Genetic Crosses

A Monohybrid cross is a cross between two homozygous individuals for the study of a character. Monohybrid crosses the inheritance of a single character at a time. Through monohybrid cross law dominance can be determined.

TT x tt

⇩

F1 generation Tt( tall) x Tt(tall)

⇩

Male/female gamete ➡

⇩

(tall)

(short)

According to the law of dominance of the two alleles of a gene, one is dominant over the other. The dominant gene expresses itself in one generation. As well in up to generation recessive genes completely mask recessive genes in the F1 generation. But in F2 generation also Expresses itself showing that the gene is not lost in the F1 generation. It is only expressed in homozygous conditions. Monohybrid cross can be in test cross where the individual of the F2 generation is crossed with a recessive parent this cross determines homozygosity or heterozygosity of the individual. If the F2 individual is homozygous dominant, the test cross ratio will be 3:1, if the F2 individual is heterozygous the test cross ratio will be 2:2.

Tt x tt

⇩

Male/female gametes➡

⇩

Tall

short

Tall

short

Dihybrid cross is a cross to study two characters at a time. Dihybrid cross between a pea plant with it and a pea plant with green wrinkled seed shot up to a ratio of 9:3:3:1. There is a parental type as well as a recombinant type of seeds. The parental type that is round yellow which were both dominant characters appeared in the 9/16 ratio. While the recombinant i.e. green round 3/16, wrinkled yellow 1/16. This show that the seed color and shape depend upon two different genes, which are inherited independently of each other in the next generation. All the possible four types of gametes are formed. Each type of gamete has a 25% possibility. This is called the law of independent segregation where genes segregate from each other independently. They combine in next-generation and result in the formation of parental type as well as recombinant individuals in the F2 generation.

Male/ female gamete

WWGG

Yellow round

WWGg

Yellow round

WwGG

Yellow round

WwGg

Yellow round

WWGg

Yellow round

WWgg

Green round

WwGg

Yellow round

Wwgg

Green round

WwGG

Yellow round

WwGg

Yellow round

wwGG

yellow wrinkled

wwGg

yellow wrinkled

WwGg

Yellow round

Wwgg

Green round

wwGg

yellow wrinkled

wwgg

Green wrinkled

Complementary gene

F2 generation ratio is 9:7 complementary genes. are those where both the gene are required for the expression of a character. As seen in the flower color of Lathyrus odoratus where two genes control the expression of flower color. When both the genes are present in dominant form only then purple flower color will appear. But if a single gene is present in the dominant form white flower color is formed. It may be understood from a two-step reaction where one gene controls the formation of the intermediate product and the intermediate product is converted into purple flower color only in presence of the product of the second gene. So, in absence of any of these genes the purple flower color is not formed.

Supplementary gene interaction

It is an interaction between two independent genes. Here two independent genes when present in the dominant form together give a different phenotype when these are present in the dominant form individually give a different phenotype.

As coat color in mice AB together gives agouti, ab gives albino, AA and B give black color give albino coat color.

Duplicate gene

It is the interaction between two different genes that have the same phenotypic effect. This interaction has F2 generation 15:1. Fruit shape of Capsella bursa pastoris (shepherded purse). Which has two types of fruit triangular and oval. The shape of the fruit is determined by two independent genes present on different chromosomes. The dominant form of both the genes gives triangular fruit shape and recessive genes give oval shape.

Incomplete dominance

This is against the law of complete dominance where heterozygous individuals show intermediate complete dominance in incomplete dominance the genotypic ratio as well as phenotypic ratios are the same as incomplete dominance. It can be studied in flower color of mirabilis Jalapa homozygous dominant has red flower color heterozygous has pink flower color and homozygous recessive show white flower color.

GENE THERAPY

Gene therapy

Gene therapy is the medical approach to correcting a gene to cure a disease or disorder. There is a number of diseases that are caused due to dysfunctional genes or due to the gain of function of a gene. Genes express themselves by synthesizing proteins. If a protein is required for a metabolic process and this protein is non-functional due to defected gene, the metabolism is affected. Curing the protein is a process of re-establishing the metabolism. This can be done by curing the gene or transferring the correct gene into the organism.

