Biotechnology MohanBio


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Biotechnology MohanBio

  1. 1. • The application of technology to improve a biological organism. natural variation: Allelic differences at genes control a specific trait. • Gene - a piece of DNA that controls the expression of a trait. • Allele - the alternate forms of a gene.
  2. 2. • Central Dogma of Molecular Genetics DNA RNA Transcription Translation Protein trait or phenotype. • The application of the technology to modify the biological function of an organism by adding genes from another organism.
  3. 3. Biotechnology. • Utilization of biological entities and their component in production of some products for human welfare is called biotechnology. • The contribution of biotechnology in different field of biology are,
  4. 4. Medical Biotechnology: • Production of human insulin using recombinant DNA technology. • Production of anti biotic like penicillin erythromycin. Etc. • Production of mono clonal antibodies using hybridoma technology. • Treating defective gene using gene therapy. • Identification of immigrants. Criminals, disputed parents, missing baby etc. using DNA finger printing technology.
  5. 5. Environmental biotechnology. • Some microbes are used to treat sewage waste in water purification. • Detoxification of industrial waste are done using microbes. • Some microbes are used to reduce the percentage of oxides of sulphur in industrial effluents. • Degradation of petroleum products and management of oil spills are done by using microbes.
  6. 6. Industrial biotechnology. • Production of useful organic compounds like ethyl alcohol, lactic acid, citric acid etc. by using microbes. • Production of enzymes like amylase, lipase, protease from microbes. • Production of bio fuel like ethanol, bio gas etc. • extraction of some minerals like copper, uranium, from low grade ore using microbes.
  7. 7. Plant biotechnology or agricultural biotechnology. • Rapid multiplication of crop plants, medicinal plants, forest plants and endangered plants using tissue culture. • Production of viral and other pathogen resistance plants. • Production of haploid or polyploidy crop plants to increase yield. • Production of transgenic plants as nitrogen fixing plants, insect resistance plants etc.
  8. 8. Animal biotechnology • To develop Genetically modified animals or transgenic animals. • Transgenic cows – increase milk supply and meat • Transgenic Chickens – more resistant to infections. • Transgenic Goats, sheep and pigs – produce human proteins in their milk • To increase the heard of specific breed using invitro fertilization and embryo transfer. • Cloning of animals. • Transgenic mice – used to study human immune system • NTBT: National biotechnology board. • DBT: department of biotechnology.
  9. 9. • Application of biotechnology varies from agriculture to industry - food, pharmaceutical, chemical, bio-products, textiles, medicine, nutrition, environmental conservation, animal sciences etc. • Admission to the integrated five year M.Tech program offered by IIT Delhi and Kharagpur is through the Joint Entrance Exam (JEE). • Jawaharlal Nehru University, New Delhi conducts a combined all India level entrance examination for MSc Biotechnology program. • Candidates with Bachelor's degree under 10+2+3 pattern of education in Physical, Biological, Agricultural, Veterinary & Fishery Sciences, Pharmacy, Engineering, Technology 4Years BS (Physician Assistant Course); OR Medicine (MBBS) OR BDS with at least 55% marks are eligible to apply for MSc (Biotechnology) offered by JNU and several other universities all over the country
  10. 10. A biotechnologist may find jobs in various quarters. In India Students can mainly explore job options in the following fields: • Drug and pharmaceutical research • Public funded laboratories • Chemicals • Environment control • Waste management • Energy • Food processing • Bio-processing industries
  11. 11. • The government institutes and organizations, such as Department of Biotechnology (DBT), several agriculture, dairy and horticulture institutes offer employment. • In private sector, Drug companies in biotechnology like Dabur, Ranbaxy, Hindustan Lever, Dr Reddy's Labs that have their R & D units offer Biotechnology professional . • Even in the food processing industry, chemical industry and the textile industry. • The major companies, which hire biotechnologists, are Hindustan Lever, Thapar Group, Indo American Hybrid Seeds, Bincon India Ltd., IDPL and Hindustan Antibiotics etc.
