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Unit 5 – Biotechnology Class 12 Biology Notes

5.1 Introduction, tissue culture, plant breeding, disease resistance plants, green manure and biofertilizer, bio-pesticide, genetic engineering and GMOs (genetically modified organisms) and application, bioengineering, food safety and food security. (7 teaching hour)

Biotechnology

The term biotechnology is derived from the fusion of 2 words biology and technology. Biotechnology consists of the controlled biological agent such microorganism, cellular compound for beneficial use (USA national science foundation).

Biotechnology is 'use of living organism in system or process for manufacture of useful products'. It may involve algae, bacteria, fungi, yeast of higher plants + animals or isolated compound from living matter. (Gibbs + Green 1983)

Tissue Culture

Plant tissue culture is one of the important aspect of biotechnology. It is a method of culturing of excised plant part called explant. In an artificial medium central + aseptic environmental condition. Tissue culture is developed on the principal of totipotency (ability of living vegetative cell to develop into complete plant). Haberlandt develops the idea of totipotency.

Types of plant tissue culture:

1. Callus culture: When callus + undifferentiated mass of cell is used as explant for the development of new mature plant is called callus culture. It can be used to regenerate different part of plant such as root, shoot + somatic embryo (unfertilized).
2. Shoot tip culture: In this technique tip of shoot either axillary or apical part is taken as explant and is grown in suitable medium. After their growth they are transfer to rooting medium + then to soil.
3. Protoplast culture: It is a living part of the cell. It can be obtain after removing cell wall from the plant. For this it is treated with many chemicals + enzymes that degrade cell wall + help in the fusion of protoplast.
4. Anther culture: Anther which pollens grains are used as explanted. The plant regenerate from anther culture are haploid. If there genome is doubled, homozygous diploid plant are produced which very much beneficial in plant breeding.
5. Meristem Culture: In meristem culture meristematic tissue of root or shoot tip is isolated + culture on the artificial media to regenerate plant. The meristem culture is very effective means of developing virus-free plant.
   Ex: Potato, banana.
6. Embryo Culture: Here embryo is used as explant. This culture is useful in overcoming the dormancy of seed for producing viable plant from the seed which fails germinating due to immature embryo.
7. Root culture: Root of several plant is cultured in liquid medium. Similarly infection of culture root by rhizobium can be done that helps in the formation of nodule (nodulation).

Method of tissue culture

The tissue culture involved following essential step:

1. Selection of plant: First step involved the selection of plant and part of plant is used as explant. Ex: shoot tip + anther culture.
2. Preparation of culture media: For the in-vitro culture of plant, artificial medium is used to be prepared because plant grown in vitro mainly depends upon nutrient content in medium. The media selected should contain the required amount of all essential elements. The principal compound of most plant tissue culture are inorganic nutrients such as N, P, Sulphur, Zinc, Copper, Carbon source such as sucrose, organic supplement such as vitamin, thiamine, growth regulator such as auxins, gibberellins. Agar is used as solidified medium + pH 5 to 6 is maintain.
3. Sterilization: The method of killing unwanted microbial organism is called sterilization. Sterilization is the process of making the material free from pathogens. In vitro plant are very susceptible to infection. If the pathogens enter into the culture vessel the whole plant inside the vessel are infected + lost. The resource of infection may be explant, instrument, glassware + hand of person or culture room. To prevent from contamination everything should be sterilized such as autoclave (dry or wet heat), chemicals to prevent from UV radiation.
   • 1) Lab: UV rays or laminar flow fumigation
   • 2) Glassware: hot air oven 160°C
   • 3) Autoclave: for petriplates, pipettes, media
4. Micropropagation: By tissue culture, any part of tissue of plant can be regenerated or multiplied in large numbers. The technique of regenerating inside glass vessels is termed as micropropagation. It involves the following stages:
   • a) Establishment of culture (Inoculation): During inoculation, the sterilized explant is transferred to the sterilized media with the help of sterilized forceps.
   • b) Multiplication: According to the type + proportion of growth hormones, the explant is kept in culture. Now it gives multiple shoot or callus (undifferentiated mass of cells).
   • c) Rooting: After development of microshoot in desired numbers they are transferred into rooting media which facilitates the rooting.
   • d) Transplantation: The in-vitro plant have tube-grown in soil. As the plants are very delicate + cannot tolerate the external environment directly so they are transplanted first in greenhouse (humidity + temp is maintained) + then to soil.

