heads namely conventional or traditional biotechnology and modern biotechnology.

1. Conventional or traditional biotechnology:
This is the kitchen technology developed by our ancestors, and it is as old as human civilization. It uses bacteria and other microbes in the daily usage for preparation of dairy products like curd, ghee, cheese and in preparation of foods like idli, dosa, nan, bread and pizza.

This conventional biotechnology also extends to preparation of alcoholic beverages like beer, wine, etc. With the advancement of the science and technology during the 18th century, these kitchen technologies gained scientifi validation.

Modern biotechnology

There are two main features of this technology, that differentiated it from the conventional technology they are

• Ability to change the genetic material for getting new products with specific requirement through recombinant DNA technology.
• Ownership of the newly developed technology and its social impact.

Today, biotechnology is a billion dollar business around the world, where in pharmaceutical companies, breweries, agro industries and other biotechnology based industries apply biotechnological tools for their product improvement.

Modern biotechnology embraces all methods of genetic modifiation by recombinant DNA and cell fusion technology. The major focus of biotechnology are:-

Fermentation
For production of acids, enzymes, alcohols, antibiotics, fine chemicals, vitamins and toxins.

Biomass
Biomass for bulk production of single cell protein, alcohol, and biofuel.

Enzymes
Enzymes as biosensors, in processing industry.

Biofuels
Biofuels for production of hydrogen, alcohol, methane.

Microbial inoculants
As biofertiliser, and nitrogen fiers.

Plant and animal cell
Culture for production of secondary metabolites, monoclonal antibodies.

Recombinant DNA technology
For production of fine chemicals, enzymes, vaccines, growth hormones, antibiotics, and interferon.

Process engineering
Tools of biotechnology is used for effluent treatment, water recycling. This unit will reveal the various aspects of modern biotechnology, its products and applications.

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Mutation – Types, Mutagenic Agents and Their Significance

Genetic variation among individuals provides the raw material for the ultimate source of evolutionary changes. Mutation and recombination are the two major processes responsible for genetic variation. A sudden change in the genetic material of an organisms is called mutation. The term mutation was introduced by Hugo de Vries (1901) while he has studying on the plant, evening primrose (Oenothera lamarkiana) and proposed ‘Mutation theory’.

There are two broad types of changes in genetic material. They are point mutation and chromosomal mutations. Mutational events that take place within individual genes are called gene mutations or point mutation, whereas the changes occur in structure and number of chromosomes is called chromosomal mutation.

Agents which are responsible for mutation are called mutagens, that increase the rate of mutation. Mutations can occur either spontaneously or induced. The production of mutants through exposure of mutagens is called mutagenesis, and the organism is said to be mutagenized.

Types of mutation

Let us see the two general classes of gene mutation:

• Mutations affcting single base or base pair of DNA are called point mutation
• Mutations altering the number of copies of a small repeated nucleotide sequence within a gene

Point mutation

It refers to alterations of single base pairs of DNA or of a small number of adjacent base pairs.

Types of point mutations

Point mutation in DNA are categorised into two main types. They are base pair substitutions and base pair insertions or deletions. Base substitutions are mutations in which there is a change in the DNA such that one base pair is replaced by another (Figure: 3.17).

It can be divided into two subtypes: transitions and transversions. Addition or deletion mutations are actually additions or deletions of nucleotide pairs and also called base pair addition or deletions. Collectively, they are termed indel mutations (for insertion-deletion).

Substitution mutations or indel mutations affect translation. Based on these different types of mutations are given below. The mutation that changes one codon for an amino acid into another codon for that same amino acid are called Synonymous or silent mutations. The mutation where the codon for one amino acid is changed into a codon for another amino acid is called Missense or non-synonymous mutations.

The mutations where codon for one amino acid is changed into a termination or stop codon is called Nonsense mutation. Mutations that result in the addition or deletion of a single base pair of DNA that changes the reading frame for the translation process as a result of which there is complete loss of normal protein structure and function are called Frameshift mutations (Figure: 3.19).

Mutagenic agents

The factors which cause genetic mutation are called mutagenic agents or mutagens. Mutagens are of two types, physical mutagen and chemical mutagen. Muller (1927) was the first to fid out physical mutagen in Drosophila.

Physical mutagens:

Scientists are using temperature and radiations such as X rays, gamma rays, alfa rays, beta rays, neutron, cosmic rays, radioactive isotopes, ultraviolet rays as physical mutagen to produce mutation in various plants and animals.

Temperature:

Increase in temperature increases the rate of mutation. While rise in temperature, breaks the hydrogen bonds between two DNA nucleotides which affects the process of replication and transcription.