  12. 12. • Institutes Offering B.Tech/M.Tech/PhD : • Admission to the integrated five year M.Tech program offered by IIT Delhi and Kharagpur is through the Joint Entrance Exam (JEE). • Indian Institute of Technology, Delhi • Courses Offered: B.Tech., M.Tech. in Biochemical Engineering and Biotechnology, M.S. (Research) in Biochemical Engineering and Biotechnology and Pre Ph.D. Courses • National Dairy Research Institute, Karnal • Courses Offered : M.Sc and M.Tech degree in Animal Biotechnology
  13. 13. • Indian Institute of Technology, kharagpur. • Courses Offered : B.Tech.(H) in Biotechnology and Biochemical Engineering, B.Tech.(H) and M.Tech. in Biotechnology and Biochemical Engineering, MS (Biotechnology), M.Tech. Biotechnology and Biochemical Engineering, Ph.D. (Biotechnology) • All India Institute of Medical Sciences (AIIMS) • Courses Offered : M Biotech
  14. 14. • Centre for Cellular and Molecular Biology imparts training to doctoral students in an academic program linked to the Jawaharlal Nehru University, New Delhi. Besides, the Centre also trains post-doctoral fellows though training programs sponsored by CSIR, Department of Biotechnology (DBT), and the Department of Science and Technology (DST), Govt. of India, New Delhi. • On an average at any given point of time there are over 100 such researchers at the CCMB, including guest workers from various institutions.
  15. 15. • The students enrolled in academic programs require to have strong motivation to pursue research in modern biology leading to a Ph.D degree. • The projects offered for Ph.D. cover specialized areas of Cell Biology, Molecular Biology, Genetics, Genomics, Developmental Biology, Nano biology, Plant Molecular Biology, Membrane Biology, Protein Structure and Function, Biology • of Macromolecules, Biology of Infection, Epigenetics, Chromatin Biology and Bioinformatics
  16. 16. Genetic Engineering (Gene Manipulation ) • The technique of transferring desired gene to an organism to manipulate its genome is called genetic engineering. Application of Genetic engineering: • Understanding biological events in biological courses. • Production of pharmaceutical compounds like insulin, growth hormone,etc • Production of transgenic animals. • Production of transgenic plants. • Production of pathogen and insect resistance plants.
  17. 17. Tools used in Genetic Engineering. • • • • • Desired gene Vector Enzymes: REN, DNA ligase. Host cell. Bio reactor.
  18. 18. • Desired gene: The functional or normal gene of our interest taken from donor cell. It is also known as foreign gene or trans gene. • Vector: The carrier DNA that act as vehicle to carry desired gene to the host cell is called vector. • The imp vectors used in Genetic engineering are, 1. Plasmid. 2. Phages. 3. Plant virus. 4. Animal virus. 5. Cosmids. 6. Artificial chromosomes.
  19. 19. • Plasmids: The extra chromosomal small circular self replicating DNA present in bacterial cell is called plasmid. The number of plasmid varies from 1 to 20 in a single bacterial cell.
  20. 20. Types of plasmids: • F+ plasmid: It is the plasmid that contains fertility factor. • R plasmid: It is the plasmid that contains antibiotic resistance gene. Ex: ampicillin and tetracycline resistance gene. • Col plasmid: it is the plasmid that contains col gene that synthesizes the protein colocin. The colocin kills the other strains of bacteria.
  21. 21. • Virulence plasmid: It is the plasmid that contains pathogenic gene. • Metabolic plasmid: It is the plasmid that contains gene for metabolic activity. –Ex: nif + gene. Common plasmids used in genetic engineering: • pBR 322. • pUC18. • Ti plasmid. ( tumor inducing plasmid) • Ri plasmid. ( root inducing plasmid)
  22. 22. pBR 322 plasmid: • It is the naturally occurring E. coli plasmid. • It has 4.3 Kbs. (kilo base pair size) • It contains one Ori site. ( origin of replication site). • It contains two antibiotic resistance genes. Amp+ and Tet + • It contains specific restriction endonuclease recognizing site.
  23. 23. pUC 18 plasmid. • pUC 18 was first constructed at university of California. • It has the size of 2.73 kbs • It contains one ori site. • The fertility factor is absent. • It contains ampicillin resistance gene. • It contains Lac promoter and lac Z gene. • The lac Z gene it contains 10 to 15 restricted sites for different REN. It is called MCS ( multiple cloning site.)
  24. 24. • multiple cloning site. (MCS): 10 to 15 restricted sites for different REN present in lac Z gene of pUC 18 plasmid is called MCS. Enzymes in genetic engineering: • The two imp enzymes used as molecular scissor and molecular stitchers are Restricted endonuclease enzyme (REN) and DNA ligase.