Significance / Importance of Tissue culture:

1. This method helps in producing disease free plant.
2. Embryo culture is useful in overcoming dormancy of seed.
3. Plant tissue culture has been an alternative mean to produce industrially important natural products such as drugs, insecticides.
4. Tissue culture technique is applied as the effective mean for ex-situ conservation.
5. Producing large amount of plant from small tissue.

Plant breeding

It is a method of improving heredity of crops so that new generation of crops variety are far better than original types in all aspect. Plant breeder are the people who do plant breeding.

Objective of plant breeding

1. To evolves higher yielding variety.
2. To produce the crops those has better taste + give quality products.
3. To obtain plant of which products can be stored for long time.
4. To obtain the varieties that are resistance to pest, insect + pathogens.

General method of plant breeding

It involved following method:

1. Introduction: This is a very simple method. Here people can take variety of plant with better qualities to new place where it has not been grown before (new habitat).
2. Selection: It is a process of choosing best one among the varieties which are fit, survive + continue its generation. This is made artificially. It is done by following process:
   • a) Mass Selection: In this method a large no. of plant of similar phenotype are selected. Cross pollinated + mixed together. This selected seed are sown + population is increase so as to get superior variety.
   • b) Pure-line selection: In this method the individual plant are selected which are better than other plant. They are allowed to self pollinate + progeny are tested for superior quality separately. Seed are kept separately + sown separately.
   • c) Clonal selection: Clonal is the progeny of a plant produced through vegetative propagation (clonal is genetically similar). In clonal selection method, the clonal with superior character are selected + multiply vegetatively. This process is repeated until individual with desire character are obtain.
3. Hybridization: It is the process of crossing or meeting of two individual of different genotype for producing hybrid i.e. new variety.

   Process of hybridization:

   • a) Emasculation: The first step is the selection of desire parent from the population then the selected plant are self crossed to obtained pure lines. To prevent self pollination in female parent, the stamen are removed before anther rip. This process is called emasculation.
   • b) Bagging: To prevent foreign pollens to come in contact with the stigma, the entire flower are kept in plastic bags + are called bagging.
   • c) Pollination: When stigma of emasculated flower is mature then bag is temporarily removed + the stigma is pollinated by mature pollen from male parent artificially. The pollen grains are dusted on stigma.
   • d) Tagging: The crossed flower are properly labelled or tagged soon after pollination + the flower again bag.
   • e) Raising of hybrid plant: The seeds are harvested + sown. The plant obtained from such seed are F1 plant (hybrid) so these hybrid plant grown several years.
   • f) Selection + testing of superior recombinant: This is a most crucial step + required careful evaluation of progeny. This superior hybrid is then pollinated until there is stage of homozygosity so that hybrid traits are not segregated + pure line is obtain.
   • g) Testing, Release + Commercialization of new cultivars: Selected line are tested at researcher field for yield + traits of quality + disease resistance + verify scientifically. Testing are done at field for 5-10 years + performance was recorded. If the plant are good they are produced in mass + distributed to the farmer.

* Mutation breeding:

Breeding done by inducing mutation to get new variety resistant to disease which is induced by mutagen either chemicals (colchicine) or physical agents X-rays, Gamma rays, Beta rays etc.
Ex: polyploid mutation increase yield of seed, seedless fruit.

Disease resistance plant + their production:

Farmer faces several problem on growing crops among them plant disease is the major disease which is caused by different agent like viruses, bacteria, fungi, protozoans. They can be treated by medicine like pesticides + biopesticides which is not effective in all cases so biotechnology has developed the techniques to produce disease resistance plant.

Disease resistance plant has capacity to resist particular disease therefore scientist have developed like hybridization, genetic breeding, mutation for the development of resistance variety of crops. Some important variety are cultivated in Nepal are:

• 1) Wheat - RR21, NP120, Kalyan-227, Po-18
• 2) Rice - Mansuli, Basmati 370, Rusia-1332, Savitri, Pokharelo masino etc.
• 3) Maize - Rampur yellow, Kakani yellow, Khumal yellow.
• 4) Potato - Kufri-Jyoti, Kufri-Red, Shakti etc.

