Category: Biology

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Basic Concepts In Plant Disuse Culture

Growing plant protoplasts, cells, tissues or organs away from their natural or normal environment, under artificial condition, is known as Tissue Culture. It is also known as in vitro (In vitro is a Latin word, it means that – in glass or in test-tube) growth of plant protoplasts, cells, tissues and organs. A single explant can be multiplied into several thousand plants in a short duration and space under controlled conditions.

Tissue culture techniques are often used for commercial production of plants as well as for plant research. Plant tissue culture serves as an indispensable tool for regeneration of transgenic plants. Apart from this some of the main applications of Plant tissue culture are clonal propagation of elite varieties, conservation of endangered plants, production of virus-free plants, germplasm preservation, industrial production of secondary metabolites. etc., In this chapter let us discuss the history, techniques, types, applications of plant tissue culture and get awareness on ethical issues.

Gottlieb Haberlandt (1902) the German Botanist proposed the concept Totipotency and he was also the first person to culture plant cells in artifiial conditions using the mesophyll cells of Lamium purpureum in culture medium and obtained cell proliferation. He is regarded as the father of tissue culture.

Basic concepts of Tissue Culture

Basic concepts of plant tissue culture are totipotency, diffrentiation, dediffrentiation and rediffrentiation.

Totipotency

The property of live plant cells that they have the genetic potential when cultured in nutrient medium to give rise to a complete individual plant.

Differentiation

The process of biochemical and structural changes by which cells become specialized in form and function.

Rediffrentiation

The further diffrentiation of already differentiated cell into another type of cell. For example, when the component cells of callus have the ability to form a whole plant in a nutrient medium, the phenomenon is called redifferentiation.

Dediffrentiation

The phenomenon of the reversion of mature cells to the meristematic state leading to the formation of callus is called dediffrentiation. These two phenomena of rediffrentiation and dedifferentiation are the inherent capacities of living plant cells or tissue. This is described as totipotency.

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Applications Of Biotechnology

Biotechnology is one of the most important applied interdisciplinary sciences of the 21st century. It is the trusted area that enables us to find the benefiial way of life.

Biotechnology has wide applications in various sectors like agriculture, medicine, environment and commercial industries.

This science has an invaluable outcome like transgenic varieties of plants e.g. transgenic cotton (Bt-cotton), rice, tomato, tobacco, cauliflwer, potato and banana.

The development of transgenics as pesticide resistant, stress resistant and disease resistant varieties of agricultural crops is the immense outcome of biotechnology.

The synthesis of human insulin and blood protein in E.coli and utilized for insulin defiiency disorder in human is a breakthrough in biotech industries in medicine.

The synthesis of vaccines, enzymes, antibiotics, dairy products and beverages are the products of biotech industries. Biochip based biological computer is one of the successes of biotechnology.

Genetic engineering involves genetic manipulation, tissue culture involves aseptic cultivation of totipotent plant cell into plant clones under controlled atmospheric conditions.

Single cell protein from Spirulina is utilized in food industries. Production of secondary metabolites, biofertilizers, biopesticides and enzymes. Biomass energy, biofuel, Bioremediation, phytoremediation for environmental biotechnology.

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Transgenic Plants / Genetically Modified Crops

Herbicide Tolerant – Glyphosate

Weeds are a constant problem in crop filds. Weeds not only compete with crops for sunlight, water, nutrients and space but also acts as a carrier for insects and diseases. If leaf uncontrolled, weeds can reduce crop yields signifiantly.

Glyphosate herbicide produced by Monsanto, USA company under the trade name ‘Round up’ kills plants by blocking the 5-enopyruvate shikimate-3 phosphate synthase (EPSPS) enzyme, an enzyme involved in the biosynthesis of aromatic amino acids, vitamins and many secondary plant metabolites. There are several ways by which crops can be modified to be glyphosate-tolerant.

One strategy is to incorporate a soil bacterium gene that produces a glyphosate tolerant form of EPSPS. Another way is to incorporate a different soil bacterium gene that produces a glyphosate degrading enzyme.

Advantages of Herbicide Tolerant Crops

• Weed control improves higher crop yields;
• Reduces spray of herbicide;
• Reduces competition between crop plant and weed;
• Use of low toxicity compounds which do not remain active in the soil; and
• The ability to conserve soil structure and microbes.

Herbicide Tolerant – Basta

Trade name ‘Basta’ refers to a non-selective herbicide containing the chemical compound phosphinothricin. Basta herbicide tolerant gene PPT (L-phosphinothricin) was isolated from Medicago sativa plant. It inhibits the enzyme glutamine synthase which is involved in ammonia assimilation.