  25. 25. Restricted endonuclease enzyme • REN is the endonuclease enzyme that cuts double stranded DNA molecule at specific palindrome sequence. It is Used as a molecular scissor in genetic engineering. • REN are the defensive enzyme for bacteria. It cuts and destroys bacteriophage DNA that infects bacterial cell. • Different types of REN are identified and isolated for different palindromic sequence.
  26. 26. • Hamilton smith discovered and isolated HIND II REN from Haemophilius influenzae in 1968. He received the Nobel Prize in Physiology or Medicine in 1978.
  27. 27. palindromic sequence • The region of DNA in which two strands are identical when read in both the direction is called palindromic sequence. Ex: palindromic sequence for Eco-I is.
  28. 28. • palindromic sequence for HIND III is 5'-A |A G C T T-3' 3'-T T C G A| A-5‘ • In bacteria specific DNA palindromic sequence are methylated periodically throughout the genome. Hence REN is not effective against bacterial genome. • Foreign DNAs which are not methylated are introduced into the cell are degraded by sequence-specific restriction enzymes and cleaved.
  29. 29. DNA ligase. • The enzyme that joins the two sticky ends of DNA is called DNA ligase. It is used as molecular sitichers in genetic engineering. • DNA ligase was discovered by H G Khorna. • Dr. Hargobind Khorana was born on 9th January 1922 at Raipur, Punjab (now in Pakistan). • Died November 9, 2011 (aged 89)Concord, Massachusetts, U.S. • In 1968, He was awarded the Nobel Prize in Physiology or Medicine for the interpretation of the genetic code and its function in protein synthesis 1922 - 2011
  30. 30. • Host cell: The cell to which desired new gene is introduced is called host cell. Any living cell can be used as host cell. Commonly E.coli bacterial cell is used as host cell in genetic engineering. Because, 1. It is a simple prokaryotic cell. 2. It is a non-pathogenic bacteria. 3. It can be cultured easily in laboratory condition. 4. It has very short life span. 5. It contains self replicating plasmid. 6. The plasmid of E.coli can be easily handled as vector.
  31. 31. Bioreactor • It is an apparatus for culturing organisms like algae, fungi, bacteria, or animal or plant cells under controlled conditions. • It is used in industrial processes to produce pharmaceuticals, vaccines, or antibodies. • It give the cells a homogeneous and controlled environment by ensuring the same temperature, pH, and oxygen levels.
  32. 32. Bioreactor Components • • • • • Bioreactors consist of: Vessel Agitator Sparger inlets to maintain – – – – Temperature. Dissolved Oxygen pH Pressure Gauge • Ports for input and output of material
  33. 33. • Bioreactor consists of vessel which holds the media and the cells. It can be made of glass, stainless steel, or a durable plastic. • An agitator or stirrer is fixed inside to mix the contents in the vessel. Mixing of the contents is to maintain a constant nutrients and oxygen to the culture.
  34. 34. • The sparger is an apparatus used to introduce gasses into the vessel. It aerate and supply oxygen to the contents in the vessel, as well as to the cells. • Bioreactors has inlets to monitor the culture in the vessel. Useful inlets are foam control system and pH control • Cooling jacket with water circulation maintains the temperature. • It contains additional ports to introduce and remove materials from vessels. The outlet is present at bottom to collect product.
  35. 35. Application of bioreactor. • It is used to culture microbes like bacteria, fungi, algae or plant cell or animal cell. • It is used for the production of single cell protein. • It is used for culturing genetically modified microbes for production of antibiotics, pharmaceutical compounds, vaccines etc. • It is in the production of primary metabolites from microbes.
  36. 36. Recombinant DNA technology: • The technology of incorporation of desired gene to the vector DNA and transferring it into host cell is called r-DNA technology. Steps involved in r-DNA technology: 1. Extraction of DNA or isolation of gene. 2. Selection of vector. 3. Gene splicing. 4. Transfer of r-DNA to the host cell. 5. Culturing of transformed host cell.
  37. 37. Extraction of DNA or isolation of gene: • The cells of organism that contains desired gene are collected. • The DNA of these cells is extracted by using refrigerated centrifuge technology. • The isolation of gene is done by shoot gun method. In this specific REN is used to cut and isolate desired gene. The isolated gene contains two sticky ends.
  38. 38. • Complementary DNA ( c-DNA) • c-DNA is used instead of isolating desired gene. In this m-RNA is transcribed from desired gene is used as templet to syntheses of DNA using reverse transcription.