Gene: DEE-GEE-WOO-GEN (Taiwan)
at IRRI (International rice research institute in Philippines).
Ex: Jaya developed from IR-8 and Taichung native.

Some of steps which is used to produced disease resistance plant are as follow:

1. Cross breeding: This is a effective way to combine 2 variety together. In this condition the hybridization technique is used to incorporate the resistance with good quality in economically important plant. Ex: Pest, drought + several disease resistance varieties have been developed.
2. Tissue Culture: The tissue culture technique has provided support for manipulating the character of the plant. Some of steps are listed as below:
   • a) Meristem culture: This technique is followed obtained virus free plant. Meristem consist of cell in dividing stage which required energy + enzymes. Viruses are obligate parasite that also need enzymes of living cell to replicate. Due to competition in enzymes in meristem, viruses can't replicate in meristematic tissue. Another reason of meristem being viruses free there is no vascular tissue developed there. Therefore plants produced by this technique are virus free and healthy.
   • b) Somatic hybridization: In this technique protoplast of commercial variety into which disease resistance gene has to be incorporated + other variety other inferior quality is fused artificially. In this way desire variety is produced.
   • c) Somaclonal variation: The plant regenerated from tissue culture may show various variation which is called somaclonal variation. The resistance against a particular disease or test is developed in such plant.
   • d) Genetic engineering: The desire gene (like disease resistance gene) are inserted into the genome of a commercially important plant. For this desire gene is isolated from an organism + it is multiplied + then transferred into the tissue of desire variety either by bacteria (Agrobacterium) or by other physical chemical method. Then the tissue with incorporated new gene is culture + the plant is desire gene is grow such plant called transgenic plant which is disease resistance.

Green manure + Biofertilizers

Plant need several kinds of nutrients for their development + grow which they get from soil. Based on their origin, manure are classified as: farmyard manure (cattle dung), compost manure (decomposing different organic matter ex leaf, rotten vegetables) + green manure.

Farmer used chemical fertilizer for the addition of particular nutrients artificially but they have disadvantages:

1. They alter chemical composition of soil.
2. They harm soil microorganism + decrease water holding capacity of soil.
3. It causes environment pollution & also changes PH of soil.

To overcome this problem organic fertilizer are used which are substance either living being or decaying matter of living being that increase fertility of soil. It is of 2 types:

a) Green manure:

These are green plant of leguminous family which can fix atmospheric nitrogen + mixed soil fertilizer if the growing green plant are directly used as manure in the field it is called green manure.

Mainly leguminous crops which are herbaceous in nature + grows fast are shown in the crop field. When they produce many leafy shoot it is ploughed + they are buried under the soil. They are soon decayed + improve the fertility of soil + increase the crops productivity. As they contain more nitrogen which is fixed by nitrogen fixing bacteria. These practice of growing green crops + mixing them into the soil by ploughing is called green manure.

The most commonly used green manure of Nepal is Dhaincha (Ghansi) - Sesbania aculeata.

Importance of green manure in agriculture:

1. They are cheap + easily available.
2. They supply essential nutrient (all type to the soil).
3. They improve aeration of soil.
4. They increases crops yield from 30 to 50 percentage.
5. They are eco-friendly because control different types environmental pollution.
6. They controls soil erosion + leaching.

B) Biofertilizers:

Biofertilizers is a substance which contains living microorganisms when supplied to soil promote the growth of the plant by increasing the supplied of nutrients to the plant.

• The main source of biofertilizer are bacteria, fungi + cyanobacteria.
• These organism improves soil fertility by adding nutrient to the soil through their metabolic activities such as nitrogen fixation, solubilization of phosphorous.
• These biofertilizer restore soil nutrient cycle build soil organic matter + increases water holding capacity of soil.