The PPT gene was introduced into tobacco and transgenic tobacco produced was resistant to PPT. Similar
enzyme was also isolated from Streptomyces hygroscopicus with bar gene encodes for PAT (Phosphinothricin acetyl transferase) and was introduced into crop plants like potato and sugar-beet and transgenic crops have been developed.

Insect resistance – Bt Crops:

(i) Bt Cotton

Bt cotton is a genetically modifid organism (GMO) or genetically modified pest resistant plant cotton variety, which produces an insecticide activity to bollworm. Strains of the bacterium Bacillus thuringiensis produce over 200 different Bt toxins, each harmful to different insects.

Most Bt toxins are insecticidal to the larvae of months and butterfles, beetles, cotton bollworms and gatfles but are harmless to other forms of life. The genes are encoded for toxic crystals in the Cry group of endotoxin. When insects attack and eat the cotton plant the Cry toxins are dissolved in the insect’s stomach.

The epithelial membranes of the gut block certain vital nutrients thereby suffient regulation of potassium ions are lost in the insects and results in the death of epithelial cells in the intestine membrane which leads to the death of the larvae.

Advantages

The advantages of Bt cotton are:

• Yield of cotton is increased due to effective control of bollworms.
• Reduction in insecticide use in the cultivation of Bt cotton
• Potential reduction in the cost of cultivation.
  

Disadvantages

Bt cotton has some limitations:

• Cost of Bt cotton seed is high.
• Effctiveness up to 120 days after that efficiency is reduced
• Ineffctive against sucking pests like jassids, aphids and whitefly.
• Affects pollinating insects and thus yield.

(ii) Bt Brinjal

The Bt brinjal is another transgenic plant created by inserting a crystal protein gene (Cry1Ac) from the soil bacterium Bacillus thuringiensis into the genome of various brinjal cultivars.

The insertion of the gene, along with other genetic elements such as promoters, terminators and an antibiotic resistance marker gene into the brinjal plant is accomplished using Agrobacterium – mediated genetic transformation. The Bt brinjal has been developed to give resistance against Lepidopberan insects, in particular the Brinjal Fruit and Shoot Borer (Leucinodes orbonalis).

(iii) Dhara Mustard Hybrid (DMH)

DMH – 11 is transgenic mustard developed by a team of scientists at the Centre for Genetic Manipulation of Crop Plants Delhi University under Government sponsored project. It is genetically modified variety of Herbicide Tolerant (HT) mustard.

It was created by using “barnase/barstar” technology for genetic modifiation by adding genes from soil bacterium that makes mustard, a self-pollinating plant. DMH – 11 contains three genes viz. Bar gene, Barnase and Barstar sourced from soil bacterium. The bar gene had made plant resistant to herbicide named Basta.

Virus Resistance

Many plants are affcted by virus attack resulting in series loss in yield and even death. Biotechnological intervention is used to introduce viral resistant genes into the host plant so that they can resist the attack by virus. This is by introducing genes that produce resistant enzymes which can deactivate viral DNA.

FlavrSavr Tomato

Agrobacterium mediated genetic engineering technique was followed to produce Flavr-Savr tomato, i.e., retaining the natural colour and flavour of tomato. Though genetic engineering, the ripening process of the tomato is slowed down and thus prevent it from softning and to increase the shelf life.

The tomato was made more resistant to rotting by Agrobacterium mediated gene transfer mechanism of introducing an antisense gene which interferes with the production of the enzyme polygalacturonase, which help in delaying the ripening process of tomato during long storage and transportation.

Golden rice – Biofortification

Golden rice is a variety of Oryza sativa (rice) produced through genetic engineering of biosynthesized beta-carotene, a precursor of Vitamin-A in the edible parts of rice developed by Ingo Potrykus and his group. The aim is to produce a fortifid food to be grown and consumed in areas with a shortage of dietary Vitamin-A.

Golden rice differs from its parental strain by the addition of three beta-carotene biosynthesis genes namely ‘psy’ (phytoene synthase) from daffdil plant Narcissus pseudonarcissus and ‘crt-1’ gene from the soil bacterium Erwinia auredorora and ‘lyc’ (lycopene cyclase) gene from wild-type rice endosperm.

The endosperm of normal rice, does not contain beta-carotene. Golden-rice has been genetically altered so that the endosperm now accumulates Beta-carotene. This has been done using Recombinant DNA technology. Golden rice can control childhood blindness – Xerophthalmia.