  39. 39. • The single stranded DNA is later converted into double stranded DNA. • The DNA synthesized by reverse transcription of m-RNA using reverse transcriptase enzyme is called c-DNA. • Artificial gene: The DNA synthesized with reference to the number and sequence of amino acids of protein chain in laboratory condition is called artificial gene
  40. 40. • Multiplication of gene: The isolated desired gene is multiplied into millions of copies using polymerase chain reaction.
  41. 41. Selection of vector: • Vector is a vehicle that carries desired gene into host cell. Depending on host cell vectors like plasmids or phages are selected. Gene splicing: • Incorporation of desired gene into vector to develop r-DNA is called gene splicing. • The REN is used to cut the vector at specific restricted site to insert desired gene. Later it is ligated by DNA ligase. • The vector with desired gene is called r-DNA.
  42. 42. Transfer of r-DNA into host cell: • The bacterial cell ( host cell) and r-DNA are made to suspend in cold (5-6 0C ) calcium chloride solution. After some interval of time, the temp of solution is suddenly raised to 42 0C and again cooled. • The increase in temperature increases the pore size of bacterial membrane. Through this pore r-DNA enters the bacterial cell .
  43. 43. Culturing of transformed host cell: • The transformed host cells are screened with antibiotic to select r-DNA transformed cells. These cells are isolated and cultured in bioreactor.
  44. 44. Human insulin • Insulin is a protein natured hormone that maintains sugar metabolism. It converts the excess of blood sugar (glucose) into glycogen to maintain normal sugar level. • This hormone is secreted by β-cells of islets of Langerhans present in pancreas. • The deficiency of insulin increases the blood sugar level and causes diabetes mellitus.
  45. 45. • The diabetic patients are treated with hypoglycemic oral drug or insulin injection. • The oral drug stimulates the β–cells to secrete insulin. • In previous years insulin extracted from cows and pigs are injected to control diabetic condition. It causes allergy to most of the patients. • The r-DNA technology gives solution to overcome this problem by producing human insulin (humulin) using human insulin producing gene.
  46. 46. Production of human insulin by r-DNA technology. • • • • • • • Tools required. Proinsulin gene. vector pUC 18. REN HIND – III DNA ligase. Host cell- E.coli. Bioreactor.
  47. 47. • m- RNA of proinsulin is used to produce complimentary DNA by reverse transcription process. • The m-RNA of proinsulin is treated with reverse transcriptase enzyme and deoxyribo nucleotide to get c-DNA. • Single stranded c-DNA hybridized to get double stranded c-DNA. • The c-DNA of proinsulin is incorporated with pUC18 with in lac Z gene using REN HIND III and DNA ligase.
  48. 48. • r-DNA and host cells E.coli are made to are suspend in cold (5-6 0C ) calcium chloride solution. After some interval of time, the temp of solution is suddenly raised to 42 0C and again cooled. • The increase in temperature increases the pore size of bacterial membrane. Through this pore rDNA enters the bacterial cell. • The E.coli are screened to ampicillin to isolate transformed cells. • The transformed E.coli are cultured in bioreactor to produce proinsulin. • The transformed E.coli produces proinsulin along with β–galactosidase.
  49. 49. • The fused proinsulin from β-galactosidase is isolated by treating with cyanobromide (CNBr). • The proinsulin is inactive form and contains α, β and c chain. • It is treated with proteiolytic enzymes trypsin and carboxy peptidase to remove c-chain • The product obtained is functional insulin having α and β - chains bounded by two disulphide bond.
  50. 50. Application of r-DNA technology. 1. 2. 3. 4. 5. 6. 7. 8. 9. In production of human insulin to treat diabetes mellitus. In production of growth hormone to treat dwarfism. In production of blood clotting factor VIII to treat hemophilia. In production of interferon's to treat viral disease and cancer. In production of vitamins, enzymes, amino acids for commercial use. In production of alcohol. In production of GMO plants as golden rice, BT plants, insect resistance, viral resistance, plants. In production of GMO microbes to clean environment pollutant. In production of GMO microbes to extract metals from low grade ore.