Gene therapy includes somatic gene therapy where genes are edited or transferred into somatic cells of the organism. This therapy cures the disease in an individual. But it is not inheritable as the gene is edited in somatic cells. This can be used for genes that are specific in their expression in the organism. Somatic gene therapy includes gene transferring and gene editing.

SOMATIC CELL GENE THERAPY

Gene editing involves the specific technique of knocking out or knocking in the new gene. This uses crisper cas 9 Technology.

The transfer of genes is used for somatic gene therapy. However, vectors can be used to deliver genes to a specific site. Even though intravenous gene transfer can be done for this virus vectors are used. These viruses will deliver the correct gene to the site of its requirement.

The embryonic gene is the gene therapy where embryonic stem cells are cured for defected genes.

Gene editing may be used in embryonic gene therapy. Here the organism is cured in the embryonic stage only. Gene transfer can also be used in the embryonic cell stage also.

RNA EDITING

The new organism that is born will have corrected gene function. However, this approach has broad applicability but it is not allowed on the ethical ground. The embryonic stem cell gene therapy may lead to human cloning and the formation of designer babies.

Uses of gene therapy

It utilizes the organism-specific approach to cure a disease. No chemical drugs or antibiotics are used in therapy to avoid their side effects.

Gene therapy genetic disorders can be corrected for which rarely any curing approach is available to cancel can be cured through gene therapy without utilizing chemotherapy or radiotherapy which is destructive to human health. Spinal muscular dystrophy can be cured through gene therapy.

Immunosuppressant diseases are also cured by gene therapy.

Leukemia can be cured through gene therapy

Crisper cas9 Technology

Crisper cas 9 has a small guide RNA along with A nucleases the guide RNA has a specific sequence. This is called PAM this sequence binds to the targeted gene nucleus and then recognizes the site for cutting the DNA. It cut the DNA to either generate knockout where a gene is rendered dysfunctional or knock-in where the gene is rendered functional Crisper cas 9 Technology initiates the cellular mechanism of gene recombination it can be done through homologous change recombination or non-homologous gene recombination. Both of these mechanisms are well established in the cell and they can generate knockout or knock-in.

Fermentation technology: Ethanol, Lactic acid, and amylase production

ETHANOL PRODUCTION

Ethanol is an organic acid. It is widely used as a solvent. Ethanol is produced from carbohydrates (sucrose and starch). Ethanol is used as fuel.

Microorganisms producing ethanol

Both bacteria and fungi produce ethanol. Bacteria used for ethanol production are Zymomonas mobilis. The yeasts used for ethanol production are Saccharomyces cerevisiae, and Kluyveromyces fragilis.

Factors affecting ethanol production

Ethanol is inhibitory in higher concentrations. So, the microorganisms used in industrial production must be tolerant to the high concentrations of ethanol.

Pure sugar solution can increase the yield of ethanol. Osmotic tolerance of microorganisms is also important for ethanol production.

Organisms must have a high specific growth rate.

pH for ethanol production is 5-7.

The temperature for ethanol production is 30ºC.

PRODUCTION OF ETHANOL

The process of ethanol production involves the following steps

1) Preparation of nutrient medium

2) Fermentation

3) Distillation of ethanol.

Preparation of medium

Substrates used for ethanol production are-

Starch-containing roots, tubers, or grains

Molasses

Wood waste

Starch used in ethanol production has a low yield.

The substrate is heated to soften the substrate. It is liquefaction.

It is enzymatically metabolized into sugars

Molasse is also used as a source of sugar for ethanol production.

Continuous culture is used to increase ethanol production.

BIOSYNTHESIS OF ETHANOL

Product recovery

Product recovery of ethanol involves two main steps.

1) Separation of biomass, which involves centrifugation.

2) Distillation of ethanol, this step of product recovery for ethanol production is the most energy demanding. It increases the cost of production of ethanol.