Radiation:

The electromagnetic spectrum contains shorter and longer wave length rays than the visible spectrum. These are classified into ionizing and non-ionizing radiation. Ionizing radiation are short wave length and carry enough higher energy to ionize electrons from atom.

X rays, gamma rays, alfa rays, beta rays and cosmic rays which breaks the chromosomes (chromosomal mutation) and chromatids in irradiated cells. Non-ionizing radiation, UV rays have longer wavelengths and carry lower energy, so they have lower penetrating power than the ionizing radiations. It is used to treat unicellular microorganisms, spores, pollen grains which possess nuclei located near surface membrane.

Sharbati Sonora

Sharbati Sonora is a mutant variety of wheat, which is developed from Mexican variety (Sonora 64) by irradiating of gamma rays. It is the work of Dr. M.S.Swaminathan who is known as ‘Father of Indian green revolution’ and his team.

Castor Aruna

Castor Aruna is mutant variety of castor which is developed by treatment of seeds with thermal neutrons in order to induce very early maturity (120 days instead of 270 days as original variety).

Chemical mutagens:

Chemicals which induce mutation are called chemical mutagens. Some chemical mutagens are mustard gas, nitrous acid, ethyl and methyl methane sulphonate (EMS and MMS), ethyl urethane, magnous salt, formaldehyde, eosin and enthrosine. Example: Nitrous oxide alters the nitrogen bases of DNA and disturb the replication and transcription that leads to the formation of incomplete and defective polypeptide during translation.

Comutagens

The compounds which are not having own mutagenic properties but can enhance the effects of known mutagens are called comutagens. Example: Ascorbic acid increase the damage caused by hydrogen peroxide. Caffine increase the toxicity of methotrexate.

Chromosomal mutations

The genome can also be modified on a larger scale by altering the chromosome structure or by changing the number of chromosomes in a cell. These large-scale variations are termed as chromosomal mutations or chromosomal aberrations. Gene mutations are changes that take place within a gene, whereas chromosomal mutations are changes to a chromosome region consisting of many genes.

It can be detected by microscopic examination, genetic analysis, or both. In contrast, gene mutations are never detectable microscopically. Chromosomal mutations are divided into two groups: changes in chromosome number and changes in chromosome structure.

I. Changes in chromosome number

Each cell of living organisms possesses fixed number of chromosomes. It varies in different species. Even though some species of plants and animals are having identical number of chromosomes, they will not be similar in character. Hence the number of chromosomes will not differentiate the character of species from one another but the nature of hereditary material (gene) in
chromosome that determines the character of species.

Sometimes the chromosome number of somatic cells are changed due to addition or elimination of individual chromosome or basic set of chromosomes. This condition in known as numerical chromosomal aberration or ploidy. There are two types of ploidy.

1. Ploidy involving individual chromosomes within a diploid set (Aneuploidy)
2. Ploidy involving entire sets of chromosomes (Euploidy) (Figure 3.20)

1. Aneuploidy

It is a condition in which diploid number is altered either by addition or deletion of one or more chromosomes. Organisms
showing aneuploidy are known as aneuploids or heteroploids. Thy are of two types, Hyperploidy and Hypoploidy (Figure 3.21).

Hyperploidy

Addition of one or more chromosomes to diploid sets are called hyperploidy. Diploid set of chromosomes represented as Disomy. Hyperploidy can be divided into three types. They are as follows,

(a) Trisomy

Addition of single chromosome to diploid set is called Simple trisomy (2n+1). Trisomics were first reported by Blackeslee (1910) in Datura stramonium (Jimson weed). But later it was reported in Nicotiana, Pisum and Oenothera. Sometimes addition of two individual chromosome from diffrent chromosomal pairs to normal diploid sets are called Double trisomy (2n+1+1).

(b) Tetrasomy

Addition of a pair or two individual pairs of chromosomes to diploid set is called tetrasomy (2n+2) and Double tetrasomy (2n+2+2) respectively. All possible tetrasomics are available in Wheat.

(c) Pentasomy

Addition of three individual chromosome from different chromosomal pairs to normal diploid set are called pentasomy (2n+3).

2. Hypoploidy

Loss of one or more chromosome from the diploid set in the cell is called hypoploidy. It can be divided into two types. They are

(a) Monosomy

Loss of a single chromosome from the diploid set are called monosomy(2n-1). However loss of two individual or three individual chromosomes are called double monosomy (2n-1-1) and triple monosomy (2n-1-1-1) respectively. Double monosomics are observed in maize.

(b) Nullisomy

Loss of a pair of homologous chromosomes or two pairs of homologous chromosomes from the diploid set are called Nullisomy (2n-2) and double Nullisomy (2n-2-2) respectively. Selfig of monosomic plants produce nullisomics. They are usually lethal.