  51. 51. DNA finger printing technology. • The technology used for identification of individual at genetic level is called DNA finger printing technology. • This technology was first developed by alec Jeffreys, Wilson and Thein in 1985. Born 9 January 1950 (age 62) Oxford, United Kingdom
  52. 52. • The principle is based on matching of VNTRs of DNA collected at crime spot with suspect person DNA. • VNTRs: Variable number of tandem repeats. It is also called as mini satellites. • The identical and repeated sequence of nucleotides present adjacent to each other in DNA is called VNTRs. • VNTRs are very specific to individual and differs from person to person. It shows some similarities between family members. • VNTRs of identical twins are same. Hence it is not possible to identify individuality in identical twins by DNA finger printing technology.
  53. 53. Application of DNA finger printing technology. 1. 2. 3. 4. 5. It is used to identify criminals and rapist. To solve parental dispute. To solve immigrant problems. To identify dead bodies of soldiers died in wars. To identify dead bodies of person died at accidents and bomb blast. 6. To identify racial groups. 7. To detect inheritable disorders. 8. To detect donor cell in case of transplantation.
  54. 54. Steps involved in DNA finger printing technology.
  55. 55. • The DNA is isolated from the sample of blood cells, hair root cells, semen or bone collected at crime spot. • The DNA of suspect also collected and isolated separately. • The isolated DNA is treated with REN to cut into number of fragments. • The DNA fragments are separated according to their length on gel slab using gel electrophoresis. • The DNA strand on gel slab is treated with alkaline solution to split double strand in to single strand.
  56. 56. • The single strand DNA is transferred to nylon sheath using southern blotting technology. • The single stranded DNA is hybridized with radioactive probes of VNTRs . The excess of probes are washed off. • Nylon sheath is X-ray photographed to get bands of VNTRs. • The bands of X-ray sheath is the DNA finger print. • Comparing the DNA finger print of sample collected at crime spot with suspect identifies the individuality.
  57. 57. • Southern blotting: The technique of transferring DNA from agar gel to nylon sheath is called southern blotting. • Probe: Single stranded polynucleotide fragment complementary to specific sequence of nucleotides of DNA is called probe. It is mainly used in identify VNTRs and desired gene
  58. 58. Gene therapy • The technique of replacement of defective gene by normal functional gene to treat genetic disorder is called gene therapy. Two ways of gene therapy: 1. Invivo approach. 2. Invitro approach. • In invivo approach normal functional gene is directly transferred to the target organ of patient. • In invitro approach the defective cells are cultured in lab condition. The normal gene is transferred to this cultured cell. The genetically modified cell or tissue is transplanted to patient.
  59. 59. The disease cured by gene therapy; 1. SCID: Sevier combined immune deficiency syndrome. 2. Cystic fibrosis. 3. Muscular dystrophy. Types of gene therapy: • Somatic gene therapy: It is the replacement of defective gene by normal gene to somatic cells. It is non heritable. • Germ line gene therapy: It is the replacement of defective gene by normal gene to germ cells. It is to be done to avoid the inheritance of defective gene to the next generation.
  60. 60. Methods of gene therapy 1. Viral method. 2. Non viral method. • Viral method: in this method retrovirus are mainly used as vector to transfer functional gene to the target cell. Steps involved in the viral method are, • Selection of specific retrovirus that infect target cell. • The virus used as vector is trimmed by removing harmful pathogenic genes. • The desired gene is incorporated into the vector. • The vector is made to infect the target cells.
  61. 61. • Non viral method: Number of non viral methods are used to transfer functional gene. Some of them are, 1. Microinjection. 2. Electroporation. 3. Calcium phosphate mediated transfer. • Micro injection: In this method functional gene is directly transferred to target cell using micro injection.
  62. 62. • Electroporation: In this method isolated cells are subjected to low voltage electric shock. It causes cell membrane to become permeable for exogenous DNA. • Calcium phosphate mediated transfer: In this method functional gene is mixed with calcium phosphate. This mixture is introduced near target cells. The calcium phosphate disturbs the cell membrane and makes permeable to exogenous DNA
  63. 63. Embryo gene therapy through IVF-ET ( invitro fertilization and embryo transfer) In this method gene therapy is given to the embryo through IVF-ET. The steps involved are, 1. The gamete ovum and sperm are collected from defective gene carrier patients. 2. The gametes are made to undergo fertilization invitro under laboratory condition. 3. The zygote formed is incubated for three days to develop in to 8cell stage. 4. The defective gene of the embryo cells are replaced by normal functional gene. 5. The embryo is implanted back to the mother uterus for further development. 6. The baby born is free from genetic disorders.