So, the cheap source of sucrose is beneficial for ethanol production.

Lactic acid production

Lactic acid was the first organic acid that was produced in 1880. Lactic acid can be produced through chemical methods or through fermentation. There is a competition between the chemical production of lactic acid and fermentation.

Lactic acid production by the chemical method is cheaper. The production of lactic acid through fermentation should be from a cheaper feedstock.

Making it more economical through fermentation technology, it is challenging for lactic acid production.

The organisms that produce lactic acid are classified as homofermentative organisms or Hetero fermentative organisms. The homofermentative organisms produce especially the lactic acid only. However, Heterofermentative organisms produce lactic acid as well as other acids.

BIOSYNTHESIS OF LACTIC ACID

Bio-synthesis of lactic acid occurs from glucose when oxygen is insufficient. As we know that glucose is metabolized to pyruvate through glycolysis. Pyruvate is a feeder molecule of the Kreb cycle, where it is oxidatively metabolized into carbon dioxide and water. However, when oxygen is limiting Pyruvate is converted into lactic acid by the enzyme lactate dehydrogenase. Lactic acid is produced maybe Levo or dextrorotatory.

BIOSYNTHESIS OF LACTIC ACID

MICROORGANISMS USED FOR LACTIC ACID PRODUCTION

Organisms those produced lactic acids are Lactobacillus brueckii and Lactobacillus leichmannii, Lactobacillus pentosus. These organisms are facultative anaerobes. Thus only reducing the presence of oxygen can induce the production of lactic acid.

Lactic acid production does not require the complete exclusion of oxygen from the fermentation vessel.

The process of lactic acid production

It requires the preparation of medium, inoculation, and extraction of product. Preparation of the medium requires calibrating the medium according to the needs of the organism. For bacterial culture, the medium contains containing 12 to 13% Glucose, Ammonium Phosphate 0.25%, Vitamin B, and calcium carbonate are used.

The vessel size can be 25 to 120 m³.

The temperature of the culture is 45 to 50ºC. The maximum production of lactic acid occurs within 72 hours. So, the product is extracted after 72 hours. The higher concentration of lactic acid in the medium is poisonous to the bacteria. The concentration of lactic acid in the culture medium becomes up to 80 grams per liter per hour.

Amylase Production

Amylase is a starch liquefaction enzyme. There are two types of amylase, α-amylase, and β-amylase. These enzymes are used in the sweetener industry for the conversion of starch into dextrins, maltose, or glucose. The conversion of starch into these products is called starch saccharification.

Both α-amylase and β-amylase are extracellular enzymes. These are produced by bacteria as well as fungi both the amylases attack on 1-4 α glycosidic bond of starch thus producing the product of different lengths. The products include dextrins which are oligosaccharides, maltose which are disaccharides, and glucose which are monosaccharides.

All these products are sweet in taste so amylase found its application in the sweetener industry, baking industry, beverage industry, paper and pulp industry, and cloth industry. The enzymes that cause starch saccharification are α- amylase, β- amylase, and o- glucoamylase. Other enzymes are isoamylase and x-pullulanase. These can metabolize amylopectin as well because they can attack 1-6 α- glycosidic bonds as well.

The microorganisms that are used for amylase production

Bacterial species used in fermentation Technology for the production of amylase are

Bacillus subtilis,

Bacillus cereus,

Bacillus amyloliquefaciens,

Pseudomonas and Thermofactor

The thermophilic species used for amylase production is Thermobacter.

It can be cultured at 53ºC.

Fungi used in amylase production are

Aspergillus,

Penicillium,

Aspergillus oryzae,

Mucor,

Candida,

and Rhizopus.

Medium for amylase production by fungi has

8% starch,

1.2% NaNO3,

0.1 MgSO4,

2.0% Malt extract,

0.05%KCl,

0.003% FeSO4,

0.08% Mg(NO3)2.