GM Food – Benefits

• High yield without pest
• 70% reduction of pesticide usage
• Reduce soil pollution problem
• Conserve microbial population in soil

Risks – believed to

• Affect liver, kidney function and cancer
• Hormonal imbalance and physical disorder
• Anaphylactic shock (sudden hypersensitive reaction) and allergies.
• Adverse effect in immune system because of bacterial protein.
• Loss of viability of seeds seen in terminator seed technology of GM crops.

Polyhydroxybutyrate (PHB)

Synthetic polymers are non-degradable and pollute the soil and when burnt add dioxin in the environment which cause cancer. So, efforts were taken to provide an alternative eco-friendly biopolymers. Polyhydroxyalkanoates (PHAs) and polyhydroxybutyrate (PHB) are group of degradable biopolymers which have several medical applications such as drug delivery, scaffld and heart valves.

PHAs are biological macromolecules and thermoplastics which are biodegradable and biocompatible. Several microorganisms have been utilized to produce diffrent types of PHAs including Gram-positive like Bacillus megaterium, Bacillussubtilis and Corynebacterium glutamicum, Gram-negative bacteria like group of Pseudomonas sp. and Alcaligenes eutrophus.

Polylactic acid (PLA)

Polylactic acid or polylactide (PLA) is a biodegradable and bioactive thermoplastic. It is an aliphatic polyester derived from renewable resources, such as corn starch, cassava root, chips or starch or sugarcane. For the production of PLA, two main monomers are used: lactic acid, and the cyclic diester, lactide. The most common route is the ringopening polymerization of lactide with metal catalysts like tin octoate in solution. The metalcatalyzed reaction results in equal amount of d and polylactic acid.

Green Fluorescent Protein (GFP)

The green florescent protein (GFP) is a protein containing 238 amino acid residues of 26.9 kDa that exhibits bright green florescence when exposed to blue to ultraviolet range (395 nm). GFP refers to the protein first isolated from the jellyfih Aequorea victoria.

GFP is an excellent tool in biology due to its ability to form internal chromophore without requiring any accessory cofactors, gene products, enzymes or substrates other than molecular oxygen. In cell and molecular biology, the GFP gene is frequently used as a reporter of expression. It has been used in modifid forms to make biosensors.

Biopharming

Biopharming also known as molecular pharming is the production and use of transgenic plants genetically engineered to produce pharmaceutical substances for use of human beings. Ths is also called “molecular farming or pharming”. These plants are different from medicinal plants which are naturally available. The use of plant systems as bioreactors is gaining more signifiance in modern biotechnology. Many pharmaceutical substances can be produced using transgenic plants. Example: Golden rice

Bioremediation

It is defined as the use of microorganisms or plants to manage environmental pollution. It is an approach used to treat wastes including wastewater, industrial waste and solid waste. Bioremediation process is applied to the removal of oil, petrochemical residues, pesticides or heavy metals from soil or ground water.

In many cases, bioremediation is less expensive and more sustainable than other physical and chemical methods of remediation. An eco-friendly approach and can deal with lower concentrations of contaminants more effectively. The strategies for bioremediation in soil and water can be as follows:

• Use of indigenous microbial population as indicator species for bioremediation process.
• Bioremediation with the addition of adapted or designed microbial inoculants.
• Use of plants for bioremediation – green technology.

Some examples of bioremediation technologies are:

• Phytoremediation – use of plants to bring about remediation of environmental pollutants.
• Mycoremediation – use of fungi to bring about remediation of environmental pollutants.
• Bioventing a process that increases the oxygen or air flow to accelerate the degradation of environmental pollutants.
• Bioleaching use of microorganisms in solution to recover metal pollutants from contaminated sites.
• Bioaugmentation a addition of selected microbes to speed up degradation process.
• Composting process by which the solid waste is composted by the use of microbes into manure which acts as a nutrient for plant growth.
• Rhizofitration uptake of metals or degradation of organic compounds by rhizosphere microorganisms.
• Rhizostimulation stimulation of plant growth by the rhizosphere by providing better growth condition or reduction in toxic materials.

Limitations

• Only biodegradable contaminants can be transformed using bioremediation processes.
• Bioremediation processes must be specifially made in accordance to the conditions at the contaminated site.
• Small-scale tests on a pilot scale must be performed before carrying out the procedure at the contaminated site.
• The use of genetic engineering technology to create genetically modifid microorganism or a consortium of microbes for bioremediation process has great potential.

Biofuel: Algal Biofuel

Algal fuel, also known as algal biofuel, or algal oil is an alternative to liquid fossil fuels, the petroleum products. This is also used as a source of energy-rich oils. Also, algal fuels are an alternative to commonly known biofuel sources obtained from corn and sugarcane. The energy crisis and the world food crisis have initiated interest in algal culture (farming algae) for making biodiesel and other biofuels on lands unsuitable for agriculture. Botryococcus braunii is normally used to produce algal biofuel.