The common biofertilizers are following types:

1. Bacteria as biofertilizers: Several symbiotic + non-symbiotic bacteria helps in nitrogen fixation such as:
   • a) Rhizobium: Rhizobium occurs symbiotically if it is inoculated in leguminous plant artificially. It promotes nodulation + crop yield due to fixation of atmospheric N2 + providing to plant.
   • b) Azotobacter + Azospirillum: Azotobacter is a asymbiotic bacteria + fix nitrogen by using organic matter present in soil where as Azospirillum occurs in association with root of many plant like wheat, millet + add about 15-25 kg N/ha/year.
   • c) Phosphate solubilizing bacteria: Some bacteria like Thiobacillus, Bacillus etc convert non available inorganic phosphorous present in soil into available form which is utilized by crops and also protect plants from other harmful bacteria.
2. Algae as fertilizer: Blue green algae as fertilizer: Some of cyanobacteria, Anabaena, Nos are important N2 fixer for rice field. Approx 20-30 kg/ha of N2 is fixed by them. Thus entire need of nitrogen supply is provide through nitrogen which is fixed by blue green algae.
3. Fungi as biofertilizers: The symbiotic association between fungus hyphae + the roots of higher plants is called mycorrhizae. The mycorrhizae may be located at root surface (ectomycorrhizae) or it penetrated inside the root (endomycorrhizae). This fungi helps in absorption of mineral and water as well as phosphorus protects the plant against soil pathogens.

Advantage:

1. They are cheap.
2. They lead to soil enrichment.
3. They are sustainable i.e as biofertilizer once use they replicate themselves and remain active for many years + maintain soil fertility.
4. They help in high quality yield.
5. They do not possess any harm to soil, water & air that is ecofriendly nature + pollution free.
6. They control and inhibit pathogenic soil bacteria.

Disadvantage:

1. They do not show quick response as chemical fertilizer.
2. Biofertilizer only result 20-30% increasing crops production.
3. They do not cause increasing in oil productivity like chemical fertilizer.

Biopesticides

Biopesticides or biological pesticides is a nature product from living organism including animal, plant, nematodes + micro organism such as bacteria, viruses, fungi + protozoans. This is widely use for controlling insect + diseases causing pathogens.

Ex: Bacillus thuringiensis (Bt), Baculoviruses + neem.

Advantage of uses biopesticides:

• They are less harmful than chemical pesticides.
• They affect only target pests.
• They are ecofriendly + do not destroy other insect, birds + mammals.
• They are often effective in small quantity.
• They decompose quickly + has no side effects.

Disadvantages of biopesticides:

• They are use in combination with normal pesticides.
• High cost of productivity.
• Limited period of activity + have low potency.

c/d) Biochemical / Herbal pesticides:

Naturally occur in substance such as plant extract, fatty acid or pheromones etc. That controls pests by non-toxic mechanism. They include substance such as insects, sex pheromones that interfere with mating as well as various scented plant extract that attract insect pest to trap. Ex: Neem etc.

* Neem as biopesticides: It contains ~35 biologically active ingredient such as nimbin, azadirachtin present in seed, leaves etc.

* Jholmal: It is a handmade biofertilizer + biopesticides that help to improve crop yield with low cost. It is prepared by mixing + fermenting materials like animal urine, water, microorganism, farmyard manure, plant in a definite ratio.
Jholmal 1 = 17 kg of decomposed cowdung + 15 lit of cattle urine + 15 lit of water + 2 lit Jeevatu.

Ex: used such as Urtica dioica (Sisnu), Azadirachta indica (Neem), Artemisia vulgaris (Titepati) etc.

Types of Pesticides:

There are 3 types:

1. Microbial pesticides: Microbial pesticides contains microorganism (fungi, bacteria, virus + protozoa) as a active ingredient. It is most commonly used as biopesticides + are cheaper. They are highly selective in nature. Ex: strains of Bacillus thuringiensis (Bt).
2. Plant-incorporated protectants: Pesticides substance that plant produce from genetic material that has been added to a plant is called genetically modified crops. And when pest feeds on such plants they eventually die.
   Eg: scientist can take the gene of Bt-pesticidal protein + intro introduce the gene into plant so the plant instead of Bt bacterium manufacture the substance that destroy the pest.
3. Cloning vectors: (Vehicle DNA or Carrier of DNA) - The DNA used as a carrier.