  64. 64. Monoclonal antibodies. (MABs). • The specific antibody produced against specific mono antigen artificially from hybridoma cells is called monoclonal antibodies. • The hybridoma cells are developed by fusion of B-lymphocytes and myeloma cells.
  65. 65. Steps involved in production of monoclonal antibodies. • The specific mono antigen to which antibodies are required is injected to the mouse. • The mono antigen stimulates the immune cells to produce specific antibody. • The B-lymphocytes that produces the specific antibody are isolated from the spleen of mouse. • The isolated B-lymphocytes and myeloma cells ( tumor cells or cancer cells) are made to suspend in polyethylene glycol (PEG) solution. In this media two cells fuses and develops in to hybridoma cells.
  66. 66. • The hybridoma has the capacity to undergo uncontrolled mitotic cell division. These cells are allowed to undergo multiplication. • The hybridoma cells are screened for the ability of monoclonal antibody production. • The hybridoma cells that produces monoclonal antibodies are cultured in hypoxanthin aminopterin thymidine (HAT) for production of MABs. • Some of the cells are frozen for future use. Note: 1. PEG – Polyethylene glycol. 2. HAT- Hypoxanthin aminopterin thymidine . 3. ELISA - Enzyme linked immune sorbent assay. 4. RIA - Radio immune assay.
  67. 67. Application of monoclonal antibodies. 1. MABs are used for identification of cancer cells, pathogens, enzymes, hormone assay etc. 2. It is used in ELISA and RIA to measure circulating level of hormones and enzymes. 3. The specific MAB is used for identification of HIV by ELISA test. 4. It is used to Identify pregnancy by assaying pregnancy hormone HCG in urine. 5. It is used to identify A, B, AB and O blood groups. 6. It is used to identify sexually transmitted diseases. 7. It is used to treat cancer. 8. It is used as immune suppresser in organ transplantation. 9. Herceptin: Genetically engineered monoclonal antibody used to treat breast cancer.
  68. 68. • U.S. govt. started Human genome project in 1986 coordinated by the Department of Energy and the National Institutes of Health. • GENOME – The whole hereditary information of an organism that is encoded in the DNA is called genome. Aims or goal of the project: • To identify the approximate 35,000 genes in the human DNA. • To determine the sequences of the 3 billion bases that make up human DNA. • To store this information in databases. • To develop tools for data analysis. • To address the ethical, legal, and social issues that arise from genome research.
  69. 69. Achievement of HGP • The project achieved to identify 35000 genes in human beings. • They sequenced about 3.2 billion base pairs in 23 pairs of chromosome • Almost all (99.9%) nucleotide bases are exactly the same in all people. • The functions are unknown for over 50% of discovered genes. • Chromosome 1 has the most genes (2968), and the Y chromosome has the fewest (231)
  70. 70. Application or Benefits of HGP • It helps in understanding human genome biology and human genetics. • It helps to identify the gene associated with genetic disorders. • It helps in improving medicine and drugs. • It helpful in giving gene therapy. • It helps in studying human migration and evolution.
  71. 71. Improvement of crop plants • India planed green revolution in 1968 to over come from the problem of scarcity of food and starvation. • It achieve its aim in 1978. • Dr. swaminathan, Dr. w.K.Jain. Dr. Partha sarthi and other agricultural scientist contributed their work in green revolution. • In this project crop plants are improved as high yielding, disease resistance drought resistance etc.
  72. 72. Indian organization for crop production. • • • • • IARI – Indian Agricultural research Institute. ICAR – Indian council of Agricultural research. CRRI – Central Rice Research Institute. GKVK – Gandhi Krushi vignana Kendra. DAU – Darwad Agricultural university
  73. 73. Plant breeding technique. 1. Introduction of crop plant. 2. Selection of crop plant.- Mass selection. pure line selection, clonal selection 3. Hybridization. 4. Polyploidy breeding. 5. Mutation breeding. 6. Tissue culture and development of transgenic plants.
  74. 74. • Hybridization: The cross made between two plants differing in one or more desirable characters. • Intra specific hybridization: It is the cross made between two plants of different breeds. • Inter specific hybridization: It is the cross made between two plants of different species but belongs to same genus. • Inter generic hybridization: It is the cross made between two plants of different genus belongs to same family. • Usually inter specific and inter generic hybrids are sterile. Polyploidy is induced to develop them into fertile.