Biological hydrogen production by algae

The biological hydrogen production with algae is a method of photo biological water splitting. In normal photosynthesis the alga, Chlamydomonas reinhardtii releases oxygen. When it is deprived of sulfur, it switches to the production of hydrogen during photosynthesis and the electrons are transported to ferrodoxins. [Fe]-hydrogenase enzymes combine them into the production of hydrogen gas.

Bioprospecting

Bioprospecting is the process of discovery and commercialization of new products obtained from biological resources. Bioprospecting may involve biopiracy, in which indigenous knowledge of nature, originating with indigenous people, is used by others for profit, without authorization or compensation to the indigenous people themselves.

Biopiracy

Biopiracy can be defined as the manipulation of intellectual property rights laws by corporations to gain exclusive control over national genetic resources, without giving adequate recognition or remuneration to the original possessors of those resources. Examples of biopiracy include recent patents granted by the U.S. Patent and Trademarks Office to American companies on turmeric, ‘neem’ and, most notably, ‘basmati’
rice. All three products are indigenous to the Indo-Pak subcontinent.

Biopiracy of Neem

The people of India used neem and its oil in many ways to controlling fungal and bacterial skin infections. Indian’s have shared the knowledge of the properties of the neem with the entire world.

Pirating this knowledge, the United States Department of Agriculture (USDA) and an American MNC (Multi Nation Corporation) W.R.Grace in the early 90’s sought a patent from the European Patent Office (EPO) on the “method for controlling of diseases on plants by the aid of extracted hydrophobic neem oil”. The patenting of the fungicidal and antibacterial properties of Neem was an example of biopiracy but the traditional knowledge of the Indians was protected in the end.

Biopiracy of Turmeric

The United States Patent and Trademark Office, in the year 1995 granted patent to the method of use of turmeric as an antiseptic agent. Turmeric has been used by the Indians as a home remedy for the quick healing of the wounds and also for purpose of healing rashes. The journal article published by the Indian Medical Association, in the year 1953 wherein this remedy was mentioned.

Therefore, in this way it was proved that the use of turmeric as an antiseptic is not new to the world and is not a new invention, but formed a part of the traditional knowledge of the Indians. The objection in this case US patent and trademark office was upheld and traditional knowledge of the Indians was protected. It is another example of Biopiracy.

Biopiracy of Basmati

On September 2, 1997, the U.S. Patent and Trademarks Offi granted Patent on “basmati rice lines and grains” to the Texas-based company RiceTec. This broad patent gives the company several rights, including exclusive use of the term ‘basmati’, as well proprietary rights on the seeds and grains from any crosses. The patent also covers the process of breeding RiceTec’s novel rice lines and the method to determine the cooking properties and starch content of the rice grains.

India had periled the United States to take the matter to the WTO as an infringement of the TRIPS agreement, which could have resulted in major embarrassment for the US. Hence voluntarily and due to few decisions take by the US patent office, Rice Tec had no choice but to lose most of the claims and most importantly the right to call the rice “Basmati”.

In the year 2002, the fial decision was taken. Rice Tec dropped down 15 claims, resulting in clearing the path of Indian Basmati rice exports to the foreign countries. The Patent Office ordered the patent name to be changed to ‘Rice lines 867’.

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Screening For Recombiants

After the introduction of r-DNA into a suitable host cell, it is essential to identify those cells which have received the r-DNA molecule. This process is called screening. The vector or foreign DNA present in recombinant cells expresses the characters, while the non-recombinants do not express the characters or traits. For this some of the methods are used and one such method is Blue-White Colony Selection method.

Insertional Inactivation – BlueWhite Colony Selection Method

It is a powerful method used for screening of recombinant plasmid. In this method, a reporter gene lacZ is inserted in the vector. The lacZ encodes the enzyme β-galactosidase and contains several recognition sites for restriction enzyme.

β-galactosidase breaks a synthetic substrate called X-gal (5-bromo-4-chloro-indolyl-β-D-galacto-pyranoside) into an insoluble blue coloured product. If a foreign gene is inserted into lacZ, this gene will be inactivated. Therefore, no-blue colour will develop (white) because β-galactosidase is not synthesized due to inactivation of lacZ.

Therefore, the host cell containing r-DNA form white coloured colonies on the medium contain X-gal, whereas the other cells containing non-recombinant DNA will develop the blue coloured colonies.
On the basis of colony colour, the recombinants can be selected.