(ii) Euploidy

Euploidy is a condition where the organisms possess one or more basic sets of chromosomes. Euploidy is classifid as monoploidy, diploidy and polyploidy. The condition where an organism or somatic cell has two sets of chromosomes are called diploid (2n). Half the number of somatic chromosomes is referred as gametic chromosome number called haploid(n).

It should be noted that haploidy (n) is diffrent from a monoploidy (x). For example, the common wheat plant is a polyploidy
(hexaploidy) 2n = 6x = 72 chromosomes. Its haploid number (n) is 36, but its monoploidy (x) is 12. Therefore, the haploid and diploid condition came regularly one after another and the same number of chromosomes is maintained from generation to generation, but monoploidy condition occurs when an organism is under polyploidy condition. In a true diploid both the monoploid and haploid chromosome number are same. Thus a monoploid can be a haploid but all haploids cannot be a monoploid.

Polyploidy

Polyploidy is the condition where an organism possesses more than two basic sets of chromosomes. When there are three, four, fie or six basic sets of chromosomes, they are called triploidy (3x) tetraploidy (4x), pentaploidy (5x) and hexaploidy (6x) respectively.

Generally, polyploidy is very common in plants but rarer in animals. An increase in the number of chromosome sets has been an important factor in the origin of new plant species. But higher ploidy level leads to death. Polyploidy is of two types. They are autopolyploidy and allopolyploidy.

1. Autopolyploidy

The organism which possesses more than two haploid sets of chromosomes derived from within the same species is called autopolyploid. They are divided into two types. Autotriploids and autotetraploids.

Autotriploids have three set of its own genomes. They can be produced artifially by crossing between autotetraploid and diploid
species. They are highly sterile due to defective gamete formation. Example: The cultivated banana are usually triploids and are seedless having larger fruits than diploids.

Triploid sugar beets have higher sugar content than diploids and are resistant to moulds. Common doob grass (Cyanodon dactylon) is a natural autotriploid. Seedless watermelon, apple, sugar beet, tomato, banana are man made autotriploids. Autotetraploids have four copies of its own genome. They may be induced by doubling the chromosomes of a diploid species. Example: rye, grapes, alfalfa, groundnut, potato and coffee.

2. Allopolyploidy

An organism which possesses two or more basic sets of chromosomes derived from two different species is called allopolyploidy. It can be developed by interspecific crosses and fertility is restored by chromosome doubling with colchicine treatment. Allopolyploids are formed between closely related species only. (Figure 3.22)

Karpechenko (1927) a Russian geneticist, crossed the radish (Raphanus sativus, 2n=18) and cabbage (Brassica oleracea, 2n=18) to produce F₁ hybrid which was sterile. When he doubled the chromosome of F₁ hybrid he got it fertile. He expected this plant to exhibit the root of radish and the leaves like cabbage, which would make the entire plant edible, but the case was vice versa, so he was greatly disappointed.

Example: 2 Triticale, the successful fist man made cereal. Depending on the ploidy level Triticale can be divided into three main groups.

(i) Tetraploidy:
Crosses between diploid wheat and rye.

(ii) Hexaploidy:
Crosses between tetraploid wheat Triticum durum (macaroni wheat) and rye

(iii) Octoploidy:
Crosses between hexaploid wheat T. aestivum (bread wheat) and rye Hexaploidy Triticale hybrid plants demonstrate characteristics of both macaroni wheat and rye.

For example, they combine the high-protein content of wheat with rye’s high content of the amino acid lysine, which is low in wheat. It can be explained by chart below (Figure: 3.23).

Signifiance of Ploidy

• Many polyploids are more vigorous and more adaptable than diploids.
• Many ornamental plants are autotetraploids and have larger flowers and longer flowering duration than diploids.
• Autopolyploids usually have higher in fresh weight due to more water content.
• Aneuploids are useful to determine the phenotypic effcts of loss or gain of different chromosomes.
• Many angiosperms are allopolyploids and they play a role in the evolution of plants.

II Structural changes in chromosome (Structural chromosomal aberration)

Structural variations caused by addition or deletion of a part of chromosome leading to rearrangement of genes is called structural chromosomal aberration. It occurs due to ionizing radiation or chemical compounds. On the basis of breaks and reunion in chromosomes, there are four types of aberrations. They are classified under two groups.