Medium used for amylase production by bacteria has

0.5% starch

0.56% NH4NO3

0.28% sodium citrate

0.13%KH2PO4

0.05% MgSO4.7H2O

0.01%CaCl2

0.05% peptone

0.2% yeast extract

Amylase is an extracellular enzyme. It is produced in the cytoplasm. It has a signal sequence that targets the enzyme to the cell membrane of the organism, where it is folded into three-dimensional structures which is the functional form of the enzyme. The enzyme is released into the extracellular matrix, where it converts starch into different sweeteners.

Glucose present in the culture medium increases the growth of the bacteria but decreases amylase production.

When nitrogen is a limiting factor in the medium, amylase production decreases. The maximum production occurs at 45ºC in 18 hours for bacterial species. Up to 3000 units/ml is produced at 27 ºC to 37 ºC.

Amylase is a heat-tolerant protein that can tolerate up to 70°C temperature.

Purification of amylase is done through protein purification Technology.

ANIMAL CLONING

Cloning is making a copy of an organism. The animal clones produced have the same genetic makeup as that of the cloned organisms.

Natural evidence of cloning is found in bacteria, yeast, and other single-cell organisms that reproduce vegetatively.

In higher animals, evidence of cloning is not found. However, artificial animal cloning is possible. It was first successfully done by cloning Dolly, the sheep. It was started in 1979. However, successful cloning of Dolly could be done only in 1996.

Producing an animal of the same genetic makeup is called cloning. Clones can not be produced by the sexual reproductive process. Gene recombination occurs in sexual reproduction. Even the siblings are not clones. They differ from one another in their genetic makeup.

Animal cloning is somehow allowed in some countries, but human cloning despite different claims has not been done. Further, it is opposed on ethical grounds.

Identical twins are clones, as they develop from the single fertilized cell, the zygote.

The technology of animal cloning: Somatic Cell Nuclear Transfer

The technology used for animal cloning is SCNT( Somatic Cell Nuclear Transfer). It is the transfer of the somatic cell nucleus in the enucleated egg cell.

Animals can not be cultured in vitro. The production of the animal clone from a Somatic cell requires stimulation of the somatic cell. This stimulation is done through the somatic nucleus transfer in enucleated egg cells.

Steps involved in animal cloning

Somatic cells from the skin of animals.

An unfertilized egg is isolated from the surrogate mother.

The egg is enucleated, through irradiation or suction of the nucleus using a micropipette.

The enucleated cell is fused with the somatic cell/nucleus, and an electric pulse is used to fuse these cells. Microinjection can also be used for SCNT.

The fused cell is then allowed to divide and develop in the embryo.

This is then transferred to the surrogate mother.

The embryo develops into a clone of the organism.

Animal cloning
( SCNT)

Uses of animal cloning

Animal cloning is used to

Improve cattle variety.

Increase milk and meat production.

Produce disease-free animals.

Studying gene expression.

Study gene therapy.

Improve food production.

Limitations of animal cloning

The clone produced is not a true copy of the animal.

As fur depends on the gene being expressed in an individual cell.

Aging occurs in the cells depending on telomer shrinking, cells cease to divide if the telomere shrinks excessively.

X- chromosome expression differs in somatic cells.

The success rate of animal cloning is very low. Dolly was produced after 276 unsuccessful attempts.

Disease-free animals are not produced through animal cloning as somatic cells become immunologically older.

Animals produced through genetic engineering are not allowed to be used as human food as their long-term effect on health is not known.

EUKARYOTIC DNA PACKAGING

DNA is the genetic material. It is a large biomolecule. Eukaryotic DNA is present in the nucleus. It has millions of base pairs. The total length of DNA is 1.8 m in a cell. Such a large molecule is to be packed in the nucleus of an 8 micrometer in size.

Metaphase DNA is packed into chromosomes that are visible through the light microscope. This packaging occurs approximately 10,000 times. The original thickness of the DNA strand is the two-nanometer. The thread of DNA is packed into a 1400 nanometer thread which is the breadth of the chromosome. This packaging requires multiple types of proteins that are associated with DNA molecules.