  75. 75. • Polyploidy breeding: The organisms having more than one set of chromosomes are called polyploidy. It is induced by spraying colchicine on seeds or seedlings. • Mutation breeding: Any change in chromosome or chromosome number or sequence of DNA leads to mutation. • Dr. Swamynathan was the first person introduced mutation breeding in India. Hence he was regarded as father of radiation genetics in India. • Mutation is induced by irradiating seedlings to xrays, ϒ-rays, α-rays, β-rays. Etc. • It is also induced by exposing seedlings to mustered gas.
  76. 76. Tissue culture and development of transgenic plants. • It is the technique of culturing cells into tissue, organ or organism on cultural media under laboratory condition. • Totipotency: The ability of a single cell develop into a tissue or organ or individual is called totipotency. • The totipotency of plant cells are more than animal cells. • Explant: Any part of the plant body or tissue that is used in tissue culture is called explant. • Usually parenchyma tissue of stem or root is used as explant. • Callus: the undifferentiated and unorganized mass of cells developed by explant during tissue culture is called callus.
  77. 77. Requirements for tissue culture: • Sterilization room: this room is used to sterilize glass equipment's and explant. It is also used to prepare media. • Incubation room: It is germ free room. The room is completely sterilized by using luminous flow bench. In this room explant is inoculated into culture media. • Culturing room: In this room culturing tubes are stored. The room is maintained by proper temp, light and optimum humidity.
  78. 78. Steps in tissue culture. • Sterilization: The sterilization of laboratory equipment's is done by washing with potassium dichromate solution. Further sterilization is done by dry heat or autoclave. • Preparation of media: The culture media is prepared as formulated by scientist. The media contains macro and micro nutrients. Essential amino acids. Vitamins. Salt and some plant hormones. • Selection of explant: The explant is a small piece of plant tissue. The parenchyma cells of stem, root, apical bud meristem, or pollen grains are used.
  79. 79. • Formation of callus: the explant selected is sterilized and cut into number of pieces. Each piece is inoculated into test-tube contain culture media. • Inoculated explant undergoes dedifferentiation and develops in to mass of undifferentiated tissue called callus. • Inducing organogenesis: the organogenesis is induced by applying different ratio of plant hormones - Auxin and cytokinin. • After organogenesis the seedlings having small roots and shoot is transferred to plastic bags containing fertile soil. • These are grown in to small plants under green chamber. Later plants are transferred into fields.
  80. 80. Application of tissue culture. • Micropropogation –production of millions of plants by tissue culture. It is applied to increase number of crop plants, medicinal plants, forest plants, endangered plants. • Production of haploid plants: anther or pollen grain culture results in development of haploid plant. The chromosomal dabbling is done to get diploid or polyploidy. • Production of viral free plants: The apical bud culture is done to develop viral free plant.
  81. 81. • Production of sec metabolic compounds: the callus culture is transferred to bio reactor for production of pharmaceutical compounds, alkaloids, colouring agent etc. • Production of transgenic plants: the desired gene is introduced into cells of callus to develop into transgenic plants.
  82. 82. Transgenic plants. • The genetically modified plants developed by transferring desired gene are called transgenic plants. • Some transgenic plants are, 1. nif plants involve in nitrogen fixation. 2. BT plants having pest resistance. 3. Golden rice plant that fulfills vitamin A deficiency. Importance of transgenic plants. • Transgenic plants are developed, 1. To improve crop plants for high yield. 2. To produce disease resistance plants. 3. To produce pest resistance. 4. To produce drought resistance. 5. To produce secondary metabolite’s.
  83. 83. Golden rice: • It is genetically modified rice plant which is rich in beta carotene a precursor of vitamin A in its endosperm. • The golden rice was first developed by Ingo Potrykus (1999). The IR-64 rice is selected to develop golden rice.
  84. 84. • The important genes transferred to IR-64 are, • PSY – Phytoene synthatase. • LYC – Lycopane cyclase. From Daffodil plant. • Ctrl-I gene – to synthesis enzymes of the biosynthetic pathway of b-carotene from Erwinia uredovora.
  85. 85. Steps in development of golden rice. • The two specific genes PSY and LYC that involves in production of b-carotene are isolated from daffodil plant. • ctrl-I gene that synthesizes necessary enzyme for production of provitamin A is isolated from the bacteria Erwinia uredovera. • These three genes are incorporated into Ti-plasmid to develop r-DNA. • This r- DNA is first transferred to bacteria Agrobacterium tumefaciens. • These bacteria are cultured to get number of cloned genes. • The transformed agrobacterium is made to infect IR- 64 rice embryo. • the infected embryos are screened for transformed genes and cultured. • The seedlings produced from these cultured embryos are called Golden rice.