A. Changes in the number of the gene loci

• Deletion or Defiiency
• Duplication or Repeat

B. Changes in the arrangement of gene loci

• Inversion
• Translocation

1. Deletion or Defiiency

Loss of a portion of chromosome is called deletion. On the basis of location of breakage on chromosome, it is divided into terminal deletion and intercalary deletion. It occurs due to chemicals, drugs and radiations. It is observed in Drosophila and Maize. (Figure 3.24)

2. Duplication or Repeat

The process of arrangement of the same order of genes repeated more than once in the same chromosome is known as duplication. Due to duplication some genes are present in more than two copies. It was first reported in Drosophila by Bridges (1919) and other examples are Maize and Pea. It is three types.

4. Translocation

The transfer of a segment of chromosome to a non-homologous chromosome is called translocation. Translocation should not
be confused with crossing over, in which an exchange of genetic material between homologous chromosome takes place.
Translocation occurs as a result of interchange of chromosome segments in non-homologous chromosomes. There are three types

• Simple translocation
• Shif translocation
• Reciprocal translocation
  

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Crossing Over, Recombination and Gene Mapping

Crossing over is a biological process that produces new combination of genes by interchanging the corresponding segments between non-sister chromatids of homologous pair of chromosomes. The term ‘crossing over’ was coined by Morgan (1912).

It takes place during pachytene stage of prophase I of meiosis. Usually crossing over occurs in germinal cells during gametogenesis. It is called meiotic or germinal crossing over. It has universal occurrence and has great significance. Rarely, crossing over occurs in somatic cells during mitosis. It is called somatic or mitotic crossing over.

Mechanism of Crossing Over

Crossing over is a precise process that includes stages like synapsis, tetrad formation, cross over and terminalization.

(i) Synapsis

Intimate pairing between two homologous chromosomes is initiated during zygotene stage of prophase I of meiosis I. Homologous chromosomes are aligned side by side resulting in a pair of homologous chromosomes called bivalents. This pairing phenomenon is called synapsis or syndesis. It is of three types,

• Procentric synapsis: Pairing starts from middle of the chromosome.
• Proterminal synapsis: Pairing starts from the telomeres.
• Random synapsis: Pairing may start from anywhere.

(ii) Tetrad Formation

Each homologous chromosome of a bivalent begin to form two identical sister chromatids, which remain held together by a centromere. At this stage each bivalent has four chromatids. Ths stage is called tetrad stage.

(iii) Cross Over

After tetrad formation, crossing over occurs in pachytene stage. The non-sister chromatids of homologous pair make a contact at one or more points. These points of contact between nonsister chromatids of homologous chromosomes are called Chiasmata (singular-Chiasma).

At chiasma, cross-shaped or X-shaped structures are formed, where breaking and rejoining of two chromatids occur. This results in reciprocal exchange of equal and corresponding segments

(iv) Terminalisation

After crossing over, chiasma starts to move towards the terminal end of chromatids. This is known as terminalisation. As a result, complete separation of homologous chromosomes occurs. (Figure 3.10)

Importance of Crossing Over

Crossing over occurs in all organisms like bacteria, yeast, fungi, higher plants and animals. Its importance is

Exchange of segments leads to new gene combinations which plays an important role in evolution. Studies of crossing over reveal that genes are arranged linearly on the chromosomes. Genetic maps are made based on the frequency of crossing over. Crossing over helps to understand the nature and mechanism of gene action.
If a useful new combination is formed it can be used in plant breeding.

Recombination

Crossing over results in the formation of new combination of characters in an organism called recombinants. In this, segments of DNA are broken and recombined to produce new combinations of alleles. This process is called Recombination.

Calculation of Recombination Frequency (RF)

The percentage of recombinant progeny in a cross is called recombination frequency. The recombination frequency (cross over frequency) (RF) is calculated by using the following formula. The data is obtained from alleles in coupling confiuration.

Genetic Mapping

Genes are present in a linear order along the chromosome. They are present in a specific location called locus (plural: loci). The diagrammatic representation of position of genes and related distances between the adjacent genes is called genetic mapping.

It is directly proportional to the frequency of recombination between them. It is also called as linkage map. The concept of gene mapping was first developed by Morgan’s student Alfred H Sturtevant in 1913.
It provides clues about where the genes lies on that chromosome.

Map distance

The unit of distance in a genetic map is called a map unit (m.u). One map unit is equivalent to one percent of crossing over (Figure 4.). One map unit is also called a centimorgan (cM) in honour of T.H. Morgan. 100 centimorgan is equal to one Morgan (M).

For example: A distance between A and B genes is estimated to be 3.5 map units. It is equal to 3.5 centimorgans or 3.5 % or 0.035 recombination frequency between the genes.

Uses of genetic mapping

• It is used to determine gene order, identify the locus of a gene and calculate the distances between genes.
• They are useful in predicting results of dihybrid and trihybrid crosses.
• It allows the geneticists to understand the overall genetic complexity of particular organism.