The first level of Packaging of DNA: Nucleosome

The 11-nanometer structure is known as a nucleosome. This structure requires a special type of protein those are known as histone proteins.

There are five types of histone proteins which are named H1, H2A, H2B, H3, and H4. The histone proteins are positively charged due to their composition of amino acids. The amino acids present in histone proteins are lysine and arginine, these give histone proteins a positive charge.

Eight histone proteins form the core of the nucleosome. The core of the nucleosome is octamer which has two subunits each of H2A, H2B, H3, and H4.

146 base pair of DNA coil around the histone octamer, H1 histone then clip both the ends of DNA to the histone octamer. This further takes 20 base pairs of DNA.

The DNA between the two histone octamer is called linker DNA this reduces the length of DNA from 1m to 14 cm which reduces the size of DNA to 1/7. The Packaging of DNA into nucleosomes, that results in the formation of a string of beads. This is visible under the electron microscope.

30 nm fiber

Even after this level of packaging DNA is too large to be packed into the nucleus of the cell for this the histone octamer is packed into a solenoid or zig-zag structure of the 30-nanometer fiber.

In the solenoid structure, the linker DNA is present in the center. It is a compact structure.

Zig-zag

In this folding, the 30 nm fiber is formed by the zig-zag arrangement of nucleosomes. Here the linker DNA is outside the center.

CHROMATIN

This 30-nanometer fiber is then packed into 300nm and the 300 nm fiber coil around proteins to form 700 nm fiber. This is called chromatin.

The 700-nanometer fiber is then packed into a chromosome that has 1400 nanometers this thickness appears in the metaphase chromosome.

SIGNIFICANCE OF DNA PACKAGING

Interphase DNA is diffusely distributed and in form of chromatin. This is in an irregular shape.

This allows a huge amount of DNA to be packed in the small nucleus of a cell.

DNA packaging in chromosomes allows the distribution of genetic material equally to the daughter nuclei during cell division.

The numerical and structural study is possible of the metaphase chromosome.

Karyotype and banding pattern study is done on metaphase Chromosomes, this helps in identifying any genetic disorder in feotus. This study is not on unpacked DNA.

BIOREMEDIATION

BIOREMEDIATION is curing the polluted soil, air, or water using living organisms. It is eradicating the pollutants. It is a practical application of biotechnology in ecology.

The basis of strategies used for bioremediation, it is basically two types:

The in situ bioremediation

The ex-situ bioremediation

The in situ bioremediation

It is eradicating pollutants at the site of pollution. In situ bioremediation is require different strategies such as

Bioventing

Biosparging

Percolation

Air sparging

Pump and treat

Bio-slurping

Bioremediation eradicates pollutants from the environment. Microorganisms are used for this purpose. The microorganisms convert pollutants into CO2 and H2O or biomass. The pollutants are degraded to non-harmful molecules. The degradation of pollutants requires oxygen. Some ions facilitate the metabolism of pollutants. Supplying these nutrients to the site of the pollution is called bio stimulating. Improving the strain of microorganisms for better metabolism of pollutants is done with the help of Biotechnology.

Bioventing

It is bioremediating the underground water. Microorganisms present at the site can eradicate the pollutants from the underground water. But their action is insufficient. To effectively eradicate the impurities oxygen is required by the microorganisms. Oxygen is supplied to the polluted site by creating a vacuum. A well is dug on the site of action. The vacuum is created by using a pump and fans. Air passes through the vent and fastens the aerobic metabolism of the pollutants.

Biosparging/ Air sparging

This is just the opposite of bioventing. In sparging air is pumped through pipes. This aerobic facilitates metabolism.

Pump and treat

It is pumping the groundwater and then treating it.

Bio-slurping

It is vacuum enhanced dewatering technique for hydrocarbon-contaminated sites. Water is sucked through a vacuum pipe and treated.

Bioslurping is the combination of both bioventing and free product recovery. Here the pollutants belong to two different categories. It is used to treat petroleum and other hydrocarbons impurities in groundwater.