Example of biopesticides:

1. Bacterial biopesticides: Crystalliferous spores
2. Fungal biopesticides: Pyrenomycetes, Zygomycetes, Streptomycetes
3. Viral biopesticides: Parvoviruses, Tetraviruses
4. Nematode biopesticides: Heterorhabditis genus, Steinernema genus
5. Protozoans biopesticides: Nosema genus, Vairimorpha genus

Principle of biotechnology

The two main technique that give modern biotechnology:

1. Genetic engineering (recombinant DNA-tech): It includes technique to alter the nature of genetic material (DNA + RNA) to introduce these into host organism + thus change the phenotype of then of the host organism to produce desire product.
2. Biochemical engineering: (Maintenance of sterile condition) - It involved maintenance of sterile microbial contamination free condition in chemical engineering process to have growth of only desire microorganism or eukaryotic cell in large quantities for the manufacture of bio-technological product such as anti-biotic, vaccine, enzyme, medicine + hormones.
3. Genetic engineering: So, it is modern branch of biotechnology which involves the manipulation of DNA of an organism that is remove + transfered of cell from one to another microorganism with the human need. It is also called gene cloning + r-DNA technology or gene-therapy.
   The first recombinant DNA was constructed by Stanley Cohen + Herbert Boyer in 1972. Berg (1979) introduce gene of SV-40 bacteriophage into a bacterium with the help of bacteriophage. Hence he is regarded as father of genetic engineering.

Tools of genetic engineering:

3 basic tools are required for recombinant DNA technology. These are DNA modification enzymes, cloning vectors, and host organism.

1. Enzymes: These are DNA modified enzymes:
   • i) Restriction endonuclease: They are know as molecular scissor or chemical scalpels. They can cut DNA at specific location from a plasmid as well as from foreign DNA in such a way that they leave single-standard portion called sticky end both in plasmid as well as foreign DNA. They are obtain from bacteria. In 1978 Arber, Smith + Nathan were awarded Nobel prize for discovery of this enzyme. Ex EcoR1.
   • ii) DNA ligase: DNA ligase help in joining DNA fragment. These acts as molecular glue. In DNA ligase is most often used enzymes in r-DNA technology.
   • iii) DNA-polymerase: They are also obtain from bacteria. They replicate DNA that is synthesis new strand of DNA from existing DNA hence they help in cloning of gene.
2. Cloning Vectors (Vehicle DNA or Carrier of DNA): The DNA used as a carrier for transferring a fragment of foreign DNA into suitable host is called vehicle DNA. It is also called cloning vectors or gene carriers. The desired gene is introduced into a vector where DNA is formed the vectors carrying r-DNA divides + forms several copies of r-DNA so five types of DNA are used as vehicle:
   • i) Plasmid DNA
   • ii) Bacteriophage DNA
   • iii) DNA of plant + animal viruses
   • iv) Transposons (Jumping gene): DNA sequence which change their location in the genome also called mobile gene.
   • v) Artificial chromosome of bacteria, yeast + fungi.

   Notes: Plasmid: Plasmid vectors are extra chromosomal self reflecting usually circular, double standard DNA molecule found naturally in bacteria. Plasmid is discovered by William Hey + Joshua Lederberg in 1952. The most widely use, versatile, easily manipulated vector pBR322 is an ideal plasmid vector (which is also called work house of gene cloning). This is first artificial cloning vector constructed in 1977 by Bolivar + Rodriguez.

   * Transposons were first discovered by Barbara McClintock in maize also called Ac-Ds elements (activator-dissociation) element. The transposons of Drosophila is known as P-element.

3. Host Organism or Competent host: DNA is a hydrophilic molecule so it can't passed through cell membrane. Therefore bacteria should be made competent to accept DNA molecules. The competency is the ability of the cell to take a foreign DNA.
   Ex: electroporation method, microinjection.

Technique of genetic engineering or Process of recombinant DNA technology

It involved:

1. Isolation of target gene: The DNA or gene which is going to transfer from one organism to another is called target gene. DNA containing target gene is isolated + cut with the enzymes called restriction endonuclease. They produce DNA that have sticky ends.
2. Selecting of suitable vector or cloning vehicle: Vector is a DNA which transfer DNA fragment into the suitable host. Vector should be small in size + must have site of replication. Cutting of DNA at specific location.
3. Cutting of DNA at specific location: A restriction endonuclease enzymes is used to cut the source of DNA at the specific site with the sticky end of fragment.
4. Formation of recombinant DNA (ligation): It involves the joining of target gene into vector. Vector is also cut with the same restriction enzymes both DNA have sticky end. The complementary sticky ends pairs + their ends are sealed with ligase. The DNA molecule which has both vector DNA + target gene (DNA) is called recombinant DNA (r-DNA) or chimeric DNA / Hybrid DNA.
5. Insertion / Transformation of recombinant DNA into host: It is the target gene which is transferred from from one organism to another by combining it with the vector. The r-DNA is inserted into plasmid of E.coli by process of electroporation.
6. Gene cloning: Target gene present in vector gets multiplied in host cell when host cell multiplies. So, large number of target gene can be produced then they are isolated, collected + purified.