Antibiotic resistant markers

An antibiotic resistance marker is a gene that produces a protein that provides cells with resistance to an antibiotic. Bacteria with transformed DNA can be identifid by growing on a medium containing an antibiotic. Recombinants will grow on these media as they contain genes encoding resistance to antibiotics such as ampicillin, chloro amphenicol, tetracycline or kanamycin, etc., while others may not be able to grow in these media, hence it is considered useful selectable marker.

Replica plating technique

A technique in which the pattern of colonies growing on a culture plate is copied. A sterile filter plate is pressed against the culture plate and then lifted. Then the filter is pressed against a second sterile culture plate. This results in the new plate being infected with cell in the same relative positions as the colonies in the original plate. Usually, the medium used in the second plate will differ from that used in the first. It may
include an antibiotic or exclude a growth factor. In this way, transformed cells can be selected.

Molecular Techniques – Isolation of Genetic Material and Gel Electrophoresis

Electrophoresis is a separating technique used to separate diffrent biomolecules with positive and negative charges.

Principle

By applying electricity (DC) the molecules migrate according to the type of charges they have. The electrical charges on different molecules are variable.

Agarose GEL Electrophoresis

It is used mainly for the purifiation of specific DNA fragments. Agarose is convenient for separating DNA fragments ranging in size from a few hundred to about 20000 base pairs. Polyacrylamide is preferred for the purifiation of smaller DNA fragments. The gel is complex network of polymeric molecules.

DNA molecule is negatively charged molecule – under an electric field DNA molecule migrates through the gel. The electrophoresis is frequently performed with marker DNA fragments of known size which allow accurate size determination of an unknown DNA molecule by interpolation. The advantages of agarose gel electrophoresis are that the DNA bands can be readily detected at high sensitivity.

The bands of DNA in the gel are stained with the dye Ethidium Bromide and DNA can be detected as visible florescence illuminated in UV light will give orange florescence, which can be photographed.

Nucleic Acid Hybridization Blotting Techniques

Blotting techniques are widely used analytical tools for the specifi identification of desired DNA or RNA fragments from larger number of molecules. Blotting refers to the process of immobilization of sample nucleic acids or solid support (nitrocellulose or nylon membranes.) The blotted nucleic acids are then used as target in the hybridization experiments for their specific detection.

Types of Blotting Techniques

Southern Blotting:
The transfer of DNA from agarose gels to nitrocellulose membrane.

Northern Blotting:
The transfer of RNA to nitrocellulose membrane.

Western Blotting:
Electrophoretic transfer of Proteins to nitrocellulose membrane.

Southern Blotting Techniques – DNA:
The transfer of denatured DNA from Agarose gel to Nitrocellulose Blotting or Filter Paper technique was introduced by Southern in 1975 and this technique is called Southern Blotting Technique.

Steps

The transfer of DNA from agarose gel to nitrocellulose filter paper is achieved by Capillary Action. A buffer Sodium Saline Citrate (SSC) is used, in which DNA is highly soluble, it can be drawn up through the gel into the Nitrocellulose membrane.

By this process ss-DNA becomes ‘Trapped’ in the membrane matrix. This DNA is hybridized with a nucleic acid and can be detected by autoradiography.

Autoradiography – A technique that captures the image formed in a photographic emulsion due to emission of light or radioactivity from a labelled component placed together with unexposed film.

Northern Blot

It was found that RNA is not binding to cellulose nitrate. Therefore, Alwin et al. (1979) devised a procedure in which RNA bands are transferred from the agarose gel into nitrocellulose filter paper. This transfer of RNA from gel to special filter paper is called Northern Blot hybridization. The filter paper used for Northern blot is Amino Benzyloxymethyl Paper which can be prepared from Whatman 540 paper.

Western Blot

Refers to the electrophoretic transfer of proteins to blotting papers. Nitrocellulose filter paper can be used for western blot technique. A particular protein is then identified by probing the blot with a radio-labelled antibody which binds on the specific protein to which the antibody was prepared.

Bioassay for Target Gene Effect

Target gene is target DNA, foreign DNA, passenger DNA, exogenous DNA, gene of interest or insert DNA that is to be either cloned or specifially mutated. Gene targeting experiments have been targeting the nuclei and this leads to ‘gene knock-out’. For this purpose, two types of targeting vectors are used. They are insertion vectors and replacement or transplacement vectors.

Insertion vectors are entirely inserted into targeted locus as the vectors are linearized within the homology region. Initially, these vectors are circular but during insertion, become linear. It leads to duplication of sequences adjacent to selectable markers.