Ex-situ bioremediation

Ex-situ bioremediation is treating the polluted soil or water away from the site of pollution. It involves the transportation of soil/water at the site of treatment. The special typeS of plants are established for the treatment of polluted soil or water.

Ex-situ bioremediation includes land farming, compost piles, and irrigation, piles.

Land farming

It is the method of treatment for contaminated soil. It utilizes volatilizing and treatment of the soil using microorganisms. Contaminated soil is extracted and spread on an open farm. The volatile pollutants are vaporized and other substances are treated with the help of microorganisms.

Compost piles and irrigation

It is remediating the polluted soil by adding manure or increasing air circulation by adding wood filings. Water holding capacity is increased in the contaminated soil. Irrigation of contaminated soil increases the microbial bioremediation of contaminated soil.

Biopiles

It may be combining phytoremediation and microbial bioremediation. The contaminated soil is treated by this method. Biopiles involve engineering too. It involves irrigation, aeration, and adding manures to the contaminated soil. Biopiles are made on a farm of non-contaminated soil. The two soils are separated with the help of thick polyethylene. Aeration is done with the help of pipes. Microbes are mixed in the soil. Alginate beads are added for bioremediation. Plants are used to treat the uppermost layer.

Thuringiensis toxin as a natural pesticide

Thuringiensis toxin is produced by Bacillus thuringiensis. The toxins are called cry proteins. These are endotoxins. The bacterium is present in the soil. Thuringiensis toxin (cry protein) has been used for protecting crops against insect pests.

Tissues sprayed with this toxin kill the insect pests. This toxin is an effective biocontrol against insect pests. Through genetic engineering, the Cry gene has been inserted into plants. The transformed plants produce their own thuringiensis toxin.

Using the chemical Pesticides

The pesticides have always been erratic in their performance as;

Their concentration is not equally distributed on the plant tissues that are sprayed with the toxin

They may not be eaten by insects.

Sprayed the toxin may be washed away by the rain, so the endotoxin performs irregularly.

The use of toxic pesticides pollutes the soil.

It may be dissolved in water that pollutes the water as well.

MECHANISM OF ACTION OF CRY PROTEIN

When sprayed on the crops it has been found that Bacillus thuringiensis produce crystalline protein. This is known as cry protein this endotoxin when entered into an insect is metabolized by insect Gut enzymes which convert the inactive cry protein into active cry protein and this protein breakdown the cells of the insect gut and causes paralysis of insect gut muscles in this way this toxin kills the insect pest.

MOLECULAR MECHANISM OF ACTION

The crystalline protein is insoluble and inactive.

It is converted into soluble protein by proteases of the insect gut.

The soluble cry AC protein bind to cell membrane protein cadherin.

This can kill the gut cells by two processes.

MOLECULAR MECHANISM OF cry1AC PROTEIN

They form pores in the cell membrane. As binding of cry oligomer to the cadherin protein activates binding it to GPI anchor protein, this causes pore formation in the cell.

The cell dies due to leakage.

Another method is a cascade of events. This initiates programmed cell death.

PLASMID FOR Bt GENE

Gene for cry protein has been isolated from the bacterium this gene is under the constitutive promoter of cauliflower mosaic virus CaMV and G7 Terminator. The gene has been inserted into the plasmid and an expression vector has been created. These genes are also associated with an antibiotic-resistant marker gene.

cry1AC plasmid (simplified)

This plasmid was inserted into Agrobacterium tumefaciens. This bacterium is selected for its transformation. The bacterium is then utilized to transfer genes into the crop of interest, in this case, it is cotton.

Production of Bt plant

Transformed plants are selected through marker-assisted methods. These Transformed plants are utilized to reduce modified plants which are known as BT cotton.

CRY PROTEINS

Cry proteins are classified basically into four classes. Each of these classes is effective against the special class of insects. Cry I is effective against Lepidoptera try to be effective against Lepidoptera and Diptera Cry 3 is effective against Colepetra and Cry 4 is effective against specific Diptera.