The Application of Biotechnology in Genetic Engineering:

Human beings are using biotechnology to improve the quality of life, especially in the field of food production + health. The application of biotechnology include:

• Therapeutics
• Diagnostics
• Genetically modified (GM) crops for agriculture
• Food processing
• Bioremediation
• Waste treatment
• Energy protection

1. Medicine:

• a) Human Insulin (Humulin): Deficiency of insulin hormone causes disease diabetes mellitus. In 1983 Eli Lilly an American company prepared two DNA sequence corresponding to A + B chain of human insulin + inserted into plasmid of E.coli to produce insulin called Humulin. (Mature human insulin).
• b) Growth hormones (Human Growth hormones): It is also produced by genetically engineered bacteria which contain the human gene for the hormones. Regular injection of the hormone restore normal height in childhood.
• c) Bovine Somatotrophin (BST) hormone: hormones helps in production of large quality small dose are injected into cows every 1 to 2 week which increase production upto 25%.
• d) Antibiotics: Antibiotics are compound produced by one micro-organism which inhibit the growth of other microorganism. This process produces transgenic strains of microbes which can produce high amount of antibiotics.
• e) Gene therapy: Gene therapy is a treatment that involves altering the gene inside our body's cell to stop disease. Gene therapy replaced a faulty gene or add a new gene to cure disease + important our body ability to fight disease ex heart diseases, hemophilia, cystic fibrosis, sickle cell anemia etc.

2. Agriculture:

The use of biotechnology in the sector of agriculture is know as green revolution. The main goal of agriculture are to grow as much as food as possible. A cheaply with minimum with pest. There are 3 options:

1. Agro-chemical base agriculture: It includes use of improved varieties of crops, use of agro chemical (fertilizers + pesticides).
2. Organic agriculture: Farmer use manure, biofertilizer, biopesticides + biocontrols to increase the crops production.
3. Genetically engineering crop-based agriculture: Crops in which foreign gene have been introduced through genetic engineering are called genetically modified crops or GM crops. Ex Bt-cotton, Bt maize, nif gene, Golden rice, Hirudin etc.
   * Father of Green Revolution is Norman Borlaug.

3. Cleaning up oils spills:

Micro-organism can be used to clear up waste. So genetically engineered bacteria are capable of cleaning of oil spills. Ex Pseudomonas (It can breakdown 4 groups of hydrocarbons present in oil: xylene, napthalene, octane).

4. DNA fingerprinting:

It is a technique that is used to identity criminals + to established parentage in case of dispute. It was discovered in England by Alec Jeffreys. This technique is based on genetic engineering.

Disadvantage of Genetic Engineering:

1. Transgenic plant pollengrains may cross pollinate with natural crops + may destroy wild varieties.
2. Manipulation of gene randomly create various types of new diseases to man.
3. Functional gene may become non-functional due to genetic engineering.
4. Genetic engineering can cause mutation in organism which may be harmful for crops.
5. Genetic engineering use antibiotic-resistance gene which may create drug resistance germs.

Genetically modified Organism (GMOs):

Plant, bacteria, fungi + animals whose gene have been altered by manipulation are called GMO.

a) Transgenic plant:

It is uses to produce transgenic plant than animal. A single cell in most plant species can regenerate a whole plant with new trait. They are useful in many ways:

1. Make crops more tolerant to abiotic stresses (cold, drought).
2. Reduces reliance on chemical pesticides.
3. Helped to reduce pest harvest loss.
4. Increase efficiency of mineral usage by plants.
5. Enhanced nutritional value of food.