Differences between Blotting Techniques

The replacement vector has the homology region and it is co-linear with target. This vector is linearized prior to transfection outside the homology region and then consequently a crossing over occurs to replace the endogenous DNA with the incoming DNA.

Genome Sequencing and Plant Genome Projects

The whole complement of genes that determine all characteristics of an organism is called genome. Which may be nuclear genome, mitochondrial genome or plastid genome. Genome of many plants contain both functional and non-expressive DNA proteins.

Genome project refers to a project in which the whole genome of plant is analysed using sequence analysis and sequence homology with other plants. Such genome projects have so far been undertaken in Chlamydomonas(algae), Arabidopsis thaliana, rice and maize plants. Genome content of an organism is expressed in terms of number of base pairs or in terms of the content of DNA which is expressed as c-value.

Evolutionary pattern assessed using DNA

In recent years the evolutionary relationship between different plant taxa is assessed using DNA content as well as the similarities and differences in the DNA sequence (sequence homology). Based on such analysis the taxa and their relationship are indicated in cladogram. Which will show the genetic distance between two taxa. It also shows antiquity or modernity of any taxon with respect to one another (See also Unit-2, Chapter-5 of XI Std.)

Genome editing and CRISPR – Cas9

Genome editing or gene editing is a group of technologies that has the ability to change an organism’s DNA. These technologies allow genetic material to be added, removed, or altered at particular locations in the genome. Several approaches to genome editing have been developed. A recent one is known as CRISPR-Cas9, which is short form of Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9.

The CRISPR-Cas9 system has generated a lot of excitement in the scientific community because it is faster, cheaper, more accurate, and more efficient than other existing genome editing methods. Rice, was among the first plants to be used to demonstrate the feasibility of CRISPR mediated targeted mutagenesis and gene replacement.

The gene editing tool CRISPR can be used to make hybrid rice plants that can clone their seed. Imtiyaz Khand and Venkatesan Sundaresan and colleagues reported in a new study which clearly shows one can re-engineer rice to switch it from a sexual to an asexual mode.

RNA Interference (RNAi)

All characters of organism are the result of expression of different genes which are regions of nuclear DNA. This expression involves transcription and translation. Transcription refers to the copying of genetic information from one strand of the DNA (called sense strand) by RNA. This RNA, as soon as it formed cannot be straight away sent to the cytoplasm to undertake the process of translation.

It has to be edited and made suitable for translation which brings about protein synthesis. One of the main
items removed from the RNA strand are the introns. All these changes before translation normally take place whereby certain regions of DNA are silence. However, there is an (RNAi) pathway. RNA interference is a biological process in which RNA molecules inhibit gene expression or translation. This is done by neutralising targetd mRNA molecules.

A simplified model for the RNAi pathway is based on two steps, each involving ribonuclease enzyme. In the first step, the trigger RNA (either dsRNA or miRNA primary transcript) is processed into a short interfering RNA (siRNA) by the RNase II enzymes called Dicer and Drosha. In the second step, siRNAs are loaded into the effector complex RNA-induced silencing complex (RISC). The siRNA is unwound during RISC assembly and the single-stranded RNA hybridizes with mRNA target. This RNAi is seen in plant feeding nematodes.

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Methods Of Gene Transfer

The next step after a recombinant DNA molecule has been generated is to introduce it into a suitable host cell. There are many methods to introduce recombinant vectors and these are dependent on several factors such as the vector type and host cell.

For achieving genetic transformation in plants, the basic pre-requisite is the construction of a vector which carries the gene of interest flinked by the necessary controlling sequences, i.e., the promoter and terminator, and deliver the genes into the host plant. There are two kinds of gene transfer methods in plants. It includes:

• Direct or vectorless gene transfer
• Indirect or vector – mediated gene transfer

Direct or Vectorless Gene Transfer

In the direct gene transfer methods, the foreign gene of interest is delivered into the host plant without the help of a vector. The following are some of the common methods of direct gene transfer in plants.

a. Chemical mediated gene transfer:

Certain chemicals like polyethylene glycol (PEG) and dextran sulphate induce DNA uptake into plant protoplasts.

b. Microinjection:

The DNA is directly injected into the nucleus using fine tipped glass needle or micro pipette to transform plant cells. The protoplasts are immobilised on a solid support (agarose on a microscopic slide) or held with a holding pipette under suction.

c. Electroporation Methods of Gene Transfer:

A pulse of high voltage is applied to protoplasts, cells or tissues which makes transient pores in the plasma membrane through which uptake of foreign DNA occurs.

d. Liposome mediated method of Gene Transfer:

Liposomes the artificial phospholipid vesicles are useful in gene transfer. The gene or DNA is transferred from liposome into vacuole of plant cells. It is carried out by encapsulated DNA into the vacuole.