Other endotoxins that are produced by Bacillus thuringiensis are- cry which are crystal proteins there is 126 type of Crystal protein that is produced by bacteria as endotoxin cyt proteins that are cytolytic there are 22 classes of this endotoxin, and the toxin is VIP which is vegetative insecticidal protein.

Bt cotton was first produced in 1987 it got permission to be grown in India only in 2003 Monsanto is a seed company that markets BT cotton seeds. Other crops that have been improved by utilizing the cry gene are Bt brinjal and Bt tobacco.

Drawbacks of using pest-resistant crop

The endotoxin produced in young tissue is efficient against the larva of insects. In older tissue effective production of endotoxin has not been achieved yet this endotoxin is effective against certain types of insects only. It has not found its universal application. Using terminator Technology has also made it controversial.

Cotton happens to be the first crop to receive environmental clearance as a genetically modified crop in Indian agriculture. Cotton bollworm belongs to Lepidoptera and these are sensitive to cry 1 endotoxin. The cotton bollworm reduces the yield of the cotton crop by up to 50% in India and this endemic effect Rajasthan Haryana and Punjab.

Benefits of using BT cotton

It helps in managing the bollworm infection without any adverse effect on the environment.

It reduced the use of pesticides the reduced the cost of production of the crop. It reduced the risk of cotton cropping. It reduced the adverse effects of utilizing pesticides on soil, it improved the yield of cotton. Bt gene has no adverse effect on human and cattle health. Seeds produced by cotton are used as cattle feed.

Seeds of Bt cotton can also be used as cattle feed, and they are not harmful to cattle these are easily digested by the cattle. The cry endotoxin is not harmful to cattle. This toxin was not found in cattle after eating reset escaping the gene into the environment is negligible for BT Cotton as the BT gene is inserted into tetraploid cotton with chromosome number 52 and this cotton does not breed with a wild variety of cotton as well cotton does not outbreed with other members of Malvaceae so, there is least of this gene escaping into the environment.

SOMATIC HYBRIDIZATION

Somatic hybridization is a fusion of two somatic cells for the production of hybrids.

The somatic hybrids are intra/inter-specific hybrids.

Here distant parental cells are fused to obtain somatic hybrids.

Plant cells have cell walls so it is not possible to fuse intact plant cells. Protoplasts are isolated before somatic hybridization

STEPS INVOLVED IN SOMATIC HYBRIDIZATION

A. Isolated protoplasts of two species.

B. Adhesion of protoplasts

C. Formation of a connection between two protoplasts.

D. Dissolution of the intervening membrane of the two protoplasts.

F. Fusion of the cytoplasm.

E. Formation of heterokaryon.

Different methods of protoplasts fusion

Somatic hybridization involves the isolation of protoplasts, the fusion of protoplasts, isolation of products of somatic hybridization, verification of hybridization, and culture of somatic hybrid.

Protoplasts can fuse spontaneously, chemical fusion, electrofusion, or through physical methods.

Spontaneous fusion is not favored. Other methods used to produce somatic hybrids are given below.

CHEMICAL FUSION

Calcium chloride (CaCl₂) is used for chemical fusion. Calcium ions form a bridge between the cells. So, calcium ions are used to fuse somatic cells.

NaNO₃ is used for the fusion of somatic cells.

Polyethylene glycol

PEG forms a bridge between the somatic cells.

In the plant, PEG is a fusogen that forms a bridge between the somatic cells. This ensures the adhesion of protoplast with each other. Once the adhesion is complete, the intervening cell membrane of the protoplast is dissolved. This results in the fusion of cytoplasm. The cell membrane is regularized and the heterokaryon is produced. This heterokaryon then undergoes mixing of the nucleus and results in the formation of somatic hybrids.