  86. 86. • The seeds of golden rice are golden yellow in colour. The density of colour depends upon richness of b-carotene.
  87. 87. Improvement of animals. • Animal husbandry: It is the science of raring breeding and caring of domestic animals ( live stock). • Number of methods are applied to improve animals using the knowledge of genetics and reproductive physiology. • The cattle breeds are mainly improved by cross breeding. • Cross breeding: mating of two parental animals of different breeds to develop a hybrid is called cross breeding.
  88. 88. In cattle's it is applied to develop a hybrid in such a way that, 1. To increase the capacity of milk production. 2. To increase lactation period up to 10 months. 3. To increase reproductive capacity. 4. To develop resistance to disease. 5. To make them to adopt tropical and sub-tropical climates. • Artificial insemination: it is injecting the semen of desired bull into female reproductive tract mechanically. • Cryopreservation: It is preservation of semen in liquid nitrogen at -70 C to -196 C. The frozen semen can be stored up to 20 years.
  89. 89. Advantage of artificial insemination. • The semen collected from single ejaculation of bull can be inseminated to five hundred cows. • The semen can transmitted easily than transporting bull. • The frozen semen can be stored upto 20 years. Hence there is no need of maintaining large bull yard.
  90. 90. MOET - multiple ovulation and embryo transfer (OR) SOET - super ovulation and embryo transfer. • Super ovulation is the technique in which female cow is forced to release large number of ovum by injecting Follicle stimulating hormone. • Embryo transfer is a technique of transferring eight cell stage embryo to surrogate mother for further development.
  91. 91. • Steps involved in SOET or MOET. • Estrus synchronization: The donor and surrogate mother cows are artificially made to have same reproductive stage. It is done by injecting gonado tropic hormone. • Super ovulation: The follicle stimulating hormone is injected to donor cow to release large number of eggs.
  92. 92. • Artificial insemination: The super Ovulated donor cow is artificially inseminated by desired bull semen. • Embryo recovery: After fertilization eight cell embryos are recovered for further development by surgical or non-surgical method.
  93. 93. • Embryo transfer: The collected embryos are transferred to surrogate mother cow uterus for further development. • By this technique desired breed yard can be increased in short period.
  94. 94. • IVF- ET : Invitro fertilization and embryo transfer: • In this technique superovulated ovum are collected and fertilized in laboratory condition. • The fertilized ovum are incubated to develop into eight cell stage. • These embryos are transferred to surrogate mother for further development.
  95. 95. Stem cells: • The undifferentiated cells that have the ability to undergo mitosis and differentiation in to tissue are called stem cells. • The two types of stem cells are 1. embryonic stem cells 2. Adult stem cells. • The inner masses of cells of blastula are embryonic stem cells. • The bone marrow cells, placental cells are the adult stem cells.
  96. 96. Application of stem cell culture: • Cultured stem cells are used to differentiate into different types of cells like liver cells, nerve cells, muscle cells and blood cells etc. • The differentiated tissue cells are used to treat nervous disorders like Parkinson’s disease, Alzheimer’s disease spinal cord injury, etc. muscular destropy, cardiovascular disorders are also treated. • Cultured stem cells are used in genetic modification. • Stem cells are used to repair damage defective tissue.
  97. 97. Hazards of biotechnology. • Genetic engineering may develop new pathogen • Genetically modified microbes may use as biological weapons. • Many transgenic food causes allergy in few people. • BT toxin transgenic plants kills and decreases the population of different useful insects. • Genetically modified crop plants for resistance to weedicide can cross naturally with weeds. It becomes difficult to control weeds. • Wide spread of transgenic plants depletes the agriculture biodiversity. • Animal genes transferred to crop plants arises ethical and religious problems.
  98. 98. Safeguard of genetic engineering. • Strict laboratory procedure should follow in biotechnology labs. • Genetically engineered microbes are crippled in such a way that they cannot survive outside the laboratory condition. • The permission from r-DNA advisor committee is taken to undergo genetic work. • The permission from genetic engineering committee is taken to release genetically modified organism. • Human cloning and transgenic human experiments should be banned.