This technique is advantageous because the liposome protects the introduced DNA from being damaged by the acidic pH and protease enzymes present in the vacuole. Liposome and tonoplast of vacuole fusion resulted in gene transfer. This process is called lipofection.

e. Biolistics:

The foreign DNA is coated onto the surface of minute gold or tungsten particles (1-3 µm) and bombarded onto the target tissue or cells using a particle gun (also called as gene gun/micro projectile gun/shotgun). Then the bombarded cells or tissues are cultured on selected medium to regenerate plants from the transformed cells. (Figure 4.16)

Indirect or Vector-Mediated Gene Transfer

Gene transfer is mediated with the help of a plasmid vector is known as indirect or vector mediated gene transfer. Among the various vectors used for plant transformation, the Ti-plasmid from Agrobacterium tumefaciens has been used extensively.

This bacterium has a large size plasmid, known as Ti plasmid (Tumor inducing) and a portion of it referred as T-DNA (transfer DNA) is transferred to plant genome in the infected cells and cause plant tumors (crown gall). Since this bacterium has the natural ability to transfer T-DNA region of its plasmid into plant genome, upon infection of cells at the wound site, it is also known as the natural genetic engineer of plants.

The foreign gene (e.g. Bt gene for insect resistance) and plant selection marker gene, usually an antibiotic gene like npt II which confers resistance to antibiotic kanamycin are cloned in the T DNA region of Ti-plasmid in place of unwanted DNA sequences. (Figure 4.17)

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Tools For Genetic Engineering

In order to generate recombinant DNA molecule, certain basic tools are necessary. The basic tools are enzymes, vectors and host organisms. The most important enzymes required for genetic engineering are the restriction enzymes, DNA ligase and alkaline phosphatase.

Restriction Enzymes

The two enzymes responsible for restricting the growth of bacteriophage in Escherichia coli were isolated in the year 1963. One was the enzyme which added methyl groups to DNA, while the other cut DNA. The latter was called restriction endonuclease.

A restriction enzyme or restriction endonuclease is an enzyme that cleaves DNA into fragments at or near specific recognition sites within the molecule known as restriction sites. Based on their mode of action restriction enzymes are classified into Exonucleases and Endonucleases.

• Exonucleases are enzymes which remove nucleotides one at a time from the end of a DNA molecule. e.g. Bal 31, Exonuclease III.
• Endonucleases are enzymes which break the internal phosphodiester bonds within a DNA molecule. e.g. Hind II, EcoRI, Pvul, BamHI, TaqI.

Restriction endonucleases: Molecular scissors

The restriction enzymes are called as molecular scissors. These act as foundation of recombinant DNA technology. These enzymes exist in many bacteria where they function as a part of their defence mechanism called restrictionmodifiation system. There are three main classes of restriction endonucleases: Type I, Type II and Type III, which differ slightly by their mode of action.

Only type II enzyme is preferred for use in recombinant DNA technology as they recognise and cut DNA within a specific sequence typically consisting of 4-8 bp. Examples of certain enzymes are given in table 5.1.

The restriction enzyme Hind II always cut DNA molecules at a point of recognising a specific sequence of six base pairs. This sequence is known as recognition sequence. Today more than 900 restriction enzymes have been isolated from over 230 strains of bacteria with different recognition sequences. This sequence is referred to as a restriction site and is generally palindromic which means that the sequence in both DNA strands at this site read same in 5’ – 3’ direction and in the 3’ – 5’ direction

Example:
MALAYALAM: This phrase is read the same in either of the directions.

Restriction endonucleases are named by a standard procedure. The first letter of the enzymes indicates the genus name, followed by the first two letters of the species, then comes the strain of the organism and fially a roman numeral indicating the order of discovery. For example, EcoRI is from Escherichia (E) coli (co), strain RY 13 (R) and fist endonuclease (I) to be discovered.

The exact kind of cleavage produced by a restriction enzyme is important in the design of a gene cloning experiment. Some cleave both strands of DNA through the centre resulting in blunt or flush end. These are known as symmetric cuts. Some enzymes cut in a way producing protruding and recessed ends known as sticky or cohesive end. Such cut are called staggered or asymmetric cuts.

Two other enzymes that play an important role in recombinant DNA technology are DNA ligase and alkaline phosphatase.

DNA Ligase

DNA ligase enzyme joins the sugar and phosphate molecules of double stranded DNA (dsDNA) with 5’-PO₄ and a 3’-OH in an Adenosine Triphosphate (ATP) dependent reaction. This is isolated from T4 phage.