Electrofusion

Where the cells are fused under the current of electricity electric current of 0.5-1.5 volt is passed through the suspension medium that contains isolated protoplast. This allows charge separation of isolated protoplast. These protoplasts now behave like a dipole. They line up between the two poles. The charge is disturbed by passing a current of high voltage for a few seconds. The intensity charge is passed between the electrodes, which is 0.125- 1kVcm^-1. The high-intensity charge causes reversible membrane breakdown. This allows the formation of a contact area between the two protoplasts. Fusion of protoplast takes less than 10 minutes in the electric field. The presence of calcium chloride 1mM in the fusion mixture increases fusion frequency protoplast. The density should be 1x10⁴ protoplasts per ml for electro-fusion of protoplasts.

CHARGE SEPARATION OF PROTOPLASTS UNDER ELECTRIC FIELD

In the microchamber or microdroplet method of protoplast culture, protoplast can be fused in the microchambers. Platinum wires are used as electrodes for electrofusion. Electrofusion is more suitable for mesophyll cells than root or callus protoplasts (Pelletier 1993)

ELECTROFUSION OF PROTOPLASTS IN MICROCHAMBER

Selection of fusion products

Morpho-physiological basis of selection

Fusion product can be selected by studying the morphology of cellular hybrid callus, where the fusion product of Solanum tuberosum to Solanum ceraceifolium where one parent forms a green color callus and the other form brown yellow color callus, and the fusion product forms intermediate callus where green color and purple color cells are present.

Hybrid vigor

Hybrid vigor where cells of Dianthus chinensis and Dianthus barpatus show hybrid vigor. Hybridized cells of these parents divide and form shoots and roots vigorously.

Markar based selection or complement selection

Where metabolic deficiencies of two fusion parents are utilized to select a hybrid. The metabolic deficient parents are eliminated by themselves in the medium that doesn't contain the metabolic component. However, hybrids will survive in this medium.

The complement selection also utilizes Herbicide resistance, antibiotic resistance, or amino acid analog genes as these can be markers.

Isolation of heterokaryon

In the low-density culture where use and products are cultured in the low-density medium so that callus of different colors and morphology are selected individually in this medium.

Morphologically isolated fusion products can be manually selected through a micropipette, where the two fused protoplasts have a different color to that of their parent. This process was first determined by Hoffmann in 1978-1979.

Fluorescent isolation of heterokaryons

Dual fluorescence labeling system where protoplast are labeled by Green pigment the Fluorescein diacetate (1 to 20mgl^-1 ) which emit green color and the other set of protoplast are labeled with red color using Rhodamine isothiocyanate (10 to 20mgl^-1). This labeling is achieved by adding enzymes and the fluorescent product to the culture mixture. Manual isolation of product was done through Pasteur pipette.

The dual-labeled products can also be isolated using FACS where cells are sorted on the basis of the wavelength they emit. Due to their staining, different wavelengths are emitted by the cells. Thus the cells are selected into the different containers by FACS.

Verification of hybrid product

It is done to study morphology where flower color or expression of leaf variegation can be used to determine the hybridization of the fusion product.

Cytological analysis

Where the number of chromosomes is estimated for hybrid cells. The chromosomes number could be multiple of the number of chromosomes that were present in parents.

Isozyme analysis

Here the isozymes that are present in two different parents that reflect different band patterns are studied through molecular isolation. Isozymes studied for hybrid verification are Phosphatases, esterases, peroxidases, and phosphoglucomutase.

DNA analysis

Restriction enzyme polymorphism studied for hybrid product establish the hybrid ability or DNA fingerprinting can be used for hybrid products.

The fate of the genome of hybrid

The fate of the genome depends upon the number and type of cells fused.

Genome segregation occurs during cell division, after fusion genome segregation during regeneration of the plant.

Inter-parent recombination of plastid-genome occurs rarely so plastids are selectively eliminated in hybrid products.

The nuclear chromosome may be selectively eliminated from the hybrid products, so this may result in the formation of hybrids and result in a novel combination of the plastid-mitochondrial genomes.

USES OF SOMATIC HYBRIDIZATION

Tomato hybrids have been developed that are resistant to TMV and spotted wilt virus.

Environment tolerance and stress-tolerant plants can be developed through hybridization.

The high-yielding plants can be developed through symmetric hybridization.