Examples:

1. Bt toxin: A number of Bt gene have been isolated + used for transformation of cotton, corn, rice etc.
2. Flavr Savr tomato: It was introduced by MacGregor. It is also transgenic crops + is genetically engineered that increase the shell life of tomato (polygalacturonase).
3. Hirudine: It is a gene was chemically synthesize + introduced in Brassica napus. The protein accumulates in seed from which it is isolated in the form of oil + used for medicine purpose (anticoagulant).
4. Nif gene: Nif gene are those gene taken from N2 fixing bacteria + inserted into non-leguminous plant. So these plant can fix N2 + solve problem of high cost of nitrogen fertilizers.
5. Golden rice: B-carotene gene is taken from daffodils flowers + inserted into rice which produces yellow rice enriched with vitamin A.

Transgenic Animals:

The organism which contains functional foreign gene experimentally introduced into their genome by genetic engineering from another species are called transgenic organism also transgenics. The foreign gene alters the host cell genetically + is called transgene. The production of transgenic organism is know as transgenesis. The transgenic organism are also called GMOs.

• Transgenic sheep if with better production of wool having human anti-haemophilic factor IX gene in their genome help to produce human clotting factor IX in their milk.
• Transgenic pigs - These pigs have human gene caning human antigen in their organs. The organs (heart, kidney, pancreas) of such pig can be transplanted into human without risk of infection.

Bio-Engineering:

Bio-engineering is the use of life plant or plant part to control soil erosion and mass movement of land in order to fulfill engineering function. It is also called vegetative structure, bio-technical engineering, vegetative soil conservation, engineering biology.

• This technique help to address road side slope stability problems.
• It is defined as use of vegetative measure small civil engineering structure in order to reduce the shallow seated instability.

There are different method of using vegetation to protect + stabilizes the slope. Ex: They are:

• Simple planting (ex grass seedling, planting trees, bamboos).
• Structure using vegetation (eg line of grasses or of shrub cutting in the form of brush layering).
• Composite system (eg plant jute netting or vegetated stone pitching).
• So commonly used bioengineering techniques includes fascine, Palisade, wattling, bamboo planting, grass layering, stone masonry etc.

Advantages of Bioengineering:

• Protect almost all slopes against erosion.
• Reducing the incident of shallow planar landslide.
• Providing support to the base of slope + trapping material moving downward.
• Cost effective.
• Provide beauty to the side.
• Food.

Food safety + Food security:

• The Food + Agriculture Organization of the United nation (FAO) state that food safety stands as the foundation of a healthy diet + life.
• It refers to the process of scientific organization that deal with manufacture + storage of food to prevent from food born disease.

Five keys principal of food hygiene:

• Prevent contaminating food with pathogen spreadly from people pest.
• Seperate raw + cook food to prevent contamination.
• Cook for the appropriate temp' to kill the pathogen.
• Store food at proper temp'.
• Use safe water + raw material.

Food safety Hazard:

There are 3 major hazard that can create the food borne illness:

1. Microbiological hazard: Food becomes contaminated by microorganism. Ex: Bacteria, Virus.
2. Chemical hazard: It occurs any time during harvesting procedure. Chemical used for pest control may cause food borne illness.
3. Physical hazard: It occurs accidental contamination + poor handling eg nail, hair, glass rod etc.

Food security:

The refers to access by all people at all times to sufficient safe + nutritious food for a healthy + active life. The component of food security are:

Food Security Tree:

• Availability: Domestic production, Commercial imports, Reserves + food aids.
• Accessibility: Household production, Financial resources to purchase food, Food prices + market.
• Utilization: Care + Feeding, Food preparation, Intro-household distribution, Biological utilization of food.
• Stability: Maintaining consistent food availability access + utilization despite various challenges + crises.

So, these two concept food safety + food security are strongly connected so there is no food security without food safety (FAO) or food safety is food security (UNSCN).

Food insecurity in Nepal:

• New constitution of Nepal 2075 + right to food act 2018 shows as a fundamental rights of every citizen.
• However the zero hunger strategic review shows that country still suffers from severe food insecurity + malnutrition around.
• Around 31% people in the country live below poverty level + wide spread poverty is the measure cause of food insecurity. The major reason of food insecurity are:
  1. low cash income
  2. Over population
  3. Marginal land / Small farms
  4. Resources degradation
  5. Unfavourable condition
  6. low production
  7. war + migration
  8. Food habits (Bhate culture)
  9. Poor storage + distribution