Alkaline Phosphatase

It is a DNA modifying enzyme and adds or removes specific phosphate group at 5’ terminus of double stranded DNA (dsDNA) or single stranded DNA (ssDNA) or RNA. Thus it prevents self ligation. This enzyme is purified from bacteria and calf intestine.

Vectors

Another major component of a gene cloning experiment is a vector such as a plasmid. A Vector is a small DNA molecule capable of self-replication and is used as a carrier and transporter of DNA fragment which is inserted into it for cloning experiments. Vector is also called cloning vehicle or cloning DNA.

Vectors are of two types:

• Cloning Vector, and
• Expression Vector. Cloning vector is used for the cloning of DNA insert inside the suitable host cell. Expression vector is used to express the DNA insert for producing specifi protein inside the host.

Properties of Vectors

Vectors are able to replicate autonomously to produce multiple copies of them along with their DNA insert in the host cell.

1. It should be small in size and of low molecular weight, less than 10 Kb (kilo base pair) in size so that entry/transfer into host cell is easy.

2. Vector must contain an origin of replication so that it can independetly replicate within the host.

3. It should contain a suitable marker such as antibiotic resistance, to permit its detection in transformed host cell.

4. Vector should have unique target sites for integration with DNA insert and should have the ability to integrate with DNA insert it carries into the genome of the host cell. Most of the commonly used cloning vectors have more than one restriction site. These are Multiple Cloning Site (MCS) or polylinker. Presence of MCS facilitates the use of restriction enzyme of choice.

The following are the features that are required to facilitate cloning into a vector.

1. Origin of replication (ori):

This is a sequence from where replication starts and piece of DNA when linked to this sequence can be made to replicate within the host cells.

2. Selectable marker:

In addition to ori the vector requires a selectable marker, which helps in identifying and eliminating non transformants and selectively permitting the growth of the transformants.

3. Cloning sites:

In order to link the alien DNA, the vector needs to have very few, preferably single, recognition sites for the commonly used restriction enzymes.

Types of vector

Few types of vectors are discussed in detail below:

Plasmid

Plasmids are extra chromosomal, self replicating ds circular DNA molecules, found in the bacterial cells in addition to the bacterial chromosome. Plasmids contain Genetic information for their own replication.

pBR 322 Plasmid

pBR 322 plasmid is a reconstructed plasmid and most widely used as cloning vector; it contains 4361 base pairs. In pBR, p denotes plasmid, Band R respectively the names of scientist Boliver and Rodriguez who developed this plasmid. The number 322 is the number of plasmid developed from their laboratory.

It contains amp^R and tet^R two different antibiotic resistance genes and recognition sites for several restriction enzymes. (Hind III, EcoRI, BamH I, Sal I, Pvu II, Pst I, Cla I), ori and antibiotic resistance genes. Rop codes for the proteins involved in the replication of the plasmid.

Ti Plasmid Bacteria

Ti plasmid is found in Agrobacterium tumefaciens, a bacteria responsible for inducing tumours in several dicot plants. The plasmid carries transfer (tra) gene which help to transfer T – DNA from one bacterium to other bacterial or plant cell.

It has Onc gene for oncogenecity, ori gene for origin for replication and inc gene for incompatibility. T – DNA of Ti – Plasmid is stably integrated with plant DNA. Agrobacterium plasmids have been used for introduction of genes of desirable traits into plants.

Competent Host (For Transformation with Recombinant DNA)

The propagation of the recombinant DNA molecules must occur inside a living system or host. Many types of host cells are available for gene cloning which includes E.coli, yeast, animal or plant cells. The type of host cell depends upon the cloning experiment.

E.coli is the most widely used organism as its genetic make-up has been extensively studied, it is easy to handle and grow, can accept a range of vectors and has also been studied for safety. One more important feature of E.coli to be preferred as a host cell is that under optimal
growing conditions the cells divide every 20 minutes.

Since the DNA is a hydrophilic molecule, it cannot pass through cell membranes, In order to force bacteria to take up the plasmid, the bacterial cells must first be made competent to take up DNA. This is done by treating them with a specific concentration of a divalent cation such as calcium.

Recombinant DNA can then be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them briefl at 420C (heatshock) and then putting them back on ice. This enables bacteria to take up the Recombinant DNA.

For the expression of eukaryotic proteins, eukaryotic cells are preferred because to produce a functionally active protein it should fold properly and post translational modifiations should also occur, which is not possible by prokaryotic cell (E.coli).

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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.